PMID- 7530354 TI - Attila the Hun versus Attila the hen: gender socialization of the American nurse. AB - Gender socialization of American nurses mirrors the socialization of women in our society. Societal influences have perpetuated the development of nursing as a predominantly female gender specific occupation. Determining how we can bridge the gap of our gender socialization and develop effective behaviors irrespective of our sex role could enhance our effectiveness as health care providers. PMID- 7530355 TI - Hepatitis B carriers. PMID- 7530356 TI - Plasma prothrombotic markers after a short- and middle term treatment with iloprost in arteriopathic patients with critical limb ischemia. AB - The authors carried out an investigation on the "short" and "middle-term" effect of the prostanoid derivate iloprost on some molecular haemostatic markers in a group of peripheral vasculopathic patients with critical limb ischemia. The series consists of 10 patients (6 males, 4 females, age 52 +/- 5) suffering from peripheral obstructive vasculopathy at the III-IV stage by Fontaine. After overnight fasting, each patient was given an intravenous infusion of iloprost lasting six hours at the rate of 2 ng/kg/min and reaching approximately the global dosage of 50 gamma; before and after the infusion a venous blood sample was withdrawn; the experiment was repeated under the same conditions after a four week treatment with the drug administered daily at the same dosage. For each sample the plasma levels of betathromboglobulin (BTG) fibrinopeptide A (FPA), tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-I) and D dimer (D-D) (ELISA, methods, kits Boehringer) were measured. The basal values of BTG, FPA, tPA, PAI-I and D-D were significantly increased compared to those of a control group; after the iloprost infusion (acute effect) significant changes of the BTG, FPA, tPA and PAI-I were not found; D-D only showed a marked reduction (p < 0.05); after the four week treatment with infusion the basal values of BTG, FPA, tPA and PAI-I resulted almost unchanged; D-D only showed a marked reduction (p < 0.05) both as regards the basal value and those after the infusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530357 TI - A "pitiful death": is it the response to a "painful life"? AB - The problem of pain and suffering in the terminal phase of illness such as cancer or Aids has not received adequate attention and has not been given valid solutions. The author tries to offer a new interpretation key for the problem starting from the Good Samaritan Novel that is considered a clear example of the capability of bringing help and assistance in concrete terms. The request for euthanasia will never be considered a superfluous problem unless a patient can receive concrete answers to his physical and emotional suffering and to the problem of his personal "quality of life". PMID- 7530358 TI - [Celioscopy in digestive oncology]. AB - In patients with suspected or patent cancer involving digestive tract organs, laparoscopy can be successfully used both as a diagnostic tool for initial diagnosis or evaluation of cancer extension and as a therapeutic tool, either in successive or in a single operation. For diagnosis, the main aim of laparoscopy is to confirm the clinical diagnosis or search for neoplastic diffusion. Diagnostic laparoscopy is particularly important in cases of advanced cancer when extension may render laparotomy unjustifiable. Needle biopsies of the liver can be obtained under visual control and non-resectable tumours can be identified. For cancer of the pancreas laparoscopic findings are highly specific, reaching 100% in cases with peritoneal carcinoma. Laparoscopy has similar advantages for staging cancer of the gall bladder which is often discovered after extension to other organs. There are few indications for cancer of the digestive tract itself although laparotomy would be more sensitive than echography or computed tomography for recognizing hepatic or peritoneal metastasis. Palliative treatment via laparoscopy is just at its beginnings and will require continued development in techniques and material. Currently, palliative hyperthermia for peritoneal carcinoma is under clinical evaluation. The future role of curative laparoscopy for abdominal cancers must be evaluated on the basis of prospective protocols. PMID- 7530359 TI - Predictive modelling of the 3-D structure of interleukin-13. AB - Several atomic structures are now available for the family of helical cytokines, which includes growth hormone as well as many of the interleukins. Using structural information from five members of this family, two alternative models of interleukin (IL)-13 are proposed. IL-13 has biological properties similar to those of IL-4 and, like the other interleukins, is a potentially important pharmaceutical target. The model of IL-13 is discussed and compared with the known interleukin structures. PMID- 7530360 TI - Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase. AB - The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+. Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E. coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E. coli RNase HI is an excellent substrate binding region because of its sequence and/or location. The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase. PMID- 7530361 TI - T cells with two functional antigen-specific receptors. AB - Although the clonal selection theory states that lymphocytes should bear only a single specificity of receptor, there is much evidence that some T cells, at least, bear two receptors. Here, we have used mice transgenic for genes encoding an autoreactive T-cell receptor (TCR) to examine the specificity of T cells bearing two functional TCRs. We find that T cells developing in mice that do not express the major histocompatibility complex (MHC) molecule recognized as self by the transgene-encoded TCR express both this TCR and a second TCR that is specific for the MHC molecules of the strain in which it arose. Thus, these T cells have two TCRs, each specific for a distinct antigen bound to a distinct MHC molecule. In contrast, when raised in mice bearing the MHC for which the receptor is specific, T cells develop that express the transgene-encoded TCR almost exclusively. Such mice are highly susceptible to autoimmune disease. Our data suggest that on most T cells bearing two TCRs, only one is specific for peptides bound to self-MHC molecules and, thus, that expression of two TCRs does not usually confer reactivity to two unrelated antigens. PMID- 7530362 TI - Cells of Escherichia coli swim either end forward. AB - Chemotactic cells of the bacterium Escherichia coli were marked asymmetrically by growth on a rich medium containing tetrazolium red. When this dye is reduced, it tends to form a refractile granule near one end of the cell, readily visualized by dark-field microscopy. In smooth-swimming cells, the marker was found with equal probability in front or behind. In wild-type cells, tumbles changed the cell orientation nearly as often as not. Some cells formed flagellar bundles at one end more frequently than at the other, but the run-interval distributions were the same either way. We conclude that the sensory system does not favor one end of the cell over the other. Thus, chemoreceptors that appear in patches at only one pole do not serve as a nose. PMID- 7530363 TI - Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. AB - We analyzed the complete mitochondrial DNA (mtDNA) sequences of three humans (African, European, and Japanese), three African apes (common and pygmy chimpanzees, and gorilla), and one orangutan in an attempt to estimate most accurately the substitution rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated with an approximately clock-like regularity. From these substitutions and under the assumption that the orangutan and African apes diverged 13 million years ago, we obtained a divergence time for humans and chimpanzees of 4.9 million years. This divergence time permitted calibration of the synonymous substitution rate (3.89 x 10(-8)/site per year). To obtain the substitution rate in the displacement (D) loop region, we compared the three human mtDNAs and measured the relative abundance of substitutions in the D-loop region and at synonymous sites. The estimated substitution rate in the D-loop region was 7.00 x 10(-8)/site per year. Using both synonymous and D-loop substitutions, we inferred the age of the last common ancestor of the human mtDNAs as 143,000 +/- 18,000 years. The shallow ancestry of human mtDNAs, together with the observation that the African sequence is the most diverged among humans, strongly supports the recent African origin of modern humans, Homo sapiens sapiens. PMID- 7530364 TI - Active gypsy/Ty3 retrotransposons or retroviruses in Caenorhabditis elegans. AB - A gypsy/Ty3-class retrotransposon (Cer1) is integrated in the DNA of Caenorhabditis elegans chromosome III. It is 8865 nt in length and has 492-nt long terminal repeats that are identical in DNA sequence. There is an exceptionally long (6819 nt) open reading frame uninterrupted by frame-shift mutations in the period since the insertion, which must therefore have been rather recent. Alignment with other gypsy-class elements and with retroviruses indicates that an env gene occupies the 3' 1.2 kb of the open reading frame. A search through GenBank has uncovered two additional gypsy-class elements from C. elegans that are very closely related in DNA sequence to this insert and are transcribed. Since gypsy of Drosophila has been shown to be an infectious element, it is possible that retrovirus-like gypsy elements are active in C. elegans. PMID- 7530365 TI - In vivo administration of endotoxin and tumor necrosis factor-alpha produce different effects on constitutive and inducible nitric oxide synthase activity in rat neutrophils and aorta ex vivo. AB - Tumor necrosis factor-alpha (TNF-alpha) inhibits release of nitric oxide (NO) in vitro by stimulating the degradation of constitutive NO synthase (cNOS III) mRNA. However, TNF-alpha is believed to be the cytokine mediator of the hypotension and upregulation of inducible NO synthase (iNOS II) produced by gram-negative bacterial endotoxin (LPS). Some in vivo effects of TNF-alpha are opposite to those which occur in vitro. This study tested the hypothesis that in vivo administration of exogenous TNF-alpha and endogenously released TNF-alpha induce iNOS II activity and inhibit cNOS III activity, and thereby mediate the acute phase effects of LPS on blood pressure and the NO system in the rat. We show that LPS produces acute phase hypotension in ketamine anesthetized rats. The hypotension was associated with elevation of biologically active TNF-alpha in plasma, increased production of RNI (NO2- and NO3- anion) in rat neutrophils (PMN) and suppression of RNI production by A23187 (1 microM) stimulated thoracic aorta (RTA) ex vivo. TNA-alpha (10(6) U/ml, iv) did not produce acute phase hypotension but initially raised arterial blood pressure and heart rate (HR), did not increase RNI production by PMN, and inhibited RNI production by A23187 stimulated RTA ex vivo. Pretreatment of rats with the immunex monomeric soluble P75 receptor binding protein for TNF-alpha (TNFsr, 0.5 mg/kg, iv) 15 min prior to LPS administration decreased circulating TNF-alpha from 92,137 +/- 12,456 U/ml to undetectable levels as determined by the L929 bioassay. However, LPS-induced increases in RNI in PMN was enhanced and LPS-induced decreases in RNI production by RTA was inhibited by TNFsr. Thus, in vivo administration of TNF-alpha does not mimic the hemodynamic and NO-inducing effects of LPS. However, TNF-alpha mediates in part LPS-induced inhibition of RNI production by RTA. Thus, endogenous TNF alpha is not required for LPS-induced acute phase hypotension or iNOS II activity. The importance of TNF-alpha in sepsis resides in systems other than iNOSII and blood pressure. PMID- 7530366 TI - Phosphatidyl-inositol-linked CD14 is involved in priming of human neutrophils by lipopolysaccharide (LPS), but not in the inactivation of LPS. AB - The results concerning LPS priming and inactivation are summarized in table 4. This table clearly shows that priming by and inactivation of LPS are mediated via different pathways, because there is no correlation whatsoever between priming and inactivation. LPS priming nicely correlates with CD14 expression. Monocytes express high levels of CD14, and are highly sensitive to LPS. Neutrophils express lower levels of CD14, and also require a higher concentration of LPS to obtain a primed state. Eosinophils express even lower levels of CD14 (if any), and these cells are not primed by LPS up to 150 ng/ml in the presence of serum (data not shown). [table: see text] The inactivation experiments were performed with LPS, but unpublished results from M. Pabst et al. show the same phenomenon with synthetic lipid A. This indicates that the inactivation of LPS is caused by a modification of the lipid A moiety of LPS. As a first step, we tested the hypothesis that the inactivation of LPS is a dephosphorylation of lipid A by alkaline phosphatase (AP). However, AP activity in isolated neutrophils of one of the PNH patients was completely undetectable. Nevertheless, these neutrophils were able to inactivate LPS. This result clearly shows that AP is not the enzyme that inactivates LPS. Secondly, we measured neutral pH 7 phosphatase both in whole cells, and in cell lysate. All cell types tested (neutrophils, eosinophils and monocytes) contained comparable levels of pH 7 phosphatase, but only neutrophils were able to inactivate LPS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530367 TI - Pulmonary injury following sepsis. PMID- 7530368 TI - The receptor(s) for endotoxin on mammalian cells. PMID- 7530369 TI - LPS, recIL1 and smooth muscle cell-IL1 activate vascular cells by specific mechanisms. AB - Vascular endothelial (EC) or smooth muscle cells (SMC) cultured in vitro respond to cytokines, such as interleukin 1 (IL1), or endotoxin (LPS) by production of IL6 or expression of adhesion molecules. Activation of the cells is mediated by specific recognition mechanisms as shown by inhibition experiments with IL1 receptor antagonists (IL1-Ra) or endotoxin antagonists in this report. IL1Ra reduced the activation of EC and SMC by recombinant IL1, as well as the activation of SMC with vascular cell-derived membrane IL1 in coculture experiments. Radiolabelled IL1 bound specifically to EC and SMC, indicating that both cell types expressed IL1 binding sites. LPS antagonists, such as lipid A precursor Ia or nontoxic LPS of Rhodobacter capsulatus reduced the LPS-stimulated IL6 production of EC and SMC and the adhesion of leukocytes to EC, but not the cytokine-induced activation. The antagonists blocked the LPS-mediated activation of vascular cells both in the presence of fetal calf or human serum. The activation of cells by LPS was dependent on the serum concentration. In contrast, activation of cells by IL1 was not serum dependent. Incubation of EC with CD14 antibodies prior to LPS stimulation in medium containing human serum reduced the activation of the cells. CD18 antibody (IB4) did not reduce activation. S-form LPS and IL1 stimulated IL6 production and adhesion differentially. Compared to IL1 stimulation (100%), S-form LPS induced adhesion to the same degree (96.4 +/- 15.5%; n = 6), however, IL6 production to a lower degree (33.7 +/- 15%; n = 6). These results show that both EC and SMC cultured in vitro respond specifically to both IL1 and LPS and that IL6 production and adhesion are regulated differentially. PMID- 7530370 TI - LPS binding protein and CD14 in the LPS dependent activation of cells. PMID- 7530371 TI - Septin, an activity in plasma that enables CD14-dependent recognition of LPS. PMID- 7530372 TI - Interaction between CD14 lipopolysaccharide binding protein and bactericidal/permeability-increasing protein during lipopolysaccharide-induced cell activation. PMID- 7530373 TI - Toxic effects of heavy metals on ionic channels. PMID- 7530374 TI - Angiogenesis inhibition: a review. AB - In this review we discuss the concept of anti-angiogenesis, which is the inhibition of neovascularization. Anti-angiogenic agents are viewed from the standpoint of their effect on various elements of the angiogenic process, including induction of vascular discontinuity, endothelial cell movement, endothelial cell proliferation, and three-dimensional restructuring of patent vessels. An effort is made to place the many different approaches to anti angiogenesis research into a comprehensible structure, in order to identify problems of evaluation and interpretation, thereby providing a clearer basis for determining promising and needed directions for further investigation. PMID- 7530375 TI - Retrovirus-mediated gene transfer in cancer therapy. AB - Retroviral vectors are one of the most promising systems for the transfer and the expression of therapeutic genes in human gene therapy protocols. This review will focus both on the advantages and intricacies of retroviral vectors themselves as well as on the application of these vector systems in experimental and clinical cancer therapy protocols. Therefore, the retrovirus life cycle and the general features of retroviral vectors, including possible targeting strategies with retroviral vectors, are overviewed. These topics are followed by the presentation of genes with emphasis on their potential as tools in somatic cell cancer therapy (cytokines, lymphokines, colony-stimulating growth factors, suppressor genes, antisense oncogenes, suicide genes). Finally, a prospect on the application of retroviral vectors will be described. PMID- 7530376 TI - 5-HT2 and D2 dopamine receptor occupancy in the living human brain. A PET study with risperidone. AB - It has been suggested that a combined blockade of 5-HT2 and D2 dopamine receptors may be superior to D2 dopamine antagonists alone in the treatment of schizophrenia. Risperidone, which has a high affinity for 5-HT2 and D2 dopamine receptors in vitro, is a new antipsychotic drug that has been developed according to this hypothesis. The aim of this study was to examine if risperidone indeed induces 5-HT2 and D2 dopamine receptor occupancy in vivo in humans. Central receptor occupancy was examined by positron emission tomography (PET) in three healthy men after oral administration of 1 mg risperidone. [11C]N-methylspiperone ([11C]NMSP) was used as a radioligand for determination of 5-HT2 receptor occupancy in the neocortex. Both an equilibrium ratio analysis and a kinetic three-compartmental analysis indicated a 5-HT2 receptor occupancy about 60%. [11C]raclopride was used as a radioligand for determination of D2 dopamine receptor occupancy in the striatum and the calculated occupancy was about 50%. This is the first quantitative determination of 5-HT2 receptor occupancy induced by an antipsychotic drug in the living human brain. The results indicate that 5 HT2 receptor occupancy should be very high at the dose level of 4-10 mg risperidone daily, as suggested for clinical use. Risperidone is thus an appropriate compound for clinical evaluation of the benefit of combined 5-HT2 and D2 dopamine receptor blockade in the treatment of schizophrenia. PMID- 7530377 TI - Interaction of antipsychotic drugs with neurotransmitter receptor sites in vitro and in vivo in relation to pharmacological and clinical effects: role of 5HT2 receptors. AB - In the introductory section an overview is given of the strategies which have been proposed in the search for side-effect free antipsychotics. Special attention is paid to the role of predominant 5HT2 receptor blockade over D2 blockade. Whereas D2 receptor blockade seems to be essential for the treatment of positive symptoms of schizophrenia, it also underlies the induction of extrapyramidal side effects (EPS). Predominant 5HT2 receptor blockade may reduce the EPS liability and can ameliorate negative symptoms of schizophrenia. We further report a nearly complete list of neuroleptics that are on the European market and eight new antipsychotics that recently entered clinical trial, 5HT2 and D2 receptor binding affinity (Ki values) and the rank order in affinity for various neurotransmitter receptor subtypes are also discussed. For the eight new antipsychotics and for six reference compounds the complete receptor binding profile (including 33 radioligand receptor binding and neurotransmitter uptake models) is reported. Furthermore, for a series of 120 compounds the relative affinity for D2 receptors and D3 receptors (a recently cloned new dopamine receptor subtype) is compared. Finally, original findings are reported for the new antipsychotic risperidone and for haloperidol and clozapine on the in vivo occupation of neurotransmitter receptors in various brain areas after systemic treatment of rats or guinea pigs. The receptor occupation by the drugs was measured ex vivo by quantitative receptor autoradiography. The receptor occupancy was related to the motor activity effects of the test compounds (measurements were done in the same animals) and to the ability of the drugs to antagonize various 5HT2 and D2 receptor mediated effects. With risperidone a high degree of central 5HT2 receptor occupation was achieved before other neurotransmitter receptors became occupied. This probably co-underlies the beneficial clinical properties of the drug. Antagonism of the various D2 receptor-mediated effects was achieved at widely varying degrees of D2 receptor occupancy, from just about 10% to more than 70%. For therapeutic application it may be of prime importance to carefully titrate drug dosages. Antipsychotic effects may be achieved at a relatively low degree of D2 receptor occupancy at which motor disturbances are still minimal. With drugs such as risperidone that produce shallow log dose effect curves, differentiation between the various D2 receptor mediated effects may be made more easily, allowing EPS-free maintenance therapy of schizophrenic patients. PMID- 7530378 TI - The dopamine-serotonin relationship in clozapine response. AB - The effects of clozapine on the dopamine and serotonin systems may underlie its atypical pharmacologic and clinical profile. To examine this hypothesis, we measured dopamine and serotonin plasma and cerebrospinal (CSF) metabolites and the relationship of these values to treatment response in 19 neuroleptic refractory and intolerant schizophrenic patients. Only a small change in the CSF and plasma homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) levels was found. However, the pretreatment CSF HVA/5HIAA ratio and, to a lesser extent, the CSF HVA level predicted treatment response. These results suggest that the modest relationship between HVA and 5-HIAA and treatment response supports the involvement of both neurotransmitters in the pathophysiology of schizophrenia. PMID- 7530380 TI - Identification of sperm antigen targets for immunocontraception: B-cell epitope analysis of Sp17. AB - The selection of immunological targets present on gametes is an important first step in the successful production of animal or human vaccines for immunocontraception. One strategy with regard to sperm antigens is to select antigens with physiological roles in gamete interaction, obtain the mRNA sequence for such an antigen, and then determine which region or domain of the molecule is available to the immune system when spermatozoa are presented to the female reproductive system. To illustrate this strategy, we have used the rabbit sperm antigen Sp17 (RSA-3), which has been cloned and sequenced. The peptide pin-block method of Chiron Mimotopes, Australia, has been used to analyse the 146 amino acids of Sp17 with various homologous and heterologous antisera. This study demonstrates that such an analysis can lead to the identification of a B-cell epitope with strong immunocontraceptive potential. PMID- 7530379 TI - Pharmacokinetics of risperidone in chronic schizophrenic patients. AB - In a randomized, crossover study, 24 schizophrenic patients received a single 4 mg dose of risperidone in caplet or tablet form. Each of the two study periods lasted 5 days. Blood samples to determine (by radioimmunoassay) plasma levels of risperidone and its major metabolite, 9-hydroxy-risperidone (9-OH-risperidone), were obtained each day. The two formulations of risperidone were bioequivalent. Following are the mean pharmacokinetics of risperidone and risperidone + 9-OH risperidone: area under the plasma concentration curve (AUC) from 0 to 96 hours; 278.0 and 716.9 ng.hr/mL; AUC from 0 to infinity, 291.9 and 762.4 ng.hr/mL; peak plasma concentration, 33.0 and 44.5 ng/mL; time to peak concentration, 1.39 and 1.78 hours; and elimination half-life, 14.93 and 23.04 hours. These results demonstrate that the active moiety (risperidone plus 9-OH-risperidone) has a half life of 23 hours and reveal a pool of risperidone (terminal half-life, 14.9 hours) that may allow twice-daily or even once-daily dosing. PMID- 7530381 TI - Human testis cDNAs identified by sera from infertile patients: a molecular biological approach to immunocontraceptive development. AB - Sera from patients with known or suspected immunological infertility were used to screen a human testis cDNA library. A total of 59 sera detected 38 unique cDNA inserts of which four were testis specific by Northern blot analyses. One of these is a testis-specific isoform of calpastatin. Five additional clones, although not testis specific, were found to be testis abundant. The number and type of clones identified by these human sera suggests a possible aetiology for immunologic infertility. The testis-specific clones will be further characterized to establish their usefulness as contraceptive vaccine candidates. PMID- 7530382 TI - Contraceptive vaccine assessment based on a murine ZP3 mini-autoantigen. AB - A summary is presented of published and some unpublished observations from studies on the immunological response of mice to a 13-mer peptide of the murine ovarian zona pellucida glycoprotein ZP3. The findings have the following implications for the design of immunocontraceptive vaccines. To be reversible, a ZP3 vaccine must not contain pathogenic T cell epitopes of ZP3, but contraception without autoimmune oophoritis may be feasible. The immune response to the ZP3 mini-autoantigen is highly variable among inbred mouse strains, suggesting that a single oophoritogenic peptide would not achieve irreversible contraception in an outbred population. The discovery of antigen mimicry at the level of T cell peptide has thrown doubt on the validity of current strategy in detecting relevant self-antigens that might cross react with vaccine immunogens and on the feasibility of fully predicting the cross-reactive autoimmunogenic potential of a peptide or polypeptide vaccine antigen. Autoantibodies directed against epitopes outside the ZP3 mini-autoantigen, produced by immunization with the pure T cell epitope, react with high affinity, with native zona pellucida, and may be useful in identifying B cell epitopes in ZP3. PMID- 7530383 TI - Self-expanding esophageal prosthesis. Effective palliation for inoperable carcinoma of the cervical esophagus. AB - Whether to palliate dysphagia in patients with inoperable cancer of the cervical esophagus is a debatable issue. We report herein a patient who underwent definitive chemoradiotherapy for cancer of the cervical esophagus, with early recurrence of dysphagia 1 month after the end of the treatment. No salvage surgery was attempted due to the poor general conditions and to the residual effects of the radiotherapy in the neck. Endoscopically, the upper esophageal sphincter (UES) was located 17 cm from the incisors, and the cranial margin of an infiltrating stricture was just 1 cm below the sphincter. After endoscopic dilatation, a self-expanding esophageal prosthesis (Ultraflex, Microvasive, USA) was placed under endoscopic and radiologic control with the cranial margin at the level of the UES. The patient promptly resumed oral feeding and 2 months later he is still on unrestricted diet. PMID- 7530384 TI - Airborne dissemination of Burkholderia (Pseudomonas) cepacia from adult patients with cystic fibrosis. AB - BACKGROUND: Burkholderia (Pseudomonas) cepacia is an increasingly important pathogen in patients with cystic fibrosis but it is unclear how it spreads from patient to patient. A study was undertaken to determine whether B cepacia could be recovered from room air occupied by colonised adult patients with cystic fibrosis. METHODS: Air samples were obtained consecutively from an enclosed room or isolation cubicle before, during, and after occupation by six patients on nine occasions using a surface air sampler incorporating contact plates with selective medium. Settle plates were also used and sputum from five patients was cultured. RESULTS: B cepacia was recovered from room air during occupation by five of six patients, the number of bacteria ranging from 1 to 158 cfu/m3 (mean 32 cfu/m3). The number of bacteria isolated was greater when patients were coughing. B cepacia persisted in room air on four occasions after the patient left the room, on one occasion for up to 45 minutes. CONCLUSIONS: The isolation of B cepacia from the air of rooms occupied by colonised patients suggests that dissemination might occur by aerosol as well as by direct physical contact with patients or contaminated environmental sites. PMID- 7530385 TI - The effects of bedrest on circadian changes in hemostasis. AB - Venous stasis occurs when people are at bedrest, because of altered venous flow characteristics. This is commonly believed to be one etiology behind the development of deep venous thrombosis (DVT). The hemostatic effects of bedrest and their possible role in DVT development have not been fully examined. We hypothesized that bedrest would lead to increases in hemostatic function and that these increases could be important in the development of DVT. Twelve non-smoking volunteers were studied during supine positioning for 36 hours. Platelet reactivity and plasma concentrations of fibrinogen, alpha 2-antiplasmin, plasminogen, thromboxane beta 2, plasminogen activator inhibitor-1, tissue plasminogen activator and neuroendocrine hormones (cortisol, epinephrine and norepinephrine) were measured at 8:00 a.m., 10:00 a.m., 4:00 p.m. and 8:00 a.m. Cortisol demonstrated an early morning increase while catecholamines were unchanged throughout. Fibrinogen, alpha 2-antiplasmin, plasminogen and platelet reactivity were no different at any time point. Fibrinolytic proteins changed over time, manifested by decreased PAI-1 antigen and activity levels at 24 h. Based upon the parameters measured, bedrest causes no increase in hemostatic function. In fact, bedrest causes the potential for enhanced fibrinolysis, that differs from that previously reported for normal activity over 24 h. This may represent a protective mechanism to counter the effects of stasis from bedrest. PMID- 7530386 TI - Desulfated hirugen (hirudin 54-65) induces endothelium-dependent relaxation of porcine pulmonary arteries. AB - Desulfated hirugen (hirudin 54-65) at concentrations from 0.1 to 2 microM was found to relax PGF2 alpha-precontracted ring segments of porcine pulmonary arteries with intact endothelium. The relaxation was associated with a pronounced increase in cGMP in the vessels. This endothelium-dependent relaxant effect depended on the extracellular calcium ion concentration and was probably due to the release of endothelium-derived NO as indicated by its susceptibility to blockade of the NO synthesis by NG-nitro-L-arginine. In the presence of indomethacin (3 microM) the maximum hirugen effect was significantly diminished by about 25%. In contrast, neither the sulfated hirugen nor recombinant desulfato hirudin at equimolar concentrations exerted endothelium-dependent relaxation. Hence, the relaxant effect did not correspond to the anticoagulant activity. Desulfated hirugen can be assigned to the group of well-known peptides causing vasodilatation via an endothelium-dependent mechanism. PMID- 7530387 TI - Nitric oxide synthase inhibition and platelet function. PMID- 7530388 TI - Dilatation of cerebral arterioles in response to lipopolysaccharide in vivo. AB - BACKGROUND AND PURPOSE: Bacterial lipopolysaccharide can increase nitric oxide (NO) production by expression of an inducible form of NO synthase. Bacterial infections of the central nervous system dilate cerebral vessels and increase blood flow. We hypothesized that topical application of bacterial lipopolysaccharide would increase production of NO, causing dilatation of cerebral arterioles. METHODS: Cranial windows were implanted in anesthetized rabbits. Windows were flushed with artificial cerebrospinal fluid, artificial cerebrospinal fluid with lipopolysaccharide, or artificial cerebrospinal fluid with lipopolysaccharide and NG-monomethyl-L- arginine (an inhibitor of NO synthase) for 4 hours. Other rabbits received either dexamethasone or indomethacin intravenously 1 hour before lipopolysaccharide treatment of cranial windows. RESULTS: Application of lipopolysaccharide in cranial windows produced marked, progressive vasodilation, with diameter increased by 58 +/- 7% (mean +/- SEM) after 4 hours. The cerebral vasodilator response was inhibited by NG monomethyl-L-arginine, dexamethasone, or indomethacin. Excess L-arginine reversed the inhibitory effect of NG-monomethyl-L-arginine. CONCLUSIONS: Inhibition of lipopolysaccharide-induced dilatation of cerebral arterioles by NG-monomethyl-L arginine and dexamethasone suggests that a portion of the vasodilation was mediated by inducible NO synthase. Indomethacin also inhibited lipopolysaccharide induced vasodilatation. These findings suggest an important role for both nitric oxide and cyclooxygenase products in lipopolysaccharide-induced cerebral arteriolar dilatation in vivo. PMID- 7530389 TI - Nitric oxide synthase inhibition and extracellular glutamate concentration after cerebral ischemia/reperfusion. AB - BACKGROUND AND PURPOSE: Transient cerebral ischemia in rats results in selective loss of neuronal viability, eg, hippocampal CA1 neurons. The neurochemical variables responsible for this selective vulnerability to ischemia/reperfusion (IR) appear to involve excitatory amino acids. In brain IR, excitatory amino acid toxicity may be modulated by endogenous nitric oxide (NO.) gas. To investigate NO. in global brain IR, we measured the effects of NO. synthase (NOS) inhibition on interstitial excitatory amino acids in rats. Changes in postischemic cerebral blood flow and blood-brain barrier function also were evaluated. METHODS: Forebrain ischemia was produced by systemic hypotension and occlusion of both carotid arteries for 15 minutes. Blood flow was restored for 60 minutes by unclamping the carotids and reinfusing with blood. A microdialysis probe was placed into the cortex and hippocampus using a stereotaxic device. Interstitial glutamate concentration was measured during IR with high-performance liquid chromatography. A competitive NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), was given intraperitoneally 30 minutes before ischemia in doses of 1, 4, and 20 mg/kg. Changes in cerebral blood flow and blood-brain barrier during IR were determined using laser-Doppler flowmetry and microdialysis with sodium fluorescein. RESULTS: Glutamate in the dialysate during IR increased transiently 10-fold and returned to baseline levels by 30 minutes of reperfusion. Animals treated with L-NAME 30 minutes before ischemia also showed increases in glutamate concentration during ischemia, but glutamate remained elevated during reperfusion. The increase in glutamate concentration during reperfusion caused by L-NAME was prevented by L-arginine. The administration of L-arginine and L-NAME together decreased extracellular glutamate concentration during ischemia. Cerebral blood flow decreased to about 5% of baseline values during ischemia but increased approximately fourfold relative to control values on reperfusion. The hyperemic responses after ischemia were not different between IR groups treated with or without L-NAME. Brain ischemia increased the permeability of the blood brain barrier to fluorescein; however, this change was attenuated by L-NAME administration at 20 mg/kg. CONCLUSIONS: NOS inhibition did not attenuate extracellular glutamate accumulation during ischemia and increased its concentration on reperfusion. The elevated glutamate concentration after IR in L NAME-treated rats did not appear to be due to either a decrease in cerebral blood flow response after ischemia or increases in local blood-brain barrier permeability. For the most part, the blood-brain barrier was spared in the immediate postischemic period by L-NAME treatment. These data suggest that NO. production may oppose synaptic excitatory amino acid accumulation and presumably excitotoxicity during IR. PMID- 7530390 TI - Identification of two independent neutralization domains on the VP4 trypsin cleavage products VP5* and VP8* of human rotavirus ST3. AB - The antigenic structure of the VP4 protein of human rotavirus (HRV) strains Wa and ST3 was studied by using a panel of Wa- and ST3-derived VP4-specific neutralizing monoclonal antibodies (NMAbs) and NMAb-resistant variants. The VP4 coding genes from three Wa and three ST3 variants were sequenced. For Wa VP4, one homotypic and one heterotypic neutralization site, at amino acids 458 and 392, respectively, were identified. For ST3 VP4, three neutralization sites were found at amino acids 72, 217, and 385 that are either homotypic or associated with limited cross-reactivity. Cross-neutralization assays using several pairs of NMAbs and resistant variants showed that Wa VP4 has at least one large neutralization domain on its larger trypsin cleavage product, VP5*, consisting of several operationally related epitopes. VP4 of ST3 has at least two neutralization domains, one located on VP5* that is operationally related to the large neutralization domains on VP5* from HRVs Wa and KU, as well as an independent neutralization domain on VP8*, the smaller trypsin cleavage product of VP4. PMID- 7530391 TI - Immunization of mice with HPV vaccinia virus recombinants generates serum IgG, IgM, and mucosal IgA antibodies. AB - To assess the utility of vaccinia virus recombinants in the development of an immune response against HPV capsid antigens, 5-week-old C57B16 female mice were administered either purified HPV 1 capsids produced by a vaccinia virus recombinant or the recombinant vaccinia virus itself. Animals were boosted at Week 4 with either agent. Mice developed a serum IgG antibody response in all the administration protocols that was directed mainly against native L1 epitopes. Mice injected initially with the vaccinia virus recombinant and boosted with purified capsids had a higher titer antibody response (P = 0.024) with more mice responding to a greater extent. All mice produced a serum IgM response that preceded the IgG response by approximately 2 weeks and lasted 1-3 weeks. The IgM response was directed against native L1 epitopes. Although no serum IgA was detected, IgA could be detected in vaginal secretions of mice that were immunized or boosted with the vaccinia virus vector. These results indicate that an extensive humoral immune response to HPV can be elicited using vaccinia virus recombinants. PMID- 7530392 TI - Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV 5 gD. AB - Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusion proteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52-126 and 165-216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV 5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92-106 (3402 epitope) and amino acids 202-213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1. PMID- 7530393 TI - Enhancement of HIV-1 proteinase activity by HIV-1 reverse transcriptase. AB - HIV-1 reverse transcriptase (RT) was found to increase the activity of HIV-1 proteinase in vitro and in eukaryotic cells. The effect of RT on proteinase activity was dose-dependent and independent of pH or salt concentration. The cleavage of sequences corresponding to all the naturally occurring cleavage sites that could be tested in vitro was enhanced. The effect of RT on cleavage was greatest at the cleavage site between RT and integrase. The enhancement of viral proteinase activity by the virus RT may contribute to regulation of the order and/or efficiency of cleavage at different sites during virus replication and maturation. PMID- 7530394 TI - Glycosylation and antigenic variation among Kunjin virus isolates. AB - Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation. PMID- 7530395 TI - T cell responses to BPV-4 E7 during infection and mapping of T cell epitopes. AB - Vaccination of cattle with the recombinant E7 protein of bovine papillomavirus type 4 (BPV-4) prior to BPV-4 infection has been shown to retard development of papillomas and accelerate their regression. To understand the mechanism of regression we have measured proliferation of peripheral blood mononuclear cells (PBM) to E7 in vitro during the course of BPV-4 infection in both vaccinated and nonvaccinated cattle. In vaccinated cattle, T cells specific for E7 could be detected at high levels shortly after challenge, whereas in nonvaccinated cattle low responses of E7-specific T cells could be detected in only a few animals at the late stages of papilloma development. Using short overlapping synthetic peptides corresponding to the E7 protein, three T cell epitopes have been identified. T1 (aa 31-59) was immunodominant and T2 (aa 70-88) and T3 (aa21-40) were minor epitopes. PMID- 7530396 TI - Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms of the RNA-dependent protein kinase PKR--determination of KD values for VAI and TAR RNAs. AB - The RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKR cDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1-243) and the full length catalytic mutant K296R(21-551) were analyzed for their abilities to bind adenovirus VAI RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pI:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1 243)] bound VAI RNA with similar affinity as the 551 amino acid residue full length catalytic mutant [K296R(1-551)]. The dissociation constant for VAI RNA was approximately 2 x 10(-9) M for both the K296R(1-551) and Wt(1-243) proteins. The K64E mutation significantly impaired the VAI RNA-binding activity as measured with the full-length double-point mutant PKR protein, K64E/K296R(1-551). Using a gel-shift competition assay, the dissociation constants of K296R(1-551) and Wt(1 243) for VAI(1-160) RNA and pI:pC RNA were comparable. By contrast, the dissociation constants of K296R(1-551) and Wt(1-243) for TAR(1-82) RNA were both about 1 x 10(-7) M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VAI RNA or pI:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity. PMID- 7530397 TI - Peptides derived from the unique region of B19 parvovirus minor capsid protein elicit neutralizing antibodies in rabbits. AB - B19 parvovirus is pathogenic in humans. The virus propagates in the bone marrow, where it is cytotoxic to erythroid progenitor cells. Antibodies appear in blood after infection and neutralize virus in vitro; infection appears to confer lasting immunity. The predominant immune response on immunoblot is to the minor capsid protein (VP1), which differs from the major capsid protein (VP2) by an additional 227 amino acids. We previously demonstrated that antisera directed to a fusion protein containing this unique region or to more limited fusion peptides of 50-100 amino acids each neutralized virus. In the current work, we tested synthetic peptides of about 20 amino acids derived from the VP1 unique region for their ability to elicit a neutralizing antibody response in rabbits. Individual peptides were covalently linked to a lysine core to produce a multivalent antigen. Animals produced antibodies to all 13 synthetic peptides, as determined by ELISA. At 12 weeks, animals injected with one of three peptides--two from the far amino terminus and the third from the center of the unique region--had produced antibodies that completely neutralized virus; by 16 weeks, antisera elicited with another four peptides also were effective. In summary, we identified regions containing neutralizing epitopes within the first 80 amino acids and amino acids 148-205 of the unique region. Our data suggest that synthetic peptides might be useful vaccine reagents for protection against parvovirus infection in humans. PMID- 7530398 TI - Linear B-cell epitopes of the NS3-NS4-NS5 proteins of the hepatitis C virus as modeled with synthetic peptides. AB - A set of 150 synthetic peptides spanning the proteins NS3-NS4-NS5 of the hepatitis C virus (HCV) was synthesized and tested with a panel of 20 sera obtained from HCV-infected patients. Of 62 peptides prepared from the NS3 region, none exhibited strong antigenic reactivity. Rather, five peptides from this region demonstrated specific reactivity with only 5-10% of anti-HVC-positive sera. Nonetheless, it is well known that the NS3 region contains strong antigenic epitopes. These epitopes appear to be modeled in a functionally active manner with recombinant proteins and cannot be mimicked properly with short synthetic peptides. This finding suggests that the major NS3 antigenic epitopes are conformationally dependent. Seven of 20 peptides prepared from the NS4 region were immunoreactive. Five peptides from this region demonstrated very strong HCV specific antigenic reactivity. Four of the five peptides belong to the recognized immunoreactive 5-1-1 region located inside the C100-3 antigen. One peptide demonstrating immunoreactivity with approximately 90% of anti-HCV-positive sera was found outside the C100-3 region at the C-terminal part of the NS4 protein. Of 68 peptides synthesized from the NS5 protein, 30 were immunoreactive. Six of the 30 demonstrated immunoreactivity with 35-50% of anti-HCV-positive sera. Thus, the NS4 and NS5 regions of the HCV polyprotein contain a large number of specific, broadly reactive, linear antigenic epitopes. The highly antigenic reactivity of the NS5 region suggests that this protein may have significant diagnostic potential. PMID- 7530399 TI - Requirement for HIV-1 TAR sequences for Tat activation in rodent cells. AB - HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process. PMID- 7530400 TI - Epitope exposure on functional, oligomeric HIV-1 gp41 molecules. AB - We have used cells infected with the HIV-1 molecular clone HX10 to study the binding of monoclonal antibodies (mAbs) to different epitopes within the extracellular domain of the HIV-1 transmembrane glycoprotein gp41. Gp41 mAb binding to the infected cells at 4 degrees was variable but weaker than the binding of an anti-gp120/V3 loop mAb and increased substantially for three of the gp41 antibodies at 37 degrees. Treatment of the cells with soluble CD4 (sCD4) at 37 degrees increased gp41 mAb binding to epitopes spanning residues 521-663, implying that these regions had probably been masked by gp120, which following interaction with sCD4 had subsequently dissociated from gp41. By contrast, the binding of a mAb to residues 662-667 which form a neutralization epitope was reduced by sCD4 binding. Another region which has been described as containing a neutralization epitope spans residues 725-750. MAbs to this region bound equally well to noninfected and HIV-infected cells, and binding was not increased in the presence of sCD4. These data strongly imply that this epitope is not exposed on the external surface of the membrane, a finding in accord with the proposed cytoplasmic localization of this region. PMID- 7530401 TI - Variation in the hypervariable region of cucumber mosaic virus satellite RNAs is affected by the helper virus and the initial sequence context. AB - The D satellite RNA (sat RNA) of cucumber mosaic virus (CMV) was previously shown to contain a region of hypervariability around nucleotide 230, in wild-type populations and in cDNA clones and progeny of one such clone (pDsat4) after passage with the subgroup I strain Fny-CMV. This hypervariable region (HVR) consists of a series of consecutive A and/or U residues. We found that variability is also generated in the HVR of transcript derived from pDsat4 after passage with the subgroup II strain LS-CMV and with tomato aspermy virus (TAV). However, the progeny differ with respect to the sequence of the HVR after passage with both LS-CMV and TAV. Another D-sat RNA cDNA clone that contains a C residue in the HVR, pDsat1, was previously shown not to develop variability in the HVR upon passage with Fny-CMV. However, when the C (position 231) was changed to an A residue, variability developed by the third passage with Fny-CMV. An additional cDNA clone derived from the B1-sat RNA, pBsat5, also contains a C residue in the region analogous to the D-sat RNA HVR and did not develop variability upon passage with either Fny- or LS-CMV. Changing this C to a U residue did not result in the development of hypervariability in the progeny of transcript from this mutant. Models to explain the generation of hypervariability are discussed. PMID- 7530402 TI - [An experimental validation of the clinical use of a nitroglycerin ointment of an original composition]. AB - Application employment of the proposed nitroglycerin ointment, original in its composition, permits the attacks of angina pectoris to be controlled, their onset to be prevented and adverse events associated with the use of nitrates to be eliminated. Therapeutic benefit becomes clinically apparent with small doses of nitroglycerin, the ointment itself is good for repeated therapeutic and prophylactic application. PMID- 7530403 TI - Regulation of the chemotactic cytokines IL-8 and MCAF and their induction in different cell types related to the skin. PMID- 7530404 TI - [Measurement of serum prostatic acid phosphatase (PAP) by Delfia PAP Kit using europium and clinical evaluation in patients with prostate cancer]. AB - Fundamental and clinical studies of serum prostatic acid phosphatase (PAP) detected by a Delfia PAP kit were performed. The system is a time-resolved fluoroimmunoassay using europium as a tracer. The lower limit of detection was 0.2 ng/ml. Sera from 54 patients with prostate cancer, 20 with benign prostatic hypertrophy, 20 with urological malignancies other than prostate cancer and 140 adult males over 46 years old were determined. From the mean + 2 S.D. of serum PAP values obtained on the adult males, 1.5 ng/ml was considered as the upper normal level of adult males. By calculating the efficiency and ROC curve using the PAP values of prostate cancer and benign prostatic cancer, 2.5 ng/ml was decided as a cut-off value of this kit. The positive rates of adult males, prostate cancer, benign prostatic cancer and urological malignancies other than prostate cancer were 0.7%, 65%, 20% and 10%, respectively. The sensitivity of stage A2, B2, C and D1 + D2 was, 0%, 0%, 64% and 83%, respectively. The efficiency of the Delfia PAP kit was 52% and that of the Markit M PA kit was 71%. The correlation between the values assayed with the Delfia PAP kit and the Dinabot PAP kit was very high; the value obtained with the Delfia PAP kit was about 80% of that obtained with the Dinabot PAP kit. PMID- 7530406 TI - Genetic heterogeneity of beta-thalassemia in southeast Sicily. AB - In this study we have defined the spectrum of the beta-thalassemia mutations, the beta-thalassemia haplotypes, and the genotype-to-phenotype correlations in a large number of patients with different beta-thalassemia conditions. Seventeen different beta-thalassemia mutations were detected which included one chromosome each with Hb Dhonburi and Hb Lepore. Five alleles, namely, codon 39 (C-->T), IVS I-110 (G-->A), IVS-I-6 (T-->C), IVS-II-745 (C-->G), and IVS-I-1 (G-->A), account for 90% of all beta-thalassemia mutations in 846 thalassemic chromosomes studied. Haplotyping for a large number of subjects showed that the five common mutations are linked to a few haplotypes. The presence of milder mutations, mainly IVS-I-6 (T C), in about 19% of our patients explains some of the clinical variables. Among the 37 patients with thalassemia of intermediate severity, only 6 were homozygous or compound heterozygous for two severe alleles. The type of beta thalassemia is the main factor responsible for differences in the phenotypic expression of the disease in patients with Hb S-beta-thalassemia; patients with Hb S-beta(+)-thalassemia are less severely affected than those with Hb S-beta(0) thalassemia. The five most frequent mutations have comparable distributions all over Sicily. PMID- 7530407 TI - Indocyanine green videoangiography of angioid streaks. AB - PURPOSE: The fluorescein angiographic features of angioid streaks are variable, and angioid streaks and their main complication, choroidal neovascularization, can sometimes be difficult to visualize in the presence of diffuse pigment migration, diffuse atrophy of the retinal pigment epithelium, or hemorrhage. The objective of the present investigation was to define the indocyanine green angiographic features of angioid streaks and to compare them with findings on fluorescein angiography. METHODS: For this prospective study, we recruited 22 consecutive patients, 21 of whom had angioid streaks and one who had typical peau d'orange appearance of the fundus. Complete ophthalmologic examination, fluorescein angiography, and indocyanine green videoangiography by the means of scanning laser ophthalmoscope were performed on all patients. RESULTS: In 21 patients with angioid streaks and in one patient with peau d'orange appearance of the fundus, indocyanine green videoangiography showed angioid streaks in the form of hyperfluorescent lines with numerous associated hyperfluorescent foci. The angioid streaks were more clearly visualized and were seen to be more numerous and larger by indocyanine green videoangiography than with red-free images or fluorescein angiography. Choroidal neovascularization was suspected in six eyes but could be precisely localized by fluorescein angiography in only three eyes. Indocyanine green angiography allowed precise localization of choroidal neovascularization in all six of these eyes. CONCLUSIONS: These findings indicate that indocyanine green videoangiography provides different information than fluorescein angiography in the evaluation of angioid streaks and can more precisely localize their neovascular complications. PMID- 7530405 TI - Combination therapy with recombinant human granulocyte colony-stimulating factor and erythropoietin in aplastic anemia. AB - Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and erythropoietin (rhEPO) were used to treat patients with aplastic anemia (AA). In terms of effects on erythrocyte recovery, the combined use of rhG-CSF and rhEPO showed a favorable response in 6 of 14 (42.9%) patients with moderate AA following 10 weeks treatment and in 3 of 14 (21.4%) patients thereafter. However, the response was poor in patients with severe AA (3/13). A favorable response in severe AA was observed in 1 of 13 (7.7%) patients following 10 weeks treatment and in 2 of 13 (15.4%) patients thereafter. The overall effect on erythrocytes was observed in 44.4% patients. A dose of 400 micrograms/m2 G-CSF was sufficient to cause an increase in neutrophil count and 100 IU/kg rhEPO appeared to be sufficient to cause an increase in erythrocyte count. In 6 of 27 (22.2%) patients, a trilineage response was observed. Interestingly, a delayed and long lasting effect was obtained in 5 of 27 (18.5%) patients. These results suggest that rhG-CSF can synergize with rhEPO in erythrocyte response, especially in patients with moderate AA. PMID- 7530408 TI - Scanning laser ophthalmoscopic analysis of the pattern of visual loss in age related geographic atrophy of the macula. AB - PURPOSE: We explored the clinical impression that geographic atrophy of the retinal pigment epithelium, a form of advanced age-related macular degeneration, is perceived by the patient as progressing gradually, even when fixation switches from foveal to extrafoveal. METHODS: We analyzed the responses of 60 patients with geographic atrophy to a questionnaire administered as part of a five-year study of the natural course of geographic atrophy, funded by the National Eye Institute. We performed scanning laser opthalmoscope perimetry on all patients. We examined two additional patients with geographic atrophy who reported abrupt visual loss. RESULTS: No eye with geographic atrophy was reported by any patient to have had sudden visual loss. Although most patients with geographic atrophy show foveal fixation until the fovea is atrophic and then show extrafoveal fixation, scanning laser ophthalmoscope perimetry in three patients with geographic atrophy showed alternation between a foveal and an extrafoveal retinal locus for fixation. Two patients with geographic atrophy who complained of abrupt visual loss were found to have occult choroidal neovascularization, which evolved in one patient to classic choroidal neovascularization. The neovascularization was difficult to detect because of the presence of geographic atrophy and its associated ophthalmoscopic and fluorescein angiographic features. CONCLUSIONS: Visual loss in geographic atrophy is nearly always perceived by the patient as being gradual, even when considerable decreases in visual acuity occur and when foveal vision and fixation are lost. A possible explanation for this perception is that there is a transitional period during which a patient uses both a foveal and extrafoveal site for fixation. The complaint of abrupt visual loss in a patient with geographic atrophy should raise the suspicion of choroidal neovascularization, which may be occult and difficult to detect. PMID- 7530409 TI - Gastrointestinal stromal tumors--value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas. AB - The term "gastrointestinal stromal tumor" (GIST) has been applied to mesenchymal tumors that represent neither typical leiomyomas nor schwannomas. In this study we analyzed immunohistochemically 67 histologically benign [< 2 mitoses/10 high power field (HPF)], six borderline (3-5 mitoses/10 HPF), and 23 malignant GIST (> 5 mitoses/10 HPF) and compared them with 10 typical leiomyomas and 5 schwannomas of the gastrointestinal tract. The benign GISTs with spindle cell pattern (67 cases) were typically negative for muscle cell markers (only 3% positive for desmin and 25% for alpha-smooth muscle actin) and S100 protein, but 70% of the cases were positive for CD34, the myeloid progenitor cell antigen also present in endothelial cells and some fibroblasts. However, none of the cases was positive for CD31 (PECAM-1), a more endothelial cell-specific antigen. The absence of CD31 in GIST separates it from Kaposi's sarcoma, a tumor known to be positive for both CD34 and CD31. Fourteen cases of benign GIST of epithelioid cell type showed an immunophenotypic profile similar to the spindle cell tumors. The small intestinal tumors were more commonly actin positive and less commonly CD34 positive than were the gastric tumors. The malignant spindle and epithelioid GIST showed features essentially similar to those in corresponding benign tumors. In contrast, all typical leiomyomas were positive for muscle cell markers and were negative for CD34 and S100 protein. Gastrointestinal schwannomas were S100 protein positive, and negative for muscle markers and CD34. Our results show that gastrointestinal mesenchymal tumors can be immunophenotypically divided in categories that correlate with light microscopically defined diagnostic entities, namely typical leiomyomas, schwannomas, and GIST, most cases of the latter representing tumors of primitive mesenchymal cells that are CD34 positive. PMID- 7530410 TI - Hyalinizing clear cell carcinoma of salivary gland: report of a case with multiple recurrences over 12 years. PMID- 7530412 TI - Cells of origin, thalamic relay and termination of the external cuneothalamocortical tract in the gerbil. AB - The present study is concerned with the connections of the external cuneate nucleus (ECN) in the gerbil following an injection of horseradish peroxidase (HRP) into the ventralis posterior pars oralis (VPLo) or adjacent nuclei of the thalamus. The number, soma size and distribution of the retrograde-labelled ECN neurons were studied and quantified. The application of two retrograde fluorescent tracers was also used to determine whether the ECN neurons would project to the thalamus as well as to the cerebellum through their collaterals. The HRP-positive ECN neurons projecting to the thalamic VPLo were confined to the contralateral caudal half of the ECN, primarily within the intermediate portion represent the forearm and arm territories with a small part of the thoracic and shoulder areas. Labelled neurons were classified into small and medium-sized cells. The majority (96%) of the external cuneothalamic neurons were of the small variety. No double-labelled cells were detected in the ECN following injections of Rhodamine-labelled latex microspheres and Fast blue into the cerebellum and thalamus respectively, suggesting that the ECN neurons projecting to the thalamus form a separate cell group different from those projecting to the cerebellum. The injected HRP into the VPLo was also transported in an anterograde direction by the thalamocortical fibers. The HRP-labelled axonal terminals were distributed within motor area 4 and the dysgranular zones (DZs) of the primary somatosensory cortex (SmI), reaching the deep layers IV and VI as well as superficial layer I. The external cuneothalamocortical pathway shown in the present study may be related to the proprioceptive feedback control of the coordinating motor activity, especially during forelimb muscle movement. PMID- 7530411 TI - Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine. AB - The presence and topographical distribution of nitrergic neurons in the enteric nervous system (ENS) of the pig small intestine have been investigated by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide dinucleotide phosphate diaphorase (NADPHd) histochemistry. Both techniques yielded similar results, thus confirming that within the pig ENS the neuronal isoform of NOS corresponds to NADPHd. Intrinsic nitrergic neurons were not confined to the myenteric plexus; considerable numbers were also present in the outer submucous plexus. In the inner submucous plexus, NOS immunoreactivity or NADPHd staining was restricted to a few nerve fibres and nerve cell bodies. The nitrergic neurons displayed a wide variety in size and shape, but could all be characterized as being multidendritic uniaxonal. Nerve lesion experiments showed that the majority of the myenteric nitrergic neurons project in an anal direction. Evidence is at hand to show that a substantial proportion of these neurons contribute to the dense nitrergic innervation of the tertiary plexus and the circular smooth muscle layer. Some of the nitrergic neurons of the outer submucous plexus were equally found to send their axons towards the circular muscle layer. In some of the nitrergic enteric neurons, VIP, neuropeptide Y, galanin or protein 10 occurred colocalized, but not calbindin or serotonin. The present findings provide morphological evidence for the presence of NOS in a proportion of the enteric neurons in the small intestine of a large omnivorous mammal, i.e. the pig. The topographical features of the staining patterns of NOS and NADPHd are in accord with the results of neuropharmacological studies and argue for the existence of distinct nitrergic subpopulations acting either as interneurons or as motor neurons. PMID- 7530413 TI - Ultracytochemistry of glycoconjugates in pig duodenal gland. AB - To elucidate the ultrastructure, glycoprotein profile and site of glycosylation of glandular cells in relation to the functional polarity of the organelles, swine duodenal tissue was embedded in glycol methacrylate and subsequently stained for periodic acid thiocarbohydrazide silver proteinate (PA-TCH-SP), high iron diamine (HID)-TCH-SP, low iron diamine (LID)-TCH-SP, ninhydrin T-TCH-SP and five peroxidase-labeled lectins. The secretory granules in the duodenal gland cells were electron lucent with a 200 nm to 500 nm electrondense core. Glycoconjugates were confined to the secretory granules and elements of the Golgi complex. Protein activity was located only in the electron dense core. Achivementestic staining pattern for Concanavalin A (Con A), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) was observed in stained secretory granules and the Golgi apparatus. A few cis cisternae were stained with SBA and UEA-I. Trans cisternae were stained with WGA and PNA. Con A reacted with seromucous granules and rough endoplasmic reticulum. These observational findings suggest that these are seromucous cells. The Golgi apparatus is the site of glycosylation and can be divided into two distinct compartments. PMID- 7530414 TI - Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+. AB - BACKGROUND: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS: Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS: Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia. PMID- 7530415 TI - First place winner of the Conrad Jobst Award in the gold medal paper competition. Nitric oxide synthase inhibitors N-monomethylarginine and aminoguanidine prevent the progressive and severe hypotension associated with a rat model of pancreatitis. AB - The objective of this study was to determine whether the observed vascular collapse and other pathologic features of severe pancreatitis may be related to the induction of nitric oxide synthase (NOS). The rat model of pancreatitis reported by Schmidt et al. was employed. Rats in the experimental groups received pretreatment with known NOS inhibitors, N-Monomethylarginine (NMMA) or Aminoguanidine (AG). Controls included sham-operated rats without pancreatic insult and a diseased control group which received pretreatment with normal saline (NS). Arterial blood pressure was continuously recorded with a femoral arterial catheter connected to a transducer and monitor. Fluid resuscitation for hypotension followed a strict protocol with the administration of 5.0 cc NS for sustained decreases in systolic blood pressure (SBP) below 90 mm Hg at 5-minute intervals. Laboratory parameters and histopathology confirmed the induction of pancreatitis, with 6 to 15-fold increases in serum amylase levels and an average of approximately 20% decrease in serum ionized Ca++ levels. Immunohistochemical studies of the pancreas revealed that pancreatic insult resulted in the induction of NOS. Rats in the saline control group (n = 5) became hypotensive (SBP less than 90 mm Hg) between 3 and 4 hours post pancreatic insult and required an average of 110.0 cc (3-4 x blood volume) of NS fluid resuscitation. Rats which were not resuscitated (n = 5) did not survive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530416 TI - Role of nitric oxide synthase, superoxide dismutase, and glutathione peroxidase in radiation-induced decrease in norepinephrine release. PMID- 7530417 TI - Nitric oxide expression and regulation in the dorsal root ganglion and spinal cord. PMID- 7530418 TI - Identification of NO-producing and -receptive cells in mesencephalic transplants in a rat model of Parkinson's disease: a study using NADPH-d enzyme- and NOSc/cGMP immunocytochemistry. PMID- 7530419 TI - Nitric oxide and long-term habituation to novelty in the rat. AB - The role of nitric oxide in learning and memory processes has been tested in the albino rat by a histochemical and a behavioral study, following behavioral habituation to spatial novelty. Histochemically, the neural consequences of behavioral testing were mapped in the brain by staining for NADPH-d, known to be a NOS, whereas behaviorally the formation of LTH has been interfered with by posttrial NOS-inhibition. In the histochemical study, adult male Sprague-Dawley rats were tested in a Lat-maze and sacrificed at different time intervals thereafter. Handled unexposed rats served as controls. The brains were perfused with aldheide and processed for NADPH-d staining. In unexposed control rats the basal expression of NADPH-d was low and scattered. It pertained to few cells in the neostriatum, cerebral cortex, and CA1 hippocampal regions. In contrast, rats that had been exposed for the first time to the maze (spatial novelty) showed NADPH-d activity in the dorsal hippocampus (granule cells, few hilar neurons, and some CA1 pyramidal cells), the caudate-putamen complex, the cerebellum, and in all layers of somatosensory cortex. The positivity was not due to activity per se, since immediately after exposure it was not different from baseline. In contrast, it was present by 2 h and decreased significantly 24 h later. In addition, a strong neuronal discharge induced by the convulsant pentylentetrazol did not induce NADPH-d 2 h afterwards. The staining was prevented by pretreatment with the NMDA receptor antagonist CPP (5 mg/kg) or with the NOS inhibitor L-NOARG (10 mg/kg). In the behavioral study, rats were given an intraperitoneal injection of 1-10 mg/kg (L-NOARG) or vehicle immediately following exposure to a Lat-maze. The highest dose used (10 mg/kg) disrupted habituation of the vertical component only, known to be mainly of emotional meaning. Conversely, both doses disrupted emotional habituation based on defecation scores. The data indicate that the formation of LTH to novelty triggers a cascade of neurochemical events also involving NOS neurons. Further, the widespread induction of NADPH-d by exposure to novelty suggests that spatial and emotional information processing activate neural networks across different organizational levels of the CNS. PMID- 7530420 TI - Suppression of glial iNOS expression by tyrosine kinase inhibitors. PMID- 7530421 TI - Nitric oxide and cerebral ischemia. PMID- 7530423 TI - Reactions of nitric oxide and hydrogen peroxide with hemoglobin-based blood substitutes. PMID- 7530422 TI - The effect of hemorrhagic hypotension and retransfusion and 7-nitro-indazole on rCBF, NOS catalytic activity, and cortical NO content in the cat. PMID- 7530424 TI - Involvement of oxygen-based radicals in methamphetamine-induced neurotoxicity: evidence from the use of CuZnSOD transgenic mice. PMID- 7530425 TI - RT-PCR assay for heme oxygenase-1 and heme oxygenase-2: a sensitive method to estimate cellular oxidative damage. PMID- 7530426 TI - Expression of both constitutive and inducible nitric oxide synthases in neuronal and astrocyte cultures. PMID- 7530427 TI - Induction of superoxide anion and nitric oxide production in cultured microglia. PMID- 7530428 TI - Oxygen free radicals and neurodegeneration in Parkinson's disease: a role for nitric oxide. PMID- 7530429 TI - Specificity of hypothalamic peptides in the control of behavioral and physiological processes. AB - This review summarizes two model systems for understanding how brain neurochemicals, in conjunction with peripheral endocrine and metabolic processes, may be active in controlling very different functions in relation to energy and nutrient balance. As proposed, these systems are unquestionably oversimplified; however, they generate testable hypotheses for future investigations that will help to advance and revise these working models, as well as those of other peptide systems in the brain. Under normal conditions, these peptide systems are behaviorally and endocrinologically specific, and they are activated at very different periods of the daily cycle and at different stages of development. However, under pathologic conditions, their specificity and rhythmicity may be greatly disturbed. This occurs in states involving hypercortisolemia along with hyperinsulinemia or insulin deficiency, when these peptide systems become chronically activated. To determine whether this increased activity actually contributes to conditions of hyperphagia and obesity, and, thus, whether a reversal of this neurochemical activity may help in the treatment of these conditions, critical studies with various pharmacological manipulations are required. Of equal importance are investigations examining the development of these pathologic conditions, from birth to maturity, and their associated disturbances in neurochemical and endocrine processes. A thorough understanding of gene expression in localized brain areas and the contribution of various transcription factors to this process should allow the identification and development of methods that are useful in the treatment, as well as prevention, of disturbed patterns of nutrient intake, fat deposition, and body weight gain. PMID- 7530430 TI - Cytokines as regulators of sleep. PMID- 7530431 TI - The power of integrative peptides to reinforce behavior by releasing dopamine. PMID- 7530432 TI - Antibody-targeted photolysis. Photophysical, biochemical, and pharmacokinetic properties of antibacterial conjugates. PMID- 7530433 TI - Existence of an optimum expression level for secretion of foreign proteins in yeast. PMID- 7530434 TI - Energy cost of translational proofreading in vivo. The aminoacylation of transfer RNA in Escherichia coli. AB - In many cases, the intrinsic binding energies of amino acids to aminoacyl-tRNA synthetases are inadequate to give the required accuracy of translation. This has necessitated the evolution of a second determinant of specificity, proofreading, or editing mechanisms that involve the expenditure of energy to remove errors. Studies of an error-editing function of bacterial methionyl-tRNA synthetase have led to the discovery of a distinct chemical mechanism of editing and to molecular dissection of the dual synthetic-editing function of the active site of the synthetase. Studies have also established the importance of proofreading in living cells and allowed direct measurements of energy costs associated with editing in vivo. An unexpected outcome of these studies was a discovery of functional and structural similarities between methionyl-tRNA synthetase and S adenosylmethionine synthetase, suggesting an evolutionary relationship between the two proteins. The mechanism of editing involves a nucleophilic attack of a sulfur atom on the side chain of homocysteine in homocysteinyl adenylate on its carbonyl carbon, yielding homocysteine thiolactone. The model of the active site of methionyl-tRNA synthetase derived from structure-function studies explains how the active site partitions amino acids between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions of active site residues Trp305 and Tyr15 with the side chain of methionine prevent the cognate amino acid from entering the editing pathway. These interactions are missing in the case of the smaller side chain of the noncognate homocysteine, which therefore enters the editing pathway. Homocysteine thiolactone is formed as a result of editing of homocysteine by methionyl-tRNA synthetase in bacteria, yeast, and some cultured mammalian cells. In mammalian cells, enhanced synthesis of homocysteine thiolactone, is, thus far, associated with oncogenic transformation. In E. coli, most of the energy cost of proofreading by methionyl-tRNA synthetase is due to editing of the incorrect product, homocysteinyl adenylate. PMID- 7530435 TI - Human olfactory receptor neurons express heat shock protein 70: age-related trends. AB - Immunocytochemical methods were used to investigate the cellular distribution and age-related trends in the expression of constitutive and/or inducible forms of heat shock protein (hsp) 70 in the human nasal mucosa of 22 subjects who ranged in age from 16 weeks prenatal to 90 years, including 3 subjects with Alzheimer's disease. The olfactory mucosa was characterized by the presence of olfactory marker protein-immunoreactive olfactory receptor neurons. The hsp 70 immunoreactivity was localized in olfactory receptor neurons and the supranuclear region of sustentacular cells in the olfactory epithelium, and in the acinar cells of the Bowman's glands in the lamina propria. A systematic age-related decrement in the expression of hsp 70 immunoreactivity was observed in the olfactory receptor neurons. This trend was not apparent in sustentacular cells and Bowman's glands. A marked decrement in hsp 70 immunoreactivity was also noted in the olfactory receptor neurons of subjects with Alzheimer's disease when compared to age-matched controls. These results suggest that the age-dependent decrease in hsp 70 in olfactory receptor neurons of older subjects and those with Alzheimer's disease may be attributable to their greater susceptibility to stress. PMID- 7530436 TI - Expression of intercellular adhesion molecule-1 during inner ear inflammation. AB - This study was designed to investigate the expression of intercellular adhesion molecule-1 (ICAM-1) on the spiral modiolar vein (SMV) with its collecting venules (CVs) and the venules of the endolymphatic sac during inner ear inflammation. These data will further elucidate the role of adhesion molecules in extravasation of inflammatory cells from blood vessels during an inner ear immune response. Labyrinthitis was induced in rats by inoculation of keyhole limpet hemocyanin into the scala tympani of animals who had been systemically sensitized to it. Expression of ICAM-1 was examined with a mouse monoclonal antibody to rat ICAM-1 by immunohistochemistry. ICAM-1 was found weakly on the epithelium of SMVs and CVs as early as 6 hours postchallenge, reaching a maximum by day 2 and then fading away gradually. The maximum influx of immunocompetent cells into the cochlea was seen between days 3 and 7. Staining for ICAM-1 was observed on the epithelium of the endolymphatic sac and perisaccular region at 12 and 24 hours, respectively, and this was associated with infiltration of cells into these areas 3 days postchallenge. By day 28, the inner ear had developed endolymphatic hydrops, but at this time it showed almost no significant staining with anti-ICAM 1. The molecule was also expressed in the mesothelium of perilymph, the perineurium of cochlear nerves, the spiral ligament, and the basal cells of the stria vascularis following immunization. Our data provide evidence that endothelial cells of the SMV and its CVs, as well as other inner sites, have the potential to express ICAM-1. This expression precedes the influx of immune cells; therefore, it is possible that this ligand plays a pivotal role in the onset of inflammation in the inner ear. This study also confirmed that the immune response results in endolymphatic hydrops as a long-term consequence. PMID- 7530437 TI - First trimester biochemical screening for trisomy 21: the role of free beta hCG, alpha fetoprotein and pregnancy associated plasma protein A. AB - The potential efficacy of screening for trisomy 21 in the first trimester, using maternal serum markers alpha fetoprotein, free beta human chorionic gonadotropin, unconjugated oestriol and pregnancy associated plasma protein A, was studied in an unselected population of women between the seventh and fourteenth week of gestation. Using a combination of alpha fetoprotein and free beta human chorionic gonadotropin, 53% of affected pregnancies could be identified at a false positive rate of 5%. Unconjugated oestriol and pregnancy associated plasma protein A levels were lower in cases of trisomy 21, but their inclusion with other markers did not significantly improve detection rate. Monitoring the same pregnancies also in the second trimester showed that screening in the first trimester identified the same cases as in the second. We conclude that first trimester screening using free beta human chorionic gonadotropin and alpha fetoprotein, is a viable possibility and will lead to detection rates in excess of 50%. Prospective studies are needed to confirm these observations. PMID- 7530438 TI - A novel method for concentrating urinary type IV collagen based on precipitation with polyethylene glycol: application to its measurement by enzyme immunoassay. AB - We investigated the efficacy of polyethylene glycol (PEG) for effective and reproducible concentration of urinary type IV collagen prior to measurement by enzyme immunoassay (EIA). Human placental type IV collagen at low concentrations (5 and 10 micrograms/L) and urinary type IV collagen were readily precipitated by PEG-4000 added at a concentration of about 150 g/L in the presence of 0.5 g/L gamma-globulin. Type IV collagen measurement by EIA from PEG-concentrated urine samples showed complete recovery and good reproducibility. Analysis of size distribution by Sephacryl S-300HR gel chromatography and Western blotting following polyacrylamide gel electrophoresis confirmed that type IV collagen in PEG-concentrated urine samples was of high molecular weight comparable to that of human placental type IV collagen. After PEG concentration, type IV collagen was detectable by EIA even in the urine of healthy subjects. Significantly higher concentrations of urinary type IV collagen were found in 30 diabetic patients with nephropathy than in 20 healthy subjects [99.5 (8.9) micrograms/L, mean (SEM) versus 21.4 (2.6) micrograms/L, P < 0.0001]. Thus, urinary type IV collagen can be measured effectively by EIA following concentration with PEG. This method has potential for the assessment of the progression of diabetic nephropathy. PMID- 7530439 TI - Stability of parathyroid hormone-related protein and parathyroid hormone at room temperature. AB - The stability of plasma parathyroid hormone-related protein (PTHrP) as measured by the Nichols Institute assay at room temperature was assessed over a period of 72 h in blood samples collected in protease inhibitor tubes and EDTA tubes at 0, 6, 24, 48 and 72 h from 10 patients with hypercalcaemia of malignancy. Mean plasma PTHrP concentrations in blood samples collected in protease inhibitor tubes remained stable for up to 48 h but had decreased by 10% at 72 h. The mean EDTA plasma PTHrP at zero time was 67% of the protease inhibitor tube value and this had fallen to 39% at 72 h. The stability of parathyroid hormone (PTH) in separated blood samples was also assessed by collection into heparin and plain tubes as well as EDTA and protease inhibitor tubes. Serum PTH concentrations progressively declined throughout the 72 h study period although the zero time values were significantly higher than corresponding plasma PTH concentrations. Plasma PTH concentrations appeared to be stable when blood was collected in heparin, EDTA and protease inhibitor tubes during the 72 h period, except in one subject with markedly elevated plasma amylase activity. PMID- 7530440 TI - Disagreement between the Roche Cobas Core and Hybritech TANDEM-E PSA assays when measuring free, complexed and total serum prostate specific antigen. AB - A comparison of the Roche Cobas Core and Hybritech TANDEM-E PSA (prostate specific antigen) assays revealed a large difference in the reactivity of each assay to the separated free and complexed forms of serum PSA in patients with prostatic carcinoma. The Roche assay was relatively much more responsive to the free form, but the Hybritech assay was relatively more responsive to the complexed form and total serum PSA. It is possible that the adoption of a universal standard for PSA will not completely resolve the disagreement between PSA assay on individual patient samples, and the use of separate assays for the free and complexed forms may be necessary for the further clinical development of PSA as a tumour marker. PMID- 7530441 TI - The effects of patients' sera with sperm-immobilizing antibodies on sperm of the Japanese monkey. AB - In order to study the interspecies cross-reactivity of sperm-immobilizing antibodies found in sera of women who were infertile for unknown reasons, we used the sperm-immobilization test to examine the effects of patients' sera with or without sperm-immobilizing antibodies on the sperm of the Japanese monkey (Macaca fuscata). Fourteen of 17 antibody-positive sera and 7 of 14 antibody-negative sera showed sperm-immobilizing activity with regard to monkey sperm. The sperm immobilizing antibody that was active against both human and monkey sperm could be absorbed only by sperm from each corresponding species. The periodate treatment of human or monkey sperm markedly diminished their antibody-absorbing capabilities. Human and mouse monoclonal antibodies having sperm-immobilizing activity with regard to human sperm showed no sperm-immobilizing activity with regard to monkey sperm. These results indicate that the sperm-immobilizing activity of the sera of infertile women against human and monkey sperm might be due to antibodies with different specificities, which recognize a unique carbohydrate antigen epitope expressed in the sperm of each species. PMID- 7530442 TI - Expression of Mycoplasma gallisepticum F-strain surface epitope. AB - The expression of a Mycoplasma gallisepticum (MG) F-strain surface epitope was studied using monoclonal antibody 6F10. Different F strain colonies were cloned based on positive or negative expression of the 6F10-recognized epitope. Clones selected for the lack of expression of this epitope (negative clones) reverted rapidly to positive expression. On the other hand, negative clones derived from F strain-vaccinated chickens expressed the 6F10-recognized epitope variably. After in vitro passages, two groups of cultures were observed. One group reverted rapidly to positive expression. The other group exhibited a very slow rate of reversion, with 30% or fewer of their colonies expressing the 6F10-recognized epitope. These results indicate that the 6F10-recognized surface epitope of F strain is variably expressed under in vivo and in vitro conditions. PMID- 7530443 TI - Response of unimmunized and sporozoite-immunized chickens to challenge with avian Eimeria species: effects of intraperitoneal injection of Sephadex. AB - Unimmunized chickens, given intraperitoneal injections of Sephadex at the same time that they were inoculated with oocysts of either Eimeria tenella or E. acervulina, had significantly lower lesion scores at 6 days postinoculation (PI) than unimmunized chickens that were injected with saline instead of Sephadex. Despite the difference in lesion scores, there was little effect on weight gain, except in one experiment, in which Sephadex-injected chickens gained significantly more weight than saline-injected chickens. In contrast, in chickens that were immunized by prolonged exposure to sporozoites of E. adenoeides, injection of Sephadex at the time of challenge with E. tenella did not reduce lesion scores or parasite development as compared with the uninjected chickens, and the weight gain of the Sephadex-injected challenged chickens fell to a level significantly lower than that of their saline-injected challenged counterparts. The data indicate that Sephadex injected at the time of oocyst inoculation 1) produces markedly different effects on lesion scores and weight gain in unimmunized and immunized chickens and 2) abrogates sporozoite-elicited immunity against E. tenella challenge. PMID- 7530444 TI - The prostate--a gland that does not age well. PMID- 7530445 TI - Identification of the in vitro phosphorylation sites on Gs alpha mediated by pp60c-src. AB - Overexpression of pp60c-src in mouse fibroblasts potentiates both agonist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttrell, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400]. In reconstitution experiments in vitro, phosphorylation of Gs alpha by immune-complexed pp60c-src resulted in enhanced rates of receptor-mediated guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding and GTP hydrolysis [Hausdorff, Pitcher, Luttrell, Linder, Kurose, Parsons, Caron and Lefkowitz (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5720-5724]. These results suggest that one mechanism by which pp60c-src affects signalling through the beta adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of Gs alpha might affect its function, we subjected phosphorylated, recombinant Gs alpha to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edman degradation to determine the cycle numbers at which radiolabelled phosphotyrosine was released. Candidate peptides that contained Tyr residues at the corresponding positions were synthesized, phosphorylated in vitro by pp60c-src, and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact Gs alpha. We report here that Gs alpha is phosphorylated on two residues by pp60c-src, namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP [Johnson, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cell Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-terminus, within a region of Gs alpha important for receptor interaction [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Nature (London) 334, 712-715]. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains. PMID- 7530446 TI - Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer. AB - In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange. PMID- 7530447 TI - Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor. AB - In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells. PMID- 7530448 TI - The independent gene amplification of electrophoretically indistinguishable B esterases from the insecticide-resistant mosquito Culex quinquefasciatus. AB - Resistance to organophosphates in Culex mosquitoes is typically associated with increased activity of non-specific esterases. The commonest phenotype involves two elevated esterases, A2 and B2, while some strains have elevation of esterase B1 alone. Overexpression of the two B esterase electromorphs is due to gene amplification. Full-length cDNAs coding for amplified esterase B genes from a resistant Cuban strain (MRES, with amplified B1 esterase) and a Sri Lankan strain (PelRR, with amplified B2 esterase) of C. quinquefasciatus have been sequenced. In addition, a partial-length cDNA coding for a B esterase from an insecticide susceptible Sri Lankan strain (PelSS) has been sequenced. All the nucleotide sequences and the inferred amino acid sequences show a high level of identify (> 95% at the nucleotide and amino acid level), confirming that they are an allelic series. The two B1 esterase nucleotide sequences (MRES and the previously published TEM-R [Mouches, Pauplin, Agarwal, Lemieux, Herzog, Abadon, Beyssat Arnaouty, Hyrien, De Saint Vincent, Georghiou and Pasteur (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2574-2578]) showed the lowest identity, and restriction fragment-length-polymorphism analysis of the two strains was different. On the basis of these data we suggest that the two electrophoretically identical B1 esterase isoenzymes from California and Cuba have been amplified independently. Alternatively, if amplification has occurred only once, the original amplification has not occurred recently. PMID- 7530451 TI - High-performance liquid chromatographic determination of benzalkonium and naphazoline or tetrahydrozoline in nasal and ophthalmic solutions. AB - A high-performance liquid chromatographic method is described for the determination of benzalkonium chloride homologues and naphazoline nitrate or tetrahydrozoline hydrochloride in ophthalmic and nasal solutions. The technique involves a one-step dilution (1:5) for sample preparation and direct injection onto a 5 mm RP C-8 column. The mobile phase is acetonitrile-diluted acetic acid (80:20, v/v) with 6 mM tetramethylammonium bromide. Detection is carried out at 260 nm with a diode array. The method is rapid, specific, reproducible and is especially useful for quality control procedures. PMID- 7530450 TI - Neuronal nitric oxide synthase contains a discs-large homologous region (DHR) sequence motif. PMID- 7530453 TI - Gastropathies in the Lundehund. II. A study of mucin profiles. AB - The mucin profiles of the gastric mucosa in Lundehunds suffering from intestinal lymphangiectasia were examined and compared to the mucin profiles in control dogs from other breeds. A previous study performed on this material had shown that all examined Lundehunds had gastritis and about 30% had gastric carcinoma. Neutral and acid mucins were identified using the periodic acid-Schiff (PAS) and Alcian blue (pH 2.5) periodic acid-Schiff (AB-PAS) methods. The acid mucins were divided into sialomucins and sulfomucins based on their reaction with high-iron diamine Alcian blue, pH 2.5 (HID-AB). In Lundehunds with chronic atrophic gastritis in the fundic and body regions the surface and foveolar epithelium showed a predominantly normal mucin profile although some Lundehunds had a reduced mucin content. The mucous neck cells extended from below the gastric foveolae towards the muscularis mucosae. Morphometric examination showed that the abnormal presence of mucous neck cells occupied 41% of the height of the gastric mucosa in Lundehunds compared to only 19% in the control dogs (p < 0.05). Of the four Lundehunds with gastric carcinoma, two possessed neoplastic cells that contained minimal or no mucins. The amount and type of mucin in the neoplastic cells of the remaining two Lundehunds varied both between individuals and within a neoplasm. This study shows that the abnormal presence of mucous neck cells and the associated pseudopyloric metaplasia comprised the predominant changes in the gastric mucin profiles of Lundehunds. PMID- 7530452 TI - Transcriptional regulation of cell line-dependent, baculovirus-mediated expression of foreign genes. AB - Cell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinant vAc beta hCG-luc virus carrying two reporter genes--beta subunit of human chorionic gonadotropin (beta hCG) and luciferase (luc)--placed under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived from Spodoptera frugiperda (Sf21 and Sf9), Bombyx mori (BmN and Bm5), and Trichoplusia ni (TN368) were used as host cells. TN368 expressed both beta hCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from the vAc beta hCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530449 TI - Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells. AB - Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor. PMID- 7530454 TI - Visualization of RNA in gel by shortwave UV epi-illumination without staining. PMID- 7530455 TI - Sequential staining of short oligonucleotides in polyacrylamide gels with ethidium bromide and methylene blue. PMID- 7530456 TI - Preparation of RNA from lyophilized tissue: a stable and reliable method for long term storage. PMID- 7530457 TI - RNA analysis using miniprep RNA in reverse transcription PCR. PMID- 7530458 TI - Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo. AB - We employ fluorescently labeled dextran beads to follow cycles of nuclear division and nuclear envelope breakdown in living Drosophila embryos. When injected into syncytial Drosophila embryos, 3000 mol wt fluorescently labeled dextran beads concentrate in the interphase nucleus. Through confocal microscopy, undisrupted multiple cycles of nuclear division are readily followed. In contrast, 40,000 mol wt fluorescently labeled dextran beads concentrate in the cytoplasm and enter the nucleus only after nuclear envelope breakdown during prophase. Once the nuclear envelope reforms during telophase, these large dextran beads are again excluded from the nuclei. The complementary behavior of the large and small dextran beads makes them applicable to a broad range of cellular studies. We employ the 40,000 mol wt fluorescein-labeled beads, in combination with rhodamine-labeled histones, to demonstrate that nuclear envelope breakdown occurs about 3.5-4.0 minutes prior to the initiation of anaphase. PMID- 7530459 TI - An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. AB - The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins. PMID- 7530460 TI - Pulse-first heterofusion of cells by electric field pulses and associated loading of macromolecules into mammalian cells. AB - Exposing cells to brief, high-intensity electrical field pulses can lead to the permeabilization of their plasma membranes. This electro-induced permeated state of the cell membrane is reversible and leads to cell fusion; i.e., the electropermeabilized state if fusogenic. The size of cells intended for fusing, however, limits the obtention of viable hybrids when there is a wide range of cell sizes. We report here that electrofusion of cells of different origins and sizes (hamster fibroblasts and red blood cell ghosts) can be easily obtained. Due to size differences, the optimum electric field conditions for their permeabilization and the maintenance of their integrity are observed to be different (10 pulses, 100 microseconds duration, 1.2 kV/cm for Chinese hamster ovary [CHO] kV/cm for erythrocyte ghosts). Cells and ghosts are electropermeabilized separately and kept at low temperature. Their fusion is then induced by the creation of contact between them by gentle centrifugation. This process takes advantage of the long-lived fusogenicity of the electropermeabilized cell membrane. An immediate consequence is the introduction of macromolecules into mammalian cells. This is obtained by fusing cells with pre loaded erythrocyte ghosts. Under optimum conditions, penetration of large quantities of FITC-dextran (70 kDa) and beta-galactosidase into 90%-95% of CHO cells while preserving their viability has been observed. PMID- 7530461 TI - The selectins and their ligands. AB - The selectins are a family of carbohydrate-binding proteins, or lectins, that have stimulated tremendous interest because of their involvement in a wide array of interactions between leukocytes and endothelial cells. Highlights of recent progress include an extension of the list of instances of selectin participation in inflammatory diseases, further definition of selectin carbohydrate specificities, and identification of their carbohydrate-based ligands. PMID- 7530463 TI - Wound repair in the context of extracellular matrix. AB - Wound repair requires a continually evolving network of interactions among cells, cytokines and the extracellular matrix. Cell-surface integrins provide a mechanical connection between matrix components and the cytoskeleton, and integrins can transduce an astonishing variety of signals along pathways that may intercept the pathways triggered by cytokine receptors. PMID- 7530462 TI - Adhesive signaling in platelets. AB - The anucleate platelet must perform its hemostatic functions in the absence of transcriptional regulation. Central among these functions is cell adhesion, which is mediated by multiple specialized plasma membrane receptors. The adhesive function of one of the key receptors, integrin alpha IIb beta 3, is regulated by intracellular signals triggered by platelet agonists and antagonists. Recent evidence indicates that adhesion receptors can transduce extracellular signals into the platelet to activate intracellular signaling pathways that affect hemostasis. PMID- 7530464 TI - Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. AB - Hyaluronate (HA) is an abundant component of extracellular matrix that is believed to be crucial in many cellular processes, including tissue remodeling, the creation of cell-free spaces, inflammation and tumorigenesis. Although several well characterized proteins within the extracellular matrix associate with HA, it is now clear that cells can also bind and respond to HA directly, via cell-surface HA-binding proteins. The cDNAs coding for two families of such proteins, CD44 and RHAMM, have been cloned and characterized. These proteins have been implicated in a number of physiological processes, including cell migration, lymphocyte activation and tumor progression. Although many of these processes depend on an association with HA, some are apparently HA-independent, suggesting that other ligands for these receptors may be involved. PMID- 7530465 TI - Fluorospectrometric analysis of heparin interaction with fibroblast growth factors. AB - The fluorescence emission of a single tryptophan residue present in both FGF-1 and FGF-2 was used as a structural probe to directly assess the interaction of the growth factors with heparin or beta-cyclodextran tetradecasulfate. About 20 25% of the fluorescence of either FGF-1 or FGF-2 is quenchable, and is dependent on sulfation of the ligands. The quenchable fluorescence is associated with about 20% of total FGF, suggesting the presence of two fluorospectrometric forms of the protein. The equilibrium dissociation constants, determined by this method, for heparin or beta-cyclodextrin tetradecasulfate binding to FGF-1 are about 1 nM, whereas the values for FGF-2 are 1 and 23 nM, respectively. The method provides a direct tool to evaluate FGF-ligand interaction and assess the structural integrity of the proteins. PMID- 7530466 TI - Bone morphogenetic proteins induce differentiation in astrocyte lineage cells. AB - Serum-free mouse embryo (SFME) cells express Glial Fibrillary Acidic Protein (GFAP), a specific marker of the astrocyte lineage, when treated with either Transforming Growth Factor Beta (TGF-beta) or calf serum. We examined the effects of the related Bone Morphogenetic Proteins (BMPs) which are expressed in the developing murine nervous system. Treatment with the heterodimers BMP-2/6 and 2/7 followed by the homodimers BMP-2, 4, 5, 6, and 7 induced higher levels of GFAP in these cells than either TGF-beta 1 or activin when tested at the same concentration. The BMP-induced cells resembled classically described astrocytes and were characterized by antibody markers as type 1 and type 2. In addition, these astrocytes also showed increased levels of the cell adhesion molecules CD44 and neural cell adhesion molecule (N-CAM), both known to be expressed by this cell type. These data clearly demonstrate that the BMPs function as differentiation factors as well as regulators of adhesion molecule expression for cells of the astrocyte lineage and suggest a key role in glial development in the nervous system. PMID- 7530467 TI - Induction of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in bone marrow and fractionated marrow cell populations by interleukin 3 (IL-3): IL-3-mediated positive feedback mechanisms of granulopoiesis. AB - Interleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitors in vitro, whereas it induces granulocytosis in vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction of IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P < 1.061) and 2(1.061 < P 1.074) of bone marrow cells fractionated by equilibrium density centrifugation, G-CSF was expressed in macrophages and fraction 2 and 3 (1.074 < P < 1.097), and interleukin 6 (IL-6) in macrophages and fraction 1 to 3. These results indicate a hierarchical regulation of cytokine production and the existence of a positive feedback mechanism in granulopoiesis. IL-6, induced by IL-3, stimulates stem cells into cycle and induces stem cells to respond to IL-3. The stem cells differentiate to granulocyte-macrophage colony-forming cells by the combined effect of IL-3 and IL 6. IL-3 also induces GM- and G-CSF expression which in turn makes granulocyte macrophage colony-forming cells differentiate to granulocytes. These factors organize a cytokine network in granulopoiesis. PMID- 7530468 TI - Economic and clinical evaluation of therapy of HIV-related non-Hodgkin's lymphoma with chemotherapy and granulocyte colony-stimulating factor (G-CSF) PMID- 7530469 TI - Long-term results of a randomised trial of short-course low-dose adjuvant pre operative radiotherapy for rectal cancer: reduction in local treatment failure. AB - A prospective randomised multicentre trial compared pre-operative radiotherapy followed by surgery with surgery alone for rectal cancer < or = 12 cm from the anal verge. Of 468 patients (mean age 67 years, range 31-94, 273 males) who met the entry criteria, 228 were randomised to radiotherapy (3 x 5 Gy over 5 days within 2 days of operation) followed by surgery, and 239 to surgery alone. Randomisation was unknown in 1 patient. Follow-up to death or 5 years was achieved in 454 (97%) patients. 31 (7%) of the 468 patients died within 30 days of operation (radiotherapy and surgery 21 [9%], surgery alone 10 [4%]; P < 0.05). Cardiovascular and thromboembolic complications were more common after radiotherapy and surgery (30, 13%) than after surgery alone (8, 3%; P < 0.005). Of the 280 patients who had curative surgery, 52% of those who had radiotherapy and surgery and 56% of those who had surgery alone survived 5 years (P = 0.88). 395 patients attended outpatients clinics at least once. Local treatment failure was identified during follow-up in 82 patients [31/185 (17%) radiotherapy and surgery; 51/210 (24%) surgery alone; P < 0.05]. It occurred in 33 of the 258 patients who had a curative resection and attended outpatients [radiotherapy and surgery, 11/120 (9%), surgery alone, 22/138 (16%); P = 0.08]. Long-term survival was unaffected, but long-term local recurrence was reduced by the addition of low dose radiotherapy to surgery. Peri-operative mortality was, however, increased. PMID- 7530471 TI - Peripheral blood progenitor cell transplantation mobilised by r-metHuG-CSF (filgrastim); a less costly alternative to autologous bone marrow transplantation. AB - In a retrospective study, we calculated the treatment costs of 63 patients who received either autologous bone marrow transplantation (ABMT) with recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) (filgrastim) (n = 13) or without r-metHuG-CSF (n = 22) or alternatively, peripheral blood progenitor cell (PBPC) transplantation mobilised by r-metHuG-CSF (n = 28). The recovery of granulocytes, platelets and reticulocytes after PBPC was markedly accelerated as compared with ABMT with or without r-metHuG-CSF. The accelerated haematopoietic recovery was associated with a reduction in platelets and red blood cell transfusion requirements, with a reduction in episodes of fever and with earlier discharge from the hospital. This resulted in the average cost per treatment of the PBPC group being almost 30% lower than the treatment costs in the ABMT groups. PMID- 7530470 TI - Accelerated chemotherapy for poor prognosis germ cell tumours. AB - A pilot study has been completed of an innovative dose intensive chemotherapy schedule for poor prognosis patients with metastatic germ cell tumours referred to the Royal Marsden Hospital between August 1989 and January 1992. The rationale underlying the regimen was the use of an extremely short intercycle interval in order to counteract the potential of these tumours for rapid proliferation. The drug combination in the first phase incorporated a combination of cisplatin and carboplatin, infusional bleomycin and vincristine and this was followed by three cycles of bleomycin, etoposide and cisplatin (C-BOP/BEP). 21 patients with adverse presentations were treated with C-BOP/BEP. The median follow-up of surviving patients is 36 months (range 18-52 months). 1 patient died of disease, 1 died of a treatment complication while in remission and 1 further patient relapsed, and is in remission after radiotherapy and surgery. The 2-year overall survival rate was 90% [95% confidence interval (CI) = 77-100%]. We conclude that this approach may represent an improvement over standard chemotherapy and should be assessed in a multicentre setting. PMID- 7530472 TI - Escalating doses of mitoxantrone with granulocyte colony-stimulating factor (G CSF) rescue plus 5-fluorouracil and high-dose levofolinic acid in metastatic breast cancer. PMID- 7530474 TI - Influenza in the world. 1 October 1993-30 September 1994. PMID- 7530473 TI - Effect of NG-monomethyl-L-arginine on kinin-induced vasodilation in the human forearm. AB - 1. We compared effects of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, on vasodilator responses to intra-arterial infusion of bradykinin and substance P in the human forearm. 2. Bradykinin (100 pmol min-1) increased forearm blood flow when infused into the brachial artery of eight healthy male volunteers, from 2.8 +/- 0.2 (mean +/- s.e. mean) to 9.3 +/- 1.0 ml min-1 per 100 ml forearm volume. 3. Co-infusion of L-NMMA (2 mumol min-1 and 4 mumol min-1) with bradykinin (100 pmol min-1) for 6 min produced respectively a 9 +/- 14% and 42 +/- 14% inhibition (compared with L-NMMA vehicle) in the response to bradykinin. 4. Substance P (1 pmol min-1) when infused into the brachial artery of a further eight male subjects increased forearm blood flow from 3.4 +/- 0.2 to 6.3 +/- 0.7 ml min-1 100 ml-1. 5. Co-infusion of L-NMMA (2 mumol min-1 and 4 mumol min-1) with substance P (1 pmol min-1) for 6 min produced respectively a 27 +/- 8% and 70 +/- 13% inhibition (compared with L-NMMA vehicle) in the response to substance P. 6. These results demonstrate that vasodilator responses to both bradykinin and substance P are mediated in part via the L-arginine/NO pathway. Bradykinin and substance P may be useful agonists with which to study endothelial function in this vascular bed. PMID- 7530475 TI - Gene rearrangement in acute non-lymphoblastic leukaemia: correlation with morphological and immunophenotypic characteristics of blast cells. AB - The presence of Ig and TCR gene rearrangement has been reported to occur in ANLL. However, most of the studies have been performed in short series of patients and, in general, not all rearranging genes have been included. We have investigated the configuration of immunoglobulin (Ig) and T-cell receptor loci (TCR) in a series of 160 untreated patients with de novo acute non-lymphoblastic leukaemia (ANLL) and correlated the results with the morphological and immunophenotypic characteristics of blast cells. IGH gene rearrangement was detected in 16/160 cases analysed (10%) and IGK was rearranged in half of them. The incidence of cases displaying TCRB, TCRG and TCRD rearrangements was 5.6%, 13.8% and 13%, respectively. Concomitant recombinatorial events including different Ig and/or TCR genes were frequently detected. Gene rearrangement was not related to the stage of cell differentiation within the myeloid lineage assessed both by morphological and immunophenotypic criteria. Regarding the correlation with the presence of lymphoid related markers, the only relevant association was between the expression of CD7 antigen and TCRG and TCRD gene rearrangement. Our results show that the incidence of gene rearrangement in ANLL may be slightly higher than previously suspected, and that it is not associated with early stages of cell differentiation nor to the expression of lymphoid markers. PMID- 7530476 TI - Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency. AB - Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40 60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen. PMID- 7530477 TI - Myelodysplasia and acute myeloid leukaemia in cases of aplastic anaemia and congenital neutropenia following G-CSF administration. AB - Myelodysplasia and acute myeloid leukaemia (MDS/AML) developed in three cases of severe aplastic anaemia (SAA) and one case of congenital neutropenia (CN, Kostmann's disease) who received recombinant human granulocyte colony-stimulating factor (G-CSF) are reported. In these four MDS/AML cases, age at diagnosis of SAA/CN was 0-13 years, the cumulative dose of G-CSF was 98 micrograms/kg to 10 mg/kg over 1-57 months, and the interval from initiation of G-CSF to MDS/AML was 25, 23, 31 and 57 months, respectively. These results suggest a link between SAA/CN and MDS/AML in relation to G-CSF administration; however, large studies are necessary to determine if such a risk is significant in patients with SAA/CN who are treated with G-CSF. PMID- 7530478 TI - Do CD34+ cell counts predict haemopoietic recovery after autologous blood stem cell transplantation (ABSCT)? PMID- 7530479 TI - Tat-expressing Jurkat cells show an increased resistance to different apoptotic stimuli, including acute human immunodeficiency virus-type 1 (HIV-1) infection. AB - Human CD4+ T lymphoblastoid Jurkat cells were stably transfected with two different plasmid vectors containing the cDNA of human immunodeficiency virus type 1 (HIV-1) tat gene under the control of either the promoter of simian virus 40 (pRPneo/tat) or the long terminal repeat region of SL3 murine leukaemia virus (pRPneo/SL3/tat). Both pRPneo/tat and pRPneo/SL3/tat Jurkat cell lines showed a constant and high production of bioactive Tat in transient co-transfection assays with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) reporter plasmid. Tat-positive and mock-transfected Jurkat cells were cultured with various cytotoxic agents, which have been associated to the progressive loss of CD4 T-lymphocytes characteristic of HIV-1 disease. In the presence of recombinant tumour necrosis factor-alpha (TNF-alpha), anti-fas antibody, Leu3a anti-CD4 antibody, the percentage of apoptosis, evaluated in a 24-72 h short-term assay, was lower (P < 0.05) in tat-positive Jurkat cells than in mock-transfected controls. The low susceptibility to the cytotoxic activity of TNF-alpha and anti fas antibody of tat-transfected cells was confirmed by counting viable cells up to 15 d of culture. Also, recombinant Tat protein was able to prevent the increase of apoptosis induced in mock-transfected Jurkat by TNF-alpha. Of note, tat-expressing cells showed a better survival with respect to mock-transfected control cells even when acutely infected with high doses (500,000 cpm of reverse transcriptase) of HIV-1 (strain IIIB) or treated with heat-inactivated HIV-1. These data demonstrate that the expression of the regulatory HIV-1 Tat protein is able to rescue Jurkat lymphoblastoid cells from apoptosis induced by a variety of cytotoxic agents. Since Tat protein expression is restricted to the initial phases of an active HIV-1 replication, the anti-apoptotic effect of Tat could have the physiological significance of selectively protecting HIV-1 producing cells from death, at least for the time necessary to allow virus production and spreading. PMID- 7530481 TI - Activation and inactivation of gene expression using RNA sequences. PMID- 7530480 TI - PIG-A, DAF and proto-oncogene expression in paroxysmal nocturnal haemoglobinuria associated acute myelogenous leukaemia blasts. AB - Failed surface expression of the complement decay-accelerating factor (DAF) due to mutation of the PIG-A gene is a hallmark of affected paroxysmal nocturnal haemoglobinuria (PNH) blood elements. Previous findings that acute myelogenous leukaemia (AML) blasts evolving in a PNH patient differed from idiopathic AML blasts in that they exhibited DAF negativity suggested that the leukaemic blasts derived from an affected PNH cell. To investigate whether these cells differ from untransformed PNH cells in PIG-A genetic alterations or in DAF mRNA processing, or are distinguishable from conventional AML blasts in proto-oncogene activation or chromosomal structure, their DNA and RNA were examined using PIG-A, DAF and proto-oncogene probes and their karyotype was analysed. Analyses of the PIG-A genome revealed dual exchanges of A1110-->G and T1130-->A resulting in conversions of T370 to R and I377 to N in the coding region but no deletions or rearrangements. Investigations of DAF mRNA processing showed mRNA species differing in 3' UT regions from those in untransformed cells but similar to those in DAF-positive leukaemia cell lines. Studies of c-myb, c-myc, c-fos and c-fms showed no gross genetic alterations, amplifications or variations in mRNA transcripts deriving from these genes. Karyotypic analysis showed no alterations. The results indicate that in AML blasts evolving in PNH: (1) the PIG-A genome exhibits multiple point mutations but no gross genetic changes; (2) DAF mRNA transcripts exhibit differentiation-dependent variations that do not affect GPI anchoring; (3) c-myb, c-myc, c-fos and c-fms activation show no differences from idiopathic AML; and (4) no karyotypic abnormalities are associated with AML transformation. PMID- 7530482 TI - Y chromosome loss in chronic myeloid leukemia detected in both normal and malignant cells by interphase fluorescence in situ hybridization. AB - Loss of the Y chromosome in bone marrow (BM) cells is a normal age-associated event. Y chromosome loss is also observed in the Philadelphia chromosome (Ph) positive BM cells of some patients with chronic myeloid leukemia (CML) in chronic phase, but at a younger age than in normal individuals. While the significance of loss of the sex chromosome in normal males is uncertain, -Y marrow cells are not believed to be of clonal origin. However, because CML is a clonal disease, CML sub-populations with Y loss may constitute a disease-related sub-clone. We used a PCR-amplified yeast artificial chromosome containing the BCR gene region for single color interphase analysis of BCR rearrangement by fluorescence in situ hybridization (FISH). Then, using two color FISH, with one fluorochrome detecting the BCR gene region and the other detecting Y chromosome repeat sequences, we surveyed peripheral and BM Y loss in both normal Ph- (BCR not disrupted) and CML Ph+ (BCR rearranged) interphase nuclei of two patients with Y loss in Ph positive cells observed by metaphase analysis. -Y was seen in a proportion of Ph+ cells in both cases, and the proportion matched that seen in Ph- cells, indicating that Y loss is probably sporadic in both normal and CML populations, and that the propensity for Y loss in normal BM cells may be a phenotype that can be retained by malignant cells in CML. PMID- 7530483 TI - Rhabdoid tumor of the kidney with primitive neuroectodermal tumor of the central nervous system: associated tumors with different histologic, cytogenetic, and molecular findings. AB - Rhabdoid tumor of the kidney (RTK) is associated with tumors of the central nervous system (CNS) in approximately 15% of cases. We describe the clinical features, histologic and cytogenetic findings, and molecular analysis of renal and CNS tumors from the same patient. The histology of the renal tumor was consistent with rhabdoid tumor. The CNS tumor was a primitive neuroectodermal tumor (PNET). The karyotype of the RTK was normal male. The PNET of the brain demonstrated monosomy 22 as the only cytogenetic abnormality, similar to reported cases of malignant rhabdoid tumor of the brain, but dissimilar to nonrandom cytogenetic findings in other CNS PNETs. Molecular cytogenetic and DNA marker studies confirmed loss of chromosome 22 in this patient's brain tumor. DNA allelotyping showed retention of both parental chromosome 22 alleles in the RTK and loss of the maternal allele in the PNET. Evaluation of additional RTKs and brain tumors occurring in the same patient may provide insight into the origins and relationships of these enigmatic tumors. PMID- 7530484 TI - Comparative genomic hybridization, allelic imbalance, and fluorescence in situ hybridization on chromosome 8 in prostate cancer. AB - Due to problems with primary tumor cell culture, conventional cytogenetics has yielded little insightful information on chromosomal alterations in prostate cancer. The primary aim of this study was to define the ability of comparative genomic hybridization (CGH) to detect and map genetic deletions in prostate tumors. A secondary aim was to apply multiple assays to individual tumors as a means of deciphering the mechanisms of genetic alterations in prostate cancer. CGH results were compared with allelic imbalance measurements at 29 distinct loci on chromosome 8 in 18 specimens (17 malignant and 1 benign). CGH detected no changes in cases where all informative PCR/RFLP loci were retained and detected all p arm deletions consisting of at least two loci. We estimate that in this study, the smallest deletions detected by CGH were approximately 20-30 cM. Physical mapping of subchromosomal arm deletions by CGH correlated well with allelic imbalance mapping by PCR/RFLP: The data agreed at 88% of loci on 8p and 92% of loci on 8q. Fluorescence in situ hybridization (FISH) with multiple centromere probes and DNA content flow cytometry (FCM) also was performed on selected specimens. FISH revealed two cases of chromosome 8 aneusomy. In these two cases and three others, CGH showed simultaneous p arm deletion and q arm gain, suggesting isochromosome 8q formation. Together, these data suggested that, simple chromosomal aberrations were responsible for allelic losses on 8p and allelic gains on 8q in a significant number of prostate tumors. We also used CGH to examine relative DNA sequence copy number throughout the genome. Changes frequently associated with 8p loss include gains of 8q and losses of 13q, 16p, 16q, 17p, 17q, 20q, and Y. Cases with 8p loss exhibited five times the number of alterations as did cases without 8p loss. PMID- 7530486 TI - Telomere length variation in normal and malignant human tissues. AB - Tissue and tumor specific length variation of telomere (TTAGGG)n repeats was studied in DNAs from various normal and malignant tissues. DNA was isolated from bone marrow and blood cells, malignant tissues, and established tumor cell lines. Nonisotopic Southern hybridization revealed a reduction of telomere repeat arrays in 14 of the 35 tumors analyzed. However, other cases (60%) showed no reduction, or even an increase, in telomeric length. Our finding of elongated telomere stretches in several tumors of different origin compared with normal tissue is in contrast to previous reports describing a general shortening of terminal repeat length in colorectal cancer and neuroblastoma. We tentatively conclude that there is no general tendency to telomere reduction in malignant tissues. PMID- 7530485 TI - Aneusomy of chromosomes 7 and 17 detected by FISH in prostate cancer and the effects of selection in vitro. AB - Twenty prostate tumor specimens, obtained from radical prostatectomies, and two lymph node metastases were examined by classical and molecular cytogenetic methods. A sample from each tumor was analyzed histologically and used for touch preparations. Adjacent samples were used for preparation of single-cell suspensions before cell culture (DirFISH) and for establishing cell cultures, which were subsequently harvested for classical G-banding analysis. Fluorescence in situ hybridization (FISH) was performed on touch preparations, DirFISH, and cells obtained from tissue culture. Biotinylated pericentromeric probes for chromosomes 7 and 17, in addition to a digoxigenin-labeled X-chromosome probe, were used in a dual-color FISH assay. The results indicated that, in uncultured tumor cells, chromosome 17 was lost in 55% of specimens, chromosome 7 was gained in 16% of specimens, and 9% of specimens showed large tetraploid populations. After cell culture, 23% of specimens showed loss of chromosome 17, no specimens showed gain of chromosome 7, and no tetraploid populations were present. This study suggests that loss of chromosome 17 may play an important role in the development of prostate cancer, and that genetic changes observed after selection in vitro may not represent those in the original tumor. PMID- 7530487 TI - Cytogenetic analysis of 63 non-small cell lung carcinomas: recurrent chromosome alterations amid frequent and widespread genomic upheaval. AB - A detailed cytogenetic analysis of 63 non-small cell lung carcinomas (NSCLCs) was carried out for identification of recurrent chromosomal alterations. Most specimens displayed very complex karyotypes with multiple numerical and structural changes (median number, 31). Losses of chromosomes 9 (65% of cases) and 13 (71%) were the most frequent numerical changes. Loss of the Y was often observed in tumors from males. Gain of chromosome 7 was also frequent (41%). Chromosome arms 1p, 1q, 3p, 3q, 6q, 7q, 8q, 9p, 11q, 17p, and 19q were particularly prone to rearrangement. The chromosome arm most often contributing to losses was 9p (79%). Other arms that were frequently lost included 3p, 6q, 8p, 9q, 13q, 17p, 18q, 19p, 21q, 22q, and the short arm of each acrocentric chromosome. The percentage of cases with loss of 3p was significantly higher in squamous cell carcinomas (94%) than in adenocarcinomas (60%). There was also a statistically significant increase in the proportion of cases with gains of 1q, 7p, and 11q in adenocarcinomas compared to squamous cell carcinomas. Several recurrent isochromosomes and unbalanced exchanges were found. Among these was i(5p), which was observed in nine tumors, eight of which displayed adenomatous features. An i(8q) was identified in six cases, including five adenocarcinomas. Double minutes and/or homogeneously staining regions were seen in seven specimens. These data indicate that numerous chromosome alterations contribute to the pathogenesis of NSCLC and that, amid this widespread genomic disarray, recurrent abnormalities exist that could have biological and clinical implications. PMID- 7530488 TI - Loss of heterozygosity in 8p is associated with microinvasion in colorectal carcinoma. AB - Loss of heterozygosity (LOH) from the short arm of chromosome 8 is frequent in a variety of malignancies, suggesting the presence of a tumor suppressor gene in this region. Previous studies suggested that this deletion may correlate with higher clinicopathologic stages in colorectal cancer, but others did not support this finding; in part, this difficulty is due to the low heterozygosity of the RFLP markers that were used. Here we report on a preliminary investigation in which we used highly informative microsatellite markers to determine whether deletions of 8p are correlated with poor prognostic features. Paraffin-embedded tumor tissue from 15 patients was analyzed with a panel of three microsatellite markers that are known to be sites of frequent LOH. Fourteen of the 15 cases were informative with at least one marker, and 7 showed LOH. Analysis of clinical features showed that there was no relation of 8p LOH with patient age or tumor stage, grade, location, or pattern of growth. However, a statistically significant correlation was seen between LOH and lymphatic, vascular, or perineural microinvasion (Fisher exact test, P = 0.01). This histologic feature is known to be a stage-independent indicator of prognosis. Our data suggest that 8p LOH may be associated with poor outcome and demonstrate the utility of these microsatellite markers for its detection. PMID- 7530489 TI - Trisomy 2, trisomy 20, and del(17p) as sole chromosomal abnormalities in three cases of hepatoblastoma. AB - Short-term cultures of three hepatoblastomas were analyzed cytogenetically. Trisomy 2, trisomy 20, and a deletion of 17p were found as the sole abnormalities, yielding the karyotypes 47,XY, + 2; 47,XX, + 20; and 46,XX,del(17)(p12)/46,XX. This is the first reported case of deletion of 17p as the sole chromosomal abnormality in a hepatoblastoma and the first reported case of trisomy 20 without double minute chromosomes as a sole chromosomal abnormality in hepatoblastoma. PMID- 7530490 TI - First trimester maternal serum alpha-fetoprotein in fetal trisomies. AB - OBJECTIVE: To evaluate the potential value of maternal serum alpha-fetoprotein concentration in the detection of fetal trisomy at 10 to 13 weeks gestation and to examine the possible association between maternal serum alpha-fetoprotein and fetal nuchal translucency thickness. DESIGN: Cross-sectional study. SETTING: Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London. SUBJECTS AND METHODS: Maternal serum alpha-fetoprotein concentration was measured at 10 to 13 weeks gestation in samples from 57 pregnancies with fetal trisomies (trisomy 21 (n = 35), trisomy 18 (n = 16), and trisomy 13 (n = 6)) in 228 matched controls in whom the fetal nuchal translucency was < 3 mm and in 114 chromosomally normal fetuses with translucency > or = 3 mm. RESULTS: In the control group maternal serum alpha-fetoprotein increased significantly with fetal crown-rump length (r = 0.451). In this group, the median maternal serum alpha-fetoprotein was not significantly different from that in the groups with trisomy 21 (median = 0.84 MoM), trisomy 18 (median = 0.86 MoM), or trisomy 13 (median = 0.94 MoM), respectively. Neither in the control group nor in the group with trisomic fetuses was maternal serum alpha-fetoprotein significantly associated with fetal nuchal translucency thickness (r = 0.01 and r = 0.03). CONCLUSION: Measurement of maternal serum alpha-fetoprotein concentration in the first trimester of pregnancy is not likely to be useful in the prediction of fetal trisomies. PMID- 7530491 TI - Interaction of alpha-latroinsectotoxin from Latrodectus mactans venom with bilayer lipid membranes. AB - alpha-Latroinsectotoxin (LIT) from Latrodectus mactans venom increased the conductance of bilayer lipid membranes (BLM) by inducing channel like activity. The channels formed had a maximal single channel conductance of 5 pS in 10 mM CaCl2 solution. This process occurred more rapidly in symmetrical 10 mM CaCl2 solution than in equimolar KCl or NaCl. The LIT induced conductance showed pronounced rectification, that was dependent upon the face of the BLM to which the LIT was applied. This suggests that the LIT molecules incorporate into the bilayer lipid membrane in an oriented manner. The ion channels formed in bilayer phospholipid membrane by LIT are cation selective. The permeability of divalent cations decreased in the order Ba2+ > Ca2+ > Mg2+ > Cd2+ > Zn2+ (Zn2+ and Cd2+ blocked effectively LIT channels with the ratio of Ca2+trans and Cd2+cis or Zn2+cis of 1:1). Selectivity of LIT to monovalent cations was not high and was Ca2+ sensitive. Our data suggest that LIT has at least two Ca(2+)-binding sites, a high affinity site and low one (pK of binding is 2.4). As a result, the binding kinetics of Ca2+ with the toxin shows a high positive cooperativity (Hill coefficient, (h) = 5.95) and that dimerization might be a prerequisite to channel formation. Temperature dependence of conductance of LIT treated lipid bilayers in 100 mM KCl and 10 mM CaCl2 solutions was also determined: 18.9 +/- 2.11 kJ/mol and 28.537 +/- 1.678 kJ/mol, respectively. PMID- 7530492 TI - Cytosolic pH regulation in chicken enterocytes: Na(+)-independent regulatory cell alkalinization. AB - The mechanisms involved in intracellular pH (pHi) recovery from an acid load have been investigated in enterocytes isolated from chicken. Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+ and bicarbonate. This Na(+)- and bicarbonate independent (NBI) regulatory cell alkalinization became negligible when the pHi has reached a value of approx. 6.85. Addition of Na+ induced a rapid pHi recovery to control values. Rotenone, DCCD, vanadate, NBD-Cl, SCH 28080 and EIPA inhibited the NBI cell alkalinization, whereas bafilomycin A1, ouabain and H2-DIDS were without effect. Na(+)-dependent pHi recovery from an acid load was inhibited by EIPA and unaffected by SCH 28080 or DCCD. The rate of NBI cell alkalinization was a linear function of the electrochemical proton gradient. In high external K+ buffer plus valinomycin the line goes through the origin. Gramicidin accelerated the rate of NBI cell alkalinization, whereas it was slightly reduced by low external potassium. The results demonstrate that in intestinal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)- and bicarbonate-independent proton transport system. This latter mechanism appears to be a proton conductance pathway. PMID- 7530493 TI - Calorimetric and theoretical studies of the effects of lindane on lipid bilayers of different acyl chain length. AB - The effects of the insecticide lindane on the phase transition in multilamellar bilayers of saturated diacylphosphatidylcholines of different acyl chain length (DC14PC, DC16PC, and DC18PC) have been studied by means of differential scanning calorimetry (DSC), as well as computer-simulation calculations on a molecular interaction model. The calorimetric data show that increasing concentrations of lindane lower the transition temperature and lead to a broadening of the specific heat in a systematic way depending on the lipid acyl chain length. Kinetic effects in the observed calorimetric traces indicate that the incorporation of lindane into multilamellar lipid bilayers is slow, but faster for the shorter lipid species. Large unilamellar vesicles do not show such kinetic effects. The transition enthalpy is for all three lipid species found to be independent of the lindane concentration which implies that the entropy of mixing is vanishingly small. This lends support to a microscopic molecular interaction model which assigns the absorbed lindane molecules to interstitial sites in the bilayer. Computer-simulation calculations on this model, which assumes a specific interaction between lindane and certain excited acyl chain configurations, lead to predictions of the lipid-water partition coefficient in qualitative agreement with experimental measurements (Antunes-Madeira and Madeira (1985) Biochim. Biophys. Acta 820, 165-172). The partition coefficient has a peak near the phase transition which is a consequence of enhanced interfacial adsorption of lindane at lipid-domain interfaces. PMID- 7530494 TI - Increasing expression of the normal human CFTR cDNA in cystic fibrosis epithelial cells results in a progressive increase in the level of CFTR protein expression, but a limit on the level of cAMP-stimulated chloride secretion. AB - Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and the consequent defective regulation of cAMP-stimulated Cl- permeability across epithelial cell apical membranes. Given that in vitro transfer of normal CFTR cDNA corrects this defect and that recombinant adenovirus (Ad) vectors can transfer the normal human CFTR cDNA in vivo, Ad vectors have significant potential in the development of effective strategies for CF gene therapy. One concern is whether CFTR overexpression achievable with Ad vectors may have untoward effects on cAMP-stimulated Cl- efflux. To address this, the CF pancreatic epithelial cell line CFPAC-1 was infected with increasing doses of AdCFTR, a recombinant Ad containing the normal CFTR cDNA, and analyzed for CFTR mRNA and protein levels and CFTR function. As the AdCFTR dose increased [multiplicity of infection (moi) 0-1,000], CFTR mRNA and protein levels increased. However, while CFTR function measured by cAMP-stimulated 36Cl- efflux was observed with low doses of the vector (moi 20), there was no further increase in CFTR function with increasing doses of AdCFTR (moi from 20 to 1,000). These data suggest that after AdCFTR-mediated gene transfer, epithelial cells limit the level of cAMP-stimulated Cl- secretion despite increasing levels of CFTR protein. PMID- 7530495 TI - Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice. AB - Human fetal lung rudiments (8-12 weeks of development) undergo considerable growth upon microsurgical ectopic implantation in the xenograft-tolerant SCID mouse, and differentiate into a lung-like tissue that includes: (i) bronchial structures lined with pseudostratified, secretory, ciliated epithelium surrounded by smooth muscle and cartilage rings, (ii) submucosal glands, and (iii) alveolar sacs. Normal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was detected by immunostaining in those grafts, and similar differentiation was observed from either normal or cystic fibrosis (CF) fetal lung rudiments. Upon microinjection into human CF or normal lung grafts in SCID mice, beta-galactosidase-adenovirus gene constructs were efficiently transduced into epithelial and glandular cells. Such an in vivo replica of the human respiratory tissue may be a useful experimental model to study normal and pathologic lung development, and to assay candidate therapeutic gene constructs preclinically. PMID- 7530496 TI - Interleukin-7 gene transfer in patients with metastatic colon carcinoma, renal cell carcinoma, melanoma, or with lymphoma. PMID- 7530497 TI - Effects of chronic cocaine treatment on rat brain: long-term reduction in frontal cortical serotonin. PMID- 7530498 TI - Preparation and properties of A-B-A type block copolymer membranes consisting of poly(N-hydroxypropyl-L-glutamine) as the A component and polyisoprene as the B component. AB - A-B-A type block copolymer (GIG(P)) membranes consisting of poly(N-hydroxypropyl L-glutamine) (PHPG) as the A component and polyisoprene (PI) as the B component were prepared by carrying out aminoalcoholysis reaction with 3'-amino-1-propanol and a crosslinking reaction with 1,8-octamethylenediamine (OMDA) on membranes of the starting block copolymer (GIG) membranes consisting of poly(gamma-benzyl L glutamate) (PBLG) and PI. It was shown that the effective crosslink density was proportional to the molar % of OMDA in the reaction mixture. The relation between their bulk structure and membrane properties was investigated, such as the swelling ratio q in a pseudo-extracellular fluid (PECF), tensile properties, and enzymatic degradation behaviour of the membranes in PECF. The tensile properties of the hydrophilic membranes were highly dependent on q in PECF, and on the hydrophobic portions in molecular chains, whose behaviour was typical of an elastomer. Biodegradation of samples in vitro by papain indicated that the rate of degradation was also highly dependent on q of membranes in PECF. PMID- 7530499 TI - Role of lindane in membranes. Effects on membrane fluidity and activity of membrane-bound proteins. AB - The influence of lindane (gamma-hexachlorocyclohexane) on fluidity of plasma membranes from rat renal cortical tubules has been investigated. Preincubation with lindane increased membrane fluidity. This effect was accompanied by (i) a decrease in the transport of glucose with regard to the controls and (ii) an inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation. However, a significant decrease of the membrane fluidity was found when rats were injected with lindane for 12 days. The injection of lindane exerted the opposite effect on the membrane proteins, the glucose transporter and the beta-adrenergic receptor, enhancing the glucose uptake and increasing the isoproterenol-stimulated cycle AMP accumulation. A possible explanation of the difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the activity of many lindane-sensitive proteins in insecticide-injected rats. PMID- 7530500 TI - Association and activation of Btk and Tec tyrosine kinases by gp130, a signal transducer of the interleukin-6 family of cytokines. AB - Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor constitute the IL-6 family of cytokines and play important roles in hematopoiesis, immune response, and nervous system. The receptors for the IL-6 family of cytokines share gp130 through which signals are generated, although the cytoplasmic region of gp130 does not contain any catalytic domain. In this study we show that in addition to Jak family tyrosine kinase, the stimulation of gp130 by IL-6 plus soluble IL-6 receptor alpha induced the activation of Btk and Tec tyrosine kinases, whereas IL-3 and granulocyte colony-stimulating factor activated Tec but not Btk in a pro-B cell line. Furthermore, both Btk and Tec kinases were associated with gp130 without the ligand stimulation. Because Btk is a critical tyrosine kinase for B lymphopoiesis and Tec is considered to be involved in hematopoiesis, the results suggest the involvement of gp130-Btk-Tec signal pathway in early lymphohematopoiesis. PMID- 7530501 TI - Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis. AB - To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors. PMID- 7530502 TI - Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form. AB - Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4 h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase. PMID- 7530503 TI - The role of the homeobox gene, HOX B7, in human myelomonocytic differentiation. AB - Homeobox genes encode transcription factors known to be important morphogenic regulators during embryogenesis. An increasing body of work implies a role for homeobox genes in both hematopoiesis and oncogenesis. We have analyzed the role of the homeobox gene, HOX B7, in the program of differentiation of the biphenotypic myeloid cell line, HL60. Induction of monocytic differentiation in HL-60 cells by vitamin D3 resulted in rapid expression of HOX B7 mRNA, but stimulation with phorbol ester or dimethyl sulfoxide (DMSO) did not. Constitutive overexpression of HOX B7 in the HL60 cell line inhibited the granulocytic differentiation associated with stimulation with DMSO or retinoic acid, but had no effect on the monocytic differentiation induced by vitamin D3. Normal human monocytes do not constitutively express HOX B7, nor are they able to be induced to do so by stimulation with colony-stimulating factor 1 (CSF-1) and gamma interferon (IFN gamma), or with vitamin D3 and lipopolysaccharide. Human bone marrow (BM) cells were found to express HOX B7 in response to granulocyte macrophage CSF (GM-CSF) and antisense oligonucleotides directed against HOX B7 inhibited the formation of colonies derived from GM-CSF-stimulated BM. These data suggest a critical role for HOX B7 in myelomonocytic differentiation. PMID- 7530504 TI - Tumor necrosis factor alpha induces endothelial galactosyl transferase activity and verocytotoxin receptors. Role of specific tumor necrosis factor receptors and protein kinase C. AB - Infections with verocytotoxin (VT) producing Escherichia coli have been strongly implicated in the epidemic form of hemolytic uremic syndrome (HUS). Endothelial damage plays a central role in the pathogenesis of HUS. In vitro studies have shown that VT can damage endothelial cells after interaction with its cellular receptor globotriaosylceramide (GbOse3cer). Cytokines, such as tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) can potentiate the toxic effect of VT by inducing a protein-synthesis dependent increase in VT receptors on endothelial cells. In this study, the mechanisms underlying the increase in endothelial VT receptors induced by TNF alpha were studied in more detail. To investigate which proteins were involved in this induction, endothelial cells were incubated with and without TNF alpha in the presence of 14C-galactose or 14C glucose. Thin-layer chromatography (TLC) analysis of the glycolipid extracts of these cells demonstrated a markedly enhanced incorporation of 14C-galactose in GbOse3cer and other galactose-containing glycolipids, suggesting that TNF alpha enhanced galactosyl-transferase activity. To examine the role of the two recently cloned TNF-receptors (TNFR-p75 and TNFR-p55) in the TNF alpha-induced increase in GbOse3cer in human endothelial cells, cells were incubated with TNF alpha, the TNFR-p55 selective R32W-S86T-TNF alpha-mutant, or the TNFR-p75 selective D143N A145R-TNF alpha-mutant. The effect of TNF alpha activation, determined by binding experiments with 125I-VT-1, could be largely, but not completely mimicked by R32W S86T-TNF alpha. Although incubation of cells with D143N-A145R-TNF alpha did not show an increase in VT-1 binding, the monoclonal antibody utr-1, which prevents binding to TNFR-p75, decreased the TNF alpha-induced VT-1 binding. Activation of protein kinase C (PKC) by phorbol ester increases the expression of VT-1 receptors; this effect was prevented by the PKC inhibitor Ro31-8220 and by homologous desensitization by pretreatment with phorbol ester. In contrast, the presence of the protein kinase inhibitor Ro31-8220 or desensitization of PKC activity reduced the TNF alpha-induced increase in VT-1 receptors maximally by 50% and 24%, respectively. Comparable reductions in overall protein synthesis and the synthesis of E-selectin and plasminogen activator inhibitor-1 (PAI-1) were observed. This suggests an effect on general protein synthesis rather than a specific effect of PKC in the signal transduction pathway, by which TNF alpha induces VT-1 receptors. Our results indicate that TNF alpha can increase the VT-1 receptors on endothelial cells by inducing galactosyl-transferase activity, that this action of TNF alpha mainly occurs via the TNFR-p55; and that PKC activation increases expression of VT-1 receptors by a separate mechanism that acts additively to the TNF alpha-induced increase in VT-1 receptors. PMID- 7530505 TI - Morphology, immunophenotype, and distribution of latently and/or productively Epstein-Barr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of Epstein-Barr virus. AB - The present study was undertaken to unequivocally demonstrate the morphology, immunophenotype, and localization of Epstein Barr virus (EBV)-infected cells as well as the type of infection (latent versus productive) in tonsils of acute infectious mononucleosis. Paraffin sections from nine cases with clinical, serologic, and morphologic evidence of EBV infection were analyzed for the detection of small transcripts, designated EBER1 & 2, and BHLF1 by in situ hybridization (ISH) using nonisotopically labeled probes. ISH was combined with immunohistology, employing a broad panel of antibodies against B-, T-, epithelial , macrophage-, and follicular dendritic cell (FDC)-antigens. All EBER-positive cells could be identified as lymphocytes, as they did not exhibit any morphologic or immunologic characteristics of epithelial cells, macrophages, or FDCs. A preferential accumulation of EBER-positive cells was noted around crypts, within surface squamous epithelium, and in the surroundings of necrosis. The majority of these lymphocytes could be shown to be B cells, which morphologically included Reed-Sternberg (RS)-like cells, immunoblasts, medium-sized lymphoid cells, as well as cells with plasmacytoid differentiation. In all cases, a varying number of EBER-positive T cells could be identified. ISH for BHLF1-RNA detection showed that almost all cases contained single positive small lymphoid cells, indicating a transition from latent to productive infection cycle. Such cells could also be detected within the crypt epithelium reaching up to its surface. Additional screening of 123 oropharyngeal mucosa samples from patients without evidence of acute EBV-infection, using the polymerase chain reaction for EBV-DNA detection combined with EBER- and BHLF1-ISH showed single latently infected lymphocytes in only one case. Our data imply that infected lymphocytes and not epithelial cells are, in fact, the reservoir for EBV infection, and that these are the cells that participate in the interindividual virus transfer. PMID- 7530506 TI - Myeloma cells express Fas antigen/APO-1 (CD95) but only some are sensitive to anti-Fas antibody resulting in apoptosis. AB - To find out which cytokines are involved in the pathogenesis of multiple myeloma, we investigated cytokine receptor expression on myeloma cells using a panel of monoclonal antibodies (MoAbs). Flow cytometric analysis of five myeloma cell lines (RPMI8226, ARH77, KMM-1, U266, and Hs) and myeloma cells freshly isolated from eight patients showed that interleukin-1 receptor (IL-1R) type I and type II, IL-2R alpha and beta chains, IL-4R, IL-6R, IL-7R, IL-8R, granulocyte macrophage colony-stimulating factor receptor (GM-CSFR), c-kit (stem cell factor receptor [SCFR]), membrane bound stem cell factor (MBSCF), and tumor necrosis factor (TNF) receptors type I and type II were not always detected on the myeloma cells. However, interferon-gamma receptor, gp130, and Fas antigen were constitutively expressed, except one sample. To determine the role of Fas antigen on myeloma cells, these cells were cultured with anti-Fas MoAb. Apoptotic changes characterized by loss of cell volume, membrane blebbing, fragmentation of nuclei, and condensed chromatin were observed in three of five myeloma cell lines. When bcl-2 expression was examined, it was seen in all the cell lines regardless of the sensitivity to anti-Fas MoAb. Furthermore, anti-Fas MoAb not only induced apoptosis of freshly isolated myeloma cells but also inhibited the DNA synthesis, although such effects varied from patient to patient. The data indicate that only some myeloma cells undergo apoptosis in response to the signal mediated by the Fas antigen. PMID- 7530507 TI - Immunoreactive interleukin-6 and acute phase proteins as prognostic factors in multiple myeloma. Finnish Leukemia Group. AB - High serum level of bioactive interleukin-6 (IL-6) is regarded as a predictor of poor prognosis in multiple myeloma (MM). On the other hand, the reported levels of immunoreactive IL-6 have been highly variable, and the prognostic value of immunoreactive IL-6 in MM is not clear. We have analyzed the prognostic significance of serum immunoreactive IL-6, as measured by a sensitive immunosorbent assay, in 210 patients with newly diagnosed MM subsequently treated with intermittent melphalan and prednisone. The serum levels of acute phase proteins C-reactive protein (CRP), alpha 1-antitrypsin (alpha 1AT), and acid alpha 1-glycoprotein (orosomucoid; OM) were evaluated as surrogates for IL-6. Serum IL-6, CRP, alpha 1AT, and OM levels were raised in 42%, 40%, 41%, and 24% of the patients, respectively. There was a significant correlation between the clinical stage of the patients and serum IL-6 (P = .006), alpha 1AT (P = .001), and OM (P = .004) levels at diagnosis. At 3 years, 52% of the patients were alive. Univariate logistic regression analysis showed that high levels of IL-6 (P = .002), CRP (P = .02), alpha 1AT (P < .001), OM (P = .007), beta 2-microglobulin (beta 2M; P < .001), and thymidine kinase (P < .05) were all associated with 3 year mortality. In multivariate regression analysis, beta 2M (P < .0001) and alpha 1AT (P = .01) had independent prognostic significance. The patients with high levels of both beta 2M and alpha 1AT or IL-6 were at very high risk of dying within 3 years from diagnosis (16% and 21% of the patients in these groups were alive, respectively). When the patients were stratified according to the clinical stage, the prognostic significance of serum IL-6 and alpha 1AT was especially evident in stage II patients. When the patients were divided into two groups according to normal or raised serum IL-6 levels, the patients with high IL-6 levels had more frequent osteolytic bone lesions (P = .03) and a more aggressive disease. We conclude that serum immunoreactive IL-6 is a significant prognostic marker in MM. PMID- 7530508 TI - Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin's disease cell lines. AB - CD40 is a member of the nerve growth factor receptor family, showing a significant homology to the Hodgkin's disease (HD)-associated antigen CD30 and is capable of transduce growth signals in a number of cell types. A series of 312 lymphoma samples, including 139 cases of HD, 32 cases of CD30+ anaplastic large cell (ALC) lymphomas, 141 cases of other non-Hodgkin's lymphomas (NHLs), and a panel of HD- or NHL-derived cell lines, were evaluated for CD40 expression by immunostaining of paraffin embedded sections, cell smears and flow cytometry. CD40 was strongly expressed with a highly distinct pattern of staining on Reed Sternberg (RS) cells and variants in 100% (139/139) of HD cases, irrespective of their antigenic phenotype (T, B, non T-non B) and histologic subtype of HD. Conversely, CD40 was immunodetected on only one third (12/32; 37%) of ALC lymphoma cases and on 105 of 127 B-cell NHLs. The relative cell density of CD40 on HD cell lines (L-428, KM-H2, HDLM-2) as assessed by flow cytometry was significantly higher than on all other lymphoma cells analyzed. Engagement of CD40 by its soluble ligand (CD40L) enhanced both clonogenic capacity and colony cell survival of HD cell lines. Such effect was potentiated by interleukin-9 costimulation in KM-H2 cells. Finally, we have shown that in vitro rosetting of activated CD4+ T cells to HD cells (L-428) is mediated in part by the CD40/CD40L adhesion pathway. Our data indicate that CD40 is a useful antigen for immunodetection and identification of tumor cells in all subtypes of HD, and suggest that it may play a role in the regulation of RS cell expansion and the contact-dependent interactions of these cells with cytokine-producing T lymphocytes. PMID- 7530509 TI - Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. AB - The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies. PMID- 7530511 TI - The many faces of lysosomal proteinases (cathepsins) in human neuropathology. A histochemical perspective. PMID- 7530510 TI - Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression. AB - We have used a combination of hematopoietic growth factors to induce in vitro granulocytic maturation. A fraction of marrow cells enriched for hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from normal human bone marrow by monoclonal antibody staining and fluorescence-activated cell sorting. Cells were cultured in a suspension system for 3 days in the presence of stem cell factor and interleukin-3 (IL-3), after which granulocyte colony-stimulating factor (G CSF) was added. Cells were harvested daily and analyzed for phenotypic maturation by morphologic criteria, and total RNA was obtained for analysis of myeloid gene expression. Maturation was observed to progress to the late metamyelocyte and band stage over a period of 10 to 12 days. Neutrophil-specific gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Induction with G-CSF resulted in sequential expression of primary and secondary granule proteins, with asynchronous expression of primary granule proteins starting from days 1 to 5, and synchronous expression of lactoferrin and transcobalamin I (secondary granule proteins) from days 7 to 8. Interestingly, myeloperoxidase (MPO) mRNA expression was easily detected in both the freshly isolated CD34+, HLA-DR+ cells and cells at all subsequent stages of induction. This suggests that MPO mRNA is expressed very early during neutrophil development, perhaps before the development of significant numbers of phenotypically recognizable granules. This recapitulation of a program of sequential expression of primary and secondary granule protein genes suggests that in vitro marrow culture suspensions to which appropriate growth factors are added can mimic normal granulocytic maturation. This system should provide an important model for the study of neutrophil-specific gene expression. PMID- 7530512 TI - Regional and seasonal variations of RNA synthesis in the brain of the green frog, Rana esculenta. AB - Changes of RNA synthesis were demonstrated in neurons and ependymal cells of the green frog Rana esculenta during the annual cycle using the Mallory's trichrome stain as histochemical marker and autoradiography. Since the higher affinity of the nuclei for aniline blue is consistent with the increase of RNA content, the increase of RNA synthesis was expressed as percentage of the blue stained nuclei (% BSN). Neuronal transcription starts slowly in March or April, reaches a maximum in July and declines from September to November or December, depending on the brain region. In the ependymal cells, RNA synthesis starts in March and lasts until October. Neuronal transcriptional activity is found mostly in the glomerular layer of the olfactory bulb, in the striatum, nucleus accumbens septi, lateral and medial septal nuclei of the telencephalon, in the habenulae and various nuclei of the diencephalon, in the tectum opticum (particularly in the stratum griseum centrale), in the molecular layer of the cerebellum and in various nuclei of the rhombencephalon. The transcriptional activity of the ependymal cells is quite uniform in the lateral ventricles and the fourth ventricle, while it shows regional symmetric distribution in the third ventricle. Seasonal differences in transcriptional activity appear to be independent of seasonal thermic and photoperiodic fluctuations. In fact, temperature and photoperiod manipulations do not modify significantly the number of active nuclei. It is likely that the increase of RNA synthesis in nerve and ependymal cells corresponds to the resumption of neurotransmitter biosynthesis after hibernation. The simple Mallory's trichrome stain provides a reliable method for revealing increased transcriptional activity in histological sections. PMID- 7530513 TI - Immunocytochemistry of carbonic anhydrase and non-radioactive detection of its RNA by in situ hydridization in the chick embryo membranous labyrinth. AB - An immunocytochemical study was carried out with the aim to localize carbonic anhydrase isozymes (CA I and CA II) in the chick membranous labyrinth. CA I and CA II were localized in the same cells and, during the early developmental stages, were diffusely present in the labyrinthine epithelium. At later stages, the sensorial epithelium of the maculae acusticae, crista ampullaris and papilla basilaris, as well as labyrinthine dark cells and the epithelium of the endolymphatic sac strongly stained by the immunocytochemical procedure. In 1-day and 1-week old chicks, CA was present in the same sites as at advanced embryonal stages. In order to detect mRNA for carbonic anhydrase II, in situ hybridization was also used. Strong hybridization signals were observed in the sensorial epithelium of the saccule, utricle and crista ampullaris, in the tegmentum vasculosum, the endolymphatic sac, the erythrocytes and the choroid plexus in embryos older than 10 days. This positivity persisted until the day after hatching. The authors discuss the significance of labyrinthine CA and, based on in situ hybridization results, suggest that melanocytes do not provide labyrinthine dark cells with enzyme protein. PMID- 7530514 TI - Immunocytochemical study of intestinal endocrine cells in germ-free mice. AB - Morphological and functional modifications have been described in the gastrointestinal tract of germ-free rodents in comparison to their conventional counterparts. These differences have been related to the absence of normal flora, but peptides and biogenic amines may also be involved. Enteroglucagon-, peptide YY-, somatostatin-, serotonin-, neurotensin-, chole-cystokinin-, and secretin producing cells were studied by immunocytochemistry in the ileum, cecum and colon of 10 germ-free, 10 conventional and 5 conventionalized CFW adult male mice. In the cecum and colon of germfree mice, enteroglucagon- and peptide YY-positive cells appeared enlarged, were globular in shape and strongly immunostained. Enlarged and strongly immunostained serotonin- and neurotensin-positive cells were also seen in some germ-free mice. Somatostatin-positive cells and all the endocrine cells of the ileum were similar in the 3 groups, and no cholecystokinin or secretin-positive cells were observed. Our results are consistent with higher enteroglucagon and peptide YY production in germ-free mice that may be related to the lower cellular renewal rates and slower gastrointestinal transit reported in these animals. Locally produced biogenic amines and peptides may be important factors in the mediation of the effect of the intestinal flora on gastrointestinal structure and function and germ-free mice seem to be a good model for the study of the mechanisms involved. PMID- 7530515 TI - Lymphoid follicles in extranodal sites: an immunohistochemical study. AB - Lymphoid follicles in extranodal sites involved in chronic inflammatory reactions are the subject of this study. Immunohistochemistry was focused on associated lymphoid formations considered as incomplete or prefollicular structures because they were composed only of some types of the germinal centre cells among demonstrated dendritic reticulum cells (DRC) and thin vessels. Paraffin embedded tissues were stained with current histological methods and immunohistochemically processed with a panel of antibodies against DRC (CD21, CD35, IgM); endothelial cells (factor VIII R. Ag., EUA); B lymphoid cells (L26, MB2); T lymphoid cells (UCHL1, MT1): intercellular adhesion molecule (ICAM-1); histiocytic/monocytic cells (KP1, PS100, delta-1-antitrypsin, delta-1-antichymotrypsin); smooth muscle (actin 1A4); basement membranes (collagen IV). Prefollicular structures showed positivity for B cells markers among a network of DRC and thin long processes. Furthermore, scattered T lymphoid and histiocytic cells such as collagen IV fragments and some pericytic cells were demonstrated. Some sections with Emat eos. were destained and submitted to immunohistochemistry analyses: in prefollicular structures, histologically referred endothelial cells were positive for dendritic reticulum markers and ICAM-1. These findings appear to favor the hypothesis that DRC could be transformed endothelial cells which therefore represent an important component in follicular formation. PMID- 7530516 TI - Protein G-horseradish peroxidase based method for light-microscope immunocytochemistry. Application to the pituitary gland of the killifish, Fundulus heteroclitus. AB - Horseradish peroxidase-protein G conjugate was used to localize anti-human luteinizing hormone and anti-human chorionic gonadotropin primary antibodies bound to gonadotropins in paraffin and Historesin embedded sections of Fundulus heteroclitus pituitaries. The sensitivity and specificity of this method and those obtained after the avidin-biotin-peroxidase complex, the streptavidin biotin-peroxidase complex and the peroxidase-antiperoxidase procedures were compared. The protein G-horseradish peroxidase method gave a clear immunostaining of gonadotrophic cells with virtually no background. Detection efficiency was reduced in comparison with the other techniques. As for the embedding medium, hydrophilic resin Historesin considerably enhanced the structural detail and the resolution of the immunostaining at light-microscope level. PMID- 7530517 TI - Immunocytochemical detection of endocrine cells in the gut of Viviparus ater (Mollusca, Gastropoda). AB - The presence of endocrine cells was investigated by immunocytochemical procedures in the gut and salivary gland of Viviparus ater, a freshwater prosobranch gastropod. The endocrine cells were scanty and both of closed and open cell type. Most of them were located in the esophagus (immunostaining with anti-gastrin, anti-insulin, anti-serotonin and anti-substance P antisera), very few in the stomach (immunoreactive only to anti-gastrin antibody) and in proximal part of the intestine (immunoreactive to anti-serotonin and anti-substance P antibodies). In the salivary glands, occasional endocrine cells scattered among the glandular cells in the adenomera stained with anti-neuropeptide Y, anti-pancreatic polypeptide and anti-somatostatin sera were detected. PMID- 7530519 TI - Chondroitin sulphates and embryonic chick skin fibroblast proliferation. AB - We have investigated the action of sulphated glycosaminoglycans (GAGs) upon primary 11-day chick embryo skin fibroblasts cultured for various periods of time in the presence or absence of fetal calf serum (FCS). Chondroitin 4- and 6 sulphate (CS) added to serum supplemented medium provoked a decrease in DNA synthesis both in sparse and crowded cultures only at high concentrations (250 micrograms/ml). Synthesis of endocellular and secreted proteins was not affected. In contrast, CS administered to fibroblasts for 24 h in serum-free medium stimulated DNA synthesis. We postulate that the CS stimulating effect seen in serum-free cultures might be antagonized by peptide regulators of cell growth normally present in serum. PMID- 7530518 TI - Effects of spermidine synthase inhibition on cytoskeletal organization in cultured chick embryo fibroblasts. AB - The administration of bis-cyclohexylammonium sulphate (BCHS), an inhibitor of spermidine synthase, to cultured chick embryo fibroblasts provoked alterations in cell morphology, a marked disorganization of microfilaments and changes in microtubule network structure. In addition, the rate of microtubule reappearance, after disrupting them with colchicine, was impaired by BCHS. These responses to BCHS were prevented by spermidine addition, which thus suggests an involvement of spermidine in microtubule and microfilament organization. PMID- 7530520 TI - Hepatotoxic effects of hexachlorocyclohexane on carbohydrate metabolism of a freshwater fish Channa punctatus (Bloch). PMID- 7530523 TI - [Biomedical studies at the Russian Academy of Medical Sciences]. PMID- 7530522 TI - Methimazole-induced agranulocytosis treated with recombinant human granulocyte colony-stimulating factor (G-CSF). AB - A 35-year-old female hematology technician with Graves' disease developed agranulocytosis a few days after starting therapy with Tapazole (methimazole). Because of a recent report of use of recombinant human granulocyte colony stimulating factor (G-CSF) in patients with propylthiouracil-induced agranulocytosis, 5 micrograms/kg/day G-CSF was administered and her granulocyte count returned to normal after three doses, on the sixth day after the last dose of methimazole. We conclude that in patients with drug-induced agranulocytosis, the use of G-CSF, in addition to discontinuation of the offending drug, hastens recovery and reduces morbidity. PMID- 7530521 TI - Connections of inferior temporal areas TEO and TE with parietal and frontal cortex in macaque monkeys. AB - Inferior temporal cortex is perhaps the highest visual processing area and much anatomical work has focused on its connections with other visual areas in temporal and occipital cortex. Here we report connections of inferior temporal cortex with regions in the frontal and parietal lobes. Inferior temporal areas TEO and TE were injected with WGA-HRP and 3H-AA, respectively, or vice versa, in 1-week-old infant and 3-4-year-old adult monkeys (Macaca mulatta). The results indicated that whereas TEO has more extensive connections with parietal areas, TE has more extensive connections with prefrontal areas. Thus, in the intraparietal sulcus, area TEO is connected with areas LIPd, LIPv, and V3A, and with the as yet undefined region between LIPv and V3A, whereas the connections of TE are predominantly with LIPd, and to a lesser extent with LIPv. In the prefrontal cortex, area TE is connected with areas 8 and 45 in the inferior limb of the anterior bank of the arcuate sulcus, with area 12 on the inferior prefrontal convexity, and with areas 11 and 13 on the orbital surface. By contrast, the connections of area TEO are limited to areas 8, 45, and 12. Furthermore, within prefrontal cortex, the projections from areas TEO and TE terminate in different layers in areas 8 and 45, such that those from TEO terminate in all layers, whereas those from TEO terminate in layers I and V/VI only. In contrast to the connections of areas TEO and TE with various medial temporal-lobe and subcortical structures, which are immature in infant monkeys (Webster et al., 1991, 1993b), the connections with parietal and prefrontal areas appear adult-like as early as 1 week of age. PMID- 7530524 TI - [Main results of the performance of the preventive medicine section of the Russian Academy of Medical Sciences in the areas of hygiene, microbiology and epidemiology]. PMID- 7530525 TI - [History of the development of the Siberian section of the Russian Academy of Medical Sciences]. PMID- 7530526 TI - [Modern pharmacology and progress of medicine]. PMID- 7530527 TI - [Academy of Medical Sciences--50 years]. PMID- 7530528 TI - [State and prospects of the development of new synthetic drugs]. PMID- 7530529 TI - [Current tasks of psychopharmacology]. AB - The basic principle of psychopharmacology--is its impact on the psychopathological syndrome, having in view its specificity, expressivity of negative disturbances, nosological entity. One of the approaches to the selection of adequate psychopharmacotherapy may be the concept of comorbidity of disturbances, determining the structure of the syndrome and perspectives of its impact. With this in view, the structure of some syndromes may be considered not as a complex of closely interrelated disturbances, but as states stemming from relatively independent pathological disturbances, connected by probable relations. The problem of differentiated psychopharmacotherapy in line with the phenomenon of comorbidity at the level of symptom-syndrome, psychopathological personality disturbances (including negative ones), may have a practical as well as a theoretical significance. An analysis of comorbid connections determines as well the selection of preventive drugs in the prophylaxis of relapses of psychoses. An eventual study of all sides of this problem has its goal to define the spectre of action of the already known drugs and new psychopharmacological preparations, a deeper understanding of psychopathology and the clinical state being under the impact of psychopharmacological therapy, a transition from an extensive understanding of a wide range of questions of psychopharmacology to an intensive study of separate facets of the problem, which seems to be most perspective. PMID- 7530530 TI - [Biomedical effects of solar activity]. PMID- 7530531 TI - [Regulation of prostatic growth, pathogenesis of prostatic adenoma and cancer]. AB - This review discusses the molecular mechanism of the regulation of prostate growth, maintenance of the prostate integrity and possible disturbances in these processes leading to the development of prostatic cancer and benign hyperplasia. The role of hormones, growth factors, oncogenes is described in more details. The participation of the angiotensin converting enzyme and renin-angiotensin systems in the regulation of polyamine synthesis in the prostate and the regulation of prostate growth is assumed. PMID- 7530532 TI - [Scientist ahead of his time (95th anniversary of the birth of the Academician V. N. Ioffe)]. PMID- 7530533 TI - [Achievements and prospects of clinical medicine]. PMID- 7530535 TI - Evaluation of palliative care. Patients must be told that treatment will be randomised. PMID- 7530534 TI - Evaluation of palliative care. Important factors are hard to measure. PMID- 7530536 TI - Evaluation of palliative care. Patients should be randomised at time of diagnosis. PMID- 7530537 TI - Evaluation of palliative care. Recruitment figures may be low. PMID- 7530538 TI - Hepatitis C virus antibodies and Graves' disease. PMID- 7530539 TI - Detection of prostate cancer. PMID- 7530540 TI - Selective stimulation of excitatory amino acid receptor subtypes and the survival of granule cells in culture: effect of quisqualate and AMPA. AB - Differentiating granule cells develop survival requirements in vitro which can be met by treatment with high K+ or excitatory amino acids. Promotion of cell survival by N-methyl-D-aspartate (NMDA) or kainate has already been established and here we report that treatment of the cells with alpha-amino-3-hydroxy-5 methyl-4-isoxazole-propionate (AMPA) or quisqualate (QA) also leads to cell rescue. In comparison with the effect of NMDA, the influence of AMPA/QA is small, resulting in a 20-30% increase in cell survival, with a peak at a very narrow concentration range (0.5-2.0 microM QA and 5-10 microM AMPA). The effect is exclusive to AMPA receptor stimulation, since stimulation of metabotropic glutamate receptors with (1S3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) has no effect. Furthermore, AMPA/QA rescue of cells is blocked by ionotropic non NMDA receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2,3 dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX). In addition, both nifedipine and dizolcipline (MK-801) interfered with the cell survival promoting effect of AMPA, suggesting that the influence of AMPA is mediated via calcium influx involving both depolarization-activated voltage sensitive calcium channels and NMDA receptors stimulated as a result of AMPA-induced release of glutamate. Possible reasons for the small cell survival promoting effect of AMPA/QA compared with the influence of high K+ or NMDA are discussed. PMID- 7530541 TI - Human immunodeficiency virus-1 reverse transcriptase heterodimer stability. AB - Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 degrees C, with a dimerization Ka value of 5.2 x 10(5) M-1 for both models. Ka values of 2.1 x 10(5) and 3.8 x 10(5) M-1 were obtained for the respective association models at 20 degrees C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 degrees C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 degrees C to 37 degrees C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer. PMID- 7530542 TI - Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor. AB - In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available. PMID- 7530543 TI - Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. AB - We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID- 7530544 TI - Identification of a molecular switch that selects between two crystals forms of bovine pancreatic trypsin inhibitor. AB - Two crystals forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2-fold axis in form III crystals, the Lys 41 side chain amino nitrogens from 2 symmetry-related molecules are only 2.72 A apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side-chain amino group is protonated and forms a salt bridge with a solvent derived phosphate group. For the Lys 41 side-chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional intermolecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals. PMID- 7530545 TI - Expression of the c-kit mRNA in human aortic endothelial cells. AB - We investigated the expression of the c-kit gene in human aortic endothelial cells. RT-PCR and Northern blot analyses showed that a human aortic endothelial cell line expressed mRNA specific for the c-kit gene. Furthermore, the addition of stem cell factor stimulated the cells to increase the amount of c-kit mRNA transcribed in response to its ligand. In situ hybridization also revealed that endothelial cells in vivo express the c-kit gene. PMID- 7530546 TI - The leg of Drosophila as a model system for the analysis of neuronal diversity. AB - The neurons innervating insect sense organs vary in number, shape, dendritic morphology, axonal projections and connectivity, providing abundant material for the genetic analysis of neuronal diversity. Here we describe the leg of Drosophila as a potential model system for this analysis. The leg of Drosophila comprises a variety of sense organs arranged in a precise and reproducible pattern. The cell bodies of the sensory neurons are located near the organ they innervate, which greatly facilitates their identification and accessibility. The development of the leg from its progenitor structure, the imaginal disc, is known in good detail. In particular, the time of appearance and of divisions of the sense organ precursors is known. The origin and mode of formation of the leg nerve (through which all sensory axons project into the central nervous system) has been described. The central projections of some of the sensory neurons have been examined by horseradish peroxidase backfill or DiI labelling. Finally, the expression of several genes that control the differentiation of various types of sensory neurons can be manipulated at will. We illustrate these different aspects, and discuss the potentials and shortcomings of this system. PMID- 7530548 TI - The relationship between synaptogenesis and expression of voltage-dependent currents in cerebellar granule cells in situ. AB - In this work we consider the ontogenetic changes of membrane currents and their relationship with synaptogenesis in cerebellar granule cells. Recordings were performed in whole-cell patch-clamp configuration from cerebellar slices obtained from 4 to 31-day-old rats. Granule cells in the external granular layer, and non connected granule cells in the internal granular layer expressed outward currents, and inconstantly also small Ca2+ currents, but no fast Na+ currents. Most connected granule cells expressed Ca2+ and Na+ currents. These data indicate that Ca2+ and Na+ current development occurs after synapse formation, while outward (K+) currents begin their development before. Mixed NMDA/non-NMDA synaptic currents were observed at all stages, while synaptic currents with a prominent NMDA component were observed exclusively at immature stages. At P4, ie 1-2 days after the arrival of the first granule cells in the internal granular layer, some granule cells already expressed mature synaptic and voltage-dependent currents, suggesting that establishment of mossy fibre synapses and development of membrane properties takes just 1-2 days to complete. Starting at P4, the probability of activating mossy fibre currents, and sizeable Ca2+ and Na+ currents increased at a similar rate, attaining a plateau level around P20. Average amplitude of Na+ and outward currents decreased until P10 and then increased attaining plateau soon beyond P20. Average amplitude of Ca2+ currents increased monotonically. The time courses of probability and average current amplitude curves are likely explained by changes in the rate of accumulation of migrating granule cells in the internal granular layer, and by changes in granule cell membrane surface extension. These data suggest a relevant role for the process of synapse formation in inducing the expression of new channels in the developing granule cells, which may involve Ca2+ influx through the NMDA channel. PMID- 7530547 TI - Glutamate receptor modulation by protein phosphorylation. AB - Glutamate-gated ion channels mediate most excitatory synaptic transmission in the mammalian central nervous system and play major roles in synaptic plasticity, neuronal development, and in some neuropathological conditions. Recent studies have suggested that protein phosphorylation of neuronal glutamate receptors by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) may regulate their function and play a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the receptors and to further examine the sites and mechanisms by which the receptors are modulated, we transiently expressed recombinant glutamate receptors in HEK 293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the proposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current voltage relation or desensitization kinetics. In other experiments, we have demonstrated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a single alternatively spliced exon in the C-terminal domain. These findings suggest that RNA splicing can regulate NMDA receptor phosphorylation and that, contrary to the previously proposed membrane topology model, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal exon with the PKC phosphorylation sites, our preliminary studies indicate that the NMDA-evoked current is potentiated by intracellular PKC. We are currently examining PKC effects on the NMDA-evoked current responses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are directly phosphorylated and functionally modulated by protein kinases. Moreover, by identifying phosphorylation sites within the receptor proteins, our results provide information about the structure and membrane topology of these receptors. PMID- 7530549 TI - Identification of genes within CpG-enriched DNA from human chromosome 4p16.3. AB - We combined the isolation of gene-enriched genomic DNA with gene prediction by computer to search for genes in a cosmid contig covering one million base pairs in the Huntington disease region on chromosome 4. Our aim was to develop a simple, robust strategy to identify genes adjacent to CpG islands without first characterizing undermethylated regions with multiple rare-cutter restriction enzyme sites. We cloned DNA adjacent to the rare-cutter restriction enzyme sites EagI and SacII, which are predicted to cut more frequently within CpG islands and relied solely on minimal sequence analysis to determine the likely coding potential of the DNA next to these sites. Our results indicated that isolating fragments with a single rare-cutter restriction enzyme site was sufficient to provide a high likelihood of identifying genes. Of the 42 CpG-selected clones analyzed, we determined that 17 contained exons as determined by sequence identity to known genes in this region, sequence identity to gene fragments isolated by direct cDNA selection in our laboratory, and/or their ability to detect transcripts on Northern blots. Analysis of the sequences with the BLAST and GRAIL programs provided additional independent evidence that 15 of these 17 clones contain coding sequences and that nine other clones are likely to contain sequences coding for portions of new genes. By mapping these clones to an EcoRI restriction map of the region, we determined a detailed localization for each of the exons and estimate that there are a minimum of seven genes that contain CpG rich DNA between D4S126 and D4S181. PMID- 7530552 TI - Population variation of common cystic fibrosis mutations. The Cystic Fibrosis Genetic Analysis Consortium. PMID- 7530551 TI - A de novo deletion in FMR1 in a patient with developmental delay. PMID- 7530554 TI - Correlation of the AUA symptom index with urodynamics in patients with suspected benign prostatic hyperplasia. AB - The AUA symptom index is widely used to access patients with suspected benign prostatic hyperplasia (BPH). In order to determine how well symptoms as assessed by this index correlate with urodynamic findings, we evaluated 83 patients referred to our urology clinics with symptoms of BPH. All patients completed the AUA symptom index and then underwent a multichannel urodynamic evaluation. Patients were classified as obstructed, unobstructed, or equivocal according to the Abrams Griffiths nomogram. The AUA symptom index was recorded as the total score and, for purposes of symptom classification, further subdivided into an obstructive score (questions 3, 5, and 6) and an irritative score (questions 1, 2, 4, and 7). The mean age of the 83 patients was 67 (45-84). The mean total AUA symptom score was 16.6 (6-34), mean obstructive score was 6.1 (0-15), and the mean irritative score 10.4 (3-20). Pressure flow analysis using the Abrams Griffiths nomogram classified 28 patients (34%) as obstructed, 17 (20%) as unobstructed, and 38 (46%) as equivocal. Using the analysis of variance procedure (ANOVA) there was no statistically significant difference in the mean total (P = 0.446), obstructive (P = 0.979), or irritative (P = 0.136) scores. Detrusor instability was present in 45 patients (54%). While total and obstructive scores were not significantly different in patients with detrusor instability vs. those with stable bladders, irritative scores were higher in patients with instability (P = 0.028) using the T-test procedure. Using ANOVA, the difference in post void residual (PVR) between the groups was not quite statistically significant (P = 0.057).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530555 TI - Urodynamic measure of urethral cross-sectional area: application for obstructive uropathy. AB - A conductance formula was developed based on pressure (P) and flow (Q) data collected from rigid tubes with a cross-sectional area in the range of the flow controlling zone of the urethra. Curve fitting of data resulted in an exponential formula and a simple proportionality constant related tube of differing cross sectional area. The resulting formula estimates cross-sectional area based on P and Q data and is in the form of a conductance ratio because Q is in the numerator and P is in the denominator. The formula is AEFm = 3.5 Q/P0.58, where AEFm is the area equivalent factor-male and Q is measured in ml/sec and P is cm H2O. To further introduce the estimate of cross-sectional area, urodynamics were retrospectively reviewed from 19 males over the age of 50 with complaints of prostatism. The AEFm was plotted on standard urodynamic records much like P and Q data. The onset of voiding was characterized by a rapid increase in urethral area. Maximal cross-sectional area obtained at maximal flow was 2.9 +/- 1.4 mm2. The maximal urethral area per unit of applied detrusor pressure, an estimate of urethral compliance, was 0.06 +/- 0.6 mm2/cm H2O. This latter measure may help to compare patients with widely different detrusor pressures. The urodynamic data from two young adult males is also presented for comparison. Their average maximal urethral cross-sectional area was 7.1 mm2. Their average area per unit of applied detrusor pressure was 0.15 mm2/cm H2O. These larger values contrast sharply with the BPH group. The AEFm clearly shows urethral function in terms of the principal factor regulating the urine flow rate, the urethral cross-sectional area. PMID- 7530556 TI - Invasive Haemophilus influenzae infections: changing patterns. PMID- 7530550 TI - Identification of a de novo insertional mutation in P0 in a patient with a Dejerine-Sottas syndrome (DSS) phenotype. PMID- 7530553 TI - Identification of two novel mutations in the CFTR gene: 3007 del G and 3271 + 1 G ->A. PMID- 7530557 TI - Tuberculosis in 1993: preliminary results of national survey. PMID- 7530558 TI - AIDS and HIV-1 infection in the United Kingdom: monthly report. PMID- 7530559 TI - [To be recognized and supported as parents. Postnatal visits. Better outfitting of nurses]. PMID- 7530560 TI - [Hereditary epidermolysis bullosa: towards classification and genetic counseling based upon identification of molecular defects]. AB - Inherited epidermolysis bullosa (EB) includes three main types depending on the precise ultrastructural level at which the split responsible for the blistering occurs; 1) simplex EB (SEB) located at the level of the basal cells; 2) junctional EB (JEB) located in the lamina lucida; 3) dystrophic EB (DEB) located in the dermis below the lamina densa at the level of the anchoring fibrils. The authors review the major recent progresses which have led to the identification of the genes and of several molecular defects in these three types, such as: mutations of the genes of keratins 5 and 14 in SEB, molecular defects in the gamma 2 chain gene of nicein/kalinin coding for the anchoring fibrils in JEB, abnormalities of the collagen VII gene coding for the anchoring fibrils in DEB. These data allow to consider a classification of EB based on molecular defects. They also have important consequences for genetic counselling and prenatal diagnosis for the families presenting an affected child. PMID- 7530562 TI - Palliation and salvation. Interview by Gael Knepfer. PMID- 7530561 TI - [Gifted children and dysharmonious development]. AB - Gifted children are exposed to psychological disturbances. How much this psychological fragility is of a constitutional origin or is acquired, resulted from their intellectual precocity, is unclear. Nevertheless it justifies an early recognition of these children, knowing that psychological disturbances may be present early in life and mask the intellectual precocity. This is illustrated by the case of a three-year-old girl. Psychometric tests should be performed on an individual basis in order to avoid the negative effects of affective and emotional factors; an IQ above 140 characterizes gifted children. However, creativity tests are equally important allowing to identify their high capacity of originality and creativity. Uneven development of intelligence, affectivity and psychomotricity is frequent, which may induce anxiety disorders, obsessional behaviour and personality disorders. It appears that the higher the IQ is, the greater the psychological fragility, this fragility usually being important for an IQ above 170. The education of gifted children should be concentrated on the development of creativity and reflection, avoiding the tediousness of learning mainly based on memorizing and the acquisition of mechanisms. Early intervention is recommended in the case of psychological and behavioural disturbances, as an attempt to prevent the risk of pathological psychological organization. PMID- 7530563 TI - Sinusoidal capillarization in small hepatocellular carcinoma. AB - The morphological and phenotypic changes of sinusoidal endothelial cells in hepatocellular carcinoma were investigated along with the hemodynamic changes. Hepatocellular carcinomas, which were 10-20 mm in diameter, were obtained by liver aspiration biopsy. Twenty specimens of well differentiated and 20 specimens of moderately differentiated hepatocellular carcinoma were used. These were classified into two groups of hepatocellular carcinoma: with and without hypervascularity. Electron microscopy, immunohistochemistry using antibodies against factor VIII-related antigen, type IV collagen and laminin and lectin histochemistry using Ulex europaeus agglutinin I (UEA-I) were performed. Factor VIII-related antigen and UEA-1 binding sites were present in the sinusoidal endothelial cells in two groups. In hepatocellular carcinoma with hypervascularity, type IV collagen and laminin were present corresponding to basement membranes along the endothelial cells, which had few fenestrae. In another group, although type IV collagen was present along the endothelial cells that had a few fenestrae, laminin and basement membranes were rarely observed. These results suggest that the sinusoidal endothelial cells tend to show phenotypic changes in the early stage of hepatocarcinogenesis. As the arterial blood supply for hepatocellular carcinoma increases, the sinusoidal endothelial cells may form basement membranes and take on the morphological appearance of capillaries. PMID- 7530564 TI - Differential immunoreactivity of transforming growth factor alpha in benign, dysplastic and malignant prostatic tissues. AB - Immunohistochemical examination of radical prostatectomy specimens from 57 patients was performed to determine the differential expression of transforming growth factor alpha in the human prostate. In addition, epidermal growth factor receptor (EGFr) immunoreactivity was assessed in each case. Stromal versus epithelial staining was determined for each histological subtype: benign prostatic hypertrophy (BPH), prostatic intra-epithelial neoplasia (PIN), and prostatic cancer (CaP) by a single pathologist reviewer. TGFa staining was predominant in stroma while EGFr was localized to the epithelial basal cell layer. Immunoreactivity of both TGFa (P = 0.002) and EGFr (P < 0.001) revealed a significant reduction in CaP compared to BPH or PIN. Autocrine stimulation of EGFr by TGFa or other unrecognized factors may be present in CaP. Conversely, altered stromal influence of CaP via TGFa may be present. These observations could form the basis for future cancer therapeutic strategies using antagonist factors. PMID- 7530565 TI - Diagnostic assessment of developmental disability: the parents' view. AB - A self-report questionnaire was used to survey parental recollections and opinions concerning: detection and diagnosis of their child's developmental disabilities; the accessibility, usefulness and the efficacy of generic and specialist services which were offered and utilized by the family during the child's first 5 years from birth; the necessity, utilization and adequacy of current generic and specialist services for the parents and their child. Ninety parents of children who were clients of two New South Wales developmental disability community services at the time of this study were contacted by mail and asked to complete the questionnaire. Eighty parents correctly completed the questionnaire. Parents of children using these two services were selected on the socioeconomic and resettlement differences between these regions (Australian Bureau of Statistics, 1989); descriptive analysis using cross-tabulation tables indicated a significant group difference with regard to the fathers' occupational status and the families' private health-care insurance membership. Group variation was also found with the responses to the availability and adequacy of services. Otherwise parents' experiences in the recognition and diagnostic assessment of their children's developmental disabilities were homogeneous. PMID- 7530566 TI - Changes of nursing organization in a surgical department: effects on work satisfaction and quality of care. AB - The aim of the study was to evaluate the effects of an organizational change programme in a surgical department in Sweden (the introduction of modular nursing) on the nursing staff's perception of job satisfaction and quality of care, and to identify factors which promote or hinder this organizational change. Planning and implementation of the change programme took about 1 year and comprised structural changes and staff training. Assessments of job satisfaction and quality of care were made immediately before, and 1 year after, implementation of the change programme. Data were collected from the staff of two wards. Virtually no statistically significant changes were found when looking at the department as a whole. However, considerable differences were noted between the two wards, particularly in the following areas: relationships with colleagues, identification and commitment, and perceived quality of care. The quality of the interpersonal relationships, and the leadership of the wards' head nurses, appeared to be crucial determinants of the outcome. PMID- 7530567 TI - Cyclothiazide unmasks AMPA-evoked stimulation of [3H]-L-glutamate release from rat hippocampal synaptosomes. AB - The effect of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) on Ca(2+) sensitive, tetrodotoxin (TTX)-insensitive K(+)-stimulated [3H]-L-glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose-dependent increase in K(+)-stimulated [3H]-L glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release. PMID- 7530568 TI - NO-dependent and -independent elevation of plasma levels of insulin and glucose in rats by L-arginine. AB - 1. L-Arginine elevates plasma insulin in man. Recent in vitro data indicate that this is based on stimulation of endogenous nitric oxide (NO) with subsequent pancreatic release of insulin by L-arginine. L-Arginine also raises plasma glucose. 2. We studied plasma levels of insulin, glucose and NO metabolites, as well as systemic blood pressure, in anaesthetized rats during i.v. infusion of L arginine (25-200 mg kg-1 min-1) or glucose (55 mg kg-1 min-1), before and after administration of the NO synthesis inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 50 mg kg-1). 3. Before L-NAME, L-arginine elevated plasma insulin from about 15 to 65 ul-1 and glucose from 5.2 to 6.7 mmol l-1. These effects of L arginine were not dose-related. 4. L-NAME alone had no effect on plasma insulin and glucose levels, but diminished the effects of a low dose (25 mg kg-1 min-1) of L-arginine on plasma insulin by about 40%, and that on plasma glucose by more than 90%. In contrast, the effects of a high dose (200 mg kg-1 min-1) of L arginine on plasma insulin and glucose levels were not affected by L-NAME. 5. L NAME elevated systemic blood pressure by about 35 mmHg. L-Arginine (25-100 mg kg 1 min-1) had no effect on systemic blood pressure, either before or after L-NAME. L-Arginine (200 mg kg-1 min-1) lowered systemic blood pressure, both before and after L-NAME. 6. Glucose infusion elevated plasma glucose from about 5.5 to 6.8 mmol l-1, and plasma insulin from about 18 to 26 ul-1. 7. The basal plasma levels of the NO metabolite nitrate (18 +/- 4 mumol l-1) were not affected by L-arginine (200 mg kg-1 min-1). Plasma nitrosohaemoglobin was likewise unaffected by L arginine (200 mg kg-1 min-1). 8. We conclude that L-arginine separately elevates plasma insulin and glucose levels, both by NO-dependent and -independent mechanisms. PMID- 7530569 TI - Regulation of vascular and gastric smooth muscle contractility by pervanadate. AB - 1. The contractile actions of vanadate (VO4) and pervanadate (PV, peroxide(s) of vanadate) were studied in rat gastric longitudinal muscle strips and in aortic rings. The roles of extracellular sodium and calcium were evaluated and the potential effects of nerve-released agonists were considered. The possibility that these responses were due to the potentiation of tyrosine kinase activity, as a result of PV-mediated tyrosine phosphatase inhibition was explored with the use of tyrosine kinase inhibitors (genistein, tyrphostin) and by Western blot analysis of phosphotyrosyl proteins in PV-treated tissues. The ability of PV to mimic the action of the tyrosine kinase receptor-associated agonist, epidermal growth factor-urogastrone (EGF-Uro), in the gastric preparation was also studied. 2. PV caused concentration-dependent contractions in both gastric and aorta derived tissues, with a potency that was 1 to 2 orders of magnitude greater than that of VO4. 3. Although repeated exposure of gastric and aortic tissues to a fixed concentration of VO4 caused reproducible contractions in both tissues, repeated exposure of gastric tissue to PV caused an increased contractile response plateauing after 3 exposures. In contrast, a single exposure of aortic tissue to PV (20 microM) caused a prolonged desensitization of the tissue to the subsequent contractile actions of PV or other agonists. 4. The contractile responses to PV were unaffected in both preparations by tetrodotoxin, atropine, yohimbine and phenoxybenzamine; and in the aortic preparation, the responses to VO4 and PV were the same in the presence or absence of a functional endothelium. 5. PV-induced contractions in both tissues were observed in the absence of extracellular sodium but required extracellular calcium and were attenuated by 1 micro M nifedipine.6. In the gastric preparation, the characteristics of the contractile actions of PV paralleled those of EGF-Uro in terms of (1) inhibition by genistein, (2) inhibition by indomethacin and (3) a requirement for extracellular calcium. These response characteristics differed from those of other contractile agonists such as carbachol.7. In both the gastric and aortic preparations genistein was able to inhibit PV-induced contractions selectively without causing comparable inhibition of KCI-induced contractions. Tyrphostin (AG18) also selectively blocked PV-induced contractions in the gastric, but not in the aortic preparation.8. In both the gastric and aortic tissue, in step with an increased contractile response, PV caused increases in tissue phosphotyrosyl protein content, as detected by Western blot analysis using a monoclonal antiphosphotyrosine antibody; the increases in phosphotyrosyl protein content were reduced when tissues were treated with PV at the same time as a tyrosine kinase inhibitor.9 PV, at sub-contractile concentrations, potentiated the contractile action of angiotensin II in both the gastric and aorta tissue.10 We conclude that the growth factor-mimetic agent, PV, is a much more potent contractile agonist than V04 in both vascular and gastric smooth muscle tissue. PV can cause enhanced tissue phosphotyrosyl protein content most likely via the inhibition of tissue protein tyrosine phosphatases. The contractile actions of PV, which require extracelullar calcium and are independent of extracellular sodium, would appear not to be due either to Na+/Ca2" exchange, promoted by Na+/K+-ATPase inhibition or to the inhibition of Ca2+-ATPase and might be best explained by the ability of PV, via tyrosine phosphatase inhibition, to potentiate a tyrosine kinase pathway linked to calcium entry and to the contractile process. PMID- 7530570 TI - Actions of cromakalim on outward currents of CA1 neurones in hippocampal slices. AB - 1. Membrane effects of cromakalim (Crom; 50-300 microM) were examined in CA1 neurones recorded mainly by intracellular, single-electrode voltage-clamping in slices (from Sprague-Dawley rats) kept in an interface chamber at 33 degrees C. 2. In 14 cells held at -63 +/- 3.5 mV, in the presence of tetrodotoxin, kynurenic acid and (in most cases) bicuculline, bath applied Crom produced no consistent change in holding current (-59 +/- 66 pA) or input conductance (GN) (-3.9 +/- 5.2%). 3. Overall there were no significant changes in instantaneous inward rectification or in Q-current inward relaxations. 4. In 18 out of 22 cells, outward currents, evoked by 0.5 s pulses to voltages > -50 and < -20 mV, were depressed by Crom (by 42 +/- 11%, for n = 22). Because this effect was consistently seen in Ca current-blocking media, containing either Mn and low Ca, or Cd (and also carbachol), the K channels depressed by Crom were probably of the delayed rectifier (IDR) type. 5. The Crom-control difference current (ICrom), obtained with slow depolarizing ramps, had a biphasic character, inward in the voltage (V) range > -50 < -20 mV (where outward currents are depressed by Crom) and tending outward for V > or = -20 mV. 6. In 10 out of 11 cells, Crom potentiated a D-like, slowly-inactivating outward current (by 88 +/- 31%, for n = 11). 7 The effects of Crom and of 2 min periods of anoxia were compared in 12 cells: unlike anoxia, Cromproduced no consistent increases in GN; the currents evoked in the same cells by anoxia differed significantly from those evoked by Crom (by 150 +/- 60 pA); the directions of current changes induced byCrom and anoxia respectively were not significantly correlated. Crom strongly depressed anoxic outward currents (by 80 +/- 12%, n = 4).8 Some Crom-induced effects (increases in D-like current and the outward current elicited at V>- 20 mV) were always reversed by tolbutamide (1 mM), but much less consistently by glibenclamide(10-30 microM).9 In conclusion, the effects of Crom, recorded with intracellular electrodes in CA1 neurones in slices,show little resemblance to the effects of activation of ATP-sensitive K channels. PMID- 7530571 TI - Effect of acute administration of the 5-HT1A receptor ligand, lesopitron, on rat cortical 5-HT and dopamine turnover. AB - 1. The involvement of presynaptic 5-hydroxytryptamine1A (5-HT1A) autoreceptors in the anxiolytic-like properties of lesopitron (E-4424) (2-(4-[4-(4-chloro-1 pyrazolyl)butyl]-1- piperazinyl)pyrimidine) was studied. Brain microdialysis was used to examine the effect of the drug on the release of 5-hydroxytryptamine (5 HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex of awake, freely moving rats. Moreover, extracellular cortical 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were also studied to assess the possible participation of dopaminergic systems. 2. Lesopitron administered at a dose which induces anxiolytic behavior in rats (30 micrograms kg-1, i.p.) markedly reduced 5-HT levels (to 45% of the basal value) in cortical perfusates, having no effect on 5-HIAA, DOPAC and HVA. The effects of lesopitron were compared with those produced by the anxiolytic, and structurally related compound, buspirone. 3. Buspirone administered at a dose inducing anxiolytic-like effects in rats (5 mg kg-1, i.p.) produced a marked decrease in cortical 5-HT levels (to 20% of the basal value), but in contrast to lesopitron, buspirone produced a pronounced increase in cortical DOPAC (to 300% of the basal value) and HVA (to 400% of the basal value) levels. Buspirone administered at a low dose (30 micrograms kg-1, i.p.) was unable to affect cortical 5-HT levels. 4. To test the hypothesis that the 5-HT decreasing effect of lesopitron could be due to 5-HT1A autoreceptor (somatodendritic)-mediated inhibition of 5-HT neurotransmission, lesopitron was administered locally into the raphe nuclei. Intraraphe administration of 10 micro M lesopitron caused a decrease incortical 5-HT levels (the effect being of the same order as that obtained after systemic injection), with no effect on 5-HIAA, DOPAC and HVA. Raphe 5-HT extracellular levels were not modified afterintraraphe administration of lesopitron, indicating the absence of 5-HT reuptake blocking properties.5 We concluded that lesopitron, at an anxiolytic dose produced a marked inhibition of 5-HT release in the frontal cortex of awake, freely moving rats. This effect was observed after systemic administration as well as after intraraphe administration of the drug, suggesting an agonistic action at raphe 5-HTIA autoreceptors controlling 5-HT release in the projecting areas. In contrast to buspirone, lesopitrontreatment had no effect on cortical DOPAC or HVA levels. PMID- 7530572 TI - Effect of nitric oxide gas on the generation of nitric oxide by isolated blood vessels: implications for inhalation therapy. AB - 1. We have investigated, using rat aortic rings, whether exogenous nitric oxide (NO) gas affects the activity or expression of the inducible, Ca(2+)-independent NO synthase. 2. Incubation of rings with lipopolysaccharide (LPS, S. typhosa) for 6 h resulted in a gradual loss of tissue tone, a time-dependent reduction in constrictor response to phenylephrine and significant expression and activity of Ca(2+)-independent NO synthase. 3. Following incubation of LPS-treated rings with NO gas, the expression of inducible NO synthase mRNA was still observed, although the enzyme activity was significantly reduced and there was no reduction in the response to phenylephrine. 4. Therefore, NO gas can inhibit the action but not the induction of an NO synthase likely to play a role in inflammatory states such as adult respiratory distress syndrome (ARDS). 5. These observations may explain the rebound phenomenon observed in some ARDS patients following inhalation therapy with NO gas. PMID- 7530573 TI - Inhibition by SR 140333 of NK1 tachykinin receptor-evoked, nitric oxide-dependent vasodilatation in the hamster cheek pouch microvasculature in vivo. AB - 1. This study investigated tachykinin-evoked vasodilatation in the microvasculature of the hamster cheek pouch in vivo. Arterioles and venules were observed by intravital microscopy with video recording, and vasodilatation and constriction, defined as changes in blood vessel diameter, measured by image analysis. All agents were applied topically by superfusion. None of the agents tested had a significant effect on venule diameter. 2. When arterioles were preconstricted (by ca. 50%) with endothelin-1 present in the superfusing medium, substance P (0.3-30 nM) was a potent vasodilator, being 10 fold more active than both neurokinin A and the NK1 receptor-selective agonist, substance P methyl ester. The NK2 receptor-selective agonist, [beta-Ala8]-NKA(4-10)(0.1-10 microM) was active only at high concentrations, and the NK3 receptor-selective agonist senktide (0.1-10 microM) was virtually inactive (n = 8 hamsters). Dilatation evoked by tachykinins and analogues was rapid in onset (< 0.5 min) and readily reversible. 3. At low concentrations (1-10 nM), the non-peptide tachykinin NK1 receptor antagonist SR140333 ((S)1-(2-[3(3,4-dichlorophenyl)-1-(3-iso propoxyphenylacetyl)pi peridin-3- yl]ethyl)-4-phenyl-1 azoniabicyclo[2.2.2]octone, chloride) had no effect on the diameter of preconstricted arterioles per se, but potently inhibited dilator responses to substance P methyl ester (apparent pKB 9.9 +/- 0.2; n = 5 hamsters, n = 10 estimates). SR140333 (10 nM) did not inhibit submaximal dilator responses evoked by human alpha calcitonin gene-related peptide (alpha CGRPh; 1.0 nM; P > 0.05; n = 5). 4 The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME; 10 microM) caused a51.3 +/- 5.4% arteriolar constriction. In the presence of L-NAME, submaximal vasodilator responses to substance P (10-I00 nM) and carbachol (0.1-1.0 microM) were significantly attenuated (n = 5 hamsters;P<0.05) as compared to responses obtained in preparations that were preconstricted to a similar extent by endothelin-l (48.0 +/- 5.6%). L-NAME (10 M) was without effect on submaximal vasodilator responses to alpha CGRPh (0.1 nM) or sodium nitroprusside (1O nM) (n = 5 hamsters; P> 0.05).5 We conclude that tachykinin evoked arteriolar vasodilatation in the hamster cheek pouch is mediated via NK, receptor activation and depends, at least in part, on the release of nitric oxide. The NKI receptors mediating vasodilatation can be blocked by topical application of SR140333; which may therefore be useful in the investigation of the role of NK1 receptors in neurogenic inflammation in the microvasculature. PMID- 7530575 TI - A distinctive electrophysiological signature from the Peyer's patches of rabbit intestine. AB - 1. Rabbit small intestinal segments containing Peyer's patches (PP) were examined in Ussing chambers using short-circuit current (Isc) recording. By comparison with control small intestinal mucosal segments, rabbit PP-containing epithelia exhibited decreased basal Isc, increased transepithelial resistance (TER) and unchanged potential difference (PD). 2. Carbachol caused a decrease in Isc in rabbit PP epithelia. Forskolin, dibutyryl cyclic GMP, histamine and the calcium ionophore, A23187, were without effect. In contrast, control epithelial segments of rabbit intestine responded to carbachol and forskolin with an increased Isc, indicative of electrogenic chloride secretion. The EC50 for carbachol was approximately 2 microM in both types of epithelia. Methacholine also caused an outward current in rabbit PP epithelia which had similar properties to that of carbachol. The effect of the cholinomimetics on rabbit PP was basolateral-sided, reversible, and sensitive to low concentrations of the general muscarinic cholinoceptor blockers, atropine, scopolamine and also to the M1 cholinoceptor blocker, pirenzepine. 3. The Isc response to cholinomimetics in rabbit PP was insensitive to bumetanide, amiloride, TEA, barium, acetazolamide, piroxicam and omeprazole, but was attenuated in the presence of ouabain. Using bilaterally substituted solutions, the carbachol effect on rabbit PP Isc was abolished in chloride/bicarbonate-free, but not in chloride-free solutions, suggestive of stimulation of electrogenic bicarbonate absorption by the agent. Substitution for sodium abolished both the basal current and the Isc response to carbachol. Part of the effect of carbachol on PP Isc appeared to be mediated by submucosal neurones because addition of tetrodotoxin reduced the effect by 60%. 4 As microfold (M) epithelial cells predominate in the PP of the rabbit, the unusual phenotype of cholinomimetic-induced outward current may be used as an electrophysiological marker for these potential sites of oral vaccine delivery, and in particular it may also be of use as a marker for rabbit M cells. PMID- 7530574 TI - Reduction by NG-nitro-L-arginine of H2O2-induced endothelial cell injury. AB - 1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) 4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production. PMID- 7530576 TI - Effect of the tachykinin NK1 receptor antagonists, RP 67580 and SR 140333, on electrically-evoked substance P release from rat spinal cord. AB - 1. The effects of the non-peptide tachykinin NK1 receptor antagonists, RP 67580, SR 140333, CP-96,345 and CP-99,994 have been investigated on electrically-evoked release of substance P-like immunoreactivity (SP-LI) from rat spinal cord slices. 2. RP 67580 (10 nM) and SR 140333 (1 nM), perfused 5 min prior to and during 8 min stimulation of the dorsal roots (20 V, 0.5 ms, 1 Hz), significantly enhanced SP-LI release by 213 +/- 43 (n = 8) and 203 +/- 31 (n = 5) % of control evoked release (187 +/- 16% of basal outflow, n = 22) respectively. Neither compound modified basal outflow of SP-LI (15.3 +/- 2.5 fmol/8 ml, n = 10). 3. RP 67580 (10 nM) did not modify electrically-evoked release of calcitonin gene-related peptide LI from rat spinal cord slices. 4. CP-96,345 (10 nM) and CP-99,994 (1 and 10 nM) did not alter electrically-evoked SP-LI release; however, they both inhibited release at 1 microM. Inhibition was also induced by 1 microM RP 67580 but not 1 microM SR 140333. 5. The effect of the NK1 receptor agonists, [Sar9 Met (O2)11]SP and [Sar9]SP, could not be tested on SP-LI release due to interference with the substance P radioimmunoassay (RIA). The other NK1 receptor agonists used, GR 73632, [Pro9]SP and septide, which did not interfere with the RIA, increased SP LI basal outflow by 1807 +/- 713% (n = 3), 1259 +/- 160% (n = 3) and 620 +/- 69% (n = 3) at 10 nM, 1 nM and 1 microM, respectively. At the same concentrations, the three agonists also enhanced electrically evoked SP-LI release by 204 +/- 38% (n = 6), 753 +/- 40% (n = 3) and 504 +/- 97% (n = 3), respectively. The GR 73632 (10 nM)-induced increase in electrically-evoked SP-LI release, was not prevented by SR140333 (100 nM). None of the agonists inhibited SP-LI release at lower concentrations (0.1 nM GR73632; 0.01 and 0.1 nM [Pro9]SP and 1-100 nM septide).6 NKA and NKB, at concentrations up to 10 nM which did not interfere with the RIA, did not modify electrically-evoked release of SP-LI.7 The ability of NKI receptor antagonists to enhance electrically-evoked SP-LI release supports the concept of an NK1 autoreceptor control mechanism at substance P nerve terminals within the dorsal horn of the rat spinal cord. PMID- 7530579 TI - Nitric oxide (NO) is an endogenous anticonvulsant but not a mediator of the increase in cerebral blood flow accompanying bicuculline-induced seizures in rats. AB - Neurons synthesize NO, which may act as a retrograde messenger, involved in either potentiating or depressing neuronal excitability. NO may also play a role in the cerebral vasodilatory response to increased neuronal activity (i.e., seizures). In this study, two questions were asked: (1) is NO an endogenous anticonvulsant or proconvulsant substance? and (2) is the cerebral blood flow (CBF) increase accompanying bicuculline (BC)-induced seizures mediated by NO? The experiments were performed in 300-400-g Wistar rats anesthetized with 0.6% halothane and 70% N2O/30% O2. CBF was measured using the intracarotid 133Xe clearance method or laser-Doppler flowmetry. EEG activity was recorded. Chronic treatment (4 days) with nitro-L-arginine (L-NA), a potent NO synthase (NOS) inhibitor (400 mg/kg total), suppressed brain NOS by > 97% and prolonged seizure duration from 6 +/- 1 (saline-treated controls) to 12 +/- 2 min. In the L-NA treated group, the CBF increase was sustained as long as seizure activity remained, indicating that CBF was still tightly coupled to seizure activity. Interestingly, the supposed inactive enantiomer of L-NA, D-NA, also showed an inhibition of brain NOS activity, ranging from 87 to 100%. The duration of seizures in this group (average 8 +/- 2 min) corresponded directly to the magnitude of reduction in NOS activity (r = 0.83, P < 0.05). Specifically, the D NA results indicated that NOS inhibition had to exceed 95% before any effect on seizure duration could be seen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530577 TI - Inhibition of substance P-induced microvascular leakage by inhaled methoxamine in rat airways. AB - 1. The effect of the inhaled alpha-adrenoceptor agonist, methoxamine (MTX), was studied on experimental airway oedema induced by injection of substance P (SP) in the rat. Sprague-Dawley rats (300-350 g) were anaesthetized with sodium thiopentone, tracheotomized and artificially ventilated. 2. MTX or its vehicle was administered by inhalation. Airway resistance and blood pressure were monitored continuously. Evans Blue dye (EB, 20 mg kg-1) was injected through a jugular catheter 1 min before SP (14.8 nmol kg-1). Airways were dissected out, weighed and placed in formamide for EB extraction and determination by spectrophotometry. 3. EB extravasation induced by SP was significantly reduced in distal intraparenchymal bronchi by inhaled MTX at doses of 50 micrograms kg-1 (58 +/- 9 vs 96 +/- 9 ng EB mg-1 tissue after vehicle, P < 0.001) and 100 micrograms kg-1 (69 +/- 11 vs 137 +/- 26 ng EB mg-1 tissue after vehicle, P < 0.01). Inhaled MTX by itself (100 micrograms kg-1) increased blood pressure: 172 +/- 6 vs 132 +/ 10 mmHg baseline (P < 0.02), but neither induced extravasation nor increased airway resistance. 4. In another set of experiments without SP, MTX was administered intravenously 1 min after EB. At 100 micrograms kg-1, i.v. MTX increased blood pressure to a similar extent as inhaled MTX (180 vs 147 mmHg baseline, P < 0.01), increased airway resistance and caused leakage of plasma proteins in distal intraparenchymal bronchi (79 +/- 7 vs 47 +/- 1 ng EB mg-1 tissue, P < 0.02). 5 Similarly, after sequential i.v. injections of doubling doses of MTX (50-800 microg kg-1), a marked EB extravasation was found in the airways. This was abrogated by pretreatment with prazosin (100 microg kg-1)but not with propranolol (2 mg kg-1).6 These results suggest that microvascular leakage and airway oedema induced by i.v. MTX may be linked to an increase in pressure in the pulmonary circulation, resulting from vasoconstriction of the pulmonary vasculature and acute cardiac dysfunction due to systemic hypertension.7 Our results with inhaled MTX show that direct deposition of MTX at the bronchial vasculature induces a reduction in SP-induced microvascular leakage in rat airways and that inhaled MTX does not share the untoward effect of i.v. MTX inducing airway oedema. PMID- 7530578 TI - Sister chromatid differentiation in 5-bromo-2'-deoxyuridine-substituted chromosomes: a study with DNA-specific ligands and monoclonal antibody to histone H2B. AB - Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy. PMID- 7530580 TI - Low environmental temperatures or pharmacologic agents that produce hypothermia decrease methamphetamine neurotoxicity in mice. AB - Recently we have reported that methamphetamine (METH) neurotoxicity in rats depends on the environmental temperature. Here, we evaluate whether a cold environment (4 degrees C) or drugs which chloride and glutamate ion channel function block METH neurotoxicity in mice. Adult male CD mice received METH i.p. (4 x 10 mg/kg METH at 23 degrees C along with saline. 2.5 mg/kg (+)-MK-801, 40 mg/kg phenobarbital or 2.5 mg/kg diazepam and either 4 x 10 or 4 x 20 mg/kg METH at 4 degrees C). Multiple injections of METH (4 x 10 mg/kg i.p.) at room temperature (23 degrees C) produced a significant depletion of dopamine (DA) in striatum at 24, 72 h, 1 and 2 weeks. Three days post 4 x 10 mg/kg METH at 23 degrees C, an 80% decrease in striatal dopamine (DA) occurred while the same dose at 4 degrees C produced only a 20% DA decrease, and 4 x 20 mg/kg METH at 4 degrees C produced a 54% DA decrease. At 23 degrees C (+)MK-801 completely blocked while phenobarbital (40% decrease) and diazepam (65% decrease) partially blocked decreases in striatal DA produced by 4 x 10 mg/kg METH. Decreases in DOPAC and HVA were similar to the decreases in DA after METH and antagonists. Multiple injections of METH (4 x 10 mg/kg, i.p.) at room temperature also produced a significant depletion of serotonin (5-HT) in striatum at 24, 72 h, 1 and 2 weeks. This depletion of 5-HT at room temperature was blocked either by changing the environmental temperature to 4 degrees C, or by pretreatment with MK 801, diazepam and phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530581 TI - Sustained tyrosine phosphorylation of p140trkA in PC12h-R cells responding rapidly to NGF. AB - The PC12h cell, a subclone of PC12 cells, has considerable activities of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) and shows an NGF-induced increase in both enzyme activities. The TH activity and its inducibility by NGF in PC12h cells were stably maintained in the passage of > 200 generations whereas the ChAT activity was not. We isolated a new cell line, PC12h-R (originally clone 8), from a long-term culture of PC12h cells. PC12h-R cells still showed the considerable TH activity, but not the ChAT activity, and maintained the inducibility of TH activity by NGF. Thus, the responses of PC12h-R cells to NGF were similar to those of chromaffin cells and sympathetic neurons. PC12h-R cells were found to extend neurites and differentiate into sympathetic neuron-like cells in response to NGF much more rapidly than PC12h cells. In addition, PC12h-R cells showed sustained NGF-induced tyrosine phosphorylation of p140trkA and several cellular proteins, including 42-, 44- and 54-kDa proteins, in comparison with PC12h cells. We suggest that the NGF-induced sustained tyrosine phosphorylation signal in PC12h-R cells may be correlated closely with their rapid NGF-induced differentiation into neuron-like cells. PMID- 7530582 TI - Lesions of the central gray block the sensitization of the acoustic startle response in rats. AB - The amplitude of the acoustic startle response (ASR) in rats is increased after administration of footshocks, a phenomenon termed sensitization. The neural circuitry underlying this kind of modulation of the ASR is only partly understood. It has been shown that the central nucleus of the amygdala (cA) and its efferent pathway to the caudal pontine reticular nucleus (PnC), an essential part of the primary startle circuit, is important for the sensitization of the ASR. It was unclear, however, whether the amygdaloreticular pathway directly transfers the effects of footshocks onto the PnC, or whether there exists a relay nucleus within this pathway. The present study tested the hypothesis that the midbrain central gray (CG) is important for the sensitization of the ASR. Neuroanatomical tracing experiments indicate that a descending projection from the medial part of the cA might form synapses in the region of the midbrain CG, where a descending projection to the PnC takes its origin. We lesioned the dorsal and lateral part of the CG with the neurotoxin quinolinic acid and measured the effects of this lesion on the sensitization of the ASR by footshocks. Lesions confined to the dorsal and lateral parts of the CG totally blocked the sensitization of the ASR, without affecting the ASR amplitude in the absence of sensitizing stimuli. These findings suggest a crucial role of the CG for the sensitization of the ASR. The present data are reconciled with other findings from our laboratory and from the literature and we discuss possible mechanisms underlying the mediation of the sensitization of the ASR in rats. PMID- 7530583 TI - Interspecific variation of isthmo-optic projections in poikilothermic vertebrates. AB - Retinopetal projections from isthmic and caudal mesencephalic regions were studied in a number of poikilothermic vertebrates. The tested animals were elasmobranchs, bony fishes, amphibians, and reptiles. Isthmo-optic projections were absent in elasmobranchs, sturgeons, most teleosts, all anuran and urodel amphibians, and in anolis lizards. Tegmental projections to the retina were found in a few fishes and in most reptiles. Interspecific variations regarding three morphological parameters were as follows: (i) isthmic projections to the contralateral eye originate from one or from two distinct nuclei; (ii) major interspecific differences exist regarding numbers and aggregation densities of cells in the nuclei of origin; (iii) the percentage of retinopetal fibres that innervate the ipsilateral retina varies significantly between species. PMID- 7530584 TI - Long-term exposure to high levels of corticosterone aggravates AF64A-induced cholinergic hypofunction in rat hippocampus in vivo. AB - Male Sprague-Dawley rats were bilaterally adrenalectomized and corticosterone (CORT) was substituted as subcutaneous pellets in two groups of animals: low- (L CORT: 1 x 25 mg pellet) or high-level of CORT (H-CORT: 4 x 100 mg pellet). Between 14 and 19 days after CORT substitution, ethylcholine aziridinium (AF64A) was intracerebroventricularly (i.c.v.) injected in the CORT long-term exposed rats and the dose- and time-dependent effect of this treatment was measured on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in hippocampus and septum and on serotonin (5-HT), 5-hydroxyindoleacetic acid (5 HIAA) and noradrenaline (NA) levels in hippocampus. Rats were killed at 2, 4, 7 and 14 days after AF64A treatment. Starting 4 days after the i.c.v. administration of 0.5 or 1.0 nmol of AF64A, an aggravation of the reduction of ChAT activity was measured in the hippocampus of the H-CORT animals compared to the L-CORT ones. In the septum of the H-CORT rats, the activity of ChAT increased within the first week after the infusion of the toxin, while no significant effect was observed in the L-CORT group. As we observed with ChAT, AF64A induced a severe inhibition of AChE activity in the hippocampus of the H-CORT rats compared to the L-CORT ones. In the septum, an increase of AChE activity was observed in both groups of CORT-exposed animals. In the hippocampus of H-CORT animals, the exacerbation of the inhibition of ChAT and AChE activity was accompanied by a parallel decrease in the content of 5-HT and 5-HIAA starting 4 days after AF64A injections. Finally, NA content in hippocampus was not affected by the toxin in the CORT-substituted animals. These data demonstrate that: (1) long-term exposure to supraphysiological levels of CORT enhances the cholinodisruption induced in hippocampus by AF64A, at doses of 0.5 and 1.0 nmol/side; (2) high circulating plasma CORT concentrations impair hippocampal cholinergic neuronal capacity to recover from damage; and (3) the degree of inhibition of the serotoninergic system is augmented in H-CORT animals, most probably due to an adaptation of the serotoninergic neurons to the larger withdrawal of cholinergic function observed in this group. PMID- 7530585 TI - Phase I/II evaluation of nevirapine alone and in combination with zidovudine for infection with human immunodeficiency virus. AB - In these Phase I/II open-label clinical trials, 62 persons with human immunodeficiency virus type 1 (HIV-1) infection and CD4+ cell counts < 400/mm3 received nevirapine at doses of 12.5, 50, and 200 mg/day, alone or in combination with zidovudine, 200 mg q8h. Nevirapine was well tolerated in the doses tested. Mean steady-state trough levels were 0.23, 1.1, and 1.9 micrograms/ml for the 12.5, 50, and 200 mg/day doses, respectively. Early suppression of p24 antigen levels and increase in CD4+ cell count were reversed following rapid emergence of virus less susceptible to nevirapine. Resistant strains were isolated from all participants by 8 weeks. Nevertheless, reduction of p24 antigen levels to < 50% of baseline values persisted for 12 weeks or more in four of seven persons who received 200 mg nevirapine/day in combination with zidovudine: these individuals had been antigenemic on long-term zidovudine therapy. This study demonstrates a direct relationship between drug resistance and effects on surrogate markers in HIV-1 infection. PMID- 7530586 TI - Strategies for prevention of perinatal transmission of HIV infection. Siena Consensus Workshop II. AB - Worldwide clinical trials are in progress or are being planned to evaluate different interventions aimed at reducing transmission of human immunodeficiency virus (HIV) from mother to infant. Several new therapeutic agents will soon be the focus of clinical trials to evaluate their safety for pregnant women and their infants and to test their efficacy in preventing perinatal transmission. Identification of the factors that influence the timing and mechanism of perinatal HIV transmission is essential to the assessment of interventions to reduce transmission. An international workshop was held on June 3-6, 1993, in Siena, Italy, to review what is known about the risk factors, timing, and mechanisms of HIV type 1 (HIV-1) transmission from mother to infant during gestation, birth, and lactation. Several possible interventions were compared with respect to their mechanism of action, their dependence upon the mode and timing of perinatal HIV transmission, and their suitability for incorporation into trials, alone or in combination, in various parts of the world. Obstetrical interventions such as caesarian section or use of a virucidal vaginal lavage during labor, administration of antiretroviral drugs to mother and/or infant, as well as active and/or passive immunization were discussed. The role of breast feeding in perinatal HIV infection and in the trials was also evaluated. Because the ability to enroll pregnant, HIV-infected women in trials is limited, it is essential that proposed trials be carefully evaluated for adequacy of the sample size, appropriateness of the type of intervention to the particular population of women to be enrolled, and feasibility of the intervention. Worldwide coordination of perinatal intervention trials is essential to prevent duplication, allow the optimal use of human and financial resources, and ensure the most rapid possible evaluation and implementation of means to prevent pediatric HIV infection. PMID- 7530587 TI - Prostate cancer screening: role of the digital rectal examination and prostate specific antigen. AB - BACKGROUND: This study was designed to determine the efficacy of digital rectal examination (DRE) and serum prostate-specific antigen (PSA) for early detection of prostate cancer in men > or = 50 years of age. METHODS: A prospective single center clinical trial was conducted to screen 644 asymptomatic men, who were elicited by newspaper and radio advertisements, with DRE and PSA. Quadrant biopsy examinations of the prostate were performed if PSA > 4 ng/ml or if DRE was suspicious. RESULTS: Thirty-seven percent of the men (n = 241) had an abnormality of DRE or elevated PSA. Of the 163 patients who underwent transrectal ultrasound and quadrant biopsies of the prostate, 77% had normal biopsies, 14 (8%) had prostatic intraepithelial neoplasia, and 24 (15%) had carcinoma of the prostate. PSAs ranged from 0.3 to 65.5 ng/ml, with a mean of 2.35 and a median of 1.6. Ninety-five patients had a PSA > 4 ng/ml, of whom 17 had a PSA > 10 ng/ml. Sensitivity of PSA was 75% and specificity 87%; for DRE the sensitivity was 75% and the specificity 69%. Clinical stage of patients who underwent radical prostatectomy was B1 in 15 and B2 in five. Fifteen of 20 patients (75%) had organ confined disease; the other five had specimen-confined disease. No patient was found to have nodal involvement. CONCLUSION: The combination of PSA and DRE seems to improve the stage of diagnosis of patients with prostate cancer. Larger, randomized studies will be necessary to evaluate the effect of screening on overall survival. PMID- 7530588 TI - Distribution of Hsp-27 and HER-2/neu in in situ and invasive ductal breast carcinomas. AB - BACKGROUND: The overexpression of heat shock protein 27 (hsp-27) in early-stage breast cancer is associated with histopathologic features of poor prognosis and clinically with an increased probability of disease recurrence. Hsp-27 is overexpressed in 25% of invasive ductal carcinomas (IDC); however, its distribution in ductal carcinoma in situ (DCIS) and DCIS associated with IDC has not been investigated. We postulated that hsp-27 might be detected and variably expressed in DCIS and, like HER-2/neu oncoprotein expression, might be a tumor specific marker worthy of future clinical investigation. METHODS: To test these hypotheses, the distribution of hsp-27 in noncomedo and comedo DCIS, and DCIS associated with IDC, was evaluated by immunohistochemistry and compared with HER 2/neu expression within the same cancers. RESULTS: Hsp-27 was overexpressed in 28 of 47 (approximately 60%) cases of DCIS; expression in pure DCIS was 16 of 24 (67%), and 12 of 23 (approximately 50%) in DCIS associated with IDC. Hsp-27 expression by in situ and invasive components of the same neoplasm were concordant in 22 of 23 (approximately 95%) cases tested. Comedo variants appeared to have somewhat higher hsp-27 expression than noncomedo DCIS, whether or not there was an associated IDC. These results are reminiscent of HER-2/neu oncoprotein expression in DCIS and DCIS associated with IDC observed by others. However, although 4 of 22 (18%) cancers containing DCIS + IDC expressed HER 2/neu, no relationship with hsp-27 expression in the same cancers was observed. CONCLUSIONS: We found a high incidence of hsp-27 overexpression in DCIS and in DCIS associated with IDC. This rate is twice that previously observed in IDC alone. Hsp-27 expression is independent of HER-2/neu expression. PMID- 7530589 TI - Effect of glutamine on tumor and host growth. AB - BACKGROUND: Oral glutamine supplementation has been found to support gastrointestinal mucosal growth and increase intestinal and systemic toxicity after chemotherapy and radiation therapy. Glutamine is also an important nutrient for rapidly proliferating tumor cells. However, it is not clear whether long-term glutamine supplementation in the tumor-bearing host has a selective benefit for host growth or tumor cell proliferation. METHODS: To study the effect of glutamine in tumor-bearing animals, 30 Lewis/Wistar rats with subcutaneous mammary tumor implants (MAC-33) were randomized to receive a 3% glutamine- or 3% glycerine-enriched (control) diet for 25 days. RESULTS: No significant difference was found in carcass weight, primary tumor weight, or spontaneous pulmonary metastasis with glutamine supplementation. Tumor cell cycle kinetics (aneuploidy, %S and %S [synthetic] + G2/M [growth fraction]) were similar between glutamine supplemented and control animals. A trophic effect of glutamine on distal ileal mucosa was seen with increased DNA content (344 +/- 68 vs. 184 +/- 38 micrograms/100 mg tissue) (p < 0.05) and RNA content (435 +/- 44 vs. 335 +/- 30 micrograms/100 mg tissue) (p = 0.06) compared with control animals. No detectable differences were observed in liver or muscle, or in tumor DNA, RNA, or protein content. CONCLUSIONS: These findings confirm the trophic effect of glutamine on small intestinal mucosa and suggest that glutamine can be administered to the tumor-bearing host over a long period of time without significantly stimulating tumor growth kinetics or metastasis. PMID- 7530590 TI - Serum prostate-specific antigen as a predictor of radiographic staging studies in newly diagnosed prostate cancer. AB - The standard staging evaluation of prostate cancer includes digital rectal examination, measurement of serum tumor markers, radionuclide bone scan, and abdominal pelvic computed tomography (CT) or nuclear magnetic resonance imaging (MRI). We retrospectively reviewed 300 cases of newly diagnosed, untreated adenocarcinoma of the prostate to evaluate the ability of serum prostate-specific antigen (PSA) to predict results of staging radiographic studies (bone scan, CT/MRI). The medical records of 300 newly diagnosed, untreated prostate cancer patients were reviewed. The following information was collected on a standard data form: age, clinical stage based on digital rectal examination, method of diagnosis, histological grade, serum PSA level, results of radionuclide bone scan and additional radiographic studies to confirm bone scan results, results of abdominal pelvic CT/MRI, and presence or absence of bone pain. The results of this review were tabulated and analyzed with regard to the ability of serum PSA level to predict positive results of radiographic staging studies. The mean PSA level of the study group was 24.6 ng/ml. Ten patients (3.6%) presented with positive bone scan results with 5 of these having serum PSA levels greater than 20 ng/ml (range 27.6 ng/ml-144 ng/ml, mean 66.3 ng/ml). The 5 remaining patients all had elevated PSA levels ranging between 4.1 and 20.0 ng/ml. No patient with a positive staging bone scan presented with a normal serum PSA. Ten patients (4.0%) presented with a positive abdominal/pelvic CT/MRI (adenopathy only; no patients had radiographic evidence of abnormalities of the upper urinary tract). Eight had serum PSA levels greater than 20 ng/ml, ranging from 30.0 to 234 ng/ml. No patient with a positive study presented with a normal serum PSA level. No patient with either positive bone scan or abdominal pelvic CT/MRI presented with bone pain. We conclude that in asymptomatic patients with newly diagnosed, untreated prostate cancer and serum PSA levels of less than 10 ng/ml, a staging radionuclide bone scan may not be necessary. Likewise, in patients with serum PSA levels of less than 20 ng/ml the likelihood of positive findings on abdominal/pelvic CT/MRI is extremely low. Abdominal/pelvic CT/MRI does not appear necessary in this setting. With over 130,000 cases of newly diagnosed prostate cancer each year in the United States, elimination of staging radiographic studies in the patients outlined above could result in economic savings on the order of 30-80 million dollars per year. PMID- 7530591 TI - Recombinant human granulocyte colony-stimulating factor infusion and/or autologous peripheral blood stem cell transplantation as a rescue for sequential high-dose combination chemotherapy: a preliminary report. AB - BACKGROUND: Sequential cycles of combination chemotherapy with high-dose cyclophosphamide, etoposide and cisplatin (sHDCEP) can largely increase the total dose (TD) of drug delivered. If granulocyte colony-stimulating factor (G-CSF) and/or autologous peripheral blood stem cell (PBSC) rescue can shorten the duration of cytopenia between cycles of sHDCEP, the dose intensity (DI) can be increased as well. In order to explore the feasibility of delivering maximal TD and DI by administration of sHDCEP with G-CSF and/or PBSC rescue, this trial is undertaken to investigate the hematologic and nonhematologic toxicity observed with sHDCEP by G-CSF and/or PBSC rescue. METHODS: Patients with refractory malignancy and well preserved physiologic function for whom no available therapy is likely to cure or prolong the survival were eligible for the study. Each cycle of high-dose chemotherapy consisted of: cyclophosphamide 5,000 mg/m2, etoposide 1,500 mg/m2 and cisplatin 150 mg/m2. G-CSF and/or PBSC were administered alternatively after each cycle as rescue for myelosuppression. The next cycle was given to patient who showed response to the previous cycle after recovery from toxicity for a maximal of 4 cycles. RESULTS: Two cases of refractory malignancy with progressive disease were treated by sHDCEP for 7 cycles, including 4 cycles with G-CSF rescue, 2 cycle with PBSC rescue, and 1 cycle with G-CSF + PBSC rescue. In the 4 cycles rescued by G-CSF alone, we observed a slightly slower granulocyte and markedly prolonged platelet recovery in the subsequent cycle. By comparing the effect of G-CSF and/or PBSC rescue on hematologic recovery with the preceding cycle in the same patient, we found that G-CSF rescue provided faster granulocyte recovery than PBSC, but PBSC rescue provided faster platelet recovery than G-CSF. Rescue by larger number of PBSCs provided only faster platelet but not granulocyte recovery than rescue by adding G-CSF to a very small number of PBSCs. However, G-CSF plus the very small number of PBSCs provided shorter duration of both granulocytopenia and thrombocytopenia than rescue by G-CSF alone. The most common nonhematologic toxicity from sHDCEP included transient nausea, vomiting, diarrhea and mild impairment of liver function but we observed no significant or irreversible major organ damage. The side effect from PBSC collection was mild and toxicity from reinfusion of the thawed PBSCs was not obvious. Using G-CSF and/or PBSC rescue, sHDCEP was delivered repeatedly in no more than 4 weeks for the next-cycles except for patient 1 who had cycle 4 delayed because of prolonged platelet recovery by only G-CSF rescue in cycle 3. CONCLUSIONS: Our initial experience has shown that the nonhematologic toxicity from sHDCEP, G-CSF and PBSC rescue was well tolerated. Prolonged platelet recovery after sequential cycles of HDCEP by only G-CSF rescue delayed the next cycle of chemotherapy. Although the next cycle was delivered within 4 weeks by only PBSC rescue, concurrent infusion of G-CSF and larger number of PBSCs should provide the most rapid hematologic recovery. Sequential high-dose chemotherapy administered by this model is likely to provide the maximal delivery of TD and DI, and is worthy of further clinical trials. PMID- 7530593 TI - Activation of spinal cord serotonergic neurons accompanies cold-induced sympathoexcitation. AB - These studies examined the hypothesis that serotonergic neurons located in central sites known to be involved with autonomic regulation are activated by cold exposure, a potent stimulator of the sympathetic nervous system. In all experiments, rats were exposed to either 3 degrees C or 22 degrees C for 24 h. Significant increases (p < 0.05) in urinary norepinephrine excretion, depletions of myocardial norepinephrine, and enhanced myocardial L-DOPA accumulation following decarboxylase inhibition provided evidence of sympathoexcitation at 3 degrees C. Accumulations of the serotonin metabolite 5-hydroxyindoleacetic acid, in saline-injected rats, and 5-hydroxytryptophan in decarboxylase-inhibited rats were increased in spinal cord and brainstem regions of cold-exposed rats. Two hours after injection of the serotonin synthesis inhibitor p-chlorophenylalanine, significantly greater depletions of serotonin in spinal cord and 5 hydroxyindoleacetic acid in spinal cord and brainstem of cold-exposed rats were noted; synthesis inhibition also caused a larger drop in body temperature in cold exposed rats. Microdissections dissections of raphe nuclei and thoracic spinal cord sites indicated that the principal sites of serotonergic activation were the dorsal and intermediate spinal regions, and the raphe magnus. Thus, cold-induced sympathoexcitation was accompanied by activation of serotonergic neurons in spinal cord and brainstem regions known to be involved in autonomic regulation. PMID- 7530592 TI - Insulin potentiates norepinephrine-induced vascular tone by activation of protein kinase C and tyrosine kinase. AB - Insulin might play a role in the hypertension occurring in insulin-resistant diabetes. In addition, insulin has recently been shown to potentiate norepinephrine (NE) induced vascular tone. We used ring segments of the rabbit facial artery mounted in a myograph to test the hypothesis that potentiation of NE-induced tone by insulin may be related to activation of protein kinase C (PKC) and tyrosine kinase (TK). NE-induced contractions in the presence of insulin (1 mU/mL) were 200% (NE 0.1 and 0.3 microM), 252% (NE 1 microM), and 129% (NE 3 microM) of control. Insulin (1 mU/mL) had no effect on NE (10 and 100 microM) induced contractions. The potentiation by insulin of NE-induced tone was not altered by endothelium removal and could be mimicked by phorbol-12-myristate-13 acetate (PMA, 0.1 microM). Histamine-induced contractions were not altered by insulin (1 mU/mL). Insulin potentiation of NE-induced tone was suppressed by pretreatment of the rabbit facial artery with the PKC inhibitor calphostin C (0.1 microM) or the TK inhibitor genistein (10 microM). 45Ca2+ influx due to NE (3 microM) did not change in the presence of insulin (1 mU/mL) or PMA (0.1 microM) despite a higher contractile response, so that wall force per unit of 45Ca2+ influx was increased by insulin (1 mU/mL) and PMA (0.1 microM). Calphostin C (0.1 microM) and genistein (10 microM) both prevented the increase in wall force per unit of 45Ca2+ influx due to insulin (1 mU/mL). Our study shows that insulin potentiates NE-induced tone through a TK- and PKC-dependent mechanism. PMID- 7530595 TI - Different angiogenic pathways characterize superficial and invasive bladder cancer. AB - We have investigated by RNase protection analysis the expression of 2 angiogenic factors in 45 primary bladder tumors and 8 normal bladders. Expression of vascular endothelial growth factor (VEGF) was 3-fold higher and that of platelet derived endothelial cell growth factor was 40-fold higher in tumors compared to normal bladder. However, the factors were differentially expressed in different stages of the cancer. Expression of VEGF in superficial tumors was 4-fold higher than in invasive tumors and 10-fold higher than in normal bladder (superficial versus invasive, P < 0.0006; superficial versus normal, P < 0.0002). Expression of platelet-derived endothelial cell growth factor in invasive tumors was 33-fold higher than in superficial tumors and 260-fold higher than in normal bladder (invasive versus superficial, P < 0.0001; invasive versus normal, P < 0.0003). In well differentiated or moderately differentiated superficial tumors which had invaded the lamina propria (pT1G1/2) VEGF expression was 4-fold higher in tumors which subsequently recurred at 3 months compared to those which did not recur (P < 0.002). Thus there are two distinct angiogenic pathways involved in different stages of bladder cancer, which is in keeping with the evidence for two different genetic pathways. Elevated VEGF expression predicts early recurrence of pT1G1/2 tumors. PMID- 7530596 TI - Autocrine growth stimulation by transforming growth factor alpha in asbestos transformed rat mesothelial cells. AB - Although the association between asbestos exposure and mesothelioma development has been established for decades, very little is known regarding the molecular mechanism(s) by which asbestos fibers induce this disease. In this series of experiments, the potential for transforming growth factor alpha (TGF-alpha) to act as an autocrine growth factor in transformed mesothelial cells was examined in rats, a model system frequently used to assess the tumorigenic potential of fibrous particulates. Both asbestos-transformed cells and spontaneously transformed cells expressed functional EGF receptors, although only the asbestos transformed cells expressed TGF-alpha. Expression of TGF-alpha transcripts was correlated with secretion of picogram amounts of growth factor into conditioned medium by the asbestos-transformed cells. In addition, whereas TGF-alpha inhibited the growth of spontaneously transformed mesothelial cells, it stimulated the growth of asbestos-transformed cells. Neutralizing antibody that recognized TGF-alpha secreted by the asbestos-transformed cells was able to inhibit the growth of these cells. Taken together, these data indicate that TGF alpha acts as an autocrine growth factor for asbestos-transformed rat mesothelial cells. Therefore, in asbestos-transformed mesothelial cells, altered production and responsiveness to TGF-alpha distinguish these cells from spontaneously transformed mesothelial cells. These data suggest that differences in mesothelioma etiology may be reflected in differences in the molecular alterations present in these tumors. PMID- 7530597 TI - A case-control study of nonrandom distribution of bleomycin-induced chromatid breaks in lymphocytes of lung cancer cases. AB - We used a case-control study design to determine the association between bleomycin-induced chromatid breaks and the risk of lung cancer in general and by specific histopathological types. Lymphocytes from primary blood cultures of 78 controls and 75 cases with 4 histopathological types of lung cancer were treated with 0.03 unit/ml bleomycin for 5 h, and the frequency of induced chromatid breakage and the locations of the breaks were determined in Q-banded preparations. After adjustment for their length, the larger chromosomes had more breaks than the smaller chromosomes in both cases and controls. The cases had significantly more breaks on chromosomes 4 and 5 than the controls did, with odds ratios (ORs) of 4.9 [95% confidence limits (CL), 2.0, 11.7] and 3.9 (95% CL, 1.6, 9.3), respectively. When the lung cancers were classified by histopathological type, adenocarcinomas had significantly more breaks on chromosomes 4 and 5, with ORs of 3.0 (95% CL, 1.0, 8.7) and 3.5 (95% CL, 1.2, 10.7), respectively. For squamous cell carcinoma, the ORs were significantly elevated for breaks on chromosomes 2, 4, and 5 with ORs of 3.5 (95% CL, 1.0, 11.7), 10.2 (95% CL, 2.5, 41.9), and 7.9 (95% CL, 1.9, 32.8). For small cell carcinoma, breaks on chromosomes 2 and 4 showed significantly increased ORs of 33.2 (95% CL, 2.2, 513.3) and 20.4 (95% CL, 1.7, 250.1), respectively. However, no specific chromatid breaks were detected in cases with large cell carcinoma. When the frequency of chromatid breaks at specific regions was calculated, breaks at 4p14, 4q27, 4q31, 5q21-q22, 5q31, and 5q33 were significantly more common in lung cancer cases than in controls. Lung cancer risk had a dose-response relationship with breaks on chromosomes 4 and 5. Cigarette smoking had a strong interaction with breaks on chromosomes 2, 4, and 5. The findings suggest that the susceptibility of particular chromosome loci to mutagenic damage may be a risk factor for specific types of lung cancer. PMID- 7530598 TI - Antibody-targeted superantigens induce lysis of major histocompatibility complex class II-negative T-cell leukemia lines. AB - CTLs bearing certain T-cell receptor V beta-regions are directed by the bacterial superantigen Staphylococcus enterotoxin A (SEA) to lyse MHC class II-positive cells. In order to extend superantigen-dependent cytotoxicity to MHC class II negative carcinoma cells, covalent conjugates of superantigen and mAbs against surface markers of these cells have been used. We now describe a novel strategy which allows rapid selection of mAb suitable for superantigen targeting against MHC class II-negative tumor cells. A recombinant fusion protein of protein A and SEA binding to the mAbs CD7 or CD38 was able to mediate T cell-dependent lysis of MHC class II-negative Molt-4 and CCRF-CEM acute lymphatic leukemia cell lines. Lysis was dose dependent and correlated with E:T cell ratio. In contrast, SEA alone did not induce any significant lysis. In order to decrease the MHC class II affinity of the protein A-SEA complex, a point mutation was introduced into SEA (protein A-SEA mu9). The mutated fusion protein had similar potency as protein A SEA against Molt-4 cells but was 100-fold less active against MHC class II positive cells. Considering the efficiency and specificity of the mutated SEA protein interacting with mAb in targeting T lymphocytes against MHC class II negative leukemia cells while only marginally affecting normal MHC class II positive cells, we suggest the development of SEA-mAb fusion proteins as a potential adjuvant therapy of leukemias. PMID- 7530594 TI - Uses of flow cytometry in virology. AB - This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed. PMID- 7530599 TI - p53 abnormalities and genomic instability in primary human breast carcinomas. AB - Abnormalities in the p53 tumor suppressor gene have been shown to affect cell cycle control and lead to genetic instability in cell lines of murine and human origin. We have examined genetic instability in 183 primary human breast carcinomas with and without p53 abnormalities. Mutation analysis was performed by constant denaturant gel electrophoresis and DNA sequencing, and abnormal protein expression was examined by immunohistochemical staining methods. Genetic instability was studied by detection of gene amplification, allelic loss, karyotype analysis, and fluorescent in situ hybridization. We found a significant association between p53 abnormalities and genetic instability detected by these methods. PMID- 7530600 TI - NM23-H1 and NM23-H2 messenger RNA abundance in human hepatocellular carcinoma. AB - nm23 was originally identified as an antimetastatic gene, the expression of which was inversely correlated with tumor metastatic potential in rodent model systems. Subsequently, two related human nm23 genes, nm23-H1 and nm23-H2, were identified. The relationship between expression of nm23-H1 and nm23-H2 in hepatocellular carcinoma specimens from 30 patients and metastatic potential was investigated with the use of a quantitative reverse transcription-PCR procedure. The abundance of nm23-H1 and nm23-H2 mRNA was compared with serum alpha-fetoprotein concentration, tumor size (maximum diameter), and histopathological parameters such as portal vein tumor thrombus, intrahepatic metastasis, capsular formation, capsular infiltration, differentiation of tumor cells, and TNM stage. The abundance of nm23-H1 mRNA showed a significant inverse correlation with intrahepatic metastasis and TNM stage. Furthermore, we confirmed that reduced expression of nm23-H1 mRNA was in accordance with a reduced amount of NM23-H1 protein using Western blot analysis. No correlation was apparent between nm23-H2 mRNA abundance and intrahepatic metastasis. These data support the conclusion that nm23-H1 may play a more important role than nm23-H2 in intrahepatic metastasis in hepatocellular carcinoma. Furthermore, nm23-H1 mRNA abundance may be a predictor of intrahepatic metastasis, the most important factor correlated with the metastatic potential of hepatocellular carcinoma. PMID- 7530601 TI - [Seroepidemiologic study on the feasibility of screening for the antibody to HCV by pooling the serum specimens]. AB - The feasibility of screening for the antibody to HCV by pooling 10 serum specimens was studied by utilizing the epidemiologic method of single blind random allocation. We changed only the ratio between the pooled serum specimen volume and the serum specimen diluent volume, with their total volume and single serum specimen volume being as much as the volume required by EIA. Other steps were the same as those of EIA. The result showed that taking single serum specimen as the control group, the sensitivity and specificity of pooled serum specimen were 100% and 97.6%, respectively. The false positive rate was 2.4%, Kappa value was 0.95 (P < 0.05). The analysis of profit found that remarkable social and economic benefit will be obtained when serum pooling method is used. These results demonstrate that pooling 10 serum specimens is feasible to screening the antibody to HCV in blood donors by EIA. PMID- 7530602 TI - [Exploration on the association between the pattern of HBV markers and infection of HCV among population]. AB - Sera from 193 cases with positive HBV markers in April 1993 were detected for infection of HCV to explore the association between the pattern of HBV markers and the infection of HCV among population. The result showed that 14 patterns were found: the pattern of positive HBsAg, HBeAb, HBcAb (43.01%) and the pattern of positive HBsAg, HBeAg, HBcAb (30.57%) were more often seen. The percentage of HCV infection among cases infected with HBV was 11.39%. The difference of the positive rate of anti-HCV among various patterns of HBV markers was significant (chi 2 = 24.48, P = 2.062 x 10(-5). PMID- 7530604 TI - [Pulmonary melioidosis. Report of 4 cases and literature reviewed]. AB - In this report, We present 4 patients with pulmonary melioidosis. The literatures on epidemiology, clinical manifestation, roentogenology, diagnosis and therapeutics of melioidosis are reviewed. PMID- 7530603 TI - Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. AB - To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 (-/-) mice by gene targeting. NF-IL6 (-/-) mice were highly susceptible to infection by Listeria monocytogenes. Electron microscopic observation revealed the escape of a larger number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 (-/-) mice. Furthermore, the tumor cytotoxicity of macrophages from NF-IL6 (-/-) mice was severely impaired. However, cytokines involved in macrophage activation, such as TNF and IFN gamma, were induced normally in NF-IL6 (-/-) mice. Nitric oxide (NO) formation was induced to a similar extent in macrophages from both wild-type and NF-IL6 (-/-) mice. These results demonstrate the crucial role of NF-IL6 in macrophage bactericidal and tumoricidal activities as well as the existence of a NO-independent mechanism of these activities. We also demonstrate that NF-IL6 is essential for the induction of G-CSF in macrophages and fibroblasts. PMID- 7530605 TI - TGF alpha differentially expressed in liver foci induced by diethylnitrosamine initiation and peroxisome proliferator promotion. AB - Transforming growth factor alpha (TGF alpha) is a mitogenic growth factor for hepatocytes that may play a role in the development of liver cancer in rodents and humans. Epidermal growth factor receptor (EGFR) is the receptor for TGF alpha; its expression is also altered in mitogenic and carcinogenic processes. Homogeneous basophilic foci (HBF), the precursor lesion to hepatocellular carcinomas in peroxisome proliferator (PP)-treated rats, have labeling indices much greater than surrounding liver (approximately 5- to 20-fold) and other types of foci (approximately 2-fold greater than eosinophilic foci; EF). To test the hypothesis that PP-induced HBF over-express TGF alpha and/or EGFR, male F344 rats were treated with the PP WY-14,643 for 22 weeks (1000 p.p.m. in the diet) with (DEN-WY) and without (WY) prior diethylnitrosamine initiation (150 mg/kg body wt i.p.). Serial paraffin sections of liver were stained for TGF alpha or EGFR and with hematoxylin and eosin. DEN-WY and WY abrogated the small amount of centrilobular TGF alpha staining observed in livers from control rats. Increased staining for TGF alpha was not observed in HBF induced by WY (0/22) or DEN-WY (0/101). Additionally, increased EGFR expression was not observed in HBF induced by WY (0/22) or DEN-WY (0/19) or in EF induced by DEN-WY (0/30). An unexpectedly large proportion of EF induced by DEN-WY (6/38) and DEN-control (3/11) were TGF alpha-positive. None of the tumors induced by WY (0/13) or DEN-WY (0/11) over expressed TGF alpha. All 13 WY-induced tumors also lacked increased expression of EGFR. TGF alpha over-expression noted in a significant proportion of EF was associated only with those regimens including DEN initiation. In conclusion, TGF alpha or EGFR over-expression is not associated with early appearing, rapidly proliferating HBF or tumors induced by PP. PMID- 7530606 TI - Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice. AB - Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific angiogenic growth factor. VEGF gene transfer strategies to stimulate focal angiogenesis could be used to ameliorate myocardial ischemia. To induce angiogenesis in vivo, we have constructed a replication-defective herpes simplex virus type 1 (HSV-1) amplicon vector that places the human VEGF-165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter (HSVhvegf). Transduction of NIH 3T3 fibroblasts with HSVhvegf resulted in the secretion of high levels of biologically active VEGF, as assayed by microvascular endothelial mitogenesis. By use of an ex vivo protocol, BLK-CL4 fibroblasts were transduced with HSVhvegf or control HSVlac virus (expressing Escherichia coli beta galactosidase), resuspended in basement membrane extract (matrigel), and coinjected subcutaneously into syngeneic C57BL/6 mice. One week later, the matrigel plugs with HSVhvegf showed a strong angiogenic response, in contrast to the plugs with HSVlac-transduced fibroblasts. These data indicate that transduction with HSVhvegf virus can induce an angiogenic response in vivo and suggest that this is a viable gene therapy approach for tissue ischemia. PMID- 7530607 TI - Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes. AB - Recent evidence suggests that protein kinase A (PKA)-activated Cl- channels in heart are encoded by an isoform of the epithelial cystic fibrosis transmembrane conductance regulator gene (CFTR). Macroscopic current measurements indicate that a similar time-independent Cl- conductance can be activated through a protein kinase C (PKC)-dependent pathway in guinea pig and feline ventricle. However, it is presently not clear whether PKC is activating the same population of channels as PKA or a separate class of Cl- channels. even though the regulatory (R) domain of CFTR is known to contain consensus phosphorylation sites for both PKA and PKC. In the present study we directly compare the properties of single Cl- channels activated by PKC and PKA in cell-attached patches of guinea pig ventricular myocytes. Pipette and bath solutions contained N-methyl-D-glucamine and Cs+ or tetraethylammonium as substitutes for Na+ and K+, respectively, and Cl- concentration in the patch pipette was either 150 mmol/L or 40 mmol/L. Bath application of phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate (PDBu or PMA; 50 nmol/L), activators of PKC, resulted in the appearance of unitary Cl- channels with a mean conductance of 9.31 +/- 0.94 pS (n = 8) and 8.8 pS (n = 2), respectively, and reversal potentials were close to predicted ECl. Addition of staurosporine (500 nmol/L) reduced open probability (Po) of channels activated by PDBu. Bath application of the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX, 500 mumol/L) resulted in the activation of Cl- channels with a conductance (mean 8.76 +/- 0.67 pS, n = 3) similar to those activated by PDBu.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530608 TI - Simultaneous determination of betaine and N,N-dimethylglycine in urine. AB - A method is described for the determination of betaine and its metabolite N,N dimethylglycine in human urine. The method involves a deamination step and a solid-phase extraction to isolate both substances from urine followed by high performance liquid chromatography (HPLC) separation on a 5-microns SCX column with UV absorbance detection. Limit of detection is about 0.9 micrograms for both substances. Recoveries were > 85%. The method appears to be suitable for monitoring betaine and its metabolite in urine of patients undergoing therapy with betaine. PMID- 7530609 TI - Salivary flow rate and acute-phase proteins in Bell's palsy. AB - The flow rate of extra-parotid and parotid saliva was compared in patients with Bell's palsy and in healthy volunteers. Samples were analysed for the concentration of total protein and seven acute-phase proteins. There was no difference between younger and older patients with regard to oral status, salivary flow rate, total protein or acute-phase proteins, in either extra parotid or parotid saliva. No significant difference in flow rate for both extra parotid and parotid saliva was found in the Bell's palsy patients in comparison with the controls. In the patients the salivary flow rate from the parotid gland on the paralysed side was slightly lower than on the healthy side, but not to a significant extent. The quantity of total protein was lower in the extra-parotid saliva in the patient group; there were no differences between the two groups with regard to parotid saliva. We were able to demonstrate small amounts of various acute-phase proteins in the control group. In the patients we found higher quantities per minute of acute-phase proteins in both extra-parotid saliva and parotid saliva than in the controls. In extra-parotid saliva there were significant differences in haptoglobulin, alpha 2-macroglobulin, C3-complement factor and ceruloplasmin; in parotid saliva the differences in haptoglobulin and ceruloplasmin were significant. However, there was a large inter individual variation in both groups studied. In the patient group no significant difference in the secretion of acute-phase proteins from the parotid gland could be demonstrated between the paralysed side and the healthy side.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530610 TI - Molecular and clinical analyses of cystic fibrosis in the south of Spain. AB - We report molecular and clinical analyses in 71 unrelated patients with cystic fibrosis (CF) from Andalusia (South of Spain). Direct mutation analysis of six mutations of the CFTR gene (delta F508, G542X, R1162X, N1303K, W1182X and 1949de184) was performed. The proportion of CF chromosomes with the above mentioned mutations was 58.5%. Haplotype analysis was performed with the marker/enzyme pairs XV2C/TaqI and KM19/PstI. A particular haplotype has been found associated with each of the studied mutations, while the pooled data for the unknown mutations are not associated with any particular haplotype. This lack of association indicates that there will not be a single predominant mutation amongst the other CF chromosomes. To assess the relationship between genotype and phenotype in these patients, we correlated the pancreatic status and the occurrence of chronic Pseudomona aeruginosa infection with the observed genotype. Pancreatic insufficiency was present in all patients in whom the analyzed mutations were found to be homozygous or compound heterozygous. We also found a higher rate of Pseudomonas colonization in the group of patients in whom the genotype was homozygous or compound heterozygous for the analysed mutations when compared with the group of patients with a different genotype, but the difference was not statistically significant. PMID- 7530611 TI - A RsaI RFLP at the human myelin protein zero (MPZ) locus. PMID- 7530612 TI - Reactivation of inactive X-linked genes. PMID- 7530613 TI - Palliation of malignant oesophageal perforation and proximal oesophageal malignant dysphagia with covered metal stents. AB - Twelve patients underwent placement of nine polyethylene-covered self-expanding metal stents (Cook), and three polyurethane-covered Wallstents (Schneider). All obstructing lesions were crossed fluoroscopically and stents placed under fluoroscopic control. Eleven of the 12 patients had recently undergone failed endoscopic stent insertion, which had resulted in oesophageal perforation. Technical success was achieved in 100% of patients, with no immediate complications. The stents were well tolerated and allowed the patients to eat within 12 h of their insertion. Covered stents are technically safe to insert, are best inserted fluoroscopically, and are particularly cost-effective in proximal oesophageal lesions and perforations, where Atkinson tubes are often not tolerated. PMID- 7530614 TI - Allergic contact dermatitis in early infancy. PMID- 7530615 TI - Assignment of the tenascin gene (HXB) to swine chromosome 1q21.1-->q21.3 by fluorescence in situ hybridization. AB - The chromosomal location of the swine tenascin gene (HXB) was determined by fluorescence in situ hybridization and simultaneous R-banding. Swine tenascin cDNA was labeled with biotin, and used as a probe. Hybridization was detected by the FITC-labeled streptavidin/biotinylated antistreptavidin antibody system. The signals revealed that the tenascin gene is localized on swine chromosome 1q21.1- >q21.3. PMID- 7530616 TI - [The spectral characters of pulsed Doppler in fetal arrhythmia]. AB - Blood flow spectra of left ventricular inflow and outflow of the fetus were measured by pulsed Doppler echocardiography in 82 cases who were diagnosed as fetal arrhythmia clinically. By analysing the order of atrio- ventricular excitation, the spectral characters and the relationship of time- phase, the classification of fetal arrhythmia was confirmed. Among the 82 cases, there were 17 cases of sinus bradycardia, 16 cases of sinus tachycardia, 28 cases of atrial premature beats, 16 cases of ventricular premature beats, 3 cases of II degrees atrioventricular conduction block, 2 cases of atrial fibrillation. These common spectral characters of fetal arrhythmia were confirmed by the examination of neonatal Doppler echocardiography and ECG. This study indicated that this method could be used as one of the diagnostic methods for fetal arrhythmia. PMID- 7530617 TI - Primordial germ cell migration. AB - Primordial germ cells are migratory cells. They arise very early in embryogenesis and have a similar pattern of migration in Drosophila, Xenopus, chick and mouse. In each case the primordial germ cells associate with the developing gut from which they migrate to the gonads during organogenesis. Germ cells proliferate mitotically from the time they begin to migrate to the time they colonize the genital ridges. From the study of mouse primordial germ cells, we now know of a number of agents which affect primordial germ cell proliferation, migration and adhesion in vitro. More recently, we have studied the interactions between primordial germ cells and the cells and extracellular matrix molecules on their migratory route. By labelling germ cells in whole-mount preparations with an antibody to the germ cell marker SSEA-1, we have studied the spatial distribution of germ cells in situ using confocal microscopy. This study has revealed that germ cells link up with each other forming extensive networks during migration. PMID- 7530620 TI - TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. AB - A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper. A subequilibrium concentration of 1 microM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. PMID- 7530619 TI - Germ plasm formation and germ cell determination in Drosophila. AB - In organisms as diverse as frogs, worms and flies germline precursor cells are set aside from the somatic cells early in development. It has been proposed that specific molecules, referred to as germ cell determinants, are deposited in the egg and direct the germ cell fate, but the molecular nature and function of these determinants is not fully understood. Genetic and molecular analysis in Drosophila melanogaster indicates that germ cell determination involves not only the synthesis of specific germ cell factors but also the proper localization and assembly of a morphologically distinct germ plasm. A pathway for germ plasm assembly has been established in which the oskar gene has a central role. The amount of oskar product in the embryo controls the number of germ cells formed and mislocalization of oskar RNA and protein in the egg cell leads to the formation of ectopic germ cells in the embryo. In addition to its role in anchoring germ cell-specific signals, the germ plasm also serves as the source of abdomen-specific signal. Such a colocalization of morphogenetic signals involved in germ cell formation and in the specification of the body axis is not unique to Drosophila but is also found in Caenorhabditis elegans and Xenopus. PMID- 7530618 TI - Interactions between migratory primordial germ cells and cellular substrates in the mouse. AB - In previous in vitro studies we found that contact between mouse primordial germ cells and other cell types (neighbouring somatic cells or established TM4 or STO cell lines) is crucial for supporting primordial germ cell survival and proliferation and for activating their motility. We have studied primordial germ cell adhesion to different cell monolayers (STO, TM4, COS and F9 cells) as an in vitro model for interactions between primordial germ cells and cellular substrates. The results suggest that these cell interactions are mediated by multiple mechanisms involving Steel factor and its receptor encoded by c-kit, carbohydrates and possibly other unknown factors. We find that Steel factor and leukaemia inhibitory factor are survival rather than proliferation factors for primordial germ cells. Both molecules prevent primordial germ cell death in culture by suppressing apoptosis. Morphological and molecular features of primordial germ cell apoptosis in vitro are reported. Activation of protein kinase C does not promote primordial germ cell proliferation, but compounds known to enhance intracellular levels of cAMP (i.e. dibutyryl cAMP and forskolin) markedly stimulate primordial germ cells to proliferate in culture. We have preliminary results indicating that neuropeptides PACAP-27 and PACAP-28 are possible physiological activators of adenylate cyclase in primordial germ cells. PMID- 7530622 TI - The in vitro hepatic metabolism of ABT-418, a cholinergic channel activator, in rats, dogs, cynomolgus monkeys, and humans. AB - The metabolism of the cholinergic channel activator [3H]ABT-418 was studied in 9,000g supernatant (S-9) fractions and precision-cut tissue slices prepared from rat, dog, monkey, and human livers. In rat S-9 fractions and tissue slices, the lactam and trans N'-oxide were detected as major metabolites. The lactam was also the major metabolite in monkey and human S-9 fractions and tissue slices, although the rate of formation was greater in monkey (Vmax' of 428 vs. 103 pmol/min/mg S-9 protein). Trans N'-oxide was not detected in either species, but low levels of the cis N'-oxide were detected in tissue slice preparations from two human subjects. In contrast, trans ABT-418 N'-oxide was identified as a major metabolite in dog S-9 fractions (Vmax' of 266 pmol metabolite formed/min/mg S-9 protein) and tissue slices. Although identified as a minor metabolite in dog S-9 fractions, the lactam metabolite was shown to account for a sizeable proportion of the total radioactivity in the corresponding tissue slice preparations (22% of the total radioactivity at 12 hr); the rank order of lactam formation by the precision-cut liver slices was monkey > human > rat > or = dog. Evidence that N' oxidation and C-oxidation (to lactam) of ABT-418 was mediated by liver microsomal flavin-containing mono-oxygenase (FMO) and cytochromes P-450 (CYPs), respectively, was obtained with the inhibitors thiobenzamide and clotrimazole. The involvement of cytosolic aldehyde oxidase (AO) was suggested by a significant correlation (r2 = 0.998, p < 0.01) between the observed rate of lactam formation and AO (N1-methylnicotinamide oxidase) activity in rat, dog, monkey, and human S 9 fractions; inhibition of lactam formation by the AO substrate N1 methylnicotinamide; and the lack of lactam formation in the absence of cytosol. Data indicate that the species-related differences in the hepatic metabolism of ABT-418 may be dependent on the relative levels and/or activity of FMO, CYP, and AO. In this regard, ABT-418 is very similar to nicotine. However, unlike nicotine, the N-demethylation of parent drug and the further products of lactam metabolism was not detected. PMID- 7530621 TI - Insulin-like growth factors and binding proteins in patients with recent-onset type 1 (insulin-dependent) diabetes mellitus: influence of diabetes control and intraportal insulin infusion. AB - Type 1 diabetes mellitus is associated with decreased insulin-like growth factor 1 (IGF-1) levels, enhanced values of growth hormone (GH) and IGF-binding protein 1 (IGFBP-1). Since the liver is the major source of IGF and IGFBP production, we have therefore examined whether levels of IGFs (IGF-1 and IGF-11) and IGFBPs (IGFBP-1 and IGFBP-3) differ when insulin is infused into the portal or peripheral vascular system. IGF, IGFBP, and GH levels were determined within 1-3 weeks of diagnosis in 36 patients (ranging in age from 18 to 22 years) with Type 1 diabetes mellitus. IGF-1 levels were low before insulin therapy administration (0.49 +/- 0.05 vs. 1.11 +/- 0.04 U/ml in controls, P < 0.01). With insulin treatment, IGF-1 levels rose to the normal range and IGF-1 normalisation depended on diabetes control and the route of insulin infusion. Diabetic patients with conventional insulin therapy (CIT; n = 12) had low IGF-1 (0.57 +/- 0.07 U/ml) compared with patients with continuous subcutaneous insulin infusion (CSII; n = 12; 0.75 +/- 0.08 U/ml; P < 0.05) and intraportal insulin infusion (IPII; n = 12; 1.07 +/- 10.05 U/ml; P < 0.05). Significant correlations were found between IGF-1 and parameters of glycemic control: HbA1c (r = -0.64; P < 0.01) and glycemia (r = -0.56; P < 0.05). The pattern of changes in IGF-11 levels was not significantly different from that of controls and was not altered by insulin therapy (0.98 +/- 0.08 and 1.01 +/- 0.04 U/ml in controls). Measured fasting 08:00 h IGFBP-1 levels were elevated 3-fold and IGFGP-3 levels were 2-fold lower in diabetic patients than in controls. Elevated IGFBP-1 levels were significantly correlated with metabolic control (glycemia, r = 0.64, P < 0.01; HbA1c, r = 0.71, P < 0.01). The mean elevated GH level before insulin administration (13.4 +/- 0.9 mg/l) was decreased by intensified insulin therapy (CSII, 8.8 +/- 0.6, P < 0.05; IPII, 5.6 +/- 0.9 mg/l, P < 0.001). There was a negative correlation between GH and IGF-1 (r = -0.72, P < 0.01). These results show the role of glycemic control and the route of insulin administration in the normalisation of IGF-1, IGFBP-1 and GH up to non-diabetic controls in patients with recent-onset Type 1 diabetes mellitus. PMID- 7530623 TI - FK 506 metabolism in human liver microsomes: investigation of the involvement of cytochrome P450 isozymes other than CYP3A4. PMID- 7530624 TI - Trough/peak ratios for antihypertensive agents. The issues in perspective. PMID- 7530625 TI - Interleukins. Clinical pharmacology and therapeutic use. AB - With interleukins (IL), a new class of potential drugs has been introduced into clinical research. These signal peptides are involved in the regulation of many physiological and pathophysiological processes. IL-1, -2, -3, -4, -6 and -11 have been tested in clinical trials. The growth promoting, growth inhibiting or immunomodulatory activities of interleukins represent the theoretical basis for large scale clinical testing, predominantly in malignant disease. Dose-dependent effects on numbers of peripheral blood cells and recovery from bone marrow failure have been demonstrated for IL-1, -3, -6 and -11. Phase III trials are in progress to determine their value for clinical practice. However, investigations on the immunomodulatory activities proved to be more difficult. This is because key mechanisms for successful treatment of malignant disease by immunomodulation are not clearly defined and the methodology for assessment of immunostimulatory effects is not well established. Besides treatment of renal cell carcinoma and malignant melanoma with IL-2, no successful trials have been reported. However, phase I clinical trials with IL-1, IL-4 and IL-6 have just been completed. Thus, it seems too early to conclude on their therapeutic potential. PMID- 7530626 TI - Multidrug-resistant Enterococcus faecium. An untreatable nosocomial pathogen. AB - The prevalence of enterococci and nosocomial pathogens has increased over the past 15 years. They have become increasingly resistant to agents traditionally useful in the treatment of invasive diseases due to enterococci. Vancomycin resistance, first described in clinical isolates in 1988, has disseminated worldwide. It is usually associated with high-level resistance to penicillins and aminoglycosides rendering the treatment of patients with vancomycin-resistant enterococci very difficult. Several investigators have reported mortality rates greater than 50% for vancomycin-resistant enterococcal bacteraemia. Risk factors associated with vancomycin-resistant enterococcal bacteraemia include prolonged hospital stay, neutropenia, prior oral or parenteral vancomycin use, and broad spectrum antibiotics. Since there is no uniformly effective antimicrobial therapy for patients infected with vancomycin-resistant enterococci, preventing of the spread of infection with the rigorous application of barrier precautions and other infectious control techniques is of paramount importance. PMID- 7530628 TI - Drug therapy of mastalgia. What are the options? AB - Severe breast pain or mastalgia is a common symptom, affecting up to 70% of the female population at some time in their lives. It accounts for approximately 50% of referrals to a specialised breast clinic, two-thirds of patients having cyclical and one-third experiencing noncyclical mastalgia, or pain arising from the chest wall deep to the breast. After exclusion of breast cancer and proper reassurance, 85% of patients can be discharged from the clinic without specific treatment. In only 15% of patients is the pain severe enough to affect their lifestyle and warrant drug therapy. Using EF-12 (gammalinolenic acid; gamolenic acid) as first-line therapy, with danazol and bromocriptine usually as second line agents, a clinically useful improvement in pain can be anticipated in 92% of patients with cyclical and 64% with noncyclical mastalgia. Patients with severe recurrent or refractory mastalgia may require treatment with tamoxifen, goserelin or testosterone, but the short and long term adverse effects of these drugs preclude their use as first-line agents. Chest wall pain is usually self limiting, but symptomatic relief can often be obtained using steroidal and local anaesthetic injections or nonsteroidal anti-inflammatory drugs. PMID- 7530627 TI - Current and potential treatment of tuberculosis. AB - A recent resurgence of interest in tuberculosis as a global health problem has accompanied the resurgence of tuberculosis in both industrialised and developing countries. It has also been demonstrated recently that tuberculosis treatment and control is one of the most cost effective of all medical interventions. The human immunodeficiency virus (HIV) epidemic and increasing resistance to antituberculous drugs complicate our response to the problem of tuberculosis. Chemotherapy with currently available agents is highly effective, not only in pulmonary tuberculosis in adults, but also in extrapulmonary disease, and in disease in children and even patients with concomitant HIV infection. Short course chemotherapy and intermittent therapy are as effective as older regimens. Measures, including directly observed therapy, to maximise compliance with therapy, are of utmost importance. An efficient programme which assures compliance with effective antituberculosis chemotherapy should be a priority for health spending even in those countries with fewest resources. PMID- 7530629 TI - Urticaria. Recognition, causes and treatment. AB - The urticarias are a complex group of disorders characterised by transient whealing or swelling of the skin. Understanding the many possible causes is the first step in assessing urticaria. Allergic and drug-induced urticaria respond to removal of the cause. The physical urticarias, particularly delayed pressure urticaria and also urticarial vasculitis, require separate consideration. For the majority of patients with chronic idiopathic urticaria, nonsedating antihistamines are the mainstay of treatment. There are several to choose from, including cetirizine, astemizole, loratadine, terfenadine and acrivastine, each with its own pharmacokinetics and antiallergic properties. When these fail, histamine H2-antagonists may help either alone or in combination with H1 antagonists. Older sedative antihistamines are still useful. Ketotifen, oxatomide and azelastine have mast cell stabilising effects that are considered an advantage in treating these disorders. Second-line therapies include a wide range of drugs such as doxepin, dapsone, attenuated androgens, calcium antagonists, antimalarials, gold and methotrexate. The most effective and regularly used second-line agents are corticosteroids. These are best limited to short term crisis management, except in severe recalcitrant cases, and in patients with pressure urticaria or urticarial vasculitis. Recent work on circulating histamine releasing autoantibodies suggests that there is scope for more aggressive immunosuppression in selected patients. However, effective treatment with immunosuppression often requires plasma exchange and more toxic agents such as cyclosporin. Such treatments are only likely to be entertained in exceptional cases. PMID- 7530631 TI - Granisetron. An update of its therapeutic use in nausea and vomiting induced by antineoplastic therapy. AB - Granisetron is a selective serotonin3 (5-hydroxytryptamine3, 5-HT3) receptor antagonist which has significant antiemetic activity against chemotherapy-induced nausea and vomiting. A single prophylactic intravenous dose is sufficient to control acute nausea and vomiting in approximately 60 to 70% of patients. In comparative studies, the acute antiemetic efficacy of granisetron is equivalent or superior to that of traditional antiemetic regimens even in patients receiving highly emetogenic cisplatin-containing chemotherapy. However, limited data have suggested that granisetron therapy offers no advantages over traditional antiemetics in terms of the control of delayed emesis. Recently, a number of large randomised studies have directly compared the efficacy and tolerability of granisetron, ondansetron and tropisetron and reported no significant differences between the 3 drugs in controlling acute nausea and vomiting, although 1 study reported a modest statistical advantage for granisetron over ondansetron but not tropisetron in the complete control of vomiting. In crossover studies, significantly more patients preferred granisetron to either ondansetron or tropisetron. The efficacy of granisetron appears to be maintained with repeated doses over several cycles of chemotherapy, although the influence of various prognostic factors affecting antiemetic response has not been adequately analysed. Concomitant administration of dexamethasone significantly improves the acute antiemetic efficacy of granisetron, increasing response rates by approximately 15%. Granisetron is an effective antiemetic in children undergoing highly emetogenic chemotherapy, and effectively controls radiotherapy-induced and postoperative nausea and vomiting. Trials using an oral formulation are scarce at present, but preliminary results suggest a similar efficacy and tolerability profile to that of the intravenous formulation. Granisetron has been well tolerated in clinical trials. The most frequently reported adverse event has been headache (14%). Extrapyramidal effects, which can limit the use of traditional antiemetics such as metoclopramide, have not been reported with granisetron. Thus, recent data confirm that granisetron is an effective and well tolerated agent for the prophylactic treatment of chemotherapy-induced acute nausea and vomiting, with efficacy equivalent or superior to that of other currently available agents. It has a promising role to play in paediatric oncology, and is an effective agent in controlling radiotherapy-induced acute emesis. Granisetron offers comparable or superior efficacy in controlling acute nausea and vomiting with a much simpler dosage regimen than that of traditional antiemetic regimens. PMID- 7530630 TI - Filgrastim. A review of its pharmacological properties and therapeutic efficacy in neutropenia. AB - Filgrastim, a recombinant human granulocyte colony-stimulating factor (G-CSF), has identical biological activity to that of endogenous human G-CSF, but differs in that it contains an N-terminal methionine residue and is not glycosylated. It principally stimulates activation, proliferation and differentiation of neutrophil progenitor cells and has been evaluated in the treatment of patients with various neutropenic conditions, both iatrogenic and disease-related. Two comparative studies have demonstrated that prophylactic administration of filgrastim 230 micrograms/m2/day significantly reduces the incidence, duration and severity of neutropenia in patients with previously untreated small-cell lung cancer receiving standard-dose chemotherapy with CDE (cyclophosphamide, doxorubicin plus etoposide). Concomitant with the amelioration of neutropenia, the incidence of febrile neutropenia was significantly reduced by 50% and there were 35 and 50% decreases in hospitalisation rates and intravenous antibiotic requirements. Since not all patients receiving standard-dose chemotherapy are at risk of infectious complications, prophylactic filgrastim use may be reserved for those patients who have developed febrile neutropenia during a previous cycle of the same regimen. This strategy may prove less costly, although potential savings must be weighed against a greater risk of patient morbidity and reduced quality of life. When combined with standard intravenous antibiotic therapy, filgrastim further decreases morbidity in patients with established febrile neutropenia and may have a positive impact on overall treatment costs by shortening the length of hospitalisation. Attention is focused on the use of haematopoietic growth factors to support dose-intensification of chemotherapy with a view to improving treatment outcomes in patients with chemo-responsive tumours. Filgrastim, used alone, permits modest increases in dose-intensity and/or dose-escalation of some standard-dose chemotherapy regimens. Moreover, the drug has proven useful as an adjunct to myeloablative chemotherapy followed by stem cell rescue with autologous bone marrow transplantation and/or peripheral blood progenitor cells. However, the impact of these dose-intensification approaches on survival remains to be determined in well-controlled clinical studies. Filgrastim is effective in increasing the neutrophil count and decreasing morbidity in patients with severe chronic neutropenia, including Kostmann's syndrome, and in idiopathic and cyclic neutropenia. In addition, filgrastim has accelerated neutrophil recovery in patients with idiosyncratic drug-induced agranulocytosis. Available data indicate that filgrastim is generally well tolerated. The most frequent adverse reaction is mild to moderate medullary bone pain, reported by approximately 20% of patients, although this can generally be controlled using simple analgesics without the need to discontinue treatment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530633 TI - Genetics of Pelizaeus-Merzbacher disease. AB - Pelizaeus-Merzbacher disease (PMD) has been recognized as a clinical entity for more than a century. It has gradually become apparent that the disorder is a dysmyelination, in distinction to demyelinating conditions such as adrenoleukodystrophy. The failure to deposit myelin is due to decreased production of its chief protein, proteolipid protein (PLP). In about 30% of patients with the diagnosis of PMD there is a mutation in the coding portion of the proteolipid protein gene, PLP. This gene is located at Xq22 so the disease in these families shows an X-linked pattern of inheritance. The expression of the mutant gene is generally recessive, but some mutations are expressed frequently in females. At least some patients with PMD that do not show mutations in the coding region of PLP demonstrate linkage between the disease and PLP. As additional mutations in PLP are discovered, it is becoming apparent that the nosology of PLP-associated disease is changing. PMD now comprises a spectrum of disorders with similar but not necessarily identical clinical pictures. Some of these disorders may be certain forms of X-linked paraplegia, SPG2. Finally, some diseases that look like PMD may not be X-linked. PMID- 7530634 TI - EEG spectral findings in healthy, elderly men and women--sex differences. AB - Computerized spectral analysis of the EEG was performed in 119 healthy, elderly men and women, ranging in age from 60 to 87 years with an x age of 70.4 years, to evaluate gender-related variance. The groups did not differ in age, education levels or Folstein score. We compared the parasagittal derivations in men (n = 53) to women (n = 66), controlling for the effect of age by decade. Compared to men, women had a significant (usually P < 0.01) increase in the parasagittal mean frequency (1-30 Hz and 4-20 Hz), beta 1 and beta 2, while alpha 2 and theta-beta were decreased. Mean frequency did not differ significantly by decade, nor were there significant sex-age interactions. Our results extend previous EEG spectral findings to older, healthy, elderly subjects. Although the basis for the gender related differences is uncertain, we suggest that quantitative EEGs establish gender-based norms in elderly adults. PMID- 7530635 TI - The clinical and prognostic relevance of the postictal slow focus in the electrocorticogram. AB - Electrocorticograms of 192 complex partial seizures which were recorded via chronically implanted subdural electrodes during presurgical evaluation of 64 patients with medically intractable epilepsy were visually analysed. The objective was to assess the diagnostic and prognostic relevance of postictal slow foci (PISF) in the electrocorticogram which were defined as focal isoelectric activity or as a focal burst-suppression pattern. The following results were obtained: (1) PISF were seen in a total of 114 of 192 seizures (59.4%), (2) 48 of 64 patients (75%) demonstrated at least 1 PISF in 3 seizures, (3) PISF were more frequent in seizures of temporal lobe origin (66%) than in those of extratemporal origin (33.3%), (4) no PISF developed if the electrographic seizure duration was < 32 sec, (5) PISF were (predominantly) localized in the lobe of seizure origin in 85% of the seizures, (6) there was a strong correlation between frequent occurrence of temporal PISF and favourable seizure outcome following temporal lobe surgery. In conclusion, PISF contribute valuable data as to the localization of the epileptogenic zone during presurgical evaluation of epilepsy and indicate favourable seizure outcome following temporal lobe surgery. PMID- 7530632 TI - Paclitaxel. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer. AB - Paclitaxel is a new anticancer agent with a novel mechanism of action. It promotes polymerisation of tubulin dimers to form microtubules and stabilises microtubules by preventing depolymerisation. In noncomparative trials, continuous infusion of paclitaxel 110 to 300 mg/m2 over 3 to 96 hours every 3 to 4 weeks produced a complete or partial response in 16 to 48% of patients with ovarian cancer and 25 to 61.5% of patients with metastatic breast cancer, many of whom were refractory to treatment with cisplatin or doxorubicin, respectively. 23 to 100% of patients with ovarian cancer achieved complete or partial responses with paclitaxel in combination with cisplatin, carboplatin, cyclophosphamide, altretamine and/or doxorubicin. Similarly, response rates of 30 to 100% were observed with paclitaxel plus doxorubicin, cisplatin, mitoxantrone and/or cyclophosphamide in patients with metastatic breast cancer. Comparative trials in patients with advanced ovarian cancer showed paclitaxel therapy to produce greater response rates than treatment with parenteral hydroxyurea (71 vs 0%) or cyclophosphamide (when both agents were combined with cisplatin) [79 vs 63%]. Paclitaxel was also more effective than mitomycin in 50 patients with previously untreated breast cancer (partial response in 20 vs 4% of patients). Paclitaxel therapy also produced promising results in patients with advanced squamous cell carcinoma of the head and neck, malignant melanoma, advanced non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), germ cell cancer, urothelial cancer, oesophageal cancer, non-Hodgkin's lymphoma or multiple myeloma, and was successfully combined with cisplatin, carboplatin and/or etoposide in patients with NSCLC, SCLC or advanced squamous cell carcinoma of the head and neck. Hypersensitivity reactions were initially a concern with administration of paclitaxel, although current dosage regimens have reduced the incidence of these events to less than 5%. The major dose-limiting adverse effects of paclitaxel are leucopenia (neutropenia) and peripheral neuropathy. Other haematological toxicity was generally mild. Cardiac toxicity was reported in small numbers of patients and most patients developed total alopecia. Several aspects of paclitaxel use remain to be clarified, including the optimal treatment schedule and infusion time, confirmation of the tolerability profile and efficacy of combination regimens in an expanded range of malignancies. Long term follow-up of paclitaxel recipients will also allow the effects of the drug on patient survival to be determined. Nevertheless, paclitaxel is a promising addition to the current therapies available, with significant activity reported in patients with advanced ovarian or breast cancer or other types of tumors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530636 TI - Epileptic seizures triggered directly by focal transcranial magnetic stimulation. AB - Focal, secondarily generalizing, epileptic seizures were released by magnetic stimulation in a patient with focal epilepsy. The stimulation induced seizures had a similar clinical appearance to the patient's spontaneous seizures. They were released exclusively by an angulated "figure-of-8" coil which stimulates the brain more focally as compared to the commonly used flat round coil. The epileptic focus could be located in the left frontal cortex by electroencephalographic recordings, by magnetic resonance and by positron emission tomography imaging. Functional assessment of the motor system by transcranial magnetic stimulation (TMS) disclosed markedly prolonged electrical silence of contralesional electromyographic activity following the early excitation. This is the first EMG-documented demonstration of a focal motor seizure directly triggered by magnetic brain stimulation. PMID- 7530637 TI - Specific tonotopic organizations of different areas of the human auditory cortex revealed by simultaneous magnetic and electric recordings. AB - This paper presents data concerning auditory evoked responses in the middle latency range (wave Pam/Pa) and slow latency range (wave N1m/N1) recorded from 12 subjects. It is the first group study to report multi-channel data of both MEG and EEG recordings from the human auditory cortex. The experimental procedure involved potential and current density topographical brain mapping as well as magnetic and electric source analysis. Responses were compared for the following 3 stimulus frequencies: 500, 1000 and 4000 Hz. It was found that two areas of the auditory cortex showed mirrored tonotopic organization; one area, the source of N1m/N1 wave, exhibited higher frequencies at progressively deeper locations, while the second area, the source of the Pam/Pa wave, exhibited higher frequencies at progressively more superficial locations. The Pa tonotopic map was located in the primary auditory cortex anterior to the N1m/N1 mirror map. It is likely that N1m/N1 results from activation of secondary auditory areas. The location of the Pa map in A1, and its N1 mirror image in secondary auditory areas is in agreement with observations from animal studies. PMID- 7530638 TI - Event-related brain potentials differentiate priming and recognition to familiar and unfamiliar faces. AB - Recent studies from our laboratory have resulted in the identification of an event-related potential (ERP) correlate of a visual memory process. This memory process is reflected by a reduction in the voltage of the visual memory potential (VMP) to repeated pictures of unfamiliar faces compared to novel pictures of faces. In the current experiment we used unfamiliar and famous faces in an identical repetition priming paradigm, while the subject differentially recognized famous from non-famous faces. Significant differences in response times were obtained between primed and unprimed familiar faces, but not between primed and unprimed unfamiliar faces. The VMP was reduced to primed unfamiliar faces and significantly diminished to primed familiar faces compared to unprimed stimuli. Priming was typically reflected by a reduction of the VMP at the occipito-temporal region, whereas recognition resulted in a diminution of the VMP at both the occipito-temporal region and at the frontal region. These data support the involvement of differential neural systems for priming and recognition of visual stimuli. PMID- 7530639 TI - Frequency and phase characteristics of slow cortical potentials preceding bimanual coordination. AB - The aim of the present study was to derive quantities which relate behavioral and neurophysiological levels of observation during a bimanual coordination task. We recorded the scalp electroencephalographic (EEG) signal preceding a sequence of 4 bimanual finger flexions of varying response rates in 12 subjects. A slow negative-going Bereitschaftspotential (BP) displayed larger mean amplitudes and earlier onset times for the faster required response rates. The amplitude of the BP was also larger for electrode locations contralateral to the side initiating the behavioral response. A Fourier transform showed two predominant frequencies (0.5 and 2.0 Hz) to be amplitude modulated as a function of the required response rate in addition to increased power on the contralateral side of the finger initiating the response. A measure of the phase relationship between the left (C3) and right (C4) hemispheres of the fronto-central cortex at each of these spectral frequencies was calculated as well as the variance in this measure and found to correspond closely to the variance in inter-response times derived from the subjects' movements. These findings indicate that changes in the stability and rate of a patterned movement are generally preceded by similar changes in the stability and amplitude of components observed on the neurophysiological level. PMID- 7530640 TI - Dynamics of EEG background activity level within quiet sleep in successive cycles in infants. AB - We investigated in infants the emergence of the trends of the EEG synchronization throughout quiet sleep (QS) as a function of the QS rank. The night sleep of 3 groups with 6 subjects each (aged respectively 9-18 weeks, 21-47 weeks, and 16-45 years) was recorded. A parameter value reflecting the degree of synchronization of the EEG background activity for successive epochs was computed by automatic analysis. For each QS phase 3 indicators of the dynamics of the time course of the EEG parameter activity were determined: the range (difference between the EEG parameter value at the beginning of the QS episode and that at the trough), the trough latency (after QS onset), and the rate of synchronization (range/trough latency). The range and the trough latency increased with age, whereas the rate of synchronization decreased. The range and the rate of synchronization decreased in the successive cycles, whereas the trough latency increased. These results provide further support for the hypothesis of the early emergence of the process S mechanisms and suggest that the framework of the 2-process model could account also for the development of both the EEG background activity dynamics and the sleep-wake organization. PMID- 7530642 TI - Refractory periods following interictal spikes in acute experimentally induced epileptic foci. AB - Under epileptic conditions, interictal epileptic events are followed by large inhibitions which prevent the transition to ictal discharges. In the present experiments the refractory period following interictal epileptic spikes was investigated in animal experiments. Interictal epileptic activity was elicited by application of penicillin onto the motor cortex of anesthetized rats. Interictal epileptic discharges were followed by an absolute refractory phase lasting 200 300 msec, in which no epileptic event could be elicited by epicortical stimulation. This was followed by a relative refractory period up to 900 msec after onset of the conditioning spike; spikes elicited with intervals between 300 and 900 msec were smaller than those with greater intervals and required higher stimulation intensities. This period ends by a sharp drop of threshold. In two thirds of the experiments, spikes were favoured in intervals of 300-500 msec due to a sag of the threshold, which possibly indicates recurrent neuronal excitations. Stimulations with frequencies of about 1/sec favoured a transition from a pattern with spikes appearing in an irregular sequence every 2-3 sec, to a discharge pattern with spikes appearing with regular intervals of about 1 sec. This change of firing pattern was associated with a drop of the spike threshold. It is concluded that interictal epileptic events are followed by a refractory period comprising different components. Alterations of the neuronal inhibitions responsible for these refractory phases may be critical for the activity of the focus and may determine the transition from interictal to ictal discharges. PMID- 7530641 TI - Positive event-related potentials to real and dummy rule-learning feedback and to perceptuomotor feedback. AB - Amplitudes of 5 event-related potentials (ERPs) were recorded at 5 sites of 10 males to real (R-) and dummy (D-) feedback (FB) over two difficulty levels of a rule-learning task, with interspersed perceptuomotor (PM) task trials. Rule learning R-FB for positive slow wave (PSW) and P3b was greater than for D-FB for both mean time-window and principal component factor score measures. FB effects varied by site for P2/P3a (mainly Fz-C4-Pz) and for a late PSW (LPSW; mainly C4 C3-Fz). A new ERP, P508, showed the greatest topographic differentiation, but no FB main effect. The following ERPs may reflect different sources: PSW versus P3b; P2/P3a versus LPSW; R-FB versus D-FB P2/P3a; R-FB versus D-FB LPSW; and P508 versus all others. LPSW was greater to simple than complex task difficulty; while the "P508" factor score trended towards being greater for complex than simple. ERP interpretation is in terms of stimulus recognition classification, comparative evaluation and development elaboration of mental models. Rule learning D-FB exceeded PM accuracy R-FB for all ERPs but P2/P3a. Strongly implicated in these differences are preparatory acts in the former task as reflected by the PSW and LPSW. PMID- 7530643 TI - Evaluation of the "monolog" spike and wave monitor: a study on patients with typical absences. AB - The aim of this study was to evaluate the effectiveness and accuracy of the "monolog," a pocket-size spike and wave monitor, in patients with typical absence seizures. Two observers were scoring independently and blindly from the monitor the number and duration of the 3 Hz spike-wave discharges from the EEG recordings of 10 selected patients having typical absences. High correlation was found between the two observers' scorings for both the number and the mean duration of events (Spearman's rank correlation (r): 0.9970, P < 0.001, and 0.9758, P < 0.001), as well as between each of them and the "monolog" for the mean event duration (r: 0.9848, P < 0.001, and 0.9788, P < 0.001). Conversely, a poor correlation was found between each of the observers and the "monolog" for the number of events (r: 0.7121, P = 0.021, and 0.6939, P = 0.026). In conclusion, the "monolog" accurately measured the duration of 3 Hz spike-wave activity; however, it failed to accurately count the number of events that occurred throughout the recordings. We suggest that this device, with appropriate modification to its memory epochs, may be a useful tool in monitoring patients with typical absences. PMID- 7530644 TI - Intraoperative monitoring of corticospinal function. PMID- 7530645 TI - Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25 (OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes. AB - Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25 (OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development. PMID- 7530646 TI - Folding of the recombinant human thyrotropin (TSH) receptor extracellular domain: identification of folded monomeric and tetrameric complexes that bind TSH receptor autoantibodies. AB - We have analyzed protein folding and disulfide bond formation in the extracellular domain of the human TSH receptor (hTSHR-ecd) expressed in Escherichia coli. This domain, which begins at the amino-terminus and ends at residue 415, is a major autoantigen in human autoimmune thyroid disease. Refolding of reduced and denatured hTSHR-ecd occurred in polyacrylamide gels treated with 0.25 M KCl for visualization of protein bands. Under conditions of partial renaturation, at least three forms of the hTSHR-ecd were resolved by reelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 1) unfolded monomers, 2) folded monomers, and 3) tetramers. Disulfide bond formation was implicated in both folding and tetramerization, as reduction of these forms produced only unfolded monomers. A natural variant of the hTSHR (v1.3), sharing 231 N-terminal amino acids with hTSHR-ecd, formed folded monomers and dimers on renaturation, but not tetramers, implicating one or more of the five cysteine residues residing between positions 231-415 in the association of dimers into tetramers. Binding of three different sources of hTSHR antibodies to these various forms of the hTSHR-ecd was assessed by immunoblotting using: 1) murine monoclonal antibodies (MAbs) generated against hTSHR-ecd, 2) rabbit polyclonal antisera generated against overlapping synthetic peptides spanning residues 37-71 of the hTSHR-ecd, and 3) human immunoglobulin G from patients with Graves' disease and detectable hTSHR-Ab. One of the MAbs, shown to recognize residues 21-35, and the rabbit polyclonal antibodies bound to all three forms of the hTSHR-ecd. Some of the hTSHR autoantibodies bound predominantly to the monomeric forms of the hTSHR, but autoantibodies were also identified that recognized tetrameric hTSHR-ecd. These data demonstrate that hTSHR-Abs recognize differing nonlinear and linear epitopes in the hTSHR-ecd and provide a methodology that should be useful for further defining the structural requirements for folding of this functionally and immunologically important domain of the hTSHR. PMID- 7530647 TI - Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation. AB - Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction. PMID- 7530649 TI - A major portion of the 150-kilodalton insulin-like growth factor-binding protein (IGFBP) complex in adult rat serum contains unoccupied, proteolytically nicked IGFBP-3 that binds IGF-II preferentially. AB - Insulin-like growth factor-II (IGF-II) binds to 150-kilodalton (kDa) protein complexes in adult rat serum that have higher affinity for IGF-II than IGF-I. We now examine the nature of these IGF-II-preferring complexes. A 150-kDa pool, isolated after neutral Sephadex G-200 gel filtration of adult rat serum, bound IGF-II with approximately 80-fold greater affinity than IGF-I, but did not contain immunoprecipitable IGF-II/mannose-6-phosphate receptors, which bind IGF II with the same preferential affinity. Exogenous IGF-II bound to the 150-kDa complex without displacing endogenous IGF-I, indicating that it bound to sites that were previously unoccupied. IGF-I affinity chromatography was used to separate unoccupied 150-kDa complexes that bound to the column and were eluted with acid from complexes that contained endogenous IGF-I, did not bind to the column, and appeared in the flow-through. In competitive binding assays, IGF binding proteins (IGFBPs) in the acid eluate bound IGF-II with higher affinity than IGF-I, whereas IGFBPs in the flow-through, after acid stripping, bound IGF-I and IGF-II with similar affinity. The acid eluate and the acid-stripped flow through predominantly formed 50-kDa complexes with [125I]IGF-II when affinity cross-linked and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; these complexes were precipitated by antibodies to rat IGFBP-3. Larger complexes (> 95 kDa) present in the nonacidified 150-kDa pool were not observed, consistent with the IGFBP-3 molecules in the flow-through and eluate having been complexed to an acid-labile subunit (ALS). By contrast, when these preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without affinity cross-linking, the flow-through contained intact IGFBP-3 (40-43 kDa), whereas the eluate contained only truncated 30-kDa IGFBP-3. We conclude that the 150-kDa fraction of adult rat serum includes 1) ternary complexes of intact IGFBP-3 (which binds IGF-I and IGF-II with similar affinity), endogenous IGF-I, and the ALS; and 2) binary complexes of proteolytically nicked IGFBP-3 and ALS that bind IGF-II preferentially. The nicked IGF-II-preferring complex is present in native serum before acidification or exposure to denaturants. PMID- 7530648 TI - Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease beta 1-integrin levels. AB - Prolonged glucocorticoid treatment causes osteoporosis in vivo and inhibits bone formation in vitro. We have previously shown that glucocorticoids inhibit calcification and alter osteoblast organization in a mineralizing bone organ culture system. In this study, the effect of glucocorticoids on osteoblast adhesion to bone matrix proteins and integrin expression was examined in primary rat osteoblasts and a transformed rat osteosarcoma-derived cell line ROS 17/2.8. After 24 h of treatment with corticosterone, these cells displayed a concentration-dependent decrease in adhesion to type I collagen and fibronectin. Adhesion was significantly decreased as early as 4 h after glucocorticoid administration. With 100 nM corticosterone treatment for 24 h, inhibition of the adhesion of ROS 17/2.8 cells and primary osteoblasts to fibronectin was 75 +/- 10% and 50 +/- 8%, and inhibition of adhesion to collagen was 31 +/- 10% and 65 +/- 5%, respectively. This effect was specific for osteoblasts, because glucocorticoids did not change the adhesion of fibroblasts. However, glucocorticoids did inhibit the adhesion of all cell types to rat osteonectin. To determine whether the change in osteoblast attachment to collagen and fibronectin was due to an alteration in integrin levels, the plasma membranes of these cells were labeled with [125I]lactoperoxidase, solubilized, and immunoprecipitated with an antibody to beta 1. A 24-h treatment with 100 nM corticosterone caused 80 +/- 2% and 64 +/- 9% decreases in beta 1 levels in primary osteoblasts and ROS 17/2.8 cells, respectively. These results were confirmed with immunofluorescence microscopy, which showed a glucocorticoid-induced decrease in beta 1 staining. Treatment of primary rat osteoblasts and ROS 17/2.8 cells for 72 h with corticosterone also decreased beta 1-integrin messenger RNA levels in a dose dependent manner. We have demonstrated that the inhibition of integrin expression by glucocorticoids is involved in the decrease in osteoblast adhesion to bone extracellular matrix proteins. These data suggest that integrin modulation may influence osteoblast function and bone formation and, thus, contribute to glucocorticoid-induced osteoporosis. PMID- 7530650 TI - Cloning and characterization of the promoter for the rat insulin-like growth factor-binding protein-3 gene. AB - Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the adult circulation, is produced by a wide range of cell and tissue types. IGFBP-3 appears to be regulated by transcriptional and/or posttranslational mechanisms in a species-, cell-, and development-specific manner. In vitro and in vivo studies suggest that a number of factors (e.g. cAMP, GH, insulin-like growth factor-I, epidermal growth factor, TSH, and FSH) can act as transcriptional regulators of IGFBP-3 in particular cell types. To address the mechanistic basis for these observations, we isolated the rat IGFBP-3 gene and began characterization and analysis of the hormonal regulation of its promoter. The rat IGFBP-3 gene is located within 2 adjacent EcoRI fragments spanning about 10 kilobases. Southern analysis indicated a single copy gene. A 1.18-kilobase fragment 5' to the translation initiation codon has been sequenced and showed 65% homology with the corresponding human IGFBP-3 sequence. The region between -100 and -1 bp relative to the transcription start site showed 85% homology. The transcription start site was 118 basepairs (bp) up-stream of the initiation codon, and a TATA box consensus was located 27 bp 5' to this CAP site. No CAAT box was present, but a CpG island was identified. Consensus sequences for a number of putative response elements (e.g. activating protein-2, insulin, TSH/insulin-like growth factor, and GH) were present within -700 bp of the CAP site. A series of 5'-truncated chloramphenicol acetyltransferase reporter constructs has been transfected into both COS-1 cells and the rat thyroid cell line FRTL-5. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. These data indicate that suitable transfected cell systems can be established in which additional investigations can be undertaken into the mechanisms of cell- and species-specific hormonal regulation of IGFBP-3 gene expression. PMID- 7530651 TI - Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells. AB - The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1 3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4. PMID- 7530653 TI - Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone. AB - LNCaP is an androgen-sensitive human prostatic cancer cell line. The effect of androgen on these cells is characterized by a bell-shaped growth response and a dose-dependent induction of prostate-specific antigen (PSA) production. The present study was carried out to gain further insight into the effect of androgen on LNCaP. Cells were cultured in phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped fetal bovine serum, with concentrations of dihydrotestosterone (DHT) ranging from 0-10(-7) M, in a 4-day culture system. A bell-shaped growth response was reproduced with a peak level of cell count at 10(-10) M DHT. PSA secretion from these cells did not increase significantly until the DHT level in the medium reached 10(-9) M. A progressive increase in PSA secretion was observed at higher DHT concentrations accompanied with a progressive decline in cellular proliferation. The results of immunocytochemical analysis of PSA localization indicated that the proportion of cells with positive staining for PSA also increased with increasing concentrations of DHT. Analysis of androgen receptors, as determined by both immunocytochemistry and Western blot analysis, showed a decline in nuclear androgen receptor at low concentrations of DHT and an increase in the amount of receptor protein at high concentrations. These results indicated that the androgen-induced bell-shaped growth response in LNCaP cells represented the manifestation of two different cellular events in dose-related manner: cellular proliferation at low DHT concentrations and increased production of PSA at high DHT concentrations. PMID- 7530652 TI - Sex difference in coexpression by galanin neurons accounts for sexual dimorphism of vasopressin in the bed nucleus of the stria terminalis. AB - Vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) are steroid sensitive and sexually dimorphic. The number of VP messenger RNA (mRNA) expressing neurons is larger in male than in female rats. This initial observation suggested that sexual dimorphism resulted from enhanced proliferation and/or survival of VP neurons after gonadal hormone exposure during the critical perinatal period. However, galanin (GAL) and VP mRNAs were recently reported to be coexpressed in the BNST of adult male rats, and GAL gene expression, unlike VP gene expression, is not sexually dimorphic. These findings are consistent with the hypothesis that the sex difference in VP cell number in the BNST results from a sex difference in the number of GAL neurons dedicated to express the VP gene. To test this hypothesis, double in situ hybridization histochemistry was performed for GAL and VP mRNAs in the BNST of adult male and female rats. For quantification, the posterior BNST was divided into its two anatomical regions: medial (BSTM) and lateral (BSTL) divisions. Extending previous results for the whole BNST, the number of GAL-expressing cells in either the BSTM or the BSTL was not sexually dimorphic. A significant sex difference was found in the number of GAL cells coexpressing VP in the BSTM (mean +/- SE, male, 124 +/- 8; female, 56 +/- 6; P < or = 0.0001), but not in the BSTL (male, 80 +/- 9; female, 83 +/- 15). Accordingly, the number of cells expressing GAL mRNA only was significantly lower (P < or = 0.002) in the BSTM of male (43 +/- 5) than in female (85 +/- 9) rats. Evidence is provided that the reduced incidence of coexpression of VP by GAL neurons in the BSTM of female rats may account for the reported sex difference in VP cell number in the entire BNST. The results suggest that gonadal hormones in the perinatal period may not influence the proliferation and/or survival of VP neurons in the BNST per se but influence, instead, the capacity of GAL neurons to synthesize VP. PMID- 7530655 TI - A re-examination of basal metabolic rate predictive equations: the importance of geographic origin of subjects in sample selection. AB - OBJECTIVE: The study of geographic differences in basal metabolic rate (BMR). DESIGN: A retrospective analysis of two large BMR databases, namely those of Schofield et al. (1985; Hum. Nutr.: Clin. Nutr. 39C, Suppl. 1, 5-41) and Henry and Rees (1988; Comparative nutrition, pp. 149-159). SUBJECTS: The pooled database from the above sources comprised 7737 individual measurements. The largest sub-set of males and females were the 18-30-year-olds (n = 2999 and n = 874 respectively), this group was selected for analysis. INTERVENTION: Least squares linear regression equations were derived for groups of subjects according to the most common geographic regions represented in the data (North Europeans and Americans, Italians, Indians and Chinese). The method of Rees & Henry (1988) was used to compare these equations over a range of body weights. RESULTS: Regression equations for Italian males and females were significantly greater than other groups over almost the entire body weight range tested. CONCLUSIONS: Italian subjects comprise 45% of the Schofield et al. (1985) database which may therefore be biased and in need of reassessment. PMID- 7530654 TI - Influence of obstacles on lipid lateral diffusion: computer simulation of FRAP experiments and application to proteoliposomes and biomembranes. AB - Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach in which a membrane lipid leaflet was mimicked using a triangular lattice obstructed with randomly distributed immobile and non overlapping circular obstacles. Influence of the radius r and area fraction c of these obstacles and of the radius R of the observation area on the relative diffusion coefficient D* (Eq. (1)) and mobile fraction M was analyzed. A phenomenological equation relating D* to r and c was established. Fitting this equation to the FRAP data we obtained with the probe NBD-PC embedded in bacteriorhodopsin/egg-PC multilayers suggests that this transmembrane protein rigidifies the surrounding lipid phase over a distance of about 18 A (approximately equal to two lipid layers) from the protein surface. In contrast, analysis of published diffusion constants obtained for lipids in the presence of gramicidin suggests that in terms of lateral diffusion, this relatively small polypeptide does not significantly affect the surrounding lipid phase. With respect to the mobile fraction M, and for point obstacles above the percolation threshold, an increase in R led to a decrease in M which can be associated with the existence of closed domains whose average size and diffusion properties can be determined. Adaptation of this model to the re-interpretation of the FRAP data obtained by Yechiel and Edidin (J Cell Biol (1987) 115:755-760) for the plasma membrane of human fibroblasts consistently leads to the suggestion that the lateral organization of this membrane would be of the confined type, with closed lipid domains of approximately equal to 0.5 microns 2 in area. PMID- 7530656 TI - Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL. AB - Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530659 TI - Reduction of cerebral blood flow with induced tachycardia in rats and in patients with coronary artery disease and premature ventricular contractions. AB - A reduction of cerebral blood flow (CBF) was observed in experimental studies in rats immediately after the onset of parasystolic rhythm or with stable, but haemodynamically compromising, tachycardias. Based on these data and with a view to studying the effects of premature ventricular contractions (PVCs) on the cerebral circulation in humans, CBF was measured using the 133-Xenon inhalation method in 24 age matched human controls (group A1: age 58.5 +/- 6.2 years; group A2: 52.2 +/- 7.8 years) in nine coronary artery disease (CAD) patients without PVCs (B), in 11 CAD patients with frequent PVCs (> 300.h-1) (C) and in nine patients, after exclusion of CAD by angiography, also with frequent PVCs (> 300.h 1) (D). Holter monitoring was performed during the CBF measurement. CBF determined in the human control groups A1 and A2 was 79.9 +/- 9.9 ml.100 g.-1 min 1 and 81.5-13.0 ml. 100 g-1 min-1, respectively. CBF was 74.1 +/- 13.6 ml . 100 g.-1 min-1 (P = 0.267 vs A1) in group B, 65.8 +/- 11.8 ml.100 g-1 min-1 (P = 0.004 vs A1) in group C and 74.2 +/- 15.6 ml.100 g.-1 min-1 (P = 0.218 vs A2) in group D. The significant reduction of CBF in CAD patients with frequent PVCs suggests that arrhythmias have a significant impact on CBF. Non-CAD patients with frequent PVCs did not show significant CBF decreases in comparison with controls. One can hypothetize that an impairment of electrical postextrasystolic potentiation, due to premature ventricular depolarization, and hence myocardial dysfunction leads to CBF reduction in CAD patients. The CBF reduction with CAD could also reflect concomitant coronary and cerebral arteriosclerosis. PMID- 7530657 TI - An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms. AB - The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony-stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a. PMID- 7530658 TI - Post-poly(Glu) cleavage and degradation modified by O-sulfated tyrosine: a novel post-translational processing mechanism. AB - Expression of bioactive peptides requires several modifications of the primary translation product. Gastrin, a vertebrate gut hormone, occurs in multiple forms, including a bioactive fragment of the predominant gastrin-17. Gastrin-17 is, however, without known cleavage sites. In order to identify the new site, we therefore isolated, from antral mucosa, fragments of gastrin-34 and -17 monitored by monospecific immunoassays. After three steps of reverse-phase chromatography, the short gastrins were identified as hepta-, hexa- and pentapeptide amides. By far the most abundant of these was tyrosine O-sulfated gastrin-6. The near complete sulfation contrasts with the larger gastrins, of which only half are sulfated. The longest N-terminal fragment of gastrin-34 was a hexadecapeptide without complementarity to the short gastrins. Instead, the predominant N terminal fragment of gastrin-17 was the decapeptide complementary to gastrin-7. Therefore the novel processing site is the Glu10-Ala11 bond that follows a poly(Glu6-10) sequence. Moreover, gastrin-7 is apparently trimmed, with subsequent accumulation of sulfated gastrin-6. Consequently, O-sulfated tyrosine ensures production of a new hormone which stimulates gastric acid secretion as potently as gastrin-17. PMID- 7530660 TI - Cardiac arrhythmia and ischaemic events after combination chemotherapy for testicular cancer. AB - The aim of the present investigation was to evaluate the type and the incidence of cardiac arrhythmias and ischaemic events in patients suffering from testicular cancer and submitted to combination chemotherapy with cisplatin, bleomycin and vinblastine (PVB) or etoposide (PEB). Forty-seven patients took part in the study; 23 were treated with PVB and 24 with PEB. Holter monitoring was performed in each patient before chemotherapy and on the 1st, 2nd and 5th day of the first cycle of drug administration. The results showed that combination chemotherapy with PVB or PEB was accompanied by the appearance of, or an increase in, the incidence of supraventricular ectopic beats. No significant difference was found between the two groups. No significant conduction disturbances were recorded. These results show that combination chemotherapy with PVB or PEB, at least during the first cycle, has no significant ventricular arrhythmogenic or ischaemic potency in young people with no history of cardiac disease. PMID- 7530661 TI - The effects of intracoronary substance P and acetylcholine on coronary blood flow in patients with idiopathic dilated cardiomyopathy. AB - Attenuation of the increase in blood flow caused by acetylcholine in the peripheral vasculature and coronary circulation of patients with heart failure has been interpreted as an impairment of endothelium-dependent vasodilation. The aim of this study was to compare in man the effects of acetylcholine, which also has endothelium-independent actions, with substance P, which appears to be a pure endothelium-dependent vasodilator, on epicardial and resistance coronary arteries in patients with idiopathic dilated cardiomyopathy. The effects of intracoronary acetylcholine (10(-7) M and 10(-6) M) and substance P (5, 10 and 25 pmol.min-1) on epicardial coronary artery diameter and coronary blood flow velocity were measured with an intracoronary Doppler flow probe and quantitative coronary angiography in 11 patients with idiopathic dilated cardiomyopathy and 10 control subjects. Epicardial coronary artery diameter did not change with acetylcholine but increased significantly with substance P in both groups (cardiomyopathy patients: 3.3 +/- 0.2 mm (mean +/- SEM) at baseline vs 3.9 +/- 0.2 mm with substance P25 pmol.min-1, P < 0.01; controls: 3.1 +/- 0.2 mm at baseline vs 3.9 +/- 0.3 mm with substance P25 pmol.min-1, P < 0.05). Coronary flow ratios with acetylcholine were lower in cardiomyopathy patients (10(-7) M: 1.4 +/- 0.1 vs 2.3 +/- 0.4, P = 0.05; 10(-6) M: 1.8 +/- 0.2 vs 3.2 +/- 0.5, P = 0.05 vs controls).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530662 TI - Effects of heterodimeric isoform of platelet-derived growth factor PDGF-AB on wound healing in the rat. AB - Earlier we reported a positive effect of platelet-derived growth factor (PDGF-BB) on rat wound healing while corresponding doses of PDGF-AA were not as effective. The present work was undertaken to study the effects of the heterodimeric form of PDGF (PDGF-AB) on wound healing in rats. Subcutaneously implanted hollow cylindrical cellulose sponges served as an inductive matrix for the granulation tissue. Fifty microliters of solutions containing 0, 5, 50 or 500 ng of PDGF-AB heterodimer were injected daily into the sponges. The analyses of the granulation tissue in the sponge cylinders were carried out 7 days after implantation. Injections of 500 ng of PDGF-AB stimulated significantly the accumulation of collagen and proteins, indicated by the elevated hydroxyproline and nitrogen content of the sponge (+51%, p < 0.01 and +48%, p < 0.001, respectively). Similarly, the amounts of DNA, RNA, hexosamines and uronic acids were significantly higher, reflecting a PDGF-AB-induced increase in the cellularity and glycosaminoglycans. Analyses of wound fluid showed a marked decrease in the number of polymorphonuclear leukocytes in response to PDGF-AB treatment. Injections of a lower dose, 50 ng, of PDGF-AB increased significantly the mean amounts of nitrogen, RNA and uronic acids but no significant changes were observed in other parameters. The lowest dose of PDGF-AB, 5 ng daily, induced no essential effect. In vitro PDGF-AB increased the number of rat granulation tissue derived fibroblasts and collagen accumulation per culture dish at concentrations of 10 and 30 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530663 TI - Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. AB - The purpose of this study was to determine whether epidermal growth factor (EGF), EGF receptor, transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), acidic fibroblast growth factor (acidic-FGF), basic fibroblast growth factor (basic-FGF), and interleukin-1-alpha (IL-1-alpha) proteins were present in cultures of human corneal cells and/or in sections of human corneal tissue. Immunohistochemistry was performed on human corneal sections. Immunofluorescent cell staining was used to evaluate corneal epithelial, stromal fibroblast, and endothelial cells in primary culture. Basic FGF production was evaluated in culture cells using immunoprecipitation. EGF, TGF alpha, TGF-beta-1, and IL-1-alpha were detected by immunohistochemistry in cells in all three layers of the cornea. EGF receptor and acidic FGF were detected by immunohistochemistry in epithelial and endothelial cells, but not in stromal fibroblast cells. Differences in distribution of the growth factors were noted within individual layers of the cornea. EGF and basic-FGF proteins were detected in all three predominant cell types of the cornea using immunocytology. IL-1 alpha protein was detected by immunocytology in corneal epithelial and endothelial cells, but not stromal fibroblasts. Immunoprecipitation confirmed the production of basic-FGF in all three cell types. IL-1-alpha protein detection in the corneal stroma by immunohistology, but not by immunocytology in first passage stromal fibroblasts, suggests that IL-1-alpha may localize to the corneal stroma after production by corneal epithelial and/or endothelial cells. PMID- 7530664 TI - Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture. AB - Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immuno-cytochemically in interferon-gamma lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-gamma or or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in gamma interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but not unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-gamma or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamma and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-gamma, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-beta was present in the culture medium, gamma-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-gamma synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta. PMID- 7530665 TI - Novel assay for the influenza virus M2 channel activity. AB - The influenza A M2 ion channel was expressed and activity characterized in Xenopus oocytes. Based on the activation properties of the channels, a high throughput, non-electrophysiological screening assay was developed in order to identify novel inhibitors of the channel. This will facilitate discovery of novel agents to treat influenza viral infections. PMID- 7530666 TI - Increased attenuation in odontogenic keratocysts with computed tomography: a new finding. AB - An increased attenuation area (IAA) is sometimes seen in the cystic cavity of odontogenic keratocysts (OKCs) on CT scans. The significance of IAA was compared radiologically and histologically in 26 cysts in which a provisional diagnosis of OKC had been made. First, the presence of IAA in the cystic cavity was assessed. Then, relationship between the presence of IAA and data obtained from the cysts, including the CT and histological findings and the visual appearance of the cyst contents, was evaluated. An IAA was frequently seen in large multilocular cysts. There was no relationship between the presence of IAA and aggressive features of the cyst on CT or the cyst contents. Histologically, subepithelial inflammation was often observed in the cysts with IAA. In order to ascertain whether the IAA was due to the keratin mass, a CT scan of a bundle of hair in a water bath was performed and shown to have a similar density. Our study demonstrated that IAA in cystic cavities results from desquamated keratin. Since this seems to occur in long-standing or inflamed multilocular OKCs, it could be used as a significant finding in the diagnosis of OKC. PMID- 7530667 TI - Effect of 5-azacytidine administration during very early pregnancy. AB - The chemotherapeutic agent 5-azacytidine (5AZ) is cytotoxic via nucleic acid hypomethylation. Malformations and embryolethality result when rats or mice are exposed to 5AZ on any of Days 9 through 12 of pregnancy. To investigate the effect of 5AZ exposure during the pre- and early postimplantation period, we administered 0.15, 0.30, 0.60, or 1.2 mg/kg 5AZ/day to rats during Days 1-8 of pregnancy and evaluated outcome on Days 9 or 20. No adverse effects were detected on Day 9; the numbers and weights of implantation sites, the numbers of resorptions, maternal body weight gains, and hormone measures were not different from those of controls. However, when pregnancy outcome was evaluated on Day 20, dose-dependent decreases in offspring survival and fetal weight were observed and the incidences of two malformations, microphthalmia and exencephaly, were increased. In a follow-up study, 5AZ was administered during the preimplantation period (Days 1-3) or during the postimplantation period (Days 4-8) and pregnancy outcome was evaluated on Day 20. When rats were exposed to 5AZ during the preimplantation period, no adverse effects were seen. Postimplantation dosing produced an increase in resorptions and a decrease in fetal survival and fetal weight, with no gross external malformations evident. At the doses used in this study, 5AZ was embryotoxic, with limited embryonic vulnerability prior to Day 4 of pregnancy but serious consequences following postimplantation exposure. This embryotoxicity is not detectable by our measures on Day 9 but is evident on Day 20. PMID- 7530668 TI - The effect of dideoxycytidine on lymphocyte subpopulations in nonhuman primates. AB - In the present study, 2',3'-dideoxycytidine (ddC), which has antiretroviral activity, was given chronically to uninfected nonhuman primates to determine whether it produces adverse immunological or hematological effects. Nine healthy adult male rhesus monkeys were divided into three groups and given the following doses of ddC in a gelatin vehicle: group A, 0.06, 6.0, 3.0, and 1.5 mg/kg; group B, 0.6 mg/kg; group C, 0 mg/kg. Blood samples were collected for hematologic analysis and flow cytometric analyses of lymphocyte subpopulations. Chronic ddC exposure did not cause significant changes in the number of red blood cells, monocytes, or reticulocytes. The number of white blood cells and neutrophils increased and these changes were observed only in group A animals at the 1.5 mg/kg dose. The most significant alterations observed were decreases in the number of T helper cells (CD4) and B cells (CD20). CD4+ and CD20+ lymphocytes exhibited dose-related shifts that were reversible over time and after drug withdrawal. The results indicate that ddC has few hematologic effects but it does have profound but transient effects on the number of cells in lymphocyte subpopulations in normal primates. PMID- 7530669 TI - Localization of nitric oxide synthase in the brain stem neural circuit controlling esophageal peristalsis in rats. AB - BACKGROUND/AIMS: The central subnucleus of the nucleus of the solitary tract has been implicated in central reflex control of esophageal peristalsis. This study determined the presence of nitric oxide synthase in the brain stem circuit controlling esophageal peristalsis by combining transsynaptic retrograde tract tracing with pseudorabies virus and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH) histochemistry. METHODS: Virus was injected into the esophagus of 10 of 15 rats. After 60-63 hours, brain sections were processed for viral immunofluorescence and NADPH histochemistry. RESULTS: Fluorescent neuronal labeling was limited to the compact formation of the nucleus ambiguus and the central subnucleus of the nucleus of the solitary tract. Most fluorescence labeled neurons in the central subnucleus stained positively for NADPH (double labeled). In the compact formation, there were almost no double-labeled neurons; however, NADPH-stained terminals surrounded fluorescence-labeled motoneurons. CONCLUSIONS: NO synthase is present in premotor neurons of the central subnucleus of the nucleus of the solitary tract that innervate esophageal motoneurons in the compact formation of the nucleus ambiguus. NADPH staining in both somata and terminals of esophageal premotor neurons suggests that NO is involved in neurotransmission in the central subnucleus and at the site of synaptic contact between esophageal premotor neurons and motoneurons in the compact formation of the nucleus ambiguus. PMID- 7530670 TI - Role of tumor necrosis factor alpha release and leukocyte margination in indomethacin-induced gastric injury in rats. AB - BACKGROUND/AIMS: Several studies have shown that polymorphonuclear neutrophil leukocyte (PMN) margination is an early and critical event in the pathogenesis of gastric mucosal injury caused by nonsteroidal anti-inflammatory drugs. Tumor necrosis factor (TNF) alpha is a proinflammatory cytokine that causes PMN margination by up-regulating expression of adhesion molecules on both PMN and endothelial cells. This study investigated whether substances that modulate TNF synthesis and release influence PMN margination and indomethacin-induced gastric damage. METHODS: Rats were treated with several doses of indomethacin alone or in association with substances known to increase (interleukin 2 and lipopolysaccharide) or inhibit (pentoxifylline, dexamethasone, granulocyte colony stimulating factor [G-CSF]) TNF synthesis and release. RESULTS: Indomethacin administration caused dose-dependent damage and increased PMN margination and plasma TNF concentrations. Pretreatment with interleukin 2 and lipopolysaccharide significantly increased TNF release, PMN margination, and gastric mucosal damage, but administration of dexamethasone, pentoxifylline, and G-CSF provided almost total protection. The administration of G-CSF alone caused a significant increase in gastric PMN margination but protected against the indomethacin-induced gastropathy. CONCLUSIONS: Agents that regulate TNF synthesis and release influence gastric susceptibility to indomethacin by modulating PMN margination. G CSF increased PMN infiltration but protected against the mucosal injury, suggesting that PMN margination alone is not sufficient to induce mucosal damage. PMID- 7530671 TI - Conformational epitopes on CYP2D6 are recognized by liver/kidney microsomal antibodies. AB - BACKGROUND/AIMS: Four linear antigenic sites have been shown on the CYP2D6 molecule that are recognized by serum positive for liver/kidney microsomal antibody (LKM) type 1. The aim of this study was to search for antibodies against CYP2D6 conformational antigenic sites in LKM-1-positive sera. METHODS: The capacity of four LKM-1-positive sera, before and after absorption with synthetic peptides representing CYP2D6 linear antigenic sites, and rabbit sera against linear antigenic sites between CYP2D6 amino acids 254-271 and 373-389 to inhibit the O-demethylation of dextromethorphan by CYP2D6 was tested in vitro. RESULTS: Inhibition of O-demethylation of dextromethorphan was not modified by absorption of antibodies against linear CYP2D6 antigenic sites. In addition, rabbit sera against two of these sites did not inhibit the reaction. These results strongly suggest that antibodies against CYP2D6 conformational antigenic sites were present in LKM-1-positive sera. CONCLUSIONS: The autoimmune response against CYP2D6 is directed against linear and conformational antigenic sites. These results strengthen the argument that the LKM-1 response is polyclonal and antigen driven. PMID- 7530672 TI - Correction of the cystic fibrosis defect by gene complementation in human intrahepatic biliary epithelial cell lines. AB - BACKGROUND/AIMS: Hepatobiliary disease is the second most common cause of mortality in patients with cystic fibrosis (CF). In the liver, only the intrahepatic biliary epithelial (IBE) cells express cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine whether human CF-derived IBE cells can be infected with adenovirus and the CF phenotype complemented. METHODS: IBE cells were isolated from 2 patients with CF and immortalized using retrovirus transduction of SV40 large T antigen. Immortalized cells were infected with the adenovirus vector Ad2/CFTR2 and assayed 2-31 days postinfection for cyclic adenosine monophosphate (cAMP)-induced halide efflux. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe 6-methoxy-N-(3-sulfopropyl)-quinolinium. RESULTS: CF derived IBE cell lines express biliary specific markers and express no cAMP inducible halide efflux. Following infection with the adenovirus vector Ad2/CFTR2, a cAMP-induced halide efflux was observed for 31 days, although the number of responsive cells decreased with time. CONCLUSIONS: Human CF-IBE cells can be infected by adenovirus and the defective CFTR complemented. The loss of responsive cells with time could be due to loss of construct and/or a reduced growth of cells that are overexpressing CFTR. These CF-IBE cell lines offer an opportunity to determine the mechanisms responsible for hepatobiliary disease in the patients with CF. PMID- 7530673 TI - The role of leukotriene C4 in biphasic release of histamine from mouse mastocytoma cell line (P-815). AB - 1. A biphasic change of compound 48/80-induced histamine release was observed in mastocytoma cell line (P-815). 2. The first and second phase of compound 48/80 induced histamine release were elicited via a different pathway. 3. The second phase of compound 48/80-induced histamine release was provoked with leukotriene (LT) C4. 4. The results suggest that this biphasic release may be one of the important regulatory mechanisms for the production of histamine. PMID- 7530674 TI - Epitope-tag vectors for eukaryotic protein production. AB - We report on the construction and use of two eukaryotic expression vectors which add well-characterized epitope tags to the N termini of proteins. The utility of these vectors is demonstrated for detecting the expression of a variety of proteins. As the addition of these epitope tags can in some cases obviate the need to generate specific antisera to each individual protein, these vectors provide a facile means both to monitor protein expression and to purify such expressed proteins. PMID- 7530675 TI - [Seroprevalence of hepatitis C virus antibodies in obstetric patients at the Nuevo Hospital Civil de Guadalajara]. AB - The hepatitis C virus is the predominant cause of parental transmission of hepatitis. The importance to recognize the prevalence of this virus in obstetrics patients is the possible vertical transmission of mother to child which has been reported by various authors. The objective of this investigation was to determine the presence of antibodies of the hepatitis C virus in the obstetrical population of the Nuevo Hospital Civil de Guadalajara. Selection was made at random; questionnaires were applied to identify factors of associated risks. Of the 244 patients studied, the prevalence was found in 2% (n = 5). The only risk factor closely related was previous blood transfusion (P < 0.00004964) with a relative risk of 29.6 for the obstetrics population with previous transfusion. PMID- 7530676 TI - Prognostic significance of serum fragments of cytokeratin 19 measured by Cyfra 21 1 in cervical cancer. AB - Pretreatment sera of 78 patients with squamous cell cervical cancer were tested for the presence of cytokeratin 19 (CK 19) fragments to determine the relationship among this parameter, tumor stage, various histopathologic characteristics, and prognosis. For the quantitative determination of CK 19 fragments in serum, the enzyme assay Cyfra 21-1 was used. This assay, based on the simultaneous sandwich principle, utilizes two different monoclonal antibodies. The test was considered positive when levels of Cyfra 21-1 were > or = 1.2 micrograms/liter. Cyfra 21-1 was positive in the majority of patients and in all patients with advanced disease (FIGO III or IVa). A highly significant relationship was found between pretreatment Cyfra 21-1 level and FIGO stage (P < 0.0001). Mean Cyfra 21-1 concentration was elevated in the case of macroinvasive disease (FIGO Ib, IIa, IIb, III, IVa). A distinct relationship was found between tumor size (P < 0.001; r = 0.73) and Cyfra 21-1 level. In the univariate Cox analysis Cyfra 21-1 level was significantly related to both disease-free interval (P < 0.0001) and survival (P < 0.0001) of patients. Patients with an increased Cyfra 21-1 level had a significantly worse prognosis. However, in the stepwise Cox regression analysis, these variables had no additional value over known prognostic factors such as FIGO stage and tumor size. It is concluded that Cyfra 21-1 may be of significance as an additional marker in the management of patients with cervical cancer, but further investigation is needed. PMID- 7530679 TI - Quality of life in progressive ovarian cancer. AB - When ovarian cancer progresses, goals change from cure to prolongation of life with the best possible quality for the patient. Criteria for futility must be established to guide the transition from active to palliative management. Pain control can be achieved by following established WHO guidelines. Continued education of the medical community, legislators, and the public is needed to assure pain control for the cancer patient. Limited surgery or radiation can be used to control symptoms from locally progressive disease. Other symptoms to be actively controlled include nausea and vomiting, nutrition, hydration, and fatigue. Support services, including home services, psychological counseling, and nutritional support need funding for both home and hospital settings. Quality of life assessment must be as specific as possible and address the patient's concerns by self-assessment techniques. Funding must be provided to develop specific quality of life tools and to then apply them clinically, both as part of research protocols and to assess success of palliative care. PMID- 7530678 TI - Endodermal sinus tumor arising from a dysgenetic gonad in a 46,XX female combined with mullerian agenesis. AB - The occurrence of neoplastic transformation in a dysgenetic gonad of a female with 46,XX karyotype is very rare. Moreover, to our knowledge, a case of endodermal sinus tumor arising from that setting combined with Mullerian agenesis has not been reported. This case suggests the possibility of development of cancer in a dysgenetic ovary of a female even with 46,XX or Mullerian agenesis. PMID- 7530677 TI - Chemoprophylaxis with oral ciprofloxacin in ovarian cancer patients receiving taxol. AB - The purpose of this study was to review the clinical outcomes and cost of administration of a prophylactic antibiotic compared to G-CSF for the prevention of neutropenic morbidity associated with taxol. The study group was composed of 62 patients with ovarian cancer who received a 24-h infusion of a taxol-based regimen at doses less than or equal to 175 mg/m2 between June 1992 and April 1994. The records were retrospectively reviewed and the patients were grouped and analyzed according to the management of their myelosuppression. Group I patients (n = 29) were observed until their absolute neutrophil count (ANC) was less than 500/microliters and then were placed on ciprofloxacin 500 mg orally twice a day until their ANC was 1,000/microliters. Group II patients (n = 15) received G-CSF from Day 2 until the ANC was greater than 10,000/microliters beginning with their first cycle. Group III patients (n = 18) received their taxol regimen without either ciprofloxacin or G-CSF. Two hundred eighty-two taxol-based chemotherapy cycles were administered to these 62 patients. There was no statistically significant difference between the groups concerning disease status as measured by age, stage, performance status, dose intensity, or number of previous regimens. There were two episodes of febrile neutropenia in Group I and three episodes in Group II. Group III had 15 episodes of febrile neutropenia. The estimated cost of the different prophylactic regimens was $5,215.00 for Group I versus $104,000.00 for G-CSF in Group II. Within the three groups, there were 27 patients with an episode of febrile neutropenia (n = 20) or prolonged myelosuppression (n = 7) that were followed for an additional 104 taxol cycles. Twenty-four of these patients received G-CSF prophylaxis with intermittent ciprofloxacin and three received only ciprofloxacin. There were eight more episodes of febrile neutropenia in the patients receiving G-CSF. There were no additional febrile episodes on cycles prophylaxed with ciprofloxacin. There was no septic mortality. For patients receiving a 24 h infusion of taxol at doses less than 175 mg/m2, ciprofloxacin given through the ANC nadir may be effective in preventing febrile morbidity. A prospective randomized trial is underway to evaluate this approach. PMID- 7530680 TI - Management of early ovarian cancer: germ cell and sex cord-stromal tumors. AB - Malignant ovarian germ cell tumors (OGCT) and sex cord-stromal tumors (OSCST), each of which account for less than 5% of all ovarian malignancies, are much less common than epithelial ovarian cancer. In young patients suspected of having an OGCT, laparotomy is initially indicated for both diagnosis and treatment. For most patients, unilateral salpingo-oophorectomy with preservation of the contralateral ovary and the uterus is appropriate. The basis for this surgical approach is retrospective studies that show an equivalent cure rate for patients who undergo unilateral or bilateral adnexectomy. No prospective studies have compared unilateral with bilateral adnexectomy. Surgical staging is also important to determine the extent of disease, to determine prognosis, and to guide postoperative management. If metastatic disease is encountered during initial surgery for OGCT, the same principles of cytoreductive surgery that have been applied to surgically manage advanced epithelial ovarian cancer are recommended, with resection of as much tumor as is technically feasible and safe. For all OGCT patients except those with well-documented stage IA grade 1 pure immature teratoma or stage IA pure dysgerminoma, postoperative chemotherapy is indicated. The current recommended regimen for OGCT is bleomycin, etoposide, and cisplatin--a combination that appears to result in at least a 95% cure rate for stage I disease and at least a 75% cure rate for advanced-stage disease. For patients with metastatic dysgerminoma, chemotherapy, which has the advantage of preserving fertility in the majority of patients, has supplanted radiotherapy as standard treatment. For patients with OSCST, no standard therapy exists. Surgery alone is currently acceptable treatment for all patients with OSCST except those who have metastatic disease or Sertoli-Leydig cell tumors with poor differentiation or heterologous elements. Currently, platinum-based combination chemotherapy is favored for these latter patients, but the activity of such regimens appears only modest. PMID- 7530681 TI - Cutaneous lymphadenoma. PMID- 7530682 TI - Carcinosarcoma of the parotid gland: immunohistochemical study of a case. PMID- 7530683 TI - Risperidone. PMID- 7530684 TI - The expression of CD59 in normal human nervous tissue. AB - The expression of CD59, a complement regulator of the formation and function of the terminal cytolytic membrane attack complex, was studied in human normal nervous tissue by immunohistochemical markers using two monoclonal antibodies 1F5 and MEM43. CD59 was present on Schwann cells, neurons and endothelial cells in the peripheral nervous system (PNS), and on Schwann cells in culture. In the central nervous system (CNS) CD59 was found predominantly on endothelial cells. There was also a diffuse staining of white and grey matter of the spinal cord and brain, presumably of microglia, oligodendrocytes, astrocytes and neurons, as these cells were CD59 positive in culture. Furthermore, CD59 was detected in the cerebrospinal fluid (CSF) of healthy individuals. CD59 in the PNS and CNS was glycosyl-phosphatidylinositol linked and had a molecular weight of 19,000-25,000. The presence of CD59 on various cells of the nervous system and in the CSF suggests that regulation of complement activation by this protein is important in neural host defence mechanisms. PMID- 7530685 TI - Characterization of monoclonal antibodies specific for equine homologues of CD3 and CD5. AB - Two monoclonal antibodies (mAb), UC F6G-3 and UC F13C-5, were characterized as being specific for the apparent equine homologues of CD3 and CD5, respectively. Both antibodies exhibited characteristics of pan-T-lymphocyte markers based upon immunohistology and two-colour flow cytometry. UC F6G-3 precipitated a complex of proteins (up to seven) with molecular weights ranging from 18,000 to 42,000, similar to the human and murine CD3 complex. Upon further dissociation of the precipitated complex, two proteins were identified with molecular weights of 22,000 and 27,000. Immobilized UC F6G-3 was effective at inducing interleukin-2 receptor (IL-2R) expression on T lymphocytes, a feature consistent with antibodies specific for the epsilon chain of human and murine CD3. Three populations of cells in the thymus were distinguishable by UC F6G-3 target antigen density, suggesting increasing stages of T-cell maturation. UC F13C-5 precipitated a 67,000 MW protein, consistent with reported values for CD5 in multiple species. While this antibody exhibited characteristics of a pan-T-cell marker, low numbers of B lymphocytes also expressed the target antigen. Phorbol esters induced variable increases in target antigen density on B lymphocytes. These two antibodies, taken together with the few equine CD markers currently available, represent a substantial resource for further defining the equine immune system in health and disease. PMID- 7530686 TI - Multiple nitric oxide synthase systems in adult rat thymus revealed using NADPH diaphorase histochemistry. AB - Nitric oxide (NO) has become recognized as a multifunctional mediator, with roles in vascular physiology, neurotransmission and non-specific immune defense. The histochemical marker associated with the neural and endothelial form of NO synthase (NOS), reduced nicotinamide adenine dinucleotide diaphorase (NADPHd), has enabled the indirect localization of potential sites of NO production. Innervation of the thymus and its immunological functions made this tissue a candidate for utilization of various NO systems. In the present study on adult rat thymus, multiple cellular sites expressing NADPHd activity, thereby implicated as sites of NOS activity, have been identified using morphological criteria alone: blood vessel endothelium, dendritic cells, deep cortical or medullary stromal cells, intrinsic neuron-like profiles, granulocytes (possibly neutrophils) and fat cells. In addition, the availability to the thymic microenvironment of another form of NOS in macrophages, which is not stained by the diaphorase technique, was supported by the observation of these cells at corticomedullary and cortical locations. These results indicate that a wide variety of possible immunomodulatory roles can be expected for NO in the thymus including the induction of tolerance, major histocompatibility complex (MHC) restriction, lymphocyte trafficking and regulation of thymic endocrine output. PMID- 7530687 TI - The in vivo production of specific human antibodies by vaccination of human-PBL SCID mice. AB - Human-PBL-SCID animals were created by intraperitoneal (i.p.) transfer of human peripheral blood lymphocytes (PBL) or PBL depleted of CD8-expressing lymphocytes (CD8dL). Analysis of human immunoglobulin levels in these animals revealed that severe combined immunodeficiency (SCID) mice receiving CD8dL produced significantly higher levels of serum human immunoglobulin than those receiving PBL. In an attempt to induce antigen-specific human antibodies these human-PBL SCID animals were vaccinated with soluble protein antigen [ovalbumin (OVA)] entrapped within liposomes as an immunological adjuvant. Vaccination produced antigen-specific human IgM and IgG in human-PBL-SCID mouse serum. The use of liposomes as adjuvant and the reconstitution of animals with CD8dL together enhanced the OVA-specific immune response as evidenced by the detection of significantly increased serum antibody titres. In the CD8dL reconstituted group, solid tumours of human B-cell origin became detectable in the peritoneal cavity of animals at 8-10 weeks post-reconstitution. These tumours were readily established in vitro and subsequent analysis of culture supernatants showed that these malignant cells continue to secrete human antibodies specific for the original immunizing antigen in vitro. We believe this vaccination of the human PBL-SCID mouse to produce antigen-specific human antibodies, may find use in the future production of human monoclonal antibodies and in the testing and development of novel vaccine/adjuvant systems. PMID- 7530688 TI - Inhibition of T-cell responses by feeding peptides containing major and cryptic epitopes: studies with the Der p I allergen. AB - H-2b mice respond to the 222 residue allergen Der p I by producing T cells sensitized to the dominant epitopes encompassed in peptides 21-49, 78-100, 110 131 and 197-212. Immunization with the synthetic peptides 120-143 and 144-169, however, revealed cryptic epitopes which could sensitize T cells for responses to the respective peptides and, providing splenic adherent cells were added to lymph node cultures, to the whole allergen. It is shown that feeding recombinant fusion peptides can markedly inhibit the ability of the whole antigen to immunize mice, as measured by the in vitro interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 release on stimulation with protein or peptides, although inhibition measured by IL-2 release was more marked. The inhibition extended to epitopes other than those in the fusion peptides used for feeding. Thus feeding peptide 101-154 inhibited responses to 110-131 and 78-100. Fusion peptides 1-14 and 188-222 did not inhibit responses, although 188-222 did contain an epitope. Inhibition was also obtained when mice were fed a fusion containing the cryptic epitope 144-169. The ability of peptides containing the cryptic epitopes to inhibit responses has significant implications for peptide based immunotherapy. PMID- 7530689 TI - Transcription of HLA-G transgenes commences shortly after implantation during embryonic development in mice. AB - We studied the pattern of transcription of a human HLA-G transgene in mice using polymerase chain reaction (PCR) techniques. Transcription of the HLA-G transgene commenced in cells derived from embryos as soon as 48 hr after implantation of embryos in the uterine wall and continued for at least a further 48 hr during embryonic development. HLA-G transcripts were also present in RNA extracted from thymus, spleen and liver of adult HLA-G transgenic mice, although transcripts were not detected in RNA from any other tissues except testes of male transgenic mice. These results demonstrate that the restricted pattern of HLA-G transcription in embryo-derived trophoblast cells during the first trimester of human pregnancy is reproducible in mice. This suggests that transcription factors required for a highly regulated pattern of gene expression during embryonic development are present in murine trophoblast cells and provide a means to investigate the factors and study the consequences of HLA-G expression during development of the embryo. PMID- 7530690 TI - IL-13 has only a subset of IL-4-like activities on B chronic lymphocytic leukaemia cells. AB - The recently described interleukin-13 (IL-13) has been shown to share many of the effects of IL-4 on normal B cells, including growth-promoting activity and induction of CD23. In this study, we compared the effects of IL-13 and IL-4 on B chronic lymphocytic leukaemias (B-CLL) cells. After anti-CD40 activation, both IL 13 and IL-4 promoted the DNA synthesis of B-CLL cells and increased the recovery of viable cells. The time kinetics of the proliferative response of B-CLL cells to IL-13 or IL-4 were superimposable and showed the long-lasting effect of both cytokines. As on normal B cells, both IL-4 and IL-13 synergized with IL-10 to enhance B-CLL DNA synthesis. Moreover, IL-13, like IL-4, was able to increase CD23 expression on anti-CD40-activated leukaemic B cells. The CD23 up-regulation and the DNA synthesis induced by IL-13 on anti-CD40-activated B-CLL cells, were significantly reduced when B-CLL cells were cultured with anti-IL-4 receptor monoclonal antibody, suggesting a common pathway for IL-13 and IL-4 signalling. However, after cross-linking of surface IgM, IL-4 strongly inhibited the IL-2 induced DNA synthesis of B-CLL cells, whereas IL-13 did not inhibit IL-2-driven proliferation of anti-IgM-activated B-CLL cells. Furthermore, while IL-4 strongly up-regulated the expression of CD23 on anti-IgM-activated leukaemic B cells, IL 13 only marginally increased it. Finally, IL-13, in contrast to IL-4, did not prevent the entry of B-CLL cells into apoptosis. Thus IL-13 and IL-4 display comparable effects on anti-CD40-activated B-CLL cells, which are blocked by anti IL-4 receptor (IL-4R) monoclonal antibodies. However, IL-13-dependent effects are absent or inefficient in non-activated or anti-IgM-activated B-CLL cells. This suggests that such cells may lack functional IL-13 receptors, though IL-13R and IL-4R on B-CLL cells share a common component. PMID- 7530691 TI - Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells. AB - In this report we show that rat retinal pigment epithelial (RPE) cells express an inducible form of nitric oxide synthase (iNOS) and secrete high levels of nitric oxide (NO.) when co-cultured with activated lymphocytes. We have previously shown that cultured rat RPE cells suppress syngeneic lymphocyte proliferation, an effect attributed to prostaglandin E2 (PGE2) secretion by the RPE cells. However supernatants from such co-cultures were also found to contain high levels of nitrite (NO2-), the stable end-product of NO. synthesis. RPE cell secretion of NO. was stimulated by the cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), an effect enhanced by endotoxin [lipopolysaccharide (LPS)], reduced by the competitive inhibitor of L-arginine metabolism, NG-monomethyl-L-arginine (L-NMMA) and inhibited by cycloheximide. These effects were dose dependent. Using reverse transcription (RT)/PCR a product of 1398 bp was amplified which showed sequence identity with iNOS cloned from rat vascular smooth muscle. Northern blot analysis of total RNA extracted from rat RPE before and after cytokine stimulation showed induction of a 4.5 kb (kilobase) transcript which hybridized with a 1398 bp (base pair) polymerase chain reaction (PCR)-generated cDNA probe derived from the sequence of rat RPE cell iNOS. These results indicate RPE cells express an inducible form of nitric oxide synthase (NOS) and that high levels of NO. may be produced locally in the eye by the RPE in the presence of activated lymphocytes. Given the cytostatic and cytotoxic properties of this molecule, NO. may play an important role as an inducible mediator of immunosuppressive mechanisms within the microenvironment of the eye at the site of lymphocyte activation. PMID- 7530693 TI - Effects of irradiation, glucocorticoid and FK506 on cell-surface antigen expression by rat thymocytes: a three-colour flow cytofluorometric analysis. AB - The expression of T-cell antigen receptor (TCR) alpha beta was investigated in rat CD4- CD8- thymocytes during thymic reconstitution after the exposure of animals to irradiation or glucocorticoid. The effect of the immunosuppressant FK506 on the expression of TCR alpha beta in rat CD4- CD8- thymocytes was also examined. The percentage of CD4- CD8- thymocytes constituted 2.6% of total thymocytes and that of CD4- CD8- TCR alpha beta high cells constituted 12.6% of CD4- CD8- thymocytes in normal adult Lewis rats. The percentage of CD4- CD8- TCR alpha beta high cells increased during thymic reconstitution after irradiation, and maximally constituted 28.6% of CD4- CD8- thymocytes on day 7. Similar results were obtained during thymic reconstitution after glucocorticoid treatment. In contrast, continuous treatment with FK506 for 7 days markedly decreased not only the percentages of CD4+ CD8- TCR alpha beta high and CD4- CD8+ TCR alpha beta high thymocytes, but also that of CD4- CD8- TCR alpha beta high thymocytes. These results indicate that rat CD4- CD8- thymocytes contain a subpopulation of mature (TCR alpha beta high) cells. The possible implications of the existence of this subpopulation with regard to thymocyte differentiation and maturation are discussed. PMID- 7530692 TI - Antibodies to distinct epitopes on the CD40 molecule co-operate in stimulation and can be used for the detection of soluble CD40. AB - The B-cell surface protein, CD40, belongs to the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family and plays a crucial role in T cell dependent B-cell activation. Ligation of this receptor with antibodies or its recently defined ligand, gp39, generates an intracellular signal that, when combined with triggering of surface immunoglobulin or the interleukin-4 (IL-4) receptor, induces a variety of stimulatory effects in B cells. In this study we provide further evidence for the importance of receptor cross-linking in generating this signal and we also report on the presence of a soluble form of CD40. A new CD40 monoclonal antibody (mAb), 17:40, was found to synergize with other CD40 antibodies (mAb89 and S2C6) in inducing proliferation as well as IgE synthesis in IL-4-treated tonsillar B cells. However, both this mAb and mAb89 failed to co-operate with a soluble construct of the CD40 ligand, whereas such co operation was seen with the S2C6 antibody. Cross-inhibition experiments showed that the 17:40 mAb recognized an epitope that was clearly distinct from that seen by S2C6 and mAb89. Although directed to separate epitopes, both 17:40 and mAb89 completely blocked binding of gp39 to its receptor, while the S2C6 mAb only partially interfered with this binding. The findings suggest a close relationship between the degree of receptor clustering and the strength of the delivered signal. With the access to antibodies recognizing distinct structures on CD40 we also established a sandwich enzyme-linked immunosorbent assay for quantitative determinations of the antigen. With this assay we could demonstrate the presence of a soluble form of CD40 (sCD40) in culture supernatants. The fact that sCD40 also retained its ligand-binding capacity indicates that it may have an important regulatory role and modulate the T cell-dependent stimulation via CD40. Both the finding of soluble receptors and the need for receptor clustering are features that CD40 share with other members of the TNF/NGF receptor family. PMID- 7530694 TI - The expression of mycobacterial heat shock protein (HSP64) on Meth A tumour cells. AB - Immunological cross-reactivity between Mycobacterium bovis BCG stress protein (heat shock protein: HSP64) and Meth A tumour cells was analysed by using anti BCG HSP64 mAb whose recognition epitopes were characterized against BCG HSP64 peptides. By indirect immunofluorescence analysis (IIFA), it was found that one of seven anti-BCG HSP64 mAb, XVIIIG1, bound to the cell surface of Meth A in BALB/c mice. This result was further confirmed by western blot analysis, demonstrating the presence of a 64 kDa protein which reacted with mAb XVIIIG1 that recognizes the 110-123 amino acid peptide of the BCG HSP64. Comparison of the amino acid sequence between the mouse HSP65 and BCG HSP64 recognized by mAb XVIIIG1 revealed 50% amino acid sequence homology. It was concluded from these results that Meth A tumour cells continuously express the stress protein HSP64 as a kind of tumour-associated antigen on the surface of tumour cells. PMID- 7530695 TI - History of development of oral rehydration therapy. PMID- 7530696 TI - Global results: 15 years of CPITN epidemiology. AB - The general acceptance and use of the CPITN system worldwide has provided, in a very short time, a vast amount of data on periodontal diseases. This data has been entered into the WHO Global Oral Data Bank, from which, after analysis, much valuable information has been derived. Overall the CPITN system is seen as having more than fulfilled the initial hopes invested in it. It has provided a simple yet effective method for measuring and monitoring the magnitude, prevalence and severity of periodontal diseases throughout the world, as well as helping in our understanding of the disease process. PMID- 7530697 TI - Human pharmacokinetics and tolerability of L-697,639, a non-nucleoside HIV-1 reverse transcriptase inhibitor. AB - L-697,639, a potent and selective non-nucleoside inhibitor of HIV-1 reverse transcriptase and HIV-1 replication in vitro, was administered to healthy male volunteers to investigate the pharmacokinetics and tolerability of single and multiple oral doses. Single doses ranging from 25 to 500 mg, and multiple doses of up to 100 mg every 12 h for ten days, produced no clinically important adverse events. Dose proportionality with respect to AUC was seen over the range of 25 100 mg administered as a single dose. Single doses of 200 mg and 500 mg resulted in an increase in AUC and Cmax that was less than proportional to the increase in dose. The mean Cmax after single doses of 25 and 500 mg were 0.9 and 5.8 microM respectively. Mean Tmax values ranged from 1.7-3 h. Mean AUCs (0-48 h) were from 6.05 to 50.3 microM h after doses from 25 to 500 mg respectively. After the 500 mg dose less than 0.7% appeared unchanged in the urine over 48 hours. During multiple doses, steady-state was reached on day 3 and slight accumulation occurred (approximately 1.5-fold). L-697,639 was well tolerated for up to ten days at doses that resulted in mean steady-state trough concentrations that exceed their in-vitro susceptibilities. PMID- 7530698 TI - Language, meaning and the immune system. PMID- 7530699 TI - CD1 expression is not affected by human peptide transporter deficiency. AB - Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP independent peptides. PMID- 7530700 TI - Lymphocyte predominant Hodgkin's disease: a clinicopathologic comparative study of histologic and immunophenotypic subtypes. AB - PURPOSE: To compare the clinicopathologic features of the histologic and immunophenotypic subgroups of lymphocyte predominant Hodgkin's disease. METHODS AND MATERIALS: A retrospective review of 64 patients with lymphocyte predominant Hodgkin's disease treated at the Peter MacCallum Cancer Institute, Melbourne, was performed. Nodular and diffuse histological subtypes were confirmed by review of hematoxylin and eosin paraffin sections. Immunophenotyping with monoclonal antibodies L26 (B-cell origin) and Leu M1 (Hodgkin's phenotype) were available in 36 patients. RESULTS: The estimated freedom from progression and estimated overall survival at 10 years was 74% standard error (SE 5.8%) and 85% (SE 5.2%), non-Hodgkin's respectively. There were no significant differences in freedom from progression or overall survival when nodular and diffuse histology were compared. Similarly the presence of B-cell markers did not influence prognosis. There was only one case of secondary non-Hodgkin's lymphoma. CONCLUSION: Our results are consistent with major reported series displaying no differences between any of the subgroups of lymphocyte predominant Hodgkin's disease. PMID- 7530701 TI - Current status of tissue markers as prognostic factors in prostatic adenocarcinoma. PMID- 7530702 TI - Gleason grade and other prognostic factors--response to Drs. Hammond and Grignon. PMID- 7530703 TI - Reply to editorial by Dr. Earle. PMID- 7530704 TI - 5-HT modulates noncholinergic contraction in guinea pig airways in vitro by prejunctional 5-HT1-like receptor. AB - 5-Hydroxytryptamine (5-HT) has been demonstrated to cause both constriction and relaxation of guinea pig airways, partly through direct action on airway smooth muscle and partly through postganglionic facilitation of cholinergic neurotransmission. We performed an in vitro study to investigate whether 5-HT can modulate the noncholinergic contraction in guinea pig airways due to release of neuropeptides from airway sensory nerves. In the presence of atropine (1 microM), ketanserin (10 microM), and indomethacin (10 microM), 5-HT (0.1-100 microM) produced concentration-dependent inhibition of electrical field stimulation induced noncholinergic contraction with maximal inhibition of approximately 72 +/ 4%. Tropisetron (ICS-205-930, 1 microM), a 5-HT3 and 5-HT4 receptor antagonist, was unable to prevent the inhibition produced by 5-HT. Methiothepin (1-100 nM), a 5-HT1 and 5-HT2 receptor antagonist, produced a concentration-dependent inhibition of the effect of 5-HT (1 microM) with a 50% inhibition concentration value of 66 nM. 5-HT (100 microM) had no effect on the cumulative concentration response relationship to exogenous substance P (10 nM-10 microM). The concentration of agonist causing 35% inhibition of the noncholinergic contraction (EC35) was calculated, and a rank order of potency was established: 5 carboxamidotryptamine (EC35 = 0.24 microM) > 5-HT (EC35 = 0.77 microM) > 8 hydroxy-2-(dipropylamino)tetralin (EC35 = 8.1 microM) > sumatriptan (EC35 = 18 microM). We conclude that 5-HT concentration dependently modulates noncholinergic contraction in guinea pig airways in vitro by a prejunctional mechanism. This effect is probably mediated through a 5-HT1-like receptor; however, the exact subtype remains to be elucidated. PMID- 7530705 TI - Effects of NO synthase inhibition on the muscular blood flow response to treadmill exercise in rats. AB - The functional role of nitric oxide (NO) release in regulating blood flow (BF) to exercising skeletal muscle was studied in conscious male Sprague-Dawley rats (603 +/- 28 g; n = 6). In this study, BF was measured using radiolabeled microspheres during treadmill exercise (10% grade, 20 m/min) before and after NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (30 mg/kg ia). After NOS inhibition, mean arterial blood pressure increased from resting baseline values and the duration of vasodilator responses to acetylcholine (ACh) injections (3.0 and 10.0 micrograms/kg ia) was diminished (P < 0.05), demonstrating reduced NOS function. During exercise, BF to the kidneys and organs of the gut was reduced after NOS inhibition. In addition, BF was reduced in 16 of the 28 individual hindquarter muscles or muscle parts. Moreover these reductions in BF were linearly correlated with the estimated sum of the percentage of fast-twitch oxidative glycolytic (FOG) and slow-twitch oxidative (SO) types of fibers found in each muscle [delta BF = -1.1 (%SO + %FOG) + 16.4; r = 0.88, P < 0.001]. These results suggest that NO-mediated vasodilation contributes to the BF responses within and among the muscles of the rat's hindquarters during exercise. PMID- 7530706 TI - Effects of inhaled substance P on airway responsiveness to methacholine in asthmatic subjects in vivo. AB - We tested the hypothesis that the inhaled tachykinin substance P (SP) can induce hyperresponsiveness to methacholine in asthmatic subjects in vivo. Nine atopic nonsmoking asthmatic males with normal forced expiratory volume in 1 s (FEV1; > 80% predicted) and increased methacholine sensitivity [provocative concn causing 20% fall in FEV1 (PC20) < 8 mg/ml] participated in a two-period placebo controlled crossover study. Dose-response curves to SP (0.25-8 mg/ml) and placebo were recorded on 2 randomized days at least 1 wk apart, and methacholine tests were done 24 h before and 2 and 24 h after these challenges. The responses were measured by FEV1 (%fall from baseline). The position of the methacholine dose response curves was expressed by the PC20 FEV1 and by the maximal response by the plateau level (MFEV1). SP caused a dose-dependent fall in FEV1 (P < 0.001). There was a slight increase in the PC20 FEV1 at 2 and 24 h, which was not significantly different between placebo and SP. Similarly, there was a reduction in MFEV1 at 2 h after both pretreatments. However, at 24 h after SP inhalation, MFEV1 increased compared with placebo. These changes in MFEV1 were significantly different between SP and placebo by 5.2 +/- 2.2% fall (SE) (P < 0.05). We conclude that 1) a bronchoconstrictive dose of SP, compared with placebo, enhances maximal airway narrowing to methacholine in asthma 24 h after inhalation and 2) tolerance develops to high doses of inhaled methacholine. These findings are suggestive of a role of SP in causing excessive airway narrowing in asthma by inflammatory mechanisms. PMID- 7530707 TI - Compared with crystalloid, colloid therapy slows progression of extrapulmonary tissue injury in septic sheep. AB - We tested the hypothesis that the type of fluid infused to chronically maintain intravascular volumes would modify both microvascular integrity and cellular structure in extrapulmonary organs in hyperdynamic sepsis. After cecal ligation and perforation, awake sheep were treated for 48 h with 10% pentastarch (n = 9), 10% pentafraction (Du Pont Critical Care; n = 8), or Ringer lactate (n = 8) titrated to maintain a constant left atrial pressure. After 48 h of fluid therapy, biopsy samples were taken from the left ventricle and gastrocnemius for electron microscopy. At this time, all groups demonstrated a similar hyperdynamic circulatory response, increased systemic O2 utilization and organ blood flows, measured by radioactive microsphere injection. However, greater capillary luminal areas with less endothelial swelling and less parenchymal injury were found in septic sheep treated with pentastarch vs. Ringer lactate infusion in both muscle types. Pentafraction showed few benefits in study end points over pentastarch. Thus, we conclude that chronic intravascular volume resuscitation of hyperdynamic sepsis with pentastarch ameliorated the progression of both microvascular and parenchymal injury. These findings indicate that microvascular surface area for tissue O2 exchange in sepsis may be better preserved with chronically infused colloid, resulting in less parenchymal injury. PMID- 7530708 TI - Sidestream cigarette smoke exposure and airway reactivity during early life. AB - We established a guinea pig model to investigate effects of in utero and neonatal exposure to sidestream cigarette smoke (SSCS) on bronchial reactivity during early life. Animals were divided into four groups: 1) room air/room air, 2) sham/sham, 3) SSCS/room air, and 4) SSCS/SSCS. Pregnant and neonatal animals of group 1 breathed room air and those of group 2 were sham treated. Pregnant animals of both groups 3 and 4 as well as neonates of group 4 were exposed to SSCS. SSCS exposure was limited to between days 28 and 55 of pregnancy and days 8 and 24 of the neonatal period. Bronchial response to acetylcholine (ACh) and substance P (SP) were determined in very young animals at 25 days of age. Maximal expiratory flow was used as an index of airway dimension. SP, but not ACh, induced a significantly larger decrease in peak maximal expiratory flow in group 4, indicating an important role of neonatal SSCS exposure in augmenting bronchial response to SP. To further investigate the role of tachykinins in cigarette smoke induced changes in bronchial reactivity, four additional groups (the same as above) of neonates were pretreated with capsaicin to deplete tachykinins. In the SSCS/SSCS group, SP-induced airway hyperreactivity was abolished by capsaicin pretreatment. Furthermore, in all four groups, capsaicin pretreatment abolished the bronchial response to SP but not the response to ACh. In additional very young animals, acute SSCS caused a nonsignificant increase in bronchial response to SP. These results indicate that chronic neonatal SSCS exposure induces bronchial hyperreactivity to SP; this hyperreactivity is abolished by capsaicin pretreatment. PMID- 7530710 TI - The groESL operon of Agrobacterium tumefaciens: evidence for heat shock-dependent mRNA cleavage. AB - The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cleavage product appeared to be unstable and subjected to degradation, while the groEL cleavage product appeared to be stable and became the major mRNA representing the groESL operon after long periods of growth at a high temperature. The polycistronic mRNA containing the groES and groEL genes was the major mRNA representing the groESL operon at a low temperature, and it reappeared when the cells were returned to the lower growth temperature after heat shock induction. These findings indicate that the cleavage event is part of the heat shock regulation of the groESL operon in A. tumefaciens. PMID- 7530709 TI - Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS. AB - The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxylation of 2 halobenzoates with concomitant release of halogenide and carbon dioxide, yielding catechol. The gene cluster encoding this enzyme, cbdABC, was localized on a 70 kbp conjugative plasmid designated pBAH1. The nucleotide sequences of cbdABC and flanking regions were determined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bind the Rieske-type [2Fe-2S] cluster was identified. In the NADH:acceptor reductase component (CbdC), a putative binding site for a chloroplast-type [2Fe 2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show significant homology to benABC, which encode benzoate 1,2-dioxygenase from Acinetobacter calcoaceticus (52% identity at the deduced amino acid level), and to xylXYZ, which encode toluate 1,2 dioxygenase from Pseudomonas putida mt-2 (51% amino acid identity). Recombinant pkT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wild-type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also allowed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Functional NADH:acceptor reductase and oxygenase components of 2-halobenzoate 1,2 dioxygenase were enriched from recombinant Pseudomonas clones. PMID- 7530712 TI - Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS. AB - CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1 152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild type bioactivity in vitro. PMID- 7530711 TI - Carbohydrate-dependent binding of the cell-free hemagglutinin of Vibrio cholerae to glycoprotein and glycolipid. AB - The carbohydrate-binding specificity of the cell-free hemagglutinin (HA) of Vibrio cholerae (K.K. Banerjee, A.N. Ghose, K. Datta-Roy, S.C. Pal, and A.C. Ghose, Infect. Immun.58:3698-3705, 1990) was studied by using glycoconjugates with defined sugar sequences. The HA was not inhibited by simple sugars including glucobiose, galabiose, and their N-acetylated derivatives. The hemagglutination of rabbit erythrocytes by the HA was inhibited moderately by fetuin, calf thyroglobulin, and human alpha 1-acid glycoprotein, all of which contain multiple asparagine-linked complex-type oligosaccharide units alone or in combination with serine/threonine-linked oligosaccharide units. The inhibitory potencies of the glycoproteins increased approximately 10-fold following removal of the terminal sialic acid and were completely destroyed by exhausative proteolysis. The HA agglutinated phosphatidylcholine liposomes containing GM1-ganglioside or its asialo-derivative in the presence of Ca2+ ions. The association constants of the complexes of the HA with asialofetuin, asialothyroglobulin, GM1-ganglioside, and asialo-GM1-ganglioside were determined by an enzyme-linked immunosorbent assay based assay and found to be 1.7 x 10(7) M-1, 1.5 x 10(7) M-1, 1.8 x 10(7) M-1, and 2.4 x 10(7) M-1, respectively. Studies using chemically modified glycoproteins and plant lectins with defined sugar specificity revealed that the HA recognized the terminal beta 1-galactosyl moiety of these glycoconjugates. There was no evidence for the presence of an extended carbohydrate-binding domain in the HA molecule or a preference of the HA for a complex, branched oligosaccharide structure. Similar to the mechanisms proposed for the binding of cholera toxin and Shiga toxin to glycolipids and neoglycoproteins, the strong interaction of V. cholerae cell-free HA with glycoconjugates appeared to be a consequence of multiple weak binding to terminal beta1-galactosyl moieties of the glycoproteins or glycolipids. PMID- 7530713 TI - Monoclonal antibodies that block the activity of leukocyte function-associated antigen 1 recognize three discrete epitopes in the inserted domain of CD11a. AB - The epitopes recognized by eight independently isolated monoclonal antibodies to the alpha chain of human and murine leukocyte function-associated antigen 1 (LFA 1), all able to inhibit receptor function, were identified. Initial localization of epitopes was accomplished using chimeric proteins constructed by splicing fragments of cDNAs encoding the alpha subunit of LFA-1 (CD11a) and the alpha subunit of the closely related leukocyte integrin, Mac-1 (CD11b). Antibody binding to CD11a/CD11b chimeras, expressed in the 293 human kidney cell line, demonstrated that the epitopes recognized by six monoclonal antibodies to human CD11a were located in a approximately 200-amino acid sequence found in all beta 2 integrin alpha subunits, termed the inserted (I) domain. Three distinct epitopes within the I domain (IdeA, IdeB, and IdeC) were identified using a series of mutants in which sequences from murine CD11a were substituted into human CD11a. A series of mutants incorporating single amino acid substitutions was used to identify individual amino acids essential for antibody binding. The location of these residues accounts for the binding specificity of LFA-1-blocking antibodies and identifies particular conserved sequences (residues 126-150) in the I domain of CD11a and homologous sequences in other beta 2-integrin alpha subunits that may be important for ligand binding. PMID- 7530714 TI - Agonist-modulated palmitoylation of endothelial nitric oxide synthase. AB - The nitric oxide synthases (NOS) comprise a family of enzymes which differ in primary structure, biological roles, subcellular distribution, and post translational modifications. The endothelial nitric oxide synthase (ec-NOS) is unique among the NOS isoforms in being modified by N-terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists such as bradykinin, which promote ecNOS translocation from membrane to cytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-deficient mutant ecNOS undergoes phosphorylation despite restriction to the cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether other post-translational modifications might regulate ecNOS targeting and now report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of endothelial cells with [3H]palmitic acid followed by immunoprecipitation of ecNOS revealed that the enzyme is palmitoylated; the label is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse chase experiments in endothelial cells biosynthetically labeled with [3H]palmitate show that bradykinin treatment promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation is dynamically regulated by bradykinin and propose that depalmitoylation of the enzyme may result in its cytosolic translocation and subsequent phosphorylation. PMID- 7530715 TI - P62 association with RNA is regulated by tyrosine phosphorylation. AB - The ras-GAP associated protein, p62, is a major tyrosine phosphoprotein in transformed and growth factor treated cells. Although its exact function is not known, it can bind directly to src-family tyrosine kinases and has been implicated as a linker protein bridging activated src family tyrosine kinases with downstream effectors. One novel feature of p62, revealed by its predicted amino acid sequence, is the presence of an RNA-binding region, the KH domain. As p62 becomes tyrosine phosphorylated when src-kinases become activated, we compared the RNA binding ability of p62 in both its phosphorylated and unphosphorylated state. The ability of p62 to bind RNA was severely impaired when p62 was tyrosine phosphorylated. This suggests that the ability of p62 to bind RNA is regulated by tyrosine phosphorylation and implicates the regulation of RNA as a component of tyrosine kinase signaling pathways. PMID- 7530716 TI - Complete and reversible inhibition of NADPH oxidase in human neutrophils by phenylarsine oxide at a step distal to membrane translocation of the enzyme subunits. AB - The effects of the trivalent arsenical phenylarsine oxide (PAO) on the activity of NADPH oxidase in human neutrophils were studied. PAO caused a rapid dose dependent inhibition of superoxide generation which was maximal at a concentration of 1 microM, irrespective of the stimulating agent. This inhibitory effect was not due to impaired transduction of activation signals since neither degranulation nor phagocytosis were modified. When cytosolic and membrane fractions from resting neutrophils were combined to reconstitute the NADPH oxidase, O2-. generation was inhibited by PAO while translocation of the NADPH oxidase components to the plasma membrane fraction was not affected. The inhibition was completely and specifically reversed by 2,3-dimercaptopropanol, not by dithiothreitol or beta-mercaptoethanol, indicating that PAO binds covalently to spatially vicinal thiol groups. PAO inhibited the plasma membrane's capacity to initiate O2-. generation while it apparently did not affect the cytosol. When PAO was added subsequently to NADPH oxidase activation, no inhibition was observed, indicating that PAO cannot reach its target once the oxidase is functionally assembled. In conclusion, PAO is the first complete and reversible inhibitor of NADPH oxidase which could provide the basis for new therapeutical approaches in inflammatory diseases. PMID- 7530717 TI - Collagen and stretch modulate autocrine secretion of insulin-like growth factor-1 and insulin-like growth factor binding proteins from differentiated skeletal muscle cells. AB - Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micrograms of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli. PMID- 7530718 TI - Biochemical properties of the ligand-binding 20-kDa subunit of the beta-glucan receptors on human mononuclear phagocytes. AB - beta-Glucan receptors are present on mammalian leukocytes and initiate phagocytosis of particulate yeast beta-glucans, such as zymosan particles. Human monocytes and U937 cells express two membrane proteins of 180 and 160 kDa, each of which binds particulate yeast glucan through a 20-kDa polypeptide constituent. In this report, the structural composition of the two beta-glucan receptors and the biochemical properties of their polypeptide constituents were examined. The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa, whereas the 160-kDa receptor was a multimer of two polypeptides of 27 and 20 kDa. Unlike other receptor constituents, the 20-kDa polypeptide was nonglycosylated and focused at two distinct isoelectric points. Immunoblots of the focused polypeptides showed the two 20-kDa variants and the 95-kDa subunit to be constitutively tyrosine-phosphorylated, a feature not previously reported for receptors on human mononuclear phagocytes. Dephosphorylation of the receptor proteins resulted in the loss of antigenic phosphotyrosine without affecting the antigenicity of either 20-kDa variant for the anti-idiotypic antibody to beta glucan receptors. Separate analysis of the 160-kDa receptor showed it contained both variants of the 20-kDa polypeptide. Thus, the 20-kDa subunit constituent of the two beta-glucan receptors is a functionally and chemically unique polypeptide with apparent microheterogeneity in its primary structure. PMID- 7530719 TI - cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation. AB - Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR. PMID- 7530720 TI - Activation and translocation of c-Src to the cytoskeleton by both platelet derived growth factor and epidermal growth factor. AB - We have examined the subcellular distribution and catalytic activity of c-Src tyrosine kinase after stimulation of A172 glioblastoma cells with peptide growth factors. Treatment of resting cells with platelet-derived growth factor resulted in an increase (3.5-fold) in the amount of c-Src protein associated with the cytoskeleton. In addition, an increase in specific c-Src kinase activity was observed in the cytoskeleton as well as in the cytosol and the membrane fraction. Similar effects on both c-Src redistribution and activity were seen after stimulation with epidermal growth factor. These data show that, like other signal transducing components, c-Src also becomes activated and associated to the cytoskeleton in response to growth factor stimulation. PMID- 7530721 TI - Tetrazole and carboxylate groups of angiotensin receptor antagonists bind to the same subsite by different mechanisms. AB - To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT1 receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys102 and Arg167 mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar1,Ile8]angiotensin II, and several carboxylate analogues of losartan. An Ala199 substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan. Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys199 is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT1 receptor involves multiple contacts with residues such as Lys199 and His256 that constitute the same subsite of the ligand binding pocket. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction. PMID- 7530722 TI - Cell spreading in Colo 201 by staurosporin is alpha 3 beta 1 integrin-mediated with tyrosine phosphorylation of Src and tensin. AB - Staurosporin, a broad-spectrum kinase inhibitor, induced cell spreading in a human colon cancer cell line, Colo 201. On collagen and laminin, cell spreading was induced in more than 90% of the cells and was dependent on very late activation antigen-3, as shown by an antibody inhibition assay. Cell spreading required divalent cations and showed the order of preference Mn2+ > Mg2+ > Ca2+. On fibronectin, only about 30% of the cells were observed to spread, and spreading occurred via a non-integrin, RGD-independent pathway. Staurosporin induced spreading was inhibited by treatment with tyrosine kinase inhibitors herbimycin A and methyl 2,5-dihydroxycinnamate. Despite the presence of staurosporin, seven proteins (220, 175, 150, 98, 62, 58, and 45 kDa) showed increased levels of tyrosine phosphorylation in association with cell adhesion. Two of these (58 and 220 kDa) were identified by immunoprecipitation as Src product and tensin, respectively. Flow cytometric analysis showed that the Colo 201 cells expressed the alpha 2, alpha 3, alpha 6, and beta 1 chains of integrin, but expression of these chains was not influenced by staurosporin. Immunofluorescence microscopy revealed that the alpha 3 chain, diffusely expressed on the cell surface in the absence of staurosporin, was concentrated at focal adhesion plaques after staurosporin treatment. Neither alpha 2 nor alpha 6 was focalized by the treatment. PMID- 7530723 TI - Prostaglandin F2 alpha stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells. AB - Human ovarian follicular fluid contains a number of insulin-like growth factor binding proteins (IGFBP) of which IGFBP-3 is the most abundant. IGFBP-3 synthesis is growth hormone-regulated. We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on IGFBP-3 secretion by cultured human granulosa-luteal cells from follicular aspirates of women participating in an in-vitro fertilization programme. The IGFBP-3 concentration was measured using a specific monoclonal immunofluorimetric assay. Contrary to a previous report on unstimulated follicles, this study demonstrated a positive correlation between follicular fluid IGFBP-3 concentration and follicular size. PGF2 alpha was found to stimulate in a dose-dependent fashion the secretion of IGFBP-3. Significant (P < 0.05) effects were found at PGF2 alpha concentrations of 10(-8), 10(-7) and 10( 6) M. Because IGFBP-3 inhibits progesterone production stimulated by insulin-like growth factor (IGF)-I, the PGF2 alpha-induced stimulation of IGFBP-3 production may be one of the mechanisms whereby PGF2 alpha exerts its luteolytic effect via the IGF system. PMID- 7530724 TI - Endometrial endothelial cell proliferation in long-term users of subdermal levonorgestrel. AB - The aim of the present study was to quantify endothelial cell proliferation (a component of angiogenesis) using immunohistochemistry, in the endometrium of users of subdermal levonorgestrel (Norplant). It was postulated that the increased endometrial microvascular density seen in Norplant users, compared to normally cycling women, was associated with an increased rate of endothelial cell proliferation. The results, however, showed that the endometrial endothelial cell proliferative index of Norplant users (0.39 +/- 0.16%; mean +/- SEM) was significantly reduced compared to that seen in normally cycling women (8.99 +/- 1.64). At the same time, total numbers of endometrial endothelial cells per mm2 in Norplant users (317.40 +/- 13.88) were significantly higher than in normally cycling women (223.35 +/- 10.31). It is possible that in the endometrium with levonorgestrel use, there is either a reduced rate of regression of the blood vessels relative to the rest of the tissue, or there is a reduced rate of endothelial cell death or turnover. Peripheral oestrogen and progesterone concentrations, bleeding pattern over the previous 90 days, and the histological appearance of the endometrium did not appear to be associated with the endothelial cell proliferative index. The results suggest that subdermal levonorgestrel use affects the mechanisms that dictate the normal relationship between endometrial blood vessel growth and regression, and the surrounding non vascular tissue. PMID- 7530725 TI - Production of granulocyte-macrophage colony-stimulating factor by human trophoblast cells and by decidual large granular lymphocytes. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has been implicated in placental growth and development. In this study, we investigated the production of GM-CSF in human first trimester pregnancy by the predominant uterine lymphocyte population of decidual CD56+ NK cells (large granular lymphocytes) and the factors that influence this production using enzyme-linked immunosorbent assays (ELISAs) and bioassays, supplemented by immunocytochemistry. We have also investigated and compared production of GM-CSF by human first trimester trophoblast and by JEG-3 and JAR choriocarcinoma cells. Our data show that appreciable amounts of GM-CSF are produced in first trimester maternal decidua and that a significant component of this secretion was from decidual large granular lymphocytes (LGL). Production of GM-CSF by LGL was constitutive and considerably greater than that of freshly isolated peripheral blood leukocytes. GM-CSF secretion by decidual LGL could be enhanced by co-culture on a monolayer of decidual stromal cells, and could also be increased in a dose-dependent manner by stimulation with interleukin-1 (IL-1) or IL-2. IL-4, IL-6, tumour necrosis factor alpha (TNF alpha), transforming growth factor beta (TGF beta), interferon alpha (IFN alpha) and IFN gamma individually had no effect on GM-CSF secretion, although IL-4, TGF beta and IFN alpha all inhibited the action of IL-2. IFN gamma had no effect on the IL-2 induced GM-CSF secretion, but did antagonize the action of IL-1. Normal human first trimester trophoblast was also found to produce GM-CSF, although no production whatsoever was seen by JEG-3 or JAR choriocarcinoma cells. These results suggest that GM-CSF from uterine lymphocytes, and from trophoblast itself, may influence placental growth and development in both a paracrine and an autocrine manner. PMID- 7530726 TI - Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and delta F508 mutations. AB - W1282X (W) and delta F508 (delta) are the two most common mutations of the cystic fibrosis Israeli population. Patients who are homozygotes (WW and delta delta) as well as compound heterozygotes (W delta) present a severe phenotype of the disease. In the present study, we have developed a polymerase chain reaction (PCR)-based method for the detection of both mutations simultaneously in a single blastomere. Unfertilized human oocytes and single polyspermic blastomeres were subjected to a two-round PCR amplification: a first round of multiplex PCR followed by a second round of nested PCR, done separately at each locus. Clear signals at both loci were obtained in 51% (47/65) of oocytes and 69% (24/35) of blastomeres. The genotype of the single cell analysed was determined by endonuclease digestion of the W products and by heteroduplex formation of the delta F products. This diagnostic system will allow the identification of affected embryos (WW, delta delta, W delta) as well as phenotypically normal carriers (W+, +delta), and therefore may be used for cystic fibrosis preimplantation diagnosis in families who carry either or both mutations. PMID- 7530727 TI - A soluble binding assay for measuring 3H-FK506 binding to the hsp56 immunophilin. AB - Heat shock protein 56 (hsp56) was previously identified as an immunophilin based on its ability to specifically bind to FK506-Affi-Gel 10. In this report, we have quantitated human Jurkat T cell hsp56 binding to 3H-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that 3H-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-Sepharose column binds 3H-FK506 with an affinity of 19.4 +/- 4.6 nM, as compared to 23.2 +/- 6.8 nM for soluble hsp56. In competition experiments, the apparent affinity constant for rapamycin was 11.6 +/- 2.8 nM, using immobilized hsp56, and 17.3 +/- 7.7 nM, using the soluble hsp56 preparation. These results demonstrate that hsp56 binds FK506 and rapamycin with similar affinities, and suggest that hsp56 may play a role in mediating the cellular function of both of these drugs. PMID- 7530728 TI - Interspecies scaling and pharmacokinetic parameters of 3TC in humans. AB - 3TC is a dideoxy-nucleoside analogue that has demonstrated in-vitro activity against human immunodeficiency virus (HIV). 3TC concentrations in humans were predicted before the initiation of clinical trials by interspecies scaling of pharmacokinetic parameters observed in animal species. Clearance and volume of distribution were estimated for humans using linear regression on a log-log scale of each parameter versus body weight for rats and dogs. The concentration-time profile and the average serum concentration at steady state after various dosage regimens were estimated as a basis for initial dose selection for clinical trials. The predicted parameters (clearance of 16.3 L/hr and volume of distribution of 40 L for a 70-kg man) were compared with that observed (mean clearance of 24 L/hr and mean volume of distribution of 96 L, mean weight of 74 kg) in 20 asymptomatic, HIV positive, volunteers after single intravenous doses of 3TC. Interspecies scaling was applied prospectively as a rationale for dose selection of 3TC in clinical trials. PMID- 7530729 TI - Glomus cell differentiation in the carotid body region of chick embryos studied by neuron-specific class III beta-tubulin isotype and Leu-7 monoclonal antibodies. AB - Development of the carotid body and the glomus cell groups in the wall of the common carotid artery and its branches was examined in chickens at various developmental stages by immunohistochemistry using three different monoclonal antibodies, i.e., anti-neuron-specific class III beta-tubulin isotype (TuJ1), anti-rat brain beta-tubulin, and anti-Leu-7 (HNK-1) antibodies. All the antibodies reacted with neurons. The carotid body anlage was first discerned at 6 days of incubation at the lateral portion of the third branchial artery. The cells and nerve fibers immunoreactive for TuJ1, brain beta-tubulin and Leu-7, which were connected with the distal ganglion of the vagus nerve, were found around the carotid body anlage at this stage. Within the carotid body anlage, no immunoreactivity yet appeared. The immunoreactive cells were accumulated around the carotid body anlage until 8 days of incubation. From 9 days of incubation, the immunoreactive cells continuing with the distal vagal ganglion began to enter into the carotid body anlage and also dispersed widely along the common carotid artery and its branches, giving rise to the glomus cells. At 12 days of incubation, a large portion of the carotid body was occupied by the immunoreactive cells. Thus, the present study evidences that the glomus cells in the carotid body and around the arteries are emigres that arrive in each residential place from the distal vagal ganglion. Immunoreactivity for TuJ1, brain beta-tubulin, and Leu-7 in the glomus cells started to decrease at late stages of embryonic development. After hatching, no TuJ1-immunoreactive cells were detected in the carotid body region. PMID- 7530730 TI - Octopamine-like immunoreactivity in the brain and subesophageal ganglion of the honeybee. AB - The organization of putative octopaminergic pathways in the brain and subesophageal ganglion of the honeybee was investigated with a well-defined polyclonal antiserum against octopamine. Five prominent groups of just over 100 immunoreactive (IR) somata were found in the cerebral ganglion: Neurosecretory cells in the pars intercerebralis innervating the corpora cardiaca via NCC I, one cluster mediodorsal to the antennal lobe, one scattered on both sides of the midline of the protocerebrum, one between the lateral protocerebral lobes and the dorsal lobes, and a single soma on either side of the central body. With the exception of the pedunculi and beta-lobes of the mushroom bodies, varicose immunoreactive fibers penetrate all parts of the cerebral ganglion. Strong labelling was found in the central complex and the protocerebral bridge. Fine networks of labelled processes invade the antennal lobes, the calyces and a small part of the alpha-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia. In the subesophageal ganglion, one labelled cell body was found in the lateral soma layer of the mandibular segment. Each of the three neuromeres contains a group of six to ten somata in the ventral median parts. Most of the ventral median cells send their neurites dorsally through the midline tracts, whereas the neurites of a few cells follow the ventral cell body neurite tracts. Octopamine-IR was demonstrated in all neuropils that contain pathways for proboscis extension learning in honeybees. Because octopaminergic mechanisms seem to be involved in the behavioral plasticity of the proboscis extension reflex, our study provides anatomical data on the neurochemical organization of an appetitive learning paradigm. PMID- 7530731 TI - Immunocytochemical analysis of bipolar cells in the macaque monkey retina. AB - Transfer of visual information from photoreceptors to ganglion cells within the retina is mediated by specialized groups of bipolar cells. At least 10 different morphological types of bipolar cells have been distinguished in Golgi studies of primate retina. In the present study, bipolar cell populations in the macaque monkey retina were identified by their differential immunoreactivity to a spectrum of antibody markers. This enabled their spatial density and photoreceptor connections to be analysed. An antibody against the beta isozyme of protein kinase C (PKCA beta) labelled many cone bipolar cells. Invaginating (presumed ON) cone bipolar cells and rod bipolar cells were preferentially labelled with a monoclonal antibody raised against rabbit olfactory bulb. Flat (presumed OFF) bipolar cells were labelled with an antiserum against the glutamate transporter protein (GLT-1). Different populations of diffuse cone bipolar cells, which contact 5-10 cones, could be distinguished. The GLT-1 antiserum preferentially labelled the flat diffuse bipolar cell type DB2 (Boycott and Wassle, 1991, Eur. J. Neurosci. 3:1069-1088) as well as flat midget bipolar cells. Antibodies to calbindin (CaBP D-28K) labelled the flat diffuse bipolar cell type DB3 and (possibly) the invaginating diffuse bipolar cell type DB5. An antibody against the alpha isozyme of PKC labelled an invaginating diffuse bipolar cell type (DB4) as well as rod bipolar cells. Comparison of the spatial density of cone bipolar cell populations with that of photoreceptors suggests that each bipolar cell class provides a complete coverage of the cone array (each cone is contacted by at least one member of every bipolar cell class). These results support the classification scheme of Boycott and Wassle (1991) by showing that different diffuse bipolar cell classes express different patterns of immunoreactivity, and they reinforce the view that different spatial and temporal components of the signal from the photoreceptor array are processed in parallel within the primate retina. PMID- 7530732 TI - Biological behaviour and morphological characteristics of a transplantable tumour (MM-KMY) derived from a malignant meningioma in an F344 rat. AB - A subcutaneously transplantable meningeal tumour (MM-KMY) was derived from a spontaneous malignant meningioma which developed in the cerebellar meninges of a female F344 rat. MM-KMY was subjected to 25 serial passages in syngeneic male and female rats. The transplants grew in 8 weeks into a nodule with an average diameter of 5.7 cm and average weight of 125.2 g. MM-KMY possessed large cysts containing fluid and necrotic tissue. Metastases frequently occurred in the lungs of MM-KMY-bearing rats. Histologically, both the original tumour and MM-KMY consisted of round to fusiform neoplastic cells of varying size, with nuclear pleomorphism. Mitotic figures occurred frequently. MM-KMY cells were positive for vimentin. Ultrastructurally, the cells showed desmosome-like structures, interdigitating processes and cytoplasmic intermediate filaments, suggesting an arachnoid cell origin. Abnormal accumulations of hyaline droplets in renal tubular epithelial cells were frequently observed in MM-KMY-bearing rats, suggesting overload of low molecular proteins in the renal tubules. The droplets gave a faint immunoreaction for lysozyme. The relation between the appearance of renal tubular hyaline droplets and the growth of MM-KMY remains to be determined. MM-KMY may prove useful for studying the biological behaviour and morphogenesis of meningeal tumours. PMID- 7530733 TI - Functional role of charybdotoxin-sensitive K+ channels in the resting state of dog basilar artery. AB - To determine the possible role of Ca(2+)-activated K+ (KCa) channels in the regulation of resting tone of cerebral arteries, the effect of agents which interact with these channels on tension and 86Rb efflux was examined in endothelium-denuded strips of dog basilar artery, and data were compared with findings in the mesenteric artery. The basilar artery maintained a myogenic tone; that is, the resting tone decreased when nifedipine was added. The addition of charybdotoxin, a blocker of large conductance KCa channels, caused a concentration-dependent contraction in the basilar artery but not in the mesenteric artery. In strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in the basilar artery than in the mesenteric artery. Nifedipine decreased the basal 86Rb efflux rate constant only in the basilar artery. The basal 45Ca influx in the resting state of the basilar artery was significantly increased when compared with the mesenteric artery and this increase in the basilar artery was reduced by nifedipine. The results suggest that the charybdotoxin-sensitive KCa channels regulate the myogenic tone in the resting state of the basilar artery. The activation of KCa channels in the basilar artery appears to be secondary to the increased transmembrane Ca2+ influx probably via the activation of L-type voltage-dependent Ca2+ channels in the resting state of this artery. PMID- 7530734 TI - Cerebrovascular sensory innervation involved in the development of cerebral vasospasm following a subarachnoid hemorrhage. AB - Cerebral vasospasm following subarachnoid hemorrhage was induced in the squirrel monkey in order to evaluate the involvement of cerebrovascular sensory nerves in the development of the vasospasm. A unilateral surgical section of the trigeminal nerve at post- but not at pre-Gasserian level caused constriction of the major ipsilateral cerebral arteries. A pre- or postganglionic trigeminal lesion induced an increased glucose uptake globally without influencing the cerebral blood flow. Following a subarachnoid hemorrhage, the decrease in cerebral blood flow was similar of that seen in control animals, while post-ganglionically lesioned animals had an additional increase in glucose uptake. Intrathecal injection of gamma-globulin against substance P prevented the occurrence of vasospasm and the decrease in cerebral blood flow, while calcitonin gene-related peptide (CGRP) anti-gamma-globulin injection significantly reduced the resting vessel diameter and did not influence spasm development. It is concluded that a nervous reflex mechanism could underlie cerebral vasospasm. The cerebrovascular sensory nerves have both a peripheral and a central function. A peripheral or axon reflex mechanism exerts a tonic effect on the cerebral arteries. Central neurotransmission seems to be involved in the regulation of cerebral metabolism and possibly in the coordination of cerebral blood flow and glucose metabolism. CGRP could be the transmitter involved in a peripheral axon reflex and substance P might be the neurotransmitter conveying information to the brainstem vascular centers. PMID- 7530735 TI - Nitric oxide synthase does not contribute to cerebral autoregulatory phenomenon in anesthetized dogs. AB - Nitric oxide (NO) is a potent vasodilator produced by nitric oxide synthase (NOS). We tested the following hypotheses: (1) cerebral blood flow (CBF) is NO dependent, (2) NO contributes to CBF autoregulation, and (3) NO participates in the neurohypophysial vasodilator response to hypotension. Three groups of sodium pentobarbital anesthetized dogs were studied using microspheres. In 7 dogs, N omega-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg, i.v.) increased mean arterial pressure (MAP) by 12%. Cerebrovascular resistance (CVR) increased more than MAP, resulting in a 20 +/- 4% reduction (range 12-67%) in baseline CBF. In unblocked conditions, actively autoregulated regions (e.g. cortex, white matter, median eminence) demonstrated a correlation between CVR and MAP whereas passive regions (neural lobe) did not. NOS block did not effect the relationship between MAP and CVR in most brain regions. However, a significant relationship between CVR and MAP developed in neural lobe after NOS block. Abrupt hypotension increased neural lobe blood flow to 239 +/- 37% control at 3 min, despite NOS block. These results show that baseline cerebral vessel tone depends upon NOS activity. Enhanced NO release cannot explain either cerebral autoregulation or the transient hyperperfusion seen in neural lobe immediately following rapid hemorrhage. PMID- 7530736 TI - The role of nitric oxide in the cerebrovascular flow response to stimulation of postganglionic parasympathetic nerves in the rat. AB - Stimulation of cerebrovascular parasympathetic nerves markedly increases cortical blood flow. Nitric oxide (NO) or a NO containing compound is present in these nerves, and its release may therefore be partly responsible for the flow increase. In addition, transmitters released from the nerves may cause synthesis and release of this compound from the endothelium. The contribution of NO synthesis to the cortical blood flow increase during parasympathetic stimulation was elucidated in rats by laser-Doppler flowmetry. Thirty min exposure to circulating N omega-nitro-L-arginine methyl ester (L-NAME) 50 mg.kg-1 eliminated most of the response (from 104 to 8% increase), whereas 10 min exposure to this dose or 30 min exposure to 5 mg.kg-1 caused a less marked reduction. The reducing effect was particularly evident after elimination of the systemic blood pressure increase caused by L-NAME (only 3% increase after the high dose). In fusion of L arginine restored the flow response. Resting cortical blood flow was not substantially affected by blockade of NO formation. Thus, release of a NO containing compound constitutes a major component of the increase in cortical blood flow caused by parasympathetic nerve stimulation, but does not seem to contribute to cortical flow regulation during resting conditions. PMID- 7530737 TI - Hepatocellular carcinoma in primary biliary cirrhosis. AB - Primary biliary cirrhosis is considered a low risk for the development of hepatocellular carcinoma. We evaluated the incidence of hepatocellular carcinoma in a cohort of 89 female primary biliary cirrhosis patients and, as a control group in 73 female patients with cirrhosis of a different etiology. The patients underwent ultrasound and alfa-fetoprotein determination every 6 months. Two patients developed hepatocellular carcinoma in the primary biliary cirrhosis and three in the control group, respectively. The Relative Risk for hepatocellular carcinoma in primary biliary cirrhosis patients was 0.7. However, when considering only stage IV, primary biliary cirrhosis patients with cirrhosis, the Relative Risk was actually 1.5 with respect to controls, with 1.4% cases of hepatocellular carcinoma per year. These data suggest that the risk of hepatocellular carcinoma is similar in female patients with primary biliary cirrhosis and in female patients with cirrhosis of a different etiology. PMID- 7530739 TI - Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns. AB - IgG, IgA, and IgE production by newborn B cells is limited both in vivo and in vitro in various activation conditions, whereas IgM production is readily detectable. It has been suggested that the Ig heavy chain switch inability could be the consequence of T and B cell immaturity. As the interaction between CD40 (expressed on B cells) and its ligand CD40-L (expressed on activated T cells) triggers a key signal required for isotype switching, we studied the expression and function of these two components in normal fetuses, newborns, and infants, compared with adults. CD40-L expression was not inducible in 28 of 30 specimens of newborn cord-blood T cells following incubation with PMA and ionomycin, whereas activation markers such as CD69 were inducible. CD40-L expression was triggered by activation of T cells from infants > 3 wk of age. Surprisingly, T cells from 19- to 28-wk-old fetuses also expressed CD40-L following activation. CD40-L expression on newborn T lymphocytes was induced on T cell lines generated in the presence of PHA and maintained with IL-2 following further stimulation with PMA and ionomycin. CD40-L mRNA transcripts and intracytoplasmic protein expression following activation of newborn T cells were strongly decreased, leading to undetectable protein membrane detection. These results point to a possible transcriptional down-regulation of CD40-L expression by neonatal T lymphocytes. In addition, fetal and cord-blood B cells were poorly able to switch to IgG or IgA by stimulation with CD40 agonists (Ab or soluble CD40-L) in the presence of IL-4 or IL-10 as also detected with surface IgD+ adult B cells. Both phenomena could contribute to the neonatal Ig switch inability, although distinct underlying regulatory mechanisms are probably involved, as suggested by different in vivo time courses. PMID- 7530738 TI - Immune response to a hepatitis C virus nonstructural protein in chronic hepatitis C virus infection. AB - The immune responses to a hepatitis C virus nonstructural protein (T3Ag) overlapping with the C100-3 antigen were examined in three groups of patients with chronic non-A, non-B hepatitis. Group I included 20 cases positive for both anti-C100-3 and the second-generation anti-HCV test (anti-HCV-II): Group II, five cases with anti-C100-3(-)/anti-HCV-II(+); and Group III, seven cases negative for both tests. HCV RNA was detectable in 20 (100%), 4 (80%) and 0 (0%) patients in each group, respectively. Proliferative responses of peripheral blood mononuclear cells to T3Ag were present in 16 (80%), 3 (60%) and 0 (0%) cases in each group, respectively (p < 0.05). Removal of CD8+ T cells from peripheral blood mononuclear cells resulted in a conversion of unresponsiveness to significant proliferation to T3Ag in the remaining cases in groups I and II, but not in group III. This change paralleled the antigen-induced production of interferon-gamma and interleukin-2, but not interleukin-4. The removal also enhanced the T3Ag stimulated anti-C100-3 antibody production from cultured peripheral blood mononuclear cells in group II patients. These results indicate that the T3Ag specific type 1 T helper cells play an important role in regulating anti-C100-3 antibody secretion in hepatitis C patients. PMID- 7530740 TI - Expression and function of the gamma c subunit of the IL-2, IL-4, and IL-7 receptors. Distinct interaction of gamma c in the IL-4 receptor. AB - IL-2R, IL-4R, and IL-7R share a common subunit referred to as gamma c and the IL 13R has been proposed to contain gamma c as a subunit. In this report we have used two novel mAbs (3E12 and 4G3) to distinct epitopes of mouse gamma c to determine its lymphoid cell distribution and to examine whether gamma c uses similar epitopes to interact with different cytokines and cytokine receptors. FACS analysis revealed that gamma c is expressed in most lymphocytes, myeloid cells, embryonic thymocytes, and lymphoid cell lines. Results from radiolabeled ligand binding studies, biochemical analysis of ligand-receptor cross-linked complexes, and cytokine bioassays indicate that the epitope defined by mAb 4G3 closely defines the IL-7 binding region of gamma c and overlaps the IL-2 binding region of gamma c. These studies also indicate that gamma c interacts with IL-4 in the context of the IL-4R in a manner that is distinct from its role in the IL 2R and IL-7R and suggest that the 3E12 epitope defines a region of gamma c that intimately interacts with the IL-4R. The B9 plasmacytoma, which proliferates in response to IL-4 and IL-13, was shown to not express gamma c. Thus, at least in some circumstances, gamma c is dispensable for signaling via the IL-4R and is not a required subunit of the IL-13R. PMID- 7530741 TI - Transplantable NK cell progenitors in murine bone marrow. AB - Differentiation of NK cells from pluripotent hematopoietic stem cells is a poorly understood process. Although it is known that NK cells are bone marrow derived and dependent upon an intact bone marrow microenvironment for complete maturation, it is not known if they arise from an intermediate lymphoid stem cell or from progenitors exclusively committed to the NK lineage. To determine whether phenotypically distinct committed NK progenitor cells exist in murine bone marrow, we sorted cells capable of repopulating recipient mice with mature NK cells upon i.v. transfer. We identified a rare population of bone marrow cells with the phenotype Ly6+ Lin- c-kit+ CD43high Fall-3high TSA-1- AA4.1low Rh123high that is highly enriched for the ability to generate NK cells after transplantation. Although these cells are relatively depleted of Rh123low pluripotent stem cells, they are highly enriched for both lymphoid and myeloid repopulating ability. Thus, we have found no evidence to support the existence of a phenotypically distinct transplantable progenitor population in mouse bone marrow that is either exclusively committed to the NK cell lineage or exhibits the functional characteristics of a common lymphoid stem cell. PMID- 7530742 TI - IL-4 renders mast cells functionally responsive to endothelin-1. AB - It has previously been shown that mouse bone marrow-derived mast cells (BMMC) synthesize and secrete endothelin-1 (ET-1) and express ETA-type endothelin receptors (ETA-R). The study presented here was designed to elucidate the influence of different cytokine conditions for cellular differentiation and maturation on the ability of primary mouse BMMC to respond to exogenous ET-1. BMMC were grown for 2 wk in IL-3 alone and then cultured for 2 to 3 wk with kit ligand (KL) and/or IL-3 in the presence or absence of IL-4. ET-1 induced a very rapid (< or = 1 min) and dose-dependent release of histamine and serotonin from BMMC cultured in the presence of both IL-3 and IL-4. The effect of ET-1 was quantitatively comparable with IgE/Ag-induced mediator release and comprised up to 20% and 16% of total cellular histamine and serotonin, respectively. In BMMC grown with KL or KL plus IL-3, a substantial effect of ET-1 on amine release was only observed when IL-4 had been included in the culture medium. These IL-4 effects could not be observed if BMMC grown in IL-3 and/or KL were preincubated for 1 or 24 h with IL-4 before activation with ET-1, suggesting that a differentiation process rather than a functional priming effect had been initiated by IL-4. In BMMC, the histamine and serotonin release induced by ET-1 (10(-6) M) was inhibited by an ETA-R-specific antagonist (cyclic [D-Asp-Pro-D-Val Leu-D-Trp]) in a dose-dependent manner, with complete inhibition at an antagonist concentration of 10(-8) M. ET-1 stimulated leukotriene C4 biosynthesis up to 4.5 fold in BMMC cultured in the presence of IL-4. No such ET-1 effect was observed in BMMC cultured in media containing IL-3, KL, or a combination of both cytokines. Peritoneal cells (containing 2 to 3% serosal mast cells) obtained from BALB/c mice released 87 +/- 2% of histamine within 1 min after challenge with ET 1. Our results demonstrate that ET-1 can directly act as a histamine and serotonin secretagogue and as a stimulator of leukotriene C4 production in mast cells. IL-4 appears to be critically involved in the differentiation of immature mast cell precursors to an ET-1-reactive phenotype.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530743 TI - The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells. AB - FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts. PMID- 7530744 TI - Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL 13. AB - Production by endothelial cells of the regulated on activation normal T expressed and secreted chemokine (RANTES) has recently been evidenced during delayed-type hypersensitivity (DTH) reactions and may contribute to the local accumulation of macrophages and CD4+ memory T lymphocytes. To document the mechanism inducing RANTES production in this condition, we analyzed the effect of cytokines known to influence the formation of DTH granulomas. Little or no RANTES was produced after stimulation of HUVEC with IFN-gamma, IL-1 beta, or TNF-alpha. However, the combination TNF-alpha+IFN-gamma induced a strong RANTES production. In situ hybridization experiments with a RANTES probe showed that this synergy was also observed at the mRNA level and that the effect of the combination was mainly to increase the amount of RANTES mRNA per cell. The expression of the luciferase gene under the control of the RANTES gene regulatory elements was analyzed; TNF alpha and the combination TNF-alpha+IFN-gamma activated the regulatory elements. Sequential treatment of HUVEC with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized HUVEC to the stimulating effect of TNF-alpha. The production of RANTES induced by TNF-alpha+IFN-gamma was partly but significantly inhibited by the Th2 type cytokines IL-4 and IL-13. In contrast, IL-10 had no effect. These results indicate that the microenvironment of DTH granulomas, containing high levels of both TNF-alpha and IFN-gamma, may be responsible for RANTES production by perigranulomatous endothelial cells. Inhibition of this production by Th2-type cytokines may be a mechanism by which these cytokines interfere with the formation of DTH granulomas. PMID- 7530745 TI - Cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) by astrocytes and astrocytoma cell lines. AB - Astrocytes in the central nervous system (CNS) associate intimately with vascular endothelial cells and are integral to the blood-brain barrier (BBB). Leukocyte transmigration across the BBB is a cardinal feature of CNS inflammation, as observed in experimental autoimmune encephalomyelitis, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1) interactions have recently been proposed as essential for this process. VCAM-1 expression by astrocytes was recently reported. We addressed the regulation of VCAM-1 expression by inflammatory cytokines in primary human astrocytes and two human astrocytoma cell lines. Astrocytoma cells up-regulated surface VCAM-1 expression in response to cytokines in the following order: IFN-gamma plus TNF-alpha > IFN-gamma plus IL-1 beta > TNF-alpha > IFN-gamma. Cytokine-activated astrocytoma cells expressed 7 domain VCAM-1, as indicated by accumulation of 3.2-kb VCAM-1 mRNA and immunoprecipitation of a 100-kDa protein with anti-VCAM-1 mAb. Lymphoblast adhesion to cytokine-activated astrocytoma cell monolayers was significantly blocked by mAb specific for VCAM-1 and VLA-4, indicating that astrocytoma cell VCAM-1 was functional. Astrocytoma cell expression of VCAM-1 could be a constituent of the astrocyte phenotype. To support this possibility, we demonstrated that cytokine-treated adult human and rat primary astrocytes expressed VCAM-1, and the rank order of cytokine potency for VCAM-1 induction in primary and neoplastic astrocytes was strikingly similar. This is the first documentation of VCAM-1 expression by adult human astrocytes. Expression of VCAM 1 by astrocytes at the BBB could play a role in mononuclear leukocyte entry into the CNS. PMID- 7530746 TI - Compartmentalized expression of RANTES in a murine model of endotoxemia. AB - Systemic exposure to LPS initiates a complex sequence of events culminating in organ-specific leukocyte recruitment and end organ injury. We hypothesized that RANTES, a C-C chemokine with potent M phi (mononuclear phagocyte) chemotactic activity, is expressed in vivo in response to endotoxemia, and that this protein may play an important role in the recruitment of M phi to the lung. CD-1 mice were challenged with LPS (200 micrograms), resulting in a maximal fourfold increase in polymorphonuclear leukocyte (neutrophils) at 6 h post LPS, and a 2.4 fold increase in numbers of M phi within lung minces at 24 h. A time-dependent increase in RANTES mRNA was detected in lung after LPS treatment, whereas minimal quantities of RANTES mRNA were detected in blood buffy coats and liver. Furthermore, treatment with LPS resulted in time-dependent increase in RANTES protein within lung homogenates, with immunolocalization to alveolar epithelial cells. The pretreatment of mice with goat anti-RANTES Ab significantly inhibited the influx of lung M phi, but not polymorphonuclear leukocyte and lymphocytes, at 24 h post-LPS challenge. Lastly, the pretreatment of animals with soluble TNF receptor: Ig construct 2 h before LPS resulted in a 60% reduction in steady state levels of RANTES mRNA within lung homogenates at 4 h post-LPS. Our observations suggest that RANTES represents an important mediator of lung M phi recruitment in the setting of endotoxemia, and that the expression of RANTES in vivo is dependent upon the endogenous production of TNF. PMID- 7530747 TI - Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients. AB - The immune response toward allergens in nonallergic healthy individuals was investigated. To boost immune responses, two injections of birch pollen extract were administered to five nonallergic volunteers. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analyzed for epitope specificity using overlapping peptides. Forty-nine T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. Comparison with TCL and TCC established from birch pollen-allergic patients was performed. All TCC revealed the Th phenotype. Epitope specificities of TCL and TCC from nonatopics were identical to those found in allergic individuals. No association between MHC class II molecules and particular epitopes could be observed. In nonallergic as well as in allergic individuals, cytokine production in response to specific stimulation revealed a majority of Th-clones producing IL-4 and IFN-gamma. However, TCC derived from atopic individuals revealed a higher IL-4/IFN-gamma ratio. Immunoblot and ELISA revealed Bet v 1-specific IgG in nonallergic individuals before and after booster injections, but no IgE could be detected. High levels of Bet v 1-specific IgG and IgE could be detected in birch pollen allergic patients. It can be concluded, that nonatopic and allergic individuals display the same repertoire of T cell specificities. In allergic individuals, the activation of allergen-specific TCC leads to a higher ratio of produced IL-4 vs IFN-gamma, which is responsible for enhanced IgE production. PMID- 7530748 TI - Immunodominant framework region 3 peptide from TCR V beta 8.2 chain controls murine experimental autoimmune encephalomyelitis. AB - Previous work has demonstrated the existence of regulatory circuitry that controls response to the dominant determinant Ac1-9 of myelin basic protein (MBP) which is highly restricted in TCR V gene usage to V beta 8.2 and V alpha 2.3. In particular, a CD4+ V beta 14+ regulatory T cell was shown to be a vital component of this circuit. In our work presented here, the peptide specificity of the response to V beta 8.2 peptides was examined. Five overlapping peptides, B1 through B5, were studied for their ability to induce a proliferative response: B2 (21-50), B4 (61-90), and B5 (76-101) each had this capacity in the B10.PL or (SJL x B10.PL)F1 mice. The determinant within the TCR peptide B5 appears dominant, whereas determinants within the B2 and B4 peptides are physiologically cryptic. Furthermore, only B5 could down-regulate the response to MBP Ac1-9 and significantly protect mice from MBP- or Ac1-9-induced EAE, whereas B2 or B4 treatment had no significant effect. Treatment of mice with B5 did not result in generalized deletion or inactivation of V beta 8.2+ T cells. The core residues of the B5 determinant lie within framework region 3 of the V beta 8.2 chain and do not include residues from the joining CDR3 region. Response to B5 was restricted by the I-Au MHC molecule. Furthermore, B5 only induced responses in mice with certain MHC alleles. It is evident that by specifically down-regulating the initial dominant response to Ac1-9, Ag-induced disease can be prevented. These data have implications for understanding induction of TCR-based regulation, as well as relevance to possible therapeutic approaches for oligoclonal responses in human autoimmune diseases. PMID- 7530749 TI - Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule. AB - Although several peptides have been found to bind to both class I and class II molecules, the basis for this binding of the same peptide to two classes of MHC molecules has not been compared previously. We have analyzed one such peptide, P18 from the V3 loop of HIV-1 gp160, which we have previously shown to be recognized by CD8+ CTL with the class I molecule H-2Dd, and by CD4+ Th cells with the class II molecule I-Ad. With the use of truncated and substituted peptides, we found that the minimal core peptides are very similar, that the residues required for class I binding precisely fit the recently identified consensus motif for peptides binding to Dd (XGPX[R/K/H]XXX(X) [L/I/F]), and that at least three of the same residues are involved in binding to class II I-Ad. In addition, several of the same residues are involved in TCR interaction when the peptide is presented by class I and class II molecules. Modeling shows results to be consistent with the crystal structure of a peptide-class II MHC complex. Thus, the recognition of this versatile peptide by CD4+ Th cells with class II MHC molecules and by CD8+ cytotoxic T cells with class I MHC molecules is remarkably similar in both the core peptide used and the role of different residues in the ternary complex. PMID- 7530750 TI - [Results of radical prostatectomies for cancer: a retrospective multicenter study on 918 cases]. AB - The author presents the results of a follow-up study on 918 cases of radical prostatectomy for prostatic cancer performed by 21 urologists. The average follow up was two years and six months, (from three months to eight years). Mortality caused by the cancer was 1.96% and from all other reasons was 3.05%. 86.82% of the patients have been operated on for clinical stage B 8.49% for stage A. 3.81% for stage C and 0.22% for stage D. Comparison between pre-operative for evaluation and for pathological features show an understaging rate of 52.03%, an overstaging rate of 4.07% and an accurate evaluation in 43.90% of the cases. Almost half on the understaged patients (208 on 422) received adjuvant therapy, but only 14 received adjuvant therapy in the group of 342 patients whose evaluation was accurate. PSA level seems to be an important component in the stadification, since 73.59% patients whose PSA level was under 50 ng/ml did receive an adjuvant therapy, while 21.24% patients whose PSA level was over 50 ng/ml remain without complementary treatment. The post-operative incontinence rate was 11.75%. The post-operative impotence rate was 83.15%. PMID- 7530751 TI - The need to define the substances measured in tests for cytokeratins. PMID- 7530752 TI - Preliminary evaluation of CYFRA 21-1 in lung cancer cytosol. PMID- 7530753 TI - TPA-cyk assay in cytosol from primary breast cancer: preliminary results. PMID- 7530754 TI - Defect in radiation signal transduction in ataxia-telangiectasia. AB - Exposure of mammalian cells to ionizing radiation causes a delay in progression through the cycle at several checkpoints. Cells from patients with ataxia telangiectasia (A-T) ignore these checkpoint controls postirradiation. The tumour suppressor gene product p53 plays a key role at the G1/S checkpoint preventing the progression of cells into S phase. The induction of p53 by radiation is reduced and/or delayed in A-T cells, which appears to account for the failure of delay at the G1/S checkpoint. We have investigated further this defect in radiation signal transduction in A-T. While the p53 response was defective after radiation, agents that interfered with cell cycle progression such as mimosine, aphidicolin and deprivation of serum led to a normal p53 response in A-T cells. None of these agents caused breaks in DNA, as determined by pulse-field gel electrophoresis, in order to elicit the response. Since this pathway is mediated by protein kinases, we investigated the activity of several of these enzymes in control and A-T cells. Ca+2-dependent and -independent protein kinase C activities were increased by radiation to the same extent in the two cell types, a variety of serine/threonine protein kinase activities were approximately the same and anti-tyrosine antibodies failed to reveal any differences in protein phosphorylation between A-T and control cells. It is not evident what is the nature of the defect in signal transduction in A-T cells. However, it is clear that the p53 response is normal in these cells after exposure to some agents and it is mediated through protein kinase C or another serine/threonine kinase. PMID- 7530755 TI - Expression of a candidate ataxia-telangiectasia group D gene in cultured fibroblast cell lines and human tissues. AB - A candidate gene for ataxia-telangiectasia group D (ATDC) has been cloned (Kapp et al. 1992), sequenced and found to be a member of a recently reported gene family (Leonhardt et al. 1994). Transcriptional behaviour of ATDC has been examined in a number of cell lines and human tissues using a 3.0 kb cloned cDNA as a probe. Three normal and two ataxia-telangiectasia (A-T) group D, non transformed fibroblast cell lines produced a 2.4 kb mRNA transcript. The size of mRNA transcripts seen and the level of expression differ in different human tissues. Many tissues have multiple transcripts of ATDC and the most prominent transcripts observed were 3.0, 2.4 and 1.6 kb. Two out of three SV-40 transformed normal and one SV-40-transformed A-T group D fibroblast cell lines demonstrated no transcription of ATDC by RNA blotting analysis. These results suggest that SV 40 transformation may affect the expression of ATDC. Reverse transcription polymerase chain reaction analysis showed that ATDC was expressed at low levels in all of these cells. Additional Northern blot analysis demonstrated that X irradiation with 10 Gy had no effect on ATDC expression at 1, 4 and 24 h after irradiation in either SV-40-transformed normal or in SV-40-transformed A-T group D fibroblast cell lines. Further understanding of ATDC will require cloning of additional transcripts and studies of ATDC protein behaviour. PMID- 7530756 TI - Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity. AB - Autoantibodies from many patients with systemic lupus erythematosus bind the Sm autoantigen B/B' polypeptide. The binding of serial serum specimens to the 233 overlapping octapeptides of Sm B/B' have shown that of the B/B'-derived octapeptides, PPPGMRPP and PPPGIRGP are early targets of the autoimmune response in some lupus patients. Rabbits immunized with PPPGMRPP and PPPGIRGP develop antibodies which not only bind these octapeptides, but also subsequently bind many other octapeptides of Sm B/B'. Eventually, the rabbits immunized with one octapeptide develop autoantibodies that bind other spliceosomal proteins including D, 70K, A, and C. Any mechanisms that operate to maintain tolerance or anergy for the spliceosome are thus overcome. Features considered typical of human systemic lupus erythematosus are also found in these peptide-immunized animals, such as antinuclear antibodies, anti-Sm precipitins, anti-double stranded DNA, thrombocytopenia, seizures, and proteinuria. This disease model provides access to a mechanism for the development of humoral autoimmunity and may provide a basis to explain the immunopathogenesis of lupus in humans. PMID- 7530757 TI - The development of T and non-T cell lineages from CD34+ human thymic precursors can be traced by the differential expression of CD44. AB - In addition to T-lineage cells, a small proportion of hematopoietic non-T cells are present in the human postnatal thymus. However, the origin of this minor non T cell thymic compartment is presently unknown. In this study we have analyzed the developmental potential of the earliest human intrathymic precursors, characterized as CD34+ cells expressing intermediate levels of CD44. We show that these CD34+CD44int thymocytes cultured with interleukin 7 were able to develop simultaneously into both T- and non-T (monocytes and dendritic cells) -lineage cells. Both developmental pathways progress through a CD1+CD4+ intermediate stage, currently believed to be the immediate precursor of double positive thymocytes. However, separate progenitors for either T or non-T cells could be characterized within CD1+CD4+ thymocytes by their opposite expression of CD44. Downregulated levels of CD44 identified CD1+CD4+ T-lineage precursors, whereas CD44 upregulation occurred on CD1+CD4+ intermediates that later differentiated into non-T cells. Therefore, commitment of human early intrathymic precursors to either T or non-T cell lineages can be traced by the differential expression of the CD44 receptor. PMID- 7530758 TI - Selective apoptosis of CD4+CD8+ thymocytes by the anti-Fas antibody. AB - Fas is a cell surface protein that mediates apoptosis. A mouse mutant, lpr (lymphoproliferation), has a mutation in the Fas gene. In this report, we studied the expression and function of Fas in various subpopulations of mouse thymocytes. Abundant expression of Fas was detected on CD4+CD8+ double positive as well as CD4+ or CD8+ single positive thymocytes in wild-type mice. Little or low levels of Fas were expressed in CD4-CD8- double negative thymocytes except for the CD4 CD8-CD3+ phenotype, which expresses Fas as abundantly as double positive or single positive subsets. On the other hand, no Fas expression was detected in any population of thymocytes from lpr mice. When the wild-type thymocytes were treated with the agonistic anti-Fas antibody, double positive cells from the wild type mice were selectively killed by apoptosis, whereas, the single positive cells were resistant to its cytolytic activity despite their abundant expression of Fas. Unlike the apoptosis of thymocytes induced by glucocorticoid or T cell activator, the Fas-induced apoptosis of thymocytes was enhanced by metabolic inhibitors such as cycloheximide. Furthermore, intraperitoneal administration of the anti-Fas antibody into mice caused rapid apoptosis of thymocytes in vivo. PMID- 7530759 TI - Intraislet release of interleukin 1 inhibits beta cell function by inducing beta cell expression of inducible nitric oxide synthase. AB - Cytokines, released in and around pancreatic islets during insulitis, have been proposed to participate in beta-cell destruction associated with autoimmune diabetes. In this study we have evaluated the hypothesis that local release of the cytokine interleukin 1 (IL-1) by nonendocrine cells of the islet induce the expression of inducible nitric oxide synthase (iNOS) by beta cells which results in the inhibition of beta cell function. Treatment of rat islets with a combination of tumor necrosis factor (TNF) and lipopolysaccharide (LPS), conditions known to activate macrophages, stimulate the expression of iNOS and the formation of nitrite. Although TNF+LPS induce iNOS expression and inhibit insulin secretion by intact islets, this combination does not induce the expression of iNOS by beta or alpha cells purified by fluorescence activated cell sorting (Facs). In contrast, IL-1 beta induces the expression of iNOS and also inhibits insulin secretion by both intact islets and Facs-purified beta cells, whereas TNF+LPS have no inhibitory effects on insulin secretion by purified beta cells. Evidence suggests that TNF+LPS inhibit insulin secretion from islets by stimulating the release of IL-1 which subsequently induces the expression of iNOS by beta cells. The IL-1 receptor antagonist protein completely prevents TNF+LPS induced inhibition of insulin secretion and attenuates nitrite formation from islets, and neutralization of IL-1 with antisera specific for IL-1 alpha and IL-1 beta attenuates TNF+LPS-induced nitrite formation by islets. Immunohistochemical localization of iNOS and insulin confirm that TNF+LPS induce the expression of iNOS by islet beta cells, and that a small percentage of noninsulin-containing cells also express iNOS. Local release of IL-1 within islets appears to be required for TNF+LPS-induced inhibition of insulin secretion because TNF+LPS do not stimulate nitrite formation from islets physically separated into individual cells. These findings provide the first evidence that a limited number of nonendocrine cells can release sufficient quantities of IL-1 in islets to induce iNOS expression and inhibit the function of the beta cell, which is selectively destroyed during the development of autoimmune diabetes. PMID- 7530760 TI - Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. AB - Mice homozygous for the lpr gene have a defect in fas (CD95), a cell surface receptor that belongs to the tumor necrosis factor receptor family and that mediates apoptosis. This genetic abnormality results in lymphoproliferation characterized by the accumulation of CD4-CD8- (double negative [DN]) T cells, autoantibody production, and background strain-dependent, end-organ disease. Our previous results suggested that major histocompatibility complex (MHC) class I may be involved in the development of DN cells. To test this hypothesis, we derived C57BL/6-lpr/lpr (B6/lpr) mice that were deficient for the beta 2 microglobulin gene (beta 2m lpr) and had no detectable class I expression. At 6 mo of age, compared with B6/lpr littermates with normal class I genes, these mice showed greatly reduced lymphadenopathy, mostly due to a dramatic decrease in the number of DN cells. Significant changes in the percentage of other T cell subsets were noted, but only gamma/delta+ T cells showed a marked increase in both percentage and absolute numbers. Analysis of T cell receptor V beta expression of the remaining DN T cells in beta 2m -lpr mice showed a shift to a CD4-like repertoire from a CD8-like repertoire in control B6/lpr mice, indicating that a small MHC class II selected DN population was unmasked in lpr mice lacking class I. We also found that the production of immunoglobulin G (IgG) autoantibodies (antichromatin and anti-single stranded DNA), total IgG and IgG2a, but not total IgM or IgM rheumatoid factor, was significantly reduced in the beta 2m -lpr mice. This work suggests that >90% of DN T cells in lpr mice are derived from the CD8 lineage and are selected on class I. However, a T cell subset selected on class II and T cells expressing gamma/delta are also affected by the lpr defect and become minor components of the aberrant DN population. PMID- 7530762 TI - Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease. AB - Mononuclear phagocytes (monocytes, macrophages, and dendritic cells) play major roles in human immunodeficiency virus (HIV) persistence and disease pathogenesis. Macrophage antigen presentation and effector cell functions are impaired by HIV-1 infection. Abnormalities of macrophage effector cell function in bone marrow, lung, and brain likely result as a direct consequence of cellular activation and HIV replication. To further elucidate the extent of macrophage dysfunction in HIV 1 disease, a critical activation-specific regulatory molecule, nitric oxide (NO.), which may contribute to diverse pathology, was studied. Little, if any, NO. is produced by uninfected human monocytes. In contrast, infection with HIV-1 increases NO. production to modest, but significant levels (2-5 microM). Monocyte activation (with lipopolysaccharide, tumor necrosis factor alpha, or through interactions with astroglial cells) further enhances NO. production in HIV infected cells, whereas its levels are diminished by interleukin 4. These results suggest a possible role for NO. in HIV-associated pathology where virus-infected macrophages are found. In support of this hypothesis, RNA encoding the inducible NO synthase (iNOS) was detected in postmortem brain tissue from one pediatric AIDS patient with advanced HIV encephalitis. Corresponding iNOS mRNA was not detected in brain tissue from five AIDS patients who died with less significant brain disease. These results demonstrate that HIV-1 can influence the expression of NOS in both cultured human monocytes and brain tissue. This newly described feature of HIV-macrophage interactions suggests previously unappreciated mechanisms of tissue pathology that result from productive viral replication. PMID- 7530761 TI - Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. AB - Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60 120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin deficient mice is completely blocked by L-selectin mAb. These data show that P selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice. PMID- 7530763 TI - TCR/CD3 coupling to Fas-based cytotoxicity. AB - We studied the coupling of the TCR/CD3 complex to a T cell effector function, namely Fas-based T-cell-mediated cytotoxicity. Encounter or re-encounter with antigen was mimicked by treating 5 d mixed lymphocyte culture cells or T cell hybridomas with anti-CD3 antibody. This TCR/CD3 engagement induced swift expression of Fas-based cytotoxicity in these cells. Induction of Fas-based cytotoxicity was Ca(2+)-dependent, while its execution was not; induction was sensitive to macromolecular synthesis inhibitors, in line with a demonstrable increase of the Fas ligand (Fas-L) message. We also used T cell hybridomas transfected with various constructs to dissect the involvement of distinct components of the TCR/CD3 complex. The cytoplasmic domain of the CD3 zeta chain was able to transduce by itself a signal leading to Fas-L expression, unless there were mutations in its activation receptor homology sequence 1 (ARH-1) motifs. On the one hand, these findings are relevant to signal transduction pathways coupled to the TCR/CD3, and on the other hand, to the involvement of Fas based T cell-mediated cytotoxicity in various physiological and possibly pathophysiological situations. PMID- 7530764 TI - Functional NF-IL6/CCAAT enhancer-binding protein is required for tumor necrosis factor alpha-inducible expression of the granulocyte colony-stimulating factor (CSF), but not the granulocyte/macrophage CSF or interleukin 6 gene in human fibroblasts. AB - Tumor necrosis factor (TNF) alpha participates in the regulation of the acute phase, immune, and inflammatory responses. Target genes known to be transcriptionally activated by TNF-alpha include the granulocyte (G)-colony stimulating factor (CSF) gene, the granulocyte/macrophage (GM)-CSF gene, as well as the interleukin (IL) 6 gene. Functional nuclear factor (NF)-IL6 recognition sites have been identified in regulatory regions of these genes by transient transfection studies using deleted promoter constructs. In addition, NF-IL6 is known to form heterodimeric complexes with the NF-kappa B transcription factor, which is also engaged in the transcriptional regulation of these genes. The indispensable importance of NF-IL6 for regulating gene expression of proinflammatory cytokine genes in response to inflammatory stimuli in vivo remains, however, unclear. We here report, by using both antisense (AS) oligodesoxyribonucleotide (ODN) and ribozyme (RZ)-mediated specific elimination of NF-IL6 transcripts in human fibroblasts, that TNF-alpha-induced synthesis of G CSF, but not of GM-CSF or IL-6, is abolished in the absence of functional NF-IL6 in vivo. Both AS ODN and RZ targeting of the NF-IL6 transcript eliminate NF-IL6 protein, as shown in Western blot analysis and electrophoretic mobility shift assays. Similarly, fibroblasts exposed to either the AS NF-IL6 ODN or the NF-IL6 RZ, but not to the sense or nonsense ODN or a mutated ribozyme, also failed to respond with functional activation of NF-IL6 as assayed in transient transfection studies using heterologous promoter constructs harboring the NF-IL6 recognition site. In contrast, protein synthesis, DNA-binding activity, and transcriptional activation capacity of the NF-kappa B transcription factor is not impaired upon exposure to either ODN or RZ. Fibroblasts that had been cultured in the presence of the AS NF-IL6 ODN or the NF-IL6RZ failed to synthesize G-CSF protein in response to TNF-alpha, while TNF-alpha-inducible transcription and release of GM CSF and IL-6 was preserved. PMID- 7530765 TI - Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma. AB - Vascular cell adhesion molecule 1 (VCAM-1) mediates extravasation of circulating leukocytes into inflamed tissues, and presumably, plays a role in the immigration of cytotoxic effector lymphocytes into tumor metastases. Since metastases are rarely cleared by blood-borne cells from the immune system, we asked whether the tumor may escape host defense by interfering with the mechanism of effector cell extravasation. Here we show that in mice and humans, VCAM-1 expression is repressed on tumor-infiltrating vascular endothelial cells in the lungs. On lung blood vessels distant from the tumor, VCAM-1 is constitutively expressed. When melanoma and endothelioma cells were cultured on either side of a Nucleopore membrane, the expression of VCAM-1 on the endothelioma cells was inhibited and VCAM-1 gene transcription was suppressed. We propose that the downregulation of VCAM-1 is a mechanism by which vascularized melanoma and carcinoma avoid invasion by cytotoxic cells of the immune system. PMID- 7530766 TI - Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers. AB - Lipid metabolites, free fatty acids and lysophospholipids, modify the function of membrane proteins including ion channels. Such alterations can occur through signal transduction pathways, but may also result from "direct" effects of the metabolite on the protein. To investigate possible mechanisms for such direct effects, we examined the alterations of gramicidin channel function by lysophospholipids (LPLs): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The experiments were done on planar bilayers formed by diphytanoylphosphatidylcholine in n-decane a system where receptor mediated effects can be excluded. At aqueous concentrations below the critical micelle concentration (CMC), LPLs can increase the dimerization constant for membrane-bound gramicidin up to 500-fold (at 2 microM). The relative potency increases as a function of the size of the polar head group, but does not seem to vary as a function of head group charge. The increased dimerization constant results primarily from an increase in the rate constant for channel formation, which can increase more than 100-fold (in the presence of LPC and LPI), whereas the channel dissociation rate constant decreases only about fivefold. The LPL effect cannot be ascribed to an increased membrane fluidity, which would give rise to an increased channel dissociation rate constant. The ability of LPC to decrease the channel dissociation rate constant varies as a function of channel length (which is always less than the membrane's equilibrium thickness): as the channel length is decreased, the potency of LPC is increased. LPC has no effect on membrane thickness or the surface tension of monolayers at the air/electrolyte interface. The bilayer-forming glycerolmonooleate does not decrease the channel dissociation rate constant. These results show that LPLs alter gramicidin channel function by altering the membrane deformation energy, and that the changes in deformation energy can be related to the molecular "shape" of the membrane modifying compounds. Similar alterations in the mechanical properties of biological membranes may form a general mechanism by which one can alter membrane protein function. PMID- 7530767 TI - Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro. AB - Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy. PMID- 7530768 TI - Polyclonal antibodies against NCAM and tenascin delay endplate reinnervation. AB - Experiments were performed to block molecules with antibodies which are upregulated in nerve and muscle following denervation. The delay in endplate reinnervation was taken as a measure for their involvement in regeneration. Gluteus maximus muscles of 86 male CBA/J mice were hemidenervated by freezing the caudal gluteal nerve at a defined position. The degree of reinnervation was evaluated in identified endplates by repeated vital staining of ACh receptors with rhodaminated alpha-bungarotoxin and of axons with 4Di-2ASP. Normally, endplates were completely reinnervated by 13-14 days (108 endplates in seven muscles). After daily application of polyclonal antibodies against NCAM or tenascin, reinnervation was significantly delayed. Preimmune serum, rabbit immunoglobulins or saline did not show this effect. Several monoclonal antibodies against NCAM (H-28) and tenascin (576, 578, 630, 633) showed a tendency but no significant effect. It is concluded that both NCAM and tenascin, upregulated after denervation, are involved in axon guidance and/or endplate reinnervation. PMID- 7530770 TI - Use of cultured human neuroblastoma cells in rapid discovery of the voltage-gated potassium-channel blockers. AB - Depolarization of human neuroblastoma cells by high concentrations of extracellular potassium ions, leads to the activation of the voltage-gated potassium channels. The activity of such potassium channels can be effectively and rapidly monitored by tracking the efflux of 86Rb from pre-loaded target cells in response to the depolarizing stimulus. The inclusion of compounds with unknown activity in the assay medium, can result in the identification of novel blockers of the voltage-gated potassium channels. Since this functional assay is performed in 96-well microtitre plates, it represents a rapid and high-volume primary screening method for the detection and identification of the voltage-gated potassium-channel blockers, which may have therapeutic utility in several indications including memory degeneration and cardiac arrhythmias. PMID- 7530769 TI - The myelin basic protein gene is not a major susceptibility locus for multiple sclerosis in Italian patients. AB - To verify whether multiallelic polymorphism adjacent to the gene encoding for myelin basic protein is associated with or linked to multiple sclerosis in Italians, we studied 54 sporadic patients, 55 control subjects and 18 families with two or more affected individuals. Allelic typing was carried out by analysis of fragment length polymorphisms after DNA amplification by the polymerase chain reaction. The presence of linkage with the disease was tested according to either autosomal dominant or autosomal recessive modes of inheritance, and with or without the introduction of liability classes accounting for the age of the individuals. Furthermore sib-pair analysis was performed in 11 siblings. No evidence for association or linkage between the myelin basic protein gene polymorphism and multiple sclerosis was found. Our data indicate that in the Italian population the myelin basic protein gene does not play a major role in conferring genetic susceptibility to multiple sclerosis, and suggest that the latter is a heterogeneous phenomenon, possibly influenced by the different ethnic origin of the populations which have been investigated. PMID- 7530771 TI - Modifications of A-current kinetics in mammalian central neurones induced by extracellular zinc. AB - 1. Whole-cell voltage clamp recordings were used to study the action of the transition ion zinc on the A-current kinetics in granule cells from rat cerebellar slices. 2. The effects of zinc have been tested in the concentration range from 1 microM to 1 mM, and fully characterized on all kinetic parameters at 100 and 300 microM. All the effects observed were rapid, concentration dependent and fully reversible. 3. Steady-state inactivation curves are strongly shifted towards depolarized potentials, with activation curves much less so. These shifts lead to an increase of the peak current amplitude around physiological resting membrane potentials and to a decrease at hyperpolarized potentials. 4. The forward 'on' rate constants are slowed by Zn2+ at a concentration of 100-300 microM by a factor from 1.5 to 4. The backward 'off' rate constants are unaffected by Zn2+. 5. The development of IA inactivation, as measured from the current decay, is not affected by Zn2+ up to 1 mM. Removal of inactivation is, on the contrary, significantly slowed. 6. The results are neither compatible with the theory of the surface charge screening effect nor with a mechanism involving channel block. It seems more likely that Zn2+ interferes with the channel gating by binding to a specific domain of the channel protein. 7. After treatment with Hg2+, which is irreversible, Zn2+ still maintains its effects, which suggest that the two divalents act at different sites. 8. In view of the widespread distribution of zinc throughout the brain, its actions on the A-current could play an important role in physiological function. PMID- 7530772 TI - Expression of a developmentally regulated epitope on fibronectins from the synovial fluid of patients with rheumatic disease. AB - OBJECTIVE: To determine if differential glycosylation of fibronectin (Fn) in inflammatory synovial fluid (SF) included expression of an oncofetal epitope (Onf Fn) heretofore detected only on Fn derived from embryonal or neoplastic tissue. METHODS: Fn were purified from plasma, SF and synoviocyte conditioned medium by affinity chromatography and were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting utilizing a monoclonal antibody (FDC-6) specifically recognizing the Onf Fn. RESULTS: The Onf Fn was not expressed on Fn isolated from normal or RA plasma but was strongly expressed on Fn from rheumatoid arthritis (RA) SF and to a lesser extent osteoarthritis SF. Onf Fn was also detected on Fn secreted by cultured RA synoviocytes. CONCLUSION: Fn present in the SF but not plasma of patients with rheumatic disease contains an epitope previously thought to be restricted to Fn produced by embryonal or malignant tissue. PMID- 7530773 TI - Derivatives of cis-2-amino-8-hydroxy-1-methyltetralin: mixed 5-HT1A-receptor agonists and dopamine D2-receptor antagonists. AB - (1S,2R)-8-Hydroxy-1-methyl-2-(dipropylamino)tetralin [(1S,2R)-3] has been previously characterized as a selective and potent but partial 5-HT1A-receptor agonist. In the present study, we have prepared derivatives of (1S,2R)- and (1R,2S)-3 in which various C8-substituents have been introduced. In addition, the enantiomers of the N-isopropyl-N-n-propylamino derivative of 3 were prepared. The new derivatives were tested in vivo by use of behavioral and biochemical tests in rats. In addition, the affinity of the compounds was studied by competition experiments with [3H]-8-OH-DPAT in rat brain tissue. The only new derivative which behaved like a selective 5-HT1A-receptor agonist was the C8-carboxamide derivative (1S,2R)-13. The other active derivatives, including (1S,2R)-3, have more complicated pharmacological profiles and may be best characterized as mixed 5-HT1A-receptor agonists/dopamine D2-receptor antagonists. PMID- 7530774 TI - Linkage and mutation analysis in an extended family with Charcot-Marie-Tooth disease type 1B. AB - Charcot-Marie-Tooth disease type 1 (CMT1) or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most families the disease segregates with a 1.5 Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with point mutations in the PMP-22 gene. In some families linkage has been found with markers located on chromosome 1q21 q25 (CMT1B) and more recently mutations have been identified in the P0 gene. We analysed an extended CMT1 pedigree (CMT-B) without the CMT1A duplication. Significant positive linkage with chromosome 1 indicated that this family is of the CMT1B subtype. Sequencing of the candidate gene P0 located in chromosome band 1q21-q23 showed a C to A point mutation at position 446 in exon 3 resulting in an Asp134Glu substitution. Since the P0 mutation cosegregated with CMT1 disease we suggest that this mutation is the primary genetic cause of CMT1B in family CMT-B. PMID- 7530776 TI - Axonal transport of type III intermediate filament protein peripherin in intact and regenerating motor axons of the rat sciatic nerve. AB - Slow axonal transport of peripherin has been studied in the motor axons of both intact and regenerating rat sciatic nerves 7 days post-crush. The studies were done by two-dimensional gel electrophoresis after intraspinal injection of 35S methionine. In the first experiment, the sciatic nerves were removed 3 weeks after the radiolabeling pulse and cut into 6 mm segments. Each nerve segment was submitted to two-dimensional gel electrophoresis and analyzed by an original procedure which allowed us to study the distribution along the nerve of the radioactivity associated with several proteins of the cytoskeleton, especially the intermediate filament proteins, peripherin, and the low molecular mass neurofilament protein, NF-L. Peripherin was transported at two main rates: 66% of the total radiolabeled peripherin moved at 1.42 mm/day and the remainder moved at 2.28 mm/day. The radioactivity associated with NF-L exhibited a similar pattern. In the second experiment, similar intraspinal injections were made 7 days after a unilateral crush of the sciatic nerve. Regenerating nerves exhibited a clear SCa wave. However, in contrast to the intact nerves, the SCb wave could not be precisely defined in the regenerating nerves. Thus, the changes in the amount of transported proteins were analyzed in the SCa wave only. Autoradiograms of 2D PAGE revealed that in the regenerating axons, the quantity of transported peripherin in SCa was increased by 3.5-fold. In contrast, the quantity of transported NF-L was decreased by 1.6-fold. The regenerating motor axons conveyed significantly greater (approximately twofold) amounts of labeled tubulins and actin than did intact motor axons. Our results suggest that peripherin, although mainly conveyed by SCa, plays a role during the elongation process in addition to actin and tubulin. PMID- 7530775 TI - The insulin-like growth factor I system in the rat cerebellum: developmental regulation and role in neuronal survival and differentiation. AB - The developmental regulation of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF-I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF-I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF-I receptor mRNA was found in adults. Levels of IGF-I and its mRNA also peaked at early ages, although immunoreactive IGF-I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF-I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF-I on developing cerebellar cells in vitro. We found a dose dependent effect of IGF-I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell-specific markers indicated that both Purkinje cells (calbindin-positive) and other neurons (neurofilament-positive) contain IGF-I binding sites. These results extend previous observations on a developmental regulation of the IGF-I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF-I in cerebellar development. PMID- 7530777 TI - Patterns of reactivity with anti-glycolipid antibodies in human primary brain tumors. AB - Antibodies against carbohydrates of three glycolipids were used to determine patterns of immunohistochemical reactivity of histologically identifiable cell subpopulations in 101 human primary brain tumors. For all tumor types fibrillary cells, polar cells, and gemistocytes (commonly seen in astrocytomas and ependymomas) stained more frequently for galactosylcerebroside with mAbO1 than small tumor cells and macrophages. Frequency of staining for sulfatide with mAbO4 was fibrillary > polar > small cells = macrophages. Gemistocytes stained more frequently with mAbO4 than polar cells in all tumors except low grade astrocytomas. These data indicate that tumors classified on histological grounds as astrocytic are often stained with antibodies that recognize oligodendrocytes and their progenitors. Thus, anti-glycolipid antibodies used in the study of developmental lineage may offer useful tools for classification of human brain tumors. Staining of fibrillary cells, polar cells, and gemistocytes for paragloboside directly with mAb F1H11 was much less common than with mAbO1, but this increased by pretreatment of the tissues with neuraminidase (F1H11 + N). Of particular note was the finding that small tumor cells frequently stained with F1H11 + N. Evidence that these were not macrophages was obtained using double immunostaining with F1H11 + N and anti-macrophage antibodies. In astrocytomas the frequency of small tumor cells immunostained with F1H11 + N was high grade > anaplastic > low grade, demonstrating a correlation of this tumor cell population with more aggressive astrocytomas. Thus, immunostaining with F1H11 + N may be of value in identifying small, anaplastic tumor cells, especially in small biopsies or tissue taken adjacent to the main tumor mass. PMID- 7530778 TI - Solubilization of full-length amyloid precursor proteins from PC12 cell membranes. AB - The amyloid beta protein (A beta) of Alzheimer disease (AD) is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane proteins. Production of A beta from a transmembrane precursor predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Here we show that incubation of a membrane fraction of PC12 cells at 37 degrees C results in the solubilization of an APP species which migrates on SDS-PAGE as full-length APP. The release of this full-length APP was pH-dependent with a peak of activity of pH 9.0. At this pH about 19% of the membrane APP was released from the active subcellular fraction. Under the same conditions other transmembrane proteins remained insoluble. Very little APP was solubilized at 4 degrees C. APP solubilization was specifically inhibited by the serine protease inhibitors aprotinin and pefabloc. Other protease inhibitors, including leupeptin and alpha 1-antitrypsin, had no effect. Several metal cations, including Ca++ and Zn++, also inhibited release of soluble full-length APP. Low levels of full-length APP were also detected in both the soluble fraction of PC12 cell extracts and in the media of PC12 cell cultures. These data suggest the involvement of a serine protease in the solubilization of membrane, full-length APP. The release of this APP could provide a soluble substrate for the proteolytic enzymes involved in the production of A beta. PMID- 7530779 TI - Colchicine toxicity--clinical features and treatment. Massive overdose case report. AB - This is a report of colchicine poisoning in a 24-year-old woman. She developed multiple organ failure and bone marrow suppression after the suicidal ingestion of 50 (1 mg) colchicine tablets. The pancytopenia responded to granulocyte colony stimulating factor 300 micrograms on days 4, 5, 6, and 8. Although anticolchicine monoclonal antibody administration is the only specific therapy described, intensive supportive care including granulocyte colony-stimulating factor administration can facilitate recovery from severe colchicine intoxication. PMID- 7530780 TI - Ultrastructure, ZIO-staining and chromaffinity of gerbil pinealocytes. AB - The ultrastructure and cytochemistry of the gerbil pineal gland were studied by the conventional electron microscopy, zinc iodide-osmium tetroxide (ZIO) staining and chromaffin reaction. Conventional electron microscopy revealed that the ultrastructure of gerbil pinealocytes are similar to other rodents, i.e., irregular cell contour with numerous cytoplasmic processes, round or oval nucleus and prominent nucleoli, elongated mitochondria with flattened and tubular cristae and dense matrix, well-developed Golgi apparatus and its associated structures, abundant elements of endoplasmic reticulum--both smooth and rough varieties, and bundles of microfilament and microtubule in the cytoplasm. Some pinealocyte processes contain numerous small clear and "slightly coated" vesicles. Numerous profiles of varicosities containing small dense-cored and clear vesicles were frequently encountered. After ZIO treatment, ZIO staining was preferentially localized in the cytoplasm of some, but not all, of the gerbil pinealocytes. Numerous small clear vesicles (30-50 nm in diameter) in the process of the pinealocytes or in the varicosities of the nerve fibers showed strong ZIO-philia. After chromaffin reaction treatment, the number and electron density of small clear and dense-cored vesicles in the profiles of nerve varicosities increased and this indicates that some of the small clear and dense-cored vesicles in the varicosities are reactive. It is thus concluded that (1) the vesicles in the pinealocytes may be rich in cystine and/or cysteine and possibly the organelle is involved in the sequestering calcium ion during the calcification of the pineal concretions, and (2) the small dense-cored and clear vesicles in the nerve fibers in the gerbil pineal parenchyma may contain both serotonin and primary biogenic amines. PMID- 7530781 TI - Tourette's syndrome. A model neuropsychiatric disorder. PMID- 7530782 TI - The role of increasing detection in the rising incidence of prostate cancer. AB - OBJECTIVE: To assess the reasons for the dramatic surge in prostate cancer incidence from 1986 to 1991. DESIGN: Population-based study of incidence rates and procedures used to detect and diagnose prostate cancer derived from Medicare claims data and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program from 1986 to 1991. SETTING: Four SEER areas (Connecticut; Atlanta, Ga; Detroit, Mich; and Seattle--Puget Sound, Wash) covering approximately 6% of the US population. PARTICIPANTS: A 5% random sample of male fee-for-service Medicare beneficiaries aged 65 years and older without cancer, and all men with prostate cancer diagnosed at 65 years of age and older residing in the four areas. MAIN OUTCOME MEASURES: The age-adjusted rates of prostate cancer incidence, prostate needle biopsy, transurethral resection of the prostate, serum prostate-specific antigen (PSA) testing, and transrectal ultrasound. RESULTS: The age-adjusted incidence rate of prostate cancer among men aged 65 years and older in the four SEER areas rose 82% from 1986 to 1991, with the largest annual increases occurring in 1990 (20%) and 1991 (19%). Prostate needle biopsy rates increased while the use of transurethral resection of the prostate declined from 1986 to 1991. The rising needle biopsy rate has been driven by an exponential increase in PSA testing in the general population from 1988 to 1991 and, to a much lesser extent, the increasing use of transrectal ultrasound since 1986. The use of PSA or transrectal ultrasound has increased across age and race groups and in different geographic areas. However, there remain wide geographic variations in the use of PSA screening. CONCLUSIONS: The recent dramatic epidemic of prostate cancer is likely the result of the increasing detection of tumors resulting from increased PSA screening. The magnitude and rapidity of the incidence rise suggest that changes in the intensity of medical surveillance is the most plausible explanation for this trend. IMPLICATIONS: The rapid diffusion of screening interventions that have the ability to detect latent asymptomatic disease leads to important concerns regarding costs and patient quality of life for men aged 65 years and older. Geographic variability in the adoption of PSA testing underscores uncertainty and disagreement about its value for reducing prostate cancer mortality. More research is required to determine the effectiveness of screening for prostate cancer. PMID- 7530783 TI - Molecular assays for chromosomal translocations in the diagnosis of pediatric soft tissue sarcomas. AB - OBJECTIVE: To compare molecular assays for characteristic chromosomal translocations with standard histopathologic and cytogenetic analysis in the differential diagnosis of pediatric soft tissue sarcomas. DESIGN: Blinded comparison with histopathologic diagnosis. SETTING: Tertiary care children's hospital. PATIENTS: A total of 79 soft tissue sarcoma patients with frozen tumor tissue and histopathologic slides available for review. METHODS: The RNA from the tumors was assayed by the reverse transcriptase-polymerase chain reaction. These assays detect PAX3-FKHR and PAX7-FKHR chimeric transcripts in alveolar rhabdomyosarcoma, EWS-FLI1 and EWS-ERG chimeric transcripts in Ewing's sarcoma, and EWS-WT1 chimeric transcripts in desmoplastic small round cell tumor. MAIN OUTCOME MEASURES: The polymerase chain reaction findings were compared with cytogenetic and histopathologic results. RESULTS: These assays detected chimeric transcripts in all cases in which translocations were found by standard cytogenetics as well as additional cases without cytogenetically detectable translocations. PAX3-FKHR or PAX7-FKHR fusions were present in 18 of 21 alveolar rhabdomyosarcomas, two of 30 embryonal rhabdomyosarcomas, and one of seven undifferentiated sarcomas. EWS-FLI1 or EWS-ERG fusions were detected in six of eight Ewing's sarcomas and one of seven undifferentiated sarcomas. The EWS-WT1 fusion was found in three of three desmoplastic small round cell tumors. CONCLUSIONS: Molecular assays for specific gene fusions provide a genetic approach to the differential diagnosis of soft tissue sarcomas. The genetic categories correspond closely to the standard histopathologic categories. The polymerase chain reaction assays for chimeric transcripts are useful tools for the rapid and objective assessment of pediatric soft tissue sarcomas. PMID- 7530784 TI - [Whipple's disease]. PMID- 7530785 TI - [AFP-producing intestinal cancer]. PMID- 7530787 TI - Isolated dihydroxyacetonephosphate acyltransferase deficiency presenting with developmental delay. AB - A boy aged 21 months who was being investigated for developmental delay and failure to thrive was found to have punctate epiphyseal calcification. He had no evidence of rhizomelic shortening of the limbs or cataracts. Investigation revealed defective plasmalogen synthesis due to isolated deficiency of dihydroxyacetonephosphate acyltransferase (DHAP-AT). The parents were consanguineous and a sister was similarly affected, suggesting autosomal recessive inheritance. Hitherto, recessively inherited isolated DHAP-AT deficiency has only been described in patients with a phenotype similar to that of rhizomelic chondrodysplasia punctata. This report indicates that the same biochemical disorder can be associated with a less severe phenotype. PMID- 7530786 TI - [Effects of ileal perfusion of carbohydrates on upper GI motility and pancreatic exocrine secretion]. AB - The aim of this study was to determine if upper gut function and pancreatic exocrine secretion are affected by the presence of unabsorbed carbohydrate in the distal ileum. In this investigation, starch or glucose was perfused into the canine ileum. This study showed that ileal perfusion of both starch and glucose delayed gastric emptying and prolonged small bowel transit time. Furthermore, glucose had greater effects on gastrointestinal transit time than starch. In contrast, amylase output was increased by ileal perfusion of starch, but was not affected by that of glucose. Our results suggest that the ileal regulation of gastrointestinal transit and amylase output could be mediated by different mechanisms. PMID- 7530788 TI - Reduced stimulability of platelet surface adhesion molecules under treatment with Lorenzo's oil. PMID- 7530789 TI - Cytokine modulation of interleukin 1 and tumour necrosis factor-alpha secretion from acute myelogenous leukaemia blast cells in vitro. AB - Spontaneous secretion of interleukin 1 (IL-1) alpha, IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) from acute myelogenous leukaemia (AML) blasts showed significant correlation, and detectable levels of all cytokines were seen for a majority of patients. IL-3 and granulocyte/macrophage colony-stimulating factor increased secretion of IL-1 alpha, IL-1 beta and TNF-alpha for a majority of AML patients, whereas IL-4 decreased cytokine secretion. The effect of IL-6 and stem cell factor on cytokine secretion varied between different patients. A wide variation in IL-1 alpha, IL-1 beta and TNF-alpha secretion between different patients was seen both for spontaneous secretion and in the presence of all cytokines. PMID- 7530791 TI - Differential effects on sympathetic neurotransmission of mast cell degranulation by compound 48/80 or antigen in the rat isolated perfused heart. AB - The aim of the present study was to investigate whether or not release of endogenous mast cell mediators modulates exocytotic noradrenaline overflow. Therefore, we perfused rat isolated hearts with the right sympathetic innervation intact and investigated the effect of mast cell degranulation on the efflux of noradrenaline. Compound 48/80 (48/80), a mast cell degranulating agent, caused a large release of histamine and serotonin and a facilitation of evoked noradrenaline overflow. When 48/80 was introduced into the perfusion medium 4 min before sympathetic nerve stimulation (SNS), evoked noradrenaline overflow was increased by about 60%. In the presence of the uptake 1-blocker cocaine, facilitation was attenuated (increase by only 30%). This effect was abolished by the histamine H2 receptor antagonist cimetidine or the inhibitor of nitric oxide synthesis NG-nitro-(L)-(-)-arginine. When the preexposure time to 48/80 was reduced to 30 s, the facilitation was less pronounced (15%) and inverted to an inhibition in the presence of cocaine (plus idazoxan) by 17% and/or cimetidine (by about 30%). The resulting inhibition of noradrenaline efflux was attenuated by the serotonin 5-HT1/2 receptor antagonist methiothepin or the 5-HT2 antagonist ketanserin. Infusion of ovalbumin into hearts of not specifically sensitized, but sham treated rats (in vivo injection of a saline-alumina mixture 10-12 days before the in vitro experiment) did not affect histamine, serotonin or (basal and evoked) noradrenaline efflux. In hearts from rats that were previously sensitized by an injection of an ovalbumin-alumina adsorbate, ovalbumin induced a marked increase of histamine and serotonin efflux. When the infusion of the antigen started 30 s before SNS, evoked noradrenaline overflow was inhibited by about 60%. The inhibition was unaffected by histamine receptor antagonists, but attenuated by purinoceptor (suramin plus 1,3-dipropyl-8-cyclopentylxanthine), or serotonin receptor (methiothepin, rauwolscine or ketanserin) antagonists. When the preexposure time to ovalbumin was prolonged to 4 min before SNS, no significant change of stimulation-induced noradrenaline overflow was observed. Basal, immunologically and non-immunologically induced histamine and serotonin efflux were not significantly affected by SNS or any of the drugs tested. The results indicate a complex influence of various mediators released upon mast cell degranulation induced by two different stimuli on exocytotic noradrenaline release from rat heart. Depending on the stimulus and on the time interval between the start of the application of the mast cell degranulating agent and SNS, a histamine- and nitric oxide-mediated facilitation, or a serotonin- and purine-mediated inhibition prevails. PMID- 7530790 TI - The effect of photogenerated site-directed free radicals on surface dihydropyridine binding sites identified with photoaffinity probe (-)-[3H] azidopine on cultured monkey renal cells. AB - With the use of the photoactivable dihydropyridine (DHP)-type calcium (Ca2+ channel antagonist (-)-[3H]-azidopine, a specific photoaffinity probe for Ca2+ channels, we tested the hypothesis of the existence of a separate subsite in the DHP receptor region on native polarized, stimulated depolarized and UV irradiated green monkey renal (GMR) cells preincubated with selected DHPs. Our results demonstrate that specific binding of (-)-[3H]-azidopine on GMR cells is of high affinity, stereoselective and dependent mainly on the inactivation of the membrane bound Ca2+ channel. Preincubation of the GMR cells with the DHP Ca2+ channel agonist BAY-K-8644 significantly reduced specific photolabeling. The site directed free radicals generated after UV irradiation in DHP-preincubated renal cells inactivated Ca2+ channels and did not significantly affect the specific photoincorporation of (-)-[3H]-azidopine. (+)-Niguldipine, a DHP with the voluminous substituent on the DHP ring, significantly reduced the photolabeling. Low affinity labeling was partially prevented in (+)-nimodipine and (+) niguldipine preincubated photoirradiated cells. The results strongly support the existence of central and peripheral subsites of the DHP region on GMR cells, with the former incorporating on photoactivation the intrinsically photoactive DHPs and with the latter labeled with a side chain bearing nitrene-generating photoreactive group, the photoaffinity probe, (-)-[3H]-azidopine. PMID- 7530792 TI - Cleaved L-selectin concentrations in meningeal leukaemia. AB - Involvement of the central nervous system has important therapeutic implications in acute leukaemia. Because the identification of blast cells in cerebrospinal fluid (CSF) is often difficult, there is a need for sensitive markers of leukaemic infiltration. Since the shed form of L-selectin (sL-selectin) is frequently increased in acute leukaemia (sL-selectin+ leukaemia), we examined whether assay of sL-selectin in CSF could improve our ability to detect such meningeal involvement. CSF sL-selectin was significantly (p < 0.001) higher in 15 patients with sL-selectin+ meningeal leukaemia (median 60 ng/mL, range 34-150) than in 20 patients with acute leukaemia without meningeal involvement (12 ng/mL, 1-39) or 88 control patients (14 ng/mL, 0-37). Serial measurements of sL-selectin in patients with sL-selectin+ leukaemic meningitis showed increased CSF concentrations of the cleaved receptor in 4 patients with therapy-resistant meningeal leukaemia and sustained normal concentrations in 9 patients in remission. Our results suggest that CSF sL-selectin may be a useful marker in the detection of meningeal involvement by blast cells in patients with sL-selectin+ leukaemia. PMID- 7530793 TI - Mother-to-infant transmission of hepatitis C virus. Lombardy Study Group on Vertical HCV Transmission. AB - To assess the risk of mother-to-infant transmission of hepatitis C virus (HCV), we followed up 116 babies of anti-HCV positive mothers, of whom 22 were coinfected with HIV and 94 had HCV alone. None of the babies whose mothers had HCV alone acquired HCV, while 8 babies (36%; p < 0.001) of mothers co-infected with HIV acquired HCV (5 babies) or HCV and HIV (3). There was no association between any specific maternal HCV genotype and enhanced risk of neonatal infection. HCV-RNA levels were significantly higher (p < 0.05) in mothers with HIV coinfection than in those with HCV alone. These data indicate that maternal HIV status correlates with enhanced level of viraemia which favours neonatal infection. PMID- 7530794 TI - Lead-210, polonium-210, and vehicle exhaust pollution. PMID- 7530795 TI - Effect of CD44v6 on survival in colorectal carcinoma. PMID- 7530796 TI - Survival after systemic therapy for metastatic colorectal cancer. PMID- 7530797 TI - Odontogenic keratocysts: a study of 50 patients. AB - The odontogenic keratocyst (OKC) is a locally aggressive neoplasm with rates of recurrence reported as high as 60%. Correlation between histopathology and the likelihood of recurrence remains a subject of controversy. In this review of the authors' experience in treating 50 patients with OKC between 1977 and 1993, 58 specimens were studied to correlate the likelihood of recurrence with the presence of the following histologic features--parakeratosis, orthokeratosis, satellite cysts, epithelial rests, or epithelial proliferation. Orthokeratinized cysts were associated with a higher recurrence rate than in previously reported studies. Disruption of the epithelial lining in the resected specimen was found to be a primary determinant of recurrence. PMID- 7530798 TI - Expression of mRNA for all five human somatostatin receptors (hSSTR1-5) in pituitary tumors. AB - Expression of mRNA for hSSTR1-5 was determined in secretory (GH, PRL, TSH, ACTH) and nonsecretory pituitary tumors, as well as normal human fetal and adult pituitary by reverse transcriptase (RT) PCR followed by Southern blots. All 5 hSSTR subtype mRNAs were expressed in fetal pituitary, while adult pituitary was positive for 4 subtypes, lacking hSSTR4 mRNA. All 15 tumors analyzed were positive for SSTR mRNA, 14 expressing more than one subtype. SSTR2 mRNA in all tissues was expressed as the 2A variant, there being no detectable transcript for SSTR2B. Amongst the 5 SSTRs, mRNA for SSTR2A was the most frequently expressed (87% of tumors) followed by SSTR1 (73%), SSTR3 (53%), SSTR5 (47%), and SSTR4 (40%). The frequency and pattern of expression of the SSTR mRNAs was virtually identical in the different tumor subclasses and did not correlate with tumor size. Since pituitary tumors are monoclonal in origin, multiple SSTR genes are expressed in individual cells. Most tumors are rich in SSTR1 and SSTR2A mRNA compared to the other subtypes. This implies that SST analogs like SMS 201-995, known to interact with SSTR2A, but not with SSTR1, act on pituitary tumors mainly via the SSTR2 subtype. PMID- 7530799 TI - The in vitro cytokinesis-block micronucleus assay: a detailed description of an improved slide preparation technique for the automated detection of micronuclei in human lymphocytes. AB - An improved slide preparation technique is described for the automated detection of micronuclei in binucleate cytokinesis-blocked human lymphocytes. This automated system (Discovery Image Analyser, Becton-Dickinson Image Cytometry Systems, Leiden, The Netherlands) searches for the pattern of two touching nuclei in binucleate cells instead of the edges of the cytoplasm. For this purpose several ratios of the fixative mixture, methanol/acetic acid, were checked. After fixation with a ratio of 25:1, touching nuclei were obtained in almost 100% of the binucleate cells. A hydrolysis treatment with 5 N HCl before staining with Romanowsky-Giemsa resulted in binucleate cells with dark-stained nuclei and micronuclei and a vaguely stained cytoplasm. The high visual contrast between cytoplasm and nuclear material obtained by this staining procedure makes an accurate automated detection of micronuclei feasible. Additionally, a 64 h culture time resulted in an optimal yield of binucleate cells. The results of manual micronucleus scoring on slides prepared with the 'manual protocol' and with the 'automation protocol' indicate no significant differences between both sets of data supporting the validity of the automation protocol. PMID- 7530800 TI - Prescriber guidelines improve initiation of anticoagulation. AB - OBJECTIVES: To improve the initiation of anticoagulation in patients with thrombotic disorders. DESIGN AND SETTING: We carried out a preliminary audit in 1991, developed and implemented strategies to address identified problems, and followed up with another audit in 1993. Medical records of inpatients at The Royal Melbourne Hospital whose anticoagulation therapy was initiated with heparin (intravenous) in therapeutic doses and subsequently converted to warfarin were prospectively assessed over 19-day periods. INTERVENTION: Pocket-sized laminated prescriber guidelines for initiating anticoagulation were disseminated after publishing the results of the first audit, together with a single prominent display of two posters in series advocating the use of the guidelines. RESULTS: The audits identified 25 eligible patients in 1991 and 27 in 1993. The mean duration of intravenous heparin therapy was reduced from 150 hours in 1991 to 97 hours in 1993 (P < 0.002). The mean delay before initiating warfarin therapy after starting heparin therapy was reduced from 69 hours in 1991 to 21 hours in 1993 (P < 0.002). The mean percentage of time the activated partial thromboplastin time was within the therapeutic range increased from 30% in 1991 to 44% in 1993 (P < 0.05) and was above the therapeutic range increased from 13% in 1991 to 24% in 1993 (P < 0.01). Although the number of adverse events was too small to draw definitive conclusions, no difference in adverse haemorrhagic effects was noted between the 1991 and 1993 groups. CONCLUSION: There was an improvement in the initiation of anticoagulation and we hypothesise that this was most likely due to a broad acceptance of the guidelines into clinical practice. PMID- 7530801 TI - Results of a randomized study of early stage Hodgkin's disease using ABVD, EBVD, or MBVD. AB - From January 1986 to December 1989, 157 previously untreated patients, with Hodgkin's disease stage I or II without bulky disease, were enrolled in a clinical comparative study. The objectives of the study were to compare the efficacy and safety of using epirubicine or mitoxantrone instead of adriamycin in the combination chemotherapy regimen ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine). The complete response rate was better in the patients treated with the ABVD or EBVD regimens compared to the MBVD arm. Also, differences in overall survival and relapse-free survival were better in the patients who received ABVD or EBVD compared to the MBVD regimen. Hematological, gastrointestinal and cardiac toxicity were similar in the three groups. Dose intensity, delays and complications were also similar in the three groups. The mitoxantrone-containing regimen was found to have less efficacy in comparison to the other regimens tested in the present study in patients with favorable stage I or II Hodgkin's disease. PMID- 7530802 TI - Primary Hodgkin's disease of the bone. AB - A primary extranodal presentation occurs in less than 0.25% of patients with Hodgkin's disease (HD) and we describe the primary clinicopathologic findings in a rare case of a 21-year-old female with primary bony involvement of Hodgkin's disease. The patient presented with microcytic, hypochromic anemia and shoulder pain. The diagnosis of HD (lymphocyte depleted type) was made by biopsy taken from a lytic lesion in the lateral third of the clavicle. Staging work-up revealed an additional lesion in the ilium, but there was no evidence of lymph node involvement. MOPP/ABV (nitrogen mustard, Oncovin, procarbazine, prednisone/adriamycin, bleomycin, and vinblastine) chemotherapy resulted in complete response lasting, to date, 36 months. To the best of our knowledge, this is only the second reported case of disseminated primary Hodgkin's disease of the bone and all have had a favourable response to combination chemotherapy. A review of the literature is also presented. PMID- 7530804 TI - The role of the pain clinic in hospice and palliative care. PMID- 7530803 TI - Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse. AB - We have previously observed similarities in the behavioral effects produced by the NH2-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl) guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro2, D-Phe7]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [3H]-DTG binding sites in the mouse brain and spinal cord in vitro: Brain; DTG = HAL >> SP = MOR = NAL >> SP(1-7) >> D-SP(1-7) >> SP(5-11): Spinal cord; DTG = HAL >> SP(1-7) = MOR = NAL >> SP >> D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [3H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1 7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site. PMID- 7530805 TI - Channel inhibition by alkanols occurs at a binding site on the nicotinic acetylcholine receptor. AB - The mechanism by which normal alkanols longer than ethanol inhibit cation flux through the transient open state of the nicotinic acetylcholine receptor (nAcChoR) is unknown. They might act nonspecifically either by perturbing the lipid bilayer or by binding to many low affinity sites. Alternatively, they might act in a mutually exclusive manner at a well defined site on the protein. To address this problem, a rapid assay of agonist-induced 86Rb+ efflux from nAcChoR rich Torpedo membrane vesicles was used that enabled the anesthetic-induced inhibition to be measured on a millisecond time scale, under conditions where the concentration of all ligands was raised in < 1 msec, thereby avoiding complications due to desensitization. By measuring the inhibition constant of one agent as a function of the fixed concentration of a second agent, it is possible to distinguish between nonspecific action and mutually exclusive action. Our data are inconsistent with the hypothesis that 1-octanol and 1-heptanol act in a nonspecific manner, but they are consistent with the hypothesis that these two alkanols act in a mutually exclusive manner at a well defined site. The data suggest that the alkanols sterically compete for the site, but experimental limitations prevented a less plausible model, in which there is a strong negative allosteric interaction between separate octanol and heptanol sites, from being ruled out. Should the latter interaction occur, the data indicate that occupation of one alkanol site would decrease the affinity of the other by about 50-fold. The local anesthetic procaine is known to act in a mutually exclusive manner with the agonist self-inhibition site. We found that octanol and procaine acted as separate sites, which exhibited a negative heterotrophic interaction such that octanol reduced the affinity of procaine 6-fold. We conclude that octanol and heptanol inhibit cation flux through the channel of the nAcChoR by binding to a site (or a set of sites of equal affinity) whose location is distinct from, but allosterically coupled to, the agonist self-inhibition site. PMID- 7530806 TI - A ryanodine receptor-like Ca2+ channel is expressed in nonexcitable cells. AB - In this study we investigated the effects of 4-chloro-3-ethylphenol on Ca2+ homeostasis, and we report that this compound induces Ca2+ release from a ruthenium red-sensitive Ca2+ release channel present in skeletal muscle terminal cisternae. When tested with bovine cerebellar microsomes, the Ca(2+)-releasing activity of 4-chloro-3-ethylphenol was reduced by ruthenium red and unaffected by heparin. In PC-12 cells, HL-60 cells, human fibroblasts, the human hepatoma cell line PLC/PRF/5, and Jurkat cells, 4-chloro-3-ethylphenol released Ca2+ from intracellular thapsigargin-sensitive stores. Although decreased, its effect was retained after treatment of intact/permeabilized cells with inositol-1,4,5 trisphosphate (IP3)-mobilizing agonists/IP3, whereas pretreatment of permeabilized cells with ruthenium red reduced the Ca(2+)-releasing activity of 4 chloro-3-ethylphenol. These results provide functional evidence for the presence of a Ca2+ channel distinct from the IP3 receptor, having pharmacological similarities to the ryanodine receptor, in the intracellular Ca2+ stores of a variety of nonexcitable cells. We also demonstrate that a monoclonal anti ryanodine receptor antibody recognizes a protein in human fibroblasts with similar apparent molecular mass the ryanodine receptor. Thus, the intracellular Ca2+ stores of mammalian cells appear to be endowed with two distinct intracellular Ca2+ channels. PMID- 7530807 TI - Differential effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the "adipose- type" and "brain-type" glucose transporters in mice. AB - One prominent symptom of acute toxicity from 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) is a loss of adipose tissue and body weight, a phenomenon known as the wasting syndrome. In the current study, we examined the effect of TCDD on glucose transport in mice. A single intraperitoneal dose of TCDD (116 micrograms/kg) resulted in a time-dependent decrease in transport activity in adipose tissue and brains of C57BL/6 mice. Reduction of transport occurred within 24 hr in both tissues. In adipose tissue a slight recovery was observed by 30 days, but in the brains of treated animals glucose transport was significantly decreased even at the latest time. A comparison of dose-response relationships for several tissues between C57BL/6 (TCDD-responsive) and DBA/2J (TCDD-nonresponsive) mice showed parallel curves, with the C57BL/6 animals showing a 10-20-fold greater sensitivity. The estimated ED50 values for reduction of transport in adipose tissue were 50 micrograms/kg and 800 micrograms/kg for the C57BL/6 and DBA/2J strains, respectively. Treatment of isolated adipose tissue in culture with TCDD and two biphenyl congeners produced a decrease in transport activity that matched the rank order of aryl hydrocarbon receptor affinity for the compounds. Immunoblotting for the adipose-type (type 4) glucose transporter (GLUT) showed a 40% decrease in the membrane fraction of adipose tissue from C57BL/6 mice treated with 116 micrograms/kg TCDD for 40 hr. A similar decrease in the brain-type GLUT1 was observed in the plasma membrane fraction of brain tissues isolated from the same animals. Analysis of RNA for the corresponding GLUT4 and GLUT1 genes showed a dramatic decrease in GLUT4 mRNA as early as 24 hr after treatment. In contrast, the level of GLUT1 mRNA increased slightly in the brains of treated mice. We conclude that regulation by TCDD of glucose transport activity in mice is an aryl, hydrocarbon receptor-dependent process and that the adipose-type GLUT4 appears to be regulated at the mRNA level, whereas the brain-type GLUT1 is affected mainly at the protein level. PMID- 7530808 TI - Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients. AB - The results of this report provide evidence that insulin-like growth factor-1 binding proteins (IGFBPs) in human sera are differentially regulated as a result of severe burn injury. Using the ligand binding technique, 125I-IGF-1 visualizes 5 different protein bands corresponding to those previously reported for IGFBP-1 to 4 with apparent sizes of 23-42 kd in serum samples prepared from severely burned patients and healthy individuals. The level of IGFBP-3 was significantly decreased within 3-5 days of injury and remained depressed for up to 20 days post injury. The average level of this binding protein reached its lowest value within 3-5 days of the injury (3.8 +/- 1.48% relative to day 0-1 value, n = 4, p < 0.01). Serum samples from 3 of 4 patients showed no recovery within 20 days post injury and the level of IGFBP-3 remained significantly depressed (p < 0.01). In contrast, the levels of IGFBP-2 and IGFBP-4 increased 2 and 3 fold in the same serum samples within 3-5 days of the burn injury, respectively. This increase returns to normal (day 0-1 value) within 7-10 days for IGFBP-2, but the level of IGFBP-4 remained elevated 4 fold relative to the day 0-1 (p < 0.01). However, the abundance of IGFBP-1 in these serum samples was not significantly altered by the burn injury. By controlling for protein loading, these apparent alterations of IGFBPs in the sera of burned patients were not due to hemodilution. Similarly, significant reductions in IGFBP-3 were not likely due to IGFBP-3 specific protease activity in the sera of burn patients since incubation of sera from burn patients and normal individuals at 37 degrees C did not alter the pattern of IGFBPs in sera obtained from normal individuals. Of interest, the level of IGF-1 protein in these samples was also markedly reduced following severe burn injury similar to IGFBP-3. The results of this study suggest that a marked reduction of serum IGF-1 seen in burn patients is associated with a significant reduction of IGFBP-3, a major IGF-1 binding protein in human serum. PMID- 7530810 TI - Decreases of plasma tryptophan concentrations following restricted feeding do not decrease serotonin and its metabolite in rat brain. AB - Rats trained on a restricted feeding (RF) schedule of 4 h/day were killed before (starved) and after (fed) the presentation of food to compare hepatic and brain tryptophan metabolism with feeding (FF) controls. The RF rats exhibited greater holo-tryptophan pyrrolase activity in the liver than FF controls. Holoenzyme activity of starved rats was greater than fed animals. Plasma levels of tryptophan higher in the FF rats were comparable in the starved and fed groups of RF rats. Plasma levels of glucose comparable in the FF and RF fed rats were smaller in the starved animals. Brain levels of tryptophan comparable in the RF fed rats and FF controls were smaller in RF starved rats. Brain concentrations of serotonin (5-hydroxytryptamine) and its metabolite 5-hydroxyindoleacetic acid comparable in the RF starved rats and FF controls were higher in the RF fed rats. Possible implication of the findings in the pathogenesis of food deprivation/starvation related disease, anorexia nervosa is discussed. PMID- 7530811 TI - Circulating leukocyte-endothelial adhesion molecules in IgA nephropathy. AB - There is accumulating evidence that leukocyte-endothelial adhesion molecules are important in inflammatory injury in being involved in the primary step of entrapment and migration of leukocytes to the site of inflammation. We have used an antigen capture ELISA to measure the levels of circulating intercellular adhesion molecule-1 (cVCAM-1), vascular cell adhesion molecule-1 (cVCAM-1) and E selectin (cE selectin) in the peripheral blood of 33 patients with IgA nephropathy (IgAN) during clinical quiescence and 24 healthy controls. The serum levels of cICAM-1 and E selectin in IgA-nephritic patients were not different from that of healthy controls but the cVCAM-1 level was significantly elevated in IgAN despite a lack of clinical activity (p = 0.008). The differential rise of circulating leukocyte-endothelial adhesion molecules in IgAN probably reflects the origins and nature of these molecules as well as the specific immunological profile of IgAN. There was no correlation between the levels of these three circulating adhesion molecules. When the patients with IgA nephropathy were stratified according to the severity of their glomerular and interstitial lesions, there was an apparent increase in cE selectin and cVCAM-1 associated with increased histopathologic grading. The changes in endothelial adhesion molecules during clinical exacerbation were studied in 10 patients. Coinciding with synpharyngitic macrohematuria, there was a significant rise of cVCAM-1 and cE selectin (p = 0.046 and p = 0.016, respectively) but no similar rise was observed in cICAM-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530809 TI - Fetal haemoglobin level--effect of gender, age and haemoglobin disorders. AB - Fetal haemoglobin (Hb F) levels shows significant variations in health and disease states. In this study we investigated Hb F level in 75 cord bloods, 1266 healthy individuals, 1582 Hb S heterozygotes, 464 sickle cell anaemia, 93 Hb S/beta(0) -thalassaemia and 65 beta-thalassemia major patients. The age range of the study groups varied from newborn to over 60 years of age. Hb F level was measured by an alkali denaturation procedure and by radial immunodiffusion. The ratio of the level of G gamma-globin chains to the level of A gamma-globin chains (G gamma/A gamma) was determined in the patients group by high performance liquid chromatography. The Hb F level was significantly higher in the sickle cell anaemia and beta-thalassemia major patients compared to the Hb S heterozygotes and the normal individuals. Within each group Hb F level was higher in the female population compared to the age-matched male groups. This difference was statistically significant (P < 0.05) in the sickle cell disease patients and beta thalassemia major patients but not in the normal individuals. After the age of 30 years, the difference in the value of Hb F in the male and female population become more apparent (P < 0.05) in the sickle cell disease and beta-thalassaemia major patients. No statistically significant sex differences were found in the G gamma/A gamma ratio in the patient groups, and the range of G gamma/A gamma ratio in the patients groups were similar to those in the control group. The results showed that age, sex and genetic disorders of haemoglobin are factors that affect Hb F level and indicate the possible involvement of an X-linked factor in control of Hb F production. PMID- 7530812 TI - A urinary marker for occult systemic coccal disease. AB - Gram-negative particles, found in 'normal' urine by an improved microscopic technique, become excessive (often > 10(5)/ml) in many systemic diseases. In these diseases they are accompanied by sparse, usually fastidious, gram-positive cocci. Antibiotics at moderate doses usually have little effect. Larger doses of antibiotics suppress, or temporarily eliminate, the particles. In this report, the particles are characterized by light microscopy for better identification. Then, by detection of muramic acid in a hydrolysate and by transmission electron microscopy, they are identified as decomposed ('exploded') gram-positive cocci. Since explodeds cannot be external contaminants, and their precursors cannot proliferate sufficiently in urine, they must have crossed renal membranes to come from within the body. They are demonstrated in tissue fluids and in synovial fluids by optical microscopy, by their muramic acid, and by transmission electron microscopy. Thus, they cross other membranes. Explodeds are excessive in several rheumatic diseases, in renal diseases, in diseases in which a coccal cause has been sought, and in some in which cocci have never been considered. There is no precedent for explodeds. Their appearance and numbers are compatible with the literature on natural and experimental systemic streptococcal diseases and with the experimental illnesses following injection of streptococcal cell walls. Urinary explodeds are likely to be the end result of the 'almost physiological' entry of streptococci into the circulation which necessitates predental antibiotic prophylaxis in mitral disease. Increased numbers of urinary explodeds probably represent excessive entry of precursors or proliferation of precursors within the host. Urinary explodeds serve as a marker for diverse systemic diseases, systemic coccal disease. PMID- 7530813 TI - Membranous nephropathy in Hodgkin's disease in complete remission. PMID- 7530814 TI - Potentiation of synaptic transmission in the rat hippocampal slice by exogenous L glutamate and selective L-glutamate receptor subtype agonists. AB - We have investigated the effects of administration of exogenous glutamate receptor agonists on the amplitude of field excitatory post-synaptic potentials (fEPSPs) evoked in the CA1 region of the rat hippocampal slice by stimulation of the Schaffer collateral-commissural fibres. L-Glutamate applied by iontophoresis or by bath perfusion (50 microM for 5 min) evoked a slowly rising increase in the amplitude of the fESPS which persisted for over 90 min. L-Glutamate induced potentiation was blocked by either D(-)-2-amino-5-phosphonopentanoic acid (40 microM) or by (RS)-alpha-methyl-4-carboxyphenylglycine (500 microM). In slices in which synaptic long-term potentiation had been saturated, iontophoretically applied L-glutamate did not induce further potentiation, but reset the fEPSP amplitude back to control levels. Iontophoretic administration of N-methyl-D aspartate (NMDA) evoked a transient potentiation which decayed back to control levels within 90 min whereas bath perfusion of NMDA (50 microM) evoked a persistent depression. Bath perfusion of alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA, 50 microM) evoked no persistent effects. Bath administration of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 50 or 100 microM) caused a short term depression of the fEPSP and no significant persistent effects. Perfusion of 100 microM ACPD in medium containing 1 microM picrotoxin caused a much smaller short term depression of the fEPSP and this was followed by a gradually developing and persistent potentiation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530815 TI - Influence of the neuropeptide galanin on active avoidance in rats. AB - It was demonstrated in experiments on rats that the injection of the neuropeptide, galanin (200, 500, and 1000 ng), into the lateral cerebral ventricles induced a dose-dependent decrease in the number of successful attempts at avoidance in rats trained preliminarily to active avoidance by jumping. The preliminary administration of the cholinolytic, atropine (1 mg/kg, intraperitoneally), to the rats also caused an acceleration of the damping of the conditioned reflex and potentiated the indicated effect of galanin during the experiment. The use of the opioid antagonist, naloxone (1 mg/kg, intraperitoneally), did not exert an influence on the animals' behavior, but blocked the galanin-provoked acceleration of the extinction of the active avoidance habit. The intraperitoneal administration of a noncompetitive antagonist of excitatory amino acids, ketamine (10 mg/kg), did not influence the character of the animals' behavior nor the indicated effects of galanin. It was concluded that galanin possesses an amnestic action in the active avoidance test, and that this effect of the peptide is determined by the suppression of cholinergic and activation of opiatergic transmission in the central nervous system. PMID- 7530816 TI - Neuronal nitric oxide synthase is induced in spinal neurons by traumatic injury. AB - Nitric oxide appears to mediate the immune functions of macrophages, the influence of endothelial cells on blood vessel relaxation, and also to serve as a neurotransmitter in the central and peripheral nervous system. Macrophage nitric oxide synthase is inducible with massive increases in new nitric oxide synthase protein synthesis following immune stimulation of macrophages. By contrast, endothelial nitric oxide synthase and neuronal nitric oxide synthase are thought to be constitutive with activation induced by calcium entry into cells in the absence of new protein synthesis. Developmental studies showing the transient expression of neuronal nitric oxide synthase in embryonic and early postnatal life in rodent spinal motoneurons and cerebral cortical plate neurons (Bredt and Snyder, unpublished observations) implies inducibility of neuronal nitric oxide synthase. Moreover, neuronal nitric oxide synthase expression is greatly enhanced in sensory ganglia following peripheral axotomy. Staining for NADPH diaphorase in spinal motoneurons is greatly increased following ventral root avulsion. In many parts of the Central Nervous System NADPH diaphorase staining reflects nitric oxide synthase. In the present study, we have combined in situ hybridization for neuronal nitric oxide synthase, immunohistochemical staining of neuronal nitric oxide synthase, and NADPH diaphorase staining to establish that neuronal nitric oxide synthase expression is markedly augmented in spinal motoneurons following avulsion. The generality of this effect is evident from augmented staining in nucleus dorsalis following spinal cord transection. PMID- 7530817 TI - Input from the amygdala to the rat nucleus accumbens: its relationship with tyrosine hydroxylase immunoreactivity and identified neurons. AB - Both tyrosine hydroxylase-positive fibres from the mesolimbic dopamine system and amygdala projection fibres from the basolateral nucleus are known to terminate heavily in the nucleus accumbens. Caudal amygdala fibres travelling dorsally via the stria terminalis project densely to the nucleus accumbens shell, especially in the dopamine rich septal hook. The amygdala has been associated with the recognition of emotionally relevant stimuli while the mesolimbic dopamine system is implicated with reward mechanisms. There is behavioural and electrophysiological evidence that the amygdala input to the nucleus accumbens is modulated by the mesolimbic dopamine input, but it is not known how these pathways interact anatomically within the nucleus accumbens. Using a variety of neuroanatomical techniques including anterograde and retrograde tracing, immunocytochemistry and intracellular filling, we have demonstrated convergence of these inputs on to medium-sized spiny neurons. The terminals of the basolateral amygdala projection make asymmetrical synapses predominantly on the heads of spines which also receive on their necks or adjacent dendrites, symmetrical synaptic input from the mesolimbic dopamine system. Some of these neurons have also been identified as projection neurons, possibly to the ventral pallidum. We have shown a synaptic level how dopamine is positioned to modulate excitatory limbic input in the nucleus accumbens. PMID- 7530818 TI - Topography of Purkinje cell compartments and mossy fiber terminal fields in lobules II and III of the rat cerebellar cortex: spinocerebellar and cuneocerebellar projections. AB - The cerebellar cortex is histologically uniform by conventional staining techniques, but contains an elaborate topography. In particular, on the efferent side the cerebellar cortex can be subdivided into multiple parasagittal compartments based upon the selective expression by Purkinje cell subsets of various molecules, for example the polypeptide antigens zebrin I and II, and on the afferent side many mossy fibers terminate as parasagittal bands of terminals. The relationships between mossy fiber terminal fields and Purkinje cell compartments are important for a full understanding of cerebellar structure and function. In this study the locations of spino- and cuneocerebellar mossy fiber terminal fields in lobules II and III of the rat cerebellum are compared to the compartmentation of the Purkinje cells as revealed by using zebrin II immunocytochemistry. Wheat germ agglutinin-horseradish peroxidase was injected at three different levels in the spinal cord and in the external cuneate nucleus, and the terminal field distributions in lobules II and III of the cerebellar cortex were compared with the Purkinje cell compartmentation. In the anterior lobe, zebrin II immunocytochemistry reveals three prominent, narrow immunoreactive bands of Purkinje cells, P1+ at the midline and P2+ laterally at each side. These are separated and flanked by wide zebrin- compartments (P1- and P2-). There are also less strongly stained P3+ and P4+ bands more laterally. The spinocerebellar terminals in the granular layer are distributed as parasagittally oriented bands. Projections from the lumbar region of the spinal cord terminate in five bands, one at the midline (L1), a second with its medial border midway across P1- and its lateral border at the P2+/P2- interface (L2), and a third extending laterally from midway across P2-. The lateral edge of L3 may align with the P3+/P3- border. The terminal fields labeled by a tracer injection into the thoracic region give a very similar distribution (T1, T2 and T3). The only systematic difference is in T2, which statistical analysis suggests may be broader than L2. In contrast, anterograde tracer injections into the cervical region label synaptic glomeruli scattered throughout the lobule with much weaker or no evidence of banding. The terminal fields of the cuneocerebellar projection have a complementary distribution to those of thoracic and lumbar spinocerebellar terminals. There are two lateral bands, Cu2 and Cu3. Cu2 lies within the Purkinje cell P1-compartment, abutting L1/T1 medially and L2/T2 laterally. Cu3 lies between L2 and L3 within the P2- Purkinje cell compartment. The medial edge of Cu3 is tightly aligned with the P2+/P2- border.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530819 TI - Evidence that glutamate is released from capsaicin-sensitive primary afferent fibers in rats: study with on-line continuous monitoring of glutamate. AB - The aim of this study is to elucidate whether the excitatory amino acid glutamate is released from capsaicin-sensitive primary afferent fibers, and to compare the releasing effect of capsaicin on glutamate with that on substance P. The release of glutamate was measured using a fluorometric on-line continuous monitoring system, in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, 2-min application of capsaicin produced an increased outflow of glutamate, as well as an increase in the release of immunoreactive substance P from dorsal horn slices of the rat. The release of glutamate was concentration-dependently increased by capsaicin at concentrations in the range of 0.1-3 microM, and the release evoked by 10 microM capsaicin was not higher than that evoked by 3 microM. On the other hand, capsaicin at concentrations of 1-10 microM produced a concentration dependent increase in the release of immunoreactive substance P, without effect at 0.1 microM. The amount of glutamate release evoked by 3 microM capsaicin was about 42.8 pmol.mg-1 protein, and 290 times that of immunoreactive substance P. The release of glutamate by 3 microM capsaicin was suppressed by the depletion of calcium from the superfusate. Capsaicin at 3 microM failed to increase the release of glutamate from the dorsal horn slices of the rats made an L4-L6 dorsal rhizotomy. These results suggest that capsaicin evoked the release of glutamate from primary afferent fibers in the dorsal horn and that glutamate may play an important role in pain transmission between primary afferent fibers and dorsal horn neurons. PMID- 7530821 TI - Correlation between objective nasal mucosal swelling and estimated stuffiness during long-term use of vasoconstrictors. AB - The correlation between nasal mucosal swelling and estimated stuffiness was evaluated in 13 healthy volunteers who entered the trial. The nasal mucosa was challenged with increasing concentrations of histamine solutions. The amount of mucosal swelling was recorded using rhinostereometry, and the stuffiness was estimated by the subjects on a visual analogue scale. Then the subjects were randomly chosen to receive either oxy- or xylometazoline nasal spray for 30 days. After 10 days on the drug, the histamine sensitivity was enhanced, and after another 20 days, it had increased further, reflecting the development of rhinitis medicamentosa. Before the subjects started the nasal spray and after 10 days on the drug, a correlation between stuffiness and swelling was seen in the individual subject only when the degree of stuffiness almost obliterated the nasal opening. At the end of the month, no correlation was present. In all histamine provocations throughout the month, a very high correlation was noted between mean mucosal swelling and mean stuffiness. It is concluded that the symptom scores for nasal obstruction are useful for determining nasal mucosal swelling in groups. However, in the individual subjects one can estimate the mucosal swelling only when the amount of swelling almost obliterates the nasal opening. This cannot be done when the mucosa is hyperreactive. PMID- 7530822 TI - [Experience with urinary conduit procedures]. AB - Between 1989 and 1993, 32 urinary conduit procedures were carried out at the Department of Gynaecological Oncology, National Institute of Oncology Budapest. Of these, 26 patients with pelvic tumour underwent total or anterior pelvic exenteration. The urinary conduit operation was performed in associated with radical hysterectomy due 2 vesico-urinary fistula in 2 patients, and as a palliative procedure in 4 instances (bladder fistula 2, bladder fistula and ureter occlusion 1 and bilateral ureteric obstruction 1). Mean age of the patients was 46, range 20-73 years. 23 patients underwent bladder replacement with "Bricker pouch" or ileal conduit, mostly in the first 2 years, and as a palliative procedure. Kock pouch was constructed in 3 and an Indiana pouch in 6 women. There was no intraoperative mortality. 3 patients died in the postoperative period, none of them due to complication of the urinary diversion procedure. Postoperative bleeding occurred in one ileal conduit that ceased spontaneously and in one Indiana pouch that required reoperation. Haematuria was a common finding in the first 3 to 5 days following surgery. Urinary leakage in the abdominal cavity lasting for 7 to 10 days postoperatively occurred in almost all instances in those who underwent a Bricker pouch. This did not require surgical intervention. 3 patients with Bricker pouch experienced pyelonephritis. Continent pouches are emptied by self-catheterization, 6-8 times daily. There were no other early complications. Techniques of urinary diversion are discussed. PMID- 7530820 TI - Penile condylomata: a gynecological epidemic disease: a review of the current approach and management aspects. AB - Viral venereal infection caused by human Papillomavirus has reached epidemic state. The proper management of this infection in men is of great benefit, because it may possibly decrease the reservoir of disease in both sexes from which genital condylomata and associated lesions may arise. We report a selection of current knowledge about the epidemiology, etiology, diagnosis, and treatment of male condyloma, occurring predominantly among male consorts of women with genital human Papillomavirus infection. In a review of 1455 affected women, compiled from the literature, 1019 (70 per cent) of their sexual partners were diagnosed as having been infected with the same viral disease. The current diagnostic and treatment modalities and their outcomes are discussed with appropriate recommendations for their use. PMID- 7530823 TI - [CHOP-Bleo therapy in high-malignancy non-Hodgkin lymphoma]. AB - The records of 85 patients were reviewed in order to study the efficacy of CHOP Bleo combination chemotherapy and to determine factors influencing prognosis. The patients were treated between 1978 and 1989, the median age was 44.4 (range, 15 68) years, the majority of them were in advanced clinical stages (92.9%). Systemic (B) symptoms were present in half of the patients and a quarter of them had bulky disease. Complete response was achieved in 36 patients (42.5%), partial response in 26 patients (30.5%), and primary treatment failure occurred in 23 patients (27.0%). The median survival was 92.6 months and 36.5% of the patients remained relapse-free at 10 years. The expected 5-year and 10-year survival rates were 47% and 39%, respectively. Using multivariate analysis B symptoms, involvation of two or more extralymphatic sites and lymphoblastic histologic subtype proved to be unfavourable prognostic factors. The most frequently observed side-effects were myelosuppression, neurotoxicity and gastrointestinal symptoms. No treatment-related death have occurred. PMID- 7530825 TI - TGF alpha and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas. AB - To assess the synergistic effect of growth and transcription factor deregulation on carcinogenesis in vivo, mating experiments were performed between transgenic mice expressing human TGF alpha or v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos, HK1.TGF alpha and HK1.fos/alpha). While HK1.TGF alpha mice exhibited mild epidermal hyperplasia resulting in a wrinkled appearance, this hyperplasia was significantly increased in HK1.fos/alpha mice which also exhibited a novel opalescent and peeling skin phenotype. HK1.fos/alpha keratinocyte differentiation was considerably deregulated with cornified cells appearing in the granular layer, granular cells in the spinous layer and a sixfold increase in BrdU labeling over normal. In addition, hyperplastic HK1.fos/alpha epidermis exhibited aberrant loricrin, filaggrin and novel K13 expression associated with v-fos expression. Unlike adult HK1.TGF alpha controls, hyperplasia persisted in HK1.fos/alpha adults which also rapidly developed autonomous squamous cell papillomas. These results demonstrate that v-fos and TGF alpha over-expression can cooperate to reprogram keratinocyte differentiation and elicit the early stages of neoplasia. Moreover, TGF alpha over-expression appeared to play an early, initiating role in HK1.fos/alpha papilloma etiology, and a promotion role in the accelerated appearance of v-fos wound-associated preneoplastic phenotypes. However, the stable persistence of HK1.fos/alpha papillomas for up to 12 months, suggests that additional events are required for malignant conversion. PMID- 7530824 TI - [Blocked atrial extrasystole simulating impaired anterograde conduction during atrial stimulation]. AB - In a 72-years-old patient with intermittent bradycardia definitive atrial pacing was considered. Surface ECG was misleading because of demonstrating 2:1 anterograde block during atrial stimulation at a rate of 100 beats per minute. Intracardiac recordings disclosed the bigeminal occurrence of premature atrial contractions. Every second stimuli fall within the premature atrial contraction's refractory period simulating impaired AV-conduction. Our report emphasizes the importance of intracardiac recordings during atrial stimulation in order to evaluate the anterograde conduction before pacemaker implantation. PMID- 7530826 TI - An in vivo model for receptor tyrosine kinase autocrine/paracrine activation: auto-stimulated KIT receptor acts as a tumor promoting factor in papillomavirus induced tumorigenesis. AB - Constitutive overactivation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in cancer cells and are thought to play a critical role in carcinogenesis. In the present report, we propose a refined in vivo model which explains the significance of these mechanisms in tumour development. We have previously established transgenic mouse lines containing human papillomavirus type 16 (HPV16) E6E7 oncogenes, in male mice of which a Leydig cell tumor developes with a very high incidence. Not only HPV transgene but also the c-kit proto-oncogene receptor tyrosine kinase and its ligand Steel Factor (SLF) were coexpressed in all tumors analysed. This coexpression of c-kit/SLF was also found in two other Leydig cell tumor lines. Moreover, the proliferation of transgenic tumor cells was attenuated by treatment with a c-kit neutralizing antibody in vitro, strongly suggesting that tumorigenesis is closely related to stimulation of receptors through ligand induction. To confirm the significance of these findings, a defective mutation of the SLF gene in a laboratory mouse, the Steel-Dickey (Sld) mutation, was introduced into a line of transgenic mice showing 100% incidence of the tumor. In Sld-E6E7 transgenic mice, tumorigenesis was initiated but numbers of tumor cells were markedly reduced compared with transgenic mice carrying both wild type SLF allele, showing that c-kit activation through the induction of SLF is essential for testicular tumorigenesis, especially in tumour promotion. This transgenic mice system should be a useful in vivo model for clarifying the implication of growth factor autostimulation in carcinogenesis. PMID- 7530828 TI - Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein. AB - The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain. PMID- 7530829 TI - Transformation of mouse cells by wild-type mouse c-Src. AB - Previous studies in which chicken and human c-Src were overexpressed in chicken and rodent cells have indicated that overexpression of wild-type c-Src can not induce complete neoplastic transformation. However, studies with v-Src mutants have demonstrated that species-specific differences can play a significant role in transforming activity. Here we show that, in contrast to chicken c-Src, overexpressed mouse c-Src can induce significant anchorage-independent growth and tumorigenicity when transfected into NIH3T3 mouse cells. The biochemical cause for this difference is unknown. In particular, the protein-tyrosine kinase activities of chicken and mouse c-Src appear to be similar. This result is consistent with the hypothesis that v-Src-induced transformation results from perturbation of signalling pathways modulated by c-Src and highlights the need for caution in controlling for potential species-specific differences in studies of c-Src function. PMID- 7530827 TI - Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site. AB - The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C terminal 50 amino acids by five unrelated residues. Examinations of individual v kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity. PMID- 7530830 TI - Adjuvant drug strategies in the treatment of recurrent respiratory papillomatosis. AB - The purpose of this study was to evaluate adjuvant drug therapies combined with standard laser excision in the treatment of recurrent respiratory papillomatosis. Previous studies have presented conflicting data on the efficacy of various treatments, including interferon and isotretinoin. A retrospective study of 34 patients with moderate to severe papillomatosis who underwent both laser surgery and adjuvant therapy was therefore performed. All patients were treated with interferon. Five interferon failures received isotretinoin, and three with recalcitrant disease received methotrexate. Interferon produced a complete response in 16 patients and partial response in 12 patients. Juvenile-onset disease had a slightly higher response to interferon than adult-onset disease. isotretinoin produced no response in all five patients. Methotrexate demonstrated a marked improvement in both severity of disease and treatment interval in all three patients. Serious side effects were limited to one interferon patient with febrile seizures, which resolved with discontinuation of therapy. We conclude that adjuvant therapy including interferon and methotrexate is clearly of benefit in the treatment of patients with respiratory papillomatosis. A detailed approach to surgery combined with an interferon dosing regimen is presented. Further study of methotrexate appears warranted. PMID- 7530831 TI - Staging of chronic hyperplastic rhinosinusitis: treatment strategies. AB - In 1990 we reported an initial prospective study of 100 patients using a four stage system for classification of chronic rhinosinusitis. Between January 1988 and July 1992, we used this system in staging an additional 1814 patients, on whom 2980 intranasal sphenoethmoidectomies were performed. In this staging system a protocol trial of medication was given for 2 weeks, followed by axial and coronal computed tomography. Medication consisted of a second-generation cephalosporin antibiotic, usually cefuroxime; a 4-day burst of intraoral steroids, usually prednisone; and an antihistamine decongestant if not contraindicated. The stages of chronic hyperplastic rhinosinusitis included the stages described in the 1990 report (i.e., stage I, single-focus disease; stage II, discontiguous disease throughout the ethmoid labyrinth; stage III, diffuse disease responsive to medication; and stage IV, diffuse disease unresponsive to or poorly responsive to medication). The results of this study have shown that the computed tomography staging system based on computed tomography extent of disease after medical therapy is a simple, easily remembered, and very effective modality for the classification of chronic sinusitis. This system provides a rationale for discussing and planning surgery with patients and physicians and is a convenient reference for the reporting of end results. More importantly, a linear relationship between disease stage and outcomes is demonstrated. This statistically highly significant feature of the staging system provides a firm basis for the production of outcomes after various treatment strategies, particularly ethmoidectomy and the treatment of sinusitis. PMID- 7530832 TI - Assessment of bone formation during osteoneogenesis: a canine model. AB - Distraction osteoneogenesis, callotasis, has been demonstrated to be an effective means of lengthening long bones. A variation of Ilizarov's technique produces a transport disk from one cut surface of bone within a defect and advances the disk to the opposite surface to close the defect. This process, previously described by Costantino et al. (Arch Otolaryngol Head Neck Surg 1990; 116:535-45), demonstrated bone formation within the distraction site. The precise mechanism of bone formation has not yet been described for the mandible. Four conditioned beagles were studied, with one control dog maintained in neutral fixation and three dogs distracted at 0.25 mm every 8 hours. A two-cm defect was closed, and dogs were kept in fixation for 1 week after closure, after which they were killed. Three sites were evaluated: (1) the distraction seam, (2) the interface of the cortical and distracted bone, and (3) the cortexes at the closed defect. Each site was bisected, and one half was decalcified for immunohistochemical and hematoxylin and eosin pathologic evaluation. The vascular basement membrane was labeled for laminin and type IV collagen. Both of these substances demonstrate the differentiation of the vascular matrix component predisposing primary bone formation. Labels were intense at the distraction seam where intense angiogenesis occurred. No hyalin cartilage was observed at the distraction site, which indicates that the fixation was stable and that ossification occurred primarily without intermediate callous formation. This model demonstrated that osteoclasts within the canine model produce bone through primary bone formation within an angiogenic matrix rich in basement membrane laminin and type IV collagen. Likewise, bone is species specific in mineral composition for dog mandible. Understanding the formation and composition of distracted bone is essential for understanding application of this technique within the clinical setting. PMID- 7530833 TI - Theileria annulata sporozoite targets. AB - Bovine peripheral blood mononuclear cells (PBM) infected in vitro with Theileria annulata sporozoites have previously been characterized as MHC class II+ mature macrophages. The ability of T. annulata sporozoites to infect different subpopulations of MHC class II+ bovine monocytes was investigated. Cells were labelled with monocyte specific monoclonal antibodies (MoAb) and isolated using magnetic cell sorting (MACS). Sporozoites infected both immature and mature monocytes, but more readily infected the mature population. A potential ligand for sporozoite entry is the elastin receptor which is expressed mainly on the immature population of monocytes and not on B cells or T cells. T. annulata sporozoites infected elastin receptor positive and negative cell populations equally well. Infected immature cells lost the expression of elastin receptors and the immature marker, subsequently expressing the mature marker. All monocytes lost the expression of CD14 (the LPS receptor) upon infection with sporozoites. The infection of specific populations and subsequent alterations in phenotype may alter the function of these cells and play an important role in disease pathogenesis. PMID- 7530834 TI - Thermophilic amylase-digested rice-electrolyte solution in the treatment of acute diarrhea in children. AB - OBJECTIVE: To compare the efficacy of an oral rehydration solution (ORS) containing short polymers of glucose derived from rice (Amylyte-ORS) and five times the caloric density of current ORS to the standard glucose-ORS (World Health Organization [WHO] = ORS) in the treatment of acute diarrhea in children. METHODS: The rice ORS (Amylyte-ORS) was obtained by adding thermophilic amylase (252,500 MW units) and salts (1.5 g NaCl, 600 mg KCl, and 150 mg CaCl2) to 100 g rice and boiling for 10 minutes in 500 mL water. This yields 250 mL Amylyte-ORS, which contains 92% to 96% short-chain glucose polymers, three to nine molecules in length, and provides 425 kcal/L, compared to 80 kcal/L for the WHO-ORS. One hundred forty-four male children, 4 months to 3 years of age, presenting with acute diarrhea and mild, moderate, or severe dehydration, were assigned by random allocation to receive either WHO-ORS or Amylyte-ORS. Data from 127 children were analyzed (57 received the WHO-ORS and 70 the Amylyte-ORS). Two children given Amylyte-ORS and 15 given the WHO-ORS were not included in the analysis because of improperly collected data or lost urine or fecal specimens. None were given antibiotics during the study. Free water and feeding were allowed after the children were rehydrated. RESULTS: The clinical characteristics of the children in the two treatment groups were comparable. Five children who received the WHO ORS and three children given Amylyte-ORS were treatment failures. Amylyte-ORS reduced diarrhea duration by 15% (41.4 +/- 2.5 vs 34.7 +/- 1.8 hours; P < .03) compared to the WHO-ORS, regardless of the severity of dehydration. In the Amylyte-treated group, ORS requirements were significantly less (234 +/- 15.2 vs 295 +/- 17.6 mL/kg; P < .01) and weight gain was significantly more (367.7 +/- 45.1 vs 199.2 +/- 38.2 g; P < .01) than in those given the WHO-ORS. The net intestinal fluid balance and total body fluid balance were similar in the two groups. CONCLUSIONS: Amylyte-ORS effectively rehydrates children with acute diarrhea, reduces diarrhea duration, decreases ORS requirements, and improves weight gain compared to the WHO-ORS. PMID- 7530835 TI - Influence of acidosis, hypoxemia, and hypotension on neurodevelopmental outcome in very low birth weight infants. AB - OBJECTIVE: We previously demonstrated that acidosis (pH < 7.15) predicts poor motor outcome in very low birth weight (VLBW) infants. The present study was undertaken to examine the association between acidosis and developmental outcome in more detail and to better understand the interrelationship of acidosis with related factors such as hypoxemia and hypotension. METHODS: The nursery records of 191 infants enrolled in our VLBW follow-up study were reviewed to identify the type of acidosis (metabolic or respiratory) present, measure the duration of single and cumulative episodes, and examine the interaction of acidosis with hypoxemia and hypotension. The Bayley Scales of Infant Development and a detailed neurologic examination were performed at 6 (n = 158) and 24 (n = 106) months corrected age. RESULTS: At 6 months, both respiratory and metabolic acidosis as well as the total duration and longest single episode of acidosis were significantly correlated with cognitive, motor, and neurologic outcome (P < .0001). By 24 months, only the association of the metabolic component of acidosis with all three outcome measures remained significant. Duration of hypotension independently correlated with outcome at both testing periods (P < .002) but isolated hypoxemia did not. The metabolic component of acidosis and isolated hypotension contributed significantly to the variance in all three outcome measures (P < .05). Duration of hypoxemia, but not hypotension, contributed significantly (53%) to the variance in the metabolic component of acidosis. CONCLUSION: We conclude that it is the metabolic component of acidosis that is important in predicting poor developmental outcome in VLBW infants. The detrimental effect of hypoxemia appears to be closely related to the occurrence of metabolic acidosis while hypotension has an independent effect on outcome. PMID- 7530836 TI - Disability and perinatal care. PMID- 7530839 TI - Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion. AB - The involvement of large-conductance, voltage- and Ca(2+)-activated K+ channels (maxi-K+ channels) in basolateral Ca(2+)-dependent K(+)-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K+ efflux was monitored by measuring the change in K+ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na+/K(+)-ATPase and Na(+)-K(+)-2Cl- cotransporter were inhibited by ouabain (1 mmol/l) and bumetanide (50 mumol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mumol/l) caused a transient large net K+ efflux, followed by a smaller K+ efflux, which gradually returned to the basal level within 10 min. These two components of the K+ efflux appear to be dependent on an increase in cytosolic Ca2+ concentration. The initial transient K+ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K+ efflux transient induced by ACh was inhibited by quinine (0.1-3 mmol/l), quinidine (1-3 mmol/l) and Ba2+ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mumol/l). Ca(2+)-dependent transient large K+ effluxes induced by substance P (0.01 mumol/l) and A23187 (3 mumol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mumol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole cell outward K+ current attributable to maxi-K+ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1-10 mmol/l) to the bath. It is concluded that maxi-K+ channels are not responsible for the major part of the Ca(2+)-dependent basolateral K+ efflux and fluid secretion by the rat submandibular gland. PMID- 7530837 TI - An experimental model of the production of early after depolarizations by injury current from an ischemic region. AB - An ischemic myocardial region contains cells with a depolarized resting membrane potential. This depolarization leads to an intercellular current flow between the ischemic region and the surrounding normal myocardial cells which has been termed an "injury current". We have devised an experimental model system in which an isolated guinea pig ventricular cell is electrically coupled to a model depolarized cell in order to evaluate the effects of this injury current on the electrical properties of a normal ventricular cell exposed to drugs which increase calcium current or decrease potassium current. Using low doses of isoproterenol, forskolin, or Bay K 8644 (or 8-bromo-cyclic adenosine monophosphate in the pipette) we found that the action potential duration of the isolated cell was lengthened, but that early afterdepolarizations (EADs) were not produced unless the cell was also coupled to a depolarized cell model representing an adjacent ischemic region. A similar prolongation of the action potential was produced by low doses of quinidine, but EADs were not produced unless coupling to a depolarized cell model was added. EADs could not be produced in any cells in the absence of the drugs even though the coupling to the depolarized cell model was increased up to the level at which the action potential was indefinitely prolonged. At higher isoproterenol concentrations, EADs or spontaneous activity were produced without coupling to the depolarized cell model. Under these conditions, coupling of the cell to a cell model with normal resting membrane potential stopped the spontaneous activity and prevented the occurrence of EADs even with high levels of resistive coupling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530838 TI - Examination of rat salivary glands for the presence of the aquaporin CHIP. AB - The aquaporin CHIP, AQP-CHIP, is a 28-kDa integral membrane protein that functions as a water channel in kidney and red blood cells. We have examined rat salivary glands for the presence of this molecule. In Northern blot analyses an AQP-CHIP cDNA hybridized to rat kidney mRNA approximately 20-fold more than to mRNA from the rat parotid gland. Western blot analyses, using antisera against human AQP-CHIP, showed that AQP-CHIP was present in crude membranes from rat salivary glands to a much lesser extent than in rat kidney membranes. A 2.8 kb cDNA encoding AQP-CHIP was also isolated from an adult rat parotid gland cDNA library. Immunolocalization studies using thin and ultrathin cryosections and light and electron microscopy, however, revealed that no glandular epithelium was labeled. In contrast, significant immunolabeling was present in microvascular endothelial cells. Non-fenestrated endothelia of capillaries and venules exhibited labeling on both their apical and basolateral membranes. The present study demonstrates that AQP-CHIP is not involved directly in fluid secretion from salivary epithelial cells. Further, this study provides strong evidence that AQP CHIP is an endothelial cell water channel found within salivary gland tissue which may contribute to water permeability in the capillary beds. PMID- 7530840 TI - CGRP-induced activation of KATP channels in follicular Xenopus oocytes. AB - The two-microelectrode voltage-clamp technique was used to monitor K+ channel activity in Xenopus oocyte follicular cells, which are electrically coupled to the oocyte itself by gap junctions. Endogenous vasodilators such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), prostaglandin E2 (PGE2) and adenosine activate glibenclamide-ATP-sensitive K+ (KATP) channels in Xenopus oocyte follicular cells. The mechanism of action of CGRP was studied in detail. CGRP effects undergo a rapid desensitization. CGRP acts via CGRPI receptors. Its effects are antagonised by the amino-truncated CGRP analog hCGRP(8 37). The second messenger for CGRP activation of KATP channels is cAMP. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine enhances the CGRP response while adenyl cyclase inhibition by either 2',5'-dideoxyadenosine or progesterone nearly completely depresses the CGRP response. Vasoconstrictors such as ACh and angiotensin II also have receptors in follicular cells. ACh strongly inhibits the CGRP activation of K+ channels as it inhibits the activation of KATP channels by P1060, but angiotensin II does not. It is concluded that as in vascular smooth muscle cells, CGRP and probably other hyperpolarizing vasodilators open KATP channels in follicular cells by protein kinase A activation. PMID- 7530841 TI - Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA- streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. AB - Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel retardation assays. PMID- 7530842 TI - Binding of Y-box proteins to RNA: involvement of different protein domains. AB - Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. PMID- 7530843 TI - Mechanism of inhibition of HIV-1 infection in vitro by guanine-rich oligonucleotides modified at the 5' terminal by dimethoxytrityl residue. AB - Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA 1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction. PMID- 7530844 TI - Color Doppler imaging of modified Blalock-Taussig shunts during infancy. AB - To assess the role of color Doppler echocardiography in the early postoperative evaluation of patients with a Blalock-Taussig shunt we examined 13 consecutive infants who underwent insertion of either a modified right (6 patients) or a modified left (7 patients) Blalock-Taussig shunt (age range 7 days to 6 months, mean age 8 weeks). Examination of the patients in a high parasternal right or left long axis was able to determine patency of the shunt in 12 patients. In the remaining patient, who did not show flow in the shunt, complete occlusion of the shunt was confirmed by angiography. From subcostal views we were able to demonstrate patency of the shunt in 75% of the infants and in all patients younger than 4 weeks of age. In our experience color Doppler echocardiography is a highly reliable method for early postoperative evaluation of infants with a Blalock-Taussig shunt. PMID- 7530845 TI - Doppler flow profiles in the right and left pulmonary artery in children with congenital heart disease and a bidirectional cavopulmonary shunt. AB - The systolic and diastolic Doppler tracings in the right and left pulmonary artery were analyzed in 10 patients with complex cyanotic congenital heart disease, aged 6 months to 12 years (median 3 years), after employment of a bidirectional cavopulmonary shunt. The postoperative interval ranged from 2 weeks to 1.7 years (median 1.3 years). In children with pulmonary atresia or severe pulmonary stenosis with minimal antegrade pulsatile pulmonary blood flow Doppler echocardiography confirmed a systolic and diastolic bidirectional shunt from the vena cava superior to both pulmonary arteries. In children with pulmonary stenosis, Doppler echocardiography confirmed a systolic shunt only to the right pulmonary artery and a diastolic bidirectional shunt into both pulmonary arteries. As the left pulmonary artery was perfused by the pulsatile transvalvular flow it was difficult to detect a concomitant systolic Glenn related flow in those patients. Quantitative analysis of the diastolic Doppler tracings revealed a significant difference in the velocity time integral in the right and left pulmonary artery indicating a dominant right lung perfusion in diastole. PMID- 7530847 TI - Pregnancy-associated plasma protein A levels in maternal serum, extraembryonic coelomic and amniotic fluids in the first trimester. AB - First trimester maternal serum levels of pregnancy-associated plasma protein A(PAPP-P) are reduced in women with a Down's syndrome pregnancy. We have examined the concentration of this molecule in the amniotic (AF) and extra-embryonic coelomic (EECF) fluids surrounding the developing fetus. Maternal serum levels of PAPP-A were elevated in all samples and steadily rose from a median of 480 mIU/1 at 8 weeks to a median of 6375 mIU/1 at 14 weeks gestation. Levels of PAPP-A were low in EECF and undetectable in the AF until 14 weeks gestation. This pattern of distribution is in contrast to that of most other trophoblast-associated antigens. This may reflect PAPP-A physiology and its specific production by the syncytiotrophoblast. PMID- 7530848 TI - Immunohistochemical localization of two endometrial proteins in the early days of human pregnancy. AB - Insulin-like growth factor binding protein-1 (IGFBP-1) [also known as placental protein 12(PP12)] and placental protein 14 (PP14) have been identified by specific immunostaining in early pregnancy specimens obtained 13-35 days of gestation. PP12 was evident in a discrete number of stromal decidual cells at the deciduotrophoblastic interface and under the endometrial surface epithelium. These cells did not have the rounded appearance of classic decidual cells but most often displayed cytoplasmic expansions. Staining for PP14 was strictly localized to the glandular epithelium of the endometrium. Implantation of the conceptus may be an important mechanism in the early expression of PP12 but not PP14. PMID- 7530846 TI - Displacement of preexisting thrombus by umbilical vein catheterization. AB - Balloon atrial septostomy is recommended as a standard palliative procedure in neonates with congenital heart defects who are dependent on intracardiac shunting. We describe an unusual and unreported complication associated with "bedside" balloon atrial septostomy. The umbilical venous catheterization resulted in displacement of a thrombus from the ductus venosus or the hepatic vein. We recommend careful two-dimensional echocardiographic monitoring during such procedures. PMID- 7530849 TI - Lymphoproliferative disorder of granular lymphocytes: nine cases including one with features of CD56 (NKH1)-positive aggressive natural killer cell lymphoma. AB - Twelve cases of large granular lymphocytosis were examined by flow cytometry and Southern blot analysis to correlate immunophenotypic and molecular genetic markers with clinical features. Nine cases fulfilled clinical criteria for the lymphoproliferative disorder of granular lymphocytes, and eight of these demonstrated the molecular features of T-cell-type lymphoproliferative disorder of granular lymphocytes including surface expression of CD3, expression of one or more natural killer (NK)-cell antigens (CD11b, CD16, or CD57), and clonal rearrangement of both the T-cell receptor beta- and gamma-chain-joining genes. One of these cases demonstrated coexisting clonal rearrangement of the immunoglobulin heavy-chain-joining genes, but none demonstrated kappa-light-chain joining gene rearrangement. The eight T-cell lymphoproliferative disorder of granular lymphocytes cases all lacked expression of the NK antigen CD56 (NKH1). In contrast, the other case of lymphoproliferative disorder of granular lymphocytes rapidly evolved into an aggressive NK-cell lymphoma which did not express CD3, did express CD56, had germline T-cell receptor gene configurations, and had multiple clonal chromosomal abnormalities. This case demonstrated nasal cavity and cutaneous tumor infiltrates consistent with previously described CD3 negative, CD56-positive NK-cell lymphoma of the upper aerodigestive tract. Three cases of transient large granular lymphocytosis demonstrated germline T-cell receptor gene configurations. This study demonstrates the usefulness of Southern blot analysis and flow cytometry in characterizing proliferations of large granular lymphocytes. The transformation of a single case into an aggressive NK cell lymphoma with blastic morphology and tissue infiltration was associated with a fatal outcome. PMID- 7530850 TI - E-cadherin expression in prostatic carcinoma biopsies: correlation with tumor grade, DNA content, pathologic stage, and clinical outcome. AB - We compared tumor grade and DNA content with expression of E-cadherin (E-CD), a cell adhesion molecule associated with cell-cell and cell-matrix interaction, leukocyte function, and tumor invasion and metastases, on 56 prostate carcinoma needle biopsies. The findings were correlated with final pathologic stage at subsequent prostatectomy, preoperative serum prostate-specific antigen level and further development of metastases during an initial 2.4-yr mean clinical follow up period (range 0.5 to 5.5 yr). E-CD expression (uvomorulin, L-CAM, cell CAM 80/120, ARC-1, Sigma, St. Louis, MO) was measured by double-linked immunoalkaline phosphatase immunohistochemistry quantified with a the Roche RPW image analyzer (Roche Image Analysis Systems, Elon College, NC). DNA ploidy was determined on formalin-fixed, paraffin-embedded Feulgen-stained 5-microns tissue sections of the narrow-bore initial prostate carcinoma biopsies with the Roche RPW image analyzer. The 51% mean positive area E-CD expression in the group of 56 adenocarcinomas was significantly less than the 76% expression level for 15 normal control prostate tissues (P < 0.001). E-CD expression was also decreased in aneuploid (39%) versus diploid tumors (54%, P < 0.001); and in high-grade (44%) versus low-grade lesions (54%; P < 0.01). The 44% E-CD expression level in patients with metastases was lower than the 52% level in the nonmetastatic cases, but this finding was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530851 TI - p53 immunolabeling in archival paraffin-embedded tissues: optimal protocol based on microwave heating for eight antibodies on lung carcinomas. AB - The prognostic value of p53 gene mutations is dealt with by several recent reports. However, retrospective assessment of p53 tumor status on archived samples has been prevented by p53 epitope alteration during routine fixation and embedding procedures. This study aimed at establishing a reproducible low-cost protocol to retrieve not only N-terminal, but also midregion and C-terminal, epitopes, with special attention to possible artifacts induced by epitope retrieval procedures. Using microwave heating, we compared the epitope retrieval efficiency of five solutions with eight commercial antibodies on 21 lung carcinomas for which frozen tissue and samples fixed with formalin and Bouin's liquid were available. All eight epitopes were retrieved, citrate buffer proving efficient for seven. PAb 240 epitope was restored by target unmasking fluid only. No false positivity was observed. Fixation-induced loss of p53 immunoreactivity was minimal for formalin (two of 10 tumors for one antibody each), more significant for Bouin (six of 10 tumors for one to five antibodies). On the other hand, staining intensity was maintained or even improved, and nonspecific staining reduced, through fixation. We conclude that p53 stabilization can be detected on routinely processed archival tumor samples with a reliability similar to that of frozen tissue by means of a microwave-based procedure and a panel of at least three antibodies, with epitopes on the N-terminal, C-terminal, and midpart of the molecule. PMID- 7530852 TI - [Immunochemical determination of saccharin]. AB - Saccharin is coupled to bovine serum albumin by spacers via the five membered as well as the six membered ring. Rabbits treated with these immunogenic conjugates give precipitating antibodies which are used for coating polystyrol tubes and microtiter plates. Saccharin is determined down to 0.1 mg/l, the detector reaction being performed by a saccharin horseradish peroxidase conjugate and hydrogen peroxide/1,2-diaminobenzen as substratum. PMID- 7530853 TI - Beta-endorphin, but not substance-P, is increased by acute stress in humans. AB - The role of neuropeptides in the psychoneuroendocrinological stress response is largely unknown. In this study the effect of acute psychological stress on beta endorphin and substance-P plasma concentrations was investigated and further the effect of different anxiety levels or control attributions on beta-endorphin or substance-P levels were determined. Blood samples were obtained from 47 inexperienced tandem-parachutists 2 h before, immediately after, and 1 h after a parachute jump and plasma concentrations of beta-endorphin and substance-P were analysed. Anxiety levels and control attributions were assessed by psychometric scales. Whereas substance-P concentrations seemed to be unaffected by the jump stress, there was a transient but significant increase in beta-endorphin levels immediately after jumping. However, subjects higher in state-anxiety at the point of jumping (exit) displayed higher substance-P values at all three time points compared to the "low-anxiety" jumpers. In addition, stress-induced beta-endorphin secretion was dependent on subjective control attributions. PMID- 7530855 TI - [Anesthesia of a patient with systemic mastocytosis]. PMID- 7530854 TI - Effects of diltiazem in convulsive states differ from those previously reported for dihydropyridine calcium channel antagonists. AB - Unlike the dihydropyridine calcium channel antagonists studied previously, the benzothiazepine calcium channel antagonist, diltiazem, increased the incidence of convulsions caused by bicuculline, N-methyl-DL-aspartate or 4-aminopyridine. However, the latencies to convulsions were also increased. Diltiazem increased the ratings of convulsive behaviour on handling after intraperitoneal administration of bicuculline, or pentylenetetrazol and after the calcium channel activator, Bay K 8644, administered ICV. When the binding of the dihydropyridine, [3H]-nitrendipine in the CNS was measured in vivo, this was increased by diltiazem. This compound therefore showed a different pattern of interaction with convulsant drugs then that previously demonstrated for other calcium channel antagonists, appearing to possess both pro- and anticonvulsant actions, and a different pattern of interaction with the dihydropyridine receptor complex. PMID- 7530856 TI - [Pleurodesis. Indications and review of symphyseal agents]. AB - Recurrent or chronic pneumothorax and neoplasm of the pleura are the major indications for pleurodesis. However certain chronic or recurrent forms of pleural affections of mechanical origin or secondary chylothorax may not respond to aetiologic treatment and could benefit from therapeutic pleural symphysis. The objective may be reached with several physical and chemical agents including cyclins and talcum powder among the most effective products devoid of major risk. The classic surgical techniques have been abandoned in favour of interventional thoracoscopy to obtain pleural abrasion and pleurectomy. Promising results have been obtained. PMID- 7530859 TI - Nitric oxide contributes to multiorgan oxidative stress in acute experimental pancreatitis. AB - BACKGROUND: Nitric oxide is a highly reactive free radical gas. The study was undertaken to determine the nitric oxide contribution to oxidative stress in acute experimental pancreatitis induced in Wistar rats. METHODS: Acute haemorrhagic pancreatitis was induced in male Wistar rats by means of a retrograde intraductal injection of 5% Na-taurocholate. The rats were treated with the nitric oxide donor, sodium nitroprusside (SNP) (0.25 mg/kg), or with N omega-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg), which is an inhibitor of nitric oxide synthase. We measured malondialdehyde and sulphhydryl group concentrations in pancreatic, lung, and liver tissue. RESULTS: In rats with acute pancreatitis treated with SNP, oxidative stress, expressed by malondialdehyde increase and sulphhydryl group depletion, was much more pronounced than in the other groups. In contrast, intensity of the oxidative stress was significantly reduced in rats treated with L-NAME. CONCLUSION: The data suggest that nitric oxide is partly responsible for oxidative stress in acute haemorrhagic pancreatitis. PMID- 7530858 TI - Induction of HLA-DR antigen expression on human colonic epithelium by tumor necrosis factor-alpha and interferon-gamma. AB - BACKGROUND: We investigated the effects of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN-gamma) on HLA-DR expression on human normal colonic epithelium, using an organ culture technique. METHODS: Biopsy specimens from normal colonic mucosa were cultured for 24 h with various concentrations of TNF-alpha and/or IFN-gamma. Epithelial HLA-DR antigens were identified by an indirect immunoperoxidase staining method. RESULTS: No expression of epithelial HLA-DR antigens was observed in specimens cultured with medium alone. Concentrations of 10(3) U/ml or higher of TNF-alpha and 10(2) U/ml or higher of IFN-gamma induced expression of HLA-DR antigens in a dose-dependent manner. Expression of HLA-DR was increased in specimens cultured with both TNF-alpha and IFN-gamma compared with those cultured with either cytokine alone. CONCLUSIONS: Our findings suggest that both TNF-alpha and IFN-gamma play important roles in regulating the expression of HLA-DR on colonic epithelium in patients with inflammatory bowel disease. PMID- 7530857 TI - Olfactory mucosal findings and clinical course in patients with olfactory disorders following upper respiratory viral infection. AB - Seventy patients with olfactory disorders following upper respiratory viral infection (URVI) were studied clinically, and the olfactory mucosa of 13 patients was biopsied using special biopsy forceps. The specimens were examined using immunohistochemical staining for neuron-specific enolase (NSE), S-100 protein (S 100), cytokeratin (CK), and proliferating cell nuclear antigen (PCNA). Although the clinical course of URVI-olfactory disorders was not very good, overall a high proportion of Alinamin intravenous injection test-positive patient's recovered their sense of smell. Immunohistochemical study of the biopsy specimens revealed a decrease in the number of olfactory receptor cells and nerve bundles. In a few cases the olfactory neuroepithelium was replaced by metaplastic squamous epithelium. Sometimes different types of degeneration were found in the same specimen. No PCNA-immunoreactivity was detected in the olfactory epithelium. The result generally correlated with the degree of degeneration of the olfactory mucosa, because regeneration of the olfactory receptor cells is suspected to be extremely slow or rare in humans. Alinamin test-positive patients had many olfactory receptor cells. These findings suggest that olfactory mucosal biopsy and the Alinamin intravenous injection test are useful methods of determining the prognosis in post-URVI olfactory disorders. PMID- 7530860 TI - Aminoacyl-RNA synthesis catalyzed by an RNA. AB - An RNA has been selected that rapidly aminoacylates its 2'(3') terminus when provided with phenylalanyl-adenosine monophosphate. That is, the RNA accelerates the same aminoacyl group transfer catalyzed by protein aminoacyl-transfer RNA synthetases. The best characterized RNA reaction requires both Mg2+ and Ca2+. These results confirm a necessary prediction of the RNA world hypothesis and represent efficient RNA reaction (> or = 10(5) times accelerated) at a carbonyl carbon, exemplifying a little explored type of RNA catalysis. PMID- 7530862 TI - Granulocyte colony stimulating factor in the treatment of alcohol abuse, leukopenia, and pneumococcal sepsis. AB - A 32-year-old woman was admitted with alcoholism, leukopenia, and pneumococcal sepsis (ALPS). Standard treatment consists of antibiotics, vitamin replacement, and intensive care unit support. Even with this treatment, the mortality rate is exceedingly high. In addition to standard therapy, this patient received subcutaneously 300 micrograms granulocyte colony stimulating factor (G-CSF) daily. Initial white blood cell count was 700 microL; by day 4 it had increased to 11,400 microL. She had a prolonged hospital course but was discharged in good condition 6 weeks after admission. G-CSF may be warranted in treating ALPS. PMID- 7530861 TI - Pathogenesis, recognition, and management of common cardiac arrhythmias. Part II: Supraventricular premature beats and tachyarrhythmias. PMID- 7530863 TI - Adjuvant chemotherapy for stage I non-seminomatous testicular cancer. AB - Developments in the treatment of stage I testicular non-seminomatous germ cell tumours have aimed primarily at reducing morbidity since the introduction of retroperitoneal lymph node dissection. Surveillance after orchidectomy, i.e. follow-up alone with chemotherapy only for relapsed disease, was found to be logistically and psychologically taxing for patients. Risk factors for relapse were, however, identified from analyses of tumour histology of the orchidectomy specimen. Between September 1988 and April 1992, 20 patients with clinical stage I testicular non-seminomatous germ cell tumours and a relatively high risk of relapse were entered into a prospective study of adjuvant chemotherapy. The chemotherapy regimen consisted of 2 cycles of cisplatin, etoposide and bleomycin. Each cycle of chemotherapy lasted 3 days. There have been no relapses at a median follow-up of 31 months (range 12-53 months). Acute and late toxicity have been modest. We have found adjuvant chemotherapy to be effective after orchidectomy in patients with stage I disease with adverse prognostic factors for relapse. PMID- 7530864 TI - Characterization of benzo[a]pyrene quinone-induced toxicity to primary cultured bone marrow stromal cells from DBA/2 mice: potential role of mitochondrial dysfunction. AB - Oral exposure of DBA/2 mice to benzo[a]pyrene (BP) has been shown to result in hematotoxicity which is manifested as aplastic anemia and leukemia. Since normal hematopoiesis is regulated by bone marrow stromal cells, in this study we have characterized the bone marrow stromal toxicity induced by BP and BP-derived metabolites, particularly quinones. Incubation of stromal cells with various concentrations of BP-1,6-, 3,6-, 6,12-, or 7,8-quinone for 24 hr resulted in a significant decrease of cell survival in a concentration-dependent manner, while cells treated with BP or BP-7,8-dihydrodiol did not exhibit any significant loss of cell survival. Among the BP quinones examined, BP-1,6-quinone was the most cytotoxic to stromal cells. The cytotoxicity induced by BP-1,6-quinone also exhibited a time-dependent relationship. Pretreatment of stromal cells with 1,2 dithiole-3-thione (D3T) resulted in a significant induction of both cellular reduced glutathione (GSH) content and quinone reductase (QR) activity in a concentration-dependent manner. However, D3T pretreatment did not offer any protection against BP-1,6-quinone-induced toxicity. Furthermore, dicumarol, a potent inhibitor of QR, or buthionine sulfoximine, a specific inhibitor of GSH biosynthesis, did not potentiate BP-1,6-quinone-induced cytotoxicity was not altered. However, incubation of stromal cells with BP-1,6-quinone resulted in a significant depletion of cellular ATP content and mitochondrial morphological changes, which preceded the loss of cell survival. In addition to BP-1,6-quinone, other cytotoxic BP quinones also exhibited a capacity to deplete cellular ATP level in stromal cells, while BP, which was not cytotoxic to stromal cells, did not elicit any significant decrease in cellular ATP level. These observations suggest that mitochondria may be a potential target of BP quinones. Overall, the above results indicate that neither cellular GSH and QR nor reactive oxygen species appear to be involved in BP quinone-induced stromal cell injury and that BP quinones may elicit cytotoxicity to stromal cells through directly disrupting mitochondrial energy metabolism. PMID- 7530865 TI - Iron(II) is a modulator of ryanodine-sensitive calcium channels of cardiac muscle sarcoplasmic reticulum. AB - Iron is examined for its ability to modify Ca2+ transport across sarcoplasmic reticulum (SR) and to alter the binding of [3H]ryanodine to its high-affinity site on the Ca2+ release channel complex of SR preparations from rat heart. Iron(III) (added as ferric chloride) has negligible activity on active Ca2+ accumulation into SR and on the binding of [3H]ryanodine. In contrast, Fe(II) (added as ferrous sulfate) is a potent inhibitor of both Ca(2+)-induced Ca2+ release (IC50 of 29 microM) and DXR-induced Ca2+ release (IC50 of 14 microM). Iron(II) enhances the rate of active Ca2+ uptake into SR vesicles, mimicking the actions of the known SR Ca2+ channel blocker ruthenium red. The underlying mechanism of Fe(II) on SR Ca2+ transport is shown to be a direct and potent action on the ryanodine receptor. Fe(II) inhibits the binding of [3H]ryanodine when assayed in the presence of 5 microM Ca2+ with an IC50 of 4 microM and in an apparently cooperative manner (nH = 1.7). In the presence of physiological (1 mM) Mg2+, Fe(II) decreases the sensitivity of ryanodine receptors toward activation by Ca2+ shifting EC50 from 18 to 35 microM in the absence and presence of 5 microM Fe(II), respectively, without significant decrease in maximum [3H]ryanodine occupancy. In the presence of 5 microM Ca2+ and 1 mM Mg2+, Fe(II) decreases the potency of doxorubicin (DXR) on [3H]ryanodine binding (shifts EC50 from 8 to 24 microM in the absence and presence of 5 microM Fe(II)). These results suggest that Fe(II) competes with Ca2+ at the activator sites on the channel complex. The actions of Fe(II) on ryanodine receptor function is not correlated with membrane lipid peroxidation of SR vesicles since Fe(II) does not produce detectable changes in malondialdehyde using the thiobarbituric acid assay. These results demonstrate a direct inhibition of the Ca2+ release channel of cardiac SR by Fe(II) which may be important in pathological states of the heart during iron overload. PMID- 7530866 TI - Changes in gap junction permeability, gap junction number, and connexin43 expression in lindane-treated rat liver epithelial cells. AB - The pesticide lindane (gamma-hexachlorocyclohexane) is a mammalian neurotoxin and hepatocarcinogen. Lindane can inhibit gap junctional intercellular communication (GJIC) and this effect may contribute to its toxic properties. The mechanism of inhibition of GJIC by lindane in WB-F344 rat liver epithelial cells was studied to determine if altered gap junction permeability, gap junction number, and/or gap junction protein (connexin43) expression were involved. GJIC was monitored by fluorescent Lucifer Yellow CH dye microinjection (dye-coupling). Gap junction number was quantified visually after indirect immunostaining of gap junctions using an anti-connexin43 monoclonal antibody. Connexin43 mRNA and protein levels were determined by Northern and Western blotting, respectively. Short-term treatment (10-30 min) with lindane (50 microM) resulted in the rapid (within 10 min), nearly complete loss of dye-coupling but no changes in gap junction number of connexin43 mRNA or protein levels. Medium-term treatment (1-4 hr) resulted in the loss of dye-coupling, gap junctions, and phosphorylated connexin43 proteins. Long-term treatment (1-14 days) led to reductions in dye-coupling, total connexin43 protein, and connexin43 mRNA. Nuclear run-on assays indicated that transcription of the connexin43 gene was reduced nonspecifically by lindane. These data indicate that reductions in gap junction permeability, number, and expression may be involved in the inhibition of GJIC depending on pesticide treatment duration. PMID- 7530867 TI - MHC class I heavy chain-dependent expression of discontinuous antigenic epitopes on beta 2-microglobulinb is inducible with peptide-ligand. AB - Previously, we reported that expression of the murine beta 2-microglobulinb (beta 2mb) antigenic epitopes defined by the mAb S19.8 and 23 (SJL [beta 2ma] anti B10.S beta 2mb]) was dependent upon association of beta 2m with MHC class I heavy chains. We have further explored the antigenic properties of beta 2m under circumstances requiring the induction of MHC class I surface expression with heavy chain-specific peptide-ligand. For the RMA-S cell line, which is class I surface null due to a defect in the TAP-2 peptide transporter, treatment with the H-2Kb-specific vesicular stomatitis virus-derived N p52-59 peptide resulted in the cell surface expression of the epitopes defined by the anti-H-2Kb mAb Y-3, as well as equally strong expression of the epitopes defined by the anti-beta 2mb mAb S19.8 and 23. Similarly, the FLU-NP p366-374 peptide induced H-2Db on the surface of RMA-S cells as determined by cytofluorometry with the mAb MKQ8; however, expression of the epitope defined by S19.8 was only partially recovered and no reactivity was observed for mAb 23. That the H-2Db heavy chain was assembled with beta 2mb on the cell surface was established from immunoprecipitation experiments with 125I-surface-radiolabeled RMA-S cells treated with FLU-NP p366-374; MKQ8 immunoprecipitated prominent heavy chain and beta 2m bands, whereas S19.8 and 23 isolated a weak beta 2m band (12-15% of that co-immunoprecipitated with MKQ8). These results are consistent with the observation that human beta 2m-deficient cells (designated FO-1) transfected with the B2mb allele were induced, in combination with the endogenous HLA class I heavy chains, to express the epitope defined by S19.8, but not mAb 23, whereas both were expressed when contransfection was performed with the H-2Kb gene. That the determinants recognized by S19.8 and 23 were formed by a discontinuous cluster of amino acids within beta 2m was established from experiments demonstrating that H-2Kb heavy chain assembled with a chimeric beta 2m molecule (comprising human beta 2m from 1-69 and mouse beta 2m from amino acid 70-99, including the polymorphic residue Ala 85) did not lead to expression of the S19.8 and 23 epitopes. The results of this study provide evidence that heavy chain polymorphism can affect the antigenic properties of beta 2m and offer insight into the basis by which CTL may react against beta 2mb when assembled with the H 2Kb molecule. PMID- 7530868 TI - Comparison of FK506- and cyclosporine-based immunosuppression in primary orthotopic liver transplantation. A single center experience. AB - FK506 has been proven effective for prevention and treatment of liver allograft rejection. Herein, we compare FK506-based immunosuppression with an effective quadruple immunosuppressive regimen, including cyclosporine and antithymocyte globulin. The results of a single center participating in the European multicenter FK506 study are reported, including immunosuppressive efficacy as well as toxicity. One-year patient and graft survival was 96.7% and 90.0% for the CsA group and 90.2% and 88.5% for the FK506 group, which is not statistically different. The incidence and severity of acute rejection episodes during the first postoperative year was similar in both treatment groups with 34.4% and 33.3% for the FK506 and CsA treatment group, respectively. Immunosuppressive potency was better for the FK506 group compared with the CsA group according to the incidence of chronic rejection. Furthermore, 5 patients (8.3%) required conversion to FK506 for immunological reasons, i.e., refractory acute or chronic rejection. The incidence of moderate and severe neurotoxicity during the early postoperative period was higher in the FK506 group (21.3%) compared with the CsA group (11.7%), while the incidence of renal insufficiency and acute renal failure was similar (18.0% and 18.3% for the FK506 and CsA treatment groups, respectively). The incidence of CMV infection was significantly higher under treatment with CsA (25.0%) than with FK506 (6.6%) (P < or = 0.05), while the incidence of pneumonia (13.1% and 13.3%), cholangitis (29.5% and 26.7%), and urinary tract infection (39.3% and 28.3% for the FK506 and CsA treatment groups, respectively) was similar in both treatment groups. However, infection was more serious in some cases treated with FK506, and evolved as the main cause of death in the FK506 treatment group. Therefore, caution should be paid to over immunosuppression and toxicity in FK506-treated patients. Regarding the monitoring of FK506, FK506 plasma level failed to be a reliable indicator, and therefore we recommend measurement of whole blood FK506 levels. Our data indicate that immunosuppressive potency of FK506 is greater than that of CsA, especially concerning the incidence of chronic rejection. PMID- 7530869 TI - Renal allograft rejection. Tubular epithelial cells present alloantigen in the presence of costimulatory CD28 antibody. AB - Renal tubular epithelial cells can be induced to express potentially immunogenic levels of class II MHC antigens but fail to stimulate the activation of allospecific T lymphocytes. The current series of experiments was performed to determine whether the failure of lymphocyte activation in this system is caused by defective T cell costimulation. It was found that cultured renal epithelial cells expressed class II MHC antigens and the immunoregulatory adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and lymphocyte function-associated antigen-3 after stimulation with IFN-gamma, but that the B7 ligand for CD28 was not expressed. Mixed cell culture experiments were set up in which lymphocytes were mixed with IFN-gamma-treated allogeneic renal cells. Lymphoproliferation and IL-2 production were only observed if bivalent anti-CD28 antibodies were titrated into these cultures. The requirement for antigen stimulation was retained by these lymphocytes, as no proliferation was observed after stimulation by class II MHC antigen nonexpressing, resting renal cells. Further experiments demonstrated that the effectiveness of the anti CD28 antibody-mediated signal was enhanced by cross-linking with a secondary anti kappa-chain antibody. These data are consistent with the concept that a costimulatory signal generated by ligation of CD28 is of central importance to the development of an immune response to alloantigen. Furthermore, these results indicate that tubular epithelial cells within a rejecting renal allograft are unlikely to initiate direct activation of infiltrating allospecific lymphocytes. PMID- 7530870 TI - Functional impairment of enteric smooth muscle and nerves caused by chronic intestinal allograft rejection regresses after FK506 rescue. AB - We have previously demonstrated that subclinical chronic rejection (CR) induces structural and functional alterations in enteric smooth muscle and nerves in a rat model of small intestinal transplantation. This study was designed to investigate the effect of prolonged FK506 rescue therapy on these sequelae of CR. Immunohistochemistry of BrdU-labeled muscle cells demonstrated that active proliferation of intestinal smooth muscle caused by CR was successfully aborted by FK506 rescue therapy after a period of 30 days (control = 0.14 +/- 0.09; CR = 30.4 +/- 1.73; rescue = 2.4 +/- 0.63 cells/jejunal cross-section, P < 0.01). However, FK506 did not reverse the already established increase in muscular thickness (control = 92 +/- 2.4; CR = 193 +/- 10.6; rescue = 188 +/- 8.1 microns) due to CR. Bethanechol stimulated circular muscle contractility was improved markedly with rescue therapy (maximal contractile force reached 39.5% of control values in CR grafts and 68.8% after rescue). Rescue therapy did not reverse the loss of NADPH-diaphorase positive myenteric ganglia (control = 37 +/- 1.4; CR = 28 +/- 2.9; rescue = 23 +/- 1.7 ganglia/cross-section). Despite the persistent loss of ganglia, inhibitory junction potentials (IJPs) improved significantly returning to control values with FK506 (control = 10 +/- 0.5; CR = 5 +/- 0.3; CR rescue = 10 +/- 0.7 mV; IJPs recorded at 1 pulse/150V/0.75 ms). Although structural changes in enteric smooth muscle and myenteric neurons induced by CR were not reversed, the progression of subclinical CR can be effectively curbed by FK506 rescue therapy. The improvement in muscular mechanics and inhibitory neural innervation is probably due to the cessation of infiltrating immunocytes and sprouting of remaining myenteric nerves. PMID- 7530871 TI - A prospective study of FK506 versus CsA and pig ATG in a porcine model of small bowel transplantation. AB - Rejection remains a major barrier to successful bowel transplantation, and immunosuppressive protocols are far from standardized. In 88 nonrelated outbred pigs, we compared the effects of two immunosuppressive regimens--one with FK506, the other with cyclosporine (CsA) and pig antithymocyte globulin (ATG)--on incidence and severity of rejection in the early, critical posttransplant period. Group A (n = 14) was nonimmunosuppressed (controls). Group B (n = 17) received pig ATG (10 mg/kg/day x 10 days), CsA (3 mg/kg/day), prednisolone (2 mg/kg/day), and azathioprine (2.5 mg/kg/day); prednisolone and azathioprine were each reduced by 50% at 8 and 15 days posttransplant. Trough CsA whole-blood concentrations were > or = 400 ng/ml for the first 7 days, > or = 200 ng/ml thereafter. Group C (n = 13) received FK506 (0.2 mg/kg/day) and prednisolone (2 mg/kg/day); prednisolone was reduced by 50% at 8 and 15 days. FK506 whole-blood concentrations were > or = 20 ng/ml. All immunosuppression in groups B and C was given intravenously. We performed orthotopic small bowel transplants with systemic venous drainage. Recipient bowel was resected distal to the second portion of the duodenum and proximal to the rectum at transplant; bowel continuity was restored by duodenojejunostomy; ileostomy was created distally to allow access for daily biopsies. We graded interstitial and vascular rejection separately, according to a scoring system (no, mild, moderate, and severe rejection). Rejection-free graft survivals at 7, 14, and 21 days posttransplant were 38%, 19%, and 0% in group A; 93%, 93%, and 62% in group B; and 100%, 91%, and 82% in group C (P < 0.001). Comparing rejection in the immunosuppressed groups, group C (FK506) had a stronger tendency toward rejection than group B (CsA-ATG); significant differences between groups B and C were, however, noted only on individual days posttransplant, not over time. The death rate due to irreversible rejection was not significantly different in groups B and C (P = 0.8), but was significantly better in both of these immunosuppressed groups than in group A (P < 0.001). Pig survival was significantly longer in group C than in B (P = 0.001) due to a lower infection rate in group C. Posttransplant serum interleukin 2 and 7 levels did not correlate with rejection grades. Graft-versus host reaction was noted only in the skin in 29% of group A, 73% of group B, and 77% of group C pigs; liver and native bowel were not involved.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530872 TI - Predictive value of inducible endothelial cell adhesion molecule expression for acute rejection of human cardiac allografts. AB - We conducted a prospective longitudinal study to determine the clinical significance of endothelial adhesion molecule expression in endomyocardial biopsies from human cardiac allografts. Ten to 18 (mean 13) consecutive allograft biopsies were obtained from 20 serial human transplant recipients over a one-year period. A total of 267 biopsies was examined. The expression of endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, as well as the presence of CD3+ T cell infiltrates was assessed by immunocytochemical staining of frozen sections. Separate specimens taken at the same time were analyzed histologically for ischemic injury or rejection. ICAM-1--and, to a lesser extent VCAM-1--was expressed at low levels in normal biopsies. E-selectin was only expressed in 15% of histologically normal biopsy specimens. Ischemic injury noted in the immediate posttransplant period was associated with increased expression of all three adhesion molecules. VCAM-1 expression increased both with the degree of CD3+ T cell infiltrates (P < 0.001) and with the degree of rejection (P < 0.05). ICAM-1 increased over constitutive levels in association with diffuse CD3+ infiltrates (P < 0.001) and with rejection (P < 0.05). E-selectin was increased on occasional vessels in association with CD3+ infiltrates (P < 0.001), but was not associated with active rejection. Increases in E-selectin were most likely to occur in biopsies just prior to rejection episodes (odds ratio 3.3), and were least likely to occur in biopsies following rejection (odds ratio 0.3). ICAM-1, but not VCAM 1, was also elevated in prerejection specimens. VCAM-1 and ICAM-1 declined in postrejection specimens. These data suggest a dynamic pattern in the expression of endothelial cell adhesion molecules during the course of cardiac allograft rejection. This study also suggests that endothelial E-selectin expression may be a useful clinical marker of impending rejection. Finally, inducible VCAM-1 expression may be a helpful adjunct in the diagnosis of ongoing acute rejection, and decreases in its expression may be indicative of successful antirejection therapy. PMID- 7530875 TI - Signaling pathways mediating axon--Schwann cell interactions. PMID- 7530876 TI - Roger W. Sperry (1913-1994). PMID- 7530877 TI - Synuclein proteins and Alzheimer's disease. AB - In Alzheimer's disease, synuclein/NAC (non-amyloid beta component of Alzheimer's disease amyloid) proteins are found in presynaptic cholinergic nerve terminals that degenerate early in Alzheimer's disease, and they are also found closely linked to beta-amyloid fibrils in senile plaques. Synuclein/NAC proteins provide a potential molecular link between the degeneration of cholinergic nerve terminals, and the formation of plaques, and might have a primary role in their development. PMID- 7530874 TI - Cytokine mRNA profiles in mouse orthotopic liver transplantation. Graft rejection is associated with augmented TH1 function. AB - Although mouse liver allografts are spontaneously accepted without immunosuppression in many strain combinations, rejection can be induced by presensitization with a donor skin graft two weeks prior to transplantation. In this study, the semiquantitative reverse transcription polymerase chain reaction (RTPCR) was used to assess the involvement of T helper (TH) cell subsets in liver allograft acceptance by determining cytokine mRNA in the graft and spleen of recipients with (A) spontaneously accepting allografts (B) rejecting liver allografts after previous skin sensitization, and (C) syngeneic controls. Spontaneously accepted liver allografts showed upregulation of TH1 (IL-2, IFN gamma) and TH2 (IL-4, IL-10) intragraft cytokine mRNA, which peaked at day 6 and tapered off thereafter, when compared with levels in syngeneic grafts, but both IFN-gamma and IL-10 mRNA persisted up to day 30. This cytokine mRNA profile correlated with the transient intragraft inflammation associated with spontaneously resolving rejection. Presensitized recipients that rejected their grafts revealed marked upregulation of TH1 (IL-2 and IFN-gamma) and TH2 (IL-4, IL 6) intragraft cytokine mRNAs compared with spontaneously accepting recipients, although IL-10 mRNA levels showed no differences between the two groups. The most striking difference was seen in IFN-gamma levels, which correlated well with the preferential deposition of IgG2a antibody isotype in the rejecting compared with the spontaneously accepting liver allograft recipients. These results suggested an association between liver allograft rejection and enhanced TH1 cytokine immune response. The ability to reject liver allografts by the adoptive transfer of splenocytes, but not serum, from a sensitized mouse ruled out preformed antibodies alone as a cause of rejection. However, spleen cytokine mRNA profiles showed no differences or trends in TH1 or TH2 expression in spontaneously accepting versus rejecting recipients, which suggested that the spleen is not a major site of alloreactive immune expansion. These data suggest that spontaneous acceptance of mouse liver allografts is associated with an insufficient intragraft TH1 cytokine response, the cause of which is currently under investigation. PMID- 7530873 TI - Abdominal multivisceral transplantation. AB - Under FK506-based immunosuppression, 13 abdominal multivisceral transplantations were performed in 6 children and 7 adults. Of the 13 recipients, 7 (53.8%) are alive and well with functioning grafts after 9 to 31 months. Six recipients died: three from PTLD, one from rejection, one from sepsis, and one from respiratory failure. In addition to rejection, postoperative complications occurring in more than isolated cases included PTLD (n = 6), abdominal abscess formation (n = 5), pancreatitis (n = 3), and ampullary dysfunction (n = 2). In addition, infection by enteric microorganisms was common during the early postoperative period. Currently, all 7 survivors are on an oral diet and have normal liver function. Two recipients (one insulin-dependent) require antidiabetes treatment, in one case following distal pancreatectomy and in the other after two episodes of pancreatic rejection. Thus, abdominal multivisceral transplantation is a difficult but feasible operation that demands complex and prolonged posttransplantation management. It is not yet ready for application and awaits a better strategy of immune modulation. PMID- 7530878 TI - The CaM kinase II hypothesis for the storage of synaptic memory. AB - Much has been learned about the activity-dependent synaptic modifications (long term potentiation and long-term depression) that are thought to underlie memory storage, but the mechanism by which these modifications are stored remains unclear. A good candidate for the storage mechanism is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) because it is localized at synapses, and its known autophosphorylation properties enable it to undergo long-term modification. In this review, John Lisman describes recent tests of the role of CaM kinase II in long-term potentiation. Experiments show that activity of CaM kinase II is increased for long periods of time after induction of long-term potentiation, that enhanced activity mimics long-term potentiation, and that enzyme activity is necessary for induction of long-term potentiation. The crucial question remaining is whether persistent enzyme activity is necessary to maintain stored information. Related issues concerning the mechanism by which synapses are weakened and the role of gene expression and structural changes are also discussed. PMID- 7530879 TI - Neural architectures for adaptive behavior. AB - How do animals use the same peripheral structures to generate different behavioral responses? Three different neuronal architectures have been proposed to mediate this task: dedicated circuitry; distributed circuitry; and reorganizing circuitry. This review will critically examine the evidence for these different architectures in invertebrate circuits, and then examine the evidence for them in more complex vertebrate circuits. The evidence suggests that these different architectures are unlikely to be found in pure form in most neural circuits, but are useful for guiding the experimental analysis of circuitry. PMID- 7530880 TI - Signalling via ATP in the nervous system. AB - Strong evidence has been provided that ATP can act as a transmitter not only in smooth muscle but also in peripheral ganglia and in brain. The cloning and molecular identification of two putative ATP receptors supports the previously established pharmacological receptor classifications. This review places into perspective the evidence for ATP as a neural signalling substance by examining sites of storage, release and hydrolysis, as well as potential actions and targets. The action of ATP is related to that of the nucleoside adenosine, and the potential of additional nucleotides to function as neural messenger is examined briefly. PMID- 7530881 TI - Rab3 proteins: key players in the control of exocytosis. AB - Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic. PMID- 7530882 TI - Modulation of spinal excitability: co-operation between neurokinin and excitatory amino acid neurotransmitters. AB - Activation of C fibres with strong 'potentially tissue damaging' chemical, mechanical or thermal stimuli produces painful sensations that are significantly enhanced during pathological conditions, such as neuropathy and inflammation. The pronounced painful symptoms of hyperalgesia and allodynia are induced, in part, by the development of spinal hyperexcitability. This involves plastic changes in synaptic transmission between primary afferents and dorsal horn neurones induced by sustained activity of peripheral nociceptors. L. Urban, S. W. N. Thompson and A. Dray describe some of the central mechanisms that account for central hyperexcitability occurring in hyperalgesia and allodynia based on evidence from experiments both in vivo and in vitro with neurokinin and N-methyl-D-aspartate receptor antagonists. PMID- 7530883 TI - [Diagnosis of sore throat. A multipractice study of 3 different ways of antigenic determination for detection of group A streptococci in throat swabs]. AB - During five months in the winter of 1992/1993, 34 general practitioners (GPs) from 18 offices participated in a clinical testing of three group A streptococcal antigen detection test (ADT) kits (Abbott TestPack Strep A Plus (Abbott), Concise Strep A, Hybritech (Concise) and Kodak SureCell Strep A (Kodak)). The GPs obtained duplicate throat swabs, one for processing with the ADT kit, the other for culture reference at The Streptococcus Laboratory (Bacteriological Department, Statens Seruminstitut, Copenhagen). A total of 1389 patients were enrolled in the study, thirty percent of whom were infected by group A streptococci. The following results were obtained: Abbott: Sensitivity: 76%, specificity: 99%, positive predictive value: 97%, negative predictive value: 91%. Concise: Sensitivity: 82%, specificity: 95%, positive predictive value: 86%, negative predictive value: 92%. Kodak: Sensitivity: 84%, specificity: 87%, positive predictive value: 73%, negative predictive value: 93%. As a follow-up to the main study, each GP filled in a questionnaire, stating his opinion about the investigated ADT kit. Considering the practical handiness, Concise scored higher than Abbott, which in turn scored higher than Kodak. In conclusion, Abbott and Concise are recommended for the diagnosis of group A streptococcal pharyngotonsillitis in general practice. PMID- 7530884 TI - [Reevaluation of antithyroid drug therapy in Graves disease]. AB - Though antithyroid medical therapy has been used for several decades in the medical treatment of hyperthyroidism, only recently has attention been drawn towards prospective controlled trials concerning the effect in relation to dosage as well as the dosage related to side effects. A review is given in relation to recent investigations on treatment strategies in various parts of the world as well as the most frequently used strategies in Europe. Special attention is given to treatment principles in relation to pregnancy, children and adolescents as well as patients with eye symptoms. Antithyroid drug therapy of hyperthyroidism is a frequent and successful treatment strategy in Europe. Globally, there are still large discrepancies in the treatment strategies, related rather to conventions than to a rational attitude. PMID- 7530885 TI - Comparison of the postoperative fibrinogen, alpha-1-antitrypsin and alpha-2- macroglobulin concentrations in the blood of wounded soldiers of Croatian Army and peacetime injured civilians. AB - In 10 wounded Croatian Army soldiers and in 10 civilians with accidental musculosceletal traumatisation, blood concentrations of the 3 acute phase proteins: fibrinogen, alpha-1-antitrypsin and alpha-2-macroglobulin on the 1st, 5th and 10th postoperative day were assessed. On the 5th day after injury, increased concentrations of all 3 acute phase proteins (APP) were found in all patients. The rise of the fibrinogen concentrations in the wounded soldiers was the steepest. On the 10th day after wounding, fibrinogen concentrations in the gorup of wounded soldiers were significantly lower than those in the group of injured civilians. On the 10th day after wounding there were no differences between the 2 groups in the concentrations of alpha-1-antitrypsin and of alpha-2 macroglobulin. The lower fibrinogen concentrations in the wounded soldiers could be explained by the modulation of the general reactivity of the organism to injury, proposed by Woloski, which is induced by stress and microtrauma on battlefield before the wounding. PMID- 7530887 TI - Oral immunization against diphtheria and tetanus infections by fluid diphtheria and tetanus toxoids. AB - Purified diphtheria toxoid incorporated in egg yolk and mixed with a medicinal plant seed was used to orally immunize rabbits against diphtheria infection. Animals were partially immunized against a lethal diphtheria toxin challenge. The immunity was complete when gastric enzyme juices were inhibited before oral vaccination by aprotinin, a natural protease inhibitor. Rabbits and monkeys were orally immunized against both diphtheria and tetanus in the same way by pre treatment with aprotinin. Adult volunteers receiving protease inhibitor before administration of oral toxoids have shown a significant rise in specific circulating antitoxins. PMID- 7530888 TI - Production of Vibrio cholerae ghosts (VCG) by expression of a cloned phage lysis gene: potential for vaccine development. AB - The protein E-specific lysis mechanism of the Escherichia coli-specific bacteriophage PhiX174 was employed to produce Vibrio cholerae ghosts (VCG). VCG consist of both rounded and collapsed cells that have lost their cytoplasmic contents through an E-specific hole in the cell envelope. These ghosts are proposed as non-living material for immunization against cholera. A specific membrane anchor sequence was used to insert the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) fusion protein into the cell envelope of V. cholerae. The identity of the expression products was confirmed by Western blot analysis employing an RT-specific monoclonal antibody. HIV-1 RT was chosen as a model for the purpose of evaluating heterologous gene expression in V. cholerae and the carrier potential of VCG. Intraperitoneal immunization of mice was used to evaluate the immunogenic potential of VCG. Preliminary results showed significant seroconversions to intact whole-cell vibrio antigens in mice immunized with VCG or a heat-killed whole-cell vibrio preparation. PMID- 7530886 TI - Enhanced immunogenicity of protein-dextran conjugates: I. Rapid stimulation of enhanced antibody responses to poorly immunogenic molecules. AB - In view of our observation that anti-immunoglobulin antibody conjugated to high molecular-weight dextran stimulates high levels of B-cell activation (Brunswick et al. J. Immunol. 1989, 143, 1239), we coupled T cell-dependent antigens to dextran. When mice were immunized, in the absence of adjuvant, with a BSA-dextran conjugate (BSA-dex), a persistent, high-titre anti-BSA IgG1 response was induced. Titres were dose-dependent and seen with as little as 10 micrograms of conjugated protein. Anti-BSA titres were detected as early as day 7, usually peaked at about day 14 and persisted for at least 4 weeks. Anti-hapten antibodies were also elicited in mice that were immunized with haptenated BSA covalently bound to dextran, and secondary responses could be induced even after inoculation of the unconjugated protein. Covalent attachment of the protein to the polymer was necessary, and the response was specific, as coinjection of BSA-dex and an unrelated antigen, goat IgG, did not elicit detectable anti-goat antibodies. The immunogenic potential of these conjugates did not depend on the ability of the dextran carrier to induce antibody, inasmuch as they stimulated high levels of anti-protein antibody in mice unresponsive to dextran. A minimum size dextran polymer was required for enhanced immunogenicity as conjugates of BSA with dextran of molecular mass 500 or 2000 kDa but not of 70 kDa gave detectable anti BSA titres. PMID- 7530890 TI - Substance P in intervertebral discs. Binding sites on vascular endothelium of the human annulus fibrosus. AB - The annulus fibrosus of the human intervertebral disc is sparsely innervated, some of the fibers containing substance P. We could demonstrate, by autoradiography, binding sites for substance P localized on the endothelium of small blood vessels in the annulus fibrosus of human intervertebral discs removed during anterior fusion for back pain. In binding inhibition studies, binding of 125I-Bolton Hunter-substance P was inhibited by unlabeled substance P and the related tachykinins neurokinin A and neurokinin B with a rank order of potency substance P > NKA > NKB. Specific binding was reduced > 75 percent by 5' guanylylimidodiphosphate, indicating G-protein coupling. These features are characteristic of an NK1 receptor through which vascular effects, i.e., vasodilation, plasma extravasation and angiogenesis of substance P, are mediated. The presence of NK1 receptors on blood vessels in the annulus fibrosus may indicate a role for substance P in tissue repair although acute proinflammatory effects may contribute to discogenic pain. PMID- 7530889 TI - Intrathecal synthesis of anti-myelin basic protein IgG in HIV-1+ patients. AB - Human immunodeficiency virus type 1 (HIV-1)-infected individuals frequently develop a broad spectrum of neurological syndromes, classified as HIV-1 associated cognitive/motor complex. Diffuse demyelination of hemispheric white matter is a commonly observed in HIV-1 infected brain, but the events leading to myelin destruction are still obscure. Since oligodendrocyte infection by HIV-1 is not proven as yet, myelin damage in HIV-1 infection may result from indirect mechanisms such as the excessive release of myelinotoxic substances or the triggering of autoimmune responses directed to myelin constituents. To verify the latter hypothesis, we searched for elevated anti-myelin basic protein (MBP) IgG levels in the cerebrospinal fluid (CSF) and serum of 25 patients with HIV-1 infection, 12 with multiple sclerosis (MS), and 9 with non-inflammatory neurological diseases (NIND). CSF, but not serum, anti-MBP IgG levels were more frequently elevated in HIV-1+ (16/25, 64%) than in MS (3/12, 25%) or NIND (0/9) patients. By using the anti-MBP IgG index, the anti-MBP IgG antibody specificity index (ASI), and the search for anti-MBP oligoclonal IgG, we ascertained that anti-MBP IgG were produced within the CNS in 13 of 25 (52%) HIV-1+, in 6 of 12 (50%) MS, and in none of NIND patients. The incidence of increased CSF anti-MBP IgG levels was higher among HIV-1+ patients at stage II-III (4/4, 100%) or at stage IV B (7/9, 78%) than among those at stage IV C-IV D (5/12, 42%). Although our data indicate that intrathecal anti-MBP IgG may occur early during HIV-1 infection and that they are more common in patients with HIV-1-associated cognitive/motor complex, the possible demyelinating role of these antibodies remains to be demonstrated. PMID- 7530891 TI - Effects of NK1 receptor antagonists on vasodilation induced by chemical and electrical activation of sensory C-fibre afferents in different organs. AB - The effects of the non-peptide NK1 receptor antagonists, CP 96,345 and RP-67,580, were investigated in a model using anaesthetized pigs. Both the blood flow in the internal maxillary and the bronchial artery (ultrasonic flowmetry) and the superficial blood flow in nasal mucosa and the skin (laser-Doppler flowmetry) were monitored simultaneously. Vasodilation induced by substance P administered i.v. systemically was blocked by pretreatment with CP-96,345, 3 mg kg-1 but not by RP-67,580. CP-96,345 had no effects on the vasodilation induced by calcitonin gene-related peptide or vasoactive intestinal polypeptide. The capsaicin-induced vasodilation in the superficial blood flow of the nasal mucosa and the skin, was reduced after the CP-96,345 pretreatment. The vasodilation induced by capsaicin infusion in the internal maxillary or the bronchial artery was not affected by the CP-96,345 pretreatment. Electrical stimulation of the vagal nerve induced a vasodilation in the bronchial circulation which was not attenuated by pretreatment with CP-96,345. In the nasal mucosa and the skin NK1 receptors seem to be involved in the vasodilation in the superficial small vessels, due to chemical activation of sensory C-fibre afferents. Furthermore, CP-96,345 is a useful tool in the evaluation of NK1 receptor-mediated responses. RP-67,580 which has been shown to have NK1 antagonistic properties in the rat has no such effects in the domestic pig. PMID- 7530892 TI - Problems in the introduction of genetically engineered microorganisms into the environment. AB - The use and release of genetically engineered microorganisms (GEMs) into the environment, usually the agricultural environment, is increasing exponentially. Potential applications of GEMs include crop production, pest management, degradation of environmental pollutants, mining and mineral recovery, and others. Several strategies of molecular and cellular biotechnology, such as recombinant DNA techniques, nuclear microinjection and cell fusion may be used to modify bacteria and fungi for useful purposes. The benefits expected from release of genetically engineered microorganisms, if safely applied, might be substantial in various fields. However, a safe introduction of GEMs into the environment requires full environmental and ecological risks assessment. Because of the wide scope of genetic engineering targets this review will focus on the application of GEMs potentially useful in agricultural practices (crop nutrition, pest and disease control) and ecological problems associated with the introduction of alien microorganisms into the environment. PMID- 7530893 TI - Insertions within iap gene of Listeria monocytogenes generated by plasmid pLIV are not lethal. AB - To carry out efficient insertional mutagenesis in Listeria monocytogenes 1040S and to facilitate the characterisation of disrupted genes, three novel derivatives of plasmid pACYC 184 were constructed, p-LIV 1, pLIV-2 and pLIV-3. The technique is simple and rapid and can be applied to most genes, even those that are essential. The method is unique and particularly effective by the use of a temperature-sensitive pE 194 replicon to facilitate the insertion of the gene. After transformation of the plasmid into L. monocytogenes it is possible to select for integration of the plasmid into the chromosome at 42 degrees C. High insertion frequency and convenience for looking for specific mutations with known sites of insertion make them the plasmid derivatives of choice for insertional mutagenesis in any bacteria that support replication of pE 194 TS. Three insertional mutants of L. monocytogenes are described. Two insertions were shown to be within iap gene, one in hly gene. The supernatants and the pellets from the iap mutants had no detectable haemolytic titer when assayed without the reducing agent. PMID- 7530894 TI - Integration host factor, a histone-like Escherichia coli protein, binds to at least four sites of the DNA fragment containing the recA gene. AB - The integration host factor (IHF) is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli. The characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. Less is known concerning the role of IHF in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression. In this paper we report data concerning the binding of IHF protein to the recA gene region. IHF binds to at least four sites of the DNA fragment containing the recA gene, as shown by gel mobility shift assays. On the basis of the ihf consensus sequences published, we have been able to identify two sequences of putative ihf sites (ihf 1 and ihf2) into the 1390 bp long sequence containing the recA gene, but only the ihf2 site was able to bind IHF, as measured by gel mobility shift experiments. The nonfunctional ihf1 sequence was found to overlap the -35 region of the recA promoter and the functional ihf2 sequence was found within the recA gene structure at nt +780 to +807 (both with three mismatches according to the consensus sequence of Kur et al., 1989). This confirms our earlier results that the IHF-DNA interaction does not depend on any very rigid sequence, but also on the suitable sequences of the neighbouring regions, together with the proper DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530895 TI - Factors affecting intra and extracellular phospholipase A production by Salmonella typhi. AB - The effect of various physico-chemical factors on the production of intra and extracellular phospholipase A by Salmonella typhi was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth, after 16h of incubation at 37 degrees C. Highest level of extracellular phospholipase A was also seen in the same medium (pH 7.0), but after 24 h of incubation at 37 degrees C. Agitation during incubation enhanced the enzyme synthesis. Addition of surfactants to the growth media significantly decreased both intra and extracellular phospholipase A production. PMID- 7530896 TI - Improvement of the strain for the rapid identification of genes encoding restriction and modification enzymes. AB - The E. coli AP1-200-9 strain for rapid identification of genes encoding restriction and modification enzymes carries a temperature sensitive lacZ gene fused to the damage-inducible dinD locus. A derivative of this strain was constructed that has a wild-type form of this locus which allows for a more efficient identification of recombinant plasmids encoding restriction and modifications enzymes. PMID- 7530897 TI - Methods of introduction of foreign DNA into mycobacteria. AB - Two methods: triparental conjugation and electrotransformation were used for introduction of plasmid DNA into mycobacterial cells. The introduction of shuttle plasmid pMY10 into M. fortuitum mutant caused the activation of its chromosomal cryptic KmR gene. The used of integration vector pUS 903 allowed to obtain a collection of mutants interesting for studies of genetic determination of steroid biotransformation and drug-resistance in mycobacteria. PMID- 7530898 TI - Postselective (directed) mutagenesis of fast-growing strain of Mycobacterium vaccae as a method of creating mutants showing changed phenotypic properties. AB - Postselective (directed) mutagenesis was used to create mutants of M. vaccae B3805 showing changed abilities for steroid biotransformations. Three morphological classes of such mutants, differing in colony colour: yellow, white and pink, were selected. Some of them can be helpful in genetic studies of steroid biotransformation. Two mutants were also shown to be hosts for foreign DNA. PMID- 7530899 TI - Mutagenicity of carboline methyl derivatives in Salmonella. AB - A series of linear, methyl-substituted derivatives of 5H-indolo[2,3-b] quinolines was tested for mutagenic activity with the battery of Ames tester strains. Mutagenic activity of indoloquinolones was strongly influenced by the position and a number of methyl groups. All compounds tested, with one exception, act like frame-shift mutagens. Only two compounds among them were mutagenic in the strain detecting oxidative and cross-linking mutagens. PMID- 7530900 TI - Problems associated with plasma albumin estimation in nephrotic syndrome using the bromocresol green method. AB - Evaluation of albumin estimation by bromocresol green (BCG) method was carried out in sixty nephrotics and twenty control subjects. In nephrotic syndrome, varies; is directly proportional, to 2-globulin and total cholesterol concentrations were significantly increased, while the mean albumin level was significantly reduced when compared with the corresponding control values. In both control and nephrotics, the determination of serum albumin by the BCG method showed good correlations with values obtained by cellulose acetate electrophoresis using the biuret method to determine the total protein, but the mean value for the nephrotics was higher by an average of 0.4g/100ml. Interference with the BCG reaction by an increased varies; is directly proportional, to 2-globulin level was suggested as a possible explanation for the higher mean albumin level obtained by the BCG method in the nephrotics. Inclusion of 0.8M NaCl in the BCG assay system did not prevent the interference by other proteins. However, this interference could to a large extent, be offset by calibrating with a pool of fresh sera previously determined by electrophoresis. PMID- 7530902 TI - Interface of malnutrition and periodontal diseases. AB - In response to periodontal pathogens neutrophils release oxidants, proteinases, and other destructive factors. The balance between these factors, the antioxidants, and endogenously synthesized antiproteinases determine the extent of periodontal damage. Malnutrition, particularly protein-energy malnutrition involving concomitant deficiencies of antioxidant nutrients, is characterized by impairment in production and cellular actions of the cytokines, diminished acute phase protein response (APR) to infections, endocrinopathies, defective metabolism of drugs, and impaired response to stress. The APR plays a central role in promoting healing. Additionally, malnutrition elicits adverse alterations in the oral microbial ecology as well as in the volume and the antibacterial and physicochemical properties of saliva. Good dietary practices and optimal nutritional status are therefore important in mitigating the severity of inflammatory periodontal lesions but are likely of limited value if the stimuli from dental plaque are not removed. PMID- 7530903 TI - Re: "Case-control study of childhood cancer and exposure to 60-HZ magnetic fields". PMID- 7530901 TI - Intravenous urography pre prostatectomy: an evaluation of its use. AB - The pre-operative intravenous urograms of 120 consecutive patients who had prostatectomy for benign prostatic hypertrophy (BPH) were studied. Of these, seventy eight patients (65%) had normal intravenous urograms (IVU) while 42 patients had abnormal IVU. In this study serum creatinine above 2.0mg/dl and blood urea above 35mg/dl proved valuable indices for possible selection of patients with BPH likely to show significant obstructive disease on IVU. This is not only cost saving, but also reduces unnecessary radiation to the patient. PMID- 7530905 TI - Interrater reliability of the Peabody Developmental Motor Scales: fine motor scale. AB - OBJECTIVE: The interrater reliability of the Peabody Developmental Motor Scales (PDMS) fine motor scale was examined in 23 children with developmental disabilities who were between 2 years and 5 years of age. METHOD: Three occupational therapists viewed videotapes of the children and scored each child's performance on the fine motor section of the test. Data were analyzed with the intraclass correlation (ICC) approach; ICC values ranged from 0.90 to 0.97 for the subskills of Grasping, Hand Use, Eye-Hand Coordination, and Manual Dexterity. RESULTS: The ICC interrater reliability value for the total fine motor score was 0.99. Reliability values were additionally computed with the PDMS fine motor scale age-equivalent scores and Z scores. The ICC values for these methods of scoring the PDMS were 0.99 and 1.00, respectively. CONCLUSION: The PDMS fine motor scale can be used consistently to evaluate fine motor delays in this population of young children. PMID- 7530904 TI - Performance of typical children on the Sensory Profile: an item analysis. AB - OBJECTIVES: The purpose of this study was to obtain data about typical children on the 99-item Sensory Profile, a newly developed tool derived from sensory history items reported in the literature and designed to assess children's responses to commonly occurring sensory events. METHOD: Parents of 64 typical children 3 to 10 years of age completed the Sensory Profile; parents used a five point Likert scale to report the percentage of time their children engaged in each behavior. Researchers then analyzed these percentages to determine differences by age and gender. RESULTS: Sixty-seven of the items on the Sensory Profile were found to be uncommon for these typical children. On further analysis with a multivariate analysis of variance and appropriate follow-up procedures, one item was more common for younger children, and four items were more common for girls. CONCLUSION: Two thirds of the items on the Sensory Profile were uncommon for typical children and thus may contribute useful information about children with disabilities who respond to these sensory events. PMID- 7530907 TI - Extracellular H+ modulates acetylcholine-activated nonselective cation channels in guinea pig ileum. AB - The effects of external H+ on the acetylcholine-induced inward current (nonselective cationic current; InsACh) in guinea pig ileal smooth muscle were investigated using the conventional whole cell patch-clamp technique. When the external pH (pHo) was lowered, the amplitude of InsACh was increased, with no significant change in the reversal potential or no detectable induction of other ionic permeabilities. The dose-response curve for this effect was best described by a Hill-type equation with an apparent pKa value of 7.4 and a Hill coefficient of approximately 1. The effect of pHo was associated with a shift of the steady state activation curve for InsACh; the half-maximum activation potential became more negative on lowering pHo. Similar results were obtained when InsACh was activated by intracellularly applied guanosine 5'-O-(3-thiotriphosphate). These results indicate that the external H+ activity is an efficient regulator of InsACh channel, and this may have a physiological importance for controlling the muscarinic receptor-mediated contractions in this muscle. PMID- 7530906 TI - The iodide channel of the thyroid. II. Selective iodide conductance inserted into liposomes. AB - An iodide channel has been previously identified in the plasma membrane of bovine throcytes [Golstein et al., Am. J. Physiol. 263 (Cell Physiol. 32): C590-C597, 1992]. The plasma membrane proteins were solubilized and ultrafiltered, and the protein fraction collected above 100 kDa was inserted in liposomes. Voltage sensitive uptake of radiolabeled I- by these proteoliposomes was studied. To this end, an outward I- gradient was set up by loading the proteoliposomes with KI and removing extraliposomal I-. I- exit from the proteoliposome induces an inside positive membrane potential, which leads to the uptake of 125I- added to the incubation medium. This uptake was abolished by valinomycin, which in the presence of K+ short circuits the liposomal membrane potential, demonstrating the conductive nature of this uptake. A double reciprocal plot of I- influx over I- concentration suggests the existence of a single population of channels in these proteoliposomes with a Michaelis-Menten constant for I- of approximately 9 microM. When the proteoliposomes were loaded with KCl or KSCN instead of I-, no conductive uptake occurred anymore, suggesting that these anions are unable to diffuse through the I- conductance, hence do not generate a diffusion potential. I- uptake by KI-loaded proteoliposomes was not inhibited in the presence of a 1,000-fold excess of extraliposomal Cl- but was completely inhibited by a 1,000 fold excess of extraliposomal SCN-, indicating that Cl- does not permeate the I- channel, whereas SCN- inhibits it. SCN- and flufenamate were both shown to be competitive inhibitors of the I- channel with an inhibitor constant of approximately 10 and 750 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530908 TI - Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells. AB - When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF) affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells. PMID- 7530909 TI - Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP dependent protein kinase. AB - The effect of the nitric oxide (NO) donor SIN-1 (3-morpholino-sydnonimine) on the calcium current (ICa) was examined in guinea pig ventricular myocytes. SIN-1 had little effect on basal ICa. After moderate stimulation of ICa with 10 nM isoproterenol (ISO), 10 microM SIN-1 caused either stimulation or inhibition of ICa; 100 microM SIN-1 consistently caused inhibition. SIN-1 (1-100 microM) inhibited ICa equally following considerable enhancement of ICa by either 1 microM ISO or 100 microM 3-isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor. SIN-1 (100 microM) also inhibited ICa equally following enhancement by either 10 microM pipette adenosine 3',5'-cyclic monophosphate (cAMP) or hydrolysis-resistant 8-bromo-cAMP. Thus the inhibitory effect of SIN-1 appears independent of PDEs. Addition of LY-83583 (a blocker of guanylate cyclase) to the pipette or superfusion with KT-5823 [a blocker of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase] suppressed the inhibitory effect of SIN-1. We conclude that NO is an important modulator of beta-adrenergic effects on ICa and that the mechanism of NO inhibition of ICa in mammalian cardiac cells involves the cGMP-dependent protein kinase. PMID- 7530910 TI - Measurement of secretogranin II release from individual adenohypophysial gonadotropes. AB - Secretogranin II (SG-II) is an acidic 86-kDa protein found in high abundance in the anterior pituitary gland. In the present studies, we investigated the secretion and the localization of SG-II using pituitary cells from female rats at all stages of the estrous cycle. Double immunofluorescence staining revealed that SG-II immunoreactivity was localized in low abundance in about half of all pituitary cells and in high abundance in all of the luteinizing hormone (LH) immunoreactive cells (which represent approximately 5% of all pituitary cells). Using a reverse hemolytic plaque assay for measurement of SG-II release from individual pituitary cells in culture, we found that SG-II secretion was strongly stimulated by gonadotropin-releasing hormone in a dose-related fashion, and the amount of SG-II secretion was also related to the stage of the estrous cycle: it was highest at proestrus and lowest at estrus. SG-II plaque assay followed by LH immunofluorescence staining further revealed that all the SG-II-secreting cells contained LH immunoreactivity. At proestrus all the LH-immunoreactive cells secreted SG-II, whereas another days of the estrous cycle only a fraction of them did so. Thus our findings demonstrate a striking resemblance between SG-II and LH with regard to cell localization and secretory regulation. PMID- 7530911 TI - The tachykinin receptors inducing contractile responses of canine ileum circular muscle. AB - This study sought to determine which tachykinin receptors were involved in contractile responses of circular muscle to tachykinins infused into isolated segments of canine ileum. Selective agonists for neurokinin receptors NK1 and NK2 as well as for substance P (SP) and neurokinin A (NKA) were infused, and selective antagonists against NK1, NK2, and NK3 receptors were tested. The responses to a submaximal concentration of NKA were reduced by a selective NK2 antagonist, SR-48968, and abolished by a combination of this antagonist with an NK1 antagonist, either CP-96,345 or RP-67580. The selective NK2 agonist, [Nle10]NKA-(4-10), had low potency. We concluded that NKA acted on typical NK2 receptors and that is action was potentiated by its additional action on NK1 receptors. Neither the contractile responses to SP nor those to [Sar9,Met(O2)11]SP given in submaximal concentrations were inhibited by CP-96,345 or RP-67580, either alone or together with SR-48968. Indeed, the two NK1 selective antagonists potentiated responses to the selective NK1 agonist, [Sar9,Met(O2)11]SP, an effect attributed to previously demonstrated prejunctional inhibitory action of the agonist. The selective NK3 agonist, succinyl-[Asp6,N-Me Phe8]SP-(6-11), was not effective as a contractile agent, even after block of nitric oxide synthase with N omega-nitro-L-arginine. The selective NK3 antagonist, R-487, was also ineffective in blocking responses to SP. Studies with an antagonist to H1 histamine receptors suggested that contractile actions of SP did not involve histamine release from mast cells. We concluded that, in addition to typical NK1 and NK2 receptors activated by NKA and a prejunctional inhibitory receptor activated by SP and [Sar9,Met(O2)11]SP, another tachykinin receptor existed on canine ileum to initiate contractions. It is not a typical NK1, NK2, or NK3 receptor. PMID- 7530912 TI - Nitric oxide pathway in cat esophagus: localization of nitric oxide synthase and functional effects. AB - In the cat lower esophageal sphincter (LES) and esophageal body, nitric oxide synthase (NOS) immunoreactive nerves were abundant in the circular smooth muscle layer, especially in the LES region. NADPH diaphorase staining showed an identical pattern. The ability to form L-citrulline from L-arginine corresponded roughly to the distribution of NOS. Confocal microscopic analysis indicated colocalization within neurons of vasoactive intestinal peptide (VIP) in 65% of NOS-positive nerves. In LES circular smooth muscle preparations, electrically induced relaxations (single train stimuli) were generally abolished by NG-nitro-L arginine (L-NNA). Continuous electrical stimulation for 2 min evoked a relaxation in the presence of L-NNA. This relaxation was inhibited by VIP antiserum and followed by a decrease in guanosine 3',5'-cyclic monophosphate, but not by any consistent change in adenosine 3',5'-cyclic monophosphate levels. K+ (124 mM) induced a biphasic relaxation, with L-NNA inhibiting the first phase but not the second. We conclude that nitric oxide (NO) has a major role as the mediator responsible for relaxation in the cat esophagus. NO seems also to initiate the release and enhance the effect of another transmitter. PMID- 7530913 TI - Rabbit cortical collecting ducts express a novel prostacyclin receptor. AB - Prostaglandin E2 (PGE2) inhibits vasopressin-stimulated water conductivity (AVP Lp) and inhibits Na+ reabsorption in the rabbit cortical collecting duct (CCD). Inhibition of Na+ reabsorption is mediated by increased intracellular calcium ion concentration ([Ca2+]i). Prostacyclin (PGI2) has also been shown to inhibit Na+ reabsorption in the CCD. The present studies were designed to examine the effect of the PGI2 agonist, Iloprost (ILP), on AVP-Lp and [Ca2+ in the isolated perfused rabbit CCD and to determine whether ILP activates different receptors than PGE2. ILP and PGE2 each maximally inhibited AVP-Lp equipotently at 10(-7) M. When CCDs were exposed to PGE2 and ILP simultaneously, or if PGE2 was added in the presence of ILP, inhibition of AVP-Lp was additive. Additivity was not observed if the PGI2 agonist, carbaprostacyclin (c-PGI2), was added with ILP, or if the PGE2 agonist, sulprostone, was added with PGE2, or if ILP was added to CCDs preexposed to PGE2. In fura 2-loaded CCD, ILP and PGE2 added separately increased [Ca2+]i. The response to c-PGI2 could be desensitized by prior exposure to ILP. ILP did not cause desensitization to PGE2, but PGE2 could desensitize the CCD to ILP. We conclude that PGI2 inhibits AVP-Lp by activation of a novel IP3 prostacyclin receptor and increases [Ca2+]i by activation of an IP1 prostacyclin receptor in the rabbit CCD. Functional evidence is presented that PGI2 cannot occupy PGE2 receptors and that PGE2 can occupy but cannot activate PGI2 receptors linked to inhibition of AVP-Lp. PMID- 7530914 TI - Insulin-induced release of plasminogen activator from human blood platelets. AB - Incubation of washed platelets in Tyrode buffer, pH 7.5, with insulin (200 microU/ml) and CaCl2 (1.2 mM) at 37 degrees C for 3 h resulted in a threefold increase of plasminogen activator activity in the supernatant over the basal level as determined by both the amidolytic assay and the proteolysis of alpha casein through the formation of plasmin from plasminogen. This plasminogen activator showed no plasmin-like activity and was inhibited by anti-tissue plasminogen activator antibody as well as by type 1 plasminogen activator inhibitor. The substrate specificity and the inhibition of the enzymic activity by various inhibitors indicated that the platelet plasminogen activator (pPA) was related to tissue-type plasminogen activator of relative molecular weight 56,000. Fibrinolytic activity of pPA and its insulin-dependent release were demonstrated by the shortening of euglobulin lysis time and by the clot lysis time of platelet rich plasma from normal and type I diabetes mellitus patients. Treatment of platelet membranes with insulin also increased the release of pPA. Increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in platelets by incubation with various agents completely inhibited the insulin-induced release of the activator. On the other hand, inhibition of platelet aggregation by aspirin had no effect on the release of pPA, indicating that the effect of cAMP was not due to the inhibition of platelet aggregation by the nucleotide. PMID- 7530915 TI - CPA enhances Ca2+ entry in cultured bovine pulmonary arterial endothelial cells in an IP3-independent manner. AB - Studies of rat aorta revealed that cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum Ca2+ pump, released endothelium-derived relaxing factor (EDRF) and relaxed the muscle. We have used CPA to elucidate how this inhibitor of Ca2+ uptake into internal stores affects K+ channels and Ca2+ entrance in cultured bovine pulmonary endothelial cells using patch-clamp techniques. CPA increased a Ca(2+)-dependent outward K+ current for many minutes, presumably as a consequence of the unbalanced leakage of Ca2+ from internal stores and Ca2+ entrance across the cell membrane. An expected consequence of this activation of the outward current change is hyperpolarization of the cell membrane and increased driving force for Ca2+ entry. CPA activated the influx of extracellular Ca2+ through nonselective cation channels. Ca2+ influx through nonselective cation channels could help maintain intracellular Ca2+ concentration elevation and EDRF release. CPA also reduced the inwardly rectifying K+ current. Inositol 1,4,5-trisphosphate (IP3) in the patch pipette also produced an increase in outward K+ currents, which were Ca2+ dependent. After depletion of Ca2+ internal stores by CPA, the response to IP3 was abolished. Heparin in the patch pipette reduced the increase in outward currents induced by bradykinin, an agonist known to raise IP3 and to release Ca2+, but did not prevent CPA-induced increases in outward current. Thus CPA acts to elevate Ca(2+)-activated currents in endothelial cells by a mechanism independent of IP3-induced release, and this may lead to EDRF release both directly and as a consequence of Ca2+ entry through nonselective cation channels driven by an increased electrical gradient for Ca2+. PMID- 7530916 TI - Mechanical determinants of left ventricular relaxation in isovolumically beating hearts. AB - Both pressure and volume have been proposed to determine the speed of left ventricular (LV) relaxation, but their relative importance is not known. Accordingly, we used isolated, buffer-perfused, isovolumically beating ferret hearts to study the effects of maximal developed pressure (Pdmax) and LV volume (V) on the speed of LV relaxation. Experiments were performed at 30 degrees C, and the hearts were paced at a baseline interbeat interval (BI) of 800 ms. Pdmax was varied independently of V by use of seven BI (75 to 133% of baseline BI), which resulted in test beats that developed a range of Pdmax due to varying degrees of restitution. Pdmax was also varied by setting V at five levels (80 to 120% of baseline V) during the test beats. Speed of relaxation was quantified as the time period of pressure decay from 75 to 25% Pdmax (T75-25). Data were analyzed by multiple linear regression. Increases in both Pdmax and V independently prolonged T75-25, and T75-25 was 1.45 times more sensitive to Pdmax than to V. However, when Pdmax and V were combined to estimate maximal wall stress (sigma max), the effects of Pdmax and V, as well as relative circumferential muscle length (estimated by V1/3), were not important determinants, and T75-25 depended on sigma max alone. Thus we conclude that 1) Pdmax and V are both determinants of the speed of LV relaxation and that Pdmax is approximately 1.5 times more important than V, and 2) the effects of Pdmax and V on relaxation act via the common mechanism of sigma max. PMID- 7530918 TI - Opioids and nitric oxide contribute to hypoxia-induced pial arterial vasodilation in newborn pigs. AB - The present study was designed to investigate the contribution of opioids and nitric oxide (NO) to hypoxia-induced pial vasodilation. Newborn pigs equipped with a closed cranial window were used to measure pial arteriolar diameter and to collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and guanosine 3',5'-cyclic monophosphate (cGMP). Hypoxia-induced pial dilation was potentiated by norbinaltorphimine, 10(-6) M, a kappa-opioid antagonist (25 +/- 2 vs. 33 +/- 3%, n = 5), but was blunted by beta-funaltrexamine, 10(-8) M, a mu opioid antagonist (28 +/- 2 vs. 19 +/- 1%, n = 5). Hypoxia-induced vasodilation was associated with increased CSF methionine enkephalin, a mu-opioid agonist (884 +/- 29 vs. 2,638 +/- 387 pg/ml, n = 5). N omega-nitro-L-arginine (L-NNA), an NO synthase inhibitor (10(-6) M), also blunted hypoxia-induced vasodilation that was further diminished by coadministration of L-NNA and beta-funaltrexamine (26 +/- 2, 14 +/- 1, and 9 +/- 1%, respectively, n = 5). Reversal of the above order of antagonist administration resulted in similar inhibition of hypoxia-induced pial dilation. Hypoxia-induced vasodilation was also associated with an increase in CSF cGMP that was attenuated by L-NNA (2.1 +/- 0.1- vs. 1.1 +/- 0.2-fold change in CSF cGMP, n = 5). Sodium nitroprusside (10(-6) M) increased CSF cGMP and methionine enkephalin concentration similar to hypoxia. These data suggest that hypoxia-induced pial arterial vasodilation, in part, is due to NO and/or cGMP induced methionine enkephalin release as well as the direct action of NO. PMID- 7530919 TI - Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. AB - Endothelium-derived nitric oxide (NO) has recently been reported to be a mediator of ischemic preconditioning in dog hearts. The aim of the present study was to determine the role of NO in ischemic preconditioning in isolated perfused rat hearts. Rat hearts were perfused at either constant pressure (80 mmHg) or constant flow. After aerobic perfusion (37 degrees C) for 10 min, hearts were treated with N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), which is an inhibitor of NO synthase, or vehicle. Ten minutes later, the hearts were preconditioned (4 episodes of 5 min of global ischemia and 5 min of reperfusion) or perfused normally before a 30-min global ischemic period. All hearts were reperfused for 30 min. Coronary flow or perfusion pressure plus heart rate and contractile function were measured continuously. Hearts perfused at constant pressure and treated with 30 microM L-NAME, a concentration that effectively inhibits endogenous NO synthesis, exhibited decreased coronary flow after 10 min, and flow remained decreased throughout the experiment. Ischemic preconditioning before 30 min of global ischemia resulted in a doubling of contractile function and a reduction of lactate dehydrogenase release at the end of the 30-min reperfusion period compared with nonpreconditioned hearts. The protective effect of preconditioning was not different in L-NAME-treated hearts. In addition, inhibition of NO synthase had no effect on the severity of ischemia in nonpreconditioned hearts. Similar results were obtained in preconditioned hearts that were perfused at constant flow, indicating that the flow reductions caused by L-NAME did not influence the results.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530917 TI - Impaired cyclic nucleotide-dependent vasorelaxation in human umbilical artery smooth muscle. AB - Activation of either the adenylate cyclase pathway with forskolin or the guanylate cyclase pathway with sodium nitroprusside fails to induce active relaxation of serotonin-precontracted human umbilical artery smooth muscle (HUASM) but causes active relaxation of serotonin-precontracted bovine carotid artery smooth muscle (BCASM). This difference in response appears to be unique to HUASM in that all other vascular muscles exhibit relaxation in response to these substances. Forskolin and sodium nitroprusside stimulation leads to respective increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations in HUASM and BCASM. cAMP- and cGMP-dependent protein kinases are both present in HUASM and can be activated in homogenates of HUASM by the addition of exogenous cAMP and cGMP, respectively. When either forskolin or nitroprusside acts in BCASM, two low molecular-weight proteins display an increase in the extent of phosphorylation. Neither protein shows such an increase when HUASM is treated with either agent. Thus the inability of HUASM to display active relaxation appears to be secondary to impaired activation of cyclic nucleotide-dependent protein kinases. The refractoriness to active relaxation may contribute to the vasospasm that occurs in the umbilical vasculature with parturition. PMID- 7530920 TI - Contribution of nitric oxide to coronary vasodilation during hypercapnic acidosis. AB - The present study was performed to evaluate the role of nitric oxide (NO) in coronary vasodilation during hypercapnic acidosis (HC). The left anterior descending coronary arteries of 17 anesthetized, open-chest dogs were perfused with normal arterial blood or with arterial blood equilibrated in an extracorporeal circuit with 90% O2-10% CO2 [arterial carbon dioxide tension (PaCO2) 72 +/- 3 mmHg, arterial pH 7.16 +/- 0.02]. Coronary perfusion pressure (CPP) was initially set at 100 mmHg. Coronary blood flow (CBF) was measured with a Doppler transducer. Studies were conducted under constant-pressure (variable CBF; n = 13) and constant-flow (variable CPP) conditions (n = 4). Steady-state changes in CBF (or CPP) during HC and during intracoronary infusions of acetylcholine (ACh, 20 micrograms/min), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP, 80 micrograms/min), an endothelium-independent vasodilator, were compared before and after intracoronary infusion of a NO synthase inhibitor, either NG-nitro-L-arginine methyl ester (L-NAME, 4.5 mg) or NG-monomethyl-L-arginine (L-NMMA, 30 mg). Under constant pressure, L-NAME blunted increases in CBF by HC (274 +/- 32 vs. 113 +/- 24%) and ACh (400 +/- 43 vs. 68 +/ 17%), whereas increases in CBF by SNP were not significantly affected (207 +/- 34 vs. 186 +/- 18%). Results with L-NMMA were similar. Under constant flow, L NAME attenuated decreases in CPP by HC and ACh, whereas it had no significant effect on decreases in CPP by SNP. In conclusion, HC elicits release of NO from coronary vascular endothelium via a direct effect rather than secondary to an increased flow rate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530921 TI - Platelet-activating factor stimulates protein tyrosine kinase in hamster cheek pouch microcirculation. AB - We studied the interactions between platelet-activating factor (PAF) and protein tyrosine kinase (PTK) in the modulation of microvascular responses in the hamster cheek pouch using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter and in integrated optical intensity (IOI; an index of vascular permeability) were measured. Fluorescein isothiocyanate-labeled dextran 150 (FITC-Dx 150) served as a tracer for macromolecular transport. Genistein and tyrphostin 25, two PTK inhibitors, were applied topically in separate experiments. Pretreatment with 10(-4), 10(-6), and 10(-8) M genistein and with tyrphostin 25 at 10(-5) and 10(-7) M attenuated the maximal increment in mean IOI (+/- SE) induced by PAF at 10(-7) M (19.9 +/- 5.3, 21.5 +/- 4.5, 58.5 +/ 11.4, 28.7 +/- 7.6, and 35.0 +/- 10.9 vs. 70.7 +/- 8.9 units, respectively). Pretreatment with PTK inhibitors resulted in vasodilation but did not inhibit PAF induced vasoconstriction. Our results suggest that PTK represents a biochemical pathway involved in the PAF modulation of microvascular permeability but not PAF modulation of arteriolar tone. PMID- 7530922 TI - Does protein kinase C play a role in ischemic preconditioning in rat hearts? AB - Protein kinase C (PKC) has been implicated in the cardioprotective effects of ischemic preconditioning in rabbits, but whether it plays a role in rats is unknown. We tested this preconditioning PKC theory by assessing whether the inhibition of PKC with calphostin C, a potent and specific inhibitor of PKC, can block the preconditioning effects in this model. Four groups of rats were studied: 1) control + vehicle, 2) control + calphostin C, 3) preconditioning + vehicle, and 4) preconditioning + calphostin C. All rats underwent 90 min of coronary occlusion followed by 4 h of reperfusion; in addition, preconditioned rats underwent three 3-min episodes of ischemia and 5 min of reperfusion before the 90 min of ischemia. Two injections of vehicle or calphostin C (0.1 mg/kg) were administered in intravenous boluses 29 min and 3 min before the 90-min coronary occlusion, i.e., one dose was given 5 min before preconditioning, and another dose was given between preconditioning and the sustained 90 min of ischemia in preconditioned rats. After 4 h of reperfusion, the area at risk (AR) was delineated by dye injection and area of necrosis was assessed by triphenyltetrazolium chloride staining. The electrocardiogram was recorded for the incidence of ventricular tachycardia (VT) and ventricular fibrillation. AR was similar in all four groups. In the nonpreconditioned control rats receiving vehicle, myocardial infarct size expressed as a percentage of the AR averaged 45.7 +/- 1.7%. Pretreatment with calphostin C had no effect on infarct size (48.9 +/- 3.4%) in nonpreconditioned control rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530923 TI - Endothelial and nonendothelial cyclooxygenase mediate rabbit pial arteriole dilation by bradykinin. AB - Aspirin (acetylsalicylic acid, ASA) was administered to rabbits in an attempt to inhibit selectively endothelial cyclooxygenase activity and therefore to determine its role in bradykinin-induced radical-mediated dilation of cerebral arterioles. With the use of the cranial window technique in anesthetized rabbits, pial arteriolar diameters were recorded in response to topically applied bradykinin, acetylcholine, and ventilation with 10% O2-9% CO2 gas mixture. Prostaglandins were measured in isolated cerebral microvessels and cerebrospinal fluid (CSF) using radioimmunoassay. Microvessel prostaglandin production was reduced significantly by 90 mg/kg i.v. ASA, whereas acetylcholine-stimulated increases of CSF prostaglandins were not similarly affected. This treatment reduced bradykinin-induced dilation of pial arterioles by 47%. After concurrent 90 mg/kg i.v. ASA plus 300 microM ASA topically applied to the brain, stimulated increases of CSF prostaglandins were reduced by 79%, while bradykinin-induced dilation was reduced by 78%. ASA did not reduce the dilator activity of either acetylcholine or ventilation with 10% O2-9% CO2. Acetylcholine- but not bradykinin-induced dilation was reduced by NG-nitro-L-arginine methyl ester. These results indicate intravenous ASA produced a relatively selective inhibition of cerebral microvascular cyclooxygenase and partial inhibition of bradykinin induced dilation. Further inhibition of dilation occurred following ASA administered both systemically and topically to the brain. This indicates two sources of cyclooxygenase, endothelial and nonendothelial, mediate the bradykinin induced dilation of rabbit pial arterioles. Furthermore, systemic doses of ASA do not eliminate brain prostaglandin formation. PMID- 7530925 TI - Protein synthesis and rapid recovery of endothelium-dependent dilation after endothelial injury of pial arterioles. AB - A laser-dye technique is known to injure selectively microvascular endothelium in situ. With the use of the arterioles on the surface of the mouse brain (pial arterioles), the endothelial injury is manifest by loss of several endothelium dependent responses, including the dilation produced by topical application of acetylcholine (ACh), bradykinin (BK), and calcium ionophore A-23187. The responses normally recover by 60 min following injury. The present study shows that inhibitors of protein synthesis prevent the recovery. Either actinomycin D (Act-D; 1 mg/kg) or cycloheximide (CX; 10 mg/kg) was injected intraperitoneally 35 min before the light-dye injury. The CX was used in separate studies with ACh, BK, and ionophore. In each study, recovery of the endothelium-dependent dilation failed to occur 60 min after injury, while recovery occurred in contemporary vehicle-treated controls. Act-D was used in separate studies of ACh and BK. Again, recovery of endothelium-dependent dilation was prevented. Neither CX nor Act-D inhibited the response to ACh in uninjured vessels. Thus response to ACh was intact in such arterioles 90 min after the injection of CX or Act-D. CX was also given to uninjured mice and was found to have no inhibitory action on the response to ionophore 90 min later. CX and Act-D inhibit protein synthesis by very different mechanisms, inhibition of translation and of transcription, respectively. Because both prevented recovery of the endothelium-dependent responses, we conclude that one or more rapidly synthesized proteins are required for the recovery. PMID- 7530924 TI - Effect of aging on nitric oxide-mediated penile erection in rats. AB - Aging is an important risk factor for impotence in men. Because nitric oxide (NO) appears to be the mediator of corpora cavernosal smooth muscle relaxation, we have examined in 5-, 20-, and 30-mo-old rats, designated "adult," "old," and "senescent," respectively, whether aging causes a decrease of erectile response that may correlate with lower NO synthase (NOS) in the penis. Electric field stimulation (EFS) of the cavernosal nerve showed that the maximum intracavernosal pressure (MIP) declined in the old and senescent rats to 80 and 51% of the adult value, respectively. A low systemic dose of the NOS inhibitor, N omega-nitro-L arginine methyl ester (L-NAME; 2 mg/kg), reduced the MIP by only 38% in the adult rats but decreased it in the old and senescent rats by 72 and 80%, respectively. In the absence of EFS, intracavernosal papaverine (phosphodiesterase inhibitor), or nitroglycerin (NO donor), caused a lower erectile response in the old and senescent rats compared with the adult animals (MIP: 41 and 14%, respectively; duration of the erection 46 and 21%, respectively). Tissue sections from old and senescent penises showed increasing degrees of sclerotic degeneration. In comparison with the adult rats, the penile soluble NOS activity per gram of tissue that is sensitive to L-NAME decreased significantly by 63% in the senescent rats but was elevated in the old rats. These results indicate that aging causes an erectile failure due to factors initially independent from an impairment of penile NO synthesis but which are compounded in the very old rats by the decrease of penile NOS activity. PMID- 7530926 TI - Influence of nitric oxide on the hemodynamic response to hemorrhage in conscious rabbits. AB - We investigated the role of nitric oxide, an endothelium-derived relaxing factor, in the hemodynamic response to acute hemorrhage in conscious rabbits. Chronically instrumented rabbits were treated with the nitric oxide synthase inhibitor N nitro-L-arginine methyl ester (L-NAME) or vehicle and hemorrhaged until mean arterial pressure fell below 40 mmHg. Control animals were treated with L-NAME or vehicle but not subjected to hemorrhage. L-NAME increased mean arterial pressure and decreased heart rate in control animals. Hindquarters and mesenteric blood flow velocity and conductance were reduced by L-NAME. Nitric oxide synthase inhibition also produced significant changes in the hemodynamic response to hypotensive hemorrhage. Mean arterial pressure was higher and regional vascular conductances were lower throughout hemorrhage and during recovery. L-NAME treatment significantly (but in some cases, subtly) altered the characteristic pattern of changes in vascular conductance associated with acute hypotensive hemorrhage and recovery. Similar experiments with other arginine analogues or phenylephrine infusion showed that L-NAME's effects during hemorrhage were due to nitric oxide synthase inhibition. We conclude that nitric oxide plays a role in the hemodynamic response to acute hemorrhage in the rabbit and is essential for the full expression of the vasodilation associated with hypotensive hemorrhage. PMID- 7530927 TI - Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. AB - We sought to determine whether expression of the inducible, calcium-independent isoform of nitric oxide synthase (iNOS) contributes to the tissue damage produced by focal cerebral ischemia. The middle cerebral artery was occluded in halothane anesthetized spontaneously hypertensive rats. Twenty-four hours later rats received intraperitoneal injections of the iNOS inhibitor aminoguanidine (100 mg/kg twice per day; n = 10) or of aminoguanidine + L-arginine (300 mg/kg four times per day; n = 7), aminoguanidine + D-arginine (n = 7), arginine alone (n = 6), or vehicle (n = 9). Drugs were administered for 3 consecutive days. Infarct volume was determined by image analysis in thionin-stained brain sections 4 days after induction of ischemia. Administration of aminoguanidine reduced infarct volume by 33 +/- 4% (P < 0.05 from vehicle; analysis of variance and Tukey's test), a reduction that was antagonized by coadministration of L- but not D arginine. Administration of L-arginine alone did not affect infarct size (P > 0.05 vs. vehicle). In separate rats (n = 10), aminoguanidine attenuated calcium independent NOS activity in the infarct (P < 0.05 vs. vehicle) without affecting calcium-dependent activity (P > 0.05). Aminoguanidine did not affect resting cerebral blood flow or the cerebrovascular vasodilation elicited by hypercapnia, as determined by laser-Doppler flowmetry (n = 4). We conclude that aminoguanidine selectively inhibits iNOS activity in the area of infarction and reduces the volume of the infarct produced by middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530928 TI - Cytokines and the acute phase response to influenza virus in mice. AB - This study characterized selected aspects of the acute phase response after intranasal inoculation of mice with two doses of mouse-adapted influenza virus differing in lethality. Mice given 140 plaque-forming units (PFU) of virus (58% survival) gradually decreased food and water intake to nearly zero over 6 days; survivors then slowly increased intakes. Declines in these behaviors were parallel to decreases in body temperature and general locomotor activity and were associated with elevated activities of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferons in lung lavage fluid. Circulating levels of these cytokines were not increased. After 55,000 PFU of virus (100% mortality), food and water intake fell to near zero within 48 h, temperature and locomotor activity decreased significantly, and activities of IL-1 and IL-6 were elevated in lung lavage fluid. These data show that cytokine activities in the lungs are elevated in a time frame that supports the hypothesis that cytokines could mediate behavioral and physiological changes in mice during acute influenza infections. PMID- 7530929 TI - Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue. AB - Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellularly in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors. PMID- 7530931 TI - Digital cell image analysis of Feulgen-stained nuclei from human papillary, medullary, colloid, lobular and comedocarcinomas of the breast. AB - The morphonuclear characteristics (nuclear size and chromatin pattern), the proliferation index and the ploidy level were characterized in a series of 46 breast tumors including medullary (5 cases), papillary (6 cases), lobular (27 cases), colloid (4 cases) and comedo- (4 cases) carcinomas. The quantitative assessments were carried out by means of digital cell image analyses of Feulgen stained nuclei from imprint smears. The results show that monovariate analyses (one-way variance analyses) were much less potent than multivariate analyses (principal components analyses followed by the canonical transformation of the data and discriminant analyses) in assessing the morphonuclear characteristics of these breast tumors. The multivariate analyses indicated that there might be a level of malignancy which increases according to the sequence papillary and medullary and colloid carcinomas-->comedocarcinomas-->lobular carcinomas. This assertion is corroborated by the ploidy-level-related results which revealed a higher proportion of highly aneuploid cases in the group of lobular carcinomas than in the group which included medullary, papillary and colloid carcinomas. However, since highly aneuploid cases were also encountered in this latter low malignancy level group, we expressed the hypothesis firstly that aneuploidy reflects two distinct biological properties, i.e. the aggressiveness of a tumor and its age, and secondly that a highly aneuploid but low malignancy tumor should correspond to old degenerating tumors. PMID- 7530930 TI - Comparative immunohistochemical study of ras-p21 oncoprotein in adenomatous hyperplasia and adenocarcinoma of the prostate gland. AB - The present study assessed the immunohistochemical expression of ras p21 oncoprotein in 41 cases of benign and cancerous lesions of the prostate gland and correlated p21 expression with survival of patients operated for prostate cancer. Our results show that p21 is detected in both categories of patients, the expression being weak in adenomatous hyperplasia and more intense in cancer. We were also able to show an inverse relation between ras p21 positivity and the degree of differentiatin. Follow-up study revealed a statistically significant (p < 0.05) correlation between 5 year survival and p21 expression. PMID- 7530932 TI - Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5' triphosphate beta enantiomers of cytidine analogs. AB - (-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5 fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5 fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3' thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate. PMID- 7530933 TI - Anti-human immunodeficiency virus type 1 activities of U-90152 and U-75875 in human brain cell cultures. AB - Antiviral activities of the reverse transcriptase inhibitors U-90152 and 3'-azido 2',3'-dideoxythymidine and the protease inhibitor U-75875 were compared in two culture models of human immunodeficiency virus type 1 brain infection. In a model involving acutely infected microglial cells, U-90152 was the most active, whereas in a model using chronically infected promonocytes, U-75875 was the most active. PMID- 7530934 TI - Inhibition of gp120-CD4 interaction and human immunodeficiency virus type 1 infection in vitro by pyridoxal 5'-phosphate. AB - Pyridoxal 5'-phosphate and related compounds were tested for their ability to inhibit gp120-CD4 interaction and human immunodeficiency virus infection in vitro. The results show that pyridoxal 5'-phosphate is a unique CD4 antagonist whose antiviral potency derives from the presence of both lysine-reactive and anionic substituents. PMID- 7530935 TI - Antibodies to three chondroitin sulfate-containing proteoglycans in squid skin recognize hexa- or longer chondroitin oligosaccharides as major antigenic determinants. AB - The reactivities of antibodies to three squid skin proteoglycans with (a) chondroitin-derived oligosaccharides and chondroitin sulfate-derived disaccharides, (b) the proteoglycans and their constituents, and (c) chondroitin, chondroitin sulfate, and hyaluronic acid were studied with enzyme-linked immunosorbant assay inhibition tests. Immunization of rabbits with two chondroitin proteoglycans (ChPG I and ChPG II) and an oversulfated chondroitin sulfate proteoglycan (CSSPG) gave rise to highly reactive antisera which were mainly reactive with the glycosaminoglycans, the oligosaccharides, and the core proteins obtained after digestion of proteoglycans with chondroitinase AC. Inhibition of binding of antibodies to ChPG I, ChPG II, and CSSPG with chondroitin-derived oligosaccharides revealed that the minimal antigenically active structure was the hexasaccharide of chondroitin and that the respective octasaccharide was more active. Sulfated delta-disaccharides were not reactive with antibodies to ChPG I and II, whereas some reactivities (30% maximum inhibition) were obtained with antibodies to CSSPG. Chondroitin chains (80 kDa) of ChPG I and II were responsible for most of the reactivity with proteoglycans (78-95% maximum inhibition). Chemically desulfated chondroitin sulfate (12 kDa) showed considerable cross-reactivity with all antisera tested (62-68% maximum inhibition), whereas the nonsulfated molecule of hyaluronic acid and a hyaluronic acid fraction of 16 kDa were not reactive. The reactivities of antibodies with the proteoglycans' oligosaccharides and core proteins obtained by chondroitinase AC digestion were mainly due to the presence of nonsulfated chondroitin sulfate structures. This study clearly shows that the major antigenic determinants recognized by antibodies to squid skin proteoglycans, each containing chondroitin sulfates with different sulfation patterns, involve hexa- or larger chondroitin oligosaccharides. PMID- 7530936 TI - Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences. AB - Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation. PMID- 7530937 TI - Aminoguanidine is an isoform-selective, mechanism-based inactivator of nitric oxide synthase. AB - Aminoguanidine produces a time-dependent inactivation of the citrulline forming activity of all three nitric oxide synthase isoforms that is blocked by arginine. Aminoguanidine inactivates both the NADPH oxidase and citrulline forming activities of GH3 pituitary constitutive nitric oxide synthase (cNOS) but does not alter its cytochrome c reductase activity. GH3 pituitary cells contain an NOS isoform identical physically, kinetically, and immunologically to cerebellar neuronal NOS (Wolff and Datto, Biochemical J. (1992) 285, 201-206). The inactivation of GH3 cNOS NADPH oxidase activity, as measured without added tetrahydrobiopterin cofactor, is saturable, is inhibited by arginine, and follows pseudo-first-order kinetics with an inactivation rate constant of 0.25 min-1 and a Ki value of 0.83 mM aminoguanidine. The inactivation of the citrulline forming activity of GH3 cNOS by aminoguanidine was not saturable by aminoguanidine. Aminoguanidine, at concentrations in the millimolar range, inhibited the citrulline forming activity of endothelial cNOS by an apparently nonsaturable mechanism. Aminoguanidine inactivates the citrulline forming activity of murine macrophage iNOS. The inactivation is saturable and follows pseudo-first-order kinetics with an inactivation rate constant of 0.46 min-1 and a Ki value of 16 microM. The inactivation of the constitutive isoforms of nitric oxide synthase by aminoguanidine required the concurrent presence of Ca2+, calmodulin, NADPH, tetrahydrobiopterin, and oxygen in preincubations and was not reversed either by dilution or dialysis. These observations support the assertion that aminoguanidine is a mechanism-based inactivator of the nitric oxide synthase isoforms and exhibits marked specificity for the inactivation of the inducible isoform. PMID- 7530938 TI - Accumulation of fibronectin in articular cartilage explants cultured with TGF beta 1 and fucoidan. AB - Fibronectin is a glycoprotein involved in cell matrix interactions. In osteoarthritis, fibronectin levels in the lesion cartilage are elevated up to 20 fold above control levels. In these experiments, explants of disease-free cartilage cultured in the presence of a combination of TGF beta 1 and the sulfated fucopolysaccharide, fucoidan, accumulated fibronectin at levels comparable to those found in osteoarthritic lesions. TGF beta 1 increased fibronectin synthesis, most of which was released to the medium. The addition of fucoidan favored retention of the newly synthesized fibronectin within the matrix. The fibronectin which accumulated as a result of these treatments was similar to the fibronectin in normal and osteoarthritic cartilage with respect to the ED-B+ alternative splice form. No change in the proteoglycan content of the cartilage explants with elevated fibronectin levels was detected. PMID- 7530939 TI - BPH treatment: urodynamic preoperative assessment and evaluation. AB - Originally prostatectomy was performed for complications to infravesical obstruction. Today 70% are performed because of lower urinary tract symptoms which might be due to obstructive BPH, but also might be a consequence of detrusor weakness or other age-related functional changes in the lower urinary tract. Of patients operated for uncomplicated BPH, approximately 30% are urodynamically unobstructed. Traditional urological measures like residual urine volume, endoscopic impression of prostate and bladder, as well as prostate size and even urinary flow rate measurements, are not able to separate this group. The symptomatic outcome after surgery is statistically significantly worse in the urodynamically unobstructed group, while urodynamic improvement is only marginal. As pressure/flow studies are the only method to diagnose infravesical obstruction, this should therefore be considered before more invasive procedures intended to treat suspected infravesical obstruction are performed. PMID- 7530940 TI - Evaluation of cervico-urethral obstruction with ambulatory pressure/flow studies: an alternative to conventional pressure/flow studies? AB - Most of the urodynamic studies are conducted in the laboratory during a brief recording time and under nonphysiological conditions and, thus, may fail to unfold the nature of existing pathological conditions of the lower urinary tract; false positives and false negatives are possible. To overcome some of the difficulties associated with conventional P/F studies we have developed, with the cooperation of Medical Measurement System company, Entschede, The Netherlands, a portable system (UDS 2000) for ambulatory monitoring of intravesical pressure, abdominal pressure and EMG connectable with a weight transducer flowmeter that permits performing Holter P/F measurements. We compared the results obtained with conventional P/F studies and with the Holter P/F studies in 58 BPH patients. During the filling phase we observed a slightly increased number of stable detrusors with Holter P/F recording (46 vs 42); conversely, the number of patients suffering from urge incontinence was the same (7 pts). During the voiding phase, out of 45 patients considered obstructed at conventional P/F study, only 42 were really urodynamically obstructed (93.3%), while 3 other patients had borderline obstruction. Four patients with borderline obstruction at conventional P/F study were considered nonobstructed after Holter P/F. PMID- 7530942 TI - Prostate cancer detection in BPH patients. AB - The first problem to be solved in the evaluation of BPH patients is surely the differential diagnosis with prostate carcinoma. We evaluated the impact of a combined approach for prostate cancer detection using DRE, PSA, TRUS and ultrasound-guided biopsy, determining the sensitivity and specificity of different tools, in order to obtain a diagnostic algorithm to be used for pretreatment differential diagnosis between prostate cancer and BPH. PMID- 7530941 TI - Imaging in BPH patients. AB - Benign prostatic hyperplasia is one of the most common pathological processes to afflict men. Strikingly, there is a large variety of methods of evaluation and therapeutic strategies for BPH in various countries. Before adequate treatment is possible, the process of benign adenomatous hyperplasia has to be distinguished from malignant or inflammatory prostatic disease. Furthermore, secondary alterations of the bladder and upper urinary tract have to be evaluated. A wide range of imaging techniques of the lower and upper urinary tract have evolved over the years. The specific benefits and restrictions of each of them are discussed. Finally, the recent developments and possibilities of computer-aided tissue characterisation and volume determination are outlined. The development of imaging techniques for BPH has just begun... PMID- 7530943 TI - Role of alpha blockers in the treatment of BPH: a critical review of clinical results. AB - Benign prostatic hyperplasia (BPH) is a benign disease characterized in a high percentage by cervico-urethral disorders. With regard to the obstructive aspect, this is the result of two components: mechanic and dynamic. The mechanic component is a direct consequence of the obstruction caused by enlargement of the prostate gland and the urethral deformation. The dynamic, on the other hand, is derived from the increase in tone of the smooth muscle following stimulation by adrenoreceptor alpha, localized at the level of the bladder neck, prostatic urethra and prostatic capsule. The use of alpha blockers in the treatment of BPH has the goal of intervening in the effects of the obstruction acting on the dynamic components. The use of alpha-blockers drugs, as explained in published literature, shows more or less satisfactorily, a significant symptomatic improvement and consequently an improvement in the quality of life. But the use of alpha blocker presently available may be considered as an unspecific or primitive, strictly symptomatic treatment for irritative symptoms for those who desire to postpone surgery, or in the cases in which surgery is not recommended or refused by the patient, and finally, to prevent acute urinary retention while the patient is waiting for surgery. PMID- 7530944 TI - Pharmacological treatment of benign prostatic hyperplasia with finasteride: a clinical review. AB - Finasteride acts by blocking the conversion of testosterone to 5 alpha dihydrotestosterone, the active androgen metabolite in the human prostate. In large, double-blind, placebo-controlled phase III studies recruiting over 1600 patients, it was shown that the administration of Finasteride, either 5 or 1 mg a day, reduced the size of the prostate by a mean of 22%, following 6 months of therapy. Despite this reduction in prostate size, urinary flow rate only improved by a mean of 1.7 ml per second and symptom score improved only marginally, but statistically significantly different from placebo. Long-term results in small series of patients have indicated a further improvement beyond 1 year. After 3 years flow was improved by 60%. The future role for Finasteride therapy is emerging, but it appears as if patients with mild to moderate symptoms would be a group who could benefit the most. Whether or not Finasteride can stop the long term natural course of benign prostatic hyperplasia has still to be demonstrated. PMID- 7530945 TI - Microwave treatment of BPH: still an option? AB - Surgery, the gold standard in the treatment of benign prostatic hyperplasia (BPH), is presently performed in the majority of patients who receive treatment. An increasing number of alternative options recently became available for treatment of BPH and especially minimal invasive treatment modalities, such as TUMT (transurethral microwave thermotherapy), have become increasingly popular. Results of TUMT have been very encouraging: both subjective and objective response have been reported on. A number of issues regarding the use of this treatment modality remains to be answered. In general, TUMT seems to be a valuable acquisition as a minimal invasive therapeutic alternative for treatment of BPH. PMID- 7530947 TI - Laser prostatectomy: which way forward? AB - A number of endoscopic laser strategies have been employed in the treatment of symptomatic benign prostatic hyperplasia at our hospital. The post-operative results in 102 patients are presented. Sidefiring fibres were coupled with the Nd:YAG laser to produce a deep coagulative necrosis within the prostatic adenoma. This approach was associated with a short operative time and a delay of up to three months before treatment effect. Contact laser techniques (with both the 1064 nm and 805 nm lasers) were used to vaporize prostatic tissue in an attempt to produce a defect similar to that following TURP. This provided an immediate treatment effect with early removal of catheters. The operative time, however, was long and this limited the procedure to glands of less than 40 ccs. The hybrid approach (combining laser prostatotomies with coagulation) provided an immediate relief of obstruction with an additional delayed effect as the coagulated cavity matured. All approaches were associated with minimal haemorrhage and limited morbidity with encouraging improvements in both symptom scores and uroflowmetry. The various advantages and disadvantages of each endoscopic laser strategy are discussed. PMID- 7530946 TI - Transurethral needle ablation of the prostate (TUNA): pathological, radiological and clinical study of a new office procedure for treatment of benign prostatic hyperplasia using low-level radiofrequency energy. AB - Many attempts have been made to develop a method for treating BPH that is minimally invasive, efficacious and low-cost. TUNA is a new, fast anesthesia free, outpatient device for selectively ablating prostatic tissue by delivering low-level radiofrequency power through a special 22 French urethral catheter positioned in the prostate via TURS and/or direct fiberoptic vision, and outfitted with adjustable needles placed in a selected prostatic area. A pilot study was performed in 70 patients to evaluate TUNA safety and feasibility via histopathological measurement and clinical outcome. Twenty-five patients were treated with TUNA prior to scheduled retropubic prostatectomy. The surgical prostatic specimens recovered from 1 day to 1 month after TUNA were step sectioned and examined histologically. The TUNA procedure averaged 30 minutes; 4 15 Watts were applied for 3-5 minutes per lesion. The central lesion temperatures were above 100 degrees C. The urethral temperature averaged 41 degrees C and rectal temperature remained unchanged. Macroscopic and MRI examination of the specimens demonstrated localized lesions averaging 12 x 7 mm and 17 x 10 mm for 3 and 5 minutes of treatment, respectively. Microscopic examination of the specimens showed sharply delineated lesions of extensive coagulative necrosis measuring up to 35 x 15 mm. The clinical efficacy of the procedure was evaluated in 25 symptomatic BPH patients treated by TUNA. Tolerance using topical anesthetic and intravenous valium was excellent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530948 TI - TUIP for infravesically obstructed BPH patients: a review of 300 cases. AB - Our experience is described in the treatment of BPH by prostatic incision. Three hundred patients were treated from 1989 to 1993; 285 had obstructive BPH (48 in complete urine retention) and 15 had prostatic carcinoma. One hundred eighty-nine patients were controlled and in 93% the results were satisfactory for disappearance of symptoms. Insignificant complications were recorded and only 9% had retrograde ejaculation. Ninety-six urodynamic controls (flowmetry) were performed which showed a considerable improvement in maximum urinary flow rate. The operation was successful even in patients with large adenomas, with a considerable reduction of the Boyarsky score (mean from 17.8 to 3.8) six months postoperatively. In conclusion TUIP is a good procedure in BPH. It is an easy technique without important complications. PMID- 7530949 TI - [Transurethral resection versus transurethral incision in benign prostate hypertrophy --critical assessment]. AB - We evaluated 665 patients who had undergone TURP and 100 patients who had undergone TUIP (50 bilateral, 50 unilateral). Transurethral incision, when indicated (less than 35 gm, no middle lobe), had the following advantages: easy to perform, short operating time (39 minutes) and hospitalization (3-4 days). There was less bleeding; 2% versus 6% for TURP. The complication rate was lower: no bladder neck sclerosis for TUIP versus 2.9% for TURP; urethral stenosis: 6% for the bilateral and 0% for the unilateral incision versus 8.7% for TURP, and the incidence of retrograde ejaculation was 24% for the bilateral and 19% for the unilateral incision versus 82% for TURP. We also evaluated the disadvantages of transurethral incision: no tissue for study 4% incidental prostate cancer for TURP, limited indications, anatomic variability, less disobstructive, reoperation rate was 10% versus 11.7% for TURP, design of comparative studies deficient and no long-term follow up. The results of bilateral and unilateral incision were compared, however no statistically significant differences were observed. The bilateral incision procedure was slightly more disobstructive: 92% clinical improvement for the bilateral and 84% for the unilateral, increased flow was 6.5% for the bilateral versus 5.6% unilateral, and the reoperation rates were 2% for the bilateral and 8% for the unilateral incision procedure. However, there were less complications for the unilateral incision procedure: no stenosis for the unilateral and 6% for the bilateral, and retrograde ejaculation was 81% for the unilateral and 76% for the bilateral incision procedure. For all the foregoing reasons, we prefer to do unilateral incision of the prostate. PMID- 7530950 TI - The use of wallstents in patients with benign prostatic hyperplasia. AB - This study was conducted to assess the clinical reliability of the prostatic Urolume Wallstent for symptomatic BPH patients. One hundred patients were treated under intravenous sedation or local anesthesia. Pre- and postoperative patient evaluation included scoring of subjective symptoms, physical examination, laboratory analysis, transrectal ultrasonography, uroflowmetry with maximum flow nomograms, intravenous pyelogram and cystourethroscopy. At the long-term follow up the vast majority of patients showed non-obstructed voiding parameters. The most frequent complications included stent malpositioning and dislocation, and persistent irritative symptomatology. The prostatic UroLume Wallstent is a safe and effective minimally invasive procedure for carefully selected BPH patients. PMID- 7530951 TI - Thermosensitive stent (Memotherm) for the treatment of benign prostatic hyperplasia. AB - Recently, more and more alternative therapeutic methods have been used for the treatment of benign prostatic hyperplasia (BPH). We will report on therapeutic results with a new thermosensitive stent system (Memotherm). This wire mesh stent has been designed especially for urological purposes. It is made of Nitinol, a thermoreactive material, and gains its maximum expansion force al body temperature. Due to the properties of the material the stent is flexible and can adapt to the anatomical conditions of the prostatic part of the urethra. Because of individual variations in the length of the prostatic part of the urethra, the system is available in lengths from 2 to 8 cm. The knitted structure for the first time allows atraumatic removal. Between April, 1992, and September, 1993, we treated a total of 54 BPH patients with the stent system. Mean patient age was 76.1 +/- 7.6 years (61-98). Mean prostatic volume was 51.9 +/- 25 ml (20-150), and the length of the applicated stents was 32.3 +/- 9.5 mm (20-70). Patient selection for stent treatment was carried out with regard to the preoperative risk status of this patient group. Fourteen (26%) of the patients treated were able to micturate before operation; in 40 (74%) urinary drainage was accomplished by means of an indwelling catheter. Following stent application, 53 out of 54 patients were able to micturate. With the first group (preoperative voiding ability), maximum flow had increased from 4.5 ml/s to a mean of 15.8 ml/s, while residual urine volume had decreased from 194.4 ml to 11.8 ml and the AUA 6 Symptoms Score had improved from 24 points to 3.5 points 6 months after stent application. All differences were statistically significant (P < 0.02). With the second group (no preoperative voiding ability), the AUA 6 Symptoms Score had improved from 25 points to 3.9 points (P < 0.02) 6 months after the operation, at which time the mean maximum flow rate was 14.8 ml/s and residual urine volume 14.8 ml. There was no statistically significant difference between the patients who were able and those who were unable to micturate before operation. One case of epididymitis was the only major complication observed after stent placement. Frequent urgency symptoms (30 out of 54 patients; 55.5%) subsided after a mean period of 1 week. The Memotherm stent offers an interesting therapeutic alternative for BPH risk patients. PMID- 7530952 TI - Hepatitis C virus antibody, viraemia and genotypes in individuals infected with HIV-1 in Cameroon. PMID- 7530954 TI - Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism. PMID- 7530953 TI - Effect of lipopolysaccharide on nitric oxide synthase activity in rat proximal tubules. AB - Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin dependent NOS activity that is increased in vivo by lipopolysaccharide. PMID- 7530955 TI - Alpha 2A-adrenoceptors mediate activation of non-selective cation channels via Gi proteins in human erythroleukaemia (HEL) cells. No evidence for a functional role of imidazoline receptors in modulating calcium. AB - Human erythroleukaemia (HEL) cells were investigated to characterize their alpha 2-adrenoceptor and imidazoline receptor sites. Membranes from HEL cells bound [3H]2-(2-methoxy-1, 4-benzodioxan-2yl)-2-imidazoline ([3H]RX821002) in a saturable and specific manner with a KD of 0.64 +/- 0.07 nM and a Bmax of 126 +/- 4 fmol/mg protein. [3H]RX821002 was displaced from HEL membranes by adrenergic drugs with the order of potency being yohimbine approximately oxymetazoline >> prazosin = 2-[2-[4-(o-methoxyphenyl)piperazin-1-yl]ethyl]-4,4-dimethyl- 1,3(2H,4H)-isochinolindione HCl (ARC 239), consistent with this site being an alpha 2A-adrenoceptor. HEL membranes also bound [3H]idazoxan in the presence of adrenaline to block alpha 2-adrenoceptors. This binding was saturable and specific with a KD of 3.5 +/- 1.0 nM and a Bmax of 31 +/- 6 fmol/mg protein. Adrenergic drugs from both the phenylethylamine and imidazoline classes increased high-affinity GTPase activity, an index of activation of regulatory heterotrimeric guanine-nucleotide binding proteins (G-proteins), and produced increases in cytosolic free calcium concentration ([Ca2+]i). The effects of these agonists in both systems were abolished by pertussis toxin pretreatment, and oxymetazoline and clonidine were antagonists. The potency of adrenergic drugs to inhibit 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304)-induced increases in [Ca2+]i was yohimbine approximately oxymetazoline >> ARC 239, consistent with the binding data and an action at alpha 2A-adrenoceptors. No evidence was found for a role of imidazoline receptors in stimulating G-proteins or modulating [Ca2+]i. The adrenergic agonist-induced increases in [Ca2+]i were due to both release of Ca2+ from intracellular stores and entry of extracellular Ca2+. Ca2+ entry was blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4 methoxyphenylethyl)-1H- imidazole hydrochloride (SKF 96365), but not by nitrendipine. Adrenaline also stimulated Mn2+ entry in HEL cells. Taken together, these results suggest that HEL cells have alpha 2A-adrenoceptors that activate non-selective cation channels via pertussis toxin-sensitive G-proteins, i.e. Gi proteins. PMID- 7530956 TI - [Experiences with the salivary tube in tumor-induced salivary fistula and stenosis after laryngectomy]. PMID- 7530957 TI - Relationship between nuclear morphology and DNA content in large cell lymphomas. AB - The relationship between DNA content and nuclear morphology in large cell lymphomas (LCLs) was investigated on lymph node imprints. Mean maximum nuclear diameter (mean MND), nuclear shape and chromatin pattern were evaluated microscopically on May-Grunwald-Giemsa-stained slides. DNA content and nuclear area were measured on Feulgen-stained slides by image analysis. Twelve of the 24 cases were DNA diploid and 12 tetraploid. The DNA diploid cases were characterized by medium large (mean MND 11.2-13.7 microns), round nuclei with a fine chromatin pattern. The DNA tetraploid cases had significantly (P < .01) larger (mean MND 13.0-19.1 microns) nuclei and a higher frequency of coarse chromatin pattern, nuclear irregularities and multilobation. One of the researchers, unaware of the DNA index, could predict the ploidy level in 80% of cases on morphology. The linear coefficient of correlation between mean MND and mean nuclear area was 0.84. We also found that nuclear area increased as the cell moved through the cell cycle. DNA content, related to ploidy and position in the cell cycle, is an important explanation for the variable nuclear morphology in LCLs. PMID- 7530958 TI - Quantification of area percentage of immunohistochemical staining by true color image analysis with application of fixed thresholds. AB - Most image analysis systems (IAS) use black-and-white cameras. However, true color IASs are considered to be useful for quantification of immunohistologically stained structures. Using a true color IAS, we evaluated two methods of segmentation for quantification of area percentage of staining: one using fixed, preset thresholds and one using thresholds interactively set per image. Furthermore, the effect of shading correction was evaluated, and measurements in both color and black-and-white mode were compared. The results of segmentation with fixed thresholds did not differ significantly from those of control percentages, established by interactive morphometry using a grid, which served as reference. Interactive segmentation was significantly different from the reference (t test, P = .0001). The effect of shading correction was negligible. Measurements with and without this procedure correlated highly (r = .99, P < .001). Comparison of the results obtained in color and black-and-white mode showed a significant difference in the latter from the reference (t test, P = .005). We conclude that it is possible to quantify, in a reliable way, area percentage of positive staining using a true color IAS with application of a segmentation method with fixed thresholds. PMID- 7530959 TI - Vimentin expression, proliferating cell nuclear antigen and flow cytometric factors. Prognostic role in breast cancer. AB - A series of 71 patients undergoing surgery for primary breast carcinoma was prospectively studied in order to evaluate the relative weight for four biologic factors (intermediate filament vimentin expression, proliferating cell nuclear antigen [PCNA], flow cytometric DNA ploidy and S-phase fraction) and of several clinicopathologic and biologic features in predicting clinical outcome (disease free interval). In univariate statistical analysis, positivity of axillary nodes, high number of mitoses, high nuclear grade, high histologic grade, positivity of vimentin, high flow cytometric S-phase fraction (FCM-S) value, high PCNA and high silver-stained nuclear organizer regions scores were significantly related to risk of relapse. In multivariate analysis (Cox's logistic regression) only histologic grade (3) and high FCM-S values (> 10.7) were independently related to risk of relapse, with hazard ratios of 9.84 and 7.98, respectively. The results of our preliminary, prospective study suggest that FCM-S, in addition to morphologic criteria (histologic grade), may be an important biologic indicator in determining breast cancer patients' prognosis. PMID- 7530960 TI - Analytical ultracentrifugation and the genetic engineering of macromolecules. PMID- 7530961 TI - Lpr and MRL background gene involvement in the control of adjuvant enhanced arthritis in MRL-lpr mice. AB - The MRL-lpr mouse strain develops a mild spontaneous arthritis which can be enhanced by the intradermal injection of complete Freund's adjuvant (CFA). In this study we examined the requirement of the lpr gene and background MRL genes on CFA-enhanced murine arthritis. MRL+, MRL-lpr, AKR/J, and B6-lpr mice (experimental) and B6 mice (control) were injected intradermally with CFA containing M. tuberculosis H37 RA. The development of swelling and erythema was monitored for 1 month after the injection, when the histopathology of the joints was investigated. It was found that while 74% of both 7-month-old MRL + and 3 month-old MRL-lpr mice and 11% of AKR/J mice displayed clinically visible arthritis, B6, B6-lpr, and 3-month-old MRL+ did not develop the condition after CFA treatment. In accordance with the clinical observations, the histopathological changes were manifested only in older MRL+, AKR/J and 3-month old MRL-lpr mice. One month after the CFA injection, milder changes were observed in the MRL+ than in the MRL-lpr mice, with the MRL+ mice developing a disease of similar severity to uninjected MRL-lpr mice. The AKR/J mice demonstrated the least severe histopathological changes. In the long term (150 days) more severe destructive changes could be demonstrated in the cartilage and bone of the MRL+ mice although the average histological scores did not show statistically significant differences from those found in the MRL+ 30 days after injection. The serological evaluation of the adjuvant-injected mice demonstrated significantly enhanced antibody production to type II collagen and M. tuberculosis, but did not correlate with the disease activity. These observations suggest that while the lpr gene causes a more severe early effect, background genes other than the lpr are more involved in the adjuvant-enhanced arthritis-afflicted mice. PMID- 7530962 TI - HLA association of anti-Ro60 and anti-Ro52 antibodies in Sjogren's syndrome. AB - The possible pathological role of antibody subsets specific for different regions of 60-KD and 52-KD Ro/SSA proteins (Ro60 and Ro52) and La protein is unclear. Previously, we have shown that in patients with Sjogren's syndrome, the fine specificity of Ro60 and Ro52 antibodies, as characterized with synthetic peptides, varied considerably according to the origin of sera. We therefore looked for possible associations of HLA class I and class II alleles with Ro and La IgG antibodies in patients with primary Sjogren's syndrome (n = 24) and secondary Sjogren's syndrome associated with systemic lupus erythematosus (n = 25). Ro60 and Ro52 antibodies were tested by ELISA with the complete parent proteins and with 10 to 24 residue-long peptides of these proteins. Anti-Ro60 antibodies were more frequent in DR3-positive patients and in DQ1-negative patients whereas the presence of Ro52 and La IgG antibodies was significantly increased in patients with A1/B8/DR3 haplotype. Certain HLA associations observed with antibodies reacting with the whole Ro52 protein were not found with antibodies reacting with certain Ro60 and Ro52 peptides and, reciprocally, certain anti-peptide antibodies were linked to particular haplotypes whereas antibodies to the respective parent proteins were not linked to these haplotypes. Thus the production of antibody subsets reacting with different parts or presentations of Ro proteins is, at least in part, influenced immunologically by different HLA haplotypes, and this predisposition may play a role in the pathological development of the disease. PMID- 7530964 TI - Callosal size in children with learning disabilities. AB - The corpus callosum (CC), the main structure subserving hemispheric collaboration, that is necessary for efficient cognitive functioning, undergoes developmental processes such as axonal retraction and myelination. Callosal growth therefore is vulnerable for adverse events such as perinatal asphyxia, but there are also genetic and epigenetic factors that determine form and thickness. MRI scans of 110 children, either with specific learning disabilities (LD), i.e. dysphasia/dyslexia, or with several degrees of general LD, showed callosa that were highly variable in size. The callosal size corrected for brain size did not vary significantly according to the severity of the LD, although it tended to be smaller in severe LD, i.e. mental retardation. Callosal size varied however, due to the likely presence of genetic influences or of adverse perinatal events. Children with familial dysphasia/dyslexia, had a thicker CC, possibly reflecting a poorly understood neurodevelopmental mechanism that inhibits the establishment of cerebral dominance. LD children (all subgroups together) with perinatal adverse events had a smaller CC than the familial cases, suggesting CC damage. Despite a multitude of developmental factors influencing the final size, this study suggests that total callosal size, supposedly linked to interhemispheric function, may contribute to the pathophysiological mechanisms that give rise to LD. PMID- 7530963 TI - Drug-induced actions on brain neurotransmitter systems and changes in the behaviors and emotions of depressed patients. AB - Despite cumulative evidence that the tricyclic drugs result in significant changes in the functioning of brain serotonergic (5-HT) and nordrenergic (NE) systems, such changes have not been found to be associated with recovery from depression. Based upon evidence that the 5-HT and NE systems were associated with different emotions, it was hypothesized that changes in these systems were associated with different components of behavior in drug-responsive patients and not with changes in the "whole" disorder. Findings from this multihospital study of 104 unipolar and bipolar depressed patients showed early drug-associated reductions in anxiety and hostility in treatment responders to precede changes in motor retardation and depressed mood. Adopting this approach of looking for relationships between changes in components of major depression and changes in neurotransmitter system function, decreases in 5-HT and NE metabolite concentrations in cerebrospinal fluid (CSF) in patients treated with tricyclics, were found to be correlated with changes in specific behaviors. Results indicated the following: (1) drug-induced changes in the 5-HT system to be associated with mood aspects, notably anxiety, and depressed mood; changes in NE primarily with the psychomotor, secondarily with the mood components of the depressed state; (2) the pattern of relationships between changes in 5-HT and in mood in the unipolar was different than that in the bipolar subtype. The results indicate that in determining the relationships of biochemical changes to behavioral ones, that it is important to take into account the type of depression (bipolar or unipolar), as well as examining individually and over time those components that make up the disorder of depression. These results support evidence that tricyclics have multiple behavioral actions, that response is mediated through changes in specific behaviors and that this approach warrants further application in prospective studies of antidepressant drug mechanisms and their therapeutic actions. PMID- 7530965 TI - Pulmonary toxicity of inhaled diesel exhaust and carbon black in chronically exposed rats. Part I: Neoplastic and nonneoplastic lung lesions. AB - This study compared the pulmonary carcinogenicities and selected noncancer effects produced by chronic exposure of rats at high rates to diesel exhaust and carbon black. The comparison was intended to provide insight into the likely importance of the mutagenic organic compounds associated with the soot portion of diesel exhaust in inducing pulmonary carcinogenicity in diesel exhaust-exposed rats. The role of the organic fraction has become important in judging the usefulness of the substantial data base on carcinogenicity in rats for predicting lung cancer risk for humans, and for determining the most appropriate method of extrapolating results across species and exposure concentrations. Rats were exposed chronically to either diesel exhaust or carbon black, which served as a surrogate for diesel exhaust soot with much reduced mutagenic activity associated with its organic fraction. The sequestration of particles in the lung and the induction of pulmonary neoplasia and non-neoplastic changes in the lung were compared in detail. Samples also were provided to collaborators to examine adduct formation in lung DNA and hemoglobin. Approximately 140 female and 140 male F344/N rats were exposed for 16 hours per day, 5 days per week for up to 24 months, beginning at eight weeks of age, to diesel exhaust or carbon black at 2.5 mg or at 6.5 mg particles/m3 of air, or to clean air as controls. The diesel exhaust was generated by light-duty engines burning certification fuel and operating on an urban-duty cycle. The carbon black was selected because it had particle size and surface area characteristics similar to those of diesel exhaust soot, but markedly less mutagenic activity associated with its organic fraction when analyzed using procedures typically used in studies of diesel soot. Rats were killed after 3, 6, 12, 18, or 23 months of exposure to measure lung and lung associated lymph node burdens of particles, lung weight, bronchoalveolar lavage indicators of inflammation, DNA adducts in whole lung and alveolar type II cells, and chromosome injury in circulating lymphocytes, and to perform histopathologic assessment. In addition, after 3 and 18 months of chronic exposure, one group of rats was acutely exposed to radiolabeled carbon black particles or to fluorescent microspheres. These exposures were conducted to examine the clearance of radiolabeled particles and the sequestration of the fluorescent microspheres in the lungs. These experiments provided information on clearance overload and particle dosimetry. The growth characteristics of lung neoplasms also were examined by transplanting neoplastic cells into athymic mice.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7530966 TI - Cyfra 21-1. A new potential tumor marker for squamous cell carcinoma of head and neck. AB - OBJECTIVE: Evaluation of Cyfra 21-1 (cytokeratin fraction 21-1) in squamous cell carcinoma of the head and neck. DESIGN: Prospective study. PATIENTS: Serum Cyfra 21-1 concentration was measured in 250 samples from patients with squamous cell carcinoma of head and neck, patients with benign tumors of head and neck, healthy control subjects, and patients in remission from squamous cell carcinoma of head and neck. RESULTS: Cyfra 21-1 concentration was elevated in 60% of the new patients with squamous cell carcinoma but only in 8% of patients with benign tumors and 3.5% of the healthy controls. At a cutoff of 1.3 ng/mL, the sensitivity of the test was 60%, the specificity was 94%, positive predictive value was 75%, and negative predictive value was 89%. The marker levels tended to follow the clinical course of the disease and were useful for therapy monitoring. Cyfra 21-1 levels were in good correlation with the tumor stage expressed by the local (T) and the lymphatic spread (N) and were inversely correlated with histologic grade, eg, higher in poorly differentiated carcinoma than in well differentiated squamous cell carcinoma. CONCLUSION: Cyfra 21-1 evaluation in head and neck squamous cell carcinoma is worthwhile for performance of an ample study that will prove and establish its routine use. PMID- 7530967 TI - Serological and structural relationships between Escherichia coli O:98 and Yersinia enterocolitica O:11,23 and O:11,24 lipopolysaccharide O-antigens. AB - The serologically related lipopolysaccharide O-antigens of Yersinia enterocolitica serotypes 11,23 and 11,24 and of Escherichia coli O:98 were analysed by composition analysis, methylation, and the use of one- and two dimensional nuclear magnetic resonance methods. They were found to be composed of the same basic linear unbranched polysaccharide of repeating tetrasaccharide units containing 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D galacturonic acid (D-GalNAcA), and 2-acetamido-2,6-dideoxy-L-glucose (L quinovosamine, L-QuiNAc), having the structure -->3)-alpha-L-QuipNAc-(1-->4) alpha-D-GalpNAcA-(1-->3)-alpha -L-QuipNAc-(1-->3)-beta-D-GlcpNAc-(1-->. The lipopolysaccharide O-antigens of E. coli O:98 and Y. enterocolitica O:11,24 were polymers of the above repeating unit, whereas that of Y. enterocolitica O:11,23 was a linear polymer of the same repeating units in which the alpha-D-GalpNAcA residues were stoichiometrically substituted by acetyl groups at O-3. PMID- 7530968 TI - [Antibodies against the structural and nonstructural components of HCV in i.v. drug abusers]. PMID- 7530969 TI - [An observational study on 8 years of admissions for HIV-related pathology at an infectious disease center]. PMID- 7530970 TI - [Animal-assisted therapy. Is there a future in Italy for pet therapy?]. PMID- 7530971 TI - Immune biological products: normal and specific immunoglobulin. PMID- 7530972 TI - [The efficacy of a new cold disinfectant-sterilizing agent: our preliminary experience]. PMID- 7530973 TI - [Sexual behavior in representative samples of young people: the methodological aspects of the research]. PMID- 7530975 TI - [The health and hygiene aspects of thermal baths]. PMID- 7530974 TI - [Falls in the frail elderly: a prospective studies]. PMID- 7530976 TI - [An estimation of the efficacy and costs of treatment, therapeutic and/or preventive, with Fiuggi water in renal and metabolic pathologies]. PMID- 7530978 TI - An efficient and high-yielding method for isolation of RNA from mycobacteria. PMID- 7530977 TI - [The opinions of physicians in the area of the USM RM/33 local health unit on the proposed implementation of a preventive medicine outpatient facility]. PMID- 7530980 TI - Handling of large (300 x 400 mm), thin (0.4 mm) polyacrylamide gels and recovery as dried gels. PMID- 7530979 TI - Isolation of total RNA from yeast and bacteria and detection of rRNA in northern blots. PMID- 7530981 TI - Quantitation of metallothionein mRNA by RT-PCR and chemiluminescence. AB - A general procedure has been developed for the determination of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR), over a wide concentration range, with quick quantitation of amplified products by luminescence. The discriminating power of this approach is the specific hybridization of PCR product to ruthenium-labeled oligonucleotide probe(s). This method is sensitive enough to detect increases in the formation of PCR product by the 6th cycle. The accumulation of PCR product was successfully modeled with a recursive relationship. This procedure was capable of accurately determining starting template copies over a 9-log dynamic range, with a sensitivity limit of 10(2) copies. Inclusion of an mRNA internal standard (identical to amplified template except for a 6-bp deletion) corrected variabilities in the reverse transcriptase as well as PCR, allowing for the expression of data as mRNA copy number/micrograms total tissue RNA. This procedure was used to detect changes in levels of winter flounder (Pleuronectes americanus) liver metallothionein mRNA. Liver metallothionein mRNA levels ranged from 1.0 x 10(6) copies/micrograms total tissue RNA in control samples to 1.0 x 10(9) copies/micrograms total tissue RNA in samples treated with Cd (a known metallothionein mRNA inducer). PMID- 7530983 TI - Detection of epithelial cancer cells in peripheral blood by reverse transcriptase polymerase chain reaction. AB - Circulating cancer cells in the blood play a central role in the metastatic process. Their number can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumour cells in haematological cancer in which abnormalities in DNA are sufficiently consistent to make this possible. For most solid tumours this not yet feasible. However, we have found that reverse transcriptase (RT)-PRC for tissue-specific gene expression is a useful technique for identifying small numbers of circulating cells in melanoma and neuroblastoma patients. In this report we describe detection of colon carcinoma cells by RT-PCR using CK 20 mRNA as a marker. Unlike other cytokeratin genes examined (CK 8 and CK 19), CK 20 was not transcribed in normal haematopoietic cells. This suggests a role for RT-PCR in the detection of colon carcinoma metastasis in blood and bone marrow, using CK 20 as the target gene. Future analysis of clinical material will determine the clinical significance of this technique. PMID- 7530985 TI - Microvessel quantification in primary colorectal carcinoma: an immunohistochemical study. AB - The vascularisation of human primary colorectal carcinomas was studied immunohistochemically using the endothelial cell markers CD31 and factor VIII related antigen. Tumour sections were systematically scanned at a magnification of x 100 to find areas of intense neovascularisation. Microvessel counts within these vascular 'hotspots' were performed at magnification x 250. Regions in which tumour cords were surrounded by a collagen IV-positive basement membrane were compared with those in which this was absent and with normal mucosa. CD31 appeared to be a more sensitive marker for endothelial cells than factor VIII related antigen (mean 185 +/- 59 and 120 +/- 38 microvessels mm-2). Within individual tumour sections microvessel counts in vascular hotspots with highest vessel density correlated significantly with microvessel counts in vascular hotspots with second highest vessel density (P < 0.01). Microvessel counts in tumour areas where collagen IV-positive basement membrane were absent exceeded those in areas where it was present (factor of 1.7) and those in normal mucosa (factor of 1.6). The differences in vessel density between individual tumours and the low variability in vessel density within individual tumours using this quantification technique allow us to investigate the prognostic value of vessel density in areas of intense neovascularisation in human primary colorectal carcinomas. PMID- 7530984 TI - A phase I trial of repeated tumour-infiltrating lymphocyte (TIL) infusion in metastatic melanoma. AB - The aim of the protocol was to evaluate the side-effects induced by repeated tumour-infiltrating lymphocyte (TIL) infusions in patients with metastatic melanoma (MM). Patients were to receive four TIL infusions at given intervals: every 3 weeks (two patients), every 2 weeks (3 patients) and weekly (4 patients). All patients were evaluated and received a total of 34 TIL infusions. The total number of TILs administered varied from 0.65 to 2.34 x 10(11) cells. TIL phenotypes were predominantly CD8+ (two patients), CD4+ (4 patients), CD4+ then CD8+ (two patients) or CD56+ (two patient). Autocytotoxicity was only observed for one culture. Six patients presented at least one WHO grade 3 side-effect: hypotension (5 patients), dyspnoea (two patients), fever (one patient), fatigue (one patient), chills (two patients), diarrhoea (one patient), agitation (one patient), locoregional pain (two patients). Hypotension was constantly seen in patients who were given TILs every week. Two cases of minor pericarditis were recorded. No objective response to treatment was observed; 1 stable disease occurred in one patient and progression in eight. However, five patients presented a partial response on a tumour site for 1-4 months. Three patients presented signs of inflammation or softening at one tumour site. Plasma tumour necrosis factor alpha (TNF-alpha) levels were increased 1.2- to 22-fold after TIL infusion. TILs could be produced in sufficient quantity to perform this study, so repetitive infusions of TIL became possible on a weekly basis. However, no objective response was observed even when TIL infusions were performed weekly. An increase in circulating TNF-alpha was noted after TIL infusion. PMID- 7530982 TI - Effect of epidermal growth factor on cadherin-mediated adhesion in a human oesophageal cancer cell line. AB - Epidermal growth factor (EGF) mediates many pleiotrophic biological effects, one of which is alteration of cellular morphology. In the present study, we examine the possibility that this alteration in cell morphology is caused in part by the dysfunction of cadherin-mediated cell-cell adhesion using the human oesophageal cancer cell line TE-2R, which expresses E-cadherin and EGF receptor. In the presence of EGF, TE-2R changed its shape from round to fibroblastic and its colony formation from compact to sparse. Vanadate, a tyrosine phosphatase inhibitor, further potentiated the EGF response, whereas herbimycin A, a tyrosine kinase inhibitor, interfered with it. Moreover, EGF enabled the cells to invade in organotypic raft culture. These phenomena were accompanied not by decreased expression of the E-cadherin molecule but by a change in its localisation from the lateral adhesion site to the whole cell surface. Both alpha- and beta catenin, cadherin-binding proteins, were also expressed at the same level throughout these morphological changes. Finally, we examined tyrosine phosphorylation of E-cadherin and alpha- and beta-catenin, and observed tyrosine phosphorylation of beta-catenin induced by EGF. These results suggest that EGF counteracts E-cadherin-mediated junctional assembly through phosphorylation of beta-catenin and modulates tumour cell behaviour to a more aggressive phenotype. PMID- 7530987 TI - Treatment of advanced medullary thyroid cancer with an alternating combination of 5 FU-streptozocin and 5 FU-dacarbazine. The Groupe d'Etude des Tumeurs a Calcitonine (GETC). AB - Combinations of 5-fluorouracil (5-FU) and streptozocin and 5-FU and dacarbazine were given alternately to 20 patients with metastatic medullary thyroid carcinoma. Three partial responses and 11 long-term stabilizations were observed. No unexpected toxicity occurred. PMID- 7530986 TI - Measurement of urinary beta core fragment of human chorionic gonadotrophin in women with vulvovaginal malignancy and its prognostic significance. AB - Tumours of the vulva and vagina are rare and there are relatively few studies of circulating markers in these conditions. The urinary measurement of the core fragment of the beta-subunit of hCG has been proposed as a useful tumour marker in non-trophoblastic gynaecological malignancies. This study describe the measurement of urinary beta-core in 50 patients with vulvovaginal malignancy. In contrast to other studies corrections were made for both the effect of urine concentration and the age of the patient. Each patient was followed up for at least 24 months, and at this time their status was correlated with their initial level of urinary beta-core. The sensitivity of beta-core was only 38%, but of those patients with elevated levels 90% had died within 24 months, while only 32% of those with normal levels had died. For both patients at initial presentation and those with recurrent disease, there was a highly significant difference in the survival curve between those with elevated beta-core levels and those with normal levels. This is similar to findings in cervical carcinoma, and suggests that for lower genital tract cancer the measurement of urinary beta-core may be valuable as a prognostic indicator, allowing a more informed approach to treatment and follow-up. PMID- 7530988 TI - Symptom relief with MVP (mitomycin C, vinblastine and cisplatin) chemotherapy in advanced non-small-cell lung cancer. AB - The role of chemotherapy in the palliation of patients with advanced stage (IIIB and IV non-small-cell lung cancer (NSCLC) remains controversial. We have carried out a chemotherapy study emphasising symptom relief, a topic not normally discussed in previous similar studies. A total of 120 patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) were treated with a moderate-dose palliative chemotherapy regimen consisting of mitomycin C 8 mg m-2 i.v. on day 1 (alternate courses), vinblastine 6 mg m-2 i.v. on day 1 and cisplatin 50 mg m-2 i.v. on day 1 (MVP), repeating every 21 days for a maximum of six courses. Thirty-eight of 118 assessable patients (32%) achieved an objective response. Patients with locally advanced disease (stage IIIB) had a significantly better response rate (52%) than those with metastatic disease (25%) (P < 0.01). In 76 out of 110 (69%) patients, with tumour-related symptoms including 24 out of 31 patients (78%) with locally advanced disease, symptoms completely disappeared or substantially improved. In only 15 patients (14%) did symptoms progress during treatment. Symptomatic improvement was achieved after one course of chemotherapy in 61% and after two courses in 96% of responding patients. The schedule was well tolerated. Only 19% developed WHO grade 3/4 nausea/vomiting, and only 3% developed significant alopecia. Other toxicities were minimal. MVP is a pragmatic inexpensive chemotherapy regimen that offers useful symptom palliation in patients with advanced NSCLC and merits a 1-2 course therapeutic trial in such patients. The schedule should also be assessed as primary (neoadjuvant) chemotherapy before radical radiotherapy for locally advanced NSCLC in a randomised trial. PMID- 7530989 TI - Cell kinetic investigations in craniopharyngioma: preliminary results and considerations. AB - In vitro determination of 3H-thymidine labeling index (LI%) was carried out in craniopharyngiomas from 16 patients submitted to repeated surgical procedures. This study showed a poor correlation between LI% values and prognosis, but allowed to detect a high LI% variation in the same tumor investigated in serial different times. The spatial distribution of S-phase proliferative cells in squamous epithelium of cystic components suggests a rationale for intracavitary treatment of cystic and mixed craniopharyngioma and may help explain its success. PMID- 7530990 TI - Surgical management of craniopharyngioma. AB - Craniopharyngiomas can be divided into sellar, prechiasmatic, retrochiasmatic and giant tumors. The initial approach to a craniopharyngioma should be an attempt at its removal. Only if surgical therapy fails to deal with the tumor should more conservative approaches utilizing radiotherapy be used. The use of intracystic injection of bleomycin in craniopharyngiomas has proven to be a valuable adjunct in helping to remove these tumors. PMID- 7530993 TI - A novel monoclonal antibody to the outer root sheath cells. AB - A novel monoclonal antibody to hair keratins (TYHF-1) which recognized a doublet of 43/44-kDa hair keratins in the immunoblot analysis was developed. TYHF-1 strongly reacted with the outer root sheath cells at the region of prekeratinization, the keratogeneous zone and the lower part of the isthmus portion in the indirect immunofluorescent examination. The cells of the inner root sheath and cuticle in the upper bulbar portion were also stained with this antibody. In addition, the tongue and nail matrix were also reacted with TYHF-1. However, TYHF-1 reacted with no epidermal keratins either in the indirect immunofluorescent examination or by immunoblot analysis using plantar epidermis. In addition, the cells of the eccrine and apocrine sweat glands, the epithelia of the buccal mucosa, larynx, tonsil, oesophagus, liver, intestine, gallbladder and urinary bladder demonstrated no positive reaction. PMID- 7530992 TI - Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: a possible role for rho protein in epidermal cells. AB - We examined the role of rho gene products (rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC 1), using recombinant botulinum C3 exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with a molecular weight of 23,000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 exoenzyme at 1, 3, 10, 30 and 60 micrograms/ml culture medium resulted in 9.0 +/- 1.8%, 20 +/- 2.9%, 26 +/- 2.3%, 50 +/- 1.4% and 40 +/- 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells. PMID- 7530991 TI - Identification of cone classes in Xenopus retina by immunocytochemistry and staining with lectins and vital dyes. AB - The aim of the present study was to determine the number of cone classes in the Xenopus retina. We examined the dimensions and staining properties of cones, utilizing two monoclonal antibodies, COS-1 and OS-2, developed by Szel and Rohlich (1985). Living cones also were reacted with the plant lectins peanut agglutinin (PNA) and wheat germ agglutinin (WGA) and with a fluorescent stilbene dye, DIDS, which binds selectively to red-sensitive cones (Kleinschmidt, 1991; Kleinschmidt & Harosi, 1992a,b). Three cone populations were distinguished based on differences in size and staining properties. Eighty-eight percent of all cones were stained strongly by COS-1, PNA, and DIDS, but weakly by OS-2. The group of cones stained by COS-1 had the largest mean dimensions of outer segment length, width, and oil droplet diameter. COS-1 negative cones were divisible into two groups: a subclass of miniature cones (approximately 4% total cones) was stained strongly by OS-2, PNA, and DIDS. The balance, constituting approximately 9% total cones, were of intermediate size, were not stained by PNA and reacted weakly to OS-2 and DIDS. WGA stained all cones. Large, COS-1+ cones appear to be red sensitive and belong to the class of anion-tunable cone pigments. We suggest that the intermediate size, COS-1 negative cones are blue-sensitive based on the finding that blue-sensitive chromatic horizontal cells connect to them preferentially (Witkovsky et al., work in progress).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7530994 TI - Identification of the epitopes on human type VII collagen for monoclonal antibodies LH 7.2 and clone I, 185. AB - Type VII collagen is the major component of anchoring fibrils, structures in human skin that mediate the adherence of the epidermis to the underlying dermis. Dystrophic forms of epidermolysis bullosa, a group of inherited mechanobullous disorders of the skin, are linked to the type VII collagen gene. Several mutations in the recessive form of this inherited disorder have been delineated. In this study, we mapped the epitopes of two commercially available monoclonal antibodies (clone I, 185 and LH 7.2) within the amino-terminal, non-collagenous domain of type VII (anchoring fibril) collagen. The precise localizations of the epitopes for these two monoclonal antibodies which are widely used to diagnose dystrophic epidermolysis bullosa, will be useful for the confirmation of gene mutations at the protein level. PMID- 7530995 TI - Blood levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin and gamma-globulins in a follow-up investigation of employees with chloracne. AB - In a follow-up investigation of 8 employees who partly contracted chloracne between 1973 and 1976, elevated concentrations of 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD, 'Seveso dioxin') were found in blood lipids. The concentrations measured were between 163 and 1935 pg TCDD/g blood lipids, other 'dioxin' levels being normal or only slightly increased in comparison to the background level in the general population. The cause of this was exposure at work during the manufacture of trichlorophenol (TCP). Apart from high cholesterol levels, there was a conspicuous reduction in gamma-globulins. PMID- 7530996 TI - Immunohistochemical evaluation of bone morphogenetic protein (BMP) in mixed tumor of skin. AB - Immunohistochemical reaction of bone morphogenetic protein (BMP) was assessed in 19 cases of skin mixed tumor and 5 cases of skin appendage tumors by using monoclonal antibody raised against BMP. All cases of skin mixed tumor showed positive staining for BMP in modified myoepithelial cells located at the periphery of tubulo-ductal or solid structures, and in plasmacytoid or tumor cells in hyalinous structures. Chondroidally changed cells also showed a strong BMP immunoreactivity. Tumors originating from sweat glands were devoid of BMP immunoreactivity. It is concluded that BMP is synthesized and produced in modified or transformed myoepithelial cells in skin mixed tumor and participates in the process of chondroid changes in the tumor. PMID- 7530998 TI - Intron-encoded small nucleolar RNAs: new RNA sequence variants and genomic loci. AB - Three small nucleolar RNAs (snoRNAs) whose 5' termini are monophosphorylated, termed E1, E2 and E3, were reported earlier, and E1 and E3 are encoded in pre mRNA gene introns. In the present work, the ends of these snoRNAs were identified by analysis of terminal mononucleotides, and heterogeneity was observed at the 3' ends of E1 and E2 RNAs. Two new E1 RNA species were detected in HeLa cells by cDNA cloning. Four novel human genomic loci were identified that have E1 or E3 sequence homology. The sequence CTAGAGCACYSAATCTGGAT (where S = C or G and Y = C or T), that is present three nucleotides downstream from the coding region of an E1 RNA-encoding gene, lies in the same location in a different human genomic locus (which has a E1-homology sequence whose expression has not been detected yet), suggesting that this sequence may be functional. PMID- 7530997 TI - Twenty-four-hour total small bowel hypothermic storage preservation and transplantation in the rat: a study of various preservation solutions. AB - Three hundred forty-eight Sprague Dawley rats were used as donors and recipients of fresh and preserved small bowel transplants in order to evaluate both the most important determinants of failure after preservation and transplantation, and the effect of various preservation solutions used for 24 h. Four different solutions were tested: saline for the control group, and Euro-Collins (EC), University of Wisconsin (UW), and albumin-dextran-adenosine-allopurinol-verapamil (ADAAV) for the experimental groups. The survival at 3 days was 80% for the control group, 66.6% for the ADAAV group, and 53.5 and 46.6% for the UW and EC groups, respectively (p < .05 saline vs EC, UW; p > .05 saline vs ADAAV). Another two groups received small bowels on which the effect of nonflushing was studied for 6 and 24 h. In general, laboratory and histologic studies did not show a significant difference among the various groups of preserved small bowels. The worst groups were the nonflushed preserved small bowels, in which no survival was obtained. Although some improvement was noted in early survival with the ADAAV solution, no true-long term survivors were seen in any of the preservation groups studied. Factors of preservation injury were related to hemorrhagic necrosis and sepsis in the majority of the experimental groups. Thus, there were no available preservation solutions that could give consistent results after 24 h of small bowel preservation in the rat. PMID- 7530999 TI - Effect of dialyser membranes on extracellular and intracellular granulocyte and monocyte activation in ex vivo pyrogen-free conditions. AB - This study examined effects of blood-contacting materials on the monocyte reaction following the first contact of human blood with hollow fibre dialyser membranes under pyrogen-free conditions. Membrane materials were the unchanged regenerated cellulose, the synthetic polysulphone (PS), a positively charged diethylaminoethyl cellulose (DEAE-C), the negatively charged carboxymethyl cellulose (CMC) and acrylonitrile copolymer (AN). The experimental system involved perfusion with human fresh venous blood through different modules containing the materials in the form of hollow fibre membranes. Extracellular and intracellular aspects of blood reactions after the first contact with the materials were investigated in Ficoll-separated granulocytes and peripheral blood mononuclear cells. Investigations were done by release reactions of platelet activating factor (PAF), oxygen radical (O2-), leukotriene B4, prostaglandin E2 (PGE2) and cytokines (IL-1 beta, TNF-alpha, IL-6). The intracellular activation of peripheral blood mononuclear cells was done by mRNA transcription of IL-1 beta, TNF-alpha, IL-6, IL-8 and beta 2-microglobulin (beta 2-MG). From the set of parameters, release reactions were only measurable for PAF, PGE2 and O2- if a second stimulus (phorbol myristate acetate, lipopolysaccharide, zymosan and calcium ionophore) was used after blood-membrane interaction. Although the extent of the release reaction was weak, negatively charged membranes were, in general, more active. All dialysers exhibited the same increase in beta 2-MG mRNA transcription, suggesting that all blood-contacting membranes initiate the gene expression of beta 2-MG at the same level. TNF-alpha, IL-6, IL-1 beta and IL-8 mRNAs were demonstrated in the AN and CMC membranes rather than the other materials, which exhibit a lower transcription than the tubing set. As has been found, an enhanced generation of PGE2 for both CMC and AN membranes supports, therefore, the concept of an effect of the negative charges of the materials in the gene expression of cytokines. However, this initiation does not lead to the generation of cytokines, even after stimulation with pyrogens. PMID- 7531000 TI - Studies on the mechanisms of action of aprotinin and tranexamic acid as plasmin inhibitors and antifibrinolytic agents. AB - Both aprotinin and tranexamic acid are effective inhibitors of fibrinolysis in vitro and in vivo and both agents can act as plasmin inhibitors in purified systems, although there is some debate on their exact mechanism of action in vivo. The studies reported here using an in vitro clot lysis system designed to provide precise inhibition constants show that aprotinin remains a very potent inhibitor of plasmin even in the presence of fibrin with Ki = 2 nM. Plasmin aprotinin interactions in solution are not affected by a number of kringle binding ligands, aminohexanoic acid, tranexamic acid or CNBr-fibrinogen fragments with Ki = 0.4 nM. The difference between these two Ki values is explained by competition for the plasmin active site between substrate (fibrin) and inhibitor (aprotinin). Inhibition of fibrinolysis by tranexamic acid is not readily analysed by a simple inhibition model which may be due to multiple overlapping ligand-kringle interactions or tranexamic-fibrin interactions. Experiments using combinations of aprotinin and tranexamic acid in the clot lysis system confirm the complementary nature of inhibitory mechanisms and suggest a slight synergism. These results support the idea that aprotinin inhibition of plasmin is a primary mode of action in vivo, and suggest that combination therapy of aprotinin with tranexamic acid might be more effective than either inhibitor alone. PMID- 7531002 TI - News on NOS: neuronal nitric oxide synthase as a drug target. PMID- 7531003 TI - Non-Alzheimer neurofibrillary tangles show beta-amyloid-like immunoreactivity. AB - As well as being a neuropathological hallmark of Alzheimer's disease (AD), neurofibrillary tangles (NFT) are present in the brain in a number of other neurodegenerative conditions. beta-Amyloid (beta/A4) plaque formation is a central event in AD, although recent reports indicate that beta/A4, and its precursor protein (beta APP), are also associated with AD NFT. Using an antibody to a beta APP epitope overlapping the beta/A4 region, we demonstrate that immunoreactivity is found in extracellular NFT in Niemann-Pick disease, Hallervorden-Spatz disease, progressive supranuclear palsy and subacute sclerosing panencephalitis. These NFT were not labelled with a beta/A4 antibody which recognizes plaque beta/A4 in AD, which supports the proposal that beta/A4 can exist in different conformations. Our results also imply a common mechanism of NFT development in different neurodegenerative conditions, including those not normally associated with substantial beta/A4 deposition or beta APP involvement. PMID- 7531004 TI - Anxiolytic effect of nitric oxide synthase inhibitors microinjected into the dorsal central grey. AB - Nitric oxide synthase (NOS) accounts for most of the NADPH-diaphorase neuronal activity in the brain. NADPH-diaphorase-positive neurones have been localized at the dorso-lateral part of the periaqueductal grey (PAG), a region related to anxiety. Microinjections of the NOS inhibitors L-NAME (10-200 nmol 0.5 microliter 1) and L-NOARG (10-100 nmol) at this site induced anxiolytic-like effects in the elevated plus maze. These effects, however, occurred only at a limited range of doses and the dose-effect curve had a bell shape, higher doses of both compounds tending to be anxiogenic. The anxiolytic effect of L-NAME was antagonized by a previous microinjection of L-arginine (50 nmol 0.5 microliter-1). These results suggest that NO may play a role in PAG neurotransmission involved in anxiety. PMID- 7531001 TI - Infusion of large quantities of autologous blood monocyte-derived macrophages in two cancer patients did not induce increased concentration of IL-6, TNF-alpha, soluble CD14 and nitrate in blood plasma. AB - In an attempt to increase the number of macrophages available for reinfusion in immunotherapy trials, GM-CSF was injected in vivo to mobilize circulating blood monocytes in 2 cancer patients. Subsequently mononuclear cells were collected by apheresis, cultured in the presence of GM-CSF and activated with IFN-gamma. This procedure resulted in the harvesting of 1.3 to 3.1 x 10(9) (mean 2 x 10(9)) macrophages per apheresis, product which was very well tolerated at autologous reinfusion. These infusions did not induce increased levels of TNF-alpha, IL-6, soluble CD14 nor nitrates in blood plasma (or urine). The lack of TNF-alpha and IL-6 release in blood plasma could explain the good tolerance of these infusions. No in vivo anti-tumoural activity of these high numbers of infused macrophages could be observed. PMID- 7531005 TI - Effects of a nitric oxide donor on glutamate and GABA release in striatum and hippocampus of the conscious rat. AB - The effects of intracerebral perfusion of the nitric oxide (NO) donor 3 morpholino-sydnonimine (SIN-1) and the nitric oxide synthase inhibitor N nitroarginine (NARG) on the extracellular concentrations of glutamate (GLU) and gamma-aminobutyric acid (GABA) in striatum and CA1 area of the hippocampus were studied. Continuous push-pull perfusions at a flow rate of 20 microliters min-1 were performed in the conscious rat. SIN-1 (100, 200, and 400 microM) and NARG (100 microM) were perfused over 20 min. In both striatum and CA1 SIN-1 increased extracellular concentrations of GLU (maximal increase 150% and 197% of baseline, respectively) and GABA (maximal increase 202% and 204% of baseline, respectively). NARG had no effects on extracellular levels of GLU and GABA in either area. These results are consistent with the hypothesis that NO acts as a modulator of GLU and GABA release in both striatum and hippocampus. This study is the first to report the potentiation of GLU and GABA release by NO in CA1 area of the hippocampus in the conscious rat. PMID- 7531006 TI - Laminar pattern of NADPH-diaphorase activity in the developing avian cerebellum. AB - The present study aimed to define the distribution of NADPH-diaphorase (NADPH-d) positive cells and fibers in the developing chick cerebellum. From embryonic day 9 (E9) until day E15 NADPH-d positive ascending axons, originating from pontine nuclei, targeted selectively the developing folia. Cerebellar cortex exhibited weak NADPH-d activity until day E15, when NADPH-d negative granule cells of external granular layer began inward migration. From that embryonic age, differentiated granule, Golgi II type cells and glomeruli of the internal granular layer exhibited gradually increasing diaphorase activity. In the molecular layer diffuse activity increased, possibly due to the development of the parallel fibers. Transient NADPH-d activity appeared on Purkinje somata (PC) at day E17 and by posthatching day 1 all PC were devoid of diaphorase activity. Our data support the hypothesis that neurons producing nitric oxide (NO) are involved in the activity dependent developmental processes for the establishment of connections and normal cytoarchitecture. PMID- 7531007 TI - Time course of inhibition of brain nitric oxide synthase by 7-nitro indazole. AB - 7-Nitro indazole (7-NI) inhibits rat striatal, cerebellar, hippocampal, cerebral cortex and olfactory bulb nitric oxide synthase (NOS) in vitro with IC50 values of 0.68 +/- 0.01 microM, 0.64 +/- 0.03 microM, 1.53 +/- 0.05 microM, 0.93 +/- 0.04 microM and 1.05 +/- 0.02 microM respectively (n = 6). Intraperitoneal (i.p.) or oral administration of 7-NI (30 mg kg-1) to rats inhibited NOS enzyme activity measured ex vivo in all five brain regions (n = 5-6). NOS inhibition (maximal effect, 0.5 h post-injection) was transient with complete recovery at either 4 h (oral administration) or 24 h (i.p. administration). Repeated i.p. injection of 7 NI (30 mg kg-1, every 4 h for 20 h) inhibited NOS enzyme activity at 24 h by 51 61% in all brain regions. The relatively transient NOS inhibitory effect of 7-NI following parenteral administration should be taken into account when using this drug to evaluate the central effects of nitric oxide. PMID- 7531009 TI - Recurrent acute pancreatitis induced by isoniazid. AB - A 31-year-old man on haemodialysis who developed recurrent attacks of acute pancreatitis during the treatment of pulmonary tuberculosis is presented. The pancreatitis rapidly subsided once isoniazid was withdrawn, but the problem recurred again upon reintroduction of the drug. There was no further recurrence during successful treatment with full doses of rifampicin and pyrazinamide for 1 year. PMID- 7531008 TI - The ribosomal intergenic spacer region: a target for the PCR based diagnosis of tuberculosis. AB - SETTING: DNA Diagnostics, The National Diagnostics Centre and University College Hospital, Galway, Republic of Ireland. OBJECTIVE: To investigate the possibility of developing a DNA probe to distinguish the members of the Mycobacterium tuberculosis complex and develop an assay for the detection of M. tuberculosis in sputum. DESIGN: The ribosomal intergenic spacer regions from the members of the M. tuberculosis complex were sequenced and analysed for the ability of this region to provide sequence diversity for DNA probe development. RESULTS: The 100% sequence homology in this region precluded the development of a probe to distinguish the individual members of the M. tuberculosis complex. However it was possible to develop a DNA probe that could specifically detect only the members of the M. tuberculosis complex. CONCLUSION: A specific DNA probe was developed for the detection of the M. tuberculosis complex and its application to M. tuberculosis detection in sputum was demonstrated. PMID- 7531010 TI - Pharmacology of histamine liberation. Cationic amphiphilic drugs and mast cells. AB - The effect of betaadrenoceptor blocking drugs atenolol and propranolol was studied in both nonstimulated and stimulated isolated rat mast cells. Atenolol did not liberate histamine from non-stimulated mast cells, decreased spontaneous secretion, inhibited 48/80 stimulated histamine release, increased 32P incorporation into membrane phospholipids, decreased membrane fluidisation and decreased arachidonic acid liberation from membrane phospholipids of stimulated mast cells. Propranolol dose-dependently liberated histamine from nonstimulated mast cells and inhibited histamine liberation, it nonsignificantly increased membrane phospholipid turnover but significantly increased membrane fluidisation and inhibited stimulated arachidonic acid liberation in stimulated mast cells. The results indicated the interaction of atenolol and propranolol with mast cell membranes, particularly with the phospholipid bilayer, resulting in a possible inhibition of phospholipase A2 activation. Histamine liberation suggested its displacement from granule binding sites after intracellular propranolol accumulation in mast cells. PMID- 7531011 TI - Activation of BCEC-channels for electrochemical therapy (ECT) of cancer. Proceedings of the IABC International Association for Biologically-Closed Electric Circuits (BCEC) in Medicine and Biology. Stockholm, September 12-15, 1993. PMID- 7531012 TI - Electrochemical aspects of treatment of tissue with direct current. AB - In the electrochemical treatment of tissue with direct current the main reactions at the anode are the formation of oxygen in addition to acidification due to liberated hydrogen ions, and formation of chloride, if platinum is used as anode material. At the cathode hydrogen is formed and hydroxide ions are liberated. Calculations have been made for the two major anodic reactions. As a first approximation just one-dimensional diffusion is considered corresponding to the use of plate electrodes. It appears from these calculations that the liberated hydrogen ions determine the extension of the locally destroyed zone around the anode. The destructive effect of chlorine probably occurs in an inner zone close to the anode. PMID- 7531013 TI - A new type of liposome for electrochemical treatment of cancer: the lipogelosomes. PMID- 7531014 TI - Electrostatic field interference with cellular and tissue function, leading to dissolution of metastases that enhances the effect of chemotherapy. PMID- 7531015 TI - BCEC--gravitational circuits and Jacobson Resonance: the basis from which to evaluate potential hazard and therapeutic benefit from extrinsic magnetic fields. PMID- 7531016 TI - Organisation and spread of electrochemical therapy (ECT) in China. PMID- 7531017 TI - Advances in the treatment of malignant tumours by electrochemical therapy (ECT). AB - A total of 4,081 patients with various malignant tumours were treated by electrochemical therapy (ECT) at 66 hospitals in China during the period 1987 1992. The clinical effectiveness of ECT was analysed in 2,516 patients who had complete hospital records. The most common malignant tumours treated with ECT was lung cancer (n = 593), skin cancer (n = 401), liver cancer (n = 388) and breast cancer (n = 228). Two thousand one hundred and twenty-four patients were followed up for 1 to 5 years. Survival rates were 84.3% for 1 year, 79.1% for 2 years, 63.5% for 3 years, 57.8% for 4 years, and 46.6% for 5 years. The 5-year survival for T1 and T2 stages was significantly better than for T3 and T4 stages (66.7% vs. 27.1%, p < 0.05). The short-term objective response (complete and partial response) was 78.1%. ECT is indicated in patients who are unsuitable for operation and radio- or chemotherapy due to old age, and who suffer from general weakness, or insufficiency of vital organs. PMID- 7531018 TI - Analysis of the clinical effectiveness of 144 cases of soft tissue and superficial malignant tumours treated with electrochemical therapy. AB - Electrochemical therapy (ECT) was used for the treatment of 144 cases of soft tissue and superficial malignant tumours. All tumours were cytologically verified and included 39 soft-tissue sarcomas, ten head-neck cancers, 21 malignant melanomas, seven skin cancers, three cervical cancers, two breast cancers, 27 recurrent cancers and 35 metastatic cancers. The TNM stage of primary cancers were: T1: n = 28, T2: n = 36, T3: n = 12, and T4: n = 6. Among these, 23 cases were lymph nodes metastases and 12 systemic metastases. There were 43 cases of localised recurrent cancer and 19 cases of generalised metastatic disease. ECT was used to treat 128 cases once, nine cases twice, four cases three times and three four times. All patients received systemic chemotherapy for one to two cycles, and three of them were also treated by radiotherapy. The short-term effectiveness rate was 86.87% (125/144) with 81 complete response (CR) (tumour disappeared completely), accounting for 58.3% of all cases, 41 partial response (PR) (tumour regressed to more than half of its original size) accounting for 28.5%. Fourteen (9.7%) no change (NC); tumour regressed to less than half of its original size) and five (4.5%) progressive disease (PD); tumour did not regress or even increased in size). The short-term effectiveness rate in relation to tumour size was 94.5% (120/127) for tumour diameters of < 7 cm and 29.4% (5/17) for tumour diameters > 7 cm (p < 0.001). The long-term effectiveness, that is the survival rates for 1, 2, 3 and 4 years, was 83.3% (120/144), 74.1% (89/120), 84.3% (75/89) and 73.3% (55/75), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531019 TI - Electrochemical therapy in the treatment of malignant tumours on the body surface. AB - From November 1988 to May 1991, 113 patients with malignant tumours on their body surface, who were at great operative risk or had refused surgery, underwent local treatment with electrochemical therapy (ECT). After 2.6 years' follow up, total effective rate of the cure was 79.7%. The clinical results were considered satisfactory. PMID- 7531020 TI - Electric treatment of human melanoma skin lesions with low level direct electric current: an assessment of clinical experience following a preliminary study in five patients. AB - In this study we have investigated the effects of negative low level direct electric current electrotherapy (current: 1 milliampere; treatment time: 30 minutes) in metastatic or primary melanoma skin lesions. After 12 applications in five patients, tumour regression was observed in all evaluable melanoma skin lesions; four of them were classified as partial responses. This study shows that locally applied electrotherapy with a negative low level direct electric current is an effective treatment that results in reduction of tumour mass of human melanoma skin lesions, and it is possible that it has a positive influence on the course of the metastatically disseminated melanoma. No serious side effects were observed. PMID- 7531021 TI - Electrochemical therapy for intermediate and advanced liver cancer: a report of 50 cases. AB - Fifty cases of primary liver cancer were treated by electrochemical therapy (ECT). Tumour size ranged from 3.5 to 21.0 cm. ECT was performed by administration of 6-10 V, 50-100 mA and an electric quantity of 300-1000 C. Duration of treatment was one and a half to four hours. There was an inverse relationship between tumour size and curative effect. There was no death within three months of treatment. Six- and 12-month survival was 88% and 69%, respectively. In conclusion, ECT is an effective treatment which prolongs the patients' life. We have found the method to be of practical value in the management of liver cancers in the intermediate or advanced stage. PMID- 7531022 TI - Electrochemical therapy of 74 cases of liver cancer. AB - Ninety-four patients with primary liver cancer have been treated with electrochemical therapy (ECT) in our hospital. Seventy-four cases were followed up for more than one year, 12 cases for less than one year, and 8 cases were lost. Seventy-four cases of stage II and III liver cancer were divided into three groups. Group A received ECT only, group B received ECT plus transcatheter hepatic artery infusion chemotherapy (TAI) and embolisation (IAE) treatment. Group C received ECT plus systemic chemotherapy. The survival rate at 12 months was 58% for group B patients, which was significantly higher than for group A or C patients. We found ECT to be relatively simple and safe. The main side effect is short-time fever after treatment. PMID- 7531024 TI - Electrochemical and megavolt treatment of rat tumours. AB - In a series of experiments, an electrolytic method of tumour destruction has been developed. When tested clinically, no tumour has been totally destroyed but substantial necroses have been achieved. To make the electrolytic method more effective it can favourably be combined with radiotherapy. This has been done experimentally with good results. It is likely that the intense inflammation around the chemical destruction creates a radio-sensitising effect. PMID- 7531023 TI - Clinical and experimental studies of anti-tumoural effects of electrochemical therapy (ECT) alone or in combination with chemotherapy. AB - The present experimental study shows that anticancer agents may accumulate around electrodes placed in saline and in lung when the electrodes are charged with direct electric current. We also observed that electrochemical therapy (ECT) in combination with chemotherapy was more effective for treatment of localized tumours (human lung cancer PC-13 transplated to nude mice) than was ECT or systemic chemotherapy alone. In a clinical study, 26 patients (27 lesions) received ECT with or without systemic chemotherapy. We observed a decrease in tumour size in more than 70% of the cases, and in two cases the use of ECT alone resulted in complete regression of the tumour. In one case there was an increase in tumour size. The main side-effects observed during treatment of lung cancer was pain during treatment, fever after treatment, and pneumothorax. We conclude that ECT may be effective in controlling localised tumours. The clinical use of ECT is, however, associated with several problems and the mechanism of this treatment has not yet been completely established. PMID- 7531025 TI - The paradigm of biologically closed electric circuits (BCEC) and the formation of an International Association (IABC) for BCEC systems. AB - Matter is condensed energy. Matter derived from electromagnetic energy, supplied with the electrically powered BCEC systems (biologically closed electric circuits) inherits prerequisites to become biological matter. This is possible because mechanisms of BCEC systems contain the capacity to initiate structuring and functioning of matter. The principle of BCEC systems, their actual and potential importance for structure and function in biology and medicine, has been presented only in the form of a survey. Only few examples of the impact of the BCEC systems have been described. For detailed information, the reader is advised to take part of the original background articles by means of the reference list, which extends beyond the selected descriptions. Thereby it is hoped that the reader may get an understanding of the central biological role of the BCEC systems. They represent in this partial theory of the biological evolution a key mechanism which may provide the important primary steps that are necessary for the transfer of non-biological into biological matter. Second, also other factors are evidently contributing to biological differentiation, including for instance the principle of differential selection of species. Due to their basic role, the BCEC systems can nevertheless be recognized to be involved in the majority of structural and functional expressions in biology. This rests on the fact that our physical world once developed from energy and specifically its representation of electric energy. It has also been emphasised that electric energy is equivalent to the remarkable Oriental concept of Qi ("life energy"). Differences are predominantly a matter of semantics. PMID- 7531026 TI - Experimental studies on electrolytic dosage of ECT for dog's oesophageal injury and clinical effects of ECT for oesophageal anastomotic opening stenosis and oesophageal carcinoma. AB - Experimental and clinical studies have been performed to explore the potential benefits of electrochemical therapy (ECT) in oesophageal cancer. In a dog experiment, the oesophageal injury induced by ECT was evaluated. The provision of 7.5 volts (V) and 25-100 coulomb (C) was associated with a slight injury of the mucosa, which was completely healed after two weeks. Ten patients with oesophageal stenosis were treated with ECT (4.5-5.5 V, 20-50 mA and 85-180 C). In all patients there was a significant dilatation of the stenotic area and the patients could eat a normal diet after three to four weeks. The clinical effectiveness in 35 patients treated with ECT for inoperable oesophagela carcinoma was found to be satisfactory. Complete and partial response was obtained in 15 cases (42.8%), and dysphagia was relieved for five to 13 months. In conclusion, oesophageal cancer may be successfully treated with a specially designed electrode and a specified dosage of electricity. PMID- 7531027 TI - Effect of electrochemical therapy on immune functions of normal and tumour bearing mice. AB - The effect of electrochemical therapy (ECT) on immune functions of normal and tumour-bearing mice was studied. In normal mice, ECT had no obvious effect on delayed type hypersensitivity (DTH) or on the mononuclear phagocytic system, but significantly reduced the production of agglutinin antibodies. In tumour-bearing mice, ECT enhanced the DTH reaction, increased the production of agglutinin antibodies and enhanced the clearance of Congo red. In conclusion, in tumour bearing mice, ECT enhanced both cellular immune functions of T- and B-lymphocytes and non-specific immune functions of the phagocytic system. PMID- 7531028 TI - Experimental study on electrochemical treatment of cancer in mice. AB - Electrochemical treatment of cancers in mice resulted in at least partial regression or disappearance of transplanted SRS solid tumours and increased the survival time. Six days after treatment, lymphocyte infiltration in the tumour tissue was found. Seventy-one days after treatment, tumour cells were rarely found in the tissue. The application of direct current was found to facilitate the destruction of tumour cells in a shear stress field. PMID- 7531029 TI - Electrochemical therapy (ECT) for thyroid adenoma during acupuncture anaesthesia: analysis of 46 patients. AB - Between February 1989 and April 1993, 46 patients with thyroid adenoma were treated by electrochemical treatment (ECT) during acupuncture anaesthesia. After three months to four years follow up period, the cure rate was 97.8%. ECT is a new technique which provides simple, effective and safe treatment of thyroid adenoma. Treatment of benign tumours with ECT represents a new application. PMID- 7531030 TI - Electrochemical therapy of pelvic pain: effects of pulsed electromagnetic fields (PEMF) on tissue trauma. AB - Unusually effective and long-lasting relief of pelvic pain of gynaecological origin has been obtained consistently by short exposures of affected areas to the application of a magnetic induction device producing short, sharp, magnetic-field pulses of a minimal amplitude to initiate the electrochemical phenomenon of electroporation within a 25 cm2 focal area. Treatments are short, fasting-acting, economical and in many instances have obviated surgery. This report describes typical cases such as dysmenorrhoea, endometriosis, ruptured ovarian cyst, acute lower urinary tract infection, post-operative haematoma, and persistent dyspareunia in which pulsed magnetic field treatment has not, in most cases, been supplemented by analgesic medication. Of 17 female patients presenting with a total of 20 episodes of pelvic pain, of which 11 episodes were acute, seven chronic and two acute as well as chronic, 16 patients representing 18 episodes (90%) experienced marked, even dramatic relief, while two patients representing two episodes reported less than complete pain relief. PMID- 7531031 TI - Dielectric spectroscopy of human blood. AB - Impedance measurement is an established technique for studying the passive electric properties of cell membranes. Dispersions can be detected by studying the electric properties (capacitance and conductance) in the radio-frequency range (kHz-mHz). The theoretical interpretation is based on the Maxwell-Wagner effect at the interface between the cytoplasm and the cell membrane. The specific electric variables of the membrane, the cytoplasm, as well as the surrounding plasma (medium) are estimated by non-linear regression fitting and appropriate equations. Using a four-electrode technique, we have measured the impedance with a commercial instrument working in the frequency range of 0.2-10 mHz interfaced to a computer. Differences were found in conductivity and capacitance of blood from 1) persons exposed to organic solvents, 2) patients with metal exposure, and 3) patients with cardiovascular disease. The effects of plasma components and haematocrit are crucial when undertaking measurements on whole blood. The results are difficult to interpret but we consider perturbations in the erythrocyte membrane to be involved. Potential clinical applications will be promoted by the development of the software. PMID- 7531033 TI - Survey of mechanisms in electrochemical treatment (ECT) of cancer. PMID- 7531032 TI - Electrostatic therapy (EST) of lung cancer and pulmonary metastasis: report of 15 cases. AB - Fifteen patients with primary lung cancer or pulmonary metastases were treated with electrostatic therapy (EST). The patients were treated with either low voltage (200 to 1200 V), high voltage (500 to 2500 V), or high voltage together with high magnetic field. At six months after EST, tumour regression was observed in two cases, relief of symptoms, but no change in tumour size, in five cases, and no effect of EST in nine patients. In conclusion, no marked effect of EST for treatment of cancer could be observed. PMID- 7531034 TI - The genetics of cystic fibrosis: a paradigm for uncovering new drug targets. AB - A broad-based approach will be required for the development of new therapies for cystic fibrosis lung disease. Recently, rapid progress has been made in identifying and testing a number of gene transfer vectors, including adenoviral vectors and liposomes. Major problems, however, have been identified with respect to the efficiency of these systems. Preliminary studies suggest that small molecules (e.g. amelioride and UTP) may normalize the clearance of secretions from the cystic fibrosis lung. The concept of recombinant protein based therapy for cystic fibrosis has now been realized with the successful application of DNase in clinical trials. PMID- 7531035 TI - Three-dimensional reconstructions of autonomic projections to the gastrointestinal tract. AB - Three-dimensional reconstruction protocols in confocal microscopy are typically considered in terms of rendering separate stacks of optical sections. Single stacks, however, include volumes that are often too small to permit descriptions of entire neurons, complete axonal arbors, or complex neural networks. Furthermore, traditional tissue preparation protocols generally yield specimens too limited to permit reconstructions of complex neural systems. For 3-D analyses of extensive networks such as the autonomic nervous system projections within the viscera, it is critical to incorporate appropriate tissue techniques, including suitable tracer protocols, into the reconstruction strategy. This report summarizes complementary technologies, including whole mount procedures, tracer techniques for identifying single fibers in situ, and methods of examining stacks of optical images, which make it practical to describe the complete terminal field of an individual axon in the gastrointestinal tract. Such methods establish that vagal motor axons travel long distances within their target organs, collateralize frequently, and ramify extensively. Vagal afferents have extensive, complex, and, in some cases, polytopic arbors within target tissues. PMID- 7531036 TI - Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane. AB - The kit ligand (KL) is one of several growth factors that are active as transmembrane molecules and can also be proteolytically cleaved to yield soluble forms. We have investigated the signals and structural determinants that control the cleavage of KL. Presentation at the membrane appears to be critical, because no cleavage occurs in variants that lack a transmembrane domain. Signals in the cytoplasmic domain do not seem to be required, because cleavage was not blocked by removal of the C-terminal valine residue, deletion of the whole cytoplasmic tail, or the replacement of the cytoplasmic tail that occurs in the Sl17H mutation. KL thus appears to differ from transforming growth factor-alpha, which apparently requires a C-terminal valine as a signal for cleavage. Although proteolysis must be tightly restricted to the correct cell surface proteins and sites within each protein, cleavage of KL does not seem to be determined entirely by a requirement for a specific substrate sequence. However, the effects of deletion or insertion variants of KL suggest that cleavage may be limited to sites within a specific range of distances from the cell membrane. PMID- 7531037 TI - Parental alcoholism and early child development. AB - In a cohort of 532 pregnant women from the general population, it was found by compilation of the results from interviews, police records, hospital records and social welfare records that 23 mothers and 51 fathers in 64 families (12%) were suffering from alcoholism/heavy drinking. In these 64 families, the mother was an addict in only 13 families, both parents were addicts in 10 families, and in the remaining 41 families only the father was an addict. Pregnancy, delivery, the newborn child and the child's development until their fourth year of life have been described using a multidisciplinary approach and a longitudinal prospective design. An hypothesis on mental and physical development, and the occurrence of psychopathological symptoms in the children was tested. None of the children of the 13 alcoholic mothers was born with foetal alcohol syndrome, but foetal hazard was indicated by lower birth weight and a higher rate of perinatal deaths. Children of alcoholic parents had retarded mental development and showed more behavioural problems until 4 years of age than controls, but the differences related to physical development during the first year of life had then disappeared. Boys were found to be more vulnerable than girls. The consequences of behaviour seemed to be more pronounced when both parents were alcoholics. No obvious deviation was found when only the father was addicted. Regarding mental development, it appears that factors related to parental alcoholism, including genetic and social factors, and the sex of the child, are of greater importance than the neonatal score on reduced optimality. PMID- 7531038 TI - The fetal alcohol syndrome in adolescence. AB - At present, alcohol is recognized as the leading teratogenic agent in long lasting CNS dysfunction. Little is known about the long-term development and outcome of children with fetal alcohol syndrome (FAS). Forty-four FAS patients who were diagnosed in early childhood were followed up for 10-14 years. This study documents the developmental changes of the manifestations of FAS from childhood to adolescence and describes a characteristic "juvenile" pattern of FAS, which may help to identify this syndrome even in adolescence. This is especially relevant for patients who were not diagnosed earlier. PMID- 7531039 TI - Amphetamine addiction during pregnancy: 10-year follow-up. AB - Sixty-five children born to women who all used amphetamine during pregnancy have been followed prospectively since birth 1976-77. They have been tested and examined at one, 4 and 8 years of age. In addition, information about the school situation was collected after 10 years. Twenty percent of the children were put in foster homes at birth, and after 10 years 70% were in foster homes. The children showed a normal intellectual capacity. However, at 10 years of age 8 children (12%) attended one class below what was normal for their biological age (normal in Sweden < 5%). At 4 years of age 35% had an aggressive behaviour and at 8 years 23%. There was a statistically significant correlation between aggressive behaviour and amphetamine exposure during fetal life. At both 4 and 8 years of age 40% were considered to have a poor adjustment, which correlated with socio environmental factors. Lower values for weight and length also showed a correlation with socio-environmental factors. The study shows that amphetamine abuse during pregnancy will influence development of exposed children at least up to the age of 10 years. PMID- 7531041 TI - Developmental outcomes of children of mothers dependent on heroin or heroin/methadone during pregnancy. AB - Developmental outcomes of infants of drug-dependent mothers (IDDM) who used heroin during preganncy, alone or in combination with methadone, were unfavorable compared with outcomes of infants of mothers who were not drug dependent or infants in the general population. Unfavorable items for IDDM were: gestational age and birth weight, physical growth, neurological development, intelligence, behavior, social competences and social state (foster care). Prenatal care with social support and methadone substitution were critical factors for IDDM. The IDDM in foster care appeared to exhibit even more unfavorable outcomes than those living with their biological parents. PMID- 7531040 TI - Cocaine use in pregnancy in Amsterdam. AB - To study the effects of cocaine use in pregnancy in Amsterdam, clinical data on cocaine-using pregnant women (n = 21) and their offspring (n = 23) were obtained retrospectively (1987-1994) at the Academic Medical Center, Amsterdam. Infants exposed to cocaine had a median gestational age of 39 weeks and a median birth weight of 3090 g. There were six preterm infants, two small-for-gestational-age infants and five infants with a small head circumference. Three infants had a congenital malformation. One infant (Potter's syndrome) died shortly before birth. One infant had congenital syphilis, four had intracerebral abnormalities on ultrasound and four had abnormal neurologic symptoms in the neonatal period. One infant died after 21 days of life. At follow-up four infants showed abnormal development. In 12 of the 23 infants (52%), one or more possible effects of cocaine were found. PMID- 7531042 TI - Development after prenatal exposure to cocaine, heroin and methadone. AB - In Amsterdam a longitudinal, prospective and multidisciplinary study on the development of infants of drug-dependent mothers (IDDM) was started in 1983: 35 IDDM and 35 reference infants were originally enrolled. The drug-dependent women had used combinations of methadone, heroin, cocaine and other drugs during pregnancy. Of the IDDM, 80% had to be treated pharmaceutically for neonatal abstinence symptoms (NAS). Physical, neurological, cognitive and the socio emotional development of the children were studied regularly from birth until 5.5 years of age. Differences between the reference group and the IDDM were found most clearly in cognitive development. The IDDM also had more behavioural problems at some of the ages studied. No group differences were seen in motor development. So far the results of the study show that IDDM and their caregivers need extra support in order to improve early communication and the children's cognitive development. PMID- 7531043 TI - Studies on long-lasting consequences of prenatal exposure to anticonvulsant drugs. AB - Based on neonatal examination at birth, it has been estimated that epileptic women have a 2-3 times greater risk of giving birth to an infant with congenital anomalies. But anticonvulsant drugs may also have more subtle influences on the developing foetus which are not visible at birth but only emerge later in life. Evidence for these functional teratogenic influences has been provided by animal research and follow-up studies in young children. This article discusses these findings in human and animal studies. In addition, the outline of a study carried out at the Department of Obstetrics and Neonatology, Academic Medical Centre, Amsterdam, is described. In this study cognitive functioning, fertility and gender role behaviour of young adults, who had been prenatally exposed to barbiturates and/or hydantoins was examined. PMID- 7531044 TI - Treatment effects in Trichuris dysentery syndrome. AB - Heavy infection with the geohelminth Trichuris trichiura causes the Trichuris dysentery syndrome (TDS). Growth retardation and anaemia are characteristic of TDS and both are associated with poor development. We have examined the growth and developmental responses to treatment in 19 children aged 27-84 months with TDS. Developmental levels (DQ) were measured with the Griffiths mental development scales. Compared with a control group matched for age, gender and neighbourhood, the TDS children initially had serious deficits in DQ (24 points, p < 0.001). After a year of anthelmintic treatment, the TDS children showed improvement in locomotor development (p < 0.001) compared with the controls. The TDS children also had initial deficits in height-for-age, weight-for-height, mid upper arm circumference and haemoglobin levels. They caught up rapidly in indices of wasting (weight-for-height and mid-upper arm circumference) and showed steady improvement in height-for-age and haemoglobin levels. Catch-up in height was comparable to that of children recovering from coeliac disease. The importance of continuing prevention after initial treatment is highlighted. PMID- 7531045 TI - School performance, nutritional status and trichuriasis in Jamaican schoolchildren. AB - Severe infections by the geohelminth Trichuris trichiura detrimentally affect young children's growth and development. There is concern that mild to moderate infections may affect older children's school performance and nutritional status. We therefore examined the relationship between varying intensities of infection and school achievement, attendance and nutritional status in 616 schoolchildren. A total of 409 children with Trichuris infection of intensities greater than 1200 eggs per gram of stool (epg) were first identified, then for every 2 infected children in a class, an uninfected child was selected. After controlling for socioeconomic status, gender, age, school and the presence of Ascaris infections, the uninfected children had higher reading and arithmetic scores than children with infections of more than 4000 epg and were taller than those with intensities greater than 2000 epg. However, there were no significant differences in spelling, school attendance and body mass index. Although a treatment trial is needed to determine causation, these results indicate that moderate levels of infection are associated with poor school achievement and growth. PMID- 7531046 TI - [New diagnostic developments in infection serology]. AB - New developments concerning the diagnostic of virus-transmitted diseases led to a strong improvement of virus security in hemotherapy. The introduction of additional virus antigens associated with the core region of the hepatitis C virus (HCV) resulted in an increase of sensitivity and especially of specificity of second-generation enzyme immuno assays (EIAs). The recombinant immunoblot (RIBA) with 4 synthetic antigens continues to be the most accepted confirmatory test in the field of HCV diagnostic. Beside the simultaneous detection of specific IgG and IgM, especially the HIV-p24 antigen test is discussed concerning HIV-1, -2 diagnostic in order to shorten the 'windows period' in the early time of infection. It is assumed that the HIV-p24 antigen test is not able to effectively improve virus security in areas with low HIV-1, -2 prevalence. PMID- 7531047 TI - [Erythropoietin]. AB - The production of red cells can be stimulated by pharmacologic doses of recombinant human erythropoietin (rHu-EPO), provided EPO-sensitive precursors and iron are available. Its side effects are negligible when used in patients with nonrenal anemia. Antibodies against rHu EPO are a rare event. Iron supplementation is routinely necessary in patients with low iron stores, since availability of iron is a rate-limiting cofactor for red cell production. The rationale for treating patients with anemia of cancer or chronic inflammation is to avoid homologous blood transfusion. However, it is not proven whether a raising of the hemoglobin concentration by 2 or 3 g% will improve the quality of life in these multimorbid patients who undergo palliative treatment. There is no evidence yet that rHu-EPO has reduced morbidity and mortality in such patients. Another question is the cost effectiveness of EPO particularly in patients who suffer from an incurable disease. EPO has also been used as an adjuvant in autologous preoperative transfusion programs and has increased the available volume of red cells for transfusion particularly in conjunction with intravenous iron supplementation. EPO given for 14 days preoperatively in patients with elective hip replacement reduced the need for transfusion by nearly 50%. A high dose of oral ferrous sulfate (300 mg) was given 11 days in advance of rHu EPO. Randomized trials are needed for patients with an initial low Hb (< 13.5 g/dl) to study the efficacy and cost effectiveness of different strategies avoiding homologous transfusion and also the risk-benefit ratio of such strategies versus homologous transfusions, since the risk of homologous transfusions has decreased considerably in recent years. PMID- 7531048 TI - [Colony stimulating factors in blood formation]. AB - The aim of this article is to review the present state of knowledge of the clinical effects of the colony-stimulating factors. Clinical experience and publications on this subject are used for this purpose. Colony-stimulating factors are being successfully used in various diseases and clinical situations, particularly in the area of hematology/oncology. However, numerous questions still remain. There are many new untested areas in which filgrastim could be employed. New indications and open questions require suitable new studies. PMID- 7531049 TI - Bovine and rodent tamm-horsfall protein (THP) genes: cloning, structural analysis, and promoter identification. AB - We have isolated bovine and rodent cDNA and genomic clones encoding the kidney specific Tamm-Horsfall protein (THP). In both species the gene contains 11 exons, the first of which is noncoding. Exon/intron junctions were analyzed and all were shown to follow the AG/GT rule. A kidney-specific DNase I hypersensitive site was mapped onto a rodent genomic fragment for which the sequence is highly conserved in three species (rat, cow, and human) over a stretch of 350 base pairs. Primer extension and RNase protection analysis identified a transcription start site at the 3' end of this conserved region. A TATA box is located at 32 nucleotides upstream of the start site in the bovine gene and 34 nucleotides upstream in the rodent gene. An inverted CCAAT motif occurs at 65 and 66 nucleotides upstream of the start site in the bovine and rodent genes, respectively. Other highly conserved regions were noted in this 350 bp region and these are likely to be binding sites for transcription factors. A functional assay based on an in vitro transcription system confirmed that the conserved region is an RNA Pol II promoter. The in vitro system accurately initiated transcription from the in vivo start site and was highly sensitive to inhibition by alpha-amanitin at a concentration of 2.5 micrograms/ml. These studies set the stage for the further definition of cis-acting sequences and trans-factors regulating expression of the THP gene, a model for kidney-specific gene expression. PMID- 7531050 TI - Species-specific expression of sialosyl-Le(x) on polymorphonuclear leukocytes (PMN), in relation to selectin-dependent PMN responses. AB - Sialosyl-Le(x) (SLex) and its positional isomer sialosyl-Le(a) are the epitopes recognized by the lectin domain of E- and P-selectins. Expression of SLex in polymorphonuclear leukocytes (PMN) plays an important role in recruitment of these cells at sites of inflammation through activation of selectins. We studied expression of SLex in PMN of seven mammalian species in comparison with that in humans. Only PMN of humans (no other species) expressed SLex or other lacto series epitopes such as Le(x) or Le(y). The observed absence of these epitopes in rat PMN seems inconsistent with recent reports that the lung inflammation process in a rat model is inhibited by perfusion of SLex oligosaccharide (Mulligan MS, et al. (1993a) Nature 364:149; (1993b) J Exp Med 178:623). Rat selectins may be able to recognize SLex, even though this epitope is absent in rat PMN. PMID- 7531052 TI - Cell line-dependent changes in sensitivity to mitomycin C by 1-methyl-3 isobutylxanthine is due to an altered intracellular dose of the anticancer drug and/or to changes in DT-diaphorase activity. AB - Sensitization by 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, of cellular sensitivity to mitomycin C (MC) was tested using various cell lines. Sensitization was seen with CHO cells and Balb/c 3T3 cells, but a decrease in sensitivity was seen with HeLa, K-balb and other cell lines. To measure the extent of crosslinks produced by MC, we used an alkaline elution assay. The extent of crosslinks was increased by MIX in CHO cells and decreased in K-balb cells. These changes in extent are well reflected by changes in sensitivity to MC in both cell lines. In CHO cells, an intracellular dose of MC was increased by MIX with no change in the rate of uptake or efflux of MC. There was a slight decrease in the dose in HeLa or K-balb cells. Among the enzymes which engage in the reductive activation of MC, we tested DT-diaphorase and NADPH cytochrome P450 reductase using CHO, HeLa and K-balb cells. MIX had almost no effect on the activity of NADPH-cytochrome P450 in each cell line, whereas it suppressed the activity of DT-diaphorase, significantly in HeLa and K-balb cells, and slightly in CHO cells. All these facts indicate that MIX caused an increase in the extent of crosslinks via an increase in the intracellular dose of MC in CHO cells, and that these increases may lead to the sensitization. On the other hand, the decrease in the sensitivity shown in HeLa and K-balb cells may be due to the suppression of DT-diaphorase activity and/or to a decrease in MC dose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531051 TI - Molecular mechanisms of immunosuppressive agents. AB - Recent data on the molecular mechanism of some immunosuppressive drugs provide strong support for the fascinating postulate that CSA and FK506 work by binding to immunophilins and then, as a drug-immunophilin complex, inhibiting the calcium activated protein phosphatase, calcineurin. This inhibition could result in an altered modification pattern of the cytoplasmic components of transcription factors, thereby disturbing their nuclear translocation, which is a prerequisite for proper IL-2 transcription. It looks as if, with the immunosuppressive microbial metabolites as molecular probes, the pieces of this complex signal transduction puzzle are starting to fit together! Once the details of the chain of events along the T-cell signaling pathways are known, the molecular structures involved will provide new tools to be used in the search for and the rational design of new and improved therapeutic agents. PMID- 7531053 TI - Tissue distribution of macromolecular conjugate, adriamycin linked to oxidized dextran, in rat and mouse bearing tumor cells. AB - Tissue distribution of the radioactivities after intravenous administration of [14C]adriamycin ([14C]ADM) or [14C]ADM linked to oxidized dextran ([14C]ADM-OXD) in mouse bearing Lewis lung carcinoma (LLC) and rat bearing Walker 256 carcinosarcoma was studied. ADM conjugated with OXD increased plasma half-life and gave high area under the plasma concentration-time curve (AUC). The AUC values were 13.0 and 5.8 times higher than those of the [14C]ADM group in mice and rats, respectively. In the tumor tissues, AUC values of the [14C]ADM-OXD group were also respectively 1.6 and 1.9 times higher than those of the [14C]ADM group. However, the AUC values in the heart of the [14C]ADM-OXD group were about half those of [14C]ADM group in both animals. Thus the distribution of ADM was changed by the conjugation with OXD. The excretion profile of ADM was also changed by the conjugation. During 6 h after administration, [14C]ADM-OXD was mainly excreted into rat urine at 45.2% of the original dose, but in the [14C]ADM group recovery in urinary excretion was 4.2%. Using [14C]ADM-OXD and ADM [14C]OXD, the respective tissue distribution of ADM and OXD portions in the ADM OXD was studied in rats bearing Walker 256. The radioactivities of both [14C]ADM OXD and ADM-[14C]OXD groups increased in tumor and liver within 1 h after administration. In the liver, both radioactivities were retained for 24 h, which suggested that ADM and OXD were retained as conjugated form, however, different behavior was observed between the two groups in tumor tissues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531055 TI - Ion channel shake-down. PMID- 7531056 TI - Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. AB - The earliest genetic alteration in human astrocytoma progression is mutation of the p53 tumour suppressor gene, while one of the earliest phenotypic changes is the stimulation of neovascularization. Here, we tested the role of p53 in the angiogenic process by introducing a tetracycline-regulated wild type p53 gene into null glioblastoma cells. The parental cells expressed strong angiogenic activity while upon induction of wild type, but not mutant, p53 expression, the cells secreted a factor able to neutralize the angiogenicity of the factors produced by the parental cells as well as of basic fibroblast growth factor. PMID- 7531054 TI - Approaches to the analysis of quality of life data: experiences gained from a medical research council lung cancer working party palliative chemotherapy trial. AB - Standardization in the choice of quality of life (QOL) instruments and their application in randomised clinical trials have been advocated and generally accepted. However, there is now an urgent need to address the problems relating to the analysis and presentation of the data thus generated. There are intrinsic difficulties associated with QOL data, namely its multidimensional nature, attrition and missing data, and there is no consensus as to how these problems should be dealt with. This paper therefore considers these problems using interim data from a large Medical Research Council randomised trial in patients with small cell lung cancer and a poor prognosis, in which attrition and compliance are major concerns. Three possible approaches to the analysis of these data, which use different subsets of patients, are examined in detail. The strengths and weaknesses of these three methods are discussed, and examples of their use in the literature are given and compared with other reported approaches. The need for a standard definition of compliance is also emphasised, and a method of presentation suggested. The best current advice is that QOL data should be analysed in a number of different ways, and conclusions reached only when consistency is seen. PMID- 7531057 TI - [The role of the information diversity of physiological processes in the development of the adaptation and correction of the body's physiological status]. PMID- 7531058 TI - [The synaptic inputs of the neurons in the cat parietal associative cortex from the motor cortical area]. AB - In anesthetised cats, antidromic activation as well as mono- and polysynaptic EPSPs to cortical motor stimulation, were shown in the parietal cortex's neurons. Functional significance of the cortico-cortical reciprocal connexion between the motor cortex and the parietal associative cortex, is discussed. PMID- 7531059 TI - [Immunophysiology as a new scientific discipline]. AB - The history of development of immunophysiology in Russia as a new branch of science, is described. New mechanisms of stress-induced immunological disorders were shown: definite effect of stress on immunogenesis in the form of cGMF lymphocytes responses to an antigen, activity of the T-suppressors, etc. The data obtained suggests a correlation between the type of stress-induced derivation of the IL-1 level the LAF activity. A 35-year long experience in immunophysiology makes it possible to define the latter as a new scientific discipline. PMID- 7531060 TI - [Feedback in the synapse realized by K+ ions. The research results]. PMID- 7531062 TI - [The role of the cholinergic system in regulating body functions]. PMID- 7531061 TI - [The role of oxytocin in forming the asymmetry of the rostral structures of the rat brain under osmotic stress]. PMID- 7531063 TI - [The problem of stimulus quality in the physiology of higher nervous activity]. PMID- 7531065 TI - [Alternative biocontrol as a psychophysiological mechanism of functional adaptive self-regulation]. AB - Study of voluntary autoregulation of the heart rate (HR) by means of artificial biofeedback (BFB) using a display, has revealed the possibility of changing the HR voluntarily within a wide range of (from 50 to 140 beats per minute). Respective fluctuations occurred in the arterial pressure. A decrease in the HR and reactive alarm, increase in the self-assessment of physical state, activity, mood and work level occurred in result of the HR-BFB training. PMID- 7531064 TI - [The mechanisms of individual adaptivity to psychoneural loads in different pathological states of the circulatory system]. AB - In patients with ischemic cardiac disease after myocardial infarction, characteristics of psychophysiological, neurodynamic and vegetative regulation levels were shown. The levels support the optimal adaptability in conditions of cardiovascular pathology. PMID- 7531067 TI - [The development of human ecological physiology at high latitudes]. AB - Regional specific features of human specific adaptation to stressful performances under Arctic and Antarctic conditions, are analysed in respect to its central and peripheral regulating mechanisms. New concepts of the adaptation process are presented. PMID- 7531068 TI - [The physiological aspects of individual human eco-adaptation]. AB - These data obtained shows the typological pattern of the brain neurodynamics to be related to functional self-controlling mechanisms as reflected in probable structure of the interlinks among the EEG basic rhythms. Three types were singled out with respective dynamics of the functional state readjustment under a prolonged effect of ambient factors. PMID- 7531066 TI - [Intersystemic functional integration under the action on the body of electromagnetic factors]. AB - Frequency zones of the brain electrical activity, heart rate and respiration were studied through certain parameters characterised by individual asymmetry and low but stable intersystemic integration. Chronic electromagnetic irradiation reduced a resistance against an emotional stress as manifested in a "decay" of individual spatial-temporal infrastructure of informational parameters of the EEG, heart rate and respiration. PMID- 7531069 TI - [The adaptation of the human central nervous system to ecological job factors]. AB - The EEG, quantity of cation proteins in neutrophils, the opioid peptides concentration in the CSF, were analysed in military personnel after a prolonged climatic and specific stress complicated by traumas, at the Kabul military hospital in Afghanistan. The brain functional reserve was significantly lower in the control subjects. A positive correlation between the brain functional reserve and the average cytochemical coefficient, was found in certain phases of traumatic disease alone. Accumulation of opioid peptides occurred under the adaptation to stress, being more evident in wounded soldiers. PMID- 7531071 TI - [The role of the ventral structures of the medulla oblongata in regulating the resistive functions of organ arterial vessels]. AB - In the rostral part of medullary ventral areas (MVA) both the pace-maker neurons forming the tonic sympathetic activity, and the relay structures integrating the baroreceptor information on its way towards vascular areas, are located. The structures of the caudal portion of this cerebral area exert a depressing effect upon the activity of neuronal pools of the brain rostral part and modulate own baroreflexes. An obvious relationship was revealed between the character the extension of changes in the resistive function of the intestine and skeletal muscles' vessels, on the one hand, and the functional state of the MVA structures (excitation or inhibition), on the other hand. PMID- 7531072 TI - [The degree of functional system integration as an indicator of human health status]. PMID- 7531070 TI - [The epileptiform reactions of the human brain to prolonged exposure to low-dose ionizing radiation]. AB - 1125 inhabitants of Slavutich city where workers of the Chernobyl Atomic Power Station and their families live, have been electroencephalographically examined. The reasons for the medical check-up involved specific complaints of a headache, dizziness, nightmares, early waking up in the mornings and sometimes seizures. Disorders of the bioelectrical activity of paroxysmal nature were revealed in 286 cases (197 children of 3-17 years, 99 adults of 18-50 years). The epileptiform findings seem to be related to the ionising radiation. The latency for a high (single or repeated) dose may be short (or minimal), and for a long-term effect of small doses--3-6 years. The latency was more or less similar and depended on a number of biophysical factors for the majority of the examined inhabitants of Slavutich city: children as well as adults (including those who work at the Chernobyl Atomic Power Station). PMID- 7531074 TI - [The principles of studying ion transport in the kidney tubules]. AB - The present overview indicates that the various nephron segments take part in the Na+Cl- reabsorption, the primary driving force for it is the (Na+, K+)-ATPase, which is localized in the basolateral membrane. The various segments have different modalities of Na+ uptake: in is by Na+/H+ exchange in the proximal tubule, by Na+/2Cl-/K+ co-transport in TAL, Na+Cl- cotransport in the distal tubule, and via Na+ channels in the principal cell of the collecting duct. In the proximal tubule bulk reabsorption occurs, but very small ionic gradients are built up, there for the transport here is so economical. In the TAL transport is already less economical, however ionic gradients are built up by this nephron segment inasmuch as Na+Cl- is reabsorbed and water cannot follow (the urinary concentrating mechanism). The distal tubule is concerned with defined control of Na+ and K+ excretion. Transport at this side is expensive, but very steep ionic gradients can be built up. The control is mediated by several hormones amongst which ADH and aldosterone are the most important ones. PMID- 7531075 TI - [A standard nomenclature for structures of the kidney. The Renal Commission of the International Union of Physiological Sciences]. PMID- 7531073 TI - [The effect of divalent cations on the kinetics of the activation and blockade of the nicotinic cholinoreceptor ion channel in rat skeletal muscle]. AB - The influence of alkaline earth metal ions calcium and magnesium on the conductance and on the kinetics of activation and atropine-induced blockade of nicotinic cholinoreceptor ion channels in cultured rat skeletal muscle were studied using patch-clamp technique. In physiological concentrations these cations influence both conductance and kinetics of blockade, but have no significant effect on the kinetics parameters of activation. The data suggest that physiological cations modulate the generation of postsynaptic potentials in the normal condition as well as in the presence of blocking drugs. PMID- 7531076 TI - [The nature of the thermomechanical reactions of the vascular wall]. AB - Thermosensitivity of the guinea pig aorta wall stretched to 1.05-1.40 of its initial length, was shown to completely depend on the connective tissue: the thermomechanical responses of the stretched aorta entailed changes in its stiffness typical for an agonist-induced vessel tissue contraction. The connective tissue component of the mechanical response of the vascular wall seems to have a universal character. PMID- 7531077 TI - [Patch-clamp methods in research on the kidney tubules]. AB - Patch-clamp studies in renal tubule cell have been very helpful in increasing our understanding of the tubule transport function. However, it has been a disappointing experience that the properties of cultured cells deviate grossly from the physiology of the intact tubule segment. Also, several important conductive pathways have not been characterized thus far on single channel level. Several ion channels, which have been found in the patch-clamp studies, such as the non-selective cation channel, do not find their equivalent in the intact tubule preparation. Therefore, much more efforts are required not only to characterize individual ion channels, but rather to integrate this knowledge for the understanding of tubule function. PMID- 7531079 TI - [The strategy for research on tubular transport in the kidney]. AB - The present publication summarises the experimental strategies that have been developed to identify, localize and characterize renal tubular transport mechanisms on the nephron, cellular and molecular level. 1) Identification and localization of transporters can be achieved on single ne (mRNA hybridization) techniques. 2) The functional properties of transporters and their regulation can be investigated on individual nephron segments in vivo or in vitro, on cell cultures, isolated cells, or on isolated membrane vesicles, applying different analysis techniques: net flux and tracer take-up studies, microscopic volume determinations, microelectrode measurements (conventional and ion-selective), patch-clamp measurements, microspectrofluorometry, X-ray microanalysis of frozen tissues, nuclear magnetic resonance, etc. 3) The primary structure (amino acid sequence) of transport proteins can be determined by protein purification with subsequent amino acid sequencing. More powerful, however, is expression cloning, e.g. screening of cDNA libraries for expression of a given transport protein in a model cell system (e. g. Xenopus laevis oocytes) with subsequent base sequence analysis. 4) The molecular function of transport properties can best be studied in heterologous expression experiments (overexpression, easy technical, e. g. in oocytes). This also allows monomeric and multimeric transporters to be distinguished and is a prerequisite for mutagenic studies in which the function of individual amino acid constituents of the protein can be determined. Such studies eventually allow detailed molecular models of transport function to be developed. PMID- 7531078 TI - [The regulation of proximal tubule function]. AB - This paper deals with the recent concept that the effectiveness of coupling between filtration and reabsorption is influenced by local and circulating hormones and by renal sympathetic nerves. The evidence supports a view of the proximal tubule as being an important regulator of extracellular fluid composition and blood pressure. PMID- 7531080 TI - [The regulation of renin synthesis]. AB - Secretion and synthesis of renin are stimulated by the lack of angiotensin II and by a low sodium intake. They are inhibited by a high sodium intake, by denervation and by propranolol. A high sodium intake can overcome the effect of the lack of angiotensin II. A low sodium balance and the lack of angiotensin II did not have additive effects. The sodium effects did not appear to depend on the presence of the macula densa, the presence of angiotensin II, or the presence of the nerve supply. It was not evaluated whether baroreceptors may be important in allowing sodium effects to be processed, but in the experiments conducted there were no significant changes in blood pressure associated with alterations in sodium balance. Sodium balance has an important effect on synthesis and secretion. This effect does not appear to be mediated by the usual set of controls and raises questions as to the signal relating sodium balance to renin synthesis. Secretion does appear to depend on macula densa. The role of the macula densa appears to control the basal level of renin secretion and to permit the conversion and processing of prorenin to renin. PMID- 7531081 TI - [The molecular physiology of the kidney and the mechanisms integrating its functions]. PMID- 7531082 TI - [Osmoregulation--integrative control of water and sodium balance]. PMID- 7531083 TI - [The renal renin-angiotensin system and its contribution to the regulation of glomerular hemodynamics]. AB - It has been demonstrated in whole kidney and micropuncture studies that angiotensin II, either delivered systemically or formed locally, has a crucial role in the regulation of both afferent and efferent arteriolar resistances throughout the cortex. The angiotensin induced constriction on preglomerular arterioles has been further confirmed by direct assessments of segmental vascular responses during systemic or topical administration of the peptide. The preglomerular vasoconstrictor effects of the peptide is not restricted only to the afferent arteriole. Angiotensin II formed even beyond the glomerular circulation in the postglomerular capillary and/or interstitial environment can reduce glomerular and proximal tubular function. Recent findings on intrarenal angiotensin formation i. e. the existence and response of angiotensinogen mRNA to different experimental and clinical conditions, the high intrarenal conversion of angiotensin I to II, the higher concentration of angiotensin I in the blood than that of angiotensin II etc., all support the idea that the effects of angiotensin II on the renal hemodynamics may be mainly due to the effects of the locally formed peptide. Therefore it is especially important to emphasize that there are some differences observed between the effects of angiotensin II formed intrarenally versus delivered systemically on renal hemodynamics. PMID- 7531084 TI - [The kidney and hypertension]. AB - The kidney and hypertension are critically linked. They appear to be linked particularly by the renin angiotensin system and the control of sodium balance. There are other mechanisms by which the kidney controls hypertension, but there are not as important as the above. In people with intrinsic renal disease hypertension increases the rate of deterioration and such hypertension should be treated energetically to prevent this deterioration. Long-standing chronic mild impairment of renal function leads to hypertension and vascular disease. This hypertension and vascular disease causes further deterioration of renal function, setting up a vicious cycle and an irreversible process unless measures are taken to interrupt the cycle. PMID- 7531086 TI - [The functional characteristics of Gs-protein in the developing mammalian kidney]. AB - Characteristics of G-proteins were studied in renal medulla of rats and mice of different ages. It was established that G-protein alpha-subunit gene expression has a specific dynamics with maximum on the 5-8-th day of life and at the end of weaning. It was shown that vasopressin does not stimulate G-protein GTPase activity in membrane preparations from immature kidneys. The differences of G protein chromatographic characteristics were revealed. It is suggested that the development of vasopressin sensitivity of the kidney is related with the changes in G-protein system. PMID- 7531085 TI - [The activity of erythrocyte membrane enzymes in different stressor exposures]. AB - The decrease in activity of acetylcholinesterase and Na,K-ATPase of erythrocyte membranes was found to be the result of stress effects. These decrease may be concerned with the changes of ratio of various erythrocyte population the appearance in blood "old" cells having 1.8-2.0 times lower activity of membrane bound enzymes. The specific digoxin-like inhibitors of Na,K-ATPase was revealed in blood, which also contribute to the inhibition of the enzyme activity. For correct interpretation of the reason of changes in activity of erythrocyte membrane enzymes in stress conditions the complex of physiological changes on the level of the whole organisms has to be taken into account. PMID- 7531087 TI - [The metabolism of heavy metals depends on the vitamin-D status of the body]. AB - The effect of vitamin D3 and its two metabolites 1,25(OH)2D3 and 24,25(OH)2D3 on the metabolism of heavy metals--Sr, Pb and Cd was studied. The experiments were carried out on chickens, the heavy metals were added to the chicken's ration. The results obtained demonstrated that vitamin D3 caused accumulation of those metals in tissues and their toxicity in organisms. When increasing the vitamin dose from 200 to 500 IU on 1 kg of ration that tendency was being heightened. On the three steroids which had been studied the metabolite 1,25(OH)2D3 displayed the greatest activity in accumulating metals in tissues, Pb in particular. The effect of 24,25(OH)2D3 on the indices being studied was comparable with that of vitamin D3. PMID- 7531088 TI - [The blockade of glutaminergic and cholinergic ion channels by adamantane derivatives]. AB - It has been shown that a homologous series of adamantane derivatives of general structure Ad-CH2-N+H2-(CH2)5-N+R3, where Ad, adamantane, R varied from H (hydrogen) to t-Bu (tertiary butyl), blocks the open state of postsynaptic activated channels. In the presence of the drugs studied the decay of evoked cholinergic postsynaptic currents in frog neuromuscular junction could be fitted by two exponentials. However, the rate constants of interaction of blocker with channel did not depend on the R structure and membrane potential. The rate of blockade of glutamatergic postsynaptic currents in insect neuromuscular junction increased as the radicals at nitrogen atom became heavier, but was independent on membrane potential. The drugs studied affected in voltage dependent manner the kinetic properties of single channels (recorded in outside-out patches excised from cultured neurones of embryonic rat brain cortex) induced by NMDA application. Each of these compounds evoked fast flickering of single channels between an open and blocked state. Drugs effectively prevented the convulsions evoked by intraventricular injection of NMDA into mouse brain. The compound IEM 1754 that was the most potent blocker in the experiments on NMDA-activated single channels possessed six times higher anticonvulsant activity than dizocilpine (MK 801). PMID- 7531090 TI - Unpasteurised milk and Streptococcus zooepidemicus. PMID- 7531089 TI - Legionnaires' disease--Edinburgh. PMID- 7531091 TI - British Paediatric Surveillance Unit: annual report for 1993. PMID- 7531092 TI - Cell properties in the epileptic hippocampus. PMID- 7531093 TI - Principal cells are the postsynaptic targets of supramammillary afferents in the hippocampus of the rat. AB - Neurons of the supramammillary nucleus are known to fire phase-locked to hippocampal theta rhythm. Stimulation of this area induces theta activity in the hippocampus via the medial septum and facilitates perforant pathway stimulation evoked population spikes in the dentate gyrus even if the medial septum is inactivated. This latter effect was suggested to be due to a direct inhibitory input from the supramammilary nucleus to hippocampal nonpyramidal cells resulting in disinhibition. In the present study, using anterograde tracing with Phaseolus vulgaris leucoagglutinin, we aimed to identify the types of neurons innervated by the supramammillary projection in the dentate gyrus and Ammons horn, with particular attention to the presumed postsynaptic inhibitory neurons, which may mediate the proposed disinhibitory action. Double-immunostaining for the tracer and different neuropeptides (somatostatin, cholecystokinin, neuropeptide Y) or calcium binding proteins (calretinin, parvalbumin, calbindin D28K) present in different subpopulations of interneurons revealed no multiple contacts between supramammillary afferents and labeled inhibitory cells at the light microscopic level. Furthermore, postembedding immunostaining of electron microscopic sections for GABA demonstrated that none of the 68 PHAL-labeled supramammillary boutons examined and none of their postsynaptic targets were immunoreactive for the inhibitory neurotransmitter. We conclude, therefore, that most if not all postsynaptic targets of the supramammillary projection are principal cells both in the dentate gyrus and in the CA2-CA3a subfields. This suggests that a mechanism other than disinhibition is responsible for the facilitatory effect of this pathway on hippocampal evoked activity. PMID- 7531094 TI - [Alpha fetoprotein in congenital hypothyroidism before and during treatment]. AB - BACKGROUND: Screening for congenital hypothyroidism (CH) is an opportunity to investigate maturation by measuring alphafetoprotein (AFP). PATIENTS AND METHODS: Blood AFP was measured in 73 full-term infants (controls), 22 infants with permanent CH and 19 with a transient form (FT) of hypothyroidism. It was also measured in mothers of the two groups with hypothyroidism. AFP was measured by RIA and its value was compared to those of FT4, TSH and bilirubin. RESULTS: Blood AFP was higher in patients with CH with significant differences between patients who had jaundice or not, but AFP was not significantly different in FT patients and controls. Initial values of AFP in both CH and FT population was correlated to the levels of AFP in their mothers. Under treatment with 7.5 micrograms/kg/day of l-T4, AFP levels remained increased at T15, then gradually normalized at T30 T60. The log of AFP was correlated to TSH levels between T15 and T60 but was not correlated to FT4 levels. Preliminary results at one year of age show that IQ seems better in infants with early normalization of AFP. CONCLUSIONS: Prolonged follow-up is necessary to assess the possibility that initial kinetics of AFP under therapy have a prognostic value for estimating the quality of outcome. PMID- 7531095 TI - Studies of gramicidin S analogues having various ring sizes by reversed-phase high-performance liquid chromatography. AB - Many gramicidin S analogues containing six to fourteen amino acid residues were synthesized and their chromatographic behaviour was investigated using reversed phase high-performance liquid chromatography. Cyclo(-Val-Leu-Orn-Leu-D-Phe-Pro Val-Orn-Leu-D-Phe-Pro-) gave double peaks in the chromatogram, whereas the other peptides gave a single peak. The influences of the concentration of this peptide, column temperature and flow-rate on the chromatographic separation were examined. The isomeric conformers were separated from each of the double peaks and were in equilibrium with each other at low temperatures. These results suggested that the presence of the additional L-Leu residue preceding the Orn residue gives rise to moderate stabilization of their conformers. PMID- 7531096 TI - Nuclear accumulation of exogenous basic fibroblast growth factor in endothelial, fibroblast, and myoblast cell lines results in diverse biological responses. AB - During studies comparing 125I-bFGF internalization between endothelial cells and other cell types, we found, unexpectedly, internalization and nuclear translocation of exogenously added 125I-bFGF in two cell lines: Chinese hamster ovary cells (CHO) and rat L6 myoblasts. These cell lines were previously reported to be devoid of FGF receptors. Furthermore, CHO cells showed a weak mitogenic response to added bFGF, while L6 cells were mitogenically unresponsive. By comparison, coronary venular endothelial cells (CVEC), BALB/c 3T3 fibroblasts, and BHK-21 cells, demonstrated internalization and nuclear translocation of added 125I-bFGF, and mitogenic responsiveness to the growth factor. Insulin alone stimulated DNA synthesis in all cell types, yet augmented bFGF-dependent DNA synthesis only in CVEC, 3T3, and BHK. All five cell types expressed FGF receptors as assessed by covalent crosslinking with 125I-bFGF and immunoblotting with anti FGF receptor antibodies. Differing rates of cytoplasmic and nuclear accumulation of 125I-bFGF and partial inhibition of internalization by pretreatment of CVEC with chlorate support a recent model that bFGF can internalize by two mechanisms. Insulin did not significantly affect 125I-bFGF internalization or metabolism in any cell type. bFGF treatment resulted in weak inhibition of RNA synthesis in L6 cells. bFGF appears firmly bound to the nuclear matrix as little nuclear-bound 125I-bFGF in CVEC is released by DNAse I or RNAse A digestion, while washes with 0.5 M NaCl result in partial release. Nuclear bFGF may thus be involved in regulation of nuclear events (e.g., gene transcription and/or DNA replication). PMID- 7531097 TI - Effect of granulocyte colony-stimulating factor on acute lung injury in the rat. AB - The effect of G-CSF pretreatment on experimental acute lung injury was studied in Sprague-Dawley rats receiving one of the following treatments: (1) G-CSF 50 micrograms/kg subcutaneously twice daily beginning 2 d prior to being killed; (2) ANTU 50 mg/kg intraperitoneally; (3) ANTU+G-CSF 50, 25, or 12.5 micrograms/kg; (4) HCl 0.6 ml of a 0.1 N solution intratracheally; (5) HCl+G-CSF 50 or 25 micrograms/kg; (6) control solutions. Lung injury was quantified by measurement of lung wet/dry weights, by histopathologic scoring, and by measurement of fluid flux during ex vivo perfusion. G-CSF pretreatment elevated the baseline blood neutrophil counts as much as 6-fold compared with Control, and it increased the numbers of lung neutrophils and caused a mild histologic lung injury, but it did not significantly alter wet/dry weight ratios or fluid flux. ANTU alone and HCl alone caused a moderate histologic lung injury, increased wet/dry weight ratio, and resulted in a small increase in flux. The combination injuries, ANTU+G-CSF and HCl+G-CSF, caused a more severe lung injury manifested by increased wet/dry weight ratios and increase in flux when compared with ANTU alone and HCl alone, respectively. We conclude that pretreatment with G-CSF potentiates ANTU- and HCl induced lung injury in non-neutropenic rats. The potential for G-CSF to aggravate acute lung injury in patients remains speculative. PMID- 7531098 TI - L-selectin expression increases on peripheral blood polymorphonuclear leukocytes during active marrow release. AB - The cell adhesion molecule L-selectin is highly expressed on mature bone marrow polymorphonuclear leukocytes (PMN), and the release of these cells from the bone marrow could produce a population of circulating PMN expressing high levels of L selectin. Because L-selectin initiates the interaction of PMN with activated endothelium, these cells could be important in the pathogenesis of multiorgan failure following sepsis and septic shock. The present study was designed to test the hypothesis that the release of PMN from the bone marrow increases the expression of L-selectin on circulating PMN. Peripheral blood and bone marrow samples were obtained immediately after sternotomy (BM1) and during (BM2) and just before closing the sternum (BM3) in five normothermic and five hypothermic cardiopulmonary bypass (CPB) procedures. L-selectin was measured using both immunocytochemistry and flow cytometry. The results showed that L-selectin expression on bone marrow PMN was greater than on peripheral blood PMN (p < 0.01) in all patients at baseline. Bone marrow release of PMN during normothermic CPB was associated with a rise in peripheral blood band cells (0.18 +/- 0.7 versus 0.56 x 10(9)/L) (p < 0.01) and an increase in the percentage of PMN expressing high levels of L-selectin (9 +/- 3.3 to 36 +/- 6.6%, p < 0.03) and the L-selectin mean fluorescence intensity (MFI) on PMN (p < 0.05). The expression of L-selectin on band cells was higher than on segmented PMN in the circulation (p < 0.01). Hypothermia (27 degrees C) prevented the release of band cells into the circulation and the increased expression of L-selectin on the PMN.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531099 TI - Soluble CD14 is increased in bronchoalveolar lavage of active sarcoidosis and correlates with alveolar macrophage membrane-bound CD14. AB - CD14 is a myeloid differentiation antigen which exists in a membrane-bound (55 kD) and a soluble (48 kD) form. This antigen is a receptor for lipopolysaccharide (LPS) structures and triggers the production of various cytokines. The aim of this study was to evaluate whether in active sarcoidosis, a disease with increased proportions of alveolar macrophages (AM) with CD14 expression in BAL fluid, the soluble form of CD14 (sCD14) is also increased. The sCD14 levels were measured in BAL fluid with an ELISA, and membrane-bound CD14 was determined by an immunoperoxidase assay, in active sarcoidosis (n = 13), inactive sarcoidosis (n = 9), idiopathic pulmonary fibrosis (IPF) (n = 6), and control subjects (n = 8). Higher concentrations of sCD14 were present in BAL fluid of patients with active sarcoidosis (58 +/- 34 ng/ml) than in those with inactive disease (13 +/- 10 ng/ml), patients with IPF (5 +/- 5 ng/ml), or control subjects (10 +/- 8% ng/ml) (p < 0.01). Similarly, the proportions of AM expressing membrane-bound CD14 were increased in active sarcoidosis (91 +/- 6%) compared with inactive sarcoidosis (82 +/- 6%), patients with IPF (76 +/- 13%), and control subjects (79 +/- 9%) (p < .05). In sarcoidosis, a significant correlation was found between the sCD14 concentration in BAL fluid and AM membrane expression of CD14 (r = 0.57, p < 0.01). We conclude that sCD14 is increased in BAL of active sarcoidosis suggesting a potential role for this substance as marker of activity and in the pathogenesis of pulmonary sarcoidosis. PMID- 7531100 TI - Neuropeptide content of lungs from asthmatic and nonasthmatic patients. AB - Tracheal and lung parenchymal SP-LI (substance P-like immunoreactivity) and VIP LI (vasoactive intestinal peptide-like immunoreactivity) content was measured in HPLC-purified tissue extracts from patients with and without asthma. We detected significantly less SP-LI in tracheal tissue from asthmatic than from nonasthmatic patients, whereas parenchymal SP-LI content was not significantly different between these groups. This finding does not support the concept that asthmatic lungs contain excessive amounts of SP. Indeed, lower SP-LI content of tracheal tissues from asthmatic patients may reflect augmented SP release followed by degradation. We detected greater quantities of VIP-LI in tracheal than in parenchymal tissue in both groups, but did not detect significant differences in VIP-LI content in tracheal or parenchymal tissues from asthmatic and nonasthmatic patients. These findings indicate that asthmatic and nonasthmatic lungs contain similar levels of VIP. PMID- 7531101 TI - Substance P receptors and signal transduction in leukocytes. AB - In this paper we demonstrate several aspects of the mechanisms of action of the neurotransmitter Substance P in the immune system. We describe how Substance P can activate T cells, B cells, monocytes, and granulocytes to, respectively, proliferation, immunoglobulin synthesis, cytokine production, and chemotaxis. However, the neurotransmitter does not trigger cells of the immune system only via the well-characterized neurokinin-1 receptor, which mediates the signaling by Substance P in the neuroendocrine system. We show that Substance P can activate T cells receptor-independently. The receptor-independent activation of T cells leads to the activation of heterotrimeric G proteins and calcium-influx into the T cell, followed by an increase in proliferation of the cell. Apart from the receptor-independent activation pathway, Substance P can also activate monocytes and B cells via a nonneurokinin Substance P receptor. Activation of this novel receptor leads to the activation of MAP kinase, which is an important second messenger in the cascade leading to cytokine production by monocytes. In contrast to the non-neurokinin Substance P receptor, triggering of the NK-1 receptor, transfected in Jurkat cells, or triggering of T cells via receptor-independent pathways does not lead to activation of MAP kinase. Combining the data, we can conclude that the interaction between the neuroendocrine system and the immune system with regard to Substance P clearly indicates that the immune system does not necessarily mirror the communication pathways that are used in the neuroendocrine system. Substance P is capable of signaling the immune system via multiple activation pathways. PMID- 7531102 TI - The enteric nervous and immune systems: interactions for mucosal immunity and inflammation. AB - A number of anatomical studies have demonstrated the presence of peptidergic nerve fibers infiltrating mucosal lymphoid tissues. The exact mechanisms of how neuropeptides are released to affect these lymphoid sites are unclear, but radiolabeled binding studies have shown that mucosal leukocytes bear a number of neuropeptide receptors on their cell surfaces capable of responding to neural signals. The presence of neuropeptide-containing fibers and the ability to receive neural signals suggest that mucosal lymphocytes can be influenced by neurogenic mediators. The objectives set forth in this review are to provide what is currently known about the ability of substance P and vasoactive intestinal peptide to promote mucosal IgA responses in the gastrointestinal tract via Th2 mechanisms and to discuss how these neuropeptides contribute to the exacerbation of the inflammatory diseases of the gastrointestinal tract. We describe how immune responses develop in the gastrointestinal immune system and emphasize how neuropeptides may influence the differentiation of lymphocytes in mucosal inductive tissues and their subsequent expression in mucosal effector sites. Finally, we discuss new techniques developed by the Mucosal Immunization Research Group that have enabled the study of mucosal immune responses. PMID- 7531103 TI - LFA-1 binding site in ICAM-3 contains a conserved motif and non-contiguous amino acids. AB - The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-3 and VCAM-1 and hence many form a common site of contact in all CAMs of this type. Other ICAM 3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggest an evolutionary relationship between ICAMs and fibronectin interactions with integrins. PMID- 7531104 TI - Acute and chronic nephrotoxicity associated with immunosuppressive drugs. AB - Although the main immunosuppressive drugs used commonly after transplantation and in the treatment of autoimmune disease are cyclosporine and FK506, both have similar nephrotoxic properties that may limit their clinical use. The complexity of both the functional and structural characteristics of cyclosporine and FK506 nephrotoxicity strongly suggests a multifactorial process involving complex physiologic and intracellular signaling processes that have obvious implications for the design and development of new immunosuppressive drugs. This review summarizes recent concepts regarding the mechanisms responsible for the renal dysfunction related to these drugs and outlines methods of possible therapeutic intervention. PMID- 7531105 TI - Results of aggressive treatment of gastric sarcoma. AB - BACKGROUND: Leiomyosarcoma and leiomyoblastoma and subtypes of gastric smooth muscle tumors. These rare tumors are usually treated with surgical resection. However, there is controversy regarding the optimal surgical management for these malignancies and little information is available on the efficacy of radiation and chemotherapy in the adjuvant or palliative setting. METHODS: The records of 32 patients with gastric leiomyosarcoma or leiomyoblastoma were reviewed. Survival data were obtained and patient outcome was analyzed with respect to the type of treatment given. Four different staging systems were compared for their ability to predict survival. RESULTS: Thirty patients with leiomyosarcoma and two patients with leiomyoblastoma were followed after surgery. All 32 patients were explored, and 21 curative and 11 palliative procedures were performed. Adjacent organs were included in 38% of resections. Only three patients did not undergo gastric resection. Local recurrence developed in eight patients after curative resection for a local control rate of 62%. Eight other patients developed metastatic disease for an overall recurrence rate of 76% after curative resection. Median survival of patients undergoing curative resection was 40 months compared with 8 months for those having a palliative procedure. The estimated 5-year survival was 34% and 10%, respectively (p = 0.05). Twenty-five patients with advanced disease received systemic, hepatic arterial, or intraperitoneal chemotherapy. Eighty percent of patients received a regimen including doxorubicin. Four partial and one complete response were noted. Seven patients received postoperative radiation therapy. Fourteen patients underwent debulking surgery of recurrent or persistent disease in conjunction with chemotherapy. Chemotherapy, radiation therapy, and debulking surgery did not result in statistically significant prolongation of survival. Seven patients remain alive, two with liver metastases. Four different staging systems for gastric sarcomas were compared, but none of them were found to be clearly superior in predicting survival. CONCLUSIONS: Curative gastric resection was achieved in 66% of patients and resulted in a significant prolongation of survival as compared with patients who had a palliative procedure. Wedge resection of tumor or partial gastric resection appears to be an acceptable surgical approach to these tumors as long as negative margins can be obtained. Chemotherapy, radiation therapy and debulking surgery did not result in significant prolongation of survival in the face of advanced disease. None of the staging systems for gastric sarcoma currently in use is completely satisfactory. Tumor grade and extent of disease seem to be the most important factors when determining prognosis or considering adjuvant therapy. PMID- 7531106 TI - Aging and the nigrostriatal dopamine system: a non-human primate study. AB - The present study examined neurochemical, morphological and functional markers of the nigrostriatal dopamine system in young, intermediate-aged and old squirrel monkeys. Striking reductions in motoric activity were observed with advancing age. significant age-related loss of dopamine occurred in the substantia nigra (70%) and the putamen (30%) but not in the caudate. There was a strong correlation between the reductions in motoric activity and the loss of putamen dopamine. However, nigrostriatal dopamine loss did not appear to be the consequence of age-related loss of dopaminergic nigral neurons since the number of tyrosine immunoreactive cells was not significantly different among the three age groups. These results suggest that the aging squirrel monkey demonstrates the age-related loss of nigrostriatal dopamine thought to occur in humans and identify this non-human primate as a useful model to further investigate the underlying mechanism(s) and functional consequences of age-related decline of the nigrostriatal dopamine system. In addition, the selective loss of dopamine in the putamen but not the caudate parallels the regional vulnerability observed in Parkinson's disease, an age-related neurodegenerative disorder, raising the possibility of a relationship between normal aging and the development of this disease. Finally, because the number of tyrosine hydroxylase (TH) positive cells remains constant with age, these results raise the possibility that therapeutic strategies aimed at increasing dopamine concentrations may benefit elderly individuals. PMID- 7531107 TI - Automatic quantification of amyloid plaque formation in human spongiform encephalopathy. AB - This study compared a panel of three different prion protein antibodies with conventional plaque staining methods--Congo Red, Periodic acid Schiff and sulphated Alcian blue--to investigate amyloid plaque formation in cases of human spongiform encephalopathy (HSE). Tissue samples were taken from the cerebellum in nine sporadic cases of Creutzfeldt-Jakob Disease, with plaque formation noted on routine histology, and one case of Gerstmann-Straussler Scheinker syndrome. Using image analysis techniques, a semi-automatic system of plaque quantification was devised to measure the relative performance of these different staining methods. A total figure of percentage tissue area stained positively was returned by the system in each case analysed. A significant statistical correlation was observed among all three antibodies (r > 0.9, P < 0.01, in all comparisons) and a significant improvement was observed when the average antibody staining figures were compared to those of the Alcian blue technique (P < 0.05). The distribution of plaques across the cerebellar layers observed here appears to confirm earlier research findings. A strong correlation was found between staining in the two cerebellar hemispheres (r = 0.97, P < 0.01). This novel image analysis system has considerable potential for objective assessment of the pathology of HSE. PMID- 7531108 TI - RANTES: a novel mediator of allergic inflammation? PMID- 7531109 TI - Do nasal mast cells release histamine on stimulation with substance P in allergic rhinitis? AB - The effects of nasal administration of increasing doses of exogenous substance P have been studied in patients with allergic rhinitis treated with placebo or with the H1 antagonist cetirizine (10 mg twice daily for 3 days). Responses to substance P were assessed by posterior rhinomanometry (measuring nasal airway resistance) and by measure of histamine, protein and albumin production and cell recovery in nasal lavage fluids before and after challenge. Substance P induced a dose-dependent increase in nasal airway resistance which was similar after treatment with either cetirizine or placebo (maximal increase in nasal airway resistance was 4.2-fold greater than the baseline with the placebo and 4.7-fold greater than the baseline with cetirizine). No histamine release was observed. Similar increases in protein and albumin production were observed after stimulation with substance P along with the placebo (protein: from 0.35 +/- 0.11 to 3.31 +/- 0.62 mg and albumin: from 0.09 +/- 0.04 to 2.08 +/- 0.39 mg) and when combined with cetirizine treatment (proteins: from 0.42 +/- 0.09 to 3.62 +/- 0.77 and albumin: from 0.17 +/- 0.04 to 2.19 +/- 0.51 mg). After stimulation with substance P, percentages of neutrophils recovered in nasal fluids increased from 26.2 +/- 11.5 to 54.5 +/- 9.5 with the placebo and from 35.5 +/- 11.0 to 53.6 +/- 9.5 with cetirizine. Eosinophils were inconsistently found after substance P stimulation during both treatments. In conclusion, nasal response to substance P is not modified by cetirizine which suggests that the effect of substance P is not secondary to histamine release in the nose in man. PMID- 7531110 TI - Discrepancies between in vitro and in vivo tests for house dust mite allergy: in domestic exposure a better predictor than sensitization? AB - We subjected seven asthmatic children to two bronchial allergen challenges, first with an extract from the house dust mite Dermatophagoides pteronyssinus (Der p) and then Dermatophagoides farinae (Der f), or vice versa. All children had elevated specific serum IgE to both species as well as reactions by crossed radioimmuno/electrophoresis (CRIE) to both group I and II allergens from both species. Immunoabsorption and subsequent analysis by CRIE showed a considerable concentration of serum IgE with specificity for epitopes common to the two species of house dust mite. Home dust sampling established that all children were exposed to Der f and only two to Der p. On bronchial provocation tests, all responded to Der f with an immediate reaction and five with a late reaction, only three of seven showed an immediate response to Der p, with four of the seven showing a late reaction. Our data could indicate that the local allergic immune reaction in the respiratory tract is sustained by ongoing exposure, and may thus have a different species specificity than the response reflected in the serum. In conclusion, our data indicates a lack of association between in vitro and in vivo tests for house dust mite allergy, which supports the continuing need for monitoring current clinical sensitization by allergen provocation tests and by measuring domestic exposure to the corresponding allergen. Extended studies are needed to support our findings. PMID- 7531111 TI - Dextran sedimentation induces a difference in the percentage of hypodense eosinophils in peripheral blood between children with allergic asthma and healthy controls. AB - Considerable differences in the percentage of hypodense eosinophils in the peripheral blood of asthmatics have been reported by different investigators. In these previous studies dextran sedimentation was used for removal of erythrocytes prior to density centrifugation. We hypothesized that the sedimentation procedure might induce the presence of hypodense eosinophils in the peripheral blood of asthmatic patients. In order to test this hypothesis, we compared eosinophil density profiles from peripheral blood of children with asthma and of age-matched healthy controls, using different procedures. In the first method (direct method) blood samples were directly layered on a discontinuous Percoll gradient. Erythrocytes were removed by isotonic lysis. In the second method (dextran sedimentation) erythrocytes were removed by sedimentation with dextran prior to gradient centrifugation. Results of the direct method show no significant difference in percentage of hypodense eosinophils between children with asthma and healthy controls (9.19% and 6.84% respectively). However, after dextran sedimentation, children with asthma had a significantly higher percentage of hypodense eosinophils than healthy controls (15.40% and 8.84% respectively; P < 0.05). The percentage of hypodense eosinophils was correlated with the number of eosinophils and with the lung function, measured as the Tiffeneau index (FEV1/VC), in the whole group of subjects when the direct method was used. We conclude that an increased percentage of hypodense eosinophils is not present in the circulation of children with asthma, but can be induced in vitro by dextran sedimentation. Therefore, in vitro generation of hypodense eosinophils in the blood of patients with asthma seems to be related with the primed state of eosinophils. PMID- 7531112 TI - Nitric oxide synthase inhibitors do not alter functional hyperemia in canine skeletal muscle. AB - To test the hypothesis that endothelium-derived products contribute to functional hyperemia in skeletal muscle, we infused nitric oxide synthase inhibitors, either 200 microM N omega-nitro-L-arginine (NNA) (N = 4) or 1 mM N gamma-monomethyl-L arginine (NMMA) (N = 4), before and during 6 min of 4 Hz stimulation of canine gastrocnemius in situ. We infused saline (N = 4) as a control. NNA significantly decreased steady-level resting flow by 3.8 +/- 0.4 mL.kg-1.s-1. The increase in flow from rest to 5 min of stimulation was not changed by the nitric oxide synthase inhibitors. We also stimulated muscles for 60 min either with saline infusion (N = 4) or with the infusion of saline during the first 15 min and NNA for the remaining 45 min (n = 4). There was no difference in the flow during contractions. To clarify the effect of these inhibitors on canine vessels, we challenged rings of canine femoral artery with and without endothelium with acetylcholine and bradykinin (both 1 microM) before and after the addition of NNA and NMMA (both 10 microM). The nitric oxide synthase inhibitors decreased the relaxation accompanying acetylcholine. Both inhibitors caused only endothelium intact rings to contract. Thus, the presence of a nitric oxide synthase inhibitor identified an endothelium-dependent contribution to the regulation of blood flow to skeletal muscle at rest but had no effect on functional hyperemia. PMID- 7531113 TI - Intraoperative irradiation after palliative surgery for locally recurrent rectal cancer. AB - BACKGROUND: In patients with locally recurrent rectal cancer, long-term disease control and survival is uncommon with single-modality therapy. This report evaluates results achieved at the Mayo Clinic (Rochester, MN) with single- or combined-modality treatment, including intraoperative irradiation. METHODS: From 1981 to 1988, 106 patients underwent palliative surgical resections at the Mayo Clinic for locally recurrent rectal cancer. None had evidence of extrapelvic disease, and 42 received intraoperative electron beam irradiation (IORT) as a component of treatment. Gross residual disease remained after maximal surgical resection in 34 of the 42 patients and 61 of the patients who did not receive IORT. The IORT dose was 15-20 Gy in 39 patients and 10, 25, and 30 Gy in the other 3. External beam irradiation (EBRT) was administered to 41 of the 42 patients (doses > or = 45 Gy to 38 patients). RESULTS: Kaplan-Meier survival estimates at 3 and 5 years were analyzed for the 106 patients. Palliative surgical resection alone (12 patients) resulted in a 3-year survival of 8% and a 5-year survival of 0%. Statistically significant factors relative to survival based on the univariate analysis of all patients included amount of residual tumor (microscopic vs. gross, P = 0.032) treatment method (P = 0.005), IORT versus no IORT (P = 0.0006), type of symptoms (P = 0.0075), type of fixation (P < 0.0001), and preoperative Eastern Cooperative Oncology Group status (P = 0.03). For patients who received IORT, 3-year survival with gross residual tumor or presentation with pain was 44% and 43%, respectively. Factors not associated with survival (univariate) included extended versus conventional surgical resection, grade, age, and sex. The 3-year cumulative probability of distant metastasis was 60% in the patients who received IORT and 54% in those who did not. The 3-year local relapse rates were 40% versus 93% in patients who received IORT versus those who did not. CONCLUSIONS: Although the addition of IORT to external irradiation and maximal surgical resection appears to improve local tumor control and survival in patients who undergo palliative surgical resection for locally recurrent rectal cancer, further gains in treatment are necessary. Considering the high rates of distant metastasis, more routine systemic therapy with 5 fluorouracil (5-FU) leucovorin, 5-FU levamisole, or all three needs to be incorporated into aggressive treatment approaches. In patients with gross residual tumor after maximum surgical resection, local tumor control is inadequate despite treatment combinations including IORT. The evaluation of radiation sensitizers or biologic modifiers during external irradiation and IORT is indicated. PMID- 7531114 TI - Forearm blood flow responses to a nitric oxide synthase inhibitor in patients with treated essential hypertension. AB - OBJECTIVE: There is evidence that basal NO mediated vasodilatation is abnormal in patients with essential hypertension. Studies in animals suggest that treatment of hypertension may restore the nitric oxide system towards normal. The objective of this study was to examine basal nitric oxide mediated vasodilatation in patients with treated essential hypertension. METHODS: The forearm blood flow response to noradrenaline and NG-monomethyl-L-arginine (L-NMMA), a stereospecific inhibitor of nitric oxide synthesis, was compared in 11 patients with treated essential hypertension and 18 normotensive healthy controls. The results in the treated hypertensive patients were also compared with those in a previously reported group of seven untreated patients with essential hypertension. Drugs were infused locally into the brachial artery and forearm blood flow measured using venous occlusion plethysmography. RESULTS: In the healthy controls noradrenaline (60, 120, and 240 pmol.min-1) and L-NMMA (1, 2, and 4 mumol.min-1) produced similar reductions in resting forearm blood flow. In the patients with treated essential hypertension, at the same doses, noradrenaline and L-NMMA also produced similar reductions in forearm blood flow. There was no significant difference in the response to either noradrenaline or L-NMMA between the healthy controls and the treated hypertensive patients. There was also no significant difference in the response to noradrenaline or L-NMMA between the treated patients and the patients with untreated essential hypertension. However, the response to L-NMMA in all subjects correlated significantly with blood pressure, such that the responses in the treated patients lay between those of the healthy controls and the untreated patients. CONCLUSIONS: Basal nitric oxide mediated vasodilatation appears to be a continuum that varies with blood pressure. Reduction in blood pressure with medication moves the L-NMMA response nearer to that seen in healthy subjects. These results suggest that treatment of hypertension may restore NO mediated vasodilatation towards normal. PMID- 7531115 TI - Production of tumor necrosis factor induced by synthetic low-toxicity lipid A analog, DT-5461a, is mediated by LPS receptor sites and tyrosine kinase-MAP kinase signaling pathway in murine macrophages. AB - The synthetic low-toxicity lipid A analog DT-5461a induces endogenous TNF production in mice. The activity of TNF so induced is probably the main contributor to the antitumor effect of this compound. In the present study, we investigated the mechanism by which DT-5461a induces TNF production in murine macrophage RAW 264 cells. DT-5461a mimicked the ability of LPS to induce TNF production in a dose-dependent manner. DT-5461a at higher concentrations inhibited specific binding of [3H]LPS to the cells and reduced LPS-induced TNF production to the level induced by DT-5461a alone. In addition, DT-5461a, as well as LPS, induced tyrosine phosphorylation of MAP kinases, the early signal transduction pathway of this production. Herbimycin A, an inhibitor of tyrosine kinase, inhibited the LPS- and DT-5461a-induced tyrosine phosphorylation, expression of TNF mRNA, and subsequent TNF secretion. These results suggest that DT-5461a and LPS induce TNF production in murine macrophages through the common receptor sites and the similar early signaling pathway. PMID- 7531116 TI - Upregulation of B7 molecules by the Epstein-Barr virus enhances susceptibility to lysis by a human NK-like cell line. AB - The original human NK-like line YT was reported to lyse K562 and several B- and T cell lines. The YT subline we are investigating, YT-INDY, does not lyse K562 or the T-cell line Molt-4. It does, however, lyse the EBV+ Burkitt lymphoma (BL) B cell line Raji and EBV-immortalized B-cell lines. Several EBV- BL lines and an EBV- pre-B-cell leukemia line that we tested were not appreciably lysed by YT INDY. To determine if EBV plays a role in TC susceptibility to lysis by YT-INDY, we compared YT-INDY's ability to lyse the EBV- BL line BL41 to its ability to lyse an EBV-infected derivative of BL41. The EBV-infected cell line was lysed, on average, at twice the level of the uninfected line. CD28/B7 interactions appeared to be involved in TC recognition by YT-INDY. Therefore, we examined the level of expression of B7 molecules on the infected and uninfected BL41 lines. An average of 15% of the uninfected BL41 cells expressed B7-1/B7-3, compared to 79% of the infected. B7-2 expression was similar in the two cell lines. Lysis of EBV infected BL41 was reduced by anti-B7-1/B7-3 (BB1) or anti-CD28 antibodies (Abs) to the level of lysis of the uninfected line, indicating that upregulation of B7 1/B7-3 by the virus may be responsible for the enhanced susceptibility. We attempted to determine the particular EBV latent protein responsible for B7-1/B7 3 upregulation by analyzing BL41 clones expressing LMP1, EBNA-2/EBNA-LP, or EBNA 1. All of the high-expressing clones showed a higher level of B7-1/B7-3 expression than the vector-transfected control cell line, with LMP1-expressing clones expressing the highest amount. EBNA-1 clones and a high-expressing EBNA 2/EBNA-LP clone had a slightly higher density of B7-2 on their surface than the remaining clones. The increased expression of molecules of the B7 family correlated with increased susceptibility of the clones to lysis by YT-INDY. Anti CD28 or a combination of anti-B7-1/B7-3 and anti-By-2 did not inhibit lysis of the clones to the level of lysis of the vector-transfected control cell line in all cases. We conclude that intact EBV enhances susceptibility to YT-INDY lysis by upregulating B7-1/B7-3. EBV proteins expressed individually also enhance susceptibility to lysis and upregulate members of the B7 family.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531117 TI - Prostaglandin E2 promotes the induction of anergy during T helper cell recognition of myelin basic protein. AB - Remission of experimental autoimmune encephalomyelitis (EAE) in Lewis rats may involve mediators such as prostaglandins (PG) that are produced within demyelinating lesions and are known to potently inhibit T cell responses. In support, this study shows that PGE2 inhibited myelin basic protein (MBP)-specific responses of proliferation and IL-2 production by continuously propagated lines of T-helper cells. Simultaneous exposure to PGE2 and immunogenic MBP rendered T cells profoundly anergic. Even after several weeks of propagation in IL-2 containing medium, anergic T cells exhibited marked reductions in MBP-stimulated proliferation and IL-2 production responses when restimulated with optimal concentrations of MBP and irradiated splenocytes (SPL). PGE2 did not block other measures of MBP-dependent activation, including induction of postactivation refractoriness in IL-2 production pathways, activation-dependent decreases in MBP reactivity, and activation-dependent increases in PGE2 sensitivity. Proliferative responses by anergic T cells were reduced in magnitude but were not altered in their sensitivity to MBP. PGE2-mediated anergy was manifest as an intrinsic deficit rather than an acquired suppressive activity and was associated with reduced mitogenic responsiveness and a block in IL-2 production pathways. Anergic T cells were responsive to IL-2 and eventually regained full antigenic reactivity after extended propagation in IL-2-supplemented medium. In summary, a limited exposure to PG had long-lasting inhibitory effects on subsequent T cell responsiveness to the target autoantigen MBP. These findings support the hypothesis that PG may promote disease remission by inducing anergy in helper T cells. PMID- 7531118 TI - Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors. AB - Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34+ cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34+ cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts. PMID- 7531119 TI - Parameters controlling the programmed death of mature mouse T lymphocytes in high dose suppression. AB - We have characterized parameters of both T cells and antigen-presenting cells (APCs) that influence high-dose suppression due to apoptosis. Blockade of interleukin-2 (IL-2) utilization is shown to inhibit both proliferation and the ensuing death. An analysis of sublines of a mature T cell clone demonstrates a correlation between IL-2 receptor alpha chain (IL-2R) induction, increased proliferation, and greater suppression at high antigen doses. Profound loss of cells at high antigen dose was found to require at least 48 to 72 hr to develop. Antigen add-back experiments showed that strong T cell receptor reengagement of activated, cycling cells was essential for proliferative suppression and cell loss. Increasing the ratio of APC:T lymphocytes to 50:1 augmented cell death. For antigen-induced death of lymph node T cells, fresh T-depleted splenocytes were more effective than splenocytes that had been irradiated or treated with mitomycin C. Thus, T lymphocyte apoptosis at high antigen doses is a function of the activation response of the T lymphocyte as well as the efficiency of antigen presentation by the APC. These results strengthen the theory that apoptosis takes part in a feedback regulatory mechanism that has been called propriocidal regulation, which limits T cell expansion at high antigen doses. PMID- 7531120 TI - Brain growth retardation due to the expression of human insulin like growth factor binding protein-1 in transgenic mice: an in vivo model for the analysis of igf function in the brain. AB - Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation. PMID- 7531122 TI - Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells. AB - It has been shown in previous studies that a subpopulation of neurons in monolayer cultures prepared from immature embryonic chicken retina acquired a series of functional properties which characterized them as GABAergic amacrine cells after 1 week in vitro. In the present study, we demonstrate that immature precursors of these cells were already identifiable by morphological criteria after 2 days in vitro (DIV). Using the whole cell patch-clamp technique we have studied the time-course of the expression of voltage-dependent and of glutamate and GABA receptor-associated conductances in these identified retinal interneurons developing in vitro. Recordings after 2 DIV revealed a very homogeneous pattern of membrane conductances. In all cells tested, whole cell responses to depolarizing voltage steps consisted solely of a sustained outward potassium current and 100% of the cells responded to the glutamate receptor agonist kainic acid (KA) and to GABA. Fast activating inward sodium currents first appeared after 3 DIV, whereas a transient component of outward potassium currents was not detectable before day 4 in vitro. N-Methyl-D-aspartate (NMDA) evoked currents were first observed at 3 DIV in the GABAergic neurons. Only 1 day later they were found in all of the GABAergic neurons. Expression of responses to quisqualic acid (QU) started at 3 DIV, but remained restricted to a subpopulation of the GABAergic cells even at later stages (59% at 4 DIV, 63% at 6-9 DIV). Antagonistic effects of QU on KA responses, however, were detectable in all cells tested, independent of the developmental stage and the presence of QU-evoked currents.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531121 TI - Development of abnormal lamination and binocular segregation in the retinotectal pathways of the rat. AB - The uncrossed retinotectal pathway of pigmented rats originates from a small fraction of the retinal ganglion cell population. This projection terminates deeply in discrete patches within the upper grey layers where crossed and uncrossed inputs overlap. However, after the experimental enlargement of the uncrossed pathway, the ipsilateral fibers are also found in a superficial tier of the upper grey layers where binocular inputs segregate [36]. We studied the development of the retinotectal projections in rats after the enlargement of the uncrossed pathway as a result of a contralateral (left) optic tract lesion (OTL) made at birth. Horseradish peroxidase (HRP) was used as an anterograde tracer. An abnormal uncrossed projection from the right eye to the collicular surface appeared at postnatal day 3 (P3). Between P5 and P10, this projection developed the bilaminar pattern seen in similar operated adults. The laminar arrangement of the aberrant terminal fields did not change significantly after an ipsilateral visual cortex ablation on the day of birth. Despite the early development of the aberrant uncrossed pathway, binocular segregation was incipient at P10. At P14, 46% of the operated rats presented gaps in the terminal labeling at the tectal surface. This figure increased to 55.5% at 6 weeks, a proportion still smaller than in adult animals of the same group (69%). Eyelid suture had no effect on segregation. This projection remains plastic for at least 3 weeks, since the removal of the ipsilateral input at either P14 or P21 resulted in the absence of gaps in the contralateral projection. We conclude that the laminar selection of retinotectal projections depends on binocular interactions and that the abnormal segregation of retinal inputs to the superior colliculus has an unusually protracted development which can be reversed long after the previously defined critical period in this system. PMID- 7531124 TI - Apoptosis in SLE--too little or too much? AB - Cells of the immune system are, most likely, programmed to die unless recruited into an immune response. An active cell death program may also be induced by a variety of soluble and surface signals, many of which have only recently been recognized. The importance of these pathways in maintaining tolerance is highlighted by the development of lupus-like diseases in three different mouse strains that have spontaneous mutations in the Fas/APO-1 receptor or its ligand. The known function of Fas/APO-1 in signalling apoptosis explains the persistence of self reactive cells in lpr mice although it is unclear, at present, whether Fas defects effect both central and peripheral tolerance. Whereas Fas/APO-1 receptor expression appears to be normal in humans with SLE, other defects in the Fas/APO-1 pathway or other key molecules in the cell death survival require further study. PMID- 7531123 TI - Biochemical and molecular characterization of stromelysin synthesized by human osteoarthritic chondrocytes stimulated with recombinant human interleukin-1. AB - OBJECTIVE: To study the biochemical and molecular characterization of stromelysin synthesized by human chondrocytes derived from osteofemoral heads. METHODS: First passage human chondrocyte cultures were incubated with recombinant human interleukin-1 alpha or recombinant human interleukin-1 beta (10-1000 pg ml-1) for either 24 or 48 hrs. The medium compartment of these cultures was assayed for stromelysin activity. Total cellular RNA was used to determine: (i) the molecular structure of the stromelysin synthesized by these cells; and (ii) whether or not these chondrocytes expressed the Type II procollagen gene (COL2A1). RESULTS: Human osteoarthritic chondrocytes released into the medium on enzyme requiring tryspin activation that possessed Substance P (SP) cleaving activity. SP cleaving activity was completely inhibited by EDTA. Casein zymography showed lysis zones produced by trypsin-activated chondrocyte culture medium that co-migrated with casein lysis zones produced by recombinant human prostromelysin. The majority of SP cleaving activity was eluted from a Zn-Sepharose column with 0.25 M glycine. Enzyme activity eluted from Zn-Sepharose produced casein lysis zones which co migrated with lysis zones produced by recombinant human prostromelysin. Immunoblotting revealed the presence of prostromelysin (M(r), 55-57 kDa) in the pooled chondrocyte culture media applied to Zn-Sepharose and in the 0.25 M glycine eluate. Trypsin-activation converted prostromelysin to a mature stromelysin form (M(r), 45-47 kDa). Polymerase chain reaction (PCR) amplification of human chondrocyte cDNA demonstrated COL2A1 transcripts. A PCR product of expected size (680 bp) was produced by amplification of chondrocyte cDNA using stromelysin-1 oligonucleotide primers. The cloned and sequenced PCR product showed 100% homology between the chondrocyte stromelysin-1 mRNA-derived cDNA and the stromelysin-1 mRNA-derived cDNA of cultured human synovial, gingival and skin fibroblasts. CONCLUSIONS: By several criteria, human osteoarthritic chondrocytes synthesized stromelysin which was biochemically and antigenically identical, and molecularly homologous with human fibroblast stromelysin-1. These results suggest that a quantitative imbalance between stromelysin-1 and endogenous stromelysin-1 inhibitors rather than the transcription of a new stromelysin gene is the mechanism underlying the increased proteoglycan degradation seen in osteoarthritic cartilage. PMID- 7531126 TI - Glomerular microproteinuria in children treated with non-steroidal anti inflammatory drugs for juvenile chronic arthritis. AB - The urinary excretion of selected glomerular (albumin, transferrin, and IgG) and tubular (alpha 1-microglobulin) protein and enzyme (N-acetyl-beta-D glucosaminidase) markers was studied in 36 patients with juvenile chronic arthritis in order to investigate whether children receiving therapeutical doses of non steroidal antiinflammatory drugs (NSAIDs) and without clinical signs of gross renal dysfunction provide evidence of sub-clinical renal injury. Forty seven age-matched healthy children as well as nine children with juvenile chronic arthritis but without NSAID therapy served as control groups. Although there was no difference between patients and controls regarding the serum creatinine and urea nitrogen levels, the urinary excretion of all three glomerular markers was significantly elevated in the patient group treated with NSAIDs (p < 0.001). In contrast, there was no difference between patients and controls concerning the urinary excretion of both tubular markers. Furthermore, no correlation was found between protein and enzyme excretion and the onset type, duration or activity of the underlying disease. Taken together, these data indicate that patients receiving NSAIDs display signs of glomerular dysfunction concerning the handling of plasma proteins. The systematic assessment of urinary protein- and/or enzyme excretion may constitute a useful tool for the early detection and monitoring of otherwise subclinical renal injury in patients treated with NSAIDs. PMID- 7531125 TI - Study of IL-2, IL-6, TNF alpha, IFN gamma and beta in the serum and synovial fluid of patients with juvenile chronic arthritis. AB - In the last few years the important role played by various cytokines in the pathogenesis of chronic inflammatory diseases has emerged. In the present study, serum and synovial fluid levels of IL-2, IL-6, TNF alpha, IFN beta and IFN gamma were evaluated in a group of 66 patients with juvenile chronic arthritis (JCA). At the same time the ESR, CRP, hemoglobin, immunoglobulins, platelet count and Ritchie index were measured. In the serum of pauciarticular patients, IL-6 and TNF alpha levels were only slightly elevated compared with controls, but there was no correlation between these cytokines and clinical and other laboratory parameters. Serum IL-2 and IFN gamma were undetectable. In contrast, in the synovial fluid IL-6 levels were very high in all of the patients examined and there was a significant correlation between synovial fluid IL-6 levels and Ritchie's articular index. TNF alpha tended to be elevated but to a lesser extent, while synovial fluid IL-2 and IFN gamma were undetectable or very low, as in the serum. In polyarticular and systemic patients, on the other hand, serum IL 6 was elevated and statistically correlated with the majority of the laboratory parameters and with the Ritchie articular index. TNF alpha levels were only slightly elevated; on the other hand, IL-2 and IFN gamma were undetectable. There was an inverse correlation between IFN beta levels and the Ritchie articular index and a significant correlation with hemoglobin levels. In conclusion, our study demonstrates that not only IL-1 (as shown in other studies), but also IL-6 and to a lesser extent TNF alpha play a central role in the pathogenesis of JCA. IFN beta on the other hand, would seem to play an anti-inflammatory role. PMID- 7531127 TI - The value of cytoreductive surgery. PMID- 7531128 TI - PCR expression analysis of the estrogen-inducible gene BCEI in gastrointestinal and other human tumors. AB - A polymerase chain reaction (PCR) assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot) analysis. Presently, by using reverse transcription-PCR analysis, a series of primary tumor tissues and established tumor cell lines were tested for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene's product. The result demonstrate the superior sensitivity of PCR by indicating the gene's expression in cases where immunohistochemical testing remained negative. PMID- 7531129 TI - Feasibility study of voice-driven data collection in animal drug toxicology studies. AB - The object of this study was to determine the feasibility of using voice recognition technology to enable hands-free and eyes-free collection of data related to animal drug toxicology studies. Specifically, we developed and tested a prototype voice-driven data collection system for histopathology data using only voice input and computer-generated voice responses. The overall accuracy rate was 97%. Additional work is needed to minimize training requirements and improve audible feedback. We conclude that this architecture could be considered a viable alternative for data collection in animal drug toxicology studies with reasonable recognition accuracy. PMID- 7531130 TI - Renin mRNA is synthesized locally in rat ocular tissues. AB - Components of the Renin Angiotensin System (RAS) have been detected in ocular tissues and fluids. The source of the ocular RAS proteins is unknown but possibilities include diffusion or leakage from the systemic circulation, specific uptake from the blood, or local synthesis. We have used RT-PCR and in situ hybridization (ISH) to show that renin mRNA is present in ocular tissues from 3 strains of rats. By RT-PCR, we found 10 of 15 ciliary body samples, 13 of 16 iris samples, and 1 of 3 retina samples were positive for renin mRNA. Also, 6 of 6 brain and 7 of 8 kidney samples were positive. Using ISH, we found renin mRNA in the ciliary muscle adjacent to the sclera extending into the choroid. Tissue near the outflow channels of the anterior chamber angle also labeled. Retinal labeling was weak but present in the nerve fiber layer. Clusters of grains, possibly representing blood vessels, were also seen in the ciliary body, iris, and retina using ISH. These results suggest the presence of a local ocular RAS. PMID- 7531131 TI - Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium. AB - The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation. PMID- 7531132 TI - Palliative endobronchial brachytherapy for central lung tumors. A prospective, randomized comparison of two fractionation schedules. AB - AIM OF THE STUDY: Remote high dose rate brachytherapy is an effective local treatment modality for central lung tumors and has the potential to improve survival time. Optimal dose and fractionation schemes have not been identified yet. We conducted a prospective randomized study to compare two treatment schedules in terms of survival time, local tumor control, and possible complications. DESIGN: Group 1 received 4 brachytherapies with a dose of 3.8 Gy (at a 10-mm depth) on a weekly basis, and group 2 received 2 treatments with 7.2 Gy (at a 10-mm depth) at a 3-week interval. At a depth of 5 mm, the calculated doses would be 8 and 15 Gy. This study is still ongoing. Here we report interim results. PATIENTS: Ninety-three patients with advanced cancer were included in the study; 44 were in group 1 and 49, in group 2. Both groups were comparable regarding age, sex, tumor stage, Karnofsky performance status, and histologic findings. INTERVENTIONS: A mean total irradiation dose of 13.4 +/- 5.2 Gy for group 1 and 13.7 +/- 4.4 for group 2 were applied (calculated at 10 mm from the source axis, equivalent to 27.9 Gy in group 1 and 28.5 Gy in group 2 at a 5-mm depth). RESULTS: The 1-year survival rate was 11.4% in group 1 and 20.4% in group 2. No significant difference in survival time was found, but mean survival was longer in group 2 (49 weeks) than in group 1 (26 weeks). Local control after 3 months was comparable in both groups. Fatal hemoptysis occurred at a similar rate in group 1 (22.2%) and in group 2 (21.1%). CONCLUSION: High-dose rate brachytherapy with 2 x 7.2 Gy with a 3-week interval is equivalent to a 4 x 3.8 Gy regimen on a weekly basis. The shorter treatment schedule is more convenient for patients, does not cause more side effects, and provides an equal local tumor control. PMID- 7531133 TI - Bronchogenic carcinoma presenting as a pseudopregnancy. AB - A young woman with an occult bronchogenic carcinoma presented with secondary amenorrhea and an elevated beta subunit of human chorionic gonadotropin that was mistakenly attributed to pregnancy. Physicians should be aware that this carcinoma may present solely with an elevated beta human chorionic gonadotropin value, and the potential exists for confusion with a pregnancy state in women of childbearing age. PMID- 7531134 TI - Caso quiz. Neurogenous arthropathy caused by syringomyelia secondary to Chiari malformative syndrome. PMID- 7531135 TI - [Studies on HDV and HCV infection in plasma donors with positive HBsAg]. AB - Studies on HDV and HCV infection were carried out in 127 plasma donors and 152 non-donors carrying HBsAg. Results showed 7.9% (10/127) of the plasma donors and 1.3% (2/152) of the non-donors carrying HBsAg infected with HDAg, 41.7% (53/127) and 2.6% (4/152) with anti-HCV positive, and with prevalence rates of hepatitis of 18.1% (23/127) and 4.6% (7/152), respectively. The studies also indicated higher rates of HDV and HCV infection and prevalence of hepatitis may be caused by cross-infection during the course of plasmapheresis. PMID- 7531136 TI - [Transurethral non-contact laser treatment of prostatic hypertrophy]. AB - 66 cases of prostatic hypertrophy have been treated by transurethral non-contact Nd:YAG laser irradiation since April 1993. Sixty patients regained unobstructed urination within 1 week and 6 within 2 weeks after operation. The symptoms of the treated group were improved with the necrotic tissues gradually peeling off in 3 to 6 weeks. The short-term follow-up indicated that the prostatic gland distinctly reduced and residual urine decreased or vanished after treatment. Three patients had one of the following complications: urinary tract infection, epididymitis or urinous infiltration. These complications were treated appropriately. This therapy is ideal for the treatment of prostatic hypertrophy. PMID- 7531137 TI - [Staining test with methylene blue during resection of bladder cancer in 30 patients]. AB - From November 1991 to March 1993, staining test with methylene blue was applied in 30 patients with the bladder cancer during operation. Twelve patients (40%) showed positive results, and 27 specimens were obtained from the stained areas. Histological examination proved that they were carcinoma in situ (11), transitional cell carcinoma (7), squamous cell carcinoma (4), dysplasia (3), chronic cystitis (1), and normal mucosa (1). The positive rate of histological results was 96.3%. 21 specimens from the unstained areas showed negative results. Six (33%) of 18 patients with single tumor and 6 (50%) of 12 patients with multiple tumor were positively stained. Follow-up from 10 to 26 months showed one patient (3.3%) had recurrence after 8 months. PMID- 7531138 TI - [Transurethral laser prostatectomy: report of 200 cases]. AB - From August 1988 to May 1993, 200 patients with benign prostatic hyperplasia (BPH) were treated by transurethral laser prostatectomy (TULP) in our hospital. 165 patients were followed up for 6 months to 4.5 years and the results showed excellent effect in 128 cases, improved 32 cases, the total effectiveness was 97%. TULP, a safe and effective therapy for BPH, have more wide operative indication range but it is not suitable for patients with carcinoma of prostate. PMID- 7531139 TI - Metal interactions with voltage- and receptor-activated ion channels. AB - Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E 115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions. PMID- 7531140 TI - Evaluation of a commercial rRNA amplification assay for direct detection of Mycobacterium tuberculosis in processed sputum. AB - A commercial assay (Amplified Mycobacterium tuberculosis Direct Test, Gen Probe) which combines transcription-mediated amplification of target rRNA with amplicon detection by a chemiluminescent DNA probe for the rapid detection of Mycobacterium tuberculosis in sputum was evaluated. The test was applied to consecutively collected, NALC/NaOH processed sputum sediments from two laboratories (H and L), each serving a different population of patients with pulmonary tuberculosis. Results were compared to those of fluorochrome staining and culture. A total of 760 specimens obtained from 246 patients were used for the study. The test was positive in 141 of 144 (98%) specimens that were fluorochrome-positive and culture-positive for Mycobacterium tuberculosis. Fifteen of 31 specimens that were fluorochrome-negative, culture-positive were also assay-positive. A total of 312 specimens (100 patients) from laboratory H (prevalence = 10%) and 448 specimens (146 patients) from laboratory L (prevalence = 34%) were analyzed. Compared to culture, test sensitivity, specificity, positive predictive and negative predictive values were 65%, 99%, 94% and 97%, respectively, for laboratory H and 93%, 99%, 99% and 97%, respectively, for laboratory L. If the results were analyzed on the basis of at least one concordant result between the amplification assay and culture in three sputum samples per patient, then the sensitivity, specificity, positive and negative predictive values for identifying infected patients was 70%, 99%, 87% and 97%, respectively, for laboratory H, and 100%, 98%, 96% and 100%, respectively, for laboratory L. PMID- 7531141 TI - Detection of Chlamydia pneumoniae in clinical specimens by polymerase chain reaction using nested primers. AB - A nested primers strategy was used to develop a two-step PCR test for the direct species-specific detection of the 16s rRNA gene of Chlamydia pneumoniae. This test was applied to 58 nasopharyngeal or oropharyngeal swab specimens collected from patients in studies of community-acquired pneumonia and in a local outbreak of respiratory disease. Twelve patients (21%) showed evidence of Chlamydia pneumoniae infection in serological tests (7/56; 13%), culture (8/58; 14%) or PCR (10/58; 17%). Nested PCR but not single-step PCR was found to be as sensitive as culture or serology for detection of infection with this organism. In summary, nested PCR can be useful in direct testing of clinical specimens for Chlamydia pneumoniae, making additional DNA purification steps unnecessary. PMID- 7531142 TI - Epstein-Barr virus isolates with the major HLA B35.01-restricted cytotoxic T lymphocyte epitope are prevalent in a highly B35.01-positive African population. AB - An influence of cytotoxic T lymphocyte (CTL) response over Epstein-Barr virus (EBV) evolution was first suggested by the finding that virus isolates from highly HLA-A11-positive Oriental populations were specifically mutated in two immunodominant A11-restricted CTL epitopes. Here we turn to a second HLA allele, B35.01 and show that B35.01-restricted CTL responses in Caucasian donors reproducibly map to a single peptide epitope, YPLHEQHGM, representing residues 458-466 of the type 1 EBV nuclear antigen 3A protein (B95.8 strain). In this case, however, most EBV isolates from a highly B35.01-positive population (in The Gambia) either retained the CTL epitope sequence or carried a mutation (P-->S at position 2) which conserved antigenicity; changes leading to reduced antigenicity (Y-->N at position 1) were found in only a minority of cases. Furthermore, CTL recognizing the YPLHEQHGM epitope could be reactivated from the blood of some B35.01-positive Gambian donors by in vitro stimulation with the synthetic peptide, indicating that epitope-specific immunity does exist in this population. Possible differences between the A11-based and B35.01-based studies are discussed. PMID- 7531143 TI - Characterization of two Epstein-Barr virus epitopes restricted by HLA-B7. AB - We have identified two epitopes for Epstein-Barr virus specific cytotoxic T lymphocytes (CTL) restricted by the common allele HLA-B7. They are EBNA 3C 881-9 (QPRAPIRPI) and EBNA 3A 379-387 (RPPIFIRRL). The epitopes conform well to the recently described motif for HLA-B7-binding peptides (Huckzo et al., J. Immunol. 1993. 151:2572). Titration of the peptides in CTL assays and detergent lysate binding assays revealed that extending the peptides at either the N or C terminus did not reduce their affinity for HLA-B7. This behavior contrasted with HLA-B51, which binds peptides with a similar motif to B7, and has identical amino acid residues at sites expected to form the "F" pocket of the peptide-binding groove. HLA-B51 also bound the peptide EBNA 3C 881-9, but was unable to bind peptides extended at the C terminus. PMID- 7531144 TI - Recombinant CD4-IgE, a novel hybrid molecule, inducing basophils to respond to human immunodeficiency virus (HIV) and HIV-infected target cells. AB - Basophils and mast cells, as the main effector cells in IgE-mediated type I hypersensitivity, are involved in the elimination of parasites and, according to recent findings, may also play an important role in the defense against bacterial and viral infections. Using a genetic engineering approach we wanted to redirect this potent IgE-mediated defense system against intruding human immune deficiency virus. We constructed a recombinant CD4-IgE molecule, consisting of the two N terminal domains of CD4 and the CH2-4 domains of the IgE heavy chain, thus providing the IgE with specificity for the gp120 of human immunodeficiency virus (HIV). The binding properties of hybrid CD4-IgE to the high-affinity receptor for IgE (Fc epsilon RI) on basophils as well as to the low-affinity receptor (Fc epsilon RII or CD23) for IgE on lymphoid cells were found to be similar to those of native IgE. At the same time, the CD4 domains of the recombinant molecule retained the gp120 binding specificity with an affinity similar to that of the native CD4. By functional tests, we demonstrated that CD4-IgE armed basophils can be triggered by free HIV and by HIV-infected cells to release their mediators. We further show that HIV-triggered basophils lead to a decreased replication of HIV in susceptible T cells. We, therefore, conclude that the type I hypersensitivity effector cells can be engaged in the elimination of HIV-infected cells, at least in vitro. Because of the strong binding of the CD4-IgE construct to the Fc epsilon RI, we assume that CD4-IgE has a short t1/2 in serum, but may similarly to IgE exhibit prolonged resident time on basophils and mast cells, which are located close to mucosal surfaces or in the connective tissue. Thus CD4-IgE could play an important role in the elimination of HIV also in vivo. PMID- 7531145 TI - Reciprocal expression of co-stimulatory molecules, B7-1 and B7-2, on murine T cells following activation. AB - The co-stimulatory B7 molecules (B7-1 and B7-2) are expressed on professional antigen-presenting cells in mice. In this study, we demonstrate that B7-1 (CD80) and B7-2 (CD86) are also expressed on murine T cells in the absence of major histocompatibility complex class II molecules. The temporal expression of these two molecules on T cells varies with the state of activation where resting T cells express B7-2 but show little or no expression of B7-1. Following activation, B7-2 expression is down-regulated and there is a concomitant increase in the expression of B7-1 on the cell surface which peaks at about 72 h. Thus these two co-stimulatory molecules are reciprocally expressed on the T cell surface. This pattern of expression of B7-1 and B7-2 on T cells suggests that these two molecules may have different roles in the generation and regulation of immune responses. PMID- 7531146 TI - Evidence for intact costimulation via CD28 and CD27 molecules in hyporesponsive T cells from human immunodeficiency virus-infected individuals. AB - In the activation of T cells, the primary signal is antigen-specific and given through T cell receptor (TcR)/CD3 ligation. Furthermore, costimulatory molecules such as CD28 and CD27, provide an essential signal for activation through interaction with their ligands, present on the membrane of antigen-presenting cells. During asymptomatic human immunodeficiency virus (HIV)-1 infection, T cell function is progressively lost. Here, we investigated whether in the presence of impaired responses of T cells from HIV-infected individuals to signal one, costimulation through CD28 and CD27 after interaction with their natural ligands CD80 and CD70 is intact. T cell proliferative responses to signal one in combination with CD80 or CD70 were decreased in a large fraction of asymptomatically HIV-infected individuals. This was due to impaired responses of signal one but not to impaired responses to costimulation, since CD80 or CD70 did enhance signal one-mediated proliferative responses to a normal extent. Moreover, in individuals with proliferative responses to signal one that were decreased to 50% of normal T cell responses, costimulation even was increased compared to controls. Our results demonstrate that in HIV-infected individuals the response to costimulation is relatively preserved compared to responses to the first signal and point to the defect in T cells in HIV infection being primarily in the CD3/TcR-mediated pathway. PMID- 7531148 TI - Reduced development of CD4-8-B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules. AB - The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4-8-B220+ T cell receptor-alpha/beta+ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with beta 2-microglobulin (beta 2m)-deficient mice. Interestingly, these lpr beta 2m-/- mice develop 13-fold fewer DNT cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lpr beta 2m-/- mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in lpr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage. PMID- 7531147 TI - Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement. AB - HIV-1, in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species-specific protection mechanism. HIV-1 was expressed in cells of non-human origin. Recombinant HIV-1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS-1 cells, mink cells and, as a control, in human HEp-2 cells and were then used to infect CD4-positive target cells. Analysis of the CAT activity of the target cells revealed that fresh HIV-1-negative human serum reduced the infectivity of HIV-1 derived from monkey and mink cells five- to tenfold, but had no effect on HIV-1 produced in human cells. In addition, human serum efficiently lysed HIV-1 produced in non-human cells in contrast to HIV-1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane-bound regulatory molecules. Two of these complement inhibitors, namely decay-accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV-1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV-1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV-1 in a species-specific way against the attack of human complement. PMID- 7531149 TI - Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta on human monocytes. AB - The activities of six synthetic CC chemokines, MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta on human blood monocytes were studied. All CC chemokines elicited a bimodal migration response in vitro. Highest numbers of migrating cells were obtained with the monocyte chemotactic proteins (MCP) and RANTES, somewhat lower numbers with MIP-1 alpha, and only weak migration with MIP-1 beta. The most potent attractants were MCP-1 and MIP-1 alpha which reached maximum efficacy at 0.1 to 1 nM. All CC chemokines also induced the release of N-acetyl beta-D-glucosaminidase from cytochalasin B-pretreated monocytes. The MCP were most effective (MCP-1 > MCP-3 > MCP-2), RANTES and MIP-1 alpha showed moderate (1/3 of MCP-1 activity), and MIP-1 beta only minimal activity. Cytosolic free Ca2+ changes and exocytosis were used to monitor receptor desensitization. Marked cross-desensitization was observed among MCP-1, MCP-2 and MCP-3 on the one hand, and RANTES, MIP-1 alpha and MIP-1 beta on the other, indicating receptor sharing within these two subgroups of CC chemokines. The responses to RANTES, MIP-1 alpha and MIP-1 beta were also moderately to markedly desensitized by pretreatment with MCP-1, MCP-2 or MCP-3, while the responses to the MCP were virtually unaffected by pretreatment with RANTES, MIP-1 alpha and MIP-1 beta. These results suggest that the MCP also interact with receptors recognized by RANTES, MIP-1 alpha and MIP-1 beta, but not vice versa. Binding studies were performed with radiolabeled MCP-1 or MIP-1 alpha. All MCP competed readily for labeled MCP-1 yielding a concentration-dependent sigmoidal displacement curve. Displacement with RANTES, MIP-1 alpha and MIP-1 beta was observed at higher concentrations, but was not complete. Radiolabeled MIP-1 alpha was displaced efficiently by MIP-1 alpha or MIP-1 beta, but only partially by RANTES. Of the MCP, only MC-3 completely displaced MIP-1 alpha, while only partial displacement was observed with MCP-1 and MCP-2. PMID- 7531150 TI - The clonal composition of myelin basic protein-reactive encephalitogenic T cell populations is influenced both by the structure of relevant antigens and the nature of antigen-presenting cells. AB - Studies of experimental autoimmune encephalomyelitis (EAE) in rodents have revealed that encephalitogenic T cell lines reactive with myelin basic protein (BP) are frequently dominated by clones expressing a restricted T cell receptor repertoire. Using the rat EAE model, we have begun to examine the basis for clonal dominance within BP-reactive T cell lines. We find that variations introduced into the standard protocol of periodic antigen stimulation produce marked shifts in the representation of different clones within encephalitogenic T cell populations. For example, altering the source of antigen-presenting cells (APC), while holding antigen (BP) constant, and substituting BP from guinea pig (GPBP) for that of the rat antigen (RBP) with constant APC, both cause shifts in the composition of the dominant clones within BP-reactive T cell lines. Our results suggest that: (i) adherence to an invariant protocol of antigen challenge may lead to an underestimation of the diversity of BP-reactive encephalitogenic T cell populations; and (ii) the minor structural differences between GPBP and RBP not only cause the weak immunogenicity of RBP but also result in the alteration of different T cell subsets. These observations indicate that apparent restrictions upon the repertoire of autoimmune T cells should be interpreted with caution when such cells are elicited by immunization with foreign antigens. PMID- 7531151 TI - A cytotoxic CD40/p55 tumor necrosis factor receptor hybrid detects CD40 ligand on herpesvirus saimiri-transformed T cells. AB - The B cell activation molecule CD40 and the p55 tumor necrosis factor receptor (p55TNFR) belong to the same family of structurally conserved proteins. We constructed a chimeric receptor consisting of the CD40 extracellular and transmembrane domains and the p55TNFR intracellular domain. This receptor hybrid retained the biological activity and the ligand specificity of the respective wild-type receptor domains. Thus it exerted a marked cytotoxic effect in three different transfected cell lines after activation not only with anti-CD40 antibody but also with CD40 ligand (CD40L) in soluble and membrane-bound forms. Using hybrid-transfected baby hamster kidney cells we demonstrated that herpesvirus saimiri-transformed human CD4+ T lymphocytes constitutively express bioactive CD40 ligand on their surface. The hybrid receptor-based assay was highly specific for CD40 activating reagents and more sensitive than an assay measuring CD40-mediated B cell rescue from apoptosis. Hence CD40/p55TNFR transfectants may be useful for dissecting CD40L-mediated events in T-B cell interactions, and also to detect a defective CD40L molecule in putative hyper-IgM syndrome patients. PMID- 7531153 TI - Constitutive or inducible overexpression of the IGF-2 gene in cells of a human colon carcinoma cell line. AB - Two types of clones have been isolated from the SW613-S human colon carcinoma cell line. Clones with a high level of amplification of the c-myc gene are tumorigenic in nude mice and can proliferate in chemically defined, serum-free medium, whereas clones with a low level of amplification are nontumorigenic and cannot multiply in defined medium. The expression level of the insulin-like growth factor type 1 (IGF-1) gene is low in tumorigenic clones and undetectable in nontumorigenic clones. Tumorigenic clones produce high levels of IGF-2 (and IGF-binding proteins), compared to nontumorigenic clones. This is the consequence of a differential transcriptional regulation of the IGF-2 gene between the two types of clones. This regulation consists of a modulation of the activity of promoters P3 and P4. Overexpression of the IGF-2 gene is constitutive in tumorigenic clones: it is stably maintained during in vitro propagation of the cells. Tumorigenic cell lines obtained after transfer of c-myc gene copies into the cells of nontumorigenic clones exhibit a high level of expression of the IGF 2 gene when they are grown in vivo, as subcutaneous tumors in nude mice. This high level of expression is lost in most of these cell lines when they are returned to in vitro culture conditions indicating that, in these cells, IGF-2 overexpression is not constitutive but inducible by in vivo growth conditions. We had previously shown that tumorigenic clones use the overproduced IGF-2 as an autocrine growth factor. The results reported here suggest than IGF-2 overexpression has an important role in the tumorigenic phenotype of these cells. PMID- 7531154 TI - Retinol-binding protein mediates uptake of retinol to cultured human keratinocytes. AB - Retinol (vitamin A) circulates in the blood bound to retinol-binding protein (RBP). The process by which target cells acquire retinol is not fully elucidated, although a cell surface receptor for RBP has recently been identified. We show here that retinol is at least an order of magnitude more efficient at blocking the terminal differentiation of cultured normal human keratinocytes when administered as a complex with RBP than when administered free in solution. This inhibition of differentiation by RBP can be reversed by monoclonal antibody P142, reactive toward the RBP-binding membrane protein p63. These results demonstrate, at least in this in vitro system, the importance of the RBP receptor in the generation of a cellular response to retinol. PMID- 7531152 TI - Effects of single and chronic treatment with tranylcypromine on extracellular serotonin in rat brain. AB - We have examined the effects of tranylcypromine, a monoamine oxidase inhibitor used as antidepressant, on the tissue and extracellular concentration of serotonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in frontal cortex and dorsal raphe nucleus using microdialysis in conscious rats. Single treatment with tranylcypromine sulphate (0.5, 3 and 15 mg/kg, i.p.) dose dependently elevated dialysate 5-HT in both areas but more markedly in the DRN. Extracellular and tissue 5-HT concentrations were affected by the drug in a different manner. The former increased sharply when tissue 5-HT reached a plateau. This may have reflected saturation of intracellular stores and overflow of the amine. In contrast, tissue and extracellular 5-HIAA concentrations--that indicate metabolic effects of tranylcypromine--were affected similarly. A 2-week treatment with 0.5 mg/kg.day of tranylcypromine sulphate increased basal extracellular 5-HT in frontal cortex and dorsal raphe nucleus (ca. 220%) whereas a further injection of 0.5 mg/kg was without effect in both areas. Thus, chronic, but not acute, treatment with low doses of tranylcypromine increases extracellular 5-HT concentration, suggesting that clinical effects of this monoamine oxidase inhibitor are related to its capacity to enhance serotonergic transmission. PMID- 7531155 TI - Protracted postnatal development of corticospinal projections from the primary motor cortex to hand motoneurones in the macaque monkey. AB - We have studied the development of corticospinal projections from the hand area of the primary motor cortex to the spinal cord using anterograde transport of WGA HRP. In the neonate, as in the adult, corticospinal projections to the intermediate zone at the C8/T1 spinal level were clearly present. However, in contrast to the adult, there was only very faint and barely visible labelling in the dorso-lateral motor nuclei which supply the hand muscles. No aberrant projections to other motor nuclei were seen. By 2.5 months, a ring of dense labelling was present around the dorso-lateral motor nuclei, but labelling was still sparse in the central region. This labelling was more pronounced at 11 months, but was still not as heavy as in the adult. There was no labelling among the ventral motoneurones at any age. The conduction velocity (c.v.) of the fastest corticospinal fibres was determined in each of the monkeys. There was an age-related increase in c.v. within the spinal cord. At birth, the fastest axons had a c.v. of only 8 m.s-1. At 11 months c.v. was still substantially slower (55 m.s-1) than the adult value of 73 m.s-1. In contrast, by 11 months, the axonal c.v. within the brain was close to the adult value, suggesting a rostro-caudal maturation of the corticospinal system. Our results demonstrate that corticospinal projections in the macaque monkey mature gradually over a period of at least 11 months, much longer than previously thought. PMID- 7531156 TI - Substance P-containing hypothalamic afferents to the monkey hippocampus: an immunocytochemical, tracing, and coexistence study. AB - In order to identify the synaptic connections of substance P-containing afferents within the hypothalamo-hippocampal projection of the monkey, we performed a combined light and electron microscopic, immunocytochemical study, made lesions of the fimbriafornix, and employed retrograde tracing using WGA-HRP. Furthermore, coexistence studies for substance P and GAD were performed to identify the putative transmitters of these hypothalamic projection neurons. A plexus of large substance P-immunoreactive terminals was identified in both the innermost portion of the molecular layer and in CA2. Axon terminals in both plexuses established exclusively asymmetric synapses with spines and dendritic shafts. Substance P immunoreactive boutons were degenerating 5 days after lesioning, and had disappeared 10 days after ipsilateral fimbria-fornix transection. Thus, these terminals were of extrinsic origin. In contrast, immunoreactive fibers in the outer third of the dentate molecular layer remained unaffected by the lesion. Retrograde tracing combined with immunostaining for substance P revealed the parent cell bodies of the extrinsic substance P-containing afferents in the supramammillary nucleus. Colocalization studies employing a consecutive semi-thin sections technique indicate that these large substance P-containing projection neurons lack GABA as an inhibitory transmitter. These results suggest that hypothalamic afferents of the monkey hippocampus contain substance P. Because these afferents lack GABA as an inhibitory transmitter and establish exclusively asymmetric synapses, this projection may excite hippocampal target neurons. PMID- 7531158 TI - The 16S/23S ribosomal spacer region of Coxiella burnetti. AB - The 16S/23S spacer region of Coxiella burnetti isolate Nine Nile, phase 1, was sequenced. Sequence analysis revealed two tRNA coding regions for tRNA(Ile) and tRNA(Ala). DNA sequence alignment demonstrated significant homology with tRNA species from Pseudomonas aeruginosa and Rhodobacter sphaeroides, respectively. The non-coding tRNA spacer region was unique to Coxiella burnetti, based on database alignment. PMID- 7531157 TI - The periaqueductal gray in the cat projects to lamina VIII and the medial part of lamina VII throughout the length of the spinal cord. AB - The periaqueductal gray (PAG) plays an important role in analgesia as well as in motor activities, such as vocalization, cardiovascular changes, and movements of the neck, back, and hind limbs. Although the anatomical pathways for vocalization and cardiovascular control are rather well understood, this is not the case for the pathways controlling the neck, back, and hind limb movements. This led us to study the direct projections from the PAG to the spinal cord in the cat. In a retrograde tracing study horseradish peroxidase (HRP) was injected into different spinal levels, which resulted in large HRP-labeled neurons in the lateral and ventrolateral PAG and the adjacent mesencephalic tegmentum. Even after an injection in the S2 spinal segment a few of these large neurons were found in the PAG. Wheat germ agglutinin-conjugated HRP injections in the ventrolateral and lateral PAG resulted in anterogradely labeled fibers descending through the ventromedial, ventral, and lateral funiculi. These fibers terminated in lamina VIII and the medial part of lamina VII of the caudal cervical, thoracic, lumbar, and sacral spinal cord. Interneurons in these laminae have been demonstrated to project to axial and proximal muscle motoneurons. The strongest PAG-spinal projections were to the upper cervical cord, where the fibers terminated in the lateral parts of the intermediate zone (laminae V, VII, and the dorsal part of lamina VIII). These laminae contain the premotor interneurons of the neck muscles. This distribution pattern suggests that the PAG-spinal pathway is involved in the control of neck and back movements. Comparing the location of the PAG-spinal neurons with the results of stimulation experiments leads to the supposition that the PAG-spinal neurons play a role in the control of the axial musculature during threat display. PMID- 7531159 TI - Rapid Alzheimer-like phosphorylation of tau by the synergistic actions of non proline-dependent protein kinases and GSK-3. AB - Tau protein from Alzheimer disease (AD) brain is phosphorylated at eleven Ser/Thr Pro and nine Ser/Thr-X sites. The former sites are phosphorylated by proline dependent protein kinases (PDPKs), the latter by non-PDPKs. The identities of both the PDPKs and non-PDPKs involved in AD tau hyperphosphorylation are still to be established. In this study we have analyzed the interactions between a PDPK (GSK-3) and several non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) in the phosphorylation of one isoform (tau 39) of human tau. We found that the rate of phosphorylation of tau 39 by GSK-3 was increased several-fold if tau were first prephosphorylated by the non-PDPKs. Further, several Alzheimer-like epitopes in tau can be induced only slowly after phosphorylation of tau by GSK-3 alone. After a prephosphorylation of tau by the non-PDPKs, however, the rate of induction of these epitopes by GSK-3 is increased several-fold. These results suggest that one role of non-PDPK-catalyzed phosphorylation is the modulation of PDPK-catalyzed phosphorylation of tau in AD brain. PMID- 7531161 TI - Intercellular channels in teleosts: functional characterization of two connexins from Atlantic croaker. AB - Gap junction channels, composed of protein subunits termed connexins, are believed to play a critical role in the process of oocyte differentiation and maturation. We have used the paired Xenopus oocyte assay to characterize functionally two connexin genes, connexin-32.2 and connexin-32.7, recently cloned from the ovary of the Atlantic croaker (Micropogonia undulatus), a species that has emerged as a useful model to study the process of maturation of the ovarian follicle. We have found that, while both connexin proteins were expressed at comparable levels in Xenopus oocytes, only one, connexin-32.2, was functionally competent to induce the formation of intercellular channels. Connexin-32.2 channels exhibited voltage-dependent closure that was similar to, but distinct from that of previously characterized mammalian connexins. In addition, the silent connexin-32.7 was unable to functionally interact with connexin-32.2, either in heterotypic channels or as dominant negative inhibitor. Because connexin-32.2 expression is strikingly regulated during oocyte maturation, these data provide further evidence for a role of intercellular channels in the control of oocyte-follicular cell interactions. PMID- 7531160 TI - The Drosophila cation channel trpl expressed in insect Sf9 cells is stimulated by agonists of G-protein-coupled receptors. AB - Structures and regulations of vertebrate channels responsible for sustained calcium elevations after hormone stimulation are largely unknown. Therefore, the Drosophila photoreceptor channels, trp and trpl, which are assumed to be involved in calcium influx, serve as model system, trpl expressed in Sf9 cells showed spontaneous activity. Hormonal stimulations of calcium influx (detected by fura 2) and of an outwardly rectifying current were observed in Sf9 cells coinfected with baculoviruses encoding trpl and various heptahelical receptors for histamine, thrombin, and thromboxane A2, all known to cause phospholipase C-beta activation in mammalian cells. Although the identity of the G-proteins and of possible second messengers involved need to be clarified, it is clear that trpl represents a receptor/G-protein regulated cation channel. PMID- 7531162 TI - N-terminal truncated forms of insulin-like growth factor binding protein-3 in the peritoneal fluid of women without laparoscopic evidence of endometriosis. Le groupe d'investigation en gynecologie. AB - OBJECTIVE: To characterize and purify peritoneal mitogens able to stimulate the proliferation of human endometrial cells in vitro. DESIGN: Peritoneal fluids (PFs) from 50 patients were collected at laparoscopy and pooled (270 mL) for purification of mitogenic activity. SETTING: University infertility clinic and endocrinology of reproduction and molecular endocrinology laboratories. PATIENTS: Fifty subjects presenting for tubal ligation, pelvic pain, mass, or infertility but otherwise having no evidence of endometriosis inflammation, infection, or tumor. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Assessment of mitogenic activity by 3H-thymidine incorporation into mouse embryo fibroblasts and into primary cultures of isolated epithelial and stromal cells of human endometrium. RESULTS: The PF mitogens were purified successively on carboxymethyl-sepharose and heparin-sepharose columns followed by fractionation on cartridges of C18 silica and reverse-phase high-performance liquid chromatography (HPLC). Four distinct bands were eluted from Sep-Pak, (Mississauga, Ontario, Canada) with molecular weights of 17 to 18, 20, 25, and 29 to 30 kd. The eluted fractions of Sep-Pak exerted preferential mitogenic activity on epithelial-derived human endometrial cells at an equimolar ratio with epidermal growth factor. Microsequencing of the 17 to 18, 20, 25, and 29 to 30 kd bands showed a homologous sequence with N-terminal amino acid sequences of insulin-like growth factor binding protein-3 (IGFBP-3). CONCLUSION: These data indicate that the PF of normal women without evidence of endometriosis contains N-terminal truncated forms of IGFBP-3 that mediate an apparent preferential mitogenic action on epithelial-derived endometrial cells. Therefore, they could play a role in the ectopic growth of endometrial cells. PMID- 7531164 TI - Cytokine stimulation of CD34+ bone marrow cells prior to cryopreservation enhances their post-thawing proliferative potential. AB - Marrow aplasia remains a significant cause of morbidity and mortality in the peri transplant period. Administration of recombinant human hematopoietic growth factors along with the marrow graft is a widely used strategy to ameliorate this problem. Though arguably effective, this approach is extremely expensive. To develop alternative strategies for stimulating marrow engraftment, we investigated the utility of stimulating CD34+ enriched bone marrow cells with cytokines prior to cryopreservation. We found that culturing these cells for 1 to 3 days in Iscove's medium supplemented with kit ligand (KL), interleukin-3 (IL 3), interleukin-1 beta (IL-1 beta) and 20% bovine calf serum before freezing doubled the proliferative capacity of both myeloid and erythroid progenitor cells after thawing. Confirmation of increased proliferative activity in vivo would suggest that this approach might significantly shorten the period of marrow aplasia post transplant at far less expensive means. PMID- 7531163 TI - Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. AB - OBJECTIVE: To examine whether placental protein 14 (PP14) may affect directly those sperm functions crucial to fertilization and early embryo development. DESIGN: In these prospective studies, we evaluated semen samples of fertile men incubated under capacitating conditions with and without PP14. SETTING: Academic tertiary institution. INTERVENTIONS: Biologically active PP14 was purified from human midtrimester amniotic fluid by anion exchange and immunoaffinity chromatography. After separation of the motile fraction, spermatozoa were incubated for 30 minutes with or without PP14 (concentration range of 0.01 to 100 micrograms/mL), washed, and then aliquots were prepared for use in the different assays. Human sperm-zona pellucida (ZP) binding was assessed using the hemizona assay (HZA) in a 4-hour gametes coincubation period. Sperm motility parameters were evaluated using a computerized semen analyzer. The acrosome reaction (AR) was determined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and indirect immunofluorescence. MAIN OUTCOME MEASURES: Sperm-ZP binding, sperm motility patterns, and AR. RESULTS: Preincubation of sperm (and not the hemizonae) with PP14 produced a significant and dose-dependent inhibition of binding in the HZA. Monoclonal antibodies generated against PP14 showed no direct effect in the HZA and partially neutralized the inhibitory activity of PP14 in the HZA. Insulin-like growth factor binding protein-1 (IGFBP-1), an endometrial stromal cell product, showed no effect in the HZA. Neither PP14 nor IGFBP-1 interfered with sperm motility parameters or the AR. CONCLUSIONS: Placental protein 14 produced a potent, fast, and dose-dependent inhibition of binding of human spermatozoa to the human ZP without affecting other prefertilization events (i.e., hyperactivated motility or AR). The detrimental effect on sperm-zona interaction seems to be specific for this endometrial epithelial protein (not observed with an endometrial stromal product) and may have fundamental bearance to the fertilization process thus providing a mechanism for endometriosis-related infertility. PMID- 7531166 TI - Urinary symptoms in older men, their investigation and management: is there an epidemic of undetected morbidity in the waiting room? AB - As in other developed countries with an ageing population, there is a growing perception in Australia that urinary symptoms are a major cause of morbidity among older men. This study aimed to determine the prevalence of urinary symptoms amongst male patients in general practice, their presentation, investigation and management. Randomly selected general practitioners' (GPs') surgeries in five mainland state capital cities in Australia were involved. A consecutive sample of male patients aged 50 years and over was asked to complete a self-administered questionnaire about urinary symptoms, allowing a symptom score to be calculated. For each participating patient, GPs completed a checklist about symptoms, investigation and management without knowledge of the patient's answers. Sixty eight per cent of GPs agreed to participate in the study and 95% of patients participated, resulting in 4268 patient questionnaires and 4255 GP checklists. Men bothered by severe symptom scores were significantly more likely than men with mild symptoms to be presenting with a urinary problem. For no city did the proportion of men with severe symptom scores who were not presenting for a urinary problem exceed 7%. Other measures of morbidity were similarly low in this group. GPs investigated 61% of urological symptoms presenting on the day and initiated treatment for less than a third. Approximately 10% of the total sample had had a transurethral resection of the prostate: 9% of these had severe symptom scores. It was concluded that almost all older men in general practice who are sufficiently troubled by urinary symptoms will see their GP for advice at some time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531165 TI - Immunohistochemical localization of neuropeptides in nerve fibers of the porcine vagina and uterine cervix. AB - The distribution of vasoactive intestinal polypeptide, substance P, Met5 enkephalin-Arg6-Gly7-Leu8, neuropeptide Y, calcitonin gene-related peptide and bombesin/gastrin releasing peptide was studied immunohistochemically in nerve fibres supplying the vagina and uterine cervix of sexually immature pigs. Nerves containing neuropeptide Y- and Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactivity were particularly numerous in the uterine cervix and vagina. Vasoactive intestinal polypeptide- and and calcitonin gene-related peptide-containing nerves were less numerous, whereas the bombesin/gastrin releasing peptide- and substance P-immunoreactive nerves were scarce in these organs. Generally, the immunoreactive fibres, nerve networks or bundles were distributed under the serous membrane, among smooth muscle of muscular membranes, in the submucosal layer and under the luminar epithelium of the uterine cervix and vagina. PMID- 7531167 TI - Immunohistochemical localization of chondroitin sulfate proteoglycan and tenascin in the human eye compared with the HNK-1 epitope. AB - BACKGROUND: A previous study revealed the HNK-1 epitope in the human ciliary body beneath the ciliary epithelium. The molecules bearing this 3-sulphoglucuronic acid-containing oligosaccharide epitope in the eye remain unknown. As chondroitin sulphate proteoglycan (CSPG) and tenascin are potential candidates as bearers of the HNK-1 epitope, their distribution in the human eye was compared with that of the HNK-1 epitope. METHODS: Fifty-five formalin-fixed, paraffin-embedded human eyes, including 20 normal eyes and 35 eyes with exfoliation syndrome or glaucoma, were studied immunohistochemically with monoclonal antibody (MAb) CS-56 to CSPG, MAb TN2 to tenascin, and MAbs HNK-1 and VC1.1 to the HNK-1 epitope. Additionally, four frozen lens capsules with exfoliation material were studied by indirect immunofluorescence. RESULTS: A population of dendritic cells in the inner connective tissue layer of the ciliary body and exfoliation material were immunoreactive with antibodies to the HNK-1 epitope, but no labelling for CSPG and tenascin was seen in them, including frozen sections. The inner surface of the nonpigmented ciliary epithelium was reactive for the HNK-1 epitope, and at the ora serrata also for CSPG. In some eyes with glaucoma, immunoreaction for CSPG and tenascin was seen beneath the epithelium and endothelium of the cornea. The nerve fibre layer of the retina was labelled for tenascin. In the sclera, all antibodies labelled the ground substance, and in some large blood vessels immunoreaction for CSPG and tenascin was seen subendothelially. CONCLUSION: Apart from the sclera, the distribution of CSPG and tenascin was different form that of the HNK-1 epitope, suggesting that this carbohydrate epitope may not be borne by these molecules in the human ciliary body. PMID- 7531169 TI - [Are there alternative therapeutic methods for breast carcinoma? Status and perspectives for treatment of metastatic breast carcinoma]. PMID- 7531171 TI - Expression of von Willebrand factor in normal and diseased rat livers and in cultivated liver cells. AB - Von Willebrand factor (vWf) is an adhesive glycoprotein known to play an important role in hemostasis and in tissue injury. Because the latter process resembles hepatic fibrogenesis, we studied the tissue distribution of vWf in diseased livers. In normal rat liver vWf was strongly expressed in the vascular endothelium and as small spots or fiber-like structures in the hepatic parenchyma. During acute liver injury, pronounced staining was observed within the area of necrosis. In fibrotic livers vWf deposits were distributed predominantly at the scar-parenchyma interface but also within the septum and in sinusoidal lining cells. Testing different liver cell populations in vitro demonstrated that vWf gene expression was limited to endothelial cells (ECs) and, therefore, the latter cell population might represent the vWf-positive cells detected in vivo. The distribution of vWf within fibrotic septa suggests that vWf becomes a component of the extracellular matrix (ECM) in fibrotic livers. PMID- 7531170 TI - Persistence of non clonal hematopoietic progenitor cells in blastic phase chronic myelogenous leukemia (CML). Working Party on Severe Aplastic Anemia (WPSAA) of the European Group of Bone Marrow Transplantation (EBMT). AB - Normal and clonal hematopoietic progenitor cells have been demonstrated to coexist in chronic-phase chronic myelogenous leukemia (CML), but few data are available on the presence of non neoplastic hematopoiesis during the blastic transformation phase. We used reverse transcription-polymerase chain reaction (RT PCR) to investigate expression of the BCR/ABL transcript of individual hematopoietic progenitors from a CML patient in blastic phase. We demonstrate that non clonal hematopoiesis is induced to re-emerge by conventional chemotherapy containing fludarabine. In addition, we confirm that some pluripotent CD34+/CD33-/DR- cells circulating in the peripheral blood are not clonal. Our data provide an encouraging basis for further studies of in vitro purification of normal hematopoietic stem cells in advanced stage CML and of their use in the context of autologous bone marrow transplantation. PMID- 7531168 TI - Transforming growth factor-beta regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway. AB - BACKGROUND: Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of many ocular diseases, including proliferative vitreoretinopathy. We examined the effect of TGF-beta on the phagocytosis of rod outer segments by retinal pigment epithelium (RPE), which is a major function of RPE, and investigated the dependence of this effect on the protein kinase C (PKC) pathway. METHODS: Phagocytotic uptake of fluoresceinated bovine rod outer segments was determined by flow cytometry. RPE cells were treated with TGF-beta 1 or TGF-beta 2 and their effects on phagocytosis were examined. The effects of various PKC inhibitors (calphostin C, staurosporine, and extended exposure to phorbol 12-myristate 13-acetate, PMA) and a stimulator (brief exposure to PMA) on RPE phagocytosis was evaluated. RESULTS: Both TGF-beta 1 and TGF-beta 2 up regulated RPE phagocytosis and PMA abolished the up-regulating effect of TGF beta. In contrast, PKC inhibition by staurosporine and calphostin C resulted in increased phagocytosis. A combination of TGF-beta and PKC inhibitor treatment did not produced any additive effect on phagocytosis. CONCLUSION: We concluded that TGF-beta up-regulates human RPE phagocytosis, but that this effect is counteracted by PKC activation. It is possible that this TGF-beta-induced effect is due, in part, to a negative modulation of the PKC-dependent pathway. PMID- 7531172 TI - Detection of Gp210 autoantibodies in primary biliary cirrhosis using a recombinant protein containing the predominant autoepitope. AB - Autoantibodies against nuclear pore membrane glycoprotein gp210 have been identified in between 10% and 25% of patients with primary biliary cirrhosis (PBC). These antibodies may be useful in diagnosing PBC and in identifying subgroups of patients. Because previous detection procedures relied on the need to purify hydrophobic proteins and perform immunoblotting, the aim of the present study was to develop a simple assay to detect gp210 autoantibodies. A recombinant polypeptide containing glutathione-S-transferase (GST) fused to the region of gp210 that contains its predominant autoepitope(s) was expressed in bacteria. This fusion protein was purified by glutathione-Sepharose chromatography and used in an enzyme-linked immunosorbent assay (ELISA). The ELISA was reproducible in detecting gp210 autoantibodies in serum samples from patients with PBC. Compared with immunoblotting, the ELISA was 93% sensitive and 96% specific for the detection of gp210 autoantibodies. In conclusion, autoantibodies against gp210 can be easily and reliably detected in patients with PBC by an ELISA that uses a purified recombinant polypeptide. PMID- 7531173 TI - Variability of hepatitis C virus. PMID- 7531174 TI - Inhibition of endothelial nitric oxide synthase activity by protein kinase C. AB - Nitric oxide (NO) is an important molecular messenger accounting for endothelium derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells. PMID- 7531175 TI - Impaired endothelium-dependent vasodilation of large epicardial and resistance coronary arteries in patients with essential hypertension. Different responses to acetylcholine and substance P. AB - Hypertensive patients have impaired endothelium-dependent coronary vasodilation evoked with acetylcholine. The aim of this study was to examine whether the impaired endothelium-dependent dilation of coronary arteries is related to a specific abnormality of the muscarinic receptor that mediates the effects of acetylcholine. Responses of the large epicardial and resistance coronary arteries were assessed in seven hypertensive patients (mean arterial pressure, 106 +/- 14 mm Hg) and seven control subjects (83 +/- 6 mm Hg, P < .01) during cardiac catheterization. To assess coronary endothelial function, we infused acetylcholine and substance P (endothelium-dependent agents that act on different receptors) and papaverine and nitrate (direct vascular smooth muscle dilators) into the left anterior descending coronary artery and determined coronary artery diameter by arteriography and coronary blood flow with an intracoronary Doppler catheter technique. In control subjects, 3 micrograms/min acetylcholine increased (P < .05) and 30 micrograms/min acetylcholine decreased (P < .05) arterial diameter, and in hypertensive patients, 1, 3, 10, and 30 micrograms/min acetylcholine decreased arterial diameter in a dose-dependent manner. Substance P at 3, 10, and 30 ng/min caused comparable increases in diameter in both groups. Increases in coronary blood flow with both acetylcholine and substance P were significantly (P < .01) blunted in hypertensive patients compared with control subjects. No significant differences were noted between the groups in the responses of large epicardial coronary artery diameter and coronary blood flow to papaverine and nitrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531176 TI - Cellular distribution of angiotensin-converting enzyme after myocardial infarction. AB - We studied the cellular distribution of angiotensin-converting enzyme (ACE) in the heart related to the cell types involved in left ventricular repair and remodeling before and after myocardial infarction by immunohistochemical techniques using monoclonal and polyclonal antibodies. In noninfarcted myocardium of both human and rat, ACE expression was confined to endothelial cells and subendocardial cell layers of the aortic valve. ACE was prominent in endothelia of small arteries and arterioles, whereas only half the coronary capillaries were immunoreactive and venous vessels were almost completely devoid of the enzyme. In a rat model of myocardial infarction, ACE distribution was determined 1, 3, and 7 days and 2, 3, and 6 weeks after coronary occlusion. Three and 7 days after infarction, endothelial cells of sprouting capillaries and macrophages in the marginal zone of necrosis revealed ACE expression. In both human and rat with the onset of fibrosis, intense staining of the enzyme was found in the marginal zone of the repair tissue. In situ hybridization for collagen type I in the rat revealed that zones with high collagen content had almost no ACE immunoreactivity. Vascular smooth muscle cells and cardiomyocytes revealed no ACE expression throughout the study. We conclude that endothelial cells are the principal source for the expression of ACE after myocardial infarction. The observed induction of ACE with the onset of fibrosis suggests a role of this enzyme that is related to tissue repair and remodeling. PMID- 7531178 TI - Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM. AB - RHAMM (Receptor for Hyaluronic Acid Mediated Motility) has been identified as a receptor for the extracellular matrix component hyaluronan (HA) and was recently shown to be essential for the locomotion of normal and transformed peripheral cells. Until now the potential role of RHAMM in the motility of neural-derived cells has not been investigated. Here, we report that cultured primary astrocytes, astrocyte cell lines, and microglia express this receptor and exhibit RHAMM-dependent motility. Immunocytochemical localization of RHAMM showed that it was often present as aggregates at the periphery of cells in contact with one another or concentrated on protruding processes of isolated cells. Glial cells contained 50 and 72 kDa forms of RHAMM, and both of these forms were found to have HA binding capacity. Time lapse imaging of cell locomotion revealed a significant inhibition of motility and process elongation by neutralizing anti RHAMM antibodies and by peptides corresponding to the HA binding domains of RHAMM. These results demonstrate that RHAMM serves a role in glial cell locomotion in vitro and provide the basis for investigations of the motile behavior of glial cells in vivo after CNS injury. PMID- 7531177 TI - Osmoregulatory changes in myo-inositol content and Na+/myo-inositol cotransport in rat cortical astrocytes. AB - Exposure of cortical astrocytes to 325, 350, or 390 mosM culture media for 48 h caused a 1.4-, 2.1-, and 3.5-fold increase, respectively, in cellular content of the compatible osmolyte myo-inositol. Elevated myo-inositol levels accounted for approximately 56-100% of the solute needed by the cells for complete volume regulation under hypertonic conditions. Myo-inositol accumulation was associated with 4-5-fold (peak rate) and 1.8-2-fold (steady-state rate) increases in the rate of Na(+)-dependent myo-inositol uptake when cells were acclimated to 390 mosM culture medium for 12 h or 24-96 h, respectively. When medium osmolality was elevated by 25 mosM, peak and steady-state increases in myo-inositol uptake of 1.7-fold and 1.3-fold, respectively, were observed. Exposure to 390 mosM medium for 12-48 h induced a 3-8-fold increase in cotransporter mRNA levels suggesting that the increase in myo-inositol uptake is brought about by increased cotransporter gene expression. Abrupt return of hypertonic cells to an isotonic medium induced a rapid increase in myo-inositol efflux and a return of cotransporter mRNA to control values in < 2 h. In contrast, the cotransporter remained fully activated at hypertonic levels for 16 h. Between 16-24 h after the transfer, the rate of myo-inositol uptake returned to control values. The remarkable sensitivity of the cotransporter to hypertonic stress indicates that upregulation of myo-inositol transport in glial cells is likely to occur in a variety of disease states that cause an elevation of plasma osmolality. Slow downregulation of the cotransporter may be responsible in part for the slow loss of myo-inositol and cerebral edema that occurs with too rapid correction of chronic plasma hypertonicity. PMID- 7531179 TI - TNF alpha, IL-1 beta and IL-6 plasma levels in neutropenic patients after onset of fever and correlation with the C-reactive protein (CRP) kinetic values. AB - Cytokines, especially tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) play an important role in the genesis and progression of the septic shock syndrome. We performed a study monitoring levels of these three cytokines in ten neutropenic oncology patients in whom an infectious syndrome was suspected. A comparison was made with a population of nine non-neutropenic patients on the intensive care unit. Unfortunately the results of this study do not allow specific profiles to be established for each cytokine in the populations studied. Levels of IL-6, TNF alpha and IL-1 beta were not statistically higher in the non-neutropenic patients when compared with the neutropenic group. However, the highest IL-6 levels were observed for four non neutropenic patients, three of whom died. High levels of C-reactive protein (CRP), haptoglobin and fibrinogen were found, reflecting the inflammatory status of each patient. CRP levels were higher in the non-neutropenic patients and correlated with IL-6 levels, indicating the importance of CRP determination in this group of patients. PMID- 7531180 TI - Prevalence of anti-HCV and risk factors for hepatitis C virus infection in healthy pregnant women. AB - The prevalence of anti-HCV antibodies and the risk factors for HCV infection were assessed in 5,672 pregnant women living in North Italy. All reactive sera were confirmed by RIBA-2 test. Anti-HCV positive pregnant women together with an anti HCV negative control group, were interviewed by standardised questionnaire to identify "known" or "potential" risk factors for HCV infection. The anti-HCV prevalence was 0.7% (40/5,672), higher than that observed among blood donors in the same geographical area (0.2%). The RIBA-2 assay was positive in 60% (24/40) of cases, indeterminate in 10% (4/40) and negative in 30% (12/40). As for "known" risk factors, considering RIBA-2 positivity, intravenous drug use was by far the main risk factor for HCV infection, resulting in a significantly higher risk than in the control group (50% versus 5.9% [O. R. 15.8, CI 5.4-45.5]). The ten RIBA-2 positive women without histories of transfusion or IV drug use had a significantly higher frequency of "sexual contacts with IV drug users" compared to controls (50% vs 4.9% [O. R. 19.0, CI 3.6-94.0]). In conclusion, our study provides evidence that in our geographical area the anti-HCV antibody prevalence is higher in pregnant women than in blood donors and that IV drug use and sexual contacts with IV drug users represent the most important risk factors for HCV infection among young women in North Italy. PMID- 7531181 TI - Low prevalence of anti-HCV antibody among Italian children. AB - The seroprevalence of anti-HCV antibody was studied among 2,749 children and teenagers (1,438 males and 1,311 females) living in Italy. Anti-HCV antibody testing was positive by both EIA and RIBA in ten (0.36%) subjects. The positivity rate increased with age, ranging from 0 among children less than 6 years of age to 0.8% among those aged 17-19 years chi 2 linear regression = 0.038). Anti-HCV prevalence ranged from 0.2% in northeastern regions and in Apulia to 0.6% in Sicily and Sardinia (p > 0.005), and no difference was seen between males (0.35%, C.I. 95%: 0.04-0.66) and females (0.38%, C.I. 95%: 0.04-0.66) (Fisher's exact test = 0.565). From these data it appears that in Italy HCV infection is an uncommon event during childhood. PMID- 7531182 TI - Transient anti-hepatitis C antibodies after influenza vaccination. PMID- 7531183 TI - George Wise and subretinal neovascularization. PMID- 7531184 TI - Human corneal and conjunctival epithelia produce a mucin-like glycoprotein for the apical surface. AB - PURPOSE: The authors have determined that the corneal and conjunctival epithelia of the rat produce a mucin-like glycoprotein at the apical surface of the epithelium. The purpose of this study was to determine if human ocular surface epithelium produces similar glycoproteins. METHODS: Because our initial attempts at production of monoclonal antibodies yielded blood type A-specific antibodies, corneal epithelia from blood type O donor eyes were used for the production of monoclonal antibodies. Screening of hybridoma products was accomplished by immunofluorescence microscopy of cryostat sections of blood type O donor eyes. The monoclonal antibody produced was used for immunofluorescence microscopy and immunoelectron microscopy to determine tissue and cellular distribution, respectively. Immunoblot analysis of SDS-PAGE-separated proteins from corneal epithelial tissue and from cultured human corneal epithelium was used to determine molecular weight range and epitope binding after periodate oxidation, N glycanase, and O-glycanase treatment. RESULTS: A monoclonal antibody, designated H185, that binds to apical cell layers of human corneal, conjunctival, laryngeal, and vaginal epithelium was produced. The antibody binds to apical membranes of apical cells, particularly at the tips of microplicae. In subapical cells, the antibody binds to small cytoplasmic vesicles. Cultured human corneal epithelium produces H185 antigen. By immunoblot analysis, H185 binds a high molecular weight protein, > 205 kD, from corneal epithelium and cultured corneal epithelium. The protein band visualized by immunoblot analysis cannot be stained by Coomassie or silver stain on SDS-PAGE, but it does stain with Alcian blue followed by silver stain, indicating that the protein is highly glycosylated. H185 binding to blotted proteins is destroyed by sodium periodate treatment and O-glycanase incubation, suggesting that the epitope of H185 is an O-linked carbohydrate. CONCLUSION: Human corneal and conjunctival epithelia produce a molecule, similar in size, cellular localization, and distribution to the mucin-like glycoprotein of rat ocular surface epithelium. These data suggest that the entire ocular surface epithelium produces mucins for the tear film. PMID- 7531185 TI - Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. AB - PURPOSE: To describe and evaluate retinal pigment epithelial (RPE) cell transdifferentiation in vitro and to determine its importance to the development of proliferative vitreoretinal disorders. METHODS: Porcine RPE cells from single animals were examined at different passages in culture. The authors examined cellular morphology, contraction of a collagenous matrix, and adhesion to fibronectin and type I collagen-coated substrata. These activities were correlated with loss of epithelial characteristics, redistribution of the actin cytoskeleton, and expression of alpha-smooth muscle actin (alpha-SMA), a marker of myoid differentiation. RESULTS: During routine culture on tissue culture plastic, porcine RPE cells lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The ability of cultured porcine RPE cells to adhere to and exert tractional forces on an extracellular matrix increases with continued passage in vitro and transdifferentiation. This correlates with the loss of the differentiated epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton, and de novo expression of alpha-SMA. CONCLUSION: Results indicate that RPE transdifferentiate in culture and that this transition is accompanied by a shift in biologic activities. Therefore, morphologic and behavioral transdifferentiation of these cells in culture are influencing factors in experimental pathology. The potential relevance of these extensive changes to the biology of proliferative vitreoretinal disorders is discussed. PMID- 7531186 TI - Nerve cells in the human ciliary muscle: ultrastructural and immunocytochemical characterization. AB - PURPOSE: Intrinsic nerve cells in the human ciliary muscle were identified and characterized by immunohistochemical and ultrastructural methods. METHODS: Serial sections through the ciliary muscle of 10 human donors (age range, 53 to 91 years) were investigated by electron microscopy, NADPH-diaphorase (NADPH-d) staining, and immunohistochemistry. Antibodies against nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), neurofilament proteins, tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. Nerve cell density per millimeter of circumferential width was analyzed in three eyes, and in one eye the total number of neurons in the entire ciliary muscle was evaluated. RESULTS: Small (70% of the total; longitudinal diameter 10 to 14 microns) and large (longitudinal diameter 20 to 30 microns) ganglion cells were identified in the inner parts of the muscles' reticular and circular portions. No nerve cells were observed in the anterior longitudinal portion. The cells were in contact with unmyelinated axons and synaptic boutons containing small agranular and large granular vesicles. Axo-somatic and axo-dendritic synapses were observed. Histochemically and ultrahistochemically, the neurons stained intensely for NADPH-d. Both cell types were multipolar and expressed long filamentous processes. Axonal processes with periodic swellings suggesting varicosities ran close and parallel to neighboring muscle bundles. Some nerve cells were connected with each other by axonal processes. No perivascular NADPH-d-positive nerves were seen around ciliary muscle vessels, but they were present in the wall of the major arterial circle of the iris. A small number of ganglion cells contributed to this perivascular network. NADPH-d-positive neurons stained for PGP 9.5 and NOS. No TH, NPY, or VIP-positive ciliary muscle neurons were observed. In double labeling experiments, 70% of the nerve cells were in contact with nerve endings expressing SP-like and CGRP-like immunoreactivity. Seventeen to 32 NADPH-d positive neurons were counted per millimeter of ciliary muscle circumferential width, with 923 in the entire ciliary muscle of one donor eye. CONCLUSIONS: The presence of intrinsic NOS-positive nerve cells concentrated in the inner parts of the ciliary muscle might indicate a physiological role of nitric oxide for disaccommodation or fluctuations during accommodation. PMID- 7531187 TI - Opaque eyes developed in transgenic mice with T-cell receptor delta gene. AB - PURPOSE: During the generation of transgenic mice (TGs) introduced with mouse T cell receptor delta (TCR delta) gene, the authors found a TG line with corneal opacity that coincided with the presence of the transgene. The authors investigated the pathogenesis and molecular mechanisms of this corneal opacity in this line. METHODS: The pathologic features and pathogenesis of the corneal opacity in TGs were examined histologically using transmission and scanning electron microscopy, as well as light microscopy. DNA and RNA blot analyses were performed to examine the copy number and the expression of the transgenes, respectively. RESULTS: Histologically, edema of the corneal epithelium and adhesion of the iris to the cornea were observed in adult TGs. In the developmental analysis, the authors first observed relative hypoplasia of the ciliary body on day 18 of gestation and dysgenesis of the anterior chamber angle from postnatal day 2. Corneal opacity was observed from postnatal day 8, coinciding with the histologic vesicular change of the epithelium. No inflammation was observed through its life. In the sublines that have different copy numbers of the transgene, the occurrence of the opacity depended on the copy number of the transgene. Expression of the transgene in the thymus was consistent with the number of the introduced transgene. CONCLUSION: In a TCR delta TG line, the overexpression of transgenes coincided with abnormal development of the ocular anterior segment and the corneal opacity. Pathogenesis is described, and possible molecular mechanisms are discussed. PMID- 7531188 TI - Increased serum levels of ICAM-1, ELAM-1 and TNF-alpha in inflammatory disorders of the peripheral nervous system. AB - Endothelial intercellular adhesion molecule-1 (ICAM-1) and glycoprotein E selectin (ELAM-1) allow the homing of leukocytes to inflammation sites. A circulating form of ICAM-1 markedly increases in inflammatory CNS disorders. In the present study, the serum levels of ICAM-1, ELAM-1 and tumor necrosis factor alpha (TNF-alpha) were measured in patients with acute (AIDP) and chronic (CIDP) inflammatory demyelinating polyneuropathies and cryoglobulinemic neuropathy (CGN). Immunoenzymometric assays revealed increased sICAM-1 levels in some of these patients; furthermore, high titres of ELAM-1 and TNF-alpha were detected in two patients with AIDP and one patient with CGN. Our data extend previous observations on inflammatory PNS disorders by showing that, in addition to ICAM 1, ELAM-1 also represents a useful marker of endothelial activation and that, taken together, the two molecules may serve as an indicator of specific pathogenetic mechanisms. PMID- 7531189 TI - [Surgical therapy of lip involvement in acanthosis nigricans maligna]. PMID- 7531190 TI - Nitric oxide synthase-containing, peptide-containing, and acetylcholinesterase positive nerves in the cat lower oesophagus. AB - The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility. PMID- 7531191 TI - Differential localization of lectin binding sites and neuropeptides in human dorsal root ganglia. AB - The subpopulations were compared of neurons in human dorsal root ganglia (DRG), as substance P, identified by somatostatin, Glycine max lectin (SBA) specific to terminal N-acetylgalactosamine, and Ulex europaeus I agglutinin (UEA-I) specific to L-fucose. The lectins and neuropeptides all bound to neurons of small diameter. Furthermore, the majority of the SBA binding neurons or somatostatin positive neurons were also UEA-I binding neurons. However, SBA binding neurons were not colocalized with somatostatin or substance P. Less than 20% of substance P positive neurons showed colocalization with L-fucosyl residues, and approximately 10% of L-fucosyl residues showed colocalization with substance P. Our results suggest that both L-fucose and terminal N-acetylgalactosamine containing neurons in the human DRG are subjected to different subpopulations from substance P or somatostatin positive neurons. PMID- 7531192 TI - Late appearance of a type I alveolar epithelial cell marker during fetal rat lung development. AB - Recent studies in fetal lung using immunological and molecular probes have revealed type I and type II cell phenotypic markers in primordial lung epithelial cells prior to the morphogenesis of these cell types. We have recently developed monoclonal antibodies specific for adult type I cells. To evaluate further the temporal appearance of the type I cell phenotype during alveolar epithelial cell ontogeny, we analyzed fetal lung development using one of our monoclonal antibodies (mAb VIII B2). The epitope recognized by mAb VIII B2 first appears in the canalicular stage of fetal lung development, at approx. embryonic day 19 (E19), in occasional, faintly stained tubules. Staining with this type I cell probe becomes more intense and more widespread with increasing gestational age, during which time the pattern of staining changes. Initially, all cells of the distal epithelial tubules are uniformly labelled along their apical and basolateral surfaces. As morphological differentiation of the alveolar epithelium proceeds, type I cell immunoreactivity appears to become restricted to the apical surface of the primitive type I cells in a pattern approaching that seen in the mature lung. We concurrently analyzed developing fetal lung with an antiserum to surfactant apoprotein-A (alpha-SP-A). Consistent with the findings of others, labeling of SP-A was first detectable in scattered cuboidal cells at E18. Careful examination of the double-labeled specimens suggested that some cells were reactive with both the VIII B2 and SP-A antibodies, particularly at E20. Confocal microscopic analysis of such sections from E20 lung confirmed this impression. Three populations of cells were detected: cells labeled only with alpha-SP-A, cells labeled only with mAb VIII B2, and a smaller subset of cells labeled by both.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531194 TI - Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia. II. Physico-chemical properties and structure elucidation. AB - Cepacidine A is a novel glycopeptide with a potent antifungal activity, which is produced by Pseudomonas cepacia AF 2001. Its molecular weight was determined by FAB-MS (m/z 1215). The compound is comprised of glycine (1), serine (2), 2,4 diaminobutyric acid (1), aspartic acid (1), beta-hydroxy tyrosine (1), beta hydroxy asparagine (1), xylose (1) and 5,7-dihydroxy-3,9-diamino-octadecanoic acid (1). Unfortunately, cepacidine A is a mixture of A1 and A2, either of which is barely distinguishable. Cepacidine A2 includes asparagine (1) instead of beta hydroxy asparagine (1) of cepacidine A1. The MS data and the NOESY, TOCSY and HMBC spectra show that cepacidine A is a cyclic peptide and xylose is connected to 5,7-dihydroxy-3,9-diaminooctadecanoic acid. PMID- 7531193 TI - Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia. I. Taxonomy, production, isolation and biological activity. AB - Cepacidine A is a potent antifungal antibiotic produced by Pseudomonas cepacia AF 2001. The compound was isolated from the fermentation broth with 1 vol isopropyl alcohol, followed by the collection of the precipitation formed upon concentration of the extract. Purification was effected by chromatography on Diaion HP-20, alumina and reversed phase C18 followed by TLC on silica gel. These techniques afforded the two closely related compounds, cepacidine A1 and cepacidine A2. A mixture of these two compounds called capacidine A, showed high in vitro antifungal activity against the various animal and plant pathogenic fungi. The activity was diminished by the presence of serum. No antibacterial activity was demonstrable. PMID- 7531195 TI - Microbial transformation of immunosuppressive compounds. IV. Hydroxylation and hemiketal formation of ascomycin (immunomycin) by Streptomyces sp. MA 6970 (ATCC No. 55281). PMID- 7531196 TI - Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62. AB - Nuclear pore complexes are constructed from a large number of different proteins, called collectively nucleoporins. One of these nucleoporins, p62, has an alpha helical coiled-coil COOH-terminal rod domain linked to an NH2-terminal domain that contains a series of degenerate pentapeptide repeats. In nuclear pores p62 forms a tight complex with at least two other proteins, p58 and p54, which can be extracted from isolated rat liver nuclei (Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). We have used a range of methods to demonstrate a strong binding between p62 and p54 in this complex and show that the rod domain of p62 appears to constitute the principal binding site for p54. Whole p62 and its rod domain expressed in Escherichia coli both bind strongly to p54 in blot-overlay assays. Most of the epitopes on the p62 rod recognized by polyclonal antisera are masked in the complex, whereas epitopes on the NH2-terminal domain of p62 are still exposed, both in the isolated complex and also in nuclear pores stained in situ by immunofluorescence in isolated rat nuclei. Moreover, it has been possible to exchange recombinant p62 rod for some of the native p62 in complexes partially dissociated by 4 M urea. Overall these results suggest a key role for the p62 rod domain in maintaining the structural integrity of the complex and also suggest a molecular model for the complex. This model is consistent with data that indicate that the analogous coiled-coil region of yeast nucleoporin NSP1 may function in a similar way. PMID- 7531197 TI - The role of the cytoplasmic domain in regulating CD44 function. PMID- 7531198 TI - Fast myosin heavy chains expressed in secondary mammalian muscle fibers at the time of their inception. AB - Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development. PMID- 7531200 TI - A novel role for E- and P-selectins: shape control of endothelial cell monolayers. AB - The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked 'rounding up' of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins. PMID- 7531199 TI - Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line. AB - We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them. PMID- 7531201 TI - Analysis of a cortical cytoskeletal structure: a role for ezrin-radixin-moesin (ERM proteins) in the marginal band of chicken erythrocytes. AB - We are studying how the cytoskeleton determines cell shape, using a simple model system, the marginal band of chicken erythrocytes. We previously identified a minor component of the marginal band by a monoclonal antibody, called 13H9 (Birgbauer and Solomon (1989). J. Cell Biol. 109, 1609-1620; Goslin et al. (1989). J. Cell Biol. 109, 1621-1631). mAb 13H9 also binds to the leading edges of fibroblasts and to neuronal growth cones and recognizes the cytoskeletal protein ezrin. In recent years, two proteins with a high degree of homology to ezrin were identified: moesin and radixin, together comprising the ERM protein family. We now show that the contiguous epitope sufficient for mAb 13H9 binding is a sequence present in each of the ERM proteins, as well as the product of the gene associated with neurofibromatosis 2, merlin or schwannomin. We used biochemical and immunological techniques, as well as PCR to characterize the expression and localization of the ERM proteins in chicken erythrocytes. The results demonstrate that radixin is the major ERM protein associated with the cytoskeleton. Both ezrin and radixin localize to the position of the marginal band. Our results suggest that the ERM proteins play functionally conserved roles in quite diverse organelles. PMID- 7531202 TI - Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican. AB - Malignantly transformed cells usually display a rosette-like morphology of substratum adhesions (called podosomes) and disorganized microfilaments, and are often associated with elevated production of chondroitin sulphate. We previously showed that many tissues and cells express alternatively spliced multiforms of the large chondroitin sulphate proteoglycan termed PG-M (versican is one of the short transcripts). Since PG-M/versican inhibits many types of cell-substratum adhesion and is found to be excluded from focal contacts of cultured fibroblasts, it is likely that this proteoglycan is generally involved in regulating cell substratum adhesion. We report here that PG-M/versican is selectively excluded from podosomes of human osteosarcoma cells and that specific inhibition of its biosynthesis by an antisense method suppresses such a malignant cell-adhesive phenotype. The results support the idea that PG-M/versican acts as an anti adhesive molecule and raise the possibility that PG-M/versican controls one type of cancer cell behaviour. PMID- 7531204 TI - [Interferons: structure, functions and therapeutic applications]. PMID- 7531203 TI - Ultraviolet radiation-induced melanogenesis in human melanocytes. Effects of modulating protein kinase C. AB - The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultraviolet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C alpha, beta I, epsilon and zeta but not the isozyme eta. Following exposure to 500 nM TPA for 24 hours, isozymes alpha, beta I and epsilon were downregulated, but zeta was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl 2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetylglycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways. PMID- 7531205 TI - Effects of persistent chlorinated hydrocarbons on fertility and embryonic development in the rabbit. AB - The commercial polychlorinated biphenyl (PCB) formulation Aroclor 1260 (4 mg/kg body weight), technical grade dichlorodiphenyltrichloroethane (DDT; 3 mg) and Lindane (gamma-hexachlorocyclohexane; 0.8 mg) were administered orally, either separately or in combination, to sexually mature female rabbits three times per week for 12-15 weeks. Oviductal and uterine luminal fluid, cleavage stage embryos (day 1 post coitum), blastocysts (day 6), fetuses, exocoelic fluid and placentae (day 11) were analysed, firstly for chlorinated hydrocarbon residues, and secondly for embryonic and fetal development. The doses applied were well tolerated by the treated animals. PCB and DDT accumulated in uterine secretions (day 6) but not in oviductal luminal fluid (day 1). Both chlorinated hydrocarbons were found in preimplantation blastocysts. Residues in day 11 fetuses were 16- (DDT) or 18-fold (PCB) higher than in day 6 blastocysts. Significant amounts were also detected in placental tissue and in exocoelic fluid. A specific accumulation of the highly chlorinated biphenyl congener no. 180 was noted in fetuses, placentae and exocoelic fluid. The clear accumulation of the chlorinated hydrocarbon compounds in luminal fluid and embryonic tissue is contrasted by rather weak effects on fertility. No statistically significant differences between treated animals and controls were observed for fertilization rate and pre and post-implantation (up to day 11 post coitum) losses. However, in females exposed to PCB, a 20% higher loss of blastocysts was noticed, as compared with controls (P > 0.05). This effect was shown on day 6 of embryonic development and may be due to the embryotoxic activities of PCB. PMID- 7531206 TI - Termination pattern of medial hyperstriatum ventrale efferents in the archistriatum of the domestic chick. AB - The chick archistriatum receives afferents from the intermediate part of the medial hyperstriatum ventrale (IMHV) and projects to the lobus parolfactorius (LPO). There is functional evidence to suggest that the IMHV and the LPO are connected, but there is no anatomical evidence for a direct connection between the two structures. The aim of the current study was to characterize the termination pattern of medial hyperstriatal afferents within the archistriatum to determine whether the archistriatum may act as a relay between the IMHV and LPO. Following iontophoresis of Phaseolus vulgaris leucoagglutinin into the medial hyperstriatum ventrale (including the IMHV) of 1-week-old domestic chicks, anterogradely labelled fibers were observed to descend through the medial neostriatum and paleostriatum to enter the archistriatum. These medial hyperstriatum ventrale afferents arborised profusely to give varicose axon branches within all except the anterior part of the archistriatum. However, the greatest density was present in the ventral part of the intermediate archistriatum. Electron microscope examination of Phaseolus lectin immunocytochemistry and Golgi impregnation revealed that medial hyperstriatum ventrale axons formed multiple asymmetric synapses with dendritic spines (head and neck regions) on the terminal and preterminal dendritic segments of densely spiny archistriatal projection neurons. Medial hyperstriatum ventrale afferents were not observed to contact calbindin immunoreactive, presumptive "local circuit" neurons, within the archistriatum, despite a spatial overlap in their distribution. These results suggest that the archistriatum may be capable of mediating the transfer of information from the IMHV to the LPO. PMID- 7531207 TI - Phase I study of atevirdine, a nonnucleoside reverse transcriptase inhibitor, in combination with zidovudine for human immunodeficiency virus type 1 infection. ACTG 199 Study Team. AB - Twenty patients were enrolled in a phase I clinical trial of atevirdine, a nonnucleoside reverse transcriptase inhibitor (NNRTI), given in combination with zidovudine for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Fifteen patients had received no previous antiretroviral therapy. HIV 1 isolates obtained at 6-week intervals were tested for sensitivity to atevirdine and zidovudine. Two patients developed a rash within 2 weeks of enrollment, and 1 of these developed concomitant fever and hepatitis. No hematopoietic, neurologic, or pancreatic toxicities were observed. Atevirdine had considerable initial interpatient pharmacokinetic variability. Forty-seven percent of patients treated with atevirdine plus zidovudine had increased CD4 lymphocyte counts, and HIV isolates from 62% of patients remained sensitive to atevirdine after 24 weeks of therapy. Atevirdine plus zidovudine was well-tolerated. Additional studies should be done to determine the role of atevirdine in the therapy for HIV infection. PMID- 7531208 TI - Cytokine secretion in acute shigellosis is correlated to disease activity and directed more to stool than to plasma. AB - Stool and plasma cytokine levels in 60 adults with acute shigellosis were studied by EIA at various intervals (0-45 days) after onset of diarrhea. Cytokine levels correlated with severity of disease. Significantly higher levels of proinflammatory cytokines peaked at onset of disease in stool of patients with severe disease (P < .05). In contrast, interferon-gamma (IFN-gamma) levels, depressed at disease onset, progressively increased during the convalescent stage. Concomitantly obtained plasma cytokine levels were 100 times lower than levels in stool. Controls in Shigella-endemic areas (n = 42) had persistently significantly higher levels of IFN-gamma and interleukin-1 receptor antagonist in both stool and plasma. The lack of host defense activity against shigellosis may be linked to delayed recovery of IFN-gamma. This cytokine may play an important role in elimination of the infection and development of immunity against shigellosis. PMID- 7531209 TI - Detection of serum antibodies to the lipopolysaccharide of Escherichia coli O103 in patients with hemolytic-uremic syndrome. PMID- 7531210 TI - [Screening for adverse perinatal outcome by biomarkers in maternal serum; a comparison of elevated maternal serum alpha-fetoprotein versus human chorionic gonadotropin]. AB - We conducted a cohort analytic study to determine whether an unexplained increase in maternal serum alpha-fetoprotein (MSAFP) or human chorionic gonadotropin (MShCG) is effective in screening for adverse perinatal outcomes. MSAFP and MShCG levels were measured in 1,097 consecutive pregnant women in whom gestation was dated by ultrasonography and who later delivered a single infant. Patients with high MSAFP or MShCG had a much higher risk of low birth weight, intrauterine growth retardation and fetal death. But a screening test with MShCG for predicting preterm birth, low birth weight and intrauterine growth retardation had an approximately twice higher sensitivity than that with MSAFP. A combination of MSAFP and MShCG had little effect on the sensitivity. We concluded that a screening test with MShCG alone might be useful in predicting adverse perinatal outcomes. PMID- 7531211 TI - Temporal alterations in regional gene expression after nephrotoxic renal injury. AB - To examine at a molecular level the repair process during recovery from acute renal failure, we determined, after nephrotoxic acute renal injury, levels for several RNAs associated with differentiated functions in the kidney. To delineate changes in different regions, we determined RNA levels separately in cortex, outer medulla, and inner medulla of adult rat kidney. Several RNAs were regionally distributed in control kidneys, including epidermal growth factor (EGF, highest in cortex and outer medulla), aldose reductase (highest in inner medulla), and prostaglandin synthase (highest in inner medulla). Futhermore, RNA for the heat shock protein Hsp70 was present at high levels in the inner medulla of controls. A single intraperitoneal dose of folic acid caused reversible, nonoliguric acute renal failure with impairment of glomerular filtration, tubular sodium reabsorption, and urinary concentration. Fractional excretion of sodium returned to normal by day 3, while serum creatinine, serum urea nitrogen, and urinary osmolality remained abnormal until day 7. During acute renal failure there was a marked, reversible decrease in the regional mRNA levels for EGF, aldose reductase, prostaglandin synthase, and Hsp70. Aldose reductase, prostaglandin synthase, and Hsp70 RNA levels returned to control levels by 7 to 10 days, while EGF remained depressed for a more prolonged period. High levels of aldose reductase, prostaglandin synthase, and Hsp70 RNAs in the inner medulla and their loss/recovery during acute renal failure with a time course similar to alterations in urinary osmolality were consistent with regulation in response to changes in inner medullary osmolality, as has been demonstrated for aldose reductase in normal animals. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531213 TI - Renal hemodynamic effects of dietary protein in the rat: role of nitric oxide. AB - The biologic mediator(s) of the renal hemodynamic effects of a high dietary protein intake (hyperfiltration and renal vasodilation) are unknown. The endogenous nitrovasodilator nitric oxide (NO) derives from the amino acid L arginine, and NO has been demonstrated to mediate the hyperfiltration and vasodilation observed during amino acid infusion in rats. We therefore hypothesized that NO may also mediate the long-term renal hemodynamic effects of variations in dietary protein intake. We studied rats maintained with low protein (6%) and high-protein (50%) diets for 2 weeks. An additional group of rats receiving a high-protein diet was also treated with the NO synthase inhibitor, L nitro-arginine-methyl ester (NAME, 100 mg per liter of drinking water). After 2 weeks a high-protein diet was associated with a significant increase in glomerular filtration rate (GFR) (50% protein group vs 6% protein group, 1.01 +/- 0.03 vs 0.61 +/- 0.03 ml/min; p < 0.05) and renal vasodilation (renal vascular resistance: 50% protein group vs 6% protein group, 11.70 +/- 0.88 vs 17.65 +/- 1.55 mm Hg/min/ml; p < 0.05) compared with a low-protein diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531212 TI - Streptokinase and rt-PA activate platelets by a different way: implications on the rethrombosis rate after their administration in myocardial infarction. AB - The natural history of acute myocardial infarction has been dramatically changed by the advent of thrombolytic treatment, with a 30% mortality reduction, a better recovery of ventricular function, and a better quality of life. This treatment notwithstanding, failure or delay in achieving reperfusion, along with reocclusion and bleeding, still worry clinicians and challenge researchers to improve thrombolytic regimens and concomitant antithrombotic treatments. Platelet activation, at least in part because of thrombolytic treatment itself, plays a pivotal role in the pathogenesis of resistance to lysis and rethrombosis. The aim of this study was to compare in vitro the effects on platelets of therapeutic concentrations of streptokinase (SK) and recombinant type plasminogen activator (rt-PA). The effects of plasmin and thrombin were also studied as a reference. Fluorescence flow cytometry was used to evaluate (1) fibrinogen binding and (2) surface expression of GMP-140, a sensitive marker of platelet release reaction. Platelet function was further studied by measuring the release of carbon 14 labeled serotonin, beta-thromboglobulin, plasminogen activator inhibitor-1 (PAI 1) and the generation of thromboxane (TxB2). We found that 10 nmol/L SK and 14 nmol/L rt-PA increased fibrinogen binding to platelets by 12 +/- 2 and 10 +/- 4 times, respectively (p = not significant). At the same concentrations, SK, but not rt-PA, also induced the platelet release reaction: surface expression of GMP 140 was increased by 6 +/- 1.5 times by SK and 1.3 +/- 0.2 times by rt-PA (p < 0.05). TxB2 production was not modified by plasmin and plasminogen activators. Our data showed that plasmin and SK stimulate fibrinogen receptor expression and platelet degranulation. Conversely, rt-PA, at concentrations up to 14 nmol/L, only promotes fibrinogen binding. These results, coupled with the less-pronounced lytic state induced by rt-PA, could explain the higher incidence of reocclusion accompanying rt-PA therapy in comparison with SK that occurs unless effective "adjunctive" antithrombotic treatment is used. Neither of the thrombolytic agents activates the arachidonate pathway. Thus aspirin is probably not an ideal agent to be used in conjuction with thrombolytic agents. We speculate that newer approaches, particularly RGD peptides and antibodies against GP llb/llla, might produce better results. PMID- 7531214 TI - Platelet activation in whole blood by artificial surfaces: identification of platelet-derived microparticles and activated platelet binding to leukocytes as material-induced activation events. AB - Because the lack of thromboresistant vascular biomaterials is in part due to platelet activation, we have attempted, by using fluorescence-activated flow cytometry, to fully characterize the platelet population after in vitro material contact with whole blood. We have used a very simple, near-physiologic system whereby whole blood, anticoagulated with D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone (thrombin inhibitor), contacts materials for 1 hour at 37 degrees C, under low shear. Unlike other tests of platelet compatibility that focus on adherent platelets, this assay evaluates the platelets in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate for the first time significant materials-induced microparticle formation. One-hour contact with Silastic, polyethylene, and polyvinyl alcohol hydrogel surfaces lead to 30 +/- 1, 33 +/- 4, and 43 +/- 4 x 10(9) microparticles/L, respectively, whereas resting blood samples contained only 10 +/- 1 x 10(9) microparticles/L. In addition, significant increases in activated platelet(s) binding to neutrophils/monocytes after material contact were noted for all surfaces tested. For polyvinyl alcohol hydrogel surfaces a greater than 500% increase in the fluorescent intensity over that of resting whole blood was attained. The addition of monoclonal antibodies to GPIIb/IIIa (A2A9), the tetrapeptide adhesion ligand RGDS (arginine-glycine-aspartate serine), or the calcium ion chelator ethyleneglycol-bis-(B-aminoethyl-ether)- N,N,N',N'-tetraacetic acid to the whole blood before material contact fully inhibited all platelet reactivity noted for all surfaces--platelet microparticles, platelet P-selectin expression, loss of platelets from bulk, and the formation of platelet/leukocyte aggregates--thereby indicating that material induced platelet activation is a calcium-dependent process involving GPIIb/IIIa receptors. PMID- 7531215 TI - Small satellite of arabis mosaic virus: autolytic processing of in vitro transcripts of (+) and (-) polarity and infectivity of (+) strand transcripts. AB - In vitro RNA transcripts of both (+) and (-) polarities were obtained from a circularly permuted dimer clone of the small satellite of arabis mosaic virus (sArMV). The transcripts show efficient self-cleavage at the two specific sites in the sequence where the monomers were joined. Autolytic processing of the full length transcript in both orientations releases promoter-proximate fragments (+) or (-) P, promoter-distal fragments (+) or (-) D, and the monomer fragments (+) or (-) M. The presence of an OH group at their 5' ends and a 2',3' cyclophosphate at their 3' ends suggests that (+) and (-) M originated via two self-cleavage reactions within the full-length transcript of corresponding polarity. Infectivity assays showed that the (+) M fragment but not the (-) M fragment initiates replication as efficiently as the natural linear sArMV in Chenopodium quinoa. Two (-) fragments were identified which are the result of religation activity: a P-D fragment formed by religation of P and D, and c-M, which is the result of efficient self-ligation of (-) M. In contrast, linear (+) M self ligates in vitro to a very limited extent but could be circularized enzymically in a wheat germ extract. PMID- 7531216 TI - Human immunodeficiency virus type 2 (HIV-2) env gene analysis: prediction of glycoprotein epitopes important for heterotypic neutralization and evidence for three genotype clusters within the HIV-2a subtype. AB - The env gene sequences of ten tissue-culture-adapted human immunodeficiency virus type 2 (HIV-2) isolates from West African patients were determined. Alignment and comparison of the gene sequences and putative translation products with database sequences revealed 11-29% diversity at the nucleotide level and 15-31% variation at the protein level. From analysis of glycoproteins of HIV-2 strains sensitive and resistant to neutralization by HIV-1 antisera, five regions were identified as putative targets for cross-neutralizing antibody. The HIV-2 equivalent of the HIV-1 V3 loop was not included in this number. However, three of the HIV-2 peptides aligned with regions identified as targets for broad neutralization of HIV-1 strains. These were the V2 and CD4-binding domains of gp120 and the Kennedy domain in gp41. Phylogenetic analysis of the env gene sequences, together with HIV-2 env gene sequences published in the Los Alamos database, support the identification of two distinct HIV-2 subtypes, HIV-2a and HIV-2b. The new sequences are located within the HIV-2a subtype and allow prediction of at least three genotypes, designated I-III. Some correlation of genotype with geographical origin of isolates was noted. Genotype I viruses originate from Guinea Bissau and group II viruses mainly originate from The Gambia. One isolate from Guinea Bissau, HIV-2CAM4, appears phylogenetically older than other viruses in the HIV 2a subtype. The possible implications of this in the light of epidemiological findings in Guinea Bissau are discussed. PMID- 7531217 TI - Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein. AB - Three linear antigenic regions on the N protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were identified by using peptides which reacted in ELISA with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the protein: Thr11 to Gly30 (N3 peptide), Ser231 to Ala250 (N25 peptide) and Thr371 to Leu391 (N39 peptide). The site represented by the N39 peptide reacted with four subgroup A-specific MAbs. There were minor variations in the amino acid epitope dependencies of each of these MAbs. Two additional antigenic regions Ser131 to Arg150 and Ala181 to Leu200, were represented by peptides that reacted with human convalescent sera, but these peptides did not differentiate between acute and convalescent sera from RSV-infected humans. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the N protein from a nucleocapsid-containing homogenate derived from extracts of RSV (subgroup A and/or B)-infected 35S-labelled cells in a radioimmuneprecipitation assay (RIPA); the sera were also used to demonstrate RSV infection in cells by immunofluorescent assay (IFA). Anti-N3 peptide sera precipitated N41, the full length (M(r) 41,000) form of N protein, in a RIPA done on a soluble protein pool. Anti-N39 (C-terminal region) peptide sera precipitated both forms, suggesting that N38 (M(r) 38,000) is an N-terminally truncated (probably at position Tyr23 located inside the N3 peptide linear antigenic region) form of N41 protein. PMID- 7531218 TI - Role of virus and host variables in virus persistence or immunopathological disease caused by a non-cytolytic virus. AB - C57BL/6 mice infected with increasing doses of the Armstrong isolate of lymphocytic choriomeningitis virus (LCMV) or a variant Cl 13-Armstrong, derived from LCMV-Armstrong, exhibited distinct phenotypes with respect to clearance of virus and to cytotoxic CD8+ T cell (CTL)-dependent immunopathological disease. Low (10(2) p.f.u.) and high doses (10(7) p.f.u.) of LCMV-Armstrong were cleared rapidly from immunocompetent mice. Inoculation of a high dose (10(7) p.f.u.) of LCMV Cl 13-Armstrong temporarily induced a partial deletion of the antiviral CTL precursors (CTL-p) leading to chronic infection in several organs. Although virus was cleared from most organs by day 90-150 post-infection, it persisted in the kidney. The few remaining CTL-p were able to expand and eventually clear the virus. Systemic viral titres correlated inversely with the number of CTL-p. However, in contrast LCMV-Docile injected at high dose was able to cause complete exhaustion of CTL-p resulting in long term viral persistence. LCMV-Aggressive, derived from the same parental LCMV-WE (UBC) as Docile, showed a phenotype comparable to wild-type virus. Doses of < 10(7) p.f.u. of both Armstrong virus and of Cl 13-Armstrong failed to exhaust CTL-p and caused lethal CD8+ T cell dependent choriomeningitis and a substantial footpad swelling after local infection. By contrast, doses > 10(3) p.f.u. of LCMV-Docile failed to cause lethal choriomeningitis in C57BL/6 mice. When Cl 13-Armstrong virus (but not LCMV Armstrong) was injected intravenously in addition to intracerebrally or into the foot, the local immunopathology was abrogated in a dose-dependent fashion. The suppression of immunopathology paralleled the extent of exhaustion of the specific CD8+ T cell response. Nucleotide sequence analysis of the viral S-RNA fragments coding for CTL epitopes in H-2b mice revealed an asparagine to serine change of amino acid 280 in the CTL epitope 275-286 of the LCMV-Docile glycoprotein (GP) in comparison to LCMV-Aggressive or wild-type WE. This change reduced overall CTL activity and thereby probably contributes to exhaustion of CTL responses in C57BL/6 (H-2b) mice. Thus, local versus systemic antigen distribution, viral characteristics and immunological parameters determine induction and exhaustion of CD8+ T cells and the course and extent of immunopathological disease. PMID- 7531220 TI - Phase II study of paclitaxel in relapsed non-Hodgkin's lymphomas. AB - PURPOSE: To assess the efficacy and toxicity of paclitaxel administered as a 96 hour infusion to patients with relapsed non-Hodgkin's lymphomas (NHLs). PATIENTS AND METHODS: Eligible patients had relapsed NHL and measurable disease and were considered incurable. Paclitaxel was infused at a dose of 140 mg/m2 every 3 weeks. Premedications to prevent paclitaxel hypersensitivity reactions were not administered and no patients received corticosteroids. Expression of the multidrug resistance (mdr-1) gene was determined in tumor from 17 patients by mRNA quantitative polymerase chain reaction (PCR). RESULTS: Thirty-one patients received a total of 99 cycles of paclitaxel. Two patients were not assessable for response. The median age was 50 years, 71% had stage IV disease, and intermediate/high-grade histology was present in 65% of patients. Patients had received a median of three prior chemotherapy regimens, and 68% of patients had responded to the previous chemotherapy (chemotherapy-sensitive). Of 29 assessable patients, five (17%) achieved a partial response (PR). With a median potential follow-up time of 17 months, the median event-free and overall survival durations were 1.6 and 7.5 months, respectively. No correlation was found between response to paclitaxel and extent of prior treatment or response. The mdr-1 gene was easily detectable in 14 of 17 tumor biopsies, but was low in all but one sample. The most serious toxicity was grade 4 neutropenia, which occurred during 14% of cycles. CONCLUSION: Paclitaxel was well tolerated, but had a low response rate in patients with relapsed NHLs. There was no clear association between response to paclitaxel and extent of our response to prior treatment. Most patients had chemotherapy-sensitive disease, which suggests that the low response rate to paclitaxel was probably not due to general chemotherapy resistance. Paclitaxel provided good palliation in a minority of patients and is a reasonable agent to consider for use in patients who have failed to respond to standard chemotherapy. PMID- 7531221 TI - Significance of normal serum prostate-specific antigen in the follow-up period after definitive radiation therapy for prostatic cancer. AB - PURPOSE: To determine the prognostic significance of a normal serum prostate specific antigen (PSA) level in patients with prostatic cancer with long-term follow-up evaluation after radiotherapy. MATERIALS AND METHODS: PSA information was available in 403 patients (38%) who were treated with pelvic lymph node dissection and retropubic radioactive iodine-125 implantation. One hundred eighty two patients had a normal serum PSA level (< or = 4.0 ng/mL) the first time this test was conducted during the follow-up period, designated PSA-1. RESULTS: Among patients with PSA-1 values < or = 1.0 ng/mL, the 5-year PSA relapse-free survival rate was 85% compared with 27%, respectively, among those with PSA values in the higher range of normal (P < .00001). Multivariate analysis demonstrated that only a PSA-1 value greater than 1.0 to < or 4.0 (P < .00001) and grade II/III histology (P = .009) had a negative impact on continued PSA relapse-free survival. The only independent variable identified by a multivariate analysis to affect local relapse-free survival (LRFS) was a PSA-1 value greater than 1.0 to < or = 4.0 ng/mL (P < .004), while high-grade histology (P < .0001) and local failure (P < .001) were the only significant variables to affect distant metastases-free survival (DMFS). CONCLUSION: Patients with PSA values < or = 1.0 ng/mL are significantly less likely to have a subsequent relapse after therapy than those with levels greater than 1.0 to < or = 4.0 ng/mL. Continuously maintained PSA levels of < or = 1.0 ng/mL after treatment may serve as an end point for early evaluation of the efficacy of experimental radiotherapy protocols in prostate cancer. PMID- 7531219 TI - Risk of relapse in childhood acute lymphoblastic leukemia is related to RBC methotrexate and mercaptopurine metabolites during maintenance chemotherapy. Nordic Society for Pediatric Hematology and Oncology. AB - PURPOSE: During maintenance chemotherapy for childhood acute lymphoblastic leukemia (ALL), the cytotoxic metabolites of methotrexate (MTX polyglutamates) and mercaptopurine (6MP) (thioguanine nucleotides [6TGN]) accumulate intracellularly, including in erythrocytes (E-MTX and E-6TGN) with large interindividual variations. In the present Nordic Society for Pediatric Hematology and Oncology (NOPHO) study, the relation of E-MTX and E-6TGN to relapse risk was explored. PATIENTS AND METHODS: Two hundred ninety-seven patients with non-B-cell ALL, aged 1 to 14 years, on oral MTX and 6MP had E-MTX and E-6TGN levels measured three to 35 (median, eight) and three to 75 (median, nine) times, respectively. For each patient, a mean of all E-MTX (mE-MTX) and E 6TGN (mE-6TGN) measurements was calculated, as well as the product of mE-MTX and mE-6TGN (mE-MTX-6TGN), since MTX and 6MP may have synergistic action. RESULTS: For patients in remission, the median mE-MTX and mE-6TGN values were 4.7 nmol/mmol hemoglobin (Hgb) (range, 0.4 to 10.3) and 173 nmol/mmol Hgb (range, 58 to 846). With a median follow-up duration of 66 months for patients in remission, 64 patients relapsed. Cox regression analysis identified mE-MTX-6TGN and sex to be the most significant parameters to predict relapse (global P = .001). Factors that predicted a better prognosis were high mE-MTX 6TGN and female sex. Patients who had a mE-MTX-6TGN less than the product of the median mE-MTX and median mE 6TGN (813 [nmol/mmol Hgb]2) had a significantly poorer event-free survival (EFS) than did patients with higher values (5-year probability of EFS [pEFS5y], 0.70 v 0.86; P = .001). CONCLUSION: The pharmacokinetics of MTX and 6MP may have significant influence on the risk of relapse. The value of dose adjustments by E MTX and E-6TGN remains to be determined. PMID- 7531222 TI - Localized prostate cancer treated by external-beam radiotherapy alone: serum prostate-specific antigen--driven outcome analysis. AB - PURPOSE: To determine the 5-year rate of survival with no evidence of disease (NED) using strict biochemical criteria in men with prostate cancer treated by external-beam radiotherapy alone and to examine possible clinical and treatment factors that predict the likelihood of NED survival. MATERIALS AND METHODS: Five hundred men with clinically localized prostate cancer consecutively treated with external-beam radiotherapy alone with no prior, concomitant, or adjuvant endocrine therapy were identified. All patients had serial serum prostate specific antigen (PSA) values determined after treatment and 451 patients had pretreatment PSA values determined. The median follow-up duration is 20 months (range, 2 to 72; mean, 36). RESULTS: The 5-year rate of overall survival in this group of patients was 80%. The 5-year rate of survival without clinical evidence of disease (cNED) was 72%. The 5-year rate of survival without evidence of clinical, radiographic, or biochemical relapse (bNED) was 51%. Multivariate analysis demonstrated that a pretreatment serum PSA level < or = 15 ng/mL was the most important predictor of bNED survival (P < .0001). Patients with early-stage (T1, T2a/b) tumors and a pretreatment serum PSA less than 15 ng/mL had a 3-year rate of bNED survival of 86%. The rate of bNED survival for patients with a pretreatment PSA level greater than 15 ng/mL was 38% at 3 years. CONCLUSION: Pretreatment serum PSA level is the most important predictor of treatment outcome in this group of patients treated with definitive radiotherapy alone. External beam radiation alone can produce acceptable early rates of bNED survival in patients with clinically organ-confined tumors and a pretreatment PSA level < or = 15 ng/mL. To produce acceptable results in those patients with pretreatment PSA levels more than 15 ng/mL, effective adjuvant treatments in addition to aggressive local treatments are necessary. PMID- 7531224 TI - Tumor angiogenesis as a predictor of recurrence in gastric carcinoma. AB - PURPOSE: We investigated the correlation between tumor angiogenic activity and progression of gastric carcinoma using immunohistochemical staining with antifactor VIII-related antigen (F-VIII RAg) antibody. MATERIALS AND METHODS: One hundred twenty-four specimens resected from patients with gastric carcinoma were investigated by staining with a monoclonal antibody against F-VIII RAg. Correlations between the microvessel count (the mean number of microvessels in the five areas of highest vascular density at 200 times magnification), various clinicopathologic factors, and prognosis were studied. RESULTS: The microvessel count increased with histologic stage. The microvessel count was significantly higher in patients with lymph node metastases than in those without such metastases. Moreover, in patients with a high microvessel count (> or = 16), prognosis was significantly poorer than in those with low count (< 16). Multivariate analysis indicated that the microvessel count is an independent prognostic factor in patients with gastric cancer. According to the mode of recurrence, the frequency of hepatic metastases was significantly increased in patients with a high count. CONCLUSION: Microvessel count may be a good prognostic indicator and may be useful as a predictor for the mode of recurrence in patients with gastric carcinoma. PMID- 7531223 TI - Importance of bleomycin in favorable-prognosis disseminated germ cell tumors: an Eastern Cooperative Oncology Group trial. AB - PURPOSE: This prospective, randomized trial was designed to determine if three cycles of cisplatin plus etoposide (PVP16) can produce therapeutic results comparable to three cycles of cisplatin, etoposide, and bleomycin (PVP16B) in patients with disseminated germ cell tumors. PATIENTS AND METHODS: One hundred seventy-eight patients with minimal- or moderate-stage disease (Indiana staging system) were randomized to receive cisplatin (20 mg/m2 on days 1 to 5) plus etoposide (100 mg/m2 on days 1 to 5) with or without weekly bleomycin (30 IU/wk for 9 consecutive weeks). Following three cycles of chemotherapy over 9 weeks, patients with residual radiographic disease underwent surgical resection. If persistent carcinoma was noted, two additional 3-week courses of chemotherapy were administered. RESULTS: One hundred seventy-one patients were fully assessable for response and survival. The two treatment groups were similar with respect to patient characteristics. The toxicities were comparable between the two arms. No clinically significant incidence of pulmonary toxicity occurred with PVP16B. Overall, 81 of 86 patients (94%) who received PVP16B and 75 of 85 patients (88%) who received PVP16 achieved a disease-free status with chemotherapy and/or surgery. However, greater numbers of treatment failures, including persistent carcinoma in postchemotherapy resected residual disease and relapses from complete remission, occurred on the arm without bleomycin (overall adverse outcome, P = .004). The failure-free (86% v 69%; P = .01) and overall survival (95% v 86%; P = .01) rates were inferior on the PVP16 arm. CONCLUSION: Bleomycin is an essential component of PVP16B therapy in patients who receive three cycles of treatment for minimal- or moderate-stage disseminated germ cell tumors. PMID- 7531225 TI - Research in palliative care populations: ethical issues. PMID- 7531226 TI - Bedside assessment of competency in palliative care. PMID- 7531227 TI - The ethics of partial palliative care. PMID- 7531228 TI - Advance directives in palliative care. PMID- 7531229 TI - Ethics of palliative care medicine: palliative care for the rich nations only! PMID- 7531230 TI - Ethical issues in palliative care: the case of dialysis and organ transplantation. PMID- 7531232 TI - Ethics of palliative care in the context of limited resources: an essay on the need for attitudinal change. PMID- 7531231 TI - More money for palliative care? The economics of denial. PMID- 7531233 TI - A day soon must come when ... PMID- 7531234 TI - From the front lines. PMID- 7531235 TI - Barriers to pain control: ethics & knowledge. PMID- 7531236 TI - Choosing palliative care: do religious beliefs make a difference? PMID- 7531237 TI - Ethical issues in palliative care research. AB - During the last 10 years interest in palliative care research has grown. This interest has brought about an awareness of the unique ethical issues related to research in this special population. Issues related to the vulnerability of patients and families, consent protocols, unstable mental status, and the invasiveness of assessments have been discussed. Unfortunately, devastating physical and psychosocial distress continue to afflict the majority of cancer patients before death. Although our current knowledge of palliative care allows us to help a significant number of patients and families, there are a number of symptoms that are currently intractable. Further, a 50% increase in the number of cancer deaths in Canada is expected between 1990 and the year 2000 (11). Therefore, one of the major ethical issues we must confront during the coming decade is whether we can afford not to make a major commitment to research in palliative care. We are aware that maintaining the status quo of patient care is unacceptable. However, until now, administrators, governments, and clinicians have planned palliative care programs to have only service and some teaching components. And traditional research-sponsoring agencies have not allocated priority to palliative care in their funding strategies. The palliative care community must take the initiative in this debate. PMID- 7531239 TI - Ethical challenges to the palliative care volunteer. PMID- 7531238 TI - "We don't do that in palliative care". AB - Five situations have been presented to consider whether there are interventions which are "not done in palliative care". No attempt was made to cover all possible circumstances. The only limits which emerged are (a) an absolute limitation on criminal action and (b) a relative limitation on non-beneficial treatment. Determination of what is non-beneficial is subject to various considerations: firstly, not recommending non-beneficial interventions; secondly, giving special allowance for experimental interventions in properly conducted trials; and, thirdly, accepting the patient's right to gamble on a trial of therapy. PMID- 7531241 TI - Transformation of congenital neutropenia into monosomy 7 and acute nonlymphoblastic leukemia in a child treated with granulocyte colony-stimulating factor. AB - A cytogenetically normal infant with Kostmann syndrome (severe congenital granulocytopenia) was treated with granulocyte colony-stimulating factor, which resulted in a rapid improvement in his neutrophil count and a resolution of recurrent infections. After 11 months of therapy, splenomegaly developed, with thrombocytopenia, anemia, circulating nucleated erythrocytes, and acquired monosomy 7, which evolved during a period of 7 months into acute nonlymphoblastic leukemia. The use of granulocyte colony-stimulating factor in patients with congenital marrow failure disorders may induce or hasten the onset of a malignant transformation. PMID- 7531240 TI - Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator. AB - OBJECTIVE: To determine whether there is an association between mutations of the cystic fibrosis transmembrane regulator (CFTR) and the predilection of patients with cystic fibrosis (CF) for Pseudomonas aeruginosa infection. METHOD: We quantified the adherence of P. aeruginosa PA01, labeled with sulfur 35 methionine, to epithelial monolayers derived from nasal scrapings of patients with specific CFTR mutations, and of carriers and normal subjects. RESULTS: Adherence of P. aeruginosa to epithelial cells from patients with CF was significantly greater than to cells from either carriers (t = 2.94; p = 0.009) or normal subjects (t = 3.32; p = 0.004). Adherence to epithelial cells from patients with CF who were homozygous for the delta F508 mutation ranged from 12% to 35% (mean, 23.7%) of the added inoculum, which was significantly greater than the binding to cells from patients with other mutations, which ranged from 3% to 18% (mean, 9.4%; t = 3.71; p = 0.002), from heterozygote carriers (3% to 11%; mean, 7.9%; t = 4.87; p = 0.002), or from normal subjects (2% to 10%: mean, 7.0%; t = 5.21; p = 0.002). CONCLUSION: Adherence to P. aeruginosa can be correlated with homozygosity for the delta 508 mutation; CFTR dysfunction may be one of the factors involved in the pathogenesis of pulmonary infection in CF. PMID- 7531242 TI - Comparisons between in vitro glial cell adherence and internalization of non encapsulated and encapsulated strains of Cryptococcus neoformans. AB - The adherence of non-encapsulated and encapsulated strains of Cryptococcus neoformans to rat glial cells in culture was compared. Like the encapsulated strain, the adherence of the unencapsulated strain was affected by the yeast culture age and growth temperature. Yeasts grown to late stationary phase at 37 degrees C were the most adherent. Neither encapsulated nor non-encapsulated strains adhered to glial monolayers when the experiments were conducted at 5 degrees C, indicating that metabolically active mammalian cells were required for yeast adherence. Additionally, the non-encapsulated strain was consistently three times more adherent than the encapsulated strain, suggesting that the non encapsulated strain either had more adhesins or more adhesins were exposed. Electron microscope studies indicated that both strains were internalized by glial cells, an event previously not reported for this mammalian cell type. The same carbohydrates (N-acetyl-D-glucosamine, sucrose and inositol) that inhibited adherence of the encapsulated strain the most, also inhibited adherence of the non-encapsulated strain, again indicating similar adhesin mechanisms. Both encapsulated and non-encapsulated strains were made non-adherent by treatment with pronase, papain, trypsin and amylase. Immobilized amylase appeared to remove an adhesin fragment that bound to glial cells and inhibited the adherence of intact cryptococci. PMID- 7531243 TI - [Whole-cell recording and perforated-patch clamping method]. PMID- 7531244 TI - The physical properties of local interactions of tyrosine residues in peptides and unfolded proteins. AB - Peptides and unfolded proteins with the sequence Xaa-Pro-Tyr or Xaa-Pro-Phe have a relatively strong local interaction, present about 64% of the time at 10 degrees C, of the aromatic ring with the side-chain of Pro and the C alpha H of residue Xaa, but only when the Xaa-Pro peptide bond is cis. With the sequence Tyr Yaa-Gly (Yaa not equal to Pro), there is a somewhat present about 26% of the time, of the aromatic ring with the Gly residue. When present together, in the sequence Xaa-Pro-Tyr-Yaa-Gly, the two interactions of the Tyr side-chain compete and have the expected strengths. Both interactions have an enthalpic basis, with enthalpies of about -3 and -2.8 kcal/mol, respectively. The two interactions responded differently to the denaturants urea and guanidinium chloride; urea had little effect on either, but the second was weakened by guanidinium chloride, whereas the first interaction was strengthened slightly. This explains why such local interactions can be observed in unfolded proteins under strongly denaturing conditions. Interactions such as these are stronger than anticipated in an otherwise disordered peptide; they are probably important for determining the conformational tendencies of unfolded polypeptide chains and may play a role in protein folding. Similar interactions probably occur in protein-ligand interactions. PMID- 7531245 TI - Structure of an antibody-lysozyme complex unexpected effect of conservative mutation. AB - The structure of the complex between the Fab HyHEL-5 and chicken lysozyme revealed a large interface region containing 23 lysozyme and 28 Fab residues. Arg68 of the lysozyme is centrally placed in this interface and theoretical studies together with binding assays of this Fab to different avian lysozymes have previously shown that this arginine residue is an important contributor to the binding. The Arg68-->Lys mutant binds 10(3) times less well to the HyHEL-5 Fab. We have examined the refined crystal structure of the complex of this mutant lysozyme with the Fab. No global changes occur, but there is an introduction of a new water molecule into the interface that mediates the hydrogen bonding interactions between the lysine and residues on the Fab. These data are compared with the effects of similar changes on the inhibition of serine proteases such as trypsin where the energetic effects of this substitution are small. PMID- 7531247 TI - Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process. AB - The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs. PMID- 7531246 TI - RNA determinants required for L4-mediated attenuation control of the S10 r protein operon of Escherichia coli. AB - We have probed regions of the S10 leader RNA to determine their role in L4 mediated, NusA-dependent attenuation control of the S10 ribosomal protein operon. Using genetic and "antisense" oligonucleotide competition approaches, we were able to distinguish between the determinants necessary for intrinsic (NusA independent) pausing by RNA polymerase at the S10 attenuation site, for NusA dependent enhancement of pausing, and for L4 stabilization of the paused ternary complex. The upper stem-loop structure in the attenuator hairpin is the major determinant for the NusA-dependent pause, while the sequence at the site of pausing is important for RNA polymerase to pause in the absence of NusA. The determinants for L4 stabilization of the paused complex include the hairpin immediately upstream of the attenuator hairpin as well as the ascending side of the attenuator structure. In conclusion, our results suggest that there are three distinct pausing activities by RNA polymerase during its transcription of the S10 leader, with three corresponding signals in the S10 leader. PMID- 7531248 TI - Protein hydration dynamics in aqueous solution: a comparison of bovine pancreatic trypsin inhibitor and ubiquitin by oxygen-17 spin relaxation dispersion. AB - Water oxygen-17 spin relaxation was used to study hydration and dynamics of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal and transverse relaxation rates was measured over the Larmor frequency range 2.6 to 49 MHz in the pD range 2 to 11 at 27 degrees C. While the protein-induced relaxation enhancement was similar for the two proteins at high frequencies, it was an order of magnitude smaller for ubiquitin than for BPTI at low frequencies. This difference was ascribed to the absence, in ubiquitin, of highly ordered internal water molecules, which are known to be present in BPTI and in most other globular proteins. These observations demonstrate that the water relaxation dispersion in protein solutions is essentially due to a few structural water molecules buried within the protein matrix, but exchanging rapidly with the external water. The relaxation data indicate that the internal water molecules of BPTI exchange with bulk water on the time-scale 10(-8) to 10(-6) second thus lowering the recently reported upper bound on the residence time of these internal water molecules by four orders of magnitude, and implying that local unfolding occurs on the submicrosecond time-scale. The water molecules residing at the surface of the two proteins were found to be highly mobile, with an average rotational correlation time of approximately 20 picoseconds. For both proteins, the oxygen-17 relaxation depended only very weakly on pD, showing that ionic residues do not perturb hydration water dynamics more than other surface residues. We believe that the present results resolve the long-standing controversy regarding the mechanism behind the spin relaxation dispersion of water nuclei in protein solutions, thus establishing oxygen-17 relaxation as a powerful tool for studies of structurally and functionally important water molecules in proteins and other biomolecules. PMID- 7531250 TI - From the Centers for Disease Control and Prevention. Encephalitis associated with cat scratch disease--Broward and Palm Beach counties, Florida, 1994. PMID- 7531249 TI - Hydrogen exchange and protein hydration: the deuteron spin relaxation dispersions of bovine pancreatic trypsin inhibitor and ubiquitin. AB - Water deuteron (2H) spin relaxation was used to study hydrogen exchange, hydration, and protein dynamics in aqueous solutions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal 2H relaxation rate was measured in the pD range 2 to 11 at 27 degrees C. In contrast to the previously reported water 17O relaxation dispersion from the same samples, the 2H dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the 2H relaxation in BPTI solutions could be quantitatively accounted for in terms of known pK values and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the 2H relaxation dispersion even in the neutral pD range. The 2H relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the 2H dispersion with the 17O dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10(-6) to 10(-4) second) than the other three (10(-8) to 10(-6) second). PMID- 7531251 TI - [G-CSF and G-CSF receptor]. AB - Relationship between leucocytosis and G-CSF blood concentration were examined. Increase in blood concentration of G-CSF of surgical patient and dialysis patient with leucocytosis were noticed. Determination of G-CSF receptor on leucocyte was performed by the flow cytometry using biotinylated G-CSF. Modulation of G-CSF receptor on leucocyte at surgery and dialysis was identified in vivo. Studies of G-CSF blood concentration and modulation of G-CSF receptor are useful in analysis of dynamics of leucocyte and analysis of infectious disease. PMID- 7531252 TI - [The clinical significance of interleukin-6 as an inflammatory marker (the studies in patients with open heart surgery)]. AB - We analyzed serum levels of interleukin-6 (IL-6) and seven acute phase proteins in CRP positive samples and in patients with open heart surgery. The concentrations of serum IL-6 were not correlated with other acute phase proteins in CRP positive samples. However, IL-6 were in inverse correlation with CRP, AAG, AAT and CER in patients with open heart surgery. These discrepancies were due to the differences in response time of each acute phase protein after the start of inflammation. Responses of acute phase proteins after open heart surgery were investigated from hour to hour. IL-6 increased rather rapidly than other acute phase proteins, and increases of CRP, TRF, AAT, AAG, CER, HAP and AMG followed. The time reached the peak were IL-6, CRP, TRF, AAT, AMG, AAG, HAP reached the peak in that order. IL-6 constantly increased seven hours earlier, and reached at maximum values forty three hours earlier than CRP in each case. The measurement of serum concentration of IL-6 may be useful for early detection of acute inflammation. PMID- 7531253 TI - [HCV antibody positive rate observed in periodic checkup in Kitakyushu]. PMID- 7531254 TI - [A case of prostatic adenocarcinoma with pulmonary metastases--diagnosis by transbronchial lung biopsy and immunohistochemistry]. AB - Metastatic pulmonary adenocarcinoma was found in a 79-year-old man, who had symptoms of general malaise and poor appetite. An extensive work-up including a transurethral resection of the prostate, failed to establish the primary site of the malignancy. By administering chlormadinone acetate for prostatic hypertrophy, the pulmonary metastases improved dramatically. The tumor cells in the lung, which had previously been obtained by transbronchial lung biopsy, stained positive for prostatic acid phosphatase and prostatic specific antigen. These data suggested that prostatic carcinoma had metastasized to the lung. The prostatic carcinoma was finally confirmed at autopsy. PMID- 7531255 TI - Studies on the diagnosis of Tyzzer's disease in laboratory rat colonies with antibodies against Bacillus piliformis (Clostridium piliforme). AB - To develop a system to be used for confirming diagnosis of Tyzzer's disease in seropositive rat colonies, 38 Mol:SPRD, BB/Wor/Mol-BB, and Stroke-Prone rats suffering from megaloileitis were examined. All affected rats had been found by a systematic examination of 5-week-old male rats from barrier-protected colonies, sero-positive to Bacillus piliformis, the agent of Tyzzer's disease. The rats were evaluated by serologic testing, histologic examination of the ileum, liver, and heart (hematoxylin and eosin and Warthin-Starry staining), and immunofluorescence staining of tissue smears of the ileum and liver. The presence of B. piliformis was verified in 24 of the rats. All animals that had the agent in the liver or heart also had it in the ileum. The sensitivity of immunofluorescence staining for identification of the agent was higher than that for Warthin-Starry staining. Thirty-seven rats had histologic changes indicative of Tyzzer's disease in the liver, and 23 had histologic changes in the myocardium. All rats had a high titer of antibodies against B. piliformis. Having verified the presence of the agent, germ-free sentinels were placed in one of the colonies. These also became infected, as did germ-free sentinels caged with the infected rats outside the barrier unit. The number of sentinels infected increased with the age of the sentinels at introduction. To prove the absence of Tyzzer's disease in a seropositive colony, it is considered necessary to examine approximately 3,000 5-week-old rats for abdominal distension and demonstrate negative staining of the ileum for B. piliformis. PMID- 7531256 TI - Quinolone arthropathy in juvenile New Zealand white rabbits. AB - Twenty juvenile New Zealand white rabbits were dosed orally once daily with 1,000 mg of nalidixic acid/kg for 3, 7, and 14 consecutive days, then for 14 days followed by a 14-day recovery period. Eighteen age-matched rabbits were allotted to four groups and given corn oil vehicle to serve as controls for the various treatment durations. The articular cartilage from the stifle joints, shoulders, and elbows was studied by gross examination, light microscopy, and toluidine blue histochemistry, and the hips were studied by gross examination and transmission electron microscopy. When examined grossly, raised vesicles could be detected in the joints of some animals after 3 days of treatment. The distal portion of the femur and proximal portion of the ulna were predilection sites for gross lesions. Histologically, the vesicles were fluid-filled clefts in the intermediate layer of the articular cartilage. After 14 days, many of the lacunae in the areas of the defects contained chondrocyte clusters. When treated for 14 days and allowed a 14-day recovery period, territorial matrix had been deposited around individual chondrocytes within the clusters, compressing the matrix between the chondrocyte clusters into thin collagenous bands. For all treatment groups, decreased metachromasia, when stained with toluidine blue, provided histochemical evidence of loss of glycosaminoglycans in the matrix around the clefts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531257 TI - Influence of slight sequence changes on the free energy of a single stranded ribonucleic acid molecule. AB - Single-stranded ribonucleic acid molecules take a variety of secondary structures. The free energy (g) of a given secondary structure of a molecule is calculated from the Boltzmann weighted summation over the states of this molecule taking this secondary structure. Likewise, the free energy (G) of a molecule is calculated from the summation over the states of this molecule, which takes various secondary structures. The g-value can be evaluated by Salser's method (1977, Cold Spring Harbor Symp. Quantum Biol. 42, 985-1002). By use of these values, the G-value can also be obtained. Computer studies utilizing this method reveal that there is a particular class of molecules whose G-values tend to increase when the sequences are slightly changed. As far as we examined, such molecules take few secondary structures that fulfill the following two conditions simultaneously: (i) The g-value is close to that of the optimal secondary structure. (ii) The structure is very different from the optimal secondary structure. Here, the optimal secondary structure means the one with the lowest g value among all the secondary structures taken by the molecule. PMID- 7531258 TI - Evolution of genetic information flow from the viewpoint of protein sequence similarity. AB - As a course of inquiry into the evolution of genetic information flow, similarity relations of amino acid sequences between the proteins involved in translation, transcription and replication are investigated. The sequence data of these proteins are mostly accumulated from Escherichia coli, and the present investigation is carried out mainly on this organism by the FASTP program. This result reveals an interesting similarity linkage extending from ribosomal proteins to the proteins participating in translational elongation process and to the proteins in transcription and replication. Although the ribosomal proteins are of relatively short polypeptide chains, our systematic comparison between these proteins finds many similarity relations, being more than 100 in terms of "overlap", reducing them to about 14 elementary ribosomal proteins from which other ribosomal proteins would have diverged. Moreover, the proteins involved in translation, transcription and replication contain the regions similar to the elementary ribosomal proteins. In particular, some initiation and elongation factors in translation process are assigned to be similar to the elementary ribosomal proteins almost over the whole regions. To such an elongation factor Tu, the alpha and sigma 70 subunits of RNA polymerase and primase also show similarity in the wider regions than the individual ribosomal proteins, and they are shown to be fundamental for the similarity linkage extending to the other polypeptide chains involved in transcription and replication processes, although the latter polypeptide chains contain regions not similar to any ribosomal protein. This divergence pattern of similarity relations strongly suggests that the proteins involved in the contemporary genetic information flow DNA-->RNA- >protein have evolved from some elementary ribosomal proteins, first by gene fusion, in a primitive organism of the RNA-protein world, and then by the addition of the mechanism of domain shuffling from other genes in the DNA-RNA protein world. PMID- 7531259 TI - Concurrent use of granulocyte colony-stimulating factor with low-dose cytosine arabinoside and aclarubicin for previously treated acute myelogenous leukemia: a pilot study. AB - We used a new chemotherapy regimen for the treatment of 18 consecutive patients with relapsed AML (median age 44 years, range 18-74). The regimen consisted of low-dose cytosine arabinoside (10 mg/m2/12 h, usually day 1 to 14), low-dose aclarubicin (10-14 mg/m2/day, day 1 to 4), and concurrent use of G-CSF (200 micrograms/m2/day) (CAG regimen). Overall, 15/18 patients (83%) achieved complete remission (CR) after one or two courses, including eight out of ten refractory patients with early relapse, second or subsequent relapses, and/or resistant relapse. Two of three patients who relapsed, achieved CR again after reinduction with a modified CAG regimen. Fourteen of the 15 complete remitters received consolidation therapy with the CAG regimen modified, followed by oral busulfan in eight cases, and by allogeneic bone marrow transplantation in two cases. At a median follow-up of 12 months, median CR duration and survival were 6 months and 17 months, respectively. Myelosuppression in the first course of induction therapy was moderate to severe. However, severe non-hematologic toxicity (WHO grade > or = 3) was characteristically rare. Although this is a preliminary study, the CAG combination seems promising for the treatment of relapsed AML, with its low toxicity contributing to a higher quality of life for the patient. PMID- 7531260 TI - In vivo thrombin and plasmin activities in patients with acute promyelocytic leukemia (APL): effect of all-trans retinoic acid (ATRA) therapy. AB - APL-associated hemostasis disorders result from at least two distinct mechanisms due to the release of procoagulant activities and plasminogen activators from the leukemic cells. These two mechanisms (thrombin activation and plasmin activation) may cleave the fibrinogen molecule, but their respective roles in low fibrinogen levels and bleeding diathesis genesis remain in dispute. In vivo ATRA therapy induces a rapid correction of both low fibrinogen level and bleeding tendency, but no clear explanation of this beneficial effect has been proposed. We prospectively investigated 27 APL patients at presentation for diffuse intravascular coagulation (DIC) markers (prothrombin activation fragment and thrombin/antithrombin complexes) and plasmin-dependent primary fibrinogenolysis markers (alpha 2 plasmin inhibitor consumption +/- plasmin/alpha 2 plasmin inhibitor complexes). Fourteen of these patients were then serially studied during the first 2 weeks of ATRA therapy. Four of them, however, developed an hyperleukocytosis requiring additional chemotherapy before the end of the 2nd week. At presentation, low level of fibrinogen was clearly associated with alpha 2 plasmin inhibitor deficiency (p < 0.01), while DIC was equally present in fibrinogenopenic and non-fibrinogenopenic patients. Moreover, was observed a rapid simultaneous correction of low fibrinogen levels and plasmin activation markers in APL patients undergoing ATRA therapy (before day 5), but a more prolonged persistence of DIC markers (until day 14). Initial bleeding syndrome seemed more frequent in patients with initial low fibrinogen level. These data indicate that plasmin-dependent primary fibrinogenolysis is the major etiologic factor of low fibrinogen level in APL patients. In vivo differentiation ATRA therapy induces a rapid decrease in the plasmin activation and a normalization of fibrinogen level, while DIC may in vivo persist for several weeks. Prospective studies evaluating antifibrinolytic agents as therapy of APL-associated hemostasis disorders should be considered. Additionally, prophylactic heparin therapy might be useful after day 5 in patients undergoing ATRA therapy, since they present a prolonged procoagulant tendency. PMID- 7531261 TI - Effects of granulocyte colony-stimulating factor therapy on in vitro hemopoiesis in myelodysplastic syndromes. AB - We evaluated the effects of 2 months of G-CSF treatment on in vitro hematopoiesis in 17 patients with myelodysplastic syndromes (MDS). Although in vitro marrow myeloid progenitor cell (CFU-GM) growth stimulated by G-CSF generally remained subnormal, in the majority of neutrophil responders significantly augmented incremental change (termed AIC) of CFU-GM numbers occurred after treatment, as did neutrophilic differentiation. The neutrophil non-responders had less prominent in vitro myeloid responses and lower basal neutrophil levels (p < 0.05). Following G-CSF treatment, the initially subnormal erythroid burst-forming unit (BFU-E) values underwent AIC in five of 11 patients along with increased reticulocyte responses in vivo, whereas four of the five patients who lacked AIC of BFU-E did not. Three patients with persisting cytogenetic abnormalities and increased neutrophilic differentiation in vitro also responded in vivo, suggesting that G-CSF induced in vivo cellular differentiation from the abnormal clone. Two of the three patients who developed blastic responses in vivo had increased CFU-GM growth pre- and post-therapy. These results indicate in vivo-in vitro correlations for myeloid and erythroid responses of MDS marrow cells which related to treatment with G-CSF. PMID- 7531263 TI - [Palliative nephrostomy. A noninvasive method in irreversible ureteral obstruction]. PMID- 7531262 TI - Chronic eosinophilic pneumonia and idiopathic bronchiolitis obliterans organizing pneumonia: comparison of eosinophil number and degranulation by immunofluorescence staining for eosinophil-derived major basic protein. AB - OBJECTIVE: To undertake additional assessment of the possible overlap between bronchiolitis obliterans organizing pneumonia (BOOP) and chronic eosinophilic pneumonia (CEP). DESIGN: We retrospectively reviewed open-lung biopsy specimens from six patients with CEP, five patients with idiopathic BOOP, and four patients with secondary BOOP, encountered during the 5-year period 1986 through 1991, for the presence of eosinophils and extent of eosinophil degranulation. MATERIAL AND METHODS: Using previously described immunofluorescence methods for detection of intact eosinophils and extracellular deposition of eosinophil-derived major basic protein, we counted the number of eosinophils per x160 microscopic field and evaluated the extent of eosinophil degranulation semiquantitatively. RESULTS: The median numbers of eosinophils were 221 (range, 26 to 343) in cases of CEP, 7 (range, 1 to 65) in cases of idiopathic BOOP, and 7.5 (range, 1 to 39) in cases of secondary BOOP. More eosinophils were found in CEP than in idiopathic BOOP or all cases of idiopathic and secondary BOOP. We found no differences in the extent of eosinophil degranulation among the three groups, although a tendency for more degranulation was noted in cases of CEP. CONCLUSION: Even though clinical and histologic overlap may exist between CEP and idiopathic BOOP, the exact relationship and the role of the eosinophil in idiopathic BOOP remain to be determined. PMID- 7531264 TI - Subcutaneous gammaglobulin for patients with secondary hypogammaglobulinaemia. PMID- 7531265 TI - Dual role for Afipia felis and Rochalimaea henselae in cat-scratch disease. PMID- 7531268 TI - [Cholangiolar carcinoma]. AB - Cholangiocarcinoma affects both sexes equally, is more prevalent in individuals age 50 to 70. Associations with many other diseases are known, in particular with primary sclerosing cholangitis, ulcerative colitis and parasitic infestation (Clonorchis sinensis, Opisthorchis viverrini). About 95% are histopathologically classified as adenocarcinoma. The localisation of the tumor determines clinical course and prognosis. If the tumor is located above the hepatic duct bifurcation, only one side of the biliary tree may be obstructed, resulting in atrophy of the corresponding liver lobe, without clinical manifestation of jaundice. Obstructive jaundice is the characteristic symptom of hilar tumor localisation. Laboratory examinations show hyperbilirubinemia and liver enzymes indicating cholestatis are elevated. Pathological levels of the tumor markers carcinoembryonic antigen (CEA) and CA 19-9 are frequently found. Ultrasonography in combination with endoscopic retrograde or percutaneous transhepatic cholangiography and cytological examination of aspirates are of main diagnostic importance. Treatment consists of curative resection or palliative decompression to relieve jaundice, the latter can be performed using endoscopic implantable self-expanding metal stents. Cytotoxic chemotherapy or liver transplantation show no satisfactory results. The prognosis is mainly poor, only few patients survive more than 6 months after diagnosis. PMID- 7531266 TI - Lack of effect of nedocromil sodium in ACE-inhibitor-induced cough. PMID- 7531267 TI - [What is the value of arterial chemotherapy in treatment of colorectal liver metastases]. PMID- 7531269 TI - [Pronounced flush symptoms in carcinoid syndrome without liver metastases]. AB - The case of a 78 years old patient with impressive flush symptoms is reported. 5 Hydroxyindoleacetic acid and serotonin were elevated. The responsible pathomorphological structure is a 8 x 4.5 x 6 cm ileocaecal neoplasm. Pre-, intra and postoperatively liver metastases could not be detected during a 18 month period. After surgery of the carcinoid flush disappeared, so that the tumour seize resulting in enormous secretion of metabolites with endocrine activity should be claimed responsible for causing this symptom. PMID- 7531270 TI - Urology. PMID- 7531271 TI - Porphyria cutanea tarda and hepatitis C. PMID- 7531272 TI - A right to die? Some medico-legal reflection. PMID- 7531274 TI - Nephrotoxicity of immunosuppressive drugs. AB - Drugs used to modify the immune response in solid organ transplantation or autoimmune disease may cause dose-related nephrotoxicity. Cyclosporine, FK506, cyclosporine G, and rapamycin have all been studied experimentally and to a more limited extent in patients. This paper summarizes this literature using data from clinically relevant animal models. PMID- 7531273 TI - Autoimmunity caused by host cell protein-containing viruses. AB - Autoreactive T cells specific for myelin basic protein (MBP), a major component of central nervous system (CNS) protein, are frequently found in blood and cerebrospinal fluid of patients with postinfectious encephalomyelitis. This autoimmune syndrome is a CNS complication after infections with a number of different enveloped viruses, e.g. mumps, measles, rubella, influenza and varicella. However, the pathophysiological mechanism leading to this breaking of natural self tolerance in the course of viral infection remains an enigma. A long lasting hypothesis has suggested that incorporation of cellular (self) proteins into the envelope of budding viruses might be a possible mechanism leading to autosensitization. In a model study we demonstrate here that vesicular stomatitis virus (VSV), grown in myelin protein-expressing cell cultures, is highly efficient in triggering T cell responses to MBP in vitro and can prime autoreactive T cell immune responses in vivo. On the basis of these findings, we suggest that incorporation of CNS membrane components into the viral envelope and subsequent priming of self-reactive immune responses might be the common pathogenic mechanism underlying the postinfectious encephalomyelitis syndrome. PMID- 7531275 TI - Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD dependent secretion comprises the C-terminal 62 amino acids of HlyA. AB - Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA. PMID- 7531276 TI - Transcriptional analysis of groEL genes in Streptomyces coelicolor A3(2). AB - In Streptomyces coelicolor A3(2), synthesis of the groES, groES-groEL1 and groEL2 transcripts is induced either by heat shock or by undefined physiological stress signals present at a certain stage of growth. Under all conditions tested, transcription of groES and groES-groEL1 originated from a unique start site upstream of groES, whereas transcription of groEL2 originated from a unique site upstream of groEL2. RNA polymerase isolated either from heat-shocked or control mycelia allowed in vitro transcription from the P1 promoter of groES/EL1 and the P2 promoter of groEL2. The fact that these two RNA polymerase preparations both initiated transcription with equal efficiency from the same sites suggested that a heat shock-specific sigma factor is not responsible for the temperature-induced transcription of groE genes. Instead, regulation of these genes from vegetative type promoters may be effected by a DNA-binding protein observed in gel retardation assays, which recognizes a motif found in the groE and dnaK promoter regions of many prokaryotic genes. PMID- 7531277 TI - Changes of cell cycle-regulating genes in interferon-treated Daudi cells. AB - Interferon (IFN) modulates the expression of several genes and some of them are considered to be responsible for the inhibition of cellular growth. However, the alterations of cell cycle-regulating genes produced by IFN still remain unclear. Accordingly, we studied the expression of cell cycle-regulating genes during IFN induced growth arrest. Cell cycle synchronized and unsynchronized Daudi Burkitt lymphoma cells were treated with IFN. Both the cell cycle distribution and the expression of cell cycle-regulating genes (cdk2, cdc2, cyclins A, B, C, D3, cdc25, and wee 1) were studied by flow cytometry and by Northern blot hybridization or the reverse-transcription polymerase chain reaction, respectively. Treated cells passed through the first G1 phase and gradually accumulated in the following G1 phase. Expression of cyclins A, B, and D3 oscillated along with the cell cycle progression in control cells, and the alterations of cyclin B expression were especially prominent. Both cdc2 and cdk2 also showed changes, but these were not so distinct as observed with cyclin B. Expression of cdc25 and wee1 was little affected by cell cycle progression. In IFN-treated cells, expression of cyclins A and B were down-regulated, while that of cyclin C was not. Cyclin D3 expression was also down-regulated at 48 h, followed by an increase at 72 h. Expression of both cdc2 and cdk2 was down regulated, especially that of the later. Wee1 expression was down-regulated by IFN but, the expression of cdc25 remained stable. These findings suggest that the modulation of cell cycle-regulating genes, particular by cyclin A and cdk2, plays an important role in IFN-induced cellular growth arrest. PMID- 7531278 TI - On the role of the invariant glutamine at position 54 in the human choriogonadotropin beta subunit. AB - The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone beta subunits from a variety of mammalian species. beta-Gin 54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single A-->G mutation in the LH beta gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH beta, with Arg replacing Gin-54, associated with the alpha subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG beta with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG beta (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine alpha, and the media were analyzed for holoproteins, which were characterized in vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG beta(Glu-54) bound to alpha almost as well as hCG beta wild-type, and the resulting heterodimer competed with [125l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED50s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG beta(Lys-54) associated very poorly with alpha. Our results suggest that while Gin-54 in hCG beta participates in receptor binding, its major function appears to involve alpha binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding. PMID- 7531281 TI - Site-specific RNA crosslinking with 4-thiouridine. PMID- 7531280 TI - Perspectives on zinc finger protein function and evolution--an update. AB - Complexity is one of the hallmarks that applies to C2H2 type zinc finger proteins (ZFPs). Structurally distinct clusters of zinc finger modules define an extremely large superfamily of nucleic acid binding proteins with several hundred, perhaps thousands of different members in vertebrates. Recent discoveries have provided new insights into the biochemistry of RNA and DNA recognition, into ZFP evolution and genomic organization, and also into basic aspects of their biological function. However, as much as we have learned, other fundamental questions about ZFP function remain highly enigmatic. This essay is meant to define what we personally feel are important questions, rather than trying to provide a comprehensive, encyclopaedic review. PMID- 7531282 TI - Serotonin-dopamine antagonist and Gilles de la Tourette's syndrome: an open pilot dose-titration study with risperidone. PMID- 7531279 TI - Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture. AB - In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collagen I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of 3[H] leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of 35[S] sulphate into total proteoglycans (PG) and a relatively higher fraction of the 35[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells on collagen gels. But in contrast to the above results, the incorporation of 3[H]-leucine into cytokeratins C8, C18 and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently. PMID- 7531283 TI - Genotoxic activity of environmentally important polycyclic aromatic hydrocarbons and their nitro derivatives in the wing spot test of Drosophila melanogaster. AB - The genotoxicity of three polycyclic aromatic hydrocarbons (PAHs) and of three of their nitro derivatives was evaluated in the wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster. Two crosses were used, i.e. the standard cross (ST) and the improved high bioactivation cross (HB) which is characterised by an increased sensitivity to the genotoxic effects of promutagens and procarcinogens. Larvae trans-heterozygous for the two recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) were fed with the test compounds for 48 h. The wings of the surviving flies were analysed for the occurrence of single and twin spots. Naphthalene, 1-nitronaphthalene and 1,5 dinitronaphthalene proved to be more genotoxic in the HB cross than in the ST cross. Anthracene showed a clear genotoxic activity only in the HB cross whereas it was negative in the ST cross. 9-Nitroanthracene gave inconsistent results in both crosses. Phenanthrene was negative in the ST cross, but weakly positive in the HB cross. These results demonstrate that the genotoxic activity of these PAHs and their nitro derivatives can be detected with the somatic cells of the wing imaginal discs of larvae with high bioactivation capacity. PMID- 7531284 TI - The effect of smoking on sister chromatid exchange rate of newborn infants born to smoking mothers. AB - Sister chromatid exchange (SCE) frequencies in lymphocytes of 21 smoking mothers and their 21 newborns were compared to those that of 10-infants whose mothers had never smoked and to those of 8 infants whose mothers were passive smokers and reported high exposure to tobacco smoke by living or working with smokers. Mothers in the first group also smoked throughout their pregnancy. Results confirm our earlier study on smoking effects reported for adults. Additionally, we saw that neonates have consistently lower SCE frequencies than adults. PMID- 7531285 TI - A pharmacokinetic study of ethanol inhibition of micronuclei induction by urethane in mouse bone marrow erythrocytes. AB - Urethane (ethyl carbamate) is a genotoxic carcinogen in fermented products and alcoholic beverages. The genotoxicity of urethane requires metabolic activation. Metabolism of urethane is mediated by multiple pathways, and ethanol is known to inhibit the esterase hydrolysis pathway of urethane, which accounts for over 95% of urethane metabolism. This report shows that ethanol also inhibits the induction of micronuclei by urethane in mouse bone marrow erythrocytes, presumably by inhibiting the minor pathway that generates genotoxic metabolite(s). In this study, male CD-1 mice were administered urethane, ethanol, or urethane co-administered with increasing amounts of ethanol in single intraperitoneal injections. Bone marrow polychromatic erythrocytes (PCE) obtained 24 h after injection were scored for micronuclei. The dose of urethane was 1000 mg/kg, and the doses of ethanol were 0, 625, 1250, 2000, 2250, 2500, 3000 and 3500 mg/kg. The blood ethanol level at each dose was determined. Two pharmacokinetic parameters, Cmax and AUC, were estimated for each dose. The observed Cmax of ethanol at doses of 1250, 2000, 2250, 2500, 3000 and 3500 mg/kg were 1.39, 2.84, 3.15, 3.69, 4.13 and 4.76 mg/ml, with AUCs of 1.37, 4.84, 5.88, 7.28, 10.76 and 13.51 mg.h/ml, respectively. Urethane treatment alone markedly increased the micronucleus frequency from 0.1% in the vehicle control to 2.47%. This magnitude of increase was suppressed when urethane was co-administered with ethanol at ethanol doses of 2500 mg/kg and above. At 2500, 3000 and 3500 mg/kg, the micronucleus frequencies reduced from 2.47% to 0.9, 0.44 and 0.28%, respectively. This study shows that ethanol inhibits the induction of micronuclei by urethane. PMID- 7531286 TI - Intensification and depletion of specific bulky renal DNA adducts (I-compounds) following exposure of male F344 rats to the renal carcinogen ferric nitrilotriacetate (Fe-NTA). AB - The effects of the renal carcinogen ferric nitrilotriacetate (Fe-NTA) on kidney DNA of male F344 rats were studied to determine whether bulky DNA oxidation products (putative intrastrand crosslinks) could be detected by 32P-postlabeling in the target organ of carcinogenesis. Rats (10-11 weeks old) were given a single dose of Fe-NTA (15 mg Fe/kg body weight) i.p. at 3:00 pm. After 5 h, renal DNA from Fe-NTA-treated and vehicle control animals was assayed by 32P-postlabeling. Thin-layer chromatography and quantitative analysis of two labeled nucleotide fractions of increasing polarity, L and C, showed that three spots (L1, L2, and C3) were intensified 3.5- to 4.2-fold in treated animals. L1 consisted of subfractions L1a, L1b, and L1c, which could be resolved chromatographically. L1c, L2, and C3 were identical to DNA oxidation products generated by the Fenton reaction in vitro, while L1a and L1b apparently did not arise by this mechanism. DNA damage and toxicity appeared reduced in younger animals and animals treated in the morning, presumably due to differences in antioxidant defenses. Liver and lung (non-target organs) DNA did not exhibit enhanced L1, L2, and C3 spots. In addition to augmenting renal I-compounds, Fe-NTA reduced the levels of three major polar kidney I-compounds (C4, C5, and C6) to 22-53% of control. This reduction did not appear to arise by direct oxidative DNA damage, resembling the previously documented loss of liver I-compounds induced by numerous hepatocarcinogens. Two of these I-compounds (C4 and C5) have been reported to exhibit positive linear correlations with median lifespan of male F344 rats. The pleiotropic response of kidney I-compound levels to Fe-NTA was consistent with different roles of different types (I and II) of I-compounds in Fe-NTA-mediated renal carcinogenesis. The results strongly support a causal relationship between oxidative DNA lesions and Fe-NTA-mediated carcinogenesis. PMID- 7531287 TI - Mutagenicity of ethylene glycol ethers and of their metabolites in Salmonella typhimurium his-. AB - Ethylene glycol ethers, their aldehyde and their acid metabolites were evaluated for their mutagenicity with the Ames test. The Salmonella typhimurium his- tester strains TA 97a, TA 98, TA 100 and TA 102 were used with and without rat S9 mix. Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol n-butyl ether and their corresponding aldehyde and acid derivatives were tested up to 10(-4) mol/plate (around 10 mg/plate) or up to cytotoxic concentrations. All tested substances gave negative results with TA 98, TA 100 and TA 102 either with or without S9 mix. In contrast, ethylene glycol n-butyl ether (EGBE) and the aldehyde metabolite of ethylene glycol monomethyl ether, methoxyacetaldehyde (MALD), displayed mutagenic potency in strain TA 97a with and without S9 mix at high concentrations. A significant number of revertants was obtained from 19 mumol/plate EGBE (2.2 mg/plate) and from 34 mumol/plate MALD (2.5 mg/plate). At these concentrations the level of revertants reached up to 7 fold and 3-fold the control values for EGBE and MALD respectively. PMID- 7531288 TI - Study of the genotoxic activity of six halogenated acetonitriles, using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test. AB - Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of six halogenated acetonitriles identified in chlorinated waters (monochloro-, dichloro , trichloro-, monobromo-, dibromo- and bromochloroacetonitrile). With the SOS chromotest, three of the chemicals studied (dichloro-, dibromo- and bromochloroacetonitrile) were found to induce primary DNA damage in Escherichia coli PQ37. In the Ames-fluctuation test, all the compounds except dibromoacetonitrile showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for all the six haloacetonitriles studied. Moreover, two structure-activity relationships were noted: (1) the genotoxic activity of haloacetonitriles containing bromine substituents appeared higher than the corresponding chlorinated acetonitriles and (2) the clastogenic activity of the chlorinated acetonitriles increased with the number of chlorine substituents. PMID- 7531289 TI - Modifying actions of solvent extracts from fruit and vegetable residues on 2 amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5 f]quinoxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98. AB - The edible parts of 13 fruits--apples, apricots, bananas, blackberries, sweet cherries, red currants, white grapes, oranges, peaches, pears, plums, raspberries, and strawberries--and of 12 vegetables--asparagus, green beans, broccoli, brussels sprouts, red and white cabbage, carrots, cauliflower, onions, green peppers, spinach, and tomatoes--were squeezed in order to separate juices and residues. The residues were washed, lyophilized, and extracted sequentially with n-hexane, dichloromethane, acetone, and 2-propanol. Solvent extracted materials were tested in Salmonella typhymurium TA 98 for antimutagenicity against IQ and MeIQx. We found antimutagenic activities in 96% of the n-hexane extracts, 64% of the dichloromethane extracts, 44% of the acetone extracts, and 36% of the 2-propanol extracts. Since no or only minor differences were seen between the mutagens IQ and MeIQx investigations were continued with IQ only. Additional antimutagenic activities were detected in a total of 29.6% of extracts tested when an enzyme preparation with glycosidase-activities (fecalase) was included in the assay. These activities were found in originally inactive or less active dichloromethane, acetone, and 2-propanol extracts, and are therefore strongly suggestive for the liberation of antimutagenic aglycones from inactive glycosides. The existence of possibly a multitude of antimutagenic factors in fruits and vegetables was further substantiated by: (1) solvent partitioning of the n-hexane extracts of cauliflower, peaches, and spinach; (2) separation of the n-hexane and dichloromethane extracts of cauliflower, peaches, and spinach into acidid, neutral, and basic compounds; (3) chromatographic analysis of the n hexane and dichloromethane extracts of spinach. Taken together, antimutagenic activities were present in 32 of 36 subfractions, corresponding to 88.9%. In the green vegetables beans, broccoli, and spinach the known antimutagen chlorophyll was proven to contribute considerably to antimutagenic potency. Other important contributions may be caused by various fibers: (I) antimutagenicity of fruit and vegetable solvent extracts was extensively heat stable; (II) heating surprisingly caused an increase of antimutagenic potencies or generated new antimutagenic activities in several solvent fractions, especially of broccoli, white and red cabbage. Indeed, mutagenicity induced by IQ was strongly reduced by lignin, weakly by alginic acid and pectin A, while cellulose, gum arabic, gum guar, and xylan were ineffective. With respect to the mechanisms of antimutagenicity binding of IQ by various fibers and inhibition of cytochrome P-450-dependent monooxygenases might be of major importance since no solvent fraction of any fruit or vegetable was able to reduce mutagenic activity induced by N-OH-IQ in S. typhimurium TA 98NR. PMID- 7531290 TI - Effects of recombinant human granulocyte-colony stimulating factor on neutropenic mice infected with Candida albicans: acceleration of recovery from neutropenia and potentiation of anti-C. albicans resistance. AB - The treatment of systemic candidal infection in neutropenic patients continues to be a major problem, and only 20% of patients survive despite treatment with amphotericin B (Amph B). Granulocyte colony-stimulating factor (G-CSF) is a haemopoietic glycoprotein that appears to control the survival, cycle, activation, proliferation and maturation of neutrophil granulocytes and promoter recovery from neutropenia. Confirming previous results, we observed that subcutaneous (s.c.) injection of recombinant human (rh) G-CSF in mice (30 micrograms kg-1 daily) increased the circulating leucocyte count (fourfold) on day 5 of treatment, and led to an expansion of the bone marrow myeloid compartment. The in vivo effect of rhG-CSF on murine resistance to systemic Candida albicans infection was also studied in neutropenic mice. Neutropenia was induced by intraperitoneal injection of a single dose of cyclophosphamide (CPA, 200 mg kg-1) 4 days before C. albicans infection and 2 days before rhG-CSF treatment. rhG-CSF administration showed a protective role on mice infected intravenously (i.v.) with one million C. albicans spores; all the untreated control mice died within 8 days after infection, whereas about 40% of mice treated with rhG-CSF remained alive for the same period. Furthermore, the survival rate was greater in host animals treated with combined Amph B and rhG CSF than in those treated with Amph B alone. The number of C. albicans colony forming units (CFU-C. albicans) in the kidney of infected mice was lower in the rhG-CSF-treated group than in the non-treated control mice. This suggests that the severity of infection is decreased in rhG-CSF-treated host animals. PMID- 7531291 TI - Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution. AB - The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents. The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily. PMID- 7531292 TI - Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin. AB - In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT2A antagonist) and methysergide (5-HT1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2 phthalimido)butyl] piperazine (NAN-190) (5-HT1A antagonist), but not by granisetron (5-HT3 antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT. PMID- 7531293 TI - Characterization of histamine H3 receptors inhibiting 5-HT release from porcine enterochromaffin cells: further evidence for H3 receptor heterogeneity. AB - The nature of the histamine receptor mediating inhibition of 5-HT release was investigated in strips of the porcine small intestine by investigating the effects of histamine ligands on the overflow of endogenous 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The overflow was measured by HPLC, combined with electrochemical detection and represents calcium-sensitive 5 HT release from enterochromaffin cells, as reported previously. The histamine H3 receptor selective agonists (R)-alpha-methyl-histamine and imetit inhibited the overflow of 5-HT maximally by 50-60%, with EC50 values of 48 and 3.2 nmol/l, respectively. Effects on 5-HT overflow were always accompanied by similar effects on the overflow of 5-HIAA. Thioperamide (100 nmol/l) shifted the concentration response curve of (R)-alpha-methyl-histamine to the right (pKB value 8.38). The inhibitory effect of 1 mumol/l (R)-alpha-methyl-histamine was antagonized in a concentration-dependent manner by thioperamide (IC50: 65 nmol/l) and dimaprit (IC50: 8.6 mumol/l); however, the effect of (R)-alpha-methyl-histamine was weakly antagonized by burimamide (by 38% at 100 mumol/l) and not significantly affected by other H3 receptor antagonists, such as impromidine, betahistine and phenyl butanoyl-histamine (each up to 100 mumol/l). In conclusion, H3 receptors mediating inhibition of 5-HT release from porcine enterochromaffin cells have a particular pharmacological profile indicating that heterogeneity of H3 receptors may exist. The data suggest that histamine H3 receptors modulating 5-HT release in pig small intestine do not belong to either H3A or H3B receptors as defined in rat tissue. PMID- 7531294 TI - Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists. AB - Galanin, a 29 amino acid peptide, has been reported to possess antinociceptive properties at the spinal site and to potentiate opioid-induced antinociception. Our aim was to investigate whether also endogenous galanin interacts with an exogenously administered opioid, morphine, in the rat spinal cord. This question was investigated by use of the recently developed galanin receptor antagonists galantide [M-15, galanin-(1-13)-substance P-(5-11) amide] and M-35 [galanin-(1 13)-bradykinin-(2-9) amide]. Nociception was assessed in the rat tail-flick test using radiant heat and the rat Randall-Selitto model of inflammatory pain using vocalization as the nociceptive criterion. Intrathecal (i.t.) injections were performed in rats under either anaesthesia. Morphine was administered either i.t. or intraperitoneally (i.p.), and the antagonists were injected i.t. [125I]Galanin binding experiments were performed on crude synaptosomal membranes of the rat spinal cord. In the rat tail-flick test, i.t. injection of 3 micrograms morphine evoked antinociception of about 75% of the maximal possible effect (% MPE). Co injection of either 2 micrograms galantide or 2 micrograms M-35 with morphine almost completely abolished the antinociceptive effect of morphine. I.p. injection of 2.15 mg/kg morphine elicited about 80% MPE when given 10 min prior to i.t. saline injection. Injection of the antagonists instead of saline antagonised the antinociceptive effect of morphine partially thus showing the spinal proportion of the overall antinociceptive effect. In the rat Randall Selitto test, 3 micrograms morphine, injected i.t., produced antinociception of almost 100% MPE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531296 TI - [A case report of a 6-year-old boy with intracranial yolk sac tumor treated by VAB-6 regimen]. AB - Several clinical trials have demonstrated that cisplatin-based chemotherapy for primary intracranial germ-cell tumors is effective as a neoadjuvant chemotherapy. In this report, we describe a 6-year-old boy, Down syndrome and Hirschsprung's disease with intracranial pure yolk sac tumor treated by combined chemotherapy with cisplatin, vinblastine, bleomycin and cyclophosphamide (modified VAB-6 regimen). He had been admitted to our hospital because of intractable vomiting, and left facial nerve palsy since 1 month before. An MRI revealed an enlarged mass, 4cm in diameter, in the left cerebello-pontine angle with uniformal enhancement by Gd-DTPA, and bilateral ventricular dilatation. He was found to have increased serum alpha-fetoprotein level (AFP 11, 786ng/ml), but not human chorionic gonadotropin beta-subunit. After a partial resection of the tumor, diagnosed as pure yolk sac tumor, and ventriculo-peritoneal shunt, three courses of combined chemotherapy with cisplatin, bleomycin, vinblastine and cyclophosphamide (modified VAB-6 therapy) were carried out. The serum AFP level returned to normal, and the tumor mass entirely disappeared (a complete response) on MRI after the second course of chemotherapy. However, cisplatin-induced vomiting and mild neutropenia and renal tubular injury developed after the third course of chemotherapy. Irrespective of administration of recombinant human G-CSF and broad spectrum antibiotics, he suffered from pneumonia and died of septic shock and multiple organ failure. Autopsy showed microscopic residual tumors. The combination chemotherapy with cisplatin, bleomycin, vinblastine and cyclophosphamide is effective for initial treatment of childhood intracranial yolk sac tumor. It is necessary, however, to reevaluate the cisplatin dosage and treatment schedule in order to reduce such side effects as bone marrow suppression and renal damage. PMID- 7531295 TI - Pharmacological characterisation of NK1 receptor antagonist, [D-Trp7]sendide, on behaviour elicited by substance P in the mouse. AB - An analogue of sendide, [D-Trp7]sendide, was newly synthetized and evaluated as a putative NK1 receptor antagonist in a mouse behavioural test. Effects of [D Trp7]sendide on the scratching, biting and licking response induced by substance P (SP), neurokinin A (NK A) and neurokinin B (NK B) was studied after intrathecal injections. When administered simultaneously with SP, an endogenous agonist for NK1 receptors, [D-Trp7]sendide inhibited the behavioural response to this tachykinin in a dose-dependent manner with ID50 value of 11.0 pmol/mouse. The behavioural response elicited by other NK1 receptor agonists, septide and physalaemin, was reduced significantly by a small dose (32.0 pmol) of [D Trp7]sendide. Large doses (nmol order) of [D-Trp7]sendide were needed to reduce the characteristic behaviour of NK A, an NK2 agonist, NK B, an NK3 agonist and eledoisin, an NK2/NK3 agonist. The duration of the antagonistic effect of [D Trp7]sendide was relatively longer. In a [3H]labeled SP binding assay using mouse spinal cord membranes, [D-Trp7]sendide potently displaced [3H] labeled SP binding with a Ki value of 0.023 +/- 0.007 nM, which was approximately 140 and 9400 times more potent than that of unlabeled SP and CP-96,345, respectively. These findings suggest that [D-Trp7]sendide interacts selectively with the NK1 receptor in the mouse spinal cord as assayed by the receptor binding and SP-induced behavioural tests. PMID- 7531297 TI - Platelet-derived growth factor (PDGF) and receptor (PDGFR) expression in human meningiomas: correlations with clinicopathological features and cytogenetic analysis. AB - PDGFs and their receptors expression were examined in a series of 46 meningiomas by using specific monoclonal antibodies. The immunostaining was quantified by an image analyser and the results correlated with clinical and morphological data (histological type and grade). In addition, since the PDGFB chain is encoded by the c-sis proto-oncogene localized on chromosome 22 and because monosomy 22 has been frequently reported in meningiomas, PDGFs and PDGFRs expression have been correlated with cytogenetic analysis performed in 29 cases. The results demonstrate PDGF A and PDGF B expression in most meningioma specimens and co expression of these growth factors in numerous cells. PDGF A and B immunoreactivity was related to histological grade. PDGFR beta expression was strong in almost all meningiomas whereas PDGFR alpha was low. PDGFR alpha expression was related to tumour location and grade and PDGFR beta to histological subtype only. The cytogenetic analysis was not related to PDGFB chain expression. Taken together these data further confirm PDGF and PDGFR expression in human meningioma; PDGF may exist as an heterodimer (AB) as well as its receptor. The lack of correlation between cytogenetic analysis and PDGF values, the low level of PDGFB in recurrent meningiomas suggests that it is unlikely that the c-sis proto-oncogene plays an important role in the genesis of meningiomas. PMID- 7531298 TI - A modified NOR-silver impregnation technique for amyloid plaques and neurofibrillary tangles: comparative assessment. AB - A modified nucleolar organizer region (NOR)-silver impregnation technique was recently introduced allowing the simultaneous staining of neurofibrillary tangles (NFT) and amyloid plaques (AP) in a single section. This impregnation technique proved to be very effective, rapid, of low cost, and to yield consistent results even in brain tissue fixed for long periods of time. In the present study, several silver impregnation and immunohistochemical techniques were applied and their reliability for the demonstration of NFT and AP was analysed. The modified NOR stain allowed the reliable and simultaneous demonstration of both amyloid plaques and neurofibrillary tangles; this method produced better results than the standard silver impregnation techniques. It is only in their early stages of formation that NFT cannot be detected by this method and, instead, immunohistochemistry for tau-2 is required. There was no significant difference in the staining properties for amyloid plaques between the modified NOR silver impregnation technique and the anti-beta-A4 immunostain. PMID- 7531299 TI - Increased hypothalamic glutamate receptors induced by water deprivation. AB - To examine the role of receptor changes in the adaptive response to physiological stimulation, the density and distribution of excitatory amino acid receptors within the hypothalamus and other brain regions were examined in rats deprived of water for 2 days. Membrane binding assay revealed an increase in glutamate receptor density and a small shift in the affinity of glutamate for the receptor. Regional analysis of these changes by receptor autoradiography specific for NMDA, non-NMDA or metabotropic glutamate receptor binding indicated that NMDA and metabotropic receptor densities are increased in the brain. Regional increases were found principally for the NMDA receptor binding within the supraoptic nucleus, anterior hypothalamus, caudate-putamen and globus pallidus with no significant changes in 24 other brain regions. No significant changes were found in any brain regions for AMPA receptors. Metabotropic and kainate receptors tended to parallel the NMDA receptor changes, although few regions reached statistical significance. These changes indicate that brain regions associated with water balance regulation show selective adaptive increases in NMDA receptors during water deprivation which may facilitate prolonged activation of these cells. PMID- 7531300 TI - The effect of tianeptine and sertraline in three animal models of depression. AB - The activity of tianeptine (2.5 and 5.0 mg/kg twice daily, i.p.) and of sertraline (5.0 mg/kg, twice daily, i.p.) were assessed in three animal models of depression. In the Behavioural Despair Test, acute treatment with sertraline or tianeptine (5.0 mg/kg) significantly reduced the immobility time. In the olfactory bulbectomized (OB) rat model, chronic treatment with tianeptine (2.5 and 5.0 mg/kg) or sertraline (5.0 mg/kg) antagonized the lesion-induced hyperactivity in the "open field" apparatus. The hypothermic response to the 5 HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.15 mg/kg, s.c.) was significantly attenuated after chronic setraline treatment, whereas tianeptine was inactive at the 2 doses tested. Neither drug affected the hypersection of corticosterone that occurs at the light:dark interface. A reduction in the serotonin metabolite 5-HIAA was found in the hypothalamus of sertraline-treated sham rats. It can be concluded that although the neurochemical properties of sertraline and tianeptine differ, they demonstrate similar antidepressant-like activities in the Behavioural Despair and OB rat models. The lack of effect of tianeptine on the 8-OH-DPAT-induced hypothermic effect indicates that it does not induce 5-HT1A subsensitivity, contrary to most antidepressants. PMID- 7531301 TI - Self-administration of neurokinin substance P into the ventromedial caudate putamen in rats. AB - There is evidence that the neurokinin substance P plays a role in learning and reinforcement processes. Reinforcing effects of substance P were found upon injection into several parts of the brain. The aim of the present study was to gauge possible reinforcing effects of microinjections of substance P into the ventromedial caudate-putamen in rats. Two different behavioral paradigms were employed. In the first experiment a two-compartment choice procedure was used and the rats could trigger substance P injections (500 pg per 5 nl injection volume) into the ventromedial caudate-putamen by entering one distinctive compartment. During the injection period, substance P-injected animals spent significantly more time in the drug-paired compartment than vehicle-injected controls. In the second experiment, nose-poking through a hole in one wall of the cage was used as the operant. Rats that could self-administer substance P (100 pg per 5 nl injection volume) into the ventromedial caudate-putamen emitted a significantly higher rate of operant responding on the first day of testing and a significantly lower rate on the third day compared to vehicle-injected animals. The experiments provide evidence that the administration of substance P into the ventromedial part of the caudate-putamen can have positive reinforcing effects, but that repeated injections can have aversive properties. These effects are discussed, firstly, with regard to the possible mechanisms of intrastriatal substance P on striatonigral and striatopallidal output systems and, secondly, with respect to their possible relevance in the study of the basal forebrain reinforcement system. PMID- 7531302 TI - Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex. AB - Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes. PMID- 7531303 TI - Neurokinin A mimics the slow excitatory postsynaptic current in submucous plexus neurons of the guinea-pig caecum. AB - Single microelectrode voltage-clamp recordings were made from submucous neurons of the guinea-pig caecum. The slow excitatory postsynaptic current was compared with the currents induced by neurokinin A and substance P. The current induced by neurokinin A (100-300 nM) was associated with a decreased membrane conductance and reversed in polarity between -90 and -100 mV. The neurokinin A current was reduced by Co2+ (1-2 mM), but was not affected by Cs+ (1-2 mM), Ba2+ (10-100 microM) or low Cl- (20-40 mM) solutions. In about 80% of the neurons, the current induced by substance P (100-300 nM) was associated with a decreased membrane conductance and did not reverse with hyperpolarization of the membrane potential up to -130 mV. The current was reduced by Co2+ (1-2 mM) and augmented by low Cl- (20-40 mM) solutions, but was not affected by Cs+ (1-2 mM) or Ba2+ (10-100 microM)-containing solutions. In about 20% of the neurons, the substance P current reversed in polarity between -100 and -120 mV. The slow excitatory postsynaptic current elicited by repetitive nerve stimulation (10-40 Hz, three to five pulses) was accompanied by a decreased membrane conductance, and reversed in polarity between -90 and -100 mV. The slow excitatory postsynaptic current was abolished by Co2+ (1-2 mM) or low Na+ (12 mM) solutions, but was not affected by Cs+ (1-2 mM), Ba2+ (10-100 microM) or low Cl- (20-40 mM) solutions. In such neurons, the neurokinin A current was reversed at approximately the same potential at which the slow excitatory postsynaptic current was reversed, while the substance P current was not reversed even by much stronger hyperpolarizations. It was concluded that the neurokinin A current was mainly due to depression of potassium conductances, while the substance P current resulted from both increased anion conductance and decreased potassium conductances. The conductance change underlying the slow excitatory postsynaptic current is similar to that caused by neurokinin A. PMID- 7531304 TI - Nitric oxide and cyclic guanosine 3',5'-monophosphate do not alter neuropeptide release from rat sensory neurons grown in culture. AB - Recent studies demonstrate that nitric oxide and cyclic guanosine 3',5' monophosphate may mediate hyperalgesia induced by N-methyl-D-aspartate at the level of the spinal cord. One possible mechanism for this action is that nitric oxide increases transmitter release from the primary afferent nociceptors that synapse in the dorsal horn of the spinal cord. To address this possibility, we investigated whether various nitric oxide donors and cyclic guanosine 3',5' monophosphate could alter the release of substance P and calcitonin gene-related peptide from rat sensory neurons in culture. Sodium nitroprusside (100 nM to 100 microM) had little effect on basal release of either peptide, but it significantly increased the release of substance P and calcitonin gene-related peptide induced by 50 nM capsaicin. In contrast, sodium nitroprusside did not alter release evoked by 100 nM bradykinin or 30 mM KCl. Two other nitric oxide donating compounds, S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine did not enhance resting or capsaicin-evoked peptide release, although they induced a marked elevation in the intracellular cyclic guanosine 3',5' monophosphate levels. Pretreating the cultures with 8-bromo-cyclic guanosine 3',5'-monophosphate, (0.5 or 0.1 mM for 30 or 60 min) did not result in the enhancement of capsaicin-induced release from sensory neurons. Moreover, pretreating the cells with the nitric oxide synthase inhibitor, NG-nitro-L arginine (100 microM), abolished the rise in cyclic guanosine 3',5'-monophosphate induced by capsaicin without altering capsaicin-stimulated release of either peptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531305 TI - The effect of atropine on the activation of 5-hydroxytryptamine3 channels in rat nodose ganglion neurons. AB - It has been suggested that changes in brain 5-hydroxytryptamine3 receptor function may contribute to some behavior disorders, such as anxiety, schizophrenia and drug abuse. We are using the whole-cell version of the patch clamp technique to study the function of 5-hydroxytryptamine3 channels in neurons freshly dissociated from rat nodose ganglion. In these cells, 5-hydroxytryptamine elicits an inward current over the concentration range of 0.25-100 microM (EC50 = 2.62 microM) by activating 5-hydroxytryptamine3 receptors. The muscarinic cholinergic antagonist atropine reduced the amplitude of 5-hydroxytryptamine activated inward current in a concentration-dependent manner. Other muscarinic antagonists, scopolamine, dexetimide, the M1 muscarinic receptor antagonist pirenzepine, the M2 receptor antagonist methoctramine and the M3 receptor antagonist 4-DAMP methiodide also inhibited 5-hydroxytryptamine-induced inward current. Atropine did not appear to change the reversal potential of this current. In the presence of 5 microM atropine, the concentration-response curve for 5-hydroxytryptamine current was shifted to the right in a parallel fashion. The EC50 value for 5-hydroxytryptamine was increased from 2.62 to 8.76 microM. Schild plots of increasing atropine and 5-hydroxytryptamine concentrations revealed a pA2 value of 5.74 for atropine (apparent KD = 1.8 microM). These observations suggest that atropine competitively antagonizes the activation of a receptor for the neurotransmitter serotonin, a novel action of muscarinic antagonists in the nervous system. This effect of atropine may contribute to the clinical symptoms seen in severe atropine intoxication. PMID- 7531306 TI - Kindling increases N-methyl-D-aspartate potency at single N-methyl-D-aspartate channels in dentate gyrus granule cells. AB - Dose-response studies of N-methyl-D-aspartate channel openings were carried out using cell-attached patches in dentate gyrus granule cells acutely isolated from control and kindled rats. The tips of the patch electrodes were first filled with regular extracellular solution, followed by backfilling through the shank with the agonist containing solution. As the two solutions joined, the agonist (N methyl-D-aspartate, 25 microM) steadily diffused to the cell membrane, and the concentration gradually built up resulting in the progressive increase in the opening probability of N-methyl-D-aspartate channels. The reliability of this cell-attached diffusional drug delivery method was tested by determining the concentration dependence of competitive antagonism of N-methyl-D-aspartate induced channel activity by D(-)-2-amino-5-phosphonopentanoic acid. The Ki for D( )-2-amino-5-phosphonopentanoic acid in the presence of 25 microM N-methyl-D aspartate was found to be 6.8 microM. Twenty-four hours following the last seizure, N-methyl-D-aspartate channels on kindled neurons were consistently activated by lower N-methyl-D-aspartate concentrations than channels on control granule cells, indicating a higher potency of agonist at epileptic N-methyl-D aspartate channels. The higher potency of the agonist is most likely a reflection of the long-term alterations in the modulation of N-methyl-D-aspartate receptor function in epileptic neurons. PMID- 7531307 TI - Type I nitric oxide synthase fully accounts for NADPH-diaphorase in rat striatum, but not cortex. AB - The novel gaseous neuromediator nitric oxide is thought to play an important role in development and plasticity. Despite this, gene-knockout mice lacking neuronal (Type I) nitric oxide synthase exhibit relatively normal brain development and behavior. The nervous system of these mice (especially the forebrain) retains some calcium-dependent nitric oxide synthesis, presumably reflecting other isozymes. Type I nitric oxide synthase has NADPH-dependent diaphorase activity. However, this stain also recognizes other isozymes, and it remains controversial whether all diaphorase-positive neurons contain Type I nitric oxide synthase. To assess whether neurons containing another isoform of nitric oxide synthase may be present in the forebrain of normal rodents, we studied co-localization of diaphorase staining with immunocytochemistry for Type I nitric oxide synthase. Co localization was complete in the striatum, but some neurons deep in cortex were diaphorase-positive and immunonegative, and therefore may contain a splice variant or novel isozyme of nitric oxide synthase. PMID- 7531309 TI - Atrial natriuretic peptide in the innervation of the bovine heart conduction system: relationship with substance P and autonomic innervation- immunohistochemical studies. AB - Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity (IR) was present not only in the Purkinje fibres, but also in nerve fibre varicosities in the conduction system of the bovine heart. These findings and previous observations that ANP is able to influence autonomic neurotransmission in the heart, lead us to elucidate the possible occurrence of ANP in the sympathetic and/or parasympathetic nervous systems and/or in various types of peptidergic innervation in the conduction system. The different parts of the conduction system of bovine hearts were dissected out and processed for immunohistochemistry including double-staining, using antisera against ANP, tyrosine hydroxylase and different neuropeptides. We observed that some of the nerve fibre varicosities exhibiting ANP-IR showed substance P-IR and that ANP was present as scattered immunoreactive granules in intracardial, presumably parasympathetic, ganglionic cells. The study shows that ANP is likely to be present in parasympathetic innervation and in afferent nerve endings in the bovine heart conduction system. PMID- 7531308 TI - Functional coupling of the 5-HT2C serotonin receptor to G proteins in Xenopus oocytes. AB - The serotonin 2c (5-HT2C) receptor mediates its cellular effects by interacting with heterotrimeric guanine nucleotide binding proteins (G proteins). To characterize which G proteins are involved in functional coupling to the receptor, a mouse 5-HT2C receptor was expressed in Xenopus oocytes, and antisense oligoncleotides complementary to the mRNA sequence of the endogenous Xenopus G protein alpha subunits were used to inhibit G protein synthesis. Antisense oligonucleotide against the Xenopus G(o) alpha subunit inhibited the 5-HT2C receptor function, and coexpression of a rat G(o) alpha subunit reversed the inhibition by the anti-Xenopus G(o) oligonucleotide. Furthermore, antisense oligonucleotides against both the G(o) and Gi1 alpha subunits inhibited the electrophysiologic response induced by stimulation of the 5-HT2C receptor. These data suggest that both G(o) and Gi1 are involved in functional coupling of the 5 HT2C receptor to phospholipase C in Xenopus oocytes. PMID- 7531310 TI - Persistently enhanced ratio of NMDA and non-NMDA components of rat hippocampal EPSC after block of A1 adenosine receptors at increased [Ca2+]o/[Mg2+]o. AB - NMDA and non-NMDA receptor-mediated components of excitatory post-synaptic current (EPSC) were studied by in situ whole-cell voltage-clamp recordings in the CA1 field of rat hippocampus. We found that the amplitudes ratio of the NMDA to the non-NMDA components can be strongly increased by blocking A1 adenosine receptors. The necessary conditions for this effect are both, increased Ca2+ and lowered Mg2+ in the external medium. The so achieved increase in the NMDA/non NMDA ratio of EPSC components is irreversible and no longer depends on the activity of A1 adenosine receptors. PMID- 7531311 TI - NMDA receptor-mediated synaptic input to nitric oxide synthase-containing neurons of the guinea pig mesopontine tegmentum in vitro. AB - Intracellular recordings were obtained from laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) neurons in a slice preparation to determine if excitatory synaptic input to the nitric oxide synthase (NOS)-containing cells was mediated by NMDA receptors. NOS cells were identified by intracellular injection of biocytin and NADPH-diaphorase histochemistry. Application of NMDA produced membrane depolarization, and EPSPs were reversibly reduced by an average of 46.3 +/- 16.6% with AP-5 (50 microM). These results indicate that post-synaptic NMDA receptors participate in synaptic transmission to NOS-containing neurons in LDT and PPT and are consistent with the hypothesis that synaptic NMDA receptors may control NO production in these cells. PMID- 7531313 TI - The pyrimidine derivative BW 1003C87 protects against excitotoxic lesions induced by kainate in the rat striatum. AB - We have investigated the neuroprotective effect of the pyrimidine derivative BW 1003C87 (5-[2,3,5-trichlorophenyl] pyrimidine-2,4-diamine ethane sulphonate) against striatal and hippocampal lesions induced by kainic acid (KA), N-methyl-D aspartate (NMDA) and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((S)-AMPA) in the rat. BW 1003C87 20 mg/kg i.p. administered pre- and post treatment (20 min prior to excitotoxic injection and again 4 h later) protects against the lesions induced by KA (1.1 nmol) in the hippocampus (CA2 pyramidal cells only; 40% protection, P < 0.05). In the striatum, the same dose of BW 1003C87 significantly reduces KA toxicity (80% protection, P < 0.001). BW 1003C87 has no significant effect on the lesions induced by NMDA (30 nmol) or S-AMPA (6 nmol) in either brain region. These results are consistent with previous studies showing that the neurotoxicity of KA occurs via an indirect mechanism involving glutamate release. PMID- 7531315 TI - Divalent metals of myelin and their differential binding by myelin basic protein of bovine central nervous system. AB - Divalent metal ions are being implicated in the compaction of myelin. Levels of Cd, Co, Cu, Hg, Mn, Pb, Zn, Ca and Mg of isolated myelin of bovine central nervous system (CNS) were measured by flame atomic absorption spectrophotometry. Binding of these metal ions by isolated myelin basic protein (MBP, M(r) 18,500) of bovine CNS was concurrently assessed by centrifugal equilibrium dialysis. Metals were bound in the order of Hg > Cu > Zn > Mg > Cd > Co, exempting Mn, Pb and Ca. The results are indicative of differential metal affinity of MBP which may account for the immobilization or anchoring of MBP in myelin by zinc and other divalent metal cations. PMID- 7531314 TI - Hydroxyl radical generation and nitric oxide synthase activity in Fisher 344 x brown-Norway F1 rat brain. AB - Hydroxyl radical production in the cerebral cortex, striatum and hippocampus of young (6 months), middle-aged (15 months) and old (28 months) Fisher 344 x Brown Norway F1 rats was quantitated by measuring the salicylate hydroxyl radical trapping product, 2,5-dihydroxybenzoic acid. The levels of hydroxyl radical between different age groups in each region examined were not statistically different. In all regions, with the exception of hippocampus from old rats which had a lower content of hydroxyl radical, the levels of hydroxyl radical were higher for middle-aged and old rats than young ones. There was no regional difference in the production of hydroxyl radicals, except that the level was significantly higher in striatum than in cortex for 15-month-old rats. The cerebral cortical nitric oxide synthase activities were similar in the three age groups studied. PMID- 7531316 TI - Nitric oxide synthase inhibitors prevent N-methyl-D-aspartic acid-induced penile erection and yawning in male rats. AB - The effect of NG-nitro-L-arginine methylester (NAME) and N-mono-methyl-L-arginine (NMMA), inhibitors of nitric oxide (NO) synthase on penile erection and yawning induced by N-methyl-D-aspartic acid (NMDA) injected in the paraventricular nucleus of the hypothalamus (PVN) was studied in male rats. NAME (75-150 micrograms) and NMMA (250-500 micrograms), but not N-monomethyl-D-arginine (D NMMA)(250-500 micrograms) prevented both responses in a dose-dependent manner when given intracerebroventricularly (i.c.v.) 15 min before NMDA (50 ng). NMDA induced penile erection and yawning was also prevented by the guanylate cyclase inhibitor methylene blue (200-400 micrograms i.c.v.), but not by the NO scavenger methemoglobin (50-100 micrograms i.c.v.). NAME (10-20 micrograms), but not Methylene blue or methemoglobin (10-20 micrograms), prevented NMDA-induced responses also when injected in the PVN 15 min before NMDA. The present results suggest that NMDA-induced penile erection and yawning is mediated by an increased NO synthesis in the PVN. PMID- 7531312 TI - Expression of c-jun and neuronal nitric oxide synthase in rat spinal motoneurons following axonal injury. AB - Expression of neuronal NOS, c-jun and c-fos in spinal motoneurons following axonal damage were investigated in the present study. Although either distal spinal root axotomy or root avulsion induced expression of c-jun, expression of c jun was predominantly found in distal root-axotomized motoneurons. In contrast, expression of NOS was exclusively observed in avulsed motoneurons. c-fos was not expressed in spinal motoneurons following either distal root axotomy or root avulsion. The different expression patterns of c-jun and NOS in the injured neurons suggest that these molecules may involve in different cellular processes and might play different roles in response to the injury. Since distal root axotomy did not cause motoneuron death and root avulsion did, expression of c-jun is likely related to regenerative process while induction of NOS may be involved in the degenerative process. PMID- 7531317 TI - [Interferon therapy in chronic active viral hepatitis]. AB - The authors give a short review of the recent data about the types of interferons and their biological activity. The role of interferons in the therapy of B, C and D chronic viral hepatitis is discussed. Interferon treatment means a substantial progress in the therapy of chronic viral hepatitis, however it represents a final recovery from chronic B or C hepatitis only in 25-40 percent or 40-45 percent of the cases, respectively. The authors refer to the combination therapy which seems to be promising in the future. PMID- 7531318 TI - Binding in vitro of phosphotyrosine-containing proteins to pp60c-src SH2 domain does not correlate with CEF transformation. AB - We have previously described pp60c-src SH2 mutants that are host-range-dependent for cell transformation; most of these mutants can transform CEF cells but not NIH3T3 cells, and other transform NIH3T3 cells more efficiently than CEF (Hirai and Varmus, 1990c). In an attempt to understand the molecular basis of these phenotypes, we analysed the ability of mutant SH2 domains in GST fusion proteins to bind to tyrosine phosphorylated proteins in lysates from CEF and NIH3T3 cells. The relative affinity of mutated versions of the SH2 domain for phosphotyrosine containing proteins from CEF and NIH3T3 cells was compared with the relative ability of the mutant Src proteins to transform the two cell types. While the affinity of the SH2 domain for phosphotyrosine-containing proteins was closely correlated with transformation in NIH3T3 cells, there was no correlation between phosphotyrosine binding and transformation of CEF cells, and none of the host range mutant SH2 domains showed significant differences in their ability to bind phosphotyrosine-containing proteins from lysates from either cell type. In addition, the SH3 domain was shown to augment the capacity of mutant SH2 domain to bind phosphotyrosine-containing proteins. PMID- 7531319 TI - Use of granulocyte colony-stimulating factor in neutropenic children. PMID- 7531320 TI - Detection of Rochalimaea henselae DNA by polymerase chain reaction from suppurative nodes of children with cat-scratch disease. AB - A polymerase chain reaction (PCR) assay was designed to amplify DNA from Rochalimaea henselae, Rochalimaea quintana and Afipia felis in the purulent material from lymph nodes in three patients with clinical cat-scratch disease (CSD) and two patients with lymphadenitis from other causes. All of the patients with CSD had positive immunofluorescent antibody serology for R. henselae, while none of the controls was positive. PCR amplification confirmed the presence of R. henselae DNA and the absence of R. quintana and A. felis DNA in the purulent material from CSD patients. PCR samples from control patients were negative. The PCR amplification of R. henselae DNA was performed quickly and with great sensitivity and specificity. It confirmed the presence of R. henselae in the CSD patients and eliminated the need for more extensive diagnostic and therapeutic procedures. PMID- 7531322 TI - Acute pancreatitis: an early manifestation of systemic lupus erythematosus. AB - Acute pancreatitis is thought to be quite uncommon in patients with systemic lupus erythematosus (SLE), especially if it is the initial manifestation of the disease. We report a 13-year-old girl with SLE whose early clinical presentation was acute pancreatitis. The disease was not related to concomitant drug therapy. It was associated with elevated serum and urine amylase levels and pancreatic enlargement by ultrasound assessment. The clinical symptoms and signs subsided after prednisolone treatment. PMID- 7531324 TI - Analysis of prostatic fluid: evidence for the presence of a prospective marker for prostatic cancer. AB - In an endeavor to identify marker(s) for prostatic cancer, proteins in prostatic fluids were analyzed by two-dimensional (2-D) gel electrophoresis. The fluids were obtained from five males who had no prostate lesions and five patients each with benign prostatic hyperplasia (BPH) and prostatic carcinoma (PCA). The specimens were collected directly over a mixture of protease inhibitors and centrifuged, and the supernatants were lyophilized and solubilized in sodium dodecyl sulfate mix. Identical amounts of proteins were pooled according to donors' prostate disease and the resulting samples were subjected to 2-D gel analysis employing the ISO-DALT system. The electrophoretograms were developed by silver or double stain. The samples of each group exhibited distinctive profiles with the exception of similar relative positions of major protein spots. A predominant protein occurring as several charge variants was consistently present in prostatic fluids of patients with PCA. This protein appeared to be a previously unknown constituent that we have called protein D (molecular weight approximately 22 kDa and isoelectric point approximately 4), and was undetectable in the fluids of "normal" men and patients with BPH. An analysis of pooled, unprocessed urine from PCA patients revealed that perhaps this protein is excreted in urine in very low quantities. These results strongly suggest that the potential of this protein as a marker for prostatic cancer should be further explored. PMID- 7531321 TI - Analysis of circadian distribution of premature ventricular contractions in patients after heart transplantation. AB - The aim of our study was to analyze circadian distribution of premature ventricular contractions (PVC) and its coupling interval (CI) in patients after orthotopic heart transplantation (HTx). Forty-two patients (5 females, 37 males) were monitored from 2 weeks to 5 years after HTx; 180 24-hour Holter ECG studies were performed. All recordings were divided into two groups: group I, within 1 month after HTX; and group II, after 1 month. Patients with more than 250 PVC/24 hours were selected for distribution of PVC and CI evaluation. CONCLUSIONS: Ventricular arrhythmias occur frequently in patients after heart transplantation. In patients with high Lown scale arrhythmias low occurrence (< 250/24 hours) of PVC was frequently observed (IVa: 81.8%; IVb: 84.7%). Similar patterns of circadian distribution (CD) of PVC and CD of HR in denervated heart after HTx suggest the influence of circulating catecholamines on their occurrence. PMID- 7531323 TI - Cellular PSA in benign and malignant prostate. AB - To determine the relationship of carcinoma of the prostate and cellular production of prostate-specific antigen, cytosol levels of PSA were measured in benign and malignant fresh prostate tissue harvested from radical prostatectomy specimens. Wedge biopsies were taken from benign (N = 21) and malignant (N = 74) prostate tissue and were immediately fixed in liquid nitrogen, and then homogenized and differentially centrifuged, and the cytosol fractions extracted. The remaining specimen was sent for routine pathologic assessment. The Hybritech methodology was used to measure the cytosol PSA and standard protein analysis was used for cytosol protein (CP) measurement. There was a significantly greater concentration of PSA in malignant tissue (P = 0.046). Also, when benign and malignant tissue were available from a single prostate (N = 17), these differences in cytosol PSA were even greater (P = 0.002). In addition, there was no significant difference when serum PSAs from the malignant tissue were ranked according to Gleason score and placed into three different histologic grades (i.e., Gleason scores 2-4, 5-6, and 7-10). PMID- 7531325 TI - A clinical and immunohistochemical study of papillary adenocarcinoma of the prostate. AB - Clinical and immunohistochemical studies were conducted to evaluate prostatic papillary adenocarcinoma and prostatic papillary hyperplasia. Subjects consisted of 5 cases of papillary adenocarcinoma and 2 cases of papillary hyperplasia. There is no conclusive clinical factor for preoperative diagnosis, but we attach importance to endoscopic findings. PSA, PAP, high molecular weight cytokeratin, and PCNA were evaluated immunohistochemically. PSA became positive in every instance but one--a case of papillary adenocarcinoma which became +/-. PAP was + in all cases, except for 1 case of papillary adenocarcinoma. Basal cells were positive for high molecular weight cytokeratin in 2 cases of papillary hyperplasia but were missing in papillary adenocarcinoma. Although PCNA was free from positive nuclei in papillary hyperplasia, positive nuclei were found in all cases of papillary adenocarcinoma. Considering these immunohistochemical results, papillary adenocarcinoma can be said to originate in the glandular epithelium of the prostate, as does ordinary prostatic carcinoma. PMID- 7531326 TI - A prospective study of plasma hormone levels, nonhormonal factors, and development of benign prostatic hyperplasia. AB - We assessed the relation of plasma hormone levels and nonhormonal factors with subsequent occurrence of surgical treatment for benign prostatic hyperplasia (BPH) among participants in the Physicians' Health Study. Frozen plasma samples, collected at the study onset, were available for 320 men who developed surgically treated BPH up to 9 years later and for 320 age-matched controls. Plasma testosterone (T), dihydrotestosterone (DHT), androstenedione, estradiol (E2), and estrone (E1) were measured for each case-control pair. In unadjusted analyses, none of the hormones or hormone ratios were associated with BPH; for example, for T and E2 the odds ratios (OR) comparing the highest quintile (Q5) with the lowest (Q1) were 0.74 (95% CI = 0.42, 1.30) and 1.07 (95% CI = 0.51, 2.22), respectively. However, in multivariate analyses controlling diastolic blood pressure, exercise, alcohol, E1, and DHT:T ratio, we observed a strong trend for increasing risk across quintiles for E2 (Q5 vs. Q1 OR = 3.56, P trend = 0.009), and a weak inverse trend for E1 (Q5 vs Q1 OR = 0.51, P trend = 0.07). The excess risk associated with E2 was confined to men with relatively low androgen levels. Three nonhormonal factors previously suspected as risk factors were independently associated with surgical BPH in these data. The OR for a 1-mm Hg difference in diastolic blood pressure was 1.04 (95% CI = 1.01, 1.07). Alcohol use and infrequent exercise were inversely associated with risk of BPH surgery; however, risk estimates were not consistent across categories of exercise and alcohol frequency. Our results indicate that normal variation in circulating androgen levels does not influence development of BPH, but that variation in estrogen levels might be important. PMID- 7531327 TI - The association between alpha-fetoprotein and beta hCG levels prior to and following chorionic villus sampling in cases that spontaneously miscarried. AB - Maternal serum alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (beta hCG) measurements taken prior to chorionic villus sampling (CVS) in 21 patients who subsequently miscarried were compared with measurements in a control group of 113 patients with uneventful pregnancies. Patients with AFP levels of 10 iu/ml or more prior to the CVS had a 4.3 times greater risk of miscarriage (95 per cent confidence interval 1.3-13.6). AFP levels obtained 1 week after the CVS in the 13 patients with late miscarriages were higher than in the control group (P = 0.06). Patients miscarrying had a greater rise in AFP (P = 0.06) and a greater fall in beta hCG levels (P = 0.04) following the CVS procedure, compared with the control subjects. Each 10-unit change in the difference between AFP or beta hCG levels prior to and 1 week following the CVS was associated with a significantly increased risk for late miscarriage. Elevated maternal serum AFP levels early in pregnancy and changes in AFP and beta hCG levels following CVS may predict an increased risk for subsequent miscarriage. PMID- 7531328 TI - Maternal serum free alpha- and free beta-human chorionic gonadotrophin in pregnancies with insulin-dependent diabetes mellitus: implications for screening for Down's syndrome. AB - A study was performed to investigate the concentrations of the alpha and beta free sub-units of human chorionic gonadotrophin (free alpha-hCG and free beta hCG) in maternal serum between 15 and 22 weeks of pregnancy in 126 pregnancies among 92 women with insulin-dependent diabetes mellitus (IDDM). Each IDDM pregnancy was matched with two control singleton pregnancies for gestational age (same completed week) and duration of sample storage (same calendar quarter). The median free alpha-hCG level in the IDDM pregnancies was 0.86 multiples of the median (MOM) for pregnancies without IDDM at the same gestational age (P < 0.002) (95 per cent confidence interval 0.80-0.94). The corresponding free beta-hCG level was 0.96 MOM (95 per cent confidence interval 0.85-1.09). These results enable free alpha-hCG values to be adjusted so that antenatal screening for Down's syndrome can be performed using this marker in IDDM pregnancies as well as in non-diabetic pregnancies. PMID- 7531330 TI - Pregnancy-associated plasma protein A (PAPP-A), a first-trimester screening test for Down syndrome and other chromosomal anomalies. PMID- 7531329 TI - Comparison of two Down syndrome screening strategies for women aged 35 and older. PMID- 7531331 TI - Prevention policy: perspectives on the critical interaction between research and policy. AB - Prevention researchers should join political and professional efforts to maximize coverage for validated preventive services and programs as part of health care reform. We have a useful framework from Healthy People 2000, the Guide to Clinical Preventive Services, and preventive clinical trials. We must anticipate the clinical and community-based questions for which prevention research can provide needed answers. Cancer prevention trials, serum cholesterol and prostate specific antigen screening, prevention of Helicobacter-related diseases, and comprehensive health promotion strategies for older adults are excellent examples of policy-laden research challenges. To assure future prevention research capability, we must invest in training in epidemiology, biostatistics, behavioral, environmental, and health services research. PMID- 7531332 TI - Epitope analysis of SPan-1 and DUPAN-2 using synthesized glycoconjugates sialyllact-N-fucopentaose II and sialyllact-N-tetraose. AB - Epitope analysis of SPan-1 and DUPAN-2 was compared with that of CA19-9 using the synthesized glycoconjugate sialyllacto-N-fucopentaose II (SLF II, sialyl Lewis(a)) and its precursor, sialyllact-N-tetraose (LSTa, sialyl-Lewis(c)), conjugated to human serum albumin. The CA19-9 and DUPAN-2 assay systems specifically recognized SLF II and LSTa, respectively. The SPan-1 assay system recognized both SLF II and LSTa, although the reactivity with the former was far stronger than that with the latter. These results were, in general, compatible with those obtained from assaying these markers in the sera of two pancreatic cancer patients with definite Lewis-negative phenotype and in the sera of 39 CA19 9-negative pancreatic cancer patients. In conclusion, DUPAN-2 is the precursor of CA19-9 and is accumulated in the sera of pancreatic cancer patients with Lewis negative phenotype and SPan-1 has an advantage over CA19-9 in the diagnosis of patients with Lewis-negative phenotype, although both markers have almost the same sensitivity for this malignancy. PMID- 7531333 TI - Effectiveness of multivariate analysis of tumor markers in diagnosis of pancreatic carcinoma: a prospective study in multiinstitutions. AB - In 403 patients suspected of having pancreatic cancer, we prospectively studied a combination assay of various serum tumor markers: CA19-9, DUPAN2, tissue polypeptide antigen, elastase 1, gamma-glutamyltranspeptidase, lactate dehydrogenase, lipase, amylase, and alkaline phosphatase. The diagnostic value of each marker was compared with a multivariate analysis (computer-aided multivariate and pattern analysis system for pancreatic cancer examine-1: CAMPAS PX1). Pancreatic cancer was subsequently identified in 47 patients. CAMPAS-PX1 had higher negative in health and positive predictability than those of each marker used alone in the diagnosis of pancreatic cancer. CAMPAS-PX1 proved the most effective marker for diagnosing pancreatic cancer, but in terms of its cost/benefit ration CAMPAS-PX1 was not superior to CA19-9 used alone. In this prospective trial, we experienced poor generalizability in the statistical models (CAMPAS-PX1). We believe that selection bias was present in samples used for model building. Based on this study a new model has been designed. PMID- 7531334 TI - The significance of CD44 in human pancreatic cancer: I. High expression of CD44 in human pancreatic adenocarcinoma. AB - The CD44 cell surface glycoprotein, which is the adhesion molecule of lymphocytes, has been suggested to be an important factor for the metastatic potential and invasive ability of cancer. We demonstrated the expression of CD44 in human pancreatic adenocarcinoma cells and normal cells by using flow cytometry (FACS-can) and immunohistological staining. CD44 was highly expressed in human pancreatic adenocarcinoma cells, while it was little expressed in normal human pancreas cells. These results indicate that the quantitative analysis of CD44 expression may be a useful diagnostic tool for pancreatic cancer. PMID- 7531335 TI - The significance of CD44 in human pancreatic cancer: II. The role of CD44 in human pancreatic adenocarcinoma invasion. AB - In our previous report (1), we showed the high expression of CD44 in human pancreatic adenocarcinomas cells. Pancreatic cancer aggressively invades surrounding tissues by penetrating basement membrane barriers. To examine the relation between the biological characteristics of human pancreatic adenocarcinoma cells and CD44 expression, we studied the function of CD44 in cell proliferation and cell invasion using a Matrigel basement membrane. The proliferation of human pancreatic adenocarcinoma cells was not affected by anti CD44 antibody. On the contrary, invasion of the cells was suppressed by anti-CD44 antibody. This result suggests that the CD44 molecule plays an important role in pancreatic cancer cell invasion of the basement membrane. PMID- 7531336 TI - Coiled-coil stutter and link segments in keratin and other intermediate filament molecules: a computer modeling study. AB - Structural discontinuities have previously been identified in four regions of the coiled-coil rod domain structure present in intermediate filament (IF) protein molecules. These include a point at which a phase shift occurs in the heptad periodicity characteristic of the sequence of polar and apolar residues in alpha helical coiled-coils, and three links that lack a heptad substructure. We have studied these regions by computer-based molecular modeling and comparative sequence analysis and conclude that the phasing discontinuity can be accommodated without significant distortion of the overall double-helical chain conformation; the L2 link has a similar conformation in all different types of IF molecules, a favorable conformation being one in which the two strands wrap tightly around each other; the L12 links vary in length between different IF types but contain important sequence similarities suggestive of a partial beta structure; the L1 links show larger variations in length, a lower degree of similarity, and probably diverse structures. Variations in the overall charges of the different links suggest that ionic interactions may play a significant role in filament assembly. The results also have general significance for other alpha-fibrous proteins in which either the characteristic heptad phasing undergoes a discontinuity or where a short non-coiled-coil sequence occurs within a coiled coil rod domain structure. PMID- 7531337 TI - Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling. AB - SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two amino-terminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression of dominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erk1) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated pp125FAK. PMID- 7531338 TI - Human immunodeficiency virus reverse transcriptase substitutes for DNA polymerase I in Escherichia coli. AB - We present evidence that human immunodeficiency virus (HIV) reverse transcriptase (RT) can substitute for DNA polymerase I in bacteria. Expression of HIV RT enables an Escherichia coli mutant, polA12 recA718, containing a temperature sensitive mutation in DNA polymerase I, to grow at a nonpermissive temperature. The plasmid pBR322 contains a DNA polymerase I-dependent origin of replication. Expression of HIV RT enables the same E. coli mutant to maintain this plasmid at a nonpermissive temperature. Furthermore, expression of HIV RT in this mutant renders it sensitive to 3'-azido-3'-deoxythymidine, a commonly used anti-AIDS drug that targets HIV RT. These combined findings on the genetic complementation of DNA polymerase I by HIV RT provide a bacterial assay to screen for drugs directed against HIV RT. Genetic complementation provides a method for positive selection of large numbers of functional HIV RT mutants for studies on structure function relationships. PMID- 7531339 TI - Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1. AB - Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients. PMID- 7531340 TI - Differentiation of canine bone marrow cells with hemopoietic characteristics from an adherent stromal cell precursor. AB - Stromal cell lines were established from canine long-term marrow cultures, cloned by limiting dilution, and maintained in stromal cell-conditioned medium. These cells grew adherent, maintained stable growth rate and morphology under standard conditions (in 20-30% conditioned medium; confluency, 70-90%), and supported hemopoiesis in long-term marrow cultures. In the presence of exogenous recombinant canine stem cell factor (rcSCF), round cells developed from the adherent layer, detached, and remained in culture as viable floating cells. Round floating cells also appeared when cultures were grown to > 90% confluency without rcSCF. Round cells were smaller than adherent cells, expressed CD34, showed basophilic plasma, and stained positive for c-kit, MHC-class II markers, and myeloid markers. In standard assays for colony formation, the detached cells produced granulocyte-macrophage colony-forming units (CFU-GM), fibroblast colony forming units (CFU-F), and less well-defined colony-forming units. In addition, on allogeneic feeder cells in long-term cultures, these cells generated hemopoietic colonies. Strikingly, the differentiation was reversible: when nonadherent cells were resuspended at lower density in serum-containing medium, they reattached and grew to confluence when, once again, round cells detached. Detached cells from this secondary cycle produced mainly CFU-F and few CFU-GM when placed in clonal assays. These results suggest that some fibroblast-like stromal cells have the potential to differentiate into cells with hemopoietic characteristics. These observations provide evidence for the existence of a quiescent precursor of hemopoietic progenitors in the bone marrow stroma of the adult dog. PMID- 7531341 TI - Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit. AB - In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the epsilon-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the alpha, beta, delta, and epsilon subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264-->Pro mutation in the epsilon subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 alpha-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders. PMID- 7531343 TI - The effect of Iloprost (ZK 36374) on isolated and transplanted pancreatic islet cells. AB - Several methods have been described for the prolongation of survival of isolated and transplanted islet cells. To investigate the effect of a stable prostacyclin analogue, ZK 36374 (Iloprost) on isolated and allotransplanted islet cell function, we studied 6 groups of rats: Group 1 (n = 7) animals underwent pancreatectomy and their islets were isolated and cultured by standard techniques. Group 2 (n = 8) animals were treated the same, except for the addition of Iloprost to the culture solutions. Group 3 (n = 7) animals were treated as group 1, but the isolated islets were transplanted to the subcapsular space of the left kidney of group 5 (n = 7) animals. Group 4 (n = 8) animals were treated as group 2, and the isolated islets were transplanted to group 6 (n = 8) animals. The insulin levels in the culture media obtained in group 1 and 2 were measured. In groups 5 and 6 blood glucose levels were measured and intraperitoneal glucose loading tests were performed. Histological examination was performed for both isolated and transplanted islets. The results showed that both insulin levels and histologic evaluation were better for group 2 than group 1. Animals in group 6 reached normoglycemia on the fifth day following transplantation while it was the ninth day for group 5. The intraperitoneal glucose loading test was tolerated better by group 6 animals. We conclude that Iloprost may be responsible for the improved results which seem to be due to its cytoprotective effect. PMID- 7531342 TI - Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. AB - Perfusion insufficiency, and the resultant hypoxia, often induces a compensatory neovascularization to satisfy the needs of the tissue. We have used multicellular tumor spheroids, simulating avascular microenvironments within a clonal population of glioma tumor cells, in conjunction with in situ analysis of gene expression, to study stress inducibility of candidate angiogenic factors. We show that expression of vascular endothelial growth factor (VEGF) is upregulated in chronically hypoxic niches (inner layers) of the spheroid and that expression is reversed when hypoxia is relieved by hyperoxygenation. Acute glucose deprivation- another consequence of vascular insufficiency--also activates VEGF expression. Notably, glioma cells in two distinct regions of the spheroid upregulated VEGF expression in response to hypoxia and to glucose starvation. Experiments carried out in cell monolayers established that VEGF is independently induced by these two deficiencies. Upon implantation in nude mice, spheroids were efficiently neovascularized. Concomitant with invasion of blood vessels and restoration of normoxia to the spheroid core, VEGF expression was gradually downregulated to a constitutive low level of expression, representing the output of nonstressed glioma cells. These findings show that stress-induced VEGF activity may compound angiogenic activities generated through the tumor "angiogenic switch" and suggest that stress-induced VEGF should be taken into account in any attempt to target tumor angiogenesis. PMID- 7531344 TI - Structural elements required for receptor recognition of human interferon-gamma. AB - Interferon (IFN)-gamma is a central factor in numerous immune responses. Recently the three-dimensional structure of human and rabbit IFN-gamma has been elucidated. This review attempts to bring together the structure and function information into a working model of IFN-gamma: receptor interaction. Based on mutagenesis studies, and corroborated by work with peptides, antibodies and proteolytic digestion, three regions have been found to be important for receptor binding: a long loop connecting the A and B helices, His111 in the F helix and a conserved section of the flexible carboxyl terminus. These three regions may form one continuous binding domain. PMID- 7531345 TI - Apoptosis regulated by a death factor and its receptor: Fas ligand and Fas. AB - Homeostasis in vertebrates is tightly regulated by not only proliferation and differentiation of cells, but also cell death or apoptosis (Ellis et al. 1991; Raff 1992). Many cytokines bind to their respective receptors to regulate proliferation and differentiation of cells. Our recent studies on the Fas ligand and Fas indicate that they work respectively as a death factor and its receptor and suggest that, in some cases, cell death or apoptosis is regulated by cytokines and their receptors. Here, I present the summary of the Fas/Fas ligand system which has been studied in my laboratory over the past 5 years, and I will discuss its physiological roles. PMID- 7531346 TI - Molecular mechanisms for B lymphocyte selection: induction and regulation of antigen-receptor-mediated apoptosis of mature B cells in normal mice and their defect in autoimmunity-prone mice. AB - Apoptosis (programmed cell death) has been suggested to be involved in clonal elimination of self-reactive lymphocytes for the normal function of the immune system. By crosslinking the antigen receptor (surface immunoglobulin; sIg) on the peritoneal B cells of normal mice, we found that strong crosslinking of sIg induces apoptosis of mature B cells, suggesting that interaction with membrane bound self-antigens may eliminate self-reactive mature B cells by apoptosis. Antigen-receptor-mediated B cell apoptosis is blocked when a signal is transduced via the CD40 molecule on the B cell surface. Because the ligand of CD40 (CD40L) is expressed on activated T helper cells, B cells may escape from apoptosis and are activated when the immune system interacts with foreign antigens, which are normally able to activate T helper cells. Moreover, sIg crosslinking fails to induce apoptosis of both bcl-2-transgenic mice and autoimmune-disease-prone New Zealand mice. In these mice, the defect in sIg-mediated apoptosis of mature B cells may allow generation of self-reactive B cells, resulting in pathogenic consequences. PMID- 7531347 TI - Fas-based d10S-mediated cytotoxicity requires macromolecular synthesis for effector cell activation but not for target cell death. AB - Two main mechanisms seem at play in T cell-mediated cytotoxicity, a process in which target cell death often follows an apoptotic cell death pattern. One of these involves Fas at the target cell surface and a Fas ligand at the effector cell surface. This allowed us to reinvestigate the long-standing question of macromolecular synthesis requirement in T cell-mediated cytotoxicity, using the d10S model cell line which is cytotoxic apparently only via the Fas molecularly defined mechanism. We showed, first, that induction of cytotoxic activity of effector cells, obtained by preincubating these effector cells with a phorbol ester and a calcium ionophore, could be inhibited by macromolecular synthesis inhibitors (cycloheximide, actinomycin D, DRB). We then investigated whether macromolecular synthesis was required, when effector and target cells were mixed, to obtain target cell death. Preincubating already activated effector cells for 30 min with macromolecular synthesis inhibitors, then adding target cells and performing the 51Cr release cytotoxicity test in the presence of these inhibitors, did not significantly decrease subsequent target cell death, indicating that already activated effector cells could kill without further requirement for macromolecular synthesis. In addition, target cell preincubation for up to 3 h in the presence of one of these inhibitors did not decrease cell death. The high sensitivity of mouse thymocytes to this type of cytotoxicity enabled us to devise the following experiment. As previously shown by others, thymocyte death induced by dexamethasone (DEX) could be blocked by coincubation with cycloheximide (CHX). Such DEX-treated CHX-rescued thymocytes, the survival of which was an internal control of efficiency of protein synthesis inhibition, were then subjected to effector cells in the presence of CHX, and were shown to die. Thus, there is no requirement for macromolecular synthesis at the target cell level in this variety of apoptotic cell death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531349 TI - Identification and characterization of an Enterococcus faecalis plasmid pAD1 encoded stability determinant which produces two small RNA molecules necessary for its function. AB - A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function. PMID- 7531348 TI - Mathematical model of temperature-sensitive plasmid replication. AB - The copy number of a series of plasmids constructed at Odense University is regulated by the lambda PR/PRM promoters and the temperature-sensitive cI857 repressor. At low temperatures, these plasmids exhibit the low copy number of the parent plasmid R1 (5-6 per cell). At high temperatures, the plasmids exhibit runaway replication, reaching copy numbers of greater than 1,000 per cell. A detailed mathematical model of the temperature-sensitive replication of these plasmids has been developed incorporating three features: replication of the parent plasmid, regulation of the lambda PR/PRM promoters by the cI repressor, and thermal denaturation of the cI857 repressor. Models of the first two of these features have been described by others. We revised and extended those models, described the thermal denaturation of the cI857 repressor, and integrated these features to give a comprehensive model of temperature-sensitive plasmid replication. Model predictions were compared to experimental measurements of both steady-state copy numbers as a function of temperature and the change in copy number following temperature shifts up and down. The model accurately describes the qualitative behavior of the system and gives reasonable quantitative results. This is particularly significant since all the parameter values used in this model were determined independently: that is, there was no adjustment of parameter values to match our experimental data. The regulatory system that gives rise to the temperature-sensitive replication of these plasmids is widely used in biotechnology applications, so the elements of the model related to this regulation should be applicable to a wide variety of systems. PMID- 7531350 TI - In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803. AB - Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication. PMID- 7531351 TI - Inhibition of neuronal nitric oxide synthase by antipsychotic drugs. AB - There is rapidly accumulating evidence that generation of nitric oxide (NO) through a Ca2+ and calmodulin-dependent pathway plays various important roles in the central nervous system. In the present study, effects of several antipsychotics on the activity of NO synthase were investigated in rat cerebellum and neuroblastoma N1E-115 cells, due to the known ability of these agents to inhibit calmodulin. In cytosolic preparations of rat cerebellum, the antipsychotic drugs inhibited the conversion of [3H]L-arginine into [3H]L citrulline by NO synthase in a concentration-dependent manner. This inhibition was noncompetitive in nature, and it exhibited an excellent correlation with blockade of calmodulin activity. Furthermore, these drugs attenuated cyclic GMP formation induced by a calcium ionophore in N1E-115 cells, a response which takes place as a consequence of NO generation. Taken together, our data demonstrate that antipsychotic drugs inhibit NO formation in vitro. It is unlikely, however, that these actions might contribute to their therapeutic and/or side effects, since they take place at relatively high concentrations. PMID- 7531352 TI - Regional brain distribution of risperidone and its active metabolite 9-hydroxy risperidone in the rat. AB - Risperidone is a new benzisoxazole antipsychotic. 9-Hydroxy-risperidone is the major plasma metabolite of risperidone. The pharmacological properties of 9 hydroxy-risperidone were studied and appeared to be comparable to those of risperidone itself, both in respect of the profile of interactions with various neurotransmitters and its potency, activity, and onset and duration of action. The absorption, plasma levels and regional brain distribution of risperidone, metabolically formed 9-hydroxy-risperidone and total radioactivity were studied in the male Wistar rat after single subcutaneous administration of radiolabelled risperidone at 0.02 mg/kg. Concentrations were determined by HPLC separation, and off-line determination of the radioactivity with liquid scintillation counting. Risperidone was well absorbed. Maximum plasma concentrations were reached at 0.5 1 h after subcutaneous administration. Plasma concentrations of 9-hydroxy risperidone were higher than those of risperidone from 2h after dosing. In plasma, the apparent elimination half-life of risperidone was 1.0 h, and mean residence times were 1.5 h for risperidone and 2.5 h for its 9-hydroxy metabolite. Plasma levels of the radioactivity increased dose proportionally between 0.02 and 1.3 mg/kg. Risperidone was rapidly distributed to brain tissues. The elimination of the radioactivity from the frontal cortex and striatum--brain regions with high concentrations of 5-HT2 or dopamine-D2 receptors--became more gradual with decreasing dose levels. After a subcutaneous dose of 0.02 mg/kg, the ED50 for central 5-HT2 antagonism in male rats, half-lives in frontal cortex and striatum were 3-4 h for risperidone, whereas mean residence times were 4-6 h for risperidone and about 12 h for 9-hydroxy-risperidone. These half-lives and mean residence times were 3-5 times longer than in plasma and in cerebellum, a region with very low concentrations of 5-HT2 and D2 receptors. Frontal cortex and striatum to plasma concentration ratios increased during the experiment. The distribution of 9-hydroxy-risperidone to the different brain regions, including frontal cortex and striatum, was more limited than that of risperidone itself. This indicated that 9-hydroxy-risperidone contributes to the in vivo activity of risperidone, but to a smaller extent than would be predicted from plasma levels. AUCs of both active compounds in frontal cortex and striatum were 10-18 times higher than those in cerebellum. No retention of metabolites other than 9-hydroxy risperidone was observed in any of the brain regions investigated. PMID- 7531354 TI - Renal failure associated with laxative abuse. AB - Eating disorder patients often abuse laxatives in an attempt to purge excess food. Laxative abuse can cause hypokalemia and volume depletion. Hypokalemia, in turn, can lead to rhabdomyolysis. Laxative-induced hypokalemia and volume depletion have been previously reported to cause renal insufficiency, but not severe enough to require hemodialysis. A 27-year-old woman with a long history of laxative abuse presented with severe renal failure associated with hypokalemia and volume depletion. She required acute hemodialysis for worsening acidosis (pH 7.05) despite assisted ventilation. A prior episode of hypokalemic rhabdomyolysis at age 23 had resulted in only mild renal insufficiency. Her later episode of severe renal failure was linked to profound volume depletion (blood urea nitrogen 135 mg/dl). This patient calls attention to a potentially life-threatening complication of laxative abuse and indicates that volume depletion can exacerbate laxative-associated renal failure. PMID- 7531353 TI - Survey on the pharmacodynamics of the new antipsychotic risperidone. AB - This review reports on the pharmacodynamics of the new antipsychotic risperidone. The primary action of risperidone is serotonin 5-HT2 receptor blockade as shown by displacement of radioligand binding (Ki: 0.16 nM), activity on isolated tissues (EC50: 0.5 nM), and antagonism of peripherally (ED50: 0.0011 mg/kg) and centrally (ED50: 0.014 mg/kg) acting 5-HT2 receptor agonists in rats. Risperidone is at least as potent as the specific 5-HT2 receptor antagonist ritanserin in these tests. Risperidone is also a potent dopamine D2 receptor antagonist as indicated by displacement of radioligand binding (Ki: 1.4 nM), activity in isolated striatal slices (IC50: 0.89 nM), and antagonism of peripherally (ED50: 0.0057 mg/kg in dogs) and centrally acting D2 receptor agonists (ED50: 0.056-0.15 mg/kg in rats). Risperidone shows all effects common to D2 antagonists, including enhancement of prolactin release. However, some central effects such as catalepsy and blockade of motor activity occur at high doses only. Risperidone is 4-10 times less potent than haloperidol as a central D2 antagonist in rats and it differs from haloperidol by the following characteristics: predominant 5-HT2 antagonism; LSD antagonism; effects on sleep; smooth dose-response curves for D2 antagonism; synergism of combined 5-HT2/D2 antagonism; pronounced effects on amphetamine-induced oxygen consumption; increased social interaction; and pronounced effects on dopamine (DA) turnover. Risperidone displays similar activity at pre- and postsynaptic D2 receptors and at D2 receptors from various rat brain regions. The binding affinity for D4 and D3 receptors is 5 and 9 times weaker, respectively, than for D2 receptors; interaction with D1 receptors occurs only at very high concentrations. The pharmacological profile of risperidone includes interaction with histamine H1 and alpha-adrenergic receptors but the compound is devoid of significant interaction with cholinergic and a variety of other types of receptors. Risperidone has excellent oral activity, a rapid onset, and a 24-h duration of action. Its major metabolite, 9-hydroxyrisperidone, closely mimics risperidone in pharmacodynamics. Risperidone can be characterized as a potent D2 antagonist with predominant 5HT2 antagonistic activity and optimal pharmacokinetic properties. PMID- 7531355 TI - Risperidone and clozapine in the treatment of drug-resistant schizophrenia and neuroleptic-induced supersensitivity psychosis. AB - 1. Supersensitivity psychosis (SSP) has emerged as a potential side effect of long-term neuroleptic therapy similar to tardive dyskinesia (TD). 2. Six schizophrenic patients with SSP, considered to be drug-resistant, were treated with risperidone, while another 5 were treated with clozapine. 3. The 6 risperidone-treated patients (all women) were rated on the Clinical Global Impression Improvement Scale as at least very much improved. Among the 5 clozapine-treated patients, all 4 men were found to have a marked response to clozapine, while the female patient was judged to be minimally improved. 4. It is hypothesized that not only TD but also SSP arise from destruction of cholinergic interneurons in the striatum as a consequence of prolonged neuroleptic administration. Thus, the drug-induced parkinsonism, which was proposed as mediating the antipsychotic effect of dopamine D2 blocking drugs, depends on the integrity of these cholinergic neurons. If these neurons are destroyed, drugs such as haloperidol lose their therapeutic effect. 5. In contrast, atypical neuroleptics like clozapine and risperidone reduce dopamine release in the striatum independently of prior production of extrapyramidal symptoms and, in this way, may be effective in psychotic illnesses unresponsive to classical anti D2 neuroleptics. 6. In the present sample of patients, it is worth noting that schizophrenic men were good responders to clozapine. In comparison, risperidone was found to be efficacious in schizophrenic women. PMID- 7531356 TI - Effects of BIM26226, a potent and specific bombesin receptor antagonist, on amylase release and binding of bombesin-like peptides to AR4-2J cells. AB - Using AR4-2J rat pancreatic carcinoma cells, the effects of a novel bombesin (BN) receptor antagonist [D-F5Phe6, D-Ala11]BN(6-13)OMe (BIM26226) on BN- or GRP stimulated amylase release and binding of radio-labeled bombesin-like peptides to these cells were examined and compared to [D-Phe6,Leu13 psi(CH2NH)Leu14]BN(6-14) (Psi Bn(6-14)), one of the most potent BN receptor antagonists presently known. BN and GRP both stimulated amylase release with EC50 values in the nanomolar range. Both antagonists were devoid of agonist activity when tested alone. BIM26226 was most potent, antagonizing BN- or GRP-stimulated amylase release with IC50 values in the nanomolar range, whereas Psi Bn(6-14) was approximately ten times less potent. With 125I-[Tyr15]GRP bound to these cells, the binding affinities were BIM26226 > GRP > Psi Bn(6-14) >> neuromedin B. BIM 22626 was not able to inhibit binding of radio-labeled CCK-33, gastrin-17 or VIP. These results suggest that BIM26226 is one of the most potent and specific bombesin receptor antagonists in vitro and seems to be a useful tool to define the physiologic role of GRP in vivo. PMID- 7531357 TI - Tachykinin NK3 receptor mediates NANC hyperpolarization and relaxation via nitric oxide release in the circular muscle of the guinea-pig colon. AB - In the presence of atropine (1 microM), guanethidine (3 microM) and of the tachykinin NK1 (SR 140,333 0.1 microM) and NK2 (GR 94,800 3 microM) receptor antagonists, the application of the tachykinin NK3 receptor selective agonist senktide, or that of neurokinin B, produced concentration-dependent sustained nonadrenergic noncholinergic (NANC) relaxation of mucosa-free circular muscle strips from the guinea-pig proximal colon. The maximal relaxant responses to senktide and neurokinin B were similar, approaching about 70% of the relaxation to 1 microM isoprenaline. Senktide (EC50 0.16 nM) was about 64-fold more potent than neurokinin B (EC50 10.3 nM). When tested in the presence of peptidase inhibitors (thiorphan 1 microM, captopril 1 microM and amastatin 10 microM), neurokinin B (EC50 0.24 nM) was equipotent to senktide (EC50 0.19 nM). At 1 nM, substance P and neurokinin A were ineffective in producing a NANC relaxation of the colon. At 1 microM substance P, neurokinin A and neurokinin B produced a NANC relaxation, which averaged 23, 40 and 79% of the maximal response to isoprenaline, respectively. In the presence of peptidase inhibitors, 1 nM substance P and neurokinin A produced threshold relaxant responses and, at 1 microM, the three natural tachykinins were equieffective (66 +/- 8, 72 +/- 5 and 75 +/- 5% of the relaxation to isoprenaline for substance P, neurokinin A and neurokinin B, respectively). The relaxant response to 1 nM senktide (producing about 70-80% of its maximal effect) was totally abolished by 1 microM tetrodotoxin and largely (> 90%) inhibited by 100 microM L-nitroarginine (L NOARG). The inhibition by L-NOARG was partially reversed by L-arginine (3 mM) but not D-arginine. Apamin (1 microM) produced a slight (about 20%) inhibition of the response to senktide. The peptide blocker of N-type calcium channels, omega conotoxin (0.1 microM) was ineffective. In sucrose gap electrophysiological experiments, superfusion with senktide (0.1 microM for 10 s) produced a slowly developing and prolonged hyperpolarization of the membrane and relaxation. Both effects were inhibited by L-NOARG while apamin had no effect. These findings indicate that a neuronal NK3 receptor mediates NANC hyperpolarization and relaxation of the circular muscle of the guinea-pig proximal colon, principally through the release of NO. NO generation/release in response to NK3 receptor stimulation does not require calcium influx through N-type calcium channels. PMID- 7531358 TI - Testing a visual display to explain small probabilities. PMID- 7531359 TI - Carbon dioxide laser bronchoscopy--a review of its use in the treatment of malignant tracheobronchial tumours in 142 patients. AB - We report our experience over an 8-yr period, 1984-1991, of the use of the carbon dioxide (CO2) laser in the treatment of otherwise inoperable malignant tracheobronchial lesions. In that period 142 patients (84 male, 58 female; median age 63 years) underwent 278 procedures. The trachea was the site of treatment in 44 patients, the carina in nine, a main bronchus in 80 and a lobar bronchus in nine. All resections were performed under general anaesthesia via a rigid bronchoscope. Symptomatic relief was obtained in 103 of the 116 patients whose main complaint was dyspnoea. Overall there was a mean improvement in forced expiratory volume in 1 s (FEV1) of 27%, in peak expiratory flow (PEF) of 22% and in forced vital capacity (FVC) of 7%. Most improvement in FEV1 and PEF was obtained by the treatment of tracheal lesions. Three patients died within 24 h of surgery and 30 day mortality was 18%. At a mean follow-up of 18.3 months the mean post-laser survival is 5 months. While the CO2 laser has limitations in the treatment of distal tumours when compared to the neodymium/yttrium aluminium garnet (Nd:YAG) laser, there was no higher incidence of complications. We have found CO2 laser bronchoscopy to be an effective palliation of inoperable malignant tumours particularly of the trachea and main bronchi. PMID- 7531361 TI - [A case from practice (315)]. PMID- 7531362 TI - Sleep deprivation in the rat: XVIII. Regional brain levels of monoamines and their metabolites. AB - Several theories have linked sleep with change in monoamine activity. However, the use of sleep deprivation to show that changes in sleep generate changes in monoamines (directly or through feedback) has produced inconsistent results. To explore whether longer sleep deprivation, better documented sleep loss, more complete controls or regional brain analyses would produce clear sleep loss induced change, eight rats were subjected to total sleep deprivation (TSD) by the disk-over-water method for 11-20 days and were guillotined along with yoked control (TSC) and home-cage control (HCC) rats. Brains were removed and dissected to obtain the caudate, frontal cortex, hippocampus, hypothalamus, midbrain and hindbrain (pons-medulla). Tissue sections were analyzed for concentrations of serotonin (5HT), its metabolite 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and either norepinephrine or, in the caudate section, the DA metabolite homovanillic acid. The ratios DOPAC/DA and 5HIAA/5HT, which under some conditions are indicators of turnover, were also calculated. Because sleep deprivation time varied across sets of TSD, TSC and HCC rats and not all eight sets were analyzed simultaneously, a repeated-measures ANOVA was performed within sets with HCC, TSC and TSD considered as successive levels of sleep deprivation treatment. In no case did TSD rats have significantly higher or lower values of amines, metabolites or ratios than both HCC and TSC rats. The most common outlying values were for TSC rats. Thus, these results failed to demonstrate sleep loss-induced regional changes in levels of major brain monoamines or their metabolites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531363 TI - The association between birth weight, placenta weight, pregnancy duration, subfecundity, and child development. AB - It is well known that very low birth weight and preterm birth are risk indicators for delayed child development. It is the purpose of this study to estimate the association between birth weight, placenta weight, and gestational age in consecutive pregnancies which survived till after 28th week of gestation. The association between fecundity and child development is also studied. Data stem from pregnant women in a well defined regional area in Denmark (Odense) who participated in a concerted action on moderate alcohol consumption in pregnancy (EuroMac). All pregnant women with an alcohol consumption of 5 drinks or more per week or more in the first trimester were selected for the study in 1988 to 1989. A one to one match of pregnant woman was selected among the remaining pregnant woman based upon expected time of delivery and age. Altogether 326 women were selected for the study and the two groups are combined since alcohol intake in the measured dose range had no association with child development. The newborn went through two psychological tests at 18 month (the Bayley test) and again at 42 months of age (the Griffiths' test). Two hundred fiftynine pairs of mothers and children participated in all parts of data collection. Birth weight and gestational age was associated with the psychological scoring in the test performed at 18 and 42 months of age, especially the psychomotor index. Especially newborns with low birth weight and high placenta weight had low score values on mental development indices. No association was seen between a measure of fecundity (waiting time to pregnancy) and reduced child development. PMID- 7531360 TI - Release of neurotransmitters in the CNS by spinal cord stimulation: survey of present state of knowledge and recent experimental studies. AB - Electric stimulation applied to the posterior surface of the spinal cord (SCS) is an established treatment in certain chronic pain syndromes resistant to conventional therapeutic procedures. Despite the clinical value of SCS, the mechanisms behind the efficacy of the method are largely unknown. Several neurotransmitters in the CNS (e.g. opioids, serotonin, noradrenaline, substance P, GABA), have been proposed to be involved in the pain-alleviating effect of SCS. However, as yet there is no evidence that these would be involved in the beneficial effects of SCS. We have studied neurotransmitter release, using microdialysis techniques, in the spinal dorsal horn and the periaqueductal grey substance (PAG) of the rat and the cat, induced by SCS applied with current parameters equivalent to those used clinically in man. Up to now dialysates have been assayed for GABA, serotonin and substance P with highly sensitive methods. Three groups of studies have been carried out: (1) dorsal horn microdialysis in rats under halothane anesthesia during acute SCS; (2) dorsal horn microdialysis in cats under barbiturate anesthesia or following decerebration, and (3) PAG microdialysis in awake, unrestrained rats with chronic SCS. In the dorsal horn studies, microdialysis probes of different sizes were implanted in the lower lumbar dorsal horns. In the PAG studies, rats had guide cannulas for microdialysis stereotactically inserted into the PAG. SCS was applied at a low thoracic level with 50 or 100 Hz; 0.2 ms and an intensity amounting to 2/3 of the motor threshold. The microdialysis probes were perfused with modified Ringer's solution. Fractions of the dialysate were collected at various intervals. GABA and serotonin were assayed by reverse-phase HPLC, while substance P was investigated using a highly sensitive radioimmunoassay. SCS induced a significant release of GABA in the dorsal horn, most marked in the fraction following the stimulation period. In the rats with PAG microdialysis, the GABA level decreased significantly following two stimulation periods, although transitional increases during SCS were noted in some animals. In the decerebrated cat, a significant release of serotonin in the dorsal horn was obtained with SCS, while the levels of the metabolite 5-HIAA were little influenced by stimulation. On the contrary, in the decerebrated preparation there was no release of substance P in the dorsal horn with SCS, although in the intact cat under barbiturate anesthesia a significant release was induced.4+ off PMID- 7531364 TI - Biophysicists re-establish a Chilean research tradition. PMID- 7531365 TI - Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases. AB - B-cell precursor (BCP) leukemia is the most common form of childhood cancer and the second most common form of acute leukemia in adults. Human BCP leukemia was treated in a severe combined immunodeficient mouse model by targeting of the tyrosine kinase inhibitor Genistein (Gen) to the B cell-specific receptor CD19 with the monoclonal antibody B43. The B43-Gen immunoconjugate bound with high affinity to BCP leukemia cells, selectively inhibited CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. At less than one-tenth the maximum tolerated dose more than 99.999 percent of human BCP leukemia cells were killed, which led to 100 percent long-term event-free survival from an otherwise invariably fatal leukemia. The B43-Gen immuno-conjugate might be useful in eliminating leukemia cells in patients who have failed conventional therapy. PMID- 7531367 TI - Modulation of chemotropism in the developing spinal cord by substance P. AB - Developing axons find their targets through direct contact with cues in the extracellular environment and in response to gradients of diffusible factors. The floor plate, a neuroepithelial structure, guides developing commissural axons in the spinal cord by release of chemoattractants. Floor plate cells express neurokinin-1 receptors, and a transiently appearing subpopulation of commissural axons contains substance P, the neuropeptide ligand for this receptor. Substance P increases the amount of axon outgrowth from dorsal horn explants cocultured with floor plate explants. Results of experiments with embryonic rats suggest that substance P released from pioneering neuronal pathways may regulate the release of chemoattractants from floor plate cells. PMID- 7531366 TI - Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. AB - Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression. PMID- 7531368 TI - Use of high-dose etoposide/ifosfamide/carboplatin/epirubicin and peripheral blood progenitor cell transplantation in limited-disease small cell lung cancer. AB - Conventional-dose chemotherapy for limited-disease small cell lung cancer has resulted in high response rates, but rarely in a cure. In an ongoing phase I-II trial, limited-disease small cell lung cancer patients received high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation as part of an early intensification strategy after two cycles of induction chemotherapy. Eligible patients (n = 18) were initially treated with two cycles of etoposide (500 mg/m2), ifosfamide (4 g/m2), cisplatin (50 mg/m2), epirubicin (50 mg/m2) and granulocyte colony-stimulating factor to combine an effective, standard-dose chemotherapy regimen with simultaneous mobilization of PBPCs. Patients who were in partial remission or complete remission after two cycles of induction chemotherapy received high-dose intensification chemotherapy with cumulative doses of 1,500 mg/m2 etoposide, 12 g/m2 ifosfamide, 750 mg/m2 carboplatin, and 150 mg/m2 epirubicin, followed by autologous PBPC transplantation and granulocyte colony-stimulating factor. The duration of the complete chemotherapy program was 9 weeks. All patients received chest irradiation posttransplantation (50 Gy), and those in complete remission received additional prophylactic cranial irradiation (30 Gy). To date, 13 patients with a median age of 49 years (age range, 34 to 62 years) have been treated within this combined-modality treatment protocol. At a median follow-up of 14 months (range, 3 to 45 months) after transplantation, 11 patients were alive and nine were still in complete remission. Nonhematologic toxicity was acceptable; World Health Organization grades 2 to 4 oral mucositis was the most frequently observed (85%) adverse event. No toxic deaths were observed, and hematopoietic reconstitution occurred rapidly after PBPC transplantation; platelet transfusion independence (> 20,000 microL) and neutrophil counts greater than 500 microL were observed at study day 12+. The median survival time was not yet reached. These preliminary data demonstrate that early, high-dose chemotherapy and PBPC transplantation followed by local radiotherapy is safe and may lead to prolonged disease-free survival in some patients. Prospective, randomized studies in a larger series of patients will be required to provide definitive proof of the role of early high dose chemotherapy in the management of limited-disease small cell lung cancer. PMID- 7531369 TI - [Modulatory effects of substance P on the membrane responses mediated by GABAA and GABAB receptors in DRG neurons]. AB - Intracellular recordings were performed on the neurons of young rat DRG to study the modulatory effects of SP on the responses mediated by GABAA and GABAB receptors. In the majority of the neurons (20/30) the GABA induced depolarization was suppressed to 50.8 +/- 20.2% by preapplication of SP (5 x 10(-6)-4 x 10(-5) mol/L), which applied alone had no effect or only caused a slight depolarization. In addition, SP could increase APD50 by 28.7 +/- 9.1% in many neurons (10/18), and the baclofen-induced shortening of 20.6 +/- 2.9% in APD50 could abolished (4/12) or even reverse to a lengthening of 19.3 +/- 8.9% in APD50 (8/12) by the preapplication of SP. Moreover, it was found that the activation of GABAB receptors could inhibit the succeeding response mediated by GABAA receptors. Since the soma of the DRG neuron is generally considered as an accessible model for the primary afferent terminals, the results suggest that SP which is released in the dorsal horn during nociceptive stimulation can antagonize the responses mediated by GABAA and GABAB receptors. PMID- 7531370 TI - Anterior cervical discectomy and fusion using a porous hydroxyapatite bone graft substitute. AB - OBJECTIVES: This study analyzed the use of a coral hydroxyapatite bone substitute for use in ACDF both with and without an anterior cervical plate. STUDY DESIGN: The healing of multilevel anterior cervical fusions was tested using a goat model. Comparisons were drawn with histologic, radiographic, and biomechanical test data. METHODS: Forty-nine mature alpine goats had three-level anterior discectomies performed. Seven treatment groups of seven goats each were used; Group I with no fusion, Group IIa having tricortical iliac crest autograft, Group IIb having autograft plus an anterior plate, Group IIIa having tricortical iliac crest fresh-frozen allograft, Group IIIb having allograft plus an anterior plate, Group IVa having rectangular-shaped implants of porous hydroxyapatite, and Group IVb having ProOsteon 500 implants with an anterior cervical plate. RESULTS: Histologically, at 12 weeks 48% of the ProOsteon (Interpore, Irvine, CA) implants were rated as incorporated, 10% as possessing a fibrous gap, 29% as collapsed, and 14% as extruded. Anterior cervical plating improved the results with 71% of the implants showing good incorporation, 24% with collapse, and 5% with a fibrous gap. These histologic results compare favorably with autogenous bone and are improved over allograft bone. Fluorochrome analysis showed that none of the implants had complete turnover with host bone, but that all possessed peripheral creeping substitution with cutting cones of new bone formation at 12 weeks. Biomechanically, the spines using the ProOsteon implant were less stiff in torsion than autograft, but equal in stiffness to allograft. Flexion-extension neutral zone stiffness was lower in the ProOsteon implant group than either allograft or autograft. CONCLUSIONS: The use of a coral-based hydroxyapatite bone graft substitute for anterior cervical fusions led to significant rates of implant collapse at 12 weeks but showed excellent biologic compatibility with good early creeping substitution of the implant by host bone. The concomitant use of an anterior cervical plate with the implant prevents extrusion. PMID- 7531371 TI - A protocol with FK 506 for inducing unresponsiveness to murine islet allografts. AB - BACKGROUND: The most favorable protocol for transplantation is inducing unresponsiveness before operation by means of nondangerous modalities. This would permit discontinuance of long-term use of immunosuppressants. In this study we developed a potential protocol for inducing unresponsiveness to islet allografts by preoperative donor spleen cell inoculation (DSI) and a single injection of FK 506. METHODS: BALB/c (H-2d) and C57BL/6 (H-2b) mice were used as islet donors and recipients, respectively. The streptozocin-induced diabetic mice that had been given DSI at a dose of 1 x 10(7) or 1 x 10(4) and a single injection of FK 506 (10 mg/kg intramuscularly) at different schedules (on day 1, 3, 5, or 7 relative to DSI on day 0) were subjected to islet allografting on day 10. RESULTS: All islet recipients returned to normoglycemia within a few days with no toxic effect of FK 506 treatment. DSI at a dose of 1 x 10(7) alone led to shortening of the mean survival time to 10.1 +/- 4.1 days, as compared with 13.5 +/- 6.3 days for the untreated animals. In contrast, marked prolongation of graft survival was induced when FK 506 was given on day 3 (> 84 +/- 27.3 days, p < 0.0001) or on day 5 after DSI (> 50.9 +/- 46.0 days, p < 0.05). Five of seven allografts given FK 506 on day 3 and three of seven allografts given FK 506 on day 5 survived indefinitely. Other time schedules of DSI and FK 506 treatment (on day 1 or 7 after DSI) or FK 506 treatment alone had no significant effect on mean survival time. With the same protocol, third-party islet allografts (C3H) were immediately rejected (10.6 +/- 2.6 days). CONCLUSIONS: Prolongation of islet allograft survival was induced by certain doses of DSI and preoperative FK 506 treatment. This modality prevents an adverse effect of FK 506 on grafted islets and can induce unresponsiveness to islet allografts, offering a protocol for successful clinical islet transplantation. PMID- 7531372 TI - [From the hospice movement to palliative medicine]. AB - The "modern" hospice movement was established at the beginning of the 1960s in Great Britain. Philosophical issues and a holistic treatment model have been emphasized in order to meet the physical, emotional, social and psychological needs of dying patients. Less time has been devoted to research. Several hospices now find themselves at crossroads. It is necessary to clarify key strategic goals. More patients will be in need of palliation in the next decades. A 35-40% increase in the incidence of cancer is predicted from now up to the year 2010. More knowledge must be obtained about patients with a limited life expectancy. The patients' needs will best be taken care of by incorporating a multidisciplinary team into the clinic. It is also important to increase the teaching about palliative medicine at medical school. The first unit of palliative has been established at the University Hospital, Trondheim. Similar clinics should be established at the other university hospitals in Norway. PMID- 7531373 TI - Toxic effects of water eutrophication on pancreatic, hepatic and osteogenic tissues of rats. AB - Pollution, industrial solvents, concentrations of metals and other environmental agents are widely related to biochemicals values which are used in disease diagnosis of environmental toxicity. A rat bioassay validated for the identification of toxic effects of eutrophication revealed increased serum activities of amylase, alanine transaminase (ALT) and alkaline phosphatase (ALP) in rats that received algae, filtered water and nickel or cadmium from drinking water. Serum Cu-Zn superoxide dismutase activity decreased from its basal level of 40.8 +/- 2.3 to 26.4 U/mg protein, at 7 days of algae and at 48 hr of nickel and cadmium water ingestion. The observation that lipoperoxide concentration was not altered in rats treated with filtered water, while amylase, ALT and ALP were increased in these rats and in those treated with nickel or cadmium, indicated that pancreatic, hepatic and osteogenic lesions by eutrophication were not related to superoxide radicals, and might be due to a novel toxic environmental agent found in filtered and non-filtered algae water. PMID- 7531375 TI - [Electrocardiographic diagnosis of atrial fibrillation and flutter]. AB - The key for the diagnosis of atrial fibrillation is the complete irregularity of the R-R intervals in the absence of P waves. In atrial flutter, the analysis of the flutter waves itself is important. In doing so, common and uncommon flutter can be distinguished. The A-V conduction and therefore the R-R intervals can be constant or irregular. If widened QRS complexes are registered intermittently, aberrant conduction has to be differentiated from ventricular premature beats. In this context die analysis of the width and the shape of the QRS complexes as well as the R-R intervals preceding and following the abnormal beats are important criteria. In principle, this holds true as well for the diagnosis of WPW syndrome in the presence of AF. If the surface ECG does not allow a clear-cut diagnosis, the indication for invasive electrophysiological testing may be given in a few cases. PMID- 7531374 TI - [Results of oral sotalol therapy in children with supraventricular and ventricular arrhythmias]. AB - Experience with oral sotalol, a beta-blocker with class III-antiarrhythmic properties, is limited in the pediatric population. Sotalol was administered to 32 patients with a mean age of 8.7 years (range 1 day-19.9 years). Mean dosage was 4.6 (1.5-9.4) mg/kg or 122.1 (52-306) mg/m2, respectively. In 27/32 patients, at least 1 antiarrhythmic agent had failed to control the dysrhythmia before sotalol was started. Cardiac diagnoses included normal heart (n = 16), status after correction of congenital heart disease (n = 13), and cardiomyopathy (n = 3). Success (based on symptoms and 24-h electrocardiogram) was achieved in 16/18 patients with reentry supraventricular tachycardia, in 7/8 patients with atrial flutter, and in 4/6 patients with ventricular tachycardia. During a mean follow up of 15.6 (2-78) months, proarrhythmia occurred in five (16%) patients (symptomatic bradycardia n = 2, QT-prolongation and ventricular extrasystoles n = 1, ventricular extrasystoles n = 1, 2 degrees av-block n = 1), requiring dosage reduction (n = 3), cessation of treatment (n = 1) and pacemaker implantation in one patient, respectively. Symptomatic hypotension was noted in two patients, in whom therapy had to be stopped. Sotalol was a very effective agent for the treatment of various pediatric cardiac dysrhythmias. However, incidence of proarrhythmic effects warrants close electrocardiographic monitoring. PMID- 7531376 TI - [Liver resection in hepatocellular carcinoma]. AB - Hepatic resection offers the only chance of cure for patients with hepatocellular carcinoma. Preoperative tumor staging is based on radiologic procedures (ultrasound, CT- and MR-imaging). Compromised hepatic function will limit resectability, leaving only palliative procedures. Unfavorable prognostic tumor factors (diameter > 5 cm, multifocal, nonencapsulated, vascular invasion) should lead to a combination of adjuvant chemotherapy/chemoembolization and surgical intervention. In irresectable situations multimodality treatment protocols (combined i.v. chemotherapy, radio-immuno-therapy) play an increasing role. PMID- 7531377 TI - [Therapeutic interventional radiology procedures in hepatocellular carcinoma]. AB - Interventional treatment of hepatocellular carcinoma is indicated if surgical treatment is impossible. Selection of the individual method of interventional treatment is determined by the size, location and number of tumors and by the grade of tumor vascularization. Tumors below 5 cm in diameter are well-suitable for aethanol ablation. In larger tumors and in tumors with high-grade vascularization transarterial chemoembolization is probably more effective. In patients with more than 3 tumor nodules chemoembolization is the treatment of choice. On the other hand chemoembolization shows reduced efficacy in tumors with low-grade hypervascularization and the aethanol ablation as a stand-alone procedure or supplemental treatment seems to be valuable. Until now there is no generally accepted opinion in terms of the indications for different methods of interventional treatment. Further comparative studies including aethanol ablation, chemoembolization and resection are necessary. PMID- 7531378 TI - [Pouches of the upper gastrointestinal tract]. AB - The own findings about the functional behavior of the gastric substitute together with the results from the literature demand a jejunal J-pouch after gastrectomy, if it is not a palliative resection. It is an open question, whether the good experience with the Roux-en-Y-pouch-reconstruction--only 15 min. more time of operation, low rate of complications--allows a wider indication for this procedure. PMID- 7531379 TI - Cytology and immunohistochemistry of anaplastic meningiomas in squash preparations. A report of two cases. AB - Cytodiagnosis is a useful adjunct to frozen section diagnosis of brain tumors during surgery and has been applied in many pathology laboratories. Although anaplastic (malignant) meningiomas are not extremely rare tumors, their cytologic features have not been well described. Cytologic details of anaplastic meningioma, papillary meningioma with a fibroblastic component and anaplastic angiomatous meningioma are presented. Papillary meningioma showed atypical cell clusters having a close relation to mature endothelial cells and that corresponded to papillary proliferation. Other cell clusters of this tumor showed an ordinary fibroblastic pattern with coarse cytoplasmic filaments and necrotic changes in the cytoplasm. Anaplastic angiomatous meningioma exhibited loose cell clusters. Some of them had coarse cytoplasmic filaments, and others were similar to epithelial cells in origin. Specific features commonly found in anaplastic meningioma were hyperchromatic and pleomorphic nuclei and cytoplasmic filaments more obscure than those of the benign counterpart. Immunocytochemically the tumor cells in the papillary area of papillary meningioma had a positive reaction to antiserum of vimentin, cytokeratin and S-100 protein. The tumor cells of anaplastic plastic angiomatous meningioma showed intracytoplasmic positivity to antivimentin and anti-epithelial membrane antigen serum. PMID- 7531380 TI - Ultrafast Papanicolaou stain. An alternative preparation for fine needle aspiration cytology. AB - The objective of this study was to develop a Papanicolaou stain as fast as Diff Quik yet with cytomorphology as exquisite as that processed by ThinPrep for the optimal evaluation of fine needle aspirates. Satisfactory results were obtained after three modifications were made: (1) rehydration of air-dried smears with normal saline, (2) use of a 4% formaldehyde/65% ethanol fixative, (3) and use of Richard-Allan Hematoxylin 2 and Cyto-stain. The first modification restored the transparency of the cells and hemolysed red blood cells, the second modification reduced the time needed for proper fixation and staining from minutes to seconds, and the third modification simplified the procedure. This 90-second protocol yields a transparent, polychromatic stain with crisp nuclear and cytoplasmic features. The cytomorphology processed by this protocol is at least equal to, if not better than, the quality of specimens prepared by ThinPrep and superior to those processed by the standard Papanicolaou procedure. PMID- 7531382 TI - Ultrastructural observation of capillary sprouts in the dental organs of rat molars. AB - The morphological events associated with the formation of capillary sprouts were examined in the dental organs to elucidate the morphological processes of angiogenesis in epithelial tissues. Mandibular first molars of 2-day-old male rats were processed for routine observation by transmission electron microscopy. In angiogenic regions, capillaries were located in the caves delineated by outer enamel epithelium, and extravasated erythrocytes were often observed in the stellate reticulum. At the capillary sprout, endothelial cells were amoeboidal in shape, and extended their filopodia to the fluffy extracellular matrix. On the other hand, each endothelial cell interdigitated with each process to form a tubule, and bulbous cell processes filled the luminal side of capillaries. Coated vesicles or pits 50-90 nm in diameter lined the luminal plasma membrane and that comprising the base of filopodial processes. Some endothelial sprouting tips (30 of 47 sprouts) were associated with pericyte-like cells. However, pericyte-like cells were observed in most of the caves where capillaries were located or destined to be located. We investigated the dynamic status of endothelial cells via parameters such as cell elongation and spreading which were supported by the additional observation of membranous components such as coated vesicles at the capillary sprouting region. It was also suggested that pericyte-like cells participated in angiogenesis of epithelial tissues by guiding the sprouts and sealing labile junctions between endothelial cells. PMID- 7531381 TI - [Treatment outcome of advanced Hodgkin's disease based on using an alternating program ChLVPP (chlorambucil, vinblastine, procarbazine, prednisone) and ABV (doxorubicin, bleomycin, vinblastine)]. AB - Twenty nine patients (7 women, 22 men) with III and IV stage of Hodgkin's disease were treated according to alternating programme ChLVPP/ABV. The results were evaluated in 27 patients. Complete remission was obtained in 41% (11 patients), partial remission in 22% (6 patients). Applied treatment revealed a relatively small toxicity. PMID- 7531383 TI - Language function following subdural grid-directed temporal lobectomy. AB - The purpose of the study was to determine the extent to which a temporal resection may be undertaken without producing risk to temporal language areas. Patients undergoing craniotomy and placement of a subdural electrode array (SEA) for evaluation of intractable epilepsy were studied to determine the variability of distance of temporal language cortex from the temporal pole. Hemisphere dominance was determined by intracarotid sodium amytal injection. Temporal lobe speech arrest (SA) was mapped with a 64 contact point SEA. Thirty-one patients had left dominant hemisphere SEAs. Thirty had SA 5 cm to 9 cm from the temporal pole (median 7 cm). One had SA at 3 cm. Twenty-one patients subsequently had temporal lobectomy (TL). Mean extent of resection was 5.7 cm (range 3 to 9 cm). In 18 TL patients who had neuropsychometric evaluation of language function pre- and post-surgery, there was no significant deterioration. Thirty-nine patients had right non-dominant SEAs placed. Eighteen had TL. Thirteen of these had pre- and post-surgery language evaluation and there was no significant change. Comparison of preoperative scores showed significant superiority of the right non dominant group over the left dominant group for naming. TL up to 5 cm without stimulation mapping of language areas would be safe in the majority of cases, but one subject (3%) had SA mapped anterior to this and a small number of cases may therefore be at risk to language function following a 5 cm TL. Extensive lateral resections up to 9 cm are possible with preservation of language function with stimulation cortical mapping. PMID- 7531385 TI - Peripheral surgical techniques for the management of trigeminal neuralgia- alcohol and glycerol injections. AB - Trigeminal neuralgia remains a difficult condition to manage. Surgery aimed at peripheral nerve branches continues to be used extensively by oral and maxillofacial surgeons. The efficacy of 68 peripheral alcohol injections (retrospective study) and 22 peripheral glycerol injections (prospective study) were assessed in comparison with peripheral cryotherapy. The results indicate that the median time for total pain control after the first alcohol block was 13 months. When individual branches were assessed it was 13 months for the infra orbital nerve and 19 months for the inferior alveolar nerve. These results compare with the published results on cryotherapy. The results for glycerol were disappointing with a mean time of pain relief of 7 months. The results suggest that peripheral alcohol nerve blocks do still have a role to play in the management of trigeminal neuralgia, particularly in the elderly, medically compromised and those unwilling to undergo more extensive surgery. They also provide a means of temporary relief. PMID- 7531384 TI - CD34 immunoreactivity in nervous system tumors. AB - CD34 is a sialylated transmembrane glycoprotein of unknown function that is present in myeloid progenitor cells, endothelial cells, and some fibroblast related mesenchymal cells. However, its tissue distribution is still incompletely characterized. In this study we evaluated the distribution of CD34 antigen in tumors of the central and peripheral nervous system. For comparison the tumors were also stained for CD31, also known as platelet-endothelium cell adhesion molecule (PECAM-1), a transmembrane glycoprotein so far considered to be endothelium specific beyond its reactivity with certain hematopoietic cells. Neurofibromas showed consistently high numbers of CD34-positive spindle cells, whereas peripheral and acoustic schwannomas were negative. A subset of meningiomas (15%) showed CD34-positive tumor cells, and some were also weakly positive for CD31. Gliomas were negative. Meningeal hemangiopericytomas were consistently CD34 positive, but CD31 negative. These results indicate a moderately widespread distribution of the CD34 antigen in nervous system tumors, and necessitate caution in making conclusions regarding endothelial cell differentiation of nervous system tumors on the basis of CD34 immunoreactivity. PMID- 7531386 TI - Anti-oedematous action of some H1-receptor antagonists. AB - The oedema disk technique was used to study the effects of orally administered H1 receptor antagonists (cetirizine, chloropyramine, clemastine, cyproheptadine, dimethindene, loratadine, mequitazine and terfenadine) on the inflammation induced with capsaicin or croton oil in the mouse ear, and the effect of topically applied dimethindene maleate gel on the inflammation induced with croton oil in the mouse ear. In rats of the Wistar strain, oedema was induced in the hind paw by the subplantar injection of dextran or compound 48/80. Preliminary antihistamine treatment inhibited the development of oedema in the mouse ear, and of oedema in the rat paw, to statistically significant extents, in a dose-dependent manner. In all experiments, the most potent drugs were loratadine and cyproheptadine. PMID- 7531387 TI - Inhibition of inflammatory angiogenesis in rats by loco-regional administration of hydrocortisone and protamine. AB - We have studied the antiangiogenetic effects of hydrocortisone and protamine given intra-arterially. The cornea of male, Sprague-Dawley rats were cauterized with silver nitrate. The following treatments were given: 30 micrograms hydrocortisone topical (t.p.), b.i.d., 50 mg/kg/day intraperitoneally (i.p.) or intra-arterially (i.a.), 10 mg/kg/day protamine i.p. or i.a. Saline was administered to the control groups. In separate experiments we also evaluated the anti-inflammatory effects of hydrocortisone, i.p., on the cauterized corneas. Five days after cauterization, the animals were killed, exsanguinated and India ink was injected to show the network of neovessels. The percentage area of the cornea covered by neovessels was measured morphometrically and evaluated statistically. Hydrocortisone t.p. (-84%), i.a. (-60%) and protamine i.a. (-44%) significantly inhibited angiogenesis in the cauterized cornea. Either drugs, i.p., had any antiangiogenetic effects, but hydrocortisone significantly reduced cell infiltration of the corneas. The results suggest that locoregional administration of antiangiogenetic drugs might be clinically useful. PMID- 7531389 TI - Viability of fresh and frozen-thawed biopsied bovine embryos. AB - Bovine embryos were biopsied using a simplified splitting technique and frozen thawed according to a standard method with glycerol as cryoprotectant. The viability of fresh and frozen-thawed biopsied and intact embryos were evaluated after in vitro culture, by means of fluorescence test or following transfer to recipients. The survival rates after in vitro culture of fresh intact and biopsied embryos and of frozen-thawed intact and zona free embryos were not significantly different (70%, 60%, 68% and 52%, respectively), but significantly reduced for biopsied frozen-thawed embryos (16%) (p < or = 0.05). The pregnancy results after transfer of biopsied frozen-thawed embryos were also significantly lower (8%) compared to fresh biopsied embryos (39%) (p < or = 0.05). Both intact and biopsied embryos fluoresced after incubation with diacetylfluorescin but with higher intensity for the intact embryos. It is suggested that the reduced survivability for the frozen-thawed biopsied embryos might be caused by combined effects of the loss of the zona pellucida and the reduction of cells as a result of the simplified biopsy technique. It is concluded that improved biopsy and/or freezing techniques must be used if biopsied embryos have to be frozen. PMID- 7531390 TI - Combined carboplatin plus bleomycin and conventional radiotherapy for advanced carcinomas of the head and neck. AB - In a previous study, we reported a 72% response rate (CR = 52%) in patients with unresectable head and neck (H&N) carcinomas treated with simultaneous carboplatin (CBDCA) and radiotherapy (RT). Bleomycin (Bleo), a known radiosensitizing agent, has been shown to increase response rates when given together with RT in similar patients. To explore the nonoverlapping toxicities of these two agents, we combined i.v. CBDCA (100 mg/m2/week), Bleo (5 units on day 1 and 4/weekly) and standard doses of RT in patients with unresectable H&N carcinomas. Chemotherapy (CT) was continued until completion of RT. Twenty-three (13 males, 10 females) previously untreated patients with stage IV squamous cell carcinoma of the H&N were treated at the University of Maryland Medical Center: 61% had oropharyngeal cancers; 26%, hypopharynx; 9%, oral cavity; and 4%, an unknown primary. Moderate to severe mucositis developed in 90%, which required RT interruptions of up to 3 weeks. After a median follow-up (FU) of 18 months, 35% achieved a complete response (CR) and 65% died from progressive disease. These preliminary data suggest that the addition of Bleo increases mucosal toxicity substantially and, while a moderate response rate was observed, it is unlikely that the CR rate will be higher than CBDCA/RT, which was also better tolerated and hence more suitable to multimodal approaches. PMID- 7531388 TI - Inhibition of relaxin-induced pubic symphyseal "relaxation" in guinea pigs by glycosaminoglycan polysulfates and pentosan polysulfate. AB - There are similarities between the actions of estrogen and relaxin on the connective tissues of the pubic symphysis and those of neutral proteases on cartilage in osteoarthritis, including cartilage hydration, proteoglycan loss, and dissolution of collagen fibers. We hypothesized that compounds known to inhibit cartilage breakdown in animal models of osteoarthritis, such as polysulfated GAGs, would also antagonize the actions of estrogen and relaxin that increase the laxity and mobility of the pubic symphyses of guinea pigs. Estrogen primed guinea pigs were injected with relaxin or with relaxin and the test compound. The pubic symphyses were manually palpated 6 h later and the degree of mobility scored. Glycosaminoglycan polysulfates and pentosan polysulfate inhibited relaxin-induced pubic symphyseal relaxation, whereas other types of agents were without effect. The guinea pig pubic symphysis assay for relaxin may thus provide a novel rapid screening test for compounds with potential chondroprotective activity. PMID- 7531391 TI - Tamoxifen for the treatment of metastatic inflammatory breast carcinoma. AB - In this series, 65 patients with metastatic inflammatory breast carcinoma were treated with neoadjuvant chemotherapy followed by local regional treatment followed by adjuvant chemotherapy. All of these selected patients developed further metastatic disease and were treated at that time with tamoxifen alone. Measurements of hormone receptor levels were available for 46 patients. Four tumors were positive estrogen receptors and eight tumors were positive progesterone receptors. The objective response rate for tamoxifen therapy was 5% (3/65). No major side effects were observed. When the metastasis-free interval was over 19 months, the overall survival and after metastases survival rates were significantly increased. Our conclusion is that tamoxifen should not play any role in the palliative treatment of metastases in inflammatory breast carcinoma. PMID- 7531392 TI - Filgrastim-mobilized peripheral-blood stem cells can be stored at 4 degrees and used in autografts to rescue high-dose chemotherapy. AB - We studied 21 filgrastim (G-CSF)-mobilized peripheral blood stem cells (PBSC) apheresis products obtained from seven patients, and stored at 4 degrees C for periods of up to 96 hr prior to their reinfusion, to rescue high-dose chemotherapy. The apheresis products contained a median of 106 x 10(8)/L mononuclear cells (MNC), 14.6% of them displaying the CD34 antigen; the viability was over 90% in all samples studied at 24, 48, and 72 hr after harvesting. These PBSC were successfully used to rescue high-dose chemotherapy; patients received a median of 4.8 x 10(8)/Kg MNC; the median time to achieve > 500 granulocytes was 14 days (range 11-26) and the median time to achieve > 20,000 platelets was 20 days (range 11-40). Since autologous transplants with nonfrozen PBSC are feasible and less costly than those using frozen PBSC, restrictions to PBSC autotransplant programs may be overcome and costs may be diminished. PMID- 7531393 TI - Combined therapy of G-CSF and prednisolone for neutropenia in a patient with Felty's syndrome. PMID- 7531394 TI - Role of granulocyte colony-stimulating factor as adjunct therapy for septicemia in children with acute leukemia. AB - The granulocyte colony-stimulating factor (G-CSF) has been shown to accelerate recovery from severe neutropenia and to decrease the incidence of documented infections after intensive chemotherapy in cancer patients. However, the routine prophylactic use of G-CSF is expensive. This study was conducted to determine the role of G-CSF as adjunct therapy for septicemia following neutropenia caused by chemotherapy in children with acute leukemia. Fifty consecutive episodes of septicemia were studied involving 34 episodes of Gram-negative, 7 episodes of Gram-positive, 5 episodes of polymicrobial bacterial septicemia, one episode of fungemia, and 3 episodes of disseminated fungal infection. In the first 25 episodes, G-CSF was not used (group A). For the next 16 episodes, G-CSF 200 micrograms per square meter per day subcutaneously was given immediately after the septicemia was documented until the absolute neutrophil count was maintained at more than 1,500 per cubic millimeter (group B). Thereafter, G-CSF at the same dose as that of group B was prophylactically used in all the children who received high-dose cytosine arabinoside-containing regimens. Nine episodes of septicemia occurred (group C). The incidences of mortality per episode of septicemia in groups A, B, and C were 12.0% (3/25), 12.5% (2/16) and 0% (0/9), respectively. Statistically, there was no difference between the three groups overall and in pair-wise comparisons (all P > 0.5). The durations of G-CSF administration in group B ranged from 6 to 26 days with a median of 12 days and the durations of G-CSF administration in group C ranged from 10 to 23 days with a median of 19 days. With or without G-CSF, there may be no significant difference in the mortality of septicemia following neutropenia caused by chemotherapy in children with acute leukemia. PMID- 7531395 TI - Morphologic and ultrastructural evidence of interleukin-6 induced platelet activation. AB - The in vitro effect of IL-6 on platelet activation was investigated. When human platelets were incubated with high (1,000 ng/ml) or low (1 ng/ml) dose IL-6, expression of GMP-140 was enhanced by 42% (N = 6; P < 0.009) and 46% (N = 6; P < 0.061) in 1 hr low and high dose IL-6-platelet incubations, respectively, as assessed by flow cytometry. In platelet specimens incubated with high dose IL-6 for 3 hr, a 70% (N = 6; P < 0.009) increase in GMP-140 expression over control was observed. Parallel high dose IL-6 incubations subjected to scanning electron microscopic studies revealed a 3.4-fold increase (N = 6; P < 0.001) in spheroid morphologic platelet forms in 1 hr incubations in comparison to control platelet preparations, whereas in 3 hr IL-6-platelet incubations, a 96% increase in dendritic platelet forms was observed (N = 6; P < 0.001). Significant increases in platelet ATP levels were observed in both 1 min and 1 hr high dose and low dose IL-6 platelet incubations. In 3 hr high dose-IL-6 platelet incubations, a significant 18% (N = 8; P < 0.001) decrease in platelet ATP was parallelled by a significant 40% increase (N = 8; P < 0.014) in plasma ATP in the same specimens. This increased plasma ATP was highly correlated with a reduction in platelet ATP when analyzed by bivariate regression analysis. Lastly, transmission electron microscopic analysis demonstrated a significant reduction in dense granule number and ratio of dense granule surface area/cell surface area in 3 hr high dose IL-6 incubations. These findings suggests that IL-6 activates platelets in vitro. PMID- 7531396 TI - Urinary iron speciation in nephrotic syndrome. AB - In nephrotic syndrome, iron is presented to the tubule fluid in a nonreactive form in association with transferrin as a result of the glomerular protein leak. At an alkaline pH, iron remains bound to transferrin throughout the nephron and is excreted as such in the urine. As urine pH decreases below 6, iron is dissociated from transferrin. In the dissociated form, iron exists in the urine in a soluble, ultrafiltrable, and labile state. It is suggested that iron is maintained in this state by chelation to a relatively small organic compound, such as citrate. This non-transferrin-bound iron is capable of catalyzing bleomycin degradation of DNA, suggesting that this labile form of iron is able to catalyze free radical formation and cause tubule cell injury. Urine from proteinuric states represents one of the few, if not only, biologic fluids containing large amounts of reactive iron species. This may explain the mechanism by which proteinuric states cause tubulointerstitial disease and renal failure. PMID- 7531397 TI - A prenatal trisomy 21 screening program using alpha-fetoprotein, human chorionic gonadotropin, and free estriol assays on maternal dried blood. AB - OBJECTIVE: The feasibility of large-scale Down syndrome maternal screening with dried blood samples and nonradioactive methods was examined. STUDY DESIGN: A prospective observation study was performed on a nonselected population of 11,241 pregnant women sampled between January 1991 and September 1992, between 14 and 24 weeks' gestation (ultrasonographic scanning available for 91.6%), through a multicenter collaborative network. Enzyme-linked immunosorbent assays for alpha fetoprotein, human chorionic gonadotropin, and free estriol were performed on dried blood samples. Risk determination was made with an in-house software implementing the multivariate gaussian log likelihood method. RESULTS: A total of 10,450 samples were eligible for the study. Mean age at term was 27.9 years. A total of 6.84% of the patients were > or = 35 years old with a prior risk of trisomy 21 > 1:350. The general positive rate of our sample was 8.15%. After calculation 31.7% with prior risk > 1:350 were still in the high-risk group; 6.36% of the low-risk group were found to be at high risk for Down syndrome. Fifteen trisomic pregnancies were observed, of which 11 had a calculated risk higher than the selected cutoff value (1:350). The overall detection rate was 73%, specificity was 92%, and positive predictive power was 1.2%. CONCLUSION: Our pilot study has shown performances within the range of conventional serum screening programs. Dried blood assays are a handy alternative to serum assays. Blot paper cards represent a simple method of sampling, well fitted for large population screening. Combined with nonradioactive methods, this method appears to be both low cost and effective. The current work apparently is the first large scale Down screening program performed with dried blood. PMID- 7531398 TI - Hepatocellular carcinoma in a pregnant woman detected by routine screening of maternal alpha-fetoprotein. AB - A 26-year-old pregnant woman was diagnosed as having hepatocellular carcinoma at 16 weeks of gestation when extremely high levels of alpha-fetoprotein were found on a routine screening examination. The pregnancy was terminated, and a right hepatectomy was performed. Although such cases are rare, this one suggests that early measurement of maternal serum alpha-fetoprotein in a pregnant woman with hepatitis B surface antigenemia may allow the timely detection of a hepatocellular carcinoma. PMID- 7531399 TI - Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term. AB - OBJECTIVE: Our purpose was to compare the level of expression of both type 1 and type 2 cyclooxygenase genes before and after labor and to localize their expression within the fetal membranes. STUDY DESIGN: The sites of type 2 and type 1 cyclooxygenase messenger ribonucleic acid synthesis were identified with in situ hybridization. Expression of both types 1 and 2 cyclooxygenase was studied by reverse transcriptase polymerase chain reaction. RESULTS: Cyclooxygenase type 2 and type 1 expression was localized within the amniotic epithelium and amniotic mesoderm. Type 1 but not type 2 enzyme was also expressed in the chorionic mesoderm. Expression of the type 2 enzyme was significantly increased with the onset of labor. Type 1 enzyme expression did not significantly change with labor. CONCLUSION: It is most likely that it is the inducible type 2 cyclooxygenase enzyme that mediates the increase in prostaglandin synthesis in amnion with the onset of labor. PMID- 7531400 TI - The maternal-fetal transfer of bisheteroypiperazine (U-87201-E) in the ex vivo human placenta. AB - OBJECTIVE: The purpose of this study was to elucidate the maternal-fetal transfer of bisheteroypiperazine (U-87201-E), a nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus-1. STUDY DESIGN: Placentas from normal term deliveries were used in this study to determine the maternal-fetal transfer of bisheteroypiperazine. The studies were conducted at several concentrations with the circulation either open-open or closed-closed. RESULTS: In this study we determined that the clearance index of bisheteroypiperazine was 0.72 +/- 0.17 at maternal concentrations of 1.0 and 20.0 micrograms/ml. This is at least twice the clearance index of 3'-azido-2',3'-dideoxythymidine and more than five times greater than that of 2',3'-dideoxyinosine. CONCLUSIONS: Bisheteroypiperazine crosses the maternal-fetal membranes by simple diffusion, in some instances almost equivalent to the reference compound antipyrine. Placental tissue concentrations were equivalent at all maternal concentrations, suggesting saturation. This high rate of maternal-fetal transfer suggests that it may be an effective prophylactic drug for fetuses of human immunodeficiency virus-infected mothers. PMID- 7531401 TI - Interleukin-2 receptor beta and CD57 expression in orbital tissues from patients with chronic, stable thyroid-associated ophthalmopathy. AB - In order to determine whether components of the interleukin-2 receptor (IL-2R) and lymphoid cells are present in extra ocular and periocular tissues from patients with chronic, stable thyroid-associated ophthalmopathy (TAO) we studied 16 specimens of extra ocular muscle and periorbital connective tissue from 14 patients with chronic, stable, TAO using an immunohistochemical assay and a panel of murine monoclonal antibodies reactive with IL-2R alpha and beta components and lymphoid cell surface markers. As controls we studied orbital tissues from 11 patients undergoing surgery for unrelated orbital disorders. All extra ocular muscle specimens from patients with TAO exhibited IL-2R beta expression primarily on the perimysium and endomysium surrounding the ocular muscle fasciculi and fibers of which nine specimens stained intensely. The Natural Killer (NK) cell marker CD57 was the most common cell surface antigen detected, in seven of nine specimens, whose localization often corresponded to that of IL-2R beta distribution. No IL-2R alpha expression was detected in any specimen. Seven of the 11 control specimens were positive for IL-2R beta but in a less intense fashion than in TAO specimens while no CD57 staining was detected. T cell, B cell, and cells of granulocyte and monocyte lineage were only occasionally found in both TAO and control specimens. The aberrant expression of IL-2R beta and CD57 which may be representative of NK cell presence in extra ocular muscle tissues from patients with stable, chronic TAO may play a role in the pathogenesis of the ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531402 TI - Metabolic hyperpolarization of liver by ethanol: the importance of Mg2+ and H+ in determining impermeant intracellular anionic charge and energy of metabolic reactions. PMID- 7531403 TI - Effects of ethanol on NMDA receptors in brain: possibilities for Mg(2+)-ethanol interactions. AB - The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N-methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5-50 mM). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg2+, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg2+ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg2+ site has been shown to increase the binding of N-[1-(2 thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(+/-)-carboxypiperazine-4-yl)-[1,2]-propyl-1 phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg2+ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg2+ can be clarified and any relationship between Mg2+ regulation and ethanol inhibition established. PMID- 7531404 TI - Effects of moderate alcohol consumption during pregnancy on child development at 18 and 42 months. AB - The aim of this study was to estimate the association between moderate alcohol consumption in pregnancy and child development to the age of 3.5 years. Furthermore, the aim was to compare development indices at 18 and 42 months of age. This study is an extension of the Danish participation in the EuroMac study. In a two-stage sampling, pregnant women in a well-defined region (Odense, Denmark) were recruited to a follow-up study according to their reported drinking habits in the first trimester of pregnancy. All among 2880 pregnant women who reported an alcohol consumption early in pregnancy (12th week of gestation) of at least 5 drinks/week were ascertained (164 women). A similar age and expected time of delivery matched group was selected from the remaining group of pregnant women. Alcohol consumption data were based on self-reported data, and child development recording was done blindly by two psychologists at 18 and 42 months after birth. Two hundred fifty-one mother-child pairs participated in all follow up studies. None of the reported levels of alcohol intake was statistically significantly associated with any of the child development indices (including measures of binge drinking). Comparing child development indices at 18 and 42 months did, however, reveal a rather large variation over time for alcohol exposures, as well as nonexposures. A large variation in the outcome measure will tend to mask effects of any exposures, but nevertheless it is unlikely that a low alcohol intake in pregnancy has any substantial impact on child development. PMID- 7531405 TI - Ethanol increases hepatocyte water volume. AB - Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as Cl-, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with ion sensitive microelectrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4 methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from -30 mV to -46 mV. Ethanol did not inhibit this osmotic stress-induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol-induced swelling of hepatocytes differs from that resulting from hyposmotic stress.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531406 TI - Transcription factors as molecular mediators in cell death. PMID- 7531407 TI - [Symbolism of the breast]. PMID- 7531409 TI - Effect of anti-calmodulin drugs on the growth and sensitivity of C6 rat glioma cells to bleomycin. AB - Antipsychotic drugs that bind to and inhibit the action of calmodulin also inhibit cellular proliferation. In addition these drugs are cytotoxic to most malignant cells and can augment the antiproliferative and cytotoxic effects of bleomycin. They are attractive candidates for use against tumors of the central nervous system since they readily pass the blood-brain barrier and accumulate in the brain. To identify more active derivatives, we studied the effect of a series of phenothiazines and a group of related compounds alone or in combination with bleomycin against rat glioblastoma cell lines. C6 cells were grown for 24 hours prior to a 48 hour exposure to anti-psychotic drug alone or to an IC20 concentration of antipsychotic drug with bleomycin. Cells were stained with methylene blue and enumerated spectrophotometrically. Eight phenothiazines were found to augment the effect of bleomycin by > or = 3-fold. These included 1 chlorpromazine (3.8x), chlorpromazine (3.2x), 3-chlorpromazine (3.0x), 4 chlorpromazine (3.4x), thiomethylpromazine (3.3x), didesmethylchlorpromazine (11x), fluphenazine (5.5x) and trifluoperazine (3.2x). Structurally similar compounds also having activity included trans-flupenthixol (6.0x), 2 chloroimipramine (6.0x), desipramine (22x), and penfluridol (24x). There was a direct correlation between the antiproliferative effect of anticalmodulin compounds and the ability of these drugs to inhibit the activation of calmodulin sensitive phosphodiesterase. However, there was no correlation between the inhibition of calmodulin and the augmentation of the antiproliferative activity of bleomycin. Penfluridol, one of the most active compounds, was chosen for further study. It increased the activity of bleomycin against L1210 leukemic cells by 90-fold and MCF-7 human breast cancer cells by 4-fold. The effect of penfluridol in combination with bleomycin was due to increased cytotoxicity as measured by clonogenic assay. PMID- 7531410 TI - Overexpression of P-glycoprotein but not its mRNA in multidrug resistant cells selected with hydroxyrubicin. AB - Previous studies have revealed that cultured cells treated with lipophilic natural products containing aromatic rings and basic amino group usually yielded multidrug resistant (MDR) variants. These MDR cells overexpress P-glycoprotein (P gp), most often due to gene amplification or transcriptional activation of mdr/P gp genes. Doxorubicin (Dox) is an anthracycline that belongs to this group of compounds. To explore the possible resistance mechanism(s) to anthracyclines that do not involve P-gp, we use a Dox analog, hydroxyrubicin (HyR) or WP159, which contains a C3' hydroxy group in replacement of the amino group in the sugar moiety of Dox thereby reducing basicity and eliminating positive charge in the parental compound to establish HyR-resistant cell lines. These resistant cells displayed the MDR phenotype and overexpressed P-gp as analyzed by Western blot analyses and immunohistochemical staining using two different anti-P-gp antibodies. Strikingly, the levels of P-gp mRNA in the majority of these MDR cells remained comparable to those in the drug-sensitive counterparts by slot blot hybridization. These results implicate that the basic center of the selecting agent is a critical determinant for generating diverse MDR variants, and that HyR may have a posttranscriptional effect on P-gp biosynthesis. This is the first report suggesting that cultured cells exposed to a particular selecting agent may give rise to particular subtype of MDR variants. PMID- 7531408 TI - Flow cytometric staining of pancreatic endocrine cells. AB - Few distinct surface markers have been identified for endocrine cells, but antibodies directed against hormonal epitopes are widely available. In this study, human fetal pancreatic beta cells were identified on the flow cytometer by intracellular staining for insulin. A paraformaldehyde based permeabilization scheme was used which preserves surface staining and helps maintain cell integrity while allowing antibodies access to compartments within the cell interior. Cells were first stained for surface antigens, fixed in paraformaldehyde, and then permeabilized in phosphate buffered saline containing 0.2 percent Tween. Intracellular staining was accomplished using a fluorescein conjugated anti-insulin monoclonal antibody. In this manner, major histocompatibility complex Class II antigen expression was documented on beta cells exposed to interferon gamma. This study demonstrates the feasibility of using hormonal content to identify endocrine cells by flow cytometry while simultaneously staining for surface antigens. PMID- 7531411 TI - Peritoneal fluid and HCG influence on steroid production from ovarian tumor tissue, in vitro. AB - Steroids are produced by malignant and benign epithelial ovarian tumor tissue in vitro, but the regulation is unknown. The effect of peritoneal fluid and beta-HCG on steroid production was analysed. Tissue from 17 malignant or benign epithelial ovarian tumors and 6 normal postmenopausal ovaries were incubated. In 11 cases tissue was incubated with and without addition of the patient's own peritoneal fluid (Part I). Tissue from 22 ovaries was incubated with and without HCG (100 IU/ml medium), (Part II). Furthermore, the release from tumorous and control ovaries of beta-HCG, progesterone, androstenedione and estradiol measured using radioimmunoassay (Part III). Peritoneal fluid stimulated the release of progesterone from both malignant and benign tissue and androstenedione or estradiol stimulated release from benign tissue (Part I). There was no stimulatory effect of HCG on steroid release from malignant tissue. HCG stimulated release of estradiol in benign tissue (Part II). All malignant tumors and 4 out of 6 benign tumors released beta-HCG (Part III). There seems to exist factors in peritoneal fluid which are able to stimulate steroid production from ovarian tumors. PMID- 7531412 TI - The effect of alpha-, beta- and gamma-interferon on the growth of breast cancer cell lines. AB - We investigated the antiproliferative effect of different concentrations (10, 100, 1000 IU/ml) of alpha-, beta- and gamma-interferons (IFN) on breast cancer cell lines. Cell lines were treated with IFN, in the absence or in the presence of estradiol for 9 days, and the effect on growth was evaluated as variation in DNA content. Inhibitory effect varied as a function of the type of interferon and cell line. Alpha-IFN and gamma-IFN were effective only at 1000 IU/ml and not in all cell lines, whereas a maximum effect was observed for beta-IFN regardless of the steroid receptor status of cell lines. In fact, a significant growth inhibition was observed at the intermediate concentration of 100 IU/ml in all but MDA-MB231 cell lines. Moreover, in both estrogen-receptor positive cell lines, beta-IFN counteracted growth stimulation induced by estradiol and showed a strong antiestrogenic activity. In conclusion, our results show that beta-IFN is the most active among the IFNs tested and suggest its usefulness in the treatment of all breast cancers, irrespective of their steroid receptor status. PMID- 7531413 TI - Altered drug sensitivities to anticancer agents in radiation-sensitive DNA repair deficient yeast mutants. AB - We studied whether cellular sensitivity to anticancer agents was correlated with a repair deficiency in three yeast epistasis groups of radiation sensitive mutants. All these mutants were hypersensitive to cisplatin and mitomycin C. By contrast, both rad51 and rad52 mutants deficient in double-strand breaks repair were hypersensitive to adriamycin and bleomycin, but the rad1 and rad10 mutants deficient in nucleotide excision repair were not. These results were confirmed by examining the cellular sensitivity of either revertants or strains carrying wild RAD+ protein expression plasmids to various drugs. Cellular damage by the above anticancer agents is discussed in relation to the DNA repair mechanisms. PMID- 7531414 TI - Effects of arginine butyrate and tributyrylxylitol on cultured human sarcoma cells. AB - The effects of arginine butyrate and tributyrylxylitol were studied comparatively in a human sarcoma cell line. Both induced important structural and functional modifications suggestive of cell differentiation, as shown by the dose-dependent increase of alkaline phosphatase activity and total protein content, at concentrations ranging from 1mM to 5 mM expressed in butyrate equivalents. hCG beta-subunit present in the culture medium increased with differentiation. Our results show that most of the differentiation changes previously reported for sodium butyrate in cancer cell lines are also produced by both drugs. Tributyrylxylitol appears to be the more potent and effective inducer of differentiation but its use is limited << in vitro >> on account of its relative toxicity at concentrations above 3 mM butyrate equivalents. PMID- 7531415 TI - Detection of apoptosis in leukemic and breast cancer cells with monoclonal antibody to single-stranded DNA. AB - In cultures of leukemic HL-60 and MOLT-4 cells treated with etoposide all nuclei with distinctive morphology of apoptosis (chromatin condensation at the nuclear periphery, nuclear fragmentation) were stained with monoclonal antibody F7-26 specific for single-stranded DNA. DNA in interphase and mitotic cells of control cultures and DNA in necrotic cells of cultures treated with sodium azide did not bind the antibody. In monolayer cultures of breast cancer cell line MDA-468 treated with tamoxifen for 4 hours all cells detached from substratum. These cells were apoptotic by nuclear morphology and stained with F7-26. Subset of cells without visible chromatin condensation but stained with F7-26 was detected among cells still attached to the substratum 1 hour after addition of tamoxifen. Thus, in breast cancer cells reactivity with F7-26 preceded chromatin condensation detected by fluorescence microscopy. Apoptotic cells stained with the antibody and non-apoptotic cells with background fluorescence were completely separated on two-parameter plots generated on a flow cytometer. Linear relation between percentage of apoptotic cells and ELISA reactivity with F7-26 in the cells attached to microtiter plates was demonstrated. These data show that apoptotic response can be measured by ELISA using staining with F7-26. Cells undergoing apoptosis can be detected by the procedure based on thermal denaturation of DNA in situ in the presence of Mg2+ with subsequent staining with the antibody specific for DNA in single-stranded conformation. Correlation between nuclear morphology typical of apoptosis in various cell types demonstrated that staining with monoclonal antibody F7-26 provides specific cytochemical marker for apoptotic cells. PMID- 7531417 TI - Cerebrospinal fluid 5-hydroxyindoleacetic acid and homovanillic acid in the pediatric opsoclonus-myoclonus syndrome. AB - To study the purported role of central monoamine disturbances in the pathophysiology of the opsoclonus-myoclonus syndrome, the serotonin metabolite 5 hydroxyindoleacetic acid and the dopamine metabolite homovanillic acid were measured in cerebrospinal fluid samples from 27 affected children and 47 age- and gender-matched control subjects by high-pressure liquid chromatography with electrochemical detection. 5-Hydroxyindoleacetic acid and homovanillic acid concentrations in the cerebrospinal fluid were approximately 30 to 40% lower in opsoclonus-myoclonus patients compared to control subjects, and the normal inverse correlation between age and monoamine metabolite concentrations in the cerebrospinal fluid of control subjects was not found in opsoclonus-myoclonus patients. Patients with the lowest values were less than 4 years old, and a subgroup had extremely low levels, but differences in older children were not significant. Cerebrospinal fluid levels of 5-hydroxyindoleacetic acid and homovanillic acid were more positively correlated in control subjects than in opsoclonus-myoclonus patients. None of the patients exhibited high levels of monoamine metabolites. Homovanillic acid levels were slightly lower in the cerebrospinal fluid of patients receiving corticotropin or steroids at the time of lumbar puncture. Clinical variables that could be excluded were paraneoplastic etiology, anesthetic for lumbar puncture, syndrome duration, age at onset, gender, response to steroids, length of time until initiation of corticotropin or steroids, presence of seizures, opsoclonus, and functional impairment. These data suggest a disturbance and possible altered ontogeny of serotonin or dopamine neurotransmission in a subpopulation of children with opsoclonus-myoclonus with low cerebrospinal fluid levels of 5-hydroxyindoleacetic acid and homovanillic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531418 TI - Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. AB - Both apolipoprotein E and its receptor, the low-density-lipoprotein receptor related protein (LRP), are associated with senile plaques in Alzheimer's disease. We examined the relationship of other LRP-related molecules to senile plaques. LRP is a multifunctional receptor that binds and rapidly internalizes at least seven ligands: apolipoprotein E, activated alpha 2-macroglobulin, tissue and urokinase-type plasminogen activators, plasminogen activator inhibitor-1, lipoprotein lipase, and lactoferrin. Using immunohistochemistry, we showed that all of these ligands, representing a diverse group of otherwise apparently unrelated proteins, accumulate on senile plaques. We also studied expression of the receptor-associated protein, a physiological inhibitor of LRP, in the hippocampal formation from normal subjects and Alzheimer's disease patients. Receptor-associated protein colocalizes with LRP on neuronal soma, but not on neuronal processes or reactive astrocytes. It is not present on senile plaques. These results suggest that senile plaque-associated LRP can bind its ligands, but clearance of these compounds may be impaired in the vicinity of senile plaques. PMID- 7531416 TI - Effects of tamoxifen on potential doubling time of human breast cancer cell line determined by image cytometry of double fluorescent BrdU and DNA labeling. AB - Tamoxifen is extensively used for the treatment of human breast cancer. However, the mechanisms by which antiestrogens regulate the growth of estrogen receptor positive tumors have not been totally defined. A new methodology, using automated image analysis BIOCOM 500, was developed for determining potential doubling time (Tpot) of tumors. This new method was checked on three different human breast cancer cell lines (MCF-7, CAL 85-1, CAL 148) in comparison with flow cytometry and then applied to determine the effects of short-term tamoxifen treatment on Tpot of MCF-7 cells. Using the resulting bivariate contour plot of blue fluorescence (DNA content) versus green fluorescence (Bromodeoxyuridine content), a labeling index (LI) value of 0.39 +/- 0.05 and a Tpot value of 21 +/- 2.09 hours were determined for MCF-7 cells. As expected, data demonstrated that 72 hours of 1 microM tamoxifen treatment decreased the LI to 35% by increasing the proportion of G0/G1 cells. It increased the Tpot to 35% compared to untreated cells (Tpot = 31.8 + 4 hours) by a lengthening of G0/G1 phase without changing the length of S phase (Ts = 10.2 +/- 1 hours). At suprapharmacological concentrations (5, 10 microM), an approximately 50% increase in Tpot was observed without modification in Ts. These data suggested a specific cell cycle action of tamoxifen which was probably mediated by mechanisms other than estrogen inhibition, since these experiments were performed in estrogen-deprived medium. In addition, the automated imaging procedure appears to provide a rapid and quantitative approach to determine Tpot in fine needle biopsies which is useful for investigating alterations in cell growth after endocrine treatment or chemotherapy. PMID- 7531419 TI - Serum anti-GQ1b IgG antibodies recognize surface epitopes on Campylobacter jejuni from patients with Miller Fisher syndrome. AB - Three patients who had diarrhea prior to the development of Miller Fisher syndrome are presented. Campylobacter jejuni was isolated from stool specimens from all patients. High titers of anti-GQ1b IgG antibodies were demonstrated in the serum of these patients by enzyme-linked immunosorbent assay and thin-layer chromatography overlay. In enzyme-linked immunosorbent assay inhibition studies the anti-GQ1b IgG antibodies bound specifically to whole bacteria of the Miller Fisher syndrome-associated C. jejuni strains. The presence of anti-GQ1b IgG binding epitopes on the surface of the C. jejuni from the patients was not exclusively associated with a specific Penner serotype. It is suggested that anti GQ1b antibodies are formed during the initial infection that elicits Miller Fisher syndrome. The cross-reactivity of anti-GQ1b IgG antibodies with surface epitopes on Miller Fisher syndrome-associated C. jejuni strains supports the hypothesis of molecular mimicry between bacteria and neural tissue. PMID- 7531420 TI - [Hepatitis B and C in pregnancy]. PMID- 7531421 TI - Successful application of the Norwood procedure for infants without hypoplastic left heart syndrome. AB - Although the first-stage Norwood procedure mostly has been used for hypoplastic left heart syndrome, there are other anomalies in which the Norwood procedure can be applied. Since 1991, 18 newborns without hypoplastic left heart syndrome underwent a first-stage Norwood procedure. All had a hypoplastic aortic annulus, ascending aorta, and transverse aorta. Ten had normally related great arteries: aortic atresia or aortic stenosis with inadequate left ventricle in 4, mitral atresia or stenosis in 4, and interrupted aortic arch in 2. The 8 others had double-outlet right ventricle with mitral atresia or complete transposition with a hypoplastic right ventricle. Age ranged from 2 to 77 days (median, 6 days) and weight from 2.4 to 4.4 kg (mean, 3.0 kg). The patients with interrupted aortic arch simultaneously underwent primary repair of the interruption. There were 17 hospital survivors (94%). There have been no late deaths in follow-up from 4 to 30 months (mean, 15.5 months). Thirteen children have had subsequent creation of a bidirectional Glenn shunt with takedown of the original systemic to pulmonary shunt. The 2 with interrupted aortic arch underwent a Rastelli-type biventricular repair. These results show that the Norwood procedure can be applied to infants without hypoplastic left heart syndrome who have hypoplastic aortas and excessive pulmonary blood flow with very low mortality and excellent palliation. PMID- 7531422 TI - Strategies for repair of congenital heart defects in infants without the use of blood. AB - Eleven infants and children with a body weight of less than 10 kg (median weight, 6.8 kg) whose parents were Jehovah's Witnesses underwent repair (n = 10) or palliation (n = 1) of congenital heart defects without the use of blood products and with (n = 9) or without (n = 2) cardiopulmonary bypass (CPB). In 1 neonate (weight, 3.2 kg) with critical aortic stenosis, moderate hypothermia and a 3.5 minute period of inflow occlusion and circulatory arrest allowed an aortic valvotomy; in another patient (weight, 7.0 kg) with tricuspid and pulmonary atresia, transposition of the great arteries, and persistent left superior vena cava, a bilateral bidirectional cavopulmonary shunt procedure was performed without CPB. Use of heparin-bonded tubing allowed reduction of the initial dose of heparin sodium to 1 mg/kg. Tissue perfusion and oxygenation on bypass were adequate, as evidenced by a mean lowest pH of 7.38 +/- 0.09 and a mean lowest venous oxygen tension of 65.0 +/- 36.2 mm Hg. Although the mean postoperative hematocrit (Hct) was lower than the mean preoperative Hct (p < 0.05, analysis of variance and Scheffe's F test), the Hct within 2 hours after CPB was restored to a value (mean Hct, 27.5% +/- 1.0%) between the preoperative Hct (mean value, 42.7% +/- 3.5%) and the lowest Hct on CPB (mean value, 18.4% +/- 1.4%). The Hct at discharge was 31.8% +/- 1.1%. The median postoperative blood loss was 9 mL/kg. There was no perioperative mortality. The median stay in the intensive care unit and the hospital was 2 days and 6 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531423 TI - Hemostatic efficacy of dipyridamole, tranexamic acid, and aprotinin in coronary bypass grafting. AB - Sixty patients (four groups of 15 patients) were entered in a randomized, controlled study to compare the efficacy of prophylactic treatment with dipyridamole, tranexamic acid, and aprotinin to reduce bleeding after elective coronary artery bypass grafting. Only patients with a preoperative platelet count of less than 246 x 10(9)/L were selected because a previous study showed that these individuals are at risk for increased postoperative bleeding. Compared to control subjects, postoperative blood loss 6 hours after operation was significantly reduced by tranexamic acid (674 +/- 411 versus 352 +/- 150 mL; p < 0.05) and by aprotinin (270 +/- 174 mL; p < 0.01). Dipyridamole did not reduce postoperative blood loss and was associated with complications in 3 patients. We conclude that hemostasis after cardiac operations can be improved with tranexamic acid and aprotinin. Dipyridamole appeared to be ineffective. PMID- 7531424 TI - Systemic-pulmonary shunt with a right retroesophageal subclavian artery. AB - A 19-day-old child suffering from cyanosis due to tetralogy of Fallot was palliated by using his right retroesophageal subclavian artery. It was anastomosed side-to-side onto the ascending aorta and end-to-side onto the right pulmonary artery. The palliation obtained with this systemic-pulmonary shunt was satisfying. The right brachial vascular flow was normal. PMID- 7531425 TI - Origin of post-depolarization hyperpolarizations in a grease-gap recording preparation. AB - In grease-gap recording preparations, depolarizing responses evoked by agonists are often followed by a hyperpolarization (post-depolarization hyperpolarization). We have investigated the origin of these post-depolarization hyperpolarizations in the rat CA1-subiculum slice. They were evoked by L glutamate, N-methyl-d-aspartate or alpha-amino-3-hydroxy-5-methyl-isoxazole-4 propionate in approximately 80% of the slices tested. The post-depolarization hyperpolarizations evoked by perfusion of N-methyl-d-aspartate through the CA1 compartment of the chamber, persisted when N-methyl-d-aspartate receptors in the subicular compartment were blocked with D-2-amino-5-phosphonopentanoate. Carbachol only blocked the post-depolarization hyperpolarizations evoked by alpha amino-3-hydroxy-5-methyl-isoxazole-4-propionate at concentrations which also blocked the depolarization. The post-depolarization hyperpolarization was selectively blocked by perfusion with Ca(2+)-free medium and by administration of ouabain, and showed a marked sensitivity to temperature. It is concluded that the post-depolarization hyperpolarizations observed in this preparation are not a consequence of diffusion of the agonist through the slice. The evidence is, however, consistent with them being generated by activation of the electrogenic Na(+)-K+ pump, although we cannot exclude an additional contribution from Ca(2+)- or voltage-dependent K+ currents. PMID- 7531426 TI - Effect of digital rectal examination on serum prostate-specific antigen in a primary care setting. The Internal Medicine Clinic Research Consortium. AB - BACKGROUND: Measurement of serum prostate-specific antigen (PSA) and digital rectal examination (DRE) are commonly used for prostate cancer screening. Clinicians have been advised to avoid measuring serum PSA after DRE because of the possibility of spurious elevation. However, studies in healthy volunteers have found no change. We sought to determine whether DRE performed by internists affects PSA levels in a primary care clinic population. METHODS: Men older than 49 years enrolled in a Veterans Affairs internal medicine clinic were eligible for the study. For all patients, prostate size and nodularity were assessed by DRE. Blood for determination of PSA levels was drawn immediately before DRE and 30 minutes after DRE. All patients completed a questionnaire regarding voiding. RESULTS: Two hundred two men aged 50 to 85 years (mean, 67 years) were enrolled and had complete data. The mean PSA level increased by 0.26 micrograms/L after DRE (P < .001). Six patients (2.9%; 95% confidence interval, 0.6% to 5.3%) had an increase in PSA level from less than 4 micrograms/L to more than 4 micrograms/L after DRE. There was a statistically significant but weak correlation between voiding score and PSA level (coefficient, .17; P = .02). Patients with an enlarged prostate gland had higher PSA levels than did patients with normal prostates or borderline results of examination. Nodularity did not predict an increased PSA level. CONCLUSIONS: We conclude, in an elderly veteran population, that DRE causes a statistically significant but clinically insignificant increase in serum PSA level. Additionally, we found that symptoms and physical examination results cannot be used to select a population for whom PSA screening would likely be useful. PMID- 7531427 TI - Modification of the fungi-fluor and the genetic systems fluorescent antibody methods for detection of Pneumocystis carinii in bronchoalveolar lavage specimens. AB - Four hundred forty-five bronchoalveolar lavage specimens from patients with the human immunodeficiency virus were preserved in Saccomano's fixative and stained for Pneumocystis carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warrington, Pa) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems Corp, Seattle, Wash). The majority of patients had been treated for suspected P carinii pneumonia for a few days prior to collection of bronchoalveolar lavage specimens. P carinii cysts were detected in 194 (43.6%) specimens. Both stains identified P carinii in 166 (37.3%) specimens and were negative in 251 (56.4%), yielding a concordance rate of 93.7%. P carinii cysts were detected in 25 (5.6%) specimens by the Genetic Systems stain only, and in 3 (0.7%) specimens by the Fungi-Fluor stain only. The sensitivity for detecting cysts of P carinii was significantly greater with the Genetic Systems stain (P < .01). PMID- 7531428 TI - Immunoreactivity of sinusoids in hepatocellular carcinoma. An immunohistochemical study using lectin UEA-1 and antibodies against endothelial markers, including CD34. AB - The reactivity of sinusoids in hepatocellular carcinoma (HCC), focal nodular hyperplasia, and nonneoplastic liver tissue with various endothelial markers was investigated to detect any differences that might be of diagnostic relevance. The lectin UEA-1 antibody BMA 120, and antibodies against von Willebrand's factor, CD31, and CD34 were used. KP1 was employed to detect Kupffer cells. In the normal liver there was only focal staining of sinusoidal endothelium in the vicinity of the portal tracts with all of the endothelial markers applied. In the cirrhotic liver a slightly greater number of sinusoids (mainly in the vicinity of the fibrous septa) stained with UEA-1 and, although to a lesser extent, with anti-von Willebrand's factor and anti-CD31. A slight increase in staining for CD34 was seen in only 1 of the 11 specimens of cirrhotic liver. In focal nodular hyperplasia, there was increased staining of sinusoids with all of the markers investigated; staining was confined mainly to the periphery of the nodules. HCC exhibited the most obvious differences in numbers of stained sinusoids and staining intensity in comparison with both normal and cirrhotic liver. UEA-1 and anti-CD34 stained large numbers of sinusoids in virtually all of the HCC investigated; UEA-1 stained a slightly greater number of sinusoids and did so with slightly greater intensity. BMA 120 and the antibodies against von Willebrand's factor and CD31 stained a smaller number of sinusoids and did so with lower intensity; they failed to stain sinusoids in some of the tumors. Because staining of the sinusoids in cirrhotic liver was minimal with anti-CD34, this antibody proved to be the best of all the markers investigated for distinguishing highly differentiated HCC from nonneoplastic liver tissue. It seems possible that the increase in immunoreactivity of sinusoids in HCC with anti-CD, unlike that with Uea-1, anti-von Willebrand's factor, and anti-CD31, is not an expression of capillarization, but rather of angiogenesis. PMID- 7531429 TI - Intrahepatic anastomoses for malignant and benign biliary obstruction. AB - OBJECTIVE: To assess the role, techniques, and outcome of intrahepatic biliary anastomoses in patients undergoing resective or palliative surgery at the hepatic hilum. DESIGN: A retrospective review over a 23-year period of all patients undergoing intrahepatic bilioenteric anastomoses. SETTING: A major university teaching hospital in France. PATIENTS AND INTERVENTIONS: Over a 23-year period, 59 patients (19 with palliative, 35 with curative, and five with benign disease) underwent intrahepatic bilioenteric anastomoses. MEASUREMENTS AND RESULTS: There was a hospital mortality of six for the palliative group and four for the curative group. A significant fall in both the serum bilirubin and alkaline phosphate levels occurred after surgery (P < .5). Long-term follow-up was possible in 44 patients. Recurrent stenosis due to recurrent disease occurred in 100% of the palliative group and 69% of the curative group. There were two stenoses in four patients with benign disease. Recurrent cholangitis developed in 37 patients, five without apparent stenosis. The median survival for the palliative group was 6 months (range, 2 days to 13 months) and for the curative group, 21.7 months (range, 0.5 to 148 months); all the benign group remain alive. CONCLUSIONS: Despite the advent of modern endoscopic and percutaneous intubation techniques, intrahepatic anastomoses after tumor resection offer the only chance of cure for obstructing hilar malignant disease. PMID- 7531430 TI - Free perforation of gastric carcinoma. Results of surgical treatment. AB - OBJECTIVE: To define by actual standards the results of treatment of free perforation of gastric carcinoma in a consecutive number of patients treated at one institution. DESIGN: Case series of patients with perforated adenocarcinoma of the stomach treated in Hong Kong between 1984 and 1992. SETTING: Urban academic medical center. PATIENTS: Thirty-four Chinese patients who were operated on for perforated gastric carcinoma. A risk scoring system was used to predict postoperative mortality. Factors with a possible influence on postoperative mortality and long-term survival were studied using univariate and multivariate analysis. INTERVENTION: All patients underwent laparotomy, which was performed for closure of the perforation in four patients and for gastrectomy in 30. MAIN OUTCOME MEASURES: Thirty-day mortality and survival times. RESULTS: The 30-day mortality rate was 20%, and the median survival time was 10 months (range, 2 to 92 months). The risk score was the only significant predictor of 30-day mortality, and the pathologic TNM staging, of long-term survival. CONCLUSIONS: A significant proportion of patients can be saved and offered good palliation with emergency gastrectomy; those likely to die can be identified before surgery. PMID- 7531431 TI - Endoscopic stenting for malignant biliary obstruction. AB - OBJECTIVE: To evaluate the results of endoscopic stenting in patients with malignant biliary obstruction. DESIGN: Retrospective review. SETTING: Surgical endoscopy unit at a tertiary referral center. PATIENTS AND INTERVENTION: Plastic endoprosthetic stents were inserted for the relief of biliary obstruction in 131 consecutive patients. Among the 104 patients with malignant biliary obstruction, 60 underwent stenting as definitive palliative treatment. MAIN OUTCOME MEASURES: Data on the type and size of stents employed, their effectiveness in relieving jaundice, their complications, and the quality of survival were analyzed. RESULTS: Median survival was 1.93 months. The majority of the stents (88.3%) were placed endoscopically, and single straight stents were most frequently used (96.7%). Jaundice was relieved in 35 patients. Early (< or = 14 days) and late acute cholangitis were found in four and 11 patients, respectively. Early and late stent migration was noticed in two and six patients, respectively. Pancreatitis occurred in one patient, and no papillotomy site bleeding was encountered. When compared with the 18 patients with proximal obstruction at or above the common hepatic duct, the 42 patients with distal ductal obstruction had longer median survival (P = .03) and more effective relief of jaundice (P = .005) and required less antibiotic treatment (P = .03). These 42 patients also enjoyed better quality of survival when it was analyzed with reference to six objective parameters (P = .0018). CONCLUSIONS: Stenting offers palliation of malignant biliary obstruction, and the results are particularly encouraging in distal obstruction. PMID- 7531433 TI - Phase II study of glycosylated recombinant human granulocyte colony-stimulating factor after HLA-identical sibling bone marrow transplantation. AB - BACKGROUND: The lengthy period of neutropenia which follows allogeneic bone marrow transplantation (BMT) results in significant morbidity and some mortality. Recombinant human granulocyte colony-stimulating factor (rhuG-CSF) effectively reduces neutropenia and morbidity when given after autologous BMT, but has not been adequately investigated in allografts. AIMS: To assess the tolerability, safety and efficacy of rhuG-CSF after allogeneic BMT. METHODS: rhuG-CSF was administered to 13 adult patients with haematological malignancies after HLA identical sibling BMT. Five micrograms/kg of rhuG-CSF was given daily by subcutaneous bolus injection, commencing four hours after marrow infusion and continuing until the neutrophil count was > or = 1.0 x 10(9)/L on three consecutive days. Graft-versus-host disease (GVHD) prophylaxis was cyclosporin and short-course methotrexate (days 1, 3, 6 and 11). Prophylactic intravenous (IV) antibiotics were administered from the onset of neutropenia. The control group consisted of patients with comparable diagnoses, transplanted before and after the current study using identical supportive care and GVHD prophylaxis policies. RESULTS: Although time to recovery of the neutrophil count to > 0.1 x 10(9)/L was similar, the rhuG-CSF-treated patients experienced accelerated recovery to > 0.5 x 10(9)/L, which occurred at a median of 15 days (range 11-21) after marrow infusion in study patients compared to 18.5 days (range 14-41) in the controls (p = 0.04). No significant differences were detected in any of the indices of transplant-related morbidity examined, including the number of days of fever, the incidence of culture-positive infections, the usage of antibiotics, the requirement for parenteral nutrition and IV morphine, the maximum severity of mucositis and GVHD, and the day of discharge. CONCLUSION: Within the context of this study, rhuG-CSF had limited impact on the clinical outcome of HLA-identical sibling BMT. PMID- 7531432 TI - Selection of patients for randomised trials: a study based on the MACOP-B vs CHOP in NHL study. AB - BACKGROUND: Selection of patients for a clinical trial is affected by awareness of the existence of the trial, interest in the study question and clinical practices and views of the clinicians. AIMS: To investigate the selectivity that may have occurred at Peter MacCallum Cancer Institute (PMCI) during the ANZ Lymphoma Group trial of MACOP-B vs CHOP in non-Hodgkin's lymphoma (NHL). METHODS: NHL patients at PMCI in the study period were assessed against the trial's eligibility criteria. Comparisons were made between eligible (except for consent) non-trial patients and all patients actually randomised into the trial. RESULTS: Of 497 patients presenting during the trial period, 320 (64%) did not meet the specified eligibility criteria, 102 (21%) were unsuitable on other grounds (age and medical) and 75 (15%) were eligible. Of those eligible, 43 (57%) were entered into the trial and 32 (43%) were not. Four non-trial patients had inappropriate application of eligibility criteria and 13 unknown reason. Eligible non-trial patients were similar to trial patients in most patient and tumour characteristics and overall survival. Significantly more non-trial patients had higher stage disease (p = 0.02). More non-trial patients had lower grade histology, but this was not significant. CONCLUSIONS: Physician selectivity occurred with respect to patient entry, but trial and non-trial patients were similar in most characteristics. Eligibility criteria should specify that patients can withstand all trial drugs and patient availability for treatment and follow-up. PMCI trial accural could have been up to 33% greater. These results suggest the trial accrual period could have been 25% shorter. Patient entry into this trial by PMCI clinicians compared favourably with other centres. PMID- 7531434 TI - Signals from the conceptus. PMID- 7531435 TI - src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase C gamma 1 structure and activity. AB - Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+] dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed. PMID- 7531436 TI - Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro. AB - Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids. PMID- 7531437 TI - Interleukin-3 facilitates glucose transport in a myeloid cell line by regulating the affinity of the glucose transporter for glucose: involvement of protein phosphorylation in transporter activation. AB - Growth factors promote cell survival and proliferation by activating signal transduction pathways that result in progression through the cell cycle and differential gene expression. Uptake of simple sugars needed for basal cell metabolism, and for macromolecular synthesis necessary for cell growth and proliferation, is thought to follow as a consequence of signal transduction to the nucleus. However, in the presence of inhibitors of DNA synthesis and respiration, growth factors can still promote cell survival responses in the short term, raising the possibility that they may also regulate critical membrane and cytosolic processes necessary for cell survival. We have tested this hypothesis directly by investigating the role of the haemopoietic growth factor, interleukin-3 (IL-3), in the regulation of glucose transport in the bone marrow derived cell line, 32D. We show that IL-3 promotes glucose transport by actively maintaining the affinity of the plasma membrane, glucose transporter for glucose (Km 1.35 +/- 0.15 mM, n = 4). Withdrawal of IL-3 for 1 h resulted in reduced affinity for glucose (Km 2.96 +/- 0.28 mM, n = 4) without an associated change in Vmax. Furthermore, glucose transporter molecules as the cell surface, as determined by cytochalasin B binding to isolated plasma membranes, did not differ significantly between control and IL-3-treated cells. Inhibition of DNA synthesis with mitomycin C or with the respiratory poison, sodium azide, did not affect the ability of IL-3 to promote glucose transport. In contrast, the tyrosine kinase inhibitors genistein and erbstatin extensively inhibited control and IL-3 stimulated glucose transport, some preference of IL-3-stimulated glucose transport, some preference for IL-3-stimulated responses being observed at low inhibitor concentrations. The light-activated protein kinase C inhibitor, calphostin C, also inhibited control and IL-3-stimulated glucose transport but without preference for IL-3 responses. Additionally, the tyrosine phosphatase inhibitor, orthovanadate, stimulated control and IL-3-dependent glucose transport by 50-80% while the protein kinase A inhibitor, KT5720, inhibited glucose transport by about 20% at plateau values. These results indicate that IL-3 is involved in continuous maintenance of glucose transporter activity by a mechanism that involves tyrosine kinases and protein kinase C, and demonstrate that this activation is not dependent on respiration or signal transduction to the nucleus. PMID- 7531441 TI - Influence of acetaldehyde on airway resistance and plasma exudation in the guinea pig. AB - In anaesthetized ventilated guinea-pig, acetaldehyde (CAS 75-07-0) (40-80 mg/kg i.v.) elicits a dose-dependent increase in intratracheal pressure accompanied by an increase in circulating histamine. When acetaldehyde is injected repeatedly at 15 min intervals in capsaicin-desensitized animals, it already loses its activity at the second administration (50% reduction; p < 0.01); this does not happen in control animals. This phenomenon is even more marked when acetaldehyde is given at the dose of 80 mg/kg i.v., since at the third injection both the bronchoconstriction and the increase in blood histamine are almost completely reduced to baseline values. The increase in intratracheal pressure caused by acetaldehyde (20, 40, 80 mg/kg i.v.) is associated with a dose related increase in microvascular permeability and leakage of protein-bound Evans blue in lower tracheal tissue. This event and the bronchoconstrictor response caused by acetaldehyde (40 mg/kg i.v.) are 87% and 35% inhibited, respectively, (p < 0.01) in tachykinin-depleated animals. On the contrary, thiorphan (2 mg/kg i.v.) remarkably potentiates both the rise in intratracheal pressure (110%; p < 0.01) and Evans blue extravasation (215%; p < 0.01) induced by acetaldehyde (20 mg/kg i.v.) in normal guinea-pigs. Furthermore, treatment with CP-96,345, a selective tachykinin NK1-receptor antagonist, only prevents plasma extravasation in lower tracheal tissue (82% inhibition; p < 0.01) without affecting the bronchoconstriction caused by acetaldehyde (40 mg/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531440 TI - Synovial tissue response to treatment with Campath-1H. AB - OBJECTIVE: Therapeutic trials in rheumatoid arthritis with the monoclonal antibody Campath-1H have demonstrated recurrent clinical synovitis in some patients, despite profound depletion of circulating lymphocytes. This study was undertaken to examine the cellular infiltrates in synovial tissue at a time of persistent peripheral lymphopenia. METHODS: Immunohistochemical staining of synovial tissue and peripheral blood lymphocyte phenotyping. RESULTS: Synovial tissues from 2 patients with recurrent synovitis after Campath-1H therapy contained significant T lymphocytic infiltrates at a time when circulating T lymphocytes were markedly depleted. CONCLUSION: These results demonstrate that peripheral blood analysis may not accurately reflect the synovial tissue response to monoclonal antibody therapy. PMID- 7531438 TI - Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters. AB - Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3' untranslated regions but differed in their 5' sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation. PMID- 7531439 TI - Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29) AB - Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195 268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp 130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected]. PMID- 7531442 TI - Effect of hyperlipidemic diets, proinflammatory agents and plasma expanders on leukocyte adhesion to the endothelium of aorta and carotid artery of rats. AB - A comparative study of leukocyte adhesion to the endothelium of the thoracic aorta and left carotid artery in rats has been performed after administration of two hyperlipidemic diets for 15 days, proinflammatory agents (thrombin, lipopolysaccharide and zymosan activated serum) and plasma expanders [dextran, polyvinylpyrrolidone (PVP), rat albumin and several bovine albumins from different sources]. Leukocytes adhered to the endothelium were demonstrated in surface preparations by esterase activity. Activation of circulating leukocytes was measured by nitroblue tetrazolium reduction and luminol enhanced chemiluminescence. Both hyperlipidemic diets produced, in all rats, more leukocyte adhesion in the aorta than in the carotid artery. All proinflammatory agents produced at 1 h, increases in leukocyte adhesion--which in all rats were greater in the carotid artery than in the aorta--and leukocyte activation, which was higher at 3 h than at 1 h. Dextran, PVP, bovine albumins 103700 and A-4503 at 18 h produced slight increases in leukocyte adhesion in the aorta but not in the carotid artery. Rat albumin and bovine albumin A-7906 determined an intense leukocyte adhesion at 18 h which was not preferential to either vessel. Adhesion produced by A-7906 was maximal at 12 h and partially inhibited by dexamethasone. This last albumin produced leukocyte activation at 3 h and was sequestered 5 min after administration, reaching normal values at 1 h. Albumins 103700 and A-4503 neither activated leukocytes nor were sequestered after administration. PMID- 7531443 TI - Polyclonal antiidiotypic antibodies mimicking gp120 of HIV-1. AB - Rabbit antiidiotypic antibodies (Ab2) were produced against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120 of HIV-1 and shows virus-neutralizing activity. The Ab2 bound specifically to the Ab1 and their binding to Ab2 was inhibited by a recombinant fragment of gp120 (PB1) or a peptide (residues 301-324 of gp120), both expressing the Ab1-defined epitope. The Ab2 induced in rats antiantiidiotypic antibodies (Ab3) that were Ab1-like in their binding reactivities to PB1, native gp120 or peptide and shared idiotopes with the Ab1. However, the Ab2 did not induce virus-neutralizing Ab3, probably a reflection of the low avidity of the Ab3 as compared to the Ab1. PMID- 7531445 TI - The ins and outs of the transmembrane 4 superfamily. AB - The recently discovered transmembrane 4 superfamily comprises a group of cell surface proteins that are characterized by the presence of four hydrophobic domains, which are presumed to be membrane spanning. At least seven of these molecules are expressed on leukocytes, and it seems likely that they mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility. PMID- 7531444 TI - A shared idiotope among antibodies against Semliki Forest virus. AB - In the present study a shared idiotope was found among antibodies against a previously defined linear B-cell epitope of Semliki Forest virus (SFV). The synthetic B-cell epitope, located at amino acid positions 240 to 255 of the E2 membrane protein, was linked to an H-2d-restricted T-helper cell epitope of either SFV or influenza virus. Colinearly synthesized peptides of T-B polarity mixed with adjuvant were used to immunize BALB/c (H-2d) mice. After one booster immunization with either chimaeric peptide high serum antibody titers were measured against both synthetic peptide (240-255) and glutaraldehyde-fixed SFV infected L cells. Against the synthetic peptide (240-255) a variety of monoclonal antibodies (MAbs) were produced that differed in reactivity with SFV, varied in heavy chain family, isotype, isoelectric point, and idiotype. Against one of the antipeptide MAbs (I02), that strongly reacted with SFV-infected L cells, an antiidiotypic MAb (ab2MAb), designated I02A3, was produced that could be inhibited in its binding to MAb I02 by the synthetic B-cell epitope. Therefore it was concluded that ab2 MAb I02A3 recognizes an idiotope closely associated with the antigen combining site of antipeptide MAb I02. This idiotope was definitively shared by two out of 15 antipeptide MAbs and by SFV-reactive antibodies present in both antipeptide sera and SFV-immune sera. PMID- 7531446 TI - Histamine modulates the acute phase response at multiple points. PMID- 7531447 TI - Determinant spreading: implications in allergic disorders. PMID- 7531448 TI - Exogenous antigen control of autoreactive T-cell MHC specificity. PMID- 7531449 TI - Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence. AB - Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7.5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3' non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 2.6 kb major transcript and two minor transcripts of approximately 2.1 and 1.8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver > kidney > lung >> heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs. PMID- 7531450 TI - Experimental podophyllotoxin (bajiaolian) poisoning: III. Biochemical bases for toxic effects. AB - The biochemical mechanism underlying the toxicity of podophyllotoxin is investigated. Previous studies from our laboratories suggested that hepatocytes were extremely sensitive to the toxicity of podophyllotoxin and a disruption of protein synthesis was suspected. Dose-response and time-course studies on the effects of podophyllotoxin on protein. RNA, and DNA syntheses on hepatocellular cultures were made. Inhibitions of protein, RNA, and DNA syntheses were demonstrated, and a direct correlated dose-response relationship on such effects was also evident. Inhibition of protein synthesis appeared to be a direct toxic effect of podophyllotoxin and occurred independently from that of RNA. The reduction of DNA synthesis was reflected by a reduction of H3-thymidine incorporation, which is believed to be the consequence of inhibition on the mitotic activity by podophyllotoxin. PMID- 7531451 TI - Cytotoxicity of captafol in mammalian cells. AB - The cytotoxicity of captafol, a phthalimide-derived fungicide, was evaluated in IB-RS-2 cells. Captafol at 0.12-1.0 microgram/ml blocks the cell multiplication. This effect is concentration-dependent, only partially reversible and the degree of inhibition increases with time. The synthesis of DNA and RNA is inhibited in parallel by increasing concentrations of the chemical. PMID- 7531452 TI - A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate. AB - We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure. PMID- 7531454 TI - Somatostatin, galanin and peptide histidine isoleucine in the newborn and adult human trigeminal ganglion and spinal nucleus: immunohistochemistry, neuronal morphometry and colocalization with substance P. AB - By means of indirect immunofluorescence the neuropeptides somatostatin, galanin and peptide histidine isoleucine were localized in cell bodies, nerve fibres and terminal-like elements in the ganglion and spinal nucleus of the human trigeminal nerve in perinatal and adult ages. No immunoreactivity to vasoactive intestinal polypeptide was observed. In the gasserian ganglion somatostatin-, galanin- and peptide histidine isoleucine-containing neurons and nerve fibres occurred frequently in pre- and full-term newborns, but were scarce to absent in adults. Somatostatin- and galanin-positive pericellular basket-like structures around non immunoreactive perikarya were observed in newborn specimens. Immunoreactivity to somatostatin, galanin and peptide histidine isoleucine labelled nerve fibers and punctate and felt-like nerve terminals in the pars interpolaris and subnucleus caudalis of the spinal trigeminal nucleus, with immunostaining and distribution patterns characteristic for each peptide. In addition, somatostatin-containing neuronal cell bodies frequently were detected. At variance with those containing somatostatin, the number of galanin- and peptide histidine isoleucine-like immunoreactive elements were dramatically reduced in the adult tissue compared to the newborn one. Double immunostaining revealed that each of the three peptides partially colocalizes with substance P, the degree of coexistence being very low for somatostatin/substance P and high for galanin/substance P and peptide histidine isoleucine/substance P both in the gasserian ganglion and in the spinal nucleus. The results obtained suggest that somatostatin, galanin and peptide histidine isoleucine may play functional roles in primary sensory neurons and at the first synaptic level of the human trigeminal sensory system. PMID- 7531453 TI - The distribution of tachykinin binding sites in the brain of an electric fish (Apteronotus leptorhynchus). AB - We mapped the distribution of tachykinin binding sites utilizing quantitative autoradiography of iodinated substance P and eledoisin as prototypic ligands for neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors, respectively. The two ligands produced highly heterogenous and quantitatively different patterns of specific binding, suggesting that they revealed different tachykinin receptor subtypes. Although [125I]substance P and [125I]eledoisin binding were correlated in most brain regions, the binding of substance P was usually denser. [125I]substance P binding and substance P-like immunoreactivity were reasonably correlated in most brain areas, although discrepancies were found in some nuclei. Dense [125I]substance P binding was found in most areas of the subpallium and in parts of the pallium related to the olfactory system, as well as in the glomerular layer of the olfactory bulb. Moderate to dense binding of both ligands was observed in preoptic area, hypothalamus, habenula, parts of the thalamus and preglomerular complex. Especially noteworthy was the presence of [125I] substance P binding in the diencephalic prepacemaker nucleus, a region involved in the control of electroncommuncatory behavior. Substance P-like immunoreactivity is sexually dimorphic in certain diencephalic nuclei, including the prepacemaker nucleus (Weld and Maler, 1992); no obvious difference was seen between [125I]substance P or [125I]eledoisin binding in the brains of male versus female fish. In the mesencephalon striking laminar patterns of binding were seen in the torus semicircularis dorsalis and the optic tectum. Dense binding was also noted in the raphe nuclei, the locus ceruleus and the sensory nucleus of the vagus. Although binding of substance P in the electrosensory lateral line lobe and nucleus preeminentialis was light, it was distributed in a discrete fashion, suggesting a role of substance P in electrosensory processing. PMID- 7531455 TI - Compartmental organization of the peptide network in the human caudate nucleus. AB - The mammalian striatum may be divided into a striosomal compartment and a surrounding matrix region. We have examined the distribution of leucine enkephalin (LENK) and substance P (SP) immunoreactivity in relation to striosomes defined by calbindin-D (CABD) staining in alternate 70 microns serial sections from the human caudate nucleus. The distribution of LENK immunoreactivity showed a transition from dorsal to ventral striatum: dorsally, LENK-rich patches were present in a lightly stained matrix; mid-ventrally, annular patches of LENK staining with a lighter core were seen. These patches corresponded to striosomal regions defined by CABD-poor zones. In contrast, in the ventral caudate and nucleus accumbens, LENK-poor zones matched CABD-defined striosomes. CABD staining in the matrix was intense in the dorsal caudate, diminishing ventrally. SP-rich zones in dorsal caudate and SP-poor areas in the mid-ventral region overlapped striosomes. In the ventromedial sector, the SP staining pattern was complex and did not consistently correlate with striosomes. Computer-assisted three dimensional reconstruction of the striosomal system in the human, based on regions of either high LENK or low CABD immunoreactivity, revealed the existence of considerable long-range order. Patches appeared aligned over several millimeters to form long, horizontal structures in the caudate nucleus, with occasional orthogonal interconnecting crossbridges. Our results are in accord with previous work in the human and in other species. These three-dimensional networks are strikingly similar across individuals and may relate to the segregation of and interactions between striatal circuits. PMID- 7531456 TI - Productive infection of normal CD40-activated human B lymphocytes by HIV-1. AB - OBJECTIVE: Antigen-driven B-cell proliferation and maturation occur in germinal centres present in lymphoid tissues. This process is highly dependent on functional interactions between B and T lymphocytes. In vitro activation of CD40 present on B cells mimics B cell-T interactions and allows the proliferation of normal Epstein-Barr virus (EBV)-negative B lymphocytes. In HIV-1-seropositive individuals, B cells become exposed to free viral particles and to infected T lymphocytes while migrating through germinal centres. The effect of HIV-1 viral exposure on CD40-activated B lymphocytes was therefore examined. METHODS: Freshly isolated B lymphocytes were cultured in vitro through activation of CD40. B-cell proliferation, HIV-1 infectivity and viral production were monitored following B lymphocyte exposure to HIV-1. In addition, HIV-mediated fusion between infected B cells and uninfected CD4+ T lymphocytes was assessed in a coculture assay. RESULTS: EBV-negative, CD40-activated human B lymphocytes were directly infected by HIV-1. The infection significantly reduced their proliferation rate. Viral production was detected in B-cell culture supernatant. Numerous fusion events indicated that HIV-1 infection of B lymphocytes could spread to T lymphocytes following HIV-1-mediated fusion of these two cell types. CONCLUSION: In view of the importance of B cell-T cell interactions in the maintenance of a functional immune system, disruption of B-lymphocyte development could have direct implications on the course of AIDS progression. PMID- 7531457 TI - Analysis of Pneumocystis carinii organism burden, viability and antigens in bronchoalveolar lavage fluid in AIDS patients with pneumocystosis: correlation with disease severity. AB - OBJECTIVES: We examined 96 bronchoalveolar lavage fluid (BALF) specimens from AIDS patients with proven Pneumocystis carinii pneumonia (PCP) in order to compare the relationship of organism burden, viability and antigen expression with disease severity at the time of clinical presentation. METHODS: Tinctorial analysis of BALF specimens with proven PCP using Diff-Quik, cresyl echt violet and erythrosin B stains to evaluate organism burden and viability. P. carinii antigen examination was performed by Western blot analysis. RESULTS: P. carinii cluster ratios were more sensitive than cyst counts as an indicator of organism burden, and correlated well with the alveolar-arterial oxygen gradient as a measure of disease severity. Erythrosin B, the vital stain used to measure P. carinii viability, displayed a wide range of values and provided little useful information. Antigens of 35-45 and 95kD, which were specific for P. carinii, were found by immunoblot analysis in BALF cellular fraction of most patients with pneumocystosis. By contrast, antigens of 52 and 66 kD, which were found in both BALF supernatant and cellular fractions of P. carinii patients and controls, most likely represented albumin and immunoglobulin G heavy chain, respectively, of host origin. The 35-45 kD antigen was found in 88% of the BALF specimens and appeared to represent an important marker of P. carinii infection. The 95 kD antigen was detected in 49% of the specimens. CONCLUSIONS: We conclude that analysis of P. carinii characteristics in BALF specimens of patients with pneumocystis may provide additional information. These data will also be helpful in developing more sensitive assays and in targeting specific P. carinii factors for future investigation. PMID- 7531458 TI - FK506 can inhibit apoptotic cell death induced by the HIV-1 envelope glycoprotein gp120. PMID- 7531459 TI - Resistance artery structure and neuroeffector mechanisms in hypertension induced by inhibition of nitric oxide synthase. AB - Oral administration of NW-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, results in blood pressure elevation. In this study we investigated resistance artery structure and neuroeffector mechanisms in rats treated with L-NAME. After 5 days of treatment the morphology of resistance arteries was unchanged, whereas after 2 or 4 weeks, when rats were hypertensive, the media was thicker and the media-to-lumen ratio was enhanced. When L-NAME administration was stopped for 2 weeks, the media-to-lumen ratio remained increased. At all time periods under L-NAME and 2 weeks after stopping treatment, the dose-response to norepinephrine was significantly more sensitive than in controls, with a concomitant slight increase in efficacy. Cocaine (3 mumol/L) or L-NAME (100 mumol/L) applied in vitro produced a leftward displacement of the dose-response of arteries from controls to norepinephrine, and abolished the difference with vessels from L-NAME-treated rats. Responses evoked by electrical field stimulation were also displaced to the left in L-NAME-treated rats and by L NAME in vitro, and exhibited a slight increase in maximal response. Dose-response curves to methoxamine, which is not subject to neuronal reuptake, were displaced to the left by L-NAME in vivo and in vitro, similarly to those of norepinephrine, but were not displaced by cocaine in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531460 TI - Nef and Gag synthetic peptide priming of antibody responses to HIV type 1 antigens in mice and primates. AB - T epitope mapping in human immunodeficiency virus proteins provides a useful tool for AIDS vaccine design. We have previously shown that four peptides selected from the Gag polyprotein of HIV-1 were able to prime mice for in vitro lymphoproliferative responses. These responses were shown to be MHC restricted, and a pool of these peptides was able to prime mice for a subsequent humoral response to HIV-1 Gag proteins. Here we show that two of these Gag peptides are able to prime the anti-HIV-1 IgG response to heat-inactivated HIV-1 in B10Sc.Cr mice. Furthermore, we extended this study in the nonhuman primate model, and show efficient priming of the IgG response to heat-inactivated HIV-1 using the pool of four Gag peptides in baboons. Further mapping of "nonself" peptides is extended to the HIV-1 Nef protein. Three potential Nef T epitopes located at positions 137 145, 98-107, and 81-95 are also shown to prime the IgG response to HIV-1 in the mouse model, although T cell proliferation to recall peptides in vitro was not detectable. Although they have not yet been defined as major helper T epitopes in humans, using classic in vitro stimulation assays, the fact that most of them are able to prime IgG responses in animals without detectable in vitro proliferative responses does not rule out their functional helper capacity in humans. PMID- 7531462 TI - Inhibition of apoptosis in T cells expressing human T cell leukemia virus type I Tax. AB - This study set out to determine whether T cell dysfunction associated with HTLV-I led to increased sensitivity of infected cells to apoptosis or, owing to their potential to develop ATL, if infected cells would become resistant to this process. To test this hypothesis we utilized the monoclonal antibody anti-APO-1, which has been demonstrated to induce apoptosis in human T cells. Human T cell lines expressing HTLV-I showed reduced susceptibility to anti-APO-1-induced apoptosis despite expression of high levels of cell surface APO-1. Cell-free supernatant of the Tax-expressing cell line C8166 and heat-inactivated supernatant of the HTLV-I-producing cell line MT2 transferred increased resistance to anti-APO-1 to susceptible Jurkat T cells. Susceptible T cells transfected with an HTLV-I Tax-expressing vector or treated with soluble Tax protein became less susceptible to anti-APO-1-induced cell death. Furthermore, primary human lymphocytes treated with soluble Tax were less susceptible to apoptosis induced by anti-APO-1. The protective effect of Tax in T cell lines and primary human lymphocytes was reversed by the addition of anti-Tax antibodies. Anti-APO-1-induced apoptosis was also found to be inhibited in Jurkat cells by the induction of protein kinase C (PKC) with 12-O-tetradecanoylphorbol-13-acetate (TPA). Resistance to apoptosis conferred by HTLV-I Tax and an active PKC pathway may be factors contributing to the survival of dysregulated HTLV-I-infected T cells prone to the development of adult T cell leukemia. PMID- 7531461 TI - Inhibition of HIV type 1 reverse transcriptase assay by nucleases produced by contaminating mycoplasmas. AB - Mycoplasmal contamination of HIV-1-infected cells has been found to induce reduction of reverse transcriptase (RT) activity; however, the exact mechanism of this phenomenon was not clearly elucidated. Our results indicate that the apparent reduction in RT activity is due to a calcium-dependent nuclease(s) that is (are) produced by contaminating mycoplasmas. The interference with the RT assay was found to be due to the degradation of products of the RT activity. Addition of EGTA at a 1 mM concentration was sufficient to remove the inhibitory effect. The particular HIV-1-producing cell line that was under study was found to be contaminated with Mycoplasma fermentans and Mycoplasma pirum and the latter was isolated in pure culture. Nuclease activity was also observed with pure cultures of mycoplasmas from different species. The activity was found to be of the endonuclease type because it was active with both supercoiled and linear DNAs. PMID- 7531464 TI - Production of a membrane-bound protein, the human gamma-glutamyl transferase, by CHO cells cultivated on microcarriers, in aggregates and in suspension. AB - Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h-1, the highest cell density (near 1.3 x 10(6) cells ml-1), and the highest enzyme activity around 300 mU ml-1, which corresponded to a specific cellular level of 20 mU 10(-5) cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems. PMID- 7531463 TI - The c-kit receptor transduces the stem cell factor-triggered growth signal in murine interleukin-3-dependent cell line. AB - The stem cell factor is a glycoprotein hormone which regulates the proliferation and differentiation of primitive hematopoietic cells through its interaction with a tyrosine kinase transmembrane receptor which is encoded by the c-kit proto oncogene. To examine whether a murine c-kit receptor can be functional in murine interleukin-3 (mIL-3)-dependent hematopoietic cell line, we introduced the murine c-kit cDNA into mIL-3-dependent pro-B cell line Ba/F3. One of the resulting clones, Ba/F3 clone BF-K96, expressed the 140 kDa protein recognized by anti-c kit monoclonal antibody and the expressed c-kit receptor protein on the cell surface bound to a radiolabeled soluble form of murine stem cell factor (mSCF) with high affinity. BF-K96 clone expressing the c-kit receptor could proliferate in response to mSCF in the absence of mIL-3. The cell clone could also grow in co culture with mouse 3T3 cells which are endogeneously expressing a membrane associated type of mSCF on their cell surfaces. These findings demonstrate that the c-kit receptor expressed on mIL-3-dependent hematopoietic cell line Ba/F3 transduce the mSCF-dependent growth signal, indicating that established cell clone will provide a unique cellular system for the study of SCF/c-kit signal transduction mechanism. PMID- 7531466 TI - [A proposal for a functional evaluation record for the hand]. AB - Proposition of a new evaluation method included an analytic cotation, a functional cotation, and a cosmetic cotation. Use is speeded by the suppression of all kind of measure instrument and facilited by pictures. Application to the corporal damage evaluation. PMID- 7531465 TI - Airborne cytotoxicity in the DiSC assay caused by solutions of treosulfan but not busulphan. AB - Treosulfan and busulphan are similar molecules, the former used in the treatment of ovarian cancer and the latter in chronic myelogenous leukaemia. We have used both in the differential staining cytotoxicity (DiSC) assay for in vitro drug sensitivity testing to aid in the choice of chemotherapy for individual patients. It was observed that occasionally the viability of control cells in one assay box was reduced compared with control cells in other boxes from the same assay. Treosulfan was suspected as the cause because cells throughout the microtitre box containing treosulfan had reduced viability in 28/62 (45%) experiments and in 9 of these, total kill of all cells in the box was observed. We tested the hypothesis that a metabolite of treosulfan might be the cause of this airborne cytotoxicity, and found that whilst 10 mg ml-1 of either methane sulphonic acid or tetrahydrofuran had no airborne cytotoxic effect, 1 mg ml-1 diepoxybutane killed over 95% of cells in all tubes in the same box. Treosulfan is another chemical (cf. azide, mafosfamide and possibly other cytotoxic agents) that can cause airborne cytotoxicity. PMID- 7531467 TI - [A comparative study of the "Levame" type extension splint and the "Low Profile" type extension splint: impact on therapeutic indications]. AB - The most widely used dynamic extension systems, both to straighten the digital flexion retractions as well as to ensure directed mobilization after surgical repair of extensor tendons, are the so-called "Levame" and "Low Profile" splints. This study analysed the application of forces for these two types of extension splints, the directional constraints which they impose and freedom of joint movement which they allow in order to more clearly define the type of motorization to be used as a function of the most frequent indications. PMID- 7531468 TI - [Use of an implanted electrode for rehabilitation after tenolysis of the flexor tendons]. AB - The electric stimulation is widely used in flexor tenolysis rehabilitation by the authors. Since 1983 implantable electrodes were used during the operative procedure. Actually they gave better access to the deep muscles (Flexor Profundus of the index), better specificity, less dysesthesia of the skin. Further more they allowed to tests the quality of the tenolysis without wrist incision. This procedure gave 76% of good or very good results over 25 finders in 20 patients. The results were especially good for the index finger which is usually unable to be stimulated by the surface electrodes and for the fingers badly damaged. In this type of situation prognostic was getting better (70% of good and very good results). PMID- 7531469 TI - [Restoration of opposition using the extensor pollicis longus. Apropos of 8 cases reviewed after 11 years]. AB - Moutet and coll. had studied in 1986 a series of 16 cases of restoration of thumb opposition by extensor pollicis longus transfer onto abductor pollicis brevis through the inter osseous membrane. Eleven years follow-up revision of 8 of those 16 patients was performed by the authors. Indication of transfer was traumatic median nerve isolated palsy or associated to ulnar nerve palsy. Objective and subjective analysed parameters allowed to check the evolution of the transfer and to justify its indications. No transfer retraction has been noted in long term follow-up. The side effects of its removal (thumb retropulsion and MP extension defects) have minimum functional consequences. The opposition has been restored at the time of thenarians recovery and as well, each time the palsy was going on. In the cases where the thenarians recovered, the transfer became an antepulsion supply. The authors suggest to perform an opposition transfer, each time it is technically possible, in severe median nerve lesions, at the time of emergency operation. PMID- 7531470 TI - [The evaluation of the Artem grip in its function assessment]. AB - The aim of our study is to assess the Artem pincers efficiency, which is both a new grip strength instrument and a hand reeducation instrument. It is original through its pleasant good looking and it offers many different programs of use. This study was carried out involving 60 healthy subjects. We compared the Artem system with two other measurement instruments of reference (Jamar for grip strength and Pinch-Gauge for pinch strength). With this Artem system we also studied two other originals tests: 1) Evolution of hand and finger strength within 15 seconds. 2) Tiredness test. Our measurements demonstrated good high correlation and high reliability between reference instruments and Artem system dealing with strength measurements. We can't conclude about the two original program's statistics, but it would probably be interesting to compare these with a group of patients in course of reeducation. PMID- 7531472 TI - [Sensory potential of the ring finger. Its value in the electromyographic diagnosis of carpal tunnel syndrome]. PMID- 7531471 TI - [A new titanium-carbon finger joint implant. Apropos of 15 initial cases]. AB - Arthroplasty of the MP and PIP joint remains a therapeutic challenge for the hand surgeon. The poor results of the currently available implants are due to their mechanical conception (non strained) or their material (silicone causing instability, ruptures and silicone synovitis). The authors present the preliminary results of a new digital articular implant (IAD). It is a strained implant with a rigid hinge allowing 105 degrees of range of movement (0 degree to 105 degrees). Three sizes are available and very soon four. The intramedullary stems are in titanium alloy (TA6V) and the gliding surfaces in pyrolytic carbon. This association allows solidity, endurance, excellent gliding and no wear debris. 15 implants were evaluated (13 PIP and 2 MP joints) with a follow-up from 4 to 1 year. A painless range of motion were restored in 11 cases and 4 articulations were only lightly painful during motion requiring strength and at the end of the movement. The gain of the range of movement was 50 degrees always in a useful sector. The grasp was 60% better in post than in pre operative conditions. No infection was found. It is only the beginning of this implant but the indications seem to be good for all the articular destructions of the digital joints what ever was the aetiology (traumatic, infection or rheumatoid arthritis). PMID- 7531473 TI - [The contribution of MRI in the study of glomus tumors]. AB - The glomus tumors of the finger tip are benign. Their diagnosis is clinic but their precise localization is quite difficult by classic imaging. MRI with gadolinium injection seemed a good method to check exactly this lesion and have been tested on 20 patients suspected suffering of glomus tumor. On 18 glomus tumors surgically and histologically confirmed MRI examination have been positive for 16. In two cases MRI and surgery were negative. A good correlation seems observed with surgery for the size (7 tumor of 2 mm) the localization and osseus extension. Addition of T2 and T1 before and after injection weighted acquisitions improved the results. Precise informations on topography and size seems improved the surgical approach. PMID- 7531476 TI - [A free transfer of the pulp of the toe with an exteriorized flap. A compromise between microsurgery and traditional surgery]. AB - Free toe pulp transfer represents the most sophisticated technique which corresponds functionally as close as possible to total restoration of the digital pulp. However, this operation presents technical disadvantages related to the very small calibre of the vessels when the vascular anastomosis is performed in the digit. When the artery and vein are anastomosed in the metacarpal region to achieve vessels with a larger calibre, the passage of the pedicle in the finger is difficult and requires complete opening of the digital canal, resulting in a finger which is often enlarged with limited mobility. The authors propose a technique which overcomes these two disadvantages. PMID- 7531475 TI - [The relation of the short radial extensor muscle of the wrist with the deep branch division of the radial nerve. Its significance in the physiopathology of elbow pain]. AB - Since Roles and Maudsley's publication, in 1972, it has been admitted that the entrapment of the posterior interosseous nerve (PION) is a possible cause of lateral elbow pain. In the radial tunnel, at least 4 or 5 compressive structures have been described. The arcade of Frohse is a well known compressive cause while the medial edge of the extensor radialis brevis (ECRB) muscle is less frequently incriminated. An anatomic study of 45 supper limbs from 40 cadavers was performed to analyse the relations between the medial edge of the ECRB and the PION. The medial edge of the ECRB was a real fibrous arch in 43 cases (95%). It crossed over the PION in 42 cases, 9 mm more proximally than the arcade of Frohse. So the relationships between the ECRB and PION are very close in 93% of cases. The proximal edge of the supinator muscle was fibrous in 40% of cases but always supple. These findings suggest that the ECRB is a possible cause of PION entrapment. Its role is underestimated in pathology. The frequent association between lateral epicondylitis and PION compression can be explained by the presence of this fibrous structure. Lateral elbow pain must be considered as a regional pathology. The treatment has to deal with every pathological aspect. PMID- 7531474 TI - [Stabilized arthroplasty of the 5th metacarpal bone. A therapeutic proposal for the treatment of old fractures-luxations of the 5th metacarpal bone]. AB - The goals of the surgical treatment of fracture-dislocations of the fifth ray are length restoration of the fifth metacarpal, release of impingement and preservation of the carpo-metacarpal mobility. Our technic of "stabilised arthroplasty" satisfies theoretically to these three goals. It consists in resection-arthroplasty of the base of the fifth metacarpal associated with stabilisation by arthrodesis between fourth and fifth metacarpal. PMID- 7531477 TI - The palmar wrist ligaments revisited, clinical relevance. AB - In fifteen cadaver wrists and ten foetus wrists palmar ligaments were studied. As a result, the palmar scapho-triquetral ligament was presented by means of gross anatomical study, MR imaging, and histological study. A kinematic study was conducted on three cadaver wrists to analyze patterns of motion on the proximal carpal row bones based on the helical axis concept. Motion analysis showed that the scaphoid has a set of helical axes bundled at the radial side of the wrist. On the contrary, the triquetrum had axes bundled on the ulnar side, approximately in the same position as the lunate. That shows that the lunate and triquetrum are coordinated in motion and opposed to that of the scaphoid. The structure possibly ruling this pattern of motion could be the palmar scapho-triquetral ligament, whose insertions on the scaphoid and triquetrum are close to the crossing-point of the helical axes of the scaphoid and triquetrum/lunate, respectively. In order to study the clinical relevance of our findings, a prospective clinical study on 53 patients after wrist injury was conducted. All patients were clinically examined, simple and semi-stress X-rays were taken, and arthrography and arthroscopy performed. Ligament tears were often found, but any correlation to clinical findings or arthrography could not be found. However, high correlation could be found between the lesion of the palmar scapho-triquetral ligament and a DISI position of the lunate combined with increased scapho-lunate angle. It can be concluded that the tear of the palmar scapho-triquetral ligament causes instability pattern that is radiologically evident at an early stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531478 TI - Cartilaginous and ligamentous degeneration of the wrist. Anatomic study. AB - The growing precision of diagnostic techniques (MRI, arthrography, arthroscopy) and the consequent increase of the diagnosis of cartilaginous and ligamentous lesions of the wrist led us to undertake a detailed anatomical study of the carpus and to extend this study to the search for correlations between these lesions and the radio-ulnar index. Fifty one cadaveric wrists were dissected from an elderly population (mean age of 76 years). Cartilaginous lesions were found in two-thirds of radioulnar joints of the wrist with a marked predominance for the lunate bone (43%). The triangular cartilage of the fibrocartilaginous complex (TFCC) was perforated in 23 wrists (46%). We established a correlation between the radio-ulnar index and perforations of the TFCC (p < 0.05), as well as the thickness of this structure (p < 0.05). The relationship between age and rupture of intrinsic ligaments (p < 0.05), and the radio-ulnar index (p < 0.05) and age was also established. We present our figures, discuss the clinical implications, and draw the following conclusions from this study. 1) The carpus is a complex joint which is subject to age-related degeneration. 2) The large number of cartilaginous lesions observed in this study must be taken into account in the interpretation of MRI and the "over" precise results of arthroscopy. PMID- 7531479 TI - [Palliative surgery in nonresectable pancreatic carcinoma]. AB - The authors report 30 cases of adenocarcinoma of the pancreas treated with palliative surgery at the 2nd Department of Surgery of S. Maria delle Croci Hospital in Ravenna, in the period 1985-1991. Biliary bypass or gastroenterostomy were performed because of severe jaundice and duodenal obstruction, and not with prophylactic intent. According to Fortner's classification 60% of the neoplasias were stage 3, 36.7% stage 2, and 3, 3% stage 1 tumors. The hospitalization mean time was 15 days, perioperative mortality was 6.6% and mean survival rate was 6.5 months. The authors conclude that palliative surgical treatment is useful in case of neoplastic biliary and duodenal stenoses, with no liver or lung metastases, and also whenever an histological evaluation of the tumor is necessary to evaluate resection chances. PMID- 7531480 TI - Regenerative capability of upper and lower jaws in the newt. AB - The regenerating amphibian jaw represents an important model for studying pattern formation and the mechanisms underlying regeneration of facial structures. We have studied regeneration of upper and lower jaws in the urodele amphibian, Notophthalmus viridescens, using whole mount preparations stained for bone and cartilage, scanning electron microscopy and immunocytochemistry to further characterize these regenerating systems. In addition, we have investigated whether lower jaws of adults and larvae display similar regenerative ability. Although in adult animals the original shape of both the lower and upper jaws is rather faithfully reproduced following amputation, and the teeth and oral mucosa with its specialized sensory organs fully regenerate, significant differences in the regenerative ability of the various skeletal elements are observed. In fact, only tooth-bearing skeletal elements ossify, while the other elements of the regenerated skeleton remain cartilaginous for as long as 5 months after amputation. In contrast, a regenerated lower jaw in the larva is indistinguishable from an unamputated one at the same stage of development. Interestingly, regenerating adult jaws form directly bicuspid teeth, which are the type of teeth normally found in the adult, rather than the monocuspid teeth characteristic of larval jaws, indicating that jaw regeneration is not a recapitulation of development, in that an adult jaw blastema directly regenerates an adult jaw. Finally, we have studied the expression of tissue specific markers in normal and regenerating upper and lower jaws to establish whether the blastemal cells, which will form the missing part of the jaw, express any of these markers of the differentiated state, or are undifferentiated as suggested by their morphological appearance. Under our experimental conditions, no expression of markers of the differentiated state, such as those for muscle, cartilage and glands is detectable in early regenerates. On the contrary, the mesenchymal marker 22/31, whose expression in normal jaws is restricted to dermal fibroblasts and the dental pulp, is expressed in at least a half of the blastemal cells. The significance of these observations in relation to the origin of blastemal cells in the jaw will be discussed. PMID- 7531481 TI - A beta 1-integrin associated alpha-chain is differentially expressed during Xenopus embryogenesis. AB - The antigen of mAb 2F10 was identified as a Xenopus beta 1-integrin associated alpha-chain by the criteria (1) that it coprecipitates with anti beta 1-antibody, (2) that it changes molecular mass upon reduction in a way that is characteristic for integrin alpha-chains and (3) that it is present on cell membranes. This alpha-chain, termed alpha 2F10, is found in small amounts in the pregastrula stages of Xenopus development and accumulates thereafter in the embryo, alpha 2F10 can be detected by immunofluorescence first at stage 17 of embryogenesis on the cell membranes of the sensorial layer of the ectoderm, the notochord and the endoderm. This characteristic pattern of distribution is maintained throughout the following embryonic stages. Timed explanation experiments indicate that all cells of the pregastrula have the potency to express alpha 2F10. This potency becomes successively restricted during gastrulation to yield the ultimate pattern of expression. PMID- 7531483 TI - Flow cytometric DNA analysis of breast cancer by two colour method using cytokeratin labeling for identification of tumour cells. AB - Flow cytometric assessment of DNA-ploidy and S-phase fraction in breast cancer is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g. tumour, stromal and inflammatory cells. To identify tumour cell subpopulations, epithelial cells were labeled by a FITC-conjugated cytokeratin antibody (CK6, CK18) prior to flow cytometric cell cycle analysis in 205 fresh specimens of primary breast cancer. We found 158/205 (77%) DNA-aneuploid tumours compared to 127/205 (62%) without identification of cytokeratin positive cells (P < 0.001). In addition, the number of detected DNA-multiploid tumours rose from 31 (15%) to 51 (25%) after gating for cytokeratin positive cells. In DNA-diploid tumours, S-phase and G2M-phase fractions were significantly higher in cytokeratin positive (tumour) cells compared to total cell populations (4.4% and 5.8% vs. 3.2% and 4.4%; P < 0.001). Cytokeratin negative cells were found in all tumours and can be used as internal standard for calculation of ploidy and for quality control (CV, linearity) of each individual sample. We conclude that at least 20% of DNA-aneuploid tumours would not have been diagnosed without cytokeratin labeling. In addition, influence of non-tumourous cell elements on cell cycle analysis can be markedly reduced. Therefore, both determination of DNA-ploidy and cell cycle analysis can be optimized by cytokeratin labeling. PMID- 7531482 TI - Xenopus cadherins: the maternal pool comprises distinguishable members of the family. AB - Three maternal cadherins have been reported to occur in the pregastrula Xenopus embryo. EP- and XB-cadherin are distinguished by their distinct cDNA sequences. U cadherin has been characterized by its reaction with a specific monoclonal antibody (mAb 6D5). Thus far, lack of specific probes that discriminate between these molecules has prevented their identification as distinct cadherins. We now demonstrate by means of RNase protection assays that both EP- and XB-cadherin mRNAs are present in oocytes and mature eggs. By use of the Xenopus cadherin proteins expressed in mammalian cell lines, we find that mAb 6D5 crossreacts with XB-cadherin, but not with EP-cadherin. The major fraction of the maternal cadherins does not contain the 6D5 epitope and probably represents EP-cadherin. A minor fraction carries the 6D5 epitope indicative for the XB- and U-type of cadherins. We have termed this fraction XB/U-cadherin. The function of maternal cadherins was examined by in vitro cell adhesion assays. A newly developed antiserum with a broad specificity for various Xenopus cadherins efficiently blocks all calcium dependent cell adhesion in the early embryo. We conclude that the maternal cadherins play a central role in interblastomere adhesion in the early embryo and comprise at least two discrete cadherin forms, EP- and XB/U cadherin. PMID- 7531484 TI - Caveats in the use of archival cell material for DNA ploidy analysis by image cytometry. AB - Monolayer preparation from paraffin-embedded tissue from archival biopsy specimens older than 10 years was shown in this study to be more difficult to deal with than recent biopsies. We have studied the disseminative effect of several enzymes on archival biopsies from laryngeal epithelium, as well as mechanical dissemination. Thirty minutes incubation in protease 0.5% in 37 degrees C without syringing provided the best cell yield. Hydrolysis and Schiff staining conditions were also studied. Cell loss was calculated by stereological methods and found to be high in most cases. Cell loss during various steps in monolayer production was examined and we found no proof of selective cell loss. Large variations in Feulgen-Schiff staining intensities were observed, especially in biopsy specimens older than 10 years. The use of reliable internal (intrinsic) control cells is advocated since interspecimen variations in the diploid integrated optical density (IOD) can be expected. Although DNA measurements on archival biopsy material should be treated with caution, we demonstrate that meticulous studies of the preparation procedures will make it possible to obtain reliable histograms from old, archival biopsies. PMID- 7531485 TI - Computerized morphonuclear analyses of Feulgen-stained nuclei from 11 chemosensitive and from 11 chemoresistant neoplastic cell lines. AB - It has previously been demonstrated that the cell nuclei from 1 mouse mammary cancer and 2 neoplastic human bladder cell lines displayed common morphonuclear characteristics when they were made resistant to different anti-neoplastic agents. In the present work this experiment was repeated with a greater number of cell lines from different kinds of tumours. The nuclei from the sensitive and resistant cells were submitted to Feulgen staining and studied by means of computerized nuclear image analysis. This methodology made it possible to characterize the cell nuclei by means of 15 parameters including 1 geometric, 2 densitometric and 12 others quantitatively describing the chromatin pattern. The results showed that the morphonuclear feature of the cell lines significantly varied according to the extent of the resistance. The majority of the observations showed that the direction of the variations seemed to be a function of the origin of the tissue. On the basis of the experimental results reported here, we think that it would be possible to establish a model for the purpose of monitoring the effectiveness of anticancer treatment from a clinical point of view. PMID- 7531486 TI - Bone marrow reconstitution after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation: effect of graft size. AB - BACKGROUND: Peripheral blood progenitor cell transplantation is rapidly replacing autologous bone marrow transplantation as hematological support after high-dose chemotherapy for lymphoma or solid tumors. Controversy exists concerning the number of progenitor cells required for rapid and sustained bone marrow recovery, and as to which of the widely available methods for estimating this number should be employed. METHODS: Forty consecutive patients with solid tumors or lymphomas received high-dose chemotherapy followed by autologous peripheral stem cell reinfusion. All stem cell harvests had been performed after mobilization with standard-dose chemotherapy followed by 300 micrograms G-CSF daily. Hematopoietic reconstitution was studied in relation to pertinent patient characteristics, to the size of the graft (in terms of the total number of mononuclear cells (MNC), the number of granulocyte/macrophage colony-forming units (CFU-GM) and the number of CD34+ cells, and to the use of G-CSF after stem cell reinfusion. RESULTS: Both the numbers of CFU-GM and CD34+ cells reinfused, but not those of the MNC, correlated with granulocyte and platelet recovery. Patients who received at least 5 x 10(6) CD34+ cells/kg body weight achieved platelet transfusion independence on day 12 after reinfusion (range: day 7-37), significantly earlier than patients who had received less (p = 0.001). Thirty patients who received G-CSF (300 micrograms s.c. daily) after reinfusion achieved granulocyte recovery (> or = 500 x 10(6)/l) on day 9 (range: day 8-12), while this took a median of 15 days (range: day 10-28) in 10 consecutive patients not receiving G-CSF (p = 0.0003). In one patient who had received 1.4 x 10(6) CD34+ cells/kg, secondary bone marrow failure developed 3 months after transplantation. Reinfusion of cryopreserved autologous bone marrow was followed by prompt recovery. CONCLUSION: Peripheral stem cells, mobilized by moderate-dose chemotherapy and G-CSF, lead to rapid and durable engraftment after high-dose chemotherapy when at least 3-5 x 10(6) CD34+ cells/kg are reinfused. Lower numbers may also be satisfactory, but are associated with slower granulocyte and platelet recoveries. A moderate dose of G CSF after reinfusion significantly hastens granulocyte recovery without interfering with platelet recovery. PMID- 7531487 TI - Vinorelbine: an active drug for the management of patients with heavily pretreated Hodgkin's disease. AB - BACKGROUND: This study evaluated the therapeutic effect of the weekly administration of vinorelbine (5'-nor-anhydrovinblastine), a semisynthetic vinca alkaloid, in heavily pretreated patients with Hodgkin's disease. PATIENTS AND METHODS: Twenty-four patients with Hodgkin's disease refractory or resistant to at least two chemotherapy regimens were enrolled in this study. Vinorelbine was administered in a weekly dose of 30 mg/m2 i.v. bolus and patients were evaluated after four courses. All but two were considered evaluable for drug response. The reasons for their exclusion were early death due to pancytopenia and loss to follow-up after two courses. In complete responders, six additional courses were administered; in all other patients, treatment was continued until their diseases progressed. Toxicity was evaluated in 23 patients according to the Common Toxicity Criteria. RESULTS: Eleven of 22 evaluable patients (50%) showed objective response (complete 14% and partial 36%). The median duration of response was six months for both complete and partial responders (range 2-10 months). Thirteen patients are still alive and five are still on therapy. Grade 3 4 granulocytopenia was documented in 53% of patients and grade 3 infections in 13%. Anemia and thrombocytopenia were negligible. Nausea and vomiting were not observed; grade 2 alopecia occurred in only one patient. There were grade 3 reactions at the injection site in the first five patients, so a venous central access was utilized in the subsequent patients. Two patients had grade 1 constipation and only one developed an adynamic ileum. Although all patients had previously been treated with vinca alkaloid analogs, peripheral neuropathy was mild. CONCLUSIONS: Our data indicate that vinorelbine is active as a single agent in heavily pretreated patients with Hodgkin's disease. The efficacy in patients pretreated with at least two vinca alkaloids suggests a possible absence of cross resistance between vinorelbine and other vinka analogs. Toxicity is mild and reversible. The inclusion of vinorelbine in secondline combination chemotherapy regimens for Hodgkin's disease is strongly recommended. PMID- 7531488 TI - Metastatic tumor of unknown primary site. PMID- 7531489 TI - Coronary vasomotor responses: role of endothelium and nitrovasodilators. AB - The endogenous nitrovasodilator endothelium-derived nitric oxide (EDNO) is continuously synthetized enzymatically by NO synthase from L-arginine and is released from endothelial cells. Enhanced, superimposed EDNO release can be stimulated by various local and circulating factors, such as bradykinin, ATP, etc., but also most importantly by viscous drag-induced shear stress of the bloodstream acting on the endothelial lining. Thus luminal release suppresses leukocyte adhesion (expression of adhesion molecules), platelet activation, platelet adhesion, and platelet aggregation, and abluminal release counteracts myogenic and neurogenic coronary constrictor tone, thereby increasing myocardial perfusion and dilating large coronary artery calibers. Thus endothelial impairment and denudation (hypercholesterolemia, atheromatosis, balloon catheter interventions) favor excessive constrictor tone and myocardial ischemia. Under these conditions EDNO can be supplemented by compounds (e.g., nitroglycerin, isosorbide dinitrate) converted by biological systems into NO. In addition, it can be supplemented by compounds that even spontaneously release NO (e.g., sydnonimines such as SIN-1 and sodium nitroprusside). EDNO and exogenously supplemented NO stimulate soluble guanylyl cyclase, increase cGMP levels, and bring about vascular relaxation, particularly in those still compliant sections in which EDNO production is impaired and cGMP levels are thus diminished. Exogenous nitrovasodilators are preferentially converted (in the presence of cysteine) enzymatically in large coronary arteries, improving coronary conductance, and in the venous bed (preload reduction), resulting in an improved O2 supply/demand ratio. During chronic, continuous application, neurohormonal counterregulation and diminished enzymatic biotransformation into NO may reduce their effectiveness, resulting in tolerance, particularly in the most sensitive vascular sections, such as veins and coronary arteries. This drawback can be overcome by applying spontaneously NO-releasing compounds, intermittent therapy, or intermittent interposition of other vasodilator principles. PMID- 7531490 TI - Alterations in expression of p56lck during myeloid differentiation of LSTRA cells. AB - The src-related tyrosine kinase p56lck is overexpressed in the mouse leukemia cell line LSTRA. Although p56lck is thought to be a specific T-cell marker, we found that LSTRA cells can be induced to differentiate towards macrophages or granulocytes by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the cyclic nucleotide analogue, dibutyryl cAMP, respectively. Treatment of LSTRA cells with 12-O-tetradecanoylphorbol-13-acetate resulted in marked alterations in morphology including increased size, adherence, and spreading on culture dishes. These cells also ceased proliferating, accumulated in G0-G1 and expressed nonspecific esterase activity. In contrast, although LSTRA cells treated with dibutyryl cAMP stopped growing and accumulated in G0-G1, these cells expressed functionally active chemotactic peptide receptors and became irregular and granular in appearance. Differentiation of LSTRA cells was also found to be associated with altered expression of p56lck. Thus, while 12-O tetradecanoylphorbol-13-acetate treatment caused the cells to produce higher molecular weight forms of p56lck, dibutyryl cAMP treatment resulted in increased expression of total p56lck mRNA as well as the more mature type II p56lck mRNA transcript. There were no major alterations in p56lck kinase activity in vitro following differentiation. Phospholipase C gamma and p21rasGAP, two putative substrates for the tyrosine kinase activity of p56lck, were found to be constitutively phosphorylated on tyrosine in LSTRA cells. Tyrosine phosphorylation of these substrates was not altered following differentiation. These results indicate that LSTRA cells are relatively early precursors that have the capacity to develop along the myeloid differentiation pathway.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531491 TI - Immunogold study of non-collagenous matrix components in normal and exfoliative iris. AB - The present investigation was undertaken to determine if some of the components of exfoliation material in iris tissue were unique to exfoliation or were part of normal iris architecture. Eleven normal iris specimens and 10 exfoliative iris specimens were processed for cryoultramicrotomy and London resin white embedding. Immunogold electron microscopy was used to investigate the fine structural distribution of amyloid P component, elastin, entactin, fibronectin, gp115, and vitronectin in normal iris and their association with exfoliation material. Exfoliation material was positive for amyloid P component and possibly gp115, neither of which were present in normal iris tissue. Elastin and fibronectin were present in the normal iris stroma but were not associated with exfoliation material. The distribution of amyloid P component in the vessel lumen and wall led to the conclusion that amyloid P is a serum contaminant. The presence of gp115 in exfoliation material represents the synthesis of a component novel to the iris vascular cell synthetic repertoire. PMID- 7531492 TI - Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C. AB - The relationship between lipid domains and enzyme activity was studied via the direct visualization and quantitation of domains by fluorescence digital imaging microscopy. The substrate used in these experiments was a basic peptide derived from a prominent cellular substrate (MARCKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, which were labeled by two different fluorophores, colocalized into domains in large vesicles (5-10 microns). Increasing the ionic strength disrupted the domains of the MARCKS peptide and phosphatidylserine, and this was accompanied by a decrease in protein kinase C activity. Dansylpolylysine, which inhibits protein kinase C, was similar to the MARCKS peptide in forming domains enriched in phosphatidylserine. The degree of enrichment of the MARCKS peptide in the phosphatidylserine domains decreased proportionally with protein kinase C activity when polylysine was added. Polylysine caused the MARCKS peptide to be displaced from the domains into the nondomain areas of the vesicles. This suggested that binding of the substrate to the vesicles was not the critical factor for protein kinase C activity, but rather it was the organization of the substrate into domains that was related to the activation of the enzyme. Gramicidin, which was chosen to represent a neutral membrane protein, was excluded from the domains with phosphatidylserine, and it had no effect on the enrichment of the domains or the enzyme activity. The results of this study show that the formation of membrane domains can be important for the activation of protein kinase C and the activity can be inhibited by disrupting the domains. PMID- 7531494 TI - Phosphotryptic peptide analysis of the human androgen receptor: detection of a hormone-induced phosphopeptide. AB - Phosphorylation of the androgen receptor (AR) in human prostate tumor cells (LNCaP) is increased by androgens. The AR is expressed as two isoforms with apparent molecular masses of 110 and 112 kDa. Metabolic labeling experiments with [32P]orthophosphate revealed that only the 112 kDa isoform is radioactively labeled. Phosphoamino acid analysis revealed only phosphorylation on serine residues. Phosphotryptic peptide analysis of human AR protein by two-dimensional peptide mapping and by reverse-phase HPLC showed phosphorylation at multiple sites. Comparison of phosphopeptide maps of AR protein from cells incubated in the absence or presence of the synthetic androgen R1881 indicated that the ligand stimulated phosphorylation is probably due to induction of phosphorylation at a new site rather than increased phosphorylation at an existing site. This result suggests that hormone-dependent AR phosphorylation might play a role in the signal transduction pathway of androgens. PMID- 7531493 TI - Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction. AB - Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction. PMID- 7531495 TI - Hydrogen peroxide-supported oxidation of NG-hydroxy-L-arginine by nitric oxide synthase. AB - The ability of murine macrophage nitric oxide synthase (NOS) to utilize peroxides in place of O2 and NADPH was investigated using hydrogen peroxide (H2O2), tert butylhydroperoxide, and cumene hydroperoxide with both L-arginine and NG-hydroxy L-arginine (L-NHA) as substrates. Of the three peroxides examined, only H2O2 was able to support product formation using L-NHA as a substrate. No product formation was observed from L-arginine with any peroxide tested. Therefore, the L NHA/H2O2 reaction was examined in greater detail. The products of the reaction were citrulline and nitrite/nitrate (NO2-/NO3-) with a stoichiometry of approximately 0.75:1 (citrulline to NO2-/NO3-). Product formation was greater in the presence of oxygen. Both the Km and Vmax of the reaction, determined under aerobic conditions, were affected by (6R)-tetrahydro-L-biopterin (H4B). Chemiluminescence experiments failed to detect nitric oxide (.NO) as a reaction product. However, spectral spectral experiments with L-NHA and H2O2 under anaerobic conditions demonstrated the appearance of a ferrous heme-.NO complex with a Soret peak at 440 nm and a broad single alpha/beta peak at 578 nm, which is believed to arise from single electron transfer of a ferric-NO- (nitroxyl) complex. Preliminary experiments detected nitrous oxide (N2O) formation by gas chromatography under anaerobic conditions. Stable isotope labeling experiments with [18O]H2O2 conclusively established incorporation of label exclusively into the ureido position of citrulline. Based on these results, a mechanism of oxidation of L-NHA by H2O2 is proposed. PMID- 7531496 TI - Chemical cross-linking of IgE-receptor complexes in RBL-2H3 cells. AB - Aggregation of the high-affinity receptors for IgE (Fc epsilon RI) on mast cells activates nonreceptor kinases and other enzymes, thereby initiating a complex biochemical cascade. We have employed a chemical cross-linker in order to stabilize the association of Fc epsilon RI with other cellular proteins that are involved in the early events. We reacted the water-soluble, membrane-impermeant chemical cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) with permeabilized rat mucosal mast cells of the RBL line and analyzed immunoprecipitates of the receptor in detergent lysates of cells that had biosynthetically incorporated [35S]cysteine. Gel electrophoresis revealed substantial amounts of nonreceptor components only when the cells had been reacted with DTSSP. Receptors isolated from cells whose receptor-bound IgE had been aggregated with antigen prior to cross-linking yielded a similar pattern of 35S-labeled proteins. However, when the cells had also been exposed to [gamma 32P]ATP, the proteins associated with the cross-linked, aggregated receptors revealed enhanced incorporation of 32P compared to those associated with cross linked, unaggregated receptors. A variety of antibodies were used to try to identify the associated proteins. Of those tested for, two, the src-like kinase p53/56lyn and the delta isoform of protein kinase C, were associated with the cross-linked Fc epsilon RI in amounts much greater than could be accounted for by nonspecific cross-linking. PMID- 7531497 TI - Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney. AB - Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins. PMID- 7531498 TI - Sequence-specific double-strand cleavage of DNA by Fe-bleomycin. 1. The detection of sequence-specific double-strand breaks using hairpin oligonucleotides. AB - A new method is described for the evaluation of sequence-specific double-strand (ds) cleavage of DNA by Fe-bleomycin (BLM). The method uses high-resolution polyacrylamide gel electrophoresis to separate single-strand (ss) and ds-cleavage products derived from hairpin oligonucleotides that have been designed to contain a specific ds-cleavage site. The BLM induced ss/ds-cleavage ratios ranged from approximately 3.3 to approximately 5.8 at 4 degrees C, with the most efficient ds cleavage involving the thymidines of a 5'-GTAC/5'-GTAC site. Double-strand cleavage was not detected at several sites that were shown to yield significant ss-breaks. A study of the ss/ds-cleavage ratio at the 5'-GTAC/5'-GTAC site revealed that the ratio remained constant over a 70-fold range in concentration of Fe-BLM and extent of DNA degradation. The ss/ds-cleavage ratios at three sites studied were not significantly affected by the presence of "inert" Co(III)-BLM. The results are consistent with the proposal of Steighner and Povirk (1990) that a single molecule of Fe-BLM is responsible for ds-cleavage. The use of these hairpin oligonucleotides has greatly facilitated quantitative analysis of the ds cleavage process (Absalon et al., 1995). PMID- 7531499 TI - Sequence-specific double-strand cleavage of DNA by Fe-bleomycin. 2. Mechanism and dynamics. AB - The mechanism of iron-bleomycin-mediated ds-cleavage of DNA has been investigated at specific sites within specific sequences using hairpin oligonucleotides (Absalon et al., 1995) and our recently developed technique for determining sequence-specific isotope effects upon oxidative degradation of DNA (Kozarich et al., 1989; Worth et al., 1993). Isotope effects upon ds-cleavage have been observed when the C-4' hydrogen of either nucleotide involved in the ds-break was substituted with deuterium. The values of the isotope effects determined for ss and ds events occurring at the same site were indistinguishable at four sites examined in detail. The results are consistent with a mechanism of ds-cleavage in which the pathways leading to ss- and ds-cleavage partition from a common intermediate subsequent to abstraction of the C-4' hydrogen from the first nucleotide involved in the cleavage. Deuterium substitution at the primary cleavage site of a ds-break failed to result in an equivalent effect on the amount of cleavage at the secondary cleavage site, suggesting that ds-cleavage may be initiated from either of the nucleotides involved in the ds-cleavage event. A kinetic preference for cleavage initiated at the 1 degree site, however, is probable. The requirement in the ds-cleavage process for O2, in addition to that needed to form "activated BLM", has been clearly demonstrated by the absence of ds-cleavage products in reactions performed under anaerobic conditions in which ss-cleavage still occurs. These results support, in part, the basic model for ds-cleavage proposed by Steighner and Povirk [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8350-8354], in which a single molecule of BLM effects ds-cleavage and requires reactivation to effect cleavage at the second strand. The essential factor establishing the ratio of ss- to ds-cleavage at a specific site may be related to the efficiency by which Fe-BLM can be reactivated and/or repositioned at a second site for cleavage. PMID- 7531500 TI - Steel factor and c-kit protooncogene: genetic lessons in signal transduction. AB - Despite extensive research on the molecular mechanisms of signal transduction by growth factors and their oncogenic receptor tyrosine kinases, the physiological relevance of these pathways, especially in mammals, remains largely unknown. A unique exception is the Steel factor (SLF) and its c-kit-encoded receptor, because many natural germ line mutations of both the ligand and the receptor exist in mice. The protooncogene c-kit encodes a cell surface receptor that belongs to the immunoglobulin gene family and carries an intrinsic tyrosine kinase activity in its cytoplasmic portion. The precursor of the Kit ligand, SLF, is also a transmembrane protein that exists as a soluble factor as well as a cell surface protein. The interaction of Kit with SLF leads to receptor dimerization, kinase activation, and tyrosine phosphorylation of cytoplasmic proteins that contain Src homology 2 motifs. Various mutations in Kit and SLF result in a defective signaling pathway and underly the complex phenotypes of W and Sl mice, respectively. The early development of at least four cell lineages is affected. These are erythrocytes, melanocytes, germ cells, and mast cells. Correlation between the behavior of these lineages and specific mutations uncovered interesting physiological aspects of the mechanism of signal transduction by a polypeptide growth factor. These include the different degrees of severity of affected lineages, indications for distinct functions during early embryonic development and at late phases, the significance of synergy between a growth factor and lymphokines, the interaction between mutant and wild-type proteins in heterozygous animals, and the possibility that a surface-anchored ligand may act differently than a soluble factor. Predictably, the lessons learned with Kit and Sl mice will be widely relevant to other pairs of ligands and receptors that control the function of different cell lineages and physiological processes. PMID- 7531501 TI - The CD44 adhesion molecule and metastasis. AB - The family of related cell surface adhesion molecules designated CD44 are multifunctional proteins displayed by a wide variety of normal and malignant tissues. CD44 properties in vitro include hyaluronate-mediated adhesion, motility, hyaluronate degradation, self-aggregation, and adhesion to lymphoid tissue. These properties are among several that are required by invasive and metastatic tumor cells. Several in vivo experiments indicate that tumor cells transfected to overexpress specific CD44 isoforms display an enhancement in metastatic potential. Numerous CD44 isoforms exist as a result of alternative splicing, and specific isoforms enhance tumor cell metastasis more efficiently than others. This suggests that regulation of CD44 alternative splicing is an important determinant of metastatic potential. Several human tumors display specific alterations in CD44 isoform expression, and the extent of clinical disease in specific tumors correlates with the CD44 isoform expression pattern. In this review we analyze these data that suggest that CD44 plays an important role in tumor metastasis. PMID- 7531502 TI - Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer. AB - CD4 is the major cellular receptor for the human immunodeficiency virus (HIV). A hybrid gene encoding the extracellular domains of CD4, linked to the sequence encoding the membrane attachment region of the glycosylphosphatidylinositol (GPI) anchored protein decay accelerating factor (DAF) was stably transfected into HeLa cells. The resultant cell line (T4HD) expressed GPI-anchored CD4DAF at high levels and was susceptible to gene transfer with a recombinant HIV vector. In an effort to expand the spectrum of cells susceptible to HIV gene transfer, CD4DAF was released from the surface of the T4HD cell line by detergent lysis, purified by immunoaffinity chromatography, and reincorporated into native HeLa cells. Incorporation occurred via the GPI anchor as evidenced by cleavage with phosphatidylinositol-specific phospholipase C. More than 95% of the CD4DAF treated HeLa cells were CD4-positive by flow cytometry, and kinetic analysis demonstrated that over 75% of the fusion protein remained anchored to the cell membrane after 90 min at 37 degrees C. The purified protein retained its ability to bind the envelope protein of HIV. When incorporated, it bound fluorescein isothiocyanate (FITC)-conjugated gp120, and in its soluble form blocked transduction of CD4-positive cells incubated with an HIV-derived vector containing the Neo gene. In contrast to the T4HD cells, exposure of CD4DAF treated cells to the Neo HIV vector yielded only transient neomycin-resistant colonies. These results suggest that endogenous synthesis of the CD4 molecule may be necessary for successful HIV genomic integration. PMID- 7531503 TI - Gene therapy for cystic fibrosis using cationic liposome mediated gene transfer: a phase I trial of safety and efficacy in the nasal airway. PMID- 7531504 TI - Retroviral-mediated gene transfer of CD34-enriched bone marrow and peripheral blood cells during autologous stem cell transplantation for multiple myeloma. PMID- 7531505 TI - Triiodothyronine inhibits proliferation and stimulates differentiation of cultured neonatal Sertoli cells: possible mechanism for increased adult testis weight and sperm production induced by neonatal goitrogen treatment. AB - Transient neonatal hypothyroidism in the rat causes prolonged Sertoli cell proliferation, delayed Sertoli cell maturation, and increased adult Sertoli cell number, testis weight, and sperm production. Conversely, neonatal hyperthyroidism decreases Sertoli cell proliferation and ultimate testis size. This suggests that thyroid hormones might normally directly inhibit Sertoli cell proliferation while promoting maturation. However, these Sertoli cell effects could be due to secondary hormonal or metabolic effects of hypo- or hyperthyroidism. In this study, we directly tested thyroid hormone effects on Sertoli cell proliferation and differentiation in vitro. Sertoli cells from 5-day-old rat testes were grown in serum-free medium alone (controls) or with additional triiodothyronine (T3; 1 200 nM) and/or FSH (1 microgram/ml). After 4 days, cultures were used to obtain RNA for Northern hybridization or for thymidine autoradiography. Labeling index (LI) for control cultures and cultures receiving 100 nM T3 alone was 5.2 +/- 0.5% and 5.0 +/- 0.4%, respectively. The LI of FSH-treated cultures increased to 8.4 +/- 0.8% (p < 0.01 vs. control). Cultures treated with FSH and 1, 10, 100, or 200 nM T3 had LIs of 8.0 +/- 0.9%, 6.1 +/- 0.4%, 5.3 +/- 0.6%, and 4.8 +/- 0.6%, respectively; the last three values were less than for cells receiving FSH alone (p < 0.01) or FSH + 1 nM T3 (p < 0.05). Northern hybridization indicated that mRNA levels for clusterin and inhibin-beta B, Sertoli cell secretory proteins whose production normally increases during postnatal differentiation in vivo, were significantly increased by T3 or FSH alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531506 TI - Role of methylation in placental major histocompatibility complex antigen expression and fetal loss. AB - The purpose of this study was to determine the role of gene methylation on major histocompatibility complex (MHC) antigen expression in the rat placenta, specifically: 1) the constitutive suppression of labyrinthine class I genes and of all class II genes, 2) genomic imprinting of class I genes, and 3) the amount of fetal loss in relationship to gene methylation. Placentas from all four mating combinations, or a subset thereof, of the inbred DA and WF strains of rats were studied simultaneously through 1) molecular assessment of their levels of methylation at various stages of pregnancy and changes in methylation after treatment with 5-azacytidine and 2) immunohistochemical determination of their MHC class I and class II antigen expression. Treatment with 5-azacytidine caused demethylation of both class I and class II genes, the level depending upon the method of administration of the drug. Treatment with 5-azacytidine did not, however, alter the expression of either the class I or class II antigens. Hence, demethylation is not involved in the constitutive suppression of labyrinthine class I genes or that of all placental class II genes, and it is not involved in the genomic imprinting of placental class I genes. The effect of 5-azacytidine on fetal loss is due to its direct cellular effects and not to an immunological mechanism. PMID- 7531507 TI - In situ localization of apoptosis in the rat ovary during follicular atresia. AB - Apoptosis is a type of physiologic cell death that occurs in many tissues and be regulated by peptide growth factors. Recent studies indicate that apoptosis occurs in the ovary during follicular atresia in several animal species, including the rat, pig, chicken, baboon, and rabbit. The purpose of this study was to demonstrate, through in situ identification of apoptotic cells in intact ovarian sections, the sites in which apoptosis occurs in the rat ovary in different functional states. We evaluated the presence of apoptosis in three models: immature rats, eCG-treated rats and adult cycling rats. Paraffin ovarian sections were pretreated with proteinase K and then end-labeled with biotinylated deoxyuridine triphosphate (dUTP) by incubation with the enzyme terminal deoxynucleotidyl transferase (TDT). They were then stained through use of avidin conjugated peroxidase with 3,3'-diaminobenzidine as the substrate. Healthy antral and preantral follicles had no staining. The nuclei of granulosa cells of preantral and antral atretic follicles were positively stained in all the animal groups. Scattered theca cells were also stained. Stromal cells were consistently negative. Positive controls were sections pretreated with DNase I; these displayed intense staining of all nuclei. Negative controls, in which either terminal TDT or its biotinylated substrate was omitted, were appropriately negative. This study represents a systematic analysis of apoptosis in the rat ovary at different functional stages and supports the hypothesis that apoptosis is involved in the process of follicular atresia. PMID- 7531508 TI - Maintenance of meiotic arrest by a phosphorylated p34cdc2 is independent of cyclic adenosine 3',5'-monophosphate. AB - The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition and is associated with a drop in intraoocyte concentrations of cAMP. Recent studies imply that tyrosine dephosphorylation of p34cdc2 is a prerequisite for entry into the M-phase of the cell cycle. Our study was designed to test the involvement of protein tyrosine phosphatase (PTPase)-regulated dephosphorylation of p34cdc2 in resumption of meiosis in rat oocytes and to explore the possible control of this event by the intraoocyte concentrations of cAMP. Isolated rat oocytes undergoing meiotic maturation spontaneously in vitro served as our experimental model. We found that sodium metavanadate, an inhibitor of PTPase, reversibly blocked the spontaneous maturation in vitro of rat oocytes (ED50 = 0.26 mM). We further demonstrated that the vanadate-sensitive event is completed by 2 h after reinitiation of meiosis. Immunoblot analysis using specific antiphosphotyrosine antibodies revealed that vanadate caused accumulation of phosphotyrosine on a 34-kDa protein, also recognized by anti-p34cdc2 antibodies. The phosphorylated form of p34cdc2 was also detected in oocytes arrested in the G2 phase by the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX). Intraoocyte concentrations of cAMP in vanadate-inhibited oocytes were similar to those in oocytes that resumed meiosis spontaneously in vitro and lower than those in oocytes maintained in meiotic arrest by IBMX (0.73 +/- 0.08, 0.84 +/- 0.09, and 1.42 +/- 0.3 fmol/oocyte, respectively) [corrected]. We conclude that a PTPase that regulates the phosphorylation state of p34cdc2 participates in the control of meiosis in rat oocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531510 TI - Parallel nucleic acid helices with Hoogsteen base pairing: symmetry and structure. AB - Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2' endo region and the phosphodiester conformations are (trans, gauche-), and the RNA helices have sugars in the C3'-endo region and the phosphodiester conformations are (gauche-, gauche-). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the bases. The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. PMID- 7531509 TI - Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle. AB - The relationship between ovarian follicular steroidogenesis and insulin-like growth factor binding protein (IGFBP) activity was evaluated during the follicular phase of the bovine estrous cycle. In experiment 1, follicles were collected from cyclic cows (n = 11) slaughtered at 48 h after administration of prostaglandin F2 alpha (PGF; 35 mg i.m.). In experiment 2, cows were injected twice daily with saline (control) or FSH (FSH cows; total dosage = 42 mg) from Day 2 to Day 6 (estrus = Day 0) and with PGF (35 mg i.m.) on Day 7; follicles were collected from control cows (n = 20) slaughtered at 0, 24, 48, or 72 h and from FSH cows (n = 8) at 0 and 48 h after PGF. Follicular fluid was assayed for estradiol (E2), androstenedione (A4), progesterone (P4), and insulin-like growth factor-I (IGF-I) by RIA and for IGFBP activity by ligand blotting and densitometry. Intensities of the 34-kDa (IGFBP-2), 29-27-kDa, and 22-kDa IGFBP bands in follicular fluid were nondetectable or were lower (p < 0.01) in the fluid of large (> or = 8 mm) E-active (E-A; E2 > 50 ng/ml and > P4) follicles than in large E-inactive (E-I), medium (5-7 mm), or small (< 5 mm) follicles. IGFBP-3 (44-40-kDa doublet) was unaffected by follicle stage in experiment 1, but IGFBP-3 was lower (p < 0.01) in follicular fluid of E-A vs. E-I large follicles in experiment 2. Profiles of IGFBP activity were similar in follicular fluid of small, medium, and E-I large follicles. In experiment 2, E2 concentrations in large E-A follicles increased (p < 0.01) from 0 to 48 h after the PGF injection for control cows but decreased (p < 0.01) for FSH cows, whereas follicular fluid IGFBP-2 binding activity decreased from 0 to 48 h after PGF in controls and increased in FSH cows (treatment x time, p < 0.05). IGFBP-3 binding was unaffected by FSH treatment or time after administration of PGF. Profiles of IGFBP activity in homogenates of granulosa or theca cells were similar to follicular fluid profiles except for the absence of IGFBP-3 binding activity. The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBPs in follicular maturation and on aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531511 TI - Nitric oxide in essential and renal hypertension. AB - L-Arginine-derived nitric oxide (NO) maintains the systemic and renal vasculature in a state of active vasodilation. Inhibition of NO synthesis increases renal vascular tone, reducing RBF and GFR. Similar effects reproduced in other vascular beds result in systemic hypertension. In addition, NO modulates natriuresis by a direct effect on renal tubular function. Abnormalities of the L-arginine:NO pathway occur in experimental hypertension and renal disease and could contribute to alterations in vascular tone; similar abnormalities are seen in essential hypertension in humans. In dialysis-dependent renal failure, the accumulation of endogenous compounds that inhibit NO synthase could exacerbate renal hypertension by inhibiting vascular and renal tubular NO synthesis and might provoke atherogenesis. PMID- 7531513 TI - An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age. AB - Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s). PMID- 7531512 TI - A primary T-cell immunodeficiency associated with defective transmembrane calcium influx. AB - We investigated a T-cell activation deficiency in a 3-month-old boy with protracted diarrhea, serious cytomegalovirus pneumonia, and a family history (in a brother) of cytomegalovirus infection and toxoplasmosis. In spite of detection of normal number of peripheral lymphocytes, T cells did not proliferate after activation by anti-CD3 and anti-CD2 antibodies, although proliferation induced by antigens was detectable. We sought to determine the origin of this defect as it potentially represented a valuable tool to analyze T-cell physiology. T-cell activation by anti-CD3 antibody or phytohemagglutinin (PHA) led to reduced interleukin-2 (IL-2) production and abnormal nuclear factor-activated T cell (NF AT; a complex regulating the IL-2 gene transcription) binding activity to a specific oligonucleotide. T-cell proliferation was restored by IL-2. Early events of T-cell activation, such as anti-CD3 antibody-induced cellular protein tyrosine phosphorylation, p59fyn and p56lck kinase activities, and phosphoinositide turnover, were found to be normal. In contrast, anti-CD3 antibody-induced Ca2+ flux was grossly abnormal. Release from endoplasmic reticulum stores was detectable as tested in the presence of anti-CD3 antibody or thapsigargin after cell membrane depolarization in a K+ rich medium, whereas extracellular entry of Ca2+ was defective. The latter abnormality was not secondary to defective K+ channel function, which was found to be normal. A similar defect was found in other hematopoietic cell lineages and in fibroblasts as evaluated by both cytometry and digital video imaging experiments at a single-cell level. This primary T-cell immunodeficiency appears, thus, to be due to defective Ca2+ entry through the plasma membrane. The same abnormality did not alter B-cell proliferation, platelet function, and polymorphonuclear neutrophil (PMN) function. Elucidation of the mechanism underlying this defect would help to understand the physiology of Ca2+ mobilization in T cells. PMID- 7531514 TI - Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria. AB - Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. PIG-A, an X-linked gene that participates in the first step of GPI anchor synthesis, is responsible for PNH. Abnormalities of the PIG-A gene have been demonstrated in all patients with PNH that have been studied to date. In this study, we analyzed 14 Japanese patients with PNH and identified 15 somatic mutations of PIG-A. The mutations included eight single-base changes and seven frame shift mutations. The single-base changes were two nonsense, three missense, and three splice site mutations. The frame shift mutations were four single-base deletions, two single-base insertions, and a replacement of two bases with one. They were all different, except for the same missense mutation being found in two patients. Moreover, these mutations were distributed in various regions of the gene. These results indicated that the mutations occurred at random sites and that there is no mutation hot spot in the PIG-A gene. All the mutations resulted in complete loss of function. Interestingly, the granulocytes in these patients contained variable proportions of mutant cells, suggesting that clonal expansion is not determined solely by mutations but is influenced by another factor(s). PMID- 7531515 TI - A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform. AB - A novel human granulocyte colony-stimulating factor (G-CSF) receptor isoform, designated SD, has been identified in which the distal C-terminal cytoplasmic region, previously shown to be essential for maturation signalling, is substituted by an altered C-terminus. The SD receptor has a high affinity for G CSF and retains the membrane-proximal cytoplasmic region known to be sufficient for proliferative signalling. Nonetheless, the SD isoform lacks the ability to transduce growth signals in murine BAF3 cells and, in contrast to the wild-type G CSF receptor, is scarcely capable of activating JAK2 kinase. Expression of SD receptor was found to be low in normal granulocytes, but was significantly increased in a patient with acute myeloid leukemia (AML). The leukemic cells of this patient harbour a point mutation in the SD splice donor site of the G-CSF receptor gene. These findings provide the first evidence that mutations in the G CSF receptor gene can occur in certain cases of clinical de novo AML. The possible contribution of defective G-CSF receptor signalling to leukemogenesis is discussed. PMID- 7531516 TI - FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver. AB - The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL-cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors. PMID- 7531518 TI - Sequential order of T and B cell epitopes affects immunogenicity but not antibody recognition of the B cell epitope. AB - Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known murine T cell epitope, both in T-B and B-T orientations, show that induction of high murine anti-MUC1 antibody titers is dependent on the presence and orientation of the T cell determinant. However, the sequential order of the epitopes does not affect binding of anti-B cell epitope antibodies to the constructs. Haplotype mismatching leads to a significant lowering of the anti MUC1 antibody responses, implicating a central role for the T cell epitope in eliciting anti-B cell epitope responses. Secondary structure analysis by circular dichroism spectroscopy reveals the T-B construct to be partially ordered, while the B-T peptide adopts a highly ordered conformation in trifluoroethanol. These studies suggest that the sequential order of epitopes may significantly alter the immunogenicity of the peptide but may not necessarily affect its antigenicity. Immunogenicity of the peptide constructs may be governed by subtle differences in secondary structure, leading to variation in the way peptides are presented or processed within cells governing immune responses. These findings have relevance for the construction of peptides to be utilized as potential synthetic vaccines and for the design of peptide immunogens. PMID- 7531519 TI - [Effect of arginine-dependent nitric oxide synthesis on regional perfusion of the eye in the anesthesized dog]. AB - The changes of the retinal and uveal perfusion after inhibition of the arginine dependent nitric oxide (NO) synthesis by systemic administration of NG-nitro-L arginine methyl ester (L-NAME; 20 mg/kg bw i.v.) were studied in four anesthetized dogs using the tracer microsphere technique. The regional perfusion rates (ml.min-1.g-1) under steady-state control conditions were: retina 0.13 +/- 0.05, choroid 8.26 +/- 1.85, iris 0.24 +/- 0.05 and ciliary body 1.11 +/- 0.26. Infusion of L-NAME over 10 min reduced the perfusion of the retina on the average by 23% (P > 0.05). The perfusion of choroid, iris and ciliary body fell by 54 +/- 8%, 58 +/- 7% and 53 +/- 8%, respectively (P < or = 0.05 for all). In five additional experiments the local activity of NO-producing enzymes (NO synthases) was determined by measuring the production rate of citrullin in tissue extracts of the different eye regions. Total NO synthase activities (pmol citrullin.min 1.g-1) were: retina 31.0 +/- 5.5, choroid 3.1 +/- 2.8, iris 7.1 +/- 2.1 and ciliary body 1.3 +/- 1.3. Differences of the total NO synthase activities of retina, iris and cilary body were statistically significant (P < or = 0.02). The results show that the uvea perfusion is largely influenced by the steady-state production of NO. The homogeneous flow reduction in the uvea after inhibition of NO synthase is contrasted by the heterogeneous NO synthase activities of the different uvea regions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531520 TI - The amiloride receptor. PMID- 7531521 TI - Water transport in renal tubules is mediated by aquaporins. AB - Water reabsorption in mammalian renal tubules is mediated by channel-forming membrane glycoproteins termed aquaporins (AQP). So far three different kinds of AQP have been described in renal tubules. AQP CHIP is localized to the luminal and contraluminal membranes of the proximal tubule and descending thin limb cells, i.e., in tubule segments that exhibit a constitutive high permeability to water that is insensitive to vasopressin. AQP-CD is present in subapical vesicles and the luminal membrane of collecting duct principal cells. Its intracellular distribution depends on vasopressin or hydration status of the animal and, thus, may represent the vasopressin-sensitive water channel. The basolateral integral protein (BLIP) may represent the vasopressin-insensitive water channel in basolateral membrane of collecting duct principal cells. The exact localization of a recently cloned homologue, WCH3, which may be either related to BLIP or represent yet another kind of AQP, is not known. Heterogeneity of aquaporins in the renal tubule may provide a molecular basis for the treatment of certain diseases with disturbances in water homeostasis. PMID- 7531517 TI - Aprotinin for prophylaxis of blood loss. PMID- 7531522 TI - [Diagnosis of the breast tumor entity with "vocal fremitus" in ultrasound diagnosis]. AB - In the published study a totally new method for the diagnosis of malign and benign tumors of the female breast is presented. During sonographic examinations with the maximum entropy method we found out, purely by chance, that artifacts produced by vibrations of the thorax can be used to differentiate malignant from benign lesions. These artifacts in form of small colour pixels can be provoked by the vocal fremitus of the internal medicine or by humming a deep sound. In case of malignancy, the colour artifacts are visible inside of the tumor, in benign lesions, however, only in the surrounding tissue. On the preoperative day, 95 patients were examined with the method described above. In 91% of the cases the diagnosis of the dignity corresponded with the histological results. Seven times a benign lesion was classified as a malignant tumor and twice malignancy was not correctly diagnosed. Our explanation of these phenomena is the following: Benign lesions of the female breast show a displacing growth and a definable boundary. Thus the vibrations are damped and not conducted into the tumor. In contrast, malignant lesions grow invasively into the surrounding tissue. Consequently, the vibrations can be conducted into the center of the tumor by this dissemination. PMID- 7531523 TI - The epidermis: rising to the surface. AB - At the skin surface, the epidermis serves an important protective function which it manifests by building an extensive cytoskeletal architecture of keratin filaments, spanning from the nuclear envelope to hemidesmosomes and desmosomes. Recent studies on epidermal proteins and their interactions have provided insights into human skin diseases, including genetic disorders of keratins, laminins, and collagen. Explorations into the regulatory mechanisms underlying epidermal genes have underscored the importance of transcription factors AP-1 and AP-2, retinoic acid receptors, and POU proteins. Transgenic and gene ablation experiments on TGF-alpha and TGF-beta genes have yielded clues as to how the epidermis maintains a balance of growing and differentiating cells. PMID- 7531524 TI - Neurotrophin signalling. AB - The neurotrophins act through their signalling competent trk tyrosine kinase receptors (trkA, trkB and trkC), and, in addition, they share a common low affinity receptor, p75. Acting alone, trk kinases can mediate neurotrophin action, including survival, fiber outgrowth, differentiation and proliferation. The p75 receptor modulates trk activity and also couples to an independent signalling mechanism involving the sphingomyelin cycle. The elucidation of pathways that couple trk receptor activation to fiber outgrowth and gene expression has made good progress. New work on signalling in postmitotic neurons is beginning to reveal that similarities and differences in these pathways exist, which depend on the neuronal type or the developmental stage. PMID- 7531525 TI - Neural recognition molecules in disease and regeneration. AB - The genetic analysis of inherited human diseases of the nervous system and the characterization of transgenic mice deficient in neural recognition molecules is opening up a new dimension in understanding the cellular and molecular mechanisms underlying neuro-developmental and -degenerative diseases, as well as in delineating the functions of recognition molecules in cell-cell interactions. Progress in identifying recognition molecules that inhibit neurite outgrowth and further characterization of the mechanisms that promote neurite outgrowth are shedding more light on the processes of regeneration in the mature nervous system. In the adult, recognition functions are fine-tuned by glycan moeities associated with neural recognition molecules, and successful neurite outgrowth is likely to depend on the delicate balance between growth-promoting and inhibitory recognition cues. PMID- 7531527 TI - Characterization and comparison of the polyclonal immune response to purified PLP and DM-20 in the Lewis rat: determination of a new antigenic determinant. AB - Lewis rat polyclonal antibodies raised against pure PLP and DM-20 were characterized by indirect and competitive ELISA assays. Anti-PLP antibodies showed a good cross-reactivity with DM-20 and vice versa, suggesting that the chief B-cell epitopes are the same on both proteolipids. Three antigenic determinants, PLP 57-70, PLP 200-218 and the carboxyl-terminal sequence PLP 262 276, were identified for both proteolipids. Peptide PLP 57-70 represents a new B cell epitope. The major hydrophilic loop (PLP 116-150), deleted in DM-20, did not show any antigenic activity. The study of polyclonal immune response to PLP and DM-20 will be helpful in resolving their potential role in the development and course of experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS). PMID- 7531526 TI - Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis. AB - N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM 4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15 triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+) groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities. PMID- 7531528 TI - Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein. AB - Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage dependent channel activity on membranes of large area. In this respect, melittin TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation. PMID- 7531530 TI - The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels. AB - Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4 acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels. PMID- 7531529 TI - Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor. AB - In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains. PMID- 7531531 TI - Compartmentalization of secretory proteins in pancreatic zymogen granules as revealed by immunolabeling on cryo-fixed and molecular distillation processed tissue. AB - Immunogold labeling of amylase obtained over zymogen granules of rat pancreatic acinar cells processed through cryofixation, molecular distillation drying and embedding in resins was found to be of high intensity and displayed a particular pattern. Indeed, it was concentrated in certain areas of the granules leaving others devoid of gold particles. This pattern of labeling reflects a strong compartmentalization of the secretory proteins within each granule. In order to assess this phenomenon, we have compared the intensities and the pattern of distribution of the labelings in tissues processed through: chemical fixation with embedding in various resins, cryo-ultramicrotomy and cryo-fixation followed by molecular distillation drying. Serials sections and double labeling experiments were performed for further evaluation of the results and for assessing artefactual displacement of proteins during tissue preparation. The results obtained indicate that the secretory proteins are indeed segregated within the granule which appears thus as a well organized structure. Cryo fixation combined with molecular distillation appears thus to be superior in terms of preservation of protein antigenicity and retention of cellular components close to their living state. PMID- 7531532 TI - Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium. AB - Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins. The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences. PMID- 7531533 TI - Structure-antigenicity relationship of peptides from the pre-S2 region of the hepatitis B virus surface antigen. AB - Peptide antigenicity against the pre-S2 region of the hepatitis B virus surface antigen was studied using a pre-S2 specific anti-hepatitis B virus mouse monoclonal antibody (H8 mAb) and synthetic peptides by competitive ELISA. The mAb showed preferences for long peptides with the sequence 120/123-145, though the mAb binding region was located in the sequence 130-145 from the analysis of a conjugation study. The N-terminal residues 120/123-129 play an important role for the maintenance of the highly antigenic structure of the B cell epitope. Among these, the N-terminal hydrophilic residues 124-126 and hydrophobic residue 127 were important, whereas residues 120-122 did not affect antigenicity. Residues 131 and 141 appeared to be critical for the mAb binding. The relationship between peptide structure and antigenicity was also investigated by probing the secondary structure of the peptides by circular dichroism. Highly antigenic peptides elicited more ordered structure in 20% trifluoroethanol than less antigenic peptides. The results suggested that peptide antigenicities against H8 mAb are closely related to the B-cell epitope conformations of peptides. PMID- 7531535 TI - Catalytic activity of refolded penicillin acylase subunits in aqueous solution and aerosol ot reversed micelles in octane. AB - Refolded alpha and beta subunits of penicillin acylase were produced by gel filtration under denaturing conditions followed by removal of urea through dialysis. Preparations of both renatured subunits hidrolysed specific substrate- phenylacetic acid p-nitroanilide in buffer and in the system of Aerosol OT reversed micelles, the alpha subunit being most catalytically active at Wo = 11.9, while the beta subunit--at Wo = 17.5. There was a good correlation between the position of the found maxima, the theoretically calculated optimal hydration degrees as well as the earlier reported profile of enzymatic activity for native enzyme in reversed micelles. PMID- 7531534 TI - Pressure effects on enzyme reactions in mainly organic media: alpha-chymotrypsin in reversed micelles of Aerosol OT in octane. AB - Biocatalytic transformations in reversed micelles formed by anionic surfactant Aerosol OT in octane have been studied at high pressures by an example of alpha chymotrypsin-catalyzed hydrolysis of N-carbobenzoxy-L-tyrosine p-nitrophenyl ester and N-succinyl-L-phenylalanine p-nitroanilide. For the first time it has been found that the enzyme retains high activity in these water-in-oil microemulsions up to a pressure of 2 kbar. The value of the activation volume (delta V*) for the enzyme reactions shows a dependence on the water content in the system. When the size of the micellar aqueous inner cavity (as evaluated at 1 atm) approaches the molecular size of alpha-chymotrypsin, delta V* becomes significantly different from the value in aqueous solution and in the micelles with a larger size. Possibilities of regulating the enzyme activity by pressure in systems with a low content of water are discussed. PMID- 7531536 TI - A crosslinked tetrameric alpha 2M that binds but incompletely entraps trypsin. AB - A crosslinked preparation of alpha-2-macroglobulin was obtained by treatment of the purified human plasma inhibitor with glutaraldehyde at low temperature. The preparation migrated as a 780 KDa polypeptide in SDS-PAGE and with mobility comparable with that of trypsinized native alpha-2-macroglobulin under nondenaturing conditions. Trypsinization of the glutaraldehyde treated alpha-2 macroglobulin further increased its electrophoretic mobility in non-denaturing gels and resulted in the association of the proteinase with the crosslinked inhibitor. Trypsin associated with crosslinked alpha-2-macroglobulin, unlike that associated with native inhibitor was incompletely protected from soybean trypsin inhibition. PMID- 7531537 TI - Temperature dependent influence of As2O3, HgHPO4 and KCl on lysosomal acid phosphatase isolated from rat liver. AB - Studies were carried out to investigate acid phosphatase activity in the presence of As2O3, HgHPO4 and KCl at 25 degrees C and 37 degrees C. In all cases examined enzyme activities measured at 25 degrees differ from those detected at 37 degrees C. When activity was measured in the presence of As2O3 at 25 degrees C a stimulation was found while at 37 degrees C activities remained within the control range. Similar results were obtained, when As2O3 was replaced by HgHPO4. In contrast to that, added amounts of KCl cause an increase of activity at both incubation temperatures, but the increment being greater at 37 degrees C. Furthermore in most cases correlation between increasing amounts of substances added and enzyme activity measured was non-linear. PMID- 7531538 TI - Molecular cloning of mouse insulin-like growth factor binding protein 4 (IGFBP4) cDNA and expression of a fusion protein with IGF-binding activity. AB - The TC-1 bone marrow stromal cell line expresses a 2.3 kb IGFBP-4 mRNA transcript. Reverse transcription/polymerase chain reaction was used to amplify the complete open reading frame of the insulin-like growth factor binding protein 4 (IGFBP-4) from poly(A)+ of a murine bone marrow stromal cell line (TC-1). Sequence analysis reveals that the murine IGFBP-4 is highly homologous to the rat IGFBP-4 and less so to the human IGFBP-4. The inferred amino acid sequence has a molecular weight of 25.7 kD. An IGFBP-4/maltose binding protein fusion peptide expression in the pMal-p2 vector produced a fusion protein exhibiting both IGFBP immunoreactivity and IGF-I binding activity with specificity characteristic of IGFBPs. PMID- 7531539 TI - The gene for diffuse palmoplantar keratoderma of the type found in northern Sweden is localized to chromosome 12q11-q13. AB - Hereditary palmoplantar keratoderma is characterized by hyperkeratosis of the skin of palms and soles. An autosomal dominant form of diffuse non-epidermolytic palmoplantar keratoderma, frequently complicated by fungal infections, is encountered in northern Sweden with a prevalence of 0.3-0.55%. We have examined two families with this type of palmoplantar keratoderma and localized the causative genetic defect to a 14 cM interval on chromosome 12q11-q13, a region known to contain the keratin type II gene cluster as well as the retinoic acid receptor gamma gene. The palmoplantar keratoderma variant investigated in this study is thus genetically different from epidermolytic palmoplantar keratoderma, which recently has been shown to result from mutations in the gene for the type I keratin 9. PMID- 7531541 TI - A novel nonsense mutation identified in the first nucleotide binding fold of the CFTR gene in a Greek patient. PMID- 7531542 TI - [The regulation of gastrointestinal motility: the neuromediator and hormonal functions of serotonin]. PMID- 7531540 TI - Fabry disease: twenty-three mutations including sense and antisense CpG alterations and identification of a deletional hot-spot in the alpha galactosidase A gene. AB - Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A gene at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes, and for precise carrier detection in Fabry families, the alpha-galactosidase A coding and flanking intronic sequences from 23 unrelated Fabry hemizygotes were analyzed. In patients with the classic phenotype, 16 new missense and nonsense mutations and four small exonic gene rearrangements were identified: C52S, C56F, E59K, L89R, R100K, R112H, L131P, A143P, G144V, C172Y, D244N, N272K, A288D, W81X, Q99X, Q157X, R301X, 25del1, 333del18, 358del6, and 1020del1. The R112H mutation at a CpG dinucleotide resulted in residual activity and a mild variant phenotype while the R112C lesion caused the classic disease manifestations, defining a genotype/phenotype correlation for sense and antisense mutations at the same CpG dinucleotide. In addition, two complex rearrangements, each involving two mutational events, occurred in classic hemizygotes. Both rearrangements resulted in missense mutations that did not change the reading frame. Notably, three of the deletions occurred within 11 codons in exon 2, thereby defining a 'hot-spot' for deletions. These studies revealed that most mutations in the alpha-galactosidase A gene causing Fabry disease were private, that codons 111-122 defined a deletion hot spot, and that different substitutions of the same codon resulted in markedly different disease phenotypes. PMID- 7531543 TI - [The 6th School on the Physiology of the Kidney and Water-Salt Metabolism]. PMID- 7531544 TI - [The creation of a Russian section of the International Academy of Sciences and of the International Institute of Social Physiology]. PMID- 7531545 TI - [The intrahemispheric contrast in transcallosal responses and its interhemispheric asymmetry in animals of both sexes]. AB - A certain dynamics of changes in intrahemisphere contrast was revealed in the process of development of the transcallosal response: in the beginning of the response the right hemisphere is more focal, then the left one and afterwards again the right hemisphere. In males, the main changes of focality are related to the left hemisphere whereas in females--to the right one. The focality of a greater value is characteristic of the males' transcallosal responses whereas the distribution of the EPs is more diffuse in females. A specifics of the contrast gradient dynamics is described for the projection and associative cortical areas. A zonal specifics of the intrahemisphere contrast was shown in animals of different sex. PMID- 7531546 TI - [The effect of plaferon on disorders in the rabbit brain induced by circulatory hypoxia]. AB - The effect of plaferon on the local cerebral blood flow and brain tissue oxygen tension during transient incomplete ischemia and following recirculation, was studied. The difference revealed between experimental and control data is, probably, due to vasodilatory action of the vasoactive neuropeptides and the effect on the systems of phosphorylation. PMID- 7531547 TI - [The effect of heparin on the permeability of the skin vessels of rats in hypobaric hypoxia]. PMID- 7531548 TI - [Respiration and blood circulation in the terminal stages of deep hypothermia]. AB - In immersion hypothermia (9 degrees C) the brain temperature was 2 degrees higher on the average than in the heart area and 3-4 degrees C higher than rectal temperature in rats. At the brain temperature below 24-25 degrees C a strong dependence of the respiration rate on the body temperature occurs followed by the same dependence for the heart rate and arterial pressure. Arrest of the respiration occurs at the brain temperature 18 degrees C on the average, heart arrest occurs within 31 min on the average (at the heart temperature 11 degrees C). The main hypothermic pathogenetic factor is a direct effect of the cold on the brain and heart added by hypoxia due to weakening and arrest of respiration. PMID- 7531549 TI - [The capacity of humans to regulate the artificial ventilation of their own lungs]. AB - The subjects were given the possibility to control the capacity of the artificial ventilation apparatus (AVA) at resting and during physical work. The subjects were able to find such a level of the artificial ventilation when he or she could delay natural respiratory movements. The subjects seem to orient themselves to afferents from the chemoreceptors in this task. The subjects could not delay the respiration during physical work, obvious hypocapnia being preserved at that. PMID- 7531551 TI - [The activity of digestive enzymes in the digestive and nondigestive organs during stress exposures]. AB - The effects of fasting and immobilization manifests itself in changes of the enzymes activity in the stomach, duodenum, jejunum, ileum, colon as well as in the liver, spleen, kidneys. In these conditions, the protective function is primarily fulfilled by the enzyme systems of the non-digestive organs whereas the enzymatic intestinal barrier is less obvious. PMID- 7531550 TI - [The respiratory and metabolic functions of the lungs under the conditions of body water immersion and the breathing of pure oxygen]. PMID- 7531552 TI - [Sex dimorphism in the formation and extinction of aversion acquired to known and unknown tastes]. AB - One group of Wistar rats were taught to consume 0.2% solution of saccharin up to stable preference of the latter to water. The aversion was established by means of an angle acceleration rotation. This was preceded by feeding the saccharin solution unfamiliar to the second group of rats. The aversion formed more obviously in the second group animals. The data obtained is discussed from the standpoint of sexual characteristics of the taste preference, defence behaviour, ecological specifics of interaction of males and females with ambient milieu. PMID- 7531553 TI - [The effect of cold adaptation and isopropylnoradrenaline on the value of the body temperature set-point in white rats]. PMID- 7531554 TI - [The firing of the preganglionic fibers of the splanchnic nerve in the temperature exposure of the nucleus magnus raphae]. PMID- 7531556 TI - [The inaugural congress of physiologists of Russia (the history of the congresses of the I. P. Pavlov Physiology Society of the Russian Academy of Sciences)]. PMID- 7531555 TI - [The chemical stability of a cataleptogenic enkephalin-like tetrapeptide in a cannula implanted in the rat striatum]. PMID- 7531557 TI - Protein metabolism in coronary wall affected by cardiac load. AB - The relation between biomechanics of conduit coronary artery and metabolic processes in its wall were studied in cardiac pressure overload, in anaesthetized dogs. Two sorts of forces and/or coronary deformations were followed: (1) radial and length deformation of ramus interventricularis anterior (RIA) due to ventricle volume changes, and (2) shear stress in ramus circumflexus (RC) ensuing the blood flow changes. Increase in aortic pressure (33.9 +/- 2.30%) accompanied an increase in RIA diameter (4.6 +/- 0.38%) and an increase in segment length (8.0 +/- 1.0%) indicating that both circumferential and longitudinal oriented muscle bundles were loaded. Increase in RC blood flow (55.6 +/- 4.0%) and diameter (3.8 +/- 0.3%) resulted in an increase of shear stress (35.15 +/- 11.16%) that affects particularly the endothelial cells. Two hours lasting cardiac load suggested a tendency, 4 h lasting load resulted in a significant increase both in RNA content (11.06 +/- 4.68% and 9.05 +/- 0.72%) and in [14C]leucine incorporation (53.59 +/- 13.75% and 48.41 +/- 16.16%) in the myocardium and RIA wall, respectively. No change was found in RC wall. The process was reversible, 2 h after the load had ceased. The heterogeneity of metabolic processes reflected closely the heterogeneity in coronary deformation. PMID- 7531558 TI - Growth of the coronary vasculature in hypertrophy: mechanisms and model dependence. AB - Coronary microvascular angiogenesis during cardiac enlargement is variable and dependent upon several factors including age, model, and duration of cardiac hypertrophy. We, and others, have documented coronary angiogenesis in several models of cardiac enlargement and in several species. Although growth of the coronary vasculature occurs during hypertension, it usually fails to compensate for the magnitude of hypertrophy. Substantial growth may occur, however, after a long period of time. In some other models of cardiac hypertrophy (excess thyroxine hormone, exercise training, and volume overload) capillary angiogenesis is greater and may occur more rapidly. An examination of a variety of models and interventions (e.g., increased myocardial perfusion, bradycardia) suggests that mechanical factors (stretch, shear stress and wall tension) associated with increases in (A) capillary perfusion, (B) diastolic interval, or (C) diastolic ventricular chamber filling may provide an initiating stimulus for capillary angiogenesis during cardiac enlargement. PMID- 7531559 TI - The effect of long-term bradycardia on heart microvascular supply and performance. AB - Bradycardia has been shown to be beneficial for the normal and ischaemic heart because it improves diastolic perfusion and oxygen supply demand balance. Experimentally, a chronically induced decrease in heart rate, either by electrical pacing or pharmacological means, was found previously to increase myocardial capillary supply in normal rabbit and rat hearts. These studies have been extended to a larger mammal, the pig, in which a direct bradycardia (approximately 30% decrease in heart rate) was induced by electrical pacing for 4 5 weeks. There was no evidence of heart hypertrophy and capillary density was found to be significantly increased in the left, but not right, ventricle. Cardiac function during dobutamine inotropic challenge was better in pig hearts which had been paced bradycardially. They performed greater stroke work-higher stroke flow output at lower heart rate--for similar coronary blood flow, thus demonstrating an improved economy of flow utilisation. Heart rate reduction may facilitate capillary growth in the absence of cardiac hypertrophy by prolonging diastolic perfusion, and/or mechanical stretch of vessels due to increased stroke volume capacity. In either case, capillaries would be exposed to increased wall tension which could trigger angiogenesis. PMID- 7531560 TI - Molecular and cellular basis of myocardial angiogenesis. PMID- 7531562 TI - Developing concise tables. AB - When faced with the dilemma of what tables to include in their manuscripts, some authors mistakenly assume the more tables the better. Others admit they put all the tables in, hoping the editor will pick the ones he or she wants. Many of these authors find the manuscripts rejected. Do not assume the editor or reader will weed through your tables or condense them. Instead, spend at least as much, if not more, time developing your tables as you spend editing the text sections. This author with experience in book, article, and research publications describes four guidelines she uses to develop tables for a successful manuscript. PMID- 7531561 TI - Coordinate TGF-beta receptor gene expression during rat heart development. AB - Transforming growth factor-beta (TGF-beta) has been implicated to participate in heart development. The receptors which transduce the signal(s) mediated by TGF beta ligand binding have only recently been cloned. One of the most prominent effects of TGF-beta is inhibition of cell proliferation, a process that is tightly regulated during heart development. Using the developing rat ventricle as a model system, we have determined the steady state expression patterns for the Type I, II, and III TGF-beta receptors (TGF-beta Rs). Using RNA isolated from ventricular chambers on day 18 of gestation through the ninth postnatal week of age, we detected a modest increase in expression levels for Type I and Type III TGF-beta Rs. In contrast, steady state transcript levels for the Type II TGF-beta R showed a profound developmental increase from nearly undetectable levels at the fetal ages examined to high levels during the first postnatal week of age. Immunoelectron microscopic localization of Type II TGF-beta R confirmed that the 3-week-old ventricular myocyte, as well as nonmyocytes, contained immunoreactive material. Immunoreactivity was found at both the cell surface as well as intracellular compartment. Regional variations (right ventricle, left ventricle, or septum) in the expression pattern of several markers of heart development, but not the TGF-beta R's, were found in RNA obtained from 3-week-old postnatal animals. These data suggest that "downstream" effectors of TGF-beta-mediated stimulation are modulated in a developmental, regional-specific manner in the neonatal/mature myocardium by the level of bioactive TGF-beta. PMID- 7531564 TI - Cholera: current epidemiology. AB - Cholera remains an important cause of morbidity and mortality worldwide. Its epidemiology has changed in the 1990s, with the spread of the seventh pandemic to the western hemisphere and the emergence of a new serogroup, Vibrio cholerae O139. The spread of cholera may be rapid and unpredictable because of aeroplane travel, international shipping, and the migration of people due to war or political unrest. Increasing amounts of largely untreated faeces from growing human populations favour cholera's survival. Most of the world has inadequate sanitation, and future prospects are undermined by the impact of international debt on ailing economies. Furthermore, because cholera is difficult to eradicate from water it is likely to remain a serious threat to public health for some time. Progress is being made in the development of oral vaccines against V. cholerae O1 and serogroup O139. PMID- 7531563 TI - Zinc-containing neurons. AB - Zinc-containing neurons are cells that sequester zinc in their presynaptic vesicles and release it in a calcium- and impulse-dependent manner. The zinc containing neurons are a subclass of the glutaminergic neurons: all known zinc containing pathways are also glutaminergic pathways. With a few exceptions, zinc containing neurons are located only in the telencephalon. The major glutaminergic systems of the brain stem, thalamus, and cerebellum, for example, all lack vesicular zinc, whereas many cerebrocortical systems are zinc containing. Within the telencephalon, the zinc-containing fiber systems form a vast associational network that reciprocally interconnects isocortical, allocortical and 'limbic' structures. Because the hippocampal, amygdalar, and perirhinal regions are prominent nodes in this network, it is presumed that vesicular zinc is involved in epileptic phenomena (in pathology) and (in normal function) in the synaptic plasticity of developmental and experiential learning. Zinc ions are potent modulators of amino acid receptors [especially the N-methyl-D-aspartate (NMDA) receptor] and corelease of zinc along with glutamate would provide a mechanism for modulating postsynaptic excitability levels. One useful hypothesis is that synaptically released zinc controls a 'window' of postsynaptic excitability, having little or no effect at physiological firing rates, but selectively depressing excitability (by NMDA receptor depression) when firing rates reach dangerous, paroxysmal levels. PMID- 7531565 TI - Influenza surveillance, England and Wales: October 1993 to June 1994. AB - This report summarises the information obtained by surveillance of influenza activity in England and Wales from 30 October 1993 to 24 June 1994 (weeks 93/44 to 94/25). A moderate epidemic of influenza occurred, which was earlier than in recent years. It followed an increase in influenza activity reported in Scotland in early October. Activity spread from north to south. Influenza A(H3N2) viruses accounted for over 98% of isolates typed, but a few strains of influenza B were identified. PMID- 7531566 TI - Malaria imported into the United Kingdom in 1992 and 1993. AB - A total of 1,629 cases of malaria were reported in the United Kingdom in 1992, and 1,922 cases in 1993, fewer than the peak of 2,332 reached in 1991. Of the 3,551 cases of malaria reported during 1992 and 1993, 74 were reported from Scotland, 45 from Wales, and 13 from Northern Ireland. Fourteen people died of falciparum malaria in the two years, 12 of whom contracted the disease in Africa and two in India. Preventive measures were inadequately followed by 12 of the 14 patients who died, eight of whom took no prophylaxis at all, and diagnosis was delayed in the other two. Over a third of the malaria cases were immigrants from endemic areas who had settled in the United Kingdom and who travelled to visit friends and relations. This was the commonest reason for travel given by cases, and correlates with a marked rise in falciparum malaria in settled immigrants from West Africa. PMID- 7531568 TI - COVER (cover of vaccination evaluated rapidly): 32. PMID- 7531567 TI - The risk of hepatitis A from sewage contamination of a water supply. AB - The drinking water supply of a town became contaminated with sewage. The town's population was surveyed to determine the baseline prevalence of hepatitis A antibodies and to establish whether an associated outbreak of hepatitis A had occurred. Samples of saliva were obtained from 540 people in 200 randomly selected households, and tested for IgG and IgM antibodies to hepatitis A. Fifty six per cent (279/495) were susceptible to hepatitis A and 43% (213/495) immune as a result of previous infection. Immunity was directly related to age; those who were immune were significantly older than those who were susceptible (mean ages: 43.5 years and 19.0 years; p < 0.0001). Six people were found to have had a recent infection with hepatitis A, but exposure to hepatitis A during the water pollution incident was possible in only one case. The results offer no evidence that this incident caused an outbreak of hepatitis A, but the study has provided useful epidemiological data on hepatitis A. PMID- 7531569 TI - Escherichia coli O 157: outbreak in a day nursery. AB - Seventeen out of 54 preschool children at a day nursery became infected with Escherichia coli O 157. The nursery outbreak formed part of a wider community outbreak, whose source was not identified. Epidemiological investigation suggested that the nursery outbreak resulted from person to person spread. Children with diarrhoea or a positive faecal specimen were excluded until their symptoms had resolved and one negative faecal specimen had been obtained. Children who were asymptomatic throughout were not excluded. This policy appeared to be effective in preventing further spread of infection within the nursery. PMID- 7531570 TI - Response to news that an obstetrician/gynaecologist has AIDS. AB - This paper summarises the response of a district health authority, in March 1993, to the diagnosis of AIDS in a consultant obstetrician/gynaecologist. A helpline was established, facilities to test for HIV antibody were arranged, and patients upon whom the infected doctor had performed surgical procedures in the preceding 10 years were identified from local records. Letters were sent to patients who did not contact the helpline, with advice and the offer of HIV antibody testing. A total of 4594 National Health Service and private patients of the obstetrician/gynaecologist were identified, 1206 of whom (26%) were tested for HIV antibody. A further patient, who had other identified risks for HIV infection, had been known to be positive for anti-HIV since 1992. An additional 475 people who had not been treated by the doctor asked to be tested as a result of the publicity given to the incident. The estimated cost to the health authority of this patient notification exercise was 150,100 pounds. PMID- 7531571 TI - Respiratory infection: "a lot of it about". PMID- 7531572 TI - Granulocyte colony-stimulating factor for neutropenia in neonatal sepsis. PMID- 7531573 TI - An adult with juvenile hyaline fibromatosis of the foot. AB - An unusual consideration in the differential diagnosis of benign, solid tumors of the foot is juvenile hyaline fibromatosis, which only superficially resembles the better known myofibromatosis. The onset of the typical cutaneous lesions of this affliction, more frequently found in the head, trunk, or upper extremity, usually begins in childhood, with a progressive increase in their size and number. No cure is known, but early surgical excision of smaller lesions will minimize bony erosion and skin ulceration that otherwise risks toe amputation, thereby preserving function and a reasonable esthetic result. Incomplete excisions are doomed to recurrence, but, fortunately, this normally is a slowly growing tumor. PMID- 7531574 TI - Production of insulin-like growth factor binding protein (IGFBP), IGFBP-3 protease, and expression of IGF-I receptors by cells of the sheep immune system. AB - Single-cell suspensions of sheep thymus cells were cultured in serum-free medium with or without polyclonal activators (phytohaemagglutinin or concanavalin A) and the resultant conditioned medium was assayed for insulin-like growth factor binding protein (IGFBP) activity by binding of [125I]IGF-I, using charcoal to separate free from bound. All cultures produced IGFBP but mitogen stimulation significantly increased IGFBP concentrations, indicating production by lymphoid cells. Conditioned medium also degraded recombinant human [125I]IGFBP-3, suggesting IGFBP-3 protease production within the thymus. This degradation was inhibited by several protease inhibitors (phenylmethylsulphonyl fluoride, aprotinin, N-alpha-p-tosyl-L-lysine chloromethyl ketone), suggesting the presence of a serine protease. Cell surface [125I]IGF-I binding was demonstrated on cells from thymus, mesenteric lymph node, peripheral blood mononuclear cells and platelets. The [125I]IGF-I binding to platelets could be inhibited by unlabelled peptides, with relative potencies IGF-I > IGF-II >> insulin. Scatchard analysis of IGF-I competitive binding revealed a Kd of 266 pmol/l and approximately 40 receptor sites per cell. The high-affinity binding of IGF-I and competition by insulin suggested that the [125I]IGF-I binding was to an IGF-I receptor rather than to a membrane-associated IGFBP, to which insulin does not bind. These data provide further support for the role of the IGF-IGFBP axis in the immune system, particularly in relation to the thymus. PMID- 7531575 TI - Mapping thyroid peroxidase epitopes using recombinant protein fragments. AB - The identification of autoantibody epitopes is important to the understanding of autoimmune thyroid diseases. In the case of thyroid peroxidase antibodies (TPO ab), recent reports have disagreed about the number and type of autoantibody epitopes found in human TPO. In order to clarify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four slightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2(Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)- were amplified by polymerase chain reaction and subcloned into the expression vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino terminal portion is maltose-binding protein, followed by the sequence of the fragment of human TPO. The plasmid constructs were transformed in Escherichia coli and, after growth, bacterial cells were harvested, lysed and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patients with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radio immunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured by radioimmunoassay (group 3) and sera from 30 patients with antigastric parietal cell antibodies without signs or symptoms of thyroiditis, 16 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531576 TI - Thyroid-stimulating antibodies in sera from patients with Graves' disease are heterogeneous in epitope recognition. AB - Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354 14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354 14 and five others were absorbed by both peptides to an almost similar extent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531577 TI - Continuous versus discontinuous B-cell epitopes on thyroid-specific autoantigens- thyrotropin receptor and thyroid peroxidase. PMID- 7531578 TI - Immunohistochemical localization of inhibin alpha- and beta A-subunits in the ovary of immature female rats. AB - Immunohistochemical localization of inhibin alpha- and beta A-subunits was examined in the ovaries of immature female rats. The granulosa cells in various sized ovarian follicles obtained from rats that were 10-24 days old exhibited positive staining for inhibin alpha- and beta A-subunits. The relative intensities of immunostaining for alpha- and beta A-subunits increased during follicular growth and maturation. Ova and internal thecal cells did not show any immunostaining for inhibin alpha- and/or beta A-subunits. These results suggest that granulosa cells of immature rat ovaries may produce inhibin from the 10th day after birth, and that an increase in the number of mature ovarian follicles results in an increase in inhibin production in the immature rat ovary during prepubertal development. PMID- 7531579 TI - Glycinergic inhibitory synaptic currents and related receptor channels in the zebrafish brain. AB - To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole cell recordings of the Mauthner cells, applications of 10(-6) M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl--dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mIPSCs). Applications of 0.5-1 microM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of -50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81 86 pS and another displaying only one opening level of 41-43 pS. These two mean conductance states had similar mean open times, of 0.6-1 and 4.5-6 ms respectively. In addition, three mean closed times were observed for the 41-43 pS level. The shortest group (0.6-1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14-22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4-5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre- and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation. PMID- 7531581 TI - [Use of audiovisual systems in teaching]. PMID- 7531582 TI - [Collecting patients' data. Nursing history in surgical patients]. PMID- 7531580 TI - Acute-phase response in early refractory rheumatoid arthritis: long-term follow up study. AB - In order to investigate the role of the acute-phase response in early rheumatoid arthritis (RA), we followed the changes of alpha 1-acid glycoprotein (AGP), alpha 1-antitrypsin (AT) and alpha 1-antichymotrypsin (ACT) in the sera of 25 patients with refractory rheumatoid arthritis (RA) during the first 3 years of the disease. Serum levels of AGP, ACT and AT were measured using rocket immunoelectrophoresis, and AGP, ACT and AT microheterogeneities were performed using two-dimensional immunoelectrophoresis with concanavalin A (Con A) as ligand. On average, serum levels of AGP, ACT and AT proteins were higher at the onset of the disease as compared with healthy controls. After 3 years, a significant decrease in serum levels of all three acute-phase proteins (APPs) was observed, but only in patients without anatomical progression was this decrease statistically significant. At the beginning of the study, only the AT reactivity coefficient (RC) was decreased in comparison with healthy subjects. However, after 3 years of disease, AGP, ACT and AT RCs all decreased. This investigation provided two new observations. The first is that only AT RC is significantly lower in comparison with normal values at the beginning of RA, whereas AGP RC and ACT RC remain within the normal range of values; a decrease in AGP RC and ACT RC appeared later. The second observation is that the highly elevated levels of APPs at the onset of RA decrease during the course of the disease. Moreover, this decrease does not depend on the disease activity, but a relationship with radiological progression was shown. PMID- 7531584 TI - [Agents of clinically significant bacteriuria]. AB - Bacterial composition of cultures isolated from 468 patients hospitalized at the urologic surgery department was analyzed to elucidate the etiologic structure of clinically significant bacteriurias. A total of 342 bacterial cultures were isolated. In 63.7% of cases monocultures were isolated, in 66.7% the isolated cultures were referred to Enterobacteriaceae and in 18.7% to Pseudomonas. The most significant agents of bacteriurias were Escherichia coli. Proteus mirabilis, and Pseudomonas aeruginosa. PMID- 7531583 TI - Modified Jeener solid-echo pulse sequences for the measurement of the proton dipolar spin-lattice relaxation time (T1D) of tissue solid-like macromolecular components. AB - Modified Jeener solid-echo pulse sequences are proposed for the measurement of the proton dipolar spin-lattice relaxation time, T1D, of motionally restricted (solid-like) components in the presence of mobile molecular species, such as encountered in biological tissue. A phase-cycled composite-pulse sequence was used for detection of the dipolar signal and cancellation of the Zeeman signal. A homospoil gradient pulse was added to the Jeener echo pulse sequence to enhance dephasing of the transverse magnetization components of mobile species, thereby aiding in elimination of the Zeeman signal during dipolar signal acquisition. A modified Jeener echo sequence incorporating water suppression is also proposed as a means to further depress the Zeeman signal arising from mobile components. The modified Jeener echo sequences were successfully used for the measurement of proton T1D values of solid 2,6-dimethylphenol and Sephadex gels of differing degrees of cross linking and hydration. PMID- 7531585 TI - Biological effects of diesel exhaust particles (DEP) on isolated cardiac muscle of guinea pigs. AB - Diesel engine-powered vehicles emit some 30 to 100 times more particles than do gasoline engine cars. We previously reported that diesel exhaust particles (DEP) could produce superoxide anions in an in vitro study. Furthermore, mice instilled intratracheally with DEP showed high mortality at low doses. The cause of death was lung edema with damage to the lung endothelial cells. In order to elucidate the mechanism of the onset of mortality induced by DEP, we examined the direct action of DEP on the isolated atrium of guinea pigs. A light-duty (2740cc), four cylinder diesel engine was used. The DEP were collected on fiberglass filter. DEP caused a negative inotropic action that was followed by the cardiac arrest of the isolated left atrium. These actions were not inhibited by propranolol, atropine, verapamil, diltiazem, diphenhydramine, indomethacin, superoxide dismutase or catalase. The precise mechanism of cardiac arrest is unknown. However, these results suggest that cardiac toxicity induced by DEP might be involved in lung edema. PMID- 7531586 TI - The role and methodology for purging tumor from autologous bone marrow and peripheral blood progenitor cells. PMID- 7531587 TI - Post-operative serial prostate-specific antigen and transrectal ultrasound for staging incidental carcinoma of the prostate. AB - OBJECTIVES: To examine the value of post-operative serum prostate-specific antigen (PSA), PSA density, incremental change in serial serum PSA (PSA slope) and transrectal ultrasound (TRUS) in the assessment of residual malignancy after the diagnosis of clinically unsuspected prostatic adenocarcinoma at transurethral resection of the prostate (TURP). PATIENTS AND METHODS: Forty-eight untreated patients with incidental carcinoma of the prostate, demonstrated at TURP for a clinically benign gland, were evaluated post-operatively with serum PSA and TRUS with multiple systematic prostatic biopsies. Prostatic volume was determined from TRUS measurements and PSA density was defined as serum PSA divided by gland volume. Those patients who did not undergo further treatment were monitored with serial PSA levels, and PSA slope was calculated as the overall annual percentage increase in serum PSA. RESULTS: Among 36 patients staged T1A (A1), 11 (31%) had histologically proven residual carcinoma, and five of the 12 patients (42%) with T1B (A2) disease had no residual disease on biopsy. Serum PSA levels following TURP were greater in those patients with residual disease than those without (P = 0.001), but at a cut-off of 4.0 ng/mL--providing a sensitivity of 89%--the specificity of PSA alone was 57%. PSA density had an 83% sensitivity and a 67% specificity with a cut-off of 0.15 ng/mL/cm3. TRUS had a sensitivity of 63% and a specificity of 52%. An incremental rise in PSA exceeding 20% per year in untreated patients gave a sensitivity of 90% and specificity of 79% for biopsy proven residual malignancy. CONCLUSION: This study demonstrates the inaccuracy of staging incidental prostatic malignancy by TURP. Although the performance of PSA density is better than that of PSA alone, the reliability of both are limited by the lack of specificity, and TRUS imaging lacks both sensitivity and specificity. The PSA slope has sufficient sensitivity and specificity to distinguish reliably most patients with biopsy proven residual malignancy. Although ultrasound-guided systematic biopsies provide a means for confirming residual malignancy, they may not be indicated in all patients with incidental carcinoma: for such patients, PSA progression may provide a rational basis for subsequent treatment. PMID- 7531588 TI - Prostate-specific antigen obtained under optimal conditions determines extracapsular adenocarcinoma of the prostate. AB - OBJECTIVE: To assess the relationship of prostate-specific antigen (PSA) obtained under optimal conditions with the presence or absence of organ-confined prostate cancer following radical prostatectomy. PATIENTS AND METHODS: The medical records of 300 consecutive patients who underwent radical retropubic prostatectomy were retrospectively reviewed. Ninety-three patients were excluded who had a pre operative PSA level potentially altered by various factors (prostate infection, manipulation or instrumentation). RESULTS: A pre-operative PSA value < 4 ng/mL accurately predicted pathologically confined disease in 42 of 51 patients (82%) which contrasted with extracapsular disease in 74 of 84 patients (88%) who had a PSA value > 10 ng/mL. One of the 53 patients with a PSA > 15 ng/mL had organ confined disease at surgery. CONCLUSION: These data demonstrate that optimal serum PSA values correlate well with pathological stage. PMID- 7531589 TI - DNA ploidy, serum prostate specific antigen, histological grade and immunohistochemistry as predictive parameters of lymph node metastases in T1 T3/M0 prostatic adenocarcinoma. AB - OBJECTIVE: To evaluate whether DNA ploidy and immunohistochemistry performed in primary prostatic carcinoma specimens give predictive information on regional lymph node metastasis in addition to T category, histological grade and serum prostate specific antigen (PSA). PATIENTS AND METHODS: Pre-treatment TURP specimens from 80 patients with prostatic carcinoma T0-T3/M0 disease were retrospectively evaluated by means of DNA ploidy and histological grade, and immunostaining for PSA, prostatic acid phosphatase (PAP), neuron-specific enolase (NSE) and p53 protein. Pelvic lymph node dissection was performed in all patients. Serum PSA was determined in 76 of the 80 patients before pelvic staging lymphadenectomy. Thirty-two (40%) of the 80 patients had pN+ disease. RESULTS: Thirty-six patients (46%) had serum PSA values below the upper reference limit (< or = 10 micrograms/L). By univariate analysis the pN category correlated with the serum PSA level (P < 0.001), histological grade (P < 0.001), tissue PSA (P < 0.001), tissue PAP (P < 0.04), T category (P < 0.005) and DNA ploidy (P < 0.02). Multivariate analysis revealed that the serum PSA level was the most powerful independent prognosticator, followed by the T category, tissue PAP and tissue PSA. Histological grade and DNA ploidy did not reach the level of significance in the multivariate analysis. CONCLUSION: These data suggest that tissue PAP and tissue PSA predict the pN status in patients with T0-T3/M0 prostate carcinoma, in addition to serum PSA and T category. Neuroendocrine differentiation and p53 protein seem to have no predictive ability. PMID- 7531590 TI - Should we reconsider the indications for ileo-obturator node dissection with localized prostate cancer? AB - OBJECTIVE: To evaluate the need for ileo-obturator node dissection in patients with localized prostate cancer who are undergoing radical retropubic prostatectomy. PATIENTS AND METHODS: Over a 5-year-period, 95 patients underwent bilateral pelvic lymphadenectomy. Ninety were performed in association with planned radical prostatectomy and five were staging procedures in clinical stage T3 patients prior to radiotherapy or hormonal therapy. The patients with localized prostate cancer were stage T1a (one patient), T1b (21), T2a (30), and T2b (38). Pre-operative biopsies in the patients with localized cancer were well differentiated in 44 patients, moderately well differentiated in 45 and poorly differentiated in one. In the patients with T3 tumours, pre-operative biopsies were well differentiated in one, moderately well differentiated in two and poorly differentiated in two. Prostate-specific antigen (PSA) levels ranged from 0.4 to 110.1 ng/mL (Hybritech assay). RESULTS: Two patients had positive lymph node dissections on fixed section. These two patients had well-differentiated T1b disease with a PSA level of 72.4 ng/mL and poorly differentiated T3 disease with a PSA level of 58.5 ng/mL. There was significant upstaging (P < 0.001) and upgrading (P < 0.001) on pathological examination. None of the 71 patients with a PSA < or = 10 ng/mL had positive lymph nodes compared with 8.3% of the 24 patients with a PSA > 10 ng/mL (P = 0.0618). Lymph node metastases were present in 1% of patients with well or moderately well-differentiated prostate cancer on pre-operative biopsy versus 33% with poorly differentiated disease (P = 0.0625). These P values strongly suggest an association, achieving significance only at the 10% level which might be the more appropriate level given the relative lack of power of the study due to the small number of patients with positive lymph nodes. CONCLUSION: These results suggest that routine ileo-obturator node dissection in patients with well or moderately well-differentiated, localized prostate cancer and a PSA level < 10 ng/mL may be unnecessary, especially as a separate procedure. PMID- 7531591 TI - The significance of abdominal straining in men with lower urinary tract symptoms. AB - OBJECTIVE: To determine the relationship between the symptom of straining to void, the presence of objectively demonstrated abdominal straining and bladder outflow obstruction in men with lower urinary tract symptoms (LUTS), and to assess the effect of straining on flow rate in these men. PATIENTS AND METHODS: The presence of straining to void as a symptom was determined from a symptom questionnaire in 61 men presenting with LUTS. Objective evidence of straining to void was assessed by rectal pressure measurement and the presence of bladder outflow obstruction was determined by pressure-flow studies. The effect of straining on flow rate was assessed in a separate group of 58 men with LUTS, by comparing the maximum flow rate on non-strain and strain voids of similar volume. RESULTS: There was poor agreement between the complaint of straining and the presence of straining as measured by rectal pressure recording. Twelve of the 53 patients who claimed they never or occasionally strained to void in fact showed straining on each of four voids. There was no significant difference in the proportion of men who complained of straining in the obstructed and non obstructed groups (P = 0.86). The probability of obstruction in those men with objective evidence of straining on all voids was 80% and in those who did not strain on all voids was 51%, but this difference was not statistically significant (P = 0.53). There was no clinically significant effect of straining on maximum flow rate in men with LUTS. CONCLUSION: As a symptom, straining is an uncommon complaint in men with benign prostatic hyperplasia (BPH), and the relationship between the symptom and objective evidence of its presence is poor. Both as a symptom and as an objectively measured voiding disorder, straining lacks sufficient sensitivity and specificity for it to be of value in the diagnosis of bladder outlet obstruction (BOO). Abdominal straining does not have a marked effect on flow rate in men with BPH. This study suggests that straining is an unreliable symptom of BOO, that it does not influence voiding function in elderly men and that its inclusion in symptom scores for BPH should be reconsidered. PMID- 7531593 TI - Intravesical treatment of interstitial cystitis with a heparin analogue. PMID- 7531592 TI - Immunohistochemical localization of neuromarkers and neuropeptides in human fetal and neonatal urinary bladder. AB - OBJECTIVE: To use immunohistochemical techniques to determine the spatial and temporal distribution of a variety of neuropeptides in the human fetal and neonatal urinary bladder. MATERIALS AND METHODS: Thirteen pre-natal specimens ranging in gestational age from 17 to 35 weeks were acquired following abortion or miscarriage. In addition two post-natal specimens aged 8 and 12 weeks were obtained at post-mortem and were included in this study. The overall innervation of each specimen was visualized using the general nerve marker protein gene product 9.5 (PGP). Localization of dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) revealed putative noradrenergic nerves. The neuropeptides studied included neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP). RESULTS: At 17 weeks a rich plexus of PGP and NPY-containing nerves was present throughout the detrusor muscle coat. As gestational age increased, VIP, SP and CGRP-containing nerves were observed with increasing frequency although SP and CGRP were mainly confined to perivascular nerve plexuses. TH- and DBH-containing nerves were first observed in the intramural ureters at 30 weeks and the detrusor muscle at 35 weeks and were relatively numerous in the intramural ureters and muscle of the superficial trigone in the two post-natal specimens. PGP-containing nerves were first observed beneath the bladder epithelium at 23 weeks and gradually became more numerous with increasing age. Occasional NPY, VIP, SP and CGRP-containing nerves were observed in the submucosa but TH- and DBH-immunostained nerves were especially numerous in the mucosa of the trigone in the two post-natal specimens, many such nerves being unrelated to the vascular supply. CONCLUSIONS: The bladder detrusor possesses a rich autonomic innervation by 17 weeks of gestation and this presumptive cholinergic innervation is associated with NPY immunoreactivity. Presumptive noradrenergic nerves appear relatively late in pre-natal development and mainly supply the intramural ureters and superficial trigone. A submucosal plexus of nerves has been demonstrated, the functional significance of which remains uncertain. PMID- 7531594 TI - Cytotoxic lymphocytes. Two ways to kill target cells. AB - Of the two pathways implicated in target-cell damage by cytotoxic lymphocytes, secretory granule exocytosis seems to dominate in anti-viral immunity whereas the Fas pathway may regulate immune responses. PMID- 7531595 TI - Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis. AB - A cardiovascular-active pressor area of medullary reticular formation was defined by mapping changes in arterial blood pressure produced by microinjections of the neuroexcitatory amino acid, L-Glutamate (L-Glu). Sites where L-Glu provoked pressor responses larger than 10 mmHg were localized to a rostral longitudinal cell column of the nucleus reticularis rostroventrolateralis (n.RVL) extending 450 microns posteriorly to the facial nucleus. Spinal projections from the ventrolateral medulla were studied with a dual retrograde transport immunocytochemical method. A striking correspondence was observed between the ventrolateral pressor area (VLPA) of n.RVL and rostrocaudal distribution of a circumscribed population of thoracic reticulospinal neurons containing tyrosine hydroxylase (TH)- or phenylethanolamine N-methyltransferase (PNMT)_immunoreactivity. Quantitative analysis revealed that 72% of the total number of retrogradely labeled neurons within the active area were immunocytochemically positive for TH; 28% of the reticulospinal projection cells were immunonegative. Deposits of L-Glu and dye through the same micropipettes verified a consistent correlation of vasopressor sites and the rostral subset of catecholaminergic neurons. Since comparable numbers of cell bodies in the VLPA contain TH and PNMT all are presumed to be adrenergic. At levels of n.RVL immediately adjacent to the VLPA commencing at a level 450 microns caudal to the facial nucleus, sites were unresponsive to Glu-stimulation or vasodepressor. At these levels, only non-adrenergic reticulospinal neurons project to cervical or thoracic spinal segments. We conclude that the VLPA is highly restricted to a narrow column of n.RVL < 0.5 mm in length and corresponds precisely with a population of predominantly adrenergic thoracic reticulospinal neurons that project exclusively to sympathoadrenal preganglionic motoneurons [cf 46]. These findings corroborate the idea that an adrenergic-spinal pathway may play a role in controlling sympathetic outflow. PMID- 7531596 TI - The N-methyl-D-aspartate channel blocker ketamine does not attenuate, but enhances, locus coeruleus-induced potentiation in rat dentate gyrus. AB - Norepinephrine (NE)-induced potentiation of the perforant path-evoked potential is blocked by N-methyl-D-aspartate (NMDA) receptor antagonism in vitro. Does this occur in vivo? A saline and a ketamine micropipette monitored perforant path evoked potentials in the dentate gyrus of urethane-anesthetized rats. Activation of locus coeruleus (LC) produced short- and long-term potentiation of the perforant path-evoked potential on both pipettes. Spike amplitude potentiation was enhanced on the ketamine pipette. In contrast high frequency-induced potentiation on the ketamine pipette was attenuated. LC-NE potentiation may not require NMDA channel activation in vivo. PMID- 7531597 TI - Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey. AB - Neurons in the brainstem and spinal cord of the lamprey were retrogradely labeled with Calcium Green-dextran, an indicator dye that increases its fluorescence when intracellular calcium levels increase. Optical signals could be recorded from these labeled neurons during spinal cord stimulation, nerve stimulation, or spontaneous activity, up to 4 days after dye application and for distances of 5 14 mm away from the application site. Optical signals were enhanced by 4-AP, a potassium channel blocker, and blocked by cadmium, a calcium channel blocker. Taken together, the results suggest that the optical signals recorded from labeled neurons were due to calcium influx during electrical activity. Thus, retrograde labeling with calcium indicator dyes may provide a general purpose method for simultaneously monitoring the activity-related changes of intracellular calcium in anatomically identified groups of neurons in the lamprey nervous system. PMID- 7531598 TI - Impairment of serotoninergic transmission is followed by adaptive changes in 5HT1B binding sites in the rat suprachiasmatic nucleus. AB - Serotonin1B (5-HT1B) receptor binding in the suprachiasmatic nucleus (SCN) following impairment of serotoninergic transmission was studied by quantitative autoradiography. Serotonin (5-HT) denervation with 5,7-dihydroxytryptamine (5,7 DHT) caused a significant increase in the density of 5-HT1B receptors in both the ventral (62%) and dorsal (53%) parts of the SCN as early as 3 days after axotomy. The magnitude of this increase did not differ 3, 15 or 21 days post-lesion. An up regulation of 5-HT1B receptors with similar magnitude was obtained in the two parts of the SCN after inhibition of 5-HT synthesis by chronic parachlorophenylalanine treatment. In this case, up-regulation was shown to be reversible after restoration of 5-HT synthesis with L-5-hydroxytryptophan. These results indicate that 5-HT1B receptor density in the SCN was inversely correlated with 5-HT levels. These plastic properties exhibited by 5-HT1B receptors in the SCN are discussed in relation to the mode of 5-HT transmission and possible localization of the receptors onto the main chemically defined cell populations of the nucleus. PMID- 7531599 TI - Body fat and RNA content of the VMH cells in rats neonatally treated with monosodium glutamate. AB - The RNA content of the ventromedial hypothalamus (VMH), the lateral hypothalamic area (LHA), and the cortical neurons of male and female rats, neonatally treated with monosodium glutamate (MSG), were investigated. MSG (2 g/kg b.wt.) was injected subcutaneously to male and female rat pups daily for 5 days after birth. At 12 weeks of age a significant decrease of RNA content in the VMH cells and significantly increased body fat in neonatally MSG-treated animals were found. Correlation of these data showed a significant negative correlation between the body fat content and the RNA content in VMH neurons. The results a) confirm a closed relationship between the body fat content and the functional activity of VMH, b) indicate that obesity of neonatally MSG-treated animals should be due to decreased functional activity of the VMH cells. PMID- 7531600 TI - Peritoneovenous shunts for palliation of the patient with malignant ascites. AB - METHODS: This study evaluated the effectiveness and morbidity associated with 116 peritoneovenous shunts inserted into 89 patients for symptomatic malignant ascites between 1981 and 1991. All patients had previously failed standard medical therapy of salt and fluid restriction, diuretics, and intermittent peritoneal aspirations. RESULTS: Symptoms from the ascites included discomfort, impaired mobility, early satiety, and dyspnea. The study population was composed of 31 men and 58 women whose ages ranged from 24 to 81 years (mean 56). Primary tumors originated from the following sites: breast, 17; ovary, 17; pancreas, 13; gastrointestinal or related organs, 21; and other sites, 21. Mean shunt patency was 83 days; however, only 31% of the patients studied maintained a patent shunt and lived for > 2 months. Symptomatic relief was achieved in only 57 patients (62%). Forty-four patients (49%) developed one or more complications related to shunt placement. The deaths of 12 patients (13%) within 30 days of surgery were directly related to a complication of shunt placement. Overall, 30- and 60-day mortality rates were 43% and 61%, respectively. CONCLUSIONS: Due to the brief and only fair relief of symptoms with a high associated morbidity and mortality, we believe peritoneovenous shunts are of very limited usefulness in the palliation of malignant ascites and suggest that alternate methods should be considered to manage these patients. PMID- 7531601 TI - T cells or active Epstein-Barr virus infection in the development of lymphoproliferative disease in human B cell-injected severe combined immunodeficient mice. AB - BACKGROUND: Severe combined immunodeficient (SCID) mice develop Epstein-Barr virus (EBV) containing human lymphoproliferative disease (LPD) tumors when reconstituted with human peripheral blood leukocytes (PBLs) from EBV-seropositive donors, but LPD tumors do not develop in the presence of immunosuppressive agents, such as cyclosporine A or corticosteroids. METHODS: Therefore, LPD development in SCID mice was used as a model to explore the relationship among B cells, T cells, and EBV in vivo. SCID mice were engrafted with PBLs isolated by leukapheresis from a single EBV-seropositive donor. Purified populations of CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) were isolated and engrafted into SCID mice. RESULTS: SCID mice engrafted with purified CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) did not develop LPD. In contrast, mice engrafted with purified B cells developed LPD if they were co-engrafted with purified T cells or if they were inoculated with infectious EBV. CONCLUSIONS: This study confirms the requirement of T cells or active EBV infection in the development of LPD in animals engrafted with B cells latently infected with EBV. A greater understanding of the cellular and viral interactions leading to transformation and malignancy may allow the development of specific interventional therapies for malignancies in the immunosuppressed host. PMID- 7531603 TI - Beneficial effects of oligotide, a novel oligodeoxyribonucleotide, in murine traumatic shock. AB - The effects of oligotide, an oligodeoxyribonucleotide analog, were investigated in an experimental model of traumatic shock. Pentobarbital-anesthetized rats subjected to Noble-Collip drum trauma and receiving only the vehicle (i.e., Krebs Henseleit solution) developed a severe form of traumatic shock characterized by marked hypotension (61 +/- 6 mmHg), a survival time of 115 +/- 21 min, endothelial dysfunction, significant increases in plasma free amino-nitrogen concentration (p < .001) as well as elevated intestinal myeloperoxidase activity. In contrast, oligotide given intravenously (15 mg/kg bolus + 10 mg/kg/h infusion for 5 h) resulted in a significant prolongation of survival time to 209 +/- 31 min (p < .01), a significant and sustained increase in mean arterial blood pressure, a significant attenuation of plasma free amino-nitrogen concentration (p < .01), and intestinal myeloperoxidase activity (p < .05). Furthermore, oligotide significantly preserved superior mesenteric artery (SMA) endothelial function as seen by the relaxation response of isolated SMA rings to acetylcholine (71 +/- 5% vs. 36 +/- 5%, p < .01 compared to untreated trauma rats). Moreover, oligotide in a concentration-dependent manner attenuated unstimulated human neutrophil adherence to either thrombin or trauma-activated SMA endothelium in vitro (p < .001). Thus, our data suggest that the mechanism of the protective effect of oligotide in traumatic shock is improving endothelial function and diminishing neutrophil accumulation leading to reduced tissue injury. PMID- 7531602 TI - Immunocytochemical assay for androgen receptors in prostate cancer: a prospective study of 63 cases with long-term follow-up. AB - BACKGROUND: Numerous problems are associated with biochemical androgen receptor (AR) assay performance and interpretation in prostatic cancer. The purpose of this study was to determine if a novel immunocytochemical AR assay performed on intact tissue sections would prove useful in prognosticating endocrine response and survival. METHODS: A prospective study was done on 63 prostatic carcinomas maintained in liquid nitrogen for over a decade. The study used the peroxidase antiperoxidase system and a polyclonal anti-AR antibody. RESULTS: Marked tissue and cellular heterogeneity of nuclear AR was apparent. A cut-off of 10% AR positive cells maximized assay prognostic efficiency. Frequency of positivity was 48% and correlated significantly with endocrine response (p = 0.03), time to progression (p = 0.0016), and survival (p = 0.02), but not with grade, stage, or ethnicity. CONCLUSIONS: This AR assay could be prognostically useful in the clinical management of prostate cancer and is suitable for use in the community hospital laboratory. PMID- 7531604 TI - Comparison of 7.5% NaCl/6% dextran-70 resuscitation of hemorrhage between euhydrated and dehydrated sheep. AB - 7.5% NaCl/6% dextran-70 (HSD) has been shown to be an effective, small volume resuscitation fluid following hemorrhage (HEM) in euhydrated (E) sheep. However, there is controversy whether hypertonic solutions would be effective in dehydrated (D) animals. Therefore, we used two groups (E and 4 days D) of chronically instrumented ewes to evaluate the responses to HSD following HEM. All sheep were bled and maintained at 50 mmHg mean arterial pressure (MAP) for 2 h, then resuscitated with a 4 mL/kg bolus of HSD. Dehydration did not affect baseline MAP, heart rate (HR), or total peripheral resistance (TPR), whereas cardiac output (CO: E, 5.28 +/- .31; D, 4.00 +/- .31 L/min), stroke volume (SV: E, 61 +/- 4; D, 44 +/- 4 mL/beat), urine flow rate (V: E, .51 +/- .11; D, .34 +/- .07 mL/min), and sodium excretion (UNa V: E, 22 +/- 8; D, 6 +/- 2 microEq/min) were reduced and plasma sodium (PNa: E, 150 +/- 3; D, 157 +/- 2 mEq/L) and protein (PTP E, 7.8 +/- .1; D, 8.8 +/- .6 g/dL) were elevated. The only difference between groups during HEM were HR (E, 98 +/- 8; D, 150 +/- 17 bpm), SV (E, 31 +/- 3; D, 14 +/- 2), and TPR (E, 23 +/- 2; D, 32 +/- 3). Resuscitation with HSD restored MAP (E, 92 +/- 3; D, 92 +/- 2), CO (E, 6.2 +/- .3; D, 4.2 +/- .2), and TPR (E, 15 +/- 1; D, 23 +/- 1) to baseline values. SV was increased above hemorrhage values but was not restored to baseline values in either group (E, 50 +/- 7; D, 27 +/- 3). HR increased further following HSD (E, 143 +/- 11; D, 158 +/- 5). PNa was raised 10 and 16 mEq/L in the E and D sheep, respectively, following HSD infusion, but no adverse effects associated with elevated PNa were observed in either group. Thus, HSD was effective in restoring MAP, CO, and TPR to baseline values in D sheep but it was at the expense of a lower SV and a higher HR than in E sheep. PMID- 7531605 TI - Controversies in shock research: hypertonic resuscitation--pros and cons. AB - The proper fluid resuscitation of hemorrhagic shock is still controversial. Hypertonic saline has been suggested for prehospital resuscitation of hemorrhagic shock, because of its superior ability to expand blood volume and elevate systemic blood pressure and cardiac output in a small volume and during a short time period. We have defined two types of hemorrhagic shock: controlled hemorrhagic shock (CHS), where the bleeding source is immediately occluded following hemorrhage, and uncontrolled hemorrhagic shock (UCHS), where bleeding is induced by injury to blood vessels that are left unoccluded. It was observed that hypertonic saline (HTS) treatment of controlled hemorrhagic shock leads to an increase in blood pressure and cardiac output, while HTS treatment of UCHS leads to increased bleeding from injured blood vessels, hemodynamic deterioration, and increased mortality. Conversion of UCHS to CHS by tourniquet, military antishock trousers, or surgical hemostasis prevented excessive bleeding and mortality following HTS. Several clinical studies have used hypertonic saline dextran (HSD) or hypertonic saline (HS) for treatment of trauma casualties, but to date no significant improvement in mortality has been demonstrated by either HS or HSD. A more favorable effect but still not statistically significant effect has been demonstrated in patients with a Glasgow Coma Scale of 8 or less. The efficacy of HS has not clearly been established in clinical trials, in all of which HS was used in combination with conventional crystalloid therapy. Further human trials are required to better define the patient population that would benefit most from the prehospital administration of HS. PMID- 7531606 TI - Nitric oxide synthase inhibition does not prevent cardiac depression in endotoxic shock. AB - Enhanced production of nitric oxide has been implicated in cardiac and vascular dysfunction associated with septic and endotoxic shock. To test this hypothesis, conscious rats were administered endotoxin. 6 h later, the rats were anesthetized, arterial pressure was measured, and hearts were removed for Langendorff perfusion in the absence and presence of .01 microM isoproterenol. Left ventricular developed pressure was 61 +/- 6 mmHg in control rats 39 +/- 5 mmHg in endotoxin-treated rats. Inotropic responses to isoproterenol were unaffected by endotoxin treatment. Administration of nitric oxide synthase (NOS) inhibitors (NG-nitro-L-arginine and aminoguanidine) prior to endotoxin did not improve left ventricular function in endotoxin-treated rats. Dexamethasone pretreatment, however, prevented endotoxin-induced cardiac depression. These results suggest that cardiac depression during endotoxemia is not caused by NOS activation and increased nitric oxide production. Furthermore, the cardioprotectant actions of dexamethasone are not related to its ability to inhibit inducible NOS expression. PMID- 7531607 TI - Prenatal cytogenetic screening. PMID- 7531608 TI - Making a spectacle of herself: reading community mental health nursing assessments. AB - The metaphor of mapping is used in this paper to examine the discursive construction of women whose nudity in public places (making a spectacle of herself) provides dilemmas for community mental health nurses required to make assessments of these women's ability to function in the community. Excerpts from stories provided by the nurses are used to demonstrate the complexity of the decision-making processes and the limits to the choices they perceive they can make. PMID- 7531609 TI - Maternal serum alpha-fetoprotein levels between 13 and 24 weeks' gestation. AB - Maternal serum alpha-fetoprotein (MSAFP) screening has been widely used and proven valuable in the prediction of a variety of fetal disorders. Any laboratory intending to provide MSAFP screening needs to establish its own reference data. In order to establish a normal MSAFP median value at our own laboratory, 5256 samples of MSAFP were collected from uncomplicated, singleton pregnant women between 13 and 24 weeks' gestation. The MSAFP median levels steadily rise with advancing gestation about 16% per gestational week in average. There were 0.17% and 4.4% of the pregnancies with serum AFP levels less than 0.25 multiple of the median (MoM) and 0.5 MoM respectively, and 4.22% and 1.66% with serum levels above 2.0 MoM and 2.5 MoM. Accurate and satisfactory interpretation of MSAFP screening should be emphasized in the establishment of a well-developed normal median value. PMID- 7531610 TI - Kluver-Bucy syndrome: report of a case with nasopharyngeal cancer after irradiation and chemotherapy. AB - A case of Kluver-Bucy syndrome associated with delayed cerebral necrosis after radiation therapy and chemotherapy is reported. The patient--a 51-year-old man who was a victim of nasopharyngeal cancer--developed a complex behavioral disturbance consisting of visual agnosia, alteration of sexual behavior, strong oral tendency, placidity, memory deficit and speech disturbance one year after two courses of irradiation and chemotherapy. Brain computed tomography disclosed bilateral temporal hypodensity with edema. The patient expired from cachexia three months later. The clinical picture fulfilled the criteria of Kluver-Bucy syndrome. It should be emphasized that this syndrome may occur after irradiation and chemotherapy in patients with nasopharyngeal cancer. PMID- 7531611 TI - Differential expression of the human metastasis adhesion molecule CD44V in normal and carcinomatous stomach mucosa of Chinese subjects. AB - BACKGROUND: CD44 is a cell surface adhesion molecule involved in cell-cell and cell-matrix interactions. Tumor cells transfected to overexpress the isoform CD44V readily gain access to lymph nodes and form distant metastases. METHODS: Monoclonal antibodies directed at epitopes common to known CD44 isoforms were used to investigate CD44V expression in 30 normal gastric mucosa tissues, 64 different gastric adenocarcinomas, 20 metastatic adenocarcinoma lymph nodes and 4 established gastric carcinoma cell lines. In addition, CD44V gene expression in six gastric adenocarcinoma tissues and four gastric cancer cell lines were investigated by northern blotting. RESULTS: Immunohistochemistry screening of 30 subjects with normal gastric mucosa did not reveal expression of CD44 variants. Areas of intestinal metaplasia, a precancerous lesion, were stained with antibodies against either V5- or V6-containing isoforms of CD44. Tubular and signet-ring cell types of adenocarcinoma were strongly positive for epitopes encoded by CD44 variants containing exons V5 (41/49 and 10/10, respectively). Some tubular type adenocarcinomas (15/49) also expressed CD44 variants containing the V6 epitope. Tumor differentiation was closely related to CD44 V5 expression (P < 0.001). In addition, 18 of 20 gastric adenocarcinomas metastatic to lymph nodes expressed the V5 epitope of CD44 and 4 of 20 expressed the V6 epitope. Analysis of four established gastric adenocarcinoma cell lines revealed that two had moderate to strong expression of exons V5 and V6 of CD44. An antibody directed against CD44 variants containing exons V8 to V10 strongly stained all gastric adenocarcinoma cell lines. Northern blotting demonstrated that all four tumor cell lines and six gastric carcinoma mucosa tissues expressed CD44V. CONCLUSIONS: Generation of CD44 splice variants may be closely linked with gastric carcinoma tumorigenesis and differentiation. In addition, expression of CD44 variants containing exons V5 and V6 may be used as an indicator of evolving gastric cancer. PMID- 7531612 TI - Controlled delivery of taxol from microspheres composed of a blend of ethylene vinyl acetate copolymer and poly (d,l-lactic acid). AB - 'Large' (30-100 microns) and 'small' (10-30 microns) size range taxol-loaded microspheres composed of a blend of biodegradable poly (d,l-lactic acid) (PLA) polymer and nondegradable ethylene-vinyl acetate (EVA) copolymer were prepared using the solvent evaporation method. Encapsulation efficiencies were between 95 100% for taxol in 50:50 EVA:PLA blend microspheres. Between 10-13% of the total taxol content of the microspheres (0.6% w/v taxol loading) was released in 50 days. Using the chick chorioallantoic membrane (CAM) model, the taxol microspheres released sufficient taxol to produce vascular regression and inhibition of angiogenesis. This taxol-loaded microsphere formulation may have potential for the targeted delivery of taxol to a tumor via arterial chemoembolization. PMID- 7531613 TI - Expression of nitric oxide synthase in human central nervous system tumors. AB - The nitric oxide synthases (NOS) are a family of related enzymes which regulate the production of NO, a free radical gas implicated in a wide variety of biological processes. Vasodilation and increased tumor blood flow, increased vascular permeability, modulation of host tumoricidal activity, and free radical injury to tumor cells and adjacent normal tissues are pathophysiological features of malignant tumors that may be mediated by NO. We examined human brain tumors for three NOS isoforms and NADPH diaphrase, a histochemical marker of NOS activity in the brain. We detected increased expression of the brain and endothelial forms of NOS [NOS I and NOS II, respectively (C. Nathan and Q. Xie. Cell, 78: 915-919, 1994)] in astrocytic tumors, and the highest levels of expression was found in higher grade tumors. Each of these two isoforms was found in tumor cells and tumor endothelial cells. The macrophage isoform of NOS (NOS III) was less frequently detected and expressed at a lower level, predominantly in tumor endothelial cells. NADPH diaphorase staining for NOS activity paralleled this pattern of NOS expression. Western blot analysis of tumor tissues for these NOS isoforms confirmed these observations. Our data indicate that malignant central nervous system neoplasms express unexpectedly high levels of NOS and suggest that NO production may be associated with pathophysiological processes important to these tumors. PMID- 7531614 TI - Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen. AB - T cells can play a central role in the immune response to cancer, with tumor specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) alpha and beta chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length alpha and beta T-cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor beta chain specific mAb. Jurkat clone 5 TCR+ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR+ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy. PMID- 7531615 TI - Prevention of hepatic metastasis of human colon cancer by angiogenesis inhibitor TNP-470. AB - The antimetastatic effect of a potent angiogenesis inhibitor, O-(chloroacetyl carbamoyl)fumagillol (TNP-470), was investigated in nude mice implanted with human colon cancer. Small pieces of tumors from three established human colon cancer cell lines (TK-3, TK-4, and TK-9), which were maintained in nude mice, were implanted into the cecal wall of nude mice via a small incision in the serosa. TNP-470 (20 or 30 mg/kg) was given s.c. every other day from day 10 after implantation, and the mice were sacrificed after 6 weeks. There was no difference in the weight of the implanted tumors (control group: 0.45 +/- 0.29 g versus treated group: 0.49 +/- 0.27 g). An antimetastatic effect of TNP-470 was clearly demonstrated in a dose-dependent manner. In the mice given 20 mg/kg TNP-470, liver metastasis developed in 3 of 10 cases. In the 30-mg/kg group, metastasis developed in only 1 of 17 mice, while it developed in 22 of 32 mice of the control group. The number of metastatic foci was significantly less in the treated groups. TNP-470 effectively prevented liver metastasis, however, but had no effect on the growth of the primary tumor. These results indicate that the angiogenesis inhibitor TNP-470 has a strong inhibitory activity against in vivo hepatic metastasis of human colon cancer. PMID- 7531616 TI - Enhanced antitumor effects of CD20 over CD19 monoclonal antibodies in a nude mouse xenograft model. AB - We used a nude mouse xenograft tumor model to compare the efficacy of unconjugated CD19 and CD20 mAbs (IgG2a subclass) in mediating antilymphoma effects. Treatment with the CD20 mAbs NKI-B20 and BCA-B20 resulted in a drastic decrease in tumor take rate (P < 0.0001) in comparison to controls, whereas the CD19 mAb CLB-CD19 was ineffective. Tumor growth rates were reduced by both CD19 and CD20 (P < 0.0001). The decrease in growth rate induced by NKI-B20 or BCA-B20 was larger than that induced by CLB-CD19 (P = 0.0022). In vitro experiments showed that NKI-B20 or BCA-B20 are more powerful than CLB-CD19 in mediating lysis by interleukin 2-activated natural killer cells. No difference was observed between different isotypes (IgG1, IgG2a, IgG2b) of the switch variants of NKI-B20 or CLB-CD19. A positive correlation between antigen density and the sensitivity to antibody-dependent cellular cytotoxicity was demonstrated with human lymphoblastoid B cells, JY, transfected with cDNA encoding the human CD19 antigen that expressed high levels of this antigen. These cells are more efficiently killed by natural killer cells when coated with CLB-CD19 mAbs than JY wildtype cells that express 1 log lower levels of the CD19 antigen. Antibody-dependent cellular cytotoxicity experiments with thioglycolate-activated macrophages show a more complex relationship between antigen density, isotype of the mAb, and cytotoxicity. BCA-B20 (IgG2a) and CLB-CD19 (IgG2a) and all isotypes of NKI-B20 mediated strong cytotoxicity, whereas CLB-CD19 isotypes IgG1 and IgG2b were associated with limited cytotoxicity. Proliferation of Daudi cells was inhibited with high concentrations of all isotypes of CLB-CD19, but not with any of the CD20 mAbs. To our knowledge this is the first report showing that the antitumor effects in vivo of unconjugated CD20 mAbs are far superior to those of CD19 mAbs. PMID- 7531617 TI - Tenascin expression in astrocytomas correlates with angiogenesis. AB - We investigated the expression and distribution of the extracellular matrix protein tenascin (TN) in 59 astrocytomas and 11 samples of normal brain by Western blot analysis and immunohistochemistry using antibodies against human TN. The tumors included 14 juvenile pilocytic astrocytomas (grade 1), 13 low grade fibrillary astrocytomas (grade II), 8 anaplastic astrocytomas (grade III), and 24 glioblastomas multiforme (grade IV). Proliferation indices were calculated by computer-based image analysis after immunostaining with the MIB-1 antibody against the Ki-67 proliferation-associated antigen. Western blot analysis for TN on fresh frozen tumor tissue from 23 of the 59 astrocytomas indicated up to 4 fold higher TN expression in glioblastomas multiforme than in nontumorous control tissues. Enhanced intercellular expression of TN was observed by immunohistochemistry in glioblastomas multiforme. More-over, TN immunostaining was consistently greater within and around the walls of hyperplastic blood vessels than nonhyperplastic vessels of both high grade tumors and juvenile pilocytic astrocytomas. Juvenile pilocytic astrocytomas with increased TN expression by Western blot analysis had vascular hyperplasia by light microscopy. Proliferation indices moderately correlated with tumor grade. Enhanced immunohistochemical expression of TN was associated with higher tumor grade with higher proliferation indices. The strong association of TN and vascular hyperplasia, regardless of tumor grade, suggests that TN may play a crucial role in angiogenesis. PMID- 7531618 TI - Regulation of apoptosis in normal and malignant ovarian epithelial cells by transforming growth factor beta. AB - Previously, we found that transforming growth factor beta (TGF-beta) inhibits proliferation of normal human ovarian epithelial cells. In addition, although only 1 of 5 immortalized ovarian cancer cell lines was inhibited, TGF-beta inhibited proliferation of 19 of 20 primary epithelial ovarian cancers. In this study, we examined whether TGF-beta induces apoptosis in normal and malignant ovarian epithelial cells. Among 5 immortalized cell lines, only OVCA 420 is markedly growth inhibited by TGF-beta, and this was the only cell line in which TGF-beta elicited DNA fragmentation characteristic of apoptosis. Induction of apoptosis in OVCA 420 was time and concentration dependent and could be partially inhibited by concurrent treatment with an anti-TGF-beta mAb. Although apoptosis was not seen in normal ovarian epithelial cells (n = 7), [3H]thymidine incorporation was inhibited in all cases [mean = 61.2 +/- 7.2% (SD) of untreated control; P < 0.01]. Similarly, TGF-beta inhibited [3H]thymidine incorporation in all 10 primary ovarian cancers (mean = 40.4 +/- 7.1% of control; P < 0.01), but only 3 of 10 (30%) were found to undergo apoptosis when treated with TGF-beta. There was no relationship between p53 status of the ovarian cancers and the ability of TGF-beta to elicit apoptosis. In conclusion, TGF-beta inhibits proliferation but does not induce apoptosis in normal human ovarian epithelial cells. In contrast, some ovarian cancers that are growth inhibited by TGF-beta also undergo apoptosis. These data are consistent with the hypothesis that malignant cells are more susceptible to apoptosis than their normal nontransformed counterparts. PMID- 7531619 TI - Flow rate and composition of whole saliva in rural and urban Tanzania with special reference to diet, age, and gender. AB - The association of flow rate and biochemical and microbiological characteristics of saliva with diet was studied in 83 12-year-old children and in 127 adults (84 in the age group 35-44 years, 43 in the age group 65-74 years) living in rural and urban communities in Tanzania. No significant differences were observed between the salivary flow rates of the rural and urban subjects. The mean salivary flow rates were slightly lower in women than in men and significantly lower in the 12-year-old children than in the two older age groups (p < 0.05). The buffer effect was higher in the rural than the urban population. Further, it was lower in women than in men (p < 0.001). Salivary protein, IgG, and sialic acid concentrations were significantly higher in the rural than in the urban population, whereas amylase activity and IgA concentrations were lower. Microbiological studies showed mutans streptococci in 97% of the rural and in 91% of the urban subjects (n.s.). Lactobacilli grew significantly less often in the rural than urban samples (p < 0.01). Analyses of 24-hour dietary recalls showed that the rural and urban diets differed. The proteins in the rural diet were largely of plant origin, while in the urban diet animal proteins dominated. The rural diet contained also less sucrose but more fibre (mainly from grain) than the urban diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531620 TI - Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana). AB - Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smooth-muscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin gene-related peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethyl-carbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase--33%, calcitonin gene-related peptide--30%, galanin--55%, neuropeptide Y--16%, substance P--35%, and vasoactive intestinal polypeptide--58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P. PMID- 7531622 TI - Expression of the alpha-subunit of glycoprotein hormones in the pars tuberalis specific glandular cells in rat, mouse and guinea-pig. AB - The nature of the hormone(s) secreted by the pars tuberalis (PT) is still unknown. This pituitary lobe is mainly formed by specific glandular cells that differ in their ultrastructural features from the other adenohypophysial cell types. Data from the literature indicate the presence of thyroid-stimulating hormone immunoreactivity in the PT-specific cells of the rat and the Djungarian hamster but not of other species, including the mouse and guinea-pig. The PT also encloses variable numbers of pars distalis cells, essentially gonadotrophs that are mainly dispersed in its caudal area. We studied the expression of the glycoprotein hormone alpha-subunit in the PT of the rat, mouse and guinea-pig by in situ hybridization and immunocytochemistry. In situ hybridization, using an oligonucleotide probe complementary to rat cDNA sequence 196-237 revealed the expression of the alpha-subunit gene throughout the PT of the rat and the mouse; in the guinea-pig, the probe labelled no pituitary cells. Light- and electron microscopic immunocytochemistry demonstrated alpha-subunit immunoreactivity in the secretory granules of the PT-specific cells in the three species examined. These cells did not react with a specific antibody against the beta-subunit of luteinizing hormone, an antibody that labelled scattered gonadotrophs. The present data suggest that hormone(s) produced by the PT-specific glandular cells are, at least partly, related to glycoprotein hormones. PMID- 7531621 TI - Selective association of nerve fibres immunoreactive for substance P or bombesin with putative cholinergic neurons of the male rat major pelvic ganglion. AB - The male rat major pelvic ganglion contains both sympathetic and parasympathetic neurons that supply the lower urinary and digestive tracts, and the reproductive organs. The aim of this study was to describe the distribution and identify potential targets of sensory and intestinofugal axons in this ganglion. Two putative markers of these projections were chosen, substance P for primary sensory axons and bombesin for myenteric intestinofugal projections. Varicose substance P-immunoreactive axons were associated only with non-noradrenergic (putative cholinergic) somata, and most commonly with those that contained vasoactive intestinal peptide. Immunoreactivity for substance P was also present in a small group of non-noradrenergic somata, many of which were immunoreactive for enkephalins, neuropeptide Y or vasoactive intestinal peptide. Bombesin immunoreactivity was found only in preterminal and terminal (varicose) axons, the latter of which were exclusively associated with non-noradrenergic somata that contain neuropeptide Y-immunoreactivity. Some varicose axons containing either substance P- or bombesin-immunoreactivity were intermingled with clumps of small, intensely fluorescent cells. These studies indicate that substance P- and bombesin-immunoreactive axons are likely to connect with numerically small, but discrete, populations of pelvic neurons. PMID- 7531623 TI - Transformation of fibroblasts into endothelial cells during angiogenesis. AB - Light- and electron-microscopic autoradiography have been used to study fibroblast transformation into endothelial cells in the formation of new blood vessels during wound healing in rabbit ear chambers. When cultured fibroblasts labeled with tritium thymidine were transplanted autologously into the chambers, newly formed blood vessels contained endothelial cells labeled with tritium thymidine. This result suggests that fibroblasts play a pivotal role in angiogenesis, as progenitors of endothelial cells in newly formed blood vessels. PMID- 7531624 TI - Depression and 18-month prognosis after myocardial infarction. AB - BACKGROUND: We previously reported that major depression in patients in the hospital after a myocardial infarction (MI) substantially increases the risk of mortality during the first 6 months. We examined the impact of depression over 18 months and present additional evidence concerning potential mechanisms linking depression and mortality. METHODS AND RESULTS: Two-hundred twenty-two patients responded to a modified version of the National Institute of Mental Health Diagnostic Interview Schedule (DIS) for a major depressive episode at approximately 7 days after MI. The Beck Depression Inventory (BDI), which measures depressive symptomatology, was also completed by 218 of the patients. All patients and/or families were contacted at 18 months to determine survival status. Thirty-five patients met the modified DIS criteria for major in-hospital depression after the MI. Sixty-eight had BDI scores > or = 10, indicative of mild to moderate symptoms of depression. There were 21 deaths during the follow-up period, including 19 from cardiac causes. Seven of these deaths occurred among patients who met DIS criteria for depression, and 12 occurred among patients with elevated BDI scores. Multiple logistic regression analyses showed that both the DIS (odds ratio, 3.64; 95% confidence interval [CI], 1.32 to 10.05; P = .012) and elevated BDI scores (odds ratio, 7.82; 95% CI, 2.42 to 25.26; P = .0002) were significantly related to 18-month cardiac mortality. After we controlled for the other significant multivariate predictors of mortality in the data set (previous MI, Killip class, premature ventricular contractions [PVCs] of > or = 10 per hour), the impact of the BDI score remained significant (adjusted odds ratio, 6.64; 95% CI, 1.76 to 25.09; P = .0026). In addition, the interaction of PVCs and BDI score marginally improved the model (P = .094). The interaction showed that deaths were concentrated among depressed patients with PVCs of > or = 10 per hour (odds ratio, 29.1; 95% CI, 6.97 to 122.07; P < .00001). CONCLUSIONS: Depression while in the hospital after an MI is a significant predictor of 18-month post-MI cardiac mortality. Depression also significantly improves a risk-stratification model based on traditional post-MI risks, including previous MI, Killip class, and PVCs. Furthermore, the risk associated with depression is greatest among patients with > or = 10 PVCs per hour. This result is compatible with the literature suggesting an arrhythmic mechanism as the link between psychological factors and sudden cardiac death and underscores the importance of developing screening and treatment programs for post-MI depression. PMID- 7531625 TI - Carbohydrate-mediated recognition of a circulating placental alkaline phosphatase immunoglobulin M complex. AB - We detected an abnormal alkaline phosphatase (AP) electrophoretically in the serum of a patient with rheumatoid arthritis, who had a macromolecular AP linked with immunoglobulin M (IgM) bearing a kappa light chain. The IgM isolated from the AP-IgM complex in the patient's serum reacted apparently with all of the AP isozymes tested, i.e. those originating in the liver, bone, intestine and placenta, but the alpha-mannosidase-treated IgM from the patient's serum bound to placental AP (PAP) alone. This suggests that untreated IgM recognizes multivalent epitopes of the AP and that the complex of AP with alpha-mannosidase-treated IgM is a specific antibody-antigen complex. In order to investigate further the multivalent binding capacity for the PAP-untreated IgM complex, we prepared a monoclonal antibody (MoAb) against PAP and identified it as an IgM with a kappa light chain. The binding affinities and their circulating half-lives of the synthetic complexes of PAP and respective MoAbs were examined with and without treatment with several glycosidases. The untreated MoAb bearing IgM had binding affinity for all of the AP isozymes tested, while alpha-mannosidase-treated IgM attached only to PAP, the same as the IgM isolated from the PAP-IgM complex in the patient's serum. The circulating clearance of the PAP-IgM complex in rabbits was faster than either component alone. In addition, the PAP-IgM complex treated with alpha-mannosidase was found to have the shortest half-life of all the complexes of PAP and Igs treated with the several glycosidases tested. These results suggest that the formation of the PAP-IgM complex as an enzyme-linked antibody and the clearance of the complex in vivo are dependent on the sugar moieties of the Igs. PMID- 7531626 TI - Acute phase proteins and recombinant IL-2 therapy: prediction of response and survival in patients with colorectal cancer. AB - Twenty-four patients with metastatic colorectal cancer were treated with recombinant IL-2 (rIL-2) by continuous intravenous infusion for 5 days (18 x 10(6) U/m2 per 24 h), followed by three injections of 5-fluorouracil (600 mg/m2) and folinic acid (25 mg/m2) at weekly intervals. The response to treatment was assessed using standard UICC criteria (partial or complete response, stasis or progression of disease). The serum concentrations of the acute phase proteins; C reactive protein (CRP), retinol binding protein (RBP), alpha 1-antitrypsin (alpha 1-AT), transferrin (TF) and albumin were measured. A response to therapy occurred in the tumours of seven (29%) of the 24 patients (two complete and five partial responses). All patients who demonstrated a response to treatment had a serum albumin level of > 37 g/l and a CRP level of < or = 10 mg/l. In contrast, of the 17 patients who did not respond to therapy, 12 (71%) had a serum albumin of less than 37 g/dl and a CRP of greater than 10 mg/l. Examination of the survival times of the 12 patients who had a pretreatment serum albumin level of less than 37 g/l revealed that all had died within 12 months of cessation of therapy. However, 58% of patients with pretreatment serum albumin levels of greater than 37 g/l survived for longer than 12 months. These results have shown that (i) patients who respond to rIL-2-based therapy and (ii) those patients who have prolonged survival times, can be identified by pretreatment measurement of serum levels of acute phase proteins. PMID- 7531627 TI - IL-8/IL-8 receptor expression in psoriasis and the response to systemic tacrolimus (FK506) therapy. AB - Recently, the keratinocyte IL-8/IL-8 receptor (IL-8R) pathway has been implicated in the pathogenesis of psoriasis, and there is evidence that the potent macrolide immune suppressant tacrolimus (formerly FK506) can inhibit this pathway in vitro. In this study, determination of the expression of cytokine mRNAs in lesional skin of patients with active disease by reverse transcriptase polymerase chain reaction revealed transcripts for IL-1 beta, tumour necrosis factor-alpha (TNF alpha), IL-6, IL-8, IL-8R, IL-10, interferon-gamma (IFN-gamma), IL-2R and transforming growth factor-beta (TGF-beta), but not IL-2 or IL-4. IL-8 was the only cytokine expressed in affected skin of all patients but not in clinically normal skin of healthy subjects. In seven CD4+ T cell clones propagated from the lesional skin of an untreated psoriasis patient, IL-8 was expressed by the skin derived T lymphocytes and not by feeder cells (irradiated autologous blood lymphocytes); IL-1 beta, IL-2, IL-6 and IL-10 were also expressed by some or all of the T cell clones. IL-8 mRNA was not detected in the skin of any patient after the start of systemic tacrolimus therapy; IL-1 beta, IL-6 and IFN-gamma transcripts were also reduced. By 12 weeks, the mean psoriasis area and severity index (PASI) had decreased from 18.8 to 3.8, a reduction of 80%. In the same post treatment biopsies, however, message for IL-8R persisted. Estimation of circulating IL-8 levels by enzyme immunoassay showed that all patients with detectable IL-8 before treatment had decreased levels in response to treatment with tacrolimus; reductions in PASI scores were accompanied by decreases in IL-8 levels, that varied both in rate and extent. Partial relapse, which in a minority of patients followed the initial period of remission, and was precipitated by drug dose reduction, was accompanied by an increase in circulating IL-8. These findings add credence to the view that the IL-8/IL-8R autocrine/paracrine pathway may be important in the pathogenesis of psoriasis. They further suggest that interference with IL-8 production and/or that of other key chemokines may be an important mechanism underlying the therapeutic efficacy of tacrolimus, and other agents such as cyclosporin A, with similar molecular actions. PMID- 7531629 TI - Immunogenetics of epitopes of the carboxyl terminus of the human 60-kD Ro autoantigen. AB - Systemic lupus erythematosus is associated with the presence of autoantibodies which bind several ribonucleoproteins, including Ro (or SS-A). We have explored the relationship of the HLA-DQ and T cell receptor alleles in patients producing autoantibodies binding the 13-kD carboxyl terminus fragment of the 60-kD Ro and with autoantibodies binding a peptide epitope within this fragment (amino acid residues 480-494). Antibodies binding the 13-kD fragment are more likely to be found in the sera of patients with particular DQA1 and DQB1 alleles, while antibodies binding the epitope at 480-494 are found almost exclusively in the sera of patients with a Bg/II 9.8-kb polymorphism of the T cell receptor beta gene. Meanwhile, in these same patient sera the level of autoantibodies binding the complete 60-kD Ro particle is associated with a distinct pattern of alleles at these same immunoregulatory loci. These data demonstrate that component parts of autoantibody responses may be under genetic control which can be distinguished from the HLA associations characteristic of the response to the intact, complete autoantigen. PMID- 7531628 TI - Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia. AB - Fas antigen (CD95) is a membrane-associated molecule that mediates apoptotic cell death and may play a role in the induction and maintenance of T cell tolerance. To elucidate the involvement of Fas antigen in human autoimmune diseases, we analysed Fas antigen expression by peripheral T cells from patients with SLE and rheumatoid arthritis (RA), using three-colour flow cytometry. Both CD4+ and CD8+ T cells from SLE patients expressed Fas antigen in a higher density than did these cells from healthy donors and from RA patients. Enhancement of Fas antigen density was noted in Fas+CD45RO+ memory T cells from SLE patients. More remarkably, a significant expression of Fas antigen was observed in CD45RO- naive T cells from SLE patients. CD4+CD45RO- T cells from SLE patients co-expressed Fas antigen and early to intermediate activation antigens such as CD25 and CD71, and late activation antigen HLA-DR in only FashiCD4+ naive T cells. Such up regulation of Fas antigen expression in SLE patients seems to be clinically meaningful, because mean fluorescence intensity (MFI) of Fas antigen on CD4+ T cell subsets inversely correlates with the absolute size of CD4+ T cell subsets in peripheral blood of SLE patients. These results suggest that T cells with increased Fas antigen expression may be highly susceptible to apoptotic cell death, in vivo. A putative mechanism for lymphopenia in SLE patients is discussed. PMID- 7531630 TI - Cytoplasmic lamellar bodies are not immunostained with rabbit anti-cystatin C serum. PMID- 7531631 TI - Expression of keratins K12, K4 and K14 during development of ocular surface epithelium. AB - The 55 kDa keratin K12 and the 59 kDa keratin K4 were used as biochemical markers of differentiated corneal and conjunctival epithelium, respectively, to follow the temporal and spatial appearance of these cell types during embryonic development of the mouse eye. K12 was first detected in corneal epithelial cells of day 15 mouse embryos in a small subpopulation of superficial cells. At later developmental stages only suprabasal corneal epithelium expressed K12, however, in post-natal and adult cornea all cell layers were K12-positive. K4 was first observed, in 14 and 15 day embryos, in a subpopulation of epithelial cells which had invaginated from surface ectoderm to form the lid buds. From embryonic day 16 on K4 was detected in all areas of developing conjunctival epithelium. Some superficial corneal epithelial cells also expressed K4 during embryonic development, but by immunofluorescence microscopic criteria, this keratin was localized exclusively to the conjunctiva in post-natal and adult eye. Expression of the 50 kDa 'basal-type' keratin K14 was also examined in this study. Similarly to K4, K14 was first noted in epithelium comprising the lid bud at embryonic day 14. Between 14 and 17 days of development some epithelial cells in the putative fornix of the conjunctiva did not express K14. Although corneal epithelial cells expressed K14 during development, in adult cornea only certain basal cells did so. These results suggest that the invagination of surface ectoderm to form the presumptive eyelid may be coupled to the initiation of differentiation of ocular surface epithelium. PMID- 7531632 TI - Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections. AB - The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three dimensional (3-D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO-1 iodide and YOYO-1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO-1 iodide and YOYO 1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO-1 iodide and YOYO-1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO-1 iodide and YOYO-1 iodide. Contrary to acriflavine, bleaching of TOTO-1 iodide and YOYO-1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531633 TI - Detection of immunocytochemically stained rare events using image analysis. AB - The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events. PMID- 7531634 TI - Distribution of mast cells in human ileocecal region. AB - The number and histochemistry of mast cells were analyzed in surgical specimens of the ileocecal junction and neighboring intestinal segments. All the basophilic cells contained tryptase and some were immunoreactive for chymase, vasoactive intestinal polypeptide, or nitric oxide synthase. The medium density of mast cells per square millimeter was 31.90, 110.38, 72.83, 29.80, and 32.70, in the mucosa, submucosa, inner circular, outer circular, and longitudinal muscle layers, respectively. Mast cell density was higher at the ileocecal junction (for all layers together, 79.29 mast cells/mm2) than elsewhere (mast cells/mm2: ileum, 52.29; cecum, 59.22; cecocolonic junction, 54.65; ascending colon, 48.63). The differences among layers and among segments were significant and might be due to layer- and region-specific mast cell roles. Mast cell richness in the muscle coat, especially in the inner circular muscle layer, might be important in regulating its motility. PMID- 7531635 TI - Changes of protein gene product 9.5 (PGP 9.5) immunoreactive nerves in inflamed appendix. AB - The existence of chronic appendicitis is controversial. In this prospective study, we investigated possible changes in the innervation of the appendix under different pathological conditions and correlated histological findings with clinical observation. Thirty appendectomy specimens and 14 appendices obtained from organ donors or patients who underwent right hemicolectomy were immediately fixed in Bouin's solution and processed for immunocytochemistry using an antiserum directed against the panneuronal marker protein gene product 9.5 (PGP 9.5). The density of PGP 9.5 immunostaining was evaluated by digitized morphometry. Significant differences in the density of the PGP 9.5-immunoreactive area were detected in the mucosal layer. In the nonacute appendicitis group, PGP 9.5 was increased (10.99 +/- 3.15%) as compared to acute appendicitis (3.89 +/- 1.77%) and controls (4.98 +/- 1.25%). The significant increase of PGP 9.5 in nonacute appendicitis may suggest axonal sprouting leading to hyperinnervation of the mucosa. This may be a neuronal factor in the pathophysiology of the disease and pain symptoms. PMID- 7531637 TI - Behavior in cocaine-exposed infants and children: association versus causality. AB - The effects of prenatal cocaine exposure on infant and child development and behavior are uncertain. This ambiguity has been enhanced by the early nature of human research and suboptimal study designs. Methodological difficulty in this research, particularly in the consideration of confounding effects, has made it difficult to ascribe causal relationships. Future research must consider more precise measures of confounding. PMID- 7531636 TI - Clinical study of IgA antibody against hepatitis C virus core antigen in patients with type C chronic liver disease. AB - Immunoglobulin A class antibody to hepatitis C virus core antigen (IgA anti-HCc) was measured in the serum of 128 patients with type C chronic liver disease. Fifty-eight patients (45.3%) were seropositive. IgA anti-HCc was detected in only one of 20 patients with chronic persistent hepatitis; however, 52.3% (46/88) of patients with chronic active hepatitis and 55% (11/20) of patients with liver cirrhosis were seropositive. Histological examination revealed that 22 (71.0%) of 31 patients with severe disease activity were seropositive compared to 35 (44.9%) of 78 patients with moderate (P < 0.05) and one (5.3%) of 19 patients with mild (P < 0.01) histological changes. IgA anti-HCc was measured sequentially in 65 patients who underwent interferon therapy. There was a significant difference between responders and other patients in the mean ratio of IgA anti-HCc titers one month after therapy. Three months after therapy, IgA anti-HCc was detectable in only two of 15 responders who were IgA anti-HCc seropositive at the start of therapy. In contrast, IgA anti-HCc reappeared three months after therapy despite a temporary decrease to undetectable levels in all nonresponders. We conclude that IgA anti-HCc is a useful marker to identify the presence of active type C liver disease and that the disappearance of IgA anti-HCc three months after interferon therapy predicts a good response in treated patients. PMID- 7531639 TI - Expression and purification of a soluble functional form of the platelet alpha IIb beta 3 integrin. AB - Platelet glycoproteins alpha IIb and beta 3 are membrane proteins that associate to form a Ca(2+)-dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, alpha IIb and beta 3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wild-type alpha IIb beta 3. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild-type alpha IIb beta 3 in COS cells or in human megakaryocytes. The truncated beta 3 subunit associated with the Pro-truncated form of the alpha IIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro-truncated alpha IIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr alpha IIb subunits associated with tr-beta 3. In contrast, tr-beta 3 was produced and secreted in excess as the free form. Immunoreactivity of the wild-type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface-located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr-alpha IIb beta 3-containing culture supernatant was adsorbed on an RGDW-affinity column and eluted with a solution of the free peptide RGDW. In the RGD-eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild-type and truncated RGD-eluted complexes to interact with soluble fibrinogen was compared using a solid-phase immunocapture assay. tr-alpha IIb beta 3 and platelet alpha IIb beta 3 exhibited similar fibrinogen-binding capacity. For both complexes, these interactions were mediated by RGD and gamma fibrinogen signals. PMID- 7531638 TI - Increased bilirubin production, ceruloplasmin concentrations and hyperbilirubinaemia in full-term newborn infants. AB - Ceruloplasmin possesses antioxidant activity in vitro, but such a property has not been substantiated in vivo so far. However, it has been suggested that the lack of factors protective against oxidative haemolysis might have a role in neonatal hyperbilirubinaemia. Ceruloplasmin and alphafetoprotein concentrations were measured in cord blood in 78 unselected full-term singleton newborn infants without G6PD deficiency and haemolytic disease of the newborn; in the same infants, the carboxyhaemoglobin level was assessed on the fourth day of life and taken as an index of bilirubin production. The relationship between these variables and maximum bilirubin level in the first 4 days was studied by multiple regression analysis. High carboxyhaemoglobin levels and low ceruloplasmin concentrations, but not alphafetoprotein resulted, associated with hyperbilirubinaemia (P < 0.001). No relationship was found between carboxyhaemoglobin and ceruloplasmin levels. These results exclude an important role for ceruloplasmin in protecting against possible oxidative haemolysis in full-term newborn infants. Ceruloplasmin levels in cord blood are most probably related to hepatic metabolism and are better predictors of hyperbilirubinaemia than alphafetoprotein concentrations. PMID- 7531640 TI - Structure of the human urokinase receptor gene and its similarity to CD59 and the Ly-6 family. AB - Urokinase plasminogen activator receptor (uPAR) gene expression has been implicated in many important biological processes including cell invasiveness and migration. The uPAR gene was cloned from a human genomic library by hybridization with a uPAR cDNA. The complete structure of the human uPAR gene, including a 21.23-kb transcription unit with 204 bp 5' and 239 bp 3' flanking sequences, was determined by comparison with the uPAR cDNA sequence. The uPAR gene is composed of seven exons and six introns. The seven exons of 101, 111, 144, 162, 135, 147 and 563 bp are separated by six introns of approximately 2.04, 2.62, 8.42, 0.906, 3.10 and 2.78 kb. Exons 1-7 encode 19, 37, 48, 54, 45, 49 and 83 amino acid residues, respectively. A CpG-rich island and sequences related to the transcription factors AP-1, AP-2, c-Jun and NF kappa B are present, but no potential TATA or CAAT boxes were found in the proximal 5' region of the uPAR gene. Comparison of the exon organization of the uPAR gene with that of human CD59 and murine Ly-6 reveals similarity to all three domains encoded by the uPAR exons (2 + 3), (4 + 5) and (6 + 7). These data enable elucidation of the mechanisms involved in regulation of the uPAR gene expression and provide further evidence that the uPAR gene belongs to the Ly-6 superfamily. PMID- 7531641 TI - Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon gamma. Analysis of gamma-interferon activation sites. AB - The acid cytokeratin K17 is inducible by interferon-gamma (IFN-gamma), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-gamma in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13), cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-gamma but not IFN-alpha in transient transfection assays. Sequence analysis revealed three putative gamma-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyl-tRNA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GAS1 (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN-gamma. Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFN-gamma treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified. PMID- 7531642 TI - T cell recognition of hepatitis B and C viral antigens. AB - The outcome of hepatitis B and C heavily depends on the appropriate virus specific T cell response. Both CD8+ and CD4+ T lymphocytes do not recognize native viral proteins but processed peptides bound to MHC class I and class II, respectively. For therapeutical intervention aimed at T lymphocytes in chronic carriers as well as for the development of new vaccines, a precise identification of immunodominant epitopes, which can be recognized by a majority of patients, is necessary. Biological features of certain viral antigens have been partly characterized in animal models, but with the availability of modern molecular technology it is possible to extend these findings to the human system. The identification of anchor residues and motifs in peptides, which are essential for binding to certain MHC class I and class II molecules, allows the prediction of MHC allele-specific epitopes within viral proteins. By the use of synthetic peptides and vaccinia expression vectors, several epitopes for cytotoxic and helper T lymphocytes have been identified in HBV and HCV antigens. In HBV infection cytotoxic T lymphocytes recognize epitopes within the polymerase protein, the envelope protein and the nucleocapsid. In HCV cytotoxic epitopes have so far been identified within the nucleocapsid, E1, E2 and NS2. Since virus specific CD8+ T lymphocytes lyse virus infected cells in vitro and seem to play an important role for viral elimination in vivo, activation of virus specific effector cells may be achieved by immunizing chronically infected patients with the MHC-allele-specific peptides. Epitopes for CD4+ T lymphocytes have been demonstrated in the majority of HBV- and HCV-proteins. Different subsets of CD4+ T lymphocytes influence the course of infection by the production of lymphokines which either support antibody production by B cells or cellular antiviral effector mechanisms. In acute and chronic HBV infection the HBcAg/HBeAg-specific T cell response is closely correlated to viral elimination and the occurrence of anti-HBe- and anti-HBs antibodies. In HCV infection the CD4+ T cell response appears to be more heterogenous, and better functional characterization of the CD4+ response to immunodominant peptide epitopes in association with certain disease stages is required. Since T cell activation, the resulting effector functions and binding of the peptide to the HLA-molecule mainly depend on the peptide structure, viral mutations leading to amino acid changes may contribute to T cell non-responsiveness or an inappropriate T cell response.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531643 TI - Platelets in ulcerative colitis and Crohn's disease express functional interleukin-1 and interleukin-8 receptors. AB - Tissue and plasma concentrations of several cytokines are increased in patients with inflammatory bowel disease (IBD). Platelets play an important role in inflammation and circulate in an activated state in patients with IBD. This study assesses the expression of IL-8 and IL-1 receptors on the surface of platelets from patients with IBD using phycoerythrin (PE)-labelled recombinant human rhIL-1 beta and rhIL-8 and flow cytometry. The percentage IL-1R expressing platelets (median and interquartile range IQR) in the IBD group was 8.7% (5.5-18.2) compared to 4.2% (2.3-6.1) in controls (P = 0.02). The percentage IL-8R expressing platelets in the IBD group was 22.5% (16.5-27.9) and 9.2% (4.3-9.6) in controls (P < 0.001). Furthermore, platelet IL-1R expression in patients with IBD was inversely related to the total daily dose of steroids (r = 0.71, P < 0.01 linear regression analysis). Finally, platelet rich plasma from healthy controls was stimulated with rhIL-1 beta and rhIL-8 and assessed for activation dependent expression of platelet aGPIIb/IIIa and CD62 (p-selectin, GMP-140). IL-1 beta and IL-8 in vitro significantly and specifically activated the platelets. The surface membrane of platelets is able to express functional IL-1R and IL-8R, the expression of which is significantly increased in IBD. Interleukin-1 beta and IL 8 modulate platelet activation in vitro indicating a target role for platelet function in inflammation. PMID- 7531644 TI - Histaminergic neurons mediate restraint stress-induced activation of central 5 hydroxytryptaminergic neurons in the rat. AB - The role of histamine in mediating restraint stress-induced increases in the activity of central 5-hydroxytryptaminergic neurons was evaluated in male rats. 5 Hydroxytryptaminergic neuronal activity was estimated by measuring concentrations of the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens and suprachiasmatic nucleus which contain terminals of these neurons. Placement of rats within restraining tubes rapidly increased (within 10 min) 5-HIAA concentrations in the nucleus accumbens and suprachiasmatic nucleus. Depletion of neuronal histamine by alpha fluoromethylhistidine or antagonism of histamine H1 receptors by mepyramine prevented stress-induced increases in 5-HIAA concentrations, whereas blockade of histamine H2 receptors by zolantidine was without effect. Neither alpha fluoromethylhistidine, mepyramine nor zolantidine affected basal 5-HIAA concentrations in either brain region. These results indicate that histaminergic neurons mediate stress-induced increases in the activity of central 5 hydroxytryptaminergic neurons via an action at histamine H1 receptors. PMID- 7531646 TI - Allosteric interactions and modulator requirement for NMDA receptor function. AB - Activation of NMDA receptors by glutamate, glycine and spermine was assessed using non-equilibrium [3H]dizocilpine binding. Glutamate and glycine mutually increased their efficacies and affinities to stimulate [3H]dizocilpine binding, which were further increased by spermine. Enhancement curves by glycine in the presence of increasing concentrations of the glycine antagonist 7 chlorokynurenate revealed that spermine markedly increased the affinity of glycine but not that of 7-chlorokynurenate, and that glycine was also required for [3H]dizocilpine binding in the presence of spermine. Spermine had a biphasic effect on [3H]dizocilpine binding, with a stimulatory phase followed by an inhibitory phase. The potency of spermine for both phases was increased by glutamate, but not by glycine. These observations indicate that there are positive cooperative interactions between the glutamate, the glycine and the stimulatory and inhibitory polyamine sites of the NMDA receptor. Moreover, glutamate and glycine, but not spermine, are required to activate the receptor. PMID- 7531645 TI - The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line. AB - The human galanin receptor has been characterized pharmacologically from the Bowes melanoma cell line. Using porcine [125I]galanin as the radioligand, a single population of non-interacting high-affinity binding sites (KD = 0.05 +/- 0.01 nM; Bmax = 135 +/- 7 fmol/mg protein) was demonstrated. Human galanin peptide competitively inhibited the specific binding of [125I]galanin (IC50 = 0.35 +/- 0.13 nM) and decreased the forskolin-stimulated cAMP production (EC50 = 0.46 +/- 0.05 nM) with a maximal inhibition of 63 +/- 2% at 10(-7) M. Rat and porcine galanin peptides and the chimeric peptides M15, M35, M32, M40 and C7 also dose-dependently inhibited the forskolin-stimulated cAMP production, while the fragment porcine galanin-(3-29) and [D-Trp2]galanin were found to be inactive. The specific binding of [125I]galanin was decreased in a dose-dependent manner by GTP and the cAMP response was inhibited by the pertussis toxin, suggesting the activation of a G-protein dependent process. The Bowes cell line thus appears to be a relevant tool for the study of human galanin receptor. PMID- 7531647 TI - Characterization of galanin and 5-HT1A receptor coupling to adenylyl cyclase in discrete regions of the rat brain. AB - We have studied the coupling of galanin and 5-HT1A receptors with adenylyl cyclase in the hypothalamus, the entorhinal cortex and the hippocampus of the rat brain. Furthermore, we have evaluated the effects of simultaneous activation of galanin and 5-HT1A receptors on adenylyl cyclase activity. Galanin-(1-29) and galanin-(1-15) showed a dose-dependent inhibitory effect on forskolin-stimulated adenylyl cyclase activity in the hypothalamus and entorhinal cortex. No clear effects were observed in the hippocampus. Neither galanin-(1-29) nor galanin-(1 15) had any effect on the basal activity of adenylyl cyclase in these regions. The selective 5-HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH DPAT) induced a dose-dependent inhibition of forskolin stimulated adenylyl cyclase activity in the hippocampus and the entorhinal cortex. 5-HT induced an inhibition in the hypothalamus. In all regions the effects could be fully counteracted by methiothepin. 5-HT was shown to stimulate the basal activity of adenylyl cyclase in the hippocampus and the entorhinal cortex. The effects could be counteracted by methiothepin. When galanin-(1-29) and 5-HT/8-OH-DPAT were incubated simultaneously additive inhibitory effects, but no synergistic interactions, could be observed on the stimulated adenylyl cyclase activity. In conclusion, galanin and 5-HT1A receptors seem to be linked to different independent pools of G proteins, indicating that the previously demonstrated intramembrane interactions between galanin and 5-HT1A receptors involve a mechanism not directly related to adenylyl cyclase. PMID- 7531648 TI - Subtype- and species-selectivity of a tachykinin receptor antagonist, FK888, for cloned rat and human tachykinin receptors. AB - We investigated the receptor-binding properties and potencies of FK888 (N2-[(4R) 4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N- phenylmethyl-3-(2 naphthyl)-L-alaninamide), a tachykinin receptor antagonist, for the rat and human tachykinin receptor subtypes (NK1, NK2 and NK3) expressed in transfected mammalian cells. In displacement analyses, using membrane preparations derived from monkey kidney COS-7 cells transiently expressing tachykinin receptor subtypes, FK888 showed a subtype selectivity for NK1 receptor and its affinity for the human NK1 receptor was 320-fold higher than that for the rat NK1 receptor, demonstrating species difference in its binding affinity. This was in marked contrast to FK224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2 pentylphenyl )- propionyl]-L-threonyl]tyrosyl-L-leucynyl]-D-phenylalanyl]-L- allothreonyl]-L-asparaginyl]-L-serine-n-lactone) that was selective for NK1 and NK2 receptors with similar affinities for the rat and human receptors. In Chinese hamster ovary cells permanently expressing the human NK1 receptor, FK888 inhibited the substance P-induced phosphatidylinositol hydrolysis and produced a parallel shift in the dose-response curve for substance P. Schild analysis of the antagonism of phosphatidylinositol hydrolysis by FK888 yielded a pA2 value of 8.9 and a slope of 0.97 of the regression line. FK888 itself showed no stimulatory effect on phosphatidylinositol hydrolysis in Chinese hamster ovary cells expressing the human NK1 receptor. Thus, FK888 is a potent, competitive and selective antagonist for human NK1 receptor. PMID- 7531649 TI - Inactivation of the infragranular striate cortex broadens orientation tuning of supragranular visual neurons in the cat. AB - Intracortical inhibition is believed to enhance the orientation tuning of striate cortical neurons, but the origin of this inhibition is unclear. To examine the possible influence of ascending inhibitory projections from the infragranular layers of striate cortex on the orientation selectivity of neurons in the supragranular layers, we measured the spatiotemporal response properties of 32 supragranular neurons in the cat before, during, and after neural activity in the infragranular layers beneath the recorded cells was inactivated by iontophoretic administration of GABA. During GABA iontophoresis, the orientation tuning bandwidth of 15 (46.9%) supragranular neurons broadened as a result of increases in response amplitude to stimuli oriented about +/- 20 degrees away from the preferred stimulus angle. The mean (+/- SD) baseline orientation tuning bandwidth (half width at half height) of these neurons was 13.08 +/- 2.3 degrees. Their mean tuning bandwidth during inactivation of the infragranular layers increased to 19.59 +/- 2.54 degrees, an increase of 49.7%. The mean percentage increase in orientation tuning bandwidth of the individual neurons was 47.4%. Four neurons exhibited symmetrical changes in their orientation tuning functions, while 11 neurons displayed asymmetrical changes. The change in form of the orientation tuning functions appeared to depend on the relative vertical alignment of the recorded neuron and the infragranular region of inactivation. Neurons located in close vertical register with the inactivated infragranular tissue exhibited symmetric changes in their orientation tuning functions. The neurons exhibiting asymmetric changes in their orientation tuning functions were located just outside the vertical register. Eight of these 11 neurons also demonstrated a mean shift of 6.67 +/- 5.77 degrees in their preferred stimulus orientation. The magnitude of change in the orientation tuning functions increased as the delivery of GABA was prolonged. Responses returned to normal approximately 30 min after the delivery of GABA was discontinued. We conclude that inhibitory projections from neurons within the infragranular layers of striate cortex in cats can enhance the orientation selectivity of supragranular striate cortical neurons. PMID- 7531650 TI - Monosynaptic innervation of facial motoneurones by neurones of the parvicellular reticular formation. AB - In order to determine whether neurones in the parvicellular reticular formation are in direct synaptic contact with motoneurones innervating facial muscles, a combined retrograde and anterograde transport study was carried out in the rat. Animals received injections of the retrograde tracer cholera toxin B conjugated to horseradish peroxidase into facial muscles and of the anterograde tracer biocytin into the parvicellular reticular formation. The facial motor nucleus was then examined for anterograde and retrograde labelling in the light and electron microscopes. Retrogradely labelled neurones were found in the facial motor nucleus with a distribution that was dependent on the muscles injected. Terminals anterogradely labelled with biocytin from the parvicellular reticular formation was observed in the motor nucleus amongst the retrogradely labelled neurones. At the electron microscope, the retrogradely labelled cells were found to receive input from unlabelled terminals and from terminals that were anterogradely labelled from the injections of biocytin in the parvicellular reticular formation. The labelled terminals were 1-2 microns in diameter at the active zone and packed with spherical vesicles. They formed both symmetrical and asymmetrical synapses with their labelled or unlabelled targets. It is concluded that neurones in the parvicellular reticular formation form direct synaptic contact with motoneurones of facial muscles. This may represent a pathway by which the basal ganglia can directly influence orofacial movement, as the substantia nigra is known to project to that part of the reticular formation. PMID- 7531652 TI - A ciliary K+ conductance sensitive to charibdotoxin underlies inhibitory responses in toad olfactory receptor neurons. AB - In olfactory neurons from Caudiverbera caudiverbera, a mixture of putrid odorants trigger an inhibitory, K(+)-selective current and a hyperpolarizing receptor potential. The current-voltage relation resembles that of a Ca(2+)-activated K+ conductance; their amplitude depends on extracellular Ca2+. 10 nM charibdotoxin, a blocker of K(+)-selective channels, including Ca(2+)-activated ones, reversibly abolished inhibitory currents and receptor potentials. Focal stimulation demonstrates that the underlying transduction mechanism is confined to the cilia. This represents the first evidence for inhibitory responses in vertebrate olfactory cells mediated by a ciliary CTX-sensitive K+ conductance, most likely a Ca(2+)-activated one. PMID- 7531653 TI - Folding intermediates are involved in genetic diseases? AB - Recent experimental data show that some human genetic diseases are due to mutations in proteins which influence their trafficking and lead to retaining of proteins in the endoplasmic reticulum or their unproper processing. In this paper a hypothesis is proposed that these mutations are connected with an incomplete protein folding, blocking it at the stage of the kinetic molten globule or even earlier. If so, the specific drugs against these diseases may be ligands and other factors which facilitate the correct protein folding. PMID- 7531654 TI - Stability and immunological reactivity of recombinant membrane CD4 electroinserted into the plasma membrane of erythrocytes. AB - Concentration-dependent electroinsertion of recombinant human membrane CD4 in human erythrocytes shows a saturation at an average of about 3,500 inserted CD4 epitopes per cell, detectable by flow cytometry. The erythrocyte recovery drops to 10% at this high level of electroinsertion. Experimentally an optimum for cell recovery and insertion rate was found at about 2,500 CD4 epitopes per red blood cell. In vitro stability assay by flow cytometry indicated a temperature- and medium-dependent decrease in the number of CD4 epitopes inserted per cell. This decrease is biphasic, with an exponential part during the first 24 h after electroinsertion followed by a much slower linear decay. PMID- 7531651 TI - GABAergic and pallidal terminals in the thalamic reticular nucleus of squirrel monkeys. AB - The ultrastructure of synaptic terminals from the external segment of the globus pallidus and of other synaptic terminals positive for gamma-aminobutyric acid (GABA) was examined in the thalamic reticular nucleus (TRN) of squirrel monkeys. Two GABA-positive terminals types were commonly encountered within the TRN neuropil. The most common type of GABAergic terminals (F terminals) are filled with dispersed pleomorphic synaptic vesicles and clusters of mitochondria. These terminals establish multiple symmetric synapses upon the somata and dendrites of TRN neurons. The external pallidal terminals, labeled with WGA-HRP, arise from thinly myelinated axons and correspond to the medium to large F terminals. A less prevalent population of smaller GABAergic synaptic profiles was also identified. The synaptic profiles in this second group contain considerably fewer pleomorphic synaptic vesicles in small irregular clusters and fewer mitochondria, establish symmetric synapses, are postsynaptic to other axonal terminals, are presynaptic to dendrites and soma, and are unlabeled following pallidal injections of WGA HRP. PMID- 7531655 TI - A twin study of polycystic ovary syndrome. AB - OBJECTIVE: To examine the role of genetic and environmental factors in polycystic ovary syndrome (PCOS) by using the classic twin model. SETTING: Outpatient clinic of the Royal Hospital for Women, Paddington, Sydney, New South Wales, Australia. PATIENTS: A group of 19 monozygotic (MZ) and 15 dizygotic (DZ) twin pairs identified from the national twin register. INTERVENTIONS: Ultrasound, clinical, and biochemical parameters were used to define PCOS. RESULTS: Eleven pairs of twins (5 MZ, 6 DZ pairs) were scan-discordant (i.e., one twin had scan-PCOS and the co-twin did not). Model-fitting analysis suggested that fasting insulin level, androstanediol glucuronide, and body mass index (BMI) were significantly influenced by genetic factors. CONCLUSION: This study suggests that PCOS is not the result of a single autosomal genetic defect, but rather environmental factors, perhaps both intrauterine and extrauterine, are involved in the pathogenesis of this disorder or that PCOS may be an X-linked disorder or the result of polygenic factors. However, fasting insulin level, androstanediol glucuronide, and BMI did appear to be under significant genetic influence. PMID- 7531656 TI - Modulation of nerve and glial function by adenosine--role in the development of ischemic damage. AB - Adenosine is released during brain ischemia and provides neuroprotection by actions on nerve and glial cells. Activation of the adenosine A1 receptor enhances the K+ and Cl- conductance in neurons, leading to membrane hyperpolarization and postsynaptic reduction of neuronal Ca2+ influx through voltage- and NMDA receptor-dependent channels. In addition adenosine A1 receptor activation decreases excitatory amino acid release, possibly via inhibition of N- and P-type Ca2+ channels. The A1 and A2 receptors, coupled to Gi/G(o) and Gs proteins respectively, often co-exist and interact with the phospholipase C dependent activation of the protein kinase C and the adenylyl cyclase. Activation of the A1 receptor may mimic metabotropic receptor stimulation in activating intracellular Ca2+ mobilization and PKC. A2 receptor mediated cAMP formation is depressed by high intracellular Ca2+ but enhanced by PKC activation. By modulating these metabolic signaling events, adenosine may influence acute cell functions, gene transcription and sustained changes of nerve and glial cells relevant for the development of ischemic damage. The neuroprotective adenosine effect seems to be amplified by treatment with propentofylline, which enhances adenosine release, influences the balance between A1 and A2 receptor mediated actions, depresses the free radical formation in activated microglia and influences astrocyte reactions. PMID- 7531657 TI - Nerve extracts and substance P activate the phosphatidylinositol signaling pathway and mitogenesis in newt forelimb regenerates. AB - We investigated the inositol phospholipid transmembrane signaling pathway as a possible mediator of neurotrophic (mitogenic) signals in the newt limb regeneration blastema. Blastema mesoderm tissues were prelabeled with myo [3H]inositol, treated with 10 mM LiCl and then exposed to substance P or to extracts of spinal ganglia, brain, or spinal cord. Stimulation with substance P resulted in a rapid dose-dependent reduction of [3H]phosphatidylinositol 4,5 bisphosphate and [3H]phosphatidylinositol 4-phosphate, correlated with a rapid accumulation of inositol 1,4,5-triphosphate. This effect was inhibited when the blastema tissue was treated with neomycin, a known inhibitor of inositol phospholipid turnover. In addition, substance P stimulated the incorporation of [3H]thymidine into DNA of blastema mesoderm cells, and this effect was also suppressed by neomycin, at a dose corresponding to that required to inhibit inositol phosphate accumulation. Extracts of neural tissues, especially spinal ganglia, induced the formation of inositol phosphates and extract activity was attenuated following treatment with heat or trypsin. These findings suggest a role for mitogen-activated inositol phospholipid signaling, initiating events that ultimately lead to cell proliferation. PMID- 7531658 TI - Neonatal seizures: long-term outcome and cognitive development among 'normal' survivors. AB - The authors report the long-term outcome and cognitive development in the late teenage years of 'normal' survivors of neonatal seizures. The outcome of the children was good, and normal in that they had attended normal schools and had normal overall intelligence test scores as adults. However, all of the sample displayed abnormal neuropsychological development in terms of intelligence test profile and subtest scatter, or development of spelling, or development of memory. This was independent of social and behavioural difficulties, which may also be increased. Neonatal seizures may be indicative of a subtle neurodevelopmental vulnerability which may manifest later in life as specific learning difficulties or poor social adjustment. PMID- 7531659 TI - [Immunocytochemical detection of tumor cells in bone marrow as a prognostic factor in breast carcinoma]. AB - In a prospective study at the University of Erlangen, Dept. Gynaecol, and Obstet., 228 patients with breast cancer during their primary surgery and 20 patients during their metastatic surgery, underwent bone marrow punctions at six punction sides, which were twice at the sternum and twice at both iliac crest. The control group was 20 patients without an invasive carcinoma. Aim of the study was to detect or exclude tumour cells in the bone marrow via examination of the biopsies with monoclonal antibodies EMA and cytokeratin and consequently to find out the meaning of the results as prognostic criteria by statistical measurements. Tumour cells in the bone marrow were detected in 46.5% (106/228) of the patients, who underwent a bone marrow biopsy during primary surgery. 21% (23/106) of the patients who were bone marrow positive, but only 5.75% (7/122) of the patients, who were bone marrow negative, developed metastases during a median follow-up of 20 months. This difference is statistically significant. 17 of the 30 patients with recurrences developed bone metastases; 16 of them were EMA positive. The median recurrence-free interval was 5 months in the bone marrow positive group and therefore noticeably shorter, than in the bone marrow negative patient group with 11 months. Of the nodal negative patients, 2 bone marrow positive patients developed distant metastases. With the knowledge of the nodal status and bone marrow biopsy result, it was possible to predict 28 of the 30 patients correctly in respect of their risk to metastasize. The result of the bone marrow puncture was proved in a multivariate analysis to be an independent prognostic factor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531660 TI - [Effect of granulocyte colony stimulating factor (G-CSF) on peripheral blood leukocytes and lymphocytes in patients with chemotherapy-induced leukopenia]. AB - In patients with extensive chemotherapy, G-CSF abrogated leukopenia following administration of cytotoxic agents. Six women with ovarian cancer and chemotherapy-induced leukopenia received 300 micrograms Filgrastrim (r-metHuG CSF, Neupogen 30; AMGEN, Germany) daily for 10 days. Leukocytes and lymphocyte subsets of peripheral blood were determined before, throughout and after subcutaneous injections of G-CSF by flow cytometry using monoclonal antibodies to CD3, CD4, CD8, CD14, CD16/56, CD19 and CD45. It could be observed that not only neutrophils (23 fold) but also lymphocytes (6 fold) and monocytes (10 fold) showed a dramatic increase in cell counts throughout and after G-CSF administration. This is in contrast to previous reports, where only effects on neutrophils were described. In spite of the increase in lymphocytes the relative percentage of CD3+, CD19+, CD3-CD16/CD56+, CD3+, CD8+ and CD3+ CD4+ lymphocyte subsets did not change throughout and after therapy, except for an increased expression of HLA-DR on CD3+ lymphocytes. PMID- 7531661 TI - [Leukocyte and lymphocyte populations in peripheral blood and malignant ascites in patients with ovarian carcinoma]. AB - Ascites and peripheral blood of 12 patients with advanced ovarian cancer (stage IV) have been investigated by two-color flow cytometry for leukocytes and lymphocyte subsets with monoclonal antibodies, against CD3, CD4, CD8, CD14, CD16/56, CD19, CD25, CD45, CD57, and HLA-DR. Ascites compared with blood showed a significant raise of CD3-positive lymphocytes (80 +/- 14% vs. 69 +/- 8%) and a significant reduction of CD57-positive lymphocytes (13.6 +/- 13% vs 24 +/- 21%). There was an increased expression of HLA-DR on CD3-positive lymphocytes in malignant ascites. The results are discussed with regard to a supposedly defective local immune defense against the tumor. PMID- 7531662 TI - [Lung metastasis after pelviscopic salpingectomy in therapy of tubal pregnancy]. AB - A 34-year old VI gravida, II para underwent a pelviscopic salpingectomy of the left fallopian tube for treatment of a repeated ectopic pregnancy. Histology revealed an invasive hydatidiform mole. The declining beta-hCG titers after surgery were not further controlled. Seven months later a secondary amenorrhoea occurred again; beta-hCG levels increased steadily. We could not obtain trophoblastic tissue by operative pelvis-copy (excision of the tubal stump on the left side and tubal sterilisation on the right side) nor by curettage of the uterine cave. A lesion of the lung was interpreted as a metastatic lesion following malignant transformation of the invasive hydatidiform mole and disappeared under cytotoxic therapy containing methotrexate with an uneventful follow-up period of now 4 years. RECOMMENDATION: In order not to overlook the presence of persistent trophoblastic tissue and the possible development of a choriocarcinoma, beta-hCG serum titers should be measured until they disappear, following the surgical therapy of an ectopic pregnancy. PMID- 7531663 TI - Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro. AB - Pasteurella piscicida grown in a glucose-rich medium produces a capsule that can be see under light and electron microscopy. The capsular polysaccharide was purified and characterized by chemical and HPLC analysis. The polymer has the composition glucose/mannose/N-acetylgalactosamine/galacturonic acid/acetic acid in the molar ratios of approximately 2.5:1.3:0.5:0.4:2.5. The polysaccharide was immunogenic in rabbits and did not cross-react with antibodies against the O antigen lipopolysaccharide. PMID- 7531664 TI - Comparison of amylase-binding proteins in oral streptococci. AB - Certain species of oral streptococci bind salivary amylase to their cell surface. The patterns of amylase-binding proteins produced by a range of streptococci have been compared by ligand blotting and several characteristics of the binding proteins investigated. Streptococcus gordonii was the most homogeneous species and almost all strains produced proteins migrating with molecular mass 82 kDa and 20 kDa. Other species were more heterogeneous, releasing proteins that resolved at 87 or 82 kDa and/or between 20 and 36 kDa. Binding of amylase to the 82/87-kDa proteins on ligand blots was prevented by amylase inhibitors, amylase substrates and periodate treatment but these had limited or no effect on amylase binding to 20-36 kDa proteins. Also, the 20 kDa protein of S. gordonii Challis was released into culture medium before the 82-kDa protein. These data suggest that there is significant variation in amylase-binding proteins among streptococci and that the high and low molecular mass proteins differ in the way they interact with salivary amylase. PMID- 7531665 TI - Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. AB - Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBP delta. Likewise, MIX is a direct inducer of C/EBP beta gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBP delta and C/EBP beta accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP alpha. Conclusive evidence has already shown that C/EBP alpha regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBP delta and C/EBP beta play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP alpha. Conditions facilitating the precocious expression of either C/EBP delta or C/EBP beta were observed to accelerate adipogenesis and, in the case of C/EBP beta, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP beta effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBP beta in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat-laden adipocytes. PMID- 7531666 TI - Prognostic factors after hepatectomy for hepatocellular carcinoma. AB - During a seven and a half year period from 1985 through June 1992, hepatic resection was carried out on 84 patients with hepatocellular carcinoma. Their ages ranged from 10 to 79, with an average of 55 years. Hepatitis B surface antigen (HbsAg) was positive in 83%, an alfafetoprotein level of more than 20 ng/dl was found in 71%, and cirrhosis was noted in 64%. Surgical mortality was 3.6% and hospital mortality 1.2%. The 1-, 3- and 5-year actuarial survival rates, excluding in-hospital deaths, were 74%, 56% and 49%, respectively. The large number of patients operated on in the early stage (52%), and Child's class A (61%) might account for the low surgical mortality and high survival rate. The favorable conditions were identified as follows; no cirrhosis, surgery in the early stage, tumor size less than 5 cm, negative HbsAg and freedom from vascular involvement. Multivariate analysis showed that only negative HbsAg had better survival. PMID- 7531667 TI - Prevalence of HBV and HCV infection in Japanese patients with hepatocellular carcinoma. AB - The prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was investigated in 63 Japanese patients with hepatocellular carcinoma (HCC). HBV infection was confirmed by measuring hepatitis B surface antigen and HBV-DNA in the serum, and HCV infection was confirmed by measuring antibody to HCV using a 2nd generation test and HCV-RNA in serum. Some 54.0% of the patients had HCV infection only, 27.0% had HBV infection only, and 9.5% had both HCV and HBV infection. Only 9.5% of HCC patients had neither HCV nor HBV markers. These results indicate that, in Japan, HCV and HBV infection is an important factor associated with HCC, and that the hepatitis virus may have a role in the carcinogenesis of HCC. PMID- 7531668 TI - Acute pancreatic injury in asymptomatic individuals after heavy drinking over the long-term. AB - Recently, high-dose short-term alcohol exposure has been observed not to induce acute pancreatic damage, as evaluated by serum pancreatic enzyme activities. In this study the effect of high-dose, long-term alcohol exposure on the pancreas was investigated in 32 consecutive alcoholics admitted to a unit to treat the problems of withdrawal after a long period of heavy drinking. None of the alcoholics complained of abdominal symptoms. The signs of clinical acute pancreatitis (pain, increased serum C-reactive protein concentration or blood white cell count) were not observed in any of the alcoholics. A significant increase in serum total amylase, pancreatic isoamylase and lipase activities developed by the second day after termination of alcohol intake. These enzyme activities remained significantly increased for one week after cessation of drinking. Seven alcoholics had signs of chronic pancreatitis at ultrasonography (pancreatic calcification, pseudocyst). These results suggest that heavy alcohol intake over the long term may frequently induce subclinical pancreatic injury. PMID- 7531669 TI - Transgenic mouse model of hemifacial microsomia: cloning and characterization of insertional mutation region on chromosome 10. AB - The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5' and 3' flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggest that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans. PMID- 7531670 TI - A rapid colorimetric assay for carcinoembryonic antigen (CEA)-mediated cell adhesion and analysis of CEA domains involved in the adhesion. AB - A colorimetric microadhesion assay that allows the quantitative determination of carcinoembryonic antigen (CEA)-mediated homophilic cell adhesion to CEA immobilized on 96-well polyvinyl chloride plates is described. Chinese hamster ovary (CHO) cells transfected with a full-length CEA cDNA were used as indicator cells. After dislodging nonadherent cells, specifically bound cells were quantified by a colorimetric analysis based on the ability of live cells to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product. The domains of CEA responsible for the homophilic cell adhesion were analyzed by inhibition assays using anti-CEA monoclonal antibodies whose reactive domains were already known. The involvement of domain N and possibly subdomain A3 of CEA in the homophilic cell adhesion has been suggested. PMID- 7531671 TI - Protein tyrosine kinase activation and protein kinase C translocation are functional components of CD40 signal transduction in resting human B cells. AB - CD40 is a 47kD glycoprotein expressed on all B cells that plays an important role in B cell development and activation. Previous investigations have focused on signal transduction events in activated B cells and B cell lines, and little information is available regarding CD40 signal transduction in resting, normal B cells. Because CD40 plays a critical role in the regulation and activation of resting B cells, we studied the signaling mechanisms involved in functional responses to CD40 ligation in these cells. Treatment of dense tonsil B cells with either protein tyrosine kinase (PTK) or protein kinase C (PKC) inhibitors, but not with an inhibitor of cyclic nucleotide dependent kinases, resulted in abrogation of CD40-mediated cell adhesion, suggesting a role for both PTK and PKC in CD40-mediated B cell adhesion. Direct evidence for CD40-mediated PTK activation was demonstrated by increased tyrosine phosphorylation as detected by anti-phosphotyrosine Western blots of cell lysates prepared from dense resting tonsil B cells stimulated with biotinylated anti-CD40 monoclonal antibody plus avidin. CD40 engagement also resulted in PKC activation, as detected by translocation of cytosolic PKC activity to the membrane-bound compartment. CD40 mediated PKC translocation was dependent on PTK activation, whereas PTK activity induced by CD40 ligation was independent of PKC activity, suggesting that PTK activation precedes PKC activation in resting B cells stimulated with anti-CD40 mAb. The results of our experiments identify PTK and PKC activation as components of CD40 signal transduction in normal, resting B cells and establish a functional role for these events. PMID- 7531672 TI - Cytokine and insulin regulation of alpha 2 macroglobulin, angiotensinogen, and hsp 70 in primary cultured astrocytes. AB - Acute-phase proteins and heat shock proteins (hsp) are upregulated following exposure to a number of conditions including bacterial infection, tissue injury, or stress. We show here that alpha 2 macroglobulin (alpha 2M), angiotensinogen (AOG), and hsp 70 are regulated by cytokines in primary cultures of astrocytes. In addition, we have found that insulin modulates the effect of cytokines on these proteins. In cells treated with lipopolysaccharide (LPS) conditioned Raw media, interleukin (IL)-6, or IL-1 beta for 24 h, there was a significant decrease of alpha 2M secretion below control levels. In the absence of insulin, however, similar treatments resulted in a significant increase in alpha 2M secretion. AOG secretion increased significantly following treatment with individual cytokines either in the presence or absence of insulin, but conditioned media did not cause a response in the absence of insulin. Hsp 73 concentrations also increased following treatment with conditioned media and IL-1 beta in the presence or absence of insulin. Following IL-6 treatment, however, hsp levels either decreased (- insulin) or did not change (+ insulin). To determine whether acute-phase proteins are regulated similarly to hsp, astrocytes were subjected to elevated environmental temperatures. Cells incubated at 43 degrees C for 90 min showed a marked increase in AOG secretion. However, alpha 2M and hsp 73 levels remained unchanged. In the absence of insulin, heat shock caused a significant increase of alpha 2M and AOG secretion. Thus, in astrocytes, alpha 2M is upregulated by cytokines and heat shock in the absence of insulin, while in the presence of insulin, cytokines function as negative regulators.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531673 TI - Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects. AB - Healing of an experimental bony defect in the rat's tibia was studied with an immunofluorescent technique to clarify when and where substance P (SP) and calcitonin gene-related peptide (CGRP) would develop. The normal tibia showed a few SP- and CGRP-immunofluorescent nerve fibres. In the experimental tibia, the number of these fibres increased on the 6th day after operation, reached a peak of proliferation on the 15th day and reverted to normal after the 24th day. The changes were associated with the development and decay of callus tissue suggesting that harmful stimuli from the injured site in a bone could be mediated by sensory nerves throughout the repair period. Most of the SP- and CGRP immunofluorescence was seen near the vessels, frequently in the same nerve fibres. The SP- and CGRP-immunofluorescent nerves seemed to take part jointly in callus formation through the enhancement of local blood flow. PMID- 7531674 TI - Evidence of specificity in the action of angiogenesis antagonists. AB - It is not known whether each type of angiogenesis reaction has a specific antiangiogenic mechanism and to what extent angiostatic agents can exert additional or synergistic effects. The objective of this study was to elucidate the way in which systemic angiostatic drug treatment by either one drug at a time or two in combination affects distinct angiogenesis reactions in one and the same vascular bed. The chemically unrelated drugs which were used were dexamethasone (DE) and ciclosporin A (CS) at nontoxic doses in the range used clinically. These drugs are known to act angiostatically in the rat. The rat mesenteric window angiogenesis assay was used to assess the systemic effect of DE, CS and DE plus CS in saline-mediated angiogenesis (SMA) and mast-cell-mediated angiogenesis (MCMA). Angiogenesis was quantified by microscopic and morphometric means, one variable being the number of vessel profiles per unit tissue length (No/UL), a measure related to the degree of microvascular branching and tortuosity. Whereas CS significantly suppressed No/UL in SMA without substantially affecting MCMA, DE plus CS significantly suppressed No/UL in MCMA, but, unexpectedly, somewhat increased No/UL in SMA. As compared with either drug given alone, the coadministration of the drugs clearly did not enhance the antiangiogenic effect in SMA. The drugs, whether alone or in combination specifically and selectively suppressed the angiogenesis reactions which were examined, thereby suggesting distinct antiangiogenic mechanisms in SMA and MCMA. PMID- 7531675 TI - Elimination of the diatrizoate-induced effects on the microcirculation by the prostacyclin derivative, iloprost. AB - RATIONALE AND OBJECTIVES: Contrast media, especially high-osmolar ionic compounds elicit drastic effects on the microcirculation. In some areas of the microcirculatory network, blood flow decreases up to total stasis; in other areas, blood velocity increases. The authors studied the effect of the stable prostacyclin derivative, iloprost, added to the diatrizoate formulation on the number of perfused microvessels in the rat intestine. METHODS: Diatrizoate (600 mg iodine/kg) and increasing concentrations of iloprost, ranging from a total dose of 0 to 560 ng/kg, were injected intravenously into groups of four anesthetized male rats. The number of unperfused microvessels in the intestine were determined by in vivo microscopy. Each animal received two treatments at an interval of 35 minutes. One treatment consisted of diatrizoate alone, the other treatment consisted of diatrizoate plus iloprost. Two animals of each group received first diatrizoate alone and then diatrizoate plus iloprost. The other two animals first received diatrizoate plus iloprost and then diatrizoate alone. RESULTS: Diatrizoate alone reduced the number of perfused microvessels by 45%. The addition of iloprost resulted in a dose-dependent improvement of microcirculation. At a dose of 280 ng/kg, the effects of diatrizoate were completely abolished. Increasing the dose further diminished this effect. CONCLUSIONS: The addition of iloprost to diatrizoate eliminates the deleterious effects of diatrizoate on microcirculation. PMID- 7531676 TI - Improvement of the T-cell response to a non immunogenic peptide by its tandem association with a highly efficient T-helper peptide. AB - The 45-69 peptide, an helper T-cell epitope derived from the HIV nef protein is strongly immunogenic. A T-cell proliferative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein of the parasite Schistosoma mansoni, requires the presence of a carrier of the use of peptidic constructs. We demonstrate here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic constructs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent association of both peptides is necessary, since the coinjection of 45 69 and 115-131 peptides is not sufficient to induce a detectable anti-115-131 T cell response. The mutual orientation between the respective tandem peptides (45 115 and 115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and immunogenicity but both allowed to reveal the existence of a specific T-cell response normally undetectable using 115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction of vaccines. PMID- 7531677 TI - Diversity and genetic differentiation among subpopulations of Gliricidia sepium revealed by PCR-based assays. AB - Randomly amplified polymorphic DNA (RAPD), and a mitochondrial marker based on amplification of the V7 region of the mitochondrial small ribosomal RNA (srRNA) gene, were used to partition genetic variation within a single population of Gliricidia sepium sampled from Guatemala. Seventeen per cent of the variation detected with RAPDs was partitioned among subpopulations and indicated a greater level of discrimination than previously detected with isozymes. Cluster analysis indicated a direct relationship between this variation and the geographical distance between subpopulations. A polymorphism identified within the maternally inherited mitochondrial V7 srRNA product, which relied on digestion with restriction endonucleases, confirmed the genetic subdivision identified with RAPDs, and suggested a relatively limited role for seed in gene dispersal. PMID- 7531678 TI - In response to Chiang et al. PMID- 7531679 TI - Non-Hodgkin's lymphomas in Turkey: eighteen years' experience at the Hacettepe University. AB - In this retrospective study, 470 patients with non-Hodgkin's lymphoma (NHL) who had been followed in the Hacettepe University Medical Oncology Department between 1973 and 1990, were evaluated to establish their epidemiologic, clinical and therapeutic characteristics. Out of 470 patients, 302 (62.2%) were male and 168 (37.8%) were female. The ages ranged from 16 to 85, with a median of 44 years. Constitutional symptoms were present in 46.4% of the patients. According to the Working Formulation, low, intermediate, and high-grade lymphomas comprised 33.4%, 54.9%, and 12.7%, respectively. The most common extranodal presentation was gastrointestinal. The chemotherapy regimens most commonly used were CVP (cyclophosphamide, vincristine, prednisone), BCNOP (bleomycin, cyclophosphamide, mitoxantrone, vincristine, prednisone), CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) and CHOP-Bleo (cyclophosphamide, doxorubicin, vincristine, prednisone, bleomycin). The response rates and the survival figures attained with these regimens were not statistically significantly different (P > 0.05). In the Cox multivariate model, pathologic grade, leukopenia, responsiveness to chemotherapy, bone marrow involvement and age were the important factors influencing the disease-free survival, while responsiveness to chemotherapy, age, presence of constitutional symptoms, pathologic grade, extranodal presentation and stage were the important factors influencing the overall survival. The distribution of NHL according to grade and stage was similar to that in western societies, while constitutional symptoms and lymphomas of the small intestine including immunoproliferative small intestinal disease were more common in Turkey. PMID- 7531680 TI - New human oral squamous carcinoma cell line and its tumorigenic subline producing granulocyte colony-stimulating factor. AB - A new human carcinoma cell line, MISK81-5, was established from a metastatic lymph node of oral squamous cell carcinoma. Immunocytochemical and ultrastructural observations revealed an obvious epithelial origin of the cell line. Chromosome analysis revealed a hypertriploid karyotype with numerical and structural anomalies. MISK81-5 cells could form a tumor mass in the subcutaneous tissue of recipient BALB/c athymic mice only when coinjected with Matrigel. A stem cell assay revealed that conditioned medium (CM) of MISK81-5 contained granulocyte colony-stimulating factor (G-CSF) or interleukin-6 activity. Quantitation by ELISA disclosed a higher concentration of G-CSF in the CM of MISK81-5 than in the CM of other squamous and gastric carcinoma cell lines. The sMISK, that was derived from MISK81-5 as a subpopulation of the cell line having higher tumorigenicity, also showed a similar hematopoietic stimulating activity to that of MISK81-5. These characteristics of the MISK81-5 cell line and its subpopulation, sMISK will be useful for studying the biological behavior of oral squamous cell carcinomas and its relation to hematopoietic stimulating factors. PMID- 7531681 TI - Immunohistochemical analysis of cyclin D1 protein in hematopoietic neoplasms with special reference to mantle cell lymphoma. AB - Immunohistochemical expression of PRAD1/cyclin D1 protein has been investigated in 106 tissue specimens of 104 cases of lymphoma, non-neoplastic lymphoid disorders and other hematologic malignancies by employing the monoclonal antibody 5D4 with formalin-fixed paraffin-embedded sections, using the microwave oven heating method. Positive neoplastic cells were found in 60 (74%) of 81 cases of non-Hodgkin's lymphoma. The positivity pattern was nuclear in 17 (85%) of 20 cases of mantle cell lymphoma in which cytoplasmic staining was also seen. This pattern of cyclin D1 positivity was in contrast to the negative staining of normal reactive mantle zones. In the other cases, positivity appeared to lie within the cell cytoplasm without nuclear staining, and most of the nodal follicular and diffuse B-cell lymphomas variously expressed PRAD1/cyclin D1. In contrast, the reaction was absent in a significant number of T-cell and extranodal B-cell lymphomas. Immunolocalization of PRAD1/cyclin D1 expression appears to be a useful diagnostic adjunct to discriminate mantle cell lymphoma from other non-Hodgkin's lymphomas. PMID- 7531683 TI - Voltage-activated K channel in luminal membrane of marginal cells of stria vascularis dissected from guinea pig. AB - Inward rectifying K channel activity was observed in the luminal membrane of the marginal cell by use of the patch clamp technique. When pipette solution contained 150 mM KCl, the current reversed at 10.2 +/- 8.8 mV (N = 27). The channel current showed multiple subconductance levels and depolarization activated the channel very slowly (order of seconds). Current-voltage relationships in cell-attached mode showed inward rectification. The calculated slope conductance of inward and outward currents were 18 +/- 7 pS and 5.4 +/- 2.0 pS (N = 27), respectively. When pipette solution contained 75 mM NaCl and 75 mM KCl, the current reversed -7.0 +/- 7.6 mV (N = 6). When pipette solution contained NaCl or Na gluconate, the channel currents were outwardly directed within a range of -60 to 60 mV. These results suggested that the channel is selective to K+. The channel showed immediate run down after excision, and this rundown was not prevented by elimination of cytoplasmic Ca2+. The channel activity was not affected by an increase of intracellular cAMP and cGMP, an application of cytoplasmic catalytic subunit of PKA with ATP. Taking its density into consideration, the channel seems to contribute considerably to the K+ conductance of the luminal membrane of the marginal cell. PMID- 7531682 TI - Risk factors related to liver metastasis in colorectal carcinoma: a multivariate analysis of clinicopathologic and immunohistochemical variables. AB - Specimens from 48 consecutive patients undergoing surgery for colorectal carcinoma and having synchronous or metachronous liver metastases (Group 1) and those from 52 consecutive patients who had no evidence of hepatic metastases within at least 5 year after colorectal resection (Group 2) were selected and compared using a multiple logistic regression analysis. A multivariate analysis using a stepwise logistic regression revealed six independent risk factors significantly related to hepatic metastases. In addition, the following logistic regression model was obtained from this analysis. P = exp a/(1 + exp a): a = 3.524(SM-V) + 2.731(Ex-V) + 2.718(E/M) + 2.562(Lo) + 1.858(p53) + 1.941(HIR) - 4.397, where P is the probability of hepatic metastasis given six independent risk factors (E/M, Ex/M ratio; Lo, location; HIR, host inflammatory cell reaction). When the estimated probability "P" in the above logistic regression model is more than 0.55 after an examination of surgical specimens, we must consider adjuvant chemotherapy and closely monitor the patient to ensure early detection of hepatic metastases. PMID- 7531685 TI - A simple staining method for dna and rna blotted on a membrane using a polyethyleneimine-enzyme conjugate. AB - We describe a new method for the detection of nucleic acids fixed on a membrane involving enzyme-conjugated polyethyleneimine. This method is based on the strong ionic interaction of the cationic polymer, polyethyleneimine (aziridine), with nucleic acids. Sensitivities of 10 pg for DNA and 1 ng for rRNA were achieved in slot-spot blotting experiments. DNA fragments blotted onto a membrane by Southern transfer can be stained after hybridization with a labeled nucleic acid probe. By combining this method with a non-isotopic detection system, the positions of probe hybridized and non-hybridized signals were visualized as distinct colors on the same membrane without superimposing any other image. This technique should have wide application for the detection of nucleic acids fixed on membranes in blot-hybridization experiments. PMID- 7531684 TI - Visualisation of domains in the avian tectorial and otolithic membranes with monoclonal antibodies. AB - The staining patterns observed with six monoclonal antibodies (mAbs) raised in vitro against a fraction derived from the utricular macula were examined with cryosections of the auditory and vestibular organs of the avian inner ear. These antibodies revealed several distinct domains within the gelatinous membranes. Three different labelling patterns were observed in the tectorial membrane. Staining was seen either throughout the entire tectorial membrane, restricted to its core, or in a narrow zone lying close to the surface of the basilar papilla. In the maculae, the mAbs stained either the striolar region of the otolithic membrane or the entire structure. One monoclonal which labelled otoconia, stained small otoconia in their entirely, whilst larger otoconia were only labelled around their periphery. Only one of the mAbs stained the cupulae of the semi circular canal ampullae and this antibody stained neither the tectorial nor the otolithic membranes. These results suggest that there are biochemically distinct regions in the gelatinous membranes of the inner ear and indicate that these matrices are not simply homogeneous extracellular structures. PMID- 7531686 TI - Characterization of gangliosides of epithelial cells of calf small intestine, with special reference to receptor-active sequences for enteropathogenic Escherichia coli K99. AB - Glycolipids were prepared from epithelial cells of the small intestine of a newborn calf and assayed for Escherichia coli K99 binding activity on thin-layer chromatograms and in microtiter wells. The bacteria did not bind to any of the non-acid glycolipids, while in the acid fraction several binding-positive glycolipids were detected. The acid glycolipids were isolated and characterized by mass spectrometry, proton NMR spectroscopy and other methods. The following gangliosides were identified, mainly from the epithelial cells from the upper part of the small intestine: NeuAc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuAc GM3), NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuGc-GM3), GalNAc beta 1 4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM2), Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM1), and NeuGc alpha 2 3Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc GD1a). A positive binding was demonstrated to NeuGc-GM3, NeuGc-GM2, and NeuGc GD1a, while NeuAc-GM3 and NeuGc-GM1 were negative. The binding pattern differed somewhat for total acid glycolipids of epithelial cells from three different parts of the small intestine. Based on binding preferences of E. coli K99 to a number of glycolipids of various origins, in comparison with calculated minimum energy conformations, a binding epitope was delineated. PMID- 7531687 TI - Cloning and functional expression of human inducible nitric oxide synthase (NOS) cDNA from a glioblastoma cell line A-172. AB - Nitric oxide (NO) is a messenger molecule with diverse functions throughout the body. The inducible type of nitric oxide synthase (NOS) is considered to be a key molecule in the immune responses to bacteria, parasites, and tumors, and its gene expression is regulated by cytokines. We isolated 3 overlapping partial inducible NOS cDNA clones from a human glioblastoma cell line A-172 induced by IL-1, TNF alpha, and IFN-gamma. The 3,963-bp human glioblastoma inducible NOS cDNA contained the longest open reading frame of 3,459 bp, which encoded a polypeptide of 1,153 amino acids with a calculated molecular mass of 131 kDa. This human inducible NOS possessed consensus recognition sites for the cofactors FMN, FAD, and NADPH and calmodulin recognition sites, and displayed 48.1% sequence identity with the endothelial type, 43.1% with the neuronal type, and 99.3% with the inducible type from hepatocytes, and 99.9% with the inducible type from chondrocytes and adenocarcinoma. An expression plasmid consisting of pSG5 expression vector and cDNA containing the entire putative coding sequence was constructed and transfected into COS-1 cells. COS-1 cells showed nitric oxide synthase activity together with a 130 kDa immunoreactive band on Western blot analysis. PMID- 7531688 TI - Isolation and characterization of a rat testis glycosphingolipid based on gangliotetraosylceramide and having a blood group B determinant extended with beta 3-linked N-acetylgalactosamine. AB - Monoclonal antibody 14.2, directed against the GalNAc beta 3Gal alpha 3Gal beta epitope, was used to monitor the isolation of a novel glycosphingolipid from rat testes. Structural analysis was performed by mass spectrometry, proton NMR spectroscopy, gas chromatography-mass spectrometry after degradation, and exoglycosidase digestion. It was concluded that the structure was [formula: see text] This is the first report on an extended blood group B determinant. PMID- 7531689 TI - Characterization of an exchange reaction between soluble FKBP-12 and the FKBP.ryanodine receptor complex. Modulation by FKBP mutants deficient in peptidyl prolyl isomerase activity. AB - FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK 590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S. (1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S. (1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP.RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 microM for wild type FKBP versus 0.6 to 2.4 microM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP.RyR complex is in equilibrium with the cytosolic pool of FKBP (approximately 3 microM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity. PMID- 7531690 TI - Epitope insertions define functional and topological features of the Escherichia coli ferric enterobactin receptor. AB - The outer membrane protein FepA of Escherichia coli is the receptor for the ferric enterobactin siderophore complex and colicins B and D. A foreign antigenic determinant inserted into selected FepA sites allowed mutational analysis of receptor function and in situ immunological tracking of specific protein domains with respect to the bacterial cell compartment. Immunoblot analysis of bacterial proteins using an epitope-specific antibody detected the peptide determinant in the receptor fusions. The impact of the insertions on FepA function was examined by ferric enterobactin-mediated iron uptake experiments and colicin sensitivity tests. In all cases, FepA retained biological activity despite introduction of the foreign sequence. To further develop the topological model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their isolated membranes. One epitope resided in a region on the exterior of the cell, at the surface of the FepA protein, while other epitopes appeared to be localized to the periplasm or within the outer membrane. PMID- 7531691 TI - Active site studies of DT-diaphorase employing artificial flavins. AB - NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6 amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones. PMID- 7531693 TI - Glyceraldehyde-3-phosphate dehydrogenase selectively binds AU-rich RNA in the NAD(+)-binding region (Rossmann fold). AB - A 36-kDa protein that binds AU-rich RNA was purified from human spleen and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH has been previously demonstrated to bind tRNA with high affinity. Competition studies suggested that cytoplasmic GAPDH binds the AU-rich elements (AREs) of lymphokine mRNA 3'-untranslated regions with higher affinity than tRNA. The AUUUA-specific RNA binding activity of GAPDH was inhibited by NAD+, NADH, and ATP in a concentration-dependent manner, suggesting that RNA binding of GAPDH might involve the NAD(+)-binding region, or dinucleotide-binding (Rossmann) fold. This hypothesis was supported by experiments that localized RNA binding to the predicted N-terminal 6.8-kDa peptide, known to be involved in the formation of the NAD(+)-binding domain. The direct demonstration of ARE-specific binding protein activity localized to the NAD(+)-binding region of GAPDH supports the general concept that enzymes containing this domain may exhibit specific RNA binding activity and play additional roles in nucleic acid metabolism. Finally, cytoplasmic GAPDH was found in the polysomal fraction of T lymphocytes. Thus, the RNA binding specificity of GAPDH as well as its localization within the cell merit its strong consideration as a protein important in the regulation of ARE dependent mRNA turnover and translation in addition to its well described role in glycolysis. PMID- 7531692 TI - Mutation of Leu25 and Val27 introduces CC chemokine activity into interleukin-8. AB - Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu25 in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val27 residue, which points out from the same side of the beta-sheet as Leu25. In RANTES, Val27 is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu25-->Tyr, Val27-->Tyr and the double mutant Leu25-->Tyr, Val27--> Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [125I]IL-8 competition assays. Additionally, the Leu25-->Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [125I]MIP-1 alpha by IL-8 Leu25-->Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1 alpha ligand with an affinity only 12-fold less than MIP-1 alpha itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines. PMID- 7531694 TI - Crk interacts with tyrosine-phosphorylated p116 upon T cell activation. AB - Products of the crk oncogene are expressed in all tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130cas and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation. PMID- 7531695 TI - Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains. AB - Src homology 2 (SH2) domains are phosphotyrosine binding modules found within many cytoplasmic proteins. A major function of SH2 domains is to bring about the physical assembly of signaling complexes. We now show that, in addition, simultaneous occupancy of both SH2 domains of the phosphotyrosine phosphatase SH PTP2 (Syp, PTP 1D, PTP-2C) by a tethered peptide with two IRS-1-derived phosphorylation sites potently stimulates phosphatase activity. The concentration required for activation by the tethered peptide is 80-160-fold lower than either corresponding monophosphorylated peptide. Moreover, the diphosphorylated peptide stimulates catalytic activity 37-fold, compared with 9-16-fold for the monophosphorylated peptides. Mutational analyses of the SH2 domains of SH-PTP2 confirm that both SH2 domains participate in this effect. Binding studies with a tandem construct comprising the N- plus C-terminal SH2 domains show that the diphosphorylated peptide binds with 60-90-fold higher affinity than either monophosphorylated sequence. These results demonstrate that SH-PTP2 activity can be potently regulated by interacting via both of its SH2 domains with phosphoproteins having two cognate phosphorylation sites. PMID- 7531696 TI - Recombinant GST/CD36 fusion proteins define a thrombospondin binding domain. Evidence for a single calcium-dependent binding site on CD36. AB - CD36 is a multifunctional cell surface glycoprotein that acts as a surface receptor for thrombospondin (TSP), and thereby may mediate adhesive interactions between cells and substrata, platelets and other cells, and macrophages and apoptotic neutrophils. The identity of the TSP binding site on CD36 is controversial and may involve more than one structural domain. We have constructed a series of recombinant bacterial GST/CD36 fusion proteins that span nearly all of the CD36 molecule and have demonstrated that fusion proteins containing the region extending from amino acid 93 to 120 formed specific, saturable, and reversible complexes with TSP. As with intact CD36, binding was calcium-dependent, was independent of which ligand was immobilized, and was blocked by monoclonal antibodies to both CD36 and TSP. Stoichiometry and affinity of the fusion proteins for TSP were consistent with that of the intact protein. We also demonstrated that these fusion proteins competitively inhibited binding of TSP to purified platelet CD36 and to cell surface CD36 on peripheral blood monocytes and CD36 cDNA-transfected melanoma cells. These data demonstrate that the region between amino acids 93 and 120 has all of the characteristics required of the TSP binding domain. PMID- 7531697 TI - Islet-specific proteins interact with the insulin-response element of the glucagon gene. AB - Glucagon gene expression is negatively regulated by insulin at the transcriptional level. G3, a DNA control element located in the 5'-flanking sequence of the rat glucagon gene mediates the inhibition of transcription, which occurs in response to insulin. We show here that two islet-specific protein complexes C1A and C1B, bind to the A domain of G3, which is critical for the insulin response. These two complexes bind to overlapping sequences of the A domain and display very similar binding specificities. Point mutations in the A domain that affect binding of C1A and C1B result in both decreased G3 enhancer activity and insulin-mediated inhibitory effects with a close correlation between diminution of binding and function. One of the two complexes, C1A, is similar or identical to B1, a protein complexes interacting with the upstream promoter element of the glucagon gene, G1, implicated in the A cell-specific expression of the glucagon gene. Our data indicate that islet-specific proteins are involved in glucagon gene regulation by insulin. PMID- 7531698 TI - The kinase domain and membrane localization determine intracellular interactions between epidermal growth factor receptors. AB - Receptor tyrosine kinases play a central role in cellular growth, differentiation, and oncogenesis. All of these responses are triggered by growth factors interacting with the extracellular domain of transmembrane-spanning receptors, leading to dimerization and activation of an intrinsic tyrosine specific kinase activity by an allosteric mechanism. Precise mechanisms of receptor dimerization remain poorly understood, and current models suggest that the ligand binding domain plays a major determining role. To examine the role of the intracellular domain in the association of juxtaposing receptor molecules, the full-length epidermal growth factor receptor was transiently co-expressed in human 293 fibroblasts with a truncated receptor that lacks the extracellular domain. After metabolic labeling with [35S]methionine, the association of these receptor constructs was monitored by co-immunoprecipitation with an extracellular domain-specific antibody. Specific interactions found between these receptors were independent of ligand binding or an intact ATP-binding site. Truncated receptors that had sequences necessary for membrane localization, and that were capable of interacting with full-length receptor tyrosine kinase, also displayed constitutive kinase activity as well as the capacity to transphosphorylate kinase negative receptors. Receptor co-immunoprecipitation occurred between constructs that comprise the intracellular domains of the epidermal growth factor and beta platelet-derived growth factor receptors, and HER-2. Subsequent deletion analysis has identified the major region of epidermal growth factor receptor intracellular interaction to be within the kinase domain. PMID- 7531699 TI - Platelet-derived growth factor signal transduction through the interferon inducible kinase PKR. Immediate early gene induction. AB - The interferon-inducible, double-stranded RNA (dsRNA)-dependent eukaryotic initiation factor-2 alpha kinase PKR has primarily been characterized as a component of the interferon-mediated cellular antiviral response. Several lines of evidence now exist that suggest that PKR plays a role in the regulation of growth in uninfected cells. The most direct examples are the finding of an oncogenic variant of PKR and the effects of activators and inhibitors of PKR phosphorylation on the expression of platelet-derived growth factor (PDGF) inducible genes. Previous reports have shown that 1) dsRNA, a direct activator of PKR, induces the genes c-myc, c-fos, and JE; 2) 2-aminopurine, a chemical inhibitor of PKR, blocks the induction of these genes by serum; and 3) activated p21ras induces a cellular inhibitor of PKR. We report here that activation of PKR was correlated with the induction of the immediate early genes c-fos, c-myc, and JE by PDGF in the following situations: 1) PDGF induction of these genes, also inducible by dsRNA, was blocked by two inhibitors of PKR activation: 2 aminopurine and v-ras; 2) PDGF induction of another immediate early gene, egr-1, which could not be induced by dsRNA, was not blocked by 2-aminopurine or v-ras; 3) agents that reverse v-ras inhibition of PKR activation also reversed the v-ras block of PDGF induction of c-myc, c-fos, and JE; 4) down-regulation of PKR protein levels by antisense inhibition of translation blocked the induction of c myc, c-fos, and JE by PDGF, but had no effect on egr-1 induction; and finally, 5) PKR was autophosphorylated in vivo in response to PDGF. These results provide direct evidence that PKR activation functions as a second messenger in a growth factor signal transduction pathway. Thus, PKR may serve as a common mediator of growth-promoting and growth inhibitory signals. PMID- 7531700 TI - Identification and characterization of a functional murine FLT3 isoform produced by exon skipping. AB - The FLT3 gene encodes an hematopoietic receptor related to the receptors for colony-stimulating factor 1, FMS, and for Steel factor, KIT. The extracellular part of these molecules is exclusively composed of five immunoglobulin (Ig)-like domains, designated 1 to 5, from the amino terminus to the carboxyl terminus of the extracellular region. We have isolated a unique murine FLT3 cDNA that codes for a variant isoform of FLT3, devoid of the fifth Ig-like domain, by comparison with the prototypic form. The corresponding mRNA is the result of a splicing event that leads to the elimination of two coding exons. mRNA coding for this variant was detected in almost all the tissues expressing the mRNA coding for the prototypic molecule, although at a lower level. Ligand-induced tyrosine phosphorylation of the two isoforms was equivalent in COS-1 transfected cells, indicating that the fifth Ig-like domain is not strictly necessary for either ligand-binding or kinase activation. PMID- 7531701 TI - c-kit ligand mediates increased expression of cytosolic phospholipase A2, prostaglandin endoperoxide synthase-1, and hematopoietic prostaglandin D2 synthase and increased IgE-dependent prostaglandin D2 generation in immature mouse mast cells. AB - We have examined the cytokine regulation of IgE-dependent prostaglandin (PG) D2 generation in mouse mast cells by assessing the changes in the levels of the transcript, translated protein, and activity of the enzymes involved in the synthesis of PGD2 from endogenous arachidonic acid. When mouse mast cells, derived by culture of bone marrow cells with WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 (BMMC), were cultured in recombinant ckit ligand (KL), sensitized with IgE, and stimulated with antigen, PGD2 generation increased 3-fold; when KL was combined with IL-3, IL-9, or IL-10, PGD2 generation increased 6-8-fold above that produced by the cells cultured in IL-3 alone. The increased IgE-dependent PGD2 generation by BMMC was apparent after 1 day of culture, reached a maximum after 2-4 days of culture, and was dose-dependent for KL and for each of the accessory cytokines. IgE-dependent generation of leukotriene C4 increased 2-fold after the cells were cultured with KL and was not increased by the addition of IL-3, IL-9, or IL-10. Assays for steady-state transcripts by RNA blotting, for protein by SDS-PAGE/immunoblotting, and for function by enzymatic activities revealed that KL alone stimulated the increased expression of cytosolic phospholipase A2 (cPLA2), prostaglandin endoperoxide synthase (PGHS)-1, and the terminal enzyme, hematopoietic PGD2 synthase, without a change in expression of 5-lipoxygenase. IL-3, IL-9, and IL-10 each enhanced the KL-induced expression of PGHS-1. In contrast, transcripts for PGHS-2, which were detected transiently after the cells had been cultured for 5 h in KL+IL-3, were not expressed during the period of subsequent increase in IgE-dependent PGD2 generation. These findings demonstrate that KL up-regulates expression of cPLA2, PGHS-1, and hematopoietic PGD2 synthase, leading to a relatively selective increase in IgE-dependent production of PGD2 from endogenously released arachidonic acid in BMMC, and they provide the first example of cytokine regulation of hematopoietic PGD2 synthase. PMID- 7531702 TI - Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus crmA gene product. AB - crmA is a cowpox virus gene that encodes a protease inhibitor of the serpin family. The only reported target for the CrmA protein is the cysteine protease interleukin-1 beta converting enzyme (ICE). ICE, by virtue of its homology to the Caenorhabditis elegans cell death protein Ced-3, has been suggested to play a fundamentally important role in mammalian apoptosis. We hypothesized that a function of crmA may be to inhibit cell death, since a major mechanism of viral clearance is the immune system-mediated induction of apoptosis of infected cells. The tumor necrosis factor receptor and the Fas antigen are two cytokine receptors which, by engaging and activating the death pathway, can eliminate virus-infected cells. Remarkably, crmA was found to be an exceptionally potent inhibitor of apoptosis induced by both these receptors, capable of blocking the cell death program even at pharmacological doses of the death stimulus. Therefore, an important new function for crmA is the inhibition of cytokine-induced apoptosis. Further, the data suggest that a protease, either ICE or a related crmA inhibitable protein, is a component of the Fas- and tumor necrosis factor-induced cell death pathways. PMID- 7531703 TI - Matrix nonenzymatic glycosylation leads to altered cellular phenotype and intracellular tyrosine phosphorylation. AB - The effect of matrix nonenzymatic glycosylation on signal transduction and the cellular phenotype was examined. Human microvascular endothelial cells were plated on control or glycated basement membrane-like matrix. Cells exhibited a decrease in their ability to adhere and spread on modified matrix. The pattern of intracellular tyrosine phosphorylation was examined by Western Immunoblotting; a band with 65 kDa mobility exhibited a marked reduction of tyrosine phosphorylation in cells adherent to modified matrix. Immunoprecipitation experiments provided evidence that this band is paxillin, a member of focal adhesion proteins. Immunoprecipitation with antibodies against focal adhesion kinase (pp125FAK), the enzyme that is thought to regulate paxillin tyrosine phosphorylation, also demonstrated a reduction in tyrosine phosphorylation of pp125FAK. To confirm these biochemical data, adherent cells were examined for the distribution of paxillin, using immunofluorescence microscopy; paxillin was seen in focal points peripherally located in cells on normal matrix, but lacked this pattern in cells on modified matrix. Actin filaments were also disorganized in cells plated on modified matrix. These data suggest that matrix nonenzymatic glycosylation can interfere with and potentially alter cellular phenotype and intracellular signaling. PMID- 7531704 TI - Distinct domains of the protein tyrosine kinase tyk2 required for binding of interferon-alpha/beta and for signal transduction. AB - tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-alpha/beta binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-alpha 8 and partial signaling. While no contribution of this protein toward interferon-beta binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-alpha and -beta binding and signaling. PMID- 7531705 TI - A single heparin binding region within the fibrinogen-like domain is functional in chick tenascin-C. AB - Tenascin-C binds to cell surface and matrix proteoglycans and to heparin. Two heparin binding regions have recently been localized per tenascin-C monomer, one in the C-terminal fibrinogen-like domain and the other in fibronectin type III repeats 3-5. Here we show that a single region in each subunit is necessary and sufficient for heparin binding by whole tenascin-C at physiological ionic strength. First, native tenascin-C was bound to heparin-agarose and digested with Pronase. A 29-kDa fragment retained on the heparin column was recognized by a monoclonal antibody against the fibrinogen-like domain. In contrast, small fragments labeled by an antibody against fibronectin type III repeats 2-5 were released. Second, mild tryptic digestion of tenascin-C yielded two related fragments of 180 and 170 kDa. The latter missed part of the fibrinogen domain and had lost affinity for heparin, in contrast to the former. Finally, chick tenascin C constructs were recombinantly expressed in human cells. Whereas the complete protein and a mutant lacking fibronectin type III repeats 1-5 bound to heparin agarose, recombinant tenascin-C missing the C-terminal fibrinogen-like globe did not. Thus, whole chick tenascin-C contains one essential heparin binding region per subunit, located in the fibrinogen-like domain within 10 kDa from the C terminus. PMID- 7531706 TI - Lck unique domain influences Lck specificity and biological function. AB - Src-family tyrosine kinases share structural and amino acid sequence homology, particularly in the catalytic domain as well as in the SH2 and SH3 domains of the regulatory region. However, each src-family member also contains a unique domain which is specific to and characteristic of each individual tyrosine kinase. These unique or specific domains may contribute to the functional specificity of each src-family kinase. To address this possibility, we analyzed the kinase activities and substrate specificities of the lymphoid src-kinase, pp56lck, and a mutant of pp56lck lacking its specific domain. Our data show that both the wild type enzyme and the specific domain-deleted mutant displayed similar affinities for ATP and the non-physiological substrate denatured enolase. However, the specific domain deleted mutant failed to phosphorylate a number of physiological substrates of pp56lck. In addition, the ability of pp56lck to mediate induction of the interleukin-2 promoter was strongly impaired upon deletion of its specific domain. Thus, the unique domain is not required for the intrinsic kinase activity of pp56lck, however, it influences substrate preference and contributes to the unique physiological function of this src-family tyrosine kinase. PMID- 7531707 TI - Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences. AB - This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to 2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene. PMID- 7531708 TI - Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells. AB - The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist specific receptor regulation. PMID- 7531709 TI - Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA. AB - 2'-5' oligoadenylate (2-5(A)) synthetase and protein kinase, RNA activated (PKR) are the only two known enzymes that bind double-stranded RNA (dsRNA) and get activated by it. We have previously identified their dsRNA binding domains, which do not have any sequence homology. Here, we report a profound difference between the two enzymes with respect to the structural features of the dsRNA that are required for their activation. The adenoviral virus-associated type I (VAI) RNA cannot activate PKR, although it binds to the protein and thereby prevents its activation by authentic dsRNA. In contrast, we observed that VAI RNA can both bind and activate 2-5(A) synthetase. Mutations in VAI RNA, which removed occasional mismatches present in its double-stranded stems, markedly enhanced its 2-5(A) synthetase-activating capacity. These mutants, however, are incapable of activating PKR. Other mutations, which disrupted the structure of the central stem-loop region of the VAI RNA, reduced its ability to activate 2-5(A) synthetase. These debilitated mutants could bind to the synthetase protein, although they fail to bind to PKR. PMID- 7531710 TI - Bigeminal rhythm and cardiac failure in rheumatic heart disease. PMID- 7531711 TI - Immunologic tolerance: development and disruption. AB - Loss of tolerance to self-antigens is thought to have a role in a variety of common diseases. Studies in animal models of such diseases as systemic lupus erythematosus have provided new insights into how self-tolerance is broken or bypassed and how it may be restored. These and other experiments also are clarifying how self-tolerance is normally established; the T lymphocyte appears to be the dominant player. PMID- 7531712 TI - Effects of caloric or protein restriction on insulin-like growth factor-I (IGF-I) and IGF-binding proteins in children and adults. AB - Serum concentrations of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1), -2, and -3 are influenced by dietary intake in normal adults. These studies were undertaken to determine the influence of dietary factors on these proteins in children and to compare the responses in children to those in adults. Eight adults and eight pubertal children underwent energy restriction (50% reduction intake) for 6 days and were refed a normal diet for an additional 6 days. Basal energy intakes during the prerestriction periods were 70 Cal/kg in children and 35 Cal/kg in adults. A second group of 8 adults and 6 children underwent protein restriction (decreased from 1.0 to 0.66 g/kg in both groups) for 6 days and were refed a normal diet for 6 days. Calorie restriction resulted in a significant decrease in nitrogen balance in adults and children. Likewise, IGF-I concentrations declined significantly in both adults and children. In contrast, IGFBP-1 concentrations were significantly increased in adults (from 40 +/- 6 to 62 +/- 4 ng/mL; P < 0.05), but not in children. Serum concentrations of IGFBP-2 did not change in either group in response to energy restriction. IGFBP-3 declined significantly in the children (3189 +/- 90 to 2843 +/- 96 ng/mL; P < 0.05), but not in the adults. Protein restriction also caused negative nitrogen balance in both children and adults and a decline in the mean IGF-I concentration in the adults. IGFBP-2 concentrations rose significantly in both adults (131 +/- 15 to 164 +/- 15 ng/mL; P < 0.005) and children (126 +/- 13 to 158 +/- 15 ng/mL; P < 0.05) in response to protein restriction and returned to normal during refeeding. IGFBP-3 was slightly, but significantly, reduced in response to protein restriction in adults (3518 +/- 180 to 3328 +/- 151 ng/mL; P < 0.05), but not children. The findings indicate that protein or energy restriction in children leads to changes in IGF-I or specific IGFBPs. Changes in IGFBP-2 are sensitive to protein restriction, and measurement of IGFBP-2 may be useful in monitoring the response to refeeding in children who have been ingesting suboptimal amounts of protein. PMID- 7531713 TI - H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. AB - The expression of H19 and insulin-like growth factor-II (IGF-II) genes is important for fetal growth, and the misexpression of these genes may also be involved in the development of some tumors. In human fetal adrenals, H19 and IGF II expression levels are very high. We show here that H19 is strongly expressed (approximately 50% of the expression in fetal adrenals and 6-fold higher than that in adult liver) in normal adult adrenals (n = 9), adrenocortical adenomas (n = 28), and hyperplastic adrenals (n = 11). In four hormonally active adrenocortical carcinomas, very low levels of H19 ribonucleic acid (RNA) were detected, whereas IGF-II was highly expressed. In cultured adrenocortical cells, ACTH, (Bu)2cAMP, and cholera toxin increased H19 RNA accumulation 2- to 5-fold (P < 0.01), but had no significant effect on IGF-II messenger RNA levels. In pheochromocytomas (n = 22), H19 expression was variable, on the average, about 13% of the expression in the adjacent adrenal cortex. In primary cultures of pheochromocytoma cells, H19 RNA was not detectable via Northern blot analysis. Our data show that H19 expression is maintained at high levels in adult human adrenals and benign neoplasms. H19 RNA is up-regulated by ACTH in adult adrenocortical cells. The very low levels of H19 expression in hormonally active adrenocortical carcinomas suggest that loss of H19 expression may be associated with malignancy in these neoplasms. PMID- 7531714 TI - Identification of transformed alpha 2-macroglobulin as a growth hormone-binding protein in human blood. AB - We have found that the human GH forms complexes with the purified proteinase inhibitor, alpha 2-macroglobulin. This inhibitor occurs in two different forms in human blood, known as native and transformed alpha 2-macroglobulin. It could be clearly demonstrated by chromatography and electrophoresis combined with autoradiography that the GH binds specifically to the transformed inhibitor, but not to the native protein. The binding was characterized as being mainly noncovalent to specific binding sites present only in the transformed inhibitor molecule. Binding analysis using antibody precipitation technique revealed two different classes of binding sites with dissociation constants of 0.49 +/- 0.12 and 61 +/- 8 mumol/L for high and low affinity binding sites, respectively. Distribution analysis of 125I-labeled GH in whole plasma suggested that the hormone is bound to different proteins: 1) to the high affinity GH-binding protein, and 2) to the low affinity GH-binding protein identified as transformed alpha 2-macroglobulin. PMID- 7531715 TI - Regulation of insulin-like growth factor-binding protein-4 in human endometrial stromal cell cultures: evidence for ligand-induced proteolysis. AB - The present study investigates the regulation of IGFBP-4 levels by insulin-like growth factor (IGF) peptides in human endometrial stromal cell cultures. A 24 kilodalton (kDa) IGF-binding protein (IGFBP) secreted by stromal cells was identified as IGFBP-4 by immunoprecipitation and Western ligand blotting. Western ligand blot analysis of conditioned medium showed that treatment of stromal cells with IGF-I or IGF-II induced a dose-dependent reduction of detectable IGFBP-4 levels. Two IGF analogs that bind type I IGF receptors, but have reduced affinity for IGFBPs, increased detectable levels of IGFBP-4, and their ability to reduce IGFBP-4 at high concentrations was positively correlated with their affinity for this binding protein. LR3-IGF-I, which has 1000-fold lower binding affinity than IGF-I, increased detectable IGFBP-4 at all concentrations tested. Des-(1-3)-IGF I, whose affinity for IGFBP-4 is 30-fold lower than that of IGF-I, increased detectable IGFBP-4 at low concentrations (0.1-10 ng/mL), but reduced its levels at 100 ng/mL, consistent with the significant binding to IGFBP-4 at this concentration. In contrast, IGFBP-4 was undetectable in cultures receiving Leu27 IGF-II, which has reduced affinity for the type IIGF receptor but unaltered affinity for IGFBPs and the type II receptor. High concentrations of insulin (100 ng/mL), which interacts with type I IGF receptors without binding IGFBPs, also increased detectable levels of IGFBP-4 in stromal cultures. The addition of IGF-I or IGF-II to cell-free conditioned medium from stromal cells cultured in the absence of IGFs resulted in the reduction of detectable endogenous IGFBP-4 levels. The effects of IGFs on IGFBP-4 levels in this cell-free system were time, temperature, and pH dependent and were prevented by the serine proteinase inhibitor, aprotinin, by the divalent cation chelator, EDTA, and by the metal ion chelator, 1,10-phenanthroline. Western immunoblotting showed that the IGF-induced reduction of intact 24-kDa IGFBP-4 was accompanied by the generation of an immunoreactive fragment of approximately 16 kDa, which was not detectable by Western ligand blotting. Cell-free conditioned medium from endometrial stromal cultures proteolyzed covalently cross-linked [125I]IGF-II-IGFBP-4 complexes in the absence of added IGFs, generating an 18-kDa radiolabeled fragment, and addition of free IGF peptide did not enhance the degradation of IGF-II-IGFBP-4 complexes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531716 TI - Development of a simple valid method for the complete removal of insulin-like growth factor (IGF)-binding proteins from IGFs in human serum and other biological fluids: comparison with acid-ethanol treatment and C18 Sep-Pak separation. AB - Insulin-like growth factor (IGF)s circulate in plasma as large mol wt proteins bound to specific proteins, termed IGF-binding proteins (IGFBPs). As IGFBPs have been shown to produce artifacts in IGF radioligand assays, various extraction procedures have been proposed to eliminate IGFBPs from biological samples before radioligand assays. Comparison of acid-ethanol and C18 Sep-Pak extraction methods, the two most widely used procedures for separation of IGFs from IGFBPs in human serum samples, with the established gold standard (Sephadex G-75 acid gel filtration) revealed that a significant amount of IGFBP activity survived the acid-ethanol extraction and C18 Sep-Pak separation techniques. We, therefore, have developed a simple novel method comprising a combination of two techniques, involving separation based on size and separation based on centrifugation. In this method, serum samples were acidified and applied to Bio-Spin columns containing BSA-pretreated Bio-Gel Polyacrylamide-10 (P-10). Upon centrifugation, IGFBPs eluted in the void volume. IGFs were then eluted with 1 mol/L acetic acid containing 0.1 mol/L NaCl upon subsequent centrifugation. The efficacy of Bio Spin P-10 separation for the complete removal of IGFBPs was determined by Western ligand blot analysis and determination of IGFBP-3 levels by RIA in the extracted serum samples. The recovery of exogenously added IGF-I to the serum samples was greater than 90%. Comparison of IGF-I and IGF-II values determined in 12 human serum samples after Bio-Spin P-10 separation with those obtained after separation with the established gold standard method (Sephadex G-75) revealed a correlation greater than 0.9. In contrast to the established gold standard method, which is tedious and time consuming, Bio-Spin P-10 separation offers the advantage of speed, such that 50 or more samples can be processed in less than 4-6 h. Application of Bio-Gel P-10 gel filtration to determine the IGF-I and IGF-II levels in 14 normal and 15 age-matched postmenopausal osteoporotic women revealed that 1) both IGF-I and IGF-II levels were reduced by 30% (P < 0.01) and 20% (P < 0.05), respectively, in osteoporotics; and 2) both IGF-I and IGF-II levels correlated with bone mineral density in the pooled data from normal and osteoporotic populations even when age was held constant (P < 0.05). PMID- 7531717 TI - Decidual histamine release and amplification of prostaglandin F2 alpha production by histamine in interleukin-1 beta-primed decidual cells: potential interactive role for inflammatory mediators in uterine function at term. AB - Inflammatory cytokines such as interleukin-1 (IL-1) have been implicated as paracrine mediators of decidual prostaglandin (PG) production during preterm labor. The aim of the present in vitro study was to investigate a similar potential role for the inflammatory autacoid histamine. Histamine action on human primary decidual cell cultures was monitored by measuring both PGF2 alpha production and PG precursor release. Histamine release from decidual cell suspensions was measured in response to a variety of putative mast cell secretagogues. Histamine stimulated a dose-dependent increase in PGF2 alpha production and [14C]arachidonate release from prelabeled decidual cells, with ED50 values around 1 and 2 mumol/L, respectively. Pretreatment of cells with IL-1 beta enhanced maximal PGF2 alpha production in response to histamine by approximately 4-fold. Mepyramine, an H1 receptor antagonist, completely blocked this PGF2 alpha response. The mean histamine content of unpurified decidual cells was approximately 81 pmol/10(6) cells. Upon challenge with antihuman immunoglobulin E, these cells exhibited a dose-dependent release of histamine. Basal release from decidual cell suspensions and release in response to antiimmunoglobulin E (1:400) or A23187 (1 mumol/L) were 7.6 +/- 2.7%, 25.5 +/- 0.9%, and 63.5 +/- 5.6% of the total histamine content, respectively. No significant changes in histamine secretion were seen in response to compound 48/80, substance-P, phorbol ester, or bacterial endotoxin. These in vitro findings support a potential role for histamine as a local regulator of phospholipase-A2 and PGF2 alpha production in human term decidua. The interaction of this autacoid with other inflammatory mediators, such as IL-1, could play a role in the control of PG production during preterm labor. PMID- 7531718 TI - Human histocompatibility leukocyte antigen-DR and heat shock protein-70 expression in eye muscle tissue in thyroid-associated ophthalmopathy. AB - To investigate the expression and localization of human leukocyte antigen (HLA) DR and heat shock protein-70 (HSP-70) in orbital tissue from patients with thyroid-associated ophthalmopathy (TAO), we carried out an immunohistochemical study using anti-HLA-DR and anti-HSP-70 monoclonal antibodies and a streptavidin biotinperoxidase detection system. Eye muscle tissues were obtained at surgery from 38 patients with TAO and 8 control subjects. HLA-DR expression on eye muscle cells was demonstrated in orbital tissue from 2 of 3 untreated patients and 2 of 35 patients who had been treated with orbital irradiation or corticosteroids, in all of whom lymphocytic infiltration was also demonstrated. HLA-DR was not detected on eye muscle cells from 8 normal controls studied. HLA-DR was expressed on endothelial cells and interstitial cells from almost all patients with TAO and all 8 control subjects. HSP-70 was detected in eye muscle cells from 31 of the patients with TAO, including all 3 untreated patients, and 3 of the controls. Although the degree of HSP-70 expression did not correlate with the severity of the ophthalmopathy, significant expression of HSP-70 in eye muscle cells was more often demonstrated in patients with eye disease of short duration (83%) than in those with disease of longer duration (33%). These results support the notion that eye muscle fiber is an important target of the orbital autoimmune reactions that characterize TAO. PMID- 7531719 TI - Novel, ultrasensitive, Q-beta replicase-amplified hybridization assay for detection of Chlamydia trachomatis. AB - A sensitive, nonisotopic hybridization assay termed "dual capture" is described. The assay rapidly and specifically detects very low levels of target nucleic acids and organisms. The assay is based on the principles of sandwich hybridization, reversible target capture, and Q-Beta replicase amplification. The assay can be completed in less than 4 h, and in the described model format, it detects Chlamydia trachomatis rRNA or rDNA. Up to 96 samples can be analyzed simultaneously. The assay employs two types of probes: a test-specific capture probe, which mediates the cycling of the target probe complex on and off derivatized magnetic beads, and a replicatable RNA detector molecule containing a sequence complementary to and adjacent to the capture probe site on the target. Following reversible target capture, detection of the signal is accomplished by replication of the detector molecule by Q-Beta replicase in the presence of propidium iodide. A specific assay signal can be detected from as few as 1,000 molecules above the background. In a limited study of 94 urogenital samples the assay detected five of the six culture-positive samples and did not detect the C. trachomatis target in 85 of the 88 culture-negative samples. PMID- 7531720 TI - Intrafamilial transmission of hepatitis C virus. AB - Family members of hepatitis C virus (HCV)-infected hemodialysis patients had a high rate (8.1%) of HCV infection and were often infected by the same genotype of HCV as the renal patients. Renal patients who had a longer history of hemodialysis or more frequent arteriovenous shunt replacement posed a higher risk to their household contacts, especially to their primary-care providers at home. PMID- 7531721 TI - Cellular and molecular effects of malnutrition and their relevance to periodontal diseases. AB - In response to periodontal pathogens, the leukocytes (PMN) elaborate destructive oxidants, proteinases, and other factors. The balance between these factors, the antioxidants and endogenously synthesized antiproteinases determine the extent of periodontal damage. Malnutrition (PEM) is characterized by marked tissue depletion of the key antioxidant nutrients, including GSH (gamma-glutamyl cysteinyl-glycine), and impaired acute-phase protein response (APR) to infections. The latter results in diminished production of the acute-phase proteins (APP). The APR plays a key role in promoting healing, and its deficit in PEM is due to impairment in the production and cellular action of the cytokines. Other features of malnutrition include inverted helper-suppressor T-cell ratio, histaminemia, hormonal imbalance with increased blood and saliva levels of free cortisol, and defective mucosal integrity. Malnutrition, particularly of the PEM type which usually involves concomitant deficiencies of several essential macro- and micronutrients, therefore has the potential to adversely influence the prognosis of periodontal infections. PMID- 7531722 TI - Central projections of the nervus terminalis and the nervus praeopticus in the lungfish brain revealed by nitric oxide synthase. AB - Lungfishes possess two cranial nerves that are associated with the olfactory system: the nervus terminalis enters the telencephalon with the olfactory nerve, and the nervus praeopticus enters the diencephalon at the level of the optic nerve. We investigated the central projections of the nervus terminalis and the nervus praeopticus in the Australian lungfish (Neoceratodus forsteri) and in the African lungfish (Protopterus dolloi) by NADPH-diaphorase histochemistry (nitric oxide synthase; NOS) and compared them with the projections of the nervus terminalis of the frog (Xenopus laevis). In Neoceratodus, NOS-positive fascicles of the nervus terminalis divide and project with a ventral component through the septum and with a dorsal component through the pallium; fibers of both trajectories extend caudally beyond the anterior commissure and join the lateral forebrain bundle. In the nervus praeopticus, about 300 fibers contain NOS; they innervate the preoptic nucleus and continue their course through the diencephalon; many fibers cross in the commissure of the posterior tuberculum. In Protopterus, ganglion cells of the nervus terminalis and of the nervus praeopticus contain NOS. NOS-positive fibers of the nervus terminalis project through the septal region but not through the pallium. Several major fascicles cross in the rostral part of the anterior commissure, where they are joined by a small number of NOS-containing fibers of the nervus praeopticus. Both nerves innervate the preoptic nucleus. The number and pathways of the fascicles of the nervus terminalis are not always symmetric between the two sides. The nervus terminalis fascicles remain in a ventral position, whereas the nervus praeopticus gives rise to the more dorsal fascicles. Many fibers of the two nerves extend throughout the diencephalon and cross in the commissure of the posterior tuberculum. These findings demonstrate many similarities but also significant differences between the contributions of the nervus terminalis and the nervus praeopticus to forebrain projections in the two lungfishes. They support the view that the nervus praeopticus is part of a nervus terminalis system comparable to that in frogs and other nonmammalian vertebrates. PMID- 7531723 TI - Distribution of galanin-like immunoreactivity in the brain of the turtle Mauremys caspica. AB - Galanin is a brain-gut peptide present in the central nervous system of fish, amphibians, birds, and mammals. For comparative studies among vertebrates, the distribution of galanin in the brain of reptiles has been investigated. We studied the localization of galanin-like-immunoreactive perikarya and nerve fibers in the brain of the turtle Mauremys caspica by using an antiserum against porcine galanin. In the telencephalon, few immunoreactive perikarya were seen in the amygdaloid complex. The diencephalon contained the majority of the immunoreactive perikarya present in the lamina terminalis, nucleus periventricularis anterior, lateral preoptic area, nuclei hypothalamicus ventromedialis and posterior, nucleus basalis of the anterior commissure, and nucleus ventralis tuberis. Many immunoreactive cells, especially in the infundibulum, contacted the cerebrospinal fluid by an apical process. In the rhombencephalon, immunopositive perikarya were restricted to a few cells in the nucleus tractus solitari. In the mesencephalon, they were absent. Immunoreactive nerve fibers were present in all regions containing labeled perikarya and in 1) telencephalon: septum, nucleus fasciculi diagonalis Brocae; 2) diencephalon: nucleus paraventricularis, nucleus supraopticus, nucleus suprachiasmaticus, subventricular grey, nucleus of the paraventricular organ, nucleus mamillaris, infundibular decussation, outer layer of the median eminence, posterior commissure and subcommissural organ region, habenula, nuclei dorsomedialis anterior, and dorsolateralis anterior of the thalamus; and 3) mesencephalon and rhombencephalon: stratum griseum periventriculare, stratum fibrosum periventriculare, laminar nucleus of the torus semicircularis, periventricular grey, nucleus interpeduncularis, nucleus ruber, substantia nigra, locus coeruleus, raphe nuclei, nuclei of the reticular formation, nucleus motorius nervi trigemini, cochlear and vestibular area, and nucleus spinalis nerve trigemini. Our results suggest that galanin may have hypophysiotropic and central roles in the turtle Mauremys caspica. PMID- 7531724 TI - A sulfated proteoglycan as a novel ligand for CD44. AB - We have identified a novel ligand for CD44, a cell surface glycoprotein implicated in tumor metastasis and lymphocyte homing. When the mouse T cell line CTLL-2 was transfected with cDNA encoding a hemopoietic form of mouse CD44, CTLL 2 cells exhibited a new self-adhesive phenotype, forming large aggregates. The aggregation was blocked by neutralizing anti CD44 monoclonal antibody but unaffected by hyaluronidase, indicating the involvement of CD44 and its non hyaluronate ligand in the cell aggregation. The ability to induce CD44-dependent aggregation was found in culture supernatants of CTLL-2 and its CD44 transfectants. The use of CD44-immunoglobulin chimeric protein revealed that CTLL 2 and its transfectants synthesized a large-molecular weight protein (gp600) which bound specifically to CD44. The gp600 was readily labeled with radioactive sulfate, and treatment of gp600 with chondroitinase ABC or ACII generated a lower molecular weight species (18-22 kDa), suggesting that gp600 consists of a small core protein with chondroitin sulfate glycosaminoglycan side chains. However, binding of CD44 to glycosaminoglycans such as chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate was undetectable, suggesting either that a novel chondroitin-type glycosaminoglycan is recognized by CD44 or that a particular configuration of the glycosaminoglycan is required for recognition by CD44. PMID- 7531725 TI - Pharmacological modulation of endothelial cell-associated adhesion molecule expression: implications for future treatment of dermatological diseases. AB - Skin diseases with an inflammatory component, regardless of their etiology, are characterized at some point by the extravasation and subsequent infiltration of leukocytes into the dermal and/or epidermal compartments. This trafficking pattern is determined by a complex series of events whereby the leukocytes interact with cell adhesion molecules (CAM), particularly those induced on endothelial cells following activation with various inflammatory mediators. Vascular CAMs belonging to the selectin family (i.e., P-selectin and E-selectin) are thought to mediate early and reversible events involving leukocyte rolling and margination along the lumenal surface of microvascular cells (post-capillary venules). Certain members of the immunoglobulin supergene family (i.e., VCAM-1 and ICAM-1) regulate later and irreversible steps which lead to firm attachment and subsequent diapedesis of leukocytes. Accumulating evidence suggests that if one blocks the ligand-binding sites between leukocytes and endothelial cells, or inhibits vascular CAM expression, hematopoietic cell extravasation and progressive inflammatory events can be greatly diminished. To identify such inhibitors we developed a cell-based Elisa using the human microvascular cell line HMEC-1. As reported in the present paper, this approach yielded a naturally occurring, low molecular weight compound which potently inhibits cytokine-induced adhesion molecule expression on cultured endothelial cells, without modulating "house-keeping" proteins. PMID- 7531726 TI - Molecular control of melanogenesis in malignant melanoma: functional assessment of tyrosinase and lamp gene families by UV exposure and gene co-transfection, and cloning of a cDNA encoding calnexin, a possible melanogenesis "chaperone". AB - Melanogenesis is a cascade of events significantly controlled by regulatory genes which are associated with the melanosomal membrane. This report introduces our current research efforts dealing with (a) the gene and protein expressions of tyrosinase and Lamp (lysosome-associated membrane protein) families by human melanoma cells after repeated exposures to UV light, (b) the coordinated alterations in the expression of the Lamp family gene and its encoding product after transfection of two genes of the tyrosinase family in human melanoma cells and (c) cloning and sequencing of a Ca(2+)-binding phosphoprotein, calnexin, which could be a candidate as a chaperone for sorting and maturation of tyrosinase and Lamp family glycoproteins in melanogenesis cascade. Our UV exposure study, as well as gene transfection and antisense hybridization experiments, has clearly indicated a marked and coordinated interaction of the Lamp-1 gene with the tyrosinase and TRP-1 genes in this process. We propose that melanogenesis is controlled at least by two major gene family products, i.e., (a) the tyrosinase family of tyrosinase, TRP-1 and TRP-2, and the Lamp family of Lamp 1, Lamp-2 and Lamp-3. These two gene families probably derived from primordial melanogenesis-associated genes which are common or closely related to each other. PMID- 7531727 TI - Exclusion of close linkage of bipolar disorder to the Gs-alpha subunit gene in nine Australian pedigrees. AB - Growing evidence suggests that guanine nucleotide binding proteins (G proteins) may be involved in both the pathogenesis and treatment of bipolar affective disorder. Both overactive G proteins and increased levels of the alpha subunit of the stimulatory form (Gs-alpha) have been demonstrated in peripheral leucocytes of manic patients while an increase of Gs-alpha subunit levels has also been found in a postmortem study of bipolar disorder. The function of Gs and Gi alpha subunits has now been shown to be affected by lithium. The present study aimed to determine whether bipolar affective disorder was linked to the Gs-alpha subunit gene which has been mapped to chromosomal region 20q13.2. Linkage analysis utilized the PCR amplification of a portion of the Gs-alpha gene that contains a dinucleotide repeat (CA repeat) polymorphism. Linkage of bipolar disorder and recurrent depression to the Gs-alpha subunit gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness. Close linkage to the Gs-alpha subunit gene was strongly excluded using each model and definition. Thus, our study indicates that a genetic defect in the Gs-alpha subunit gene is unlikely to be the cause of bipolar disorder. PMID- 7531728 TI - Histamine release from human basophils induced by platelet activating factor: the role of extracellular calcium, interleukin-3, and granulocyte-macrophage colony stimulating factor. AB - Although we have demonstrated that platelet activating factor (PAF) directly induces histamine release from human basophils, other studies have failed to report similar effects. In an attempt to understand the variability of these results, we examined the effect of some factors that could influence the basophils' response to PAF such as, extracellular Ca2+ and cytokines (interleukin 3 and granulocyte-macrophage colony-stimulating factor [GM-CSF]). The secretion of histamine induced by PAF was optimal when the cells were incubated in Ca2+ for 2 to 5 minutes, whereas it declined at longer time intervals up to 15 minutes. If cytochalasin B (5 micrograms/ml) was coincubated with PAF (1 mumol/L) to enhance the secretory response, histamine release was maximal at time 0 and decreased in parallel with the time of the basophils' exposure to Ca2+, like 0.1 microgram/ml anti-IgE-induced histamine secretion but unlike 1 mumol/L formyl-methionyl-leucyl phenylalanine-induced histamine secretion. We found that there is synergy between interleukin-3 (1 to 3 ng/ml) and PAF (1 mumol/L) for secretion of histamine from human basophils (p < 0.05) and that GM-CSF (10 ng/ml) significantly (p < 0.02) potentiates the secretion of histamine activated by PAF (1 mumol/L). Our results demonstrate that: (1) the kinetics of the interaction between Ca2+ and the activation pathway that leads to histamine secretion are central events in the release reaction elicited by PAF in human basophils, and (2) interleukin-3 and GM CSF can potentiate the secretory response of human basophils stimulated by PAF. PMID- 7531729 TI - Chemokines of the alpha, beta-subclass inhibit human basophils' responsiveness to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. AB - Monocyte chemotactic and activating factor (MCAF) is the most potent cytokine that activates basophils to release histamine. The response of human basophils to either simultaneous or sequential addition of the chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, platelet factor (PF)4, connective tissue activating peptide III (CTAP-III), interleukin (IL)-8, and inflammatory protein (IP)-10 on MCAF-induced histamine release was studied. Simultaneous addition of MCAF and any of the chemokines studied evoked an augmented response as measured by histamine release, whereas preincubation of leukocytes or purified basophils (80%) with these chemokines decreased MCAF-induced histamine release in a dose-dependent manner. Histamine release by anti-IgE remained unchanged. When tested at 5 x 10(-9) mol/L, the decrease in histamine release by RANTES was 69.2% +/- 3.5%, by MIP-1 alpha 48.8% +/- 3.1%, by MIP-1 beta 42.9% +/- 3.1%, by PF4 56.5% +/- 2.9%, by IL-8 41.2% +/- 2.2, by CTAP III 27% +/- 4.4%, and by IP-10 15.3% +/- 2.6%. The peak inhibition of histamine release by the chemokines was reached within 10 minutes of preincubation with basophils and remained unchanged thereafter. Washing basophils after preincubation with chemokines abolished the inhibition, with the exception of desensitization by low concentrations of MCAF. With the exclusion of MCAF and RANTES, none of the chemokines (at the concentration range of 5 x 10(-8) to 5 x 10(-11)) induced significant (> 10% above spontaneous) histamine release from basophils. Preincubation of basophils with C5a (5 x 10(-10) mol/L) did not affect histamine release, whereas preincubation with granulocyte-macrophage colony-stimulating factor (10 ng/ml) or IL-5 (10 ng/ml) enhanced MCAF-induced histamine release by 121.8% +/- 10.1% and 108% +/- 10.8%, respectively. We have therefore characterized RANTES, MIP-1 alpha, MIP-1 beta, CTAP III, PF4, IL-8, and IP-10 as inhibitors of MCAF-induced histamine release. Although the results are consistent with receptor blockade, the alpha and beta chemokines appear to interact with separate receptors linked to G proteins; thus, a mechanism of receptor class desensitization is proposed. Interaction of this group of cytokines at the site of allergic inflammation may modulate a function of basophils to initiate, augment, or inhibit histamine release. PMID- 7531730 TI - IgE+ cells in the peripheral blood of atopic, nonatopic, and bee venom hypersensitive individuals exhibit the phenotype of highly differentiated B cells. AB - We have analyzed IgE+ cells in peripheral blood of atopic donors, donors hypersensitive to bee venom, and nonatopic control donors with two- and three color flow cytometry. Although the percentage of IgE+ cells varied among these groups, the overall phenotypic patterns were similar. Most IgE+ cells do not display typical B-cell markers, such as CD19, CD20, and CD21. A significant proportion of these cells stain for CD38, indicating that they are more differentiated. IgE+ cells express Fc gamma RII and CD45RO, an isoform associated with an advanced level of differentiation. The majority of IgE+ cells do not coexpress other surface immunoglobulin isotypes. In the case of bee venom hypersensitive donors, we have been able to identify a small population of IgE+ cells with a specificity for phospholipase A2, a major immunogenic component of bee venom. The phospholipase A2+ cells display a phenotype similar to that of the IgE+ cells. PMID- 7531731 TI - Epitope specificity of the major peanut allergen, Ara h II. AB - The antigenic and allergenic structure of Ara h II, a major allergen of peanuts, was investigated with the use of four monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h II. Our previous studies with monoclonal antibodies generated to peanut allergens showed this method to be useful for epitope mapping. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive peanut challenge responses. The Ara h II monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified two different antigenic sites on Ara h II. In similar studies with pooled human IgE serum from patients with positive challenge responses to peanuts, we identified two closely related IgE-binding epitopes. These characterized monoclonal antibodies to Ara h II will be useful for future studies to immunoaffinity purify the Ara h II allergen and to use in conjunction with recombinant technology for determining structure-function relationships. PMID- 7531732 TI - Cetirizine reduces inflammatory cell recruitment and ICAM-1 (or CD54) expression on conjunctival epithelium in both early- and late-phase reactions after allergen specific challenge. AB - BACKGROUND: Allergen-specific conjunctival challenge (ASCC) is a safe and reproducible experimental model of allergic conjunctivitis and a useful tool in the evaluation of effectiveness and possible mechanisms of action of drugs commonly used in the treatment of allergic diseases. OBJECTIVE: The protective effect of cetirizine on inflammatory changes after ASCC was assessed in 12 patients with rhinoconjunctivitis caused by Parietaria judaica in a double-blind study. METHODS: After a screening ASCC was performed, patients were randomized into two treatment groups; each patient was given cetirizine (oral tablets) 10 mg twice daily, or matching placebo for 3 1/2 days in off-pollen season. Clinical evaluation (itching, hyperemia, lacrimation, and swelling of eyelids) and cytologic assessment (number of inflammatory cells in conjunctival scraping and evaluation of intercellular adhesion molecule-1 (ICAM-1)/CD54 expression on epithelial cells) were performed at baseline, 30 minutes (i.e., early-phase reaction [EPR]), 6 hours, and 24 hours (i.e., late-phase reaction [LPR]) after ASCC, before and after treatment. RESULTS: The EPR clinical events and the EPR total number of inflammatory cells were significantly reduced by cetirizine compared with placebo. The LPR clinical events and inflammatory cell recruitment were reduced by cetirizine in a similar manner. Both eosinophil and neutrophil numbers were decreased by active drug in EPR and LPR. Furthermore, ICAM-1/CD54 expression was significantly reduced by cetirizine in both the EPR and LPR compared with placebo. CONCLUSIONS: This study shows that cetirizine has a protective effect on clinical and cellular EPR and LPR events (including ICAM 1/CD54 expression on epithelium) induced by ASCC. PMID- 7531733 TI - Probable grade IV dextran-induced anaphylactic reaction despite hapten inhibition. PMID- 7531736 TI - The assessment of constipation in terminal cancer patients admitted to a palliative care unit: a retrospective review. AB - Constipation is a frequent and distressing complication in patients with advanced cancer. However, very few studies have reviewed the assessment and management of these patients. The purpose of this study was to review the documentation and assessment and diagnosis of constipation in patients admitted to a Palliative Care Unit, and the correlation between those findings and radiological evidence of stool in the colon. The records of 122 consecutive patients admitted to the Palliative Care Unit, Edmonton General Hospital were reviewed in order to assess the physician's and the nurse's record of symptoms, physical findings, and diagnosis and treatment of constipation. All patients also underwent a flat abdominal radiograph that scored for the presence of stool in the colon (0 = no stool; and 12 = stool occupying all the lumen of the four quadrants of the colon). The radiograph was scored blindly by two different physicians. Of 103 evaluable patients, a rectal exam was reported only in 42. Correlation between the assessment by the two physicians' radiograph score was high (0.78, P nd nurses' diagnosis of constipation, the presence of laxative treatment, the number of days since the last bowel movement, and the source of the admission (hospital vs home) were not associated with higher radiological scores for constipation. Assessment is insufficient in this population at high risk for severe constipation. Radiological examination may be necessary for adequate diagnosis in some patients. More research is needed in this area. PMID- 7531735 TI - The benzodiazepines as adjuvant analgesics. AB - There has been long-standing debate regarding whether benzodiazepines possess analgesic properties that are independent of their effects on mood and alertness. A careful review of the literature reveals insufficient evidence to support the contention that the benzodiazepines have meaningful analgesic properties in most clinical circumstances. Treatment with the benzodiazepines may reduce complaints of pain, but this seems to be an indirect effect related to their psychotropic properties, such as alleviation of anxiety and, in selected cases, depression. In the absence of definitive data, clinical experience suggests a potential role for treatment with benzodiazepines for acute muscle spasm, concomitant chronic pain and anxiety, and lancinating neropathic pain, in which case clonazepam and alprazolam may be the agents of choice. They should probably not be considered as first-line choices even for the above indications, since potential benefits must be considered in the context of potential for the development of cognitive impairment, physical and psychological dependence, worsening depression, overdose, and other side effects. PMID- 7531734 TI - Local anesthetics as adjuvant analgesics. AB - Local anesthetics administered to block nerve conduction for surgical anesthesia and to provide analgesia in management of acute pain have become a standard of anesthesiology practice. These drugs have had an important role in the multimodality management of chronic pain as well, and this role is expanding since the revival of systemic administration. Local anesthetics are analgesics, albeit not in the traditional clinical and pharmacologic sense. Evidence suggests that intravenous administration is an effective treatment in chronic neuropathic pain syndromes. There is also evidence that intravenous local anesthetics can relieve acute pain. Furthermore, the novel idea that acute procedural and postprocedural pain control with local anesthetics could prevent the development of chronic pain syndromes, including chronic neuropathic pain syndromes, adds another important potential dimension to the role of local anesthetics in pain management. PMID- 7531737 TI - Organic brain syndromes and opioid administration for cancer pain. AB - To clarify the range of potential etiologies that may contribute to organic brain syndrome in patients receiving systemic opioids for cancer pain, we describe 15 patients who presented this complication. In 11 cases, concomitant conditions were found that could contribute to the onset of organic brain syndrome. These data illustrate that multiple causes often play a role in the development of mental status changes in advanced cancer. Opioids are seldom the only causal factor implicated. PMID- 7531738 TI - The use of corticosteroids in the management of bilateral malignant ureteric obstruction. AB - Ureteric obstruction is not an uncommon complication of malignant disease or its treatment. We describe the effectiveness of a corticosteroid in a patient with bilateral ureteric obstruction from metastatic colon cancer causing acute renal failure. We presume that the effectiveness was because of its activity in reducing edema related to the tumor. Other modalities of treatment are discussed but each case requires individualization of treatment with due ethical consideration. PMID- 7531739 TI - Delayed sympathetically maintained pain caused by electrical burn at the current's entry and exit sites. AB - It is not uncommon for sympathetically maintained pain (SMP) to follow electric burns at the site of current entry. The occurrence of SMP at the exit point has not been reported. This report describes a patient who was exposed transiently to a 220W electrical current. After a delay of 3 months, the typical manifestations of SMP appeared on the right hand (entry point); symptoms appeared on the left foot (exit point) after 11 months. Ultrasonography was helpful in identifying the bony and soft tissue changes that occurred with SMP. Serial sympathetic blocks, oral phenytoin, and an intensive physical rehabilitation program were useful in treating this electrically induced SMP. PMID- 7531740 TI - [Structure and expression of cancer associated carbohydrate antigens: sialyl Le(a) and Tn antigens]. PMID- 7531741 TI - [Mother-to-infant transmission of hepatitis C virus]. AB - Anti-HCV antibody was examined in 4,801 pregnant women by using the first or second generation radioimmunoassay systems. Antibody positive patients were further tested for HCV-RNA by RT-semi nested polymerase chain reaction. Anti-HCV antibody was found to be positive in 59 women (1.23%) and HCV-RNA in 25 women (0.52%). Fourteen newborn babies born to anti-HCV antibody positive mothers were all positive for anti-HCV antibody but negative for HCV-RNA at delivery. HCV-RNA was detected in 3 of 13 children (23.1%) at 3, 5 and 9 months of age, respectively. All of their mothers had mild transaminase elevations in the third trimester. One of them was positive for both HCV and HIV. Although three mothers had HCV-RNA in the milk, their children were still sero-negative for HCV-RNA. These results suggest that mother-to-infant transmission of HCV may occur at delivery and transaminase elevation in the third trimester is one of the risk factors in mother-to-infant transmission of HCV. PMID- 7531742 TI - A study on cell proliferation in cultured human tendons--time dependence, and labeling of 5-bromodeoxyuridine. AB - Cells which proliferated on cultured human tendon were studied using scanning and transmission electron microscopy, and by immunohistochemical staining. Cell proliferation was continuously observed after 3, 6, 9 and 14 days in culture. Electron microscopy revealed the proliferation and differentiation of fibroblasts and macrophages on the cut surface of the tendon. Immunohistochemical staining by 5-bromodeoxyuridine demonstrated increased cellular activity of the proliferation in the epitenon and endotenon, and also in some mature tenocytes. This activity reached a peak on the 9th day of incubation. PMID- 7531743 TI - Validation of chromosome painting. II. A detailed analysis of aberrations following high doses of ionizing radiation in vitro. AB - Fluorescence in situ hybridization with chromosome-specific composite DNA probes ('chromosome painting') is useful for quantifying radiation-induced cytogenetic damage. Recently we showed that the frequency of aberrations observed with painting is similar to that seen with conventional cytogenetic methods, at least at doses of < or = 2 Gy. Above this dose, however, the agreement was not as good. We describe here the results of additional work designed to clarify our earlier findings, and provide a detailed analysis of the type and frequency of aberrations induced in human peripheral lymphocytes following acute exposure to 137Cs at doses of 0 (unexposed control), 1, 2, 3 and 4 Gy. The newly-developed nomenclature for chromosome aberrations detected by painting (Protocol for Aberration Identification and Nomenclature Terminology, 'PAINT') was used to classify all aberrations. Our results indicate that if the guidelines of the PAINT system are followed, chromosome painting can provide meaningful biodosimetry at high doses, and that the observation of complicated rearrangements not only does not interfere with dose estimation, but also the information provided by these exchanges can be easily broken down into the component aberrations and included in the dose estimate. We also show that the inequality between translocations and dicentrics that we previously observed can be explained by an excess of one class of translocated chromosomes, specifically those in which the centromere is from an unpainted chromosome. Translocated chromosomes in which the centromere is painted were found to occur at a frequency equal to dicentrics. These results should help clarify the use of painting for radiation biodosimetry by improving our understanding of the frequencies of various types of stable aberrations observed shortly after exposure. This will improve our ability to perform meaningful biodosimetry long after the frequencies of unstable aberrations have ceased to be informative. PMID- 7531744 TI - Effects of field size on the survival of pig epidermal colony-forming in situ after electron irradiation. AB - The survival of colony-forming cells in pig epidermis after irradiation was measured using different electron field sizes. The sensitivity of the colony forming cells was characterized by D(o) = 2.5-3.0 Gy. There was an effect of field size, described approximately by: Dose (to give five colonies/cm2) (Gy) = (31 +/- 2) x Area-(0.048 +/- 0.015). The effect of field size was less than found previously using tolerance to skin reactions (area exponent = 0.16). This work indicates for the first time that the effects of different large field sizes in skin can be detected at the level of colony-forming cell survival. PMID- 7531745 TI - Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG. AB - Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed-type hypersensitivity (DTH) in guinea pigs injected with liver Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis. PMID- 7531746 TI - Modulation of rat neutrophil functions by administration of granulocyte colony stimulating factor. AB - Neutrophil function was examined in rats treated with recombinant human granulocyte colony-stimulating factor (G-CSF) using peripheral blood neutrophils (PBNs) and peritoneal exudate neutrophils (PENs) as sources of cells examined in vitro. Adherence to plastic plates containing fetal calf serum of nonstimulated or N-formylmethionyl-leucyl-phenylalanine (fMLP)- or tumor necrosis factor-alpha stimulated PBNs obtained from G-CSF-injected rats was lower than that of control rats. In contrast, this adherence was higher in G-CSF-treated rats than in the control group when PENs were used. Neutrophil adherence of G-CSF-injected and noninjected groups was identical when phorbol myristate acetate was used to stimulate neutrophils. Superoxide production of PBNs stimulated with fMLP in vitro was lower in G-CSF-treated rats than in control rats but higher than in the controls when PENs were used. Furthermore, in vitro tumor cell growth inhibition by PBNs was lower in G-CSF-treated rats than in control rats, but when PENs were used inhibition was higher than in the controls. PMID- 7531748 TI - Regulation of L-selectin and CD18 on bovine neutrophils by glucocorticoids: effects of cortisol and dexamethasone. AB - The responsiveness of bovine neutrophil L-selectin and CD18 to in vivo glucocorticoid administration was characterized by flow cytometric analysis. Blood was sampled intensively from dairy cows treated for 3 days with placebo, cortisol, or dexamethasone. Immunostaining was performed on whole blood (100 microliters) that was left unstimulated or was stimulated with platelet activating factor (PAF; 1 microgram/ml blood) prior to incubation with fluorescein isothiocyanate-conjugated monoclonal antibodies against L-selectin and CD18. Results were expressed as the percentage of positive-staining cells and as mean fluorescence intensity (MFI) of those cells. Total leukocyte count and leukocyte differentials were also performed on all blood samples. Dexamethasone caused nearly complete down-regulation of L-selectin (P < .01) on the surface of gated cells and reduced to half the MFI of CD18 (P < .01). Compared with values for the placebo group, dexamethasone began to cause L-selectin down-regulation within 8 h after the first injection and these effects persisted until 48 h after the third injection. This was correlated in time with an acute reduction in the proportion of cells that stained positive for L-selectin (from 98% before dexamethasone injections to a low of 17% by 40 h after the first injection). Dexamethasone also caused leukocytosis and neutrophilia during this time interval. In contrast, CD18 down-regulation was delayed until 16 h after the second dexamethasone injection and persisted for roughly 8 days. However, at no time during the experiment did dexamethasone influence the proportion of gated cells staining positive for CD18 (always 100%). Effects of cortisol were generally similar in pattern to those of dexamethasone but were more subtle and more readily detected when PAF was added to blood prior to immunostaining. These results strongly suggest that one mechanism of the anti-inflammatory action of glucocorticoids is to induce dramatic down-regulation of L-selectin and CD18 adhesion molecules on blood neutrophils. PMID- 7531747 TI - Expression of cytokine mRNA in murine splenic dendritic cells and better induction of T cell-derived cytokines by dendritic cells than by macrophages during in vitro costimulation assay using specific antigens. AB - Among antigen-presenting cells dendritic cells (DC) have the unique ability to generate primary T cell response. The reasons for the superior inductive property of DC still remain obscure. The explanations offered include higher expression of CD80, MHCII, and ICAMI on DC surface which allows effective cluster formation with T cells. It is also possible that additional cellular characteristics of DC, i.e., their ability to release critical mediators involved in the induction of effective immune response, are important. We examined the ability of DC to express IL-1, IL-6, and IL-12 using the highly sensitive reverse transcription quantitative polymerase chain reaction. Our data show that highly purified (97 99% pure) murine splenic DC were capable of expressing IL-1, IL-6 and IL-12 mRNA upon stimulation with lipopolysaccharide. We also compared the ability of DC and macrophages to induce T cell-derived cytokines IL-2 and IFN-gamma in an in vitro antigen-specific costimulation assay. In naive T cells stimulated with antigen presented via DC or macrophages, the levels of mRNA for IL-2 and IFN-gamma were 2 to 4-fold higher when cells were stimulated with DC. Overall, our data add additional support to the description of DC as superior antigen-presenting cells for the activation of naive T cells. PMID- 7531750 TI - The involvement of insulin-like growth factor-I in local control of steroidogenesis and DNA synthesis of Leydig and non-Leydig cells in the rat testicular interstitium. AB - Insulin-like growth factor-I (IGF-I) peptide, receptors and binding proteins are present in the rodent testis, which strongly implies that IGF-I has one or more testicular functions. In the present study we provide further information to support the concept that IGF-I is an important local mediator in the testis. High concentrations of IGF-I were measurable in interstitial fluid by radioimmunoassay, and IGF-I-binding proteins (IGFBPs) were readily detectable in interstitial fluid by ligand blotting, the predominant type being IGFBP-2. In vitro, IGF-I bound to testicular interstitial cells which did not have 3 beta hydroxysteroid dehydrogenase (3 beta-HSD) activity and which were resistant to ethane dimethanesulphonate treatment. In vitro, IGF-I receptor-mediated actions increased both steroidogenesis and DNA synthesis. Insulin stimulated DNA synthesis at concentrations appropriate to cross-react with the IGF-I receptor, and this effect was greater in a testicular interstitial Leydig cell-depleted cell population compared with a Leydig cell-enriched cell culture. Furthermore, combinations of epidermal growth factor or transforming growth factor -alpha together with insulin appeared to act synergistically, causing extremely large increases in [3H]thymidine incorporation in the interstitial cells. These results support a paracrine and/or autocrine role for IGF-I in interstitial cell growth and development. PMID- 7531749 TI - Activation of multiple protein kinases induced by cross-linking of Fc gamma RII in human neutrophils. AB - Several neutrophil protein kinases that undergo changes in activity during Fc gamma RII activation have been investigated. These kinases include calcium/calmodulin-dependent protein kinase II (CAMPKII), mitogen-activated protein kinase (MAPK), and histone H4 protein kinase (PKH4). They are rapidly and transiently activated in a dose-dependent manner by the cross-linking of Fc gamma RII. The activation of CAMPKII but neither PKH4 nor MAPK was inhibited by treating the cells with either a tyrosine kinase inhibitor, genistein, or an intracellular calcium chelator, BAPTA/AM. The superoxide production induced by cross-linking Fc gamma RII can be inhibited partially by various protein kinase inhibitors: 33% by protein kinase C inhibitor calphostin C, 30% by CAMPKII inhibitor KN-62, and 62% by tyrosine kinase inhibitor genistein. These results indicate that cross-linking of Fc gamma RII induces multiple signaling pathways that lead to the activation of various protein kinases. The activation of these kinases may be involved directly or indirectly in the regulation of superoxide production. PMID- 7531751 TI - The inhibitory effect of noradrenaline on thyrotrophin-stimulated 3,5,3'-tri iodothyronine and thyroxine release is mediated through a Ca(2+)-dependent process in the thyroid gland of the mouse. AB - We examined the effect of noradrenaline on the release of 3,5,3'-tri iodothyronine (T3) and thyroxine (T4) from perifused mouse thyroid. Noradrenaline suppressed the thyrotrophin (TSH)-stimulated release of T3 and T4. The addition of prazosin, which is a specific alpha 1 antagonist, or the depletion of Ca2+ from the perifusion buffer completely abolished the inhibitory effect of noradrenaline on TSH-stimulated T3 and T4 release. Noradrenaline did not inhibit TSH-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) release in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibits both cAMP-specific and calmodulin-sensitive phosphodiesterases. Noradrenaline significantly suppressed the TSH-stimulated release of T3 and T4 in the presence of IBMX. These results suggest that the inhibitory effect of noradrenaline on TSH-stimulated T3 and T4 release is not mediated through a cAMP-dependent process or the activation of a calmodulin-sensitive phosphodiesterase, and that this inhibition is mediated through a Ca(2+)-dependent process regulated by the alpha 1-adrenergic system in the mouse thyroid. PMID- 7531752 TI - Sequence analysis of proviral HIV RT amplified directly by a semi-quantitative technique from AZT treated patients. AB - To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the National Academy of Sciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV-1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (Ile), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild-type "AZT sensitive" RTs co-existed with mutated "AZT-resistant" RTs in the same patient sample. PMID- 7531753 TI - Diagnosis of hepatitis C virus-associated chronic liver disease in India: comparison of HCV antibody assay with a polymerase chain reaction for the 5' noncoding region. AB - The relative value of an anti-hepatitis C virus (HCV) serological assay and reverse transcriptase-nested polymerase chain reaction assays (RT-PCR) were investigated for the constant 5' putative noncoding region of HCV for the diagnosis of HCV-associated chronic liver diseases in India. One hundred fifteen patients with biopsy proven chronic active hepatitis and 140 cases of cirrhosis of the liver were investigated for anti-HCV antibody using a second generation commercial enzyme-linked immunosorbent assay (ELISA). A proportion of these patients: 42 with chronic hepatitis and 27 with cirrhosis of the liver were analysed further for HCV RNA in the serum using RT-nested PCR assay. Thirty-three (12.9%) of the 255 patients were positive for anti-HCV antibody and 23 of 69 (33.3%) patients were positive for HCV RNA in serum. Fifteen of the 33 (45.5%) anti-HCV positive patients had HCV RNA in the serum. Eight of 36 (22.2%) HCV seronegative patients tested were found with HCV RNA. This indicates that the diagnosis of HCV infection is not possible if it is based solely on the available serodiagnostic tests. Inclusion of both assays improved the diagnostic efficiency, 18.8% (13/69) were negative for all virological markers associated with HBV and HCV infection. Since a majority of the chronic liver disease patients (143/255 [56%]) were seronegative for either HBV or HCV infection, it is significant that HCV RNA was detected in 38% (8/21) of a randomly selected group from these patients. The antibody assay and PCR were compared using interclass correlation (kappa statistics).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531754 TI - Application of commercial assays to detect IgG antibodies to hepatitis C virus in urine: a pilot study from autopsy cases. AB - Hepatitis C virus (HCV), the main cause of non-A, non-B hepatitis in the United States and possibly in the world, is believed to be transmitted primarily through parenteral exposure. Many screening and supplemental tests are available to detect antibodies to HCV in serum. The ability to use commercial assays to detect antibodies to HCV in urine was investigated in this study. A total of 229 serum/urine matched samples were collected sequentially from forensic autopsy cases examined at the Office of the Chief Medical Examiner, State of Maryland. Testing was performed using the Ortho, Innogenetics, and Abbott second generation HCV screening tests and the INNO-LIA HCV Ab supplemental assay. Sample volumes were increased for urine testing. Forty-six of 229 serum samples were positive by screening and confirmed by supplemental tests. The urine samples produced positive results on 44-45 of the same 46 by screening tests and all 46 positives by the supplemental test. There were no false positive samples using urine when compared with the serum pairs. The one false negative sample using urine was still nonreactive when the urine volume was increased to 200 microliters using the screening tests. Generally, five times the serum volume was required for the screening tests to be optimal for urine samples. The urine samples were stored under different conditions prior to testing to determine the influence on antibody stability in urine. PMID- 7531755 TI - Viral specificity of hepatitis E virus antigens identified by fluorescent antibody assay using recombinant HEV proteins. AB - A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169-7126). PMID- 7531756 TI - Hepatitis C virus and its genotypes in patients suffering from chronic hepatitis C with or without a cryoglobulinemia-related syndrome. AB - Recently, evidence has been presented for a possible association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). Eleven consecutive patients with EMC and two with cryoglobulinemia type I were examined for the presence of markers of HCV infection. Eleven of 13 patients (10 with EMC and 1 with type I cryoglobulinemia) had anti-HCV antibodies (as determined by a second generation anti-HCV assay) and HCV-RNA in plasma or serum. HCV-RNA was also detected in liver biopsies of five patients. Genotyping showed that HCV genotype 1 was found in 10 of 11 patients with HCV-RNA (9 genotype 1b and 1 genotype 1a) and only one patient had HCV genotype 2. However, a similar high prevalence of genotype 1b (100%) was found in a group of 14 consecutive patients with chronic hepatitis C, who had no clinical evidence of cryoglobulinemia. Concomitant infection was present in three patients with genotypes 2, 3 and 4, respectively. These findings stress the high prevalence of HCV infection in patients with EMC and further study shows that a difference in genotype prevalence was not found between HCV-related EMC and chronic hepatitis C without clinical manifestations of EMC. PMID- 7531757 TI - Genotyping of hepatitis C virus isolates by a modified polymerase chain reaction assay using type specific primers: epidemiological applications. AB - A polymerase chain reaction (PCR)-based assay using primers against the hepatitis C core gene has been described [Okamoto et al. (1992a): Journal of General Virology 73:673-679]. Within the two major HCV genotypes 1 and 2, the Okamoto system identifies two subtypes each (1a, 1b and 2a, 2b, respectively). Typing is achieved by a primary PCR with consensus primers followed by a nested PCR with type specific primers. The original assay was modified by addition of a parallel second PCR identifying the recently described major genotype 3. The assay also identifies in duplicate subtype 1b (type II by Okamoto), suggested to respond poorly to interferon. Reaction conditions were reviewed and melting temperatures of all typing primers equalised to increase strigency. The modified system functioned well and typing results were supported by partial core sequencing. The following distribution of genotypes was found in 53 hepatitis C virus (HCV) infected Swedish blood donors: genotype 1a (57%), 3 (19%), 1b (13%), and 2b (11%). In six recipients of HCV infected blood identified in a retrospective study, the recipient HCV genotype was identical to donor HCV genotype. Furthermore, in HCV positive couples identical genotype was observed when only one partner had an external risk factor; whereas genotypes were often diverse if both sex partners had parenteral risk factors. Finally, a cluster of hepatitis C cases in a haemodialysis unit was evaluated retrospectively. Eight patients had genotype 1b, two had mixed 1a and 1b, and one had type 1a. The modified HCV genotyping assay was of value in examining different epidemiological situations and can be expanded presumably to include future genotypes. PMID- 7531759 TI - Expression of a blood group B antigen-related glycoepitope in human dorsal root ganglion cells. AB - Carbohydrate epitopes of glycoconjugates are expressed on sensory neurons of dorsal root ganglion (DRG). A possible role of antibodies directed at carbohydrate determinants of the glycoconjugates has been suggested in some patients with sensory neuropathy. We investigated expression of blood group antigen-related epitopes in human DRG immunohistochemically using monoclonal antibodies to A, B, and H antigens. A blood group B determinant [Gal alpha 1 3(Fuc alpha 1-2)Gal beta-]-related glycoepitope was demonstrated in the neurons and surrounding satellite cells of DRG obtained from subjects with any ABO blood group phenotype. The treatment with trypsin or chloroform/methanol prior to the immunostaining suggested that the glycoconjugate exhibiting the blood group B determinant-related epitope consisted mainly of glycoprotein and included glycolipid. The glycoconjugates with the blood group B determinant-related epitope may play a role in the physiological function and pathophysiology of human DRG neurons. PMID- 7531758 TI - Hepatitis C virus infection in patients with chronic liver disease or chronic renal failure and blood donors in Thailand. AB - Hepatitis C virus (HCV) RNA and genotypes, as well as markers of hepatitis B virus infection, were surveyed in 171 patients with chronic liver disease, 276 patients with chronic renal failure, and 961 blood donors in Thailand. HCV RNA was detected in 30 (23%) of 128 patients with non-alcoholic chronic liver disease and hepatitis B surface antigen (HBsAg) in 60 (47%), and both HCV RNA and HBsAg in 3; the cause of liver disease was not established in 41 (32%) patients. HCV RNA was detected in 44 (20%) of 221 patients on maintenance hemodialysis or with kidney transplantation, but in none of 55 patients on peritoneal dialysis. Antibodies to synthetic HCV core peptides were detected in 39 (4.1%) of sera from 961 blood donors, and HCV RNA was detected in 8 (0.8%). Of the 90 HCV RNA samples from patients and donors, genotype V prevailed (46%) followed by II (22%), I (14%), III (3%), and VI (2%); genotypes were not classifiable into any of I-VI in the remaining 10%. There were six sera which contained HCV RNA, but were without antibody to HCV detectable by the second-generation enzyme immunoassay. HCV RNA titers were high in four patients with kidney transplantation, but low in one patient with chronic liver disease and one patient on maintenance hemodialysis. HCV RNA at high titer (> or = 10(4)/ml) was not classifiable in one patient. These results indicate HCV of novel genotypes in Thailand, seronegative HCV infection in patients with kidney transplantation, and a low risk of HCV infection in patients treated by peritoneal dialysis. PMID- 7531760 TI - Reactive astrocytes in viral infections of the human brain express endothelin like immunoreactivity. AB - In order to investigate the expression of endothelin-like immunoreactivity in astrocytes of viral infections of the human brain the avidin-biotin peroxidase complex method and a polyclonal antiserum were used. Autopsy material was obtained from 5 cases of herpes simplex encephalitis, two of progressive multifocal leukoencephalopathy (PML) and two of subacute sclerosing panencephalitis (SSPE). All the 5 herpes simplex encephalitis cases presented groups of immunoreactive astrocytes around necrotic, inflammatory lesions. The PML cases exhibited a large number of immunoreactive astrocytes in and around lesions of the white matter. The cases of SSPE disclosed numerous, markedly stained fibrillary immunoreactive astrocytes; they were most abundant in degenerated regions of the white matter. The processes and peripheral cytoplasm of giant astrocytes in the PML cases contained immunoreactive material but the perinuclear region was devoid of such material. In the herpes simplex and the SSPE cases immunoreactivity was present throughout the cytoplasm and processes of reactive fibrillary astrocytes. Many nerve cells in the cerebral cortex, hippocampus, cerebellum and pons of control cases exhibited endothelin-like immunoreactivity but this occurred in only exceptional astrocytes of control cases. Endothelin-like immunoreactivity was not present in the oligodendrocytes and vascular endothelial cells of controls and cases of virus infection. The expression of endothelin-like immunoreactivity in astrocytes in human viral diseases reflects probably an increased intracellular content of endothelin. If this peptide is released from such astrocytes, it may act as a mitogen and by inducing constriction of arterioles it may influence the microcirculation. PMID- 7531761 TI - Cerebellar ataxia and polyneuropathy in a patient with IgM M-protein specific to the Gal(beta 1-3)GalNAc epitope. AB - A 79-year-old man with sensory dominant polyneuropathy, cerebellar ataxia, and palatal myoclonus had serum IgM M-protein that specifically bound to GM1, GD1b, and asialo-GM1. IgM with the same specificity was detected in his cerebrospinal fluid. Results of immunohistochemical studies showed specific binding of this monoclonal IgM to the cerebellar granular layer, dentate nucleus, inferior olive, and gray matter of the cerebrum and spinal cord. Monoclonal antibody GGR12, monospecific to GD1b, had an immunostaining distribution similar to that of the patient's IgM M-protein. The binding of M-protein may be associated with the development of cerebellar ataxia and palatal myoclonus in this patient. PMID- 7531762 TI - [4-Dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline] is a novel ligand to the picrotoxin site on GABAA receptors, and decreases single-channel open probability. AB - U-93631,[4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline], represents a GABAA receptor ligand of novel chemical structure, and has been shown to induce a rapid, time-dependent decay of GABA-induced whole-cell Cl- currents in recombinant GABAA receptors (Dillon et al., 1993). In this study, we found that the drug competitively inhibited [35S]t-butylbicyclophosphorothionate binding to the picrotoxin site on a cloned GABAA receptor, alpha 1 beta 2 gamma 2, and preempted the action of picrotoxin and [35S]t butylbicyclophosphorothionate on GABA-induced Cl- currents. We further examined the effect of U-93631 on GABA-induced single channel openings in outside-out patches. U-93631 (5 microM) showed no effect on single channel conductance, the duration of channel open states or the short closed state, but increased the duration of the long closed state and its relative contribution to total closed time. In addition, closings of very long duration (> 500 msec), albeit rare, occurred more frequently in the presence of U-93631. Thus, the probability of single channel openings decreased from 0.12 +/- 0.02 to 0.04 +/- 0.01 in the presence of U-93631 (5 microM). These properties of U-93631 are analogous to those of picrotoxin and [35S]t-butylbicyclophosphorothionate reported in earlier studies with native neurons. We conclude that U-93631 at least shares overlapping binding domains with picrotoxin and [35S]t-butylbicyclophosphorothionate, and the ability to stabilize the GABAA receptor/Cl- channel complex in an inactivated state(s).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531763 TI - The paralytic agent 2,4-dithiobiuret decreases open time of murine skeletal muscle acetylcholine receptor channels. AB - 2,4-Dithiobiuret (DTB) causes a delayed-onset neuromuscular weakness when given chronically to rats. Mechanisms underlying this effect involve disruptions of acetylcholine (ACh) release and possibly effects on the ACh receptor channel complex. Previous experiments demonstrated a decrease in decay time constants for end-plate currents and miniature end-plate currents of muscles from rats exhibiting DTB-induced muscle weakness compared with those of controls. The purpose of the present study was to determine whether the alteration in rise and decay times for synaptic currents was due to direct effects of DTB on ACh receptor channels. Currents carried through single ACh-activated channels were recorded using patch voltage-clamp techniques in G8 mouse myotubes exposed to DTB in their growth medium and from intact hemidiaphragm preparations of rats treated with DTB by examining fluctuations in membrane noise during iontophoresis of agonist. Exposure of myotubes to DTB (1 or 10 microM) decreased the mean channel open time induced by suberyldicholine for short durations of exposure, whereas longer exposures (24-48 h) to DTB were required in order for decreased open times for ACh as an agonist to be observed. In the absence of DTB, closed times for single channels of G8 cells were described by a two-exponential fit reflecting intraburst and interburst closures. At 1 microM DTB, the duration of gaps within bursts and of gaps between bursts increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531764 TI - Effects of typical and atypical antipsychotic drugs on response decrement patterns in rats. AB - Within-session decrements in instrumental responding are a characteristic effect of certain neuroleptic drugs including haloperidol and pimozide. There is little consensus, however, as to whether these effects can best be explained as motivational or motor deficits. A number of recently developed drugs have been described as atypical antipsychotics and are claimed to produce fewer motor side effects than more traditional neuroleptics. Little work has been done to investigate whether such atypical agents also give rise to within-session response decrements. The present experiment used the operant responding of rats to study the effects of a range of antipsychotic drugs. Rats were trained to lever press for food during daily sessions (FR10 schedule) and responding was recorded during each 3-min period of the 15-min sessions. Dose-related decreases in overall response rates were produced by all the drugs studied; ED50 values were: haloperidol 0.17 mg/kg, risperidone 0.18 mg/kg, setoperone 0.23 mg/kg, remoxipride 1.2 mg/kg, sertindole 4.8 mg/kg, thioridazine 7.6 mg/kg, clozapine 8.9 mg/kg, amisulpride 34 mg/kg, sulpiride 74 mg/kg. The presence of within session response decrements was confirmed for haloperidol and demonstrated with remoxipride, but similar effects were not observed with clozapine, thioridazine, risperidone, sertindole, setoperone, sulpiride and amisulpride. As a number of these drugs were known to have high affinity for 5HT2 receptors, the effects of the 5HT2 antagonist ritanserin (1 and 10 mg/kg) were studied alone and in combination with haloperidol. Ritanserin had no effect on response rates, and did not antagonize but rather potentiated the effect of haloperidol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531765 TI - Role of hypothermia in the mechanism of protection against serotonergic toxicity. I. Experiments using 3,4-methylenedioxymethamphetamine, dizocilpine, CGS 19755 and NBQX. AB - High doses of 3,4-methylenedioxymethamphetamine (MDMA) have been shown to cause long-lasting depletions of central serotonin (5-HT) which are indicative of neuronal toxicity. The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (DZ) attenuates depletions of 5-HT induced by MDMA. Because DZ has been shown to induce hypothermia in rat models of ischemia, the purpose of this study was to assess whether DZ and two other glutamate antagonists, CGS 19755 (CGS) and 2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(F)quinoxaline (NBQX), protect against MDMA-induced 5-HT depletions by induction of hypothermia. Male Sprague-Dawley rats were injected with either saline (SAL), DZ (2.5 mg/kg), CGS (25.0 or 50.0 mg/kg x 2 injections) or NBQX (30.0 mg/kg x 2 injections or 55.0 mg/kg x 3 injections) followed by either MDMA (40.0 mg/kg) or SAL. Core body temperature (TEMP) was monitored for 4 h or longer using radiotelemetry. Base-line TEMP was between 37.0 and 37.6 degrees C. Administration of DZ with MDMA decreased TEMP to 34.0 +/- 0.39 degrees C within 2 h of the MDMA injection, and also protected against serotonergic toxicity. Neither SAL/MDMA nor DZ/SAL had an effect on TEMP over the same period. When rats were treated with DZ/MDMA and TEMP was maintained between 38.4 degrees C and 40.4 degrees C for 4 h, protection against 5-HT depletion was abolished. Coadministration of the competitive NMDA antagonist CGS with MDMA-resulted in a decrease in TEMP to 34.5 +/- 0.27 degrees C, and provided partial protection against 5-HT depletions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531766 TI - Previous exposure to bradykinin unmasks an endothelium-dependent relaxation to the converting enzyme inhibitor trandolaprilat in isolated canine coronary arteries. AB - Experiments were designed to investigate the mechanism underlying the endothelium dependent relaxation to trandolaprilat, a converting enzyme inhibitor, in canine coronary arteries previously exposed to bradykinin. Rings suspended in organ chambers were exposed to bradykinin for 3 min and washed repeatedly for 150 min. Trandolaprilat caused relaxations [IC50(-log M)8.59] in rings with endothelium previously exposed to bradykinin. This response was observed already at concentrations where trandolaprilat did not augment relaxations to bradykinin. When the rings were exposed to acetylcholine or to Des-Arg9-bradykinin (B1 agonist) trandolaprilat caused only a minimal response. Carboxypeptidase B but not aprotinin impaired the relaxation to trandolaprilat, suggesting a contribution of bradykinin. After exposure to [3H]-bradykinin, no detectable amounts of the peptide were released by trandolaprilat or found in the preparations. The relaxation to trandolaprilat was not affected by the B1 antagonist Leu8-des-Arg9-bradykinin. HOE-140, a B2 antagonist impaired the maximal response to trandolaprilat, while exhibiting competitive antagonism against bradykinin (pA2 9.00). The maximal relaxation to trandolaprilat was impaired in the presence of nitro-L-arginine and methylene blue. The potency, but not the maximal effect of bradykinin was reduced by these inhibitors. Exogenous bradykinin, but not trandolaprilat, caused an endothelium-dependent hyperpolarization. At equipotent submaximal concentrations, bradykinin released both nitric oxide and endothelium-derived hyperpolarizing factor although trandolaprilat stimulated the production of nitric oxide only. These findings suggest that acute endothelium-dependent relaxations to trandolaprilat in preparations previously exposed to bradykinin are mediated by nitric oxide and may be due partially to protection of bound bradykinin but also to some other unresolved mechanism(s) as well. PMID- 7531767 TI - Nitric oxide synthase inhibitor alters papillary muscle force-frequency relationship. AB - We provide evidence for an immediate effect of NG-monomethyl-L-arginine (L-NMMA) on the force-frequency relationship in isolated hamster papillary muscles. L-NMMA (competitive inhibitor of nitric oxide synthase) reversed the force-frequency relationship (staircase effect) in isolated hamster papillary muscles from negative to positive (P < .01; ANOVA; n = 6). The addition of L-arginine (substrate for nitric oxide synthase) blocked the L-NMMA effect (P < .01; ANOVA; n = 6). The addition of the nitric oxide (NO) donor, sodium nitroprusside (NTP), significantly increased the level of cGMP in the tissue bath (P < .01; test; n = 6) and reversed the positive inotropic effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). The addition of 8-Br-cGMP to the bath resulted in a concentration dependent decrease in tension generated by the papillary muscles (n = 6). Methylene blue (known inhibitor of cGMP) mimicked the effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). L-NMMA also significantly blunted the negative inotropic effect of ryanodine (SR calcium release channel regulator) (P < .01; ANOVA; n = 6). The positive inotropic effect of Bay K 8644 (sarcolemmal, L-type calcium channel regulator) was not affected by L-NMMA (P = NS; ANOVA; n = 6). L NMMA had no effect on either [3H]ryanodine or [3H]PN200-110 (sarcolemmal, L-type calcium channel regulator) binding to cardiac membranes. These findings support a cGMP-dependent role for endogenous NO in myocardial E-C coupling. PMID- 7531768 TI - Contribution of chloride conductance increase to slow EPSC and tachykinin current in guinea-pig myenteric neurones. AB - 1. Single electrode voltage clamp recordings were obtained from myenteric neurones of guinea-pig ileum in vitro. Slow excitatory postsynaptic currents (sEPSCs) were elicited by focal stimulation of interganglionic nerve strands in twenty-four of thirty neurones more than 30 min after impalement. In seventeen of twenty-four neurones, sEPSCs were associated with a conductance decrease and reversed polarity at -96 +/- 3 mV (near the reversal potential for potassium, EK); this response was due to inhibition of resting potassium conductance, gK. In seven of twenty-four neurones, there was either no net conductance change or a biphasic conductance change during the sEPSC; a reversal potential for peak currents could not be determined. 2. Application of senktide (3 microM), a neurokinin-3 receptor agonist, caused an inward current in forty-one of fifty three neurones more than 30 min after impalement. In twenty of forty-one neurones, senktide-induced currents were due to inhibition of resting gK. In eleven of forty-one neurones there was either no net conductance change or a biphasic conductance change; a reversal potential for peak currents could not be determined. In ten out of forty-one neurones, senktide-induced currents were associated with a conductance increase (ginc); the estimated reversal potential was -17 +/- 3 mV. 3. Application of forskolin (1 microM) caused an inward current that occluded the decrease in gK caused by senktide and the sEPSC. In neurones in which sESPCs and senktide responses were associated with an unclear or biphasic conductance change, forskolin did not reduce the peak current and residual currents were usually associated with a ginc. 4. In neurones in which senktide induced currents were associated with a ginc, reducing extracellular Cl- to 13 mM reduced senktide-induced currents by 79%. Reducing extracellular Na+, or adding tetraethylammonium (TEA, 50 mM), cobalt (2 mM) or picrotoxin (30 microM) did not change senktide-induced currents. The chloride transport/channel blockers niflumic acid and mefenamic acid (both at 100 microM) blocked senktide-induced currents. It was concluded that senktide increases chloride conductance (gCl). 5. Chord conductance measurements made between -70 and -90 mV during sEPSCs were used to determine the contribution of an increase in gCl to sEPSCs. These measurements indicated that the peak sEPSC is composed of a 90% decrease in gK and a 10% increase in gCl. Similar data were obtained from measurements made during senktide responses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531769 TI - Ionic conductances contributing to spike repolarization and after-potentials in rat medial vestibular nucleus neurones. AB - 1. Intracellular recordings were made from 123 tonically active medial vestibular nucleus (MVN) neurones in a horizontal slice preparation of the dorsal brainstem of the rat. On the basis of their averaged action potential shapes, the cells were classified as either type A, having a single deep after-hyperpolarization (AHP; 40/123 cells, 33%), or type B, having an early fast AHP and a delayed slow AHP (83/123 cells, 67%). The two cell types were distributed throughout the rostrocaudal extent of the MVN. 2. In type A cells TEA reduced the single deep AHP and decreased the rate of spike repolarization. Depolarizing current pulses from a hyperpolarized membrane potential elicited spikes with short plateau potentials in TEA. These persisted in Ca(2+)-free medium but were abolished along with the spontaneous activity in TTX. Ca(2+)-free medium did not affect the initial rate of repolarization but reduced the deep AHP. Apamin and carbachol had little effect. 4-Aminopyridine (4-AP) slowed spike repolarization and the AHP amplitude by a small amount. Thus, in type A cells spike repolarization and AHP appear to be mediated largely by a TEA-sensitive potassium current (presumably IK) and an apamin-insensitive Ca(2+)-activated potassium current (presumably IC). 3. The early fast AHP in type B cells was readily abolished in TEA. In seven of ten type B cells tested, the spontaneous spikes developed plateau potentials of 100-120 ms duration in 10 mM TEA, which then became 7-9 s long in Ca(2+)-free medium. In the remaining three cells, the spontaneous plateaux were 1.75-2 s long in TEA, and were reduced to 30-100 ms in Ca(2+)-free medium. TTX abolished the spontaneous spikes and plateaux. The delayed AHP was abolished by apamin, which induced irregular firing. 4-AP slowed spike repolarization and abolished the fast AHP, but did not induce plateaux. Thus, in type B cells spike repolarization involves a TEA-sensitive current (presumably IK) as well as IC and the 4-AP sensitive potassium current IA, while the apamin-sensitive potassium current IAHP is responsible for the delayed AHP. 4. The tonic activity in type B cells appears to be regulated mainly by interactions between a persistent Na+ current, which in most cells is large enough to generate plateaux when repolarization is impeded in TEA, and the hyperpolarization mediated by IAHP. About 30% of type B cells have an additional inward Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531770 TI - Ca2+ inhibition of inositol trisphosphate-induced Ca2+ release in single smooth muscle cells of guinea-pig small intestine. AB - 1. Single smooth muscle cells from the longitudinal muscle layer of guinea-pig small intestine were voltage clamped using patch pipettes in the whole-cell mode. 2. When D-myo-inositol 1,4,5-trisphosphate (InsP3) was released at intervals, by photolysis of 'caged' InsP3 within the cell, increases in [Ca2+]i in many cells, as judged from Ca(2+)-activated K(+)-current, were all-or-none; release of InsP3 before a critical interval had elapsed, which was quite stable for an individual cell, resulted in no response. After Ca(2+)-induced Ca2+ release had been evoked by depolarization, the InsP3 response was inhibited. Oscillations in [Ca2+]i evoked by muscarinic receptor activation were unaffected by Ruthenium Red; during these oscillations exogenous InsP3 was not effective close to, or shortly after, peak [Ca2+]i but was effective at other times. 3. Reproducible release of Ca2+ and elevation of [Ca2+]i could be produced by brief (up to 0.5 s) pressure applications of 10 mM caffeine at intervals of 10 s or greater but caffeine itself rarely evoked oscillations in [Ca2+]i. Responses to flash release of InsP3 were reduced after caffeine-induced responses and recovery of caffeine-induced Ca2+ release was faster than recovery of InsP3-induced Ca2+ release. 4. The results support the idea that InsP3-induced Ca(2+)-store release can be inhibited by a certain level of [Ca2+]i at a time when Ca2+ stores have refilled and can be released by caffeine; they also support the suggestion that during oscillations of [Ca2+]i evoked by muscarinic receptor activation, Ca2+ inhibition of InsP3 induced Ca2+ release at some critical level of [Ca2+]i allows Ca2+ stores to refill and leads to a fall in [Ca2+]i so contributing to the oscillations which are observed. PMID- 7531771 TI - An atomic force microscope for cytological and histological investigations. AB - An atomic force microscope (AFM) specifically designed for cytological and histological studies and able to operate on the same scale of the highest optical magnification is described. The AFM is a non-invasive instrument; it operates on samples which do not require any kind of treatment and it can produce information that supplements and completes the information given by traditional microscopical methods. The apparatus has been used to image fixed human chromosomes and to investigate the action of trypsin during the staining for banding. First results showed that banding patterns very similar to G-banding pre-exist to staining and to trypsin treatment in human metaphase chromosomes, and that the trypsin treatment induces a structural collapse in the chromatin. The instrument was also used on thin sections of plant tissue and gave promising results. Experience confirmed that AFM is a suitable tool for this kind of investigation, and proved the importance of developing AFM microscopes specifically designed for routine use in cytology and histology, conceived for non-specialized users. PMID- 7531772 TI - Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. AB - Ribonuclease H is an endonuclease that hydrolyzes the RNA moiety of RNA-DNA duplex molecules. Escherichia coli ribonuclease H is involved in DNA replication, and retroviral ribonuclease H is essential for reverse transcription of the viral genome. To characterize the intramolecular dynamical properties of E. coli ribonuclease H, spin-lattice relaxation rate constants, spin-spin relaxation rate constants and steady state nuclear Overhauser effects for the 15N nuclear spins were measured by using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed by using a series of dynamical models in conjunction with a statistical model selection protocol. Ribonuclease H exhibits a complex array of dynamical features, most notably in the parallel beta-strands of the principal five-stranded beta-sheet, the coiled-coil helical interface, the active site, and the loop regions surrounding the active site. The dynamical properties are correlated with local structural environments of the 15N spins and suggest possible relationships to the functional properties of ribonuclease H. Results for E. coli ribonuclease H are compared to previously reported results for the human immunodeficiency virus type 1 ribonuclease H domain of reverse transcriptase. PMID- 7531773 TI - Prostate-specific antigen as predictor of prostate cancer in black men and white men. AB - BACKGROUND: The increasing incidence of prostate cancer creates complex issues in health care management and cost containment. There is a need to evaluate serial measurements of prostate-specific antigen (PSA) as a marker for long-term risk of clinically important prostate cancer (stages B through D). PURPOSE: We used a nested case-control design within a retrospective cohort study to evaluate serial PSA concentrations in relation to subsequent prostate cancer diagnoses. METHODS: Participants included 40 black and 96 white men with subsequent diagnoses of prostate cancer and 84 black and 100 white men without such diagnoses (control subjects) in a multiphasic health screening program conducted by the Kaiser Permanente Medical Care Program of Northern California. Serial serum samples were collected 1.5-23 years before prostate cancer diagnosis. RESULTS: Median serum PSA concentrations, specific for age and subsequent cancer status, were similar in blacks and whites. Concentrations in control subjects increased exponentially with age, with a doubling time of 24.9 years. Concentrations in men with stage A cancer were similar to those in control subjects. Until about 13 years before diagnosis, PSA in men with subsequent cancer stages B through D increased exponentially with age, with a doubling time similar to that of control subjects. Thereafter, the PSA concentrations increased exponentially, with a doubling time of 4.3 years. Rapid increase in PSA concentration started about 1.5 years earlier for men with stage D cancer than for men with stage B or C cancer. The single PSA measurement drawn closest to diagnosis was a more sensitive marker of stages B through D cancer within the next 7 years than was any index of change that also took account of earlier PSA readings. CONCLUSIONS: These data suggest that 1) age specific PSA concentrations are similar in black men and white men and 2) current PSA concentration, specific for age, outperforms changes in past concentrations in identifying the man who will develop stage B, C, or D cancer within 7 years, albeit at the cost of a slightly higher rate of false-positive results. This interpretation needs confirmation in other data containing many serial PSA measurements within a few years of diagnosis. PMID- 7531774 TI - Association with clinical outcome of expression of VLA-4 in primary cutaneous malignant melanoma as well as P-selectin and E-selectin on intratumoral vessels. AB - BACKGROUND: The process of tumor growth and metastasis is a complex multistep cascade. The ability of tumor cells to adhere to and detach from extracellular matrix and endothelial cells may be crucial in the metastatic process and may dramatically alter the clinical prognosis and outcome for patients with certain cancers. A number of adhesion molecules have been detected on human melanoma cells and have been associated with various properties in vitro including invasiveness. Recent findings from our laboratory have indicated an ordered change in integrin expression during the process of tumor progression. PURPOSE: This study was designed to identify molecular markers present on human melanoma cells and in intratumoral vessels that have prognostic significance regarding disease-free interval and survival time. METHODS: Specimens of primary cutaneous malignant melanoma were obtained from 60 patients who had been followed for at least 36 months, with development of metastases in 29 patients during that period of time, and were analyzed for their expression of VLA-4, VLA-6, ICAM-1, ELAM-1 (E-selectin), CD62 (P-selectin), and CD44v6 molecules on tumor and endothelial cells by immunostaining. Light microscopy was used to evaluate and categorize the number of positively stained cells. Statistical analyses were done to determine the relationship of the expression of individual adhesion molecules with time to disease progression (i.e., disease-free interval) and overall survival time. RESULTS: In each case, positive staining for ELAM-1 and CD62 on intratumoral vessels and for VLA-4 on human melanoma cells was negatively associated with disease-free interval (P < .01) and overall survival time (P < .01). The presence of VLA-6, CD44v6, and ICAM-1 on melanoma cells was not associated with clinical outcome. CONCLUSIONS: Immunohistochemical screening and detection of ELAM-1, CD62, and VLA-4 may help to define a subgroup of melanoma patients at risk of developing metastases. PMID- 7531775 TI - Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: a critical review of the literature. AB - Chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) is a broad spectrum organophosphorus (OP) insecticide. Anticipated increases in the already extensive use of this compound have prompted this reassessment of its neurotoxicity. Because chlorpyrifos and other OP insecticides are designed to produce acute cholinergic effects through inhibition of acetylcholinesterase (AChE) and some OP compounds can cause OP compound-induced delayed neurotoxicity (OPIDN) via chemical modification of neurotoxic esterase (neuropathy target esterase, NTE), this review focuses on the capacity of chlorpyrifos to precipitate these and other adverse neurological consequences. Chlorpyrifos exhibits only moderate acute toxicity in many mammalian species, due largely to detoxification of the active metabolite, chlorpyrifos oxon, by A-esterases. Rats given large doses of chlorpyrifos (sc in oil) have prolonged inhibition of brain AChE, possibly due to slow release of the parent compound from a depot. Associated cognitive and motor deficits return to normal well before recovery of AChE activity and muscarinic receptor down-regulation, as expected from classic tolerance. Controlled studies of OP compound exposures in humans also indicate that cognitive dysfunction requires substantial AChE inhibition. Information is relatively sparse on neurological dysfunction that is secondary to theoretical reproductive, developmental, or immunological effects, but the best available data indicate that such effects are unlikely to result from exposures to chlorpyrifos. In accord with the much greater inhibitory potency of chlorpyrifos oxon for AChE than for NTE, clinical reports and experimental studies indicate that OPIDN from acute exposures to chlorpyrifos requires doses well in excess of the LD50, even when followed by repeated doses of the OPIDN potentiator phenylmethanesulfonyl fluoride (PMSF). Likewise, studies in hens show that subchronic exposures at the maximum tolerated daily dose do not result in OPIDN. Although exposure to chlorpyrifos as a result of normal use is unlikely to produce classical OPIDN, a recent report stated that mild reversible sensory neuropathy had occurred in eight patients who had been exposed subchronically to unknown amounts of chlorpyrifos. It is not clear whether these cases represent an incorrect linkage of cause and effect, a newly disclosed reversible sensory component of OPIDN, or an entirely new phenomenon. The question of the potential for chlorpyrifos to cause this mild sensory neuropathy could be resolved by the use of quantitative tests of sensory function in animal experiments and/or prospective studies of humans with known exposures to chlorpyrifos. PMID- 7531776 TI - Comparative developmental and maternal neurotoxicity following acute gestational exposure to chlorpyrifos in rats. AB - Chlorpyrifos (CPF), an organophosphorus (OP) insecticide, exerts toxicity through inhibition of acetylcholinesterase (AChE). In the present study, pregnant Sprague Dawley rats were given CPF (200 mg/kg, sc) as a single dose on gestation d 12 (GD12) and then sacrificed on either GD16, GD20, or postnatal d 3 (PND3) for measurement of maternal and developmental indicators of toxicity. While most CPF treated rats exhibited no overt signs, a subset (4/28) showed moderate to severe signs of "cholinergic" toxicity at 2-3 d after treatment, and these rats were omitted from further studies. Extensive AChE inhibition (82-88%) was noted in maternal brain at all three time points following acute exposures. At GD16 and GD20, fetal brain AChE activity was inhibited 42-44%. While some degree of recovery in AChE activity was noted in pup brain by PND3, AChE activity was still inhibited (30%) in treated pups cross-fostered to control dams. In vitro inhibition of maternal and fetal (GD20) brain AChE activity by the active metabolite, chlorpyrifos oxon, suggested that the prenatal brain AChE activity was somewhat more sensitive (IC50 at 37.0 degrees C, 20 min: dam, 26.6 +/- 1.8 x 10(-9) M; fetus, 6.7 +/- 0.4 x 10(-9) M). Maternal brain muscarinic receptor binding was more extensively reduced (30-32%) at GD20 and PND3 as compared to the developing brain at GD20 (16%) and PND3 (11%). A simple postnatal reflex test (righting reflex) was transiently altered by CPF. The results suggest that CPF exposure to dams during gestation produces more extensive neurotoxicological effects in the dam relative to the developing fetus. PMID- 7531777 TI - Gender-dependent differences in hepatic tumor promotion in diethylnitrosamine initiated infant B6C3F1 mice by alpha-hexachlorocyclohexane. AB - Chronic exposure of B6C3F1 mice to phenobarbital (PB), subsequent to a single initiating dose of diethylnitrosamine (DENA) at 15 d of age, has been previously shown to inhibit hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice (Weghorst & Klaunig, 1989). In the present study, the effects of another hepatic tumor promoter, alpha-hexachlorocyclohexane (alpha HCH), in similarly initiated B6C3F1 mice was investigated. Male and female mice received a single intraperitoneal (ip) injection of either DENA or saline at 15 d of age. Beginning at 28 d of age, the mice received either alpha-HCH in the diet (250 ppm) or untreated basal diet. Like PB, alpha-HCH inhibited hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice following chronic exposure. In an additional experiment, already formed preneoplastic hepatic foci in male and female B6C3F1 mice were examined for their responsiveness to the induction of DNA synthesis by alpha-HCH treatment. The mice received a single ip injection of DENA at 15 d of age to induce hepatocellular foci. Beginning at 24 wk of age, mice received either basal diet or diet containing 250 ppm alpha-HCH for 7 consecutive d. DNA synthesis was assessed by continuous [3H]thymidine infusion via subcutaneously implanted osmotic minipumps. In female mice treated with alpha-HCH, DNA synthesis in hepatocellular foci was increased substantially compared to untreated females. In contrast, male mice receiving alpha-HCH showed no increase in DNA synthesis in hepatocellular foci from that seen in non-alpha-HCH-treated males. Based on these results, we postulate that the gender-dependent differences in hepatic tumorigenesis observed in B6C3F1 mice initiated during infancy may be related to chemical tumor promoter modulation of the normal hormonal environment, or to differences in the ability of hepatocellular foci to respond to the induction of DNA synthesis by the tumor promoter. PMID- 7531778 TI - In vitro effects of tachykinins on the smooth musculature of horse gut. AB - The contractile effects of the tachykinins eledoisin, substance P and neurokinin A and B were investigated in vitro on circular and longitudinal muscle strips from horse duodenum, ileum and colon. Circular smooth muscle of the small intestine was highly responsive, large intestine circular smooth muscle less so, while longitudinal muscle from all gut segments was much less sensitive. pD2 values and intrinsic activities on small intestine circular muscle indicated differences in receptor distribution between the duodenum and ileum: NK3 and a smaller number of NK2 receptors being present in the duodenum, and NK2 receptors predominating in the ileum. Notwithstanding this, eledoisin and neurokinin B were the most active substances on duodenum and ileum, respectively. These findings suggest that tachykinins may play a role in equine gastrointestinal pathophysiology. PMID- 7531779 TI - Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus. AB - Synthetic peptides representing T- and B-cell epitopes from the fusion (F) protein of measles virus (MV) were tested for their ability to induce a protective immune response against intracerebral challenge with neuroadapted strains of MV and canine distemper virus (CDV) in mice. Of the panel of peptides tested, only a chimeric peptide consisting of two copies of a promiscuous T-cell epitope (representing residues 288 to 302 of MV F protein) synthesized at the amino terminus of a B-cell epitope (representing residues 404 to 414 of MV F protein) was able to induce a protective response against challenge with MV and CDV in inbred mice. The protective response induced by this peptide (TTB) was associated with a significant reduction in mortality, histological absence of acute encephalitis, and greatly reduced titers of virus in the brains of TTB immune mice following challenge compared with the results for nonimmunized controls. A chimeric peptide comprising one copy of the T-cell epitope and one copy of the B-cell epitope (TB) did not induce a protective response. A comparison of the antibody responses induced by the two chimeras suggested that differences in protective efficacy following immunization may be a result of the higher affinity of the antibody induced by the TTB peptide than that of the antibody induced by the TB peptide. In addition, differences in the immunoglobulin G subclass of the antipeptide antibody responses were observed, and these may play a role in the differences in protection observed. These results indicate that appropriately designed synthetic peptides have potential as vaccines for the induction of cross-reactive protection against morbilliviruses. PMID- 7531782 TI - A potpourri of new observations about prostate cancer. PMID- 7531781 TI - Measurement of serum prostate specific antigen levels in women and in prostatectomized men with an ultrasensitive immunoassay technique. AB - Serum prostate specific antigen (PSA) was measured in 89 prostate cancer patients with no evidence of relapse after radical prostatectomy, in 387 hospitalized women with various diseases and in 674 apparently healthy female blood donors, using an ultrasensitive time resolved immunofluorometric assay with a biological detection limit of 0.01 microgram./l. Of the prostatectomized cancer patients 50% had measurable PSA (0.010 microgram./l. or greater) and 21% had levels of 0.050 microgram./l. or greater. Postoperative PSA was not associated with year of surgery, preoperative PSA or histological grade of the tumors. The distribution of PSA in 1,064 female sera is also reported, of which 83% had no detectable serum PSA, and in 15% PSA was between 0.010 and 0.049 microgram./l. Only 27 subjects (2.5%) had PSA levels of 0.050 microgram./l. or greater, including 16 (1.5%) with PSA of 0.10 microgram./l. or greater. High serum PSA in women was associated with age 50 years or older. Further studies are needed to examine if the difference in PSA levels between women and prostatectomized men is due to residual or recurrent tumor or to other reasons associated with gender. PMID- 7531780 TI - Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55). AB - A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment. PMID- 7531783 TI - Variable histology of anastomotic biopsies with detectable prostate specific antigen after radical prostatectomy. AB - Progressive elevation of the prostate specific antigen (PSA) level after radical prostatectomy for adenocarcinoma is generally considered as irrefutable evidence of recurrent tumor. We assessed the results of 62 biopsies of the vesicourethral anastomosis in 41 men who had 3 or more consecutive PSA levels of 0.4 ng./ml. or greater after radical prostatectomy and no evidence of metastatic disease. The median PSA at the time of the first biopsy was 2.2 ng./ml. (range 0.4 to 50). Histological confirmation of recurrent cancer was established after 1 biopsy procedure in 39% of the patients and after 1 or more biopsy procedures in 59%. Biopsy was positive in 78% of 23 patients with an abnormal digital rectal examination, 40% of 5 with an abnormal transrectal ultrasound only, and 23% of 13 with a normal digital rectal examination and ultrasound. Among the patients with and without biopsy proved tumor recurrence there were no significant differences between the pathological stage or histological grade of the primary tumors, the month after surgery of the first detectable PSA level, the PSA doubling time, the month after surgery of the positive biopsy or the last negative biopsy, and the PSA level at the time of the positive biopsy or the last negative biopsy. In 6 cases benign prostatic tissue only was recovered from 1 or more biopsy specimens. This experience demonstrates that in patients with a detectable PSA after radical prostatectomy recurrent cancer may be difficult to document by biopsy of the vesicourethral anastomosis. PMID- 7531785 TI - Withdrawal phenomenon with the antiandrogen casodex. AB - Total androgen blockade with castration and antiandrogens has become the primary therapy in metastatic prostate carcinoma. Several reports have been published on the flutamide withdrawal syndrome with a favorable response in patients with progression of disease after lengthy remission while taking combined hormone therapy. The experience with withdrawal of the experimental nonsteroidal antiandrogen casodex in 3 patients is reported. All patients received casodex monotherapy initially, then luteinizing hormone-releasing hormone analogues were added on progression of disease (bone scan and prostate specific antigen [PSA]) and eventually casodex was withdrawn on further progression of disease (PSA and bone scan). Two patients had declines in PSA levels of 42% and 75% sustained for 3 to 6 months, and 1 patient had a stable condition for 2 months. These responses were similar to those of withdrawal of flutamide, which were limited to patients who had received combination therapy at the onset of treatment. Thus, the observations in this report would suggest that the initial and sustained exposure to an antiandrogen is the more important factor in the withdrawal phenomenon rather than the low androgen environment alone that occurs with initial luteinizing hormone-releasing hormone analogue monotherapy, orchiectomy or combination luteinizing hormone-releasing hormone analogue and antiandrogen. PMID- 7531787 TI - [LDL-receptor-related protein]. AB - The LDL-receptor-related protein is a multifunctional endocytotic receptor that clear apo E- and/or lipoprotein lipase-enriched lipoproteins, proteases such as alpha 2-macroglobulin plasminogen and activator inhibitor-1 (PAI-1), Pseudomonas exotoxin A and probably vitellogenin from blood circulation. Its role in plasma chylomicron remnant clearance has been postulated, and recently has been demonstrated by using its inhibitor; receptor-associated protein (RAP) in LDL receptor knockout mice. PMID- 7531786 TI - Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. AB - PURPOSE: Recent experimental studies have established the feasibility of therapeutic angiogenesis; in all cases, this has been achieved with local administration of angiogenic growth factors. This study was designed to investigate the hypothesis that systemic administration of an angiogenic growth factor specifically mitogenic for endothelial cells--vascular endothelial growth factor (VEGF)--could augment collateral vessel development in a rabbit ischemic hindlimb model. METHODS: Ten days after the ligation of the external iliac artery and excision of the common and superficial femoral arteries in one limb of New Zealand white rabbits, heparin (800 IU, n = 13), VEGF (1 mg, n = 3; 5 mg, n = 5), heparin (800 IU) + VEGF (1 mg, n = 5; 5 mg, n = 7), or saline solution (n = 8) was injected as a single bolus in a marginal ear vein. Collateral vessel formation and limb perfusion were assessed 10 and 30 days after treatment. RESULTS: Animals in both VEGF-treated groups had a significantly higher (p < 0.01) increase in calf blood pressure ratio at day 10 (control, 0.44 +/- 0.02; heparin, 0.47 +/- 0.02; VEGF, 0.60 +/- 0.01; heparin+VEGF, 0.61 +/- 0.02) and day 30 (control, 0.49 +/- 0.05; heparin, 0.48 +/- 0.02; VEGF, 0.70 +/- 0.03; heparin+VEGF, 0.73 +/- 0.03). Both VEGF-treated groups had a significantly higher (p < 0.05) angiographic score at day 30 (control, 0.28 +/- 0.01; heparin, 0.28 +/ 0.01; VEGF, 0.37 +/- 0.01; heparin+VEGF, 0.38 +/- 0.02). Maximum flow reserve at day 30 in the ischemic limb was higher (p < 0.05) in VEGF-treated rabbits (control, 1.87 +/- 0.07; heparin, 1.92 +/- 0.08; VEGF, 2.42 +/- 0.16; heparin+VEGF, 2.33 +/- 0.12). Capillary density was higher (p < 0.01) in the ischemic muscles of VEGF-treated rabbits (control, 156 +/- 10/mm2; heparin, 178 +/- 8/mm2; VEGF, 230 +/- 10/mm2; heparin+VEGF, 233 +/- 8/mm2). CONCLUSIONS: This series of in vivo experiments demonstrates that intravenous administration of VEGF, with or without heparin, results in both anatomic and physiologic evidence of enhanced collateral vessel formation in the rabbit ischemic hindlimb. Single bolus systemic administration of VEGF may be a feasible therapeutic strategy in patients with lower-extremity ischemia. PMID- 7531784 TI - Detailed preliminary analysis of 125iodine implantation for localized prostate cancer using percutaneous approach. AB - Recent developments have permitted accurate seed placement and dosimetry for interstitial brachytherapy of selected patients with localized prostate cancer. We present our experience with 76 patients divided into 2 groups. Group 1 included 45 patients with smaller, more well differentiated tumors, usually less than 2 cm. in diameter on digital rectal examination or transrectal ultrasound and a Gleason score of less than 7 who were treated with 125iodine alone. Group 2 consisted of 31 patients with localized tumors greater than 2 cm. in diameter and/or a Gleason sum equal to or greater than 7 who were treated with low dose external beam radiation followed by 125iodine boost 4 weeks later. Complete clinical progression-free survival, including prostate specific antigen, digital rectal examination and biopsy, was 51% for group 1 and 63.3% for group 2, with a mean followup of 26.3 months. Prostate specific antigen progression-free survival was 97.7% for group 1 and 94.7% for group 2. These results appear to be superior to external beam radiation only although longer followup is needed to substantiate these favorable early results. The procedures were well tolerated with good potency sparing. They were performed on an outpatient or short stay basis and provided a good alternative to external beam radiation only or hormonal treatment for select patients with localized prostate cancer who may not be candidates for radical prostatectomy. PMID- 7531789 TI - Fish oil supplementation and essential fatty acid deficiency reduce nitric oxide synthesis by rat macrophages. AB - Both fish oil-derived omega-3 polyunsaturated fatty acid (omega 3 PUFA) supplementation and essential fatty acid (EFA) deficiency have been shown to exert anti-inflammatory effects and, hence, to ameliorate immune-mediated glomerulonephritis. The mechanisms underlying these effects include alterations in the production of eicosanoids, cytokines (that is, tumor necrosis factor, TNF alpha) and reactive oxygen species by blood borne cells. Because, in addition to these mediators nitric oxide (NO) is also implicated in glomerular injury, we have examined if both diets affected macrophage NO production as well. Rats were fed a standard chow, an omega 3 PUFA-supplemented diet, or an EFA-deficient diet for six weeks before resident peritoneal macrophages were isolated. These cells were exposed to lipopolysaccharide (LPS) and the NO metabolite, nitrite (NO2-), was measured in the medium using the Griess reagent. Release of NO2- was enhanced by LPS in a dose-dependent manner. With 10 ng/ml LPS challenge, NO2- release was reduced by 37% and 57% by omega 3 PUFA supplementation and EFA deficiency, respectively. NO2- returned to control levels two weeks after the end of diet. Macrophage production of TNF-alpha responded in a similar manner. Diet-induced reduction of NO2- release was neither attributable to a reduction of inducible NO synthase mRNA levels as shown by Northern blot analysis, nor to an increased competition of NO synthase and arginase for the substrate (L-arginine). Indeed, arginase activity of macrophages was even slightly reduced by both omega 3 PUFA supplemented diet and EFA-deficient diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531788 TI - [3 cases of elderly malignant lymphoma of the thyroid gland]. AB - Chemotherapy and radiotherapy were performed on patients over 70 years of age with malignant lymphoma of the thyroid gland and complete remission (CR) was achieved in three patients who are reported here. Case 1: An 87-year-old woman who had been treated for Hashimoto's disease for 10 years as an outpatient was diagnosed as having a diffuse-small cell, B-cell type lymphoma by an aspiration biopsy of the tumor in the left thyroid. COP-BLAM chemotherapy followed by radiotherapy achieved CR. Case 2: A 71-year-old woman was referred to our department because of a right cervical tumor. A biopsy of the tumor revealed a diffuse medium-sized, B-cell type lymphoma. She also has been proved to have hypothyroidism. She entered CR after COP-BLAM chemotherapy combined with a radiotherapy. Case 3: A 76-year-old male patients with Hashimoto's disease who had been treated for 12 years at another outpatient clinic, presented with a right cervical tumor. Aspiration biopsy of the tumor yielded a diagnosis of B cell lymphoma. COP-BLAM chemotherapy combined with radiotherapy achieved a CR. All three patients entered CR after chemotherapy combined with radiotherapy. Moreover, the microsome test and thyroid test also became negative after the combined modality therapy. In the future, there will probably be increases in the incidence of malignant lymphoma of the thyroid gland, due to the aging of society, and it will be necessary to establish methods of diagnosis and treatment for this disease. PMID- 7531790 TI - Tetrahydrobiopterin is a limiting factor of nitric oxide generation in interleukin 1 beta-stimulated rat glomerular mesangial cells. AB - Treatment of mesangial cells with recombinant human interleukin 1 beta (IL-1 beta) triggers the expression of a macrophage-type of nitric oxide (NO) synthase and the subsequent increase of cellular concentration of cGMP and nitrite production. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase, and in the present study we investigated its impact on inducible NO synthesis in mesangial cells. Inhibition of GTP-cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis, with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) potently suppresses IL-1 beta-induced nitrite production and elevation of cellular cGMP levels. This inhibitory effect of DAHP is reversed by sepiapterin, which provides BH4 via the pterin salvage pathway. Most importantly, sepiapterin dose-dependently augments IL-1 beta-stimulated NO synthesis, indicating that the availability of BH4 limits the production of NO in cytokine-induced mesangial cells. N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, completely abolishes IL-1 beta-stimulated nitrite production, whereas methotrexate, which inhibits the pterin salvage pathway, displays only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize BH4 by de novo synthesis from GTP. In conclusion, these data demonstrate that BH4 synthesis is an absolute requirement for, and limits IL-1 beta induction of NO synthesis in mesangial cells. Inhibition of BH4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions involving increased NO formation. PMID- 7531791 TI - Increased stem cell factor release by hemangioma-derived endothelial cells. AB - BACKGROUND: Capillary hemangiomas, the most common tumors in young children, consist of proliferating capillary vessels and endothelial cells. These tumors also contain large numbers of mast cells, compared with the normal surrounding skin or tissue. We have recently shown that stem cell factor (SCF), the gene product of the murine steel locus, can act as a chemoattractant for mast cells. In this study, we investigated whether SCF might be involved in the recruitment and maintenance of mast cells in hemangiomas. EXPERIMENTAL DESIGN: Cultured endothelial cells derived from a murine hemangioma were compared with normal vascular endothelial cells for the ability to produce and release SCF, a mitogen for mast cells. RESULTS: Conditioned medium from hemangioma-derived endothelial cells stimulated the proliferation of cultured mast cells. This proliferative activity was potentiated by interleukin-3. The same conditioned medium was unable to stimulate proliferation of mast cells expressing a defective receptor for SCF. The medium was also unable to stimulate proliferation when it was preincubated with neutralizing antibodies specific for SCF. Immunoprecipitation and Western blot analysis of the conditioned media from hemangioma cells and normal endothelial cells demonstrated the 31,000 molecular weight SCF in hemangioma conditioned medium only. In addition, proliferative activity for mast cells could not be demonstrated in the conditioned medium of the normal endothelial cells, although Northern blot analysis indicated that both normal and hemangioma-derived endothelial cells express SCF mRNA. Reverse transcriptase-polymerase chain reaction techniques were used to amplify the DNA sequence coding for the proteolytic cleavage site used for release of SCF. Results indicated that both normal and hemangioma-derived endothelial cells express the same transcript for SCF. CONCLUSIONS: Our data suggest that increased release of SCF is a property of hemangioma-derived endothelial cells that may account for the high numbers of mast cells observed in hemangioma tissue. This increased release of SCF is not due to alternate splicing of SCF transcripts by hemangioma cells. PMID- 7531792 TI - Decreased expression of the CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients. AB - BACKGROUND: In normal adult pseudostratified human nasal surface epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) is localized to the apical domain of the ciliated cells, whereas in cystic fibrosis (CF), the mutated delta F 508 CFTR exhibits an abnormal cytoplasmic localization. Frequent airway injuries either in CF or non-CF patients may induce a remodeling of the surface epithelium characterized by a change in the morphological structure from normal columnar pseudostratified epithelium to either basal cell hyperplasia, mucous cell hyperplasia, or squamous metaplasia. EXPERIMENTAL DESIGN: The localization of CFTR parallel to markers of cell differentiation, such as cytokeratin 14 (CK14, a marker of basal cells), cytokeratin 18 (CK 18, a marker of ciliated and mucous cells), cytokeratin 13 (CK13, a marker of squamous metaplasia cells), and desmoplakins (DP) 1 and 2 (markers of desmosomes) was analyzed by indirect immunofluorescence. RESULTS: In normal pseudostratified epithelium, CFTR was detected at the apical plasma membrane of the ciliated cells, CK14 was identified in basal cells of focal areas, CK18 was localized in both ciliated and mucous cells, CK 13 was detected in all basal cells, and DP 1 and 2 were preferentially detected at the interface between columnar and basal cells. In basal cell hyperplasia, CFTR was poorly expressed in the cytoplasm of the more superficial cells, CK14 and CK13 were localized in basal cell multilayers, CK18 labeling was present in the more superficial cell layers, and DP 1 and 2 were preferentially detected at the interface between the more basal cells. In squamous metaplasia, CFTR labeling was either very low or even undetectable, CK14 was found in focal areas of the more basal cell layers, CK18 labeling was either very low or undetectable, CK13 expression was restricted to the flattened cells toward the epithelial surface, and DP 1&2 were intensively present between all the epithelial cells. CONCLUSIONS: These results suggest that the localization of CFTR in human nasal surface epithelium is related to the differentiation state of this epithelium. Abnormally low expression of the CFTR protein may not only be caused by CFTR gene mutations but can also be associated with airway surface epithelium dedifferentiation and remodeling. PMID- 7531793 TI - Expression of vascular cell adhesion molecule-1 mRNA and protein in rheumatoid synovium demonstrated by in situ hybridization and immunohistochemistry. AB - BACKGROUND: Vascular cell adhesion molecule-1 (VCAM-1) is expressed in synovial tissue of patients with rheumatoid arthritis. VCAM-1-protein has been demonstrated in nonvascular cells beside a vascular expression of this molecule. There are conflicting results about the nonvascular cell types expressing VCAM-1. EXPERIMENTAL DESIGN: For the evaluation of VCAM-1 expression in rheumatoid synovium, this molecule has been demonstrated by alkaline phosphatase anti alkaline phosphatase (APAAP) technique. Furthermore, VCAM-1 mRNA has been demonstrated by in situ hybridization to evaluate de novo synthesis of this molecule in vivo. To elucidate the nature of the cell types expressing VCAM-1 mRNA, this molecule has been shown by combined in situ hybridization for VCAM-1 and immunohistochemistry in the same tissue section. Double labeling has been performed with anti-collagen type IV monoclonal antibodies to delineate endothelial cells and pericytes and with anti-CD68 antibodies to elucidate the expression of VCAM-1 mRNA in fibroblast-like (type B) or macrophage-like (type A) synoviocytes. RESULTS: Although it has been reported that VCAM-1 occurs on endothelial cells after cytokine stimulation, we show that vascular expression of VCAM-1 mRNA and protein was minimal and restricted to small vessels beneath the lining cell layer. Further expression of VCAM-1 mRNA could be demonstrated in pericytes outside the collagen type IV containing vascular basement membrane. With respect to the expression of VCAM-1 in the synovial lining layer, we could clearly demonstrate by combined in situ hybridization and immunohistochemistry that CD68 positive cells of the monocyte/macrophage lineage in the lining layer (type A cells) do not express VCAM-1 mRNA and that the expression of VCAM-1 mRNA in the lining layer was restricted to fibroblast-like synoviocytes (type B cells). Scattered stromal cells revealing VCAM-1 mRNA were also CD68 negative. CONCLUSIONS: The strong expression of VCAM-1 in the fibroblast-like cells of RA synovium and the lack of expression in the vascular endothelium suggest that the major role of VCAM-1 appears to be associated with the proliferating synovial cells prone to attach and subsequently invade articular cartilage. PMID- 7531794 TI - Human cutaneous mast cells express basic fibroblast growth factor. AB - BACKGROUND: The normal response to tissue injury involves a complex sequence of inflammation and repair of epithelial and mesenchymal structures through a coordinated process of chemotaxis and cellular proliferation directed by specific chemical mediators. One mediator that is probably important to these processes is basic fibroblast growth factor (bFGF), a polypeptide present in diverse tissues and a potent chemotactic, mitogenic, and angiogenic factor in vitro. Although many cells have receptors for bFGF, the precise role of this factor as a paracrine mediator in vivo remains disputable because a mechanism for its secretion has not been elucidated. bFGF is known to be capable of binding to heparin, and our data demonstrate bFGF in mast cells (MCs) that also produce many other mediators (including heparin) important for wound healing and angiogenesis. EXPERIMENTAL DESIGN: We used nucleic acid in situ hybridization to detect bFGF specific mRNA transcripts within MCs in formalin-fixed, paraffin-embedded specimens of nonlesional human skin and skin containing increased numbers of MCs, including granulation tissues, benign cutaneous mastocytosis, and solitary neurofibromas. Immunohistochemistry was used to confirm bFGF protein expression in each tissue studied. RESULTS: All tissues examined contained MCs that expressed bFGF mRNA transcripts. bFGF protein was also detected in MCs, but the intensity of labeling was dependent on prior treatment of the tissues with heparinase, suggesting that epitopes recognized by the bFGF-specific antiserum were masked by endogenous heparin. CONCLUSIONS: The results further elucidate the mechanism of involvement of MCs in tissue repair and angiogenesis and suggest that bFGF produced by MCs is bound to intracellular heparin. Thus, it seems possible that release of heparin by degranulation from MCs also may result in the release of biologically active bFGF in vivo. PMID- 7531795 TI - Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells. AB - BACKGROUND: It has been hypothesized that carcinoma in situ of the testis (CIS), which is the precursor of invasive testicular germ cell tumours, may arise from fetal germ cells during fetal development rather than later in life. In order to corroborate this hypothesis, we undertook the present study. EXPERIMENTAL DESIGN: Normal human germ cells from 10 first-trimester fetuses and 76 second- and third trimester testes were investigated for the immunohistochemical expression of the markers of testicular carcinoma in situ. The panel of markers included in the study consisted of placental-like alkaline phosphatase, the protooncogene c-kit protein product, and the antigens for the monoclonal antibodies TRA-1-60 and M2A. The relative numbers of fetal germ cells that demonstrated positive reaction with the markers were calculated. RESULTS: The vast majority of the germ cells (75 100%) in the first-trimester gonads were positive for placental-like alkaline phosphatase, TRA-1-60, and M2A. The c-kit protein was detected in three out of the ten first-trimester gonads. The relative number of germ cells positive for all the markers studied declined rapidly during the first part of the second trimester, and the decrease continued with the fetal age. CONCLUSIONS: The expression of adult carcinoma in situ markers in normal fetal germ cells is consistent with the hypothesis that CIS cells may arise from fetal germ cells, although re-expression of the antigens in postnatally arising CIS cells could provide an alternative explanation. However, we speculate that a transformation of normal fetal germ cells into CIS cells may take place before the end of the 9th week of fetal development. Furthermore, the expression of c-kit in early human fetal germ cells indicates that the c-kit and its ligand play a role in the early human testicular development. PMID- 7531796 TI - Improved results of lung transplantation for patients with cystic fibrosis. AB - Patients with cystic fibrosis pose particular challenges for lung transplant surgeons. Earlier reports from North American centers suggested that patients with cystic fibrosis were at greater risk for heart-lung or isolated lung transplantation than other patients with end-stage pulmonary disease. During a 3 1/2 year period, 44 patients with end-stage lung disease resulting from cystic fibrosis underwent double lung transplantation at this institution. During the same interval, 18 patients with cystic fibrosis died while waiting for lung transplantation. The ages of the recipients ranged from 8 to 45 years, and mean forced expiratory volume in 1 second was 21% predicted. Seven patients had Pseudomonas cepacia bacteria before transplantation. Bilateral sequential implantation with omentopexy was used in all patients. There were no operative deaths, although two patients required urgent retransplantation because of graft failure. Cardiopulmonary bypass was necessary in six procedures in five patients and was associated with an increased blood transfusion requirement, longer postoperative ventilation, and longer hospital stay. Actuarial survival was 85% at 1 year and 67% at 2 years. Infection was the most common cause of death within 6 months of transplantation (Pseudomonas cepacia pneumonia was the cause of death in two patients), and bronchiolitis obliterans was the most common cause of death after 6 months. Actuarial freedom from development of clinically significant bronchiolitis obliterans was 59% at 2 years. Results of pulmonary function tests improved substantially in survivors, with forced expiratory volume in 1 second averaging 78% predicted 2 years after transplantation. Double lung transplantation can be accomplished with acceptable morbidity and mortality in patients with cystic fibrosis. PMID- 7531797 TI - Primary small-cell cancer of the esophagus. AB - Primary small-cell cancer of the esophagus is a rare tumor that disseminates early with a uniformly poor prognosis if untreated. Sixteen patients with malignant dysphagia referred to the Thoracic Surgical Unit, City Hospital, Edinburgh, within a 10-year period had a diagnosis of primary small-cell cancer of the esophagus. Seven patients underwent subtotal esophagectomy or esophagogastrectomy, either alone or with adjuvant chemotherapy or radiotherapy, with a mean survival of 20 months (standard deviation 35.4 months, range 2 weeks to 96 months). The remaining nine patients had disseminated disease when they were first seen and were treated symptomatically by intubation alone (1 patient), intubation and palliative chemotherapy or radiotherapy (3 patients), palliative chemotherapy (2 patients), palliative radiotherapy (1 patient), or no therapy (2 patients), with a mean survival of 4.8 months (standard deviation 2.6 months, range 2 to 9 months). Patients seen with this aggressive tumor should be assessed urgently for evidence of metastatic spread and then offered resection in combination with chemotherapy if they are otherwise fit for operation. This treatment regimen has given us one long-term survivor (96 months) who, we believe, is the only patient to have been cured of this condition. Patients seen with disseminated disease should have symptomatic treatment of the dysphagia combined with palliative chemotherapy. PMID- 7531798 TI - Intermediate results after complete repair of tetralogy of Fallot in neonates. AB - From July 1988 through September 1993, 30 neonates with symptomatic tetralogy of Fallot underwent complete repair. Sixteen patients had tetralogy and pulmonary stenosis, 9 had pulmonary atresia, 3 had nonconfluent pulmonary arteries, and 2 had both pulmonary atresia and nonconfluent pulmonary arteries. The median age at operation was 11 days (mean +/- standard error of the mean, 12.6 +/- 2.9 days), with a mean weight of 3.1 +/- 0.1 kg (range 1.5 to 4.4 kg). Preoperatively, 14 patients were receiving an infusion of prostaglandin, 13 were mechanically ventilated, and 6 required inotropic support. Right ventricular outflow tract obstruction was managed by a limited transannular patch in 25 patients, infundibular muscle division with limited resection in 15, and insertion of a right ventricle-pulmonary artery valved aortic homograft conduit in 5 patients. Follow-up was complete at a median interval of 24 months (range 1 to 62 months). There were no hospital deaths and two late deaths, for 1-month, 1-year, and 5 year actuarial survivals of 100%, 93%, and 93%, respectively. The hazard function for death had a rapidly declining single phase that approached zero by 6 months after the operation. Both late deaths occurred in patients with tetralogy of Fallot and pulmonary atresia who had undergone aortic homograft conduit reconstruction, so that the only independent risk factor for death was the use of a valved homograft conduit (p < or = 0.005). Eight patients required reoperation, resulting in 1-month, 1-year, and 5-year freedom from reoperation rates of 100%, 93%, and 66%, respectively. Indications for reoperation were branch left pulmonary artery stenosis in 5 patients, residual right ventricular outflow tract obstruction in 2 patients, and severe pulmonary insufficiency in 1 patient. Independent risk factors for reoperation included an intraoperative pressure ratio between the right and left ventricles of 0.75 or greater (p = 0.01), Doppler residual left pulmonary artery stenosis of 15 mm Hg or more, or Doppler right ventricular outflow tract obstruction gradient of 40 mm Hg or more at hospital discharge (p = 0.002 and 0.02, respectively). This series demonstrates the safety of early hemodynamic repair of symptomatic tetralogy of Fallot in neonates. It also emphasizes the importance of relieving all sources of right ventricular outflow tract obstruction at the initial operation, particularly that located at the site of insertion of the ductus arteriosus, which may be difficult to diagnose in the neonate before ductal closure occurs. The safety and efficacy of valved aortic homograft conduits in neonates requires further investigation. PMID- 7531800 TI - Invited letter concerning: topical aprotinin. PMID- 7531799 TI - Topical effect of aprotinin on the surgical wound in cardiac surgery. PMID- 7531801 TI - [Effects of tachykinin antagonists on pain]. PMID- 7531802 TI - Follow-up of preterm children: important to document dropouts. PMID- 7531803 TI - Rapid and stable gene expression in hippocampal slice cultures from a defective HSV-1 vector. AB - Stable transfer of genetic information into neurons is a powerful strategy to elucidate specific mechanisms of neurophysiology and to develop therapies for neurological disorders. To evaluate the optimal parameters for efficient gene delivery of defective herpes simplex virus type one (HSV-1) vectors into a specific brain region, an HSV-1 vector expressing E. coli beta-galactosidase was used to infect organotypic cultures of hippocampal slices. beta-Galactosidase was expressed as early as 2 h after infection in a dose-dependent manner as measured on immunoblots, and reached a maximum level after approximately 35 h. Expression of the RNA and the antigen was still evident after the longest time sampled (11 12 days), whereas no beta-galactosidase was ever detected in cultured slices infected with a control virus lacking the reporter gene. Hippocampal cells expressing the reporter gene outlined the contour of the neuronal cell body layers in fields CA3 and dentate gyrus; such correspondence was less evident in field CA1. Anatomical, morphological, and immunohistochemical criteria also confirmed that the majority of these infected cells were neurons. beta Galactosidase was also detected in the somata and processes of infected interneurons. Tests for synaptic pathology associated with virus infection showed no changes in pre- and postsynaptic markers. PMID- 7531804 TI - Ion permeation properties of the cloned mouse epsilon 2/zeta 1 NMDA receptor channel. AB - The heteromeric mouse epsilon 2/zeta 1 N-methyl-D-aspartate (NMDA) receptor was expressed in Xenopus oocytes, and its channel properties were studied using both the outside-out patch-clamp and two-microelectrode voltage-clamp techniques. In the cloned receptor channel, permeation properties of monovalent and divalent cations, and voltage-dependent block by Mg2+ were similar to those reported previously in the native NMDA receptor channels. The sequence of single-channel conductances for alkali metals was Rb+ > Cs+ approximately K+ > Na+ > Li+, whereas the sequence of relative permeabilities was Cs+ > Rb+ > K+ approximately Na+ > Li+. The single-channel conductances measured in isotonic Ca2+, Sr2+ and Ba2+ solutions were almost equal, and approximately one-fifth of the value in the Na+ solution, although the permeabilities for these alkaline earth cations were higher than for Na+. It is likely that Ca2+, Sr2+ and Ba2+ would enter the NMDA receptor channel more easily than Na+, but would bind to a site in the channel more tightly, the net effect being a reduced value of the current. PMID- 7531805 TI - Automated speech recognition in medical applications. PMID- 7531806 TI - Transcription of the principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase. AB - The rpoS gene encodes the second principal sigma factor of RNA polymerase in stationary-phase cells in Escherichia coli. We examined the transcription of Pseudomonas aeruginosa rpoS as to the growth of cells. The results of quantitative S1 nuclease mapping of rpoS and rpoD, encoding the principal sigma factor, indicated that the transcription of rpoS is induced in stationary-phase cells, whereas that of rpoD is induced in exponential-phase cells. By high resolution S1 nuclease mapping, the 5'- and 3'-ends of rpoS mRNA were determined. The results indicated that rpoS is transcribed as a monocistronic mRNA. The sequence preceding the 5' end of rpoS mRNA showed poor homology to the consensus sequences of the previously known promoters. P. aeruginosa rpoS was not transcribed in E. coli. By in vitro transcription assaying, P. aeruginosa rpoS was shown to be transcribed by the RNA polymerase fraction containing the principal sigma (sigma 70)-RNA polymerase of P. aeruginosa. PMID- 7531807 TI - Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack. AB - RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology. PMID- 7531808 TI - Detection of hydroquinone-induced nonrandom breakage in the centromeric heterochromatin of mouse bone marrow cells using multicolor fluorescence in situ hybridization with the mouse major and minor satellite probes. AB - Fluorescence in situ hybridization with a mouse major satellite probe and CREST staining were used to characterize the origin of micronuclei occurring in mouse bone marrow erythrocytes following administration of the benzene metabolite hydroquinone. Hydroquinone was administered to male CD-1 mice by i.p. injection on three consecutive days and the bone marrow cells were harvested 24 h later. A pronounced difference in the results was observed using the two approaches: 63% of the micronuclei induced by hydroquinone labeled with the major satellite probe whereas only 28% labeled with the CREST antibody. To determine whether the observed difference was due to a disruption of the kinetochore or a result of breakage within centromeric heterochromatin, we developed a tandem label multicolor hybridization assay, which requires the presence of both the mouse major and minor satellite probes in a micronucleus for a classification of chromosomal loss. The minor probe targets a centromeric region physically linked to the short arm of mouse chromosomes, whereas the major probe hybridizes to the centromeric heterochromatin adjacent to the long arm. Using this approach, 29% of the micronuclei induced by hydroquinone hybridized with both the major and minor satellite probes, indicating chromosome loss; an additional 37% labeled with only the major satellite probe indicating breakage within the centromeric heterochromatin. Although the region targeted by the major satellite probe comprises only 5-10% of the mouse genome, these major-probe containing micronuclei represent 53% of the micronuclei formed as the result of chromosome breakage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531809 TI - Microcirculation-derived factors in airway epithelial repair in vivo. AB - Airway epithelial repair, by cell migration over a denuded, intact basement membrane, occurs rapidly in vivo. The present study examines microcirculation derived factors in the reepithelialization process in the guinea pig. A well defined tracheal zone was gently deepithelialized; no bleeding occurred and the basement membrane was left intact. Plasma exudation was visualized by use of iv colloidal gold (diameter: 5 nm) or fluoresceinisothiocyanate-labeled dextran. Scanning and transmission electron microscopy confirmed the migration of epithelial cells and, additionally, allowed us to examine the presence of an extracellular matrix gel and leukocytes on the denuded basement membrane. Fibronectin was analyzed by immunocytochemistry. Following epithelial removal plasma promptly extravasates and produces a fibrin-fibronectin gel to cover the denuded basement membrane. Epithelial cells dedifferentiate, flatten, and migrate rapidly (several micron/min) beneath the plasma-derived gel. Within 30 min the gel contains numerous leukocytes, some of which are eosinophils. Plasma exudes into the gel until about 8 hr by which time the entire denuded zone (800 microns) is covered by squamous epithelium. The fibrin-fibronectin gel is suggested to be exclusively plasma-derived. In conclusion, reepithelialization in vivo occurs beneath a gel containing adhesive plasma proteins and leukocytes. We suggest that a plasma exudate provides immediate cover of denuded airway basement membrane and that plasma- and leukocyte-derived factors contribute essentially to reepithelialization in vivo. PMID- 7531810 TI - Elevated plasma levels of P-selectin (GMP-140/CD62P) in patients with Plasmodium falciparum malaria. AB - The plasma concentration of soluble P-selectin (GMP-140/CD62P/PADGEM), a selectin produced by activated platelets and endothelial cells, was quantitated in a group of adults and East African negro children presenting with either non-severe or severe (cerebral) malaria caused by Plasmodium falciparum. Sixty percent of adults with non-severe malaria had immunoreactive levels of P-selectin above 200 ng/ml (the maximum recorded for any normal healthy adult in the assay) and 86% of all African children with malaria had concentrations above normal irrespective of their clinical categorization, and most exceeded the maximum limits of the assay (> 640 ng/ml). There was no correlation between P-selectin levels and parasitemia. These results raise the possibility that elevated soluble P-selectin in malaria may have an important beneficial anti-inflammatory function. PMID- 7531811 TI - Pulsed field gel electrophoresis analysis of Borrelia burgdorferi sensu lato isolated in Japan and taxonomic implications with Lyme disease spirochetes. AB - Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates. PMID- 7531812 TI - Localised prostate cancer: which way forward? PMID- 7531813 TI - A case of Whipple's disease in Missouri. AB - Whipple's disease is a rare disorder which has fascinated physicians since its description in 1907. The onset is usually indolent, the symptoms are protean and multisystemic, and disease has often been present for years before a diagnosis is made. Although rare, its identification is important since therapy is simple and effective. We report a case of Whipple's disease in a lifelong resident of Missouri and review the important features of the disease. PMID- 7531814 TI - Factors regulating cryIVB expression in the cyanobacterium--Synechococcus PCC 7942. AB - The expression of the larvicidal Bacillus thuringiensis subsp. israelensis cryIVB gene in cyanobacteria has been suggested to be an effective means of controlling mosquito populations. Using a variety of cryIVB constructs, in this study we have examined the effect of Synechococcus PCC 7942 culture age on intracellular toxin levels and have attempted to determine the mechanisms by which cryIVB gene expression is regulated. The data suggest that specific degradation of the cryIVB mRNA limits toxin production; however, the addition of cyanobacterial 3' untranslated DNA sequences to the cryIVB gene did not improve mRNA stability or toxin levels. An analysis of the cryIVB sequence and comparison of codon usage patterns with highly expressed cyanobacterial genes suggest that inefficient translation and intragenic ribosomal binding sites impede protein synthesis and result in rapid turnover of the toxin mRNA. PMID- 7531815 TI - Transcription termination of the streptokinase gene of Streptococcus equisimilis H46A: bidirectionality and efficiency in homologous and heterologous hosts. AB - In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes. PMID- 7531816 TI - Allosteric effects of monoclonal antibodies on human growth hormone. AB - We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation of 125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case performed 125I MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to 125I-MAb AC8 or 125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531817 TI - Purification and characterization of a low molecular weight zinc binding protein from human placenta. AB - A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively). PMID- 7531818 TI - Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red. AB - Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347-368, which yields small amounts of material having an apparent molar extinction coefficient of approximately 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of approximately 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca2+ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca2+ release through the permeability transition pore. Crude ruthenium red is 7 10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I50 values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca2+ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium red per se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations. PMID- 7531820 TI - Activation of transcriptionally inactive human estrogen receptors by cyclic adenosine 3',5'-monophosphate and ligands including antiestrogens. AB - We show that some transcriptionally inactive human estrogen receptor (ER) mutants can be activated by 17 beta-estradiol (E2), and sometimes by antiestrogens, in the presence of elevated levels of intracellular cAMP. ER-deficient Chinese hamster ovary or 3T3 mouse fibroblast cells were transfected with mutant ERs (the point mutant L540Q, the frameshift mutant S554fs, or the carboxy-terminal truncated receptor ER1-530) and various estrogen response element-containing reporter genes. Individual treatments with E2, the antiestrogens trans hydroxytamoxifen and ICI 164,384, or with 3-isobutyl-1-methyl-xanthine plus cholera toxin (IBMX plus CT) which raise intracellular cAMP, generally do not activate the mutant receptors. However, cotreatment with IBMX/CT and one of the three ligands (E2, trans-hydroxytamoxifen, or ICI164,384) results in the unexpected recovery of strong activation of the L540Q or S554fs receptors, the magnitude of which is dependent upon promoter- and cell-contexts. Unlike L540Q and S554fs, the transcriptionally inactive ER1-530 is not activated by any combination of ligands and IBMX/CT. These data demonstrate that some ER mutants that form transcriptionally nonproductive ER-E2 complexes can be successfully activated by the combination of an agonist or antagonist ligand and an agent thought to act via phosphorylation pathways. Also highlighted is the promoter- and cell-specific nature of the transcriptional response to different ligand-ER complexes. Lastly, the enhanced transcriptional activity of wild type ER and some ER mutants in the presence of antiestrogens and elevated intracellular cAMP may provide a partial explanation of the ability of some estrogen-dependent human breast tumors to resist antiestrogen therapies currently employed. PMID- 7531819 TI - Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland. AB - Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP) mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTP gamma S in PT treated membranes was much larger than that in normal membranes. ANP dose dependently inhibited adenylate cyclase stimulated by GTP gamma S in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10(-7)M) inhibited cAMP accumulation stimulated by forskolin (10(-6)M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells amylase release stimulated by isoproterenol (10(-6)M) and forskolin (10(-6)M) were also depressed by ANP (10( 7)M) by 27 and 30% respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP ribosylated 41-KDa protein by incubation of parotid membranes with PT and [alpha 32P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. PMID- 7531822 TI - Protein modules and signalling networks. AB - Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted. PMID- 7531821 TI - Enhancer-mediated high level expression of mouse pituitary glycoprotein hormone alpha-subunit transgene in thyrotropes, gonadotropes, and developing pituitary gland. AB - The pituitary hormones LH, FSH, and TSH are heterodimers composed of a common alpha-subunit and unique beta-subunits. We demonstrate that 4.6, 2.7, 1.49 or 0.48 kilobases (kb) mouse alpha-subunit 5'-flanking sequences are sufficient for transgene expression in both gonadotropes and thyrotropes but not in inappropriate pituitary cells. In contrast, transgenes with bovine or human alpha subunit flanking sequences have been shown to confer reporter gene expression only to gonadotrope cells, suggesting that the elements regulating cell-specific expression may differ between species. Equal levels of reporter gene expression were conferred by 5.0 and 0.48 kb in transiently transfected thyrotrope tumor derived cells. In contrast, in transgenic mice, high level expression was only obtained with 4.6 kb 5'-flanking sequences, indicating the presence of an enhancer element between 4.6 and 2.7 kb. The 4.6 kb of 5'-flanking sequences are sufficient for both hormonal and developmental regulation of transgene expression. Mice rendered hypothyroid by radiothyroidectomy had significantly higher levels of transgene expression than either hyperthyroid or euthyroid animals. The temporal and spatial pattern of transgene expression in Rathke's pouch paralleled that of the endogenous gene; the onset of transgene expression occurred by embryonic day 9.5. Low level expression of both the transgene and the endogenous alpha-subunit gene were detected in some unexpected peripheral sites, such as the embryonic extraocular and olfactory regions, suggesting that alpha subunit may have a more diverse role in development than previously considered. PMID- 7531824 TI - Endoscopic ND:YAG laser treatment of rectosigmoidal cancer. AB - Over a period of 7 years 126 patients with rectosigmoid carcinoma considered unsuitable for surgery underwent endoscopic Nd:YAG laser treatment for palliation of symptoms and tumor eradication, if feasible. Altogether 72 (59%) of the lesions had distal margins less than 7 cm from the anus and 32 (23%) above the peritoneal reflection. In 14 patients with tumor less than 3 cm in diameter, symptomatic improvement was achieved in all. In the remaining 112 patients with larger tumors (81 greater than 3/4 circumferential, mean length 5.5 cm) symptomatic improvement was achieved with repeated treatments (average 3.7) in 58 (71%). All treatment failures occurred in patients with extensive tumors (17 initial, 12 late). Bowel perforation did not occur and there was no treatment related mortality. The average stay in hospital for all laser patients was 9 days (32% of patients were outpatient attendance). These results suggest that laser therapy may be the palliative treatment of choice in patients with rectosigmoid carcinoma unsuitable for surgery. PMID- 7531823 TI - The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor. AB - E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E selectin on myeloid cells, using a recombinant antibody-like form of mouse E selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin. PMID- 7531825 TI - High-grade B-cell cerebral lymphoma in a patient with anti-myelin-associated glycoprotein IgM paraproteinemic neuropathy. AB - A 74-year-old woman with a sensory neuropathy and IgM M-protein monoclonal gammopathy of undetermined significance developed a fatal B-cell cerebral lymphoma. CSF protein immunofixation revealed intrathecal secretion of a paraprotein of the same heavy- and light-chain isotypes as the serum monoclonal component (IgM-lambda). Reactivation of Epstein-Barr virus was present in the lymphoma cells. Different factors may be involved in the preferential malignant development of the monoclonal B-cell clone within the CNS. PMID- 7531826 TI - Agitated behavior relieved following treatment of cervical dystonia in dementia. PMID- 7531827 TI - A novel mutation in exon 3 of the proteolipid protein gene in Pelizaeus Merzbacher disease. PMID- 7531828 TI - Effects of organochlorine pesticides on 36Cl- flux in primary neuronal cultures. AB - Primary cultures from cortical neuronal cells were used to study the effect of organochlorine pesticides on GABA-dependent Cl- fluxes. In this system cyclodienes inhibited GABA-induced 36Cl- uptake with similar potencies to those described using brain synaptoneurosomes whereas lindane showed greater potency than that described using other in vitro systems. Basal Cl- uptake was not affected by these compounds although alpha-endosulfan exhibited a small inhibition. DDT, which mechanism of neurotoxic action has been postulated through activation of Na+ channels, did not inhibit GABA-induced 36Cl- uptake. These results clearly demonstrate that the stimulant neurotoxic effects of cyclodienes and lindane are produced by their interaction with the Cl- channel coupled to the GABAA receptor. Furthermore, these results show the usefulness of cortical neuronal cultures to study the effects of toxic agents on GABAA receptor. PMID- 7531829 TI - Anticonvulsant activity of calmodulin antagonist W-7 in convulsions induced by lindane and BayK-8644: effects in c-fos expression. AB - The anticonvulsant activity of calmodulin antagonist W-7, was investigated on convulsions induced in mice by the insecticide lindane and by the calcium channel agonist BayK-8644. We also studied the inhibitory effect of W-7 on on c-fos mRNA expression induced by both convulsants. We observed a good correlation between doses and the acute convulsive effects of lindane and BayK-8644. The incidence rate and time to onset were clearly dose-dependent. W-7 antagonized the convulsive effects of lindane and BayK-8644 in all the parameters studied. A significant decrease in the incidence rate and time to onset were observed when they are compared with the values obtained with the ED100 of lindane- and BayK 8644 induced seizures. Both were able to activate the mRNA expression of the proto-oncogene. The pattern of this expression displayed by in situ hybridization was very similar. A dramatic increase was found in dentate gyrus and high levels of mRNA expression also occurring in hippocampal fields and cortical regions. In accordance with the behavioural results, W-7 antagonized also the c-fos expression induced by lindane and BayK-8644. Our results suggest that lindane as BayK-8644 may activate voltage-dependent calcium channels leading to calmodulin activation. PMID- 7531830 TI - Poster presentations: a tool for evaluating nursing students. AB - Developing effective means to evaluate students taking undergraduate nursing research and stimulating interest in a course that is typically viewed by nursing majors as "irrelevant" is challenging and problematic. Traditional examinations and proposal assignments do not inspire undergraduate nursing students to appreciate research. The author outlines the steps taken to design and use poster presentations to evaluate students' progress and offers recommendations for adapting this innovative evaluation tool to clinical content. PMID- 7531831 TI - Fishbone strategy: a TQM strategy applied to nursing education. PMID- 7531832 TI - Evaluating P.S.A. levels. PMID- 7531833 TI - Understanding urologic stents. PMID- 7531834 TI - New drugs. PMID- 7531837 TI - Pain characteristics of advanced lung cancer patients referred to a palliative care service. AB - Sixty consecutive lung cancer patients referred to a palliative care service were followed until death to obtain specific information about the prevalence, characteristics and localization of pain. To determine the course of treatment, an Opioid Escalation Index and Effective Analgesic Score were calculated. The prevalence of pain was almost 90%. Chest and lumbar pain were the most common sites with a clear correlation between site and metastases for the chest. Somatic incident pain did not achieve good pain relief while patients with neuropathic pain did not show any particular disadvantage compared to those exhibiting somatic or visceral pain. Mean Opioid Escalation Index and percentage of pain control observed in lung cancer patients were similar to those recorded in the general cancer population. PMID- 7531835 TI - Usefulness of granulocyte colony-stimulating factor on neutropenia in patients of invasive mole and choriocarcinoma. AB - The effect of human granulocyte colony stimulating factor (hG-CSF) was investigated on the proliferation of choriocarcinoma cells in vitro. No growth stimulating effect was observed. Then, 22 patients with invasive mole and 9 patients with choriocarcinoma who received combination chemotherapy were treated with hG-CSF when white blood cell counts decreased below 2,000/mm3. The duration of neutropenia was reduced significantly by 3-4 days with the use of hG-CSF. No side effects were observed. These results indicate safety and usefulness of hG CSF as an adjunct to chemotherapy in the treatment of gestational trophoblastic disease. PMID- 7531836 TI - Antigen immunohistochemistry of renal cell adenomas in autopsy cases: relevance to histogenesis. AB - Eighty-three kidneys from autopsy cases, all more than 60 years of age, were used in the present studies. Three millimeter-thick step slices from all kidneys were embedded in paraffin, and serial sections from all blocks used for the immunohistochemical demonstration of Leu M1 (leukocyte membrane antigen) and LTA (Lotus tetragonolobus agglutinin) in cells of proximal convoluted tubular origin, and PNA (peanut agglutinin) and EMA (epithelial membrane antigen) in cells of distal convoluted tubular origin. The ABC staining method was used in all cases. A total of 65 renal cell adenomas found in 31 of the 83 kidneys consisted of 40 papillary, 20 tubular and 5 solid type lesions. The sizes of these renal cell adenomas were from 0.6 to 5 mm in diameter and compression of neighboring tissues was characteristic. Papillary renal cell adenomas were positive in their cytoplasms for Leu M1 and LTA in 7 cases and at their cell membranes for PNA and EMA in 33 cases. The respective figures for tubular renal cell adenomas were 6 cases for Leu M1 and LTA and 14 cases for PNA and EMA. All solid renal cell adenomas were positive in their cytoplasms for PNA and EMA. The immunohistochemical results thus indicated 13 of 65 lesions to have a proximal convoluted tubular cell origin and 52 to be possibly derived from distal convoluted tubules or collecting ducts. A role for metaplasia, however, could not be ruled out. PMID- 7531838 TI - [Antitumoral electrochemotherapy]. PMID- 7531839 TI - [A family of receptors with tyrosine kinase activity for growth factors of fibroblasts]. PMID- 7531840 TI - Understanding and helping families with neurodevelopmental and neuropsychiatric special needs. AB - Children with persistent neurodevelopmental and neuropsychiatric problems have extensive treatment, care, and community service needs. The child's disabilities can create new responsibilities, emotions, roles, and goals for the family. This article provides a framework for identifying and categorizing children with special needs. In addition, methods for assessing the family's psychoeducational, social, and emotional concerns are presented. PMID- 7531841 TI - Effects of tachykinins on the splanchnic circulation: a comparison of the dog and rat. AB - Responses to the administration of graduated doses of tachykinins were examined in various vascular beds of the dog. Among the tachykinins, all vascular beds studied demonstrated that the greatest vasodilation responses was produced by substance P. However, each tachykinin demonstrated a specific pattern of response. With substance P the greatest sensitivity was seen in the superior mesenteric artery (SMA), with neurokinin B in the celiac axis, and with neurokinin B a prolonged vasodilation response of the SMA compared to the other two vessels was seen. The response to substance P in the SMA of the rat was considerably less than that of the dog. PMID- 7531842 TI - Chronic cerebroventricular galanin does not induce sustained hyperphagia or obesity. AB - Acute central administration of galanin has been reported to increase fat consumption. These experiments were designed to test the hypothesis that repeated injections of galanin would elicit hyperphagia and weight gain and that this response would depend on the available diet. Male Sprague-Dawley rats were fed high (56% energy) or low (10% energy) fat diets. Galanin (300 pmol) or saline vehicle was injected into the third ventricle twice daily for 7 days and three times daily for another 6 days. On both the high-carbohydrate and high-fat diets, twice daily galanin increased daytime food intake, but there was a compensatory decrease in nighttime intake. The addition of a third, nighttime injection of galanin was ineffective in producing an increase in total 24-h intake. There was no significant increase in body weight during galanin treatment in rats eating either diet although animals eating the high-fat diet gained more weight as reflected by a significant increase in epididymal fat pad weight. Galanin treatment had no effects on serum insulin, glucose or corticosterone concentrations, measured at the end of the experiment. However, animals fed the high-fat diet had significantly higher insulin concentrations at the time of sacrifice. Although repeated central infusions of galanin reliably stimulated daytime intake of both diets, they failed to increase total daily energy intake or body weight. PMID- 7531843 TI - Intraventricular administration of galanin does not affect behaviors associated with locus coeruleus activation in rats. AB - The 29 amino acid peptide galanin (GAL) coexists with norepinephrine in rat locus coeruleus (LC) neurons to a remarkably high degree. The effects of central administration of GAL were examined in three behavioral paradigms that putatively involve increases in the activity of LC neurons. GAL did not affect behavioral signs associated with naloxone-precipitated withdrawal in rats treated chronically with morphine, a condition in which the firing rate of LC neurons is dramatically increased, although the behavioral signs of withdrawal were abolished by clonidine. Foot shock induced freezing behavior was similarly unaffected by either dose of GAL but was significantly diminished by clonidine and the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF. GAL did not influence the decrease in exploratory activity in a novel open field induced by idazoxan. The behavioral activity of the peptide and route of administration were confirmed in a feeding paradigm. Doses of GAL that were inactive in the three paradigms were active in stimulating intake of a palatable food to a similar degree as clonidine-stimulated intake. These results suggest that intraventricularly administered GAL may not influence behaviors thought to be mediated by activation of neurons in the LC. PMID- 7531845 TI - Evaluation of three different AgNOR counting methods in advanced carcinoma of the prostate. AB - We applied the silver staining of nucleolar organizer regions (AgNOR) technique to the pretreatment biopsies of 50 cases of advanced prostate cancer. Three different counting methods were utilized in the enumeration of AgNORs. All methods yielded statistically significant differences of mean AgNOR counts of groups defined by high and low WHO, and by Gleason grades. However, there was overlap among groups, and further analysis of counts by grouping of patients according to their stage, response to treatment, and prognosis was not conclusive. Replicate counts were performed in 10 cases. While intraobserver reproducibility was high by all methods, only the second counting method yielded nonsignificant interobserver variability. There was a significant intratumoral heterogeneity of AgNOR scores. Lack of technical standardization, low reproducibility, and lack of correlation with prognosis limit the use of AgNOR counts in advanced carcinoma of the prostate. PMID- 7531844 TI - [Selected clinical and laboratory tests during specific immunotherapy of patients hypersensitive to insect bites]. AB - The study aimed at determining selected immunologic parameters of the cellular histamine secretion and skin reactivity in the group of 9 patients hypersensitive to insect poison with effective immunotherapy following 2 years of maintenance treatment. It has been shown that the continuation of the specific immunotherapy for 2 years maintains and even increases favourable changes in humoral and cellular immunity induced already in the early phase of desensitization. An assay of the specific IgE and IgG4, cellular histamine secretion, and skin reactivity is of value in monitoring specific allergy to insect poison immunotherapy. PMID- 7531846 TI - Comparison of the effects of new specific azasteroid inhibitors of steroid 5 alpha-reductase on canine hyperplastic prostate: suppression of prostatic DHT correlated with prostate regression. AB - Four new azasteroid inhibitors of steroid 5 alpha-reductase were compared to the benchmark compound finasteride, each at a dose level of 1 mg/kg/day, as well to placebo and to castration, in seven groups of mature male beagle dogs with enlarged prostates. Prostate volumes were measured repetitively by a volume MRI method over 15 weeks of treatment. The study probed the obverse of the familiar relation between DHT and prostate growth, and provides the first documentation of a tight negative correlation between prostate regression and the prostatic concentration of DHT across a range of treatment regimens (r = -0.982). In this first direct comparison study of structure vs. in vivo activity for several azasteroids in the dog model of BPH, relative efficacy for induction of shrinkage of the dog prostate did not correlate at all with the inhibitor's relative activity against the dog 5 alpha-reductase in vitro. On the basis of the relative IC50 values it would not have been predicted that, at the dose tested, the analogue MK-434 (17 beta-benzoyl-4-aza-5 alpha-androst-1-en-3-one) was distinguished from the other inhibitors with respect to the induction of faster and more complete regression (69%) as well as greater reduction in prostatic DHT (95%), both of which approached the castrated dog levels of 75% prostatic shrinkage and > 98% reduction in DHT. Treatment with any one of the five azasteroids induced two- to five-fold increases in prostatic testosterone. However, total androgen was conserved at the placebo control level. Despite the differences noted, each azasteroid tested induced a highly significant decrease in prostatic volume that correlated tightly with a decreased prostatic DHT level in canine spontaneous BPH. PMID- 7531847 TI - IGF-binding proteins in human prostate tumor cells: expression and regulation by 1,25-dihydroxyvitamin D3. AB - The expression of the six known insulin-like growth factor binding proteins (IGFBPs) and their corresponding messenger RNAs has been examined in three cell lines established from surgical and biopsy specimens of human prostate carcinoma. All three cell lines produced both IGFBP-4 and IGFBP-6 and the respective mRNAs; expression of IGFBP-6 has not been previously demonstrated in human prostate tumor cells. No other binding proteins were detected. The levels of IGFBP mRNAs were not regulated by androgens or IGF-1, but the level of IGFBP-6 mRNA was sharply increased by 1,25-dihydroxyvitamin D3 (1,25(OH)D3). The stimulation was dose-dependent with a maximum effect at 10 nM 1,25(OH)D3 and a clearly discernible effect at 0.1 nM. The results support a role for vitamin D in the control of prostate tumor growth, mediated at least in part by interaction with IGFs and specific IGFBPs. PMID- 7531848 TI - Differential expression of uPA in an aggressive (DU 145) and a nonaggressive (1013L) human prostate cancer xenograft. AB - Prostate cancer has a slow growing noninvasive phase, but, in general, is invasive on diagnosis. An initial step in the invasion of surrounding normal tissue is the activity of proteolytic enzymes such as components of the plasminogen activator system (PA). In cell culture, the primary human prostate cancer cell line 1013L expressed no urokinase type-PA (uPA), while DU 145, a cell line derived from a metastatic lesion, expressed high levels of uPA. The DU 145 cells grew easily as xenografts but the establishment of 1013L in the SCID mice was possible only with the aid of a gelatin sponge (Spongostan). The latency period was 42-64 days, followed by a slow growth phase before a fast growth phase occurred. This fast growth phase was characterized by rapid degeneration of tumor tissue, while high proliferation occurred around the blood vessels. On serial transplantation of tumor material, the growth pattern was similar. Furthermore, the 1013L tumor was encapsulated by connective tissue and no invasiveness could be detected. We found that 1013L tumor homogenates had hardly detectable levels of uPA, i.e., 300-fold lower than we found in the invasive prostate xenograft DU 145. In addition, no expression of uPA was found in the plasma of 1013L tumor bearing mice whilst uPA antigen was detected in the plasma of DU 145 tumor bearing mice. In conclusion, the 1013L cell line, which exhibits a nonaggressive pattern, could be a good model for studying progression of prostate cancer to a more aggressive phenotype in vivo and in vitro. PMID- 7531850 TI - Assessing child development. AB - The family practitioner has an important role in the detection of infants and young children with developmental delays. The physician may be the first or only professional in regular contact with infants and young children. Interpretation of a child's developmental status is best done with a clear understanding of the child's family and social environment, and the family practitioner is in a unique position to possess this knowledge by virtue of his or her established relationship with the family. PMID- 7531849 TI - Interferon-gamma-activated immature macrophages exhibit a high Trypanosoma cruzi infection rate associated with a low production of both nitric oxide and tumor necrosis factor-alpha. AB - Murine peritoneal macrophages (MPM) can be subdivided into two subpopulations of mature and immature macrophages. In contrast to mature macrophages, immature ones were highly susceptible to Trypanosoma cruzi infection. This highly susceptibility was associated with a low production of alpha 2-macroglobulin. Interferon-gamma (IFN-gamma)-activated immature macrophages also exhibited a higher infection rate than did IFN-gamma-activated mature ones. This higher rate of infection was associated with a low production of both nitric oxide (N = O) and tumor necrosis factor-alpha (TNF-alpha). In contrast, mature MPM showed a lower rate of infection and produced higher levels of N = O and TFN-alpha. Taken together, these results show a clear-cut difference in the course of T. cruzi infection in relation to the macrophage maturation state. PMID- 7531851 TI - Age-related behavioural, neurochemical and radioligand binding changes in the central 5-HT system of Sprague-Dawley rats. AB - Mature (3-4 months) and aged (18-19 months) Sprague-Dawley (SD) rats were treated with 5-HT receptor agonists and drug-induced behaviours monitored. The 5-HT2/1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), induced wet dog shakes and back muscle contractions which were significantly increased in aged, compared to mature, rats, suggesting an age-related enhancement of 5-HT2 receptor function. In contrast, the selective 5-HT1A agonist 8-hydroxy-2-(di-n propylamino) tetralin (8-OH-DPAT) induced forepaw treading, flat body posture, hypothermia and hyperactivity which were not significantly different in aged compared to mature rats. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus and frontal cortex were measured using high performance liquid chromatography with electrochemical detection. There were no age-related changes in hippocampal 5-HT or 5-HIAA. However both 5-HT and 5-HIAA were increased in the frontal cortex of aged SD rats. 8-OH-DPAT reduced 5-HIAA in both regions examined in mature rats, an effect which was attenuated in the aged rats, suggesting an age-related reduction in presynaptic 5-HT1A receptor function. DOI did not induce any changes in 5-HT or 5-HIAA in either of the regions examined. Radioligand binding studies with [3H] ketanserin showed there to be no significant age related changes in cortical 5-HT2 receptor density or affinity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531853 TI - Effect of lesions of the ascending 5-hydroxytryptaminergic pathways on timing behaviour investigated with the fixed-interval peak procedure. AB - Twelve rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei; 12 rats received sham lesions. The rats were then trained for 60 sessions under a discrete-trials fixed-interval schedule (peak procedure). In half the trials, a reinforcer became available 40 s after trial onset, and the trial was terminated upon reinforcer delivery; the remaining trials were 120 s in duration, and reinforcement did not occur in these trials. Performance during the 120-s trials was characterized by increasing response rate during the first 40 s of the trial, declining response rate between 40 s and 80 s, and a secondary increase in response rate during the final 40 s of the trial. The lesioned group showed a broader "spread" of the response rate function than the control group (time between attainment of 70% of the peak response rate and subsequent decline of response rate below this level); however, the peak response rate and the time from trial onset until attainment of the peak response rate did not differ significantly between the groups; the spread/peak-time ratio was significantly greater in the lesioned group than in the control group. The levels of 5 hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, amygdala, nucleus accumbens and hypothalamus were markedly reduced in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected by the lesion. The results confirm the involvement of 5HTergic function in timing behaviour. PMID- 7531852 TI - Effects of diazepam and hydromorphone in triazolam-trained humans under a novel response drug discrimination procedure. AB - Seven healthy normal male and female volunteers (21-31 years) were trained to discriminate between the benzodiazepine triazolam (0.32 mg/70 kg, PO; e.g., drug A) and placebo (e.g., drug B) under a three-choice, instructed novel response drug discrimination procedure. Once the criterion for discrimination was met (i.e., > 85% correct responding on four consecutive sessions), dose-effect curves were determined for triazolam (0.1-0.56 mg/70 kg), the benzodiazepine diazepam (10-32 mg/70 kg) and the opioid agonist hydromorphone (1-6 mg/70 kg). Subjects met the criterion for discrimination within four to six sessions. Triazolam and diazepam produced dose-related increases in triazolam-appropriate responding and no novel-appropriate responding at any dose tested. In contrast, hydromorphone generally increased novel-appropriate responding in a dose-related manner with placebo-appropriate responding and some triazolam-appropriate responding at intermediate doses occurring also. Triazolam and diazepam produced qualitatively similar increases on several measures of sedative drug effects; hydromorphone increased ratings of "like novel" and sedative-like effects in subjects who discriminated hydromorphone as novel relative to those who did not. These results indicate that the novel response drug discrimination procedure enhances the specificity of the triazolam-placebo discrimination. PMID- 7531854 TI - Plasma protein binding of risperidone and its distribution in blood. AB - The plasma protein binding of the new antipsychotic risperidone and of its active metabolite 9-hydroxy-risperidone was studied in vitro by equilibrium dialysis. Risperidone was 90.0% bound in human plasma, 88.2% in rat plasma and 91.7% in dog plasma. The protein binding of 9-hydroxy-risperidone was lower and averaged 77.4% in human plasma, 74.7% in rat plasma and 79.7% in dog plasma. In human plasma, the protein binding of risperidone was independent of the drug concentration up to 200 ng/ml. The binding of risperidone increased at higher pH values. Risperidone was bound to both albumin and alpha 1-acid glycoprotein. The plasma protein binding of risperidone and 9-hydroxy-risperidone in the elderly was not significantly different from that in young subjects. Plasma protein binding differences between patients with hepatic or renal impairment and healthy subjects were either not significant or rather small. The blood to plasma concentration ratio of risperidone averaged 0.67 in man, 0.51 in dogs and 0.78 in rats. Displacement interactions of risperidone and 9-hydroxy-risperidone with other drugs were minimal. PMID- 7531855 TI - The putative anti-addictive drug ibogaine is a competitive inhibitor of [3H]MK 801 binding to the NMDA receptor complex. AB - Ibogaine is a putative anti-addictive drug with potential efficacy for the treatment of opiate, stimulant, and alcohol abuse. We now report ibogaine is a competitive inhibitor (Ki, 1.01 +/- 0.1 microM) of [3H]MK-801 binding to N-methyl D-aspartate (NMDA) receptor coupled cation channels. Since MK-801 can attenuate the development of tolerance to morphine and alcohol as well as sensitization to stimulants in preclinical studies, the reported ability of ibogaine to modify drug-seeking behavior in man may be attributable to a blockade of NMDA receptor coupled cation channels. PMID- 7531856 TI - Production and action of interferons: new insights into molecular mechanisms of gene regulation and expression. PMID- 7531857 TI - [Persistence of RNA]. PMID- 7531859 TI - [Evolution of proteins viewed on their three-dimensional structures]. PMID- 7531858 TI - [Origin and evolution of genetic code: an idea on the mechanism of emergence of unusual genetic codes]. PMID- 7531860 TI - [Origins of life and RNA world]. PMID- 7531861 TI - [Retroposon as temporal landmarks of evolution]. PMID- 7531862 TI - [Peopling of the Americas, founded by four major lineages of mitochondrial DNA]. PMID- 7531863 TI - [Evolutionary molecular engineering]. PMID- 7531864 TI - Inter-species extrapolation of pharmacokinetic data of three prostacyclin mimetics. AB - Cica-, eptalo- and iloprost are chemically and metabolically stabilized derivatives of prostacyclin which maintain the pharmacodynamic profile of the endogenous precursor. While iloprost is still subject to beta-oxidative degradation of the upper side chain, cicaprost is highly metabolically stable. Eptaloprost was synthesized to realize the pro-drug concept in PGI2-mimetics and was designed to be activated to cicaprost by single beta-oxidation. All three prostacyclin-mimetics were studied in various animal species (mouse, rat, rabbit, monkey, dog and pig) and in man to determine their pharmacokinetic profiles. Based upon this data, it was of interest whether an inter-species extrapolation of pharmacokinetic parameters can be performed to show the predictive value of animal experimentation. Allometric inter-species extrapolation is performed by modelling pharmacokinetic data (Y) as exponential functions (x) of species characteristics (e.g. body weight, W) as: Y = .aWx. For total clearance and volumes of distribution at steady state, a clear-cut correlation with x-values of 0.6-0.8 and 1.0-1.1 could be shown for all three compounds. For cicaprost, which was excreted unchanged in several species, renal and non-renal clearance was also mathematically scalable. Due to the use of different compartment models to describe plasma disposition, different sets of half-life data were obtained and could not be extrapolated reasonably. However, mean residence time showed a dependency on body weight with 0.25 as power function. In case of cicaprost, only the dog, which extensively metabolizes the compound, could not be enrolled in inter-species extrapolation. Excretion half-lives or residence times did not show a significant correlation to body weight or maximum life time potential. The present inter-species extrapolation showed a dependency from species body weight for model-independent pharmacokinetic data, e.g. clearance, volume of distribution at steady state and correspondingly mean residence time. The disposition profile of these compounds can therefore be predicted. Preliminary information on bio-degradation is an additional prerequisite for extrapolation. These data demonstrate that basic physiologically determined processes, which show some evolutionary allometric dependency, also influence the disposition of prostacyclin-mimetics. An extrapolation of data from animal to man could easily be realized giving additional justification for animal studies in pharmacology, toxicology and pharmacokinetics. PMID- 7531865 TI - Role of L-arginine-derived NO in ischemic acute renal failure in the rat. AB - Nitric oxide (NO) is involved in the regulation of renal perfusion and glomerular hemodynamics under basal conditions. We examined the hypothesis that L-arginine derived NO modifies ischemic acute renal failure (ARF) in the rat. After a basal period ischemia was induced by clamping of both renal arteries (40 min). Thereafter, in the reperfusion period, we intravenously infused L-arginine (Arg, 300 mg/kg/60 min), or L-monomethylarginine (MeArg, 30 mg/kg/60 min), or Arg + MeArg (300 mg/kg/60 min, 30 mg/kg/60 min, resp.). Besides monitoring of urinary flow rate and arterial blood pressure, and determination of sodium excretion, glomerular filtration rate (GFR, mL/min/100 g) was estimated at the end of the infusion period and again after another 30 and 120 min by inulin clearance (fluorescence-marked inulin). In the basal period GFR showed no differences between the groups (Arg: 0.86 +/- 0.07, MeArg: 0.92 +/- 0.06, Arg + MeArg: 0.89 +/- 0.08, control: 0.84 +/- 0.07). At 180 min after the beginning of the reperfusion period, GFR was 0.13-0.02 in the control group. After administration of Arg, a remarkable and persistent increase in GFR was observed (0.28 +/- 0.03), whereas infusion of MeArg showed no significant effects (0.13 +/- 0.04). Combined administration of Arg + MeArg revealed a moderate increase of GFR (0.19 +/- 0.05), ranging between the Arg and the control group. Also, 60 and 90 min after the beginning of the reperfusion period, the highest values for GFR were obtained in the Arg group. We conclude that in this model of ischemic ARF in the rat, L arginine-derived NO is capable of improving renal function. These data underline the regulatory role of the L-Arg-NO pathway for renal function, not only under normal conditions, but also in ARF. PMID- 7531866 TI - [Elimination program and information systems]. PMID- 7531867 TI - [Soluble antigens released by Trypanosoma cruzi trypomastigotes used in ELISA to detect cure in chagasic patients following specific treatment]. AB - Two soluble antigens isolated from T. cruzi were evaluated by ELISA for the diagnosis of chronic infection and assessment of cure after specific chemotherapy, namely, supernatants from parasite cell-cultures (SpAg) and trypomastigote excretory/secretory antigens (ESAg). Among the treated patients, a group defined as "dissociated" had been monitored for 3 to 10 years and displayed negative hemocultures and tests of trypomastigote complement-mediated lysis persistently negative although positive by conventional serology (indirect fluorescence with epimastigote, mainly). A negative lysis test indicates parasite elimination by the patient. Our ELISA results showed that among the non-treated chagasic controls the SpAg e ESAg detected 93% and 100%, of the cases, respectively. However, only 28% of sera from the dissociated group, considered as cured, were positive by ELISA using SpAg whereas all of such sera were negative using ESAg. Therefore ELISA with ESAg seems to be ideal to replace the complement mediated lysis test with the aim to define cure after treatment of the chronic infection by T. cruzi. PMID- 7531868 TI - A comparison of the effectiveness of Therevac SB and bisacodyl suppositories in SCI patients' bowel programs. AB - The evaluation described in this article compared the Therevac SB "mini-enema" with bisacodyl suppositories in the bowel management programs of patients with spinal cord injury (SCI). Of particular interest were (a) determining whether the additional costs of Therevac SB could be justified and (b) identifying the degree of bowel program improvement possible. Fourteen SCI patients were selected from the SCI inpatient unit and the clinic of a Department of Veterans Affairs facility. All patients experienced a delay of at least 45 minutes between insertion of a suppository and the beginning of stool evacuation. Each patient used bisacodyl suppositories for five bowel programs, then Therevac SB for five programs, finally repeating the bisacodyl suppositories for five more programs. Each patient maintained a bowel program log. Ten patients completed the evaluation. Using a MANOVA, the authors found a significant difference between bisacodyl and Therevac SB mean evacuation times for this group. An analysis of direct and indirect costs related to bowel care with the two regimens is also presented. PMID- 7531869 TI - Pathogenesis and medical management of benign prostatic hyperplasia. AB - This is simultaneously an exciting and confusing time for those caring for patients with BPH. For years we thought we had the answer, TURP, but costs and complications have opened this area for further study in alternative methods for the management of BPH. The pathogenesis and the maintenance of BPH is poorly understood and the natural history of BPH has not been well characterized. The bothersome level of symptoms to individual patients varies widely as well as the expectations of patients to the outcome of treatment. Satisfaction and cure are not necessarily identical, especially in patients with BPH, in which there seems to be great variability in the clinical course. Perhaps, in the future, this will be able to be related to the equally variable histological picture of BPH. There are problems in designing appropriate clinical trials for the evaluation of new treatments for BPH to take into account these variabilities in patient cases and histology (Table 9). The proper evaluation of new approaches to the management of patients with BPH takes years to complete, and physicians must resist the temptation to rush into any new treatment without reasonable caution for proven safety and efficacy when compared with existing modalities of treatment. PMID- 7531870 TI - [Local intratumor chemotherapy. Its indications, performance and risks]. PMID- 7531871 TI - A 22-year-old man with a liver mass and markedly elevated serum alpha fetoprotein. AB - Our patient presented with a large liver mass, an extremely elevated AFP level, and an almost certain diagnosis of HCC. However, extensive evaluation and biopsies failed to demonstrate malignancy, and the available evidence strongly suggests that the patient has an adult polycystic disease without renal involvement, and that the mass was the result of hemorrhage and degenerative changes in one of his cysts. Polycystic diseases can involve only one lobe, as it appears in this case. Only about 10-15% of patients with polycystic disease have symptoms due to the liver disease, while 30-50% have associated renal disease. Thus, our patient is unusual in several respects. However, his liver mass has decreased in size, he feels well, and his biochemical abnormalities have returned to normal. Despite a classic presentation for HCC, this case underscores the necessity for a thorough evaluation, especially for patients without major risk factors for hepatocellular carcinoma. PMID- 7531873 TI - Alterations in mRNA levels of D2 receptors and neuropeptides in striatonigral and striatopallidal neurons of rats with neuroleptic-induced dyskinesias. AB - Chronic neuroleptic treatment in rat produces vacuous chewing movements (VCMs), analogous to TD in humans. We hypothesized that these hyperkinetic movements were due to alterations in striatonigral and striatopallidal GABAergic spiny II neurons. Rats were treated for 36 weeks with haloperidol decanoate and withdrawn for 28 weeks. Striatonigral and striatopallidal neurons were assessed using in situ hybridization histochemistry for mRNA levels of D1 and D2 dopamine receptors, preproenkephalin (ENK), prodynorphin (DYN), protachykinin (substance P), and glutamate decarboxylase (GAD67) in the dorsolateral and ventromedial striatum as well as the nucleus accumbens. Rats that did not develop VCMs (-VCM) had increased D2 receptor and DYN mRNA, and reduced substance P mRNA in the dorsolateral striatum. Rats with persistent VCMs (+VCM) had increased D2 receptor, ENK, and DYN mRNA in both striatal regions, and increased ENK and DYN mRNA in the nucleus accumbens, compared with controls. Relative to -VCM rats, however, +VCM rats only had increased ENK mRNA in the nucleus accumbens. Considering the overall pattern of mRNA changes, the data suggest that alterations in both the D1-mediated striatonigral and the D2-mediated striatopallidal pathways play a role in the expression of the VCM syndrome. To the extent that gene expression parallels changes in neuronal activity, this implies that the VCM syndrome is associated with increased activity in both pathways. PMID- 7531872 TI - Depressed specific and nonspecific immune responses in secondary Brugia pahangi infection in jirds. AB - The immune responsiveness to specific antigens or mitogens was examined in jirds after primary and secondary infections with Brugia pahangi. When spleen cells were obtained from secondarily infected jirds, their proliferative responses to mitogens such as Con A or LPS, or to specific antigens prepared from infective larvae or adult worms were significantly lower than those of spleen cells obtained from primarily infected jirds. The proliferative responses of the peripheral blood mononuclear cells obtained from animals undergoing primary and secondary infections also showed a similar tendency. The depressed proliferative responses of the secondary infected spleen cells to Con A or LPS was partially restored by removing adherent/phagocytic cells from the original cell populations. After deletion of the adherent cells, however, antigen-specific proliferative responses were not altered and remained at low level. These results suggest that at least two different mechanisms of depression, namely adherent cell-mediated antigen-nonspecific suppression and unresponsiveness of lymphocytes to filarial antigens, are induced in jirds in the secondary infection. PMID- 7531875 TI - Isolation and characterization of single-chain protein S. AB - Protein S is a vitamin K-dependent cofactor of activated protein C in the proteolytic cleavage and concomitant inactivation of the coagulation factors Va and VIIIa. Protein S is sensitive to proteolysis by thrombin which reduces its functional activity. Uncontrolled proteolytic breakdown, leading to the generation of a two-chain molecule, is commonly encountered during the purification of both human and bovine protein S. In this study we demonstrate that human, single-chain, intact protein S can be isolated from plasma in a single step by affinity chromatography using a monoclonal antibody, CLB PS 52, directed to an epitope located within the thrombin-sensitive region of protein S. The product of purification was readily cleaved by thrombin after Arg49, resulting in a two-chain molecule which demonstrated a lower reactivity towards CLB-PS 52 than the parent single-chain protein. This study for the first time shows that intact protein S can be isolated directly from plasma using a monoclonal antibody selected for its ability to recognize uncleaved protein S. PMID- 7531874 TI - Haemostatic factors and inhibitors and coronary artery bypass grafting: preoperative alterations and relations to graft occlusion. AB - Graft closure remains a major problem after coronary artery bypass surgery. While a number of graft characteristics influencing the risk of occlusion have been defined, the role of haemostatic factors and inhibitors has not been studied in detail. The present study examined the time course of changes in blood coagulation and fibrinolytic function after coronary artery bypass grafting in 20 consecutive patients. Pre- and postoperative determinations of haemostatic factors and inhibitors were also related to the presence of graft occlusion assessed by angiography at three months after surgery. A broad panel of haemostatic tests was used preoperatively, on the first, third and eight postoperative days, and at three months after surgery. A particular emphasis was placed on fibrinogen, factor VII activity, von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) activity, anticoagulant proteins C and S, thrombin-antithrombin complex and D-dimer. A marked activation of the coagulation cascade was noted postoperatively along with enhanced degradation of cross-linked fibrin. The degree of activation of blood coagulation and fibrinolysis differed widely between individuals and appeared to relate only partly to the acute phase reaction produced by the surgical trauma. Preoperative values of haemostatic factors and inhibitors showed fairly weak associations with the levels of postoperative determinations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531876 TI - A pilot study of pro-urokinase in the treatment of deep vein thrombosis. AB - Safety and efficacy of the thrombolytic agent pro-urokinase (pro-UK) in the treatment of deep vein thrombosis of the lower limbs (DVT) have been investigated in an open, uncontrolled, pilot study. Fifteen patients were infused with 800.000 IU (5 mg)/h of pro-UK over 24 h (120 mg), together with unfractionated heparin adjusted to maintain the activated partial thromboplastin time between 1.5 and 2.5 times the basal value. Efficacy was assessed comparing venographic changes in the 11 evaluable limbs before and after pro-UK infusion. The Marder score decreased from a median pre-thrombolysis value of 28 (range 4-40) to 16 (3-38) (p < 0.05). One major hemorrhagic event (retroperitoneal bleeding 4 days after the end of the pro-UK infusion) occurred. Fibrinogen, alpha 2-antiplasmin and plasminogen significantly decreased from baseline values after 12 and 24 h, fibrin(ogen) degradation products significantly increased. Changes in hemostasis parameters were unrelated to thrombolytic efficacy. The results of this pilot study indicate that pro-UK is thrombolytic in DVT and that it can be administered simultaneously with conventional heparin treatment. PMID- 7531877 TI - Postoperative hemostasis and fibrinolysis in patients undergoing cardiopulmonary bypass with or without aprotinin therapy. AB - Intra- and postoperative blood loss during open heart surgery is reduced by approximately 50% when aprotinin, a potent inhibitor for plasmin and kallikrein, is administered during surgery. But whether aprotinin increases the risk of thrombotic complications remains controversial. The aim of this study was to evaluate the effects of aprotinin administration on coagulation and fibrinolysis during and after cardiopulmonary bypass (CPB). Thirty patients undergoing CPB were randomly assigned to two comparable groups for a double-blind study (16 patients receiving high-dose aprotinin, 14 patients receiving placebo). Patients' plasma levels of ATM (thrombin-induced modified antithrombin III), FbDP (fibrin degradation products, D-Dimers), t-PA (tissue-type plasminogen activator) and PAI 1 (plasminogen activator inhibitor type 1) were measured at regular intervals. In both groups, ATM level increased during surgery (from less than 30 to 90-110 ng/ml) and returned to normal 24 h after surgery and remained unchanged thereafter. Aprotinin reduced this increase in ATM levels (p = 0.02 at 30 min after the start of CPB). The FbDP generated during surgery was greatly reduced in the aprotinin group (945 ng/ml) in comparison with the placebo group (1889 ng/ml, p = 0.004). After surgery, FbDP levels decreased in both groups with nadirs at 2nd day (placebo group: 940 ng/ml and aprotinin group: 865 ng/ml) indicating a hypofibrinolytic period. Then, the FbDP level in both groups started to increase up to the 9th day, in an identical manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531878 TI - Polymorphonuclear leukocyte-platelet interaction: role of P-selectin in thromboxane B2 and leukotriene C4 cooperative synthesis. AB - In PMN/platelet suspensions stimulated by fMLP giant mixed aggregates are formed and TxB2 and LTC4 are synthesized as the result of the cooperation in the arachidonic acid (AA) metabolism during cell/cell contact. PMN-derived cathepsin G induced the expression of P-selectin on platelet surface. GE12, an antibody against P-selectin, significantly reduced mixed cell aggregates. GE12 did not affect platelet aggregation induced by PMN-derived supernatants, indicating that the inhibitory effect of GE12 on mixed cell aggregation depends on inhibition of PMN/platelet adhesion. GE12 significantly reduced TxB2 and LTC4 production in PMN/platelet mixed cell suspensions stimulated by fMLP. As previously reported, synthesis of 3H-TxB2 in 3H-AA-labeled PMN/unlabeled platelets indicates that platelets utilize 3H-AA from PMN. 3H-LTC4 production in unlabeled PMN/3H-AA labeled platelets indicates that bidirectional routes are utilized in this system for LTC4 synthesis. GE12 significantly reduced 3H-TxB2 and 3H-LTC4 synthesis. These results show that cathepsin G released by activated PMN induces the expression of P-selectin on platelet membrane: this adhesive glycoprotein modulates cell-cell contact and transcellular metabolism of AA. PMID- 7531880 TI - Regulation of CFTR channel gating. AB - The debilitating symptoms of cystic fibrosis stem from the reduced Cl- permeability of epithelial cells owing to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. In cells with normal CFTR channels, receptor-mediated activation of cyclic-AMP-dependent protein kinase causes phosphorylation of several serines in the regulatory domain of CFTR, permitting channel opening and closing via cycles involving ATP hydrolysis. Cellular phosphatases rapidly dephosphorylate the channels, inactivating them. Here we discuss recent advances in our understanding of this complex mechanism for regulating channel gating. PMID- 7531881 TI - The cloning of a mechano-gated membrane ion channel. PMID- 7531879 TI - Malformin A prevents IL-1 induced endothelial changes by inhibition of protein synthesis. PMID- 7531882 TI - How do developing cortical neurones know where to go? PMID- 7531883 TI - David Whitteridge (1912-1994). PMID- 7531884 TI - Extending the neuron doctrine: Carl Ludwig Schleich (1859-1922) and his reflections on neuroglia at the inception of the neural-network concept in 1894. AB - Since the term 'neuroglia' was called into existence in 1856, the vast majority of neuroscientists dispensed with an integration of glial function into nervous system operation. In the shadow of the neuron doctrine, glial cells have been regarded as a static and functionally passive filling matter. Currently, new insights in glial physiology and glia-neuron interactions increasingly force a radical revision of this view. Meanwhile, some neurobiologists even profess a dynamic and active role for glial cells in the function of the nervous system. As early as the 1890s, Carl Ludwig Schleich (1859-1922), a surgeon and anaesthetist in Berlin, pleaded for this. PMID- 7531885 TI - Infrared videomicroscopy: a new look at neuronal structure and function. AB - Brain slices were introduced as a standard preparation for neurophysiological experiments some 20 years ago. A drawback of this preparation compared with cell culture has been the difficulty to visualize individual neurones in standard thick slices. This problem has been overcome by the use of infrared videomicroscopy. Neurones in slices can now be visualized in great detail, and neuronal processes can be patch-clamped under direct visual control. Infrared video-microscopy has also been applied successfully to other fields of neuroscience such as neuronal development and neurotoxicity. A further development of infrared videomicroscopy enables one to visualize the spread of excitation in slices making the technique a tool for the direct investigation of neuronal function. PMID- 7531886 TI - Brain atlases--a new research tool. AB - Conventional brain atlases are collections of micrographs or schematic drawings of brain sections from one or a few brains in which anatomical structures are identified, for example, nuclei, cortical areas and fibre tracts. Conventional brain maps have now been replaced with modern computer-based brain atlases. The structures in computerized atlases are deformable so as to fit the sizes and shapes of individual brains, and transform three-dimensional reconstructions or images of brains into a standard brain format. In order to make generalizations about localization of function and structure at both the macroscopical and microscopical level computerized brain atlases are needed. Computerized brain atlases are also used to compensate for the shrinkage and distortions during sectioning and embedding of post-mortem brains, to study structural-functional relationships in the human brain at both the macroscopical and microscopical level, and variations in gross morphology and microstructure of the human brain, and for establishing a three-dimensional human-brain database for all of the above and also for topographically defined data from the literature. PMID- 7531887 TI - Carnosine and free-radical defence mechanisms. PMID- 7531889 TI - Perspectives on axonal regeneration in the mammalian CNS. AB - In the CNS of mammals, axonal regeneration is limited by inhibitory influences of the glial and extracellular environment. Myelin-associated inhibitors of neurite growth, as well as some properties of so-called 'reactive astrocytes' which make the environment non-permissive for axonal growth, contribute to the inhibitory nature of the mammalian CNS. Furthermore, mechanisms for effective removal or neutralization of inhibitory components of cell debris are lacking in the mature mammalian CNS. However, in a permissive environment, mammalian CNS axons are able to regrow, to recognize target areas and to re-establish functional synapses with target neurones. Moreover, recent observations suggest that guiding molecules, like those required for axon guidance in the developing CNS, become expressed after lesions. Regenerating CNS axons seem to be able to recognize such guidance cues. Thus, even regenerating CNS axons of mammals might ultimately succeed in re establishing topographically ordered functional synapses in their target regions. PMID- 7531888 TI - Building synapses: agrin and dystroglycan stick together. AB - A major effort of the past decade for those studying synaptic development has been to identify the molecular signals whose carefully choreographed exchange between pre- and postsynaptic cells regulates the local differentiation of each cell to form the mature synapse. Now that several of these factors [agrin, ACh receptor inducing activity (ARIA) and calcitonin gene-related peptide] have been identified and isolated, efforts have moved toward understanding their receptors and the intracellular signaling pathways by which the factors achieve their effects. One of the most intensively studied of the synaptic signaling molecules is agrin, a large protein synthesized and released by motor neurons that induces ACh receptors and other synaptic molecules in muscle cells to accumulate at the sites of nerve contact. Recent efforts to discover the agrin receptor have led to a surprising conclusion: the only agrin-binding component so far detected in muscle cells is dystroglycan, an extracellular protein that is part of the complex of proteins associated with dystrophin, and its homologue, utrophin. Because dystroglycan binds laminin, and dystrophin binds actin, the complex containing these two proteins is thought to link the extracellular matrix to the cytoskeleton. Those interested in synapses are now pondering whether dystroglycan has a new and unexpected role as a signaling receptor for agrin-induced ACh receptor clustering, whether it serves as an auxiliary for another receptor, or whether it serves as a receptor for an entirely different agrin-mediated function. PMID- 7531890 TI - The behaving brain of a fly. AB - The use of Drosophila as a suitable system to answer behavioural questions is usually based on the availability of mutant phenotypes. Indeed, the 'single-gene' approach to behaviour was a very illuminating strategy in practical and conceptual terms, and served to prove that the genetic mechanisms sustaining behaviour could be analysed. However, the essence of neurogenetics goes for beyond the utilitarian use of mutants as tools to dissect behavior. Our main contention is that the study of the genetic basis of behaviour requires the study of genomes, rather than single genes, and their functional organization. Here, we use two aspects of behaviour, olfaction and movement control, as examples to illustrate the intricate, albeit understandable, relationship between the genome and behaviour. At present, these examples offer only a glimpse into this relationship. Further progress might be reached if studies on the regulation of functionally related genes are undertaken. On these grounds, it appears that the answer to many fundamental questions about behaviour that are amendable to experimentation might come from the work on Drosophila, providing that the required multidisciplinary efforts are focused on this organism. PMID- 7531891 TI - Role of neurotrophins in cholinergic-neurone function in the adult and aged CNS. AB - Cholinergic neurones in the CNS undergo complex changes during normal aging. In recent years, considerable attention has focussed on the neurotrophins and, in particular, nerve growth factor, as potential maintenance factor for cholinergic neurone function, and as therapeutic agents for use in a variety of neurodegenerative disorders including Alzheimer's disease. While brain cholinergic neurones from the neonate to the aged respond to nerve growth factor with enhanced expression of transmitter phenotype, there appears to be an age related, region-specific decline in responsiveness. This age-related decrement in neurotrophin action might play a role in dysfunction of cholinergic neurones, and cognitive loss, and could limit the use of these factors as therapeutic agents. PMID- 7531892 TI - Neurotrophins and brain insults. AB - Epileptic, hypoglycaemic, ischaemic and traumatic insults to the brain induce marked changes of gene expression for the neurotrophins, nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, and their high-affinity receptors, TrkB and TrkC, in cortical and hippocampal neurones. Release of glutamate and influx of Ca2+ are the most important triggering factors. The major hypotheses for the functional effects of the insult-induced neurotrophin changes are protection against neuronal damage and stimulation of sprouting and synaptic reorganization. More insight into the regulation and role of the neurotrophins after brain insults should increase our understanding of pathophysiological mechanisms in, for example, epileptogenesis and cell death, and could lead to new therapeutic strategies. PMID- 7531893 TI - Anoxic LTP sheds light on the multiple facets of NMDA receptors. AB - Hippocampal neurones in the CA1 region have become a model system to study the mechanisms of long-term potentiation (LTP) and memory processes. The CA1 region is also highly vulnerable to ischaemic or anoxic episodes which induce a selective and delayed degeneration of pyramidal neurones. In CA1 neurones, anoxic episodes generate a novel form of LTP to which we refer as anoxic LTP. In common with tetanic LTP, the induction of anoxic LTP is voltage- and NMDA receptor dependent. However, in contrast with tetanic LTP, the expression of anoxic LTP is mediated exclusively by NMDA receptors. These observations suggest that anoxic ischaemic episodes trigger a switch in favour of NMDA receptor-operated synaptic transmission. We suggest that the multiple forms of NMDA receptor-dependent LTPs are determined by extracellular and intracellular modulatory sites of this receptor. PMID- 7531894 TI - Long-spacing collagen and proteoglycans in pathologic tissues. AB - The presence of proteoglycans in fibrous long-spacing collagen (FLSC) was assessed in various pathologic tissues using the highly selective proteoglycan stains cuprolinic blue and polyethyleneimine. Two types of FLSC could be distinguished: one that contained proteoglycans and one that did not. The conditions in which these types occurred suggested a completely different physiologic significance. Also, their morphologies were different: The FLSC containing proteoglycans constituted compact, often fusiform structures long known in diagnostic electron microscopy as Luse bodies. In accordance with the literature, this compact type of FLSC was found especially in schwannomas and other neurogenic tumors. Enzymatic digestion experiments indicated that the proteoglycan present was dermatan sulfate proteoglycan. The average periodicities measured ranged from 101 to 147 nm. The type of FLSC lacking proteoglycans, on the other hand, formed dispersed aggregates. This dispersed FLSC had periodicities ranging from 79 to 103 nm (ie, just below those of the compact type). It was found only under circumstances in which there was high collagen breakdown and/or turnover. That dispersed FLSC is a marker for collagen degradation was further supported by its presence inside fibroblasts engaged in collagen phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531895 TI - Influence of digital examination, cystoscopy, transrectal ultrasonography and needle biopsy on the concentration of prostate-specific antigen. AB - The influence of various prostatic manipulations, including digital rectal examination, cystoscopy, transrectal ultrasonography and transrectal needle biopsy, on the serum prostatic-specific antigen (PSA) levels in 170 men, were examined. We found that digital rectal examination, cystoscopy and transrectal ultrasonography had no significant effect on PSA levels, except for transrectal needle biopsy, which caused an immediate increase of serum PSA in 96.2% of the patients lasting more than 2 weeks in 42.3% of the cases. In conclusion, serum PSA determination after digital rectal examination, after cystoscopy and after transrectal ultrasonography is accurate and reliable. On the other hand, we must wait about 6 weeks after needle biopsy before measuring PSA in the serum of patients with prostatic diseases. PMID- 7531897 TI - Second Stanford Conference on International Standardization of Prostate-Specific Antigen Immunoassays: September 1 and 2, 1994. PMID- 7531898 TI - Lasers in the treatment of benign prostatic hyperplasia. AB - Laser treatment of men with BPH remains in its infancy. To date, a large number of techniques and devices have been developed and investigated to varying degrees. Each laser system that is utilized in a unique fashion must be evaluated individually, since tissue effects may vary significantly with minor changes in technique or technology. Overall, it appears that the majority of men treated by laser prostatectomy experience objective and subjective improvement with short term follow-up. In most cases, further direct comparisons with TURP with longer follow-up are needed to assess adequately the relative efficacy and morbidity of laser therapy. Although early results are promising and technologic advances are likely to improve further the results seen with laser treatment of men with BPH, it may be premature to relegate TURP to the history books. PMID- 7531896 TI - Radical prostatectomy in patients with incidental prostate carcinoma. AB - Radical prostatectomy was carried out in 30 patients with an incidental carcinoma of the prostate. A tumor stage T1a (A1) was present in 7 cases and a tumor stage T1b (A2) in 23 cases. None of the 30 patients died postoperatively. As intraoperative complication, a rectum perforation occurred in 1 case. In the 23 patients with a T1b tumor, a pT2pN0M0 lesion was found in 10 cases and pT3pN0M0 lesion in 11 cases on pathohistological examination after radical prostatectomy. In 5 out of 30 patients the carcinoma could no longer be detected in the radical prostatectomy specimen. In 1 patient with a T1a tumor, pelvic lymphadenectomy revealed a single lymph node metastasis. Tumor progression did not occur in any of the 7 patients with a T1a tumor after an observation period of 9 months up to 16 years (average 4.6 years). The percentage of progression-free patients in stage T1b was calculated as 81.4% 10 years after surgery. The calculated overall survival time of patients in stage T1b was 76.3% after 10 years and the tumor related survival was 87.5%. PMID- 7531899 TI - Transurethral ultrasound-guided laser-induced prostatectomy: clinical outcome and data analysis. AB - OBJECTIVES: To analyze efficacy and side effects of the transurethral ultrasound guided laser-induced prostatectomy (TULIP) procedure for the treatment of bladder outlet obstruction due to benign prostatic hyperplasia (BPH). Based on the results obtained so far, the influence of preoperative and intraoperative parameters on clinical outcome is analyzed. METHODS: Between May 1991 and March 1994, 89 men with symptomatic and obstructive BPH underwent a TULIP procedure and were followed after 3 and 6 weeks, and 3, 6, and 12 months postoperatively. RESULTS: On average, mean urinary peak flow increased from 7 to 15 mL/s, postvoidal residual urine decreased from 215 to 40 mL and symptoms (assessed by modified Boyarsky score) decreased from 17 to 5 at 12 months after TULIP: In about half of the patients peak flow increased to more than 20 mL/s on average, and approximately 85% of all men reported that their symptoms improved on average 80% during this time. Because of insufficient micturition, 7 patients subsequently underwent conventional transurethral resection of the prostate (TURP) and 1 had an open prostatectomy. Either partial or complete retrograde ejaculation was reported by 13 of 58 sexually active men. No blood transfusion was required and no post-transurethral resection syndrome occurred in any case. Analyses of preoperative and intraoperative parameters and clinical outcome with respect to prostate volume, urinary retention, type of BPH enlargement, sleeve size, and good and poor responders did not reveal any significant influence of any factor. There is, however, a trend toward a lower laser energy delivered in patients who failed or may be considered to be poor responders. CONCLUSIONS: In this unselected group of patients with BPH, who otherwise would have been TURP candidates, the TULIP procedure demonstrated the efficacy of this technique to relieve bladder outlet obstruction. Compared with TURP, the TULIP procedure proved to be advantageous in regard to less blood loss and rate of retrograde ejaculation. In addition, TULIP can be performed under analgesic sedation, which is especially advantageous in high-risk patients. Disadvantages, however, are that it takes a considerably longer time to obtain substantial improvements in subjective and objective symptoms and there are more irritative symptoms in the early postoperative phase. PMID- 7531900 TI - Transition zone volume and transition zone ratio: predictor of uroflow response to finasteride therapy in benign prostatic hyperplasia patients. AB - OBJECTIVES: The aim of this study was to: (1) determine effects of finasteride on transition zone (TZ) volume, TZ ratio (TZ volume/total prostate volume), and total prostate volume; (2) analyze differences in TZ and total volume reduction among patients who improve peak urinary flow rates following finasteride therapy with those who do not; (3) investigate which parameters correlate with improvement in peak urinary flow rate and urinary symptom score; and (4) establish if there is any predictive value of these parameters for response to therapy. METHODS: Twenty-three patients with symptomatic benign prostatic hyperplasia (BPH) were treated with finasteride (5 mg/d) for 12 months and underwent transrectal ultrasound (TRUS) evaluation of total and TZ volume of prostate and measurement of peak flow rate and modified Boyarsky symptom score at baseline and at 12 months. Statistical analysis was done by unpaired t, Mann Whitney, and Spearman rank correlation tests among responders (more than 3 cc/s improvement in peak flow rate) and nonresponders (less than 3 cc/s improvement in peak flow rate) to therapy. RESULTS: (1) Responders had substantial reduction in TZ volume (44.8% versus 16.05%; P < 0.03) and TZ ratio (25% versus 5% increase, P < 0.02) compared with nonresponders. (2) There was a significant correlation between reduction in TZ volume (r = 0.50; P < 0.03) and TZ ratio (r = 0.60; P < 0.006) with improvement in peak flow rates. No similar correlation was seen with total prostate volume changes. (3) Pretreatment TZ ratio helped in predicting peak flow improvement following finasteride therapy (r = 0.52; P < 0.01) and there was a 2.5-fold increased chance of improvement if baseline TZ ratio was more than 0.51. The modified Boyarsky symptom score decreased by 3.1 (mean), but there was no correlation with changes in peak urinary flow rate, total prostate volume, TZ volume, and TZ ratio. TZ ratio did not have significant predictive value for improvement in symptom score. CONCLUSIONS: This study provides data that simple measurable parameters are available which may be used prior to therapy to predict uroflow response to finasteride and similar agents. PMID- 7531901 TI - Pelvic lymphadenectomy can be omitted in selected patients with carcinoma of the prostate: development of a system of patient selection. AB - OBJECTIVES: The prevalence of pelvic lymph node metastases in men with clinically localized prostate cancer has decreased dramatically over the past decade, possibly due to efforts at early detection. With a significantly lower incidence of pelvic node involvement, it may be possible to identify a segment of patients for whom pelvic lymph node dissection (PLND) may be omitted. This study was conducted to develop a method to select patients for whom PLND could be omitted. METHODS: We analyzed serum prostate-specific antigen (PSA), clinical stage, biopsy Gleason score, and final pathologic stage in 481 men with clinically localized prostate cancer. These variables were compared to the risk of positive pelvic lymph nodes. RESULTS: Logistic regression analysis determined that combining all three variables provided the best determination of final pathologic stage. A series of probability curves have been created to estimate the risk of positive lymph nodes in a given patient. Based on the distribution of patients in this study and using these probability functions, PLND could be avoided in up to 50% of patients with localized prostate cancer diagnosed by contemporary methods. CONCLUSIONS: In properly selected patients, pelvic lymphadenectomy can be omitted in the staging and treatment of localized prostate cancer. PMID- 7531903 TI - Equine hoof hardener. PMID- 7531902 TI - Luteinizing hormone-releasing hormone antagonist cetrorelix as primary single therapy in patients with advanced prostatic cancer and paraplegia due to metastatic invasion of spinal cord. AB - OBJECTIVES: To assess the clinical response to luteinizing hormone-releasing hormone (LH-RH) antagonist cetrorelix (SB-75) in patients with advanced carcinoma of the prostate and paraplegia due to metastatic invasion of spinal cord. METHODS: Cetrorelix was given at two different dose regimens to 5 patients with prostatic cancer Stage D2 and paraplegia. Urologic and neurologic examinations, laboratory studies, radiography (myelography), and prostate ultrasonography were carried out. Prostate-specific antigen (PSA) and free testosterone were also measured. RESULTS: In all patients, the neurologic symptoms regressed. The recovery of the thermic and vibratory sensation and motility of the toes was observed. The neurologic improvement continued during the treatment and at 3 months all the patients were able to walk with the aid of a cane. In 1 patient, the myelography showed that the spinal cord compression had disappeared and prostate volume assessed by ultrasonography showed a significant decrease. The bladder function greatly improved in all 5 patients during the treatment with cetrorelix. Baseline levels of luteinizing hormone fell from 9.28 to 1.0 IU/L and those of follicle-stimulating hormone (FSH) fell from 18.28 to 12 IU/L (P < 0.05) after the first day of therapy with cetrorelix. Mean levels of free testosterone were reduced from 52.4 to 14.7 pmol/L (P < 0.005) at 12 hours and to 13.1 pmol/L (P < 0.005) 3 days after the first injection of cetrorelix. A persistent inhibition of gonadotropins and testosterone was maintained during the subsequent 3 months of therapy. The high levels of PSA gradually decreased. CONCLUSIONS: Our results show that LH-RH antagonist cetrorelix causes an immediate lowering of the serum testosterone levels in patients with prostate cancer and metastases in the spinal cord, in whom the LH-RH agonists cannot be used as single drugs because of the possibility of flare-up and appears to be appropriate for long-term therapy. PMID- 7531904 TI - Analysis of monoclonal antibodies reactive with the porcine CD2 antigen. AB - As result of the First International Swine CD Workshop, six monoclonal antibodies (mAbs) (numbers 014, 023, 024, 057, 128, and 130) clustered closely to the internal standard anti-porcine CD2 mAb, MSA4. Despite the close clustering, the cluster was split into two subgroups. To further characterize the relationship between these mAbs, they were used in flow cytometry to inhibit binding of MSA4 to porcine lymphocytes. mAbs 014 (1038-8-31), 023 (MAC83), 024 (MAC80), 057 (PG168), and 128 (MSA4) completely inhibited the binding of MSA4, mAb 130 (MSA2) failed to inhibit MSA4 binding. On dual parameter flow cytometry comparing MSA2 with MSA4, all MSA2+ cells were MSA4+, two thirds of the MSA4+ cells were MSA2-. We conclude that five of the mAbs bind to the same or a closely related epitope on porcine lymphocytes. mAb 130 appears to have aberrantly clustered with the CD2 group of mAb. PMID- 7531905 TI - Analysis of monoclonal antibodies reactive with the porcine CD4 antigen. AB - As result of the First International Swine CD Workshop, four monoclonal antibodies (mAb) (#002, 054, 118, and 127) clustered closely to the internal standard anti-porcine CD4 mAb, 74-12-4. To further characterize the relationship between these mAb, they were used in flow cytometry to inhibit binding of 74-12-4 to porcine lymphocytes. mAb #002 (74-12-4) and #054 (PT90) completely inhibited, while mAb #127 (10.2H2) and #118 (b38c6) partially inhibited (57% and 77% respectively), the binding of 74-12-4. Furthermore, none of the mAbs bound to a 74-12-4 negative strain of pigs. We conclude that the four mAb bind to the same, or a closely related, epitope on porcine lymphocytes. PMID- 7531906 TI - Analyses of monoclonal antibodies reactive with porcine CD5. AB - Among all monoclonal antibodies (mAbs) analyzed in the first porcine CD workshop, six mAbs showed reactivity to the porcine CD5 antigen (workshop mAbs 067, 068, 069, 070, 071 and 119). Because of lack of immunoprecipitation studies for five (067, 068, 069, 070 and 071) out of the six mAbs, only one mAb (119) could be definitely characterized as mAb against the monomeric 63 kDa porcine CD5 antigen. The five other mAbs included in this cluster are characterized by an identical labelling pattern in FCM and competition of the CD5 epitope recognized by mAb 119. These mAbs were allocated to the wCD5 subcluster. PMID- 7531907 TI - Analyses of monoclonal antibodies reactive with porcine CD6. AB - Amongst the monoclonal antibodies (mAbs) submitted to the first porcine CD workshop, two mAbs (workshop numbers 055 and 120) could be identified to recognize the porcine CD6 analogue. Both mAbs seemed to be highly T-cell specific and showed neither reactivity with cells of the myeloic lineage nor with B lymphocytes. The observed molecular mass of the antigen precipitated by mAb 120 of 110 kDa confirmed this classification. Without molecular analyses of the antigen recognized by mAb 055, but similar staining pattern in FCM compared with 120, mAb 055 was allocated to the wCD6 subcluster. PMID- 7531908 TI - Analyses of mAb reactive with porcine CD8. AB - Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051, 052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051, 052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109. PMID- 7531910 TI - Competitive binding analysis of monoclonal antibodies reactive with porcine alveolar macrophages using anti-CD14 and anti-CD18. AB - Four monoclonal antibodies (mAb) from the myeloid subset panel of the First International Swine CD Workshop (74-22-15, DH59B, PM16-6, and MUC21A) were analyzed using competitive inhibition studies with anti-human CD14 (My4) and anti human/anti-porcine CD18 (MHM23) on porcine alveolar macrophages. Results suggested that none of the mAb tested recognized CD14 or CD18 on porcine alveolar macrophages. Additionally, the cross-reactivity of My4 with porcine alveolar macrophages was established. PMID- 7531909 TI - Analysis of mAb reactive with the porcine SWC1. AB - Among 54 mAb determined to be reactive with porcine T lymphocytes and/or activation antigens, eight mAb (workshop Nos. 005, 031, 080, 091, 092, 093, 094 and 110) derived from different laboratories grouped together in the T11 cluster and were ordered into the SWC1. One mAb (No. 111) which belong also to this group was lost during the workshop. The SWC1 antigen is a molecule expressed on the majority of leukocytes, resting T lymphocytes, monocytes and granulocytes, but not on B lymphocytes. On T lymphocytes it is down-regulated after activation. The molecular mass of the antigen is unknown. Epitope analyses revealed that seven out of the nine mAb recognized similar epitopes on the SWC1 molecule. PMID- 7531911 TI - CD11/CD18 panel report for swine CD workshop. AB - Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W #078), MUC93A (W #079) and MHM23 (W #136) of CD11/18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD18 epitope defining antibody resulted H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD18a mAbs. PMID- 7531912 TI - Analyses of monoclonal antibodies reactive with porcine CD44 and CD45. AB - Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system. PMID- 7531913 TI - Biochemical analysis of molecules reactive with monoclonal antibodies specific for porcine CD45. AB - The apparent molecular weight of molecules reactive with eight mAbs, which were identified during the First International Swine CD Workshop as putatively specific for porcine CD45, was determined by immunoprecipitation analysis of 125I surface-labeled peripheral blood mononuclear cells. Four putative CD45 specific mAbs (K252.1E4, MAC323, 74-9-3 and 2A5) precipitated three polypeptides of 226, 210 and 190 kDa. Two putative CD45R specific mAbs (MAC326 and MAC327) precipitated two polypeptides with an apparent molecular weight of 226 and 210 kDa, while another two (3a56 and -a2) precipitated a single polypeptide of 210 kDa. Sequential immunoprecipitations analyses indicated that the comigrating molecules precipitated by the CD45 and CD45R were the same. The results from this study confirmed the assignment of mAbs K252.1E4, MAC323, 74-9-3 and 2A5 as CD45, and mAbs MAC326, MAC327, 3a56 and -a2, as CD45R. These CD45 and CD45R specific mAbs should prove to be valuable reagents for the study of the porcine immune system. PMID- 7531914 TI - Replication of cymbidium ringspot virus satellite RNA mutants. AB - Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA. PMID- 7531915 TI - A cell fusion-inhibiting monoclonal antibody binds to the presumed stalk domain of the human parainfluenza type 2 virus hemagglutinin-neuraminidase protein. AB - Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) protein proved to be identical to that of wild-type F protein, and furthermore, protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN protein, which may play an important role in promoting cell fusion. PMID- 7531916 TI - Cellular tropism of human T-cell leukemia virus type II is enlarged to B lymphocytes in patients with high proviral load. AB - To establish the in vivo cellular tropism of human T-cell leukemia virus type II (HTLV-II) in peripheral blood, subpopulations of mononuclear cells isolated from patients with a history of drug abuse and with high proviral load were analyzed by polymerase chain reaction for the presence of the proviral sequences. After purification of cellular subsets by immunomagnetic fractionation of blood cells of an infected patient, HTLV-II DNA was detected in CD4+ and CD8+ T-cells as well as in CD19+ B-cells. A positive PCR signal was obtained for purified B-cells also at limiting dilutions. This observation was confirmed by purifying the B-cell fraction by a two-step immunomagnetic procedure from the peripheral blood of another patient with very high HTLV-II copy number and quantifying the B-cell proviral load by means of competitive PCR. A proviral copy number of 90/100 B cells was found, demonstrating that the great majority of these cells were infected by HTLV-II in this subject. The results indicate that HTLV-II has a broad host range in some infected individuals, showing an enlargement of cellular tropism to B lymphocytes and suggesting that this expression is associated with an increase in proviral load. PMID- 7531917 TI - Molecular analysis of the essential and nonessential genetic elements in the genome of peanut chlorotic streak caulimovirus. AB - The DNA genome of caulimoviruses contains a set of essential genes: I (movement gene), IV (major capsid protein gene), V (reverse transcriptase gene), and VI (gene coding for a post-transcriptional activator of the expression of other virus genes). In peanut chlorotic streak caulimovirus (PCISV), three ORFs, A, B, and C, are located between genes I and IV. They are dissimilar to other caulimovirus ORFs. ORF VII of PCISV is a homolog of ORF VII of soybean chlorotic mottle caulimovirus (SoCMV), but is not similar to the nonconserved ORF VII in other caulimoviruses. The sequence complementary to a portion of tRNA(Met), thought to be essential for the priming of minus-strand DNA synthesis in caulimoviruses, is located within the coding sequence of ORF A. To explore the functional significance of ORFs VII, A, B, and C, various mutations were engineered into an infectious DNA clone of PCISV. ORFs VII and B are shown to be dispensable, while ORFs A and C are essential. ORF C is a possible functional equivalent of gene III in other caulimoviruses. Sequences within ORF A that are required for efficient priming of minus-strand synthesis are likely to extend beyond the 12-bp tRNA-binding site. Complete deletion of ORF VII was correlated with severe symptoms, notably with the necrosis of apical meristems. Significance of these observations for the understanding of replication and pathogenesis of plant pararetroviruses and for the improvement of caulimovirus-based expression vectors is discussed. PMID- 7531918 TI - Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. AB - HIV-1 vpr encodes a 96-amino acid, nuclear protein whose function is not well understood. Unlike the other lentivirus regulatory proteins, Vpr is present in virions at relatively high copy number. In cells, Vpr is localized to the nucleus. Possible functions for vpr consistent with these findings include the nuclear import of preintegration complexes, transactivation of cellular genes, or induction of cellular differentiation. We show here, using both replication competent, macrophage-tropic virus and a sensitive, single-cycle luciferase HIV-1 reporter vector, that vpr is important for efficient viral replication in primary monocyte/macrophages, but appears to play no role in activated or resting T cell infection. The block to infection in monocytes was localized by PCR analysis of newly synthesized viral DNA and with the luciferase reporter vector to a stage in the viral life cycle after entry and reverse transcription, yet prior to, or at the time of, proviral transcription. In addition, infection of mononuclear phagocytes with virions that had been loaded with Vpr molecules in the producer cells by trans-complementation still showed a vpr-phenotype. These data suggest a role for vpr molecules produced in newly infected cells, in addition to its presumed function in the virion. PMID- 7531919 TI - Requirement for the G1 protein of California encephalitis virus in infection in vitro and in vivo. AB - A role for the large glycoprotein (G1) of California encephalitis (CE) virus was examined in the infection of baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cell lines and the mosquito Ae. dorsalis using G1 monoclonal antibodies (MAbs) and selective protein cleavage. Five MAbs neutralized CE viral infectivity in both cell lines. One MAb, 7D4.5, efficiently neutralized the peroral infection of Ae. dorsalis females fed CE virus in artificial bloodmeals. To determine if MAbs to G1 neutralized CE virus by sterically hindering the small glycoprotein (G2), portions of G1 were trypsinized, and viral infectivity was assayed in vivo and in vitro. Cleavage of G1 resulted in a complete loss of infectivity both in mosquitoes and in culture, even though a significant amount of G2 remained intact. The loss of infectivity by both neutralization with G1 MAbs and trypsinization indicates that the G1 protein of CE virus is required for infection of mosquito and mammalian cells in vitro and of mosquitoes by the peroral route. PMID- 7531921 TI - Antibody-binding sites on truncated forms of varicella-zoster virus gpI(gE) glycoprotein. AB - Varicella-zoster virus (VZV)-seropositive human sera were shown to be reactive with the truncated VZV gpI(gE) candidate subunit vaccine (TgpI-511). To identify the location of antibody-binding sites (epitopes) on TgpI-511, three truncated forms of TgpI-511 glycoprotein (TgpI-124, TgpI-160, TgpI-316) DNA encoding the N terminal region of this glycoprotein with amino acid residues of 124, 160 and 360, respectively, were inserted into the vaccinia virus genome. Infection of cells with recombinant vaccinia viruses resulted in the secretion of all three truncated gpI(gE) as well as TgpI-511 from the infected cells. Immunoprecipitation of these truncated glycoproteins with VZV-seropositive human sera and gpI(gE)-specific monoclonal antibodies identified the location of four new antibody-binding sites on the VZV TgpI-511 glycoprotein. In addition, tunicamycin treatment and O-glycanase digestion revealed the presence of both N linked and O-linked oligosaccharides on TgpI-511. These results revealed the location of new epitopes on VZV TgpI-511 and demonstrated that the epitopes on TgpI-511 were recognized by human sera from VZV-seropositive individuals. PMID- 7531920 TI - Lysis pneumonopathy associated with the use of fludarabine phosphate. PMID- 7531922 TI - Characterization of the N-terminal part of the neutralizing antigenic site I of coxsackievirus B4 by mutation analysis of antigen chimeras. AB - Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Ag1 of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4 specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar. PMID- 7531923 TI - Nucleotide and deduced amino acid sequences of the matrix (M) and fusion (F) protein genes of cetacean morbilliviruses isolated from a porpoise and a dolphin. AB - Morbilliviruses have been isolated from stranded dolphins and porpoises. The present paper describes the cloning and sequencing of the porpoise morbillivirus (PMV) F gene and of the dolphin morbillivirus (DMV) M and F genes and their flanking regions. The gene order of the DMV genome appeared to be identical to that of other morbilliviruses. A genomic untranslated region of 837 nucleotides was found between the translated DMV M and F gene regions. The predicted DMV M protein were highly conserved with those of other morbilliviruses. Both the deduced PMV and DMV F0 proteins exhibited three major hydrophobic regions as well as a cysteine rich region, a leucine zipper motif and a cleavage motif allowing cleavage of the F0 protein into F1 and F2 subunits. Apparently the DMV F0 cleavage motif was not modified by adaptation of DMV to Vero cells. The predicted PMV and DMV F proteins were 94% identical. Comparisons with the corresponding sequences of other morbilliviruses demonstrated that the cetacean morbillivirus does not derive from any known morbillivirus but represents an independent morbillivirus lineage. PMID- 7531924 TI - Cloning of the MCF1233 murine leukemia virus and identification of sequences involved in viral tropism, oncogenicity and T cell epitope formation. AB - MCF1233 is an oncogenic C57BL-derived retrovirus of the Murine Leukemia Virus (MuLV) family, that causes T and B lymphomas in an MHC-associated fashion. In this study, we cloned MCF1233, determined its nucleotide sequence and, by comparison with its MuLV relatives, identified the sequences that relate to the leukemogenic character of this virus. MCF1233 was found to have an ecotropic backbone, and carried acquired polytropic sequences in the 3' pol and 5' env region. The gag-region contained six specific nucleotides, determining the viral B-tropism. Short sequences within the U3 LTR shared specific homology with the xenotropic Bxv-1 MuLV, which is the U3 donor for leukemogenic MCF MuLV of AKR origin. These sequences, in combination with specific ecotropic sequences present in env p15E, most likely determine the viral oncogenicity. Currently, the deduced MCF1233 amino sequence is being exploited for T cell epitope analysis, which in this paper is discussed with respect to antigenically distinct Friend/Moloney/Rauscher types of MuLV. Identification of these T cell epitopes will contribute to our understanding of the fundamental aspects of immune control on MCF1233-induced lymphomagenesis. It will help to elucidate the mechanisms that underlie immune escape of T lymphomas, rarely arising in immunoresistant mice, and allow the development of vaccination protocols for tumor therapy. PMID- 7531925 TI - [Mechanosensing in the cell--SA channels as a mechanotransducer]. PMID- 7531926 TI - [The factors in Klebsiella persistence]. AB - The data on the study of antilysozyme and anti-interferon properties of 375 Klebsiella strains are presented. The heterogeneity of strains with respect to their antilysozyme and anti-interferon activity is described. As revealed in this study, these properties occur more frequently and considerably to a greater extent in strains isolated from patients with Klebsiella infections and from carriers. Strains, incapable of inhibiting lysozyme and interferon or capable to digest them poorly, prevail among strains isolated from healthy persons. The data on the relationship between the level of the persistence factors of Klebsiella and the source of their isolation are presented. PMID- 7531927 TI - [The microbiological diagnosis of pyelonephritis in children under the control of bacterial persistence factors]. AB - The study of antilysozyme and anti-interferon activity in 474 urological strains of opportunistic bacteria isolated from the urine of pyelonephritis patients and in 302 strains isolated from the urine of healthy children was made. The occurrence and the average amount of bacterial persistence factors were found to be directly related to the isolation source of cultures. The determination of the antilysozyme and anti-interferon activity of urological strains made it possible to confirm the etiological importance of enterobacteria and Pseudomonas in the development of renal infections and to differentiate the causative agent of pyelonephritis from the concomitant microflora. PMID- 7531928 TI - [A comparative evaluation of the persistence characteristics of Escherichia isolated from different econiches]. AB - The multiple evaluation of the persistence characteristics, including antilysozyme, anti-interferon and anticomplement activity, as well as other biological properties, such as adhesiveness, colicinogenicity and resistance to antibiotics, was carried out in 173 E. coli strains isolated from water, healthy and sick children. This evaluation revealed that each group of E. coli, depending on the source of its isolation, had its characteristic set of properties (or bioprofiles) to be analyzed, making it different from other bacterial populations. The comparative intergroup analysis showed differences between E. coli isolated from children with pathological conditions (enteric coli bacteriosis, pyelonephritis) and E. coli isolated from water and feces of healthy children. These differences were manifested by more pronounced persistence characteristics. Dispersion analysis, having confirmed this feature, revealed that the most labile characteristics of E.coli, subject to the influence of ecological conditions, were their markers of persistence and antibiotic resistance. The results of factor analysis made it possible to unite the above mentioned properties which determined, together with adhesiveness, pathogenic potential of these bacteria. PMID- 7531929 TI - [Markers of the persistence of the staphylococcal air microflora in a hospital]. AB - The presence of persistence markers of some staphylococcal species isolated from the air at an obstetric hospital was established. The presence of such markers was most pronounced in S. aureus, which was indicative of its absolute value for characterizing the sanitary state of the environment. Staphylococci of coagulase negative species were characterized by the presence of antilysozyme activity, as well as by their ability to inactivate the antibacterial component of the preparation of interferon, most pronounced in microorganisms of the species S. epidermidis and S. haemolyticus. Disinfecting measures, carried out with the use of ozone, made it possible to normalize the quantitative and qualitative characteristics of bacterial aerosol, which was mainly manifested by the air elimination of staphylococci having a set of persistence markers, as well as the capacity for producing hemolysin. PMID- 7531930 TI - Towards a standard method to demonstrate adenylate cyclase activity at the electron microscopical level. AB - The ventral epidermis of the frog Rana fuscigula is a typical tight epithelium which acts as a functional syncytium in the active transepithelial transport of sodium ions. Transport across this epithelium is regulated by cyclic adenosine monophosphate (cAMP). This study was undertaken to formulate an optimal protocol for the localization, within this epithelium, of adenylate cyclase; the enzyme involved in cAMP synthesis. The ventral epithelium of R. fuscigula was collagenase treated and processed using five different fixation/incubation protocols. The components of a basal incubating medium were modified by changing the localizing agent, adding adenylate cyclase stimulators and inhibitors of other enzymes. Control incubations undertaken included a) leaving the substrate out, b) prior heat inactivation of the enzyme, c) specific blockers and d) incubation for alkaline phosphatase as an alternative enzyme. The samples were then processed for electron microscopy. Localization of adenylate cyclase was best obtained, when fixing the tissue after incubation for 30 min at 37 degrees C. The medium that gave the best and most consistent localization contained magnesium chloride; as a required ion, theophylline, dithiothreitol, ouabain, levamisole; as enzyme inhibitors, forskolin; as a stimulator of adenylate cyclase, lead nitrate; as the capture agent and column purified adenylyl imidodiphosphate; as the substrate. PMID- 7531931 TI - The damaging effect of UV rays (with the wavelength shorter than 320 nm) on the rabbit anterior eye segment. I. Early changes and their prevention by catalase aprotinin application. AB - In experiments carried out in rabbit eyes, UV rays of 254 or 312 nm wavelength damaged the anterior eye segment, whereas those of 365 nm wavelength did not. Two min irradiation with 254 nm UV rays led to a decrease of catalase activity in the corneal epithelium. After 5 min irradiation the catalase activity in the epithelium was not detectable at all. Catalase activity was also diminished in the corneal endothelium and lens epithelium. In this stage the changes were accompanied by decreased activities of Na(+)--K(+)-dependent adenosine triphosphatase, gamma-glutamyl transpeptidase and increased activities of lysosomal enzymes in the corneal and lens epithelium as well as in the corneal endothelium. The transparency of the cornea and lens was decreased. Plasmin activity appeared in the tear fluid. The irradiation with UV rays of 312 nm caused similar disturbances, however, a longer exposure was necessary. In contrast, irradiation with UV rays of 365 nm did not produce any changes. The described corneal disturbances were prevented by dropping of catalase solution on the eye surface during the irradiation or shortly after it. However, after a protracted irradiation aprotinin had to be added to catalase to achieve the healing. The decrease of catalase activity and its prevention by a local application of catalase suggests a key role of oxyradicals in the damage of the eye by UV rays. PMID- 7531932 TI - Unusual immunostaining pattern of chromogranin in normal urothelium and in transitional cell neoplasms. AB - We report an unusual pattern of immunostaining for chromogranin A in 24 transitional cell carcinomas (TCC). Positive immunostaining was seen with chromogranin A antisera in 10 of 16 urinary bladder TCC, neither of 2 prostatic TCC, 3 of 3 ureteral TCC and 2 of 3 metastatic TCC. This staining was demonstrated within the cytoplasm of transitional cells at all levels of the neoplastic epithelium, including cells near the basement membrane and at the free surface (umbrella cells). In 5 of 7 cases there was also immunoreactivity of the non-neoplastic urothelium. Immunostaining with neuron-specific enolase and synaptophysin was negative in all of these cases and no neuro-secretory granules were identified in 7 cases examined by electron microscopy. This pattern of immunostaining is probably due to reactivity with chromogranins or certain chromogranin-like proteins in the transitional cells, particularly in the umbrella cells. PMID- 7531933 TI - Fluorescence of bisazo dye reaction products from the coupled tetrazonium method for proteins. AB - The coupled tetrazonium reaction is a well known histochemical method for proteins. This method, using Fast Blue B salt and 1-naphthol, has been applied on paraffin sections of grasshopper testis and rabbit trachea, as well as on horse blood smears. Microscopic observation under bright field illumination revealed the expected purple staining of protein-rich cell and tissue structures, which also showed a strong red fluorescence under ultraviolet, violet, violet-blue and green exciting light. Some weakly stained cell components (e.g., meiotic spindles) were easily visualized by fluorescence microscopy. Control preparations lacking either the tetrazonium or naphthol treatment, and spectroscopic studies on the bisazo dye produced in vitro (showing an emission peak at 660 nm) confirmed that the red fluorescence of stained structures arises from the protein tetrazonium-naphthol reaction product formed in situ. PMID- 7531934 TI - When NADPH diaphorase (NADPHd) works in the presence of formaldehyde, the enzyme appears to visualize selectively cells with constitutive nitric oxide synthase (NOS). AB - The NADPH diaphorase (NADPHd) reaction for nitric oxide synthase (NOS) visualization suffers from the circumstance that the diaphorase activity of NOS represents only part of the total diaphorase activity, and so far all efforts to make the reaction more specific for routine studies failed. The present investigation describes a simple procedure for mouse tissue, which allows the selective staining primarily of neurons, vascular endothelial cells and macula densa cells, those cells where constitutive NOS has been described reliably. In this method unfixed cryosections and 0.5-1% phosphate-buffered formaldehyde containing 0.5 mg NADPH/1 ml and 1 mg nitro BT/1 ml are used. Compared to strong prefixation with formaldehyde after which many additional cells are still positive for NADPHd, presence of formaldehyde in the incubation medium obviously allows the selective reaction of NOS-positive cells. In conclusion, compared with the original technique a more specific method for the visualization of the NADPHd activity of NOS appears to be available now and can be used for NOS studies in all kinds of mammalian species. PMID- 7531935 TI - Depressed serum levels of human placental lactogen in first trimester vaginal bleeding. AB - BACKGROUND: It has previously been shown that serum levels of pregnancy associated plasma protein A (PAPP-A) are depressed in patients with early pregnancy bleeding and a live gestation. We examined whether human placental lactogen (hPL) levels show a similar pattern. METHOD: Prospective study in the 8th-14th gestational week comparing serum levels in 69 women with early pregnancy bleeding with those in 93 control pregnancies. RESULTS: Serum levels of hPL were significantly depressed in women with early pregnancy bleeding (p < 0.0001). CONCLUSIONS: The findings suggest a primary affection of the placenta in early pregnancy bleeding. PMID- 7531936 TI - Declining an alpha-fetoprotein test in pregnancy, why and who? AB - OBJECTIVE: The aim of the study was to elucidate the questions: 1) Why are some women declining an alpha-fetoprotein (AFP)-test? and 2) Do women who decline an AFP-test differ in background and in attitudes towards abortion issues from women who accept the test? Furthermore the study was aimed at clarifying some aspects of the decision process. DESIGN: Questionnaire study over a one-year period from October 1, 1988 to September 30, 1989. SETTING: Hvidovre University Hospital, Copenhagen, and the county hospitals of Sonderjylland, Denmark. SUBJECTS: 336 women who declined the AFP-test and 3331 women who had the test performed. MAIN OUTCOME MEASURES: Main questions concerned reasons for declining AFP-testing, attitudes to abortion issues and how the decision was made. RESULTS: The main reasons for declining were found to be ethical/religious reasons or the view that the test was not reliable enough. The frequencies of women with a high school degree (46.4% versus 30.8%) and of women who had had a previous spontaneous abortion (24.1% versus 14.8%) were both significantly higher in the declining group. Regarding abortion issues, women who declined the test were significantly more restrictive in their acceptance of abortion than women who had taken the AFP test. 5.7% of the women who declined reported that they had perceived pressure to accept the offer to have the test. CONCLUSION: The study has clarified the questions as to who declines the AFP-test and why. It is discussed whether the AFP-programme and its consequences are, in fact, too difficult for some women to grasp and understand in detail. From the Health Service's point of view, it is noteworthy that a considerable number of women who declined the test offer might make a different choice if the AFP-programme were improved. PMID- 7531937 TI - Serum levels of hPL, PAPP-A and PP14 in patients with early pregnancy bleeding and subchorionic hemorrhage. AB - OBJECTIVE: To investigate possible endocrine changes in patients with early pregnancy bleeding and subchorionic hemorrhage. DESIGN: Prospective study in first trimester of pregnancy comparing circulating levels of human placental lactogen (hPL), pregnancy-associated plasma protein A (PAPP-A) and endometrial secretory protein PP14 (PP14) in women with and without subchorionic hemorrhage. SUBJECTS: Sixty-nine women with early pregnancy bleeding, who in week 7-14 had a blood sample and an ultrasound study with both determination of fetal crown-rump length and a search for subchorionic hemorrhage. RESULTS: There was no difference between serum levels of hPL, PAPP-A and PP14 in 29 women with and 40 women without subchorionic hemorrhage, and no correlation between serum levels and the size of the hematoma. CONCLUSION: Judged from the serum levels of the studied substances there were no indications of an impaired function of the trophoblast or the decidua that could explain the development of subchorionic hemorrhage. PMID- 7531938 TI - Precision and accuracy of monocyte counting. Comparison of two hematology analyzers, the manual differential and flow cytometry. AB - The Coulter STKS (Coulter, Hialeah, FL), the Abbott CD3500 (Abbott Diagnostics, Abbott Park, IL), a 400-cell manual differential, and flow cytometry using double staining with fluorescence-labelled monoclonal antibodies (CD45-FITC and CD14-PE) on a Coulter Epics Profile II were evaluated for precision and accuracy in relative monocyte counting. STKS, CD3500, and Profile II achieved a precision analogous to a 3,542-, 1,835-, and 11,998-cell differential, respectively, demonstrating the superiority of automated methods. Analysis of 156 normal and abnormal samples revealed that the mean relative monocyte counts of the manual differential, CD3500 and Profile II were not significantly different. Only the STKS results showed a positive bias (0.79% +/- 1.65), which was increased in lymphocytic samples. Linear regression between the Profile II as independent viable, and the other techniques yielded acceptable correlation coefficients (STKS: 0.861, CD3500: 0.844, manual differential:0.833). Profile II results were also compared to those of a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA), which yielded an excellent correlation (r = 0.991) but a slightly smaller relative monocyte count (bias-0.39% +/- 0.60) of the latter. On the basis of these data, the authors recommend the use of monoclonal antibodies as a new reference method, but also indicate the need for further methodological investigations. PMID- 7531939 TI - In vivo effects of recombinant human stem cell factor treatment. A morphologic and immunohistochemical study of bone marrow biopsies. AB - Bone marrow (BM) aspirate and biopsy specimens from seven female patients with advanced or metastatic breast cancer and preserved marrow function treated on a phase I trial of recombinant methionyl human stem cell factor (r-metHuSCF; SCF) were evaluated by immunohistochemical staining before and after treatment with SCF. Doses of SCF included 10 g/kg/day in 2 patients, 25 micrograms/kg/day in 2 patients, and 50 micrograms/kg/day in 3 patients administered as subcutaneous bolus injections for 14 days. Following treatment, bone marrow cellularity increased up to 1.6-fold (P = NS), with an increased frequency of promyelocytes (P < .002), but an unchanged relative frequency of other marrow hematopoietic cells. The mean relative frequency of BM CD34+ progenitor cells increased from 0.9% to 1.8% (P < .001). The mean proportion of BM cells stained by Ki-67/MIB 1 and PCNA/PC10, monoclonal antibodies (MoAb) recognizing proliferation-associated nuclear proteins, increased from 18.6% to 35.4% (P < .003) and from 32.4% to 49.4% (P < .01), respectively. Most of the Ki-67 and PCNA positive cells were represented by promyelocytes, proerythroblasts, and myeloblasts. SCF therapy was not associated with marrow fibrosis or increases in the number of macrophages. Peripheral white blood cell counts increased 1.3- to 3.6-fold following SCF. The mean absolute neutrophil counts increased from 3.9 x 10(9)/L (range 2.6-5.3) to 7.2 x 10(9)/L (range 4.7-12.3), and reticulocyte counts increased by a mean of 1.5 fold (range 1.2-fold to 2.0-fold). No consistent difference in platelet counts was seen. These results suggest that SCF given in vivo is effective in increasing the frequency of CD34+ BM progenitor cells, and has the capacity to increase the proliferation and differentiation rate of hematopoietic precursor cells. These effects indicate that SCF may represent a cytokine capable of affecting multiple hematopoietic lineages. PMID- 7531940 TI - Preparation of dextran 70 injection labeled with technetium 99m for use as a cardiac blood-pool imaging agent. AB - A means of compounding dextran 70 injection labeled with technetium Tc 99m by using readily available pharmaceutical components is described, and the compound's biological distribution is evaluated. The radiopharmaceutical was prepared by mixing 10 mg (0.17 mL) of dextran 70 in sodium chloride with 0.33 mL of 0.9% sodium chloride injection in a 1-mL syringe. This solution was added to a sterile, pyrogen-free vial containing stannous chloride, and the steps were repeated until five vials had been prepared. The contents of each vial were mixed by swirling until the solids were dissolved. The mixture was incubated for five minutes at 22 degrees C, then 1.48 gigabecquerels of sodium pertechnetate Tc 99m injection in a volume of 0.5 mL was added to each vial. The final mixture was incubated for 15 minutes at 22 degrees C and then stored at room temperature. Thin-layer chromatography was performed after zero, three, and six hours of storage to assess radiochemical purity. Five more vials were prepared as above, and five male volunteers were given 185 megabecquerels of the radiopharmaceutical by i.v. push, and scintigraphic images of the anterior chest were taken immediately and 1, 2, and 24 hours after injection. Immediately after preparation, a mean +/- S.D. of 99.0 +/- 1.0% of the 99mTc was bound to dextran 70. Mean +/- S.D. binding was 98.1 +/- 3.7% and 95.8 +/- 7.5% at three and six hours, respectively. Scintigraphy in the five volunteers yielded high-contrast images of the cardiac blood pool with little uptake of the radionuclide by the lungs. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531941 TI - Sterility of unit dose syringes of filgrastim and sargramostim. PMID- 7531942 TI - Child with oral, facial, digital, and skeletal anomalies and psychomotor delay: a new OFDS form? AB - We report on a male infant with oral, facial, digital, and skeletal anomalies in association with severe psychomotor delay. This may represent a new oral-facial digital syndrome. PMID- 7531943 TI - Iatrogenic gastroschisis in the treatment of diaphragmatic hernia. PMID- 7531944 TI - Palliative fetal surgery for diaphragmatic hernia. PMID- 7531945 TI - Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development. AB - Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role. PMID- 7531946 TI - A mucin-like glycoprotein identified by MAG (mouse ascites Golgi) antibodies. Menstrual cycle-dependent localization in human endometrium. AB - Human endometrial glands synthesize and secrete a high molecular weight mucin like glycoprotein in a menstrual cycle-dependent fashion. A novel moiety within this Golgi-associated glycoprotein is strongly reactive with IgG antibodies in numerous murine ascites, and has been termed MAG (mouse ascites Golgi). Immunohistochemical staining of 201 endometrial biopsies revealed the following patterns: MAG first appeared in the Golgi on cycle day 5, peaked on day 15, was present on the surface of the luminal epithelium between days 17 and 19, and was no longer detectable after day 19. MAG was also present in cervical, prostate, seminal vesicle, and lacrimal glands, pancreatic acinar cells, gall bladder and bile duct epithelium, and certain cells of the salivary and sweat glands. Interestingly, only tissues from blood group A individuals exhibited this staining. As a common link among all these cell types is the expression of mucins, we speculated that the MAG epitope could be a mucin-associated blood group A-related epitope. This hypothesis was tested by absorption experiments with a variety of glycoconjugates and erythrocytes and by immunoblots of MAG-rich material. The absorption studies demonstrated that only type III porcine mucin (< 1% sialic acid) and blood type A or AB erythrocytes were able to absorb the anti MAG antibody. Inasmuch as N-acetyl-galactosamine alone, the terminal blood group A carbohydrate, did not block MAG antibody binding, the MAG epitope appears to involve N-acetylgalactosamine plus other determinants. Immunoblots of endometrial extracts and saliva from blood type A individuals revealed MAG-reactive material with a molecular weight > 200 kd under reducing conditions. Because the MAG epitope appears on the endometrial surface during the purported implantation window, we speculate that mucin-like epitopes could play a role in the earliest apposition phases of conceptus-endometrial interaction. PMID- 7531947 TI - Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. AB - We studied the expression of the angiogenic factor vascular permeability factor) (VPF, also called vascular endothelial growth factor), in human melanoma cells in vitro and in vivo. Melanoma lines that develop tumors with a low metastatic potential in nude mice were found to have low expression levels of VPF in vitro, and the VPF expression levels in melanoma lines that yield highly metastatic xenografts were high. However, in vivo the correlation between VPF mRNA levels and the frequency of metastasis was lost; in all xenografts equally high levels of VPF mRNA were found, independent of the parental cell line. Hence, in vivo VPF gene expression was upregulated in the low expressing lines. The external factor responsible for this induction may be hypoxia, given that we found that low oxygen tension caused a (reversible) increase in the VPF mRNA levels in otherwise low expressing melanoma lines in vitro. A melanoma line with an inducible VPF expression was engineered into a line with a constitutive VPF expression. In the xenografts from this line a change in the vascular architecture was seen, indicating that the pattern or the level of VPF expression is important for tumor angiogenesis in melanoma xenografts. PMID- 7531951 TI - Nitric oxide synthase in the human pituitary gland. AB - Nitric oxide (NO) is generated by the NO synthase family of isozymes, which is present in many mammalian cells. The constitutive NO synthase isozymes generate NO, which acts via signal transduction mechanisms in the regulation of many functions including vascular tone and blood pressure, and the inducible isozymes mediate immunological mechanisms by cytotoxic and cytostatic effects. To determine whether NO has a role in anterior pituitary cell function, immunohistochemistry and in situ hybridization analyses were used to study NO synthase expression in normal and neoplastic human pituitary tissues. Brain NO synthase was localized in the anterior pituitary in secretory and in folliculo stellate cells and in the posterior pituitary. Pituitary adenomas had higher levels of brain NO synthase protein and mRNA compared with normal pituitaries. Endothelial NO synthase was also present in anterior and posterior pituitary cells and in endothelial cells of the pituitary. Immunoblotting studies with brain NO synthase antibodies detected a slowly migrating approximately 155-kd band and more rapidly migrating approximately 90-kd and approximately 60-kd bands. Endothelial NO synthase, but not macrophage NO synthase, was also detected in the pituitary by immunoblotting studies, confirming the immunohistochemical observations. These findings indicate that NO synthase is expressed in normal and neoplastic human pituitary tissues with increased levels of brain NO synthase protein and mRNA in adenomas compared with non-neoplastic pituitary cells and suggest that NO may play a regulatory role in hormone secretion in anterior pituitary cells. PMID- 7531956 TI - Treatment of intracranial alphavirus infections in mice by a combination of specific antibodies and an interferon inducer. AB - Finding an effective treatment for viral infections that cause encephalitis remains an important problem. A model of human alphavirus infections, Semliki Forest virus, causes lethal encephalitis in weanling mice. Mice are viremic within 24 hr of an intraperitoneal challenge with the equivalent of three 75% lethal doses of Semliki Forest virus. Virus reaches the brain by 48 hr, and mortality results in all mice in 5-7 days. Introduction of virus intracranially accelerates the course of the infection. Neither anti-Semliki Forest virus hyperimmune serum nor the potent interferon inducer poly I:CLC given intraperitoneally are protective when used therapeutically after an intracranial virus infection, but a combination of 1,000 U hyperimmune serum and 80 micrograms/mouse of poly I:CLC results in a 50% survival rate. This combination treatment of intracranial Semliki Forest virus infection eliminates detectable viremia and reduces virus load in the brain over the course of the infection. These data show that when combined, specific antibody and an interferon inducer can interact synergistically to protect mice from alphavirus infections of the central nervous system even when given after the virus is replicating in the target organ. PMID- 7531948 TI - In vitro effects of oxidized low density lipoprotein on CD11b/CD18 and L-selectin presentation on neutrophils and monocytes with relevance for the in vivo situation. AB - Oxidized LDL (oxLDL) has been identified as a potent stimulus of leukocyte adhesion to endothelium, a hallmark of early atherogenesis. A cytofluorometric study was performed to further characterize the mechanisms by which oxLDL stimulates the rapid adhesion of leukocytes to endothelium in vitro and in vivo. Incubation (30 minutes at 37 C) of whole blood (diluted with buffered saline to 1 x 10(6) leukocytes/ml) with oxLDL (0.85 mg LDL cholesterol/ml; oxidized by 7.5 mumol/L Cu2+ for 18 hours) but not native LDL stimulated the upregulation of CD11b/CD18 adhesion receptors on neutrophils (anti-leu-15 binding: 178 +/- 16% of baseline, P < 0.01, means +/- SD of n = 10 experiments) and on monocytes (169 +/- 34% of baseline, P < 0.01). This phenomenon was almost entirely inhibited by n butanol or the vasoactive drug pentoxifylline (PTX), which also significantly reduced oxLDL-induced leukocyte adhesion to venular and arteriolar endothelium, as assessed by intravital microscopy on the dorsal skinfold chamber in hamsters (venules: 49 +/- 19 versus 120 +/- 34 cells/mm2, P < 0.05; arterioles: 9 +/- 4 versus 52 +/- 7 cells/mm2, P < 0.01) 30 minutes after intravenous injection of oxLDL (4 mg/kg body weight; means +/- SD of n = 7 hamsters per group). Butanol and PTX also significantly reduced the upregulation of CD11b/CD18 by f-methionyl leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) but not by phorbol myristate acetate (PMA). Whereas fMLP and PAF stimulate leukocytes via binding to specific cell surface receptors and triggering complex signal transduction pathways, PMA bypasses these pathways and directly activates intracellular protein kinase C. By analogy, we propose that oxLDL upregulates CD11b/CD18 through its previously documented ability to stimulate the generation of second messengers. The effect of n-butanol and PTX on receptor presentation cannot be explained by changes in plasma membrane fluidity, as both agents failed to reverse the decrease in plasma membrane fluidity of neutrophils after stimulation with oxLDL, as assessed by fluorescence anisotropy measurement of the membrane marker diphenylhexatriene. Incubation of isolated neutrophils but not of whole blood with oxLDL resulted in a significant loss of L-selectin from the neutrophil surface (anti-TQ-1 binding: 40 +/- 13% of baseline, P < 0.01). A significant loss of this adhesion receptor on neutrophils and monocytes was also observed after stimulation of isolated neutrophils and whole blood with fMLP, PAF, and PMA.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7531950 TI - Detection of apoptosis and expression of apoptosis-related proteins during human intrahepatic bile duct development. AB - We investigated apoptosis by nick end labeling and the expression of apoptosis related proteins by immunohistochemistry in fetal development of human intrahepatic bile ducts and hepatocytes. During intrahepatic bile duct development, apoptosis was present at all stages, and its positive ratio was high in the remodeling ductal plate, moderate in the ductal plate, and relatively low in remodeled ducts. The cell proliferative activity as determined by proliferating cell nuclear antigen was also high in the remodeling ductal plate, and relatively low in the ductal plate and remodeled ducts. fas antigen and c-myc protein were constantly positive in the ductal plate, remodeling ductal plate and remodeled ducts. Bcl-2 protein was negative or faintly positive in the ductal plate and remodeling ductal plate, but was apparently positive in remodeled ducts. Lewisy as detected by the BM-1 antibody was present in the ductal plate, remodeling ductal plate, and remodeled ducts. p53 protein was not found in any cell types in the liver development. During hepatocyte development, many apoptotic and proliferating cell nuclear antigen-positive hepatocytes were noted. The developing hepatocytes expressed c-myc protein and fas antigen. Bcl-2 protein and Lewisy antigen were also weakly positive in the developing hepatocytes. These findings showed that balanced cell proliferation and apoptosis are involved in the normal development of intrahepatic bile ducts and hepatocytes, and suggest that c-myc protein, fas antigen, Bcl-2 protein, and Lewisy antigen modulate apoptosis of fetal intrahepatic biliary cells and hepatocytes, probably by stimulative (c-myc protein and fas and Lewisy antigens) or inhibitory (Bcl-2 protein) effects. PMID- 7531955 TI - Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets. AB - The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants. PMID- 7531953 TI - New characterization of infectious mononucleosis and a phenotypic comparison with Hodgkin's disease. AB - Recent nucleic acid hybridization studies have implied that Reed Sternberg/Hodgkin (RS/H) cells are infected with Epstein-Barr virus (EBV) before malignant transformation, and hence, that Hodgkin's disease could develop as a consequence of malignant transformation of an EBV-infected cell. This study is a detailed immunohistochemical and in situ hybridization characterization of the various lymphoid cells in nine cases of infectious mononucleosis (IM), the acute manifestation of EBV infection. The RS/H-like cells of IM were similar in most respects to their morphologically identical counterparts in Hodgkin's disease; they expressed the EBV-encoded protein LMP1, EBV EBER1 transcripts, and CD30 and rarely, if ever, expressed CD45/LCA or T cell markers. Dissimilarities were limited to CD15 negativity and the absence of a collarette of T cells around the RS/H-like cells of IM compared with their Hodgkin's counterparts. Expression of the immortalizing bcl-2 oncoprotein was variable in the RS/H-like cells of IM, as has been demonstrated in the RS/H cells of Hodgkin's disease by other investigators. An apoptosis assay suggested that many apoptotic cells in IM were EBV-infected T cells, in keeping with the previous in vitro observation that IM derived T cells succumb to apoptosis. Additionally, the apoptosis assay suggested that RS/H-like cells of IM can succumb to programmed cell death, reminiscent of the mummified RS/H cells seen in Hodgkin's disease. The accumulation of evidence suggests that RS/H-like cells of IM are more similar to true RS/H cells than previously recognized. PMID- 7531957 TI - Em18, a new serodiagnostic marker for differentiation of active and inactive cases of alveolar hydatid disease. AB - We determined whether detection of antibody response against a newly detected epitope, designated Em18, among Echinococcus multilocularis antigens could be a reliable marker for differentiation of active cases of alveolar hydatid disease (AHD) from inactive cases. Fifteen Alaskan patients with either active or inactive lesions of AHD previously confirmed clinically, pathologically, and serologically by the Em2-enzyme-linked immunosorbent assay (ELISA) were used for a blind test by Western blotting. Ten and five cases were considered to be active and inactive cases, respectively. One of the 10 cases classified serologically as active was judged to be inactive based on clinical and pathologic criteria; the patient had a recognizable parasitic lesion, and following short-term treatment with albendazole, a biopsy of the liver showed a degenerated lesion that did not grow in rodents. The five cases judged to be inactive included two confirmed inactive cases with cicatrized lesions and three active cases that showed the weakest values in the Em2-ELISA. The most predominant IgG subclass responding to Em18 was IgG4. In general, there were good correlations between 1) the antibody response against Em18 and the presence of active lesions and 2) the antibody response against Em18 and the Em2-ELISA values. PMID- 7531954 TI - Identification of tissue-infiltrating lymphocytes expressing PEN5, a mucin-like glycoprotein selectively expressed on natural killer cells. AB - PEN5 is a carbohydrate epitope selectively expressed on peripheral blood natural killer cells. We have used a monoclonal antibody reactive with PEN5 to survey the expression of PEN5+ large granule lymphocytes in a variety of human tissues. PEN5+ cells are scattered throughout lymphoid (eg, lymph node, tonsil, thymus, spleen, and intestine) and nonlymphoid (eg, liver, esophagus, lung, and uterus) organs. Due to their relatively abundant cytoplasm, these cells are somewhat larger than resting tissue lymphocytes. The majority of splenic (87 +/- 13%, n = 5) and hepatic (92 +/- 6%, n = 5) PEN5+ lymphocytes coexpress TIA-1, a cytotoxic granule protein found in natural killer cells. In some tissues (eg, tonsil and Peyer's patch), however, relatively few PEN5+ lymphocytes coexpress TIA-1, possibly reflecting different stages of activation or differentiation. Our results indicate that PEN5 may be a useful marker of tissue-infiltrating natural killer cells and reveal these cells to be surprisingly abundant in lymphoid tissues. PMID- 7531958 TI - A pilot study of the prevalence of hepatitis C virus antibodies and hepatitis C virus RNA in southern Cameroon. AB - Information is lacking on the prevalence of hepatitis C virus (HCV) infection in most African countries. An algorithm based on a combination of enzyme immunoassays (EIAs) with different formats (a commercial test, an HCV antibody [Ab] III test, and an HCV core Ab EIA) was used to estimate the prevalence of HCV infection in different population groups from southern Cameroon. An overall high prevalence was observed, with a significant increasing trend for both sexes with respect to age. A high proportion (67.4%) of HCV-positive sera were viremic as demonstrated by the reverse transcription-polymerase chain reaction. We conclude that the prevalence of HCV is high in southern Cameroon and increases linearly with age. PMID- 7531949 TI - Ovine lentivirus expression and disease. Virus replication, but not entry, is restricted to macrophages of specific tissues. AB - To better define the relationship between lentivirus infection and lymphoproliferative or inflammatory disease, we studied postmortem specimens of 38 sheep naturally infected with ovine lentivirus (OvLV) and with different clinical manifestations of OvLV-associated disease. Immunohistochemistry, in situ hybridization, and virus isolation were used to localize viral protein, viral RNA, and infectious virus to specific cells and tissues. Viral protein or infectious virus was found in cells morphologically and histochemically compatible with macrophages (M phi s), but only in lung, bone marrow, mammary gland, lymph node, spleen, synovium, brain, and spinal cord, frequently in association with lymphocyte infiltrates. In contrast, viral RNA was found in a variety of cell types, including epithelium, M phi s, and M phi-like cells, and in a wider range of tissues, with or without OvLV-associated lesions. In summary, these findings suggest that in vivo: 1), OvLV can enter a variety of cell types, 2), productive infection is restricted to cells of M phi lineage, and 3), cells expressing viral proteins are limited to specific tissues, those associated with OvLV-induced diseases. PMID- 7531959 TI - Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections. AB - We describe a generally applicable and easily reproducible method for the cloning of differentially expressed RNA, amplified from small numbers of enriched cell populations, obtained by microdissection from single cryostat sections. The procedure involves homopolymeric A tailing of cDNA synthesized from released RNA using an anchored (NN)T12 primer. Subsequent entire cDNA population polymerase chain reaction amplification was carried out using a biotinylated (X)nT16 primer adaptor in the presence of biotin-dATP. This biotinylated driver cDNA was then twice hybridized in 50-fold excess to heterologous target cDNA made with nonbiotinylated (Y)nT16 primer; common hybrids and excess driver cDNA were magnetically removed following the addition of streptavidin-coated magnetospheres which bound biotinylated strands, leaving enriched target population sequences. These were then directly amplified through the tails using a primer containing only the target-specific (Y)n sequence. Insertion into a lambda-phage vector was facilitated by means of an EcoR1 site incorporated in the (Y)n primer. Subsequent packaging and transformation into Escherichia coli NM522 resulted in cDNA libraries containing approximately 5 x 10(3)-10(4) pfu. Screening of these primary libraries with cDNA derived from the starting populations yielded a large number of differentially hybridizing clones which are currently under analysis. PMID- 7531960 TI - An improved method to do dot blot analysis. PMID- 7531961 TI - [Comparison of two serum alpha-fetoprotein assays: Amerlite AFP and AFP-2T]. PMID- 7531962 TI - Pancreatic injuries from blunt trauma. AB - Pancreatic injuries from blunt trauma are infrequent, and their diagnosis and management can be extremely difficult. Over the past 10 years we treated 13 patients with major pancreatic injuries from blunt trauma. Twelve had been involved in motor vehicle collisions, none of whom were wearing seat belts. One patient was injured in an assault. Only five patients had physical findings suggesting intra-abdominal injury. Serum amylase levels were elevated in seven of the nine patients (78%) in whom this test was obtained. Computed tomography scans demonstrated pancreatic injury in three of the four patients in whom it was performed. The five patients in whom peritoneal lavage was performed had free intraperitoneal blood from injuries to other abdominal viscera. One injury was diagnosed by ultrasound. Injuries were equally distributed throughout the pancreas, with two injuries in the head, three in the body, five in the tail, and three with injuries in both the head and the body. Five patients had ductal injuries. All patients underwent celiotomy. Eight patients required no operative management of their pancreatic injuries other than drainage. Injury Severity Score averaged 28.5 +/- 2.6 (mean +/- standard error), and mean hospital stay was 31 +/- 9.8 days. One patient with delayed diagnosis of a pancreatic injury developed a pseudocyst, but all other complications and prolonged hospitalizations were due to injuries to the head, chest, other abdominal organs, or extremities. All patients survived. The diagnosis of blunt pancreatic injury requires a high index of suspicion, and diagnostic studies may demonstrate only subtle signs of injury. Most injuries can be managed by localized resection and/or drainage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531965 TI - Intralesional bleomycin for acquired immunodeficiency syndrome-associated cutaneous Kaposi's sarcoma. PMID- 7531963 TI - [High-dose chemotherapy with peripheral blood stem cell transplantation as a consolidation therapy for testicular cancer with poor prognosis]. PMID- 7531964 TI - [GM-CSF.G-CSF]. AB - The basic characteristics of granulo-macrophage colony stimulating factor (GM CSF) and granulocyte colony stimulating factor (G-CSF) are reviewed. The structure of the proteins and genes have been clarified. The clinical use of these hematopoietic growth factors are also discussed. Diseases for which these growth factors will be effective are follows, the granulocytepenic state after anti-cancer chemotherapy, aplastic anemia, bone marrow transplantation and so on. As these growth factors increase the granulocyte function, these proteins will be effective on the immunodifective state. PMID- 7531968 TI - [The antiviral aerosol aprotinin. General effect on the body after inhalation administration]. AB - A new medicine for the treatment of respiratory viral infections is described. It contains aprotinin, a natural inhibitor of proteases, and is used in the form of a fine aerosol for inhalation or intra-respiratory instillation. To estimate its innocuousness, the inhalation effect of the aerosol was studied on animals of two species. The experiments included examination of the functional state of the central nervous and cardiovascular systems, determination of the blood cell composition and biochemical blood count, morphological and histological investigation of the internal organs. The toxicological studies showed that aprotinin inhalation induced no adverse reactions which made it possible to recommend the aprotinin inhalation for the use in the treatment and prophylaxis of infections caused by a wide variety of respiratory viruses in humans. PMID- 7531967 TI - Segmental occlusion of the pancreatic duct with prolamine to prevent fistula formation after distal pancreatectomy. AB - OBJECTIVE: The authors used prolamine (Ethibloc, Ethicon GmBH, Norderstedt, Germany) for segmental obstruction of the pancreatic duct to prevent pancreatic fistula development after distal pancreatectomy combined with total gastrectomy for gastric malignancies. SUMMARY BACKGROUND DATA: Although the initial clinical application of prolamine was pancreatic duct obstruction for patients with pancreatitis and undergoing pancreatic transplantation and pancreaticoduodenectomy for pancreatic cancer, there are no reports on prevention of pancreatic fistula formation after distal pancreatectomy. METHODS: Prolamine (0.2 mL) was injected into the distal segment of the main duct in the remaining pancreata of 51 patients. Small pancreatic ducts on the cut surface, from which prolamine extravasates, were closed by ligation, the main duct was ligated doubly, and the transected pancreatic margin was closed 15 minutes after phenylpropanolamine hydrochloride injection. RESULTS: No patient developed a pancreatic fistula or the complication of arterial bleeding due to prolonged infection. CONCLUSION: Segmental obstruction of the pancreatic duct with prolamine is useful for preventing pancreatic fistula development after distal pancreatectomy. PMID- 7531969 TI - Electrovesicography in benign prostatic hyperplasia. AB - The electric activity of the urinary bladder (UB) as demonstrated by electrovesicogram (EVG) was studied in 26 patients with benign prostatic hyperplasia (BPH) and in 10 healthy volunteers. Ten of the BPH patients were in the compensated stage and 16 were in the decompensated stage. Three electrodes were applied to the skin of the hypogastric area and one reference electrode was applied to the lower limb. Recordings were made from the full and empty UB and after the residual urine had been evacuated. In healthy volunteers, pacesetter potentials (PPs) were recorded as triphasic waves from the full and empty UB. Amplitudes were lower in the empty than in the full UB (p < .05), and were reproducible in the individual subject. The EVG of the compensated prostatic patients showed, in both the full and empty UB, PPs of higher frequency, amplitude, and velocity than that of the controls (p < .01 and p < .05, respectively), a condition called "tachyvesica." In the decompensated prostatic patients, the EVG showed a bradyarrhythmic pattern in the full UB and a silent pattern in the empty UB. It would appear that EVG recorded for BPH could be used to differentiate the compensated from the decompensated hyperplastic prostate. The technique is simple, easy, noninvasive, nonradiologic, and without complications when compared with other investigative methods. PMID- 7531952 TI - Angiogenic growth factors in neural embryogenesis and neoplasia. AB - "Blood vessels have the power to increase within themselves which is according to the necessity whether natural or diseased. As a further proof that this is a general principle, we find that all growing parts are much more vascular than those that are come to their full growth; because growth is an operation beyond the simple support of the part. This is the reason why young animals are more vascular than those that are full grown. This is not peculiar to the natural operation of growth, but applies also to disease and restoration." PMID- 7531971 TI - Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell. AB - The replication of a RNA template catalyzed by Q beta replicase was obtained in oleic acid/oleate vesicles simultaneously with the self-reproduction of the vesicles themselves. This was accomplished by entrapping the enzyme Q beta replicase, the RNA template, and the ribonucleotides ATP, CTP, GTP, and UTP inside the vesicles. The water-insoluble oleic anhydride was then added externally. It binds to the vesicle bilayer where it is catalytically hydrolyzed yielding the carboxylate surfactant in situ, which then brings about growth and reproduction of the vesicles themselves. This experiment is presented as a first approach to a synthetic minimal cell, in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously. PMID- 7531972 TI - RFLP analysis of human chromosome 11 region q13 in multiple symmetric lipomatosis and multiple endocrine neoplasia type 1-associated lipomas. AB - Six lipomas from patients affected by Multiple Symmetric Lipomatosis (MSL) and by Multiple Endocrine Neoplasia Type 1 (MEN 1) were analyzed for loss of heterozygosity on chromosome 11 region q12-13 using four RFLPs. Allelic loss for the D11S146 locus was found only in one visceral MEN 1-associated lipoma. Lipomas that exhibited a lack of allelic lesions were analyzed for an eventual abnormal amount or a defective function of the Gs protein by studying the Gs alpha subunit gene, codons 201 and 207, by PCR and TGGE techniques. All the samples were negative for activating mutations. PMID- 7531974 TI - Defective phospholipase D activation in Ki-ras-transformed NIH3T3 cells: evidence for downstream effector of PLC-gamma 1 in PDGF-mediated signal transduction. AB - Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts and many other cell types, was used to examine phosphatidylcholine-specific phospholipase D (PLD), phosphoinositide-specific phospholipase C (PI-PLC) and tyrosine phosphorylation in NIH3T3 fibroblast and its Ki-ras-transformed derivative, DT. When cells prelabeled with [3H] myristic acid were stimulated by 10 and 50 ng/ml of PDGF in presence of 0.3% butanol, formation of phosphatidylbutanol (PtdBut) was increased three to six fold in NIH3T3 fibroblasts whereas it was marginal in DT cells. Myo-[3H]inositol-labeled cells showed higher inositol phosphate production in nontransformed control fibroblasts, indicating higher phospholipase C activity compared to the transformed DT cells. PDGF caused increase in tyrosine phosphorylation of a group of proteins with 110-130 kDa, PLC-gamma 1 and PDGF receptor in NIH3T3 cells. There was no significant increase in tyrosine phosphorylation in both PDGF receptor and PLC-gamma 1 in DT cells. These results suggest that PLD acts as a downstream effector molecule of PLC-gamma 1 in the PDGF-mediated signal transduction pathway. PMID- 7531970 TI - [Palliative treatment of tetralogy of Fallot by percutaneous dilatation of the right ventricular outflow tract. 40 cases]. AB - This study was undertaken to assess the value of percutaneous dilatation of the right ventricular outflow tract as a substitute for surgical systemic pulmonary anastomosis in varieties of tetralogy of Fallot with severe irregularity or major pulmonary arterial hypoplasia unsuitable for complete repair of first intention. Fifteen neonates aged 3 to 23 days with severe desaturation (SaO2 = 73 +/- 11%) and twenty five children aged 1.2 to 174 months with anoxic crises or severe desaturation (SaO2 = 67 +/- 15%) fulfilled these criteria. They underwent right heart catheterisation completed by an attempted pulmonary dilatation. This manoeuvre failed in 6 cases, all neonates, because it was not possible to cross the annulus or maintain the balloon in position, or because of infundibular perforation. There were complications in 5 cases, all neonates: 2 tamponades with 1 death, one dissecting aneurysms of the annulus, 1 gastroenteritis and 1 caval thrombosis. Of the 34 patients dilated, 8 were poor results, mainly in the older age group (7 cases) and 26 were successful as judged by a significant improvement in arterial saturation. However, 7 patients rapidly deteriorated due to anoxic crises (6 infants and children) or fatal secondary tamponade (1 neonate). Therefore, there remained 19 stable successful procedures, 49% of attempts, representing 56% of successful attempts and 73% of primary successes. In neonates, these proportions were respectively: 47%, 78% and 87%, and in the older children: 48%, 48% and 63%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7531975 TI - Enhanced expression of inducible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: possible mediation via protein kinase C. AB - Increased blood flow and vascular permeability of early diabetes have been associated with increased nitric oxide formation in diabetic rats, but the specific nitric oxide synthase responsible is unknown. We examined the modulation of the induction and activity of the inducible NOS isoform by high glucose concentration in a murine macrophage cell line, RAW 264.7, and murine glomerular mesangial cells. Culturing both cell types in high glucose concentration led to significant increases in nitrite production and the mRNA encoding iNOS upon stimulation with LPS plus interferon-gamma, as compared with normal glucose concentration. High glucose also modestly enhanced LPS/IFN-gamma-induced stimulation of the iNOS promoter in transient transfection experiments in mesangial cells. Protein kinase C activation led to enhanced mRNA expression of iNOS, and inhibitors of protein kinase C blocked nitrite accumulation in mesangial cells. These findings suggest that high glucose in combination with stimulation by LPS plus IFN-gamma enhances iNOS expression, and protein kinase C activation may be playing a role in this enhancement. PMID- 7531973 TI - Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori. AB - Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates. PMID- 7531976 TI - Growth inhibitory effects of FK506 and cyclosporin A independent of inhibition of calcineurin. AB - The ability of the immunosuppressive agent FK506 to affect growth of the epidermal growth factor-receptor (EGF-R) overexpressing cell line, A431, was compared with that of the structurally unrelated immunosuppressive compound, cyclosporin A (CyA). Both were shown to inhibit growth, although neither of them caused down-regulation of the EGF-R or affected epidermal growth factor (EGF) induced tyrosine phosphorylation of the EGF-R. Inhibition of growth was not specific to EGF-R pathways, as both FK506 and CyA also inhibited EGF- and platelet-derived growth factor (PDGF)-induced DNA synthesis in fibroblasts. In all assays FK506 was less potent than CyA even though it is 10-100 times more potent as an immunosuppressive agent. The role of calcineurin in CyA- or FK506 induced growth inhibition was investigated using the synthetic pyrethroid insecticides: cypermethrin, deltamethrin and fenvalerate, which are known calcineurin inhibitors. Failure of these agents to block cell growth or influence growth factor-induced mitogenesis indicated that the biochemical pathway(s) by which CyA or FK506 inhibited cell growth did not depend solely on inhibition of calcineurin. PMID- 7531966 TI - Current management of pancreatic carcinoma. AB - OBJECTIVE: The author seeks to provide an update on the current management of pancreatic carcinoma, including diagnosis and staging, surgical resection and adjuvant therapy for curative intent, and palliation. SUMMARY BACKGROUND DATA: During the 1960s and 1970s, the operative mortality and long-term survival after pancreaticoduodenectomy for pancreatic carcinoma was so poor that some authors advocated abandoning the procedure. Several recent series have reported a marked improvement in perioperative results with 5-year survival in excess of 20%. Significant advances also have been made in areas of preoperative evaluation and palliation for advanced disease. CONCLUSION: Although carcinoma of the pancreas remains a disease with a poor prognosis, advances in the last decade have led to improvements in the overall management of this disease. Resection for curative intent currently should be accomplished with minimal perioperative mortality. Surgical palliation also may provide the optimal management of selected patients. PMID- 7531977 TI - Gene expression for interleukin-2 and tumor necrosis factor-alpha in the spleen of old rats under physiological condition and during septic shock. Possible pharmacological modulation. AB - Older individuals are more susceptible to infectious agents than younger and this is related to the disrepair of the immune defence mechanisms associated with aging. In this study we evaluated the activity of a new biological response modifier (BRM), pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl)carbonyl] thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6) in relation to the expression of some cytokine genes. We utilized 24 month-old Sprague-Dawley rats (n = 24), randomly divided into 4 groups: controls (n = 6), pidotimod-treated (n = 6; 200 mg/kg i.p., for 10 days), infected (n = 6; i.p. infection of E. coli CH 198) and pidotimod-treated + infected (n = 6). Poly(A+)RNA purified from the spleens of the animals killed 48 h after the infection was probed with Interleukin-2 (IL-2) and Tumor Necrosis Factor-alpha (TNF-alpha) cDNA clones. Northern blot analysis showed a slight signal of the IL-2 steady state mRNA in the groups of control, pidotimod-treated and infected animals, with an increase (20%) evident only in pidotimod + infected rats, 48 h after E. coli injection. On the contrary, the TNF-alpha mRNA levels were easily detectable in controls and infected rats and lower (20%, 40%) following the drug treatment, independent of i.p. infection. These results account for the BRM activity of pidotimod. PMID- 7531978 TI - Distribution of D1- and D2-dopamine receptors, and dopamine and its metabolites in the human brain. AB - Densities and distribution of D1-dopamine and D2-dopamine receptors were investigated in vitro using [3H]SCH 23390 and [3H]raclopride in receptor binding assays and autoradiography on human post mortem whole hemisphere slices to serve as anatomical correlates to PET studies using [11C]SCH 23390 and [11C]raclopride. In addition, the levels of dopamine and its metabolites were determined by HPLC in various brain regions. Both dopamine receptor subtypes, as well as dopamine, HVA and DOPAC, were primarily found in the basal ganglia. Very high densities of D1-dopamine receptors were found particularly in the medial caudate nucleus, whereas D2-dopamine receptors were evenly distributed throughout the caudate. The densities of D1- and D2-dopamine receptors were similar in the caudate nucleus and the putamen, whereas there were 4 to 7 times higher densities of the D1- than of the D2-dopamine receptors in several limbic and neocortical regions. The receptor distribution in the autoradiographic study was consistent with that demonstrated in the living human brain using [11C]SCH 23390 and [11C]raclopride. PMID- 7531980 TI - Similar effects of glycine, zinc and an oxidizing agent on [3H]dizocilpine binding to the N-methyl-D-aspartate receptor in neocortical tissue from suicide victims and controls. AB - This study used [3H]dizocilpine (MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine glycine, redox and zinc modulatory sites in membranes derived from the frontal and parietal cortex of control subjects (n = 8) and suicide victims (n = 6). [3H]dizocilpine binding in the presence of glutamate and glutamate plus glycine was similar in control and suicide subjects. The sulphydryl redox site was assessed using the oxidizing agent 5,5'-dithio-bis (2 nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. Both redox and zinc sites were unaffected in the frontal and parietal cortex of suicide victims. These data indicate that the NMDA receptor and its glycine, redox and zinc subsites are preserved in the neocortex of suicide victims. PMID- 7531981 TI - Development of pre-implantation mouse embryos under the influence of pineal indoles. AB - The developmental toxicity of pineal hormones on mouse embryos was examined both in vitro and in vivo. Pregnant ICR mice were divided into groups which received at 1.5 days post-coitum (p.c.) and again at 2.5 days p.c. a subcutaneous injection of one of the following pineal indoles: hydroxyindoleacetic acid (HIAA), melatonin (MEL), methoxytryptophol (MTP) or methoxytryptamine (MTA). Mice treated with the injection vehicle served as the control. The animals were sacrificed at 17.5 days p.c. The pineal indole treatment did not cause changes in the gravid uterine weight, numbers of implants, early resorption, late resorption, dead fetuses and live fetuses, fetal weight or fetal crown-rump length, and did not produce embryos with external or visceral defects. However, some mice treated with MTP or MTA produced litters in which all embryos underwent resorption. Cultured embryos at the 4-cell stage were treated with the aforementioned pineal indoles and examined after 24, 48, 72 and 96 hours. It was found that MTA retarded embryonic development at all time points studied. HIAA also produced a slight inhibitory effect on embryonic development. Some embryos underwent degeneration in response to the MTA and HIAA treatments. However, MEL- and MTP-treated embryos were in general developmentally similar to control embryos. When cultured embryos were treated at the 8-cell to compacting stage, it was found that MTA exerted only a slight retarding effect on embryonic development, while other indoles were devoid of any conspicuous effects. PMID- 7531979 TI - Immunosuppression in insulin-dependent diabetes mellitus: from cellular selectivity towards autoantigen specificity. PMID- 7531982 TI - Inhibitory effects of salmon calcitonin on the tail-biting and scratching behavior induced by substance P and three excitatory amino acids. AB - We have examined the effects of salmon calcitonin (SCT), injected into the cerebral ventricle (i.c.v.), on the tail-biting and scratching behavior induced by the intrathecal injection of different types of nociceptive agents, i.e., substance P, N-methyl-D-aspartate (NMDA), kainate (KA), and quisqualate (Quis). Tail-biting and scratching behavior induced by the 4 substances was significantly inhibited by SCT (i.c.v.) in the same manner: the dose-response curves were U shaped, and the most effective dose was 0.1 IU/mouse in all cases. SCT did not, however, completely inhibit tail-biting and scratching behavior. At its most effective dose, the percent inhibition of substance P-, NMDA-, KA- and Quis induced behavior were 77.9%, 40.2%, 49.4%, and 52.9%, respectively. These results suggest that SCT has the inhibitory effects of substance P- and glutamate receptor agonists-induced nociceptive response in vivo. PMID- 7531983 TI - Evaluating the empirical support for the Geschwind-Behan-Galaburda model of cerebral lateralization. AB - The Geschwind-Behan-Galaburda (GBG) model of cerebral lateralization provides a complex but testable theory of the origins and associates of cerebral lateralization. An overall evaluation of the model suggest that it is not well supported by empirical evidence and that in the case of several key theoretical areas, the evidence that does exist is inconsistent with the theory. In particular: the concept of "anomalous dominance" is shown to be theoretically and methodologically flawed; a meta-analysis of the relationship between handedness and immune disorders finds a marginal overall association, and while three conditions (allergies, asthma, and ulcerative colitis) do show significant associations with left-handedness, two other conditions (myasthenia gravis and arthritis) show significant negative associations with left-handedness. Finally, a review of the origins of the neural crest, and its associations, suggests there is almost no empirical support for the GBG theoretical model in this area. PMID- 7531984 TI - Assessing the legacy of the GBG model. AB - A critique of the article by Bryden, McManus, and Bulman-Fleming ("Evaluating the empirical support for the Geschwind-Behan-Galaburda model of cerebral lateralization") is presented. Bryden et al. expose some of the principal weaknesses of the cerebral lateralization model put forth by Geschwind and colleagues and contribute an important meta-analysis of findings concerning the model's predicted relationship between immune disorders and handedness. Nonetheless, the theory put forth by Geschwind and colleagues retains an important legacy despite a multitude of faulty assumptions and predictive failures. Also offered is a brief synopsis of a new model that explains the many positive relationships found among various neurodevelopmental disorders and medical conditions, as well as why abnormal handedness distributions occur in some but not other neurodevelopmental disorders. PMID- 7531985 TI - The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. AB - Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing. PMID- 7531986 TI - Laminin and fibronectin guideposts signal sustained but opposite effects to passing growth cones. AB - Guidepost cells are known to alter the behavior of growth cones in vivo, yet the nature of communication and the type of signals employed are largely undefined. The present study demonstrates that model guideposts, composed of a single molecular species, are sufficient to change the navigation and the behavior of advancing growth cones well beyond the time of contact. Laminin on model guideposts caused a sustained increase in growth cone velocity, whereas fibronectin led to a sustained decrease. A spatially discrete array of multiple laminin-model guideposts maintained increased growth rates on fibronectin, as expected for homogeneous laminin, and also provided unambiguous directional guidance information. Laminin-evoked growth cone responses required activation of protein kinase C-dependent intracellular signalling mechanisms. PMID- 7531987 TI - Modulation of calcium currents by a D1 dopaminergic protein kinase/phosphatase cascade in rat neostriatal neurons. AB - In rat neostriatal neurons, D1 dopamine receptors regulate the activity of cyclic AMP-dependent protein kinase (PKA) and protein phosphatase 1 (PP1). The influence of these signaling elements on high voltage-activated (HVA) calcium currents was studied using whole-cell voltage-clamp techniques. The application of D1 agonists or cyclic AMP analogs reversibly reduced N- and P-type Ca2+ currents. Inhibition of PKA antagonized this modulation, as did inhibition of PP1, suggesting that the D1 effect was mediated by a PKA enhancement of PP1 activity directed toward Ca2+ channels. In a subset of neurons, D1 receptor-mediated activation of PKA enhanced L-type currents. The differential regulation of HVA currents by the D1 pathway helps to explain the diversity of effects this pathway has on synaptic integration and plasticity in medium spiny neurons. PMID- 7531988 TI - Receptor insertion into factor-dependent murine cell lines to develop specific bioassays for murine G-CSF and M-CSF and human GM-CSF. AB - cDNAs encoding the receptors for murine G-CSF, M-CSF or human GM-CSF were inserted into the murine hemopoietic continuous cell lines Ba/F3 or FDC-P1 and sublines selected that were then responsive to proliferative stimulation by these growth factors. When used in microwell assays the Ba/F3 G-CSF receptor-expressing cell line was able to detect 100 pg G-CSF per ml, the Ba/F3 M-CSF receptor expressing cell line 100-400 pg M-CSF per ml and the FDC-P1 line expressing the alpha- and beta-chains of the human GM-CSF receptor detected 5-10 pg/ml of GM-CSF in test material. These cell lines appear satisfactory for use as selective bioassays for these colony stimulating factors in material potentially containing a mixture of growth factors. PMID- 7531990 TI - Hexokinase autophosphorylation: identification of a new dual specificity protein kinase. AB - Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of other protein substrates. The amino acid specificity of rat brain autophosphorylation was analyzed with monoclonal antibodies directed against phosphotyrosine and by acid hydrolysis of the phosphorylated enzyme. The results show that serine, threonine, and tyrosine residues are phosphorylated after incubation with ATP. The stoichiometry of this phosphorylation was 0.2 mole phosphate per mole hexokinase after 30 min of incubation. Evaluation of freshly isolated HK1 with monoclonal anti phosphotyrosine antibody indicates that the enzyme is phosphorylated at a basal level in its native state. We concluded that rat brain HK1 is a dual specificity protein kinase that is phosphorylated physiologically. PMID- 7531989 TI - Methylated purine bases in hepatic and colonic RNA of rats treated with 1,2 dimethylhydrazine. AB - Administration of 1,2-dimethylhydrazine (DMH) to rats produces colon cancer. The mechanism by which this agent induces colon cancer is unclear. This investigation was conducted to assess the effect of DMH on the hepatic RNA and the colonic RNA of rats. DMH (300 mg/kg body wt) was administered to rats by ip injections. After 24 h, the hepatic RNA and the colonic RNA were isolated and their component purine bases were analyzed by HPLC. DMH treatment resulted in the formation of 1 methyladenine, 1-methylguanine, N2-methylguanine, O6-methylguanine, and 7 methylguanine in RNA. These methylated products may play a role in cellular injury produced by DMH. PMID- 7531991 TI - Colony-stimulating factor-producing tumours: production of granulocyte colony stimulating factor and interleukin-6 is secondary to interleukin-1 production. AB - We have reported that tumours producing colony-stimulating factor (CSF) also secrete interleukin-1 (IL-1) and IL-6. In the present study, we characterised the role played by IL-1 in CSF production. IL-1 receptor antagonist inhibited the production of granulocyte (G)-CSF and IL-6 by approximately 90% in CSF-producing human lung carcinoma cell lines. Similar results were obtained with hydrocortisone, which suppresses IL-1 gene expression. In contrast, 15 non-CSF producing human lung carcinoma cell lines did not show detectable IL-1 production, although seven of them were induced to produce G-CSF and IL-6 by exogenous IL-1 alpha. Cell lines that responded to IL-1 alpha, including the CSF producing lines, expressed receptors for IL-1 alpha. These results indicate that CSF-producing tumours can be characterised by their constitutive IL-1 production, IL-1 receptor expression and IL-1-dependent excess production of G-CSF and IL-6. Furthermore, transcription factor(s) may be involved in the abnormal IL-1 alpha production. PMID- 7531992 TI - The relative roles of vitronectin receptor, E-selectin and alpha 4 beta 1 in cancer cell adhesion to interleukin-1-treated endothelial cells. AB - Adhesion of cancer cells to endothelium is thought to be a prerequisite to extravasation during the haematogenous phase of metastasis, and is enhanced after perturbation of the endothelium by interleukin-1 (IL-1). The inducible endothelial adhesion molecules, E-selectin, VCAM-1/alpha 4 beta 1 and vitronectin receptor have been reported to mediate attachment of cancer cells to IL-1-treated endothelial cells. We have examined the relative contribution of these molecules by quantifying the adhesion of a panel of 22 human, 125I-labelled cancer cells and the rat W256 tumour to untreated and IL-1-treated endothelial monolayers in the presence of relevant neutralising antibodies. Antibodies against E-selectin inhibited the adhesion of HL-60 leukaemia cells and two colon carcinomas. Anti alpha 4 beta 1 antibodies blocked adhesion of four melanomas, five sarcomas and one lung carcinoma. Anti-vitronectin receptor antibodies inhibited adhesion of 14 of the 22 human cell lines to IL-1-treated endothelial cells. Adhesion of seven cell lines was inhibited by more than a single antibody. In contrast, adhesion of one of the cancer cell lines was unaffected by any of the antibodies, suggesting involvement of other IL-1-inducible endothelial adhesion molecules. Moreover, none of the antibodies altered the attachment of cancer cells to unstimulated endothelial monolayers. We conclude that the mechanisms of cancer cell adhesion to the endothelium are influenced by endothelial activation and by the adhesive repertoire of the cancer cell. PMID- 7531994 TI - Interactions between adenylyl cyclase, CAP and RAS from Saccharomyces cerevisiae. AB - The adenylyl cyclase complex from Saccharomyces cerevisiae contains at least two subunits, a catalytic subunit of M(r) 200,000, encoded by CYR1 and a cyclase associated subunit, of apparent M(r) 70,000, encoded by CAP. The complex is a major effector of RAS proteins in S. cerevisiae. The interactions between CAP, adenylyl cyclase and RAS were explored in a strain of yeast that lacked CAP and contained an epitope tagged adenylyl cyclase. Adenylyl cyclase activity in this strain was not immunoprecipitated with anti-CAP antibodies, but was immunoprecipitated with anti-epitope antibodies. Two anti-CAP polyclonal antisera and five anti-CAP monoclonal antibodies were used in these studies. Like CAP bound adenylyl cyclase, the CAP-free adenylyl cyclase was fully activated by yeast RAS2. Transformation of cap strains with plasmids expressing portions of CAP allowed the adenylyl cyclase binding sites on CAP to be mapped by immunoprecipitation experiments. In other experiments, deletion mutations of adenylyl cyclase were used to map the CAP binding site on adenylyl cyclase. The adenylyl cyclase binding site localized to the amino one third of CAP (amino acids 1-168), and the CAP binding site localized to the carboxyl terminus of adenylyl cyclase (amino acids 1768-2026). PMID- 7531993 TI - Inhibition of platelet aggregation by transdermal glyceryl trinitrate. AB - OBJECTIVE: To determine the optimum conditions for the demonstration of an antiplatelet effect of nitric oxide and to use these conditions to elucidate the effects of a transdermal glyceryl trinitrate patch on platelet aggregation in normal volunteers. METHODS: An open prospective crossover study. The effects of nitric oxide on platelet aggregation in whole blood and platelet rich plasma as stimulated by adenosine diphosphate and U46619 was assessed in the presence and absence of iloprost and MB22948. Optimum conditions for the demonstration of an antiplatelet effect of nitric oxide were then applied to whole blood from normal volunteers in the presence and absence of a transdermal glyceryl trinitrate patch. SETTING: University hospital. SUBJECTS: Eight normal volunteers. MAIN OUTCOME MEASURES: Platelet aggregation in the presence and absence of transdermal glyceryl trinitrate. RESULTS: The optimum conditions for the demonstration of an antiplatelet effect of nitric oxide in whole blood were collecting blood into a tube containing MB22948 and citrate and stimulating platelet aggregation with adenosine diphosphate in the presence or absence of iloprost. Using this method a significant effect of transdermal glyceryl trinitrate on platelet aggregation was shown (P < 0.03) in the presence and absence of iloprost. CONCLUSIONS: These results are consistent with an inhibitory effect on platelet aggregation of nitric oxide liberated by transdermal glyceryl trinitrate. Optimum test conditions are needed to show this effect. The clinical significance of the antiplatelet effect of transdermal glyceryl trinitrate is unknown. PMID- 7531995 TI - Box I and II motif from myelin basic protein gene promoter binds to nuclear proteins from rodent brain. AB - The box I and II motif located within the promoter of the myelin basic protein gene contains a simian virus 40 T-antigen-binding site, a MyoD/E2a-binding site, and a glucocorticoid receptor-binding site. We have found proteins within nuclear extracts from adult mouse brain, rat embryonic cerebral cortex in culture, and a mouse oligodendrocyte-like cell line that bind to a 32P-labeled synthetic DNA fragment containing the sequences of the box I and II motif. Three major complexes (A, B, and C) were seen in gel-shift assays. Only complexes A and B were competed out by the unlabeled box I and II fragment or by another synthetic DNA fragment that also contains sequences similar to a glucocorticoid receptor binding site. Therefore, complexes A and B were thought to be specific. The expression pattern of the proteins responsible for the formation of these complexes was also assessed during development in mouse brain. PMID- 7531996 TI - Small-subunit rRNA sequences and whole DNA relatedness concur for the reassignment of Pasteurella piscicida (Snieszko et al.) Janssen and Surgalla to the genus Photobacterium as Photobacterium damsela subsp. piscicida comb. nov. AB - The taxonomic status of Pasteurella piscicida (strain NCIMB 2058T [T = type strain] and a strain isolated from the environment) was investigated by performing phylogenetic analyses of small-subunit rRNA sequences, DNA-DNA hybridization analyses, and biochemical characterization analyses. The results of the phylogenetic analyses and the levels of DNA-DNA complementarity demonstrated conclusively that Pasteurella piscicida is extremely closely related to Photobacterium damsela ATCC 33539T. Since the two taxa exhibited a level of DNA DNA relatedness of 80%, they are members of the same species. The high level of DNA relatedness and the presence of specific morphological and biochemical characteristics support the hypothesis that two subspecies should be recognized. On the basis of its phylogenetic position, we concluded that Pasteurella piscicida should be renamed Photobacterium damsela subsp. piscicida comb. nov. PMID- 7531997 TI - Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis). AB - To evaluate the role of filaggrin in keratin filament aggregation in epidermolytic ichthyosis (epidermolytic hyperkeratosis, EH), we studied EH skin by light and electron microscopic immunohistochemistry, and biochemical analysis using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting. Immunohistochemical staining showed an increased number of filaggrin-immunoreactive cell layers, but the reaction was still confined to the mid- and upper epidermal layers, whereas an abnormal granular pattern of staining for K10 began in the lower suprabasal cell layers. This suggests that the aggregation of keratin filaments precedes, and occurs independently of, profilaggrin synthesis during epidermal differentiation. Although keratohyalin granules were frequently associated with clumped filaments, immunoelectron microscopy showed that K10 labelling was confined to keratin filaments (including clumped filaments), and that antifilaggrin antibodies stained only keratohyalin granules, at least in the living cells. Certain keratin aggregates in the cornified cells were still devoid of filaggrin staining. However, in some cells which appeared partially cornified, filaggrin immunoreactivity occurred over the aggregated keratin filaments. Immunoblotting showed a clear increase of filaggrin/profilaggrin expression, without evidence for a qualitative abnormality. It seems unlikely, therefore, that filaggrin is primarily involved in the keratin filament clumping in EH, but that in some EH cases it interacts with keratins in a defective manner, possibly due to premature cell death and profilaggrin processing and/or altered keratin filament structure involving the interaction points of keratin with filaggrin. PMID- 7531998 TI - In cutaneous T-cell lymphoma, class II MHC molecules on CD1+ antigen-presenting cells are upregulated in involved compared with uninvolved epidermis. AB - CD1+ antigen-presenting cells in involved epidermis of patients with cutaneous T cell lymphoma exhibit and enhanced functional capacity to activate autologous CD4+ T cells compared with CD1+ antigen-presenting cells from uninvolved and normal epidermis. Class II major histocompatibility complex molecules are involved in antigen presentation, and their expression on CD1+ Langerhans cells is known to vary. The expression of all three class II (HLA-DR, -DQ, -DP) molecules was therefore determined on CD1+ epidermal cells from both involved and uninvolved epidermis, using flow cytometry. The involved CD1+ epidermal cells exhibited a 1.5-1.6-fold, statistically significant increase in fluorescence intensity after staining of the class II molecules (HLA-DR, -DQ, -DP) compared with CD1+ epidermal cells from uninvolved epidermis. The autologous CD4+ T cells, activation was almost completely blocked by anti-HLA-DR, and partly by anti-HLA DQ and anti-HLA-DP. In contrast, an antibody against class I, and an irrelevant control antibody, had no blocking effect. In a pokeweed mitogen assay it was demonstrated that autologous CD4+ T cells, activated by involved epidermal cells, demonstrated suppressor activity rather than helper activity. The suppressor activity was dependent on the presence of HLA-DR-positive epidermal cells. Thus, in cutaneous T-cell lymphoma, class II molecules on the individual CD1+ antigen presenting cell are upregulated in clinically involved compared with uninvolved epidermis, and these molecules are crucially involved in activation of CD4+ T cells. PMID- 7532000 TI - Immunohistochemical analysis of cytokeratin expression in eccrine spiradenoma: similarities to the transitional portions between secretory segments and coiled ducts of eccrine glands. AB - Despite light and electron microscopic and histochemical studies, there is no consensus on the cellular differentiation of eccrine spiradenoma. In the present study, eight specimens of eccrine spiradenoma were analysed by immunohistochemical techniques, using a panel of monoclonal antibodies against cytokeratins. Two types of epithelial cells were identified in tumour nodules: large, pale epithelial cells in the centre, and small, dark epithelial cells situated at the periphery. These nodules frequently contained tubular structures lined by cuboidal, columnar or, less commonly, flattened epithelial cells. Cytokeratin expression in eccrine spiradenoma was compared with expression in normal eccrine glands. Immunohistochemistry revealed that large, pale epithelial cells expressed immunophenotypes similar to those of luminal cells in the transitional portions between the secretory portions and the coiled ducts. The small, dark cells expressed immunophenotypes similar to those of basal cells in the transitional portions. Tubular structures observed in eccrine spiradenoma showed staining patterns similar to those of the luminal cells in the transitional portions. Eccrine spiradenoma may, therefore, differentiate towards the transitional portions between the secretory portions and coiled ducts of eccrine glands. Some of the large, pale epithelial cells in eccrine spiradenoma differentiate towards tubular structures, forming a lumen lined by a cuticle. PMID- 7531999 TI - Evidence that regression in keratoacanthoma is immunologically mediated: a comparison with squamous cell carcinoma. AB - Recent research observations suggest that the keratoacanthoma (KA) is a form of resolving squamous cell carcinoma (SCC). The mechanism by which this resolution takes place has not been fully explored, although it may have an immunological basis. To investigate this, we compared 15 clinically and histologically diagnosed KAs and 15 SCCs with regard to cellular infiltrate and keratin expression. We found that KAs have significantly higher numbers of CD3+ and CD4+ cells invading their epidermal component than SCCs. The T lymphocytes infiltrating KAs were more immunologically active, as greater numbers expressed the interleukin-2 receptor (IL-2R) than those in SCCs. It is of interest that CD36 was expressed by a significantly greater proportion of tumour cells within KAs than SCCs. This was also the case for the intercellular adhesion molecule ICAM-1, and the differentiation marker keratin 10. Overall, these findings suggest that KA regression is immunologically mediated, with activated (IL-2R+) CD4+ T lymphocytes and adhesion molecules playing a pivotal role in the immune response. PMID- 7532001 TI - Investigations of the 'active' edge of plaque psoriasis: vascular proliferation precedes changes in epidermal keratin. AB - We have investigated markers of epidermal proliferation and differentiation in terms of keratin expression, the morphology of the cutaneous vasculature, and numbers of cutaneous mast cells, in patients with chronic plaque psoriasis. Using the phenomenon of the 'active edge', we have studied these features in the psoriatic plaque itself, and in the clinically normal active and inactive edges of the same plaque. Our results confirm the anticipated changes in keratin profiles, mast cell numbers and psoriatic morphology of the vasculature within the plaque itself. They further indicate that the vascular changes precede the epidermal and mast cell features at the active edge, and that the inactive edge is inactive for all of these variables. Mediators responsible for the vascular proliferation and elongation must be present in increased amounts at the active edge when compared with the inactive, and include locally produced and circulating factors. PMID- 7532002 TI - 1 alpha, 25-Dihydroxyvitamin D3 decreased ICAM-1 and ELAM-1 expressions on pulmonary microvascular endothelial cells and neutrophil motivation. AB - Preincubation of pulmonary microvascular endothelial cells (PMVECs) with platelet activating factor (PAF) for 3.5 h increased the adhesion rate of polymorphonuclear leukocytes (PMNs) to PMVECs from 57.3 to 72.8% (P < 0.01). Coincubations of 1 alpha, 25-dihydroxyvitamin D3 (VD) or MC903 (VD analog) decreased the ability of PAF to increase PMN-PMVEC adhesion rate significantly. Preincubation of PMNs with PAF also increased PMN-PMVEC adhesion rate. Coincubation of VD but not MC903 blocked PAF-induced adherence of PMNs to PMVECs. Both VD and MC903 decreased PAF-increased expression of ICAM-1 on PMVECs, PMN chemotaxis to zymosan activated serum and histamine, and PMN aggregation induced by PAF significantly. VD but not MC903 decreased the expression of ELAM-1 on PMVECs. VD and MC903 did not affect PAF-induced release of acid phosphatase from PMNs. The results suggest that VD may be a good drug to inhibit PMN motivation by acting on both ECs and PMNs. PMID- 7532005 TI - Investigation of the mechanism of phosphoribosylamine transfer from glutamine phosphoribosylpyrophosphate amidotransferase to glycinamide ribonucleotide synthetase. AB - Phosphoribosylamine (PRA) is a product of glutamine phosphoribosylpyrophosphate amidotransferase (PRPP-AT) and a substrate for glycinamide ribonucleotide synthetase (GAR-syn), the first two enzymes in the de novo purine biosynthetic pathway. PRA has a half-life of 5 s under physiological conditions, hydrolyzing to ribose 5-phosphate. The instability of this purine precursor brings to question how the efficiency of transfer from one active site to the next is ensured: Is PRA transferred by free diffusion, or is it transferred directly from one enzyme to the next through a process defined as substrate channeling? Kinetic investigations of reactions containing both enzymes monitoring the appearance of the intermediate PRA and/or the product GAR were performed and compared with the predicted kinetics assuming a free diffusion mechanism of transfer. A significant discrepancy exists between the free diffusion model and the experimental data when the ratios of the two enzymes are varied. To accommodate this discrepancy, a direct transfer mechanism is proposed that is facilitated by protein-protein interactions. Experiments to provide evidence for these stable protein-protein interactions including gel chromatography, fluorescence spectroscopy, chemical cross-linking, and affinity gel chromatography; however, have all been unsuccessful. These results suggest that the requisite channeling interaction between PRPP-AT and GAR-syn, which is indicated by the kinetic results, must be a transient one. PMID- 7532003 TI - Solution structure of the human pp60c-src SH2 domain complexed with a phosphorylated tyrosine pentapeptide. AB - Human pp60c-src is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-p YEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 A for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the non-hydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/ 0.04 A for the N, C alpha, C' and 0.73 +/- 0.08 A for all the non-hydrogen atoms. The angular order parameter calculated for the phi, psi angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel beta-strands that traverse a compact core with an alpha-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532004 TI - Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels. AB - CHIP28 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP28 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP28. In purified native CHIP28 from erythrocytes, approximately 50% of CHIP28 molecules were glycosylated; each mole of glycosylated CHIP28 contained 5.4 kDa of monosaccharides consisting of 2 mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl beta D-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP28 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP28 eluted as an apparent dimer, whereas deglycosylated CHIP28 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP28 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.2 x 10(-14) cm3/s for native CHIP28. Circular dichroism of native and deglycosylated CHIP28 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP28 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532006 TI - Mechanism of inhibition of HIV reverse transcriptase by toxiusol, a novel general inhibitor of retroviral and cellular DNA polymerases. AB - Toxiusol, a natural product isolated from the Red Sea sponge Toxiclona toxius, has been shown to be a potent inhibitor of various viral reverse transcriptases (RT) [i.e., of human immunodeficiency virus (HIV-1), equine infectious anemia virus, and murine leukemia virus] and cellular DNA polymerases (i.e., of DNA polymerases alpha and beta and Escherichia coli DNA polymerase I). A thorough investigation of the mode of inhibition was conducted with HIV-1 RT-associated DNA polymerase activity. The inhibition is unaffected by the nature of template primer used. The inhibitory active site of toxiusol is attributable to the polar moieties at the benzene ring. The presence of either sulfate groups in the natural lead compound or hydroxyl groups in the corresponding hydroquinone is critical, because both compounds are equally effective at low micromolar concentrations. Conversely, the presence of acetyl groups in the same position in the derivative toxiusol diacetate lowers significantly or abolishes the inhibitory activity. Toxiusol binds the HIV-1 RT irreversibly and in a noncompetitive way with high affinity (Ki = 1.2 microM), probably through polar groups. The replacement with acetyl moieties in the analog toxiusol diacetate hampers the binding of the inhibitor to the enzyme (Ki increases to about 26 microM). Still, the compound binds irreversibly, probably through its hydrophobic structure skeleton. Toxiusol diacetate loses its ability to inhibit the first step in the DNA polymerization process (that is, the formation of the DNA-enzyme complex as measured by a gel retardation assay), which contributes to its poor inhibitory capacity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532007 TI - Molecular interactions of 3',5'-cyclic purine analogues with the binding site of retinal rod ion channels. AB - Photoreceptor outer segments transduce information about incoming light levels through a class of ion channels that respond directly to changes in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C8 positions was used to examine molecular interactions between the nucleotide and the channel. The maximal current activated by C2-altered analogues in excised membrane patches was less than the current activated by cGMP, and the K0.5, the concentration which activates 50% of the current in a patch, was increased. Nonpolar C8-substituted cAMP analogues activated more current than the parent cAMP with lower K0.5 values. This was in contrast to 8-amino-cAMP, which exhibited greatly reduced activity. The rank order of activity, based on K0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-benzylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N6-Etheno-cAMP and N6-monobutyryl-cAMP activated a small fraction of the total possible current with high K0.5 values. Other analogues with alterations at N1 or C6 positions including N1-oxide-cAMP, 2 aminopurine riboside 3',5'-monophosphate, and N6-monosuccinyl-cAMP do not bind to the channel, suggesting that interactions with the channel in this region are essential for binding. In order to help interpret the changes in maximal current and K0.5 values compared to cGMP, molecular models of the active analogues were constructed and then docked into a molecular model of the cyclic nucleotide binding site of the retinal channel. This model, proposed by Kumar and Weber [(1992) Biochemistry 31, 4643-4649], was based on the crystal structure of cAMP bound to catabolite activator protein. Our modeling showed that the analogues were sterically accommodated within the binding site. No hydrogen bonds were predicted between the purine rings of cAMP and the pocket; however, Phe 533 on the beta 5 strand was predicted to form weak electrostatic interactions with C6 substituents on both cAMP and cGMP. The importance of contacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6. PMID- 7532008 TI - A case of profound iron deficiency anemia owing to corrosive esophagitis in a 20 year-old developmentally delayed male. AB - The level of severe compensated iron deficiency anemia incompatible with life is not defined in the pediatric or adolescent literature. A hemoglobin of 1.5 gm/dl in an older adolescent with few physical symptoms is distinctly unusual. A case of profound iron deficiency anemia in a 20-year-old developmentally delayed male is the subject of this brief report. There were only subtle physical findings in spite of this severe anemia. The anemia was the result of corrosive esophagitis associated with a hiatal hernia and reflux. Physicians dealing with developmentally delayed adolescents should be aware of the fact that a severe anemia may develop, and such individuals should be periodically screened for anemia, melena, hematochezia, and hematemesis. PMID- 7532009 TI - Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats. AB - We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion. PMID- 7532010 TI - Rapid protein tyrosine phosphorylation in the cytoskeleton of stimulated human platelets. AB - Upon activation platelets show elevated protein tyrosine phosphorylation, and translocation of the protein tyrosine kinase pp60c-src from the plasma membrane to the cytoskeleton occurs. We therefore investigated whether tyrosine phosphorylation also increases in the cytoskeletal compartment. Here we show that almost identical patterns of phosphotyrosine-containing proteins are detectable in the cytoskeleton after platelet stimulation with compounds that directly (phorbol 12-myristate, 13-acetate) or indirectly (thrombin, vasopressin, collagen, ADP) activate protein kinase C. The apparent molecular masses of the proteins phosphorylated at tyrosine residues are 145, 130, 100, 85, 80, 60, 56, 54 and 38 kDa. Elevation of cyclic AMP by prostaglandin E1 had no effect. Concentrations of thrombin as low as 0.01 units per ml are able to cause tyrosine phosphorylation of multiple proteins. The time course of protein tyrosine phosphorylation for thrombin- and vasopressin-stimulated platelets revealed a rapid increase in the cytoskeleton within 5 to 20 s following activation consistent with a role in early events of platelet function. PMID- 7532011 TI - Differential activation of Gi and Gs proteins by E- and I-type prostaglandins in membranes from the human erythroleukaemia cell line, HEL. AB - The group of prostaglandin (PG) E2- and prostacyclin receptors consists of different subtypes, which exhibit different affinities for prostaglandins and synthetic analogues. PGE2 activities the E-type PG receptor subtypes EP1, EP2 and EP3, whereas the PGE2 analogue, sulprostone, binds only to the EP1 and EP3 receptor subtypes. The stable PGI2 analogues, iloprost and cicaprost, both activate the PGI2 receptor (IP) and iloprost, additionally, bind to the EP1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with Gs and Gi-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE2, iloprost and cicaprost were only partially inhibited by PTX. PGE2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified Gi protein alpha subunits. Furthermore, PGE2, iloprost and cicaprost enhanced GTP azidoanilide labelling of Gs protein alpha subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP1 and EP3 receptor subtypes activate G1 proteins, that (2) the EP2 receptor subtype activates Gs proteins and that (3) the IP receptor activates both Gi and Gs proteins. PMID- 7532012 TI - Lessons in living from a dying patient. PMID- 7532013 TI - Molecular fingerprinting of Pasteurella multocida associated with progressive atrophic rhinitis in swine herds. AB - Ninety-six nasal isolates of Pasteurella multocida from swine herds with progressive atrophic rhinitis were characterized by restriction endonuclease analysis (REA) of whole-cell DNA, ribotyping, and plasmid analysis. For REA, bacterial DNA was digested with SmaI and electrophoresed in 0.7% agarose, and fragments were visualized with UV light. For ribotyping, EcoRI-digested and electrophoresed restriction fragments of whole-cell DNA were transferred to nitrocellulose membranes, hybridized with gamma-32P-labeled Escherichia coli ribosomal RNA, and visualized by autoradiography. Phenotypes of isolates were toxigenic capsular type D (n = 51), nontoxigenic type D (n = 28), nontoxigenic type A (n = 16), and toxigenic type A (n = 1). Plasmids of various sizes were evident in 92.2% and 17.9% of toxigenic and nontoxigenic D strains, respectively, but were absent from all type A strains. Among the 4 phenotypes, there were 17 REA profiles and 6 ribotypes. For 3 of 17 REA patterns, multiple ribotypes were evident, and several REA types were evident in 5 of 6 ribotypes. Thirty-seven isolates of toxigenic capsular type D from Australian herds were either SmaI type B or C and ribotype 2, whereas 14 toxigenic D isolates from the USA and other countries were more heterogeneous (7 REA types and 6 ribotypes). The fingerprinting results provided evidence in support of the hypothesis of a single source infection in Australia associated with the introduction of breeding pigs from overseas. PMID- 7532014 TI - Canine mast cell tumors: a comparison of staining techniques. AB - Twelve histochemical methods; affinity staining with avidin peroxidase, wheat germ agglutinin, and concavalin-A agglutinin; and an immunohistochemical stain with Kp1 (CD68) antibody were compared for their relative effectiveness in staining canine mast cell tumors. Stains were compared in 28 mast cell tumors and 19 histiocytomas. The effectiveness of the histochemical methods and the lectins decreased as the mast cells became less differentiated. None of the staining methods were positive on histiocytomas. Periodic acid-Schiff (PAS) gave positive results in a few cases of mast cell tumors where other histochemical stains were negative. Although avidin peroxidase and Kp1 antibody stained more mast cell tumors than any other method, they did not differ significantly from Luna's method, toluidine blue pH 0.5, toluidine blue pH 4.5, alcian blue pH 2.5, safranin O, Unna's method, and Giemsa. No stain was ideal for the diagnosis of canine mast cell tumors; however, this study suggests that the use of avidin peroxidase, Kp1 antibody, and PAS may give additional information for individual poorly differentiated tumors without substantial increase in time or cost. PMID- 7532015 TI - Cholelithiasis during pregnancy and lactation. Prospective study. AB - OBJECTIVE: To assess the incidence of formation of gallstones during pregnancy and lactation, the time when gallstones first appear, and any changes in predisposing factors. DESIGN: Prospective study. SETTING: District hospital, Greece. SUBJECTS: 669 pregnant women seen between 1988 and 1991. MAIN OUTCOME MEASURES: Body mass index (kg/m2); real time ultrasonography; and measurements of serum concentrations of bilirubin, total cholesterol, low density lipoprotein cholesterol, and triglycerides, and of activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and amylase. All measurements were made at the beginning of pregnancy; at the third, sixth and ninth months; and 7 days and 6 months after delivery. RESULTS: 6 women (1%) mean age 26 years, had pre-existent gallstones and 14 further women (2%) mean age 27 years, developed gallstones; 13 were discovered during pregnancy (8 during the second trimester, and 5 during the third) and one during lactation. Women who had 3 or more pregnancies (6/110, 5%) were significantly more likely to develop gallstones than those who had not been pregnant before (3/304, 1%, p = 0.01). Gallbladder volume more than doubled during pregnancy from a mean (SD) of 12 (4) to 26 (4) ml. Total cholesterol, low density lipoprotein cholesterol and triglyceride concentrations, and alkaline phosphatase activity increased gradually, and reverted to the value found at the beginning of pregnancy immediately after delivery. CONCLUSIONS: The risk of developing gallstones increased with the number of pregnancies, and most of the stones developed during the second or third trimester. PMID- 7532016 TI - Carcinoid tumour of the thymus gland. PMID- 7532017 TI - Effect of tumor necrosis factors, interferons, interleukins, and growth factors on the activation of NF-kappa B: evidence for lack of correlation with cell proliferation. AB - The nuclear transcription factor NF-kappa B has been identified as a critical component in signal transduction pathways. We used an electrophoretic gel mobility shift assay to examine the activation of NF-kappa B in human U-937 cells treated with tumor necrosis factor (TNF), lymphotoxin (LT), interferons (IFN) alpha, IFN-beta, and IFN-gamma, interleukins (IL)-1 beta, IL-4, and IL-6, leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-beta (TGF-beta). Only TNF, LT, and IL-1 activated NF-kappa B. Since interferons have been shown to induce TNF receptors and potentiate TNF-mediated cellular responses, we also measured the effect of interferons on TNF-induced activation of NF-kappa B. Under our conditions, all three IFNs potentiated the cytotoxic effects of TNF but had no effect on the TNF-dependent NF-kappa B activation. These results suggest overall that the activation of NF-kappa B is not a generalized mediator of signal transduction of most cytokines and also that NF-kappa B activation is not sufficient for antiproliferative effects mediated through certain cytokines. PMID- 7532018 TI - CD5-positive and CD5-negative plaque-forming cells against poly-L-lysine-treated sheep erythrocytes in patients with systemic lupus erythematosus. AB - While attempting to evaluate CD5+ and CD5- anti-DNA-secreting plaque-forming cells (PFC) in patients with systemic lupus erythematosus (SLE), significant numbers of PFC against control sheep erythrocytes (ShE) treated with poly-L lysine (PLL) but not further conjugated with single-stranded (ss) or double stranded (ds) DNA were noted. Numbers of PFC obtained using PLL-ShE, ssDNA-ShE and dsDNA-ShE were not significantly different, all reactivity to DNA apparently being accounted for by binding of antibodies to PLL-treated ShE. Nevertheless, anti-PLL-PFC could be inhibited by soluble dsDNA included in the plaque assay. These findings might be explained by cationic anti-DNA antibodies binding non specifically to anionic PLL. Control healthy subjects gave few PFC against PLL ShE, ssDNA-ShE or dsDNA-ShE. Anti-PLL-PFC appeared to be related to disease activity, with higher numbers of both CD5+ and CD5- PFC in patients with clinically active SLE. PMID- 7532020 TI - Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel. AB - A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance. Therefore, the use of paramagnetic centers such as spin labels or metal ions with long electron T1 values provides a distance measurement that reflects a dynamically averaged structure where the averaging process heavily weights short distances. The results of such measurements, when combined with other structural information, may provide particularly clear evidence for the magnitude of structural fluctuations involving distances greater than 10 A. PMID- 7532022 TI - The up-to-date molecular genetics of cystic fibrosis. PMID- 7532019 TI - Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties. AB - To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation. PMID- 7532021 TI - Phosphate stimulates CFTR Cl- channels. AB - Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains. PMID- 7532024 TI - Comparison of expression of a humanized monoclonal antibody in mouse NSO myeloma cells and Chinese hamster ovary cells. AB - We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically. PMID- 7532023 TI - Anti-Tn human monoclonal antibodies generated following active immunization with partially desialylated ovine submaxillary mucin. AB - Partially desialylated ovine submaxillary mucin plus an immunological adjuvant has been used by us to induce a humoral immune response to Tn antigen and sialylated Tn in colon cancer patients at risk of recurrence. Peripheral blood lymphocytes were purified from one of these patients with a high IgM titer reactive with Tn antigen transformed with Epstein-Barr virus and subsequently fused with a human-mouse heteromyeloma cell line, HMMA2.11TG/O. Clones were screened and subcloned using an enzyme-linked immunoassay and four stable IgM secreting clones were tested for reactivity with a variety of natural and synthetic antigens, paraffin-embedded colon carcinomas and normal colonic mucosa to determine their specificity. Four human IgM monoclonal antibodies reactive predominantly with Tn antigen were produced and shown to react with a mucinous human colon carcinoma cell line, LS-174T, and with paraffin-embedded human colon carcinomas. We conclude that modified ovine submaxillary mucin is an effective vaccine for generating a humoral immune response directed to Tn antigen present on human colon cancer. PMID- 7532025 TI - Plasmodesmata: composition, structure and trafficking. AB - Plasmodesmata are highly specialized gatable trans-wall channels that interconnect contiguous cells and function in direct cytoplasm-to-cytoplasm intercellular transport. Computer-enhanced digital imaging analysis of electron micrographs of plasmodesmata has provided new information on plasmodesmatal fine structure. It is now becoming clear that plasmodesmata are dynamic quasi organelles whose conductivity can be regulated by environmental and developmental signals. New findings suggest that signalling mechanisms exist which allow the plasmodesmatal pore to dilate to allow macromolecular transport. Plant viruses spread from cell to cell via plasmodesmata. Two distinct movement mechanisms have been elucidated. One movement mechanism involves the movement of the complete virus particle along virus-induced tubular structures within a modified plasmodesma. Apparently two virus-coded movement proteins are involved. A second movement mechanism involves the movement of a non-virion form through existing plasmodesmata. In this mechanism, the viral movement protein causes a rapid dilation of existing plasmodesmata to facilitate protein and nucleic acid movement. Techniques for the isolation of plasmodesmata have been developed and information on plasmodesma-associated proteins is now becoming available. New evidence is reviewed which suggests that plasmodesmatal composition and regulation may differ in different cells and tissues. PMID- 7532027 TI - Green circuits--the potential of plant specific ion channels. PMID- 7532028 TI - Field evaluation of transgenic potato plants expressing an antisense granule bound starch synthase gene: increase of the antisense effect during tuber growth. AB - Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content. PMID- 7532030 TI - Serum stem cell factor levels in patients with aplastic anemia. AB - Immune suppression of hematopoiesis has been implicated in the pathogenesis of acquired aplastic anemia. Similarly, abnormalities of T cells and bone marrow stromal cells have been reported in aplastic anemia, as has abnormal cytokine production. Stem cell factor (SCF) (also known as kit ligand, mast cell growth factor and Steel factor) is an early acting hematopoietic growth factor that is produced by a variety of mesenchymal cells including bone marrow stromal cells. To determine whether abnormalities in the production of stem cell factor occur in aplastic anemia, we evaluated serum levels of SCF in 25 patients with aplastic anemia. The mean serum levels of SCF in aplastic anemia patients were significantly lower (2.7 +/- 1.1 ng/ml) than those found in a comparable population of 257 normal controls (3.3 +/- 1.1 ng/ml) (P = 0.011). The SCF level did not correlate with patient age, the duration of aplastic anemia or with white blood cell count, platelet count or hematocrit. Although there is no direct evidence that lower SCF serum levels contribute to the pancytopenia seen in this disorder, identification of underlying abnormalities that can result in the deficient production of stromally derived hematopoietic growth factors will be important. PMID- 7532031 TI - Treatment with cytosine arabinoside and granulocyte colony-stimulating factor in patients with myelodysplastic syndrome and its leukemic phase. AB - Twenty-one patients with myelodysplastic syndrome (MDS) or overt leukemia resulting from MDS were treated with recombinant human granulocyte colony stimulating factor (rhG-CSF) and cytosine arabinoside (Ara-C). Ara-C was administered in a dose of 20 mg/m2 every 12 h for 5 days and after 2 days 125 micrograms of rhG-CSF was administered for 10 days. After recovery of the leukocyte count the therapy was repeated, doubling the dose of Ara-C serially when possible. Of 13 patients with MDS, four achieved complete remission (CR), two good response (GR), two minor response (MR), and five no response (NR). Of eight patients with overt leukemia from MDS, only one with hyperplastic bone marrow achieved a partial response (PR) and the remaining seven achieved NR. The efficacy of the combination of rhG-CSF and Ara-C in the treatment of MDS and its leukemic phase is discussed, including at which time rhG-CSF should be administered: before, after or concomitantly with Ara-C. Multicenter randomized studies are needed in the evaluation of this combination therapy. PMID- 7532029 TI - Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs. AB - Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells. PMID- 7532026 TI - Molecular genetics of auxin and cytokinin. PMID- 7532032 TI - In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single-cell culture. AB - Uni- or multi-lineage suppression of hematopoiesis is observed in the majority of acquired immunodeficiency syndrome (AIDS) patients. The mechanism(s) underlying these abnormalities is not understood: particularly, the human immunodeficiency virus (HIV) infection of hematopoietic progenitor and stem cells (HPCs/HSCs) is highly controversial. We report that CD34+ HPCs from adult peripheral blood (PB) are in part CD4+ and susceptible to in vitro HIV infection. Primitive CD34+ HPCs were approximately 80% purified from PB. Double labeling for CD34 and CD4 membrane antigens was shown for 5% to 20% of the purified cells, thus suggesting their potential susceptibility to HIV-1 infection. The enriched HPC population, challenged with purified or unpurified HIV-1 strains, was cloned in unicellular methylcellulose culture. The single colonies generated by erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) were analyzed for the presence of HIV, ie, for gag DNA, tat mRNA, and p24 protein by PCR, reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay, respectively. In the first series of experiments incubation of HPCs with HIV-1 at multiplicities of infection (MOI) ranging from 0.01 to 10 TCID50/cell consistently yielded an 11% to 17% infection efficiency of BFU-E-generated colonies, thus indicating the sensitivity of HPCs to in vitro HIV infection. An extensive series of experiments was then performed on HPCs challenged with HIV at 0.1 MOI level. In the initial studies proviral gag sequences were detected in 9.2% of 121 analyzed CFU-GM colonies. In further experiments tat mRNA was monitored in 17% and 23% of BFU-E and CFU-GM colonies, respectively, but never in CFU-GEMM clones. Finally, 12% of CFU-GM clones and rare erythroid bursts were shown to be positive for the p24 viral protein. In control studies, purified HPCs grown in liquid suspension culture were induced to terminal unilineage erythroid, monocytic, or granulocytic differentiation: monocytes were consistently HIV infected, whereas mature-terminal erythroblasts and granulocytes were not. Our observations indicate that a minority of primitive HPCs, but not of the multipotent type, is susceptible to in vitro HIV infection. These observations may reflect on the in vivo hematopoietic impairment in AIDS patients; more important, they provide an experimental model for studies on HIV hematopoietic infection and in vitro tests for anti-HIV HSC gene therapy. PMID- 7532034 TI - Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs. AB - We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells. PMID- 7532035 TI - Cell-to-cell interaction of cytokine-dependent myeloblastic line constitutively expressing membrane-bound stem cell factor abrogates cytokine dependency partially through granulocyte-macrophage colony-stimulating factor production. AB - Stem cell factor (SCF) is a cytokine for hematopoietic progenitor cells and plays an important role in megakaryocyte proliferation. The UT-7 cell line was established from a patient with megakaryoblastic leukemia, and its growth and survival are strictly dependent on interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (Epo), or IL-6. In this study, we showed that SCF also supported the growth of UT-7 in the absence of other cytokines and downregulated the cell surface c-kit receptors. Constitutive expression of SCF by introducing SCF expression vector made UT-7 grow factor independently in liquid medium, but not in semisolid medium. This SCF-expressing factor-independent UT-7 (UT-7scf9) expressed the membrane bound form of SCF on their surface, but did not secrete detectable amounts of soluble SCF. UT-7scf9 formed aggregates as they grew in the absence of cytokines, and this aggregation was inhibited by adding soluble SCF into the medium. UT-7 cultured with SCF and UT-7scf9 cultured without cytokines expressed GM-CSF, and anti-GM-CSF neutralizing antibody partially inhibited their growth. These results suggest that SCF stimulated UT-7 proliferation partially through the autocrine-loop of GM CSF, and UT-7scf9 expressed SCF mostly as a membrane-bound form, which transduces its growth signal through c-kit receptor as they aggregate by cell-to-cell interaction. PMID- 7532033 TI - Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC resistant rat leukemic cell line of clonal origin fully restores drug sensitivity. AB - The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1 ADCK) that were stably transfected and monitored over a period of 14 weeks. Northern and Western blot analysis showed restoration of dck mRNA and protein expression. Initial rate measurements of DCK activity showed that Km values for dck were only slightly altered as a result of transfection, whereas strongly increased Vmax values were observed, resulting in a 12-fold increased phosphorylation efficiency for both dC and AraC, compared with the AraC-sensitive parental cell line RO/1 from which the RO/1-A was originally derived. In vitro sensitivity to AraC- and DAC-mediated cytotoxicity was fully restored in RO/1 ADCK. The data pinpoint acquired DCK deficiency caused by mutations of the dck gene as the major cause of AraC resistance in this model. PMID- 7532036 TI - The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformation-sensitive epitope on glycoprotein IX: implications for the target antigen of quinine/quinidine-dependent autoantibodies. AB - The monoclonal antibody SZ1 is of interest for two reasons: it was used to define complex formation between glycoprotein (GP) Ib and GP IX, and its epitope is likely to be identical to that recognized by most quinine- and quinidine dependent autoantibodies that cause thrombocytopenia. To determine the location of the epitope for SZ1 within the GP Ib-IX complex (which consists of three subunits: GP Ib alpha, GP Ib beta, and GP IX), we tested the ability of the antibody to bind transfected cells that expressed different combinations of complex subunits, and compared this binding to the binding of antibodies of known specificity. SZ1 bound to cells that expressed the entire GP Ib-IX complex in the same pattern as did AN51 (an antibody specific for GP Ib alpha). However, unlike AN51, SZ1 did not bind alpha beta cells (ie, cells that express GP Ib alpha and GP Ib beta, but not GP IX), but did bind to beta IX and alpha IX cells. We then compared the binding patterns of SZ1 and FMC25, an antibody specific for GP IX. Both bound virtually identically to cell lines that expressed every combination of two of the three GP Ib-IX complex subunits. However, the epitopes of the two antibodies were not identical, because fixation with 4% paraformaldehyde of cells that expressed GP IX destroyed the SZ1 epitope while maintaining the FMC25 epitope. Because of the ability of SZ1 to block the binding of many quinine- and quinidine-dependent antibodies, these data strongly suggest that GP IX is the component of the GP Ib-IX complex recognized by those antibodies. PMID- 7532037 TI - The antineoplastic bryostatins affect human basophils and mast cells differently. AB - Bryostatins, macrocyclic lactones from the marine bryozoan Bugula neritina, are potent antineoplastic agents and multi-potential stimulators of immune cells. We have examined the effects of bryostatins on mediator release from human basophilic leukocytes and human tissue mast cells. Bryostatins 1, 2, and 5 (10 to 3,000 nmol/L) induced histamine secretion from purified and unpurified peripheral blood basophils, whereas they caused no release of peptide-leukotriene C4 from these cells. The rate of histamine release caused by bryostatin 1 was slower than that caused by anti-IgE (t1/2 +/- SEM = 38.2 +/- 4.7 minutes v 8.9 +/- 0.2 minutes; P < .01), whereas the temperature dependence was similar (optimum release at 37 degrees C, approximately 30% less at 30 degrees C, and no release at 22 degrees C or 4 degrees C). The addition of increasing concentrations of extracellular Ca2+ to the medium caused histamine release in the presence of bryostatins. Subeffective concentrations of bryostatins and anti-IgE produced a synergistic effect on histamine release from basophils. Staurosporine, chelerythrine, and calphostin C (0.1 to 10 nmol/L), which are protein kinase C inhibitors, inhibited the histamine secretion activated by bryostatin 1 and tetradecanoylphorbol-acetate (TPA). Preincubation with granulocyte-monocyte colony-stimulating factor (GM-CSF; 1 and 5 nmol/L) and interleukin-3 (IL-3; 10 ng/mL) potentiated the activation of human basophils induced by bryostatin 1. Neither bryostatin 1 nor bryostatin 2 induced the release of histamine from mast cells isolated from human lung or skin tissues. However, brief (10 minutes) preincubation with bryostatin 1 (3 to 300 nmol/L) potently inhibited the histamine secretion induced by anti-IgE from skin or lung mast cells. Bryostatin 1 was a more potent (by approximately 30 times) inhibitor of IgE-mediated histamine release than was TPA. The heterogeneous effects exerted by bryostatins on human basophils and mast cells can be of interest for those designing therapeutic trials using these agents. PMID- 7532038 TI - Long-term culture of chronic myelogenous leukemia marrow cells on stem cell factor-deficient stroma favors benign progenitors. AB - Long-term culture of marrow from patients with chronic myelogenous leukemia (CML) has been reported to favor the outgrowth of bcr/abl- progenitor cells in some patients. We examined the effect of the presence of soluble or transmembrane forms of stem cell factor (SCF) in long-term cultures of CML marrow. CD34 enriched cells from CML patients in advanced chronic phase or accelerated phase were plated on immortalized fetal liver stromal cells from homozygous SCF deficient SI/SI mice (SI/SI4) with or without the addition of soluble human SCF, SI/SI4 cells expressing high levels of the transmembrane form of human SCF (SI/SIh220), or primary human allogeneic stroma. Cells were removed from cultures and plated weekly in colony assays. The clonagenic cell output from cultures completely lacking SCF was lower over the first 2 to 3 weeks, but by 5 weeks was similar to the clonagenic cell output from the other culture conditions. Analysis of bcr/abl transcripts from individual colonies showed a lower percentage of malignant progenitors present in long-term cultures completely deficient in SCF than under the other culture conditions, particularly compared with primary human stroma-containing long-term cultures. SCF may specifically favor malignant versus benign progenitor cells present in the marrow of CML patients, and an abnormal proliferative response to SCF in very primitive cells may be an underlying defect in the pathophysiology of this disease. PMID- 7532039 TI - Infection with Nippostrongylus brasiliensis induces invasion of mast cell precursors from peripheral blood to small intestine. AB - Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site. PMID- 7532041 TI - Paroxysmal nocturnal hemoglobinuria clone in bone marrow of patients with pancytopenia. AB - The lack of glycosylphosphatidylinositol (GPI)-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59 on blood cells has a diagnostic value in paroxysmal nocturnal hemoglobinuria (PNH). Because PNH often develops in patients with aplastic anemia (AA), we attempted to detect a PNH clone in the bone marrow (BM) of patients with AA and pancytopenia before affected cells were evident in the peripheral blood (PB). We used flow cytometry with monoclonal antibodies against DAF and CD59 for the detection of the clone. Affected cells were observed in the BM of 3 of 7 patients with AA and 1 of 3 patients with pancytopenia of unknown origin, but not in their PB. All 8 patients with apparent PNH had affected cells in their BM and PB. On the basis of the early appearance of the PNH clone in the BM, a prospective 4-month follow-up study of the PB cells was performed. The study showed the release of affected mature cells first in granulocytes, then in monocytes, and finally in lymphocytes. Ham's test was positive before affected erythrocytes were detected by flow cytometry. Our findings indicate that detection of the PNH clone in BM could be predictive of the development of PNH in patients with AA and pancytopenia. PMID- 7532043 TI - A simple procedure to demonstrate the presence of the O-antigen capsule in enteropathogenic Escherichia coli. AB - Enteropathogenic Escherichia coli O111 bacteria very often produce a capsule-like polysaccharide exhibiting the same chemical composition as the O-specific chains of their lipopolysaccharide and being designated as O-antigen capsule. In this paper, a new simple procedure is described for the detection of this capsule polymer. O-antigen capsule polysaccharide and lipopolysaccharide of bacterial extracts were electrophoretically separated in agarose cetavlon gel, blotted onto nitrocellulose membranes and visualized by immunological methods. As a result of this procedure, the O-antigen capsule can be detected as a spot completely separated from the LPS (Figs. 1 and 2). Consequently, this method allows a clear discrimination of K+ and K- strains. PMID- 7532040 TI - Antilymphocyte globulin, cyclosporin, and granulocyte colony-stimulating factor in patients with acquired severe aplastic anemia (SAA): a pilot study of the EBMT SAA Working Party. AB - Patients with severe aplastic anemia (SAA) and a neutrophil (PMN) count of less than 0.5 x 10(9)/L are exposed to a high risk of early mortality when treated with antilymphocyte globulin (ALG) and steroids, with the major problem being infectious complications. The addition of human recombinant granulocyte colony stimulating factor (rhG-CSF) to ALG may reduce early mortality by improving neutrophil counts in the short term. To test the feasibility of this approach, the SAA Working Party of the European Group for Blood and Marrow Transplantation (EBMT) designed a pilot study that included rhG-CSF (5 micrograms/kg/d, days 1 through 90), horse ALG (HALG; 15 mg/kg/d, days 1 through 5), methylprednisolone (2 mg/kg/d, days 1 through 5, then tapering the dose), and cyclosporin A (CyA; 5 mg/kg/d orally, days 1 through 180). Patients with newly diagnosed acquired SAA (untreated) and with neutrophil counts of < or = 0.5 x 10(9)/L were eligible. Forty consecutive patients entered this study and are evaluable with a minimum follow up of 120 days: the median age was 16 years (range, 2 to 72 years), the interval from diagnosis to treatment was 24 days, and the median PMN count was 0.19 x 10(9)/L. Twenty-one patients had hemorrhages, and 19 were infected at the time of treatment. Overall, treatment was well tolerated: the median maximum PMN count during rhG-CSF administration was 12 x 10(9)/L (range, 0.4 x 10(9)/L to 44 x 10(9)/L). There were three early deaths (8%) due to infection. Four patients (10%) showed no recovery, whereas 33 patients (82%) had trilineage hematologic reconstitution and became transfusion-independent at a median interval of 115 days from treatment. Median follow up for surviving patients is 428 days (range, 122 to 1,005). Actuarial survival is 92%: 86% and 100% for patients with PMN counts less than 0.2 x 10(9)/L or between 0.2 x 10(9)/L and 0.5 x 10(9)/L, respectively. This study suggests that the addition of rhG-CSF to ALG and CyA is well tolerated, is associated with a low risk of mortality, and offers a good chance of hematologic response. This protocol would appear to be an interesting alternative treatment for SAA patients with a low PMN count who lack an HLA identical sibling. PMID- 7532042 TI - Influence of the timing of FK 506 (Tacrolimus) administration on recovery of renal function from warm ischemic injury in rats. AB - The influence of timing of FK 506 (Tacrolimus) administration on renal function and recovery from renal warm ischemia was studied in Sprague-Dawley rats. Animals were administered FK 506 and subjected to 60 min of renal warm ischemia by temporary occlusion of the renal artery and vein. No significant differences in serum creatinine levels among rats subjected to renal ischemia, FK 506, or FK 506 vehicle (methanol and 5% dextrose in water) were demonstrated. In contrast, FK 506 administration (4 mg/kg intraperitoneally) in combination with renal warm ischemia resulted in significant deterioration of renal function with peaking of serum creatinine on day 2. The timing of FK 506 administration relative to renal ischemia did not significantly affect serum creatinine levels. Rats that received FK 506 either 24 hr pre-ischemia, 4 hr pre-ischemia, 4 hr post-ischemia, or 24 hr post-ischemia all showed similar serum creatinine levels on day 2 (3.85 +/- 0.9, 4.7 +/- 0.5, 3.8 +/- 0.9, and 5.1 +/- 0.6 mg/dl, respectively, p = NS). In all animals, serum creatinine returned to baseline values by day 10. Histopathologic examination of kidneys revealed tubular atrophy and dilatation with tubular calcifications at the corticomedullary junction in FK 506 treated animals with or without ischemia. Our data suggest the timing of FK 506 administration in rats subjected to renal warm ischemia does not influence the extent of renal injury with an equally deleterious effect seen when administered within a 24 hr period of an ischemic event. Changes in kidney morphology, however, were seen in all FK 506 treated rats, with or without a period of warm ischemia. PMID- 7532044 TI - Fibronectin and vitronectin binding of Bacteroides fragilis and eight other species of the genus. AB - The fibronectin and vitronectin bindings of 152 strains belonging in 9 Bacteroides species of different origins were tested by means of latex agglutination. 23% of the strains isolated from faeces exhibited fibronectin binding, as did 46% of the strains obtained from severe infections. Most of the strains displaying fibronectin binding belonged to the species Bacteroides fragilis or Bacteroides vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. Vitronectin binding was less common, but was always observed in parallel with fibronectin binding. PMID- 7532046 TI - Prevalence of markers of hepatitis B virus infection among the general population of the municipality of Slavonski Brod. AB - To assess the prevalence of hepatitis B viral infection in general population of the Community of Slavonski Brod, sera from 2,142 apparently healthy individuals were tested for the hepatitis B virus (HBV) markers: surface antigen and antibody (HBsAg and anti-HBs), and core antibody (anti-HBc), by radio and enzyme immunoassay. The HBsAg results showed a general prevalence of 1.8%(38/2,142): for males 1.9% and for females 1.6%. The highest HBsAg incidence rates (2.1% and 1.9%) were found in young adults aged 21-30, respective in older children and adolescents, from 11 to 20 years. The lowest HBsAg prevalence (1.0%) was proved in children from 1 to 5 years, while in sera of 68 examined sucklings HBsAg was not detected. The HBsAg incidence was 1.9% (21/1132) in urban and 1.7% (17/1010) in rural inhabitants. The overall prevalence rate of total HBV infection in 13.7% (294/2,142) examinees was found. There was no difference in the prevalence of HBV infection in males 15.7% (177/1130) and females 11.6% (117/1012), as well as in persons living in rural 12.5% (126/1010) or in urban 14.8% (168/1132) areas. The prevalence rate of anti-HBs and anti-HBc gradually increased with increasing age, starting at about 3.2% in the youngest, and reaching 15.5% in adults over 50 years. The results obtained have place the Community of Slavonski Brod among areas with an intermediate endemicity of HBV infection. PMID- 7532047 TI - Does the human placenta differentiate structurally during the 10th lunar month? AB - The placenta is the central organ of fetomaternal exchange, which metabolically provides for the growth of the fetus and its membranes as a whole. The main structural components of the human placenta develop until the beginning of the second trimester of gestation, and continue to grow and differentiate. The dynamics of the histomorphological development of the placenta lasts until birth. The aim of this stereological investigation was to analyze and compare structural components of placental parenchyma during two periods of the 10th lunar month: from the 38th to 39th week, and in the 40th week of gestation. The results show that during the last four weeks of gestation the trophoblast of the chorionic villi changes the most. At the beginning of the 10th lunar month, alpha zones, bearers of transplacental gas transport, prevail. At the end of the 10th lunar month beta zones dominate (P < 0.005). These are metabolically active parts of trophoblast. These quantitative results of stereological analysis have to be assumed as structural evidence of the physiological maturity of human placenta. PMID- 7532045 TI - Prostatitis in benign prostatic hyperplasia: a histological, bacteriological and clinical study. AB - The paper deals with the incidence of prostatitis in benign prostatic hyperplasia (BPH) and its effect on the postoperative course in the patients subject to the transvesical prostatectomy. The samples of urine and expressed prostatic secretion were bacteriologically analysed before the operation, and samples of urine on three occasions following the operation. Specimens of BPH tissue obtained by surgery were subjected to pathohistological and microbiological examination. The incidence of prostatitis was found to be 90.3%. In the specimens of BPH tissue obtained by transversal prostatectomy, Gram-positive microorganisms were isolated in 32.8% of the tissue. In particular, Staphylococcus epidermidis was found in 26.6% of BPH tissue. Gram-negative microorganisms were isolated in 30.8% of BPH tissue, and fungi in 2.9%. In 27.9% of BPH tissue a microorganism could not be isolated although pathohistological examination evidenced prostatitis. Therefore, in these cases etiology remained unclear. Patients with Gram-negative prostatitis had the highest number of complications and the longest period of postoperative hospitalization. Preoperative and postoperative antibiotic therapy reduced the incidence of postoperative complications and shortened the period of hospitalization in the patients with significant Gram negative bacteriuria before operation, that is, in the patients with Gram negative prostatitis in BPH. PMID- 7532048 TI - War wounds management--early reconstruction of soft tissue defects. AB - The authors emphasize that the knowledge of terminal ballistics is important for understanding the pathophysiology of war wounds. They present their own experiences in the treatment of war wounds in 504 casualties treated at the Institute of Plastic and Reconstructive Surgery, Department of Surgery, Clinical Hospital Center in Zagreb. The locations of soft-tissue defects were: head and neck, 103; trunk, 90; and extremities, 903. War wounds were divided into four main categories with regard to the type of the injury and the extension of soft tissue defect, thus showing the differences in primary excision and reconstruction of the wounds. About 30% of head and neck injuries were treated by primary or delayed primary reconstruction. All thoraco-abdominal wounds were type I or II, and most of them (53.3%) were reconstructed with split-thickness skin grafts. The greatest number of sophisticated reconstructions were performed on extremity injuries (63 wounds were reconstructed by local flaps, while free flaps were used in 40 cases). The authors emphasize the importance of proper primary treatment, which is the condition for early reconstruction. This results in significantly shorter hospitalization, so that 62% of the patients were cured in 20 days and then discharged to early rehabilitation. PMID- 7532049 TI - Reprocessing and multiple use of hemodialyzers: a critical appraisal. AB - Economic considerations have made the reuse of hemodialyzers desirable, particularly with the introduction of expensive devices, such as large surface area or high-flux dialyzers, hemodiafilters and hemofilters. Recent studies have shown that certain reprocessing techniques confer improved biological properties on dialyzers compared with new membranes. Several investigators observed certain symptoms which occurred with greater frequency in patients receiving first use dialyzers as opposed to patients receiving dialysis treatment with reused dialyzers. Despite the potential advantages of more biocompatible membranes, and less expensive dialyzers, the safety and efficiency of reuse is the subject of some controversy. The article critically reviews the available scientific information on the risks and benefits of reprocessing. PMID- 7532050 TI - The fetus as a patient. AB - This article highlights the concept, history and development of fetal medicine. The first attempts of fetal medicine date from the stone age and have continued throughout the medieval and modern ages. Attempts to influence fetal health and pregnancy outcome at first were purely theoretical and passive, limited by ignorance, fear, and religion. With the development of the medical sciences, the role of the obstetrician became more active. Today, not only obstetricians, but many other experts in the field of antenatal care as well, deal with the great number of medical and technological advances. The limitations arising today are mainly ethical. The main question that still has not been answered is to what extent the physician is allowed to interfere with the process of fetal development and pathology, based upon real fetal and maternal benefit. In order to answer this question and to coordinate many activities by different experts in this field, an international society antitled The Fetus as a Patient was formed in 1985. Croatian experts have played a leading role in this concept. PMID- 7532051 TI - Preparation of respiratory syncytial virus subgroup A and B antigens for enzyme immunoassay antibody detection. AB - A simplified method was described for purification of respiratory syncytial virus (RSV) subgroup A and B aimed to be used as antigens in enzyme immunoassay (EIA). The titer of each RSV subgroup and the amount of protein was determined from the visible band in 45% sucrose gradient. The quality of prepared RSV subgroup antigens for EIA was described in terms of the achievable final titer, the amount of protein, and EIA criss-cross titration. The RSV subgroup A and B antigens, diluted as 1:100 (low opalescent band in 45% sucrose layer) or 1:800 (high opalescent band in 45% sucrose layer) produced a positive reaction in EIA criss cross titration with IgG antibodies from the patient's serum (convalescent phase) diluted as 1:25,600 (for RSV A) and 1:6,400 (for RSV B). This method offers shorter and more simplified steps of viral antigen purification, and provides acceptable quantity and quality of viral antigens appropriate for use in EIA. PMID- 7532052 TI - Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits. AB - Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer. PMID- 7532053 TI - Amniotic fluid alpha-fetoprotein levels during midtrimester of trisomy pregnancies. AB - To investigate the association between low amniotic fluid alpha-fetoprotein (AFP) and trisomy pregnancies, we retrospectively reviewed 26 trisomy pregnancies including 18 fetuses with Down's syndrome and eight with trisomy 18. The amniotic fluid AFP median values of Down's syndrome, trisomy 18, and the study groups were 0.73 MoM, 1.15 MoM, and 0.85 MoM, respectively. There was a significant difference between the mean values of the Down's syndrome-affected fetuses (0.78 +/- 0.29 MoM) and that of the control group (p < 0.001), whereas no such difference was found for that of trisomy 18-affected fetuses (1.16 +/- 0.38 MoM). Only three patients in the study group (3/26, 11.5%) had an amniotic fluid AFP value below 0.5 MoM, including the two cases of Down's syndrome (2/18, 11.1%) and one case of trisomy 18 (1/8, 12.5%). Most of the values for the trisomy pregnancies were within the normal range, thereby precluding the possibility of using this measurement as an alternative to fetal karyotyping as a screening test for Down's syndrome or other trisomy pregnancies. PMID- 7532054 TI - Insulin-like growth factor-I and insulin stimulate the synthesis of IGF-binding protein-2 in a human embryonic kidney cell line. AB - Insulin and insulin-like growth factor (IGF)-I are thought to be major metabolic regulators of IGF-binding protein-2 (IGFBP-2). We have examined the regulation of IGFBP-2 expression by IGF-I and insulin in 293 cells, a cell line derived from human embryonic kidney. The predominant 34 kDa IGFBP in media conditioned by unstimulated 293 cells was identified as IGFBP-2 by immunoprecipitation. IGFBP-2 levels were increased 6 to 7-fold following incubation with IGF-I, IGF-II or insulin for 48 h. A corresponding increase in IGFBP-2 mRNA was not observed, suggesting that regulation occurred at the translational or post-translational level. The stimulation of IGFBP-2 by IGF-I and insulin was reversibly abolished by incubation with protein synthesis inhibitors such as cycloheximide. Biosynthetic labeling of quiescent 293 cells using [35S]cysteine indicated that incubation with insulin or IGF-I for 24 h increased the synthesis of total cell proteins (predominantly intracellular) and IGFBP-2 (predominantly secreted) to a similar extent (2- to 4-fold). These results suggest that the increase in IGFBP-2 secreted by 293 cells after incubation with IGF-I or insulin largely results from a general stimulation of protein synthesis. PMID- 7532055 TI - Insulin-like growth factors and their binding proteins in normal and abnormal human fetal growth. AB - There is now a well recognized series of findings which suggests that the insulin like growth factors (IGFs) and their binding proteins (IGFBPs) may play an important role in both normal and abnormal human fetal growth: (1) IGFs are detectable in many fetal tissues from the first trimester onwards; (2) the levels of the IGFs in the fetal circulation increase during pregnancy, and at term the levels of IGF-I are directly related to birthweight; (3) in mice, disruption of the IGF gene leads to severe growth retardation; (4) in the first trimester the levels of IGFBP-1 are higher in the coelomic fluid than in amniotic fluid or maternal serum; (5) at 9-12 weeks there is a striking increase in IGFBP-1 and IGFBP-2 levels in amniotic fluid; (6) the major binding proteins in the human fetus are IGFBP-1 and IGFBP-2; (7) from as early as 16 weeks there is an inverse correlation between fetal levels of IGFBP-1 and birthweight; (8) in the mother, circulating levels of IGF-I and IGFBP-1 increase during pregnancy; (10) maternal levels of IGFBP-1 are elevated in severe pre-eclampsia and intrauterine growth retardation; (11) fetal levels of IGFBP-1 are elevated in cases of intrauterine growth retardation, especially those associated with specific evidence of reduced uteroplacental bloodflow; and (12) fetal levels of IGFBP-1 are elevated in labour, especially if there is evidence of fetal hypoxia. In conclusion, levels of IGFBP-1 appear to be a sensitive indicator of fetal nutrition, and of the short- or long-term response to reduced fetal nutrition. PMID- 7532057 TI - Interleukin-11. AB - Interleukin-11 (IL-11), a stromal cell-derived cytokine, has been known to act widely in hematopoietic and non-hematopoietic systems. IL-11 supports the growth of certain types of plasmacytoma and hybridoma cells, acts with interleukin-3 (IL 3) in shortening the Go period of early progenitors. IL-11 supports megakaryocyte colony formation and maturation, and acts as an autocrine growth factor in megakaryoblastic cell lines. In addition, IL-11 stimulates erythrocytopoiesis, enhances antigen-specific antibody responses, induces the synthesis of acute phase proteins, inhibits lipoprotein lipase activity and adipocyte differentiation, and promotes neuronal development. Administration of rhIL-11 to mice resulted in an increase of neutrophils and platelets. The human IL-11 gene is localized at 19q13.3-13.4, and codes 199 amino acids and 23 kDa with no N glycosylation. Its receptor and signal transduction share partially those of interleukin-6 (IL-6). Further analysis of its role in normal and pathological state is necessary to determine the exact function and its application for clinical uses. PMID- 7532058 TI - CD34 immunophenotyping of blasts in myelodysplasia. AB - Immunological analysis of bone marrow cells in myelodysplasia using immunofluorescence did not allow accurate morphological identification of blast cells. However, improvement of the immunoperoxidase technique allows one to realize the diagnostic potential of immunocytochemistry. CD34 immunotyping of blasts in normal human bone marrow showed 0.8 +/- 0.4% CD34 positive blasts and these cells had the morphology of type 1 blasts. The increase of bone marrow blasts in RAEB patients is related to CD34 negative type II and III blasts. A clone of undifferentiated CD34 positive blasts is characteristic of RAEB-T and acute myeloid leukaemia evolving from myelodysplasia. The detection of CD34 positive bone marrow blasts allows a better discrimination between RAEB and RAEB T. PMID- 7532056 TI - Effect of granulocyte colony-stimulating factor in patients with diffuse large cell lymphoma treated with intensive chemotherapy. AB - We investigated whether Granulocyte colony-stimulating factor (G-CSF) could prevent myelotoxicity or accelerate hematopoietic recovery after intensive chemotherapy in previously untreated patients with diffuse large cell lymphoma (DLCL). Forty-two patients were included in a prospective clinical trial in which alternating chemotherapy ESAP (etoposide, Solu-Medrol, cytosine arabinoside, cis platinum), m-BECOD (low doses methotrexate, bleomycin, epirubicin, cyclophosphamide, vincristine, dexamethasone), MVPP-Bleo (mitoxantrone, vincristine, prednisone, procarbazine, bleomycin) were administered by 9 cycles. Each cycle was followed by 10 days of G-CSF (5 micrograms/kg/day) started five days after chemotherapy compared to a control group which received chemotherapy without G-CSF support. Leucocytes and granulocytes were significantly higher in patients receiving G-CSF compared to the control group. The total number of days of leukopenia (WBC counts below 2.0 x 10(9)/L and absolute granulocytes below 1.0 x 10(9)/L) were longer in the patients without G-CSF compared to those who received G-CSF (14.1 days versus 1.9 days). Delays in treatment were most frequent in the control group: 38% versus 4% in all cycles. Infection episodes occurred in 41 out of 168 cycles (25%) in the control group compared to 7 out of 172 (4%) in the G-CSF arm. Complete response was achieved in 12 out of 22 (54%) in the control group compared to 16 out 20 (80%) in the patients who received G CSF. Toxicity secondary to G-CSF was mild. G-CSF can be administered safely to patients with DLCL and results in improved hematologic recovery after intensive chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532059 TI - Combined chemotherapy and granulocyte colony-stimulating factor (G-CSF) mobilise large numbers of peripheral blood progenitor cells in pretreated patients. AB - The aim of this study was to examine the effect of G-CSF given after salvage chemotherapy on the mobilisation of peripheral blood progenitor cells (PBPC) in pretreated patients. Seven patients with relapsed or refractory non-Hodgkin's lymphoma (NHL) were treated with methotrexate, cyclophosphamide, cytarabine, etoposide and dexamethasone. G-CSF was given at a dose of 3.8-7.2 micrograms/kg (1-2 ampoules) daily by subcutaneous injection from the onset of neutropenia (< 1.0 x 10(9)/L). A median of 3 leukaphereses was performed when the white cell count was recovering. The median number of granulocyte-macrophage colony-forming cells (GM-CFC) collected was 99 x 10(4)/kg per leucapheresis (range 19-800) or 260 x 10(4)/kg in total per patient (110-1800). Six patients underwent myeloablative chemotherapy with PBPC rescue. No autologous bone marrow or growth factors post-PBPC infusion were administered. The median duration of severe neutropenia (< 0.5 x 10(9)/L) was 8.5 days (range 5-10) and to recovery of neutrophils post-PBPC infusion was 11.5 days (10-15). Severe thrombocytopenia (< 20 x 10(9)/L) was present for 4 days (range 1-5) and the median number of days post-infusion to platelet-transfusion independence was 9 (6-12). In conclusion, G CSF combined with chemotherapy mobilised large numbers of PBPC for subsequent autotransplantation in pretreated patients with NHL. A single leukapheresis procedure may be sufficient following this protocol. PMID- 7532060 TI - Long-term outcome of postremission chemotherapy for adults with acute myeloid leukemia using different dose-intensities. AB - The long-term results of postremission chemotherapy for 122 consecutive, unselected adults (15-65 years) with acute myeloid leukemia (AML) were assessed in two sequential prospective studies involving an identical 3/7-type induction regimen, and in those achieving remission, another course for early consolidation using 1 day of daunorubicin instead of three. Forty-one patients reaching C.R. during the first study period, were treated with an intensive ablative maintenance ("IM") program for a period of 9 months. They were randomized to either 6 cycles of induction-type regimen or to 6 cycles of an alternating-type regimen consisting of high-dose (HD)-Ara C/AMSA or 5-azacytidine/AMSA every 6 weeks. There was no difference in disease-free survival (DFS) or survival. Results are compared with 27 patients reaching C.R. on the subsequent protocol where IM was replaced by intensive, short-term consolidation ("IC") using 1 cycle of intermediate-dose Ara C plus AMSA and 1 cycle of HD-AraC/AMSA. Fifteen patients received both courses of IC as scheduled, 12 refused the second cycle. There was no significant difference in DFS or survival. Seventeen out of the 122 patients refused either IM or IC following early consolidation ("refusals"). They received no further treatment and served as control. Fourteen percent of all patients underwent autologous or allogeneic bone marrow transplantation (BMT) at different stages of their disease, equally distributed amongst the IM and IC group. Median DFS was 3.3 months in the refusal group, 12.4 months in the IM group, and 18.4 months in the IC-group when censored for BMT (p = 0.01) with 6%, 12%, and 40% in C.C.R. at 50 months. Accordingly, median survival was 5.4, 20 and 47 months (p = 0.001) with 6%, 15%, and 45% of patients alive at 5 years. There was a definite trend (p = 0.14) for a higher proportion of long-term survivors in the IM-group when BMT was performed (not censored), while long-term survival was identical in the IC-group whether BMT was considered for analyses (not censored) or not (censored). Median follow-up for both studies is 5.6 years, the longest, 10 years. In conclusion, progressive increments in the intensity of postremission therapy yields in a graded, significant improvement of remission duration and survival. PMID- 7532061 TI - [Formation of parallel RNA-RNA duplexes in vitro]. PMID- 7532062 TI - [Aprotinin slows the effect of cycloheximide on DNA synthesis in heterodikaryon nuclei, obtained during fusion of resting and stimulated NIH 3T3 cells]. PMID- 7532063 TI - Clinical implications of qualitative and quantitative polymerase chain reaction analysis in the monitoring of patients with chronic myelogenous leukemia. The European Investigators on Chronic Myeloid Leukemia Group. AB - Recent progress in the development of diagnostic techniques has greatly facilitated the monitoring of minimal residual disease (MRD) in patients with hematologic neoplasia. The presence of genetic markers, such as the BCR/ABL rearrangement in chronic myelogenous leukemia (CML), has allowed highly sensitive detection of residual leukemic cells by polymerase chain reaction (PCR). However, complete eradication of the leukemic clone may not be a necessary prerequisite for long-term remission or cure. This observation limits the value of qualitative PCR analysis for prediction of progressive disease and highlights the need to monitor the proliferative activity of the malignant clone in order to permit timely detection of impending relapse. We have developed a quantitative PCR protocol based on the principle of competitive enzymatic amplification and demonstrated its applicability to the monitoring of treatment efficacy and early assessment of clonal expansion in patients with CML. We have introduced the term 'PCR relapse' for the detection of a proliferating leukemic clone by serial quantitative PCR (Q-PCR) analyses. At a recent meeting of the group of European Investigators on Chronic Myelogenous Leukemia (EICML Group) the use of Q-PCR investigation has been recommended for the monitoring of MRD, and the detection of PCR relapse was accepted as a basis for therapeutic decisions. This article discusses problems in the interpretation of MRD by PCR and presents guidelines for the clinical use of qualitative and quantitative PCR analyses. PMID- 7532065 TI - Change of serum alpha-1 microglobulin and beta-2 microglobulin following allogeneic bone marrow transplantation. AB - By serially measuring serum levels of alpha-1 microglobulin and beta-2 microglobulin following allogeneic bone marrow transplantation (BMT), we tried to define their relationship to renal dysfunction, acute graft-versus-host disease (GVHD) and infection as complications of the transplantation. The study involved a total of 25 patients with leukemia, myelodysplastic syndrome and aplastic anemia who received BMT in this department; one patient received re transplantation, thus bringing the total number of transplants to 26. Twenty-four patients received BMT from HLA-identical siblings while two others received BMT from unrelated donors. Alpha-1 microglobulin was within normal limits in all patients before BMT; among various complications such as nephrotoxicity, acute GVHD and infection which took place after transplantation, a raised alpha-1 microglobulin level was found only in nephrotoxicity; however, the increase was not significant compared with the pre-transplantation level. The pre transplantation beta-2 microglobulin level was higher than normal in some patients; it was significantly increased in all of the above complications compared with the pretransplantation level (1.57 +/- 0.57 mg/l). A significant correlation was found between the serum creatinine level and the beta-2 microglobulin level (r = 0.849) in patients with renal dysfunction. In some patients, however, the beta-2 microglobulin level increased earlier than the serum creatinine level, and this finding was considered useful for the early diagnosis of renal dysfunction following allogeneic BMT. PMID- 7532064 TI - Large scale ex vivo expansion and activation of human natural killer cells for autologous therapy. AB - Immunotherapy with recombinant interleukin-2 (rIL-2) activated natural killer cells (ANK) may be useful in the treatment of malignancies. Difficulties in large scale cultivation of purified ANK have hampered clinical trials. In a first set of experiments designed to characterize ANK precursors in blood we demonstrated that both FACS purified CD56+/CD3- and CD56-/CD3- cultured with rIL-2 give rise to an expanded cell population bearing the CD56+(bright)/CD3- phenotype and having both NK and lymphokine-activated killer (LAK) activity. Cultivation of NK was markedly enhanced by autologous monocytes. We next demonstrated that panning of peripheral blood stem cells on CD5/CD8 coated flasks yielded a starting population enriched for monocytes and NK precursors which after cultivation resulted in production of 4 x 10(10) highly cytotoxic ANK adequate for in vivo clinical trials. Finally, we demonstrated that ANK generated in high dose IL-2 maintain NK and LAK activity for up to 6 days when cultured in as little as 1 U/ml rIL-2. This may allow infusion of ANK with a rIL-2 dose achievable in vivo that does not produce significant systemic toxicity. We plan to test the efficacy of ANK to prevent relapse in a minimal residual disease state following autologous bone marrow transplant. PMID- 7532066 TI - Effects of ramipril on contractile oscillations in arteries from genetically hypertensive rats. AB - We have tested the hypothesis that altered vascular reactivity, specifically the appearance of spontaneous and BayK 8644 (L-type voltage gated calcium channel agonist)-induced oscillations in the carotid artery and the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA)-induced oscillations in the aorta from stroke-prone spontaneously hypertensive rats (SHRS), are dependent upon angiotensin II production early in life. SHRSP and normotensive Wistar-Kyoto (WKY) rats were treated from 6-10 weeks of age with vehicle, hydralazine/hydrochlorothiazide (used as a control for lowered blood pressure) or the angiotensin converting enzyme inhibitor ramipril (3 mg/kg/day). Systolic blood pressures were measured weekly in rats from 6 to 17 weeks of age. In SHRSP (at 17 weeks of age), ramipril-treatment but not hydralazine/hydrochlorothiazide attenuated the long term expression of elevated systolic blood pressure in adult SHRSP while blood pressures of all adult WKY rats were unaffected by any treatment. At 17 weeks, rats were killed and arteries removed for in vitro measurement of isometric contractile activity. Only the incidence of spontaneous oscillations (carotid artery) was affected by ramipril treatment; ramipril did not change the frequency of BayK 8644-induced oscillations in the artery or the frequency of CPA-induced oscillations in aorta from either SHRSP or WKY. These data indicate that while spontaneous oscillations in the carotid artery may be dependent on an angiotensin II-sensitive mechanism during development, agonist induced oscillations (CPA and BayK 8644) appear not to be angiotensin II dependent. Thus, not all of the contractile oscillations which appear in vascular smooth muscle from SHRSP are angiotensin II-dependent, suggesting that some of these vascular abnormalities may develop at a time separate from that in which increased blood pressure is firmly established and may not be associated with the for maintenance of elevated blood pressure. PMID- 7532067 TI - [Current data on rheumatoid factors]. AB - Rheumatoid factors are autoantibodies against the Fc fragment of IgGs. The polyclonal rheumatoid factors found in autoimmune diseases are distinct from the monoclonal rheumatoid factors produced in lymphoproliferative disorders in that they rarely express public idiotypes, are relatively specific for their Fc, and exhibit increased affinity. They may be derived from natural rheumatoid factors. The role of the latter is unclear but may involve presentation of immune complex antigens to T cells. PMID- 7532068 TI - [Paget disease, complicated by Ki-1 lymphoma with G-CSF growth factor secretion]. AB - Development of lysis within an area of pagetic bone suggests a sarcoma. We report the case of a 61-year-old woman who had increasingly severe pain in her right hip and a decline in general health. The roentgenogram of the pelvis showed previously known pagetic lesions of the right half of the pelvis and osteolysis of the roof of the acetabulum. Additional imaging studies (ultrasonography, computed tomography, and magnetic resonance imaging) disclosed a pelvic tumor extending on either side of the right ilium. The peripheral white blood cell count was markedly elevated (93,000/mm3) as a result of inappropriate secretion of granulocyte-colony-stimulating factor. The tumor was an anaplastic large cell lymphoma of T-cell origin with CD30 positivity, i.e., a Ki-1 lymphoma. Most CD30 positive lymphomas are high-grade tumors. There has been only one previous report of Ki-1 lymphoma with production of granulocyte-colony-stimulating factor and hyperleukocytosis. It has been suggested that cytokines may influence tumor growth. Unusual features in our case include development of the Ki-1 lymphoma in an area of pagetic bone and presence of a leukemic syndrome due to increased production of a growth factor. PMID- 7532069 TI - Endoscopic and ultrasonographic observation of the canine prostate after transurethral balloon laser therapy (PROSTALASE). AB - We investigated the healing process and changes in the canine prostate after transurethral balloon-equipped laser (PROSTALASE) prostatectomy using both endoscopy and transrectal ultrasonography (TRUS). On TRUS, the prostate volume was significantly increased (123%) just after irradiation, decreasing to 106% at 1 week. Cavity formation was observed in the prostate 1 week after irradiation, and the median volume was 1.5 cm3 at 4 weeks. A TRUS study performed immediately after irradiation revealed a 1- to 2-mm deep hyperechoic lesion around the urethra. Urethrocystoscopy disclosed red and markedly edematous mucosa of the prostatic urethra, but no hemorrhage. The hyperechoic mass was also observed in the cavity on TRUS images 1 week later. Urethrocystoscopy disclosed abundant necrotic tissue within the cavity, and reddish granulomatous sections were observed on a portion of the cavity surface. At 4 weeks, the hyperechoic mass in the cavity was no longer observed on TRUS images. The prostate parenchyma was thin but uniformly isoechoic. At urethrocystoscopy, a smooth surface without redness and with the small craters, which were histologically identified as the duct openings of the prostate gland, were observed. PMID- 7532070 TI - [Role of biological and anatomo-pathologic criteria in the prognosis evaluation of patients before and after radical prostatectomy]. AB - Radical prostatectomy is the treatment of choice for organ-confined prostatic cancers (T1-T2). However, it has been reported to improve the long-term survival of patients and achieve its oncological objectives in only one half of patients despite a serious morbidity. Based on a consecutive series of one hundred patients, the authors performed univariate statistical analysis to determine the predictive value of eight preoperative criteria for biological progression and capsular effraction: clinical stage, PSA (using a highly sensitive test derived from the Yang Proscheck and lowering the limit of detection to 0.1 ng/ml), PSA density (PSAD), percentage and topography of positive biopsies, capsular effraction, perineural spaces and Gleason's score. The predictive value for progression of five postoperative histological criteria (Gleason's score, capsule, perineural spaces, seminal vesicles and resection margins) was also studied in the same group of patients. The presence of capsular effraction on the biopsy was shown to have a positive predictive value of 90%. The three preoperative criteria most closely correlated with tumour progression were PSA, PSAD and percentage of positive biopsies (p < 0.001). Analysis of the combined predictive value of PSAD, percentage of positive biopsies and capsular effraction revealed that progression was always present when two of these criteria were positive. Seminal vesicle invasion on the operative specimen is the most pejorative element for progression. In conclusion, analysis of the histological features of biopsies associated with PSA and PSAD allows a more accurate selection of patients with a high risk of progression and seminal vesicle invasion is so pejorative that it could be detected in subjects with a preoperative PSA greater than 25 ng/ml. PMID- 7532071 TI - [Development and validation of a quality of life scale associated with health status, specific for benign hypertrophy of the prostate and including a sexuality evaluation scale]. AB - In order to develop a quality of life scale related to the state of health, specific for benign prostatic hypertrophy, a group of French specialists constructed a self-administered questionnaire, designed to complete the usual evaluations of the efficacy and safety of new medical treatments for this disease. This questionnaire was well accepted, reliable, clinically valid and sensitive to clinical changes occurring in a given patient. It includes questions concerning sexuality. An abbreviated form of the questionnaire was then developed in order to obtain a tool suitable for use in daily practice, in combination with the International Prostate Symptom Score (IPPS). PMID- 7532072 TI - Diabetes in the Americas. AB - Diabetes mellitus is an important cause of disability and death throughout the Americas. Of the three main types (insulin-dependent, noninsulin-dependent, and malnutrition-related), virtually all cases in the Americas are either insulin dependent (generally assessed in terms of incidence and usually occurring in subjects under 30) or noninsulin-dependent (generally assessed in terms of prevalence and usually occurring in subjects over 30). Data on noninsulin dependent diabetes mellitus (NIDDM) in various parts of the Americas point to prevalences ranging from 1.4% (among the Mapuche Indians in Chile) to 14.1% (among residents of Mexico City). However, the use of different methods and standards to gather and analyze these data renders comparison of the NIDDM situations in different countries uncertain. A fair amount of comparable data on insulin-dependent diabetes mellitus (IDDM) have been gathered in various countries of the Region. These point to marked differences in annual incidence- ranging from 0.7 cases per 100,000 in Peru to 27 among males on Prince Edward Island, Canada--that have not been adequately explained, underlining the need for additional comparable data. Considering the seriousness of the disease, it is important to know how many people have and develop diabetes, so as to be able to take preventive and therapeutic measures and guide public health actions. Hence, further cooperation directed at effective standardization of procedures and goals is indicated. Such cooperation, which should also come to include standardized national and hemispheric diabetes programs, must be achieved in accordance with the resources available to each country. PMID- 7532073 TI - Long-term outcome after severe brain injury in preschoolers is worse than expected. AB - OBJECTIVE: To determine the long-term outcome after severe brain injury at preschool age. DESIGN: Follow-up until adult life. SETTING: A centralized guidance center for all traffic-associated injuries. PATIENTS: Children with severe brain injury at preschool age between January 1959 and December 1969. MAIN OUTCOME MEASURE: Final evaluation in adulthood was performed by our team. The capability to work and live independently was rated. RESULTS: Twenty three (59%) of 39 children attended a typical school, eight (21%) attended a school for the physically disabled, and seven (18%) attended a school for the mentally retarded; information was not available for one child. In adulthood, nine patients (23%) were able to work full-time, 10 (26%) worked at sheltered workplaces, 14 (36%) lived independently at home, and six (15%) needed physical and/or psychotherapeutic support. The difference between normal school performance (59%) and capability to work full-time (23%) was significant (P < .05). In evaluating different aspects after the severe brain injury, the sense of identity was the best indicator of final outcome. CONCLUSIONS: The final evaluation of severe brain injury at preschool age should be performed in adulthood. Normal school performance or normal intelligence functioning is not a guarantee for good long term prognosis. To let the child develop a firm identity is essential for good outcome. PMID- 7532075 TI - Sinus histiocytosis with massive lymphadenopathy. PMID- 7532074 TI - A comparison of educational interventions. Multimedia textbook, standard lecture, and printed textbook. AB - OBJECTIVE: To compare the instructional effectiveness and efficiency of a pediatric multimedia textbook (MMTB) with that of a standard lecture and of a printed textbook in a prospective, interinstitutional study. DESIGN: Randomized, prospective cohort. SETTINGS: An urban and a rural medical school affiliated with tertiary care hospitals. POPULATION: Third- and fourth-year medical students from June 1992 to June 1993. INTERVENTIONS/OUTCOME MEASURES: Students were randomized to one of four treatment groups: (1) computer-aided instruction with MMTBs (n = 39), (2) traditional lecture (n = 39), (3) printed textbook (n = 39), or (4) a control group (n = 62). Only the control group was pretested. Following their randomized instruction, all groups were tested via a 26-question multiple-choice test. Statistical analysis was accomplished by analysis of variance of mean post test scores. The amount of time that students spent with each educational intervention was recorded. RESULTS: Three hundred two students were eligible for the study, 267 entered the study, and 179 completed the study. The instructional effectiveness of the MMTB was greater than that of the lecture (P < .05), and it was the same as that of the printed textbook. All instructional methods were more effective than the control group (P < .05). The instructional efficiency of the MMTB was equal to that of the lecture and of the printed textbook. The subjective response to the MMTB instruction was positive. CONCLUSION: The MMTBs constitute an educationally sound alternative instructional method and have a promising future in medical education. PMID- 7532076 TI - Microbore high-performance liquid chromatographic method for the measurement of dopamine and its metabolites: recommendations for optimal sample collection and storage. AB - A microbore high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of small quantities of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in microdialysis samples from rat striatum. The limit of detection of dopamine was in the low nmol/l range (10 fmol/40 microliters dialysate). Standard mixtures of dopamine and its metabolites were stored in tubes made of polypropylene, soda lime glass or borosilicate glass. Dopamine and homovanillic acid were stable in all three types of container for 24 h providing they were kept at an acidic pH. However, 3,4-dihydroxyphenylacetic acid was only stable in tubes made of polypropylene or borosilicate glass. Basal levels of dopamine in the dialysate obtained from rats which had been chronically implanted with a microdialysis probe in the anterior striatum were stable for one to three days following probe implantation. However, the levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the dialysate decreased rapidly with time after the first day. This indicates that the synthesizing capacity of the neurons is compromised by the microdialysis probe after day one, and that microdialysis experiments in freely moving animals should be confined to the day immediately following probe implantation. PMID- 7532077 TI - 'It's the little things that count': the hidden complexity of everyday clinical nursing practice. AB - This article reports on one aspect of a phenomenological study of everyday experience in nursing and how it contributes to the development of nursing expertise. The study was carried out with 10 'excellent, experienced' surgical ward sisters in two Scottish teaching hospitals. The ward sisters were found to help individual patients towards recovery by making the ward work for all. When moment-by-moment nursing practice, the context of nursing experience, is examined, it is found to be patient-centred and complex, geared towards multilayered goals. Three distinct yet inextricably intertwined processes- noticing, understanding and acting--characterize how nurses practise nursing. The quality of these processes contributes to the effectiveness of nurses' caring practices. The 'little things' of nursing practice make a difference to patients because they are imbued with nursing knowledge and skill. PMID- 7532078 TI - Induction of nitric oxide synthase activity in phagocytic cells inhibited by tricyclodecan-9-yl-xanthogenate (D609). AB - 1. The synthesis of nitric oxide (NO) by immune-stimulated murine phagocytic cells (J774) and the modulation of this synthesis by tricyclodecan-9-yl xanthogenate (D609), a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), was investigated. D609 dose-dependently suppressed production of NO, as measured by the release of nitrite and nitrate, in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in intact cultured cells with an IC50 of approximately 20 micrograms ml-1. D609 at 40 micrograms ml 1 completely abrogated immune-stimulated nitrite production. 2. The inhibitory effects of D609 on nitrite production were time-dependent and restricted to the first 18 h post-stimulation. D609 did not inhibit nitrite production in the cytosol of immune-stimulated phagocytes. 3. These findings indicate that the xanthogenate, D609, is a potent inhibitor of the induction of NO-synthase activity in immune-stimulated phagocytes. Furthermore, since D609 has been demonstrated to inhibit PC-PLC specifically, our findings suggest that the activation of this enzyme by LPS and IFN-gamma is a proximal step in the signal transduction of inducible NO-synthase in phagocytic cells. PMID- 7532079 TI - Contribution of ATP and nitric oxide to NANC inhibitory transmission in rat pyloric sphincter. AB - 1. Changes in isometric tension were recorded from circular muscle strips of rat pyloric sphincter in vitro, in response to electrical field stimulation and exogenously applied muscle relaxants. 2. Concentration-response relationships were studied for relaxation to exogenously applied adenosine 5'-triphosphate (ATP) and two analogues, 2-methylthioATP (2-MeSATP) and alpha,beta-methylene ATP (alpha,beta-MeATP). These drugs evoked concentration-dependent relaxation of rat pyloric sphincter with an order of potency 2-MeSATP > ATP >> alpha,beta-MeATP, indicating the presence of P2y-purinoceptors. The IC50 value of each nucleotide was: 2-MeSATP, 5.0 x 10(-8); ATP, 7.9 x 10(-6) M; alpha,beta-MeATP showed only slight activity at a concentration of 0.1 mM. 3. Frequency-response relationships for relaxations evoked by electrical field stimulation (EFS) were studied in the absence and presence of 10 microM NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide (NO) synthesis) and 20 microM reactive blue 2 (a P2y purinoceptor antagonist). It was found that these substances significantly reduced the relaxant response of rat pyloric sphincter to EFS by 40% and 50% respectively. In the presence of both L-NAME and reactive blue 2 the responses were reduced by 75%. 4. Concentration-response relationships were studied for ATP and 2-MeSATP in the presence of L-NAME. It was found that L-NAME did not significantly inhibit the relaxant responses to these drugs. 5. Concentration response relationships for ATP and noradrenaline were studied in the presence of reactive blue 2 (20 microM); the P2y-antagonist significantly inhibited the relaxant response to ATP, but not that to noradrenaline. 6. The distribution of nitric oxide synthase in rat pyloric sphincter was investigated immunohistochemically,with immunoreactive nerve fibres found throughout the circular muscle layer and myenteric plexus of the sphincter.7. While abundant vasoactive intestinal polypeptide (VIP)-containing nerve fibres were demonstrated immunohistochemically in the pyloric sphincter, relaxations to VIP (1 nM-0.3 micro M) were not observed in this preparation.8. It is concluded that ATP, acting through P2y-purinoceptors, and NO contribute to NANC inhibitory neurotransmission in rat pyloric sphincter. NO appeared to contribute to the later component of NANCrelaxation. The action of ATP was not mediated by NO, and VIP did not contribute to the NANCinhibitory responses in this preparation. PMID- 7532080 TI - Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy. AB - 1. The profiles of cyclo-oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin-induced pleurisy model of acute inflammation. 2. The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3. COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6-keto prostaglandin F1 alpha) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4. NOS activity was based on the conversion of [3H]-L-arginine to [3H]-L-citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5. Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6. Western blot analysis demonstrated that the inducible isoform of COX (COX-2), was the predominant enzyme at all time points. Low levels of COX-2 were seen in PBLs. In pleural exudate cell pellets maximal COX-2 protein levels were seen at 2 h. 7. Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX-2. In cell pellet smears from carrageenin induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX-2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX-2.8. Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6 h and greatly reduced by 24 h. This activity was solely attributable to iNOS.9. The present results illustrated a similar profile of COX and NOS activity in the carrageenin-induced pleurisy model of acute inflammation. It was demonstrated that COX-2 and iNOS were the predominant isoforms of their respective enzymes. PMID- 7532081 TI - Antagonistic properties of McNeil-A-343 at 5-HT4 and 5-HT3 receptors. AB - 1. This study describes the in vitro interaction of the muscarinic ligand McNeil A-343 with two 5-hydroxytryptamine (5-HT) receptor subtypes, the 5-HT4 and 5-HT3 receptors, using functional as well as radioligand binding studies. 2. In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil-A-343 was a competitive antagonist (pA2 6.2) of the 5-HT4 receptor which mediates the relaxation induced by 5-HT. The compound per se relaxed the oesophagus at high concentration only (> or = 10 microM), an effect unchanged by desensitization of the 5-HT4 receptor with 10 microM 5-methoxytryptamine. In the same preparation in the absence of tone, McNeil-A-343 displaced the carbachol concentration-response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3. In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil-A-343 caused a concentration-dependent depolarization that was unaffected by 100 nM ondansetron. The concentration-fast depolarization curve to 5-HT, mediated by the 5-HT3 receptor, was displaced to the right by McNeil-A-343, which showed an apparent affinity (pA2) of 4.8 for the 5-HT3 subtype. 4. In binding studies, McNeil-A-343 recognized a single population of 5-HT4 receptors in pig caudate nucleus, with a pKI of 5.9. The binding affinity of McNeil-A-343 for 5-HT3 receptors in NG 108-15 cells was approximately four times lower (pKI 5.3). Binding affinities (pKI) for muscarinic receptor subtypes in rat tissues were 5.3 (M1, cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively. 5. McNeil-A-343 is an antagonist at 5-HT4 and 5-HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5-HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors. PMID- 7532084 TI - The peculiar scope of "supportive care in cancer"! PMID- 7532083 TI - Modulatory effect of neuropeptide Y on acetylcholine-induced oedema and vasoconstriction in isolated perfused lungs of rabbit. AB - 1. The modulatory role of neuropeptide Y (NPY) on pulmonary oedema induced by acetylcholine and capsaicin was investigated. The effects of NPY on the haemodynamic response to acetylcholine, phenylephrine and substance P were also investigated. 2. Isolated, ventilated, exsanguinated lungs of the rabbit were perfused with a constant flow of recirculating blood-free perfusate. The double/arterial/venous occlusion method was used to partition the total pressure gradient (delta Pt) into four components: the arterial gradient (delta Pa), the pre- and post-capillary gradients (respectively delta Pa' and delta Pv') and the venous pressure gradient (delta Pv). Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). 3. Acetylcholine (10(-8) M to 10(-4) M) and substance P (SP, 10(-10) M to 10(-6) M) induced a concentration dependent increase in the Kf,c. Capsaicin (10(-4) M) and 5-hydroxytryptamine (5 HT) (10(-4) M) also increased this parameter. NPY (10(-8) M) completely inhibited the effects of acetylcholine and capsaicin on the Kf,c, without preventing the effects of substance P and 5-HT. 4. Acetylcholine induced concentration-dependent vasoconstriction in the precapillary segment. The effect was inhibited by NPY and aspirin, an inhibitor of cyclo-oxygenase, while ketanserin, a 5-HT2 receptor antagonist, and SR140333, a new NK1 antagonist, had no protective effect. Phenylephrine increased delta Pa at high concentration, an effect also inhibited by NPY and aspirin. Substance P had no significant haemodynamic effect. When injected together with NPY, substance P (10(-6) M) induced a significant increase in the total pressure gradient. 5. It was concluded that NPY can protect the lung against acetylcholine- and capsaicin-induced oedemavia a prejunctional modulatory effect on the C-fibres. NPY also inhibits acetylcholine-evoked precapillary and phenylephrine-induced arterial vasoconstriction, probably by interfering with cyclo-oxygenase products synthesis. PMID- 7532085 TI - Retroelements, reverse transcriptase and evolution. AB - Retroelements are genetic elements that can exist as DNA or RNA or DNA/RNA duplexes. Although retroviruses are the best known retroelements, there are many other types, including close relatives of retroviruses like LTR retrotransposons, more distant relatives like non-LTR retrotransposons, caulimoviruses and hepadnaviruses and elements with virtually no similarity, like retrons. Virtually all retroelements are 'selfish DNAs' with no involvement with the normal development or maintenance of their host cells, the only known exception being telomereres/telomerases which maintain the ends of chromosomes. Virtually all retroelements use tRNA, or RNA with strong secondary structure, to initiate their reverse transcription. The coincidence between the use of tRNA, a molecule central to the conversion of RNA to protein, with reverse transcriptase, an enzyme which is crucial for the conversion of RNA to DNA is striking, because RNA probably preceded DNA and protein in evolution. It seems plausible that retroelements were present at the genesis of living systems. PMID- 7532082 TI - The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites. AB - 1. We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with gamma-interferon (IFN), showing that polyamines may act as suppressants of NO-mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action. 2. Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine >> putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant. 3. Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L-arginine to L-citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS. 4. Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum. 5. The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxylamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10(-5) M) inhibits nitrite formation by IFN-activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro. 6. The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS-induced production of prostaglandin F2 alpha or tumour necrosis factor. PMID- 7532086 TI - Immunotoxins composed of monoclonal antibody to alpha-fetoprotein and gelonin as a potent hepatoma-targeted drug delivery system. AB - This study was carried out to evaluate our monoclonal antibody (MoAb) to alpha fetoprotein (AFP), 80G, as a carrier for targeting AFP-producing hepatoma. Pharmacokinetic analysis showed that the MoAb 80G was actively incorporated into AFP-producing HuH-7N cells (xenograft of human hepatoma cell line, HuH-7) in nude mice. Four conjugates composed of MoAb 80G, and a type 1 ribosome-inactivating protein, gelonin, were prepared. They involve two disulfide-linked and two thioether-linked conjugates. The binding activity of conjugates against AFP remained as high as that of intact 80G according to enzyme-linked immunosorbent assay. The in vitro cytotoxic effects of all the conjugates were specific against AFP-producing HuH-7 cells. Of these conjugates, two containing gelonin modified with 2-iminothiolane were more potent than the others. They showed significant antitumor activity upon AFP-producing HuH-7N cells in nude mice. However, the disulfide conjugate was more toxic to mice than the thioether conjugate judging from the loss in body weight and the liver damage. These results suggest that our MoAb 80G is a suitable carrier for targeting AFP-producing hepatoma cells, and that the noncleavable thioether conjugate is promising as an AFP-producing hepatoma-targeted drug delivery system. PMID- 7532087 TI - Efficacy of aprotinin in different doses and autologous blood transfusions in cardiac surgery. AB - A study was undertaken to compare the two doses of aprotinin and the amount of autologous blood transfused in order to determine the optimal dose of this inhibitor for use in cardiac surgery. A total of 120 patients undergoing elective cardiac surgery from November 1990 to April 1992 took part in this randomized double-blind study. Two groups of 60 patients were treated. Patients in the high dose group were given the dose of aprotinin recommended by the Hammersmith group (6 million kallikrein inactivator units), the other 60 (the low-dose group) received half the dose. Blood loss from thoracic drains in the postoperative period showed a statistically significant difference between the two groups between 6 and 12 h (a mean loss of 69 ml in the high-dose group versus 109.5 ml in the low dose, P = 0.003). The overall postoperative drainage losses were very similar (537.2 ml in the high-dose group versus 610.9 ml in the low dose). The blood and clotting markers did not differentiate between the high- and low-dose patients. In combination with autologous blood transfusions, a low dose of aprotinin appears as efficient in reducing postoperative blood loss as the high dose regimen. PMID- 7532088 TI - Thrombosed coronary artery fistula as a cause of paroxysmal atrial fibrillation and ventricular arrhythmia. AB - This case report presents a 68-year-old woman with a symptomatic coronary artery fistula. Usually these rare congenital malformations are symptom-free but in this patient the fistula began to cause various arrhythmias after it thrombosed. Her symptoms resolved completely after connective surgery. PMID- 7532089 TI - The nephrotoxicity of FK506 as compared with cyclosporine. AB - FK506, a new macrolide immunosuppressant agent, is approximately 100 times more potent than cyclosporine. Early clinical trials demonstrated FK506 to be effective in reversing refractory rejection in liver, kidney, and heart transplantation. Like cyclosporine, FK506 has significant nephrotoxicity. The clinical presentation and morphology of FK506 nephrotoxicity are identical to those of cyclosporine. Many animal and in vitro studies suggest that FK506 may be less nephrotoxic than cyclosporine. Studies in humans after transplantation have not confirmed this advantage. FK506 has pursued the same pattern in drug development as cyclosporine, with progressive dose reductions over the years. Studies from this early developmental period suggest that the nephrotoxicity of FK506 and cyclosporine in clinical use are approximately equivalent. Further refinement in the clinical use of FK506 will likely reduce its toxicity further. Appropriate studies conducted at that stage will determine which drug possesses less nephrotoxicity. PMID- 7532091 TI - Endothelial cell activation in patients with systemic vasculitis. AB - Vascular endothelial cells respond in vitro to a number of stimuli, and in particular to cytokines, by undergoing functional and morphological alterations which endow them with the capacity to promote inflammatory reactions. We studied this process of endothelial cell activation in 20 skin biopsies from 18 patients with systemic vasculitis. At sites of cutaneous inflammation, blood vessels were lined with swollen endothelial cells which expressed increased levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and were associated with a mononuclear cell inflammatory infiltrate. Neutrophil infiltration was only found in the presence of endothelial leucocyte adhesion molecule-1 (ELAM-1), which was expressed in 15/20 biopsies. ELAM-1 and VCAM-1 were associated with the presence of inflammatory cytokines which induce expression of these molecules in cultured endothelial cells. Endothelial activation in vivo appears to parallel that observed in vitro, and is likely to be important in determining the nature of an inflammatory response. PMID- 7532090 TI - Nephrotoxicity of immunosuppressants in rats: comparison of macrolides with cyclosporin. AB - The nephrotoxic potential of the macrolide immunosuppressants, FK506 and rapamycin, was compared with that of cyclosporin (CsA) in male Wistar rats. FK506 induced a reduction of creatinine clearance, hypomagnesemia and hyperuricemia as previously described for CsA. In contrast, equidosed rapamycin did not alter the glomerular filtration rate. FK506 caused proximal tubular epithelial changes consisting of atrophy, vacuolization, inclusion bodies, microcalcification and focal mononuclear interstitial infiltrate as described for CsA. The most striking alteration was hypertrophy of the juxtaglomerular apparatus (JGA). The percentage of renin-containing JGA and the extent of renin immunoreactivity along afferent vessels were significantly increased in FK506- and CsA-treated rats. By contrast, no renal morphologic lesions were found in rapamycin-treated animals. Renal cortical extracts contained abundant cyclophilin and FK506-binding protein (FKBP), the main intracytoplasmic receptors for CsA and FK506, respectively. Furthermore, we demonstrated that receptor bound CsA and FK506, but not rapamycin, formed complexes with the phosphatase calcineurin, as shown previously for lymphocytes. Thus, it is hypothesized that both the immunosuppressive and toxic effects of FK506 and CsA, but not of rapamycin, are mediated through an immunophilin-drug-calcineurin complex. The renal substrate of calcineurin, which mediates renal vasoconstriction is yet to be identified. PMID- 7532092 TI - The endogenous substrate of low molecular weight acid phosphatase in the brain is an epidermal growth factor receptor. AB - We have recently reported that low molecular weight (LMW) acid phosphatase, which is supposed to possess phosphotyrosine protein phosphatase activity, showed a significant decrease of activity in Alzheimer brains compared to control brains [Ann. Neurol., 33 (1993) 616-621]. In the present study, we investigated the endogenous substrate of LMW acid phosphatase in the brain. LMW acid phosphatase was purified from bovine brain, and the enzyme was obtained with both a high specific activity and a good yield. The bovine brain enzyme was a monomer with a molecular mass of 17 kDa. We used a specific monoclonal anti-phosphotyrosine antibody to detect phosphotyrosine protein in rat brain extracts. The LMW acid phosphatase from bovine brain dephosphorylated a M(r) 170 kDa phosphotyrosine protein in rat brain extracts. This M(r) 170 kDa protein was considered to be the epidermal growth factor (EGF) receptor using a specific antibody. These results suggest that LMW acid phosphatase in the brain may regulate EGF receptor dependent transmembrane signalling by dephosphorylating the phosphorylated receptor. PMID- 7532093 TI - Primary culture of circumventricular organs from the rat brain lamina terminalis. AB - A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes. PMID- 7532094 TI - Changes in monoamine turnover in forebrain areas associated with masculine sexual behavior: a microdialysis study. AB - This report compares the changes in the main dopamine (DA) and serotonin (5-HT) metabolites, respectively dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy indoleacetic acid (5-HIAA) in three relatively close brain regions, namely the nucleus accumbens (ACB), the medial preoptic area, and the medial basal hypothalamus (MBH), as well as DA in the ACB, of copulating male rats. All these neurochemicals remained fairly stable when the animals were exposed to non sexual social stimuli (castrated females), and they increased during mating with receptive females. There were regional differences in these copulation-related changes, however, with those in the MBH being shorter-lived. There were also differences in the time-course of the changes in DOPAC and 5-HIAA the latter being slower. It is suggested that they reflect the involvement of the DA and 5 HT innervation of diencephalic structures in, respectively the appetitive and consummatory/satiation mechanisms of sexual behavior. The physiological relevance of these neurochemical changes is supported by the lack of differences between the standard measures for sexual behavior recorded before surgery and during the dialysis session. PMID- 7532095 TI - Evidence that Ca(2+)-activated K+ channels participate in the regulation of pituitary prolactin secretion. AB - We evaluated the role of Ca(2+)-activated K+ channels in the regulation of prolactin (PRL) secretion with a perifusion system using acutely dispersed rat anterior pituitary cells. Apamin, which blocks Ca(2+)-activated K+ channels, induced PRL secretion in a dose-dependent fashion between 1 and 300 nM (r = 0.99, P < 0.01). Charybdotoxin, another Ca(2+)-activated K+ channel-blocker, also induced PRL secretion at 20 nM concentration. These were not non-specific toxic effects, since stimulation of PRL secretion by 10 nM thyrotropin-releasing hormone (TRH) was not different before and after applying the channel-blockers. Both 10 microM dopamine and 2 microM nifedipine significantly, but incompletely, depressed PRL secretion induced by 100 nM apamin; 10 microM dopamine completely blocked PRL secretion induced by 20 nM charybdotoxin. Our data indicate that Ca(2+)-activated K+ channels may play an important role in the regulation of PRL secretion. PMID- 7532096 TI - The vascular response to nerve transection: neovascularization in the silicone nerve regeneration chamber. AB - The rat sciatic nerve regeneration chamber was used to study the spatial and temporal response of the endoneurial vasculature during regeneration. Proximal and distal stumps of a transected rat sciatic nerve were placed in opposite ends of a silicone tube and allowed to regenerate for periods of 2, 3, 4 or 52 weeks after the surgery. Serial, transverse sections of nerve were studied at each time point to quantitate the number of vessels, capillary density and the vessel luminal perimeter per nerve area. The results indicate that the vascular growth relative to the existing tissue in the chamber increases to a peak beyond normal levels and later decreases to values associated with control tissue. While this growth occurred from both the proximal and distal stumps, it appeared predominantly as a traveling wave in the proximal-distal direction preceding the major thrust of neuritic outgrowth from the proximal stump. Morphologic measurements of angiogenesis were paralleled in other animals by measurements of nerve blood flow using laser Doppler flowmetry at corresponding time-points. These data differ somewhat from previous reports of angiogenesis following nerve crush injury and are useful in formulating a general mathematical model of regeneration in the peripheral nervous system. PMID- 7532097 TI - Neurotrophic factor gene expression in astrocytes during development and following injury. AB - Astrocyte cultures were utilized to examine regulation of the expression of several trophic factor genes. Regulation by the beta-adrenergic receptor was demonstrated by exposure of striatal and cortical astrocytes to isoproterenol, which resulted in increased content of mRNAs for nerve growth factor (NGF), brain derived neurotrophic factor, and proenkephalin (PE), as well as NGF and Met enkephalin. Developmental regulation was analyzed by preparing cortical astrocytes from animals of four different ages--embryonic day 20, postnatal days 3 and 8, and adult. Because both the PE and NGF genes showed developmental downregulation, we asked whether we could prepare reactive astrocytes from lesioned adult brain and see expression turned back on. Astrocytes prepared from 6-hydroxydopamine-lesioned rat striatum or MPTP-lesioned mouse striatum contained increased GFAP and NGF mRNA. Comparable changes in GFAP and NGF could be achieved by treatment of control cultures with interferon-gamma or interleukin-1 beta. These results suggest that locus coeruleus neurons could control astrocyte synthesis of neurotrophic factors through release of of norepinephrine, but that in injured brain other factors, such as cytokines, may become equally important. PMID- 7532098 TI - Degeneration of locus coeruleus axons in stress-induced depression model. AB - Antidepressants such as desipramine induce axonal regeneration of brain noradrenergic neurons. This novel action of antidepressants suggests the involvement of degeneration or retraction of brain noradrenergic axons in the pathophysiology of clinical depression. The present study was designed to further confirm this view in an animal model of stress-induced depression. The depression model was produced by exposing rats to prolonged forced walking stress. To see if axonal degeneration of noradrenergic neurons occurred in the depression model, the density of noradrenergic axons in the cerebral cortex was assessed by three different methods, antidromic stimulation technique, retrograde tracing with horseradish peroxidase and immunohistochemical staining with dopamine-beta hydroxylase antiserum. These methods all assured of degenerative changes of noradrenergic axon terminals in the depression model. Furthermore, it was found that repeated treatments of the depression-model rats with imipramine could cause regeneration of cortical noradrenergic axons. These findings support the view that degeneration or retraction of noradrenergic axons is involved in the pathophysiology of depression. PMID- 7532101 TI - Detection of proteins and sialoglycoproteins in polyacrylamide gels using eosin Y stain. AB - An eosin Y staining technique that permits detection of various proteins, including membrane sialoglycoproteins, in polyacrylamide gels is described. The sensitivity of the eosin Y staining method is comparable to silver staining. In addition, there is an added advantage of the antigenicity of the stained proteins being retained in a Western blot. Details of the procedure to obtain optimal staining results are described. PMID- 7532100 TI - In vitro assessment of compounds for anti-HIV activity. AB - Human immunodeficiency virus types 1 and 2 (HIV-1, -2), the etiological agents of AIDS, are retroviruses that replicate in CD4+ T lymphocytes, monocytes, and macrophages. Early anti-HIV therapies were directed at the step in the virus life cycle that was considered the most readily blocked: the transcription of viral RNA into its DNA copy by the viral enzyme reverse transcriptase (RT). Unfortunately, to date, patient therapies have been relatively unsuccessful and hampered by toxicological problems. This has promoted further research into inhibitors of HIV replication, which may act at alternative stages in the viral replicative cycle as well as more effective RT inhibitors. In order to facilitate such research, simple and accurate in vitro assays are highly desirable. Here we describe such assays that measure components of the HIV replicative cycle and are suitable for use within antiviral experiments. PMID- 7532099 TI - Neither whole inactivated virus immunogen nor passive immunoglobulin transfer protects against SIVagm infection in the African green monkey natural host. AB - Attempts to protect against infection with simian immunodeficiency virus (SIV)mac grown in rhesus peripheral blood mononuclear cells (PBMCs) using whole inactivated virus immunogen or passive transfer of antibody have so far universally failed. However, such experiments have succeeded in the closely related human immunodeficiency virus (HIV)-2/cynomolgus system. To determine whether the failure in the SIVmac system is typical of primate lentiviruses we performed vaccination and passive transfer experiments using SIVagm (genetically distinct from SIVmac and HIV-2) in the natural African green monkey (AGM) host. To maximize the chances of success, both immunogen and challenge material were prepared using the molecular SIVagm3 clone. Two AGMs were immunized four times with whole inactivated SIVagm3 in RIBI adjuvant and two received intravenously a high dose of immunoglobulin (Ig) purified from a mixture of plasma from AGM, either naturally infected or infected with the homologous SIVagm3 clone. All were challenged with 20 median minimum infective doses of the SIVagm3 grown in AGM PBMCs and previously titrated in AGMs. Despite a strong humoral immune response in all monkeys at the time of challenge, including measurable neutralizing and antibody-dependent cell-mediated cytotoxicity antibodies, none were protected from infection. PMID- 7532103 TI - Quantitative and qualitative analysis of exogenous gene expression by the S1 nuclease protection assay. AB - A method for determining the beginning and end of gene transcripts along with the position of intron/exon boundaries by S1 nuclease protection is described. Probe strategies and hybridization conditions are considered in detail. Under conditions of probe excess the method is quantitative. PMID- 7532102 TI - Nonradioactive labeling and high-sensitive detection of PCR products. AB - The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin (DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin labeled amplification products as hybridization probes, sensitivities in the 0.1 pg range are obtained in Southern blot procedures. PMID- 7532105 TI - [Hyperthyroidism, the cardiovascular apparatus and physical exercise]. PMID- 7532104 TI - Protein (western) blotting. AB - The different steps involved in protein (Western) blotting and subsequent analysis of the proteins are reviewed. Electrophoretic separation of proteins, procedures of transfer to membranes, immunological and nonimmunological protein detection systems, and characterization of protein-nucleic acid and protein protein interactions are described. Emphasis is on the sensitivity of the methods described and on possible variations that allow the individual steps of Western blotting to be adapted to specific questions. PMID- 7532106 TI - The imidazole antimycotics econazole and miconazole reduce agonist-evoked protein tyrosine phosphorylation and evoke membrane depolarisation in human platelets: cautions for their use in studying Ca2+ signalling pathways. AB - In many cell types depletion of the intracellular Ca2+ stores promotes divalent cation entry across the plasma membrane, although the mechanism of such store regulated Ca2+ entry remains unclear. It has been suggested that cytochrome P-450 plays a role in the communication from intracellular stores to plasma membrane in human platelets and other cells. These studies involved the use of the imidazole antimycotics, econazole and miconazole, which are inhibitors of cytochrome P-450. Here we report additional effects of the imadazole antimycotics, which we show to inhibit agonist-evoked protein-tyrosine phosphorylation and to evoke plasma membrane depolarisation in human platelets. Both of these effects might be expected to influence agonist-evoked Ca2+ entry in these and other cells. These data suggest that great caution is required in interpreting the results from studies using imadazole antimycotics and in particular imply that the effects of these inhibitors cannot be taken as evidence for a role for cytochrome P-450 in the Ca2+ entry pathway. PMID- 7532107 TI - [Superinfection of hepatitis viruses]. AB - Using ELISA method, anti-HAV-IgM and anti-HCV were determined in 493 blood donors and 192 patients with HBV infection. The positive rate of anti-HCV in blood donors was 1.62%. The superinfection rates of HBV and HAV, HBV and HCV, HBV, HAV and HCV in patients with HBV infection were 16.7%, 3.13% and 0.52%, respectively. The incidence of hepatitis B viral replication marker was found to be lower in patients with superinfection of types A and B and/or C hepatitis viruses than in those with HBV infection alone. The prolonged course was found to be longer and the degree severer in patients with superinfection of types A and B and/or C hepatitis viruses than in those with HAV or HBV or HCV infection alone. The results suggest that those patients with superinfection of types A and B and/or C hepatitis viruses showed poor prognosis and prolonged course, and many patients with chronic Type A hepatitis resulted from superinfection HBV and/or HCV. PMID- 7532108 TI - [An investigation on the present situation of HCV infection patients with liver diseases and in blood recipients in Baise District of Guang Xi]. AB - ELISA was used to detect anti-HCV antibody in 179 serum samples of patients with various types of liver diseases and 41 serum samples of blood recipients in Baise district of Guang Xi. The results showed that 17.9% of patients with various types of liver diseases and 31.7% of blood recipients were anti-HCV positive. Among patients with liver diseases, the anti-HCV positive rate was 4.3% (1/23) in acute hepatitis (AH), 12.8% (10/78) in chronic hepatitis (CH), 28.6% (12/42) in liver cirrhosis (LC) and 25.0% (9/36) in hepatocellular carcinoma (HCC). As liver diseases became chronic, the anti-HCV positive rate tended to rise. The anti-HCV positive rate in LC was significantly higher than that in AH or CH (P < 0.05). The anti-HCV positive rate in HCC was not significantly different from that in AH or CH (P > 0.05 or 0.1). It was found that the anti-HCV positive rate in HBsAg negative patients was significantly higher than that in HBsAg-positive patients (P < 0.05). The anti-HCV positive rate in liver diseases was not related to ALT (P > 0.05). In blood recipients, the anti-HCV positive rate was closely related to the number of transfusion and the activity of ALT. PMID- 7532109 TI - [Epidemiology of melioidosis in China]. AB - From 1975 to 1989, a total of 73 strains of P. pseudomallei was isolated from the water samples and the pathological samples of human and domestic animals in 13 counties and cities located different latitude from four provinces Qiong, Yue, Gui and Xiang in China. Serological investigation demonstrated that the geographical distribution of the organism had a significant correlation with the positive rate of antibodies against P. pseudomallei and the native foci of the organisms distributed over the southern subtropical zone and the edge of tropical zone in Qiong, Yue and Gui. In endemic areas, the positive rates of antibodies against P. pseudomallei in human-beings, horses, oxen and pigs are 3.8%-15.2%, 9.1%-18.4%, 6.6%-33.0% and 35% respectively. The investigation results showed the horses and mules infected by the organism would interfere with quarantine of the animals, meanwhile, the meat contaminated by the bacterium would endanger the public health. In Sept. and Oct. of 1989, three cases in Zhanjiang and Sanya of Hainan were reported, two cases died of acute melioidosis with septicemia, another case was the chronic leg ulcers. So, it was predicated that there could have some cases of melioidosis which were misdiagnosed or missed out. PMID- 7532110 TI - alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. AB - Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli. PMID- 7532112 TI - Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger. AB - Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression. Both genes were found to be under complex regulatory control. The expression of the two genes could be turned off by the presence of good nitrogen or carbon sources in the media, and external protein sources did not induce expression of either gene under conditions of carbon and nitrogen repression. The pH of the medium also played a major role in their regulation as the expression of both genes was completely turned off under alkaline conditions, even when grown in media lacking good nitrogen and carbon sources but containing proteins. We isolated clones containing 5' non-coding sequences of the pepA gene from a lambda genomic library with a pepA specific probe. Analysis and comparison of the promoter sequences of the pepA and pepB genes revealed that both contain several putative AREA- and CREA-binding sites and they also share an 18-bp-long sequence which is 83% identical in these two genes. PMID- 7532113 TI - [Cytokeratin expression in spinocellular carcinoma of the uterine cervix]. AB - Expression of cytokeratins 7, 8, 10, 14, 18 and 19 was studied in 48 cases of advanced (stage II and III) squamous cell carcinomas of the uterine cervix. Despite of the degree of differentiation, the expression of simple epithelia cytokeratins 8 (72.9%) and 19 (97.9%) was high. A subset of eight (16.6%) predominantly poorly differentiated tumours had the expression restricted to simple epithelia pattern (cytokeratin 7 and/or 8 and 18 and/or 19). In twenty cases (41.7%) the cytokeratin 14 was added to this pattern, representing an intermediate differentiation level, while the other twenty tumours, usually exhibiting more pronounced squamous differentiation had the most complete cytokeratin pattern including class 10. This grouping was of no prognostic significance but might represent a valuable tool in the classification of cervical squamous cell carcinoma. PMID- 7532114 TI - Development of bioactive functions in hydrangeae dulcis folium. III. On the antiallergic and antimicrobial principles of hydrangeae dulcis folium. (1). Thunberginols A, B, and F. AB - From the less polar fraction of Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla Seringe var. thunbergii Makino, Eight antiallergic and antimicrobial principles were isolated together with several known compounds. Among the newly isolated bioactive constituents, the chemical structures of thunberginols A, B, and F have been determined on the basis of chemical and physicochemical evidence. Thunberginols A, B, and F were found to exhibit more potent antiallergic activity than phyllodulcin, hydrangenol, disodium cromoglycate (DSCG), and tranilast. In addition, these thunberginols showed antimicrobial activity against oral bacteria. PMID- 7532111 TI - PMA and calcium ionophore induce myosin and F-actin rearrangement during histamine secretion from RBL-2H3 cells. AB - Rat basophilic leukemia (RBL-2H3) cells undergo morphological and cytoskeletal changes during antigen-induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA+A23187 causes extensive F-actin rearrangements during secretion of [3H] 5-HT. We also describe for the first time the association of myosin with F-actin during this secretory process. In unstimulated cells, myosin and F-actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F-actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F-actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two-fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin-decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed. PMID- 7532115 TI - Retinoid regulation of cell differentiation in a series of human papillomavirus type 16-immortalized human cervical epithelial cell lines. AB - Retinoids are important regulators of cervical epithelial cell differentiation and have been used in the treatment of cervical cancer. In the present study we evaluate the effects of retinoic acid on expression of biochemical markers of differentiation and expression of human papillomavirus reading frames encoding the early gene products E6 and E7 in normal and HPV16-immortalized cervical epithelial cell lines. Our results indicate that the differentiation markers cytokeratins K5 and K16 and transglutaminase type 1 are suppressed by all-trans retinoic acid (RA). A marked concentration-dependent reduction in the level of of each mRNA is observed with maximal suppression at 1 microM. Each of the HPV16 immortalized cell lines (ECE16-1, ECE16-D1 and ECE16-D2) are more sensitive to the effects of RA than normal cells. The level of HPV16 transcript encoding E6/E7 is not significantly suppressed by 1 microM RA in ECE16-1 cells, but is suppressed in ECE16-D1 and ECE16-D2 cells. In addition, an increase in HPV transcripts encoding E6/E7 is observed at intermediate (10 and 100 nM) retinoic acid concentrations in ECE16-1 and ECE16-D2 cells, but not in ECE16-D1 cells. Our results show that retinoids regulate E6/E7 transcript levels in some cervical cell lines but not in others, suggesting that different cervical tumors may respond to retinoids via different mechanisms. PMID- 7532117 TI - Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. AB - Multiple growth factors can stimulate quiescent vascular smooth muscle cells to exit from G0 and reenter the cell cycle. The macrolide antibiotic rapamycin, bound to its cytosolic receptor FKBP, is an immunosuppressant and a potent inhibitor of cellular proliferation. In the present study, the antiproliferative effects of rapamycin on human and rat vascular smooth muscle cells were examined and compared with the effects of a related immunosuppressant, FK520. In vascular smooth muscle cells, rapamycin, at concentrations as low as 1 ng/mL, inhibited DNA synthesis and cell growth. FK520, an analogue of the immunosuppressant FK506, is structurally related to rapamycin and binds to FKBP but did not inhibit vascular smooth muscle cell growth. Molar excesses of FK520 blocked the antiproliferative effects of rapamycin, indicating that the effects of rapamycin required binding to FKBP. Rapamycin-FKBP inhibited retinoblastoma protein phosphorylation at the G1/S transition. This inhibition of retinoblastoma protein phosphorylation was associated with a decrease in p33cdk2 kinase activity. These observations suggest that rapamycin, but not FK520, inhibits vascular smooth muscle cell proliferation by reducing cell-cycle kinase activity. PMID- 7532116 TI - A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases. AB - The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins. PMID- 7532118 TI - Inhibition of nitric oxide synthesis causes myocardial ischemia in endotoxemic rats. AB - Inhibitors of nitric oxide (NO) synthesis have been used in the treatment of septic and endotoxic shock. However, several studies question the beneficial effect of inhibiting NO production in sepsis and endotoxemia. We have investigated the effect of inhibition of NO synthesis after endotoxemia in the isolated perfused rat heart. In hearts from endotoxin-treated animals, coronary flow was elevated 64% and oxygen consumption was elevated 20% compared with control hearts. NADH fluorescence imaging was used as an indicator of regional hypoperfusion. A homogeneous low-surface NADH fluorescence, indicative of adequate tissue perfusion, was observed in both control and endotoxin-treated hearts. The increase in coronary flow and oxygen consumption could only partially be prevented by pretreatment of the animals with dexamethasone. Addition of N omega-nitro-L-arginine (NNLA), an inhibitor of NO synthesis, to the perfusion medium eliminated differences in coronary flow and oxygen consumption between normal and endotoxin-treated hearts. However, NADH surface fluorescence images of endotoxin-treated hearts after NNLA revealed areas of high fluorescence, indicating local ischemia, whereas the control hearts remained without signs of ischemia. The ischemic areas were present at various perfusion pressures and disappeared after the infusion of L-arginine, the natural precursor of NO, or the exogenous NO donor sodium nitroprusside. Methylene blue (MB), an inhibitor of soluble guanylate cyclase, the effector enzyme of NO, also eliminated differences in coronary flow and produced similar areas of local myocardial ischemia in endotoxin-treated hearts but not in control hearts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532119 TI - Essential role for nitric oxide in neurogenic inflammation in rat cutaneous microcirculation. Evidence for an endothelium-independent mechanism. AB - The possible modulatory role of nitric oxide (NO) in neurogenic edema formation in rat paw skin, induced by electrical stimulation of the saphenous nerve, was investigated by using two NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both L-NAME (100 mg/kg IV, P < .05) and 7-NI (10 mg/kg IV, P < .05) caused an L-arginine (100 mg/kg IV, P < .01) reversible inhibition of neurogenic edema as measured by 125I-albumin accumulation, whereas D-NAME (inactive enantiomer of L-NAME) and 6-aminoindazole (structurally similar to 7-NI) were without inhibitory effect. L-NAME produced the predicted vasopressor effect (before, 115 +/- 18 mm Hg; 5 minutes after, 174 +/- 18 mm Hg; n = 6; P < .05), whereas 7-NI showed no significant increase in blood pressure (before, 96 +/- 9 mm Hg; 5 minutes after, 102 +/- 10 mm Hg; n = 6), and neither L-NAME nor 7-NI had any effect on basal or vasodilator calcitonin gene-related peptide (CGRP, 10 pmol per site)-stimulated local blood flow in rat skin, as measured by laser Doppler flowmetry. Furthermore, systemic and local 7 NI had no effect on edema formation induced by local administration of substance P (with or without CGRP) and histamine (with or without CGRP) in rat skin. Since 7-NI blocks edema produced by stimulation of the saphenous nerve, it is suggested that release of NO is involved in neurogenic edema formation, but the vasodilator action of NO is unimportant in this context.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532120 TI - T and B cell responses to mycobacterial 65-kDa heat-shock protein in sheep infected with maedi visna virus. AB - Sheep infected with maedi visna virus were tested for immune reactivity to recombinant HSP65 and tuberculin PPD from mycobacteria. The results showed that both naturally and experimentally infected animals had elevated IgM but not IgG or IgA antibodies to HSP65 from Mycobacterium leprae or M. bovis. In experimentally infected animals, the elevated IgM antibodies appeared in blood from about 3 to 4 weeks postinfection. Increased T cell proliferative responses to HSP65 and PPD were also found in both naturally and experimentally infected sheep. The T cell responses to HSP65 were substantially inhibited by antibodies to ovine major histocompatibility complex class II molecules, indicating that the responses were class II restricted. Increased expression of a putative HSP65 molecule was observed in synovial membranes from sheep infected with maedi visna virus and goats infected with the related, caprine arthritis encephalitis virus. The results thus show that lentivirus infection induces T and B cell anti-HSP65 immune responses and suggest that synovial inflammation may be due, at least in part, to T and B cell recognition of HSP65-like molecules expressed in joints. PMID- 7532121 TI - Immunobiology of lyssaviruses: the basis for immunoprotection. PMID- 7532122 TI - Immunoprotection by rabies virus nucleoprotein. PMID- 7532123 TI - [Angiogenesis and its effect to the cell proliferation during experimental buccal carcinogenesis]. PMID- 7532125 TI - Persistent hepatomegaly: an index of severity in sickle cell anaemia. AB - Eighty sickle cell anaemia patients, 29 with persistent hepatomegaly and 51 without, were evaluated with respect to clinical severity score. Haemoglobin F (HbF) was also determined in 52 patients, 20 with persistent hepatomegaly and 32 without. The severity scores were computed from the steady state haematocrit, number of transfusions per year, and number of crises per year. The HbF level was determined by the alkali denaturation method. The patients with persistent hepatomegaly had significantly higher clinical severity scores (P < 0.001) and significantly lower levels of HbF (P < 0.0001). The implication of the finding is that patients with persistent hepatomegaly may have a more severe clinical course than those without, and clinicians managing them may have to pay more particular attention to them. In effect, persistent hepatomegaly may be a useful indicator of severity in sickle cell anaemia. PMID- 7532124 TI - Observations on aflatoxins and the liver status of Ghanaian subjects. AB - To find out if the presumed intake of dietary aflatoxins (AFB1 and AFG1) has adverse effect on the liver of Ghanaians, the toxins were measured in serum, urine and faecal specimens obtained from a group of apparently healthy Ghanaian adults. Liver status of the subjects was monitored with serum alphafeto protein (AFP), alpha-l-antitrypsin (AAT) and direct: total bilirubin ratio. Aflatoxin G1, AFB1 and AFQ1, AFM1 (both metabolites of AFB1) were detected in one or more of the body specimens in 35% of the subjects (AFB1+ group). Sixty-five percent (26 out of 40) of the subjects had only AFG1 in their body specimens (AFB1- group). Serum levels of AFP (greater than 20.0 ng/ml, AAT (greater than 170.0 mg/dl) and direct: total bilirubin ratio (greater than 0.5) which indicate absence of predisposition to liver cancer in all the subjects but suggestive of liver inflammation were noted in both the AFB1+ and AFB1- subjects. The pattern of distribution of the aflatoxins in the subjects suggests that the suspected liver inflammation may involve other factors and may not only be due to the present intake levels of aflatoxins. PMID- 7532126 TI - Self-expanding metal stents for palliative treatment of malignant biliary and duodenal stenoses. AB - Patients with malignant biliary stenosis due to pancreatic head cancer often have the associated problem of duodenal obstruction. We report here the case of a 78 year-old woman with this clinical situation, who was treated with self-expanding Gianturco metal stents placed in the distal common bile duct and, nine months later, placed in the descending duodenum. The patient's frequent vomiting resolved, and she was able to tolerate peroral solid food; jaundice also decreased (bilirubin pre-stenting: 7.8; post-stenting: 1.2). Self-expanding metal stents therefore appear to be effective for the palliative treatment of malignant duodenal stenosis as well as biliary stenosis. PMID- 7532127 TI - Differential splicing of the glycophorin A mRNA. AB - Two truncated mRNAs, in contrast to the full length of mRNA associated with the glycophorin A gene (GpA-TI, GpA-TII), were isolated from erythroid cells cultured by the selective two-phase liquid culture system for erythroid progenitors in peripheral blood from a normal individual. The GpA-TI mRNAs displayed a direct transition from exon I to exon III, so that the deletion of exon II resulted in the deletion of 33 amino acids encoded by this exon. Furthermore, the GpA-TII showed two direct transitions from exon I to exon III and from exon III to the exon V of the GpA gene. This mRNA lacked both exons II and VI, resulting in the deletion of 46 amino acids. Is is concluded that these truncated mRNAs are transcribed from the same gene as the GpA gene and correspond to splicing isoforms lacking different exons. PMID- 7532128 TI - Analysis of the inter-alpha-trypsin inhibitor polymorphism in west Africa: description of a new allele, ITI*7. AB - Inter-alpha-trypsin inhibitor (ITI) phenotypes were classified in the West African population of Cabo Verde by polyacrylamide gel isoelectric focusing, followed by immunofixation and silver staining. Gene frequencies of the alleles ITI*1, ITI*2, ITI*3, and ITI*4 were calculated to be 0.532, 0.153, 0.307 and 0.002, respectively. A new rare allele, ITI*7, was found, providing evidence for further genetic variability of the ITI protein. The ITI*7 allele frequency has been determined to 0.006. The assumption that allele ITI*3 may be used to characterize populations of African origin is supported by our data. PMID- 7532129 TI - Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: an interlaboratory comparison. AB - An intra- and interlaboratory comparison of positional reproducibility of protein spots in two-dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG-DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4-8, for the first dimension and homogeneous SDS-PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well-resolved spots distributed across the 2-D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG-dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along the x-axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation approximately 1.5 mm). Nevertheless, IPG-DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes. PMID- 7532130 TI - An aspartate residue of the Yersinia pseudotuberculosis invasin protein that is critical for integrin binding. AB - The Yersinia pseudotuberculosis invasin protein mediates bacterial entry into mammalian cells by binding multiple beta 1-chain integrins. Invasin binding to purified alpha 5 beta 1 integrin is inhibited by Arg-Gly-Asp (RGD)-containing peptides, although invasin contains no RGD sequence. Fifteen mutations that diminished binding and bacterial entry were isolated after mutagenesis of the entire inv gene. All of the mutations altered residues within the C-terminal 192 amino acids of invasin, previously delineated as the integrin binding domain, and 10 of the mutations fell within an 11 residue region. This small region was subjected to site-directed mutagenesis and almost half of the 35 mutations generated decreased invasin-mediated entry. D911 within this region was the most critical residue, as even a conservative glutamate substitution abolished bacterial penetration. Purified invasin derivatives altered at this residue were defective in promoting cell attachment and this defect was reflected in a 10-fold or greater increase in IC50 for integrin binding. D911 may have a function similar to that of the aspartate residue in RGD-containing sequences. PMID- 7532132 TI - Targeting gene expression to haemopoietic stem cells: a chromatin-dependent upstream element mediates cell type-specific expression of the stem cell antigen CD34. AB - The ability to target heterologous gene expression to haemopoietic stem cells will allow biological manipulation of this compartment and facilitate gene therapy of blood disorders. To identify regulatory elements with this potential, we have analysed the transcriptional regulation of the murine stem cell antigen CD34. Within haemopoiesis CD34 is expressed in stem and early progenitor cells, but it is also expressed in certain non-haemopoietic cell types, including fibroblasts. Comparison of CD34 chromatin in haemopoietic progenitor cells (416B and M1) and fibroblasts (Swiss 3T6) revealed several DNase I hypersensitive regions, one of which, a cluster of sites centred 3 kb upstream of exon 1, was specific to haemopoietic progenitors. This element stimulated expression by approximately two orders of magnitude in CD34+ haemopoietic progenitors, but not in CD34+ fibroblasts or in CD34- haemopoietic cells (18.8). Enhancer function was dependent upon chromosomal integration, although position-independent expression was not obtained. However, largely position-independent expression was conferred by addition of a downstream element which was hypersensitive in all the cell types analysed. We conclude that the murine CD34 gene is regulated by a chromatin dependent, upstream enhancer element which acts in conjunction with a downstream domain-controlling element to confer high level gene expression in haemopoietic progenitor cells. PMID- 7532134 TI - Interaction between FK 506 and clarithromycin in a renal transplant patient. PMID- 7532133 TI - The cognitive outcome of very preterm infants may be poorer than often reported: an empirical investigation of how methodological issues make a big difference. AB - The effects of relying on outmoded IQ-test norms and the use of arbitrary classifications of developmental delay on estimates of cognitive impairment of very preterm infants (VPI) was evaluated in a prospective population study. Cognitive assessments included the Griffiths test at 5 and 20 months and the Columbia Mental Maturity Scales (CMM) and a vocabulary test (Aktiver Wortschatz Test, AWST) at 56 months of age. Rates of cognitive impairment of 321 very preterm infants (VPI; < 32 weeks gestation or < 1500 g birth weight) were determined according to the published test norms, to scores of a full-term control group (FC n = 321), and to scores from a representative sample of children (NC n = 431) of the same birth cohort. IQ-scores were higher in the FC and NC children than in the original standardisation sample (SS). Using the concurrent test norms (FC, NC) up to 2.4 times more VPI were identified as seriously impaired (<-2 SD) than if the published (outdated) norms were used. Serious developmental delay was underestimated when arbitrary (e.g. DQ < 70) rather than across age comparable definitions (DQ <-2 SD) were used. VPI study drop-outs had mothers with lower educational qualifications and poorer cognitive developmental scores at 5 or 20 months of age. In conclusion, a lack of appropriate control groups and use of arbitrary criteria for judging serious delay leads to large underestimations of cognitive impairment in VPI. Findings from previous uncontrolled studies of VPI need re-interpretation. PMID- 7532135 TI - Role of nitric oxide in PGF2 alpha-induced ocular hyperemia. AB - The effect of nitric oxide synthase inhibition on prostaglandin F2 alpha (PGF2 alpha)-induced ocular hyperemia in the rabbit has been studied. PGF2 alpha was administered topically as the isopropyl ester (PGF2 alpha-IE) unilaterally, with the other eye serving as a control. The regional blood flow in the eye was determined with radioactively-labelled microspheres in conscious animals. Synthesis of nitric oxide (NO) was blocked by L-NMMA (200 mg kg-1 b.w., i.v.). PGF2 alpha-IE induced marked hyperemia of the surface structures of the eye (conjunctiva, eye lids, nictitating membrane, anterior sclera), as well as increased blood flow of the anterior uvea. L-NMMA blocked the hyperemia of the surface structures but not completely the increase in blood flow of the anterior uvea. PhXA41 (13,14-dihydro-17-phenyl-18,19,20-trinor-PGF2 alpha-isopropyl ester), a selective prostaglandin FP-receptor agonist, had no significant effect on the ocular blood flow. These results indicate that PGF2 alpha causes surface hyperemia of the eye by activating nitric oxide synthase, but this mechanism seems to be only partly involved in the increase in blood flow of the ciliary processes and the iris. The PGF2 alpha-induced ocular hyperemia is unlikely to be mediated by FP receptors. PMID- 7532131 TI - Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells. AB - The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3 kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion. PMID- 7532136 TI - Epidemiological typing of Legionella pneumophila with ribotyping. Report of two clinical cases. AB - The ribotyping method, adapted to the strains of Legionella pneumophila in our possession, was tested in two separate cases of legionellosis and in the associated finding of Legionella pneumophila in the water, from different sources, with which these patients had come into contact. Determination of the serogroup enabled us to carry out a preliminary analysis of the strains, which was then confirmed by application of the ribotyping procedure: the ribosomal profile of the strains found in the two patients correspond to that of the strains isolated from the water with which they had come into contact. These results provide important information concerning the probable sources of infection involved in these two cases of Legionnaires' disease. We consider ribotyping to be a very useful tool, which is easy and simple to perform and is applicable to the Legionella genus as the method of choice for epidemiological studies. PMID- 7532140 TI - Leukemic nodules at the site of G-CSF injection in acute myelomonocytic leukemia. PMID- 7532139 TI - Thymic stromal cells support differentiation of natural killer cells from CD34+ bone marrow cells in vitro. AB - Natural killer (NK) cells are CD3-CD56+ lymphocytes characterized by exhibiting non-MHC restricted cytotoxicity. A developmental relationship between NK cells and T lymphocytes has been proposed, and, moreover, the thymus has been shown to contain NK cell precursors. In this study we utilized an in vitro assay, devised to study T-lymphocyte development from bone marrow progenitors, to investigate the ability of thymic stromal cells to support generation of NK cells from CD34+ bone marrow cells. CD34+ cells purified from healthy adults were seeded on adherent thymic stromal cells. The cells emerging after culture were phenotypically characterized by flow cytometry. We show that lymphocytes expressing the phenotypical characteristics of NK cells were generated from CD34+ bone marrow cells, and that these cells represented 1% of the cells recovered from the cultures. Furthermore, this was accomplished without supplement of exogenous interleukin 2 which is required for NK cell differentiation in bone marrow cultures. PMID- 7532137 TI - Hepatitis C viraemia and antibody to core epitopes in anti-HCV ELISA negative blood donors. PMID- 7532138 TI - Changes in adhesion molecule expression and function in B-cell chronic lymphocytic leukaemia after in vitro interferon-alpha stimulation. AB - Peripheral blood mononuclear cells (PBMCs) from 10 B-CLL patients were investigated after 24 hours of in vitro interferon-alpha (IFN-alpha) stimulation. The constitutional expression of the L-selectins (LECAM-1), LFA-1/CD11a, VLA alpha-4/CDw49d and ICAM-1/CD54 adhesion molecules was detected, and changes in their density after IFN-alpha stimulation were compared to results obtained by the high endothelial venule (HEV)-binding assay and a carbohydrate (phosphonomannan core polysaccharide: PPME and fucoidin) immobilization test. The LECAM-1 and ICAM-1 molecules were expressed on the great majority of CLL cells, while the LFA-1 and VLA-4 alpha-chains were expressed by only a small number of cells. Statistically significant changes (p < 0.001) were observed in LECAM-1 antigen density (changes in mean cell fluorescence), as well as in functional tests (HEV-, PPME- and fucoidin-binding; p < 0.01) after in vitro IFN-alpha stimulation. Based on a prior study (Jewell et al., Leukemia 1992: 6: 400-404) and on the present findings, not only an increased expression but also an enhanced function of the L-selectins seem to be well substantiated after IFN alpha stimulation, which may explain the therapeutic effect of IFN-alpha in reducing the accumulation of leukaemic B cells in the blood. The remarkably high expression of ICAM-1 in this series necessitates further studies to clarify the exact expression rate and role of this molecule. PMID- 7532142 TI - Steroidogenic adrenocortical cells synthesize alpha 2-macroglobulin in vitro, not in vivo. AB - We previously identified alpha 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of alpha 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of alpha 2-macroglobulin immunoreactivity whereas the proportion of alpha 2-macroglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co localization of alpha 2-macroglobulin and the steroidogenic enzyme P-450SCC. Treatment of 5-day-old cultures (expressing alpha 2-macroglobulin) for 24 h by either ACTH (10(-9)-10(-6) M) or alpha 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of alpha 2-macroglobulin. These data indicate that ACTH and plasmatic alpha 2-macroglobulin could physiologically repress alpha 2-macroglobulin expression in the adrenal cortex in vivo. PMID- 7532141 TI - Monoclonal antibodies that recognize the native human thyrotropin receptor. AB - Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor. PMID- 7532143 TI - Two distinct glutamic acid decarboxylase auto-antibody specificities in IDDM target different epitopes. AB - Although most individuals with insulin-dependent diabetes mellitus (IDDM) have autoantibodies to glutamic acid decarboxylase (GAD), antibodies to GAD are also present in some individuals with a low risk of developing diabetes. The GAD autoantibodies of IDDM are specific for the GAD65 isoform, do not bind denatured GAD protein, and target epitope(s) dependent on conformation of the protein. However, the IDDM epitopes have been difficult to further define because the antibodies do not bind GAD protein fragments or synthetic peptides. Since the GAD67 isoform is highly homologous to GAD65 but is usually not a target of the GAD autoantibodies in IDDM sera, we created six GAD65/GAD67 chimeric proteins to maintain the overall GAD protein conformation and used these chimeric proteins to map conformation-dependent epitopes of GAD65 targeted by IDDM sera. We find that the GAD binding present in most IDDM sera (n = 11 of 12) is composed of two distinct GAD antibody specificities that target different conformation-dependent regions of the GAD65 protein, one that is located between amino acids 240 and 435 (termed IDDM-E1) and one that is located between amino acids 451 and 570 (termed IDDM-E2). One IDDM serum (n = 1 of 12) bound only the IDDM-E1 region. Identification of epitopes targeted by IDDM sera may allow one to distinguish between GAD antibody-positive individuals at high and low risk of developing IDDM and to determine if differences in the autoimmune repertoire directed at GAD are present. The chimeric GAD65/GAD67 proteins may also be useful in designing GAD assays specific for IDDM. PMID- 7532144 TI - Small bowel involvement by Mycobacterium avium complex in a patient with AIDS: endoscopic, histologic, and radiographic similarities to Whipple's disease. PMID- 7532145 TI - Palliation of malignant dysphagia by ethanol induced tumor necrosis. PMID- 7532146 TI - Medical legal and ethical questions in palliative medicine and euthanasia. AB - The increasing importance of legal and ethical questions in palliative medicine and euthanasia due to the increased technical possibilities for extending life will be considered. In palliative medicine, the choice of the best therapy will be discussed, especially in the case of oncological diseases. Here, consideration of the prospects of success, for example, in chemotherapy, is faced with partly serious side-effects. The requirements of palliative medicine that the patient has to be fully informed of the fatal prognosis of his disease is equally debated as the optimum pain therapy. In this respect, the modification of the Narcotics Act of 1 February 1993 is also under discussion. In the field of euthanasia, the technical development of life extension versus dying has raised considerable legal and ethical problems regarding termination of therapy. Additionally, fiscal considerations are of increasing relevance. The common development of the legal and ethical discussion, for example, with regard to the publicity of the work of the so-called 'Gesellschaft fur humanes Sterben', the public discussion leading up to a hearing of the 'Bundestag' regarding active euthanasia leads to a realization of the subject. The proposals for an active termination of life by discontinuing therapy for adults and also for malformed newborns are discussed. A dispute concerning the new legal regulation of active euthanasia in the Netherlands of February 1993 is also discussed. There, around 2% of all deaths per year result from active termination of life and also cases where persons are not able to consent. This also has enormous consequences for the position of the physician.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532147 TI - Endocrine-paracrine cell types in the prostate and prostatic adenocarcinoma are postmitotic cells. AB - Neuroendocrine (NE) differentiation frequently occurs in common prostatic malignancies and has potential prognostic and therapeutic implications. In a recent study we were able to provide immunohistochemical evidence that endocrine paracrine cell types represent an androgen-insensitive cell population in prostate cancer, documented by the consistent lack of the pertinent receptor. In this study we investigated the proliferative activity of endocrine-paracrine cell types in normal, hyperplastic, and neoplastic prostate tissue. Using double-label techniques for the endocrine marker chromogranin A (chr A) and the proliferation associated MIB-1 antigen, we evaluated the proliferative status of endocrine paracrine cell types in the prostate and prostatic adenocarcinoma showing marked NE differentiation. In this series of carcinomas and in nonneoplastic tissue the proliferative activities were exclusively restricted to nonendocrine cell populations, whereas endocrine-paracrine cell types characterized by Chr A consistently lack MIB-1 immunoreactivity. This may indicate that prostatic endocrine-paracrine cell types do not participate in the cell cycle during normal, hyperplastic, and neoplastic prostatic growth. Based on the present information, the endocrine phenotype can be considered to be an androgen insensitive, postmitotic subpopulation in the prostate and prostate cancer. PMID- 7532148 TI - Expression of the 85-kd membrane protein in primary human breast cancer: relationship to hormone receptor levels, DNA ploidy, and tumor grade. AB - Using the monoclonal antibody (MAb) MRK20 the immunohistochemical expression of the 85-kd membrane protein (MP)/cell adhesion molecule (CAM) CD36 was investigated in tumors from 41 patients with untreated breast adenocarcinoma. DNA ploidy as measured by flow cytometry and estrogen (ER) and progesterone receptor (PgR) tumor contents also were determined. Immunohistochemistry on frozen tissue showed expression of 85-kd MP localized to the carcinoma cells in tumors from 16 patients (39%). These samples also displayed variable staining of the capillary endothelium. Twenty-one samples (51%) showed 85-kd MP expression in small blood vessels. Thus, a total of 37 of 41 tumors (90%) were MAb MRK20 positive in carcinoma and/or mesenchymal cells. Expression of 85-kd MP in carcinoma cells was negatively associated with expression of ER (P < .05) and PgR (P < .01) receptors and positively associated with nondiploid tumors (P < 0.05) and tumor grade (P < .05). Axillary lymph node status, tumor size, and (patient) age were not associated with expression of 85-kd MP. In cytological smears obtained by fine needle aspiration (FNA) cytology from 19 of the samples, expression of 85-kd MP in carcinoma cells showed a good correlation with tumor cell positivity in the corresponding histological section. Expression of 85-kd MP in breast carcinoma cells thus was associated with some recognized adverse prognostic factors. Future studies of its pathophysiological and prognostic roles in breast cancer seem warranted. PMID- 7532150 TI - Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected. AB - Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (delta E115). A second new mutation was found in exon 17b, viz., an A --> C substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P << 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (delta F508/L206W, delta F508/R74W + D1270N, R117H/712-1G --> T), highlighting L206W, R74W/D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%-10% necessary for normal protein function. PMID- 7532151 TI - The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11. AB - The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11. PMID- 7532149 TI - Supernumerary marker 15 chromosomes: a clinical, molecular and FISH approach to diagnosis and prognosis. AB - Seventeen patients presenting with either de novo or familial supernumerary marker (mar) 15 chromosomes were shown by fluorescent in situ hybridization techniques (FISH) to have markers derived from and composed entirely of chromosome 15 material. Using a combination of conventional cytogenetics, FISH, Southern blotting and multiplex polymerase chain reaction (PCR) methods, it was possible to sub-classify the 17 mar(15)s into six distinct morphological and molecular groups. Analysis of DNA and metaphase spreads from the probands and their parents using probes and primers from the pericentromeric and Prader Willi/Angelman syndromes critical regions (PWS/AS), clearly differentiated between marker 15s which included the PWS/AS critical regions and those which did not. A direct correlation between the presence of the PWS/AS region in the mar(15) and severe mental retardation was observed. Based on these results, a system of classification of supernumerary marker 15 chromosomes is proposed. PMID- 7532153 TI - Production of mouse monoclonal anti-idiotypic antibodies specific to epitopes of tumor-associated mucin TAG-12. AB - The tumor-associated mucin-glycoprotein TAG-12 is strongly expressed in approximately 96% of all breast cancer patients and nearly 68% of all ovarian cancers. The experimental results of this work indicated that humoral immune response against TAG-12 is possible. Immunization with anti-idiotypic monoclonal antibodies produces this response. In this experiment, anti-idiotypic monoclonal antibodies represent the internal image of a specific epitope on TAG-12. Monoclonal antibody (MAb) 12H12 was selected to produce anti-idiotypic antibodies (anti-Ids) because of its high reactivity with TAG-12. Syngeneic murine anti-Ids were developed by immunization of BALB/c mice with the 12H12-Fab-KLH conjugate. A competitive assay with purified TAG-12 was utilized to identify anti-Ids with mirror image function. Two MAbs with "internal image" specificity were selected, 5H8 and 5H2. Two New Zealand White rabbits were immunized with 5H8. Serum samples tested 6 weeks after the initial immunization showed comparable titers against TAG-12. The binding capacities of the rabbit sera to different human breast as well as nonbreast cancer cell lines demonstrated strong binding with TAG-12 positive breast cancer cell lines. Competitive inhibition assays demonstrate that Ab3 and purified TAG-12 totally inhibit the binding of 12H12 antibody to TAG-12 positive cells. No inhibition was detectable with unrelated MAbs or normal mouse immunoglobulin. Binding assays with polyclonal Ab3 serum and several human cancer cell lines showed reactivity to nearly every tested cell line. Soluble TAG-12 showed no inhibition, indicating that this binding is due to a different set of idiotypes. Anti-Id 5H8 elicited an immune response to TAG-12. Utilization of anti Id as a vaccine against the breast cancer-associated tumor antigen TAG-12 was successfully demonstrated in a xenogeneic animal model. PMID- 7532154 TI - Production and partial characterization of monoclonal antibodies against erythrogenic toxins type A and C from Streptococcus pyogenes. AB - Hybridoma cell lines producing monoclonal antibodies against streptococcal erythrogenic toxins type A and C were established from fusions of immunized BALB/c mice splenocytes with P3X63-Ag8.653 myeloma cells. Six MAbs recognize ETA and 11 MAbs bind to ETC. Two MAbs (designated ETA-2 and ETC-10) were produced in ascitic fluid and further characterized. ETA-2 (IgG2a) binds to ETA with an affinity constant of 1.8 x 10(10) M-1 and ETC-10 (IgG1) binds to ETC with an affinity constant of 3.5 x 10(9) M-1. The specificities of the MAbs were evaluated by ELISA and immunoblotting. Both MAbs ETA-2 and ETC-10 are useful in developing specific double-sandwich ELISAs, in which the MAbs were used as solid phase capture antibodies for the quantitative determinations of ETA and ETC. PMID- 7532152 TI - Common CFTR mutations are not likely to predispose to chronic bronchitis in northern Germany. AB - The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common delta F508 mutation. R553X, G542X, G551D, N1303K and 621 + 1G-->T were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely. PMID- 7532155 TI - Recognition of murine integrin beta 1 by a rat anti-stromal cell monoclonal antibody. AB - Previous studies with the rat monoclonal antibody KMI6 had localized its antigen in vivo to a discrete subpopulation of marrow stromal cells. The KMI6 antigen has now been identified as the murine homolog of integrin beta 1 by amino acid sequence analysis and by cross-reactivity with antiserum to the avian integrin beta 1. The relative tissue abundance of murine integrin beta 1 was determined by Western blot. Although immunoperoxidase staining of fixed murine hematopoietic tissues demonstrated an abundance of intracellular beta 1, few primary-derived cells of lymphohematopoietic origin were surface positive as assessed by immunofluorescence and flow cytometry. Fetal erythroblasts provided the only exception. In contrast, the antigen was readily detected on the surface of several cultured cell lines in association with a variety of alpha chains. The biochemical properties of the surface labeled murine integrin beta 1 were similar to those of its human counterpart, exhibiting an altered electrophoretic migration under reduced conditions or following N-glycanase treatment. The antibody recognition of the protein was insensitive to glycosylation state, presence of divalent cations, detergents, or transfer to a nitrocellulose membrane. However, on Western blot, the epitope was lost on reduction of the protein, suggesting that it is conformation dependent. These data indicate that although KMI6 epitope is widely distributed, its surface expression in vivo may be restricted within lymphohemopoietic tissues. PMID- 7532158 TI - The role of hyaluronan in tumour neovascularization (review). AB - Tumour growth and metastasis are totally dependant upon neovascularization. The target cell for tumour neovascularization is the blood-vessel endothelial cell, and specific angiogenic molecules produced or induced by the tumour are believed to initiate the process. In this report, we review one of these angiogenic molecules, the glycosaminoglycan hyaluronan (HA), which appears to have differing roles in neovascularization depending on its molecular mass. High-molecular-mass HA is anti-angiogenic whereas oligosaccharides of HA, of specific size, actively stimulate endothelial-cell proliferation and migration, 2 of the key events associated with neovascularization, and induce angiogenesis in vivo. We provide details of the action of HA oligosaccharides on endothelial cells, from binding to cell-surface receptors, through activation of signal transduction pathways and gene expression to protein synthesis, cell proliferation and cell migration. We also suggest a model to account for HA of differing molecular mass being present, at different locations, within a single tumour and how this HA aids both general tumour growth and tumour metastasis. PMID- 7532156 TI - Immunohistochemical localization of the plasminogen activator inhibitor-1 in breast cancer. AB - We used an immunohistochemical assay with an antigen-retrieval technique to study plasminogen activator inhibitor type-1 (PAI-1) expression in paraffin-embedded breast tissue samples at different stages of malignant transformation. We detected PAI-1 in 15/20 invasive tumors. In several cases staining was localized to the stromal component. PAI-1-positive fibroblasts could be seen surrounding tumor nodules or at tumor margins. In addition, tumor-infiltrating macrophages (13 cases) and endothelial cells (5 cases) were positive. In 11 specimens PAI-1 positive cancer cells were also detected. In 2 strongly positive cases secreted PAI-1 was visible in the extracellular matrix surrounding the cells. Six of 9 samples of carcinoma in situ (DCIS) were weakly positive. No staining of endothelial cells was visible in DCIS. Only a few positive adenomatous epithelial cells could be seen in 3 of 7 papillomas. All biopsies of normal breast tissue were negative, with the exception of one sample, obtained from a patient with a previous segmental mastectomy for DCIS. PAI-1 production by invasive breast cancers could reflect a general upregulation of the plasminogen activation system in proliferating cancer cells, as suggested by the finding that normal mammary epithelium cultures expressed PAI-1 in all cases examined. In addition, production of PAI-1 by the tumor stroma could protect the tumor itself from excessive proteolysis. PMID- 7532157 TI - Reduction of the systemic toxicity of cisplatin by intra-arterial hepatic route administration for liver malignancies. AB - Cisplatin (CDDP) administration by the intra-arterial hepatic route (i.a.h.) in patients with primary or metastatic liver malignancies could enhance the anti tumor activity of the drug and reduce its systemic toxicity. The aim of the present study was to compare Pt pharmacokinetics and the toxicity of the circulating drug after i.a.h. versus intravenous (i.v.) administration. CDDP pharmacokinetics was followed-up in 11 i.a.h. courses given to 7 patients with liver malignancies and compared with 19 i.v. courses in 15 patients with cancer of different origins. The Pt level in blood was monitored by sensitive atomic absorption spectrometry. The dose given was in the range of 25-80 mg/m2/treatment. For analysis and for comparison purposes, the data from both CDDP treatments were normalized to a standard dose of 35 mg/m2. The mean peak Pt level for i.a.h. treatment was found to be about half of the mean peak value for i.v. administration with a similar dose-independent bi-exponential rate of elimination i.a.h. CDDP treatment was relatively well tolerated with no symptoms of either nephro- or neurotoxicity. For in vitro evaluation of peripheral CDDP toxicity, a sensitive ovarian carcinoma cell line, OV-1063, was used. A cytotoxic effect was recorded only within 2 hr following high-dose i.v. CDDP treatment. A substantial fraction of the drug given by the i.a.h. route was found to be extracted by the liver in the first passage, with reduced drug level in the peripheral blood plasma relative to the dose given. This may explain the apparent diminution of side-effects following i.a.h. CDDP treatment. PMID- 7532159 TI - Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones. AB - Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix. PMID- 7532160 TI - Modulation of intra-epithelial expansion of human T24 bladder-carcinoma cells in murine urothelium by growth factors and extracellular-matrix components. AB - The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and out-growth of transformed urothelial cells, we recently developed an in vitro co-cultivation model. With this model system we studied the effects of growth factors and extracellular-matrix components on the intra-epithelial expansion of human T24 bladder-carcinoma cells in primary mouse-bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further. PMID- 7532162 TI - Phase II study of fludarabine phosphate for gastric adenocarcinoma. An Illinois Cancer Center trial. AB - In an Illinois Cancer Center phase II trial, fludarabine phosphate was administered to a total of 14 patients (9 men, 5 women) with advanced, measurable, gastric adenocarcinoma. Fludarabine phosphate was given as a rapid intravenous (IV) bolus at a starting dose of 20 mg/m2/d for the first 5 days of a 28-day cycle. For subsequent cycles, the dose was escalated in increments of 2 mg/m2/d, provided that no toxicities greater than grade 1 were noted. In cases of grade 3 toxicity, dose reductions of 2 mg/m2/d were required, and patients who experienced grade 4 toxicities were removed from study. Receiving one complete 5 day course of fludarabine phosphate and surviving for 4 weeks on study were required for a patient to be evaluable for response. None of the patients responded to treatment. Although fludarabine phosphate was ineffective against gastric adenocarcinoma in this study, toxicity was acceptable at the 20 mg/m2/d times 5 every 28 days dose and schedule. PMID- 7532161 TI - E-selectin expression induced by pancreas-carcinoma-derived interleukin-1 alpha results in enhanced adhesion of pancreas-carcinoma cells to endothelial cells. AB - Cellular adhesion of sialyl-Lewis-a(SLea)-positive pancreas carcinoma to endothelial cells (EC) is augmented by activation of EC via up-regulated E selectin expression on EC. Co-cultivation of pancreas-carcinoma cells, PCI-24, with human umbilical-vein endothelial cells (HUVEC) for 5 hr at the PCI-to-HUVEC ratio of 1:10 induced E-selectin expression on the endothelial-cell surface, augmenting SLea-positive pancreas-carcinoma cell attachment with HUVEC. Culture supernatants of 6 tested pancreas-carcinoma cell lines contained soluble, E selectin-inducing factor(s). The E-selectin-inducing effect by the supernatants was blocked by the protein-kinase-C inhibitor, H7. Antibodies against SLea and E selectin but not SLex or ICAM-1 blocked the increased pancreas-carcinoma-to endothelial attachment. Paraformaldehyde(PFA)-fixed PCI-24 cells also induced E selectin on vascular endothelial cells upon direct contact with endothelial cells, indicating the presence of a membrane-bound form. The 6 pancreas-carcinoma lines all produced IL-1 alpha mRNA and protein but not IL-1 beta or TNF-alpha protein and/or mRNA. Absorption of IL-1 alpha from the supernatants by IL-1 alpha specific antibody almost completely abolished E-selectin-inducing activity. Anti IL-1 alpha antibody also abolished the E-selectin-inducing activity of PFA-fixed PCI. IL-1 alpha production by PCI cells was up-regulated by TNF-alpha. These observations suggest that substance(s) produced by pancreas-carcinoma cells, in this case, IL-1 alpha, may contribute to pancreas-carcinoma-cell colonization in non-inflamed, distant locations in vivo, by activating vascular endothelial cells. PMID- 7532164 TI - The effect of high-dose methylprednisolone combined chemotherapy on CD34-positive cells in acute lymphoblastic leukemia. AB - The expression of CD34 antigen on the surface of bone marrow cells during remission induction was studied in 27 selected acute lymphoblastic leukemia (ALL) patients who were CD34 negative at presentation and were stratified to receive high-dose methylprednisolone (30 mg/kg/day po) or conventional-dose prednisolone (2 mg/kg/day po). Patients received either induction with L-Asparaginase, vincristine (VCR), and high-dose methylprednisolone [HDMP, 30 mg/kg/day po for 1 week, 20 mg/kg/day po for 1 week, and 20 mg/kg/day po every other day for 2 more weeks (20 patients)], or identical induction in which HDMP was replaced by prednisolone 2 mg/kg/day by mouth for 4 weeks (8 patients). Bone marrow cells from all patients were studied 1,2, and 4 weeks after initiation of treatment for expression of the CD34 antigen using a three-step indirect immunoperoxidase staining technique. In the 20 patients with ALL who received HDMP the percentage of normal bone marrow cells expressing CD34 was significantly higher (p < 0.05) than in the 8 patients who did not receive HDMP. The mean percentage of CD34 positive bone marrow cells during the fourth week was 17.2% in patients with ALL who received HDMP, whereas patients who received 2 mg/kg prednisolone per day had only 6.1% CD34 cells in the marrow. Absolute polymorphonuclear leukocyte (pmnl) count was also significantly higher in the patients who received HDMP in the second and third week of therapy [(absolute pmnl count was 2197.7/mm3 in the second week and 4091.8/mm3 in the fourth week in the patients who received HDMP compared to 974.4/mm3 and 1556.5/mm3 in the patients who did not receive HDMP) (p < 0.05)].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532163 TI - Fludarabine phosphate. A new anticancer drug with significant activity in patients with chronic lymphocytic leukemia and in patients with lymphoma. AB - Fludarabine phosphate is a purine antimetabolite approved for use in the management of patients with chronic lymphocytic leukemia. Fludarabine works primarily by inhibiting DNA synthesis. The compound also possesses lymphocytotoxic activity with preferential activity toward T-lymphocytes. Initial preclinical studies demonstrated antitumor activity with fludarabine against L1210 murine leukemia. In phase I studies, myelosuppression was identified as the dose-limiting toxicity in patients with solid tumors and fatal neurotoxicity as the dose-limiting toxicity in adult patients with acute hematologic malignancies. The recommended dose and schedule was determined to be 18-25 mg/m2/d for five days, repeated every 28 days. Unlike preclinical studies, phase II trials showed a lack of significant effect when fludarabine was given to patients with solid tumors. However, phase II investigations have confirmed the efficacy of fludarabine in lymphoid malignancies, including non-Hodgkin's lymphoma, mycosis fungoides, prolymphocytic leukemia, and chronic lymphocytic leukemia. The place of fludarabine in the management of leukemias in children is under investigation. Early results indicate an unusual degree of antitumor activity when the agent is used in combination chemotherapy for patients with refractory disease. Fludarabine is an effective antitumor agent in the management of lymphoid malignancies. Studies are ongoing to more completely define the role of fludarabine in these malignancies as well as in the pediatric leukemias. Additional studies evaluating the activity of fludarabine as an immunomodulator are warranted, due to the lymphocytotoxic properties associated with this agent. PMID- 7532165 TI - Antibody to hepatitis-C virus in multitransfused thalassaemics--Indian experience. PMID- 7532166 TI - Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers. AB - Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified. PMID- 7532167 TI - Structurally variant classes of pilus appendage fibers coexpressed from Burkholderia (Pseudomonas) cepacia. AB - One or more of five morphologically distinct classes of appendage pili were determined to be peritrichously expressed by Burkholderia (formerly Pseudomonas) cepacia isolated from disparate sources. B. cepacia-encoded cblA pilin gene hybridization-based analysis revealed that one associated class, cable (Cbl) adhesin type IIB. cepacia pili, correlates with epidemically transmitted strains from a single cystic fibrosis (CF) center. When only phenotypic assays were available, correlations between the source and the pilus type were nonetheless observed: filamentous (Fil) type IIIB. cepacia pili correlated with CF-associated nonepidemic isolates, spine (Spn) type IVB. cepacia pili correlated with clinical (non-CF) isolates, and spike (Spk) type VB. cepacia pili correlated with environmental isolates. Further, Cbl, Fil, or Spk pili typically appear as an internal framework for constitutively coexpressed, peritrichously arranged dense mats of fine, curly mesh (Msh) type IB. cepacia pili. Constitutive coexpression of dense mats of Msh type IB. cepacia pili in association with a labyrinth of either Cbl, Fil, or Spk pili suggests possible cooperative pilus interactions mediating adhesion-based colonization in the differing environments from which the strains were isolated. Despite such correlations, phylogenetic analyses indicate that with the exception of the epidemically transmitted clusters of isolates, the remaining B. cepacia strains from the other three sources exhibited an equal degree of genetic relatedness independent of origin. As previously found for Escherichia coli, this discrepancy could be accounted for by selection driven, in vivo horizontal transfer events between distantly related members of the species B. cepacia, leading to the genetic acquisition of environmentally appropriate adhesion-based colonization pilus operons. PMID- 7532168 TI - Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. AB - The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223 2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L [35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase). PMID- 7532169 TI - Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454. AB - By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate. PMID- 7532170 TI - Architecture of the cell envelope of Chlamydia psittaci 6BC. AB - The cysteine-rich envelope proteins of the elementary body form of chlamydiae are thought to be located in the outer membrane on the basis of their insolubility in the weak anionic detergent N-lauryl sarcosinate (Sarkosyl). We found, however, that the insolubility of the small (EnvA) and the large (EnvB) cysteine-rich proteins of Chlamydia psittaci 6BC in Sarkosyl is dependent on the maintenance of a supramolecular disulfide-cross-linked complex and is unlikely to be a valid indicator of outer membrane location. Consequently, we used other methods to characterize the architecture of the cell envelope of C. psittaci 6BC. We found that disulfide-reduced EnvA, previously shown to be a lipoprotein, segregated into the detergent phase during Triton X-114 partitioning experiments and was recovered from the membrane fraction of elementary bodies lysed by nondetergent means. In contrast, disulfide-reduced EnvB segregated to the aqueous phase in partitioning experiments and was found in the soluble fraction of elementary bodies lysed in the absence of detergents. The hydrophobic affinity probe 3 (trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine labeled the major outer membrane protein and EnvA but did not label EnvB. Treatment of intact elementary bodies of C. psittaci with trypsin had no effect on the cysteine-rich proteins, although the major outer membrane protein was partially degraded. On the basis of these and other observations, we propose that EnvA is anchored to the outer membrane by its lipid moiety, with a hydrophilic peptide portion extending into the periplasm, and that EnvB is located exclusively within the periplasm. We further propose that disulfide-cross-linked polymers of EnvB are the functional equivalent of peptidoglycan, forming a disulfide-cross-linked network with the periplasmic domains of EnvA and other membrane proteins, which accounts for the osmotic stability of elementary bodies. PMID- 7532171 TI - Cell size regulation, a mechanism that controls cellular RNA accumulation: consequences on regulation of the ubiquitous transcription factors Oct1 and NF-Y and the liver-enriched transcription factor DBP. AB - Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level. PMID- 7532172 TI - Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain. AB - Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso-1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain. PMID- 7532173 TI - Myogenin is required for late but not early aspects of myogenesis during mouse development. AB - Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD-lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts. PMID- 7532174 TI - P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P selectin. AB - Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses. PMID- 7532178 TI - Down-regulation of bradykinin receptors and bradykinin-induced Ca2+ mobilization, tyrosine phosphorylation, and DNA synthesis by autocrine factors, tumor necrosis factor alpha, and interferon beta in Swiss 3T3 cells. AB - Preincubation of quiescent Swiss 3T3 cells in fresh synthetic medium caused a reduction of the lag period prior to bradykinin-stimulated DNA synthesis as well as a leftward shift in the dose-response curve (half-maximum effect at 2 nM and 8 nM for preincubated cells and control cells, respectively). These enhancing effects were selective for bradykinin since vasopressin-stimulated DNA synthesis was not affected by preincubation in synthetic medium. Preincubation in synthetic medium also caused a marked enhancement (five- to sixfold increase) of bradykinin induced Ca2+ mobilization from intracellular stores. This enhancement was time dependent, peaked after 12 h of preincubation, and was prevented by inhibition of RNA or protein synthesis. Furthermore, preincubation in synthetic medium did not enhance the Ca2+ mobilization by bombesin, vasopressin, or PDGF. Additionally, bradykinin-induced tyrosine phosphorylation was also enhanced by prior incubation in fresh medium. Scatchard analysis of [3H]bradykinin binding revealed a doubling of the number of bradykinin receptors without any significant change of affinity after preincubation, thus providing an explanation for the increased cellular responsiveness to bradykinin. This enhancement of responsiveness to bradykinin was caused by the removal of an inhibitory factor present in conditioned medium which is produced by the cells and accumulates gradually in the medium. Addition of tumor necrosis factor alpha or interferon beta to synthetic medium substituted for conditioned medium in preventing the increase in responsiveness to bradykinin. These findings demonstrate a novel mechanism that regulates cellular sensitivity to bradykinin via an autocrine factor(s). PMID- 7532175 TI - Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon. AB - The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8-10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin-like alternative exon inserts. We also demonstrate that the majority of endogenous GAG modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression. PMID- 7532180 TI - [Palliative gastrojejunal anastomosis by celioscopy]. PMID- 7532181 TI - [Non-surgical treatment of benign prostatic hypertrophy]. PMID- 7532179 TI - Cell density-dependent regulation of PLC gamma 1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC-6). AB - Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC-6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC-6 cells with EGF provoked a significant increase in the hydrolysis of PtdIns 4,5-P2 by immunoisolated PLC gamma 1. The tyrosine phosphorylation state of PLC gamma 1 and the association of PLC gamma 1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2-containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLC gamma 1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLC gamma 1, therefore, is regulated by physiological factors dependent on cell density in IEC-6 cells. PMID- 7532177 TI - Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment. AB - The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532182 TI - Sequence 104-117 of myelin proteolipid protein is a cryptic encephalitogenic T cell determinant for SJL/J mice. AB - Immunization of animals with myelin proteolipid protein (PLP) causes experimental autoimmune encephalomyelitis (EAE), a disease model that shares many features with human multiple sclerosis (MS). The SJL/J (H-2s) mouse is widely used in EAE studies because of its high disease susceptibility. Previous studies have shown that sequences 139-151 HCLGKWLGHPDKF and 178-191 NTWTTCQSIAFPSK represent distinct co-immunodominant encephalitogenic determinants of PLP for SJL/J mice. In the present study, we identify a third distinct PLP encephalitogenic peptide for SJL/J mice. Following immunization with PLP 104-117 KTTICGKGLSATVT, 10/14 SJL/J mice developed clinical and histological EAE with a mean time of onset of 38 days (18-65 days). T cell lines generated from SJL/J mice immunized with p104 117 were predominantly (> 90%) CD3+, CD4+, alpha beta TCR+, CD8dim, gamma delta TCRdim T cells and responded in an Ag-specific, I-A(s)-restricted manner to p104 117. Upon adoptive transfer of 16-40 x 10(6) T line cells, EAE was produced in naive SJL/J recipients 20-34 days after transfer. The delayed onset of both active and passive disease may be related to the non-immunodominant, cryptic nature of p104-117 in SJL/J mice. Lymph node cells from SJL/J mice immunized with either whole PLP or with pooled encephalitogenic PLP peptides responded to challenge with the immunodominant PLP determinants p139-151 and p178-191 but did not respond to p104-117. The existence of three distinct PLP encephalitogenic T cell determinants for SJL/J mice suggests that susceptibility to EAE and perhaps MS may be related to promiscuous T cell recognition of multiple myelin protein determinants. PMID- 7532176 TI - CD44 isoforms containing exon V3 are responsible for the presentation of heparin binding growth factor. AB - Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall. PMID- 7532183 TI - Effect of granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor on melatonin secretion in rats in vivo and in vitro studies. AB - The study was performed in order to clarify whether granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) affect melatonin production and release. We have found that both factors (GM-CSF at doses of 10.0 and 100.0 micrograms/kg, and G-CSF at doses of 1.0, 10.0, and 100.0 micrograms/kg) stimulate melatonin secretion in rats in vivo. Positive correlations between tested doses of GM-CSF and G-CSF and plasma melatonin levels were observed (P < 0.01). Moreover, GM-CSF at doses of 2.0 and 20.0 ng/ml activated in vitro the pineal gland to melatonin release (P < 0.05) in a dose dependent manner. PMID- 7532184 TI - The ability of synoviocytes to support terminal differentiation of activated B cells may explain plasma cell accumulation in rheumatoid synovium. AB - To understand the accumulation of plasma cells within RA synovium, the ability of rheumatoid synoviocytes to support the differentiation of B cells into plasma cells was explored. Tonsillar B lymphocytes cultured over confluent monolayers of synoviocytes, secreted threefold more Igs (mainly IgM) than B cells cultured directly on plastic well. More importantly, synoviocytes enhanced by 14-fold the production of Igs (mainly IgG) by B cells costimulated with Staphylococcus aureus Cowan (SAC) particles. IL-10 and, in a lower extent, IL-2 increased Ig secretion in cocultures, and their combination was synergistic. In the presence of SAC, IL 2, and IL-10, synoviocytes increased by 13-884-fold the production of IgG, which reached 0.19 ng/cell per day. RA as well as normal synoviocytes were more potent than other adherent cell lines to support terminal B cell differentiation. Synoviocyte activity involved both a support of B cell survival, and an induction of the terminal differentiation of B cells into mature plasma cells with typical morphology, high levels of intracytoplasmic Igs, and CD20- CD38high surface expression. The present observation should permit the identification of molecules involved in the maturation of B cells into plasma cells, and in their accumulation in rheumatoid synovium. PMID- 7532185 TI - Signaling through CD40 rescues IgE but not IgG or IgA secretion in X-linked immunodeficiency with hyper-IgM. AB - The ligand for CD40 (CD40L) is a membrane protein on activated T cells that induces B cell proliferation and differentiation. Several mutations of the CD40L gene were reported responsible for defective class switching of B cells in an X linked immunodeficiency with hyper IgM (X-HIM). We studied four affected males from three families and found three independent mutations including new mutations of CD40L gene. In every X-HIM patient tested, however, anti-CD40 plus IL-10 did not induce class switching from IgM to IgG or IgA, even in the presence of Staphylococcus aureus Cowan I strain (SAC). CD4+ T cell clones, expressing CD40L on their surface, also did not rescue IgG or IgA induction by X-HIM peripheral blood B cells in vitro. But signaling through CD40 induced both B cell proliferation and IgE secretion when IL-4 was added to the culture. Taken together, these results show that in vitro signaling through CD40 rescues IgE but not IgG or IgA secretion by peripheral blood X-HIM B cells and suggest that in vivo CD40 and CD40L interaction might be necessary for IgG and IgA differentiation in X-HIM. PMID- 7532188 TI - Nitric oxide mediates cytotoxicity and basic fibroblast growth factor release in cultured vascular smooth muscle cells. A possible mechanism of neovascularization in atherosclerotic plaques. AB - To define the pathophysiological role of nitric oxide (NO) released from vascular smooth muscle cells (VSMC), we examined whether NO released from VSMC induces cytotoxicity in VSMC themselves and adjacent endothelial cells (EC) using a coculture system. Prolonged incubation with interleukin-1 (IL-1) induced large amounts of NO release and cytotoxicity in VSMC. NG-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited both NO release and cytotoxicity induced by IL-1. In contrast, DNA synthesis in cocultured EC was not inhibited but rather stimulated by prolonged incubation with IL-1 or sodium nitroprusside (SNP), a NO donor. However, IL-1 and SNP did not stimulate but inhibited DNA synthesis in EC alone. On the other hand, conditioned medium from VSMC incubated for a long period with IL-1 or SNP stimulated DNA synthesis in EC alone. Furthermore, the concentration of basic fibroblast growth factor in the conditioned medium was increased and correlated with the degree of cytotoxicity in VSMC. These results indicate that NO released from VSMC induces VSMC death, which results in release of basic fibroblast growth factor, which then stimulates adjacent EC proliferation. Thus, NO released from VSMC may participate in the mechanism of neovascularization in atherosclerotic plaques. PMID- 7532187 TI - Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator. AB - It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process. PMID- 7532186 TI - Sialoadhesin binds preferentially to cells of the granulocytic lineage. AB - Sialoadhesin is a macrophage-restricted, sialic acid-dependent receptor of 185 kD that binds to the oligosaccharide sequence NeuAc alpha 2,3Gal on cell surface glycoconjugates. Recent cDNA cloning has shown that sialoadhesin is a new member of the immunoglobulin superfamily with sequence similarity to CD22, a sialic acid dependent receptor of B lymphocytes. Sialoadhesin has been implicated in cellular interactions of stromal macrophages with developing myeloid cells. In this study, direct evidence for this interaction was obtained in cell-cell binding assays using both native and recombinant forms of the protein. In all assays, sialoadhesin exhibited specific, differential binding to various murine cell populations of hemopoietic origin. In rank order, sialoadhesin bound neutrophils > bone marrow cells = blood leukocytes > lymphocytes > thymocytes. Single-cell analyses confirmed that sialoadhesin selectively bound myeloid cells in complex cell mixtures obtained from the bone marrow and blood. In comparison, a recombinant Fc-chimeric form of murine CD22 showed high binding to B and T lymphocytes, but very low binding to immature and mature myeloid cells. These results are consistent with the notion that sialoadhesin in involved in interactions with granulocytes at different stages of their life histories. PMID- 7532189 TI - The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta. AB - Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies. PMID- 7532190 TI - The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro. AB - Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions. PMID- 7532193 TI - Thalamic connections of the primary motor cortex (M1) of owl monkeys. AB - To determine the relative contributions of transthalamic cerebellar and pallidal projections to the primary motor cortex (M1) of owl monkeys, we examined the thalamic labeling resulting from injections of fluorescent tracers and wheatgerm agglutinin/horseradish peroxidase conjugate (WGA-HRP) into regions of M1 identified by intracortical microstimulation. Injections were placed in the major somatotopic divisions of M1 (the hindlimb, trunk, forelimb, and face representations) and in the caudal and rostral M1 subareas. In most cases, we injected several differentiable tracers into different parts of M1. Our results indicate that the strongest connections of M1 are with subdivisions of the ventral lateral thalamus (VL); other connections are mainly with intralaminar nuclei (the central lateral, paracentral, and center median nuclei) and the reticular nucleus. Most projections are reciprocal and topographically organized. M1 is strongly connected with the principal (VLp), medial (VLx), and anterior (VLa) subdivisions of the VL complex but has at most weak connections with the dorsal division (VLd). Each of the major somatotopic divisions of M1 is connected with an anteroposteriorly elongated territory within the VL complex. The connections are somatotopically organized such that the M1 hindlimb representation is connected with a band of cells in the lateral and anterior portions of the VL complex (spanning VLa and VLp), whereas the trunk, forelimb, and face representations are connected with successively more medially and posteriorly situated cell bands (spanning VLa, VLp, and VLx). There is some degree of overlap between the somatotopic territories within VL, although the absence of double-labeled cells in cases with injections of adjacent somatotopic divisions of M1 suggests that individual thalamic neurons project to single somatotopic regions. In addition to somatotopic differences, the connections of the caudal and rostral subdivisions of M1 differ to some extent. Caudal M1 is connected most strongly with VLp, a target of cerebellar projections, but it is also connected with VLa, which receives pallidal inputs. In complementary fashion, rostral M1 is most strongly connected with VLa, but it is also connected with VLp. VLx, a target of cerebellar projections, has significant connections with both caudal and rostral M1. These results indicate that all parts of M1 are influenced by both the cerebellum and globus pallidus in owl monkeys, as has been suggested in some recent studies of macaque monkeys. PMID- 7532194 TI - Characterization of trout galanin and its distribution in trout brain and pituitary. AB - Galanin was purified from an extract of the stomach of the rainbow trout, Oncorhynchus mykiss, and its primary structure was established as Gly-Trp-Thr-Leu Asn-Ser- Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Gly-Ile-Asp-Gly-His-Arg20- Thr-Leu-Ser Asp- Lys-His-Gly-Leu-Ala. Trout galanin shows six amino acid substitutions compared with pig galanin, but the N-terminal region (residues 1-14) has been fully conserved. The distribution of galanin-immunoreactive (GAL-IR) structures in the trout brain and pituitary was studied via immunohistochemistry. GAL-IR cell bodies were observed only in the caudal telencephalon, the preoptic region, and the mediobasal hypothalamus. GAL-IR fibers, however, are widely distributed throughout the brain, with a much lower density in the midbrain and posterior brain than in the tel- and diencephalon. Particularly dense innervation of the mediobasal hypothalamus, the ventral and supracommissuralis parts of the caudal telencephalon, and the region above and below the anterior commissure was observed. A heavy innervation of the pituitary was consistently detected. GAL-IR fibers were present in neurohypophyseal digitations of both the anterior and intermediate lobes with highest density in the region of the proximal pars distalis, where growth hormone and gonadotropic cells are located. Fibers were also seen in digitations of the rostral pars distalis, in particular between the prolactin follicles. The distribution of GAL-IR neurons in the central nervous system and pituitary of the trout suggests that the peptide may exercise an important role in the regulation of neuroendocrine functions, particularly those related to reproduction. PMID- 7532192 TI - Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen. AB - The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses. PMID- 7532195 TI - Contra biology: a polemic. AB - The philosophical basis of the prevalent biological model of psychiatry is examined critically, with particular reference to the reductionism inherent in such an approach. The pharmacological response initiated by the biological approach is then considered, and the perfidious nature of the complicity between psychiatry and the pharmacological/industrial nexus drawn out. Biological psychiatry is then situated within the prevailing political and economic ideology of the 1990s, and the move towards more community-based psychiatry examined from this perspective. Finally, the stresses necessitated by the incompatibility of many nursing models with the biological paradigm are addressed. This is, as the title suggests, a deliberately polemical and provocative paper, which is offered to stimulate debate both on the future of psychiatric/mental health nursing as a soundly theory-based speciality, and also on the uncritical acceptance of the biological paradigm. Wholesale adoption of this paradigm is considered to be inimical to many of the values notionally espoused by nursing, as well as posing dangers in the larger social field, in terms of the culturally acceptable notions of how we define ourselves, and how such definitions affect our conceptions of freedom, free will and agency. PMID- 7532191 TI - The origin of the myofibroblasts in breast cancer. Recapitulation of tumor environment in culture unravels diversity and implicates converted fibroblasts and recruited smooth muscle cells. AB - The origin of myofibroblasts in stromal reaction has been a subject of controversy. To address this question definitively, we developed techniques for purification and characterization of major stromal cell types. We defined a panel of markers that could, in combination, unequivocally distinguish these cell types by immunocytochemistry, iso-electric focusing, immunoblotting, and two dimensional gel electrophoresis. We then devised an assay to recapitulate in culture, within two weeks of incubation, critical aspects of the microenvironment in vivo including the typical tissue histology and stromal reaction. When confronted with tumor cells in this assay, fibroblasts readily converted into a graded pattern of myogenic differentiation, strongest in the immediate vicinity of tumor cells. Vascular smooth muscle cells (VSMC), in contrast, did not change appreciably and remained coordinately smooth muscle differentiated. Midcapillary pericytes showed only a slight propensity for myogenic differentiation. Analysis of ten primary tumors implicated converted fibroblasts (10/10), vascular smooth muscle cells (4/10), and pericytes (1/10) in the stromal reaction. Tumor cells were shown to specifically denude the venules both in culture and in vivo, explaining the VSMC phenotype in the stroma. The establishment of this assay and clarification of the origin of these cells pave the way for further analysis of the mechanisms of conversion, and of the consequence of such heterogeneity for diagnosis and treatment. PMID- 7532196 TI - Relative amounts of keratin 17 are higher than those of keratin 16 in hair follicle-derived tumors in comparison with nonfollicular epithelial skin tumors. AB - Specimens of trichilemmal cyst, malignant trichilemmoma, keratoacanthoma, and epidermal cyst were examined to characterize keratin peptides in hair-follicle derived tumors. Keratins were extracted from the specimens and analyzed by two dimensional gel electrophoresis and densitometry; the results were then compared with those for normal epidermis, the outer root sheath of hair follicles, psoriatic epidermis, and various nonfollicular cutaneous epithelial tumors. The specific nonfollicular tumors examined were squamous cell carcinoma, Bowen disease, actinic keratosis, eccrine porocarcinoma, and sebaceous carcinoma. Immunohistochemistry also was performed with a few anti-keratin monoclonal antibodies. As a general rule, K6 and K16 were expressed in hyperproliferative conditions, such as epidermal tumors, and K17 was coexpressed in the same lesions. The ratio of K16 to K17 in many epithelial skin tumors has been unclear until now. K17 content exceeded K16 content in most follicular tumors, whereas in almost all the nonfollicular tumors and the psoriatic epidermis, K17 levels were less than or about equal to K16 levels. There was a significant difference in the ratio of K16 to K17 between follicular and nonfollicular skin tumors. These results indicate that alterations in the content of these keratins may be associated with follicular differentiation. PMID- 7532197 TI - The appearance, density, and distribution of Merkel cells in human embryonic and fetal skin: their relation to sweat gland and hair follicle development. AB - The density and distribution of Merkel cells in human embryonic and fetal skin were studied using an immunolabeling technique on epidermal and dermal sheets obtained by ethylenediamine tetraacetic acid separation. Merkel cells were identified by the known cytokeratin markers CK20 and CK18. Merkel cells showed CK20 immunoreactivity as early as 56 d estimated gestational age (EGA) in the palmar epidermis (133.11 +/- 44.27 cells/mm2). The density increased rapidly, reaching a maximum of more than 1400 cells/mm2 at 80-90 d EGA. At this stage, the cells became distributed along the primary epidermal ridges. In the palmar epidermis of fetuses older than 100 d EGA, the distribution of Merkel cells showed the same pattern, but the density then decreased gradually. Merkel cells were not observed in ductal and glandular portions of eccrine sweat glands. In the epidermal sheets of hairy skin, a few cells were first seen in the fetus at 75 d EGA. At 100 d EGA, only a few Merkel cells were observed, mostly in the hair pegs and bulbous hair pegs. In the older fetus, ring-like arrangements and aggregates of Merkel cells were prominent in the infundibulum and bulge of hair follicles, respectively. Merkel cells were both globular and dendritic in shape. The ratio of dendritic to globular cells increased gradually until the period of highest Merkel cell density in both the glabrous and hairy skin. All Merkel cells located in the dermis were globular in shape. In accord with the results obtained, we postulate that Merkel cells may have some functional role in the formation and proliferation of eccrine sweat glands and hair follicle anlagen in developing skin. PMID- 7532198 TI - Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma. AB - Palmoplantar keratoderma of Voerner type (or epidermolytic palmoplantar keratoderma) is an autosomal dominant inherited disorder of keratinization with histologic features of epidermolytic hyperkeratosis. We studied members of two large unrelated kindreds with epidermolytic palmoplantar keratoderma, and biopsy specimens of lesional palmar skin from both families confirmed the histologic changes of epidermolytic hyperkeratosis. Whorls of abnormally aggregated keratin filaments were seen ultrastructurally to be associated with signs of cellular disintegration in spinous and granular cells. Direct sequencing of genomic DNA samples obtained from several members of each family established the substitution of a highly conserved arginine by tryptophan (R162W) in the 1A region of the alpha-helical rod domain of keratin 9. This arginine residue in a highly conserved region of keratins 1 and 10 is affected by disruptive missense point mutations in many patients with bullous ichthyosiform erythroderma. An equivalent position in the sole and palm restricted keratin 9 appears to be the mutation hot spot in epidermolytic palmoplantar keratoderma. To date, R162W is the most prevalent genetic defect reported in this genodermatosis. PMID- 7532199 TI - Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma. AB - Epidermolytic palmoplantar keratoderma is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles. Ultrastructurally the disease exhibits abnormal keratin filament networks and tonofilament clumping like that found in the keratin disorders of epidermolysis bullosa simplex and epidermolytic hyperkeratosis. The disease has been mapped to chromosome 17q11-q23 in the region of the type 1 keratin gene locus and more recently mutations have been found in the palmoplantar specific keratin, keratin 9. We have analyzed six unrelated incidences of epidermolytic palmoplantar keratoderma for mutations in their keratin 9 genes. In two of these, we have identified mutations that alter critical residues within the highly conserved helix initiation motif at the beginning of the rod domain of keratin 9. In a three-generation Middle Eastern kindred we found a C to T transition at codon 162 that results in an arginine to tryptophan substitution at position 10 of the 1A alpha-helical domain, thus confirming this codon as a hot spot for mutation in keratin 9. The other mutation found involves a T to C transition at codon 167 that results in the expression of a serine residue in place of the normal leucine at position 15 of the 1A segment and is the first documentation of this mutation in this gene. The identification of these substitutions extends the current catalog of disease causing mutations in keratin 9. PMID- 7532200 TI - Prompt decrease of circulating hepatitis C virus in patients with chronic hepatitis C after treatment with interferon. AB - Patients with chronic hepatitis C were treated with interferon (IFN) and followed for hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum. The response was correlated with decrease in serum levels of HCV RNA, as well as HCV genotypes and liver histopathology. Response to IFN, estimated by clearance of HCV RNA and normalization of aminotransferase levels at 6 months after the withdrawal of IFN, was observed in 11 (31%) of 35 patients infected with HCV of genotype II/1b, 13 (72%) of 18 with genotype III/2a, and 2 (33%) of 6 with genotype IV/2b; a single patient with genotype I/1a responded while the one doubly infected with HCV of genotypes II/1b and IV/2b did not. Response was seen in 10 (71%) of 14 patients with chronic persistent hepatitis, 14 (39%) of 36 with chronic active hepatitis 2A, and 3 (27%) of 11 with 2B. Response was achieved less often in patients with high than low pretreatment levels of HCV RNA. HCV RNA dropped sharply on a day after the start of IFN, and continued to decrease during the 2 weeks, irrespective of the response to IFN or HCV genotypes. In contrast, anti-HCV decreased more gradually and only in responders to IFN. These results support the rapid development of an IFN-mediated antiviral effect on HCV, and support therapeutic effects of IFN dependent on histopathology of liver as well as HCV RNA titers and genotypes. PMID- 7532202 TI - Modulation of interferon-mediated inhibition of human immunodeficiency virus type 1 by Tat. AB - Recently, we have shown that in acutely infected T cells interferons (IFNs) effectively inhibit the human immunodeficiency type 1 (HIV-1) proviral DNA synthesis during a single replication cycle. In the present study, we have evaluated the relative effectiveness of IFNs in restricting HIV-1 expression at post-transcriptional level. Treatment of HeLa cells with IFNs A* and B (up to 1,000 U/ml) did not result in a reduction in HIV-1 RNA and protein synthesis encoded by the transfected HIV-1 proviral clone. Interestingly, IFN treatment reduced significantly the HIV-1 mRNA levels encoded by the transfected tat defective HIV-1 provirus, and this inhibition could be overcome by transfection with Tat- and Rev-expressing plasmids. These results suggest that HIV-1-encoded Tat and Rev can overcome the inhibitory effects of IFNs on HIV-1 replication. PMID- 7532201 TI - Characterization of an interferon-induced 48-kD protein immunologically related to the double-stranded RNA-activated protein kinase PKR. AB - Polyclonal antibodies raised against purified and urea-denatured double-stranded protein kinase (PKR) from human origin cross-reacted by immunoblotting with a 48 kD protein (p48) induced by the three types of interferon (IFN), alpha, beta, and gamma. The induction of p48 is IFN dose dependent and its accumulation occurs a few hours after the addition of IFN. The induction of p48 is blocked by actinomycin D. Analysis by two-dimensional gel isoelectric-focusing, revealed p48 as a single spot with an isoelectric point (pI) of 6.8. In the same experiment the PKR was revealed as several subspecies with pI values in the pH range of 7.4 8.0. Cell fractionation experiments indicated that PKR and p48 have different subcellular localizations: PKR was found to be associated with the microsomal pellet as shown previously whereas p48 was recovered in the microsomal supernatant fraction. In addition to these differences, PKR and p48 were found to be differentially expressed in some human cells treated with the three types of IFN. For example, in HeLa cells, IFN-alpha or IFN-beta induced similarly both PKR and p48 whereas IFN-gamma induced mainly p48. In U937 cells in which PKR was not expressed with or without IFN treatment, p48 was strongly induced by all three types of IFN. These results suggest different mechanisms for the induction of PKR and p48. In view of its presence in different types of human cells and its induction by different types of IFN, it is possible to suggest that p48 might play an important role in mediating some of the action of IFN. PMID- 7532203 TI - Selective interferon-alpha/beta effects on platelet-derived growth factor stimulated processes in quiescent BALB/c-3T3 fibroblasts. AB - Interferon-alpha/beta (IFN-alpha/beta) suppresses cell cycle activation by platelet-derived growth factor (PDGF) as well as the induction of the 31-kD (pI) and the 35-kD (pII) proteins in density-arrested BALB/c-3T3 cells. We report that elevation of [Ca2+]i by ionomycin induces the synthesis of the 31-kD protein, but not that of the 35-kD protein. Since IFN blocks the PDGF-induced elevation of [Ca2+]i, these results suggest that IFN treatment may suppress pI induction by impairing this PDGF-activated signal transduction pathway. In contrast, because ionomycin did not induce the 35-kD protein, the suppression by IFN of PDGF induced pII appears to be mediated via a pathway distinct from that operating in the suppression of pI. In BALB/c-3T3 cells, IFN-alpha/beta did not itself affect the turnover or de novo synthesis of inositol phospholipids and the cellular content of diacylglycerol, nor did IFN block the enhancement of these parameters by PDGF. PMID- 7532204 TI - Interleukin-4 and interleukin-10 as antagonists of interferon-gamma. PMID- 7532206 TI - Okadaic acid and cultured frog sciatic nerves: potent inhibition of axonal regeneration in spite of unaffected Schwann cell proliferation and ganglionic protein synthesis. AB - Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury-induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 nM) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating-dephosphorylating reactions are critical for regeneration of peripheral nerves. PMID- 7532205 TI - Broad-range polymerase chain reaction for detection and identification of bacteria. AB - Detection and identification of fastidious pathogenic bacteria have traditionally presented an obstacle to the clinical and laboratory microbiologist. The diagnosis of disease caused by these bacteria is often empiric relying on clinical observations or indirect laboratory tests. Recently, a technique called broad-range polymerase chain reaction (PCR) has been integrated into studies designed to detect and identify previously uncharacterized bacterial pathogens. By using regions of the bacterial 16S ribosomal RNA (rRNA) gene that are highly conserved to prime synthesis of the remainder of this gene, PCR amplification can be performed directly from clinical samples which may contain small numbers of bacteria. The resulting PCR-amplified DNA can be sequenced to identify variable regions of the 16S rRNA gene that are bacteria-specific. This technique has proven valuable in identifying new fastidious bacterial pathogens that have resisted detection and identification by traditional microbiological methods. PMID- 7532210 TI - Protein kinase C modulates calcium sensitivity of nitric oxide synthase in cerebellar slices. AB - The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (+/-)-1-aminocyclopentane-trans 1,3-dicarboxylate (trans-ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans-ACPD were independent of extracellular calcium blocked by N omega-nitro-L-arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans-ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 nM free calcium, whereas at 25-70 nM free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations. PMID- 7532209 TI - N-linked glycosylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4 propionate (AMPA)-selective glutamate receptor channel alpha 2 subunit is essential for the acquisition of ligand-binding activity. AB - The N-linked glycosylation of the alpha 2 subunit of the mouse alpha-amino-3 hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N glycosylation sites. The recombinant receptor proteins were identified by [35S]methionine/[35S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [3H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 102 kDa and a minor species of approximately 98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N-glycosylation site is required for the formation and maintenance of the GluR alpha 2 subunit protein in an active conformation for ligand binding. Possible roles of N-glycosylation of GluR alpha 2 subunit protein are discussed. PMID- 7532207 TI - Similar rates of phosphatidylinositol hydrolysis following activation of wild type and truncated rat neurokinin-1 receptors. AB - The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild type and truncated receptor types. This decrease in rate of substance P stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization. PMID- 7532208 TI - Neocortical dialysate monoamines of rats after acute, subacute, and chronic liver shunt. AB - Intracerebral microdialysis was applied to monitor the neocortical extracellular levels of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, the neurotransmitters dopamine (DA), noradrenaline (NA), and serotonin (5-HT), and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3 acetic acid (5-HIAA) in rats with various forms of experimental hepatic encephalopathy (HE). The extracellular aromatic amino acid levels were clearly increased in acute, subacute, and chronic HE. No changes compared with controls in the neocortical DA release could be detected in the three experimental HE rat models investigated. The NA release showed a significant increase only in the subacute HE group. These data suggest that HE may not be associated with any major reduction of neocortical DA or NA release as previously suggested. In acute and subacute HE, decreased extracellular DOPAC but elevated 5-HIAA concentrations were seen. In chronic HE, elevations of both DOPAC and 5-HIAA were observed. Neocortical 5-HT release did not change in subacute and chronic HE, whereas it decreased in acute HE compared with control values. Significant increase in extracellular concentrations of 5-HIAA and of the 5-HIAA/5-HT ratio in the present study are in agreement with previously reported increases in 5-HT turnover in experimental HE. However, a substantially increased 5-HT turnover in experimental HE does not appear to be related to an increase in neuronal neocortical 5-HT release. PMID- 7532211 TI - The 84-kDa form of human matrix metalloproteinase-9 degrades substance P and gelatin. AB - Matrix metalloproteinase-9 (MMP-9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP-9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP-9 (94 kDa) was purified from the human cell line HL-60 and converted to an intermediary active form (84 kDa) with p aminophenylmercuric acetate. This active form of MMP-9 degraded SP with a kcat/Km of 170 mM-1 min-1, which is 30-fold greater than the previously reported value for a representative collagen-derived peptide. The major digestion products were identified as SP and SP, which were derived from cleavage of the Gln6-Phe7 bond. Minor products were also generated from cleavage of the Gly9-Leu10 bond. The other representative tachykinin peptides were cleaved at rates > 10-fold slower than that of SP. The 84-kDa peptidase was also active as a gelatinase. Longer treatment of MMP-9 with p-aminophenylmercuric acetate caused the conversion of the 84-kDa enzyme to the established 68-kDa active form; however, the rate of SP degradation did not increase. Because MMP-9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in interacellular communication by altering the availability of bioactive peptides. PMID- 7532212 TI - Effects of non-NMDA receptor modulators on [3H] dopamine release from rat mesencephalic cells in primary culture. AB - The effects of AMPA and kainate on [3H]dopamine release from fetal (embryonic day 15) rat mesencephalic neurons in primary culture were enhanced markedly in a dose dependent fashion by cyclothiazide, a recently described inhibitor of AMPA receptor desensitization. The EC50 value for cyclothiazide was 2.2 +/- 0.8 microM. The release of [3H]dopamine induced by both AMPA (or kainic acid) and the combination of AMPA (or kainic acid) with cyclothiazide was antagonized by specific antagonists like 6-cyano-7-nitroquinoxaline-2,3-dione or the noncompetitive benzodiazepine GYKI 52466. Unlike cyclothiazide, the lectin concanavalin A did not stimulate [3H]dopamine release. These results established the involvement of AMPA-preferring receptors on [3H]dopamine release from rat mesencephalic neurons in primary culture and provided further evidence for the existence of regulatory allosteric sites on AMPA receptor subunits. PMID- 7532213 TI - Phosphorylation of tau protein by casein kinase-1 converts it to an abnormal Alzheimer-like state. AB - The microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease. Both proline-dependent protein kinases (PDPKs) and non-PDPKs are involved in this hyperphosphorylation of tau. Several PDPKs can phosphorylate tau in vitro and induce Alzheimer-like epitopes to many phosphorylation-dependent antibodies. A similar induction has not been reported with non-PDPKs. In this study we have evaluated six non-PDPKs [cyclic AMP-dependent (A-kinase), calcium/phospholipid-dependent (C-kinase), casein kinase-1 (CK-1), casein kinase 2 (CK-2), calcium/calmodulin-dependent protein kinase II, and calcium/calmodulin dependent protein kinase from rat cerebellum] for their abilities to induce Alzheimer-like epitopes on tau. Such epitopes were induced by A-kinase, C-kinase, CK-1, and CK-2, but the degree of induction achieved by CK-1 was much greater than with the other kinases. These results suggest that CK-1 may play an important role in the conversion of tau from the normal to the abnormal phosphorylation state in Alzheimer's disease. PMID- 7532214 TI - Over-expression of the DM-20 myelin proteolipid causes central nervous system demyelination in transgenic mice. AB - We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM-20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at approximately 3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3-4 months. These symptoms progress in severity with age. Death occurs by 8-10 months. Myelination in 2-month-old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2-month-old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM-20 as normal animals. In 7-month-old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM-20 are greatly reduced relative to the 2-month-old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (> 2 yr). Mice with low copy numbers of the transgene (2-4 copies) appear to be unaffected and have normal life spans. PMID- 7532215 TI - Distribution and expression of developmentally regulated phosphorylation epitopes on MAP 1B and neurofilament proteins in the developing rat spinal cord. AB - The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstration in vivo of a mAb 150 axonal gradient similar to that seen previously in vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies. PMID- 7532216 TI - Drug-mediated changes in the susceptibility of human lymphoblastoid B cells to NK mediated cytolysis: studies with a triazene compound. AB - Previous studies have shown that treatment of leukemia-bearing mice with triazene compounds results in a profound alteration of the immunological properties of leukemic cells. These cells become highly immunogenic and susceptible to natural immunity (NI). Moreover, in a pilot clinical study, dacarbazine was found to suppress bone marrow blasts in patients with acute non-lymphoblastic leukemias. The cytotoxic mechanism involved could be of biochemical and immunological origin as well. Therefore experiments were carried out to test whether triazenes could influence the susceptibility of blast cells to human NI effector lymphocytes (represented, at least in part, by NK cells). The results obtained with target Epstein-Barr virus (EBV)-immortalized B cells and effector cells of different donors, showed that: (a) multiple in vitro treatments of lymphoblastoid cells with methyl-triazene-benzoic acid (MTBA, a triazene compound active in vitro), gave origin to lines that were more resistant than the parental lines to the antitumor effects of MTBA; (b) MTBA-treated lines were more susceptible (37.5% of cases), or less susceptible (31.2% of cases) to NI than parental cells. Effector lymphocytes of various donors recognized different changes in susceptibility to natural killer (NK)-mediated lysis; (c) treatment of parental or MTBA-treated lines with interferon-beta reduced target cell susceptibility to NK-mediated cytolysis, but increased NK activity and lymphoblast chemosensitivity to MTBA. PMID- 7532218 TI - Primary intraosseous Kaposi's sarcoma of the maxilla in human immunodeficiency virus infection: review of literature and report of case. PMID- 7532217 TI - Cancer and noncancer risk to women in agriculture and pest control: the Agricultural Health Study. AB - The Agricultural Health Study is a collaborative effort involving the National Cancer Institute, the US Environmental Protection Agency, and the National Institute of Environmental Health Sciences. A goal of this investigation is to establish a large cohort of men and women that can be followed prospectively for 10 years or more to evaluate the role of agricultural exposures in the development of cancer, neurologic disease, reproductive difficulties, childhood developmental problems, and other chronic diseases. The study also will provide an opportunity to assess the role that diet, cooking methods, and other lifestyle factors have on the cause of cancer and other diseases. The cohort will be composed of approximately 112,000 adult study subjects, including 42,000 women, making this one of the largest cohorts of women ever assembled for an epidemiologic investigation of environmental and occupational exposures. Children of farm families also will be enrolled. The study will be conducted in Iowa and North Carolina. Enrollment will begin in December 1993 and continue for 3 years. PMID- 7532221 TI - An unusual variant of keratoameloblastoma. AB - An unusual case of ameloblastoma which depicts cystic follicles containing orthokeratin, parakeratin, desquamated epithelium and necrotic material with dystrophic calcification is presented. The presence of ameloblast-like cells confirms the diagnosis of an ameloblastoma. However, certain features resembled those of the keratoameloblastoma and others, less convincively, the papilliferous keratoameloblastoma. The extensive keratinisation in this tumour and in the aforementioned neoplasms raises the question whether they represent variants of the acanthomatous ameloblastoma. PMID- 7532220 TI - Immunocytochemical localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in oral squamous cell carcinoma (SCC). AB - The localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in human oral squamous cell carcinoma (SCC) was examined by immunohistochemical techniques using anti-FGF-1 and anti-FGF-2 monoclonal antibodies. Immunofluorescence staining of two oral SCC cell lines revealed that growing cancer cells were intensely positive for both FGF-1 and FGF-2, but confluent cells showed a faint immunostaining. In addition, two molecular mass species of FGF-1 (16 and 18 kDa) and one of FGF-2 (18 kDa) were identified by Western blot in cell extracts derived from growing SCC cells, but not from confluent SCC cells. The growing cell extracts significantly stimulated the proliferation of human umbilical vein endothelial cells. Immunoperoxidase staining of 13 oral SCC cases showed that both well-differentiated and poorly-differentiated cancer cells were positive for FGF-1 and FGF-2 with high frequency and intensity as compared to normal oral epithelium. These results indicate that SCC cells express high levels of endogenous FGF-1 and FGF-2, and suggest that these growth factors may contribute to cancer cell growth. PMID- 7532219 TI - Distribution of tenascin in oral premalignant lesions and squamous cell carcinoma. AB - The distribution of the extracellular matrix (ECM) protein tenascin (Tn) was studied in oral premalignant lesions and squamous cell carcinoma by using the monoclonal antibody (Mab) 143DB7. In normal buccal and palatal mucosa, in ventral tongue, in floor of mouth and in gingiva, immunoreaction for Tn was seen to be distributed as a continuous thin, delicate line merely in the basement membrane region. Hyperkeratosis without dysplasia showed a distinct zone of enhanced Tn immunoreactivity immediately beneath the epithelium. In dysplasias of various degree, enhancement of the stromal Tn content could be observed, being most conspicuous in carcinoma in situ lesions. In most invasive carcinoma cases the Tn immunoreactivity was intense, extending deeply into the underlying stroma. In such lesions Tn-reaction often covered the total stroma. Notably, the strongest immunoreaction was seen at the advancing edges of the tumor. The triggering factor for stromal Tn enhancement seems to be of epithelial origin. The enhanced expression of Tn suggests that Tn plays a role in organizing and remodelling the stroma to support active epithelial proliferation and migration. However, it also seems that inflammation is associated with Tn expressions. PMID- 7532222 TI - [Zonal anatomy of the prostate using endorectal MRI]. AB - The development of an endorectal surface coil now permits a partial study of the anatomical model developed by McNeal. Axial and coronal views, which were used to establish the model can be obtained in a short period of time with fast spin echo sequences. Axial views are performed along the proximal urethra and coronal views are performed along the axis of the distal urethra and the ejaculatory ducts. Anatomical boundaries of the transitional zone are well delineated on axial views, illustrating the concept of "inner gland". The prostatic capsule and the neuro-vascular bundles, pathways of extension of the cancer out of the prostate are also well delineated. Coronal sections allow a very good anatomical study of the caudal junction of the vas deferens and the seminal vesicles (the so called weak space), pathway of tumor extension to the seminal vesicles. Differences in signal of the prostatic zones make the outer gland cancers very conspicuous as well as some transitional cancers which can show, in some cases, an homogeneous hyposignal. PMID- 7532224 TI - Expression of a rat vasopressin transgene in rat testes. AB - The rat vasopressin gene contains two transcriptional promoters; the activity of one is confined to the hypothalamus, while the other is testis specific. To define the sequences mediating the cell-specific expression of the vasopressin gene, we introduced rat vasopressin transgenes into the rat germ line. Neither transgene 1.5-V beta gal-0.2, which consists of the entire vasopressin structural gene containing a 3 kbp beta-galactosidase reporter element in exon III, flanked by 1.5 kbp upstream of the start of hypothalamic transcription and 0.2 kbp downstream of the polyadenylation site, nor transgene 3-V beta gal-0.2, which consists of the entire VP structural gene containing a 3 kbp beta-galactosidase reporter element in exon III, flanked by 3 kbp upstream of the start of hypothalamic transcription and 0.2kbp downstream of the polyadenylation site, were expressed in the hypothalamus. This contrasts with a previously described transgene consisting of the rat vasopressin structural gene containing a reporter in exon III, flanked by 5 kbp of upstream and 3 kbp of downstream sequences, which is expressed in vasopressinergic hypothalamic neurons. Both the 3-V beta gal-0.2 and 1.5-V beta gal-0.2 transgenes were expressed in testicular germ cells using a promoter located within the beta-galactosidase reporter element. Transgene RNA was most abundant during the late stages of meiosis. Rats bearing vasopressin-beta-galactosidase transgenes provide new models for the study of the mechanisms whereby an epigenetic choice is made between the use of a germ cell or a somatic promoter, and the stage-specific transcriptional regulation of a germ cell promoter during spermatogenesis. PMID- 7532223 TI - Differential expression of the integrin subunits in human fetal membranes. AB - The distribution of the alpha 1, alpha 3-alpha 6, beta 1, beta 3 and beta 4 integrin subunits in fetal membranes at term was examined using an indirect immunofluorescence technique and confocal laser scanning microscopy. In the amniotic epithelium, beta 4 integrin (alpha 6 beta 4) exhibited distinct basal localization, whereas beta 1 integrins (alpha 3 beta 1, alpha 5 beta 1) were localized basolaterally. This finding suggests that integrins, especially alpha 6 beta 4 which is a structural component of the hemidesmosomes, may function as basement membrane receptors. Integrins localized laterally may play a role in cell-cell interactions. beta 1 (alpha 1 beta 1, alpha 5 beta 1) integrins are probably involved in cell-matrix interactions in the connective tissue layers which are rich in collagens and fibronectin. Cytotrophoblasts, located predominantly towards the chorionic basement membrane, mainly expressed alpha 6 beta 4, while those located predominantly in the vicinity of decidua expressed alpha 5 beta 1, alpha 3 beta 1 and alpha 1 beta 1. Decidual cells expressed alpha 3 beta 1 and alpha 1 beta 1, whereas alpha 1, alpha 5, alpha 6, beta 1 and beta 4 were expressed in blood vessels. This pattern of integrin expression reflects the reported difference in composition of the extracellular matrix at these locations and obviates an important role for alpha 5 beta 1 at the chorio-decidual interface. The differential integrin expression at the cell-basement membrane interfaces demonstrated in this study (at amniotic epithelium, cytotrophoblasts, decidual cells and blood vessels) indicated a differential recognition of basement membranes by these cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532225 TI - Effect of exogenous insulin on plasma and follicular insulin-like growth factor I, insulin-like growth factor binding protein activity, follicular oestradiol and progesterone, and follicular growth in superovulated Angus and Brahman cows. AB - Angus (n = 14) and Brahman (n = 14) cows were used to evaluate the effects of insulin administered concomitantly with FSH in a superovulation regimen. Cows were allotted to four pen replicates by treatment and breed, and received FSH (i.m.) twice a day for 5 consecutive days (first day of injections = day 0 of study) plus concomitant administration of either saline (control) or long-acting bovine insulin (0.25 iu kg-1 body mass; s.c.). Blood samples were collected at intervals of 6 h during the injection period and analysed for plasma insulin, glucose, insulin-like growth factor I (IGF-I) and IGF-I binding protein (IGFBP) activity. Cows were ovariectomized on day 5. The number and diameter of follicles were recorded. Follicular fluid was aspirated for determination of IGF-I, IGFBP activity, oestradiol and progesterone. Mean plasma concentration of glucose was lower in insulin-treated than in control cows averaged over days 1-5 (56 +/- 3 versus 82 +/- 3 mg dl-1; P < 0.01). Plasma concentration of IGF-I and IGFBP activity were not affected (P > 0.10) by treatment, but were higher in Brahman than in Angus cows (IGF-I: 41 +/- 6 versus 19 +/- 6 ng ml-1, P < 0.05; IGFBP activity: 17.5 +/- 0.4 versus 15.8 +/- 0.04% (10 microliters)-1; P < 0.03). Insulin treatment did not affect the number of small (1.0-3.9 mm), medium (4.0 7.9 mm) or large (> or = 8.0 mm) follicles. Brahman cows had a greater (P < 0.01) number of medium and total follicles (19.4 +/- 2.5 and 60.5 +/- 5.5, respectively) than did Angus cows (7.5 +/- 2.6 and 30.5 +/- 5.6, respectively). Diameter of large follicles was greater in insulin-treated than in control cows (11.4 +/- 0.2 versus 10.6 +/- 0.1 mm; P < 0.05). Follicular fluid IGF-I concentration in large follicles was higher in insulin-treated Brahman cows (60 +/- 2 ng ml-1) than in control Brahman cows (37 +/- 2 ng ml-1), but was lower in insulin-treated Angus cows (31 +/- 3 ng ml-1) than in control Angus cows (38 +/- 2 ng ml-1; treatment x breed interaction, P < 0.01). IGFBP activity in fluid from large follicles was not affected by insulin treatment in Brahman cows but was reduced (P < 0.05) by insulin treatment in Angus cows.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532226 TI - 6-(4-pyridinyl)-1H-1,2,3-triazolo[4,5-d]-pyrimidin-4(5H)-one: a structurally novel competitive AMPA receptor antagonist. PMID- 7532227 TI - Association of serum immunoglobulin G antibodies against human papillomavirus type 16 capsids with anal epidermoid carcinoma. AB - BACKGROUND: Anal epidermoid carcinoma is a relatively rare tumor, but its incidence has been increasing rapidly during the past few years. Genetic material from the major oncogenic types of human papillomavirus (HPV), types 16 and 18, has regularly been demonstrated in a substantial proportion of anal cancers, suggesting an etiologic role of HPV infection. Recently, serum antibodies against HPV type 16 capsids were shown to be a serologic measure of HPV16 infection. PURPOSE: We investigated whether serum antibodies against HPV16 capsids are associated with an increased risk of developing anal cancer. METHODS: Serum samples from 64 patients (48 women and 16 men) with untreated anal epidermoid cancer and from 79 age- and sex-matched healthy blood donors were analyzed for the levels of serum immunoglobulin G (IgG) against capsids of HPV16 by the enzyme linked immunosorbent assay. The levels of serum IgG against HPV type 6 and bovine papillomavirus (BPV) capsids, as well as against HPV16 peptide antigens, were also measured. RESULTS: Whereas antibodies against HPV6 or BPV capsids were not significantly associated with anal cancer, the presence of IgG against HPV16 capsids exceeding the anti-BPV antibody levels was demonstrated among 55% (35 of 64) of the case patients but only among 4% (three of 79) of the control subjects (odds ratio [OR] = 30.4; 95% confidence interval [CI] = 8.4-161.5). Antibodies against HPV16 E2 and E7 peptides were also more common among case patients (OR = 12.8 and 95% CI = 5.4-31.5 for E2; OR = 3.0 and 95% CI = 1.4-6.7 for E7). CONCLUSION: The results suggest that HPV16 capsid antibodies are serologic markers for anal cancer. IMPLICATION: Exposure to HPV16 or related viruses appears to be a major risk factor in the majority of anal cancers. PMID- 7532228 TI - Alterations in gastric mucin with malignant transformation: novel pathway for mucin synthesis. AB - BACKGROUND: Mucins are high-molecular-weight glycoproteins produced by both normal and cancer cells. However, in cancer cells, abnormal mucins are synthesized and potentially can be used as markers for the development and progression of certain malignancies. In a previous study, we reported the production of a new monoclonal antibody directed against a mucin antigen termed F1 alpha, an O-linked oligosaccharide similar to sialyl Tn and Thomsen Friedenreich (T) antigens, that has not been previously detected in human cancers. F1 alpha is expressed in a high percentage (80.2%; 89/111) of gastric cancers. PURPOSE: In the present study, we compared the expression of F1 alpha with that of sialyl Tn and T antigens in human gastric cancer tissues to determine how differences in the expression of these cancer-associated antigens correlated with the biological properties of cancer cells. METHODS: A total of 141 cases of gastric cancer were studied. Sections of formalin-fixed, paraffin embedded tissue were immunostained for F1 alpha, sialyl Tn, and T antigens. The relationship between the expression of these antigens and the patient's clinicopathologic characteristics was studied. The chi-square test (two-sided) was used for statistical analyses. RESULTS: F1 alpha was expressed in a high percentage of the cases of early to advanced cancers, irrespective of the degree of malignant progression. The rate of expression of sialyl Tn antigen in early carcinoma was low, but it increased significantly as depth of invasion increased (P < .05) and was significantly higher in patients with hepatic or lymph node metastasis than in those without such metastasis (P < .01). Expression of T antigen significantly increased with depth of invasion (P < .01) and was significantly higher in patients with hepatic metastasis (P < .05), lymph node metastasis (P < .05), or peritoneal dissemination (P < .01) than in those without such metastasis or dissemination. In consecutive sections of the same specimen, the sites of staining for F1 alpha and sialyl Tn antigens seldom coincided. In many cases, F1 alpha staining was predominant, but the sialyl Tn-dominant region tended to increase as gastric cancer progressed. Regions of T-antigen staining were usually circumscribed by those of F1 alpha staining. CONCLUSION: Our findings indicate that the expression of F1 alpha begins almost at the same time as does carcinogenesis in gastric epithelial cells. Moreover, in association with progression of gastric carcinoma, synthetic pathways for sialyl Tn antigen and T antigen probably are activated independently. PMID- 7532230 TI - Impact of symptoms of prostatism on level of bother and quality of life of men in the French community. AB - The impact of symptoms of prostatism on level of bother and quality of life of French men was assessed nationwide, in a representative community sample of 2,011 subjects 50 to 84 years old. Bothersome level and quality of life associated with 12 urinary symptoms were assessed by face-to-face interviews, using a previously validated questionnaire for benign prostatic hyperplasia (BPH). The international prostate symptom score and the American Urological Association (AUA) bothersome index were also computed. Urgency was by far the most bothersome symptom in French men. Nocturia and wetting underclothes ranked second in subjects who did not undergo prostate surgery and among symptomatic patients, respectively. Overall, symptom frequency accounted for 72% of the variability of symptom bother but the form and strength of this correlation varied among symptoms from 0.18 to 0.43. Among the symptomatic subjects who had not yet undergone prostate surgery 11%, 10% and 79%, respectively, were dissatisfied, neutral and satisfied with the current urinary condition. Satisfaction with the urinary condition was positively linked to symptom frequency (p < 0.001) and symptom bother level (p < 0.001). Nocturia, dysuria, daytime repeat voiding, wetting clothes and urgency, when severely bothersome, were independent predictors of decreased satisfaction with the urinary condition (p = 0.01). Approximately half of the patients with severe BPH symptoms expressed serious worries and concerns with the urinary condition, and sizable levels of interference by the symptoms with daily activities, which are potentially affected by urinary troubles. The AUA bother index was the best determinant of subject level of worry about the urinary condition and of interference with daily life. This study supported the concomitant use of the international prostate symptom score and the AUA bother index in further research studies of BPH related impairments in quality of life. PMID- 7532229 TI - Sensitive detection of prostatic hematogenous tumor cell dissemination using prostate specific antigen and prostate specific membrane-derived primers in the polymerase chain reaction. AB - We developed a polymerase chain reaction based assay enabling sensitive detection of hematogenous tumor cell dissemination in patients with prostate cancer. We performed "nested polymerase chain reaction," amplifying messenger ribonucleic acid sequences unique to prostate specific antigen (PSA) and to the prostate specific membrane antigen, and compared the respective results. Prostatic tumor cells were detected in 2 of 30 patients (6.7%) by polymerase chain reaction with PSA derived primers, while prostate specific membrane primers detected tumor cells in 19 (63.3%). All 16 negative controls had negative PSA and prostate specific membrane polymerase chain reaction. Assays were repeated to confirm results, and polymerase chain reaction products were verified by deoxyribonucleic acid sequencing and Southern analysis. Patients harboring circulating prostatic tumor cells as detected by prostate specific membrane and not by PSA polymerase chain reaction included 7 of 13 previously treated by radical prostatectomy who had nonmeasurable serum PSA levels at the time of this assay. The significance of these findings with respect to future disease recurrence and progression will be investigated. PMID- 7532232 TI - A possible condition of pre-instability in prostatic obstruction. AB - There were 40 patients with benign prostatic hypertrophy (mean age 66 +/- 9 years) who had an obstructed stable bladder on a pressure-flow study. After a mean of 18 months (range 5 to 64) urodynamic studies were repeated and 20 men (group 1, mean age 60 +/- 7 years) had a stable bladder, while 20 (group 2) who were older (mean age 71 +/- 7 years) had detrusor instability. In the latter 20 patients greater outflow obstruction was noted at the second assessment. What seems most interesting is that at time 1 group 2 patients had a significantly higher rate of increase in opening pressure, which would possibly characterize an early stage in the development of structural changes leading to detrusor instability. This finding might prove a reliable clue to predict the latter condition in men with a proximally obstructed stable bladder. PMID- 7532231 TI - Correlation of American Urological Association symptom index with obstructive and nonobstructive prostatism. AB - The precise role of the American Urological Association (AUA) symptom index in the management of benign prostatic hyperplasia (BPH) is not well established. The AUA symptom index has been recommended only for quantifying the symptoms of BPH but not for its diagnosis. However, to our knowledge the ability to discriminate obstructive from nonobstructive BPH using the AUA symptom index has never been investigated. To establish the relationship between the AUA symptom index and prostatic obstruction 125 men (mean age 67.7 +/- 8.4 years) with voiding dysfunction presumably related to BPH were analyzed. Patients were given the AUA symptom questionnaire, following which video urodynamic studies were done, including micturitional urethral pressure profilometry for specifically diagnosing outlet obstruction. The patients were divided into 2 groups: group 1 78 with primary BPH dysfunction and group 2-47 with prostatism of ambiguous etiology. The mean AUA symptom index in group 1 (15.5 +/- 7.1) was not statistically different from that in group 2 (14.8 +/- 7.9). In both groups the mean AUA symptom index in the patients with obstruction (15.3 +/- 7.2 for group 1 and 13.9 +/- 7.9 for group 2) was not statistically different from that in the nonobstructed group (17.0 +/- 5.4 and 16.1 +/- 7.9, respectively). Of the severely symptomatic patients 22% did not have obstruction whereas all mildly symptomatic patients did. No significant correlations were found between the severity of obstruction and the AUA symptom index in either group. These observations indicate that the AUA symptom index cannot discriminate obstructed from nonobstructed BPH cases, not all severely symptomatic BPH patients will have outlet obstruction, a significant proportion of mildly symptomatic BPH patients can have outlet obstruction and voiding dysfunctions in elderly men, regardless of the etiology, produce similar symptoms. PMID- 7532233 TI - Acute urinary retention in men: a comparison of voiding and nonvoiding patients after prostatectomy. AB - Multiple parameters were examined preoperatively to determine if any could predict successful voiding after prostatectomy in male patients who present in acute urinary retention due to benign prostatic hypertrophy. A total of 50 men 50 to 85 years old (mean age 69.5 years) who presented with this clinical picture was investigated with multichannel urodynamic studies, and completed the American Urological Association (AUA) symptom score for benign prostatic hypertrophy preoperatively and postoperatively. All patients underwent prostatectomy. Mean retention volume was 1,172 cc (range 500 to 2,100). Mean preoperative and postoperative AUA symptom scores were 15.5 and 5.0, respectively. At 3 months postoperatively 45 patients (90%) were able to void without catheterization. At 16.6 months 5 patients still required clean intermittent catheterization to empty the bladder. Postoperatively, there was a statistically significant improvement in AUA symptom score, opening voiding pressure and peak flow rate. The AUA symptom score was not predictive of either impending acute retention or normal voiding after prostatectomy. In our patients no preoperative parameters were statistically different between those voiding and those on clean intermittent catheterization. However, poor sensation, large retention volumes, lack of instability and no voluntary detrusor contractions were more common in the nonvoiding men. PMID- 7532235 TI - Symptoms and urodynamics. PMID- 7532234 TI - Reasons for the weak correlation between prostate volume and urethral resistance parameters in patients with prostatism. AB - In an attempt to increase our understanding of the clinical syndrome of benign prostatic hyperplasia (BPH) an analysis was made of the association between prostate volume as measured by transrectal ultrasound and several reported urodynamically determined urethral resistance parameters. Two types of obstruction can be recognized on the basis of urodynamic data: a compressive type characterized by a high urethral opening pressure and a prolonged isovolumetric contraction phase before urine flow can start, and a constrictive type characterized by a normal opening pressure and an increased slope of the urethral resistance relation. A combination of both types is often seen in BPH. In our study, parameters that selectively quantify compression correlate weakly to moderately with prostate volume, whereas parameters that mainly quantify constriction do not correlate at all with prostate volume. Parameters that combine a measure for compression and constriction correlate less well with prostate volume than parameters that mainly quantify compression. The variation in prostate volume was found to determine the variation in urethral resistance by 15% or less depending on the parameter used, which implies that the different pathophysiological mechanisms that can increase urethral resistance in the complex process of clinical BPH are mainly determined by factors other than the volume of the prostate. Thus, despite the lack of correlation between prostate volume and urethral resistance, pressure-flow studies and the determination of urethral resistance parameters provide a valuable contribution to the understanding of the pathophysiology of voiding dysfunction in men with symptoms of prostatism. PMID- 7532236 TI - Transurethral resection of the prostate versus open prostatectomy: long-term mortality comparison. AB - To determine whether transurethral prostatectomy results in higher long-term mortality rates than open prostatectomy, we reviewed retrospectively 1,125 patients treated by transurethral and 190 treated by nonperineal open prostatectomy for benign disease at 1 institution from 1978 through 1987. Patients in whom prostatic cancer was found were excluded. We identified age, preoperative medical illnesses and urinary retention, American Society of Anesthesiologists category, type of anesthesia, length of followup, health status and cause of death. For statistical analysis the study cohort consisted of 527 patients in whom the charts were complete and followup was adequate (421 in the transurethral prostatectomy and 106 in the open prostatectomy groups). Mean age for the 2 groups was 66.3 and 67.5 years, respectively. With an average followup of 70.7 months 77% of the transurethral prostatectomy group were alive, compared to 78% of the open prostatectomy group at an average followup of 71.4 months. We found no supportive evidence that transurethral prostatectomy results in higher long-term mortality rates than does an open operation (log-rank test p = 0.74). Also, there was no significant survival difference in patients who required a preoperative Foley catheter. We also examined a subset of patients with adequate followup who had no significant medical history (for example hypertension, diabetes, heart disease and so forth) and compared them to patients with medical illnesses at prostatectomy. There was a significant survival difference between those with and without preoperative medical conditions (Wilcoxon test p = 0.047) in the transurethral prostatectomy group but not in the open group (p = 0.58). However, there was no significant survival difference between procedures among the healthiest subset of patients (p = 0.16). PMID- 7532237 TI - Re: Genetic susceptibility of benign prostatic hyperplasia. PMID- 7532238 TI - Re: The inability of prostate specific antigen index to enhance the predictive value of prostate specific antigen in the diagnosis of prostatic carcinoma. PMID- 7532239 TI - Regional concentration of basic fibroblast growth factor in normal and benign hyperplastic human prostates. AB - Basic fibroblast grown factor (bFGF) is a potent mitogen for mesenchymal cells, including fibroblasts cultured from prostate, and has been postulated to play a role in the development of benign prostatic hyperplasia (BPH). If this is the case, it might be expected that bFGF levels would be elevated in the adenomas of BPH and in the periurethral region of the prostate where BPH is believed to arise. This study was undertaken to test this hypothesis. The concentration of bFGF was evaluated in 31 prostates, 13 normal glands and 18 with BPH. A method for quantitating bFGF by radioimmunoassay was developed that enabled growth factor levels to be correlated to the geographic region of the prostate and the histopathology of the specimen. A 2- to 3-fold higher concentration of bFGF (ng./g. of tissue) was noted in the benign hyperplastic prostates when compared with the adult normal glands. Pubertal specimens demonstrated low growth factor levels comparable to those observed in the normal adult group. Two prepubertal prostates analyzed had high levels similar to those measured in the hyperplastic glands. While the levels of bFGF in the normal adult prostates were highest in the periurethral region, statistical analysis failed to demonstrate a significant difference. Similarly, quantitative morphometric evaluation failed to demonstrate any significant differences in bFGF concentration related to the proportion of stromal, epithelial, or lumenal elements in the tissue sections. PMID- 7532240 TI - Cytokines modulate in vitro invasiveness of renal cell carcinoma cells through action on the process of cell attachment to endothelial cells. AB - Cytokines play important roles in adhesion between various cells and endothelium. Cell-cell adhesion is also a critical step in the invasion of interstitial tissue by cancer cells. Using an in vitro invasion assay modified by cultured human umbilical venule endothelial cells (HUVEC) and an extracellular matrix (Matrigel) membrane system, we studied how cytokines affect the invasiveness of human renal cell carcinoma cells through action on the endothelium. When HUVEC were treated for 6 hours with tumor necrosis factor (TNF, 100 or 1000 U/ml.), interleukin-1 (IL-1, 10 ng./ml.) or IL-6 (1.0 or 10 ng./ml.), the treatment significantly increased in vitro invasiveness of the carcinoma cells. Enhancement of carcinoma cell invasiveness reflected the enhancement by the cytokine treatments of the ability of HUVEC to express vascular cell adhesion molecule-1 (VCAM-1). Application of anti-VCAM-1 monoclonal antibody (mAb) suppressed in vitro invasion of the carcinoma cells when HUVEC were treated with the cytokines at the above concentrations. Moreover, an adherence assay demonstrated that a larger number of carcinoma cells adhered to the endothelium treated by the cytokines than to endothelium not receiving such treatment and that anti-VCAM-1 mAb application inhibited the adhesion. The cytokine treatment of HUVEC did not affect type IV collagenolysis. These results indicated that cytokines can enhance the in vitro invasiveness of carcinoma cells through their action on endothelium (that is, augmentation of VCAM-1 expression) in the in vitro invasion assay modified with HUVEC-Matrigel-reconstituted membrane. PMID- 7532242 TI - Pinacidil attenuates positive inotropic but not chronotropic responses to norepinephrine in isolated dog atrial and ventricular preparations. AB - We investigated whether pinacidil, a K+ATP channel opener like acetylcholine and adenosine, attenuated the positive chronotropic and inotropic responses to norepinephrine in isolated, blood-perfused dog atrial and ventricular preparations. Pinacidil (0.01-0.3 mumol) decreased atrial and ventricular contractile force to a much greater extent than sinus rate in a dose-related manner. Pinacidil dose-dependently attenuated increases in atrial and ventricular forces induced by norepinephrine but not increases in sinus rate. Pinacidil similarly attenuated the positive atrial and ventricular inotropic responses to Bay k 8644 and CaCl2. The pinacidil doses producing a fifty percent decrease (ED50) of the atrial and ventricular contractile force were not significantly different from the respective pinacidil doses producing a fifty percent inhibition (ID50) of the positive inotropic responses to norepinephrine, Bay k 8644 and CaCl2. Ouabain (5 and 15 nmol) did not affect the decreases in atrial and ventricular contractile force in response to pinacidil. These results suggest that the K+ATP-channel activator pinacidil, unlike acetylcholine or adenosine, functionally attenuates increases in ventricular and atrial contractile force in the responses to norepinephrine and other cardiotonics due to shortening of the action potential duration induced by K+ATP-channel activation in the dog heart. PMID- 7532243 TI - A novel modified tissue-type plasminogen activator (t-PA), E6010, gradually increases coronary blood flow after thrombolysis compared with native t-PA, urokinase and balloon catheter occlusion-reperfusion. AB - In a canine copper coil-induced coronary thrombosis model, the differences in frequency of reperfusion arrhythmias (premature ventricular complexes: PVC) and mortality rate after thrombolysis by intravenous bolus injection of a novel modified tissue-type plasminogen activator (t-PA), E6010, and by continuous intravenous infusion of native t-PA or urokinase were evaluated. Rapid coronary occlusion and reperfusion were produced with a balloon catheter in another group of dogs, and the findings were compared with those in the thrombolysis groups. Reperfusion occurred gradually after the administration of E6010, but was significantly more rapid after administration of native t-PA and urokinase (P < 0.05). PVC were observed more frequently in native t-PA, urokinase and balloon occlusion-reperfusion groups than in the E6010 group. The mortality rate due to ventricular fibrillation was 0.0% in the E6010 group, 50.0% in the native t-PA and balloon occlusion-reperfusion groups, and 33.3% in the urokinase group. These results suggest that the more gradual reperfusion of the coronary artery at an earlier period after drug administration led to the lower frequency of reperfusion arrhythmias and low mortality rate in the E6010 group than in the native t-PA, urokinase and balloon occlusion-reperfusion groups. PMID- 7532241 TI - Treatment guidelines for patients with hyperthyroidism and hypothyroidism. Standards of Care Committee, American Thyroid Association. AB - OBJECTIVE: To develop a set of minimum clinical guidelines for use by primary care physicians in the evaluation and management of patients with hyperthyroidism and hypothyroidism. PARTICIPANTS: Guidelines were developed by a nine-member ad hoc Standards of Care Committee of the American Thyroid Association (the authors of this article). The participants were selected by the committee chair and the president of the American Thyroid Association on the basis of their clinical experience. The committee members represented different geographic areas within the United States, in order to take into account different practice styles. EVIDENCE: Guidelines were developed on the basis of expert opinion of the participants, as well as on available published information. CONSENSUS PROCESS: Input was obtained from all of the participants, each of whom wrote an initial section of the document. A complete draft document was then written by three participants (P.A.S., D.S.C., and E.G.L.) and resubmitted to the entire committee for revision. The revised document was then submitted to the entire membership of the American Thyroid Association for written comments, which were then reviewed (mainly by P.A.S., D.S.C., and E.G.L.). Many of the suggestions of the American Thyroid Association members were incorporated into the final draft, which was then approved by the Executive Council of the American Thyroid Association. The entire process, from initial drafts to final approval, took approximately 18 months. CONCLUSIONS: A set of minimum clinical guidelines for the diagnosis and treatment of hyperthyroidism and hypothyroidism were developed by consensus of a group of experienced thyroidologists. The guidelines are intended to be used by physicians in their care of patients with thyroid disorders, with the expectation that more effective care can be provided, and at a cost savings. PMID- 7532245 TI - Electron microscopic studies on the inhibition of degranulation of rat mast cells by a novel anti-allergic agent, PTPC. AB - When rat mast cells sensitized by IgE antibody were exposed to antigen, transmission electron microscopy revealed alteration of the granules, cavity formation by fusion of the perigranular membrane and granule release by the fusion of the cavity membrane with the mast cell membrane. Scanning electron microscopy disclosed the extrusion of smooth and round bodies from pores formed on the cell surface. These changes were accompanied by the release of histamine. The inhibition of this degranulation by a novel anti-allergic agent, 6-(1 pyrrolidinyl)-N-(1H-tetrazol-5-yl)-2-pyrazinecarboxamide (PTPC), was evaluated quantitatively as an inhibition of the granule alteration and cavity formation. At a concentration of 100 nM, PTPC inhibited the granule alteration and cavity formation as well as histamine release. In the same concentration, PTPC significantly increased the cyclic AMP content in the mast cells. These results suggest that the inhibition of the morphological changes in mast cells by PTPC might be due to the increased cyclic AMP caused by the agent and plays an important role in the suppression of chemical mediators release. PMID- 7532246 TI - Antiallergic effects of ZCR-2060: effect on allergic cutaneous reactions and rhinitis models in mice and rats. AB - The antiallergic action of 2-[2-[4-(diphenylmethyl)-1-piperadinyl] ethoxy] benzoic acid maleate (ZCR-2060) was investigated on allergic cutaneous reactions and nasal vascular permeability in mice and rats. ZCR-2060 markedly inhibited immediate allergic cutaneous reactions, including passive cutaneous anaphylaxis (PCA) in rats and mice; histamine-, compound 48/80- and calcium ionophore A 23187 induced cutaneous reactions in rats; and biphasic skin reactions mediated by monoclonal IgE antibody and epicutaneous challenge with antigen in mice, but did not affect 5-hydroxytryptamine-induced cutaneous reaction in rats. The antigen induced nasal vascular permeability increase in actively and passively sensitized rats and histamine-induced nasal vascular permeability increase in rats (allergic rhinitis model) were clearly inhibited in a dose-dependent fashion by ZCR-2060. Moreover, ZCR-2060 significantly inhibited antigen-induced anaphylactic histamine release from rat peritoneal mast cells and carrageenin-induced paw edema in rats. These results suggest that ZCR-2060 has antiallergic effects on allergic cutaneous reactions and experimental rhinitis, probably due to histamine H1 receptor blockage and the inhibition of histamine release. PMID- 7532244 TI - Acteoside, a component of Stachys sieboldii MIQ, may be a promising antinephritic agent (2): Effect of acteoside on leukocyte accumulation in the glomeruli of nephritic rats. AB - We investigated the effect of acteoside in comparison with that of cyclosporin A on leukocyte accumulation in the glomeruli of rats with crescentic-type anti glomerular basement membrane (GBM) nephritis. Acteoside given p.o. at a dose of 30 mg/kg once a day for 15 consecutive days after treatment with anti-GBM serum markedly suppressed the urinary protein as well as glomerular histological changes. Acteoside given p.o. for 5 or 15 consecutive days markedly suppressed the accumulation of total leukocytes, ED-1-positive cells (monocytes/macrophages), CD4-positive cells, CD8-positive cells, interleukin-2 receptor-positive cells (activated T cells) and Ia-positive cells in the glomeruli. These effects of cyclosporin A (20 mg/kg/day, p.o.) were also as potent as those of acteoside (30 mg/kg/day, p.o.). Cyclosporin A also strongly suppressed the elevation of plasma antibody level against rabbit gamma-globulin. However, in this dose, acteoside did not significantly suppress the antibody formation. It can be concluded from these results that acetoside may exert its antinephritic action by suppressing the accumulation of leukocytes in the glomeruli. PMID- 7532247 TI - Expression of insulin-like growth factor and binding protein genes during nephrogenesis. AB - To study the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in human nephrogenesis, we examined the temporal and spatial pattern of expression of these genes using in situ hybridization. The uninduced metanephric blastema (MB) expressed abundant IGF-II mRNA. With induction by the ureteric duct (UD), the aggregated MB additionally expressed IGFBP-2 and IGFBP-4 mRNAs. The mature UD expressed IGFBP-3 mRNA while the ampulla in contact with the MB lacked IGFBP-3 mRNA and expressed IGFBP-2 exclusively. Upon formation of the S shape nephron, IGFBP-2 mRNA was expressed in the committed glomerular and epithelial cells which also expressed IGF-II and IGFBP-4, and the mesenchyme of the vascular cleft expressed IGFBP-5 mRNA. In the maturing glomerulus, the glomerular epithelial cells expressed IGF-II mRNA together with IGFBP-2 and IGFBP 4 mRNAs, while IGFBP-5 mRNA was localized to the mesangium and supporting mesenchyme. As the proximal tubule was formed the epithelium expressed less of IGFBP-2 mRNA and more of IGFBP-4 mRNA. The renal mesenchyme in the cortex and medulla expressed abundant IGF-II mRNA, and lower levels of IGFBP-4 and -5 mRNAs. The epithelium of the collecting ducts and pelvicalyceal system expressed abundant IGFBP-3. In contrast, IGF-I, IGFBP-1, and IGFBP-6 mRNAs were expressed at low levels. The specific temporal and spatial pattern of expression of IGFBP genes on the background of abundant IGF-II gene expression suggests that the IGFBP peptides, as modulators of IGF action, are expressed locally at specific points of nephrogenesis to interact with IGF-II to regulate mesenchymal induction, renal epithelial cell commitment, differentiation and growth. PMID- 7532248 TI - Nitric oxide production by human proximal tubular cells: a novel immunomodulatory mechanism? AB - It is believed that human proximal tubular cells may possess immunological function and play an important role in a variety of renal disease states such as interstitial nephritis, allograft rejection and drug induced nephrotoxicity. The role of cytokines and nitric oxide in the human forms of these disease states is not clear. In this study we examined the effect of stimulation with the cytokines IL-1 beta. TNF-alpha and IFN-gamma, individually and in combination, upon primary cultures of human proximal tubular cells. Nitric oxide production increased significantly within 24 hours following cytokine stimulation. This response was inhibited, in a dose dependent manner, by L-NMMA. PCR amplification of mRNA extracted from control and cytokine stimulated human proximal tubular cells revealed a NOS product with a > 97% homology with human hepatocyte inducible nitric oxide synthase. The results of this study clearly show that human proximal tubular cells, in primary culture, are capable of producing nitric oxide in response to an immune challenge secondary to the induction of nitric oxide synthase. PMID- 7532250 TI - Hepatitis C: advances in diagnosis. PMID- 7532249 TI - Effects of complement activation products on the synthesis of decay accelerating factor and membrane cofactor protein by human mesangial cells. AB - We previously demonstrated that activation of terminal complement components (C8 and/or C9) increases the synthesis and expression of decay accelerating factor (DAF) on human glomerular cells. DAF is a cell membrane-associated complement regulatory protein that inhibits complement activation on cell surfaces. In the present studies we evaluated, first, the mechanisms by which complement activation stimulates DAF synthesis, and second the effect of complement activation on the synthesis. and expression of membrane cofactor protein (MCP), another complement regulatory protein, by human mesangial cells (HMC) in culture. Complement activation by immune complexes resulted in increased DAF mRNA levels by at least two mechanisms: deposition of activated C3 on HMC and generation of soluble complement activation products, specifically C5a. The increase in DAF mRNA levels induced by activated C3 or C5a was short lived (less than 4 hr). In contrast, the up-regulation of DAF mRNA levels induced by activation of the complete complement cascade persisted for at least eight hours. The effect of complement activation on DAF mRNA levels was not affected by cycloheximide, a protein synthesis inhibitor. However, cycloheximide alone resulted in a significant up-regulation of DAF mRNA levels on HMC. In contrast to those findings complement activation did not cause an up-regulation of MCP mRNA, nor an increase in the synthesis of this protein. However, by FACS, complement produced a small but significant increase of MCP protein levels on HMC. In conclusion, both MCP and DAF are present on HMC. Several activated complement components are capable of increasing DAF mRNA levels, but DAF protein levels increase only after activation of the whole complement cascade.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532251 TI - [Current status of radiotherapy treatment in esophageal cancer]. PMID- 7532253 TI - Dilemmas of organ transplantation from anti-HCV-positive donors. PMID- 7532252 TI - [Use of colony stimulating factors in the treatment of aplastic anemia]. PMID- 7532255 TI - Intraluminal treatment of inoperable oesophageal tumours by intralesional photodynamic therapy with methylene blue. PMID- 7532254 TI - A controlled study of hepatitis C transmission by organ transplantation. The New England Organ Bank Hepatitis C Study Group. AB - Hepatitis C virus (HCV) can be transmitted by transplantation of cadaver organs from donors with antibody to HCV (anti-HCV); therefore, transplantation of organs from anti-HCV positive donors to anti-HCV-negative recipients has been discouraged. We have looked at outcomes in recipients of organs from anti-HCV positive and negative donors to determine whether this advice is well-founded. Stored sera from 716 consecutive cadaver organ donors procured by the New England Organ Bank between 1986 and 1990 were tested for anti-HCV by a first-generation ELISA (ELISA1); 13 (1.8%) were positive. 29 recipients who received organs from these donors were the study group. 37 donors were randomly selected from 703 ELISA1-negative cadaver organ donors. 74 recipients of organs from these 37 donors were the control group. Clinical records were reviewed and recipient sera were tested for anti-HCV with a second-generation ELISA (ELISA2), and HCV RNA was tested for by polymerase chain reaction. Median post-transplant follow-up was 42 and 49 months for study and control groups. Post-transplantation prevalence of anti-HCV and HCV RNA was 67% and 96% among recipients from anti-HCV-positive donors, and 20% and 18% among recipients from anti-HCV-negative donors (p < 0.001). Post-transplantation non-A, non-B hepatitis, graft loss, and death were observed in 55%, 52%, and 31% among recipients of organs from anti-HCV-positive donors, and 16%, 53%, and 33% among recipients from anti-HCV-negative donors. In a proportional hazards model, the relative risks for non-A, non-B hepatitis, graft loss, and death among recipients from anti-HCV-positive donors were 4.37 (95% CI 1.97-9.70), 0.93 (0.51-1.70), and 0.89 (0.41-1.93). Transmission of HCV infection by organ transplantation increased the risk of liver disease among recipients. However, after 3.5 years, donor HCV infection did not adversely affect patient survival or graft survival. PMID- 7532256 TI - Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C. AB - The entry, and possibly the exit, of rickettsiae from eukaryotic cells, as well as erythrocyte lysis by some members of this group of organisms, is thought to be mediated by a phospholipase A activity even though the enzyme has not been isolated from these organisms. Evidence for phospholipase C, on the other hand, has not been reported for the genus Rickettsia. In this study, in a preliminary attempt to demonstrate the presence of phospholipase A2 and phospholipase C in the virulent Sheila Smith strain of Rickettsia rickettsii, we performed immunoblotting and immuno-gold electron microscopy using anti-phospholipase A2 and anti-phospholipase C IgG antibodies (raised against mammalian enzymes). We provide evidence for cross-reactivity of the antibodies with proteins present in R. rickettsii. Western blots showed a higher staining intensity with anti phospholipase C antibody than with anti-phospholipase A2. According to the results obtained with the immuno-gold labeling of phospholipase A2 and phospholipase C reactive epitopes, most of the phospholipase A2 cross-reactive material appears to be associated with the membrane of the organism while the phospholipase C cross-reactive material appears to be randomly distributed throughout the cell. PMID- 7532258 TI - Cell membrane potential analysis. PMID- 7532257 TI - Flow cytometry crossmatching for solid organ transplantation. PMID- 7532259 TI - Measurement of intracellular pH. PMID- 7532260 TI - Cellular protein content measurements. PMID- 7532262 TI - Staining of DNA in live and fixed cells. PMID- 7532261 TI - Lysosomal proton pump activity: supravital cell staining with acridine orange differentiates leukocyte subpopulations. PMID- 7532263 TI - Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis. PMID- 7532265 TI - Analysis of intracellular proteins. PMID- 7532264 TI - Cell-cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33358 ethidium bromide bivariate flow cytometry. PMID- 7532266 TI - "Washless" procedures for nuclear antigen detection. PMID- 7532267 TI - Simultaneous analysis of cellular RNA and DNA content. PMID- 7532269 TI - Immunophenotyping using fixed cells. PMID- 7532268 TI - Acid-induced denaturation of DNA in situ as a probe of chromatin structure. PMID- 7532271 TI - Communication skills in palliative care: development and modification of a rating scale. AB - A complex rating scale has been used to evaluate the verbal communication skills of health professionals before and after training. The scale has been modified in order to decrease the useage time, to improve its accuracy and to develop a rating tool available to teachers. This paper describes the process of the modification and discusses the consequent validation of the scale. PMID- 7532270 TI - Characterization of the anti-HIV-1 activity of 3,4-dihydro-2-alkoxy-6-benzyl-4 oxopyrimidines (DABOs), new non-nucleoside reverse transcriptase inhibitors. AB - Novel 3,4-dihydro-6-benzyl-4-oxopyrimidines (DABOs), variously substituted at both the C-2 and C-5 positions of the pyrimidine ring, proved to be specific inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro. Some compounds showed potency at micromolar doses, no cytotoxicity at the maximum testable doses and selectivity indexes comparable to that of 2'-3'-dideoxyinosine (ddI). Mode of action studies suggested that DABOs interfered with a step of the virus multiplication cycle following adsorption and preceding integration. Enzyme assays indicated that DABOs targeted HIV-1 reverse transcriptase: they inhibited the RNA-dependent DNA polymerase activity in a template-dependent manner and, to a lesser extent, the DNA-dependent DNA polymerase activity. No inhibition of the RNase-H associated activity was observed. When DABOs were assayed in combination with 3'-azido-3'-dideoxythymidine (AZT) or ddI against HIV-1 in cell cultures, a slightly synergistic inhibitory effect was observed. The combination of DABO 546 and AZTTP in enzyme assays showed that the two compounds were kinetically mutually exclusive. PMID- 7532272 TI - Use of an amino-cupric-silver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma. AB - A new amino-cupric silver protocol is described for detection of neuronal degeneration. We describe its selectivity in visualizing both early and semiacute degeneration after intracerebral or systemic administration of a variety of neurotoxicants in rats, and after transient ischemic episodes in gerbils. As early as 5 min after physical trauma, or 15 min following either intrastriatal injections of glutamate analogs or exposure to ischemic episodes, neuronal silver staining was evident at primary sites of trauma (i.g. injection sites) and at hodologically related secondary sites. With intoxication by peripheral injections of trimethyltin (IP) or intracerebral injections of Doxorubicin, reproducible patterns of degeneration are demonstrable after 24 h or after 9-13 days, respectively. The amino-cupric silver method permits simultaneous detection of all neuronal compartments against a clear background. Degeneration in the neuronal cell bodies, dendrites, axons and terminals, as well as the recruitment of new structures in a progressive pathologic process, could be accurately followed. The inclusion of new reagents increased the sensitivity vis-a-vis previous versions of the cupric-silver method. The advantages and disadvantages of the current method in comparison with other means of neurotoxic assessment are discussed in detail, with special emphasis on its unique ability to discriminate irreversible degenerative phenomena and degeneration of axonal components in cases where the cell body remains apparently intact. The amino-cupric silver method is an especially useful tool for surveying neuronal damage in basic neuroscience investigations and in neuropathologic and neurotoxic assessment. PMID- 7532273 TI - Responses of the fetal cerebral vasculature to prenatal cytotoxic brain damage. AB - Prenatal exposure to the drug methylazoxy-methanol acetate (MAM Ac) has been commonly used as an experimental model of prenatal cytotoxic brain damage. We have examined the effects of this prenatal brain damage on the development of vasculature in the fetal neocortex, because vasculature is likely to play an important role in fetal brain repair. Pregnant Wistar strain rats received either MAM Ac (25 mg/kg) or saline on embryonic day (E) 13 by IP injection. At E15, E17, E19, and and E21, the rats were overdosed with pentobarbitone (100 mg/kg) and fetuses removed for paraffin embedding, lectin histochemistry, and electron microscopy. For lectin histochemistry, sections were exposed to a horseradish peroxidase-conjugated B4 isolectin from Griffonia simplicifolia. Fetuses exposed to MAM Ac showed significant deficits in cortical vessel branch point density at E15 and E17 (46% and 20%, respectively). There were also significant reductions in stem vessel density at these ages (34% and 26%, respectively) among MAM Ac exposed animals, but there were no differences in growing tip density, capillary internal diameter, or stem vessel internal diameter. The only consistent differences between the two groups at the electron microscope level were an increase in vacuolisation and an irregular luminal surface in MAM Ac animals at E15. Tight junction formation, basement membrane formation, and loss of fenestrations were not delayed in experimental animals. The results indicate that there is no increase in angiogenic activity coincident with the fetal neocortical repair that follows MAM Ac induced damage. PMID- 7532274 TI - Immunoenzymometric analysis for expression and shedding of intercellular adhesion molecule-1 on human endothelial cells stimulated with cytokines or lipopolysaccharide. AB - Unstimulated endothelial cell (EC) cultures express low levels of intercellular adhesion molecule-1 (ICAM-1) and their expression can be enhanced by inflammatory cytokines such as tumor necrosis factor alpha (TNF). Three monoclonal antibodies (MoAbs) highly reactive with TNF-stimulated human ECs were established and defined to recognize a 95 kDa cell surface protein specifically expressed on cytokine-activated ECs, which was immunochemically identified as ICAM-1. The quantitative immunoassay of soluble and insoluble ICAM-1 could be performed with two different MoAbs. Secretion of fibronectin or the von Willebrand factor, was not significantly enhanced with TNF stimulation. Cellular expression of ICAM-1 was drastically induced by TNF or interleukin-1 stimulation, and the moderate expression with delayed-action was observed only by lipopolysaccharide stimulation. A maximal amount of soluble ICAM-1 was released from ECs stimulated only by TNF, apparently in a dose dependent manner, but no significant release of ICAM-1 was induced by thrombin interleukin-2, or lipopolysaccharides. Released levels of soluble ICAM-1 from interleukin-1-stimulated ECs were apparently diminished as compared with those from TNF-stimulated cells. These results suggest that release of soluble ICAM-1 from EC surfaces can be most significantly enhanced by TNF-specific signaling, and prospectively, should be a sensitive indicator of intravascular inflammation in acute endothelium injury. PMID- 7532275 TI - Differential effects of phosphotyrosine phosphatase expression on hormone dependent and independent pp60c-src activity. AB - pp60c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443-23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependent activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+ cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+ cells, but the effect was smaller in PTPase+ cells. In an in vitro assay, hormone-stimulated pp60c-src autophosphorylation from PTPase+ cells was decreased 64 +/- 22%, and substrate phosphorylation by pp60c src was reduced 54 +/- 16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, an in vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition, in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was an in vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions. PMID- 7532276 TI - Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A. AB - Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes. PMID- 7532278 TI - Role of STAT2 in the alpha interferon signaling pathway. AB - We have isolated U6A, a mutant cell line which lacks the STAT2 subunit of the transcription factor interferon (IFN)-stimulated gene factor 3 (ISGF3). The response of U6A cells to IFN-alpha is almost completely defective, but the response to IFN-gamma is normal. Complementation of U6A cells with a cDNA encoding STAT2 restores the IFN-alpha response, proving that STAT2 is required in this pathway. Binding of IFNs to their receptors triggers tyrosine phosphorylation and activation of the receptors, JAK family kinases, STAT1, and STAT2. In IFN-alpha-treated U6A cells, phosphorylation of the essential tyrosine kinases TYK2 and JAK1 is normal, but the phosphorylation of STAT1 is weak. A mutant STAT2 protein in which the phosphorylated tyrosine at position 690 is changed to phenylalanine does not restore normal phosphorylation of STAT1 in response to IFN-alpha. The dependence of STAT1 phosphorylation on the presence of STAT2 but not vice versa (T. Improta, C. Schindler, C. M. Horvath, I. M. Kerr, G. R. Stark, and J. E. Darnell, Jr., Proc. Natl. Acad. Sci. USA 91:4776-4780, 1994) indicates that in the formation of ISGF3, these two proteins may be phosphorylated sequentially in response to IFN-alpha and that phosphorylated STAT2 may be required to allow unphosphorylated STAT1 to bind to the activated IFN-alpha receptor. PMID- 7532277 TI - Single-chain antibody-mediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling. AB - ErbB-2 becomes rapidly phosphorylated and activated following treatment of many cell lines with epidermal growth factor (EGF) or Neu differentiation factor (NDF). However, these factors do not directly bind ErbB-2, and its activation is likely to be mediated via transmodulation by other members of the type I/EGF receptor (EGFR)-related family of receptor tyrosine kinases. The precise role of ErbB-2 in the transduction of the signals elicited by EGF and NDF is unclear. We have used a novel approach to study the role of ErbB-2 in signaling through this family of receptors. An ErbB-2-specific single-chain antibody, designed to prevent transit through the endoplasmic reticulum and cell surface localization of ErbB-2, has been expressed in T47D mammary carcinoma cells, which express all four known members of the EGFR family. We show that cell surface expression of ErbB-2 was selectively suppressed in these cells and that the activation of the mitogen-activated protein kinase pathway and p70/p85S6K, induction of c-fos expression, and stimulation of growth by NDF were dramatically impaired. Activation of mitogen-activated protein kinase and p70/p85S6K and induction of c fos expression by EGF were also significantly reduced. We conclude that in T47D cells, ErbB-2 is a major NDF signal transducer and a potentiator of the EGF signal. Thus, our observations demonstrate that ErbB-2 plays a central role in the type I/EGFR-related family of receptors and that receptor transmodulation represents a crucial step in growth factor signaling. PMID- 7532279 TI - Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins. AB - We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins. PMID- 7532280 TI - Clustering of Syk is sufficient to induce tyrosine phosphorylation and release of allergic mediators from rat basophilic leukemia cells. AB - In mast cells, antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, stimulates tyrosine phosphorylation and activation of multiple signaling pathways leading to the release of several classes of mediators of the allergic response. Early events induced upon cross linking of Fc epsilon RI include tyrosine phosphorylation of Fc epsilon RI subunits and activation of the tyrosine kinase p72syk (Syk), which binds to tyrosine-phosphorylated Fc epsilon RI. Clustering of Syk, as a result of its interaction with aggregated Fc epsilon RI, may play a role in activating one or more of the signaling pathways leading to mediator release. To test this possibility, Syk was introduced into a model mast cell line (rat basophilic leukemia cells) as part of a chimeric transmembrane protein containing the extracellular and transmembrane domains of CD16 and CD7, respectively. Clustering of the Syk chimera, using antibodies against CD16, was found to be sufficient to stimulate early and late events normally induced by clustering of Fc epsilon RI. Specifically, aggregation of Syk induced degranulation, leukotriene synthesis, and expression of cytokine genes. Induction of mediator release was dependent on the kinase activity of Syk. Consistent with this finding, clustering of Syk also induced the tyrosine phosphorylation of a profile of proteins, including phospholipase C-gamma 1 and mitogen-activated protein kinase, similar to that induced upon clustering of Fc epsilon RI. These results strongly suggest that Syk is an early and critical mediator of multiple signaling pathways that emanate from the Fc epsilon RI receptor and give rise to the allergic response. PMID- 7532281 TI - Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. AB - Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity. PMID- 7532284 TI - Burkholderia cepacia in cystic fibrosis. PMID- 7532285 TI - Burkholderia cepacia in cystic fibrosis. PMID- 7532282 TI - Transcription-independent turnover of I kappa B alpha during monocyte adherence: implications for a translational component regulating I kappa B alpha/MAD-3 mRNA levels. AB - We identified I kappa B alpha/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the I kappa B alpha protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the I kappa B alpha protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-kappa B can regulate I kappa B alpha/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady state I kappa B alpha/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-kappa B-dependent transcriptional stimulation of the I kappa B alpha/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1 beta (IL 1 beta) gene, were transcriptionally activated during monocyte adhesion, the rate of I kappa B alpha/MAD-3 gene transcription remained constant. The adherence dependent increase in I kappa B alpha/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1 beta mRNA increased during adherence whereas those of I kappa B alpha/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic I kappa B alpha/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of I kappa B alpha/MAD-3 mRNA, thereby increasing mRNA levels without stimulating I kappa B alpha/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of I kappa B alpha/MAD-3 mRNA without stimulating I kappa B alpha/MAD-3 gene transcription, we suggest that low cytoplasmic levels of I kappa B alpha/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism. PMID- 7532286 TI - Burkholderia cepacia in cystic fibrosis. PMID- 7532287 TI - Dynorphin A-(1-13) potently improves galanin-induced impairment of memory processes in mice. AB - The present study examined the effects of intracerebroventricular injection of dynorphin A-(1-13) on memory processes by using the passive avoidance task in mice. Galanin (0.3 microgram) significantly shortened the step-down latency when given 15 min before retention tests. Although dynorphin A-(1-13) (1 or 3 micrograms) did not prolong the step-down latency induced by weaker electroshocks, it inhibited the galanin (0.3 micrograms)-induced shortening of step-down latency. The effects of dynorphin A-(1-13) (3 micrograms) on the galanin-induced shortening of step-down latency were almost completely reversed by pretreatment with nor-binaltorphimine (4 micrograms), a kappa-selective opioid antagonist. These results strongly suggest that dynorphin A-(1-13) attenuates galanin-induced impairment of memory processes through the mediation of kappa opioid receptors. PMID- 7532283 TI - Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes. AB - The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP 1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed. PMID- 7532288 TI - Immunohistochemical identification of galanin and leucin-enkephalin in the porcine lacrimal gland. AB - The presence and distribution of galanin (GAL) and leucine-enkephalin (LEU-ENK) in the pig lacrimal gland was investigated using immunohistochemical techniques. Lacrimal gland segments obtained from the abattoir were fixed in paraformaldehyde picric acid solution, cut into 40-50 microns thick sections and processed for immunohistochemistry using polyclonal antisera against GAL and LEU-ENK. GAL- and LEU-ENK-immunoreactive intrinsic multipolar neurons were observed in the interacinar and interlobular areas of the pig lacrimal gland. From these regions these intrinsic neurons send branches to the acinar and other tissues of the gland. Immunoreactivity was confined solely to the neural elements. These two potent biologically active neuropeptides may play important roles in the regulation of lacrimal fluid secretion. PMID- 7532289 TI - Neurochemical effects of neuropeptide Y (NPY) and NPY2-36. AB - Our previous in vivo structure-activity studies suggested that the putative receptors mediating the effects of NPY and NPY2-36 on food intake and body temperature following ICV administration are pharmacologically different. In the present study, we examined and compared dose related effects of NPY and NPY2-36 on levels of norepinephrine (NE), dopamine (DA) and its main metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA), as well as serotonin (5-HT) and its metabolite, 5-hydroxyindolacetic acid (5-HIAA), in several brain regions of the rat, including: frontal cortex, hypothalamus, amygdala, septum, nucleus accumbens, corpus striatum, globus pallidus, substantia nigra and hippocampus. NPY and NPY2-36 (10 or 20 micrograms) were administered intraventricularly and the regional levels of the amines and metabolites were assessed 30 min following administration. Results indicate that both doses of NPY decreased NE levels within the hypothalamus. Furthermore, DOPAC concentrations were increased in this region while DA and HVA remained unchanged. The most pronounced neurochemical effects of NPY were found in the hippocampus, where the peptide produced dose related increases in DA, DOPAC and HVA. On the other hand, NPY2-36 significantly increased NE, DA and its metabolite DOPAC in both the amygdala and septum. The metabolism of DA was most obviously affected in the hippocampus and frontal cortex where levels of DA and DOPAC were significantly increased. 5-HT was affected in both the hypothalamus and globus pallidus where DA and its metabolite HVA were also increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532292 TI - Clinicopathologic correlation of spontaneous retinal pigment epithelial tears with choroidal neovascular membranes in age-related macular degeneration. AB - BACKGROUND: A correlation of the clinical and histopathologic features of age related macular degeneration (AMD), complicated by choroidal neovascular membrane (CNVM) with spontaneous tears of retinal pigment epithelial (RPE) detachment, has not been reported. METHODS: Two patients with vision loss from AMD and spontaneous pigment epithelial detachment tears, involving the fovea with serous or hemorrhagic neurosensory retinal detachments, were examined clinically. The surgically excised CNVM from each patient was examined with light microscopy. RESULTS: The authors present two patients with clinicopathologic correlation of the ophthalmoscopic, fluorescein angiographic, and pathologic findings of spontaneous pigment epithelial detachment tears. In the surgical specimens from these two eyes, the removed tissue, presumed to be the spontaneous RPE tear with CNVMs, demonstrated a fibrovascular membrane surrounding RPE with adjacent abnormally thickened basement membrane. The RPE-basement membrane complex was folded redundantly and scrolled beneath itself. CONCLUSION: The authors found RPE basement membrane abnormalities and CNVMs associated with clinically evident spontaneous pigment epithelial detachment tears. These findings support the current theories of their pathogenesis. PMID- 7532291 TI - Treatment of retinal breaks with autologous serum in an experimental model. AB - PURPOSE: The standard treatment for retinal breaks is thermal adhesion. Breaks in the posterior pole (i.e., macular holes) recently have been treated using vitrectomy and the recombinant cytokine transforming growth factor-beta. This has been shown to achieve closure of the retinal breaks by stimulating localized fibrocellular proliferation. Serum has been shown to contain chemoattractants and mitogens for many types of cells. The authors studied the clinical and histologic effect of autologous serum application to retinal breaks in an experimental model. METHOD: Twenty-four rabbits underwent pars plana lensectomy, vitrectomy, retinectomy, fluid-air exchange, application of test solution (12 with Hank's buffered salt solution and 12 with autologous serum), and air-gas exchange. Clinical examination with indirect ophthalmoscopy was performed, and animals were killed 5, 14, and 28 days after treatment. Tissue sections through the retinectomy were studied by light microscopy, electron microscopy, and immunocytochemistry. RESULTS: None of the serum-treated eyes showed retinal detachment at the site of the retinectomy by evaluation with indirect ophthalmoscopy at each of the time points. In contrast, in control eyes retinal detachment developed at the retinectomy site from 0% at day 5 to 50% at day 14 and 75% at day 28. By light microscopy, serum-treated eyes contained multilayers of fibroblast-like cells adhering the retinectomy edges to the underlying retinal pigment epithelium and choroid. The control eyes had nonadherent retinal edges at the retinectomy site with little sign of fibrocellular response. Results were confirmed by electron microscopy. The fibroblast-like cells by immunocytochemistry contained vimentin, cytokeratin 18, and/or glial fibrillary acidic protein. CONCLUSION: This study suggests that serum induces a localized fibrocellular response at the retinectomy edges compared with control eyes. This response, characterized by light microscopy, electron microscopy, and immunocytochemistry, appears to involve a mixed population of glial, retinal pigment epithelial, and/or fibroblastic cells. These cells seem to enhance adhesion and subsequent reattachment of the edges of the retinectomies at the time points studied when compared with controls. PMID- 7532295 TI - The inappropriate use of school "readiness" tests. American Academy of Pediatrics Committee on School Health and Committee on Early Childhood, Adoption and Dependent Care. PMID- 7532294 TI - Antibody-dependent neutrophil-mediated parasite killing in non-lethal rodent malaria. AB - The effects of administrating recombinant human granulocyte colony-stimulating factor (rhG-CSF) and passively transferring immune serum on infection with an attenuated variant of Plasmodium berghei XAT (Pb XAT), in severe combined immunodeficiency (SCID) mice were examined. In immune competent (C.B-17) mice, the attenuated parasite infection was inevitably self-resolving and degenerating forms inside erythrocytes appeared, coinciding with the drop in parasitaemia, whereas SCID mice were unable to control parasite growth and all the mice died. Continuous administration with rhG-CSF caused neutrophilic granulocytosis in both SCID and C.B-17 mice. The effect of rhG-CSF on the infection in C.B-17 mice was to suppress the course of the parasitaemia at an early phase whereas it had no effect in SCID mice. When immune serum was transferred on the day of infection, the prepatent period was prolonged two days in both SCID and C.B-17 mice. When administration with rhG-CSF was combined with transfer of immune serum, SCID mice showed four days delay in patency and degenerating parasites were seen during the course of parasitaemia, although the infection was ultimately fatal. C.B-17 mice similarly treated showed a seven day delay in the onset of the patent parasitaemia which was of a lesser magnitude and shorter in duration compared with control mice. On the other hand, when C.B-17 mice were splenectomized three weeks before infection and then treated with rhG-CSF and immune serum, no degenerating parasites were seen during the infection and all mice died with high parasitaemias.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532293 TI - Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent. AB - Several signal transducers bind through their SH2 domains to phosphotyrosine containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates. PMID- 7532290 TI - The diagnosis and management of sinusitis. AB - Sinusitis, a very common health care complaint, is one of the most frequently overlooked and misunderstood diseases in clinical practice. An understanding of the pathophysiology is essential for health care providers to treat sinusitis effectively and to teach clients and families regarding management and possible complications. This article addresses clinical signs and symptoms and the differences between adults and children, as well as the subtle distinctions between acute and chronic sinusitis, and appropriate diagnostic techniques. Emphasis is placed on the diagnostic profiles of acute sinusitis relative to the different sinuses with progression to chronic sinusitis. Bacterial etiology of sinusitis is reviewed as well as management goals of sinusitis. Traditional management of sinusitis focuses on appropriate use of antibiotics, decongestants, steroids, antihistamines, and saline washes. A section on implications for practice discusses the important role of the health care provider relevant to populations at risk and possible complications, with an emphasis on primary and secondary prevention. PMID- 7532296 TI - Alexithymia and overeating. AB - While obesity is a complex, multidetermined disorder, a significant subgroup of overeaters suffer from alexithymia, which contributes to weight gain and difficulties in losing weight. For these individuals, food is used to regulate tension and inner-feeling states. The authors advocate psychotherapy aimed toward helping clients differentiate feelings and develop the capacity for symbolization as the most effective treatment. PMID- 7532298 TI - A theory of transformed parenting: parenting a child with developmental delay/mental retardation. AB - The purpose of this grounded theory study was to examine the parenting process when a child has developmental delay including mental retardation (DD/MR). The sample consisted of parents (29 mothers, 13 fathers) of 31 children with DD/MR. A semistructured interview guide was used to collect data during 1- to 1.5-hour interviews. The basic social process of Transformed Parenting emerged from the data. Transformed Parenting consists of an initial entrance process and a subsequent ongoing performance process. The entrance process encompasses receiving the child's diagnosis and responding to the child's diagnosis. The performance process consists of three areas: reality construing processes, contextual processes, and operating processes. At the core of the reality construing process is construing reality loops, which describes the ongoing process of interpreting the child's problems. PMID- 7532297 TI - Purine-purine mismatches in RNA helices: evidence for protonated G.A pairs and next-nearest neighbor effects. AB - Thermodynamic parameters are presented for 12 different RNA duplexes containing A.A, A.G, G.A and G.G mismatches flanked by C-G base pairs. UV melting studies were conducted under three different buffer conditions in order to evaluate the effects of salt concentration and pH on the stability of each mismatch-containing duplex. The main findings are: (i) the mismatches have a wide range of effects on duplex stability, decreasing delta G degrees 37 of denaturation by approximately 0-7 kcal/mol; (ii) the nearest-neighbor assumption commonly used to calculate helix stability breaks down for G.A mismatches; and (iii) G.A mismatches separated by 2 bp form a protonated structure. PMID- 7532299 TI - Diagnosing and treating bone metastases. AB - There can be little that is more distressing for a nurse to witness than the anguish and despair of patients and their relatives caused by unrelieved pain. In spite of increasing doses of morphine, some patients remain in pain. Why is this and, as nurses, what can we do about it? It is the nurse who spends time with the patients and relatives, both at home and in hospital. We are, therefore, in the front line, supporting patients and assessing their needs. PMID- 7532300 TI - Challenging conventional roles in palliative care. AB - In October 1991, North Humberside was offered its own in-patient palliative care service. This was a natural development of the existing Macmillan Nurse Service and the hospice day centre. This paper discusses how the role of the nurse practitioner has developed. PMID- 7532301 TI - Dietary L-homoarginine has no lysine bioactivity in chicks. AB - A chick growth assay was conducted to investigate the effect of dietary L homoarginine supplementation on performance of chicks fed a Lys-deficient corn feather meal diet. Weight gain, feed intake, and feed efficiency increased linearly (P < .01) as Lys was added at .1 and .2% from feed-grade L-Lys.HCl. Adding homoarginine at .2% resulted in less (P < .05) weight gain and feed intake than observed in chicks fed the unsupplemented basal diet. These data suggest that dietary homoarginine had no Lys bioactivity, but may also have antagonized Lys utilization from the basal diet. PMID- 7532302 TI - Ionic regulation of human basophil releasability. III. Effects of Na+ and Ca2+ on histamine release induced by different stimuli. AB - The effects of Na+ and Ca2+ ions on histamine release from human basophils stimulated by anti-IgE, N-formyl-methionyl-leucyl-phenylalanine (FMLP), 4 beta phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophore A23187 were evaluated. Isosmotic replacement of Na+ in the extracellular medium with the nonpermeant Na+ analogue choline+ or with glucose led to a significant increase in anti-IgE- (1/5000: 43.7 +/- 7.3% in high Na+ vs 68.9 +/- 7.3% in low Na+, mean +/- SEM, n = 8, P < 0.001), FMLP- (1 microM: 37.9 +/- 2.3% vs 49.5 +/- 4.3%, n = 8, P < 0.01) and PMA-(160 nM: 12.7 +/- 0.9% vs 27.3 +/- 4.3%, n = 8, P < 0.05) induced histamine release, whereas A23187-induced histamine release was reduced (1 microM: 90.4 +/- 2.4% vs 45.4 +/- 3.4%, n = 8, P < 0.0001). The progressive increase in extracellular Na+ concentration was accompanied by a decrease of basophil response to anti-IgE, FMLP and PMA; in contrast, A23187-induced histamine release was up-regulated by Na+. The Na+/H+ exchanger monensin, in the concentration range of 10(-8)-10(-4) M, exerted a dose-dependent inhibitory effect on anti-IgE-, FMLP- and PMA-induced histamine release, but not on A23187 induced histamine release. Extracellular Ca2+ up-regulated the histamine release induced by all the above stimuli. Removal of extracellular Na+ lowered the requirement of extracellular Ca2+ for anti-IgE, FMLP- and PMA-induced histamine release. In contrast with previous observations showing that Na+ supports histamine release from rat peritoneal mast cells and rat basophilic leukaemia cells, these results indicate that Na+ strongly inhibits histamine release from human basophils stimulated by anti-IgE, FMLP and PMA, whereas it enhances Ca2+ ionophore A23187-induced histamine release. The effects of Na+, which are probably related to modulation of membrane potential and/or intracellular pH, vary depending on the cell type and the stimulus employed for cell activation. PMID- 7532303 TI - Vasodilatation induced by capsaicin in rat mesenteric vessels is probably independent of nitric oxide synthesis. AB - Vasal relaxation induced by capsaicin was investigated on perfused mesenteric vascular bed prepared from Wistar rats. Bolus infusion of capsaicin, from 3.5 to 16 nmol, elicited a dose-dependent vasal relaxation effect, which was antagonized by pretreatment with 3 x 10(-6) M calcitonin gene-related peptide (CGRP) (8-37), an antagonist of CGRP. In order to test whether NO-release is involved in vasorelaxant response to capsaicin, a preparation of mesenteric vascular bed was perfused and superfused for 1 h by N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-3) M), an NO-synthase inhibitor. Vasodilatation induced by capsaicin remained unchanged, while that induced by acetylcholine, used as control, was significantly reduced. The results indicate that in the mesenteric bed, capsaicin induced vasodilatation is probably independent of the NO-synthesis mechanism and possibly mediated by CGRP. PMID- 7532304 TI - Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. AB - Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned whose expression appears to be limited to the collecting duct. Previously, we immunolocalized this water channel to the apical plasma membrane (APM) and to intracellular vesicles (IVs) of collecting duct cells. Here, we test the hypothesis that vasopressin increases cellular water permeability by inducing exocytosis of AQP-CD-laden vesicles, transferring water channels from IVs to APM. Rat collecting ducts were perfused in vitro to determine water permeability and subcellular distribution of AQP-CD in the same tubules. The collecting ducts were fixed for immunoelectron microscopy before, during, and after exposure to vasopressin. Vasopressin exposure induced increases in water permeability and the absolute labeling density of AQP-CD in the APM. In parallel, the APM:IV labeling ratio increased. Furthermore, in response to vasopressin withdrawal, AQP-CD labeling density in the APM and the APM:IV labeling ratio decreased in parallel to a measured decrease in osmotic water permeability. We conclude that vasopressin increases the water permeability of collecting duct cells by inducing a reversible translocation of AQP-CD water channels from IVs to the APM. PMID- 7532306 TI - The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1. AB - The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. (1992) Science 256, 1783 1790] and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. & Arnold, E. (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324] reveals changes associated with ligand binding. The enzyme is a heterodimer (p66/p51), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (approximately 33 degrees rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small beta-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound. PMID- 7532305 TI - Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. AB - It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia. PMID- 7532307 TI - Two closely linked but separable promoters for human neuronal nitric oxide synthase gene transcription. AB - In this report we demonstrate that the human cerebellum contains neuronal nitric oxide synthase (nNOS) mRNAs with two distinct 5'-untranslated regions that are encoded through use of closely linked but separate promoters. nNOS cDNA clones were shown to contain different 5' terminal exons spliced to a common exon 2. Genomic cloning and sequence analysis demonstrate that the unique exons are positioned within 300 bp of each other but separated from exon 2 by an intron that is at least 20 kb in length. A CpG island engulfs the downstream 5'-terminal exon. In contrast, most of the upstream exon resides outside of this CpG island. Interestingly, the upstream exon includes a GT dinucleotide repeat. A fusion gene with a 414-bp nNOS genomic fragment that includes a portion of the upstream 5' terminal exon and its immediate 5'-flanking DNA is expressed in transfected HeLa cells. Also expressed is a fusion gene that contains the luciferase reporter under transcriptional control by a 308-bp genomic fragment that includes the region separating both 5'-terminal exons. These results indicate that expression of these exons is subject to transcriptional control by separate promoters. However, the proximity of these promoters raise the possibility that complex interactions may be involved in regulating nNOS gene expression at these sites. PMID- 7532308 TI - Thymidine phosphorylase is angiogenic and promotes tumor growth. AB - Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies. PMID- 7532310 TI - Functional and biochemical evidence for altered serotonergic function in the fawn hooded rat strain. AB - Administration of various doses of DOI (a 5-HT2A/5-HT2C agonist) produced hyperthermia that was significantly less in the FH rat strain relative to the Wistar rat strain. Similarly, administration of various doses of ipsapirone (a 5 HT1A agonist) produced hypothermia that was significantly less in the FH rat strain relative to the Wistar rat strain. Furthermore, m-CPP (a 5-HT agonist) induced increases in growth hormone levels were also significantly less in the FH rat strain relative to the Wistar rat strain. There was no significant difference in the levels of either 5-HT or 5-HIAA between the two rat strains in the frontal cortex, hippocampus, hypothalamus, and striatum. In the brain stem, however, both 5-HT and 5-HIAA levels were significantly lower in the FH rat strain relative to the Wistar rat strain. On the other hand, 5-HT turnover rate was significantly higher in the hypothalamus and striatum and significantly lower in the hippocampus in the FH rat strain relative to the Wistar rat strain. These findings provide further evidence for altered serotonergic function in the FH rat strain and, in addition, suggest that the FH rat strain may prove to be a useful genetic model for some neuropsychiatric disorders with possible abnormalities in serotonergic function such as depression, obsessive-compulsive disorder, and the eating disorders. PMID- 7532309 TI - Methylenedioxyamphetamine: a selective effect on cortical content and turnover of 5-HT. AB - This study examined the effects of the hallucinogen, MDA, on brain content of monoamines and their metabolites in the rabbit. A single 1.8 mg/kg dose of MDA produced 30 to 64% increases in the 5-HT content of frontal cortex from 30 to 120 min after injection and a decrease in 5-HT turnover from 30 min to 8 h, but had no effect in hippocampus, caudate nucleus, or hypothalamus. A single 3.6 mg/kg dose of MDA also reduced the turnover of 5-HT in frontal cortex, but this was accompanied by a decrease in 5-HIAA with no increase in 5-HT. The 1.8 and 3.6 mg/kg doses of MDA had no significant or consistent effects on the contents of DA, DOPAC, HVA, and NE in any brain area examined. Chronic administration of MDA (3.6 mg/kg/day for 4 days) failed to produce any evidence of a neurotoxic action on 5-HT neurons. Higher doses could not be employed because the LD50 of MDA was approximately 5 mg/kg. This study has demonstrated that behaviorally effective and nonneurotoxic doses of MDA produce increases in the content and decreases in turnover of 5-HT in frontal cortex that resemble those of other hallucinogens such as LSD and DOM. PMID- 7532311 TI - Contractile hyperreactivity and alteration of PKC activity in gastric fundus smooth muscle of diabetic rats. AB - Contraction dose dependently induced in gastric smooth muscle of diabetic rats by Bay K 8644 in the presence of 20 mM KCl was about two times that induced in controls, and was inhibited more than 50% by 1-(5-isoquinolinesulfonyl)-2 methylpiperazine (H-7). Contraction was caused in diabetics but usually not in controls by 10(-5) M phorbol 12-myristate 13-acetate (PMA). In diabetics, this contraction was about 2.5 times that in controls. Protein kinase C (PKC) activity in the soluble fraction was depressed by H-7 or staurosporine, and depended on PMA concentration, but was greater in diabetics than in controls at any PMA concentration. PKC activity in the soluble fraction was inhibited by lower Ca2+ concentration, and was greater in diabetics than in controls. Affinity and density of binding sites of a Ca2+ channel antagonist ligand, [3H]PN200-110, were the same in plasma membrane-enriched fractions isolated from either controls or diabetic preparations. Thus, hyperreactivity in diabetic fundus may depend, in part, on alteration of PKC properties, but not on the density of Ca2+ channels. PMID- 7532312 TI - Depletion of brain serotonin by 5,7-DHT: effects on the 8-OH-DPAT-induced changes of sleep and waking in the rat. AB - The effect of 5-HT1A receptor agonist 8-OH-DPAT on sleep and wakefulness was studied in rats with selective serotonin depletion after ICV administration of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Injection of 8-OH-DPAT to vehicle-treated animals induced biphasic effects, such that low doses (0.010 mg/kg) which act perferentially on the somatodendritic autoreceptor decreased wakefulness (W) and increased slow wave sleep (SWS), while higher doses (0.375 mg/kg) which stimulate postsynaptic receptors caused opposite effects. REM sleep was suppressed irrespective of the dosage given. Injection of the 0.010 mg/kg dose in the 5,7-DHT-treated rats did not result in significant changes in sleep or W. On the other hand, the 0.375 mg/kg dose produced changes in sleep variables which were similar to those described in the vehicle-treated animals. Our findings tend to indicate that increased SWS after low doses of 8-OH-DPAT depends upon the activation of inhibitory somatodendritic 5-HT1A receptors, while increased W after higher doses of the compound is related to stimulation of postsynaptic receptors. PMID- 7532314 TI - Inflammatory arthritis: imaging of endothelial cell activation with an indium-111 labeled F(ab')2 fragment of anti-E-selectin monoclonal antibody. AB - PURPOSE: To use imaging performed with an indium-111-labeled F(ab')2 fragment of monoclonal antibody that recognizes E-selectin (1.2B6) to evaluate inflammation. MATERIALS AND METHODS: After the in vivo immunoreactivity of In-111-F(ab')2 was verified, imaging experiments were conducted on pigs with induced arthritis of the right knee. In-111-labeled 1.2B6-F(ab')2, technetium-99m hexamethylpropyleneamine oxime-labeled polymorphonuclear leukocytes (PMNs), and iodine-125-MOPC 21-labeled F(ab')2 (the control) were administered by means of intravenous injection. Scintigrams were obtained until 20 hours after injection, and tissue sampling was performed. RESULTS: Scintigrams showed uptake of 1.2B6 in the right knee but only faint filling of the joint space with PMNs. The right knee-to-left knee ratios on images obtained 20 hours after injection of the tracers were 10.8 +/- 3.6 for 1.2B6 and 1.1 +/- 0.4 for PMNs (P < .05). CONCLUSION: Endothelial cell immunotargeting with a radiolabeled anti-E-selectin F(ab')2 monoclonal antibody is feasible for use in imaging of inflammation. PMID- 7532313 TI - Effects of two stressors on behaviour in the elevated X-maze: preliminary investigation of their interaction with 8-OH-DPAT. AB - Effects of water deprivation and restraint were compared in the rat elevated X maze. Water deprivation for 12-48 h increased corticosterone and had a duration dependent "anxiolytic" effect in the elevated X-maze, increasing the ratio of open/total arm entries (OTR) and the proportion of time spent on the open arms (% time) without affecting total entries. Brain 5HIAA/5HT was increased only after 24 or 48 h deprivation. Restraint for 15 min also increased plasma corticosterone and brain 5HIAA/5HT but had no effect on behaviour in the elevated X-maze when rats were tested immediately afterwards. However, 1 h restraint was "anxiogenic" in the elevated X-maze immediately after release, reducing OTR and % time, but with a less consistent reduction in total entries; reductions in OTR and % time were still present 24 h later. The 5HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.1-0.2 mg/kg), administered 10 min before testing in the elevated X-maze, had "anxiogenic" actions in non-stressed rats. The effect of 0.1 mg/kg 8-OH-DPAT was not significantly altered by 24 or 48 h water deprivation but was abolished by restraint for 1 h immediately beforehand, despite the "anxiogenic" effect of restraint alone. Similar mutual antagonism of 8-OH-DPAT and restraint occurred when the dose of 8-OH-DPAT was increased to 0.2 mg/kg. Twenty-four hours after restraint, restrained rats which had received 8-OH-DPAT (0.1-0.2 mg/kg) still did not show any significant "anxiogenic effect" compared with non-restrained vehicle treated controls. Restraint-induced deficits in elevated X-maze exploration may prove a useful model with which to study the pharmacology of depression-related anxiety. However, the effects of the stressors examined, and their interaction with 8-OH-DPAT in the elevated X-maze, appear to depend on the nature of the stressor. PMID- 7532316 TI - Biogenic amine metabolites in delusional (psychotic) depression and melancholia subtypes of major depression. AB - 1. The levels of the urinary main metabolites of norepinephrine 3-methoxy-4 hydroxyphenylglycol (MHPG), of dopamine homovanillic acid (HVA) and of serotonin 5-hydroxyindoleacetic acid (5-HIAA) were measured in 84 patients with major depressive disorder, 34 delusional and 50 nondelusional. Melancholia subtype was also defined (N = 62). 2. MHPG was significantly higher in the delusional depressed group (p = 0.023). Female patients with delusional major depression also had significantly higher HVA excretion than female patients with non delusional major depression (p = 0.036). 5-HIAA excretion was similar in the two patient subgroups. 3. No significant differences in the three monoamine metabolites were found between the melancholic and nonmelancholic depressed patients. PMID- 7532317 TI - Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor. AB - The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment. PMID- 7532315 TI - Polynucleotide recognition and degradation by bleomycin. PMID- 7532318 TI - Increased mRNA expression of IL-10 in mononuclear cells in multiple sclerosis and optic neuritis. AB - The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the Th2 related IL-10. We describe the use of in situ hybridization with cDNA oligonucleotide probes to detect and enumerate mononuclear cells (MNC) expressing mRNA for IL-10, which is known to down-regulate Th1 cell related cytokines such as interferon-gamma. Expression of IL-10 was studied in blood MNC of MS and blood and cerebrospinal fluid (CSF) MNC of optic neuritis (ON) patients without culture and after culture in the presence of myelin basic protein (MBP), the control antigen acetylcholine receptor (AChR), and without antigen. Numbers of IL-10 mRNA expressing MNC were elevated in the MS patients' blood both when enumerated without culture and after culture with MBP. Control patients with myasthenia gravis had elevated numbers of AChR-reactive IL 10 mRNA expressing cells, while numbers of MBP-reactive IL-10 positive cells did not differ from numbers registered in cells without antigen. Patients with ON, in many instances representing early MS, had IL-10 positive blood MNC that were elevated to the same extent as in clinically definite MS, and further increased in the CSF. ON patients examined within 1 month after onset had lower numbers of MBP induced IL-10 mRNA expressing blood MNC compared with patients examined later suggesting that IL-10 is related to the degree of inflammation and outcome in ON. PMID- 7532319 TI - Tenidap: a novel cytokine-modulating antirheumatic drug for the treatment of rheumatoid arthritis. AB - Tenidap is a novel, once-daily, cytokine modulating antirheumatic drug indicated for the treatment of rheumatoid arthritis (RA). In vitro, tenidap significantly inhibits the production of the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tumour necrosis factor in human cell lines, and inhibits cytokine-mediated processes such as cartilage degradation, bone resorption, metalloprotease synthesis, endothelial cell adhesion and monocyte differentiation. Tenidap also inhibits cyclo-oxygenase. In RA patients, tenidap 120 mg/day is clinically equivalent to the combination of disease-modifying antirheumatic agents plus non-steroidal anti-inflammatory drugs (NSAIDs) and significantly more effective than NSAIDs. Tenidap also produces rapid, profound and sustained reductions in the serum levels of the acute phase proteins, C reactive protein and serum amyloid A, an effect suggestive of disease modifying properties. In addition, tenidap reduces circulating levels of IL-6 in RA patients. Tenidap is well tolerated. PMID- 7532320 TI - Cytokines and acute phase proteins in rheumatoid arthritis. AB - Cytokines are extracellular signalling glycoproteins that play an important pathological role in rheumatoid arthritis (RA) where they mediate acute inflammation, chronic inflammation and connective tissue destruction. In RA the macrophage-derived cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor (TNF), colony stimulating factors (CSFs) and growth factors play a key role in amplifying and perpetuating inflammation. IL-1 and TNF activate cartilage and bone degrading enzymes, while IL-8 recruits inflammatory cells into the joint. IL-1 and TNF play an important role in the acute phase response in that they potently induce IL-6, itself the major mediator and regulator of hepatic synthesis of acute phase proteins (APPs). The acute phase response is signalled by the rapid elevation of APPs such as C reactive protein (CRP) and serum amyloid A (SAA) in the blood, and these can be used as good surrogate markers of disease activity. In health, the activity of cytokines such as IL-1 or TNF is checked by inhibitory molecules such as receptor antagonist molecules or soluble receptor molecules. In disease, cytokine activity appears to be relatively unopposed, leading to the recent development of cytokine inhibitory molecules as potential anti-RA therapies. However, while cytokines are mediators of disease, they probably do not provide the initial stimulus for RA to develop, although polymorphisms in TNF, IL-1 and IL-1 receptor antagonist genes which have been recently found may represent important genetic modifying factors of disease severity in RA. PMID- 7532322 TI - Acute myelogenous leukemia in the older patient. PMID- 7532323 TI - New agents in the therapy of hormone-refractory patients with prostate cancer. PMID- 7532321 TI - Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors. AB - The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS. PMID- 7532324 TI - Topography and subdivision of the pelvic connective tissue in human fetuses and in the adult. AB - We investigated epoxy resin impregnated sections through the pelves of 9 to 37 week-old fetuses, of newborn infants, and of adults to study the topography and subdivision of the pelvic connective tissue. Fetal and adult preparations show that the pelvic connective tissue can be subdivided into a presacral, a perirectal and a paravisceral compartment. Whereas the presacral and the perirectal compartment contain connective tissue, adipose tissue and supplying structures, the paravisceral compartment is mainly composed of adipose tissue. While only a very thin rectal fascia was found at the border of the perirectal compartment, no further visceral pelvic fascia can be seen in the impregnated sections. Moreover it is shown that the ligaments of the pelvic cavity are only composed of the sacrouterine ligaments and the pubovesical ligaments in the female and the puboprostatic ligaments in the male. Our data show that sectional anatomy provides new insights into the organization of the pelvic connective tissue, that may be of clinical importance. PMID- 7532325 TI - Flow and antibody binding properties of hydrated fibrins prepared from plasma, platelet rich plasma and whole blood. AB - Previous studies, using cross-linked fibrin prepared from purified fibrinogen, showed low binding of a fibrin-specific monoclonal antibody designated T2G1 (Procyk et al., Blood 77:1469-75, 1991). In this study we investigated the binding of T2G1 and one other antibody to clots prepared from platelet poor plasma (PPP), platelet rich plasma (PRP) and whole blood. In contrast to our previous study, we used unlabelled antibodies and quantitated the level bound by ELISA, measuring antibody concentration in the non-adsorbed fraction. Antibody T2G1 bound 1.35 +/- 0.10 pmol/pmol fibrin (n = 11) to whole blood columns, 1.64 +/- 0.18 (n = 10) to PRP columns and 1.58 +/- 0.13 (n = 8) to PPP columns. The binding of T2G1 to columns made from purified fibrinogen was 0.78 +/- 0.05 pmol/pmol fibrin (n = 15). An antibody to a conformation-dependent epitope on Fragment D (Fd4-7B3) bound in comparable amounts to the different fibrins. Flow data show that whole blood columns, and also, but to a lesser extent those made with plasma, had a higher flow rate, permeability and fiber mass-length ratio than columns prepared from fibrinogen indicating a more coarse fibrin network. These data show that the presence of other proteins and blood cells, similar to what might occur in vivo, not only lead to an increase in the permeability of gels but also allow for better exposure of some epitopes. PMID- 7532327 TI - Effects of a nitric oxide synthase inhibitor on vasopressin and atrial natriuretic hormone release, thermogenesis and cardiovascular functions in response to interleukin-1 beta in rats. AB - To assess whether nitric oxide (NO) formed by IL-1 beta affects vasopressin (AVP) and atrial natriuretic hormone (ANH) release and the regulation of blood pressure and body temperature, intravenous infusion of either N omega-nitro-L-arginine methyl ester (L-NAME) alone (50 micrograms/kg.body weight.min for 135 min), human recombinant interleukin 1 beta (IL-1 beta) alone (750 ng/kg.body weight.min for 120 min), or L-NAME (50 micrograms/kg.body weight.min for 135 min) with IL-1 beta (750 ng/kg.body weight.min for 120 min), was performed following priming doses of L-NAME (2 mg/kg.body weight) and IL-1 beta (7.5 micrograms/kg.body weight) into conscious rats (n = 6 each). In the control group, saline alone was administered. Plasma AVP and ANH, mean arterial blood pressure (MABP), heart rate (HR) and rectal temperature (RT) were determined. In response to L-NAME, plasma AVP significantly increased, but plasma ANH did not change, despite increases in MABP and decreases in HR. In response to IL-1 beta, both plasma AVP and ANH increased with decreases in MABP and RT without any changes in HR. With L-NAME and IL-1 beta, both plasma AVP and ANH increased, and depressor response to IL-1 beta was partly attenuated by L-NAME, without any changes in RT. With saline alone, none of these parameters changed during the study. These results suggest that NO may directly affect the release of AVP and ANH and the regulation of body temperature and blood pressure, but NO formed by IL-1 beta may not have direct effects on the release of these hormones, and the regulation of blood pressure and temperature. PMID- 7532328 TI - Effect of hexachlorocyclohexane (HCH) on testicular plasma membrane of rat. AB - The study deals with the analysis of residue of hexachlorocyclohexane (HCH) and its possible damaging potential on testicular plasma membrane of rats. In vitro studies were conducted by exposing plasma membrane of testis with 1.46 x 10(-5) M, 2.92 x 10(-5) M, 4.38 x 10(-5) M, 7.30 x 10(-5) M, 1.46 x 10(-4) M and 2.92 x 10(-4) M HCH at 37 degrees C for 15 min. The results indicate inhibition in the activity of the Ca(2+)-ATPase, Na(+)+K(+)+Mg(2+)-ATPase and 5'Nucleotidase. In vivo studies were carried out following repeated dermal exposure to HCH at a dose level of 50 or 100 mg/kg/day for 60 days to male rats. The results show significant decrease in the activities of 5'-Nucleotidase, Ca(2+)-ATPase, Na(+) + K(+)-ATPase and Mg(2+)-ATPase in the plasma membrane of testis following exposure to HCH. The analysis of the residues of HCH reveals the presence of significant quantities of its different isomers viz., alpha, beta, gamma and delta in the testicular plasma membrane of rats given in vivo dermal exposure of this pesticide. These results suggest that the presence of HCH residue may be a factor in inhibiting the marker enzymes of the plasma membrane of testis. PMID- 7532326 TI - Serotonergic measures in cyclosporine A treated rats. AB - Whole blood and plasma serotonin (5-HT), its major metabolite--5 hydroxyindoleacetic acid (5-HIAA), renal cortical blood flow, serum creatinine and whole blood cyclosporine A (CyA) levels were investigated in rats administered with CyA at a dose of 5 mg/kg b.w. or 10 mg/kg b.w. for 14 consecutive days. Serum creatinine remained unaltered during CyA treatment and no apparent changes in excised kidneys were found. Dose-dependent increases in whole blood and plasma 5-HT as well as whole blood 5-HIAA levels were observed. Renal cortical blood flow declined significantly and correlated inversely with whole blood 5-HT and 5-HIAA as well as with plasma 5-HT. Whole blood 5-HT was positively related to whole blood CyA levels. Taking all these data into account and considering the fact that 5-HT is a potent vasoconstrictor, a possible role of this amine in the pathogenesis of renal ischemia during CyA administration is suggested. PMID- 7532329 TI - Thymus involvement in murine acquired immunodeficiency (MAIDS). AB - Due to self-renewal of the peripheral pool of T-cells, adult thymectomy has normally little influence on immunocompetence. However, thymus might play a more important role in the setting of viral-induced cytopathic effects on T-cells in the periphery. Therefore, thymus weight, cell numbers, and subset distribution were sequentially analysed after infection with RadLV-Rs, a viral mixture known to induce murine retrovirus induced immunodeficiency (MAIDS). Infection induced thymic atrophy (concerning organ weight as well as total cell number) which culminated seven weeks after inoculation. The atrophic process mostly reflected the depletion of double positive CD4+ CD8+ cells since their proportion sharply decreased around week 6. Single positive T-cells were less affected by the process. The proportion of B-cells progressively increased. Surprisingly, there was a strong correlation between the extent of atrophy and the frequency of B cells in the thymus. Finally, an abnormal CD4+ T-cell subset lacking Thy-1 and previously described in the periphery also appeared in the thymus and its frequency was strongly correlated with the expansion of B-cells in this organ. PMID- 7532330 TI - Effects of FK506 on surface antigen expression by regenerating thymocytes after sublethal irradiation in the rat. AB - To determine the effects of FK506 on the intrathymic maturation of T cells, we analyzed changes in surface antigen expression by regenerating thymocytes after sublethal irradiation. Two-color flow cytofluorometric analysis of the regenerating thymocytes revealed that FK506 had few effects on the maturation of rat thymocytes from CD4-8- to CD4+8+, and from TCR alpha beta- MHC class I- to TCR alpha beta low MHC class I+ (TCR alpha beta, alpha and beta chains of the T cell antigen receptor). However, defects in the maturation of CD4+8+ cells to CD4+8- cells and of TCR alpha beta low MHC class I+ cells to TCR alpha beta high MHC class I+ cells were observed after FK506 administration. Furthermore, three color analysis also detected a maturational defect of CD4-8+ TCR alpha beta high cells. Immunohistochemical analysis using MoAbs specific for MHC and TCR alpha beta revealed marked atrophy of the thymus medulla, suggesting disturbances in thymocyte regeneration and maturation. These results suggest that the maturation of CD4-8- TCR alpha beta- MHC class I- cells to CD4+8+ TCR alpha beta low MHC class I+ cells may be FK506 resistant, whereas FK506 may interfere with the maturation of CD4+8+ TCR alpha beta low MHC class I+ thymocytes not only to CD4+8 TCR alpha beta high MHC class I+ cells but also to CD4-8+ TCR alpha beta high MHC class I+ cells. We also demonstrated the possibility of enrichment of CD4-8- cells after sublethal irradiation and the existence of small subpopulations of CD4+8- TCR alpha beta- and CD4-8+ TCR alpha beta- cells which might mature to CD4+8+ cells in rat thymus. PMID- 7532331 TI - Walter Heiligenberg (1938-1994). PMID- 7532332 TI - Protons: small stimulants of capsaicin-sensitive sensory nerves. AB - The data reviewed in this article suggest that protons should no longer be considered simply as an unwanted by-product of anaerobic respiration that results from either an accumulation of inflammatory cells or a reduced oxygenated blood supply during ischaemia. A fall in extracellular pH can stimulate a subpopulation of sensory nerves by activation of ion channels. The available evidence indicates that most, if not all, of the activated neurones are also stimulated by capsaicin, and that protons and capsaicin share a common mechanism of neuronal activation. A proton should be viewed as a mediator that elicits a protective response with reflex cardiovascular and respiratory responses, which modulate systemic blood flow, and with the local release of sensory neuropeptides, which vasodilates the microvasculature and stimulates extravasation. PMID- 7532333 TI - The memory storehouse. PMID- 7532334 TI - Beyond 'pet' methodologies to converging evidence. PMID- 7532335 TI - NADPH dehydrogenase/NOS revisited. PMID- 7532336 TI - Bridging the cleft at GABA synapses in the brain. AB - A fragile balance between excitation and inhibition maintains the normal functioning of the CNS. The dominant inhibitory neurotransmitter of the mammalian brain is GABA, which acts mainly through GABAA and GABAB receptors. Small changes in GABA-mediated inhibition can alter neuronal excitability profoundly and, therefore, a wide range of compounds that clearly modify GABAA-receptor function are used clinically as anesthetics or for the treatment of various nervous system disorders. Recent findings have started to unravel the operation of central GABA synapses where inhibitory events appear to result from the synchronous opening of only tens of GABAA receptors activated by a saturating concentration of GABA. Such properties of GABA synapses impose certain constraints on the physiological and pharmacological modulation of inhibition in the brain. PMID- 7532338 TI - Modulation of ion-channel function by G-protein-coupled receptors. AB - Neurotransmitters acting through G-protein-coupled receptors change the electrical excitability of neurons. Activation of receptors can affect the voltage dependence, the speed of gating, and the probability of opening of various ion channels, thus changing the computational state and outputs of a neuron. Each cell expresses many kinds of receptors, and uses several intracellular signaling pathways to modulate channel function in different ways. It has become possible to dissect these pathways by combining pharmacological and biophysical experiments. Recent patch-clamp work in sympathetic neurons will be summarized to illustrate the mechanisms underlying modulation and its significance. PMID- 7532340 TI - Ultrastructural appearance of hyaline droplet-laden cells in the glomerular mesangium of pigs. PMID- 7532339 TI - Cellular and synaptic localization of NMDA and non-NMDA receptor subunits in neocortex: organizational features related to cortical circuitry, function and disease. AB - Excitatory amino acid (EAA) receptors are an important component of neocortical circuitry as a result of their role as the principal mediators of excitatory synaptic activity, as well as their involvement in use-dependent modifications of synaptic efficacy, excitoxicity and cell death. The diversity in the effects generated by EAA-receptor activation can be attributed to multiple receptor subtypes, each of which is composed of multimeric assemblies of functionally distinct receptor subunits. The use of subunit-specific antibodies and molecular probes now makes it feasible to localize individual receptor subunits anatomically with a high level of cellular and synaptic resolution. Initial studies of the distribution of immunocytochemically localized EAA-receptor subunits suggest that particular subunit combinations exhibit a differential cellular, laminar and regional distribution in the neocortex. While such patterns might indicate that the functional heterogeneity of EAA-receptor-linked circuits, and the cell types in which they operate, are based partly on differential subunit parcellation, a definitive integration of these anatomical details into current schemes of cortical circuitry and organization awaits many further studies. Ideally, such studies should link a high level of molecular precision regarding subunit localization with synaptic details of identified connections and neurochemical features of neocortical cells. PMID- 7532337 TI - Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease. AB - Recent evidence indicates that apolipoprotein E (ApoE) plays a central role in the hippocampal response to injury. The co-ordinated expression of ApoE and its receptor, the ApoE/ApoB [low density lipoprotein (LDL)] receptor, appears to regulate the transport of cholesterol and phospholipids during the early and intermediate phases of the reinnervation process. During dendritic remodeling and synaptogenesis, neurons progressively repress the synthesis of cholesterol in favor of cholesterol internalization through the ApoE/LDL receptor pathway. The discovery that the epsilon 4 allele is strongly linked to both sporadic and familial late-onset Alzheimer's disease (AD) raises the possibility that a dysfunction of the lipid-transport system associated with compensatory sprouting and synaptic remodeling could be central to the AD process. The role of ApoE in the CNS is particularly important in relation to the function of the cholinergic system, which relies to a certain extent on the integrity of phospholipid homeostasis in neurons. Recent evidence suggests that the epsilon 4 allele has a direct impact on cholinergic function in AD. PMID- 7532342 TI - Anti-HCV seroconversion in multitransfused and immunocompromised patients. A long term longitudinal study. AB - In a series of 6 multitransfused, immunocompromised patients, the diagnosis of posttransfusion hepatitis C was based upon the analysis of long-term follow-up serum samples. The HCV RNA was detected by a nested PCR assay using primers located in the 5' noncoding region (5'NCR), and anti-HCV antibodies were assayed with third-generation tests. The interval between the first rise in alanine aminotransferase and seroconversion varied from less than 5 months to more than 38 months. Five out of 6 patients seroconverted after 14 months or later. In most cases, the anti-NS3 and anti-NS4 antibodies appeared first. In such patients, the etiology of chronic liver disease may thus be overlooked for 1 or more years, a definitive diagnosis requiring the detection of HCV RNA. PMID- 7532341 TI - Hepatitis C virus antibodies and virus replication in asymptomatic blood donors. AB - We assessed hepatitis C virus (HCV) infection in 99 asymptomatic blood donors positive using a first-generation HCV antibody assay. When tested with second generation assays, 86 (87%) donors were reactive (group 1), 2 (2%) were indeterminate (group 2), and 11 (11%) were non-reactive (group 3). Viraemia was revealed by polymerase chain reaction in all group 1 cases. The 2 group 2 cases and 6 (55%) group 3 cases were also viraemic. Viraemia was confirmed by a branched DNA assay in the 2 group 2 cases and 4 (36%) group 3 cases. Serum HCV RNA levels were further studied using a competitive reverse transcription polymerase chain reaction assay. All cases in groups 2 and 3 were low viraemic (range 10(4)-10(5.5) copies/ml) compared with the 9 group 1 cases examined (range 10(7)-10(9) copies/ml). No correlation was evident between viraemic levels and antibody cut-off index in the first-generation assay. These findings indicate the possibility that low levels of viraemia can occur in individuals non-reactive in second-generation HCV antibody assays. PMID- 7532343 TI - A novel agglutination method for screening of HIV and HCV antibody testing with 5 microliters reagents: reduction of cost and time with high sensitivity. PMID- 7532344 TI - [Pathophysiology, therapy and prognosis of hypoxic-ischemic brain damage]. AB - Generally accepted treatment regimens of hypoxic-ischemic brain damage have not been established so far. Therefore, therapeutic measures are oriented to the pathophysiological mechanisms known at present, including ischemic calcium cascade, excitotoxicity, NO overformation, and disturbances of re-circulation (e.g., no reflow phenomenon). Bioelectric changes in the brain parenchyma evolving during hypoxia-ischemia become successively apparent as hyperpolarization, failure of synaptic transmission, massive depolarization of cells resembling the spreading depression of Leao, neuronal K+ loss and uptake of large amounts of Na+, Cl-, Ca++, accompanied by H2O, causing cell swelling. Up to now, the rapid progress of these pathological events has hardly permitted an efficacious treatment. If any therapy, the combination of NMDA receptor antagonists, glucocorticosteroids, GABAergic drugs and heparin could be helpful in preventing the delayed postischemic injury that often occurs after initial apparent recovery. The therapeutic role of lazaroids, NO donators, and endothelin antagonists still has to be defined. An early assessment of the brain damage subsequent to hypoxia-ischemia is possible by means of somatosensory evoked potentials (SSEP) and serum concentration of neuronspecific enolase (NSE), respectively. NSE values exceeding 120 ng/ml during the first 5 days after hypoxia-ischemia point to an unfavorable outcome. In contrast, NSE concentrations below 35 ng/ml mostly indicate a good recovery. PMID- 7532345 TI - Distribution of complement regulatory proteins, decay-accelerating factor, CD59/homologous restriction factor 20 and membrane cofactor protein in human colorectal adenoma and cancer. AB - To clarify the events related to complement-mediated immune responses in human colorectal cancers, we immunohistochemically examined the distribution of decay accelerating factor (DAF), CD59/homologous restriction factor 20 (HRF20), membrane cofactor protein (MCP) and terminal complement complex (TCC) in human colorectal adenomas and cancers, and then compared the findings with their distribution in normal colonic mucosa. In the normal mucosa, TCC was not present on epithelial cells. Whereas DAF and CD59/HRF20 were present only occasionally on the apical surfaces of normal epithelial cells, MCP was diffusely distributed on the basolateral surfaces of most epithelial cells of the colon. These findings suggest that MCP has a primary role in the regulation of complement activation on these cells. In adenoma cells, the expression of both DAF and CD59/HRF20 was enhanced. In cancer cells, the expression of CD59/HRF20 and MCP was diminished, whereas DAF expression was markedly enhanced. Since DAF was frequently stained in the lumen of the cancer glands, it was suggested that DAF was released into the colonic lumen in patients with colorectal cancer. PMID- 7532348 TI - Differential distribution of insulin-like growth factors and their binding proteins within bone: relationship to bone mineral density. AB - To evaluate the possibility that insulin-like growth factors (IGFs) and their binding proteins (BPs) in bone play a role in regulating cortical bone formation in growing animals, we compared changes in IGF and IGF BP levels with changes in bone mineral density (BMD) at three different regions (proximal, middle, and distal) along the rabbit femoral shaft. BMD measured by dual-energy x-ray absorptiometry decreased progressively from proximal to distal regions of the shaft, from 0.449 +/- 0.005 to 0.354 +/- 0.002 g/cm2 (mean +/- SEM; n = 9), respectively; total protein concentrations also decreased toward the distal region. We extracted the IGFs and their BPs from bone by demineralization in 10% EDTA and 4 M guanidine-HCl (pH 4.5). The IGFs were then separated from their BPs by size exclusion HPLC. The pH of the extraction buffer profoundly influenced the recoveries of the IGFs and, to a lesser extent, the total protein; at least 100% more IGFs were recovered at acid (4.5) pH than at neutral (7.5) or basic (10.5) pH. The levels of IGF-I decreased markedly from proximal to distal regions, from 273 +/- 27 to 100 +/- 38 ng human IGF-I equivalent/g bone (or 103 +/- 10 to 52 +/ 11 ng human IGF-I equivalent/mg protein), respectively. IGF-II was uniformly distributed (385 +/- 17 ng human IGF-II equivalent/g bone; mean of all three regions). Levels of the predominant 28-32 kD IGF BP doublet increased by about 100% from proximal to distal segments, regardless of whether the data were expressed per unit mass or protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532347 TI - Skeletal unloading induces resistance to insulin-like growth factor I. AB - In previous studies with a hindlimb elevation model, we demonstrated that skeletal unloading transiently inhibits bone formation. This effect is limited to the unloaded bones (the normally loaded humerus does not cease growing), suggesting that local factors are of prime importance. IGF-I is one such factor; it is produced in bone and stimulates bone formation. To determine the impact of skeletal unloading on IGF-I production and function, we assessed the mRNA levels of IGF-I and its receptor (IGF-IR) in the proximal tibia and distal femur of growing rats during 2 weeks of hindlimb elevation. The mRNA levels for IGF-I and IGF-IR rose during hindlimb elevation, returning toward control values during recovery. This was accompanied by a 77% increase in IGF-I levels in the bone, peaking at day 10 of unloading. Changes in IGF binding protein levels were not observed. Infusion of IGF-I (200 micrograms/day) during 1 week of hindlimb elevation doubled the increase in bone mass of the control animals but failed to reverse the cessation of bone growth in the hindlimb-elevated animals. We conclude that skeletal unloading induces resistance to IGF-I, which may result secondarily in increased local production of IGF-I. PMID- 7532346 TI - Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes. AB - There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation. PMID- 7532349 TI - [Assessment of quality of life after transurethral resection of prostate for benign prostatic hyperplasia]. AB - The quality of life (QOL) after transurethral resection of the prostate (TURP) was studied. A total of 113 patients were operated and released, and 86 (76%) cases responded to the TURP follow-up survey. The average age was 69 years and the average time elapsed after the operation was 220 days. Data regarding the patients undergoing TURP was gathered from a questionnaire consisting of 22 questions concerning the preoperative condition and 28 about the post-operative state. Performance status was not changed post-TURP. The patients showed improvements in nocturia, pollakisuria and dysuria. These urological symptoms were in accordance with the findings of uroflowmetry and American Urological Association Symptom Index. Ten questions addressing the patients mental and physical conditions revealed that good quality was generally retained. Sexual activity showed a tendency of gradual decrease in relation to increasing age. Ejaculatory function and actual satisfaction with the sexual act were obviously damaged by the operation. TURP showed no changes in regard to morning erection, sexual desire in an arousing atmosphere, penile hardness at sex and total satisfaction with the sexual life. Social life, family life and mental status were not influenced, but the physical status of 21 (26%) of the patients was decreased by the operation and hospital stay. Over all, 78% of the patients could maintain a good quality of life in post-TURP. PMID- 7532350 TI - Palliative treatment of malignant pleural effusions: value of small-bore catheter thoracostomy and doxycycline sclerotherapy. AB - OBJECTIVE: This study evaluates small-bore catheter thoracostomy combined with doxycycline sclerotherapy for palliative treatment of presumed malignant pleural effusions. SUBJECTS AND METHODS: Forty-seven consecutive patients referred from the medical oncology department to the thoracic radiology section with known primary malignant tumors and symptomatic pleural effusions over a 2-year period were treated with small-bore pleural catheter drainage followed by doxycycline sclerotherapy. Of the 47 patients, 20 (43%) died less than 30 days after sclerotherapy, one (2%) died without radiographic follow-up, and five (11%) were lost to follow-up. Twenty-one patients formed the study group. Response to treatment was defined based on findings on follow-up chest radiographs obtained 30 days after sclerotherapy as complete (no reaccumulation of pleural effusion), partial (accumulation above postpleurodesis level but below that at presentation), or as a failure (reaccumulation to the amount seen at presentation). RESULTS: Seventeen (81%) evaluable patients showed complete response to sclerotherapy, three (14%) showed a partial response, and one (5%) showed no response. All complete and partial responders were clinically improved with resolution of their shortness of breath. Therefore, 95% of evaluable patients had clinically and radiographically successful treatment. Six patients underwent sclerotherapy when their tube output was greater than 100 ml/24 hr. Five of the six had completely successful pleurodesis, and one failed to respond. Two (10%) of the 21 patients had greater than 150 ml drainage in the 24 hr after initial doxycycline administration and were therefore given a second dose of intrapleural doxycycline. Both of these patients subsequently had less than 150 ml drainage in an additional 24-hr observation period and went on to complete response. Complications included three patients (14%) with mild discomfort at the chest tube site during drainage, one patient (5%) with pain during instillation of doxycycline, and one patient (5%) with transient fever (38.3 degrees C body temperature) one day after sclerotherapy. CONCLUSION: Small-bore catheter thoracostomy followed by doxycycline sclerotherapy successfully resolves symptomatic pleural effusion in patients with known primary malignant tumors. PMID- 7532352 TI - Effect of propranolol on circadian variation of ventricular arrhythmias in elderly patients with heart disease and complex ventricular arrhythmias. PMID- 7532351 TI - Vascular endothelium: an integrator of pathophysiologic stimuli in atherosclerosis. AB - Vascular endothelium, the single-cell-thick lining of the cardiovascular system, is an important functional component of the blood vessel wall, actively participating in normal vascular physiology as well as the pathogenesis of vascular diseases such as atherosclerosis. The localized modulation of vascular endothelium to a non-adaptive functional state can be termed "endothelial dysfunction." This article provides a brief overview of endothelial dysfunction, especially as it relates to mononuclear leukocyte recruitment during atherosclerotic lesion formation. Potential diagnostic and therapeutic implications are also considered. PMID- 7532354 TI - Bone marrow biopsy imprints (touch preparations) for assessment of iron stores. AB - Noninvasive evaluation for iron deficiency is compromised in many individuals due to the presence of chronic inflammatory processes and/or malignancy, thus necessitating bone marrow examination for definitive diagnosis. However, bone marrow aspiration is not obtainable or is inadequate for interpretation (dry tap) in some individuals, and decalcified bone marrow biopsies require 24-48 hr to prepare, and may falsely indicate absence of iron. We evaluated the accuracy of bone marrow biopsy imprints (touch preparations) compared with aspirate particle smears for semiquantitation of bone marrow iron stores. Results indicate that Prussian blue-stained bone marrow biopsy imprints accurately reflect the quantity of iron, compared with bone marrow aspirate particle smears, allowing for rapid determination of iron stores in individuals in whom a bone marrow aspirate cannot be obtained. PMID- 7532353 TI - Gender and haplotype effects upon hematological manifestations of adult sickle cell anemia. AB - In Africa, the beta-globin gene cluster haplotype may be associated with variation of Hb F levels in subjects with sickle cell anemia (SS). These observations have not yet been conclusively confirmed in SS out of Africa, perhaps because of small sample sizes, the predominance of haplotype heterozygotes, and diverse influences, including gender, upon Hb F levels. We studied 384 adult African-American SS patients (mean age, 31 years) and explored the relationship of gender, beta-globin gene cluster haplotype, and alpha thalassemia to hematological values and Hb F levels. Both haplotype and gender influenced Hb F concentration. In the total sample, Hb F was higher in females than in males (8.2 vs. 6.5%). In 35 males who were either homozygous for the Senegal chromosome or had the Senegal/Benin haplotype, the mean percent Hb F (8.0%) was equivalent to the Hb F level in females with Benin and Bantu haplotypes (approximately 7.5%). Both females and males homozygous for the Senegal haplotype chromosome or with the Senegal/Benin combination had a significant increase in Hb F compared to other groups. In 44 Senegal/Senegal or Senegal/Benin females the Hb F was 10.9%, or 1.0 g/dl, the highest value observed in all primary analysis groups. Preliminary analyses suggested that the presence of a Bantu chromosome blunted the gender-associated difference in Hb F, but Hb F differences between females with the Senegal/Benin haplotype (11.2%) and the Senegal/Bantu haplotype (8.8%) were not statistically significant. Hemoglobin concentrations were higher in males than in females except in subjects with at least one Senegal haplotype chromosome, where hemoglobin levels were equal. As expected, alpha thalassemia reduced the MCV, increased hemoglobin concentration, and lowered reticulocyte counts, regardless of haplotype. Hb F levels were not affected by the presence of alpha thalassemia in any group. We conclude that gender and beta-globin gene cluster haplotype interact significantly in the modulation of Hb F and anemia in adults with SS. PMID- 7532355 TI - Successful treatment of CD7-positive, undifferentiated acute leukemia with recombinant interferon alpha. PMID- 7532356 TI - Tetrasomy 21 pter-->q22.1 and Down syndrome: molecular definition of the region. AB - Down syndrome is usually caused by complete trisomy 21. Rarely, it is due to partial trisomy of the segment 21q22. We report on a 33-month-old girl with tetrasomy 21 pter-->q22.1 resulting from an extra chromosome idic(21)(q22.1). She has craniofacial traits typical of Down syndrome, including brachycephaly, third fontanel, upward slanting palpebral fissures, round face, and protruding tongue. Speech development is quite delayed whereas motor development is only mildly retarded. The molecular content of the extra isodicentric chromosome was defined by molecular genetic investigations using 13 single copy probes unique to chromosome 21, and SOD1 expression studies. The child was found to have 4 copies of the region defined by D21S16 (21cen) through D21S93 on 21q22.1 and two copies of the remaining region defined by SOD1-->D21S55-->D21S123. In view of the recent assignment of Down syndrome facial characters to the 21q22 region, defined in part by D21S55, it is significant that this child shows a subset of Down syndrome facial manifestations, without duplication of this region. These results suggest that genes contributing to the facial and some of the hand manifestations of Down syndrome also exist in the chromosomal region proximal to D21S55 in band 21q22.1. PMID- 7532357 TI - Small terminal deletions of the long arm of chromosome 2: two new cases. AB - We report on 2 girls with small de novo terminal deletions of the long arm of chromosome 2 and breakpoints within q37. Four cases with similar or more extensive deletions have been previously reported in full. Hypotonia and psychomotor retardation were the only manifestations common to all 6 cases. The phenotype associated with small terminal 2q deletions is variable and clearly not always as mild as indicated in previous reports. The abnormality may also be more common than has been assumed. PMID- 7532358 TI - Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells. AB - We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30 pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously. PMID- 7532359 TI - Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP. AB - Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8 (p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells. PMID- 7532360 TI - Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence. AB - The process of active intestinal inflammation can be modeled by the migration of neutrophils [polymorphonuclear leukocytes (PMN)] across monolayers of the human intestinal cell line T84. We now evaluate the polarity of early adhesive events occurring between human PMN and ligands on intact T84 monolayers. Transient Ca2+ depletion (tr-Ca-depl) of confluent monolayers grown on permeable supports (2 mM EDTA, 12 min, 37 degrees C) resulted in disruption of tight junctions separating apical and basolateral domains (resistance fell from 973 +/- 35 to 57.4 +/- 3.8 omega.cm2 after 12 min). The columnar T84 phenotype was not grossly altered by tr Ca-depl. Access to basolateral membrane proteins from the apical surface after tr Ca-depl was confirmed by enzyme-linked immunosorbant assay and immunofluorescence, using antibodies to the basolateral proteins E-Cadherin and beta 1-integrin. The effects of tr-Ca-depl on early events in PMN adhesion were next assessed. Neutrophils (2 x 10(6)) were applied to the apical surface of monolayers followed by stimulation with the chemotactic peptide N-formyl methionyl-leucyl-phenylalanine (100 nM, 5 min, 37 degrees C) and quantitation. In comparison with control monolayers, tr-Ca-depl increased subsequent PMN adhesion (1.7 +/- 0.24 vs. 3.1 +/- 0.28 x 10(4) adherent PMN/monolayer; P < 0.005), which was inhibited 80% by anti-CD11b/CD18 antibodies. These experiments suggest that PMN adhere to subjunctional ligands on T84 cells in a CD11b/CD18-dependent fashion. PMID- 7532361 TI - Impaired EDRF production by endothelial cells exposed to fibrin monomer and FDP. AB - Fibrin and fibrinogen and their proteolytic degradation products, found within the atheroma, may contribute to vascular dysfunction. We monitored the production of endothelium-derived relaxing factor (EDRF) by bovine aortic endothelial cells (BAEC) after exposure to fibrinogen, fibrin monomer (FM), and fibrin and fibrinogen degradation products (FDP). Cells incubated with FM and FDP, compared with cells incubated with fibrinogen, were less able to inhibit platelet aggregation, and this effect correlated with a concentration-dependent decrease in EDRF production: BAEC incubated with FM or FDP, but not fibrinogen, produced significantly less nitric oxide (NO), as determined using a photolysis chemiluminescence system. Northern analysis of BAEC incubated with fibrinogen, FM, or FDP and probed for constitutive bovine endothelial NO synthase mRNA demonstrated decreased expression in cells exposed to FDP or FM. These data show that FM and FDP reduce EDRF produced by BAEC and attenuate constitutive NO synthase expression. These effects may represent a mechanism by which thrombotic determinants adversely affect endothelial function and thereby potentially impair vasomotor responses and contribute to atherothrombogenesis. PMID- 7532363 TI - Is nitric oxide the final mediator regulating the migrating myoelectric complex cycle? AB - The main objective was to study the role of nitric oxide (NO) in the conversion of migrating myoelectric complexes (MMC) to the irregular electrical activity characteristic of the postprandial state. Both rats and chickens were implanted with electrodes for electromyography in the small intestine. Intravenous infusion of NG-nitro-L-arginine (L-NNA), a NO synthase inhibitor, induced an organized MMC like pattern in fed rats. Infusion of sodium nitroprusside, a NO donor, disrupted the MMC, inducing a postprandial-like motor pattern in fasting rats. Similarly, in chickens L-NNA mimicked the fasting pattern, consisting of a shortening of phase II, enlargement of phase III, orad displacement of the origin of the MMC, and an increase in the speed of phase III propagation. An inhibition of NO synthesis seems to be involved in the induction of the fasting motor pattern, whereas an increase of NO mediates the occurrence of the fed pattern. It is suggested that NO might be the final mediator in the control of small intestine motor patterns. PMID- 7532362 TI - Muscle growth in response to mechanical stimuli. AB - The relative merits of the separate and combined uses of stretch and electrical stimulation at 10 Hz in influencing the rates of protein synthesis in vivo, proteolysis, and the growth of the extensor digitorum longus muscle have been investigated after 3 days in the rabbit. Continuous electrical stimulation failed to change muscle protein turnover or growth. Static stretch caused significant adaptive growth, with increases in c-fos, c-jun, and insulin-like growth factor I (IGF-I; 12-fold) mRNA levels, and protein (19%), RNA (128%), and DNA (45%) contents. Both the fractional (138%) and total (191%) rates of protein synthesis increased with stretch, correlating with increased ribosomal capacities. Combining stretch and electrical stimulation increased the mRNA concentration of IGF-I (40-fold). The adaptive growth was greater (35%), with massive increases in the nucleic acids (185 and 300%), ribosomal capacities (230%), and the rates of protein synthesis (345 and 450%). Large increases (i.e., 200-400%) in cathepsins B and L and dipeptidyl aminopeptidase I activities during stretch, with or without stimulation, suggest a role for these enzymes in tissue remodeling during muscle hypertrophy. PMID- 7532364 TI - Influence of acid and angiogenesis on kinetics of gastric ulcer healing in rats: interaction with indomethacin. AB - Indomethacin delays healing of experimental gastric ulcers. We investigated whether inhibition of gastric acid secretion by omeprazole or stimulation of angiogenesis by basic fibroblast growth factor (bFGF) may reverse this delay. Rats with gastric ulcers induced by cryoprobe were treated subcutaneously with either placebo, indomethacin (2 x 0.5 mg/kg), bFGF (2 x 100 micrograms/kg), omeprazole (1 x 40 mumol/kg), indomethacin plus omeprazole, or indomethacin plus bFGF given daily for 8, 10, 15, and 22 days. Ulcer size, epithelial cell proliferation, angiogenesis, and maturation of granulation tissue were sequentially quantified. Omeprazole significantly accelerated ulcer healing in an early phase (days 3-8). In contrast, bFGF accelerated healing in a late phase (days 10-15). Indomethacin significantly delayed ulcer healing in late phase and decreased prostaglandin generation, cell proliferation, angiogenesis, and maturation of granulation tissue. Despite stimulation of angiogenesis, bFGF did not reverse indomethacin-induced delay in ulcer healing. In contrast, omeprazole reversed indomethacin-induced effects on angiogenesis, cell proliferation, maturation of granulation tissue, and ulcer healing rate. PMID- 7532365 TI - Presynaptic inhibition by serotonin of nerve-mediated secretion of pancreatic amylase. AB - Enteric cholinergic and serotonergic neurons innervate pancreatic ganglia. Enteropancreatic cholinergic neurons are secretomotor, bu the function of the serotonergic cells is unknown and was investigated. Postganglionic cholinergic nerve-mediated amylase secretion was evoked by veratridine in isolated pancreatic lobules. This concentration-dependent response was inhibited by tetrodotoxin (1.0 microM), atropine (5.0 microM), 5-hydroxytryptamine (5-HT; 5.0 microM), 5 hydroxyindalpine (5-OHIP; 10.0 microM; a 5-HT1P agonist), and 8-hydroxy-2-(di-n propylamino)tetralin (8-OH-DPAT; 0.1 microM), but not by hexamethonium (100.0 microM), 2-methyl-5-HT (10 microM), or 5-carboxyamidotryptamine (10 microM). The effects of 5-HT and 5-OHIP were blocked by the 5-HT1P antagonist N-acetyl-5 hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP; 100.0 microM). Carbachol (5.0 microM)-evoked secretion was not affected by 5-HT or 5-OHIP. Veratridine induced c-fos expression in pancreatic neurons and acinar cells. This expression was inhibited by tetrodotoxin, 5-HT, and 5-OHIP. These observations suggest that the serotonergic enteropancreatic innervation inhibits pancreatic secretion via presynaptic receptors on cholinergic nerves. Although the data are consistent with the hypothesis that the inhibitory receptor is a 5-HT1P site, positive identification awaits further study. PMID- 7532366 TI - Halothane reduces force and intracellular Ca2+ in airway smooth muscle independently of cyclic nucleotides. AB - Halothane relaxes airway smooth muscle in part by a direct effect on the smooth muscle cell. The purpose of this study was to investigate the possible role of cyclic nucleotides in this direct effect. Strips of canine tracheal smooth muscle in vitro were contracted with acetylcholine (ACh) and then exposed to 0.7-2.6% halothane. Isometric force and the intracellular concentrations of adenosine cyclic 3',5'-monophosphate ([cAMP]i) guanosine cyclic 3',5'-monophosphate ([cGMP]i), and free calcium ([Ca2+]i, using the fluorescent Ca(2+)-sensitive dye fura 2) were measured. ACh caused significant increases in force, [cAMP]i, [cGMP]i, and [Ca2+]i. Subsequent exposure of the strips to halothane caused an additional increase in [cAMP]i, decreases in force and [Ca2+]i, and no effect on [cGMP]i. The additional increase in [cAMP]i was similar to that produced by a concentration of isoproterenol (0.03 microM) that caused equipotent relaxation. Indomethacin abolished the increase in [cAMP]i produced by ACh and abolished the additional increase in [cAMP]i produced by halothane. In contrast, indomethacin had no effect on the decreases in force and [Ca2+]i. These findings suggest that in canine tracheal smooth muscle contracted with ACh 1) halothane increases [cAMP]i by a cyclooxygenase-dependent mechanism and 2) the increase in [cAMP]i produced by halothane is not responsible for the relaxation or the decrease in [Ca2+]i. PMID- 7532367 TI - TGF-beta regulates expression of tenascin alternative-splicing isoforms in fetal rat lung. AB - Two distinct mRNA-splice isoforms of tenascin (TN) are expressed differentially during rat lung development. The unique temporal expression pattern of two TN isoforms suggests the expression of tenascin is strictly developmentally regulated in rat lung tissue. We investigated molecular mechanisms which modulate alternative-splicing expression of TN in lung development. The effect of transforming growth factor-beta 1 (TGF-beta 1) on regulation of expression of TN isoforms was examined by in vitro lung explant culture. Immunoblotting with anti TN antibody detected two TN polypeptides in rat lung explant culture, the larger [relative molecular weight (M(r)) 230, TN230] polypeptide and the smaller (M(r) 180, TN180). TGF-beta 1 markedly induced the TN180 isoform and caused only a moderate increase of the TN230 isoform. The effects of TGF-beta 1 were shown to be dose dependent over a physiological range of TGF-beta 1 protein concentration. The induction of TN isoform biosynthesis by TGF-beta 1 was detected 12 h after addition of the growth factor, and the effects endured for up to 48 h at a dose of 5 ng/ml. By reverse transcriptase-polymerase chain reaction through amplification of the entire fibronectin type III (FN-III) splicing domain, two distinct TN isoforms were detected in total RNA isolated from gestational day 21 rat lung explant culture treated with TGF-beta 1 and from postnatal day 8 rat lung. The larger isoform contained five FN-III alternative splicing repeats [1,420 base pairs (bp)], but the shorter splicing isoform lacked four FN-III alternative splicing repeats (340 bp).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532369 TI - Intratracheal administration of endotoxin and cytokines. VI. Antiserum to CINC inhibits acute inflammation. AB - Cytokine-induced neutrophil chemoattractant (CINC), a chemotactic molecule of the interleukin (IL)-8 family, is known to be induced in the rat in response to tumor necrosis factor (TNF), IL-1, and lipopolysaccharide (LPS). Intratracheal injection of endotoxin (LPS) is shown to cause CINC mRNA expression in pulmonary tissue, peaking after 2 h, and CINC protein expression in bronchoalveolar lavage (BAL) fluid, peaking after 2-4 h. Intratracheal injection of synthetic CINC causes acute inflammation that is abrogated by coinjection of antiserum to purified natural rat CINC. Intratracheal injection of antiserum to CINC inhibits intratracheal LPS- and IL-1-induced neutrophil emigration into BAL fluid by approximately 60-70%. Despite the anti-inflammatory activity of anti-CINC antiserum, TNF is elevated in the lavage fluid of rats receiving anti-CINC, suggesting that CINC may act in a negative feedback loop to downregulate TNF expression. Intratracheal injection of antiserum to CINC combined with intravenous injection of anti-E-selectin antibody inhibits intratracheal LPS- and IL-1-induced neutrophil emigration into BAL fluid by approximately 75-85%. CINC mediated chemotactic activity and E-selectin-mediated adherence of neutrophils to endothelium contribute significantly to the pathogenesis of LPS-initiated acute inflammation. PMID- 7532371 TI - Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis. AB - An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways. PMID- 7532368 TI - Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle. AB - Hypoxia dilates airways in vivo and reduces active tension of airway smooth muscle in vitro. To determine whether hypoxia impairs Ca2+ entry through voltage dependent channels (VDC), we tested the ability of dihydropyridines to modulate hypoxia-induced relaxation of KCl- and carbamyl choline (carbachol)-contracted porcine bronchi. Carbachol- or KCl-contracted bronchial rings were exposed to progressive hypoxia in the presence or absence of 1 microM BAY K 8644 (an L-type channel agonist). In separate experiments, rings were contracted with carbachol or KCl, treated with nifedipine (a VDC antagonist), and finally exposed to hypoxia. BAY K 8644 prevented hypoxia-induced relaxation in KCl-contracted bronchi. Nifedipine (10(-5) M) totally relaxed KCl- contracted bronchi. Carbachol contracted bronchi were only partially relaxed by nifedipine but were completely relaxed when the O2 concentration of the gas was reduced from 95 to 0%. These data indicate that hypoxia can reduce airway smooth muscle tone by limiting entry of Ca2+ through a dihydropyridine-sensitive pathway, but that other mechanisms also contribute to hypoxia-induced relaxation of carbachol-contracted bronchi. PMID- 7532370 TI - Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung. AB - Throughout intrauterine life, Cl(-)-rich liquid is secreted by the pulmonary epithelium. To evaluate the role of the most distal epithelium in liquid secretion, we measured bioelectric properties of monolayers composed of epithelial cells from acinar structures of postmortem human fetal lung (mean gestation, 22.3 wk; range, 18-24 wk). These monolayers formed high-resistance (R) barriers (mean R = 363 Ohm/cm2) when cultured in hormone-supplemented, serum-free medium. The transepithelial electrical potential difference (4.0 mV, lumen negative), was similar to that of whole fetal sheep lung in vivo. Equivalent short-circuit current (Ieq) was inhibited by apical amiloride (-20%), 5-(N-ethyl N-isopropyl)-amiloride (-33 to -49%), or diphenylamine-2-carboxylate (DPC; -26%), and by basolateral ouabain (-77%), whereas apical 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) had no effect. Bumetanide added to the basolateral bath did not affect resting Ieq, but inhibited Ieq (-19%) in monolayers pretreated with apical amiloride, basolateral terbutaline, and apical ATP, and also inhibited Ieq (-22%) of monolayers pretreated with basolateral amiloride and DIDS. Ieq was stimulated by terbutaline (90-128%), ATP (70-186%), and ionomycin (141%). Stimulation of Ieq by these agents is compatible with induction of Cl- secretion through two pathways: channels that are opened by a rise in adenosine 3',5'-cyclic monophosphate, and channels that are opened by a rise in intracellular Ca2+. Inhibition of Ieq by apical DPC implies that Cl- secretion may contribute to basal Ieq. PMID- 7532373 TI - Shear stress increases hydraulic conductivity of cultured endothelial monolayers. AB - To examine the effect of shear stress on hydraulic conductivity (Lp) of bovine aortic endothelial cell monolayers grown on polycarbonate filters, we developed a rotating disk system, which imposed a defined shear stress while Lp was measured. A 10-cmH2O pressure differential was applied to monolayers, and baseline Lp was established between 1.65 +/- 0.85 and 4.94 +/- 1.05 x 10(-7) cm.s-1.cmH2O-1. One hour exposure to 10 dyn/cm2 shear stress caused a significant (P < 0.05) increase in Lp by 2.16-fold (+/- 0.42), and Lp remained elevated when shear stress was removed. Three-hour exposure to shear stresses between 0.1 and 20.0 dyn/cm2 revealed a threshold for shear-induced increase in Lp of 0.5 dyn/cm2. At 20 dyn/cm2, Lp initially decreased by 30% (+/- 13.4%, P < 0.05) and then increased to a level 3.76-fold (+/- 0.83, P < 0.05) greater than baseline Lp at 3 h. The shear-induced increase in Lp was reversed with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM) and could be significantly (P < 0.05) inhibited when monolayers were preincubated with 0.3 mM DBcAMP, a concentration that did not significantly affect baseline Lp. Furthermore, preincubation with a general phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (1 mM), completely blocked the shear-induced increase in Lp. On the basis of these results, we conclude that shear stress alters endothelial Lp through a cellular mechanism involving signal transduction, not by a purely physical mechanism. PMID- 7532372 TI - Role of spinal NK1 receptors in cardiovascular responses to chemical stimulation of the gallbladder. AB - The present study examined the role of substance P (SP) as a sensory neurotransmitter in cardiovascular responses to bradykinin applied on the gallbladder. Experiments were performed in anesthetized cats in which sympathetic chains were transected at the T5-T6 level, and the tip of the intrathecal catheter was positioned at T6-T7 to limit the injectate between T6 and L2. Bradykinin (10 micrograms/ml) was applied onto the gallbladder before and after intrathecal injection of [D-Pro2,D-Phe7,D-Trp9]SP (100-200 micrograms, NK1/NK2 receptor antagonist), CP-99,994 (50-100 micrograms, selective NK1 antagonist), MEN-10,376 (100-500 micrograms, selective NK2 antagonist), or vehicle. Intrathecal injection of NK1 but not NK2 antagonist significantly reduced increases in mean arterial pressure, heart rate, and maximal rate of left ventricular pressure change by 28 +/- 2 mmHg (33 +/- 4%), 4 +/- 1 beats/min (42 +/- 5%), and 497 +/- 46 mmHg/s (36 +/- 4%), respectively. Intrathecal injection of NK1 or NK1/NK2 antagonist had no effect on cardiovascular responses evoked by electrical stimulation in the rostral ventral lateral medulla. These data suggest that endogenous SP, acting as a sensory neurotransmitter, is involved in the excitatory cardiovascular reflex caused by chemical stimulation of the gallbladder through its action on NK1 receptors in the spinal cord. PMID- 7532375 TI - Coronary neovascularization of embryonic rat hearts cultured in oculo is independent of thyroid hormones. AB - We tested the hypothesis that neovascularization of embryonic hearts is independent of thyroid hormones. To dissociate the effects of hemodynamic load from the direct influence of thyroid hormones on coronary neovascularization, we grafted avascular, gestational day 12 rat hearts to the anterior eye chamber of adult rats receiving daily injections of thyroxine (T4; 0.25 mg/kg sc), propylthiouracil (PTU; 10 mg/kg ip), or saline (control). After 21 days, grafts were perfuse fixed and prepared for transmission electron microscopy. Neither T4 nor PTU treatment affected size or mass of the unloaded graft, or the volume densities of vessels, myocytes, or the extracellular matrix, which were determined by standard point-counting techniques. Thus neither exogenous administration of T4 nor the induction of hypothyroidism in host rats produced a significant effect on the growth or vascularity of the unloaded hearts. We conclude that vascular growth in embryonic rat hearts cultured in oculo is independent of thyroid hormones and that the angiogenic effect of thyroid hormones in loaded hearts is a consequence of increased work-related events. PMID- 7532374 TI - Protamine inhibits coronary collateral development in a canine model of repetitive coronary occlusion. AB - Protamine has been demonstrated to inhibit angiogenesis in vitro and in vivo; however, its effect on coronary collateral development has not been examined. The present investigation tested the hypothesis that subcutaneously administered protamine inhibits canine coronary collateral development in response to chronic myocardial ischemia. Dogs underwent daily, repetitive, 2-min, left anterior descending coronary artery (LAD) occlusions for 22 consecutive days. Regional myocardial blood flow (radioactive microspheres), LAD segment shortening, and coronary flow debt repayment were measured in saline-treated (n = 7) and protamine-treated (n = 6) dogs on days 1, 8, 15, and 22. Coronary collateral development in saline-treated dogs was demonstrated by time-dependent significant (P < 0.05) increases in collateral blood flow to ischemic myocardium [day 1 0.10 +/- 0.01 vs. day 22 0.88 +/- 0.05 (SE) ml.min-1.g-1], progressive normalization of myocardial contractile function during LAD occlusion, and successive reduction in flow debt repayment. In contrast, protamine treatment significantly attenuated, increases in collateral perfusion (day 1 0.13 +/- 0.02 vs. day 22 0.36 +/- 0.03 ml.min-1.g-1). Regional contractile dysfunction and postocclusive reactive hyperemic responses were sustained over time in protamine-treated compared with saline-treated dogs. The results demonstrate that protamine inhibits coronary collateral development in response to chronic myocardial ischemia. PMID- 7532376 TI - Effects of hypertonic saline and dextran 70 on cardiac functions after burns. AB - The effects of hypertonic saline-dextran (HSD) on cardiac contractility and hemodynamics after burns were studied in anesthetized animals with full-thickness 50% total body surface area burns that were resuscitated with HSD or lactated Ringer solution (LR) alone. No significant difference in cardiac contractility during 6 h postburn was observed between the two groups, as assessed by the end systolic pressure-volume relationship and the stroke work-end-diastolic volume relationship. An additional bolus of HSD at 6 h postburn caused no significant changes in the end-systolic pressure-volume relationship and stroke work-end diastolic volume relationship in the burned and sham-burned animals, both of which were resuscitated with HSD. Ten minutes of hemodynamic changes following HSD infusion at 30 min postburn revealed a sudden increase in stroke volume with biphasic responses in left ventricular systolic pressure, which first decreased, then increased, and finally returned to the pre-HSD value. End-diastolic volume was maintained at approximately 110% of the pre-HSD value during this period. We concluded that HSD does not enhance cardiac contractility after severe burns but does produce direct effects on postburn circulation to reduce afterload and augment preload, resulting in a short-lived increase in cardiac output. PMID- 7532377 TI - Plasma proteins as biomarkers of the aging process. AB - This study was designed to characterize the rat serum proteins as biomarkers of the normal aging process. Crossed immunoelectrophoresis or electroimmunodiffusion quantitation of proteins was performed in rats aged 6, 12, 24, and 30 mo. Selection of healthy animals was based on confrontation of crossed immunoelectrophoresis patterns with those of experimentally inflamed young adults and with individual anatomopathological data. Convergence of inflammatory patterns and severe histological lesions was the exclusion criterion. Senescence induced decrease was demonstrated for eight proteins [negative senescence reactants (SRs-)] and increase for six proteins [positive SRs (SRs+)]. Most SRs belonged to the class of proteins responsive to acute inflammation [acute phase reactants (APRs)]. One SR+, the thyroxine-binding globulin, a high-affinity thyroid hormone binder, emerged as a particularly reliable senescence biomarker, showing the highest aging-related variation (8-fold increase from 6 to 30 mo) and not belonging to the APR class. Chronic treatment with perindopril, an angiotensin I-converting enzyme inhibitor used in heart and renal disease therapy, significantly enhanced thyroxine-binding capacity, possibly by preventing age-related alterations of serum lipids. Serum protein patterns prove valuable both as indexes for selecting aging animals free from superimposed pathologies and as parameters of senescence-induced changes in protein biosynthesis. PMID- 7532378 TI - Hyalinizing spindle and epithelioid cell nevus. A study of five cases of a distinctive histologic variant of Spitz's nevus. AB - Five cases are described of a distinctive histologic variant of benign spindle and epithelioid cell nevus characterized by extensive and prominent stromal hyalinization. The lesions consisted of a proliferation of spindle or epithelioid nevocytes scattered singly or in small clusters in the dermis and surrounded by abundant paucicellular hyalinized or collagenous stroma. Three patients were men and two were women. Their age range was 23 to 45 years (mean, 32). Two of the lesions were located in the head and neck region, two in the lower extremities, and one in the trunk. Immunohistochemical strains showed positive staining of the spindle or epithelioid cells with S-100 protein and vimentin; stains for keratin, EMA, CEA, actin, and desmin were all negative. Van Gieson and trichrome histochemical reaction demonstrated the collagenous nature of the hyalinized intercellular matrix; Congo red, crystal violet, and alcian blue stains were all negative. The etiology and pathogenesis of the intercellular hyalin deposits are unknown, but they probably represent a regressive phenomenon in longstanding or involuting lesions. Hyalinizing Spitz nevus must be included in the differential diagnosis of cutaneous lesions exhibiting a prominent hyalinized stroma and must be differentiated from other dermal neoplasms, particularly cutaneous metastases from occult internal malignancies and malignant melanoma. PMID- 7532379 TI - Nail matrix hypergranulosis. AB - Nail pathology shares some common features with skin pathology, but it also has its own peculiar aspects. The anatomical and physiological characteristics of the nail unit probably play a major role in determining these pathological differences. Although the presence of keratohyaline granules is a normal feature of the skin, there is no granular layer in the normal nail matrix. As a consequence, nail matrix hypergranulosis should be considered a separate entity from skin hypergranulosis. In our review of 150 longitudinal nail biopsy specimens, keratohyaline granules were seen in the nail matrix of 24 cases of lichen planus, 29 cases of spongiotic trachyonychia, 10 cases of psoriasis, and three cases of Hallopeau acrodermatitis. In all cases, the presence of keratohyaline granules was associated with the absence of the normal keratogenous zone. Similar nail matrix features were detectable in three cases of malignant melanoma, two cases of primary systemic amyloidosis, and one case of histiocytoid hemangioma compressing the nail matrix. Our data suggest that inflammatory and compressive insults to the nail matrix cause both disappearance of the keratogenous zone and matrix keratinization with the formation of keratohyaline granules. Skin hypergranulosis reflects a hyperplasia of a normal skin component. In the nail matrix, however, hypergranulosis represents the appearance of structures not normally present. Nail matrix hypergranulosis should be considered a pattern of nail matrix reaction to different inflammatory insults. It is therefore more analogous to epidermal parakeratosis than to epidermal hypergranulosis. PMID- 7532380 TI - Juvenile hyaline fibromatosis. A case report of a localized form? AB - Juvenile hyaline fibromatosis is a rare soft-tissue, tumor-like condition that usually arises in children. We report a case characterized by localized lesions and very slow progression. We postulate the existence of two distinct forms of juvenile hyaline fibromatosis--a localized form with very slow growth and a diffuse form with large and rapidly growing tumors. PMID- 7532381 TI - A method to isolate RNA from gram-positive bacteria and mycobacteria. PMID- 7532382 TI - Discharge instructions: do illustrations help our patients understand them? AB - STUDY OBJECTIVE: To determine whether the addition of illustrations to discharge instructions improves patient comprehension. DESIGN: Randomized, blinded, prospective study. A blinded investigator asked a series of questions designed to test the participant's comprehension of the discharge instructions. There were 10 possible correct responses. SETTING: Emergency department of a rural Level I trauma center. PARTICIPANTS: Convenience sample of 101 patients discharged with the diagnosis of laceration. INTERVENTIONS: Patients were randomly assigned to receive discharge instructions with (n = 54) or without (n = 47) illustrations. RESULTS: The median number of correct responses was five. Patients with illustrations were 1.5 times more likely to choose five or more correct responses than those without illustrations (65% versus 43%; P = .033). The effect of illustrations varied by demographic group. Among nonwhites (n = 51), patients with illustrations were more than twice as likely to choose five or more correct responses (P = .032). Among patients with no more than a high school education (n = 71), patients with illustrations were 1.8 times more likely to choose five or more correct responses (P = .038). Among women (n = 48), patients with illustrations were 1.7 times more likely to chose five or more correct responses (P = .006). CONCLUSION: The addition of illustrations to discharge instructions for patients who have sustained lacerations improves patient comprehension. There is a large effect among patients who are nonwhite, female, or have no more than a high school education. PMID- 7532383 TI - [Sexuality and genito-urinary cancers]. AB - In mythological terms, cancer symbolizes death and suffering, and sexuality, on the other hand, symbolizes life and pleasure. This diagnosis whether explicit or not, calls for two-fold reflection. Firstly the individual is faced by the inexorable approach of death and is called to bury his fantasies of immortality. Secondly, it also constitutes a threat to the body-image, and this may have profound repercussions on sexuality. The author investigates the impact of genito urinary cancers and also cancers of the breast, which are included on the basis of its symbolic nature, on the patient's personal life: conjugal dynamism, socio professional life and, in particular, physiological changes of sexual function. PMID- 7532385 TI - Antioxidative roles of metallothionein and manganese superoxide dismutase induced by tumor necrosis factor-alpha and interleukin-6. AB - Antioxidative roles of metallothionein (MT) and manganese superoxide dismutase (Mn-SOD) induced by tumor necrosis factor (TNF) and interleukin 6 (IL-6) have been studied. Since pretreatment of rat with dexamethasone, an inhibitor of cytokine production, prevented MT synthesis induced by paraquat which is a typical superoxide generator, MT synthesis by oxidative stress may be, at least partly, mediated through cytokines. Pretreatment of rat with TNF or IL-6 prevented liver damage and lipid peroxidation caused by carbon tetrachloride. Administration of TNF increased activity of mitochondrial Mn-SOD and concentrations of cytoplasmic MT, but not activities of glutathione peroxidases and Cu,Zn-SOD in the liver. The increment of the Mn-SOD activity and MT was due to the de novo protein synthesis, because gene expression of mRNAs of Mn-SOD and MT in the liver was also induced by TNF and IL-6. These data strongly suggest that MT and Mn-SOD in the liver cooperatively play antioxidative roles. Pretreatment with TNF, however, did not affect the increased levels of plasma fibrinogen and liver MT induced by the following paraquat treatment, although it did prevent lipid peroxidation in the liver. The data suggest that MT is not directly induced by oxygen free radicals. Cytokines may be released by paraquat in tissues other than the liver and induce hepatic synthesis of acute phase proteins including fibrinogen, MT, and Mn-SOD. MT and Mn-SOD induced by cytokines in the liver exert an antioxidative role during acute phase response, therefore preventing tissues from injury by oxidative stress. PMID- 7532384 TI - Regulation of hepatic nitric oxide synthase by reactive oxygen intermediates and glutathione. AB - Regulation of induced nitric oxide synthase in rat hepatocyte primary cultures was explored. Nitric oxide synthase (NOS) induction by tumor necrosis factor alpha (TNF alpha) is synergized by interferon-gamma, and both NOS activity and gene expression are maximal by 10 h and maintained through 24 h. Glutathione depletion by diethylmaleate, which conjugates reduced glutathione, 1,3 bis(chloroethyl)-1-nitrosourea (BCNU), a glutathione reductase inhibitor, or buthionine sulfoxamine, a glutathione synthesis inhibitor, abolishes or reduces NOS induction in TNF alpha-treated hepatocytes, whereas N-acetylcysteine has little effect. Thus, reduced glutathione is critical to NOS mRNA induction and activity in TNF alpha-treated hepatocytes. NOS induction in TNF alpha-treated cells is reduced by rotenone, a mitochondrial complex 1 inhibitor. Concurrent treatment with TNF alpha and the antioxidant, Trolox, or the iron-chelating agent, desferrioxamine, also reduces NOS activity. Dithiothreitol, a thiol antioxidant, reduced TNF alpha induction of NOS. Trolox and BCNU, combined, blocked TNF alpha stimulation of NOS greater than either agent alone. These results suggest that TNF alpha increases mitochondrial production of reactive oxygen intermediates (ROI), which contributes to NOS induction. Hepatocytes exposed to extracellular ROI generation through a xanthine/xanthine oxidase superoxide-generating system expressed increased NOS activity and mRNA levels. NOS induction by superoxide also requires reduced glutathione since diethylmaleate blocks induction by xanthine/xanthine oxidase while N acetylcysteine elevates NOS expression. Thus, the generation of ROI by cytokines or other physiological processes stimulates the induction of NOS and this process is regulated by cellular levels of reduced glutathione. PMID- 7532386 TI - Peyssonols A and B, two novel inhibitors of the reverse transcriptases of human immunodeficiency virus types 1 and 2. AB - Two new sesquiterpene hydroquinones, peyssonol A and peyssonol B, of the Red Sea algae Peyssonelia sp., have been shown to be potent inhibitors of the RNA directed DNA synthesis of the reverse transcriptases (RTs) of human immunodeficiency virus (HIV)-1 and HIV-2. The DNA-dependent DNA polymerase activity is inhibited to a lesser extent, whereas the RNase H activity is unaffected. The inhibition of the DNA polymerase activities is independent of the nature of the template primers used. Peyssonol A probably binds the RT at a site distinct from those occupied by the substrates of the RNA-directed DNA synthesis, since the mode of inhibition is noncompetitive with respect to both dNTP's and template primer. This is partially true for peyssonol B, which is noncompetitive with respect to only dNTP, but is competitive with respect to the template primer. We have speculated that, since peyssonol B and the template primer bear no apparent structural resemblance, the competitive pattern of inhibition can be explained by an indirect steric hindrance or by the overlap of the inhibitor and the substrate distinct binding sites of the enzyme. Alternatively, the binding of the inhibitor to a distinct site induces conformational changes that distort the binding of the template primer. Furthermore, we have shown that both peyssonol A and peyssonol B interfere with the direct binding of the RT to the template primer, offering an explanation for the mechanism of the enzyme inhibition. The insensitivity of DNA polymerase beta and the poor response of DNA polymerase alpha to peyssonol A make this inhibitor more attractive for the future development of a potent anti-HIV RT drug. PMID- 7532387 TI - p-nitrosophenol reduction by liver cytosol from ADH-positive and -negative deermice (Peromyscus maniculatus). AB - Liver cytosolic fractions are known to catalyze the reduction of certain C nitroso compounds to their corresponding hydroxylamines and amines. Alcohol dehydrogenase (ADH), NAD(P)H:quinone oxidoreductase, and xanthine and aldehyde oxidases have been implicated as C-nitroso reductases. To probe the role of these cytosolic enzymes in the reduction of C-nitroso compounds we have studied the effects of classical inhibitors of these enzymes on the ability of liver cytosolic fractions from ADH+ and ADH- deermice to reduce p-nitrosophenol to p aminophenol. Pyrazole, a potent inhibitor of ADH, inhibited NADH-p-nitrosophenol reduction by ADH+ cytosol by > 85%. Thus, ADH contributes substantially to NADH-C nitroso reduction by cytosol from ADH+ deermice. The NAD(P)H:quinone oxidoreductase inhibitor, dicumarol, inhibited NADH-dependent p-aminophenol formation by about 25%; however, dicumarol potently inhibited the NADPH-dependent formation (90-95%). As expected, cytosol from ADH- deermice did not catalyze pyrazole-sensitive (ADH-dependent) C-nitroso reduction with NADH as the cofactor. Both NADPH- and NADH-p-nitrosophenol reduction by ADH- cytosol were inhibited > 90% by dicumarol. The xanthine oxidase/aldehyde oxidase inhibitor, allopurinol, was without effect on NAD(P)H cytosolic p-nitrosophenol reduction from ADH- and ADH+ deermice under either aerobic or anaerobic conditions. Our findings suggest that in the ADH+ animal, ADH contributes significantly to NADH-dependent C nitroso reduction by cytosol relative to NAD(P)H:quinone oxidoreductase. NADPH dependent p-nitrosophenol reduction by liver cytosol of ADH+ animals is mostly dicumarol-sensitive, which implicates NAD(P)H:quinone oxidoreductase as the major NADPH-dependent activity. In ADH- deermice, both NADH- and NADPH-dependent p nitrosophenol reduction are essentially dicumarol-sensitive (NAD(P)H:quinone oxidoreductase-dependent). Because the toxic expression of C-nitroso compounds is mediated by hydroxylamine intermediates, the present data indicate the importance of considering the role of ADH in the toxic sequelae of nitro and nitroso arenes. PMID- 7532388 TI - Distributional changes of Langerhans cells in human skin during irritant contact dermatitis. AB - We used light and electron microscopic immunocytochemistry to study distributional changes in the human Langerhans cell (LC) system during the first 14 days of a mild irritancy caused by sodium lauryl sulphate (SLS). A marked initial decrease in epidermal LC was noted possibly resulting from migration from the epidermis to the dermis and from irreversible cell damage. Several studies have previously found an unchanged number of LC in SLS-induced contact irritant dermatitis, but these studies may not have taken into account the fact that SLS is effectively absorbed from the test chamber. Unless certain precautions are taken the SLS concentration rapidly falls to topical levels that have no effect on the LC system. Simultaneously with the decrease in the epidermis we observed an increase in dermal CD1a+ cells, confirming an often reported finding. There is, however, no consensus as to the identity of these cells, and several authors have reported that such cells lack LC granules and thus these cells have often been classed as indeterminate cells. We found that, during irritant contact dermatitis, provided an adequate number of sections were scrutinized in the electron microscope, all dermal CD1+ cells contained Birbeck granules. PMID- 7532390 TI - Can fibroblast growth factors substitute for bovine pituitary extracts in culture systems for hair apparatus cells? AB - The effects of acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) on proliferation and differentiation of cultured hair apparatus cells were examined using bovine pituitary extract (BPE)-free media. The importance of FGFs as growth factors in BPE was also examined. Both FGFs and BPE stimulated cell proliferation and induced cell differentiation into several subpopulations corresponding to those of in vivo cell layers of the hair apparatus. Heparin enhanced the mitogenic activity of aFGF, but it did no affect that of bFGF. Heparin also partially inhibited or postponed the occurrence of cell differentiation induced by aFGF. Both aFGF- and bFGF-like proteins were detected in BPE by Western immunoblotting. BPE treated with anti-aFGF and/or anti bFGF antibodies retained a significant mitogenic activity, although the mitogenic activities of FGFs were inhibited by their own specific antibodies. These results suggest that growth factors other than FGFs for cultured hair apparatus cells exist in BPE, although FGFs might substitute for BPE in this culture system as they stimulated cell proliferation and induced cell differentiation. PMID- 7532389 TI - Repeated tape stripping of normal skin: a histological assessment and comparison with events seen in psoriasis. AB - The aim of the present study was to investigate the response of normal human skin to repeated courses of Sellotape stripping. The skin of healthy volunteers was stripped five times at 24-h intervals. Skin biopsies were taken before stripping (day 0) and on days 2, 4, 7 and 10. The responses were studied using H & E staining and an immunohistochemical analysis of several aspects of epidermal proliferation and keratinization. Although increased proliferation (nuclear binding to Ki-67 binding), acanthosis and parakeratosis were observed, the overall histological picture did not resemble psoriatic histology completely: no micropustules of Kogoj and no thinning of the suprapapillary plate were observed. Involucrin staining followed the recruitment of cycling epidermal cells showing a statistically significant elevation of positive cell layers from day 2 onwards. Filaggrin expression showed an increase from day 2 onwards, which was statistically significant on day 7 and day 10. Using the anti-keratin antibodies KS8.12 (K13 and K16) and RKSE60 (K10) we observed a fast induction of K13/K16 expression, while the staining of keratin 10 showed the same overall intensity at different time intervals. In conclusion, the response to repeated courses of tape stripping provides an adequate model for studies on epidermal proliferation, hypergranulosis and hyperkeratosis. This approach causes a more prolonged induction of these phenomena than a single course of stripping. In contrast to the situation following a single course of stripping, repeated tape stripping induced the expression of filagrin. Therefore the repeated tape stripping model is less compatible with psoriasis than a single course of stripping. PMID- 7532391 TI - [Inverted papilloma of the prostatic urethra. Histopathogenetic considerations]. AB - We present a rare case of inverted papilloma of the prostatic ureter whose presenting features were gross hematuria and variable dysuria. Endoscopy revealed a polypoid appearance with a fine pedicle arising from the prostatic urethra. Treatment was by transurethral resection of the neoformation. The diagnosis was made on the histopathological findings. The different histopathogenic considerations published in the literature are discussed. PMID- 7532392 TI - New diagnostic and treatment guidelines for benign prostatic hyperplasia. Potential impact in the United States. AB - BACKGROUND: The Agency for Health Care Policy and Research (AHCPR) recently released the clinical practice guidelines for the diagnosis and treatment of benign prostatic hyperplasia. Prevalence estimates from a population-based cross sectional study, the baseline component of a cohort study of the natural history of prostatism, were used to assess their potential impact in the United States. METHODS: The study group comprised a population-based sample of white men aged 50 to 79 years who were randomly selected within age- and residence-specific strata from the Olmsted County, Minnesota, population (1990 census, 105,720). These 1317 men completed symptom assessments and diagnostic evaluations that paralleled the AHCPR guidelines, including the measurement of urinary flow rates and, for a subset (n = 303), ultrasonic determination of postvoiding residual urine volume. RESULTS: The application of the AHCPR benign prostatic hyperplasia diagnostic guidelines to the study cohort (American Urologic Association Symptom Index > 7 and peak urinary flow rate < 15 mL/s) suggests that 17% of men aged 50 to 59 years, 27% of men aged 60 to 69 years, and 35% of men aged 70 to 9 years are eligible to discuss treatment options. Application of these percentages to the 1990 US white population suggests that approximately 5.6 million men aged 50 to 79 years are eligible to discuss treatment options. This number will double by the year 2020 owing to the aging of the population. CONCLUSION: The projected number of men potentially meeting AHCPR guidelines to discuss treatment options for benign prostatic hyperplasia could have a substantial impact on the health care system; this will be compounded by the aging of the population. PMID- 7532394 TI - The impact of the macular photocoagulation study results on the treatment of exudative age-related macular degeneration. AB - OBJECTIVE: To determine the percent of cases with all primary forms of exudative age-related macular degeneration that are eligible for treatment by the Macular Photocoagulation Study (MPS) guidelines in a retina clinic serving both as a primary care center and as a referral center. DESIGN: Fluorescein angiograms of patients with age-related macular degeneration examined at the Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer, Israel, during a 5-year period (1985 to 1990) were randomly selected and reviewed. Angiograms showing all forms of exudative age-related macular degeneration were eligible. The first 100 eligible angiograms identified constituted the study series and were classified into four groups: active choroidal neovascularization (CNV), pigment epithelium detachment, hemorrhage, and disciform scars. The active CNVs were further divided into three subgroups: classic (well defined), occult (poorly defined), and combined. Eligibility for laser treatment was determined exclusively with use of the published MPS recommendations for treatment of extrafoveal, juxtafoveal, and subfoveal membranes. RESULTS: There were 10 cases in the pigment epithelium detachment group, 11 cases in the hemorrhagic group, and 16 cases with disciform scars. All of these 37 cases were ineligible for treatment by MPS guidelines. Sixty-three eyes had active membranes; 37 were classic CNVs (eight extrafoveal, seven juxtafoveal, and 22 subfoveal). All extrafoveal and juxtafoveal CNVs were found to be eligible for laser treatment. Eleven membranes of the subfoveal group were larger than 2 disc areas, a size for which the MPS did not demonstrate benefit from laser treatment. There were 19 membranes in the strictly occult CNV subgroup, all of them untreatable by MPS criteria. Seven cases had both occult and classic CNV, and all were larger than 3.5 disc areas and therefore ineligible for treatment. Overall, 26 cases were eligible for treatment by strict MPS criteria; these constitute 26% of the whole series and 41% of the active CNV cases in the series. CONCLUSIONS: Our results indicate that the MPS guidelines for laser treatment are applicable only to a minority of the cases with exudative age-related macular degeneration presenting to our clinic. Further studies should be conducted to identify additional treatment modalities for this common eye disease. PMID- 7532393 TI - Treatment of subfoveal choroidal neovascularization. PMID- 7532396 TI - Patient education. Your enlarged prostate. PMID- 7532395 TI - The influence of treatment extent on the visual acuity of eyes treated with Krypton laser for juxtafoveal choroidal neovascularization. Macular Photocoagulation Study Group. AB - PURPOSE: To examine the direct relationship between the extent of treatment with krypton red laser photocoagulation and visual acuity loss in eyes with choroidal neovascularization secondary to ocular histoplasmosis or age-related macular degeneration. PATIENTS AND METHODS: Photographic and visual acuity records from 129 eyes treated in the Ocular Histoplasmosis Study--Krypton Laser and 224 eyes treated in the Age-Related Macular Degeneration Study--Krypton Laser were reviewed. The proportion of eyes with severe visual acuity loss (6 or more lines of loss) was examined for subgroups of eyes based on the distance of the neovascular lesion from the center of the foveal avascular zone and on the extent of laser treatment to the lesion. Differences in the proportions with severe visual acuity loss were evaluated by longitudinal data analysis methods. RESULTS: Among eyes in the Ocular Histoplasmosis Study--Krypton Laser with lesions less than 200 microns from the center of the foveal avascular zone, only 5% of eyes with laser treatment that covered the foveal side and had a narrow (< or = 100 microns) border of treatment to adjacent uninvolved retina experienced severe visual acuity loss compared with approximately 25% of eyes with either some of the foveal side of the lesion left untreated or a wide border of treatment on the foveal side. Treatment extent had little influence on severe visual acuity loss in eyes in the Ocular Histoplasmosis Study--Krypton Laser with neovascular lesions 200 to 500 microns from the center of the foveal avascular zone or in eyes in the Age-Related Macular Degeneration Study--Krypton Laser with lesions in either distance category. CONCLUSIONS: Accurate, complete treatment of choroidal neovascularization close to the foveal center is required to provide the patient with the best chance of avoiding further severe visual acuity loss, especially in patients with ocular histoplasmosis. Even among experienced retinal specialists, the required accuracy of treatment is difficult to achieve. PMID- 7532397 TI - Structural domains of interleukin-2 receptor beta critical for signal transduction: kinase association and nuclear complex-formation. AB - The structural domains of interleukin-2 receptor beta (IL-2R beta) were examined, characterizing the protein domains, associated phosphoproteins and nuclear complexes of IL-2-induced signal transduction. A series of IL-2R beta cytoplasmic deletion mutants were constructed and transfected into a murine pre-B-cell line, Ba/F3. The proliferative response of characterized clones was determined. A minimal linear cytoplasmic sequence required for proliferation and a sequence motif (PQPLXP) needed along with Box1-Box2 for IL-2-induced proliferation were identified. Anti-phosphotyrosine Western-blot analysis of a stimulated biologically active clone showed several IL-2-induced tyrosylphosphorylated proteins with molecular masses ranging from 45 to 116 kDa. In vitro kinase studies of biologically active clone-receptor complexes showed a 116 kDa protein (p116) to be the major tyrosine-phosphorylated component. The presence of the p116 kinase in the receptor complex correlates with IL-2-induced proliferation. An IL-2-inducible p116 kinase has recently been characterized as a Jak kinase family member and named Jak3. Nuclear complexes were formed with the GRR oligomer only when the IL-2R beta mutant supported proliferation. This led us to conclude that Box1-Box2 and PQPLXP motifs associate with Jak3 and that this association is an essential element in the IL-2 signal-transduction pathway culminating in the formation of a nuclear complex. PMID- 7532399 TI - Oxidized low-density lipoprotein induces the expression of P-selectin (GMP140/PADGEM/CD62) on human endothelial cells. AB - It is now well established that monocytes adhere to endothelial cells activated by oxidized low-density lipoproteins (LDL). However, the adhesive receptors on endothelial cells involved in binding monocytes, following an insult by oxidized LDL, remains to be elucidated. In this study we have looked at the effect of native or oxidized LDL on the expression of P-selectin. Native LDL (N-LDL) was oxidized by incubation with either endothelial cells (EC-LDL) or copper (Cu-LDL), or in culture medium as a control (C-LDL). Expression of P-selectin was assayed with an anti-P-selectin (CD62) monoclonal antibody (LYP20). Results show that EC LDL and Cu-LDL, but not N-LDL or C-LDL, induce the expression of P-selectin by human umbilical-vein endothelial cells (HUVECs). Induction of P-selectin by low concentrations (20 micrograms/ml) of LDL is directly related to the state of oxidation of the LDL particles. In addition, high concentrations (100 micrograms/ml) of N-LDL also activate HUVECs by inducing P-selectin expression. This expression was sustained for a period of over 1 h on LDL-activated endothelial cells, in contrast with thrombin- or histamine-activated endothelial cells, whose P-selectin levels fall within 15-20 min after induction. E-selectin, in contrast with P-selectin, could not be induced by endothelial cells treated with low or high concentrations of oxidized LDL. Results in this study show that P-selectin expressed by oxidized-LDL-treated endothelial cells are involved in mediating the adhesion of a monocytic cell line (U937) or monocytes in peripheral blood mononuclear cells. An anti-P-selectin monoclonal antibody (LYP20) inhibited the binding of U937 cells and monocytes. These results strongly suggest that P selectin is involved in the early stages of atherogenesis. PMID- 7532398 TI - Regulation of purified hepatic PC-1 (phosphodiesterase-I/nucleotide pyrophosphatase) by threonine auto(de)phosphorylation and by binding of acidic fibroblast growth factor. AB - The plasma cell differentiation antigen PC-1 was purified to homogeneity from rat liver membranes. Denaturing electrophoresis revealed polypeptides of 118 and 128 kDa, which were both recognized by antibodies against recombinant murine PC-1. During gel filtration PC-1 migrated as a protein of about 500 kDa, suggesting a tetrameric structure. Purified PC-1 displayed a phosphodiesterase-I/nucleotide pyrophosphatase activity that could be completely blocked by EDTA, dithiothreitol and acidic fibroblast growth factor (extrapolated Ki = 1.3 nM). Purified PC-1 was also capable of threonine autophosphorylation and of phosphorylation of histone IIa. The autophosphorylation of PC-1 was inhibited by addition of histone IIa, and it was blocked by phosphodiesterase-I inhibitors (acidic fibroblast growth factor, dithiothreitol), by nucleotides (ATP, ADP, AMP), and by vanadate. When added to autophosphorylated PC-1, these compounds caused a prompt dephosphorylation. However, the same agents did not affect the (de)phosphorylation of histone IIa, which is not a substrate for the PC-1 phosphatase. These data indicate that phosphodiesterase-I inhibitors, nucleotides and vanadate affect the (de)phosphorylation of PC-1 by stimulating the PC-1 phosphatase and/or by shielding the autophosphorylation site from the PC-1 kinase. The rate of dephosphorylation of PC-1 was independent of the dilution, suggesting an autocatalytic intramolecular process. We propose that the autophosphorylation of PC-1 serves to block its nucleotide pyrophosphatase activity when extracellular ATP becomes scarce. PMID- 7532400 TI - Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi. AB - Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps. PMID- 7532403 TI - Replacement of conserved threonines by alanine residues in high mobility group protein HMG-I(Y): effect on DNA binding affinity. AB - A threonine residue at the beginning of each DNA-binding domain of HMG-I (residue numbers 21, 53, and 78) is conserved among mammalian species and proposed to help stabilize the A.T-hook DNA-binding motif. Phosphorylation of threonines number 53 and 78 of human HMG-I(Y) both in vivo and in vitro leads to a 20 fold reduction in the proteins DNA binding affinity. Recombinant human HMG-I proteins were engineered to contain alanine instead of the conserved threonine in each DNA binding domain. The DNA dissociation constant of each protein was assayed at various salt concentrations by competition with the fluorescent dye Hoechst 33258 for an AT-rich DNA substrate. Replacement of these threonines did not affect the equilibrium binding of these proteins to DNA as compared with wild-type HMG-I and HMG-Y. Molecular modelling of analogous peptides supported this finding. We conclude that these threonines are not directly important for A.T-hook DNA binding and are conserved phosphorylation sites for down regulation of DNA binding by the A.T-hook motif in the HMG-I(Y) proteins. PMID- 7532402 TI - Specific aspartic acid-rich sequences are responsible for silver staining of nucleolar proteins. AB - Ag-NOR proteins are silver-stainable proteins in the nucleolar organizer regions and are used to distinguish benign from malignant tumors. B23 and C23 are the two major Ag-NOR proteins. This study shows that only one of the two acidic clusters of B23 is responsible for its silver staining property. Fusion of this region of B23 (amino acids 161-188) to glutathione S-transferase produced an Ag-NOR positive fusion protein. The same result was obtained when amino acids 233-277 of C23 was fused with glutathione S-transferase. The aspartate residues, but not the glutamate residues, were found to be primarily responsible for the silver staining of the acidic clusters. PMID- 7532401 TI - Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in N1E-115 neuroblastoma cells. AB - Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacological properties defining the Ang II receptor subtype 2 (AT2): high affinity for 125I-labelled AT2 selective agonist CGP 42112 (Kd 91 +/- 19 pM); expected rank order of potency (CGP 42112 = (Sar1,Ile8)Ang II > or = Ang II > PD 123319 >> DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagent dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecular cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals nucleotide sequence identity with the AT2 subtype expressed in fetal tissue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E-115 cells. Taken together, these data reveal the exclusive presence of the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cells with Ang II leads to a marked decrease in the level of tyrosine phosphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-specific agonist CGP 42112. The response to both effectors is rapid and transient, showing a maximum between 5 and 10 min, and returning to basal levels after 20-30 min. In both cases, tyrosine dephosphorylation can be prevented by co-incubation with an excess of the antagonist Sarile. These data thus establish that AT2 receptor activation leads to protein tyrosine dephosphorylation in N1E-115 cells, and support a possible role for AT2 receptors in the negative regulation of cell proliferation. PMID- 7532404 TI - Monoclonal anti-FLAG antibodies react with a new isoform of rat Mg2+ dependent protein phosphatase beta. AB - The FLAG peptide has been widely used as a multi-purpose tag for the identification and detection of recombinant FLAG fusion proteins. The practicability of this approach depends on specific detection of FLAG fusion proteins with no or very little cross-reactivity to cellular proteins. We have isolated a rat cDNA clone coding for a new splicing isoform of Mg2+ dependent protein phosphatase beta (MPP beta) by screening a rat brain expression library with monoclonal antibody Anti-FLAG M2. MPP beta reacts strongly both as a MPP beta-beta-galactosidase- and as a glutathione S-transferase fusion protein with anti-FLAG M2 antibodies. Sequence analysis of MPP beta revealed a sequence motif with five out of eight amino acid residues identical to the FLAG peptide hitherto believed to be mono-specific. PMID- 7532405 TI - Sensitive assay for detection of hepatocellular carcinoma associated gene transcription (alpha-fetoprotein mRNA) in blood. AB - The sensitivity of RT-PCR and nested RT-PCR for detection of circulating hepatocellular carcinoma was assessed by demonstrating the tumor cell-associated gene transcription, alpha-fetoprotein mRNA, in the nuclear cells of peripheral blood. When HepG2 cells were mixed in blood, 100-1000 tumor cells/5ml of blood could be detected by RT-PCR, in contrast to 1-10 tumor cells/5ml of blood by nested RT-PCR. In addition, 2 x 10(4) copies of AFP mRNA were found in one HepG2 cell when analyzed by the quantitative nested RT-PCR assay. Thus, the nested RT PCR assay could provide a useful tool for detecting a tiny amount of circulating tumor cells in patients with hepatocellular carcinoma presenting extra-hepatic metastasis. PMID- 7532406 TI - Effects of 2,5-di(tert-butyl)-1,4-hydroquinone, an endoplasmic reticulum Ca(2+) ATPase inhibitor, on agonist-stimulated phasic myometrial contractions. AB - Phasic myometrial contractions utilize mechanisms involving the cycling of calcium into and out of intracellular calcium stores. These studies were performed to determine the effects of 2,5-di(tert-butyl)-1,4-hydroquinone (tBHQ), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on in vitro isometric myometrial contractions. These studies demonstrated that low concentrations of tBHQ (eg. 10 microM) appear to inhibit intracellular calcium cycling, whereas higher concentrations also inhibit extracellular calcium influx. These combined tBHQ effects markedly suppressed myometrial contractions stimulated in response to various agonists including oxytocin, PGF2 alpha, KCl, ionomycin, and Bay K 8644. PMID- 7532407 TI - HIV-1 expression induced by anti-cancer agents in latently HIV-1-infected ACH2 cells. AB - The expression of human immunodeficiency virus type 1 (HIV-1) in infected cells is induced (or enhanced) by a number of agents including phorbol myristate acetate (PMA), phytohemagglutinin (PHA), certain infectious agents, certain cytokines, and ultraviolet light. ACH2 cells represent latently HIV-1-infected T cells, which produce only a low level of HIV-1 in vitro. We found that various anti-cancer agents including 5-azacytidine (5-AZC), 5-fluorouracil (5-FU), methotrexate, cytosine arabinoside, and vinblastine potentiated the expression of HIV-1 in ACH2 cells. There was no evidence of altered DNA methylation patterns in ACH2 cells cultured with 5-FU unlike with 5-AZC. The NF-kappa B binding activity was found to be enhanced in ACH2 cells exposed to 5-FU (but not in those exposed to 5-AZC) as assessed by the mobility shift assay using an oligonucleotide containing two NF-kappa B binding sites. These data suggest that the use of certain anti-cancer agents may induce (or enhance) the expression of HIV-1. PMID- 7532408 TI - Characterization of the 5' region of the CD10/neutral endopeptidase 24.11 gene. AB - We have characterized the 5' region of the CALLA/CD10 gene which has been shown to be identical to the membrane-associated enzyme neutral endopeptidase 24.11 (NEP). There is no CAAT or TATA box in the 5' flanking region, upstream of exon 1, but a GC rich region with several Sp1 binding sites. We have detected several putative initiation transcription sites by primer extension and by nuclease S1 analysis. Moreover by reverse transcriptase-polymerase chain reaction, we demonstrated the existence of a new exon: exon 1bis. This exon can be alternatively spliced as has already been described for exon 1 and exon 2. PMID- 7532410 TI - Quantitative computerized analysis of silver-stained and Coomassie Blue-stained two-dimensional protein maps. AB - Using the two-dimensional gel electrophoresis and two systems for computer analysis of the resulting maps, we obtained a good quantitative reproducibility of individual spots of silver-stained and Coomassie Blue-stained gels from mouse fetus liver samples. The GelScan-XL system was established for the evaluation of silver-stained gels. The Gel-Image software has shown to be capable of the analysis of Coomassie Blue-stained gels and autoradiographs. For these systems the linear relationship between defined concentrations of protein loaded on the gel and the resulting integrated intensities of spots were examined. The results show that both systems are efficient to detect quantitative changes in protein expression correlated with malformations in mouse fetuses. PMID- 7532411 TI - [Detection of antigen structures in blood cells in various prepared plasma transfusions]. AB - We investigated the content of antigen-bearing cells and cell fragments in Fresh Frozen Plasma (FFP) from blood centers, in Octaplas (virus-inactivated fresh plasma produced with the solvent/detergent technique by the Octapharma Company) and in MB-plasma (virus-inactivated fresh plasma after photodynamic treatment with methylen blue coming from the German Red Cross in Springe, Lower Saxony). With the aid of an immunoassay (MAIPA-test) these plasmas were tested regarding Rhesus-D-antigen, HLA-class-I- and HLA-class-II-antigens, platelet specific antigens HPA-1a/HPA-1b and granulocyte specific antigens NA1/NA2. In Octaplas (n = 10) we did not find cells or cell fragments and no antigen-bearing blood cell structures. In FFP (n = 28) there were platelet specific antigens in 27 cases (96.4%) and HLA-class-I-antigens in 4 cases (14.3%). In MB-plasma (n = 14) we found platelet specific antigens in all cases, HLA-class-I-antigens in 4 cases (18.6%), HLA-class-II-antigens and granulocyte specific antigens in 1 case (7.1%) and Rhesus-D-antigen in 3 cases (21.4%). Plasma derived from whole blood showed lower levels of cells and antigens than plasma which was produced with the aid of the cell separator. PMID- 7532412 TI - Effects of restraint stress on components of adenylyl cyclase signal transduction in the rat hippocampus. AB - Chronic stress can injure hippocampal neurons as well as alter hippocampal function. The adenylyl cyclase (AC) signal transduction system is an important modulator of neurotransmission in this brain region. This study was conducted to begin to understand the effects of chronic stress on the hippocampal AC system. To assess dependence of type I and type II AC mRNA expression on adrenal integrity, total RNA was prepared from the hippocampus of nonstressed rats 7 days following either sham surgery or adrenalectomy (ADX). Adrenalectomy resulted in a 60% diminution in steady-state expression of type II AC mRNA (p < .005) and a nonsignificant fall in type I AC mRNA expression. Both sham and adrenalectomized rats were then exposed to 1-hour sessions of restraint, twice per day (0800 to 0900 and 1500 to 1600 hours) for 4 days. Following the stress paradigm, RNA was prepared from the hippocampus and type I and type II AC mRNA levels determined by Northern blot. Densitometic analysis showed that in comparison to unstressed sham rats, stressed sham rats had a significant twofold increase in steady-state levels of type I and type II mRNA. Stressed adrenalectomized rats also had increased expression in type II AC mRNA but no significant stress-induced change in expression of type I AC mRNA. To assess dependence of hippocampal membrane G Proteins on adrenal integrity, hippocampal membranes from nonstressed rats 7 days following either sham surgery or adrenalectomy (ADX) were analyzed by immunoblot.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532409 TI - Tyrosine kinase inhibitors enhance cGMP production in rat pinealocytes. AB - The role of tyrosine kinase(s) in the regulation of cGMP accumulation in rat pinealocytes was investigated using three tyrosine kinase inhibitors. Treatment with genistein, erbstatin or the active analogues of tyrphostin selectively increased basal cGMP accumulation in a dose-dependent manner without a concomitant increase in cAMP. In contrast to the norepinephrine- and vasoactive intestinal peptide-stimulated cGMP responses, the stimulatory effect of genistein was not blocked by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Furthermore, in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, neither genistein nor erbstatin had an effect on cGMP accumulation. These results indicate that tyrosine kinase inhibitors increase pineal cGMP accumulation through inhibition of the metabolism of cGMP. PMID- 7532413 TI - Cerebrospinal fluid levels of monoamine metabolites. A preliminary study of their relation to menstrual cycle phase, sex steroids, and pituitary hormones in healthy women and in women with premenstrual syndrome. AB - The cerebrospinal fluid (CSF) levels of the serotonin metabolite 5 hydroxyindoleacetic acid (5-HIAA), the noradrenaline metabolite 3-methoxy-4 hydroxyphenylethylene glycol (MHPG), and the dopamine metabolite homovanillic acid (HVA) were measured in a group of drug-free non-depressed women with premenstrual syndrome (PMS) (late luteal phase dysphoric disorder) (n = 13) and in controls with no premenstrual complaints (n = 13). In six patients and eight controls, CSF samples from both the luteal and the follicular phase were obtained, whereas in the remainder of the subjects, samples from either the follicular phase (patients: 4, controls: 2) or the luteal phase (patients: 3, controls: 3) were taken. The following observations were made: (1) Neither in the follicular phase nor in the luteal phase did the mean concentrations of CSF monoamine metabolites in the PMS group differ from the corresponding values in the control group. (2) Neither in the PMS group nor in the control group did the mean concentrations of monoamine metabolites in CSF samples obtained in the luteal phase differ from the corresponding values obtained in the follicular phase. (3) The intraindividual, intersample variations of CSF HVA and 5-HIAA concentrations were significantly smaller in the PMS group than in the control group. (4) CSF HVA correlated strongly to CSF 5-HIAA in the luteal phase of both patients and controls whereas in the follicular phase, particularly in controls, this correlation was much weaker. (5) In the luteal phase, the CSF HVA/5-HIAA ratio correlated negatively to serum levels of estradiol, progesterone, and testosterone. (6) The CSF HVA/5-HIAA ratio was significantly lower in PMS patients than in controls. (7) A positive correlation between CSF MHPG and serum luteinizing hormone was observed in the follicular phase. (8) A positive correlation between CSF HVA and serum prolactin was observed in the luteal phase. Because the study was comprised of a small number of subjects, the reported findings until replicated should be interpreted with caution. PMID- 7532415 TI - Ethanol suppresses LPS-induced mRNA for nitric oxide synthase II in alveolar macrophages in vivo and in vitro. AB - Alcohol abuse increases the incidence and severity of opportunistic lung infections and pneumonias. Inducible nitric oxide (NO) synthase (iNOS II) and NO may be a pivotal system in the intracellular bactericidal activity of macrophages. We tested the hypothesis that acute administration of ethanol (ETOH) suppressed Escherichia coli endotoxin lipopolysaccharide (LPS) mediated upregulation of the iNOS II system in the lung of the rat, in vivo. We also tested the effect of ETOH on alveolar macrophage (AM) production of free NO using microelectrodes. Male Sprague-Dawley rats were given ETOH (5.5 g/kg, IP) 30 min. before giving intratracheal sterile phosphate buffered saline solution (PBS, 0.5 ml) or LPS (1 mg/kg in a total volume of 0.5 ml PBS). The isolated lungs were subjected to bronchoalveolar lavage (BAL) 3.5 hr. later. Aliquots of the BAL fluid were assayed for tumor necrosis factor alpha TNF alpha and reactive nitrogen intermediates (nitrate and nitrite) (RNI) with chemiluminescence. Aliquots of AM were incubated 1 hr ex vivo for spontaneous production of RNI or frozen and assayed for iNOS II mRNA with competitor exchange reverse transcriptase polymerase chain reaction (cERT-PCR). The lung was homogenized and assayed for RNI. LPS increased BAL fluid TNF alpha and RNI, lung RNI, and the spontaneous production of RNI by AM, ex vivo. These effects were inhibited by in vivo administration of inhibitors of iNOS II. LPS increased iNOS mRNA in AM. This was unaffected by iNOS inhibitors. ETOH suppressed LPS-induced BAL fluid TNF, iNOS mRNA and RNI production by AM and the lung.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532414 TI - Monomorphic and polymorphic carbohydrate antigens on pig tissues: implications for organ xenotransplantation in the pig-to-human model. AB - The existence of the alpha Gal epitope in 137 pigs belonging to 23 different breeds suggests that this antigen is either monomorphic or occurs at a high incidence in the porcine species. Its histological location at the surface of pig vascular endothelial cells makes it a target for human natural anti-alpha Gal antibodies and complement, which may be responsible for the hyperacute vascular rejection of transplanted pig organs. The precursor carbohydrate chain (N acetyllactosamine) and NeuAc-substituted epitopes are also exposed at the surface of pig vascular endothelium and were found in all pigs in this study. However, humans also have these two epitopes on vascular endothelium and, consequently, have not made natural antibodies against these carbohydrate antigens. Therefore, these two pig epitopes cannot be the main target of the hyperacute vascular rejection process. Three pig phenotypes-A+ (51%), A:H+ (38%), and A-H- I+ (11%) were identified among 37 Large-white pigs by the presence of polymorphic A, H, and I carbohydrate antigens on the brush border of the surface epithelium of small intestine. These antigens were also present in other exocrine secretions but were not detected on vascular endothelium of the same pigs, suggesting that they are not involved in the hyperacute vascular rejection, although the pig A tissue antigen can induce an immune response in 0 or B blood group recipients. Once the problem of the initial hyperacute vascular rejection directed against the alpha Gal epitope is overcome, typing donor pigs for A, H, and I, as well as for the protein swine leukocyte antigens (SLA) and other pig antigens, may help in elucidating antigens involved in acute or chronic xenograft rejection. PMID- 7532417 TI - Clinical significance and detection of individual differences and changes in transperitoneal transport. AB - A review is given on the pathophysiology of the transport of solutes and fluid during continuous ambulatory peritoneal dialysis. Special attention is paid to the assessment of peritoneal permeability in individual patients, its inter- and intraindividual variability, the effect of systemic disease, some regulatory mechanisms, and alterations observed during long-term continuous ambulatory peritoneal dialysis. PMID- 7532416 TI - Systemic phencyclidine administration is associated with increased dopamine, GABA, and 5-HIAA levels in the dorsolateral striatum of conscious rats: an in vivo microdialysis study. AB - In vivo microdialysis was used to study the effects of systemically administered phencyclidine (PCP, 10 mg/kg) on the extracellular levels of dopamine, dihydroxyphenylacetate (DOPAC), homovanillate (HVA), 5-hydroxy-indolacetate (5 HIAA), gamma-aminobutyrate (GABA), glutamate, and aspartate in the rat dorsolateral striatum. In order to demarcate the effects of anesthesia, tissue trauma and gliosis, the effect of PCP was studied in both anesthetized rats with long and short probe implantation periods and in conscious rats with a long probe implantation period. PCP significantly increased the extracellular levels of dopamine in all experimental groups, though the post-implantation interval and anesthesia modulated the degree of increase. PCP increased 5-HIAA levels in both conscious and anesthetized rats after a long post-implantation period and HVA only in anesthetized rats after a long post-implantation period. Glutamate, aspartate, and DOPAC were not affected by PCP challenge but our study indicated for the first time that systemic PCP elevates extracellular GABA in conscious rats. PMID- 7532421 TI - Administration of anti-TNF-alpha or anti-CD11a antibodies to normal adult mice decreases lung and bone collagen content: evidence for an effect on platelet consumption. AB - Administration of anti-tumor necrosis factor-alpha (anti-TNF-alpha) or anti-CD11a (LFA-1) antibodies to normal adult mice for about 10 days markedly decreased the hydroxyproline content in lung and femur. Similar observation was made using the recombinant soluble TNF receptor (rsTNFR) as tumor necrosis factor-alpha (TNF alpha) antagonist. This decrease in collagen content was most likely due to a decrease of collagen synthesis, as evidenced by a decrease of the 3H-proline incorporation in lung and bone and by a decrease of collagen alpha 1 (I) mRNA levels in the lung RNA. The localization of labeled platelets in the lung of normal mice was decreased by anti-TNF-alpha but not by anti-CD11a antibodies. Thrombocytopenia increases the localization of labeled platelets in the lung, and, in this condition, both anti-TNF-alpha and anti-CD11a antibodies decreased pulmonary trapping. TNF-alpha mRNA was detected in the lung of normal adult mice, suggesting that this cytokine is released in the absence of inflammation. These results indicate that in vivo, endogenous TNF-alpha stimulates collagen synthesis, most likely by its influence on platelet trapping, which appears to involve the platelet CD11a since it is decreased by anti-CD11a antibody. PMID- 7532418 TI - Effect of blood contact on the transport properties of hemodialysis membranes: a two-layer membrane model. AB - The effects of blood-membrane interactions on hydraulic permeability, sieving coefficients, and diffusive permeabilities of Cuprophan and AN69 polyacrylonitrile hemodialysis membranes were studied using polydisperse dextrans (MW 5,000-20,000). Blood contact had no effect on the transport characteristics of the Cuprophan, but it caused a significant reduction in all of the transport parameters for the AN69 membrane due to the formation of a layer of adsorbed plasma proteins on the upper surface of the membrane. The transport characteristics of the membrane after exposure to blood are described using a two layer membrane model, with this model able to accurately account for the different effects of blood contact on the sieving coefficients and diffusive permeabilities as well as the observed asymmetry in solute transport through the blood-contacted membrane. These results have important implications for solute transport during clinical hemodialysis. PMID- 7532422 TI - Significance of increased neutrophils in patients with advanced colorectal cancer. AB - We examined the ratio of neutrophils to lymphocytes (N/L ratio) in the peripheral blood in patients with colorectal cancer. The ability to produce active oxygen and phagocytosis of neutrophils, G-CSF, sIL-2R and IAP (immunosuppressive acidic protein) were also measured. The N/L ratios were significantly higher in the advanced stages of cancer than in normal controls. The ability to produce active oxygen in the terminal stage was 33% lower than in the control group. The G-CSF levels had no relationship with the neutrophil counts. IAP levels increased with cancer stage, and were inversely related to the ability to produce active oxygen. The IAP levels correlated well with the sIL-2R levels and the N/L ratio. These findings suggest that the ability to produce active oxygen, N/L ratio and IAP reflect anticancer mechanisms and that they may be useful when considering treatment or prognosis of patients with advanced stages of cancer. PMID- 7532419 TI - Hepatocyte growth factor stimulates DNA synthesis in alveolar epithelial type II cells in vitro. AB - Hepatocyte growth factor (HGF) and its receptor, the product of c-MET proto oncogene, are highly expressed in both fetal and adult lung, though their physiologic functions in the lung are largely unknown. In the present study, we examined whether alveolar type II cells in the lung are the target of HGF and whether HGF has any effects on growth of these cells. The alveolar epithelial type II cells were isolated from the lungs of adult male Sprague-Dawley rats by elastase digestion, and the cells were used to determine whether they express HGF and c-MET mRNAs and whether recombinant HGF has any effect on their DNA synthesis in primary culture. The effects were further compared with those induced by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis and in situ hybridization revealed that type II cells express c-MET mRNA but not HGF mRNA. HGF stimulated [3H]thymidine incorporation into type II cells in primary cultures. An increase was also seen in labeling index as determined by nuclear immunostaining of bromodeoxyuridine incorporated DNA. While aFGF (200 ng/ml) exerted an effect comparable to HGF (25 ng/ml) on DNA synthesis in type II cells, EGF (20 ng/ml) and TGF-alpha (100 ng/ml) had lesser effects. TGF-beta 1, a potent inhibitor of epithelial cell proliferation, at 0.25 to 2 ng/ml, did not inhibit HGF-induced [3H]thymidine incorporation into type II cells. The results indicate that HGF exerts its effects on type II cells as a potent mitogen by a paracrine mode of action. PMID- 7532420 TI - Characterization of the mechanism involved in bleomycin-induced increased hyaluronan production in rat lung. AB - The molecular mechanisms behind the accumulation of hyaluronan during bleomycin induced lung injury in rats were investigated. The stimulatory effects of bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM)-conditioned media on hyaluronan synthesis in normal rat lung fibroblast cultures were studied as well as the hyaluronan binding activity on AM. BALF obtained on days 1 and 5 after bleomycin instillation exhibited hyaluronan stimulatory activity similar to that of 10% fetal serum; the activity returned to control values on day 14 after bleomycin treatment. Conditioned media from cultures of AM obtained from bleomycin-treated rats exhibited stimulatory effects higher than that of media from AM of control rats and equal to or higher than that of 10% fetal calf serum. The stimulatory activity in BALF was significantly inhibited by neutralizing antibodies against transforming growth factor-beta; the activity in AM conditioned media was only partially affected. Neutralizing antibodies against platelet-derived growth factor-BB or -AA had no such inhibiting effect. Interestingly, AM from bleomycin-treated rats exhibited low hyaluronan binding activity. [3H]Hyaluronan binding by AM on days 1 and 5 after bleomycin administration was about 2-fold and 4-fold lower, respectively, compared with that by AM derived from saline-treated rats. This decrease was normalized 14 days after bleomycin treatment. In conclusion, our results indicate that factors with high potential to stimulate hyaluronan synthesis in rat lung fibroblasts are accumulated in BALF from bleomycin-treated rats and that AM are likely to be one source of such stimulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532423 TI - Cat scratch disease: past and present. PMID- 7532424 TI - Medico-legal aspects of birth asphyxia: a paediatrician's perspective. PMID- 7532425 TI - Epitope specificity of MHC restricted cytotoxic T lymphocytes induced by candidate HIV-1 vaccine. PMID- 7532426 TI - HIV-1-specific cytotoxic T lymphocyte response in healthy, long-term nonprogressing seropositive persons. PMID- 7532427 TI - Cytotoxic T lymphocytes induced by liposomal antigens: mechanisms of immunological presentation. AB - It is known that liposomes can deliver encapsulated substances, including drugs and antigens, to lysosomes in macrophages. Because of this it has been assumed that although liposomes might be useful for induction of humoral (class II) immunity, they would not be capable of cytoplasmic delivery of antigen for introduction into the class I pathway leading to induction of cytotoxic T lymphocytes (CTLs). However, experiments conducted by numerous laboratories, including our own, have demonstrated the ability to induce CTLs either in vitro with cultured cells incubated with liposome-associated antigen, or in vivo after immunization of mice or monkeys with liposomes containing associated antigen. Using a monoclonal antibody that recognizes repeating sequences of tetrapeptide epitopes derived from the circumsporozoite protein of Plasmodium falciparum, it has been shown by immunogold electron microscopy that liposomal antigenic epitopes can actually spill from endosomes into the cytoplasm of cultured macrophages. On the basis of this observation, a theoretical intracellular pathway is proposed whereby liposomal antigen is processed by macrophages through a cytoplasmic process that results in delivery of antigenic epitopes to the Golgi apparatus and the endoplasmic reticulum. The liposomal antigenic epitopes would then have the opportunity to associate with class I MHC molecules and undergo vesicular transport to the surface of the cells for presentation and induction of CTLs. PMID- 7532428 TI - [Experimental alkaline reflux esophagitis. Role of the refluxed material]. AB - Alkaline reflux is new recognised to play an important place in gastroesophageal reflux disease. In order to investigate the role of refluxate in the development of esophagitis we have performed an experimental study in laboratory animals submitted to alkaline reflux inducing operations. We produced esophagitis in two experimental groups of Sprague-Dawley rats: group I, end-to-side esophagojejunostomy (EY) and group II, esophagojejunostomy with partial derivation of afferent loop by an omega-like conduit (EO) designed to attain lesser reflux. Group III consisted of sham laparotomized animals (L). RESULTS. At day 14, all the animals of experimental groups have developed esophagitis, but the macroscopic extent and histological grade in group EO (med: 27 mm; grade 2) were significantly lower than in group EY (med: 35 mm; grade 3). Biochemical measurements (med) in esophageal washout revealed: a) pH were alkaline in all groups with a decrease in experimental groups that was only significant in group EY; b) Amylase, lipase and bile acids in groups EY (8090; 498; 32) and EO (12840; 449; 50) were similar, but significantly more higher than in group L (920; 24; 0). The analysis of correlation between esophagitis parameters and biochemical measurements revealed no significant ones except for pH (r = -.437 and -.417). CONCLUSIONS. 1- The alternative model (EO) presents lesser grade and extent of esophagitis. 2) The refluxate was rich in bile acids and pancreatic enzymes what is proof of the actual occurrence of reflux and supports its determinant contribute to the development of esophagitis. 3) Esophagitis has developed at alkaline pH but with no alkalinity increase in the experimental groups what indicate that "alkaline reflux esophagitis" is not an appropriate denomination. 4) The discrepancy between the biochemical analysis in esophageal washout and the extent and grade of esophagitis suggests that others than the nature and amount of refluxate (e.g. mucosal resistance) could play a role in reflux esophagitis. PMID- 7532429 TI - Intraorbital injection of amphotericin B for palliative treatment of Aspergillus orbital abscess. AB - We report successful palliative treatment of an Aspergillus fumigatus orbital mass in a patient with acquired immunodeficiency syndrome by direct injection of amphotericin B into the abscess cavity. This case presents intraorbital injection of amphotericin B as an alternative to surgical debridement of Aspergillus orbital infection. In patients who are unable or unwilling to undergo more aggressive treatment, this procedure appears to limit morbidity while still providing effective palliative therapy. PMID- 7532431 TI - Differential inhibition of T and B cell responses to individual antigenic determinants in orally tolerized mice. AB - Immunological tolerance of systemic immunity can be induced by the oral administration of an exogenous antigen, which is termed oral tolerance. We examined whether there was a difference in the degree of tolerance between individual antigenic determinants in oral tolerance. Feeding bovine alpha s1 casein, a major protein in cow's milk, as a constituent of the diet induces oral tolerance in mice. However, a weak antibody response can be elicited in the alpha s1-casein-fed animals by subsequent immunization with the antigen. We examined the fine specificity of such anti-alpha s1-casein antibodies produced in alpha s1 casein-fed mice. The results demonstrated that there was a difference in the inhibition of antibody response between different B cell determinants. Differential inhibition could also be observed for T cell responses. T cells specific for dominant determinants were preferentially tolerized, while those for cryptic determinants escaped oral tolerance. Our results imply the importance of antigen presentation for this differential inhibition of antigenic determinants. We suggest that orally administered antigen does not induce tolerance to some of the B and T lymphocyte repertoire which could potentially induce harmful food hypersensitivity. PMID- 7532430 TI - NADPH diaphorase staining suggests a transient and localized contribution of nitric oxide to host defence against an intracellular pathogen in situ. AB - Nitric oxide (NO) is formed constitutively in neurons by the constitutive enzyme NO synthase (cNOS) and acts as a neurotransmitter. It has already been shown that cNOS-containing neurons are identical to neurons staining for NADPH diaphorase and vice versa. Effector cells of the immune response produce high NO levels after appropriate stimulation and this NO is formed by inducible NO synthase (iNOS). The NO produced by macrophages is considered an important effector molecule of antimicrobial host defence. We have applied NADPH diaphorase staining for the detection of NO producing cells in situ during infection with an intracellular pathogen. Macrophages which produce NO in vitro are stained for NADPH diaphorase. Expression of iNOS mRNA and macrophage NADPH diaphorase staining was inhibited by iNOS-specific antisense oligonucleotides. These data suggest coincidental similarity between NADPH diaphorase activity and NO production by macrophages. Cells staining for NADPH diaphorase were identified in cryostat frozen sections of livers from mice infected with the intracellular pathogen, Listeria monocytogenes, and co-localized with cells labelled by MAC-1 mAbs. The purple-blue reaction product of NADPH diaphorase staining was visible in discrete granulomatous lesions but was absent from the liver parenchyma. Our results provide direct evidence for localized and transient participation of NO in antimicrobial immunity in the infected organ. This restriction may focus NO production to lesions, leaving unrelated tissue sites unaffected. PMID- 7532432 TI - Regulation of apoptosis and T cell activation by Fas-specific mAb. AB - Fas was initially described as a molecule expressed on the surface of certain cell lines that could mediate programmed cell death (apoptosis) subsequent to ligation by specific mAb. To determine whether mAb to other epitopes on the Fas molecule might mediate other functions, we generated a panel of mAb to the extracellular portion of human Fas. Significant lysis of Fas-expressing target cells was only observed when the new mAb were first bound to a solid-phase support and not when the mAb were added in solution. However, several of these mAb inhibited the killing of target cells induced by the prototypic Fas-specific mAb, CH-11. Those mAb that inhibited apoptosis of target cells mediated by the CH 11 mAb also blocked lysis of target cells mediated by cells expressing Fas ligand. Finally, some of the Fas-specific mAb were found to co-stimulate proliferation of peripheral blood T cells in the presence of immobilized CD3 mAb. Thus, the data indicate the existence of a complex set of interactions mediated by Fas in both normal and transformed lymphoid cells that may have important implications regarding the role(s) of this molecule in regulation of immune responses. PMID- 7532433 TI - The expression of beta 1 integrin and intercellular adhesion molecule-1 on mesangial cells and its modulation in glomerulonephritis. AB - OBJECTIVES: This study was performed to observe the effects of conditioned media (CM) of cultured mononuclear cells (MNC) on the expression of beta 1 integrin and intercellular adhesion molecule(ICAM)-1 on mesangial cells and the modulation by TGF-beta, TNF alpha, or hydroxyl radical Also these were examined in anti-Thy mesangial proliferative GN (MsPGN) or puromycin aminonucleoside nephrosis(PAN). METHODS: The expression of beta 1 integrin and ICAM-1 on mesangial cells was examined by ELISA and the modulation by TGF-beta, TNF alpha, or hydroxyl radical was investigated by using neutralizing antibody and dimethylthiourea. [.OH] scavenger. RESULTS: The CM of MNC from Control suppressed the beta 1 integrin expression and thymidine incorporation and increased ICAM-1 expression on mesangial cells, compared to media alone. Especially the significant increase in ICAM-1 expression was reversed by anti-TNF alpha antibody. beta 1 integrin expression on mesangial cells was higher by the addition of CM of MNC in MsPGN than in Control, which was prevented by anti-TGF beta antibody or dimethylthiourea. ICAM-1 expression was not different among groups. Thymidine incorporation by adding CM of MNC was lower in MsPGN and PAN, which was mitigated by anti-TGF beta and anti-TNF alpha. Thus the ratio of the expression of ICAM-1 to thymidine incorporation was higher in MsPGN and PAN. CONCLUSION: The expression of beta 1 integrin and ICAM-1, and the thymidine incorporation on mesangial cells are directly regulated by MNC, maybe through factors such as TGF beta or TNF alpha. In MsPGN, the overexpression of beta 1 integrin induced by MNC through TGF beta or hydroxyl radical on day 3 may be related to the pathogenesis. PMID- 7532434 TI - Non stochastic distribution of single channels in planar lipid bilayers. AB - The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5 7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface. PMID- 7532435 TI - Monovalent cation selective channel in the apical membrane of rat inner medullary collecting duct cells in primary culture. AB - Ion channels in the apical membrane of rat inner medullary collecting duct (IMCD) were investigated by the patch clamp technique. Owing to the histological heterogeneity of IMCD, cells were cultured from the lower half of the inner medulla of Wistar rat kidney. Channel activity was rarely seen in cell attached patch, but membrane excision activated multiple units of 28.2 +/- 0.7 pS cation selective channel. A Na or K selective channel was not found. The 28 pS channel showed membrane voltage dependency, no rectification, almost equal permeability to monovalent cations (Na/K/Li/Cs/Rb/NH4 = 1:1.00:0.82:0.97:1.10:1.71) and no significant permeation to anions or divalent cations. Calcium of the cytoplasmic side from 10(-7) M to 10(-4) M affected the mean number of open channels (nPo) dose-dependently in excised patch (IC50 = 5 x 10(-6) M). 1 mM of ATP, ADP, AMP and gadolinium reversibly suppressed nPo to near zero whereas amiloride, cAMP or cGMP had no effect. Multiple conductance substates were frequently observed. These results suggested that this channel belongs to the nonselective cation channels which has been identified in other epithelia and is not responsible for amiloride sensitive Na transport through IMCD cells. PMID- 7532440 TI - Serum total sialic acid and acute phase proteins in elderly subjects. AB - Serum total sialic acid has gained recent interest as a cardiovascular risk factor. We measured serum total sialic acid and three acute phase proteins; C reactive protein, alpha 1-antichymotrypsin and alpha 1-acid glycoprotein in 37 geriatric patients (age 80.1 +/- 7.0 years) and 50 younger subjects (age 40.3 +/- 11.4 years). Serum total sialic acid was higher in the geriatric subjects 2.41 +/ 0.39 mmol/l versus 2.04 +/- 0.35 mmol/l, P < 0.04. Serum alpha 1-acid glycoprotein, alpha 1-antichymotrypsin and C-reactive protein were also elevated in the geriatric patients; serum alpha 1-acid glycoprotein being 1.16 +/- 0.32 g/l versus 0.41 +/- 0.28 g/l, P < 0.0001, serum alpha 1-antichymotrypsin being 0.80 +/- 0.20 g/l versus 0.52 +/- 0.10 g/l, P < 0.0001 and serum C-reactive protein being 9.71 +/- 21.0 mg/l versus 4.73 +/- 1.30 mg/l, P < 0.04. There was a correlation with serum total sialic acid and serum alpha 1-acid glycoprotein and alpha 1-antichymotrypsin in the geriatric subjects and with alpha 1-acid glycoprotein, alpha 1-antichymotrypsin and C-reactive protein in the younger group. PMID- 7532437 TI - Growth properties of a feline immunodeficiency virus mutant which lacks an AP-1 binding site in primary peripheral blood mononuclear cells. AB - Thirty-one base pairs (bp) containing putative AP-1 and AP-4 binding sequences in the U3 region of feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted from an infectious molecular clone of FIV for construction of a mutant virus, and the replication rate and the cytopathogenic activity of the virus were compared with those of the wild type virus in concanavalin-A (Con-A) stimulated primary feline peripheral blood mononuclear cells (fPBMCs). It was found that the replication rate and cytopathogenic activity of the mutant were almost the same as those of the wild type. The deletion of the mutant virus was stable during the infection experiments. From these data, we concluded that the 31 bp fragment in the LTR is not required for the replication of FIV in Con-A stimulated primary fPBMC. PMID- 7532438 TI - Inhibition of viral multiplication by hammerhead ribozymes targeted against the polymerase gene of mouse hepatitis virus. AB - We designed and constructed two hammerhead ribozymes targeted against the polymerase gene of mouse hepatitis virus (MHV). They consisted of a 22-nucleotide (nt) ribozyme core sequence and antisense sequences of different lengths, 243-nt (S-ribozyme) and 926-nt (L-ribozyme). In cell-free reactions, the constructed ribozymes cleaved the target RNA at a specific site. Vectors that directed the expression of ribozymes by a promoter of human elongation factor 1 alpha were introduced into DBT cells, and the resulting several cell lines constitutively expressing the ribozymes were selected by Northern blot analysis and examined for intracellular multiplication of MHV. The production of infectious progeny virus particles was significantly reduced in the transfected cell lines expressing either S-ribozyme or L-ribozyme. Although the in vitro cleavage process of L ribozyme was slower than that of S-ribozyme, no difference was observed in inhibitory effects on MHV multiplication between S- and L-ribozymes in the transfected cells. PMID- 7532439 TI - Endothelial modulation of vascular tone in isolated porcine and bovine basilar arteries. AB - Endothelium-dependent modulation of vascular tone was investigated in isolated porcine and bovine basilar arteries. L-Nitro-arginine (NO synthase inhibitor) and methylene blue (soluble guanylate cyclase inhibitor) increased, but indomethacin (cyclooxygenase inhibitor) decreased the vascular tone in the basilar arteries from both species. Bradykinin evoked relaxation of precontracted porcine basilar artery, but not bovine basilar artery. Sodium fluoride (endothelial G-protein activator) produced relaxation of precontracted basilar arteries from both species. The effects of bradykinin and sodium fluoride were completely abolished by endothelial denudation and markedly inhibited by L-nitro-arginine and methylene blue, but not by indomethacin. Sodium nitroprusside (guanylate cyclase activator) evoked relaxation of precontracted endothelium-denuded basilar arteries from both species. These results suggest that there is species variation in endothelium-dependent modulation of vascular tone in the basilar artery. PMID- 7532441 TI - Plasma glutathione peroxidase activity as an index of renal function. AB - The kidney is a major source of the plasma enzyme glutathione peroxidase. We measured plasma glutathione peroxidase activity in 130 patients affected with different renal diseases at various stages, and compared it with the following indices of kidney function: serum creatinine, creatinine clearance, and urinary excretion of alpha 1-microglobulin, beta 2-microglobulin, albumin and N-acetyl beta-D-glucosaminidase. Plasma glutathione peroxidase activity appeared significantly reduced in most of the renal diseases considered, and showed a significant correlation with most of the renal function indices. Linear discriminant analysis showed that the set of indices composed of plasma glutathione peroxidase activity, serum creatinine and creatinine clearance allowed the best classification of renal diseases. During treatment with the nephrotoxic aminoglycoside, tobramycin, plasma glutathione peroxidase activity showed an early and progressive decrease. We suggest the measurement of plasma glutathione peroxidase activity as an adjunctive index for the assessment of kidney alterations. PMID- 7532442 TI - Comparison of a time-resolved immunofluorimetric assay with two immunoenzymatic methods for alpha-foetoprotein in human serum. AB - We compared two commercial methods with a time-resolved immunofluorimetric assay for alpha-foetoprotein in human serum using the europium complex of 4,7 bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label. The correlation coefficients were r1 = 0.94 and r2 = 0.96. PMID- 7532436 TI - Antigenicity of hepatitis B surface antigen proteins reconstituted with phospholipids. AB - Hepatitis B surface antigen (HBsAg) has been reconstituted with different phospholipid classes. All epitopes defined by a panel of monoclonal antibodies which recognize both group- and subtype-specific antigenic determinants showed specificity for acidic phospholipids. Electrostatic interactions between HBsAg proteins and acidic phospholipids are partly responsible for the complete recovery of the antigenic properties. In addition to the nature of the polar head group, the fatty acid composition of the phospholipid also influenced the recovery of the antigenic activity. Negatively charged phospholipids must bear at least one unsaturated fatty acid in order to be effective in recovering full antigenic activity of HBsAg. The results reported herein support the conclusion that the antigenic activity is dependent on the physical state of the phospholipid moiety. The appropriate membrane fluidity is required for optimum conformation but, once this conformation is established, additional interactions imparted by the various phospholipids give a difference in the patterns of antigenicity. The analysis of binding of the monoclonal antibodies allowed the classification of the epitopes into two groups according to their dependence on the lipid moiety. Of all the antigenic determinants only those close to the lipid protein interface would change upon direct interaction with the phospholipids. The rest would depend on the correct protein conformation determined by the appropriate phospholipid composition. PMID- 7532443 TI - Mononuclear phagocytic cells in human milk: HLA-DR and Fc gamma R ligand expression. AB - The study of the cellular immune components of human milk is essential in the understanding of the role human milk may play in protecting the nursing infant against infection. We have investigated some phenotypic characteristics of breast milk macrophages (BMM) and have compared them to the characteristics of adult peripheral blood monocytes (PBM) by using dual parameter flow microfluorometry. Most BMM expressed the monocyte/macrophage markers Leu-M3 and Leu-M5. The latter marker was present in high density (bright) on BMM, but the density of expression of Leu-M3 was higher on PBM than on BMM [median fluorescence intensity (MFI) 409 +/- 105 versus 203 +/- 106, p = 0.02]. The percentage of BMM (98 +/- 2) that expressed the HLA-DR antigen did not differ significantly from PBM, but the density of expression was higher on BMM (MFI 318 +/- 56 versus 264 +/- 41, p = 0.03). The HLA-DR expression of BMM was further enhanced after incubation with interferon-gamma for 36 h; however, receptor for interleukin-2 could not be induced on BMM by this treatment. The expression of the three classes of Fc gamma R was lower on BMM than on PBM, in percentage (Fc gamma RI 56 +/- 23 versus 79 +/ 17%, p = 0.02), density of expression (Fc gamma RIII MFI 71 +/- 20 versus 153 +/ 73, p = 0.002), or both (Fc gamma RII 74 +/- 22% versus 94 +/- 12%, p = 0.02, and MFI 115 +/- 53 versus 202 +/- 59, p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532444 TI - Comparison of colony stimulation factors on in vitro rat and human neutrophil function. AB - The effects of rhCSFs on in vitro polymorphonuclear leukocyte (PMN) function were studied in Sprague-Dawley neonatal and adult rats and adult and umbilical cord derived human PMN to compare species response. Following in vitro exposure to buffer or rhCSFs (50-100 micrograms/ml), PMN oxidative burst, chemotactic activity and adherence protein expression were measured. RhG-CSF increased the oxidative burst of adult rat PMN as measured by chemiluminescence and altered CD11b/CD18 in resting neonatal rat but not adult rat cells. RhGM-CSF had no effect on adult rat PMN function in vitro, but led to modest changes in adult rat PMN diapedesis across rat peritoneum. No responses were noted to rhM-CSF. Human PMN responded best to GM-CSF (particularly in the neonate), intermediately to G CSF and none to M-CSF. These experiments show that the profile of cytokine effects is not similar in adult and neonatal rat PMN when compared to human cells. The diversity of actions in other species must be considered when using rhCSFs in animal models. PMID- 7532446 TI - The effect of recombinant aprotinin on t-PA-induced bleeding in rats. AB - High dose aprotinin administered during cardiopulmonary bypass (CPB) has been shown to reduce post-operative bleeding substantially. The exact mechanism of action is still debated. A reduction in fibrinolytic activity by inhibition of plasmin generated during CPB may be the primary mode of action. However, this hypothesis has been questioned as the apparent inhibitory constant of aprotinin for plasmin is orders of magnitude lower than the clinically effective plasma concentration of aprotinin. In the present study the effect of various plasma levels of aprotinin on a plasmin-induced bleeding in rats was investigated. The mean time of bleeding was 10 min in rats receiving only saline whereas a prolonged bleeding of up to > 45 min was seen in rats given t-PA and saline. The steady-state plasma concentration of recombinant aprotinin during infusion of 11 mg/kg/h was approximately 2 microM which roughly corresponds to the clinical use of aprotinin. This dose reduced the t-PA-induced bleeding to a mean value of 11 min, whereas no effects were observed with lower doses. The effect of aprotinin in clinical care of blood loss in CPB in man may therefore be caused by direct inhibition of plasmin. PMID- 7532445 TI - Basal serum gastrin concentrations before and after eradication of Helicobacter pylori infection measured by sequence specific radioimmunoassays. AB - BACKGROUND: Helicobacter pylori infection of the antral mucosa is responsible for an increase in basal and stimulated serum gastrin. In the present study we have investigated whether gastritis induced by H. pylori is responsible for abnormalities in the processing of gastrin in dyspeptic patients. METHODS: Basal serum gastrin was measured by radioimmunoassay before, 5 weeks, and 1 year after anti-H. pylori therapy in 73 H. pylori positive functional dyspeptic patients. Three region-specific antisera were used, specific for the biologically active carboxy-terminal part, the biologically inactive amino-terminal part of gastrin 1 17, and for the non-sulphated tyrosyl residue in gastrin 1-17. RESULTS: Basal serum gastrin levels were markedly (P < 0.01) decreased 5 weeks and 1 year after successful eradication of H. pylori (n = 39) but not in the patients in whom treatment failed (n = 34). A decline of gastrin was observed for each of the three radioimmunoassays. CONCLUSION: The decrease of serum gastrin levels in all three radioimmunoassays after a successful eradication of H. pylori does not point to major changes in the processing of gastrin. These results suggest that G cells in the antral mucosa are not functionally affected by the inflammation. PMID- 7532449 TI - Plasma endotoxin concentration and endotoxin binding capacity of plasma acute phase proteins in cirrhotics with variceal bleeding: an analysis by new methods. AB - Plasma endotoxin levels in 12 cirrhotics with bleeding from oesophageal varices and 50 cirrhotics without bleeding were measured by the chromogenic assay after the pretreatment of sample by perchloric acid (HClO4) and triethylamine. Endotoxin in cirrhotics with bleeding from varices was significantly higher than those without bleeding. In patients with bleeding, endotoxin increased for 3 days after the bleeding, first in the supernatant fraction and then in the precipitate fraction by HClO4 treatment. Peak plasma alpha 1-acid glycoprotein and haptoglobin were observed 3 days after the bleeding. Alpha 1-antitrypsin gradually increased for 14 days. Transferrin did not markedly change. The endotoxin-binding capacity of transferrin and alpha 1-acid glycoprotein increased immediately after bleeding and thereafter decreased, but that of alpha 1 antitrypsin tended to increase in the recovery period. In summary, the plasma endotoxin concentration and endotoxin-binding capacity of alpha 1-acid glycoprotein and transferrin were shown to have increased after bleeding from varices by this new method. There may be a close relationship between endotoxaemia and acute phase reaction in this situation. PMID- 7532450 TI - High rate of mixed genotypes of hepatitis C virus in patients of an epidemic area in Japan. AB - The subjects of this study were 151 patients (69 males and 82 females) who underwent examination and liver biopsy owing to liver dysfunction in an epidemic area with hepatitis C. Second generation hepatitis C virus antibody (HCV Ab) was positive in 116 (76.8%) of 151 cases. HCV-RNA was detected in 120 (79.5%) by polymerase chain reaction (PCR). In 7 (4.6%) cases, HCV Ab could not be found, but HCV-RNA was detected. Three (2.0%) cases were positive for HCV Ab but negative for HCV-RNA. On the basis of variation in nucleotide sequence within a restricted region in the putative core gene of HCV, HCV genotypes were classed into four types of I, II, III and IV by PCR. The genotypes were identified in 120 cases. Ninety-eight (81.7%) cases carried one of the four types. Type II was found in 76 (63.3%) cases and type III in 22 (18.3%). The other 22 (18.3%) carried simultaneously two different genotypes (mixed type): 21 (17.5%) cases with type II + III and one (0.8%) case with type II + IV. In comparison with the incidence of HCV mixed types in cases with hepatitis C in a non-epidemic area, carriers of mixed types were found at a significantly higher rate in the epidemic area. Liver biopsy of 120 cases with identified HCV genotypes in the epidemic area showed 93 cases of chronic active hepatitis, nine of chronic lobular hepatitis, 10 of chronic persistent hepatitis and eight of liver cirrhosis. No significant correlation could be detected between liver histology and HCV genotypes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532448 TI - Involvement of N-acetyl-lactosamine-containing sugar structures in the liver metastasis of mouse colon carcinoma (colon 26) cells. AB - Histochemical aspects of the process of experimentally induced metastasis were examined by light and electron microscopy with labelled lectins employed as a probe. Mouse colon carcinoma cells (colon 26) were injected into the spleen of Balb/c mice and liver metastasis was induced. Among the lectins tested, Erythrina cristagalli agglutinin (ECA) stained the metastasized colon 26 cells strongly compared with the heterogeneous and faint staining in non-metastasized tumour foci in the spleen or in the subcutaneous space. Other lectins, such as Phaseolus vulgaris leucoagglutinin (PHA-L), Phaseolus vulgaris erythroagglutinin (PHA-E) and Datura stramonium agglutinin (DSA), having specificity for branched complex type sugar chains, did not show any differences between metastasized cells and non-metastasized tumour foci. In addition, N-acetyl-lactosamine, a specific inhibitor of ECA binding, significantly inhibited the attachment of suspended colon 26 cells to sectioned unfixed normal liver tissue. These results indicate that the expression of galactose (Gal) beta 1-4 N-acetyl-glucosamine (GlcNAc) residues of branched complex type sugar chains having specificity for ECA are important for the interaction process of carcinoma cells with hepatic cells in the process of liver metastasis. PMID- 7532447 TI - Modulation of protein C inhibitor activity. AB - Protein C inhibitor (PCI), antithrombin, and heparin cofactor II are members of the serine proteinase inhibitor (serpin) superfamily that inhibit proteinases at rates which increase in the presence of the glycosaminoglycan heparin. These studies were undertaken to understand how PCI activity is modulated by various substances that are found in or interact with the vascular endothelium/basement membrane. The effects of antithrombin-heparin, thrombomodulin, vitronectin and leukocyte elastase on PCI-thrombin and PCI-activated protein C (APC) interactions were investigated. Antithrombin, which does not inhibit APC but which does bind to heparin/heparan sulphate with higher affinity than PCI, caused only a small decrease in the inhibition rate of PCI-APC in the presence of unfractionated heparin. Thrombomodulin, a chondroitin sulphate-containing proteoglycan, accelerated PCI inhibition of thrombin and APC. PCI-thrombin in the presence or absence of heparin bound plastic absorbed vitronectin, but neither PCI alone nor PCI-APC bound. Vitronectin also decreased the inhibition rate of PCI-thrombin and PCI-APC in the presence of low concentrations of heparin. Leukocyte elastase proteolytically inactivated PCI in a reaction that was accelerated by heparin. Overall, these results indicate that PCI activity is modulated by these endothelial cell/basement membrane-based substances in similar ways as other heparin-binding serpins, especially antithrombin. PMID- 7532451 TI - Pathomorphological study of HCV antibody-positive liver cirrhosis. AB - A morphological investigation was carried out to study the pathological features of liver cirrhosis caused by hepatitis C virus (HCV) infection. The materials consisted of liver specimens taken from 47 cases of anti-HCV antibody-positive liver cirrhosis (37 by surgery for hepatocellular carcinoma and 10 by autopsy), and from 21 cases of hepatitis B surface antigen-positive liver cirrhosis as the control. Liver specimens containing more than 10 regenerative nodules were examined. In addition, a histometric study was conducted to determine the degree of fibrosis and the size of regenerative nodule using a computer image-analysis system. The results showed that the histological characteristics of HCV antibody positive liver cirrhosis are: (i) broadly expanded fibrous septa and small regenerative nodules; (ii) relatively strong inflammatory reaction and prominent lymphoid aggretation in the fibrous septum; and (iii) mild regenerative activity of the liver parenchyma, and infrequent liver cell dysplasia. These findings may facilitate better understanding of the pathology of HCV antibody-positive liver cirrhosis and more accurate pathological diagnosis by needle biopsy. PMID- 7532452 TI - Amyloid beta protein-induced irreversible current in rat cortical neurones. AB - The effect of amyloid beta protein (A beta P) was examined in neurones dissociated from rat cortex using the nystatin perforated patch-clamp technique. A beta P at concentrations > 10 nM induced an irreversible slow inward current associated with an increase in membrane conductance. The time lag until the appearance of the effect of A beta P shortened in a concentration-dependent manner. When extracellular Na+ and Cl-, and intracellular K+ were replaced by equimolar N-methyl glucamine, isothionate- and Cs+, respectively, the membrane conductance and the reversal potential were not affected. Even when an internal solution including 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (20 mM) was used, the effect of A beta P did not alter. It is suggested that A beta P binds to the neuronal membrane and opens non-selective ion channels, resulting in neuronal degeneration. PMID- 7532453 TI - Noxious stimulation decreases substance P binding in rat spinal dorsal horn: competition by endogenous ligand? AB - To determine the participation of NK-1 receptors in mediating inputs from noxious thermal stimulation, sustained noxious stimulation was applied to anaesthetized rats by immersing one hind paw in water for 1.5 min at different temperatures. After sacrificing the animal, lumbar spinal cords were removed and 20 microns sections were incubated with [125I]BH-substance P. Compared with unstimulated controls, binding was the least in rats given a 55 degrees C stimulus and sacrificed 1 min after the stimulus; the greatest reduction was found in the superficial dorsal horn. Rats sacrificed at 10 min showed intermediate binding levels. Groups given less intense stimuli showed smaller decreases in binding. It is suggested that the decrease in binding was due to occupation of receptors by endogenous ligand, which is consistent with the idea that noxious stimulation evokes the release of substance P at the spinal level. PMID- 7532454 TI - Synergy between diazepam and NBQX in preventing neuronal death caused by non-NMDA agonists. AB - The non-N-methyl-D-aspartate (NMDA) glutamate receptor agonists kainate and L alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), when injected into the rat dorsal hippocampus, cause neuronal death directly by activating non-NMDA receptors and as a consequence of initiating seizure activity. Co-injection of the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX; 12.5-95 nmol) was partially effective in preventing up to about 60% of the direct excitotoxicity. On the other hand, diazepam (6 x 5 mg kg-1, i.p.) had only a minor protective effect against the direct neuronal damage, but was effective in preventing almost all the extra-hippocampal loss of neurones caused by seizure activity. The combination of intracerebral NBQX and systemic diazepam reduced the toxicity of kainate or AMPA to a greater extent than that found in the presence of either protectant alone. At optimum doses the neuronal cytotoxicity caused by non-NMDA agonists in the hippocampus was completely prevented. PMID- 7532455 TI - NBQX prevents contralateral but not ipsilateral seizure-induced cytotoxicity of kainate but not AMPA-reversal at high doses of NBQX. AB - The non-N-methyl-D-aspartate (NMDA) glutamate agonists are potent convulsants, and cause neuronal loss in many regions of the limbic system in the brain. Kainate or L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX) were concomitantly injected unilaterally into the rat dorsal hippocampus. Kainate with low doses of NBQX (12.5-25 nmol) almost completely prevented damage to the contralateral but not ipsilateral limbic areas (distal toxicity). In most animals, damage was still found in the contralateral midline thalamic nuclei. In contrast, high doses of NBQX (95-190 nmol) gave no protection to either side. AMPA showed a trend for a reduction in limbic damage to both sides with increasing dose of NBQX. At the highest doses, NBQX prevented damage to both sides. PMID- 7532456 TI - The expansive role of CD40 and its ligand, gp39, in immunity. AB - The interactions between CD40 and its ligand, gp39, are central to the development and maintenance of immunity. The role this receptor-ligand pair plays in B cell activation, isotype switching and memory generation has been firmly established through the study of in vitro and in vivo murine models, as well as human in vitro systems and immunodeficiencies. In this review, the potential role of gp39 in the regulation of co-stimulatory molecules and their implications on thymic education and peripheral tolerance are discussed. PMID- 7532457 TI - Structural characteristics of CD40 ligand that determine biological function. AB - CD40 ligand (CD40L) is a 33 kDa type II glycoprotein which is transiently expressed on the surface of T cells following activation. The demonstration that signals delivered by CD40L are essential for the process of affinity maturation and immunoglobulin isotype switching following antigenic challenge came from the study of X-linked hyper-IgM patients whose T cells cannot express functional CD40L. While some of the biological activities of CD40L, especially on B cells, can be mimicked by monoclonal antibodies (MAb) specific for CD40, it is becoming increasingly clear that CD40L also mediates various functional effects on other cell types. Not only are there distinctions between the activities of CD40L and CD40 MAb, but the manner in which CD40 is ligated appears to play an important part in the biological outcome of signaling through this receptor. In this review, we compare and contrast the activities which can currently be ascribed to CD40L and CD40 MAb and consider the role that ligand oligomerization plays in CD40-mediated signal transduction. PMID- 7532460 TI - CD40 ligation in B cell activation, isotype switching and memory development. AB - Interaction of CD40 on B cells with its ligand on activated T helper cells is crucial for the generation of mature T-dependent antibody responses. Experimental observations suggest that CD40 ligation acts at several points in B cell differentiation; however, it is not clear whether the consequences of ligation are direct or indirect. While its role in B cell activation is clearly direct we discuss the several ways in which it might drive isotype switching, the influence that it has on the development of memory B cells and the possibility that certain types of T-dependent antibody response are CD40L-independent. Experiments in which the ligation of CD40 is blocked in vivo reveal that early CD40 signals are important for the development of memory B cells but this may only reflect the ability of 'memory precursors' generated by CD40 ligation to accept subsequent programming or survival signals. PMID- 7532458 TI - CD40 and its ligand in the regulation of humoral immunity. AB - T cells provide help for B cell antibody production by cell-cell contact and by soluble factors. CD40L is the predominant protein induced on activated T cells which constitutes contact-dependent help, and lack of CD40L or blocking CD40 CD40L interactions leads to severely impaired antibody production. In addition to CD40-CD40L, B and T cells express costimulatory, accessory molecules which amplify T and B cell function and allow for reciprocal dialogues during antigen presentation. Interactions between costimulatory counter-receptors can determine lymphocyte activation or nonresponsiveness, and provides a means for regulating self-tolerance. PMID- 7532459 TI - CD40 ligand-CD40 interaction in Ig isotype switching in mature and immature human B cells. AB - The CD40 ligand (CD40L) is a member of the TNF family, and has emerged as a key molecule in the contact-mediated signal required for B cell activation and differentiation. The cloned CD40L expressed on heterologous cells, or in the form of soluble multimeric molecules, can directly activate B cells and, in conjunction with cytokines, can induce Ig isotype switching in naive B cells. Patients with hyper-IgM syndrome, which results from defective CD40L expression, generally have no circulating Ig, except for IgM, indicating that the CD40L is also important for Ig isotype switching, in vivo. CD40L does not play a role in B cell development and appears not to be required for human activation and differentiation. The presence of CD40L on cells other than T cells, the relatively broad distribution of its ligand CD40, and the ability of T cells to be co-stimulated via CD40L, indicates a broader role for CD40L-CD40 mediated intercellular communication. PMID- 7532462 TI - The role of CD40L (gp39)/CD40 in T/B cell interaction and primary immunodeficiency. AB - The interaction between the CD40 ligand (gp39), expressed by activated T cells, and CD40, constitutively expressed by B cells, is critical for an effective antibody response to T cell dependent antigens. Patients with X-linked hyper IgM (HIM) syndrome fail to express a functional CD40 ligand due to a mutation within the gene for gp39. As a direct consequence, HIM patients, when immunized with T dependent antigens, produce only small amounts of IgM antibody without the development of immunologic memory, amplification and switch from IgM to IgG. Mutations affecting the gene for the HIM syndrome are localized throughout the coding region of gp39 and consist predominantly of point mutations. The resulting amino acid substitutions interfere directly with the receptor binding site or lead to stop codons or deletions secondary to splice site mutations. Expression of gp39 by activated T cells from patients with common variable immunodeficiency (CVI) is low in approximately half of the patients and is associated with depressed expression of IL-2. These findings suggest that inefficient signaling via CD40 may be responsible in part for failure of B cell differentiation in CVI. PMID- 7532463 TI - [Preparation of appropriate diagnostic systems of monoclonal antibodies based on human thyroid hormone, and their use in clinical practice]. PMID- 7532461 TI - In vivo T-B cell interactions and cytokine-production in the spleen. AB - T-B cell interactions have a central role in the development of humoral immunity. The binding of a 39 kDa protein (gp39), selectively expressed on activated Th cells, to its receptor CD40, on B cells, results in the initial B cell activation. Thereafter, Th cell derived cytokines regulate the differentiation of B cells into antibody-forming cells. Most of these data are derived from in vitro experiments. This article discusses in vivo experiments dealing with T-B interactions. First, the immunohistochemical analysis of T cell activation (gp39 expression), cytokine and antibody production in murine spleens after injection of model antigens (TNP-Ficoll, TNP-KLH, and rabbit anti-IgD antibodies). Second, the in vivo role of gp39 and cytokines in these immune responses. Finally, by combining these in vivo experiments with in vitro data we present an in vivo model for T cell dependent antibody responses. PMID- 7532465 TI - Expression of insulin-like growth factor binding proteins by T-47D human breast cancer cells: regulation by progestins and antiestrogens. AB - We have used ligand blotting and Northern blotting techniques to examine the effects of progestins and antiestrogens on expression of insulin-like growth factor binding proteins (IGFBPs) by T-47D human breast cancer cells under conditions where these agents are growth inhibitory. Under basal conditions, conditioned medium from T-47D cells was found to contain IGFBPs of 39, 33, and 27 kDa. Northern blot and/or Western blot analysis have identified these as IGFBP 2, 5, and 4, respectively. Medroxyprogesterone acetate (MPA) treatment resulted in a time- and dose-dependent decrease in IGFBP 4 and 5 mRNA abundance and secretion of these proteins, while little if any effect was observed on IGFBP 2 expression. A decrease in the steady state mRNA levels for IGFBP 4 and 5 was observed with as little as 0.1 nM MPA. Using 10 nM MPA a maximum decrease in IGFBP 4 and 5 mRNA levels was observed between 12 and 24 hours. While RU 486 alone had little or no effect on IGFBP 4 expression, it inhibited the effect of MPA. However, in the same samples, IGFBP 5 expression was inhibited by RU 486, and RU 486 was unable to reverse the effects of progestins on the expression of IGFBP 5. Furthermore, another synthetic progestin, Org 2058, but not dexamethasone, inhibited IGFBP 4 and IGFBP 5 expression. The antiestrogen ICI 164384 also transiently decreased the steady state mRNA levels of both IGFBP 4 and IGFBP 5. Regulation of expression of the IGFBPs by these agents suggests a potential role for the IGFBPs in the growth response of T-47D cells to these agents. PMID- 7532466 TI - High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: a preventive mechanism of malignant tumor cells against complement attack? AB - GB24 is a mouse monoclonal antibody raised against a common trophoblast lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation. PMID- 7532464 TI - Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant. AB - The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells. PMID- 7532470 TI - Rapid HPLC assay with coulometric detection for norepinephrine and 3-methoxy-4 hydroxyphenylglycol in the mouse brain. AB - For the rapid assay of norepinephrine (NE) and its major metabolite, 3-methoxy-4 hydroxyphenylglycol (MHPG) in the mouse brain, we developed a simple method using isocratic HPLC with coulometric detection. This method permits NE and MHPG assay within 5 min in one chromatographic run. Within-run coefficients of variation for NE and MHPG in the working standard solution were 0.8% and 0.6% (n = 50), respectively. The detector responses were linear from 0.025 to 100 pmol for NE and from 0.05 to 100 pmol for MHPG in the working standard solution. Using this method, the NE and MHPG concentrations were measured in discrete brain areas of the mouse prior treatment with or without alpha-methyl-p-tyrosine or N-(2 chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). PMID- 7532469 TI - Technique for assessment of leukocyte adherence to human umbilical vein endothelial cell monolayers. AB - The interaction between endothelial cells and immune/inflammatory cells plays an important role in the pathogenesis of vascular diseases. Inflammatory cells also activate endothelial cells and release both proliferative and cytotoxic mediators. In order to examine the interaction between leukocytes and endothelial cells and the effect of various drugs, we established the methodology for isolating and culturing the endothelial cells from human umbilical vein. Endothelial cells were harvested by using 0.1% collagenase within 48 hr of collecting the cord. Cells were grown to confluency in 96-well plates in Medium 199 containing 20% fetal calf serum, endothelial cell growth supplement, heparin, and antibiotics. Using this method, we obtained a confluent layer of the cells in all the 96 wells within 48 hr. We then examined the effect of peptides, endothelin-1, substance P, and neurokinin-A on the adherence of human blood neutrophils (purity and viability > 98%) to endothelial monolayers. All the peptides enhanced (p < 0.05) the adherence of neutrophils to endothelial cells in a time-dependent manner. This method of endothelial cell culturing is reliable, reproducible, and effective in evaluating the role of various mediators and drugs on the adherence of various white blood cells to endothelium. PMID- 7532468 TI - Detection of prostate-specific antigen immunoreactivity in breast tumors. AB - Prostate-specific antigen (PSA) is a glycoprotein produced by the epithelial cells of the prostate. PSA is currently used in clinical practice to facilitate diagnosis and monitoring of prostate carcinoma. The prostate is an organ that possesses androgen, estrogen, and progesterone receptors, and in this respect is similar to the breast. We postulated that breast tumors might also have the ability to produce PSA. We performed these studies on a collection of 525 tumor specimens collected for routine biochemical determination of estrogen and progesterone receptors. Using a highly sensitive immunofluorometric procedure, we measured the p53 tumor suppressor gene product and PSA. Twenty nine percent of the breast tumor extracts contained detectable levels of PSA immunoreactivity (> 0.05 microgram/L). The immunoreactive PSA content was associated with estrogen and/or progesterone receptor-positive tumors (P < 0.002). No association was found between PSA immunoreactivity and levels of the p53 tumor suppressor gene product (P = 0.37). High performance liquid chromatography and Western blot analysis revealed that the PSA immunoreactivity in the tumor had a molecular weight of 30 kDa, similar to that of seminal PSA. Immunoreactive PSA-positive tumors were associated with younger women (P = 0.012) and earlier disease stage (P = 0.064). We postulate that PSA immunoreactivity may be an additional marker of steroid hormone receptor-ligand action. PMID- 7532467 TI - Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines. AB - We demonstrate that the steroid hormone receptor-positive breast carcinoma cell lines T-47D and MCF-7 can be induced by androgens, progestins, mineralocorticosteroids, glucocorticosteroids, and antiestrogens, to produce prostate specific antigen (PSA). Estrogens failed to induce such stimulation in both cell lines and, in addition, were able to block the induction by androgens in the cell line T-47D. These data support and extend our previous report on PSA production by breast tumors and describe an in vitro system which can be used to study the phenomenon for possible application in prognosis and design of new therapy. PMID- 7532471 TI - Human p80-coilin is targeted to sphere organelles in the amphibian germinal vesicle. AB - Cultured vertebrate cells often display one or more coiled bodies in their nuclei. These are spherical structures approximately 0.5-1.0 micron in diameter that contain high concentrations of small nuclear ribonucleoproteins (snRNPs); they are distinct from nuclear speckles and nucleoli, the other major sites of snRNP concentration. Coiled bodies in human cells contain a unique protein, p80 coilin, that has an M(r) = 80 kDa. Cloned p80-coilin cDNA encodes 576 amino acids with a calculated molecular weight of 62.6 kDa. To determine which of several snRNP-containing structures in the amphibian germinal vesicle (GV) might be the homologue of coiled bodies, we injected myc-tagged transcripts of full-length human p80-coilin into the cytoplasm of Xenopus oocytes and followed the fate of the translated proteins with an antibody specific for the tag. Western blots of GV proteins showed rapid appearance of both full-length and truncated p80-coilin in the nucleus. Immunofluorescent staining of spread GV contents demonstrated specific uptake of p80-coilin by the sphere organelle within 1 h after injection. Similar experiments were performed with a series of deletion constructs that lacked progressively longer segments from the carboxy terminus. A construct that contained only the first 102 amino acids (18% of the molecule) was specifically targeted to the sphere organelle. Conversely, a construct lacking the first 92 amino acids failed to localize, although it was imported into the GV. Thus, a relatively short region at the amino terminus of human p80-coilin is both necessary and sufficient for localization in the sphere organelle. Sphere organelles in the GV and coiled bodies in somatic nuclei are clearly related in composition. We suggest that they are homologous organelles with similar functions in preassembly and sorting of RNA processing components. Differences in their composition suggest functional specialization in the two cell types. PMID- 7532472 TI - Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment. AB - Integrin receptors localize to focal contact sites and interact with the cytoskeleton via the beta 1 cytoplasmic domain. To study the role of this domain in adhesion, we have expressed in NIH 3T3 cells a cDNA consisting of the interleukin 2 receptor alpha subunit extracellular and transmembrane domains, connected to the integrin beta 1 cytoplasmic domain (IL2R-beta 1). Since the extracellular domain of the chimeric protein has no role in adhesion, this protein could uncouple adhesion from intracellular events. As expected, in a cell line expressing IL2R-beta 1, this chimera was directed to focal contact sites. Unexpectedly, the cells exhibited normal adhesion to fibronectin (FN). However, when a rapid reorganization of the cytoskeleton was induced using lysophosphatidic acid (LPA), IL2R-beta 1 cells detached from FN in contrast to wild-type cells. The detachment in response to LPA could be prevented with cytochalasin D, an inhibitor of actin polymerization. These results imply that a beta 1 cytoplasmic domain, which is uncoupled from adhesion, can compete with the cytoplasmic domain of native integrin beta 1 for cytoskeletal proteins. As a consequence, the IL2R-beta 1 protein acts as a dominant negative effector of adhesion by disrupting the integrin-cytoskeleton connection. PMID- 7532474 TI - Inhibition of AIDS-Kaposi's sarcoma cell induced endothelial cell invasion by TIMP-2 and a synthetic peptide from the metalloproteinase propeptide: implications for an anti-angiogenic therapy. AB - In the initial phases of angiogenesis, endothelial cells must degrade and cross the vessel basement membrane, as do tumor cells during invasion and metastasis formation. Various metalloproteinases have been implicated in tumor cell invasion, in particular MMP-2 (72 kDa collagenase IV, gelatinase A), which has been demonstrated to be associated with tumor metastasis formation. Supernatants from AIDS-Kaposi sarcoma (KS) cells induce normal endothelial cells to invade through a reconstituted basement membrane (Matrigel) in vitro, which correlates with the angiogenic potential of KS cells in vivo. Here we demonstrate that two specific inhibitors of MMP-2, TIMP-2 and a peptide from the MMP-2 propeptide region (peptide 74), inhibit endothelial cell invasion induced by AIDS-KS cell supernatants. Smooth muscle cells were much less sensitive to these inhibitors. These data suggest that MMP-2 activation is a key event in endothelial cell invasion, the initial phase of tumor-associated neoangiogenesis. Inhibition of this enzyme could be an effective treatment for KS and tumor-associated angiogenesis. PMID- 7532473 TI - The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183. AB - Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition. PMID- 7532476 TI - Assessing functional communication in aphasia: clinical utility and time demands of three methods. AB - A variety of methods has recently been used to assess everyday communication abilities in aphasic adults. This study compares three such methods for their clinical utility and the amount of a therapist's time they use. The three methods employed a standard rating schedule completed by relatives, analysis of speech elicited through role-play and a partial analysis of everyday conversation samples. The utility of these assessments as a clinical tool was measured in terms of the therapist's time needed, and the assessment's ability to show stability or change of communicative effectiveness on test-re-test measures and to illuminate areas for therapeutic intervention. Eight aphasic adults (five acute and three chronic) were tested on all three assessments, then re-tested after a period of 3 months. The results suggested that, although more time consuming, the partial conversational analysis was a more sensitive measure of stability or change of communicative effectiveness over time than the other two measures, and had the potential advantage for indirect intervention of revealing conversational strategies used by the partner as well as those used by the aphasic individual. PMID- 7532475 TI - A prospective, randomized trial of FK-506 in renal transplantation--a comparison between double- and triple-drug therapy. AB - Previous clinical evaluation of FK506 in renal transplantation has demonstrated equivalent patient and graft survival when compared with cyclosporine-based regimens. However, lower steroid and anti-hypertensive mediation requirements and lower serum cholesterol levels have been seen in patients receiving FK506. In August, 1991, a prospective, randomized trial was begun, comparing FK506/prednisone with FK506/azathioprine/prednisone. Two-hundred-and-four adults were entered into this trial between August 1, 1991, and October 11, 1992. The mean recipient age was 43.8 +/- 13.7 years, with a range of 17.6-78.0 years. Sixty-one (30%) recipients received a 2nd, 3rd or 4th transplant, while 35 (17%) had a PRA greater than 40% at the time of transplant. Thirty-three (16%) of the transplants were in recipients over 60 years of age, Thirteen percent of the kidneys were from living donors; 13% of the cadaveric kidneys were from pediatric donors less than 3 years of age and were transplanted en bloc. The mean cold ischemia time was 31.4 +/- 8.4 hours, and the mean donor age was 34 +/- 2.10 years, with a range from 4 months to 75 years. With a mean follow-up of 9 +/- 4 months, the 1-year actuarial patient survival is 93%; for the two-drug group it is 95%, and for the three-drug group it is 91% (p = NS). One-year actuarial graft survival is 86%; in the two-drug group it is 90%, while in the three-drug group it is 82% (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532477 TI - Validational data on the Warwick Symbolic Play Test. AB - This paper examines the validity of a recently developed test of symbolic play. The Warwick Symbolic Play Test (WSPT) was administered to 60 normal children, 30 girls and 30 boys aged 3-6 years. In addition the children's expressive and receptive language skills were assessed using the Renfrew Action Picture Test and the British Picture Vocabulary Scale respectively. Significant correlations were found between scores obtained on the play test and both language measures. Exploratory statistics indicated that, when age was taken into account, partial correlations between play and language remained moderate. Further analysis of the results also suggested that the relationship between play and language ability as measured by the tests was stronger for the younger children in the sample than for the older children. These results suggest that the WSPT may indeed be a useful assessment instrument, in conjunction with measures of functional play, for assessment of language ability, diagnosis and possible treatment of language disorders, especially for children under the developmental age of 5 years. PMID- 7532478 TI - The clinical characteristics of patients with synchronous squamous cell carcinoma of the esophagus and hepatocellular carcinoma. AB - An ongoing analysis of 762 patients with esophageal cancer revealed 4 (0.52%) male patients with synchronous hepatocellular carcinoma (HCC). A long history of habitual alcohol intake and heavy cigarette smoking was recognized in all four patients and, therefore, the possibility of these two factors being independent risk factors for this double cancer was suggested. Palliative treatment was undertaken since either one or both cancers were too far advanced, or because liver function was poor even in those patients with resectable cancers. The prognosis correlated more closely to the TNM stage of esophageal cancer rather than the HCC and the causes of death were related to the esophageal cancer in all four patients. These findings suggest that, in patients with this combination of double cancer, the state of the esophageal cancer may be a more reliable prognostic factor than that of the HCC and thus, the curability of esophageal cancer is of primary importance. PMID- 7532479 TI - Combination chemotherapy with mitomycin C and cisplatin for advanced gastric cancer with multiple liver metastases. AB - A patient with advanced gastric cancer with multiple liver metastases was treated by reduction surgery at the primary site as well as by the intraarterial administration of mitomycin C (MMC) and cisplatin (CDDP) through a reservoir catheter inserted into the proper hepatic artery. After a palliative subtotal gastrectomy, MMC 8 mg/m2 was administered intraarterially (i.a.) followed by the administration of CDDP 80 or 40 mg/m2 i.a. with an interval of less than 1 week. After the completion of five courses of this regimen, a complete reduction of the hepatic tumors was achieved, while the level of serum carcinoembryonic antigen decreased to the normal range. The patient is currently alive with signs of disease recurrence at 17 months after initial diagnosis, while additional therapy with MMC + CDDP was continuously undergone until 17 months' after initial diagnosis with various interval. Although thrombocytopenia occurred during the treatment, it resolved within a few weeks after completing the combination chemotherapy without any specific treatment. The present case showed a better prognosis than we had expected, which suggested that combination chemotherapy with MMC and CDDP might thus be clinically useful because of its excellent antitumor activity and low toxicity. PMID- 7532480 TI - Genetics of alkane oxidation by Pseudomonas oleovorans. AB - Many Pseudomonads are able to use linear alkanes as sole carbon and energy source. The genetics and enzymology of alkane metabolism have been investigated in depth for Pseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid. The so-called alk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis. This has led to the identification and characterization of of the alkBFGHJKL and alkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives. These then enter the beta-oxidation-cycle, and can be utilized as carbon- and energy sources. Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized. The alkane-oxidizing enzymes used by some of these strains (e.g. two P. aeruginosa strains, a P. denitrificans strain and a marine Pseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid. PMID- 7532482 TI - [Clinical study on comparison of tonifying kidney replenishing vitality and removing blood stasis and resolving mass in the treatment of prostatic hyperplasia]. AB - 70 patients with prostatic hyperplasia were observed. Among them 34 cases were treated with the regime of tonifying Kidney-replenishing Vitality (TKRV) and the other 36 cases were treated with removing blood stasis and resolving mass (Xiao Jin Dan). The dysuria of the patients in both groups was improved after treatment. The results of the transrectal ultrasonotomography showed as follows: (1) The effect on the volume of residual urine: the average values of the TKRV group were 43.29 +/- 14.03 ml before treatment and 17.24 +/- 5.13 ml after treatment (P < 0.05), that of Xiao Jin Dan were 21.17 +/- 5.57 ml and 7.37 +/- 2.87 ml respectively (P < 0.01). (2) The effect on the size of the prostate gland: only 10 cases in the TKRV group were measured both before and after treatment, the average size of the prostate gland were 46.30 +/- 8.23 ml and 43.60 +/- 4.52 ml respectively (P < 0.05), and 4 cases were decreased in the size after treatment. 35 cases in Xiao Jin Dan group were measured before and after treatment and their size of prostate gland were 47.83 +/- 2.50 ml and 43.45 +/- 2.24 ml (P < 0.01) respectively. 27 out of the 35 cases were decreased in their size of prostate gland. The results showed that the therapeutic effect of Xiao Jin Dan was slightly better than that of the TKRV, which suggested that the prostatic hyperplasia was closely related to the syndrome of Kidney-Deficiency as well as to the syndrome of Blood Stasis. So it is important that we combined the reinforcement and elimination in the treatment of prostatic hyperplasia. PMID- 7532481 TI - Circulating adhesion molecules ICAM-1, VCAM-1 and E-selectin in systemic vasculitis: marked differences between Wegener's granulomatosis and systemic lupus erythematosus. AB - Inflammatory systemic disorders with renal tissue damage require the adherence of polymorphonuclear leukocytes to the endothelium, which is mediated by cell surface adhesion molecules. This study measured the serum concentrations of circulating adhesion molecules [intercellular adhesion molecule 1 (cICAM-1), vascular cell adhesion molecule 1 (cVCAM-1), cE-selectin] by sandwich enzyme linked immunosorbent assay in Wegener's granulomatosis (n = 25) and systemic lupus erythematosus (n = 50). Active Wegener's granulomatosis with cellular crescent formation was associated with significantly raised cICAM-1 levels, an elevated histological activity index, and a high rate of hemodialysis in comparison to Wegener's granulomatosis with fibrocellular crescents or lupus nephritis. The activity index and cICAM-1 levels can be used as parameters to predict renal outcome in Wegener's granulomatosis. cVCAM-1 levels in Wegener's granulomatosis were significantly higher in patients with hemodialysis compared to those without hemodialysis. In lupus nephritis only cVCAM-1, not cICAM-1 or cE selectin, were significantly higher (P < 0.01) than controls. cVCAM-1 levels in both diseases were significantly higher than in controls. Only in Wegener's granulomatosis did cICAM-1 and cVCAM-1 levels decrease with clinical remission; in systemic lupus erythematosus the levels remained unchanged. cE-selectin levels were not significantly elevated in systemic vasculitis or collagen disease although individual patients revealed high serum concentrations. These data suggest that levels of circulating adhesion molecules reflect different pathophysiological processes in systemic vasculitis and collagen disease and may be used as markers of disease activity and renal outcome. PMID- 7532484 TI - [Mechanisms responsible for protective effect of Sini decoction on ischemic myocardium in mice]. AB - The possible mechanisms responsible for the protective effect of Sini decoction (SND) on the ischemic myocardium were studied on the mice model of pituitrin induced myocardial ischemia in terms of free radical. Results indicated that SND significantly decreased the concentration of oxygen free radical and the content of malondialdehyde (MDA), increased nutritional blood flow and the activity of superoxide dismutase (SOD) in ischemic myocardium. These results suggested that three mechanisms whereby SND exerted its protective effects existed; improving the perfusion of ischemic myocardium, attenuating the injurious action of free radical and strengthening the free radical defense. PMID- 7532483 TI - [Effects of dachengqi decoction on gut hormones and intestinal movement after cholecystectomy]. AB - In order to elucidate the mechanism of postoperative intestinal dysfunction and the effects of Dachengqui Decoction (DCQD) on it, somatostatin (SS), gastrin (GAS), vasoactive intestinal polypeptide (VIP), substance P (SP), motilin (MOT) and atrial natriuretic peptide (ANP) were determined, frequency and spectrum of peristaltic sound were simultaneously analyzed in 31 subjects undergoing cholecystectomy, the value of pre-, post-operation and post-medication were compared. Plasma SS and MOT decreased postoperatively (P < 0.05), DCQD elevated SS and MOT to higher level than preoperation, VIP, SP increased for half fold (P < 0.05). Gurgling sound diminished after operation, whereas DCQD normalized it. Peak frequency (Fmax) of gurgling ranging from 234.4 to 468.2 Hz preoperatively, mean frequency (FA) was 341.8 +/- 30.9 Hz postoperatively. FA reduced to 322.3 +/ 79.4, DCQD elevated it to 374.2 +/- 57.1 Hz. The result suggested that intestinal motility was disturbed after cholecystectomy, bowel was in dystonic status, accompanying with decreased plasma MOT, DCQD promoted intestinal peristalsis and enhanced it's tonus. The influence of gut peptides might be one of the important pathway that DCQD works. PMID- 7532486 TI - [Epidemiologic aspects of human herpes virus infection type VI]. AB - The paper presents a review of foreign publications (35 references) regarding the history of isolation and investigation of one of the herpesviruses--a new lymphotropic human herpesvirus type 6 (HHV-6). The wide spread of the virus in the human population is demonstrated. Both patients with manifestative types of the disease and asymptomatic HHV-6 carriers are supposed to provoke the infection. The disease can be transmitted by the droplet and genital routes of infection, via hemotransplantation, and by the vertical transmission of the agent. HH-6 was found to play an etiological role in the development of sudden exanthema in early childhood. PMID- 7532485 TI - [Hospital respiratory infections in children]. AB - Acute respiratory superinfections (ARS) induced by secondary nosocomial respiratory pathogens and characterized by the subsequent generalization of the process have been reported as one of the main contributions to pediatric inpatient mortality. The recurrent infections were observed in all types of hospitals; they might be induced by the whole group of respiratory pathogens, but adeno-, coronaviral and pneumococcal ones are predominating. The ARS clinical picture showed various patterns determined both by the etiology of basic and recurrent infection and by a child's immunity. There was a correlation between the severity of ARS and the level of specific immunoglobulins, especially of secretory origin. PMID- 7532487 TI - [Production of monoclonal antibodies to the human respiratory syncytial virus]. AB - The paper describes the procedure for producing monoclonal antibodies to human respiratory asyncytial virus using hybridoma technology. Two groups of antibodies were identified in the study of their specific activity. The representatives of the first group preferably reacted with purified virus antigens. Those of the second one binded mostly to unpurified virus antigens from the cultural fluid. The antibodies of these groups appear to be different in their epitope specificity. PMID- 7532488 TI - [Genetic predisposition to latent influenza A virus in children with blood type B(III) as a possible cause of new epidemiologic strains in the countries of South Eastern Asia]. AB - Regular clinical and epidemiological surveys of two groups of healthy 14-16-year old boys were carried out for 3 years from September 1989 to May 1992 to investigate the latent circulation of influenza A viruses in the human population. Once or twice a quarter clinical materials were laboratorily studied to detect virus antigens, virus-specific NA-sequences and to determine changes in humoral immunity. The latent circulation of influenza A viruses might be identified in 10-20% of cases long before (2-4 months) the onset of epidemic development. The incidence of epidemic isolations was found to reach 33-36%, gradually decreasing from 29 to 9 or 3.5% in the postepidemic period. The long term persistence (for 5 months) of virus antigens was reported among healthy children who had chiefly blood group B(III). The new hypothesis that the genetic predisposition of children with blood group B(III) to latent persistence of influenza A viruses may be a possible cause of the emergence of new epidemic strains in the South-East Asian countries has been forwarded. PMID- 7532490 TI - [Influenza A virus antigens in children with congenital pathology of central nervous system]. AB - The latent circulation of influenza A virus in nursing home children with congenital central nervous abnormality was studied during 1989-1993 by the present-day diagnostic methods for detection of viral antigens. The influenza A virus antigen detection rates correlated with age, genetic predisposition, prior acute respiratory diseases in mothers during their pregnancy. There is evidence for long-term (up to 17 months) detection of influenza virus antigens in nasal secretions, as well as for prolonged (up to 7 months) detection of the same genomic sequences of HA gene both in leukocytes and nasal washes. PMID- 7532489 TI - [Long-term carriers of genomic structures of the influenza virus in the blood leukocytes of children with congenital pathology of central nervous system]. AB - Children with central nervous abnormality were followed up for a long time (180 days). The clinical samples (nasal swabs, blood samples) were investigated for influenza virus antigens or RNA detection by virologic and molecular biological techniques. No viral isolate was found throughout the follow-up. The use of the polymerase chain reaction made it possible to reveal the long-term persistence (for 180 days) of NS- and genes of influenza A (H1N1) viruses in the leukocytes and of HA-gene in the nasal swabs. No NS-gene was found in the nasal swabs. The polymerase chain reaction appears to be more effective for the diagnosis of persistent influenza infection that the conventional techniques- immunofluorescence and enzyme-linked immunosorbent assay (ELISA). PMID- 7532492 TI - [Pathogenesis of severe influenza]. AB - The paper analyzes the contribution of active oxygen radicals whose formation was observed in the inflammatory foci, as well as in generalized influenza infection. There is a correlation between the inactivation of protease inhibitors (alpha-1 antitrypsin) and the increase in the efficacy of virus protein proteolysis, in particular hemagglutinin and hence the acceleration of the infection activation of viral particles. The proteolytic provision of infection viral particle activation occurs due to cell membrane lipid peroxidation, by impairing their barrier function, this is an important condition for the generalization of infection and development of endemic processes. With this, involvement of St. aureus that secretes serine proteases accelerates the closing of the vicious circle of pathogenetic processes and further generalization of infection. The scheme of pathogenesis is based on the data on the molecular mechanisms of formation of free oxygen radicals, as well as on the structure of cell inhibitors of virus proteins. PMID- 7532491 TI - [Influenza: current status and epidemiologic situation]. AB - In the period of 1993 to 1994, influenza activity caused by circulation of viruses antigenically structurally related to the strain A/Beijing/32/92 (H3N2) began in the countries of North America and West Europe in October-November 1993, by spreading to the countries of East Europe, the Urals, and West Siberia. Epidemic events in China and in the Far East were evoked mainly by the influenza B virus. The influenza A (H1N1) viruses did not widely spread. Single isolates (about 1% of all the influenza A virus isolates, as evidenced by the CDC, Atlanta, USA) of this subtype were etiological agents of sporadic morbidity. A fourteen-year period (1977-1991) of the epidemic activity of the influenza A (H1N1) virus seems to be over. The last epidemic of the influenza A (H3N2) virus has a moderate intensity. Among all the age-group populations, children were largely afflicted. The highest incidence in Russia was recorded in Arkhangelsk, Barnaul, Nizhny Novgorod, Omsk, Samara, and Smolensk where approximately 16-18% of children under 14 years of age and 3.5-5% of the population of 15 years or more fell ill. The highest incidence was recorded in Minsk and Vilnus too. PMID- 7532495 TI - [Mortality rate of influenza, acute respiratory diseases and acute pneumonia as a health status factor]. AB - The rates of mortality due to influenza, acute respiratory disease and acute pneumonia were studied in relation to the patients' age, sex, epidemic etiology. Quantitative characteristics of excess mortality might be used in the assessment of the influenza epidemic intensity. The most vulnerable groups of the population were identified in whom a comprehensive complex of measures should be made to reduce ARD mortality rates. PMID- 7532493 TI - [Polymorphism of current human influenza A and B virus population]. AB - During the past years, the etiological situation has been significantly complicated. It is characterized by simultaneous circulation of A(H1N1), A(H3N2) and influenza B viruses and by the isolation of reassortant strains and viruses, which are atypical in relation to the process of their natural variability. The antigenic properties of epidemic strains and unusual isolates were investigated. The marked heterogeneity of the A(H3N2) influenza viruses was demonstrated. It was determined by the circulation of several antigenic variants during the epidemic. Two separate antigenic lineage of the influenza B viruses- b/Victoria/2/87 and B/Yamagata/16/88--cocirculated in our country in 1991. Since 1986, all the influenza A(H1N1) viruses have been considered to be varieties of the reference strain A/Taiwan/1/86. A direct correlation was found between some atypical viruses and the vaccine strains previously used. PMID- 7532494 TI - [Radiologic characteristics of lung pathology in children with acute respiratory diseases]. AB - Analysing the X-ray picture of childhood pneumonias from 8 X-ray parameters chosen by the authors has ascertained that bacterial pneumonias are most characterized by 2 X-ray signs: total segmentation and homogenicity of the lesion, whereas viral pneumonias by 4-5 or more out of the 8 chosen signs, among which there is partial segmentation and unhomogeneity of the lesion with increased lung outline within the lesion focus. This enables at least 96% probability to differentiate viral and bacterial pneumonias. The differences between pneumonic changes in viral respiratory infections are not so well defined. But at the same time the X-ray pattern of RS infection most commonly includes an obstructive component (most infrequently in influenza and parainfluenza), partial segmentation and a limited area of infiltration (only 1-2 segments are involved). Adenoviral infections are most frequently characterized by increased lung outline beyond the pneumonic focus, indicating concomitant bronchitis beyond the pneumonic area, as well as by the atelectasis and extensive infiltration (involving 3-5 segments). Thus, the X-ray technique enables etiological rapid diagnosis during the first three days of the onset of the disease, providing timely initiation of etiothropic therapy. PMID- 7532496 TI - [Photochemotherapy in the treatment of acute pneumonias]. AB - The application of photochemotherapy in acute pneumonia is based on pneumonia pathogenesis and the mechanism of ALIB- (autotransfusion of laser irradiated blood) and AUVBI- (autotransfusion of ultraviolet blood irradiation) techniques. The ALIB is preferable since the technique requires no device sterilization. The complex treatment of acute pneumonia with ALIB and AUVBI was found to enhance therapeutic efficacy, to decrease the severity of the disease course, to reduce its duration and hospital stay in inpatients. No considerable impact of ALIB on the duration of pneumonia was observed, but X-ray examination revealed a definite reduction in pulmonary microcirculatory disorders. Laser therapy promotes the normalization of functional T-lymphocytic activity. ALIB should be applied individually and in relation to the severity of the disease, to the distribution of an inflammatory pulmonary process and to the duration of the disease course. PMID- 7532498 TI - [Pharmacologic effect of anthropogenic air pollutants on children and prognosis for correction of these disorders]. PMID- 7532497 TI - [Remantadine in the treatment of influenza in children]. AB - The therapeutical efficacy of remantadine was studied in influenza in- and out patients aged 3-14 years during consecutive influenza epidemics. The drug was given to ill children on the first or second day from the onset of the disease at a dose of 1.5 mg/kg of body weight 3 times a day for 3 days. The curative effect of remantadine was established not only in influenza A, but also in influenza B infections, as well as in mixed infections with other respiratory agents and with pathogens of non-influenza etiology, which might be the result of its antitoxic activity. Remantadine application decreased fever and complication rates and reduced the duration of intoxication symptoms and the diseases as a whole. A more pronounced drug activity was observed when given to children on the first day of the diseases. Remantadine exerted no toxic effect on the basic functions of the child's body. Ill children displayed no inhibitory activity on cell and humoral immunity, as well as on a serum antibody response to hemagglutinin of influenza virus. PMID- 7532499 TI - [Some characteristics of circulation and respiratory viruses in the country]. AB - The 17-year observation of changes in the etiological pattern of acute respiratory diseases (ARD) in the country has demonstrated that different types of adenoviruses are prevalent in the circulation on the whole territory. Adenoviral infection was found to prevail among non-influenza infections in the Ukraine, RSV infection in the north of the Russian Federation and in the Far East; piconaviral infections caused by Echo and Coxsackievirus B in the Urals and Byelarus Republic. However, some years were marked by a change in various serotypes of adenoviruses and picornaviruses in different climatic and geographical zones and in different age population groups. There was a seasonal variability in the spread of respiratory viruses and herpes simplex virus type I. Adenovirus infection is characterized by fall-winter-spring seasonal patterns. RSV-infection and herpes infection are characterized by fall-winter seasonal patterns. In contrast, the acute respiratory infections of picornavirus etiology show a well-defined summer seasonal pattern. PMID- 7532500 TI - Need for rigorous assessment of palliative care. PMID- 7532502 TI - Unusual cause of pseudomonal infection. PMID- 7532501 TI - Evaluation of a palliative care service: problems and pitfalls. AB - OBJECTIVE: To evaluate a palliative care home support team based on an inpatient unit. DESIGN: Randomised controlled trial with waiting list. Patients in the study group received the service immediately, those in the control group received it after one month. Main comparison point was at one month. SETTING: A city of 300,000 people with a publicly funded home care service and about 200 general practitioners, most of whom provide home care. MAIN OUTCOME MEASURES: Pain and nausea levels were measured at entry to trial and at one month, as were quality of life for patients and care givers' health. RESULTS: Because of early deaths, problems with recruitment, and a low compliance rate for completion of questionnaires, the required sample size was not attained. CONCLUSION: In designing evaluations of palliative care services, investigators should be prepared to deal with the following issues: attrition due to early death, opposition to randomisation by patients and referral sources, ethical problems raised by randomisation of dying patients, the appropriate timing of comparison points, and difficulties of collecting data from sick or exhausted patients and care givers. Investigators may choose to evaluate a service from various perspectives using different methods: controlled trials, qualitative studies, surveys, and audits. Randomised trials may prove to be impracticable for evaluation of palliative care. PMID- 7532503 TI - Biological influences on criminal behaviour: how good is the evidence? PMID- 7532504 TI - Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius. AB - The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species. PMID- 7532505 TI - Detection of mip-like sequences and Mip-related proteins within the family Rickettsiaceae. AB - The Mip surface protein, a prokaryotic analog of the FK506-binding proteins, enhances the ability of Legionella pneumophila to infect macrophages and protozoa. Using mip-specific probes and low-stringency Southern hybridizations, we have detected DNA sequences homologous to mip within Coxiella burnetii and Rochalimaea quintana. Using specific anti-Mip antisera and immunoblot analysis, we also detected Mip-related proteins within these bacteria as well as within Rickettsia and Ehrlichia species. These data suggest that Mip-related proteins have broad significance for host-parasite interactions. However, they also indicate that care must be exercised when using mip probes or anti-Mip antibodies for the detection of Legionella organisms in water or clinical samples. PMID- 7532506 TI - [Brief definitions of concepts in medical ethics. 33. Assisted suicide--the problem of the "double effect"]. PMID- 7532508 TI - The possible role of G-CSF in the pathogenesis of Sweet's syndrome. AB - Sweet's syndrome (SS) is characterized by the clinical features of fever, leucocytosis, neutrophilia and the sudden onset of asymmetric, often very painful skin lesions and dense dermal infiltrates of mature neutrophils without signs of vasculitis. Apart from idiopathic cases the disease is frequently associated with hematological malignancies, but it may also be observed in patients with solid tumors, mainly tumors of the genito-urinary tract. In the past, numerous theories have been proposed to explain the pathogenesis of this rare disease. SS has been interpreted as a direct response to mechanical and chemical irritants, an infectious disease or a disorder of neutrophilic chemotaxis and/or phagocytosis, but most often it has been described as a hypersensitivity reaction. Each of these theories can account for particular symptoms, but none of them reconciles the dominating clinical and laboratory features of the disease. Furthermore recently published casuistic observations suggest the involvement of certain cytokines in particular G-CSF and Il-6 in the pathogenesis of the disease, which might explain many of the observed clinical and laboratory findings. The following article summarizes these data and gives a review of the current literature. PMID- 7532507 TI - Antibody-induced modulation and intracellular transport of CD10 and CD19 antigens in human malignant B cells. AB - Antibody-induced antigenic modulation (AIAM) is a complex biological phenomenon closely resembling other receptor-ligand interactions. Following exposure to specific antibodies, surface antigens are usually rapidly redistributed on the cell surface and internalized. A subsequent intracellular processing results in dissociation of the antigen-antibody complexes, degradation, exocytosis and recycling. AIAM plays an important role in MoAb-targeted therapy of hematopoietic malignancies contributing to escape of tumor cells from immunodestruction. On the other hand, internalization of MoAbs used as carriers of toxins and drugs is a prerequisite of therapeutic efficacy. Even though MoAbs directed against CD10 and CD19 have been used in immunotherapy of B cell malignancies, some aspects regarding AIAM of these Ags are not yet fully understood. Both Ags are modulated by specific MoAbs and internalized through the same pathway, however, the kinetics of AIAM vary from one Ag to another and from one cell type to another. Recent studies with malignant B-cell lines show that, under certain experimental conditions, the extent and rate of surface clearing, uptake and intracellular transport are considerably higher in the case of CD19 than in CD10 and higher in less mature cells compared with more mature cells. These observations may be useful in the selection of MoAbs for immunotherapy, although they need to be confirmed with fresh malignant B cells. PMID- 7532509 TI - [A case of juvenile embryonal germ cell tumor of the mediastinum]. AB - A rare case of a embryonal germ cell tumor of the mediastinum is presented in a 22 year old male. A radical excision of the tumor was performed, followed by chemotherapy. A five year survival was observed without evidence of a recurrence. PMID- 7532510 TI - [Lung fibrosis after bleomycin therapy]. AB - A case of lung fibrosis is presented in a 44 year old male in whom interstitial pulmonary changes were observed in the course of bleomycin therapy of malignant granulomatosis. Following introduction of corticosteroids a radiological, clinical and functional improvement was observed. The functional changes were analyzed during the 7 month period of treatment. This case suggests that post bleomycin induced injury can be reversed with appropriate high doses of corticosteroids. PMID- 7532511 TI - Cardiovascular effects of L-arginine as physiological precursor of nitric oxide. AB - In this study the cardiovascular effects of L-arginine were investigated. Perfusion with sorbitol L-arginine 5% solution induced a significant decrease of the systolic blood pressure in the human arterial hypertension. The hypotensive effects of L-arginine are accompanied by an increase of the ventricular ejection fraction. In vitro, L-arginine as physiologic precursor of nitric oxide induced a smooth muscle relaxation more evident in the coronary vessels than in the thoracic aorta rings. Selective inhibition of NO-synthetise with L-NAME reduced the L-arginine vasodilation and intensified the vasoconstrictor effect of phenylephrine. PMID- 7532512 TI - Trypanosoma brucei brucei and T. b. gambiense: stumpy bloodstream forms express more CB1 epitope in endosomes and lysosomes than slender forms. AB - CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle. PMID- 7532514 TI - A novel canine model of partial outlet obstruction secondary to prostatic hypertrophy. AB - Benign prostatic hyperplasia (BPH) is a major medical problem in the United States. The primary medical complication of BPH is progressive obstruction of the urethra and a subsequent in reduction the ability or the bladder to empty efficiently. The urodynamic characteristics associated with BPH include hyperreflexia, increased bladder capacity, increased frequency, decreased flow rate, and increased residual volume. Although there currently are individual animal models of prostate enlargement and animal models of partial outlet obstruction, there is no model of progressive obstruction secondary to prostate enlargement. The primary objective of the current study was to develop a canine model of BPH that would secondarily result in partial urethral obstruction and impaired urodynamics. Our model consists of encapsulating the prostate in a nylon mesh to prevent the growth of the prostate into the peritoneal cavity and then treating the dog with steroids to induce prostate growth and subsequently produce urethral constriction. The results demonstrate that encapsulation of the dog prostate and administration of steroids results in an increase in prostate mass simultaneously with an increase in urethral pressure and in changes in bladder contraction consistent with the presence of partial outlet obstruction. This preliminary study demonstrates that by preventing the outward growth of the steroid-stimulated prostate, urethral obstruction resembling BPH can be produced. PMID- 7532518 TI - Toxoplasmosis in England and Wales 1981 to 1992. AB - We have examined laboratory reports of toxoplasmosis received by the PHLS Communicable Disease Surveillance Centre between January 1981 and December 1992 in order to describe epidemiological trends in the three main clinical manifestations of toxoplasmosis-lymphadenopathy, eye disease, and neurological disease; and the two most important risk groups-fetuses and people whose immunity is impaired. The total numbers of reports each year did not change significantly between 1981 and 1992 and were similar to the numbers between 1976 and 1980, but different trends emerged in each subgroup. Reports of acute lymphadenopathic toxoplasmosis declined in children and young adults and eye disease associated with toxoplasmosis fell markedly in all age groups. Reports of neurological disease and severe toxoplasmosis complicating impaired immunity, due mainly to HIV infection or transplant surgery, rose. Reports of infections diagnosed in pregnancy rose steeply in the late 1980s although reports of congenital toxoplasmosis were no more common than in 1975 to 1980. Reports of acute toxoplasmosis came mainly from southern England. The emergence of these diverse trends from apparently unchanging totals emphasises the importance of surveillance systems capable of discrimination. PMID- 7532519 TI - A survey of health education material for the primary prevention of congenital toxoplasmosis. AB - We have assessed health education material for the prevention of congenital toxoplasmosis in terms of its epidemiological and clinical information and preventive advice. Nineteen leaflets and booklets were evaluated: fourteen were produced locally, ten for patients and four for professionals; and five were distributed nationally, including two sponsored booklets and one produced by the Department of Health. Material was compared with 'standards' derived from recently published reviews of toxoplasmosis and the Department of Health's booklet. The amount of information about toxoplasmosis, and the advice on how to avoid it, differed widely. Some booklets contained factual errors and some of the advice was impractical and even hazardous. Booklets contained on average between a fifth and a half of the standard desired for epidemiological and clinical information and preventive advice. Leaflets devoted to toxoplasmosis were more informative than general guides. Presentation was assessed informally: medical terminology inappropriate for a 'lay' audience was used, booklets were published in English only, and the date of publication was not printed. Health education is an important means of primary prevention of disability caused by congenital toxoplasmosis. Our study suggests that it has not received the scientific appraisal and commitment needed to make it effective. PMID- 7532520 TI - Mycobacterial infections: guidelines for reporting and revised format for presentation. AB - A new format for the presentation of mycobacterial infections appears in the accompanying CDR Weekly (Communicable Disease Report 1995; 5:22). To ensure accuracy and consistency in the collation of information about mycobacterial infections, the PHLS Communicable Disease Surveillance Centre has developed guidelines for reporting and has adopted definitions for the classification of sites of infection. These guidelines and definitions are published here for the first time. PMID- 7532516 TI - Nitric oxide synthase and nitric oxide-mediated effects in lower urinary tract smooth muscles. AB - In the lower urinary tract smooth muscles, both excitatory and inhibitory non adrenergic, non-cholinergic (NANC) nerves and neurotransmission can be demonstrated. An inhibitory, relaxation-mediating system may serve not only the detrusor, the trigone, and the bladder neck/urethra, but may also be of importance for their integrated function. Available data suggest that nitric oxide synthase (NOS) is localized in nerve fibres of the lower urinary tract, preferably in the outflow region, and evidence has accumulated that L-arginine derived nitric oxide (NO) is responsible for the main part of the inhibitory NANC response. Coinciding localization of NOS positive nerves with nerves expressing acetylcholine esterase, vasoactive intestinal peptide, and neuropeptide Y, suggests that NO may have a role both as a directly acting transmitter and as a modulator of efferent neurotransmission. In addition, NO may be involved in afferent neurotransmission. Theoretically, NO released from nerves in the detrusor, could be one factor keeping the bladder relaxed during filling. However, the detrusor has a low sensitivity to NO and agents acting via cyclic GMP, which makes it less likely that NO has a role as a relaxant neurotransmitter. This does not exclude that NO may modulate the effects of other transmitters, or that it has an afferent function. NO effectively relaxes isolated smooth muscle preparations from the outflow region, suggesting that it may be involved in the decrease in intraurethral pressure associated with normal micturition, and with the excessive urethral pressure variations ("unstable urethra"), which may be associated with certain voiding disturbances in women.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532517 TI - Physiology of the vas deferens. AB - Seminal emission occurs in response to rhythmic contractions of male secondary sex organs, including the vas deferens. Although contraction of the vas is directly due to adrenergic mechanisms, numerous substances modulate the release of norepinephrine from sympathetic pathways. These substances include local endogenous factors and neurotransmitters such as acetylcholine and NPY. Many substances are capable of altering the contractility of the vas deferens by modulating neural transmitter release or the basal tone of this smooth muscle. Because multiple pathways and substrates are capable of affecting its contractility, it is not surprising that drugs and metabolic disorders influence the function of the vas deferens and, ultimately, male fertility. PMID- 7532521 TI - Current respiratory infections. PMID- 7532515 TI - Regulation of tone in penile cavernous smooth muscle. Established concepts and new findings. AB - Since Benson, in 1983, reported on a potent nonadrenergic, noncholinergic (NANC) transmitter postulated to relax penile vessels and the corpus cavernosum, much new information on the mechanisms of contraction and relaxation of corporeal smooth muscle and penile vasculature has been obtained. The information currently available suggests that NANC transmitters may be involved in both contractile and relaxant responses of penile erectile tissues. There is good experimental evidence to allow the assumption that neurogenic nitric oxide (NO) is a mediator of penile erection, but even if NO probably is the most important factor for relaxation of penile vessels and the corpus cavernosum, this does not exclude the possibility that other agents released from nerves may have a modulatory function in this process. However, the roles of, for example, vasoactive intestinal polypeptide and related peptides as neurotransmitters and/or neuromodulators in the nervous control of penile erection have yet to be established. The restricted availability of human penile erectile tissues has led to the use of cavernous tissue and penile vessels from animals, both for screening and for detailed analysis of mechanisms previously demonstrated to exist also in human tissues. When interpreting the results obtained, it is important to stress that there may be important differences between human and animal tissues, that each of the tissues only gives a piece of information on the complex process of penile erection, and that the physiological and clinical importance of results from such experiments may be limited. The differing responses in different parts of the vasculature within the penis and the multiplicity of putative transmitters present in the corpus cavernosum and in perivascular nerves make further investigations necessary, as do the interactions between transmitters and neuromodulators at the neuromuscular junction, and between the neural and endothelial control of vascular tone. PMID- 7532522 TI - Simultaneous determination of tryptophan, 5-hydroxytryptophan, 5 hydroxytryptamine, 5-hydroxyindoleacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 3-methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid. AB - Serotonin (5-hydroxytryptamine) metabolism may be influenced by its precursor tryptophan. A method utilizing reversed-phase high-performance liquid chromatography and electrochemical and ultraviolet detection with a mobile phase composed of acetate buffer and methanol has been developed for determination of tryptophan, its metabolites 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, as well as 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and 3 methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid (CSF). The electrochemical potential is set at 0.6 V in order to reduce the background current. Since tryptophan is not electroactive at this potential, it is detected by ultraviolet absorbance. The present method is simple, rapid, specific and accurate as compared with a previously reported method. No sample pretreatment is necessary and it takes ca. 20 min to run a sample. The concentrations of the compounds measured in CSF are similar to those obtained by HPLC in previous reports, although there are still arguments about the true level of serotonin in CSF. PMID- 7532523 TI - Partitioning of high-density lipoproteins in charge-sensitive two-phase systems. AB - Aqueous polymer two-phase systems characterized by a difference in the electrical potential between the upper and the lower phase (charged systems) are useful tools for the detection of changes in the surface charge and hydrophobicity of high-density lipoproteins (HDL). While the large particle size of low-density lipoproteins (LDL) leads to accumulation at the interface, the smaller diameter and the higher surface charge density of the native HDL particles allows partitioning without aggregation at the interface. Charged two-phase systems can be used to check the native state of HDL samples. Moreover, these systems would be suitable for investigating the hydrophobicity and surface charge of modified HDL. PMID- 7532524 TI - Hepatitis C virus appears to replicate not only in hepatocytes but also in hepatocellular carcinoma cells as demonstrated by immunostaining. AB - Persistent infection with hepatitis C virus is a major risk factor for the development of hepatocellular carcinoma (HCC). It has been unclear whether hepatitis C virus replicates in HCC. A total of 39 hepatitis B surface-antigen negative HCC were resected surgically at Nihon University Itabashi Hospital between January 1990 and December 1992. Of them, serum anti-hepatitis C was positive in 28 cases, negative in three and not examined in eight. The indirect immunoperoxidase technique was used for detection of the hepatitis C core antigen on formalin-fixed, paraffin-embedded sections. Positive staining was found within non-tumor hepatocytes in 22 (78.6%), one and four cases, respectively. Fourteen (58.3%) of these 24 cases, which were positively immunostained in non-tumor hepatocytes (three were excluded because of complete necrosis of HCC), were also stained for the core antigen in HCC cells. Core antigen was stained in both hepatocytes and HCC cells within the cytoplasm, diffusely or focally with varying intensity in a local or patchy distribution. Hepatitis C virus appears to replicate in HCC cells and the significance of such viral replication in hepatocarcinogenesis remains to be clarified. PMID- 7532525 TI - New approaches to and current treatment of leptomeningeal metastases. AB - Leptomeningeal metastases (LM), a common problem in neurooncology, occur in approximately 5% of all patients with cancer. Aside from frequent focal signs and symptoms, LM affects the entire neuraxis; therefore, staging and treatment must encompass all cerebrospinal fluid compartments. Central nervous system staging of LM includes contrast-enhanced cranial computed tomography or magnetic resonance imaging, contrast-enhanced spine magnetic resonance imaging or computed tomographic myelography, and radionuclide cerebrospinal fluid flow study. Treatment of LM includes involved-field radiotherapy of bulky or symptomatic disease and intracerebrospinal fluid drug therapy. At present, intracerebrospinal fluid drug therapy is confined to three chemotherapeutic agents (methotrexate, cytosine arabinoside, and thiotepa) and is administered in a variety of schedules by either intralumbar or intraventricular drug delivery. Although treatment of LM is palliative, with an expected median patient survival of 6 months, it often provides stabilization and protection from further neurologic deterioration in patients with LM. PMID- 7532513 TI - Non-adrenergic, non-cholinergic control of the urinary bladder. AB - In the urinary, bladder, ATP is an excitatory neuromuscular transmitter, possibly a cotransmitter with acetylcholine from postganglionic parasympathetic nerves, which activates P2X-purinoceptors. The synthesis of prostaglandins is closely linked to the activation of P2X-purinoceptors, and these compounds make a significant contribution to non-cholinergic neurogenic responses. Many neuropeptides, such as NPY, VIP, somatostatin, SP and CGRP, are found in nerves innervating the lower urinary tract, but it is unlikely that any is a neuromuscular transmitter in the detrusor; rather, they may act as potent modulators of sympathetic and parasympathetic transmission. Modulatory actions are shown by GABA par excellence; this compound is also well represented in vesicular neurons and, via activation of GABAA- or GABAB-receptors, can potentiate or inhibit parasympathetic transmission. Although not discussed in depth in this review, the urinary bladder shows extraordinary plasticity in expression of nerves and of their transmitters and receptors under pathophysiological conditions, including pregnancy and ageing as well as disease states. Finally, the accessibility of the urinary bladder and the enormous range of chemoreceptors that it possesses has led to its being used extensively for pharmacological investigations of transmitter and drug receptors and their subclasses. PMID- 7532527 TI - Thoracoscopically assisted esophagectomy with gastric pull-up for esophageal cancer. AB - Thoracoscopically assisted esophagectomy with gastric pull-up was done in a 56 year-old man with esophageal cancer. The thoracoscopic technique, including the use of intraoperative ultrasound, is discussed. The addition of thoracoscopy offers safer and more complete esophageal dissection than transhiatal blunt esophagectomy and is less invasive than conventional thoracotomy. PMID- 7532526 TI - Enhanced vascular responsiveness to Bay K 8644 in mineralocorticoid- and N-nitro arginine-induced hypertension. AB - The present study evaluates the response to the L-type voltage gated calcium channel agonist Bay K 8644 in two forms of experimental hypertension (mineralocorticoid- and hypertension induced by the nitric oxide synthase inhibitor N omega-nitro-L-arginine (N-Nitro arginine)) and under conditions of acute stretch. These studies test the hypothesis that increased L-type calcium channel activity in vasculature is a hallmark or general characteristic of hypertension. Male Sprague-Dawley rats were made hypertensive by subcutaneous implantation of deoxycorticosterone acetate (200 mg/kg DOCA) and given normal or high salt water (1% NaCl + 0.2% KCl); other rats were made hypertensive by ingestion of N-Nitro arginine (2% in water). Systolic blood pressures (SBP) were taken by the standard tail cuff method. Following development of hypertension, rats were anesthetized, and aortae or mesenteric arteries were isolated for measurement of isometric contractile force. Cumulative concentration response curves to Bay K 8644 (10(-10) to 10(-6) M), KCl (6 to 100 mM), or phenylephrine (10(-10)-3 x 10(-7) M) were evaluated. Isolated mesenteric arteries from rats given both DOCA and salt were most sensitive to Bay K 8644 (SBP = 191 +/- 6 mmHg, -log EC50 = 7.78 +/- 0.13), followed by rats receiving high salt alone (SBP = 118 +/- 6 mmHg, -log EC50 = 7.30 +/- 0.17), DOCA alone (SBP = 152 +/- 2 mmHg, -log EC50 = 7.25 +/- 0.15), and finally normal sham rats (SBP = 111 +/- 5 mm Hg, -log EC50 > or = 6.80 +/- 0.10). These data indicate that both DOCA and salt intake can independently influence responsiveness to Bay K 8644.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532530 TI - On developing a mind. PMID- 7532528 TI - Alteration of the p53 locus in benign hyperplastic prostatic epithelium associated with high-grade prostatic adenocarcinoma. AB - Recent evidence suggests that the tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic mutations. The genomes of cells lacking normal p53 function may become hypermutable, a condition that might result in the accumulation of multiple genetic alterations as the affected cells proliferate. Such cells may then become more susceptible to malignant transformation. We hypothesized that some high-grade prostate cancers might arise from foci of morphologically benign cells that had previously sustained p53 lesions. As an initial test of this hypothesis, we employed a microdissection technique to isolate morphologically benign cells within hyperplastic glands located near foci of high-grade adenocarcinoma. Genomic DNA from these cells was subjected to polymerase chain reaction amplification and single-stranded conformational polymorphism analysis for detecting alterations in the p53 locus. With use of this approach, gross alterations in the p53 locus were demonstrated in benign cells in 1 of 20 (5%) specimens harboring high-grade malignancy (Gleason grade 7 or higher). Thus, in some cases, hyperplastic prostatic epithelium harbors preneoplastic genetic alterations that could possibly give rise to high-grade malignancies. PMID- 7532529 TI - Detection of ptc in archival formalin-fixed, paraffin-embedded tissues. Comparison of radiolabeled DNA hybridization and direct incorporation of digoxigenin-11-dUTP into RT-PCR products. AB - Archival pathological specimens are a source of RNA and DNA for clinical surveillance or retrospective studies. We employed a modification of the acid guanidium thiocyanate-phenol-chloroform extraction method for the recovery of total RNA from formalin-fixed, paraffin-embedded neoplastic thyroid tissue. The extracted RNA was used for reverse transcription of ptc and subsequent amplification of the complementary DNA (cDNA) by the reverse transcription polymerase chain reaction (RT-PCR). In lieu of 32P-labeled DNA for hybridization studies, we supplemented the nucleotide pool in the amplification reaction with a modified pyrimidine, digoxigenin-11-dUTP. Digoxigenin-11-dUTP was incorporated directly into the PCR product, eliminating the need for hybridization, posthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels, Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonradioisotopic method has expedited and reduced the cost for molecular investigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionuclides without the associated biological hazards. PMID- 7532532 TI - Health technology assessment and gastroenterology. AB - Too much clinical practice is based on unproved opinion. Health technology assessment (HTA) encourages critical review of the literature in a particular area, from which an objective assessment can be made so that clinical practice may be more scientifically based. Only a small number of centers undertake this type of work, usually at a national level. We have been able to set up a more local HTA system (the Wessex Development and Evaluation Committee) with links to clinicians and both providers and purchasers of health care within a region of 3 million people. All aspects of medicine may be reviewed and discussed by the committee; 3 examples from within the field of gastroenterology are summarized herein. Active participation of a university-based institute to provide the reviews is essential. A local HTA facility confers a number of advantages. PMID- 7532531 TI - Adenovirus proteins that subvert host defenses. AB - Adenovirus encodes numerous products that counteract host defenses. A virus encoded RNA, VA RNA1, prevents interferon-mediated shut-off of protein synthesis. Other protein products inhibit interferon-induced gene transcription, prevent cell killing by cytotoxic T cells or block apoptosis, and three sets of proteins independently block the cytolysis and inflammation induced by tumor necrosis factor. Studies of these factors are providing insights into viral pathogenesis. PMID- 7532533 TI - 2.2 A resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains). AB - BACKGROUND: HIV-1 reverse transcriptase (RT) catalyzes the transformation of single-stranded viral RNA into double-stranded DNA, which is integrated into host cell chromosomes. The molecule is a heterodimer of two subunits, p51 and p66. The amino acid sequence of p51 is identical to the sequence of the amino-terminal subdomains of p66. Earlier crystallographic studies indicate that the RT molecule is flexible, which may explain the difficulty in obtaining high-resolution data for the intact protein. We have therefore determined the structure of a fragment of RT (RT216), which contains only the amino-terminal half of the RT molecule ('finger' and 'palm' subdomains). RESULTS: The crystal structure of RT216 has been refined at 2.2 A resolution to a crystallographic R-value of 20.8%. The structure is very similar to that of the corresponding part of the p66 subunit in the p66/p51 heterodimer, although there is a small difference in the relative orientation of the two subdomains compared with the structure of an RT-DNA antibody fragment complex. There are a large number of stabilizing contacts (mainly hydrogen bonds and hydrophobic interactions) between the subdomains. The locations of conserved amino acids and the position of some important drug resistant mutations are described. CONCLUSIONS: The RT216 structure provides detailed three-dimensional information of one important part of HIV-1 RT (including the critical active site residues). We propose a model to explain the inhibitory effect of non-nucleoside inhibitors, which partially accounts for their effect in terms of conformational changes of active site residues. PMID- 7532534 TI - Effects of long-term treatment with anthranoids and sodium picosulphate on the contents of vasoactive intestinal polypeptide, somatostatin and substance P in the rat colon. AB - OBJECTIVE: To examine the effects of chronic treatment and a single high-dose application of anthranoids and sodium picosulphate on the neuropeptide content of the rat colon. DESIGN AND METHODS: Over a 6-month period, eight groups of rats were each given one of the following: sennosides or sodium picosulphate in low daily doses (10 and 2.5 mg/kg/day, respectively), in high daily doses (40 and 10 mg/kg/day, respectively), and in high twice-weekly doses (30 and 7.5 mg/kg/day, respectively); high daily doses of danthron (500 mg/kg/day); and vehicle (tragacanth 0.5%) only. Four further groups of rats each received a single dose of vehicle or a high dose of one of the three laxatives. All rats were killed 48 h after the last dose. The ascending and descending colon were removed and separated into mucosa, submucosa, and muscularis externa. Vasoactive intestinal polypeptide (VIP), somatostatin, and substance P were extracted by boiling and homogenizing the tissue in acetic acid, and their levels were determined using validated radioimmunoassays. RESULTS: After long-term treatment with high doses of sennosides and danthron, but not after a single high-dose administration, there was a significant reduction in mucosal levels of VIP and somatostatin and in submucosal levels of somatostatin of both colonic segments, as well as in the level of VIP in the muscularis externa of the descending colon. Substance P levels remained unaffected. Sodium picosulphate had no effect. CONCLUSIONS: Chronic treatment with anthranoids in high doses, but not with sodium picosulphate, reduces VIP and somatostatin levels in the rat colon. This may represent damage to the enteric nervous tissue or a pharmacological effect of the anthranoids, causing decreased synthesis or increased breakdown of these peptides. PMID- 7532535 TI - Neural elements in the normal and experimentally injured porcine intervertebral disk. AB - There is increasing evidence that-back pain may originate from degenerated or damaged disks, even in the absence of disk herniation. For a study of the pattern of innervation in injured disks, the anterior part of the annulus fibrosus of a lumbar disk in 11 domestic pigs was incised with a scalpel through a retroperitoneal approach. The animals were killed 2 weeks, 1, 2, 3, and 5 months postoperatively, and the whole anterior annulus of each injured disk and corresponding tissue from intact animals were excised. Cryostat sections 20 microns thick were cut from the surface downward, fixed, and stained with different antisera. Antisera to neurofilament triplet protein (R39), protein gene product (PGP) 9.5 and synaptophysin were used as general neural markers. Antiserum to substance P (SP) and calcitonin gene-related peptide (CGRP) were used to localize nerves mainly of the sensory type, and C flanking peptide of neuropeptide Y (CPON) to visualize nerve fibers of the sympathetic type. It was observed that in the intact porcine disk, the outer and middle parts of the anterior annulus were innervated to a depth of 7 mm from the annular surface, but the innermost annular layers showed no immunoreactivity to any of the neural antibodies. Disk injury did not cause any major changes in the nerve topography of the wound area, even though there were granulation tissue and neovascularization in this area. PMID- 7532536 TI - Lumbar muscle fiber size and type distribution in normal subjects. AB - The macroanatomy of the clinically important lumbar muscles has recently been investigated in detail, whereas the information about their microscopic structure in healthy persons is still scanty. In this study we have analysed lumbar multifidus and erector spinae muscles from 21 previously healthy persons who died suddenly and were of working age (range 23-65 years, mean 44.7 years). The microscopic structure of myofibers within the lumbar muscles was found to be regionally uniform, which renders in vivo biopsies representative of the whole muscle bulk. Somewhat surprisingly, age did not significantly influence fiber type composition, fiber size, or proportion of nonmuscular tissue. There was a slight predominance of type 1 fibers. The size of type 1 fibers (mean lesser diameter 54.0 microns) corresponded to that in other skeletal muscles, with no significant sex difference (55.1/51.6 microns male/female). Selective type 2 fiber atrophy was a common finding in both sexes, but significantly less so in men (mean diameter 38.8/28.4 microns male/female). We suggest that the values of fiber type proportions and fiber size in the deep multifidus presented in this study can be used as reference values for healthy adults in the present society with limited physical activity. PMID- 7532537 TI - Surgical management of vertebral neoplasia: who, when, how and why? AB - To determine the role of surgery in vertebral neoplasia, we conducted a retrospective review of patients undergoing surgery for vertebral neoplasia in the Royal Orthopaedic Hospital, Birmingham, and Coventry and Warwickshire Hospital, Coventry. Surgery included decompression, stabilisation or both. The neurological status was assessed by Frankel grading before and after surgery. Of 70 patients undergoing surgery, 14 were neurologically intact preoperatively, and a further 25 were weak but ambulatory. Following surgery, 35 were intact, and a further 22 were ambulatory. Sixty-six patients (94%) obtained good pain relief. Survival correlated with histology and younger age at presentation, but not with level, neurology at presentation or type of surgery. We conclude that neurological status, pain relief and mechanical stability are better after appropriate surgery than after radiotherapy or inappropriate surgery. Failure to consider the surgical option may deny the chance of significant neurological recovery. PMID- 7532538 TI - Nuclear antisense RNA. An efficient new method to inhibit gene expression. AB - We describe an efficient new antisense RNA method to inhibit gene expression. Antisense RNAs that are retained in the nucleus bind to target transcripts and appear to lead to the degradation of their targets. Antisense RNAs can be expressed and accumulated specifically in the nucleus if they are not polyadenylated at their 3' ends. In antisense expression vectors we use a cis acting ribozyme to generate 3'-ends independently of the polyadenylation machinery and thereby inhibit transport of RNA molecules from the nucleus to the cytoplasm. We have evaluated this method in the mouse polyoma virus model system, where nuclear antisense transcripts to the viral early transcription region efficiently reduced the level of viral early-strand RNAs. Nonspecific antisense RNA had no effects on viral gene expression. In comparative studies, nuclear antisense RNAs were significantly more effective in downregulating polyoma virus early RNAs than were conventional antisense molecules, which were processed by polyadenylation. PMID- 7532539 TI - Efficient elution of purified proteins from polyvinylidene difluoride membranes (Immobilon) after transfer from SDS-PAGE and their use as immunogens. AB - Following separation of proteins by SDS-PAGE, they are electroblotted onto polyvinylidene difluoride membranes (Immobilon). Protein bands of interest are excised, and the proteins are eluted from the membrane with detergent-containing buffers at pH 9.5. The method routinely yields recovery of 70-90%, and this is independent of protein molecular weight. PMID- 7532540 TI - The effects of bisphosphonates on the resorption cycle of isolated osteoclasts. AB - Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1 hydroxypropylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl2MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl2MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl2MBP treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl2MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl2MBP slices did not exceed 5 x 10(3) microns 2, whereas on the control slices, resorption pits bigger than 15 x 10(3) microns 2 were frequently discovered. PMID- 7532541 TI - Enrichment of generated murine osteoclasts. AB - Biochemical and molecular studies of osteoclasts generally require cells in a reasonable degree of purity. The chicken has been extremely useful in this regard, as abundant avian osteoclasts can be generated in vitro entirely from pure populations of marrow macrophage precursors. Propagation of murine osteoclasts is, in contrast, far less efficient, demanding the presence of stromal cells. The aims of this study were to develop a method by which murine osteoclasts generated in culture, can be effectively enriched while maintaining viability and, to explore the mechanisms by which stromal cells promote murine osteoclast generation and survival. We find that 10(6) fractionated murine marrow cells enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000 4000 tartrate-resistant acid phosphatase (TRAP)-expressing multinucleated giant cells when cultured for 12 days with ST-2 stromal cells. These cells are osteoclasts as evidenced by their ability to "pit" bone slices, resorb radiolabeled bone particles, and generate cyclic AMP in response to calcitonin. Treatment of these generated osteoclast cultures with bacterial collagenase for 2 hours at 37 degrees selectively removes virtually all ST-2 cells, yielding a > 60% pure population of TRAP and calcitonin receptor-expressing cells, 90% of which are viable. These cells continue to respond to calcitonin and survive for 24 hours in the absence of ST-2 cells. We also found that murine osteoclast generation depends upon contact of osteoclast precursors with viable ST-2 cells. Furthermore, the stromal cells secrete macrophage colony-stimulating factor (CSF 1), and the anti-CSF-1 antibody 5A1 inhibits murine osteoclastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532543 TI - Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood. AB - Dendritic antigen-presenting cells are considered to be the most effective stimulators of T cell immunity. The use of dendritic cells has been proposed to generate therapeutic T cell responses to tumor antigens in cancer patients. One limitation is that the number of dendritic cells in peripheral blood is exceedingly low. Dendritic cells originate from CD34+ hematopoietic progenitor cells (HPC) which are present in the bone marrow and in small numbers in peripheral blood. CD34+ HPC can be mobilized into the peripheral blood by in vivo administration of granulocyte-colony-stimulating factor. The aim of the current study was to determine whether functional dendritic cells could be elicited and grown in vitro from CD34+ HPC derived from bone marrow or granulocyte-colony stimulating factor-mobilized peripheral blood. Culture of CD34+ HPC with granulocyte-macrophage-colony-stimulating factor and tumor necrosis factor alpha yielded a heterogeneous cell population containing cells with typical dendritic morphology. Phenotypic studies demonstrated a loss of the CD34 molecule over 1 week and an increase in cells expressing surface markers associated with dendritic cells, CD1a, CD80 (B7/BB1), CD4, CD14, HLA-DR, and CD64 (Fc gamma RI). Function was validated in experiments showing that cultured cells could stimulate proliferation of allogeneic CD4+ and CD8+ T lymphocytes. Antigen-presenting capacity was further confirmed in experiments showing that cultured cells could effectively stimulate tetanus toxoid-specific responses and HER-2/neu peptide specific responses. The derivation and expansion of dendritic cells from cultured bone marrow or granulocyte-colony-stimulating factor-mobilized CD34+ HPC may provide adequate numbers for testing of dendritic cells in clinical studies, such as vaccine and T cell therapy trials. PMID- 7532542 TI - Phenotype and differentiation patterns of the oval cell lines OC/CDE 6 and OC/CDE 22 derived from the livers of carcinogen-treated rats. AB - An electron microscopic, immunocytochemical, and enzyme cytochemical analysis of the previously established oval cell lines OC/CDE 6 and OC/CDE 22 was performed to characterize the phenotype and differentiation patterns of long-term cultures of oval cells. It was found that alpha-fetoprotein, albumin, and cytokeratin 19 are present in all cultured cells. This indicates that oval cells constitute a population of immature cells expressing features of the antigenic phenotype of both the hepatocyte and bile ductular cell lineages. An electron microscopic examination revealed a gradual alteration in the ultrastructure of oval cells toward hepatocyte-like cells. The majority of the oval cells were positive for glucose-6-phosphatase activity. A particularly striking observation was that oval cells were heterogeneous in terms of peroxisome content. Only about 50% of the oval cells had peroxisomes in the cytoplasm, these cells probably being part of the hepatocyte lineage. The other cultured cells did not reveal catalase activity and probably represented cells committed to the bile ductular cell lineage. An addition of clofibrate to the culture medium resulted in a marked peroxisome proliferation in all oval cells, indicating that oval cells might be able to change their differentiation pathway depending on environmental influence toward the hepatocyte lineage. It is most intriguing that in oval cells with abundant cytoplasm peroxisome proliferation was accompanied by proliferation of the smooth endoplasmic reticulum (this is a morphological marker of mature hepatocytes). Taken together, our findings suggest that within the oval cell lines OC/CDE 6 and OC/CDE 22 cells undergoing a morphological and functional differentiation along the hepatocyte and bile ductular cell lineages are present. PMID- 7532544 TI - TA1, a highly conserved oncofetal complementary DNA from rat hepatoma, encodes an integral membrane protein associated with liver development, carcinogenesis, and cell activation. AB - Hepatocellular carcinoma is characterized by changes in gene expression associated with cell growth and differentiation. Cell surface antigenic changes have also been described based on differential antibody reactivity between normal and neoplastic liver. We obtained a novel tumor-associated cDNA designated TA1 on the basis of its differential expression between hepatoma cells and normal liver. Sequence analysis predicted a 723-base pair open reading frame with the deduced amino acid sequence encoding an integral membrane protein containing multiple hydrophobic transmembrane domains. Database searches revealed TA1 as the likely rat homologue of E16, a recently cloned human cDNA associated with lymphocyte activation. Although noncoding sequences diverged significantly, the 95% conservation of the predicted proteins between species strongly suggests an important, although as yet undefined, function in normal cells. TA1 transcripts were detected in normal adult rat tissues including testes, brain, ovary, spleen, mammary gland, and uterus with the highest steady-state expression in placenta. Although no expression was detected in normal liver, all rat hepatomas examined expressed an abundant 3.2-kilobase transcript. TA1 expression was closely associated with progression in this tumor model and suggests this molecule, originally linked to cell activation, also plays a role in the malignant phenotype. PMID- 7532545 TI - Expression of vascular endothelial growth factor and its possible relation with neovascularization in human brain tumors. AB - To examine which growth factors correlate with neovascularization in human brain tumors, the mRNA levels of transforming growth factor alpha, transforming growth factor beta, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand factor. We normalized the growth factor mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and 16 meningiomas examined, the mRNA of transforming growth factors alpha and beta, basic fibroblast growth factor, and VEGF were expressed at various levels. Among those 4 growth factors, the mRNA levels of VEGF, but not those of transforming growth factors alpha and beta and basic fibroblast growth factor, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive factor in tumor angiogenesis in both human gliomas and meningiomas. PMID- 7532546 TI - Clinical relevance of alphafetoprotein microheterogeneity in alphafetoprotein secreting tumors. AB - The importance of the oncofetal glycoprotein antigen alphafetoprotein (AFP) as a tumor marker is well documented. Structural heterogeneity of AFP molecules due to its associated carbohydrate moieties has also been demonstrated. In the present study, molecular variants of AFP, Concanavalin A reactive (R Con A) and Concanavalin A nonreactive (NR Con A) were quantified in five cases of hepatocellular carcinoma (HCC), three cases of hepatoblastoma, five gonadal and two extragonadal germ cell tumors, and two suspected liver secondaries by employing crossed immunoaffino electrophoresis (CIAE). AFP peaks were localized using anti-AFP antibodies conjugated to alkaline phosphatase. Characteristic patterns of AFP R Con A and NR Con A fractions were obtained in different AFP secreting malignancies. Serum samples of HCC and hepatoblastoma were predominantly composed of R Con A AFP, while gonadal and extragonadal germ cell tumors showed significant reduction of R Con A AFP and elevation of NR Con A AFP. Analysis of AFP variants in sera from two patients of suspected liver metastasis with elevated AFP confirmed liver secondaries arising from germ cell tumor in one patient and HCC in the other patient. The present study highlights the importance of AFP microheterogeneity analysis not only as diagnostic aid for the differential diagnosis of AFP-secreting tumors, but also in providing better management and prognosis. PMID- 7532549 TI - Noncytotoxic human CD4+ T-cell clones presenting and simultaneously responding to an antigen die of apoptosis. AB - Activated T-cells expressing MHC class II surface antigens are able to present antigen and thus function as peptide-presenting cells (T-APCs). In this study we investigated whether antigen presentation by T-cells induced programmed cell death. As a model we used tetanus p30 peptide (aa 947-967)-specific, noncytotoxic CD4+ T-cell clones (C11 and C31). For experimental purposes these T-cell clones were stimulated (a) with p30 peptide-pulsed and fixed EBV-transformed antigen presenting cells (B-APCs), (b) with p30-pulsed and fixed activated T-cells as APCs (as T-APCs we used either the T-cell clones themselves or an autologous T cell clone (CT3) with p30 unrelated specificity), or (c) with soluble p30 peptide. The efficiency of antigen presentation was monitored by measuring proliferation as [3H]thymidine uptake. Apoptosis was measured by quantifying fragmented, cytoplasm DNA with the fluorescent dye 4,6-diamidino-2-phenylindole or by visualizing fragmented DNA by gel electrophoresis. Stimulation with p30 pulsed and fixed B-APCs or T-APCs induced proliferation but no apoptosis of the responding T-cells. However, stimulation of cloned T-cells with soluble peptide induced up-regulation of the FAS surface molecules and apoptosis, which was dependent on the peptide doses. Because cloned T-cells express HLA class II molecules, they can theoretically exert both functions at once: antigen presentation and antigen response when they are stimulated with soluble peptide. Because death by apoptosis is only seen under such circumstances, we suggest that T-cells simultaneously presenting and responding to an antigen die of apoptosis and thus contribute to the down-regulation of the immune response. Such phenomena might occur in HIV infection when activated CD4+ T-cells take up gp120 via their CD4 molecules, present it on their HLA class II surface antigens, and are simultaneously stimulated via their TCR. PMID- 7532547 TI - Stimulation of migration of human aortic smooth muscle cells by vitronectin: implications for atherosclerosis. AB - OBJECTIVE: Migration of smooth muscle cells into the neointima has been implicated in atherogenesis. Vitronectin, a serum factor that promotes cell spreading and attachment, accumulates in atherosclerotic human tissues. The aim of this study was to determine the role of vitronectin and its receptor (integrin alpha V beta 3) in migration of smooth muscle cells. METHODS: Human aortic smooth muscle cell migration was studied in modified Boyden chambers. Expression of vitronectin receptor was determined by northern blotting of receptor mRNA and immunoprecipitation of receptor protein. RESULTS: Vitronectin dose dependently increased smooth muscle cell migration by an amount comparable to that induced by platelet derived growth factor, (PDGF)-BB. Antiserum to alpha V beta 3 diminished vitronectin driven migration. Northern blot analysis showed low constitutive expression of alpha V and beta 3 mRNA by smooth muscle cell and rapid induction with transforming growth factor beta (TGF-beta) and thrombin. Immunoprecipitation confirmed increased synthesis of the alpha V beta 3 vitronectin receptor complex by TGF-beta or thrombin. Smooth muscle cells pretreated with TGF-beta or thrombin showed increased vitronectin driven migration. cAMP suppressed induction of migration, but inhibition of protein kinase C increased it. CONCLUSIONS: These results show that vitronectin-induced human aortic smooth muscle cell migration is mediated by alpha V beta 3 vitronectin receptor and expression of the receptor is induced by TGF-beta and thrombin, which in turn induce vitronectin driven, vitronectin receptor modulated smooth muscle cell migration. PMID- 7532548 TI - A novel form of Epstein-Barr virus latency in normal B cells in vivo. AB - We have developed a PCR assay that can detect a single Epstein-Barr virus (EBV) genome in the presence of 10(6) uninfected cells. Using this assay, we demonstrate that EBV persists, in the peripheral blood of all seropositive individuals tested, in CD19+, CD23-, and CD80 (B7)- B cells. We further show that the virus in these cells is latent, but readily reactivated to produce infectious immortalizing virus; therefore, these cells represent a true site of latent persistence. EBV was not significantly detected in monocytes or T cells. The frequency of infected cells in nine healthy donors varied from 23 to 625 per 10(7) B cells, but was relatively stable for each individual over the course of 2 years. We conclude that the EBV-infected cells in vivo are B cells with a nonactivated phenotype. This represents a novel form of latency in normal B cells. PMID- 7532550 TI - Focal adhesion kinase (pp125FAK) is tyrosine phosphorylated after engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins on human T-lymphoblastic cells. AB - pp125FAK is a novel protein tyrosine kinase associated with focal adhesions. It has been shown that ligation of VLA beta 1 integrins on a number of cell types enhanced tyrosine phosphorylation and kinase activity of pp125FAK. Cellular transformation by retroviral oncogene products or mitogenic neuropeptides also result in the activation of this kinase. On the basis of these observations, pp125FAK has been proposed to be a key regulatory molecule connecting cell adhesion, transformation, and growth. We have previously shown that ligation of VLA beta 1 integrins induced CD3-dependent T cell proliferation and stimulated tyrosine phosphorylation of a molecular mass with a 105-kDa protein (pp105). Here we report that engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins by adherence to their respective ligands stimulated tyrosine phosphorylation of 105- to 120-kDa proteins (pp105 and pp120, respectively) in human H9 T-lymphoblastic cells. At least one component of the 105- to 120-kDa proteins was found to be tyrosine-phosphorylated pp125FAK. While kinetics of adherence-dependent tyrosine phosphorylation of pp120/pp125FAK and pp105 are closely similar, pp105 appeared to be distinct from pp125FAK. Given T cell costimulation induced by VLA beta 1 integrins and the putative functional role of pp125FAK in cell growth, tyrosine phosphorylation of these two distinct proteins may be involved in T cell activation and proliferation. PMID- 7532551 TI - Antibody response of murine B1 cells to hen eggwhite lysozyme. AB - Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer of purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1 kappa anti-HEL, with no significant IgM or lambda-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited. PMID- 7532553 TI - Intravenous cocaine induces platelet activation in the conscious dog. AB - BACKGROUND: Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo. METHODS AND RESULTS: Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91 +/- 3 to 128 +/- 7 mm Hg [P < .01]) and heart rate (87 +/- 9 to 125 +/- 11 beats per minute [P < .01]). A similar change (P = NS) in mean arterial pressure followed norepinephrine infusion (100 +/- 5 to 137 +/- 13 mm Hg [P < .04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14 +/- 7% [P < .08], 31 +/- 12% [P < .04], and 55 +/- 22% [P < .04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23 +/- 7% and 53 +/- 15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538 +/- 55 ng/mL at 2 minutes) but declined rapidly (185 +/- 22 and 110 +/- 25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from < 25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46 +/- 4 and 71 +/- 11 ng/mL at 17 and 32 minutes, respectively, after infusion). CONCLUSIONS: Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation. PMID- 7532552 TI - Hypoxaemia and hypotension after intravenous codeine phosphate. AB - This report describes a case of accidental intravenous administration of codeine phosphate (1 mg.kg-1) to a previously healthy five-year-old boy, who was undergoing strabismus surgery. Hypoxaemia (SpO2 85% with FIO2 of 1) and hypotension (systolic BP 65 mmHg) resulted, which responded to resuscitation with lactated Ringers' (20 ml.kg-1) and phenylephrine (2 micrograms.kg-1). The degree of hypoxaemia observed in this case was severe, but was not associated with clinical evidence of bronchospasm. Possible mechanisms for this reaction might have included direct myocardial depression and histamine release. This case adds further support to the recommendation that codeine phosphate should never be administered intravenously. PMID- 7532554 TI - Early balloon dilatation of the pulmonary valve in infants with tetralogy of Fallot. Risks and benefits. AB - BACKGROUND: Balloon dilatation, an established treatment for pulmonary valve stenosis, remains a controversial procedure in tetralogy of Fallot. METHODS AND RESULTS: Balloon dilatation of the pulmonary valve was performed in 19 infants with tetralogy of Fallot. Its effects on the severity of cyanosis, the growth of the pulmonary valve and pulmonary arteries, and the need for transannular patching were evaluated. Clinical, echographic, angiographic, hemodynamic, and operative data were analyzed. The procedure was safe in all, without significant complications. After balloon dilatation, systemic oxygen saturation increased from a mean value of 79% to 90%. This increase proved to be short-lasting in 4 patients, who required surgery before the age of 6 months. Balloon dilatation increased pulmonary annulus size in each case, from a mean value of 4.9 to 6.9 mm (P < .001). This gain in size remained stable over time, with a mean Z score of 4.8 SD before dilatation, -3.1 SD immediately after the procedure, and -2.7 SD at preoperative catheterization (P < .001). Pulmonary artery dimensions remained unchanged immediately after balloon dilatation but increased at follow-up from a Z score mean value of -2.5 to -0.06 SD and from -2.2 to 0.04 SD for right and left pulmonary arteries, respectively (P < .001). At the time of corrective surgery, the pulmonary annulus was considered large enough to avoid a transannular patch in 69% of the infants. This represented a 30% to 40% reduction in the need for a transannular patch compared with the incidence of transannular patch expected before balloon dilatation. CONCLUSIONS: Pulmonary valve dilatation in infants with tetralogy of Fallot is a relatively safe procedure and appears to produce adequate palliation in most patients. It allowed the growth of the pulmonary annulus and of the pulmonary arteries, resulting in a mean gain of 2 SD for those structures. PMID- 7532555 TI - Gadolinium attenuates the upward shift of the left ventricular diastolic pressure volume relation during pacing-induced ischemia in dogs. AB - BACKGROUND: The mechanism of the reversible upward shift of the left ventricular diastolic pressure-volume relation during demand ischemia is controversial. To assess the possibility that cation influx through stretch-activation channels may contribute to the upward shift, we asked whether gadolinium, a blocker of the stretch-activated channels, attenuates the upward shift of the diastolic pressure volume relation during pacing-induced ischemia in 5 dogs. METHODS AND RESULTS: To produce pacing-induced ischemia, we constricted the left anterior descending and circumflex coronary arteries to reduce their flows by approximately 30% and paced the left atrium at 150 to 180 beats per minute for 3 minutes. We measured left ventricular pressure, volume, and two segment lengths with micromanometers, a conductance catheter, and ultrasonic crystals, respectively. We recorded these variables during baseline, coronary stenosis, and pacing-induced ischemia (immediately after rapid pacing). After injecting 20 mg/kg (76 mumol/kg) gadolinium, we repeated the measurements during coronary stenosis (gadolinium experiment) and pacing-induced ischemia (pacing-induced ischemia plus gadolinium experiment). For each measurement, we recorded the variables in steady state to obtain diastolic pressure-volume and pressure-segment length loops and then during a brief (within 25 seconds) inferior vena caval occlusion to obtain the left ventricular end-diastolic pressure-volume relation. We found that left ventricular diastolic pressure-volume and pressure-segment length loops in steady state beats shifted upward from coronary stenosis to pacing-induced ischemia. After injection of gadolinium, the upward shift from gadolinium to pacing-induced ischemia plus gadolinium was smaller than the shift from coronary stenosis to pacing-induced ischemia. Similarly, the left ventricular end-diastolic pressure volume relation obtained during vena caval occlusion shifted upward (by 2.2 +/- 0.6 [SE] mm Hg) from coronary stenosis to pacing-induced ischemia. After injection of gadolinium, the upward shift from gadolinium to pacing-induced ischemia plus gadolinium was smaller (by -2.1 +/- 0.4 mm Hg). CONCLUSIONS: These results indicate that gadolinium, a blocker of stretch-activated channels, attenuates the upward shift of the diastolic pressure-volume relation during pacing-induced ischemia, suggesting that the cation influx through stretch activated channels may contribute to this upward shift. PMID- 7532556 TI - Identification of a 14 kDa FK-506/rapamycin binding immunophilin from calf thymus. AB - Specific binding proteins (immunophilins, 12-17 kDa) have been described in the cytosol for the immunosuppressant drugs cyclosporine, FK-506, and rapamycin. We describe the identification of a low abundant minor immunophilin (14 kDa) from calf thymus. Saturation experiments using dihydro[3H]-FK-506 gave a Kd of 2 nM and the Bmax of 72 nmol/mg protein. At saturation, 71.3 nmol of FK-506 is bound per mg of the 14 kDa protein (71 nmol) giving a drug/protein ratio of 1.0. Competition experiments using 3H-dihydro FK-506 and rapamycin showed displacement of rapamycin, with Kd in the range of 40 nM. The 14 kDa immunophilin does not have peptidyl-prolyl cis-trans isomerase activity with any of the four substrates investigated. Initial amino acid analysis and protein sequencing data indicate that the immunophilin is different from both the 12 kDa FK-506 binding protein and any other known protein. PMID- 7532557 TI - Partial characterization of a 52 kDa CsA/FK506/rapamycin binding protein. AB - Identification and characterization of the cellular proteins that specifically bind to the immunosuppressive drugs, cyclosporine (CsA), FK506, and rapamycin is necessary to understand their mechanism of action. We have isolated and partially characterized a 52 kDa binding protein (BP) from calf thymus. Using 12 peptide substrates we observed very low or no cis-trans peptidyl prolyl isomerase activity. We further tested the protein for catalytic activity including kinase activity, phosphatase activity, protein kinase C regulation, and LCK tyrosine kinase regulation. The 52 kDa BP was capable of blocking the cyclic AMP dependent, protein kinase mediated, phosphorylation of histones and casein. The protein did not demonstrate kinase activity, nor did it affect the activity of protein kinase C or LCK tyrosine kinase. Microsequencing of the 52 kDa BP was performed. A comparison of known sequences indicated that the protein is unique and has not been previously characterized. PMID- 7532558 TI - Immunohistochemical appearance of corticosteroid contact hypersensitivity reactions. AB - We have studied, immunohistochemically, hypersensitivity reactions to corticosteroids and compared them with allergic contact dermatitis from nickel and appropriate controls. We could find no qualitative differences between nickel and corticosteroid contact reactions, providing further evidence that hypersensitivity to corticosteroids is an immunologically mediated reaction. PMID- 7532560 TI - Attenuation of shock-induced hepatic microcirculatory disturbances by the use of a starch-deferoxamine conjugate for resuscitation. AB - OBJECTIVE: To determine the effects of a hydroxyethyl starch-deferoxamine conjugate on hepatic microcirculation in an isobaric, anesthetized rat model of hemorrhagic shock and asanguineous resuscitation. DESIGN: Prospective, randomized, controlled study. SETTING: Laboratory at a university hospital. SUBJECTS: Twenty-three female, inbred Lewis rats (190 to 215 g). INTERVENTIONS: After anesthesia (pentobarbital-sodium; 50 mg/kg), tracheotomy, and cannulation, animals were assigned to a hemorrhagic shock (mean arterial pressure at 40 +/- 3 mm Hg for 45 mins) or a time-matched sham protocol. Rats in the shock groups received either hydroxyethyl starch or a starch-deferoxamine conjugate for resuscitation. Liver microcirculation was assessed in vivo 60 mins after onset of volume therapy by epifluorescence microscopy. MEASUREMENTS AND MAIN RESULTS: Conventional resuscitation with the starch-vehicle failed to restore sinusoidal blood flow compared with either time-matched controls (71% of control value; p < .01) or the starch-deferoxamine-treated animals (89% of control value; p < .05 compared with starch-vehicle), although a comparable restoration of central hemodynamics was achieved with both starch preparations. Additionally, treatment with the starch-deferoxamine conjugate resulted in a significant attenuation of sinusoidal leukocyte margination (sham 72.4 +/- 11.0/mm2; starch-vehicle 194.5 +/ 19.0/mm2 [p < .01 compared with controls]; starch-deferoxamine conjugate 135.9 +/- 12.1/mm2 [p < .02 compared with sham and starch-vehicle]). CONCLUSIONS: Asanguineous resuscitation with conventional hydroxyethyl starch failed to restore hepatic microvascular blood flow, despite otherwise effective resuscitation. In contrast, the starch-deferoxamine conjugate improved volumetric blood flow and attenuated leukocyte margination in hepatic sinusoids compared with starch-vehicle, suggesting involvement of iron-dependent, oxygen-derived radicals in shock-induced hepatic microcirculatory disturbances. PMID- 7532559 TI - Methylene blue administration in septic shock: a clinical trial. AB - OBJECTIVE: A release of nitric oxide has been incriminated in the cardiovascular alterations of septic shock. Since guanylate cyclase is the target enzyme in the endothelium-dependent relaxation mediated by nitric oxide, we studied the acute effects of methylene blue, a potent inhibitor of guanylate cyclase in patients with septic shock. DESIGN: Prospective clinical trial. SETTING: Medical-surgical intensive care unit in a university hospital. PATIENTS: Fourteen patients with severe septic shock requiring adrenergic therapy. INTERVENTIONS: Short-term intravenous infusion of methylene blue. MEASUREMENTS AND MAIN RESULTS: Hemodynamic measurements were obtained at baseline, and 30, 60, and 90 mins after the infusion of 2 mg/kg of methylene blue. Methylene blue administration was followed by a progressive increase in mean arterial pressure (from 61.1 +/- 7.6 to 71.7 +/- 12.0 mm Hg at 60 mins, p < .01). Pulmonary arterial pressure, cardiac filling pressures, cardiac output oxygen delivery, and oxygen consumption were not significantly affected. Left ventricular stroke work increased from 42.5 +/- 17.9 to 48.9 +/- 14.5 g.m after 60 mins (p < .05). Arterial lactate concentration decreased from 3.4 +/- 1.4 to 2.7 +/- 1.3 mmol/L (p < .05). Since these effects were transient, a second dose of methylene blue was administered 90 mins later to six patients and was followed by a similar response. No adverse effect was observed. CONCLUSIONS: In septic shock patients, the administration of methylene blue results in a transient and reproducible increase in arterial pressure, associated with an improvement in cardiac function, but does not increase cellular oxygen availability. The significant reduction in blood lactate concentration is probably related to the reductor effect of methylene blue, rather than to an improvement in tissue oxygenation. PMID- 7532561 TI - Hypertonic saline-dextran resuscitation from hemorrhagic shock induces transient mixed acidosis. AB - OBJECTIVE: To evaluate the magnitude and mechanism of potential metabolic acidosis after resuscitation with 7.5% sodium chloride/6% dextran-70. DESIGN: Blinded, randomized, control trial. SETTING: Laboratory setting. SUBJECTS: Sixteen healthy Yorkshire swine. INTERVENTIONS: Anesthetized, mechanically ventilated swine underwent 90 mins of hemorrhagic hypotension (mean arterial pressure of 50 to 55 mm Hg), and a lactic acid infusion (1.5 to 2.4 mmol/kg) was given during the last 60 mins of hemorrhage to produce pretreatment acidosis. The pigs were then given either 4 mL/kg of intravenous normal saline (n = 8) or 7.5% sodium chloride/6% dextran-70 (n = 8). Groups then received isotonic lactated Ringer's solution to restore and maintain cardiac output for 120 mins. MEASUREMENTS AND MAIN RESULTS: There was no difference between groups during baseline or shock for any parameter. At the end of shock, arterial pH and base balance were below baseline values. During resuscitation, cardiac output was reached and maintained in both groups. One minute after infusion of hypertonic saline/dextran, there was a significant but transient decrease in arterial pH (from 7.407 +/- 0.015 to 7.339 +/- 0.025) and base balance (from -6.5 +/- 0.7 to 9.9 +/- 1.0 mmol/L). These changes returned to shock levels by 10 mins and then normalized to baseline levels. Hypertonic saline dextran resulted in an immediate hypernatremia, hyperchloremia, and hypokalemia, a decrease in inorganic strong ion difference (calculated as sodium plus potassium minus chloride concentrations), and no immediate change in anion gap. The normal saline group did not show an initial transient decrease in pH and base balance during resuscitation. Plasma lactate, total protein, and hemoglobin concentrations decreased equally in both groups, although they decreased more quickly with hypertonic saline/dextran. CO2 temporarily and insignificantly increased in arterial blood slightly more after the administration of hypertonic saline/dextran. By 120 mins, acid-base, electrolyte and protein changes were normalizing with hypertonic saline/dextran, while pH, base balance, and protein were decreasing below shock values in animals initially treated with normal saline. CONCLUSIONS: Hypertonic saline/dextran caused an immediate, transient acidemia, which was primarily due to a hyperchloremic, hypokalemic, metabolic acidosis with normal anion gap and decreased inorganic strong ion difference, but which was partially due to a mild transient respiratory acidosis. The acidemia was transient because of the offsetting alkalotic effects of decreasing serum protein, normalization of electrolytes, and transient nature of the increase in CO2. Lactic acidosis was not the cause of the acidemia. Over time, the acid-base status appeared to be improved more effectively with hypertonic saline/dextran than with isotonic saline resuscitation. PMID- 7532562 TI - Pentafraction for superior resuscitation of the ovine thermal burn. AB - OBJECTIVE: To determine if a new hydroxyethyl starch, pentafraction, will cause better capillary retention of fluid in thermally burned and nonburned tissues when compared with some currently used volume expanders. DESIGN: Randomized, controlled, experimental study. SETTING: University research laboratory. SUBJECTS: Twenty-one healthy adult range ewes. INTERVENTIONS: Sheep, surgically prepared for chronic study, were randomly assigned to one of three colloid groups for the resuscitation of thermal injuries: a) ovine fresh-frozen plasma group; b) pentastarch group; and c) pentafraction group. Twenty-one sheep were subjected to a 40% total body surface, third-degree flame burn under anesthesia (1.5% to 2% halothane). When awakened, the sheep received 15 mL/kg of one of the above colloids, and then lactated Ringer's solution (2 mL/hr/kg). All animals survived and were killed at 48 hrs after burn injury. MEASUREMENTS AND MAIN RESULTS: We found that cardiac index decreased and systemic vascular resistance increased in the pentastarch and plasma groups. However, cardiac index increased in the pentafraction group, while systemic vascular resistance increased by only half as much as in the other two groups. Cardiac index in the plasma group decreased significantly for the first 8 hrs, and did not return to the baseline value for 48 hrs. Systemic vascular resistance increased significantly for the first 8 hrs in both the plasma and pentastarch groups. Prefemoral lymph flow was significantly more increased in the pentastarch group, while the lung lymph/plasma oncotic gradient was maintained only by the pentafraction group. CONCLUSIONS: Pentafraction is as good, or even superior, for volume resuscitation in the burn patients, when compared with pentastarch and plasma. This conclusion is made most evident by the attenuated changes in systemic vascular resistance and cardiac index. Pentafraction decreases the fluid flux and potential subsequent edema significantly in burned tissues and effectively maintains the pulmonary microvascular integrity. PMID- 7532563 TI - Nitric oxide synthesis inhibition in septic shock. PMID- 7532565 TI - [Is there hepatitis C virus associated glomerulonephritis?]. AB - Hepatitis C virus (HCV) infection is one of the important causes of chronic and severe hepatitis in China. In an attempt to understand if there is any relationship between HCV infection and glomerulonephritis (GN), serum samples from 570 GN patients and 100 normal volunteers were screened for anti-HCV antibody (HCV-Ab) with ELISA method. Among the cases with positive HCV-Ab, serum HCV-RNA was tested with nested RT-PCR method. The incidence of serum HCV-Ab was 2 in 100 normal volunteers (2%) and 34 in 570 GN patients (6%). The incidence of positive serum HCV-RNA was 0 in normal volunteers whereas 21 in GN patients. The main clinical manifestation of GN patients with serum positive HCV-Ab was an unique proteinuria with/without nephrotic syndrome or renal failure, whereas the pathologic lesions in GN patients with serum positive HCV-Ab or HCV-RNA consisted of different disease entities. There was no close link between MPGN and active HCV infection. From the data observed, it seems that there is a coincidence between glomerular diseases and HCV infection rather than a matter of cause and consequence. PMID- 7532564 TI - [Localization of extracellular matrices in dental pulp in permanent teeth in youth]. PMID- 7532566 TI - Markers for metastatic adenocarcinoma in serous effusion specimens. AB - A variety of stains have been proposed as useful adjuncts to the morphologic diagnosis of serous effusion specimens. This study evaluates the sensitivity and specificity of nine stains for the detection of metastatic adenocarcinoma in effusions. Mucin cytochemistry with the Periodic acid-Schiff reaction after diastase digestion (PASd) and the mucicarmine method, as well as immunochemical staining for five glycoprotein antigens, was undertaken on 153 effusion specimens with histological and/or clinically confirmed diagnoses. Immunochemical stains included three antibodies to CEA (Dako polyclonal, 4E7 and A5B7) and antibodies to HEA (Ber-EP4), TAG 72 (B72.3), CD15 (LeuM1) and Epithelial membrane antigen (EMA) were evaluated. The sensitivity of the stains for adenocarcinoma (102 cases) was: PASd 37%; mucicarmine 24%; polyclonal anti-CEA 69%; 4E7 52%; A5B7 21%; EMA 91%; B72.3 44%; Ber-EP4 32%; LeuM1 24%. With the exception of EMA, none of the stains reacted with any cases of benign mesothelial cells (11 cases), reactive mesothelial cells (34 cases), or with six cases of mesothelioma. However, EMA staining was present in two cases (6%) of reactive mesothelial cells and all cases of mesothelioma. The optimal combination of stains for use in a panel was polyclonal anti-CEA/B72.3/PASd. Combined results from these three stains yielded a sensitivity of 83% for adenocarcinoma with no false positive results. It is concluded that special staining may provide valuable information to assist in the classification of difficult effusion cases. PMID- 7532567 TI - Dendritic development in normal lumbar motoneurons and following neonatal nerve crush in the rat. AB - Motoneurons from the rat were retrogradely labelled with cholera toxin horseradish peroxidase at intervals during normal postnatal development and following nerve crush at birth. Normal cells displayed a relatively steady increase in total visible dendritic density which was largely confined to the dorsomedial direction. After nerve crush at birth, dorsomedially orientated dendrites failed to achieve normal density, resulting in a significantly smaller dendritic tree by adulthood. There was also a transient, abnormal extension of dendrites in the medioventral direction which had regressed to normal levels by maturity. The predominance of changes in the dorsally directed region of the dendritic tree suggests that dendritic development of motoneurons is influenced by synaptic inputs in the dorsal horn. PMID- 7532569 TI - Somatostatin- and substance P-like immunoreactivity in rat neostriatal cultures. AB - In the normal adult neostriatum, somatostatin immunoreactive interneurons constitute a few percent of the total neuronal population whereas substance P immunoreactive neurons, which project to the substantia nigra, constitute nearly half of the total. Primary monolayer neostriatal cultures derived from E17 rat brains displayed both somatostatin-like and substance P-like immunoreactivity (SOM-IR and SP-IR). However, the proportions of SOM-IR and SP-IR neurons in vitro were significantly different from those in vivo. At 4 days in vitro (DIV), SOM-IR neurons comprised 19% of all neurons and this percentage increased to 30% at 25 DIV. In contrast, SP-IR neurons were less common than expected at 4 DIV (20%) and declined in percentage to 13% at 27 DIV. These results suggest that survival in target-deprived neostriatal cultures is favored for SOM-IR interneurons. PMID- 7532570 TI - Negative myoclonus during valproate-related stupor. Neurophysiological evidence of a cortical non-epileptic origin. AB - We retrospectively reviewed clinical and neurophysiological data of 6 epileptic patients who developed negative myoclonus and stupor a few days after introduction of valproate (VPA). Prompt remission of clinical signs and symptoms followed valproate withdrawal. We attempted to elucidate the pathophysiological mechanism of VPA-induced stupor and provide further polygraphic and backaveraging EEG documentation of negative myoclonus. During VPA-induced stupor electroencephalograms revealed posterior background slowing in all patients. Interictal epileptiform discharges were present in 3 patients. In all 6 patients close examination using simultaneous video-polygraphic recording showed negative myoclonus which was not time-related to lateralized spike discharges. In 2 of 3 patients with no spikes on conventional EEG who underwent backaveraged EEG recordings we detected a large (5 microV) cortical positive-negative wave time locked (30-40 msec) with the postural modification of the contralateral wrist. This cortical potential was similar to that observed in patients with asterixis secondary to metabolic or toxic encephalopathies. In one patient i.v. administration of 10 mg diazepam did not modify this cortical potential and did not reverse the clinical manifestations. In all patients the only abnormal laboratory finding was an increased level of venous ammonemia. Our findings are against an epileptic origin of VPA encephalopathy and provide further argument in favour of a cortical non-epileptic mechanism mediating negative myoclonus. Benzodiazepines should be avoided in the management of this condition. PMID- 7532568 TI - Changes in retrogradely labelled neurones in the red nucleus and cortex after depletion of motoneurones by axotomy in neonatal rats. AB - Following nerve injury to the sciatic nerve in neonatal rats many motoneurones die, so that the target of the rubrospinal and corticospinal cord is altered. Retrograde labelling of neurones terminating in this region shows that, in the red nucleus as well as cerebral cortex, there is a significant reduction in labelled neurones on the side contralateral to the nerve injury. Thus neurones in the brain that give rise to descending fibres are influenced by the altered environment in the spinal cord following axotomy at birth. PMID- 7532571 TI - MEG and ECoG localization accuracy test. AB - We tested the localization accuracy of magnetoencephalography (MEG) and electrocorticography (ECoG) for a current dipole in a saline filled sphere at depths ranging from 1 to 6 cm at 1 cm intervals. We used standard neuromagnetometer placements and subdural electrode grids, previously employed for patient studies, with precise measurements of sensor and electrode locations with a 3-dimensional spatial digitizer. MEG and ECoG had comparable accuracy with mean errors of 1.5 and 1.8 mm, respectively. It appears that use of the spatial digitizer increases accuracy for both MEG and EGoG localizations. The larger errors in the ECoG with increasing depths could be attributed to under-sampling of the spatial pattern of the field which spreads out with deeper sources. It should be noted that in clinical applications a grid of the dimensions used here would most typically be used for superficial sources on the cortex with depth recordings being preferred for investigations of deep epileptogenic activity. Results are encouraging for continued development of non-invasive MEG methods for further definition of epileptogenic zones in the brain. PMID- 7532572 TI - The location of preceding stimuli affects selective processing in a sustained attention situation. AB - Event-related potentials (ERPs) were recorded to visual and auditory stimuli in a situation where subjects were required to attend selectively to the left or right side for an entire experimental block and to detect occasional target stimuli at attended locations. Stimuli were presented randomly at attended and unattended locations. In exp. 1, visual and auditory stimuli were presented in separate blocks, while in exp. 2, they were presented together and subjects had to detect visual targets at attended locations. Stimuli at attended positions elicited enlarged sensory-evoked potentials and an enhanced negativity at midline electrodes as compared with unattended stimuli. The latter effect was, however, modulated by the location of the preceding stimulus. At frontocentral electrodes, it was larger for stimuli that were preceded by stimuli at the contralateral side as compared with stimuli preceded by stimuli at the same location. It is argued that this effect may be due to a different amount of processing required for the preceding stimulus. When the predecessor is at a to-be-attended location, it has to processed more intensively which may interfere with the processing of the next stimulus. PMID- 7532573 TI - Regional modulation of high resolution evoked potentials during verbal and non verbal matching tasks. AB - Nine subjects performed a cued S1-S2 matching task in which two sequentially presented visual stimuli (either letter strings or non-verbal graphical patterns) were compared according to verbal (phonemic, semantic, syntactic) or non-verbal (graphic identity) criteria. The Laplacian derivation was used to spatially enhance the topography of averaged evoked potentials (EPs) recorded from 59 scalp electrodes. Several effects distinguished the non-verbal from the verbal conditions. For example, following S1 a P250 EP that reached maximum amplitude over the occipital area was larger for the non-verbal patterns, whereas word and word-like letter strings (but not unfamiliar characters) elicited an N470 in the left temporal region. In anticipation of S2, a CNV-like slow potential was enhanced over posterior regions for the non-verbal stimuli. During the matching interval following S2, a P475 peak was observed to be larger for non-verbal patterns than for letter strings over right frontal and temporal regions. Other effects distinguished the verbal conditions from one another. In particular, following S1 a left frontal P445 potential was enhanced to closed class versus open class words, and following S2 a P620 potential in the left temporal region was enhanced for phonological matching relative to semantic matching. These results suggest that processing of verbal and non-verbal stimuli depends on a network of subprocessors that are regionalized to functionally specialized cortical areas and that operate both sequentially and in parallel in order to extract and synthesize multiple forms of attribute-specific information. In contrast to neuropsychological approaches to the study of pattern recognition and reading, the fine-grain temporal resolution of EP measurements, in combination with the improved spatial resolution obtained through computation of Laplacian derivation wave forms from a large number of electrodes, permits characterization of both the regionalization of subprocesses and the subsecond dynamics of their engagement. PMID- 7532574 TI - Effects of hemi-thalamic damage on K-complexes evoked by monaural stimuli during midafternoon sleep. AB - Polygraphic recording of midafternoon sleep were made in two patients with unilateral hemorrhagic infarction in the thalamus. One patient had massive signs of reduced attention in the contralesional space following right lateral thalamic lesion. The other patient had infarction centered in the left posterior ventrolateral thalamus with no signs of hemineglect. In both cases, waking and sleep records showed symmetric background EEG at C3 and C4. In contrast, monaural sounds (clicks) elicited bilaterally symmetrical K-complexes only in the second patient. In the other patient with right thalamic lesion, both left- and right ear clicks failed to elicit K-complexes in the right cerebral hemisphere, whereas normal K-complexes continued to appear at C3 as well as at Fz. In neither patient did the frequency of evoked K-complexes depend on the side of the stimulated ear. Unlike K-complexes, sleep spindles were bilaterally absent in patient 1. The right hemisphere does not seem to have a special role in control of circuits generating K-complexes. PMID- 7532575 TI - Thoughts about the IFCN recommendations for the recording of short latency somatosensory evoked potentials. PMID- 7532576 TI - Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid. AB - The network of androgen-dependent growth factors regulating the growth and function of normal or neoplastic prostate epithelium is largely unknown. To facilitate studies directed at investigating this issue, androgen receptor negative (AR-) PC3 prostate carcinoma cells were stably transfected with the expression plasmid CMV3 containing a constitutively active AR construct that is truncated at its hormone-binding domain (CMV-ARCA). The major characteristic of the resulting cell line (PC3-ARCA) was a growth rate approximately 35% slower than that of a control mock-transfected cell line (PC3-Neo). Of the several growth factors known to be present in the prostate, the current studies focused on the insulin-like growth factor (IGF) axis, specifically the IGF-binding proteins (IGFBPs), several of which are known to be abnormally produced by prostate cancer. Northern analysis showed that IGFBP-1 and -5 are not expressed by PC3-ARCA and -Neo cells. Western ligand and immunoblot analysis of medium conditioned by PC3-Neo and PC3-ARCA cells revealed that equal amounts of IGFBP-2, -4, and -6 were secreted. In contrast, IGFBP-3 was undetectable in the conditioned medium of PC3-ARCA cells, but normally produced by the AR- cell line PC3-Neo. IGFBP-3 disappearance from the conditioned medium of PC3-ARCA cells was transcriptionally regulated, as a marked decrement in IGFBP-3 messenger RNA was detected by S1 protection analysis. We investigated the responses of these cells to exogenously added IGF-I, IGF-II, or IGFBP-3. IGF-I and IGF-II stimulated the proliferation of PC3-ARCA cells, but not of PC3-Neo cells. IGFBP-3 had no effect when given alone. When IGFBP-3 was administered together with IGF-I or IGF-II, it further increased the mitogenic response observed in PC3-ARCA cells, but no effect on PC3-Neo cells was observed. In conclusion, our studies suggest that the presence of an active AR modulates the proliferation of transfected PC3 prostate cancer cells, and that this phenomenon occurs at least in part through the regulation of IGFBP-3 production. PMID- 7532577 TI - Glutamic acid decarboxylase-67 (GAD67): expression relative to GAD65 in human islets and mapping of autoantibody epitopes. AB - Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets. PMID- 7532578 TI - Modulation of glucocorticoid-regulated transcription by purines: novel characteristics and implications for tissue specificity of steroid responses. AB - Treatment of the T47D(A1-2) mammary carcinoma cell line with the nonspecific kinase inhibitor 2-aminopurine (2AP) has an unusual effect on induction of the mouse mammary tumor virus promoter by glucocorticoids. 2AP initially abrogates the dexamethasone-mediated increase, but after about 16-20 h, this effect is reversed, and continued incubation with 2AP potently stimulates the hormone induction. The biphasic kinetics of 2AP action displayed cell specificity. No inhibitory phase was seen in fibroblasts. Cell type specificity is also seen upon treatment of cells with isobutylmethylxanthine (IBMX). Dexamethasone action is inhibited in mammary cells, but potentiated in fibroblasts. Unlike 2AP treatment, IBMX continues to suppress the hormone response through at least 48 h of treatment. IBMX is commonly used as a phosphodiesterase inhibitor, and indeed, it stimulates a cAMP-dependent promoter in both mammary carcinoma cells and fibroblasts. Because elevating cAMP will potentiate dexamethasone-mediated induction in mammary cells, IBMX must be interventing in another pathway that elicits IBMX-dependent suppression of the hormone response. Pharmacological studies suggest that this interaction is not mediated through adenosine receptors, histamine receptors, or imidazoline receptors. The five-member imidazole ring plays a critical role in the suppressive activity; substitutions at the 7 position inhibit suppression. These results give further indication of coupling of steroid receptor action to other cellular signaling pathways. This complex spectrum of interactions may play a central determining role in the tissue specificity of the response to steroid hormones. PMID- 7532579 TI - The colony-stimulating factors induce expression of insulin-like growth factor-I messenger ribonucleic acid during hematopoiesis. AB - Murine bone marrow cells cultured in the presence of colony-stimulating factor-1 (CSF-1) showed coordinate induction of insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) during the differentiation process, and these transcripts increased approximately 50- to 75-fold over the virtually negligible levels measured in freshly isolated bone marrow. In contrast, transcripts for the IGF-I receptor were evident in freshly isolated rat bone marrow cells and showed a 50% down-regulation during differentiation. Addition of a variety of single lineage and multilineage CSFs, including CSF-1, interleukin-3, granulocyte-macrophage CSF, and granulocyte-CSF to mouse bone marrow cultures revealed that induction of IGF-I mRNA is a universal feature of differentiation with these CSFs, although IGF-I transcripts are at least 10- to 20-fold higher in CSF-1- and interleukin-3 differentiated lineages than in other cultures. The IGF-I induced by CSF-1 was biologically active because a natural ligand of IGF-I, IGF-binding protein-3, caused significant down-regulation of cellular proliferation, and this could be reversed by the addition of exogenous IGF-I. In addition, whereas IGF-I mRNA could be detected in resident peritoneal macrophages, these transcripts were increased 6-fold after a local injection of thioglycollate, a stimulus that induces macrophage proliferation and differentiation in vivo. These results show that CSFs induce expression of the growth factor IGF-I during differentiation of hematopoietic cells into multiple myeloid lineages and that this endogenously produced IGF-I is also a growth factor for hematopoietic cells. The induction of IGF-I mRNA during hematopoiesis should provide a new approach to understanding the expression of this gene during development and differentiation. PMID- 7532580 TI - Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol. AB - The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer. PMID- 7532582 TI - The role of glycosylation in regulating the glycoprotein hormone free alpha subunit and free beta-subunit combination in the extraembryonic coelomic fluid of early pregnancy. AB - The extraembryonic coelomic fluid (EECF) represents a major compartment in the fetal-placental unit during the first trimester of pregnancy. The compartment is composed of the fluid contained between the chorionic and amniotic membranes. The levels of glycoprotein hormone free alpha-subunit and free beta-subunit in the EECF far exceed those in the amniotic fluid or maternal serum. Furthermore, the level of free alpha in this compartment is twice that of intact hCG. We purified the glycoprotein hormone free alpha-subunit from a pool of EECF. This free alpha subunit was found to be larger in size than the alpha-subunit of intact hCG. The size difference was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and denatured conditions. The carbohydrate composition of the EECF free alpha-subunit indicated a higher degree of oligosaccharide branching, as evidenced by larger amounts of fucose, sialic acid, galactose, and N-acetylglucosamine than were present on combined hCG alpha. These differences in size and carbohydrate composition argue strongly against the concept that free alpha-subunits might originate from dissociation of intact hCG or "nicked" hCG. The free subunits of the EECF were evaluated for their ability to combine with the corresponding subunit obtained by dissociation of intact hCG. EECF free beta was able to combine with hCG alpha to form intact hCG. In contrast, EECF free alpha was unable to combine with hCG beta to form intact hCG. However, after removal of the asparagine-linked glycans by treatment with N glycanase, most of the previously uncombinable free alpha-subunits were able to combine with hCG beta. These data demonstrate that the N-linked oligosaccharide(s) of EECF free alpha function to prevent the molecule from combining with the available and combinable free beta-sub-units that coexist in the same physiological compartment during early pregnancy. In view of the large amount of free alpha that is present in the EECF and the observation that, in vitro, free alpha can stimulate uterine decidual cell PRL secretion, together with the close apposition of free alpha-producing cells to decidual cells, it is likely that EECF free alpha has a function in early pregnancy. Carbohydrate modifications generated during the biosynthesis of EECF free alpha-subunit ensure that a population of free alpha molecules can exist in the presence of substantial quantities of free beta-subunits, and correspondingly, these same carbohydrate modifications function to permit the existence of free beta-subunits in the same gestational compartment with free alpha molecules.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532581 TI - Regulation of insulin-like growth factor system components by osteogenic protein 1 in human bone cells. AB - Bone morphogenetic proteins (BMPs) have the unique ability to convert mesenchymal cells into matrix-producing osteoblasts. To understand the mechanism(s) by which a BMP produces a multitude of effects on bone cells, we examined the effects of recombinant human osteogenic protein (OP)-1 (referred to as BMP-7) on the insulin like growth factor (IGF) regulatory system, an important growth factor system in bone. After 48 h of treatment, OP-1 increased the level of IGF-II (3- and 2-fold, respectively, at 100 ng/ml) in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cells with osteoblastic characteristics, whereas IGF-I levels were low to undetectable in the CM of either cell type. OP-1 treatment had no significant effect on the messenger RNA (mRNA) level for type 1 and type 2 IGF receptors. In TE85 and SaOS-2 cells, 100 ng/ml OP-1 increased the level of IGF binding protein (BP)-3 more than 10-fold, decreased the IGFBP-4 level by 50%, and increased the level of the 29-32.5 kDa IGFBP-5 3-fold in the CM as determined by analysis with Western ligand blot, Western immunoblot, and RIA. The effect of OP 1 on IGFBP production was time and dose dependent. The OP-1 induced changes in the levels of IGFBPs were associated with decreased IGFBP-3 and -5 protease activity (29% and 71%, respectively) and proportional changes in IGFBP mRNA levels. OP-1 increased the level of IGFBP-3 mRNA (2- and 10-fold, respectively, after 4 and 24 h of treatment at 100 ng/ml) and of IGFBP-5 mRNA (more than 5-fold after 24 h of treatment) but decreased the level of IGFBP-4 mRNA (> 50% after 24 h at 100 ng/ml). OP-1 treatment had no effect on IGFBP-4 protease activity. These results collectively demonstrate that OP-1 can act locally by modulating the IGF regulatory system, suggesting that the mitogenic/differentiative effect of OP-1 on human bone cells may in part be mediated via IGF-II by increasing its secretion, and by regulating the balance between the stimulatory (e.g. IGFBP-5) and inhibitory (e.g. IGFBP-4) classes of IGFBPs both at the level of production (mRNA) and at the level of degradation but not by up-regulating the IGF receptor. PMID- 7532583 TI - Methanethiolation of the liberated cysteine residues of human alpha 2 macroglobulin treated with methylamine generates a derivative with similar functional characteristics as native alpha 2-macroglobulin. AB - The thiol-modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human alpha 2-macroglobulin with methylamine. This methanethiolation generates a derivative of alpha 2 macroglobulin, with an 'open trap' and slow mobility in non-denaturing PAGE, similar to native alpha 2-macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native alpha 2-macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native alpha 2 macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice. In contrast to native alpha 2 macroglobulin, which can bind 2 mol chymotrypsin/mol, alpha 2-macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when alpha 2-macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group. The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half-life of 9 h as compared to a half-life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification. PMID- 7532584 TI - RNase mitochondrial RNA processing cleaves RNA from the rat mitochondrial displacement loop at the origin of heavy-strand DNA replication. AB - Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition. PMID- 7532585 TI - Substrate specificity and detailed characterization of a bifunctional amylase pullulanase enzyme from Bacillus circulans F-2 having two different active sites on one polypeptide. AB - Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on alpha-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed alpha-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and alpha-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both alpha-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro- Gln-Gln-Gln-Phe- Asp-Ala-Leu-Trp-Ala-Ala Gly-Ile-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both alpha-1,6 and alpha-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term 'amylase-pullulanase enzyme' to refer to enzymes which act on starch and cleave both alpha-1,6-bonds in pullulan and alpha-1,4 bonds in amylose at different active sites. PMID- 7532586 TI - The human inter-alpha-trypsin inhibitor genes respond differently to interleukin 6 in HepG2 cells. AB - The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL-6, suggesting that IL-6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL-6-treated cells confirmed these data. The nuclear run-on assay supports the regulatory effect of IL-6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL-6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre-alpha trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL-6. PMID- 7532587 TI - Lipopolysaccharide induces Ca(2+)-independent nitric oxide synthase activity in rat gastric mucosal cells. AB - Ca(2+)-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.). Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction. Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca(2+)-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion). Ca(2+)-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system. PMID- 7532589 TI - Evidence that carboxyl-reduced heparin fails to potentiate acidic fibroblast growth factor activity due to an inability to interact with cell surface heparin receptors. AB - Recently we reported that carboxyl-reduced heparin (CR-heparin), despite binding acidic fibroblast growth factor (aFGF) as effectively as native heparin, was much less potent at augmenting aFGF-induced mitogenesis. This paper describes experiments which examined this phenomenon in more detail in the hope that it would shed light on the mechanism by which heparin potentiates aFGF activity. Initial studies confirmed that heparin, with 60% of its carboxyl groups reduced, although binding aFGF with the same affinity as native heparin (Kd 35 +/- 5 nM), was a poor potentiator of aFGF-induced mitogenic activity. Proteolysis protection experiments also revealed that CR-heparin was as effective as native heparin at protecting aFGF from proteolytic degradation. In contrast, CR-heparin was considerably less effective than native heparin at enhancing the binding of aFGF to the fibroblast growth factor receptor (FGFR) on 3T3 cells. Furthermore, CR heparin only bound to a subset (approximately 1/3) of heparin receptors on 3T3 cells. Based on these data, it is proposed that CR-heparin is less efficient than heparin at facilitating the formation of a quaternary complex among aFGF, the FGFR, and cell surface heparin receptors. PMID- 7532588 TI - Mechanisms for pancreatic hypertrophy induced by long-term administration of bethanechol. AB - Mechanisms for the hypertrophy of rat pancreas induced by long-term administration of bethanechol were investigated. The administration of bethanechol, an acetylcholine receptor agonist, to male Wistar rats for 14 days induced significant increases in the pancreatic weight and contents of protein, amylase and RNA in the pancreas without altering the content of DNA and the incorporation of [3H]thymidine into DNA. Simultaneous administration of atropine with bethanechol suppressed the bethanechol-induced pancreatic hypertrophy. Long term administration of other acetylcholine receptor agonists also showed similar effects as produced by bethanechol. CR1505 (loxiglumide; D,L-4-(3,4 dichlorobenzoyl-amino)-5-(N-3-methoxypropyl-pentylam ino)-5- oxopentanoic acid), an antagonist of cholecystokinin receptors, inhibited pancreatic growth induced by long-term administation of pentagastrin, whereas pancreatic hypertrophy induced by bethanechol was not inhibited by CR1505. These results suggest that long-term administration of bethanechol induces pancreatic hypertrophy through direct activation of muscarinic receptors in the pancreas. PMID- 7532591 TI - Behavioral and biochemical consequences of combined lesions of the medial septum/diagonal band and nucleus basalis in the rat when ibotenic acid, quisqualic acid, and AMPA are used. AB - Combined lesions in the medial septum/diagonal band and nucleus basalis magnocellularis (NBM) in rats were produced using three excitotoxins, ibotenate (Ibo), quisqualate (Quis), and AMPA. Reductions in choline acetyltransferase (ChAT) activity differed in the cortical regions for the three toxins (AMPA > Quis > Ibo), but were fairly similar in the hippocampus. ChAT activities were not reduced in the globus pallidus, but AMPA reduced ChAT in the amygdala. Lesions with all three toxins produced similar decrements in hippocampal and posterior cortical serotonin levels. A small reduction in posterior cortical norepinephrine was detected for Quis and Ibo lesions. Spatial memory impairments were found for all three toxin groups compared with controls in acquisition, platform reversal, and a spatial probe in the water maze. The learning deficit was greatest with the Quis lesion and equivalent for the Ibo and AMPA lesions. There was no deficit in single trial passive avoidance retention for the Ibo and AMPA groups. The AMPA group was slower than controls on both training and retention trials to enter the dark compartment. This group also showed a tendency to hypoactivity as measured in an open-field test. Excitotoxic infusions into medial septum/diagonal band and NBM produced spatial mnemonic deficits which do not parallel reductions in overall ChAT activity and do not resemble the profile of behavioral changes previously reported for NBM lesions alone using these toxins. PMID- 7532590 TI - The Ca2+ signaling capacity of the beta 2-integrin on HL60-granulocytic cells is abrogated following phosphorylation of its CD18-chain: relation to impaired protein tyrosine phosphorylation. AB - The phosphorylation state of the CD18-chain of beta 2-integrins have been shown not to mediate changes in the avidity of these receptors (i.e., inside-out signaling); however, no alternative functional significance has been proposed. Our study focused on how changes in the phosphorylation state of beta 2-integrin receptors on HL60-granulocytic cells are related to its intracellular signal transduction properties (i.e., outside-in signaling). Engagement of beta 2 integrins on differentiated HL60 cells induced a transient increase in the cytosolic free Ca2+ concentration and an increased tyrosine phosphorylation of three major protein bands (70, 115, and 140 kDa). These signaling events occurred without any detectable phosphorylation of the CD18-chain. However, a strong phosphorylation of the CD18-chain by preexposure to phorbol myristate acetate (PMA) coincided with an abolishment of both the beta 2-integrin-induced Ca2+ signal and the protein tyrosine phosphorylations. By comparison, none of these effects were exhibited by 4-alpha-PMA, an analogue that does not activate protein kinase C. Thus, phosphorylation of the CD18-chain of beta 2-integrins is not required for outside-in signal transduction by these receptors, but it could constitute an effective mechanism by which the signaling properties of beta 2 integrins can be modulated by exogenous factors and possibly also by intracellular signals induced by other receptors. The fact that both the cytosolic free Ca2+ signal and protein tyrosine phosphorylations were abrogated by PMA suggests an intimate relationship between these two intracellular signals. To explore this possible relationship, we chelated the beta 2-integrin-induced Ca2+ signal with BAPTA. The beta 2-integrin-induced protein tyrosine phosphorylations were blocked by BAPTA but not by abolishment of the Ca2+ signal due to chelation with MAPT or by pretreatment with thapsigargin. These findings and the observation that pretreatment of cells with methyl-2,5-dihydroxycinnamate (a tyrosine kinase inhibitor) blocked the beta 2-integrin- but not the fMet-Leu Phe-induced Ca2+ signal suggest that beta 2-integrin-induced tyrosine kinase activation occurs prior to and is a prerequisite for the subsequent Ca2+ signal. PMID- 7532592 TI - Retrograde axonal transport and lesion-induced upregulation of the TrkA high affinity NGF receptor. AB - Long-term physiological responses of nerve growth factor (NGF) and other neurotrophins require gene regulation and likely depend on retrograde axonal transport of NGF or a signaling molecule activated by ligand-receptor interaction. The low-affinity neurotrophin receptor p75LANR is retrogradely transported, but this receptor is not sufficient for NGF-dependent cell survival or differentiation. In this study we examined the distribution and transport of the TrkA NGF receptor using two anti-peptide polyclonal antibodies and a monoclonal antibody, all of which are TrkA specific. We find that (1) in the adult rat brain TrkA-like immunoreactivity is similar with all antibodies in striatal and basal forebrain neurons, (2) TrkA is upregulated in neuronal and nonneuronal cells near the sites of injury, and (3) TrkA immunoreactivity builds up within the proximal and distal segments of transected fimbrial axons, which is consistent with its transport in the anterograde and retrograde directions. Thus, TrkA may itself be, or be a component of, the neurotrophic intraaxonal messenger by which NGF regulates gene expression in sensitive neurons. PMID- 7532593 TI - Detection and regulation of tyrosine hydroxylase mRNA in catecholaminergic terminal fields: possible axonal compartmentalization. AB - Reverse transcriptase coupled with nested polymerase chain reaction amplification (RT/nested-PCR) was used to detect mRNA encoding tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in adult rat cerebellum, striatum, and pituitary neurointermediate lobe (NIL). These regions receive catecholaminergic innervation from the locus coeruleus, substantia nigra, and arcuate and periventricular nuclei of the hypothalamus, respectively, but are devoid of catecholamine-synthesizing cells. The RT/nested-PCR products, which were generated using primers located on different exons of the tyrosine hydroxylase gene, indicate that the tyrosine hydroxylase mRNA detected is devoid of introns and, hence, is processed. These findings raise the possibility that tyrosine hydroxylase mRNA may be axonally transported. Using the same RT/nested PCR protocol, we were unable to detect mRNA encoding dopamine beta-hydroxylase, a different catecholaminergic biosynthetic enzyme, in either cerebellum, striatum, or NIL pituitary tissue. Thus, the detection of tyrosine hydroxylase mRNA in catecholamine terminal regions is biochemically specific. We were unable to detect tyrosine hydroxylase mRNA in optic nerve, indicating some degree of anatomical specificity as well. Expression of tyrosine hydroxylase mRNA in the cerebellum was markedly increased by subcutaneous administration of the catecholamine-depleting agent, reserpine, suggesting that tyrosine hydroxylase mRNA in catecholamine terminal regions may be functionally important. This finding also indirectly supports the hypothesis that tyrosine hydroxylase mRNA can be axonally transported since the ability of reserpine to induce expression of this transcript in conventional catecholamine cell groups is considered secondary to catecholamine depletion, and cerebellar cells do not synthesize catecholamines. Finally, lesions of the nigrostriatal pathway significantly decreased levels of tyrosine hydroxylase mRNA in the striatum, providing strong additional support for this hypothesis. PMID- 7532594 TI - Cloning and expression of an isoform of the rat mu opioid receptor (rMOR1B) which differs in agonist induced desensitization from rMOR1. AB - A novel rat mu opioid receptor (rMOR1B) has been isolated. It shows identity to the recently published sequence of rMOR1 [Chen, et al., Mol. Pharmacol., 44 (1993) 8-12] up to amino acid 386 and differs only in length and amino acid composition at the very carboxy-terminal tail. Both mu opioid receptor isoforms, when stably expressed in CHO-K1 cells, show similar affinities to opioid compounds and are equally effective in the inhibition of forskolin-induced cAMP formation. Reverse transcription polymerase chain reaction (RT-PCR) revealed that rMOR1B displays a similar distribution as rMOR1 in various rat brain areas. Studies measuring the inhibition of adenylate cyclase in cells that had been pre exposed to the mu opioid agonist DAMGO indicated that rMOR1B is much more resistant to agonist-induced desensitization than rMOR1. PMID- 7532595 TI - Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. AB - Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). BHAP-resistant HIV-1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline-to-leucine substitution at amino acid 236 (P236L) of HIV-1 RT. To understand the basis for the in vitro sensitization resistance phenomenon, single base pair mutations at amino acid P236 in HIV-1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV-1 RT mutants. Active HIV-1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV-2 RT, were also cloned, expressed, and purified. Three BHAPs (U-88204E, U-87201E, and U-90125S) and the pyridinone L-697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L-697,661 and resistance to the BHAPs. The HIV-1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV-2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV-1 RT mutants demonstrated a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L-697,661. PMID- 7532596 TI - Systemic production of foreign peptides on the particle surface of tobacco mosaic virus. AB - By using a new tobacco mosaic virus (TMV) vector [Hamamoto, H., et al. (1993) Bio/Technology 11, 930-932], we have constructed TMV particles which present three different kinds of epitopes, two of them from influenza virus hemagglutinin (HA), and one from human immunodeficiency virus type I (HIV-I) envelope protein, on the surface of the particles. Each of these TMV particles reacted with each anti-peptide antiserum. These results suggest that this TMV vector can be used as an antigen presentation system. PMID- 7532597 TI - Inhibition of arginase by NG-hydroxy-L-arginine in alveolar macrophages: implications for the utilization of L-arginine for nitric oxide synthesis. AB - The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, NG-hydroxy-L-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC50 > or = 15 microM), and attenuated L-[3H]arginine transport (IC50 > or = 500 microM) in rabbit alveolar macrophages. Moreover, up to 37 microM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of L-arginine for NO biosynthesis. PMID- 7532598 TI - Screening for ophthalmic manifestations of sickle cell disease in the United Kingdom. AB - There are marked variations in the manifestations of sickle disease in different populations. The ocular complications of this condition amongst the Afro Caribbeans living in the United Kingdom have not previously been reported. We present the preliminary results of an ophthalmic screening programme at King's College Hospital, London. One hundred eyes of 50 patients with sickle cell disease were assessed. Full ocular examination was performed including fundus fluorescein angiography. We have looked at the haematological and clinical profile of the patients involved as well as the number of days spent in hospital during the year preceding the eye examination. The incidence of grade II retinopathy was found to be significantly higher than grade I in SC disease. This concurs with the results of the Jamaican screening and confirms that these patients are at higher risk of visual impairment than those with SS disease. Our results also agree with the Jamaican experience which suggest that visual morbidity is mostly due to complications of proliferative sickle retinopathy (PSR). However, the findings in patients without proliferative changes are different; in particular, angioid streaks leading to disciforms are an important cause of visual loss in Jamaica, but were not seen in any of the 98 eyes examined in this study. No correlation was found between the grade of retinopathy and age, sex, systemic complications and various haematological parameters except for the percentage of haemoglobin F, which was significantly higher in patients with grade I (7.6) compared with grade II (4.2) retinopathy (p = 0.0127). PMID- 7532601 TI - Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development. AB - Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult. PMID- 7532599 TI - Reduced insulin-like growth factor-I mRNA content in liver, adrenal glands and spinal cord of diabetic rats. AB - Circulating insulin-like growth factor I (IGF-I) concentrations are known to be reduced in experimental and clinical diabetes mellitus. The IGF-I mRNA content was measured in several tissues of rats treated with streptozotocin to determine whether a correlation with neuropathy could be found. IGF-I mRNA content was sharply reduced relative to total and poly(A)+ RNA in diabetic liver and adrenal glands. In contrast, histone 3.3 mRNA content was not significantly reduced relative to poly(A)+ RNA in liver, and alpha-tubulin mRNA content instead was increased in adrenal glands, showing that the decline in IGF-I mRNAs in these tissues was selective. In addition, spinal cord IGF-I mRNA content was significantly reduced per tissue, total RNA, and poly(A)+ RNA after 1 and 2 weeks of diabetes. This was correlated with a concurrent and significant decrease in conduction velocity in both spinal cord and peripheral nerves in a separate study. The decline in liver and spinal cord IGF-I mRNA was not due to streptozotocin toxicity, because it was significantly opposed by insulin which was continuously infused beginning the day after diabetes induction. These results, when taken together with those of others, indicate that the reduction in IGF-I mRNA content may be widespread among diabetic tissues, and might contribute in part to certain syndromes of diabetes, such as neuropathy. PMID- 7532600 TI - Cyclophosphamide treatment of female non-obese diabetic mice causes enhanced expression of inducible nitric oxide synthase and interferon-gamma, but not of interleukin-4. AB - In pancreatic lesions of non-obese diabetic (NOD) mice the expression of inducible nitric oxide synthase (iNOS) and of the cytokines interferon-gamma and interleukin-4 were studied. Strong iNOS expression as determined at the level of transcription, translation and of enzyme activity was associated with destructive insulitis as seen 8-10 days after cyclophosphamide treatment of 70- to 80-day-old female NOD mice. Immunohistochemistry showed iNOS associated with infiltrating macrophages but not in endocrine cells. The enhancement of iNOS after cyclophosphamide correlated with an increase of T-helper type 1 (Th1) associated interferon-gamma expression while T-helper type 2 (Th2) associated interleukin-4 was the dominant cytokine prior to cyclophosphamide and after diabetes onset. We conclude that insulitis in young NOD mice is carried by Th2 cells while cyclophosphamide enhanced insulitis is determined by Th1 cells. Macrophages show two different functional states in insulitis; strong iNOS expression in macrophages is associated with destructive insulitis. PMID- 7532603 TI - Comparative analysis of normal and psoriatic skin both in vivo and in vitro. AB - The morphological and biochemical characteristics of psoriasis are well documented, but the pathogenesis of this disease is not clearly understood. A variety of in vitro models of psoriasis have been developed in attempts to identify the trigger factors, but no model so far reproduces the stable psoriatic phenotype accurately. In the present work, we initially checked the immunohistochemical distribution of proliferation/differentiation markers in psoriatic skin in vivo, and our results largely confirm previously reported data. However the study was performed using a new series of monoclonal antibodies to keratin. Subsequently we took normal or psoriatic skin biopsies, reconstructed skin equivalents using a recently described model and analysed the proliferation/differentiation status of the resulting epidermis. Dramatic morphological and antigenic differences were found between normal and psoriatic skin in vivo, but whatever the source of the initial biopsy, a unique in vitro phenotype was obtained in the reconstructed epidermis. This phenotype was marked by mild hyperproliferation and an altered distribution of differentiation associated antigens suggesting a need for extracutaneous stimuli to maintain the psoriatic phenotype in vitro. PMID- 7532602 TI - Existence of trichohyalin-keratohyalin hybrid granules: co-localization of two major intermediate filament-associated proteins in non-follicular epithelia. AB - Trichohyalin is a protein of relatively high molecular weight (approximately 200 kDa), associated with intermediate filaments, that was for many years thought to be expressed only in the inner root sheath and medulla of the hair follicle. We show here, however, that this protein is expressed in association with (pro)filaggrin in the granular layer of many non-follicular, keratinizing, stratified epithelia which also express keratins K6/K16, including those of the filiform papillae of dorsal tongue epithelia. In this epithelium, which elaborates morphologically heterogeneous keratohyalin granules in its upper cell layers, trichohyalin forms hybrid granules with filaggrin, the major intermediate filament associated protein found in keratohyalin granules, which is normally expressed in advanced epidermal differentiation. These two intermediate filament associated proteins remain physically segregated in the hybrid granules, but they share the same fate, as they both become dispersed in transitional cells, and are undetectable in cornified cells. Trichohyalin was also detected in nail matrix epithelia, the epithelium of Hassal's corpuscles of the thymus, and newborn foreskin epidermis. It is essentially absent from normal trunk and scalp epidermis, but is expressed in a few scattered cells of the granular layer that are also filaggrin-positive. In addition, trichohyalin is expressed in the epidermis in a number of hyperplastic skin diseases. These findings demonstrate that trichohyalin is not peculiar to a small number of hair follicle cells, but is expressed in a number of normal and pathological epithelia where it is uniquely associated with filaggrin. In addition, since all these trichohyalin expressing keratinocytes also synthesize keratins K6 and K16 (the markers for an "alternative" pathway of keratinocyte differentiation), this raises the possibility that the trichohyalin protein is specifically (or preferentially) involved in aggregating intermediate filaments containing the K6/K16 keratins. PMID- 7532604 TI - d-Limonene induced hyaline droplet nephropathy in alpha 2u-globulin transgenic mice. AB - d-Limonene is a hyaline droplet inducing agent and produces nephrotoxicity in male rats when the 1,2-epoxide metabolite binds to alpha 2u-globulin. Mice, which do not synthesize alpha 2u-globulin, are resistant to hyaline droplet nephropathy. In this study, the ability of d-limonene to cause hyaline droplet nephropathy in a transgenic mouse engineered to express alpha 2u-globulin was evaluated. The C57BL/6-derived mice excreted 0.4 +/- 0.1 mg alpha 2u globulin/day, or approximately 16 mg alpha 2u-globulin/kg body wt. This represents about 30% of the amount excreted by adult male rats (11.9 +/- 1.1 mg/day or approximately 48 mg/kg). Transgenic mice excreted less mouse urinary protein (9.3 +/- 1.2 mg/day) than normal mice (15.1 +/- 1.6 mg/day). Unlike normal male rats, untreated transgenic mice did not show significant spontaneous hyaline droplet formation. Liver microsomes from naive transgenic mice oxidized d limonene to the cis- and transisomers of the 1,2-epoxide, and following oral treatment with [14C]d-limonene reversible binding of d-limonene equivalents to renal cytosolic proteins was observed. Furthermore, with d-limonene treatment, hyaline droplets were observed in the transgenic mouse kidneys. These droplets, however, were much smaller in size than those seen in d-limonene-treated male rats. The accumulation of alpha 2u-globulin in the kidneys of transgenic mice and normal male rats before and after d-limonene treatment was analyzed by Western blotting. These results indicated that alpha 2u-globulin was present in the kidneys of the control transgenic mice, despite the lack of spontaneous hyaline droplet formation. After d-limonene treatment, approximately a three-fold increase in alpha 2u-globulin in the transgenic mouse kidney was observed, a response similar in magnitude to that seen in d-limonene-treated male rats. These results indicate that expression of alpha 2u-globulin in a species that does not normally develop hyaline droplet nephropathy is necessary and sufficient to render that species sensitive to this renal toxicity. PMID- 7532606 TI - The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid. AB - The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements. PMID- 7532605 TI - A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. AB - We identify a function-controlling O antigen chain length for a plasmid-borne gene, cldpHS-2, harboured by Flexneri strains of Escherichia coli known to cause reactive arthritis. The predicted amino acid sequence of the gene product is very similar to those of other cld genes and that of fepE, thought to be part of the enterobactin iron uptake system of E. coli. The predicted proteins are compared with rfb-associated chain length determinants as a family of related genes. PMID- 7532607 TI - Transcriptional regulation in the Chlamydia trachomatis pCT plasmid. AB - We have analyzed transcriptional regulation of the chlamydial plasmid pCT. Transcription of a full-length 2.9-kb ORF1-ORF2 mRNA is likely to be regulated by the sigma 66 transcription factor which recognizes the TATAAT and TNGNCA sequences at the -10 and -35 DNA regions, respectively. RNA synthesis starts 39 nucleotides (nt) upstream from the ATG start codon of ORF1 and terminates within the downstream ORF3 DNA region. A 2.8-kb transcript transverses the ORF3-6 DNA region, while two transcripts of 2.2 and 1.9 kb cover the ORF4-6 DNA region. These mRNAs overlap two abundant transcripts which regulate the expression of the ORF3 and ORF4 genes. The accumulation of transcripts associated with these ORFs is likely to be regulated at the level of RNA synthesis by an unknown sigma factor which could select the RTTTAAA and TTYTTR sequences located at the -10 and -35 DNA regions, respectively. This new promoter consensus sequence could be unique to the gene expression machinery of Chlamydiae. PMID- 7532609 TI - Modulation of glutathione S-transferase activity and isozyme pattern in liver and small intestine of rats fed goitrin- and T3-supplemented diets. AB - Goitrin is a potent goitrogen that has been shown to induce glutathione S transferase (GST) activity and to increase aflatoxin detoxification. In the present study with rats, dietary goitrin (200 mg/kg diet) produced a hypothyroid state and significantly increased levels of hepatic GSSG (1.4-fold), GST protein (1.4-fold) and GST activity against chlorodinitrobenzene (CDNB) (1.7-fold). Cotreatment with dietary triiodothyronine (T3) reversed these effects in a dose related manner. Intestinal GST activities against CDNB and epoxynitrophenoxypropane did not change with goitrin or T3 treatment. HPLC analysis showed that, in the liver, goitrin treatment increased the levels of GST 1b and -7 by 3.5- and 5-fold, respectively, and decreased the level of GST-3 by 50%. Cotreatment with T3 returned levels of GST-7 and -3 to control levels but only partially reduced the level of GST-1b. In the small intestine, goitrin increased the level of GST-1b by 28% and decreased the level of GST-7 by 34% compared with those of controls; thyroid hormone treatment produced no additional effect on GST in this organ. Selenium deficiency altered thyroid hormone status but significantly affected the level only of hepatic GST-3, which was reduced by 30% compared with that of controls. These results indicate that a modified thyroid hormonal status plays an important role in the GST-inducing effects of goitrin. A possible mechanism of thyroid-dependent GST induction by goitrin is discussed. PMID- 7532610 TI - Chlorpyrifos: hazard assessment based on a review of the effects of short-term and long-term exposure in animals and humans. AB - Analyses of potential dietary exposure to chlorpyrifos residues were conducted by the Department of Pesticide Regulation (DPR). Potential acute dietary ingestion of chlorpyrifos for all labelled uses was based on the 95th percentile of user day exposures. Margins of safety (MOSs) for potential acute dietary exposure to chlorpyrifos residues were based on a no-observed-effect level (NOEL) for cholinergic signs in a human study, and ranged from 52 to 205 for all population subgroups. MOSs for potential chronic dietary exposure to chlorpyrifos residues were based on a NOEL for inhibition of brain cholinesterase activity in rats and dogs, and ranged from 2198 to 8065 for all population subgroups. The limitations on toxicity, consumption and residue data are discussed, with the assumptions necessitated by those limitations. PMID- 7532611 TI - [Prevalence of hepatitis C virus infection among chronic hemodialysis patients]. AB - A high prevalence of HCV (hepatitis C virus) antibodies have been reported in chronic hemodialysis (HD) patients. In this study, sera from 584 hemodialysis patients (378 males and 206 females) were examined for the 2nd generation HCV antibody by enzyme immunoassay (EIA) and for HCV-RNA by the reverse transcription polymerase chain reaction (RT-PCR) method. The positive rates of HCV antibody and HCV-RNA were 22.0% (129/584) and 12.3% (72/584), respectively. In HD patients, HCV infections were considered to be caused by the contact with contaminated blood samples, the main cause of which is blood transfusions. However, a high prevalence of HCV markers even in HD patients without a history of blood transfusions indicates that there may be other ways of HCV transmission other than blood transfusions. While HCV-RNA was detected in about 50% of the HD patients with the HCV antibody, this rate was lower than that found in the general population with the HCV antibody. And the abnormality rate of serum alanine aminotransferase (ALT) in HD patients with HCV-RNA was lower than that of the general population with HCV-RNA. On the other hand, the amounts of HCV-RNA which were semiquantified by RT-PCR using the end-point dilution method showed a correlation with serum ALT levels in both HD patients and chronic HCV infections. Moreover, HCV-RNA titers measured immediately after hemodialysis showed a marked decrease compared with those measured before hemodialysis. These results suggest that HCV was eliminated to certain extent with hemodialysis and that it might also be the cause of the low abnormality rate of serum ALT in HD patients with HCV-RNA. PMID- 7532608 TI - Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation. AB - Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury. PMID- 7532612 TI - Insulin-like growth factor binding proteins in the human circulation: a review. AB - The actions and bioavailability of the insulin-like growth factors (IGFs) are regulated by a family of six IGF binding proteins. IGFBP-3, the major circulating IGFBP, is unique in combining with a glycoprotein, the acid-labile subunit (ALS), to form a ternary complex with IGF-I or IGF-II. Each component of this trimer is growth hormone-dependent, and the hypoglycemic potential of circulating IGFs appears neutralized in this form. IGFs in the complex have a greatly extended circulating half-life, their stability being conferred by the presence of ALS, which is itself very stable in the circulation. IGFBP-1 does not appear to be a carrier of IGFs, but to act as an acute modulator of their metabolic activities. In this role it can be viewed as an insulin counter-regulator, blocking 'free' insulin-like activity during fasting or hypoglycemia. IGF-I administration causes complex changes in circulating IGFBPs, so that a detailed knowledge of their regulation is essential if the therapeutic potential of IGF-I is to be optimised. PMID- 7532614 TI - CD34 immunoexpression in stromal tumours of the gastrointestinal tract and in mesenteric fibromatoses. AB - The aim of this study was to explore whether CD34 immunoreactivity can distinguish between different types of gastrointestinal stromal tumour, i.e. smooth muscle and neurogenic. We studied 87 stromal tumours from different sites in the gastrointestinal tract, as well as the omentum and mesentery, using a monoclonal antibody to CD34 (QBEND10). We also determined the immunoexpression of smooth muscle and muscle specific actins, S-100 protein, cytokeratin, desmin and vimentin. In addition, 15 cases of mesenteric fibromatosis were tested for CD34. Immunoexpression of CD34 was observed in 40 of the 87 stromal tumours and correlated with evidence of differentiation towards a smooth muscle phenotype. Large intestinal stromal tumours were less likely than gastric lesions to be CD34 positive. None of 15 cases of mesenteric fibromatosis was positive for CD34. We conclude that CD34 immunoexpression is seen in a proportion of stromal tumors of the gastrointestinal tract, mesentery and omentum, particularly those of smooth muscle type, and it may be useful as part of an immunohistochemical panel in the differential diagnosis of these neoplasms. PMID- 7532613 TI - Hormonal and nutritional regulation of IGF-I and its binding proteins. AB - The influence of nutrition on growth seems likely to be mediated in part through IGF-I. Restriction of dietary nutrients adversely effects IGF-I synthesis and action at multiple steps, including decreased GH receptors, postreceptor defects in GH action, decreased steady state levels of IGF-I mRNA, and attenuation of IGF I action. In addition, undernutrition affects the IGF binding proteins in ways that vary from one tissue to another. Understanding the alterations in IGFBPs that are caused by nutritional insufficiency may provide insight into the actions of the IGFBPs. PMID- 7532615 TI - Basement membrane and extracellular interstitial matrix components in bladder neoplasia--evidence of angiogenesis. AB - Immunohistochemical studies to localise collagen IV, laminin, fibronectin and tenascin were undertaken in 59 bladder biopsies. These were non-neoplastic or contained foci of flat dysplasia or of transitional cell neoplasia. Collagen IV and laminin were reliably demonstrated in basement membranes but focal loss of reactivity for collagen IV was found in the urothelial basement membrane in some cases of inflammation, dysplasia, and non-invasive papillary transitional cell tumour. Basement membranes were rarely demonstrated around invasive transitional cell carcinomas. Fibronectin and tenascin were found in the sub-urothelial lamina propria and in some urothelial basement membranes. The stroma of invasive tumours reacted strongly for both. Increased numbers of sub-urothelial capillaries were present in association with some cases of cystitis, flat dysplasia or non invasive transitional cell neoplasia suggesting an angiogenic stimulus in these circumstances. Immunohistochemical staining of basement membrane components is unlikely to be of value in the routine histopathological assessment of transitional cell neoplasms of the bladder. PMID- 7532617 TI - Natural antibodies and human xenotransplantation. PMID- 7532616 TI - Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population. AB - Seven different restriction fragment length polymorphisms (RFLPs) at the N acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or non-carrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible. PMID- 7532619 TI - Alpha 1,3galactosyltransferase: a target for in vivo genetic manipulation in xenotransplantation. PMID- 7532618 TI - Gal alpha (1,3)Gal, the major xenoantigen(s) recognised in pigs by human natural antibodies. AB - The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection- mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation. PMID- 7532620 TI - Isolation, identification, and culture of normal mouse colonic glia. AB - We have developed a novel method of isolating and culturing murine colonic mucosal glial cells. Two morphologies are appreciated, a small flat bi or tri polar cell and a larger multipolar cell. The glial cultures have been freed of contaminating fibroblasts and epithelial cells and have been passaged by trypsinization. By intermediate filament (IF) typing, the glial cells have been further characterized as astrocyte-like. All cells expressed glial fibrillary acid protein but not neurofilament 160 protein. The glial cultures expressed the neuropeptides, substance P and substance K. Central nervous system astrocytes synthesize neuropeptides, prostaglandins and cytokines, and can express major histocompatibility class II antigens. It is likely that enteric mucosal glia will also prove to have varied functions. These cultures can now be used to define the role of enteric mucosal glia and to further study their complex interaction with other cells of the colonic mucosa. PMID- 7532621 TI - Characterization of C6-10A glioma cells highly responsive to beta-adrenergic receptor agonist-induced NGF synthesis/secretion. AB - A subline of rat C6 glioma cells, C6-10A cells, in which epinephrine can induce nerve growth factor (NGF) synthesis/secretion, was isolated. C6-10A cells have retained their sensitivity to epinephrine for more than 2 years in a medium containing 0.5% fetal calf serum (FCS) but easily lose it in 10% FCS. C6-10A cells are S-100- and glial fibrillary acidic protein-positive, and the doubling time is about 60 h in the medium containing 0.5% FCS and about 20 h in 10% FCS. Epinephrine induced NGF synthesis/secretion prominently in serum-free cultures of C6-10A cells and in cultures with a high cell density, but not in serum containing cultures. The induction did not occur with parent C6 cells under the appropriate conditions in C6-10A cells. NGF secretion was induced by catecholaminergic compounds in the following order isoproterenol > epinephrine = norepinephrine >> dopamine. The induction caused by epinephrine was blocked by propranolol (beta-blocker) but not by phentolamine (alpha-blocker). Various compounds that activate the adenylate cyclase system also induced NGF synthesis/secretion. These results indicate that C6-10A cells are astrocytes that are highly responsive to beta-adrenergic receptor agonists, which stimulate NGF synthesis/secretion via receptors coupled with adenylate cyclase machinery. These cells may be a useful aid in studying the mechanism of NGF synthesis/secretion. PMID- 7532622 TI - Regulation of an oligodendrocyte progenitor cell line by the interleukin-6 family of cytokines. AB - We report pleiotropic actions of the interleukin-6 family of cytokines on a rat cerebral cortical oligodendrocyte cell line, Central Glia-4 (CG-4). This is a bipotential oligodendrocyte type-2 astrocyte (O-2A) progenitor cell line that can be manipulated in vitro to become either a type-2 astrocyte or to follow a linear sequence of events into becoming a mature oligodendrocyte. Using Northern and Western analyses in conjunction with immunocytochemistry we have demonstrated that ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and interleukin-6 (IL-6) cause a transient increase in glial fibrillary acidic protein (GFAP) in oligodendrocyte type-2 astrocyte (O-2A) progenitor cells. At maximal cytokine concentrations, the largest increase in GFAP protein levels were observed for CNTF and LIF; albeit, IL-6 did increase GFAP but the order of magnitude was 6-7 times less. Moreover, in trophic factor deprived medium, CNTF and LIF protected immature (O4+/MBP-) and mature (MBP+) oligodendrocytes from the apoptotic mode of cell death, while IL-6 had no effect in enhancing oligodendrocyte cell survival. Analysis of the cytokine-induced early response genes (ERGs) revealed a strong degree of overlap for CNTF and LIF. The effect of IL-6 was different in the degree to which the ERGs were up-regulated and in their temporal patterns of expression. These findings suggest that ERGs may be important, at least in part, for determining the extent of functional overlap observed within this cytokine family. Our findings clearly demonstrate differential regulation of oligodendrocyte survival and differentiation by the IL 6 family of cytokines. PMID- 7532624 TI - Role of a carboxy-terminal site of toxic shock syndrome toxin 1 in eliciting immune responses of human peripheral blood mononuclear cells. AB - Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1) is involved in the pathogenesis of toxic shock syndrome and perhaps other staphylococcal diseases. Recently, the C-terminal part of the TSST-1 toxin has been shown to be responsible for mitogenic activity in animal models. We studied the role of the C terminal structural unit of TSST-1 with regard to proliferation, cytokine release (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], and IL-8), mRNA expression for IL-6, IL-8, IL-10, TNF-alpha, and CD40 ligand (CD40L), synthesis of immunoglobulin E (IgE), IgA, IgG, and IgM, CD23 expression, and soluble CD23 (sCD23) release from human peripheral blood mononuclear cells (PBMC). For this purpose, we used the recombinant wild-type TSST-1 (p17) mutant toxin Y115A (tyrosine residue modified to alanine) and toxin H135A (histidine residue modified to alanine). Unmodified toxin p17 and mutant toxin Y115A, at a concentration below 5 ng, to a lesser degree, induced a strong proliferation. Toxin p17 followed by toxin Y115A was the most pronounced inducer for mRNA expression for IL-10 and CD40L and cytokine generation (mRNA and protein) for TNF alpha, IL-6, and IL-8. Mutant protein H135A failed to activate human PBMC. Both toxins p17 and, to a lesser degree, Y115A significantly suppressed IL-4- and anti CD40-induced synthesis of all four Igs as well as IL-4-induced CD23 expression and sCD23 release. Mutant toxin H135A failed to do so. Thus, our data show that a region in the C terminus of TSST-1 is responsible not only for mitogenic activity but also for additional immunomodulating biological activities of TSST-1. More specifically, histidine residue H135A of the 194-amino-acid toxin appears to be critical for the expression of biological activities in a human in vitro model. PMID- 7532625 TI - Poliovirus hybrids expressing neutralization epitopes from variable domains I and IV of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies. AB - Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes from the variable domains of the major outer membrane protein are candidates for vaccine development. We have constructed hybrid polioviruses expressing sequences from major outer membrane protein variable domains I and IV. Antisera to the hybrids could, in combination, strongly neutralize 8 of the 12 C. trachomatis serovars most commonly associated with oculogenital infections and weakly neutralize the others. PMID- 7532623 TI - Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14. AB - During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14. PMID- 7532626 TI - Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages. AB - Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages. PMID- 7532627 TI - Tumor necrosis factor induction by an aqueous phenol-extracted lipopolysaccharide complex from Bacteroides species. AB - The stimulation of macrophages and monocytes by lipopolysaccharide (LPS) results in the secretion of tumor necrosis factor (TNF), a cytokine which is thought to play a pivotal role in subsequent host responses. Its induction is thought to be facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. The LPS of Bacteroides species was considered a weak endotoxin; however, in a recent study we have shown that the biological activity and chemical composition of the LPS from Bacteroides species are dependent on the extraction method. The present study determines the capacity of LPS extracted by aqueous phenol (the method for producing an LPS of high endotoxic activity) from four species of Bacteroides to induce TNF. Induction was investigated from human mononuclear leukocytes (MNL), THP-1 cells (with and without enhancement by vitamin D2 for CD14), and peritoneal macrophages from C3H/HeJ (LPS nonresponder) and C3H/HeN (LPS responder) mice. Escherichia coli O18K- LPS, a typical smooth LPS of heterogeneous molecular mass, was used as a control throughout. The stimulation of TNF production by E. coli LPS was between two- and fourfold more than that by Bacteroides LPS in MNL, in THP-1 cells (with enhancement for CD14), and in peritoneal macrophages from C3H/HeN mice. In THP-1 cells (without enhancement for CD14), there was no significant difference in TNF production between E. coli and Bacteroides LPSs. In peritoneal macrophages from C3H/HeJ mice, E. coli LPS stimulated no TNF production, but there was no significant difference in TNF production from peritoneal macrophages from C3H/HeJ and C3H/HeN mice by Bacteroides LPS. In all cell populations, there was a peak of TNF production after approximately 4 h of stimulation with all LPSs tested. However, other peaks of TNF production were seen in MNL and THP-1 cells (with enhancement for CD14) after stimulation with E. coli LPS only. In stimulation assays in which Bacteroides LPS was together with but in excess of E. coli LPS, it was found that TNF production from MNL and THP-1 cells (with and without enhancement for CD14) was comparable to that of Bacteroides LPS alone and not E. coli LPS alone. An anti-CD14 monoclonal antibody did not inhibit Bacteroides LPS-stimulated TNF production. However, E. coli LPS-stimulated TNF release was inhibited by an anti CD14 monoclonal antibody, most noticeably in MNL and THP-1 cells (with enhancement for CD14).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532628 TI - Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi. AB - The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix. PMID- 7532631 TI - Pardes and PaRDeS: towards a psychotherapeutic theory. AB - The Hebrew term Pardes has two meanings in Jewish study: the Paradise entered by four eminent Rabbis in search of mystical truth, and PaRDeS, four levels of interpreting Biblical texts. By combining the legend of Pardes with the hermeneutic system of PaRDeS, a psychological theory is presented as a set of propositions which endeavor to demonstrate the following: 1) The combination of Pardes and PaRDeS may be used as a bridge between the rational and mystic approaches to life and death. 2) The rational and the mystic stages each contain two phases: the negative break and the positive possibility of the symbolic "forty years old" more mature active remedy. 3) The "Acher-Akiva" and the "Ben Zoma-Ben Azai" dimensions do not necessarily represent four psychological personality types, but rather inner psychological struggles which may be experienced by one person in part or in full during different phases of his or her life cycle. The successful resolution of these inner psychological struggles may be facilitated by people's ability and motivation to find their one or more personally suitable "faces" from the "seventy" that are available for them. Thus, the road to "Pardes-paradise" is paved by people's own intentions. PMID- 7532629 TI - Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library. AB - The mouse monoclonal antibody 2A10 (immunoglobulin G), which recognizes the (NANP)n repeat of Plasmodium falciparum circumsporozoite surface protein, was used to screen a filamentous phage epitope library expressing random amino acid hexamers. The sequences obtained were TNRNPQ, SNRNPQ, NND-NPQ, SNYNPQ, and QNDNPQ (single-letter amino acid designation). These peptides showed 50% homology with the native epitope (PNANPN) and therefore were considered to mimic its structure (mimotopes). Two of these mimotopes (TNRNPQ and NNDNPQ) inhibited the binding of monoclonal antibody 2A10 to the recombinant protein R32LR, which contains the amino acid sequence [(NANP)15NVDP]2. Immunization of mice and rabbits using the peptide (TNRNPQ)4 induced a humoral response that recognized R32LR by an enzyme linked immunosorbent assay and P. falciparum sporozoites by an immunofluorescence assay. These results suggest that phage epitope libraries can be exploited to screen for mimotopes in the design of subunit vaccines against infectious agents. PMID- 7532632 TI - Culturally sensitive therapy with ultra-orthodox patients: the strategic employment of religious idioms of distress. AB - The article deals with the problem of administering therapy in multicultural settings where the therapist and the patient hold divergent explanatory models in regard to the patient's symptoms. Different conceptualizations of the universal structure of symbolic healing stress the importance of therapist-patient compatibility for therapeutic success. In order to reach this compatibility, strategic therapists seek to join the patients' explanatory models and employ metaphors and symbols derived from their cultural world. From a psychodynamic perspective, strategic techniques are often presented as superficial treatments limited to the symptomatic level. In order to deal with this argument, we present a case study of an ultra-orthodox patient with a major depressive episode and describe the treatment which was based on a strategic, culturally sensitive approach. We use the case to discuss theoretical issues arising in the context of multicultural therapy such as the translatability of culturally divergent idioms of distress and the possibility to bring about significant, nonsymptomatic changes through strategic employment of culturally congruent metaphors and symbols. PMID- 7532630 TI - Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines. AB - Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed. PMID- 7532634 TI - Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies. AB - Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100 degrees C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated. PMID- 7532635 TI - Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study. AB - Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19 OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib. PMID- 7532633 TI - Hypersensitivity to substance P in the etiology of postlumbar puncture headache. AB - OBJECTIVE: Postlumbar puncture headache may represent a model which could be used to test the hypothesis that headache pain is caused by the release of substance P in patients who are predisposed to headache due to hypersensitivity to substance P. METHODS: We measured substance P in CSF and plasma in 37 patients undergoing diagnostic lumbar puncture. In 9 patients, plasma samples were obtained before lumbar puncture, in 28 patients plasma was obtained after lumbar puncture. Patients were followed up by telephone to determine if they developed postlumbar puncture headache. Patients were also asked about a history of chronic or recurrent headaches. Substance P was determined by radioimmunoassay. RESULTS: The mean plasma substance P levels obtained before lumbar puncture was 1.0 +/- 0.1 pg/mL and 1.3 +/- 1.2 after lumbar puncture (P < 0.0005). The mean plasma substance P levels in subjects who developed postlumbar puncture headache was 0.6 +/- 0.6 pg/mL compared with 1.4 +/- 1.5 in subjects who remained headache-free (P < 0.05). The mean CSF substance P levels in subjects who developed postlumbar puncture headache was 0.7 +/- 0.5 pg/mL compared with 1.2 +/- 0.8 in subjects who remained headache-free (P < 0.05). There were no significant differences in substance P levels between chronic headache sufferers and nonheadache subjects. CONCLUSIONS: Postlumbar puncture headache may be mediated by the release of substance P triggered by lumbar puncture, in patients predisposed to headache by a hypersensitivity to substance P. Hypersensitivity to substance P may also represent a mechanism for headache pain in other headache disorders. PMID- 7532636 TI - Application of X-ray microanalysis to study of the expression of endothelial adhesion molecules on human umbilical vein endothelial cells in vitro. AB - A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV with scanned areas of 200 x 400 microns. The semi-quantitative data indicated that in LPS-treated groups both adhesion molecules were expressed at a significantly higher level than in all other groups (P < 0.01). In addition, after a 4 h treatment the expression levels of E-selectin in all groups were higher compared to ICAM-1. The experimental data from X-ray microanalysis were compared with data obtained from an enzyme-linked immunosorbent assay (ELISA) and similar values were found for both types of preparation. This microanalytical method is relatively simple and seems to be suitable for immunogold labelling studies on different types of endothelial cells in vitro. PMID- 7532637 TI - Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung. AB - The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1+/SP-A+ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages. PMID- 7532639 TI - IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study. AB - Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2 like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin- and glucagon-immunoreactive cells. A varying proportion (0-70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system. PMID- 7532640 TI - Improved methodology for detecting bromodeoxyuridine in cultured cells and tissue sections by immunocytochemistry. AB - Detection of DNA synthesizing cells may often be achieved by immunocytochemical detection of bromodeoxyuridine (BrdU), which is rapid and appears to give similar results to those found using tritiated thymidine. However, the methodology for detection of BrdU involves a denaturation or digestion step to allow access of the antibody to BrdU incorporated into single- rather than double-stranded DNA. We wished to determine if microwave treatment could be used to enhance the detection of BrdU without the need for any other digestion/denaturation steps. An important consideration was to investigate whether such treatment produces a similar quantitative result, since BrdU detection is usually assessed on the basis of cell number rather than topographical distribution. We have found that microwave treatment can allow considerably lower antibody concentrations and eliminates the need for any other denaturation step. It also reduces the non specific background staining found when using monoclonal antibodies on mouse tissue. We have performed cell counts and found that the number of BrdU positive cells remains constant for a range of different immunocytochemical parameters. We also report conditions where immunopositivity is adversely affected by changes in technique and describe the optimised conditions for obtaining reproducible results. PMID- 7532642 TI - Trauma and symbol. Instinct and reality perception in therapeutic work with victims of incest. AB - With the help of the conceptualizations of Amati and Bleger, and of Jung, the loss of instinctual experience and reality sense in the victims of incestuous abuse is described as an extreme regression to a primary undifferentiated stage of development: that of 'ambiguity' and 'identity'. At this stage the identity of the victim is taken over by that of the aggressor and becomes petrified in a form of mimicry. Differentiation processes are partially blocked. Through the therapeutic work with a woman who was sexually abused as a child, and whose daughter was also being incestuously traumatized, it is demonstrated how, through the use of fantasy, the instinctive reaction of thanatosis can be given up, together with the attempt to find meaning through self-sacrifice, which continually places the victim in renewed danger. By means of activating the transcendent function, the positive pole of the archetype can be set up as a counterforce in the internal world which was once feared like an abuser, and thus as a balance to the images of actual historical violation. The positive pole of the archetype then allows the inner world to loosen itself from the tangled aggressive and auto-aggressive layers of identity. This leads to a revival of protective instinctual reactions and to an improvement in reality functioning. It is emphasized that it is not the recall of actual abuse that promotes therapeutic progress but the evolving symbolizations. The analysis of the symbolic experience leads to the following conclusion: the hypothesis that, on the grounds of positive feelings, the children 'have also wanted' the incestuous activity, cannot be upheld. PMID- 7532638 TI - Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH diaphorase staining in rat and human pancreas: influence of fixation. AB - In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NA-DPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas. PMID- 7532641 TI - Peripheral blood reduction of memory (CD29+, CD45RO+, and "bright" CD2+ and LFA 1+) T lymphocytes in Papillon-Lefevre syndrome. AB - A Papillon-Lefevre patient with characteristic chronic periodontal disease and palmoplantar keratoderma was studied over a 4-year period. An abnormal T-cell phenotype was steadily observed in peripheral blood; both low numbers of CD29+ and CD45RO+ cells and a low density surface expression of CD2 and LFA-1 molecules were found. T-cell activation through CD3, CD2 and ConA, PWM and IL-2 receptors was normal; however, there was impairment in the activation via CD28. CD2, LFA-1 and CD45 molecules were normal in charge and molecular weight. There was no tissue sequestering of T lymphocytes in periodontal lesions, but rather a relative T-cell reduction. It is suggested that an important decrease of the so called "memory/hyperreactive" (CD45RO-positive) T cells does exist; therefore, hyperreactive T cells would not be available in sufficient numbers to leave the bloodstream through blood vessel endothelium, and the periodontium would be left without these important defenses and thus exposed to chronic infections. A disregulated factor affecting the transition from "naive" to "memory" T cells and the increase in certain surface molecules expression (i.e., CD2, LFA-1, CD29, and CD45RO) or the reversion from memory to naive T cells may be responsible for the disease pathogenesis. CD2 and LFA-1 molecule synthesis might be conjointly regulated on T lymphocytes. PMID- 7532643 TI - TAN-1323 C and D, new concanamycin-group antibiotics; detection of the angiostatic activity with a wide range of macrolide antibiotics. AB - We detected potent angiostatic activity in a MeOH extract from the mycelia of microbial strain S-45628 in the chick chorioallantoic membrane (CAM) assay. The producer was taxonomically characterized as Streptomyces purpurascens. Active principles designated TAN-1323 A-D were isolated and determined to be 18-membered macrolide antibiotics; components C and D are new members of this group, while components A and B are identical to concanamycins C and A, respectively. When tested in the CAM assay, components B and D gave huge avascular zones at the extremely low doses of 10-100 ng/disk, although components A and C showed far weaker activity due to their preferential tissue-damaging effect on the CAM. The discovery that these 18-membered macrolide antibiotics are angiostatic substances prompted us to examine other types of macrolide antibiotics, leading to the discovery that 16-membered macrolide antibiotics such as bafilomycin C1, tylosin and leucomycin also show angiostatic activity on the CAM. Thus, angiostatic potential is widely distributed among macrolide antibiotics. The mechanism of action of these macrolide antibiotics is also discussed. PMID- 7532645 TI - Susceptibility in cell culture of feline immunodeficiency virus to eighteen antiviral agents. AB - The in-vitro susceptibilities of two strains of feline immunodeficiency virus to 18 antiviral agents were determined in two cell lines. In terms of inhibiting p24 antigen production, the nucleoside-analogue reverse transcriptase inhibitors were the most effective compounds. Inhibition was also observed with aurintricarboxylic acid, phosphonoformate and butyldeoxynorjirimycin, but not with the other agents tested. PMID- 7532646 TI - "Aerosolized deferoxamine prevents lung and systemic injury caused by smoke inhalation". PMID- 7532644 TI - The tartrolons, new boron-containing antibiotics from a myxobacterium, Sorangium cellulosum. AB - New antibiotics were isolated from the culture broth of the myxobacterium, Sorangium cellulosum, strain So ce 678. The antibiotics were active against Gram positive bacteria and mammalian cells. They were named tartrolon A and B. Tartrolon B contains a boron atom. The boron binding region of tartrolon is identical with that of boromycin and aplasmomycin. PMID- 7532647 TI - Aerosolized deferoxamine prevents lung and systemic injury caused by smoke inhalation. AB - We assessed the role of oxidant release at the airway mucosal surface on airway injury and systemic response to a severe smoke insult. Adult sheep (n = 20) were insufflated with well-characterized smoke from burning cotton toweling. A standardized dose of 12 breaths of smoke with a tidal volume of 20 ml/kg was given under anesthesia. Sheep were awakened, monitored for 24 h, and killed; data were compared with control sheep. Sheep were given 1) humidified oxygen, 2) continuous aerosol of 10% deferoxamine (DFO)-pentastarch solution beginning after smoke, 3) DFO-alone aerosol, or 4) pentastarch-alone aerosol. DFO has antioxidant properties directly and chelates iron. Severe respiratory failure occurred in all but DFO-pentastarch group. Shunt fraction increased from a control of 4%. Histological assessment revealed severe airway mucosal edema, ulceration, bronchorrhea, and severe atelectasis but only moderate alveolar edema. Increased lipid peroxides were also noted in free airway fluid and in bronchoalveolar lavage fluid. In addition, oxygen consumption increased by 75%, fluid requirements increased threefold, and protein-rich systemic soft tissue lymph flow doubled, all significant increases compared with control sheep. No significant physiological or histological changes were noted in DFO-pentastarch aerosol group. We conclude that 1) oxidants possibly initiated through free iron release are involved in severe smoke-induced airway injury and resulting systemic inflammatory response, probably through an amplified oxidant injury and 2) an aerosol of a DFO-pentastarch complex prevents the injury process, whereas DFO alone is not effective as an aerosol. PMID- 7532648 TI - Glucocorticoids inhibit sulfur mustard-induced airway muscle hyperresponsiveness to substance P. AB - To explore the mechanisms of airway hyperreactivity to aerosolized substance P observed in guinea pigs 14 days after intratracheal injection of sulfur mustard (SM), we studied the effects of epithelium removal and inhibition of neutral endopeptidase (NEP) activity on airway muscle responsiveness. Tracheal rings from SM-intoxicated guinea pigs expressed a greater contractile response to substance P than rings from nonintoxicated guinea pigs. After epithelium removal or incubation with the NEP inhibitor phosphoramidon, the contractile responses of tracheal rings to substance P did not differ in guinea pigs injected with SM or ethanol (SM solvent). Treatment of the guinea pigs with betamethasone for 7 days before measurement abolished the airway muscle hyperresponsiveness observed in untreated SM-intoxicated guinea pigs and partially restored tracheal epithelium NEP activity. In addition, the tracheal epithelium height and cell density of SM intoxicated guinea pigs treated with betamethasone were significantly greater than in those without betamethasone. These results demonstrate that SM intoxication induces airway muscle hyperresponsiveness to substance P by reducing tracheal epithelial NEP activity and that glucocorticoids might inhibit this hyperresponsiveness by increasing this activity. PMID- 7532649 TI - Cardiopulmonary effects of granulocyte colony-stimulating factor in a canine model of bacterial sepsis. AB - We investigated the effects of recombinant granulocyte colony-stimulating factor (G-CSF) in a canine model of septic shock. Awake 2-yr-old beagles were studied before and after intraperitoneal placement of an Escherichia coli-infected clot. Nine days before and until 3 days after clot placement, animals received daily high-dose (G-CSF (5 microgram/kg body wt; n = 17), low-dose G-CSF (0.1 microgram/kg body wt; n = 17), or a control protein (5 micrograms/kg body wt; n = 20). Survival rate was greater (P < 0.04, Wilcoxon test) in the high-dose G-CSF group (14/17) than in the low-dose G-CSF (10/17) and control (12/20) groups. High dose G-CSF improved cardiovascular function, as evidenced by increased left ventricular ejection fraction (day 1 after clot; P < 0.001) and mean arterial pressure (day 2; P < 0.02) compared with low-dose G-CSF and control groups. High dose G-CSF increased (P < 0.001) mean peripheral neutrophils before (-3 days) and after (2 h to 4 days) clot and produced a more rapid (P < 0.001) rise (day 2) and fall (day 4) in mean alveolar neutrophil numbers compared with the low-dose G-CSF and control groups. High-dose G-CSF decreased mean serum endotoxin (2-8 h; P < 0.002) and tumor necrosis factor (2 h; P < 0.02) levels and lowered blood bacteria counts (2-6 h; P < 0.04) compared with the low-dose G-CSF and control groups. Thus, in this canine model, G-CSF sufficient to increase peripheral neutrophils before and during peritonitis and septic shock enhances host defense, reduces cytokine (tumor necrosis factor) levels, and improves cardiovascular function and survival. PMID- 7532650 TI - Separate detection of the two complementary RNA strands of hepatitis A virus. AB - The minus strand of hepatitis A virus can be detected specifically by reverse transcription and polymerase chain reaction amplification in infected cell culture extracts. Several controls gave evidence that the amplified fragment actually used the minus strand as initial template. Non-thermostable reverse transcriptase was not efficient for this purpose because of self-priming of the positive-stranded viral RNA during the reverse transcription step. This problem was overcome by the use of the thermostable rTth DNA polymerase that also has reverse transcriptase activity in the presence of Mn2+. PMID- 7532651 TI - Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli. AB - The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection. PMID- 7532653 TI - Increased tenascin expression in melanocytic tumors. AB - Tenascin is a large extracellular matrix glycoprotein which is widely distributed in normal, hyperplastic and neoplastic tissues. Its function is unknown but it has been associated with the epithelial-stromal interactions, such as cell adhesion and movement which take place, e.g. in morphogenesis, cellular proliferation and neoplasia. In this study, we investigated tenascin expression in 70 benign, dysplastic and malignant melanocytic tumors by using immunohistochemistry and monoclonal anti-tenascin 143DB7C8 antibody on paraffin sections. In all types of benign nevi, both intradermal, compound and junctional, there was a moderate expression of tenascin at the dermoepidermal junction and in the papillary dermis. In dysplastic nevi, the fibrotic areas in the papillary dermis also showed a moderate staining for tenascin. Invasive malignant melanomas showed the strongest expression of tenascin. In addition to the staining at the dermo-epidermal junction and in the papillary dermis, there was a variable expression of tenascin in the reticular dermis. Intracytoplasmic tenascin was detected both in primary melanomas and melanoma metastases. In conclusion, we have shown that tenascin expression is moderately increased in benign and dysplastic melanocytic tumors and greatly increased in malignant melanomas and melanoma metastases. The function of tenascin may be related to the cellular stromal interactions and it is possibly associated with the proliferation and spread of the melanocytic tumors. PMID- 7532654 TI - Cutaneous focal mucinosis--a histopathological and immunohistochemical analysis of 11 cases. AB - The histogenesis of cutaneous focal mucinosis (CFM) is controversial. Eleven cases of CFM (5F, 6M; mean age 51 years) from our routine files between 1986 and the present time have, therefore, been examined histopathologically and immunohistochemically. Histology revealed an increased number of fibroblast-like cells in early lesions, whereas they were diminished or predominantly at the margin in advanced ones. The myxomatous areas showed slight to absent reticulum formation. Similarly, elastic fibers were almost absent, and collagen fibers were fragmented and replaced by variable amounts of mucin. One specimen revealed an epithelial component within the lesion reminiscent of a poorly induced trichofolliculoma. Immunohistochemically, vimentin was consistently present and correlated with the number of fibroblast-like cells. A few (< 5%) CD34+ dermal dendritic cells (DDs) were focally seen within CFM. In contrast, FXIIIa+ DDs accounted for up to 30%. Fibroblast-like cells were negative for S-100 protein, Leu7, desmin and alpha-SMA. The epithelial component within one of our specimens seems to have been induced by CFM and is a feature also seen in (angio)-myxomas. CFM appears to be a mesenchymally derived lesion composed predominantly of fibroblasts. DDs do not form the major cell component but rather seem passively incorporated. PMID- 7532652 TI - Recurrent gestational trophoblastic disease following in-vitro fertilization. AB - Recurrence of gestational trophoblastic disease (GTD) following two attempts at in-vitro fertilization (IVF)/embryo transfer is reported in a childless couple after 17 years of unsuccessful trials of ovulation induction. The diagnosis of bilateral tubal obstruction was finally established, indicating IVF treatment. After the first IVF/embryo transfer treatment, the woman developed GTD and was treated with methotrexate. After a second IVF attempt, GTD was again diagnosed. This time there was no response to methotrexate, thus necessitating second-line chemotherapy. Etoposide, methotrexate, actinomycin D, cyclophosphamide, oncovine was used, and after only four treatment cycles the beta-human chorionic gonadotrophin (HCG) dropped to < 5 mIU/ml. After 26 months of follow-up, the beta HCG continues to be undetectable. Preimplantation evaluation and ovum donation are described as measures to minimize the risk for GTD recurrence in a future IVF/embryo transfer. PMID- 7532655 TI - Identification of rat pancreatic duct cells by their expression of cytokeratins 7, 19, and 20 in vivo and after isolation and culture. AB - Cells from the excretory ducts of the pancreas are thought to be capable of differentiating into exocrine and endocrine cells. To study this in rat models, markers must be found to identify the cells under different experimental conditions. We tested antibodies to different cytokeratins (CKs) by immunocytochemical staining on pancreatic tissue sections from normal rats, after partial pancreatectomy, and after isolation and culture of duct fragments. Monoclonal antibodies to human CK7, CK19, and CK20 were found to react specifically on rat pancreas tissue, as shown by Western blotting. CK20 and CK19 were immunocytochemically detected only in cells of the ductal system, from centroacinar cells to main ducts. CK7 was expressed by islets of Langerhans and by duct cells from main, inter-, and intralobular ducts, but not by centroacinar and terminal duct cells. CKs 7, 19, and 20 were also expressed in proliferating duct cells during tissue regeneration and after isolation and different periods of culture. We conclude that CKs 7, 19, and 20 are very useful markers to study the differentiation of rat duct cells under experimental conditions in vivo and in vitro. PMID- 7532656 TI - An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. AB - Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters. PMID- 7532658 TI - Engagement of the vitronectin receptor (alpha V beta 3) on murine T cells stimulates tyrosine phosphorylation of a 115-kDa protein. AB - The murine vitronectin receptor (VNR, alpha V beta 3) is expressed on T cell hybridomas expressing both the alpha beta and the gamma delta TCR as well as on TCR-alpha beta cells activated for prolonged periods of time by mitogens or alloantigens. The VNR functions as a costimulatory molecule for the activation of a subset of gamma delta T cells that express the V gamma 1.1 C gamma 4 V delta 6 TCR and that may recognize a ubiquitously expressed autoantigen. To characterize further some of the signal transduction parameters observed after engagement of the VNR in stimulated T lymphocytes, we have examined the effect of ligation of the VNR by RGDS-containing proteins on the pattern of protein phosphorylation. We demonstrate the appearance of a 115-kDa, tyrosine-phosphorylated protein (pp115) after engagement of the VNR with its ligand, RGDS. pp115 was shown to be immunologically distinct from focal adhesion kinase, did not possess inherent kinase activity, and may represent an as yet unidentified substrate in the integrin signal transduction pathway. Although induction of pp115 was independent of TCR expression, because it was seen in the TG40 hybridoma, which expresses neither the alpha beta nor the gamma delta TCR, pp115 could also be induced by cross-linking of the TCR in a murine TCR gamma delta hybridoma in the absence of any extracellular matrix proteins. This result raises the possibility that induction of pp115 is a common biochemical step in signal transduction by both the TCR and the VNR. PMID- 7532657 TI - In situ localization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections. AB - Assessments of RNA integrity and its hybridizability are essential for successful implementation of in situ hybridization (ISH) for mRNA or viral RNA, particularly when paraffin-embedded specimens from surgical, biopsy, and autopsy cases are used. In this study, we examined the suitability of ISH of 28S ribosomal RNA (rRNA) for this purpose. Oligo-DNA with nucleotide sequences complementary to a well-conserved segment of 28S rRNA with auxiliary adenine-thymine-thymine (ATT) repeats at the 3' and 5' ends was synthesized. The oligo-DNA was made antigenic by converting the adjacent thymines to T-T dimers by UV irradiation and was used as a probe for ISH of 28S rRNA. The T-T dimers were detected by enzyme immunohistochemistry. When the results of ISH rRNA staining and that of total RNA staining by methyl green/pyronin Y were compared for various types of sections prepared from rat and human tissues, the staining intensities of total RNA did not always match those of ISH rRNA staining. In paraffin sections of formalin fixed tissues, the degree of proteinase digestion influenced the ISH rRNA staining intensity, whereas it had no effect on the total RNA staining intensity. The intensities of ISH rRNA staining agreed well with those of various types of mRNA staining by ISH in 10 cases of paraffin-embedded pathological specimens. We therefore believe that ISH rRNA staining is a convenient parameter for evaluation of levels of hybridizable RNAs in tissue sections. PMID- 7532659 TI - Bcl-2 blocks degranulation but not fas-based cell-mediated cytotoxicity. AB - The ability of bcl-2 in target cells to block cell-mediated cytotoxicity by allospecific CTL was tested. The blocking effect was variable. Because killing by CTL involves two different pathways, degranulation (perforin plus granzymes) and fas, we examined the effect of bcl-2 on these pathways independently. Bcl-2 in target cells blocked apoptotic cell death induced either by cytotoxic granule extracts or by CTL killing under conditions in which the fas pathway is blocked. On the other hand, bcl-2 had no effect on target cell-killing either by Fas specific mAb or by CTL capable of killing only via the fas pathway. These data suggest bcl-2 may block apoptotic lysis induced by perforin plus granzymes, but not apoptotic lysis induced via the fas pathway. Thus, any analysis of the effect of bcl-2 on apoptotic cell death in target cells killed by CTL must take into account the relative contributions of the degranulation vs fas pathways. PMID- 7532661 TI - Expression of a novel integrin beta 1 chain epitope and anti-beta 1 antibody mediated enhancement of fibronectin binding are dependent on the stage of T cell differentiation. AB - Beta 1 integrins are a family of alpha beta heterodimers that serve as cell surface receptors for extracellular matrix proteins. We demonstrate that the anti mouse integrin beta 1 chain mAb KMI6 selectively recognizes a beta 1 epitope that is constitutively expressed by certain immature thymocytes and is induced only slightly on mature thymocytes and peripheral T cells by activation with Con A. Because virtually all cells examined expressed beta 1 integrins on their surface, expression of the KMI6 epitope is T cell differentiation stage specific. Most CD3 4-8- thymocytes were KMI6+, with the lowest level of staining observed on the earliest CD44+IL-2R- cells within this subset. Expression was down-regulated during the CD3-4-8- to CD3-4-8+ transition, and lost by the CD4+8+ stage. Mature single positive thymocytes and resting peripheral T cells were also KMI6-. In contrast with the loss of the epitope before TCR expression by other thymocytes, most CD3+4-8- and certain CD8+ gamma delta TCR+ thymocytes were KMI6+ Addition of KMI6 to cell adhesion assays enhanced CD4-8- thymocyte, but not activated mature thymocyte or peripheral T cell, binding to fibronectin (via alpha 4 beta 1 and alpha 5 beta 1), whereas laminin binding (via alpha 6 beta 1) was unaffected. These properties distinguish the KMI6 epitope from other epitopes involved in beta 1 integrin activation in mice and other species. The unique selectivity of KMI6 recognition of beta 1 integrins, and its selective enhancement of ligand binding suggest that beta 1 integrin structure and factors that regulate beta 1 integrin binding are correlated with the stage of T cell differentiation. PMID- 7532662 TI - Isolation, primary structure, and evolution of the third component of chicken complement and evidence for a new member of the alpha 2-macroglobulin family. AB - Although the third component of complement, C3, has been isolated and its primary structure determined from most living classes of vertebrate, limited information is available on its structure and function for aves, which represent a significant stage in complement evolution. In this study, we present the complete cDNA sequence of chicken C3, the cDNA sequences of the thioester region for two chicken alpha 2-macroglobulin (alpha 2M)-related proteins, a simplified method for purifying chicken C3, and an analysis of the C3 convertase and factor I mediated cleavages in chicken C3. Using the reverse-transcriptase PCR, with degenerate oligonucleotide primers derived from two conserved C3 sequences (GCGEQN/TM, TWLTAY/FV) and liver mRNA as template, we isolated three distinct 220 bp PCR products, one with a high degree of sequence similarity to C3 and two to alpha 2M and pregnancy zone protein from other species. The complete cDNA sequence of chicken C3 was obtained by screening a chicken liver lambda gt10 library with the C3 PCR product and probes from the 5' end of the partial-length C3 clones. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides of purified chicken C3. Chicken pro-C3 consists of an 18-residue putative signal peptide, a 640-residue beta-chain (70 kDa), a 989-residue alpha-chain (111 kDa), and an RKRR linker region. It contains an internal thioester and three potential N-glycosylation sites, all in the alpha chain. The convertase cleavage site, predicted to be Arg-Ser, was confirmed by sequencing the zymosan-bound C3 fragments generated upon complement activation. NH2-terminal sequencing of the purified C3 chains showed that 1) pro-C3 is indeed cleaved at the RKRR linker sequence to generate the mature two-chain molecule, and 2) the beta-chain of chicken C3 is blocked. The deduced amino acid sequence shows 54, 54, 54, 53, 52, 57, and 55% amino acid identities to human, mouse, rat, guinea pig, rabbit, cobra, and Xenopus C3, respectively, and an identity of 44, 31, and 33% to trout, hagfish, and lamprey C3, respectively. The identities to human C4, C5, and alpha 2M are 31, 29 and 23%, respectively. A phylogenetic tree for C3, C4, C5, and alpha 2M-related proteins was constructed based on the sequence data and is discussed. PMID- 7532660 TI - Antigen-independent, integrin-mediated T cell activation. AB - The "outside-in" signals produced by the interaction of integrin molecules with the extracellular matrix (ECM) trigger a multitude of cellular events. The vitronectin receptor (VNR), an alpha v beta 3 heterodimer, functions as a costimulatory molecule for the activation of a subset of V gamma 1.1/C gamma 4 bearing gamma/delta T cells, which have been postulated to recognize a ubiquitous self-antigen. We addressed the question of whether stimulation of these T cells requires both engagement of the VNR by ECM proteins and engagement of the TCR by its Ag. We introduced into a TCR- but VNR+ mutant T cell hybridoma, TG40 (derived from 2B4), a chimeric molecule that contains the cytoplasmic tail of the TCR zeta chain fused to the cytoplasmic and transmembrane region of either human CD8 or human CD25. The transfectants expressing the chimeric molecules secreted IL-2 constitutively when the VNR was engaged with a ligand, e.g., provided by ECM proteins present in FCS. This constitutive cytokine secretion could be blocked with mAb directed against the VNR, with or the peptide RGD, or by growth in serum free medium. VNR-mediated cell activation also induced the phosphorylation of the zeta-chain. Signaling through the zeta-chain was required, as cells transfected with a chimera containing only a 22 amino-acid long, truncated zeta-chain did not secrete IL-2 constitutively. Thus, we demonstrated that the binding of the VNR to ECM protein in the presence of the zeta-chain is sufficient to induce cytokine secretion by T cells and does not require the recognition of an Ag by the TCR. Such integrin-mediated, Ag-independent activation of T cells may play a critical role in the potentiation of inflammatory responses. PMID- 7532664 TI - Activation of human neutrophils through L-selectin and Mac-1 molecules. AB - The effect of selective cell activation through L-selectin and Mac-1 molecules cross-linking on neutrophil superoxide anion (O2-) generation and changes in intracellular calcium [Ca2+]i was investigated. Cross-linking of L-selectin using different mAbs induced a rapid and transient increase in [Ca2+]i and O2- generation by neutrophils that were dependent on the extent of L-selectin cross linking and mAb epitope binding. In addition, cross-linking of L-selectin induced an up-regulation of surface Mac-1 expression on neutrophils. Cross-linking of Mac 1 molecules with mAb also induced significant changes in [Ca2+]i levels and O2- generation by neutrophils, which was also dependent on the epitope binding by mAb. Pretreatment of neutrophils with LPS caused a marked decrease in the expression of L-selectin molecules and significant inhibition (i.e., 59 +/- 4%) of anti-L-selectin mAb-dependent O2- generation and [Ca2+]i signal. In contrast, LPS pretreatment caused a significant up-regulation of Mac-1 molecules on neutrophil and enhanced O2- generation by neutrophils. The data indicate that cross-linking of L-selectin and Mac-1 initiates changes in [Ca2+]i and O2- production in neutrophils and suggest that these distinct adhesion molecules independently may play important regulatory roles in modulating neutrophil endothelial cell interactions, transmigration, and neutrophil function at sites of tissue injury. PMID- 7532663 TI - Resistance of melanoma cell lines to interferons correlates with reduction of IFN induced tyrosine phosphorylation. Induction of the anti-viral state by IFN is prevented by tyrosine kinase inhibitors. AB - Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN. PMID- 7532665 TI - Pyrrolidine dithiocarbamate inhibits intercellular adhesion molecule-1 biosynthesis induced by cytokines in human fibroblasts. AB - Intercellular adhesion molecule-1 (ICAM-1), the ligand of lymphocyte function associated antigen-1, plays an important role in the interactions of a variety of hemopoietic and nonhemopoietic cells, including leukocytes, fibroblasts, and endothelial cells. ICAM-1 is known to be involved in the onset of several diseases such as inflammation, allograft rejection, and so on. In this report, we investigated the effects of dexamethasone, cyclosporin A, FK506, and pyrrolidine dithiocarbamate (PDTC) on the induction of the ICAM-1 gene by cytokines in fibroblasts. PDTC, a potent inhibitor of NF-kappa B, was shown by ELISA and FACS analysis to prevent dramatically the expression of the ICAM-1 gene stimulated by IL-1 alpha, IFN-gamma, and PMA, although the other reagents inhibited it only slightly. Ribonuclease protection assay revealed that PDTC blocked the expression of the ICAM-1 gene at the mRNA level. To elucidate the mechanism of this inhibition, we constructed a series of ICAM-1 promoter deletion mutants linked to the chloramphenicol acetyl transferase gene and analyzed the effect of PDTC on their activities. Transient transfection analysis indicated that the critical region for inhibition by PDTC is an NF-kappa B binding site-like motif (GGGAGGATTCC, ICAM-1 kappa B) that is located at position-540. Electrophoresis mobility shift assay revealed that PDTC actually inhibits the binding of NF-kappa B (or NF-kappa B-like) protein to the ICAM-1 kappa B site. These findings suggest that PDTC inhibits ICAM-1 gene expression by inhibiting the association of NF kappa B (or NF-kappa B-like) protein with the ICAM-1 kappa B site. PMID- 7532667 TI - Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF-alpha-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. AB - Asthma is a disease of airway inflammation and hyper-reactivity associated with lymphocytic infiltration in the bronchial submucosa. We recently demonstrated that human airway smooth muscle (ASM) cells express the cell adhesion molecules ICAM-1 and VCAM-1, which are up-regulated by cytokines such as TNF-alpha, and which mediate binding of activated T lymphocytes. In this study, we examined whether an increase in [cAMP]i, presumably via activation of cAMP-dependent protein kinase, modulates TNF-alpha-induced ICAM-1 and VCAM-1 on ASM. We found that treatment of ASM with either forskolin, which directly activates adenylyl cyclase, or with cholera toxin, which activates the heterotrimeric GTP-binding protein, Gs, inhibited TNF-alpha-induced cell adhesion molecule expression. In addition, treatment with either isoproterenol or prostaglandin E2, which activates receptors coupled to Gs and increases [cAMP]i, also inhibited TNF-alpha induced expression of ICAM-1 and VCAM-1 on ASM. Furthermore, adhesion of activated T cells to TNF-alpha-stimulated ASM was inhibited by treating the ASM cells with either forskolin or PGE2. These data suggest that cAMP-dependent protein kinase activation decreases cytokine-induced expression of cell adhesion molecules on ASM cells, modulates T cell binding to airway myocytes and, thus, suggests novel therapeutic approaches to airway inflammation. PMID- 7532666 TI - Macrophage inflammatory protein-1 alpha enhances growth factor-stimulated phosphatidylcholine metabolism and increases cAMP levels in the human growth factor-dependent cell line M07e, events associated with growth suppression. AB - The immunoregulatory C-C chemokine, macrophage inflammatory protein-1 alpha (MIP 1 alpha) has suppressive activity on proliferation of stem cells and early subsets of myeloid progenitor cells. A receptor for C-C chemokines that binds MIP 1 alpha has been characterized, cloned, and shown to be related structurally to neuropeptide receptors that couple through G-proteins to phospholipase-C and adenyl cyclase. Yet, very little information on the intracellular mechanisms of action of MIP-1 alpha is available. We show here that the human factor-dependent cell line M07e is responsive to the cell cycle-suppressive effects of MIP-1 alpha, has specific membrane-binding sites for MIP-1 alpha, and that treatment of these cells with this chemokine increases the phosphatidylcholine (PC) and phosphocholine turnover rates in cells that are synergistically stimulated by the combination of granulocyte-macrophage colony-stimulating factor and steel factor but not these factors acting singly. Additional, MIP-1 alpha treatment induces a dose- and time-dependent increase in intracellular cAMP levels in M07e cells. Both exogenous PC and dibutyryl cAMP were found to suppress the proliferation of M07e colony-forming cells to a level similar to that of MIP-1 alpha, further implicating cAMP and PC metabolism in MIP-1 alpha-induced M07e suppression. RANTES, a related chemokine, with weak or incomplete binding to the cloned MIP-1 alpha receptor, did not suppress M07e colony-forming cells, nor did it increase intracellular cAMP levels, but it did enhance growth factor-induced PC turnover, further supporting the involvement of cAMP in MIP-1 alpha suppression while demonstrating that increased PC turnover alone is not sufficient for suppression. These findings support the idea that the human MIP-1 alpha receptor is coupled to phospholipid and cAMP metabolism in a manner similar to other 7-transmembrane, G protein-linked receptors and suggest that a phosphatidylcholine hydrolytic cycle and an associated increase in cAMP are part of the mechanisms of action of MIP-1 alpha. PMID- 7532668 TI - Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. AB - Apoptosis leads to swift recognition, ingestion, and degradation of intact senescent neutrophils by macrophages. This protects tissues from leakage of noxious contents from dying cells and may promote resolution of inflammation. However, little has been known of the mechanisms that regulate macrophage capacity for apoptotic cells during an inflammatory response. We examined whether proinflammatory cytokines modulated phagocytosis of senescent neutrophils undergoing apoptosis by human monocyte-derived macrophages at 4 days maturity (4d M phi), an in vitro model of neutrophil "disposal" by apoptosis. Pretreatment of 4d M phi with granulocyte-macrophage-CSF increased the proportion of 4d M phi taking up apoptotic PMN in a concentration-dependent fashion by up to approximately 240%. This was by a rapid effect detectable by 4 h and exerted on the M phi, not the PMN. Granulocyte-macrophage-CSF also increased the number of apoptotic PMN taken up by each M phi. IFN-gamma, IL-1 beta, TNF-alpha, and TGF beta 1 also enhanced phagocytosis, but IL-4 and IL-6 had no effect. In each case, the cytokine-expanded phagocytic subpopulation employed the thrombospondin (TSP) dependent recognition mechanism defined for mature M phi, in which M phi vitronectin receptor and CD36 cooperate. However, commensurate increases in M phi expression of VnR, TSP, or CD36 were not detectable, indicating that TSP-mediated recognition can be recruited by other mechanisms. Cytokines did not recruit phosphatidylserine-dependent recognition, the other major mechanism by which some macrophage populations ingest apoptotic cells. Thus, M phi phagocytosis of apoptotic neutrophils can be potentiated by proinflammatory cytokines, suggesting a mechanism for negative feedback control of neutrophil number at inflamed sites. PMID- 7532669 TI - Bone marrow-derived murine mast cells migrate, but do not degranulate, in response to chemokines. AB - We have determined that several chemokines induce mast cell migration in vitro. This directed migration is dependent on the presence of particular extracellular matrix proteins and the activation status of the cells. Mast cell haptotactic responses were observed in response to various chemokines on vitronectin-, laminin-, and fibronectin-coated filters. Unstimulated mast cells were chemoattracted only by monocyte chemotactic protein-1 and RANTES on vitronectin coated and, to a lesser extent, laminin-coated filters, whereas IgE-activated mast cells migrated in response to monocyte chemotactic protein-1, regulated on activation normal T expressed and secreted, platelet factor-4, and macrophage inflammatory protein-1 alpha on all three matrix proteins. No significant migration was observed on collagen type IV-coated or uncoated filters. Mast cell migration in response to chemokines on extracellular matrices and its enhancement by IgE-dependent activation provide a mechanism by which cells may be drawn to sites of inflammation. Chemokine-induced mast cell recruitment may be particularly relevant in host defense responses to parasitic infections, allergic reactions, Jones-Mote reactions, and in wound healing. PMID- 7532670 TI - A MHC class I B locus allele-restricted simian immunodeficiency virus envelope CTL epitope in rhesus monkeys. AB - In light of the importance of virus-specific CTL in the control of the spread of the AIDS virus, it will be important to assess the generation of these effector cell responses in trials of novel vaccine strategies for the prevention of AIDS virus infections. To facilitate such studies in the simian immunodeficiency virus (SIV)/macaque model for AIDS, we have defined a rhesus monkey SIVmac CTL epitope carboxy terminus to both the CD4-binding and V4 regions of the envelope glycoprotein. We also used one-dimensional isoelectric focusing to characterize the MHC class I molecule of the rhesus monkey that binds this 9-amino-acid SIVmac envelope fragment. Cloning and sequencing of the cDNA encoding this rhesus monkey MHC class I molecule demonstrated that it is a newly described HLA-B homologue, Mamu-B*01. The definition of this viral CTL epitope and its restricting MHC class I molecule will facilitate the use of the SIVmac/rhesus monkey model for studies of envelope-based vaccine strategies for the prevention of AIDS. PMID- 7532671 TI - The international standard for granulocyte colony stimulating factor (G-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study. AB - Three ampouled preparations of granulocyte colony stimulating factor (G-CSF) have been evaluated by 29 laboratories in 11 countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/502) was established as the international standard for G-CSF. PMID- 7532672 TI - [A coherent radiotherapeutic-surgical approach, based on PSA, for the management of cancer of the prostate]. AB - The presence of cancer of the prostate does not mean that a patient will succumb to the disease. In fact, prior to the advent of prostate-specific antigen (PSA) testing, the cause of death in 50% of men with detectable extraglandular cancer was intercurrent disease. No studies have conclusively demonstrated a definitive benefit of surgery or radiation over simple observation; therefore treatment complications must absolutely be restricted to a bare minimum while demonstrating curative effect. This review presents a balanced perspective on the management of prostate cancer which results from non-adversarial, extensive, and continuous interactions between two very active Departments (Urology and Radiation Oncology). Such a multidisciplinary approach fosters relevant research projects and ultimately benefits patient care. PMID- 7532673 TI - Intraoperative radiation therapy for pancreatic carcinoma. The choice of treatment modality. AB - Ninety patients with carcinoma of the pancreas treated between 1976 and 1990 were reviewed retrospectively. Intraoperative radiation therapy (IORT) in combination with external beam radiation therapy (EBRT) for localized but unresectable tumors (n = 29) prolonged survival significantly more than IORT alone (n = 16) (p < 0.01); it seems EBRT enhanced or contributed to the better results obtained with IORT plus EBRT. Moreover, IORT, alone or in combination, relieved pain. Adjuvant IORT for residual tumors (n = 20) might not effectively prolong survival, because the difference in survival rate between noncurative resection plus IORT and nonresection plus IORT in combination with EBRT was not significant. Curative tumor resection of stage III disease in combination with IORT (n = 9) resulted in significantly longer survival as compared with curative tumor resection alone (n = 8) (p < 0.05). It may be advisable to administer IORT in combination with EBRT to patients with advanced pancreatic carcinoma, avoiding aggressive tumor resection, when curative tumor resection cannot be performed. PMID- 7532674 TI - Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes. AB - Autoantibodies specific against fibrillarin, a 34-kD nucleolar protein associated with U3-snRNP, are present in patients with systemic sclerosis (SSc). To understand the mechanisms involved in the induction of these autoantibodies, we prepared a series of human fibrillarin recombinant proteins covering the entire molecule and analyzed their interaction with the autoantibodies present in various connective tissue diseases. Our results showed that antifibrillarin autoantibodies are present not only in SSc, as previously reported, but also in a variety of other connective tissue diseases. Patients with SSc (58%), mixed connective tissue diseases (60%), CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, and telangiectasia syndrome) (58%), systemic lupus erythematosus (39%), rheumatoid arthritis (60%), and Sjogern's syndrome (84%) showed presence of antifibrillarin autoantibodies. Results obtained from competitive inhibition radioimmunoassay and Western blot analyses with purified recombinant fusion proteins revealed that these autoantibodies react primarily with epitope(s) present in the NH2- (AA 1-80) and COOH-terminal (AA 276-321) domains of fibrillarin. Autoantibodies reacting with internal regions of fibrillarin are less frequent. Analysis of the hydrophilicity profiles of reactive peptides showed presence of three potential antigenic sites in the NH2- and two in the COOH-terminal regions. While a hexapeptide sequence NH2 terminus of fibrillarin is shared with an Epstein-Barr virus-encoded nuclear antigen, the COOH-terminal region shares sequence homology with P40, the capsid protein encoded by herpes virus type 1. Interestingly, these two regions of fibrillarin also contain the most immunodominant sequences, as predicted by surface probability and the Jameson and Wolf antigenic index. These observations suggest that molecular mimicry might play an important role in the induction of antifibrillarin autoantibodies. PMID- 7532675 TI - The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. AB - Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis. PMID- 7532676 TI - FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells. AB - Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester and ionomycin, the dual signal that is necessary for proliferation of untransformed lymphocytes. Furthermore, c-rel induction is blocked by the immunosuppressive drug FK506 that is known to inhibit B and T cell activation. c-rel-dependent transactivation of the interleukin-2 receptor alpha chain (IL-2R alpha) promoter is augmented by coexpression of calcineurin, suggesting the involvement of a calcineurin-dependent intracellular pathway. Our results identify c-rel as a target of immunosuppressive agents and illustrate the similarity of activation pathways in both B and T cells. PMID- 7532677 TI - The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. AB - Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones. PMID- 7532678 TI - Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. AB - Insulin-dependent diabetes mellitus (IDDM) is thought to be an immunologically mediated disease resulting in the complete destruction of the insulin-producing islets of Langerhans. It has become increasingly clear that autoreactive T cells play a major role in the development and progression of this disease. In this study, we examined the role of the CD28/B7 costimulation pathway in the development and progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse model. Female NOD mice treated at the onset of insulitis (2-4 wk of age) with CTLA4Ig immunoglobulin (Ig) (a soluble CD28 antagonist) or a monoclonal antibody (mAb) specific for B7-2 (a CD28 ligand) did not develop diabetes. However, neither of these treatments altered the disease process when administered late, at > 10 wk of age. Histological examination of islets from the various treatment groups showed that while CTLA4Ig and anti-B7-2 mAb treatment blocked the development of diabetes, these reagents had little effect on the development or severity of insulitis. Together these results suggest that blockade of costimulatory signals by CTLA4Ig or anti-B7-2 acts early in disease development, after insulitis but before the onset of frank diabetes. NOD mice were also treated with mAbs to another CD28 ligand, B7-1. In contrast to the previous results, the anti-B7-1 treatment significantly accelerated the development of disease in female mice and, most interestingly, induced diabetes in normally resistant male mice. A combination of anti-B7-1 and anti-B7-2 mAbs also resulted in an accelerated onset of diabetes, similar to that observed with anti-B7-1 mAb treatment alone, suggesting that anti-B7-1 mAb's effect was dominant. Furthermore, treatment with anti-B7-1 mAbs resulted in a more rapid and severe infiltrate. Finally, T cells isolated from the pancreas of these anti-B7-1 treated animals exhibited a more activated phenotype than T cells isolated from any of the other treatment groups. These studies demonstrate that costimulatory signals play an important role in the autoimmune process, and that different members of the B7 family have distinct regulatory functions during the development of autoimmune diabetes. PMID- 7532680 TI - P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow. AB - This report examines the adhesive interactions of human CD4+ T lymphocytes with tumor necrosis factor alpha-activated human endothelial cell monolayers in an in vitro model that mimics microcirculatory flow conditions. Resting CD4+ T cell interactions with activated endothelium consisted of initial attachment followed by rolling, stable arrest, and then spreading and transendothelial migration. P selectin, but not E-, or L-selectin, mediated most of this initial contact and rolling, whereas beta 1-integrins (alpha 4 beta 1), interacting with endothelial expressed vascular cell adhesion molecule 1, participated in rolling and mediated stable arrest. In contrast, beta 2-integrins were primarily involved in spreading and transmigration. These findings highlight an important role for P-selectin and suggest discrete functions for beta 1- and beta 2-integrins during lymphocyte recruitment to sites of immune-mediated inflammation. PMID- 7532679 TI - Breakdown of B cell tolerance in a mouse model of systemic lupus erythematosus. AB - Anti-DNA antibodies, specifically those that stain nuclei in a homogenous nuclear (HN) fashion, are diagnostic of systemic lupus erythematosus (SLE) and the MRL lpr/lpr SLE murine model. We have used a heavy chain transgene that increases the frequency of anti-HN antibodies to address whether their production in SLE is the consequence of a defect in B cell tolerance. Anti-HN B cells were undetectable in nonautoimmune-prone transgenic mice, but in MRL-lpr/lpr transgenic mice their Ig was evident in the sera and they were readily retrievable as hybridomas. We conclude that nonautoimmune animals actively delete anti-HN-specific B cells, and that MRL-lpr/lpr mice are defective in this process possibly because of the lpr defect in the fas gene. PMID- 7532681 TI - Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms. AB - In human and experimental models of arthritis, blood monocytes migrate into the inflamed synovium and joint space. The mechanisms required for monocyte migration across the vascular endothelium in joints is poorly defined. Radiolabeled rat blood monocytes were used to measure monocyte migration to the inflamed joints of rats with adjuvant arthritis, and the role of monocyte adhesion molecules was analyzed. Monocyte accumulation in the inflamed joints was maximal 14-21 d after immunization with adjuvant, when arthritis had fully developed. Blocking mAbs to lymphocyte function-associated antigen 1 (LFA-1), Mac-1, and very late activation antigen 4 (VLA-4) were used to evaluate the role of these integrins in the migration. Migration to the joints was not inhibited by treatment of the animals with mAb to LFA-1, Mac-1, or VLA-4 alone, and was partially (50%) inhibited in only the most arthritic joint, the talar joint, by the combination of mAb to LFA 1 plus Mac-1. In contrast, this combination inhibited migration to dermal inflammation induced by C5ades Arg, endotoxin, tumor necrosis factor alpha, and polyinosine-cytosine by 60-70%. When mAbs to LFA-1 and VLA-4 were combined, migration to all the inflamed joints was strongly inhibited (80-98%, depending on the joint). Treatment with the combination of the three mAbs to LFA-1, Mac-1, and VLA-4 completely eliminated monocyte migration to all joints and dermal inflammation. The results show that 51Cr blood monocytes can be used to quantify monocyte migration to arthritic joints in the rat. LFA-1 alone or VLA-4 alone is sufficient to mediate most of this migration, and either LFA-1 or VLA-4 is required for monocyte migration to joint inflammation. These results indicate that both the VLA-4 and LFA-1 integrins should be therapeutic targets for suppression of monocyte infiltration of joints in arthritis. PMID- 7532682 TI - Fas-mediated cytotoxicity by freshly isolated natural killer cells. AB - The expression of Fas ligand on natural killer (NK) cells and Fas-mediated cytotoxicity by NK cells was investigated. Fas ligand mRNA was expressed in freshly isolated NK cells but not in T cells. Furthermore, the Fas ligand was detected on the cell surface of NK cells by staining with soluble Fas molecule. We analyzed the cytolytic activity of NK cells against thymocyte targets from normal and lpr mice, and found that the NK cells killed thymocytes from normal mice but not from lpr mice. On the other hand, splenic T cells did not show any cytotoxicity against either of the thymocyte targets. Similarly, NK cells exhibited cytotoxicity against transfectants expressing Fas antigen but not against parental cells or transfectants expressing a mutant Fas antigen with deleted cytoplasmic region. These results demonstrated that NK cells express Fas ligand and possess the capability of killing target cells expressing Fas antigen on their surface. This finding suggests that NK cells play an important role by eliminating Fas-expressing cells either constitutively or inducibly in peripheral lymphoid organs. PMID- 7532683 TI - Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide. AB - A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS. PMID- 7532684 TI - The T cell response of HLA-DR transgenic mice to human myelin basic protein and other antigens in the presence and absence of human CD4. AB - Analysis of HLA class II transgenic mice has progressed in recent years from analysis of single chain HLA class II transgenes with expression of mixed mouse/human heterodimers to double transgenic mice expressing normal human heterodimers. Previous studies have used either HLA transgenic mice in which there is a species-matched interaction with CD4 or mice which lack this interaction. Since both systems are reported to generate HLA-restricted responses, the matter of the requirement for species-matched CD4 remains unclear. We have generated triple transgenic mice expressing three human transgenes, DRA, DRB, and CD4, and compared HLA-restricted responses to peptide between human-CD4+ (Hu-CD4+) and Hu-CD4- littermates. We saw no difference between Hu-CD4+ and Hu CD4- groups, supporting the notion that for some responses at least the requirement for species-matched CD4 may not be absolute. Evidence for positive selection of mouse T cell receptors in HLA-DR transgenic mice came both from the acquisition of new, HLA-restricted responses to various peptides and from an increased frequency of T cells using the TCR V beta 4 gene segment. An important goal with respect to the analysis of function in HLA transgenic mice is the clarification of mechanisms which underpin the recognition of self-antigens in human autoimmune disease. As a first step towards 'humanized' disease models in HLA transgenic mice, we analyzed the responses of HLA-DR transgenic mice to the human MPB 139-154 peptide which has been implicated as an epitope recognized by T cells of multiple sclerosis patients. We obtained T cell responses to this epitope in transgenic mice but not in nontransgenic controls. This study suggests that HLA transgenic mice will be valuable in the analysis of HLA-restricted T cell epitopes implicated in human disease and possibly in the design of new disease models. PMID- 7532687 TI - Dealing with the special patient. AB - It is important to prevent the spread of disease in the dental office, both from the immunocompromised patient to the healthcare providers and/or other patients, and vice versa. This paper reviews some of the standard infection control practices, also called universal precautions, that ensure the safety of all patients, immunocompromised or not. PMID- 7532688 TI - Nutrition update for the dental health professional. AB - The relationship between nutrition and dentistry has been longstanding. Today, dental healthcare providers need to broaden the definition of "dental nutrition" from a narrow focus on the relationship of foods to caries, periodontal disease and oral lesions, to concerns about the overall nutritional adequacy of a patient's diet. This article discusses some of the latest nutrition recommendations put forth by major United States' health organizations and comments on their applicability to dental patients. PMID- 7532690 TI - Winning relationships through customer service: initial contact. AB - First impressions last, and can have an impact on all future contact with a new patient. By using each initial contact with a new patient to begin building a strong relationship, a practice can be positioned for success. This article explores relationship building techniques. PMID- 7532689 TI - Transform practice information into team action. AB - To keep a dental office moving in the direction desired by the dentist/owner requires the active participation of all members of the dental team. This article discusses methods of measuring effectiveness in achieving practice goals. PMID- 7532686 TI - Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. AB - K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells. PMID- 7532691 TI - Light-cured glass ionomers. AB - Light-cured glass ionomers are a promising new class of restorative material, with beneficial properties such as adhesion to tooth structure and fluoride release. Their wide range of clinical applications includes use as bases and liners and in Class V cavity restoration. However, disadvantages such as plastic deformation and poor dimensional stability may limit their usefulness. PMID- 7532685 TI - T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection. AB - The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes. PMID- 7532693 TI - A new information base: integrated and networked computers. PMID- 7532694 TI - Improving aesthetic dentistry through high technology. PMID- 7532692 TI - Cephalosporin vs penicillin. AB - The correlation between late infection of an orthopedic prosthesis and a dental bacteremia remains controversial. Transient bacteremia does occur after dental treatment, usually involving streptococcal bacteria, but many orthopedic surgeons choose an anti-staphylococcal agent as their prophylactic antibiotic of choice. This article reports the results of a recent survey designed to determine, from orthopedic surgeons, antibiotic premedication coverage and, specifically, the rationale for a chosen regimen for patients having dental surgery. The responses showed no common agreement for the choice of a specific antibiotic and little rationale regarding the need for prophylaxis. PMID- 7532696 TI - Future focus: creative computer communication. PMID- 7532695 TI - Treatment room computers: enhancing scheduling, examination and recordkeeping. PMID- 7532697 TI - Advances in air abrasive technology. PMID- 7532698 TI - Dentistry in two thousand. 1895. PMID- 7532699 TI - Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF 1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis. AB - Cerebellar granule cells deprived of depolarizing concentration of extracellular potassium, [K+]o, undergo apoptosis. We here report that this apoptotic process is associated with an immediate and permanent decrease in the levels of free intracellular calcium, [Ca2+]i. Although forskolin and IGF-1 are both able to prevent apoptosis, only forskolin is able to counteract the instantaneous decrease of [Ca2+]i. However, the early effect of forskolin on [Ca2+]i is lost after longer incubation in low [K+]o. The calcium antagonist nifedipine is able to inhibit the survival effect of high [K+]o, while not affecting forskolin and IGF-1 promoted survival, as assessed by viability and genomic DNA analysis. Accordingly, the L-type calcium channels agonist Bay K8644 significantly enhanced the survival of low KCl treated neurons. To temporally characterize the signal transduction events and the essential transcriptional step in cerebellar granule cells apoptosis, we determined the time course of the rescue capacity of high [K+]o, forskolin, IGF-1, and actinomycin D. Addition of high KCl, forskolin, or IGF-1 6 hr after the initial KCl deprivation saves 50% of cells. Remarkably, 50% of neurons loss the potential to be rescued by actinomycin D after only 1 hr in low [K+]o. Finally, we show that the survival promoting activities of high [K+]o, forskolin, and IGF-1 do not require RNA synthesis. We conclude that [Ca2+]i is involved in the survival promoting activity exerted by high [K+]o but not in those of forskolin and IGF-1, and that all three agents, although rescuing neurons from apoptosis through distinct mechanisms of action, do not necessitate RNA transcription. PMID- 7532700 TI - delta-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. AB - We have recently developed antisera which recognize epitopes of the cloned delta opioid receptor (DOR; Dado et al., 1993). In the present report we have further characterized these antisera, and raised additional antisera in rats. We used these antisera to determine the distribution of DOR-like immunoreactivity (-Ll) in rat spinal cord and brainstem in relation to serotoninergic, noradrenergic, and enkephalinergic neurons. We found DOR-Ll in fibers and varicosities distributed throughout the spinal cord gray matter, with highest densities in the superficial dorsal horn, in autonomic regions, around the central canal as well as in the ventral horn motor nuclei. In the brainstem a dense innervation of DOR immunoreactive (-IR) fibers was found in several nuclei such as spinal trigeminal nuclei, midline raphe nuclei, parabrachial nuclei, periaqueductal gray matter (PAG), interpeduncular nucleus, ans substantia nigra. A group of DOR-positive cells was seen in the laterodorsal tegmental nucleus. In addition, a few DOR-IR cell bodies were demonstrated in the parabrachial nuclei, interpeduncular nucleus, PAG, and superior and inferior colliculi as well as around the central canal in the spinal cord. All DOR-positive cells showed a punctuate staining pattern within the cytoplasm of the cell body and in primary dendrites. No plasma membrane staining of cells or dendrites could be demonstrated using the DOR antisera. Double-labeling experiments for DOR and 5-hydroxytryptamine (5HT, serotonin) revealed that some 5HT-IR neurons in the raphe complex were surrounded by DOR-IR fibers. In the spinal cord a high degree of coexistence was found between DOR and 5HT in nerve fibers and varicosities in the neuropil around the motoneurons and in lamina V of the dorsal horn. In autonomic regions of the spinal cord, a low degree of colocalization was seen between DOR and 5HT; in the superficial dorsal horn no coexistence was found. Tyrosine hydroxylase (TH) positive neurons in the brainstem (in the A5 area, locus coeruleus, and A7 area) were apposed by DOR-positive fibers. However, no coexistence could be seen between DOR and TH in any part of the spinal cord. A close relation, but no coexistence, was observed between DOR- and enkephalin (ENK)-IR fibers in the spinal cord ventral horn; in the intermediolateral nucleus a low degree of colocalization was observed. Thus, a delta-opioid receptor may affect the activity of descending serotoninergic and noradrenergic neurons by means of modulating the release of neurotransmitters from afferents to these neurons.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532702 TI - Regeneration of the auditory midbrain intercommissural projection in organotypic culture. AB - The aim of this study was to determine whether postnatal mammalian central neurons retain the capacity for axonal regeneration across a lesion site in organotypic cultures of the auditory midbrain. Brain slices from the gerbil inferior colliculus (IC) were obtained from postnatal 6-8 d old animal and cultured for 6-15 d in vitro. IC explants containing an intact commissural projection exhibited robust axonal and dendritic morphologies as assessed with biocytin labelling. In transected explants, the two lobes of the inferior colliculus were cut at the midline and then reapposed to one another in vitro. There was a robust regeneration of commissural fibers across the lesion site in 78% of the biocytin-labeled explants. Massive axonal regeneration was also revealed by immunostaining explants for Tau (100% of sections), an axon-specific microtubule-associated protein. Ultrastructural analyses demonstrated that biocytin-labeled regenerating fibers established de novo synaptic profiles in the contralateral lobe of the inferior colliculus. Finally, the distribution of astrocytes and oligodendrocytes were assessed by staining for glial fibrillary acidic protein (GFAP) and myelin-associated glycoprotein (MAG), respectively. GFAP-positive astrocytes were more widely distributed than in vivo, and oligodendrocytes remained immature, and evenly distributed in all explants. Taken together, these data demonstrate that the postnatal mammalian auditory midbrain can be maintained in vitro, and that central axons are capable of regenerating across the site of injury without the aid of an artificial substrate. PMID- 7532701 TI - Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine. AB - Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses. PMID- 7532704 TI - Structural basis for explaining open-channel blockade of the NMDA receptor. AB - The open-channel structure of the N-methyl-D-aspartate (NMDA) receptor was investigated to explain apparently conflicting interpretations about ionic interactions within the pore. Patch-clamp techniques were applied to tissue cultured rat hippocampal neurons from the CA1 region. A wide range of ammonium derivatives was studied to learn about the structure of the pore from permeability and open-channel blocking characteristics. We conclude that the pore is asymmetric, having high-affinity binding for organic cations from the outside and having a larger external entrance. The minimum cross-sectional area of the pore is rectangular (approximately 0.45 x 0.57 nm) and is the single-occupancy binding site(s) for small permeant cations. The narrow region extending from this minimum cross section is short, and its shape underlies the voltage dependencies of blocking cations. While occupying the blocking site, some open-channel blockers can interact with permeant cations at their binding site in the minimum cross section. A structurally based hypothesis is presented, explaining that the electrostatic interactions between the blocking site and permeant-ion site produce a high voltage dependence for blockade by some cations. PMID- 7532707 TI - Rapid method for purification of CD56+ natural killer cells with preferential enrichment of the CD56bright+ subset. AB - A rapid method for the purification of CD56+ natural killer (NK) cells with the preferential enrichment of the CD56bright+ subset is described. The method is based on the adsorption of CD56+ cells indirectly stained with a biotinylated antibody on an avidin-coated column. The adsorbed CD56+ cells can then be squeezed out mechanically without damaging the viability or the function of the cells. Starting from peripheral mononuclear cells from adult donors, the recovery of the CD56+ cells was 11.9 +/- 9.0% (n = 8), the purity 93.0 +/- 4.9% (n = 10), and the enrichment factor 7.5 +/- 1.7 (n = 8). Further phenotypic classification of the CD56+ cells into CD56dim+ and CD56bright+ cells showed a preferential enrichment of the CD56bright+ phenotype with a recovery of 40.5 +/- 19.6% (n = 8), a purity of 30.3 +/- 13.2% (n = 8), and an enrichment factor of 29.8 +/- 7.2 (n = 8). In conclusion, the described method allows the rapid purification of CD56+ NK cells with the preferential enrichment of the CD56bright+ subset. PMID- 7532703 TI - Synaptic interactions between primary afferent terminals and GABA and nitric oxide-synthesizing neurons in superficial laminae of the rat spinal cord. AB - The superficial laminae (I and II) of the spinal dorsal horn receive small caliber primary afferent fibers responsive to noxious stimulation, and contain local circuit neurons that modulate afferent input. Many of these neurons are GABAergic; about a third of these also synthesize nitric oxide. We identified three main morphological types of primary afferent terminals in superficial laminae after injections of a tracer selective for small caliber afferents into the sciatic nerve of rats. The relative densities of the three types varied through the dorsoventral extent of laminae I and II. Synaptic contacts of each type with GABA- and nitric oxide synthase (NOS)-containing dendrites and axon terminals were determined by preembedding and postembedding immunocytochemistry. Nonglomerular primary afferent terminals, likely to originate from peptidergic unmyelinated fibers, were not seen in synaptic contact with either GABA- or NOS containing neurons. Primary afferent terminals at the center of type 1 glomeruli (C1) and at the center of type 2 glomeruli (C2) are likely to originate from unmyelinated and small myelinated fibers, respectively. GABAergic terminals contacted more C2 than C1 terminals, suggesting more effective presynaptic inhibition of C2 terminals. Many GABAergic terminals were also positive for NOS, but all GABAergic terminals presynaptic to primary afferent terminals were negative for NOS. Only C2 terminals established frequent synapses with NOS positive dendrites. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532705 TI - Target regulation of a motor neuron-specific epitope. AB - In the adult rat nervous system, motor neurons are recognized specifically by a monoclonal antibody, MO-1. Because binding by MO-1 is lost following axotomy, contact with the target may regulate this motor neuron-specific epitope. To test this hypothesis, we examined the recovery of MO-1 immunoreactivity in hypoglossal neurons following unilateral damage to the hypoglossal nerve. During the first week following nerve crush, neurons in the ipsilateral hypoglossal nucleus lost all immunoreactivity for MO-1. Antibody binding returned with time, and by 4 weeks, 80% of the injured neurons had recovered the MO-1 epitope. Since motor neurons reinnervate their original targets readily following nerve crush, it appears that MO-1 binding is recovered when motor neurons return to their original target muscles in the tongue. When the hypoglossal nerve was cut and inserted into a foreign muscle nearby (the sternomastoid muscle), the MO-1 epitope was not detected in the injured neurons, even when examined 6 weeks after surgery. However, if the sternomastoid muscle was denervated prior to insertion of the hypoglossal nerve, thus allowing the hypoglossal nerve to synapse with this foreign target, increasing numbers of hypoglossal neurons reacquired MO-1 immunoreactivity with time. Our results suggest that the MO-1 epitope is only expressed in motor neurons that are in synaptic contact with skeletal muscle. Thus, a property that distinguishes mature motor neurons from other neuronal phenotypes appears to be regulated by direct synaptic interaction with the postsynaptic target. PMID- 7532708 TI - Administration of granulocyte colony-stimulating factor to neutropenic low birth weight infants of mothers with preeclampsia. AB - Nine low birth weight infants with neutropenia born to mothers with preeclampsia were treated with granulocyte-colony stimulating factor, 10 micrograms/kg intravenously, within 24 hours of birth and at 24-hour intervals for a maximum of three doses if neutropenia persisted. The absolute neutrophil count increased significantly in eight of the nine infants within 6 hours, and neutrophilia was sustained for at least 72 hours after administration of a single dose of granulocyte-colony stimulating factor. PMID- 7532706 TI - FORSE-1: a positionally regulated epitope in the developing rat central nervous system. AB - We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1-positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5-10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high-molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development. PMID- 7532709 TI - Effect of granulocyte colony-stimulating factor on preeclampsia-associated neonatal neutropenia. AB - Absolute neutropenia lasting longer than 72 hours after birth occurred in four very low birth weight neonates with a maternal history of severe pregnancy induced hypertension, and was treated with recombinant granulocyte colony stimulating factor for 3 days. Absolute neutrophil counts increased nearly four fold within 48 hours; maximal values were recorded on the ninth day after the infusion was started. Total leukocyte counts subsequently decreased but remained in the normal range. It appears that recombinant human granulocyte colony stimulating factor promotes a rapid increase in circulating neutrophils in these patients despite the possible presence of a circulating preeclampsia-associated inhibitor of neutrophil production. PMID- 7532710 TI - Neurodevelopmental outcome of infants with hypoplastic left heart syndrome. AB - The neurodevelopmental outcome of hypoplastic left heart syndrome in infants remains unclear. All 11 survivors of staged surgical repair of hypoplastic left heart syndrome received standardized neurodevelopmental assessments at one regional children's hospital. Seven children (64%) had major developmental disabilities. Quality-of-life outcomes must be considered when management options for children with hypoplastic left heart syndrome are evaluated. PMID- 7532711 TI - Benzodiazepines influence melatonin secretion of the pineal organ of the trout in vitro. AB - The effect of benzodiazepines (BZP) on melatonin release was investigated in the pineal gland of the rainbow trout, Oncorhynchus mykiss, maintained under in vitro perifusion culture conditions. Melatonin and the methoxyindoles 5 methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5 methoxytryptamine (5-MT) were determined directly in samples of the superfusion medium by HPLC with electrochemical detection. Melatonin release was significantly increased by addition of diazepam and clonazepam in a dose-related and reversible manner. The effects of benzodiazepines were more pronounced in light-adapted pineal organs, when melatonin secretion is low, than under scotopic conditions. When the perifusion medium was replaced by a medium containing low calcium, high magnesium concentrations, melatonin release was considerably decreased by 70% in light-adapted and 20% in dark-adapted pineal organs. Addition of diazepam to low Ca2+, high Mg(2+)-medium reversed the decrease of melatonin release and produced a clear rise in its secretion rate. Addition of the BZP antagonist flumazenil to the perifusion medium slightly decreased melatonin release in the light- and dark-adapted state, whereas the peripheral receptor antagonist PK 11195 did not alter melatonin release. The effect of diazepam is reduced by simultaneous addition of flumazenil to the superfusion medium, suggesting that the effects of diazepam are receptor-mediated. The methoxyindoles 5-MTOL, 5-MIAA, and 5-MT showed no significant changes of their release pattern after diazepam application in light- and dark-adapted pineal organs. These results suggest that BZP can influence melatonin production and release by an intrapineal action possibly on the melatonin synthesizing photoreceptor cell. PMID- 7532712 TI - Three cation influx currents activated by purinergic receptor stimulation in rat megakaryocytes. AB - 1. Simultaneous patch clamp and fura-2 fluorescence measurements were used to study ATP-evoked membrane currents and intracellular [Ca2+] ([Ca2+]i) changes in rat megakaryocytes. 2. At negative potentials, under conditions that blocked K+ currents, 20 microM ATP activated a biphasic inward current and a concurrent biphasic increase in [Ca2+]i. The initial [Ca2+]i increase was due to Ca2+ influx whereas the delayed (1.70 +/- 0.13 s, mean +/- S.D.) increase was at least partly due to the release of internal Ca2+ stores. 3. The initial current was activated within 100 ms, inactivated within 1-4 s and was carried by both Na+ and Ca2+. 4. The delayed current was also transient and carried mainly by Na+ when Ca2+ buffering in the pipette was low. This Na+ conductance did not require an increase in [Ca2+]i for activation, but was triggered by inositol 1,4,5 trisphosphate (IP3), or a metabolite of IP3. 5. Buffering of [Ca2+]i changes with BAPTA revealed a third current activated by Ca2+ release from internal stores. This channel was selective for divalent cations with the permeability sequence Ca2+ >> Ba2+ > Mn2+, Mg2+. 6. Adenosine-5'-O-3-thiotriphosphate (ATP gamma S), like ATP, evoked all three influx currents, whereas ADP only stimulated Ca2+ release and the two currents associated with it. Increasing the external divalent cation concentration abolished the ATP-evoked Ca2+ release and delayed currents but not the initial transient current. 7. We conclude that rat megakaryocytes express two types of purinergic receptor. One type, activated by ATP, is closely coupled to a non-selective cation channel.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532715 TI - Total parenteral nutrition modifies the acute phase response to Crohn's disease. AB - Measurements of the acute phase proteins, C-reactive protein (CRP) and orosomucoid are widely used to monitor the activity of Crohn's disease. The effect of TPN upon the levels of acute phase proteins is unknown. Serum levels of CRP and orosomucoid were measured simultaneously over a four year period in 13 patients receiving TPN for Crohn's disease, nine patients with noninflammatory causes of intestinal failure, and 16 patients with Crohn's disease treated without TPN. An acute phase response was found with a similar frequency in both groups of patients with Crohn's disease (73.6% and 83.9% for Crohn's with and without TPN respectively), but was less prevalent in patients receiving TPN for non-inflammatory causes of intestinal failure (56.1%, P < 0.01). In this latter group, the acute phase response consisted primarily of an isolated elevation of orosomucoid (78.4%), compared with patients with Crohn's disease alone (21.1%, P < 0.001) and with Crohn's disease and TPN (46.6%, P < 0.05). Liver function abnormalities were seen on 68.8% of occasions in patients with noninflammatory causes of intestinal failure who had elevated levels of orosomucoid, compared with 34.9% of occasions on which orosomucoid levels were normal (P < 0.001). TPN may lead to isolated elevation of serum levels of orosomucoid, reducing the value of this acute phase protein in monitoring the activity of Crohn's disease in patients receiving TPN. PMID- 7532714 TI - Vascular bed-dependent roles of the peptide CGRP and nitric oxide in acid-evoked hyperaemia of the rat stomach. AB - 1. Acid back-diffusion through a disrupted gastric mucosal barrier is known to increase gastric mucosal blood flow via a neural mechanism. The present study examined how the acid-evoked change in the gastric microcirculation compares with blood flow changes in the left gastric artery, one of the major arteries supplying the stomach, and whether the dilator mediators in the left gastric artery are identical to those in the gastric mucosa. 2. The experiments were performed on rats anaesthetized with urethane. Blood flow in the left gastric artery was measured by the ultrasonic transit time shift technique, and blood flow in the gastric mucosa was assessed by the hydrogen gas clearance method. 3. Gastric acid back-diffusion evoked by perfusion of the stomach with 15% ethanol in 0.15 M HCl increased blood flow in the left gastric artery by a factor of 4.7, which was significantly larger than the 2.9-fold increase in blood flow through the gastric mucosa. Blood pressure and heart rate were not altered appreciably. 4. The acid-evoked hyperaemia in the left gastric artery was left unaltered by atropine and the substance P receptor antagonist RP-67580. 5. The calcitonin gene related peptide (CGRP) antagonist CGRP (8-37) had no effect on gastric blood flow but prevented the dilator action of CGRP and inhibited the acid-evoked hyperaemia in the gastric mucosa to a larger degree than the hyperaemia in the left gastric artery. 6. Blockade of nitric oxide synthesis by N omega-nitro-L-arginine methyl ester (L-NAME) caused constriction of the left gastric artery and the gastric mucosal microvessels. The acid-evoked vasodilatation in the gastric mucosa was blocked by L-NAME, whereas the dilator response in the left gastric artery was not significantly depressed. 7. The data show that the gastric hyperaemic response to acid back-diffusion results from dilatation of mucosal microvessels and extramural arteries. The dilator mechanisms, however, differ between the two vascular beds. CGRP and nitric oxide are important vasodilator mediators in the gastric mucosa but are of less relevance in the left gastric artery. PMID- 7532713 TI - Bleached pigment activates transduction in isolated rods of the salamander retina. AB - 1. We have used suction electrode recording together with rapid steps into Li+ solution and 0.5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10(-6) to 10(-7) times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization. PMID- 7532716 TI - Immunohistological and functional analysis of adhesion molecule expression in the rheumatoid synovial lining layer. Implications for synovial lining cell destruction. AB - OBJECTIVE: It has previously been shown that the adhesion of lymphocytes to microvascular endothelium mediates lymphocyte extravasation within inflamed synovium. After passing the endothelial barrier, binding of lymphocytes to matrix proteins and synovial lining cells may further lead to synovial membrane hyperplasia and subsequent cartilage destruction. Thus, we have explored the molecular basis of T cell-synovial lining cell interaction in the synovial membrane of patients with rheumatoid arthritis (RA). METHODS: Using an immunohistochemical staining technique and an in vitro frozen section assay we studied the expression and the role of several adhesion molecules in T lymphocyte synovial lining cell interaction in the inflamed synovial membrane. RESULTS: In RA the macrophage-like (type A) synovial lining cells express high levels of intercellular adhesion molecule 1 [ICAM-1 (CD54)], whereas the fibroblast-like (type B) synovial lining cells predominantly express vascular cell adhesion molecule 1 (VCAM-1), in addition to moderate levels of ICAM-1. Both cell types express low levels of fibronectin. Unstimulated and anti-CD3 stimulated peripheral blood T cells bear the respective ligands lymphocyte function associated antigen 1 [LFA-1 (CD18/11a)], and very late antigen 4 and 5 [VLA-4 (CD29/49d) and VLA-5 (CD29/49e)]. T lymphocytes predominantly bound to type B synovial lining cells. Inhibition studies with monoclonal antibodies revealed that this binding involves the VLA-4/VCAM-1 and VLA-5/fibronectin (FN), but not the VLA-4/CS1 pathway. LFA-1 is also involved in this interaction via its ligand ICAM-1. CONCLUSION: These results show that the molecular basis of T lymphocyte binding to rheumatoid synovial lining cells is different from that described for T lymphocyte binding to synovial membrane vascular endothelium which involves the VLA-4/VCAM-1 and VLA-4/CS-1 pathways, but not the LFA-1/ICAM-1 pathway. PMID- 7532719 TI - In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy. AB - A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In-vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300-350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post-transplantation, the animal was anaesthetized and prepared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortousity index of the neovasculature. PMID- 7532717 TI - A pilot study of anti-CD5 ricin A chain immunoconjugate in systemic lupus erythematosus. AB - OBJECTIVE: To determine the safety and clinical and biological effects of a murine monoclonal anti-CD5 ricin A chain immunoconjugate (CD5 Plus) in patients with systemic lupus erythematosus (SLE). METHODS: An open label phase I study of CD5 Plus. A dose of 0.1 mg/kg was administered intravenously on 5 consecutive days. A second course of immunoconjugate was given to patients who failed to show any clinical response one to 2 months later. RESULTS: Six patients (4 with glomerulonephritis and 2 with thrombocytopenia) were studied. Improvement was documented in 2 patients with nephritis; no effect on thrombocytopenia was observed. Adverse effects were mild and transient. Relative to pretreatment lymphocyte counts, the mean reduction in CD3+ T cell count was 69% at 2 weeks, 32% at one month, and 34% at 6 months following initial treatment. A comparable decrease in all subpopulations of mature T cells was noted, using a variety of surface markers, including CD4 and CD8. The mean percentage of T cells expressing the activation markers HLA-DR and interleukin 2R (IL-2R) was high before treatment, and remained so. There was a transient decrease in CD5+ B cells, but no persistent depletion of total B cell numbers. There was no consistent change in natural killer cell populations. CONCLUSION: Anti-CD5 ricin A chain immunoconjugate is well tolerated in patients with SLE, causes modest T cell depletion which may persist for months, and may have some clinical efficacy in lupus nephritis. PMID- 7532718 TI - In situ analysis of microbial consortia in activated sludge using fluorescently labelled, rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy. AB - Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 microns. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior. PMID- 7532720 TI - The crystal structures of the SH2 domain of p56lck complexed with two phosphonopeptides suggest a gated peptide binding site. AB - Src homology-2 (SH2) domains are protein modules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl containing protein sequences. In order to develop SH2 inhibitors that contain phosphotyrosyl analogues resistant to cellular phosphatases, we have solved the crystal structures of the SH2 domain of p56lck in separate complexes with two high-affinity p-(phosphonomethyl)phenylalanine-containing peptides. The structures have been determined at 2.3 A and 2.25 A, and refined to crystallographic R-factors of 19.2% and 18.5%, respectively. The conformation of the SH2 domain of p56lck is essentially similar to that observed in Src and Lck complexed with a phosphotyrosine-containing peptide except in some loops and especially in the loop that connects the second and third beta-strands. This loop, which was involved in hydrogen-bond interactions with the phosphotyrosine moiety, has moved away in the phosphonopeptide complexes as a rigid body by about 7 A on two hinges leaving the tyrosine phosphate mimetic moiety accessible to the solvent. Some intramolecular hydrogen bonds with other residues of the third and fourth beta-strands stabilize an open conformation of the lid, suggesting a flap mechanism for peptide binding. PMID- 7532721 TI - Internal mobility of the basic pancreatic trypsin inhibitor in solution: a comparison of NMR spin relaxation measurements and molecular dynamics simulations. AB - Order parameters as well as longitudinal and transverse relaxation rates are calculated for the backbone 15N and 13C alpha nuclei of the basic pancreatic trypsin inhibitor (BPTI) from a 1000 ps molecular dynamics trajectory in explicit water at 277 K using the "model free" approach of Lipari and Szabo. New NMR relaxation data at 277 K are presented, and a comparison is made between NMR relaxation measurements and molecular dynamics relaxation data. It is found that the relaxation processes determining the longitudinal (T1) relaxation rates are inadequately sampled even during this length of simulation. In effect, the calculated relaxation rates are determined almost solely by the order parameters and the overall rotational correlation time of the protein, which appears to be in clear contrast to experimental relaxation rates. PMID- 7532723 TI - Delayed hypertonic saline dextran administration after burn injury. AB - OBJECTIVE AND DESIGN: Experimental studies in our laboratory showed that hypertonic saline dextran (HSD; 7.5 NaCl in 6% dextran 70) given as a small bolus (4 mL/kg) immediately after burn injury in guinea pigs improved cardiac contractile performance and reduced the total fluids requirements. Although these data confirm the cardioprotective effects of HSD given immediately postburn, prehospital and early inhospital management of severely burned patients consists of aggressive crystalloid fluid resuscitation to correct intravascular volume deficits. The question arose as to whether delaying HSD for several hours after initiating crystalloid resuscitation would provide cardioprotection. MATERIALS AND METHODS: Third-degree scald burns comprising 45 +/- 1% of the total body surface area (burn groups, n = 40) or 0% for controls (group 1, n = 12) were produced; in groups 2 to 5, lactated Ringer's (LR) resuscitation was initiated immediately postburn according to the Parkland formula, 4 mL/kg/% burn. In group 2, (n = 12), LR was continued for 24 hours. HSD was administered as an i.v. bolus at either 1 hour (group 3, n = 10), 4 hours (group 4, n = 9), or 8 hours postburn (group 5, n = 9); immediately after HSD administration, LR was continued (1 mL/kg/% burn) until 24 hours postburn. RESULTS: Compared to sham burn controls, hearts from burned animals treated with LR alone had significant cardiac dysfunction, as indicated by a lower left ventricular pressure and +/- dP/dt. Compared with hearts from LR-treated animals, hearts from burned animals treated with HSD 1 hour (HSD-1) and 4 hours (HSD-4) after burn injury had significantly higher LVP and +/- dP/dt. Ventricular function curves calculated for HSD-1 and HSD-4 groups were comparable to those calculated for hearts from sham burns. Delaying HSD administration until 8 hours after burn provided little cardioprotection. CONCLUSIONS: Our data indicate that HSD effectively maintains cardiac function and reduces overall total fluid requirements if administered within 4 hours after burn injury. PMID- 7532722 TI - Differentiation-specific demethylation of myelin associated glycoprotein gene in cultured oligodendrocytes. AB - The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin. The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure. PMID- 7532724 TI - Review of peptide growth factors in benign prostatic hyperplasia and urological malignancy. PMID- 7532725 TI - The use of age-specific reference ranges for serum prostate specific antigen in men 60 years old or older. AB - Several investigations have determined that the serum prostate specific antigen (PSA) concentration is dependent upon patient age and, as a result, reference ranges wider than 0.0 to 4.0 ng./ml. have been suggested for men 60 years old or older. To determine the clinical usefulness of the age-specific reference ranges- 0.0 to 4.5 ng./ml. for men 60 to 69 years old and 0.0 to 6.5 ng./ml. for men 70 years old or older--the medical records of 2,988 men 60 years old or older who presented to a single urological practice were examined. All patients were evaluated with a serum PSA determination, digital rectal examination and transrectal ultrasound. A total of 1,686 prostate biopsies was performed (biopsy rate 56%) and 608 cancers were diagnosed (cancer detection rate 20%). By using the age-specific reference ranges as compared to the 0.0 to 4.0 ng./ml. reference range, the sensitivity of PSA for detecting early prostate cancer decreased by 9%, while the specificity and positive predictive value increased by 11% and 5%, respectively. If the age-specific reference ranges had been used 92 prostate biopsies (5.5%) performed could have been avoided, while 19 men in the study population (0.6%) would not have had prostate cancer diagnosed. Of the nondetected cancers 13 (67%) occurred in men 70 years old or older and 18 (95%) were small tumors of favorable pathological status unlikely to be of clinical consequence in these older men. These preliminary findings support the clinical usefulness of the wider age-specific reference ranges in men 60 years old or older. A prospective randomized clinical trial is currently underway to confirm the appropriateness of age-specific reference ranges compared to the reference range of 0.0 to 4.0 ng./ml. PMID- 7532726 TI - Histocultures of human prostate tissues for pharmacologic evaluation. AB - This study evaluated the growth of human prostate tumors in histoculture, an in vitro culture technique that maintains three-dimensional tissue structure and organization. Eighty-six percent of 50 tumor specimens from 50 patients were successfully cultured. The histocultures showed proliferation of epithelial tumor cells and stromal cells. Prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) were detectable by immunohistochemistry for at least 8 weeks. Synthesis of PSA was further confirmed by its presence in medium. The mean thymidine labeling index (LI) of the neoplastic cells was 62%. There was no correlation between thymidine LI and tumor grade. The explants maintained their characteristics for at least 8 weeks as indicated by unchanged thymidine LI, PSA and PAP immunohistochemistry after 2 and 8 weeks in culture. A 24 to 48 hour delay in processing the tissues for culture did not reduce the thymidine LI. The thymidine LI and PSA and PAP staining in tumors cultured in a Minimal Essential Medium (MEM)-based or a PFMR-4-based culture medium were similar, suggesting an insignificant effect of the medium on cell proliferation. Migration of neoplastic cells from the tissue fragments into the collagen gel matrix was noted in 1 of 10 samples cultured in MEM-based medium versus 8 of 10 samples cultured in PFMR-4 medium (p < 0.01). Exposure of 13 patient tumors to suramin, doxorubicin and 5 fluorouracil at clinically relevant concentrations and duration showed tumor sensitivity in 23%, 31% and 15% of the specimens. These values approximate the historical clinical response rates. These data suggest that the histoculture system holds promise for short-term culture of human patient prostate tumor specimens for biologic and pharmacologic studies. PMID- 7532727 TI - [Summary and evaluation of genome and antibody diagnosis in hepatitis C virus infection]. AB - Hepatitis C virus (HCV) infection can be diagnosed by antibody assay systems using recombinant antigens since the HCV genome has been identified. It is still impossible to detect viral antigens associated with HCV. Therefore, the polymerase chain reaction (PCR) has been widely used in practical laboratory examinations. Because HCV is an RNA virus and the reverse transcription process is required prior to the PCR reaction, the process of HCV RNA detection has a risk of contamination and the detection rate may differ with the PCR condition. There are several genome diagnostic methods for HCV infection; that is, genome detection by nested PCR, HCV subtyping using mixed primer, quantitation of HCV genome using competitive PCR, Quant-Amp and branched DNA probe. To clarify what factors are responsible, the efficacy of interferon therapy against chronic hepatitis C was examined. The normalization rate of serum ALT level and clearance rate of HCV RNA in serum at six months after the end of the treatment were correlated to the titer of HCV RNA genome and the HCV subtype. The patients with HCV RNA titers of less than 10(4) copies/50 microliters and with subtype group III showed a high response to interferon administration. On the other hand, the results of quantitation of the HCV core antibody titer were closely associated with the presence of HCV RNA using core protein with the recombinant vaccinia expression system or recombinant E. coli system (JCC-2). Especially, in the group of high responders to interferon treatment, the antibody titer against core protein was apparently decreased after the end of interferon therapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532730 TI - Beneficial circulatory effect of L-arginine. AB - L-Arginine is an essential amino acid for infants and growing children. This amino acid is a substrate for at least five enzymes identified in mammals, including arginase, arginine-glycine transaminase, kyotorphine synthase, nitric oxide synthase (NOS) and arginine decarboxylase. L-Arginine exerts antihypertensive and antiproliferative effects on vascular smooth muscles. NOS and arginine decarboxylase appear to be important for the effect of L-arginine on the circulatory system, since each produces nitric oxide (NO), a potent vasodilator, and agmatine, an endogenous noncatecholamine ligand for central alpha-2 adrenoceptors, from L-arginine. Several issues must be clarified before the mechanisms by which L-arginine exerts its effects on the circulatory system can be fully understood. PMID- 7532728 TI - Inability of Ca2+ influx through nicotinic ACh receptor channels to stimulate catecholamine secretion in bovine adrenal chromaffin cells: studies with fura-2 and SBFI microfluorometry. AB - The concentration of cytosolic Ca2+ ([Ca]in) and catecholamine (CA) secretion were examined in bovine adrenal chromaffin cells to determine whether Ca2+ influx through nicotinic ACh receptor (nAChR) channels contributes to CA secretion induced by nAChR stimulation. Nicotine added under Na(+)-free conditions caused a marked increase in [Ca]in and quenching of fura-2 fluorescence in the presence of Mn2+, suggesting the stimulated entry of divalent cations through nAChR channels. However, nicotine-induced increase in CA secretion occurred only at a non physiologically high external Ca2+ concentration under Na(+)-free conditions. Both the nicotine-induced increase in [Ca]in and CA secretion under Na(+)-free conditions were reduced in the presence of hexamethonium, methoxyverapamil (D600), nifedipine, Bay-K-8644, clonidine, and guanethidine. All of these agents inhibited the nicotine-induced increase in cytosolic Na+ concentration in a dose dependent manner, as measured by SBFI microfluorometry. The present results suggest that Ca2+ influx through nAChR channels under physiological conditions may not contribute to CA secretion. PMID- 7532729 TI - Lipid interference with fluorometric assay of DNA in adipose tissues under various conditions. AB - Interference by lipids with fluorometric assay of DNA in adipose tissues using Hoechst 33258 was investigated. Mixed glycerides shifted the emission maximum of standard DNA and induced a dose-dependent increase in fluorescence intensity. Glycerides in the samples containing a known concentration of DNA yielded erroneously higher DNA concentrations. The DNA concentrations obtained from acetone-defatted white and brown adipose tissues (WAT and BAT) were lower than those of non-defatted ones, while DNA content did not differ in low lipid containing skeletal muscle between defatted and non-defatted samples, indicating that large amounts of lipids interfere with DNA measurement using Hoechst 33258 and that acetone defatting is a simple method to avoid this interference. Using this defatting method, the cellularity of WAT and BAT was estimated in rats under various experimental conditions. Cold-acclimation and repetitive immobilization stress decreased the body weight gain and the epididymal WAT weight. Sucrose overfeeding increased WAT weight but not body weight. These treatments of 4 weeks' duration did not induce any significant difference in WAT cell number from controls, while cold-acclimation increased the tissue cell number as well as the BAT weight. PMID- 7532731 TI - Possible participation of histamine H3-receptors in the regulation of anaphylactic histamine release from isolated rat peritoneal mast cells. AB - Anaphylactic histamine release from isolated rat peritoneal mast cells was concentration-dependently blocked by a 5-min treatment with exogenous histamine at 0.9 and 9 microM and enhanced by a 20- to 30-min treatment with thioperamide (H3-antagonist) at 3 microM with significance, but little affected by mepyramine (H1-antagonist) and cimetidine (H2-antagonist) at the cell concentration of 10(6) mast cells/ml. At a low concentration of mast cells (10(4) mast cells/ml), (R) alpha-methylhistamine (alpha-MH), an H3-agonist, at 0.9-90 microM also inhibited the release in a concentration-dependent fashion. Thioperamide, but neither mepyramine nor cimetidine, significantly restored the decreased release by alpha MH. However, the complete restoration by thioperamide could not be achieved because the drug itself slightly but concentration-dependently inhibited anaphylactic histamine release. On the other hand, not only betahistine and dimaprit but also alpha-MH did not suppress histamine release from the mast cells induced by compound 48/80. In rat plasma, considerable levels of histamine were detected. From these results, it is strongly suggested that histamine H3-like receptors are largely responsible for the negative feedback regulation of the anaphylactic histamine release from rat peritoneal mast cells. PMID- 7532732 TI - Effects of risperidone on phencyclidine-induced behaviors: comparison with haloperidol and ritanserin. AB - In this study, we investigated whether risperidone, a serotonin-S2A (5 HT2A)/dopamine-D2 (D2)-receptor antagonist, inhibits phencyclidine (PCP)-induced stereotyped behaviors in comparison with haloperidol and ritanserin. Moreover, we also attempted to investigate the effects of these antipsychotics on the contents of dopamine, serotonin (5-HT) and their metabolites in rat striatum and frontal cortex. In rats, PCP (5 mg/kg, i.p.) caused hyperlocomotion and stereotyped behaviors, including sniffing, head-weaving, backpedalling and turning. Both resperidone (0.8-2.4 mg/kg, p.o.) and haloperidol (0.3-1.0 mg/kg, p.o.) inhibited these behaviors, except for backpedalling, in a dose-dependent manner. PCP (10 mg/kg, i.p.) produced hyperlocomotion and stereotyped behaviors, including rearing, sniffing head-twitch, backpedalling and turning. Risperidone (0.8-2.4 mg/kg, p.o.) inhibited both hyperlocomotion and PCP-induced behaviors, except for backpedalling, while ritanserin (3-10 mg/kg, p.o.) inhibited only the head twitch. These results suggest that risperidone may have an antipsychotic effect on schizophrenia as well as PCP psychosis in humans by exerting a mixed 5-HT2A/D2 antagonism. Neurochemically, the increasing effects of risperidone on the content of DOPAC and the ratio of DOPAC to dopamine in the striatum were lower than those of haloperidol. These findings may support the view that the extrapyramidal side effects of risperidone are lower than those of haloperidol in clinical situations. PMID- 7532733 TI - Datura stramonium agglutinin released histamine from rat peritoneal mast cells that was inhibited by pertussis toxin, haptenic sugar and N-acetylglucosamine specific lectins: involvement of glycoproteins with N-acetylglucosamine residues. AB - The N-acetyl glucosamine (GlcNAc)-specific lectin Datura stramonium agglutinin (DSA) rapidly and sugar-specifically released histamine from rat peritoneal mast cells, and pertussis toxin (IAP) inhibited it, suggesting that DSA activated mast cells via an IAP-sensitive G protein pathway. The additive effects of DSA and basic secretagogues such as compound 48/80 that activate IAP-sensitive G protein directly suggest that they shared the same mechanism of action including involvement of the IAP-sensitive G protein. Using lectin-blotting, blots of the corresponding glycoproteins detected by DSA diminished by haptenic sugar or pretreatment of the cells with N-glycosidase F, suggesting that the binding of DSA was responsible for the mast cell activation. The other GlcNAc-specific lectins such as Phytolacca americana mitogen, Solanum tuberosum agglutinin and wheat germ agglutinin (WGA) inhibited the histamine release induced by DSA, suggesting that these lectins were antagonists, but DSA was an agonist. Sialic acid-specific Macckia amurensis mitogen (MAM) inhibited the histamine release, and neuraminidase-treatment decreased mast cell activation induced by DSA. At least four mast cell glycoproteins that have affinity to DSA, WGA and MAM and are sensitive to neuraminidase-treatment were detected by lectin-blotting. Some of them may be binding sites coupled to histamine release including the IAP sensitive G protein pathway. DSA is a useful tool for studying signal transduction of mast cells including the involvement of the IAP-sensitive G protein. PMID- 7532734 TI - An initial signal of activation of rat peritoneal mast cells stimulated by Datura stramonium agglutinin: a confocal fluorescence microscopic analysis of intracellular calcium ion and cytoskeletal assembly. AB - A confocal fluorescence microscope using fluo-3 and 9-(dicyanovinyl)- julolidine (DCVJ) was used to study the mast cell activation by the N-acetyl glucosamine oligomer specific lectin Datura stramonium agglutinin (DSA) and inhibition by antagonist lectins having affinity to N-acetyl glucosamine (GlcNAc). DSA induced a transient increase in intracellular free calcium concentration ([Ca2+]i) followed by cytoskeletal disassembly and reassembly in rat peritoneal mast cells. These changes induced by DSA resulted in histamine release. The time course of fluorescence intensity in mast cells loaded with fluo-3- or DCVJ and activated by DSA resembled those activated by the basic polymer compound 48/80. Inhibition of [Ca2+]i rise by antagonist lectins was responsible for the inhibition of cytoskeletal assembly and the consequent histamine release induced by DSA. At the level of the individual cell, a mast cell stimulated by DSA responds in an all-or none fashion. DSA possible induced intracellular calcium mobilization and cytoskeletal change by recognizing the GlcNAc-oligomer residues of specific glycoproteins of mast cells. PMID- 7532735 TI - Visualization of renal microcirculation in isolated Munich-Wistar rat kidneys: effects of endothelin-1 on renal hemodynamic activity. AB - The aim of the present study was to visualize the superficial glomeruli of the Munich-Wistar (MW) rat and to characterize the responses of the renal microvasculature to endothelin-1 (ET-1). We first examined the distribution of superficial glomeruli of the MW rat compared to that in a control strain (Wistar rat). Secondly, we examined the effects of ET-1 on the renal microcirculation of the MW rat. The right kidney was perfused with a Krebs-Ringer solution containing fluorescein isothiocyanate dextran (FITC-dextran) and was visualized under an epi illuminated fluorescence microscope system. Changes in perfusion pressure and diameter of the microvessels accompanying the administration of ET-1 (10 fmole 300 pmole) were measured. The number of superficial glomeruli was greater in the MW rat than in the Wistar rat. ET-1 had long-lasting and dose-dependent pressor effects. Perfusion pressure showed a 3.5-fold increase compared with the control, and the afferent arterioles showed greater dose-dependent vasoconstriction than the efferent arterioles. These findings suggest that the MW rat is a useful animal model for the study of renal microcirculation and that the renal microcirculation is extremely sensitive to ET-1. PMID- 7532736 TI - Alteration of liver glutathione S-transferase and protease activities by cobalt chloride treatment of rats. AB - Effects of cobalt chloride on liver glutathione S-transferase and protease activities were studied. When cobalt chloride (60 mg/kg) was given to rats, liver microsomal glutathione S-transferase and protease activities were significantly increased 24 hr after the injection, whereas glutathione peroxidase activity in microsomes was decreased. The increase in glutathione S-transferase by N ethylmaleimide was similar to that of the control, indicating that the increase in the transferase activity by cobalt chloride is not due to a modification of the sulfhydryl group of the enzyme. Immunochemical analysis of the liver microsomes did not detect any proteolytic product of microsomal glutathione S transferase. In puromycin- or actinomycin D-treated rats, an increase in the transferase activity caused by cobalt chloride treatment was depressed. Thus it was suggested that liver microsomal glutathione S-transferase is induced by cobalt chloride treatment, but not activated by limited proteolysis via microsomal protease. PMID- 7532738 TI - [Transurethral microwave thermotherapy for benign prostatic hyperplasia: a 1-year follow-up study]. AB - The authors studied a 1-year effect of transurethral microwave thermotherapy (TUMT) using the PROSTCARE apparatus (Bruker Spectrospin Wissembourg, Frace) on 35 patients with symptomatic benign prostatic hyperplasia (BPH). The device is equipped with a unique non-invasive system "radiometry" for the measurement of the intraprostatic temperature to regulate the microwave emission power and to eliminate classic surface controls. All of the 35 patients received a single thermotherapy session (60 minutes), the average intraprostatic temperature was 43.6 +/- 1.2 degrees C (mean +/- SD) and the average power output was 43.9 +/- 4.1 Watt. The clinical effects were evaluated at 2 months, 6 months and 1 year after TUMT by a specially designed score scale for subjective symptoms and objective findings as compared with the pretreatment score. Assuming that more than a 25% reduction of the total score indicates "effective", 71.4%, 71.4, and 48.6% of the treatments remained effective at 2 months, 6 months, and 1 year after TUMT, respectively. The maximum prostatic urethral pressure profile decreased from 64.1 +/- 17.1 to 51.7 +/- 15.6 cmH2O (p < 0.05) at 2 months after TUMT. The thermotherapy by PROSTCARE is effective in approximately 50% of patients at least 1 year and there were no major complications associated with TUMT during the follow up period. PMID- 7532737 TI - Two-phase increment of Ca2+ uptake, intracellular Ca2+ concentration, and histamine release following antigen stimulation in mouse bone marrow-derived mast cells (BMMC). AB - The relationship between the influx of Ca2+ into cells or cytosolic Ca2+ concentration ([Ca2+]i) and the histamine release following antigen stimulation in mouse bone marrow-derived mast cells (BMMC) was examined, and the results were compared with those from human lung mast cells (HLMC) and rat peritoneal mast cells (RPMC) in some experiments. Anaphylactic histamine release from BMMC as well as HLMC, but not that from RPMC, was dependent on the extracellular Ca2+. When BMMC were challenged by antigen following radioactive 45Ca2+ addition, two phases of 45Ca2+ influx into the cells were observed. The first phase, which was initiated and completed within 30 sec and 2 min, respectively, after antigen treatment, appeared to be related to anaphylactic histamine release. The second influx began 30 sec subsequent to the first one and lasted for at least 2 min, and this occurred after the completion of the histamine release; So far, it is not known how this second influx participates in the intracellular event(s). On the other hand, only one sustained elevation of [Ca2+]i occurred that reached its maximum within 2 min after antigen stimulation. Following stimulation of BMMC with antigen in the absence of Ca2+, Ca2+ addition 1 to 5 min later time dependently enhanced the histamine release, although the release was deteriorated by further extension of Ca2+ addition. In contrast, the releasability of HLMC was rapidly decreased. These results indicate that extracellular Ca2+ not only is prerequisite for anaphylactic histamine release from BMMC, but also may modulate the release and participate in some intracellular event(s) which has yet to be focused upon. PMID- 7532740 TI - New markers for analyzing the cause of hematuria. PMID- 7532739 TI - Diagnostic strategies in urinalysis. AB - The traditional methods in urinalysis (visual microscopy, qualitative test strip screening) were compared with automated microscopy (UA-1000, TOA-medicals, Japan) and quantitative single protein analysis in 562 fresh morning urine samples. Albumin served as "glomerular" and alpha 1-microglobulin as "tubular" markers measured by turbidimetry. The test strip delivered at least one positive result in 60% of the urine for blood (21%), leukocytes (27%), or protein (34%). In only 4% casts or renal cells were found by traditional microscopy, whereas automated microscopy was positive for these findings in 28% of the urine. Quantitative urine protein analysis alone exhibited results outside the reference interval in 52% of the urine. Combination of the test strip procedure for blood and leukocytes with urine protein analysis increased the number of positives to 73%. Thirteen percent of these additional findings were classified as glomerular (64%) and tubular (72%) proteinurias. In 7% of the urine a false positive protein test strip result was confirmed by quantitative albumin determination. Of 157 urine samples, positive in mechanized video recorded screening, 60 (38%) were normal in single protein analysis. The results allow for the conclusion that the advanced techniques are superior to traditional screening procedures in detecting abnormal urine composition. It is suggested that traditional urinalysis should be supported or replaced by quantitative determination of albumin and alpha 1 microglobulin. This recommended strategy is able to exclude or detect tubulo interstitial nephropathies or microalbuminuria in earlier phases of renal complications, such as in diabetes mellitus, hypertension or in nephrotoxic injury. A fully mechanized version is suggested to meet appropriate quality criteria and economic needs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532741 TI - Urinary protein 1 or Clara cell protein: a new sensitive marker of proximal tubular dysfunction. AB - Protein 1 or Clara cell protein (CC16) is a 16 kD protein secreted predominantly by Clara cells in terminal bronchioles and from puberty on in the male urogenital tract. The sensitivity of CC16 in urine as an index of proximal tubule dysfunction was compared to that of retinol-binding protein, beta 2-microglobulin and alpha 1-microglobulin. These microproteins were measured by latex immunoassay in the urine from 114 pregnant women, 126 diabetics (65 men and 61 women), 80 workers exposed to cadmium (36 men and 44 women), and from healthy subjects matched for age and sex. In women, CC16 appeared consistently as a much more sensitive index of tubular dysfunction than other microproteins. In female diabetics, for instance, the prevalence of elevated values of CC16 in urine (53%) largely exceeded that of other microproteins (< 30%) and even of albumin (35%). In men, however, the existence of a post-renal secretion contaminating the urine limits the sensitivity of CC16 which was revealed to be higher than that of other microproteins, in diabetics only. The assay of urinary CC16 has the potential, especially in women, to detect very subtle defects of the proximal tubule which pass completely unseen with other microproteins. We postulate that this unique sensitivity of CC16 is due to its very low concentration in tubular fluid which, combined with its anionic character, strongly hinders its access to brush border binding sites. PMID- 7532742 TI - Clinical value of fructose 1,6 bisphosphatase in monitoring renal proximal tubular injury. AB - The usefulness of the gluconeogenic key enzyme fructose 1,6 bisphosphatase (FBPase), which is localized exclusively in the proximal nephron segment, as a marker compound to monitor injury of the proximal nephron segment during nephrotoxic therapy, was tested in a collective model of male patients treated for testicular cancer. These patients with normal kidney function were submitted to therapy with the nephrotoxic chemotherapeutics carboplatinum and a combination of cisplatinum, etoposide, bleomycin and ifosfamide. The release of FBPase activities into the urine was monitored during the initial two treatments over a period of eight days. The urinary enzyme activities measured were compared to the excretion of the "proximal tubular injury markers" N-acetyl-beta-D glucosaminidase (NAG) and alpha 1-microglobulin (alpha 1m). The presence of glomerular damage was determined by measurement of urinary excretion rates of albumin (ALB) and IgG. In addition, protein excretion patterns following chemotherapy were monitored. The combined administration of cisplatin, etoposide and ifosfamide resulted in a pronounced proximal tubular injury as shown by the release of FBPase into the urine. This is substantiated by simultaneously increased excretion rates for NAG and alpha 1m. Proximal tubular toxicity was found to be less severe when cisplatin was combined with etoposide and bleomycin and was nearly absent following carboplatinum monotherapy. Carboplatinum only affected glomerular function and resulted in an elevated ALB and IgG excretion. From this model investigation it can be delineated that determination of urinary FBPase activities ensures a sensitive and reliable identification of proximal nephron damage. PMID- 7532743 TI - Charge selectivity and urine amylase isoenzymes. AB - The urinary excretions of salivary and pancreatic amylase were studied in 718 type I diabetic patients and 51 control subjects, as part of a multicenter study on diabetic nephropathy in 15 Spanish hospitals. It was found that the urinary ratio of salivary to pancreatic amylase (S/P ratio), that in normal subjects is always below 1, was elevated in 35.4% of diabetic patients, whereas microalbuminuria was present in 19.8%. The prevalence of elevated S/P ratio was also higher than that of microalbuminuria at the first years from the onset of the disease, but the prevalence of microalbuminuria was higher in patients with a long duration of the disease. alpha 1-microglobulin and microalbuminuria paralleled their prevalences during the disease, when measured in a group of patients. Overnight urine samples were obtained on three consecutive weeks from the diabetic patients, and a nested ANOVA analysis showed that the intra individual variation of the urine parameters measured (albumin, salivary and pancreatic amylase, and beta-NAG) was very small and not statistically significant. All these findings suggest that in type I diabetes mellitus, loss of negative charges of GBM would induce preferential excretion of the anionic salivary amylase over the more cationic pancreatic amylase, and that this phenomenon is more frequent and appears earlier than microalbuminuria. The mechanisms for the increased excretion of salivary amylase and albumin into urine seem to be at least partly different. On the contrary, increase in urinary excretion of albumin and alpha 1-microglobulin in these patients are correlated, suggesting a tubular participation in the mechanisms of production of microalbuminuria. PMID- 7532744 TI - Structural features related to the nephropathogenicity of Bence-Jones proteins. PMID- 7532745 TI - Contraction band necrosis: its modification by the free radical scavenger N-2 mercaptopropionyl glycine. AB - Contraction band necrosis (CBN) may represent infarct extension from free radical generation during reperfusion. We sought to limit CBN with the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 20 mg/kg). Sixteen chronically instrumented Beagles (8 control, and 8 MPG treated) underwent 90-min left anterior descending coronary artery (LAD) occlusion followed by 6-h reperfusion. Coronary blood flow (CBF) was measured by the radioactive microsphere technique. The dogs were killed, and the hearts were perfused with red and blue dyes to determine area at risk (AAR), stained with nitroblue tetrazolium for infarct localization, and sectioned for histologic analysis and BF measurements. In controls and MPG-treated animals, infarct/risk ratios were 40 +/- 5 and 38 +/- 6%, and epicardial collateral BFs were 0.21 +/- 0.037 and 0.15 +/- 0.034 ml/g/min, respectively (p = NS). Hemodynamic measurements did not differ between the two groups. However, CBN as a percentage of total infarct was reduced in controls (22 +/- 3%) as compared with MPG-treated animals (35 +/- 2%, p = 0.002). Thus, MPG altered the histologic composition of infarcts in this model, surprisingly increasing the amount of CBN without altering overall infarct size (IS). These results raise questions about the role of free radical scavengers in generation of CBN and suggest that a population of cells exists in which treatment with MPG may alter the mechanism of cell death. PMID- 7532746 TI - Nitroprusside infusion improves arterial baroreflex control of heart rate in dogs with chronic congestive heart failure. AB - To determine if nitroprusside improves arterial baroreflex responsiveness in chronic congestive heart failure (CHF), we administered nitroprusside to 11 conscious dogs with pacing-induced CHF. Baroreflex sensitivity was determined by plotting the R-R interval against systolic aortic pressure after a bolus injection of phenylephrine (PE). At baseline, dogs with CHF had higher heart rate (HR), increased left atrial blood pressure (BP), and reduced left ventricular (LV) dP/dt as compared with 10 sham-operated normal animals. Baroreflex sensitivity index was significantly lower in CHF dogs, (8.3 +/- 1.3 ms/mm Hg) than normal dogs (25.1 +/- 1.2 ms/mm Hg, p < 0.001). Intravenous (i.v.) administration of nitroprusside (1 microgram/kg/min) to CHF dogs decreased left atrial BP (23 +/- 1-17 +/- 1 mm Hg) and HR (131 +/- 4-115 +/- 4 beats/min), but had no significant effect on either cardiac output (CO) or systolic aortic BP. This resulted in a 58% increase in baroreflex sensitivity index to 13.1 +/- 1.3 ms/mm Hg (p < 0.001); and the change correlated significantly with magnitude of decrease in left atrial BP (r = 0.884, p < 0.001) but not with the increase in R R interval (r = 0.390, p > 0.10). In contrast, administration of nitroprusside sufficient to decrease left atrial BP (9.0 +/- 1.4-6.4 +/- 1.2 mm Hg) did not alter baroreflex sensitivity (26.4 +/- 3.4-26.4 +/- 3.9 ms/mm Hg) in 5 normal dogs. The results suggest that nitroprusside infusion increases arterial baroreflex sensitivity only in dogs with CHF and that this effect is probably functionally linked to the reductions of cardiac filling pressure. PMID- 7532749 TI - Effects of magnesium on polymorphic ventricular tachycardias induced by aconitine. AB - We examined the effects of Mg2+ on aconitine-induced polymorphic ventricular tachycardias (PVT) in excised rabbit hearts under Langendorff perfusion and in Purkinje-muscle preparations. Local electrograms using bipolar electrodes and transmembrane potentials with the microelectrode technique were recorded from Langendorff hearts and Purkinje-muscle preparations, respectively. In Langendorff preparations, intracoronary application of 0.1 microM aconitine induced PVT 28.8 +/- 3.4 min after development of regular monomorphic ventricular tachycardias (MVT) in all 18 preparations. Application of 5 and 10 mM Mg2+ restored aconitine induced PVT to sinus rhythm after 26.8 +/- 3.4 min (n = 9), but < 3 mM Mg2+ was not effective in restoring of sinus rhythm. Increased Mg2+ concentrations < or = 5 mM in the coronary perfusate prevented development of PVT by aconitine. Intracoronary application of 10 microM tetrodotoxin (TTX) also restored aconitine induced PVT to sinus rhythm after 3.2 +/- 0.8 min (n = 4). Although applications of 50 microM lidocaine, 10 microM flecainide, or 1 microM verapamil could change PVT to MVT, they were not effective in restoring sinus rhythm. In Purkinje-muscle preparations, spontaneous action potentials (AP) from slow diastolic depolarization appeared after aconitine at the maximum diastolic potential of 75.0 +/- 3.7 mV in Purkinje fibers and were conducted to ventricular muscles (n = 5). Spontaneous activity gradually increased in rate and then developed triggered activity arising from early after depolarization (EAD). EAD induced by aconitine always appeared first in Purkinje fibers and later in muscle fibers. Once triggered activities started from EAD, rate, rhythm and amplitudes of APs became fast and variable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532748 TI - Epicardial mesothelial cells synthesize and release endothelin. AB - Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demonstrate remarkable plasticity of phenotype accompanied by a significant increase in cardiocyte contractile protein content, suggesting that a factor with growth-promoting properties may take part in EMC-adult rat ventricular cardiocyte interactions. Endothelin (ET) has been shown to induce cell hypertrophy, including enhancement of expression of muscle-specific genes. We investigated the ability of EMC to synthesize and release ET. By light microscopy, specific immunostaining, with either ET-1 or Big ET-1 antibodies, was visualized in EMC as a fine punctate distributed throughout the cytoplasm. Reverse phase-high performance liquid chromatography (HPLC) of epicardial mesothelial cells conditioned medium showed several peaks of immunoreactive ET. The major peak eluted with the same retention time as that of ET-1. By Northern blot analysis, a specific 2.3-kilobase (kb) mRNA species was detected by hybridization to a cDNA insert encoding for rat prepro-ET-1. ET accumulated in the culture medium in a time-dependent manner, whereas cell content remained comparatively low. Angiotensin II (AII) dose-dependently stimulated release of immunoreactive ET into the culture medium. PMID- 7532750 TI - Hemodynamic, neurohumoral, and myocardial energetic effects of pimobendan, a novel calcium-sensitizing compound, in patients with mild to moderate heart failure. AB - In contrast to cyclic AMP-dependent positive inotropes, the calcium-sensitizer and partial phosphodiesterase (PDE) inhibitor pimobendan may induce beneficial effects in heart failure. However, its effect on relaxation, myocardial energetics and neurohormones are unknown. Twelve patients with heart failure, New York Heart Association (NYHA) classification II-III, due to ischemic cardiomyopathy, were studied for 1 h after they received 5 mg pimobendan intravenously (i.v.). Pimobendan progressively reduced systemic resistance and left ventricular end-diastolic pressure (LVEDP) (22 and 50%, respectively) and improved isovolumetric contractility and relaxation parameters by 30% (all p < 0.05 vs. control). LV end-diastolic and end-systolic volumes (LVEDV, LVESV) decreased significantly by 20 and 19%, respectively. Cardiac output (CO) increased by 17% due to a simultaneous increase in heart rate (HR) from 75 +/- 3 to 86 +/- 5 beats/min (mean +/- SEM, p < 0.05). Pimobendan did not change coronary hemodynamics, but myocardial O2 extraction and consumption were decreased significantly by 18 and 20%, respectively. Catecholamines, angiotensin II (AII), and aldosterone levels did not change significantly. In contrast, arterial and coronary venous renin increased significantly from 57 +/- 17 and 53 +/- 14.7 microM/h at control to 69 +/- 20 and 69 +/- 20 microM/h, respectively, 60 min after pimobendan administration. Simultaneously, cardiac renin uptake at baseline (0.449 +/- 0.185 mumol/min) changed to release (-0.071 +/- 0.145 mumol/min, p < 0.05). Serious side effects did not occur. Thus, pimobendan had progressive positive inotropic and lusitropic effects, diminished preload and afterload despite modest stimulation of plasma renin activity (PRA), and reduced systemic vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532747 TI - Antiarrhythmic agent amiodarone possesses calcium channel blocker properties. AB - Amiodarone possesses multiple pharmacologic properties, including peripheral and coronary vasodilatation, negative inotropy, and negative chronotropic and dromotropic effects. These properties are shared by the group of drugs termed calcium channel blockers. We examined the interaction of amiodarone with receptors for the 1,4-dihydropyridine (DHP) calcium blockers in rat and rabbit myocardial membrane particulates. Amiodarone displaced specifically bound [3H]nitrendipine in both rat and rabbit preparations in a competitive, concentration-dependent manner at a single class of binding sites (Ki approximately 0.27 micxroM). Calcium channel activity was determined pharmacologically in a tissue bath with electrically stimulated rabbit right ventricular strips, KCl-induced aortic ring contraction, and 45Ca2+ uptake in K(+)-depolarized cultured rat cardiomyocytes. Amiodarone completely inhibited myocardial contraction (EC50 = 1.7 microM), completely antagonized depolarization induced aortic ring contraction (EC50 = 24 nM), and significantly reduced (29% vs. control) 45Ca2+ uptake into cultured cells. The calcium channel blocking effects of amiodarone may contribute significantly to its pharmacologic profile. PMID- 7532752 TI - Role of alpha 1A- and alpha 1B-adrenoceptors in phenylephrine-induced positive inotropic response in isolated rat left atrium. AB - We determined the distribution of alpha 1A- and alpha 1B-adrenoceptor subtypes and their functional roles in phenylephrine (PE)-induced positive inotropic responses in rat atrium. Radioligand binding assays in membrane preparations of rat atria showed that 62% of [125I]BE 2254 binding sites were irreversibly inactivated by pretreatment with 20 microM chloroethylclonidine (CEC). Inhibition curves for WB 4101 and 5-methyl-urapidil (5-MU) were better fit by a two-site model, comprising 29-35% high-affinity sites and 65-71% low-affinity sites, suggesting that rat atria contains both alpha 1A and alpha 1B subtypes in a ratio of approximately 1:2. In isolated perfused atria, pretreatment with CEC inhibited the maximum PE-induced positive inotropic response by 55%, and pA2 values for WB 4101 and 5-MU in inhibiting this response were 8.26 +/- 0.4 and 7.85 +/- 0.07, respectively, between the KD values for alpha 1A and alpha 1B subtypes. PE shifted the concentration-contractile response curve for Ca2+ to the left and upward. Pretreatment with CEC completely abolished whereas 1 nM WB 4101 did not alter, the effect of PE on Ca2+ sensitivity. These results demonstrate that both alpha 1A- and alpha 1B- adrenoceptor subtypes are involved in the PE-induced positive inotropic response. Only activation of the alpha 1B subtype potentiates the positive inotropic effect induced by increasing extracellular Ca2+, however, which suggests that the mechanisms involved in the action of the two subtypes may differ at least in part. PMID- 7532753 TI - New classification of moricizine and propafenone based on electrophysiologic and electrocardiographic data from isolated rabbit heart. AB - Because the classification of propafenone and moricizine is not clear, we measured in 20 specifically equipped isolated rabbit hearts QRS duration, QT interval, action potential duration at 90% of repolarization (APD90), effective refractory period (ERP), conduction time (CT), and rise velocity (Rv) of the monophasic action potentials (AP) during exposure to moricizine and propafenone in comparison with procainamide. Propafenone and procainamide prolonged APD90 and JT. All drugs increased ERP. Propafenone demonstrated marked tonic sodium channel block. Onset of use-dependent kinetics (tau on), defined as change in Rv as fraction per beat at 300 ms cycle length (CL), were 0.047 +/- 0.004/beat for procainamide, 0.050 +/- 0.004/beat for propafenone, and 0.022 +/- 0.003/beat for miricizine. Recovery kinetics, defined as recovery of Rv after cessation of pacing, had a time constant of 5.3 +/- 0.7 s for procainamide, 6.3 +/- 0.8 s for propafenone, and 25.0 +/- 1.3 s for moricizine. Based on these data, moricizine must be classified as a Ic agent, whereas propafenone demonstrates pronounced tonic sodium channel block, in addition to its phasic block, which is similar to class Ia kinetics. PMID- 7532751 TI - Action of ATP on ventricular automaticity. AB - ATP is an effective treatment of supraventricular tachycardia when the atrioventricular (AV) node is part of the reentrant circuit. However, the lower a pace-maker in the pacemaker hierarchy, the more sensitive it is to adenosine. Therefore, we investigated the effects of ATP on ventricular automaticity in in vivo and in vitro conditions. Wide and narrow QRS complex tachycardia in 46 patients was treated with 6, 12, and 18 mg ATP as sequential intravenous (i.v.) bolus. ATP terminated tachycardias in 67%. Bolus infusion ATP caused < or = 6.4-s asystole that was self-limited. Perfusion of isolated spontaneously beating guinea pig heart with 100 microM ATP completely suppressed ventricular automaticity. After ATP-infusion was discontinued, the first ventricular beat was evident after 3.1 +/- 0.9 s and sinus node activity recovered with a time constant of 3.0 +/- 1.1 s. Because sinus node and ventricular automaticity recovered within seconds after ATP infusion was discontinued in vitro, recovery in vivo is also likely to be determined by the short half-life (+1/2) of ATP. PMID- 7532754 TI - Effects of MDL 28,133A, a 5-HT2 receptor antagonist, on platelet aggregation and coronary thrombosis in dogs. AB - We wished to characterize MDL 28,133A (1-(4-fluorophenyl)-2-[4-[(4 methanesulfonamidophenyl)carbonyl]-1- piperidinyl-ethanone HCl), a potent 5-HT2 receptor antagonist, on platelet aggregation and platelet thrombosis and determined its effect on thrombolysis with streptokinase (SK) in dogs with coronary thrombosis. MDL 28,133A and ritanserin, 0.3-1 microM, competitively inhibited 5-HT-induced platelet aggregation in dog platelet-rich plasma (PRP) in vitro. The pA2 values and slopes were 6.29 +/- 0.09, -0.96 +/- 0.14, and 6.58 +/- 0.09, =1.64 +/- 0.34 for MDL 28,133A and ritanserin, respectively, suggesting that both agents are similar in potency to 5-HT2 receptor antagonists. MDL 28,133A (0.01 mg/kg, p.o.) produced inhibition of arachidonic acid-induced platelet aggregation in whole blood from conscious dogs ex vivo for > or = 2 h, indicating oral bioavailability. The magnitude and duration of the effect of MDL 28,133A on platelet aggregation in whole blood was similar to that of ketanserin (2.5 mg/kg, p.o.). MDL 28,133A (0.001-0.03 mg/kg, i.v.) completely abolished cyclic flow reductions (CFRs) in stenosed and partially deendothelialized left anterior descending coronary arteries of anesthetized dogs for at least 120 min without affecting systemic blood pressure (BP) and heart rate, thus indicating that MDL 28,133A is a potent antithrombotic agent. Ketanserin (0.5 mg/kg, i.v.) also abolished CFRs, but produced a significant decrease in systemic BP as well. MDL 28,133A (0.001 mg/kg, i.v.) shortened time to reperfusion (15 +/- 1 vs. 36 +/ 8 min), prolonged time to reocclusion (112 +/- 6 vs. 85 +/- 6 min), and increased total volume reflow (20 +/- 2 vs. 10 +/- 2%) in dogs with coronary artery thrombosis undergoing thrombolysis with SK (1,000 U/min, i.a.). Reocclusion rate was not affected by MDL 28,133A (4 of 5 vs. 4 of 6). Treatment with heparin (150 U/kg, i.v. every hour for 2 h) alone or in combination with MDL 28,133A did not improve time to reperfusion but enhanced total volume reflow and prevented reocclusion. MDL 28,133A is a potent 5-HT2 receptor antagonist that inhibits platelet thrombosis and facilitates thrombolysis in vivo. The impeding effect of MDL 28,133A in coronary thrombosis and the lack of hemodynamic effects suggests that MDL 28,133A may be of benefit in treatment of hyperthrombotic conditions without having hemodynamic side effects. PMID- 7532755 TI - Effect of hemicholinium-3 on the hypothalamic concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase-stimulating substance. AB - Hypothalamus and plasma of salt-loaded rats, spontaneously hypertensive rats (SHR), and hypertensive reduced renal mass rats (RRM), and the plasma of patients with essential hypertension and of Milan hypertensive rats contain an increased concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase (G6PD)-stimulating substance that has properties similar to that of a possible choline derivative di-methyl methylene immonium ion. Intracerebroventricular (i.c.v.) administration of hemicholinium-3 (HC-3) selectively blocks high affinity neuronal choline uptake, inhibits brain acetylcholine (ACh) synthesis, and decreases arterial pressure in SHR through an inhibiting effect on hypothalamic cholinergic function. The experiments were performed to study the effect of centrally administered HC-3 on the content of the cytochemically detectable cholinelike substance in hypothalamus and plasma of SHR. HC-3 or saline was infused into the lateral cerebral ventricle for 6 days with a minipump in 14 SHR. On day 7, the hypothalamic and plasma concentration of the cytochemically detectable substance was significantly reduced in rats that received HC-3. The hypothalamic concentration was 225 +/- 95.6 x 10(8) G6PD U per hypothalamus (range 38.2-775) in SHR that received saline and 1.037 +/- 0.45 x 10(8) G6PD U (range 0.112-3.61) (p < 0.05) in SHR that received HC-3. The respective plasma concentrations were 284.9 +/- 26 U/ml (range 192-374) and 72.7 +/- 14.7 U/ml (range 24-119) (p < 0.05). The findings are consistent with the physicochemical evidence, which suggests that the cytochemically detectable substance is a choline derivative.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532756 TI - Platelet-derived adenosine contributes to the cardioprotective effects of platelets against ischemia-reperfusion injury in isolated rat heart. AB - Platelets have been shown to protect isolated perfused rat hearts from injury and dysfunction after ischemia and reperfusion. We examined the role of platelet derived adenosine in the cardioprotective effects of platelets against reperfusion injury. Isolated perfused rat hearts were subjected to 40-min global ischemia followed by 30-min reperfusion. The buffer-perfused hearts developed dysfunction, as indicated by 40 +/- 4% decrease in force of cardiac contraction (FCC) and 24 +/- 3% increase in coronary perfusion pressure (CPP). Creatine kinase (CK) was released in coronary effluent during reperfusion, indicating myocardial injury. At the end of reperfusion, myocardial CK content and superoxide dismutase (SOD) activity were lower than in sham ischemia-reperfused hearts. Perfusion of hearts with washed platelets resulted in protection against myocardial dysfunction and injury after ischemia and reperfusion, indicated by preservation of FCC (-2 +/- 5%) and CPP (-3 +/- 2%) (both p < 0.01 vs. buffer perfused hearts). Myocardial CK and SOD activity were also preserved, and release of CK in the coronary effluent was minimal (all p < 0.05 vs. buffer-perfused hearts). The cardioprotective effects of platelets were attenuated by preincubation of platelets with adenosine deaminase. Perfusion with adenosine or the adenosine2 receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2- methylphenyl) ethyl]adenosine (DPMA) also protected heart from myocardial dysfunction and injury after ischemia and reperfusion. Both adenosine and DPMA had salutary effects on indexes of cardiac injury. Platelet-derived adenosine contributes at least in part to the cardioprotective effects of platelets against ischemia and reperfusion in isolated rat heart. PMID- 7532757 TI - Alterations of ex vivo vascular reactivity in intraperitoneal sepsis. AB - We examined vascular reactivity to vasoconstrictors [phenylephrine (PE), serotonin (5-HT), and high K+] and vasodilators [acetylcholine (ACh), A23187, L arginine, and nitroglycerin (NTG)] in isolated mesenteric arterial rings from control and septic rats. Sepsis was induced by cecal ligation and puncture (CLP). A possible mechanism underlying CLP-induced alteration in vascular reactivity was also investigated with N omega-nitro-L-arginine (L-NNA 50 microM), methylene blue (MB 10 microM), and indomethacin (5 microM). In vivo, septic rats manifested two distinct hemodynamic phases, a hyperdynamic state during early (9 h after CLP) phase, followed by a hypodynamic state during late (18 h after CLP) phase. Therefore, we examined ex vivo vascular reactivity in these two phases. Results demonstrated that CLP operation caused hyporesponsiveness to contractile agents and hyperresponsiveness to vasodilator agents. After endothelium removal, most of the contractile responses were enhanced in both CLP-operated (9 and 18 h after operation) and sham-operated rats, whereas enhancement of high-K(+)-induced contraction was observed only in denuded rings from CLP 18-h rats. In addition, augmentation of relaxation induced by ACh at 9 or 18 h after CLP was abolished by N omega-nitro-L-arginine or MB but not by indomethacin. A possible mechanism responsible for alterations of vascular reactivity may be overproduction of nitric oxide (NO) which is blocked by L-NNA or MB. PMID- 7532759 TI - Relationship between structure and sodium channel blockade by lidocaine and its amino-alkyl derivatives. AB - We examined the relationship between the physicochemical properties and the sodium channel-blocking actions of lidocaine and four of its amino-alkyl derivatives. The homologues differ in lipid solubility (log p 2.7-4.1), pKa (6.9 9.0), and molecular weight (248.5-290.7). Macroscopic sodium currents were measured in rabbit atrial myocytes by the whole-cell configuration of patch-clamp technique; single-channel currents were measured by the cell-attached configuration. Lidocaine and its homologues produced two patterns of block: tonic block and frequency-dependent block. Tonic block was highly correlated with lipid solubility and pKa. The single-channel studies suggest that tonic block results when the drug interacts with channel state(s) that precede opening. Block of open channels does not appear to play a prominent role in tonic block. The rate of recovery from block was the major determinant of the magnitude of frequency dependent block. Highly lipid-soluble homologues showed rapid recovery from block and little frequency-dependent block. Drugs with lower lipid solubility and high pKa showed slower recovery from block and greater frequency-dependent block. The seemingly different requirements for tonic and frequency-dependent block can be explained by drug interaction at a single receptor site. PMID- 7532758 TI - Characterization of the adrenoceptor antagonistic and antihypertensive activity of oral amosulalol, a combined alpha- and beta-adrenoceptor antagonist, in hypertensive rats. AB - The adrenoceptor antagonistic and antihypertensive effects of amosulalol, 5-[1 hydroxy-2-[[2-(o- methoxy)ethyl]-2-ethylbenzenesulfonamide HCl, a combined alpha- and beta-adrenoceptor antagonist, were examined in hypertensive rats. Oral administration of amosulalol (1-30 mg/kg) produced a dose-dependent antihypertensive effect without reflex tachycardia in conscious spontaneously hypertensive rats (SHR) with a duration > 10 h after the higher doses (10 and 30 mg/kg). Amosulalol was approximately threefold more potent than labetalol and arotinolol in decreasing blood pressure (BP) in conscious SHR. Oral (p.o.) administration of amosulalol 10 mg/kg produced equally potent reductions in mean arterial BP (MBP) without reflex tachycardia in deoxycorticosterone acetate-salt rats (DHR) and renal hypertensive rats (RHR) as it did in SHR. Repeated oral administration (1, 4, 8, or 12 weeks) of amosulalol 10 mg/kg elicited an antihypertensive effect without evidence of tolerance in conscious SHR and produced a rightward shift in phenylephrine (PE)-induced vasopressor and isoproterenol (ISO)-induced positive chronotropic responses with dose ratios of 3.3-12.5 and 3.7-6.4, respectively, in pithed SHR. In addition, single p.o. administration of amosulalol 10 mg/kg produced a rightward shift in these responses with dose ratios of 12.1 and 3.5, respectively, in pithed SHR. Amosulalol exerted antihypertensive activity without tachycardia through blockade of vascular alpha- and cardiac beta-adrenoceptors, and its activities were constant even after repeated p.o. administration. PMID- 7532761 TI - Leucine enkephalin effects on paracellular and transcellular permeation pathways across brain microvessel endothelial cell monolayers. AB - Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances. PMID- 7532760 TI - Effect of rapamycin on rat aortic ring vasomotion. AB - Rapamycin (RAPA) is an antifungal antibiotic with interesting new immunosuppressive properties. We evaluated RAPA's effects in vitro on basal and stimulated tension of isolated intact or denuded rat aortic rings. Rings were prepared in an organ chamber and contracted with 40 mM KCl (reference 100%). Some rings were treated with either RAPA's polysorbate/polyethylene glycol-based (PEG) vehicle (0.8% vol/vol) or with different concentrations of RAPA (10, 100, and 1,000 ng/ml) diluted in PEG; untreated rings were used as controls. Variation in tension with time (2 h) and the dose-response to thromboxane A2 analogue (U46619) and phenylephrine (PE) were measured in controls and treated rings. PEG potentiated the increase in basal tension in rings with endothelium after 2-h treatment (44.66 +/- 3.59 vs. 14.82 +/- 2.43% for controls, p < 0.05, n = 10). RAPA antagonized the contraction induced by its own vehicle dose dependently. At 1,000 ng/ml, RAPA caused relaxation of intact rings below the control level (4.29 +/- 2.20 vs. 14.82 +/- 2.43%, p < 0.05, n = 10), but not in rings without endothelium. RAPA did not modify the response to PE or U46619 in rings with endothelium. RAPA relaxed the vessels by an endothelium-dependent mechanism, and this effect can be modulated by its vasoconstrictive PEG vehicle. PMID- 7532762 TI - Epicardial administration of ibutilide from polyurethane matrices: effects on defibrillation threshold and electrophysiologic parameters. AB - Polymer-drug composites known as controlled-release systems have been used effectively to prevent and treat ventricular arrhythmias in experimental studies. We wished to determine if such systems could be useful in reducing ventricular defibrillation energy requirements in an acute canine model without producing undesirable electrophysiologic effects. Ibutilide-polyurethane monolithic controlled-release matrices were formulated with ibutilide fumarate and a polyether polyurethane. In vitro drug-release characteristics of the drug matrices were determined. Two formulations were investigated: (a) 20% ibutilide by weight in polyether polyurethane, and (b) 4% ibutilide/16% dimethyl tartrate in polyurethane. Based on in vitro release studies, 20% ibutilide matrices (25 mg) would provide a 25-kg dog with a dose of 25 micrograms/kg ibutilide in a 2-h acute experimental period, and 4% ibutilide matrices were estimated to provide 3.5 micrograms/kg. We used each of these types of matrices in acute open-chest dog studies to assess electrophysiologic effects and the influence of epicardial controlled-release ibutilide, as compared with intravenous (i.v.) administration, on defibrillation energy thresholds (DFTs), using epicardial defibrillation electrodes. In monophasic defibrillation waveform studies, 20% matrices significantly decreased DFT as compared with a predrug control period [2.54 +/- 0.59 (mean +/- SEM) vs. 7.23 +/- 1.73 J, respectively, p = 0.038]. Administration of the same dose i.v. did not cause significant reduction in energy requirement. With a biphasic defibrillation waveform, 4% ibutilide matrices significantly decreased DFT as compared with control (2.53 +/- 0.34 vs. 3.42 +/- 0.46 J, respectively, p = 0.003). Administration of an equivalent i.v. dose did not cause a significant reduction in biphasic energy requirement. Both types of controlled release systems significantly prolonged refractoriness and conduction times of ventricular extrastimuli as compared with vehicle. No proarrhythmia events were observed. Epicardial polymeric controlled-release ibutilide significantly prolonged ventricular refractoriness and conduction and thus may enhance antiarrhythmia activity. In addition, controlled-release ibutilide formulations significantly decreased DFT requirements. Thus, ibutilide-polymeric controlled release matrix systems may be useful in conjunction with implantable defibrillators in preventing ventricular arrhythmias and reducing defibrillation energy requirements. PMID- 7532763 TI - Efficacy of anipamil, a phenylalkylamine calcium antagonist, in treatment of angina pectoris. AB - To evaluate the efficacy of anipamil, a phenylalkylamine calcium antagonist, in treatment of stable angina pectoris, we performed a randomized, double blind placebo-controlled, cross-over study. Inclusion criteria were (a) stable angina pectoris for at least 2 months, (b) an exercise test with > or = 0.1-mV horizontal or downsloping ST-segment depression limited by angina, and (c) at least 10 attacks of angina pectoris in a single-blind 3-week run-in period. Nineteen patients were randomized to enter the study. In 3-week periods, they received either anipamil 80 mg once daily (o.d.), anipamil 160 mg o.d., or placebo. At the end of each period, an exercise test was performed. The number of angina pectoris attacks was significantly reduced during treatment with anipamil 80 mg (p < 0.05) and anipamil 160 mg (p < 0.001) as compared with placebo. Glycerol nitrate consumption was significantly reduced during treatment with anipamil 80 mg (p < 0.01) and 160 mg (p < 0.001) as compared with placebo. During exercise testing, the load (W) at start of angina was significantly increased during treatment with anipamil 80 mg (p < 0.01) and 160 mg (p < 0.05) as compared with placebo. Heart rate (HR) at 0.1 mV ST-segment depression was increased during treatment with anipamil 80 mg (p < 0.001). Few adverse events were reported. PMID- 7532765 TI - On the origin of protein synthesis: a speculative model based on hairpin RNA structures. AB - A speculative model for the origin of protein synthesis based on aminoacylated hairpin RNAs is presented. The model shows how dimerization of these hairpin structures might have triggered the initial synthesis of oligopeptides. The evolution of these dimers seems to lead naturally both to the origin of the tRNA molecule and to the origin of protein synthesis. This co-evolution seems to be confirmed in the determinants of the tRNA identity that might represent the vestiges of the hypothesized model. Finally, the model lends itself to direct experimentation. PMID- 7532764 TI - Nucleoside transport inhibitors enhance the infarct size-limiting effect of ischemic preconditioning. AB - We recently observed that dipyridamole pretreatment significantly enhanced the infarct size (IS)-limiting effect of preconditioning (PC), which was attenuated by adenosine receptor antagonist. This potentiation of PC was interpreted to result from inhibition of nucleoside transport by dipyridamole, but contribution of other pharmacologic actions of dipyridamole could not be excluded. To confirm that inhibition of nucleoside transport leads to PC enhancement, we assessed alteration of mild PC by two different nucleoside transport inhibitors, dilazep and R75231, which, unlike dipyridamole, lack action on phosphodiesterase (PDE) and prostacyclin. Myocardial infarction was induced in rabbits by 30-min coronary occlusion and 72-h reperfusion. IS and area at risk (AAR) were determined by histology and fluorescent particles, respectively. Rabbits either were untreated or received dilazep (0.34 mg/kg intravenously, i.v.) or R75231 (0.05 mg/kg i.v.) before coronary occlusion. In other groups of rabbits, PC was conducted with 2 min ischemia and 5-min reperfusion with or without injection of the nucleoside transport inhibitor (0.34 or 0.10 mg/kg dilazep or 0.05 mg/kg of R75231) before PC. IS expressed as percentage of AAR (%IS/AAR) was 43.9 +/- 2.3% (SE) in untreated controls; dilazep (0.34 mg/kg) and R75231 alone did not modify IS (%IS/AAR = 50.6 +/- 4.7 and 42.7 +/- 11.9%, respectively). PC tended to reduce IS (%IS/AAR = 33.3 +/- 3.5%), but the combination of dilazep or R75231 with PC significantly limited %IS/AAR (%IS/AAR = 22.5 +/- 5.0% after low-dose dilazep plus PC, 27.6 +/- 4.9% after high-dose dilazep plus PC, and 19.9 +/- 3.6%, after R75231 plus PC).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532766 TI - De novo acute myeloid leukaemia in patients over 55-years-old: a population-based study of incidence, treatment and outcome. Northern Region Haematology Group. AB - A 4-year prospective study of de novo acute myeloid leukaemia in patients aged 56 years and over was undertaken in the Northern Region of England (population 3.09 million). The study was conducted to assess the incidence and outcome of treatment in all elderly patients diagnosed between January 1, 1988 and December 31, 1991. Two hundred cases de novo AML were confirmed, giving an incidence of 6.05/10(5) per annum (age specific population) (95% Cl, 5.2-6.9). Acute promyelocytic leukaemia was rare. Erythroleukaemia, monocytic leukaemia and AML with trilineage myelodysplasia were more common than in younger patients. Karyotypic abnormalities classically associated with response to therapy were present in only six of 91 patients where cytogenetic data was available. Treatment was at the discretion of the physician in charge: if given, specific treatment was recorded and clinical outcome assessed. Only 84 (42%) of patients received treatment with curative intent. Forty-four of 84 achieved a complete remission, usually of brief duration. A normal karyotype in leukaemic cells was associated with a survival advantage in this group (p < 0.05). Actuarial overall survival at 4 years for the entire group was 2.5%. Even with aggressive treatment, the outcome is poor. The pattern of disease and its lack of response to conventional treatment would support the hypothesis that AML in the elderly may differ biologically from that observed in younger patients. Karyotyping appears to predict those patients likely to benefit from intensive therapy and decisions about management in otherwise fit patients should, if possible, be delayed until a result is obtained. Every effort should be made to give such patients optimal treatment. However, most patients are unsuitable for aggressive treatment and, since long-term survival is rare, cure should not be offered as an inducement to accept such treatment and improving quality of life outside hospital should be the aim of treatment in this group. PMID- 7532768 TI - Acute monocytic leukemia: a myeloid leukemia subset that may be sensitive to methotrexate. AB - Impaired polyglutamylation of methotrexate (MTX) and thus poor retention is believed to be the basis of intrinsic resistance in blasts from patients with acute myeloid leukemia (AML) to MTX. We studied additional samples from patients with this disease, and confirmed that polyglutamylation of MTX was poor in ANLL blast cells. However, in one subset of ANLL, acute monocytic leukemia, (M5) leukemia blasts were found to be capable of accumulating and forming long-chain MTX polyglutamates. An acute monocytic leukemia cell line, THP-1 also was found to accumulate high levels of MTX polyglutamates and was relatively sensitive to MTX, strengthening the concept that M5 blasts may be sensitive to this drug. MTX may be an overlooked drug for the treatment of acute monocytic leukemia. PMID- 7532767 TI - CD34 expression is associated with major adverse prognostic factors in adult acute lymphoblastic leukemia. AB - Seventy-five adult patients with newly diagnosed acute lymphoblastic leukemia (ALL) were analyzed for CD34 expression on leukemic cells. CD34 was significantly associated with B-cell lineage ALL (p = 0.0002). In B-lineage ALL, CD34 positivity was significantly associated with expressions of CD9 (p = 0.001), CD19 (p = 0.00001) and CD22 (p = 0.002). CD34 was more expressed in B-ALLs with higher WBC cell count (p = 0.04), and higher percentage of peripheral blood leukemic cells (p = 0.005), total or partial monosomy of chromosome 7 (p = 0.0001) or Ph+ chromosome (p = 0.01); and less expressed in cases with hyperdiploidy (> or = 50 chromosomes) (p = 0.03). CD34 was more expressed in poor risk B-ALLs patients, defined according to Hoelzer criteria (p = 0.01). In T-lineage ALL, CD34 positivity was inversely correlated with the expression of CD10 (p = 0.05). After intensive induction therapy, 58 of 73 evaluable patients (79%) achieved a complete remission (CR). CD34 positivity was correlated with the persistence of blast cells in day 15 bone marrow aspirates (p = 0.001) and after one course of induction chemotherapy (p = 0.01). With a median follow-up of 11 months, no statistical differences were seen in leukemia-free survival and overall survival between CD34 positive and negative cases, even when stratifying by immunophenotype. We conclude that CD34 expression is associated with features of poor prognosis in adult ALL. Its study might therefore become useful in the design of future prognostic models. PMID- 7532769 TI - IL-4R alpha and gamma chain expression in LPS- and IL-4-stimulated MONO-MAC-6 cells. AB - Short-term stimulation of peripheral blood monocytes (PBMo) and cells of the monocytic cell line MONO-MAC-6 with lipopoly-saccharide (LPS) induces high tumor necrosis factor (TNF)alpha mRNA levels. In contrast to the results obtained with primary cells, this effect could not be inhibited by preincubating the cell line with recombinant human interleukin-4 (rh IL-4). This deficiency in response to the cytokine was not caused by a general unresponsiveness of MONO-MAC-6 cells to IL-4. Thus, the expression of the monocyte-associated differentiation markers CD14 and monocyte-specific esterase (MSE), upregulated by long-term stimulation with LPS, could be decreased by IL-4. Long-term LPS treatment apparently induced IL-4 responsiveness of the cell line. While IL-4R alpha mRNA was upregulated about 3-fold, this positive effect was not apparent at the cell surface protein level. In contrast to the constitutive alpha chain expression, the IL-4R gamma chain expression could not be detected with a specific mAb nor by Northern blot analysis. However, reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated the presence of low-level IL-4R gamma chain mRNA in the cell line. We suggest that the low reactivity of the cells to IL-4 might be correlated with the low expression of the gamma chain. PMID- 7532770 TI - [The follow-up of the mobilization of circulating hematopoietic progenitor cells by granulocyte growth factor (G-CSF) using flow cytometry]. AB - BACKGROUND: The aim of the present study was to investigate the characteristics of the mobilization of hematopoietic precursor cells CD34+ in peripheral blood following stimulation with recombinant granulocytic colony stimulating factor (G CSF). METHODS: Fourteen patients (10 males, 4 females: mean age 33 years; range 14-58 years) diagnosed with oncohematologic neoplasms, in complete remission were studied. The patients had not received antineoplastic for at least four weeks prior to inclusion in the study. Recombinant G-CSF (8 micrograms/kg) was administered subcutaneously over a minimum of 4 days. Peripheral blood control were performed prior to administration of G-CSF (day 0), the third (day +3) day, and the sixth day (day +6). Daily leukapheresis was initiated at day +3 in 5 patients and at day +4 in 9 patients. The CD34+ cell content was determined in both peripheral blood and leukapheretic material by flow cytometry with an anti CD-34 monoclonal antibody conjugated with fluorescein. RESULTS: No significant differences were observed between the mononuclear cells and CD34+ counts obtained at the first apheresis and those performed at days +3 or +4 (32 +/- 14 x 10(9) vs 29 +/- 19 x 10(9) and 240 +/- 125 x 10(6) vs 162 +/- 160 x 10(6), respectively). The content of the apheresis products in CD34+ cells correlated positively with the number of these cells circulating in peripheral blood (r = 0.53, p = 0.001). In the second apheresis, the presence of mononuclear cells decreased approximately 20% with respect to the first, remained constant in later collections. To the contrary, a constant maintained decrease was observed in the collection of CD34+ on each leukaphesis in that the fourth apheresis only contributed in approximately 10% of the total quantity of CD34+ cells collected. CONCLUSIONS: Maximum mobilization of precursor cells was achieved on the third day at a dosage of 8 micrograms/kg/day, with the data found suggesting that three leukapheretic procedures are enough to collect most of the CD34+ cells mobilized. PMID- 7532771 TI - [Communication and information]. PMID- 7532772 TI - [Elevation of muscular enzymes and idiopathic hypoparathyroidism]. PMID- 7532773 TI - On being a complementary therapist in palliative care. PMID- 7532776 TI - Growth hormone secretion and activation of cyclic AMP by growth hormone releasing hormone and gamma-aminobutyric acid in the neonatal rat pituitary. AB - The effect of factors influencing pituitary growth hormone secretion may be mediated by a combination of several intracellular mechanisms. The involvement of cyclic AMP (cAMP) in the GH stimulatory effect of tau-aminobutyric acid (GABA) and of growth hormone releasing hormone (GHRH) was studied in neonatal rat pituitaries. In the pituitaries of the newborn rats GH secretion was stimulated by forskolin and by isobutylmethylxantine (IBMX). GHRH but not GABA elevated pituitary cAMP concentration, whereas both drugs increased GH secretion from 2 day old pituitaries. IBMX did not augment the cAMP stimulating effect of GHRH in 2-day old, but potentiated it in older (7, 14 and 21-day old) pituitaries. The results indicate the presence of a functioning, but relatively immature intracellular signal transmission system in the 2-day old rat pituitary. PMID- 7532774 TI - The herbal medicine sho-saiko-to induces nitric oxide synthase in rat hepatocytes. AB - We have examined the effects of the herbal medicine sho-saiko-to (SST) on nitric oxide (NO) biosynthesis in rat hepatocytes by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN) by itself failed to induce NO synthesis (IFN: 1-1,000 u/ml). SST also did not elicit NO synthesis at concentrations up to 300 micrograms/ml when administered alone, but dose-dependently induced NO production in the presence of IFN. Whereas SST or IFN induced barely detectable levels of iNOS mRNA when administered alone, a combination of SST and IFN markedly induced iNOS mRNA in the cells. SST also modestly increased NO synthesis caused by interleukin-1 or bacterial lipopolysaccharide as a single agent, or in combination with IFN. On the other hand, SST had no effects on the NO synthesis produced by iNOS which were already induced. Thus, we found that SST stimulates cultured hepatocytes to produce NO by inducing iNOS gene expression under appropriate conditions. The capability of SST to induce NO biosynthesis might be related to the therapeutic efficacy of SST on the liver diseases. PMID- 7532775 TI - Risperidone prevents the development of supersensitivity, but not tolerance, to phencyclidine in rats treated with subacute phencyclidine. AB - We investigated whether risperidone, a 5-HT2/dopamine-D2 receptor antagonist, inhibits the development of tolerance and supersensitivity to PCP and whether subacute administration of PCP with risperidone affects the [3H]MK-801 binding in rat brain, in comparison with dopamine-D2 receptor antagonist haloperidol and 5 HT2 receptor antagonist ritanserin. In rats treated with PCP (10 mg/kg, i.p.) for 14 days, PCP (10 mg/kg, i.p.)-induced hyperlocomotion, rearing and sniffing were potentiated (supersensitivity), and head-weaving, head-twitch, backpedalling and turning were diminished (tolerance). The development of supersensitivity to PCP was blocked by oral co-administration of risperidone (2.4 mg/kg, p.o.) and haloperidol (1.0 mg/kg, p.o.) for 14 days, but not ritanserin (10 mg/kg, p.o.) and risperidone (0.8 mg/kg, p.o.), while no drugs prevented the development of tolerance to PCP. Both risperidone (2.4 mg/kg, p.o.) and haloperidol (1.0 mg/kg, p.o.) also inhibited the cross-supersensitivity to methamphetamine (MAP; 2.5 mg/kg, i.p.)-induced rearing in rats treated with PCP for 14 days. The profiles of [3H]MK-801 binding in discrete brain areas did not change after subacute administration of PCP alone or in combination with risperidone, haloperidol or ritanserin for 14 days. Therefore, it is suggested that subacute administration of PCP may cause functional changes in the dopaminergic neuronal transmission under conditions where the binding of [3H]MK-801 in discrete brain areas is unchanged, and that co-administration of risperidone may block these PCP-induced changes in neuronal function. PMID- 7532777 TI - Angiogenic activity of anterior pituitary tissue and growth hormone on the chick embryo chorio-allantoic membrane: a novel action of GH. AB - A useful system to evaluate the angiogenic activity of hormones and growth factors is the chorioallantoic membrane (CAM) of chick embryos. The present studies examined the angiogenic activity of chicken anterior pituitary glands and both fibroblast growth factor (FGF) and growth hormone (GH). Grafts of anterior pituitary gland evoked an angiogenic response on the CAM which was lost if the adenohypophyseal tissue was first boiled. The magnitude of the angiogenic response to anterior pituitary glands increased with the age of the donor (from a minimum 15 days of embryonic development to a maximum between 2 and 6 weeks old). In view of the similarity of the profile of the angiogenic response and the reported changes in GH secretion, the angiogenic activity of GH was then examined. Considerable angiogenic responses were observed with GH; there being increases (P < 0.05) in number of new blood vessels on the CAM of chick embryos on which native chicken GH or native bovine GH or recombinant bovine GH were added. These data support GH having an angiogenic action. PMID- 7532778 TI - High-calorie total parenteral nutrition reduces hepatic insulin-like growth factor-I mRNA and alters serum levels of insulin-like growth factor-binding protein-1, -3, -5, and -6 in the rat. AB - High-calorie total parenteral nutrition (TPN) is associated with hepatic dysfunction and steatosis. Because TPN-induced steatosis might compromise hepatic expression of insulin-like growth factor-I (IGF-I) and thereby limit its potential nutritional benefit, we examined hormonal and IGF-I responses in male Sprague-Dawley rats (270 to 300 g) fed by continuous intravenous infusion with high-calorie, high-dextrose (350 kcal/kg) TPN solutions for O (control), 2, 4, and 8 days. Since IGF-binding proteins (IGFBPs) are thought to modulate the biological effects of IGFs in target tissues, we also determined serum levels of IGFBPs. Animals developed hepatic steatosis after 2 to 8 days of TPN, as reflected by a sevenfold to 15-fold increase in hepatic triacylglycerol content (P < .001 v control on each day). Serum corticosterone and insulin levels were significantly higher after 2 and 4 days of TPN, whereas serum growth hormone levels were reduced after 4 and 8 days. Serum IGF-I levels were not significantly different during TPN. However, there was a coordinate reduction in the three major hepatic IGF-I transcripts (7.0, 1.9, and 1.0 kb) after 2, 4, or 8 days of TPN, and IGF-I transcripts corresponding to multiple initiation sites within exons 1 and 2 were coordinately downregulated with TPN. Western ligand blotting indicated that serum levels of 38K to 43K, 30K to 34K, and 24K IGFBPs were increased approximately twofold after 4 and 8 days of TPN as compared with control values.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532780 TI - Neuroendocrine abnormalities in human obesity. AB - Recent research has indicated that visceral obesity is associated with multiple endocrine disturbances. Insulin resistance, as well as visceral fat accumulation, may be consequences of these abnormalities. The complex endocrine aberrations are probably of central origin, and suggest a neuroendocrine background with a "hypothalamic arousal" syndrome. Such a syndrome has been found after excess alcohol intake, tobacco smoking, and certain types of stress reactions. Subjects with visceral obesity might be characterized by a high prevalence of such factors, although only indirect evidence is available for the stress component, maybe caused by a poor socioeconomic and psychosocial situation. In primate experiments, a submissive stress reaction is followed by a syndrome essentially identical to that seen in humans with visceral obesity, including visceral fat accumulation. These observations strongly support a similar chain of events in humans. Recent studies have indicated several abnormalities in cerebrospinal fluid (CSF) concentrations of catecholamines and neuropeptides. In particular, serotonin metabolites and corticotropin-releasing factor (CRF) concentrations are apparently lower than normal. In women with visceral obesity, these low concentrations are associated with food choices that indicate a preference for carbohydrates. This finding emphasizes the importance of serotonin agonists in the treatment of human obesity. It seems possible that such drugs may have effects on metabolic and other symptoms particularly prevalent in abdominal obesity, and that these effects might be independent of the decrease in energy intake. It would seem highly desirable to explore these possibilities further. Such observations may also provide a link between the abnormalities of low serotonin and CRF concentrations in the central nervous system on one hand and peripheral metabolic and other abnormalities on the other. PMID- 7532779 TI - Galanin counteracts the inhibitory effects of glucocorticoids on growth hormone secretion in the rat. AB - The aim of our study was to investigate the effect of galanin on baseline and growth hormone (GH)-releasing hormone (GHRH)-stimulated GH concentrations in conscious, freely moving rats receiving long-term glucocorticoid treatment. Animals were treated for 7 days with an intraperitoneal injection of either vehicle or dexamethasone ([dex] 40 micrograms/d). Rats underwent the following experimental trials: at -15 minutes animals received an intravenous injection of saline or galanin (12.5 micrograms/kg), and at 0 minutes rats received a second intravenous injection of saline or rat GHRH (500 ng/kg). Blood samples were drawn every 5 minutes from -15 to +15 minutes and then at 30 minutes. The GH response to saline + GHRH alone was significantly higher (P < .05) in chronically vehicle treated rats as compared with chronically dex-treated ones. In contrast, galanin + saline increased serum GH levels in a similar fashion in both chronically vehicle- and dex-treated rats. The response to galanin + GHRH was similar to galanin + saline in chronically vehicle-treated rats, but was significantly enhanced in chronically dex-treated rats. These results suggest that galanin mediated GH release in rats may involve somatostatinergic pathways. PMID- 7532782 TI - Lipofection of synthetic oligodeoxyribonucleotide having a palindromic sequence of AACGTT to murine splenocytes enhances interferon production and natural killer activity. AB - A synthetic 22-mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC-22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC-22, having an ACCGGT palindrome, did not. The binding of AAC-22 to splenocytes was not different from that of ACC-22. Lipofection of AAC-22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC-22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC-22 to the cell surface receptors, but its penetration into the spleen cells. PMID- 7532781 TI - Determination of the agretopic residues of a peptide co-restricted to different class II isotypes, I-Au and I-Eu, and its application for preparation of a synthetic peptide vaccine against influenza virus A/Aichi/2/68. AB - We have defined that residues 46 and 54 on a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58) work as agretopes (sites bound to an MHC molecule) in I-Ab mice. Substitution of amino acid residues on these positions altered the peptide to bind with the other MHC molecules. Furthermore, by substituting the agretopic residues with a variety of amino acids, we could determine the class II binding motif for each MHC molecule. In the present study, immunogenicity of a peptide, 46R50V54A, carrying valine (V) at epitopic (site bound to TCR) position 50, arginine (R) and alanine (A) at agretopic positions 46 and 54 of the p43-58, respectively has been analyzed in B10.PL (H-2u) mice. We found that this peptide bound to two different class II isotypes, I-Au and I-Eu. Arginine at position 46 or alanine at position 54 of the 46R50V54A was shown to be critical for binding to I-Au or I-Eu, respectively. Further, on the basis of this class II binding motif we could prepare potent peptide vaccines against influenza A/Aichi/2/68 virus in B10.PL mice. PMID- 7532783 TI - [Nutritional and acute phase proteins in preterm newborns: reference standards and interrelations]. AB - The serum concentrations of 5 "nutritional" and 5 "acute phase" proteins were prospectively studied in 3 groups of newborns with nephelometric methods. Group A: 22 healthy breast fed term newborns aged 4 days; group B: 28 healthy enterally fed preterm newborns (mean gestational age 33.3 weeks); group C: 49 preterm newborns (mean gestational age 29.5 weeks) on parenteral nutrition (PN). Infants with surgical procedures, sepsis and liver or renal diseases were excluded. The serum concentrations of almost all proteins were similar or only slightly different among the 3 groups and never related to the weight and chronological or post-conceptional age. Only prealbumin, apolipoprotein A and B and C4 levels were significantly different between term and preterm newborns. Enterally fed preterm infants had lower concentrations of alpha 1 acid glycoprotein and higher albumin, transferrin and apolipoprotein A than PN fed infants. Since the observed differences were usually quite small, we suggest that--at least in clinical practice--common serum reference values of these proteins should be adopted for all healthy growing newborns, whether preterm or at term, enterally or parenterally fed. The inter-relationships between different proteins were studied. Four of the five nutritional proteins were highly correlated one with another and the same was observed for the 5 acute phase proteins. Nutritional proteins as a group did not correlate with acute phase proteins, with the only exception of alpha 1 acid glycoprotein and apolipoprotein A. Thus, the 2 groups of proteins seem to be regulated by different metabolic systems. PMID- 7532784 TI - Molecular determinants of the species selectivity of neurokinin type 1 receptor antagonists. AB - Most nonpeptide neurokinin (NK)1 antagonists display a marked difference in affinity for rat versus human NK1 receptors. The molecular basis for the species selectivity of RP67580 and CP96,345 has been previously addressed [J. Biol. Chem. 267:25668-25671 (1992); J. Biol. Chem. 268:2319-2323 (1993)]. We are extending these previous results to additional NK1 antagonists, which are members of different chemical families. Included is a new perhydroisoindolol, RPR100893, which unlike its parent compound (RP67580) is human receptor selective. Chimeric rat/human NK1 receptors, as well as rat and human mutant NK1 receptors, were constructed and expressed in COS-1 cells, and affinities for substance P and the various antagonists were determined in binding studies. With human receptor selective antagonists, the rat R290(S-->I) mutation was the most effective in increasing antagonist affinity (from 7- to 23-fold). Combination with the R116(L- >V) mutation led to an additional increase in affinity for trans-4-hydroxy-1-(1H indol-3-ylcarbonyl)-L-prolyl-N- methyl-N-(phenylmethyl)-L-tyrosineamide (a derivative of FK888) and to nearly full human receptor affinity for RPR100893 and (+/-)-CP99,994. Based on the gains in affinities, these results confirm and extend the role of residues 116 and 290 of the NK1 receptor in the species selectivity of these three new human receptor-selective NK1 antagonists. In comparison, the affinity of RP67580, the least selective molecule, was most affected by changes at position 116, and combination with mutations at either position 97 (V-->E) or position 290 led to the human receptor phenotype. For the heterosteroid KAN610857, modifications of the rat receptor at positions 97 and 290, and to a lesser degree position 116, were the most effective in reducing affinity. Two double-mutants [R(97,290) and R(116,290)], although different from those identified for RP67580, also displayed human receptor-like affinity. Therefore, the molecular determinants of the species selectivity appear to be different, in part, between rat and human receptor-selective compounds, even between closely related chemical families. PMID- 7532785 TI - Rheumatoid-factor-reactive sites on CH3 established by overlapping 7-mer peptide epitope analysis. AB - Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis and six monoclonal IgM RF were isolated from monomeric IgG affinity columns and studied for their reactivity with the entire CH3 domain of IgG synthesized as overlapping 7-mers using a pin-ELISA assay. All ten polyclonal IgM RF showed similar profiles of reactivity which included peptides with solvent accessible residues PREPQVY (residues 343-349), PQVYTLP (residues 346-352), TLPPRSE (350-356), DGSFFLY (401-407), WQQGNVF (417-423), CSVMHEG (425-430), EGLHNHY (430-436) and KSLSLSP (439-446) of the CH3 domain. Substitution of a neutral glycine or alanine for each residue within these RF-reactive epitopes indicated that tyrosine at position 349, prolines at 343, 346 and 352, glutamine 347, valine 348, threonine 350, leucine 351, arginine 354, aspartic acid 401, tyrosine 407, serine 426, histidine 429, leucine 432, tyrosine 436 and lysine 439 represented important single amino acids within CH3 for RF reactivity. Regions of CH3 primary sequence with and without the single allotype-specific amino acid substitutions of glycine for alanine 431 (Gmx) or aspartic acid for glutamic acid (356) and leucine for methionine (358) (Gma) often showed considerable differences in reactivity with individual polyclonal and monoclonal RF. However, these differences in RF reactivity did not correlate with the individual anti-Gm RF specificity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a much more restricted spectrum of reactive CH3 epitopes. 7-mer peptides representing RF-reactive sites on CH3 preincubated with polyclonal IgM RF showed strong inhibition (55-66%) of RF binding to whole IgG on the ELISA plate. These studies indicate that it is possible to define portions of the IgG CH3 domain participating in the reaction with IgM RF using reactive epitope-mapping with sequential linear peptides derived from the primary IgG CH3 sequence. PMID- 7532786 TI - Inhibition of T cell activation with a humanized anti-beta 1 integrin chain mAb. AB - The murine anti-CD29 mAb K20 (Mu-K20) is known to bind to the beta 1 chain of the human integrins and to inhibit activation and proliferation of T cells, implying an important potential for in vivo immunosuppression. However, use of K20 as an immunosuppressant drug would be impaired by the immunogenicity of mouse mAbs in man. We have therefore engineered K20 into (1) a mouse/human chimeric mAb (Ch K20) that comprises the human kappa/gamma 1C regions and the K20 V regions; and (2) a humanized mAb (Hu-K20) combining the complementarity-determining regions (CDRs) of the K20 mAb with human framework (FR) and kappa/gamma 1 C regions. Both chimeric and humanized Abs were able to reproduce a range of functional properties of the original mouse mAb K20 (Mu-K20), namely, specific binding of CD29, inhibition of T cell proliferation and elevation of second messenger phosphatidic acid (PA) induced via CD3 in a soluble form, and activation of T cell proliferation in a cross-linked form. When compared to Ch-K20, the avidity of Hu-K20 was only slightly reduced. This demonstrates the feasibility of a successful humanization performed on the sole basis of the primary amino acid sequence analysis of the original mouse antibody V regions. PMID- 7532788 TI - Reconstructing the evolutionary history of the artiodactyl ribonuclease superfamily. AB - The sequences of proteins from ancient organisms can be reconstructed from the sequences of their descendants by a procedure that assumes that the descendant proteins arose from the extinct ancestor by the smallest number of independent evolutionary events ('parsimony'). The reconstructed sequences can then be prepared in the laboratory and studied. Thirteen ancient ribonucleases (RNases) have been reconstructed as intermediates in the evolution of the RNase protein family in artiodactyls (the mammal order that includes pig, camel, deer, sheep and ox). The properties of the reconstructed proteins suggest that parsimony yields plausible ancient sequences. Going back in time, a significant change in behaviour, namely a fivefold increase in catalytic activity against double stranded RNA, appears in the RNase reconstructed for the founding ancestor of the artiodactyl lineage, which lived about 40 million years ago. This corresponds to the period when ruminant digestion arose in the artiodactyls, suggests that contemporary artiodactyl digestive RNases arose from a non-digestive ancestor, and illustrates how evolutionary reconstructions can help in the understanding of physiological function within a protein family. PMID- 7532787 TI - Mutations induced by dacarbazine activated with cytochrome P-450. AB - The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots. PMID- 7532789 TI - Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor. AB - Mutational analysis of the tachykinin NK-1 (refs 1-7), NK-2 (ref. 8) and angiotensin AT-1 (refs 9, 10) receptors indicates that non-peptide antagonists act through residues located between the seven transmembrane segments, whereas natural peptide agonists bind mainly to residues scattered in the exterior part of the receptor. The presumed contact points for the prototype NK-1 antagonist CP96,345 cluster on opposing faces of the outer portions of transmembrane helices V and VI (refs 1-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-1 receptor gradually converts it into a high-affinity metal-ion-binding site without affecting agonist binding. In a double mutant with histidine residues substituted at the top of transmembrane segments V and VI, respectively, Zn2+ inhibits binding of radiolabelled agonist peptide and efficiently blocks phosphoinositol turnover induced by substance P. We propose that Zn2+ and CP96,345 act as 'allosteric competitive' antagonists by stabilizing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of helix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins. PMID- 7532790 TI - Effect of omega-conotoxin on cholinergic and tachykininergic excitatory neurotransmission to the circular muscle of the guinea-pig colon. AB - The aim of this study was to compare the stimulus-response characteristics of the cholinergic and tachykininergic excitatory transmission to the circular muscle of the guinea-pig proximal colon and their susceptibility to inhibition by the N type calcium channel blocker omega-conotoxin (CTX). All experiments were performed in the presence of guanethidine (3 microM), indomethacin (10 microM), L nitroarginine (L-NOARG, 30 microM) and apamin (0.1 microM). In the presence of the tachykinin receptor antagonists, FK 888 (10 microM0 and GR 94,800 (3 microM), to block NK1 and NK2 receptors, respectively, electrical field stimulation (EFS) produced frequency-dependent atropine- (1 microM) sensitive contractions. In the presence of atropine (1 microM), EFS produced tachykininergic contractions which were abolished by the combined administration of FK 888 (10 microM) and GR 94,800 (3 microM). The maximal responses produced by cholinergic and tachykininergic neurotransmission ranged between 80 and 100% of the maximal contractile response to 80 mM KCl. The frequency of stimulation, pulse width and voltage required to produce 50% of the maximal cholinergic and tachykininergic contraction were not different from each other, although cholinergic transmission appeared more efficient in producing twitch contractions in response to single pulse EFS. Furthermore, cholinergic transmission was more efficient than tachykininergic transmission in producing contraction in response to short periods of EFS. CTX (0.1 microM for 30 min) produced a large and comparable rightward shift of the cholinergic and tachykininergic frequency-response curve (19 and 17 fold increase in the frequency of stimulation producing 50% of the maximal response, respectively) and markedly depressed (51 and 43% inhibition, respectively) the maximal concentrations response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532792 TI - Characterization of a pan-lymphocyte monoclonal antibody useful in differentiating leukemic from normal myeloid progenitors and monocytes from macrophages. AB - A monoclonal antibody (McAb) designated 3A2 that recognizes a 51 kDa epitope having surface density of 37 x 10(8) per MOLT-4 cells is described. This epitope appears to be expressed on (i) lymphocytes at all stages of differentiation; (ii) leukemic myeloid progenitors; (iii) peripheral blood monocytes (MO). The epitope is specifically absent from normal myeloid progenitors and macrophages. The McAb may, therefore, be useful in studying myeloid lineage leukemias and, as a marker for monocyte to macrophage (MO + MAC) differentiation. PMID- 7532791 TI - Nitric oxide induces acetylcholine-mediated contractions in the guinea-pig small intestine. AB - In longitudinal muscle/myenteric plexus preparations of the guinea-pig ileum, exogenous nitric oxide (NO) induced concentration-dependent relaxations. In tissues at basal tone, NO (3 x 10(-6) M) induced a moderate relaxation followed by a pronounced contraction, consisting of a quick and sustained component. Tetrodotoxin (5 x 10(-7) M) abolished both phases of the contraction. Atropine (5 x 10(-7) M) abolished the quick component and reduced the sustained component of the contraction; the latter was further suppressed by the selective NK1 receptor antagonist CP 96,345. Hexamethonium (5 x 10(-5) M) failed to affect the contractile response to NO. It is concluded that administration of exogenous NO in the guinea-pig ileum can lead to activation cholinergic and to a lesser degree tachykininergic neurones. PMID- 7532793 TI - Differential effects of insulin-like growth factor I and platelet-derived growth factor on growth response, matrix formation, and cytosolic free calcium of glomerular mesangial cells of spontaneously hypertensive and normotensive rats. AB - In this study, we compared the cellular functions of cultured glomerular mesangial cells (MC) from spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto rats (WKY) in response to the growth factors insulin like growth factor I (IGF-I) and platelet-derived growth factor (PDGF). IGF-I and PDGF at a concentration above 2 ng/ml and a combination of both tested growth factors exerted a highly elevated growth response of SHR MC versus WKY MC. The total RNA synthesis induced by IGF-I and PDGF was increased in SHR MC as compared with WKY MC, while the overall protein synthesis showed no differences between both strains. Analysis of cell-associated fibronectin accumulation and incorporation of proline into collagenous proteins revealed an enhanced basal and PDGF-stimulated matrix formation of SHR MC which was not dependent on the increased production of autocrine matrix-stimulatory mediators by SHR MC. Changes of cytosolic free calcium - [Ca2+]i - could not be correlated with the enhanced responsiveness of SHR MC to the tested growth factors. The described differences of cellular functions between SHR and WKY MC may contribute to pronounced glomerular alterations such as glomerulosclerosis seen in primary and secondary forms of hypertension. PMID- 7532794 TI - Abnormalities of coagulation in experimental nephrotic syndrome. AB - The human nephrotic syndrome is accompanied by important alterations of the coagulation system related proteins. The purpose of the present study was to examine the activity of coagulation- and fibrinolysis-related proteins in plasma and urine of control and puromycin aminonucleoside injected rats on days 2 (prenephrotic stage) and 10 (nephrotic stage). We measured the prothrombin time (PT), the activated partial thromboplastin time (aPTT), and the activities of (1) the coagulation factors (CFs) I, II, V, and VII-XII; (2) the inhibitor of coagulation antithrombin III (ATIII), and (3) the component of the fibrinolytic system alpha 2-antiplasmin (alpha 2-APL). PT and aPTT and the activities of CF, ATIII, and alpha 2-APL were not measurable in the urine of control and puromycin amino-nucleoside injected rats on day 2. On this same day, plasma ATIII and CF VIII decreased. On day 10 (1) PT and aPTT decreased in plasma and were not measurable in urine; (2), plasma CFs I, II, V, VII, VIII, X, and XI increased; (3), plasma ATIII decreased; (4), plasma CFs IX and XII and alpha 2-APL did not change, and (5) ATIII and CFs II, VII, VIII, IX, X, XI, and XII, but not CFs I and V and alpha 2-APL, appeared in urine on day 10. ATIII deficiency was secondary probably to the urinary losses; however, the plasma activity of CFs II, VII, VIII, X, and XI increased and that of CFs IX and XII remained unchanged in spite of their urinary losses which suggests that other mechanisms such as deranged catabolism and altered hepatic synthesis may be involved.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532796 TI - Liver histology in hepatitis B and C co-infection on hemodialysis patients. PMID- 7532795 TI - Hepatitis C viraemia is spread by dialysis--the need for an isolation policy. PMID- 7532798 TI - Effect of hypoxia on proliferation of microvascular endothelial cells derived from Mongolian gerbil brain. AB - This study investigated the effect of hypoxia on the proliferation of microvascular endothelial cells in the brain. Endothelial cells derived from microvessels of Mongolian gerbil brain and control cells (3T3) were incubated for 7 days under various hypoxic and normoxic conditions at 37 degrees C. The proliferation of endothelial cells was inhibited by severe hypoxia, but stimulated by moderate hypoxia. No significant effect of hypoxia on the proliferation of control cells was observed. Endothelial cell-conditioned medium cultured under moderate hypoxia did not affect the growth of endothelial cells. The stimulatory effect of moderate hypoxia on the proliferation of cultured endothelial cells is due to an inherent property of brain endothelial cells. PMID- 7532797 TI - Prevalence of HCV-RNA-positive patients in a dialysis unit in Germany. PMID- 7532799 TI - Phosphorylethanolamine content of human brain tumors. AB - Phosphorylethanolamine (PEA) is the major component of the phosphomonoester peak detected by phosphorus-31 magnetic resonance spectroscopy, but the absolute concentration has not been determined. This study measured the PEA concentration in biopsy specimens of brain tumors and lobectomized cerebral cortex using high performance liquid chromatography. The concentration of PEA was 118.5 +/- 10.0 mumol/100 g wet wt in cortex, and was significantly higher in malignant gliomas, metastatic pulmonary adenocarcinoma, and neurinoma. The concentration of PEA was especially high in pituitary adenoma, malignant lymphoma, and medulloblastoma. PMID- 7532800 TI - Emerging symptoms of growing skull fracture after secondary trauma in an adult- case report. AB - A 20-year-old female presented with dysphasia and slight hemiparesis following a head trauma, who had a non-treated growing skull fracture which had remained asymptomatic for about 18 years, despite an encephalocele in the left parietal region. Neuroimaging demonstrated secondary brain damage and herniated brain resulting in gliosis. Electroencephalography revealed epileptic discharge in the affected region. Dural repair and cranioplasty resolved her neurological deficits. Early corrective surgery should be performed for growing skull fracture to prevent secondary brain damage. PMID- 7532801 TI - Traumatic intracranial foreign body embolization--case report. AB - A 27-year-old male presented with intracranial embolization due to accidental penetration of his neck by an iron fragment at work. Cerebral angiography revealed foreign body embolization of the left middle cerebral artery. The foreign body was successfully removed via craniotomy and arteriotomy. The extended period from onset to surgical treatment prevented acute hemodynamic reconstruction. However, he had good collateral circulation and was discharged with mild hemiparesis and moderate motor aphasia. The possibility of intracranial embolization should be considered in patients presenting with penetrating injury of the neck. PMID- 7532802 TI - Pleomorphic adenoma of the lacrimal gland manifesting as exophthalmos in adolescence--case report. AB - A 16-year-old girl presented with a pleomorphic adenoma of the lacrimal gland manifesting as left painless exophthalmos which had persisted for 3 years. Computed tomography revealed a tumor about 15 mm in diameter in the superolateral site of the left orbit. The tumor was removed completely by combined orbitofrontal craniotomy through a transcranial approach. Histological examination demonstrated the growth of tumor cells as glandular cavities or sheets, with myxoid and partly chondroid connective tissue stroma. Pleomorphic adenoma of the lacrimal gland is unusual in adolescents. PMID- 7532803 TI - Malignant trigeminal schwannoma associated with xeroderma pigmentosum--case report. AB - A 46-year-old male with xeroderma pigmentosum developed an intracranial malignant schwannoma originating from the second branch of the left trigeminal nerve. The tumor was subtotally removed and postoperative radiation therapy given, but the tumor recurred twice over 3 years, and extended to the third branch of the left trigeminal nerve and the ipsilateral facial nerve. Radical surgery and radiation therapy finally achieved a cure. This is the first case of malignant trigeminal schwannoma with xeroderma pigmentosum, although various other internal neoplasms including central nervous system tumors have been reported in xeroderma pigmentosum patients. Radical surgery and radiation therapy are effective for treating intracranial malignant schwannoma. Radiation therapy is considered safe for xeroderma pigmentosum patients. PMID- 7532804 TI - Multiple cerebral aneurysms associated with aortitis syndrome--case report. AB - A 48-year-old female presented with four aneurysms in the anterior half of the circle of Willis associated with aortitis syndrome. All the aneurysms were successfully clipped. In general, intracranial hemodynamic change, due to stenosis or occlusion of carotid arteries, is considered to initiate aneurysm growth in the vertebrobasilar system in this syndrome, but renal hypertension was probably involved in our patient. Careful screening for multiple aneurysms, even in the anterior circulation, should be undertaken in patients with aortitis syndrome who present with an aneurysm. PMID- 7532805 TI - Multiple cerebral vascular malformations and spontaneous regression--case report. AB - A female infant manifested a rare case of spontaneous regression of a vascular malformation in the occipital lobe after removal of another arteriovenous malformation in the frontal lobe. She was born with multiple nevi on the face, body, and upper and lower extremities. She demonstrated developmental retardation at 8 months of age. Computed tomography at 11 months of age demonstrated ventricular enlargement and a mass in the subdural portion of the left anterior fossa. Magnetic resonance images demonstrated signal void signs in the left frontal lobe, which suggested vascular malformation. Cerebral angiograms disclosed two vascular malformations. The malformation in the frontal lobe was totally removed. Cerebral angiograms 25 days after the operation failed to demonstrate either vascular malformation previously observed. Hemodynamic change following the removal of the arteriovenous malformation may have contributed to the occlusion of the remaining malformation. PMID- 7532806 TI - Giant intracranial aneurysm with rapid thrombus formation and intramural hemorrhage--case report. AB - A 50-year-old female presented with an unusual giant intracranial aneurysm that showed rapid, spontaneous thrombus formation and subsequent intramural hemorrhage. The thrombus appeared as a homogeneous area on magnetic resonance images, in contrast to the usual heterogeneous appearance. Two months after thrombus formation, the aneurysm had grown and developed intramural hemorrhage. The growth of giant intracranial aneurysms is related to neovascularization and recurrent intramural hemorrhage. The rapid formation of an intra-aneurysmal thrombus may stimulate neovascularization, resulting in intramural hemorrhage and aneurysmal growth. PMID- 7532808 TI - Neuroprotective effect of acidic fibroblast growth factor on seizure-associated brain damage. AB - The neuroprotective effect of acidic fibroblast growth factor (aFGF) has been analysed in a rat model of seizures-associated brain damage. We report that after treatment with a convulsivant dose of Kainic acid, systemically administered aFGF prevents neuronal degeneration in specific brain areas, mainly in the hippocampal formation. Our findings extend the potential pharmacological use of fibroblast growth factors and afford new data to understand the neurophysiology of these proteins. PMID- 7532807 TI - Assessment of the brain areas perfused by superselective intra-arterial chemotherapy using single photon emission computed tomography with technetium-99m hexamethyl-propyleneamine oxime--technical note. AB - The brain areas perfused by superselective intra-arterial (i.a.) chemotherapy were assessed using single photon emission computed tomography (SPECT) with technetium-99m-hexamethyl-propyleneamine oxime (99mTc-HMPAO). A superselective catheter was introduced into the anterior, middle, or posterior cerebral artery of patients with malignant glioma for i.a. chemotherapy. 99mTc-HMPAO was subsequently injected via the same catheter used for chemotherapy, and a higher dose of 99mTc-HMPAO was injected intravenously to obtain adequate background brain images. Comparison of the SPECT images with magnetic resonance images could confirm complete perfusion of the tumor tissue. In two patients with malignant glioma, regions of interest were selected in the peritumoral brain area and a reference brain area, and the radioactivity was measured. The concentration of 99mTc-HMPAO was about 50 times higher in tissue perfused by superselective injection into anterior or middle cerebral artery compared to intravenous injection. 99mTc-HMPAO SPECT is readily available in many institutions and the information provided is useful for planning more effective and safe i.a. chemotherapy. PMID- 7532809 TI - Plasticity of NO synthase expression in the nervous and endocrine systems. AB - Using immunohistochemistry and in situ hybridization the effect of nerve injury and of hormones was analysed in sensory and hypothalamic systems and in the pituitary gland. After peripheral axotomy a marked increase in NOS protein and mRNA levels was observed in dorsal root ganglia, the trigeminal ganglion and a less dramatic effect in the nodose ganglia. This effect lasted in the dorsal root ganglion neurons for at least 10 weeks. In the hypothalamic magnocellular neurons a transient increase was observed in the paraventricular and supraoptic nuclei. A similar effect was also seen after salt loading. In the anterior pituitary gland NOS was expressed in gonadotrophs and folliculo-stellate cells. Castration markedly increased NOS levels in the anterior lobe, and this could be counteracted by steroid hormone replacement. Thus, the present results show that the constitutive, neuronal NOS can be dramatically regulated in response to various manipulations, suggesting an important involvement of NO in these situations. PMID- 7532810 TI - Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity. AB - We have examined the regulation of neuronal nitric oxide synthase (NOS) by phosphorylation with cyclic-GMP (PKG) and cyclic-AMP-dependent (PKA) protein kinases. In vitro phosphorylation studies indicate that both PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid analysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphorylation of purified NOS by either PKG or PKA diminishes catalytic activity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected with the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8 bromo-cGMP or dibutyryl-cAMP reduces nitrite release in response to stimulation with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals. PMID- 7532811 TI - Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole. AB - 7-Nitro-indazole (7-NI) has been described as novel nitric oxide synthase (NOS) inhibitor with in vivo selectivity for the neuronal isozyme [Moore et al. Br. J. Phaarmac. 110, 219-224 (1993)]. In the present study we have used purified porcine brain NOS to investigate the molecular mechanisms of enzyme inhibition by 7-NI. The drug was competitive with L-arginine, exhibited a kinetic KI of 2.8 microM, and additionally induced a slight reduction in Vmax. As a cytochrome P 450, NOS catalyzes a heme-mediated reduction of molecular oxygen, resulting in the formation of H2O2 in the absence of L-arginine. 7-NI turned out as a potent inhibitor of H2O2 formation (IC50 = 0.28 +/- 0.096 microM) but did not affect flavin-mediated electron transfer. Thus, 7-NI resembled imidazole, a known heme site inhibitor of NOS. We found that imidazole was a purely competitive inhibitor of L-citrulline formation (KI = 263 microM) and blocked H2O2 formation at similar concentrations (IC50 = 280 +/- 38 microM). In accordance with their L-arginine competitive effects in the citrulline assay, both drugs antagonized binding of radiolabeled NG-nitro-L-arginine (L-NNA), a high affinity probe for reversible labelling of the substrate site of NOS [Klatt et al., J. Biol. Chem. 269, 14781 14787 (1994)]. The calculated KI values for 7-NI and imidazole were 0.09 +/- 0.024 microM and 200 +/- 63 microM, respectively. Finally, binding of radiolabelled tetrahydrobiopterin, a NOS cofactor with unknown function, was also antagonized by 7-NI with a KI of 0.12 +/- 0.023 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532812 TI - Structure-activity relationships of arginine analogues on nitric oxide synthase activity in the rat brain. AB - Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from L-arginine (Arg) which has a guanidino group in its molecule. We examined the effect of 23 different Arg analogues on NOS activity in the rat brain. Though homoarginine, epsilon-guanidinocaproic acid and canavanine act as substrates of NOS, production of NO from them was lower than that from Arg. alpha-Guanidinoglutaric acid (2 GGA) and arcaine inhibited NOS activity at levels equal to NG-monomethyl-L arginine (MeArg), a well known NOS inhibitor. Though almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, the guanidino nitrogens of arcaine and 2-GGA were not substituted. Furthermore, 2-GGA is a known endogenous convulsant in mammals, and arcaine, which was isolated from a marine mollusc, is also a convulsive substance. Hence, 2-GGA and arcaine will be excellent drugs to investigate not only the chemical nature of NOS but also the physiologic function of NO. PMID- 7532813 TI - Blockade of nitric oxide synthesis by tyrosine kinase inhibitors in neurones. AB - In striatal neurones in culture, N-methyl-D-aspartate-(NMDA), kainate-(Kai) and K(+)-dependent cGMP production is entirely mediated via nitric oxide (NO). Low concentrations of lavendustin-A (< or = 0.3 microM), a highly specific tyrosine kinase inhibitor, reduced irreversibly and in a time-dependent manner NMDA stimulated cGMP production. After a preincubation period of 20 min with lavendustin-A (0.3 microM), the inhibition of NMDA-induced cGMP production was equal to 56 +/- 8% (n = 6). After the same preincubation period, the IC50 of the lavendustin-A blockade was 30 +/- 15 nM. Genistein, another tyrosine kinase inhibitor also inhibited NMDA-dependent cGMP production with high potencies (< or = 3 microM). Whatever the tyrosine kinase inhibitor tested, the basal cGMP production remained unaffected. Kai-, K(+)-, and ionomycin-induced cGMP production was also inhibited by lavendustin-A, and genistein. In contrast, tyrosine kinase inhibitors were unable to block NO donor-induced cGMP production. Using patch clamp experiments, we have also found that lavendustin-A (0.3-1 microM), the most potent tyrosine kinase inhibitor used, (a) did not reduce the NMDA receptor-mediated current, (b) only slighly affected Kai receptor-mediated current (16.4 +/- 3.4% inhibition) and (c) had a marked effect on voltage sensitive Ca2+ channel- (VSCC) mediated currents (44.4 +/- 4.9% inhibition). A reduction in VSCC activity certainly explains the inhibition of K(+)-, Kai- and possibly part of the NMDA-induced cGMP production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532815 TI - NOS-containing neurons in the rat gut and coeliac ganglia. AB - Nitric oxide (NO) is considered an important inhibitory neurotransmitter in the gut. Nitric oxide synthase (NOS)-containing neurons were visualized by immunocytochemistry using antibodies against neuronal NOS in the oesophagus, the gastrointestinal tract and the coeliac ganglion of rat. NOS-containing nerve cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut. Synthesis of NOS in enteric nerve cell bodies was confirmed by in situ hybridization, demonstrating the presence of NOS mRNA. Varicose nerve fibers formed extensive networks in the circular smooth muscle and the myenteric ganglia. The pyloric sphincter contained abundant NOS-containing nerve fibers. NOS-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals occurred in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. The neuronal cell bodies in the coeliac ganglion were non-immunoreactive but frequently surrounded by baskets of NOS-immunoreactive nerve fibers. Double staining for NOS and neuropeptides in oesophagus, stomach and small and large intestine revealed that a small subpopulation of the NOS-containing nerve cell bodies stored in addition vasoactive intestinal peptide (VIP), and in oesophagus, stomach and small intestine also neuropeptide Y (NPY). However, NOS-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained VIP throughout the gut; in the oesophagus, stomach and the small intestine they contained also NPY.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532816 TI - Involvement of endogenous nitric oxide in non-adrenergic, non-cholinergic contraction elicited by [Met5]-enkephalin in rat isolated duodenum. AB - To investigate the possible neuromodulatory role of nitric oxide (NO) in the gastrointestinal tract, an examination was made of the effects of NG-nitro-L arginine (L-NOARG), an inhibitor of NO synthase, on the intestinal response to [Met5]-enkephalin (ENK) by recording the mechanical activity of the isolated duodenum from rats. [Met5]-enkephalin elicited a biphasic response of the duodenum, i.e. transient relaxation followed by contraction. The relaxation induced by ENK was blocked by naloxone, an opioid receptor antagonist, but not by tetrodotoxin (TTX). The contractile response of the duodenum to ENK was blocked by TTX but not by naloxone. The contractile response was not affected by hyoscine, a muscarinic antagonist, or guanethidine, an adrenergic neuron blocking agent, indicating mediation by non-adrenergic, non-cholinergic (NANC) nerves. The contractile but not the relaxant response to ENK was blocked by L- but not D NOARG. The contractile response was also inhibited by methylene blue, an inhibitor of both NO synthase and guanylate cyclase, and by indomethacin, a cyclooxygenase inhibitor. Thus, endogenous NO and prostaglandins are involved in the contractile response to ENK. Endogenous NO may modulate the release of excitatory NANC transmitters via a prejunctional mechanism. PMID- 7532814 TI - Expression of cyclic nucleotide-gated cation channels in non-sensory tissues and cells. AB - Using a combination of PCR based cloning and Northern blot analysis we have investigated the tissue expression of cyclic nucleotide-gated (CNG) cation channels in a variety of non-excitable tissues and cells. Partial sequences of all three known CNG channels and of an auxiliary subunit of the rod photoreceptor channel were determined. The expression of CNG channel genes is both tissue and species specific. Southern blot analysis with human genomic DNA revealed specific patterns of hybridization with probes derived from the different CNG channel types indicating that they are encoded by distinct genes. Analysis of human genomic sequences showed that all three genes are derived from a common ancestral gene and might have a similar gene structure. We were not able to identify additional genes encoding CNG channels. The CNG3 channel, which was originally cloned from bovine kidney may be expressed also in bovine cone photoreceptor cells. These data suggest that some of the effects of cGMP in peripheral tissues and cells might involve the activation of CNG channels. PMID- 7532818 TI - Diverse roles for nitric oxide in synaptic signalling after activation of NMDA release-regulating receptors. AB - NMDA receptors regulating transmitter release were studied in three model systems to investigate whether their activation involves the NO transduction system. In superfused slices of rat brain, the release of [3H]D-aspartate, [3H]noradrenaline and [3H]GABA evoked by NMDA could be modulated by nitrergic drugs. Tetrodotoxin (0.1 microM) exerted differential effects in the three systems indicative of the NMDA receptors (and hence sites of NO generation) being pre- or extra-synaptic, or a combination of both types of localization. L-Arginine (100 microM) enhanced NMDA-evoked release of [3H]GABA (110%), [3H]NA (120%) and [3H]D-ASP (700%). Exogenous NO donors could increase NMDA-induced release of [3H]NA and [3H]D-ASP from hippocampal slices, although differential effects were noted, whilst inhibitors of NO synthase (NG-nitro- and NG-amino-L-arginine, both 100 microM) attenuated (60-85%) the release. NMDA-evoked release of [3H]GABA from striatal slices were insensitive to exogenous NO donors, but NG-nitro- and NG-amino-L arginine produced 100% increases. In all cases, the NMDA receptors regulating release are linked to a NO system, although the link to the receptors modulating release of [3H]GABA appeared different. The actions of the nitrergic drugs may depend upon the redox state and/or cellular milieu of the individual NMDA receptors involved. PMID- 7532820 TI - Intracerebroventricular injection of a nitric oxide synthase inhibitor does not affect long-term slope potentiation in vivo. AB - Although there is evidence from in vitro studies to suggest that NO synthesis may be involved in the induction of hippocampal LTP, other in vitro studies and experiments conducted in vivo have provided conflicting results. In agreement with previous work conducted in this laboratory using an i.p. route of administration, this paper reports that i.c.v. injections of the NO synthase inhibitor, N omego-nitro-L-arginine methyl ester (L-NAME), at a dose sufficient to inhibit hippocampal NO synthase by 90-95%, failed to block the induction of LTP in the dentate gyrus in vivo (as measured by the change in the slope of the early rising phase of the field EPSP). The failure to block LTP occurred following both a strong and a weak tetanus. L-NAME injections did, however, result in a small but transient increase in the baseline slope of the field EPSP, a more prolonged enhancement of the baseline population spike, and a significant attenuation of spike potentiation induced by a strong tetanus. These results offer no support for the hypothesis that NO synthase is required for the induction of the synaptic component of LTP, but do suggest a role for NO in the control of cell excitability in the hippocampus. PMID- 7532819 TI - Differential effects of nitric oxide gas and nitric oxide donors on depolarization-induced release of [3H]norepinephrine from rat hippocampal slices. AB - The NO-generating compounds sodium nitroprusside (NP), nitroglycerin (NTG), and isosorbide dinitrate (ISDN) all significantly inhibited N-methyl-D-aspartate (NMDA)-stimulated release of tritiated norepinephrine ([3H]NA) from preloaded hippocampal slices of adult male Sprague-Dawley rats with IC50's of 114 microM, 1.2 mM, and 1.7 mM respectively. NTG and ISDN also inhibited KCl-stimulated release, while NP had no significant effect on KCl-stimulated release. Although these results suggest that the inhibitory effects of these compounds were mediated by release of NO, NTG and ISDN did not generate detectable levels of NO, and iron-cyanide complexes similar in structure to NP but lacking NO also inhibited release. In contrast, both S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and authentic NO gas significantly enhanced NMDA-stimulated release of [3H]NA (EC50's: 331 and 3.4 microM respectively). This enhancement was not selective for NMDA-stimulated release, since both SNAP and NO potentiated KCl-stimulated release as well. In addition, NO gas significantly enhanced NMDA-stimulated release of tritiated dopamine ([3H]DA) from striatal slices and [3H]NA from cortical and cerebellar slices. Analogs of cyclic guanosine monophosphate (cGMP) had no significant effect on NMDA-stimulated transmitter release, suggesting that the observed increase in release is via a cGMP-independent mechanism. While exogenous NO enhanced both NMDA- and KCl-stimulated neurotransmitter release, it appears that endogenous NO does not play a role in this depolarization-induced release since NO synthase inhibitors did not significantly reduce NMDA-stimulated [3H]NA release. The possibility remains that endogenous NO could modulate neurotransmitter release in other circumstances. PMID- 7532817 TI - NMDA-dependent nitric oxide release in the hippocampus in vivo: interactions with noradrenaline. AB - Nitric oxide appears to act as a novel intercellular messenger activating soluble guanylyl cyclase to cause an increase in cGMP in target cells. In the nervous system, NMDA receptor activation has often been shown to result in activation of NO synthase, and many of the actions of NMDA are thought to be mediated by NO. We have recently combined intracerebral microdialysis with a sensitive assay for the NO oxidation products nitrite and nitrate, to assess NO release directly in awake, freely-moving animals. In the present study, we have applied this method to the hippocampus, where we have found that local NMDA application increases NO release. This was prevented by prior administration of an NMDA receptor antagonist or a nitric oxide synthase inhibitor. These pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there may be a tonic glutamate-induced NO production in the hippocampus in awake, freely moving animals. Previous work on NMDA-induced increases in cGMP suggested a possible role for noradrenaline. We found that pretreatment with the alpha 1A antagonists WB4101 or 5-methyl-urapidil, reduced the basal levels of NO oxidation products, however, NMDA still induced an increase in extracellular NO in the presence of these alpha 1A antagonists. In contrast, prior lesion of the central noradrenergic system with DSP4 prevented the increase in hippocampal NO release seen following local NMDA application. These results indicate that in vivo microdialysis can be used to monitor NO release in the hippocampus in response to NMDA receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532821 TI - Cellular locus of the nitric oxide-synthase involved in cerebellar long-term depression induced by high external potassium concentration. AB - The cellular location of the NO-synthase involved in long-term depression (LTD) of parallel fiber (PF)-mediated EPSCs induced by raising the external potassium (K) concentration has been investigated by using both whole-cell patch-clamp recordings (WCR) of Purkinje cells (PCs) in thin slices in vitro, and reverse transcription followed by polymerase chain reaction (PCR) applied to mRNAs harvested from these single PCs during WCR. In all tested cells in the control group, a large LTD of PF-mediated EPSCs was induced by perfusing the slices for 3 min with a high (30 mM) K perfusing medium. In a second group of cells for which the protein kinase C (PKC) inhibitor peptide 19-36 was added to the intrapipette solution at a concentration of 10 microM, the LTD following complete wash out of the high K solution was significantly less prominent than in the control group. Very similar results were also obtained when 30 microM NG-methyl-L-arginine (L NMMA) was added to the perfusing medium. In contrast, when both the PKC inhibitor peptide 19-36 and L-NMMA were added to the intrapipette solution at a concentration of 10 and 30 microM respectively, no LTD was revealed following wash out of the high K solution. Finally, the PCR amplification of mRNAs harvested from these single PCs during WCR, as well as from granule cells from the same slices, confirms that mRNAs encoding the NO-synthase are expressed by granule cells, whereas they are not detected in PCs. PMID- 7532822 TI - Inhibition of nitric oxide synthase alters light responses and dark voltage of amphibian photoreceptors. AB - We studied the effects of competitive inhibitors of nitric oxide synthase (L-NMMA and L-NNA) on dark voltage and flash responses of retinal rods of the frog. Substances were applied intracellularly via whole-cell patch-clamp electrodes while the membrane voltage was recorded simultaneously. During recording the exchange of substances by diffusion between cytosol and pipette medium affects the cell's function. Under control conditions this exchange is reflected by a slow hyperpolarization of the dark voltage with time and a prolongated flash response recovery, which is mainly due to a loss of nucleotides. Application of L NMMA and L-NNA accelerated the spontaneous hyperpolarization of the membrane voltage during the course of an experiment, while the recovery of the flash responses was slowed down. The effects observed upon intracellular application of NO-synthase inhibitors were opposite to those observed previously upon application of sodium nitroprusside. Sodium nitroprusside was much less effective when the intracellular calcium level was decreased by application of EGTA at the same time. It is reasonable to assume that the observed effects are linked to nitric oxide synthase and to a NO-dependent soluble guanylate cyclase. The results suggest that the activity of NO-synthase in photoreceptor cells has an influence on concentration and metabolic flux of cGMP in photoreceptors, which may be of relevance for flash response recovery and adaptation processes. It is likely that the regulation of the soluble guanylate cyclase requires a physiological level of calcium. PMID- 7532823 TI - The role of nitric oxide in the transformation of visual information within the dorsal lateral geniculate nucleus of the cat. AB - We have shown that application of an inhibitor of the enzyme nitric oxide synthase (NOS) effectively suppresses the visual responses of relay cells in the dorsal lateral geniculate nucleus (dLGN) of the anaesthetized paralysed cat. Such suppression seems to result from a specific reduction in transmission via N methyl-D-aspartic acid (NMDA) receptors, since iontophoretic application of the inhibitor of NOS selectively and in a dose-dependent manner decreased the responses to exogenously applied NMDA. Responses to other exogenously applied amino acid agonists, such as quisqualate (Quis), kainate (Kain) and alpha-amino-3 hydroxy-5-5-methyl-4-isoxazole-propionic acid (AMPA) were largely unaffected. Furthermore, the excitatory action of acetylcholine (ACh), normally co-localized with NOS in axonal terminals within the dLGN arising from the brainstem, was also unaffected. Unlike some other actions of nitric oxide (NO), this role seems not to involve an increase in production of cyclic guanosine-3',5'-mono-phosphate (cGMP), since application of the membrane permeable cGMP analogue 8-bromo-cGMP did not alter the suppressive effect of NOS inhibitors on either visual or NMDA evoked responses. We conclude that the normal function of NO at this level of the visual system is permissive, allowing full expression of NMDA mediated visually elicited information. PMID- 7532824 TI - Modulation of inducible nitric oxide synthase expression in astroglial cells. AB - Nitric oxide is produced in the CNS by both constitutive and inducible isoforms of nitric oxide synthase. Once nitric oxide synthase is transcriptionally induced in astrocytes in vitro, these cells release large amounts of nitric oxide tonically. Glial cell-derived nitric oxide can be toxic to neurons and oligodendrocytes and is implicated in a variety of neuropathologies, suggesting that the expression of nitric oxide synthase in glia must be finely regulated. From northern and western blot analysis we have identified various agents (transforming growth factor-beta, nitric oxide, receptor agonists) that modulate cytokine-induced expression of nitric oxide synthase mRNA in astrocytes. This suggests that the magnitude and duration of nitric oxide production from activated astrocytes in vivo may be determined by signals from adjacent neurons and microglial cells. PMID- 7532825 TI - Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. AB - Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity. PMID- 7532826 TI - Nitric oxide does not mediate acute glutamate neurotoxicity, nor is it neuroprotective, in rat brain slices. AB - Nitric oxide (NO), generated upon glutamate receptor activation, elicits cyclic GMP accumulation through stimulation of guanylyl cyclase. NO is also a potential cytotoxin that has been suggested, on the basis of tissue culture experiments, to mediate neuronal damage associated with excessive activity of the N-methyl-D aspartate (NMDA) subtype of glutamate receptor. We have investigated the involvement of NO in the toxicity of glutamate receptor agonists in brain slice preparations. Slices of cerebellum and hippocampus from the developing rat exhibited neuronal necrosis following exposure (5-30 min) to NMDA (100 microM or 1 mM). When the exposures were carried out in the presence of NO synthase inhibitors, at concentrations suppressing NMDA-induced NO formation (as judged by measurements of cyclic GMP accumulation), the extent of injury was unaffected. To determine if exogenous NO is able to replicate NMDA toxicity, the slices were exposed to high concentrations of NO donating compounds for up to 2 hr. No damage was detectable. NO donors, moreover, neither reduced NMDA toxicity, nor potentiated the degeneration caused by just suprathreshold NMDA concentrations. The toxicities of non-NMDA agonists, or of glutamate itself, were also unaltered by NO synthase inhibitors or NO donors. Similar results were obtained using hippocampal slices from more mature animals. We conclude that the acute neurodegeneration mediated by NMDA or non-NMDA receptors in the slice preparations is not mediated by NO, nor is NO neuroprotective under these conditions. PMID- 7532827 TI - Potentiation of NMDA-mediated toxicity on nigrostriatal neurons by a low dose of 7-nitro indazole. AB - Recent evidence suggests that an excitotoxic mechanism may play a role in the etiology of Parkinson's disease. Previously, we have shown that the nigrostriatal dopaminergic neurons are sensitive to focal infusions of an N-methyl-D-aspartate (NMDA) receptor agonist; this toxicity was potentiated by systemic administration of the nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester. The present investigation was undertaken to assess the role of the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI) on the neurotoxicity elicited by NMDA receptor activation in vivo. Single injections of 7-NI (0-125 mg/kg, i.p.) into male Sprague-Dawley rats resulted in a dose-dependent decrease in both nigral and cerebellar NOS activity measured 30 min post-injection. Maximal NOS inhibition was obtained with 20 mg/kg 7-NI (nigra: 90.2 +/- 3.7%, cerebellum: 86.7 +/- 6.3%). In addition, it was found that 7-NI (80 mg/kg, i.p.) did not cause an increase in mean arterial blood pressure over a 48 hr period. Vehicle pretreatment of animals prior to stereotaxic infusion of NMDA (15 nmol) into the substantia nigra compacta resulted in a 56.1 +/- 5.1% decrease in striatal tyrosine hydroxylase (TH) activity from the contralateral side. Pretreatment with 7-NI (5 and 80 mg/kg) produced a 76.9 +/- 3.2% and 49.8 +/- 5.6% decrease, respectively, in striatal TH activity. Thus, a significant increase in NMDA toxicity was observed at the lower but not higher dose of 7-NI. It was also observed that 7-NI (20 and 80 mg/kg) produced a decrease in locomotor activity over a 2 hr period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532828 TI - Dual role of nitric oxide in focal cerebral ischemia. AB - The importance of nitric oxide (NO) in the pathophysiology of cerebral ischemia was examined following middle cerebral artery occlusion in rats. A significant increase in infarct size developed following inhibition of NO synthase (NOS) activity by L-arginine analogues whereas intravenous L-arginine dose-dependently decreased infarct volume in the same models. Protection after L-arginine administration was associated with enhanced blood flow within the perinfarct zone as demonstrated by simultaneous recording of rCBF and electrocorticogram activity within subjacent brain. Selective NOS inhibition by 7-nitroindazole (7-NI) significantly reduced infarct volume at doses of 25 and 50 mg kg and in amounts that did not decrease the response of pial vessels to topical acetylcholine. Together these data suggest that enhanced NO production within the cerebrovasculature protects brain tissue during focal ischemia via hemodynamic mechanisms whereas neuronal overproduction may facilitate or mediate neurotoxicity. Recent data using transgenic animals lacking NOS activity support the latter conclusion. PMID- 7532829 TI - Nitric oxide and adenosine mediate vasodilation during functional activation in cerebellar cortex. AB - Activation of the cerebellar parallel fibers (PF) releases glutamate and leads to depolarization of Purkinje cells and interneurons. These cells, in turn, release GABA. We have studied the role of glutamate, GABA, nitric oxide (NO) and adenosine in the increases in cerebellar cortex blood flow (BFcrb) elicited by PF stimulation. In anesthetized rats (halothane 1%) the cerebellar vermis was exposed and the site was superfused with Ringer (37 degrees C, pH 7.4). The PF were stimulated electrically (50-100 microA; 30 Hz) and the increases in BFcrb were recorded using a laser-Doppler flowmeter. Field potentials were recorded using glass microelectrodes. During Ringer superfusion, PF stimulation increased BFcrb by 58 +/- 5% (P < 0.001; analysis of variance; n = 6). Superfusion with the broad spectrum glutamate receptor antagonist kynurenic acid (Kyn; 5 mM) abolished the negative component of the field potential (n = 4), a finding reflecting lack of depolarization of Purkinje cells and interneurons, and blocked the increase in BFcrb (P > 0.05 from Ringer; n = 6). In contrast, Kyn did not influence the increase in BFcrb evoked by hypercapnia (pCO2 55.4 +/- 1.1 mmHg) or by superfusion with the NO donor SIN-1 (0.1, 1 mM; P > 0.05; n = 6). Superfusion with the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT; 100 microM) reduced the elevation in BFcrb by 45 +/- 4% (P < 0.05; n = 6) and co application of 8-SPT and of the NO synthase inhibitor nitro-L-arginine (L-NA; 1 mM) attenuated the vasodilation further (-82 +/- 4% from Ringer; P < 0.01 from 8 SPT alone).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532831 TI - The possible role of glia in nociceptive processing and hyperalgesia in the spinal cord of the rat. AB - Recent studies have suggested that glia might play a more active role in synaptic function than previously thought. Therefore, the present studies have evaluated the potential role of spinal cord glia in acute nociceptive processing and in the thermal and mechanical hyperalgesia produced by peripheral injury. In the present experiments, we found that: (1) selective inhibition of glia metabolism with intrathecal (i.t.) administration of fluorocitrate (1 nmol) results in a marked, but reversible, attenuation of the persistent thermal and mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (2) selective inhibition of the inducible form of nitric oxide synthase (iNOS) with i.t. aminoguanidine (1 pmol-1 nmol) resulted in a dose-dependent inhibition of the persistent thermal, but not mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (3) i.t. coadministration of interleukin 1 beta (IL1 beta; 10 ng) and interferon gamma (IFN; 1000 U) resulted in expression of the message for iNOS 8 hr after administration assessed using reverse-transcription polymerase chain reaction (RT PCR) and Southern blot analysis; and (4) i.t. administration of lipopolysaccharide (LPS; 150 micrograms) produced a time-dependent thermal hyperalgesia compared with saline treated-rats (15 microliters). There was no change in mechanical withdrawal thresholds over time following any treatment, except fluorocitrate. We have previously shown that NO plays a significant role in mechanisms of hyperalgesia. In the present experiments we have extended these observations and have now shown a role for iNOS, expressed by glia, in mechanisms of hyperalgesia. These results suggest an unexplored avenue for the development of potential new and novel therapies for pain control. PMID- 7532830 TI - Nitric oxide and opioid tolerance. AB - Under conditions in which NG-nitro-L-arginine (NOArg) treatment prevents morphine tolerance, NOArg induces a slow progressive inhibition of nitric oxide synthase (NOS), starting at approx. 20% after a single treatment and increasing to approx. 65% after 10 days. Studies designed to examine potential changes in NOS levels with chronic morphine administration reveal no change. Total NOS activity in both brainstem and cerebellum homogenates is unchanged, as are levels of NOS mRNA in a variety of brain regions. L-Arginine, the precursor of nitric oxide (NO), accelerates tolerance when coadministered with morphine and when given alone L arginine decreases morphine's potency. Administration of L-arginine alone for 3 10 days shifts morphine's dose-response curve over 2-fold to the right while D arginine is without effect, as is daily administration of L-arginine along with the NOS inhibitor NOArg. Thus, chronic L-arginine induces "tolerance" in opioid naive mice through NOS. Together, our data indicate an important role for NO in the modulation of opioid analgesia. PMID- 7532832 TI - Glutamate spinal retrograde sensitization of primary sensory neurons associated with nociception. AB - In the present investigation we have tested the hypothesis that spinal glutamate release by inflammatory stimuli causes hyperalgesia through sensitization of the primary sensory neurons associated with nociception. In these experiments, the rat paw hyperalgesia pressure test in which inflammatory hyperalgesia is blocked by the intraplantar administration of morphine (MPH) or SNAP, a NO donor was used. Glutamate and glutamatergic ionotropic agonists such as NMDA or AMPA injected intrathecally (i.t.) caused a dose-dependent hyperalgesia. Quisqualate or ACPD, both of which are glutamate metabotropic receptor agonists, had no hyperalgesic effect. The hyperalgesic response to glutamate and NMDA injected i.t. was antagonized by the intraplantar (i.pl.) injection of either MPH or SNAP. This observation indicates that the hyperalgesia induced by glutamate acting through an NMDA pre-synaptic receptor causes sensitization of the primary sensory neurons. Confirming that the analgesia by i.pl. injection of SNAP or MPH was due to an action in primary peripheral sensory neurons, it was shown that pretreatment of the paws with methylene blue (MB, an inhibitor of guanylate cyclase) or with MB and L-NMMA (an inhibitor of NO synthase) abolished their respective analgesic effect. AMPA i.t. induced hyperalgesia was not inhibited by either i.pl. administration of MPH or SNAP, indicating that its hyperalgesic capacity results from an action at a site other than the primary sensory neuron.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532833 TI - The role of nitric oxide in spinal nociceptive reflexes in rats with neurogenic and non-neurogenic peripheral inflammation. AB - This in vivo electrophysiological study concerns the role of nitric oxide (NO) in mechanical and thermal spinal nociceptive reflexes in alpha-chloralose anaesthetized rats. The effects of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 5-40 mg/kg i.v.) on reflexes were compared both in normal rats and in those with peripheral inflammation induced neurogenically (mustard oil) and non-neurogenically (carrageenan). Methoxamine (0.1 mg/kg i.v.) was used to mimic the marked hypertension caused by L-NAME. Thermal nociceptive reflexes were equally reduced by methoxamine and L-NAME in both normal and inflamed rats, implying that NO has no role in mediating thermal reflexes. However, L- (but not D-) NAME dose dependently and significantly inhibited mechanical reflexes in both carrageenan inflamed (to 37 +/- 12% control) and mustard oil inflamed rats (to 75 +/- 8% control). Moreover, these reductions were greater than those by methoxamine. In contrast, L-NAME did not reduce mechanical reflexes in rats with no inflammation or in spinalized rats with inflammation. The inhibition of mechanical reflexes with L-NAME in carrageenan inflamed rats was reversed and prevented by pre- or post-treatment with L- (but not D-) arginine (50-200 mg/kg i.v.). These data imply a supraspinal role for NO in mediating mechanical (but not thermal) nociceptive reflexes only in those rats with peripheral inflammation. PMID- 7532834 TI - The roles of nitric oxide in sexual function of male rats. AB - Nitric oxide (NO) may mediate penile erection by inhibiting smooth muscle of the corpora cavernosa, thereby allowing vasodilation of the corpora. In order to test the role of NO in the sexual function of intact male rats, either the precursor of NO (L-arginine, L-Arg) or an inhibitor of its synthesis (NG-nitro-L-arginine methyl ester, NAME) was administered systemically before tests of copulation, ex copula genital reflexes, or sexual motivation/motor activity. NAME impaired copulation in a dose dependent manner. It also decreased the number of ex copula erections, but it increased the number of ex copula seminal emissions and decreased the latency to the first seminal emission. L-Arg marginally increased the number of penile reflexes, but had no other effects. NAME had no effect on sexual motivation or motor activity. The results indicate that nitric oxide promotes erection in intact male rats, probably by mediating filling of the corpora cavernosa. The data also suggest that NO inhibits seminal emission, probably by decreasing sympathetic nervous system activity; this may help prevent premature ejaculation. PMID- 7532835 TI - Nitric oxide synthase inhibition reduces wakefulness. AB - The effect of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthase and L-arginine, a precursor of NO, was examined on the sleep-waking pattern in rats. L-NMMA (3.75-15 mg/kg, i.p.) reduced wakefulness with a corresponding increase of slow wave sleep and rapid eye movement sleep. The effect of L-NMMA on vigilance was limited to the first hour following drug administration. The effect of L-NMMA was abolished by intracerebroventricular administration of L-arginine (600 micrograms). This indicates that the inhibitory effect of L-NMMA on wakefulness is mediated by decreased NO synthesis and that central NO exerts an excitatory role in vigilance. It further implicates that factors facilitating a release and/or synthesis of NO might lead to increased wakefulness and sleep disturbances. PMID- 7532837 TI - Autoradiographic characterization of [3H]6-cyano-7-nitroquinoxaline-2,3-dione binding sites in adult chick brain. AB - The non-N-methyl-D-aspartate binding sites have been characterized in chick brain using quantitative autoradiography, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) as a radioligand. [3H]CNQX binding sites were densely localized in the molecular layer of cerebellum. Other areas of prominent binding were the superficial layers of optic tectum, one of the isthmic nuclei, the hippocampus, the hyperstriatum accessorium and the archistriatum ventrale. Analysis of equilibrium binding data in the cerebellar molecular layer indicated that [3H]CNQX bound to a single class of sites (Kd = 78.9 +/- 11.8 nM and Bmax = 41.2 +/- 3.0 pmol/mg protein). Competition experiments in six different regions of chick brain gave the Ki and Bmax values for the inhibition of [3H]CNQX binding by various standard compounds and indicated that: (i) [3H]CNQX labelled non-N-methyl D-aspartate binding sites with high affinity of (RS)-alpha-amino-3-hydroxy-5 methyl-4-isoxazole propionic acid (AMPA) and kainate, (ii) the displacement curves for AMPA and kainate were biphasic in all regions studied, and (iii) the potencies of AMPA and kainate in displacing [3H]CNQX binding were different in the regions studied. Our results indicate that [3H]CNQX labelled non-N-methyl-D aspartate binding sites in chick brain. In the cerebellar molecular layer, these sites were more sensitive to kainate than AMPA, as were the binding sites in the superficial layers of optic tectum and nucleus isthmi magnocellularis. However, non-N-methyl-D-aspartate binding sites in forebrain regions such as hippocampus and hyperstriatum ventrale appeared to be different in being equally sensitive to AMPA and kainate. PMID- 7532836 TI - Reduced transport of [125I]nerve growth factor by cholinergic neurons and down regulated TrkA expression in the medial septum of aged rats. AB - Basal forebrain cholinergic neurons atrophy and degenerate in aging and Alzheimer's disease for unknown reasons. In this study, aged male Sprague-Dawley rats (26-30 months old) showed a significant 31% reduction in the number of septal cholinergic neurons which take up and retrogradely transport 125I-labelled nerve growth factor injected into their target hippocampus, as compared with young adult rats (three to six months old). In aged rats, cholinergic neurons not transporting nerve growth factor were severely atrophied and had a significant 60% reduction in mean cross-sectional area as compared with [125I]nerve growth factor transporting neurons. These changes were accompanied by a significant 43% decline in relative levels of messenger RNA encoding the high affinity nerve growth factor receptor TrkA, in the septal region of aged rats. There was no difference between young and aged rats in messenger RNA levels encoding the low affinity nerve growth factor receptor, p75NGFR. These findings suggest that aged basal forebrain cholinergic neurons exhibit a reduced capacity to sustain receptor mediated uptake and retrograde transport of target-derived neurotrophin. This reduced capacity is associated with severe neuronal atrophy and may contribute to the pronounced vulnerability of these neurons to degeneration in aging and Alzheimer's disease. PMID- 7532839 TI - Incidence of seroconversion for hepatitis C virus in chronic haemodialysis patients: a prospective study. AB - We conducted a prospective study in HD patients of our unit to evaluate the incidence of seroconversion for HCV in this high-risk group. Two hundred and thirty-five patients were observed during the average follow-up of 29.4 months: 183 were seronegative and 52 seropositive for anti-HCV antibodies at the start of the study. During the observation period two of 183 patients developed anti-HCV antibodies late in the study, while the other 181 patients remained seronegative throughout the observation period; anti-HCV antibodies persisted through the follow-up in the 52 HCV-positive patients at the beginning of the study. Our results showed a very low incidence of HCV seropositivity (0.44% per year) after implementation of our operative protocol including 'universal precautions' and other infection control procedures. Once infected, there is no disappearance rate of anti-HCV. The 4-RIBA results did not change during the follow-up period. Prevalence of HCV RNA by PCR technique was 41% (22 of 54) among anti-HCV-positive patients. Future investigations are warranted to clarify the exact route of transmission of HCV among HD patients and to reduce the rate of HCV transmission in this clinical setting. PMID- 7532840 TI - Characteristics of chronic hepatitis C virus infection in patients on maintenance haemodialysis. PMID- 7532841 TI - Terminal distribution of projections from the retrosplenial area to the retrohippocampal region in the rat, as studied by anterograde transport of biotinylated dextran amine. AB - The terminal distribution of projections from the retrosplenial area to the retrohippocampal region was examined in the rat with anterograde transport of biotinylated dextran amine. Projections from the retrosplenial granular area (RSG) to the retrohippocampal region terminate predominantly ipsilaterally in layers I, III, V and VI of the presubiculum, layers I and IV-VI of the parasubiculum, the molecular and pyramidal cell layers of the subiculum, and layers I, III, V and VI of the entorhinal area. On the other hand, projections from the retrosplenial agranular area (RSA) terminate predominantly ipsilaterally in layers I and III of the presubiculum and layers V and VI of the entorhinal and perirhinal areas, and ipsilaterally in layers IV-VI of the parasubiculum. The results show that projections from the RSG to the retrohippocampal region are as massive as those from the RSA, and that each retrosplenial area has distinct projection fields in the retrohippocampal region. This suggests that each retrosplenial area may play some distinct functional roles in memory and learning processes such as spatial behavioral learning. PMID- 7532838 TI - Non-endotoxinic tumour necrosis factor-alpha-inducing factors in haemodialysis. AB - A transmembrane passage of endotoxins may account for the dysfunction of cytokine production which has been often reported in haemodialysis. We developed an assay based on the ability of patient serum to stimulate tumour necrosis factor alpha (TNF alpha) secretion in normal peripheral blood mononuclear cells. Three groups of subjects were investigated: normal controls (n = 14), patients with chronic renal failure, CRF (n = 15), and patients dialysed with polyacrylonitrile (n = 7), polysulphone (n = 8), and cellulose acetate (n = 7). Sera from dialysed patients displayed a significantly higher TNF alpha-inducing activity than those of controls and CRF patients. The ability of serum to elicit TNF alpha secretion was neither modified during the dialysis session nor influenced by the type of haemodialysis membrane. TNF alpha-inducing activity in serum was not inhibited by polymyxin B, known to impair endotoxin-dependent cell responses, thus suggesting that it was not related to circulating endotoxins. We conclude that non endotoxinic factors are present in serum from dialysed patients and are able to induce cytokine secretion. PMID- 7532843 TI - [Results of combined multicycle locoregional therapy in the palliative treatment of multiple liver metastases]. AB - Twenty eight patients with multiple liver metastases were treated by locoregional intraarterial chemotherapy including 118 (3-7, mean 4.2) cycles. In the majority of patients the treatments were carried out in the form of combined cytostatic infusions into the hepatic artery using 5-FU, doxorubicin, cisplatin and mitomycin-C and were complemented by transcatheter embolisation, chemoembolisation and lipiodolisation. In some patients the lipiodolisation was choosen as the primary treatment. In patients with metastases from breast cancer (n = 11) 3 CR, 6 PR, 2 MR occured, the mean survival was 18.4 months, while the one- and two-year survival was 7/11 and 4/11, respectively. In the colorectal group (n = 10) 0 CR, 6 PR, 4 MR were achieved with a mean survival of 13.6 months; one-year survival was 5/10. In the third group (n = 7) consisting of also patients with the poorest prognosis a mean survival of 11.5 months (referring to 6/7 patients) could be accomplished. A patient with metastases from a retroperitoneal anaplastic carcinoma is still alive and disease-free for 92 months. Authors emphasise the importance and effectiveness of locoregional treatments in improving the quality of life as well as survival of patients with unresectable liver metastases. PMID- 7532842 TI - The role of arachidonic acid metabolism in somatostatin and substance P effects on inward rectifier K conductance in rat brain neurons. AB - Somatostatin enhances an inward rectifier K conductance in cultured locus coeruleus neurons, while substance P reduces an inward rectifier K conductance in cultured nucleus basalis and locus coeruleus neurons. The role of arachidonic acid metabolites in these responses was studied. The somatostatin-induced response was reduced by phospholipase A2 inhibitors, non-specific lipoxygenase inhibitors and specific 5-lipoxygenase inhibitors. A cyclooxygenase inhibitor and a 12-lipoxygenase inhibitor had no effect. 5(S)-HPETE occasionally increased the K conductance, but failed to occlude the somatostatin response. The substance P response was suppressed by a 5-lipoxygenase inhibitor but not by a 12 lipoxygenase inhibitor. These results suggest that the 5-lipoxygenase pathway is not a specific messenger of either one of these responses, but that it plays a more general role in maintaining or enhancing the effectiveness of both somatostatin and substance P responses. PMID- 7532844 TI - [Neuromuscular atrophy]. AB - Author reports a case of neural muscular atrophy of a 59-year-old female patient, verified by autopsy and histologic examination. The histologic picture corresponded to the both I. and III. type of sensory-motor polyneuropathyes. PMID- 7532845 TI - [Survey of one hundred patients with chronic granulocytic leukemia from the view of recent developments]. AB - Data of 100 selected CGL patients were considered. There were 50 male and 50 female patients with an average 38.1 and 41.5 years of age, respectively. Seventy nine patients were in stable, chronic and 21 patients were in accelerated phase. Patients first admitted in blastic phase were excluded. Twenty two patients were subjected to bone marrow transplantation. Characteristically low levels of neutrophil alkaline phosphatase were absent in about 10 to 25% of early cases. It was considered that the level of positive cells could eventually point to the extent of normal population. In the group of the accelerated patients the neutrophil alkaline phosphatase scores were regularly high and the serum cholesterol values lower than those among stable-phase patients. The role of G CSF (granulocyte colony stimulating factor) was considered. Secondary myelofibrosis was frequently associated with low serum cholesterol. Average survival time was 41 month among non-transplanted and 63+ months among identically-transplanted patients. In accordance with the literature, authors point out that in the presence of any factor not permitting transplantation, prolonged high-dose interferon-therapy could be the first choice, because unlike chemotherapeutic agents used till now, it prolongs survival. Apart from this autotransplantation with marrow or with circulating blood derived stem cells should be considered. PMID- 7532846 TI - [Transrectal ultrasonography in the diagnosis of prostatic cancer]. AB - There is a summary of experience with 771 transrectal ultrasound examinations. According to the authors, this picture forming method is one of the best diagnostical methods in the malignant tumors of the prostate. In every case, when the digital rectal examination is positive and the serum prostate specific antigen level is above 4 ng/ml, the transrectal ultrasonography should be done as well as the ultrasound guided biopsy of prostate. Besides of these facts, the transrectal ultrasound is useful in the following process of the drug- and/or the radiotherapy for the measurement of the therapeutical effect and the perceptions of recidives. PMID- 7532847 TI - [Detection of hepatitis C viral RNA by polymerase chain reaction]. AB - The authors describe the development and the application of polymerase chain reaction (PCR) in the detection of hepatitis C virus. Based on the sequence data available in the literature, a conservative motif at the 5' end of the virus genome has been selected for amplification. Using the flanking synthetic oligonucleotides the specificity of the highly sensitive RNA-PCR assay was proved by serologically positive and negative serum samples. HCV-RNA-PCR studies were applied to start interferon treatment in patients from National Institute of Rheumatology and Physiotherapy (Budapest) with anti-HCV positive serum samples and this method was used to evaluate the efficacy of interferon therapy, as well. PMID- 7532848 TI - [Diagnostic errors in symptom-poor hyperthyroidism in the aged]. AB - The diagnosis of hyperthyreoidism manifesting in the elderly meets difficulty in many cases. Lack of some characteristic symptoms observable in younger patients can be misleading. The authors summarize the data of ten clinical cases where the hyperthyreoidism appeared in occult form, reminding rather for heart disease or malignant tumours. The assay of thyroid hormones and the level of antithyreoglobulin and anti-thyreoid peroxidase antibodies was unavoidable in order to establish correct diagnosis. Though the early diagnosis and treatment may enhance the life expectancy of the patients and may considerably decrease the cardial and the consecutive cerebral complications. The authors analyse the reasons and possible pathomechanism of the formation of atypical hyperthyreoidism occurring in the elderly. PMID- 7532849 TI - Localization of cytokines in cholesteatoma tissue. AB - Acquired cholesteatoma is associated with an intense inflammatory reaction with resultant tissue and bone destruction. Cytokines are molecules released by inflammatory cells at the site of infection and are potent mediators of inflammation and the immune response. Five cytokines, tumor necrosis factor alpha, transforming growth factor-beta 1 and 2, and interleukin-1 and 6, were immunolocalized in human cholesteatoma epithelium and subepithelial stroma, with greater intensity of staining compared with noninflamed external auditory canal skin. Increased interleukin-6 activity in cholesteatoma epithelium and stroma correlated significantly with the presence of ossicular and bony erosion and granulation tissue noted intraoperatively. Transforming growth factor-beta 2 activity in cholesteatoma epithelium correlated significantly with bony erosion at surgery. Additionally, transforming growth factor-beta 1 activity in cholesteatoma epithelium correlated significantly with increased length of disease. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 appear to be involved in the inflammation and resultant remodeling associated with cholesteatoma. We hypothesized a protective function of transforming growth factor-beta 1 and 2 in the presence of cholesteatoma. The antiinflammatory and osteoclast and keratinocyte inhibitory actions of the transforming growth factor beta s could potentially slow the proliferation and resultant tissue destructiveness associated with cholesteatoma. PMID- 7532850 TI - Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement fixing. AB - We have studied the properties of 16 newly derived monoclonal antibodies (MoAbs) against Pfs230, a gamete surface protein of Plasmodium falciparum and a target of transmission-blocking antibodies. All 16 MoAbs recognized Pfs230 by immunoprecipitation from non-ionic detergent extracts of the protein radio labelled with 125Iodine. The MoAbs also recognized this protein on Western blots under non-reducing conditions but none of them recognized the protein under reducing conditions. Using an immunoradiometric assay the MoAbs appear to define nine different epitope regions. The MoAbs were tested for their ability to lyse extra-cellular female gametes of P. falciparum isolate 3D7. Eight of the MoAbs, all of isotype IgG2a, mediated complement-dependent lysis of the gametes; seven of the MoAbs, all isotype IgG1, failed to lyse the gametes in the presence of active complement. The eight complement-fixing MoAbs mediated almost total suppression of infectivity of gametocytes of P. falciparum 3D7 to mosquitoes; where tested this suppression was mainly complement-dependent. The seven non complement-fixing MoAbs had no significant effect on the infectivity of gametocytes of P. falciparum 3D7 to mosquitoes. When tested by immunofluorescence the target epitopes of all the MoAbs were conserved in each of the five different isolates of P. falciparum which were tested. PMID- 7532851 TI - Induction of protective immunity against murine secondary hydatidosis. AB - Significant protective immunity against secondary hydatidosis in mice was achieved by immunization with a preparation of surface molecules of E. granulosus protoscoleces with Freund's incomplete adjuvant (PSEx-IFA). Study of PSEx-IFA immunogenicity demonstrates that glucidic epitopes evoked mainly IgM responses while peptidic epitopes evoke mainly IgG responses; however both types of epitopes elicit both types of responses. Analysis of the possible association between susceptibility or resistance to infection and antibody responses after challenge was also performed. Cyst fluid antigen (CFAg) specific antibody titres on month eight after challenge correlated with number and size of cysts. On the other hand no correlation was observed between protection and PSEx specific IgG titres either on the day of challenge or one month later. Nonetheless, immunoblot analysis revealed that some PSEx molecules were recognized on day 30 after challenge only by sera from immunized mice. PMID- 7532852 TI - Communicative responsibility and semantic task in aphasia and "schizophasia". AB - A probe technique requiring convergent and divergent semantic behavior and representing five levels of communicative responsibility served as the research tool. Stimuli were presented to 29 asphasic adults (13 Broca's, 7 Wernicke's, and 9 anomic), 26 adults with chronic undifferentiated schizophrenia, and 32 normal elderly control subjects. Within each group significant differences were observed on the semantic task (convergent and divergent) and on level of communicative responsibility. Among subjects with aphasia, differences appeared to relate more to severity than type. Differences between unclassified aphasic and "schizophasic" groups occurred only when multiword responses were required. We conclude that continued use of the term "schizophasia" may be unwarranted and that the linguistic behaviors we observed in aphasia and the language of schizophrenia may contribute to differential diagnosis. PMID- 7532853 TI - Motor performance in aphasia and ideomotor apraxia. AB - Motor performance in 11 patients with ideomotor apraxia, 11 with aphasia without such apraxia, and 11 normal controls was compared. These three groups were matched on age, sex, education, severity of aphasia, intelligence, and size of lesion. Measures of aiming, tapping, line-following, and steadiness developed by Schoppe in 1974 were used. Both apraxic and aphasic groups showed difficulties with motor performance, and the data of the apraxic group were poorer than those of the aphasic group. These results were consistent with Liepmann's theory. PMID- 7532854 TI - Car ownership and suicide by car exhaust in nations of the world. PMID- 7532855 TI - Efficacy of part- and full-time early intervention. AB - The effectiveness of an early intervention program to remediate developmental delays in children age birth to 3 years was examined in part- and full-time groups (Study 1). Significant improvements on age-appropriate measures of developmental standing were observed for both groups, with the greatest gains observed for the full-time group. In Study 2, the stress of parents with developmentally delayed children was measured on the Parental Stress Inventory. Reductions in stress related to children's characteristics and dysfunctional parenting skills were observed on some subscales, supporting prior research which indicated extension of the outcomes of early intervention beyond the child was desirable. PMID- 7532856 TI - A transcriptional repressor obtained by alternative translation of a trinucleotide repeat. AB - Triplet nucleotide repeats are ubiquitous and rapidly evolving sequences in eucaryotic genomes. They are sporadically found in coding regions of transcription regulators where they become translated in different homopolymeric aminoacid (HPAA) stretches, depending on the local frame. Poly(CAG) yields three different HPAAs (poly Gln, Ser or Ala). Current sequence databases indicate a clear bias in the size and frequency of these HPAAs according to the rule: (Gln)n > (Ser)n >> (Ala)n. Aiming to understand the reasons of this bias, we changed the translational reading frame of the highly polymorphic CAG-repeat that normally encodes poly-Gln in the N-terminal portion of the rat glucocorticoid receptor (GR). The GR mutant in which the CAG repeat is translated to poly-Ala (called GR[Ala]) is incapable of transactivation, but maintains competence for hormone binding, nuclear translocation and specific DNA binding. We show that GR desactivation is obtained only when a very precise threshold length of the repeat is reached. GR[Ala] displays a strong negative dominance when tested for transcriptional activation in vivo and may become useful for selective competition of receptor dependent activities in tissue culture cells and transgenic animals. We discuss the implications of our findings for the understanding of the evolutionary behaviour of trinucleotide repeats in coding sequences. PMID- 7532857 TI - Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15. AB - The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures. PMID- 7532858 TI - Antisense 2'-O-alkyl oligoribonucleotides are efficient inhibitors of reverse transcription. AB - Reverse transcription is one step of the retroviral development which can be inhibited by antisense oligonucleotides complementary to the RNA template. 2'-O Alkyl oligoribonucleotides are of interest due to their nuclease resistance, and to the high stability of the hybrids they form with RNA. Oligonucleotides, either fully or partly modified with 2'-O-alkyl residues, were targeted to an RNA template to prevent cDNA synthesis by the Avian Myeloblastosis Virus reverse transcriptase (AMV RT). Fully-modified 2'-O-allyl 17mers were able to specifically block reverse transcription via an RNase H-independent mechanism, with efficiencies comparable to those observed with phosphodiester (PO) and phosphorothioate oligonucleotides. Sandwich 2'-O-alkyl/PO/2'-O-alkyl oligonucleotides, supposed to combine the properties of 2'-O-alkyl modifications (physical blocking of the RT) to those of the PO window (RNase H-mediated cleavage of the RNA) were quasi-stoichiometric inhibitors when adjacent to the primer, but remained without any effect when non-adjacent. They were not able to compete with the polymerase and inhibited reverse transcription only through RNase H-mediated cleavage of the target. PMID- 7532859 TI - Contribution of developmental disabilities to childhood mortality in the United States: a multiple-cause-of-death analysis. AB - Although developmental disabilities are among the major chronic health problems affecting children in the United States, the contribution of developmental disabilities to childhood mortality is unknown. To investigate the magnitude of this contribution, multiple cause-of-death data were examined for US children, aged 1-19 years, for 1980 and 1983-1989. The following conditions were included as developmental disabilities: autism, attention deficit disorder, learning disorders, mental retardation, cerebral palsy, epilepsy, muscular dystrophy, blindness and deafness. Based on underlying cause only, it was found that developmental disabilities were the fifth leading cause of nontraumatic death for children between 1 and 14 years of age and the third leading cause of non traumatic death for children between 15 and 19 years. When a multiple cause approach was used to define developmental disability-related deaths (i.e. when contributing as well as underlying cause was considered), the number of such deaths nearly doubled. On the basis of both underlying- and multiple-cause analyses, cerebral palsy was the developmental disability most frequently cited as a cause of death. Mental retardation ranked second according to the multiple cause approach but only fourth according to the underlying-cause approach. The least frequent causes of death (autism, attention deficit disorder, learning disorders, blindness, and deafness) were the ones most likely to be coded as contributing rather than underlying causes. Developmental disability-related mortality rates were highest among children aged 1-4 and 15-19 years, highest among blacks and lowest among racial groups other than blacks and whites, and higher among males than females. Although results of multiple-cause-of-death analyses more accurately reflect the proportion of deaths related to developmental disabilities, even this approach may underestimate the degree to which mortality is associated with a developmental disability. PMID- 7532860 TI - High-dose intravenous gammaglobulin therapy for acquired von Willebrand disease. AB - Patients with acquired von Willebrand disease may present with severe bleeding, which is usually difficult to manage. Adequate haemostasis in acquired von Willebrand disease may be achieved with the infusion of factor VIII/von Willebrand factor concentrates or with the administration of desmopressin. We report a case of acquired von Willebrand disease with severe postoperative bleeding, responding poorly to classical von Willebrand factor replacement therapy but successfully treated with high-dose intravenous gammaglobulins. This new treatment mode of acquired von Willebrand disease is discussed in the light of a critical analysis of the literature. PMID- 7532861 TI - Preparation of sterically stabilized human serum albumin nanospheres using a novel Dextranox-MPEG crosslinking agent. AB - Human serum albumin (HSA) nanospheres with a size less than 200 nm in diameter were prepared using a modified coacervation method and crosslinking with methyl polyethylene glycol modified oxidized Dextram (Dextranox-MPEG) which created a sterically stabilizing polyethylene oxide surface layer surrounding the nanospheres. The crosslinking efficiency and the surface characteristics of glutaraldehyde and Dextranox-MPEG crosslinked HSA nanospheres were determined and compared. The zeta potential of the Dextranox-MPEG crosslinked particles was significantly lower than that of glutaraldehyde stabilized particles. The existence of a hydrated steric barrier surrounding the nanospheres was confirmed by an electrolyte and pH induced flocculation test. The Dextranox-MPEG crosslinked nanospheres showed a significantly reduced plasma protein adsorption on the particle surface compared with glutaraldehyde crosslinked nanospheres. PMID- 7532862 TI - Effect of diazepam on cortical 5-HT release and behaviour in the guinea-pig on exposure to the elevated plus maze. AB - Previous studies have used the elevated plus maze to test for "anxiolytic" drugs in rats. The present study demonstrates that guinea-pigs handled daily from birth exhibit similar behaviour to rats on the plus maze. Pretreatment with diazepam (1.0 mg/kg) significantly increased the time the animals spent in the open arms and amount of entries into the open arms. Using intra-cortical microdialysis on exposure of the guinea-pig to the elevated plus maze resulted in increased extracellular 5-HT in the frontal cortex. Diazepam reduced, but not significantly, the increase in extracellular 5-HT and produced an "anxiolytic" profile of behaviour. Pretreatment with the benzodiazepine antagonist flumazenil (10.0 mg/kg) fully antagonised the behavioural effects of diazepam. Flumazenil also reduced the effect of diazepam on the increase in extracellular 5-HT observed on exposure of the guinea-pig to the elevated plus maze. Flumazenil alone decreased basal extracellular cortical 5-HT but had no effect on behaviour in the elevated plus maze. The results show that an increase in extracellular 5 HT occurs in the guinea-pig exposed to aversive conditions. While it remains to be determined whether the "anxiolytic" effects of diazepam in the guinea-pig are causally associated with decreased extracellular 5-HT, it is of interest that the selective benzodiazepine antagonist also prevented the increase in basal extracellular 5-HT produced by the exposure to the elevated plus maze but had no effect on behaviour. Results indicate that there is no simple relationship between inhibition of 5-HT release and the "anxiolytic" action of benzodiazepines. PMID- 7532863 TI - Changes induced by sodium cromoglycate on brain serotonin turnover in morphine dependent and abstinent mice. AB - This study was designed to explain the action of sodium cromoglycate (CRO) on the brain serotonergic system in control, morphine tolerant (by SC implantation of a 75 mg morphine pellet), and also in morphine dependent mice just before naloxone precipitated withdrawal. After SC injections of CRO in control mice, morphine tolerant mice (day 4 of addiction), and 1 h before abstinence (withdrawal was induced by SC injection of 1 mg/kg naloxone on day 4 of addiction), animals were decapitated and various brain areas were rapidly removed. 5HT (Serotonin) and 5HIAA (5-hydroxyindole-3-acetic acid) were measured by high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). The ratio 5HIAA/5HT provided one index by which the turnover of the indoleamine was measured. CRO increased the turnover of 5HT in most of the brain areas studied in both control and morphine dependent mice. Furthermore, previous administration of CRO prior to naloxone challenge induced a significant increase in the 5HIAA/5HT ratio in the hypothalamus and striatum. These results are discussed as the reason for the preventive effect of CRO on jumping behaviour in morphine abstinent mice. PMID- 7532866 TI - Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase. AB - Effects of nitric oxide synthase (NOS) inhibitors (L-NG-nitroarginine, L-NG nitroarginine methyl ester) on precipitated opioid withdrawal were studied in morphine-dependent rats given naloxone, in order to assess the involvement of nitric oxide (NO) in opioid dependence. L-NG-Nitroarginine (7.5 mg/kg, IP, 1 h before naloxone or b.i.d. on days 4-7 of an 8-day morphine treatment) reduced wet dog shakes and weight loss; when given by osmotic pumps (15 mg/kg per day), the drug reduced wet dog shakes but not weight loss. L-NG-Nitroarginine methyl ester (60 mg/kg, 1 h before naloxone) also reduced wet dog shakes and weight loss. The results indicate that NOS inhibitors warrant further study as potential treatments of the opioid withdrawal syndrome. PMID- 7532864 TI - Mnemogenic effects of injecting RA-octil, a CE-inhibitor derivate, systemically or into the basal forebrain. AB - The aim of this study was to investigate the effects of systemically or intracerebrally administered RA-octil, a derivative of the angiotensin converting enzyme (CE)-inhibitor ramipril, on memory and reinforcement and to compare its effectiveness with that of the neurokinin substance P (SP). In the first experiment systemic post-trial application of RA-octil and SP in the rat enhanced habituation, a learning task which does not require motivational treatments. Unlike SP, injection of RA-octil did not have reinforcing effects as measured with a conditioned place preference task. In the second experiment, a facilitation of inhibitory avoidance learning was obtained by injection of RA octil or SP unilaterally into the basal forebrain immediately after the learning trial. In contrast, a 5 h delayed injection of RA-octil had no effects on learning. The results demonstrate memory-enhancing effects of RA-octil after systemic application as well as after injection into the basal forebrain. Furthermore, the mnemogenic effects of SP after central and peripheral administration were confirmed. Since RA-octil, although being structurally closely related to CE-inhibitors, does not affect plasma CE, yet exhibits mnemogenic effects, it is possible that "cognition-enhancing" actions of CE inhibitors are dissociable from their action within the renin-angiotensin system. PMID- 7532867 TI - Demonstration of an association among dietary cholesterol, central serotonergic activity, and social behavior in monkeys. AB - Epidemiologic studies link plasma cholesterol reduction to increased mortality rates as a result of suicide, violence, and accidents. Deficient central serotonergic activity is similarly associated with violence and suicidal behavior. We investigated the relationship among dietary and plasma cholesterol, social behavior, and the serotonin system as a possible explanation for these findings. Juvenile cynomolgus monkeys (eight female and nine male) were fed a diet high in fat and either high or low in cholesterol. We then evaluated their behavior over an 8-month period. Plasma lipids and cerebrospinal fluid metabolites of serotonin, norepinephrine, and dopamine were assessed on two occasions, at 4 and 5.5 months after the initiation of behavioral observations. Animals that consumed a low-cholesterol diet were more aggressive, less affiliative, and had lower cerebrospinal fluid concentrations of 5 hydroxyindoleacetic acid than did their high-cholesterol counterparts (p < .05 for each). The association among dietary cholesterol, serotonergic activity, and social behavior was consistent with data from other species and experiments and suggested that dietary lipids can influence brain neurochemistry and behavior; this phenomenon could be relevant to our understanding of the increase in suicide and violence-related death observed in cholesterol-lowering trials. PMID- 7532869 TI - [Urinary excretion of alpha-1-microglobulin and complement components in patients with chronic glomerulonephritis]. AB - The urinary excretion of alpha-1-micro-albumin (alpha 1m) and complement components (CCs) was evaluated in the urine of 49 patients suffering from chronic glomerulonephritis (GN). Nephrotic syndrome (NS) was shown in 18 cases and increased serum creatinine level (Pcr: 115.0-159.1 mumol/l) in 9 patients. The most frequent CCs presence and the highest values of alpha 1m excretion were found in patients with membranous GN. In the early phase of the disease the alpha 1m urinary excretion was higher in subjects with NS than in those not showing the feature of it, independently of the morphological basis of the disease. Also CCs were detected mainly in the nephrotic patients. As the glomerular filtration improved a significant decrease in the urinary alpha 1m excretion was observed. The application of steroid immunosuppressive therapy resulted in the decrease of alpha 1m as well as CCs excretion. The results seem to point out that the increased alpha 1m and CCs excretion may be secondary to the activity of glomerular alternations as well as to the disturbances in glomerular blood flow. PMID- 7532868 TI - Clozapine and new-onset ECG abnormalities. PMID- 7532865 TI - Sequence-specific effects of neurokinin substance P on memory, reinforcement, and brain dopamine activity. AB - There is ample evidence that the neurokinin substance P (SP) can have neurotrophic as well as memory-promoting effects. This paper outlines a recent series of experiments dealing with the effects of SP and its N- and C-terminal fragments on memory, reinforcement, and brain monoamine metabolism. It was shown that SP, when applied peripherally (IP), promotes memory (inhibitory avoidance learning) and is reinforcing (place preference task) at the same dose of 37 nmol/kg. Most important, however, is the finding that these effects seemed to be encoded by different SP sequences, since the N-terminal SP1-7 (185 nmol/kg) enhanced memory, whereas C-terminal hepta- and hexapeptide sequences of SP proved to be reinforcing in a dose equimolar to SP. These differential behavioral effects were paralleled by selective and site-specific changes in dopamine (DA) activity, as both SP and its C-, but not N-terminus, increased extracellular DA in the nucleus accumbens (NAc), but not in the neostriatum. The neurochemical changes lasted at least 2 h after injection. These results show that the reinforcing action of peripheral administered SP may be mediated by its C terminal sequence, and that this effect could be related to DA activity in the NAc. Direct application of SP (0.74 pmol) into the region of the nucleus basalis magnocellularis (NBM) was also memory-promoting and reinforcing, and again, these effects were differentially produced by the N-terminus and C-terminus, supporting the proposed structure-activity relationship for SP's effects on memory and reinfrocement. These results may provide a hypothetical link between the memory modulating and reinforcing effects of SP and the impairment in associative functioning accompanying certain neurodegenerative processes. PMID- 7532870 TI - [New immunosuppressive drugs in transplantation]. PMID- 7532871 TI - Glycoconjugates in the mandibular salivary gland of adult dogs revealed by lectin histochemistry. AB - The glycosidic residues in the mandibular glands of five adult dogs were studied by using seven different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. The mucous acinar cells contained oligosaccharides with alpha- and beta-N-acetylgalactosamine, N acetylglucosamine and fucose residues, whereas the demilunar cells contained glycoconjugates rich in sialic acid linked to the penultimate disaccharide galactosyl-(beta 1-->3) N-acetylgalactosamine. PMID- 7532872 TI - Human anti-Ro autoantibodies bind peptides accessible to the surface of the native Ro autoantigen. AB - The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60 kDa Ro are variably bound by anti-Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti-Ro precipitin negative lupus patients. The specific activities of anti-peptide antibodies and of anti-native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity-enriched anti-native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti-Ro positive sera. Two peptides (residues 274-290 and 480-494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity-enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti-Ro octapeptide autoantibodies are part of the population of anti-native Ro autoantibodies in this naturally arising autoimmune response. PMID- 7532873 TI - Monospecific polyclonal anti-anti-idiotypic antibodies to the carboxyterminal undecapeptide of the SV40 large tumour antigen. AB - The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698) Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40-transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti-idiotypic antibodies (Ab-2). Ab-2 beta (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab-2 with ab-1. Murine anti-anti idiotypic antibodies (ab-3) were obtained by immunization of mice with ab-2 beta. Both ab-1 and ab-3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab-3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab-3 is of the ab-3 subtype and specific for only one epitope which is represented by KT and defined by ab-1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti-idiotypic antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour-associated antigen which represents a B- and T cell epitope, this model is of great interest for further tumour immunological studies. PMID- 7532874 TI - Endothelial prostacyclin production, synergistic effect between adrenergic stimulating and blocking drugs. AB - Endothelial cells (EC) produce prostacyclin (PGI2) in high quantities which at the luminal surface decreases platelet aggregation and adhesion and basal to the cell relaxes smooth muscle cells (SMC). Connections have been reported between prostacyclin production, hypertension and the degree of adrenergic activation. The present study tested the hypothesis that prostacyclin production by EC could be regulated by adrenergic mechanisms. EC were isolated from human umbilical cord veins. Washed cells were seeded and grown to confluency on tissue culture dishes. The test drugs were simultaneously added to parallel dishes. Samples were collected from the conditioned medium and analyzed for 6-keto-PGF1a with RIA technique. Endothelial cells pretreated with the betaadrenoceptor blocking drugs metoprolol or propranolol synergistically increased basal prostacyclin production when exposed to betaadrenergic stimulation. However, using isomers with high or low betaadrenoblocking effect, this synergism was demonstrated not to be associated to the betaadrenoceptor blocking effect of the drugs per se. These findings may have implications on the arterial hypertensive state characterized by high sympathetic tonus and low PGI2 production. The data may offer an explanation why hypertensive individuals react with increased PGI2 production, upon betaadrenoceptor blocking therapy. PMID- 7532875 TI - Characterization of a novel bovine leukocyte protein involved in cell-cell adhesion. AB - Preliminary characterization of an apparently novel bovine leukocyte adhesion protein is described. Two IgG1 monoclonal antibodies, UC-C1 and UC-H5, raised against established cultures of IL-2-dependent bovine peripheral blood lymphocytes (PBL) were found to react with an antigen expressed by the majority of bovine peripheral blood leukocytes. Immunoprecipitation and polyacrylamide gel electrophoresis of the antigen produced a distinct protein band of molecular weight 160,000, and additional diffuse protein bands of approximate molecular weight 180,000, 175,000, and 150,000. Two-color flow cytometric analyses showed that the antigen was expressed at low density on a small proportion of circulating B lymphocytes, but was highly expressed on all circulating T lymphocytes. The majority of monocytes and all granulocytes expressed the antigen at a density lower than that of T lymphocytes. Peripheral blood lymphocytes stimulated with concanavalin A had an approximately 3-fold increased expression of the antigen, which was apparent within 18 h and remained stable in long-term cultures. Expression of the antigen in thymus, analyzed by the immunoperoxidase technique, was predominantly restricted to thymocytes in the immediate subcapsular cortex and medulla; expression in lymph nodes and spleen was predominantly confined to lymphocytes in T-cell areas. Flow-cytometric analysis demonstrated that thymocytes and the majority of peripheral and mesenteric lymph node-derived T cells had relatively low surface density of antigen compared to circulating T cells. Binding of UC-C1 or UC-H5 to the antigen on lymphocytes induced homotypic aggregation. UC-C1 completely blocked binding of FITC conjugated UC-H5 to blood mononuclear cells, suggesting that the antibodies recognize the same epitope or proximal epitopes. PMID- 7532877 TI - Growth kinetics of enzyme-altered liver foci in rats treated with phenobarbital or alpha-hexachlorocyclohexane. AB - A quantitative method based upon a stochastic model for the appearance of initiated cells and their clonal growth was used to estimate cell birth and death rates in enzyme-altered liver foci (EAF). gamma-Glutamyltranspeptidase (gamma-GT) positive foci were initiated in livers of female SPF Wistar rats by a single application of N-nitrosomorpholine. Serial terminations during and after stop of promoter treatment with either phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH) provided information on the growth and regression of the EAF. Simultaneous labeling index (LI) measurements were obtained via single injections with [3H]thymidine. No significant increases of the LI were observed with PB or alpha-HCH treatment. Since both agents are strong liver promoters we conclude that the growth of gamma-GT-positive foci is mainly due to a decrease in the rate of apoptosis. Indeed, our analysis supports this conclusion but determines that the abrogation of homeostatic control during promoter treatment is subtle. The ratio of cell death and cell birth rate is found to be decreased only slightly during promoter treatment and slightly increased after stop of promotion. For the mathematical analysis, two distinct focal growth scenarios were employed: (i) volume growth, i.e., all cells within individual foci cycle actively with the same rate, and (ii) surface growth where only cells on the surface of foci cycle actively while interior cells are resting. The model based upon scenario (ii) provides a better fit to the data and is more consistent with the experimental observations indicating heterogeneity of cell division rates within individual foci. PMID- 7532876 TI - Lindane inhibition of gap junctional communication in myometrial myocytes is partially dependent on phosphoinositide-generated second messengers. AB - The ability of environmental contaminants to modulate gap junctional communication between uterine smooth muscle cells is generally unknown, despite recognition that myometrial gap junctions may play a role in synchronizing uterine contractions during parturition. The present study tested the hypothesis that the organochlorine pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junctional communication in myometrial myocytes due to the release of phosphoinositide-dependent second messengers. The effect on gap junctional communication by lindane was tested in cultured rat myometrial smooth muscle cells by monitoring transfer of the fluorescent dye Lucifer yellow. A rapid, concentration-dependent, but reversible inhibition of dye transfer was noted with 4-min exposures, and inhibition was complete with 10 microM lindane. Lindane also stimulated the production of the Ca(2+)-releasing species inositol 1,4,5 trisphosphate which peaked at 5 min (100 pmol/mg protein) and remained elevated after a 15-min exposure. To examine the possible inhibitory role of Ca2+ on gap junctions, the Ca2+ ionophore 4-br-A23187 was used. Although A23187 also inhibited gap junctional communication, inhibition was not complete even at concentrations that appeared cytotoxic (70% inhibition at 2 microM A23187). Cells were then loaded with the Ca2+ chelator BAPTA-AM, which blocked the lindane induced rise in calcium, and dye transfer experiments with lindane were repeated in Ca(2+)-free medium. Inhibition of dye transfer was still complete under these conditions, showing that increased intracellular calcium was not required for lindane-induced inhibition of gap junctional communication. Subsequently, 10 microM lindane was shown to produce a sustained increase in protein kinase C (PKC) activity (31, 17, and 15 pmol of PKC peptide phosphorylated/min/mg protein for 2-, 5-, and 10-min exposures, respectively). Known activators of PKC, 12-O tetradecanoylphorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, abolished gap junctional communication at nanomolar concentrations. Although use of the PKC inhibitor staurosporine failed to reverse lindane's inhibitory action, depletion of PKC activity through prolonged exposure to TPA partially reversed lindane's effect. This suggests that PKC activation potentiates but does not solely mediate lindane's inhibitory action on gap junctional communication. PMID- 7532878 TI - Changes in the contact system during orthotopic liver transplantation with and without aprotinin. AB - The main cause of nonsurgical bleeding during orthotopic liver transplantation has been attributed to be hyperfibrinolysis due to high plasma levels of tissue plasminogen activator. The aim of this study was to investigate contact activation and its possible contribution to fibrinolysis during OLT with and without aprotinin. Aprotinin or placebo was given to 20 patients undergoing OLT as part of a randomized double-blind trial. Plasma samples were collected before, during, and after OLT. There were decreased preoperative levels of prekallikrein and factor XIIa (P < 0.05), with a trend for kallikrein and factor XIIa activity to increase during OLT peaking on reperfusion (P < 0.05). Kallikrein inhibition, C1 esterase inhibitor, and alpha-2-macroglobulin levels were normal before surgery, with low normal levels of antithrombin III and alpha-2-antiplasmin; these levels decreased during OLT with no specific change on reperfusion. In the aprotinin-treated group, kallikrein inhibition levels increased (P < 0.05) from preoperative mean (+/- SD) values of 101 +/- 47% to 154 +/- 42% and antiplasmin levels increased (P < 0.05) from 72 +/- 28% to 243 +/- 53% during the anhepatic phase, reflecting the effect of aprotinin. The antifibrinolytic effect of aprotinin was demonstrated by decreased levels of D-dimer on reperfusion (P < 0.05) and at the end of OLT (P < 0.001) in the aprotinin-treated group. We have shown that contact activation during OLT is minimal and that aprotinin does not alter the pattern of contact activation, but provides an antikallikrein effect. PMID- 7532879 TI - Effects of rapamycin on growth factor-stimulated vascular smooth muscle cell DNA synthesis. Inhibition of basic fibroblast growth factor and platelet-derived growth factor action and antagonism of rapamycin by FK506. AB - Rapamycin (RPM) is a potent and effective immunosuppressant which we have shown previously to inhibit intimal thickening in rat allograft and balloon-injured arteries. In this report, we have examined the effects of RPM on growth factor induced vascular smooth muscle cell (VSMC) DNA synthesis. RPM potently inhibited platelet-derived growth factor (PDGF) (IC50 = 5 x 10(-9) M) and basic fibroblast growth factor (bFGF) (IC50 = 8 x 10(-10) M)-induced VSMC DNA synthesis. In contrast, only the highest concentrations of FK506 and CsA significantly altered PDGF- or bFGF-induced VSMC DNA synthesis. Addition of RPM (10(-9) M) at as late as 46 hr after growth factor addition still effectively suppressed bFGF- or PDGF induced DNA synthesis by 76% and 54%, respectively. The extent of the antagonism of RPM's inhibition of bFGF-induced VSMC DNA synthesis by FK506 was inversely proportional to RPM concentration and directly proportional to FK506 concentration. PMID- 7532880 TI - [The correction of the immune disorders in patients with chronic pyelonephritis and chronic kidney failure]. AB - The trial entered 41 patients with chronic renal failure. Group I (15 patients) received conventional therapy involving infusion detoxication, antibiotics, immunostimulators, anabolic agents. 26 patients of group II in addition to standard therapeutic measures underwent plasmapheresis. The effect was assessed by immunological and renal function parameters. Group I patients have benefited from conventional treatment, but signs of immunodeficiency remained: B lymphocyte, IgG and IgA levels were lowered and elevated, respectively. Patients of group II improved clinically, got rid of uremia and immunodeficiency. PMID- 7532881 TI - [The preoperative anti-inflammatory and postoperative therapy of patients with calculous pyelonephritis and prostatic adenoma with chronic prostatitis under outpatient polyclinic conditions]. AB - Such wide-spread urological diseases as nephrolithiasis and prostatic adenoma requiring surgical management are often associated with chronic infection or inflammation (pyelonephritis, prostatitis, adenomitis). Relevant antiinflammatory treatment as a rule is conducted after the patient hospitalization which may induce unwanted emotional stress, occasional hospital infection, additional material expenditures. The authors have the experience of bactericidal and antiinflammatory treatment of the kidneys (143 patients with nephrolithiasis) and prostate (287 patients with adenoma) in the outpatient setting. Three-stage system of the patients' care is recommended: district outpatient clinic consultative outpatient department of the Research Urological Center-Hospital of the above Center. Such an approach noticeably improved the treatment outcomes: the frequency of inflammatory postoperative complications reduced 2-fold, no more lethal outcomes occurred, the duration of the hospital stay decreased two-fold. The authors suggest to introduce the above three-stage system of pre- and posthospital outpatient antiinfectious and antiinflammatory treatment of nephrolithiasis-affected kidney and prostate in adenoma into the practice of all national, regional and local urological centers. PMID- 7532882 TI - Evaluation of an autotutorial-simulator program for instruction of hollow organ closure. AB - Forty students were randomly assigned into two study groups (traditional, T; and simulator, S) of 20 students each for a core operative practice laboratory. Students were randomly paired and their group assignment and identity remained anonymous to the evaluators throughout the study. Questionnaires were distributed to students to evaluate prior surgical experience and obtain learning resource use information. Before the evaluation sessions, both groups were given identical learning resource opportunities except students in Group S received hollow organ simulators and practice materials for gastrotomy closure. All students were forewarned that surgical instruction would not be available during the evaluation sessions. In the first live animal evaluation session, all student pairs were videotaped after which stomachs were harvested for gross evaluation of the surgical site. Group T performed an additional gastrotomy for video and gross evaluation 2 weeks later. Questionnaire, and gross and video evaluation results were compared statistically between groups and sessions. The hollow organ model did not suitably simulate live stomach tissue; the material was more fragile and stiff and suture cut-out was a problem even with appropriate suture tension and technique. The model was effective for teaching needle placement, instrument usage, creating proper tissue inversion, and methods to minimize instrument handling of tissue during gastrotomy closure. Prior practice with models did not boost student confidence during their live gastrotomy session. The autotutorials (ATs) were well received by students but did not sufficiently address how to manage mucosal eversion, suture tension, and bleeding encountered during live gastrotomy. AT viewing time positively correlated with mean total video score for Group T during both sessions. None of the students had prior experience performing hollow organ closure and no significant difference in experience level was evident between groups. Mean closure time was not significantly different between groups for session one (Group T, mean, 31.5 minutes, range, 18.4 to 53.4; Group S, mean, 28.2 minutes, range, 16.8 to 36), but was significantly reduced for session two (Group T, mean, 21.3 minutes, range, 13.9 to 31). This AT/simulator program does not significantly influence students' overall gastrotomy closure technique; gross and video evaluation scores were not significantly different between groups. Without instructor supervision, an additional gastrotomy experience did not improve surgical technique appreciably for Group T; however, these students performed the second procedure with more confidence and speed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532883 TI - Restriction of HIV-1 replication in intestinal cells is genetically controlled by the gag-pol region of the HIV-1 genome. AB - The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis, HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment BssHII255-EcoRI4183) genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level. PMID- 7532884 TI - Measles virus antisense sequences specifically cure cells persistently infected with measles virus. AB - Vectors expressing antisense mRNAs complementary to the measles virus (MV) nucleoprotein N or hemagglutinin H genes were used to transfect MV-permissive Vero cells, MV-nonpermissive C6 rat glioma cells, and C6 cells persistently infected with measles/SSPE virus (C6/SSPE cells). Transfected Vero cells infected with MV showed a drastically reduced yield of infectious virus (90-99.99%). In plaque assays, plaque numbers and plaque size were significantly reduced compared with untransfected Vero cells. With an unrelated control virus, VSV, no effects were seen in the transfected Vero cells, underlining the specificity for MV. Following stable transfection with MV antisense vectors, C6 rat glioma cells, which are normally suitable to establish persistently MV-infected lines, can no longer be infected with the virus. In this case also, VSV infection was not influenced. Furthermore, antisense transfection of already persistently infected C6/SSPE cells leads to a loss of MV-specific immunofluorescence, concomitant with a disappearance of viral RNA. Single cell clones from the antisense-transfected C6/SSPE cells appear to be totally free of virus in cocultivation with Vero cells, suggesting that they are really cured. The effectiveness of even low amounts of antisense sequences suggests that they are good candidates for antisense oligonucleotide therapy in tissue culture and might eventually also be useful for in vivo application. PMID- 7532886 TI - [Morphologic changes of peripheral lymphoid tissue and liver in experimental animals under food deprivation]. AB - Morphologic examinations of liver, spleen, mesenteric lymph nodes and groups of lymphatic follicles of small intestine (Peyer's patchs) of rats in condition of starvation were shown. Starvation of animals for 48 hours and most for 96 hours was caused statistic reliable decrease of body mass, concerning mass of thymus and liver. Centrolobular fatty infiltration and decrease of RNA and glycogen contents in hepatocytes of liver were shown. Decrease of content of small lymphocytes of T-dependent zones and alteration of cell's representation in B dependent zones were discovered. PMID- 7532887 TI - [Assisted communication]. AB - The basic rules of communication are discussed in order to find out whether communication is also possible via technical media, such as audiovisual conferences, and via the dissimination of data in printed media. With some restrictions audiovisual media may be helpful as a means of communication as they allow a detailed discussion over the individual patient and his particular problem. Printed media are helpful as a means of communication if the information given there is discussed with those colleagues who are familiar with the patient in order to establish whether it can be applied in this particular case. If this information cannot be applied because of a problem between the doctor and his patient, a discussion in a Balint-group may be helpful. PMID- 7532885 TI - An immunological analysis of Ty1 virus-like particle structure. AB - We present an immunological characterization of the Ty1 virus-like particle (VLP). A panel of monoclonal and polyclonal antibodies were raised against the TYA particle-forming protein. Using these antibodies in epitope availability assays two N-terminal regions of the TYA protein were mapped projecting from or at the surface of the proteinaceous shell of the VLP. Two different C-termini of the TYA protein, corresponding to the C-terminus of the full-length and truncated forms, were seen to be buried within the particle core and not available for antibody binding. RNase accessibility studies demonstrated a difference in the porosity of the protein shell surrounding the Ty1 nucleic acid between different particle types, suggesting differences in subunit organization. PMID- 7532888 TI - A phenolic antioxidant extracted from olive oil inhibits platelet aggregation and arachidonic acid metabolism in vitro. PMID- 7532890 TI - Sequence of MKT1, needed for propagation of M2 satellite dsRNA of the L-A virus of Saccharomyces cerevisiae. AB - MKT1 is required for maintenance of K2 above 30 degrees C in strains with the L-A HN variant of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. We report that MKT1 encodes a 92,979 Da protein with serine-rich regions and the retroviral protease signature, DTG, but with no substantial homology to proteins presently in the databases. PMID- 7532891 TI - [Media and prevention: possibilities and limitations of obtaining experience]. PMID- 7532889 TI - Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences. AB - We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively. PMID- 7532892 TI - [Whipple disease with negative duodenal histology--a case report and review of the literature]. AB - The arthritis of Morbus Whipple is an early clinical manifestation of this rare disease. As the arthritis precedes the gastrointestinal symptoms, very often it is an important early diagnostic clue in this potentially fatal disease. The classical method in establishing the diagnosis is to show the characteristic histologic features in the affected tissues or in small bowel biopsy. Employed in the latter are usually multiple directed biopsies during duodenal endoscopy. This case report gives an example that the duodenum may be spared during active Whipple's disease and even affected tissues must not show the characteristic features of Morbus Whipple. PMID- 7532893 TI - Iron uptake by plasma cells in haematological disorders. AB - Bone marrows of 38 patients have been investigated for iron-containing plasma cells. Two patterns emerged. One consisting of iron-laden plasma cells involving myeloma, iron overload and megaloblastic anaemia patients. The second showing no iron in plasma cells of patients with anaemia of chronic disease (ACD). Electron microscopy was done on 3 patients who had excess iron in their bone marrow particles and plasma cells. Perls' stain and scanning transmission electron microscopy were used to confirm the presence of iron in bone marrow plasma cells. The results show that the uptake of iron by plasma cells is dependent on marrow iron stores and the availability of the iron for uptake. PMID- 7532894 TI - Improved recovery of myelosuppression following chemotherapy in mice by combined administration of PSK and various cytokines. AB - Granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were used in combination with PSK, a protein-bound polysaccharide extracted from mycelium of Coriolus versicolor (strain CM101), in myelosuppressed mice. The myelosuppression model consisted of BDF1 mice who received 150 mg/kg 5-fluorouracil (5-FU) intravenously. The peripheral blood leukocyte count during the recovery stage was significantly increased when these cytokines were administered with PSK compared to when the cytokines were used individually. In vitro colony assay revealed that the combination of PSK and any of GM-CSF, IL-3 or stem cell factor (SCF) showed a greater increase in colony numbers than when these materials were administered individually, although G-CSF did not show a synergistic effect with PSK. When bone marrow cells were obtained from mice which had been given PSK or IL-3, the colony assays were made in the presence of PSK or IL-3 in vitro. The greatest increase in the numbers was observed in colonies of the cultured group in the presence of IL-3 after the PSK priming. However, the colony formation potential of PSK was not inhibited by addition of anti-SCF antibody. The above results indicate that the combined administration of PSK with G-CSF, GM-CSF or IL-3 increased the hematological recovery of myelosuppressed mice. Moreover, the phase at which PSK has effects on hematopoietic cells seems to be at a more immature level than with IL-3. The combined administration of PSK and the above cytokines may improve myelosuppression after chemotherapy in patients with malignancy. PMID- 7532895 TI - A case of tetrasomy 9p. AB - A case of mosaic 9p tetrasomy (46,XX/47,XX, + dic[9] [q21]) is reported. Clinical manifestations of the patient were generalized hypotonia, severe mental retardation and characteristic dysmorphic features of 9p tetrasomy. A brief review of the literature is also included. PMID- 7532896 TI - Nitric oxide-like activity in guinea pig colon as determined by effector responses, bioassay and chemiluminescence analysis. AB - The role of nerve-induced release of nitric oxide (NO) as a modulator of neuroeffector transmission was studied in the longitudinal muscle of the guinea pig colon. The biological activity of a vascular relaxing factor released by nerve stimulation was examined in a bioassay cascade system. Furthermore, biochemical measurements of nerve-induced release of the NO metabolite nitrite (NO2-) were made with a chemiluminescence technique. Transmural nerve stimulation elicited contractile responses that were partly blocked by atropine and further inhibited after additional application of the tachykinin receptor antagonist CP 96, 345. The NO-synthase inhibitor N omega-nitro-L-arginine (NOARG) enhanced the nerve-induced contractions and concomitantly increased the basal degree of contraction ('tone'). The relaxations obtained by nerve stimulation after treatment with atropine and histamine were inhibited by NOARG. Electrical stimulation of the guinea pig colon released a non-adrenergic non-cholinergic (NANC) vascular relaxing factor into the tissue superfusate. The half-life of this factor down the cascade was the same as that observed with exogenous application of NO NOARG and tetrodotoxin (TTX) inhibited the release of the relaxing factor. During transmural nerve stimulation there was a significant increase in NO/NO2- release. This increase was inhibited by TTX and N omega-nitro L-arginine methyl ester (L-NAME). In conclusion, pharmacological analysis as well as bioassay and biochemical measurements suggest that NO is released during nerve stimulation in the guinea pig colon, where it mediates smooth muscle relaxation. PMID- 7532898 TI - Ca(2+)-activated K(+)-channels from isolated type I carotid body cells of the neonatal rat. PMID- 7532897 TI - [Interventions in symbolic psychotherapy]. AB - Symbolic Psychotherapy is a psychotherapeutical technique based on the use of images produced in a patient's mind during a guided daydream. Later on, a symbolic interpretation of daydream is performed by patient. A description of a psychotherapist's tasks during patient's imaginary re-creation--bases and aims of such tasks--are discussed. PMID- 7532899 TI - Nitric oxide synthase occurs in neurons and nerve fibers of the carotid body. PMID- 7532900 TI - No mimics O2 in the carotid body chemoreception. PMID- 7532901 TI - Mechanisms of carotid body inhibition. PMID- 7532902 TI - Substance P inhibits ventilation in the goat. PMID- 7532903 TI - Central glutamate and substance-P in the hypoxic ventilatory response. PMID- 7532904 TI - Carotid chemoreceptor activity and heart rate responsiveness to hypoxia after inhibition of nitric oxide synthase. PMID- 7532905 TI - Ionic channels in type I carotid body cells. PMID- 7532906 TI - Role of intracellular pH and [Ca2+]i in acid chemoreception in type-I cells of the carotid body. PMID- 7532907 TI - Fas and Fas ligand: a death factor and its receptor. PMID- 7532908 TI - Bradycardia- and tachycardia-dependent termination of ventricular bigeminy: mechanism of ventricular extrasystoles with fixed coupling. AB - Fourteen men with intermittent ventricular bigeminy were selected for this study because coupling intervals of the extrasystoles were considerably long and usually fixed, and bradycardia-dependent (10 cases) and/or tachycardia-dependent (12 cases) termination of bigeminy occurred. In all cases, when the heart rate ranged between two certain values, ventricular bigeminy with fixed-coupled extrasystoles was sustained. In all cases showing bradycardia-dependent termination, bigeminy was suddenly terminated with no changes in coupling of the preceding extrasystoles when the heart rate was decreased below a certain lower value. In all cases showing tachycardia-dependent termination except one, when the heart rate increased beyond a certain higher value, coupling intervals gradually lengthened until bigeminy was terminated. These findings strongly suggest the possibility that, in a considerably large number of clinical cases, ventricular extrasystoles with fixed coupling are caused by longitudinal dissociation of conduction in the reentrant pathway of extrasystoles. PMID- 7532909 TI - Biologic characterization of hereditary non-polyposis colorectal cancer. Nuclear ploidy, AgNOR count, microvessel distribution, oncogene expression, and grade related parameters. AB - The identification of hereditary non-polyposis colorectal cancer (HNPCC) is important not only for the patient, but also for family members who are at increased risk of developing cancer. To determine if measuring various pathobiologic features of the colon carcinomas is useful in separating sporadic from HNPCC tumors, the authors studied tumor tissues from 46 patients with HNPCC and compared them to 70 with sporadic colorectal carcinoma. Parameters investigated included DNA ploidy (flow cytometry), AgNOR count (by silver staining), microvessel density (immunohistochemistry), p53 and K-ras expression, and grade-related parameters. Diploid tumors were more frequent in patients with HNPCC (65% vs 40%, P < .02), thus confirming previous observations concerning such an association. Higher AgNOR counts and greater AgNOR areas were observed in sporadic tumors than in HNPCC (5.2 +/- 1.5 vs 4.5 +/- 1.8, P < .01). Hereditary tumors tended to be less vascularized, whereas oncogene expression and grade related parameters did not show appreciable differences between the two types of tumors. In conclusion, some of the investigated parameters may contribute to defining the biologic profile of HNPCC. In addition, these findings support the clinical impression of a more favorable outcome that is frequently seen in HNPCC patients. PMID- 7532910 TI - Chondroid chordoma. A hyalinized chordoma without cartilaginous differentiation. AB - "Chondroid chordoma" is a controversial and confusing entity that was originally described by Heffelfinger and colleagues as a biphasic malignant neoplasm possessing elements of both chordoma and cartilaginous tissue. Because the premise for this distinction was based strictly on histomorphologic criteria, the light microscopic, immunohistochemical, and electron microscopic features of the chondroid and chordoid areas of five chondroid chordomas of the skull base were evaluated separately, and compared to five typical chordomas and six low grade chondrosarcomas. Using light microscopy, chondroid chordoma revealed areas that resembled typical chordoma (chordoid areas) and areas that resembled low grade chondrosarcoma (chondroid areas). However, both the chordoid and chondroid areas had an epithelial phenotype and stained strongly for cytokeratin and EMA as well as S-100. 5'-nucleotidase, an enzyme that has been described in chordoma but not in chondrosarcoma, was found in both the chordoid and chondroid areas of one chondroid chordoma. Electron microscopic studies of both the chordoid and chondroid areas in four of the tumors demonstrated both tonofibrils and desmosomes. Chordoma demonstrated immunohistochemical and electron microscopic features that were nearly identical to chondroid chordoma. Chordoma was cytokeratin, EMA, S-100, and 5'-nucleotidase positive. Ultrastructurally, chordoma exhibited variably-sized vacuoles, abundant rough endoplasmic reticulum (RER), and desmosomes with tonofilaments. In contrast to chondroid chordoma, chondrosarcoma consistently stained for only S-100 protein and was cytokeratin, EMA and 5'-nucleotidase negative. Ultrastructurally, chondrosarcoma demonstrated a flocculogranular matrix, glycogen, abundant RER, and scalloped cellular outlines, but lacked desmosomes with tonofilaments. These findings indicate that "chondroid chordoma" is a variant of chordoma with histologic features that may mimic chondrosarcoma. Despite the resemblance of these hyalinized areas to cartilaginous tissue, these tumors retain their epithelial phenotype. Biphasic differentiation is not present. These findings undermine the original premise for distinguishing "chondroid chordoma" from typical chordoma. The authors propose that these tumors be classified as "hyalinized chordomas," rather than "chondroid chordoma," to clarify their histogenesis and avoid confusion with chondrosarcomas of the base of the skull. PMID- 7532913 TI - Ectopic sebaceous glands in the esophagus. PMID- 7532911 TI - Clinical evaluation of the slide centrifuge (cytospin) gram's stained smear for the detection of bacteriuria and comparison with the FiltraCheck-UTI and UTIscreen. AB - The cytospin Gram's stained smear method for the detection of bacteriuria was compared with the FiltraCheck-UTI, the UTIscreen and culture of urine specimens from adult male and female inpatients and outpatients in a tertiary care medical institution. At a level of > or = 10(5) CFU/mL, the sensitivity of all three methods was at least 93% for organisms identified as potential pathogens. Investigation of false-negative screening tests by chart review demonstrated that only 5 of 10 combined misses in this category were clinically significant. At a level of > or = 10(4) CFU/mL, the sensitivity of the cytospin method remained essentially unchanged, whereas that of the FiltraCheck-UTI and the UTIscreen decreased to 89% and 85%, respectively. False positives were most frequent with the FiltraCheck-UTI, which was due in part to the presence of white blood cells in the urine. All three tests performed well in this population of patients. PMID- 7532914 TI - Palliation of malignant dysphagia: carvers versus plumbers. PMID- 7532912 TI - Tacrolimus (FK 506), a treatment for primary sclerosing cholangitis: results of an open-label preliminary trial. AB - Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the liver that is characterized by progressive cholestasis and the development of secondary biliary cirrhosis. There is no widely recognized therapy for this disease, although anti-inflammatory agents (steroids), immunosuppressive agents (methotrexate), anti-fibrotics (colchicine), and choleretic agents (ursodeoxycholic acid) have been used in various small series. In the present study, Tacrolimus (FK 506), a new and powerful immunosuppressive macrolide antibiotic, has been used to treat 10 patients with PSC. Each subject had a liver biopsy, ERCP with visualization of the intra- and extrahepatic biliary tree, and a panel of hematological, serological, and biochemical laboratory tests before the initiation of the FK 506 therapy. The FK 506 was administered orally at 12-h intervals and was monitored by serial plasma FK 506 trough levels. After 360 days of treatment, the median serum bilirubin level was reduced by 75%, and the serum alkaline phosphatase was reduced by 70%. Moreover, the serum ALT and AST levels were reduced by 80 and 86%, respectively. No change in the serum level of BUN and creatinine levels occurred as a consequence of the FK 506 treatment. These data demonstrate that: 1) FK 506 can be used to treat PSC; 2) the response to FK 506 by patients with PSC is rapid; and, 3) no adverse effect on the serum BUN and creatinine levels was observed. It is anticipated that FK 506 will become an important agent for the treatment of patients with PSC because of its powerful immunosuppressive activity. PMID- 7532915 TI - Cryoglobulinemic glomerulonephritis: a membranoproliferative glomerulonephritis induced by hepatitis C virus. AB - Mixed cryoglobulins (MCs) are proteins that precipitate from cooled serum, and are composed of a polyclonal immunoglobulin G (IgG) bound to another immunoglobulin that acts as an anti-IgG rheumatoid factor (RF). In type II mixed cryoglobulinemia, the antiglobulin component, usually of the IgM class, is monoclonal; it is polyclonal in type III mixed cryoglobulinemia. The majority of MCs are found in patients with connective tissue diseases, infectious or lymphoproliferative disorders, hepatobiliary diseases, or immunologically mediated glomerular diseases (secondary MCs). The etiology is not clear for 30% of all MCs, and this type of cryoglobulinemia is called "essential." There is a common clinical syndrome in types II and III essential mixed cryoglobulinemia (EMC) characterized by purpura, weakness, and arthralgia. In type II EMC only, in which an IgMk is the monoclonal RF, a membranoproliferative glomerulonephritis (MPGN) occurs with some peculiar morphologic and clinical features; this is termed "cryoglobulinemic GN." Glomerulonephritis can be differentiated from idiopathic MPGN, especially in the acute stage, which is characterized by an acute nephritic syndrome, by the following findings: (1) the presence of large deposits filling the capillary lumen that sometimes are shown to have a characteristic fibrillar or crystalloid structure by electron microscopy; (2) the extent of the exudative component consequent to the frequently massive infiltration of monocytes; (3) a more diffuse and evident thickening of the glomerular basement membrane, which has a double-contoured appearance that is mainly due to the peripheral interposition of monocytes, with less evident mesangial expansion; and (4) possibly some vasculitis in small and medium-sized renal arteries without concomitant features of segmental necrotizing GN or crescentic GN.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532916 TI - Hepatitis C virus infection among chronic dialysis patients in the south of France: a collaborative study. AB - During the last quarter of 1992, 984 patients from 13 dialysis centers in the Provence-Alpes-Cote-d'Azur region in France participated in a multicenter cross sectional study to determine the prevalence, the risk factors, and the clinical consequences of infection by the hepatitis C virus (HCV). Serum samples were tested for anti-HCV antibodies using second-generation enzyme-linked immunosorbent assay (ELISA). In the case of a positive result, a combination test was performed using second-generation recombinant immunoblot (RIBA) or direct detection of HCV-RNA by nested polymerase chain reaction (PCR). Collected data included the patient's age, gender, cause of the kidney disease, type of dialysis treatment, number of years on dialysis, weekly dialysis time, drug addiction, co infection with hepatitis B virus and human immunodeficiency virus (HIV), number of kidney transplants, number of blood transfusions, and history of acute or chronic hepatitis. Chronic HCV infection was detected in 232 (23.6%) patients, whereas only 71 (7.2%) were infected by HBV. Logistic-regression analysis showed that HCV infection was associated with dialysis over a long period, numerous blood transfusions, female gender, kidney grafts, HBV infection, hemodialysis, and acute as well as chronic hepatitis. Multiple-correspondence analysis confirmed that the contamination was both transfusional and nosocomial. These results underscore the need for a strict compliance with "universal precautions" (Centers for Disease Control [CDC], Atlanta) in dialysis units and raise the question as to whether anti-HCV-positive patients should be isolated. PMID- 7532917 TI - Interferon treatment of Mooren's ulcers associated with hepatitis C. AB - PURPOSE/METHODS: The cause of Mooren's ulcer is unknown. We examined a patient with chronic hepatitis C who had a corneal ulceration that resembled Mooren's corneal ulcer. RESULTS/CONCLUSION: The corneal ulceration progressed despite appropriate medical and surgical interventions. Recombinant interferon alpha 2b finally led to resolution of the process. The resolution correlated with normalization of the patient's liver function tests. Corneal ulcerations that resemble Mooren's ulcer may be associated with hepatitis C. Recombinant interferon alpha 2b seems to induce remission in the corneal as well as the systemic condition. PMID- 7532918 TI - The utility of basal cell-specific anti-cytokeratin antibody (34 beta E12) in the diagnosis of prostate cancer. A review of 228 cases. AB - Basal cell-specific anti-cytokeratin antibody (34 beta E12) decorates the basal cells of benign prostatic epithelium by standard immunohistochemical techniques, whereas adenocarcinoma of the prostate lacks immunoreactivity with this antibody. We reviewed our experience with this antibody to determine its utility in the diagnosis of adenocarcinoma of the prostate as well as its pattern of usage at a tertiary medical center. In all, 7,242 prostate specimens from 5,262 men were seen at Johns Hopkins Hospital between 1/89 and 4/93. Immunostaining for basal cell-specific cytokeratin (34 beta E12) was originally used for diagnostic purposes in 289 questionable area from 228 cases; 45% of these cases were seen in consultation. The distribution of cases using 34 beta E12 was 52% needle biopsies, 32% transurethral prostatic resections (TURPs). 13% radical prostatectomies, and 3% open enucleations. These procedures constituted 2.8% of all needle biopsies, 7.2% of all TURPs, 1.7% of all radical prostatectomies, and 3.5% of all enucleations seen during this time period. For this study the hematoxylin and eosin stain was reviewed without knowledge of the original diagnosis, a diagnosis was favored, the 34 beta E12 stain was examined, and a final diagnosis was determined. The 34 beta E12 stain established (14%), confirmed (58%), or changed (2%) our favored diagnoses, while 18% remained or became equivocal. The 34 beta E12 stain was of no use in 8% of the cases, yet we felt we were still able to render a final diagnosis even without the help of the stain. The differential diagnoses in the questionable foci using 34 beta E12 were cancer versus focus of atypical glands (44%), adenosis (39%), prostatic intraepithelial neoplasia (PIN) (8%), basal cell hyperplasia (5%), and atrophy (4%). However, 34 beta E12 was used in only 15-20% of all cases of adenosis and basal cell hyperplasia and in < 2% of PIN and atrophy cases seen during this time. Reasons for equivocal results were loss of suspicious glands on cut downs used for staining (49%), too few glands to rely on negative staining (23%), technical problems (15%), limited number of positive staining glands in a small focus (7%), and cautery artifact (6%). Although equivocal cases tended to have fewer negative stained glands than cases diagnosed with cancer, there was no minimum number of negative stained glands required to establish a diagnosis of cancer. From these data we conclude that 34 beta E12 staining is a useful tool in confirming, establishing, or changing the diagnosis in questionable foci seen in the everyday practice of surgical pathology.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532919 TI - Clinicopathologic study of CD56 (NCAM)-positive angiocentric lymphoma occurring in sites other than the upper and lower respiratory tract. AB - The expression of the neural cell adhesion molecule (NCAM) (CD56, NKH-1) is a rare phenomenon in malignant lymphoma. Recently, several authors, including our group, described the clinicopathologic, phenotypic, and genotypic features of NCAM-positive tumors as a unique subgroup within a larger category of hematolymphoid malignancies. Ten cases of CD56+ angiocentric lymphoma occurring in sites other than the upper aerodigestive tract were studied for evaluating their characteristics. The disease occurred in six men and four women varying from 24 to 85 years (mean age, 53 years) who often exhibited a striking predilection for extranodal sites of involvement, such as the skin, gastrointestinal tract, and muscle, usually in the absence of peripheral lymphadenopathy. Although the cytologic appearances and immunophenotypic profile varied from case to case, these tumors often exhibited azurophilic granules, an angiocentric growth pattern, and surface CD3-, T-cell receptor (TCR) antigens-, and CD56+ phenotype without B-cell phenotype, except for a single case of CD3+, TCR alpha/beta+, and CD56+ phenotype. Genotype investigation exhibited germline configuration of the TCR beta and gamma chain genes and the immunoglobulin heavy chain gene in all five cases of surface CD3- phenotype examined, whereas the case of CD3+ phenotype showed rearrangement of TCR beta. They seem to constitute a distinct entity of the lineage spectrum spanning from natural killer (NK) cell to NK-like T cell. PMID- 7532920 TI - CD-34 expression. PMID- 7532921 TI - Evaluation of an enzyme immunoassay for the detection of Cryptosporidium spp. in stool specimens from infants and young children in field studies. AB - Diagnosis of Cryptosporidium is made by the identification of oocysts in stool specimens. Screening in field studies relies mainly on acid-fast staining followed by microscopic examination. The more sensitive immunofluorescent antibody (IFA) staining method is time-consuming, may involve technical difficulties, and is extremely costly as a screening procedure in field studies. We evaluated the diagnostic utility of a commercially available enzyme immunoassay (EIA), which detects Cryptosporidium-specific antigen, in 204 unprocessed stool specimens obtained from patients less than three years of age from a field study in southern Israel. When compared with the routine screening procedure applied in this field study (screening by acid-fast staining and microscopy after concentration, and confirmation of positive results by IFA), both the sensitivity and specificity were 98%. Of 139 specimens negative by microscopy, 13 (9.3%) were positive by the EIA. Eleven of these were confirmed by inhibition with antibody to Cryptosporidia-specific antigen. The EIA is an important tool for identifying Cryptosporidium in fecal specimens in field studies since it is sensitive, specific, simple to use, and unaffected by the presence of a preservative. PMID- 7532922 TI - [Use of Diprivan in allergic patient]. AB - Propofol may be indicated in patients with a history of allergy provided some precautions are taken. At present, the only contra-indication for propofol are patients with a known allergy to muscle relaxants. Any anaphylactoid reaction has to be systematically reported (e.g. to the Drug Safety Department of Zeneca Pharma) and the patient tested according to the standard protocol, in order to further disclose the underlying mechanisms and evaluate the risk factors. Periodical analysis of notifications of adverse reactions will facilitate regular epidemiological studies and the update of the information on the drug for the anaesthetic community. PMID- 7532924 TI - Antithyroid drugs and radioiodine therapy. PMID- 7532925 TI - Radioiodine therapy in Graves disease. PMID- 7532923 TI - Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation. AB - The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm2 for 1 min at 37 degrees C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm2, whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin alpha v beta 3) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm2; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same beta 3 subunit as GP IIb-IIIa, plays no role in SIPA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532926 TI - [Palliative treatment of malignant effusions by thoracoscopy under video assistance: morbidity and survival]. AB - Survival after surgical palliation of patients with malignant pleural effusions is expected to be of several months. From a group of 75 patients operated by video-assisted thoracic surgery (VATS) from September 1991 through March 1993, twenty-two of 38 patients with malignant disease underwent palliative surgery and operative morbidity and length of survival were studied. Palliation consisted in 20 talc pleurodesis and 2 pericardial windows for malignant effusions. In the 16 other patients with malignancy, video-thoracoscopy was done for diagnostic purposes. Thirty-day operative mortality was 13.6% (3/22) and the rate of operative morbidity was 31.8% (7/22). The follow-up was complete for all patients with a mean length of 5 months (+/- sd 4 months). During the follow-up period, 14 of 19 (74%) patients, alive 30 days after the procedure, were deceased at a mean of 3.4 +/- 4 months (6 patients < 3 months) after the operation. In all, 9 of 22 patients (40%) survived less than 3 months (median survival 4 months). Operative morbidity and mortality of video-thoracoscopy are high in the palliative group and only 60% of patients survived more than 3 months. More stringent criteria for selection of patients for palliative endosurgery should be defined. PMID- 7532927 TI - The inhibition of apoptosis by alpha-fetoprotein (AFP) and the role of AFP receptors in anti-cellular senescence. AB - The mechanism of paradoxical growth enhancement by alpha-fetoprotein (AFP), a nonmitogenic factor, was explored in HL-60 cells using novel AFP agonists, the 167H.1 and 167H.4 monoclonal antibodies (MAbs) to AFP receptor (AFPr) isoforms. The conditions underwhich AFP led to increases in thymidine incorporation were found to promote activation induced cell death (AICD) associated with adherence in 96 well plates. The ensuing death was judged to be apoptosis based on morphology, shrinkage of dying cells, inducibility, reversibility, a sensitivity to levels of FCS, kinetics, DNA fragmentation pattern and internal program (passage number) of the cells investigated. Both AFP and the 167H.1 MAb but not the 167H.4 MAb blocked the induction of cell death under these conditions indicating apparent growth enhancement by AFP relates to the abrogation of cell death and not that AFP contains bona fide growth factor-like activity. We additionally report that HL-60 cells spontaneously senescence during normal propagation in vitro which correlated with an increased susceptibility to and earlier kinetics of AICD. In substantiation of this was the correlation of the loss of expression of the AFPr with increasing passage number and the induction of AICD. Overall, our findings lend further support to the recent proposals that AFP by binding to AFP receptors may block cellular senescence and that there may be differential expression of AFPr isoforms related to commitment (167H.1 reactive) or resistance (167H.4 reactive) of cells to apoptosis/programmed cell death (PCD). PMID- 7532928 TI - Effects of doxorubicin on pp60c-src kinase activity in polyoma virus MT antigen transformed cells. AB - Interaction between polyoma virus (Py) encoded middle tumor antigen (MTAg) and the cellular src gene product, pp60c-src, has been shown to be correlated with the enhancement of pp60c-src kinase activity and the transforming ability of the virus. In the present study, FR3T3 cells were transfected with plasmids encoding MTAg and used to study the effects of the antineoplastic agent, doxorubicin (DOX), on pp60c-src kinase activity. Our results showed that transfection of FR3T3 cells with MTAg results in relatively high levels of MTAg expression and enhancement of pp60c-src kinase activity several folds over that observed in the control cells. Treatment of the transformed MTF4 cells with DOX up to 5 x 10(-6) M was shown to have no effect on the in vitro phosphorylation of MTAg and pp60c src in the drug-treated cell lysates. On the contrary, treatment of src immunocomplexes with DOX resulted in a reduction in kinase activity. The inhibition appears to be a dose-dependent competition with respect to ATP concentrations. PMID- 7532929 TI - Significance of the tumour markers CA 125 II, CA 72-4, CASA and CYFRA 21-1 in ovarian carcinoma. AB - We compared the tumour marker CA 72-4 and the new markers CASA and CYFRA 21-1 with the established marker CA 125II in follow-up care and control of efficacy of treatment in ovarian cancer in order to determine whether there are differences in recognizing ovarian carcinomas of different histological type. The investigation was done retrospectively on serum samples frozen at -80 degrees C obtained from 262 subjects, among them 50 healthy women, 53 sera with benign gynecological diseases and 159 sera with ovarian cancer, 72 of them at the time of primary diagnosis. We used commercially available kits: CA 125 II: Centocor RIA, CASA: Medac EIA, CA 72-4: Centocor RIA and CYFRA 21-1: Boehringer EnzymunR ELISA. Fixing specificity at 95% versus benign gynecological diseases as a clinically relevant reference group, we found cut-off values of 160 U/mL for CA 125II, 6.5 U/mL for CASA, 6.8 U/mL for CA 72-4 and 2.4 ng/ml for CYFRA 21-1. Based on this specificity we can compare the corresponding sensitivities at the time of primary diagnosis (n = 72): CA 125 II 47%, CA 72-4 47%; CASA 31% and CYFRA 21-1 44%. Regarding histological types of ovarian carcinomas, we found a sensitivity of 50% for CA 125 II and CASA, 36% for CA 72-4 and 33% for CYFRA 21-1 in serous ovarian cancer (n = 53), 21% for CA 125 II and CASA, 36% for CYFRA 21-1 and 43% for CA 72-4 for mucinous ovarian cancer (n = 27). Serous ovarian carcinomas were classified by higher FIGO-stages than mucinous ovarian carcinomas. Additional sensitivities were found at the time of primary diagnosis for the combination of CA 125 II and CA 72-4 and in serous ovarian cancer for CA 125 II and CASA. According to our results, at the time of primary diagnosis the combined determination of CA 125 II and CA 72-4 is useful. If both are negative, determination of CASA can be helpful. For follow-up care and control of efficacy of treatment the preoperative positive or leading marker is sufficient. PMID- 7532931 TI - Diabetes mellitus in pancreatic cancer follow-up. AB - The aim of this study was to assess the behavior of fasting serum glucose, C peptide levels and OGTT in pancreatic cancer follow-up. We studied 49 patients with pancreatic cancer (stage I = 8 pts; II = 16 pts; III = 12 pts; IV = 13 pts). At diagnosis 13/49 patients had fasting serum glucose levels of above 140 mg/dL. Of the remaining 36 pts, 22 underwent OGTT, which indicated diabetes mellitus in 9/22 (41%) and impaired glucose tolerance in 7/22 (32%) cases. C-peptide basal values were within the normal range (0.8-2.0 micrograms/L) in 14/49 (28%), above 2.0 micrograms/L in 6/49 (13%) and below 0.8 micrograms/L in 29/49 (59%) of the cases. No significant correlation was found between tumor stage or size and the presence of diabetes or of a reduced glucose tolerance. Twenty-four patients underwent curative resection (group 1) and 16 palliative resection, while the remaining nine did not undergo surgery (group 2). Group 1 and 2 patients had a follow-up of 2 to 40 months (mean = 14 months) and from 1 to 7.5 months (mean = 3.5 months) respectively. In group 1 patients no significant difference was found between pre- and post-operative fasting serum glucose levels. However, in 11/15 (73%) patients who underwent OGTT before and after surgery, an improvement in glucose tolerance was observed after tumor resection. In group 2 patients, a significant increase in fasting serum glucose levels was found during follow-up. In neither of the groups studied were significant variations found in C-peptide levels during the follow-up, although a slight increase was observed in patients who did not undergo surgery. In conclusion, the reduced glucose tolerance or frank diabetes mellitus, which frequently occurs during the onset of pancreatic cancer, does not seem to be related to tumor stage or size. Curative resection ameliorates glucose intolerance, while tumor persistence can enhance serum glucose levels. PMID- 7532932 TI - Introduction of the cytological grading, the nuclear area, the DNA index and the DNA histogram type in the setting up of a score for ductal breast carcinoma. AB - The present study describes the setting up of a new score which makes it possible objectively to grade ductal breast carcinomas, i.e. not-otherwise-specified (NOS) cancers, on cellular material from fine-needle aspirations (FNAs). For this purpose FNAs from 252 patients--with NOS breast cancers--were smeared onto histological slides, fixed in an ethanol-formolacetic acid mixture, Feulgen stained and analysed by means of a cell image processor. Four parameters were taken into account in setting up the score, namely the cytological prognostic grade (CPM) of malignancies similar to the Scarff-Bloom-Richardson (SBR) grading, the nuclear area (NA), the DNA index (DI) and the DNA histogram type (DHT). Each of these four parameters was considered as a "sub-score" which may take three values, i.e. 1, 2 and 3. The final result may thus range from 4 to 12. Subscores of 4 and 5 correspond to a cytological score of I, subscores of 6, 7 and 8 to a cytological score of II, sub-scores of 9 and 10 to a cytological score of III, and sub-scores of 11 and 12 to a cytological score of IV. In the present study, the results show 17% of CPM grade 1.52% of CPM grade II and 31% of CPM grade III cancers. All the cases exhibiting a cytological score of IV (5%) fully fit in with the CPM grade III cancers. In the same way, none of the cases exhibiting a score of I fit in with CPM grade III cancers. The cancers with a CPM grade II fit in with the scores of II and III. It thus seems possible to convert a three-value malignancy grading system (CPM and/or SBR grading) into a four-value one (cytological score). The main advantage in this latter type of system is that it becomes possible to split up the over-large group of CPM grade II cancers. As things stand, we are unable to give any prognostic value for the score proposed here because our study is prospective only. A study of this type has been necessary so as to provide against problems connected with ways of preserving specimens that might be used in a retrospective study. The bank of clinical and biological data now in existence must be allowed to mature for a number of years before the prognostic worth of the cytological score can be established, always assuming that such a value exists. PMID- 7532930 TI - Balloon occluded arterial infusion (BOAI) treatment of patients with locally advanced cervical carcinoma. AB - Nineteen patients with locally advanced inoperable carcinoma of the uterine cervix (8 with stage IIb, 8 with stage IIIb and 3 with stage IV) were treated with a cisplatin (100 mg/m2), aclacinomycin (30 mg/m2) and bleomycin (20 mg/m2) combination using balloon occluded arterial infusion (BOAI), and 5 patients (2 stage IIb, 1 stage IIIa and 2 stage IIIb) received the same drugs intravenously (i.v.). Partial response was observed in 5 patients (26%) and minor response was observed in 11 patients (58%) after one course of BOAI treatment. The free platinum concentration achieved by BOAI was significantly greater than that achieved by i.v. administration. The intratumoral concentration of total-platinum was significantly higher in the BOAI-treated group than in the i.v. infusion group. Higher concentrations of total-platinum were found in the uterine corpus and cervical tumor as compared with those in the ovaries or lymph nodes. The antitumor effect of BOAI was confirmed by histological examination of tumor tissues. These results demonstrated that BOAI, which delivers the drug locally in a high concentration, provides an effective means for the treatment of locally advanced cervical cancer. PMID- 7532933 TI - Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro. AB - The effect of three macrolide antibiotics, midecamycin acetate, josamycin, and clarithromycin, on human T-cell function was investigated in vitro. Midecamycin acetate and josamycin suppressed the proliferative response of peripheral blood mononuclear cells stimulated by polyclonal T-cell mitogens at concentrations between 1.6 and 8 micrograms/ml. At higher concentrations (40 to 200 micrograms/ml), all these drugs showed a marked inhibitory effect. At concentrations of 1.6 to 40 micrograms/ml, these drugs suppressed interleukin-2 (IL-2) production induced by mitogen-stimulated T cells, but not the expression of IL-2 receptor (CD25), in a dose-dependent manner. Therefore, the suppressive action on T-lymphocyte proliferation seems to be based on the ability of these drugs to inhibit IL-2 production by T cells. The drug also inhibited mixed lymphocyte reaction at the same concentrations. Combined treatment with these macrolides and the known immunosuppressants such as FK506 and cyclosporin A resulted in an increased inhibition of T-cell proliferation. The immunomodulatory properties of the antibiotics may have clinical relevance for modulation of the immune response in transplant patients and in patients with inflammatory diseases. PMID- 7532934 TI - Production and analysis of transgenic mice with ectopic expression of parvalbumin. AB - Transgenic mice expressing rat parvalbumin under the control of the human metallothionein IIA (MTII A), SV-40 early, and neuron-specific enolase (NSE) promoters were produced. Ectopic expression was analyzed by RNA polymerase chain reaction and RNase protection in combination with immunohistochemistry. From a total of 25 transgenic lines 18 were found to express the transgene. Expression strength and tissue specificity were dependent upon the promoter used and varied considerably among animal lines produced with the same construct. Highest constitutive MT IIA-driven expression was found in lung, liver, heart, and kidney, as well as in brain, and lower amounts of transgene expression were found in spleen, testis, and muscle. Immunohistochemistry of tissue sections of metallothionein-parvalbumin transgenic strain 29 in the non-induced state revealed that ectopic PV mRNA is translated into protein. Short-term induction of the MT IIA promoter by CdSO4 or CdCl2 leads to a shift in tissue specificity and does not increase ectopic expression in tissues where the transgene is active in the noninduced state. As expected the NSE promoter showed highest activity in brain. However, NSE-driven expression could also be detected to various degrees in all investigated tissues. SV-40-dependent PV expression showed no tissue preference and varied considerably among different strains. Except for the observation that the SV-40-PV construct showed lower yields in transgenic production and reduced numbers of positive offspring no obvious impairment of growth or behavior as a consequence of transgenic PV expression could be detected. PMID- 7532936 TI - Clozapine therapy and increases in homovanillic acid. PMID- 7532935 TI - [Immunohistochemistry in pathology of the breast. Current diagnostic and prognostic contributions]. AB - In breast pathology, immunohistochemistry (IHC) allows the resolution of differential diagnostic problems regarding a primary tumor. With a basic antibody panel, it allows precise identification of undifferentiated, morphologically similar or unusual tumors. In case of doubt on invasiveness, IHC reveals the epithelial basement membrane and myoepithelial cells. A specific marker of breast tissue does not exist at the present time, but IHC guides the physician in the direction of breast in case of unknown secondary tumor or to confirm the primary breast site or to indicate the breast as primary site in case of two morphologically similar tumors. For the prognosis, IHC improves the detection of occult metastases (serous fluids, node, bone marrow, etc.). During the last few years, its indications have increased considerably with the possibility of performing, with paraffin slides, the detection of estrogen and progesterone receptors, growth factors and their receptors, oncoproteins, cell proliferation related proteins, etc. The effects on the prognosis and chemotherapy sensitivity evaluation are obvious. But the pathologists must be very demanding because the clinical use of these new data by the physicians will be possible only after an excellent methodological and clinical validation in relation to the classical prognostic factors. PMID- 7532937 TI - Synthesis of 15N omega-hydroxy-L-arginine and ESR and 15N-NMR studies for the elucidation of the molecular mechanism of enzymic nitric oxide formation from L arginine. AB - N omega-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15N omega-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15N omega-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed. PMID- 7532938 TI - Clinical efficacy of Tranilast on otitis media with effusion in children. AB - In this open randomized study, we evaluated the efficacy of Tranilast, one of the anti-inflammatory drugs, on otitis media with effusion in children. Sixty-two patients (103 ears) were divided into two groups: Group A was given Tranilast and local treatment (nasal and tubal); Group B only received local treatment (control for Group A). The overall improvement rating assessed as "moderately improved or above" for Group A was 63.6%, Group B 47.9%. There was a significant improvement in Group A as compared to Group B (p < 0.05). In subjects who suffered from otitis media with effusion for over 2 months. Group A exhibited 50.0% of efficacy while Group B only 15.4% (p < 0.05). PMID- 7532939 TI - Binding thermodynamics of adenosine A2a receptor ligands. AB - The thermodynamic parameters delta G degree, delta H degree, and delta S degree of the binding equilibrium of seven adenosine agonists and five xanthine antagonists binding specifically to adenosine A2a receptors were determined by means of affinity measurements at six different temperatures (0, 10, 20, 25, 30 and 35 degrees) and van't Hoff plots. Affinity constants were measured on rat striatum membranes by saturation experiments for the selective A2a agonist 2-[p (carboxy-ethyl)-phenethylamino-]5'-(N-ethyl)carboxamidoadenos ine ([3H]CGS 21680) and by inhibition assays of [3H]CGS 21680 binding for all other compounds. Scatchard plots were monophasic in the full range of temperatures, indicating a single class of high affinity binding sites whose receptor density, BMAX, is essentially temperature independent. Van't Hoff plots were linear in the temperature range 0-30 degrees for agonists and 0-35 degrees for antagonists; their thermodynamic parameters fall, respectively, in the ranges 7 < or = delta H degree < or = 50 kJ/mol and 177 < or = delta S degree < or = 278 J K-1 mol-1 and 36 < or = delta H degree < or = -7 kJ/mol and -33 < or = delta S degree < or = 94 J K-1 mol-1, showing that agonist binding is entropy-driven while antagonist binding is enthalpy-driven. The results are compared with those already reported for the binding of the same compounds to rat brain minus striatum adenosine A1 receptors obtained by displacing [3H]CHA as A1 selective radioligand (Borea PA et al., Mol Neuropharmacol 2: 273-281, 1992). The comparison suggests that the two receptors are very similar as far as their binding sites are concerned and possibly philogenetically related. The analysis of thermodynamical data makes it possible to propose an analogical model of drug-receptor interaction which may account for both affinity and intrinsic activity properties. PMID- 7532940 TI - Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin. AB - The effect of natural polyphenols on three isoforms of NO-synthase was investigated. Among the compounds tested, tannin was the most potent, inhibiting endothelial constitutive NO synthase (eNOS) with an IC50 of 2.2 microM. Other NOS isoforms (i.e. neuronal constitutive NOS and smooth muscle inducible NOS) were also inhibited but at much higher concentrations (selectivity ratio of approx. 20 30). Quercetin was also an effective but less potent inhibitor of eNOS (IC50 = 220 microM). The kinetics of tannin inhibition were investigated to gather information on the mechanism of action. Tannin did not interfere with the interaction of the enzyme with the co-substrates L-arginine and NADPH nor with the cofactor tetrahydrobiopterin. The inhibition level was also independent of free Ca2+ concentration as well as of the presence of high exogenous calmodulin concentrations. PMID- 7532941 TI - The modulated receptor hypothesis revisited from the viewpoint of myocardial interstitial potential. AB - Time- and voltage-dependent interaction of antiarrhythmic agents with target cardiac ion channels is termed the modulated receptor hypothesis. Actually class I agents suppress the maximum upstroke rate (Vmax) of intracellular potential (Vic) depending on the pacing cycle length (PCL) and external potassium concentration ([K+]e). We examined this concept from the aspect of interstitial potential (Vis), since Vis reflects the second time derivative of Vic. Vic and Vis were recorded sequentially using standard microelectrode applied to the paced and superfused guinea pig papillary muscles. In the steady state, the greatest negative deflection of Vis (Vmin) was suppressed by quinidine (10 microM) in both PCL- and [K+]e-dependent manner, just like Vmax. However, quinidine-induced greater inhibition of Vmin than Vmax was evident at shorter PCL and greater [K+]e. Based on the sequential alteration of PCL and exposure to ouabain (10 microM), different quinidine sensitivity between Vmax and Vmin is most likely accounted for by the activity-dependent K+ efflux and Na(+)-K+ pump-mediated K+ uptake (i.e., [K+]e fluctuation). Thus, the modulated receptor hypothesis is concluded to be valid in terms of Vis. PMID- 7532944 TI - B-cell epitopes of allergens determined by recombinant techniques; use for diagnosis and therapy of type I allergy. AB - In the present manuscript, the immunological and functional in vitro properties of recombinant plant allergens are summarized. Recombinant tree pollen allergens (major birch pollen allergen-BetvI, birch profilin-BetvII) and recombinant timothy grass pollen allergens (PhlpI, PhlpV, and PhlpII) were compared with the natural counterparts regarding IgE-binding properties and capacity to release histamine from patients' basophils. In addition, experimental in vivo models of Type I allergy, based on recombinant allergens, are discussed. The major conclusion is that recombinant allergens can be seriously considered as candidates for diagnosis of Type I allergy allowing to establish specific allergograms for the individual patients. The in vivo data obtained in mouse and primate systems indicate that recombinant allergens can be used to set up close to-man models of Type I allergy. Such in vivo models are useful to test the effects of already established therapeutic approaches and also allow to develop therapeutical concepts which are based on the use of recombinant allergens. Examples of specific therapeutical concepts are presented. PMID- 7532943 TI - Molecular and functional characterization of allergens: basic and practical aspects. AB - Well-defined allergen preparations are, in the first line, a prerequisite for exact diagnosis, but will be supposedly useful tools in immunotherapy of Type I (IgE-mediated) allergic diseases. The allergens have to be available in standardized and highly purified form in sufficient quantities. By applying recombinant DNA techniques this goal can be achieved with respect to both, characterization and reproducibility of allergen preparations. As an example, purified recombinant non-fusion Betv1 revealed identical immunological properties with respect to interaction with both, anti-Betv1 antibodies and Betv1-specific T cell clones when compared with natural Betv1 purified from birch pollen. Moreover, cloning of allergens yielded a number of deduced primary structures of allergens, which allows computer-aided comparisons with already known amino acid sequences. Significant sequence similarities with well-described proteins may point at a biological and biochemical function of the cloned allergen, which might be of interest for considerations why a certain protein within an extract represents an allergen. Furthermore, the interaction of small peptides synthesized according to amino acid sequences of cloned allergens with allergen specific T and B cells can be investigated. Respective results will yield information about the regulation of IgE synthesis and, thus, might point at new concepts of immunotherapy. PMID- 7532942 TI - Antinucleolar antibodies and their disease association. AB - The prevalence of the antinucleolar antibodies (ANoA) demonstrated by indirect immunofluorescence technique in 1,662 sera of patients with a known or suspected rheumatic disease increased from 1.97% when mouse kidney (MK) was used as substrate to 4.9% when HEp-2 cells were used as substrate. However, an appropriate commercial HEp-2 substrate must be selected in order to increase the sensitivity of ANoA positivity. There were 3 distinct staining patterns of the nucleolar immunofluorescence: homogeneous speckle, and clumpy. Irrespective of the patterns, the most common diagnoses among patients who had ANoA were systemic sclerosis (PSS) and systemic lupus erythematosus (SLE); 36% and 35%, respectively). On the contrary, the incidence of these antibodies in PSS was 41% while it was only 3% in SLE patients. Almost all patients with speckled nucleolar staining had PSS as their underlying disease while most of the patients with homogeneous nucleolar staining had SLE. No distinct correlation between the different nucleolar staining patterns and specific organ involvements in our lupus and PSS patients was found except for the higher frequency of clumpy staining in male scleroderma with no joint involvement. This study demonstrates that: 1) ANoA are uncommon in unselected sera although use of a cell line substrate doubles the rate of positivity; 2) the proper HEp-2 substrate is critical in the detection of ANoA; 3) PSS and SLE are the most frequent diseases associated with ANoA but the frequency of these antibodies in SLE patients was very low.; 4) there are 3 distinct nucleolar staining patterns which may be associated with different rheumatic diseases; and 5) compared with ANoA negative scleroderma, clumpy nucleolar staining had significantly higher incidence in men with no joint involvement but a tendency towards more lung manifestations. PMID- 7532945 TI - T-cell epitopes of allergen molecules. PMID- 7532946 TI - Synthetic T cell epitope peptides used for desensitization: theoretical and clinical considerations. PMID- 7532947 TI - Clinical use of recombinant allergens and epitopes. PMID- 7532948 TI - Enkephalin-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal innervation of the ventrolateral dendritic bundle in the cat sacral spinal cord: an ultrastructural study. AB - The distribution and synaptic arrangement of thyrotropin-releasing hormone-, substance P- and enkephalin-immunoreactive axonal boutons have been studied in the ventrolateral nucleus (Onuf's nucleus) of the upper sacral spinal cord segments in the cat. For this purpose, the peroxidase-antiperoxidase immunohistochemical technique was used. Immunoreactive axonal boutons were traced in complete series of sections in order to reveal synaptic contacts with the bundled dendrites of the ventrolateral nucleus. As judged from the cross sectional diameter of the postsynaptic dendrites, the distribution of immunoreactive boutons was non-random. Enkephalin-immunoreactive axonal boutons, presumed to be mostly of segmental origin, displayed a rather restricted distribution to mainly (> 80%) medium-to-large dendrites. Thyrotropin-releasing hormone-immunoreactive boutons, that derive from supraspinal levels, were also found to impinge on medium-to-large dendrites (> 80%), indicating a proximal location within the dendritic trees. The skewness toward large postsynaptic dendrites was even more marked for thyrotropin-releasing hormone- than for enkephalin-immunoreactive boutons. Substance P-immunoreactive boutons, that are of either supraspinal or spinal origin, showed a more even distribution throughout the dendritic trees, including both thin distal branches and thick proximal dendrites. In view of the well-known fact that virtually all thyrotropin releasing hormone-immunoreactive boutons in the ventral horn co-contain substance P (and serotonin) it was assumed that substance P-immunoreactive boutons in synaptic contact with the finest-calibre dendrites as well as most of those with a very proximal juxtasomatic location on the dendritic trees were of segmental origin, while those impinging on medium-to-large dendrites could be of either spinal or supraspinal origin. Fine-calibre dendrites (< 1 micron) represent about 25% of the dendritic branches in the ventrolateral nucleus, but receive, with the exception of substance P (8%), very little (< 3%) peptidergic or GABAergic (Ramirez-Leon and Ulfhake, 1993) input, although the degree of dendritic membrane covering by bouton profiles in the ventrolateral nucleus does not seem to vary much with the calibre of the postsynaptic dendrite (Ramirez-Leon and Ulfhake, 1993). Both substance P- and enkephalin-immunoreactive axonal boutons established synaptic contact with more than one dendrite. Furthermore, one and the same bouton could be found in contact with two dendrites that were coupled to each other by a dendro-dendritic contact of desmosomal or puncta adherentia type.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7532949 TI - Distribution of secretoneurin-like immunoreactivity in comparison with that of substance P in the human brain stem. AB - Secretoneurin is a peptide of 33 amino acids generated in the brain by proteolytic processing of secretogranin II which is a member of the chromogranin/secretogranin family. The distribution of this newly characterized peptide was investigated by immunocytochemistry in the human brain stem. The staining pattern of secretoneurin-like immunoreactivity was compared with that of substance P in adjacent sections. Secretoneurin-like immunoreactivity appeared mainly in dot- and fiber-like structures with densities varying from low to very high. Only a low number of secretoneurin-immunoreactive perikarya was found. Pericellular staining of both secretoneurin-immunopositive and immunonegative cells was frequently observed in the area of the central gray, in the reticular formation and in the solitary nuclear complex. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, the raphe complex and the inferior olive displayed a high density of secretoneurin-like immunoreactivity. Furthermore, a very prominent staining was found in the medial, dorsal and gelatinous subnuclei of the solitary tract and the dorsal motor nucleus of vagus. The substantia gelatinosa of the caudal trigeminal nucleus and spinal cord were also very strongly secretoneurin immunopositive. The staining patterns of secretoneurin- and substance P-like immunoreactivities were to a certain extent overlapping in several areas. The highest degree of coincidence was found in the substantia gelatinosa. This study demonstrated that secretoneurin is distinctly distributed in the human brain stem. Its distributional pattern indicates a role particularly in the modulation of afferent pain transmission and in the regulation of autonomic functions. PMID- 7532950 TI - Virulence factors and O groups of Escherichia coli strains isolated from cultures of blood specimens from urosepsis and non-urosepsis patients. AB - Ninety-six Escherichia coli strains isolated from cultures of blood specimens taken from both urosepsis patients (n = 48) and non-urosepsis patients (n = 48) were examined for the production of alpha-haemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), the expression of P-fimbriae and mannose resistant haemagglutination (MRHA). Twenty-seven (56%) of the E. coli strains from urosepsis showed some of the virulence factors investigated, whereas only 15 (31%) of the strains associated with non-urosepsis possessed virulence factors (P < 0.05). By contrast, only 16% (P < 0.001) of the faecal isolates from healthy individuals were virulent. Of the bacteremic E. coli strains, 56 (58%) belonged to one of 8 serogroups (O1, O2, O4, O6, O8, O9, O18 and O83). Virulence factors were concentrated in strains belonging to serogroups O2, O4, O6, O18 and O83. Thus, 23 (72%) of the 32 strains of these 5 groups showed virulence factors, but only 19 (30%) of the 64 strains belong to other serogroups (P < 0.001). The majority of bacteremic O2, O4, O6 and O83 E. coli strains were Hly+CNF1+ and expressed P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly+CNF1- and had P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly+CNF1- and had P-fimbriae. We conclude that strains from urosepsis show more virulence factors than bacteremic strains isolated from non-urosepsis. PMID- 7532951 TI - Topical capsaicin for chronic neck pain. A pilot study. AB - Substance P is thought to be the principle neurotransmitter of nociceptive impulses in type C sensory neurons. Prolonged repeated applications of capsaicin cream depletes the sensory C-fibers of substance P. In an open-labeled prospective pilot study, 23 patients with chronic neck pain (greater than 3 mo) completed the study. Patients applied topical capsaicin (0.025%) cream four times a day to painful areas in the neck and shoulder girdle for a 5-wk treatment period. One patient dropped out because of intolerable burning. Statistically significant improvement was obtained in two primary outcome variables, the visual analog pain scale (P = 0.00013) and the pain relief scale (P = 0.002). Paired t tests failed to show a significant improvement in the McGill Pain Questionnaire. This study demonstrated that topically applied capsaicin cream may decrease subjective neck pain. A double-blind, placebo-controlled trial is needed to confirm this treatment effect. PMID- 7532953 TI - Continuous cocaine induces persisting alterations in dopamine overflow in caudate following perfusion with a D1 agonist. AB - The present study examined whether exposure to 5 days of continuous cocaine in rats would produce any persisting alterations of the decrease in striatal dopamine (DA) overflow produced by local infusion of a D1 receptor agonist. Using a microdialysis probe in striatum, changes in DA, DA metabolites, and GABA were assessed 14 to 21 days following a 5-day continuous cocaine treatment. There were no differences in baseline levels of DA and it's metabolites. SKF 38393 (10(-6) infusion into the striatum decreased striatal DA levels in the controls and this effect was attenuated in cocaine-pretreated rats. This result, together with other observations, supports the hypothesis of a persistently altered D1-mediated negative feedback produced by previous exposure to continuous cocaine. PMID- 7532952 TI - Ammonium acetate inhibits ionotropic receptors and differentially affects metabotropic receptors for glutamate. AB - The effects of ammonium salts in concentration similar to those found in plasma in course of hepatic encephalopathy (2-4 mM) were studied in brain slices in order to clarify how glutamate synapses are affected by this pathological situation. Electrophysiological (mice cortical wedge preparations) and biochemical techniques (inositol phosphates and cyclic AMP measurements) were used so that the function of both the ionotropic and metabotropic glutamate receptors was evaluated. Ammonium acetate (2-4 mM), but not sodium acetate reduced the degree of depolarization of cortical wedges induced by different concentrations of N-methyl-D-aspartic acid (NMDA) or (S)-alpha-Amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid (AMPA). This reduction was non-competitive in nature and did not reverse during the experimental period (90 min). In a similar manner, ammonium acetate reduced the formation of inositol phosphates induced by (1S,3R)-1-amynocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100 microM), the prototype agonist of metabotropic glutamate receptors. When the metabotropic glutamate receptors negatively linked to the forskolin-stimulated cyclic AMP formation were evaluated, ammonium acetate significantly hampered forskolin effects and its actions were additive with those of the metabotropic glutamate receptor agonist 1S,3R-ACPD. In conclusion, our results suggest that toxic concentrations of ammonium impair the function of glutamate receptors of NMDA and AMPA type and of the metabotropic glutamate receptors linked to inositol phosphate formation while they functionally potentiate the action of glutamate agonists on the receptors negatively linked to adenylyl cyclase. PMID- 7532954 TI - [The tuberculin screening of boys in the 3rd grade of secondary schools located in the area of Local Health Unit No. 18, Empoli, Tuscany]. PMID- 7532955 TI - [The characterization of the health risks in a pathological anatomy and cytodiagnosis laboratory and the remedial measures]. PMID- 7532957 TI - [The relationships between alcohol consumption and arterial pressure values in a general population of northern Italy]. PMID- 7532956 TI - [Life style and the prevention of ischemic cardiopathy]. PMID- 7532958 TI - [Alcohol, smoking and the plasma lipid picture: the results of a study conducted on a general adult population]. PMID- 7532959 TI - [The ozonization of urban waste waters as tertiary treatment]. PMID- 7532961 TI - [The control of anesthetic gas contamination by the use of a closed-circuit gas delivery apparatus]. PMID- 7532960 TI - The national guidelines for supervision checklist: a tool for monitoring supervision activities at district level in Tanzania. PMID- 7532962 TI - [The circulation of microorganisms in an intensive care unit]. PMID- 7532963 TI - [Indoor air quality in buildings. Conference proceedings. Rome, Italy, 12 November 1993]. PMID- 7532965 TI - [Indoor-outdoor environmental quality. Comfort requirements in relation to building design and installation aspects]. PMID- 7532964 TI - [The activity of the National Research Council on indoor air quality]. PMID- 7532966 TI - [The numerical simulation of indoor pollutant dispersion]. PMID- 7532967 TI - [The repercussions of outdoor versus indoor environmental degradation in a residence: the methodological considerations and the first indications]. PMID- 7532968 TI - [Radon pollution]. PMID- 7532970 TI - [The air quality in residences]. PMID- 7532969 TI - [Biological pollution: the microbiological and mycological aspects]. PMID- 7532971 TI - [Air quality in completely air-conditioned offices in Rome]. PMID- 7532972 TI - [The air quality in indoor hospital environments]. PMID- 7532973 TI - [The air quality in operating rooms]. PMID- 7532975 TI - Comparison of M-MLV reverse transcriptase and Tth polymerase activity in RT-PCR of samples with low virus burden. PMID- 7532974 TI - [A synthesis of the principle effects of building materials on indoor pollution. The results from the Unita Operativa Aria]. PMID- 7532976 TI - Selective phosphoamino acid enrichment by organic solvent fractionation. PMID- 7532977 TI - Fidelity of nucleic acid amplification with avian myeloblastosis virus reverse transcriptase and T7 RNA polymerase. AB - The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases. PMID- 7532978 TI - Cytoplasmic loading of dyes, protein and plasmid DNA using an impact-mediated procedure. AB - We describe a method and apparatus designed to rapidly and reproducibly produce transient, survivable plasma membrane disruptions--"wounds"--in order to gain access to the cytoplasm of eukaryotic cells growing in culture. Compressed gas is used to propel glass beads, dispersed as a uniform aerosol, at adherent cells growing on a culture substratum. The impact of beads with the cells creates plasma membrane wounds. Macromolecules, such as dyes, proteins and plasmid DNAs, diffuse from the extracellular environment directly into the cytoplasmic compartment of the cell through these wounds. Resealing of the plasma membrane, necessary for cell survival, traps macromolecules within the cytoplasm of the cell. PMID- 7532980 TI - Magnetic resonance imaging of prostate cancer: update. AB - Prostate cancer is the most common cancer in the world, the most frequently diagnosed in the United States, and the second most lethal cancer in U.S. men. Earlier diagnosis implies better prognosis. However, prognosis may be dependent upon the stage of the malignancy at the time of diagnosis and implementation of appropriate therapy. Clinical staging, even with the development of serum prostate-specific antigen and other studies, has not proven to be highly accurate, particularly to identify and quantitate local disease and extension. Imaging has, in the past, also had limited success. With the development of computed tomography (CT), endorectal ultrasound, and magnetic resonance imaging (MRI), there was great expectations for improvement. However, CT and ultrasound have not been as accurate as hoped. MRI, because of its multiorientation and multiparameter abilities, has been the most definitive imaging tool for staging of local extension, yet still has limitations. The prostate capsule, the neurovascular bundles, the seminal vesicle, and other regions prone to initial attack by cancer extension can be seen exquisitely clearly by the newer approaches to MRI. Cancer extension, however, cannot be consistently identified when it is microscopic. MRI is an accurate identifier of macroscopic, even subtle macroscopic disease, but there are still limitations in its ability to diagnose all pathology. PMID- 7532979 TI - Comparison of human eosinophil and neutrophil ligands for P-selectin: ligands for P-selectin differ from those for E-selectin. AB - Eosinophils (EOS) and neutrophils (PMN) display different patterns of accumulation during various inflammatory reactions. We hypothesized that EOS and PMN may differ in their ligands for P-selectin, and that these ligands may differ from those previously identified for E-selectin. Recombinant human P-selectin was immobilized on plastic surfaces and adhesion of 51Cr-labeled human EOS or PMN was compared. EOS and PMN adhered in a concentration-dependent fashion, with similar maximal net adhesion. Preincubation with a blocking P-selectin antibody inhibited adhesion of both cell types, whereas a non-blocking antibody did not. To determine if the counterligands were sialylated proteins, cells were treated with various glycosidases and proteases before testing adhesion. Neuraminidase treatment markedly inhibited binding of both cell types, while endo-beta galactosidase had no significant effect. Pretreatment with several proteases reduced adhesion of both cell types, although they consistently caused a greater inhibition of PMN binding than EOS binding. To determine whether the P-selectin ligands were surface structures whose expression or function may be altered by cell activation, leukocytes were pretreated with various stimuli; only platelet activating factor (PAF) treatment reduced the capacity of leukocytes to adhere to P-selectin. Thus, the counterligands for P-selectin on EOS and PMN are similar sialylated, protease-sensitive, endo-beta-galactosidase-resistant structures, whose function and/or expression is reduced following treatment with PAF. These characteristics are clearly different than those reported for EOS and PMN ligands for E-selectin, and suggest disparate roles for P-selectin and E-selectin during EOS and PMN recruitment during inflammatory responses in vivo. PMID- 7532982 TI - Dengue type 3 infection. Nicaragua and Panama, October-November 1994. PMID- 7532981 TI - Recognition of cryptic sites in human and mouse laminins by rat osteoclasts is mediated by beta 3 and beta 1 integrins. AB - Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat beta 3 and alpha v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is alpha v beta 3. Antiserum to beta 1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that beta 1 integrins(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of alpha v beta 3 integrin was confirmed using a recombinant human alpha v beta 3 solid phase binding assay, alpha v beta 3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact laminins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7532983 TI - Childhood onset cyclic neutropenia: G-CSF therapy restores neutrophil count but does not influence superoxide anion and cytokine release by neutrophils. AB - In this paper we describe the case of a 16-year-old boy with childhood onset cyclic neutropenia (CN) with a 21 d cycle who was successfully treated with recombinant granulocyte-colony stimulating factor (G-CSF). Cyclic therapy with G CSF (5 micrograms/kg/d s.c. for 1 week every 21 d) maintained peripheral neutrophil count above the normal range, reduced the incidence of severe infections and significantly improved the patient's performance status throughout an 18-month follow-up. Phenotypic analysis of circulating lymphocytes demonstrated that G-CSF treatment does not modify the phenotypic profile of circulating B, T and NK cell populations. Circulating neutrophils released normal amounts of cytokines (including IL-1 beta, IL-8, TNF alpha) and superoxide anion during G-CSF therapy. PMID- 7532984 TI - The vascular cell adhesion molecule (VCAM-1) is expressed on a subset of lymphoid and myeloid leukaemias. AB - The expression of the vascular cell adhesion molecule (VCAM-1), recently identified as cytokine-inducible ligand of the beta 1-integrin VLA-4, was investigated on normal and malignant haemopoietic precursors as well as on haemopoietic cell lines. VCAM-1 was demonstrated on > 20% blasts in 4/22 acute myeloid leukaemia (AML) and 6/10 acute lymphoblastic leukaemia (ALL) specimens but was absent from CD34+ normal bone marrow precursor cells. Interestingly, the VCAM-1+ AMLs classified as M1 and M5 simultaneously expressed N-CAM (CD56), a member of the immunoglobulin family. In ALL, VCAM-1 expression was restricted to Calla+ CD19+ precursors of the c-ALL subtype. VCAM-1 was also found on some cell lines, mainly of the B-lymphocytic type. Furthermore, in 13/20 chronic lymphocytic leukaemia (CLL) samples > 20% of the CD19+ B-lymphocytic precursors carried VCAM-1, which seemed to correlate with more advanced disease. Therefore VCAM-1 expression appeared to characterize leukaemic cells of the B-cell lineage as well as a CD56+ subset of AML. Since its expression was clinically correlated with dermal infiltrates of leukaemic cells in AML and with advanced Rai stages in CLL, VCAM-1 may play a role in enhanced adhesion of the malignant cells to tissues and/or to each other. PMID- 7532985 TI - Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML. AB - In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117+ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients. Our results show that most of normal BM CD117+ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117+ cells are CD34+, these cells displaying a different FSC/SSC distribution when compared to the CD117+/CD34- cells. No CD117+/CD15+ and CD117+/CD10+ cells were detected and very few CD117+ cells (< 1 x 10(-3) expressing the HLADR-/CD34-, CD33+/HLADR- and CD34+/HLADR- phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117+/CD15+ blast cells and eight had the CD117+ phenotypes detected at low frequencies (< 1 x 10(-3)) in normal BM. In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117+/CD15+ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies. PMID- 7532987 TI - Adrenergic receptors: unique localization in human tissues. AB - The final point to be made is that RNA studies are only the first step in localizing the distribution of adrenergic receptors in human tissues. Although RNA levels tend to correlate well with receptor protein expression in many tissues, this must be confirmed with studies aimed at receptor protein. Selective antibodies are being developed currently by various researchers and selective ligands by several pharmaceutical companies. In the next few years, not only should it be possible to confirm or modify results of adrenergic receptor subtype distribution studies, it also should be possible to design and test specific hypotheses related to adrenergic receptor diseases in whole animal models with newly developed subtype-selective ligands. Because species heterogeneity in adrenergic receptor tissue distribution exists, final testing of adrenergic receptor subtype-selective drugs will have to occur in humans. This is a potentially exciting possibility for anesthesiologists, for what better clinical laboratory is there than the operating room? Hence, anesthesiologists are in a key position to help redefine human adrenergic physiology once new adrenergic receptor subtype-selective agents become available. PMID- 7532986 TI - Mechanical consequences of calcium channel modulation during volatile anesthetic induced left ventricular systolic and diastolic dysfunction. PMID- 7532990 TI - Progress in the treatment and palliation of advanced breast cancer. Symposium proceedings. Jerusalem, 1993. PMID- 7532989 TI - Stabilization in vitro of the untransformed glucocorticoid receptor complex of S49 lymphocytes by the immunophilin ligand FK506. AB - Untransformed steroid receptors are large heteromeric complexes which have been shown to contain the mammalian heat shock proteins hsp56, hsp70 and hsp90. Based on functional and sequence homology studies, it was recently discovered that hsp56 also belongs to the FKBP class of immunophilin proteins, which are thought to mediate the actions of the immunosuppressive drugs FK506 and rapamycin. This discovery has led to the speculation that FK506 and related drugs could influence the actions of steroid receptors. In this work, we have examined the effects of FK506 on the transformation and hormone-binding properties of glucocorticoid receptors (GR) present in the cytosolic fraction of mouse S49 lymphocyte cells. Based on immunoprecipitation studies, it was found that hsp56 was indeed a component of untransformed GR complexes in S49 cytosols. It was also found that the untransformed but not the transformed GR was retained following affinity chromatography with FK506-affigel resin, reinforcing the possibility that hsp56 within the untransformed GR complex could be a target for the actions of FK506. Using a DNA-cellulose-binding assay, FK506 exhibited a 60% inhibition of dexamethasone (Dex)-induced transformation of the GR to the DNA-binding state, while sodium molybdate, a transition metal oxyanion known to stabilize GR complexes, was 100% effective. This inhibition of GR transformation by FK506 was shown to correlate with an inhibition of Dex-induced GR/hsp90 dissociation, with 10 microM FK506 preventing 48% of the GR/hsp90 complexes from dissociating. Scatchard analysis of GR hormone-binding function was performed, with FK506 treatment of cytosols causing Kd values to decrease (3.36 nM) as compared to vehicle (8.42 nM) and no-addition (9.82 nM) controls. Taken together, our results suggest that FK506 can stabilize the untransformed GR complex of S49 cells and that this stabilization in turn results in an increase in GR ligand-binding affinity. Although we speculate that these actions of FK506 on the GR complex are mediated by the associated hsp56 component, other possible mechanisms are also discussed. PMID- 7532988 TI - Over-expression of human sex steroid-binding protein (hSBP/hABP or hSHBG) in insect cells infected with a recombinant baculovirus. Characterization of the recombinant protein and comparison to the plasma protein. AB - Human sex steroid-binding protein (hSBP/hABP or hSHBG) was over-expressed in High Five and Sf9 cells adhered to plates and in suspension. The adherent cells expressed to levels of 2.3 mg/l and 1.4 mg/l after 4 and 6 days, respectively, while Sf9 cells grown in suspension yielded 4.67 mg/l after 6 days. Recombinant hSBP/hABP, purified to homogeneity by immunoadsorption, was found to fold similarly to native plasma hSBP/hABP and to display similar sequence epitopes after heat denaturation. The recombinant protein binds dihydrotestosterone, testosterone, and 17 beta-estradiol with KdS of 0.6, 2.4, and 14.2 nM, respectively, which are similar to plasma hSBP/hABP. The recombinant protein contains N-linked and O-linked oligosaccharide side-chains but the monomer exhibits a slightly lower molecular weight than plasma hSBP/hABP (40 kDa vs 44 kDa) which may be due to the absence of one N-linked side-chain or to shorter oligosaccharide side-chains. The partial N-terminal sequence LRPVLP(T)Q of recombinant hSBP/hABP is identical to plasma hSBP/hABP but appears to be less heterogeneous. These results indicate that recombinant baculovirus SBP represents a good model for investigating the structure of plasma hSBP/hABP. The expression system will allow the isolation of preparative amounts of SBP mutants generated by combinatorial site-directed mutagenesis to advance investigations on structure function relationships and undertake crystallization trials for X-ray diffraction analyses. PMID- 7532991 TI - Pamidronate in the treatment of bone metastases: results of 2 dose-ranging trials in patients with breast or prostate cancer. AB - Four intravenous regimens of pamidronate (Aredia) were evaluated for palliative treatment of bone metastases in 2 randomized open-label trials in patients with breast cancer (n = 61) or prostate cancer (n = 58). In breast cancer patients, administration of pamidronate 60 mg every 4 weeks, 60 mg every 2 weeks, or 90 mg every 4 weeks for 3 months resulted in statistically and clinically significant reductions in bone pain, with accompanying decreases in biochemical markers of bone turnover; a regimen of 30 mg every 2 weeks was not effective. Healing of bone lesions was observed in 25% of breast cancer patients. In prostate cancer patients, the same regimens of pamidronate produced reductions in bone pain, but no dose-response relationship was apparent. Moreover, there were no consistent changes in biochemical indices in these patients, and no healing of bone lesions occurred. The different response to pamidronate in those 2 patient populations may reflect the different severity of metastatic disease at baseline. Side effects of pamidronate were mild and transient in both studies. PMID- 7532992 TI - Role of pamidronate in the management of bone metastases from breast cancer: results of a non-comparative multicenter phase II trial. Aredia Multinational Cooperative Group. AB - Bone metastases are a common cause of morbidity in patients with breast cancer. In an open, phase II, non-comparative trial to investigate the effects of repeated infusions of pamidronate (Aredia) on pain, mobility, analgesic consumption, bone healing and bone metabolism, 69 patients with breast cancer and bone metastases received pamidronate 60 mg intravenously in 250 ml normal saline over 1 or 4 hours every 2 weeks for a total of 13 infusions, until either progressive disease or a serious adverse event. Improvement in pain score was seen in 33 of 54 evaluable patients (61%) as measured by a linear analogue pain scale, and in 28 of 56 evaluable patients (50%) as measured on a 6-point pain scale: 18 (30%) of 60 evaluable patients showed reduction in a 6-point analgesic score, while 28 patients (50%) showed some improvement in mobility, as assessed by a questionnaire. Sclerosis appeared in > 25% of bone lesions in 2 patients and in < 25% of bone lesions in 12 patients. Urinary calcium/creatinine ratios fell dramatically during therapy. One patient developed symptomatic hypocalcemia, 1 showed deterioration in pre-existing renal insufficiency. Fever occurred in 19% of patients, and less than 20% developed flu-like symptoms. We conclude that intravenous infusions of pamidronate at a dose of 60 mg every 2 weeks produces a marked reduction in pain in patients with extensive bone metastases from breast cancer. PMID- 7532993 TI - Progress in the treatment and palliation of advanced breast cancer: does the dose of pamidronate determine its effects? AB - Bisphosphonates are the treatment of choice in tumor-induced hypercalcemia. Pamidronate (Aredia) is effective in this indication, but few studies have addressed the question of its dose-effect relationship. Review of the published literature suggests that a clinically relevant dose-response relationship exists in the dose range 30-60 mg; most hypercalcemic patients will achieve normocalcemia with a dose of 60 mg pamidronate or more. More recent publications have reported the emerging role of bisphosphonates in the treatment of malignant osteolytic bone disease. Several non-randomized studies have shown beneficial effects from continuous administration of clodronate or pamidronate, though the magnitude of such effects was moderate. Two dose escalation trials with pamidronate showed that regimens with a dose intensity below 20 mg/week and single doses of 30 mg or less were ineffective. One randomized, prospective study compared pamidronate 60 mg with 90 mg given intravenously every 3 weeks. Both regimens showed a significant palliative effect. Patients treated with 90 mg had a greater reduction in pain intensity compared to those receiving 60 mg. Optimal schedules for different patient populations require further investigation. PMID- 7532994 TI - Radiotherapy for bone pain: is a single fraction good enough? UK Multicentre Bone Pain Trial Collaborators. PMID- 7532995 TI - Roles of serum vitronectin and fibronectin in initial attachment of human vein endothelial cells and dermal fibroblasts on oxygen- and nitrogen-containing surfaces made by radiofrequency plasmas. AB - Fluoropolymers modified by plasma modification were studied for their suitability as surfaces for the adhesion of cells. We compared films made by plasma modification of fluoroethylenepropylene (FEP) using nitrogen-containing gases (ammonia or dimethyl acetamide) with films deposited using oxygen-containing monomers (methanol, methyl methacrylate or sequential treatment with toluene then water). The surfaces were compared for the attachment and spreading of human vein endothelial cells and human dermal fibroblasts. The initial attachment and spreading of cultured fibroblasts and endothelial cells onto films deposited using nitrogen-containing gases were equivalent to that onto films deposited using oxygen-containing monomers, but there were some differences in the mechanism of attachment. With films deposited using oxygen-containing monomers, the initial attachment and spreading of endothelial cells failed when the medium contained 15% (v/v) serum from which both fibronectin (Fn) and vitronectin (Vn) had been removed. Similarly, initial attachment and spreading of endothelial cells onto films deposited using oxygen-containing monomers were reduced by 62 86% when the cells were seeded in medium containing Vn-depleted serum (which contained Fn). Endothelial cells attached and spread onto films made using oxygen containing monomers, when seeded in medium containing Fn-depleted serum (which contained Vn). On films deposited using nitrogen-containing gases, the adhesion of endothelial cells was only slightly reduced in Vn-depleted medium (as compared to attachment in medium containing unmodified serum). Furthermore, surfaces which had incorporated nitrogen were more effective than were oxygen-containing films in adsorbing sufficient serum Fn as to promote endothelial cell attachment. Similar results were seen for the attachment and spreading of fibroblasts as for the endothelial cells. For fibroblasts, attachment and spreading onto oxygen containing films and onto nitrogen-containing films were not simply dependent upon either the Vn content or the Fn content of the medium. Maximal attachment and spreading of fibroblasts were, however, dependent upon adsorption of both serum Vn and Fn. PMID- 7532996 TI - Interactions of plasma proteins with a novel polysaccharide surfactant physisorbed to polyethylene. AB - A polysaccharide surfactant, dextran-[1,6 bis(2-hydroxypropyl-1-amine)hexane] dextran, (D-H-D) was prepared by reacting dextran (Mw = 8200) with epichlorohydrin followed by reaction with 1,6-hexanediamine. The D-H-D polymer product was characterized by gel permeation chromatography (GPC), and 13C-nuclear magnetic resonance spectroscopy (13C-NMR). D-H-D was physisorbed on polyethylene (PE) from aqueous solution, and the adhesion stability and resistance to protein adsorption was examined under static and dynamic flow conditions, using a modified rotating disk system. Modified surfaces were characterized by attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), electron spectroscopy for chemical analysis (ESCA) and by water contact angles. Under applied shear stresses of up to 73 dyn cm-2, the adhesion of D-H-D on PE was sufficient to inhibit desorption by water (> 90% D-H-D on PE was retained) and 5% SDS surfactant solution (approximately 83% D-H-D retained), as determined by ATR FTIR. Under similar shear stress conditions, albumin adsorption on D-H-D modified PE was reduced by over 90%, and protein adsorption from fresh human plasma was reduced by approximately 70% compared with unmodified PE. The results are discussed in terms of interfacial forces, and the suitability of this approach for studying protein-surface interactions and for developing a novel class of protein-resistant biomaterials. PMID- 7532997 TI - Stereochemistry of tRNA(m5U54)-methyltransferase catalysis: 19F NMR spectroscopy of an enzyme-FUraRNA covalent complex. AB - The catalytic mechanism of tRNA(m5U54)-methyltransferase (RUMT) involves the formation of a covalent adduct between Cys324 of RUMT and C6 of Ura54 in tRNA. The covalent adduct is subsequently methylated at C5 by S-adenosyl-L-methionine (AdoMet). We used an RNA substrate analog containing 5-fluorouracil (FUra) in place of Ura54 to trap the covalent complex and analyzed the adduct by 19F NMR spectroscopy. The 19F NMR spectrum of the adduct consisted of an overlapping doublet of quartets, with an H6-F coupling constant of 4 Hz and a CH3-F coupling constant of 22.4 Hz. On the basis of the magnitude of the H6-F coupling constant, we determined that Cys324 of RUMT and the methyl moiety from AdoMet added across the 5,6-double bond of FUra54 in cis fashion. We deduced that the nucleophilic addition was also cis in the normal enzymatic reaction and that the subsequent beta-elimination of the 5-H and catalytic cysteine was trans. Further, on the basis of chemical considerations, we proposed several conformational adaptations of enzyme-substrate complexes that must occur on the reaction pathway. Together with previous studies, this study enables the proposal of the complete stereochemical pathway for the RUMT-catalyzed methylation of Ura54 in tRNA. PMID- 7532998 TI - Defining a smaller RNA substrate for elongation factor Tu. AB - A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA were obtained that bound T. thermophilus EF-Tu as well as Ala-tRNA(Ala). Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA. PMID- 7532999 TI - Interactions of HIV-1 envelope glycoproteins with derivatized dextrans. AB - The present study demonstrates that derivatized dextrans, such as carboxylmethyl dextran benzylamide and carboxymethyl dextran benzylamide sulfonate, specifically interact with HIV-1 envelope glycoproteins (rgp160 and rgp41) with significantly higher affinities than those observed for dextran sulfate (MW 8 kDa). These results suggest the possible involvement in HIV infectivity of surface membrane molecules which may bind the virus at pre or post-CD4 binding steps. They also suggest the possible use of these compounds in anti-HIV therapy. PMID- 7533000 TI - The shortest isoform of human vascular endothelial growth factor/vascular permeability factor (VEGF/VPF121) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability. AB - Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a multifunctional cytokine that is expressed as four isoforms having 206, 189, 165, and 121 amino acids in humans. We constructed a system that produces the shortest isoform of human VEGF/VPF in Saccharomyces cerevisiae (yVEGF/VPF121). Active yVEGF/VPF121 was secreted from the yeast cells as a glycosylated dimeric protein. Various biological activities of the purified yVEGF/VPF121 were examined. It bound to cell surface receptor(s) and stimulated the growth of human umbilical vein endothelial cells in culture at a dose similar to that of native VEGF/VPF. Purified yVEGF/VPF121 also induced angiogenesis in mice, and promoted the extravasation of plasma proteins from the blood vessels. These observations demonstrated that the shortest isoform of VEGF/VPF with an amino acid sequence of 121 residues contains enough information necessary to trigger both angiogenesis and the induction of vascular permeability upon binding to its cognate receptor(s). PMID- 7533001 TI - Ricin A-chain cytotoxicity depends on its presentation to the cell membrane. AB - Anti-ricin-A-chain monoclonal antibodies (mAbs) were raised in order to study epitopes of the A-chain involved in the initiation of its transmembrane passage. Five nonoverlapping epitopes were selected by cluster formation using a cross inhibition assay of mAbs from 172 specific hybridomas. For each cluster, representative high affinity mAbs were selected. These mAbs were disulfide-linked to F(ab')2 fragments of a mAb directed against the CD5 antigen. The cytotoxicity of ricin A-chain bound by affinity through different epitopes to these hybrid mAbs was explored on the CD5-positive CEM cells. Without any enhancer, the hybrids displayed cytotoxicity varying in IC50 within a range of 1-10; in the presence of the enhancer NH4Cl, this variation of activity was maintained ranging from 1 to 20; with the enhancer monensin, the variation of cytotoxicity of hybrids extended over a 1-80-fold range. These differences of cytotoxicity were not correlated with mAb properties such as the inhibition of A-chain enzymatic activity, the A-chain binding affinity, the capacity for releasing A-chain at low pH, or the capacity of hybrids for delivering A-chain on the cell surface. This lack of correlation strongly suggests that the different presentations of A-chain epitopes to the cell membrane may be the essential reason for cytotoxicity variations. PMID- 7533002 TI - Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences. AB - The synthesis of three new photoactive RNA phosphoramidites, 5-bromouridine, 5 iodouridine, and O4-triazolouridine, is reported. The 5' OH of bromouridine and iodouridine were protected as dimethoxytrityl ether using dimethoxytrityl chloride and pyridine. Selective protection of 2' OH was achieved as the corresponding tert-butyldimethylsilyl ether. Protected ribonucleosides were converted to phosphoramidites using 2-cyanoethyl N,N diisopropylchlorophosphoramidite. O4-Triazolouridine phosphoramidite monomer was prepared in one step from uridine phosphoramidite. These phosphoramidites were used to incorporate photoprobes at any chosen sites in the RNA sequences during chemical syntheses. The modified monomers were incorporated into RNA oligomers with coupling yields > 98%. After chemical synthesis, O4-triazolouridine was converted to 4-thiouridine by the addition of thiolacetic acid during standard deprotection methods. The extent of thiation and incorporation of modified nucleotides into RNA sequences were confirmed by nuclease digest, HPLC, and gel electrophoresis. PMID- 7533003 TI - RNA degradation by bleomycin, a naturally occurring bioconjugate. PMID- 7533004 TI - Expression of oligohistidine-tagged ricin B chain in Spodoptera frugiperda. AB - DNA encoding ADPGH6G was fused to the 5'-end of RTB DNA and subcloned as a BamHI EcoRI DNA cassette into the baculovirus transfer vector, pAcGP67A. Spodoptera frugiperda Sf9 cells were cotransfected with pAcGP67A-ADPGH6G-RTB DNA and BaculoGold AcNPV DNA, and recombinant baculovirus was isolated by two cycles of limiting dilution assay followed by dot blot analysis with 32P-dCTP random primer labeled RTB DNA. Recombinant virus was purified and amplified to obtain stocks at titers of 10(7) infectious particles/mL. Sf9 cells grown in serum-free medium were then infected at an moi of 3 in the presence of 25 mM alpha-lactose. After 5 days, supernatants and cell pellets were harvested and assayed by an asialofetuin ELISA for recombinant RTB protein. Fusion RTB protein was produced in the supernatant at 5 mg/L and in the cell pellet at 1 mg/L. Recombinant protein was purified to > 80% homogeneity using either a monoclonal antibody affinity matrix with alkaline elution or a Ni(2+)-NTA matrix with imidazole elution. The purified protein bound asialofetuin similarly to plant RTB. N-terminal sequencing confirmed the oligohistidine tag. SDS-PAGE confirmed the 1,000 Da increase in mass relative to "wild-type" recombinant RTB produced in Sf9 cells. Immunoblots confirmed reactivity with polyclonal and monoclonal antibodies to plant RTB. The fusion protein reassociated with plant RTA similarly to plant RTB.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533005 TI - Arabinogalactan for hepatic drug delivery. AB - Arabinogalactan, a polysaccharide from the tree Larix occidentalis, has been purified and its biological and physical properties described. Intravenous injection of radiolabeled arabinogalactan (4 mg/kg) in rats resulted in 52.5% of the dose being present in the liver, while prior injection of asialofetuin (100 mg/kg) reduced hepatic radioactivity to 3.54%. Gel chromatography indicates arabinogalactan is a single species of 19 kDa, while light scattering gave a molecular weight of 40 kDa. Glycosyl linkage analysis of arabinogalactan is consistent with a highly branched structure comprising a backbone of 1,3-linked galactopyranose connected by 1,3-glycosidic linkages, comprised of 3,4,6-,3,6-, and 3,4- as well as 3-linked residues. In the carbon-13 NMR spectra, the major resonances of arabinogalactan are assigned to beta-galactopyranose, beta arabinofuranose, and beta-arabinopyranose. Arabinogalactan produced no adverse reactions in single intravenous dose (mouse, 5000 mg/kg) and repeat dose toxicity studies (rats, 500 mg/kg/day, 90 days). When tritiated arabinogalactan was injected, radioactivity cleared from the liver with a half-life of 3.42 days. Arabinogalactan has properties that make it suitable as a carrier for delivering diagnostic or therapeutic agents to hepatocytes via the asialoglycoprotein receptor. PMID- 7533006 TI - [Regulation of catalytic activity and supramolecular structure of angiotensin converting enzyme in reversed micelles of aerosol OT in octane]. AB - Regulation of the catalytic activity and the supramolecular structure of the angiotensin-converting enzyme isolated from bovine lungs has been studied in a system of reversed micelles of aerosol OT (AOT) in octane. The curve for the dependence of the enzyme catalytic activity on the degree of the surfactant hydration (micellar size) has two maxima at the hydration degrees of [H2O]/[AOT] 27 and 31. Data from velocity sedimentation suggest that depending on the hydration degree, the angiotensin-converting enzyme occurs in the system of reversed micelles in both monomeric and dimeric forms, the latter being catalytically active. In contrast with aqueous media, in the reversed micelle system the angiotensin-converting enzyme does not require chloride anions for its catalytic activity. In the system of reversed micelles of AOT in octane the holoenzyme is stable, while the apoenzyme rapidly and irreversibly loses its activity. Under these conditions the apoenzyme shows an ability to incorporate the Zn2+ ions into the enzyme active center; however, only in the presence of a substrate or an inhibitor. PMID- 7533007 TI - [Structure of lipopolysaccharides from gram-negative bacteria. III. Structure of O-specific polysaccharides]. AB - Data on the composition and structure of O-antigens which represent polysaccharide chains of outer membrane lipopolysaccharides of gram-negative bacteria defining cell immunospecificity are reviewed with special reference to some structural features of O-antigens, such as masked regularity and occurrence in one microorganism of lipopolysaccharides with two structurally distinct polysaccharide chains. Antigenic relationships between the microorganisms belonging to different taxonomic groups are discussed. PMID- 7533008 TI - Identification of CD34+ cord blood cells and their subpopulations in preterm and term neonates using three-color flow cytometry. AB - Three-color flow cytometry was used to identify CD34+ cord blood (CB) hematopoietic progenitors of preterm (n = 13) and term (n = 18) neonates. The frequency of CD34+ CB cells gated on the lymphocyte fraction in term neonates is about half the frequency of preterm neonates (mean 1.84 +/- 0.8 vs. 3.49 +/- 1.8, p < 0.005). The difference of absolute CD34+ cells between the 2 groups did not reach statistical significance (premature 169 +/- 161 vs. mature 108 +/- 62.6 x 10(6) cells/l). The proportion of myeloid lineage-committed CD34+ cells coexpressing the CD33 antigen in preterm neonates does not significantly differ from that found in term neonates. However, in preterm neonates a higher fraction of erythroid lineage-committed CD34+ cells coexpresses the CD71 antigen as compared with term neonates (mean 28.7 +/- 14.1 vs. 11.4 +/- 5.7, p < 0.001). The positive correlation between gestational age and the ratio of myeloid/erythroid lineage-specific progenitor cells (r = 0.61, n = 31, p < 0.0005) suggests gestational changes in lineage commitment of CD34+ cells. PMID- 7533009 TI - Effects of oral IGF-I or IGF-II on digestive organ growth in newborn piglets. AB - To study whether colostrum-borne growth factors are responsible for the rapid GI tissue growth in naturally suckled newborn animals, newborn unsuckled piglets were bottle-fed for 24 h with infant milk formula with or without addition of 2 micrograms/ml of recombinant human insulin-like growth factor-I (IGF-I) or insulin-like growth factor-II (IGF-II), a level which approximated that of porcine colostrum. The animals were then sacrificed for measurements of their digestive organ weights and contents of protein, RNA and DNA in the organs. The treatment with IGF-I or IGF-II failed to show any significant effect on the weight of the esophagus, stomach, small intestine, large intestine, mandibular glands, kidneys and the spleen, and had no effects on the contents of protein, RNA and DNA in the small intestinal mucosa, the liver and the spleen. However, piglets fed with infant formula containing IGF-I (n = 7) or IGF-II (n = 7) had a heavier pancreas (p < 0.05) compared to formula-fed controls (n = 7). The DNA content in the stomach and the pancreas were greater in animals treated with IGF I or IGF-II than in controls. Using a cell labelling technique it was shown that both IGF-I and IGF-II stimulated cell proliferation in the small intestinal crypts. The results indicate that the substantial GI tissue growth reported in newborn animals is unlikely due to colostrum-borne IGF-I or IGF-II alone. On the other hand the study does suggest that oral IGF-I and IGF-II are capable of stimulating cell proliferation in the GI tract. PMID- 7533010 TI - Rate of antigen entry into the circulation in experimental versus naturally occurring immune complex glomerulonephritis. AB - This study tested the hypothesis that the rate of antigen entry into the circulation in systemic lupus erythematosus (SLE), a naturally occurring immune complex glomerulonephritis (IC-GN), is slow compared with that of traditional experimental models of IC-GN, in which the antigen is delivered rapidly as a daily iv bolus. This hypothesis was tested by comparing rates of decline of the third component of complement (C3) and of circulating neutrophils (PMN) in SLE patients with active disease with those of cynomolgus monkeys (CYN) undergoing induction of experimental IC-GN by means of a daily bolus infusion of antigen. It has previously been shown that, as antigen enters the circulation and forms circulating IC, C3 levels and circulating PMN decline acutely. Thus, acute changes in these parameters can be surrogates for the rate of antigen entry into the circulation. In the CYN undergoing induction of IC-GN (N = 11), infusion of the antigen (bovine gamma-globulin) over 10 min resulted in acute declines in C3 levels (25 +/- 6.6%; P = 0.0018 and PMN counts (59 +/- 9%; P < 0.0002). In addition, the CYN experienced the onset of acute respiratory distress and hypotension. By contrast, in patients with active SLE (N = 9), C3 and white blood cell counts measured at 24-h intervals did not change significantly, and episodes of acute hypotension or respiratory distress were not observed. In the CYN, the onset of visible vasculitic lesions in the omentum were also documented within minutes of the infusion of bovine gamma-globulin. The rapidity of onset of these vascular lesions suggests that the tempo at which lesions develop in experimental models of IC disease is faster than that of naturally occurring IC diseases. It was concluded that, in naturally occurring IC diseases, antigen probably enters the circulation slowly over prolonged periods of time, rather than as large boluses over short periods of time, as in traditional experimental models of IC GN. Thus, models of IC-GN involving a daily bolus infusion of antigen may not be clinically relevant, particularly when IC clearing mechanisms are tested, because the efficiency of these mechanisms may be markedly influenced by the rate at which IC form in the circulation. PMID- 7533011 TI - Recurrent pyogenic granuloma with satellitosis--a localized variant of bacillary angiomatosis? AB - A 27-year-old male patient had recurrent pyogenic granuloma with satellitosis. Histologically, Warthin-Starry staining of the lesions revealed clumps of dark bacilli as found in patients with bacillary angiomatosis. IgG antibodies against Bartonella (Rochalimaea) henselae were elevated as shown by an indirect immunofluorescence assay. The patient did not present an obvious risk for HIV infection or immunosuppression, and no antibodies against HIV-1 and HIV-2 were found. Recurrent pyogenic granuloma with satellitosis may be a localized variant of bacillary angiomatosis. PMID- 7533012 TI - Nitinol prostheses for the treatment of inoperable malignant esophageal obstruction. AB - PURPOSE: This uncontrolled prospective study was undertaken to evaluate a new nitinol stent in the treatment of dysphagia due to inoperable malignant esophageal obstruction. PATIENTS AND METHODS: Eighteen consecutive patients (15 men, three women; mean age, 66 years +/- 15) with inoperable malignant obstructions of the esophagus or esophagogastric junction due to esophageal carcinoma (n = 14) or extrinsic obstruction (n = 4) were treated with self expanding nitinol endoprostheses (diameter, 18 mm). Dysphagia score and activity index were assessed before and every 4 weeks after stent insertion. RESULTS: Eighteen stents were deployed successfully. One stent expanded insufficiently and was removed inadvertently during retrieval of the application system. Mean time until complete expansion of the stent was 7.1 days +/- 5. Stent placement resulted in a significant decrease in the dysphagia score (2.7 +/- 0.7 before vs 0.6 +/- 0.7 after stent placement, P < .00005 by Wilcoxon matched-pairs test) and a significant increase in the activity index (2.6 +/- 1 before vs 1.7 +/- 0.9 after stent insertion; P < .005). There were no procedure-related complications. Follow-up period (mean, 155 days +/- 100) revealed recurrent dysphagia in four patients. Two patients experienced food bolus impaction, another two had tumor ingrowth through the mesh after 170 and 186 days. Fourteen patients died after a mean survival time of 158 days +/- 106, all with patent stents. CONCLUSIONS: Self expanding nitinol esophageal stents are safe and effective in the treatment of malignant esophageal obstructions. PMID- 7533014 TI - [Possibilities and limits of anti-HCV antibody screening in the blood donation service]. AB - BACKGROUND: Hepatitis C virus (HCV) is responsible for the majority of post transfusion hepatitis cases. We compared the correlation and reproducibility of different screening and confirmatory tests. MATERIAL AND METHODS: 1,406 samples of voluntary blood donors were tested in parallel using 3 enzyme-linked immuno sorbent assays (ELISA) for HCV antibodies. Those samples that were positive in at least 1 of the 3 tests were additionally tested in a 3rd-generation ELISA as well as in 3 different confirmatory tests. RESULTS: 13 samples (0.92%) were repeat reactive in at least 1 of the ELISAs with different results in the confirmatory tests. Only 3 samples (0.21%) with high sample/cutoff ratios in the ELISAs were positive in all 3 confirmatory tests. CONCLUSIONS: The reproducibility of the tested ELISAs and the correlation with confirmatory tests were good only in samples with a high signal to cutoff ratio. Two different high-positive ELISA results can be regarded as confirmation. PMID- 7533013 TI - Genomic structure of the mouse apurinic/apyrimidinic endonuclease gene. AB - A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains. One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites. The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents. This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex). The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons. Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon. Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1. A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains. These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2. Data are also presented that suggest the presence of two pseudogenes in the mouse. PMID- 7533015 TI - [Characterization of 24-hour survival rate and duration of survival of hydroxyethyl starch cryopreserved erythrocytes after autologous transfusion in the dog]. AB - BACKGROUND: Cryopreservation of erythrocytes using hydroxyethyl starch (HES) as cryoprotecting additive could result in a nearly unlimited storage stability of preserved red cells. In addition, it would allow its immediate use for transfusion. In order to assess the therapeutic efficacy of erythrocytes cryopreserved with HES, their 24-hour post-transfusion survival and long-term survival was evaluated. MATERIALS AND METHODS: The experiments were carried out with dog erythrocytes as an animal model for human erythrocytes. To each of 6 German shepherd dogs a 15-ml sample of erythrocyte suspension, labeled with 51Cr (25 microCi) after thawing, was autologously injected. Caused by hemolysis 29% of the formerly cryopreserved erythrocytes have not been labeled. To each of 6 control animals 15 ml of a suspension of freshly drawn and 51Cr-labeled erythrocytes was injected. The 51Cr radioactivity in later taken blood samples was a measure for the number of injected erythrocytes having remained in the circulation until the moment of blood withdrawal. The effect of cryopreservation was assessed by comparison of the test group with the control group. RESULTS: In both groups 30% of the applied cells left the circulation within 30 min. This was effected by pharmacological enlargement of the dogs' spleen and not by hemolysis of the erythrocytes. After the first 24 h all of the cryopreserved labeled erythrocytes had survived to the same amount (> 95%) as the labeled fresh red cells. Between 12 h and 20 days after injection, in both groups the 51Cr activity decreased exponentially by 4.8 and 4.5%/d. This difference was not significant. The area under the curve amounted to 1253 and 1257% d, respectively. CONCLUSIONS: There exists a subpopulation of red cells that is destroyed by freezing stress. As a result the freed stroma would be a serious transfusion risk. All erythrocytes having survived the cryopreservation procedure resemble the fresh erythrocytes with regard to the in-vivo survival; their therapeutic efficacy is not impaired. In the context of in-vitro results with human erythrocytes it can be expected that at the present developmental state of the cryopreservation procedure at least 93% of the human erythrocytes cryopreserved with HES have a normal 24-hour and long-term post-transfusion survival. PMID- 7533018 TI - Two novel types of bistratified amacrine cells in the chick retina: a Golgi study. AB - 1. The correlation between neurotransmitter substances in specific cell types has renewed interest in the morphology of amacrine cells. In this paper we describe the morphology of two types of amacrine cells in Golgi-stained chick retinas. 2. The first cell type was classified as an asymmetric bistratified amacrine cell suggested to play a role in the formation of complex ganglion cell receptive fields. 3. The second was classified as a bistratified amacrine cell. Their processes were stratified at sublayers 1 and 4 of the inner plexiform layer and their morphological features were similar to those of dopaminergic cells in the chick retina. PMID- 7533017 TI - The role of stem cell factor in mobilization of peripheral blood progenitor cells. AB - Stem cell factor (SCF) is a hematopoietic growth factor which acts on both primitive and mature progenitors cells. In animals, high doses of SCF alone stimulate increases in cells of multiple lineages and mobilize peripheral blood progenitor cells (PBPC). Phase I studies of rhSCF have demonstrated dose related side effects which are consistent with mast cell activation. Based upon in vitro synergy between SCF and G-CSF we have demonstrated the potential of low doses of SCF to synergize with G-CSF to give enhanced mobilization of PBPC. These PBPC have increased potential for both short and long term engraftment in lethally irradiated mice and lead to more rapid recovery of platelets. On going Phase I/II studies with rhSCF plus rhG-CSF for mobilization of PBPC, demonstrated similar increases in PBPC compared to rhG-CSF alone. These data suggest a clinical role of rhSCF in combination with rhG-CSF for optimal mobilization of PBPC. PMID- 7533016 TI - Determination of structural elements of the L2/HNK-1 carbohydrate epitope required for its function. AB - The L2/HNK-1 carbohydrate epitope has been shown to carry an unusual 3' sulfoglucuronic acid linked O-glycosidically through a neolactosyl-type backbone to a ceramide residue. Using monoclonal antibodies, the same or a closely related epitope has also been detected N-glycosidically linked to glycoproteins, amongst them several neural cell adhesion molecules. We used synthetic glycolipids carrying sulfated or non-sulfated glucuronic acid attached to ceramide through glycans of different length to show that not only the sulfated glucuronic acid but also the neolactosyl-type backbone is essential for the recognition of the L2/HNK-1 carbohydrate by a monoclonal antibody, its binding to laminin and its role in neural cell migration and outgrowth of processes from neurons and astrocytes. PMID- 7533019 TI - Effect of alpha-linolenic acid on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine release in RBL-2H3 cells. AB - We examined the effect of alpha-linolenic acid (18:3 (n-3)) pretreatment on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine content and release of RBL-2H3 cells. RBL-2H3 cells grew without reduction in number when incubated with subculture media for 3 d and then placed again in serum-free medium with bovine serum albumin (BSA) and epidermal growth factor (EGF). Cholesterol pullulan (10 micrograms/ml) emulsified alpha-linolenic acid (20 micrograms/ml) was recommended as an additional form serum free medium. We determined the fatty acid composition in all neutral lipids, free fatty acids and all phospholipids in alpha-linolenic acid-treated cells. In all cases the concentration of alpha-linolenic acid and docosahexenoic acid (DHA, 22:6 (n-3)) was increased, while linolenic acid (18:2 (n-6)) was slightly and arachidonic acid (20:4 (n-6)) was markedly decreased. Content of histamine in alpha-linolenic acid-treated cells was remarkably lower than that of untreated cells. Accordingly, net histamine release stimulated by antigen or A23187 was also markedly decreased in the alpha-linolenic acid-treated cells, as was the percent histamine release stimulated by antigen. Results from our in vitro experiment suggest that the anti-allergic effect of alpha-linolenic acid may be caused either by the decrease in histamine content or by inhibition of the release of chemical mediator resulting from changes in the fatty acid composition. PMID- 7533020 TI - Effects of surfactants and protease inhibitors on nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats. AB - The effects of various surfactants and protease inhibitors on the nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were examined in rats. No effects of bile salts and acids such as sodium glycocholate or taurocholic acid, amphoteric surfactants such as lauryldimethyl betaine, or anionic surfactants such as sodium lauryl sulfate on the absorption were found at a concentration of 1%. But non-ionic surfactants with hydrophile/lipophile balance (HLB) of 13 to 18 increased the total leukocyte numbers maximally by about 250% as a relative increase ratio to the control without surfactants. The increase in the plasma rhG-CSF concentration was obviously observed only in the presence of non-ionic surfactants, and in particular, the effects of Laureth-9 on the increase in total leukocyte numbers and plasma rhG-CSF concentration were maximal. In the presence of various kinds of protease inhibitors, the increasing effect of rhG-CSF on the total leukocyte numbers was not changed. Consequently, it is considered that the permeation of rhG-CSF through the nasal epithelium can be improved by non-ionic surfactants, but the effect of a protease inhibitor is smaller than that of the surfactant. PMID- 7533021 TI - A protective effect of glutathione-dextran macromolecular conjugates on acetaminophen-induced hepatotoxicity dependent on molecular size. AB - Glutathion (GSH) was covalently attached to dextrans with various molecular weights of 2, 5, 10, 40, and 70 kDa by the cyanogen bromide activation method. The conjugates obtained synthetically were white or pale yellowish powders containing 6-10% (w/w) of GSH. The average molecular weights of the conjugates were estimated to be larger and the molecular weight distribution was a little broader than that of each original dextran. The conjugates significantly stabilized GSH and liberated it gradually under physiological conditions (t1/2 = 0.99-1.6h). Mice depleted of GSH by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic GSH level after intravenous injection of the conjugates. In mice given a hepatotoxic dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugates, whereas a small improvement was found when free GSH was given. The conjugate of GSH attached to dextran with the molecular weight of 40 kDa exhibited the highest prophylactic effect on acetaminophen-induced hepatotoxicity in mice. The prolonged retention of the conjugates of larger molecular weight in the circulation would cause a higher hepatic accumulation. These results suggested that molecular size would be the most critical factor in the delivery of GSH, as a dextran conjugate, into the liver. PMID- 7533022 TI - A comparison of drug transport through cultured monolayers of bovine brain capillary and bovine aortic endothelial cells. AB - To examine how efficient primary cultured brain capillary endothelial cells are as an in vitro blood-brain barrier (BBB) system with regard to drug transport, we compared bovine brain capillary endothelial cells (BBCEC) with bovine aortic endothelial cells (BAEC). Paracellular and transcellular transport were examined. [14C]Sucrose and fluorescein isothiocyanate-dextran (FITC-dextran)(average molecular weight about 4400 and 71200) were used as indices of paracellular transport. The permeability coefficients of [14C]sucrose and FITC-dextran through BBCEC monolayers were about 3 and 30-40 fold lower than those through BAEC monolayers, respectively. Transcellular transport was examined by means of [3H]leucine and [3H]alanine uptake studies. The Km value (Michaelis constant) of the amino acid uptake was lower in BBCEC than in BAEC. BBCEC had higher alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP) activity than BAEC when cultured in vitro. These results suggest that BBCEC are suitable for drug transport studies across the BBB in vitro. PMID- 7533023 TI - Possible functional groups responsible for inhibition of in vivo angiogenesis by herbimycin A. AB - Six herbimycin A (HBM) derivatives were examined for their anti-angiogenic effects in a bioassay system involving chorioallantoic membranes (CAMs) of growing chick embryos on the basis of our previous observation that HBM is a potent angiogenesis inhibitor. 17-Cyclopropylamino-HBM dose-dependently inhibited embryonic angiogenesis. The ID50 value was 0.1 microgram (160 pmol) per egg and thereby lower than that of the parent compound HBM (ID50 = 0.15 micrograms (260 pmol) per egg). In contrast, 19-dimethylamino-, N-acetyl-, 2,3,4,5-tetrahydro- and 7-decarbamoyl-HBM at doses of 0.01-10 micrograms/egg failed to affect angiogenesis in CAMs. These results strongly suggest as follows: (1) C-19 position, amino group between positions C-1 and C-20 and carbamoyl group in C-7 are essential for the anti-angiogenic action of HBM; (2) HBM needs certain fixed conformation for expression of angiogenesis inhibition; (3) it is expected that the modification of C-17 with a suitable functional group results in increased anti-angiogenic potency of HBM--that is, a more potent angiogenesis inhibitor than the parent compound would be developed. PMID- 7533025 TI - E-selectin polymorphism and atherosclerosis: an association study. AB - Adhesion molecules like the members of the selectin family participate in the interaction between leukocytes and the endothelium. They are also involved in the pathogenesis of atherosclerotic processes. To contribute to the analysis of the genetic background of atherosclerosis we searched for DNA polymorphisms in the genes encoding adhesion molecules especially E-selectin which seems to be expressed only in activated endothelium. An adenine to cytosine substitution for cDNA position 561 resulting in an amino acid exchange from serine to arginine (position 128) was detected in the epidermal growth factor like domain. A significantly higher mutation frequency (P = 0.02) was observed in 97 patients aged 50 years or less with angiographically proven severe atherosclerosis (allele frequency of arginine 0.155) compared with an unselected population (allele frequency of arginine 0.088) as well as in 40 patients aged 40 years or less (allele frequency of arginine 0.21, P = 0.0025). These data suggest that the 128 serine/arginine polymorphism is associated with a higher risk for early severe atherosclerosis. PMID- 7533024 TI - Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126. AB - Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA. The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharide of E. coli O126. It was found that, amongst the monosaccharides, beta-D-N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure alpha-D-Galp (1-->3)-beta-D-GlcpNAc(1-->2)-D Manp. PMID- 7533027 TI - An MspI RFLP of the human AHR gene. PMID- 7533028 TI - Age of the delta F508 cystic fibrosis mutation. PMID- 7533026 TI - Identification of a CFTR frameshift mutation (1013 delAA) in trans to delta F508 in a pancreatic sufficient cystic fibrosis patient. PMID- 7533031 TI - Improving the postoperative care of acutely-confused older adults. AB - Delirium (acute confusional states) is the most common organic mental problem in older hospitalized surgical patients. In this review, clinical features, postoperative etiologies, associated negative outcomes, barriers to recognition and treatment, and principles of nursing management are described. An empirically based, practical resource for surgical nurses is presented to improve safe patient management. PMID- 7533029 TI - PBDX is the XG blood group gene. AB - We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG. PMID- 7533033 TI - Invasive meningococcal infections. PMID- 7533032 TI - Defining medical-surgical nursing practice. PMID- 7533030 TI - [Single-blind study of the effect of doxazosin mesylate in benign prostatic hypertrophy]. AB - The purpose of this single blind study was to verify the efficiency of Doxazosin Mesylate, an alpha-1 adrenergic blocker, in patients with benign prostatic hyperplasia (BPH). This study involved 20 patients non placebo responders. The duration of treatment was 45 days with administration of Doxazosin Mesylate increased every 15 days from 1 to 2 mg and from 2 to 4 mg respectively. At the end of each dosage cycle the following investigations were performed: a) peak urinary flow, b) residual urinary volume, c) funneling of the prostatic urethra by means of permictional transrectal echography, d) Boyarsky's score. The analysis of these data, applying a two way analysis of variance (ANOVA), showed that Doxazosin Mesylate resulted in improvements in both urodynamic and symptomatic parameters. The statistical analysis proved also that there was a good correlation between the dosage of the drug up until 2 mg and the results from each parameter considered. PMID- 7533034 TI - Cholera in England and Wales, 1994. PMID- 7533036 TI - [The morphofunctional characteristics of the heart under the joint action of hexachlorane and physical loading of varying intensity]. AB - In the experiments, performed in 125 male Wistar rats the animals were sacrificed by oral treatment with 1/20 LD50 of pesticide hexachloran with the simultaneous modelling of physical load of 3 levels (minimum, medium, maximum). Horizontal treadmill was used to obtain the dynamic load. Duration of experiment made 30 days. The data obtained demonstrated the direct cardiotropic effect of the drug Dynamic loads of the medium and maximum levels amplify the toxic effect, leading to the hypertrophy of the left heart, decrease of the coefficient of the mitochondrial energetic efficiency, aerobic oxidation and growth of the contracture myocardial degeneration and aerobic glycolysis. PMID- 7533038 TI - Therapy of chronic viral hepatitis. AB - Interferon is currently the only established therapeutical option for chronic viral hepatitis. Sustained response can be achieved in approximately 25% of patients with chronic HBV or HCV infection. Results in chronic HDV infection are disappointing. Whether combination of interferon with other lymphokines or antiviral drugs will lead to higher response rates, remains to be established. The argument that interferon will only place spontaneous seroconversion on an earlier date has not been disproved yet. Long-term follow-ups are necessary to show that therapy with interferon will improve survival and reduce the incidence of hepatocellular carcinoma in patients with chronic viral hepatitis. PMID- 7533035 TI - Biosynthesis of mucin derived from a 60-kDa precursor protein in the human stomach. AB - We studied the biosynthesis of mucin in the human stomach using an anti-mucin core peptide monoclonal antibody, 3G12. Human stomach mucosa was labeled with [35S]methionine, and chased for 3 h. An approximately 60-kDa subunit of human gastric mucin precursor protein was detected in the intracellular product. Under nonreducing conditions, dimer, trimer, and tetramer mucin precursor protein (120, 180, 240 kDa) were detected. Treatment with tunicamycin or endo-beta-N acetylglucosaminidase H had no effect on the 60-kDa subunit and its oligomers. Extracellular products contained only the high molecular weight mucin, and the secretion was not affected by tunicamycin. By treatment with monensin or brefeldin A, the mature mucin was not secreted extracellularly. These findings suggested that a 60-kDa subunit of the mucin precursor protein was biosynthesized into mature mucin after oligomerization to tetramers, and that neither the oligomerization nor the intracellular transport of the mucin in the human stomach was associated with N-glycosylation. PMID- 7533037 TI - Anaphylactic histamine release from human gastric and duodenal mast cells. AB - Mast cells from human gastric and duodenal walls were isolated using a collagenase dispersion technique. The reactivity of both mast cell populations with anti-human IgE antibodies and specific antigens was tested in an in vitro model of anaphylactic reaction. Mast cell populations were sensitive to the action of anti-IgE, and histamine release was 17.4-27.4% (duodenal) and 19.3 29.3% (gastric mast cells). No significant differences between both mast cell populations of the same individuals were observed. Gastric and duodenal mast cells obtained from patients with peptic ulcer and positive intradermal test with allergens (grass pollen, tomato, cocoa) released histamine after challenge with adequate antigens. The reaction was dose-dependent. Gastric mast cells were more reactive than duodenal cells to challenge with antigen. PMID- 7533039 TI - gp120 neurotoxicity in primary cortical cultures. AB - The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in a nitric oxide dependent manner in primary cortical cultures at low picomolar concentrations. gp120 neurotoxicity also requires calcium and glutamate and is blocked by glutamate receptor antagonists. In addition, superoxide anions play a role in gp120 neurotoxicity since superoxide dismutase also attenuates neurotoxicity. PMID- 7533040 TI - HIV infection of human brain capillary endothelial cells--implications for AIDS dementia. AB - We have demonstrated that human brain capillary endothelial (HBCE) cells, unlike umbilical or aortic endothelial cells are permissively infected by HIV. HIV infection of HBCE cells is noncytolytic and is mediated by a CD4- and GalCer independent mechanism, implying that HBCE cell tropic strains utilize a unique receptor. The V3 loop of gp120 appears to be important in this reaction. T-cell tropic but not brain-derived macrophage tropic HIV strains selectively infect brain endothelium suggesting that T-cell tropism is important for HIV entry through the blood-brain barrier (BBB). The ability of HIV to infect cells that compose the BBB implies that the virus may be directly involved in the BBB dysfunction observed in AIDS patients. HIV infection of HBCE cells may allow the flow of cytokines or toxic metabolites from the circulating blood into the brain parenchyma either by disrupting tight junctions or by altering the ability of the cells to regulate transport of substances across the BBB by transcytosis. HIV infection may also result in endothelial cell-induced astrocytosis by release of cytotoxic substances or modulation of abluminal surface antigens which contact astrocytic foot processes. Finally, HIV infection of the brain endothelium could facilitate virus entry to the CNS either by infection of HBCE cells or via entry of HIV-infected leucocytes. The establishment of our in vitro HIV-HBCE cell system will allow us to explore the potential mechanisms which mediate AIDS dementia. PMID- 7533041 TI - The role of the blood-brain barrier in HIV infection of the central nervous system. AB - The blood-brain barrier (BBB) functions to regulate the entry of macromolecules, microbial pathogens, and circulating leukocytes into the central nervous system (CNS). It consists, in part, of the microvascular endothelium and associated astrocyte foot processes, found in close apposition to the abluminal side of the vascular endothelial cells (EC). During the pathogenesis of certain nervous system diseases with inflammatory components, the BBB may function to facilitate the entry of leukocytes into the CNS parenchyma. A common histologic observation in human immunodeficiency virus type-1 (HIV) encephalitis is the localization of HIV proteins to multinucleated giant cells that co-immunolabel with antibodies specific for cells of the monocyte/macrophage lineage, suggesting that HIV can enter the CNS as cell-associated virus. We previously characterized a tissue culture model of the BBB that consists of the co-culture of autologous EC and astrocytes. In this presentation, we used this model to examine the expression of adhesion molecules by both the EC and astrocyte components of this BBB model, and to characterize the interactions between HIV-infected monocytes and EC. The data presented in this review of our work demonstrates that astrocytes upregulate the expression of intercellular adhesion molecule (ICAM-1) by EC. In a parallel study, western blot analysis demonstrated that ICAM-1 is also expressed in the developing human CNS. When exposed to the proinflammatory cytokine tumor necrosis factor alpha (TNF), both EC cocultured with astrocytes and astrocytes cultured alone expressed the adhesion proteins IG9, ICAM-1, vascular cell adhesion molecule 1 (VCAM) and E-selection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533042 TI - Case report: intestinal perforation--an unusual complication of endoscopic biliary stenting. AB - The endoscopic placement of stents to decompress biliary obstruction is accepted practice in the management of malignant biliary strictures. In our patient, a 73 year-old man with a presumed malignant common hepatic duct stricture, palliation of his obstructive jaundice has been achieved. However, his clinical course was complicated by a colonic perforation--a previously unreported complication of distal stent migration. PMID- 7533043 TI - Use of complementary peptides and their antibodies in T-cell-mediated autoimmune disease: experiments with myelin basic protein. AB - In this article we review the field of idiotype (Id) network and experimental allergic encephalomyelitis, focusing on the generation of monoclonal antibody anti-Id by using complementary peptide and the investigation of anti-Id immunoregulatory effects on immune responses of B and T cells to specific myelin basic protein peptides in vitro and in vivo. PMID- 7533045 TI - Antiepileptic effects of inhibitors of nitric oxide synthase examined in pentylenetetrazol-induced seizures in rats. AB - The effects of intraperitoneal NG-methyl-L-arginine and N omega-nitro-L-arginine methyl ester, specific inhibitors of nitric oxide (NO) synthase, were examined on the pentylenetetrazol (PTZ)-induced seizures in rats. The incidence and latency for the onset of myoclonic jerks, clonic seizures, and tonic generalized extension were observed as specific parameters among PTZ-induced seizures. Both drugs preferentially suppressed the tonic generalized extension and prolonged the latency for the onset of each parameter, suggesting NO has a significant effect on the PTZ-induced seizure. PMID- 7533044 TI - Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. AB - BACKGROUND: Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein. RESULTS: We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes. CONCLUSIONS: Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family. PMID- 7533047 TI - Human nasal absorption of 51Cr-EDTA in smokers and control subjects. AB - Passive exposure to cigarette smoke has emerged as a significant risk factor in the development of asthma and allergic airways disease. The pathogenetic mechanisms are not known, but increased absorption across the airway epithelial lining has been suggested as one possible mechanism of this effect of cigarette smoke. This study examines the absorption-permeability of the nasal epithelial lining in cigarette smokers and non-smokers. For comparison, the effect of a detergent, dioctylsodium sulfosuccinate (DS), is also examined. A solution containing 51Cr-EDTA (51-chromium labeled ethylene diamine tetraacetic acid) (mol. wt. 372 Da) was instilled and maintained in the nasal cavity in six smokers and 12 non-smokers for 15 min. Urine was collected for 24 h after the instillation. The accumulated amount of excreted 51Cr-EDTA was measured and expressed as millilitre nasal instillate. In six non-smokers the procedure was repeated when DS has been added to the instillate. The median recovered amount of 51Cr-EDTA in smokers 0.07 ml (range 0.04-0.32) was not significantly different from that in non-smokers 0.16 ml (0.01-1.22). The recovered amount of 51Cr-EDTA increased from a median of 0.18 ml (0.01-1.22) to 1.13 ml (0.53-1.80) after addition of the detergent (P = 0.028). We conclude that the nasal airway absorption-permeability is not increased in smokers. Hence, passive exposure to cigarette smoke may not produce an impairment of airway barrier functions. PMID- 7533046 TI - Estimation of Der p and Der f I quantities in the reference preparations of Dermatophagoides mite extracts. AB - A monoclonal antibody-based enzyme-linked immunosorbent assay (MoAb-ELISA) was developed to measure the major Dermatophagoides mite allergens, Der p I and Der f I. The assay was highly species-specific and sensitive. Using this assay system, the absolute mass unit of Der p I and Der f I in the reference preparations of the extracts was estimated. The primary standards used were the purified Der p I and Der f I preparations. The reference preparations of the D. pteronyssinus and D. farinae extracts (92-Dp and 92-Df), which had been prepared from the same amount of mite bodies of both species, were found to contain the same levels of the Der I allergens, 10.1 micrograms/ml of Der p I and 10.0 micrograms/ml of Der f I, respectively. A histamine release assay with leucocytes from mite-allergic donors showed that the total allergenic potency of 92-Dp and 92-Df was comparable. This results indicates that the estimated Der I levels in these extracts seem to be valid, at least, in the balance between the two species, although further comparisons of the absolute quantities by several different laboratories are needed. The Der I levels in the WHO/IUIS international reference preparation of D. pteronyssinus and the CBER standard mite extracts, E4-Dp and E5 Df, were also estimated using this assay system. They were found to contain 4.4 micrograms/vial and 13.3 micrograms/ml of Der p I and 9.5 micrograms/ml of Der f I, respectively. PMID- 7533049 TI - Developing a poster about a clinical innovation. Part I: Ideas and abstract. AB - This is the first in a series of three articles developed to assist the CNS in planning, producing, and presenting a clinical poster. Choosing an idea that is likely to be accepted by reviewers and then planning and writing the abstract are described in this article. Practical instructions, specific to the clinical rather than the research poster, are given along with illustrative examples. A sample of reviewer evaluation criteria is given to help the nurse recognize a standard for excellence in innovative poster presentations and to provide reviewers with an objective evaluation tool. PMID- 7533048 TI - Striatonigral degeneration: iron deposition in putamen correlates with the slit like void signal of magnetic resonance imaging. AB - We report three patients with striatonigral degeneration highlighting the correlation between magnetic resonance imaging (MRI) and the pathological changes. The "slit-like void signal" observed in the putamen is typical of striatonigral degeneration and can be used to assist diagnosis during life. Our histochemical studies support the concept that increased iron deposition in the putamen is responsible for this MRI picture. PMID- 7533050 TI - Novel neuroleptics in schizophrenia: theory and clinical relevance. PMID- 7533051 TI - Risperidone: neurochemical, pharmacologic and clinical properties of a new antipsychotic drug. AB - Risperidone, a novel antipsychotic with high binding affinity for serotonin 5-HT2 and a lesser affinity for dopamine D2 receptors, is described and clinical studies are reviewed. In two large-scale trials in North America and Europe, risperidone 6 mg and 4 mg to 8 mg/day compared favourably with haloperidol 20 mg or 10 mg/day in controlling the positive symptoms of schizophrenia. In the North American trial, risperidone produced a significant improvement in negative symptoms; no comparable improvement was reported among haloperidol-treated patients. Risperidone would, thus, appear to offer a potential advantage over other neuroleptics, although the place of this new compound in the pharmacological armamentarium of schizophrenia must be clarified in clinical practice. PMID- 7533052 TI - Pathophysiology and treatment of negative symptoms. AB - The assessment and clinical characterization of the negative symptoms of schizophrenia are reviewed. While negative symptoms may be secondary to positive symptoms, extrapyramidal side-effects, depressive illness, chronic institutionalization or other factors, they may also be primary, i.e., integral to the disease process itself. Recent literature has suggested that negative symptoms may represent a distinct pathophysiological process mediated in part by serotonergic mechanisms. Atypical neuroleptics, which have dual effects on D2/5 HT2 receptors, have demonstrated clinical efficacy on both negative and positive symptoms of schizophrenia, and might help to clarify the pathogenesis of these symptom clusters. PMID- 7533053 TI - Pharmacotherapy of schizophrenia: a review. AB - Traditional antipsychotic medications have targeted the dopamine system. Yet, the isolation and cloning of multiple dopamine receptor subtypes and the recognition of non-dopaminergic pathways (for example, serotonin) in schizophrenia have led to the development of new pharmacological strategies in pharmacotherapy. The author summarizes recent biochemical and pharmacological data and the clinical experience with novel antischizophrenic compounds. PMID- 7533054 TI - Re: Hypomania associated with risperidone. PMID- 7533055 TI - [The anti-HCV assay in viral hepatitis and hepatoma and the relationship between HCV infection and blood transfusion]. AB - Two hundred cases of various kinds of viral hepatitis and hepatocellular carcinoma were tested for serum anti-HCV. The positive rates of anti-HVC in patients with severe hepatitis and patients with cirrhosis were 42.86% and 46.15%, respectively. They were significantly higher than those in patients with other kinds of hepatitis (P < 0.05). The positive rate of anti-HCV was 67.5% in patients with posttransfusion hepatitis, 20.47% in healthy blood donors. In posttransfusion hepatitis B it was only 2.5%. Our results demonstrated that blood transfusion played an important role in transmitting HCV. Our findings also indicated that dual infection of HBV and HCV was important in the course of chronic hepatitis, cirrhosis and severe hepatitis. 50% of the anti-HCV positive patients with chronic hepatitis had slightly elevated serum alanine aminotransferase level. This showed that liver damage caused by HCV may be a chronic course. PMID- 7533056 TI - Homologous maturase-like proteins are encoded within the group I introns in different mitochondrial genes specifying Yarrowia lipolytica cytochrome c oxidase subunit 3 and Saccharomyces cerevisiae apocytochrome b. AB - A mitochondrial cox3 gene in the alkane yeast, Yarrowia lipolytica, encodes a subunit-3 protein of cytochrome c oxidase, and contains a 1044 base-pair-long intron, as compared with the corresponding intronless gene in Saccharomyces cerevisiae. The intron belongs to a group I intron as determined by the cDNA sequence for the splicing sites as well as the predicted RNA secondary structure. Remarkably, this intron could code for a protein of 206 amino-acid residues which showed 63% similarity with an RNA maturase encoded by the second intron of the mitochondrial apocytochrome b gene in S. cerevisiae. Both introns occurred within the conserved exon sequence, 5'-TT(G/C)AGGTGC-3', suggesting the possible transposition of a common ancestral intron. PMID- 7533058 TI - Genetic organization of a linear mitochondrial plasmid (mF) that promotes mitochondrial fusion in Physarum polycephalum. AB - The mF plasmid which promotes mitochondrial fusion in Physarum polycephalum is a linear molecule with complex terminal inverted repeats (TIRs). Its nucleotide sequence was determined. The mF plasmid is 14,503 bp in size, and contains ten open reading frames (ORFs). All of the ORFs except one are encoded on the same DNA strand (coding strand). The number of amino-acid residues in the putative proteins derived from the nine ORFs on the coding strand are 231, 163, 640, 235, 118, 1130, 366, 309, and 547 from left (5' end) to right (3' end) on the map. The amino-acid sequences of newly-identified ORFs on the mF plasmid did not show significant homology to any amino-acid sequences in the databases. A brief transcriptional map of the mF plasmid was constructed, and the following features were noted. (1) The transcription initiation site was located just inside the end of the left TIRs, but not within the TIRs themselves. (2) Three major transcripts of 1.0, 3.4 and 4.6 knt corresponded to the left region of the mF plasmid, and long, low-abundance (more than 4.6 knt), heterogenous transcripts corresponded to almost the entire mF plasmid. A low-abundance, 3.5-knt transcript corresponding to the coding region of ORF1 130 (a 1 130-amino-acid polypeptide) was also detected, and may be derived from the long transcripts. (3) The quantity of transcripts which included the region near the transcription initiation site was about 500-times more than that which included the region near the inner end of the right TIRs. PMID- 7533060 TI - [The expression of C-FOS, C-JUN and phosphotyrosine gene products in lung cancer]. AB - An immunohistochemical study on C-FOS, C-JUN and phosphotyrosine (P-Tyr) cancer gene products was performed. The results showed that C-FOS had the lowest frequency of expression and P-Tyr had the highest. The positive reactions of the three cancer gene products were observed in the nucleus, nuclear membrane and cytoplasm. The expression of C-FOS in normal bronchial and alveolar tissue was 3.8% and 1.6% respectively. But in lung cancer it was 60%. The simultaneous positive expression of C-FOS and C-JUN was 56% (54 cases). Negative C-FOS and positive C-JUN was 32%. Positive C-FOS and negative C-JUN was less than 4% (4 cases). Although C-FOS and C-JUN formed a hetero-dimer by zipper structure, the C FOS had the ability of single expression. PMID- 7533059 TI - Transcription of potato mitochondrial 26S rRNA is initiated at its mature 5' end. AB - Transcription initiation sites in plant mitochondria can be located by in vitro capping of primary 5' transcript termini. Direct sequencing of a cap-labelled mitochondrial RNA from potato shows its sequence to be identical to the 5' terminal part of the 26S rRNA. Primer extension analysis indicates the mature 5' end to be the sole detectable 5' transcript terminus. In potato mitochondria the mature 5' end of the 26S rRNA is thus created by transcription initiation without any further 5' processing. The nucleotide sequence surrounding this transcription initiation site shows only limited similarity to other putative promoter sequences from dicot plant mitochondria suggesting the possibility that divergent RNA polymerases, and/or transcription initiation factors, are present in plant mitochondria. PMID- 7533061 TI - Prostate cancer, prostate-specific antigen, and the polymerase chain reaction. PMID- 7533057 TI - Organization and transcription of the mitochondrial ATP synthase genes in the yeast Yarrowia lipolytica. AB - Mitochondrial gene organization was studied in a dimorphic yeast, Yarrowia lipoltica. The gene order in a sequenced 6.6-kilobase region closely resembles that of the human mitochondrial genome in that ATP synthase subunit 8 and 6 genes are followed by genes for cytochrome c oxidase subunit 3 (which contains an intron), NADH-ubiquinone oxidoreductase subunit 4, and ATP synthase subunit 9. This region also contains tRNA genes decoding AUA, UGA, CUN and CCN codons, suggesting a unique mitochondrial translation. All the above genes are transcribed from the same DNA strand into multigenic RNAs, starting from a nonanucleotide sequence, 5'-ATATAAATA-3', similar to other yeast mitochondrial promoters. PMID- 7533062 TI - Detection of prostatic cells in peripheral blood: correlation with serum concentrations of prostate-specific antigen. AB - We have studied the expression of prostate-specific antigen (PSA) mRNA by reverse transcriptase-polymerase chain reaction in peripheral blood of 25 patients with cancer of the prostate (CAP), four with benign prostatic hyperplasia (BPH), two with renal stones, three with other types of cancer, and six healthy male and three female controls. Expression of mRNA specific for a certain tissue in peripheral blood is thought to indicate the presence of circulating cancer cells and metastatic spread of a tumor originating from this tissue. We detected PSA mRNA in 9 of 18 CAP patients with metastatic disease but in none of 7 patients without metastases. Negative results in patients with metastatic disease were associated with successful endocrine therapy and low concentrations of serum PSA, and the correlation between serum concentrations of PSA and the presence of PSA mRNA in peripheral blood was statistically significant. PSA mRNA was not found in patients with BPH, other types of cancer, or in healthy controls. Thus the occurrence of PSA mRNA in peripheral blood is associated with metastatic CAP. PMID- 7533063 TI - Prostate-specific antigen immunoreactivity in amniotic fluid. AB - We examined whether the 33-kDa serine protease prostate-specific antigen (PSA) is present in amniotic fluid and, if so, whether its concentration changes with gestational age. Analyzing 115 amniotic fluids with a highly sensitive immunofluorometric procedure, we found PSA in all the amniotic fluids examined and established that its concentration increases with increasing gestational age from 11 to 21 weeks, decreasing at delivery. PSA in amniotic fluid is present predominantly in the free (33 kDa) form; a minor fraction (< 20%) is present bound to alpha 1-antichymotrypsin. No significant correlation was seen between PSA and alpha-fetoprotein (AFP) in amniotic fluid or maternal serum in samples with high AFP. Amniotic fluid PSA was also measurable by two different established methods for PSA. Pregnant women had higher concentrations of serum PSA than nonpregnant women. The highest PSA concentration in amniotic fluid was associated with a pregnancy that was complicated by the Rhesus incompatibility syndrome but the source of the PSA was not established. From recent literature reports, and the association of PSA with prostate and breast tumors, we think PSA may serve as a growth factor regulator in cancer and in normal fetal development during pregnancy. PMID- 7533064 TI - Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a). AB - Lipoprotein(a) [Lp(a)] has been measured in numerous clinical and epidemiological studies by a variety of immunochemical methods. However, little, if any, consideration has been given to the confounding effect of the size heterogeneity of apolipoprotein(a) [apo(a)] on the measurement of Lp(a). We developed three direct-binding enzyme-linked immunosorbent assays (ELISAs) with detecting antibodies of different specificities to evaluate the effect of apo(a) size on Lp(a) measurement. The three assays used the same monoclonal antibody to capture the apo(a)-containing particles and were calibrated (in nanomoles per liter) with a serum containing apo(a) with 21 kringle 4 domains. Using all three ELISAs, we measured Lp(a) in a group of 723 subjects selected to have a single apo(a) band, as determined by a high-resolution phenotyping system. Essentially identical results were obtained by the two methods that measured Lp(a) by use of either a polyclonal antibody against apo B or a monoclonal antibody against apo(a) that does not recognize the kringle 4 type 2 repeats. In contrast, the ELISA using a monoclonal antibody specific for apo(a) kringle 4 type 2 repeats overestimated Lp(a) concentration in samples containing apo(a) with more than 21 kringle 4 domains and underestimated Lp(a) samples containing apo(a) with fewer than 21 kringle 4 domains. Thus, these differences in Lp(a) values varied as a function of apo(a) size. We conclude that antibody specificity and apo(a) size heterogeneity can significantly affect Lp(a) measurements. PMID- 7533065 TI - 1H NMR determination of urinary betaine in patients with premature vascular disease and mild homocysteinemia. AB - Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts to help assign 1H NMR spectral peaks. The betaine calibration curve linearity was excellent (r = 0.997, P = 0.0001) over the concentration range 0.2-1.2 mmol/L, and CVs for replicate betaine analyses ranged from 7% (n = 10) at the lowest concentration to 1% (n = 9) at the highest. The detection limit for betaine was < 15 mumol/L. Urinary DMG concentrations were substantially lower than those of betaine. Urinary betaine and DMG concentrations measured by 1H NMR spectroscopy from 13 patients with premature vascular disease and 17 normal controls provided clinically pertinent data. We conclude that 1H NMR provides unique advantages as a research tool for determination of urinary betaine and DMG concentrations. PMID- 7533066 TI - Aspirin does not affect the flow cytometric detection of fibrinogen binding to, or release of alpha-granules or lysosomes from, human platelets. AB - 1. Aspirin inhibits the conversion of arachidonic acid to thromboxane A2 which reinforces the effects of weak agonists such as ADP in platelets. 2. In this study the effect of aspirin (300 mg/day) on platelet agonist response was measured by whole blood flow cytometry of unfixed blood samples from normal subjects (n = 10), an assay that investigates aggregation-independent changes in the platelet. 3. Fibrinogen binding to unstimulated platelets or to platelets stimulated with ADP or thrombin was unaffected by aspirin. 4. Under the conditions of this assay, platelets undergo a partial degranulation of alpha granules and lysosomes (evidenced by expression of P-selectin and CD63, respectively) in response to ADP, and full degranulation in response to thrombin. P-selectin expression was paralleled by release of beta-thromboglobulin. None of these events was affected by aspirin. 5. Thromboxane formation was totally prevented by the aspirin treatment, as shown by Born aggregometry in which the platelet aggregatory response to arachidonic acid was abolished and secondary aggregation by ADP was inhibited. 6. The flow cytometric assay can therefore be used to investigate platelets in patients, regardless of aspirin therapy. 7. These findings suggest that platelet fibrinogen binding and the release of platelet alpha-granule and lysosomal contents, in response to stimulation with physiological agonists, can continue in patients despite aspirin therapy. This may help to explain why aspirin is only partially effective in preventing thrombotic events. PMID- 7533067 TI - Increased serum concentrations of adhesion molecules after coronary angioplasty. AB - 1. Reocclusion is still a significant complication after percutaneous transluminal coronary angioplasty. The injury of coronary arteries resulting from PTCA plays an important role in the pathophysiology of both abrupt closure and late restenosis after an initially successful procedure. Cytokines play a pivotal role in the accumulation of circulating blood cells at the endothelium and are known to regulate their interaction with the vessel wall. 2. To obtain further information about this interaction, serum concentrations of soluble endothelial leukocyte adhesion molecule 1 (sELAM-1), leucocyte endothelial cell adhesion molecule 1 (sL-selectin), intercellular adhesion molecule 1 (sICAM-1), interleukin 2 receptor (sIL-2R) and interleukin 8 (IL-8) detected by enzyme linked immunosorbent assay were monitored in 30 consecutive patients referred for elective PTCA. Fifteen patients who underwent elective coronary angiography without PTCA served as controls. 3. All patients underwent successful first PTCA. Within 24 h the serum concentrations of sELAM-1 increased gradually from 21.7 (SD 7.1) to 48.2 (SD 8.6) ng/ml (P < 0.01); levels of sL-selectin rose from 982.1 (SD 128.7) to 1541.3 (SD 104.6) ng/ml after 48 h (P < 0.01). Serum levels of IL-8 remained stable initially, but peaked at the end of the observation time of 72 h (9.4, SD 3.8, versus 16.1, SD 4.9 ng/ml; P < 0.05). A positive correlation was found between the number of dilatations and the rise in these parameters (P < 0.01). No significant changes were found in the serum concentrations of sICAM-1 and sIL-2R after PTCA or in any of the parameters in patients after coronary angiography. 4. We conclude that PTCA induces a significant rise in the concentration of certain adhesion molecules in serum. Thus, we provide preliminary data on the potential role of cytokines for blood cell-endothelium interaction after PTCA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533068 TI - N-acetylprocainamide intoxication with torsade de pointes treated by high dialysate flow rate continuous arteriovenous hemodiafiltration. PMID- 7533069 TI - Esophageal cancer. AB - Esophageal cancer is an important problem in the United States. It results in more deaths (over 10,000 annually) than rectal cancer. Furthermore, the incidence of esophageal adenocarcinoma is increasing at a rate faster than that of nearly any other cancer and the reasons for the increase are not well understood. A variety of tumor-suppressor genes (including p53, APC, DCC and Rb) and proto oncogenes (including prad1, EGFR, c-erb-2 and TGF alpha) may be involved in the development and progression of esophageal cancer. Clinical prognostic factors include stage, Karnofsky performance status, sex, age, anatomic location of the tumor, and degree of weight loss. A new staging system based on depth of wall penetration and lymph node involvement correlates well with prognosis for patients undergoing esophagectomy. Newer staging procedures including endoscopic ultrasound as well as the use of minimally invasive surgery, such as thoracoscopy and laparoscopy, may allow accurate staging without esophagectomy. Surgical resection provides excellent palliation; however, the chance for cure with esophagectomy alone is only 10% to 20%. Adjuvant treatment with pre- or postesophagectomy radiation may improve local-regional control but does not improve survival. Nor has preoperative chemotherapy been shown to improve survival; however, it remains an active area of investigation. Multimodality therapy, namely, chemotherapy and radiation (chemoradiation), given concurrently prior to surgical resection shows promise, with one study indicating a 5-year survival of 34%. A complete pathologic response to chemoradiation correlates with improved survival. Chemoradiation has been shown to be superior to radiation as primary management of esophageal cancer. There has been no successfully completed randomized trial of surgery versus definitive radiation or chemoradiation. However, chemoradiation represents a reasonable alternative to esophagectomy in the primary management of squamous cell carcinoma of the esophagus and chemoradiation also appears to be effective in the treatment of patients with adenocarcinoma of the esophagus, offering significant palliation and a chance for long-term survival as well. Randomized studies of preoperative chemoradiation versus surgery or versus chemoradiation alone are needed. The treatment of advanced esophageal cancer must be directed toward palliation of symptoms. Newer endoscopic techniques, including the use of expansile metal stents, laser ablation, intraluminal high-dose rate brachytherapy, BICAP tumor probe, or photodynamic therapy, offer selected patients short-term palliation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533070 TI - Efficacy of tracheal and bronchial stent placement on respiratory functional tests. AB - Stent placement is the only available treatment in patients presenting either a localized external compression or a malacia of the tracheobronchial tree. To assess the functional benefit of prosthesis insertion in these indications, we compared functional respiratory values before, immediately after (48 h), and at sometime after (mean, 10.1 months) operation in 24 patients presenting with a bronchial lesion (B group, n = 5) or a lesion of the intrathoracic part (ITT group, n = 9) or of the extrathoracic part of the trachea (ETT group, n = 10). Before treatment, airflow was severely impaired in most patients without significant differences among the groups. After prosthesis insertion, airflow parameters increased [change in forced expiratory volume in 1 s (delta FEV1 = 440 mL; delta peak expiratory flow (PEF) = 0.92 L.s-1; delta maximum expiratory flow 25/75 (delta MEF25/75) = 0.47 L.s-1; and delta forced inspiratory volume in 1 s (delta FIV1 = 310 mL)] and airway resistances (Raws) decreased (delta Raw = -0.43 kPa.s-1.s-1) without any significant variation in either forced vital capacity (FVC) or total lung capacity. Airflow improvement was more apparent in ITT and ETT groups than in the B group. Moreover, inspiratory flow increase and decrease of FEV1/PEF ratio were only observed in the ETT group. This airflow improvement was maintained for a long time after and was associated with a good clinical tolerance. This study supports the clinical and functional benefits of prosthesis placement both in benign and malignant airway compressions for palliative treatment. PMID- 7533071 TI - Substance P-induced contraction of airway smooth muscle in young and adult rabbits. Effects of epithelium removal and neutral endopeptidase inhibition. PMID- 7533072 TI - Tumor necrosis factor-alpha (TNF-alpha) stimulates mucin secretion and gene expression in airway epithelium in vitro. PMID- 7533073 TI - Exhaled nitric oxide is increased in asthma. PMID- 7533074 TI - A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique. AB - In the present paper a CD38/propidium iodide double staining technique is described which separately assesses the cell cycle distribution of myelomatous plasma cells from that of the residual normal hemopoietic cells. For this purpose, bone marrow (BM) cells from a group of 42 untreated multiple myeloma patients were analyzed. Of these, 23 cases were aneuploid (55%) and 19 diploid (45%). The use of the CD38/propidium iodide double staining method allowed a clear separation between CD38 strong positive cells from the remaining bone marrow populations, cell sorting experiments confirming that plasma cells were almost exclusively contained in the former fraction where they represented 97 +/- 2% of the total cells sorted. In all cases, the S-phase in plasma cells and in the remaining normal hemopoietic bone marrow cells was assessed, being higher in normal hemopoietic cells (8.0 +/- 6.3%) than in plasma cells (3.3 +/- 2.6%, P < 0.002). In addition, there was no correlation between the S-phase of the neoplastic and normal bone marrow cells (r = 0.22; P > 0.10); this work therefore shows that the assessment of the total proliferative rate of bone marrow samples does not reflect either the proliferation of normal cells or that of neoplastic plasma cells but will depend on the proliferative rate and the percentage of each population within the sample, which can be assessed by the technique described here. PMID- 7533075 TI - Nicardipine may impair glucose metabolism in hypertensive diabetic patients. AB - The respective effects of 6 month's administration of beta-blockers (atenolol, metoprolol, carteolol and arotinolol), calcium-channel blockers (nicardipine, diltiazem) and angiotensin converting enzyme inhibitor (enalapril) on hemoglobin A1c (HbA1c) levels were evaluated in hypertensive patients with non-insulin dependent diabetes mellitus (NIDDM), using a retrospective method. NIDDM patients with stable HbA1c and body weight were selected for this study. The following results were obtained. (1) The administration of nicardipine or beta-blockers significantly elevated HbA1c levels. (2) The administration of diltiazem or enalapril did not have any influence on HbA1c levels. These findings suggest that not only beta-blocker but nicardipine (dihydropyridine type calcium-channel blocker) may cause deterioration in glucose metabolism in NIDDM patients. PMID- 7533076 TI - Developmental profiles of spontaneous movements in infants. AB - Spontaneous movements were observed for 1-h sessions in each of 50 normal full term infants aged from 2 weeks to 50 weeks in their own homes using videoanalysis. Movements were categorised according to the classifications of Thelen [17] and Hopkins and Prechtl [10]. Developmental profiles for the frequency of occurrence of the different types of movements were determined. These were found to be dependent on the posture and mobility (P < 0.05) of the infant. Developmental profiles for infants up to 6 months of age were also produced for the temporal characteristics of leg kicks and arm waves. The speed of the flexion and extension phases of both arm waves and leg kicks were found to increase significantly (P < 0.05), with a corresponding decrease in variability over age (P < 0.05). These developmental profiles may be useful in determining normal and abnormal motor behaviour in early infancy. PMID- 7533077 TI - Bleomycin sulfate-induced micronuclei in human, rat, and mouse peripheral blood lymphocytes. AB - The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 micrograms/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37 degrees C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species' concentration response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations > or = 20 micrograms/ml. The nucleation index was significantly decreased in all species at either 80 or 160 micrograms/ml. PMID- 7533078 TI - Colour flow Doppler for gestational trophoblastic neoplasia. AB - Twenty-five consecutive patients referred to the Department of Gynecology and Obstetrics, Karolinska Hospital, Stockholm, because of gestational trophoblastic neoplasia (GTN) had colour flow Doppler performed. The examinations were performed transabdominally in the initial ten patients and transvaginally thereafter. Tumour vessels within the uterus were detected in eighteen patients (72%). A negative colour Doppler finding in patients with persistent disease could imply a diagnosis of choriocarcinoma rather than invasive mole, but all five patients belonging to this group responded promptly to chemotherapy. There seems to be a rough correlation between extent of uterine disease, as judged by colour flow Doppler, and response to chemotherapy. PMID- 7533079 TI - Pelvic exenteration in gynecologic oncology. Review. AB - Pelvic exenteration is still indicated in the treatment of gynecological cancer, in patients with pelvic resectable disease, after the failure of standard treatment. The reported survival ranges from 20 to 60% and has been increasing in the last 15 years; the prognostic factors more frequently described are margin status, time from diagnosis or radiotherapy, lesion size, preoperative side-wall fixation. The role of palliative exenteration in patients with non-resectable disease and/or nodal metastases is discussed. Survival and morbidity in elderly patients are comparable to the younger group and age cannot be considered as an exclusion criteria. Gastrointestinal, urinary and infectious complications are still considerable, but morbidity and mortality have been reduced by surgical and intensive care developments. New surgical techniques, i.e. vaginal reconstruction and continent urinary diversions, have improved quality of life, especially in younger patients, with longer expected survival. PMID- 7533080 TI - Vascularisation of ocular coralline hydroxyapatite implants. AB - Vascularisation of coralline hydroxyapatite used to replace the enucleated bulb is of critical importance for the uncomplicated implantation of a motility peg connecting the implant with the cosmetic prosthesis. Technetium-99m diphosphopropanedicarboxylic acid (DPD) single-photon emission tomography (SPET) was used to evaluate the rate of vascularisation as well as the time required for completion of vascularisation. Twenty-four patients were enrolled in the study, which was designed to evaluate vascularisation 10 days, 2 months and 4 months after implantation of a coralline implant. Nineteen patients completed the study and the visual impression of the completion of the vascularisation was scored from 0 (no vascularisation) to + (complete vascularisation) for each patient. No tracer accumulation was detected in any patient at the 10-day examination. Increasing vascularisation was demonstrated with time, and full vascularisation of the coralline implant was seen in all but one case by 4 months after implantation. We conclude that vascularisation of ocular coralline hydroxyapatite implants occurs early and is completed by 4 months after implantation in most cases, but should be confirmed at this time by 99mTc-DPD SPET. PMID- 7533081 TI - Human circulating specific antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers. AB - Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and alpha 4 integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans. PMID- 7533084 TI - B7-1 expression of Langerhans cells is up-regulated by proinflammatory cytokines, and is down-regulated by interferon-gamma or by interleukin-10. AB - Langerhans cells (LC) act as potent antigen-presenting cells (APC) for primary and secondary T cell-dependent immune responses. LC express several costimulatory and/or adhesion molecules such as B7/BB1, which has been implicated as one of the important determinants for professional APC. Recent studies have shown that B7/BB1 antigens comprise three distinct molecules termed B7-1, B7-2, and B7-3. We have examined the regulatory properties of B7-1 expression in LC using various cytokines including interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, interferon (IFN)-gamma, granulocyte/macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha. We have demonstrated: 1) that the B7-1 expression of LC is reproducibly up-regulated by either GM-CSF, TNF-alpha, IL-1 alpha, IL-1 beta, or IL-4 in a dose- and time dependent manner, 2) that GM-CSF exhibits the most active effect on B7-1 up regulation in each experiment, 3) that IFN-gamma or IL-10 profoundly inhibits the B7-1 expression of LC in a dose- and time-dependent manner, and 4) that the down regulatory ability of IFN-gamma or IL-10 neutralizes the activity of up regulatory cytokines. The enhancing or inhibitory action of these cytokines on B7 1 expression occurs selectively because none of the cytokines consistently affects I-A expression of LC. These data suggest that the B7-1 expression of LC may be dynamically regulated by these up- and down-regulatory cytokines in normal and inflammatory epidermal microenvironment. PMID- 7533082 TI - Expression of cell interaction molecules by immature rat thymocytes during passage through the CD4+8+ compartment: developmental regulation and induction by T cell receptor engagement of CD2, CD5, CD28, CD11a, CD44 and CD53. AB - Rat thymocytes of the T cell receptorlow (TcRlow) CD4+8+ subset which is the target of repertoire selection are heterogeneous with respect to expression of the cell interaction (CI) molecules CD2, CD5, CD11a/CD18 (LFA-1), CD28 and CD44. We show that this heterogeneity is due to the developmental regulation of these CI molecules during passage through the CD4+8+ compartment, and to up-regulation by TcR engagement. Thus, cohorts of CD4+8+ cells differentiating synchronously in vitro from their direct precursors, the immature CD4-8+ cells, were homogeneous with regard to CI molecule expression. Upon entry into the CD4+8+ compartment, they expressed relatively high levels of CD2 and CD44, and moderate levels of CD5, CD28 and CD11a. CD2, CD28 and CD44 were slightly down-regulated during the following 2 days, whereas CD5 slightly increased and CD11a remained constant. TcR stimulation using immobilized monoclonal antibodies resulted in rapid and dramatic up-regulation of CD2, CD5 and CD28 and, to a lesser extent, of CD11a and CD44. Finally CD53, a triggering structure absent from unstimulated CD4+8+ thymocytes was also rapidly induced by TcR stimulation. Inclusion of interleukin (IL)-2, IL-4, or IL-7 in this in vitro differentiation system did not affect the levels of CI molecules studied. Since the high levels of CI molecules induced by TcR-stimulation correspond to those found in vivo on TcRintermediate thymocytes known to be undergoing repertoire selection, these results suggest that upregulation of CI molecules by TcR engagement provides a mechanism by which thymocytes that have entered the selection process gain preferential access to further interactions with stromal and lymphoid cells in the thymus. PMID- 7533083 TI - CD28 functions as an adhesion molecule and is involved in the regulation of human IgE synthesis. AB - Activated T cells induce IgE switching in B cells via a combination of lymphokines and direct T:B cell contact. As CD28-deficient mice have reduced basal levels of IgG1 and IgG2a and diminished Ig class switching, we investigated whether the CD28/B7.1 (CD80) ligand pairing might also be involved in human IgE regulation. Co-incubation of an allergen-specific, human T cell clone with tonsillar B cells caused a marked up-regulation of CD28 expression, whereas, in contrast, CD45 RB expression was unaffected. To test whether blocking the CD28: B7.1 interaction affected IgE synthesis, a dialyzed anti-CD28 monoclonal antibody (mAb) was added to cultures containing tonsillar B cells, pre-activated T cell clones and interleukin-4. Anti-CD28 treatment caused a reproducible, dose dependent inhibition of IgE, but not IgG synthesis that was accompanied by a visible decrease in cell aggregate formation. Conversely, an anti-B7.1 mAb had no effect in this system. The effect of blocking CD28-ligand interactions on lymphocyte adhesion was formally assessed on human T cell clones and B cell lines using dual intracellular staining and flow cytometry. Co-incubation with an anti CD28 mAb, but not control IgG or anti-B7.1 mAb, resulted in a marked impairment of conjugate formation that correlated well with T cell surface expression of CD28. Using this system we found that an anti-CTLA-4 mAb but not an anti-B7.2 mAb inhibited T:B cell conjugate formation. Lastly, in addition to a direct effect of anti-CD28 mAb on conjugate formation, 14-day culture of T and B cells in the presence of anti-CD28 caused a marked decrease of ICAM-1 (CD54) expression on aggregated lymphocytes. In contrast, LFA-1 (CD18) expression was unaffected. We, therefore, conclude that the T cell co-stimulatory molecule CD28 is involved in the regulation of IgE synthesis in vitro. CD28 may act to a limited extent as an adhesion molecule, though apparently not by pairing with B7.1 or B7.2. It is more likely that ligation of CD28 under certain conditions modulates the expression of other T and B cell surface molecules. PMID- 7533085 TI - Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells. AB - Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages. A well-defined HLA-B27 restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (Th) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E. coli and Salmonella typhimurium. Macrophages infected with both recombinant bacteria presented the Th epitope to the specific CD4+ T cell clone, but failed to present the CTL epitope to the specific CD8+ T cell clone. Presentation of the Th epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II. Presentation of the Th epitope to the CD4+ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation. The efficiency of macrophages to process and present the inserted Th epitope was similar for both the recombinant E. coli and S. typhimurium strains. PMID- 7533086 TI - Acute rejection of vascular heart allografts by perforin-deficient mice. AB - To study the role of perforin in cell-mediated graft rejection, vascularized hearts were grafted to perforin-deficient C57BL/6 and control C57BL/6 recipient mice. Fully allogeneic heart grafts (BALB/c) were acutely rejected by both recipients within 6 days. Peritoneal exudate lymphocytes from control mice but not from perforin-deficient mice exhibit a strong alloreactive cytotoxic activity in vitro. Histological analysis of the rejected tissues demonstrated extensive mononuclear cell infiltrates in both recipients. Flow cytometry analysis and immunohistology of graft-infiltrating cells showed similar proportions of lymphocyte subsets (CD8 >> CD4). Collectively, these data indicate that perforin is not essential in the cell-mediated acute rejection of a fully mismatched heart allograft. However, perforin-dependent effector mechanisms appeared to be limiting in the T cell-mediated rejection of heart allografts differing only at a single major histocompatibility complex class I antigen (bm1), because these grafts survived longer (mean 87.8 days) in perforin-deficient than in control mice (mean 31.5 days). PMID- 7533087 TI - Role of CD44 cytoplasmic domain in hyaluronan binding. AB - The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T-lymphoma cells was evaluated by flow cytometry using fluorescein-conjugated HA (Fl-HA). Previous studies showed that wild-type hematopoietic CD44 bound Fl-HA when expressed in AKR1, but that truncated "tailless" CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide-bonded dimer of CD44, formed by substituting the transmembrane region of CD3 zeta chain for that of CD44, binds Fl-HA, even when the cytoplasmic domain of the CD44 dimer is absent. We conclude that dimerization of CD44 abrogates the requirement for the cytoplasmic domain, suggesting that the cytoplasmic domain of CD44 may contribute to HA binding by promoting CD44 clustering. These results suggest that changes in the distribution of CD44 on the cell surface, induced by molecular interactions either from within the cell or from outside, may regulate its role as a receptor. Further studies sought to localize the region of the CD44 cytoplasmic domain contributing to HA binding by the construction of a series of cytoplasmic domain truncation mutants and internal deletion mutants. All of the mutant CD44 molecules bound Fl-HA similarly to wild-type CD44. Thus, it was not possible to assign the function mediating HA binding to a specific region of the cytoplasmic domain, suggesting either that multiple regions of the cytoplasmic domain can promote enhancement of HA binding, or that the role of the cytoplasmic domain in mediating this function does not require a specific amino acid sequence. PMID- 7533088 TI - Antibody ligation of CD7 leads to association with phosphoinositide 3-kinase and phosphatidylinositol 3,4,5-trisphosphate formation in T lymphocytes. AB - The CD7 40-kDa glycoprotein is present on a major subset of human T cells and in the presence of phorbol esters mediates an accessory pathway of T cell activation. Hitherto, the intracellular events elicited by CD7 have been ill defined. This report demonstrates that cross-linking of CD7 results in the formation of phosphatidic acid in the absence of phosphatidylinositol-4,5 bisphosphate metabolism and also the formation of D-3 phosphoinositides lipids which have been postulated to act as intracellular regulatory molecules. The magnitude of D-3 phosphoinositide formation was similar to that induced by CD3. Both the CD7- and CD3-induced elevation of phosphatidylinositol 3,4,5 trisphosphate approximately 5-10 fold less than that elicited by ligation of the costimulatory molecule CD28 by its counter receptor CD80. The formation of D-3 phosphoinositides following ligation of CD7 coincided with the co-association of CD7 with phosphoinositide 3-kinase, the enzyme which mediates the formation of D 3 phosphoinositide lipids. In contrast, ligation of another reported T cell accessory molecule CD5, failed to elicit formation of D-3 phosphoinositides, implying that phosphoinositide 3-kinase is not coupled to all T cell molecules with accessory functions. Since D-3 phosphoinositides have been suggested to play a pivotal role in T cell costimulatory signals induced by CD28, the results presented in this study suggest that CD7 may also influence T cell activation via this pathway. PMID- 7533089 TI - Location and sequence of rearranged immunoglobulin genes in human thymus. AB - Thymic B cells are a proliferating B cell population concentrated in normal thymic medulla. They are large cells, some with dendritic morphology, and are not associated with any organized follicular structure. Previous work in this laboratory has shown that most of these B cells are surrounded by tightly adherent thymocytes. The literature on human thymic B cells contains many inconsistencies. There is no consensus on whether they express CD5. Even the existence of thymic B cells has been questioned. In this study we have undertaken the first analysis of rearranged immunoglobulin (Ig) genes, looking in particular for evidence of affinity maturation. The Ig VH genes of human thymic B cells in this study are those of the fetal repertoire, though the resemblence to fetal Ig genes is limited in other respects. They are mostly unmutated, but the presence of mutated sequences suggests that this is not a uniform population, as has been previously indicated by phenotypic studies. PMID- 7533091 TI - Antigen-binding B cells and polyreactive antibodies. AB - The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigen-binding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and beta-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%-57%) by both homogeneous and heterogeneous antigens. Both CD5+ and CD5- antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5+ antigen-binding (85%) as compared to the CD5- antigen binding (50%) population. Comparison of CD5+ B cells that bound antigens with CD5+ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells. PMID- 7533090 TI - FK506 binding protein 12 mediates sensitivity to both FK506 and rapamycin in murine mast cells. AB - The immunosuppressive drugs FK506 and rapamycin bind to a family of intracellular proteins termed FK506-binding proteins (FKBP). FK506 and rapamycin inhibit lymphocyte-activation pathways by forming complexes with an FKBP; subsequently, the drug/FKBP complexes interact with target molecules involved in signal transduction. A key target of FK506/FKBP12 complexes is calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. In mammalian cells, rapamycin treatment is associated with inhibition of the activity of several cellular serine/threonine kinases, including p70 S6 kinase. These kinases may function in signaling pathways involving TOR gene producs, which have been shown to interact with rapamycin/FKBP12 complexes in vitro. To determine if FKBP12 mediates the effects of both FK506 and rapamycin in mammalian cells, we overexpressed FKBP12 in a murine mast cell line. Increased expression of FKBP12 resulted in increased sensitivity to FK506 and rapamycin, as measured by inhibition of calcineurin activity and p70 S6 kinase activity, respectively. In contrast, overexpression of FKBP25 had no effect on sensitivity to either drug. Two distinct point mutations in FKBP12, one altering a hydrophobic residue within the drug-binding pocket and the other changing a charged surface residue of FKBP12, abrogated its ability to mediate sensitivity to FK506 and rapamycin. These results establish that FKBP12 can mediate sensitivity to both FK506 and rapamycin in mammalian cells. PMID- 7533094 TI - Encephalitogenic, myelin basic protein-specific T cells from naive rat thymus: preferential use of the T cell receptor gene V beta 8.2 and expression of the CD4 CD8- phenotype. AB - Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the native thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor V beta 8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4+CD8- phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co receptor. Some clones were completely CD4-CD8-, while others included CD4-CD8- subpopulations along with CD4+CD8- T cells. In the one mixed population examined in detail, the CD4-CD8- and CD4+CD8-T cell subpopulations used a T cell receptor with identical beta chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus. PMID- 7533095 TI - Regulation of an anti-polysaccharide immune response in BALB/c mice through a tight T and B lymphocyte idiotypic connection. AB - The isotype expression in the J558 idiotype-associated humoral immune response against alpha(1-->3)-dextran in BALB/c mice is controlled by idiotype-specific T cells which silence in situ B lymphocytes primed and committed to an IgG response. This leads to a restriction of the type II thymus-independent response to the sole production of IgM antibodies. The availability of the T cell receptor (TcR) alpha and beta sequences for such a regulatory T cell clone allows the investigation of the degree of heterogeneity of the TcR usage of these T cells. It is found that all alpha(1-->3)-dextran-primed BALB/c mice use a very similar, possibly identical TcR. This suggests a tight, possibly genetically programmed, interaction between the J558 idiotype-bearing dextran-specific B cells and their idiotype-specific regulatory T cell counterparts. PMID- 7533093 TI - Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants. AB - The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCD14). Previous studies indicate that monocytes produce sCD14 by limited proteolysis of the membrane-bound receptor. In this study we demonstrate that human monocytes also produce sCD14 by a protease independent mechanism. To investigate the molecular nature of this second pathway we studied sCD14 formation in the monocytic cell line Mono Mac 6 (MM6) and in CD14 transfectants. Both MM6 and the CD14 transfectants constitutively produce sCD14 by a protease-independent mechanism. Structural analysis of sCD14 produced by the CD14 transfectants reconfirmed the presence of the COOH terminus predicted from the cDNA. Since glycosylphosphatidylinositol anchor attachment is associated with the removal of a hydrophobic C-terminal signal peptide, our finding demonstrates that the transfectants secrete sCD14 which escaped this posttranslational modification. Identical results obtained for sCD14 derived from peritoneal dialysis fluid of a patient with kidney dysfunction show the in vivo relevance of this pathway for sCD14 production. PMID- 7533092 TI - Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses. AB - Interactions between T and B cells are dynamic and regulated by interacting receptor: co-receptors. Interactions between CD40 and its ligand, gp39, and the CD28/CTLA-4 and B7 family members play a decisive role in regulating the progression of cognate interactions. The interdependence of gp39-CD40 and CD28/CTLA-B7 expression and function was studied in vitro during an antigen induced immune response using T cells from mice expressing a transgenic T cell receptor (TCR). gp39 was induced on pigeon cytochrome c (PCC)-transgenic T cells in the presence of antigen and antigen-presenting cells. The antigen-induced expression of gp39 on transgenic T cells was inhibited by antibodies to class II major histocompatibility complex, CD4 and LFA-1, but not by CTLA-4 Ig, anti-B7-1 or anti-B7-2. These data established that the antigen-induced expression of gp39 was not dependent on co-stimulation via CD28/CTLA-4. The addition of PCC also resulted in the modest expression of B7-1 and a more robust expression of B7-2 on the cognate B cells. The addition of anti-gp39 blocked the up-regulated expression of B7-1 and partially blocked the up-regulated expression of B7-2. The addition of anti-gp39 and anti-interleukin-4 inhibited antigen-induced expression of B7-2 on B cells to near background levels. Studies on the up-regulation of B7 1 and B7-2 on resting B cells showed that soluble gp39 up-regulated B7-1 and B7-2 expression on B cells. In addition, interleukin-4 and interferon-gamma up regulated B7-2 expression on B cells. Taken together, these data demonstrate that the antigen-induced expression of gp39 is dependent on TCR-derived signals, yet independent of CD28/CTLA-4 co-stimulatory signals. Cognate interactions also resulted in the modest enhancement of B7-1 expression and a more profound expression of B7-2 which were completely or partially dependent on gp39-CD40 interactions. PMID- 7533096 TI - Differential inhibition of foot tapping and chromodacryorrhoea in gerbils by CNS penetrant and non-penetrant tachykinin NK1 receptor antagonists. AB - The inhibition of GR73632-induced foot tapping in gerbils by central nervous system (CNS) penetrant of tachykinin NK1 receptor antagonists was investigated. Intracerebroventricular infusion of the highly selective tachykinin NK1 receptor agonist GR73632 (3 pmol) induced a vigorous repetitive hind foot tapping response which was inhibited by CP-99,994 (ID50 = 0.06 mg/kg i.v.) but not by its less active enantiomer, CP100,263 (10 mg/kg i.v.). Similarly, the poorly CNS penetrant quaternised compound, L-743,310, failed to inhibit foot tapping at doses up to 3 mg/kg i.v. In contrast, all three compounds inhibited chromodacryorrhoea induced by systemic administration of GR73632 (0.5 nmol i.v.) (ID50 = 0.06, 2.95 and 0.004 mg/kg i.v., respectively). These findings confirm that foot tapping and chromodacryorrhoea in gerbils provide simple assays for the central and peripheral activation of tachykinin NK1 receptors. PMID- 7533097 TI - A highly sensitive enzyme immunoassay for G-CSF in human plasma. AB - We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to measure granulocyte colony-stimulating factor (G-CSF) in human plasma. This ELISA employs a combination of a mouse monoclonal antibody (MAb) as the first antibody and an affinity-purified sheep polyclonal antibody conjugated with beta D-galactosidase as the second antibody. The coefficients of intra- and interassay variations were 2.2 to 3.6% and 8.3 to 10.2%, respectively. The assay had no cross-reactivity with four other human cytokines. Plasma G-CSF levels in healthy volunteers could easily be measured because the detection limit was 0.5 pg/mL. The mean plasma G-CSF concentration in 57 healthy volunteers (aged 19 to 47; 27 males and 30 females) was 10.5 +/- 4.5 pg/mL standard deviation [SD]). There was no statistically significant relationship between plasma G-CSF concentration and absolute neutrophil counts in those healthy volunteers (r = 0.259; p = 0.052). PMID- 7533098 TI - Effect of stem cell factor on myelopoiesis potential in human Dexter-type culture systems. AB - Hematopoiesis is influenced by the presence of the hematopoietic microenvironment, and Dexter-type liquid culture systems represent an in vitro representation of some aspects of the microenvironment that are optimal for the propagation of myeloid progenitors. Marrow stromal layers, which constitute part of these culture systems, produce growth factors, including stem cell factor (SCF), a ligand for the c-kit proto-oncogene that has been found to increase detection of myeloid, erythroid, and megakaryocytic progenitors in short-term marrow colony assays. In this work, the role of SCF in Dexter-type culture systems was examined to better define its contribution to steady-state myelopoiesis. When cultured in the continued presence of 100 ng/mL SCF, both primary and recharged cultures demonstrated significantly greater CFU-GM output, with quantitative differences noted throughout culture duration (up to 6 weeks). This increase in CFU-GM could be inhibited specifically with the addition of 1:1500 SR-1, a neutralizing anti-c-kit monoclonal antibody (MAb) that neutralizes the biological effects of SCF, and the increase was noted both with recharged light-density marrow cells and purified CD34+ progenitor cells. On the other hand, when primary or recharged marrow cultures were established in the absence of exogenous SCF, but in the continuous presence of SR-1, no inhibition of CFU-GM output was observed. When light-density marrow cells were purged of pre-existing CFU-GM by 4-hydroperoxycyclophosphamide (4-HC) and were seeded over irradiated stromal layers, exogenous SCF resulted in detection of CFU-GM from 4-HC-treated cells as early as 1 week of culture, as compared to the lack of significant emergence of CFU-GMs at 4 weeks in the control cultures. This SCF effect was also inhibited by SR-1. Purified CD34+ progenitor cells did not adhere to SCF immobilized to tissue culture plates, and the adhesion of such progenitors to murine Steel lines transfected with membrane-bound SCF was not greater than to the parent nontransfected Steel line, suggesting that the effect of SCF was not on CD34+ cell adhesion. These studies confirm the action of SCF at a pre-CFU level, and they demonstrate the ability of SCF to stimulate increased production of myeloid progenitors in long-term liquid culture systems. PMID- 7533099 TI - Effects of rhIL-1 alpha, rhIL-1 beta, and rhIL-1 receptor antagonist on erythroid progenitors (CFU-E and BFU-E) in human bone marrow. AB - Interleukin-1 (IL-1) is known to promote the production of colony-stimulating factor (CSF) and to possess the ability to protect the bone marrow suppression in granulocyte-macrophage (GM) series associated with radiotherapy or chemotherapy. There are conflicting reports concerning the action of IL-1 on erythroid progenitors, however, and no consensus has been established. In the present study, the influences of recombinant human IL-1 alpha (rhIL-1 alpha), rhIL-1 beta, and rhIL-1 receptor antagonist (rhIL-1ra) on erythroid progenitors (colony forming units-erythroid, CFU-E; and burst-forming units-erythroid, BFU-E) in human bone marrow were studied. rhIL-1 alpha and rhIL-1 beta (1-1000 pg/mL) enhanced the CFU-E and BFU-E growth in human nonadherent (NA) bone marrow cells. rhIL-1 alpha and rhIL-1 beta also stimulated the formation of CFU-E and BFU-E in the NA and T cell-depleted bone marrow fraction. Moreover, rhIL-1 alpha and rhIL 1 beta enhanced the CFU-E and BFU-E in the CD34+ bone marrow cell fraction. These data and the results of limiting dilution analysis indicate that the stimulatory effect of IL-1 may consist of direct actions on erythroid progenitors. The enhancing effect of rhIL-1 alpha and rhIL-1 beta on erythroid progenitors was inhibited by rhIL-1ra. These data suggest that the stimulatory effect of IL-1 on CFU-E and BFU-E is mediated via the IL-1 receptor. PMID- 7533100 TI - High-frequency granuloid colony-forming ability of G-CSF receptor possessing CD34 antigen positive human umbilical cord blood hematopoietic progenitors. AB - The presence of granulocyte colony-stimulating factor receptor (G-CSFR) in CD34+ hematopoietic progenitors is demonstrated by flow cytometry. Cord blood mononuclear cells (MNCs) contain about 1.09 +/- 0.2% CD34+ cells, 0.32 +/- 0.1% CD34+/G-CSFR+ (G-CSFR+) cells, and 0.77 +/- 0.1% CD34+/G-CSFR- (G-CSFR-) cells. The colony-forming ability of G-CSFR+ cells in the presence of G-CSF, granulocyte macrophage CSF (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo) was higher than that of G-CSFR- cells (29.5 vs. 9.8%; p < 0.01). In the fraction of G CSFR+ cells, the most frequently formed colony type was CFU-G/GM, while burst forming unit-erythroid (BFU-E) or colony-forming unit-macrophage (CFU-M) were rare. On the other hand, the incidence of BFU-E and CFU-G/GM was similar in the fraction of G-CSFR- cells. This indicates that most granuloid colonies of CD34+ cells were derived from G-CSFR+ cells. These results suggest a lineage commitment for the vast majority of G-CSFR+ hematopoietic progenitors. PMID- 7533102 TI - Enhancement of human hematopoiesis by mast cell growth factor in human-sheep chimeras created by the in utero transplantation of human fetal hematopoietic cells. AB - We have previously described a unique model of long-term, multilineage, human hematopoietic chimerism in sheep created by the in utero transplantation of human hematopoietic stem cells (HSC) into pre-immune fetal lambs. In this study, we examined the effect of chronic administration of recombinant human mast cell growth factor (rhMGF) on 1) human cell engraftment in pre-immune sheep and 2) human cell expression in human-sheep chimeras at 2-years posttransplant. rhMGF (25 micrograms/kg) or saline was administered in utero via chronic intraperitoneal (IP) catheters to three separate sets of twin fetuses on alternate days for 10 doses following transplantation of human HSC. Flow cytometric and karyotype analyses of peripheral blood from two sets of twins at 45-days posttransplant and of peripheral blood from the remaining set of twins at birth revealed a significant increase in percentages of donor (human) progenitors and cells in rhMGF-treated lambs. rhMGF (60 micrograms/kg/day) was also administered by IP injection to two, 2 year-old, human-sheep chimeras for 18 consecutive days. Flow-cytometric analysis of peripheral blood and bone marrow revealed a six- to seven-fold increase in human cell expression. The effect on early human progenitors (i.e., colony-forming unit-mix [CFU-Mix], CFU granulocyte/macrophage [CFU-GM], and burst-forming unit-erythroid [BFU-E]) was determined by karyotype analysis of individual colonies grown under conditions favoring human cell growth. A three- to five-fold increase in human CFU-Mix and BFU-E occurred with a minimal increase in CFU-GM. This in vivo study supports in vitro data suggesting that MGF is a powerful regulator of human hematopoiesis and preferentially stimulates early hematopoietic progenitors. It also supports the potential value of the human-sheep model for the in vivo study of normal and abnormal human hematopoiesis. PMID- 7533101 TI - Decreased expression of protein phosphatase type 2A in HL-60 variant (HL-60RAr) cells resistant to induction of cell differentiation by all-trans retinoic acid. AB - To evaluate the molecular basis for susceptibility of the cell differentiation induced by all-trans retinoic acid (ATRA), we examined biochemical activities and expression of protein phosphatases type 1 (PP1) and type 2A (PP2A) from HL-60 cells that are susceptible to differentiation induced by ATRA and HL-60RAr cells, HL-60 variant cells that are resistant to such induction. One nM of calyculin-A (CAL-A) achieved the enhancement of granulocytic differentiation in ATRA-treated HL-60 (1 microM) cells. ATRA exerted no differential action in HL-60RAr cells, but when used in combination with CAL-A, the differential activity was partly resumed at functional and phenotypic levels without change in morphology. The phosphatase activity in the cytosol from HL-60RAr cells was 50% of that from parental HL-60 cells, but the enzyme activities in either membrane or nuclear fractions showed similar values. The decreased phosphatase activity in the cytosol of HL-60RAr cells was mainly due to the decreased expression of the PP2A catalytic subunit. This low level of PP2A protein was reflected at a relative deficiency in expression of the PP2A beta gene in HL-60RAr cells. The exposure to 1 microM ATRA resulted in downregulation of PP2A catalytic subunit protein in HL 60 cells, but ATRA did not affect PP2A expression in HL60RAr cells. Both cell lines expressed the proteins of each PP1 catalytic subunit isozyme (i.e., PP1 alpha, PP1 gamma, and PP1 delta) at comparable levels. ATRA treatment had no effect on the levels of PP1 isozymes. Our results show a correlation between the extent of PP2A expression and the response of HL-60 and HL-60RAr cells to the differentiative effects of ATRA. PMID- 7533103 TI - Increased type I procollagen mRNA transcripts in the lungs of mice during the development of bleomycin-induced fibrosis. AB - In this study, in situ hybridization has been used to investigate the localization of type I procollagen messenger ribonucleic acid (mRNA) in normal lung, and in the lungs of mice during the development of bleomycin-induced pulmonary fibrosis. Lung fibrosis was induced by a single intratracheal instillation of bleomycin sulphate (6 mg.kg-1 body weight), and tissues examined at times up to 35 days thereafter. Tissue transcripts of alpha 2(I) procollagen mRNA were visualized after hybridization with 35S-labelled riboprobes. Hybridization signals were associated with alveolar interstitial cells throughout the normal lung, with additional areas of dense hybridization signals observed subpleurally. Three days following administration of bleomycin, there was no apparent change in the pattern of hybridization. By 10 days, foci of intense hybridization signals indicative of gene activation were observed associated with individual cells in the alveolar interstitium. At 21 days, the increase in hybridization signals appeared to be associated with greater numbers of cells rather than highly activated cells. These results demonstrate that procollagen genes are normally expressed in the mouse lung, and that during bleomycin-induced pulmonary fibrosis hybridization signals increase, suggesting that both enhanced gene expression by individual cells and increased numbers of cells expressing the type I procollagen gene are involved in the pathogenetic mechanism. PMID- 7533105 TI - Cytokine-mediated production of nitric oxide in isolated rat hepatocytes is dependent on cytochrome P-450III activity. AB - To investigate the role of the cytochrome P-450 system in NO synthesis, cytochrome P-450IIIA, IIE and IA activities were specifically inhibited by cimetidine (IIIA), clotrimazole (IIIA), benzoflavone (IA) and disulfiram (IIE) in a model of cultured rat hepatocytes. Cytokine-induced NO synthesis was significantly decreased in the presence of cimetidine and clotrimazole. Kinetic analysis revealed a non-competitive mode of inhibition (Ki = 21 mM, cimetidine; Ki = 13 microM, clotrimazole). Reverse transcriptase-PCR and immunoblot analysis revealed no significant change in steady state levels of iNOS mRNA and protein expression with P-450IIIA inhibition. Purified iNOS enzyme activity was not altered. These data suggest that cytokine-mediated hepatocyte synthesis of NO is dependent upon P-450IIIA activity, which functions in a post-translational capacity. PMID- 7533104 TI - The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. AB - One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition. PMID- 7533106 TI - Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes. AB - LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them alpha 2 macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand- and immunoblotting the participation of LRP/alpha 2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density 1.10 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRP/alpha 2MR is physiologically meaningful as RAP avidly binds to LRP/alpha 2MR and by that shuts off all ligand binding function. In endosomal fractions we found one single binding protein for 125I-labelled Lf. With a specific anti LRP/alpha 2MR antibody and ligand blotting with 125I labelled RAP this endosomal Lf binding site was verified to be LRP/alpha 2MR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 min prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/alpha 2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/alpha 2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation. PMID- 7533108 TI - Annual Scientific Program Summary. Medical Society of Delaware, Saturday, November 19, 1994. PMID- 7533109 TI - Pepsin hydrolysis of the adherent mucus barrier and subsequent gastric mucosal damage in the rat: effect of diosmectite and 16,16 dimethyl prostaglandin E2. AB - BACKGROUND AND OBJECTIVE: The gastroduodenal mucus layer is progressively eroded at its luminal surface as a consequence of pepsin mucolysis. Diosmectite binds to gastric mucus and modifies its rheological properties. Prostaglandins are well known mucus secretagogues. The aim of this study is to describe interactions of diosmectite and 16,16 dimethyl prostaglandin E2 on adherent gastroduodenal mucus and pepsin mucolysis in the rat. METHODS: Instillation of pepsin (1 or 2 mg.mL-1 at pH1 or pH2) into the pylorus ligated stomach of anaesthetised rats resulted in progressive disruption of the adherent mucus layer and a large, significant, increase in soluble degraded mucin compared to that following instillation of HCl pH1 or pH2. Pepsin (2 mg.mL-1), instillation over 2 hours, but not HCl alone, consistently resulted in small focal, haemorrhagic mucosal lesions, significant bleeding into the lumen and histologically, localised punctate ulcers in an otherwise intact epithelium. Diosmectite (500 mg.kg-1) and 16,16 dimethyl prostaglandin E2 were given by oro-gastric intubation. RESULTS: Diosmectite, given 30 minutes beforehand, inhibited breakdown of the adherent gastric mucus barrier by pepsin in vivo. When administered up to 16 hours prior to the experiment, diosmectite prevented pepsin induced gastric mucosal haemorrhage and histological epithelial damage. Substantial amounts of diosmectite (39.6 micrograms/mm2, equivalent in volume to layer 93 microns thick) were bound to the gastric mucosa 30 minutes after administration. Diosmectite (100:1 by weight to enzyme) completely inhibited pepsin hydrolysis of protein in vitro. Topical 16,16 dimethyl prostaglandin E2, 5 micrograms.kg-1 increased the thickness of the adherent mucus layer by two-fold. Both doses of the prostaglandin prevented pepsin induced gastric mucosal haemorrhage and histological epithelial damage. CONCLUSIONS: These results show that both diosmectite and prostaglandin increase the effectiveness of the mucus barrier against mucosal damage by pepsin in vivo. PMID- 7533111 TI - Potential role of nitric oxide in a model of chronic colitis in rhesus macaques. AB - BACKGROUND/AIMS: Excess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis. METHODS: NO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction. RESULTS: Plasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity. CONCLUSIONS: It is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model. PMID- 7533110 TI - Up-regulation of insulinlike growth factor I binding sites in experimental colitis in rats. AB - BACKGROUND/AIMS: The gastrointestinal tract is a major target of insulinlike growth factor (IGF) I. IGF-I binds to two different receptors and to binding proteins (IGFBPs), which act as carriers and mediators. This study investigated the regulation of IGF-I binding sites in rat colitis. METHODS: Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. IGF-I binding sites in colon sections were localized by incubation with 125I-IGF I. The contribution of binding to the IGF-I receptor was estimated by competition with unlabeled IGF-I, IGF-II, and insulin. Colonic RNA was screened for IGFBPs by Northern hybridization. RESULTS: IGF-I binding sites were increased more than two fold in the muscularis propria of inflamed colon as soon as 12 hours and up to 1 week after injury. Insulin could not displace this elevated level of binding, even though it could displace IGF-I from the mucosa and muscularis mucosa. Northern hybridization showed a 2-3-fold increase in IGFBP-4 and IGFBP-5 messenger RNA from inflamed colon. CONCLUSIONS: Experimental colitis in rats causes an increase in IGF-I binding to the muscularis propria, which represents increased levels of IGFBP-4 and IGFBP-5. These data suggest an important role for IGFBPs in modulating IGF effects during inflammation and tissue repair. PMID- 7533107 TI - Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction. AB - The cytokine interleukin-6 (IL-6) rapidly activates a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate early gene induction by IL-6 and, as we show here, stimulation of the promoters of the genes for alpha 2-macroglobulin, Jun-B, and intercellular adhesion molecule-1 (ICAM-1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL-6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL-6 signal transduction leading to target gene induction. PMID- 7533114 TI - Significance of the secreted form of IpaC, a 45 kDa protein of Shigella dysenteriae 1, in the invasive process as determined by monoclonal antibodies. AB - Invasion plasmid antigen C (IpaC), a 45 kDa plasmid encoded protein, is associated with the virulence of virulent Shigella spp. In S. dysenteriae type 1 the 45 kDa IpaC protein is secreted to a greater extent into the surrounding medium in comparison to other Shigella spp. Monoclonal antibodies (mAbs) to the secreted form of IpaC protein were raised in this study. Of the four secretory hybrid cells, one (3G4) was found to have a very high antibody titre as determined by ELISA. The specificity of 3G4 was confirmed by immunoblotting of whole cell extract of Escherichia coli strain MC1061 carrying the plasmid pHW756 which synthesizes both the IpaB and C proteins. The effect of the mAbs on plaque formation by virulent Shigella dysenteriae 1 was determined and it was found that the clone 3G4 substantially (55%) reduced plaque formation on HeLa cell monolayer. The epitope specificity of the mAb 3G4 was competitively inhibited by the convalescent phase sera from human, suggesting that the epitope recognized by clone 3G4 was expressed during the natural course of infection and also indicating that the 45 kDa (IpaC) protein in secreted form has a definite role in the invasive process. PMID- 7533112 TI - Serotonin, catecholamines, and spontaneous midgut carcinoid flush: plasma studies from flushing and nonflushing sites. AB - BACKGROUND/AIMS: Serotonin is the diagnostic hallmark of midgut carcinoids, but the pathophysiology of spontaneous flushing is unknown. The aim of this study was to assess to what extent serotonin and catecholamine blood levels are related in time with spontaneous midgut carcinoid flush. METHODS: Using specific radioenzymatic assays, we measured prospectively before, during, and after spontaneous flushing platelet-poor plasma and whole blood serotonin and plasma catecholamines and their metabolites in 10 patients with primary midgut carcinoids. Blood was drawn simultaneously from a forearm vein and an external jugular vein draining the flushing area. RESULTS: During flushing, plasma serotonin and norepinephrine levels increased (P < 0.001) over preflush levels at both sampling sites. Intraflush serotonin and norepinephrine were twice as high (P < 0.01) in external jugular (9.57 +/- 1.40 ng/mL and 857 +/- 33 pg/mL, respectively) than in antecubital plasma (4.59 +/- 0.73 ng/mL and 471 +/- 26 pg/mL). Preflush and postflush levels were similar at both venous sites. CONCLUSIONS: Vein plasma serotonin and norepinephrine levels do increase during midgut carcinoid flush, especially in the flushing area. This may reflect a local release secondary to flush but also suggests a role for these bioamines in the pathogenesis of flushing. PMID- 7533113 TI - Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy. AB - BACKGROUND/AIMS: Hepatitis C virus (HCV) genome heterogeneity by sequence analysis in association with interferon (IFN) inefficacy has been reported. This study was performed to establish a convenient method for detecting the HCV quasispecies complexity and to determine the correlation between the complexity and the responsiveness to IFN therapy in patients with chronic hepatitis C. METHODS: The quasispecies complexity of HCV hypervariable region 1 in patients treated with IFN-alpha was analyzed by polymerase chain reaction-mediated single strand conformation polymorphism (SSCP). RESULTS: Seven of 25 patients (28%) with low complexity (SSCP band number of < or = 2) were HCV RNA negative after treatment, whereas in 24 patients with high complexity (SSCP band number of > or = 3), the response to IFN was almost insignificant because only 1 patient (4.5%) remained HCV RNA negative after treatment (P < 0.05). Among type 1b patients, IFN therapy was only effective for patients with low amounts of HCV RNA (< or = 10(7.5) copies/mL serum) and low complexity. In contrast, most type 2a patients tended to respond to the therapy with exceptions being those with high amounts of HCV RNA and high complexity. CONCLUSIONS: The complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C. PMID- 7533115 TI - Hepatitis C virus core protein: synthesis, affinity purification and immunoreactivity with infected human sera. AB - The genomic region encoding the core (C) protein (amino acids 1-162) of hepatitis C virus (HCV) was expressed in Escherichia coli as a recombinant (re-) protein with the maltose-binding protein (MBP) using the prokaryotic expression vector pMAL-CR1. The fusion protein (C::MBP) was identified as a approx. 62-kDa polypeptide by immunoblot analysis using antiserum to MBP and HCV-infected human sera. The size of C::MBP corresponded to the calculated combined molecular mass of the approx. 20-kDa HCV C protein and the approx. 42-kDa MBP. The approx. 62 kDa C::MBP was purified using amylose resin as a matrix in affinity chromatography, and showed specific reactivity with HCV-infected human sera. These results suggest that C::MBP may serve as a source of the core antigen for immunological studies on HCV infection. PMID- 7533116 TI - Characterization of a LINE-1 cDNA that originated from RNA present in ribonucleoprotein particles: implications for the structure of an active mouse LINE-1. AB - Full-length, sense-strand, long interspersed element-1 (LINE-1 or L1) RNA is found as an RNA-protein complex in mouse embryonal carcinoma cells. Since this complex is a likely intermediate in LINE-1 transposition, its RNA may be enriched for the functional, or active, subset of mouse L1 sequences. For this reason, a cDNA library was constructed from RNA prepared from these ribonucleoprotein particles. The isolation and complete DNA sequence of one clone that is a strong candidate to be a functional version of mouse L1 is reported here. The structure of this element suggests a revision of the predicted sequence of an active mouse L1 and provides a tag that can be used to isolate its locus in the genome. PMID- 7533117 TI - Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones. AB - The gene encoding the RNA-dependent protein kinase (PKR) was cloned from mouse genomic DNA and characterized by restriction mapping, Southern blot analysis and sequencing. The Southern analyses were consistent with the presence of a single copy of the Pkr gene in the mouse haploid genome. The genomic nucleotide (nt) sequence, when compared to that of previously determined cDNA nt sequences, revealed 16 exons encoding the 515-amino-acid PKR. PMID- 7533118 TI - Sequence of the functional human keratin K14 promoter. AB - The K14 keratin is an intermediate filament produced in squamous epithelia. This tissue-specific expression is directed by the promoter (pK14) of the K14 gene which has been used extensively to direct the expression of transgenes to the skin. Human K14 was cloned and the upstream sequence is presented. In transient transfections, pK14 directs expression of a luciferase reporter in keratinocytes much more potently than in breast cancer cells. PMID- 7533119 TI - Studies of gene expression in liver of insulin-like growth factor (IGF)-I, IGF binding protein-3 and growth hormone (GH) receptor/GH binding protein in rats treated neonatally with monosodium glutamate. AB - We studied the gene expression of the insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and growth hormone (GH) receptor (GHR)/GH binding protein (GHBP) in liver of rats treated neonatally with monosodium glutamate (MSG). The MSG-treated rats showed severe growth retardation and obesity compared to saline-injected controls. Serum IGF-I levels in MSG-treated rats were significantly lower than in the controls after 6 weeks of age (p < 0.01). IGF-I gene expression increased with age and was significantly lower in MSG-treated rats than in the controls (p < 0.01). IGFBP-3 gene expression was unaffected by age in both MSG-treated male rats and the controls, but was less in MSG-treated female rats than in their controls between 6 to 8 weeks of age. In our study of GHR/GHBP gene expression, MSG-treated rats of both sexes displayed a distinct 1.5 kbase band encoding GHBP RNA. We speculated that these changes reflect disruption of GH secretion in in vivo experimental models. Thus, MSG-treated rats are useful as in vivo models for study of the effect of GH disruption on developmental gene expression. PMID- 7533121 TI - Persistent hepatitis C viremia after acute self-limiting posttransfusion hepatitis C. AB - Persistent viremia after clinical or subclinical hepatitis C virus (HCV) infection is believed to occur in patients with chronic hepatitis C, but little is known about the duration of HCV replication in patients with acute hepatitis who have recovered or the relation of HCV viremia with the kinetics of antibodies to HCV (anti-HCV). We tested HCV-RNA and anti-HCV in serial serum samples from 41 patients with posttransfusion non-A, non-B hepatitis, followed for an average of 6 years after transfusion. Serum HCV-RNA was measured by nested polymerase chain reaction, which used primers from the 5' untranslated region of the HCV genome. Anti-HCV were tested with first- and second-generation enzyme-linked immunosorbent assays (ELISA 1 and ELISA 2), and with a second-generation recombinant immunoblot assay. Of the 41 patients, 10 recovered and 31 progressed to chronic liver disease. HCV-RNA was detected in serum before or simultaneously with the onset of hepatitis in all cases, and lasted between 2 and 6 weeks in 5 of the 10 patients who recovered, whereas it persisted for the entire follow-up period in every case with chronic hepatitis and in the remaining 5 patients with self-limiting hepatitis. Anti-HCV were detected with ELISA 2 in the first serum sample, with raised serum transaminases in 57% of patients, but in only 6% with ELISA 1. In the sample obtained 1 month after the onset of hepatitis, anti-HCV were detected with ELISA 2 in 94% of patients, but in 34% with the ELISA 1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533120 TI - High prevalence of serological markers of autoimmunity in patients with chronic hepatitis C. AB - The advent of specific antiviral therapy for chronic hepatitis C has increased the importance of establishing the correct etiology of chronic hepatitis in patients, especially because interferon alfa (IFN-alpha) has been reported to exacerbate autoimmune hepatitis (AIH), whereas corticosteroids increase viral replication in chronic hepatitis C. In our medical center, we have treated many patients with apparent chronic hepatitis C and serological or clinical evidence of autoimmunity. Our aim was to estimate the prevalence of this association and to learn whether demographic or clinical features distinguished between patients with or without autoimmune markers. We performed a retrospective review of the records of 244 unselected patients seen at the Clinics and Hospital of the University of Massachusetts between May 1991 and November 1993, who had elevated serum aminotransferases. One hundred seventeen patients had chronic hepatitis C defined by elevations of serum alanine transaminase (ALT) for at least 6 months, positive serum antibodies to hepatitis C virus (HCV; second-generation enzyme immunoassay [EIA2] or recombinant immunoblot assay [RIBA]), and absence of hepatitis B surface antigen in the serum. Records were reviewed for results of autoimmune markers in sera, including anti-nuclear antibodies (ANAs), anti-smooth muscle antibodies (SMAs), rheumatoid factor (RF), antimitochondrial antibodies (AMAs), anti-liver and kidney microsomal (LKM) antibodies, and cryoglobulins. We found a high prevalence of positivity, particularly for anti-SMAs (66%) and RF (76%) in both men and women. Forty of 41 patients tested negative for anti-LKM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533122 TI - Growth hormone, insulinlike growth factor-1, and insulinlike growth factor binding proteins 1 and 3 in chronic liver disease. AB - The liver is the major source of circulating insulinlike growth factor-I (IGF-I) and has been suggested as a major source of at least two of the major binding proteins that modify its bioavailability. We aimed to assess the direct effects of liver dysfunction on serum levels of IGF-1 and its major binding proteins by measuring fasting levels of growth hormone, IGF-1, IGFBP-1, IGFBP-3, insulin, C peptide, and glucose in 35 patients with cirrhosis and during an oral glucose tolerance test in 16 of those patients. Serum levels of growth hormone (GH) were high in the patients: median, 12.0 mU/L (range, 1 to 87) compared with normals, 0.95 mU/L (0.1 to 20) (P < .0005) and serum IGF-1 levels were low: 81 ng/mL (38 to 153) versus 193 ng/mL (151 to 235) (P < .0001). Serum IGFBP-3 levels were low in the patients: 1.59 mg/L (0.46 to 4.43) compared with normals, 5.41 (4.34 to 6.11) (P < .0001), and there was a significant negative correlation between IGFBP 3 levels and Childs Pugh score (r = .63 P < .0001). Fasting IGFBP-1 levels were significantly higher in the patients 31 ng/mL (11 to 92) than normals, 14 (7 to 20) (P < .0001). There was no correlation between fasting insulin and IGFBP-1 levels despite high fasting insulin levels. A decrease in IGFBP-1 levels was seen during the glucose tolerance test (GTT) in all patients. In conclusion, there are significant changes in the levels of two of the major IGF-1 binding proteins that may further limit the bioavailability of already low circulating IGF-1 levels. Substrate availability appears to be a stronger influence on fasting IGFBP-1 levels than does insulin, and the close correlation of IGFBP-3 with liver function indicates a dominant regulatory role of the hepatocyte. PMID- 7533123 TI - Extramedullary erythropoiesis in human liver grafts. AB - Extramedullary erythropoiesis in the adult is very rare and is generally confined to situations of severe bone marrow irritation or replacement. In this study, we describe the occurrence of intrahepatic erythropoiesis in patients who have received a liver allograft and who have no evidence of bone marrow dysfunction. By routinely performed transplant aspiration cytology (TAC), marked intrahepatic erythropoiesis could be detected in 39 of 312 patients (12.5%) with liver allograft. In 19 patients, including 5 of 8 (63%) after combined liver and kidney transplantation, intrahepatic erythropoiesis occurred within the first 3 weeks after surgery. Twenty patients showed intrahepatic erythropoiesis between 3 weeks and 4 months after transplantation. Erythropoiesis was usually transient, lasting between 1 and 3 weeks. Cytologically, mature as well as immature erythroblasts of GlyA+ CD36+ CD45- phenotype could be detected in the grafts, whereas they were absent in blood; histologically, the cells could be localized to the sinusoids of the liver. There was no clear correlation of preoperative or postoperative hemoglobin levels, graft function, kidney function, and immunosuppressive medication with the presence or absence of erythropoiesis. Moreover, serum levels of erythropoietin (EPO) and stem cell factor (SCF) in patients with and without intrahepatic erythropoiesis in the early postoperative phase did not show significant differences. These findings show that intrahepatic erythropoiesis can occur transiently in human liver allografts and suggest that systemic stimuli as well as local factors may contribute to it. PMID- 7533124 TI - Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. AB - Reovirus type 3 has been implicated in the origin and pathogenesis of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but routine detection of this virus in hepatobiliary tissues from affected infants by culture and histological techniques has been unsuccessful. In this study, oligonucleotide primers specific to the M3 genome segment of reovirus 3 (Dearing) were used in a reverse transcriptase-mediated polymerase chain reaction technique to develop a sensitive and specific assay for the detection of reovirus 3 RNA in formalin fixed, paraffin-embedded patient samples. Optimal reaction conditions were determined by testing infected murine tissues and preserved human liver tissue supplemented with reovirus 3. Archival specimens from 50 infants, including 14 with extrahepatic biliary atresia, 20 with idiopathic neonatal hepatitis, and 16 age-matched controls, were evaluated. Successful amplification of human albumin complementary DNA from the preserved tissues confirmed the presence of intact RNA in every patient specimen tested. Analysis of the amplification reactions by agarose gel electrophoresis and Southern blot hybridization detected the presence of reoviral RNA only once in a single patient sample. These results do not support a strong role for reovirus 3 in the development of neonatal cholestatic liver disease. The recent association of other RNA viruses of the Reoviridae family with murine liver disease and human extrahepatic biliary atresia indicates that continued investigation into a viral cause for idiopathic neonatal hepatobiliary disease is warranted. PMID- 7533125 TI - Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis C virus antigens detected by using immunopurified polyclonal spontaneous human antibodies. AB - Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather than experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies. PMID- 7533126 TI - Nitric oxide is released in regenerating liver after partial hepatectomy. AB - The induction of hepatic nitric oxide synthase (NOS) and the biosynthesis of nitric oxide (NO) were studied in liver after partial hepatectomy (PH). NOS activity in the liver remnant was observed 4 to 6 hours after PH, and no differences were evidenced between the proximal and distal surgical areas. The form of NOS expressed in liver was independent of calcium and calmodulin, and the messenger RNA levels were first detected 2 hours after hepatectomy using a probe corresponding to the cytokine-induced macrophage NOS. The seric concentration of nitrites remained unchanged after hepatectomy, whereas the content in nitrates and in S-nitrosylated proteins progressively increased in parallel with the NOS activity. The spectra of hemoglobin in the 400-to 460-nm region failed to exhibit the characteristic shift caused by the formation of the nitrosyl-hemoglobin complex, suggesting that NO was rapidly metabolized in liver. Treatment of the animals with substrate analogue NOS inhibitors blocked the pattern of DNA ploidy elicited after hepatectomy, suggesting a role for NO in the regenerative process. Peritoneal resident macrophages were used as an alternative reporter cell system for the assessment of NOS expression. Incubation ex vivo of peritoneal macrophages from animals that underwent hepatectomy induced the expression of NOS in a cytokine-modulated fashion, suggesting that macrophages were primed as a result of the hepatectomy. When peritoneal macrophages from control rats were incubated with the sera of animals that underwent hepatectomy, a time-dependent induction of NOS was observed, with a maximal induction corresponding to sera collected 2 hours after PH. These results indicate that NO might be involved in the control of early responses after PH. PMID- 7533127 TI - Epithelial-mesenchymal transition in cultured neonatal hepatocytes. AB - When hepatocyte-enriched fractions from neonatal rat livers were cultured for different times in the absence of added growth factors, a population of highly proliferating and migrating fibroblastlike cells appeared. Double immunofluorescence with antibodies to cytokeratin and to vimentin showed a progressive reduction in the number of cytokeratin-positive cells parallel to an increase in the vimentin-positive cells. Some cells with transitional epithelial or migrating morphology coexpressed both intermediate filament proteins. Immunofluorescence with antibodies against hepatocyte differentiation markers showed that shortly after seeding most of the cells were positive to anti-albumin antibodies, but after 1 week in culture, only 10% were positive. Cells presenting albumin and cytokeratin appeared morphologically epithelial. Fibroblastlike cells were not positive for albumin, but some cells with transitional epithelial morphology presented some labels for albumin and for vimentin. Immunofluorescence with antibodies to glutathione-S-transferase subunit Pi and vimentin showed that many fibroblastlike cells were positive for both markers, some of them binucleate. Cultures performed in the presence of dexamethasone, absence of arginine, or on collagen type I matrix had no effect on the behavior of neonatal hepatocytes. The appearance of fibroblastlike cells was ontogenically regulated because the highest increase in the percentage of vimentin-positive cells was observed in cell cultures from livers of 7- and 15-day-old animals. These data provide evidence that neonatal hepatocytes in culture have the potential to dedifferentiate by epithelial-mesenchymal transition and contribute to an understanding of hepatic growth development. PMID- 7533128 TI - Is hepatitis B virus smarter than the immune system? PMID- 7533130 TI - Hemangiopericytoma of bone--a case report. AB - A case of low grade hemangiopericytoma of three years duration occurring in the upper end of the femur of a 70 year old man is described. Radiologically, the upper third of the right femur showed a destructive expansile osteolytic lesion with soft tissue extension. The tumour was partially encapsulated. Mitoses were 0 1 per 10 high power fields, suggesting the low grade malignant nature of the lesion. It is felt that all hemangiopericytomas occurring in bone should be considered biologically malignant. PMID- 7533129 TI - Post orchiectomy management in stage II testicular seminoma. AB - Twenty eight patients with stage II A and twenty patients with stage II B testicular seminoma were treated at this institute between January 1982 and December 1988. The three year crude survival observed in this retrospective analysis was 82% and 75% respectively. Post orchiectomy infradiaphragmatic radiotherapy was the mainstay of the treatment. In stage II A 4 patients were administered adjuvant chemotherapy as well. Prophylactic Mediastinal Irradiation (PMI) was not employed as a routine in this subgroup. Eight patients (28%) relapsed (Mediastinal Nodes--4, Pulmonary--3, Scrotal--1). In stage II B twelve patients were treated with primary abdominal radiotherapy and of them 4 were delivered PMI as well. Induction chemotherapy was administered in remaining 8 patients. Seven patients (35%) relapsed (Pulmonary-4, Mediastinal Nodes-3). Mediastinal recurrence was noted only in those who were treated with abdominal radiotherapy alone. Though salvage chemotherapy proved successful in 5 of the seven patients (70%) with nodal relapse, none of the patients with extranodal relapse responded to subsequent chemotherapy. For stage II A we recommend abdominal radiotherapy alone and for stage II B Induction chemotherapy is advised keeping radiotherapy reserved for residual mass. We do not advocate PMI as a routine in stage II testicular seminoma as no survival benefit is observed. PMID- 7533131 TI - Three cases of extrapulmonary small cell carcinoma occurring in the prostate, stomach, and pancreas. AB - Small cell carcinomas are fairly common neoplasms in the lung, but tumors featuring similar histological profiles may occur in extrapulmonary organs. Three cases of small cell carcinomas occurring in the prostate (case 1), stomach (case 2), and pancreas (case 3) are presented. Production of hormones was demonstrated immunohistochemically in all cases. In case 2 alpha-fetoprotein (AFP) was elevated in the serum and observed immunohistochemically in tumor cells. Production of AFP is a distinctive feature, which has not been reported in the pulmonary and extrapulmonary cases of small cell carcinoma. Amplification and/or expression of myc gene family have been suggested to be related to the prognosis of pulmonary small cell carcinoma. Amplification of myc genes was not detected in any of our cases, but c-myc protein was demonstrated immunohistochemically in tumor cells of case 1. PMID- 7533132 TI - A self-reactive class I-restricted T-cell response of H-2b mice to determinants of the V beta 8.2 domain of the T-cell receptor for antigen. AB - We studied the induction of a self-reactive cytotoxic T lymphocyte (CTL) response to determinants of the variable V beta 8.2 region of the beta-chain of the T-cell receptor (TCR) for antigen in C57BL/6 (H-2b) mice. A CTL response was elicited in vivo by TCR peptide vaccination, and detected in vitro using syngeneic transfectants expressing a rearranged V beta 8.2+ TCR beta-chain. The first series of experiments used a 15-mer peptide representing residues 68-82 of the V beta 8.2 domain and containing Kb and Db allele-specific motifs. Immunization with this peptide stimulated an autoreactive CTL response that cross-reacted with V beta 8.2 epitopes presented by transfectants endogenously processing a V beta 8.2+ TCR beta-chain. These transfectants expressed a construct derived from a murine, rearranged V beta 8.2/D beta 2/J beta 2.3/C beta 2 TCR beta-chain cDNA. The V beta 8.2+ T-cell subset of peptide-primed mice was not deleted but its proliferative response to stimulation by the V beta 8.2-specific monoclonal antibody (mAb) F23.2 was suppressed. In a second series of experiments we immunized mice with a 23-mer peptide representing residues 41-63 of the V beta 8.2 domain that does not contain putative, allele-specific H-2b class I restricted motifs. This TCR peptide vaccination stimulated a CD8+ CTL response reacting against syngeneic, peptide-pulsed targets but not cross-reacting against transfectants processing/presenting epitopes of the beta-chain. V beta 8.2+ T cells of these peptide-primed mice were not anergized. These data demonstrate that vaccination with an immunogenic peptide representing a naturally processed epitope of the V beta 8.2 domain of the TCR beta-chain induces a self-reactive CD8+ CTL specific for this V beta 8.2 epitope; and anergizes (but does not delete) V beta 8.2+ T cells. PMID- 7533134 TI - IgE receptors, IgE content and secretory response of mast cells in athymic rats. AB - Athymic, nude (Lewis rnu/rnu) and normal (Lewis +/+) rats were used to study the T-cell dependence of the regulation of IgE receptors and IgE content on mast cells in vivo. We estimated the number of IgE receptors and the IgE content on peritoneal mast cells using a cytofluorometric technique. The secretory capacity of the mast cells was measured in terms of histamine release as a function of anti-IgE concentration by incubation with antibodies in vitro. Two groups of rats, aged 6 and 13 weeks, were examined. The mast cells of the rats belonging to the older age group (both normal and athymic) had a higher total protein (equivalent to dry mass or size) and mediator content (heparin, histamine and 5 hydroxytryptamine) in accordance with previous findings. Mast cells of the athymic rats of both age groups contained similar levels of IgE receptors and IgE and did not differ in these respects from those of the normal rats. Of the IgE receptors available for binding, about 80% were occupied by IgE in mast cells of normal and of athymic rats in both age groups. The anti-IgE-induced histamine release of the mast cells was, however, significantly lower in athymic rats compared to the normal controls. A change in housing from barrier-maintained to conventional conditions for 2 weeks enhanced the IgE-receptor and IgE content, as well as the releasability of histamine of the mast cells of both athymic and normal rats. The basal level of IgE occupancy of the receptors on mast cells was therefore not impaired in the athymic rats, as might be inferred from previous findings of a T-cell dependence of the IgE immune response. The results further indicate that T-lymphocyte-derived cytokines appear not to be required for either the expression of IgE receptors or for their ability to acquire IgE on mast cells, but such factors seem to enhance the release of histamine following the cross-linkage of the IgE receptor on mast cells in normal conditions. PMID- 7533133 TI - Therapeutic effect of transforming growth factor-beta 2 on actively induced EAN but not adoptive transfer EAN. AB - A possible effect of transforming growth factor type-beta 2 (TGF-beta 2) on autoimmune inflammation of the peripheral nervous system (PNS) was evaluated in experimental autoimmune neuritis (EAN) in Lewis rats, a disease model of the human Guillain-Barre syndrome. First, EAN was actively induced by immunization with a neuritogenic peptide corresponding to amino acids 53-78 of the bovine P2 protein. Intraperitoneal (i.p.) administration of 5 micrograms TGF-beta 2 per day after onset of clinical disease shortened the duration and ameliorated the severity of EAN compared to sham-injected control animals. Inflammatory infiltration and demyelination was significantly reduced in sciatic nerves of TGF beta-treated animals, although expression of major histocompatibility complex (MHC) class II antigens was not down-regulated. Second, EAN was induced by adoptive transfer (AT) of activated P2-specific T-line cells (AT-EAN). Daily injections of 5 micrograms TGF-beta 2 i.p., beginning on the day of first clinical signs, failed to modify the clinical course of AT-EAN, although the antigen-induced activation of the neuritogenic T-line cells used for induction of disease was found to be partially sensitive to the inhibitory effect of TGF-beta in vitro. The experiments indicate that TGF-beta 2 holds promise as a therapeutic agent to combat autoimmunity in the PNS. They also suggest that the therapeutic efficacy of TGF-beta on rapidly developing disease such as AT-EAN is limited, as with other non-specific immunosuppressive drugs. PMID- 7533135 TI - Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells. AB - Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2 ) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses. PMID- 7533136 TI - Modulation of lipopolysaccharide binding to human granulocytes. AB - Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation. PMID- 7533138 TI - Increased plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 in patients with Plasmodium falciparum or P. vivax malaria and association with disease severity. AB - Increased serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble endothelial leucocyte adhesion molecule-1 (sELAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were detected in Danish malaria patients infected with sequestering Plasmodium falciparum or non sequestering P. vivax parasites, as well as in patients with sepsis or meningitis. Levels of soluble adhesion molecules remained elevated in the P. falciparum patients for several weeks after initiation of treatment. Plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 were higher in Gambian children with severe P. falciparum malaria than in children with mild malaria. Plasma levels of sVCAM-1 and sELAM-1 were significantly correlated. Plasma levels of sELAM-1 and sVCAM-1 may reflect endothelial inflammatory reactions and these reactions may be harmful for humans infected with malaria parasites. PMID- 7533137 TI - Human IgG subclass responses and subclass restriction to Schistosoma mansoni egg antigens. AB - In areas endemic for schistosomiasis, there is great heterogeneity in antibody isotype responses to parasite antigens amongst infected individuals. At the population level, the isotype composition of antibody responses undergoes dynamic changes which are associated with the age of infected individuals. Here we examine the IgG subclass responses to Schistosoma mansoni eggs (soluble egg antigens; SEA) of infected individuals by immunoblot and ELISA. By controlled treatment of SEA-coated ELISA plates and immunoblot nitrocellular strips with sodium periodate, in order to oxidize terminal carbohydrate residues selectively, we were able to relate individuals subjects' isotype responses to the different antigens that they responded to, and to the presence of putative carbohydrate and peptide epitopes on those antigens. IgG2 responses were restricted strictly to sodium periodate-sensitive carbohydrate epitopes and antigens of relatively high molecular weight. These antigens were not usually recognized by other isotypes and, therefore, they were only recognized by individuals who had high levels of IgG2. IgG1 and IgG3 responses were directed against both carbohydrate and peptide epitopes, whereas IgG4 responses were restricted to periodate-resistant epitopes. This suggests that the fall in IgG2 responses, and reciprocal rise in IgG4 antibodies, seen in young children as their intensities of schistosome infection increase, is not the result of isotype switching, and that, if these two subclasses are involved in blocking immunity to schistosomiasis, they are operating independently. PMID- 7533139 TI - Testosterone regulation of renal alpha 2B-adrenergic receptor mRNA levels. AB - Androgens regulate blood pressure and renal alpha 2-adrenergic receptors in a parallel fashion in the spontaneously hypertensive rat (SHR). The present studies investigated whether this regulation of renal alpha 2B-adrenergic receptors occurs at the mRNA level. Male and female SHR were gonadectomized at 4 weeks of age. The gonadectomized rats were implanted with or without testosterone propionate. Sham-gonadectomized rats served as controls. Total kidney RNA was purified, and alpha 2B-adrenergic receptor mRNA was quantified with a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The assay uses a mimic RNA added at known concentrations to the sample RNA. The mimic was constructed from the target sequence in the alpha 2B-adrenergic receptor mRNA plus a 20-bp insertion of a random nucleotide sequence. The amount of alpha 2B-adrenergic receptor mRNA present in each sample was obtained by determining the equivalence point between the amount of RT-PCR product formed in the target band versus the mimic band, which were resolved by gel electrophoresis. Intact males had more than two times as much alpha 2B-adrenergic receptor mRNA as intact females. Castration of males reduced the male-female difference by more than 60%. Ovariectomy slightly increased the alpha 2B-adrenergic receptor mRNA level compared with that of intact females. Treatment with testosterone elevated alpha 2B-adrenergic receptor mRNA levels of gonadectomized males and females to the level of intact males. The alpha 2B-adrenergic receptor mRNA levels correlated remarkably well with renal alpha 2-adrenergic receptor density. We conclude that testosterone regulates renal alpha 2B-adrenergic receptor gene expression at the mRNA level in the SHR. PMID- 7533140 TI - Regulation of endothelial constitutive nitric oxide synthase by protein kinase C. AB - Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. The promoter region of the endothelial constitutive nitric oxide synthase (ecNOS) gene contains a transcriptional factor AP-1 binding element. In the present study, we sought to determine the effect of PKC inhibition on the expression of ecNOS in cultured bovine aortic endothelial cells (BAEC). The PKC inhibitor staurosporine (10 to 100 nmol/L) increased the expression of ecNOS mRNA, assessed by Northern analysis, in a dose-dependent manner. A newly developed, more specific PKC inhibitor, chelerythrine (1 to 3 mumol/L), also increased the level of ecNOS mRNA. Incubation of BAEC with phorbol 12-myristate 13-acetate (100 nmol/L) for 24 hours, which downregulates PKC, increased ecNOS mRNA expression. The protein content of ecNOS, assessed by Western analysis, was also increased in staurosporine-treated or chelerythrine-treated BAEC. The release of nitrogen oxides from staurosporine-treated or chelerythrine-treated cells both under basal conditions and in response to calcium ionophore A23187 was significantly increased (P < .05). In conclusion, the present study suggests that regulation of ecNOS is mediated by PKC. The increased release of nitric oxide induced by PKC inhibition may play a protective role against atherogenic process. PMID- 7533141 TI - Regulation of neuronal nitric oxide synthase in rat adrenal medulla. AB - Neuronal nitric oxide synthase (nNOS) has been suggested to be involved in cardiovascular homeostasis. We studied the regulation of nNOS expression, determining nNOS mRNA expression levels in various tissues in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We also investigated the effects of antihypertensive treatment with the angiotensin II antagonist hydralazine or reserpine on nNOS mRNA expression. The expression levels of nNOS mRNA and nNOS protein were determined by Northern and Western blot analysis, respectively. NADPH-diaphorase histochemistry was used to identify cells in the adrenal medulla that expressed nNOS. No significant differences in expression levels in SHR and WKY were observed in the cerebellum and brain stem. nNOS mRNA expression levels in the decapsular portion of the adrenal gland were developmentally modulated and in a 24-week-old WKY were 2.5 times higher than in an age-matched SHR. This reduced expression of nNOS mRNA in the decapsular portion of the adrenal gland of SHR seemed to be a result of hypertension in the SHR, because administration of either an angiotensin II antagonist (TCV-116) or hydralazine upregulated nNOS mRNA expression in both SHR and WKY. Marked augmentation of nNOS mRNA expression in the decapsular portion of the adrenal gland by reserpine treatment suggested an intimate relation between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system. Reserpine treatment also increased the expression of nNOS protein; however, reserpine treatment did not affect the distribution pattern of nNOS-positive cells (NADPH diaphorase-positive cells) in the adrenal medulla.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533144 TI - Landau-Kleffner syndrome in cerebral cysticercosis. PMID- 7533143 TI - Migraine in children. AB - A descriptive study was carried out to find out the clinical profile, and electroencephalographic (EEG) changes in children with migraine. Screening for urinary excretion of 5-hydroxy indole acetic acid (5-HIAA) was carried out. Fifty children suffering from migraine as per Prensky's criteria were recruited over a period of 1 year. Forty six children were suffering from common migraine and 4 had classic migraine. The most common precipitating factors were physical strain and psychological stress like examination fear, fear of teacher and fights with friends. Abnormal EEG recordings were seen in 35 out of 50 patients. Urine samples taken during the headache free period were negative for 5 HIAA. Among the samples taken during the episode of headache, only 1 was positive for 5-HIAA. It is concluded that common migraine is more prevalent than classic migraine. Clinical criteria is the only way of diagnosing migraine. Since EEG changes are non-specific, this cannot be used as a diagnostic test. Biochemical analysis is expensive and less sensitive. PMID- 7533142 TI - Screening test battery for assessment of psychosocial development. AB - A multicentric cross-sectional collaborative study was undertaken in 3 centres in India with the main aim of developing simple and reliable indicators for the early detection of developmental disabilities in children under 6 years of age and to compare the age of attainment of developmental milestones in children in the three regions. The study provided a simple low-cost and culture-appropriate psychosocial developmental screening test battery which can be used with ease by trained public health grass-roots functionaries. This instrument was standardized on a large rural, tribal and urban sample comprising more than 13,000 children from 3 regions in India. The procedure for sampling, selection of items and methodology for standardization of the instrument in the Hyderabad region detailed in this paper were replicated in other centres as well. Quality control of data was ensured through inter-rater and test-retest measures of reliability. During pre-testing, 66 culture-appropriate milestones were selected finally from a larger item pool. The 50th centile age reference values of the Hyderabad study children and those obtained by other 2 centres were comparable. PMID- 7533145 TI - [Bacillary angiomatosis]. AB - Bacillary angiomatosis (BA) is a rare infectious disease usually associated with HIV infection. Recent molecular biologic investigations confirm that both Rochalimaea henselae and Rochalimaea quintana can cause BA. The bacteria can be identified by Warthin-Starry staining and electron microscopy. The typical clinical signs are solitary or multiple dermal or subcutaneous nodules. Bone, liver, spleen and other organs may also be involved. We describe the clinical and histological features of a 39-year-old HIV-infected patient with cutaneous and bony lesions of BA. All manifestations of BA disappeared during therapy with erythromycin. PMID- 7533146 TI - [Epidermolysis bullosa hereditaria]. PMID- 7533147 TI - [The value of PSA follow-up in recurrent prostate carcinoma after radical prostatovesiculectomy]. AB - We report our experience in evaluating PSA-follow-up of patients treated with radical prostatectomy. Also PSA is the most sensitive available screening test for relapse after radical prostatectomy, it is not 100% specific for cancer relapse while this serum marker is not specific for prostate cancer. In our opinion, patients with biochemical relapse (PSA > 0.5 ng/l) should undergo an early restaging including biopsy of the anastomosis. In 57% of our patients with PSA > 1.0 ng/l two months after surgery we found clinical recurrence 27 months later on. PMID- 7533148 TI - [Long-term follow-up of serum prostate-specific antigen after radiotherapy of prostatic carcinoma]. AB - The role of prostate-specific antigen (PSA) in the follow-up of patients after radiation therapy for prostate cancer is not well defined. There are no reports on long-term follow-up with PSA and serial PSA determinations, respectively. We followed 113 patients after radiotherapy for clinical stages A through D1 prostate cancer with multiple PSA measurements. External beam radiation has been applied to 98 patients and 15 received 125-iodine implantation. Eighty-eight of the 113 patients (78%) had a precipitously rising PSA with median doubling times of 14 (stage A), 15 (stage B), 7 (stage C), and 8 (Stage D1) months, respectively, at a mean follow-up of 5 years after radiotherapy. Twenty-three of the 113 patients (20%) appeared cured with a PSA of 1.7 ng/ml or less at a mean follow-up of 9 years. Two of the 113 patients (2%) continued to have a decreasing PSA 3 years after irradiation. Among the cured patients there was no relationship to clinical stage or histological grade. We conclude that about 20% of patients with clinical stages A-D1 prostate cancer can be cured by radiotherapy. They can be identified by a very low serum PSA level which seems to persist indefinitely. The remaining 80% who fail radiation therapy appear to have an accelerated growth rate suggesting tumor clonogen repopulation during radiotherapy. PMID- 7533149 TI - [Localized fibrous tumor of the pleura. 2 case reports and differential diagnoses]. AB - The localized fibrous tumors of the pleura are rare neoplasms, also known as "benign pleural fibromas" or "localized mesotheliomas", but those names are inaccurate. The tumors are composed of undifferentiated mesenchymal cells, intermediate and differentiated fibroblasts and collagenous interstitial tissue. Their origin is normally the submesothelial layer of the visceral pleura. Mesothelial differentiation is not present. The grading of malignancy doesn't correlate with final outcome, adequacy of surgical excision being the most important factor. This neoplasm may recur but retain its basical histologic features. The positive results for vimentin and negative results for cytokeratin antibodies help to distinguish the localized fibrous tumors from the mesothelioma. We present too cases and their diagnostic possibilities. PMID- 7533150 TI - [Combination of surgical therapy and interventional radiology in a recurrent myxoid liposarcoma with a 37-year history]. AB - Extreme long-term survivals of low grade liposarcomas are rare. The authors present a case of a 75 year old man with a 37 year history of recurrent myxoid liposarcoma. The tumor dynamics are obviously related to the histology subtype. The clinical and radiological findings of the sixth tumor recurrence are discussed. The combination therapy of surgical tumor reduction and interventional radiology with implantation of endovascular prostheses for iliaco-femoral vein compression due to tumor recurrence is described. PMID- 7533151 TI - Modified MACOP-B chemotherapy for intermediate and high grade non Hodgkins lymphomas. AB - Combination chemotherapy consisting of methotrexate with leucovorin rescue, doxorubicin, cyclophosphamide, vincristine, prednisolone and bleomycin (MACOP-B) has been extensively used for the treatment of Non Hodgkins Lymphoma. However, different results have been reported. The aim of this study was to assess the feasibility of administration of this regimen on an out patient basis and to confirm the efficacy of MACOP-B. 51 patients with intermediate--and high--grade lymphoma were treated with this regimen in a single institute study. Numerous clinical features predictive of response and disease free survival were analysed. The Median age was 48 years (range 14-77). Diffuse large cell lymphoma was seen in 65%, diffuse small cleaved in 10% and diffuse mixed in 15%. Eight patients (15%) had Stage I disease, 18 (35%) Stage II, 12 (23%) Stage III and 13 (25%) Stage IV. Complete remission was achieved in 65% of the patients. With a median follow up of 18 months, 40% of the patients are alive at 40 months. Sixty percent of the complete responders are disease free at 40 months. Response rates did not differ significantly for age, sex, stage, histology, bone marrow involvement and extranodal disease. However patients with absence of B' symptoms, non bulky disease at presentation and diffuse large cell histology had a higher percentage of complete remission. Hematological toxicity occurred in 90% and was grade IV in 14% patients. Three patients died of sepsis. Severe mucositis occurred in 40% of the patients. In conclusion, while it is possible to give aggressive chemotherapy at the out patient basis in India we failed to confirm the high response rates as originally reported. PMID- 7533152 TI - Chimeras of human complement C9 reveal the site recognized by complement regulatory protein CD59. AB - CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex, thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective and is most effective toward C9 derived from human or other primate plasma. By contrast, rabbit C9, which can substitute for human C9 in the membrane attack complex, mediates unrestricted lysis of human cells. To identify the peptide segment of human C9 that is recognized by CD59, rabbit C9 cDNA clones were isolated, characterized, and used to construct hybrid cDNAs for expression of full-length human/rabbit C9 chimeras in COS-7 cells. All resulting chimeras were hemolytically active, when tested against chicken erythrocytes bearing C5b-8 complexes. Assays performed in the presence or absence of CD59 revealed that this inhibitor reduced the hemolytic activity of those chimeras containing human C9 sequence between residues 334-415, irrespective of whether the remainder of the protein contained human or rabbit sequence. By contrast, when this segment of C9 contained rabbit sequence, lytic activity was unaffected by CD59. These data establish that human C9 residues 334-415 contain the site recognized by CD59, and they suggest that sequence variability within this segment of C9 is responsible for the observed species-selective inhibitory activity of CD59. PMID- 7533153 TI - The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function. AB - A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 degrees C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups. PMID- 7533154 TI - Malarial circumsporozoite protein is a novel gene delivery vehicle to primary hepatocyte cultures and cultured cells. AB - In this report we describe a novel gene delivery system using malaria circumsporozoite (CS) protein as a specific ligand. The CS protein covers the entire surface of sporozoites of malaria parasites. Previous studies have demonstrated that intravenously injected CS protein binds specifically to the basolateral surface of hepatocytes within minutes, indicating the high hepatocyte specificity of CS protein. This characteristic of CS protein prompted us to explore the possibility of using this protein as a liver-specific ligand for hepatic gene delivery vehicle. As an initial step, we investigated the efficacy of CS protein-mediated gene transfer into primary hepatocytes as well as established cell lines. Recombinant CS proteins were chemically conjugated to poly(L-lysine). The CS conjugates were complexed with recombinant plasmid DNA carrying a reporter gene. When the DNA complex was used to transfect primary hepatocytes, a very low level of expression of the reporter gene was observed. The level of expression was greatly enhanced when the cells were cotransfected with adenovirus, which presumably releases the internalized DNA from endosomal entrapment. The CS-mediated gene transfer into the cells required region II+, an evolutionarily conserved amino acid sequence conferring the binding of CS protein to its receptor. CS protein also efficiently mediated gene transfer into a number of cell lines, i.e. HepG2, HeLa, NIH3T3, and K562, but not HL-60, which contains low levels of receptor. Thus, the CS conjugate can be used to deliver DNA into many different cultured cells. Most importantly, the CS conjugate has a potential to be further developed into a liver-specific gene delivery vehicle in vivo. PMID- 7533156 TI - IV3 alpha (NeuGc alpha 2-8NeuGc)-Gg4Cer is restricted to CD4+ T cells producing interleukin-2 and a small population of mature thymocytes in mice. AB - Monoclonal antibody YK-3 was established by immunization with IV3 alpha (NeuGc alpha 2-8NeuGc)-Gg4Cer (GD1c-(NeuGc-NeuGc-)), and its epitope was determined to be NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1. Thin layer chromatography immunostaining with YK-3 detected only GD1c-(NeuGc-NeuGc-) among the gangliosides of mouse thymocytes and splenocytes. Immunohistochemical staining with YK-3 visualized the medulla of mouse thymus and T cell-dependent areas of mouse spleen and mesenteric lymph nodes. Two-color flow cytometry demonstrated that GD1c (NeuGc-NeuGc-) was expressed on a quarter of CD3+ mature thymocytes and strongly expressed on three quarters of CD4+ T cells in the spleen, lymph nodes, and peripheral blood but not on CD8+ T cells or B cells. GD1c (NeuGc-NeuGc-)-positive cells and negative cells were separated by panning with YK-3 on Petri dishes into adherent and nonadherent fractions. Following stimulation with concanavalin A, adherent cells, predominantly GD1c (NeuGc-NeuGc-)+, produced more interleukin-2 (IL-2) and markedly less interleukin-4 (IL-4) than nonadherent cells. This conclusion is supported by data obtained by lysis of cells by YK-3 and complement. These data indicate that the cell surface expression of GD1c (NeuGc NeuGc-) is restricted to a small number of mature thymocytes and a subset of CD4+ T cells, which produce abundant IL-2 and very little IL-4, suggesting that GD1c(NeuGc-NeuGc-) is an excellent marker for mouse naive T or T helper 1-like cells in vivo. PMID- 7533155 TI - Control of vascular cell adhesion molecule-1 gene promoter activity during neural differentiation. AB - Here we demonstrate that vascular cell adhesion molecule-1 (VCAM-1) is expressed in the developing central nervous system on neuroepithelial cells, which are the precursors of neurons and glia. As these cells differentiate, VCAM-1 is restricted to a subset of the glial population. An understanding of mechanisms responsible for this restricted pattern could provide insights into how lineage specific gene expression is maintained during neural differentiation. As a model of neural differentiation, we turned to the P19 embryonic carcinoma cell line, which in response to retinoic acid will differentiate along a neural pathway. We show that VCAM-1 expression on the differentiating P19 cells resembles that in the central nervous system. Transfection of VCAM-1 gene promoter constructs into P19 cells revealed that the VCAM-1 gene is controlled sequentially by negative and positive elements during differentiation. We present evidence that early during differentiation, POU proteins block VCAM-1 gene activity; however, later in differentiation coincident with the appearance of VCAM-1 the pattern of POU proteins changes and the VCAM-1 gene promoter is activated. This activation is mediated through the NF kappa B/rel complex p50/p65, which forms during P19 cell differentiation. PMID- 7533158 TI - Flanking and intragenic sequences regulating the expression of the rabbit alpha globin gene. AB - Despite their descent from a common ancestral gene and the requirement for coordinated, tissue-specific regulation, the alpha- and beta-globin genes in many mammals are regulated in distinctly different ways. Unlike the beta-globin gene, the rabbit alpha-globin gene is transiently expressed at a high level without an added enhancer in transfected erythroid and non-erythroid cells. By examining a series of alpha/beta fusion genes, we show that internal sequences of the rabbit alpha-globin gene (within the first two exons and introns) are required along with the 5' flank for this enhancer-independent expression. Furthermore, deletion of the introns of the alpha-globin gene, or replacement by introns of the beta globin gene, results in severely decreased expression of the transfecting genes. Hybrid constructs between segments of the alpha-globin gene and a luciferase gene confirm that internal alpha-globin sequences are needed for high level production of RNA in transfected cells. The flanking and internal sequences implicated in regulation of the rabbit alpha-globin gene coincide with a prominent CpG-rich island and may comprise an extended promoter (including both flanking and intragenic sequences) that is active in transfected cells without an enhancer. PMID- 7533157 TI - Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies. AB - Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146 40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81 23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146 40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation. PMID- 7533160 TI - Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase. AB - The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E. (1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta delta 1 9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha chains much more strongly and generate a 50% active enzyme. Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity. PMID- 7533159 TI - Interleukin-4 induces expression of the integrin alpha v beta 3 via transactivation of the beta 3 gene. AB - Osteoclastic bone resorption is dependent upon cell-matrix recognition. This process is mediated by the integrin alpha v beta 3 whose expression is enhanced, in avian osteoclast precursors, by bone-seeking steroids. The purpose of this study was to determine if bone-modulating cytokines impact on alpha v beta 3 expression by mouse marrow macrophages (BMMs), known to differentiate into osteoclasts. Of the cytokines tested. Interleukin-4 (IL-4) is most effective in increasing beta 3 mRNA levels by a mechanism involving transactivation of the beta 3 gene. Moreover, IL-4 augmented beta 3 mRNA is mirrored by plasma membrane appearance of alpha v beta 3. As IL-4 induces beta 3 and not alpha v mRNA, the beta 3 chain appears to regulate surface expression of the heterodimer. The functional significance of IL-4-induced alpha v beta 3 is underscored by the fact that, while attachment to fibronectin is unaltered, treatment of BMMs with the cytokine enhances alpha v beta 3-mediated binding to vitronectin 5-fold. Expression of this heterodimer by BMMs driven along a non-osteoclastic lineage suggests alpha v beta 3 may play a role in the inflammatory response of macrophages. PMID- 7533161 TI - Cleavage analysis of insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants. AB - Cultured human fibroblasts and osteoblast-like cells secrete an insulin-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP 4) into two fragments of approximately 18 and 14 kDa. Edman degradation of the isolated proteins established the amino termini of the reaction products. Sequence analysis of the 14-kDa carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine at position 135 of the mature protein. Four variant IGFBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed. Wild-type and mutant IG-FBPs-4 bound IGF-I and IGF-II with equivalent affinities and, in the intact state, were equally effective inhibitors of IGF-I action. However, the IGFBP-4 mutants were relatively resistant to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected at this time. After a 24-h incubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa fragments. Pre exposure of human fibroblasts in serum-free conditioned medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of DNA synthesis. When added with IGF II, the protease-resistant mutant IG-FBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of IGF-I-stimulated DNA synthesis. These biological studies suggest that the IGFBP-4/IGFBP-4 protease system may play a role modulating local cellular response to IGF-I. PMID- 7533162 TI - alpha 1-Microglobulin destroys the proteinase inhibitory activity of alpha 1 inhibitor-3 by complex formation. AB - The immunoregulatory plasma protein alpha 1-microglobulin (alpha 1-m) and the proteinase inhibitor alpha 1-inhibitor-3 (alpha 1I3) form a complex in rat plasma. In the present work, it was demonstrated that the alpha 1I3.alpha 1-m complex has no inhibitory activity, the bait region was not cleaved by low amounts of proteinases, and it was unable to covalently incorporate proteinases. The results also indicated that the thiolester bond of the alpha 1I3.alpha 1-m complex was broken. The alpha 1I3.alpha 1-m complex was cleared from the circulation much faster than native alpha 1I3, with a half-life of approximately 7 min. Structurally, however, the alpha 1I3.alpha 1-m complex was similar to native alpha 1I3 rather than alpha 1I3 cleaved by proteinases. It is speculated that the role of alpha 1-m is to destroy the function of alpha 1I3 by blocking the bait region and breaking the thiolester and causing its physical elimination by rapid clearing from the blood circulation. It is also possible that the formation of complexes between alpha 1-m and alpha 1I3 may serve as a mean to regulate the function of alpha 1-m since its complex with alpha 1I3 is taken up rapidly by cellular receptors for alpha-macroglobulins. PMID- 7533164 TI - Distinct domains of the CD3-gamma chain are involved in surface expression and function of the T cell antigen receptor. AB - The T cell antigen receptor (TcR) is a multisubunit complex that consists of at least six different polypeptides. We have recently demonstrated that the CD3 delta subunit cannot substitute for the CD3-gamma subunit in TcR cell surface expression, in spite of significant amino acid homology between these two subunits. To identify CD3-gamma-specific domains that are required for assembly of the complete TcR and for surface expression and function of the TcR, chimeric CD3-gamma/CD3-delta molecules were constructed and expressed in T cells devoid of endogenous CD3-gamma. Substitution of the extracellular domain of CD3-gamma with that of CD3-delta did not allow cell surface expression of the TcR. In contrast, substitution of the transmembrane and/or the intracellular domains of CD3-gamma with those of CD3-delta did allow TcR cell surface expression. These results conclusively demonstrate that the extracellular domain of CD3-gamma plays a unique role in TcR assembly. Functional analyses of the transfectants demonstrated that the intracellular domain of CD3-gamma is required for protein kinase C-mediated down-regulation of TcR but is dispensable for the pattern of tyrosine phosphorylation observed following activation through TcR. PMID- 7533165 TI - Accessibility of epitopes on human transcription factor IIB in the native protein and in a complex with DNA. AB - Transcription factor IIB (TFIIB) plays a central role in the assembly of the RNA polymerase II initiation complex. Monoclonal antibodies (mAbs) that react with human TFIIB were prepared and used as probes to identify portions of TFIIB that are accessible when the factor is in solution and when it is contained in a complex with DNA. Seven mAbs were examined and were mapped to three regions of the TFIIB molecule. Only the mAbs that mapped to residues 52-105 inhibited transcription, immunoprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extract (NE), and supershifted a complex containing TFIIB, the TATA binding protein, and DNA. The mAbs that mapped to residues 1-51 and the mAb that mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 1-51), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa cell NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation complex and that some far N-terminal epitopes are accessible on the purified protein, but become blocked when TFIIB is in HeLa cell NE or in the preinitiation complex. PMID- 7533163 TI - Tenascin-C binds heparin by its fibronectin type III domain five. AB - Two sites on tenascin mediate interactions with glycosaminoglycan chains of proteoglycans. One is situated on the fibrinogen-like domain, whereas the other lies within the fibronectin type III homology region (Aukhil, I., Joshi, P., Yan, Y.Z., and Erickson, H.P. (1993) J. Biol. Chem. 268, 2542-2553.). We now characterize the latter binding site more closely by means of recombinant protein fragments derived from the type III homology region of tenascin. Using a heparin Sepharose column, we localize the second heparin binding site to the fifth fibronectin type III domain. This is confirmed in solid phase assays by incubation of fusion proteins with biotin-labeled heparin. In addition, we demonstrate the binding of heparan sulfate and dermatan sulfate to domain five. Molecular modelling of this domain reveals a conserved heparin-binding motif that we propose as the putative binding site. The fact, that different glycosaminoglycans may bind to this domain, implies that different classes of proteoglycans may in vivo compete for the same site. PMID- 7533166 TI - Distinct mechanisms regulate 5-HT2 and thrombin receptor desensitization. AB - We have compared the desensitization of two receptors, the thrombin receptor which displays dual coupling to both pertussis toxin-sensitive (Gi) and insensitive (Gq) proteins and the serotonin type 2 (5-HT2) receptor which selectively couples to Gq. In the case of the thrombin receptor, cleavage induces activation and irreversible receptor modification followed by rapid (T1/2 = 3 min) and extensive desensitization of the receptor's ability to modulate phospholipase C (Gq). 5-HT-induced desensitization of its receptor is markedly slower (T1/2 = 10 min) and by 60 min only 50% of the phospholipase C response is lost. This effect occurs with a parallel disappearance of 5-HT receptors from the cell surface. Whole cell phosphorylation studies showed that the thrombin receptor is rapidly phosphorylated upon activation. In contrast, the 5-HT2 receptor displays a low basal level of phosphorylation which is not increased upon agonist treatment. The cytoplasmic tail of the 5-HT2 receptor which contains several protein kinase consensus sequences was found not to be involved in receptor activation or desensitization. However, a chimeric receptor having the core of the 5-HT2 receptor and the cytoplasmic tail of the thrombin receptor was able to undergo 5-HT-induced desensitization and phosphorylation. These results indicate that (i) both 5-HT2 and thrombin receptors have unique shut-off mechanisms, and (ii) that sequences in the carboxyl terminus of the thrombin receptor are sufficient to trigger rapid uncoupling of the receptor from its G protein(s) and downstream effector(s). PMID- 7533167 TI - Nevirapine alters the cleavage specificity of ribonuclease H of human immunodeficiency virus 1 reverse transcriptase. AB - The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3' terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA primer terminus allowing cleavage at more sites. The change in specificity occurred whether the primer terminus was at the end or internal on the template. Experiments with RNA primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of primers that underwent synthesis and the processive elongation of those primers. The significance of these results with respect to viral replication and recombination is discussed. PMID- 7533168 TI - Tachykinin receptors involved in the contractile effect of the natural tachykinins in the rat gastric fundus. AB - 1. The receptors involved in mammalian tachykinin-induced contractions of longitudinal smooth muscle strips of the rat gastric fundus were characterized pharmacologically. 2. Substance P (SP), neurokinin A, neurokinin B and senktide contracted the strips in a concentration-dependent manner with a potency order of neurokinin A > or = senktide > neurokinin B > substance P. The contractions were not influenced by tetrodotoxin and atropine. 3. L 659877, a NK2B-receptor preferring antagonist reduced neurokinin A- and neurokinin B-induced contractions (estimated pKB 6.9 and 6.3, respectively) but had less pronounced effects on SP induced contractions and none on contractions induced by senktide. MEN 10376, an NK2A-receptor-preferring antagonist, reduced the neurokinin A-induced contractions (estimated pKB 5.2), while dactinomycin, reduced the neurokinin A induced contractions only to a minor extent at 10(-4) M. 4. CP 96345, an NK 1 receptor antagonist, reduced substance P- and neurokinin A-induced responses, but also reduced the contractions induced by KCl and methacholine. RP 67580, another non-peptide NK1-receptor antagonist had no effect on the substance P-, neurokinin A- and neurokinin B-induced contractions up to a concentration of 3 x 10(-6) M. 5. These results suggest that the mammalian tachykinins induce contractions of the longitudinal smooth muscle strip of the rat gastric fundus by direct action at muscular NK2B- and NK3-receptors. PMID- 7533169 TI - A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. AB - We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro. PMID- 7533172 TI - Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44 expressing osteoprogenitor cells. AB - In the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealed that a large amount of hyaluronan was present in the lacunae located in the zone of hypertrophy, but it was greatly reduced or absent from the zone of erosion. In addition, hyaluronan was present in the cytoplasm of osteoprogenitor cells located in the zone of erosion. These cells also expressed CD44 on their surfaces, as revealed by double-label immunohistochemistry. These results suggested that the osteoprogenitor cells may use CD44 to bind and internalize hyaluronan, and subsequently degrade it with lysosomal enzymes. To test this possibility, we examined the human cell line, MG 63, which closely resembles osteoprogenitor cells. These cells produced several different forms of CD44, as determined by western blotting (85, 116 and 150 kDa). In addition, the binding of isotopically labeled hyaluronan to detergent extracts of these cells was blocked by a monoclonal antibody to CD44. Similarly, the degradation of hyaluronan by these cultured cells was also inhibited by a monoclonal antibody to CD44. To determine if these cells could remove hyaluronan from the growth plate, the cells were cultured directly on top of thin sections of the epiphysial region of long bone. After 16 hours, the sections were stained for hyaluronan. The MG-63 cells removed significant amounts of hyaluronan present in the zone of hypertrophy, and this effect was blocked by an excess of soluble hyaluronan and by a monoclonal antibody to CD44.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533173 TI - The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line. AB - Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions. PMID- 7533170 TI - Lymphocyte adhesion-dependent calcium signaling in human endothelial cells. AB - Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo 3-labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino-phenoxylethane N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion-dependent EC signals. mAb studies indicate that the beta 2 integrin-intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1-mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion. PMID- 7533174 TI - Effect of anticancer drugs on the release of TGF-beta in vitro. AB - Some conventional and experimental anticancer drugs were tested for their effect on Concanavalin A (Con A)-induced Transforming Growth Factor-beta (TGF-beta) release from BALB/c splenocytes, lipopolysaccharide (LPS)-induced TGF-beta release from Nude mouse splenocytes and BALB/c peritoneal exudate cells stimulated by LPS in vitro. When 5 x 10(6) BALB/c and Nude mouse splenocytes/ml stimulated with 1 microgram/ml Con A, 50ng/ml LPS respectively, and 5 x 10(6)/ml peritoneal exudate cells stimulated with 50ng/ml LPS were incubated with various non-cytotoxic concentrations of the vinca alkaloid Vincristine, there was an inhibition of the release of TGF-beta in culture supernatants of both BALB/c splenocytes and peritoneal exudate cells. But, in Nude mouse Vincristine did not alter the release of TGF-beta. The antitumour antibiotic Bleomycin, the immunoactive peptide FK565 and the immunosuppressive agent Cyclosporin A (CsA) were found to have no effect on the release of TGF-beta from all culture supernatants. PMID- 7533171 TI - Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts. AB - A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations. PMID- 7533175 TI - Antibody to silicone and native macromolecules in women with silicone breast implants. AB - Silicone implants have been associated with the development of multiple organ system abnormalities, including rheumatic disorders, nervous system, pulmonary dysfunction associated with autoantibodies and abnormalities of cellular immunity. In this regards a number of case reports and series of articles have been described. We hypothesized that an immune reaction to silicone breast implants would include the host reactivity against silicone and the macromolecules within the microenvironment of the implant, and these autoantibodies may react with other tissue antigens far from the site of the implant. To test this hypothesis 520 Symptomatic women with Silicone Implants which have developed Silicone related Immunological disorders and have typically complained of breast pain, Myalgia-Arthralgia, fatigue, or generalized pain, were examined by their physician. Blood samples were obtained and examined for the presence of Silicone antibodies, Myelin Basic Protein and human serum albumin antibodies. These samples were then compared to 520 matched controls without implants. At least at the level of two standard deviation silicone specific antibodies, IgG, IgA IgM, IgE and IgG+IgA+IgM antibodies were detected above the mean of normal controls. When these antibodies were classified based on the specialty of the examining physician, the % of patients with Silicone Antibodies were varied; general practice 51.6, Rheumatology 58.7, and Plastic Surgery 83.3, which may relate to the severeness of the disease. Being that a large % of patients demonstrated very high levels of Myelin Basic Protein Antibodies, possible cross reactive antibodies were sought. However, absorption of highly positive sera for Silicone Antibodies with MBP did not change the levels of Silicone Antibodies. On the other hand, Silicone-HSA was able to reduce the antibody values significantly. This reduction in antibody levels by Silicone is the best indication for the specificity of these antibodies. Moreover when data for silicone antibodies and MBP antibodies was analyzed in patients some with high and others with medium or low levels of silicone antibodies, MBP antibodies did not correspond to the silicone antibody levels. Similarly human serum albumin antibodies which was significantly higher in patients with silicone implants did not correlate with levels of silicone antibodies. These results indicate that immune reaction to silicone and different tissue antigens do occur and they are initiated through different mechanisms. And since predominant antibody class against silicone, MBP and HSA was IgM, clonal activation of IgM is possible which certainly warrants further investigation. PMID- 7533176 TI - Pancreatic enzyme elevation in measles. AB - During an outbreak of measles in the period from May 1993 through February 1994, a 23-year-old woman with measles was admitted because of abdominal pain and vomiting. Moderately elevated levels of serum and urinary amylase were found. We investigated prospectively the next nine consecutive young adults hospitalized with severe measles. Pancreatic and other organ involvement was determined by serum and urinary amylase, serum lipase, and additional appropriate biochemical and hematological data. Four patients had elevated amylase levels in both serum and urine, whereas in one, serum amylase alone was increased. Serum lipase determined in eight patients was elevated in seven. In all patients elevated serum levels of aspartate aminotransferase and alanine aminotransferase or lactate dehydrogenase were found. In seven patients serum calcium concentrations were below the lower limit of normal. Four patients had mild to moderate thrombocytopenia. This is the first detailed report of pancreatic involvement in young adults with measles. This abnormal finding, its possible underlying mechanisms, and the clinical significance are discussed. PMID- 7533177 TI - Endogenous IL-6 and IL-10 contribute to the differentiation of CD40-activated human B lymphocytes. AB - This study was initiated to explore the contribution of endogenous cytokines to CD40-induced B cell proliferation and differentiation. First, both CD40 and Ag receptor (AgR) cross-linking induced purified tonsillar human B lymphocytes to secrete the same pattern of cytokines, including IL-1 beta, IL-6, IL-10, granulocyte-macrophage-CSF, and TNF-alpha, whereas IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-7, granulocyte (G)-CSF, or IFN-gamma were not detected. Second, cotriggering of CD40 and AgR resulted in additive secretion of both IL-6 and IL 10. Addition of IL-4 to CD40-activated B cells increased IL-6 levels but decreased IL-10 levels. In contrast, exogenous IL-10 diminished IL-6 levels. Neutralization of IL-6 and IL-10 using blocking Abs did not alter CD40-induced B cell growth. In contrast, IL-6 neutralization markedly inhibited the IL-4-induced IgE secretion (57 +/- 10%) as well as the IgG and IgM production resulting from AgR and CD40 cotriggering (49 +/- 16.5 and 29.5 +/- 4.5%, respectively). Blocking IL-10 inhibited the IgA secretion (25 +/- 2.7%) obtained after CD40 activation and the production of IgG, IgA, and IgM (24.1 +/- 5.6, 25 +/- 8, and 42 +/- 6.5%, respectively) by B lymphocytes undergoing dual ligation of CD40 Ag and AgR. Simultaneous neutralization of both endogenous IL-6 and IL-10 resulted in an increased inhibition of Ig secretion for B cells cotriggered by CD40 Ag and AgR (65-75%). Thus, endogenously produced IL-6 and IL-10 are involved in the differentiation of CD40-activated B cell. PMID- 7533178 TI - Distinct differentiative stages of CD4+CD8+ thymocyte development defined by the lack of coreceptor binding in positive selection. AB - Cortical CD4+CD8+ thymocytes mature into CD4+ or CD8+ thymocytes through a process termed positive selection. To better define differentiative stages of CD4+CD8+ thymocyte development in positive selection, we performed a phenotypic analysis of CD4+CD8+ thymocytes from H-Y mice mated to various genetic backgrounds. We have previously shown that coordinate binding of the H-Y TCR and the CD8 coreceptor to the restricting Db MHC class I molecule is required for the efficient positive selection of this TCR. In this study we have used TCR, CD5, and CD45 expression levels as markers for thymocyte maturation. Lack of CD8/Db interaction was achieved by introducing a mutation that abrogates CD8 binding in the alpha 3 domain of Db. We found that the absence of coreceptor ligation prevented TCR up-regulation in CD4+CD8+ thymocytes and resulted in a developmental arrest characterized by low levels of TCR and CD45. We have previously shown that deletion of CD4+CD8+ thymocytes expressing the H-Y TCR is facilitated by CD8 coreceptor ligation. Here we show that expression of the deleting ligand in the absence of coreceptor ligation caused CD5 up-regulation without concomitant TCR or CD45 up-regulation in CD4+CD8+ thymocytes. In a beta 2 microglobulin null background, introduction of the H-Y TCR caused the majority of CD4+CD8+ thymocytes to express an unusually low level of of the CD5 activation marker, suggesting that a low-affinity or noncognate TCR/MHC interaction may be required for initial CD5 up-regulation to intermediate levels. Collectively, these observations favor a maturational process in positive selection in which CD5 up-regulation precedes CD45 and TCR up-regulation. PMID- 7533179 TI - Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence for the integration of efficacious and nonefficacious signals during T cell antigen recognition. AB - A central question of TCR function is based on the observation that some MHC ligands may bind TCR without stimulating biologic activity. To address the role of CD4 in this mechanism, we studied the interactions of the anti-CD4 mAb W3/25 with an encephalitogenic line of T helper cells. Proliferative responses to guinea pig (GP) myelin basic protein (GPMBP) were mediated by distinct W3/25 sensitive and W3/25-insensitive mechanisms whereas responses to autologous rat (R) MBP (RMBP) were exclusively mediated by W3/25-sensitive pathways. In assays of IL-2 production, RMBP was a partial agonist that stimulated an intermediate level of IL-2 production but antagonized high levels of GPMBP-stimulated IL-2 production to that intermediate level. In the presence of W3/25, RMBP lacked stimulatory activity but instead exhibited inhibitory activity that completely blocked GPMBP-stimulated proliferative responses. The inhibitory mechanism did not involve antigenic competition for MHC glycoproteins, a blockade of mitogenic signaling, or induction of high zone tolerance. Rather, the mechanism involved specific occupancy of TCR with antagonistic MHC ligands derived from the 72-86 region of RMBP. In proliferative assays, GPMBP was approximately 10-fold more active than RMBP. In the presence of W3/25 however, GPMBP-induced agonism and RMBP-induced antagonism exhibited overlapping dose-response curves. RMBP and W3/25 not only fully inhibited GPMBP-stimulated proliferation, this synergistic combination also elicited an extended phase of T cell energy. In conclusion, RMBP derived MHC ligands occupy TCR to exhibit full efficacy for CD4-dependent signaling pathways while simultaneously lacking efficacy for W3/25-resistant signaling pathways. These data support an "integrative" model of T cell Ag recognition whereby MHC glycoproteins actively guide specific clustering of MHC ligated TCR to enable quantitative comparisons of efficacious (nonself) and nonefficacious (self) signals by T cells. PMID- 7533180 TI - Psoriatic skin-derived dendritic cell function is inhibited by exogenous IL-10. Differential modulation of B7-1 (CD80) and B7-2 (CD86) expression. AB - Regulation of immune responses depends on interactions between APCs and T cells. Such cellular interactions are mediated by surface molecules including MHC class II Ags (DR) and CD28 ligands B7-1 (CD80) and B7-2 (CD86). Recent evidence indicates that the presence or absence of costimulatory molecules on APCs significantly influences the qualitative and quantitative nature of an immune response. In this report, we analyze two relevant cytokines in skin immunobiology, granulocyte-macrophage (GM)-CSF and IL-10, and demonstrate their effects on cultured dendritic cells obtained from dermis (DDCs) of normal skin and psoriatic lesions. For comparison, the effects on these professional APCs were contrasted with cultured blood-derived monocytes. Normal and psoriatic skin derived DDCs express high levels of CD86 over CD80, and the overall hierarchy is DR > CD86 > CD80, whereas cultured monocytes express low and equivalent levels of CD80 and CD86. If Ab is added to GM-CSF at the initial period of cultivation, DDCs that emigrate have lower levels of CD86 without any detectable effect on CD80 or DR expression and display a reduced capacity to stimulate either superantigen-driven or alloantigen-responsive T cells. Conversely, by adding GM CSF to monocytes, CD86 levels are enhanced. When IL-10 was added at the beginning of culture, DDCs had significantly lower levels of CD86, without any effect on CD80 or DR expression, and like anti-GM-CSF-treated cells, these DDCs had approximately a 50% reduction in their T cell-stimulating capacity. In contrast, when monocytes were treated identically with exogenously added IL-10, they retained their relatively low levels of CD80 and CD86 with no detectable change in APC function. Blocking studies of DDC:T cell interaction indicated that CD86 was more important than CD80. Thus, differential expression patterns and functional cytokine responses involving these APC populations may be relevant to skin disorders such as psoriasis, in which discordant patterns of CD28 ligand expression and disordered cytokine networks are present. PMID- 7533181 TI - Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. AB - Fas/Apo-1 molecule is an apoptosis-signaling cell surface Ag belonging to the TNFR family. To investigate the possibility that soluble forms of the Fas receptor are expressed in human cells, we analyzed Fas mRNA transcripts obtained from activated peripheral mononuclear cells of healthy donors and from human tumor cell lines. We identified and characterized three human mRNA Fas variants: FasTMDel, FasDel2, and FasDel3. To determine whether the three transcripts were derived by alternative splicing, the Fas genomic intron/exon organization of the regions surrounding the deleted sequences was analyzed in Fas clones isolated from a human genomic library. Expression of the transcripts was studied in COS cells transiently transfected with the FasTMDel, FasDel2, and FasDel3 cDNAs. Immunocytochemical and in vitro apoptosis inhibition studies suggest that the transcripts are expressed as soluble Fas proteins that may play a functional role in the regulation of apoptosis. PMID- 7533182 TI - CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1. AB - Vaccine development in animal models depends on ability to recognize epitopes seen by human T cells. In this work, we show that CTL responses in transgenic mice expressing human HLA-A2.1 prospectively predict the same four of 11 hepatitis C virus (HCV) structural protein-derived peptides, expressing a sequence motif for HLA-A2.1 binding, that are actually recognized by human A2.1 restricted CTLs. The CTLs also recognized targets endogenously expressing these proteins. Human CTLs from HCV-infected patients, tested by using the same peptides, revealed a virtually identical response repertoire. A highly conserved HCV core peptide was the most immunogenic, and may be a valuable component of a vaccine against a broad range of HCV isolates in HLA-A2-positive patients. These results suggest that, in spite of species differences, the T cell repertoire is plastic enough to allow a similar response when the same class I MHC molecule is presenting the peptide. Thus, the HLA molecule plays the primary role in determining which peptides are recognized by CTLs. This transgenic mouse model is important for the study of HLA-restricted CTL determinants and for an approach to design a potential HCV vaccine. PMID- 7533183 TI - Antitumor immunity elicited by tumor cells transfected with B7-2, a second ligand for CD28/CTLA-4 costimulatory molecules. AB - We have examined the role of the B7-2 costimulatory molecule, a second ligand for CD28/CTLA-4 counter-receptors, in the induction of antitumor immunity. A plasmid containing murine B7-2 cDNA was transfected into the immunogenic mouse mastocytoma P815 of DBA/2 origin. In contrast to the lethal growth of the wild type (wt) P815 tumor, B7-2-positive (B7-2+) P815 cells inoculated into syngeneic mice regressed, and immunization of mice with such tumor cells protected them against the challenge of wt P815 tumor. Depletion of CD8+, but not of CD4+, lymphocytes in vivo by specific Abs abolished the regression of B7-2+ P815 tumors. CD8+ cytolytic T cells could be generated from mice immunized with B7-2+ P815. They were found to be MHC class I-restricted and specific for the P815 tumor. In contrast, transfection of the B7-2 gene into the nonimmunogenic MCA102 fibrosarcoma of C57BL/6 origin induced neither tumor regression nor protective immunity. Co-expression on MCA102 cells of B7-2 together with the related costimulator B7-1 also failed to induce immunity to MCA102 tumor. Our results indicate that transfection of B7-2 into tumor cells can improve host response to some tumors, and that the effects seen are similar to those previously observed for B7-1. PMID- 7533184 TI - IL-4 induction of VCAM-1 on endothelial cells involves activation of a protein tyrosine kinase. AB - IL-4 triggers tyrosine phosphorylation of a single major substrate (M(r) 145,000) in cultured human endothelial cells (EC) as detected by Western blot of whole cell lysates or of anti-phosphotyrosine immunoprecipitates. Phosphorylation of this substrate depends on IL-4 concentration (appearance at 10 U/ml, maximal at 300 to 1000 U/ml) and time of treatment (onset by 1 min, peak at 5 to30 min, duration of 60 to 120 min). Immunoprecipitation with specific mAb identified the phosphorylated substrate as the IL-4R. Treatment of EC with IL-4 alone causes only a small increase in the expression of vascular cell adhesion molecule-1 (VCAM-1), but IL-4 significantly augments the level of VCAM-1 expression induced by PMA. Pretreatment of EC with herbimycin A (0.5 to 1.0 microgram/ml) for 12 to 18 h abrogates both IL-4-induced tyrosine phosphorylation and IL-4-augmented VCAM 1 expression. This concentration of herbimycin A does not inhibit and may augment PMA-induced VCAM-1 expression in replicate wells. These observations suggest that IL-4 induction of VCAM-1 in EC involves the activation of an as yet unidentified protein tyrosine kinase that phosphorylates the IL-4R. PMID- 7533185 TI - Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization. AB - We have isolated, partially purified, and characterized the mast cells from human heart tissue. The histamine content of left and right ventricles and septum of hearts obtained from 25 patients undergoing heart transplantation was 5.4 +/- 0.6, 5.3 +/- 0.5, and 5.6 +/- 0.5 micrograms/g of wet tissue, respectively. Ultrastructural study of cardiac mast cells revealed scroll, crystal, and mixed granules, homogeneously dense granules, and lipid bodies in the cytoplasm. A mild collagenase digestion was used to disperse the heart mast cells; the average yield was 3.2 +/- 0.6% (range: 0.8 to 13.6%). The average histamine and tryptase content/heart mast cells was 3.3 +/- 0.2 pg (n = 25) and 24.2 +/- 4.3 micrograms/10(6) cells (n = 11), respectively. Survival of cardiac mast cells after overnight culture was 71.9 +/- 5.4% (n = 23). The purification of human heart mast cells can be brought from less than 0.1 to 12% by a combination of low speed centrifugation over albumin (2%) solution and Percoll gradient. Viability as shown by trypan blue exclusion was greater than 90%. Heart mast cells released histamine in response to immunologic (anti-IgE, anti-Fc epsilon RI, and C5a) and nonimmunologic stimuli (recombinant human stem cell factor, A23187, and compound 48/80) but did not respond to substance P, FMLP, 12-O-tetradecanoylphorbol-13 acetate, or acetylcholine. There was a linear correlation between the percentage of release caused by anti-IgE and anti-Fc epsilon RI, whereas there was no correlation between the release caused by C5a and anti-IgE-mediated stimuli. Cross-linking with anti-IgE of IgE on heart mast cells induced the release of tryptase (10.1 +/- 2.1 micrograms/10(7) cells; n = 10) and the de novo synthesis of PGD2 (17.3 +/- 4.3 ng/10(6) cells; n = 10) and of leukotriene C4 (19.1 +/- 4.5 ng/10(6) cells; n = 10). There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.67; p < 0.001) induced by cross linking of Fc epsilon RI. similarly, there was a significant correlation between percentage of histamine secretion and PGD2 (r = 0.63; p < 0.001) and LTC4 (r = 0.64; p < 0.001) release. Immunoelectron microscopy demonstrated the presence of chymase in cardiac mast cells. Mast cells isolated from human heart can be a useful model with which to study the role of these cells and their mediators in cardiac anaphylaxis and cardiovascular diseases. PMID- 7533186 TI - Molecular basis for a familial defect in phagocyte expression of IgG receptor I (CD64). AB - Four individuals have been identified, within a single family, who lack phagocyte expression of the high affinity type I IgG receptor (CD64). As a result, their monocytes are unable to support mouse IgG2a anti-CD3-induced T cell mitogenesis (nonresponder individuals). Southern blotting proved all three human Fc gamma receptor I (hFc gamma RI) genes to be present in nonresponders without major structural changes. Nucleotide sequencing showed identical hFc gamma RIA promoter regions in all individuals. At the message level, a distinct difference was noted between monocytes from control (responder) donors and from nonresponders. Both a 1.7- and 1.6-kb message were found in responders, whereas in nonresponders only the 1.6-kb species was detectable. Reverse transcriptase-PCR analyses showed the hFc gamma RIa transcript (encoding a receptor with three extracellular Ig-like domains) to be present at a approximately 15- to 20-fold lower level in nonresponder monocytes. Importantly, we found a single nucleotide difference (C - > T) within the extracellular domain exon 1-encoding region of hFc gamma RIA in nonresponders, resulting in the change of codon 92 (encoding an arginine) into a termination codon. This change likely affects mRNA stability and, thereby, leads to undetectable expression of phagocyte-hFc gamma RIa. Despite this defect, these individuals are apparently healthy, suggesting that hFc gamma RIa is dispensable for phagocyte functioning. PMID- 7533187 TI - Vesicle membrane association of nitric oxide synthase in primary mouse macrophages. AB - The isoform of nitric oxide synthase (NOS) whose activity is independent of elevated Ca2+ and exogenous calmodulin (iNOS; NOS type II) is inducible in a wide variety of cells and plays a major role in pathophysiology. The notion that iNOS is predominantly cytosolic is based on studies of a transformed cell line; almost nothing is known about the subcellular localization of iNOS in primary cells. Accordingly, we undertook a combined immunoelectron microscopic and biochemical analysis of iNOS in primary mouse macrophages. Approximately one-half of their iNOS activity and protein could be sedimented from 1 M KCl at 100,000 x g. The morphologic counterpart of particulate iNOS was a population of 50 to 80 nm vesicles that did not correspond to lysosomes nor peroxisomes. Vesicular iNOS arose from cytosolic iNOS by undergoing a post-translational modification that increased its apparent molecular mass by 4.5 kDa and promoted its salt-, detergent-, acid- and urea-resistant association with membranes, in the absence of detectable alternative splicing, myristoylation, palmitoylation, acetylation, glycosylation, or COOH-terminal truncation. Although primary macrophage iNOS underwent phosphorylation, ubiquitinylation, and binding of calmodulin tightly enough to resist boiling in SDS, these modifications did not allow us to distinguish between the cytosolic and particulate variants. The apparently novel iNOS-positive vesicles may translocate to phagosomes containing appropriately opsonized particles. PMID- 7533188 TI - Patterns of cytokine secretion by autoreactive proteolipid protein-specific T cell clones during the course of multiple sclerosis. AB - To determine whether cytokine secretion patterns change with disease status in patients with multiple sclerosis (MS), we measured IFN-gamma, TNF-alpha beta, IL 4, IL-6, IL-10 and TGF-beta secretion in a panel of T cell clones (TCCs) specific for proteolipid protein (PLP) after stimulation with PLP peptides or polyclonal activators. During acute attack, the predominant pattern of cytokine secretion resembled that of murine Th1 cells; i.e, IFN-gamma and TNF-alpha beta, and appeared to be restricted to PLP-reactive TCCs. None of the TCCs isolated during acute attack produced TGF-beta in response to PLP, Con A, or anti-CD3 Ab. Half of these TCCs were, however, capable of TGF-beta secretion and mRNA expression upon stimulation with PMA and the calcium inonphore A23187, suggesting a possible defect in activation through the TCR/CD3 pathway. During remission in the same patients all but two PLP-TCCs showed patterns of cytokine secretion resembling that of murine Th0, Th1, and Th2 subsets. The levels of IL-10 secreted by these TCCs were significantly higher than those of TCCs isolated during acute attacks and those derived from normal subjects and patients with other noninflammatory neurologic diseases. Furthermore, 50% of these TCCs were capable of producing TGF beta after Ag-specific or polyclonal stimulation. All TCCs isolated from control subjects exhibited a Th0 like secretion profile. These data indicate that different stages of disease in MS are characterized by different patterns of cytokine secretion by PLP-specific TCCs, suggesting a role for cytokines in clinical events during the course of MS. PMID- 7533189 TI - Peri-islet infiltrates of young non-obese diabetic mice display restricted TCR beta-chain diversity. AB - To define the clonal diversity of autoreactive T cells associated with the induction of type 1 diabetes, we characterized TCR expression in the earliest detectable islet infiltrates of non-obese diabetic (NOD) mice. The islets of young NOD females were examined for V beta and J beta germ-line gene usage and V(D)J beta junctional sequence diversity. The results from 7-wk-old mice corroborate prior studies demonstrating that the T cell repertoire of islet infiltrates diversifies early in the inflammatory process. In contrast, examination of 4-wk-old NOD mice showed that TCR-beta expression in the peri islet infiltrates was restricted both in V beta and J beta gene utilization and, most significantly, in V(D)J junctional sequence diversity. Islet-infiltrating T cells from young mice included V beta 3+ T cells, despite the presence of a mammary tumor virus-3-associated superantigen that deletes the majority of immature V beta 3+ thymocytes in NOD mice. Few other TCR V beta types were repeatedly detectable in early stage infiltrates. V(D)J junctional sequence diversity was evaluated in cDNA libraries made from the islets of young NOD mice. Analysis of these clones revealed limited junctional CDR3 diversity in early infiltrating T cells, as compared with lymph node T cell libraries. Evaluation of TCR expression in individual islets revealed CDR3 sequence conservation between animals and among islets from a single animal. These results suggest that T cells bearing limited TCR-beta-chain diversity contribute to the inductive phases of autoimmune diabetes. PMID- 7533190 TI - Molecular mimicry, anti-coxsackievirus B3 neutralizing monoclonal antibodies, and myocarditis. AB - Molecular mimicry has been suggested as one mechanism to explain chronic myocarditis in some murine strains in the postinfectious period following induction of acute myocarditis by coxsackievirus B3 (CVB3). To test this hypothesis, neutralizing mAbs were generated against a highly myocarditic CVB3 virus (CVB3m). These mAbs neutralized several myocarditic and amyocarditic CVB3 variants by cytopathic effects inhibition assays. Data from several experiments suggest that these mAbs recognize discontinuous epitopes on CVB3m capsid proteins. Several mAbs were found to induce cardiopathologic alterations subsequent to i.p. inoculation of normal adolescent male CD-1 or C3H/HeJ mice. Immunocytochemical assays demonstrated significant binding of two mAbs to the surface of normal cultured murine cardiac fibroblasts. Also, several mAbs were shown to participate in C-mediated lysis of normal cardiac fibroblasts, but this property did not correlate well with cardiopathogenic potential. The two properties of a mAb that were the best predictors for cardiopathogenic potential were the capacity for stimulation of normal murine fibroblasts to produce a chemoattractant activity for unelicted murine peritoneal macrophages, and the capacity for recognition of an epitopes(s) on murine or human cardiac myosins. These data show that some anti-CVB3m neutralizing mAbs can participate in proinflammatory reactions in vitro and induce cardiopathologic alterations in vivo, suggesting one mechanism by which CVB3-induced chronic inflammation in murine heart tissues can be sustained in the absence of continued virus replication. PMID- 7533191 TI - Mechanism of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo. AB - Treating activated CD4+ T cells with DNA methyltransferase inhibitors modifies gene expression and induces autoreactivity. Adoptive transfer of viable polyclonal autoreactive cells causes a lupus-like disease, most likely because of one or more effector functions expressed by the autoreactive cells. However, the number of potential effector mechanisms expressed by polyclonal cells is large. To more readily identify responsible mechanisms, we asked if autoimmunity can be induced by using the conalbumin-reactive, cloned Th2 cell line D10.G4.1, treated with 5-azacytidine (5-azaC) or procainamide (Pca). Treated, but not untreated, cells responded to syngeneic APCs without Ag, overexpressed LFA-1, spontaneously lysed syngeneic macrophages, and secreted relatively large amounts of IL-6, small amounts of IL-4, and no detectable IL-2 nor IFN-gamma. Adoptive transfer of treated, but not untreated, cells induced a severe immune complex glomerulonephritis, pulmonary alveolitis, central nervous system abnormalities including fibrinoid necrosis, karyorrhexis, and meningitis, and bile duct proliferation with periportal inflammatory cell infiltration resembling primary biliary cirrhosis. Anti-ssDNA, anti-dsDNA, and anti-histone Abs were also found. These experiments demonstrate that modification of this cloned T cell line with DNA methyltransferase inhibitors is sufficient to cause an autoimmune disease, with features of lupus as well as autoimmune liver disease. The results also raise the possibility that macrophage lysis, IL-6 secretion, and LFA-1 overexpression could contribute to the disease process. This system may be useful in testing the role of these and other pathologic mechanisms in the development of specific autoimmune lesions. PMID- 7533193 TI - Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells. AB - Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This colorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 x 10(4) lymphoid cells. The development of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity. PMID- 7533192 TI - The international standard for macrophage colony stimulating factor (M-CSF). Evaluation in an international collaborative study. AB - Three ampouled preparations of macrophage colony stimulating factor (M-CSF) were evaluated by 23 laboratories in nine countries for their suitability to serve as international standards for this material. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), the preparation in ampoules coded 89/512 was established as the international standard for M-CSF. PMID- 7533194 TI - The production of large 'signalling competent' myeloid cells from circulating CD34+ cells in neonatal blood. AB - A method is described for the production of large myeloid cells, expressing functional formylated peptide receptors. CD34+ cells were isolated from neonatal cord blood by a two stage cell sorting method. Inclusion of SCF, together with IL 3, GM-CSF or both cytokines stimulated growth of these cells over 14 day period. The resultant cells, which ranged from 30 microns to 100 microns in size, were maintained in culture for up to 5 weeks, during which time the cell population increasingly displayed myeloid characteristics, including expression of formylated peptide receptors, phagocytosis and oxidase activation. These large cells had a functioning Ca2+ signalling system in response to the chemotactic peptide, f-Met-Leu-Phe. The large size of the cells enabled the rise in cytosolic free Ca2+ which resulted from either transmembrane influx of extracellular Ca2+ and release of Ca2+ from intracellular stores to be visualised. These large myeloid cells thus provide a model system for investigating the spatial characteristics of Ca2+ signalling by formylated peptide receptors on human myeloid cells. PMID- 7533195 TI - A rapid method for the isolation of analogues of human CD59 by preparative SDS PAGE: application to pig CD59. AB - A method for the rapid isolation of functionally active analogues of human CD59 from erythrocytes (E) is described. The method, here applied to pig E, is based on the fractionation of a butanol extract of E ghosts by preparative SDS-PAGE followed by gel filtration on Superose 12. Purification was monitored using a functional complement inhibition assay. SDS-PAGE analysis of the product of this procedure indicated a single protein band with apparent M(r) of 20 kDa under reducing and non-reducing conditions. The preparation could be incorporated into guinea pig E to inhibit both CVF-reactive lysis and lysis through C8 and C9 using preformed C5b-7 sites, demonstrating that it contained a CD59-like activity. PIPLC treatment of the isolated protein abolished the inhibition. In contrast to SDS-PAGE analysis, amino-terminal sequence analysis of the preparation showed that it comprised two components. One was identified from databank searches as a fragment of pig glycophorin. These two components could not be separated by either standard or affinity chromatographic techniques. The second component was novel and had high sequence homology with human CD59, identifying it as the pig analogue. Further functional studies showed that the pig analogue of human CD59 was effective in the protection of guinea pig E against lysis by serum from a variety of species, including human. PMID- 7533196 TI - Synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using Fmoc chemistry. AB - Multiple antigen peptides (MAP) consist of lysine residue cores with branching peptide arms and have been demonstrated to be efficient immunogens as well as useful antigens for ELISA. Synthesis of diepitope MAPs with two different branching peptides has previously been described using combined Boc and Fmoc chemistry. Here, the synthesis of a tetrameric diepitope MAP based on Fmoc chemistry is described. A lysine core was synthesized with N alpha- and N epsilon amino groups othogonally protected by Fmoc and a recently described protection group, Dde, respectively. On the N alpha-amino groups, a sequence from the Plasmodium falciparum antigen Pf332 was synthesized with a capped N-terminus. After removal of Dde, a sequence from the P. falciparum circumsporozoite protein was synthesized on the core. Amino acid analysis of the MAP displayed equimolar amounts of the two peptide sequences, indicating the reliability of the protection group Dde. In ELISA, antibodies specific for either of the two malarial sequences reacted with the MAP. The major advantages of this approach for synthesis of diepitope MAPs are that only a panel of Fmoc-amino acid derivatives is required and that the more complicated cleavage procedure for Boc chemistry can be avoided. PMID- 7533198 TI - Effects of Staphylococcus aureus leukocidins on inflammatory mediator release from human granulocytes. AB - The secretion of the Panton-Valentine leukocidin (Luk-PV) but not of another leukocidin (Luk-R) from Staphylococcus aureus strains is correlated with severe pyodermic infections (dermonecrosis). The effects of both Luk-PV and Luk-R in amounts of 0-5000 ng on inflammatory mediator release from human leukocytes were studied. Luk-PV but not Luk-R induced a pronounced release of the vasodilator histamine from human basophilic granulocytes (up to 55% +/- 7%) and of enzymes (beta-glucuronidase, up to 45% +/- 10%; lysozyme, up to 35% +/- 7%), chemotactic components leukotriene B4 (42 +/- 8 ng/10(7) cells) and interleukin-8 (up to 33 +/- 5 ng/10(7) cells), and oxygen metabolites from human neutrophilic granulocytes. The results indicate that granulocytes play a central role in dermonecrosis; these in vitro data account for the histologic picture of Luk-PV infections, characterized by local vasodilation, infiltration of granulocytes, and a central necrotic area. PMID- 7533199 TI - Increased circulating soluble CD14 is associated with high mortality in gram negative septic shock. AB - The soluble glycoprotein sCD14 binds lipopolysaccharide, a complex that activates endothelial cells and that may be crucial in gram-negative sepsis. Therefore, serum sCD14 was analyzed in 54 patients with gram-negative septic shock and in 26 healthy controls. sCD14 was tested by ELISA and Western blotting. Patients had higher sCD14 concentrations than controls (median, 3.23 vs. 2.48 micrograms/mL, P = .002). Increased levels were associated with high mortality (median, 4.2 micrograms/mL in nonsurvivors vs. 2.8 micrograms/mL in survivors, P = .001). sCD14 was found in two isoforms (49 and 55 kDa) in monocyte cultures. In sera only one of either form was detectable. Controls had the 49-kDa form, and patients had either the 49- or 55-kDa form, but patients with high levels of sCD14 had only the 55-kDa form. Twenty-one (53%) of 39 with the 55-kDa form and 8 (57%) of 14 with the 49-kDa form died. Thus, the level of sCD14 but not its biochemical form had a prognostic value in patients with gram-negative septic shock. PMID- 7533197 TI - High-dose nevirapine: safety, pharmacokinetics, and antiviral effect in patients with human immunodeficiency virus infection. AB - Nevirapine, a potent nonnucleoside reverse transcriptase inhibitor, produces a transient antiviral effect at < or = 200 mg/day due to the selection of resistant virus. To examine if higher levels of nevirapine could produce sustained antiviral activity, its safety, pharmacokinetics, and antiviral activity at 400 mg/day were studied in 21 patients. There was a rapid reduction in immune complex dissociated p24 antigen and serum human immunodeficiency virus RNA concentration in all patients, and 8 of 10 patients had > 50% reduction at 8 weeks. Nevirapine resistant virus was isolated from all subjects tested at 12 weeks: The mean plasma trough level (4.0 micrograms/mL [15.8 microM]) exceeded the mean IC50 of resistant virus. Rash developed in 48% of patients and was a dose-limiting toxicity factor in 6. These data suggest that clinical testing of potent antiviral compounds that select for drug-resistant virus is justified to determine if serum levels of drug sufficient to overcome resistant virus can be attained. PMID- 7533200 TI - Hepatitis C virus genotypes HCV-1a and HCV-1b: the clinical point of view. PMID- 7533201 TI - Seroprevalence of hepatitis B and C viruses in eastern Nepal. AB - The seroprevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) in 303 serum samples collected from that apparently healthy population inhabitating different areas in eastern Nepal was studied. Samples were collected at Dharan Municipality, Sunsari85), Pancha Kanya Village Development Committee, Ilam86), Dhankuta Hile, Dhankuta82) and Basantapur Village Development Committee, Tehrathm50). HBsAg and anti-HBsAg antibody was screened by reverse passive haemagglutination (RPHA) and passive haemagglutination (PHA) respectively and positivity was confirmed by enzyme linked immunosorbent assay (ELISA). Anti-HCV antibody was detected by ELISA. None of the samples were positive for HBsAg. Anti HBsAg antibody was positive in 1.9% (6/303). The positive rate increased with age reaching 25% positivity among the elderly. The anti-HBsAg antibody positivity was 2.35, 2.32, 1.22 and 2.00 in Dharan, Ilam, Dhankuta and Tehrathum respectively Anti-HCV antibody was detected only in one sample (15-year-old boy) collected in Dharan. These findings indicate that the HBV and HCV infections are not active in eastern Nepal. PMID- 7533202 TI - The localization and specificity of guinea pig inner ear antigenic epitopes. AB - In this study, we investigated the relative localization of some antigenic epitopes in the inner ear. The inner ear protein antigens were extracted from various parts of the guinea pig inner ear. Brain, kidney, lung, heart and liver extracts were also obtained. We found by SDS-polyacrylamide gel electrophoresis that total inner ear extracts separated into three high concentration polypeptide bands with molecular weights of approximately 30, 42, 58 kd and three low density bands of 20, 25 and 35 kd. The 30 kd band was found mainly in the extract of the spiral ganglion and the acoustic nerve in the modiolus. The 42 and 58 kd bands were detected in the extract of the spiral ligament and the stria vascularis. The Organ of Corti and the basilar membrane extract gave rise to three bands of 30, 42 and 58 kd. Twenty-eight of the 75 sera from patients with inner ear disease reacted with the 30 and 58 kd bands of the inner ear protein extracts by immunoblotting. Sixteen of these 28 positive sera were then used to probe immunoblots of the brain, kidney, lung, heart and liver extracts. The 58 kd band was also found in protein extracts of the brain, the lung and the liver. This study suggests that the 30 kd antigenic epitope may be mainly related to the acoustic nerve and that the 58 kd antigenic epitope is not cochlear specific. PMID- 7533203 TI - Changes in white blood cells and myeloperoxidase activity in rhG-CSF prophylactic patients receiving cytotoxic chemotherapy. AB - The patterns of white blood cell parameters and mean peroxidase index (MPXI) changes in recombinant human granulocyte colony-stimulating factor (rhG-CSF) prophylactic patients, receiving myelosuppressive chemotherapy, have been studied in 8 cases, using flow cytochemistry blood autoanalyser (Technicon R H*1). No severe neutropenia (absolute neutrophil count, ANC < 0.50 x 10(9)/L) appeared in 6 rhG-CSF primary prophylactic patients, but severe neutropenia was noticed in 2 rhG-CSF secondary prophylactic patients for a period less than 1 week. In most of the cases the significant increase of neutrophil during leukocytosis occurred within 24 hours after starting rhG-CSF prophylaxis, and decreased within 24 hours after the end of rhG-CSF prophylaxis. There was a small degree of lymphocytosis, monocytosis, and basophilia in some cases. From this study, there were no significant changes of MPXI during rhG-CSF prophylaxis, the neutrophils produced during proliferative response to rhG-CSF possessed normal myeloperoxidase (MPO) activity. We concluded that the information obtained from automated blood cell analyser Technicon R H*1 especially MPXI and ANC, could be very useful for monitoring rhG-CSF prophylactic patients receiving myelosuppressive chemotherapy. The advantages of the MPXI over other methods is its simplification when performed as part of a routine complete blood count (CBC) on an automated hematology instrument, and its ability to measure even severe neutropenic samples. PMID- 7533204 TI - Terazosin for BPH. PMID- 7533205 TI - Neurotoxic reaction to lindane in an HIV-seropositive patient. An old medication's new problem. AB - Scabies is a common infestation for patients of all ages throughout the world. One of the standard therapies for scabies is 1% lindane lotion. Lindane has been associated with neurotoxic reactions, specifically seizures. We describe a case of a middle-aged adult man with human immunodeficiency virus (HIV) infection who was found to have typical scabies and was treated with a single topical application of lindane. Two hours after the application, the patient experienced a new-onset generalized seizure. We believe that the triad of HIV infection, medications that reduce seizure thresholds, and percutaneous absorption factors, in the aggregate, induced the seizure. We believe that lindane should not be prescribed for patients with HIV infection. PMID- 7533206 TI - Distinguishing ingested ethanol from microbial formation by analysis of urinary 5 hydroxytryptophol and 5-hydroxyindoleacetic acid. AB - During the metabolism of ethanol, the metabolic conversion of serotonin (5 hydroxytryptamine) is altered, and, as a consequence, the ratio of 5 hydroxytryptophol (5HTOL) to 5-hydroxyindole-3-acetic acid (5HIAA) excreted in urine increases appreciably. The ratio of metabolites remains elevated for several hours after ethanol is no longer detectable. In the present study, urine specimens were supplemented with glucose and Candida albicans, a common human pathogenic yeast, and the formation of ethanol and the changes in the 5HTOL/5HIAA ratio were examined during one week of storage. Despite the production of high concentrations of ethanol (peak level 171 mmol/L, or 788 mg/dL), the 5HTOL/5HIAA ratio remained constant. The urinary 5HTOL/5HIAA ratio was also compared with urinary and blood ethanol levels in specimens selected at random during forensic autopsies. Elevated 5HTOL/5HIAA ratios were found in all specimens with detectable urinary ethanol. Some specimens showed elevated ratios of serotonin metabolites even though no ethanol was detected, indicating that these subjects had consumed ethanol prior to death but that the concentration had already returned to zero or was below the detection limit. In one case, postmortem ethanol formation was suspected, because blood ethanol concentration was 16.8 mmol/L (77 mg/dL) whereas urinary ethanol was zero. The urinary 5HTOL/5HIAA ratio fell within normal limits, which confirmed the suspicion of postmortem ethanol synthesis in the blood specimen. The present results indicate that the 5HTOL/5HIAA ratio in urine provides a useful method to distinguish between ethanol that might have been synthesized postmortem, or generated in vitro, from ethanol excreted in urine as a result of drinking. PMID- 7533207 TI - Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin. AB - Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2' disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4' isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS insensitive Cl conductance turns on at an Em of approximately -40 mV. PMID- 7533208 TI - The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions. AB - B7 is a costimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferon-gamma. B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-1, LFA-1 and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimer's disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions. PMID- 7533209 TI - Vitronectin and integrin vitronectin receptor localization in multiple sclerosis lesions. AB - Vitronectin (Vn) is a multifunctional plasma and extracellular matrix glycoprotein involved in cell attachment, coagulation, phagocytosis, and the protection of bystander cells from complement- and T cell-mediated lysis. To determine where Vn is localized and where cells expressing integrin Vn receptors may recognize it in central nervous system (CNS) lesions of multiple sclerosis (MS), CNS tissue samples were immunostained for Vn and the alphav, beta 1, and beta 3 integrin Vn receptor subunits. By light and electron microscopy, Vn was localized within dystrophic, demyelinated axons in active but not chronic lesions, normal or other neurologic disease controls. This localization is distinct from that of other plasma proteins in MS lesions and it differs from the pattern of neuron cell body localization found in other conditions. Microvascular Vn was increased and small numbers of reactive astrocytes were also Vn-positive in active plaques. Endothelial cell expression of the alpha v subunit was increased over controls and that of the beta 1 subunit was decreased whereas both the alpha v and beta 1 subunits were prominently expressed on macrophages and glia in active lesions. The beta 3 integrin subunit was expressed on platelets within and around vessels and was more prominent on endothelial cells in active plaques. The precise functions of Vn in situ are not presently known. These results indicate, however, that the regulation of expression of integrin Vn receptors is complex and that Vn may be recognized and have multiple functions in different microanatomic sites as MS lesions evolve. Intravascular Vn could participate in clotting, thereby contributing to leukocyte extravasation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533210 TI - Nutritional modulation of the somatotropin/insulin-like growth factor system: response to feed deprivation in lactating cows. AB - Mid-lactation Holstein cows (n = 4) were used to evaluate the effects of complete feed deprivation on circulating insulin-like growth factors (IGF) and their binding proteins (IGFBP). Comparisons were made between cows in fed and unfed conditions and included response to a single injection of bovine somatotropin (bST). Two days of feed deprivation decreased milk yield 66%. Concentrations of bST nearly doubled during feed deprivation, whereas glucose and insulin steadily decreased. Circulating concentrations of IGF-I decreased nearly 50%, IGF-II decreased 28% and IGFBP-2 increased 49% during the 2 d of feed deprivation. In contrast, IGFBP-3 was not affected. Eighteen to 24 h after the bST challenge, circulating concentrations of IGF-I rose 100% during the fed period, but there was no response during the unfed period. Blood concentrations of IGF-II decreased 17% after bST challenge in the fed period but were unchanged in the unfed period. More moderate undernutrition (80% of requirements) did not affect circulating IGFBP-2 but did alter the response of IGFBP-2 to bST. A conceptual model was developed that illustrates that nutritional regulation of the ST/IGF system represents a key component signaling the appropriate use of nutrients. Overall, results demonstrate that nutritional modulation of the ST/IGF system plays a key role in animal performance and well-being over a wide range of physiological situations. PMID- 7533211 TI - Vascular adhesion molecules and initial development of inflammation in clinically healthy human keratinized mucosa around teeth and osseointegrated implants. AB - Expression of vascular cell adhesion molecules (HEV-CAMs) by capillary loops represents an early step necessary for leukocyte extravasation and subsequent migration to sites of inflammation. The purpose of this investigation was to compare, the presence and distribution of ICAM-1, ELAM-1, VCAM-1 and PECAM-1 expression in the microvasculature of healthy gingiva and peri-implant keratinized mucosa. The selected HEV-CAMs were detected by a three stage immunoperoxidase technique in serial sections from clinically characterized sites. Biopsies were qualitatively and quantitatively analyzed. All biopsies displayed a small inflammatory infiltrate subjacent to the most coronal extension of the junctional epithelium. HEV-CAMs were expressed both in the sulcular and oral aspects of biopsies. Intensity of ICAM-1, ELAM-1 and VCAM-1 staining, however, was consistently higher in the region of the infiltrated connective tissue subjacent to the most coronal extension of the junctional epithelium. Only a fraction of microvascular loops were positive for ICAM-1, ELAM-1 or VCAM-1 when compared to the constitutively expressed PECAM-1. No significant differences were observed between gingiva and keratinized peri-implant mucosa. It was concluded that in healthy gingiva or peri-implant mucosa 1. HEV-CAM expressing capillary loops were in close topographic association with the inflammatory infiltrate; 2. only a fraction of capillary loops are activated to express ICAM-1, ELAM-1 and VCAM-1 at any given time; and 3. HEV-CAM expression in the periodontium may be influenced by local factors. PMID- 7533212 TI - Immunohistological study of cytokeratin 19 expression in regenerated junctional epithelium of rats. PMID- 7533214 TI - Serotonin in attempted suicide. PMID- 7533215 TI - High-affinity antigen binding by chelating recombinant antibodies (CRAbs). AB - We have developed a strategy for making antibody fragments with high binding affinities by harnessing the chelate effect. We create a bispecific antibody fragment (Chelating Recombinant Antibody or CRAb) that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously. Here the strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozyme. Computer graphic modelling indicated that two single-chain antibody fragments (scFv) derived from antibodies D1.3 (Ka = 10(8) M 1) and mutant HyHEL-10 (Ka = 10(6) M-1) could be linked together on the surface of lysozyme by a flexible and hydrophilic polypeptide between the C terminus of one fragment and the N terminus of the other. The CRAb gene was assembled and the CRAb expressed by secretion from bacteria. The purified CRAb was shown to have a much higher affinity than either of the scFv fragments, as shown by competition ELISA (Kd > 10(9) M-1), bandshift on gels (Ka > 2 x 10(9) M-1) and fluorescence quench (Ka > 1.3 x 10(10) M-1). PMID- 7533213 TI - Substance P: an immunohistochemical and biochemical study in human gingival tissues. A role for neurogenic inflammation? AB - Substance P has been studied in relation to its distribution within gingival tissues as well as its effect on cultured human gingival fibroblasts. The tissue distribution was varied depending on the degree of inflammation present. In healthy tissues substance P was found in the connective tissues interspersed between the collagenous elements and was particularly prominent in the areas immediately subjacent to the epithelial rete pegs. In inflamed tissues, substance P was markedly increased particularly around the blood vessels as well as in close association with much of the inflammatory cell infiltrate. The effect of substance P on human gingival fibroblast proliferation was monitored by [3H] thymidine incorporation and indicated substance P to be mitogenic for these cells at low concentrations (1 x 10(-9) M) and tended towards an inhibitory effect at higher concentrations (1 x 10(-4) M). Substance P did not have any effect on the release of either total proteins or proteoglycans into the culture medium. However, exposure of the cells to substance P did cause a greater accumulation of both total protein and proteoglycan with the cell layer material. These findings suggest a potential role for substance P on gingival tissues and in particular their resident fibroblastic cells and can therefore be used as a basis for more detailed studies into the relationship between neuropeptide release associated with neurogenic inflammation and periodontal pathology. PMID- 7533216 TI - Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters. AB - The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples. Excellent agreement among the three methods was obtained. With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed. Non-specific binding to DNA ends was common. The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization. With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end. Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees. Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions. PMID- 7533218 TI - Expression of C-terminal src kinase in human colorectal cancer cell lines. AB - C-terminal src kinase (CSK) is a cytoplasmic protein which decreases activities of the Src family protein-tyrosine kinases. We produced a polyclonal antibody specific for CSK and analyzed the expression of CSK by immunoblotting in two human colorectal normal cell lines and eighteen cancer cell lines. CSK was detected in both the two normal and all the eighteen cancer cell lines. The expression of CSK obtained from human colorectal cancer cell lines was greater than that from human colorectal normal cell lines in most cases. The elevated expression of CSK in human colorectal cancer cell lines appeared to correspond to the elevated p60c-src (c-Src) and p62c-yes (c-Yes) protein-tyrosine kinase activities found in other studies. Thus, CSK may not have an anti-oncogenic role to play through the negative regulation of Src family kinases in colorectal carcinogenesis. PMID- 7533219 TI - Feature analysis of singleton consonant errors in developmental verbal dyspraxia (DVD). AB - The aim of this study is to quantify diagnostic characteristics related to consonant production of developmental verbal dyspraxia (DVD). For this, a paradigmatic and syntagmatic feature-value analysis of the consonant substitution and omission errors in DVD speech was conducted. Following a three-step procedure, eleven clear cases were selected from a group of 24 children with DVD. The consonants produced in a word and nonsense-word imitation task were phonetically transcribed and transferred to confusion matrices, which allows for a feature and feature-value analysis. The analysis revealed that children with DVD (a) show low percentages of retention for place and manner of articulation and voicing, due to high substitution and omission rates; (b) show a particularly low percentage of retention of place of articulation in words, which, together with error rate, is strongly related to severity of involvement; (c) are inconsistent in their feature realization and feature preference; and (d) show a high syntagmatic error rate. These results form a quantification of diagnostic characteristics. Unexpectedly, however, very few qualitative differences in error pattern were found between children with DVD and a group of 11 age-matched children with normal speech. Thus, although the children with DVD produced higher substitution and omission rates than children with normal speech, the speech profiles of both subject groups are similar. This result stresses the importance of interpreting profiles, not isolated symptoms. The hypothesis to consider DVD as a deficit in the phonological encoding process is discussed. PMID- 7533217 TI - The 1.6 A structure of Kunitz-type domain from the alpha 3 chain of human type VI collagen. AB - The C-terminal Kunitz-type domain from the alpha 3 chain of human type VI collagen (C5), a single 58 amino acid residue chain with three disulfide bridges, was cloned, expressed and crystallized in a monoclonic form, space group P2(1), with a = 25.7 A, b = 38.2 A, c = 28.8 A and beta = 109 degrees. The structure was resolved by molecular replacement, using Alzheimer's protein precursor inhibitor and bovine pancreatic trypsin inhibitor three-dimensional structures as search models. The molecule with one sulfate ion and 43 associated water molecules was refined by XPLOR to an R-factor of 18.9% at 1.6 A. The molecule was not degraded by trypsin and did not inhibit trypsin or tested serine proteases. As opposed to the other Kunitz family members, C5 demonstrates left-handed chirality of the Cys14-Cys38 disulfide bond. Inversion of the Thr13 carbonyl and bulky side-chains at the interface with trypsin in a model of the C5-trypsin complex may explain the lack of inhibition of trypsin. PMID- 7533220 TI - Selective stimulation of I1-imidazoline receptors as a novel mechanism for antihypertensive action. Focus on moxonidine. PMID- 7533221 TI - Selective antihypertensive action of moxonidine is mediated mainly by I1 imidazoline receptors in the rostral ventrolateral medulla. AB - The rostral ventrolateral medulla (RVLM) is the primary region maintaining vasomotor tone, and a site of action for central antihypertensive agents. In vitro [125I]p-iodoclonidine binding studies showed that moxonidine was selective for I1-imidazoline over alpha 2-adrenergic receptors in the RVLM. We identified efaroxan and SK&F 86466 as selective I1- and alpha 2-antagonists, respectively. We tested moxonidine's action within the RVLM of spontaneously hypertensive rats (SHRs) on I1-imidazoline or alpha 2-adrenergic receptors, and determined whether the RVLM mediates the action of systemic moxonidine. SHRs were anesthetized, paralyzed, and ventilated and the RVLM was localized by testing for a pressor response to 2 nmol glutamate. To test whether I1 or alpha 2 mediates hypotensive effects of moxonidine, the I1/alpha 2 antagonist efaroxan (4 nmol) or the alpha 2 blocker SK&F 86466 (10 nmol) was administered 15 min before 4 nmol moxonidine. Efaroxan elevated blood pressure and abolished the action of moxonidine, whereas alpha 2-blockade with SK&F 86466 slightly lowered blood pressure and only partially attenuated moxonidine's effect. The depressor effect of intravenous moxonidine (40 micrograms/kg) was reversed within 10 min by microinjection of 10 nmol efaroxan into the RVLM. Prior bilateral microinjections of efaroxan (10 nmol in 80 nl/site) into the RVLM prevented the hypotensive action of moxonidine given i.v. (40 micrograms/kg). Pharmacokinetic studies showed that at the peak vasodepressor response (8 min post-injection), [3H]moxonidine spread less than 1 mm from the injection site. Moxonidine is a centrally acting antihypertensive with a selective action on I1-imidazoline receptors in RVLM. PMID- 7533222 TI - Mechanisms of cardiac cell damage due to catecholamines: significance of drugs regulating central sympathetic outflow. AB - A chronically increased rate of catecholamine release has various deleterious actions. Isoproterenol injections (80 mg/kg body weight) resulted in depressed Ca2+ transport in the sarcolemma (ATP-dependent Ca2+ uptake, Na(+)-dependent Ca2+ uptake) and sarcoplasmic reticulum (Ca2+ uptake) of rat heart. The formation of malondialdehyde owing to lipid peroxidation was increased. Pretreatment with vitamin E (10-25 mg/kg/day) strongly inhibited the membrane damage. The toxic effects of catecholamines arise most probably from their oxidation, and it is therefore important either to reduce the central sympathetic outflow or to prevent the oxidation. An inappropriately high sympathetic outflow is a typical feature of Western affluent societies, and is linked to psychosocial stress and hypercaloric nutrition. However, established pharmacologic interventions to reduce sympathetic outflow have proven not practicable because of marked side effects. Using radiotelemetry for monitoring cardiovascular parameters of spontaneously hypertensive rats treated with clonidine or moxonidine, we showed that clonidine, unlike moxonidine, resulted in rebound hypertension after drug withdrawal. Because the rebound blood pressure and the typical side effects of clonidine associated with low patient compliance are mainly mediated by alpha adrenoceptors, it can be inferred that the I1-imidazoline agonist moxonidine does not exhibit the side effects commonly seen with clonidine and therefore represents a promising approach for reducing an inappropriately high central sympathetic outflow. PMID- 7533223 TI - Moxonidine and hydrochlorothiazide in combination: a synergistic antihypertensive effect. AB - This study was designed as a multicenter, double-blind, placebo-controlled, parallel-group, prospectively randomized study comparing, after a 4-week placebo run-in phase, moxonidine 0.4 mg once daily (o.d.), hydrochlorothiazide 25 mg o.d., and the combination of the two with placebo. A total of 160 patients were analyzed in an intent-to-treat analysis. Moxonidine 0.4 mg o.d. was effective in significantly lowering blood pressure in this group of mild-to-moderate hypertensive patients in comparison with placebo. The efficacy and the side effect profile of moxonidine were comparable to those of the first-line antihypertensive agent hydrochlorothiazide. The combination of moxonidine and hydrochlorothiazide in the same dosage as a monotherapy improves efficacy significantly without additive effects on the safety profile. Response rate after monotherapies was calculated with 70.3 and 70.0%, respectively, after combination treatment in 87.8% of all patients in the treatment group. The trial gives support to a recommended dosage regimen of moxonidine 0.4 mg o.d. This profile of moxonidine is highly comparable to a standard first-line antihypertensive drug such as hydrochlorothiazide, without sacrificing tolerance and safety for increased efficacy, in combination with hydrochlorothiazide. PMID- 7533224 TI - Twenty-four-hour blood pressure profiles in patients with mild-to-moderate hypertension: moxonidine versus captopril. AB - In 26 patients with mild-to-moderate hypertension, 80.8% of whom had a history of concomitant diseases, the effect of moxonidine (0.2 mg b.i.d.) on the 24-h ambulatory blood pressure profile (ABPM) was compared with captopril (25 mg b.i.d.) in a double-blind, parallel-group study. After 4 weeks of treatment with placebo, ABPM was performed and the patients were treated with moxonidine (n = 14) or captopril (n = 12) for a further 4 weeks. ABPM was then repeated. Both moxonidine and captopril reduced systolic and diastolic blood pressure sufficiently and to the same extent. Mean 24-h pulse rate and standard laboratory parameters were not changed by active treatment. After drug withdrawal for 5 days, sitting blood pressure did not differ from baseline values in both groups. Serious adverse events did not occur, the most frequent complaints were nausea (2 of 14 patients receiving moxonidine) and dizziness (3 of 12 patients receiving captopril). We concluded that the blood pressure-lowering effects of moxonidine (0.2 mg b.i.d.) and captopril (25 mg b.i.d.) are comparable in patients with mild to-moderate hypertension. PMID- 7533225 TI - Therapy of hypertensive cardiac hypertrophy and impaired coronary microcirculation. AB - In arterial hypertension, cardiac remodeling comprises myocyte hypertrophy, interstitial fibrosis, and functional and structural alterations of the coronary microcirculation. This leads to diastolic and systolic dysfunction of the left ventricle and impairment of coronary flow reserve. Consequently, antihypertensive treatment should aim at repairing hypertensive cardiac remodeling through reversing myocyte hypertrophy, restoring myocardial structure, and improving coronary flow reserve along with blood pressure normalization. Although it has been shown that regression of left ventricular hypertrophy (LVH) can be achieved by suitable antihypertensive therapy, more insight regarding the ability to repair coronary microcirculation is needed. In spontaneously hypertensive rats (SHRs), it has been shown that coronary reserve was enhanced after hydralazine administration without concomitant regression of LVH. Likewise, administration of the calcium-channel blocker felodipine led to a reversal of medial hypertrophy in coronary resistance vessels. The angiotensin-converting enzyme inhibitor lisinopril was shown to improve coronary reserve and to reserve both medial hypertrophy and myocardial fibrosis in SHRs. Increase in length density of capillaries with either nifedipine or moxonidine treatment was also found in experimental hypertension. First clinical data indicate that, after prolonged antihypertensive treatment, coronary flow reserve can be improved in hypertensive patients with microvascular disease. Further studies are warranted to elucidate whether improved coronary flow reserve after medical treatment for arterial hypertension is due to an influence of myocardial factors, such as LVH or myocardial fibrosis or to repair of the structurally remodeled microcirculation. PMID- 7533226 TI - I1-imidazoline-receptor agonists in the treatment of hypertension: an appraisal of clinical experience. AB - Although essential hypertension is usually defined as a hemodynamic disorder, it is expressed differently among individuals and varies during progression of the disease state. Therefore, various types of treatment can be envisioned. The use of selective I1-imidazoline-receptor agonists to modulate I1-imidazoline receptors involved in the central regulation of blood pressure has led to the introduction of a novel class of centrally acting antihypertensive drugs. Moxonidine, a representative molecule of this class, dissociates between a 10% alpha 2-adrenoceptor-agonist action linked with side effects such as fatigue or dry mouth, and a 90% specific antihypertensive action resulting from its selective agonistic action at I1-imidazoline receptors. Clinical experience is based on more than 2,000 patients and volunteers, and long-term efficacy has been demonstrated in about 500 patients who received a daily dose of moxonidine 0.2 0.4 mg. Moxonidine produces a pronounced reduction in peripheral vascular resistance without reflex tachycardia, accompanied by reduced plasma norepinephrine concentration and plasma-renin activity. Cardiovascular responses to exercise and standing remain nearly normal, and serious or life-threatening side effects, particularly the sympathetic overactivity that can occur on sudden withdrawal of other centrally acting agents, are never observed. In addition, moxonidine behaves neutrally with respect to plasma levels of cholesterol, potassium and glucose, glucose and lipid metabolism, and renal function, and can be administered without complication to patients with asthma or certain other diseases. Studies with magnetic resonance imaging have shown that moxonidine significantly reduces left ventricular mass, an indicator of left ventricular hypertrophy (LVH), within a 6-month treatment period, an effect that coincided with decreased plasma concentrations of catecholamines and renin. Comparisons between moxonidine and other well-established antihypertensive drugs such as nifedipine, atenolol, or angiotensin-converting enzyme inhibitors showed equal effectiveness in lowering blood pressure, whereas the adverse events profile always favored moxonidine. Considering its efficacy, safety, and specific effects (e.g., its ability to reduce LVH), moxonidine meets the criteria satisfied by other currently prescribed antihypertensive drugs. Because of its especially favorable benefit-to-risk ratio, moxonidine should be recommended as first-line treatment of hypertension and may also be useful in treating related problems such as LVH, coronary artery disease, and ventricular premature beats. PMID- 7533227 TI - Effect of moxonidine on arrhythmias induced by coronary artery occlusion and reperfusion. AB - The aim of the present study was to investigate the influence of moxonidine, a representative of I1-imidazoline-receptor agonist, on arrhythmias induced by myocardial ischemia or reperfusion. Acute myocardial infarction was produced by tightening a previously placed loose silk loop around the coronary artery in conscious rats. Moxonidine (0.01, 0.03, or 0.10 mg/kg i.v., 10 min before coronary ligation) significantly decreased the incidence of ventricular tachycardia during the first 15 min of infarction (70 versus 100% in controls), and the number of animals that survived without developing any arrhythmia was increased (15, 20, and 25%, respectively, versus 0%). Reperfusion-induced arrhythmias were produced by releasing a snare after 6 min of myocardial ischemia in anesthetized, artificially ventilated rats. Reperfusion rapidly induced severe dysrhythmias in all of the control animals. Moxonidine pretreatment (0.03 and 0.10 mg/kg) decreased the incidence of ventricular fibrillation (25 and 30% versus 64%) and increased the number of animals that survived without developing any arrhythmia (20 and 25% versus 0%). We conclude that moxonidine offers significant protection against the development of arrhythmias induced by acute regional myocardial ischemia in conscious rats. Moxonidine pretreatment also provides a beneficial effect during reperfusion-induced arrhythmias that appear after a brief period of myocardial ischemia. PMID- 7533228 TI - [Palliative medicine. Reflections from the point of view of medical oncology]. PMID- 7533229 TI - Management of thyroid nodules during pregnancy. AB - Guidelines for the management of thyroid nodules discovered during pregnancy have not yet been established. The authors reviewed the records of 23 patients with thyroid nodules that were first detected during pregnancy. These patients were divided into three groups according to how they were managed. Seven patients who presented early in pregnancy had their work-up completed during pregnancy, 11 patients underwent biopsy after delivery, and 5 patients were managed with observation alone. The incidence of malignancy in the series was 39%. Four patients underwent surgery during pregnancy, and 7 patients were operated on in the postpartum period. No fetal morbidity or mortality occurred. The authors recommend that fine-needle aspiration be performed in patients who present before 20 weeks of gestation with rapidly enlarging thyroid nodules, nodules associated with palpable cervical adenopathy, solid nodules larger than 2 cm, or cystic nodules larger than 4 cm. Growth of a nodule while a patient is receiving thyroid hormone suppression therapy is highly suspicious for malignancy; in this situation, consideration should be given to performing biopsy later in gestation. PMID- 7533230 TI - [Esophageal replacement--indications, technique, results]. AB - Between November 1985 and January 1994 a total of 239 patients were operated for replacement of the esophagus and primary reconstruction. Of these 3 had benign disease and 236 had malignancy. Continuity of the alimentary tract was restored in 202 cases by stomach transposition, in 16 cases by colon interposition and in 21 cases by free jejunal autograft. Complication rate of surgical resection was 36.8%, lethality 5.1%. Lethality of coloninterposition was as low as for gastric transposition (6.3 vs 5.5%). None of the patients with free jejunal autograft died during hospital course (p < 0.001). Survival rates by life-table analysis for the whole group were 65%, 42%, 36%, 32%, and 22% after 1, 2, 3, 4 and 5 years, respectively. The malignant tumors consists of 111 esophageal carcinomas, 104 carcinomas of the esophagogastric junction and 21 carcinomas of the hypopharynx. 15.1% of the tumors were staged I, 42.7% II, 35.7% III and 6.5% IV. The collectives for esophageal carcinoma and cardia carcinoma were equal for age, sex, and distribution of tumor stages. The survival rates for both groups were similar. Differences occurred in a three times higher rate for concomittant hiatal hernia and history of reflux esophagitis in the cardia carcinoma group. PMID- 7533231 TI - Cytokeratin inclusions in alcoholic liver disease and their relation to the amount of alcohol intake. AB - In the present study, the frequency and the distribution pattern of immunoreactive hepatocytic cytokeratin (CK) inclusions was investigated using the monoclonal antibody (MAb) CAM 5.2 detecting CKs 8, 18 and 19. The CK antigenicity of the inclusions was confirmed on frozen sections with MAbs for the CKs 7, 8, 17, 18 and 19. The frequency of hepatocytic CK aggregates was compared to the presence of non-alcoholic and alcoholic liver disease (ALD) as well as to the average all-year daily ethanol intake of 195 consecutive males subjected to medico-legal autopsy. Hepatocytic CK inclusions were infrequent in patients with normal liver histology, portal fibrosis and/or portal inflammation. In ALD, however, inclusions were observed in 35.6% of patients with fatty liver, in 63.2% of patients with alcoholic hepatitis, in 30.8% of patients with bridging fibrosis and in 60.0% of patients with liver cirrhosis. In all specimens studied, the inclusions were reactive only for CKs 8 and 18, CKs 7, 17, and 19 being unreactive. The frequency of inclusion bodies increased, paralleling increasing average all-year daily alcohol consumption. Compared to non-drinkers, a daily intake of between 40 and 80 g of absolute alcohol was associated with a statistically significantly (relative risk, RR = 6.6) increased risk of formation of aggregates. Similarly, daily consumption exceeding 80 g was related to increased (RR = 6.0) frequency of CK aggregates. The frequency of full-blown "classical" Mallory bodies (MBs) was, however, increased (RR = 8.9) only in patients with a daily intake exceeding 160 g.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533232 TI - Interleukin-1 beta increases basic fibroblast growth factor mRNA expression in adult rat brain and organotypic hippocampal cultures. AB - In situ hybridization was used to study the effect of IL-1 beta on acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) mRNA expression in rat brain. Intraventricular injection of recombinant human IL-1 beta did not affect hybridization to aFGF mRNA but did induce significant and widespread increases in hybridization to bFGF mRNA. IL-1 beta induced increases in bFGF mRNA were bilaterally distributed and appeared to correspond with the distribution of non-neuronal cells. Thus, hybridization was increased in regions of both gray and white matter (e.g., corpus callosum), the ependymal lining of the third ventricle, and the pia matter. In hippocampus of IL-1 beta injected rats, hybridization was markedly increased in the molecular layers but not significantly increased in the neuronal cell layers. Elevations in bFGF mRNA were transient, peaking at 8 h postinjection in most areas. To determine if IL-1 beta effects were independent of activation of the hypothalamo-pituitary-adrenal axis, and to compare the cellular localization of increases in bFGF mRNA expression induced by IL-1 beta and bFGF, the regulation of bFGF expression was also studied in organotypic hippocampal slice cultures. Treatment of cultures with either IL-1 beta or bFGF stimulated the same general distribution of increases in bFGF mRNA as seen after IL-1 beta treatment in vivo with an additional effect on immature neurons within the hilar side of stratum granulosum; hybridization of bFGF mRNA was not increased in association with the more mature neurons of stratum pyramidale or stratum granulosum. Colocalization of bFGF cRNA hybridization with immunostaining for glial fibrillary acidic protein demonstrated that increases in bFGF mRNA induced both by IL-1 beta in vivo and in vitro and by bFGF in vitro were largely associated with astroglial cells. These findings suggest that IL-1 beta induction of bFGF contributes to the coactivation of these substances following various forms of insult to the CNS and initiates a cascade of trophic interactions that regulates processes of glial proliferation, neurotrophic factor expression, and neuroprotection. PMID- 7533233 TI - Regional expression of inducible heat shock protein-70 mRNA in the rat brain following administration of convulsant drugs. AB - Expression of inducible heat shock protein-70 mRNA (hsp-70 mRNA) was studied in the rat brain following systemic administration of different convulsant agents: an L-type voltage-dependent calcium channel agonist, (+/-)-BAY K 8644 (BAY-K); the excitotoxic glutamate agonists kainic acid and N-methyl-D-aspartic acid (NMDA); and the GABAA receptor complex antagonists pentylenetetrazole (PTZ) and lindane (gamma-hexaclorocyclohexane). BAY-K induced minimal hsp-70 mRNA expression in the hippocampus of convulsant rats, localized in the dentate gyrus and the pyramidal cell layer of Ammon's horn. Kainic acid treatment in rats, showing severe limbic convulsions, caused intense expression of hsp-70 mRNA and protein (HSP-70). Expression was localized in select cerebral regions, notably the pyramidal cell layer of the hippocampal CA3 field of Ammon's horn and the piriform cortex, and also the subicular complex and the amygdala, and, to a lesser extent, the entorhinal cortex, the pyramidal cell layer of CA1, several thalamic nuclei, and the parietal cortex. In contrast, systemic administration of NMDA, PTZ or lindane led to no detectable induction of hsp-70 mRNA in the rat brain, despite producing convulsions. Histological examination revealed cell injury only following kainic acid treatment. Damage was most apparent in the piriform and entorhinal cortices, pyramidal cell layer of the CA1 field, and cortical amygdaloid nuclei. BAY-K, NMDA, PTZ and lindane did not lead to any observable histopathological changes. These results show that convulsions of different aetiology do not inevitably induce hsp-70 mRNA expression or cell damage. Intense expression of hsp-70 mRNA was generally associated with regions that later showed variable degrees of nerve cell damage, although hsp-70 mRNA expression was not always predictive of subsequent cell death or survival. PMID- 7533234 TI - RNA transcription in axotomized dorsal root ganglion neurons. AB - Levels of RNA transcription were examined in L5 sensory ganglion neurons after unilateral crush injuries of the sciatic nerve using an in situ autoradiographic technique. Neuronal transcription increased in a biphasic pattern on the lesioned side within the first two weeks after injury. The timing and pattern of increases in transcription corresponded to previously reported increases RNA synthesis. The data demonstrate a dynamic regulation of RNA transcription in regenerating neuronal populations. This technique may prove to be useful in the study of factors controlling transcriptional activity in injured neurons. PMID- 7533235 TI - Nitric oxide synthase expression in single hippocampal neurons. AB - The presence of nitric oxide synthase (NOS) in CA1 pyramidal cells of the rat hippocampus was demonstrated by single-cell PCR. NOS-specific primers were used to amplify mRNA isolated from single hippocampal neurons. The sequence of the major amplification-product obtained was identical to that of the constitutively expressed brain-isoform of NOS. These results confirm immunocytochemical data that NOS is present in CA1, and, therefore, nitric oxide could function as a retrograde messenger in long-term potentiation. PMID- 7533236 TI - A flow cytometric technique for detection of DNA repair in mammalian cells. PMID- 7533237 TI - Measurement of micronuclei by flow cytometry. PMID- 7533238 TI - Sperm chromatin structure assay: DNA denaturability. PMID- 7533239 TI - Fine-needle cytopuncture and flow cytometry in the characterization of human tumors. PMID- 7533240 TI - Functional NK assays using flow cytometry. PMID- 7533241 TI - Antibodies to intermediate filament proteins as probes for multiparameter flow cytometry of human cancers. PMID- 7533242 TI - Analysis and sorting of hematopoietic stem cells from mouse bone marrow. PMID- 7533243 TI - Reticulocyte analysis and reticulocyte maturity index. PMID- 7533244 TI - Analysis of platelets by flow cytometry. PMID- 7533245 TI - Flow cytometry in malaria detection. PMID- 7533247 TI - Large-scale chromosome sorting. PMID- 7533248 TI - Cell sorting of biohazardous specimens for assay of immune function. PMID- 7533246 TI - Glutathione and cellular resistance to anti-cancer drugs. PMID- 7533249 TI - High-gradient magnetic cell sorting. PMID- 7533251 TI - Assay of cell resistance to ara-C. PMID- 7533250 TI - HIV infection: diagnosis and disease progression evaluation. PMID- 7533252 TI - Cell-cycle analysis of Saccharomyces cerevisiae. PMID- 7533253 TI - Staining and measurement of DNA in bacteria. PMID- 7533254 TI - Detection of specific microorganisms in environmental samples using flow cytometry. PMID- 7533255 TI - Strategies for flow cytometric analysis of marine microalgae and sponge cells. PMID- 7533256 TI - Detection of mRNA species by flow cytometry. PMID- 7533257 TI - Phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime. PMID- 7533258 TI - Spectra of fluorescent dyes used in flow cytometry. AB - Flow cytometry uses a relatively small set of dyes for immunochemistry and nucleic acid detection, most of which have been known and used reliably in flow cytometry for several years. These can usually be combined to make simultaneous two-color measurements of multiple cell-surface antigens and nucleic acid content. Because of the overlap of dye spectra and difficulties in finding dyes with substantial Stokes shifts that can be excited by the argon laser, simultaneous three-color or more detection can be more difficult. A basic knowledge of the factors that go into producing the fluorescent signal, including the spectra of dyes and their overlap, is necessary in planning multicolor experiments. By contrast, there have been a number of new fluorescent probes developed for detecting ions, membrane potential, metabolism, organelles, and other properties of living cells as well as for determining cell viability, proliferation, and cell tracking. So far most of these physiological probes are being used only for fundamental research rather than for cell classification. However, as research activity expands in this area, its diagnostic potential is likely to be increasingly appreciated. PMID- 7533259 TI - Pulmonary toxicity associated with bleomycin. PMID- 7533261 TI - Intracellular receptors. New wrinkles in retinoids. PMID- 7533260 TI - Induction of estrogen independence of ZR-75-1 human breast cancer cells by epigenetic alterations. AB - Antagonists of steroid hormones are clinically important in the management of breast cancer. However, the duration of response is limited due to the development of hormone-independent tumors in virtually all cases. In an attempt to obtain insight into the mechanisms underlying antiestrogen resistance, the consequences of epigenetic changes in gene expression were studied in vitro. Estrogen-dependent ZR-75-1 human breast cancer cells were treated with 5 azacytidine, an inhibitor of DNA methylation, and cultured in the absence of estradiol or in the presence of antiestrogens. Estrogen-independent cell colonies developed within 3 weeks at high frequency in 5-azacytidine-treated cultures (0.7 x 10(-3), in contrast to control cultures (< or = 10(-8). The derived cells (ZR/AZA) were resistant to 4-hydroxytamoxifen and ICI 164,384, independent of the selection protocol, but had lost the ability to grow anchorage-independent. Whereas expression of estrogen receptor, progesterone receptor, and pS2 were down regulated, expression of epidermal growth factor (EGF) receptor and HER2/neu were increased in ZR/AZA cells. In contrast to the stable altered expression patterns of estrogen receptor and EGF receptor, transient keratin 7 expression was observed. Transforming growth factor-alpha mRNA was identified in ZR-75-1 cells and ZR/AZA cells and EGF-like peptides were secreted in the culture medium. Proliferation of ZR/AZA cells could be partially inhibited with an EGF receptor blocking antibody. Presence of both growth factor receptors and possible ligands suggests the development of an autocrine growth mechanism. Our data show that epigenetic alterations of gene expression result in rapid progression of breast cancer cells to hormone independence. PMID- 7533262 TI - Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor. AB - Although pharmacological doses of retinoic acid (RA) have a wide variety of actions in vivo, experimental difficulties have prevented a definitive assignment of its physiological functions. We recently made a dominant-negative retinoic acid receptor (RAR) by a single amino-acid substitution which creates a dominant negative thyroid hormone receptor. The mutated RAR efficiently inhibited the endogenous activities of RARs (alpha, beta, gamma). Thus, targeted expression of the mutated receptor should reveal RA functions during organogenesis by blocking RA signalling in the tissues concerned. To address this possibility, we expressed the dominant-negative RAR in the epidermis, a potential target organ of RA. We report here that the resultant transgenic mice exhibited dramatic suppression of epidermal maturation, demonstrating the requirement of RA in normal skin development. PMID- 7533263 TI - Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells. AB - Cytotoxic CD4+ Th1-cells induce cell death by triggering a Fas-dependent apoptotic pathway. Potential targets include activated B cells, but it is not known whether the mode of B-cell stimulation influences susceptibility to Th1 mediated cytotoxicity. Here we report that CD40-ligand-stimulated B cells were extremely sensitive, whereas anti-IgM-stimulated B cells were resistant, to Fas mediated apoptosis. B cells stimulated by both CD40L and anti-IgM were not susceptible to cytolysis, demonstrating that anti-IgM-mediated protection is an active, dominant process. Resistance to Th1-mediated cytotoxicity was similarly observed in CD40L-stimulated 3-83 (anti-H-2Kk,b) transgenic B cells co-cultured with H-2Kk or H-2Kb (but not H-2Kd) splenocytes. These results indicate that B cells can participate in regulating their own destruction. Protection against Fas dependent apoptosis afforded by immunoglobulin-receptor engagement may constitute a fail-safe mechanism that eliminates bystander B cells activated by CD40L expressing T cells, but ensures survival of antigen-specific B cells. PMID- 7533264 TI - RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation. AB - Although polyadenylation has commonly been regarded as a special feature of eukaryotic messenger RNA, there are many reports of polyA tails on bacterial RNA (for example, refs 3-8). In Escherichia coli, adenylation mediated by the pcnB gene greatly accelerates decay of RNA I, an antisense repressor of replication of ColE1 type plasmids that resembles highly structured transfer RNA but shows the rapid turnover characteristic of mRNA. Here we report that both 3' adenylation and 5' phosphorylation affect the rate of digestion of RNA I by the 3' exonuclease, polynucleotide phosphorylase; conversely, mutation of the polynucleotide phosphorylase-encoding pnp gene affects ribonuclease acting at the 5' end. Together these findings indicate that enzymes attacking RNA I at its separate termini can interact functionally. Additionally, our discovery that adenylation-mediated degradation by polynucleotide phosphorylase imparts an mRNA like half-like to RNA I suggests a possible mechanism to account for the rapid decay of mRNA in E. coli. PMID- 7533265 TI - Effect of angiogenesis inhibitor TNP-470 on vascular formation in pituitary tumors induced by estrogen in rats. AB - TNP-470 (TNP), an analog of fumagillin, inhibits cell proliferation and tumorigenesis in estrogen-induced pituitary tumors in rats. This study found that treatment with TNP or TNP and bromocriptine suppressed vascular formation in these pituitary tumors more than bromocriptine alone. TNP had a greater suppressive effect on vascular formation in pituitary tumors than in normal pituitary gland. Laminin and basic fibroblast growth factor (FGF) were detected around new blood vessels in normal pituitary gland and pituitary tumor. TNP did not completely suppress laminin or basic FGF expression. The effect of TNP on pituitary tumor may involve inhibition of vascular formation by mechanisms mediated by other factors in addition to basic FGF. Treatment with bromocriptine and TNP acts indirectly and directly on vascular formation and may provide a useful chemotherapeutic modality for pituitary tumors. PMID- 7533266 TI - Experimental analysis of brain surface elastance in cats. AB - Brain surface elastance, defined as the pressure needed to compress the cortex 3 mm, was measured using the ophthalmodynamometer in six cats using three burr holes (frontal, parietal, and occipital) on each side. An intracranial mass was then used to compress the right side for 3 hours, and cardiac arrest was induced after the mass was removed. Elastance was measured four times: before insertion of the mass, 10 and 70 minutes after removal of the mass, and 60 minutes after cardiac arrest. The results showed that: brain surface elastance does not change between sides, but varies among regions with the parietal region having the highest elastance; elastance increases after compression by an intracranial mass, but not after cardiac arrest; and stiff brain tends to restore poorly. Elastance is apparently increased by the formation of edema. Measuring brain elastance may be useful in predicting brain restoration subsequent to removal of mass lesions. PMID- 7533268 TI - Follow-up study of patients with "unilateral" moyamoya disease. AB - Sixty-four patients with "unilateral" occlusive disease of the circle of Willis were studied to evaluate progression to bilateral disease. Seventeen patients developed bilateral lesions during a period of 1-7 years after the diagnosis of unilateral lesion. Most of these patients had ischemic attack as the initial episode and had repeated attacks before admission. Young children, mostly less than 10 years of age, tended to develop bilateral lesions within 1-2 years, but adults tended to have only a unilateral lesion. Children or young adults with unilateral occlusive lesion of the terminal portion of the internal carotid artery are likely to develop bilateral disease within 1-5 years. PMID- 7533267 TI - Symptomatic Chiari malformation and associated pathophysiology in pediatric and adult patients without myelodysplasia. AB - The clinical characteristics of eight pediatric and five adult patients with Chiari malformation were evaluated. Six pediatric and five adult patients had associated syringomyelia. All patients initially underwent a suboccipital craniectomy with upper cervical (C-1 and/or C-2) laminectomy and duraplasty, and/or shunting procedures. The clinical characteristics of the pediatric and adult groups were compared. The mean interval between onset of symptoms and operation was shorter in the pediatric group (3 yrs 6 mos) than in the adult group (7 yrs 1 mo). Pediatric patients without syringomyelia had the shortest mean interval of 1 year 8 months. Preoperatively, the clinical features were more severe in the adult patients than in the pediatric patients. Postoperatively, seven of eight pediatric patients improved and one stabilized, while two of five adult patients improved, one stabilized, and in two the disease continued to progress despite multiple corrective procedures. Cine magnetic resonance imaging revealed correction of the abnormal cerebrospinal fluid (CSF) flow at the craniovertebral junction and decreased to-and-fro movement in the syrinx after posterior fossa decompression, which were closely correlated with the improvement of clinical features in pediatric patients. However, adult patients required further procedures because of the multifactorial nature of the disease. Evaluation of abnormal CSF pathways at the craniovertebral junction is important for investigating the pathogenesis of Chiari malformation and associated syringomyelia. PMID- 7533269 TI - Unusual presentation of brain metastasis from hepatocellular carcinoma--two case reports. AB - Two unusual cases of brain metastasis from hepatocellular carcinoma (HCC) are described. A 15-year-old boy presented with intracerebral hemorrhage from brain metastasis from HCC, and died of rebleeding 1 month after surgery. Cerebral metastatic HCC in a child is quite rare, and has not previously been reported. A 65-year-old male with a 2-year history of treatment for HCC presented with a brain metastasis from HCC without intracerebral hematoma manifesting as gradual onset of headache. Brain metastases from HCC presenting without intracerebral hemorrhage are rare. PMID- 7533270 TI - Central nervous system metastases from Ewing's sarcoma--case report. AB - A 12-year-old boy presented with a rare case of intracranial metastasis 33 months after surgical treatment for Ewing's sarcoma of the rib. His chief complaints were headache, right hemiparesis, and disturbance of speech. Computed tomography revealed a large metastatic lesion in the left occipitoparietal region. The tumor was totally removed through an emergency operation. Seven months later the intracranial tumor recurred. Another operation was performed, but he showed spinal cord involvement at the T-10 level and died. The possibility of central nervous system metastasis should be considered in long-term follow-up survivors with Ewing's sarcoma. PMID- 7533271 TI - "Undifferentiated" cerebral primitive neuroectodermal tumor in a young adult- case report. AB - A 21-year-old female presented with a cerebral primitive neuroectodermal tumor (PNET) without either glial or neuronal differentiation, who has survived for 27 months without neurological deficit following total removal of the tumor, radiotherapy, and intensive chemotherapy. Both immunohistochemical and ultrastructural studies are important for the diagnosis of PNET. "Undifferentiated" PNET may be a distinct entity from common PNET which shows either glial or neuronal differentiation or both. PMID- 7533272 TI - Ruptured distal anterior inferior cerebellar artery aneurysm--case report. AB - A 62-year-old female presented with a distal anterior inferior cerebellar artery (AICA) aneurysm showing severe involvement of the facial and acoustic nerves manifesting as progressive left hearing disturbance and left facial nerve paresis. She was admitted to our hospital 2 months after the onset. A saccular aneurysm arising from the meatal loop of AICA was found between the facial and acoustic nerves which were damaged directly by the aneurysm. Magnetic resonance imaging demonstrated a vascular anomaly, differentiating this disorder from cerebellopontine angle tumor, and evidence of an old subarachnoid clot. These characteristics were extremely helpful for the differential diagnosis. Neck clipping and aneurysmectomy were performed to achieve decompression of the cranial nerves. One year and 2 months later full function of the facial nerve returned but the hearing disturbance persisted. PMID- 7533273 TI - Large cystic cavernous angioma of the cerebellum--case report. AB - A 40-year-old female presented with gait disturbance and dysarthria. Computed tomography revealed a large cystic tumor in the cerebellar vermis with a mural nodule located in the deepest portion of the cyst. The magnetic resonance (MR) imaging appearance suggested cavernous angioma. The solid nodule was completely removed through the suboccipital approach. The cyst was filled with transparent yellowish fluid which showed positive Froin's sign. The histological diagnosis of the mural nodule was cavernous angioma. The cyst wall consisted of gliosis and contained no angiomatous tissues. Postoperative MR imaging demonstrated that the nodule was totally removed and the cyst size was reduced. The neurological deficits improved postoperatively. The mechanism of formation of the large cyst was assumed to be repeated peritumoral hemorrhage. PMID- 7533274 TI - Cavernous sinus cavernoma treated with radiation therapy--case report. AB - A 71-year-old female presented with a syncopal attack. She underwent surgery for what appeared to be a meningioma. However, a small incision in the dura mater caused severe bleeding. Histological examination of the biopsy specimen showed sinus cavernoma with an incomplete pseudocapsule. The dura mater encapsulated the cavernous sinus cavernoma, explaining the severe bleeding from the dural incision. She was treated with Linac irradiation (40 Gy) which resulted in a decrease in tumor size. Radiation therapy is indicated for the treatment of cavernous sinus cavernoma, especially if associated with severe intraoperative bleeding. PMID- 7533275 TI - Evidence for a possible neuroanatomical basis for lexical processing of nouns and verbs. AB - Neuropsychological studies have revealed that brain-damaged patients may show impairments of specific word categories. This study reports the performance of three patients with impairments of the categories noun and verb. The first and second patients, with left frontal lobe atrophy, were impaired in naming and comprehension of verbs. The third patient, with striking atrophy of the left temporal lobe, was disproportionately impaired in naming and comprehension of nouns. These findings suggest that anatomically distinct neural systems in the temporal and frontal lobes of the dominant hemisphere might play a critical role in lexical processing of nouns and verbs, respectively. PMID- 7533276 TI - Selective attention and aphasia in adults: preliminary findings. AB - Thirteen patients with left-hemisphere stroke and history of aphasia and 13 normal controls were administered the covert orientation of visual attention task (COVAT). This task presents targets to the right or left of a central fixation point after a cue (84% of trials) or with no cue (16% of trials). Left-hemisphere damaged patients also received tests of language function at the time of the study. For targets presented 100 msec after cue onset, normal controls demonstrated equivalent responding for targets to the left and to the right of a central fixation point. Patients with left-hemisphere damage showed slower reaction times when responding to targets on the right as opposed to the left side of space when attention was first cued to the opposite side of space (invalid trials) or when attention was focused on a central fixation point (uncued trials), but they did not show slower reaction times on the right side when attention was first cued to the right (valid trials). For left-sided targets, no differences between valid, invalid, and uncued trials existed. Slower responding to right- as opposed to left-sided targets on invalid and uncued trials was correlated with impaired performance on six of seven language measures for patients with left-hemisphere damage. Implications for the relationship between language and selective attention systems in the left hemisphere are discussed. PMID- 7533277 TI - Involvement of clathrin light chains in the pathology of Pick's disease; implication for impairment of axonal transport. AB - Clathrin, which constitutes coated vesicles and plays important roles in neuronal functions, has been reported to be involved in the pathology of Alzheimer's disease. In the brains of the patients with Pick's disease, distribution of clathrin was immunohistochemically investigated using monoclonal antibodies binding to different epitopes of clathrin light chain a and b. All the antibodies intensely labeled Pick's body and some perikarya of neurons, indicating impairment of slow axonal transport b (SCb). Antibodies against neurofilament, kinesin and synaptophysin also labeled Pick's body. These observations suggested impairment of axonal transport in the brains with Pick's disease, and might contribute to elucidating the pathology of Pick's body forming. It is implied that common pathological processes might lie in Alzheimer's disease and Pick's disease. PMID- 7533278 TI - Conjugated deferoxamine reduces blood-brain barrier disruption in experimental optic neuritis. AB - The purpose of this paper was to investigate the role of deferoxamine (DFO) scavenging of hydroxyl radical (.OH) on disruption of the blood-brain barrier (BBB) and demyelination in experimental optic neuritis. Eighteen strain-13 guinea pigs were sensitized for experimental allergic encephalomyelitis. Nine animals received 100 mg/kg of hydroxyethyl starch-conjugated (HES) DFO by daily intraperitoneal injection commencing the day of antigenic sensitization. Nine paired litter mates received daily IP injections of HES. Serial fat-suppressed magnetic resonance imaging of the optic nerves was obtained with a T2 weighting (T2w) to evaluate demyelination and after intravascular administration of Gd-DTPA to evaluate BBB disruption. The intensity of Gd-DTPA enhancement and T2w signal of the optic nerves was quantitated 3, 7, 10 and 14 days after antigenic sensitization. Animals were then sacrificed and the optic nerves processed for light and transmission electron microscopy with ultracytochemical localization of endogenous hydrogen peroxide (H2O2) and immunogold colocalization of extravasated serum albumin. The area of the optic nerve head, intensity of toluidine blue staining, and the cellular infiltrate were digitized and quantitated. Administration of HES-DFO significantly reduced the intensity of Gd-DTPA enhancement in the optic nerves of HES-DFO-treated animals compared to paired control HES animals (p = 0.0236), with the mean difference between control and treated animals of 19.39. The difference in T2w signal was not significant (p = 0.39), with a mean difference between control and treated animals of -5.51. The intensity of toluidine blue staining of optic nerve specimens was slightly less with HES-DFO compared to untreated animals (mean pair difference 2.48), and the inflammatory infiltrate was reduced with HES-DFO compared to untreated animals (mean pair difference = 61.57); these differences were not statistically significant. In the optic nerve specimens of both groups cerium perhydroxide derived H2O2 reaction product was evident in a predominantly perivascular and perineural distribution. Immunogold-labeled serum albumin showed extravasation at foci of perivascular inflammation in both the presence and absence of H2O2 derived reaction product. Conjugated DFO reduces disruption of the BBB, as measured by Gd-DTPA enhancement, suggesting the .OH radical generated from perivascular H2O2 may play a role in alterations of vascular permeability in experimental optic neuritis. PMID- 7533279 TI - Viral type and CD1+ cells in HPV-induced lesions. AB - In vivo, HPV infection can display varying symptom complexes. At present 60 types of HPV are known, and some seem to be more efficient oncogenic agents than others. In particular, HPV 16 and 18 appear the types with the greatest oncogenic potential, being frequently found in cases of intraepithelial cervical neoplasia (CIN) and/or invasive carcinoma of the cervix. Many authors suggest that they might induce cell-mediated immunodeficiency into the lesions. This investigation was conducted on 15 women aged 17 to 40 (mean, 27.8). On the basis of cytological examination, colposcopy and biopsy, HPV-induced lesions were diagnosed. Biopsies were performed periodically for six months for HPV detection and typing. In addition, the cells of Langerhans were imaged by the indirect immunoperoxidase method. Variations in their number according to the HPV type involved were observed. PMID- 7533281 TI - Chromatographic and antigenic properties of Echinococcus granulosus hydatid cyst derived glycolipids. AB - The neutral and acidic fraction glycolipids of Echinococcus granulosus metacestode tissue compartments were isolated, defined by their chromatographic and antigenic properties, and assessed as to their efficacy as antigens in the serodiagnosis of human hepatic cystic and alveolar echinococcosis, and other helminthiases. Analyses were accomplished by thin-layer chromatography immunostaining and ELISA. The neutral glycolipid fraction's major carbohydrate epitope was the same as or very similar to that of Taenia crassiceps neutral glyco(sphingo)lipids, as represented by the 'neogala'-series core structure. The blood group-active, carbohydrate epitope P1 was expressed by a number of neutral fraction glycolipid component bands. The reverse-phase, thin-layer chromatography isolated neutral fraction glycolipid component, designated Ag1, was efficient in the serological discrimination of cystic echinococcosis medium to high-titred sera. Ag1 did not specifically discriminate low-titred sera, i.e., other human helminthiases. The detected sialic acid residues of the acidic fraction glycolipids, on enzymatic cleavage, were identified as N-acylneuraminic acid and terminal. The acidic fraction glycolipids exhibited the paradox of only chemically minor components being antigenic towards cystic and alveolar echinococcosis infection sera. The combined acidic fraction glycolipid components Ra and Rx were capable of serological discrimination between cystic echinococcosis, alveolar echinococcosis and other helminthiases. PMID- 7533282 TI - Age-dependency of serum isotype responses and antigen recognition in human whipworm (Trichuris trichiura) infection. AB - The present study examines the age-dependency of parasite-specific isotype responses and antigen recognition profiles of individuals within a Trichuris trichiura endemic community, in order to evaluate the significance of serum antibodies as determinants of observed age-related patterns of infection intensity. A high degree of individual heterogeneity is observed in isotype responses to separated T. trichiura antigens by Western blot. Recognition by IgG1 antibodies exhibits marked age-dependency. The age-profiles of IgG1 responses to selected antigens of 16-17 kDa and 90 kDa molecular weight reflect the age related changes in current infection intensity at the population level. Similarly, mean age patterns of IgG2 responses to a 90 kDa antigen, and mean IgG4 responses to a 16-17 kDa antigen reflect mean infection levels. IgG3 responses are negligible, and for methodological reasons, both IgE and IgM specificities are not presented. IgA responses to separated antigens of 16-17 kDa and 90 kDa, exhibit age-profiles which may suggest the development of an IgA-mediated acquired resistance to T. trichiura with age. IgA levels remain elevated throughout early adulthood, when infection intensity levels markedly decrease, supporting the hypothesis that IgA antibodies may be significant in generating the convex nature of the age-infection profile of T. trichiura. PMID- 7533280 TI - Mice immunized with a synthetic peptide construct corresponding to an epitope present on a Plasmodium falciparum antigen are protected against Plasmodium chabaudi challenge. AB - Inhibitory monoclonal antibody (MoAb) 8E7/55 recognizes a parasitophorous vacuole membrane (PVM) antigen in Plasmodium falciparum. Previous studies have identified the epitope, DNNLVSGP, recognized by the MoAb. A synthetic peptide containing this sequence was synthesized and coupled to diphtheria toxoid (DT) and was found capable of generating antibodies when used as an immunogen in mice which recognize the native antigen exp-1. In this study we demonstrate the ability of the MoAb and antisera generated against the peptide construct to recognize a 54 kD PVM antigen in Plasmodium chabaudi. The P. chabaudi antigen is synthesized in trophozoites and released to the surrounding culture media outside the parasitized erythrocyte. Mice immunized with the peptide conjugate are protected when challenged with a lethal strain of P. chabaudi. Protection in the mice correlated with the antibody titre prior to challenge. If the PVM antigen from P. chabaudi is a homologue of exp-1 from P. falciparum, then these experiments may provide a guide to the antibody titres required in human trials before antibody mediated protection could be expected. The discovery that a PVM localized antigen is secreted into the surrounding in vitro culture media provides us with a valuable model system for further investigation of protein trafficking pathways in malaria-infected erythrocytes. PMID- 7533284 TI - Trauma and the development of borderline personality disorder. AB - Prolonged and severe trauma, particularly trauma that occurs early in the life cycle, tends to result in a chronic inability to modulate emotions. When this occurs, people can mobilize a range of behaviors that are best understood as attempts at self-soothing. Some of these attempts include clinging and indiscriminate relationships with others in which old traumas are re-enacted over time, as well as more self-directed behaviors such as self-mutilation, eating disorders, and substance abuse. Usually, these behaviors will coexist. Patients with complicated trauma histories often repetitively attempt suicide or engage in chronic self-destructive behavior, and need to address issues of childhood trauma, neglect, and abandonment, both in the past and as re-experienced in current relationships. When treating these patients, therapists must anticipate that painful affects related to interpersonal safety, anger, and emotional needs may give rise to dissociative episodes, which may, in turn, be accompanied by increased self-destructive behavior. The therapy must clarify how current stresses are experienced as a return of past traumas and how small disruptions in present relationships are seen as a repetition of prior abandonment. As part of this, it is essential that the therapist provide validation and support, and avoid participating in a re-enactment of the trauma. Fear needs to be tamed in order for people to be able to think and be conscious of current needs. This bodily response of fear can be mitigated by safety of attachments, security of meaning schemes, and by a body whose reactions to environmental stress can be predicted and controlled. One of the great mysteries of the processing of traumatic experience is that as long as the trauma is experienced as speechless terror, the body continues to keep score and react to conditioned stimuli as a return of the trauma. When the mind is able to create symbolic representations of these past experiences, however, there often seems to be a taming of terror, a desomatization of experience. As Ducey and van der Kolk found in the Rorschachs of Vietnam veterans, patients were unresponsive to outside influences (good or bad) as long as they remained in a state of psychic numbing. Faced with intrusions of past trauma in their current emotional life, patients' initial sense of being overwhelmed was mastered only when a link between past trauma and current perceptions became understood.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533283 TI - Synthesis of tumour necrosis factor-alpha and interferons by mononuclear cells from Theileria annulata-infected cattle. AB - Bovine macrophage-derived tumour necrosis factor-alpha/cachectin (TNF-alpha) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naive and Theileria annulata-infected cattle were incubated in vitro with concanavalin A (Con-A) or bovine recombinant interferon gamma (Bo rIFN gamma). TNF-alpha production was also induced when adherent PBMC were cultured with T. annulata macroschizont-infected cells. In contrast, non-adherent PBMC from sublethally infected cattle produced interferon (IFN) when incubated with Hu rIL-2, Con-A, phytohaemagglutinin (PHA) or T. annulata macroschizont-infected cells growing as cell lines in vitro. Whilst PBMC from lethally infected cattle spontaneously produced IFN-gamma during advanced stages of infection, the sera of such animals contained type 1 IFN (alpha/beta). IFN was also produced by T. annulata macroschizont-infected cell lines maintained in vitro. This work suggests that cytokines serve as crucial links between proliferating Theileira infected cells and the characteristic clinical symptoms of tropical theileriosis. PMID- 7533287 TI - Maternal serum free beta hCG screening: results of studies including 480 cases of Down syndrome--problems with this approach. PMID- 7533285 TI - Serum PAPP-A and free beta-hCG are first-trimester screening markers for Down syndrome. AB - Serum measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta-human chorionic gonadotrophin (hCG) subunit were made in 13 women with Down syndrome (DS) pregnancies and six other women with fetal aneuploidy ascertained at chorionic villus sampling (CVS), as well as 89 women with contemporaneous normal control pregnancies. Median serum PAPP-A measurements (0.31 MOM, 95 per cent confidence interval (CI) 0.22-0.65 vs. normal 1.06, 95 per cent CI 0.89 1.20) were lower and free beta-hCG subunit measurements (1.13 MOM, 95 per cent CI 0.93-2.63 vs. normal 0.91, 95 per cent CI 0.79-1.03) were higher at statistically significant levels. Receiver operator characteristics (ROC) curves showed that the highest sensitivity for detection, 71.2 per cent (95 per cent CI 54.7-87.6 per cent), was for depressed PAPP-A levels; the combination of low serum PAPP-A levels, maternal age, and elevated free beta-hCG levels yielded a detection rate of 78.9 per cent (95 per cent CI 64.9-92.8 per cent) of the affected pregnancies at 8-12 weeks' gestation. PMID- 7533286 TI - MSAFP levels and oesophageal atresia. AB - This study was undertaken to evaluate the relationship between maternal serum alpha-fetoprotein (MSAFP) levels and oesophageal atresia (OA). OA occurred in 16 fetuses of mothers who had an MSAFP test in the study interval. The multiple of the median (MOM) value for MSAFP averaged 1.54 +/- 0.65 (range 0.5-2.9 MOM), which was significantly higher than the value seen in controls. The median MOM was 1.35. Using a cut-off of 2.5 MOM, the sensitivity of MSAFP for detecting OA was 19 per cent. Although OA should be considered in the differential diagnosis of an elevated MSAFP level, MSAFP cannot be considered an appropriate screening test for OA given the low sensitivity. PMID- 7533288 TI - Maternal serum free beta hCG screening. PMID- 7533290 TI - Theoretical predictions of folding pathways by using the proximity rule, with applications to bovine pancreatic trypsin inhibitor. AB - We propose a phenomenological theory that accounts for entropic effects due to loop formation to predict pathways in the kinetics of protein folding. The theory, the basis of which lies in multiple folding pathways and a three-stage kinetics, qualitatively reproduces most of the kinetic measurements in the refolding of bovine pancreatic trypsin inhibitor. The resulting pathways show that nonnative kinetic transients are involved in the productive routes leading to the formation of native intermediates. Our theory emphasizes the importance of the random origin of chain folding initiation structures in directing protein folding. PMID- 7533289 TI - Nitric oxide signaling to iron-regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts. AB - Iron-regulatory protein (IRP) is a master regulator of cellular iron homeostasis. Expression of several genes involved in iron uptake, storage, and utilization is regulated by binding of IRP to iron-responsive elements (IREs), structural motifs within the untranslated regions of their mRNAs. IRP-binding to IREs is controlled by cellular iron availability. Recent work revealed that nitric oxide (NO) can mimic the effect of iron chelation on IRP and on ferritin mRNA translation, whereas the stabilization of transferrin receptor mRNA following NO-mediated IRP activation could not be observed in gamma-interferon/lipopolysaccharide stimulated murine macrophages. In this study, we establish the function of NO as a signaling molecule to IRP and as a regulator of mRNA translation and stabilization. Fibroblasts with undetectable levels of endogenous NO synthase activity were stably transfected with a cDNA encoding murine macrophage inducible NO synthase. Synthesis of NO activates IRE binding, which in turn represses ferritin mRNA translation and stabilizes transferrin receptor mRNA against targeted degradation. Furthermore, iron starvation and NO release are shown to be independent signals to IRP. The posttranscriptional control of iron metabolism is thus intimately connected with the NO pathways. PMID- 7533291 TI - Angiogenesis: role of calcium-mediated signal transduction. AB - During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo. PMID- 7533292 TI - A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: partial purification and role in recognition of oxidatively damaged cells. AB - The binding and uptake of oxidatively modified low density lipoprotein (OxLDL) by mouse peritoneal macrophages occurs, in part, via the well characterized acetyl LDL receptor. However, several lines of evidence indicate that as much as 30-70% of the uptake can occur via a distinct receptor that recognizes OxLDL with a higher affinity than it recognizes acetyl LDL. We describe the partial purification and characterization of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that specifically binds OxLDL. This receptor is shown to be distinct from the acetyl LDL receptor as well as from two other macrophage proteins that also bind OxLDL--the Fc gamma RII receptor and CD36. We suggest that this OxLDL-binding membrane protein participates in uptake of OxLDL by murine macrophages and also represents a receptor responsible for macrophage binding and phagocytosis of oxidatively damaged cells. PMID- 7533293 TI - A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation. AB - The mutant mouse waved-2 (wa-2) is strikingly similar to transforming growth factor alpha-deficient mice generated by gene targeting in embryonic stem cells. We confirm that wa-2 is a point mutation (T-->G resulting in a valine-->glycine substitution at residue 743) in the gene encoding the epidermal growth factor (EGF) receptor. wa-2 fibroblastic cells lack high-affinity binding sites for EGF, and the rate of internalization of EGF is retarded. Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation. PMID- 7533294 TI - Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain. AB - The Src-homology (SH) 2 domain, found in a variety of proteins, has a binding site for phosphotyrosine-containing peptides. In adaptor proteins such as Grb2, the SH2 domain plays an important role in the assembly of signal transducer complexes. Many nonreceptor tyrosine kinases--e.g., Abl and Src--also contain SH2 domains. Without a functional SH2 domain, these tyrosine kinases retain catalytic activity but lose their biological function. This result suggests that the SH2 domain may be involved in the selection of biologically relevant substrates. We have previously shown that the carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl but not the Src tyrosine kinase. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated [Tyr(P)] CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2 or exchanging it with that of Src, which does not bind the Tyr(P)-CTD, abolished processivity and reduced the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity. PMID- 7533295 TI - Long-term culture system for selective growth of human B-cell progenitors. AB - We describe a simple reproducible system for enrichment and long-term culture of human B-cell progenitors. Enriched CD34+ cord blood mononuclear cells are seeded onto a murine stromal cell line to establish a biphasic culture system. These cultures are characterized by transient growth of myeloid cells followed by outgrowth of cells highly enriched for early B-cell progenitors. Cultures consisting of > 90% early B-lineage cells [expressing CD10, CD19, CD38, and CD45 but lacking CD20, CD22, CD23, and surface IgM] are maintained for > 12 weeks without growth factor addition. Cells remain predominantly germ line at the immunoglobulin locus and express only low levels of cytoplasmic mu chain, terminal deoxynucleotidyltransferase, and recombination-activating gene 1 product. They are unresponsive to the pre-B-cell growth factors interleukin 7 or stem cell factor, or both, suggesting that growth support is provided by a cross reactive murine stromal cell factor. Cultured B-cell progenitors are generated in large numbers ( > 10(8) cells from a typical cord blood specimen) suitable for use in biochemical analysis and gene-transfer studies. This system should be useful for study of normal and abnormal early human B-lymphopoiesis. PMID- 7533297 TI - Autocrine regulation of toxin synthesis by Staphylococcus aureus. AB - Staphylococcus aureus is a major human pathogen causing diseases which range from minor skin infection to endocarditis and toxic shock syndrome. The pathogenesis of S. aureus is due primarily to the production of toxic exoproteins, whose synthesis is controlled by a global regulatory system, agr. We show here that agr is autoinduced by a proteinaceous factor produced and secreted by the bacteria and that it is inhibited by a peptide produced by an exoprotein-deficient S. aureus mutant strain. The inhibitor, RIP, competes with the activator, RAP, and may be a mutational derivative. Our results suggest two possible approaches, independent of antibiotics, to the control of S. aureus infections. RIP may prove useful as a direct inhibitor of virulence and RAP as a vaccine against the expression of agr-induced virulence factors; either could interfere with the ability of the bacteria to establish and maintain an infection. PMID- 7533296 TI - Opposing mechanisms of regulation of a G-protein-coupled inward rectifier K+ channel in rat brain neurons. AB - In locus coeruleus neurons, substance P (SP) suppresses an inwardly rectifying K+ current via a pertussis toxin-insensitive guanine nucleotide binding protein (G protein; GnonPTX), whereas somatostatin (SOM) or [Met]enkephalin (MENK) enhances it via a pertussis toxin-sensitive G protein (GPTX). The interaction of the SP and the SOM (or MENK) effects was studied in cultured locus coeruleus neurons. In neurons loaded with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), application of SOM (or MENK) evoked a persistent increase in the inward rectifier K+ conductance. A subsequent application of SP suppressed this conductance to a level less than that before the SOM (or MENK) application; the final conductance level was independent of the magnitude of the SOM (or MENK) response. This suppression by SP was persistent, and a subsequent SOM (or MENK) application did not reverse it. When SP was applied to GTP[gamma S]-loaded cells first, subsequent SOM elicited only a small response. In GTP-loaded neurons, application of SP temporarily suppressed the subsequent SOM- (or MENK)-induced conductance increase. These results suggest that the same inward rectifier molecule that responds to an opening signal from GPTX also responds to a closing signal from GnonPTX. The closing signal is stronger than the opening signal. PMID- 7533298 TI - The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death. AB - We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA 1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death. Consistent with this possibility, we have found that the expression and intracellular localization of TIAR change dramatically during Fas mediated apoptosis. TIAR moves from the nucleus to the cytoplasm within 30 min of Fas ligation. Redistribution of TIAR precedes the onset of DNA fragmentation and is not a nonspecific consequence of nuclear disintegration. Cytoplasmic redistribution of TIAR is not observed during cellular activation triggered by mitogens such as concanavalin A or phytohemagglutinin. Our results suggest that cytoplasmic redistribution of TIAR may be a general feature of the apoptotic program. PMID- 7533299 TI - Hindrance of binding to class II major histocompatibility complex molecules by a single amino acid residue contiguous to a determinant leads to crypticity of the determinant as well as lack of response to the protein antigen. AB - The immune system has evolved the potential to respond to a wide variety of antigens, yet unresponsiveness to many foreign determinants is encountered frequently. Here, we report a lack of response to a particular determinant, hen egg lysozyme (HEL)-(46-61)-peptide (p46-61), in C57BL/6 (H-2b) mice, whereas a strong T-cell response to this determinant is obtained in major histocompatibility complex (MHC)-identical C3H.SW mice. However, (C3H.SW x C57BL/6)F1 mice respond well to p46-61, suggesting the absence of a p46-61 specific "hole" in the T-cell repertoire in C57BL/6 mice. We further show that p46-61 cannot bind the I-Ab class II MHC molecule, whereas p46-60 lacking Arg61 exhibits good binding and is immunogenic in both strains. Thus, the presence of the hindering residue, Arg61, renders p46-61, a dominant determinant in C3H.SW, into a silent, cryptic determinant in C57BL/6 mice. Upon i.p. immunization with HEL, no T-cell responses to either HEL or p46-61 could be demonstrated in spleens of HEL-primed C57BL/6 mice, whereas a predominant response to p46-61 and HEL was demonstrated in C3H.SW mice. Evidently, C57BL/6 mice differ from C3H.SW in their ability to process p46-61 into an actual I-Ab binding determinant, indicating a putative enzymatic defect in the C57BL/6 strain. Furthermore, our results suggest that the inability of C57BL/6 mice to respond in the spleen to HEL is based upon its failure to generate a dominant immunogenic determinant from HEL, coupled with its pattern of susceptibility to regulatory effects. PMID- 7533300 TI - Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. AB - The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance. PMID- 7533302 TI - [Symbolic process in intellectual activities of so-called psychotic adolescents]. AB - Based on his experience as psychopedagogue in a special education facility for adolescents with psychopathological disturbances, the author develops the idea that the learning difficulties cannot be reduced to intellectual deficits due to constitutional causes or traumatic experiences. The observation and analysis of the difficulties suggest that symbolization processes are deeply involved. This research is based on the assumption that symbolization processes of so-called psychotic adolescents can be assessed through various psychological techniques: the Borel-Maisonny language test, tests derived from those of Meljac on the knowledge and spontaneous use of numbers, and the EDEI (Differential scales of intellectual efficiency) by Perron-Borelli. Symbolization is defined in reference to theoretical approaches along three differentiated modes: the long option, the confused option and the short option. Different modalities of symbolization, the conceptual, the referential and the confused, are described and analyzed throughout the performances of the subjects, utilizing a differential and clinical method. PMID- 7533301 TI - Picornavirus inhibitors. AB - Picornaviruses are among the best understood animal viruses in molecular terms. A number of important human and animal pathogens are members of the Picornaviridae family. The genome organization, the different steps of picornavirus growth and numerous compounds that have been reported as inhibitors of picornavirus functions are reviewed. The picornavirus particles and several agents that interact with them have been solved at atomic resolution, leading to computer assisted drug design. Picornavirus inhibitors are useful in aiding a better understanding of picornavirus biology. In addition, some of them are promising therapeutic agents. Clinical efficacy of agents that bind to picornavirus particles has already been demonstrated. PMID- 7533303 TI - [Pathologic cognitive dysharmonies (nosographical pleasure or cognitive analysis)]. AB - In this article we question the validity of the notion of pathological cognitive dysharmonies as developed by B. Gibello on the basis of Piaget's conception of development. We wish to emphasize the following points: 1) that it constitutes in fact a deviation from Piagetian theory; 2) that recent research has lead to a new perspective on development which relativizes the so-called abnormality of cognitive discordances. The very idea of classifying intellectual disturbances is criticized. We also thoroughly analyze the notion of container and emphasize its potential danger when applied in a syncretic manner. PMID- 7533304 TI - High dose rate intraluminal radiotherapy for carcinoma of the bronchus: outcome of treatment of 406 patients. AB - In April 1988 the Christie Hospital started using the microSelectron-HDR machine to deliver intraluminal radiotherapy (ILT) to inoperable bronchial carcinomas causing symptoms due to endobronchial disease. Results of treatment in the first 406 patients with primary non-small-cell carcinoma are presented. Three main categories of patient were defined. Category 1 consisted of 324 patients (79.8%) who were previously unirradiated and received a single fraction of ILT as their primary treatment, mostly to a dose of 1500 cGy (76%) or 2000 cGy (23%) at 1 cm from the centre of the iridium-192 treatment source. The percentage of these patients whose symptoms or signs were improved at 6 weeks following ILT were as follows: stridor 92%, haemoptysis 88%, cough 62%, dyspnoea, 60%, pain, 50% and pulmonary collapse, 46%. Approximately two-thirds of these patients (67.3%) derived long lasting palliation and required no further treatment during their lifetime. The other third of patients needed subsequent treatment at some stage because of recurrence of their symptoms and in this situation external beam radiotherapy (EB) or a repeat ILT treatment was effectively utilised. Category 2 consisted of 65 patients (16%) who had previously received EB but required ILT when their tumour recurred. At 6 weeks post-ILT levels of symptom palliation were broadly similar to those obtained if ILT was used in previously unirradiated individuals, although the improvement was not so well sustained with time and only 7% showed improvement in pulmonary collapse at 6 weeks. Category 3 consisted of 17 patients (4.2%) in whom ILT was used concurrently with EB as a combined initial treatment. Similar levels of palliation were seen when compared with patients who received a single ILT treatment only. Overall, ILT was well tolerated in terms of early and late morbidity. In conclusion, the efficiency of a single ILT treatment in palliating symptoms due to endobronchial tumour in previously unirradiated individuals is comparable with that reported in series where treatment for advanced lung cancer combines a prolonged course of EB concurrently with several ILT treatments. PMID- 7533305 TI - [Radiotherapy of limited pulmonary microcytoma in current clinical practice]. AB - The use of thoracic irradiation in the treatment of "limited disease" small-cell lung cancer yields better local control and survival rates than chemotherapy alone, according to meta-analysis studies of randomized clinical trials. Outside experimental studies, however, the role radiotherapy can currently play in the management of this type of cancer is difficult to assess because treatment modalities and patient selection criteria differ greatly. We report on the treatment outcome obtained in the Radiotherapy Department of the University of Siena in a series of 86 patients with small-cell lung cancer consecutively referred, January 1986 to January 1992; after a thorough staging, 46 of them were diagnosed as having a "limited disease". A "sequential" chemo-radiotherapy combination was used: irradiation was delivered after the completion of the initial drug treatment. Twenty-four patients (52.5%) achieved a complete and 22 (47.5%) a partial objective remission after chemotherapy, with acceptable early toxicity rates and severity. Twenty-eight of them received irradiation according to the following selection criteria: objective remission after chemotherapy (19 of 24 complete responders, excluding those with initial pleural effusion or worsening medical status during chemotherapy) and initial large tumor bulk (9 of 22 patients in partial remission). The overall treatment outcome rate (median survival: 18 months, 2-year survival: 28%) is in agreement with that of similar previous studies; toxicity rates are also similar (2% of treatment-related deaths). Survival analysis, according to "performance status" score, chemotherapy schedule and the achievement of complete remission with the initial drug management, exhibited significant differences only relative to the latter parameter. Many recent clinical trials suggest that combined chemo-radiotherapy could improve these results: toxicity is however reported as heavy, with this approach. Some guidelines are here considered, which could make this combination reliable also for current clinical use. PMID- 7533306 TI - Intracellular Ca(2+)-Mg2+ interactions. AB - To ascertain possible interactions of intracellular Mg2+ and Ca2+, the concentration of intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) were experimentally increased within physiological ranges and it was measured by means of fura-2 and mag-fura-2 whether the increased concentration of one divalent cation affected the concentration of the other. A four-fold increase of [Ca2+]i in rat thymocytes by concanavalin A or thapsigargin had no significant effect on [Mg2+]i. Incubation of rat thymocytes in Na(+)-free medium increased [Mg2+]i from 0.35 to 0.7 mM. This increase in [Mg2+]i did not affect [Ca2+]i or the action of thapsigargin on [Ca2+]i. PMID- 7533307 TI - Low-molecular-weight protein competition for binding sites on renal brush border membranes. AB - Immunoglobulin light chains, beta 2-microglobulin, insulin, and lysozyme are low molecular-weight proteins (LMWP) shown to bind to renal brush border membranes. Competition among these proteins and the role of electrical charge in binding to brush border membranes have not been resolved. To investigate these factors, we performed displacement experiments with [125I]-labeled beta 2-microglobulin (pI = 5.6) using six species of LMWP over a pI range of 4.4-11.0. The inhibition constants, Ki, of these six competing ligands, kappa- and lambda-light chains, lysozyme, insulin, cytochrome c, and myoglobin, determined from the log displacement curves, ranged from 4 x 10(-5) to 8 x 10(-4) M. These experiments show marked cross-competition among LMWP for binding to brush border membranes. There was no correlation between Ki and pI indicating that the molecular structure is a more important determinant of LMWP binding to brush border membranes than net electrical charge. PMID- 7533308 TI - Effect of myeloma light chains on phosphate and glucose transport in renal proximal tubule cells. AB - Primary cultures of cells derived from the rat proximal tubule were exposed to up to 200 microM lambda- or kappa-light chain obtained from myeloma patients. Light chains inhibited the uptake of both phosphate and glucose by the cells while albumin had no effect. The half-maximal inhibitory concentration (IC50) of both the lambda- and kappa-light chains on phosphate transport were similar, 34 and 35 microM respectively. The IC50 of the kappa-light chain on glucose transport was 360 microM. The inhibitory effect of light chains was dose-dependent (r = 0.90, p < 0.01 for the lambda-light chain and r = 0.93, p < 0.001 for the kappa-light chain, on phosphate transport; and r = 0.93, p < 0.001 for glucose transport). Dixon and Line-weaver-Burk plot analyses were characteristic for noncompetitive inhibition. The inhibition constant 89 microM for phosphate uptake derived from the Dixon plot was similar to the IC50 calculated from the dose-response curves. These findings indicate that light chains, at concentrations found in the tubule fluid of a typical myeloma patient, are potent inhibitors of phosphate and glucose transport in proximal tubular cells, and that direct cell toxicity is a major mechanism of light chain nephrotoxicity. PMID- 7533309 TI - Evidence for an inhibitory effect of kallikrein on collecting duct bicarbonate secretion in rats and rabbits. AB - The luminal membrane of collecting duct cells, especially the intercalated cells, is normally exposed to active kallikrein. This is the consequence of the specific localization of this renal enzyme in the connecting tubule cells and its principal route of secretion being into the tubular lumen. It is conceivable that kallikrein acts downstream on a transporter involved in distal bicarbonate handling. To investigate this possibility, we estimated bicarbonate concentration and measured kallikrein amidolytic activity in urine fractions collected after a classical stop-flow experiment in rabbits. A highly significant inverse correlation was found between these parameters (r = -0.94, p < 0.001) in the peak kallikrein fractions. Neither sodium nor potassium concentration were correlated to kallikrein. This suggests that the physiological role of renal kallikrein may be to regulate extracellular fluid pH by inhibiting collecting duct bicarbonate secretion. To test the hypothesis that tubular fluid kallikrein activity and bicarbonate secretion are causally related, we developed a novel in vivo stop flow injection model ('orthograde stop-flow'). A hog-kallikrein containing solution (0.5 microgram/ml) was injected through the abdominal aorta into the renal tubular system of one kidney of barbiturate-anesthetized rats, while the renal blood supply was interrupted. The ureter was then occluded and renal blood perfusion reinitiated. After a 2-min contact time five 125-microliters urine fractions were collected. Bicarbonate secretion was clearly detected in the second and third fractions (i.e. those coming from the collecting ducts) of the control animals, which had received only the vehicle. There was no bicarbonate secretion peak in the corresponding urine fractions collected from kallikrein injected animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533311 TI - Abnormal gene expression of matrix metalloproteinases and their inhibitor in glomeruli from diabetic rats. AB - The steady state levels of mRNA encoding for metalloproteinase (MMP)-1, -2, -3, and -9 and tissue inhibitor of metalloproteinase (TIMP)-1 were examined in glomeruli at 4, 12, and 24 weeks after the injection of streptozocin (STZ) in rats. The mRNA levels for MMP-1 and MMP-3 decreased with age in STZ-induced diabetes. At 24 weeks after STZ injection, mRNA levels for MMP-1 and MMP-3 fell to 40% (p < 0.01) and 20% (p < 0.01), respectively, in the glomeruli of diabetic rats when compared with control rats. In contrast, mRNA levels for TIMP-1 increased significantly with age in the diabetic glomeruli and reached an 8-fold (p < 0.01) increased at 24 weeks after STZ injection. mRNA levels for MMP-2 were not altered in glomeruli from diabetic and control rats throughout the experimental period, whereas those for MMP-9 were not detected in glomeruli from either group of rats. Insulin treatment partially ameliorated the decrease in mRNA levels for MMP-1 and MMP-3 and the increase in those for TIMP-1 in the glomeruli of diabetic rats. These data indicate that abnormal gene regulation of MMPs and TIMP-1 in the glomeruli of diabetic rats may contribute to the progression of glomerular lesions and that hyperglycemia or insulin deficiency may be associated with abnormal MMPs and TIMP-1 gene regulation. PMID- 7533312 TI - Multifrequency impedance in the assessment of body water losses during dialysis. AB - Multifrequency bioelectrical impedance was used to predict changes in body water compartments during renal dialysis. Weight loss during dialysis was assumed to be water loss. Predicted total body water (TBW) from impedance after dialysis did not differ significantly from TBW determined by deuterium oxide dilution. However, the predicted change in TBW from bioelectrical impedance largely exceeded the observed weight (water) loss. The predicted change in extracellular water was slightly but significantly lower compared to the observed weight loss. The ratio of impedance at 1-100 kHz increased in all subjects during dialysis, and may be a simple tool to assess body water distribution. PMID- 7533310 TI - Ammonia production from hippurate by the rat kidney in vitro. AB - Hippurate is known to be synthesized from benzoate and glycine in the liver and kidney. It takes part in renal ammoniagenesis by modulating the activity of gamma glutamyl transpeptidase (gamma GT). Due to its chemical structure, however, hippurate might also serve as a substrate of renal ammoniagenesis. Hippurate may yield ammonia either having been cleaved by hippuricase or by Erlenmeyer's reaction after condensation with an aldehyde. In order to elucidate the possibility of hippurate being a substrate of renal ammoniagenesis, experiments were carried out on cortical kidney slices and on isolated tubular segments of the rat. The incubation medium (pH 7.1) was enriched with 10 mmol/l hippurate spiked with 15N-hippurate, some of the known competitive inhibitors of hippuricase, acivicin and different aldehydes. Factors known to affect hippuricase or gamma GT did not interfere with renal ammonia production. Glyceraldehyde (up to 1.0 mmol/l) but not glycerate had a stimulating effect, especially on the ammoniagenesis from hippurate. In normal rats fed a vegetarian diet, 1% of the added 15N moiety was found to be 15NH3. Renal 15NH3 production was significantly greater if, prior to the experiments, the animals were either acidotic or had a reduced renal mass or were fed animal proteins. These results indicate that hippurate may, to a certain extent, serve as substrate for ammoniagenesis. PMID- 7533313 TI - Laminin and fibronectin content of mouse glomerular and tubular basement membrane. AB - Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 +/- 0.001 micrograms/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 +/- 0.1 micrograms/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 +/- 0.8 and 7.7 +/- 1.0 micrograms/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components. PMID- 7533315 TI - Dibutyryl cyclic adenosine monophosphate stimulates the sodium pump in rabbit renal cortical tubules. AB - The elevation in oxygen consumption (QO2) observed following addition of the sodium ionophore nystatin in suspensions of rabbit renal proximal tubules was significantly increased by 1 mM dibutyryl cyclic adenosine monophosphate (db cAMP). The QO2 after subsequent addition of strophanthidin to block the sodium pump was unaffected by db-cAMP. However, 10 microM forskolin in the presence of 100 microM IBMX had no significant effect on the QO2 observed following addition of either nystatin or strophanthidin. Nevertheless, we can conclude that db-cAMP does stimulate the sodium pump activity independently of sodium transport mechanisms in the rabbit renal proximal tubule. PMID- 7533314 TI - Characterization and control of expression of cell surface alkaline phosphodiesterase I activity in rat mesangial glomerular cells. AB - Membrane-bound nucleotidases and phosphodiesterases are critical regulators of extracellular nucleic acid processing. We previously demonstrated that mesangial cell 5'-nucleotidase was an ectoenzyme, the expression of which was stimulated by macrophage-secreted products. We show in the present study that rat mesangial cell alkaline phosphodiesterase I is also an ectoenzyme characterized by a Km value of 0.41 mM and a Vmax of 20.8 nmol min-1 mg-1. Treatment of mesangial cells by dexamethasone increased alkaline phosphodiesterase I activity in a dose- and time-dependent manner. Maximal increase (x1.5) occurred after treatment with 1 microM dexamethasone for 5 days. Cycloheximide and RU 38486, a glucocorticoid receptor antagonist, suppressed the dexamethasone-induced increase in alkaline phosphodiesterase I activity. 5'-Nucleotidase activity was not modified by dexamethasone under similar conditions of study. In contrast with 5' nucleotidase, alkaline phosphodiesterase I expression remained unchanged in the presence of macrophage-conditioned medium or during cocultures of mesangial cells with macrophages. Interleukin-1, tumor necrosis factor, cyclic adenosine monophosphate and adenosine analogues also activated 5'-nucleotidase whereas they were inactive on alkaline phosphodiesterase I. These results suggest that extracellular DNA trapped in the mesangial area of the glomerular capillaries may be processed in part at the cell surface by alkaline phosphodiesterase I and that such an event may be regulated by glucocorticoids. They also show that alkaline phosphodiesterase I and 5'-nucleotidase obey a different regulation. PMID- 7533316 TI - Tubuloglomerular feedback in rats with chronic partial bilateral ureteral obstruction. AB - Rats with chronic partial unilateral ureteral obstruction (UUO) have a paradoxical resetting of the tubuloglomerular feedback (TGF) mechanism. During extracellular fluid volume expansion (VE) in control animals, a decrease in TGF sensitivity is normally noted. In rats with partial UUO, however, TGF sensitivity in the obstructed kidney was increased, associated with a relative reduction in single nephron glomerular filtration rate. In the present study, we examined the tubular and interstitial pressures, whole kidney function and the TGF system in rats with chronic partial bilateral ureteral obstruction. The rats were divided into preferentially ipsilaterally and preferentially contralaterally obstructed, as judged by pelvic volume. Measurements were performed both during hydropenia and during VE. During hydropenia TGF characteristics were the same in the 2 groups. During VE, however, TGF sensitivity was unchanged in the most obstructed kidneys (ipsilateral), while if the obstruction was preferentially contralateral, TGF-sensitivity decreased. This opposite change in TGF sensitivity resulted in higher electrolyte and water excretion from the least obstructed kidney. The change in TGF sensitivity was not correlated to renal interstitial pressure. IN CONCLUSION: rats with moderate chronic partial bilateral ureteral obstruction have an almost preserved function with regard to renal hemodynamics and excretion during hydropenia. During volume expansion, however, different responses were found in the least and most obstructed kidneys, with a decreased sensitivity of TGF in the least obstructed kidney, while TGF sensitivity was unchanged in the most obstructed kidney. PMID- 7533318 TI - Prostaglandin-independent decrease of sheep ureter contractility induced by bradykinin. AB - The mechanisms that mediate the actions of bradykinin on ureteral motility are poorly defined and mediation via prostaglandins has not been examined. Therefore, the effects of bradykinin on contractility and the possible mediator role of prostaglandins have been investigated in sheep ureter. At the concentrations of 10(-8), 10(-7) and 10(-6) M, bradykinin elicited marked reductions in contractile force. When ureteral strips were treated separately with 10(-6) M indomethacin, 2 x 10(-6) M sodium salicylate and 10(-5) M aspirin, each drug produced a significant decrease in contractile force. In strips in which prostaglandin synthesis was inhibited by the above concentrations of indomethacin, sodium salicylate and aspirin, 10(-7) M bradykinin significantly decreased the contractility. From these data, we concluded that in ureter bradykinin decreases contractility via a mechanism not involving prostaglandin generation. PMID- 7533317 TI - Influence of captopril on 24-hour balances and the diurnal patterns of urinary output, blood pressure, aldosterone and atrial natriuretic peptide in conscious dogs. AB - The diurnal time course of urinary flow rate (UV), urinary sodium (UNaV), and potassium (UKV) excretion, and of hormones such as atrial natriuretic peptide (ANP) and aldosterone, was investigated during 5 days of continuous captopril infusion (15 micrograms.kg body weight-1.min-1) in 4 conscious dogs on a high sodium diet (14.5 mmol Na.kg body weight-1.24 h-1). All food and water was given once daily at 8.30 a.m. On the control day and on days 1, 3, and 5 of captopril infusion, urine was collected by an automated system at 20-min intervals over 24 h, blood was taken every 4 h. Mean arterial blood pressure (MABP) and heart rate were evaluated as 5-min averages. Time courses of UNaV, UV, and UKV were compared with the individual control day without captopril. With captopril, 24-hour balances for Na and H2O were slightly negative, while the K balance was slightly positive for 2-3 days. Thereafter, all 24-hour balances were restored. MABP continued to decrease even after Na and water intake and output had come into balance again. Captopril treatment changed the diurnal excretion pattern for UNaV and UV characteristically. In the postprandial period until 5 p.m., less Na and urine were excreted than on the control day, whereas during the evening and night more Na and urine were excreted. The changes in the excretion pattern persisted for the entire observation period. The results indicate that disturbances in the regulating systems, induced by converting-enzyme blockade, bring about complex reactions of, e.g., MABP, ANP and aldosterone that finally restore Na and water 24-hour input/output balances. PMID- 7533320 TI - Bladder ultrasonography. PMID- 7533319 TI - Correlation of prostate-specific antigen and ultrasonography in the evaluation of patients with carcinoma of the prostate. PMID- 7533321 TI - Ultrasonography of the kidney. PMID- 7533322 TI - [Angiogenesis--mechanisms and therapeutic approaches]. PMID- 7533323 TI - [Cat scratch (?) disease]. AB - The case of a 24 year old female student who developed typical cat scratch disease following an insect sting invited to review the literature. Over the last two years, Rocalimaea henselae could be identified and is now considered to be responsible for the majority of infected cases. It is related to Rochalimaea quintana that provokes trench fever and to Bartonella bacilliformis, known as the inciting agent of the verruca peruana and of Oroya fever. Recently, it has been proposed to integrate Rochalimaea and Bartonella into the family of Bartonellaceae. The diverse and fascinating array of clinical syndromes caused by R. henselae as well as their treatment are described. Capability to affect organs other than skin and lymph nodes is primarily but not exclusively associated with immunodeficiencies. In countries like Switzerland little information on these clinical pictures is available. However, it is important to recognize the typical symptoms and signs in order to initiate the appropriate and necessary examinations. PMID- 7533324 TI - [Lectin and ion channel]. PMID- 7533325 TI - [Effect of acidic fibroblast growth factor on neuronal cells in vitro]. PMID- 7533326 TI - The Fas death factor. AB - Fas ligand (FasL), a cell surface molecule belonging to the tumor necrosis factor family, binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells. Various cells express Fas, whereas FasL is expressed predominantly in activated T cells. In the immune system, Fas and FasL are involved in down-regulation of immune reactions as well as in T cell-mediated cytotoxicity. Malfunction of the Fas system causes lymphoproliferative disorders and accelerates autoimmune diseases, whereas its exacerbation may cause tissue destruction. PMID- 7533328 TI - Type II cryoglobulinemia. PMID- 7533327 TI - Involvement of CRAF1, a relative of TRAF, in CD40 signaling. AB - CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor alpha (TNF-alpha) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-alpha receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes. PMID- 7533329 TI - [Changes in the functional characteristics of acetylcholine activated channels of hippocampal neurons at different periods of culture]. AB - Patch-clamp technique was used to examine activation of single acetylcholine receptor channels of hippocampal neurons of neonatal rat at different periods of culture. The conductance values showed substantial variation from patch to patch. Some of channels have the same conductance but are kinetically different. Changes in the electrophysiological behaviour of ACh-evoked channels were dependent on the duration of culture. In the earlier period (1-2 days), the 20-pS state was predominant. Isolated short openings were much more common with mean open times usually less than 2.0 ms. In the late period of culture (18-21 days), the 31-pS state was predominant. The channel openings displays either as single events (tau o1, 0.35 ms; tau o2, 1.29 ms) or as bursts of events (tau b1, 1.15 ms; tau b2, 9.6 ms) in different patches. However, 20-pS state in the late period of culture could also be observed mostly as constituting events during the long burst. PMID- 7533330 TI - [Inhibition of voltage-gated K+, Na+ and Ca2+ currents in neuroblastoma x glioma hybrid cells by paeonol]. AB - The effects of Paeonol (25 micrograms/ml to 400 micrograms/ml) on the membrane currents of NG108-15 cells were observed by means of whole cell voltage-clamp technique. The results showed that PA inhibited delayed outward K+ current (IK), and to a less extend, that the Na+ current (INa). Two Ca2+ currents (T and L type) were also inhibited significantly. All of the effects mentioned above were dose-dependent and reversible. PMID- 7533331 TI - [Inhibitory effect of SP on GABA-activated currents in freshly isolated rat DRG neurons]. AB - The whole-cell patch-clamp technique was used to record GABA-activated currents in freshly isolated DRG neurons. It was found that application of SP (10(-7)-10( 5) mol/L) could induce concentration dependent inward currents in some cells (4/26), while no such current was detectable in the other neurons. However, preapplication of SP could inhibit the inward currents mediated by GABAA receptor (18/22). The inhibitory effect of SP was concentration dependent and reversible, and the desensitization of GABA-activated current could also be enhanced by SP. PMID- 7533332 TI - Endoscopic patch closure of malignant esophagotracheal fistula using Histoacryl glue. AB - Esophagotracheal fistula is one sequel of advanced carcinoma of the esophagus. Although the pneumatic cuffed tracheoesophageal fistula stent provides satisfactory palliation for fistulas, high fistulas remain a major problem. We report a case of a 64-year-old gentleman with a high fistula that was treated successfully with endoscopic patch closure using Histoacryl glue. PMID- 7533333 TI - Effects of pentafraction and hetastarch plasma expansion on lung and soft tissue transvascular fluid filtration. AB - BACKGROUND: Hetastarch and pentafraction are high molecular weight starch solutions designed to augment plasma oncotic pressure. Although clinical utilization of hetastarch has been limited by reported coagulation abnormalities, pentafraction is a newer derivative that appears to have few adverse hemostatic effects. We examined the ability of pentafraction to modulate lung and soft tissue transvascular fluid filtration under hypoproteinemic conditions compared with hetastarch and Ringer's lactate (LR). METHODS: Awake, protein-depleted sheep (n = 19) were prepared with lung and soft tissue lymph fistulas, and comparable infusions of 5% pentafraction (n = 6), 6% hetastarch (n = 6), or LR (n = 7) were administered. Plasma and lymph samples were collected during 24-hour period to determine changes in protein concentrations, plasma-to-lymph oncotic gradients, and lung (QL) and soft tissue (QS) lymph flows. RESULTS: QL and QS rose nearly twofold after protein depletion alone. LR infusion increased QL and QS to 8.7 +/- 1.7 and 3.1 +/- 0.6 times normoproteinemic baseline, respectively (p < 0.05). In contrast, hetastarch and pentafraction infusion limited the increase in QL to 4.2 +/- 1.1 and 4.0 +/- 0.8 times normoproteinemic baseline, respectively (p < 0.05 versus LR) and did not significantly increase QS. Hetastarch and pentafraction infusions increase plasma oncotic pressure by nearly 6 mm Hg, which significantly widened the plasma-to-lymph oncotic pressure gradients above preinfusion baseline by 4.7 +/- 0.7 and 3.4 +/- 0.4 mm Hg in lung and 4.6 +/- 0.7 and 3.2 +/- 0.4 mm Hg in soft tissue, respectively (p < 0.05). CONCLUSIONS: Both hetastarch and pentafraction limit transvascular fluid filtration under hypoproteinemic conditions by augmenting plasma oncotic pressure and the plasma-to-lymph oncotic pressure gradient. Because of fewer adverse hemostatic effects pentafraction may be an improvement over current therapies in critical care fluid management. PMID- 7533334 TI - Fibreoptic bronchoscopic electrosurgery under local anaesthesia for rapid palliation in patients with central airway malignancies: a preliminary report. AB - BACKGROUND: Obstruction of a major airway by tumour causes serious morbidity. There is still scope for a widely applicable, simple and effective treatment to provide rapid palliation. METHODS: A fibreoptic bronchoscope prototype with an insulated inner sheath was used under local anaesthesia in 17 patients with locally advanced tracheobronchial malignancies. An insulated flexible electro surgery probe was used to coagulate intraluminal tumour mass using standard electrosurgery equipment. RESULTS: Immediate reopening of the airway was obtained in 15 of the 17 patients. Two appeared to have extraluminal disease. Eleven patients had an obvious bronchoscopic response in whom a > 75% reopening of the normal airway diameter was achieved. Eight patients had subjective improvement of their dyspnoea, but only in four cases was there an objective improvement in physiological parameters. Haemoptysis resolved in four. There were no deaths resulting from treatment. Minor bleeding occurred in one patient and an aspiration pneumonia occurred in one. Three patients received additional treatment. CONCLUSIONS: Fibreoptic bronchoscopic electrosurgery is a simple technique for rapid palliation and immediate tumour debulking in patients with central tracheobronchial tumours. Further work is needed to compare its efficacy with other techniques. PMID- 7533336 TI - Amyloid beta-protein precursor-rich platelet microparticles in thrombotic disease. AB - We investigated the association of amyloid beta-protein precursor (APP) and platelet derived microparticles in 20 normal controls and 91 patients with various diseases causing a thrombotic tendency. Compared with the controls, the mean percentage of APP-positive microparticles was significantly greater in the patients with cerebral infarction (39.1 +/- 17.7%, p < 0.001), diabetes (31.1 +/- 12.6%, p < 0.001), and uremia (30.1 +/- 14.7%, p < 0.01), but not in those with hypertension (8.2 +/- 6.3%, p = NS). Sixteen patients with cerebral infarction, 20 with diabetes, and 11 with uremia had microparticles with very high APP levels. In normal controls, 7.2 +/- 3.7% of the microparticles were positive for P-selectin, while the percentage in cerebral infarction, diabetes, uremia, and hypertension was respectively 43.5 +/- 15.1%, 40.0 +/- 12.8%, 31.8 +/- 12.2%, and 11.6 +/- 7.3%. There was a significant correlation between P-selectin and APP positivity of microparticles. Our results suggest that microparticle APP may have a regulatory influence on coagulation abnormalities. PMID- 7533337 TI - Inhibition of platelet recruitment to arterial lesions by predeposition of platelets containing encapsulated iloprost. AB - Drugs can be encapsulated within blood platelets by reversible electroporation and can be haemostatically targeted to vessel wall injury sites. Initial studies with iloprost-loaded pig platelets and pig aorta tunica media in perfusion circuits are presented. After autologous reconstitution into blood, no significant difference was observed in the deposition of 111Indium labelled sham loaded and untreated platelets onto the tunica media during perfusion under low and high shear conditions. In paired experiments (n = 10 pairs), the deposition of iloprost-loaded platelets was significantly lower (mean 61%) after 5 min perfusion than the deposition from blood containing sham-loaded (control) platelets. A similar significant reduction (mean 54%) was seen after 10 min perfusion. Pre-perfusion of iloprost-loaded platelets for 10 min under low shear conditions (212/s), followed by 5 min perfusion of 111Indium labelled normal platelets, significantly reduced the secondary platelet deposition (p < 0.01) when compared with the deposition seen when control untreated platelets were preperfused. Significant differences (p < 0.001) in secondary deposition were also observed when primary and secondary platelet perfusions were made under high shear (1690/s). Histology of the tunica media segments post perfusion, supported the inhibitory effect of predeposited iloprost-loaded platelets on secondary platelet recruitment. By exploiting their natural haemostatic propensity, drug loaded platelets can be targeted to vessel wall injury sites. Appropriate drugs could be packaged that may passivate the carrier platelets at the lesion inhibiting thrombus formation or they may act as a depot for sustained drug release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533338 TI - A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14). AB - TrJ14 is a cytotoxic human IgG1 lambda hybridoma mAb that recognized a novel HLA A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the alpha 1-domain helix. These amino acids were shared exclusively by all the identified TrJ14+ alleles. PMID- 7533339 TI - In vitro inhibitory effects of 16-methyl-substituted steroids on 5 alpha reductase in rat and human prostates. AB - The inhibitory effects (IC50) of 16-methyl steroids on 5 alpha-reductase were studied. The in vitro experiments were carried out with homogenates of rat and human prostates. The investigated 16-methyl steroids were found to be weak inhibitors. In comparison with the known 5 alpha-reductase inhibitor 4-MA (17 beta-N, N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), the relative IC50 values of the studied compounds are 4.7 times or more greater than 4-MA in human prostate and 23.5 times or more greater than 4-MA in rat prostate. The IC50 values increase in the sequence 16 alpha-, 16 beta- and 16,16-dimethyl derivatives. In human prostate homogenates IC50 varies between 0.6 and 120, while in rat it ranges from 1.6 to 1000 microM. This shows that the enzyme of the human prostate is more sensitive than that of the rat prostate to the methyl substituted compounds. PMID- 7533335 TI - Macroaggregation of platelets in plasma, as distinct from microaggregation in whole blood (and plasma), as determined using optical aggregometry and platelet counting respectively, is specifically impaired following cardiopulmonary bypass in man. AB - We determined changes in platelet aggregability following cardiopulmonary bypass, using optical aggregometry to assess macroaggregation in platelet-rich plasma (PRP), and platelet counting to assess microaggregation both in whole blood and PRP. Hirudin was used as the anticoagulant to maintain normocalcaemia. Microaggregation (%, median and interquartile range) in blood stirred with collagen (0.6 micrograms/ml) was only marginally impaired following bypass (91 [88, 93] at 10 min postbypass v 95 (92, 96] prebypass; n = 22), whereas macroaggregation (amplitude of response; cm) in PRP stirred with collagen (1.0 micrograms/ml) was markedly impaired (9.5 [8.0, 10.8], n = 41 v 13.4 [12.7, 14.3], n = 10; p < 0.0001). However, in PRP, despite impairment of macroaggregation (9.1 [8.5, 10.1], n = 12), microaggregation was near-maximal (93 [91, 94]), as in whole blood stirred with collagen. In contrast, in aspirin treated patients (n = 14), both collagen-induced microaggregation in whole blood (49 [47, 52]) and macroaggregation in PRP (5.1 [3.8, 6.6]) were more markedly impaired, compared with control (both p < 0.001). Similarly, in PRP, macroaggregation with ristocetin (1.5 mg/ml) was also impaired following bypass (9.4 [7.2, 10.7], n = 38 v 12.4 [10.0, 13.4]; p < 0.0002, n = 20), but as found with collagen, despite impairment of macroaggregation (7.2 [3.5, 10.9], n = 12), microaggregation was again near-maximal (96 [93, 97]). The response to ristocetin was more markedly impared after bypass in succinylated gelatin (Gelofusine) treated patients (5.6 [2.8, 8.6], n = 17; p < 0.005 v control), whereas the response to collagen was little different (9.3 v 9.5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533340 TI - Characterization of ALVA-41 cells, a new human prostatic cancer cell line. AB - The ALVA-41 cell line was derived from a bony metastasis from a human prostatic carcinoma. The line has a number of distinct, advantageous properties that should make it useful as a tool for the study of prostate cancer. It grows rapidly and is easy to work with. It has receptors for androgens and glucocorticoids but not for estrogens. Its growth is enhanced by physiological concentrations of dihydrotestosterone. It does not secrete prostate specific antigen, but does secrete prostatic acid phosphatase. Further, the secretion of prostatic acid phosphatase is enhanced by dihydrotestosterone. PMID- 7533341 TI - Changes in peripheral blood CD5 (Bla) B-cell populations and autoantibodies following blood transfusion. AB - BACKGROUND: CD5 B cells and the natural autoantibodies they produce play a role in antigen presentation, tolerance induction, and maintenance of an idiotypic immune network. The effects of transfusion on autoantibodies and peripheral blood CD5 B cells were studied. STUDY DESIGN AND METHODS: Eight previously transfused patients with sickle cell anemia and five patients who underwent orthopedic surgical procedures with transfusion were enrolled in the study. Patients in both groups received 1 to 2 units of allogeneic packed red cells. Ten untransfused healthy adults and five patients who underwent orthopedic surgery without transfusion were enrolled as controls. Peripheral blood CD5 B cells, serum levels of IgM, antinuclear antibodies, rheumatoid factor, and anticardiolipin IgM were quantitated either at the beginning of the study (baseline sample), before transfusion, or before surgery and either at 1-, 2-, 4-, 6-, and 8-week intervals after transfusion, after surgery, or after the baseline sample was obtained. RESULTS: IgM levels and the absolute number of B cells that coexpressed CD5 rose to twice pretransfusion levels in six of eight transfused sickle cell anemia patients and in four of five transfused orthopedic surgery patients. No comparable increases in CD5 B cells were noted in untransfused controls. Preexisting rheumatoid factor and antinuclear antibody levels increased in four of five transfused orthopedic surgery patients. One sickle cell anemia patient developed anti-Fya despite receiving Fya-negative blood. Increasing titers of anti-Fya paralleled the increases in IgM and CD5 B cells after transfusion. One patient who developed a positive direct antiglobulin test after transfusion had large increases in serum anticardiolipin IgM. Anticardiolipin IgM was subsequently eluted from direct antiglobulin test-positive red cells obtained after transfusion. Antibodies with anti-Fya-like activity and anticardiolipin IgM were produced in vitro by CD5 B cells and not by conventional CD5-negative B cells. CONCLUSION: An association was found between transfusion-induced increases in CD5 B cells and increased autoantibody production. These data may have implications for immunologic intervention to prevent the induction of red cell antibodies and other changes in the immune system caused by exposure to foreign antigens via blood transfusion. PMID- 7533342 TI - Location of WESb on decay-accelerating factor. PMID- 7533344 TI - An increased incidence of Epstein-Barr virus infection and lymphoproliferative disorder in young children on FK506 after liver transplantation. AB - The incidence of Epstein-Barr virus (EBV) infection and lymphoproliferative disorder (LPD) was determined in a pediatric liver transplant population consisting of 51 children treated with FK506 and 91 treated with cyclosporine. The incidence of symptomatic EBV infection was 21.9% (23 of 105 cases) in children < 5 yr old and 10.8% (4 of 37 cases) in children 5 to 17 yr old as compared with 2.7% (9 of 323 cases) in adults (P < 0.0001). In the under 5 yr old group on cyclosporine, the incidences of EBV infection and LPD were 9 of 68 (13.2%) and 2 of 68 children, (2.9%), respectively. In contrast, in children under 5 yr old group on FK506, the incidences of EBV infection and LPD in the FK506 group were 14 of 37 (37.8%) and 7 of 37 children (18.9%), respectively. The difference between these two groups was statistically significant (P < 0.02). There were no cases of LPD in the 5-17 yr-old children on either cyclosporine (n = 23) or FK506 (n = 14). The incidence of EBV infections in the 5 to 17 yr age group, 17.4% on cyclosporine and 0% on FK506, was less than for the younger children on FK506 (37.8%). A total of 39% (9 of 23) of children under 5 yr old who had symptomatic EBV infections developed LPD, and 44% (4 of 9) with LPD died. The higher incidence of EBV infections and LPD in the younger children treated with FK506 was probably related to a greater intensity of immunosuppression for patients on FK506 than those on cyclosporine. PMID- 7533343 TI - A prospective randomized trial of FK506-based immunosuppression after renal transplantation. AB - A group of 204 adult patients was entered into a prospective, randomized trial comparing FK506/prednisone with FK506/azathioprine/prednisone after renal transplantation between August 1, 1991 and October 11, 1992. The purpose of the study was to see if the addition of azathioprine would reduce the incidence of rejection and improve graft survival. The recipient population was unselected, with 61 (30%) patients undergoing retransplantation, 37 (18%) having a panel reactive antibody greater than 40%, and 33 (16%) over 60 years of age. The mean recipient age was 43.8 +/- 13.7 years (range 17.6-78). The mean donor age was 34.0 +/- 20.1 years (range 0.3-75); 13% of the cadaveric kidneys were from pediatric donors less than 3 years of age and were transplanted en bloc. The mean cold ischemia time was 31.4 +/- 8.4 hr. Living donors were the source of 13% of the kidneys. The mean follow-up was 22 +/- 4 months (range 12-29). Overall one year actual patient survival was 94%. Overall one-year actual graft survival was 87%. Patients starting on double therapy had a one-year actual patient survival of 96% and a one-year actual graft survival of 92%. Patients starting on triple therapy had a one-year actual patient survival of 91% (P = ns compared with double therapy), and a one-year actual graft survival of 82% (P < 0.02, compared with double therapy). Overall results with first cadaver transplants included a one-year actual patient survival of 94% and one-year actual graft survival of 88%, with no differences between double and triple therapy. The overall incidence of rejection was 48%, with 54% in the double therapy group and 41% in the triple therapy group (P < .07). The incidence of steroid-resistant rejection requiring antilymphocyte therapy (OKT3 or ATGAM) was 13%, and was not different between the double and triple therapy groups. The mean serum creatinine was 1.8 +/- 0.8 mg/dl. The mean BUN was 33 +/- 21 mg/dl, with no significant difference between the therapy groups. The mean serum cholesterol was 192 +/- 49 mg/dl. A total of 56% of the patients are off prednisone, and 35% of the patients are not taking any antihypertensive medications. Other complications included cytomegalovirus- 14%; new-onset diabetes--16% (half of which was reversible); and posttransplant lymphoproliferative disorder--1%.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533345 TI - FK506 (tacrolimus) compared with cyclosporine for primary immunosuppression after pediatric liver transplantation. Results from the U.S. Multicenter Trial. AB - We report on the efficacy and safety of FK506 (tacrolimus) compared with a cyclosporine (CsA)-based immunosuppressive regimen after 1 year of treatment in pediatric liver allograft recipients (< 12 years) participating in a multicenter U.S. randomized trial. Patients received either FK506 or CsA as primary immunosuppression following a first ABO-compatible liver transplant. Intravenous FK506 was initiated at 0.1 mg/kg per day, followed by oral FK506 beginning at 0.3 mg/kg per day. The dose was adjusted to maintain plasma trough levels of 0.5-2.0 ng/ml. The CsA group was treated according to each center's usual protocol. Both groups received the same initial doses of corticosteroids. All rejection episodes were biopsy-proven and a standardized algorithm was adopted for the treatment of rejection. Thirty patients were randomized to the FK506 group and 20 to the CsA group. After twelve months of follow-up 20 patients remained in the FK506 group and 13 in the CsA group. Patient survivals were 80% and graft survival 70% in the FK506 group compared with 81% and 71% respectively, in the CsA group. 48% of the FK506 group remained rejection-free compared with 21% of the CsA group, and 79% of FK506-treated patients did not require OKT3 compared with 68% of CsA treated patients. The cumulative corticosteroid dose was less at each time point throughout the first year in the FK506 group. The incidence of serious and minor infections was similar in both groups. Nephrotoxicity, neurotoxicity, and gastrointestinal disturbances were the major toxicities reported. Differences did not reach statistical significance between the two groups although major neurologic events, diarrhea and dyspepsia were more often reported in the FK506 group. There was no difference in mean serum creatinine at 12 months between the two groups. There was a tendency toward lower mean serum cholesterol in the FK506 group. There was no hirsuitism in the FK506 group compared with a 30% incidence in the CsA group. In conclusion, compared with CsA, there is a trend toward less rejection in FK506-treated pediatric allograft recipients, while both drugs have a similar spectrum of side effects. PMID- 7533346 TI - Soluble CTLA4Ig modifies parameters of acute inflammation in rat lung allograft rejection without altering lymphocytic infiltration or transcription of key cytokines. AB - The efficacy of CTLA4Ig in blocking immune activation and allograft rejection (AR) was tested in an aggressive and rapid model of rat lung AR (Brown Norway [BN]-->Lewis [LEW]). CTLA4Ig is a recombinant soluble protein that binds with high affinity to rat B7/BB1 and other surface molecules on APCs, subsequently blocking the binding of B7/BB1 to CD28/CTLA4 on T cells. This interrupts the costimulatory pathway critical for complete T cell activation and completion of the AR process. Left single-lung transplants were performed between BN-->Lew. Five allograft recipients were examined in each group. At transplantation, animals received 250 micrograms of CTLA4Ig or 250 micrograms of control Ig intraperitoneally daily until sacrifice. Animals were sacrificed on days 2, 4, and 7 after transplant. Control (BN-->Lew) grafts show irreversible rejection by day 7. Syngeneic (Lew-->Lew) grafts show no AR on day 7. AR episodes were graded histologically (stages 0-IV) and pathologic intensity of inflammation was graded on percentage of involvement. Cytokine transcript levels were measured in control and CTLA4Ig-treated animals (n = 5 in each group) on day 7 using reverse transcriptase polymerase chain reaction techniques. The most profound differences were found on day 7 after transplant. The degree of lymphocytic infiltration was greater in the CTLA4Ig group (perivascular: 4 +/- 0 vs. 2.6 +/- 0.6, peribronchial: 4 +/- 0 vs. 2.2 +/- 0.4, and peribronchiolar: 3.6 +/- 0.5 vs. 2 +/ 0.3, P < 0.01). However, in striking contrast, the stage of AR (3 +/- 0 vs. 4 +/ 0, P < 0.01), vasculitis (1 +/- 0.7 vs. 2.6 +/- 0.6, P < 0.05), hemorrhage (0.4 +/- 0.6 vs. 3.2 +/- 0.4, P < 0.01), and necrosis (0 +/- 0 vs. 2.4 +/- 0.5, P < 0.005) were significantly reduced in animals treated with CTLA4Ig. Since CTLA4Ig blocks Th1 cell activation in vitro, we compared the levels of Th1 inflammatory cytokines IL-2, gamma-IFN, and TNF-alpha in the two models. The intragraft ratios (CTLA4Ig/control) were IL-2:0.77, gamma-IFN: 1.29, and TNF-alpha:1.33. Thus, CTLA4Ig did not significantly block intragraft production of Th1 cytokines on day 7.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533348 TI - Low frequency of infiltrating cells intensely expressing T cell cytokine mRNA in human renal allograft rejection. AB - Immunosuppressive drugs used in clinical transplantation block cytokine mRNA transcription in vitro, but clinical rejection episodes are common. An understanding of what cytokine message is transcribed would be helpful in determining what contributes to the success of immunosuppression and provide directions for further research aimed at targeting specific cytokines. Previous studies have examined cytokine mRNA in rejecting solid organ biopsies by the reverse transcriptase polymerase chain reaction (RT-PCR) with variable results. We used nonradioactive in situ hybridization with cytokine-specific riboprobes to determine the frequency of cells expressing cytokine mRNA in the allograft infiltrate. Kidney biopsies were obtained from patients receiving protocol biopsies and with clinical evidence of rejection. Fourteen biopsies with a pathologic diagnosis of rejection were studied. Eight showed no cytokine staining, 2 expressed IL-2, and 3 expressed IL-4 and IFN-gamma. The positive cells were present at a low frequency (mean 2, range 1-5 per 10 high-power fields). The proportion of kidney biopsies expressing detectable message for interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-gamma (IFN-gamma) by in situ hybridization were similar to those reported using RT-PCR. The novel finding is that these cytokines are expressed in a few strongly positive cells in the allograft infiltrate. The vast majority of infiltrating cells are negative. This suggests that either the biopsies were performed when cytokine message was not expressed at a high level or that in human allograft recipients the sustained expression of the cytokines IL-2, IL-4, and IFN-gamma may not be necessary for graft rejection. PMID- 7533347 TI - Upregulation of cytokines associated with macrophage activation in the Lewis-to F344 rat transplantation model of chronic cardiac rejection. AB - Lewis-to-F344 rat cardiac allografts develop chronic rejection and arteriosclerotic lesions rich in mononuclear cells (especially macrophages). This study was performed to determine whether cytokine pathways associated with macrophage activation are upregulated in hearts undergoing chronic rejection. Gene transcript levels for IFN-gamma, monocyte chemoattractant protein-1 (MCP-1), and IL-6 were measured with reverse-transcription PCR assays optimized for each gene. Gene products were confirmed by immunohistology. For all three genes, transcript levels in rat cardiac allografts increased significantly on day 7 and remained elevated on days 14 and 28 posttransplantation, as compared with naive hearts, paired host hearts, and syngrafts (P < 0.006). For the inducible genes IFN-gamma and MCP-1, high transcript levels in cardiac allografts were in contrast with low levels in host spleens. On the other hand, transcript levels for the basally expressed gene IL-6 were elevated in both organs. Immunostaining confirmed allograft-specific expression for all three cytokines and localized the gene products to infiltrating mononuclear cells in the interstitium and vasculature. The sustained expression of these cytokines in cardiac allografts undergoing chronic rejection supports the widely held hypothesis that the intimal changes associated with transplant arteriosclerosis are mediated by cellular activation and cytokine production. PMID- 7533349 TI - Differential patterns of circulating intercellular adhesion molecule-1 (cICAM-1) and vascular cell adhesion molecule-1 (cVCAM-1) during liver allograft rejection. AB - During allograft rejection, adhesion molecules play an integral role in infiltration, activation, and binding of effector cells to target tissue. Some adhesion molecules, including ICAM-1 and VCAM-1, exist in soluble, circulating forms that retain ligand-binding activity. In the present study the levels of circulating ICAM-1 (cICAM-1) and VCAM-1 (cVCAM-1) were compared in the serum and bile of pediatric liver recipients. The cICAM-1 was significantly elevated in the serum during allograft rejection and infection relative to periods when no rejection was apparent. Biliary cICAM-1, however, was specifically elevated during rejection and not during infection or when no rejection was apparent. The cVCAM-1 levels were elevated in the serum during rejection compared with levels when no rejection was evident. In contrast, cVCAM-1 was not detected in the bile. Serum levels of both cICAM-1 and cVCAM-1 decreased rapidly following successful treatment for rejection, whereas elevated levels persisted, or increased, in ongoing rejection. The differential patterns of the circulating forms of ICAM-1 and cVCAM-1 were consistent with the membrane expression of these molecules during graft rejection. ICAM-1 expression was extensive on bile duct epithelium, endothelium, hepatocytes, and infiltrating leukocytes during rejection, while VCAM-1 was restricted to endothelium. These findings indicate that the release of circulating adhesion molecules is a prominent feature of liver allograft rejection. Measurement of these markers may be useful in distinguishing rejection from infection and in determining the efficacy of treatment for rejection. PMID- 7533350 TI - Posttransplant Epstein-Barr virus infection is associated with elevated levels of CD19+ B lymphocytes. PMID- 7533351 TI - Patterns of expression of endothelial leukocyte adhesion molecules upon revascularization of transplanted kidneys in correlation with allograft pathology. PMID- 7533352 TI - Prospective serial evaluation of cell adhesion molecule expression in transplanted kidneys. PMID- 7533354 TI - FK 506 as treatment of late acute rejection in liver transplant patients. PMID- 7533353 TI - One thousand consecutive primary orthotopic liver transplants under FK 506: survival and adverse events. PMID- 7533355 TI - FK 506 in liver transplantation: correlation of whole blood levels with efficacy and toxicity. The US Multicenter FK 506 Dose Optimization. PMID- 7533356 TI - Pharmacokinetics and pharmacodynamics of FK 506 in pediatric patients receiving living-related donor liver transplantations. PMID- 7533357 TI - OKT3 vs FK 506 rescue management of acute steroid-resistant and chronic rejection. PMID- 7533359 TI - The superior immunosuppressant according to diagnosis: FK 506 or cyclosporine A. PMID- 7533358 TI - Comparing nephrotoxicity of FK 506 and cyclosporine regimens after liver transplantation: preliminary results from US Multicenter trial. U.S. Multicenter Liver Study Group. PMID- 7533361 TI - Whole-blood and plasma levels of FK 506 after liver transplantation: results from the US Multicenter Trial. FK506 Multicenter Study Group. PMID- 7533360 TI - Hyperlipidemia after liver transplantation: long-term results of the FK506/cyclosporine A US Multicenter Trial. US Multicenter Study Group. PMID- 7533363 TI - Weight gain and lipid profile changes in liver transplant recipients: long-term results of the American FK506 Multicenter Study. PMID- 7533362 TI - FK 506 improves recovery of bile secretion following orthotopic liver transplantation in man. PMID- 7533364 TI - Glucose metabolism following liver transplantation and immunosuppression with cyclosporine A or FK 506. PMID- 7533365 TI - Influence of long-term cyclosporine or FK 506 therapy on glucose and lipid metabolism in stable liver graft recipients. PMID- 7533366 TI - Evidence for transformation of lipocytes to myofibroblast-like cells as source of transplant fibrosis in chronic human allograft rejection. PMID- 7533367 TI - Distinct patterns of Th2 cytokine production during immune activation in pediatric liver allograft recipients. PMID- 7533368 TI - Circulating intercellular adhesion molecule-1 and vascular cell adhesion molecule 1 in pediatric liver recipients. PMID- 7533369 TI - Recovery of delayed-type hypersensitivity following liver transplantation. PMID- 7533370 TI - Common and sequential overexpression patterns of T-helper cytokines during acute (cellular) rejection, and correlation of proinflammatory cytokine expression with chronic (ductopenic) rejection of human liver allografts: a study under cyclosporine, FK 506, and quadruple BT 563 immunosuppression. PMID- 7533372 TI - Molecular markers of Epstein-Barr virus infection in the circulation of transplant recipients. PMID- 7533375 TI - New criteria for diagnosing cytomegalovirus hepatitis in liver transplant patients. PMID- 7533374 TI - Alpha-interferon for acute hepatitis C in liver transplant patients. PMID- 7533371 TI - Metabolic liver function and lipoprotein metabolism after orthotopic liver transplantation in patients on immunosuppressive therapy with FK 506 or cyclosporine. PMID- 7533373 TI - Liver transplantation in hepatitis B surface antigen positive patients with postoperative long-term immunoprophylaxis. PMID- 7533376 TI - Analysis of risk factors for recurrence of hepatocellular carcinoma after orthotopic liver transplantation. PMID- 7533377 TI - In vitro induction of endothelial adhesion molecule and MHC antigen expression by cytomegalovirus-activated CD4+ T cells. PMID- 7533378 TI - Endothelial adhesion molecules in human heart and lung transplants. PMID- 7533380 TI - Isolated pancreas rejection in combined kidney-pancreas transplantation: results of percutaneous pancreas biopsy. PMID- 7533379 TI - Serum levels of pancreatic amylase and pancreas-specific protein as markers of pancreatic graft rejection episodes. PMID- 7533381 TI - Effect of rescue therapy using FK 506 on relapsing rejection after combined pancreas and kidney transplantation. PMID- 7533383 TI - FK 506-based immunosuppression for prevention of graft versus host disease after unrelated donor marrow transplantation. PMID- 7533382 TI - Low-dose ultraviolet B pretreatment of human islets reduces graft immunogenicity through the induction of antigen-presenting cell apoptosis without adversely affecting insulin secretion. PMID- 7533385 TI - Some combinations of anti-CD2 MAb induce apoptosis of activated CD8+/CD57+ T cells. PMID- 7533384 TI - Immunologic monitoring after organ transplantation: relationship between Epstein Barr virus infection and CD19+ B cells measured by flow cytometry. PMID- 7533386 TI - Bone marrow augmentation for heart, liver, and small bowel transplantation: prolongation of graft survival and incidence of graft-versus-host disease. PMID- 7533387 TI - Evidence for the presence of hematopoietic stem cells in the adult liver. PMID- 7533388 TI - Serial evaluation of immune profiles of simultaneous bone marrow and whole organ transplant recipients. PMID- 7533390 TI - Long-term nonresponsiveness to a liver allograft may be cytokine mediated. PMID- 7533389 TI - Factors influencing long-term operational tolerance in dogs treated with fractionated lymphoid irradiation, donor bone marrow cell infusion and FK506. PMID- 7533391 TI - Efficacy of various immunosuppressive drugs in preventing pig-to-rat islet xenograft rejection. PMID- 7533392 TI - Effect of anticomplement agent K-76 COOH in hamster-to-rat and guinea pig-to-rat xenotransplantation. PMID- 7533393 TI - Prevention of sensitization and hyperacute rejection in liver and heart xenografts by FK 506 plus donor antigens. PMID- 7533394 TI - Prolonged survival of hamster-to-rat pancreas xenografts by FK 506 and splenectomy. PMID- 7533395 TI - Mechanisms of protection from humoral rejection by a xenografted liver. PMID- 7533397 TI - Comparative analysis of human DAF expression in the tissues of transgenic pigs and man. PMID- 7533396 TI - Human complement regulatory proteins in transgenic animals regulate complement activation in xenoperfused organs. PMID- 7533399 TI - Production of pigs transgenic for human regulators of complement activation. PMID- 7533398 TI - Expression of human DAF and MCP on pig endothelial cells protects from human complement. PMID- 7533400 TI - Endothelial expression of human decay accelerating factor in transgenic pig tissue: a potential approach for human complement inactivation in discordant xenografts. PMID- 7533401 TI - Effect of transfected MACIF (CD59) on complement-mediated swine endothelial cell lysis, compared with those of membrane cofactor protein (CD46) and decay accelerating factor (CD55). PMID- 7533402 TI - Evidence that double transfection to xenoendothelial cells using GPI-anchoring complement regulatory factor (decay-accelerating factor and homologous restriction factor 20) gene is useful for the inhibition of human complement mediated cytolysis. PMID- 7533403 TI - Mice transgenic for human CD46 and CD55 are protected from human complement attack. PMID- 7533404 TI - Permanent expression of human CD59 and/or decay-accelerating factor by rat endothelial cells confers protection from human complement-mediated lysis. PMID- 7533405 TI - Nerve regeneration of vascularized rat limb allograft and functional recovery of long-term graft survivals treated by short course of FK 506 or cyclosporine. PMID- 7533406 TI - Significance of newly developed liposomal FK 506 in canine liver transplantation. PMID- 7533407 TI - Induction of unresponsiveness to islet allografts with donor spleen cell inoculation followed by a single injection of FK 506. PMID- 7533408 TI - Synergism of castanospermine and FK 506. PMID- 7533409 TI - Inhibition of insulin production by FK 506 is caused at the transcriptional level in pancreatic beta cell when FK BP-12 content is relatively high. PMID- 7533410 TI - Protective effect of FK 506 on hepatic injury following cold ischemic preservation and transplantation: influence on hepatic microcirculation. PMID- 7533411 TI - Therapeutic synergism between low-dose FK 506 and antimetabolites in rat allogeneic heart transplantation. PMID- 7533412 TI - Comparative study of toxicity and efficacy of cyclosporine, cyclosporine G (OG37), FK 506, and rapamycin in BALB/c mice with fitted skin grafts. PMID- 7533413 TI - Immunosuppressive effects of FK 506 on rat renal allograft survival, in comparison with cyclosporine. PMID- 7533414 TI - Immunologic characterization of murine cardiac allograft recipients with long term graft survival due to anti-VCAM-1 or anti-CD4 monoclonal antibody therapy. PMID- 7533415 TI - Comparison of FK 506- and cyclosporine-based immunosuppression: FK 506 therapy significantly reduces the incidence of acute, steroid-resistant, refractory, and chronic rejection whilst possessing a comparable safety profile. European FK 506 Multicenter Liver Study Group. PMID- 7533416 TI - Indirect recognition of native HLA alloantigens and B-cell help. PMID- 7533417 TI - Graft endothelial VCAM-1 is not necessary for acute cardiac allograft rejection in mice. PMID- 7533418 TI - Reversal of presensitization by platelet-antibody complex is not determinant specific. PMID- 7533419 TI - Japanese multicenter studies of FK 506 in renal transplantation. Japanese FK 506 Study Group. PMID- 7533420 TI - The pattern of rejection and susceptibility to different kinds of immunosuppression after combined liver, duodenum, and pancreas transplantation in swine--immunosuppressive drugs versus ex vivo immunomodulation of the allograft. PMID- 7533421 TI - Monitoring for engraftment of rat orthotopic liver transplantation by semiquantitative PCR. PMID- 7533423 TI - Role of endothelin in FK 506-induced renal hypoperfusion in rats. PMID- 7533422 TI - The minimum graft size for successful orthotopic partial liver transplantation in the canine model. PMID- 7533424 TI - Spontaneous afferent reinnervation after lung transplantation in the rat. PMID- 7533426 TI - Changes in cell adhesion molecules after small bowel transplantation in rats. PMID- 7533429 TI - Hepatocyte transplantation into solid supports in the rhesus monkey: the influence of acidic fibroblast growth factor on prevascularization and hepatocyte survival. PMID- 7533428 TI - Expression of intrahepatic inducible nitric oxide synthetase mRNA correlates with production of nitric oxide during intraportal isogeneic and allogeneic rat islet transplantation. AB - Intrahepatic NO production is related to the islet mass transplanted. Nitric oxide production is higher in recipients of allogeneic rather than syngeneic islets. In addition, in allogeneic recipients a possible second peak of NO production was observed at 120 hours corresponding to the time of cellular rejection of the islet grafts (P = .22 vs 96 hours). Finally, the time to rejection of Wistar rat donor islets transplanted into Lewis rat diabetic recipients treated with NMA was not affected. However, inhibiting NO production in the minimal islet transplant model decreased the time to islet function, it does not affect the time to clinical rejection in recipients of a high number of allogeneic islet, which functions immediately. High-level NO has been shown to inhibit T-cell activation in vitro, and thus decreasing the levels by administrating NMA may accentuate the rejection response, canceling out the beneficial effect that might otherwise have occurred on islet function. Further experiments are required to clarify these issues. PMID- 7533427 TI - Islet allografting in the dog model: potential therapies for tolerance induction. PMID- 7533430 TI - Major histocompatibility complex class II-mediated inhibition of hemopoiesis in vitro and in vivo is abrogated by c-kit ligand. PMID- 7533425 TI - Graft coronary arteriosclerosis in rat heart transplant model with FK 506 short term administration. PMID- 7533431 TI - Phase II FK 506 multicenter concentration control study: one-year follow-up. PMID- 7533434 TI - Sodium-losing nephropathy and distal tubular damage of transplant kidneys with FK506 administration. PMID- 7533433 TI - Comparison of plasma vs whole blood as matrix for FK 506 drug level monitoring. PMID- 7533432 TI - A prospective, randomized trial of FK 506/prednisone vs FK 506/azathioprine/prednisone in renal transplant patients. PMID- 7533435 TI - Phase III study of FK 506 in kidney transplantation. Japanese FK 506 Study Group. PMID- 7533437 TI - MRP8/14-positive macrophages as early acute cellular rejection markers, and soluble MRP8/14 and increased expression of adhesion molecules following renal transplantation. PMID- 7533436 TI - Renal transplantation under cyclosporine and FK 506 for hemolytic uremic syndrome. PMID- 7533438 TI - Expression of adhesion molecules and their ligands in acute rejection of human kidney allografts. PMID- 7533440 TI - Long-term impact of hepatitis virus infection on kidney transplant recipients and a pilot study of the effects of interferon alpha on chronic hepatitis C. PMID- 7533439 TI - Liver biopsy is essential in anti-HCV (+) renal transplant patients irrespective of liver function tests and serology for HCV. PMID- 7533441 TI - The association of increased soluble VCAM-1 levels with CMV disease in human kidney allograft recipients. PMID- 7533442 TI - Definition of prostatic urethral obstruction. AB - Prostatic urethral obstruction as defined by urodynamic criteria is only present in about 70% of patients undergoing surgical treatment for benign prostatic hyperplasia (BPH). Infravesical obstruction can only be diagnosed by simultaneous recording of detrusor pressure (intravesical pressure minus abdominal pressure) and urinary flow rate. The interpretation of these recordings should be based upon the pressure-flow relationship. The possible clinical implications of the objective diagnosis of prostatic urethral obstruction in BPH are discussed. PMID- 7533443 TI - High-affinity specific [3H]tamsulosin binding to alpha 1-adrenoceptors in human prostates with benign prostatic hypertrophy. AB - The binding of a novel radioligand, [3H]tamsulosin, to human prostatic membranes with benign prostatic hypertrophy (BPH) has been characterized. [3H]Tamsulosin rapidly associated with its binding sites in human prostatic membranes with BPH, and the binding reached steady state by 30 min at 25 degrees C. The rate constants for association and dissociation of [3H]tamsulosin binding were calculated to be 0.21 +/- 0.05/nM per minute and 0.01 +/- 0.004/min, respectively. The specific binding of [3H]tamsulosin in human prostatic membranes was saturable and of high affinity (Kd = 0.04 +/- 0.01 nM). The density of [3H]tamsulosin-binding sites (Bmax) was 409 +/- 28 fmol/mg protein. The Kd and Bmax values for [3H]tamsulosin binding in human prostates were significantly lower than those for [3H]prazosin binding. [3H]tamsulosin binding was remarkable for its significantly lower degree of nonspecific binding. Six alpha-adrenoceptor antagonists competed with [3H]tamsulosin for the binding sites in the rank order: tamsulosin > WB4101 > prazosin > S-(+)-isomer > naftopidil > yohimbine. The binding affinities (pKi) of these antagonists for [3H]tamsulosin binding in human prostates closely correlated with their pharmacological potencies (pA2) in prostates. In conclusion, [3H]tamsulosin selectively labels alpha 1-adrenoceptors in human prostates, and thus may become a useful radioligand for the further analysis of these receptors. PMID- 7533444 TI - Nitric oxide synthase activity in the human urogenital tract. AB - Nitric oxide (NO) has been suggested as a nonadrenergic non-cholinergic neurotransmitter in the urogenital tract and has previously been shown to have a smooth muscle relaxing effect in the urogenital organs both in various animals and in humans. It has been shown that NO is a mediator of the erection and the dilatation of the bladder neck and urethra. The aim of the study was to analyse nitric oxide synthase (NOS) activity in the human urogenital tract. NOS activity was measured by the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. In the upper urinary tract there was Ca(2+)-dependent NOS activity in the renal pelvis, but no significant NOS activity could be found in the ureter. In the lower urinary tract we found high Ca(2+)-dependent NOS activity in the urethra, intermediate activity in the bladder neck and comparatively low activity in the detrusor muscle. In the male genital tract the testis and epididymis had no significant NOS activity. The vas deferens, prostate, seminal vesicle and corpus cavernosum were found to have high levels of Ca(2+)-dependent NOS activity. Ca(2+)-independent NOS activity was not obtained in the urogenital tract. Our results correspond well with previous functional studies indicating NO to be an important nerve-induced mediator of erection and in the micturition reflex, but also suggest that NO may be involved in several other functions in the human urogenital tract. PMID- 7533446 TI - [Benign prostatic hyperplasia]. PMID- 7533445 TI - Serum TPS, PSA, and PAP values in relapsing stage D2 adenocarcinoma of the prostate. AB - Serum tissue polypeptide-specific antigen (TPS), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP) concentrations were serially measured in 31 prostate cancer patients with bone metastases who had relapsed following hormonal therapy. Of these subjects 7 had well-differentiated cancer (G1), 13 patients were assessed to have moderately differentiated tumor (G2) while in 11 subjects poorly differentiated tumor (C13) was found. With increasing tumor grade (G1 to G3), a proportional increase in mean TPS value was found while the increase in respective PAP serotest values was not linear. Simultaneously measured mean PSA values showed a curved effect. Both PSA and PAP serotest concentrations depend on the respective hormone-dependent gene expressions that gradually decrease with tumor dedifferentiation. Therefore, in progressive hormonally treated stage D2 prostate cancer patients an androgen-independent TPS serotest seems to be a useful clinical addition for monitoring protocols. The combined use of TPS, PSA, and PAP seems to give a better reflection of tumor status. According to the bone scan data metastatic tumor mass in G3 carcinomas was virtually equal to cancer burden in G2 tumors. Hence, the marked elevation of TPS serotest values in G3 adenocarcinomas could not be attributed to greater tumor mass but was most likely due to an increase in proliferation rate. Some authors have recently proposed cytokeratins 8, 18, and 19 to be the origin of TPS serum findings. However, cytokeratin content has been proven to be lower in G3 tumors than in better differentiated neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533447 TI - [Thermotherapy of benign prostatic hyperplasia]. AB - Transurethral microwave thermotherapy (TUMT) differs from hyperthermia in the higher intraprostatic temperatures reached and in the irreversible damage it causes to intraprostatic tissue. Both subjective symptoms and objective data, such as peak flow and residual urine, are influenced differently by TUMT and by sham treatment. As expected, no improvement of the objective data was documented with TUMT, in contrast to transurethral resection of the prostate (TURP), whereas the improvement in subjective symptoms was comparable. The complication rate in terms of morbidity is clearly lower with TUMT than with TURP. TUMT can be performed as an outpatient procedure without a general anaesthetic. TUMT had a specific effect on outflow obstruction, but it was less pronounced than that achieved with TURP. Therefore, TUMT is indicated for patients with typical symptoms of BPH and a mild mechanical obstruction. In this patient group TUMT seems to be the optimal treatment, whereas TURP would constitute "overtreatment" owing to its higher complication rate and the unnecessary tissue resection. At this stage of BPH, TURP is justifiable only with reservations; it should be reserved for BPH with moderate and severe mechanical obstruction. PMID- 7533448 TI - [Transurethral laser therapy of benign prostatic hyperplasia]. AB - During the last 4 years, transurethral laser therapy for benign prostatic hyperplasia (BPH) has become an increasingly popular alternative to transurethral resection of the prostate (TURP) for the treatment of obstructive BPH symptoms. Clinical data so far published prove the efficacy of this new therapeutic modality, although the results are up to now possibly inferior to those achieved with TURP. On the other hand, the incidence of severe perioperative complications is significantly lower in patients treated with laser prostatectomy than in those undergoing TURP. Therefore, laser prostatectomy is a reasonable adjunct to the therapeutic spectrum for BPH in high-risk patients. Nevertheless, its role in the treatment of obstructive BPH in low-risk patients cannot be defined until long term follow-up data are available. PMID- 7533449 TI - [Therapy of benign prostatic hyperplasia with alpha receptor blockers]. AB - The therapeutic efficacy of alpha-blockers in disorders of bladder emptying has been known for 20 years. Substantial side effects initially prevented their wide use in benign prostatic hyperplasia. The situation has changed with the development of the very much better tolerated selective alpha 1-adrenoceptor antagonists. In numerous countries, alpha 1-blockers are also licensed for use in the treatment of benign prostatic hyperplasia in addition to the main indication, vascular hypertension. Of those available in Germany, the slow-acting antihypertensive agents terazosin and doxazosin are the most suitable for use in this condition. The modern alpha 1-blockers have proved their effectiveness in large-scale placebo-controlled studies. However, the effects consist solely in improvement of the subjective symptoms and a moderate increase of urinary flow and are not comparable to those achieved with transurethral resection. alpha 1 Blockers are indicated in patients with moderate to fairly severe symptomatic benign prostatic hyperplasia (as an alternative, for example, to phytotherapeutic agents) and in patients who have severe symptoms but for whom surgical treatment is not desired, needs to be postponed or is contraindicated. Side effects arise from the concomitant vasodilatation, so that contraindications for alpha-blockers are cardiac and renal failure, a prior history of cerebrovascular incidents and a tendency to hypotension. Further attenuation of these side effects appears possible with the future development of "uroselective" alpha 1-blockers. PMID- 7533450 TI - [Hormonal therapy of benign prostatic hyperplasia]. AB - In the last 30 years transurethral prostatectomy (TURP) has been the standard treatment for benign prostatic hyperplasia. While the mortality rate of TURP has been reduced to zero, the postoperative morbidity rate has remained unchanged at 15-18% for several decades. Increasing costs and the preference of many patients for non-surgical treatment have now led to the development of new pharmacological treatment options. All hormone therapeutic modalities known to date, such as GnRH agonists, cyproteronacetate and flutamide, have acted on the male hypothalamic pituitary-gonadal system. Because of their side effects they are rarely used for the treatment of symptomatic benign prostatic hyperplasia. The 5-alpha-reductase inhibitor finasteride is a new endocrinological treatment option. It can be used to reduce the volume of enlarged prostates and to improve flow rates without severe side effects. According to the available data, finasteride is a safe treatment option for symptomatic patients with benign prostatic hyperplasia if surgery is not yet indicated. It should not, however, be regarded as an alternative to surgical treatment of benign prostatic hyperplasia. PMID- 7533451 TI - Effect of demographic factors, urinary peak flow rates, and Boyarsky symptom scores on patient treatment choice in benign prostatic hyperplasia. AB - OBJECTIVES: To determine the effect of patient's age, race, Boyarsky score, and urinary flow rate on type of treatment selected for benign prostatic hyperplasia (BPH) and to evaluate maintenance of treatment at 1-year follow-up. METHODS: Subjects in this prospective study, conducted in a referral center prostate clinic, were 174 consecutive patients, aged 67.6 +/- 10.8 years (mean +/- SD), with previously untreated BPH. Patients underwent structured interviews and uroflowmetry, then completed the Boyarsky Symptom Index. Blinded to these data, one physician described four treatment categories in a nonjudgmental fashion, always using the same order: watchful waiting; finasteride and alpha-blocker; thermal therapy, balloon dilation, and a prostate stent; and transurethral prostate resection. Treatment choice was entirely that of the patient. Independent variables were patient's age, race, symptom score, and uroflow; the dependent variable was treatment choice. At 1 year, the same physician interviewed patients to evaluate maintenance of therapy. RESULTS: Only symptom score and flow rate were predictive of treatment choice, high scores and low flow being associated with more aggressive treatment choices (P = 0.001). Most patients, regardless of symptom severity, chose interventions less aggressive than surgery and more aggressive than watchful waiting. At 1 year, 85% of patients continued to be maintained on their original treatment. CONCLUSIONS: Men with mild or moderate BPH prefer interventions of moderate aggressiveness; race and age make little or no difference. If maintenance of treatment indicates patient satisfaction, most patients appear to remain satisfied with therapy they select. PMID- 7533453 TI - Laser prostatectomy: initial experience and urodynamic follow-up. AB - OBJECTIVES: An evolving technology for the treatment of bladder outlet obstruction due to benign prostatic hyperplasia (BPH) is the use of the side firing neodymium: yttrium-aluminum-garnet (Nd:YAG) laser to achieve prostatic tissue ablation. The purpose of this study was to determine the short-term efficacy of this procedure in both an objective and subjective manner. METHODS: We examined this technique by carefully evaluating our first 25 men undergoing the procedure. Each patient was subjected to careful symptom score analysis using the American Urological Association symptom index and multichannel urodynamics, including pressure-flow studies both preoperatively and at 3 months postoperatively. RESULTS: At the 3-month follow-up, symptom scores improved from a preoperative mean of 11.4 to 7.2 and the mean maximum flow rate improved from 6.1 to 14.5 cc/s. These are both significant at P < 0.001. Statistically similar improvement was seen in detrusor pressure at opening and at maximum flow. Eighty percent of the men studied had at least a 50% reduction in symptom score and a 50% improvement in flow rate. CONCLUSIONS: We conclude that laser prostatectomy is a promising minimally invasive treatment for bladder outlet obstruction secondary to BPH and deserves further evaluation at longer terms of follow-up. PMID- 7533454 TI - A radiofrequency method of thermal tissue ablation for benign prostatic hyperplasia. AB - OBJECTIVES: To evaluate the safety, feasibility, and efficacy of transurethral ablative prostatectomy (TURAPY), a radiofrequency method of thermal tissue ablation of benign prostatic hyperplasia (BPH). METHODS: Twenty men, ages 55 to 81 years (mean, 67), with symptomatic BPH and with peak flow rate 12 mL/s or less, were treated with the TURAPY device (Direx Medical Systems). A 2 cm long heating element and 2 thermoprobes (for simultaneous prostatic temperature monitoring) are mounted on a Foley-like catheter and used for TURAPY treatment administration. This TURAPY catheter was modified by placing an extra set of thermoprobes in the sphincter region to allow sphincteric temperature monitoring. The treatment was administered at a maximum temperature of 70 degrees to 75 degrees C for 1 hour under local anesthesia as a day case. RESULTS: All 20 patients completed the treatment. The maximum recorded rectal temperature was 39.5 degrees C. The maximum sphincter temperature was not allowed to exceed 42 degrees C. The post-treatment morbidity included dysuria and minor urethral bleeding in 12 patients. Three patients developed urinary infection requiring antibiotic treatment. All 20 patients were followed up 3 months after treatment. The mean International Prostate Symptom Score (IPSS) improved from 19.4 to 6.2 (68%), the mean peak flow rate increased from 7.9 to 12.9 mL/s (63%), and the mean postvoid residue decreased from 222 to 81 mL (64%). Overall, 80% of patients exhibited at least a 50% improvement in either the IPSS or the peak flow rate. There was a mean reduction in prostatic volume measured by transrectal ultrasound of 14 mL (29% reduction). The subjective and objective improvements, in 8 patients followed up 6 months after treatment, have been maintained. There was extensive coagulative heat necrosis of periurethral tissue with sparing of subcapsular tissue in prostate biopsy specimens taken from 1 patient 5 days after treatment. There was endoscopic and sonographic evidence of canalization of the obstructed prostatic urethra 3 months after treatment. CONCLUSIONS: Treatment with the TURAPY device was found to be safe, feasible, and effective in improving both subjective and objective measurements of benign prostatic obstruction in this pilot study on 20 patients. PMID- 7533452 TI - Long-term efficacy and safety of terazosin in patients with benign prostatic hyperplasia. Terazosin Research Group. AB - OBJECTIVES: To evaluate long-term efficacy and safety of terazosin, a selective alpha 1 blocker, in the treatment of benign prostatic hyperplasia (BPH). METHODS: This was a long-term (42 months), open-label, multicenter study with patients evaluated at 1- to 6-month intervals. Twenty-three outpatient clinics throughout the United States and Canada participated in the study. A total of 494 men with symptomatic BPH, lacking absolute indications for surgery, were enrolled in this study; 298 were transferred into the study from randomized, placebo-controlled studies of terazosin and 196 had no prior terazosin therapy. Terazosin was given starting at 1 mg/d and titrated upward until symptoms were relieved or a maximum dose of 20 mg/d was achieved, whichever came first. RESULTS: Peak urinary flow rates at all visits were significantly higher than baseline values, with mean improvements ranging from 1.0 to 4.0 mL/s. At 3 months, 40% of patients exhibited a 30% or greater improvement in peak flow rate; this improvement was maintained through 42 months. Boyarsky symptom scores improved significantly at all visits; mean total score improved by at least 4.0 points (40%) at all visits beyond 3 months. The most common adverse events resulting in premature termination from the study were dizziness (6.7%), asthenia (3.8%), and somnolence (2.0%). CONCLUSIONS: This study suggests that terazosin is well tolerated and effective in longterm treatment of patients with BPH. PMID- 7533455 TI - Characterization and localization of nitric oxide synthase in the human prostate. AB - OBJECTIVES: To characterize nitric oxide synthase (NOS), which catalyzes nitric oxide (NO) production, in the human prostate using biochemical and immunohistochemical techniques. METHODS: NOS catalytic assay and NOS immunohistochemistry were performed on histologically verified nonmalignant prostate tissue obtained from the peripheral and transition zones of seven radical prostatectomy specimens. RESULTS: Biochemical analysis revealed NOS activity in the human prostate, with a greater amount in the peripheral zone than in the transition zone (P < 0.01). In both prostate zones, NOS was immunohistochemically localized to nerve fibers and ganglia coursing throughout the smooth musculature of the stroma and to subepithelial nerve plexuses. NOS immunoreactivity was also localized to glandular epithelium. CONCLUSIONS: The presence, activity, and distribution of NOS were described in two regions of the human prostate. The present evidence implicates NO in the automatic innervation and physiology of the human prostate. It is proposed that NO may modulate smooth muscle tone and secretory functions in the human prostate, although functional studies are needed to support these hypotheses. PMID- 7533456 TI - Stability of serum prostate-specific antigen determination across laboratory, assay, and storage time. AB - OBJECTIVES: To understand the comparability of serum prostate-specific antigen (PSA) determinations across assays and storage time. METHODS: Serum PSA levels were determined for men aged 40 to 79 years from the clinical subset of the Olmsted County Study of Urinary Symptoms and Health Status Among Men on fresh samples and after a median of 32 months on banked samples, frozen at -70 degrees C. Baseline serum PSA levels were determined by Tandem-R PSA assay. Follow-up levels on the banked samples were determined by the IMx PSA assay and a repeat Tandem-R PSA assay in a different laboratory and by an immunofluorometric PSA assay at another site. RESULTS: The median serum PSA level determined by Tandem-R assay at baseline was 1.0 ng/mL (25th percentile, 0.6; 75th percentile, 1.7). The distributions of determination made by follow-up Tandem-R, IMx, and immunofluorometric analyses were essentially identical. Overall, the assays were highly correlated. The correlations between the baseline serum PSA determination and repeated Tandem-R, IMx, and immunofluorometric determinations were 0.96, 0.96, and 0.97, respectively (all P < 0.001). The median duration of frozen storage was 32 months (range, 26 to 39 months), and the correlations between baseline and follow-up determinations did not change when stratified by duration of storage. CONCLUSIONS: These data provide important reassurance about the use of serum PSA determinations obtained by different assays, in different laboratories, and in properly stored samples across time. PMID- 7533457 TI - Evaluation of age-specific normal ranges for prostate-specific antigen. AB - OBJECTIVES: To compare the traditional normal range (TNR) of 0.0 to 4.0 ng/mL for serum prostate-specific antigen (PSA) to age-specific normal ranges (ASNRs). METHODS: An autopsy series of completely sectioned, clinically benign prostates from 171 consecutive Caucasian men over the age of 40 years was selected. These patients were divided into those having no prostate cancer at autopsy, prostate cancer less than 1 cc in volume, and prostate cancer at least 1 cc in volume. The PSA values of each group were compared using both the TNR and the ASNR. RESULTS: Twenty-three of 105 (21.9%) patients with no cancer had elevated PSA values by the TNR, whereas only 18 (17.1%) were elevated using the ASNR. Nine of 54 (16.7%) with cancer less than 1 cc were elevated using the TNR, and 7 of 54 (13.0%) using the ASNR. Of 12 patients with cancer at least 1 cc, all had elevated PSA levels using the TNR and 11 (91.7%) were elevated using the ASNR. All discrepancies between the TNR and ASNR occurred in the 60- to 79-year age range. CONCLUSIONS: Use of ASNRs appears helpful in increasing the specificity of PSA by eliminating some elevated values in patients in their 60s and 70s. PMID- 7533458 TI - Citrate as an in vivo marker to discriminate prostate cancer from benign prostatic hyperplasia and normal prostate peripheral zone: detection via localized proton spectroscopy. AB - OBJECTIVES: This study was designed to determine whether citrate levels detected by localized 1H spectroscopy could reliably discriminate regions of prostate adenocarcinoma from surrounding regions of normal peripheral zone and benign prostatic hyperplasia (BPH). METHODS: In 28 patients and 5 volunteers stimulated echo proton spectroscopy was used in conjunction with endorectal surface coils to obtain water-suppressed 1H spectra from regions of normal prostate peripheral zone, BPH, and prostate cancer. 1H spectra from prostate cancer patients were correlated with pathologic areas identified on T2-weighted endorectal coil magnetic resonance (MR) images and histologic study of the step-sectioned gland after surgery. RESULTS: The major finding of in vivo studies was consistently lower citrate levels in prostate cancer compared with BPH and normal prostate peripheral zone. This was reflected by significantly (P < 0.05) lower mean citrate/(creatine plus choline) peak area ratio observed for regions of cancer (0.67 +/- 0.17) compared with BPH (1.21 +/- 0.29) and normal peripheral zone (1.46 +/- 0.28). Moreover, there was no overlap of individual cancer and normal peripheral zone citrate ratios and no significant difference between citrate ratios in regions of normal peripheral zone in young volunteers (1.28 +/- 0.14) and age-matched patients (1.46 +/- 0.28). The observed alterations in vivo citrate levels were supported by citrate concentration data obtained from extracts of histologically proven samples of normal, benign, and malignant prostatic tissues removed at surgery. In vitro citrate levels in the normal peripheral zone (30.9 +/- 8.5 mumol/g wet weight) and BPH (46.3 +/- 5.4 mumol/g wet weight) were significantly higher than those for prostate cancer (3.74 +/- 0.54 mumol/g wet weight). CONCLUSIONS: These studies further demonstrate the potential of citrate as an in vivo marker for discriminating prostate cancer from surrounding regions of normal peripheral zone and BPH. PMID- 7533460 TI - Do the current subclassifications of stage T3 adenocarcinoma of the prostate have clinical relevance? AB - OBJECTIVES: To compare the outcome of patients with T3a and T3c adenocarcinoma of the prostate and determine the utility of these substages as defined in the current American Joint Committee on Cancer and the International Union Against Cancer (AJCC/UICC) staging system. METHODS: An analysis was performed of patients with T3 (clinical) prostate cancer treated with definitive irradiation at the Fox Chase Cancer Center between 1986 and 1993. The series was composed of 66 patients with T3a tumors and 44 patients with T3c tumors. The endpoints studied included freedom from biochemical relapse (bNED) and rates of clinical local and distant failure. RESULTS: No statistically significant differences in freedom from biochemical relapse were observed when comparing patients with T3a and T3c disease (3 years bNED, 41%; difference not significant). Similarly, there was no difference in the patterns of clinical failure at 3 years when comparing patients with T3a and T3c disease (21% clinically detected distant metastases; < 10% local failure in either group). In a multivariate analysis, only a low baseline prostate-specific antigen (PSA) (eg, 20 ng/mL or less) independently predicted the likelihood of remaining biochemically free of disease. CONCLUSIONS: Anatomic substaging that is based on the findings of the digital rectal examination does not distinguish meaningful prognostic substages among patients with T3 disease. PSA should be used to establish biochemical substaging of patients who present with T3 prostate cancer. PMID- 7533459 TI - Radiation therapy for T1 and T2 prostate cancer: prostate-specific antigen and disease outcome. AB - OBJECTIVES: To evaluate disease outcome using serum prostate-specific antigen (PSA) as an outcome measure in patients with T1 or T2 prostate cancer treated with radiation therapy in the PSA era. METHODS: We reviewed the outcome for 461 patients with T1 (n = 205) or T2 (n = 256) prostate cancer followed for a median of 31 months after radiation therapy as the sole initial treatment. Univariate and multivariate analyses were used to delineate significant prognostic factors. RESULTS: The freedom from relapse or rising PSA rate was 70% at 6 years and the survival rate was 83%. Although T stage, Gleason grade, serum prostatic acid phosphatase level, and serum PSA level were each significant determinants of outcome in univariate analysis, pretreatment PSA level was the only clearly independent covariate (P < 0.0001) in multivariate analysis. The 5-year actuarial freedom from relapse or from rising PSA levels is shown according to the pretreatment PSA level: 4 ng/mL or less (117 patients), 91%; more than 4 but 10 ng/mL or less (169 patients), 69%; more than 10 but 20 ng/mL or less (118 patients), 62%; and more than 20 ng/mL (57 patients), 38%. PSA doubling times in 75 patients with rising post-treatment profiles ranged from 1.3 to 78.2 months (mean, 14.4; median 11.3). Faster doubling times correlated significantly with adverse pretreatment prognostic factors (high-grade, high pretreatment PSA, and aneuploidy). To date, the survival rate of patients with rising PSA profiles was not depressed below the expected. CONCLUSIONS: Radiation therapy is an acceptable modality for treating T1 or T2 disease and produces results comparable to those following radical prostatectomy when patients are stratified according to their pretreatment PSA value. The rapid PSA doubling times observed in patients with relapsing disease are more consistent with a "selective" rather than an "aggravation" mechanism. PMID- 7533461 TI - Treatment with finasteride following radical prostatectomy for prostate cancer. AB - OBJECTIVES: The objective of this study was to evaluate the effect of finasteride (10 mg/d) or placebo on serum prostate-specific antigen (PSA) and recurrence rates in men with detectable PSA levels after radical prostatectomy. METHODS: A total of 120 men, 48 to 89 years old, previously treated with radical prostatectomy for prostate cancer within the past 10 years, with serum PSA levels between 0.6 and 10.0 ng/mL, with no evidence of skeletal metastasis on bone scan, and with no previous androgen deprivation therapy, were treated with 10 mg finasteride or placebo in a double-blind fashion for 12 months. After the first year, all patients were treated with finasteride for an additional 12 months. Primary endpoints were serum PSA levels and recurrence rates defined as positive bone scan or positive biopsy. RESULTS: Patients treated with finasteride had a delayed increase in serum PSA compared with placebo of approximately 9 months in the first year and 14 months by the end of the second year. Patients with baseline PSA levels less then 1.0 ng/mL had no significant increase in serum PSA during the 2 years of treatment. Fewer recurrences were observed in the finasteride group, but these differences were not statistically significant. Finasteride was well tolerated, and side effects were balanced between treatment groups. CONCLUSIONS: The results of this study indicate that treatment with finasteride delays but does not prevent the rise in serum PSA observed in untreated patients with detectable PSA levels after radical prostatectomy. The reduction in local and distant recurrences in the finasteride group suggests that the effect on PSA reflects a direct effect on tumor growth without affecting the initial response to subsequent hormonal therapy. These data require confirmation by studies that are longer and larger, focused on demonstrating significant differences in progression rates and survival before the use of finasteride can be considered as an option for men with detectable PSA levels after radical prostatectomy. PMID- 7533462 TI - Pressure flow studies for prostatism. PMID- 7533463 TI - Evidence that the agent of equine grass sickness may reach neurons by retrograde axonal transport. AB - Sera from acute and chronic cases of natural grass sickness or normal horses were injected into the parotid salivary gland of ponies. This gland receives its sympathetic innervation from the ipsilateral cranial cervical ganglion. None of the ponies showed any local or systemic signs of illness. After one week the cranial cervical ganglia, stellate and coeliaco-mesenteric ganglia were removed for histological study. Pathological changes were found only in the cranial cervical ganglion ipsilateral to a parotid salivary gland which had received an injection of grass sickness serum. Four out of five batches of test sera from cases of acute natural grass sickness were associated with chromatolytic changes in neurons; the remaining batch of serum produced no abnormalities. The most severe chromatolytic changes were induced by two samples obtained from horses whose signs of grass sickness had been present for less than 12 hours. A serum sample from a chronic case of grass sickness of three weeks duration did not produce chromatolysis but was associated with a moderately severe inflammatory infiltrate and neuronophagia in the ipsilateral cranial cervical ganglion. One batch of serum was size fractionated to separate components with molecular weights above or below 30 kDa. Only the fraction containing components above 30 kDa induced chromatolytic changes. PMID- 7533464 TI - [The immunostimulating action of lactobacteria on the cytotoxicity of natural killer cells and interferon production]. PMID- 7533465 TI - [The development of Russian nutrient media for isolating the recombinant surface antigen of the hepatitis B virus]. PMID- 7533466 TI - [New approaches to the screening of interferon inducers]. PMID- 7533467 TI - [Localization and regulation of cytokeratin 8 mRNA expression in rat prostate epithelia]. AB - The effects of androgen on cytokeratin (CK) 8 mRNA expression in rat ventral prostate (VP) were studied by in situ hybridization with an antisense RNA-probe. It was found that 1) the CK 8 antisense RNA-probe was accumulated only within prostatic epithelial cells, indicating that the CK 8 mRNA is a specific and sensitive marker for prostate epithelia. 2) After castration, the signals of CK 8 mRNA in VP sections were significantly increased. Administration of androgen to the castrated male host repressed the elevated expression of CK 8 mRNA in VP. This effect can be antagonized by the simultaneous administration of an antiandrogen. 3) Unlike other androgen-repressed gene, the elevated expression of CK 8 mRNA in VP was persisted even 2 months after the process of glandular involution was completed. 4) During prostate development, the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, when the levels of serum androgen were gradually increased, the shift of CK 8 mRNA staining to peripheral region and decreased level of overall CK 8 mRNA were noted. These data support the notion that the CK 8 gene of prostate epithelia is a new class of androgen repressed one. The data also indicated that the expression of CK 8 gene is closely related with the proliferation and differentiation of prostate stem cells, and excessive expression of CK 8 mRNA is a characteristic for prostatic stem cells. PMID- 7533468 TI - [The intermediate filament-lamina-nuclear matrix system of ES-M13 cells]. AB - Sequential extraction and DGD embedment-free section TEM techniques were used as new methods to study the intermediate filament-lamina-nuclear matrix (IF-L-NM) system of cells. Murine embryonic stem cells (ES-M13) were investigated by means of electron microscopy, immunofluorescence microscopy and Western-blot analysis. There existed a delicate nuclear matrix network in the nucleus domain of detergent-extracted ES cells. The filaments of the nuclear matrix were found in close association with the nuclear lamina. Some intermediate filaments were also observed in the cytoplasm. In immunofluorescence microscopy, a bright, slightly granular cytoplasmic fluorescence, showing no polar concentration, was revealed with keratin monoclonal antibody AF5. We could not detect any significant fibrillar staining in the ES cells by indirect immunofluorescence method. With antibodies to vimentin and desmin, the ES cells showed only a nonspecific, weak fluorescence, similar to that seen in the control. In Western-blot analysis of electrophoretically separated polypeptides, three polypeptides with molecular weight of 65 KD, 62 KD and 52 KD, reactive with keratin monoclonal antibody were detected in the ES cells. PMID- 7533469 TI - NO-synthase-containing neurons of the pig inferior mesenteric ganglion, part of them innervating the ductus deferens. AB - The presence of nitric oxide-synthase (NOS) in neurons of the porcine inferior mesenteric ganglion (IMG) has been investigated. A minority (about 1-3%) of the neurons were immunoreactive (IR) for NOS, the vast majority of which stained for neuropeptide Y (NPY) but not for tyrosine hydroxylase (TH). A small subpopulation of prevertebral neurons, 1% of which stained for NOS or NADPH-diaphorase (NADPHd), projected to the ductus deferens, as demonstrated by retrograde tracing. Within the wall of the ductus deferens, NOS- or NOS/NPY-IR nerve fibres were found to innervate the smooth muscle or were closely associated with blood vessels. It is therefore suggested that nitric oxide might be involved in the regulation of local blood flow and muscular tone in the wall of the pig ductus deferens. PMID- 7533470 TI - Cytoplasmic inclusions of astrocytic elements of glial tumors: special reference to round granulated body and eosinophilic hyaline droplets. AB - Round granulated body (RGB) and eosinophilic hyaline droplets (EHDs) have been described as cytoplasmic inclusions of certain astrocytic tumors. In the previous literature, however, these inclusions have been described using various terms or regarded as nosologically the same entity. Light microscopically, RGB appeared as a round discrete body filled with fine uniform granules, while EHDs demonstrated a cluster of bright eosinophilic, round objects of various size. They could be clearly distinguished even by conventional histochemical staining such as the Masson trichrome stain and the phosphotungstic acid hematoxylin preparation. Both RGB and EHDs expressed positive immunoreactions for glial fibrillary acidic protein, several lysosomal markers, and some stress-response proteins. The ultrastructural appearances of these inclusions were distinct, however, one common feature was that they consisted of aggregations of numerous membrane-bound electron-dense bodies. Thus, both inclusions appear to be produced by neoplastic astrocytes and are possibly related to the lysosomal system. We examined the presence of RGB and EHDs in 138 astrocytic tumors. Both inclusions occurred most frequently in pleomorphic xanthoastrocytomas, followed by gangliogliomas and pilocytic astrocytomas. Subependymal giant cell astrocytomas exhibited only RGBs. RGBs and EHDs were not seen in any abundance in glioblastomas, gliosarcomas, fibrillary astrocytomas, protoplasmic astrocytomas, or oligo-astrocytomas. Some glioblastomas, however, showed only EHDs in small numbers. Several anaplastic astrocytomas were associated with a large number of RGBs and/or EHDs, and they revealed only rare mitosis despite marked cellular pleomorphism. Although RGB and EHDs have different morphological features, the presence of these inclusions in abundance may represent either a degenerative change, a long-standing lesion, or an indolent growth of the astrocytic tumors. PMID- 7533471 TI - Ruthenium red selectively attenuates capsaicin induced vasodilation in pig nasal mucosa. AB - The effect of ruthenium red, a blocker of transmembrane Ca2+ fluxes, on vasodilation in pig nasal mucosa induced by capsaicin, nicotine, bradykinin or histamine was evaluated in an in vivo preparation. To decrease toxic systemic effects pretreatment with ruthenium red was performed locally, intra-arterially in a nasal artery. Pretreatment with two doses of ruthenium red was evaluated (0.25 and 2.5 mg kg-1). Ruthenium red, in the low dose, resulted in a marked attenuation of the capsaicin-induced nasal vasodilation while the effect on the nicotine-induced vasodilation was not as prominent. Pretreatment with the high dose significantly blocked the vasodilatory effects of capsaicin, nicotine, bradykinin and histamine. Our series indicates that low concentration ruthenium red selectively modulates capsaicin induced vasodilation in pig nasal mucosa in vivo, probably via a direct blocking effect on cation channels opened by capsaicin. A high concentration of ruthenium red may exert a general inhibitory effect on transmembrane Ca2+ transport. PMID- 7533473 TI - SH2 and SH3 domains in signal transduction. PMID- 7533472 TI - Recent advances in the pharmacotherapy of schizophrenia. AB - The major advance in the psychopharmacology of schizophrenia has been the rediscovery of clozapine and the development of other novel antipsychotic drugs, all of which are superior to typical neuroleptic drugs with regard to extrapyramidal symptoms. Clozapine, the best studied of these agents, is also superior in efficacy with regard to psychopathology and cognitive function and has been shown not to cause tardive dyskinesia. A variety of other novel agents, e.g., risperidone, olanazpine, amperozide, seroquel, sertindole, zaprisidone and melperone, must be further studied to establish their efficacy relative to clozapine or the typical neuroleptics, or both. It is likely that these novel agents will displace the typical neuroleptic drugs as the primary treatment of schizophrenia. PMID- 7533474 TI - Prostatic specific antigen. AB - PSA is a 34-kDa 240-amino-acid glycoprotein produced exclusively by prostatic epithelial cells. PSA is a serine protease, is a member of the kallikrein gene family, and has a high sequence homology with human glandular kallikrein. It has chymotrypsin-, trypsin-, and esterase-like activities. In the serum it is present mainly in a complex form with alpha 1-antichymotrypsin. It is secreted in the seminal plasma and is responsible for liquefaction of the seminal coagulum. The production of PSA proteins appears to be under the control of circulating androgens acting through the androgen receptors. The PSA gene is up-regulated predominantly by androgens at both the protein and mRNA levels. DRE causes minimal changes in the PSA level, while prostate massage, ultrasonography, systoscopic examination, and prostate biopsy can all cause clinically significant elevations. Other conditions, such as prostatitis, prostate intraepithelial neoplasia, acute urinary retention, and renal failure can also elevate the PSA level. The value of PSA as a screening tool is questionable because of the great deal of overlap in PSA levels between BPH and prostate cancer. However, if used in men over 50, in conjunction with DRE and/or ultrasonography, it may become a vital part of the early detection program. PSA's role in determining the clinical and pathological stage is also limited, in spite of the direct correlation between the pathological stage and the PSA level, because of great overlap in the PSA levels in various stages. The most important clinical utility of PSA is in monitoring patients after definitive therapy. PSA is most sensitive and reliable in the detection of a residual tumor, possibly recurrence, or disease progression following treatment, irrespective of the treatment modality. PSA can accurately predict the tumor status and can detect recurrence several months before its detection by any other method. PSA is also a very sensitive and specific immunohistochemical marker for tumors of prostatic origin. Compared to PAP, PSA is a more precise and meaningful marker in all clinical situations. With the development of ultrasensitive assays and the adoption of an international standard PSA calibrator, so that results from multicenter studies can be compared, PSA could become one of the most useful tumor marker in cancer biology. PMID- 7533475 TI - Lymphatic mast cell response and effect of compound 48/80 on popliteal lymph node reaction in rats following intracutaneous injection of Staphylococcus aureus. AB - To investigate the significance of mast cells in the popliteal lymph node during the development of an inflammatory response, rats were inoculated with 12 x 10(7) colony-forming units of Staphylococcus aureus in the hind foot pad. Numerical changes in mast cells were then measured in the corresponding popliteal lymph node. Six days after inoculation, despite the enlargement of the responding lymph node, a marked decrease in granulated mast cell number, relative to the contralateral node, was observed in the cortical and medullary compartments. Popliteal lymph nodes from rats treated with compound 48/80 and then inoculated with S. aureus showed a higher cortical and medullary hypertrophic response and a significant increase in degranulated/weakly basophilic mast cell number in the lymph node tissue. The findings suggest that (1) Staphylococcus aureus induces a reduction in granulated mast cell number in the cortical and medullary compartments of regional lymph nodes; (2) pretreatment with compound 48/80 appears to contribute to the lymphoid cell proliferation and the hypertrophic response of lymph nodes induced by S. aureus; and (3) granulated mast cells have a regulatory role on lymphoid cell proliferation. PMID- 7533476 TI - Study of the activation mechanism of adriamycin on rat mast cells. AB - Adriamycin (ADR) induces nonimmunological and noncytotoxic histamine release from peritoneal and pleural rat mast cells. This secretion is unaffected by the pretreatment with pertussis toxin, cholera toxin and benzalkonium chloride. Histamine release induced by compound 48/80, was markedly inhibited by pertussis toxin, cholera toxin, benzalkonium chloride and neuraminidase. The ADR dose response curve is significantly shifted to the right when cells were preincubated with the unspecific phosphodiesterase inhibitor IBMX. The activation of protein kinase C (PKC) with the phorbol esther TPA increases the response to ADR, while PKC inhibition with trifluoperazine decreases histamine release. The pretreatment of mast cells with okadaic acid did not modify the response to ADR. These results suggest that ADR elicits histamine release with a mechanism notably different from compound 48/80. PMID- 7533478 TI - Interferon production and immune response induction in apathogenic rabies virus infected mice. AB - Pathogenic parental rabies virus strain CVS (challenge virus standard) and its apathogenic variant RV194-2 were shown to differ in their ability to induce interferon (IFN) and immune response of the host. After intracerebral inoculation, IFN and antibody production was higher in the RV194-2 virus-infected mice than in the CVS infection. The enhancement of 2-5A synthetase activity, an IFN-mediated enzyme marker, showed biochemical evidence that IFN is active in both apathogenic and pathogenic infections. On the other hand, spontaneous proliferation in vitro of thymocytes and splenocytes from CVS virus-infected mice was strongly inhibited in contrast to the RV194-2 infection. In the CVS infection, the thymocyte proliferation was more affected than the splenocyte proliferation. However, in the RV194-2 infection, the thymocyte proliferation was higher than of the splenocytes. These results suggest a better performance of T cell response to the RV194-2 infection than to the CVS infection. This fact can be critical for an enhancement of antibody production in the apathogenic infection and subsequent virus clearance from the brain of RV194-2 virus-infected mice. PMID- 7533477 TI - Effects of beta 2-adrenergic agonists on isolated guinea pig lung mast cells. AB - The mast cell protective effects of the newly developed long-acting beta 2 agonists salmeterol and formoterol were compared with those of conventionally used beta 2-agonists, non-specific beta-agonists, disodium cromoglycate (DSCG) and theophylline. With the exception of DSCG, all the test agents inhibited ovalbumin-induced histamine release from enzymically dispersed guinea pig lung mast cells in a dose-dependent fashion. At the maximum concentration tested, theophylline produced the highest level of protection, inhibiting up to 90% of ovalbumin-induced histamine release whereas DSCG produced only 10% inhibition. The maximum inhibition produced by all the beta 2-agonists tested was around 45%. While salmeterol was equipotent with salbutamol, formoterol was at least a 100 fold more potent. Hence the present study confirmed the previously reported mast cell stabilizing actions of conventional beta 2-agonists and extended the observation to the newly developed long-acting analogues. PMID- 7533479 TI - Antibody response to hidden epitope of influenza A haemagglutinin elicited by anti-idiotypic antibodies. AB - Monoclonal antibody (MoAb) IIF4 defines an epitope on the HA2 part of influenza haemagglutinin (HA) (Russ et al., 1987). It was also found this epitope becomes fully accessible after pH 5 treatment of the antigen and is shared by strains of H3 subtype. In this study we found binding of MoAb IIF4 also to some strains belonging to H2, H4, H7, and H10 subtypes. We prepared rabbit polyclonal anti IIF4 anti-idiotype (anti-Id) antibody. In competitive assays, the inhibition potential of anti-Id was considerably higher than that of native HA. Anti-Id was used for the preparation of mouse Ab3 (anti-anti-IIF4) serum. Reactivity pattern of Ab3 with influenza virus strains differed from Ab1 in (i) appearance of binding to some strains of H2 and H7 subtype and (ii) decreased dependency of Ab3 binding on the pH forms of antigen. The reactivity of Ab1 and Ab3 with two amantadine-resistant virus mutants indicates that IIF4 epitope (and its related region recognized by Ab3) becomes accessible in consequence of destabilization of trimeric arrangement of HA and it also correlates with expulsion of N-terminus of HA2. PMID- 7533480 TI - Compatibility of hydromorphone hydrochloride with haloperidol lactate and ketorolac tromethamine. PMID- 7533481 TI - Automated extraction of acetylgestagens from kidney fat by matrix solid phase dispersion. AB - A new extraction method for the acetylgestagens medroxyprogesterone acetate (MPA), chloromadinone acetate and megestrol acetate, from kidney fat, has been developed. The method is a combination of matrix solid phase dispersion and solid phase extraction and is simpler and safer than previous methods, especially as it can be automated. The recovery was estimated as 59 +/- 5% (mean +/- standard deviation) for MPA. For screening purposes detection can be achieved using a commercially available enzyme immunoassay kit giving detection limits in the range of 1.0-2.0 ng g-1. PMID- 7533482 TI - Integrin and phosphotyrosine expression in normal and migrating newt keratinocytes. AB - BACKGROUND: Cells interact with the extracellular matrix through a family of cell surface receptors known as integrins. Ligand specificity of a given integrin is determined in part by the type of alpha and the type of beta subunit comprising it. Accumulating evidence suggests that integrin-ligand binding in some systems influences cell behavior through tyrosine phosphorylation of intracellular proteins. METHODS: In this study, we utilized immunohistochemistry to examine the expression of beta 1 and beta 4 integrin subunits as well as tyrosine phosphorylation in normal keratinocytes and in keratinocytes migrating to form a wound epithelium. An adhesion assay was used to determine if freshly isolated keratinocytes could interact with fibronectin and collagen. Polyacrylamide gel electrophoresis followed by immunoblotting was employed to compare beta 1 integrins in migrating and nonmigrating keratinocytes. RESULTS: In normal epidermis, beta 1 and beta 4 localized primarily to basal cells, where both subunits were generally distributed over all parts of the cell periphery. Except for a modest presence in suprabasal cells and a minimal presence adjacent to the epidermal basement membrane, phosphotyrosine (ptyr) had a similar distribution. In migrating keratinocytes, beta 1, beta 4, and ptyr localized most heavily at the interface between the forming wound epithelium and the wound bed. Adhesion assays using keratinocytes from normal epidermis revealed a population of cells that could specifically adhere and spread on fibronectin and type I collagen. Immunoblots of beta 1 subunits from normal and migrating keratinocytes showed no increase in amount of beta 1, nor did the apparent size of beta 1 change in migrating compared to normal cells. CONCLUSIONS: The heavy accumulation of beta 1 and beta 4 at the wound bed interface in migrating cells suggests that these subunits may be involved in attachments of migrating cells to extracellular matrix proteins in the wound. The accumulation of ptyr in the same region further suggests that integrin-ligand interaction in keratinocytes modulates cell behavior through phosphorylated proteins. The fact that freshly isolated newt keratinocytes could adhere and spread on fibronectin or collagen shows that these cells are constitutively activated. This view is supported by the absence of any evidence that the beta 1 in migrating keratinocytes is larger and therefore more mature than beta 1 in normal keratinocytes. By comparison, beta 1 integrins on human keratinocytes are not constitutively activated (Takashima and Grinnell, 1985; Toda et al., 1987; Guo et al., 1990, 1991), a difference that may explain why epidermal wound healing is faster in newts than in humans. PMID- 7533484 TI - Intravenous versus epidural administration of hydromorphone. Effects on analgesia and recovery after radical retropubic prostatectomy. AB - BACKGROUND: It remains unclear whether epidural administration of hydromorphone results in spinal analgesia or clinical benefit when compared with intravenous administration. Therefore, we undertook this study to determine whether epidural administration of hydromorphone resulted in decreased opioid requirement, improved analgesia, reduced side effects, more rapid return of gastrointestinal function, or shorter duration of hospital stay than intravenous administration. METHODS: Sixteen patients undergoing radical retropubic prostatectomy were randomized in a double-blind manner to receive either intravenous or epidural hydromorphone via patient-controlled analgesia (PCA) for postoperative analgesia. All patients underwent a standardized combined epidural and general anesthetic and all received ketorolac for 72 h postoperatively. To decrease variability, patients were cared for according to a standardized protocol and were deemed ready for discharge according to prospectively defined criteria. RESULTS: Patients in the intravenous PCA group required approximately twice as much opioid than the epidural PCA group (P < 0.008), but there were no differences between groups in pain scores or patient satisfaction. Epidural administration resulted in a greater incidence of pruritus (P = 0.02). Gastrointestinal function recovered quickly in all patients with little variation, and there were no differences between groups. All patients were deemed ready for discharge by the third postoperative day, and removal of surgical drains was the last discharge criterion reached in all patients. CONCLUSIONS: Our results indicate that epidural administration of hydromorphone results in spinally mediated analgesia. However, epidural administration did not provide significant benefits in terms of postoperative analgesia, recovery of gastrointestinal function, or duration of hospitalization. Furthermore, we suggest that radical retropubic prostatectomy no longer be used as a model to assess the effects of analgesic technique on postoperative recovery, because control of discharge criteria revealed that hospital discharge was primarily dependent on removal of surgical drains. PMID- 7533483 TI - Expression of cytokeratin mRNAs in normal human esophageal epithelium. AB - BACKGROUND: The cytokeratin (CK) pattern is accepted to be characteristic of a given epithelial cell or tissue. Specific changes in the CK pattern or in the expression of individual CKs may be characteristic in the early development of particular epithelial pathologies. Up to now no systematic hybridohistochemical study on the expression of CKs in normal human esophageal epithelium has been performed. Nevertheless, this knowledge may be of great importance for further research concerning the understanding of the structure and differentiation of normal esophageal epithelium and of the development of non-neoplastic and neoplastic esophageal malignancies. Therefore, we investigated the expression and distribution of nine different CK mRNAs throughout the normal human esophageal mucosa. METHODS: A non-radioactive in situ hydridization protocol was used to study the expression of CK mRNAs in fixed and paraffin-embedded human esophageal mucosa. Digoxigenin-labelled cRNA probes were produced by in vitro transcription of cDNA clones, coding for human CKs. RESULTS: In situ hybridization and immunodetection of the hybrids revealed a distinct but different distribution pattern for each specific CK mRNA. The described signal pattern was consistently found at all levels of the esophagus. We observed differences in the expression of some CK mRNAs between the interpapillar and papillar compartment of the esophageal epithelium. Mainly in the papillar regions some mRNAs are already expressed in more basally located cells in comparison with the interpapillar regions. Our results substantiate the hypothesis concerning the formation of papillae in the esophageal mucosa. We have also described some observations on the expression of CK mRNAs in fortuitous sections through excretory ducts of esophageal submucosal glands. CONCLUSIONS: The distinct, characteristic, and reproducible distribution pattern observed for each specific CK mRNA indicates that the expression of the genes encoding CKs in the esophageal epithelium as well depends on the cell proliferation, on vertical cell migration and differentiation, and on detachment from the basal lamina. The results presented should be considered as complementary to the already existing immunohistochemical results concerning the distribution of esophageal CK proteins. PMID- 7533486 TI - Effect of intravenous administration of hydroxyethyl-starch-deferoxamine on oxygen-derived free radical generation in cancellous bone specimens obtained from dogs. AB - The ability of IV administered hydroxyethyl-starch-deferoxamine to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. A core cancellous bone specimen 10 mm long and 5.6 mm in diameter was obtained, using aseptic technique, from the proximal portion of the humerus of 30 adult mixed-breed dogs. After procurement of the initial bone specimen, 10 dogs received a 10% solution of hydroxyethyl-starch-deferoxamine in 0.9% NaCl (50 mg/kg of body weight, IV), 10 dogs received an equivalent volume (5 ml/kg, IV) of a 10% solution of hydroxyethyl-starch in 0.9% NaCl, and 10 dogs received 0.9% saline solution (5 ml/kg, IV). A second core cancellous bone specimen was obtained from the contralateral humerus of each dog 45 minutes after treatment. All specimens were individually incubated in the spin trap alpha-phenyl-N-tert butylnitrone in Eagle's minimum essential medium, at 26 C for 45 minutes, then were frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum, which was analyzed for hyperfine splitting constants for radical identification. Each first-derivative spectrum was digitally double-integrated to obtain an area; these areas were used to compare intensities of the spin adducts. Difference in the area obtained before and after treatment for each dog was expressed as a ratio of that dog's pretreatment area ([pretreatment - posttreatment])/pretreatment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533487 TI - [The prevention of disorders due to congenital hypothyroidism: the experience of a neuropsychological follow-up]. AB - Early diagnosis and treatment of congenital hypothyroidism (CH) changed the prognosis for affected subjects. However it is still matter of controversy whether the neuropsychological development of early treated CH children is completely normal. In our study, even if the cognitive development is within the normal range, several disturbances were repeatedly observed in the neurological area, in language and in behavioural aspects. Severe neonatal hypothyroidism and low socio-cultural level of the family were risk factors for these disturbances; our results emphasize the relevance of a neuropsychological follow-up of early treated CH children. PMID- 7533488 TI - [The prevention of disorders due to iodine deficiency: the experience of neuropsychological screening]. AB - In this research we investigated some neuropsychological parameters in school-age children living in two areas in which mild iodine deficiency has been found in the past. In Study 1 no significant difference was found in cognitive (WISC-R Block Design test), visuo-motor (WISC-R Coding test modified) and fine distal movements tasks. On the other hand in a Simple Reaction Time task children living in iodine deficient area were significantly slower than controls. In Study 2 the same results were obtained in the group of children born before an extensive iodine prophylaxis program, but not in younger children born later. Our data show that exposure to mild iodine deficiency in fetal/neonatal life does not affect general cognitive performance but slows the speed of motor responses, which may be due to an impairment concerning neural transmission mechanisms. PMID- 7533489 TI - [Phase III clinical trial for esophageal cancer using radiotherapy]. AB - Phase III clinical trials using radiotherapy for esophageal cancer are reviewed. Concerning preoperative radiotherapy, results of 6 of 7 clinical trials failed to show its usefulness. Two randomized trials evaluating postoperative radiotherapy were performed. Both of them, however, proved negative. Chemoradiation has been used for inoperable esophageal cancer instead of radiotherapy alone. Combined modality therapy with radiation and Bleomycin was found to be useless. In early 1980, multiple drugs including 5-FU and radiation were compared with radiation alone, and 2 out of 3 clinical trials were positive. CDDP was then included in the chemoradiation regimen, and CDDP+5-FU and radiotherapy were found to yield survival benefits. Preoperative chemoradiation was tested in 4 randomized clinical trials. However, the results were entirely negative. Based on the results of these clinical trials, problems of treatment strategy for esophageal cancer in Japan are discussed in this article. PMID- 7533485 TI - Time-dependent efficacy of bacterial filters and infection risk in long-term epidural catheterization. AB - BACKGROUND: Epidural infection represents a serious albeit infrequent complication of long-term epidural catheterization. The catheter hub is regarded as the main point of entry for microorganisms among the three possible routes (hematogenous, insertion site, hub) of microbial colonization of the inserted catheter. The current study was aimed at evaluating whether frequent changing of antimicrobial filters carries an increased risk of catheter hub contamination and the time-dependent efficacy of commonly used antimicrobial filters after prolonged use. METHODS: In the first part of the study, a microbiologic survey (skin, filter, hub, and catheter tip) was performed weekly in a group of 47 patients with cancer bearing subcutaneously tunneled catheters managed at home. Subsequently, the time-dependent efficacy of 96 micropore filters (32 Portex, 32 Sterifix-Braun, 32 Encapsulon TFX-Medical) differing in surface areas and/or composition of the filtering membrane was evaluated in a laboratory study. Filters were perfused, under the usual conditions of clinical use (flow resistance, injection pressure, temperature), every 8 h up to 60 days, with 5 ml of two different analgesic solutions, either sterile or containing 1.5 x 10(5)/ml of Streptococcus milleri I. Eight filters of each type subsequently were flushed with a S. milleri suspension (0.5 McFarland) after 7, 14, 28, and 60 days of continuous perfusion, and the resulting filtrates were cultured. RESULTS: In 16 of 19 positive hub cultures, the same microorganisms (species, biotype, antibiotype) were cultured from skin and filters. A statistically significant positive trend was found between the number of filter changes and the rate of positive hub cultures (chi 1(2) trend 5.11; P = 0.02). A high correlation coefficient was found between number of positive skin cultures and number of positive filtrates (r = 0.88; P = 0.01) and between number of positive filtrates and number of positive hub cultures (r = 0.93; P = 0.003). Cultures obtained from Portex and Sterifix-Braun filters yielded no bacterial growth (64/64) throughout the study period. Cultures from Encapsulon TFX-Medical filters showed bacterial growth 2/8 at seventh day, 7/8 at the 14th day, and 16/16 from the 28th day onward. CONCLUSIONS: Our data indicate significant correlation between the incidence of catheter hub colonization and the filter-change frequency, when the skin close to the filter-hub connection is contaminated. Our results also show that Portex and Sterifix-Braun bacterial filters, when perfused with reduced volumes at low injection pressures, maintain an unmodified antimicrobial function for at least 60 days. Based on these data, it appears clinically feasible to reduce the frequency of filter changes during long-term epidural catheterization, with a consequent possible decrease of epidural catheter colonization. PMID- 7533492 TI - [Structure of O-specific polysaccharide chains of the Yersinia bercoviery O:10 lipopolysaccharide]. AB - O-specific polysaccharide composed of D-rhamnose and 3,6-dideoxy-4-C-(L- glycero 1-hydroxyethyl)-D-xylo-hexopyranose (yersiniose A) residues was obtained on mild acid degradation of the Yersinia bercovieri lipopolysaccharide. On the basis of 1H- and 13C-NMR data, methylation studies and Smith degradation, the following structure was suggested for the polysaccharide repeating unit: [formula: see text] PMID- 7533491 TI - Significance of tumour markers in colorectal carcinoma associated with Schistosomiasis. AB - Schistosomiasis as well as Colorectal Carcinoma are equally prevalent in Egypt. However, the role of Schistosomiasis as a risk factor for Colorectal Carcinoma is not well established. Three tumour markers have been assessed in 69 patients with large bowel disease. They were classified into five groups. Group 1 (A) included 21 cases with Schistosomal hepatic fibrosis. Group 2 (B) included 6 cases of ulcerative colitis. Group 3 (C) included 10 cases of adenomatous polypi of which 12 cases had Schistosomiasis. Group 4 (D) consisted of 21 cases of colorectal carcinoma, 12 cases had schistosomiasis in association with colorectal carcinoma constituting group 5 (E). Elevated CEA was observed in benign tumours but showed non significant difference in G4 and G5. Significantly increased AFP levels were evident in G1, G4, and G5. Significant increase of B-HCG was observed only in G4 and G5 indicating its significance as diagnostic index in case of malignancy. It has been observed that Schistosomal hepatic fibrosis induced increased levels of some of the tumour markers. Therefore, the factor of Schistosomal hepatic fibrosis should be considered during the assessment of tumour markers in colorectal carcinoma cases. PMID- 7533490 TI - Natural killer cells and gamma delta T cells in scleroderma: relationship to disease duration and anti-Scl-70 antibodies. AB - OBJECTIVES: To examine the expression of the natural killer (NK) antigen CD56, and T cell receptor delta chain antigen (TCR delta), expressed on the gamma delta T cell subset, in patients with scleroderma, and to correlate levels of expression with clinical characteristics. METHODS: Peripheral blood mononuclear cells (PBMCs) from 15 patients with scleroderma and 11 controls were obtained from heparinised blood on a ficoll/hypaque gradient, stained with monoclonal antibodies, and examined by flow cytometry for expression of CD56 and TCR delta. RESULTS: Overall, the proportion of PBMCs expressing CD56 in the patient group (14.4 (SEM 2.6)%) was not significantly different from controls (8.75 (2.6)%). The greatest levels of expression were found in patients late (more than three years) in their disease course (18.1 (3.3)%) and in patients who did not express anti-Scl-70 antibodies (17.1 (3.5)%). The proportion of gamma delta T cells was significantly lower in the patient group (1.61 (0.52)% v control 2.61 (0.46)%) (p < 0.05). Patients early in their disease or with anti-Scl-70 antibodies accounted for the reduction in gamma delta T cells (0.71 (0.29)% and 0.96 (0.41)% (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: This study emphasis that NK and gamma delta T cell numbers vary depending upon patient characteristics and may help explain prior contradictory reports. Decreased numbers of gamma delta T cells were seen in scleroderma patients, especially those with anti-Scl-70 antibodies and a disease duration of less than three years. PMID- 7533493 TI - Major histocompatibility complex class II gene associations with anti-U1 small nuclear ribonucleoprotein antibody. Relationship to immunoreactivity with individual constituent proteins. AB - OBJECTIVE: To better define immunogenetic associations with the anti-U1 ribonucleoprotein (U1 RNP) autoantibody response. METHODS: HLA class II alleles were determined by genotyping in 49 Japanese rheumatic disease patients with anti U1 RNP antibody and 43 race-matched healthy controls. Immunoreactivities to U1 RNP constituent proteins (70K, A, B/B', and C) were detected by immunoblots using purified HeLa cell Sm antigen, and antibody titer was determined by passive hemagglutination assay. RESULTS: DQB1*0302 was significantly more frequent in anti-U1 RNP-positive patients than in controls (43% versus 14%; odds ratio [OR] = 4.6, corrected P = 0.03). All anti-U1 RNP-positive patients had either a DQB1*0601, *0602, *0301, *0302, or *0303 allele, which share tyrosine at position 30, and the amino acid sequence Thr, Arg, Ala, Glu, Leu, Asp, and Thr at positions 71-77 in the DQB1 beta 1 domain. In contrast, one of these alleles was found in 81% of the controls (OR = 24, P = 0.002). In addition, anti-U1 RNP antibody was associated with unique DQB1*0302; DRB1*0401 haplotype. Anti-70K reactivity and antibody titer were positively associated with a basic amino acid residue, arginine or histidine, at position 13 (DR2 or DR4) and were negatively associated with the amino acid sequence Ile, Leu, Glu, and Asp at positions 67 70, which was present in some of the DR5-, DR6-, and DR8-associated alleles, in the DRB1 beta 1 domain. Anti-C reactivity was strongly associated with DR2, particularly with DRB1*1502. CONCLUSION: The several shared epitopes located on HLA-DRB1 and DQB1 genes control the anti-U1 RNP autoantibody response. PMID- 7533496 TI - Acquired epileptic aphasia: neuropsychologic follow-up of 12 patients. AB - A study of specific neuropsychologic, neurolinguistic, and behavioral features of acquired epileptic aphasia or Landau-Kleffner syndrome was conducted in a group of 12 patients followed-up for 2-15 years (mean: 8 yr). Seventy-five percent had exhibited some language disturbance prior to acquired epileptic aphasia. Even when 9 patients had normal electroencephalographic findings in the long-term course of the disease, only 3 achieved normal language. No patient with persisting electroencephalographic abnormalities recovered normal or near normal language. The need to perform detailed neurolinguistic and neuropsychologic evaluations in the work-up and follow-up of children with acquired epileptic aphasia is stressed. An adapted neuropsychologic profile battery proved to be practical and objective for the follow-up of these patients. PMID- 7533494 TI - E-selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis. Role of resident mast cell-derived tumor necrosis factor alpha. AB - OBJECTIVE: To assess endothelial cell activation in patients with systemic sclerosis (SSc). METHODS: Concomitant study of salivary gland biopsy tissues and sera for expression of E-selectin and its potent activator tumor necrosis factor alpha (TNF alpha), using immunostaining and enzyme-linked immunosorbent essay. RESULTS: E-selectin was overexpressed in SSc patients, but not in controls. TNF alpha was detected in mast cells. CONCLUSION: Mast cell-derived TNF alpha may contribute to endothelial cell activation in SSc. PMID- 7533495 TI - Discoordinate gene expression of aggrecan and type II collagen in experimental osteoarthritis. AB - OBJECTIVE: To quantify the gene expression of aggrecan core protein and type II collagen in an experimental animal model of osteoarthritis (OA). METHODS: Total RNA was extracted from the articular cartilage of unoperated knee joints and from OA joints produced by anterior cruciate ligament transection. The relative amounts of type II collagen and aggrecan core protein messenger RNA (mRNA) were evaluated by Northern blot analysis. RESULTS: Total RNA was elevated 2.5 times, aggrecan mRNA was elevated 2 times, and type II collagen mRNA was elevated 8 times, in OA knees compared to unoperated controls. CONCLUSION: Chondrocytes are activated metabolically in response to joint injury. Discoordinate gene expression of aggrecan and type II collagen is characteristic of early experimental OA, and we speculate that it may contribute to its pathogenesis. PMID- 7533497 TI - Effects of different irrigation liquids and times on articular cartilage: an experimental, biomechanical study. AB - To characterize the suitability of different solutions [6% Dextran, 5% sorbitol (I); 5% fructose (II); 5% mannitol (III); Ringer's solution (IV)] for arthroscopy, bovine knee articular cartilage specimens (n = 52) were immersed for 0, 2, 4, or 20 h before indentation creep testing, known as a sensitive probe for tissue degeneration. Immersion in liquid I for up to 20 h produced significant softening of articular cartilage [p < 0.05, Friedman two-way analysis of variance (ANOVA)]. Liquids II-III produced no statistically significant changes in the deformational characteristics of articular cartilage. After 2 h of immersion in liquid IV deformation increased and remained elevated over the observation period (p < 0.05, Friedman two-way ANOVA). Based on these results, the first and most remarkable softening of cartilage took place with Ringer's solution as compared with nonionic solutions. Therefore, the nonionic solutions, such as 5% fructose or mannitol, may have potential for use as an irrigation liquid during arthroscopic procedures. PMID- 7533498 TI - Fas and Fas ligand: lpr and gld mutations. AB - Fas ligand (FasL) is a death factor that binds to its receptor, Fas, and induces apoptosis. Two mutations that accelerate autoimmune disease, lpr and gld, are known to correspond to mutations within genes encoding Fas and FasL, respectively. Here, Shigekazu Nagata and Takashi Suda summarize current knowledge of Fas and FasL, and discuss the physiological role of the Fas system in T-cell development, cytotoxicity and cytotoxic T lymphocyte (CTL)-mediated autoimmune disease. PMID- 7533499 TI - Combinatorial libraries: new insights into human organ-specific autoantibodies. AB - The recent application of immunoglobulin (Ig) gene combinatorial library technology has led to a logarithmic increase in information concerning human, disease-associated, organ-specific autoantibodies of the IgG class. As reviewed here by Basil Rapoport, Stefano Portolano and Sandra McLachlan, the molecular cloning, analysis and expression of the genes for increasing numbers of these human, monoclonal autoantibodies is providing new insight into the genetic background and epitopic repertoires of such molecules. PMID- 7533500 TI - Decay-accelerating factor is expressed in the human endometrium and may serve as the attachment ligand for Dr pili of Escherichia coli. AB - PROBLEM: We evaluated the hypothesis that different tissue substructures in uteri may express decay accelerating factor (DAF), a complement regulatory protein that also may serve as ligand for bacterial attachment. METHOD: Purified Dr pili, anti Dr pili IgG, anti-DAF (SCR-3) IgG, and fluorescein-isothiocyanate-conjugated secondary IgG were used for binding and inhibition experiments. RESULT: We observed staining of endometrial glands, spiral arterioles, and myometrial arteries with Dr adhesin (pili) and anti-DAF (SCR-3) IgG, and found variation in distribution and amount of Dr ligands in different individuals. Anti-DAF (SCR-3) IgG blocked the binding of Dr pili to the endometrium. CONCLUSION: Presence of DAF in endometrium may protect tissues from complement-induced damage. Differences between individuals in DAF density in the endometrium may affect sensitivity to attachment of Dr-bearing E. coli and/or complement activation. PMID- 7533501 TI - The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term. AB - PROBLEM: Subclinical microbial invasion of the amniotic cavity occurs in 18.8% of women with term labor and intact membranes and in 34% of patients with term PROM and is a risk factor for the development of puerperal infection related morbidity. Although amniotic fluid white blood cell count, interleukin-6 determination, and Gram stain examination have been used for the diagnosis of intrauterine infection in patients with preterm labor and preterm premature rupture of membranes, no information is available about the accuracy and specific cut-off values for these tests in patients at term. The purpose of this study was to compare the performance of the amniotic fluid Gram stain examination, white blood cell count, and interleukin-6 determination in the identification of microbial invasion of the amniotic cavity in patients at term with and without PROM. METHOD: Amniotic fluid was retrieved from 148 patients with term gestations (90 patients with spontaneous labor and intact membranes and 58 patients with PROM). Samples were cultured for bacteria and Mycoplasma species. Amniotic fluid Gram stain, white blood cell count, and interleukin-6 determinations (ELISA, sensitivity: 43 pg/ml) were performed in all samples. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Analysis was conducted using Mann-Whitney U test, Fisher's exact test, receiver operating characteristic curves and logistic regression. RESULTS: Patients with spontaneous labor and intact membranes: The prevalence of microbial invasion of amniotic cavity in this group was 15.6% (14/90). The most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determination (sensitivity for: interleukin-6 > or = 5.7 ng/ml = 86%, white blood cell count > or = 20 cells/mm3 = 64%, Gram stain = 28%). The most specific test was the Gram stain of the amniotic fluid (specificity for: Gram stain = 84%, interleukin-6 = 79% and white blood cell count = 63%). Multiple logistic regression demonstrated that amniotic fluid interleukin-6 concentration was the only covariate that retained statistical significance when intrauterine infection was used as outcome variable. Patients with PROM: The prevalence of a positive amniotic fluid culture in this group was 39.7% (23/58). Logistic regression demonstrated that only interleukin-6 retained a significant relationship with the results of amniotic culture when all variables were entered simultaneously into a model to predict amniotic fluid culture results. The most sensitive tests for the detection of intrauterine infection were interleukin-6 determination and white blood cell count (sensitivity for interleukin-6 > or = 3.4 ng/ml and white blood cell count > or = 20 cells/mm3 = 69.6% for both). The most specific test was Gram stain (97.1%). CONCLUSIONS: Amniotic fluid interleukin-6 determination is the best rapid test for the detection of microbial invasion of the amniotic cavity in patients at term with and without PROM. When this test is not available, amniotic fluid Gram stain and white blood cell count represent valid diagnostic tools to assess the microbial state of amniotic cavity. PMID- 7533502 TI - Structure and function of the mouse insulin-like growth factor binding protein 5 gene promoter. AB - The actions of insulin-like growth factors I and II (IGF-I and -II) are modulated by interactions with one or more of a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of the six known IGFBPs, is a 252-amino acid protein that has been shown both to potentiate and inhibit IGF action. In previous studies, we have cloned and characterized the mouse IGFBP-5 gene and demonstrated that it is expressed in a hierarchical pattern in different adult mouse tissues and during rodent embryonic development. In this report, we describe the initial analysis of the IGFBP-5 gene promoter. By transient gene transfer studies, we show the orientation-specific activity of DNA fragments containing from 31 to 4,100 bp from the 5'-flanking region of the mouse IGFBP-5 gene in directing expression of the heterologous reporter gene luciferase in Hep G2 cells. DNA fragments with only 156 bp of 5'-flanking sequence mediated over 60% of maximal promoter activity, and a segment containing the TATA box and the first 120 bp of exon 1 still conferred some promoter function. Within the highly active 156-bp region, we identified a 37-bp segment from -70 to -34 that exhibited specific binding in DNase I footprinting and gel-mobility shift experiments with Hep G2 nuclear protein extracts. The footprinted region, which is almost completely conserved in the rat and human IGFBP-5 genes, was responsible for at least 70% of the activity of the intact promoter, as evidenced by the deleterious consequences of small internal deletions within this sequence on promoter function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533503 TI - Enhancer sharing in a plasmid model containing the alpha-fetoprotein and albumin promoters. AB - It has been proposed that the alpha-fetoprotein (AFP) enhancers also regulate the adjacent albumin gene, since the -10 kbp albumin enhancer is inactive in a number of cell lines that express albumin. In transfection experiments, the AFP enhancers strongly stimulate the albumin promoter in cells that silence the AFP promoter. These observations led us to develop a plasmid model of AFP-albumin gene switching, in which the albumin and AFP promoters would compete for the three AFP enhancers. However, when AFPCAT + ALBgal genes were combined with the AFP enhancers in one plasmid, both genes were driven at full activity. There was no change in the relative promoter expression over a wide range of transfected DNA concentrations, demonstrating that relative promoter activity was independent of DNA concentration and of promoter concentration, and that neither promoter was limiting the expression of the other. In contrast, a control plasmid containing two albumin promoters showed mutual inhibition, indicating the expected promoter competition. The albumin and AFP promoters noncompetitively shared the three enhancers on this plasmid, resulting in high levels of transcription from both promoters. PMID- 7533507 TI - Anionic sites on the free surface of the peritoneal mesothelium: light and electron microscopic detection using cationic colloidal iron. AB - To examine charged sites on the peritoneal free surface, cationic and anionic colloidal iron methods were applied. Light microscopy of the human and mouse specimens using cationic colloidal iron staining at pH 1.5-7.3 and successive ferrocyanide treatment resulted in a distinct Prussian blue reaction on the free surface of the parietal, mesenteric and visceral peritonea at all the examined pH values. In transmission electron microscopy of specimens stained with cationic colloidal iron at pH 1.5, colloidal particles accumulated in a dotted fashion on the free surface of the mesothelial cells. At pH 7.3, colloidal particles were deposited as numerous fine strands (100-300 nm in length), whose ends often attached to the luminal aspect of the mesothelial cell membrane. Anionic colloidal iron stain at pH 7.2 gave no deposition of colloidal particles on the mesothelial free surface. Priorly methylated samples lost stainability of the peritoneal free surface to cationic colloidal iron staining at pH 1.5 or 2.5, while methylated-saponified sections recovered it. Pretreatment with neuraminidase inhibited the cationic colloidal iron staining at pH 1.5 on the mesothelial free surface. These results indicate that the mesothelial surface anionic sites ionizing at pH 1.5 are mostly due to the carboxyl group of sialic acid. It is suggested that the peritoneal free surface substance stained with the cationic colloidal iron may be membrane-associated sialomucin, whose rich negative-charged sites may repulse each other to prevent peritoneal adhesion or to maintain peritoneal cavity. PMID- 7533505 TI - Administration of high-dose aprotinin during nonprimary cardiovascular surgery: case reports and review of the literature. AB - The perioperative management of two patients undergoing complex "redo" cardiac surgical procedures are presented. The management of both patients included the prophylactic administration of aprotinin via a "compassionate use" protocol. Aprotinin, a serine protease inhibitor, has been shown to limit the exposure to blood and blood products in patients undergoing high-risk cardiac surgical procedures. In late December 1993, the Food and Drug Administration approved aprotinin for administration to cardiac surgical patients considered at high risk for post-cardiopulmonary bypass coagulopathies. Indications for the administration of aprotinin, as well as a brief review of the literature relating to the perioperative administration of aprotinin, are included. PMID- 7533506 TI - Identification of a pharmacophore for nucleoside analog inhibitors directed at HIV-1 reverse transcriptase. AB - An 'active analog' approach to receptor mapping was used to identify a pharmacophore for a set of thymidine nucleoside analog inhibitors of HIV-1 reverse transcriptase. The preliminary results indicate that the O2, O4', and O5' atoms are capable of adopting a unique pharmacophoric pattern which may be the key to their recognition by reverse transcriptase. PMID- 7533508 TI - Second-line chemotherapy in epithelial ovarian carcinoma: platinum again? Taxanes? How to choose? PMID- 7533509 TI - Third meeting of the DCIS Working Party of the EORTC (Fondazione Cini, Isola S. Giorgio, Venezia, 28 February 1994)--Conference report. PMID- 7533510 TI - Architecture of native human alpha 2-macroglobulin studied by cryoelectron microscopy and three-dimensional reconstruction. AB - The architecture of the native human alpha 2-macroglobulin was studied by cryoelectron microscopy and image processing techniques. The lip, padlock, doughnut, and four-petaled flower views of this homotetrameric proteinase inhibitor were observed in the frozen-hydrated specimen, and a new view, termed eye view, was also characterized. The present three-dimensional reconstruction demonstrates that all these electron microscope views derive from a single three dimensional structure. The molecule is composed of two horizontal bodies and of two oblique arches, which border a large central cavity. The polymorphism and the flexibility of the native alpha 2-macroglobulin are discussed. PMID- 7533511 TI - Aprotinin therapy. PMID- 7533512 TI - Is 51W89 an improvement compared with atracurium? PMID- 7533504 TI - Hypoplastic left heart syndrome: anesthetic care prior to transplantation or surgical palliation. AB - Hypoplastic left heart syndrome is the most common lethal cardiac defect in neonates. Options for treatment include cardiac transplantation and surgical palliation. When cardiac transplantation is chosen as the preferred option, a considerable delay may occur until a suitable donor is available. During this time, anesthetic care may be required for various surgical procedures. Associated anomalies seen in these infants and the anesthetic implications imposed by the abnormal cardiac anatomy are discussed. PMID- 7533513 TI - Intrabronchial injection, an unusual complication of interpleural analgesia. PMID- 7533514 TI - Nitric oxide and focal cerebral ischemia: multiplicity of actions and diverse outcome. AB - The original observation that inhibitors of nitric oxide (NO) synthesis can antagonize glutamate toxicity in cell culture has led to extensive investigation of the role of NO in the pathophysiology of cerebral ischemic injury in vivo. However, studies of the efficacy of NO synthase inhibitors in models of focal cerebral ischemia have generated widely disparate findings, ranging from dramatic neuroprotection to exacerbation of ischemic damage. This review summarizes these studies and proposes that their apparently contradictory findings can be reconciled by viewing the results as a continuum of response that reflects the many and diverse physiological actions of NO. Thus, differences in experimental design between studies can alter the balance between these NO-controlled processes and result in the transformation of an overt neuroprotective effect of NO synthesis inhibition into one of exacerbation of ischemic injury. Thus, this review also identifies some of the most important physiological and pathophysiological functions of NO (and the consequences of their inhibition by NO synthase inhibitors) that may interact to determine outcome after a focal cerebral ischemic insult. A clearer appreciation of the potential therapeutic utility of both NO synthesis inhibitors and NO donors will emerge only when the complexity of their effects on mechanisms that interact to determine the extent of ischemic damage in vivo are more fully defined and understood. PMID- 7533515 TI - Site-specific implantation in the milky spots of malignant cells in peritoneal dissemination: immunohistochemical observation in mice inoculated intraperitoneally with bromodeoxyuridine-labelled cells. AB - To investigate the site-specific implantation of cancer cells in peritoneal dissemination, we inoculated CDF1 mice intraperitoneally with mouse P388 leukaemia cells labelled with bromodeoxyuridine (BrdU) and then observed immunohistochemically the distribution of the cells in the greater omentum taken from the mice using an anti-BrdU antibody. We found the BrdU-labelled cells infiltrating selectively into the milky spots in the omentum. Furthermore, we intraperitoneally inoculated the BrdU-labelled P388 cells at 10(5), 10(6) and 10(7) cells per mouse into three groups of ten CDF1 mice and then quantified the distribution of the BrdU-labelled cells by counting the number of the labelled cells per unit area at each milky spot and non-milky spot site in the omentum. Inoculations of 10(5), 10(6) and 10(7) BrdU-labelled P388 cells per mouse resulted in 15.8 +/- 13.3, 120 +/- 46.5 and 504 +/- 208 cells mm-2 respectively in the milky spot sites and 9.14 x 10(-3) +/- 1.58 x 10(-2), 1.14 x 10(-1) +/- 7.82 x 10(-2) and 7.07 x 10(-1) +/- 5.98 x 10(-1) cells mm-2 respectively in the non-milky spot sites. The ratios of the mean labelled cell numbers in the milky spot sites vs those in the non-milky spot sites were 1728:1, 1049:1 and 713:1 respectively. In all cases, there were statistically significant differences in the number of BrdU-labelled cells mm-2 between milky spot sites and non-milky spot sites. However, the ratios decreased as the numbers of inoculated cells increased. In addition, we inoculated C57/BL mice intraperitoneally with B-16 PC melanoma cells, which were easily differentiated from the other cells by the intrinsic black melanin, and examined the distribution of the cells macro- and microscopically. The B-16 PC melanoma cells were also found to be infiltrating preferentially into the milky spots in the omentum. These results suggest that cancer cells seeded intraperitoneally specifically infiltrate the milky spots in the early stages of peritoneal dissemination. PMID- 7533517 TI - Randomised comparison of fluorouracil, epidoxorubicin and methotrexate (FEMTX) plus supportive care with supportive care alone in patients with non-resectable gastric cancer. AB - A phase III randomised study, comparing treatment with fluorouracil, epidoxorubicin and methotrexate (FEMTX) with the best supportive care, was conducted in patients with unresectable or metastatic gastric cancer. During the period from July 1986 to June 1992, 41 patients were randomised to receive FEMTX or best supportive care. MTX was given in a dose of 1500 mg m-2 intravenously (i.v.) followed after 1 h by 5-FU 1500 mg m-2 i.v. on day 1; leucovorin rescue was started after 24 h (30 mg orally every 6 h for 48 h) and epidoxorubicin 60 mg m-2 i.v. was administered on day 15. In addition both groups received tablets containing vitamins A and E. Response rates for FEMTX were as follows: complete response (CR), 19% (4/21); partial response (PR), 10% (2/21); no change (NC), 33% (7/21); and progressive disease (PD), 24% (5/21). Response rates in the control group were: NC, 20% (4/20); and PD, 80% (16/20). Increased pain was observed in one patient in the treated group and in 11 patients in the control group within the first 2 months. WHO grade III/IV toxicity in the chemotherapy group was as follows: nausea/vomiting 40%, diarrhoea 10%, stomatitis 15%, leucopenia 50% and thrombocytopenia 10%. One possible treatment-related death was due to sepsis. The median time to progression in the FEMTX group was 5.4 months [95% confidence interval (CI) 3.1-11.7 months], but only 1.7 months in the control group (95% CI 1.2-2.7 months) (P = 0.0013). Similarly, the FEMTX group displayed significantly (P = 0.0006) prolonged survival compared with the control group, i.e. median survival 12.3 months (95% CI 7.1-15.6 months) vs 3.1 months (95% CI 1.6-4.6 months). In conclusion, FEMTX combined with vitamin A and E is a fairly well tolerated treatment, giving a response rate of 29% in patients with advanced gastric cancer, and also prolonging patients' survival. It can be used as a reference treatment in testing new investigational combinations. PMID- 7533518 TI - The use of granulocyte colony-stimulating factor to deliver four cycles of ifosfamide and epirubicin every 14 days in women with advanced or metastatic breast cancer. AB - Twenty patients with locally advanced or metastatic breast cancer were treated with four cycles of ifosfamide/mesna 5 g m-2 and epirubicin 60 mg m-2 every 14 days with granulocyte colony-stimulating factor (G-CSF, Filgrastim). Complete remission occurred in six out of the 20 patients (30%, 95% confidence interval 12 54%) and there were 12 partial responders (60%, 95% confidence interval 37-81%), thus giving an overall response rate of 90% (95% confidence interval 63-97%). Two patients had progressive disease. The median duration of response for those patients with metastatic disease was 7.3 (1.3-20.1+) months. The median survival time for these patients was 15 (5.3-27.9+) months. Of the four patients treated with locally advanced disease three achieved a complete clinical response and one a partial response. Three out of four of these patients subsequently underwent a mastectomy, and in one of these no viable tumour was seen. Our conclusion is that this regimen is excellent palliation for metastatic disease and possibly useful neoadjuvant treatment. PMID- 7533520 TI - Symptoms at presentation for treatment in patients with lung cancer: implications for the evaluation of palliative treatment. The Medical Research Council (MRC) Lung Cancer Working Party. AB - The ten most frequently reported pretreatment symptoms on the Rotterdam Symptom Checklist, which was completed by more than 650 patients entering two MRC Lung Cancer Working Party multicentre randomised trials, included general symptoms (tiredness, lack of appetite) and psychological distress (worry, anxiety) in addition to disease-related chest symptoms (cough, shortness of breath). Although the number and severity of symptoms increased with worsening performance status, the commonest symptoms were found to be virtually the same for patients with small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), and for different grades of performance status. Women with NSCLC reported more psychological symptoms than males, but this difference was much less evident in patients with SCLC. Thus, in order to assess fully the benefit of palliative treatments in patients with lung cancer, account must be taken of all symptoms at presentation, in addition to the traditionally recognised chest symptoms. PMID- 7533519 TI - Treatment with para-chlorophenylalanine antagonises the emetic response and the serotonin-releasing actions of cisplatin in cancer patients. AB - To test the role of serotonin in chemotherapy-induced nausea and emesis, ten cancer patients were pretreated with the serotonin synthesis inhibitor para chlorophenylalanine (PCPA). PCPA (2 g 8 hourly for 2 or 3 days prior to cisplatin) reduced the spontaneous urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), inhibited the increase in urinary 5-HIAA induced by cisplatin and markedly attenuated the acute period of nausea and vomiting associated with the cytotoxic drug. These results indicate that gastrointestinal serotonin mediates cisplatin-induced emesis and that the amount of serotonin released by cisplatin is a major factor in determining the severity of the acute period of emesis experienced by the patient. PMID- 7533516 TI - Molecular cloning of a cell-surface glycoprotein that can potentially discriminate mesothelium from epithelium: its identification as vascular cell adhesion molecule 1. AB - It has long been a practical problem for surgical pathologists to distinguish mesothelium from epithelium in order to make a positive diagnosis of mesothelioma. In this study, we developed a new monoclonal antibody, designated MS-2761 (IgG1, k), against cultured non-neoplastic mesothelial cells. Immunohistochemistry and slot-blot analysis revealed that this monoclonal antibody reacted with 100% (12/12) of benign and malignant mesothelioma tissues and a mesothelioma cell line, but not with 99% (77/78) of epithelial tumour tissues and 97% (33/34) of epithelial tumour cell lines. A gene encoding the cell surface antigen defined by this monoclonal antibody was isolated from a mesothelial cell cDNA library constructed with a mammalian cell expression vector through transfection of Cos-7 cells and immunoselection by panning. DNA sequencing and a database search revealed that the gene was identical to vascular cell adhesion molecule 1 (VCAM1, also referred to as INCAM110). The prominent VCAM1 transcript in mesothelium was 3.2 kb in size with seven Ig-like domains, in addition to a minor transcripts with six Ig-like domains. This monoclonal antibody potentially discriminates mesothelium from epithelium and may become a tool for differential diagnosis of mesothelioma. PMID- 7533522 TI - Have expandable metallic stents replaced operation for malignant biliary obstruction? PMID- 7533521 TI - Acute phase response in patients with uncomplicated and complicated endoscopic retrogradic cholangiopancreaticography. AB - Acute phase response after endoscopic retrogradic cholangiopancreaticography (ERCP) was studied in 42 patients with suspected pancreatic or biliary diseases. In uncomplicated cases acute phase response determined by serum C-reactive protein levels was rare and did not parallel the serum amylase or lipase levels. In three of the these 42 patients, post-ERCP pancreatitis developed and CRP levels elevated sharply and paralleled the degree of pancreatitis. Six additional patients outside of this prospective study with post-ERCP-pancreatitis and daily CRP determinations were used to determine the CRP-response curve in post-ERCP pancreatitis. PMID- 7533523 TI - Chromosome analysis of brain tumors in childhood. AB - We performed a chromosome analysis of 26 pediatric brain tumors, including 20 primitive neuroectodermal tumors (PNETs). 5 astrocytomas, and 1 immature teratoma. Specimens were treated with collagenase, placed in overnight or short term cultures, and harvested for chromosome analysis. Numerical and/or structural abnormalities were noted in 14 of the 20 PNETs and 4 of the 5 astrocytomas. In 13 PNETs, so-called medulloblastoma in the cerebellum, an i(17q) was the most frequent structural abnormality, accounting for 30% (4/13). Double minute chromosomes (dmin) were observed in one tumor. Near-diploidy was demonstrated in three of these PNETs, hyperdiploidy in three, and near-tetraploidy in three. We could not find any correlation of these cytogenetic findings with the prognosis. In the remaining seven PNETs other than medulloblastoma, the karyotypes of five PNETs demonstrated a variety of numerical and structural abnormalities. As to the astrocytomas, losses of chromosomes 7 and 9 with dmin were observed in two, and structural abnormalities of chromosomes 1 and 17 were also observed in two tumors. In our limited cases, however, we could not find the same chromosome abnormalities that are well known in adult astrocytomas. A congenital immature teratoma showed hyperdiploidy with increased numbers of chromosomes 3, 6, and 12. We conclude that i(17q) is an important chromosome abnormality in medulloblastomas, and that the oncogenesis of pediatric astrocytomas might be different cytogenetically from that of adult astrocytomas. PMID- 7533524 TI - Integrated YAC contig containing the 3p14.2 hereditary renal carcinoma 3;8 translocation breakpoint and the fragile site FRA3B. AB - An extended YAC contig has been developed for the 3p14 region containing the hereditary renal carcinoma 3;8 translocation breakpoint and the 3p14.2 fragile site FRA3B. This region of chromosome 3 has been implicated by chromosomal translocation, deletion, and loss of heterozygosity in the pathogenesis of several malignant diseases. The contig allows accurate positioning of candidate genes, polymorphic markers, and other 3p rearrangements within this region. The contig, spanning approximately 6 Mb of DNA, contains 51 YACs identified by 27 markers, including a subset of CA repeats located in the 3p14.1-14.2 interval. The order of CA microsatellites, derived from marker content of the YACs, is in agreement with the order previously determined by genetic linkage studies. We find that the protein-tyrosine phosphatase gamma gene, PTPRG, is located minimally 1 Mb proximal to the t(3;8) breakpoint. The more proximal 3p homozygous deletion in the small-cell lung cancer cell line, U2020, is more than 5 Mb from the site of the 3;8 translocation. This integrated physical and genetic map provides a framework for further investigations of malignant diseases associated with proximal 3p loss. In addition, the positioning of separate 3p14.2 aphidicolin-induced breakpoints suggests that FRA3B may represent a region rather than a single site. PMID- 7533525 TI - Delineation of two distinct deleted regions on chromosome 9 in human non-melanoma skin cancers. AB - The mapping of the naevoid basal cell carcinoma syndrome (NBCCS) and the Ferguson Smith syndrome to the same region on chromosome arm 9q has led to speculation that the two conditions may reflect different mutations within the same gene. Loss of heterozygosity of 9q alleles in both familial and sporadic basal cell carcinomas (BCCs) suggests that the NBCCS gene on 9q is acting as a tumour suppressor gene. Although LOH of 9q markers has not been studied in squamous cell neoplasms from patients with the Ferguson-Smith syndrome, chromosome 9 allele loss has been reported in sporadic squamous cell carcinomas (SCCs) of the skin. In order to characterise further the deleted region on chromosome 9 in BCCs and SCCs of the skin we have examined a series of non-melanoma skin cancers using a panel of highly informative microsatellite markers. Forty-four BCCs and 49 SCCs were studied. Loss of heterozygosity of one or more 9q markers was seen in 33 of the 44 BCCs. Only 4 of the 33 BCCs with 9q loss showed loss of 9p markers. Twenty two BCCs showed loss of all informative 9q markers. Partial or interstitial 9q deletions were seen in 5 BCCs, and in 3 of these 5 BCCs the breakpoint occurred within the currently defined NBCCS locus. Chromosome 9 loss was seen in 16 of 49 SCCs. In contrast to the low frequency of 9p loss in BCCs, LOH of 9p markers was a common finding in SCCs, occurring in 15 of the 16 SCCs with chromosome 9 loss. In 5 SCCs 9p loss occurred with retention of 9q alleles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533526 TI - AML1 fusion transcripts in t(3;21) positive leukemia: evidence of molecular heterogeneity and usage of splicing sites frequently involved in the generation of normal AML1 transcripts. AB - The t(3;21)(q26;q22) is associated with chronic myelogenous leukemia in blast crisis (CML-BC), leukemia evolving from (therapy-related) myelodysplasia, and with leukemia following other hematopoietic proliferative diseases. Molecular cytogenetic analysis and cloning of a few t(3;21) cases indicate that the breakpoints are quite heterogeneous even within a specific clinical phenotype. Interestingly some of the (3;21) breakpoints involve the AML1 gene previously found rearranged in the t(8;21) associated with acute myelogenous leukemia. AML1 is related to the Drosophila gene runt and is the human counterpart of the gene for the alpha subunit of the nuclear polyoma enhancer binding protein (PEBP2) also known as the core binding factor (CBF). In the t(3;21) AML1 was found rearranged with EAP, a gene on chromosome 3 encoding a small ribosomal protein, as well as with EV11, another gene on chromosome 3. Here we report our study of six cases of t(3;21). By using fluorescence in situ hybridization (FISH) analysis and AML1 probes we could conclude that at least in two CML-BC cases the breakpoint occurred in the AML1 intron that is disrupted by the t(8;21). An AML1/EAP fusion transcript, different from the one described in a therapy-related myelodysplasia, was detected in both CML-BC cases. This transcript is expected to result in a predicted protein containing the AML1 nuclear binding domain with an attached stretch of 17 amino acids unrelated to the EAP small ribosomal protein. In the other t(3;21) patients we could not detect an AML1/EAP transcript or an AML1/EV11 transcript. This result suggests heterogeneity of the t(3;21) at the molecular level. The AML1 chimeric transcripts identified so far, both in the t(3;21) and in the t(8;21), diverge from the normal transcripts either after exon 5 or exon 6. Here we show that in normal AML1 transcripts different splicing events are seen to occur after AML1 exon 5 as well as exon 6. PMID- 7533527 TI - Nonrandom loss of human chromosome 3 fragments from mouse-human microcell hybrids following progressive growth in SCID mice. AB - Microcell hybrid lines of A9 mouse fibrosarcoma containing complete or partially deleted human chromosomes 3 (chr. 3) were inoculated into SCID mice. Cell lines derived from the tumors were examined by fluorescent in situ hybridization for the status of the transferred human chromosome and by PCR for marker loss. The SCID tumors arising after the inoculation of 10(5) cells were passaged serially in vivo and regularly showed loss of four markers; D3S1029 (3p21.3-21.2), AP20R (3p22-21.3, D3S32 (3p21.3-p21.2), and THRB (3p24). This regularly deleted region is bordered by markers GNA12 (3p21.1-p21.3) and VHL (3p25) that were maintained in a fraction of tumors. Fragments derived from the long arm of chromosome 3 and corresponding markers in the 3q26-q28 region were retained in all tumors. Our findings may be related to the postulated presence of tumor suppressor genes in the 3p24-p21 region as indicated by the frequent deletion of this region in renal and small cell lung carcinomas and other solid tumors. The technically cumbersome identification of suppressor genes may be supplemented by an "elimination test" based on analogous principles. PMID- 7533528 TI - Molecular cloning of a t(11;14)(q13;q32) translocation breakpoint centromeric to the BCL1-MTC. AB - In B-cell malignancies, the t(11;14)(q13;q32) at the 11q13 BCL1 locus is characterized by a scattering of breakpoint sites along a 100 kb genomic region, between the BCL1 major translocation cluster (MTC) and the PRAD1 (also termed cyclin D1 or CCND1) gene. Recently, the 11q13 breakpoint region was extended on both sides, centromeric to the MTC and telomeric to PRAD1. We report here the molecular cloning of a new t(11;14) breakpoint site, 20 kb centromeric to the MTC, from a patient with prolymphocytic leukemia. We subcloned a non-repetitive DNA fragment near the breakpoint and mapped this new 11q13 probe (pHO11c) relative to already identified breakpoint sites, using long- and short-range physical mapping within the BCL1 locus. Rearrangements in the BCL1 locus are associated with deregulation of the PRAD1 gene, which is often overexpressed, particularly in mantle-cell malignancies. The detectable but weak PRAD1 expression in the case we present suggests that this breakpoint centromeric to the MTC still lies inside the BCL1 locus boundaries. We think that attention should be focused on this region centromeric to the BCL1-MTC, where the investigation of previously unidentified translocations may increase understanding of the PRAD1 gene deregulation in t(11;14) associated pathologies. PMID- 7533531 TI - Chromosome aberrations in choroid plexus papillomas. AB - Karyotypic data on choroid plexus papillomas are scarce and, to date, have revealed no consistent aberrations. We karyotyped a choroid plexus papilloma which was characterized by a stemline of 52,XX, + 7, + 11, + 12, + 12, + 15, + 18. Additional copies of chromosomes 16, 17, and 20 were also observed in a significant proportion of the metaphase cells analyzed. Based upon this index case, we retrospectively analyzed eight additional choroid plexus papillomas by fluorescence in situ hybridization by using pericentromeric probes to chromosomes 7, 11, 12, 15, 16, 17, 18, and 20. Extra hybridization signals were observed in five of the eight cases examined. All five cases had extra signals with the chromosome 7 probe, four cases had extra signals with the chromosome 12 probe, and three cases had extra signals with the chromosome 15, 17, and 18 probes. The overall DNA content of these same cases (as determined by image analysis) suggests that gains of additional chromosomes other than those examined may be present. PMID- 7533530 TI - Physical mapping of the uterine leiomyoma t(12;14)(q13-15;q24.1) breakpoint on chromosome 14 between SPTB and D14S77. AB - Uterine leiomyoma is the most common tumor of smooth muscle cell origin and is often associated with the recurrent balanced translocation t(12;14)(q13-15;q24). As an initial step toward finding the gene or genes that are interrupted by the translocation breakpoint, a somatic cell hybrid carrying the derivative 14 as the single t(12;14) translocated chromosome was constructed from a leiomyoma cell line with this translocation. Sequence tagged sites (STS) whose locations on the genetic map of chromosome 14 were known were used to map the breakpoint in the translocated chromosomes. The results of this analysis place the translocation breakpoint on the long arm of chromosome 14 between the proximal marker SPTB and the distal marker D14S77, narrowing the chromosomal translocation breakpoint to a region of approximately 7 cM. The identification of flanking markers on chromosome 14 lays the foundation for efforts to clone the breakpoint and to identify the genes involved in the formation of leiomyoma. PMID- 7533529 TI - Fusion of the FUS gene with ERG in acute myeloid leukemia with t(16;21)(p11;q22). AB - It has been shown that the gene ERG in 21q22 is rearranged in the t(16;21)(p11;q22) associated with acute myeloid leukemia (AML). ERG is a member of the ETS gene family and is fused with EWS in a subset of Ewing's sarcomas. EWS in 22q12 has a very high homology with FUS (also called TLS) in 16p11; the latter gene is rearranged in the t(12;16)(q13;p11) that characterizes myxoid liposarcoma. To investigate whether FUS is involved in the t(16;21) of AML, we used the Southern blot technique and polymerase chain reaction (PCR) to examine the bone marrow of a 3-year-old boy with a t(16;21)(p11;q22)-positive AML. Hybridization of Southern blot filters containing digested DNA with probes for FUS and ERG showed both germline and aberrant fragments. Using specific primers for the 5' part of FUS and the 3' part of ERG, we amplified a 4.4 kb genomic FUS/ERG DNA fragment from the leukemic sample. In a second PCR experiment, in which we used primers upstream of the 5' part of ERG and downstream of the 3' part of FUS, a 5.6 kb fragment was amplified. Blotting and hybridization with specific probes for FUS and ERG revealed that the amplified fragments consisted of FUS/ERG and ERG/FUS hybrid DNA. Both PCR fragments, when used as probes, detected germline ERG and FUS as well as aberrant fragments on Southern blot filters. The results suggest that the t(16;21) in AML leads to rearrangement and fusion of the FUS and ERG genes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533532 TI - Clonal structural chromosome aberrations in fibrous dysplasia. AB - Cytogenetic analysis of short-term cultures from a case of monostotic fibrous dysplasia in a 14-year-old girl revealed multiple clonal structural rearrangements with evidence of clonal evolution. The karyotype was 46,XX,del(3)(q27),add(10)(q22),add(12)(p13)/46,idem,t(3;8)(p21;q13 ),add(10) (q26),der(15)del(15)del(15)(q15q22)ins(15;?) q15;?)/46,id em, X,+2,t(3;8),add(10),der(15). The finding of clonal structural aberrations suggests that fibrous dysplasia is a neoplastic lesion which develops as the result of somatic mutations. PMID- 7533533 TI - Human scleral elastic system: an immunoelectron microscopic study. AB - An immunocytochemical study was conducted on elastic components in the sclera of seven aged human eyes. By conventional electron microscopy, elastic tissue consists of three distinct fibre types--elastic fibres, elaunin fibres, and oxytalan fibres. The distribution of six components associated with the elastic system (elastin, amyloid P component, laminin, fibronectin, gp 115, and vitronectin) were studied by immunogold transmission electron microscopy. The codistribution of amyloid P component and laminin was further studied by double immunolabelling. Both elastic and elaunin fibres contained elastin. The microfibrillar sheaths of elastic fibres labelled for amyloid P component, those of elaunin fibres for amyloid P and laminin, and those of oxytalan fibres for laminin only. No labelling was observed for fibronectin, gp 115, and vitronectin. In terms of the proteins investigated, the biochemical profile of the three fibre types was not completely identical and was manifest as different affinities in the binding of serum amyloid P component and an association with laminin. PMID- 7533535 TI - A periodic table of symmetric tandem mismatches in RNA. AB - The stabilities and structures of a series of RNA octamers containing symmetric tandem mismatches were studied by UV melting and imino proton NMR. The free energy increments for tandem mismatch formation are found to depend upon both mismatch sequence and adjacent base pairs. The observed sequence dependence of tandem mismatch stability is UGGU > GUUG > GAAG > or = AGGA > UUUU > CAAC > or = CUUC approximately UCCU approximately CCCC approximately ACCA approximately AAAA, and the closing base pair dependence is 5'G3'C > 5'C3'G > 5'U3'A approximately 5'A3'U. These results differ from expectations based on models used in RNA folding algorithms and from the sequence dependence observed for folding of RNA hairpins. Imino proton NMR results indicate the sequence dependence is partially due to hydrogen bonding within mismatches. PMID- 7533534 TI - Expression of cellular fibronectin and tenascin in the rabbit cornea after excimer laser photorefractive keratectomy: a 12 month study. AB - An indirect immunohistochemical technique was used to monitor the expression of cellular fibronectin (cFN) and tenascin (TN) in the rabbit cornea after photorefractive keratectomy (PRK) in a 1 year follow up study. Rabbits received a 5.0 D myopic PRK, and were killed 3 days, 1, 3, 6, or 12 months after the operation. In most corneas, secondary epithelial defects appeared after the primary healing (mean 6.3 (SD 1.2) days). Corneal haze appeared a few weeks after PRK and was observed throughout the follow up. Three days after wounding an immunoreaction for cFN was observed as a bright narrow subepithelial line, but no immunoreaction for TN could be seen in the anterior third of the corneal stroma. However, at 1-6 months a similar location of immunoreactions for both cFN and TN was observed. Both were found in the anterior stroma at depths of 30-50 microns. At 12 months, only a trace of cFN immunoreaction but no TN immunoreaction could be discerned. Our results suggest that subepithelial scar tissue contains both cFN and TN up to 12 months. PMID- 7533536 TI - Role of sterols in the functional reconstitution of water-soluble mitochondrial porins from different organisms. AB - Experiments were performed on lipid bilayer membranes with water-soluble mitochondrial porins from different eukaryotic organisms, such as Dictyostelium discoideum, Paramecium, and rat liver, to study the requirements of functional reconstitution of the porins. The water-soluble porins lost their associated lipids and sterols and are unable to form channels in lipid bilayer membranes. We demonstrate that the water-soluble porins regain their channel-forming ability after preincubation of the polypeptides with sterols in the presence of detergents. Mitochondrial porin from Dictyostelium discoideum maintained after this procedure its original properties, in particular the voltage dependence. Water-soluble mitochondrial porins from Paramecium tetraurelia and from rat liver were also activated upon preincubation with different sterols in detergent but showed voltage-dependences that were different from those of detergent-purified porins. Furthermore, the voltage dependence depended on the sterol used for preincubation. Interestingly, the preincubation with sterols can likewise be used to activate detergent-purified mitochondrial porins that may have lost associated sterol during isolation and purification procedures. PMID- 7533537 TI - Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein. AB - A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100 107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP. PMID- 7533538 TI - Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. AB - Magainin peptides, isolated from Xenopus skin, kill bacteria by permeabilizing their cell membranes whereas they do not lyse erythrocytes. To elucidate the rationale for this membrane selectivity, we compared the effects of the membrane lipid composition and the transmembrane potential on the membrane-lytic power of magainin 2 with that of hemolytic melittin. The activity of magainin to zwitterionic phospholipids constituting the erythrocyte surface was extremely weak compared with that of melittin, and acidic phospholipids are necessary for effective action. The presence of sterols reduced the susceptibility of the membrane to magainin. The generation of an inside-negative transmembrane potential enhanced magainin-induced hemolysis. We can conclude that the absence of any acidic phospholipids on the outer monolayer and the abundant presence of cholesterol, combined with the lack of the transmembrane potential, contribute to the protection of erythrocytes from magainin's attack. PMID- 7533539 TI - Selective phosphorylation of cationic polypeptide aggregated with phosphatidylserine/diacylglycerol/Ca2+/detergent mixed micelles by Ca(2+) independent but not Ca(2+)-dependent protein kinase C isozymes. AB - Mixed micelles containing Nonidet P40 (NP-40) (829 microM or 4.8 mM), phosphatidylserine (PS) (14.5 or 8 mol%), and 1,2-diacylglycerol (DG) (0.5 or 1 mol%) when preincubated with protein kinase C (PKC) assay mixture containing cationic substrate and CaCl2 (400 microM) formed aggregates in a time-, temperature-, and substrate concentration-dependent manner with a t1/2 approximately 3-12 min (22 degrees C). Concomitant with the formation of these aggregates there was a substantial loss of substrate phosphorylation catalyzed by the Ca(2+)-dependent PKC alpha, beta, and gamma but not the Ca(2+)-independent PKC, delta and epsilon. All cationic PKC substrates tested, neurogranin peptide analog, neurogranin, and histone III-S, formed aggregates with PS/DG/NP-40/Ca2+ mixed micelles in a time-dependent fashion. The poly(cationic-anionic) PKC substrate protamine sulfate also forms aggregates with the mixed micelles in the presence of Ca2+, but without affecting the substrate phosphorylation by the kinase. Under similar conditions, but at 4 degrees C, neither aggregation nor loss of cationic substrate phosphorylation was observed. Another nonionic detergent, octyl glucoside, behaved similarly to NP-40. Phosphatidylinositol (PI) and phosphatidylglycerol like PS, were effective in forming aggregates with NP 40/cationic polypeptide/DG/Ca2+ as monitored by light scattering, yet without affecting substrate phosphorylation. Phosphorylation of cationic substrates by M kinase, derived from trypsinized PKC beta, was also greatly diminished by the aggregation. In contrast, [3H]phorbol 12,13-dibutyrate binding to PKC beta was unaffected. Formation of the aggregates that were selectively utilized by the Ca(2+)-independent PKCs was dependent on the ratio of cationic substrate to the number of mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533540 TI - C-terminal amino groups facilitate membrane incorporation of gramicidin derivatives. AB - Gramicidin derivatives with (positively-charged) C-terminal amino groups are found to incorporate readily and refold quickly when added to dioleoylphosphatidylcholine lipid vesicles from concentrated methanol solutions. Neutral and negatively-charged derivatives do not. PMID- 7533542 TI - The properties of ion channels formed by the coumarin antibiotic, novobiocin, in lipid bilayers. AB - The coumarin antibiotic novobiocin forms ion channels of varying conductances in lipid bilayers. The conductances (about 20, 22, 14, 7 and 2 pS for 100 mM NH4Cl, CsCl, KCl, NaCl and LiCl, respectively) and selectivities (cation transference numbers in the range of 0.97-0.98) of one type of novobiocin-induced channel are similar to those found for channels formed by gramicidin A, an antibiotic of very different structure. The conductance of novobiocin channels of this type was independent of the species of the membrane lipid. This observation suggests that novobiocin molecules directly form these channels, and that channels are not formed through defects in lipid structure. The similarity in conductance and ion selectivity between channels induced by novobiocin and those formed by gramicidin A suggests that these structurally different molecules form channels with comparable internal diameter and internal surface charge distribution. Using HPLC purification we argue that the channel-forming activity of novobiocin is related to the activity of the novobiocin molecule itself, and not to a contaminant of the commercially available novobiocin sodium salt preparation. PMID- 7533541 TI - Developmental changes in intestinal glycosylation: nutrition-dependent multi factor regulation of the fucosylation pathway at weaning time. AB - Developmental changes in the fucoglycoproteins of the intestinal brush-border membranes were determined by lectin affinoblotting after electrophoresis. Whereas only two alpha(1-6)-fucoglycoproteins were detected in brush-border membranes from suckling rats, a large number of N-fucoglycoproteins with alpha(1-2)- and/or alpha(1-6)-linked fucose residues were detected in rat membranes after weaning. Dietary manipulations at weaning time were used to investigate the effect of nutritional factors in the development of fucosylation in the small intestine of prolonged-nursed rats fed with milk (a high-fat, low-carbohydrate diet) compared to rats weaned normally with a standard high-carbohydrate diet. The fucose content of the mucosa glycoproteins was lower in 22-day-old prolonged-nursed rats than in 22-day-old rats weaned normally with the standard diet. The appearance of fucoglycoproteins in the brush-border membranes, which was delayed by prolonged nursing, was accompanied by a concomitant delay in the increase of intestinal fucosyl-transferase activity and in the decrease of GDP-fucose substrate breakdown. The developmental decrease in the activity of the inhibitory protein which regulates the fucosyl-transferase activity was also delayed by prolonged nursing. The intestinal fucosylation of brush-border membrane glycoproteins (which include many digestive enzymes) displayed ontogenic changes on which were superimposed dietary influences at the time of weaning. The complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, is a developmental event which thus seems to be strongly influenced by the manipulation of nutritional factors during the weaning period. PMID- 7533543 TI - Multiple deficiencies of mitochondrial DNA- and nuclear-encoded subunits of respiratory NADH dehydrogenase detected with peptide- and subunit-specific antibodies in mitochondrial myopathies. AB - Antibodies have been raised against synthetic peptides corresponding to several computer-predicted epitopes of three mtDNA-encoded subunits, ND4, ND5 and ND6, of the human respiratory chain NADH dehydrogenase (Complex I). Antibodies were characterized by a sensitive immunoblotting assay using proteins from human skeletal muscle mitochondria and by immunoprecipitation of radio-labeled HeLa cell mitochondrial translation products. Only antibodies against two of six selected peptides of the ND4 subunit, i.e., the C-terminal peptide and an internal peptide close to the C-terminus, reacted in both assays with the subunit. Antibodies raised against an internal peptide close to the N-terminus of the ND5 subunit and antibodies raised against an internal epitope of the ND6 subunit also reacted in both the immunoblotting and immunoprecipitation assays. The antibodies described above and other Complex I subunit- or holoenzyme specific antibodies were used to investigate the subunit deficiencies of the respiratory NADH dehydrogenase in the skeletal muscle of patients affected by mitochondrial myopathies associated with Complex I defects. The reduction in enzyme activity correlated in an immunoblot assay with a decrease of four mtDNA encoded subunits of the enzyme, as well as with a decrease of other subunits of Complex I encoded in the nDNA. The present work provides the first evidence of a decrease in NADH dehydrogenase subunits encoded in the mitochondrial genome in myopathy patients. PMID- 7533545 TI - [Management of the prostate patient: from the primary care perspective?]. PMID- 7533544 TI - Heavy-chain immunoglobulin gene rearrangement and cytoplasmic immunoglobulin expression in acute monocytic leukemia following primary germ cell tumor. AB - A case of acute monocytic leukemia with rearrangement of the immunoglobulin heavy chain gene and strong cytoplasmic immunoglobulin expression in a young patient treated with multi-drug chemotherapy for primary seminomatous germ cell tumor 13 months earlier is reported. The short latency period from the beginning of therapy for primary germ cell tumor and the abrupt onset of leukemia with no identifiable prodrome bear similarities to podophyllotoxin-related leukemias. PMID- 7533546 TI - Helix formation of poly (L-glutamic acid) in the presence of alkaline earth metal cations in aqueous alcohol solutions. AB - Effects of divalent counterions (Mg2+, Ca2+, Sr2+, Ba2+) on the helix formation of poly(L-glutamic acid) are investigated in aqueous alcohol (MeOH, EtOH, 2PrOH, tBuOH) solutions. To observe helix formation inherent to the divalent counterions, the chlorides were added to a poly(L-glutamate) having an inert monovalent counterion, tetrabutylammonium cation. Marked counterion- and alcohol specificities were observed for the dependence of helix content, theta, on the alcohol content. The theta values for most Mg2+ and Ca2+ systems decreased with increasing alcohol content in a higher alcohol region, while those for Sr2+ and Ba2+ systems were almost constant. A specific helix stabilization was observed for mixed divalent counterion systems. These results are self-consistently interpreted by taking into account contact ion-pair formation between the counterions and the polymer charges on the coil conformation. PMID- 7533548 TI - Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens. AB - Microwave oven antigen retrieval has been developed to extend the range of antibodies that can be used upon sections of fixed and processed tissue. It has the additional advantages of improving immunostain intensity and reducing background positivity. It can also be employed as an alternative to proteolytic digestion. In this study the effects of microwave oven heating upon immunochemical staining of cytopathological specimens with a range of selected antibodies have been investigated. Microwaving did not result in loss of cells, and there was no need to use adhesive-coated slides. The method improved the staining intensity and reduced background with antibodies against a variety of antigens that are difficult or impossible to detect in formaldehyde-fixed cytological material. Microwave heating was also used successfully as an alternative to trypsin digestion, and had the advantage of reduced morphological distortion. The technique was useful in demonstrating the soluble formalin sensitive antigen p19 on cytospins fixed in formaldehyde vapour. Microwave oven heating thus shows promise of extending the scope of immunostaining in clinical cytopathology. PMID- 7533547 TI - Influence of ligands on the fluorescence polarisation anisotropy of ethidium bound to DNA. AB - The decay of the fluorescence polarisation anisotropy (FPA) of the ethidium-DNA complex has been measured by multifrequency phase fluorometry, in order to study the perturbations induced by the presence of different ligands on the torsional dynamics of DNA, a moderately flexible polymer that undergoes bending (flexure of the helix axis) and torsional (twisting of base pairs) motions. Two probes have been used together with ethidium: an intercalator, chloroquine, and a minor groove binding dye: hoechst 33258. Chloroquine is found to substantially modify the DNA torsional dynamics both in linear and in circularly closed DNAs only at high binding ratios, in agreement with previous reports [Wu et al. Biochem. 27 (1988) 8128]. The effective elastic constant becomes approximately three times larger when the dye/base pairs binding ratio is higher than 0.14. The minor groove ligand hoechst 33258, on the other hand, greatly increases the effective elastic constant to the point that at dye/base pairs ratios larger than 0.5, the effective elastic constant becomes stiffer by several orders of magnitude, suggesting a progressive hindering of internal motions. The results reported here show that DNA torsions are more effectively influenced by groove-binding molecules than by intercalators and it is expected that the large perturbation of the former ligand may be useful when describing the change in the dynamical properties induced by DNA binding proteins. FPA in the frequency domain, the technique adopted throughout this work, has proved to be very sensitive to changes of the elastic constant that describes DNA torsional dynamics. Several computer simulations performed in order to predict the FPA time decay of intercalated ethidium have led to good agreement with the observed results. PMID- 7533549 TI - Interleukin-6 regulation of CCK/gastrin receptors and amylase secretion in a rat pancreatic acinar cell line (AR4-2J). AB - In necrotizing pancreatitis a high interleukin-6 (IL-6) serum level has been proposed as a prognostic criterium. However, literature does not report any information about the role of IL-6 in the function of pancreatic acinar cells. Cholecystokinin, gastrin binding, amylase release and intracellular calcium measurement were performed on a rat pancreatoma cell line, AR4-2J, which has been recognized as a useful tool for studies on the long-term regulation of pancreatic acinar cells. The addition of IL-6 (400 pM) for 48hrs to the AR4-2J cells induced no change in the binding affinities but there was a 2 fold increase in the binding capacity of cholecytokinin (CCKA R) and gastrin (CCKB R) receptors. Although IL-6 treatment did not change directly the secretory capacity and did not activate the intracellular calcium mobilization of AR4-2J, it indirectly increased the sensitivity of the cells to the stimulation of amylase release and the intracellular calcium mobilization by cholecystokinin and gastrin. It is most likely this effect was due to the IL-6-induced increase in the numbers of CCKA R and CCKB R. Therefore this report suggests that the cytokine IL-6 acts on the CCK regulation of pancreatic enzyme secretion. PMID- 7533550 TI - Pro: antiarrhythmic drugs should be used to suppress ventricular ectopy in the perioperative period. PMID- 7533551 TI - Con: antiarrhythmic drugs should not be used to suppress ventricular ectopy in the perioperative period. AB - The alterations in autonomic tone imposed by the conduct of anesthesia and surgery predispose patients to ventricular ectopy. It is important to initially view any ectopy as a warning sign and promptly check the adequacy of ventilation and oxygenation. Most commonly an inadequate depth of anesthesia, surgical manipulation or electrolyte abnormality will be causative. Treatment of this underlying problem will usually suffice to terminate the ectopy. Importantly, many patients have preexisting, chronic, complex ventricular ectopy that gets revealed because of perioperative electrocardiographic monitoring. All available pharmacologic agents have significant adverse side effects. To date, all investigations examining outcome of suppression of ventricular ectopy show that successful suppression of ventricular ectopy was associated with an increased mortality. Although the prognosis for patients is worse when complex ventricular ectopy is associated with cardiac structural abnormalities, the optimal therapeutic approach to such patients remains undefined. Unless new data supporting the use of antiarrhythmic drugs in the perioperative setting become available, the risk to benefit ratio is considered unfavorable. PMID- 7533554 TI - L-arginine induces dopamine release from the striatum in vivo. AB - Recent reports indicate that induction of nitric oxide (NO) evokes dopamine (DA) release from the striatum in vitro. In this study, we used L-arginine (L-Arg) to demonstrate the in vivo stimulation of DA release from the striatum of Mongolian gerbils using microdialysis. The content of DA in the striatal extracellular fluid (ECF) increased 7-15-fold in the presence of L-Arg in the perfusate as compared with that of the controls (DA level in drug-free perfusate varied from 0.050 +/- 0.009 to 0.092 +/- 0.023 pmol 10 microliters-1). Simultaneous perfusion of L-Arg with nitro-L-arginine (NLA), an inhibitor of nitric oxide synthase, markedly reduced the L-Arg effect on DA release from the striatum. The NLA perfused animals contained DA levels significantly lower than those observed in the control striatal dialysate. These findings indicate for the first time that DA release in vivo can be induced by L-Arg, the precursor of NO. The data strongly suggest that NO may modulate striatal DA release. PMID- 7533555 TI - Abscisic acid potentiates NMDA-gated currents in hippocampal neurones. AB - NMDA receptor functioning underlies the basic mechanism of synaptic plasticity of the central nervous system. The NMDA receptor is a subject for modulation by certain substances: facilitation of this receptor is thought to be crucial for the induction of plasticity phenomena. In the present investigation evidence for a potentiation of the NMDA-receptor by trans,trans-abscisic acid (postulated endogenous substance) is provided. Pre-exposure to trans,trans-abscisic acid induced a marked increase mainly of the fast component of NMDA-gated currents in isolated rat hippocampal neurones. The potentiating effect depended on an extracellular trans,trans-abscisic acid concentration with an EC50 value about 440 microM, Hill coefficient 2. Such a facilitatory effect developed rather slowly with a time constant of about 3 s. The results obtained imply the existence of a novel mechanism of NMDA receptor potentiation. The data presented indicate a possible neurophysiological significance of abscisin. PMID- 7533552 TI - The differential mechanisms of mild irritants on adaptive cytoprotection. AB - The protective action of mild irritants has been established. However, the mechanisms as to how they antagonize the injurious action produced by the subsequent challenge with an ulcerogenic stimulus are still unclear. The present study examined the different protective mechanisms of an oral administration of the three mild irritants, 20% ethanol, 0.3 mol/L HCl or 5% NaCl against the gastric injurious actions of absolute ethanol in rats. In an attempt to clarify the pathways and mediators involved in the adaptive cytoprotection, [D-Pro2, D Trp7,9]-substance P (substance P antagonist), Nw-nitro-L-arginine methyl ester (L NAME), indomethacin, capsaicin, lidocaine, atropine or hexamethonium was given. The protective action of 20% ethanol but not the other two mild irritants, was antagonized by L-NAME, indomethacin and capsaicin, which are the inhibitors of nitric oxide (NO) and prostaglandins (PG) synthesis, and afferent sensory neuron blocker, respectively. Substance P antagonist, lidocaine or atropine given alone, prevented mucosal damage; however, only substance P antagonist enhanced the anti lesion action of 20% ethanol, while atropine and lidocaine increased the protective effect of NaCl and HCl. The three mild irritants increased the residual gastric secretion. Only 20% ethanol and 5% NaCl but not 0.3% HCl significantly increased the basal adherent mucus and also attenuated the mucus depletion by absolute ethanol. It is concluded that the cytoprotective action of either ethanol or NaCl seems to be mediated through the increase of residual gastric secretion and adherent mucus. In the ethanol-treated group, these actions could act through the afferent sensory fibres, with NO and PG as the possible mediators.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533553 TI - The mechanism underlying stimulation of gastric HCO3- secretion by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester in rats. AB - We investigated the mechanism underlying stimulation of gastric HCO3- secretion by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME) in anaesthetized rats. A rat stomach was mounted in an ex vivo chamber, superfused with saline, and HCO3- secretion was measured in the absence of acid secretion (omeprazole pretreatment). Intravenous administration of L-NAME (1-5 mg/kg) increased gastric HCO3- secretion dose dependently with concomitant rise in arterial blood pressure and decrease in heart rate, and these effects were all antagonized by simultaneous administration of L-arginine (200 mg/kg). Vagotomy did not affect the increased blood pressure response but significantly inhibited the decrease in heart rate and increase of HCO3- secretion caused by L-NAME. The HCO3- stimulatory action of L-NAME was also inhibited by pretreatment with either yohimbine (5 mg/kg s.c.) or prazosin (0.5 mg/kg s.c.). These agents alone caused a decrease in blood pressure, and reduced the magnitude of blood pressure response caused by L-NAME, leading to inhibition of heart rate changes. When the change in HCO3- output induced by L-NAME was plotted against the change in blood pressure (from basal values) under various conditions, a significant relationship was found between these two parameters. These results suggest that L-NAME stimulates gastric HCO3- secretion in association with the inhibition of endogenous NO production, and this mechanism may be in part mediated by a neural reflex through vagal efferent nerves, resulting from the pressor response to L NAME. PMID- 7533556 TI - Nitric oxide promotes seizure activity in kainate-treated rats. AB - L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7 nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures. PMID- 7533557 TI - Aluminium reduces glutamate-activated currents of rat hippocampal neurones. AB - The actions of aluminum on glutamate-activated currents of acutely isolated hippocampal neurones were investigated. N-methyl-D-aspartate (NMDA), alpha-amino 3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and glutamate mediated currents were reduced by 50% in the presence of 1.4 micrograms ml-1 aluminium. Higher concentrations (> or = 2.7 micrograms ml-1) inhibited all currents completely and irreversibly. Additionally, successive application of agonists in the presence of 2.7 micrograms ml-1 aluminium resulted in non-specific membrane currents followed by the loss of the seal resistance. Application of aluminium per se had no influence on resting membrane current or voltage-activated sodium currents. The estimation of the concentration-response relationship of the action of aluminium on NMDA-activated currents revealed a threshold concentration < 0.27 micrograms ml-1. Our data indicate that glutamate receptors are putative sites of action in aluminium neurotoxicity. PMID- 7533559 TI - Early phosphorylation of tau in Alzheimer's disease occurs at Ser-202 and is preferentially located within neurites. AB - Within the neurofibrillary tangles (NFTs) and dystrophic neurites (DNs) of Alzheimer's disease (AD), the cytoskeletal protein tau is abnormally hyperphosphorylated. In this study we evaluate the phosphorylation of specific residues on tau within different phases of the formation of NFTs. Two monoclonal antibodies, AT8 and PHF-1, were used to selectively recognize phosphorylated Ser 202 and Ser-396 of PHF-tau protein, respectively. We found that abnormal phosphorylation of tau appears to occur first at Ser-202 in DNs, then at Ser-202 in the soma and finally at Ser-396 in DNs and NFTs. These results suggest that abnormal phosphorylation at Ser-202 of PHF-tau in DNs represents one of the earliest neuropathological changes within the neurites of vulnerable neurons and may have a pivotal role in the initial pathogenesis of AD. PMID- 7533558 TI - GAP-43 persists in adulthood and coexists with SP and CGRP in human trigeminal sensory neurones. AB - Immunohistochemistry was used to examine the occurrence and colocalization of the growth-associated protein GAP-43 with substance P (SP) and calcitonin gene related peptide (CGRP) in the human trigeminal ganglion and sensory nucleus at perinatal and adult life stages. The results obtained show that: GAP-43-like immunoreactive (LI) material persists in trigeminal primary sensory neurones of the normal adult; the GAP-43-LI ganglionic population partially overlaps with those immunoreactive to SP and CGRP; the distribution pattern of the protein in the spinal nucleus varies with age; in the adult subnucleus caudalis GAP-43 is co distributed with SP and CGRP. It is suggested that the trigeminal GAP-43-LI neuronal system may retain the capacity for structural and functional plasticity in adult life. PMID- 7533560 TI - Growth factor targeted and conventional therapy of breast cancer. AB - Sustained breast cancer growth and metastasis requires paracrine signals between the tumor cells and the normal surrounding host tissue. One crucial function of these signals is to recruit endothelial cells and thus new blood vessels for the nourishment of the expanding tumor mass. This proliferation and migration of endothelial cells in the vicinity of progressing tumors contrasts with the extremely low turn-over rate of endothelial cells in the healthy adult. A blockade of tumor-induced endothelial cell proliferation should inhibit tumor growth and potentially metastasis with only few adverse effects. Different therapeutic approaches that take advantage of this situation are discussed with respect to their interaction with conventional therapies of breast cancer. PMID- 7533561 TI - Breast carcinoma: a collective disorder. AB - The development and differentiation of the epithelial component of glandular tissues such as the breast is regulated by two apparently unrelated processes. One of these is presumed to be epithelial cell collective autonomous, that is, it is mediated by gene products which act directly on the epithelial cells. An important component of autonomous regulation is the functional expression of homotypic cell-cell adhesion molecules such as cadherins. The second process is non-autonomous and involves an inductive effect of the neighboring mesenchymal cell collective. An important component of non-autonomous regulation is the aggregation/condensation of mesenchyme closely associated with the epithelium. We propose that molecular alterations in autonomous and non-autonomous pathways are important causes and indicators respectively of breast cancer progression and that these two fundamental regulators of epithelial collective organization are in fact inter-dependent. For example, we show that the expression of hepatocyte growth factor (HGF), an epithelially targeted mesenchymally derived morphogenic factor is regulated by mesenchymal cell density (condensation) and by factors released from epithelial cells. Breast epithelial cells produce factors which inhibit and stimulate HGF expression. The inhibitory factor is transforming growth factor beta (TGF-beta) and the activation state of TGF-beta is a crucial element in HGF homeostasis. The balance of negative and positive HGF regulators is markedly affected by the growth conditions and differentiation state of the epithelial cells. The expression of the HGF receptor, met, is high in normal breast epithelial cells and in dedifferentiated (ER negative) tumor cells but is reduced or lost in ER positive well differentiated epithelial cells. Our results indicate that the expression of at least one epithelial morphogen, HGF, is inter dependently regulated by mesenchymal condensation and by factors released by neighboring epithelial cells. PMID- 7533562 TI - The potential role of the heparin-binding growth factor pleiotrophin in breast cancer. AB - We propose that the secreted protein pleiotrophin (PTN) is a major factor in the malignant progression of breast cancer. This hypothesis is based on the growth stimulatory effects of PTN on cells in vitro and in vivo and on its high levels of expression in 60% of tumor samples from breast cancer patients. The stimulation of proliferation and tube formation of endothelial cells by PTN suggests that it can serve as an angiogenesis factor during tumor growth. We hypothesize that PTN has the potential to support growth of breast cancer at its primary site and to enhance the ability of tumor cells to metastasize. Furthermore, we suggest that specific endocrine signals interact to regulate the expression of PTN in vitro and in vivo. Finally, we propose that understanding the functions of PTN and its hormonal regulation can lead to the development of novel therapeutic strategies for breast cancer. PMID- 7533563 TI - Stage-specific expression of alkaline phosphatase during neural development in the mouse. AB - The expression pattern of tissue nonspecific alkaline phosphatase (TNAP) in the developing neural tube of mouse is reported. Homogeneous AP activity in the neuroepithelium becomes prominent at E8.5. At E9.5, distinctly AP-positive cells appear in the brain and spinal cord area. At stages E10.5 to E12.5, AP positivity is observed between the mesencephalon and the rhombencephalon, along the entire spinal cord and cranial nerves emerging from the myelencephalon. At E13.5, strongly AP positive fibers become prominent in the pons. At E14.5, AP expression in brain tissue is considerably reduced and there is a complete absence of AP activity in the nerve cells and glial cells of adult brain. The choroid plexus remains distinctly positive for AP expression until the adult stage. Northern blot analysis and reverse-transcriptase polymerase chain reaction amplification of RNA indicate that this AP activity results from the expression of the Akp-2 locus. This AP expression pattern is distinct from those reported for the expression of GD3, nestin, Hox 2.3, and Wnt-1 during brain development. We conclude that AP is a useful marker of a subpopulation of neuroectodermal cells present in the neural tube as early as E8.5, at which stages there are no other AP positive intraembryonic cells except PGCs. PMID- 7533564 TI - Effect of iron-deficiency anaemia on cognitive skills in infancy and childhood. AB - Animal experimentation has shown that early iron deficiency irreversibly affects brain iron content and distribution, resulting in neutransmitter and behavioural alterations. Even though extrapolation of animal data is often misleading, iron deficiency anaemia has been consistently shown to be associated with psychomotor delays in infancy. The areas most involved are language and body balance. In these infants iron therapy, in most cases was not sufficient to reverse psychological effects even after complete correction of haematological measures. These findings may imply that the impact of iron-deficiency anaemia during infancy may be associated with irreversible adverse effects on cognitive performance. Careful follow-up studies of these infants at 5-6 years of age has shown that cognitive disadvantages persist, now assessed with a comprehensive set of psychological tests that reliably predict future competence. Thus, if once anaemia ensues, even timely and adequate iron therapy seems to be ineffective in reversing these behavioural and cognitive disadvantages; the only practical way to approach this problem is by prevention of iron deficiency in infancy. Health authorities, having been shown that treatment of iron deficiency anaemia is already too late to reverse potential deficits, should strive to prevent iron deficiency with adequate food fortification strategies or by supplementing targeted population groups. PMID- 7533566 TI - Determination of 1-nitropyrene retained in leaves of roadside trees. PMID- 7533565 TI - Significant formation of 8-hydroxydeoxyguanosine through interaction of diesel particulate matter with deoxyguanosine. PMID- 7533567 TI - Possible mechanisms of pharmacological neuronal protection. PMID- 7533568 TI - A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: application to gramicidin-S. AB - A global optimization method for intensity-restrained structure refinement, based on variable target function (VTF) analysis, is illustrated using experimental data on a model peptide, gramicidin-S (GS) dissolved in DMSO. The method (referred to as VARTIGO for variable target intensity-restrained global optimization) involves minimization of a target function in which the range of NOE contacts is gradually increased in successive cycles of optimization in dihedral angle space. Several different starting conformations (including all trans) have been tested to establish the validity of the method. Not all optimizations were successful, but these were readily identifiable from their large NOE R-factors. We also show that it is possible to simultaneously optimize the rotational correlation time along with the dihedral angles. The structural features of GS thus obtained from the successful optimizations are in excellent agreement with the available experimental data. A comparison is made with structures generated from an intensity-restrained single target function (STF) analysis. The results on GS suggest that VARTIGO refinement is capable of yielding better quality structures. Our work also underscores the need for a simultaneous analysis of different NOE R-factors in judging the quality of optimized structures. The NOESY data on GS in DMSO appear to provide evidence for the presence of two orientations for the ornithine side chain, in fast exchange. The NOESY spectra for this case were analyzed using a relaxation rate matrix which is a weighted average of the relaxation rate matrices for the individual conformations. PMID- 7533569 TI - Sequential backbone assignment of uniformly 13C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment. AB - A new 1H-13C-31P triple resonance experiment is described which allows unambiguous sequential backbone assignment in 13C-labeled oligonucleotides via through-bond coherence transfer from 31P via 13C to 1H. The approach employs INEPT to transfer coherence from 31P to 13C and homonuclear TOCSY to transfer the 13C coherence through the ribose ring, followed by 13C to 1H J-cross polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of 31Pi coherence via C4'i and C4'i-1, because of the relatively large JPC4' couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4'i and C4'i-1 coherences are subsequently transferred to, amongst others, H1'i and H1'i-1, respectively, leading to a 2D 1H-31P spectrum which allows a sequential assignment in the 31P 1H1' region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a 13C-labeled RNA hairpin with the sequence 5'(GGGC-CAAA-GCCU)3'. PMID- 7533573 TI - Nutritional advice and support for individuals with incurable diseases. AB - Having an incurable illness raises a number of special nutritional issues for both the patient and the health professional. This review covers two of these issues: the use of unproven dietary products to improve immune function, and the role of nutritional support in palliative care. The usage of unproven dietary strategies, particularly vitamin and mineral supplements, is extremely popular in patients with incurable diseases such as HIV infection. A daily multivitamin and mineral supplement plus an additional supplement of beta-carotene may be beneficial in terms of slowing progression to AIDS, but megadosing of vitamins, minerals and Chinese herbs, and the use of 'special diets' should be cautioned against. The two main areas where nutritional support appears to be important in palliative care are symptom control and aggressive feeding as a means of stabilising body weight. The psychological benefits of appropriate nutritional advice and support are highlighted. PMID- 7533570 TI - Sequential correlation of anomeric ribose protons and intervening phosphorus in RNA oligonucleotides by a 1H, 13C, 31P triple resonance experiment: HCP-CCH TOCSY. AB - A three-dimensional 1H, 13C, 31P triple resonance experiment, HCP-CCH-TOCSY, is presented which provides unambiguous through-bond correlation of all 1H ribose protons on the 5' and 3' sides of the intervening phosphorus along the backbone bonding network in 13C-labeled RNA oligonucleotides. The correlation of the complete ribose spin system to the intervening phosphorus is obtained by adding a C,C-TOCSY coherence transfer step to the triple resonance HCP experiment. The C,C TOCSY transfer step, which utilizes the large and relatively uniform 1J(C,C) coupling constant (approximately 40 Hz for ribose carbons), efficiently correlates the phosphorus-coupled carbons observed in the HCP correlation experiment (i.e., C4' and C5' in the 5' direction and C4' and C3' in the 3' direction) to all other carbons in the ribose spin system. Of the additional correlations observed in the HCP-CCH-TOCSY, that to the relatively well-resolved anomeric H1',C1' resonance pairs provides the greatest gain in terms of facilitating assignment. The gain in spectral resolution afforded by chemical shift labeling with the anomeric resonances should provide a more robust pathway for sequential assignment over the intervening phosphorus in larger RNA oligonucleotides. The HCP-CCH-TOCSY experiment is demonstrated on a uniformly 13C, 15N-labeled 19-nucleotide RNA stem-loop, derived from the antisense RNA I molecule found in the ColE1 plasmid replication control system. PMID- 7533571 TI - Muscle sodium channel inactivation defect in paramyotonia congenita with the thr1313met mutation. AB - Mutations of the skeletal muscle sodium (Na) channel have been reported in families with paramyotonia congenita (PC), an autosomal dominant disorder with cold and/or exercise induced stiffness and myotonia. Functional consequences of specific Na channel mutations responsible for PC have not been described. Patch clamp recording of single Na channels were made in cultured myotubes at 22 and 34 degrees C from a PC patient with the thr1313met mutation. Cell-attached and outside-out recordings of mutant PC channels contained long duration and late openings. The mean open time was increased and the ensemble average showed a prolonged inward Na current. This membrane depolarization could cause repetitive action potentials and the clinical syndrome. PMID- 7533574 TI - Diagnostic value of histological special stains. PMID- 7533575 TI - Induced mutagenesis by bleomycin in the purple sulfur bacterium Thiocapsa roseopersicina. AB - Cell death and mutagenesis in bleomycin-treated cells of Thiocapsa roseopersicina (a purple sulfur bacterium) was studied by cultivation in a semisolid medium (agar-shake technique). This technique has also proven useful in assessing the frequency of antibiotic mutations by detecting and counting individual colonies of Thiocapsa roseopersicina. The frequencies of spontaneous mutants resistant to ampicillin, rifampicin, cloramphenicol, tetracycline, kanamycin, streptomycin, and neomycin were also studied: they ranged between 2 x 10(-9) and 9 x 10(-8). Bleomycin (4 micrograms/ml) sharply increased the frequency of ampicillin resistant mutants, from 10(-8) (spontaneous) to 4 x 10(-4) (induced), in 17 h. An inducible, error-prone mechanism of DNA synthesis seems to be responsible for this enhancement of the mutagenic effect. this is the first report on the sensitivity to several antibiotics, and capacity of lethality and mutagenesis by bleomycin has been studied in a purple sulfur bacterium. PMID- 7533577 TI - CD34+ selected cells in clinical transplantation. AB - The CD34 antigen is expressed by early hematopoietic stem cells and progenitors and is detected on the surface of approximately 1% of bone marrow mononuclear cells [1-3]. Several monoclonal antibody-based methods have been developed to isolate these cells from clinical samples of bone marrow or peripheral blood based on their expression of this antigen, utilizing either biotin-avidin affinity, panning or immunomagnetic beads. Roughly 50% of CD34+ cells, with 20 90% purity, are recovered from clinical samples using these methods. Several clinical trials have demonstrated hematopoietic recovery using CD34+ selected cells to support high dose therapy. CD34+ cells may be useful in several areas of clinical stem cell transplantation, including purging of tumor cells, T cell depletion, stem cell expansion and gene therapy. This paper reviews the current methods for purification of CD34+ cells from clinical samples and discusses potential uses of these cells in transplantation. PMID- 7533576 TI - The expanding role of fludarabine in hematologic malignancies. AB - The major clinical experience with fludarabine has been obtained in patients with chronic lymphocytic leukemia (CLL). In the initial studies in previously treated patients with CLL, the complete and partial response rate (CR + PR) was over 50%, and in previously untreated patients with CLL, a CR + PR rate of 75-80% was noted with or without the addition of prednisone. Subsequent clinical trials have also demonstrated major activity with fludarabine in Waldenstrom's macroglobulinemia. Fludarabine was noted to be an active agent in indolent lymphoma in phase I/II studies. Approximately 60% of patients with follicular lymphoma respond to fludarabine when it is administered as a single agent. Many of these remissions are complete remissions despite patients having received extensive prior therapy. Combination programs are being developed for application in CLL and indolent lymphoma. Because of the activity of fludarabine in inhibiting DNA repair, it has been combined with cisplatinum and cytosine arabinoside and plans are in place to explore the radiation sensitizing effect of fludarabine in clinical trials. A combination of fludarabine plus ara-C is now being used in patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) and a combination of fludarabine, ara-C, and G-CSF (FLAG) has been combined with idarubicin for the management of these conditions. Many of these activities of fludarabine are associated with its interaction with many enzymes which are important in DNA and RNA metabolism and in DNA repair. It is anticipated that these actions will be explored in a wider range of studies subsequently. PMID- 7533578 TI - Growth factor stimulation of cryopreserved CD34+ bone marrow cells intended for transplant: an in vitro study to determine optimal timing of exposure to early acting cytokines. AB - Stimulating CD34+ hematopoietic cells with growth factors prior to transplantation may decrease the duration of post-transplant aplasia. The optimal time in which to deliver this stimulus remains unclear. We therefore sought to determine if progenitor cell cloning efficiency, as reflected by colony forming units-granulocyte-macrophage (CFU-GM) colony formation, differed when growth factor stimulation was carried out prior to freezing or after thawing. To address this issue, CD34+ marrow cells were suspended in Iscove's medium with 20% bovine calf serum. They were then stimulated with kit ligand (KL), interleukin-3 (IL-3), and interleukin-1 beta (IL-1 beta) for 36 h either prior to cryopreservation or immediately after thawing. We observed that cells stimulated prior to freezing formed more CFU-GM colonies than cells stimulated after thawing. We also found that CD34+ cells stimulated with growth factors either before or after freezing gave rise to more CFU-GM colonies than thawed cells plated without prestimulation. Thus, the cloning efficiency of stored marrow, as reflected by CFU-GM colony formation, may be effectively enhanced by pretreating cells with hematopoietic growth factors. Our data further suggest that the optimal time for this treatment is before cryopreservation as opposed to immediately after thawing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533579 TI - Trilineage recovery by combination therapy with recombinant human granulocyte colony-stimulating factor and erythropoietin in patients with aplastic anemia and refractory anemia. AB - Ten patients with aplastic anemia (AA) and seven patients with refractory anemia (RA) were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and erythropoietin (rhEpo) in combination. rhG-CSF (5-20 micrograms/kg) and rhEpo (120-720 U/kg) were administered by s.c. injection three times a week for at least six months, and the administration was continued as maintenance therapy for as long as possible when hematological responses were observed. Six (60%) of the ten AA patients and four (58%) of the seven RA patients showed multilineage responses. Of these responders, six patients achieved trilineage recovery. While all of the responders were dependent on red blood cell transfusions and eight of them required platelet transfusions before treatment, they now no longer need transfusions of either red blood cells or platelets. A median treatment duration of 9 (range 1 to 28) months was required to achieve multilineage recovery. The responders showed an ability to maintain the multilineage recovery for 9+ to 47+ months and to tolerate long-term treatment. These results indicate that the long-term treatment with rhG-CSF and rhEpo may benefit a substantial percentage of patients with AA and RA and provide an optional therapy for these patients. PMID- 7533580 TI - Modulation of human cord blood progenitor cell growth by recombinant human interleukin 3 (IL-3), IL-6, granulocyte-macrophage colony stimulating factor (GM CSF) and stem cell factor (SCF) in serum-supplemented and serum-free medium. AB - We evaluated the growth of cord blood myeloid progenitors or colony forming units granulocyte-macrophage (CFU-GM) and their response to various recombinant growth factors or colony stimulating factors (CSFs): interleukin 3 (IL-3), IL-6, granulocyte-macrophage CSF (GM-CSF) and stem cell factor (SCF). Using classical stimulant (human placenta conditioned medium or HPCM), we observed a significantly higher day-14/day-7 CFU-GM ratio in CB than in bone marrow (BM). The association of IL-3, IL-6, GM-CSF and SCF induced significantly more CB day 14 CFU-GM than HPCM. This effect is significantly greater in CB than in bone marrow. Since fetal calf serum (FCS) is known to contain inhibitors, we have compared the ability of CSFs to induce CFU-GM formation in FCS-supplemented and FCS-free culture. In CB, using HPCM, we obtained significantly more CFU-GM in FCS free medium than in FCS-supplemented medium. This difference was corrected by the addition of anti-transforming growth factor-beta (TGF-beta) neutralizing antibody. However, with the association of the four CSFs, no significant difference between FCS and FCS-free culture was observed. IN CONCLUSION: a) day 14/day-7 CFU-GM ratio was higher in CB than in BM indicating that CB CFU-GM are more primitive than BM CFU-GM; b) FCS can be successfully replaced by serum-free medium; c) FCS contains inhibitors of day-14 CFU-GM and among them TGF-beta; and d) the association IL-3, SCF, GM-CSF and IL-6 seems able to totally overcome the inhibitory effect of FCS. PMID- 7533581 TI - Production of functional myeloid cells from CD34-selected hematopoietic progenitor cells using a clinically relevant ex vivo expansion system. AB - There is increasing clinical interest focused on ex vivo manipulation and expansion of hematopoietic cells. In this study, we demonstrate that a simple combination of growth factors can expand progenitors to yield functional myeloid cells. Furthermore, this system can produce mature, functionally competent cells in the absence of fetal bovine serum (FBS), which will enhance the clinical utility of this approach. Hematopoietic progenitor cells obtained from normal bone marrow and from leukapheresis products were studied. The mononuclear fraction was enriched for CD34 cells using the Ceprate CD34 biotin kit (CellPro #LC34-1 or LC34-2). The selected cells were expanded for two weeks in Iscove's medium supplemented with 20% FBS and various combinations of interleukin-3 (IL 3), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and interleukin -6 (IL-6) added either simultaneously or sequentially. The optimal combination of these factors identified for myeloid expansion was simultaneous addition of IL-3, SCF and G-CSF (at 50 ng/ml each), resulting in an average 773 +/- 133-fold expansion of nucleated cells (n = 5). When corrected for the purity of CD34 cells in the starting population, the mean fold expansion with IL-3, SCF and G-CSF was 2,265 +/- 729. A mean of 74.7 +/- 10.5% (n = 3) of the expanded cells was positive for CD11b; 86-91% (n = 2) of the cells were promyelocytes or more mature granulocytes. Functional assays demonstrated normal phagocytosis and intracellular killing of Staphylococcus aureus (S. aureus) by the expanded cell population. Studies performed using cells expanded in defined serum-free media demonstrated that fold expansion was decreased and that the cells produced were less mature and functionally less competent than cells expanded with FBS. The decreased expansion could be partially reversed, and the functionality almost completely restored by the addition of autologous plasma. PMID- 7533582 TI - Interleukin 4 alters human bone marrow stroma and modulates its interaction with hematopoietic progenitors. AB - To investigate the functional activity of interleukin 4 (IL-4) on human marrow stroma formation, normal bone marrow (BM) samples were cultured in "Dexter-type" long-term cultures in the presence and absence of IL-4. IL-4 (0.001 to 1.0 micrograms/ml) added at the initiation of culture and once weekly when the cultures were fed effaced the culture architecture. In four-week old confluent cultures smooth muscle-like and endothelial-like cells were rare, the fibronectin network and cobblestone areas were absent, and a preponderance of monocyte macrophages characterized the adherent layer. Exposure to IL-4 reduced the numbers of CD34+ cells, colony-forming unit granulocyte-macrophage (GFU-GM) cells and burst-forming unit-erythroid (BFU-E) cells in the adherent layer, and increased their numbers in the nonadherent layer. In five of eight IL-4 containing cultures the concentrations of macrophage colony-stimulating factor (M CSF) were increased and in two of eight IL-4-treated cultures the concentrations of tumor necrosis factor-alpha (TNF-alpha) were significantly elevated as compared to those in control cultures, whereas there were no consistent differences in the levels of either IL-6 or transforming growth factor-beta (TGF beta). IL-1 beta and granulocyte-macrophage CSF (GM-CSF) were not detected in any culture. These data suggest that IL-4 suppresses stroma formation and alters its structure and cellular composition. PMID- 7533583 TI - The effect of orgasm on prostate-specific antigen. AB - To identify the effect of orgasm on serum prostate-specific antigen (PSA) levels, a prospective trial before and after orgasm was performed in 14 healthy colleagues aged 32-62 years (mean, 44.4 years) with no evidence of prostatic disease. PSA determinations were performed on serum samples obtained before and after orgasm. Significant changes in PSA levels after orgasm were found (P = 0.002, analysis of variance). We conclude that the impact of orgasm on PSA levels should be taken into account when the latter are used for the detection of prostatic disease. PMID- 7533584 TI - Transurethral microwave thermotherapy: past, present and future. PMID- 7533586 TI - AIDS and HIV-1 infection in the United Kingdom: monthly report. PMID- 7533585 TI - Risperidone: review of its pharmacology and therapeutic use in schizophrenia. AB - The discovery of antipsychotic medications has revolutionized the treatment of schizophrenia and other psychotic disorders. However, side effects such as extrapyramidal symptoms (EPS) and tardive dyskinesia have been limiting factors in treatment. The introduction of atypical drugs such as clozapine and risperidone has ushered in a new era. This article provides an overview of the pharmacology, efficacy, and techniques for the clinical use of risperidone, which has recently become available. PMID- 7533587 TI - Hepatitis C and blood transfusion. PMID- 7533589 TI - Unlinked anonymous monitoring of HIV prevalence in England and Wales: data to the end of 1993. PMID- 7533588 TI - Register of 'HIV seroconverters' in the United Kingdom. PMID- 7533591 TI - AIDS and HIV-1 infection in the United Kingdom: monthly report. PMID- 7533590 TI - An outbreak of Salmonella agona due to contaminated snacks. PMID- 7533592 TI - Burkholderia (formerly Pseudomonas) cepacia porin is an oligomer composed of two component proteins. AB - The 81 kDa protein (designated OpcPO) which forms a diffusion pore in the outer membrane of Burkholderia (formerly Pseudomonas) cepacia has a unique characteristic in that when the purified protein is heated it yields a major 36 kDa protein (designated OpcP1) and a minor 27 kDa protein (designated OpcP2). Moreover, incubation of OpcPO in citrate buffer at pH 3.0 produced an unusual dissociation into 72 kDa and 27 kDa proteins. For the characterization of OpcPO and its derivatives, OpcP1 and OpcP2 from purified OpcPO were isolated by preparative SDS-PAGE. Reconstitution of OpcPO using purified preparations of OpcP1 and OpcP2 indicated that these derivatives were not proteolytic fragments of OpcPO. Moreover, immunoblot assays with murine polyclonal antisera specific for OpcP1 and OpcP2 yielded the following results: (i) OpcP1 and OpcP2 are immunologically distinguishable proteins; (ii) the unusual dissociation of OpcPO in citrate buffer at pH 3.0 resulted in the release of OpcP2 from OpcPO, and the resulting 72 kDa protein was probably an oligomer of OpcP1; (iii) purified OpcP1 itself produced two additional 53 kDa and 72 kDa proteins spontaneously following elution from the bottom of the SDS-PAGE gel. From these findings, it was concluded that OpcPO is formed by the non-covalent association of OpcP2 with an oligomer of OpcP1 that has the ability to self-assemble. PMID- 7533593 TI - Transcription analysis of the Streptomyces coelicolor A3(2) rrnA operon. AB - Transcription start sites and processing sites of the Streptomyces coelicolor A3(2) rrnA operon have been investigated by a combination of in vivo and in vitro transcription analyses. The data from these approaches are consistent with the existence of four in vivo transcription sites, corresponding to the promoters P1 P4. The transcription start sites are located at -597, -416, -334 and -254 relative to the start of the 16S rRNA gene. Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S. coelicolor and other eubacteria. The P1 promoter is likely to be recognized by the RNA polymerase holoenzyme containing sigma hrdB, the principal sigma factor in S. coelicolor. P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus -35 region that is also present in the -35 regions of P3 and P4. The -35 sequence common to P2, P3 and P4 is not similar to any other known consensus promoter sequence. In fast-growing mycelium, P2 appears to be the most frequently used promoter. Transcription from all of the rrnA promoters decreased during the transition from exponential to stationary phase, although transcription from P1 and P2 ceased several hours before that from P3 and P4. PMID- 7533595 TI - Helicobacter pullorum sp. nov.-genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis. AB - Campylobacter-like organisms were isolated from the liver, duodenum and caecum of broiler and layer chickens, and from humans with gastroenteritis. They formed a unique DNA homology group and a polyphasic taxonomic analysis was made of 16 strains. Analysis of the nucleotide sequence of the 16S rRNA gene from seven of the strains identified them as belonging to a single species, within the genus Helicobacter. This conclusion was supported by the studies of relative DNA homology and of total protein electrophoretic patterns. The new species could be biochemically differentiated from other helicobacters and its ultrastructure in the electron microscope was typical of the genus except that the flagellum was not sheathed. We propose the name Helicobacter pullorum sp. nov. for this group. Like H. fennelliae or H. cinaedi it represents another non-gastric urease negative Helicobacter species colonizing the lower bowel. Its isolation from the livers of chickens with vibrionic hepatitis is significant. We describe a species specific PCR assay for H. pullorum sp. nov. which will facilitate its identification and further studies of its epidemiology. PMID- 7533594 TI - The dynamic progression of evolved character states for aromatic amino acid biosynthesis in gram-negative bacteria. AB - A systematic analysis of the evolution of aromatic amino acid biosynthesis in the Proteobacteria, previously focussed mainly upon the gamma subdivision, has now been extended to the beta subdivision. Five lineages were studied, represented by Neisseria gonorrhoeae, Nitrosomonas europaea, Alcaligenes faecalis, rRNA Group III pseudomonads/Rubrivivax gelatinosus, and rRNA Group-II pseudomonads/Rhodocyclus tenuis. Within the phenylalanine pathway, the bifunctional P-protein (chorismate mutase/prephenate dehydratase) was present in each lineage and must have evolved in a common ancestor of the beta and gamma subdivisions. Each P-protein was found to be subject to activation by L-tyrosine, and to feedback inhibition by L-phenylalanine. Phenylalanine-inhibited (DS-phe) and tyrosine-inhibited (DS-tyr) isoenzymes of 3-deoxy-D-arabino-heptulosonate-7 phosphate synthase probably existed in the common beta-subdivision ancestor, with DS-tyr being lost in N. gonorrhoeae and A. faecalis. The participation of DS-phe in a dissociable multienzyme complex with one or more other common-pathway enzymes is known to exist in N. gonorrhoeae. The same complex is indicated by two peaks of DS-phe seen in chromatographic profiles of Group-III pseudomonads and A. faecalis. It is concluded that the contemporary DS-phe species present in subdivision gamma and beta must have had independent origins. Tyrosine biosynthesis was found to be quite diverse within the beta subdivision. Nit. europaea possessed an arogenate dehydrogenase which was specific for NADP+. In all other lineages, a broad-specificity cyclohexadienyl dehydrogenase (CHD) was present. In N. gonorrhoeae the CDH was specific for NAD+ while the remaining CDH species could utilize either NAD+ or NADP+. Only the CDH species within the rRNA Group-II pseudomonad/R. tenuis lineage was feedback-inhibited by L-tyrosine, and this correlated with an allosteric pattern where activation of the prephenate dehydratase component of the P-protein by L-tyrosine was relatively poor. However, the CDH enzyme present in N. gonorrhoeae and A. faecalis was subject to inhibition by 4-hydroxyphenylpyruvate, this being competitive with respect to the cyclohexadienyl substrate. The monofunctional species of chorismate mutase (CM-F) and cyclohexadienyl dehydratase, widely distributed among the gamma-subdivision assemblage and recently shown to be periplasmic enzymes, were demonstrated in Pseudomonas pickettii, a member of rRNA homology Group-II. PMID- 7533596 TI - Characterization of Thiobacillus caldus sp. nov., a moderately thermophilic acidophile. AB - Two isolates of a novel, moderately thermophilic Thiobacillus species have been studied. The isolates, KU and BC13, are Gram-negative, motile bacteria having a pH optimum for growth of 2-2.5 and an optimum growth temperature of 45 degrees C. Both isolates are capable of chemolithotrophic growth on reduced sulfur substrates. They can also use molecular hydrogen as an electron donor. These two isolates can grow mixotrophically with sulfur or tetrathionate and yeast extract or glucose. The G+C content is 63.1-63.9 mol% and the isolates exhibit no significant DNA homology to any other Thiobacillus species. Strains KU and BC13 both contain ubiquinone Q-8. 16S rRNA analysis indicates that these strains belong to a group of bacteria which includes other chemolithotrophic sulfur oxidizers such as T. ferrooxidans and T. thiooxidans. These characteristics distinguish KU and BC13 from any other species described previously and they thus represent the first acidophilic, thermophilic Thiobacillus species, named T. caldus sp. nov., to be described. The type strain, referred to as strain KU in this paper, has been deposited in the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, with the accession number DSM 8584. PMID- 7533597 TI - Glycosaminoglycans enhance phorbol ester-induced proteolytic activity and angiogenesis in vitro. AB - It has been reported that endothelial cells suspended in three-dimensional type I collagen gels can be induced to undergo tube formation by 12-o-tetradecanoyl phorbol 13-acetate (TPA). In this report, we show that TPA-induced endothelial cell tube formation can be further enhanced by the addition of other matrix components in the collagen gels. In the presence of TPA, both high molecular weight hyaluronate and chondroitin sulfate elicit a dose-dependent stimulation of tube formation. The enhanced tube formation appears to be due to an increase in the number of cells undergoing morphogenesis as the average length per tube is not obviously increased. Concomitant with the increased cell morphogenesis, there is an increase in proteolytic activity secreted by the cells. Treatment of cells with cycloheximide suppresses hyaluronate- and chondroitin sulfate-enhanced cell morphogenesis and proteolytic activity suggesting that new protein synthesis, perhaps proteases, is necessary for endothelial cell morphogenesis. The possible role of the production of proteolytic activity in endothelial cell tube formation is discussed. PMID- 7533599 TI - Expression of human tumor mucin-associated carbohydrate epitopes, including sialylated Tn, and localization of murine monoclonal antibodies CC49 and B72.3 in a syngeneic rat colon carcinoma model. AB - Using immunohistochemical techniques and whole-cell enzyme-linked immunosorbent assay, we have determined that monoclonal antibodies (mAbs) B72.3 and CC49, which are widely used in the diagnosis and treatment of several human epithelial cancers, are expressed in a transplantable rat colon carcinoma cell line, K12 TRb. MAbs B72.3 and CC49 react with tumor-associated glycoprotein-72 (TAG-72) which is a carcinoma mucin molecule expressed in colon, breast, pancreatic, ovarian, lung, and gastric cancers. The carbohydrate epitope for mAb B72.3 is sialylated Tn (sTn), whereas CC49 reacts with an unknown carbohydrate epitope. K12-TRb is a transplantable rat colon carcinoma cell line derived from a dimethylhydrazine tumor which grows as progressive tumors in syngeneic BD IX rats. We found that the carbohydrate epitopes for mAbs B72.3 and CC49, including sTn, were more tumor-restricted in the rat than in humans. The only binding these had mAbs to normal rat tissue was to small-intestinal mucosa. MAbs B72.3 and CC49 were radiolabeled with iodine-125 (125I) and injected intravenously into BD IX rats containing subcutaneously grown syngeneic K12-TRb tumors. Biodistribution experiments were conducted by dissecting groups of three rats on days 2, 4, 7, and 14 after injection of radiolabeled mAbs. These experiments confirmed that maBs B72.3 and CC49 localize to K12-TRb tumors in vivo, and that the higher affinity mAb CC49 localized better than mAb B72.3. Gamma-camera imaging of subcutaneous K12-TRb tumors was successfully performed using 125I-labeled mAb CC49. The importance of this model is that mAbs B72.3 and CC49, immunoconjugates of these mAbs, and vaccines containing their corresponding carbohydrate epitopes, including sTn, can be studied in a relevant immunocompetent syngeneic rat colon carcinoma model. PMID- 7533598 TI - Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth. AB - A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd = 360 pM, 28,000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 microM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 micrograms/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells. PMID- 7533600 TI - Cytokines in the airway mucosa of subjects with asthma induced by toluene diisocyanate. AB - To determine the status of activation of lymphocytes and the role of cytokines on the inflammatory response of the bronchial mucosa in toluene diisocyanate (TDI) asthma, we performed a quantitative analysis of bronchial biopsies obtained from 15 subjects with TDI-induced asthma and seven normal control subjects. Markers of activation of lymphocytes (CD25 and Very Late activation Antigen-1, VLA-1) and expression of Tumor Necrosis Factor-alpha (TNF alpha) and interleukin-1 beta (IL 1 beta) were determined by immunohistology in the submucosa. Moreover, expression of adhesion molecules on endothelium of submucosal vessels was assessed. Asthmatic subjects had increased numbers of cells expressing CD25 and VLA-1 compared with the control group (p < 0.05). TNF alpha and IL-1 beta immunoreactivity was increased in asthmatics compared with control subjects (p < 0.01), whereas the expression of adhesion molecules, ICAM-1 and E-selectin, on vascular endothelium was not significantly different. No significant differences in the morphologic quantifications were observed between the asthmatics who had biopsies taken 2 d after TDI challenge (n = 7) and those with longer interval (21 +/- 8 d) between TDI challenge and biopsy (n = 8), suggesting that the increase in CD25, VLA-1, TNF alpha, and IL-1 beta was not due to an acute effect, but could be considered a part of the chronic inflammatory process of the airways. We conclude that the inflammatory response of the airways in TDI-induced asthma is characterized by persistent activation of lymphocytes and by chronic expression of proinflammatory cytokines. PMID- 7533602 TI - Increased nitric oxide in exhaled gas as an early marker of lung inflammation in a model of sepsis. AB - Nitric Oxide (NO) has been implicated in the pathologic vasodilation of sepsis. Because NO can be measured in the exhaled gas of animals and humans, we hypothesized that increases in exhaled NO would occur in a septic model. Using a blinded design, 10 male Sprague-Dawley rats (300 to 400 g) were anesthetized, paralyzed, tracheotomized, and randomized (5/group) to receive an intravenous injection of either lipopolysaccharide (LPS) (Salmonella typhosa, 20 mg/kg) or placebo (equal volume of saline). Thereafter, exhaled gas was collected and measurements of NO concentration were made using chemiluminescence every 20 min for 300 min during ventilation (RR 40 breaths/min, VT 3 ml; PEEP 0, FIO2 0.21). Another group of 10 animals (5 LPS; 5 control) were treated in the same fashion and then killed at 240 min and an arterial blood sample obtained for blood gas and TNF alpha determinations. Pressure volume (PV) curves were constructed and lungs removed, preserved, and submitted for histologic evaluation. LPS-treated rats had lower mean arterial pressures than the control group, p < 0.0001. No significant differences in static lung compliance and PV curves were found in the two groups. TNF alpha levels were greater in the LPS group (1.40 +/- 0.24 ng/ml) versus control group (0.09 +/- 0.04 ng/ml), p < 0.001. By contrast to the control group, exhaled NO concentration rose in all LPS-treated rats at approximately 100 min and at about 160 min reached a plateau that was 6 times greater than control levels (p < 0.0001). There was greater interstitial, airspace, and total lung injury in the LPS group (p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533601 TI - Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. AB - In experimental studies, tachykinins, especially substance P (SP), cause many of the pathophysiological features of neurogenic inflammation. It is unclear whether these peptides are involved in human airway inflammation in diseases such as asthma and chronic bronchitis. To elucidate the relation between neurogenic inflammation and airway inflammatory diseases, we examined the SP concentration in sputum after hypertonic saline inhalation challenge in patients with asthma, patients with chronic bronchitis, and normal volunteers. SP concentration was measured by radioimmunoassay. The sputum SP concentration was significantly higher in patients with asthma (mean +/- SEM, 17.7 +/- 2.4 fmol/ml; p < 0.01) and patients with chronic bronchitis (25.6 +/- 5.5 fmol/ml; p < 0.01) than in normal volunteers (1.1 +/- 0.4 fmol/ml). In patients with asthma, the SP concentration was significantly related to the eosinophil cell count in induced sputum. In all subjects, the SP concentration in induced sputum correlated with FEV1/FVC. These data suggest that neurogenic inflammation may be involved in the airway inflammatory process and subsequent airway narrowing not only in asthma but also in chronic bronchitis. PMID- 7533603 TI - Role of substance P in cough during bronchoconstriction in awake guinea pigs. AB - To examine the role of substance P (SP) in cough during bronchoconstriction, we studied the effects of an aerosolized beta-adrenoceptor agonist, procaterol, and a specific inhibitor of SP (NK1) receptor, FK 888, on bronchoconstriction and cough induced by aerosols of histamine and acetylcholine (ACh) in unsensitized guinea pigs and by those of ovalbumin (OA) antigen in guinea pigs sensitized to OA. Intensity of bronchoconstriction was evaluated by the time to onset of bronchoconstriction after the inhalation of bronchoconstrictors. Both procaterol (10(-6) to 10(-4) M, 2 min) and FK 888 (10(-7) to 10(-5) M, 2 min) dose dependently decreased the number of coughs and increased the time to onset of bronchoconstriction induced by histamine (10(-2) M, 15 s). Procaterol attenuated histamine-induced cough only at the concentrations effective to inhibit bronchoconstriction. However, FK 888 at concentrations of 10(-7) and 10(-6) M decreased the number of coughs without effect on bronchoconstriction. Likewise, the inhibitory effects of procaterol (10(-5) M, 2 min) on the number of coughs were parallel to those on bronchoconstriction induced by ACh (10(-1) M, 15 s) and OA antigen (0.1% concentration, 30 s), but FK 888 (10(-6) M, 2 min) decreased the number of coughs without effect on bronchoconstriction induced by them. The number of coughs induced by histamine (10(-2) M, 15 s) was inhibited by systemic capsaicin treatment and enhanced by phosphoramidon (10(-5) M, 5 min) without effect on bronchoconstriction. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533604 TI - Normal sweat chloride values do not exclude the diagnosis of cystic fibrosis. AB - Since its introduction in 1959, the sweat test has remained the "gold standard" diagnostic test for cystic fibrosis (CF). It is apparent that CF encompasses a wide spectrum of disease, from meconium ileus and severe respiratory compromise in infants to the presentation of mild pulmonary symptoms and no evidence of gastrointestinal disease in adults. In patients with lung disease that might otherwise be consistent with CF, normal sweat chloride (Cl-) values have tended to exclude the diagnosis. In this report we describe two patients from two families with the compound heterozygotic CF mutations delta F508/3849 + 10 kb C- >T. These patients had mild manifestations of disease, including clinical pancreatic sufficiency (normal growth without pancreatic enzyme supplementation) and absence of azoospermia. Sweat Cl- values were in the normal range. However, both patients developed bronchiectasis, progressive obstructive lung disease, and colonization with Pseudomonas. The diagnosis of CF was made using nasal transepithelial voltage measurements and genotyping. These cases emphasize the need to maintain a high index of suspicion of CF in atypical cases, and to pursue alternative diagnostic tests to confirm a diagnosis of CF suspected on clinical grounds, despite normal sweat test results. PMID- 7533605 TI - The cystic fibrosis transmembrane conductance regulator gene. AB - Since identification of the gene responsible for cystic fibrosis (CF) in 1989, significant progress has been made in elucidating the mutational basis for this severe, autosomal recessive disease. Such advances have been of major importance in furthering our understanding of the basic defect in CF. Studies of the protein product of the CF gene, referred to as the CF transmembrane conductance regulator (CFTR), indicate that the protein is a Cl- channel but may have additional functions. The most common mutation of the CF gene (delta F508), which leads to the deletion of a single amino acid on the protein molecule, occurs in approximately 70% of CF chromosomes, but the CF Genetic Analysis Consortium has documented over 300 other sequence alterations of the CF gene. In addition to single amino acid deletions, other types of mutations include missense, nonsense (stop codon), frameshift, and splice-junction mutations. Studies of potential correspondences among these different mutational types and predicted functional domains of the CFTR molecule may provide important clues to the physiologic role of normal CFTR and how a defective protein might lead to the CF phenotype. Analyses of genotype-phenotype relationships have shown strong correlations between particular mutations and pancreatic function. However, associations between genotype and severity of CF pulmonary disease are not clear-cut, suggesting that the pulmonary phenotype is strongly influenced by other genetic or environmental factors. PMID- 7533606 TI - Functions of the cystic fibrosis transmembrane conductance regulator protein. AB - Cloning of the cystic fibrosis (CF) gene through genetic linkage analysis has led to new discoveries concerning the function of ion channels and disease mechanisms. Current understanding of CF indicates that epithelial cells from CF patients have reduced Cl- permeability, which impairs fluid and electrolyte secretion and results in luminal dehydration. There is also evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) is the cyclic AMP dependent ion channel whose activation is defective in CF cells. The CFTR is composed of 1480 amino acids that reveal a structural homology to a family of transport proteins termed the transport ATPases. The nucleotide-binding domains of CFTR are the locus of many disease-causing mutations; the common mutation in CFTR is deletion of a phenylalanine at position 508. In addition, CFTR contains a regulatory domain with consensus sites for phosphorylation by protein kinases. Reversible phosphorylation is a regulatory feature of the signal transduction pathway in which the CF defect lies. The phosphorylated channel requires the continuous presence of ATP, whether in the form of ATP binding or hydrolysis, to maintain channel activity. Channel activation requiring ATP can be inhibited by simultaneous presence of ADP, showing that this nucleotide diphosphate competes with ATP for activation. Studies of mutant CFTR expression indicate that at least two basic mechanisms are responsible for the CF phenotype, including CFTR protein dysfunction and inappropriate protein targeting. If mechanisms for bringing this partially functional protein to the plasma membrane can be found, the airway disease of the vast majority of patients with CF could be improved. PMID- 7533607 TI - A murine model of cystic fibrosis. AB - We have generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been mutated by gene targeting. Like human cystic fibrosis (CF) patients, mice lacking a functional CFTR gene, referred to as CFTR(-/-) mice, show increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. The obstruction of glands often results in the destruction of gland-containing tissues in these animals. However, unlike the case in human CF patients, the most severe pathological changes in these mice were found, on preliminary analysis, to be confined to the intestinal tract and gallbladder. Although respiratory failure is the primary cause of death among humans with CF, we found only minor pathological alterations in the lungs and upper airways of our CFTR(-/-) animals. Possible explanations for the apparent lack of respiratory disease are the young age at which the animals were examined and the pathogen-free environment in which they were housed. In this manuscript, we examine the respiratory and other organ systems of CFTR(-/-) mice that have survived to adulthood. We also report on initial experiments in which CFTR(-/-) mice have been exposed to bacterial pathogens, and we present data on a single animal that displayed severe respiratory disease. PMID- 7533608 TI - Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis. AB - Cystic fibrosis (CF) is a recessive genetic disease with thickened airway secretions that result from abnormal airway epithelial ion transport, including defective cyclic AMP-mediated Cl- (liquid) secretion and excessive Na+ (liquid) absorption. These abnormalities reflect mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which normally functions as a cyclic AMP-regulated Cl- channel. Aerosolized pharmacologic agents are being tested as novel treatment for these genetic ion transport defects. Amiloride aerosol inhibits excessive Na+ absorption, and pilot studies in adult patients with CF show improved biorheology and mucociliary clearance of airway secretions, as well as slowing of the decline in lung function. Phase III studies of amiloride in adults and adolescents are underway, and short-term safety studies in children are under way. Aerosolized uridine triphosphate (UTP) induces Cl- secretion in CF airway epithelia via non-CFTR Cl- channels. Initial safety studies suggest that acute aerosolized UTP is well tolerated, and acute studies of the effect on mucociliary clearance are underway. Pharmacotherapy that targets abnormal ion transport holds promise for the treatment of CF airway disease. PMID- 7533609 TI - Gene therapy for the respiratory manifestations of cystic fibrosis. AB - Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major manifestations are on the airway epithelial surface, with purulent mucus, recurrent infections, chronic inflammation, and loss of lung function. Consequent to mutations in both parental genes, airway epithelial cells have insufficient CFTR function. Because this can be corrected in vitro by transfer of the normal CFTR gene into airway epithelial cells, it is reasonable to hypothesize that the respiratory manifestations of CF could be prevented by transfer of the normal human CFTR cDNA to the airway epithelium in vivo. Over the past 6 years, our laboratory has developed a strategy to accomplish this goal using a replication deficient E1-E3- recombinant adenovirus (Ad) serotype 5 vector containing the normal human CFTR cDNA (AdCFTR). Studies with experimental animals demonstrate that with administration of such a vector to the airways, the human CFTR cDNA could be transferred to the airway epithelium, with expression of the human CFTR cDNA for at least 6 weeks. Extensive preclinical studies in vitro and in vivo demonstrated that the risks to humans were sufficiently low to initiate a Phase I trial using the AdCFTR vector to treat the respiratory manifestations of CF in humans. Following approval by the National Heart, Lung, and Blood Institute Institutional Review Board, the National Institutes of Health Biosafety Committee, the National Institutes of Health Recombinant DNA Advisory Committee, and the Food and Drug Administration, we initiated the first human trial of gene therapy for CF on April 17, 1993. The clinical study is still ongoing, with safety and efficacy data being evaluated, but there is clear evidence that it is feasible to transfer and express the normal CFTR cDNA to the airway epithelium in vivo in individuals with CF. PMID- 7533610 TI - Increased striatal dopamine efflux in vivo following inhibition of cerebral nitric oxide synthase by the novel monosodium salt of 7-nitro indazole. AB - The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7-nitro indazole (7-NINA). Using 7-NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7-NINA (1 mM) increased striatal dopamine efflux. The effect of 7 NINA was partially antagonized (67%) by co-perfusion with L-arginine (1 mM), the precursor of NO formation in vivo. This suggests that 7-NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo. PMID- 7533612 TI - Local cardiac effects of substance P: roles of acetylcholine and noradrenaline. AB - 1. The local cardiac actions of substance P were examined in isolated perfused hearts and atria of the guinea-pig. 2. In both hearts and right atria, substance P caused negative inotropic and chronotropic effects. 3. Atropine (10(-6) M) or depletion of acetylcholine, by electrical stimulation and hemicholinium-3 perfusion, significantly attenuated the negative inotropic and chronotropic effects of substance P. alpha- and beta-adrenoceptor blockade by nadolol and phentolamine (10(-6) M each) did not prevent the negative inotropic and chronotropic effects of substance P. This indicates that cholinergic neurones, but not adrenergic neurones, partially mediate the effects of substance P. 4. There was no significant difference in the effects of substance P observed between groups with acetylcholine depletion and with cholinoceptor blockade. This suggests that substance P elicits its effects mainly through release of acetylcholine. 5. These results indicate that substance P has negative inotropic and chronotropic effects in guinea-pig hearts and right atria mediated partly by release of acetylcholine. Substance P also appears to have direct effects on cardiac tissue. PMID- 7533611 TI - Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A. AB - 1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/ 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation. However, co-administration of these two peptides to a single sponge together caused a significant increase in the rate of 133Xe clearance and angiogenesis similar to that seen with the high dose of VEGF165 (250 ng) acting alone. This VEGF/bFGF neovascular response was also blocked by daily co-administration of lavendustin A (10 jig),suramin (3 mg) or a monoclonal anti-bFGF antibody (DG2, I jig), but not lavendustin B (10 g).6 These results suggest that selective inhibition of PTK could have therapeutic potential in angiogenic diseases where VEGF plays a dominant role. Furthermore, blockade of the angiogenic activity of VEGF and VEGF,/bFGF by suramin reveals an alternative strategy in angio suppression. PMID- 7533613 TI - Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues. AB - 1. Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH-groups. 2. The NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) dose-dependently increased L-arginine-induced insulin release. D-Arginine did not influence L arginine-induced insulin secretion. However, D-NAME which reportedly has no inhibitory action on NO synthase, modestly increased L-arginine-induced insulin release, but was less effective than L-NAME. High concentrations (10 mM) of D arginine as well as L-NAME and D-NAME could enhance basal insulin release. 3. The intracellular NO donor, hydroxylamine, dose-dependently inhibited insulin secretion induced by L-arginine and L-arginine+L-NAME. 4. Glucose-induced insulin release was increased by NO synthase inhibition (L-NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine-1 (SIN-1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose-stimulated insulin release. SIN-1 did not influence insulin secretion induced by L-arginine or the adenylate cyclase activator, forskolin. 5. The intracellular 'hydroperoxide donor' tert-butylhydroperoxide in the concentration range of 0.03 3 mM inhibited insulin release stimulated by the nutrient secretagogues glucose and L-arginine. Low concentrations (0.03-30 microM) of tert-butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). 6. Islet guanosine 3':5'-cyclic monophosphate (cyclic GMP) content was not influenced by 10 mML-arginine or tert butylhydroperoxide at 3 or 300 micro M but was markedly increased (14 fold) by a high hydroxylamine concentration (300 micro M). In contrast, islet adenosine 3':5'-cyclic monophosphate (cyclicAMP) content was increased (3 fold) by L arginine (10 mM) and (2 fold) by tert-butylhydroperoxide(300 micro M).7. Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues L-arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase-cyclic GMP system but may rather be exerted by the S-nitrosylation of critical thiol groups involved in the secretory process. Similarly the inhibitory effect of tert butylhydroperoxide is likely to be elicited through affecting critical thiol groups. The mechanism underlying the secretion promoting action of tert butylhydroperoxide on IBMX-induced insulin release is probably linked to intracellular Ca2+-perturbations affecting exocytosis.8. Taken together with previous data the present results suggest that islet production of low physiological levels of free radicals such as NO and H202 may serve as important modulators of insulin secretory processes. PMID- 7533614 TI - Modulation by nitric oxide of prostaglandin biosynthesis in the rat. AB - 1. Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)-induced paw oedema and measuring both the foot volume and the amount of 6 keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2. Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor L-NG-nitro arginine methyl ester (L-NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 mumol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 mumol/paw) and L-arginine (15 mumol/paw). 3. The NO-donors (10 mumol/paw) 3-morpholino-sydnonimine hydrochloride (SIN-1), S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4. SIN-1 (2.5, 5 and 10 mumol/paw) produced a significant dose dependent increase of the oedema induced by AA which was correlated with increased amounts of 6-keto-PGF1 alpha in the fluid recovered from inflamed paws. 5. Both oedema and prostaglandin biosynthesis induced by the combination AA+SIN-1 were greatly suppressed by either Hb (30 mumol/paw) or indomethacin (3 mumol/paw or 5 mg kg-1 s.c.) but unaffected by Mb (3 mumol/paw). 6. In LPS-treated rats (6 mg kg-1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by L-NAME or Hb, unaffected by Mb and increased by L arginine.7. These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3': 5'-cyclic monophosphate (cyclic GMP) independent mechanism and suggest that the interaction between NO synthase and cyclo-oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response. PMID- 7533616 TI - Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells. AB - 1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13 dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved. PMID- 7533615 TI - Effects of nitric oxide synthase inhibition combined with nitric oxide inhalation in a porcine model of endotoxin shock. AB - 1. The present investigation compares the effects of intravenous infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) with that of an inhalation with NO gas in a porcine model of endotoxin (lipopolysaccharide, LPS) shock. In addition, the effects of the combination of these two treatments were also investigated. 2. Male pigs were anaesthetized and instrumented for the measurement of haemodynamic parameters. Blood samples were withdrawn at different time intervals for determination of blood gases, pH, and plasma levels of nitrite/nitrate and tumour necrosis factor. 3. Endotoxin infusion (15 micrograms kg-1 h-1 for 3 h) caused a progressive fall in mean arterial blood pressure (MABP) and cardiac output (CO) and a biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR). A continuous infusion of L-NMMA (0.1 mg kg-1 min-1) significantly attenuated the fall in MABP, but did not affect MPAP, CO and PVR. NO-inhalation (50 p.p.m.) did not affect MABP, but significantly blunted the biphasic increase in MPAP and PVR and significantly delayed the fall in CO. The combination of L-NMMA infusion (0.1 mg kg-1 min-1) with NO-inhalation (50 p.p.m.) completely prevented the fall in MABP, significantly improved CO, and attenuated the biphasic increase in MPAP and PVR. 4. Endotoxin also caused a decline in PaO2 and a rise of PaCO2. Infusion of L NMMA neither affected the fall in PaO2 nor the increase in PaCO2. In contrast, inhalation with NO gas alone as well as the combined administration of L-NMMA infusion and NO-inhalation completely prevented the fall in Pao2 and significantly protected against the increase in Paco2.5. Infusion of endotoxin for 180 min resulted in a mortality of 58%, which was not affected by L-NMMA (63%). In contrast, treatment of LPS-animals with either NO-inhalation alone or NO inhalation plus L-NMMA completely prevented mortality.6. This investigation demonstrates that treatment with NO-inhalation, in order to prevent the dramatic increase in MPAP, PVR and the alterations in peripheral blood gases combined with systemic L-NMMAto improve systemic MABP and thus organ perfusion, may be a new therapeutic regimen in the treatment of septic shock. PMID- 7533617 TI - Pharmacological evidence that nitric oxide may be a retrograde messenger in the enteric nervous system. AB - 1. The effects of inhibition of nitric oxide synthase on neuro-neuronal and neuromuscular transmission during motility reflexes in the small intestine of the guinea-pig were examined. 2. Isolated segments of intestine were secured in a three chambered organ bath so that different parts of the reflex pathways could be independently exposed to drug-containing solutions. Reflexes were evoked by distension or compression of the mucosa in two adjacent chambers and reflex responses were recorded from the circular muscle with intracellular microelectrodes in the third chamber. Thus, the actions of drugs at connections between sensory neurones and interneurones, between interneurones and other interneurones and at motor neurones could be distinguished. 3. NG-monomethyl-L arginine (L-NMMA; 100 microM), an inhibitor of nitric oxide synthase, did not affect the ascending excitatory reflex when added to either the central stimulation chamber or the recording chamber. 4. In contrast, L-NMMA (100 microM) enhanced the descending inhibitory reflex when added to the chamber in which stimuli were applied. This effect was prevented by prior exposure to L-arginine (100 microM), which had no effect by itself. Conduction of reflexes between the stimulus chamber and the recording chamber was unaffected by the presence of L NMMA in an intervening chamber. 5. L-NMMA (100 microM) added to the recording chamber depressed the descending inhibitory reflex, an effect that was prevented by previous exposure to L-arginine. 6. The nitric oxide donor, sodium nitroprusside (100 microM), added to the stimulus chamber, depressed both ascending excitatory and descending inhibitory reflexes. When added to the middle chamber,sodium nitroprusside had no effect on conduction of reflexes through this chamber.7. It is deduced that nitric oxide, released from the cell bodies of descending interneurones, suppresses transmission from synaptic connections made with them by enteric sensory neurones. PMID- 7533618 TI - Necessity of protein kinase C activity for maintenance of acetylcholine receptor function at snake twitch fibre endplates. AB - 1. The extent of recovery of endplate sensitivity following a 5 or 10 min exposure to carbachol was determined from measurements of miniature endplate current (m.e.p.c.) amplitudes in voltage-clamped snake twitch fibre endplates. M.e.p.c. amplitude recovery was dependent on the carbachol concentration (0.27 5.4 mM) and duration of application. Staurosporine pretreatment (0.5 microM for approximately 15 min) further decreased the extent of m.e.p.c. amplitude recovery. 2. The decrease in m.e.p.c. amplitude at control endplates exposed to high concentrations of agonist (5.4 mM carbachol for 10 min) was due to an apparent decrease in postsynaptic receptor density, not to a change in the conductance of the acetylcholine (ACh)-activated channels. 3. Pretreatment with either 1 microM lavendustin A or 50 microM KN-62 had no effect on m.e.p.c. amplitude recovery, whereas pretreatment with either 0.5 microM staurosporine, 50 microM sphingosine, or 0.5 microM calphostin C significantly reduced m.e.p.c. amplitude recovery following carbachol exposure. 4. Sphingosine and staurosporine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery, but had no effect on m.e.p.c. characteristics in the absence of carbachol. In addition, this decrease in m.e.p.c. amplitude was not due to the presence of a subpopulation of small amplitude m.e.p.cs. 5. Prolonged treatment (18-20 h) of muscles with 200 nM phorbol 12-myristate 13-acetate (PMA), to down regulate protein kinase C, resulted in a significant reduction in m.e.p.c. amplitudes following exposure to carbachol. Conversely, treatment with 200 nM 4 alpha PMA, an inactive analogue, had no effect on m.e.p.c. amplitude recovery. 6. Only large amplitude ACh-activated channels (~50 pS) were recorded from fibres either in the presence of 50 micro M sphingosine or from fibres chronically exposed to PMA. However, following recovery from a 10 min exposure to 540 micro M carbachol, both small conductance (-25 pS) and large conductance ACh-activated channels were recorded in both sphingosine- and phorbol-treated preparations. The conductance of these two populations of channels was virtually identical to those seen in staurosporine treated fibres following carbachol exposure.7. We conclude that protein kinase C is required for full recovery of AChR sensitivity following carbachol-induced receptor inactivation. Exposure to high concentrations of agonist for prolonged periods appears to result in the inactivation of a subpopulation of receptors. These receptors must be replaced or reactivated by a process involving protein kinase C. When this phosphorylation step is inhibited, the AChRs remain in an activatable form, but with a reduced conductance. PMID- 7533619 TI - Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery. AB - 1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance. PMID- 7533620 TI - Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. AB - 1. FPL 67156 (6-N,N-diethyl-beta, gamma-dibromomethylene-D-ATP), is a newly synthesized analogue of ATP. 2. In a rabbit isolated tracheal epithelium preparation, measuring P2U-purinoceptor-dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5 fold in the presence of 100 microM FPL 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3. FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma 32P]-ATP dephosphorylation. The compound inhibited [gamma 32P]-ATP degradation with a pIC50 of 4.6. 4. FPL 67156 was then tested for its effects on ATP and alpha, beta-methylene-ATP responses at P2X purinoceptors in the rabbit isolated ear artery. In the concentration range 30 microM-1 mM, the compound potentiated the contractile effects of ATP but not those of alpha, beta-methylene-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/[A] curves. 5. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analyzed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pKi for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X- and P2T-purinoceptors (pA2 ~ 3.3 and 3.5 respectively), and weak agonist effects at P2u purinoceptors(p[A 50]~ 3.5).7. The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.8.This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors. PMID- 7533621 TI - Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages. AB - 1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. PMID- 7533623 TI - The fasciovascular flap: a new vehicle for islet transplantation. AB - In order to determine whether pancreatic islets could be neovascularized by a fasciovascular flap (FVP), islet transplant studies were conducted in Lewis rats. Islets from two donors were isolated by collagenase digestion and discontinuous gradient centrifugation on Ficoll. These islets were injected in syngeneic recipients either into random groin SC fat as a control, or into a flap composed of fascia and fat elevated from the groin based on the superficial inferior epigastric vessels. After two wk, islet viability was assessed by histological analysis. The degree of neovascularization of the islet tissue was evaluated with India ink injection through the vascular pedicle. Whereas control islets degenerated and did not show clear signs of neovascularization, FVP-islets showed rich neovascularization and viability as a large sheet of islet clusters. These results have demonstrated that the FVP-flap is a novel recipient site which can support a large quantity of islet tissue. This model constitutes a unique neo endocrine pancreas flap, which can be subsequently transplanted at will to transfer the established neo-endocrine pancreas to a desired site using microvascular surgical technique. PMID- 7533622 TI - Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity. AB - 1. The induction of a calcium-independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S-substituted isothioureas, S methylisothiourea (SMT), S-(2-aminoethyl)isothiourea (aminoethyl-TU), S ethylisothiourea (ethyl-TU) and S-isopropylisothiourea (isopropyl-TU) potently inhibit iNOS activity in J774.2 macrophages activated with bacterial endotoxin with EC50 values 8-24 times lower than that of NG-methyl-L-arginine (MeArg) and 200-times lower than that of NG-nitro-L-arginine (L-NO2Arg). 2. The inhibition of iNOS activity by these S-substituted isothioureas is dose-dependently prevented by excess of L-arginine suggesting that these isothioureas are competitive inhibitors of iNOS at the L-arginine binding site. 3. Ethyl-TU and isopropyl-TU are 4-6 times more potent than MeArg in inhibiting the constitutive NOS activity in homogenates of bovine aortic endothelial cells (eNOS) and are more potent pressor agents than MeArg in the anaesthetized rat. SMT is equipotent with MeArg, whereas aminoethyl-TU is 6-times less potent in inhibiting eNOS activity in vitro. Both SMT and aminoethyl-TU, however, elicit only weak pressor responses (approximately 15 mmHg at 10 mg kg-1, i.v.) in vivo. 4. A comparison of the potencies of ethyl-, iso-propyl-, n-propyl-, t-butyl- and n-butyl-isothioureas on iNOS activity shows that the inhibitory activity of S-substituted isothioureas declines sharply if the side chain exceeds 2 carbon atoms in length. Similarly, substitution of the ethylene side chain of ethyl-TU also results in a diminished potency. Substitution of either one or both nitrogens of SMT with either amino or alkyl groups also substantially reduces its NOS inhibitory potency.5. In conclusion, isothioureas represent a new class of NOS inhibitors which includes the most potent inhibitors of iNOS activity reported to date. Some members of this class (ethyl-TU and isopropyl-TU)are potent inhibitors of eNOS and iNOS with little selectivity towards either isoform, while others (SMT and aminoethyl-TU) are relatively selective inhibitors of iNOS activity. These latter agents may become useful tools for studying the role of iNOS in various disease models and may be useful in the therapy of diseases that are associated with an enhanced formation of NO due to iNOS induction, such as inflammation, circulatory shock or cancer. PMID- 7533625 TI - Immunocytochemical detection of the carbohydrate antigen, Sialyl Lewis(x), in normal human skin and during irritant contact dermatitis. AB - Sialys Lewis(x) (SLex) is a ligand for the E-selectin and the interaction of E selectin on the endothelium and SLex on T cells may be important for T-cell migration into the skin. We investigated the expression of SLex on Langerhans cells (LC) in normal skin and on LC repopulating epidermis deprived of LC due to a preceding irritant contact dermatitis. SLex was visualized by fluorescence and light microscopic immunocytochemistry using the monoclonal antibody, CSLEX-1. The results showed that about 40% of LC in normal epidermis express SLex. In the repopulation phase, most of the epidermal cells were CD1a+/SLex. We suggest that SLex is present on epidermal LC that have recently immigrated from the dermis. PMID- 7533624 TI - CD36(+)-dendritic epidermal cells: a putative actor in the cutaneous immune system. AB - In the present study we have investigated by indirect immunofluorescence staining and mixed lymphocyte reaction methods, the localization, distribution, percentage, and the immunological involvement of CD36(+)-dendritic epidermal cells (CD36(+)-DECs) in normal human skin. Human epidermal cell suspensions were obtained from skin specimen of healthy persons. First, an indirect immunofluorescent staining method was performed on frozen skin sections, freshly isolated cells, nonadherent and adherent cells and second, the allogeneic mixed epidermal cell-lymphocyte reaction (ELR) method was performed with human peripheral blood mononuclear cells and irradiated CD36(+)-DECs plus CD-1a+ (Langerhans cells) and/ConA (at 10 micrograms/mL). We found that CD36(+)-DECs were localized in the epidermis mainly in the basal layer. They were non adherent cells. The percentage of these CD36(+)-DECs was of about 2%. These CD36(+)-DECs were AE3 (which recognizes keratin normally expressed by keratinocytes) positive cells. Our immunoreactivity study using allogeneic mixed ELR, showed that CD36(+) DECs stimulated allogeneic lymphocyte proliferation. Their stimulatory effects were important when Langerhans cells and ConA were added separately or together to the PBMCs culture. The above results suggest that CD36(+)-DECs may contribute to the immunological role of skin and could be involved in cutaneous allograft recognition and rejection. ABBREVIATIONS: DECs: dendritic epidermal cells; ConA: concanavalin A; DPM: disintegrations per minute; ELR: epidermal cell-lymphocyte reaction; LC: Langerhans cells; PBMCs: peripheral blood mononuclear cells. PMID- 7533626 TI - Gender-related differences in expression of murine glutathione S-transferases and their induction by butylated hydroxyanisole. AB - The basal levels of mu and pi class glutathione S-transferases RNA were 18-fold higher in the male mouse liver as compared with the female. When 0.75% (w/w) BHA was included in the diet it altered the RNA levels of alpha, mu, pi GST classes and mGSTA4-4 in a tissue and sex specific manner. The most marked induction of RNA was seen for the mu class GSTs of female liver, lung and kidney (52, 10 and 8 fold, respectively), and of male liver and kidney (25 and 3.5-fold, respectively), the pi class GSTs of female liver, lung, and kidney (11, 10, and 5 fold, respectively), and mGSTA4-4 of female liver (4-fold). The effect of BHA on the induction of the mu and pi class GST RNA was 2-9 fold greater in female as compared with male tissues. The degree of induction of GST RNA did not correlate directly with changes in GST protein indicating that post-transcriptional events regulating GST expression may be affected by BHA particularly for GST mu and mGSTA4-4. PMID- 7533627 TI - Effect of lindane on galactose and leucine transport in chicken enterocytes. AB - Lindane (gamma-hexachlorocyclohexane) influence on the in vitro intestinal transport of D-galactose and L-leucine has been studied in isolated chicken enterocytes. Animals were injected i.p. with 30 mg/kg b.w. of the pesticide over 7 days. Total uptake of D-galactose and L-leucine was significantly decreased by lindane action. There was no alteration in the non-mediated component, but the mediated transport was markedly inhibited in both cases. Furthermore, the exit of D-galactose across the basolateral membrane, as well as (Na(+)-K+)-ATPase activity, was significantly decreased in pesticide-treated chickens. PMID- 7533629 TI - Concentrations of soluble vascular cell adhesion molecule-1 and E-selectin in breast cyst fluid and their relation with cyst type. AB - Gross cystic breast disease may be associated with a higher risk of breast cancer. There are two groups of mammary cysts: those lined by apocrine epithelium (intracystic sodium to potassium (Na:K) ratio < 3) and those lined by flattened epithelium (intracystic Na:K ratio > 3). Cell adhesion molecules may be involved in various stages of tumour metastasis. Intracystic levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble E-selectin (sE-selectin) were measured. Significantly greater concentrations of sVCAM-1 and sE-selectin occurred in the high-electrolyte ratio group than in the low (P < 0.001). A positive correlation was found between sVCAM-1 and sE-selectin (rs = 0.827, P < 0.001) which may be a chance observation or suggest a common mechanism of entry into mammary cysts. The significance of these soluble cell adhesion molecules in breast cyst fluid requires elucidation. PMID- 7533628 TI - Multiple calcium signaling pathways in Mardin-Darby canine kidney cells. AB - Effects of various receptor agonists on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura-2-loaded Mardin-Darby canine kidney (MDCK) monolayer cells. Carbachol (100 microM) increased [Ca2+]i which slightly declined to a sustained increase in [Ca2+]i. On the other hand, [Ca2+]i elevated by 100 nM bradykinin (BK) declined to a resting level of [Ca2+]i even in the presence of BK. After washout of BK, the subsequent addition of a higher concentration of BK (1 microM) caused a smaller increase in [Ca2+]i than that induced by 100 nM BK. Isoproterenol (100 microM) did not increase [Ca2+]i by itself but caused an transient increase in [Ca2+]i in the presence of 1 mM isobutyl-methylxanthine (IBMX). Prostaglandin E1 (1 microM) resulted in a slight increase in [Ca2+]i which was potentiatedin the presence of 1 mM IBMX. The microsomal Ca(2+)-ATPase inhibitor thapsigargin (100 nM) caused a sustained increase in [Ca2+]i. These results suggest that MDCK cells have multiple Ca2+ signaling pathways which may regulate epithelial cell functions. PMID- 7533630 TI - Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb. AB - The distribution of NADPH-diaphroase activity was examined in the accessory olfactory bulb of the rat using a direct histochemical technique. Labeled fibers and somata were found in all layers of the accessory olfactory bulb. The entire vomeronasal nerve and all vomeronasal glomeruli were strongly labeled, contrary to the main olfactory bulb, where only dorsomedial olfactory glomeruli displayed NADPH-diaphorase activity. NADPH-diaphorase positive neurons were identified as periglomerular cells in the glomerular layer and external plexiform layer, horizontal cells in the internal plexiform layer, and granule cells and deep short-axon cells in the granule cell layer. The labeled dendrites of the granule cells formed a dense neuropile in the granule cell layer, internal plexiform layer and external plexiform layer. The staining pattern in the accessory olfactory bulb was more complex than what has been previously reported, and demonstrated both similarities and differences with the distribution of NADPH diaphorase in the main olfactory bulb. PMID- 7533631 TI - Contribution of potassium channels to the discharge properties of rat aortic baroreceptor sensory endings. AB - The expression of several types of membrane potassium channel at the cell body and central synaptic terminal of the rat aortic arch baroreceptor has been reported by others. It is not known if any of the same channels function at the peripheral sensory terminal of these afferent nerves. Our study examined the effect of three potassium channel blocking agents on the pressure-evoked discharge of such baroreceptors. Thirty-one single unit, regularly discharging baroreceptors were studied using an in vitro aortic arch-aortic nerve preparation. Discharge thresholds and suprathreshold pressure sensitivities were derived from responses of receptors to slowly rising ramps of pressure applied to the aortic arch. Vessel diameter was recorded along with receptor discharge to assess any drug-induced changes in vascular smooth muscle. The blocking agents tested have a range of specificities for classes of potassium channels: tetraethylammonium (TEA), 4-aminopyridine (4-AP) and charybdotoxin. TEA depressed the pressure sensitivity of all baroreceptors tested (n = 3) in a dose-dependent manner. Baroreceptor responses to 4-AP were complex (n = 22) and varied widely across individuals. Three were unaffected by 5 mM 4-AP. Most baroreceptors were generally depressed by 4-AP. Some of the 4-AP effects appeared to be related to actions at vascular smooth muscle. None of the baroreceptors tested (n = 6) was affected by charybdotoxin. The results of selective potassium channel blockade are generally consistent with what would be expected from a sustained depolarization of baroreceptor endings such as has been reported with raising extracellular potassium and probably includes effects of inactivation of other voltage-dependent channels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533632 TI - Tachykinin NK3 receptor agonist blocks sodium deficiency-induced shift in taste reactivity. AB - Intracerebroventricular (i.c.v.) injections of tachykinin NK3 receptor agonists suppress NaCl intake by sodium deficient rats. The brief exposure, taste reactivity test was used to examine the effect of tachykinins on the immediate, oral reinforcing properties of NaCl (0.5 M) in sodium replete and sodium deficient male rats. In sodium replete rats, intraoral infusions of NaCl elicited a mixed response comprised of a similar number of ingestive and aversive responses following i.c.v. injections of saline and succinyl-[Asp6,N-Me Phe8]substance P, (6-11), (SENK), a NK3 receptor agonist. When sodium deficient, saline injected rats showed a reliable increase in ingestive and a decrease in aversive taste reactivity responses. Lateral i.c.v. injections of SENK blocked the shift in taste reactivity responses by sodium deficient rats, indicating that concentrated NaCl retained its aversive taste property. PMID- 7533633 TI - Interactions of dextromethorphan with the N-methyl-D-aspartate receptor-channel complex: single channel recordings. AB - The actions of dextromethorphan (DXM) on the 50 pS conductance state of the N methyl-D-aspartate (NMDA) receptor-operated channel were studied using outside out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5-50 microM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 microM, with the patch potential held at -60 mV. An IC50 value of 4 microM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca2+]i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 x 10(6) M-1.s-1 (at -60 mV). The estimated unblocking rate constant was about 10 s-1, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel. PMID- 7533635 TI - Nitric oxide-synthesizing neurons in the hamster suprachiasmatic nucleus: a combined NOS- and NADPH- staining and retinohypothalamic tract tracing study. AB - Neuronal nitric oxide (NO), thought to be a neuroactive substance of high potency, is produced by the enzyme nitric oxide synthase (NOS) which has been demonstrated to additionally exhibit a so-called NADPH-diaphorase (NADPH-d) activity. Since physiological results pointed to the involvement of NO in circadian regulation, and morphological descriptions are not available, we sought to study the distribution of NO-producing cells in the hypothalamic suprachiasmatic nucleus (SCN) in Djungarian hamsters (Phodopus sungorus) by means of histochemistry and immunohistochemistry (IHC). In the SCN, NADPH-d stained perikarya of varying intensity and number were found predominantly in the ventrolateral subdivision. Diaphorase staining combined with the IHC demonstration of NOS revealed a complete overlapping of both. The combination of NADPH-d staining with the demonstration of the retinohypothalamic tract using the anterograde neuronal transport of cholera toxin B (CTB) following intraocular injection showed CTB terminals accumulating at NADPH-d cell bodies mainly in the ventrolateral region of the SCN. These data provide morphological evidence for the involvement of nitric oxide in the mediation of photic stimulation of the circadian oscillator located in the SCN. PMID- 7533634 TI - The involvement of substance P and neurokinin-1 receptors in the responses of rat dorsal horn neurons to noxious but not to innocuous mechanical stimuli applied to the knee joint. AB - In 29 anesthetized rats, the involvement of substance P and neurokinin-1 receptors in the spinal processing of mechanosensory innocuous and noxious information from the knee and ankle joint was investigated. In 21 rats, multibarrel electrodes were used to record from 46 spinal cord neurons with afferent input from the knee joint and to administer agonists and antagonists by microinophoresis. In 35 of 46 nociceptive neurons, substance P (ejected at 20-120 nA) caused an excitation and/or an increase in responses to innocuous and noxious pressure applied to the knee and ankle. These effects were reduced by ionophoretic application of the specific neurokinin-1 receptor antagonist CP96,345 (ejected at 25-80 nA) but not by CP96,344, its inactive enantiomer. CP96,345 dose-dependently reduced the responses to noxious pressure applied to the knee joint in 28/28 substance P-sensitive neurons but not those to innocuous pressure in 23/23 substance P-sensitive wide dynamic range neurons. CP96,345 did not affect responses to pressure in substance P-insensitive neurons and the inactive enantiomer CP96,344 had no effect in any of the neurons tested. Using microprobes coated with antibody to substance P, intraspinal release of immunoreactive substance P was found to be evoked by noxious pressure applied to the knee but not by innocuous pressure in 8 rats. Both sets of data suggest a role for substance P and neurokinin-1 receptors in the neuronal mechanisms in the spinal cord related to nociception and pain in the normal joint. PMID- 7533637 TI - A sensitive double immunostaining protocol for Fos-immunoreactive neurons. AB - Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k. PMID- 7533636 TI - Infrequent co-localization of nitric oxide synthase and calcium binding proteins immunoreactivity in rat neocortical neurons. AB - Immunoreactivity to nitric oxide synthase (NOS-IR) was detected in a small population of rat neocortical neurons scattered throughout the frontal, parietal, temporal and occipital cortices. Two-color double-labeling studies revealed that the number of NOS-IR neocortical neurons expressing immunoreactivity to the calcium binding proteins parvalbumin (PV-IR), calbindin-D28K (CB-IR) or calretinin (CR-IR), was low. The absence or low level of calcium binding proteins may imply a reduced Ca2+ buffering capacity in NOS-containing neurons, thereby contributing to their vulnerability to Ca2+ influx through the activated AMPA/kainate receptors reported by others. PMID- 7533638 TI - NMDA, kainate, and AMPA depolarize nondopamine neurons in the rat ventral tegmentum. AB - The possible existence of N-methyl-D-aspartate (NMDA) and non-NMDA receptors on electrophysiologically identified nondopamine neurones in the ventral tegmental area (VTA) was tested in rat midbrain slice preparations. NMDA, kainate (KA), and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) depolarized the membrane potential of nondopamine neurons in a dose-dependent manner. The NMDA effect was blocked by the selective NMDA receptor antagonist, CGS 19755 (cis-4 phosphonomethyl-2-piperidine carboxylate), but not by the non-NMDA receptor antagonist, NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline]. In contrast, the effects of KA and AMPA were antagonized by NBQX, but not by CGS 19755. The rank order potency of the three agonists was AMPA > KA > NMDA, with thresholds of 0.1, 0.3, and 3 microM, respectively. These results provide clear electrophysiological evidence that nondopamine neurons in the ventral tegmental area possess both NMDA and non-NMDA receptors. PMID- 7533639 TI - [The significance of prostate markers in the orthology of the female prostate]. AB - In addition to knowledge gained in the first half of the 8th decade, the evidence of cross-antigenicity between male prostate and Skene's glands by means of PSA and PSAcP demonstrations in Skene's glands and ducts justifies utilization of the term prostate in both sexes. The authors compared the results of immunohistochemical examination of prostatic markers by means of the PAP method which was used at the beginning of the 8th decade, with that of BSAP technique. Prostatic tissues of 11 females and children at the age ranging from 5 to 71 years were examined. The results gained by means of the BSAP method were identical with those gained by means of the PAP method. Prostatic markers PSA and PSAcP were expressed on the surface and in apical cytoplasm of cells lining the prostatic ducts, and in prostatic glands. The authors proved the expression also in membranes of the stratified cylindrical epithelial cells of the ducts, and in female prostatic fluid in ducts and glands, especially PSAcP. Even though both immunohistochemical methods brought identical results, the authors recommend to prefer the BSAP method to the PAP method due to optically more contrast expression. (Fig. 8, Ref. 36.) PMID- 7533640 TI - Genotypic evolution of HIV-1 isolates from patients after a switch of therapy from zidovudine to didanosine. AB - The existence of zidovudine (ZDV)-resistant and didanosine (ddI)-resistant human immunodeficiency-1 (HIV-1) variants mutated in the reverse transcriptase (RT) gene has been previously demonstrated. In this study, we tried to follow up the genotypic changes in the RT after the switch of therapy from ZDV to ddI. We studied HIV-1 isolates from 11 patients undergoing ddI therapy. Genotypic data were obtained with differential polymerase chain reaction (PCR) and with direct sequencing after PCR. The prevalence of ZDV resistance-related mutations showed a very slow decrease, particularly when patients had been treated with ZDV for a long time. The appearance of a mutation at codon 74 seemed to be independent of the presence or absence of ZDV resistance-related mutations. The broad genotypic heterogeneity of the isolates and the complexity of the evolution in one patient's isolates plead for large sequencing studies of the RT genome in new therapeutic approaches. PMID- 7533641 TI - Major contribution of the beta chain to the antigen specificity of a T cell receptor. AB - T cell receptors (TCR) recognize peptides presented by major histocompatibility complex (MHC) molecules on the surface of cells. Sequence homology between the variable regions of the T cell receptor and of antibodies suggests that similarly folded domains participate in ligand binding in both cases. However, most current models assume that both TCR chains (alpha and beta) are required for specific binding, whereas the heavy chain alone can confer specificity on many antibodies. We have therefore constructed chimeric molecules with alpha and beta from two different TCR, one restricted by the class II MHC protein, Ek, and the other by the class I MHC, Kd. The beta chain alone was sufficient for specific recognition of a peptide, Cw3, bound to Kd, but the alpha chain contributed to the overall avidity. These results suggest that other TCR may recognize their ligands primarily through the beta chain. PMID- 7533642 TI - 12-lipoxygenase mRNA expression by cultured neurons. AB - 12-hydroxy 5,8,14-cis 10-trans eicosatetraenoic acid (12-HETE) and its derivatives are the principal lipoxygenase (Lox) products of the mammalian brain. These metabolites have been proposed to play a key role as second messengers in synaptic transmission and might function as retrograde messengers in learning and memory processes: the long-term potentiation. The exact source(s) of 12-HETE and neuronal implication have not been definitively established. The present work was therefore designed to study 12-Lox mRNA expression in neural cell cultures. Detection of this mRNA from cellular extract was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and localization in neurons by in situ RT-PCR. These results argue for 12-Lox neuronal production. PMID- 7533643 TI - Fatal interactions: Fas-induced apoptosis of mature T cells. PMID- 7533644 TI - Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. AB - To study the contribution of putative perforin-independent mechanism in the antigen-specific target destruction by cytotoxic T lymphocytes CD8+ CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-/-) embryonic stem cells into blastocysts of RAG-2(-/-) mice. When tested on normal concanavalin A blasts, these perforin-deficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas mediated pathway in antigen-specific cytolysis. PMID- 7533645 TI - The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. AB - The role of a cell death-associated gene, fas, in T lymphocyte development and responses to antigen has been analyzed by breeding a transgenic T cell receptor specific for the 81-104 peptide of pigeon cytochrome c into fas-defective MRL lpr/lpr and control MRL+/+ mice. Transgene-expressing T cells mature normally in both strains and populate peripheral lymphoid tissues in normal numbers. Mature CD4+ T cells from the lpr/lpr mice are resistant to suppression by high doses of antigen and to apoptotic cell death. In vivo administration of peptide antigen causes deletion of thymic T cells in both MRL-lpr/lpr and MRL+/+ strains. By contrast, antigen-induced deletion of peripheral T cells occurs in the MRL+/+ but not in the MRL-lpr/lpr strain. Therefore, the fas gene plays an essential role in activation-induced cell death in mature T lymphocytes, but not in the negative selection of immature cells in the thymus. PMID- 7533646 TI - A negative regulatory function of B7 revealed in B7-1 transgenic mice. AB - To analyze the functions of T cell costimulators in vivo, we have constructed a transgenic mouse strain that constitutively expresses murine B7-1 on mature B cells. Antibody responses to T-dependent hapten-protein conjugates and serum immunoglobulin levels are markedly depressed in B7-1 transgenic mice. This immune deficiency is not due to an intrinsic B cell defect, as antibody responses to T independent hapten conjugates are normal. Furthermore, treatment with anti-B7-1 restores the capacity of transgenic mice to respond to hapten-protein conjugates, demonstrating that the deficient antibody responses are directly attributable to the expression of B7-1. These results suggest that the temporally regulated expression of costimulators such as B7-1 may contribute to either initiation or down-regulation (feedback inhibition) of T-dependent immune responses in vivo, and that the inhibitory function is dominant in transgenic mice that constitutively express high levels of this costimulator. PMID- 7533647 TI - MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. AB - The use of E1-deleted recombinant adenoviruses in gene therapy has consistently been associated with transient gene expression and inflammation due to immune based destruction of the infected cells. We have used murine models of adenovirus mediated gene transfer to liver to investigate these immunologic mechanisms. Adoptive transfer experiments, as well as studies involving genetic knockout mice, confirmed the original hypothesis that cell-mediated immunity induced by E1 deleted adenovirus destroyed trans-gene-expressing hepatocytes and defined MHC class I-restricted CD8+ cytolytic lymphocytes as the primary immune effectors for hepatocyte destruction. Responses mediated by CD4+ cells per se were insufficient to mediate destruction of hepatocytes in vivo, despite the activation of virus specific T helper cells of Th1 subsets. A better understanding of the response of the host to in vivo gene therapy is important in evaluating its usefulness in humans. PMID- 7533648 TI - Effects of synthetic cyclic AMP analogs on amylase exocytosis from rat pancreatic acini. AB - Diastereomers of adenosine 3',5'-phosphorothioate activate cAMP-dependent protein kinases (cAMP-PK) in vitro. We found that these compounds are highly selective tools to monitor cAMP-dependent PKA activation and its effect on amylase exocytosis from pancreatic acini. In permeabilized rat acinar cells, (Sp)-cAMPS dose-dependently stimulated amylase secretion, while (Rp)-cAMPS inhibited (Sp) cAMPS-induced amylase release. In intact rat acini, 8-Br-(Rp)-cAMPS reduced the secretory responses to secretin, vasoactive intestinal polypeptide (VIP), 8-Br cAMP, and 8-Br-(Sp)-cAMPS, but not to cerulein. Another derivative, dibutyryl (Rp)-cAMPS, induced a small inhibitory effect against 8-Br-(Sp)-cAMPS and VIP, which was overlapped by an unspecific stimulatory effect on amylase exocytosis induced by the degradation product butyrate. Furthermore, (Sp)-5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole-3',5'- monophosphorothioate ((Sp)-5,6-DCl cBIMPS), a specific cAMP-PK activator, induced a maximal induction of cAMP-PK activity, but its stimulation of amylase secretion was less than that by secretin. (Sp)-5,6-DCl-cBIMPS regulated the phosphorylation of several proteins, which were also affected by secretin. However, secretin had additional effects. Its action was most likely mediated by a dual effect on the cAMP and the calcium pathway. Our results indicate that the cAMP-dependent pathway is involved in amylase exocytosis from rat pancreatic acini. PMID- 7533649 TI - Isoproterenol exerts cyclic AMP independent effects on DNA synthesis in cultured adventitial fibroblasts from spontaneously hypertensive and Wistar-Kyoto rats. AB - Understanding the mechanism behind the growth response evident in the vasculature of the spontaneously hypertensive rat (SHR) remains elusive. Fibroblasts from the aortic adventitial layer of the SHR manifest the heightened proliferative rate in vitro relative to Wistar-Kyoto (WKY) rats that is conspicuous in cultured aortic smooth muscle cells. The adenylylcyclase/cyclic AMP signal transduction pathway is believed to be altered in hypertensive people and animals such that responses to beta-adrenoceptor activation are blunted. The present study examined the effects of beta-adrenoceptor-mediated versus direct activation of adenylylcyclase on intracellular cyclic AMP accumulation and subsequent DNA synthesis in cultured aortic fibroblasts. We hypothesized that elevation of cyclic AMP levels by both isoproterenol and forskolin would normalize the proliferative capacity of SHR fibroblasts. Forskolin increased intracellular cyclic AMP levels and inhibited epidermal growth factor stimulated thymidine incorporation in an equivalent manner in both SHR and WKY adventitial fibroblasts, implying that there is no difference in adenylylcyclase activity. Isoproterenol elevated cyclic AMP levels to a significantly greater degree in the SHR than did forskolin, and yet, relative to forskolin, attenuated growth factor induced DNA synthesis to a lesser extent. These data suggest that isoproterenol, via beta-adrenoceptor activation, exhibits both cyclic AMP dependent and cyclic AMP independent effects in adventitial fibroblasts. The cyclic AMP independent effects of isoproterenol oppose the expected observations due to cyclic AMP and may offer an explanation to the blunted responses to beta-adrenoceptor activation evident both in vitro and in vivo. PMID- 7533650 TI - Effect of the calcium channel agonist Bay K8644 on mechanical and electrical responses of frog skeletal muscle. AB - The effects of Bay K8644, a Ca2+ channel agonist, on the mechanical and electrical properties of frog skeletal muscle fibers were investigated. At relatively low concentrations, such as 10(-6) and 10(-5) M, Bay K8644 significantly potentiated the maximum amplitudes of twitch responses, and this effect was not reversed in the presence of the calcium channel antagonist nitrendipine. At higher concentrations, such as 10(-4) M, Bay K8644 depressed the amplitudes of twitch responses, and nitrendipine did not change this effect. At all concentrations, Bay K8644 greatly reduced the area under the tetanic force versus time curve, and this effect was not modified by the concomitant application of Bay K8644 and nitrendipine. Intracellular recordings revealed that the depressing effect of Bay K8644 on tetanic contractions was due to the blockade of sodium action potentials. In conclusion, the present results suggest that the modulation of twitch responses by calcium channel agonist and antagonists, at the concentration range used, is not related to the expected modulation of voltage-sensitive slow calcium channels in frog skeletal muscle fibers, and tetanic contractions are depressed by the calcium channel agonist Bay K8644 through its effect on sodium channels. PMID- 7533651 TI - Nurses are at patients' deathbeds. PMID- 7533652 TI - Developing a poster about a clinical innovation. Part III: Presentation and evaluation. AB - This is the third in a series of three articles developed to assist the CNS in planning, producing, and presenting a clinical poster. In this article, actions that the poster presenter needs to take before, during, and after the conference and poster session are discussed. An evaluation tool to gauge participants' reactions is suggested. Examples given are specific to the clinical innovations project. PMID- 7533653 TI - Patients with ultrasonic coarse-nodular cirrhosis who are anti-hepatitis C virus positive are at high risk for hepatocellular carcinoma. AB - BACKGROUND: The relationship between echosonographic patterns of patients with cirrhosis who are antihepatitis C virus (HCV)-positive, the DNA synthesis of hepatocytes, and the risk for HCC were studied. METHODS: Thirty-eight patients with anti-C-100 antibody-positive and Child's grade A posthepatitic cirrhosis were studied. DNA synthesis activity was measured by a bromodeoxyuridine (BrdU, a thymidine analogue)-labeling index (LI), using the BrdU-anti-BrdU in vitro method, and the patients were followed prospectively by frequent liver ultrasonography for 3 years. The ultrasound patterns were classified into fine, coarse, and coarse-nodular (CN) patterns, and the reproducibility of the classification in practical use also was confirmed. RESULTS: Of the 21 patients with high DNA synthesizing cirrhosis (BrdU LI > or = 1.5%), 10 (48%) showed coarse-nodular, 5 (24%) coarse, and 6 (29%) fine pattern in ultrasonography. Conversely, of the 17 patients with low DNA synthesizing LC (BrdU LI < 1.5%), only 1 (6%) showed coarse-nodular, 2 (12%) coarse, and 14 (82%) fine pattern. A significant relationship was found between the two groups of BrdU LI and ultrasound imaging patterns (P < 0.05). The incidence of CN pattern was significantly higher (P < 0.01) in the high DNA synthesizing group than in low DNA synthesizing group. Of the 11 patients with CN pattern by ultrasound imaging, 10 (91%) were in the high DNA synthesizing group, and 9 (82%) developed HCC during the follow-up period, compared with 3 of 7 (43%) with coarse, and only one of 20 (5%) with fine pattern developed HCC. The incidence of HCC was significantly higher (P < 0.01) in patients with a CN cirrhosis pattern than in those with a fine pattern. CONCLUSIONS: In patients with cirrhosis who are anti HCV-positive, the CN pattern by ultrasound imaging indicates increased DNA synthesis of hepatocytes and a high risk for developing HCC. PMID- 7533654 TI - Enhanced expression of GM2/GD2 synthase mRNA in human gastrointestinal cancer. AB - BACKGROUND: The content of the GM2 ganglioside and the activity of UDP-GalNAc: GM3 beta-1,4N-acetylgalactosaminyltransferase (beta-1,4GalNAcT), which synthesizes GM2, increased in gastric cancer tissues and gastric cancer cell lines as compared with that in normal gastric mucosa. METHODS: Expression of beta 1,4GalNAcT mRNA and a concentration of GM2 in the human gastrointestinal tissues were examined. Beta-1,4GalNAcT mRNA in human surgical specimens, which was not detectable with Northern blotting because of the paucity of absolute amounts expressed, was detected with competitive reverse transcription-polymerase chain reaction (PCR) method using an internal standard cRNA that could be amplified by the same primers as target mRNA in PCR. The quantification of GM2 was examined using immunostaining of thin-layer chromatography. RESULTS: In 10 of 10 gastric carcinomas and 6 of 13 colonic carcinomas, mRNA expression was more enhanced than that in the normal mucosa of each patient. The alteration of GM2 content in carcinoma from normal tissue generally was correlated to the change in the expression of beta-1,4GalNAcT mRNA with a few exceptions. One gastric cancer sample had a higher level of mRNA with a lower GM2 content than the corresponding normal tissue, and two colonic carcinoma tissue specimens had a lower level of mRNA with a higher GM2 content. CONCLUSIONS: These results suggest that expression of the beta-1,4GalNAcT gene is a key step in the molecular mechanisms underlying the regulation of cancer-associated GM2 expression in the stomach and the colon. PMID- 7533655 TI - Radiation therapy in the management of patients with malignant carcinoid tumors. AB - BACKGROUND: The purpose of this study was to analyze the effectiveness of radiotherapy for symptomatic, metastatic, and/or unresectable carcinoid tumors. METHODS: From 1975 to 1991, 18 patients with histologically proven carcinoid tumors, including 7 with symptomatic hepatic involvement, received radiotherapy to a total of 31 anatomic sites of metastatic or unresectable carcinoid tumors. Symptomatic response to treatment, date of symptomatic or radiographic progression, and survival after diagnosis and completion of radiotherapy were determined. Clinical improvement was defined as symptomatic relief and/or objective reduction in size of tumor mass sustained for at least 4 weeks. RESULTS: After radiotherapy of 31 sites in 18 patients, 3 patients died in less than 3 months. Of the remaining 15 patients, survival from initial radiotherapy ranged from 8 to 108 months (median, 23 months), and 8 (53%) died without symptomatic progression in the treated region. Clinical improvement occurred in 27/31 sites (87%). Acute side effects were mild, and no late side effects were recorded. Median survivals from diagnosis were: 39 months for patients with primary tumors of the lung, 33 months for patients with brain metastases, and 32 months for patients with hepatic involvement. Patients with the carcinoid syndrome responded in 19/22 sites (86%) compared with 8/9 sites (89%) for patients without the syndrome (P = not significant). CONCLUSIONS: Radiation therapy can achieve symptomatic palliation for patients with metastatic/unresectable malignant carcinoid tumors, and it is well tolerated. Clinical improvement occurs after irradiation in patients with or without the carcinoid syndrome. PMID- 7533656 TI - Retinoids, interferon alpha, 1,25-dihydroxyvitamin D3 and their combination inhibit angiogenesis induced by non-HPV-harboring tumor cell lines. RAR alpha mediates the antiangiogenic effect of retinoids. AB - Retinoids combined with interferon alpha-2a (IFN alpha) or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] have shown marked synergistic inhibitory effects on angiogenesis induced by tumor cell lines harboring DNA of oncogenic human papillomaviruses (HPV) type 16 or 18. This report demonstrates comparable effects of these compounds on angiogenesis induced by non-HPV-bearing transformed cell lines, including breast carcinoma (T47D) and vulval carcinoma (A431) cell lines. Systemic treatment of mice with all-trans retinoic acid, 13-cis retinoic acid, 9 cis retinoic acid, IFN alpha or 1,25(OH)2D3 significantly decreased tumor cell induced angiogenesis (TIA). In vitro pretreatment of T47D and A431 cells with these compounds also led to inhibition of their angiogenic capability when tested in the TIA assay. The inhibitory effects of retinoids could be counteracted by a selective antagonist of the nuclear retinoic acid receptor RAR alpha, suggesting a RAR alpha mediated mechanism of angiogenesis inhibition. The antiangiogenic effect of retinoids could be significantly enhanced by combination with IFN alpha or 1,25(OH)2D3. The results provide a further basis for the use of combinations of retinoids with IFN alpha or 1,25(OH)2D3 in the treatment of angiogenesis dependent malignancies. PMID- 7533657 TI - Effect of tumor necrosis factor-alpha on a cell line transformed by a secreted form of human fibroblast growth factor-1 gene and on its parental cell line. AB - In the present study we characterized the responses of the cell line HST-alpha, stably transformed by a secreted form of human fibroblast growth factor-1 (acidic FGF) gene, and its parental NIH 3T3 cell line to recombinant murine (rMu) TNF alpha. Treatment of HST-alpha cells with rMu TNF-alpha can significantly reduce the number of foci formed in the in vitro transformation assay. In the presence of the RNA polymerase inhibitor actinomycin-D, the transformed HST-alpha cell is more susceptible to the cytotoxicity of rMu TNF-alpha than is its parental NIH 3T3 cell. The median lethal concentrations (LC50) of rMu TNF-alpha to HST-alpha cell and NIH 3T3 cell were 0.35 ng/ml and 4.56 ng/ml respectively. These results demonstrated that the introduction of a single oncogene or a growth factor gene to a cell can change the cell's response to cytokines. PMID- 7533658 TI - Correlation of expression of CD44 isoforms and E-cadherin with differentiation in human urothelial cell lines and transitional cell carcinoma. AB - Using immunostaining, immunoblot, reverse-transcriptase polymerase chain reaction and Southern blot, we found that expressions of CD44 isoforms and E-cadherin were very closely linked and were correlated with the differentiation status in human urothelial cell lines and clinical specimens of transitional cell carcinoma. Normal urothelium, well to moderately differentiated cell lines and surgical samples expressed E-cadherin and large CD44 isoforms containing exon v6, which was pivotal in metastasis of rat pancreatic cell line model. Poorly differentiated cell lines and surgical samples, were E-cadherin-negative and expressed primarily standard form CD44, which did not contain exon v6. We concluded that CD44v6 isoforms and E-cadherin were both down-regulated during the carcinogenesis of urothelium. The large exon v6 containing CD44 isoforms were readily detected in normal urothelium, therefore, were not likely linked to cancer metastasis. E-cadherin and CD44v6 may be used as differentiation markers for human urothelial tumors. Immunohistochemical study solely with antibody against epitopes encoded by exon v6 alone is not informative enough as other alternatively spliced exons may change the function of CD44v6 isoforms. PMID- 7533659 TI - Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor. AB - Cell adhesion molecules have been suggested to function as tumor suppressor molecules. We have been studying one of the epithelial cell adhesion molecules (C CAM), which belongs to the immunoglobulin gene superfamily. Transfection of a C CAM cDNA expression vector into a highly tumorigenic human prostate cancer cell line (PC-3) suppresses tumor formation in nude mice. Alternatively, reducing C CAM expression levels in the nontumorigenic rat prostate epithelial cell line NbE by the antisense expression vector markedly increases tumorigenicity of NbE cells in nude mice. These results suggest that C-CAM may be a tumor suppressor in prostate cancer. In this study, we examined the relationship between C-CAM expression during human prostate development and neoplastic progression by immunohistochemical staining of frozen sections. C-CAM predominantly localized on the plasma membrane of the basal cell layer in both the fetal and normal adult prostate gland. However, an overall decreased staining was seen in benign prostatic hyperplasia and high grade prostatic intraepithelial neoplasia. Furthermore, C-CAM was not detected in prostate carcinomas. Thus, a decrease in C CAM expression may be an early event in hyperplastic/neoplastic transformation. These observations support the suggestion that C-CAM is a tumor suppressor in prostate cancer progression. PMID- 7533660 TI - Apoptosis induced by serum deprivation of PC12 cells is not preceded by growth arrest and can occur at each phase of the cell cycle. AB - Previous studies have shown that PC12 cells undergo apoptosis (programmed cell death) when deprived of serum. In the present study, we examined the relationship of this death process to the cell cycle. PC12 cell populations synchronized at different, specific phases of the cell cycle exhibit similar kinetics of cell death following deprivation of serum. Flow cytometry analysis was used to examine the levels of apoptotic death in these cell populations in relationship to their progression in the cell cycle during the course of serum deprivation. Such analysis revealed that the cells die during the G0-G1, S, and perhaps G2-M phases and at the G2 to G1 transition. These results, therefore, suggest that the death of synchronized, serum-deprived PC12 cells occurs throughout the cell cycle and is not dependent on growth arrest. Flow cytometry methodology (acridine orange staining), which determines the RNA content of cells in relationship to their position in the cell cycle, was used to address these questions in nonsynchronized cells. These experiments revealed that, upon serum deprivation, an immediate loss of RNA occurred from cells in G1, S, and G2-M phases. This loss is accompanied by a slower appearance of cells with degraded DNA content. These results show that cells from all phases of the cell cycle are damaged upon serum deprivation and thus suggest that the apoptotic cell death of nonsynchronized PC12 cells may occur from each phase of the cell cycle. PMID- 7533661 TI - Up-regulation of vascular endothelial growth factor and its receptors in von Hippel-Lindau disease-associated and sporadic hemangioblastomas. AB - Capillary hemangioblastoma is the most frequent manifestation of the autosomal dominantly inherited von Hippel-Lindau (VHL) disease but also presents as a nonfamilial, sporadic vascular tumor. Hemangioblastomas are characterized by a dense network of capillaries in association with cysts. To investigate the mechanisms underlying neovascularization and cyst formation, we analyzed eight VHL disease-associated and five sporadic hemangioblastomas. Histologically, both tumor types showed a similar phenotype. The capillaries expressed the endothelial cell markers von Willebrand factor and CD31 antigen. We investigated the expression of vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen which is also known to induce vascular permeability in vivo, and its high affinity tyrosine kinase receptors flt-1 and KDR. Northern blot and in situ hybridization analysis revealed significant up-regulation of VEGF and VEGF receptor expression in VHL disease-associated and sporadic hemangioblastomas compared to normal brain and tumor stromal cells as sites of abundant VEGF transcription. Endothelial cells did not express detectable amounts of VEGF mRNA but coexpressed flt-1 and KDR. By immunohistochemistry, VEGF protein was detectable in the tumor interstitium and was found to be concentrated around capillaries. Performing reverse transcription-PCR, we demonstrated that VEGF121 and VEGF165 were the splice variants predominantly expressed, whereas mRNA encoding VEGF189 was present at smaller amounts. Our findings suggest that, in VHL disease-associated and sporadic hemangioblastomas, VEGF121 and VEGF165 are secreted by stromal cells and interact with the corresponding VEGF receptors expressed on tumor endothelial cells. This paracrine mechanism may mediate neovascularization and cyst formation in capillary hemangioblastomas. PMID- 7533662 TI - Immunohistochemical quantitation of polycyclic aromatic hydrocarbon-DNA adducts in human lymphocytes. AB - The formation of polycyclic aromatic hydrocarbon-DNA adducts was studied in peripheral blood lymphocytes obtained from men with occupational and environmental exposure. Subjects included coke factory workers, residents from the vicinity of the cokery, and rural region inhabitants (16 individuals in each exposure group). Adducts were determined by immunohistochemical analysis using a polyclonal antiserum recognizing benzo(a)pyrene and related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts, a biotinylated secondary antiserum, and streptavidin-conjugated FITC. Propidium iodide was used to quantitate nuclear DNA. Dual fluorescence intensities were simultaneously measured with a Zeiss Axiovert microscope and a Bio-Rad MRC-600 argon laser scanning confocal attachment. Adducts were significantly elevated (P < 0.001) in both occupational and environmental groups, as compared to the rural control group by Mann-Whitney U test. The distribution of the data indicated the existence of cells with relatively higher adduct levels. The percentages of these so called "higher adduct-level cells" were 13.6, 11.5, and 3.7 in cokery workers, environmentally exposed individuals, and rural controls, respectively. The immunohistochemical method allows visualization and relative quantitation of polycyclic aromatic hydrocarbon-DNA adducts in individual lymphocytes. It requires a much smaller amount of blood than the previously used 32P-postlabeling and ELISA methods, which used isolated bulk DNA. It can also be used for adduct quantitation in biopsy material. The results of this pilot study indicate that this technique is a promising addition to biomonitoring studies. PMID- 7533663 TI - Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers. AB - Linomide, a quinoline-3-carboxamide, has the ability to inhibit the growth of prostatic cancer in vivo but not in vitro (T. Ichikawa et al., Cancer Res., 52: 3022-3028, 1992). The reason for this discrepancy is that linomide inhibits tumor growth not directly but indirectly in vivo via its ability to inhibit the angiogenic response induced within the growing prostatic cancer (J. Vukanovic, et al., Cancer Res., 53: 1833-1837, 1993). Tumor associated macrophages can stimulate angiogenesis via their ability to secrete various cytokines, particularly tumor necrosis factor alpha (TNF-alpha). Treatment of rats with linomide decreases significantly (P < 0.05), by more than 50%, the number of tumor associated macrophages within both locally invasive (i.e., from 20-40 to 10 macrophages/high power field) and highly metastatic primary prostatic cancers (i.e., from 60-70 to 15-37 macrophages/high power field). Monocytes/macrophages isolated from linomide treated rats had a decreased ability to secrete TNF-alpha when challenged in vitro with the bacterial endotoxin, lipopolysaccharide [i.e., 702 +/- 76 (SEM) ng of TNF-alpha/10(5) monocytes/macrophages from control versus 401 +/- 2 ng of TNF-alpha/10(5) monocytes/macrophages from linomide treated rats]. In addition, when rats were treated with linomide and than challenged with lipopolysaccharide in vivo, the resulting elevation in serum TNF-alpha was inhibited by approximately 50% (i.e., 4.56 +/- 1.8 ng/ml of TNF-alpha in control versus 2.9-2.2 ng/ml depending upon the dose of linomide). The ability of linomide to decrease monocyte/macrophage secretion of TNF-alpha is not immediate, however, since the secretion of TNF-alpha induced by lipopolysaccharide challenge of monocytes/macrophages isolated from untreated animals is not decreased by acute (i.e., < 4 h) linomide treatment in vitro. These results demonstrate that the ability of linomide to inhibit the secretion of TNF-alpha by monocytes/macrophages requires either additional time or host factors. To test if natural killer (NK) cells might be one of the additional host factors required for the in vivo abilities of linomide, prostatic cancer bearing rats were treated with appropriate antiserum to deplete NK cells and then tested for their response to linomide treatment. These studies demonstrated that the antitumor, antimetastatic, and antimacrophage effects of linomide were unaffected by NK cell depletion.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533665 TI - Induction of human breast cancer-specific antibody responses in cynomolgus monkeys by a murine monoclonal anti-idiotype antibody. AB - We have generated and characterized a murine monoclonal anti-idiotype (Id) antibody, designated 11D10, which biologically and antigenically mimics a distinct and specific epitope of the high molecular weight human milk fat globule primarily expressed by human breast and some other tumor cells at high density. This epitope is identified by mAb BrE1, which was used as the immunizing antibody or Ab1 to generate the anti-Id (Ab2) 11D10. 11D10 induced antitumor immune responses across species barriers, i.e., in mice and rabbits. In preclinical studies, cynomolgus monkeys were immunized with 2 mg of either 11D10 or the isotype- and allotype-matched control Ab2 3H1 after precipitation with aluminum hydroxide. All monkeys developed high titers of antibodies against the immunizing mouse immunoglobulin. Immunization with 11D10 induced anti-anti-idiotype antibodies (Ab3) which reacted with breast cancer cell lines but not with control T-cell and melanoma cell lines. The Ab3 shared idiotypes with BrE1 (Ab1), as demonstrated by their ability to inhibit 11D10 binding to BrE1. The Ab3 obtained with 11D10 bound specifically to human milk fat globule antigen and competed with BrE1 for binding to breast cancer cell lines, suggesting that Ab1 and Ab3 may bind to the same epitope. In addition, Id-specific cellular immune responses were demonstrated in monkeys immunized with 11D10 by T-cell proliferation assays. These results indicate that aluminum hydroxide-precipitated anti-Id 11D10 can induce breast cancer-specific antibodies in nonhuman primates and can serve as a potential network antigen for breast cancer patients. PMID- 7533664 TI - Cytotoxicity, metabolism, and mechanisms of action of 2',2' difluorodeoxyguanosine in Chinese hamster ovary cells. AB - The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2' difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues. PMID- 7533666 TI - Transcatheter arterial embolization of unresectable renal cell carcinoma with a mixture of ethanol and iodized oil. AB - PURPOSE: The effectiveness of renal transcatheter arterial embolization (TAE) with a mixture of ethanol and iodized oil (Lipiodol) was analyzed retrospectively in 27 patients with unresectable renal cell carcinoma. METHODS: Symptomatic or permanent palliation by TAE was performed with the intent to achieve total or partial ablation of the kidney with 10-28 ml of embolic material in a 3:1 mixture of ethanol and iodized oil. Clinical, angiographic, and computed tomographic (CT) follow-up were analyzed. Cumulative survival was estimated with the Kaplan-Meier method. RESULTS: Partial (5) or complete (22) ablation of the embolized kidneys was achieved in all cases. Follow-up CT after 1 month revealed Lipiodol uptake in the tumor area (14/14), decreased size of the tumor (6/14), and gas formation (7/14). Pulmonary metastasis improved significantly in one patient after transcatheter arterial embolization and adjunctive chemotherapy. Overall median survival of the 27 patients was 8.5 months. The median survival was 23 months in 10 patients with stage III, three of whom are still alive, and was 7 months in 15 patients with stage IV tumors. CONCLUSION: The mixture of ethanol and Lipiodol is not only a convenient embolic material to use, but is also effective for selective or nonselective devascularization for unresectable renal cell carcinoma. PMID- 7533667 TI - [Undifferentiated carcinoma of the thyroid gland]. AB - Anaplastic carcinoma of the thyroid is a tumour of advanced age, with a female/male ratio of 2/1. Its incidence is of 1-2 cases/million/year. The tumour is one of the most aggressive neoplasms affecting humans. It spreads very rapidly to the regional lymph nodes and causes distant metastases, in particular in the lungs and bone. Surgery, external radiation or chemotherapy are usually ineffective when used alone. Better results are obtained by combinations of these modalities, in particular by a combination of radiotherapy and chemotherapy followed by surgery. In our Centre we have, since 1991, adopted a multimodal treatment based on radiotherapy preceded by Cisplatin administration, followed by surgery and then by adjuvant chemotherapy with Adriamycin and Bleomycin. The initial results on a limited number of cases seem encouraging. PMID- 7533668 TI - [Teleradiotherapy in the treatment of thyroid neoplasms]. AB - The role of external radiotherapy in the treatment of thyroid neoplasms is not yet well defined. The indications for loco-regional treatment in the case of: anaplastic carcinomas (alone or in combination with surgery or chemotherapy); differentiated and medullary carcinomas at diagnosis or relapse; nodes or osseous metastases not otherwise curable (surgery, hormones, brachytherapy); cerebral metastases; are accepted by everyone. Results, from the various clinical reports published, are positive. The use of this methodology is not accepted by everyone as "adjuvant" in cases "at risk" for micro or macroscopic residuals after surgery for papillary, follicular or medullary carcinomas. From the analysis of the literature, even a recent publication in relation to an apparent growing local control, we have no data in favour of increased survival. Even from our experience we cannot give definitive data (78.5% of local control in case of papillary or follicular carcinoma, survival of 57% in 5 years and 36% in 10 years). The question could be solved only by randomised trials but the difficulties due to the need for a sufficient number of cases and to the long "natural" survival, even in presence of disease, appear to be insuperable. We suggest a prudent approach, in controversial cases, because of the difficulties linked to the optimal loco-regional treatment (high doses, proximity of organs "at risk"). PMID- 7533670 TI - Direct evaluation of intracellular accumulation of free and polymer-bound anthracyclines. AB - Nanoparticulate carriers of anthracyclines are being developed with the aim of improving the pharmacokinetic or pharmacodynamic behavior of these drugs. To understand how the drug reaches its nuclear targets, we have developed two methods that allow the quantification of the interaction between an anthracycline and cellular DNA: (1) by direct evaluation of the quenching of anthracycline fluorescence due to the intercalation of the drug into DNA and (2) by the measurement of Hoechst 33258 fluorescence associated with its displacement from DNA-binding sites for which it competes with the anthracycline. We show that the intracellular accumulation and DNA binding of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres (dox-NS) and of daunorubicin bound to polyglutamic acid are reduced by 30%-40% in comparison with those obtained for free doxorubicin (dox) and daunorubicin, respectively. The results obtained with dox or NS-dox are not modified by prior incubation with either of these compounds. The two methods yielded similar results, and we conclude that either technique is applicable to the evaluation of the interaction of carrier-bound anthracyclines with cellular DNA. PMID- 7533669 TI - Synergistic and additive combinations of several antitumor drugs and other agents with the potent alkylating agent adozelesin. AB - Adozelesin is a highly potent alkylating agent that undergoes binding in the minor groove of double-stranded DNA (ds-DNA) at A-T-rich sequences followed by covalent bonding with N-3 of adenine in preferred sequences. On the basis of its high-potency, broad-spectrum in vivo antitumor activity and its unique mechanism of action, adozelesin has entered clinical trial. We report herein the cytotoxicity for Chinese hamster ovary (CHO) cells of several agents, including antitumor drugs, combined with adozelesin. The additive, synergistic, or antagonistic nature of the combined drug effect was determined for most combinations using the median-effect principle. The results show that in experiments using DNA- and RNA-synthesis inhibitors, prior treatment with the DNA inhibitor aphidicolin did not affect the lethality of adozelesin. Therefore, ongoing DNA synthesis is not needed for adozelesin cytotoxicity. Combination with the RNA inhibitor cordycepin also did not affect adozelesin cytotoxicity. In experiments with alkylating agents, combinations of adozelesin with melphalan or cisplatin were usually additive or slightly synergistic. Adozelesin-tetraplatin combinations were synergistic at several different ratios of the two drugs, and depending on the schedule of exposure to drug. In experiments using methylxanthines, adozelesin combined synergistically with noncytotoxic doses of caffeine or pentoxifylline and resulted in several logs of increase in adozelesin cytotoxicity. In experiments with hypomethylating agents, adozelesin combined synergistically with 5-azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza 2'-CdR). Combinations of adozelesin with tetraplatin or 5-aza-2'-CdR were also tested against B16 melanoma cells in vitro and were found to be additive and synergistic, respectively. The synergistic cytotoxicity to CHO cells of adozelesin combinations with tetraplatin, 5-aza-CR, or pentoxifylline was not due to increased adozelesin uptake or increased alkylation of DNA by adozelesin. PMID- 7533672 TI - Ultrasensitive assay of prostate-specific antigen used for early detection of prostate cancer relapse and estimation of tumor-doubling time after radical prostatectomy. AB - We used an ultrasensitive prostate-specific antigen (PSA) assay with a detection limit of 0.02 microgram/L for long-term monitoring of PSA changes in 5 patients who were cured by radical prostatectomy and in 10 patients who had failed prostatectomies; 5 patients who underwent cystoprostatectomy were also evaluated with one sample after surgery. Relapse-free periods, determined on the basis of criteria designed specifically for the ultrasensitive assay or proposed for other currently available PSA assays, were calculated for the patients with failed prostatectomies. Tumor-doubling times were also calculated, postsurgery, according to a model that assumes exponential tumor growth over time. We found that prostate cancer relapse, on average, could be diagnosed 420 or 883 days earlier with the ultrasensitive assay than with assays having detection limits of 0.1 or 0.3 microgram/L, respectively. Tumor-doubling times, calculated after radical prostatectomy, ranged from 67 to 568 days among the 10 patients. We also present evidence that even more-sensitive PSA assays might be able to further reduce the relapse-free periods in approximately 50% of the prostate cancer patients who ultimately relapse. PMID- 7533671 TI - Restriction fragment length polymorphisms of apolipoprotein B gene in Chinese population with coronary heart disease. AB - Three restriction fragment length polymorphisms (RFLPs), EcoRI (R), Xbal (X), and Mspl (M) of the apolipoprotein (apo)B gene, were studied to determine their distribution frequencies and influence on the lipid profiles in 148 Chinese patients with documented coronary heart disease (CHD) and in 153 healthy subjects. The plasma concentrations of cholesterol and apoB showed no difference between the CHD patients and controls. However, CHD patients had significantly higher concentrations of low-density lipoprotein cholesterol and triglyceride and lower concentrations of high-density lipoprotein cholesterol than the controls. The frequencies of these three apoB RFLPs did not differ between the CHD patients and controls. Compared with South Asians and Caucasians, the Chinese in Taiwan showed a much lower frequency of R-, X+, and M- alleles. There was no evidence of an association between lipid profiles and RFLPs in either CHD patients or controls. The weak association of EcoRI, Xbal, and Mspl polymorphisms of the apoB gene with CHD indicates that the three RFLPs cannot be used as a predictor for the risk of CHD in the Chinese population. PMID- 7533674 TI - Measurement of methemoglobin in neonatal samples containing fetal hemoglobin. AB - Because of the potential for methemoglobinemia during nitric oxide therapy in newborns, methods are needed to accurately quantify methemoglobin (MetHb) in the presence of the high concentrations of fetal hemoglobin (Hb F), bilirubin, and lipids seen in these patients. Spectral differences between fetal and adult Hbs invalidate assumptions of conventional multiwavelength Hb photometry, so we evaluated an "overdetermined" system (Ciba-Corning Model 270), in which absorbances at seven wavelengths are measured to quantify four Hb derivatives. Adult and umbilical cord blood (Hb F 96%) samples were prepared to contain known MetHb fractions. Measured MetHb was linear in cord blood to > or = 15% MetHb. Within-run precision (CV) was < 2.2% (n = 10) at each of seven MetHb fractions between 5% and 100%. Measured (y) and expected (x) MetHb fractions in cord blood were in good agreement (y = 1.0200x + 0.100, Sylx = 0). Added bilibrubin (200 mg/L serum) and lipid (30 g/L) did not interfere. No significant differences were seen for adult and cord blood samples with identical MetHb fractions (P = 0.72), whereas a significant difference was noted with an exactly determined system (P = 0.0033). At clinically relevant MetHb fractions (< 15%), a trend towards increased values in cord blood was noted with an exactly determined system (y = 1.0520x + 0.7600). We conclude that this overdetermined system measures MetHb accurately in samples from patients with large concentrations of Hb F. PMID- 7533673 TI - Discriminant function based on serum analytes differentiates hepatocarcinoma from secondary liver neoplasia. AB - Hepatocellular carcinoma (HC) is often difficult to distinguish from secondary liver neoplasia (SLN) by physical and imaging diagnostic procedures alone. To this aim we have extended and improved a laboratory approach based on a serum lactate dehydrogenase isoenzyme ratio (LD4:LD5) by adding the carcinoembryonic antigen: alpha-fetoprotein ratio, alkaline phosphatase, and serum iron concentrations to obtain a highly efficient discriminant function. In two successive cohorts, for a total of 102 patients, all histologically diagnosed, with a prevalence of HC vs SLN of 3:1, we correctly classified 96% of cases (100% of SLN cases). Subsequent verification with the jackknife reallocation statistical algorithm confirmed these results. In conclusion, this discriminant function based on simple laboratory assays of a few analytes is an important tool in solving a diagnostic dilemma in cases of liver neoplasia. PMID- 7533676 TI - Hepatitis delta virus infection: molecular biology and treatment. AB - Hepatitis delta virus is a defective human infectious agent which causes severe hepatic damage in hepatitis B virus-infected patients. Although prophylactic steps and the development, in certain countries, of efficient vaccination programs against hepatitis B virus have led to a diminution of the incidence rate of this disease, hepatitis D is still an important health problem. Hepatitis D infection is therefore associated with a worse clinical evolution than hepatitis B infection alone. On the other hand, the different therapeutic strategies being assayed at present including the use of interferon, have shown to be inefficient in the treatment of this disease. In this article, different aspects of the molecular biology and natural history of the infection, specially focused on those unknown aspects of this virus, are discussed. Finally, we have included a revision of the current state of hepatitis D treatment, as well as a comment of the new experimental strategies, like the use of antisense probes and trans ribozyme activities. PMID- 7533677 TI - The role of blood flow in gastric mucosal defence, damage and healing. AB - Blood flow plays an important role in protection of normal gastric mucosa and in the protection and healing of damaged mucosa. Blood flow contributes to protection by supplying the mucosa with oxygen and HCO3-, and by removing H+ and toxic agents diffusing from the lumen into the mucosa. Low mucosal blood flow predisposes to injury, whereas high blood flow protects against injurious agents. Superficial mucosal damage is followed by increased blood flow which supports the healing process and prevents superficial lesions from developing into deep ones. The hypermic response increases the supply of HCO3- to the mucosa and increases the resistance of the injured mucosa against back diffusing H+ and aggressive drugs such as ethanol (adaptive protection). Mucosal ischemia contributes to gastric ulceration in various clinical conditions such as hemorrhagic shock, stress, aorta aneurysm and after proximal gastric vagotomy. Blood vessels are damaged in gastric ulcers. During ulcer healing blood flow returns to normal. Stimulated or inhibited angiogenesis in the granulation tissue effects the healing of a gastric ulcer. PMID- 7533675 TI - Hook effect in immunometric assays for prostate-specific antigen. PMID- 7533678 TI - Regulatory effects of IL-13 on synovial fluid macrophages and blood monocytes from patients with inflammatory arthritis. AB - Activated macrophages are central to the destructive processes of chronic inflammatory arthritis. In this study, it was hypothesized that IL-13, a product predominantly of 'Th2-type' lymphocytes, may be used therapeutically to down regulate monocyte/macrophage activities at sites of chronic inflammation. Synovial fluid mononuclear cells were isolated from 12 patients with chronic inflammatory arthritis. Peripheral blood mononuclear cells (PBMC) were isolated at the same time as synovial fluid cells from all 12 patients. IL-13 significantly inhibited lipopolysaccharide (LPS)-induced tumour necrosis factor alpha (TNF-alpha) production by mononuclear cells from peripheral blood, but not synovial fluid. In contrast, IL-13 inhibited LPS-induced IL-1 beta production by all cells, and as a positive response to IL-13, CD23 expression was increased on both cell populations. Blood monocytes cultured for 7 days with granulocyte macrophage colony-stimulating factor (GM-CSF) or M-CSF responded to IL-13 in a manner similar to that detected for synovial fluid-derived cells, with suppression of LPS-induced IL-1 beta, but not TNF-alpha, production. In all experiments, the responses to IL-13 were very similar to those detected to IL-4, but differed from those measured with IL-10. Thus, the responses to IL-13 by synovial fluid cells and cultured monocytes are not equal to those of blood monocytes. The similar responses to IL-4 and IL-13 support claims of a common element for signalling from the IL-4 and IL-13 receptors. Furthermore, the activity of a common receptor chain may be altered by monocyte activation and differentiation. PMID- 7533679 TI - Recognition of a unique peptide epitope of the mycobacterial and human heat shock protein 65-60 antigen by T cells of patients with recurrent oral ulcers. AB - T cell epitopes of the 65-kD heat shock protein (hsp) were investigated in patients with recurrent oral ulcers (ROU). Peripheral blood mononuclear cells were stimulated with overlapping synthetic peptide (15ers), derived from the sequence of the 65-kD hsp of Mycobacterium tuberculosis. Specific lymphoproliferative responses were stimulated only with peptide 91-105 in ROU, compared with healthy or disease controls (P < 0.01). This was confirmed by studying 760 short term cell lines generated with the 65-kD hsp and then stimulated with the peptides. The frequency of short term cells lines responding to peptide 91-105 in ROU was significantly greater than in healthy (P < 0.0001) or disease controls (P < 0.01). A comparative investigation with the homologous human 60-kD hsp peptide 116-130 also showed significantly greater lymphoproliferative responses in ROU than in healthy (P < 0.01) or disease controls (P < 0.001). The potential involvement of the T cell epitope 91-105 in the pathogenesis of ROU is supported by finding a significant increase in the lymphoproliferative responses stimulated with peptide 91-105 during the stage of ulceration, compared with remission in 9/11 patients studied sequentially (P < 0.05). The results suggest that oral ulceration might be initiated by the microbial hsp peptide 91-105 stimulating the mucosal Langerhans cells, which may generate autoreactive T cell clones primed to the homologous peptide 116-130. PMID- 7533680 TI - Anti-V3 antibody reactivity correlates with clinical stage of HIV-1 infection and with serum neutralizing activity. AB - During HIV-1 infection, the current tenet is that only anti-gag antibodies decline, while those directed at env remain stable. Among the latter antibodies, those directed to the V3 domain of gp120 are assumed to play a role in the immune surveillance against HIV-1. We investigated the correlation between anti-V3 antibody levels and the clinical stage of infection and the ability to neutralize syncytium formation. Using a V3-specific antigen-limited ELISA, we analysed the antibody levels of a panel of 93 HIV-1+ sera to V3 peptides derived from different HIV-1 strains and from the North American/European consensus sequence V3(Cs). Sera preferentially recognized V3 peptides from the representative V3(MN) strain and V3(Cs). Antibody reactivity to V3(MN) or V3(Cs) actually declined in relation with progression to AIDS, while antibodies against whole recombinant gp160 or gp41 immunodominant epitope remained stable. There was a strong correlation (P < 0.0001) between anti-V3 (Cs)/V3(MN) antibody levels and serum titres that neutralized HIV-1MN-mediated syncytia. Serology based on V3-specific antigen-limited ELISA indicates that anti-V3 antibody reactivity may decline during the course of HIV infection. PMID- 7533682 TI - Decreased levels of complement receptor 1 (CD35) on B lymphocytes in persons with HIV infection. AB - Previous studies have shown that complement receptor 1 (CR1) expression on erythrocytes is decreased under several conditions including HIV infection and autoimmune diseases. The goal of this study was to determine whether expression of CR1 on peripheral blood B cells, where this receptor plays a role during immune responses, is altered in persons with HIV infection. The B cells from rheumatoid arthritis (RA) patients were also assessed since this represents a group with known complement and B cell abnormalities. The CD19+ B cells from persons with either HIV infection or RA had significantly reduced levels of CR1 when compared with control donors (75 and 72% CR1+ versus 94% CR1+ for control donors). The reduction of B cell CR1 occurred in both the percentage of B cells positive for CR1 and the levels of CR1 found on positive cells. In contrast, CR1 on monocytes was not reduced. As shown in previous studies, CR2 was also found to be reduced on B cells from the HIV-infected persons and there was extensive overlap between the B cell subsets which lacked expression of CR1 and CR2. The complement receptor-negative B cells found in HIV-infected persons were not immature or activated as defined by their lack of expression of CD10 or B7, respectively. Elevated levels of C4d, a classical complement pathway-activation product, were detected in plasma from both HIV-infected and RA patients. These studies suggest that chronic complement activation occurring in persons with HIV infection or RA can affect the complement receptor phenotype of peripheral blood B cells. Since complement receptors are involved in activation of B cells, the subset that lacks CR1 may represent cells that have encountered immune complexes and may therefore be stimulated. Additionally, the downregulation of complement receptors may have significant effects on the ability of B cells to capture and present opsonized antigens. PMID- 7533681 TI - The effect of cyclosporin A, FK506, and rapamycin on the murine chronic graft versus-host response--an in vivo model of Th2-like activity. AB - We have evaluated the effects of three potent immunosuppressive agents: cyclosporin A, FK506, and rapamycin, on a murine chronic graft-versus-host response (chronic GVHR). The chronic GVHR has previously been described to be a Th2-like response, and is characterized by a marked splenomegaly and hyper-IgE production in the early stages of the response. The effects of the immunosuppressive agents on both splenomegaly and hyper-IgE were measured 3 weeks after the induction of the chronic GVHR. Rapamycin was found to inhibit both splenomegaly and the hyper-IgE response in a dose-dependent manner. Unexpectedly cyclosporin A and FK506 were found to potentiate markedly both the splenomegaly and hyper-IgE response at low doses before exhibiting an inhibitory effect at higher doses. We propose the differences of activity seen with rapamycin compared with cyclosporin A and FK506 may be explained by their different mechanisms of action, and also by the selectivity of low dose cyclosporin A and FK506 for Th1 like lymphocytes. The implications of these observations are discussed in relation to the use of these immunosuppressives for the treatment of Th2-like diseases. PMID- 7533684 TI - Circulating immune complexes in chronic hepatitis related to hepatitis C and B viruses infection. AB - Circulating immune complexes (CIC) may be involved in tissue damage and/or viral clearance in viral hepatitis. To assess the frequency of raised CIC in chronic hepatitis related to hepatitis B and C, IgM, IgG, and hepatitis B surface antigen (HBsAg) containing CIC were determined, by conglutinin (K) and C1q assays, in 101 patients with chronic hepatitis B alone, 24 patients with chronic hepatitis B and C, 48 patients with chronic hepatitis C alone, and 54 healthy controls. Compared to patients with hepatitis B alone, patients with dual infection had higher frequency of raised IgM-C1q CIC (P < 0.001) and IgM-K CIC (P < 0.01). There is no difference in the prevalence of HBsAg-CIC between patients with hepatitis B alone and those with dual infection. Among patients with chronic hepatitis C alone, conglutinin-binding CIC is the predominant type of raised CIC and correlated with more severe liver damage. In conclusion, CIC may play a role in the pathogenesis of chronic hepatitis C virus infection. PMID- 7533685 TI - Structural characterization of peptides that bind synovial fluid antibodies from RA patients: a novel strategy for identification of disease-related epitopes using a random peptide library. AB - Phage epitope library technology has become a powerful tool for identifying determinants recognized by homogenous antibodies. Screening of human sera or fluids with random peptide phage libraries is a novel approach that can dissect common features at the amino acid level in individuals infected by the same etiological agent(s). In this study we have analyzed the specificity of antibodies in synovial fluids (SF) obtained from patients with rheumatoid arthritis (RA). We found that a high proportion of the peptides binding SF antibodies differ from those binding serum antibodies, suggesting that synovial B cells are expanded as a result of local antigen stimulation. When all the selected phages (enriched library) that bind the SF antibodies from a seropositive RA patient were further screened by the use of different SF antibodies with or without rheumatoid factor activity, we identified common SF antibody specificities (IRRSETPRA, RRRVRNSDT, PVSSTRGNM). Antibodies against these common specificities are also found in SF from other RA patients. Taken together, the present results open the possibility of defining the entire set of synovium antibody specificities and also common SF specificities between RA patients. PMID- 7533683 TI - Identification and characterization of a tolerogenic T cell determinant within residues 181-209 of chick type II collagen. AB - The murine model of collagen-induced arthritis is characterized by the development of an immune response against joint cartilage. Arthritis can be significantly suppressed by the administration of type II collagen (CII) or one of the CNBr peptides, CB11 (CII 124-402) as a tolerogen prior to immunization. We have previously shown that two synthetic peptides, representing sequences CII 260 270 and CII 181-209, are effective tolerogens. In this paper, we now characterize the T cell determinant with CII 181-209. A series of synthetic peptides overlapping CII 181-209 and analogs of chick CII 181-209 containing site-directed amino acid substitutions was developed and cultured with T cells from DBA/1 mice immunized with CII. Supernatants were collected and analyzed for the presence of the T cell lymphokine IFN-gamma. These data indicate the critical T cell determinant to be located within CII 190-200. This conclusion is further supported by the observation that an unodecapeptide representing CII 190-200 was just as effective as CII 181-209 in suppressing arthritis and anti-CII antibody response when tested as a tolerogen. Analogs containing single amino acid substitutions at residues 191, 194, 197, 198, or 200 were significantly less effective in inducing T cell responses. Each of these peptide analogs was then given as neonatal tolerogens to DBA/1 mice. Mice were subsequently immunized and observed for the development of arthritis. These studies identified residues 194, 197, 198, and 200, and probably residue 191, as critical for tolerance and the suppression of arthritis. Elucidation of the fine structures of T cell determinants which are critical for suppression of arthritis should allow these techniques to be used for developing specific immunotherapeutic approaches to autoimmune arthritis. PMID- 7533688 TI - Staging prostate cancer. PMID- 7533686 TI - T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice. AB - In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1+ T cells but not in B220+ B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4+ cells as CD8+ cells at 24 hr and after 48 hr the CD4+ subset predominated. The CD4+ T lymphocytes were CD44+ CD45RBlo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-gamma mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-gamma steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th2-like cytokines and the accumulation of eosinophils in bronchial fluids. PMID- 7533687 TI - Inverse expressions of the N-myc oncogene and beta 1 integrin in human neuroblastoma: relationships to disease progression in a nude mouse model system. AB - A nude mouse model for human neuroblastoma has been developed to examine possible relationships between amplification/over-expression of the N-myc oncogene and altered regulation of expression of specific integrin subunits during tumor progression. Subcutaneous (ectopic) or intra-adrenal (orthotopic) injection of the neuroblastoma cell lines SK-N-SH or IMR-32 has generated a number of derivative tumor cell lines. Tumor cell lines derived from SK-N-SH cells (which do not express N-myc) or IMR-32 cells (which over-express N-myc) produce tumors at higher rates when re-injected into the subcutaneous space of nude mice. Moreover, cell lines derived from tumors initiated by IMR-32 cells exhibit shorter latent periods than do IMR-32 cells direct from tissue culture. With regard to integrin subunit expression, SK-N-SH and related cell lines express high levels of beta 1 integrin, which is associated with the alpha 2 and alpha 3 integrin subunits (predominantly alpha 3). IMR-32 cells display reduced beta 1 expression, and that which is produced is not associated with common alpha subunits. LaN1 cells, which express N-myc at even higher levels than do IMR-32 cells, express even less beta 1. Interestingly, the tumor-derived cell lines (especially those from tumors initiated in adrenal glands) also exhibit reduced integrin expression compared with the parental cell lines; this reduction is associated with the enhanced tumor take rate observed when the cells are re injected into nude mice. Our results raise the possibility of a relationship between over-expression of N-myc and down-regulation of beta 1 integrin expression (possibly some alpha subunits also). In addition, the data suggest that human neuroblastoma-derived cell lines which exhibit reduced integrin expression display more aggressive tumor growth in nude mice. PMID- 7533689 TI - Benign prostatic hyperplasia: diagnosis and treatment options. AB - BPH is a complex condition effecting millions of adult male patients. The pathophysiology is a complex interrelated sequence of events involving stromal and epithelial growth changes. Within the past 20 years, major pharmacologic advances have been made that allow reversal of some of the hyperplastic changes and, thereby, improve patients voiding patterns. With this increase in medical options, there has also come an onslaught of other interventions, such as laser, prostatic stents, balloon dilators, and microwave hyperthermia--some of these have shown promise in the early clinical trials. As with all of these treatment options, further long term assessment must be scientifically proven to be of significant benefit to the patient. Would the patient be better off with observation or a TURP? PMID- 7533690 TI - Medical management of benign prostatic hypertrophy. PMID- 7533691 TI - Evaluation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy in granulopoetic patients complicated with sepsis. AB - A study was carried out to investigate the efficacy of therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in 24 patients with granulocytopenia and sepsis who had failed to respond to antibiotics. The mean leukocyte count at the start of the study was 911 +/- 334/microliter. Patients were injected subcutaneously with 75 micrograms rhG-CSF once daily for a mean of 5.2 days. The plasma G-CSF concentration was measured by ELISA. The leukocyte count increased approximately 9-fold after 1 week in 19 patients and the percentage of granulocytes rose from 46.2% to 78.9%. These 19 patients survived, while the 5 patients with no leukocyte response to rhG-CSF died. High plasma G CSF levels were found in patients with granulocytopenia. Plasma G-CSF levels decreased as levels of granulocyte increased in survivors. A high plasma G-CSF concentration persisted in the 5 non-responding patients resulting in a fatal outcome. This study suggests that rhG-CSF both increased the leukocyte count and was a useful therapeutic manoeuvre for sepsis. PMID- 7533692 TI - [Studies on electrophysiology of lysophosphatidylcholine and evaluation of its clinical significance]. AB - The cellular electrophysiological effects of lysosphatidylcholine (LPC) were investigated by using patch-clamp whole cell recording and conventional microelectrode technique, LPC(10 microns) suppressed the sodium, background potassium and calcium current and the effect of LPC on activity of ionic channels was nonselective. Resting potential, action potential amplitude and maximal rate of rise of phase of action potential were decreased with LPC (50 microns) perfusing. The incidence of abnormal automaticity, early after depolarization (EAD) and delayed after depolarization (DAD) was higher. It is clearly shown that LPC accumulates at early time of myocardial ischemia and thus may induce ischemic arrhythmias. PMID- 7533693 TI - Prospects for pharmacological male contraception. PMID- 7533694 TI - Angiotensin converting enzyme inhibitors in Raynaud's phenomenon. AB - Patients with Raynaud's phenomenon exhibit reversible digital vasospasm, often in response to cold. While this condition often responds to simple physical measures, in severe cases, symptoms may require drug treatment. Arterial vasodilators have usually been tried in clinical practice, but recently ACE inhibitors have been tested in clinical trials of this condition. Case studies, noncomparative and placebo-controlled studies have shown mixed results of ACE inhibitor therapy in Raynaud's phenomenon. While these drugs reduced symptoms in some patients, the results were not consistent. On objective measures of improvement, such as evaluation of digital blood flow patterns, consistent statistically significant changes have also not been shown. However, well conducted dose-titration studies have not been performed in patients with Reynaud's phenomenon, and the objective methods of assessing this condition require refinement. The mode of action of ACE inhibitors is promising and these agents do not have significant adverse effects in this population. While at present ACE inhibitors cannot be recommended for the routine treatment of Reynaud's phenomenon, further controlled studies with newer ACE inhibitors, which may have improved peripheral activity, may change this view. PMID- 7533696 TI - Botulinum toxin in clinical practice. AB - Over recent years botulinum toxin type A has emerged as a safe and effective treatment for a number of previously refractory conditions associated with excessive muscle activity. The list of indications is expanding, but at present it is generally considered to be the treatment of choice for focal dystonias such as blepharospasm, torticollis, laryngeal dystonia, and oromandibular dystonia, as well as hemifacial spasm, strabismus, and some forms of limb spasticity. Carefully targeted intramuscular injections of a small amount of the toxin block the release of acetylcholine at the neuromuscular junction, producing a chemical denervation, with the aim of reducing excessive muscle activity without producing significant functional weakness. In some situations electrophysiological assessment and localisation of the muscles for injection is necessary. Treatment is symptomatic, with effects lasting 3 to 4 months and most patients requiring up to 4 injections per year to maintain the beneficial effect. Appropriate use of the toxin requires both an understanding of the physiological action of the potential muscles involved in each situation, together with a knowledge of the likely dose necessary to reduce muscle activity to the required level. Botulinum toxin represents a major advance in the management of these conditions, many of which responded poorly to previously available forms of therapy. PMID- 7533697 TI - Dornase alfa. A review of its pharmacological properties and therapeutic potential in cystic fibrosis. AB - By cleaving neutrophil-derived DNA present in the infected lungs of patients with cystic fibrosis (CF), dornase alfa (recombinant human deoxyribonuclease I) reduces the adhesiveness and viscoelasticity of CF sputum. Well designed clinical studies performed in patients with CF and mild to moderate pulmonary disease [forced vital capacity (FVC) > or = 40% of predicted value] show that aerosolised dornase alfa improves lung function, achieving a 6 to 7% increase from baseline in forced expiratory volume in 1 second (FEV1) after 6 months' therapy. Improvements in general well-being and CF-related symptoms were also noted by patients. Importantly, dornase alfa reduced the relative risk of respiratory exacerbations requiring parenteral antibiotics by 22 to 34% compared with placebo. Short term studies with dornase alfa in patients with more severe pulmonary disease (FVC < 40% of predicted value) and in those with acute infectious exacerbations did not reveal any significant improvements in pulmonary function, although long term studies are required to fully determine efficacy. Voice alteration, laryngitis or rash may develop with dornase alfa therapy, although more clinical experience with the agent is required to define its tolerability profile. Anaphylaxis has not been reported with dornase alfa to date. In summary, aerosolised dornase alfa offers modest improvements in lung function and, importantly, a reduced risk of respiratory exacerbations in patients with CF and an FVC > or = 40% of the predicted value, thus representing an important adjunct agent in this patient group. PMID- 7533698 TI - Piroxicam-beta-cyclodextrin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in rheumatic diseases and pain states. AB - Piroxicam-beta-cyclodextrin is a complex of the established nonsteroidal antiinflammatory drug (NSAID) piroxicam and an inert cyclic macromolecule, beta cyclodextrin. In clinical trials in patients with rheumatic diseases or pain arising from other conditions, it was as effective an analgesic as standard piroxicam, and showed a faster onset of action on the first day of treatment. In short term pharmacodynamic studies in healthy volunteers, piroxicam-beta cyclodextrin was equivalent to or tended to show less gastrointestinal mucosal toxicity than standard piroxicam, as assessed by endoscopy and faecal blood loss. However, no data are available on its comparative gastrointestinal mucosal effects from long term clinical trials using similar measures. Preliminary findings from a clinical study suggest piroxicam-beta-cyclodextrin caused fewer gastroduodenal lesions than tenoxicam. As with other NSAIDs, the majority of adverse events associated with piroxicam-beta-cyclodextrin in clinical trials were gastrointestinal in origin, with epigastric pain, heartburn and nausea the most common. Thus, piroxicam-beta-cyclodextrin is an effective agent in patients with rheumatic diseases or other pain states. When rapid analgesia is required in the initial treatment of acute pain, the faster onset of action of piroxicam-beta cyclodextrin may be an advantage over the parent compound; however, this is unlikely to be important during long term therapy. The results of further long term trials are awaited before firm conclusions can be reached regarding the gastrointestinal tolerability of piroxicam-beta-cyclodextrin compared with that of standard piroxicam and other NSAIDs. PMID- 7533695 TI - Pharmacological management of hypertension in paediatric patients. A comprehensive review of the efficacy, safety and dosage guidelines of the available agents. AB - The prevalence of hypertension in children, although lower than in adults, is still significant. An underlying cause is often identified in the younger patient, with essential hypertension accounting for the majority of cases in adolescents. The natural history of hypertension in childhood is still not well delineated. Previous Task Force recommendations are addressed to reflect current experience with the newer classes of agents, namely the angiotensin converting enzyme (ACE) inhibitors and the calcium channel blockers (CCBs) where either limited or no experience was previously available. In addition, the current treatment recommendations of Joint National Committee V (JNCV) are reflected in our discussion. The current drug classes are reviewed with respect to dosage guidelines, adverse effects and potential drug-drug interactions. The advantages and disadvantages of a tailored or individualised therapeutic approach as opposed to rigid stepped care therapy will be presented. Clearly, more long term data need to be obtained with respect to the safety and efficacy of the newer classes of drugs. PMID- 7533700 TI - Impact of nifedipine on vascular smooth muscle cell differentiation. Implications for atherogenesis. AB - Although vascular smooth muscle cells (SMCs) play a key role in the development of atherosclerotic lesions, they are not a homogeneous cell type. Myosin has been used as a marker of SMC differentiation in order to identify distinct SMC populations in the adult rabbit aorta. The medial layer of the normal adult aorta contains predominantly 'adult' type SMCs, which express smooth muscle (SM) myosin isoforms exclusively, and a minority of 'immature' type SMCs, which coexpress SM and nonmuscle (NM) myosin isoforms. The size of this latter SMC subpopulation, showing the 'immature' pattern of myosin isoform expression, increases markedly in the aortic media during experimental atherogenesis, and represents a major SMC type in the atherosclerotic plaque. The dihydropyridine derivative, nifedipine, has a marked effect on NM myosin expression and SMC differentiation in vitro. In vivo, short term administration of nifedipine resulted in the disappearance of 'immature' type SMCs from the aortic media of both normocholesterolaemic and hypercholesterolaemic adult rabbits. Moreover, in a model of atherosclerosis prevention, nifedipine significantly reduced the area of aortic intimal thickening and reduced the size of the 'immature' type SMC population both in the aortic media and intima of the hypercholesterolaemic rabbit. PMID- 7533701 TI - Hypertension treatment and prevention of new atherosclerotic plaque formation. AB - The effect of hypertension on the arterial vascular wall is characterised primarily by morphological changes to the endothelium and hypertrophy of smooth muscle cells within the arterial media. Endothelial dysfunction is manifest through increased permeability to high molecular weight compounds as well as mitogenic and vasoactive substances. At the same time, denudation of the vascular endothelium promotes platelet aggregation and subsequent release of platelet derived growth factor (PDGF). In conjunction with endothelium- and monocyte derived growth factors, this mitogen stimulates subintimal smooth muscle cell proliferation and migration and arterial wall thickening, resulting in a haemodynamically important increase in vascular resistance, particularly at the precapillary level. In addition, focal endothelial dysfunction allows entry of lipids into the vascular wall, thereby promoting formation of a lipid-rich fatty streak, the primary 'early' atherosclerotic lesion. Most of these changes, including endothelial injury, subintimal lipid-binding, cellular proliferation and migration, platelet aggregation and PDGF release are common to both hypertensive and early atherosclerotic processes and involve the participation of calcium ions as 'second messengers'. Thus, antihypertensive treatment with calcium antagonists may not only lead to a protective decrease in wall shear stress through a reduction in blood pressure, but may also inhibit those cellular processes within the vascular wall that are responsible for initiating atherosclerosis. Indeed, experimental as well as human studies have demonstrated a beneficial suppressant effect of calcium antagonists on the early stages of atherosclerosis. PMID- 7533703 TI - The 24-hour efficacy of a new once-daily formulation of nifedipine. Italian Nifedipine GITS Study Group. AB - Nifedipine GITS (Gastro-Intestinal Therapeutic System) is a recently launched long-acting formulation of nifedipine. The aim of the Italian Nifedipine GITS Study was to determine the duration of the antihypertensive effect of once-daily nifedipine GITS in outpatients with essential hypertension. After a 2-week placebo run-in period, 126 patients with mild to moderate essential hypertension (diastolic BP95 to 114mm Hg) were randomised to receive nifedipine GITS 30mg (n = 42), nifedipine GITS 60mg (n = 42) or placebo (n = 42), once daily in a double blind fashion for 4 weeks. At the end of the run-in and treatment periods, ambulatory BP monitoring was performed (Spacelabs 90202 or 90207 device) to provide regular 15-minute BP readings for 24 to 36 hours. In the 81 patients with at least 24 hours of valid ambulatory BP data, the average systolic and diastolic BP changes after treatment with nifedipine GITS 30mg (n = 25), nifedipine GITS 60mg (n = 28) and placebo (n = 28) were -16.5/-10.8, -16.3/-10.0 and +0.4/+0.9mm Hg, respectively. BP changes with nifedipine GITS differed significantly (p < 0.01) from those with placebo. Heart rate was not significantly altered by nifedipine GITS. The effects of nifedipine GITS and placebo on ambulatory BP 24 hours and 36 hours after the last dose were evaluated in 56 patients with complete 36-hour ambulatory BP profiles. After 24 hours, both doses of nifedipine GITS caused significant (p < 0.01) reductions in systolic and diastolic BP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533702 TI - Pharmacokinetic profile of nifedipine GITS in hypertensive patients with chronic renal impairment. AB - 25 hypertensive patients with normal or impaired renal function underwent pharmacokinetic and safety studies after single and multiple dose administration of nifedipine GITS (Gastro-Intestinal Therapeutic System) 60mg tablets. Complete pharmacokinetic data were obtained from 23 of these patients. Blood pressure and heart rate changes were compatible with the known properties of the drug. Impaired renal function did not affect the maximum plasma concentrations or bioavailability of nifedipine after single or multiple dose administration of nifedipine GITS, nor was there any evidence of excessive drug accumulation in the presence of renal impairment. PMID- 7533705 TI - Nifedipine inhibits accumulation of low density lipoprotein and cholesterol in the aorta of normocholesterolaemic rabbits. PMID- 7533706 TI - Monitoring biological effects of contamination in marine fish along French coasts by measurement of ethoxyresorufin-O-deethylase activity. AB - The use of bioindicators to evaluate exposure to the biological effects of chemical pollutants in marine organisms constitutes a new tool in the monitoring field. The establishment of a North Sea monitoring network in 1991, involving such international organizations as the North Sea Task Force, the International Council for the Exploration of the Sea, and the Intergovernmental Oceanography Commission, led French researchers to develop an enzymatic biomarker to monitor biological effects within the National Observation Network. The biomarker, ethoxyresorufin-O-deethylase (EROD), dependent on the CP450 system, has been monitored biannually since 1992 in several species of fish (Callionymus lyra, Limanda limanda, Serranus sp., Mullus barbatus) in two coastal sites particularly exposed to industrial and domestic pollution. A rapid method is used to assay EROD enzymatic activity determined along a pollution gradient, and results are interpreted on a microplate reader. The strategy of this approach is to assess the effects on the marine ecosystem during prolonged exposure to specific pollutants such as polyaromatic hydrocarbons, polychlorinated biphenyls, and dioxins. PMID- 7533707 TI - Heavy metal content (Hg2+, Cd2+, Pb2+) in various body parts: its impact on cholinesterase activity and binding glycoproteins in the grasshopper Aiolopus thalassinus adults. AB - Various toxicological symptoms were observed in Aiolopus thalassinus (Fabr.) adults which resulted from either contaminated soil or from being fed HgCl2-, CdCl2-, and PbCl2-treated diet. Many insects had abnormal wings which were present until the F3 untreated generation. Heavy metals were stored in different body parts, most of Cd2+ and Hg2+ were found in the testes, followed by the gut. Lead was enriched in all organs and body parts of the treated grasshoppers compared to the control. The highest lead concentrations were found in testes, wings, gut, and ovaries. In the treated generation ChE activity was reduced to about 23% compared to the untreated grasshoppers. In contrast to this, lead increased ChE activity to about 14%. In the following F2 untreated adults, ChE activity was normal; no long-term effect was found. In the supernatant of treated adults a Cd-type glycoprotein was found which can bind Cd ions; its MW was determined by electrophoresis and reference proteins at about 14,450 Da. PMID- 7533708 TI - Combined effect of tin and lead on heme biosynthesis in rats. AB - The aim of the present study was to investigate the combined effect of tin (SnCl2) and lead Pb(CH3COO)2 on activity of heme biosynthesis enzymes [delta aminolevulinic acid synthetase (ALA-S) and heme oxygenase] in liver and kidneys, as well as iron (Fe) and copper (Cu) concentration in serum of rats. The experiment was performed on female rats which received 2 mg Sn/kg and 3.5 mg Pb/kg separately and jointly intraperitoneally (ip) for 5 days and per os (po) at single dose (100 mg Sn/kg and 17.5 mg Pb/kg). Lead induced ALA-S in liver and kidney both after ip and po administration; tin, however, induced ALA-S only after ip administration in liver of rats. The activity of heme oxygenase was induced after Sn po and ip administration in liver and kidneys and Pb administration (ip) in kidneys. Sn and Pb administered jointly caused a significant increase of Cu (ip), whereas Sn (po) decreased this metal level in serum of rats. Kidneys proved to be the organ in which the highest degree of examined enzyme induction took place. Pb is responsible for ALA-S, whereas Sn is responsible for induction of heme oxygenase activity in this organ, especially after per os administration. No additive effect on ALA-S and heme oxygenase activities of Pb and Sn combined was noticed. PMID- 7533699 TI - Leuprorelin. A review of its pharmacology and therapeutic use in prostatic cancer, endometriosis and other sex hormone-related disorders. AB - Leuprorelin (leuprolide acetate) is a gonadotrophin-releasing hormone (GnRH) analogue used to treat a wide range of sex hormone-related disorders including advanced prostatic cancer, endometriosis and precocious puberty. It acts primarily on the anterior pituitary, inducing a transient early rise in gonadotrophin release. With continued use, leuprorelin causes pituitary desensitisation and/or down-regulation, leading to suppressed circulating levels of gonadotrophins and sex hormones. Clinical trials in men with advanced prostatic cancer demonstrate that leuprorelin (usually monthly depot injections of 3.75 or 7.5 mg) is less likely to cause serious adverse cardiovascular effects than diethylstilbestrol, and has comparable efficacy to bilateral orchiectomy or other GnRH analogues. Therefore, the choice between leuprorelin and orchiectomy may be made on the basis of the patient's treatment preference, along with specific patient characteristics and cost implications. Monthly intramuscular or subcutaneous administration of depot leuprorelin 3.75 mg was superior to placebo, and comparable to oral danazol 800 mg/day or intranasal buserelin 900 micrograms/day, in achieving objective and subjective responses in women with endometriosis. Thus, leuprorelin is an effective alternative to other treatments for women with endometriosis, but the recommended duration of its use in this clinical setting is limited to 6 months because it reduces bone mineral density. In children with central precocious puberty, leuprorelin (usually monthly intramuscular or subcutaneous injections of depot leuprorelin 3.75 to 15mg) decreases mean growth velocity and signs of sexual maturation and increases predicted adult height compared with baseline measurements. Although effects on final adult height are predicted from available data and require confirmation in long term follow-up studies, the absence of effective alternatives to GnRH analogues makes leuprorelin a first-line therapy for children with this rare disease. In women with uterine leiomyomata, monthly intramuscular administration of depot leuprorelin 3.75 mg for 6 months markedly reduces uterine volume and fibroid-related symptoms, but, as with other GnRH analogues, these effects dissipate following discontinuation of the drug. As adjuvant therapy in women undergoing in vitro fertilisation or gamete intrafallopian transfer, leuprorelin (usually 0.5 to 1 mg/day subcutaneously) reduces the risk of cancelled cycles for oocyte retrieval by preventing premature luteinisation. While some studies demonstrate an improvement in intermediate end-points such as increased number of mature oocytes retrieved and embryos available for transfer, a significant effect has not been demonstrated on the rate of live births per stimulated cycle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533709 TI - Biomagnification of PCBs, p,p'-DDE and HCB in the River Po ecosystem (northern Italy). AB - Polychlorinated biphenyls (PCBs) and chlorinated pesticides were determined in several organisms and in the sediment sampled in the final stretch of the River Po (Italy). Bioconcentration models were used in order to test the equilibrium condition between biotic and abiotic compartments. The oligochaetes-sediment model fitted very well with the experimental results. The only exception was hexachlorobenzene. The results regarding the fish species demonstrate that most PCB congeners and p,p'-DDE are biomagnificated in the River Po ecosystem but to a lesser extent than expected on the basis of a four-step food chain model. In particular, most species approached to the third trophic level, while Perca fluviatilis concentrations correspond to a higher level but accumulate less than predicted by the model for a top predator. PMID- 7533710 TI - The effect of copper on the blood chemistry of Clarias gariepinus (Clariidae). AB - Environmental stressors, both natural and humanly induced, could cause changes in cellular function which alter the physiology of organ systems in fish. The need to comprehend and predict the condition which stress metals will pose on fish, and extrapolate the effect of pollutants from laboratory to population levels, have forced scientists to search for physiological and biochemical indicators of health and sublethal toxicant effects. Hematological evaluation of fish blood provides valuable facts concerning the physiological response of fish to changes in the external environment. Furthermore, hematological variables are well known for their clinical value in prognosis and diagnosis. Sublethal responses after exposure to toxicants can be determined by commonly applied techniques. Measurements for a number of hematological and carbohydrate metabolic variables were recorded at winter and summer temperatures after fish, acclimated for 3 months to experimental conditions, were exposed to sublethal concentrations of copper for 96 hr in a continuous-flow experimental system. Controls were run at the same time to establish essential baseline hematological values. Fish were exposed to the mean copper concentration as was found in the Olifants River, Kruger National Park, during summer (0.05 +/- 0.032 mg liter-1) and winter (0.085 +/- 0.032 mg liter-1) to establish the effect thereof on the variables mentioned. The results proved that the concentration of copper in the river exerts a physiological effect on Clarias gariepinus at 21 +/- 1 and 28 +/- 1 degrees C which manifests in changed blood chemistry. Pathological conditions, such as erythrocytopenia, leucocytosis, hyperglycemia, and hyperprotonemia, are evident. The fish physiologically adapted to the environmental change, which does not necessarily reflect a state of normality. PMID- 7533704 TI - Future prospects for calcium antagonists. AB - In hypertensive disease, calcium antagonists (CA) affect the limiting step of increased vascular resistance. In the future, it is likely that the use of calcium antagonists will be extended to the early stage of hypertension, which is characterised predominantly by preglomerular vasoconstriction and only discrete impairment of renal function. In the advanced stages of hypertension they will be used to prevent vascular remodelling and to limit renal and cardiac tissue damage. By preventing excessive calcium overload, calcium antagonists preserve tissue integrity and reverse the progression of specific vascular pathologies such as arteriosclerosis and microangiopathy through mechanisms independent of their blood pressure-lowering effect. The protective effect demonstrated by nifedipine against toxic and radiation-induced changes in nonvascular tissue in animal models merits specific attention. PMID- 7533711 TI - Mechanism-based comparisons of acute toxicities elicited by industrial organic chemicals in procaryotic and eucaryotic systems. AB - Comparisons of toxicities elicited by nonpolar and polar narcotics, weak acid uncouplers of oxidative phosphorylation, and bioreactive chemicals between the eucaryotic systems Pimephales promelas and Tetrahymena pyriformis and the procaryotic systems Escherichia coli and Photobacterium phosphoreum were performed. Each chemical had been a priori assigned a mechanism/mode of action based on the results from previous studies with eucaryotic systems. Hydrophobicity-dependent QSARs for nonpolar narcosis for both the E. coli and the P. phosphoreum endpoints was developed. However, due to the lack of a significant relationship between P. phosphoreum toxicity and log Kow, such a QSAR for polar narcosis was developed only for the E. coli endpoint. Except for 4-nitroaniline (the only chemical in the examined group that required activation to become the Michael receptor), all chemicals containing reactive substructures revealed excess toxicity over polar narcosis QSAR for E. coli endpoints. Moreover, chloroacidic acid and ethyl chloroacetate in this system also appear to be bioreactive. The only mechanism that seemed to not exist in the procaryotic system was uncoupling of oxidative phosphorylation. Chemicals from this group, except 2,4-dinitroaniline, did not exhibit excess toxicity over polar narcosis QSAR. This was thought to be explained by the lack of mitochondria in procaryotes, the target site of uncoupling agents in eucaryotes. In addition, evaluation of toxicities of halogen-substituted short-chain carboxylic alcohols indicated that their mechanisms vary, depending upon the type of substitution and the system. PMID- 7533712 TI - Effects of copper concentration on mineral nutrient uptake and copper accumulation in protein of copper-tolerant and nontolerant Lotus purshianus L. AB - One copper-tolerant and one copper-sensitive inbred line of Lotus purshianus L. derived from a copper mine waste site in Northern California and one inbred line of the same species derived from a pasture next to the mine waste were examined for the effects of excessive copper concentrations on mineral nutrient uptake and accumulation of copper in protein fractions. Plants were grown from seeds for a period of 24 days in a modified Hoagland nutrient solution culture supplemented with 3, 6, and 10 microM copper as copper sulfate. The basal nutrient solution without copper amendment was used as the control treatment. The uptake of Cu found in the roots was 100 times or more than that in the leaves. The root tissue copper concentrations reached a plateau under 6 microM copper treatment. The leaf tissue copper concentrations increased with the increase of copper concentration in the solution culture. No difference in pattern of copper uptake was detected between the copper-tolerant and nontolerant plants. The effects of excessive copper concentrations caused reduction of Ca uptake in the leaf tissue and P uptake in both the root and leaf tissues, and no difference was found between the copper-tolerant and nontolerant plants. Increased tissue copper concentration caused greater reduction of Fe, Mn, and Zn uptake in the nontolerant plants than in the tolerant plants; this difference may be important for the growth of the tolerant plants under conditions of excessive copper concentrations. Protein extracted from the roots and leaves of both the copper tolerant and nontolerant plants was subjected to Sephadex G-75 column separation. Two major peaks of protein fractions were detected. Under low (normal level) copper concentration treatment, the copper-tolerant and nontolerant plants had similar Cu/protein ratios. However, under high copper concentration challenged conditions the copper tolerant plant had a considerably greater Cu/protein ratio (peak II protein) than the nontolerant plants. The amino acid composition of the copper-rich protein fraction (peak II) extracted from both the tolerant and nontolerant plants demonstrated a high asparate (about 25%) content. The contents of glutamate, cystine, and glycine were about 11, 2.5, and 10%, respectively, and the rest of the amino acids were in a range of 2 to 6%. This pattern of amino acid composition is different from the amino acid composition of the phytochelatin metallothionein-like proteins found in copper-tolerant plants which are very high in cysteine.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533713 TI - Mining in northern Canada: expanding the industry while protecting Arctic fishes- a review. AB - Northern Canada has a long tradition of mining activity with a poor record of environmental protection. Pollution is acknowledged as the biggest issue currently facing the mining industry. The arctic and subarctic fishes of this region are very sensitive to heavy metals, trace elements, and other contaminants in mine wastes. High sensitivity, combined with an inherently low capacity for recovery, make this aquatic resource particularly vulnerable to impacts. At least 22 fish species with major commercial, recreational, or subsistence value may be affected as the mining industry expands. The number of mines in northern Canada is steadily increasing and a metals-mining boom is expected to take place during the next decade. Prudent planning based on comprehensive mine-site evaluation, biological risk assessment, and research is essential to reduce the threat of environmental damage. There are several progressive mining techniques that can help make mining compatible with sensitive northern fisheries while also maintaining the profitability and growth of the mining industry. Thus far, this technology has been used very little in northern Canada. PMID- 7533714 TI - Some consideration of the impact of energy and chemicals on the environment. PMID- 7533715 TI - Sensitivity and specificity of needle electromyography: a prospective study comparing automated interference pattern analysis with single motor unit potential analysis. AB - In this prospective study, automated interference pattern analysis (IPA, "Willison analysis", modified by Stalberg et al. 1983) was compared to the quantitative evaluation of mean motor unit potential duration (QMUP) in 239 muscles from consecutive, unselected patients. The sensitivity and specificity of both methods were calculated with respect to the clinically derived final neurological diagnosis, with histology available for 120 examinations. Whereas specificities were not different for the methods, the sensitivity for detection of abnormal vs. normal was 49% for QMUP and 74% for IPA (P < 0.001). The sensitivity for detection of myopathy or neuropathy was 46% or 38% for QMUP and 75% (P < 0.001) or 53% (P < 0.05) for IPA. Thus, in all instances, IPA had superior sensitivity with unchanged specificity as compared to QMUP. The results of a rapid and purely qualitative visual MUP assessment were statistically not different from QMUP. Although widely used, neither of these methods has been evaluated for its reliability in unselected patients with various grades of disease. Our results indicate that in a routine setting, the best diagnostic strategy might be the automated IPA, which can be quickly obtained in several muscles, followed by muscle biopsy in unclear cases. PMID- 7533716 TI - Conditioning effects of sural nerve stimulation on short and long latency motor evoked potentials in lower limb muscles. AB - The effects of conditioning sural nerve stimulation on motor evoked potentials (MEPs) in relaxed muscles of the lower limb were examined in 11 healthy adults. The study tested the hypothesis that cutaneous afferent stimulation, in the absence of muscle afferent input, facilitates the short latency MEPs evoked in lower limb muscles following transcranial magnetic stimulation of motor cortex. Non-painful (3.6 x sensory threshold) percutaneous electrical stimulation was delivered to the sural nerve at conditioning (C)-test (T) intervals of 0-150 msec. MEPs were elicited bilaterally in the tibialis anterior (TA) and lateral gastrocnemius (LG) muscles with and without preceding cutaneous stimulation. Mean MEP amplitudes were facilitated in the ipsilateral TA by 258% (n = 10 of 11), the ipsilateral LG by 275% (n = 7 of 8) and the contralateral TA by 313% (n = 7 of 7) within C-T: 60-100 msec. These results establish that cutaneous afferent stimulation per se does lead to a facilitation of short latency MEPs. In addition, two subjects who were examined at higher stimulation intensities both exhibited late responses (70-95 msec) at C-T: 0-15 msec. These responses, which were independent of the amplitude and appearance of short latency MEPs, suggest a convergence of the cutaneous stimulation with late arriving descending cortical or cortico-bulbospinal inputs to the target motoneuron pool.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533717 TI - Influence of physiotherapeutic facilitation techniques on motor evoked potentials in centrally paretic hand extensor muscles. AB - In the rehabilitation of stroke patients, various facilitation techniques are applied to reduce weakness in centrally paretic muscles and to improve functional motor capacity. The present investigation compared the facilitatory effect of 5 different physiotherapeutic approaches onto the centrally paretic extensor carpi radialis muscle in 30 stroke patients classified into 3 groups according to the individual degree of paresis. In order to quantify the influence of the respective facilitation manoeuvre, single transcranial magnetic stimuli were applied before and during the application of cutaneous/proprioceptive stimuli, a weight bearing task, contraction of the affected and the non-affected extensor carpi radialis muscle and during proximal preinnervation on the affected side. All procedures, indeed, enhanced the frequency of occurrence of muscular response potentials and their amplitudes while diminishing their response latencies. The most prominent effects were observed when the muscle itself was voluntarily activated. A similarly strong facilitation was obtained in the most severely affected patients with cutaneous and proprioceptive stimuli, but such stimuli had inhibitory effects in the healthy control group. The present study illustrates the interaction of cortically evoked motor potentials with peripherally or centrally generated inputs, contributes to the understanding of the neurophysiological mechanisms underlying physiotherapeutic facilitation techniques and helps in providing rational criteria to decide about the most appropriate facilitation method. PMID- 7533718 TI - Biomechanical analysis of simple jaw movements in Friedreich's ataxia. AB - The jaw movement kinematics in relation to the EMG activity of two antagonistic jaw muscles (the masseter and the digastricus pars anterior) were examined in healthy subjects and patients with Friedreich's disease. Dysarthria and ataxia are the main characteristics of this disease. Jaw movement was monitored with a magnetometer system, and bipolar surface electrodes were used to record EMG activity. Unidirectional opening, unidirectional closing and opening-closing movements of the mandible were performed in a simple reaction time situation. The data were compared with those for normal control subjects. The kinematic and electromyographic characteristics were: (a) prolonged total movement duration resulting from increased acceleration and deceleration durations; (b) decreased maximum velocity, and a secondary peak in the velocity profile; (c) tonic EMG activity in muscles supposedly at rest; (d) prolonged EMG bursts. Premotor reaction time was also increased. These characteristics are similar to those, previously described, of limb movements in subjects with cerebellar dysfunctions and suggest that the alterations of jaw movement in Friedreich's ataxia could be due to a deficit in cerebellar control. PMID- 7533719 TI - Magnetic brain stimulation: safety studies. AB - We describe short-term and long-term safety studies after low repetition rate magnetic brain stimulation in 10 normal subjects. We obtained quantitative EEG data, psychometric test results, serum prolactin and cortisol levels before and after brain stimulation. EEG and psychometric data were also obtained in 5 of these subjects 16-24 months after the initial experiment. Short- and long-term studies did not show any deleterious effects. Randt delayed recalls, however, showed a transient reduction in the score immediately after stimulation which resolved on retesting in 2 weeks. To address the question of fatigue we repeated Randt tests in 4 subjects before and after magnetic brain stimulation but without the other extensive psychometric, EEG and blood tests. Pre- and post-stimulation scores on this occasion showed no significant difference in these 4 subjects suggesting that the transient changes in the previous Randt score were related to fatigue. We conclude that single-pulse magnetic brain stimulation has no deleterious effects after magnetic brain stimulation. PMID- 7533720 TI - Effect of hemisphere-selective repetitive magnetic brain stimulation on middle cerebral artery blood flow velocity. AB - The changes of cerebral hemodynamics following repetitive hemisphere-selective magnetic stimulation over the motor cortex were investigated in 8 healthy volunteers. Bilateral simultaneous monitoring of middle cerebral artery blood flow velocity was done using transcranial Doppler ultrasonography during magnetic stimulation with single, double and triple stimuli with interstimulus intervals of 100 msec. Magnetic cortex stimulation was followed by a significant increase of ipsilateral flow velocity ranging from 5.3% +/- 2.7% for single stimuli up to 8.5% +/- 4.1% for triple stimuli and by a smaller increase of contralateral flow velocity. The ipsilateral increases are comparable to those measured previously during voluntary finger movements and indicate a physiological cortex stimulation. In parallel, the average sum of the baseline to peak hand motor response amplitudes increased from 1.5 +/- 0.7 mV (single stimuli) to 4.5 +/- 3.2 mV (triple stimuli). The increase of flow velocity in the non-stimulated hemisphere occurred during the absence of motor responses and might reflect a transcallosal activation of inhibitory neuronal structures. The occurrence of maximum velocity responses within 2-3 heartbeats following the first cortex stimulus points to a fast adjustment of cerebral perfusion in response to transcranial brain stimulation. No significant change of flow velocity was observed following motor responses evoked by unilateral magnetic stimulation of the brachial plexus or following acoustic artifacts of the coil discharge. PMID- 7533721 TI - Characteristics and variability of lower limb motoneuron responses to transcranial magnetic stimulation. AB - The characteristics and variability of the short latency responses to transcranial magnetic stimulation (TMS) were examined in 188 single lower limb motor units. Information about the cortically evoked postsynaptic potentials was derived from peristimulus time histograms generated from the discharge times of single, rhythmically active, motor units. Tibialis anterior (TA) motoneurons were almost exclusively facilitated and response magnitudes were consistent within a motoneuron pool as indicated by low coefficients of variation (CV). The net cortical effect on soleus (SOL) and medial gastrocnemius (MG) motoneurons was significantly weaker than in TA (P < 0.001) and the responses were non homogeneous within a motoneuron pool, both in terms of response amplitude and direction. The occurrence of strong facilitatory and inhibitory responses or an absence of a response within a given MG motoneuron pool was associated with CVs about 13 times that of TA and 4 times that of SOL. The large CVs may be indicative of the existence of subgroups of motoneurons differentiated by their response to TMS. Examination of only excitatory responses resulted in comparable CVs across the muscles tested. It is suggested that the degree of non-homogeneity within a motoneuron pool reflects the ability of the cortex to modulate other inputs. Selective cortical activation of specific groups (or subgroups) of motoneurons may set up appropriate conditions suited to a particular volitional task. PMID- 7533722 TI - An investigation of the late excitatory potential in the hand following magnetic stimulation of the motor cortex. AB - Magnetic stimulation of the motor cortex gives rise to a motor evoked potential (MEP) followed by a silent period (SP) during which a late excitatory potential (LEP) may occur in the surface EMG. To elucidate the mechanism of the LEP we investigated the effect of muscle contraction, stimulus intensity and stimulation site on the LEP recorded from the abductor pollicis brevis muscle. The amplitude of the LEP increased with increasing levels of muscle contraction and decreased with increasing stimulus intensity. There was no direct relationship between the amplitude of the LEP and the MEP, but there was an inverse relationship between LEP amplitude and SP duration. The latency of the LEP was unaffected by the level of muscle contraction, but increased with increasing stimulus intensity. Topographic mapping with stimulation at multiple scalp sites yielded a LEP at sites partially encircling but not including the centre of the APB motor area. These results are consistent with the LEP being due to reflex alpha motoneurone firing as a result of gamma motoneurone activation or with a period of disinhibition at cortical level allowing breakthrough of voluntary activity. PMID- 7533723 TI - Inhibition of hand muscle motoneurones by peripheral nerve stimulation in the relaxed human subject. Antidromic versus orthodromic input. AB - In active muscle, a supramaximal conditioning stimulus to peripheral nerve produces a classic silent period in the EMG. The present experiments examined the effect of this type of conditioning stimulus on motoneurone excitability in relaxed muscle. EMG responses evoked by transcranial magnetic stimulation of the brain were recorded from the first dorsal interosseus muscle (FDI) in 10 healthy subjects and 5 patients with sensory neuropathy. These responses (motor evoked potentials) were conditioned by supramaximal peripheral nerve stimuli given 0-150 msec beforehand. In the normal subjects, the classic silent period in the FDI lasted about 100 msec. The same conditioning stimulus only abolished motor evoked potentials when the conditioning-test interval was so short that the antidromic peripheral nerve volley collided with the orthodromic volley set up by magnetic brain stimulation. At longer conditioning-test intervals, although remarkably inhibited (65% mean suppression between 10 and 40 msec), the test motor potential was never completely abolished and gradually recovered by 100 msec. Inhibition of cortically evoked motor potentials did not depend upon activity set up by the conditioning stimulus in peripheral nerve sensory fibres. The patients with complete peripheral sensory neuropathy had the same extent and time-course of inhibition as the normal subjects. We conclude that in relaxed subjects the inhibitory effect of peripheral conditioning results almost exclusively from the motoneuronal inhibitory mechanisms consequent to antidromic invasion. PMID- 7533724 TI - The effect of hyperventilation on motor cortical inhibition in humans: a study of the electromyographic silent period evoked by transcranial brain stimulation. AB - We studied the effects of hyperventilation under control of the end-tidal PCO2, on the electromyographic silent period evoked by transcranial magnetic brain stimulation and by peripheral nerve stimulation. We also studied the effects of hyperventilation on the threshold, latency and amplitude of motor potentials. Hyperventilation significantly reduced the duration of the cortical silent period, but did not affect the length of the peripheral silent period. Neither did it alter the latency, amplitude or threshold of the motor potentials. These findings suggest that hyperventilation selectively depresses motor cortical inhibition in humans. PMID- 7533725 TI - HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA. AB - Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA. PMID- 7533726 TI - Pyrrolidine dithiocarbamate selectively prevents the expression of the inducible nitric oxide synthase in the rat aorta. AB - Exposure of rat aortic rings without endothelium to interleukin-1 beta for 5 h significantly attenuated the contractions due to phenylephrine and increased the tissue content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) due to the induction of nitric oxide synthase. The presence of pyrrolidine dithiocarbamate, a specific inhibitor of nuclear transcription factor kappa B activation, during the exposure of the rings to interleukin-1 beta prevented these responses to interleukin-1 beta. Rat aortic rings which had been incubated for 5 h with interleukin-1 beta in the absence and presence of pyrrolidine dithiocarbamate prior to the organ chamber experiment had a similar concentration-dependent relaxation curve for acetylcholine in rings with endothelium, and for 3 morpholino-sydnonimine (SIN-1) in rings without. Pyrrolidine dithiocarbamate applied acutely did not alter the tone elicited by phenylephrine in rings with or without endothelium and had no effect on the subsequent relaxation induced by acetylcholine in rings with endothelium or by SIN-1 in rings without endothelium. These observations suggest that pyrrolidine dithiocarbamate prevents the interleukin-1 beta-mediated expression of the inducible nitric oxide synthase without affecting the activity of the constitutive enzyme in the rat aorta. PMID- 7533727 TI - Endothelin-1, one of the most potent histamine releasers in mouse peritoneal mast cells. AB - Whether endothelin-1 or -3 is capable of inducing histamine release from the peritoneal mast cells of BALB/c mice was investigated in vitro and compared to the release induced by compound 48/80. In contrast to the mouse bone marrow derived mast cells, which originated from the same strain and have been reported upon previously, the (both crude and purified) peritoneal mast cells potently secreted histamine in response to either endothelin-1 or endothelin-3 in a concentration-dependent fashion. Even at a concentration as low as 10 nM, endothelin-1 induced a histamine release of more than 50% from the peritoneal mast cells. Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), an endothelin ETA receptor antagonist, markedly suppressed not only the histamine released induced by endothelin-1 but also that due to endothelin-3 at a similarly low concentration range. Treatment with islet-activating protein (IAP) for 3 h, an inactivator of guanosine triphosphate (GTP) (Gi)-protein, markedly reduced the histamine release induced by endothelin-1. Neomycin at 0.1 and 1 mM or ethylenediaminetetraacetic acid (EDTA) at 0.1 mM in the absence of Ca2+, neither of which affected the histamine release induced by endothelin-1, substantially reduced histamine release caused by compound 48/80. On the other hand, treatment with O-O'-bis(2 aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, completely inhibited the release induced by both endothelin-1 and compound 48/80. These results indicate that endothelin-1 is one of the most potent histamine releasers in mouse peritoneal mast cells discovered so far.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533728 TI - Delta-endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers. AB - Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, not very K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2-40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis delta-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors. PMID- 7533729 TI - Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. AB - We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17 beta-estradiol and testosterone for 8 48 h. Treatment with 17 beta-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17 beta-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens. PMID- 7533730 TI - Peptide mapping of recombinant proteins. PMID- 7533731 TI - Hybridoma stability. AB - Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product. MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones. The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated. Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks. Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N terminal sequencing, carbohydrate composition and binding assays. Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed. Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs. Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process. PMID- 7533733 TI - Renin response to 12-hydroxyeicosatetraenoic acid is increased in diabetic rats. AB - Eicosanoids (prostaglandins) can alter renin secretion and angiotensin (ANG) II action. We have studied the effects of both prostacyclin and a lipoxygenase (LO) product, 12-hydroxyeicosatetraenoic acid (12-HETE), on renin in normal and streptozotocin-induced diabetic rats. 12-HETE is not only a potent inhibitor of basal renin secretion but also a key mediator of ANG II-induced renin inhibition. We have also examined the effects of ANG II on 12-HETE formation in normal and diabetic animals. Both plasma (3.9 +/- 0.9 vs. 0.8 +/- 0.1 ng ANG I.ml-1.h-1, P < 0.01) and tissue (38 +/- 6 vs. 21 +/- 2 ng ANG I.mg tissue-1.h-1, P < 0.05) renin activity levels were markedly reduced in diabetic animals. Iloprost (10(-6) mol/l), a stable analog of prostacyclin, had similar stimulatory effects on renin secretion in both normal and diabetic tissues, but the response was enhanced by LO inhibition in diabetic tissue. 12-HETE (10(-7) mol/l) had an exaggerated effect on renin inhibition in diabetic tissue (78 +/- 2% normal vs. 65 +/- 4% diabetic, P < 0.05). Similarly, ANG II (10(-8) mol/l) inhibition of renin was significantly enhanced in diabetic rats (P < 0.001). However, ANG II did not produce an exaggerated increase in 12-HETE in diabetic renal tissue. Insulin reversed the inhibitory effects of ANG II on renin in normal rats, but it blunted the effect of ANG II in diabetic rats. These studies suggest that, while the capacity of renal cortical tissue to synthesize 12-HETE in response to ANG II is not altered, 12-HETE and ANG II actions are exaggerated in diabetes, and this may contribute to reduced renin production. PMID- 7533734 TI - Expression of the co-stimulator molecule B7-1 in pancreatic beta-cells accelerates diabetes in the NOD mouse. AB - B7-1 is a co-stimulatory molecule that signals T-cells that recognize antigen to proliferate and differentiate into effector T-cells. The same cell must present antigen and express co-stimulatory molecules, such as B7-1, to activate naive T cells. Thus, tissues that do not express co-stimulatory molecules would not be expected to induce immune responses, while expression of a co-stimulator on tissue cells may convert them into effective antigen-presenting cells and induce autoimmunity. To test this, transgenic mice have been generated that express B7-1 on the beta-cells of the pancreatic islets of Langerhans. On a B6 genetic background, B7-1 expression on beta-cells does not predispose to diabetes. B6 mice are resistant to diabetes. However, when B7-1 is expressed on the beta-cells of B6 mice backcrossed once to the genetically susceptible NOD strain, the onset of diabetes is accelerated and the autoimmune attack intensified. This illustrates that B7-1 is a very potent co-stimulatory molecule in vivo and that its presence on the surface of tissue cells can potentiate the autoimmune process. PMID- 7533735 TI - Nitric oxide synthesis and the effect of aminoguanidine and NG-monomethyl-L arginine on the onset of diabetes in the spontaneously diabetic BB rat. AB - Nitric oxide (NO) synthesis and the effect of aminoguanidine (AG) and NG monomethyl-L-arginine (NMMA) (inhibitors of NO synthase) on the onset of diabetes were studied in the spontaneously diabetic BB rat. To measure in vivo NO production, 20 male 50-day-old diabetes-prone BB (BBdp) rats and age-matched non diabetes-prone BB (BBn) rats were individually placed in metabolism cages. The animals had free access to a casein-based semipurified diet and deionized and double-distilled water. Urine excretion was collected every other day for 70 days, and urinary excretion of nitrate was measured as an index of in vivo NO synthesis. The urinary excretion of nitrate was enhanced by 150-200% in BBdp rats 4-6 days before the onset of diabetes, compared with aged-matched BBn rats. There was no difference in urinary excretion of nitrate between BBn rats and those BBdp rats that did not develop diabetes by the age of up to 120 days. To determine a role of NO in the development of spontaneous diabetes, 40-day-old male BBdp rats (30 rats per group) received daily subcutaneous injections of NMMA (acetate salt) (5 mg/kg body wt) or equal amounts of acetate (control) or oral administration of AG (0 or 3 g/l of drinking water) for 80 days. Both NMMA and AG delayed the onset of diabetes in BBdp rats by 13-15 days without altering the rate of incidence of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533732 TI - Morphological and functional characterization of beta TC-6 cells--an insulin secreting cell line derived from transgenic mice. AB - Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in beta TC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic beta-cells. We observed by immunohistochemistry and confocal microscopy that beta TC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that beta TC-6 cells at passage 30-45 secrete insulin in response to glucose. In static incubations, maximal stimulation was achieved for glucose concentrations > 2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times greater than HIT-T15 cells at passage 72-81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose in perifusion was 1,734 +/- 384 pmol.l-1. min and was 4.6-fold greater in the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Gi alpha 2, Gi alpha 3, and Go alpha 2. These results indicate that beta TC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms. PMID- 7533736 TI - N omega-nitro-L-arginine methyl ester reduces the incidence of IDDM in BB/E rats. AB - Evidence that nitric oxide (NO) is involved in cytokine-mediated islet beta-cell dysfunction and destruction in vitro has led to the hypothesis that increased production of NO may contribute to the pathogenesis of insulin-dependent diabetes mellitus (IDDM). This study demonstrates that oral administration of N omega nitro-L-arginine methyl ester (an inhibitor of NO synthase) from 30 to 150 days of age significantly reduced (P < 0.05) the incidence of IDDM in diabetes-prone BB/E rats. This supports the idea that NO plays a significant role in the pathogenesis of IDDM in this animal model. PMID- 7533737 TI - An immunocytochemical study of the respiratory system of Podarcis hispanica (Reptilia). AB - The endocrine cells and nerves of the respiratory tract of the reptile Podarcis hispanica were investigated by immunocytochemistry under light microscopy. Immunoreactivities were more numerous in the lung than in the trachea. In the tracheal epithelium, endocrine cells immunoreactive to PHI, PYY, and Leu enkephalin were detected, while immunoreactivity to serotonin, calcitonin, CGRP, PHI, and Leu-enkephalin was found in pulmonary endocrine cells. Numerous nerve fibers positive to NSE, PGP9.5, chromogranin, tyrosine hydroxylase, calcitonin, CGRP, bombesin, substance P, VIP, NPY, and PYY were found in the lungs. In addition, neurons positive to NSE and PGP9.5 were also found. Immunoreactivities to PHI and PYY in cells and to NSE, PGP9.5, chromogranin, tyrosine hydroxylase, calcitonin, CGRP, and PYY in nerves, were reported first in the respiratory system of reptiles. PMID- 7533738 TI - [The effect of the pleiotropic action of single-locus (pp) and dual-locus (aapp) mutation of mink fur color genes on serotonin metabolism in the brain]. AB - Specific features of serotonin (5-hydroxytryptamine, 5-HT) metabolism were studied in the brain of mutant sapphire minks, homozygous at genes determining silver-blue and Aleutian fur color (ppaa), and silver-blue minks (pp) in comparison with the standard-colored minks (++/++). In the midbrain of sapphire mutants, the activities of tryptophan hydroxylase (TPH) and monoamine oxidase (MAO), key enzymes of 5-HT biosynthesis and catabolism, were significantly higher; an increase in the 5-HT level was statistically nonsignificant due to considerable variation in this parameter; and the level of 5-hydroxyindole-3 acetic acid remained unchanged. A similar increase in TPH and MAO activities was also found in the midbrain of silver-blue pp minks, suggesting that activation of key enzymes of 5-HT metabolism in sapphire aapp minks is associated with the mutant alleles pp. PMID- 7533739 TI - DNA rearrangements located over 100 kb 5' of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and cause female sterility by disrupting ovarian follicle development. AB - The Steel (Sl) locus is essential for the development of germ cells, hematopoietic cells, and melanocytes and encodes a growth factor (Mgf) that is the ligand for c-kit, a receptor tyrosine kinase encoded by the W locus. We have identified the molecular and germ cell defects in two mutant Sl alleles, Steel panda (Slpan) and Steel-contrasted (Slcon), that cause sterility only in females. Unexpectedly, both mutant alleles are shown to contain DNA rearrangements, located > 100 kb 5' of Mgf-coding sequences, that lead to tissue-specific effects on Mgf mRNA expression. In Slpan embryos, decreased Mgf mRNA expression in the gonads causes a reduced number of primordial germ cells in both sexes. However, Mgf expression and spermatogenesis in the postnatal mutant tests is normal, and spermatogonial proliferation compensates for deficiencies in germ cell numbers. In Slpan and Slcon homozygous females, decreased Mgf mRNA expression causes sterility by affecting the initiation and maintenance of ovarian follicle development. Thus, regulated expression of Mgf is required for multiple stages of embryonic and postnatal germ cell development. Surprisingly, other areas of the Slcon female reproductive tract displayed ectopic expression of Mgf mRNA. We propose that the Slpan and Slcon rearrangements alter Mgf mRNA abundance through position effects on expression that act at a distance from the Sl gene. PMID- 7533740 TI - Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli. AB - The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by proteins extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by approximately 80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA. PMID- 7533741 TI - Analysis of DNA flanking the xlnB locus of Streptomyces lividans reveals genes encoding acetyl xylan esterase and the RNA component of ribonuclease P. AB - Nucleotide sequencing revealed the gene (axeA) encoding acetyl xylan esterase (AxeA) downstream from xlnB in the Streptomyces lividans DNA insert of plasmid pIAF42. AxeA consists of a catalytic- and a substrate-binding domain separated by a Gly-rich linker. The N terminus showed no significant homology with published esterases and acetyl xylan esterases, but some homology was found with the xylanases XylA and XylD and the NodB protein of Rhizobium species which is involved in the biosynthesis of root nodulation factors. The C terminus of AxeA is highly homologous to the C-termini of xylanases XlnB and TFXA, corresponding to the xylan-binding domain of these enzymes. Furthermore, the RNaseP RNA component was found immediately upstream from xlnB gene. PMID- 7533743 TI - The types of neurons in the neostriatum of bison bonasus. Nissl and Golgi study. AB - The caudate nucleus and the putamen of bison bonasus have been investigated. The Golgi procedure and Nissl stain had been used to reveal their cellular organization. In the Golgi preparation striatal neurons belong to the two fairly divergent main groups in which several types of cells can be distinguished. The first class, comprising the bulk of neuronal population, consists of only one type of efferent cells with the long axons. The spines cover dendrites except its proximal portion. The second one is composed of four types of interneurons which perikarya and dendritic processes are devoid of spines: aspiny neurons with tortuous dendrites, aspiny cells with dendritic swellings, large aspiny nerve cells, and long-axoned neurons with sparsely distributed but regularly branching dendritic processes. PMID- 7533742 TI - Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes. AB - The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes. PMID- 7533744 TI - Monoclonal antibodies against Ogawa specific & Ogawa-Inaba common antigenic determinants of Vibrio cholerae O1 & their diagnostic utility. AB - Monoclonal antibodies to Ogawa-Inaba common antigenic determinant and Ogawa specific antigenic determinant of V. cholerae belonging to the serogroup O1 were generated from BALB/c mice immunized with V. cholerae O1 Eltor Ogawa strain. Reactivity and specificities of the monoclonal antibodies were examined by slide agglutination method. The monoclonal antibodies agglutinated all the V. cholerae O1 strains tested but did not agglutinate with any of the other currently recognized 140 serogroups of V. cholerae non-O1 as well as with a variety of other enteric pathogens. Diagnostic utility of the MAbs produced in this study as compared to polyclonal O1 and monospecific antisera showed that the MAbs were as good as the latter for serological diagnosis of the two serotypes of V. cholerae O1. PMID- 7533745 TI - Umbilical cord blood as a source of CD34 positive haematopoietic stem cells. AB - In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood. PMID- 7533746 TI - Urinary red cell morphology to detect site of hematuria. AB - We studied the urinary RBC morphology in 87 consecutive cases of significant hematuria by 3 commonly used methods: (a) light microscopy of the unstained urinary sediment; (b) phase contrast microscopy of the unstained urinary sediment; and (c) Wright's staining of the urinary sediment, in order to compare the sensitivity of these methods in detecting dysmorphic RBCs and thus predicting the site of hematuria. The clinical data and the relevant investigations were made available after the morphology of RBCs in the urine was identified. Out of the 87 patients, 45 had a glomerular and 42 had a nonglomerular cause o hematuria. Phase contrast microscopy showed a sensitivity of 91.1%, Wright's stain of 82.2% and light microscopy of 66.7% in detecting a glomerular source of hematuria. Nonglomerular hematuria could be detected in 92.9% cases by each of the 3 methods. It is concluded that phase contrast microscopy is most sensitive for the detection of dysmorphic RBCs in the urine, Wright's stain nearly as sensitive whilst light microscopy of the unstained sediment is least sensitive. Urinary RBC morphology is a useful adjunct in the diagnosis of hematuria and saves the patients from unnecessary investigations. PMID- 7533747 TI - In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine. AB - A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C 8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent. PMID- 7533748 TI - Selection and the origin of information. PMID- 7533749 TI - [Chemotherapy in colorectal carcinoma]. PMID- 7533751 TI - Immunohistochemical localization of amylase in peri- and tele-insular acinar cells of the human exocrine pancreas. AB - An immunohistochemical localization of amylase was demonstrated in human pancreatic acinar cells using a commercial anti-human pancreatic amylase antibody. The immunofluorescence was mainly localized in the cell apices, and some differences in the intensity of the fluorescence was observed among the acinar cells in respect to their location from the islets of Langerhans. The peri insular acinar cells showed a brighter fluorescence than the cells of tele insular acini. This inhomogeneity of pancreatic amylase distribution in the human exocrine pancreas adds a further clue to the concept of insulo-acinar interaction. PMID- 7533750 TI - Low-dose radiotherapy for multicystic benign lymphoepithelial lesions of the parotid gland in HIV-positive patients: long-term results. AB - BACKGROUND: Multicystic benign lymphoepithelial lesions of the parotid gland (BLL) seen in patients with human immunodeficiency virus (HIV) can produce considerable cosmetic deformity as well as physical discomfort. We previously reported our preliminary results with low-dose radiotherapy in this disease, and all 8 patients were satisfied with the initial improvement in their appearance. We now report the long-term follow-up of those patients and additional patients. METHODS: Twelve HIV-positive patients with BLL were treated with 8-10 Gy of external radiation using 2-Gy daily fractions. Objective responses and subjective duration of patient-defined cosmetic control were recorded. RESULTS: All 12 patients (100%) had at least a 50% decrease in the size of their parotid masses. Five of 12 (42%) had a complete response and 7 (58%) had a partial response. Persisting complete response was achieved in only 1 patient, however, with relapse in the other 11 patients. Cosmetic palliation, as judged by the patients, was achieved for a median of 9.5 months. Eight patients were subsequently retreated with doses of 6-16 Gy (median and mode: 10 Gy). None (0%) of the 8 patients retreated achieved local control. CONCLUSIONS: Very low-dose radiation (8-10 Gy) provides reliable but temporary cosmetic palliation for BLL. Retreatment was unsatisfactory, and we are now investigating higher initial doses of radiation to prolong palliation and eliminate recurrences. PMID- 7533753 TI - Identification of the gene encoding a novel HLA-B39 subtype. Two amino acid substitutions on the beta-sheet out of the peptide-binding floor form a novel serological epitope. AB - Serological analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B*3904) and B*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B*3904 and B*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B*3901 to alanine in B*3904 whereas another difference at codon 12 changes valine in B*3901 to methionine in B*3904. The residues 11 and 12 are located on the beta sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B*3901 genes in the Japanese population showed that both B*39011 and B*39013 were observed in the Japanese population. The present study suggests that B*3904 may have evolved from B*39011 rather than B*39013. PMID- 7533754 TI - Rapid communication: SacI restriction fragment length polymorphism in a porcine vascular cellular adhesion molecule (VCAM1) gene. PMID- 7533755 TI - Effects of high molecular weight solutes on fluid flux across the arterial wall. AB - To investigate the mechanisms controlling the flux of plasma proteins into and through the walls of blood vessels, we have studied the effects of two inert protein analogues, Dextran 500 and Poly(ethylene)oxide (PEO) on fluid transport across the walls of intact rabbit common carotid arteries. Transmural fluxes were first measured in vessels pressurized to 150 cmH2O with a solution containing 10 mg/ml albumin alone (control solution) and then with one containing 10 mg/ml albumin plus 10 or 50 mg/ml dextran, or 10 or 30 mg/ml PEO (test solutions). The macromolecule solutions caused a decrease in transmural filtration; the ratios of fluxes with the test solutions to those with the control solutions were 0.89 +/- 0.11 (7), 0.63 +/- 0.08 (8), 0.69 +/- 0.24 (9) and 0.41 +/- 0.09 (4), respectively (Mean +/- SD (n)). These reductions in fluid movement through the vessel wall may be explained quantitatively in terms of the formation of concentration-polarized layers of the macromolecules at the luminal surface or interactions of the macromolecules with the endothelial glycocalyx, causing a decrease in its permeability. PMID- 7533752 TI - Human protein HC (alpha 1-microglobulin) and inter-alpha-trypsin inhibitor in connective tissue. AB - The presence of protein HC (alpha 1-microglobulin) and inter-alpha-trypsin inhibitor was investigated in different human tissues. Inter-alpha-trypsin inhibitor is a complex protein composed of bikunin and two heavy polypeptide chains. Protein HC and bikunin are transcribed from a common gene. Inter-alpha trypsin inhibitor immunoreactivity was detected in mast cells. The positive reaction could be blocked by antisera absorption with bikunin, indicating that mast cells contain only bikunin. Protein HC immunoreactivity was revealed on elastic fibres in connective tissue of skin, colon and lung, and on the internal elastic lamina of blood vessels. In the testis, the basement membrane of the seminiferous tubules reacted positively with protein HC antibodies. PMID- 7533756 TI - ACE inhibitor induced cough. PMID- 7533757 TI - The S-layer from Bacillus stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase. AB - The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase. PMID- 7533759 TI - Identification of the domain which determines the g,m serotype of the flagellin of Salmonella enteritidis. AB - Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed and screened with a g,m-specific monoclonal antibody. Results showed that the g,m epitope is localized between amino acids 258 and 348 of the flagellin. The fliC gene, encoding the flagellin of S. enteritidis, was proven to be the only flagellin gene present in S. enteritidis. PMID- 7533758 TI - Relationships between rfb gene clusters required for biosynthesis of identical D galactose-containing O antigens in Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16. AB - The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [- >3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; the resulting polymer is known as D galactan I. In K. pneumoniae serotype O1, the genes responsible for the synthesis of D-galactan I are found in the rfb gene cluster (rfbKpO1). We report here the cloning and analysis of the rfb cluster from S. marcescens serotype O16 (rfbSmO16). This is the first rfb gene cluster examined for the genus Serratia. Synthesis of D-galactan I is an rfe-dependent process for both K. pneumoniae serotype O1 and S. marcescens serotype O16. Hybridization experiments with probes derived from each of the six rfbKpO1 genes indicate that the cloned rfbSmO16 cluster contains homologous genes arranged in the same order. However, the degree of homology at the nucleotide sequence level was sufficiently low that hybridization was detected only under low-stringency conditions. rfbABSmO16 genes were subcloned and shown to encode an ABC-2 (ATP-binding cassette) transporter which is functionally identical to the one encoded by the corresponding rfb genes from K. pneumoniae serotype O1. The amino acid sequences of the predicted RfbA and RfbB homologs showed identities of 75.7% (87.9% total similarity) and 78.0% (86.5% total similarity), respectively. The last gene of the rfbKpO1 cluster, rfbFKpO1, encodes a bifunctional galactosyltransferase which initiates the formation of D-galactan I. RfbFKpO1 and RfbFSmO16 are 57.6% identical (with 71.1% total similarity), and both show similarity with RfpB, the galactosyltransferase involved in the synthesis of Shigella dysenteriae type I O-polysaccharide. The G+C contents of the rfbAB genes from each organism are quite similar, and values are lower than those typical for the species. However, the G+C content of rfbFSmO16 (47.6%) was much higher than that of rfbFKpO1 (37.3%), despite the fact that the average for each species (52 to 60%) falls within the same range. PMID- 7533760 TI - A gene encoding a putative membrane protein homologous to the major facilitator superfamily of transporters maps upstream of the beta-glycosidase gene in the archaeon Sulfolobus solfataricus. AB - We have identified a gene encoding a putative membrane protein homologous to the major facilitator superfamily, mapping upstream of the lacS gene in Sulfolobus solfataricus. Permeases from this family mediate secondary transport and are widely distributed among eubacteria and eukaryotes; the finding of an archaeal member suggests that this mechanism of transport evolved before the divergence of the three living domains. We also report a transcriptional mapping of the gene cluster. PMID- 7533762 TI - Epitopes and biological activities of two monoclonal antibodies to platelet integrin alpha IIb beta 3. AB - We have developed two monoclonal antibodies (MAbs), B6A3 and C4G1. The whole molecules of the two MAbs inhibited in vitro human platelet aggregation induced by either ADP, collagen or thrombin, and their F(ab')2 fragments inhibited ex vivo platelet aggregation induced by ADP in monkey. The concentrations necessary for complete inhibition were 5 and 1 microgram/ml for B6A3 and C4G1, respectively. The Fab fragment of C4G1 but not B6A3 inhibited platelet aggregation. B6A3 and C4G1 bound to activated platelets with dissociation constants of 0.25 and 0.82 nM, respectively. B6A3 recognized an epitope on beta 3, which was sensitive to reduction and alkylation of cystine residues, and C4G1 recognized a conformational epitope on the alpha IIb beta 3 complex, which was sensitive to EDTA. The binding of fibrinogen to activated platelets was inhibited by both MAbs. However, the binding of fibrinogen to isolated alpha IIb beta 3 was inhibited by the whole molecule of C4G1 but not B6A3, although both MAbs bound to the isolated alpha IIb beta 3. The binding of these MAbs to the isolated alpha IIb beta 3 was not inhibited by either Arg Gly Asp Ser (RGDS) or fibrinogen gamma peptide. In addition, B6A3 but not C4G1 bound to human endothelial cells. These MAbs should contribute to the elucidation of the mechanism of platelet aggregation. PMID- 7533761 TI - cDNA sequencing of mouse alpha 1-microglobulin/inter-alpha-trypsin inhibitor light chain and its expression in acute inflammation. AB - A cDNA encoding alpha 1-microglobulin (alpha 1mG)/inter-alpha-trypsin inhibitor light chain (ITI-LC) was cloned from mouse liver by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis of the cDNA showed that the basic molecular structure of the proprotein was similar to that in other animals, so that two mature proteins, alpha 1mG and ITI LC, could be produced from the proprotein translated from the mRNA. Since ITI-LC is known as a positive acute phase reactant and since ITI-LC is genetically identical with mast cell proteinase inhibitor, trypstatin, we examined the mRNA level in the liver of parasite-infected mice showing extensive mastocytosis. The mRNA level was, however, not significantly changed during inflammatory processes, except for a slight increase on day 8 post-infection. PMID- 7533764 TI - Tranilast, a selective inhibitor of collagen synthesis in human skin fibroblasts. AB - Effects of tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 microM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor beta 1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of pro alpha 1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF beta 1-mediated increase in pro alpha 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGF beta 1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug. PMID- 7533763 TI - Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor. AB - Ladsin is a large cell-adhesive protein with potent cell-scattering activity, which was recently identified in the culture of a malignant human gastric carcinoma cell line [Miyazaki, K. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11767-11771]. It is a heterotrimeric protein, containing a 140-kDa subunit similar or identical to the laminin B2t chain. Ladsin is similar to the keratinocyte-derived matrix proteins, "epiligrin" and "kalinin." In the present study, the cell-adhesion and cell-migration activities of ladsin were examined in comparison with those of three cell adhesion proteins, laminin, fibronectin, and vitronectin. Ladsin showed high cell-adhesion activity toward rat liver cell line BRL at concentrations 4-20-times lower than in the case of the other three proteins. In a monolayer culture, ladsin stimulated the migration of BRL cells about 2-times more strongly than the others, as compared at the minimal concentrations required for the maximal cell-adhesion activity. In Boyden chambers, ladsin stimulated both the chemotactic and chemokinetic migration of BRL cells. When the effect of anti-integrin antibodies on the adhesion of human fibrosarcoma cell line HT1080 was examined, the adhesion to ladsin was effectively inhibited by both the anti-integrin alpha 3 and beta 1 antibodies, but not the anti-integrin alpha 6 antibody, indicating that the primary receptor of ladsin is integrin alpha 3 beta 1. These results demonstrate that ladsin is a unique extracellular matrix component which may play a major role in cell migration. PMID- 7533766 TI - Specificity in recognition of phosphopeptides by src-homology 2 domains. AB - SH2 domains and SH3 domains, found in a number of protein-tyrosine kinases and substrates of protein-tyrosine kinases, provide specificity in downstream signaling. Both of these domains bind to relatively short linear sequences of peptides to provide specific interactions between proteins. The SH2 domains directly bind to phosphotyrosine residues of proteins in a specific sequence context. We have devised a phosphopeptide library technique that allows us to rapidly determine the sequence specificity of individual SH2 domains on the basis of amino acids selected at position +1, +2 and +3 C-terminal of the phosphotyrosine. The optimal motif for 22 distinct SH2 domains has been determined and used to predict likely sites of in vivo interaction. A second phosphopeptide library was devised in which the amino acids N-terminal of the phosphotyrosine were also varied. The residues N-terminal of phosphotyrosine had little influence on binding to the N-SH2 domain of the 85 kDa subunit of phosphoinositide 3-kinase. These results indicate that for this SH2 domain, specificity is determined by sequences carboxy-terminal of the phosphotyrosine moiety. Knowledge of the specificity of SH2 domains allows predictions about likely downstream targets on the basis of primary sequence of proteins. Some of these predictions will be discussed. PMID- 7533765 TI - DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells. AB - DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G2M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%; P < 0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis. PMID- 7533767 TI - Thyroid peroxidase autoantibody fingerprints. II. A longitudinal study in postpartum thyroiditis. AB - It is not known whether epitopes recognized by autoantibodies in an individual remain constant or change over time, especially during perturbations of the humoral immune response. To address this question, we studied the epitopic profile ("fingerprint") of autoantibodies to thyroid peroxidase (TPO) in the sera of 19 women during the postpartum period. Fingerprints were determined in competition studies using 4 recombinant F(ab). At delivery and at 3 time intervals over the subsequent 9-12 months, the pool of F(ab) inhibited autoantibody binding to TPO by 80-100%, consistent with the definition by these F(ab) of a TPO immunodominant region (A1, A2, B1, and B2 domains). Despite a wide spectrum among individuals, the TPO epitopic fingerprints for all 19 women were relatively unchanged throughout the postpartum period. Fingerprint constancy occurred regardless of fluctuations in serum TPO autoantibody levels. When assessed numerically as a ratio of inhibition by the A domain F(ab) to inhibition by the B domain F(ab), the A/B domain ratios in individual women ranged from 0.2 (predominantly B domain) to more than 3.0 (predominantly A domain). However, for each individual woman, the A/B epitopic ratio was conserved throughout the study interval. Our TPO autoantibody epitopic fingerprint data have potential implications for understanding the humoral autoimmune response in man. First, the present study indicates a remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr. Second, and perhaps more important, despite marked variations in TPO epitopic profiles among different individuals, their constancy over time suggests that TPO autoantibody fingerprints may be inherited. PMID- 7533768 TI - Expression of fibroblast growth factors in thyroid cancer. AB - We have examined immunoreactive fibroblast growth factor-1 (FGF-1) and FGF-2 in thyroid sections from normal tissue, follicular adenoma, differentiated follicular and papillary carcinoma, and anaplastic carcinoma. Polyclonal primary antibodies (Dr. A. Baird, Whittier Institute, La Jolla, CA) to FGF-1 and FGF-2 and fluorescein-conjugated secondary antibodies were used with confocal microscopy to allow quantitation and subcellular localization of the antigens. Staining for FGF-1 and FGF-2 was intense in the differentiated malignant tumor specimens, whereas staining in the normal thyroid tissue controls was not detectable above background fluorescence. Staining for FGF-1 and FGF-2 was intracellular and was not found in the nucleus. Staining using either antibody was enhanced in follicular adenomas, but was less intense than that in the malignant tumors. Sections from anaplastic carcinomas also stained positively. In primary cultures of thyroid cells derived from a papillary carcinoma, staining for FGF-2 was 10-fold greater than that from normal thyroid cells from the same patient. The data suggest a possible role for FGFs in the etiology of thyroid carcinoma. PMID- 7533769 TI - Alpha 2-macroglobulin production by the human endometrium. AB - alpha 2-Macroglobulin (A2M) is a broad spectrum plasma protease inhibitor previously described in uterine effluents and recently demonstrated to bind and possibly modulate the functions of cytokines. As cytokines, proteases, and protease inhibitors are important in implantation and endometrial physiology, we sought to investigate and characterize the pattern of production of A2M in the endometrium. Endometrial tissues from different phases of the menstrual cycle were analyzed for A2M production. Tissues were incubated in methionine-free Minimum Essential Medium with [35S]methionine for 12 h at 37 C in 5% CO2. Conditioned media were immunoprecipitated with a polyclonal antibody to human A2M. Recovered proteins were resolved under reducing and nonreducing conditions by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by autoradiography. Immunohistochemistry was performed on both cryostat sections of OCT-embedded tissue and formalin-fixed paraffin-embedded tissue. The intensity of staining was evaluated, and a histochemical score was assigned. Comparisons between histochemical scores were performed using the nonparametric Wilcoxon rank sum test. Immunoprecipitation with A2M antibody yielded a single 320-kilodalton protein band under nonreducing conditions and a single 182-kilodalton band under reducing conditions. Both physical and immunological properties of the recovered protein were consistent with A2M. A2M was identified in all endometrial samples and represented approximately 2% of the total radiolabeled proteins produced. Immunohistochemical analysis revealed prominent stromal, but no glandular, staining for A2M throughout the menstrual cycle. The stromal staining in secretory endometrial samples was significantly more intense than that in proliferative samples. In the proliferative phase, staining was more intense in the spongiosum and basalis layers, and less intense in the superficial compactum layer. In the secretory phase, it remained very intense in the spongiosum, but less so in the basalis layer. These findings indicate that A2M is predominantly produced by the stromal component of endometrial tissue. This production is menstrual cycle dependent, with zonal differences in A2M expression within the endometrium, which may indicate a functional significance relevant to the process of implantation that merits further investigation. PMID- 7533770 TI - Therapeutic controversies: clinical treatment of benign prostatic hyperplasia. PMID- 7533771 TI - Lack of stimulation of 24-hour growth hormone release by hypocaloric diet in obesity. AB - Obesity is associated with a marked reduction in the spontaneous secretion of GH. To investigate the effect of acute alterations in calorie intake on GH release, 24-hr spontaneous GH release was measured during habitual calorie intake as well as during a short term, very low calorie diet (VLCD) in 6 obese subjects, 5 obese subjects after weight loss, and 5 normal, age- and sex-matched control subjects. Integrated 20-min samples were obtained over 24-h on two occasions in each subject using a constant blood withdrawal technique. In addition, basal levels of serum insulin-like growth factor-I (IGF-I), IGF-binding protein-1 (IGFBP-1), IGF binding protein-3 (IGFBP-3), insulin, pro-insulin, and blood glucose were measured during habitual energy intake as well as during the hypocaloric diet. Twenty-four-hour GH release profiles and IGFBP-1 were decreased, and insulin as well as proinsulin levels were elevated in obese subjects compared to those in normal age- and sex-matched controls. No differences between obese subjects and normal controls were present regarding IGF-I, IGFBP-3, or IGF-I/IGFBP-3 molar ratio. In the last 24 h during the 96-h VLCD, an increase in 24-h GH release and basal IGFBP-1 levels and a decrease in basal insulin levels occurred in the normal controls, whereas no such changes were observed in the obese subjects. After caloric restriction 24-hr GH release, IGFBP-1 levels and insulin levels were similar in control subjects and obese subjects after weight loss. This suggests a reversible defect in GH release, rather than a persistent preexisting disorder. It is hypothesized that enhanced bioavailability of IGF-I, acting in concert with elevated proinsulin and insulin levels, may account for the lack of stimulation of 24-hr GH release by the hypocaloric diet in obese subjects. We conclude that the increase in 24-h spontaneous GH release and IGFBP-1 levels observed in normal subjects during the last 24 h of a 96-h VLCD is abolished in obese subjects. The lack of short term hypocaloric stimulation of spontaneous GH release may promote the retention of body fat and perpetuate the obese state. PMID- 7533772 TI - Recombinant human insulin-like growth factor-I enhances whole body protein anabolism and significantly diminishes the protein catabolic effects of prednisone in humans without a diabetogenic effect. AB - To investigate whether recombinant human insulin-like growth factor-I (rhIGF-I) could serve as a protein-sparing nondiabetogenic agent, 21 healthy volunteers (mean age, 25 +/- 1 yr) were studied in 3 similar clinical models: rhIGF-I alone, rhIGF-I and prednisone, and prednisone alone. In study A, 6 subjects received infusions of [14C]leucine and [2H2]glucose before and after 5-7 days of rhIGF-I (100 micrograms/kg, sc, twice daily, followed by 16 h of a 10 micrograms/kg.h continuous sc infusion). The rate of appearance (Ra) of leucine, an estimate of whole body proteolysis, did not change significantly, whereas leucine oxidation decreased (-31 +/- 4%; P = 0.001), hence the nonoxidative leucine disposal (NOLD) increased significantly (P = 0.04). These effects are similar to those reported with high dose hGH. Plasma glucose concentrations did not change despite a significant reduction in circulating insulin concentrations (-58 +/- 11%; P = 0.01) and an increase in the glucose Ra (+12 +/- 5%; P = 0.04). After rhIGF-I treatment, plasma IGF-I, IGF-binding protein-1 (IGFBP-1), and IGFBP-2 all increased significantly, whereas IGFBP-3 did not change. In study B, seven subjects received rhIGF-I combined with oral prednisone (0.8 mg/kg.day) for 5 days. Group C (n = 8) received only prednisone. In group B, both the leucine Ra and oxidation increased (Ra, +7 +/- 3%; oxidation, +45 +/- 13%), but this increase was significantly less than that seen in group C (Ra, +25 +/- 5%; oxidation, 117 +/- 17%; P < 0.005 vs. group B). Group B showed no significant changes in postabsorptive glucose concentrations despite marked reductions in circulating insulin levels, in contrast to the increase in insulin and glucose concentrations observed in group C. In conclusion, 100 micrograms/g rhIGF-I, given twice daily, 1) has GH-like effects on whole body protein metabolism, 2) markedly diminishes the protein catabolic effect of glucocorticosteroids, and 3) is nondiabetogenic in prednisone-treated humans. This agent offers promise in the treatment of protein catabolic states. PMID- 7533773 TI - T-cells recognize multiple epitopes in the human thyrotropin receptor extracellular domain. AB - In Graves' disease (GD), the TSH receptor (TSHR) is believed to be the major target of an autoimmune response. T-Lymphocytes regulate the immune system. To assess the interaction of T-cells with TSHR in the pathogenesis of GD, we tested the T-cell response of peripheral blood mononuclear cells and T-cell lines to the recombinant human TSHR extracellular domain (rhTSHR-ECD) and 31 synthetic peptides corresponding to the entire TSHR-ECD in 20 patients with GD, 8 patients with Hashimoto's thyroiditis, 7 with colloid nodular goiter (CNG), and 20 normal controls. Comparing patients from different groups with normal subjects, there was a significant response to rhTSHR-ECD and thyroglobulin in GD patients (P < 0.001) and HT patients (P < 0.05), but not in CNG patients (P > 0.1). All 20 patients with GD responded to at least one peptide. The reactivity in GD patients was heterogeneous and spanned the entire TSHR-ECD. However, the reactivity was significantly different from that in controls for peptide regions 44-88, 119-176, 227-263, and 343-376, and the stimulation index (SI) values were significantly different for peptides 272-291 and 301-320. Significant differences were confined to peptides 158-176 and 343-362 and the region 227-263 for comparison of the number of positive responses in patients and controls to individual peptides. Forty-six percent of human leukocyte antigen-DQA1 0501 allele-positive Graves' patients responded to peptides 158-176 and 248-263 (SI = 3 or more) compared to 14% of allele-negative patients. In HT and CNG patients, the response was mainly to peptides in the carboxy-terminal half of the TSHR-ECD. Concordance of the reactivity in T-cell lines and peripheral blood mononuclear cells was observed in 36% of direct comparisons in GD. Eighty-five percent and 90% of GD patients were positive for microsomal antibody and TSHR antibody, respectively, and 59% of microsomal antibody-positive and 67% of TSHR antibody-positive patients responded to rhTSHR-ECD (SI = 2 or more). However, there was no significant correlation between antibody-positive patients and reactivity to specific peptides. Using multiple criteria to define immunodominance, peptides 158-176, 237-252, 248-263, and 343-362 seem to be important epitopes and may be critical for T-cell triggering in GD. PMID- 7533774 TI - Serum insulin-like growth factor-binding protein-3 levels in the diagnosis of acromegaly. AB - Insulin-like growth factor-binding protein-3 (IGFBP-3) is a GH-dependent protein that binds insulin-like growth factor-I (IGF-I) in the circulation and modulates its action at the tissue level. Because IGFBP-3 is a stable and specific marker of somatotroph function, we hypothesized that it would be a useful biochemical marker in the diagnosis of patients with acromegaly and the assessment of surgical cure. We, therefore, investigated the sensitivity of serum IGFBP-3 levels to detect GH excess in 44 patients with clinical acromegaly and pathologically confirmed somatotroph adenomas, including a cohort of 18 patients with untreated disease evaluated before transsphenoidal surgery and medical therapy. IGFBP-3 levels were compared to IGF-I and random GH levels before and after transsphenoidal surgery. Concordance among IGFBP-3, IGF-I, and GH suppressibility by glucose was also determined. In addition, the response of IGFBP-3 to glucose suppression was investigated. All 18 patients with untreated acromegaly had elevated serum IGFBP-3 levels, ranging from 4,186-10,026 micrograms/L (mean +/- SD, 6566 [plusm] 1800 micrograms/L). There was no overlap with the age-adjusted normative ranges (P = 0.0001 in patients 18-55 yr old and P = 0.0176 in patients > 55 yr old) or with the levels obtained in age-comparable controls (P = 0.0001). In 11% of untreated patients with clinical findings of acromegaly and a pathologically confirmed adenoma, IG-FBP-3 levels were elevated, although GH was suppressed to less than 2 micrograms/L with glucose. In these patients, IGF-I levels were either normal or minimally elevated and considered nondiagnostic. IGFBP-3 and IGF-I levels were correlated in patients with untreated acromegaly (r = 0.650; P = 0.0162) and after transsphenoidal surgery (r = 0.644; P = 0.0001). Neither IGF-I nor IGFBP-3 correlated with random GH levels before surgery. However, both IGF-I (r = 0.471; P = 0.0001) and IGFBP-3 (r = 0.259; P = 0.041) correlated with random GH levels in patients studied more than 1 month after transsphenoidal surgery. IGFBP-3 and IGF-I levels were concordant with GH suppressibility by glucose (P = 0.0039) and IGFBP-3 decreased with glucose suppression in 7 of 10 patients. These data indicate that IGFBP-3 is a sensitive physiological marker of somatotroph function and is concordant with glucose suppression and IGF-I levels before and after transsphenoidal surgery.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533775 TI - Monoclonal thyroglobulin autoantibodies: variable region analysis and epitope recognition. AB - A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity. Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb. Three other monoclonals bound to a different site and were defined as type II TgAAb. Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD). Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera. Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used. Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies. No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes. Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different. TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level. PMID- 7533776 TI - Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells. AB - Insulin-like growth factor I (IGF-I) seems to play an important role in prostate cell growth and its actions may be modified by IGF-binding proteins (IGFBPs) secreted by prostate epithelial cells. The IGFBP system was studied in two human prostate carcinoma cell lines, PC3 and LNCaP. Androgen receptor-negative PC3 cells secrete IGFBP-3, IGFBP-4, and IGFBP-5, as determined by immunoprecipitation of the serum-free conditioned medium with specific IGFBP antibodies. Androgen receptor-positive LNCaP cells secrete IGFBP-2 and IGFBP-3. At neutral pH, there was little or no effect of a 24-h, 37 C cell-free incubation of PC3 and LNCaP conditioned media on IGFBP. On the other hand, when media was incubated at pH 3 for 24 h, [125I]IGFBP-3 hydrolysis and the virtual elimination of endogenous IGFBP detected by Western ligand blotting were observed. This loss was not due to the acid treatment, per se, since IGFBPs remained intact if the incubation at pH 3 was carried out at 4 C. The acid-activated IGFBP protease in LNCaP and PC3 cell conditioned media was identified as cathepsin D based on acidic pH optimum and immunoblotting. Furthermore, immunoadsorption of cathepsin D from the media attenuated the acid-activated IGFBP hydrolysis [125I]IGF-I binding to prostate cancer cells was reduced in the presence of LNCaP conditioned media that had been incubated at neutral pH for 24 h (i.e. containing intact IGFBP) but not by acid incubated conditioned media (i.e. cathepsin D-mediated hydrolyzed IGFBP). These data indicate that prostate carcinoma cells secrete specific IGFBPs, as well as a general IGFBP protease, cathepsin D. In the proper environment, cathepsin D is capable of hydrolyzing all endogenous IGFBP and, thus, modifying IGF-I action in prostatic cells. PMID- 7533777 TI - Thyroid peroxidase autoantibody fingerprints in hypothyroid and euthyroid individuals. I. Cross-sectional study in elderly women. AB - Human monoclonal immunoglobulin G-class autoantibodies to thyroid peroxidase (TPO), expressed as recombinant F(ab), are powerful tools for analyzing the individual components of polyclonal serum TPO autoantibodies. Four TPO-specific F(ab) interact with epitopes in two closely related domains (A and B) in the immunodominant region on TPO. In the present study, these TPO F(ab) were used to compete for serum autoantibody binding to [125I]TPO to determine the "epitopic fingerprints" in two groups of carefully controlled individuals. All individuals (14 hypothyroid and 32 euthyroid) were elderly women (60-71 yr old) with similar genetic and environmental backgrounds as well as comparable levels of serum TPO autoantibodies. Using the pool of four F(ab), serum TPO autoantibody binding was inhibited to the same extent (approximately 90%) in hypothyroid and euthyroid individuals, demonstrating that the majority of TPO autoantibodies in both groups recognize the TPO immunodominant domain. When tested individually, the F(ab) produced a spectrum of inhibition patterns, ranging from sera preferentially inhibited by domain A F(ab) to sera preferential inhibited by domain B F(ab). The ratio of inhibition by domain A F(ab) to inhibition by domain B F(ab) was similar in hypothyroid (0.11-1.39) and euthyroid (0.21-1.79) women. In conclusion, no difference in TPO autoantibody epitopes was observed in this cross-sectional study of hypothyroid and euthyroid individuals. Longitudinal studies are required to address the question of whether TPO autoantibody epitopic fingerprints are stable over time. PMID- 7533778 TI - rRNA gene restriction patterns of Streptococcus pyogenes: epidemiological applications and relation to serotypes. AB - The use of rRNA gene restriction fragment length polymorphism analysis (ribotyping) to subtype Streptococcus pyogenes strains was investigated. Sixty eight S. pyogenes strains, including 17 reference strains and 51 isolates from blood, acute or chronic pharyngitis, and food-borne outbreaks, were characterized by determination of both their rDNA restriction fragment length polymorphism profiles and their serotypes (T and M). Total DNA was cleaved with five selected restriction enzymes and then probed with a digoxigenin-labeled stretch of 1,063 bp hybridizing with 16S rRNA genes. Fifteen and nine distinct patterns were generated with SacI and XhoI, respectively, and five patterns were generated with each of the three additional restriction enzymes. With the combination SacI-XhoI, a total of 21 distinct ribotypes were obtained among the 68 isolates. This number was not increased by the results obtained with the other restriction enzymes. All strains tested were typeable. All isolates from each food-borne outbreak belonged to the same ribotype, and all isolates (pre- and posttreatment) from each child with chronic pharyngitis also belonged to the same ribotype, suggesting antibiotic treatment failures. A discriminatory index was calculated for the 47 isolates which were epidemiologically unrelated, using the Hunter-Gaston formula. This index reached 0.955 when the combination SacI-XhoI was used, showing the good discriminatory power of this typing method. Therefore, ribotyping proved to be a molecular method of interest to subtype S. pyogenes. Moreover, there was some correlation between ribotyping and serotyping, as several ribotypes were related to a unique distinct M serotype. PMID- 7533779 TI - Genetic diversity in human-derived Pneumocystis carinii isolates from four geographical locations shown by analysis of mitochondrial rRNA gene sequences. AB - The opportunistic fungal pathogen Pneumocystis carinii is a frequent cause of pneumonia in immunocompromised hosts. In this study, we have compared the DNA sequences of a portion of the mitochondrial large-subunit rRNA gene of P. carinii (an informative locus showing up to 27% differences among isolates of P. carinii from human-, rat-, mouse-, ferret-, rabbit-, and horse-infected lungs) obtained from human-derived isolates from widely disparate geographical areas, including Britain, the United States, Brazil, and Zimbabwe. A single-base polymorphism which varied among samples was identified. Apart from this nucleotide, the DNA sequences of all samples were identical. The sequences of the British samples were shown to be stable over a period of 4 years. These data suggest that there is relatively low genetic diversity among isolates of human-derived P. carinii from different global regions. PMID- 7533780 TI - Antigenic, morphologic, and molecular characterization of new Ehrlichia risticii isolates. AB - Ehrlichia risticii causes an acute infectious disease in horses called Potomac horse fever. To investigate the biological diversity of E. risticii organisms, nine E. risticii isolates derived from the peripheral blood monocytes of clinically sick horses in Ohio and Kentucky during the summers of 1991 and 1993 were compared with Illinois and Virginia isolates originally obtained from horses in Maryland in 1984. Seven of the nine isolates (081, 606, 380, 679, As, Co, and Ov) formed large morulae (tightly packed inclusions of ehrlichial organisms). The remaining isolates, including 1984 isolates, were individually dispersed or formed small morulae in the cytoplasm of P388D1 cells. In Western blot (immunoblot) analysis with four equine and one rabbit polyclonal anti-E. risticii sera, these recent E. risticii isolates showed patterns of antigenic proteins distinct from those of the 1984 isolates and could be divided into three groups: (i) 081; (ii) 606, 022, 067, 380, and 679; and (iii) As, Co, and Ov. By indirect fluorescent antibody labeling with two panels of murine anti-E. risticii (Illinois and Maryland isolates) monoclonal antibodies, isolate 081 was not labeled with any of 20 monoclonal antibodies tested. The remaining isolates were not labeled with several monoclonal antibodies. The digestion pattern with one of the restriction enzymes, AvaII, of the PCR-amplified partial 16S rRNA gene of E. risticii from all Kentucky isolates (As, Co, and Ov) was different from that of Illinois, Virginia, and six Ohio isolates. These results indicate the presence of distinct variants of E. risticii which vary significantly in morphology, antigenic composition, and the base sequence of the 16S rRNA gene. PMID- 7533781 TI - Heterogeneity of rRNA gene restriction patterns of multiresistant serotype 6B Streptococcus pneumoniae strains. AB - Three multiresistant serotype 6B Streptococcus pneumoniae strains were isolated from the middle ear fluids of children undergoing tympanostomy in Atlanta. Because multiresistant 6B pneumococci have been reported to spread from a single clone, the three isolates were compared with 13 other multiresistant 6B pneumococci by hybridization of endonuclease-restricted DNA fragments with a digoxigenin-labeled cDNA probe complementary to 16 and 23S rRNAs (ribotyping). The ear isolates were heterogeneous, whereas six of the other pneumococcal isolates were alike, indicating a need for additional studies to determine the possibility of clonal spread. PMID- 7533782 TI - Genotyping of Clostridium difficile isolates. PMID- 7533783 TI - Molecular basis of CD36 deficiency. Evidence that a 478C-->T substitution (proline90-->serine) in CD36 cDNA accounts for CD36 deficiency. AB - CD36 deficiency is divided into two subgroups: neither platelets nor monocytes express CD36 (type I deficiency), and monocytes express CD36 in spite of the lack of platelet CD36 (type II deficiency). We have already demonstrated that a 478C- >T substitution (proline90-->serine) in platelet CD36 cDNA predominates in type II deficiency (Kashiwagi, H., S. Honda, Y. Tomiyama, H. Mizutani, H. Take, Y. Honda, S. Kosugi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1993. Thromb. Haemostasis. 69:481-484). In this study, we revealed that monocyte CD36 cDNA from two type II deficient subjects was heterozygous for C478 and T478 form, while platelet CD36 cDNA of these subjects consisted of only T478 form. In a type I deficient subject, both platelet and monocyte CD36 cDNA showed only T478 form. Expression assay using C478 or T478 form of CD36 cDNA transfected cells revealed that there was an 81-kD precursor form of CD36, and that the maturation of the 81 kD precursor form to the 88-kD mature form of CD36 was markedly impaired by the substitution. The mutated precursor form of CD36 was subsequently degraded in the cytoplasm. These results indicate that the 478C-->T substitution directly leads to CD36 deficiency via defects in posttranslational modification, and that this substitution is the major defects underlying CD36 deficiency. PMID- 7533785 TI - Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM. AB - The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers. PMID- 7533784 TI - Cartilage and bone metabolism in rheumatoid arthritis. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism. AB - Serum concentrations of specific cartilage and bone molecules reflecting tissue turnover were measured in two well-defined patient groups with early rheumatoid arthritis with distinctly different disease outcome to see if early differences in their levels are prognostic of the rate of joint destruction. Compared with a matched normal population, increased concentrations of cartilage oligomeric matrix protein (COMP) were found in all patients who developed rapid hip joint destruction. In contrast, levels of a putative marker of cartilage aggrecan synthesis, the chondroitin sulfate epitope 846, were increased only in patients with slow joint destruction. Levels of bone sialoprotein (BSP) were increased in both groups, as were levels of the C-propeptide of type II procollagen (CPII), a marker of collagen II synthesis. The increased concentrations of the 846 epitope in patients with slow joint destruction suggest increased aggrecan synthesis. The low levels of the 846 epitope in patients with rapid joint destruction, concomitant with elevated levels of CPII, suggest a selective increase in collagen synthesis. The elevated BSP levels indicate an increased bone turnover in both groups. Thus elevated serum levels of COMP may indicate an unfavorable prognosis for rapid joint destruction, whereas elevated 846 epitope indicates a more favorable prognosis. PMID- 7533786 TI - Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility. AB - In liver injury, perisinusoidal cells known as lipocytes (Ito cells) undergo "activation," acquiring smooth muscle-like features and a contractile phenotype. To assess whether contraction of these cells is regulated by nitric oxide (NO), we examined the production of NO by lipocytes and the effect of NO on lipocyte contractility. Cultured lipocytes were exposed to cytokines and/or LPS. Single agents had little or no effect on the level of inducible NO synthase (iNOS) mRNA. However, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or LPS in combination with interferon-gamma (IFN-gamma) stimulated iNOS mRNA, which was present within 4 h after exposure. iNOS mRNA levels were paralleled by changes in nitrite (a metabolic product of NO). Intraperitoneal administration of IFN-gamma, TNF-alpha, and LPS led to rapid induction of iNOS mRNA in lipocytes, confirming in vivo the culture findings. Ligation of the common hepatic bile duct, which induces periportal-based liver injury, stimulated iNOS mRNA in lipocytes. Transforming growth factor-beta 1 decreased IFN-gamma/TNF-alpha- stimulated iNOS mRNA and nitrite. Finally, the effect of NO on lipocyte contractility was examined. In cells incubated with IFN-gamma and TNF-alpha, the contractile response to either serum or endothelin-1 was blocked. Contraction was restored entirely by an inhibitor of NO synthase, NG-monomethylarginine. Furthermore, 8-bromoguanosine 3':5'-cyclic monophosphate and sodium nitroprusside inhibited lipocyte contractility, consistent with the effect of NO induced by cytokines. We conclude that NO is a potent modulator of lipocyte contractility and may regulate this function by autocrine (or intracrine) mechanisms. Moreover, NO may play an important role in liver injury, countering the effect of contractile agonists on lipocytes. PMID- 7533787 TI - Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. AB - Early features in the pathogenesis of atherosclerosis include accumulation of oxidized LDL (oxLDL) and endothelial expression of the vascular adhesion molecule VCAM-1. Because antioxidants inhibit endothelial VCAM-1 expression, we tested the hypothesis that oxLDL functions as a prooxidant signal in atherogenesis to augment VCAM-1 activation by inflammatory signals. Cultured human aortic endothelial cells (HAECs) or human umbilical vein endothelial cells (HUVECs) were incubated with unmodified LDL, oxLDL, or glycated LDL for 48 h. No change in VCAM 1, intercellular cell adhesion molecule-1 (ICAM-1), or E-selectin expression from control was observed by ELISA. However, dose-response and time course studies demonstrated that oxLDL enhanced VCAM-1 expression induced by the cytokin tumor necrosis factor alpha (TNF alpha) 63% in HAECs and 45% in HUVECs over unmodified LDL or control. Using flow cytometry analysis, oxLDL augmented TNF alpha-induced VCAM-1 expression in a uniform HAEC population. oxLDL had no effect on E selection induction. oxLDL augmented TNF alpha-induced ICAM-1 expression 44% in HAECs but not in HUVECs. Glycated LDL augmented TNF alpha-induced VCAM-1 expression 35% in HAECs but not HUVECs. Similar results were obtained with 13 HPODE or lysophosphatidylcholine, significant components of oxLDL. 13-HPODE augmented TNF alpha-induced mRNA accumulation and transcriptional activation of VCAM-1 in HAECs. These results suggest that as long-term regulatory signals, specific oxidized fatty acid and phospholipid components of oxLDL augment the ability of vascular endothelial cells to express cytokine-mediated VCAM-1. These studies link oxidant signals conferred by oxLDL to oxidation-sensitive regulatory mechanisms controlling the expression of endothelial cell adhesion molecules involved in early atherosclerosis. PMID- 7533788 TI - Epstein-Barr virus-induced autoimmune responses. I. Immunoglobulin M autoantibodies to proteins mimicking and not mimicking Epstein-Barr virus nuclear antigen-1. AB - In previous studies of infectious mononucleosis, we found IgM autoantibodies which react with hematopoietic cell antigens. Many of these were inhibited by synthetic glycine/alanine peptides representing the glycine/alanine repeat of Epstein-Barr virus nuclear antigen-1. We have cloned and expressed fragments of genes encoding two of these autoantigens. One gene (p542) encodes a protein containing a glycine-rich 28-mer, which is its chief autoantigenic epitope and which represents a newly identified class of evolutionarily conserved autoepitopes. The other gene (p554) encodes a protein that is not demonstrably cross-reactive with Epstein-Barr virus nuclear antigen-1 or with any other EBV protein, but forms complexes with other proteins. Immunoaffinity-purified anti p542 and anti-p554 have relatively high binding affinities, as evidenced by inhibition at 10(6)-10(8) M-1, and neither autoantibody showed polyreactivity with other common antigens. The data thus suggest that neither autoantibody is simply an expression of polyclonal B cell activation. We conclude that the two autoantigens stimulate autoantibody synthesis by different mechanisms. One autoantigen shares homology to a viral protein which generates cross-reacting antibodies to the autoantigenic epitope. The other has no recognizable cross reaction with the infecting pathogen and may become immunogenic through complexing with other proteins. PMID- 7533789 TI - Epstein-Barr virus-induced autoimmune responses. II. Immunoglobulin G autoantibodies to mimicking and nonmimicking epitopes. Presence in autoimmune disease. AB - During infectious mononucleosis, IgM autoantibodies are generated to a protein, p542, which contains a glycine-rich 28-mer epitope cross-reactive with the Epstein-Barr nuclear antigen-1 through Epstein-Barr nuclear antigen-1's glycine/alanine repeat. In normal individuals it is uncommon to find IgG anti p542, but among patients with progressive systemic sclerosis, systemic lupus erythematosus, and ulcerative colitis high IgG anti-p542 (> 3 SD above the mean of normal 20-50 yr controls) occurred frequently. Lesser elevations occurred in Sjogren's syndrome, rheumatoid arthritis, ankylosing spondylitis, and Crohn's disease, but none with chronic hepatitis B infection. The reactive epitopes on p542 were mapped with deletion mutants, which indicated that the glycine-rich 28 mer was the major antigenic determinant, with lesser antibody responses to other epitopes. We conclude that normally there is an inability to generate IgG autoantibodies to the cross-reactive (mimicking) epitope of the p542 host protein, but that this inability is overcome in a proportion of patients with autoimmune disease. We conclude also that non-cross-reactive autoepitopes exist on p542 protein, to which IgG autoantibodies can commonly be formed in autoimmune disorders. The mechanisms responsible for the latter must involve different mechanisms than those responsible for autoantibodies to the mimicking epitope. PMID- 7533790 TI - Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. AB - Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/ 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ ( 65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+) mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis. PMID- 7533791 TI - Increased expression of mitochondrial-encoded genes in skeletal muscle of humans with diabetes mellitus. AB - Screening subtraction libraries from normal and type II diabetic human skeletal muscle, we identified four different mitochondrially encoded genes which were increased in expression in diabetes. The genes were cytochrome oxidase I, cytochrome oxidase III, NADH dehydrogenase IV, and 12s rRNA, all of which are located on the heavy strand of the mitochondrial genome. There was a 1.5- to 2.2 fold increase in the expression of these mRNA molecules relative to total RNA in both type I and type II diabetes as assessed by Northern blot analyses. Since there was approximately 50% decrease in mitochondrial DNA copy number as estimated by Southern blot analyses, mitochondrial gene expression increased approximately 2.5-fold when expressed relative to mitochondrial DNA copy number. For cytochrome oxidase I similar changes in mitochondrial gene expression were observed in muscle of nonobese diabetic and ob/ob mice, models of type I and type II diabetes, respectively. By contrast there was no change or a slight decrease in expression of cytochrome oxidase 7a, a nuclear-encoded subunit of cytochrome oxidase, and the expression of mitochondrial transcription factor 1 in human skeletal muscle did not change with type I or type II diabetes. The increased mitochondrial gene expression may contribute to the increase in mitochondrial respiration observed in uncontrolled diabetes. PMID- 7533794 TI - Schizophrenia and severe tardive dyskinesia responsive to risperidone. PMID- 7533792 TI - Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome. AB - Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers. PMID- 7533793 TI - Human alveolar macrophages present antigen ineffectively due to defective expression of B7 costimulatory cell surface molecules. AB - Alveolar macrophages, resident phagocytic cells in the lung that derive from peripheral blood monocytes, are paradoxically ineffective in presenting antigen to T cells. We found that antigen presentation by alveolar macrophages could be restored by the addition of anti-CD28 mAb to cultures of T cells and macrophages, indicating that costimulation by alveolar macrophages via the CD28 pathway was defective. In addition, we found that alveolar macrophages activated with IFN gamma failed to express B7-1 or B7-2 antigens, which normally ligate CD28 on T cells and provide a costimulatory signal required for the activation of T cells. These observations are the first to demonstrate the inability of a "professional" antigen-presenting cell type to effectively express the costimulatory molecules B7-1 and B7-2. Inasmuch as immune reactions within the lung are inevitably associated with inflammatory injury to pulmonary tissue, these observations suggest that reduced expression of B7-1 and B7-2 by alveolar macrophages may be advantageous, as a critical mechanism involved in the induction of peripheral tolerance to the abundance of antigens to which mucosal tissues are continuously exposed. PMID- 7533795 TI - Acetylcholinesterase activity in neurons of crayfish abdominal ganglia. AB - Acetylcholine is known to be a neurotransmitter in crustacean central nervous systems, but the numbers and distribution of cholinergic neurons in the segmental ganglia have not been described. To begin a census of cholinergic neurons in these ganglia, we used a histochemical assay for acetylcholinesterase to map neurons that contained this enzyme in the six abdominal ganglia of crayfish. In each abdominal ganglion, about 47 cell bodies were stained. The distributions of these stained cells in individual ganglia were similar, and the numbers were not significantly different. None of these stained cell bodies could be identified from their structures or locations as previously identified motor neurons or sensory neurons with central cell bodies. The process of one unpaired midline neuron that occurred only in the first three abdominal ganglia divided to send a pair of axons anteriorly into both halves of the connective. The central projections of afferent axons from many peripheral sensory neurons stained clearly as they entered each ganglion. Terminals of these axons were heavily stained in the horseshoe neuropil and the lateral neuropils. We labeled both gamma-aminobutyric acid (GABA) and acetylcholinesterase in individual ganglia. Only a few neurons in each ganglion were double-labeled. The unpaired midline neurons in the three anterior ganglia that stained for acetylcholinesterase did not show GABA-like immunoreactivity, but cells with similar shapes did label with the GABA antiserum. Acetylcholinesterase is not a definitive marker of cholinergic neurons, but its presence is often associated with the cholinergic phenotype. These stained cells should be considered as putative cholinergic neurons. PMID- 7533796 TI - Primate cingulostriatal projection: limbic striatal versus sensorimotor striatal input. AB - The organization of the projections from the cingulate cortex to the striatum in the monkey was studied using the retrograde tracers Lucifer Yellow conjugated to dextran amines and horseradish peroxidase conjugated to wheat germ agglutinin. These tracers were injected into the different regions of the ventral (limbic) striatum and the dorsal (sensorimotor) striatum. The shell region of the nucleus accumbens was defined using calbindin-D28K immunohistochemistry. Following injections into the ventral striatum, there are numerous retrogradely labeled neurons in the various regions of the primate cingulate cortex, most of which are derived from layer V. The cytoarchitectural subdivisions of cingulate cortex include the anterior cingulate cortex, areas 25, 24a-c, and 24a'-c', and the posterior cingulate cortex, areas 23a-c, 29, 30, and 31. There is a topographical organization of the projections from these different cingulate areas to the ventral and dorsal striatum. The medial ventral striatum receives input from the rostral part of the anterior cingulate cortex (areas 25 and 24a,b). The shell region of the nucleus accumbens receives fibers from areas 25, 24a,b, and 24a',b'. Projections to the central ventral striatum including the core of the nucleus accumbens are derived primarily from areas 25, 24a, 24b, and the medial part of area 24c. However, few labeled cells are detected in areas 24c and 24c'. The lateral ventral striatum receives input primarily from areas 24b, 24b' and 23b and medial portion of area 24c. The medial ventral striatum and the shell of the nucleus accumbens have a similar distribution of labeled cells, such that these regions derive their input almost entirely from the rostral anterior cingulate cortex. In contrast to the ventral striatum, the dorsal sensorimotor striatum receives projections from areas 24c, 24c' 23c and 31. These arise primarily from the lateral portion of lower bank and the fundus of the cingulate sulcus. Our results demonstrate that areas 24c, 24c' and 23c, the lateral portion of the lower bank and the fundus of the cingulate sulcus project to the dorsal sensorimotor striatum. The medial portion of the lower bank of the cingulate cortex projects to the ventral striatum including the core of the nucleus accumbens. Different projections to striatum from discrete subdivisions of cingulate cortex indicate that these areas are heterogeneous and have different functions such that the fundus of the cingulate sulcus is related to skeletomotor function, whereas the medial portion of the lower bank of the cingulate sulcus is associated with the limbic-related and association cortical function.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533797 TI - Serotoninergic and nonserotoninergic neurons in the medullary raphe system have axon collateral projections to autonomic and somatic cell groups in the medulla and spinal cord. AB - Fluorescent double retrograde-tracing studies combined with fluorescent immunostaining for serotonin were carried out to determine the potential patterns of divergence in axonal projections to autonomic and somatic motor sites from medullary raphe and parapyramidal neurons. Injections (20-60 nl) of combinations of fluorescent retrograde tracers (Fast Blue, fluoro-gold, green latex microspheres, Diamidino Yellow) were made into the intermediolateral cell column (IML) of the spinal cord and the brainstem lateral tegmental field or ventral horn of the lumbar spinal cord of male Wistar rats. The animals were perfused after a 7-10-day survival period, and the brains were removed, sectioned (50 microns), and immunostained for serotonin. Following injections of different retrograde-tracer substances into the IML of the thoracic cord and the ventral horn of the lumbar cord, 36% of the neurons with axon collateral projections to the IML and the lumbar ventral horn were serotoninergic. Following injections of different retrograde-tracer substances into the IML and the lateral tegmental field, 26% of the neurons with axon collateral projections to the IML and the lateral tegmental field were serotoninergic. Many of the medullary neurons with projections to the lateral tegmental field and the lumbar cord were located dorsal and lateral to those neurons with projections to the IML. The results indicate that serotoninergic and nonserotoninergic neurons of the midline raphe system and parapyramidal region have axon collateral branches to the IML and the lateral tegmental field or the IML and the lumbar ventral horn. These projection neurons may form the anatomical substrate for the integration of autonomic and somatic motor activity. PMID- 7533798 TI - Evidence for survival of the central arbors of trigeminal primary afferents after peripheral neonatal axotomy: experiments with galanin immunocytochemistry and Di I labelling. AB - Studies employing axoplasmic transport techniques have suggested that the central arbors of vibrissae-related primary afferents are rapidly and permanently lost from the trigeminal (V) brainstem complex after transection of the intraorbital nerve (ION). The present study reexamined this issue using immunocytochemistry for galanin (GAL) and anterograde labelling with Di-I to evaluate V brainstem organization in rats that sustained damage to the ION or individual vibrissae follicles in infancy or adulthood. After adult nerve damage, GAL-positive fibers are increased in layers I and II of V subnucleus caudalis (SpC). This was apparent by 3 days after the lesion. In rats that sustained nerve damage at birth (P0), GAL immunoreactivity (IR) appeared throughout the V brainstem complex and had a patchy distribution similar to that of vibrissae-related V primary afferents in normal rats. Increased GAL-IR in rostral portions of the V brainstem complex was observed in rats that sustained ION damage as late as P14. Additional experiments in which nerve damage was followed by destruction of the V ganglion demonstrated that this GAL-IR was contained in primary afferents. Damage to single vibrissa follicles or to a row of follicles produced a single patch or row of GAL-IR terminals in the somatotopically appropriate portion of the ipsilateral V brainstem complex. Di-I labelling in neonatally nerve-damaged rats demonstrated that primary afferent axons filled the central territory normally innervated by this nerve and that their terminal distribution was patchy. These results suggest that the V ganglion cells that survive neonatal axotomy may retain somatotopically organized projections to the V brainstem complex for at least a limited postnatal period. PMID- 7533799 TI - Pyramidal and nonpyramidal callosal cells in the striate cortex of the adult rat. AB - The aim of this study has been to determine the neuronal types (pyramidal and nonpyramidal) within the rat's visual cortex, which project through the corpus callosum. To this end, the morphology and laminar distribution of callosal cells have been investigated by combining Diamidino Yellow retrograde tracing with intracellular injection of Lucifer Yellow in slightly fixed tissue slices. The visual callosal projection arises from pyramidal cells of diverse morphology in layers II to VIb, as well as from several modified pyramids located mainly in layers II, IV (star pyramids) and VIb (horizontal or inverted pyramids and related forms of spiny stellate cells). Our results indicate that in rats, as in other mammals, several types of nonpyramidal neurons also contribute to the contralateral projection. Bitufted cells in layers II-III and V were found to project contralaterally. Moreover, a spine-free layer V cell and a sparsely spiny multipolar neuron of layer IV were also labeled. In both stellate cells, partial axonal labeling reveals that these callosal cells display a local axonal arborization. Finally, our results of retrograde transport with Diamidino Yellow and with another sensitive retrograde tracer, the beta subunit of the cholera toxin, demonstrate for the first time that the two main neuronal types of layer I participate in the callosal projection. In layer I, several small horizontal cells of the inner half of layer I and a large subpial cell displaying long radiating dendrites were also injected. The latter cell may correspond to the Cajal-Retzius cell of the adult rat. In spite of the important differences in the organization of the visual system between rodents and cats, the callosal projection in both mammals is composed of a large variety of pyramidal cells and several nonpyramidal neurons. This high morphological diversity suggests that the callosal projection is much more physiologically complex than the extracortical efferents of the visual cortex, resembling other cortico-cortical connections. The roles that the different callosal cells may play in the processing of visual information are discussed in relation to the known functions of the corpus callosum. PMID- 7533800 TI - Morphological evidence for the presence of ipsilateral inferior olivary neurons during postnatal development of the olivocerebellar projection in the rat. AB - The presence of ipsilateral inferior olivary neurons during postnatal development of the olivocerebellar projection in the rat was investigated by two in vitro axonal tracing methods and by the axotomy of one olivocerebellar tract. The experiments were carried out before (P1), during (P5-P10) and after (P20) the period of multiple innervation of Purkinje cells by climbing fibers. According to present results: (1) ipsilateral inferior olivary neurons are distributed, on all analyzed days, throughout the entire inferior olive; (2) cell counts after axotomy experiments demonstrated that they represent a small population of inferior olivary neurons, whose number oscillated between 271 +/- 30 in young animals (pedunculotomized at P1 and killed at P7) and 26 +/- 12 in older ones (pedunculotomized at P20 and killed at P40). This experiment confirmed that most of these neurons are eliminated during the regressive events that take place during normal development of the olivocerebellar projection; and (3) few ipsilateral inferior olivary neurons, however, survive at P40, but their significance is still unclear. PMID- 7533801 TI - Immunohistochemical distribution pattern of intermediate filament proteins and muscle actin in feline and human mammary carcinomas. AB - Thirty-seven feline and 38 human spontaneous mammary gland carcinomas were studied immunohistochemically. Commercially available antibodies directed against high and low molecular weight keratins (RCK-102 and NCL-5D3), vimentin, desmin, glial fibrillary acidic protein (GFAP), neurofilament (NF) proteins and muscle actin (HHF35) were used in the avidin biotin peroxidase complex (ABC) technique on formalin-fixed paraffin wax-embedded tumour tissue samples. Healthy feline and human mammary gland tissue adjacent to the neoplasms was also examined. The distribution pattern of intermediate filament proteins and muscle actin was comparable in healthy mammary gland tissue of the two species: both RCK-102 and NCL-5D3 antibodies reacted with luminal epithelial cells of ducts and acini, but basal/myoepithelial cells were stained by RCK-102 exclusively. In addition, basal/myoepithelial cells expressed vimentin and muscle actin in both species, and GFAP was found in some feline basal/myoepithelial cells. No immunoreactivity to desmin and NF proteins was observed. Feline mammary gland carcinoma cells reacted with RCK-102 (89%), NCL-5D3 (62%), vimentin (76%) and GFAP (30%) antibodies, while human mammary gland carcinoma cells reacted with RCK-102 (95%), NCL-5D3 (100%) and vimentin (13%) antibodies. HHF35 immunoreactivity was observed in stromal cells only. These results indicate that mammary gland carcinomas of both species share a heterogeneous immunophenotype with respect to intermediate filament proteins, which adds to the list of known similarities between mammary gland carcinomas of both species. PMID- 7533802 TI - Intrathecal capsaicin enhances one-kidney renal wrap hypertension in the rat. AB - Afferent renal nerves (ARN) have been implicated in the development of one-kidney renal wrap (1K-WRAP) hypertension. The role of renal nerves in desoxycorticosterone acetate-salt (DOCA) hypertension, a low-renin model of hypertension, is controversial. The present study was designed to determine if spinal substance P (SP) and/or calcitonin gene-related peptide (CGRP) in ARN affects the development of 1K-WRAP or DOCA hypertension in adult rats. Selective long-term partial depletion of spinal SP and CGRP within small primary afferent nerve fibers including unmyelinated ARN was achieved by intrathecal administration of capsaicin. After capsaicin treatment, 1K-WRAP hypertension was induced by removing the right kidney and wrapping the left kidney with a figure-8 ligature. In a second group of rats, DOCA hypertension was induced by subcutaneous application of desoxycorticosterone pellets after unilateral nephrectomy. Systolic arterial pressure was monitored for 8 weeks by tail cuff plethysmography after which direct blood pressure measurement was performed followed by immunohistochemistry. Intrathecal capsaicin administration had no significant effect on SP-ir and CGRP-ir of ARN soma located within thoracic dorsal root ganglia whereas immunoreactivity against these peptides was reduced by one third to one half in the dorsal horn, indicating effective long-term spinal depletion of these neuropeptides. Intrathecal capsaicin enhanced the development of 1K-WRAP hypertension, since arterial pressure was greater in the treated group. In contrast, DOCA hypertension was unaffected by capsaicin pretreatment. Considering the neurotoxic action of capsaicin for SP-ir and CGRP ir unmyelinated primary afferent neurons, we hypothesize that spinal SP, CGRP and/or related peptides existing in ARN and other capsaicin-sensitive unmyelinated primary afferent neurons in the lower thoracic spinal cord may ameliorate 1K-WRAP hypertension, but not DOCA hypertension. PMID- 7533803 TI - Role of nitric oxide in regulation of baroreceptor reflex. AB - The possible role of nitric oxide (NO) on modulating sympathetic nerve activity through its action on baroreceptor reflex arc was investigated. L-Arginine, a precursor of NO, and NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase, were separately infused intravenously in increasing doses in 126 pentobarbital-anesthetized rabbits. Mean arterial pressure (MAP), heart rate (HR), aortic nerve activity (ANA), cervical (CSNA) and renal sympathetic nerve activities (RSNA) were recorded. L-Arginine infusion decreased MAP (P < 0.05), ANA (P < 0.05), CSNA (P < 0.05) and RSNA (P < 0.05) without changes in HR. Infusion of D-arginine, an enantiomer of L-arginine, and simultaneous infusion of L-arginine and L-NMMA, did not elicit such changes. L-NMMA infusion increased MAP (P < 0.05) and ANA (P < 0.05) and decreased HR (P < 0.05), while it tended to increase CSNA and RSNA without significance. Infusion of L-arginine or L-NMMA did not alter the slope of ANA, CSNA, RSNA, or HR in relation to MAP. These results suggest that NO modulates efferent sympathetic nerve activity, not by altering the afferent or efferent limbs of the baroreceptor reflex arc, but by interacting with the sympathetic pathway in the central nervous system. PMID- 7533805 TI - Antineutrophil cytoplasmic antibodies in IBD are the same in Ashkenazi and Sepharadi Jews in Israel. PMID- 7533804 TI - Nitric oxide formation is involved in vagal inhibition of the stomach of the trout (Salmo gairdneri). AB - Stimulation of the vagus nerves to isolated stomach preparations from trout (Salmo gairdneri) caused both excitation and inhibition of smooth muscle activity. Excitatory responses were partly reduced by atropine, suggesting that cholinergic neurons are involved. The atropine-resistant excitation could not readily be ascribed to a 'rebound' from preceding inhibition, to the production of prostaglandins, or to the activation of either adrenergic or capsaicin sensitive fibres. Inhibitory responses were reduced by the nicotinic antagonist d tubocurarine and more profoundly reduced by an inhibitor of nitric oxide synthase, NG-nitro-L-arginine. The latter effect was reversed by excess L arginine. The nitric oxide donors sodium nitroprusside and nitroglycerine caused inhibition of the muscle. It is concluded that the vagal inhibitory postganglionic neurons are nitergic in this tissue. PMID- 7533807 TI - Electric heating pad burns. AB - Patients with sensory deficits are especially prone to heating pad burns. Two cases are reported of patients with anesthetic skin who received partial and full thickness burns of their feet from an electric heating pad. These burn injuries could have been prevented if the patients understood the potential hazard of heating pads. PMID- 7533806 TI - Airway compromise and delayed death following attempted central vein injection of propylhexedrine. AB - Propylhexedrine is a potent alpha-adrenergic drug available as a nasal decongestant, which drug abusers sometimes extract and inject into a central vein. A 25-year-old white male presented to a local emergency department 32 h after attempting to inject his right internal jugular vein with "home-made crank." Following injection, he noted right neck pain, followed by fever and chills. On emergency department admission, he had inspiratory stridor and respiratory distress. Massive edema of his right neck extended from his anterior chest to the right parotid. Neck radiographs showed extensive paracervical swelling with displacement of the trachea. The patient was taken to surgery for nasotracheal intubation with fiberoptic guidance and surgical exploration. The neck contained extensive necrotic tissue that was surgically debrided. In spite of treatment with antibiotics, he developed progressive renal failure and hypotension unresponsive to fluid therapy, followed by cardiopulmonary arrest and death. PMID- 7533808 TI - Hypothalamic projections to the spinal segments and preganglionic regions by means of choleratoxin subunit B conjugated HRP in the chicken. AB - Hypothalamo-spinal projections were studied using the method of retrograde axonal transport of choleratoxin subunit B conjugated to horseradish peroxidase (CTb HRP) in chickens. CTb-HRP was injected into the segmental and preganglionic regions of spinal segments 23-24 and segments 31-32 which are sympathetically and parasympathetically connected with the cloaca, respectively. Many labeled SP neurons (segmental projection neurons) were found in the paraventricular nucleus (PVN), except in its lateral protrusion. The distribution of these labeled neurons continued to the more caudal areas such as the vicinity of the nucleus periventricularis hypothalami (PHN), areas medial to the stratum cellulare externum (SCE), the nucleus interstitialis (IS), and the posterior hypothalamic areas. Some labeled SP-neurons were also found in the lateral hypothalamic area. These labeled SP-neurons were distributed in almost the same areas in both cases of injection into segments 23-24 and 31-32. However, the number of labeled neurons was somewhat different rostrocaudally according to the spinal levels injected. In contrast, after microinjections into the spinal preganglionic regions, a small number of PP-neurons (preganglionic projection neurons) were found only in the ventral part of the PVN. These PP-neurons showed the rostrocaudally discernible segregation; PP-neurons in the rostral PVN and those in the caudal PVN were predominantly labeled by microinjections into segments 23 24 and segments 31-32, respectively. The present study suggests that the rostral PVN is more sympathetic and the caudal PVN is more parasympathetic in chickens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533809 TI - Localization of NADPH-diaphorase/nitric oxide synthase activity in the rat cerebellar cortex: a light and electron microscopical study. AB - The activity of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined histochemically in the rat cerebellar cortex at the light and electron microscopical level. Different staining patterns were observed depending on the technique used. Electron dense reaction product was seen on distinct membrane portions of the endoplasmic reticulum including the nuclear envelope in most of the neurons and in endothelial cells. Electron microscopically no activity staining was seen in glial cells, including Bergmann cells. The addition of the detergent Triton X-100, usually applied in the light microscopical diaphorase histochemistry, led to a striking diminution in membrane staining. In such preparations inspected electron microscopically formazan formed small granules which were evenly distributed over the cytoplasm. PMID- 7533810 TI - Localization of NADPH-diaphorase positive neurons in the pancreas of the mouse, rat, chick, kitten and monkey. AB - In the present study, nicotinamide adenine dinucleotide hydrogen phosphate diaphorase (NADPH-d) histochemistry has been used as a marker for nitric oxide synthase. NADPH-d positive neuronal cell bodies and fibres have been localized in the pancreas of the mouse, rat, chick, kitten and monkey. In all these species, most of the NADPH-d labelled neuronal cell bodies in the ganglia were found in the interlobular and interacinar connective tissue while some were intimately associated with pancreatic ducts and blood vessels. There was, however, a gradation of innervation amongst the species. Only in the pancreatic islets of the mouse and monkey were NADPH-d positive neuronal cell bodies and nerve fibres detected in the formation of neuro-insular complexes. Besides labelling of neuronal cell bodies, nerve fibres and endothelial cells, the islet cells of the mouse, kitten and chick pancreas also gave a light positive reaction for NADPH-d activity. The nerve fibre and the outer core of the connective tissue of the Pacinian corpuscles in the kitten pancreas were also labelled for NADPH-d activity. It is concluded that nitric oxide may play an important role in the neural regulation of both exocrine and endocrine secretion and in controlling the activity of the blood vessels and ducts of the pancreas. PMID- 7533811 TI - Projections from the cerebral cortex to the accessory oculomotor nuclei of the rat: a neuroanatomical and immunohistochemical study. AB - The projections from the cerebral cortex to the accessory oculomotor nuclei, i.e. the nucleus of posterior commissure (NPC), the nucleus of Darkschewitsch (NDK) and the interstitial nucleus of Cajal (INC) and the putative neurotransmitters subserving this pathway have been studied in adult rats. Retrograde labeling with Fluoro-Gold showed that only the more medial part of the agranular cortex, which is considered the rat's analogous to the frontal eye fields of the monkey, sends axon to the ipsilateral accessory oculomotor nuclei. The retrogradely-labeled cells were located primarily in the fifth layer of this cortical region. Following an injection of horseradish peroxidase conjugated with wheat germ agglutinin in this cortical area, we observed corticofugal labeled fibers reaching the accessory oculomotor nuclei and terminating as a fine dust-like terminal labeling in the NDK, in the dorso-lateral division of the INC and in the NPC, as well as in the medial oculomotor accessory nucleus, the red nucleus, the superior colliculus and, even though to a lesser extent, in the mesencephalic reticular formation and the central gray. With regard to the immunohistochemical approach, we observed that all the cells retrogradely labeled from the accessory oculomotor area were also stained by using glutamate or aspartate antisera. PMID- 7533812 TI - Motoneuronal location of external laryngeal and hyoid muscles involved in primate phonation. AB - In seven squirrel monkeys (Saimiri sciureus), horseradish-peroxidase injections were made into a number of extralaryngeal muscles shown to be involved in vocal control. Retrogradely labeled motoneurones were found in the case of the strap muscles from a position ventrolateral to the caudal hypoglossal nucleus down into the ventral horn of C2. The thyrohoid muscle was represented most rostrally, the sternothyroid most caudally; the sternohyoid took an intermediate position. There was partial overlap between sternothyroid and sternohyoid motoneurones. The thyrohyoid motoneurones were located not only rostrally but also laterally to the sternohyoid motoneurones. Omohyoid motoneurones overlapped with those of the sternohyoid and sternothyroid in the caudal medulla; in the cervical cord, they were located laterally to them. Inferior pharyngeal constrictor motoneurones were found in the nucl. retrofacialis and rostral nucl. ambiguus. Mylohyoid and anterior digastric motoneurones were restricted to the medialmost part of the trigeminal motor nucleus, with the mylohyoid being represented ventrally to the anterior digastric. PMID- 7533814 TI - [Report of a severely deformed and calcified equine pericardial patch using for intracardiac repair of tetralogy of Fallot]. AB - An 11-year-old boy with tetralogy of Fallot had undergone palliative right ventricular outflow tract reconstruction twice previously. After the second palliative surgery recurrent mediastinitis persisted. At the age of 5 years and 11 months, he underwent closure of VSD and reconstruction of the right ventricular outflow tract with an equine pericardial patch after removal of the infected prosthetic material. The postoperative course was uneventful, however, severe pulmonary stenosis developed 6 years later. The stenosis was caused by the severely deformed and calcified equine pericardial patch used for intracardiac repair. The reason for severe calcification seemed to be the accentuated calcium metabolism in children, hemodynamic stress and persistent infection. PMID- 7533815 TI - Haematopoietic growth factors in the treatment of therapeutic and accidental irradiation-induced bone marrow aplasia. AB - Bone marrow aplasia is one of the main syndromes following a high dose accidental exposure of ionizing radiation. Although both transfusion and bone marrow transplantation have been used with some success since the first treatments of patients, other therapeutic strategies are needed. The strategies involving haematopoietic growth factors for the treatment of radiation victims have been explored in vivo mainly in animal models and it is hoped that new therapeutic regimens will be elucidated from such approaches. The growth factors stimulate proliferation and/or differentiation of haematopoietic progenitor cells and possible stem cells. Furthermore, they act on the functions of mature cells. They now have specific uses in haematology, related to their role in the regulation of growth and differentiation of haematopoietic progenitor cells. The results of the clinical trials, performed with numerous patients and often randomized bring important clues about what to expect from growth factor therapy. Other factors are only entering the preclinical or clinical trials now. Although numerous in vitro or in vivo experiments suggest a benefit from their effects, their possible uses in therapy are still questionable. Some growth factors have already been used for the treatment of accidental radiation-induced aplasia and lessons have been learned from their medical management and follow-up. PMID- 7533813 TI - Development of intra-arterial hyperthermia using a dextran-magnetite complex. AB - Dextran-magnetite complex (DM) is a colloidal sol of subdomain magnetite particles (i.e. a 'magnetic fluid'). The specific absorption rate of DM in an AC magnetic field is much higher than those of multidomain ferrite particles due to its different mechanism of heat generation. We designed two DM-containing embolic materials (a DM/Lipiodol emulsion and a DM/degradable starch microsphere suspension) to heat target tissues with the use of an external AC magnetic field. In vitro experiments showed that the heat-generating capacity of DM was not reduced in these mixtures. When these materials were injected into the renal arteries of Japanese white rabbits, they both achieved embolization of the artery and selective heating of the embolized kidney by exposure to a 100 kHz AC magnetic field of approximately 15,000 A/m. Histological examination showed a homogeneous distribution of DM in the embolized kidney. These results suggest the possibility of using DM-containing embolic materials for inductive hyperthermia. PMID- 7533816 TI - Action of recombinant interferons and interleukin 2 in modulating radiation effects on viability and cytotoxicity of large granular lymphocytes. AB - We have evaluated in vitro the modulating effect of interferon (IFN) alpha, beta and gamma as well as interleukin 2 (IL-2) on the radiosensitivity of large granular lymphocytes (LGL) having natural killer cell activity. LGL were treated with IFNs or IL-2 in concentrations from 1 to 1000 U/ml before or after a single or a split dose of irradiation. The viability of LGL was measured by intracellular ATP, and cytotoxicity by a 51Cr release assay. Both viability and cytotoxicity were clearly higher when IFNs and IL-2 were used before irradiation. Some IFNs were slightly radiosensitizing in ATP studies. Only IFN gamma in a concentration of 1000 U/ml was significantly radioprotective in cytotoxicity tests when used before irradiation. IL-2 had a significant concentration dependent radioprotective effect in cytotoxicity when used before or after irradiation, and in the viability of preincubated LGL. No combination of IFNs and IL-2 was more radioprotective than IL-2 used alone. IL-2 retarded the time dependent decrease of ATP and 51Cr release levels after irradiation. According to our results, IL-2 is a radioprotective substance for LGL. PMID- 7533819 TI - LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14. AB - In human monocytes, superoxide (O2-) generation accompanies phagocytosis and is important for bactericidal activity. It also contributes to tissue damage in inflammation. In the present study we investigated, whether lipopolysaccharide (LPS) directly stimulates monocyte O2- production with kinetics known for other LPS effects and, if so, by which mechanism. LPS caused a time- and dose-dependent O2- release in nonadherent purified monocytes. The effect appeared after 5 min, peaked at 30 min, and disappeared after 2 h. It was maximal with 10 ng/ml lipid A (+148 +/- 22%, P < .001), 1 ng/ml LPS Escherichia coli Re (+226 +/- 68%, P < .001), and 100 ng/ml LPS Salmonella abortus equi sm (+272 +/- 52%, P < .001), respectively. The effect was not observed in buffer, even when using 10 micrograms/ml LPS. It was dependent on the presence of heat-inactivated AB serum, with a maximal effect at > or = 0.5%. Serum could be replaced by LPS-binding protein (LBP). Polymyxin B and anti-LBP antiserum, respectively, blocked the LPS effect. LPS-induced O2- generation was also completely blocked by anti-CD14 antibodies (3C10 and 63D3) and by their corresponding F(ab')2 fragments. Monocytes treated with phosphoinositol-specific phospholipase C and monocytes from patients with paroxysmal nocturnal hemoglobinuria, lacking the phosphatidylinositol-anchored CD14, did not respond to LPS stimulation with O2- production. Similarly to LPS, E. coli caused stronger O2- production with heat inactivated serum than without, and this effect was blocked by anti-CD14 antibodies. In conclusion, these data indicate that LPS directly stimulates O2- production in human monocytes via CD14 depending on LBP. PMID- 7533818 TI - Retinoic acid inhibits basal and interferon-gamma-induced expression of intercellular adhesion molecule 1 in monocytic cells. AB - Retinoic acid (RA) and 1,25-(OH)2-vitamin D3 (1,25-D3) induced U937 cell maturation into distinct monocytic phenotypes, as demonstrated by up-regulation of CD23 by RA and CD14 by 1,25-D3. Differentiation by RA but not by 1,25-D3 was associated with reduction of basal and complete suppression of interferon-gamma (IFN-gamma)-stimulated intercellular adhesion molecule 1 (ICAM-1) expression. Induction of cyclooxygenase activity by RA and attenuation of basal ICAM-1 expression exhibited similar kinetics. Treatment with indomethacin prevented and prostaglandin E2 (PGE2), dibutyryl-cAMP, or forskolin mimicked reduction of basal ICAM-1 expression by RA, indicating that this effect of RA is mediated by PGE2 synthesis and subsequent cAMP elevation. In contrast, suppression of IFN-gamma induced ICAM-1 expression by RA was only partly reversible by indomethacin, suggesting that inhibition of IFN-gamma stimulation was not completely due to cyclooxygenase induction. RA did not always counter-act IFN-gamma, as it cooperated with IFN-gamma in down-regulating very late activation antigen 4. Specific polymerase chain reaction and Northern blotting of ICAM-1 mRNA revealed that RA suppressed ICAM-1 induction by IFN-gamma at the transcriptional level. RA also blocked ICAM-1 induction by IFN-gamma in isolated human blood monocytes. In conclusion, inhibition of basal and stimulated ICAM-1 expression in monocytic cells may provide a mechanism for beneficial anti-inflammatory effects of retinoids. PMID- 7533817 TI - Radiation-induced and free radical-mediated inactivation of ion channels formed by the polyene antibiotic amphotericin B in lipid membranes: effect of radical scavengers and single-channel analysis. AB - This paper is part of a study on the effect of ionizing radiation on ion channels in biological membranes. Ion channels formed by polyene antibiotics amphotericin B or nystatin represent clusters of conjugated double bonds. As a consequence of this structural peculiarity, the conductance of lipid membranes--in the presence of polyene channels--has been found to decrease by several orders of magnitude at comparatively small doses of ionizing radiation. The phenomenon shows an inverse dose-rate behaviour similar to that of radiation-induced lipid peroxidation. We report on experiments performed in the presence of various radical scavengers, at varying cholesterol concentrations, and with different lipids. They support the view that channel inactivation is due to free radical-induced peroxidation of the polyenes leading to a destabilization of the barrel-like structure of the ion channels. Radiation-induced channel closing is shown for the first time at the level of single-ion channels. PMID- 7533820 TI - Mature polymorphonuclear leukocytes express high-affinity receptors for IgG (Fc gamma RI) after stimulation with granulocyte colony-stimulating factor (G-CSF). AB - The high-affinity receptor for the constant region of immunoglobulin G IgG (Fc gamma RI; CD64) is virtually undetectable on mature polymorphonuclear neutrophils (PMNs) in healthy individuals but is expressed on PMNs in patients with certain infections and in patients treated with recombinant human granulocyte colony stimulating factor (rhG-CSF). The induction of Fc gamma RI by rhG-CSF has previously been reported to result from effects on immature granulocyte progenitors. To evaluate the G-CSF effect on mature PMNs, we studied the correlation between G-CSF plasma concentration and expression of Fc gamma RI on PMNs in vivo as well as the effect of G-CSF on Fc gamma RI expression on mature PMNs in vitro. Fc gamma RI expression on PMNs correlated (R = 0.79; p < .001) with plasma concentrations of endogenous or recombinant G-CSF in healthy volunteers and in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation. PMNs exhibited a unimodal distribution for elevated Fc gamma RI expression, suggesting that G-CSF induced increased expression of Fc gamma RI on mature as well as on immature PMNs. In vitro, incubation of mature PMNs with G-CSF induced mRNA for Fc gamma RI. Significant Fc gamma RI surface expression was induced in a time- and dose-dependent manner. Thus, G-CSF can act on mature PMNs to increase Fc gamma RI expression and may be useful for stimulating antibody mediated immune functions of PMNs in vivo. PMID- 7533821 TI - Oocyte and zygote zona pellucida permeability to macromolecules. AB - Zona pellucida intact mouse pre- and post-ovulatory oocytes and zygotes were investigated for permeability to macromolecules using graded neutral fluorescein isothiocyanate (FITC) dextrans (molecular weight range 3.84-170 kDa) and a range of galactose-binding lectins (molecular weight range 40-247 kDa). The use of both FITC neutral dextrans and FITC galactose-binding lectins gave good agreement in the zona pellucida permeability studies. Zona pellucida intact pre- and post ovulatory oocytes were permeable to both markers up to a molecular weight range of 170 kDa although movement across the zona pellucida was diminished between 100 to 150 kDa with no detectable permeability at molecular weight range of 170 kDa. Zygotes demonstrated a decreased zona pellucida permeability at around 110 kDa. Two galactose-containing oligosaccharide-rich zones in the zona pellucida were identified, and changes in zygote vitelline membrane galactose-binding lectin affinity were detected. PMID- 7533822 TI - Nitric oxide synthase in the brain: light and electron microscopical findings based on the NADPH-diaphorase reaction. AB - The recent discovery of the identify of nitric oxide synthase with the reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) has powerfully stimulated the anatomical localization of sites of nitric oxide synthesis in the nervous system. In the present study the widely used light microscopical technique for NADPH-d staining was adapted to the electron microscopical level by applying the tetrazolium salt 2-(2'-benzothiazolyl)-5-styryl-3-(4' phthalhydrazidyl)tetrazolium chloride (BSPT) which produces an electron-dense reaction product, BSPT-formazan. Predominantly membranes of the endoplasmic reticulum were stained. Apart from singular heavily labeled neurons, a majority of nerve cells, light microscopically "unstained", shows sporadically formazan deposits, and, likewise, but regionally different, a few astroglial cells. Lesions induced by the glutamate agonists quinolinic acid and alpha-amino-3 hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) display surviving neurons, which are predominantly stained for NADPH-d. Astroglial cells within lesioned areas exhibit increased amounts of reaction product, apparently as a consequence of enzyme induction. PMID- 7533823 TI - Interactions of neurotoxins with non-NMDA glutamate receptors: an autoradiographic study. AB - Neurotoxic substances are discussed to cause neurodegeneration by acting as excitotoxins on glutamate receptors. We investigated the properties of L-beta oxalyl-amino-alanine (L-BOAA) and 3,4, 6-trihydroxyphenlyalanine (6-OH-Dopa) at the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) glutamate receptor and that of L-BOAA and domoic acid at the kainate glutamate receptor in human hippocampus. (3H)AMPA binding in hippocampal subfields was inhibited by L BOAA and 6-OH-Dopa with mean IC50-values in the low micromolar range. (3H)Kainate binding was inhibited by L-BOAA with similar potency as (3H)AMPA binding and by domoic acid with mean IC50-values in the low nanomolar range. These results support the notion that symptoms like anterograde amnesia and epileptic seizures seen in domoic acid intoxication and limbic symptoms, e.g. cognitive and mood impairment observed in neurolathyrism may be caused by excitotoxic action on non NMDA receptors. The potent interaction of 6-OH-Dopa with the AMPA-receptor may point to a possible dopaminergic-glutamatergic interaction in the development of neurodegenerative diseases like Parkinson's and Huntington's disease. PMID- 7533825 TI - Can cytotoxic dose-intensity be increased by using granulocyte colony-stimulating factor? A randomized controlled trial of lenograstim in small-cell lung cancer. AB - PURPOSE: The use of granulocyte colony-stimulating factor (G-CSF) to increase cytotoxic dose-intensity was assessed in a randomized trial in better-prognosis small-cell lung cancer (SCLC). Both control and G-CSF arms were subject to the same dose-intensification strategy. PATIENTS AND METHODS: Patients with newly diagnosed SCLC and either no or one adverse prognostic factor were randomized to receive vincristine, ifosfamide, carboplatin, and etoposide (VICE) alone or with recombinant human (rHu)G-CSF (lenograstim) 5 micrograms/kg/d between cycles. Six chemotherapy cycles were given, with prophylactic cranial irradiation after cycle 1 and thoracic irradiation after cycle 3. There was no fixed dose interval. In both arms, patients were eligible for re-treatment when the WBC count was > or = 3 x 10(9)/L and platelet count was > or = 100 x 10(9)/L. No dose reductions were permitted. Dose-intensity was expressed relative to standard every-4-weeks VICE. RESULTS: Sixty-five consecutive patients in one institution were randomized to control (n = 31) or G-CSF (n = 34). WBC and neutrophil counts were consistently higher in G-CSF patients than in the control group, but there were no significant differences in the incidence of febrile neutropenia, antibiotic or transfusion requirements, or days in hospital. In both treatment arms, the median dose intensity was greater than one for each cycle (control group, P = .0009; G-CSF group, P = .0001). The G-CSF group received a significantly higher dose-intensity than the control group, with the greatest difference in the first three cycles (1.34 v 1.17, P = .001). There were more chemotherapy-related deaths in the G-CSF group than in the control group (six v one), but this group had a better 2-year survival rate (32% with G-CSF, 95% confidence interval [CI], 16 to 48; 15% with controls, 95% CI, 2 to 27). CONCLUSION: The dose-intensity of VICE chemotherapy was increased in both groups. Patients randomized to receive G-CSF achieved a significantly higher dose-intensity than controls. Despite early toxicity, they had a better 2-year survival rate. PMID- 7533824 TI - Multicenter phase II study of weekly oral vinorelbine for stage IV non-small-cell lung cancer. AB - PURPOSE: We initiated a large multicenter phase II trial in stage IV non-small cell lung cancer (NSCLC) to evaluate the activity and safety of an oral gelatin based formation of vinorelbine. PATIENTS AND METHODS: Twenty-three centers participated in this uncontrolled phase II study, which accrued patients between August 1991 and March 1992. Eligible patients had previously untreated measurable or assessable stage IV NSCLC, age more than 18 years, and Karnofsky performance status > or = 70%. The treatment plan initially was to administer 100 mg/m2/wk of oral vinorelbine or 80 mg/m2/wk for patients who had received prior radiation therapy. After the observation of grade IV granulocytopenia in six of the first 25 patients, subsequent doses were reduced by 40 mg (one capsule) in all patients. RESULTS: One hundred sixty-two patients were treated: 138 with measurable and 24 with assessable disease. One hundred two patients were men and 60 women. The mean age was 62 years (range, 36 to 83). The overall response rate was 14.5% for patients with measurable disease (95% confidence interval, 9.3% to 21.7%). The median time to treatment failure (TTF) for all patients was 9 weeks. The median survival time was 29 weeks; the 1-year survival rate was 22%. Toxicities included grade 3 or 4 neutropenia in 40%, which was dependent on the vinorelbine dose. Other toxicities included mild to moderate nausea/vomiting, diarrhea, and stomatitis. The mean dose intensity of vinorelbine was 53 mg/m2. CONCLUSION: Oral vinorelbine administered once weekly is an active agent in stage IV NSCLC. The median survival time of 29 weeks is similar to that achieved with single-agent intravenous vinorelbine and more aggressive cisplatin-based combinations. Further studies of this compound in the palliative-intent care setting appear to be indicated. PMID- 7533826 TI - Plasma chromogranin A: a marker of serotonin release and of emesis associated with cisplatin chemotherapy. AB - PURPOSE: Emesis is a common side effect of cancer chemotherapy. Serotonin released from gastrointestinal enterochromaffin cells (ECC) may mediate chemotherapy-induced emesis. Since chromogranin A (CgA) is colocalized in ECC storage granules with serotonin, we tested the hypothesis that plasma CgA could mark emesis and serotonin release from ECC. PATIENTS AND METHODS: The relationships between plasma CgA, serotonin release, and the development of vomiting following the first course of cisplatin chemotherapy were evaluated in 60 patients. RESULTS: CgA levels increased in 59 of 60 patients (245% +/- 18% increase above baseline levels, P < .001). The time course of the increase in plasma CgA matched that of emesis and of urinary 5-hydroxyindoleacetic acid (5 HIAA). Significant (P < .001) positive correlations were found between the dose of cisplatin and the increases in plasma CgA, and between the changes in plasma CgA and urinary 5-HIAA after cisplatin (r = .54, n = 39, P < .001). The increase in plasma CgA after cisplatin did not correlate with changes in serum lactic dehydrogenase (LDH) activity, a marker of cell toxicity or lysis. CONCLUSION: Plasma CgA marks emesis and serotonin release induced by cisplatin. Since both CgA and serotonin are costored in ECC granules, we suggest that the source of release of each may be the ECC. Increases in plasma CgA are not explained by drug cytotoxicity. Exocytosis appears as the main mechanism by which cisplatin releases serotonin. This work further supports the role of serotonin as a mediator of emesis associated with cisplatin and suggests that plasma CgA level is a valuable tool in studies of chemotherapy-induced emesis. PMID- 7533827 TI - Predictive factors for peripheral-blood progenitor-cell collections using a single large-volume leukapheresis after cyclophosphamide and granulocyte macrophage colony-stimulating factor mobilization. AB - PURPOSE: (1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC. PATIENTS AND METHODS: Twenty-three patients with breast cancer received cyclophosphamide (4 g/m2) and granulocyte-macrophage colony-stimulating factor ([GM-CSF] 5 micrograms/kg/d x 15 days) for PBPC mobilization. A single leukapheresis was performed 15 days after cyclophosphamide administration. Then, patients received high-dose cyclophosphamide and thiotepa followed by reinfusion of PBPC and 4-hydroperoxycyclophosphamide (4HC)-purged bone marrow. PBPC concentration was measured in serial peripheral-blood samples and in the leukapheresis product. Correlation analysis between PBPC dose and engraftment and between leukapheresis yield and patient characteristics was attempted. RESULTS: A single leukapheresis processed a median 36 L (range, 24 to 46) blood and collected 5 x 10(6) CD34+ cells/kg (< 0.3 to 24) and 6.2 x 10(5) colony-forming units granulocyte-macrophage (CFU-GM)/kg (< 0.001 to 29). All sixteen patients (70%) reinfused with > or = 2.9 x 10(6) CD34+ cells/kg reached a level of greater than 1,000 leukocytes/microL by day 13 and greater than 50,000 platelets/microL by day 15. All of these patients had a percentage of peripheral-blood CD34+ cells > or = 0.5%, and all but one, a level of greater than 100,000 platelets/microL, on the day of leukapheresis. The bone marrow CD34+ cell percentage at study entry predicted the number of CD34+ cells collected after PBPC mobilization (R2 = .42, P = .002). All patients with > or = 2.5% bone marrow CD34+ cells experienced early engraftment. CONCLUSION: Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies. PMID- 7533828 TI - Survey of the provision of supportive care services at National Cancer Institute designated cancer centers. AB - PURPOSE: The purpose of this survey was to determine the scope of supportive care services (SCS) designed to promote quality of life during cancer therapies at National Cancer Institute (NCI)-designated cancer centers. METHODS: A survey was mailed to the medical directors and nursing directors of 52 NCI-designated comprehensive (n = 26), clinical (n = 11), and planning cancer centers (n = 15) in the United States. Only one survey was completed from each institution. Survey questions identified services provided such as pain management, terminal care, psychosocial programs, and spiritual care. RESULTS: Thirty-nine questionnaires were received for a total response rate of 75%. Of the respondents, 45% were comprehensive cancer centers, 24% clinical cancer centers, and 29% planning centers. One center did not identify their NCI designation. Sixty-one percent of the centers reported research programs in supportive care. Outside funding was reported in 51% of the respondents, with 39% having American Cancer Society (ACS) or National Institutes of Health (NIH) funding and 28% having private industry funding. Overall SCS self-ratings improved from a 21% rating of excellent to very good 5 years ago to the current 54% rating. CONCLUSION: Survey results provide data on SCS across a representative sample of NCI cancer centers and can be used to develop standards for future cancer control programs. PMID- 7533829 TI - Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. AB - PURPOSE: To review prognostic and therapeutic applications of angiogenesis research in breast carcinoma. METHODS: We reviewed the (1) biologic role of angiogenesis, particularly in transformation, progression, and metastasis of breast cancer; (2) methods to detect angiogenic activity in human pathology; (3) clinical studies relating clinical outcome of patients with breast cancer to the assessment of angiogenesis; (4) predictive value of angiogenesis for response to anticancer therapies; and (5) pharmacologic characteristics of current antiangiogenic drugs. RESULTS: There is mounting evidence that angiogenesis plays a relevant role in the biologic aggressiveness of breast cancer. Using either immunohistochemical or biochemical methods, several studies have shown a worse prognosis for those patients with tumors with high angiogenic activity. In some studies angiogenesis has an independent prognostic value. The most promising angiogenic inhibitors are under early-phase clinical evaluation in patients with tumors resistant to conventional therapies. Novel therapeutic strategies for breast cancer patients are presented and discussed. CONCLUSION: The majority of the retrospective studies show that angiogenesis is an important new prognostic indicator in early-stage breast carcinoma. This marker should be evaluated in prospective controlled clinical trials to demonstrate whether adjuvant therapies may improve the prognosis of those patients at high risk, eg, those with highly vascularized tumors. Since invasive breast carcinoma has a well defined stromal vascular component and produces angiogenic factors, it seems reasonable to postulate that this tumor may be one of the most responsive to angiogenesis inhibitors given alone or in combination with conventional anticancer treatments. PMID- 7533831 TI - Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. AB - Consumption of a plant-based diet can prevent the development and progression of chronic diseases that are associated with extensive neovascularization. To determine whether prevention might be associated with dietary derived angiogenesis inhibitors, we have fractionated urine of healthy human subjects consuming a plant-based diet and examined the fractions for their abilities to inhibit the proliferation of vascular endothelial cells. One of the most potent fractions contained several isoflavonoids, which we identified by gas chromatography-mass spectrometry and subsequently synthesized. Of all synthetic compounds, the isoflavonoid genistein was the most potent and inhibited endothelial cell proliferation and in vitro angiogenesis at half maximal concentrations of 5 and 150 mumol/L, respectively. Moreover, genistein inhibited the proliferation of various tumor cells. Genistein excretion in urine of subjects consuming a plant-based diet is in the micromolar range, which is 30 fold higher than that of subjects consuming a traditional Western diet. The high concentrations of genistein in urine of vegetarians and our present results suggest that genistein may contribute to the preventive effect of plant-based diet on chronic diseases, including solid tumors, by inhibiting neovascularization and tumor cell proliferation. Thus genistein may have important applications in the treatment of solid tumors and angiogenic diseases. PMID- 7533832 TI - Lack of neuronal nitric oxide synthase in nerve fibers of aganglionic intestine: a clue to Hirschsprung's disease. AB - The lack of nonadrenergic, noncholinergic (NANC) inhibitory innervation in aganglionic intestine is typical of Hirschsprung's disease. Several neuropeptides participating in the intestinal NANC innervation are greatly reduced in aganglionic intestine. However, these findings do not fully explain the pathophysiology of the disease. Recently, nitric oxide (NO) has been presented as a potent smooth muscle relaxant, and the enzyme responsible for its formation, nitric oxide synthase (NOS) has been demonstrated in neuronal elements in both the central and peripheral nervous system. In our study, nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a marker for NOS, and NOS immunohistochemistry revealed a dense innervation of the smooth muscle layers and the myenteric ganglia in ganglionic non afflicted intestine from patients with Hirschsprung's disease. By contrast, there was an almost complete lack of NOS immunoreactive and NADPH-diaphorase-positive nerve fibers in the afflicted aganglionic bowel. NOS and vasoactive intestinal peptide were found to be partially colocalized in nerve fibers and neuronal cell bodies in the ganglionic but not in the aganglionic intestine. The lack of NO-producing nerve fibers in the aganglionic intestine probably contributes to the inability of the smooth muscle to relax, thereby causing lack of peristalsis in Hirschsprung's disease. PMID- 7533833 TI - Absence of NADPH-diaphorase activity in internal anal sphincter (IAS) achalasia. AB - We studied the internal anal sphincter (IAS) muscle from 10 patients with achalasia and five normal controls using histochemical staining for NADPH diaphorase and acetylcholinesterase (AChE). Normal control IAS muscle contained occasional AChE-positive nerve fibers, whereas IAS achalasia specimens demonstrated prominent AChE-positive nerve fibers in muscle layers. NADPH diaphorase activity was strongly expressed in nerves in the normal IAS muscle but was absent or scanty in the muscle of patients with IAS achalasia. Our findings of increased AChE-positive nerves and the absence of NADPH-diaphorase activity taken in conjunction with reports of abnormal peptidergic innervation indicate that complex neural abnormalities occur in IAS achalasia. The primary event remains obscure, but it is possible that a single defect, such as nitrergic nerve depletion, may lead to compensatory changes in the other nerve fibers. PMID- 7533834 TI - Infantile Refsum disease: neonatal cholestatic jaundice presentation of a peroxisomal disorder. PMID- 7533830 TI - Modulation of gap-junction channel gating at zebrafish retinal electrical synapses. AB - 1. Transmission at electrical synapses is modulated by a variety of physiological signals, and this modulation is a potentially general mechanism for regulating signal integration in neural circuits and networks. In the outer plexiform layer of the retina, modulation of horizontal-cell electrical coupling by dopamine alters the extent of spatial integration in the horizontal-cell network. By analyzing the activity of individual gap-junction channels in low-conductance electrical synapses of zebrafish retinal horizontal cells, we have defined the properties of these synaptic ion channels and characterized the functional changes in them during modulation of horizontal-cell electrical synapses. 2. Zebrafish horizontal-cell gap-junction channels have a unitary conductance of 50 60 pS and exhibit open times of several tens of milliseconds. The kinetic process of channel closure is best described by the sum of two rate constants. 3. Dopamine, and its agonist, (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN), modulates electrical synaptic transmission between horizontal cells predominantly by affecting channel-gating kinetics. These agents reduced the open probability of gap-junction channels two- to threefold by reducing both the duration and frequency of channel openings. Both time constants for channel open duration were reduced, whereas the duration of shut periods was increased. Similar changes in open-time kinetics were observed in power spectra of higher conductance gap junctions. 4. These results provide a description of rapid electrical synaptic modulation at the single channel level. The description should be useful in understanding the mechanisms of plasticity at these synapses throughout the vertebrate central nervous system. PMID- 7533836 TI - [Analytic method for ion-channel noise of patch-clamp technique]. PMID- 7533835 TI - Molecular weight dependent tissue accumulation of dextrans: in vivo studies in rats. AB - The effects of molecular weight (M(r)) on the serum and urine pharmacokinetics and tissue distribution of dextrans, potential macromolecular carriers for drug delivery, were studied in rats. A single 5 mg/kg dose of fluorescein-labeled dextrans (FDs) with a M(r) of 4000 (FD-4), 20,000 (FD-20), 70,000 (FD-70), or 150,000 (FD-150) was administered into the tail vein of separate groups of rats. At different times after the administration of each FD, animals were sacrificed, and blood, urine, and various tissues were obtained. The concentrations of FDs in the samples were subsequently determined by using a sensitive and specific high performance size exclusions chromatographic method. Among the tissues studied, high accumulation of dextrans was found only in the liver (liver:serum AUC ratios < or = 29) and spleen (spleen:serum AUC ratios < or = 10), with high concentrations in these tissues persisting even at the last sampling time (96 h). In contrast, the serum concentrations of the studied FDs were not measurable beyond 12 h. The serum and urine kinetics and the liver, spleen, and kidney accumulation of FDs demonstrated a significant degree of M(r) dependency. The total and renal clearance of FDs consistently decreased with an increase in M(r). However, the effects of M(r) on the tissue accumulation of dextrans was tissue dependent. For the liver, the tissue:serum AUC ratios increased from 0.346 for FD 4 to 15.2 for FD-20 and 28.8 for FD-70, while a further increase in M(r) to 150 kDa (FD-150) resulted in lowering the ratio to 8.59 in this tissue. For the spleen, the ratios increased from 0.095 for FD-4 to 9.56 for FD-150.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533837 TI - Addressing emerging social needs of patients treated with new neuroleptics. AB - 1. Clozapine and risperidone are revolutionary new neuroleptic drugs that appear to alleviate psychotic symptoms in many patients who are unresponsive to other neuroleptic medications. 2. Alleviation of negative symptoms, although beneficial in many ways, also may create fears, changes, and unknown challenges for both the patient and caregivers. 3. Thorough assessment and diligent community planning may assist patients in fulfilling the basic human need for love and belonging. PMID- 7533838 TI - 0-7-21 radiation therapy for the palliation of advanced cancer in dogs. AB - The 0-7-21 radiation therapy protocol was investigated as a palliative treatment in dogs with advanced malignancies. Twenty-four dogs with a variety of tumor types were irradiated using 800 cGy fractions given on days 0, 7, and 21. Twenty three dogs were evaluated. Palliative response was assessed using a quality of life instrument developed for veterinary use. This pain score was based on owner response to questions regarding analgesic requirement, activity level, appetite, and degree of lameness in the affected dogs. Seventeen (74%) of the 23 dogs experienced complete pain relief, and 3 (13%) obtained partial relief. Of the 17 dogs that achieved a complete response, pain recurred in 8 at a median time of 70 days. Six dogs were alive and free of pain up to 557 days after irradiation. The 0-7-21 protocol was well tolerated; pain relief occurred quickly, and acute radiation reactions were negligible. PMID- 7533839 TI - Mapping of murine Th1 and Th2 helper T-cell epitopes on fimbriae from Porphyromonas gingivalis. AB - Th1- and Th2-derived cytokine production in response to synthetic peptides of the fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was assessed in spleen mononuclear cells (MNC) of BALB/c mice (H-2d haplotype) immunised with the fimbrial protein antigen and adjuvant GM-53 in Freund's incomplete adjuvant (FIA). Sixty-seven sequential overlapping 10-mer peptides covering the complete 337 amino-acids (AA) protein of P. gingivalis fimbrilin were synthesised. Stimulation of spleen MNC in vitro with these 10-mer peptides resulted in the production of murine interleukin-2 (IL-2), gamma-interferon (IFN gamma), IL-4, IL-5 and IL-6. Peptides 13 (AA 61-70), 24 (AA 116-125), 31 (AA 151 160) and 64 (AA 316-325) markedly induced IL-2 production. In particular, peptide 24 (DPLKIKRVHA), which contained I-Ad, I-Ed and I-Ak binding motifs, was the most potent stimulator of IL-2, IFN-gamma, IL-4, IL-5 and IL-6 production. Spleen MNC from C3H/HeN mice (H-2k) followed by BALB/c mice (H-2d) immunised with peptide 24 were high responders to peptide 24 in terms of both IFN-gamma and IL-4 production, whereas A/J mice (H-2a) and C57BL/6 mice (H-2b) were very low responders, P. gingivalis fimbriae evoked higher delayed-type hypersensitivity (DTH) reactions in B10.D2 (H-2d) and B10.BR (H-2k) mice followed by C57BL/10 (B10, H-2b) and B10.A (H-2a) and in guinea-pigs immunised with the fimbriae and GM-53 in FIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533840 TI - Influence of various immunosuppressive agents on the occurrence of endogenous bacteraemia in mice. AB - The influence of six immunosuppressive agents on the occurrence of endogenous bacteraemia in mice was evaluated. The mortality rates in conventional ddY mice given cyclophosphamide (CY), fluorouracil (5-FU), methotrexate (MTX), cisplatin (CDDP) or FK-506 intraperitoneally, or dexamethasone (DXM) subcutaneously were 70, 100, 100, 100, 0 and 0%, respectively. Pseudomonas aeruginosa was isolated from 70% of mice treated with CY but from only 10% of mice treated with 5-FU and 30% treated with MTX. Enterobacteria were isolated from 90% of mice treated with 5-FU. Specific-pathogen-free (SPF) mice fed P. aeruginosa were also treated with these agents. All mice in the CY, 5-FU, MTX and CDDP groups died whereas mice treated with DXM and FK-506 showed 20% and 0% mortality, respectively. Pure cultures of P. aeruginosa were obtained from all of the mice treated with CY. Polymicrobial bacteraemia with P. aeruginosa and enterobacteria occurred in 5, 25, 5 and 5% of mice treated with 5-FU, MTX, CDDP and DXM, respectively. Enterobacterial bacteraemia was observed in 70% of mice treated with CDDP and in 5% of the DXM group. Different types of bacteraemia were induced by different immunosuppressive agents. The mechanism of immunosuppression may affect the frequency of bacteraemia and the causative organism. PMID- 7533841 TI - Basolateral K channel activated by carbachol in the epithelial cell line T84. AB - Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues. PMID- 7533842 TI - Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases. AB - Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues. PMID- 7533843 TI - Beta/A4-evoked degeneration of differentiated SH-SY5Y human neuroblastoma cells. AB - beta/A4 peptides are known to induce neurodegeneration in cultures of rat brain cells and rat neural cell lines (Yankner et al: Science 250:279-282, 1990; Behl et al: Biochem Biophys Res Commun 186:944-950, 1992). The current data show that these peptides induce similar neurodegeneration in SH-SY5Y neuroblastoma cells, extending characterization of beta/A4 toxicity to a human nerve cell line. Human SH-SY5Y cells respond to aggregated beta/A4 with changes in cell shape, membrane blebbing, antigenic modification, loss of attachment to the substrate, and cell death. beta/A4 peptides require aggregation for maximum toxic effects, as cellular degeneration is evoked by aggregated beta/A4 1-42 and 4-41 cysteine but not by monomeric beta/A4 1-40. Aged (pre-aggregated) beta/A4 1-40 also evoked neurodegeneration. Antigenic changes comprise upregulation of Alzheimer's-type tau epitopes, recognized by the PHF-1 and Alz-50 monoclonals. These particular changes in tau support the connectivity between this in vitro model and mechanisms leading to neurodegeneration in Alzheimer's disease. A significant feature of the SH-SY5Y response is that cells must be differentiated before they become sensitive to the degeneration evoked by beta/A4. Signaling pathways leading to beta/A4-evoked neurodegeneration thus are under experimental control, becoming complete only when proliferating cells withdraw from the cell cycle and develop a postmitotic phenotype. PMID- 7533845 TI - Expression and distribution of functional integrins in rat CNS glia. AB - In previous studies, we have reported on the expression of beta 1 integrins in type 1 astrocytes and their function in cell-substratum attachment (Tawil et al., J Cell Biol 120:261-271, 1993). Here we extend those findings by providing evidence that type 1 astrocytes express integrins of the beta 3 and possibly beta 4 subclasses and that the former (alpha v beta 3) functions in attachment by recognizing the peptide, Arg-Gly-Asp, in vitronectin. In addition, we have examined immunocytochemically the expression of beta 1 integrins on type 2 astrocytes and oligodendrocytes. The pattern of expression of integrins on these two cell types is distinct from type 1 astrocytes; most notably type 1 astrocytes but not oligodendrocytes or type 2 astrocytes express alpha 1 beta 1 heterodimers. Since type 2 astrocytes and oligodendrocytes originate from a common precursor (O-2A), the alpha 1 beta 1 heterodimer may be a functional marker which distinguishes O-2A-derived cells from those of the type 1 astrocyte lineage. PMID- 7533844 TI - Duration of expression of inducible nitric oxide synthase in glial cells. AB - Lipopolysaccharide (LPS) or a combination of interleukin (IL)-1 beta and interferon (IFN)-gamma cause transcriptional induction of a calcium-independent nitric oxide synthase (NOS) in astrocytes and C6 glioma cells. LPS induction of NOS in C6 cells was evidenced by a small amount of nitrite accumulation as compared with cells exposed to IL-1 beta/IFN-gamma, but the maximal NOS activity achieved (as revealed by cGMP formation) was the same. The NOS activity induced by LPS in C6 cells was maximal at 4 to 8 hr and then rapidly decreased, while NOS activity induced by IL-1 beta/IFN-gamma slowly decreased after 4 hr. In addition, the effects of re-presenting IL-1 beta/IFN-gamma to both astrocytes and C6 cells after maximal induction of activity of the inducible form of NOS were studied. The re-addition of cytokines prolonged both NOS mRNA expression and also enzyme activity, suggesting effects at either the transcriptional (further induction) or translational level (mRNA stability). These results imply that the time course of NO production by induced astrocytes depends both upon the nature of the inducing stimulus and the frequency of the cells' exposure to it. PMID- 7533846 TI - GRASP: a novel heparin-binding serum glycoprotein that mediates oligodendrocyte substratum adhesion. AB - Cell-substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphorylation of myelin basic protein; no such phosphorylation takes place in the non-adhered cell. We postulated that horse serum provides an adhesion molecule. Laminin, fibronectin, collagen and native vitronectin failed to replace horse serum. Hence, we set out to fractionate horse serum by screening with an adhesion assay. We report here the identification, purification and partial characterization of a novel, heparin binding horse serum glycoprotein that we have termed Glycine-Rich Adhesion Serum Protein--GRASP--to stress the fact that this protein has a high content of glycine and functions, in vitro, as an adhesion molecule for OLGs. There is 61% similarity at the N-terminus between GRASP and histidine-rich glycoprotein precursor (HRGP), an alpha 2-glycoprotein from human plasma. However, our data suggest that GRASP is not the horse serum homolog of HRGP. First, the two Gps are functionally distinct: HRGP does not promote the adhesion of OLGs. Second, the amino acid compositions differ significantly, e.g., GRASP is not histidine- but rather glycine-rich. Third, the region of sequence similarity between GRASP and HRGP is conserved throughout the cystatin superfamily. Fourth, anti-Gp55 polyclonal Abs recognize a similar set of polypeptides--save for slight differences in M(r)-in human serum as in horse serum, indicating that HRGP and GRASP are two distinct but related proteins and are both present in human and horse sera. GRASP is a dimer trimer of seemingly identical subunits of M(r) approximately 55,000 ; the native protein has an M(r) x 10(-3) approximately 120 140, of which 24-27% is contributed by carbohydrate. Using GRASP as a substratum allows the growth of OLGs in serum-free medium. GRASP is as good an effector of myelin basic protein phosphorylation as 20% horse serum. We conjecture that the mechanism of GRASP function features: 1) exposure of a cryptic sequence--after a change in conformation induced upon binding to polylysine--with affinity for an OLG signal-transducing receptor; and 2) interaction of its heparin-binding domain with OLG surface heparin sulfate proteoglycans and/or the aforementioned receptor. PMID- 7533847 TI - Inhibitors of free radical formation fail to attenuate direct beta-amyloid25-35 peptide-mediated neurotoxicity in rat hippocampal cultures. AB - The direct neurotoxic action of the beta-amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic-mediated neuronal death induced by beta-amyloid appears to reside in its ability to form Ca(2+)-permeable pores in neuronal membranes resulting in an excessive influx of Ca2+ and the induction of neurotoxic cascades. It is possible that during beta-amyloid exposure a Ca(2+) mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of beta-amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to "aged" beta-amyloid25-35 peptide (5-50 microM) induced a slow, concentration-dependent apoptotic neurotoxicity (25-85%) during a 6 day exposure. Co-incubation of cultures with beta-amyloid25-35 peptide (25 microM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG monomethyl-L-arginine [1 mM), N omega-nitro-L-arginine [1 mM], oxypurinol [100 microM], allopurinol [100 microM]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate beta-amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase-A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 microM), indomethacin (50 microM), and nor-dihydroguaiaretic acid (0.5 microM), respectively, did not reduce the proclivity of beta-amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 microM), vitamin C (100 microM), glutathione (100 microM), L-cysteine (100 microM), N-acetyl-cysteine (100 microM), deferoxamine (5 microM), or haemoglobin (35 micrograms/ml) failed to attenuate the neurotoxic action of beta-amyloid. On the other hand, pre-treatment of cultures with subtoxic concentrations of beta amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that beta-amyloid peptide renders neurons more sensitive to free radical attack. However, a potential beta-amyloid-mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of beta amyloid in vitro. PMID- 7533849 TI - Thyroid proliferative lesions induced by anti-thyroid drugs in rats are not always accompanied by sustained increases in serum TSH. AB - To examine changes in serum TSH and determine whether the sustained excess is necessary for the development and/or progression of thyroid tumors, male F344 rats were administered drinking water containing thiourea (TU), at 0.1 or 0.05%, or sulfadimethoxine (SM), at 0.025 or 0.0125%, for one week in Experiment I. All of the treated animals showed decreased serum levels of T3 and T4 and an increased TSH. In Experiment II, male rats were given a s.c. injection of N-bis(2 hydroxypropyl) nitrosamine (DHPN:1500 mg/kg BW) and, starting one week later, received drinking water containing the same doses of TU or SM as in Experiment I for the following 20 weeks. Thyroid follicular proliferative lesions were induced in most rats treated with TU and SM. However, these treated animals did not demonstrate any consistent alterations in serum T3, T4 and TSH levels, except for the high dose TU group. The present studies thus suggest that thyroid tumors can grow even under conditions of fluctuating serum TSH levels during the progression phase, although TSH stimulation might be an absolute requirement in the early phase of tumor development. PMID- 7533848 TI - Tacrolimus (FK506)-induced nephrotoxicity in spontaneous hypertensive rats. AB - To clarify the profile of the tacrolimus (FK506)-induced nephrotoxicity and its mechanism, 1, 2 and 4 mg/kg/day of tacrolimus was administered intramuscularly (i.m.) to spontaneous hypertensive rats (SHR) for 2 weeks, and biochemical and pathological parameters were studied in the animals. The acute nephrotoxicity of tacrolimus was characterized as increase of blood urea nitrogen (BUN) and plasma creatinine (P-Cr) levels in the groups of 1 mg/kg/day and more, decrease of creatinine clearance (CCr) value in the groups of 2 mg/kg/day and more, and histopathologically luminal narrowing of the arteriole adjacent the glomerulus in the groups of 1 mg/kg/day and more. These changes were associated with an increase of plasma renin activity (PRA) and urinary thromboxane B2 content and decrease of 6-keto-prostagrandinF1 alpha (6-keto-PGF1 alpha) content. Nilvadipine, which is one of the Ca2+ antagonist and is known to have renal vasodilating activity, prevented both biochemical and histopathological changes due to tacrolimus. The results indicated that the acute nephrotoxicity of tacrolimus was derived from impairment of glomerular function associated with the constriction of the renal arteriole brought about by the drug. All of these renal disorders induced by tacrolimus recovered completely or partially when the drug was withdrawn for 2 or 4 weeks. Consequently, the acute nephrotoxicity of tacrolimus in SHR was considered to be reversible. PMID- 7533850 TI - A visual representation system for drug abuse counselors. AB - Node-link mapping is a technique that consists of drawing spatial-verbal displays to visually represent interrelationships between ideas, feelings, facts, and experiences. These multirelational maps are drawn during ongoing counseling sessions to represent a variety of personal and nonpersonal topics. This article provides an overview of this technique, including rationale, related and supporting research, and an example of its use during counseling. In addition, this article describes the potential impacts of mapping and comments on practical considerations related to the effective use of this tool in drug abuse counseling. PMID- 7533851 TI - Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus. AB - Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7 B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7533852 TI - Inhibition of vesicular stomatitis virus infection by nitric oxide. AB - Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system. PMID- 7533853 TI - Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. AB - Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells. PMID- 7533855 TI - Optimal lymphocytic choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex class I molecules and presented to cytotoxic T lymphocytes. AB - Infection with lymphocytic choriomeningitis virus induces the generation of CD8+ cytotoxic T lymphocytes (CTL). In the H-2b mouse, this cellular immune response is directed against three viral structural epitopes (GP1, GP2, and NP) presented by the major histocompatibility complex (MHC) class I H-2Db molecules. This study was undertaken to delineate which sequence of each of these three epitopes is optimal for MHC binding and CTL recognition. The first step was to synthesize the relevant peptides truncated at the N or C terminus and flanking the crucial H-2Db anchoring Asn residue in position 5. These peptides were then tested (i) for their binding properties in two H-2Db-specific assays with viable cells (upregulation of H-2Db expression on the surface of RMA-S cells and competition against the Db-restricted peptide 125I-gp276-286 on T2-Db cells) and (ii) for their abilities to sensitize H-2b target cells for CTL lysis in vitro. For optimal antigenic presentation, all three epitopes required the MHC-anchoring Asn residue at position 5 of their sequences. The results clearly and unambiguously delineated optimal lengths for two of the epitopes and two options for the third. NP appeared as a conventional 9-amino-acid (aa)-long peptide, np396-404 (FQPQNGQFI). GP2 was defined as a longer peptide (11 aa), gp276-286 (SGVENPGGYCL). Characterization of the GP1 epitope was more complex: the 9-aa long peptide gp33-41 (KAVYNFATC) and the carboxyl-extended 11-aa-long peptide gp33-43 (KAVYN FATCGI) were both established as possible optimal sequences depending on the cell line used to test binding and lysis. PMID- 7533854 TI - Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain. AB - A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products. PMID- 7533856 TI - Longitudinal assessment of feline immunodeficiency virus kinetics in plasma by use of a quantitative competitive reverse transcriptase PCR. AB - Cats infected with feline immunodeficiency virus (FIV) develop a disease syndrome similar to that caused by human immunodeficiency virus type 1 (HIV-1) infection in humans. HIV-1 replication has been shown to correlate with the disease stage and progression. To assess replication kinetics and disease progression in early FIV infection, we developed a quantitative competitive reverse transcriptase PCR to measure the plasma virus load at serial time points after virus exposure. We found that an early peak viremia immediately preceded the onset of acute-phase symptoms in infected cats. Plasma virus levels remained high throughout the symptomatic phase of infection, which lasted for 8 to 10 weeks, and then declined as clinical symptoms resolved; however, all cats maintained significant plasma virus titers through 36 weeks postinfection. Early peak viral replication coincided with the initial precipitous decline in circulating CD4+ T lymphocytes. These results indicate that FIV kinetics are similar to those of HIV-1 during the acute and secondary phase of infection and that the plasma FIV load correlates with the disease stage. These results serve to further develop the FIV model and to enhance its usefulness for pathogenesis, vaccine development, and therapeutic studies related to HIV. PMID- 7533857 TI - Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro. AB - To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV 1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV 1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system. PMID- 7533859 TI - Turnover of circulating virion RNA and of cell-associated viral DNA reflects active viral replication in human immunodeficiency virus type 1-infected individuals. AB - A quantitative assessment of human immunodeficiency virus type 1 turnover in patient cell-free virion and infected-cell compartments under the dynamic conditions imposed by an effective antiviral therapy was performed. The turnover was rapid, and following a temporal lag, the extent of viral population replacement was eventually similar in both compartments. Each compartment therefore reflects considerable active virus replication. PMID- 7533858 TI - Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. AB - The baculovirus GP64 envelope fusion protein (GP64 EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, and membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I, nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role of region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP. In addition, a deletion that removed region II and the predicted alpha-helix was defective for oligomerization, whereas a larger deletion that retained region II and the predicted helix was oligomerized. These results indicate that region II is required for oligomerization and transport and suggest that the predicted helical structure of this region may be important for this function. Thus, by using mutagenesis, functional assays, and antibody inhibition, two functional domains were localized within the baculovirus GP64 EFP: a fusion domain located at amino acids 223 to 228 and an oligomerization domain located at amino acids 327 to 335 within a predicted amphipathic alpha-helix. PMID- 7533860 TI - Naturally occurring variants of human T-cell leukemia virus type I Tax protein impair its recognition by cytotoxic T lymphocytes and the transactivation function of Tax. AB - There is a high degree of intraisolate sequence heterogeneity in the tax gene of human T-cell leukemia virus type I (HTLV-I), although the sequence variation between patients is small compared with that of human immunodeficiency virus type 1. In the present study, we investigated whether naturally occurring amino acid substitutions changed the properties of the Tax protein in two respects: first, recognition of the protein by cytotoxic T lymphocytes (CTL), and second, the ability of the Tax protein to transactivate various promoters. We found that (i) all of the observed amino acid substitutions that occur in known CTL epitopes abolished the recognition of the synthetic peptide representing the respective epitope; (ii) these substitutions occurred significantly more frequently in subjects carrying HLA-A2; and (iii) most of the amino acid substitutions severely reduced the ability of Tax protein to transactivate three promoters: the HTLV-I long terminal repeat, the c-fos promoter, and the interleukin-2 receptor alpha chain promoter. PMID- 7533861 TI - Antiviral protective immunity induced by major histocompatibility complex class I molecule-restricted viral T-lymphocyte epitopes inserted in various positions in immunologically self and nonself proteins. AB - Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein. PMID- 7533863 TI - [Abnormality of CD4 molecule--OKT4 epitope deficiency]. AB - The individuals with OKT4 epitope deficiency are identified as incomplete (heterozygote carriers) and complete (homozygotes) by the difference in the number of OKT4 epitopes on the surface of lymphocytes. The incidence of homozygotes in the Japanese population was found to be 0.47% by examination of 1,478 random samples, and on the basis of this value, the incidence of heterozygotes was estimated to be 12.8% by the Hardy-Weinberg's formula. This deficiency was transmitted as an autosomal codominant trait. DNA from the lymphocytes with complete OKT4 epitope deficiency from the members of three families was sequenced, and a single nucleotide base substitution (CGG-->TGG) was found. This mutation was confirmed to be responsible for OKT4 epitope deficiency by using the mouse L cells transfected with mutant CD4 cDNA. The mutation results in arginine being replaced by tryptophan. Analysis showed different hydrophobicity at positions 239 and 240 from the control, probably giving rise to a conformational change in CD4 accounting for lack of reactivity with the OKT4 monoclonal antibody. The lymphocytes with OKT4 epitope deficiency did not show any abnormality in their susceptibility to HIV infection, the internalization of CD4 molecules by TPA-treatment, the capability of producing IL-2 in vitro, and the expression of IL-2 receptors (alpha/beta-chain) by PHA-stimulation. PMID- 7533862 TI - Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells. AB - The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV. PMID- 7533864 TI - Characterization of cholecystokinin receptors and messenger RNA expression in rat pancreas: evidence for expression of cholecystokinin-A receptors but not cholecystokinin-B (gastrin) receptors. AB - It has been previously demonstrated that guinea pig pancreas possesses both cholecystokinin-A (CCK-A) receptors and CCK-B (gastrin) receptors. In contrast to guinea pig pancreas, it is not known whether CCK receptors in rat pancreas are CCK-A receptors, CCK-B (gastrin) receptors, or both. Thus, in the present study, we characterized CCK receptors in rat pancreas at the receptor and mRNA level. 125I-Bolton-Hunter-labeled CCK octapeptide (125I-BH-CCK-8), the specific CCK-A and CCK-B (gastrin) receptor antagonists L364,718 and L365,260, and 125I-labeled gastrin-I were utilized to characterize CCK receptors in normal rat pancreas. Additionally, we utilized 32P-labeled cDNA probes of the CCK-A receptor and CCK-B (gastrin) receptor coding regions in order to examine the expression of CCK receptor subtypes in normal rat pancreas at the mRNA level. The dose-inhibition curve of CCK-8 inhibiting binding of 125I-BH-CCK-8 was significantly best fit by a two-site model with a high-affinity site (Kd = 0.68 +/- 0.13 nM) and a low affinity site (Kd = 656 +/- 289 nM). L364,718 inhibited binding of 125I-BH-CCK-8 with high affinity, whereas no high-affinity inhibition for L365,260 to inhibit binding of 125I-BH-CCK-8 was detected. L364,718 was 627 times as potent as L365,260 in inhibiting binding of 125I-BH-CCK-8. No saturable binding was present for 125I-labeled gastrin-I. Gastrin-17-I did not inhibit binding of 125I-BH-CCK 8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533865 TI - Sensitivities of AML blast stem cells to idarubicin and daunorubicin: a comparison with normal hematopoietic progenitors. AB - The objective of this study was to determine whether or not cell culture studies could contribute to the correct choice between idarubicin (IDA) and daunorubicin (DNR) for combination with ara-C in remission induction therapy of AML. Two growth factor-sensitive AML cell lines and the peripheral blood blast cells from 10 patients with AML were studied in culture for sensitivity to IDA and DNR under four culture conditions; cells were grown either in methylcellulose or suspension culture in the presence of G-CSF or GM-CSF. Normal bone marrow cells were cultured in methylcellulose in the presence of IDA or DNR under conditions suitable for the detection of BFU-E and CFU-C. Dose-response curves for AML blast cells were characterized by an initial shoulder and then an exponential decrease in survival. Marked patient-to-patient variation was observed for both portions of the survival curves. IDA was significantly more toxic to blast cells than DNR, especially for more sensitive cell populations. Consistent differences in drug sensitivity in the four culture conditions were not observed. BFU-E and CFU-C dose-response curves of normal marrow progenitors resembled those of AML blast cells but in contrast fell within a narrow range. The culture studies support the clinical finding in AML of modest superiority of IDA over DNR. The heterogeneity in sensitivity of AML blasts in culture suggests an opportunity to individualize treatment. Preclinical studies may help in developing such a therapeutic approach. PMID- 7533866 TI - Cytokine treatment of human bone marrow activates anti-leukaemia effector cells: monitoring of purging by polymerase chain reaction and DNA analysis. AB - The aims of this study were to investigate the role of cytokines (tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-2 (IL-2) in augmenting graft-versus-leukaemia (GVL). We have investigated the effector cells involved in GVL, by studying the role of these cells in purging of the cell line K562 in short-term bone marrow cultures. The effect of the addition in vitro of rGCSF was also studied. Monitoring of purging was achieved by cytotoxicity assays, DNA analysis and the use of the polymerase chain reaction for the detection of bcr/abl transcripts in the Philadelphia positive (Ph+) K562 cell line. Supernatants from IL-2-treated and non-treated bone marrow were tested for cytokine production (TNF alpha and IFN gamma). The results have shown that the main cytotoxic effector cells in the bone marrow generated by IL-2 have the CD56+ CD8+ phenotype. Overnight incubation of bone marrow was sufficient to generate cytotoxic cells as measured by Chromium51 (Cr51) release assays. Measurable levels of TNF alpha but not IFN gamma were also detected in supernatants. Addition of TNF alpha and IFN gamma to the IL-2 in the bone marrow cultures augmented the cytotoxicity but tended to inhibit progenitor cell growth as measured by granulocyte-macrophage colony-forming unit (GM-CFU) and erythroid blast-forming unit (BFU-e) assays. An estimate of the purging of the marrow could also be achieved by DNA analysis of K562 DNA in bone marrow. The bcr/abl transcript could still be detected by PCR analysis in marrow containing 1% K562 and treated with IL-2 for 24 h, but by 6 days of incubation the bcr/abl transcript was weak or undectable. The results suggest that although reduction in the proportion of leukaemia in contaminated marrow can be detected after incubation with IL-2 for 24 h, complete elimination of minimal residual disease requires longer incubation times. PMID- 7533867 TI - Residual normal, highly proliferative progenitors can be isolated from the CD34+/33- fraction of AML with a more differentiated phenotype (CD33+). AB - Since in AML differentiation is abnormal but not absent, a hierarchy of stem cells, progenitor cells and more differentiated cells is postulated. The leukemic stem cell might also be characterized by the expression of CD34 and the absence of differentiation markers. Bone marrow samples of 33 AML patients, including 10 patients both at presentation and after relapse, were double labeled for CD34 and CD33. In 14/33 AML less than 1% of the labeled cells were found in the CD34+/33- fraction. After relapse a certain shift towards a more primitive phenotype was observed, but in 4/5 relapsed AML the CD34+/33- fraction remained below 1%. Single cells from the different subfractions were cultured and showed heterogeneous cluster and colony growth in both the CD34-/33+ and CD34+/33+ fraction. More colonies were observed in the CD34+/33- fraction. In AML with a more 'mature' phenotype (low number of CD34+/CD33- cells), highly proliferative myeloid, erythroid and mixed colonies could be cloned exclusively from this small CD34+/33- fraction. In five patients with numerical chromosomal abnormalities all these highly proliferative colonies appeared disomic using in situ hybridization (ISH) with centromeric probes. Based on these data we conclude that the CD34+/33- cell fraction in AML with a more mature immunophenotype (small fraction of cells CD34+/33-) comprise residual normal progenitors, while no primitive leukemic progenitors could be identified. PMID- 7533868 TI - Description of a novel FR1 IgH PCR strategy and its comparison with three other strategies for the detection of clonality in B cell malignancies. AB - PCR amplification of IgH gene V-D-J junctional variability (IgH PCR) is increasingly replacing Southern analysis for the detection of clonal lymphoid populations in cases presenting diagnostic difficulties. In order to determine the most efficient strategy, we have compared three known methods, using consensus primers against the VH FR3 or FR2 (FR256) regions, or a mix of six primers against the FR1 region (FR1f), with a new approach using a consensus primer against FR1 (FR1c), never previously described for diagnostic purposes, on DNA from 89 monoclonal B-cell proliferations (16 ALL, 28 CLL/PLL, 15 myelomas, 30 NHL). We obtained a detection rate of 70% for FR3, 64% for FR1f and 77% and 78% for FR256 and FR1c, respectively. Polyclonal lymphocytes and mature T cell malignancies tested negative for all systems. Differences in the detection rate were related not only to the choice of VH primer but also the JH primer(s) used and the pathological subtype. All strategies led to adequate detection of leukaemic DNA, whereas the detection rate in myeloma varied between strategies from 47 to 80% and that of follicular lymphoma from 13 to 63%. The lowest detection rates were observed in follicular lymphoma and in mature CD5 negative proliferations, reflecting the probable correlation between somatic mutation and PCR false-negativity. The combined use of FR1c and FR256 allowed detection in at least one system of 92% of cases overall and at least 75% in all pathological subtypes, thus providing a simple, reliable and rapid non-radioactive system for the detection of B cell clonality. PMID- 7533869 TI - Glycine release from hippocampal slices in developing and ageing mice: modulation by glutamatergic receptors. AB - The release of preloaded [3H]glycine from hippocampal slices in developing and ageing mice (from 7 days to 22 months) was characterized using a superfusion system. The release was Ca(2+)-independent in each age group studied. The basal release and the responses to potassium stimulation were fairly constant during the whole life span. The release was potentiated by the glutamate receptor agonists kainate, N-methyl-D-aspartate (NMDA), tetrazolylglycine, quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) in developing mice, but only kainate was effective in adult and aged animals. The kainate effect was not modified by the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in adult and old mice, indicating that glutamatergic systems may not be involved in the release. On the other hand, hippocampal glycine release in immature mice seems to be subject to regulation by both NMDA and non-NMDA (kainate and AMPA) receptors. The potentiations by NMDA and AMPA were antagonized by dizocilpine (MK 801) and CNQX, respectively. The modulation of glycine release by glutamatergic receptors could be of importance in the regulation of synaptic glycine levels in the developing hippocampus. PMID- 7533870 TI - Effect of dietary high doses of vitamin E on lymphocyte subsets in young and old CBA mice. AB - Different theories of ageing exist. Oxidative stress by generating neo-antigens and affecting cellular communications may precipitate immune disregulation, and both may concur in the whole ageing process. In the present study, we have analysed the effect of dietary high doses of vitamin E, a free radical scavenger, on blood and spleen leukocytes and lymphocyte subsets, in young (3-month-old) and old (23-month-old) CBA mice. Age per se induced an increase in blood leukocytes, especially phagocytic cells, and a splenic atrophy, affecting both weight and cellularity. A differential effect of age on blood or spleen lymphocyte subsets was observed. In blood, there was a decrease in T cells, particularly CD4+ cells (T helper cells) and CD5+ cells without surface IgM (conventional T cells), along with a corresponding increase in B cells, principally B1 CD5+ cells (polyreactive autoimmunity-prone B cells). In the spleen, the relative percentage of each subset remained unchanged. Vitamin E supplementation for 7 weeks before sacrifice in young animals resulted in a decrease in CD4+ and CD5+ SIgM+ (putative Fc receptor positive) T cells and B1 cells in blood. Similar changes in the splenic lymphocyte subsets were found, while more leukocytes could be recovered from less weighty spleens. Considering the free radical scavenging properties of vitamin E, it was assumed that oxidative stress might play some role in the ontogenesis of the immune system in young adulthood. Vitamin E for 20 months before sacrifice in old animals did not prevent splenic atrophy and had no effect on splenocytes. In blood, the ratio of SIgM+ (assumed Fc-R+) to SIgM- CD5+ T cells decreased and the B1:B2 B-cell ratio increased. In this setting, oxidative stress seemed to play a lesser role in post-adulthood immune senescence. It must be stressed that no functional test was realized that could have uncovered qualitative changes in immune reactivity. Vitamin E supplementation had complex effects on the weight of our animals, so that influences on physical activity, energy metabolism and hormonal status should be considered in interpreting its impact on the immune system. However, the observed age-related changes in T-cell and B-cell subsets are consistent with diminished immune function and increased autoimmunity in senescence. Vitamin E, by a direct anti-oxidant effect or some other mechanisms, can prevent some changes and seems to decrease the potential for autoimmune reactivity. PMID- 7533871 TI - Tumor cell-enhanced sensitivity of vascular endothelial cells to photodynamic therapy. AB - Effective antitumor photodynamic therapy (PDT) may be related to damage of vasculature within the tumor. The purpose of this study was to determine if tumor cells secrete factors that stimulate proliferation of human umbilical vein endothelial cells (HUVEC) and result in enhanced sensitivity of HUVEC to aluminum sulfonated phthalocyanine (AlSPc)-PDT. Three human tumor cell lines--pharyngeal squamous carcinoma, colonic carcinoma, and mammary carcinoma--were used in this study. Co-culture of HUVEC and either squamous carcinoma or colonic carcinoma, but not mammary carcinoma, significantly increased HUVEC proliferation and AlSPc PDT mediated cell damage. In addition, supernatant from squamous carcinoma and colonic carcinoma cultures also stimulated HUVEC proliferation and sensitivity to AlSPc-PDT. Both supernatant and cell lysate from squamous carcinoma cells contained angiogenic factors consistent with basic and acidic fibroblast growth factors, as evidenced by Western blot analysis and BALB/c 3T3 fibroblast cell proliferation assays. Collectively, these results suggest that selected tumor cell lines produce angiogenic factors that induce HUVEC proliferation and subsequently enhance sensitivity to AlSPc-PDT. PMID- 7533873 TI - Effects of fluoxetine on basal and K(+)-induced tritium release from synaptosomes preloaded with [3H]serotonin. AB - Synaptosomes from rat brain cortex and spinal cord were preloaded with [3H]serotonin ([3H]5-HT), superfused and exposed to fluoxetine and/or 15 mM K+. In both regions 10 microM, but not 1 microM fluoxetine evoked a marked tritium overflow, about 2 min later than the immediate [3H]5-HT release induced by K+, and mainly (73%) due to the efflux of a tritiated metabolite of 5-HT, possibly [3H]5-hydroxy-indoleacetic acid. These findings confirm previous data in the rat hippocampus and are probably due to fluoxetine interacting with the 5-HT storage vesicles. One microM fluoxetine significantly reduced the d-fenfluramine-induced [3H]5-HT overflow, in accordance with its action as 5-HT uptake blocker, but did not affect the K(+)-induced [3H]5-HT overflow. This latter finding does not confirm that fluoxetine inhibits the depolarization-induced Ca(2+)-influx, suggested to involve a drug interaction with the L-type Ca(2+)-channels. Thus, the overflow induced by 10 microM fluoxetine was additive with the depolarization induced overflow, when the two stimuli were applied together. When 10 microM fluoxetine was added 7 min before 15 mM K+, there was no depolarization-induced overflow. Such inhibition might be only apparent and due either to the fluoxetine induced loss of vesicular 5-HT or to a fluoxetine-induced alterations of synaptic vesicles. The in vivo relevance of the fluoxetine releasing effect remains to be assessed. PMID- 7533872 TI - Protein kinases and phosphatases modulate c-fos expression in rat hepatocytes. Effects of angiotensin II and phorbol myristate acetate. AB - In isolated rat hepatocytes angiotensin II and phorbol 12-myristate 13-acetate (PMA) induce the expression of c-fos. We studied the possible transduction pathway(s) involved in this effect using inhibitors of serine-threonine and tyrosine protein kinases. Calphostin and staurosporine, inhibitors of protein kinase C and other serine-threonine protein kinases, block in a dose-dependent manner the effect of angiotensin II and PMA. Interestingly, genistein also blocks the induction of this proto-oncogene, suggesting a role for tyrosine protein kinases. Inhibitors of serine-threonine protein phosphatases, such as okadaic acid, microcystin LR and calyculin also induce c-fos expression. These data suggest that protein phosphatases exert a tonic inhibitory control of c-fos expression. The effect of these phosphatase inhibitors were not blocked by staurosporine, calphostin or genistein. Our results suggest that the expression of c-fos in rat hepatocytes is regulated by complex phosphorylation dephosphorylation cascade(s) probably involving serine/threonine and tyrosine protein kinase and protein phosphatase activities. PMID- 7533874 TI - [Palliative surgery of esophageal cancer in Djibouti: the value of colonic esophagoplasty]. AB - Because of delayed diagnosis in Black Africa, esophageal cancer is often seen at an advanced stage when dysphagia is total and eating becomes impossible. At this stage, palliative surgery is the only alternative to enable the patient to eat. Over a 27 month period in Djibouti, palliative therapy for esophageal cancer was indicated in 49 patients and surgery was performed in 37 of these patients. Out of 26 patients to whom gastrostomy was proposed, there were 12 that refused and 14 who underwent the procedure with a mean survival of 40 days. Esophageal bypass was performed in 23 patients. In 22 of these patients, the procedure was colon interposition and in the remaining case a gastric tube was used. There was one postoperative death. Excluding 3 patients that suffered cervical fistulas and one patient who required redo to remove a textiloma, postoperative recovery was uneventful. Patients resumed eating and were able to return home less than one month after the procedure. This study demonstrates that colon interposition can be performed in Black Africa despite limited availability of preoperative and postoperative intensive care facilities. It is a useful alternative for the management of malignant and benign stenosis. PMID- 7533875 TI - Drug mixture which improves survival of ischemic rabbit epigastric skin flaps. AB - The chief aim of this study was to maximize flap survival by counteracting the pathophysiological changes occurring during ischemia-reperfusion. Rabbit epigastric skin flaps given 21 hours of ischemia were infused intra-arterially with selected drugs at the start of reperfusion. Compared with control infused ischemic flaps, which had a 33% survival rate on day 7 post-ischemia, significant improvement was found with vasodilators nitrendipine (61%) and prostacyclin (65%) and the thrombolytic agent urokinase (65%); marginal improvement with the free radical scavenger desferrioxamine (53%); but no change with streptokinase (44%), heparin (21%), and ATP-MgCl2 (35%). A drug mixture comprising all of these agents except streptokinase and urokinase produced 87% survival, suggesting an additive effect. Biochemical assays on skin homogenates and blood implicated oxygen free radicals, neutrophil infiltration, and thromboxane in flap failure. These results imply that multiple factors are responsible for ischemic flap failure and that a mixture of drugs needs to be infused to counteract all of the detrimental changes. PMID- 7533876 TI - Physiological and pathophysiological roles of nitric oxide. AB - Nitric oxide (NO), identified as the biochemical messenger of endothelial dependent relaxation, is of obvious chemical simplicity, but the range and complexity of its biological actions are only now emerging. NO is an important determinant of vascular resistance, it reduces thrombogenicity of the vascular endothelium, contributes to non-specific, host-defence mechanisms, and is a neurotransmitter in the peripheral and central nervous systems. In addition to these physiological roles, there is now convincing evidence that excessive, prolonged production of NO contributes to tissue damage in septicemia, ischemia/reperfusion injury, and other inflammatory conditions. PMID- 7533877 TI - Nitric oxide synthase inhibitor, nitro-iminoethyl-L-ornithine, reduces ischemia reperfusion injury in rabbit skeletal muscle. AB - Nitric oxide (NO), originally identified as the mediator of endothelial-dependent relaxation of vascular smooth muscle, is now known to also have cytotoxic effects under certain conditions. Thus, we have investigated the effects of inhibition of NO synthesis on ischemia/reperfusion injury in the rabbit rectus femoris muscle. Three and a half hours of ischemia and 24 hours of reperfusion resulted in a 56% loss of viability. In muscles receiving an infusion of the nitric oxide synthase inhibitor, L-NIO (30 microM), the loss of viability was reduced to 15%. Post ischemic blood flow was increased in muscles receiving a saline infusion, whereas there was a marked decrease in blood flow for at least the first 60 minutes of reperfusion in muscles treated with L-NIO (30 microM). The increase in myeloperoxidase levels (indicative of neutrophil accumulation) following 24 hours of reperfusion was attenuated with L-NIO infusion by approximately 50% and the reperfusion-induced edema was also attenuated in L-NIO treated muscle. These findings suggest that endogenous NO production during ischemia/reperfusion injury may be deleterious to muscle survival. PMID- 7533878 TI - Nitric oxide synthase inhibitors improve skin flap survival in the rat. AB - The ability of nitric oxide (NO) synthase inhibitors to reduce ischemia-induced skin flap necrosis was assessed using a modified McFarlane flap in the rat. Flap survival was significantly improved in L-NIO treated (86 +/- 2%), L-NAME-treated (84 +/- 2%), and aminoguanidine-treated (76 +/- 2%) animals compared to the saline-treated group (54 +/- 2%), P < 0.005. Inhibition of NO synthase significantly decreased the hyperemia and edema within the flaps at 24 hours post elevation. These findings suggest that endogenous NO production contributes to ischemic necrosis and that inhibition of NO synthase may prove useful in extending survival of tissues subjected to ischemia. PMID- 7533879 TI - Use of free interpositional vein grafts as pedicles for prefabrication of skin flaps. AB - Pedicles created from a long vein graft increase the scope and applications of prefabricated skin flaps. This study reports the survival and pattern of neovascularization of lower abdominal skin flaps in rabbits based on a pedicle formed by interposition of a long vein graft between the divided ipsilateral femoral artery and vein. Flaps were elevated 2-5 weeks after pedicle implantation and the surviving area quantitated and vascular patterns examined 1 week later. Only 8 out of 35 flaps were greater than 50% alive, the most frequent cause of flap failure being pedicle non-patency. If the pedicle remained patent, complete flap survival was possible as early as 2 weeks after implantation. In non-patent pedicles, recanalization or formation of a new vascular network may, given at least 4 weeks, be sufficient to ensure partial flap survival. The findings indicate that implantation of a long, skeletonized vein graft is an unreliable method of prefabrication of abdominal skin flaps in this model. PMID- 7533880 TI - The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation. AB - Most of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F-like conjugative plasmids. Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system. The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression. Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target. Using electrophoretic mobility shift assays, we have shown that FinO is an RNA-binding protein that binds to one of the two stem-loops in FinP and to its complementary structure in traJ mRNA. This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP traJ mRNA duplex fivefold. Thus, TraJ expression appears to be influenced by a unique RNA-protein interaction that precedes duplex formation between the FinP antisense RNA and its target traJ mRNA. PMID- 7533881 TI - The copR gene product of plasmid pIP501 acts as a transcriptional repressor at the essential repR promoter. AB - The amount of the rate-limiting replication initiator protein RepR of plasmid pIP501 is negatively controlled by an antisense RNA (RNAIII) and a dispensable protein (CopR). Deletions or mutations in either component cause a 10-20-fold copy number increase. RNAIII induces transcription attenuation of the repR mRNA; the mode of CopR action remained unclear. To test the function of CopR, transcriptional fusions of promoters pI, pII and pIII with lacZ were integrated into the Bacillus subtilis chromosome. CopR and/or RepR were supplied in trans, and LacZ synthesis measured. The results show that CopR represses the repR promoter pII. Neither CopR nor RepR autoregulate their promoters. Gel mobility shift assays indicate that CopR binds to a 44 bp DNA fragment comprising the inverted repeat upstream of pII. PMID- 7533882 TI - Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. AB - The rfbKpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfbKpO1 cluster encode RfbAKpO1 and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan I-substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane-spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis ot the transport process. PMID- 7533883 TI - Mode of action of LciA, the lactococcin A immunity protein. AB - Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic, the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A. The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA. The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right side-out vesicles by proteinase K, it is located at the outside of the cell. The immunity protein contains a putative alpha-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell. Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin. PMID- 7533885 TI - An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma. AB - A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasma proteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol developed in our experiments involves coating well with 10 micrograms/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 micrograms/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levels in small samples of mouse plasma. PMID- 7533884 TI - The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site. AB - Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6-dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co-ordinate regulation of genes for erythromycin production and resistance. ermEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site-directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome binding site to give rise to the full-length 23S rRNA methylase. Deletion of the 35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the 'extended -10' class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their -10 regions and which initiate transcription seven nucleotides downstream of the -10 region. PMID- 7533886 TI - Report of the Meeting on Genetic Vaccines (Naked DNA/RNA) WHO Geneva, May 17-18, 1994. PMID- 7533888 TI - Update: Vibrio cholerae O1--Western Hemisphere, 1991-1994, and V. cholerae O139- Asia, 1994. AB - The cholera epidemic caused by Vibrio cholerae O1 that began in January 1991 has continued to spread in Central and South America (Figure 1). In southern Asia, the epidemic caused by the newly recognized strain V. cholerae O139 that began in late 1992 also has continued to spread (Figure 2). This report updates surveillance findings for both epidemics. PMID- 7533887 TI - [Cyfra 21-1--a new tumor marker of the cytokeratin series in differential diagnosis of lung diseases]. AB - BACKGROUND: Cyfra 21-1 is a novel marker for non small cell lung cancer. Up to now only few data about the value of Cyfra 21-1 in the diagnosis of malignant and non-malignant pulmonary diseases are available. Further the effect of long-time cigarette consumption on serum concentrations of Cyfra 21-1 has not been described. Aim of this study therefore was the determination of Cyfra 21-1 in different malignant and non-malignant pulmonary diseases and in healthy smokers and nonsmokers. PATIENTS: Sera of healthy individuals (control group, n = 121; 63 smokers and 58 nonsmokers), patients with chronic bronchitis (n = 50), lung fibrosis (n = 38), exogen allergic alveolitis (n = 32), lung tuberculosis (n = 45), sarcoidosis (n = 30), small cell lung cancer (n = 60), squamous cell carcinoma (n = 53), non-small cell carcinoma (n = 29) and adenocarcinoma (n = 52) were analyzed. RESULTS: Within the control group no significant differences of the Cyfra 21-1 serum concentration were observed between smokers and nonsmokers. Serum concentrations of Cyfra 21-1 were similar in the control group, patients with non-malignant pulmonary diseases and patients with small cell lung cancer whereas serum concentrations were significantly increased in patients with non small cell lung cancer, adenocarcinoma and squamous cell carcinoma. CONCLUSIONS: The data confirm the high sensitivity and specific of Cyfra 21-1 for the differential diagnosis between malignant and non-malignant pulmonary diseases as well as small cell and non-small cell lung cancer. PMID- 7533889 TI - [Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids]. AB - The interactions of HIV-I reverse transcriptase with some alkaloids were studied. Among nine compounds tested three--berberine, palmatine and sanguiritrine- inhibited RT. The dependence of the inhibition on the type of template-primer was also demonstrated. The kinetic analysis as well as circular dichroism experiments suggest the complex mechanism of RT inhibition by alkaloids. This mechanism includes both enzyme-alkaloid and alkaloid-template interactions; the latter effect also results in RT inhibition. PMID- 7533890 TI - [Features of the structure of transfer RNA coded by bacteriophage T5]. AB - Nucleotide sequence of 24 genes for the bacteriophage T5 tRNAs specific for all amino acids involved in protein synthesis was determined. All of them, except tRNA(Pro), were shown to differ significantly from the generalized "clover-leaf" structure consisting in the displacement of invariant and semi-invariant residues, the absence of pairing in the stems and some other deviations from the canonical parameters of the model. Basing on the available information on the functional activity of the phage-specific tRNAs, one can put forward a suggestion that, at least for some of them, the above anomalies do not essentially influence their activity. A comparison of phage T5 tRNAs with the Escherichia coli and phage T4 counterparts was carried out. The majority of the known elements determining the specificity of E. coli tRNA aminoacylation was also found in all phage T5 tRNAs, except tRNAPhe). PMID- 7533891 TI - Sensitive nonradioactive detection of UV-induced cyclobutane pyrimidine dimers in intact mammalian cells. AB - In this paper we present a sensitive procedure to determine specifically the induction as well as the removal of cyclobutane pyrimidine dimers in intact mammalian cells without radioactive labeling of the DNA. This technique allows the detection of DNA damage by UV doses as low as 0.1 J/m2. The method consists of gentle lysis of cell monolayers, high-salt treatment and incubation with the cyclobutane pyrimidine dimer-specific repair enzyme T4 endonuclease V, followed by alkaline unwinding, hydroxyapatite chromatography and fluorimetric DNA analysis. The number of T4 endonuclease V-sensitive sites correlates well with the amount of UV-induced cyclobutane pyrimidine dimers reported in the literature, indicating that these cyclobutane pyrimidine dimers are recognized quantitatively by the system. The assay is easily transferable to the detection of other types of DNA adducts by applying different damage-specific repair enzymes, providing a sensitive method to investigate the induction and the repair of DNA lesions without the use of radioactive labeling. PMID- 7533892 TI - Evaluation of the genetic toxicity of the organophosphate insecticide chlorpyrifos. AB - The genetic toxicity of chlorpyrifos [O,O,-diethyl-O-(3,5,6-trichloro-2- pyridinyl)phosphorothioate, C.A.S. Number: 2921-88-2)], an organophosphate insecticide, was examined by employing several end points such as gene mutations in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells both in vitro (rat lymphocyte chromosomal aberration test, RLCAT) and in vivo (mouse bone marrow micronucleus test) and induction of DNA damage and repair in rat hepatocytes in vitro. There was no indication of genotoxic activity for chlorpyrifos in any of these assays. These results are consistent with the reported lack of carcinogenic potential for chlorpyrifos in both mice and rats. PMID- 7533893 TI - The induction of dominant somatic mutations at the Dlb-1 locus. AB - In the small intestine of heterozygous mice (Dlb-1b/Dlb-1a), the Dlb-1b allele results in a stainable epithelium. The mutation or loss of the dominant Dlb-1b allele in a stem cell results in a non-staining ribbon of cells on a villus of the small intestine. To determine if dominant mutations resulting in the gain of staining--the induction of a Dlb-1b-like allele--could also be detected, we examined Dlb-1a homozygous mice (SWR) 2 weeks after a single treatment with 250 mg/kg ethylnitrosourea. Mutations to the dominant allele should appear as brown ribbons on unstained villi. Such ribbons were observed in the treated group but not in controls. The mutant frequency was low compared to the frequency of Dlb-1a like mutations reported at the Dlb-1b allele in heterozygous mice. PMID- 7533894 TI - Use of three-way differential staining and liquid holding for the assessment of individual repair capacity. AB - Many studies on DNA repair mechanisms in mammalian cells have used liquid holding (LH) recovery to evaluate premutational damage repair. We used human peripheral lymphocytes (HPL) to assess damage reduction during the G0 phase. This technique was matched with the three-way differential (TWD) staining that allows identification of sister-chromatid exchanges (SCE) per cell cycle in third metaphases. By adopting this approach, the persistence of diepoxybutane (DEB) induced lesions during subsequent cycles and individual repair capacity in LH conditions were measured. Our results show that most DEB-induced damage was repaired during the first cell cycle; a large part of lesions were removed during LH recovery, demonstrating G0 HPL repair capacity. PMID- 7533895 TI - Cdk-activating kinase complex is a component of human transcription factor TFIIH. AB - Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery. PMID- 7533897 TI - Significance of elevated thrombin-antithrombin III complex and plasmin-alpha 2 plasmin inhibitor complex in the acute stage of nontraumatic subarachnoid hemorrhage. AB - Thrombin-antithrombin III complex (TAT) and plasmin-alpha 2-plasmin inhibitor complex (PIC) were examined in the acute stage in 51 patients with nontraumatic subarachnoid hemorrhage. TAT and PIC values were correlated with severity at the time of onset and with outcome. In the patients whose TAT levels were 25 ng/ml or more and PIC levels were 3.0 micrograms/ml or more (n = 16), only 25% had a good or fair outcome. In the patients with TAT levels less than 25 ng/ml or PIC levels less than 3.0 micrograms/ml (n = 35), on the other hand, 82.9% had a good or fair outcome. There were no significant differences in TAT and PIC levels between patients who experienced arterial spasm and those who did not. These results indicate that TAT and PIC values may reflect the severity of the brain damage induced by subarachnoid hemorrhage. It is speculated that marked coagulation and fibrinolytic disorders occur in the acute stage of subarachnoid hemorrhage. PMID- 7533896 TI - Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops. AB - The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites. PMID- 7533899 TI - Cloning and functional expression of a Shaker-related voltage-gated potassium channel gene from Schistosoma mansoni (Trematoda: Digenea). AB - We have isolated a cDNA (SKv1.1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56.5 kDa) contains 6 putative membrane spanning domains (S1-S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64-74% identity in the core region (S1-S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2.8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1.1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdotoxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths. PMID- 7533898 TI - Preteaching developmentally delayed preschoolers to aid vision screening. AB - Early identification and treatment of vision problems is a key element in learning. For preschool children already identified with developmental delay, vision screening and visual correction are particularly essential. Participants drawn from a public Special School District were 105 preschoolers ages 3 to 5 years identified as having developmental delay. Examples included autism, Down syndrome, physical handicap, or cognitive impairment. A partnership was created between the public preschool and the University Schools of Nursing and Optometry to implement vision screening. Teachers/teacher assistants were asked to characterize the children before and after preteaching and after vision screening. Using a semantic differential scale of bipolar descriptors, planned comparisons within a repeated measures MANOVA were statistically significant for all pairs before teaching vs. the mean of after teaching/after screening (p = 0.027) but not statistically significant for after teaching vs. after vision screening (p = 1.000). Results of this preliminary study suggest preteaching could be an important part of a successful vision screening partnership in that 102 (97%) of developmentally delayed children successfully completed screening for vision problems. PMID- 7533900 TI - Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF 1, -3 and -4 in the liver-specific enhancer for the human alpha-1 microglobulin/bikunin precursor. AB - Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, 4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4. PMID- 7533902 TI - Human fibroblast growth factor 1 gene expression in vascular smooth muscle cells is modulated via an alternate promoter in response to serum and phorbol ester. AB - We have previously isolated the human FGF-1 gene in order to elucidate the molecular basis of its gene expression. The gene spans over 100 kbp and encodes multiple transcripts expressed in a tissue- and cell-specific manner. Two variants of FGF-1 mRNA (designated FGF-1.A and 1.B), which differ in their 5' untranslated region, were identified in our laboratory. Recently, two novel variants of FGF-1 mRNA (designated FGF-1.C and 1.D) have been isolated. In this study we used RNase protection assays to demonstrate expression of FGF-1.D mRNA in human fibroblasts and vascular smooth muscle cells and to show that promoter 1D has multiple transcription start sites. A single-strand nuclease-sensitive region has also been identified in the promoter 1D region that may have implications in chromatin conformation and transcriptional regulation of this promoter. Using Northern blot hybridization analyses, a previous study demonstrated a significant increase of FGF-1 mRNA levels in cultured saphenous vein smooth muscle cells in response to serum and phorbol ester. Here we confirm these results by RNase protection analysis and show that FGF-1.C mRNA is significantly increased in response to these stimuli. RNase protection assays indicate that promoter 1C has one major start site. The phorbol ester effect suggests that a protein kinase C-dependent signalling pathway may be involved in this phenomenon. Our results point to a dual promoter usage of the FGF-1 gene in vascular smooth muscle cells. Thus, normal growing cells primarily utilize promoter 1D. In contrast, quiescent cells, when exposed to serum or phorbol ester, utilize a different FGF-1 promoter, namely promoter 1C. Overall, these phenomena suggest mechanisms for increased production of FGF-1 that may play a role in inflammatory settings, wound healing, tissue repair, and neovascularization events and processes via autocrine and paracrine mechanisms. Our findings suggest that different FGF-1 promoters may respond to different physiological conditions and stimuli, in reference to the cell type or tissue milieu, resulting in ultimate production of the FGF-1 protein. PMID- 7533901 TI - A genetic algorithm based molecular modeling technique for RNA stem-loop structures. AB - A new modeling technique for arriving at the three dimensional (3-D) structure of an RNA stem-loop has been developed based on a conformational search by a genetic algorithm and the following refinement by energy minimization. The genetic algorithm simultaneously optimizes a population of conformations in the predefined conformational space and generates 3-D models of RNA. The fitness function to be optimized by the algorithm has been defined to reflect the satisfaction of known conformational constraints. In addition to a term for distance constraints, the fitness function contains a term to constrain each local conformation near to a prepared template conformation. The technique has been applied to the two loops of tRNA, the anticodon loop and the T-loop, and has found good models with small root mean square deviations from the crystal structure. Slightly different models have also been found for the anticodon loop. The analysis of a collection of alternative models obtained has revealed statistical features of local variations at each base position. PMID- 7533904 TI - Overview of antiretroviral therapy. AB - All of the agents that are available for the treatment of human immunodeficiency viral infection belong to the class of drugs called nucleoside analogs that act on the virus's reverse transcriptase enzyme. As their use expanded for increasing cohorts of patients and stages of disease, it became clear that additional agents were required that would act at different points in the virus's life cycle. Several different classes of drugs have been identified and evaluated in the laboratory and the clinic. At this point, one of the most promising that is undergoing clinical trials is the proteinase inhibitors. It is important to assess the data for currently available drugs and use that information to determine the most appropriate role for proteinase inhibitors. To appreciate their potential role best, we must also glance over the horizon at other experimental treatments. PMID- 7533903 TI - Recognition of duplex DNA by RNA polynucleotides. AB - We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence. PMID- 7533905 TI - Analysis of the UV-induced melanogenesis and growth arrest of human melanocytes. AB - Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm2 UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G2-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 microM alpha-melanocyte stimulating hormone (alpha-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of alpha-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers. PMID- 7533907 TI - [Hematopoietic stem cells of the human. Function and morphology]. AB - Pluripotential haematopoietic stem cells and their progeny, the so-called committed precursor cells, i.e., progenitor cells which are already lineage restricted, may be identified by the membrane-bound expression of CD34. In accordance with this peculiar property it became possible to enrich and characterize primitive precursor cells by using different methods of cell separation techniques, which involved fluorescence staining or ferro-magnetic particles bound to CD34 antibodies. Recently conducted studies demonstrate that CD34-positive (CD34+) stem cells of the peripheral blood represent a relatively uniform cell population with almost round nuclei, a finely dispersed chromatin pattern and a small portion of weakly basophilic cytoplasm. From the cytological viewpoint they resemble so-called large stimulated lymphocytes (virocytes). Ultrastructural studies are compatible with a paucity of organelles and a lymphoid character of these progenitors. In comparison, the stem cell population, derived from the bone marrow consists of more heterogeneous elements. These are generally larger and reveal an admixture of fairly immature as well as more differentiated cells, sharing bean-shaped or indented nuclei with prominent nucleoli and a more extended cytoplasm. CD34+ progenitors from the peripheral blood and those from the bone marrow display a co-expression of CD43 (MT1) and CD45 (LCA). Furthermore, different subpopulations exhibit--dependent on their origin (blood/bone marrow) and to a various extent--lineage-restricted markers like CD33, CD38, CD61, CD20, CD11a/c, glycophorin C und CD15 (LeuM1). The recently developed immuno- and ferromagnetic enrichment methods for CD34+ progenitor cells are considered innovative tools for modern oncology. These techniques play an important role in the treatment of haematological malignancies and advanced tumours in the context of autologous and, although so far rarely applied, heterologous stem cell transplantation procedures. PMID- 7533906 TI - Role of beta 1 integrins in cell spreading and migration of human nevomelanocytes and dysplastic nevi cells on collagen type IV and laminin. AB - We characterized beta 1 integrin subunit expression on three different cultures of benign human nevomelanocytes (NMC) and on four different cell cultures of human dysplastic nevus (DN) cells by flow cytometry analysis and examined their role in mediating cell spreading and migration on collagen type IV (CN IV) and laminin (LN) coated substrates by using a quantitative video image analysis system. The seven human NMC and DNC cultures expressed heterogeneous levels of beta 1, alpha 2, alpha 3 and alpha 6 integrin subunits. Image analysis showed that a significant increase (P < 0.001) in cell spreading and migration of the DN cells was induced on increasing coating concentrations of CN IV and LN. However, the NMC did not show an increase in cell spreading or migration on these substrates when compared to the substrates coated with denatured BSA only. The CN IV-induced cell spreading of the DN cells was significantly inhibited by anti beta 1 mAb (AIIB2), anti-alpha 2 mAb (P1E6), or anti-alpha 3 mAb (P1B5), but not by mAb against alpha 6 integrin subunit (GoH3). The DN cell spreading on LN was not significantly inhibited by these mAbs. In contrast, the migration of the DN on CN IV and LN was significantly inhibited by anti-beta 1 mAb, anti-alpha 2 mAb, anti-alpha 3 mAb and anti-alpha 6 mAb. These data suggest that the alpha 2 and alpha 3 subunit are important for cell spreading of the DN on CN IV, although they are less important in cell spreading on the extracellular matrix component LN.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533909 TI - Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line. AB - A cloned Drosophila gamma-aminobutyric acid GABA receptor subunit (Rdl) has been stably expressed as a functional homo-oligomeric ion channel in a Drosophila cell line. Stably-transfected clonal cell lines which expressed high levels of GABA receptor were identified by specific [3H]-muscimol binding. Expression of functional GABA-gated ion channels in these cell lines was demonstrated by electrophysiological recording. Rapid and pronounced rundown of responses to GABA during whole-cell patch clamp recordings was overcome by the inclusion of EGTA in the pipette solution, indicating a possible role for calcium-dependent processes in the functional regulation of this GABA receptor. Relative agonist potencies of the expressed receptor were found to be in the order GABA = TACA > CACA. We have observed a reversible block of the receptor by the convulsant antagonists, picrotoxinin and EBOB, and by the insecticide fipronil. Potentiation of GABA responses was seen with the anaesthetic steroid 5 alpha-pregnan-3 alpha-ol-20 one. No significant effects (either agonist, antagonist or modulatory) were observed with bicuculline (a vertebrate GABAAR antagonist), benzodiazepines or barbiturates (vertebrate GABAAR modulators), or with glycine agonist of the closely related vertebrate glycine receptors). The suitability of this Drosophila stable expression system for the characterization of receptors and ion channels is discussed. PMID- 7533908 TI - Serotoninergic and peptidergic nerve elements in the protoscolex of Echinococcus granulosus (Cestoda, Cyclophyllidea). AB - The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and neuropeptides in the nervous system of the protoscolex of the hydatid organism Echinococcus granulosus were determined by an indirect immunofluorescence technique. Nerve-cell bodies immunoreactive for 5-HT occurred in the lateral ganglia and in association with the lateral longitudinal nerve cords. 5-HT immunostaining was also evident in the central nerve ring, in the rostellar nerves and in the nerve plexus innervating the suckers. Of the antisera used to screen the protoscolex for neuropeptide immunoreactivity (IR), immunostaining was obtained with those raised against pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP). The most extensive pattern of IR occurred with antisera to PP and PYY. Immunoreactive nerve elements were evident in the lateral ganglia, central nerve ring, rostellar nerves, rostellar ganglia, sucker plexus and longitudinal nerve cords. The distribution of SP-, PHI- and VIP-IRs was more restricted: SP-IR occurred in the lateral ganglia and sucker nerves, whilst PHI- and VIP immunoreactive nerve elements were associated with the lateral longitudinal nerve cords. Protoscoleces cultured in vitro for 29 days were also examined and neuroanatomical changes noted. A greater development of the longitudinal nerve cords and their cross-connectives in the body of the worm was evident, and a group of nerve cells were seen to develop at the posterior end of the main lateral nerve cords. PMID- 7533910 TI - Effects of intraventricular injection of morphine and beta-endorphin on serotonin release from the spinal cord in rats. AB - Effects of intraventricular (third ventricle) injection of morphine and beta endorphin on the release of serotonin (5-HT; 5-hydroxytryptamine) and 5-HIAA (5 hydroxy indolacetic acid) from the spinal cord were studied using urethane anesthetized spinally perfused rats. Intraventricular injection of morphine (25 micrograms) increased the 5-HT level in the perfusate about threefold. The increase of 5-HT release reached at peak between 30 and 60 min after the first injection of morphine. However, the levels of 5-HIAA, a metabolite of 5-HT, was not significantly altered by intraventricular injection of morphine. Furthermore, second intraventricular injection of morphine at the same dose did not increase 5 HT level in the spinal perfusate. In contrast to the results with morphine, beta endorphin (10 micrograms) administered intraventricularly did not alter the release of 5-HT and 5-HIAA from the spinal cord. In addition, acute antinociceptive tolerance to intraventricular morphine induced by a prior intraventricular injection of morphine was studied in pentobarbital anesthetized rats. Acute tolerance was induced by intraventricular pretreatment with morphine (20 micrograms) for 120 min and the same dose of morphine was injected intraventricularly. The tail-flick test was used as an antinociceptive test. Pretreatment of rats with morphine intraventricularly reduced inhibition of the tail-flick response to intraventricularly injected morphine. The results support our previous hypothesis that beta-endorphin and morphine administered supraspinally activate separate descending systems. Spinopetal serotonergic descending pathway is selectively activated by intraventricularly injected morphine but not beta-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533911 TI - Axonal transport of the trkA high-affinity NGF receptor. PMID- 7533912 TI - Neurotransmitters and cytokines in CNS pathology. AB - In summary, we have demonstrated an in vitro model for oligodendrocyte cell death that may be relevant to events in formation of lesions in MS. It involves cell contact to oligodendrocytes with activated, viable microglia (or inflammatory macrophages), surface TNF-alpha, surface adhesion molecules, and production of NO. Precise mechanisms of TNF-alpha and ICAM-1/LFA-1 participation and the nature of the susceptibility of the oligodendrocyte are currently being studied. PMID- 7533913 TI - Receptor mediated retrograde axonal transport of neurotrophic factors is increased after peripheral nerve injury. PMID- 7533914 TI - Nitric oxide: cellular regulation and neuronal injury. PMID- 7533915 TI - The role of free radicals in NMDA-dependent neurotoxicity. PMID- 7533917 TI - [Human mitochondria and mitochondrial genome function as a single dynamic cellular unit]. PMID- 7533916 TI - Distribution of acidic and basic fibroblast growth factors in the mature, injured and developing rat nervous system. PMID- 7533918 TI - [Scene and symbol as driving forces. The unconscious fantasy in the transference]. AB - Scene and Symbol as Motive Forces. Unconscious fantasy in transference.- Psychoanalysis regards unconscious fantasy as the motive force in human life. The analytic situation provides a framework for the crystallisation of unconscious fantasies. In the transference process fundamental scenes from childhood are reenacted, organised in terms of symbols and charged with meaning. With reference to a number of actual cases, the author demonstrates that scenically condensed, repressed conflicts thus develop psychic virulence and that in this scenic context the so-called Zeigarnik effect brings the unconscious fantasy to a standstill, allowing it to live on in the memory as an unconcluded action. Conclusion is however a necessary condition for forgetting. Only when this standstill is overcome via transference is psychic growth possible again. PMID- 7533919 TI - Serotonergic activity in rat cerebral arteries depends on dorsal raphe nucleus but not on median raphe nucleus. AB - The effect of Chlorimipramine and Muscimol on serotonergic activity in rat cerebral arteries and in dorsal and median raphe nuclei were used to study the presence of a serotonergic innervation in the cerebral blood vessels functionally dependent on the brainstem nuclei activity. Serotonergic activity was appraised in rat cerebral arteries from 5-hydroxyindoleacetic acid (5-HIAA) disappearance rate or 5-hydroxytryptophan (5-HTP) accumulation after inhibiting monoamine oxidase (MAO) or aromatic amino acids decarboxylase, respectively. In dorsal and median raphe nuclei the decay with time of 5-HIAA after MAO inhibition was used to estimate serotonergic activity. Chlorimipramine significantly reduced serotonergic activity in cerebral blood vessels and in both raphe nuclei. 5-HIAA basal levels in these blood vessels were not altered by treatment with the drug. Muscimol evoked only a decrease in the serotonergic activity of the median raphe nucleus. These results suggest that rat cerebral arteries receive serotonergic fibers functionally active arising mainly from dorsal raphe nucleus. PMID- 7533920 TI - The "Sanchez-Medal" Lecture Award: nitric oxide 1994. PMID- 7533921 TI - [Palliative care. Various concepts revisited]. PMID- 7533922 TI - [What new is there in drug therapy? In the years 1992-1994. 2]. PMID- 7533924 TI - Prostatism symptoms in a randomly selected population: introduction of a patient administered population based symptom evaluation score. AB - A questionnaire concerning micturition symptoms and bother was answered by 2559 (66%) randomly selected males. Voiding problems increased with age from about 4 to 40%. The symptoms were weakly but significantly correlated to each other (rs < 0.55). The ability of a particular symptom question to predict whether a subject actually would seek a doctor was generally low. A score system with a maximum score of 33 points was constructed. If prostatism was defined as a certain score or higher, a change in the definition in the score interval 4-10 would change the number of subjects with this syndrome with approximately 10%/score point. As a consequence of the results, the use of a symptom score as a criterion for the decision to treat patients suffering from prostatism is challenged. A patient administered symptom evaluation from is strongly recommended to obtain a more objective symptom registration. PMID- 7533926 TI - Five year follow-up after radical prostatectomy for localized prostate cancer--a study of the impact of different tumor variables on progression. AB - Fifty-one patients with clinically localized prostate cancer stages A and B, who underwent radical prostatectomy have been followed for a minimum of 5 years. The impact of age, stage, capsular penetration, total tumor volume, Gleason score, seminal vesicle invasion and lymph node metastases on progression has been evaluated. Progression free survival was calculated according to the Kaplan-Meier method. Uni- and multivariate analyses were performed according to the Cox proportional hazards model. During the observation period 16 patients (31%) experienced progression. Tumor volume, grade and seminal vesicle invasion emerged as statistically significant predictors of tumor progression in the survival analyses while age at surgery, preoperative stage and different levels of capsular penetration were not statistically significant. The findings in the Cox models were in accordance with those at actuarial survival analyses though tumor volume was the only variable proven to have an independent statistically significant influence on progression. PMID- 7533925 TI - Influence of benign prostatic hyperplasia, testosterone and age on serum levels of prostate specific antigen. AB - Prostate specific antigen (PSA) and PSA-density (PSAd) were studied in 120 symptomatic patients with benign prostatic hyperplasia at digital rectal examination. PSA and PSAd were measured before and 3 months after transurethral resection. Comparisons were made between patients in whom histologic examination showed benign hyperplasia and those with histologically demonstrated prostatic cancer. The influence of age and of serum testosterone on PSA and PSAd was determined. PSAd in benign prostatic hyperplasia was the same in the transition zone (the adenomas) and the peripheral zone (mean 0.065), indicating no increased synthesis or leakage of PSA. PSAd with cut-off value 0.10 ng x ml-1 x cc-1 showed higher sensitivity and positive predictive value (75% vs 50% and 0.33 vs 0.15, respectively) in nonpalpable prostatic cancer than did PSA with cut-off at 4 ng x ml-1. No statistically significant influence of testosterone and/or age on serum PSA or PSAd was found. PMID- 7533923 TI - The role of the L-arginine-nitric oxide pathway for peristalsis in the opossum oesophageal body. AB - BACKGROUND: The aim of the study was to determine the effect of NG-nitro-L arginine (L-NNA), an inhibitor of nitric oxide (NO) synthesis, on primary peristalsis in the oesophageal body. METHODS: Peristalsis was induced by pharyngeal stroking in 14 lightly anaesthetized opossums. Oesophageal pressures were monitored with a four-channel, perfused catheter assembly and registered with external transducers 1, 4, 7, and 10 cm proximal to the oesophagogastric junction. Propagation time was the time taken for a contraction to travel between two recording sites and was determined in the proximal, middle, and distal parts of the oesophagus (propagation time between 10 and 7 cm, 7 and 4 cm, and 4 and 1 cm recording sites, respectively). RESULTS: L-NNA (10(-7)-10(-5) mol/kg) dose dependently reduced propagation time of the contraction in the distal oesophagus from 1.13 +/- 0.24 sec to 0.27 +/- 0.19 sec, whereas propagation in the proximal and middle parts of the oesophagus was unaffected. NG-nitro-D-arginine (D-NNA; 10(-5) mol/kg) had no influence on propagation time. In animals treated with L NNA (10(-5) mol/kg) atropine (50 micrograms/kg) had no influence on propagation time in any part of the oesophagus. L-Arginine (10(-4) mol/kg) had no influence on the propagation time in animals treated with L-NNA (10(-5) mol/kg) and atropine (50 micrograms/kg). Neither D-NNA (10(-5) mol/kg) nor L-NNA (10(-7)-10( 5) mol/kg) influenced the amplitude of the contractions at any of the recording sites. In animals given L-NNA (10(-5) mol/kg) atropine (50 micrograms/kg) reduced the amplitude of the contraction significantly only at the distal recording site (1-cm recording site) from 62.0 +/- 4.9 mmHg to 34.5 +/- 5.3 mmHg. L-Arginine (10(-4) mol/kg) had no effect on the amplitude of contractions. CONCLUSION: The L arginine-NO pathway plays a role in the control of primary peristalsic contractions of the oesophagus. PMID- 7533928 TI - Canine distemper virus and myelin mRNAs in oligodendrocytes. PMID- 7533927 TI - Segmental pruritus and intramedullary vascular malformation. AB - Pruritus remains a medical mystery. Generally related with dermatological diseases, it is rarely associated with a neurological cause. We report a case of segmental pruritus (right T2 dermatome) related to an intramedullary vascular malformation. An anatomicophysiological explanation is presented. It is the first demonstration in man that pain receptors are not involved in pruritus, and suggests pruritus is under the control of descending inhibitory pathways. PMID- 7533930 TI - Up-regulation of clusterin (apolipoprotein J) and apolipoprotein E in cultured rat spinal cord neurones. PMID- 7533929 TI - Heterologous gene expression of a novel brain-specific protein tyrosine kinase. PMID- 7533932 TI - Myelin proteolipid protein mutation in the rabbit: a new model of Pelizaeus Merzbacher disease. AB - Proteolipid protein (PLP) is a major myelin protein of the central nervous system. Mutations of the Plp gene are responsible for a number of sex-linked disorders in humans (Pelizaeus-Merzbacher disease) and in animals. We have identified a novel mutation of the Plp gene which gives rise to the paralytic tremor (pt) phenotype in rabbit. Pt rabbits are hypomyelinated and present very low levels of PLP protein and its mRNA. Sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. Histidine36 is positioned at the boundary of the first transmembrane domain. Therefore, its position can be crucial for the efficient interaction of PLP with other proteins and lipids, and for correct incorporation into the membrane. PMID- 7533931 TI - Expression patterns of human PNS myelin proteins in neuropathies associated with anti-myelin antibodies. PMID- 7533933 TI - Mature oligodendrocytes in three-dimensional brain cell culture respond to protein kinase C stimulation by dedifferentiation, proliferation and remyelination. PMID- 7533934 TI - Autoantibodies in neurological paraneoplastic diseases. AB - Paraneoplastic syndromes such as the subacute sensory neuronopathy (SSN) and paraneoplastic cerebellar degeneration (PCD) are associated with autoantibodies directed against various neural antigenic structures. SSN is characterised by autoantibodies against a neuronal intranuclear component called Hu (M. W. 35 to 40 kD), whereas in PCD these antibodies are directed against a Purkinje cell cytoplasmic component called (M. W. 34 and 65 kD). Neuroblastoma cell lines maintained in culture have been shown to contain neuronal antigens. We have demonstrated the presence of the Hu antigen in neuroblastoma cell lines such as SKN-SH, LAN-1 and IMR-32 by both immunocytochemistry and immunoblots of nuclear extracts. The Yo paraneoplastic antigen has been found to be expressed in HeLa cells. These methods may be used for screening of patients with suspected paraneoplastic disease and/or malignancy. Western blot analysis appears to be superior to immunohistochemistry alone. In our experience only 11 out of 122 SCLC patients were positive for the anti-Hu antibody, whereas 5 out of 5 patients with SSN/SCLC were positive, and all of the neurological controls were negative. The availability of cell lines expressing paraneoplastic antigens offers an easy diagnostic assay which may complement or replace conventional immunohistochemistry. PMID- 7533935 TI - Production and function of IL-10 in the central nervous system. AB - Our data suggest an important regulatory role for IL-10 within the central nervous system by a) reducing the antigenpresenting capacity of astro- and microglia and thereby lowering or preventing, antigen-specific proliferative T cell responses, and b) inhibition of inflammatory cytokine production by the resident parenchymal cells as well as from infiltrating monocytes/macrophages. In the latter instance the signals triggering cytokine production are crucial since IL-10 interferes with bacterial but not viral or cytokine-induced activation of expression of cytokine genes. PMID- 7533936 TI - Short and longterm fluctuations of generational MS risk. AB - According to the hypothesis that childhood is an important age stage in view of MS predisposition, alterations of MS risk may be expected in respect of generational succession. Age-period-cohort-analysis in the appropriate statistical analysis model in this connection. After having shown considerable fluctuations of generational MS risk in the long term view we have tried to derive additional informations from corresponding short term fluctuations: descriptive informations as well as results in relation to the short term fluctuations of infectious diseases' mortality. While applying again age-period cohort-analysis the results are questioned by the fact that the underlying cohort estimates represent 5-years-moving-averages. Computerized individual death records of the Swiss mortality statistics available since 1969 will enable us to check the recent results. PMID- 7533938 TI - Evidence for a nonneuronal receptor for axonin-1 and NG-CAM. PMID- 7533937 TI - Evaluation of a computer-based attention retraining program for patients with multiple sclerosis. PMID- 7533939 TI - [Cardiac side effects and ECG changes with lamotrigine?--A clinical study]. AB - After a patient treated with lamotrigine (LTG) had suffered from thoracal pain and repolarisation deficiencies in the electrocardiogram (ECG), we prospectively investigated 20 patients with localization-related epilepsies to assess cardiovascular side effects of LTG. During the study, LTG was added to a stable dosage of one to three first line antiepileptic drugs (AED). Physical examinations and ECG recordings were performed regularly. Clinical cardial side effects did not occur. Only in single ECG traces of six patients non specific abnormalities were seen. Only in one patient, the abnormalities (supraventricular extrasystoles and a first grade atrioventricular block) unequivocally appeared initially under LTG. However, in the same patient two later ECG recordings under higher LTG dosages were normal. The statistical analysis of the ventricular activation times did not reveal any significant changes under LTG (p = 0.05) with the exception of an extended PQ time when comparing a patient group before LTG (n = 11) with another patient group under LTG (n = 11). Since this observation was not confirmed by the statistical analysis of a third patient group (n = 10) with ECG data prior to and under LTG, we believe that the statistically significant changes mentioned above were based mainly on structural differences between the patient groups and not on true LTG effects. Our results do not give evidence for an elevated risk of clinically relevant cardial side effects under LTG. PMID- 7533940 TI - Paroxetine in the treatment of inpatients with non-delusional endogenous or neurotic depression. AB - The clinical course of 35 depressed inpatients (19 women, 16 men; 19 endogenous, 16 neurotic depression (ICD-9 diagnoses); mean age 46.7 years) who were treated with paroxetine 20 mg/d was observed over 21 days using the Hamilton Depression Scale (HAMD). For both types of depression there was a significant decrease from day 1 to day 21 in the mean HAMD total score and in a subscore of cognitive and somatic items. The mean value of the HAMD suicide item increased in 3 cases and decreased in 25. PMID- 7533941 TI - [New psychopharmacologic alternatives in treatment of schizophrenia]. AB - Clozapine, risperidone and remoxipride are three neuroleptics that represent an interesting alternative in the psychopharmacological treatment of schizophrenia. Pharmacodynamic and pharmacokinetic properties, efficacy, dosages as well as the indication of these three substances are studied. In certain particular situations, a complementary treatment is of important therapeutic use, the addition of benzodiazepines, lithium, carbamazepine or beta-blockers are discussed. PMID- 7533942 TI - [Personal reaction and understanding in interactions with the psychotic patient]. AB - The understanding of the psychotic individual is represented as a reciprocal process between patient and doctor, which is founded on emotional assumption; concerning also the psychiatrist's self-experience. Psychodynamic and phenomenological aspects of this interpersonal process are demonstrated. PMID- 7533943 TI - [Alcoholism: from the molecular to public health]. AB - The current knowledge on the interactions between alcohol and the central nervous system moves very quickly. Moreover the scientific advances follow the three axes of our understanding, biological, psychological and sociological, but this implies some dispersion. The present paper focuses on recent advances in neurobiology of alcoholism and shows new perspectives in pharmacological interventions, with new drugs. In the same time, the psychodynamic and systemic understandings are evoked, in a purpose of integration and of public health policy. PMID- 7533944 TI - [Social control, social support and therapy. The tension field of (social )psychiatric treatment]. AB - As generally in psychiatry the treatment concepts in social psychiatry base to a great extend on disease- and deficit models. These models, however, have their limits and run the risk to promote exactly that, what social psychiatry is fighting against: chronification. The question shall be discussed, if the systemic approach could be helpful to reduce (social) psychiatry's contribution on the process of chronification. Since ever psychiatric institutions have the mandate of social control and exclusion of social disturbing people from society. On the other hand, social support has become a cornerstone of rehabilitation in psychiatry. There the therapist becomes the manager of the disability. The (case )manager subsequently takes over the responsibility for the future course. So he also gets power over the system to be treated. He decides, how much of social support and control is necessary. But then he gives up the aim of profoundly changing something in the sense that the patient can give up his symptoms and take more self-responsibility for his acting. The consequence is that the patient and psychiatry keep up each other: a coevolution develops. This leads to rigidity and makes changes difficult. By taking over responsibility the therapist becomes part of the system to be treated. So he looses his neutrality and also the possibility of therapeutical influence and change. The whole system quickly moves into a vicious circle of helplessness. Finally it will be reflected how someone, working in an institution, still can do therapy in this field of tension of (social) psychiatry. PMID- 7533945 TI - [Changed perception in depressive diseases--an overview]. AB - This paper attempts to give a comprehensive survey of the disturbed perception in depressive disorders. Therefore results from clinical psychopathological research as well as from experimental studies are presented. Introductory remarks consider problems of this task that arise from the missing consistent definition of perception and the manifold classifications of affective disorders. Phenomenology and anthropological interpretations of the disturbed time- and space-experience are the main topic. Among the alterations of the subjective time-experience disturbances of "erlebnisimmanente" time and reference to the future (according to Straus and v. Gebsattel) are of special importance. Up to now studies on the time estimation of depressive patients that can be located at the interface between phenomenologically deducible subjective and experimentally determinable objective time-experience have shown different results.--The detailed description and analysis of the disturbed space-experience goes back to Tellenbach, who demonstrates applicable structures for this as well as consequences for the existential determination of life.--Furthermore disturbances of self-perception in patients with depressive disorders and their alienation of the perceptual world in its wholeness are demonstrated. The discussion of the so far reported results takes special account of their diagnostic value and calls for clinical and experimental procedures first of all at continuing the differentiated and by means of replication studies substantiated apprehension of perceptual disturbances. PMID- 7533947 TI - Dicumarol content in alcoholic herb elixirs: one of the factors at risk induced IVKD-I. AB - Nine medicinal plants known to be the ingredients of the traditional herbal medicinal elixir, and seven popular commercial alcoholic herb elixirs were investigated for the content of dicumarol by using high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) methods. Umbelliferae (Conioselinum Univittatum) were the only medicinal plants found to contain dicumarol 0.04 mg/dl. Dicumarol content was also found in three out of seven brands of commercial alcoholic herb elixirs with the concentration of 0.58, 1.86 and 6.00 mg/dl. These findings indicated that the traditional herbal medicinal elixirs containing dicumarol in varying amount may play a role in inducing bleeding diathesis in breast-fed infants of mothers known to consume the elixir. PMID- 7533948 TI - Beacons, breasts, symbols, sex and cancer. AB - Since the 1950's effective control of conception has allowed modern men and women to differentiate procreational from recreational sexual exchange. What is considered highly erotic has differed widely through time and in various cultures. In the U.S. the female breast has come to mean far more than nurturing an infant. Sexuality symbolizes youth, attractiveness, desirability and as such is used for effective commercial marketing. The reality of cancer remains to be dealt with in health care at a physical level but more is needed to assist the emotional adjustment of affected women and their partners. Brief sex therapy for sex symptoms is outlined. PMID- 7533946 TI - Prevalence of antibodies to hepatitis C virus in relation to surrogate markers in a blood donor population of Singapore. AB - Prevalence of antibodies to HCV is studied among a blood donor population in Singapore and its relationship to surrogate markers was examined. Sequential serum samples from 4,091 blood donors were tested for the presence of anti-HCV using the second generation immunoassay (Abbott). 275 random serum samples were tested for anti-HBc and ALT. All the samples positive for anti-HCV were also tested for anti-HBc and ALT. Only 22 of the 4,091 donor samples (0.54%) were repeatedly reactive for anti-HCV. Of the 275 random samples tested, 43 samples (15.6%) were positive for anti-HBc and 24 (8.7%) had ALT levels more than 45 IU/l. None of these 67 samples were positive for anti-HCV. Only 3 of the 22 anti HCV positive samples (13.6%) were positive for anti-HBc and only 6 samples (27.2%) had ALT level more than 45 IU/l. The prevalence of anti-HCV among the donors is only 0.54% which is much lower than the prevalence of HBV. An important finding is that about 60% of the donors positive for anti-HCV had no detectable surrogate markers. Exclusion of blood donors positive for anti-HBc, if implemented in an area where the prevalence of HBV infection is relatively high will result in the loss of blood donors estimated to be 15.6% and the use of raised ALT will result in a further loss of 6.1% of the blood donors. PMID- 7533949 TI - Multiple brushings with immediate Riu's stain via flexible fibreoptic bronchoscopy without fluoroscopic guidance in the diagnosis of peripheral pulmonary tumours. AB - BACKGROUND: Accurate diagnosis of peripheral pulmonary lesions usually relies on fluoroscopic guided procedures. As fluoroscopy is not routinely available in many respiratory units, an approach not using fluoroscopy but with a high diagnostic yield is highly desirable. METHODS: Immediate cytological examination of multiple brushings using Riu's stain, a modified Wright's stain, was performed in 38 patients with peripheral pulmonary lesions not visible at bronchoscopy. The results were compared with the final diagnoses determined by histological examination or subsequent Papanicolaou staining of cytological specimens and clinical course. RESULTS: Of the 38 patients 29 were subsequently confirmed to have a malignant tumour. Our method provided a diagnosis of malignancy in 86% of these lesions. The accuracy (91%) and sensitivity (88%) were higher for lesions > 3 cm in diameter than for those of diameter < or = 3 cm (87% and 83%). There were no false positive results. The 29 lesions correctly diagnosed as malignant by Riu's stain required significantly fewer brushings (mean (SD) 3 (2)) than the nine benign lesions (5 (4)). CONCLUSIONS: This technique provides a high diagnostic yield, avoids the need for fluoroscopy, and is probably safer than percutaneous biopsy. PMID- 7533950 TI - Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA2 subunit of crotoxin and neutralize its pharmacological activity. AB - Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A2. Polyclonal antibodies against three synthetic peptides selected from the C terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin. PMID- 7533952 TI - Use of anticoagulants in patients with acute ischemic stroke. PMID- 7533953 TI - A clinico-pathological study of cutaneous leishmaniasis in British troops from Belize. AB - Thirty-four cases of cutaneous leishmaniasis contracted by British soldiers in Belize were studied. Pre- and post-treatment biopsies were taken from all patients. The range of histological appearances is described and the value of histological examination (including Giemsa staining and immunohistochemistry), cytological preparations and microbiological culture in diagnosis and clinical management assessed. Histology and culture were found to be complementary techniques in reaching a positive diagnosis, whilst cytological preparations were of no additional value. Histological examination of post-treatment biopsies merely confirmed the clinical impression of healing or non-healing whilst culture identified viable organisms in apparently healed lesions, which were subsequently re-treated. PMID- 7533951 TI - Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus. AB - The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+) activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa. PMID- 7533954 TI - Thiacetazone--use with care. PMID- 7533955 TI - Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement. AB - Rapamycin (RPM) and mycophenolic acid (MPA) inhibit immune responses by antagonizing IL-stimulated lymphocyte activation. These 2 drugs, used alone or preferably in combination, also significantly reduced the response of vascular cells to balloon-catheter arterial injury in rats. When rats were treated for 2 weeks with both drugs starting the day of injury, intimal thickening was significantly reduced (P < 0.001) 14 days after injury; however, by 44 days after injury, intimal thickening had progressed to the extent measured in arteries of untreated control rats. When RPM and MPA were administered for 3 days before and 13 days after injury, arterial intimal thickening was significantly (P = 0.024) reduced and endothelium had regrown in vessels analyzed 44 days after injury. Compared with initiation of treatment on the day of injury, starting the administration of RPM plus MPA before injury appears to limit the activation of cells or actions of factors responsible for the progression of intimal thickening that occurred after the administration of the drugs was terminated. RPM and MPA prevented the development of arterial intimal thickening in a model not dependent upon a rejection response. This direct antiproliferative action on smooth muscle cells by RPM and MPA, in vivo, may prevent the development of arterial intimal thickening associated with chronic rejection. PMID- 7533956 TI - Insulin-like growth factor-I improves mucosal structure and function in transplanted rat small intestine. AB - The transplanted small intestine develops significant mucosal atrophy, impaired nutrient and water absorption, and increased bacterial translocation to mesenteric lymph nodes in rats maintained on elemental diets or total parenteral nutrition. This study determined the effects of administration of an peptide growth factor (insulin-like growth factor-I[IGF-I]) on the mucosal structure and barrier function of rat small bowel isografts. Thirty-six adult Lewis rats underwent either resection of the distal 60% of the small bowel and proximal colon followed by a 40-cm orthotopic jejunal isograft or proximal small bowel transection and distal small bowel resection to leave an analogous length of small intestine in control animals. All rats received an isocaloric, isonitrogenous, polymeric diet (200 kcal/kg/day, 2 gN/kg/day) by gastrostomy and were infused with either IGF-I (2.4 mg/kg/day) or vehicle by osmotic pumps subcutaneously. After 10 days of treatment, jejunal crypt cell production, mucosal morphometric indices, glucose and water absorption, body weight, and bacterial translocation to mesenteric lymph nodes (MLN) were measured. Jejunal mRNA content for IGF-I, IGF-I receptor, and IGF-binding proteins 3 and 4 (IGFBP 3,4) were determined by Northern blotting. Crypt cell production, villus height, crypt depth, and villus surface area were significantly increased in control and transplanted jejunum of rats infused with IGF-I when compared to animals given vehicle alone. Additionally, jejunal glucose absorption and water absorption were significantly improved in both IGF-I groups when compared with their respective vehicle controls. IGF-I infusion increased body weight in transplanted and control animals and markedly reduced bacterial translocation to MLN after small bowel transplantation. Jejunal levels of IGF-I mRNA were significantly increased in transplanted animals when compared to transected controls. IGF-I treatment significantly increased IGFBP-3 tissue mRNA levels in both transected and transplanted animals. These results demonstrate that IGF-I administration, after small bowel transplantation, improves mucosal structure and absorptive function and reduces bacterial translocation to MLN. IGF-I may have important effects in transplanted small bowel both as an endogenous and administered growth factor. PMID- 7533958 TI - Isolated alkaline phosphatemia following pediatric liver transplantation in the FK506 ERA. PMID- 7533957 TI - Optimization of the magnetic field used for immunomagnetic islet purification. AB - Purification of islets based on the physical differences in density between exocrine and islet tissue reduces islet yields and remains one of the factors limiting islet transplantation. Immunomagnetic cell separation methods provide an attractive, highly specific alternative capable of rapid, gentle, high volume cell separation, but they require modification to be applied effectively to separation of the much larger tissue fragments involved in islet purification. In this study, mAb to rat exocrine tissue were coupled to 4.5-microns magnetic beads (M450 Dynabeads), before incubation with standard aliquots of rat pancreatic digest. The effect on immunomagnetic islet purification of modifications in the magnetic field and the method of digest release into the field were investigated. The results showed that using vibration to maintain the immunomagnetically labeled digest in suspension in tissue culture medium whose density had been increased by the addition of BSA, significantly improved the purification process. When the digest suspension was slowly released and allowed to drift under gravity through a magnetic field applied across a narrow tube, the use of a quadripole of permanent magnets improved results compared with bipolar or unipolar magnetic fields. By modifying immunomagnetic cell separation techniques in this way, a median islet yield of 77% could be reliably achieved while removing 88% of the contaminating exocrine tissue. The use of such methods in human islet purification would significantly increase the yield of islets from each donor pancreas and increase the success rate of transplantation from single donors. PMID- 7533959 TI - Central Vein Occlusion Study: photographic protocol and early natural history. PMID- 7533961 TI - Analysis of the variability in expression of Mycoplasma gallisepticum surface antigens. AB - The in vitro expression of surface epitopes for different strains of Mycoplasma gallisepticum (MG) was studied with a panel of monoclonal antibodies (mAbs) using indirect colony immunostaining and Western blot (WB) analyses. Immunostaining of colonies with mAbs showed that five epitopes had different degrees of variable expression, while one epitope was permanently expressed in vitro. Colonies that failed to express the studied epitopes had the potential of phenotypically switching the expression of these epitopes in vitro. Variable and permanently expressed epitopes were associated with more than one protein and not all mAb defined proteins were responsible for the immunostaining of intact MG colonies. The ability of MG to variably express their surface epitopes maybe the mechanism utilized by the microorganism to avoid the host immune response. PMID- 7533960 TI - Biomicroscopic and histopathologic considerations regarding the feasibility of surgical excision of subfoveal neovascular membranes. AB - An understanding of the difference between the growth pattern of choroidal neovascularization within the subsensory retinal space, usually encountered in children and young adults, and that within the subpigment epithelial space, typically in older patients with age-related macular degeneration, is of fundamental importance in considering whether to surgically remove these subfoveal neovascular membranes. Their removal in young patients has some chance of restoring useful central visual function. Their removal in older patients will be associated with removal of the native pigment epithelium and little likelihood of restoration of function in the area of the membrane, unless some means are found to resurface the area with pigment epithelium. PMID- 7533962 TI - Preliminary analysis of the polypeptides of the salmon leukemia virus (SLV) and evidence for development of a bimodal viremia following SLV infection. AB - A retrovirus, known as salmon leukemia virus (SLV), was purified from farm-reared chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukemia (PL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified SLV revealed the presence of 9 virus-associated polypeptides with molecular weights from 82 kDa to 15 kDa. Endoglycosidase digestion and alcian blue staining of viral polypeptides separated by SDS-PAGE, and immunoprecipitation experiments using hyperimmune antisera suggest that the non glycosylated 27 kDa polypeptide may represent a capsid-associated protein and the 82 kDa glycoprotein may represent an envelope-associated protein, which appears to be composed of a 67 kDa protein moiety. Fish injected with PL-positive tissue homgenate developed a bimodal viremia, as indicated by the presence of cell-free, virus-associated reverse transcriptase activity and SLV in serum of fish from 1 to 3 wk post-injection and again from 7 wk on through the rest of the study. If horizontal transmission of SLV and PL occurs in infected chinook salmon, it is most likely to occur after the second viremic period begins. PMID- 7533963 TI - Listeria innocua isolated from a case of ovine meningoencephalitis. AB - This paper reports a naturally occurring case of meningoencephalitis associated with Listeria innocua in a Polled-Dorset ewe. The ewe was one of a housed group of twenty-five, fed ad lib. on wrapped baled silage. L. innocua was isolated after one week from cold enrichment culture of brain and pituitary tissue. Its identity was confirmed by conventional biochemical tests, API Listeria (BioMerieux UK Ltd), the absence of hly and prfA genes using PCR assay and sequencing two variable regions of 16S rDNA. Histological examination demonstrated lesions of vasculitis and perivascular cuffing in the midbrain which were consistent with listeriosis although limited in distribution and severity. PMID- 7533964 TI - Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA. AB - A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both the 5'- and 3' termini of the pRNA are proximate and crucial for biological function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging. Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA. As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure. Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure. PMID- 7533965 TI - Identification of a neutralization site in the major envelope glycoprotein (GL) of equine arteritis virus. AB - A panel of six neutralizing monoclonal antibodies (MAbs), neutralization resistant variant (escape mutant [EM]) viruses, and individual viral proteins derived from a vaccinia virus expression system were used to identify the neutralizing determinants of equine arteritis virus (EAV). The neutralizing MAbs recognize a single neutralization site on the 29-kDa envelope glycoprotein of EAV (U. B. R. Balasuriya et al., 1993, J. Gen. Virol., 74, 2525-2529). Vaccinia virus recombinants which express either the GL protein or the M protein of EAV, and a GL-specific antipeptide serum were used to prove that the 29-kDa glycoprotein recognized by the MAbs is the GL protein. The MAbs were used to select a panel of seven EM viruses, whose phenotypic properties were characterized by neutralization and Western immunoblotting assays. The neutralizing MAbs segregated into three groups on the basis of these assays, indicating that they define three interactive epitopes on the GL protein. Sequencing of the entire open reading frame (ORF) 5, which encodes the GL protein, from each EM virus identified the nucleotide mutations responsible for the altered phenotypic properties exhibited by the EM viruses. Compared to the sequence of ORF 5 of the parent strain (EAV-UCD), all nucleotide changes occurred within a span of 17 nucleotides (11423 to 11439). Phenotypic alterations in the EM viruses probably were the result of amino acid substitutions within a region of six amino acids (99 to 104), all of which focally altered the predicted hydrophobicity and/or secondary structure of the GL protein. We conclude that this region constitutes an important neutralization domain of EAV. PMID- 7533966 TI - Carcinoembryonic antigen, cytokeratin expression and mucin composition in hyperplastic and neoplastic polyps of the colorectum. AB - We examined the expression of carcinoembryonic antigen (CEA) and cytokeratin (CK) as well as the sialo- and sulphomucin content of 40 hyperplastic polyps (HPs), 6 mixed hyperplastic-adenomatous polyps, 30 adenomas and 40 adenocarcinomas of the colorectum. HPs showed a positive CEA expression in 95% of cases and a decreased sialo- and sulphomucin content compared with normal mucosa. Similar changes were observed in adenomas with low-grade dysplasia. The increase in CEA expression from HPs and adenomas to carcinomas was accompanied by a reduction of sialo- and sulphomucins with about three fourths of carcinomas being sialo- and sulphomucin negative. Oncofetal antigen expression concomitant with mucin changes observed in HPs may indicate impaired cellular maturation at a functional level before dysplastic changes become visible. CEA and CK positive macrophages were found in carcinomas predominantly at sites of tumor disruption and necrosis as well as within veins and lymphatic vessels. Our findings suggest that macrophages may play a role in CEA and CK release into the circulation and thus may be determinants of tumor marker serum levels. PMID- 7533968 TI - [Rudolf Virchow prize 1994 from the German Society of Pathology]. PMID- 7533967 TI - Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-S1 and pre-S2 antigens as compared with conventional yeast-derived vaccines. AB - The widely used hepatitis B virus (HBV) vaccines consist of the small hepatitis B surface (SHBs) protein produced in transfected yeast cells. The frequency of non responders, especially among immunocompromised patients, has increased the demand for a more immunogenic vaccine. We evaluated the immunogenicity of recombinant HBs 20 nm particles secreted by transfected Chinese hamster ovary (CHO) cells, Bio-Hep-B (BioTechnology General Ltd, Israel), and compared it with yeast-derived vaccines. The CHO-derived vaccine contains the small hepatitis B surface antigen (SHBs protein) as the major component, as well as the middle HBs (MHBs, pre-S2) and the large HBs (LHBs, pre-S1) antigens. Nine groups of ten female Balb/c mice, 4-6 weeks old, were injected once intraperitoneally (i.p.) with 0.09, 0.27 or 0.81 micrograms of each of three vaccines: Bio-Hep-B or two conventional yeast derived recombinant vaccines, Engerix-B (SmithKline Beecham, Belgium) and H-B-Vax II (Merck, Sharp & Dohme, USA) containing only non-glycosylated SHBs antigen. After 30 days, 40% of the mice injected with 0.09 microgram Bio-Hep-B had seroconverted, but none of the mice receiving the same dose of the other vaccines. The immunogenic dose in 50% of the mice at day 14 after injection was 0.13 microgram for Bio-Hep-B, but over 0.81 microgram for the other two vaccines. Mice of the strain B10/M (which are unresponsive to SHBs and MHBs antigens at the T-cell level) developed 100-fold higher anti-HBs titres after immunization with 1 microgram of Bio-Hep-B i.p., as compared with mice receiving the same amount of yeast-derived HBsAg vaccines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533969 TI - Diagnostic application of T cell receptor and immunoglobulin gene rearrangements in lymphoproliferative diseases. PMID- 7533970 TI - [Pathology in Zurich]. PMID- 7533971 TI - [Molecular pathology: fashion? Necessity?]. PMID- 7533973 TI - [Chromosomal in situ hybridization and interphase cytogenetics in single cell and tissue section preparations: new methods in tumor diagnosis and clinical cytogenetics]. AB - To overcome the shortcomings encountered in classical cytogenetics, a variety of in situ hybridization techniques have been developed enabling metaphase and interphase cytogenetics on routinely processed tissues and cells. These techniques comprise the detection of numerical chromosome aberrations, the delineation or "painting" of whole chromosomes or certain chromosome regions and the visualization of structural chromosome rearrangements in interphase nuclei. Some of these new methods can reliably be applied also on paraffin-embedded tissues, among them the comparative genomic in situ hybridization (CGH) technique suitable for providing a survey of over- or underrepresented genetic material on the chromosomal level even if only tumour DNA is available. PMID- 7533972 TI - [Detection of bacterial DNA using the polymerase chain reaction (PCR)]. AB - Enzymatic amplification of DNA using the polymerase chain reaction (PCR) is a very sensitive and rapid way of detecting specific DNA sequences. Bacterial DNA can be detected in a wide variety of samples provided at least partial sequence information is available. For a great number of bacteria PCR detection methods have been published. Most important for the pathologist are mycobacteriae (M. tuberculosis, avium, etc.). Borellia burgdorferi, Listeria monozytogenes and chlamydiae (Ce. trachomatis, C. psittaci). Fresh or fixed paraffin embedded tissues, exfoliated cells, whole blood, serum, sputum, urine, ascites or pleural fluid etc. can be analyzed. The time needed to perform the analysis varies between 5 hours and 2 days mostly depending on the DNA extraction method. Several potential pitfalls have to be avoided. The most common problem is contamination of reagents with target DNA. Amplification of DNA from biological samples may be prevented by enzyme inhibitors (salts, proteins). This problem can at least partially be avoided by changing the DNA purification method. Several additional problems may arise if bacterial DNA has to be amplified. Bacterial walls may have to be disrupted using heat or detergent for accessibility of target DNA. Positive results have to be judged carefully. Unlike the situation in retroviral infections with the virus sometimes present in the absence of disease, in the majority of bacterial infections the presence of bacteria signals manifest disease. A possible exception may be the finding of mycobacterial DNA in sarcoidosis patients who can be treated with steroids without provoking tuberculosis. PCR is especially useful in situations where rapid results are necessary or only fixed tissue is available.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533974 TI - In situ hybridization in pathology. AB - The technique of in situ hybridization (ISH) is presented with special reference to its use in the practice of pathology. Methodological and theoretical aspects of ISH are first considered with regard to tissue fixation, probe selection and preparation, various types of labeling reactions and aspects of hybridization conditions as they relate to hybridization protocols. This is followed by a discussion about the interpretation of the results of ISH with regard to sensitivity and specificity and the importance of, and various types of, controls. Lastly, specific applications of ISH in pathology are discussed, concluding with possible future applications for ISH in research and diagnosis. PMID- 7533975 TI - [Polymerase chain reaction in diagnostic pathology]. AB - DNA is a very stable molecule and fixation as well as routine histological processing does not destroy its molecular structure. Hence the possibility exists to use this material for molecular genetic analyses. The polymerase chain reaction (PCR) opens the chance to amplify DNA to huge amounts from pathological paraffin and plastic blocks and to use this DNA for further examinations, such as detection of mutations or translocations in malignant tumors, e.g. t(14;18) in follicular lymphomas, bcr/abl in chronic myeloic leukemia, t(11;22) in Ewing sarcomas or of the ras-gene in colon cancer, overexpression of tumor related mRNA, e.g. mdr1-mRNA of the multidrug associated P-Glycoprotein, or detection of foreign DNA from viruses or bacteriae, as well as analysis of hereditary diseases. PCR in its various forms (conventional PCR, PCR with direct sequencing, reverse transcriptase (RT)-PCR, nested primer PCR, quantitative RT-PCR, inverse PCR, degenerated primer (DOP) PCR, in situ PCR, in situ RT-PCR etc.) has proven to supplement routine diagnostic work in many occasions, however, it has to be emphasized that up to now there exists no example for a complete replacement of histological or immunhistological examination by PCR. As consequence, histology remains the first and most important step towards a relevant diagnosis supplemented e.g. by PCR and other techniques of molecular biology. PMID- 7533976 TI - In situ polymerase chain reaction: general methodology and recent advances. AB - In situ PCR is a new molecular technique, that combines the extreme sensitivity of PCR with the cellular localization provided by in situ hybridization (ISH), through the amplification of specific gene sequences within intact cells or tissue sections and increasing copy numbers to levels detectable by ISH or immunohistochemistry. In addition to the detection of viral DNA (CMV, HBV, HIV), we have used this technique for the study of DNA rearrangements, chromosomal translocations (t14;18) and viral RNA (HCV) in cells in suspension, cytocentrifuge preparations or archival tissue sections. We compared different approaches to in situ amplification of target sequences and visualization of PCR products by either subsequent ISH (indirect in situ PCR) or by direct detection of labeled nucleotides, which have been incorporated during PCR (direct in situ PCR). Our results indicate, that in situ PCR includes a number of different techniques, which are not equally applicable to all types of samples. In situ PCR appears to be most effective for the detection of DNA in single cell preparations with controlled fixation and pretreatment, although the quantification of results remains problematic. Artifacts caused by diffusion and extracellular generation of PCR products are a significant problem potentially leading to false positive results. In situ PCR works less efficiently in archival tissue sections due to poor quality of nucleic acids and retention of PCR products. Direct in situ PCR yields less specific results than indirect in situ PCR and requires additional controls such as omission of primers in the reaction mixture to detect artifacts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7533977 TI - [Relevance of apoptosis for carcinogenesis]. AB - Apoptosis is a type of active, genetically programmed cell death. It occurs under specific conditions and is characterized by it's morphology. It is controlled by genes, hormones and other factors regulating the growth of organs and cells in the organism. In the liver and some other epithelial tissues transforming growth factor beta 1 and related peptides seem to be involved in the homeostasis of cell multiplication and cell death. In the course of carcinogenesis, initiated, preneoplastic and neoplastic cells and cell foci in the liver show enhanced DNA synthesis and also enhanced apoptosis. Tumor promoters inhibit apoptosis and increase cell replication and can thereby shift the balance between birth and death of cells accelerating tumor development. Fasting can have the opposite effect. PMID- 7533978 TI - Equipment and organization of a frozen tissue bank--an update. AB - The growth of molecular diagnostics and its application in various clinical laboratories has made it necessary to standardize the methods used to freeze and store tissues used in molecular testing. It may now be advantageous to preserve fresh tissues and other specimen types in a central frozen tissue bank so that sample preparation and storage conditions are appropriate for molecular applications and so that the specimen inventory can be efficiently managed. The pathology laboratory is a logical site for this facility since the professional and technical expertise available is focused on the complex scientific and managerial aspects of laboratory medicine. Organizationally, the tissue bank program should be overseen by a surgical pathologist in order to integrate it into routine surgical pathology activities. A member of the laboratory technical staff can serve as the tissue bank coordinator with responsibility for systematic storage and retrieval of specimens and routine maintenance of equipment and supplies. In order to facilitate the tissue freezing procedure as well as efficient storage of multiple types of specimens, 2.0 ml cryogenic vials are used as the uniform storage container. All specimens are stored at -140 to -150 degrees C in the vapor phase of liquid nitrogen. The specimen inventory data are maintained with a computerized program specifically designed to manage complex specimen storage. A frozen tissue bank is easily implemented in a pathology laboratory and is a valuable institutional asset for diagnostic and research purposes. PMID- 7533979 TI - [Criteria for molecular biological diagnosis in pathology]. AB - Since its initial description in 1985 (Saiki et al., 1985), the polymerase chain reaction (PCR) has become one of the leading techniques. This approach is not only able to clarify many questions in basic research, but also diagnostical issues. The application of PCR in the field of tissue diagnosis is still at its infant stage, although it has already proved to be of great value. However, the slow introduction of PCR in the field of Pathology is the cause of much misunderstandings for many clinician. Of the possible reasons for this, one could be the fact that the tissue diagnosis is carried out by the investigation of formalin-fixed material. One solution to this situation would be to propose a frame-work of conditions for the introduction of PCR as a diagnostically valuable method of investigation. PMID- 7533980 TI - [Virus-induced immune deficiency disease AIDS: direct pathogenic effect of the virus or immunopathology?]. AB - Cytotoxic effectors-CD8+ T cells protect hosts against cytopathic viruses. CD8+ T cell mediated lysis of host cells infected with non-cytopathic viruses may lead to tissue disease causing damage as is the case in hepatitis B virus infections in men or lymphocytic choriomeningitis virus (LCMV) infections in mice. In a mouse model it was demonstrated that virus induced immunodeficiency by LCMV was not caused by the virus directly but rather by immunopathological consequences of anti-LCMV CD8+ T cells destroying LCMV-infected antigen-presenting cells and macrophages. These results may suggest that HIV induced immunodeficiency could similarly be caused by immunopathology because 1. HIV has not been demonstrated to be cytopathic in vivo. 2. CD8+ T cells control HIV infection efficiently for a longtime and 3. only few CD4+ T cells but many macrophages, monocytes and dendritic cells are productively HIV infected. Therefore AIDS may be the result of specific CD8+ T cells destroying antigen presenting cells and effector/macrophages/monocytes thereby causing immunosuppression and inability to control intracellular infections. Compared to the view that HIV is cytopathic, the immunopathological view of AIDS suggests obviously different vaccine and therapy strategies. PMID- 7533981 TI - Genetic modification of cells by receptor-mediated adenovirus-augmented gene delivery: a new approach for immunotherapy of cancer. AB - Most concepts of gene therapy of cancer are based on the generation of an enhanced immune response against the cancer by means of vaccination with gene modified cancer cells. We have investigated the applicability of a new gene transfer technique which uses the receptor-mediated endocytosis pathway and the endosome disruption activity of adenovirus for the generation of a cancer vaccine consisting of interleukin-2 (IL-2)-transfected, irradiated murine melanoma cells (clone M-3). This technique resulted in very high IL-2 expression (in the range of 30,000 Units IL-2/10(6) cells/24 hrs) in the transfected cells without the need to selection of stably expressing cell clones. We found that this high IL-2 expression of the melanoma cells correlates with high efficacy of the vaccine. Immunization of animals with this vaccine elicits a systemic T-cell-mediated immune response which protects from tumor development after implantation of highly tumorigenic doses of wild-type melanoma cells. PMID- 7533982 TI - [Viral antigen induced autoimmunity: an animal model for diabetes mellitus type I]. AB - To address the mechanisms of tolerance to extrathymic proteins, we have generated transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) in the beta islet cells of the pancreas. The fate of LCMV-GP specific T cells was followed by breeding the GP transgenic mice with T cell receptor transgenic mice, specific for LCMV-GP and H-2Db. These studies suggest that "Peripheral tolerance" of self-reactive T cells does not involve clonal deletion, clonal anergy, or a decrease in the density of T cell receptors or accessory molecules. Instead, this model indicates that potentially self-reactive cytotoxic T cells may remain functionally unresponsive, owing to a lack of appropriate T cell activation. Infection of transgenic mice with LCMV readily abolishes peripheral unresponsiveness to the self LCMV-GP antigen, resulting in a CD8+ T cell-mediated diabetes. These data suggest that similar mechanisms may operate in several so called "T cell-mediated autoimmune diseases". A synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) was used to prime or to tolerize CD8+ T cells in vivo, dependent on the mode of immunization. Peptide-specific tolerance was then examined in transgenic mice expressing LCMV-GP in the beta islet cells of the pancreas; these mice develop CD8+ T cell-mediated diabetes within 8-14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of beta islet cells and diabetes in this transgenic mouse model. PMID- 7533983 TI - [Neurotoxicity of human foamy virus in transgenic mice]. AB - Human foamy virus (HFV) is a human retrovirus originally isolated from patients with various neoplastic and degenerative diseases. Similarly to other human retroviruses, HFV encodes the three structural retroviral genes, gag, pol and env, and an additional region containing three open reading frames, bel-1 to bel 3. Bel-1 activates transcription of the long terminal repeat of HFV and also of HIV. Until today it has not been possible to identify HFV as the causative agent of any disease. In order to identify a possible pathogenic potential of HFV, we have introduced parts of the HFV genome into the germ line of mice. Our studies with transgenic mice have shown that HFV transgenes encompassing the bel region are transiently transcribed between midgestation and birth at moderate levels in various tissues. Expression is then suppressed, but resumes after a latency of several weeks in a restricted range of tissues and accumulate extensively in single cells. This is associated with a progressive degenerative disease of the central nervous system and of striated muscle. These findings provide the first evidence of a disease induced by HFV and suggest that HFV might also act as a human pathogen in neurological diseases. Moreover, the transgenic mouse model will be useful for studying the molecular basis of HFV-induced neurotoxicity, the role of individual disease-associated HFV genes, and the regulation of retroviral latency. PMID- 7533984 TI - [Detection of mycobacterial DNA from paraffin-embedded tissue in lung and lymphoid epithelial granulomas]. AB - Standard techniques for the detection of mycobacteria in granulomatous diseases can be inadequate. We analysed 71 formalin fixed and paraffin-embedded tissue blocks from 68 non-immunocompromised patients with caseating, non-caseating, scarred, and miliary granulomas of lung and lymph nodes. A reamplification PCR protocol was established to detect a 123 bp product of the repetitive insertion sequence IS986/IS6110. After exclusion of 6 PCR-negative cases with clinical sarcoidosis 97% of lung tissue blocks with more than 10% caseating and non caseating granulomas contained mycobacterial DNA. By routine microbiology mycobacteria could be detected in 78% of the patients. Scarred granulomas were PCR-negative. All miliary granulomas were PCR-positive. Lymph nodes showed comparable results. We think that this method facilitates aetiologic analysis of granulomatous diseases especially when the suspicous tissue is fixed and microbiology is not available. PMID- 7533986 TI - [Non-radioactive SSCP for the detection of androgen receptor gene mutation--a diagnostic tool for androgen resistance]. AB - Point mutations in the androgen receptor gene cause androgen insensitivity syndromes, clinically characterized by masculinization defects in karyotypic males due to endorgan resistance to androgenic steroids. Characterization of these mutations with single strand conformation polymorphism analysis utilizing radioactive PCR can serve as a diagnostic tool for molecular subclassification of these syndromes. It is the basis for genetic counseling and for therapeutic decisions. Here we report an improved non-radioactive single strand polymorphism analysis for rapid detection of androgen receptor gene mutations in affected individuals. In addition to previously reported mutations, 9 patients with clinical features of androgen resistance were studied. While one insertion mutation was detected, in all other patients different point mutations initiating amino acid substitutions were characterized. PMID- 7533985 TI - [Molecular biological analysis of unclassified and uncultured bacteria]. AB - With the advent of comparative rRNA sequence analysis and the introduction of molecular genetic methods, such as nucleic acid hybridization and amplification techniques, microbiology is gaining new grounds. Taxonomy based upon phylogenetic analysis leads to a reliable classification. Culture-independent identification procedures allow for the analysis of complex microbial ecosystems and the description of new, as-yet uncultivable infectious disease agents. PMID- 7533987 TI - [Detection of amplified DNA sequences by comparative genomic in situ hybridization with human glioma tumor DNA as probe]. AB - Comparative genomic hybridization (CGH) provides a new possibility for the investigation of genetic alterations in tumour genomes. In our experiments CGH was carried out using genomic DNA from human glioblastoma multiforme (GBM) as a probe for chromosomal in situ suppression hybridization. Amplified DNA sequences contained in the tumour DNA showed specific signals, revealing the chromosomal positions of these sequences. Using this approach we detected amplifications of different chromosomal segments in individual GBM specimens. In accordance with the results from Southern analysis demonstrating amplification of the EGFR gene in 45% of human GBM, CGH signals in different GBM mapped to the region of this gene on chromosome 7p. Other signals detected by CGH involved chromosome 12q and 8q. Our data demonstrate CGH as a novel comprehensive and rapid approach for the analysis of complex genomic alterations in glial tumours. PMID- 7533988 TI - [Intensification of sensitivity of monoclonality determination of B-cells in autoimmune thyroiditis and low malignancy B-NHL of the MALT type using temperature gradient gel electrophoresis (TGGE)]. AB - Malignant non-Hodgkins lymphoma with primary involvement of the thyroid is a rare disease. Nearly all these lymphomas are of B-cell phenotype, and they represent a broad morphological spectrum of high and low grade entities. We investigated 18 cases of Hashimotos disease and 95 cases of thyroid non-Hodgkins lymphoma including 16 cases of MALT-lymphoma. This type of lymphoma is often hardly distinguishable from reactive lesions, even by immunohistochemistry. Therefore, we introduced a new molecular genetic technique based on the polymerase chain reaction (PCR) combined with temperature-gradient gel electrophoresis to demonstrate monoclonal populations of B cells in a polyclonal background of reactive B lymphocytes. With this approach we found monoclonality in 4 of 18 cases of Hashimotos disease. In our opinion, these 4 cases demonstrate clearly the transition from autoimmune disease into non-Hodgkins lymphoma. PMID- 7533990 TI - [Tumors--disorders of cell adhesion]. AB - Cell adhesion molecules are of pivotal importance during organogenesis and fulfil important functions in maintaining normal organ architecture in the adult. Furthermore, they are involved in diverse and specialized functions such as migration and activation of leukocytes in immunosurveillance and inflammation, and homophilic as well as heterophilic interactions of cells with others and the extracellular matrix. In tumors, various cell adhesion molecules play a role in the complex process of tumor dissemination including metastatic spread. In addition, immunohistochemical detection of certain cell adhesion molecules in tumors has been shown to be of value in predicting the clinical course of the tumor disease. This short review focuses on the polysialic acid of the neural cell adhesion molecule NCAM, integrins and CD44. PMID- 7533989 TI - [Detection of EWS-/FLI-1 gene fusion transcripts by RT-PCR as a tool in the diagnosis of tumors of the Ewing sarcoma group]. AB - Recent cloning of the chromosome breakpoint regions of the reciprocal chromosomal t(11;22) (q24;q12) has revealed that the breakpoints were localized within the EWS gene (Ewings sarcoma gene) on chromosome 22 and the FLI-1 gene on chromosome 11. Thus, molecular genetic techniques were applicable for the detection of this genetic aberration, which occurs as a consistent feature of the Ewings tumor family. By reverse transcription and polymerase chain reaction technique (RT-PCR) in 78% of Ewings sarcoma derived cell lines, and in 91% of primary Ewings tumor tissue t(11;22) specific EWS/FLI-1 fusion transcripts were detected. Furthermore, in bone marrow samples from an Ewings sarcoma patient contaminating tumor cells could be shown by RT-PCR. Our results indicate that molecular genetic detection of the t(11;22) translocation opens a new modality for the differential diagnosis and the staging of Ewings tumor patients. PMID- 7533991 TI - [Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies]. AB - To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods. PMID- 7533992 TI - [Quantification of messenger RNA expression in tumors: which standard should be used for best RNA normalization?]. AB - Alterations in gene expression are frequently encountered in malignant tissues. Quantification of the often quite subtle changes in mRNA content is routinely performed by northern blot hybridization. Practical experience shows that the methodology is subject to considerable error, necessitating the use of an internal mRNA standard for quantification. Many different control mRNAs have been employed over the years, but no systematic study has proven their applicability to comparisons made between tumor cells and normal tissues. The validity of these control RNAs, usually housekeeping genes, must be established to exclude the possibility that they themselves are expressed at different levels in tumor and non-tumor cells. Our experience has revealed that serious discrepancies may arise when incorrect standard genes are used to compare tumor and normal tissues. PMID- 7533993 TI - [Elevated p53 protein expression in normal and neoplastic light-exposed epidermis]. AB - p53 protein levels are frequently elevated in basal (BCC) and squamous (SCC) cell carcinomas of UV-exposed sites. As recent findings in benign epidermal neoplasia (BEN) and normal epidermis (NE) are controversial, our aim was to look by immunohistochemistry for elevated p53 levels in NE (n = 52), reactive hyperplastic epidermis (REH, n = 8), BEN (n = 48), actinic keratosis (AK, n = 60), SCC (n = 13), and BCC (n = 42) of sun-exposed sites. 81.7% of AK, 100% of SCC, 73.8% of BCC as well as 29.2% of BEN, 37.5% of REH, and 26.9% of NE showed p53 positivity. The results further support that p53 elevated levels are an early marker of UV-mediated epidermal DNA damage. PMID- 7533994 TI - p53 mutations in oropharyngeal carcinomas: a comparison of solitary and multiple primary tumours and lymph node metastases. AB - Despite the steadily increasing number of patients suffering from squamous cell carcinomas of the oropharyngeal region, little is known about the molecular steps involved in the induction of these neoplasms. The aim of this study was to determine the incidence and type of p53 mutations in oropharyngeal squamous cell carcinomas. Five of 38 patients had lymph node metastases, three patients had multiple primary carcinomas and two patients presented with multiple primary tumours and lymph node metastases. Exons 5 through 8 of the p53 gene were screened by single-strand conformation polymorphism analysis followed by direct DNA sequencing. A total of 16 tumours (42%) contained point mutations scattered throughout exons 5 to 8. Most mutations (56%) were transitions, predominantly G- >A. G-->T mutations prevailed among the transversions that have also been found in smoking-related lung cancer. One carcinoma of the soft palate showed a mutation which was retained in a lymph node metastasis. The status of p53 differed in primary carcinomas of patients with multiple tumours indicating an independent generation of these neoplasms. The frequent occurrence of p53 mutations in oropharyngeal carcinomas supports the view that this gene plays a role in initiation or progression of the malignant phenotype. PMID- 7533995 TI - [p53 alterations and HPV status in oral squamous cell carcinomas]. AB - Immunohistochemical examination of p53 protein accumulation, analysis of the p53 gene using amplification of the exons 5-8 followed by TGGE, and PCR based HPV typing were carried out on 40 oral squamous cell carcinomas. 28 of 40 oral carcinomas (70%) contained DNA of the oncogenic HPV types 16 and 18. A p53 accumulation was present in 20 of 37 tumors (54%). Mutations of the p53 gene were detected in 15 of 39 carcinomas (38%). 15 of 20 oral carcinomas with abnormal p53 expression and 11/15 tumors with mutations of the p53 gene were HPV positive. Our results demonstrate a frequent occurrence of p53 abnormalities in HPV positive oral squamous cell carcinomas. This pattern is divergent from HPV related genital carcinomas and could reflect differences in the etiology of squamous cell carcinomas of different sites. PMID- 7533996 TI - Where pathology meets molecular biology: N-myc amplification in human neuroblastoma as a paradigm for the clinical use of an oncogene alteration. AB - Increase of the dosage of cellular oncogenes by DNA amplification is a frequent genetic alteration of cancer cells. The presence of amplified cellular oncogenes is usually signalled by conspicuous chromosomal abnormalities, "double minutes" (DMs) or "homogeneously staining chromosomal regions" (HSRs). Some human cancers carry a specific amplified oncogene at high incidence. In neuroblastomas the amplification of N-myc has been found associated with aggressively growing cancers and is an indicator for poor prognosis. N-myc amplification is of predictive value for identifying neuroblastoma patients that require specific therapeutic regimens and for identifying patients that do not benefit from chemotherapy. PMID- 7533997 TI - [Detection of human herpesvirus type 6, human herpesvirus type 7, cytomegalovirus and human papillomavirus in cutaneous AIDS-associated Kaposi's sarcoma]. AB - In order to evaluate a possible role of viral infections in the pathogenesis of AIDS-associated Kaposi's sarcoma (KS), we investigated 26 cutaneous AIDS associated KS by polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry. By PCR we detected human papilloma viruses (HPV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and for the first time human herpesvirus 7 (HHV-7) in the KS. The prevalence of HPV, HHV-6, and HHV-7 was similar to or lower in KS than in normal skin tissues of AIDS patients without KS, but higher than in normal skin of HIV-seronegative patients. All HHV-6 found in KS were identified as HHV-6 variant B. In addition to the known HPV types 16 and 18 described in KS, we also found HPV types 6 and 33 in KS specimen. By immunohistochemistry HHV-6 could be localized in macrophages in KS, in the adjacent stroma as well as in normal skin of control cases. In situ hybridization for CMV and HPV gave negative results in KS and controls. PMID- 7533999 TI - [Molecular diversity of desmosomal cadherins and their potential as markers in the histodiagnosis of carcinomas]. AB - Desmosomal cadherins, transmembrane glycoproteins of the cadherin family of cell adhesion molecules, comprise two subfamilies, the desmogleins and the desmocollins, each of which consist of at least 3 distinct proteins encoded by individual genes. We have analyzed the expression of desmogleins Dsg1 (Pemphigus foliaceus antigen), Dsg2 ("simple epithelium type") and Dsg3 (Pemphigus vulgaris antigen) in various normal tissues and diverse carcinomas, using RNase protection assays. We found that the gene encoding Dsg2 was expressed in most epithelial tissues and carcinomas whereas mRNAs encoding Dsg1 and Dsg3 were primarily restricted to stratified squamous epithelia and certain (mostly squamous cell) carcinomas. Antisera raised against recombinant polypeptides corresponding to various parts of Dsg2 produced immunostaining along intercellular borders of simple epithelia and basal cells of non-cornifying stratified squamous epithelia but were negative with most cells of epidermis. Preliminary analyses of carcinomas revealed comparable patterns. These cell type-specific differences in the molecular composition of desmosomes, which are also reflected in carcinomas, open new possibilities for the histological classification and subtyping of carcinomas. Moreover, the functional importance of desmosomal cadherins in the adhesion of carcinoma cells and during metastasis makes them a promising marker system for the assessment of the biological behaviour of carcinomas. PMID- 7533998 TI - [Thymus-like expression and molecules accessory to division and of thymocyte stroma interactions in thymomas: a prerequisite for the establishment of an abnormal T-cell repertoire?]. AB - Costimulatory stromal receptors were identified in thymuses and myasthenia gravis (MG)-associated thymic epithelial tumors (TETs) by immunohistochemistry. ICAM-1 occurred in areas with medullary or cortical differentiation of TETs and thymuses. B7 and LFA-3, absent in the normal cortex, were hyperexpressed in cortical type TETs. VCAM-1, confined to endothelium and dendritic cells in cortex and medulla, was focally expressed by epithelial cells of a medullary thymoma. We suggest that aberrantly expressed costimulatory molecules may contribute to the pathogenesis of paraneoplastic MG by an intratumorous activation of mature T cells. PMID- 7534000 TI - [Coincidence of hepatic angiosarcoma and insulin-resistance diabetes mellitus with high-dose intraperitoneal insulin therapy]. AB - Angiosarcoma of the liver was diagnosed at autopsy in a 50 year old female with insulin-resistant diabetes mellitus. Over the course of 27 years, the patient received 27 l of Insulin-preparations applied subcutaneously and by an intraperitoneal pump-system. The preparations contained 20 g phenol. Known carcinogens for angiosarcoma of the liver-thorotrast, monovinylchloride or arsenic-were excluded. Phenol is a well known carcinogen of the skin and its tumor-promoting potential has been demonstrated in vitro. We suggest phenol as a potential aetiologic factor for angiosarcoma of the liver. PMID- 7534001 TI - [Characterization of clonal B-cell populations in gastric MALT lymphomas and chronic gastritis by means of the polymerase chain reaction]. AB - Amplification of the CDR3-region of the immunoglobulin (Ig) heavy chain gene rearrangement by means of the PCR yielded clonal products in the tumor DNA of 12 high or low grade gastric B-cell lymphomas of MALT-type. In four cases, additional clonal bands were found in different areas of tumor free mucosa diagnosed as chronic gastritis associated with Helicobacter pylori (HP). Most of these small clonal populations were found to share identical DNA sequences in their clone specific CDR3-regions with the main lymphoma in each patient; a finding consistent with the multifocal character of the disease. In two cases however, single clonal populations with different CDR3-regions revealed the existence of rare additional clonal B-cell-populations not related to the lymphoma and therefore possibly representing further independent foci. PMID- 7534002 TI - [Detection of mixed lymphoid chimerism after allogeneic bone marrow transplantation: demonstration by interphase cytogenetics in paraffin-embedded tissue]. AB - In bone marrow transplantation (BMT) the detection of residual host lymphoid or haematopoietic cells surviving conditioning therapy is because of its association to graft-versus-host disease, graft-versus-leukemia reaction, and relapse of leukemia a matter of great interest. We studied the occurrence of this mixed lymphoid chimerism (MC) in the formol-fixed lymphatic tissue of lymph nodes and spleen from 21 autopsies after allogeneic sex-mismatched BMT (5 females, 16 males, survival 5 to 1140 days after BMT). In situ hybridisation with biotinylated centromer-specific anti-X- and anti-Y-chromosome probes was performed on pepsin-digested paraffin sections. The number of double X-, single X , and Y-chromosome bearing cells was analysed microscopically. Because of artefacts only 14 cases remained for valid investigation. MC was detected in 6 cases (5 out of 11 males 5 days to 840 days and 1 out of 3 females 76 days after BMT). MC occurred after whole body irradiation with 10 Gy (n = 5) and 7 Gy (n = 1). In 1 autopsy relapse of leukemia caused host cell infiltration. Cases with MC did not express histological signs of acute or chronic graft-versus-host disease, but 5 out of 8 with complete lymphoid chimerism did. The sensitivity of interphase cytogenetics on paraffin embedded tissue is low. PMID- 7534003 TI - [Correlation of organ pathology and distribution of virus replicating cells, demonstrated with RNA in situ hybridization of SIVmac infection of Macaca mulatta]. AB - 22 juvenile rhesus macaques were infected i.v. with SIVmac and killed at defined timepoints after infection. Productively infected cells were detected by RNA in situ hybridization in the paraffin material. Their number was correlated with the pathology of lymph nodes, thymus, extranodal lymphatic parenchyma and other organs. In the first weeks all lymphatic tissues and compartments got infected, as well as the brain, the bone marrow and other organs. The high virus replication during this first phase disappeared with the onset of the seroconversion and remained low during all stages of atrophy of the lymphatic parenchyma. The atrophy of the lymphatic parenchyma and its microenvironment was not correlated with virus replication. This may implicate that a virostatic therapy might be more successful in the first weeks of infection. PMID- 7534004 TI - [Detection of bcr-abl transcripts in "minimal residual disease" in chronic myelogenous leukemia by nested polymerase chain reaction (PCR)]. AB - We compared two non-radioactive PCR methods, a single step PCR and a nested PCR, for detecting bcr-abl transcripts in patients with chronic myelogenous leukemia (CML). The nested PCR assay was about thousand times more sensitive than the single step PCR. In 75 clinical samples tested in parallel, the sensitivity for the single step PCR and the nested PCR was 43.2% and 91.9%, respectively. In all 17 samples of 9 patients before bone marrow transplantation (BMT), bcr-abl transcripts were detected by the single step PCR. After BMT, 5 (9.8%) of 51 samples of 4 patients were positive by the single step PCR and 17 (33.3%) of 7 patients by the nested PCR. These data indicate that single step PCR may miss minimal residual disease whereas nested PCR is a sensitive alternative to the use of radioactive probes. PMID- 7534005 TI - [Oral non-Hodgkin lymphomas and Epstein-Barr virus]. AB - The oropharynx is the site of primary infection and further propagation of the Epstein-Barr virus (EBV). From here, virus is shed to saliva and infects peripheral blood lymphocytes. Eight oral Non-Hodgkin lymphomas (NHL) were investigated for the presence of EBV both by immunohistochemistry for the latent membrane protein (LMP) and a PCR-strategy for general and subtype-specific viral sequences. All but one NHL turned out to be negative both by LMP and PCR. EBV general sequences and of the two viral subtypes A and B were found in an HIV-1+ patient. It is concluded that it is not the localisation which predetermines NHLs to EBV-positivity but merely the underlying disease (this study) or the type of tumour (previous studies). PMID- 7534008 TI - Alpha B crystallin expression in response to hormone, oncogenes and stress. AB - alpha B crystallin is one of the major eye lens proteins and it is abundantly expressed in many non-lenticular tissues as well. The alpha B crystallin protein sequence is similar to that of small heat shock proteins and we could demonstrate that alpha B crystallin does indeed accumulate in response to heat shock and other forms of stress. Moreover, ectopic expression of alpha B crystallin establishes a state of thermotolerance. Glucocorticoid hormones activate the alpha B crystallin gene with a delayed kinetics. Induced expression of the Ha-ras oncogene accelerates hormone-mediated gene induction. A shorter promoter sequence is required for the ras supported hormone response. However, sustained Ha-ras oncogene expression attenuates glucocorticoid-triggered induction of the alpha B crystallin gene completely. Whereas the small heat shock protein Hsp27 is overexpressed in many human mammary carcinomas, we observed the frequent absence of alpha B crystallin in these tumors but not in the normal breast tissue. In contrast, overexpression of alpha B crystallin has been frequently observed in non-neuronal brain tumors. PMID- 7534006 TI - [Internal deletions of the latent membrane protein oncogenes of Epstein-Barr virus in Hodgkin's disease are almost identical with those of Asiatic nasopharyngeal carcinoma]. AB - The latent membrane protein (LMP) oncogene of the Epstein-Barr virus (EBV) is 1300 base pairs (bp) long and expressed in Hodgkin and Reed-Sternberg (HRS) cells of about 50% of Hodgkin's lymphomas and in tumor cells of about 60% of nasopharyngeal carcinomas (NPC). The LMP sequences of EBV variants isolated from two NPC (NPC 1510 and NPC CAO) have recently been published. Compared to the standard EBV sequence (EBV B95-8) they both show deletions of 30 bp near the 3' end. These mutations render the LMP oncogene more aggressive in NPC. In 52 Hodgkin's lymphomas expressing the LMP oncogene was amplified the coding sequence by the polymerase chain reaction. In five tumors deletions within the coding region for the intracytoplasmic LMP domain have been found. DNA sequencing revealed three 30 bp deletions almost identical to those observed in NPC 1510 and NPC CAO. In a forth case, a 70 bp deletion encompassing the regions of the 30 bp deletions was found. Histologically, all five tumors with such deletions showed abundant HRS cells. It is likely that these mutations of the LMP oncogene in a similar way as in NPC also favour the proliferation of HRS cells in Hodgkin's disease. PMID- 7534007 TI - [Hodgkin lymphomas ar negative with regard to bcl-2/JH gene rearrangement, but partly express BCL-2 protein: analysis of 83 cases from the German Hodgkin Trial]. AB - Conflicting data on the presence of bcl-2/JH gene rearrangement and BCL-2 protein expression in Hodgkin's disease (HD) have been reported. To find out the reason for these differences, well characterized cases from the German National Trial were examined by PCR for the bcl-2 gene rearrangement and immunohistochemically for BCL-2 protein expression. No bcl-2/JH rearrangement could be detected among the HD analyzed from paraffin sections, as well as another 24 cases of HD investigated in fresh tissue. Contrasted with this, immunohistochemistry demonstrated BCL-2 protein expression in all types of HD. In conclusion, our results suggest that the bcl-2/JH gene rearrangement does not play a role in HD, but BCL-2 protein is frequently expressed by RS/H cells of non-lymphocyte predominance subtypes. PMID- 7534009 TI - [Proliferative activity and defective replication in breast cancer]. AB - The highly proliferating phenotype of mammary carcinoma is known to be associated with a particularly aggressive clinical course. We have been interested in the underlying molecular causes that give rise to the increased proliferative activity Proliferative activity was determined immunohistochemically by the detection of topoisomerase II-alpha (Ki-S1). The subgroup of highly proliferating tumors with a Ki-S1 index exceeding 30% was characterized by a high frequency of c-myc amplification and aberrant p53 expression, whereas tumors, with a low mitotic activity rarely exhibited gene amplification or an altered p53 expression. We conclude, that the highly proliferating phenotype is not capable of regular replication and tends to develop gene amplifications. One of the causes might be a defective cell cycle control by p53. PMID- 7534010 TI - [Molecular cloning of a new AP-2 transcription factor, AP-2beta, and its function in cell differentiation]. AB - Transcription factor AP-2 has been previously shown to play an important function in embryonal development and cell differentiation. We have investigated the possibility that AP-2 function in embryonic development is exerted by a multigene family of AP-2 related transcription factors. Here we describe the molecular cloning of such an AP-2 related gene, AP-2 beta, and prove that it encodes for a functional transcription factor. In situ hybridizations of murine embryo sections revealed a temporally restricted and tissue-specific expression pattern that indicates a function of AP-2 beta in the development of the midbrain in the differentiation of sensory neurons for taste, olfaction and palpation. PMID- 7534011 TI - [Alleles in chromosome 10p21-26 in malignant gliomas]. AB - Loss of genetic material on chromosome 10 is regarded as a prominent feature in the genesis of glioblastomas. To use chromosome 10 deletions as diagnostic markers for glioblastomas we investigated, if the loss of chromosome 10 material could be restricted on the region 10q21-26. By PCR microsatellite analysis on frozen tissue and paraffin material from the ZULCH brain tumor collection we found (1) loss of heterozygosity in 10q21-26 in 75% of the investigated DNA from frozen tissue and (2) an interstitial loss in the region of the microsatellite marker D10S186. The combined immunohistochemical analysis of overexpression of EGFR, EGF and TGF alpha with LOH on chromosome 10 showed that chromosome 10 deletions are not exclusively bound to EGFR overexpression. PMID- 7534012 TI - [Increased amounts of IL-10 mRNA in anaplastic astrocytomas and glioblastoma multiforme]. AB - Interleukin 10 (Il-10) was initially discovered on the basis of its ability to suppress cytokine synthesis. Additionally, it can exert immunosuppressive effects on a variety of cell types. Since patients with malignant gliomas present with a general impairment of the immune system, we sought to investigate if IL-10 is expressed in the glioma tissue. Using RT-PCR, IL-10 mRNA levels were determined in 37 glial tumors of different grades including 2 recurrencies, 3 specimens from normal brain tissue and 3 glioblastoma cell lines. Expression of IL-10 mRNA was demonstrable in all tumors as well as in normal brain. High grade tumors and recurrent cases expressed significantly higher amounts of IL-10 specific mRNA compared to low grade tumors, while 2 out of 3 cell lines showed only weak constitutive expression. We suggest, that IL-10 may contribute to the progression of astrocytomas by allowing the tumor cells to attenuate the T-cell immune response and evade immune detection. PMID- 7534013 TI - [Progression-associated gene regions in astrocytomas: a candidate locus on chromosome 19q]. AB - A study was carried out to examine whether genetic loci which have been shown to exhibit loss of heterozygosity in gliomas are involved in the process of malignant progression from low grade astrocytomas to anaplastic variants. We have analyzed 18 well differentiated astrocytomas WHO grade II (A II) and 26 anaplastic astrocytomas WHO grade III (A III) for loss of heterozygosity (LOH) on chromosomes 1p, 1q, 9p, 9q, 10p, 10q, 11p, 13q, 17p, 19p, 19q and 22q and for amplification of the EGFR receptor. A PCR-based assay with microsatellite repeat sequences was employed for the detection of polymorphisms on silver-stained polyacrylamide gels. LOH on 9p was seen in 1/18 (6%) informative cases of A II and 4/25 (16%) informative cases of A III. LOH on 17p was observed in 10/17 (53%) informative cases of A II and 15/28 (54%) informative cases of A III. LOH on 19q was detected in 2/18 (11%) informative cases of A II and in 12/26 (46%) informative cases of A III. Thus, the majority of chromosomal regions examined in this study do not appear to play a role in malignant progression of astrocytomas. LOH 17p is a frequent event in astrocytoma but has not been detected at a higher incidence in anaplastic variants. However, a putative tumor suppressor gene on chromosome 19q emerges as an interesting novel candidate for a progression associated gene in human astrocytic gliomas. PMID- 7534014 TI - [Computer assisted grading of gliomas of the astrocytoma/glioblastoma groups]. AB - For automated astrocytoma grading morphometric parameters are determined by means of an image analysis system and a special Ki-67(MIB1)/Feulgen-staining method allowing the quantification of the essential characteristics of malignant gliomas: growth pattern, cellularity, proliferation index and nucleus pleomorphism. Based upon a cluster analytical approach a grading scale resembling the WHO-scheme is established which is suitable for automatic glioma grading purposes (HOM-scale). For automatic glioma grading backpropagation neural networks are employed. The results are compared with those of a classical multivariate discriminant classificatory analysis. The presented approach can also be employed for automatic grading of other tumour entities. PMID- 7534016 TI - [Coexpression of hepatocyte growth factor-scatter factor (HGF-SF) and HGF-SF receptors (c-met proto-oncogene) in mammalian brain]. PMID- 7534015 TI - Genetic alterations associated with glioma progression. AB - Among tumours of the nervous system, mutations of the p53 tumour suppressor gene are largely restricted to neoplasms of astrocytic origin. These are the most common human brain tumours and span a wide range of biologic behavior, from the slowly growing low-grade astrocytoma (WHO Grade II) to anaplastic astrocytoma (WHO Grade III) and, ultimately, the glioblastoma multiforme (WHO Grade IV). In low grade astrocytomas, p53 mutations with or without loss of heterozygosity on chromosome 17p are the principle detectable change. Anaplastic astrocytomas contain p53 mutations in approximately one third of cases and further display loss of heterozygosity on chromosome 19q and homozygous loss of 9p21, tentatively identified as multiple tumour suppressor 1 (MTS-1). In addition to these genetic alterations, glioblastomas show loss of chromosome 10 and amplification of the EGF receptor gene at an incidence of > 60% and > 40%, respectively. The type and distribution of p53 mutations are not suggestive of specific environmental carcinogens operative in their etiology. PMID- 7534018 TI - [Morphological findings in temporal lobe epilepsy: experience with 216 consecutive surgical specimens]. AB - We have examined 216 consecutive surgical specimens of patients with chronic pharmacoresistant temporal lobe epilepsy. Neoplasms were detected in 75 cases (34.7%). All but two of these tumors were of low histopathological grade (WHO grade I or II). There were 34 gangliogliomas, 17 pilocytic astrocytomas, 9 oligodendrogliomas, 6 fibrillary astrocytomas, and 6 dysembryoplastic neuroepithelial tumors. Fifty-one specimens contained non-neoplastic focal lesions and an additional 13 cases had both tumors and non-neoplastic focal lesions within the same specimen. The most frequent non-neoplastic focal lesions were microscopic glioneuronal hamartias (32 cases), glioneuronal hamartomas (7 cases) and vascular malformations (13 cases). The hippocampal formation was structurally well preserved in 71 specimens and in 51 of these (71.8%) there was Ammon's horn sclerosis. The findings document that intractable temporal lobe epilepsy is associated with significant structural alterations in the great majority of patients. PMID- 7534017 TI - [Morphology and development of neural transplants of AMOG-deficient mice]. AB - The adhesion molecule on glia (AMOG) has been reported to function as cell adhesion molecule and also to constitute the beta 2-subunit of the murine Na,K ATPase. In order to elucidate these functions in vivo, Magyar et al. have generated mice carrying a targeted deletion of the AMOG gene. These mice exhibit behaviourally normal development till postnatal day P16. At this time, they develop muscular weakness, incoordination, and tremor. Death invariably occurs 24 36 hours after onset of the symptoms. Histological and ultrastructural examination of brain sections show enlarged ventricles, brain edema, and swelling of astrocyte end feet. However, no disturbances of the architecture or cell migration in the brain can be detected. In order to identify long-term consequences of AMOG deficiency which might not yet be detectable at the time of death, we have established a CNS grafting model. The embryonal brain anlage (E10.5-E13.5) was grafted into the caudoputamen of wild type mice. The graft recipients are sacrificed up to 7 months after the procedure. Both wild type and AMOG deficient grafts develop and form solid neural tissue with neurons, myelinated axons, glial cells, and ventricular structures, as shown by histological and immunocytochemical analysis. However, no differences in grafts derived from wild type, heterozygous, and AMOG-deficient donors can be detected. Proliferation has been examined by BrdU immunocytochemistry. The blood-brain barrier as examined by repeated magnetic resonance imaging after injection of Gadolinium-DTPA has been shown to be largely reconstituted five weeks after grafting. PMID- 7534022 TI - [List of members]. PMID- 7534020 TI - [Expression of the extracellular matrix proteoglycan, versican, in human skin]. AB - Extracellular matrix chondroitin/dermatan sulfate proteoglycans are present in a wide variety of tissues including cartilage, placenta, aorta, tendon, brain and skin. They possibly participate in cellular processes such as cell adhesion, migration and proliferation. Recently, we have determined the entire primary structure of the large fibroblast proteoglycan, versican, on the basis of its cDNA sequence. Versican belongs to the family of large aggregating proteoglycans. Other members of the family, which have been characterized in terms of their primary structure, are aggrecan in cartilage and neurocan isolated from brain tissues. Due to the extensive sequence similarities between these three proteoglycans in the N- and C-terminal domains and due to the high degree of carbohydrate substitution, the generation of antibodies monospecific for versican has been difficult. To avoid cross-reactivity with aggrecan and neurocan, we therefore prepared unique portions of versican in a bacterial expression system and used them to immunize rabbits (Zimmermann et al., 1994). The affinity purified anti-fusion protein antibodies specifically reacted with intact versican from an osteosarcoma cell line. First immunohistochemical experiments on cryo sections of human skin revealed anti-versican staining in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. By indirect immunofluorescence, Northern and Western blotting we could demonstrate that both, dermal fibroblasts and keratinocytes express versican in primary cultures. A striking inverse correlation between versican expression and cell density was observed. Analogous to the in vivo situation, keratinocytes induced to terminally differentiate ceased to express versican.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534021 TI - [Bestowal of the Rudolf Virchow Medal of the German Society of Pathology]. PMID- 7534019 TI - [Molecular pathology of mitochondrial disorders]. AB - In the last few years morphological-cytochemical investigations and molecular genetic analysis have considerably increased the knowledge about disorders of mitochondrial functions. Molecular genetic studies have revealed that specific mutations of mitochondrial DNA are associated with specific clinical diseases and may therefore be of value in genetic counseling. Since the mitochondrial DNA is present in a high copy number in the cells a threshold exists for the biochemical manifestation of mutated mitochondrial DNA and there is no strict correlation between geno- and phenotype. Biochemical-cytochemical studies therefore are still the standard diagnostic procedures for demonstrating the clinical importance of mitochondrial mutations. During ageing similar mutations of mitochondrial DNA accumulate especially in postmitotic organs - although at a lower rate than in mitochondrial diseases - and probably contribute to the reduced organ functions in senescence. PMID- 7534023 TI - Desmosomes--dual junctional principles of intra- and supracellular order in epithelial differentiation and tissue formation. AB - The cells of most normal and malignantly growing tissues are connected by "adhering junctions", i.e. distinct sites of "homotypic" contact between the plasma membranes of two cells of the same or a similar kind, associated on the cytoplasmic side by a dense plaque at which often bundles of cytoskeletal filaments anchor. Of the various types of adhering junctions desmosomes are characteristic of epithelia and carcinomas but also occur in some other cell types. Their molecular components have recently been identified and characterized by cDNA-cloning and sequencing. Unexpectedly, the molecular complement of desmosomes has been found to show certain differences in different epithelia, with particularly complex patterns in stratified squamous epithelia as well as in tumors and cultured cell lines derived therefrom. In addition, molecular principles important in the assembly of desmosomes and in the specific anchorage of intermediate-sized filaments (IFs) at desmosomal plaques have been elucidated. The possible value of cell type-specific isoforms of desmosomal components as markers for the subtyping of carcinomas and the role of desmosomal cadherins during invasion and metastasis of carcinomas are discussed. PMID- 7534024 TI - [Hepatitis B and C: biology and molecular diagnosis]. AB - Viral infections are major causes of chronic liver diseases. The etiologic agents are hepatitis B virus (HBV), hepatitis C virus (HCV) and the HBV-associated hepatitis delta virus (HDV). The biology and molecular structure of these viruses have been studied in great detail. In general, these viral infections can be detected by serological tests and molecular techniques, including hybridization analyses (dot blot, Southern blot or Northern blot and in situ hybridization) as well as polymerase chain reaction (PCR) amplification and in situ PCR. The sensitive and specific identification of HBV and HCV is clinically relevant both for epidemiologic-preventive as well as for therapeutic reasons. PMID- 7534025 TI - Immunohistochemical properties of malignant mesothelioma cells in histologic and cytologic specimens. AB - Although there is general agreement that immunohistochemical methods can aid in the pathologic diagnosis of malignant mesothelioma, some studies have produced conflicting results. To obtain comparable and reproducible results, unequivocal malignant mesotheliomas were studied with the biotin-streptavidin-peroxidase complex method in 14 formalin-fixed, paraffin-embedded tissue specimens, 5 ethanol-fixed smear slides and 3 cold acetone-fixed smear slides. The expression of CA-125, epithelial membrane antigen (EMA) and vimentin by malignant epithelial mesothelioma cells was hindered by their poor preservation in formalin fixative. Cytologic specimens fixed in cold acetone were the best type for immunohistochemistry. The majority of malignant epithelial mesothelioma cells in the smear slides fixed in cold acetone were positive for CA-125, EMA, low molecular-weight cytokeratin and vimentin, but none of them were positive for carcinoembryonic antigen, CA-19-9, epithelial antigen, high-molecular-weight cytokeratin or Leu-M1. PMID- 7534026 TI - Perivascular neuropeptides (NPY, VIP, CGRP and SP) in human brain vessels after subarachnoid haemorrhage. AB - INTRODUCTION--Cerebral blood vessels are innervated by sympathetic nerve fibres storing neuropeptide Y (NPY), parasympathetic nerves storing acetylcholine, vasoactive intestinal peptide (VIP) and sensory afferent fibres containing calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A. In experimental studies on subarachnoid haemorrhage (SAH) there are indications that perivascular peptides are involved. In the present study we have in man measured the levels of NPY, VIP, SP and CGRP in brain vessels of patients that have suffered a fatal SAH and compared this with the levels encountered in subjects that died of an extracerebral cause. MATERIAL AND METHODS--Vessels from patients who have died from SAH or nonSAH were obtained during autopsy performed within 24 hrs after death. The peptides were extracted and fractionated with reversed phase liquid chromatography (HPLC). The levels of NPY, VIP, SP, and CGRP were measured with radioimmunoassay. Vasomotor responses of human cerebral arteries were performed using a sensitive in vitro system. RESULTS--Human cerebral vessels contained NPY, VIP, CGRP and SP which eluted at the same positions as the authentic peptides. The level of CGRP was significantly lower (p < 0.01) in arteries removed from SAH patients as compared to control subjects. The level of SP was not changed, if anything it tended to be increased after SAH. The levels of NPY and VIP were not significantly altered after SAH. In isolated brain vessels alpha-CGRP was a potent vasodilator of arteries precontracted with whole blood, prostaglandin F2 alpha or endothelin. It had a poor effect on vessels precontracted with 60 mM potassium. CONCLUSION--The evidence suggest that the trigemino-cerebrovascular system, storing CGRP and SP, is to a differential degree involved in the pathophysiology of SAH in man and supports the hypothesis of an exhaustion of CGRP as one important factor in the development of late spasm occurring after SAH. PMID- 7534027 TI - Interferon alpha-2a in the treatment of exudative senile macular degeneration. AB - We performed a pilot study of 10 patients with the objective to determine the efficacy of interferon alpha-2a in the treatment of age-related macular degeneration with freshly diagnosed choroidal neovascular membrane. Interferon was given at a dose of 3 MIU/m2 three times a week for 8 weeks. Mean age of the patients was 74 years. In 3 patients we combined laser photocoagulation of extrafoveal part of the choroidal neovascular membrane to interferon therapy. The patients were followed up for 1 year after termination of interferon treatment. In 1 patient a visual improvement of two Snellen lines was noted, while in 7 patients visual acuity remained the same during the treatment (includes 3 patients treated with combined Krypton-laser photocoagulation). In 2 patients there was a decrease in acuity of one line. Six months later visual acuity had deteriorated in 7 patients and 1 year later all the patients had visual acuity < or = 20/200. We did not find any regression of choroidal neovascular membrane in fluorescein angiography in any of the patients during the treatment except the laser-treated area. At the 6-month follow-up there was still leakage or choroidal neovascular membrane growth in 6 patients. Combined interferon-laser therapy did not prove to be more effective in preventing the membrane growth. Side-effects included weakness, apathy, and fatigue in this elderly population. Our results indicate that treatment with 3 MIU/m2 interferon for 8 weeks is not an effective treatment for subfoveal choroidal neovascular membranes. PMID- 7534030 TI - Reciprocal modulation of aconitase activity and RNA-binding activity of iron regulatory factor by nitric oxide. PMID- 7534031 TI - Mechanism of production of the serum transferrin receptor. PMID- 7534028 TI - The role of cytokines in the regulation of ferritin expression. PMID- 7534032 TI - Dual effects of taurine on membrane ionic conductances of rat skeletal muscle fibers. PMID- 7534029 TI - Stimulation of IRE-BP activity of IRF by tetrahydrobiopterin and cytokine dependent induction of nitric oxide synthase. PMID- 7534033 TI - Taurine release from mouse hippocampal slices: effects of glutamatergic substances and hypoxia. PMID- 7534034 TI - Taurine protects against oxidant-induced lung injury: possible mechanism(s) of action. AB - It is thought that oxidant-induced tissue damage is not a direct effect of the oxidant per se, but rather results from the inflammatory response that occurs thereafter. As a result of inflammation following oxidant exposure, there are neutrophils, monocytes, and macrophages with myeloperoxidase-H2O2-halide activity in the lung. Leukocytes and especially neutrophils contain high intracellular concentrations (22-50mM) of taurine (6, 8, 11, 20). Taurine acts as a trap for toxic hypochlorous acid (HOCl) and forms the less reactive metabolite, N chlorotaurine (5-6). Thus, the biological activity of halide-dependent myeloperoxidase may be regulated by endogenous taurine. Although taurine had no effect in the present study, polymorphonuclear leukocytes have an active myeloperoxidase system capable of producing N-chlorotaurine (9, 19) and would be present at the site of inflammation in oxidant-exposed lungs. Our data suggest that taurine via N-chlorotaurine formation may protect the lung from oxidant injury, at least in part, by inhibiting production of nitrite and TNF-alpha. Moreover, lavage cells isolated from rats pretreated with taurine and exposed to O3 have a significant decrease in the production of nitrite and TNF-alpha, compared with lavage cells from rats exposed to O3 without taurine supplementation (preliminary studies). Both the concentration of taurine and the effects of N-chlorotaurine strengthen the potential impact of this chlorinated amine in vivo. N-Chlorotaurine may protect against oxidant-induced lung injury by inhibiting production of nitrite and the release of TNF-alpha which are both known to be directly linked to tissue injury. PMID- 7534037 TI - [Choroidal neovascularization with indocyanine green infrared fluorescence angiography and vascular cast preparation]. AB - Indocyanine green infrared fluorescence angiography (ICG angiography) was compared with the fluorescein angiography (FAG) to evaluate its benefit. Experimentally induced choroidal neovascularization (ChNV), by laser photocoagulation of monkey eyes was studied by means of cast preparation correlating with findings of ICG angiography. FAG could detect ChNV more clearly than ICG angiography. However, some (2/35 sites) ChNVs with subretinal hemorrhage could be detected only with ICG angiography. In the early phase, the densely distributed part or thick vessels of ChNV could be detected with ICG angiography. On the other hand, the loose part could not be detected. Cast preparation did not show morphologic difference between leaky and non-leaky lesions with ICG angiography. These results showed that ICG angiography could not produce clearer results than FAG. However, ChNVs covered with subretinal hemorrhage could be detected only with ICG angiography. ICG angiography has complementary value in conjunction with FAG in order to detect ChNVs. PMID- 7534035 TI - Specific CD45 isoforms regulate T cell ontogeny and are functionally distinct in modifying immune activation. PMID- 7534038 TI - Ribozymes: RNA as a therapeutic agent. AB - RZs, like ASO agents, are several years from approval by the Food and Drug Administration. Although many problems must be solved before they can be used effectively as therapeutic agents, results from a wide variety of in vitro and in vivo experiments are encouraging. The advantages of RZs are their very high specificity of action, their catalytic mechanism, their potential for targeted therapy, and the lack of side effects with their use. These advantages, coupled with the wide applicability of ribozyme technology, will continue to drive the research necessary to develop RZs into useful therapeutic agents. PMID- 7534036 TI - The role of CD40 ligand in human disease. PMID- 7534039 TI - Epidermolysis bullosa simplex: a keratin 5 mutation is a fully dominant allele in epidermal cytoskeleton function. AB - To explore the relationship between abnormal keratin molecules, 10-nm intermediate filament (IF) organization, and epidermal fragility and blistering, we sought to determine the functional consequences of homozygosity for a dominant keratin defect. We describe a family with an autosomal dominant skin-blistering disorder, epidermolysis bullosa simplex, Koebner subtype (EBS-K), that has a novel point mutation, occurring in the keratin 5 gene (KRT5), that predicts the substitution of an evolutionarily conserved lysine by an asparagine residue (K173N). Unlike previous heterozygous mutations located within the initial segment of domain 1A of keratin molecules, K173N heterozygosity did not result in severe disease or clumping of keratin filaments. One family member was found to be homozygous for the K173N allele, having inherited it from each of her affected first-cousin parents. Despite a lack of normal keratin 5 molecules, and an effective doubling of abnormal molecules, available for heterodimerization with keratin 14 during IF formation, there were no significant differences in the clinical severity or the ultrastructural organization of the keratin IF cytoskeleton of the homozygous individual. These data demonstrate that the K173N mutation behaves as a fully dominant allele and indicate that a limited number of abnormal keratin molecules are sufficient to impair cytoskeletal function and elicit epidermal fragility and blistering. PMID- 7534040 TI - A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype. AB - mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA- >G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. PMID- 7534041 TI - Anomalous expression of P-cadherin in breast carcinoma. Correlation with E cadherin expression and pathological features. AB - Previous studies on the cell-cell adhesion molecules P- and E-cadherin have shown that P-cadherin is not expressed in breast cancer. In contrast, the expression of E-cadherin is a normal event in these tumors, but a reduction in the levels of this molecule in neoplastic cells is associated with the histological type, high histological grade, greater tumor size, and metastasis. The expression pattern of P- and E-cadherin were immunohistochemically studied in tissue sections from normal breast tissue, benign breast lesions, and 57 infiltrating breast carcinomas. Cadherin expression was analyzed in parallel with pathological features and the immunohistochemical expression of estrogen and progesterone receptors in breast carcinomas. P-cadherin was detected in the myoepithelial cells and E-cadherin in luminal epithelial cells from normal breast and benign breast lesions. P-cadherin expression was detected in 9 of 45 cases (20%) of infiltrating ductal carcinomas of no special type; none of the special histological types that were analyzed (7 infiltrating lobular carcinomas, 3 colloid carcinomas, and 2 infiltrating papillary carcinomas) expressed P cadherin. In infiltrating ductal carcinomas, P-cadherin expression correlated significantly with a reduction in E-cadherin expression, histological grade (all cases were grade III tumors), and hormone receptor content (8 of 9 cases were estrogen and progesterone receptor negative). Although E-cadherin was not found in the 7 infiltrating lobular carcinomas, it was present in the remaining histological types and was preserved in 15 infiltrating ductal and 3 colloid and 2 papillary carcinomas and was reduced in 30 infiltrating ductal carcinomas. In addition, a reduction in E-cadherin expression was significantly associated with high histological grade and a lack of steroid hormone receptors in infiltrating ductal carcinomas. No apparent relationship was found between P- and E-cadherin expression and tumor size and axillary lymph node metastasis. The distinct patterns of P- and E-cadherin expression observed in this study strongly suggest a differential role for these cadherins in human breast carcinogenesis. PMID- 7534042 TI - Immunohistochemical analysis of Bcl-2 protein regulation in cutaneous melanoma. AB - Cutaneous melanoma is becoming increasingly common. Genetic and environmental factors are thought to play a role in its pathogenesis. We have previously shown that normal human melanocytes strongly express the oncoprotein, Bcl-2. To determine the role of Bcl-2 in melanocytic tumors, we studied human benign nevi and melanomas for expression of Bcl-2 protein using immunohistochemistry. Our results show that benign melanocytes from 3 of 4 normal skin biopsies and 5 of 7 common acquired nevi strongly express Bcl-2. Conversely, only 3 of 23 primary cutaneous melanomas and 3 of 9 metastatic melanomas showed strong staining in comparison with melanocytes from normal skin and common acquired nevi (chi 2, P = 0.0021). Interestingly, 0 of 6 dysplastic nevi, a precursor of melanoma, demonstrated strong staining as compared with melanocytes and nevi (8 of 11; chi 2, P = 0.02), but similar expression to that of melanoma (6 of 32; chi 2, P = 0.6). We conclude that Bcl-2 expression decreases in malignant melanoma and suggest that this may be related to the autonomous growth characteristics of malignant melanoma. PMID- 7534045 TI - CD40 antigen expression on Reed-Sternberg cells. A reliable diagnostic tool for Hodgkin's disease. PMID- 7534043 TI - Expression of prolactin and prolactin receptor in human breast carcinoma. Evidence for an autocrine/paracrine loop. AB - The neuroendocrine hormone prolactin is a growth factor required for the proliferation and terminal differentiation of the human breast. These effects are mediated by the prolactin receptor, a member of the growth factor receptor family. Three prolactin receptor isoforms (long, intermediate, and short) have been identified in the rat, which differ in the length of their intracytoplasmic domains. In humans, however, only the long prolactin receptor isoform had been identified previously. The expression of the human intermediate prolactin receptor is demonstrated and preliminary evidence for a human short isoform is presented. Heterogeneous expression of prolactin receptor, at the immunoblot and immunohistochemical levels was observed in breast carcinoma specimens. A statistically significant correlation between prolactin receptor and estrogen receptor expression was noted. An autocrine/paracrine role for prolactin within breast tissues was further examined by performing reverse transcription polymerase chain reaction on RNA isolated from cell lines and clinical specimens with prolactin-specific primers. A 585-bp product was observed and found to be identical to human prolactin. The synthesis of prolactin by breast epithelium was confirmed by in situ hybridization analysis of breast tissues and the detection of bio- and immunoreactive prolactin in breast cancer lines. These analyses indicate that the principal site for prolactin expression within the normal or malignant breast residues within the epithelium. These data indicate that prolactin may participate in an autocrine/paracrine stimulatory loop within breast tissues and suggest a role for this growth factor in the pathogenesis of breast cancer. PMID- 7534046 TI - Dot blot analysis of rat gastric mucin using histochemical staining methods. AB - Rat gastric mucins blotted on various membranes were detected using the periodate Schiff staining method. The use of a polyvinylidene difluoride (PVDF) membrane gave the best results compared to nitrocellulose, nylon, positively charged nylon, and positively charged PVDF membranes. Alcian blue, high-iron diamine, galactose oxidase-cold thionin Schiff, and paradoxical concanavalin A stainings were also performed to detect mucins on PVDF membranes. Mucins were quantitatively detected by this analysis using each staining method with a range of detection from 0.02 to 1 microgram. Mucins extracted from rat gastric mucosa were detected by these dot blot assays at the position of void volume during Sepharose CL-4B chromatography. Mucins, after being purified with cesium trifluoroacetate centrifugation, were also mainly excluded from a Sepharose CL-2B column and detected by the dot blot assay using each staining method. In contrast, mucins after reduced alkylation were separated into two fractions during Sepharose CL-2B chromatography; one was excluded but the other was included in the column. Both fractions were detected using the periodate-Schiff method, but only the excluded fraction was stained using the paradoxical concanavalin A staining method. Thus, the dot blot assay using histochemical staining methods is useful for detecting individual mucins during gel chromatography. PMID- 7534044 TI - Decrease of heparan sulfate staining in the glomerular basement membrane in murine lupus nephritis. AB - Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined. PMID- 7534047 TI - A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail. AB - Expression of newly cloned cDNAs in mammalian cell lines is an essential tool for the functional analysis of the proteins encoded by these cDNAs. In many instances, however, evaluation of the protein is difficult because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins on Western blots or for immunocytochemistry or immunoprecipitation. A number of gene fusion systems have been employed in which a known peptide is fused to the expression product of interest and the fusion protein is purified using affinity chromatography and identified in extracts or by immunocytochemistry using antibodies directed against the affinity handle peptide. The DNA-binding domain of the yeast transcription factor GAL4 is widely used to construct fusion proteins with putative transcription factors to evaluate potential trans-acting domains. Because of the lack of commercially available anti-GAL4 antibodies, the further biochemical characterization of these fusion proteins has remained difficult. We describe the construction of two mammalian expression vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for pM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast transcription factor GAL4 with a Flag peptide (consisting of the 11-amino-acid leader peptide of the gene 10 product from bacteriophage T7) at the NH2-terminus and a tail of six histidines at the COOH terminus. Unique restriction sites allow both the construction of fusion proteins with the GAL4 DNA-binding domain and the replacement of the GAL4 fragment with another insert.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534048 TI - Cholate and pH reduce interference by sodium dodecyl sulfate in the determination of DNA with Hoechst. AB - The use of the fluorescent dye 33258 Hoechst (Hoe) to quantitatively determine DNA in cell culture in the presence of lysing agents like sodium dodecyl sulfate (SDS) is limited by the masking effect of high levels of nonspecific fluorescence, caused by the binding of Hoe to micelles. The masking effect can be reduced substantially by increasing the concentration of the counterion, the addition of cholate, or the pH of the buffer. An optimized method was developed, combining the antimasking effects of sodium chloride, cholate, and pH to accurately determine DNA concentrations as low as 15 ng/ml in the presence of up to 6.9 mM (0.2%) SDS. The effectiveness of SDS in cell dissolution can now be combined with the specificity and sensitivity of Hoe to determine cellular DNA. PMID- 7534049 TI - A staining procedure using Coomassie brilliant blue G-250 in phosphoric acid for detection of protein bands with high resolution in polyacrylamide gel and nitrocellulose membrane. PMID- 7534050 TI - Quantification of proteins dissolved in an electrophoresis sample buffer. PMID- 7534051 TI - Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development. AB - The lungs of neonatal foals contain many nerves immunoreactive for substance P and calcitonin gene-related peptide. These nerves are closely associated with the epithelium, bronchial and pulmonary vessels and the airway smooth muscle of all intrathoracic airways, including non-cartilaginous bronchioles. Activation of sensory nerves in the respiratory epithelium could thus potentially affect, via local axon reflexes, vascular and respiratory smooth muscle in neonatal equine airways. Nerves immunoreactive for these peptides are much more widely distributed within the lung than in adult horses; they may thus play a trophic role before birth, or contribute to the post-natal adaptation to breathing. PMID- 7534052 TI - Use of filamentous cyanobacteria for biodegradation of organic pollutants. AB - Biodegradation is increasingly being considered as a less expensive alternative to physical and chemical means of decomposing organic pollutants. Pathways of biodegradation have been characterized for a number of heterotrophic microorganisms, mostly soil isolates, some of which have been used for remediation of water. Because cyanobacteria are photoautotrophic and some can fix atmospheric nitrogen, their use for bioremediation of surface waters would circumvent the need to supply biodegradative heterotrophs with organic nutrients. This paper demonstrates that two filamentous cyanobacteria have a natural ability to degrade a highly chlorinated aliphatic pesticide, lindane (gamma hexachlorocyclohexane); presents quantitative evidence that this ability can be enhanced by genetic engineering; and provides qualitative evidence that those two strains can be genetically engineered to degrade another chlorinated pollutant, 4 chlorobenzoate. PMID- 7534053 TI - Application of ribotyping for differentiating Vibrio cholerae non-O1 isolated from shrimp farms in Thailand. AB - A collection of 143 Vibrio cholerae non-O1 strains isolated from shrimp farms in Thailand were characterized and grouped by ribotyping. Sixty-four ribotypes were distinguished following digestion of chromosomal DNA with the restriction enzyme BglI, and the reproducibility of the method was 100%. There was no correlation between specific ribotype distributions and the locations of the shrimp farms. Ribotype similarity was examined by cluster analysis, and two main groups with 10 and 54 ribotypes, respectively, were found. Correlation between ribotype and O antigen expression was shown to exist among those isolates tested. Ribotyping appears to be a suitable method for differentiating environmental V. cholerae non O1 strains, and comparison of ribotype patterns showed a high degree of genetic divergence within V. cholerae non-O1. PMID- 7534054 TI - Molecular cloning and transcriptional analysis of the Aspergillus terreus gla1 gene encoding a glucoamylase. AB - The Aspergillus terreus gla1 gene, coding for a glucoamylase, has been cloned by heterologous hybridization. The gene is interrupted by four introns and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The expression of the gene is induced by starch and maltose and repressed by glucose. PMID- 7534055 TI - Eosinophil granule proteins in cardiopulmonary bypass with and without heparin coating. AB - Extracorporeal circulation with exposure of blood to foreign surfaces causes activation of different defense systems, eg, white cells. Several potent mediators are released into plasma, capable of causing harmful effects to different organs, contributing to postoperative morbidity after operations using cardiopulmonary bypass. The eosinophil granulocyte has not previously been investigated in this respect. We studied two of its activation products, eosinophil cationic protein and eosinophil protein X in coronary bypass patients. In 17 control patients, plasma levels of eosinophil cationic protein and eosinophil protein X increased considerably during cardiopulmonary bypass. In 19 patients with heparin-coated cardiopulmonary bypass equipment the levels were significantly reduced, indicating improved biocompatibility of the cardiopulmonary bypass circuit. The heparin-coated surface causes less activation of eosinophils; also released eosinophil cationic protein is bound to the heparinized surface. PMID- 7534057 TI - CD34 positivity in fibrosarcomas which arise in dermatofibrosarcoma protuberans. AB - OBJECTIVE: On rare occasion, fibrosarcomatous change may arise in a dermatofibrosarcoma protuberans (DFSP), and this alteration is of prognostic significance. Histologically, this change may be subtle and difficult to recognize. The goal of this study was to evaluate the pattern of CD34 immunoreactivity in DFSP with fibrosarcomatous areas (DFSP-FS). DESIGN: Of 100 cases of DFSP identified at a large metropolitan hospital, 7 were found to have fibrosarcomatous change. Thirty cases of typical DFSP and seven cases of DFSP-FS were stained with an antibody for CD34. RESULTS: All 30 cases of typical DFSP stained intensely for CD34. In the seven cases of DFSP-FS, all cases showed a similar staining pattern in the DFSP portion. However, focal CD34 immunoreactivity was noted in only one (14%) of seven cases in the fibrosarcomatous portion of these tumors. CONCLUSIONS: CD34 immunoreactivity is rare in the fibrosarcomatous portions of DFSP-FS, and this immunophenotype may be diagnostically useful in those cases in which it is difficult to recognize this change histologically. PMID- 7534058 TI - Intravascular bronchioloalveolar tumor of the lung presenting as pulmonary thromboembolic disease and pulmonary hypertension. AB - Intravascular bronchioloalveolar tumor, the pulmonary counterpart of epithelioid hemangioendothelioma, typically presents as bilateral pulmonary nodules in young women. We report a case of intravascular bronchioloalveolar tumor that clinically mimicked acute pulmonary thromboembolic disease initially and was subsequently proven to have pulmonary hypertension with right ventricular dysfunction by angiography. The diagnosis of intravascular bronchioloalveolar tumor was confirmed by immunohistochemical and ultrastructural studies after it was suspected on routine histologic examination. In addition, the tumor cells expressed glycoprotein cell adhesion molecule CD44, which has been implicated in increased tumor invasiveness and metastasis in various carcinomas and several aggressive non-Hodgkin's lymphomas. PMID- 7534056 TI - Chronic hepatitis C. Analysis of host immune response by immunohistochemistry. AB - The pathogenesis and perpetuation of hepatocellular injury in hepatitis C viral infection remains unclear. It has been proposed that a direct viropathic effect, the host immune response, or both mediate cell damage. To address this issue, the immunophenotype of the inflammatory infiltrate in the liver of 18 patients with abnormal liver function tests and serologically detectable hepatitis C virus antibodies was compared with seven control patients (three cases with hepatitis B virus infection, two with alcoholic hepatitis, and one patient each with primary biliary cirrhosis and autoimmune hepatitis). The immunohistochemical markers included UCHL1, L26, Ham-56, Mac-387, CD68, Leu-M1, and cathepsin B. We found that T cells represent the predominant cell type in both histopathologic patterns of hepatitis C, ie, chronic active hepatitis and chronic persistent hepatitis, but the intensity of the T-cell infiltrate displayed marked differences. B-cell infiltrates were only seen in the germinal centers of lymphoid follicles in portal tracts. Furthermore, significant numbers of CD68-positive macrophages/monocytes were seen in the more aggressive form of hepatitis C viral infection. These data suggest that the T-lymphocyte-mediated host immune response is similar in chronic active and chronic persistent hepatitis patterns of hepatitis C viral infection, but varies in its intensity. In addition, macrophages/monocytes may play a role in hepatocyte and bile duct injury in chronic hepatitis C. PMID- 7534059 TI - Aggressive surgical resection for cholangiocarcinoma. AB - OBJECTIVES: To review the spectrum of cholangiocarcinoma in patients treated by a single team of hepatobiliary surgeons over an 8-year period, to evaluate the predictors of survival, and to assess the results of an aggressive approach to surgical resection. DESIGN: Retrospective review of all clinical records of patients referred for treatment of cholangiocarcinoma, with univariate analysis of clinical and pathologic factors in relation to patient survival. SETTING: New England Deaconess Hospital, Boston, Mass. PATIENTS: Eighty-eight consecutive patients referred with the established diagnosis of cholangiocarcinoma, from December 31, 1985, to April 15, 1994. INTERVENTIONS: Seventy-five of 88 patients were treated surgically, with 59 undergoing major resection for cure. Of the 29 patients treated palliatively, 16 had operations and 13 had wire mesh stents placed nonoperatively. MAIN OUTCOME MEASURES: Morbidity, mortality, and patient survival. RESULTS: Survival correlates directly with the pathologic stage (TNM). Tumor location had no impact on survival. Patients undergoing resection survived significantly longer (median, 23.2 months) than palliated patients (median, 7.7 months; P = .0015). Nonoperative palliation resulted in better survival than surgical palliation (P = .045). Major hepatic resection was used alone in eight patients with predominating intrahepatic lesions, while 18 patients with hilar lesions underwent en bloc skeletonization in conjunction with major hepatic resection. Resection with microscopically free margins significantly improved survival. Only patients undergoing major resection enjoyed survival greater than 2 years. CONCLUSIONS: Patient survival can be significantly improved by aggressive surgical resection. Hepatic resection should be used aggressively to achieve disease-free margins to optimize survival. Hepatic resection can be performed with low morbidity and mortality. Liver transplantation should be avoided as a treatment for cholangiocarcinoma. The best palliation for unresectable disease remains debatable. We advocate nonoperative treatment with endobiliary expandable wire mesh stents for patients with unresectable disease. PMID- 7534060 TI - Five-year incidence and disappearance of drusen and retinal pigment epithelial abnormalities. Waterman study. AB - PURPOSE: To obtain 5-year longitudinal data on age-related macular degeneration (AMD) that might be useful for disease prognosis, public health planning, and clinical trial development. PATIENTS AND METHODS: Baseline (1985) and 5-year follow-up (1990) fundus photographs of 483 watermen over 30 years of age who participated in a cohort study conducted on the eastern shore of Maryland were graded independently in a reliable, standardized fashion. Eyes in which AMD appeared or disappeared also were graded in a side-by-side fashion. RESULTS: Development of definite choroidal neovascularization and/or disciform scarring occurred in one of 50 participants over 70 years of age, specifically one of 15 participants over 70 years of age with AMD-3 (defined as large or confluent drusen focal hyperpigmentation of the retinal pigment epithelium [RPE], and/or nongeographic atrophy of the RPE). Appearance of large drusen, focal hyperpigmentation, or AMD-3 was age related, occurring in 5%, 1%, and 7%, respectively, of participants aged 50 to 59 years; 17%, 3%, and 14%, respectively, of participants aged 60 to 69 years; and 17%, 9%, and 26%, respectively, of participants aged 70 years or more. Disappearance of large drusen, hyperpigmentation, or AMD-3 occurred in 16 (34%) of 47 participants, 11 (58%) of 19 participants, and 17 (28%) of 61 participants, respectively, who had each feature photographically present in 1985. Among the 47 eyes identified in which AMD-3 developed by independent gradings, 38 cases of AMD-3 (81%) were confirmed on side-by-side grading. Among the 16 eyes identified as having AMD-3 that disappeared, nine disappearances (56%) were confirmed. Borderline differences in appearance of pigment, drusen size, drusen location, or photographic quality may have accounted for disappearance in seven cases (44%). CONCLUSIONS: Prospective studies on the nonneovascular features of AMD (including large drusen and abnormalities of the RPE) must account for the appearance and disappearance of these features and support the idea that side-by-side gradings can complement independent gradings identifying appearance or disappearance of features of AMD. PMID- 7534061 TI - Immunohistochemical analysis of the pathogenesis of posterior polymorphous dystrophy. AB - The pathogenesis of posterior polymorphous dystrophy was analyzed by immunohistologic methods. Sections of corneal buttons from two patients undergoing transplantation owing to posterior polymorphous dystrophy were stained with 2B4.14.1, a monoclonal antibody that reacts with human corneal endothelium, and with a cocktail of antihuman cytokeratin monoclonal antibodies that do not react with normal corneal endothelium. Single-stained sections revealed a variegated, intermittent staining pattern of antibody reactive and nonreactive cells. Double-stained sections revealed some cells that stained with only one of the antibodies and many cells that stained with both antibodies. The presence of cells staining positively for both 2B4.14.1 antigen and cytokeratins supports the hypothesis that the cytokeratin-expressing epithelial-like cells found in corneas with posterior polymorphous dystrophy arise via a metaplastic process in which the phenotype of endothelial cells becomes progressively abnormal. PMID- 7534062 TI - Laser photocoagulation of the choroid through experimental subretinal hemorrhage. AB - OBJECTIVE: To study the differential abilities of diode infrared, krypton red, and argon blue-green laser energies to penetrate experimental subretinal hemorrhage and coagulate the underlying choroid. METHODS: Autologous, heparinized whole blood was injected beneath the neurosensory retina of pigmented rabbit eyes. After 30 to 60 minutes, confluent patches of moderate or severe diode, krypton, or argon laser burns were applied to adjacent healthy retina and continued into the region of the subretinal hematoma without varying the power setting or focal plane. Histopathologic evaluation of early lesions was performed in a masked fashion, and subretinal hemorrhage thickness was determined with computer-assisted image capture and analysis. RESULTS: Retina overlying treated subretinal hemorrhage showed no ophthalmoscopically visible signs of photocoagulation with diode energy, a faint gray reaction with krypton energy, and an intense white reaction with argon energy. Histopathologic analysis revealed photocoagulative inner choroidal damage beneath a mean (+/- SD) maximum blood thickness of 0.56 +/- 0.14 mm with severe diode burns, 0.42 +/- 0.09 mm with severe krypton burns, and 0.22 +/- 0.04 mm with severe argon burns. CONCLUSIONS: These data demonstrate that laser penetration of subretinal blood increases with longer wavelengths in vivo. Diode infrared laser energy is capable of penetrating subretinal blood to coagulate the choroid in the absence of ophthalmoscopically visible changes in the overlying retina. PMID- 7534063 TI - The characterization of two diazepam binding inhibitor (DBI) transcripts in humans. AB - We have investigated the expression of diazepam binding inhibitor (DBI) (also called acyl-CoA-binding protein or endozepine) transcripts in different human tissues and tissue culture cell lines by reverse-transcriptase assisted PCR and RNase protection assay. Two different DBI transcripts capable of encoding polypeptides of 86 and 104 amino acids were detected in all the human tissues and cell lines studied. The transcript coding for the 86 amino acid DBI polypeptide was found to represent the majority of the total DBI transcript pool. PMID- 7534065 TI - Alterations in the frequency and shape of Ca2+ fluctuations in GH4C1 cells induced by thyrotropin-releasing hormone and Bay K 8644. AB - We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+) induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux. PMID- 7534064 TI - Integrin alpha 2 beta 1-independent activation of platelets by simple collagen like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity. AB - The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell recognition sequences. PMID- 7534066 TI - Tyrosine phosphorylation induced by cross-linking of Fc gamma-receptor type II in human neutrophils. AB - Neutrophils express several receptors for the Fc region of IgG molecules. Specific cross-linking of the type II receptor (Fc gamma RII) can be achieved by treating neutrophils with the Fab fragment of a specific monoclonal antibody IV.3 against the receptor followed by goat anti-mouse IgG F(ab')2 fragment. Such treatment initiates a number of neutrophil responses including the release of O2 . and increased protein tyrosine phosphorylation. The increase in tyrosine phosphorylation is rapid and transient and correlates with O2-. release. Both responses are inhibited by pretreatment of neutrophils with a protein tyrosine kinase inhibitor, genistein. The increase in protein tyrosine phosphorylation is not inhibited by pretreatment of neutrophils with pertussis toxin or an intracellular Ca2+ chelator, but is enhanced by a phosphoprotein phosphatase inhibitor, okadaic acid. The activity of a neutrophil Ca2+/calmodulin-dependent protein kinase II (CAMPKII) is also stimulated by cross-linking Fc gamma RII. The increase in CAMPKII activity is inhibited by pretreatment with either genistein or Ca2+ chelator. The results suggest that the increase in protein tyrosine phosphorylation induced by cross-linking of Fc gamma RII requires neither pertussis-toxin-sensitive G-proteins nor a rise in intracellular Ca2+ but can be regulated by protein phosphatases. Furthermore, protein tyrosine phosphorylation may be an early signal functionally linked to Fc gamma RII-mediated signal transduction leading to CAMPKII activation and O2-. release in human neutrophils. PMID- 7534067 TI - The three heavy-chain precursors for the inter-alpha-inhibitor family in mouse: new members of the multicopper oxidase protein group with differential transcription in liver and brain. AB - The inter-alpha-inhibitor (I alpha I) family is comprised of the plasma protease inhibitors I alpha I, inter-alpha-like inhibitor (I alpha LI), pre-alpha inhibitor (P alpha I) and bikunin. I alpha I, I alpha LI and P alpha I are distinct assemblies of bikunin with one of three heavy (H) chains designated H1, H2 and H3. These H chains and bikunin are respectively encoded by a set of three H genes and an alpha 1-microglobulin/bikunin precursor (AMBP) gene. All four gene products undergo maturation steps from precursor polypeptides. The full-length cDNAs for the H1-, H2- and H3-chain precursors were cloned from a mouse liver cDNA library and sequenced. Extensive searches of amino acid sequence similarities to other proteins in databanks revealed (i) a highly significant similarity of the C-terminal sequence in the three H-chain precursors to the multicopper-binding domain in the group of multicopper oxidase proteins and (ii) the presence of von Willebrand type-A domains in the mature H chains. Amino acid sequence comparisons between the three mouse H1-, H2- and H3-chain precursors and their human counterparts allowed us to appraise the timing and order of occurrence of the three H-chain genes from a shared ancestor during mammalian evolution. Owing to a multiple alignment of the six mouse and human nucleotide sequences for these H-chain precursors, a reverse transcriptase PCR assay with degenerate oligonucleotides was designed, allowing us to (i) present evidence that no mRNAs for further H genes exist in mouse liver and (ii) demonstrate a previously undescribed transcription of the H2- and H3-chain mRNAs in mouse brain, which contrasts with the expression of all four, H1, H2, H3 and AMBP, mRNAs in liver. PMID- 7534068 TI - Fibrillogenesis in Alzheimer's disease of amyloid beta peptides and apolipoprotein E. AB - A central event in Alzheimer's disease is the conformational change from normally circulating soluble amyloid beta peptides (A beta) and tau proteins into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. The apolipoprotein E (apoE) gene locus has recently been associated with late onset Alzheimer's disease. It is not know whether apoE plays a direct role in the pathogenesis of the disease. In the present work we have investigated whether apoE can affect the known spontaneous in vitro formation of amyloid-like fibrils by synthetic A beta analogues using a thioflavine-T assay for fibril formation, electron microscopy and Congo Red staining. Our results show that, under the conditions used, apoE directly promotes amyloid fibril formation, increasing both the rate of fibrillogenesis and the total amount of amyloid formed. ApoE accelerated fibril formation of both wild-type A beta-(1-40) and A beta-(1-40A), an analogue created by the replacement of valine with alanine at residue 18, which alone produces few amyloid-like fibrils. However, apoE produced only a minimal effect on A beta-(1-40Q), found in the Dutch variant of Alzheimer's disease. When recombinant apoE isoforms were used, apoE4 was more efficient than apoE3 at enhancing amyloid formation. These in vitro observations support the hypothesis that apoE acts as a pathological chaperone, promoting the beta-pleated sheet conformation of soluble A beta into amyloid fibres, and provide a possible explanation for the association of the apoE4 genetic isoform with Alzheimer's disease. PMID- 7534070 TI - Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells. AB - We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay. PMID- 7534071 TI - Growth signalling through the alpha 5 beta 1 fibronectin receptor. AB - This study demonstrates that perturbation of the fibronectin receptor (FNR), a member of the integrin family of adhesion receptors, can stimulate growth of non transformed epithelial cells but not of transformed epithelial cells. Using the non-adherent cell line FA-K562 we demonstrate that growth stimulation via FNR ligands occurs rapidly and independently of any effects on cell adhesion. Low valence FNR ligands such as glycine-arginine-glycine-aspartate-serine (GRGDS) are the most potent stimulators of the cell cycle regulatory kinase cdc2. Partial synchronization and Western blotting studies suggest that GRGDS affects cdc2/cyclin A complexes in cells in S/G2 phase of the cell cycle. These studies suggest that FNR-mediated growth control appears to be a common feature of transformation. These data suggest that the FNR may be physiologically important in growth control, especially in the presence of low valence, proteolytic degradation fragments of FN. Furthermore, escape from FNR-mediated growth control may be a common feature of transformation. PMID- 7534069 TI - bFGF and aFGF induce membrane ruffling in breast cancer cells but not in normal breast epithelial cells: FGFR-4 involvement. AB - Acidic and basic fibroblast growth factors (aFGF and bFGF) are growth factors which may have a physiological role in the normal breast and in breast cancer. A study of the effects of aFGF and bFGF on a variety of breast cell lines and epithelial cells purified from normal breast organoids showed that whereas normal breast cells did not exhibit membrane ruffling in response to either of these growth factors, some breast cancer cell lines did. This difference was not due to lack of receptor since all the cell lines tested were mitogenically stimulated by bFGF. Dominant negative mutations of FGF receptor 3 (FGFR-3) and the small GTP binding protein p21rac inhibited membrane ruffling, showing that receptor dimerization and phosphorylation and p21rac activation are prerequisites for membrane ruffling in response to aFGF and bFGF. Transient transfection of individual FGFRs into cos-7 cells showed that FGFR-1, FGFR-2 and FGFR-3 could not mediate a membrane ruffling response whereas FGFR-4 could. These studies elucidate one signalling mechanism of FGF and point to differences in the response of normal and cancer breast epithelial cells which may be important in cell motility. PMID- 7534072 TI - Differential phosphorylation of a 57-KDa protein tyrosine kinase during egg activation. AB - Fertilization results in activation of many protein kinases which function during egg activation. We have used metabolic labelling and immunoprecipitation to study changes in the phosphorylation state of a 57-KDa src-family protein tyrosine kinase during fertilization of the sea urchin egg. The kinase was phosphorylated on serine at all periods studied but it was also phosphorylated transiently on tyrosine at 5 minutes post insemination and then on threonine at 90 minutes after fertilization. These data indicate that the 57-KDa PTK may be under complex regulatory control during the first cell cycle. PMID- 7534073 TI - Epidermal growth factor stimulates mitogen-activated protein kinase by a PKC dependent pathway in human keratinocytes. AB - Epidermal growth factor (EGF), 20 ng/ml, stimulated myelin basic protein (MBP) phosphorylation in crude extracts from human keratinocyte primary cultures. In order to identify the involved kinases, we separated by fast protein liquid chromatography proteins participating in MBP phosphorylation. We detected three MBP kinase activities in the keratinocyte crude extracts. The first MBP kinase activity was the only one stimulated by EGF and reacted with anti-mitogen activated protein kinase (MAPK) antiserum recognising p42mapk and p44mapk isoforms. However, when protein kinase C (PKC) was either inhibited by the PKC inhibitor GF 109203X or depleted by a prolonged TPA treatment, the stimulation of MBP phosphorylation by EGF was strongly inhibited. The second MBP kinase activity eluted was due to a PKC isoform reacting with an anti-PKC zeta antibody, and the third was not identified. With this work, we have thus shown that, in human keratinocytes, EGF activates MAPK activity by a PKC-dependent pathway. PMID- 7534074 TI - Phosphatidylinositolmannoside-based liposomes induce no synthase in primed mouse peritoneal macrophages. AB - Liposomes prepared from phosphatidylinositolmannosides (extracted from BCG) and cholesterol are efficiently endocytosed by macrophages. Phagocytosis of particles or microbes modifies macrophage metabolism and in some cases, delivers potent stimulating signals to macrophages. We examined the effect of phosphatidylinositolmannoside-based liposomes on three macrophage functions especially important for host defenses: nitric oxide production, oxidative burst and TNF-alpha secretion. Phosphatidylinositolmannoside-based liposomes, added as empty vesicles, induced a strong NO synthase activity in mouse peritoneal macrophages primed either by interferon-gamma or by trehalose dimycolate. They also induced a moderate production of TNF-alpha. Phosphatidylinositolmannosides conferred activating properties to pH-sensitive liposomes. In contrast, liposomes composed of phosphatidylcholine and phosphatidylserine were unable to activate primed macrophages. PMID- 7534077 TI - Leucovorin and folic acid regimens for selective expansion of murine 5,10 methylenetetrahydrofolate pools. AB - Mice bearing subcutaneously implanted EMT6 mammary adenocarcinoma were treated with leucovorin or folic acid by continuous subcutaneous infusion or bolus intraperitoneal injection. (6R)-5,10-Methylenetetrahydrofolate pools in cytosolic extracts of the tumor, marrow, and gut were measured by analysis of the ternary complex with thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C methyltransferase, EC 2.1.1.45) and 5-fluoro-2'-deoxyuridylate, and the polyglutamate distribution in the (6R)-5,10-methylenetetrahydrofolate pool was analyzed by native gel electrophoresis. Bolus intraperitoneal administration of either leucovorin or folic acid caused dose-dependent expansion of the (6R)-5,10 methylenetetrahydrofolate pool in the tumor, but not in the marrow or gut. For example, the AUC (0-5 hr) in the tumor increased from a baseline value of 8.2 to 20 nmol/mg protein.hr after a bolus dose of 1.5 mmol/kg of leucovorin or folic acid, whereas the increase in marrow and gut was 2- to 4-fold lower. Continuous subcutaneous infusion at the same total dosage over 3 days gave AUC (0-96 hr) values of 134 nmol/mg protein.hr for controls as compared with 347 nmol/mg protein.hr for the leucovorin group and 254 nmol/mg protein.hr for the folic acid group. In contrast to bolus treatment, the increase in (6R)-5,10 methylenetetrahydrofolate in the marrow and small intestine with both leucovorin and folic acid infusion was similar to the increase in the tumor. Thus, intraperitoneal bolus injection was tumor selective, but subcutaneous continuous infusion was not. Longer-chain polyglutamates of (6R)-5,10 methylenetetrahydrofolate in the tumor after bolus treatment with 0.375 and 0.75 mmol/kg of leucovorin or folic acid increased relative to controls. At higher doses of 1.5 and 2.25 mmol/kg, an increase was observed only in the mono/diglutamate fraction. In marrow, on the other hand, the mono/diglutamate fraction, but not the longer-chain polyglutamates, increased at all doses. In the constant infusion regimen, longer-chain polyglutamates increased in all three tissues, though in gut and marrow the mono/diglutamate fraction increased more than in tumor. Leucovorin and folic acid were converted to (6R)-5,10 methylenetetrahydrofolate more efficiently but less selectively during a 3-day subcutaneous infusion than after an intraperitoneal bolus. Longer-chain polyglutamates were selectively increased in tumor by both regimens of leucovorin administration. PMID- 7534075 TI - Autocatalytic inactivation of lysosomal cathepsins is associated with inhibition of protein breakdown by insulin-like growth factor-1 (IGF-1) in myotubes. AB - Protein breakdown was monitored in C2C12 myotubes as the rate of release of radioactivity after prelabeling cell protein with [3H] tyrosine. IGF-1 (13 nM) and insulin (100 nM) prolonged the half-life of long-lived proteins. Enzymatic activities of cathepsins B and B+L were inhibited by the addition of IGF-1 or insulin. Immunoblotting of cathepsins B and L revealed extensive degradation of heavy chain forms by IGF-1. However, neither expression of cathepsins B and L genes nor expression of cystatin beta, an intrinsic inhibitor for cathepsins, was influenced. The addition of E-64, L-3-carboxy-trans-2,3-epoxypropionyl leucylamide-(4-guanidin o) butane, a inhibitor of cathepsins B and L, increased protein contents of heavy chains of cathepsins B and L in the IGF-1 treated cells. Inhibition of protein breakdown by IGF-1 is mediated by autocatalytic inactivation of lysosomal cathepsins B and L. PMID- 7534076 TI - High affinity chimeric human granulocyte-macrophage colony-stimulating factor receptor carrying the cytoplasmic domain of the beta subunit but not the alpha subunit transduces growth promoting signals in Ba/F3 cells. AB - Granulocyte-macrophage colony-stimulating factor receptor (GMR) is composed of two distinct subunits alpha and beta, and the cytoplasmic domains of both subunits are essential to transduce signals. We further analyzed the role of the cytoplasmic domain of each subunit by constructing chimeric subunits, designated alpha/beta and beta/alpha, by exchanging cytoplasmic domains of the alpha and beta subunits of hGMR. Reconstituted high-affinity chimeric hGMRs, hGMR(alpha/beta,beta/alpha) and hGMR(alpha/beta,beta), as well as the wild type hGMR(alpha,beta), transduced signals in Ba/F3 cells. These observations indicate that the original configuration between the extracellular and the cytoplasmic domains of the hGMR(alpha,beta) subunits is not obligatory, and that hGMR(alpha/beta,beta) transduced signals through the cytoplasmic domain of the beta subunit in an oligomeric form, without involvement of the cytoplasmic domain of the alpha subunit. PMID- 7534078 TI - Professional communicative paradigms in family-centered service delivery. PMID- 7534079 TI - Delayed kinetics of T lymphocyte anergy and deletion in lpr mice. AB - The Fas/APO-1 (Fas) antigen is a cell surface protein that mediates apoptosis and belongs to the tumor necrosis factor receptor family. The lymphoproliferative (lpr) anomaly in mice results from a retroviral disruption within the fas gene. Mice that are homozygous for the lpr mutation accumulate large numbers of T lymphocytes and exhibit an autoimmune syndrome resembling systemic lupus erythematosus. A possible explanation for this process is that in the absence of Fas antigen, lpr T cells may be resistant to normal peripheral deletional signals. The bacterial superantigen staphylococcal enterotoxin B (SEB) rapidly induces anergy and deletion by apoptosis of reactive T lymphocytes in normal mice. Administration of SEB to adult lpr mice results in the delayed induction of both unresponsiveness and deletion of V beta 8+ lymph node cells. This is not due merely to an increased thymic output in lpr mice; the delayed induction of tolerance and elimination of reactive lpr T cells by superantigens are intrinsic properties of the cells. The progressive lymphadenopathy in lpr mice may reflect a process of lymphoaccumulation rather than lymphoproliferation. A delay in tolerance induction and elimination of self-reactive T cells could have profound influence on the autoimmune diathesis of lpr mice. PMID- 7534080 TI - Cell adhesion molecules: a selective therapeutic target for alleviation of IDDM. AB - Selective homing of autoreactive lymphocytes to the pancreatic islets of Langerhans is essential for triggering the cascade of molecular and cellular interactions which culminate in the specific destruction of the insulin-producing beta-cells. Based upon the sequential multistep model of lymphocyte adhesion to the endothelium, we investigated the possibility of preventing the progression of insulin-dependent diabetes mellitus (IDDM) by selectively blocking L-selectin and alpha 4-integrin homing receptors, which function at different stages of the adhesion process. Treatment of NOD mice with mAb specific for L-selectin or alpha 4-integrin resulted in a significant inhibition of lymphocytic infiltration (insulitis). Both spontaneous development and acute transfer of IDDM were completely prevented by administration of anti-alpha 4-integrin antibody and partially inhibited by anti-L-selectin antibody. The protective effect was of long duration. Interestingly, the autoimmune T cell responses to a panel of beta cell autoantigens and the lymphocytic infiltration of salivary glands (sialadenitis) appeared unaffected by anti-L-selectin or anti-alpha 4-integrin treatment. These data suggest that prevention of lymphocyte homing to the pancreatic islets may provide a selective target for prevention/treatment of IDDM in patients. PMID- 7534082 TI - Ribavirin therapy for hepatitis C infection following liver transplantation. AB - Hepatitis C infection following orthotopic liver transplantation may lead to progressive chronic graft dysfunction. In this study, seven liver transplant recipients with chronic allograft dysfunction due to hepatitis C infection (one acquired and six recurrent infections) were treated with oral ribavirin for 6 months. Symptoms of lethargy, nausea and anorexia improved in all patients within 2 weeks of starting the drug, with a fall in serum AST of at least 40% by this time. Ribavirin-induced haemolysis was clinically significant in three patients, necessitating a reduction in the daily dose of ribavirin from 1.2 g to 0.2 g. Comparison of the pre- and post-treatment biopsy specimens in the four patients who tolerated the full dose of ribavirin and who had normal AST levels at the end of 6 months of treatment showed significant histological improvement with reduction in either lobular or periportal inflammation in all of the patients and a reduction in periportal fibrosis in one patient. HCV RNA remained detectable in serum in all of the patients at the end of the study. PMID- 7534081 TI - Acute pancreatitis after liver transplantation: incidence and contributing factors. AB - In order to assess the incidence and possible predisposing and contributing factors in the development of acute pancreatitis after liver transplantation, we reviewed the medical records of all 1832 adult patients who underwent 2161 orthotopic liver transplantation (OLTx) procedures in our center between January 1987 and September 1992. Of these patients, 55 (3% incidence) developed clinical pancreatitis and 247 (13.4% incidence) developed hyperamylasemia (biochemical pancratitis). Overall mortality in cases of clinical pancreatitis was 63.6%. The mortality in cases of hyperamylasemia was similar to that found in the general liver transplant population (i.e., 23%). A strong correlation was found between pancreatitis after liver transplantation and end-stage liver disease due to hepatitis B (30% of the cases, P = 0.00001). Extensive surgical dissection around the pancreas (P < 0.05), the type of biliary reconstruction following liver transplantation (P < 0.05), and the number of liver grafts received by the same patient (P = 0.00001) appeared to be possible contributing factors as did the duration of venovenous bypass and the quantity of IV calcium chloride administered intraoperatively. PMID- 7534083 TI - Regulation of apoptosis in germinal centre B cells. PMID- 7534084 TI - Comment on Dorak and Burnett. PMID- 7534085 TI - Immunologically ignorant autoreactive T cells, epitope spreading and repertoire limitation. AB - The factors that may cause antigen-presenting cells to alter the pattern of protein processing and presentation to autoreactive T cells, and thereby stimulate autoimmune disease, are currently under debate. In this article, Chris Elson and colleagues suggest that cytokines associated with T helper 1 (Th1) cells alter the processing of proteins and that this effect can be counteracted by Th2-associated cytokines. PMID- 7534086 TI - Immunoreactivity of apolipoprotein B-100 in oxidatively modified low density lipoprotein. AB - Thirteen monoclonal antibodies (MAbs) against apolipoprotein B-100 (apo B) were used to analyze changes in immunoreactivity of human LDL resulting from oxidation mediated by cupric ions and oxygen. Decrease in immunoreactivity of oxidized LDL was demonstrated by competitive ELISA with MAbs 5F8, BL3, Mb43, 2G8, B3, B5, and BL7 for which the epitopes are located within residues 1-1297, 4235-4355, 4027 4081, 3728-4306, 2239-2331, 1854-1878, and in the vicinity of residue 2331, respectively. Immunoreactivity of the epitope B6 (2239-2331) increased during first 4 hours of oxidation and then diminished gradually. Epitope B1 (405-539) had slightly reduced immunoreactivity during first 8 h of LDL oxidation and then its minor increase was observed. MAb 12G10, specific to the epitope within apo B thrombin-digest fragment T4 (1-1297), displayed either weak or strong binding to LDL. LDL with weak binding pattern demonstrated significant increase in immunoreactivity upon oxidation. In contrast, LDL with strong binding pattern showed little to no change. Epitopes Mb47 (3441-3569) and 8G4 (1-1297) remained unchanged in oxidized LDL. Immunoreactivity of apo B-100 epitope recognized by MAb 4C11 (residues 2377-2658) was shown to be a function of oxidation time: it increased progressively up to 16 h and was stabilized for another 24 h of LDL oxidation. This epitope may be unmasked by LDL oxidation and may provide a useful immunochemical marker to monitor the extent of LDL oxidation. PMID- 7534087 TI - Human immunodeficiency virus type 1 proteinase is rapidly and efficiently inactivated in human plasma by alpha 2-macroglobulin. AB - Human plasma impairs the activity of the human immunodeficiency virus (HIV-1) proteinase to cleave the HIV-1 gag-polyprotein precursor. The inhibition is due to the entrapment of the proteinase by plasma alpha 2-macroglobulin (alpha 2M). In methylamine-treated plasma, where alpha 2M is inactivated, HIV proteinase is not blocked. The interaction of alpha 2M and HIV-1 proteinase resulting in covalent complexes of proteinase and alpha 2M was demonstrated by immunoblotting with antiserum either to alpha 2M or to the HIV proteinase. We suggest if HIV-1 proteinase would be released in vivo from infected patients' cells, alpha 2M entrapment may prevent or minimize a conceivable cleavage of extracellular matrix or plasma proteins by the HIV-1 enzyme. PMID- 7534088 TI - Age-related fibrillar deposits in brains of C57BL/6 mice. A review of localization, staining characteristics, and strain specificity. AB - The present article reviews findings regarding the age-related occurrence of clusters of unusual granules in the brains of C57BL/6 (B6) mice and discusses the potential relevance of this phenomenon as a model of specific aspects of brain aging in humans. The granules occur predominantly in the hippocampus of B6 mice and represent aggregations of fibrillar material that are mostly associated with astrocytes. The deposits become evident at about 4 to 6 mo of age, and increase markedly in both number and size thereafter. Similar structures have been observed in adult senescence accelerated mice (SAM) and have been noted, although very rarely, in older mice from other strains. The deposits appear to manifest dominant genetic heritability. Heparan sulfate proteoglycan and laminin or related molecules have been identified as components of the granular material. Although the deposits do not represent senile plaques with beta-amyloid deposition, they might mimic the deposition of extracellular matrix molecules that is thought to be an early event in amyloidogenesis in the aged brain and in Alzheimer's disease. PMID- 7534090 TI - Phase I AIDS Clinical Trials Group (075) study of adriamycin, bleomycin and vincristine chemotherapy with zidovudine in the treatment of AIDS-related Kaposi's sarcoma. AB - OBJECTIVE: To determine the toxicity and maximum tolerated dose of doxorubicin (adriamycin) in combination with fixed doses of bleomycin, vincristine (ABV) and zidovudine in patients with advanced AIDS-related Kaposi's sarcoma. PATIENTS AND METHODS: Twenty-six HIV-seropositive men with Kaposi's sarcoma were treated daily with 100 mg zidovudine orally every 4 h, along with combination chemotherapy using bleomycin 10 U/m2 and vincristine 1.4 mg/m2 (maximum, 2 mg) given intravenously in 2-week cycles. In addition, three successive cohorts of eight patients received escalating doses of doxorubicin each beginning with no doxorubicin (level I), doses of 10 mg/m2 (level II), and 15 mg/m2 (level III). RESULTS: The major dose-limiting toxicity experienced with the combination therapy was severe neutropenia in eight patients, four of whom received level III doxorubicin (15 mg/m2). Therefore, 10 mg/m2 of doxorubicin in combination with zidovudine and BV chemotherapy was defined as the maximum tolerated dose. Other dose-limiting toxicities included neuropathy (n = 2), cutaneous toxicity associated with bleomycin (n = 1), and diarrhea (n = 1). Seventeen patients (71%; 95% confidence interval, 46-85) experienced either partial (n = 13) or clinical complete remission (n = 4) to therapy after a median of five cycles (range, 2-9). CONCLUSION: The maximum tolerated dose of doxorubicin is 10 mg/m2 when given in combination with zidovudine and BV chemotherapy. Response rates observed with the combined antiretroviral and chemotherapy regimen are similar to those previously reported with ABV chemotherapy alone. PMID- 7534089 TI - Studies of neuroimmune markers in Alzheimer's disease. AB - Based on a suspected role of the immune system in the pathophysiology of Alzheimer's disease (AD) and the new discoveries of neuroimmune networks, the investigation of certain neuroimmune markers was performed in AD patients, healthy controls, and disease controls. In agreement with our previous immunological research on AD, the assessment of additional immune parameters revealed abnormalities of both cellular and humoral immunity in several AD patients. These include: 1. Enhanced production of cytokines, such as interleukin 1 (IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6); 2. Increase plasma level of CD8-positive lymphocyte derived soluble CD8 (sCD8) antigen; and 3. Increased incidence of autoantibodies to brain myelin basic protein (MBP) and thymic cells. As analyzed by flow cytometry and enzyme immunoassay, the peripheral blood immunocytes from AD patients showed a significant increase in the expression of the brain-derived S-100 protein. In the cell proliferation assay, the blood immunocytes from healthy subjects responded to stimulation with beta-amyloid protein (beta AP), but this response was absent in AD patients. The initial results of our research suggest that the studies of specific markers of the neuroimmune axis may be potentially important for the new development of diagnostic and therapeutic strategies for AD. PMID- 7534092 TI - Immunoelectrophoretic characterization and cross-reactivity of Rochalimaea henselae, Rochalimaea quintana and Afipia felis. AB - The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species. PMID- 7534091 TI - Treatment of HIV-infected fibroblasts with human leukocyte antigen (HLA)-DR inductive cytokines leads to infectious virions with newly acquired HLA-DR. PMID- 7534093 TI - Antibody response in rabbits infected with Rochalimaea henselae, Rochalimaea quintana and Afipia felis. AB - Antibody responses in three pairs of rabbits inoculated with live Rochalimaea henselae, Rochalimaea quintana and Afipia felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens. PMID- 7534094 TI - Characterization of two antigenically related integral membrane proteins of the guinea pig sperm periacrosomal plasma membrane. AB - The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35 kD and 52-kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase-separation analysis with Triton X-114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore-induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. PMID- 7534095 TI - Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction. AB - Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. PMID- 7534096 TI - Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates. AB - Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance. PMID- 7534097 TI - Evidence of no change in V3 loop antibody recognition pattern in HIV type 1 infected Ethiopians between 1988 and 1993. AB - To determine the antibody reactivity against a V3 sequence based on local HIV-1 strains in Ethiopia, 635 serum samples derived in 1988 and 1993 were analyzed by peptide enzyme immunoassays. V3 peptides were produced according to the Ethiopian subtype C V3 consensus sequence (RKSIRIGPGQTFYAT), the HIV-1MN and HIV-1IIIB strains (subtype B), and the consensus sequences of subtypes A, D, and E. Initial analyses showed that Ethiopian anti-V3 positive sera cross-reacted between subtype A and subtype C peptides, and displayed much lower reactivities to the other peptides. Using inhibition experiments, it was found that the reactivities in the Ethiopian samples were specific for subtype C. A strong reactivity to the Ethiopian V3 consensus sequence was found in the majority of the Ethiopian samples (59%), independent of geographical origin or year of sampling. In Swedish HIV-1-positive sera, the high reactivities were restricted to the subtype B HIV 1MN peptide. A low prevalence (10%) of strong reactivity to the HIV-1MN V3 peptide was found among the Ethiopian samples. Using substitution peptide analogs it was found that a lack of cross-reactivity between subtype B and C peptides was dependent on the Arg-322 to Gln-322 substitution. The present data show that a similar antibody recognition pattern was present in sera sampled during 1993 as in sera sampled during 1988, suggesting that subtype C of HIV-1 has remained the dominant subtype in Ethiopia. PMID- 7534099 TI - The uptake of Na-selenite in rat brain. Localization of new glutathione peroxidases in the rat brain. AB - Polyunsaturated fatty acids (PUFAs) occur in phospholipids of synapses of central nervous system (CNS). PUFAs may thus determine the fluidity of synaptosomal membranes and regulate neuronal transmission. It was therefore tempting to suggest an oxidative system in CNS protecting the membrane function, e.g., glutathione peroxidase (GSH-Px). In order to trace GSH-Px Wistar rats were loaded with 4800 kBq of 75Se sodium selenite. By means of gradient ultracentrifugation, particulate fractions of CNS were isolated and radioactivity as well as selenium dependent GSH-Px were estimated. The following data were obtained: 1. All fractions (myelin, synaptic vesicles, synaptosomes, mitochondria, and microsomes) contained 75Se. 2. After acetone precipitation of GSH-Px activity, fractionation on Sephadex G-150 revealed in all particulate fractions at least two peaks of radioactivity with GSH-Px activity. 3. The two GSH-Px peaks from the Sephadex filtration were freeze dried and applied on a hydrophobic T-gel column and eluted with decreasing molarity of ammonium sulfate from 1.5 to 0.05M. The first Sephadex peak with GSH-Px activity from myelin and the second peak with GSH-Px activity from synaptic vesicles could now be resolved into two different fractions of radioactivity on the T-gel. The remaining Sephadex G-150 peaks could only be resolved into one peak of radioactivity. 4. SDS-polyacrylamide gel electrophoresis of the T-gel peaks from all fractions showed a protein band with a mobility identical with that of human erythrocyte GSH-Px. The T-gel elution of myelin, synaptic vesicles and mitochondria gave rise to nearly pure CNS GSH-Px activity. The data presented support the idea that CNS fractions have membrane bound GSH-Px activity that may function as protecting enzymes towards oxidative stress in the brain. PMID- 7534101 TI - Clinical trials referral resource. Prostate cancer. PMID- 7534100 TI - Measurement of blood concentrations of FK506 (tacrolimus) and its metabolites in seven liver graft patients after the first dose by h.p.l.c.-MS and microparticle enzyme immunoassay (MEIA). AB - 1. Blood and urine concentrations of the macrolide immunosuppressant FK506 and its metabolites were measured in seven orthotopic liver transplant patients after the first oral dose of FK506 (0.04 +/- 0.02 mg kg-1) used as primary immunosuppressant. A specific h.p.l.c.-MS assay was used, allowing the measurement of parent drug and eight metabolites. Results were compared with those obtained using a microparticle enzyme immunoassay (MEIA). 2. Blood drug concentrations were described by an open two compartment model with first-order absorption giving the following mean data: tmax: 1.9 (h), Cmax: 17.4 (microgram l 1), AUC: 328.1 (microgram l-1 h), t1/2,1: 0.74 (h). The terminal elimination half life was estimated at about 26 h using the h.p.l.c.-MS assay. 3. The metabolites found in blood were demethyl-FK506 and demethyl-hydroxy-FK506, while in urine FK506 and eight of its metabolites were detected. PMID- 7534098 TI - Effect of selenite on cell surface fibronectin receptor. AB - Incubation of cells with selenite, under conditions in which there is no effect on cell viability, results in a decrease in the rate of their subsequent attachment to extracellular matrix proteins such as fibronectin (1). The attachment was inhibited by a pentapeptide containing the RGD sequence and by antibody against the cellular fibronectin receptor (alpha 5 beta 1 integrin), indicating that it is receptor-mediated. To investigate whether exposure to selenite has an effect on fibronectin receptors, we assayed for their presence on the cell surface by measuring the ability of cells to attach to a surface that had been coated with antibodies to the receptor. Brief exposure of cells to low concentrations of selenite resulted in a significant decrease in their ability to attach to monoclonal antibodies against the alpha 5 or beta 1 subunits of the fibronectin receptor, as well as to polyclonal antibodies against the complete receptor. This indicates that exposure to selenite results in a decrease in receptors that are present at the cell surface. Exposure of the cells to selenate, selenocystine or selenomethionine did not result in a significant decrease in cell surface receptors. Preincubation of the cells with selenite was required for the effect, indicating that selenite does not directly interfere with receptor structure or function. PMID- 7534102 TI - Expression of the c-kit receptor in hypomelanosis: a comparative study between piebaldism, naevus depigmentosus and vitiligo. AB - In order to investigate possible alterations in c-kit protein expression on epidermal melanocytes in different hypopigmentary disorders, we have examined skin specimens from one patient with piebaldism, one patient with naevus depigmentosus, and five patients with vitiligo. Cryosections were examined by immunohistochemistry using monoclonal antibodies against the c-kit protein (YB5.B8) and melanosomes (TA99). In piebaldism, hypomelanotic epidermis contained only a few TA99-positive epidermal melanocytes and no detectable c-kit protein, whereas in naevus depigmentosus the expression of c-kit protein was strong, and TA99 immunoreactivity was faint. In vitiligo lesions, no epidermal immunoreactivity for melanosomes or c-kit protein was found. Normally pigmented skin of all patients showed immunoreactivity of epidermal melanocytes for both c kit protein and melanosomes. Different hypomelanotic lesions can thus be differentiated by absent melanocyte c-kit protein and low or no expression of melanosomal marker in piebaldism, normal c-kit but low melanosome expression in naevus depigmentosus, and the absence of all melanocyte markers in vitiligo. PMID- 7534105 TI - A constitutional BWS-related t(11;16) chromosome translocation occurring in the same region of chromosome 16 implicated in Wilms' tumors. AB - Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder with a varying spectrum of clinical manifestations including macroglossia, omphalocele, hemihypertrophy, and a predisposition to a subset of embryonal tumors, most frequently Wilms' tumor (WT). A variety of cytogenetic, genetic linkage, and molecular mapping data implicate a gene or genes on chromosome band 11p15.5 in BWS and its related tumors. However, some families with BWS do not show linkage to 11p15, and other alterations have been found in Wilms' tumors as well. One such alteration is loss of heterozygosity (LOH) for chromosome arm 16q. Here we have analyzed a balanced t(11;16)(p15;q13) chromosomal translocation associated with the BWS phenotype and mapped the breakpoint positions for both chromosomes 11 and 16 by using somatic cell hybrids and polymorphic markers. The chromosome 11 breakpoint was found to lie distal to the D11S12 locus, but proximal to TH on 11p15.5, a region shown previously to contain other BWS-related chromosomal events. The chromosome 16 breakpoint was distal to D16S290 in 16q13, but proximal to loci D16S265, D16S267, and D16S164 in band 16q21. This area encompasses the region of LOH occurring through mitotic recombination in sporadic WT. This raises interesting possibilities for the genetic and epigenetic involvement of both chromosomal regions (11p15 and 16q13) in the pathogenesis of BWS and Wilms' tumor. PMID- 7534104 TI - CD8+ dermal T cells from a sulphamethoxazole-induced bullous exanthem proliferate in response to drug-modified liver microsomes. AB - There is evidence that T lymphocytes play a critical role in the pathogenesis of drug-induced bullous exanthems. Sulphonamides are known to be among the most frequent aetiological agents in these severe drug-induced cutaneous hypersensitivity reactions. Several studies indicate that cytochrome P450 dependent metabolites of sulphonamides act as the nominal allergens. A 70-year old woman with a severe blistering exanthem caused by cotrimoxazole (sulphamethoxazole and trimethoprim) was studied. We employed an in vitro approach to determine whether cytochrome P450-dependent enzymes activated drug specific T lymphocytes from this patient. Immunohistochemical analysis of involved skin revealed a majority of epidermal CD8+ T lymphocytes, whereas the dermal infiltrate was composed of both CD4+ and CD8+ T cells. Dermal T lymphocytes isolated from lesional skin proliferated in response to sulphamethoxazole, but not to trimethoprim, in the presence of autologous mononuclear cells used as antigen-presenting cells. The antigen-specific response of sulphamethoxazole-specific T cells was significantly augmented in the presence of murine liver microsomes with P450-dependent catalytic activities. Our observations suggest that some cutaneous hypersensitivity reactions to sulphamethoxazole are due to drug-specific T lymphocytes. Cytochrome P450 dependent enzymes may play a critical role in the formation of the nominal antigen, which is recognized by antigen-specific T cells. PMID- 7534103 TI - Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C terminal end of the protein. Evidence using a 55 kDa recombinant protein. AB - In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes. PMID- 7534107 TI - Allelic imbalance on chromosome 1 in human breast cancer. II. Microsatellite repeat analysis. AB - We have determined regions of allelic imbalance in human breast cancer cells using highly polymorphic microsatellite markers, which can be rapidly typed by the polymerase chain reaction (PCR) using very small amounts of DNA. It appears that there are several regions of chromosome I which may be the targets of allelic imbalance, including some regions which have been identified previously by different groups. The detail with which we have mapped these regions of imbalance is, however, much greater than has been previously reported, and we have been able to localise these regions to small intervals of the genome. In addition we have identified previously uncharacterised regions of allelic imbalance on chromosome arm 1p, one of which (at 1p22-31) is lost in a high proportion of malignant lesions. We are currently attempting to analyse this latter region in detail in order to identify and characterise the sequence(s) involved. Study of such regions should help us understand some of the mechanisms underlying the development and progression of breast cancer. PMID- 7534106 TI - Allelic imbalance on chromosome 1 in human breast cancer. I. Minisatellite and RFLP analysis. AB - In order to characterise the role of chromosome 1 more fully in breast cancer, polymorphic markers mapping along the length of the whole chromosome were used to assess a panel of 71 tumour-lymphocyte pairs for allelic imbalance. Complex patterns of alterations were established that are consistent with cytogenetic data in the literature. Deletion mapping of individuals with loss of heterozygosity identified five independent smallest common regions of deletion, two of which are novel. There are also three discrete regions showing a gain in copy number of one homologue. The two arms of the chromosome may be subject to different events; the short arm primarily undergoes interstitial deletions, whereas the long arm is subject to whole arm events (as both gains and losses) as well as regional deletions. PMID- 7534108 TI - Diffuse large cell lymphomas exhibit frequent deletions in 9p21-22 and 9q31-34 regions. AB - We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series of non-Hodgkin's lymphomas (NHLs), which suggested loss of candidate tumor suppressor genes (TSGs). In order to define these deletions at the molecular level, we performed an LOH analysis of a panel of paired normal and tumor DNAs comprising 13 cases of diffuse lymphoma with a large cell component (DLLC) and 18 cases of Burkitt's lymphoma (BL). The loci tested comprised eight polymorphic probes mapped to 9p (D9S33, D9S25, IFNB, IFNA, IFNW, D9S126, D9S3, and D9S19) and seven polymorphic probes mapped to 9q (D9S29, ASS, AKI, ABL, D9S10, D9S7, and D9S14). In this analysis, among cases informative for all loci in each subset, 5/13 (38%) DLLC and 4/18 (22%) BL showed LOH at 9p loci, whereas 5/13 (38%) DLLC and 3/18 (16%) BL showed LOH at 9q loci. Among the 9p loci partial homozygous or heterozygous losses were observed in 20-50% of informative cases of DLLC at D9S25, IFNB, IFNA, IFNW, D9S126, and D9S3, whereas in BL, losses at these loci ranged from 0% to 11%. Among the 9q loci, heterozygous losses were observed in > 20% of informative cases of DLLC at D9S7 (23%) and D9S29 (27%), whereas no losses were seen at these two loci in BL. These data demonstrate a high level of molecular deletion in DLLC, but not in BL, suggesting that loss of one or more TSGs on chromosome 9 plays an important role in DLLC development. PMID- 7534109 TI - Analysis of the joining sequences of the t(15;17) translocation in human acute promyelocytic leukemia: sequence non-specific recombination between the PML and RARA genes within identical short stretches. AB - Molecular analysis of the t(15;17) translocation in 70 patients with acute promyelocytic leukemia (APL) confirmed that the breakpoints of chromosome 15 were located in two regions of the promyelocytic leukemia (PML) gene, mainly introns 3 and 6, whereas the breakpoints of chromosome 17 were consistently in intron 2 of the retinoic acid receptor alpha (RARA) gene. To study the reason for the clustering of the breakpoints and the underlying mechanism of the chromosomal translocation, we characterized the joining sequences of der(15) and der (17) by polymerase chain reaction in samples from eight patients with APL. There was no cluster of the breakpoints within the introns, and no consensus sequence-motif was found around them. One or nine extra nucleotides were inserted into two joining sites. There were identical stretches of one to seven nucleotides between the PML and RARA genes in the majority of the joining sequences. These data provide a potential model of the t(15;17) translocation: random DNA double strand cleavage, modification of DNA ends by enzymes including terminal deoxynucleotidyl transferase, and single strand base-pairing within identical short stretches. Furthermore, APL develops only when the PML and RARA genes are rearranged, within restricted genomic regions and a functional PML-RARA chimeric product is produced, and this might lead to a clustering of the breakpoints. PMID- 7534110 TI - Changes in mean telomere length in basal cell carcinomas of the skin. AB - Human telomeres consist of arrays of the sequence TTAGGG up to 15-20 kb in length, which are essential for the maintenance of normal chromosomal stability. It has been suggested that genomic instability observed in tumours may be due to loss of telomere sequences. Somatic cells that are dividing continuously appear to progressively lose telomere sequences, and it would therefore be anticipated that cell type specific differences in mean telomere length may exist within an individual. Previous reports have suggested that mean telomere length may be different in human neoplasia when compared to control. Basal cell carcinomas are epidermal derived tumours and in order therefore to make valid cell type specific comparisons we have measured mean telomere length in 20 basal cell carcinomas as well as in both adjacent epidermis and dermis. Mean telomere length was significantly reduced in epidermis in comparison with dermis, from clinically normal skin immediately adjacent to the tumours (mean difference 2.5 kb). This result is not related to the presence of the tumour as similar results were obtained from skin samples of healthy volunteers. Basal cell carcinomas showed increased mean telomere length in 13/20 samples in comparison with matched epidermis (mean difference 3.1 kb), whereas in 7/20 mean telomere length was reduced (mean difference 2.2 kb). These results showing that mean telomere length varies from cell type to cell type underpin the importance of performing cell type specific controls when assessing changes in tumour telomeres. PMID- 7534111 TI - Molecular evidence that childhood monosomy 7 syndrome is distinct from juvenile chronic myelogenous leukemia and other childhood myeloproliferative disorders. AB - The observation that juvenile chronic myelogenous leukemia (JCML) and childhood bone marrow monosomy 7 syndrome (Mo 7) are similar in many clinical and epidemiologic respects suggests a shared pathogenic basis and raises the possibility that the bone marrows of patients with JCML might lose chromosome 7 alleles by mechanisms that do not result in detectable cytogenetic deletions. We used a series of polymorphic markers mapped to chromosome 7 to test this hypothesis in 22 children with MPS and MDS, including 19 with JCML. All MPS and MDS samples demonstrated allelic heterozygosity with at least one chromosome 7 marker; 16 were heterozygous with probes from both 7p and 7q. Furthermore, the percentage of patient bone marrow samples heterozygous at each locus tested was similar to the frequency observed in the normal population. Whereas these data demonstrate that submicroscopic loss of large segments of chromosome 7 alleles is uncommon in children with MPS and MDS who do not have Mo 7, they do not exclude small deletions around an uncharacterized tumor-suppressor locus. Our results suggest that a number of distinct molecular events contribute to leukemogenesis, and we propose a multistep model to explain the similarities and differences between the major subtypes of childhood MPS and MDS. PMID- 7534112 TI - Inversion-associated translocations in acute myelomonocytic leukemia with eosinophilia. AB - Cytogenetic studies of three acute myelomonocytic leukemias with bone marrow eosinophilia (M4EO) revealed chromosome 16 inversion associated with additional abnormalities. The inverted chromosome 16 was involved in two patients. Fluorescence in situ hybridization (FISH) experiments with a YAC probe detecting inv(16) showed that the translocation breakpoints involving chromosome 16 did not implicate the inversion breakpoints. FISH can thus distinguish between true variant translocations and translocations with other breakpoints on chromosome 16 in M4EO. PMID- 7534113 TI - Amplification of the MET gene in glioma. AB - We have previously reported the finding of MET amplification linked to double minutes (dmins) in a human glioblastoma (TX3095). Because dmins are found in approximately 50% of glioblastomas, 18 gliomas were analyzed for MET amplification. Three grade IV glioblastomas and one grade II astrocytoma showed amplification. We could also localize the MET amplicon to dmins in glioblastoma TX3095 by fluorescence in situ hybridization. PMID- 7534115 TI - Translocation t(3;12)(q28;q14) in parosteal lipoma. AB - Parosteal lipoma is a rare primary benign bone tumor demonstrating histopathologic features similar to those seen in the commonly occurring lipoma of soft tissue. Cytogenetic studies of soft tissue lipoma have demonstrated frequent abnormalities of 12q13-15. To the best of our knowledge the cytogenetic findings in parosteal lipoma have not yet been described. Cytogenetic analysis of a parosteal lipoma of the femur of a 51-year-old female revealed a t(3;12)(q28;q14), indicating that bone and soft tissue lipomas are cytogenetically similar and likely share a common histopathogenesis. PMID- 7534114 TI - Identification of cytogenetically undetected 12p13 translocations and associated deletions with fluorescence in situ hybridization. AB - We performed fluorescence in situ hybridization (FISH) on bone marrow or peripheral blood cells thought to contain a del(12p) or an unbalanced 12p11-12 translocation from 17 patients who had various hematologic malignant diseases. We used 11 cosmid, phage, and plasmid probes which we had previously ordered on 12p. Cells from three patients with myeloid disorders were shown to have 12p13 translocations that involved chromosome 2 in two of them. Moreover, in all patients, FISH showed that the translocations were associated with proximal interstitial deletions which contributed to the difficulty in identifying these translocations. Our data suggest that some rearrangements of 12p which have been described previously as deletions or unbalanced translocations may, in fact, represent 12p13 translocations accompanied by an interstitial deletion. PMID- 7534116 TI - Allelic losses on chromosome band 11q13 in aldosterone-producing adrenal tumors. AB - We examined loss of heterozygosity (LOH) in 14 aldosterone-producing adrenal tumors, with six linearly ordered restriction fragment length polymorphism (RFLP) markers that map within a 12-cM region containing the MEN1 locus on 11q13. Among 11 tumors that were informative for at least one marker, five showed LOH at one or more loci, and two distinct regions of deletion were identified. The proximal region overlapped with the location of the MEN1 locus previously predicted by linkage analyses in MEN1 families and the commonly deleted region in hyperparathyroid tumors. This suggests that one of the genes associated with development of aldosterone-producing adrenal tumors may coincide with the MEN1 locus, and that a second gene, distal to the MEN1 locus, may also play a role in the development of this type of tumor. PMID- 7534117 TI - Frequent loss of chromosome arms 8p and 13q in collecting duct carcinoma (CDC) of the kidney. AB - Collecting duct carcinoma (CDC) is a malignant renal neoplasm that is believed to arise from the epithelium of the ducts of Bellini in the distal nephron. These tumors are clinically aggressive and more often occur in a younger population than is typical of the more common clear cell renal carcinoma (RCC). Using highly informative polymorphic microsatellite markers on chromosome arms 3p, 5q, 6q, 9p, 9q, 11p, 13q, 17p, and 18q, we analyzed DNA from nonmalignant and tumor tissue in 6 cases of CDC. We found no evidence of 3p loss of heterozygosity (LOH) in these renal tumors by using multiple markers, a finding that distinguishes CDC from RCC in which 3p LOH has frequently been observed. We found LOH of 8p in 50% of the tumors examined; in addition, we observed LOH of 13q in 50% of the tumors studied. Interestingly, 8p LOH may be associated with high stage and poor clinical prognosis. These data suggest that the molecular events responsible for the development of CDC differ from those associated with the origin of RCC, and that tumor suppressor genes on 8p and 13q may be involved in the pathogenesis of CDC. PMID- 7534118 TI - Definition of a new entity of malignant extragonadal germ cell tumors. AB - Two malignant extragonadal germ cell tumors are reported, histologically classified as immature teratomas, having pseudodiploid karyotypes with complex structural rearrangements but lacking isochromosome 12p or other rearrangements involving 12p. The absence of 12p material in structural rearrangements was confirmed by chromosome painting. In the two tumors the following common chromosomal breakpoints were found: 6p21, 6p22, 6q23, and 11q13. Exactly the same chromosomal regions, 6p22::6q23 and 6p21::11q13, were involved in fusions. The two tumors belong to a new entity of extragonadal immature teratomas of adults which may be located in the retroperitoneum and posterior mediastinum and are prone to blood borne metastasis. PMID- 7534119 TI - Parent preferences and prenatal testing for neural tube defects. AB - Previous analyses of prenatal screening for neural tube defects have generally found benefits to exceed costs. The usual screening battery follows an elevated maternal serum alpha-fetoprotein level with high-resolution ultrasound and/or amniocentesis. Current thinking focuses on weighing the risk of a false-negative (an abnormality missed) against the risk of an amniocentesis-induced fetal loss. This thinking neglects the risk of a false-positive (an unaffected fetus labeled abnormal) and individual parents' preferences concerning a false-negative vs a fetal loss. With these risks included, we find that high-resolution ultrasound is appropriate for all women with elevated serum alpha-fetoprotein. Women with moderately elevated serum alpha-fetoprotein who have negative ultrasound scans need no further testing, nor do women with highly elevated serum alpha fetoprotein and positive ultrasound scans. Further testing using amniocentesis to confirm the ultrasound result is appropriate for women with moderately elevated serum alpha-fetoprotein and positive ultrasound scans, and for women with highly elevated serum alpha-fetoprotein and negative ultrasound scans. The actual cutoffs defining normal, moderately elevated, and highly elevated serum alpha fetoprotein depend on several parameters, particularly the underlying prevalence of neural tube defects and the parents' preferences. PMID- 7534120 TI - Methodology-dependent variations in reticulocyte counts using a manual and two different flow cytometric procedures. AB - This paper describes a comparison between the microscopic brilliant cresyl blue and the flow cytometric thiazol orange (FACScan) and auramine O (Sysmex) methods for enumeration of reticulocytes. The mean intra-assay coefficients of variation for the microscopic, FACScan and Sysmex methods were established to be 33.9 and 5% respectively. A rather poor correlation was observed between the microscopic count and both flow cytometric methods (FACScan r = 0.61; Sysmex r = 0.57). However, the correlation between the flow cytometric methods was satisfactory (r = 0.79). Reference ranges for the reticulocyte count, corresponding with the 2.5th and 97.5th percentile, were determined for the microscopic (8-30/1000), FACScan (11-27/1000) and Sysmex (8-18/1000) methods. Sysmex R-3000 methodology definitely revealed the lowest and narrowest reference range. In conclusion, because of higher reproducibility, flow cytometric analysis of reticulocytes is an attractive alternative procedure for the microscopic enumeration method. PMID- 7534121 TI - Characterization of a monoclonal antibody to a human intra-acrosomal antigen that inhibits fertilization. AB - Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm. PMID- 7534122 TI - A novel glycoprotein of the aspartic proteinase gene family expressed in bovine placental trophectoderm. AB - The pregnancy associated glycoproteins (PAG 1) that appear in the maternal serum of cattle and sheep soon after implantation are apparently inactive members of the aspartic proteinase family. Here we describe the isolation of a highly abundant cDNA (PAG 2 cDNA) that represents a second member of this gene family which is structurally related to bovine PAG 1, ovine PAG 1, and pepsin (58%, 58%, and 51% amino acid sequence identity, respectively). The bovine PAG 2 cDNA was identified in two ways. First, the bovine placental library was screened under relatively nonstringent conditions with an ovine PAG 1 cDNA. The second fortuitous approach employed immunoscreening with an antiserum raised against a partially purified factor that competed with bovine LH for binding to the LH receptor on the CL of the ovary. The full-length cDNA (1258 bp) codes for a polypeptide of 376 amino acids. Bovine PAG 2, unlike bovine PAG 1, has a catalytic center with a consensus sequence of amino acids. Its mRNA is expressed in fetal placenta but not in other fetal organs, and is localized to both the mononucleate and binucleate cells of the trophectoderm, whereas PAG 1 is expressed only in binucleate cells. PAG 2 is synthesized by placental explants as a 70-kDa glycoprotein that is processed to several smaller molecules. Western blot analysis of culture media developed with epitope-selected antibodies to PAG 2 reveals several bands ranging in apparent M(r) from 31,000-70,000, which correspond in size to the polypeptides present in the preparation used for immunization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534123 TI - Insulin-like growth factor-I and -II binding and action on DNA synthesis in rainbow trout spermatogonia and spermatocytes. AB - Radio-labeled recombinant human insulin-like growth factor (125I-rhIGF-I) bound specifically to total testicular cells and to spermatogonia plus primary spermatocytes (Go+CI) that had been prepared from trout testes at various maturation stages by centrifugal elutriation and then cleared of somatic cells by preculture in the presence of 2% Ultroser G. Binding sites showed high affinity (Ka = 0.5 +/- 0.2 x 10(10) M-1) and low capacity (1.1 +/- 0.8 fmol/10(6) testicular cells) for 125I-IGF-I. (Gln3, Ala4, Tyr15, Leu16)IGF-I ([QAYL]IGF-I) was equipotent to IGF-I in competing with 125I-IGF-I for site occupancy on Go+CI. Insulin-like growth factor-II (IGF-II) was to 10-fold less potent than IGF-I or (QAYL)IGF-I, while bovine insulin competed only about 300-fold higher concentrations. Go+CI were cultured for 3 days in the presence or absence of mammalian IGF-I, IGF-II, (QAYL)IGF-I, and salmon or bovine insulin. All these molecules stimulated the incorporation of [3H]thymidine (added during the last 24 h in culture) by Go+CI in a dose-dependent manner. The mean ED50, independent of testicular maturation stage, was 5.9 +/- 4.9 ng/ml and 29.1 +/- 15.8 ng/ml for IGF-I and IGF-II, respectively. (QAYL)IGF-I was as potent as IGF-I. Concentrations of salmon or bovine insulin 100- to 300-fold higher were required to produce effects similar to those of IGF-I. While recombinant human IGF binding protein (IGFBP-3) had no effect by itself of basal [3H]thymidine incorporation, it inhibited the effect of IGF-I in a dose-dependent manner; however, it had no effect on the stimulation by (QAYL)IGF-F. Although combinations of low concentrations of IGF-I and IGF-II or salmon insulin had additive effects, combinations of maximum concentrations did not. We conclude that, in vitro, IGFs stimulate DNA synthesis of trout male germ cells by interacting directly with these cells through one IGF receptor. PMID- 7534124 TI - Characterization of bovine ovarian surface epithelium and stromal cells: identification of secreted proteins. AB - The majority of ovarian cancers are derived from a single layer of epithelial cells that covers the surface of the ovary termed the ovarian surface epithelium (OSE). Ovarian surface stromal cells underlie the OSE and have a morphology distinct from that of the interstitial stromal cells between follicles. Because of the similarities between bovine and human ovarian physiology, and to allow an adequate supply of tissue and cells, bovine OSE and stromal cell cultures were established to investigate the cell biology of these cell types. Morphological analysis of bovine ovarian sections revealed that several layers of ovarian surface stromal cells can be identified and are structurally distinct from interstitial stromal cells. Both OSE and stromal cells can be isolated and cultured for weeks in the absence of presence of serum. The cell populations were found, through use of a keratin immunocytochemical stain for OSE, to be highly purified. To investigate the functional properties of the two cell types, radiolabeled secreted proteins were collected and electrophoretically analyzed. The radiolabeled secreted protein profiles of OSE and stromal cells were found to be distinct with a discrete number of secreted proteins. Major OSE secretory products were obtained from serum-free concentrated conditioned medium, electrophoretically separated, blotted, and sequenced. Two OSE secretory products of 28 kDa and 40 kDa were sequenced and found to match a sequence in the computerized database. The 28-kDa OSE protein was identified as a tissue inhibitor of metalloproteinase, TIMP. The 40-kDa OSE protein was identified as the insulin-like growth factor (IGF) binding protein-2 (IGFBP2).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534125 TI - Cutaneous larva migrans in travelers: synopsis of histories, symptoms, and treatment of 98 patients. AB - The symptoms, medical history, and treatment of 98 patients with cutaneous larva migrans (creeping eruption) who attended a travel-related-disease clinic during a period of 4 years are reviewed. This condition is caused by skin-penetrating larvae of nematodes, mainly of the hookworm Ancylostoma braziliense and other nematodes of the family Ancylostomidae. Despite the ubiquitous distribution of these nematodes, in the investigated group only travelers to tropical and subtropical countries were affected; 28.9% of the patients had symptoms for > 1 month, and for 24.5% the probable incubation period was > 2 weeks. The efflorescences typically were on the lower extremities (73.4% of all locations). The buttocks and anogenital region were affected in 12.6% of all locations, and the trunk and upper extremities each were affected in 7.1%. Only a minority of patients presented with eosinophilia or an elevated serum level of IgE. No other laboratory data appeared to be related to the disease. Therapy with topical thiabendazole was successful for 98% of the patients. Systemic antihelmintic therapy was necessary in two cases because of disseminated, extensive infection. PMID- 7534126 TI - [Effective expression of the apolipoprotein A-I gene, transferred to growing human cells in vitro]. PMID- 7534127 TI - [Seasonal dynamics of serotonin of the duodenum in hibernating animals]. PMID- 7534128 TI - [Ultrastructure of hepatocytes and sinusoidal endothelial cells during use of concurrently interacting lysosomotrophic substances for treating iron deficiency anemia]. PMID- 7534129 TI - Peripheral blood stem cells for allografting. PMID- 7534130 TI - Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. AB - Murine bone marrow cells were fractionated by fluorescence-activated cell sorting into Rh123lo Lin- c-kit+ Ly6A+, Rh123hi Lin-c-kit+ Ly6A+, and Lin- c-kit+ Ly6A- populations within which most, if not all, of the hematopoietic activities of the marrow resided. The Rh123lo Lin- c-kit+Ly6A+ cells, which consist exclusively of small- or medium-sized lymphocyte-like cells, are highly enriched for long-term hematopoietic in vivo repopulating cells. The enrichment factor for these cells from the marrow was estimated as 2,000-fold. The Rh123hi Lin- c-kit+ Ly6A+ cells, although also highly enriched for day-12 spleen colony-forming units, were relatively depleted of long-term in vivo repopulation capacity. Most, if not all Lin- c-kit+ Ly6A- cells were Rb123hi. In contrast to both Rh123lo and Rh123hi Lin c-kit+ Ly6A+ stem cell populations, the Lin- c-kit+ Ly6A- cells can be stimulated to proliferate in vitro in the presence of single cytokines, which is a characteristic of committed progenitor cells. No marked synergistic interactions between individual cytokines were observed with this cell population. Both Rh123hi Lin- c-kit+ Ly6A+ mature stem cell and Lin- c-kit+ Ly6A- progenitor cell populations displayed in vivo repopulation kinetics resembling those of the putative short-term hematopoietic repopulating cells. PMID- 7534131 TI - Cell cycle and functional differences between CD34+/CD38hi and CD34+/38lo human marrow cells after in vitro cytokine exposure. AB - The proliferation kinetics and clonogenic activity of CD34+/38hi (CD38hi) and CD34+/38lo (CD38lo) human marrow cells were measured before and after culturing the cells in vitro over a 6-day period in serum-deprived medium containing recombinant growth factors (interleukin-1 [IL-1], IL-3, IL-6, granulocyte colony stimulating factor [G-CSF], granulocyte-macrophage [GM]-CSF, kit ligand, and erythropoietin). Before in vitro culture, 3% +/- 3% of the CD38lo and 13% +/- 2% of the CD38hi cells were in the S-phase of the cell cycle. The clonogenic activity of CD38hi cells was twofold greater than that of the CD38lo cells, as measured by colony-forming units (CFU) in short-term assays. However, CD38hi cells contained fewer pre-CFU than did the CD38lo cells, generating only 3 +/- 2 colonies per 1,000 cells after 4 weeks of culture on competent stromal layers, compared with 107 +/- 46 colonies per 1,000 cells from the CD38lo population. CD38hi and CD38lo cells exhibited distinctly different responses when cultured in serum-deprived medium supplemented with recombinant growth factors. After culturing cells for 24 hours, CD38lo cells essentially remained a noncycling population with only 5.1% +/- 3.0% of the cells cycling, whereas 44.2% +/- 6.9% of the CD38hi cells were in DNA synthesis. Gradually CD38lo cells were recruited into cycle, such that by 72 hours, approximately 28% of the CD38lo cells were in S-phase. However, during 6 days of culture, the percentage of cycling CD38lo cells never exceeded the proliferative response observed for CD38hi cells. Phenotype analysis conducted at day 6 indicated that 86% of the CD38hi population were no longer phenotypically CD34+/38hi, while 60% of CD38lo cells maintained a CD34+/38lo phenotype. Long-term cultures initiated with 6-day in vitro-expanded CD38lo cells showed approximately a twofold decrease in clonogenic activity attributable to a loss of erythroid precursors and a decrease in GM colonies. Thus, a proportion of CD38lo cells capable of generating CFU was maintained even after exposure to growth factors. PMID- 7534133 TI - Inactivation of factor XIa in human plasma assessed by measuring factor XIa protease inhibitor complexes: major role for C1-inhibitor. AB - From experiments with purified proteins, it has been concluded that factor XIa (FXIa) is inhibited in plasma mainly by alpha 1-antitrypsin (a1AT), followed by antithrombin III (ATIII), C1-inhibitor (C1Inh), and alpha 2-antiplasmin (a2AP). However, the validity of this concept has never been studied in plasma. We established the relative contribution of different inhibitors to the inactivation of FXIa in human plasma, using enzyme-linked immunosorbent assays (ELISAs) for the quantification of complexes of FXIa with a1AT, C1Inh, a2AP, and ATIII. We found that 47% of FXIa added to plasma formed complexes with C1Inh, 24.5% with a2AP, 23.5% with a1AT, and 5% with ATIII. The distribution of FXIa between these inhibitors in plasma was independent of whether FXIa was added to plasma, or was activated endogenously by kaolin, celite, or glass. However, in the presence of heparin (1 or 50 U/mL), C1Inh appeared to be the major inhibitor of FXIa, followed by ATIII. Furthermore, at lower temperatures, less FXIa-C1Inh and FXIa a1AT complexes but more FXIa-a2AP complexes were formed. These data demonstrate that the contribution of the different inhibitors to inactivation of FXIa in plasma may vary, but C1Inh is the principal inhibitor under most conditions. PMID- 7534132 TI - High expression of c-kit in K562YO cells due to the prolonged half-life of its mRNA: the effects of modification with serine/threonine kinase signals. AB - We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c-kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light-dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c-kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA. PMID- 7534134 TI - Enhanced production of B lymphocytes after castration. AB - Castration has long been recognized to stimulate thymic growth and augment cellular immunity. We sought to determine whether castration affects B lymphopoiesis by analyzing the phenotype of bone marrow and spleen cells from animals postcastration. In this report, we show that the bone marrow cells from castrated male mice show a sustained, twofold to threefold increase in numbers of B220+/IgM- cells and of newly formed B220+/IgM+ B cells. Most of the expanded B220+/IgM- cell population consisted of small, HSAhi, CD43- cells characteristic of pre-B cells. The castrated animals also showed increased numbers of splenic B cells, primarily consisting of small IgM+, IgDlo, B220lo, HSAhi cells. Taken together, these results show that castration causes dramatic, long-lived enhancement of B lymphopoiesis in bone marrow and increased numbers of mature B cells in the periphery. PMID- 7534135 TI - Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes. AB - Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well represented perforin-positive NK cells, which were shown to efficiently lyse NK sensitive target cells. PMID- 7534136 TI - Recognition of human T-leukemia virus (HTLV-1) envelope by human CD4+ T-cell lines from HTLV-1 seronegative individuals: specificity and clonal heterogeneity. AB - Because T-helper cells are critical for immune responses in retroviral infections, CD4+ T-cell lines specific for the human T-leukemia virus type 1 (HTLV-1) envelope have been generated from peripheral T lymphocytes of nonimmune donors to study their naive repertoire. Recombinant fragments (RE1, amino acids [aa] 26-200; RE3, aa 165-307; RE5, aa 308-401; and RE6, aa 165-401) of HTLV-1 envelope, whole envelope glycoprotein, and synthetic peptides were used to induce T-cell lines. CD4+ T-cell lines specific for one or more fragments were obtained from seven of eight individuals tested. T-cell lines generated against envelope glycoprotein from five of five donors did not cross-react with the RE fragments and vice versa. The lines specific for RE and env were mapped with overlapping peptides. The lines with single peptide (narrow) specificity contained a variety of clones that used different T-cell receptor V beta genes. These data (1) suggest that most of the normal individuals carry T-helper precursors specific for epitopes on HTLV-1 envelope; (2) indicate that heterogeneity of HTLV-1 envelope-specific T cells can be detected in the naive repertoire; and (3) define optimal antigenic preparations to be used to assess cellular immunity in HTLV-1 infected individuals. PMID- 7534138 TI - CD5-expressing B-cell non-Hodgkin's lymphomas with bcl-1 gene rearrangement have a relatively homogeneous immunophenotype and are associated with an overall poor prognosis. AB - Mantle cell lymphomas (MCLs) are typically CD5-expressing B-cell non-Hodgkin's lymphomas (NHLs) that frequently harbor the chromosomal translocation t(11;14) or bcl-1 gene rearrangements. Insufficient data are available on the biologic features and clinical behavior of rigorously characterized MCL. As these NHLs have been reported to exhibit various histologic and cytologic expressions, and in order to avoid using somewhat arbitrary and subjective morphologic definitions, we chose to study cases of MCL selected on more objective grounds. Specifically, 15 samples (from 14 patients) of CD5-expressing B-cell NHLs with detectable bcl-1 gene rearrangement were included. Overall, these patients had relatively uniform clinical manifestations. Most were older men (mean age, 67 years) who presented with lymphadenopathy, high-stage disease, and bone marrow involvement. All but two patients relapsed, demonstrated residual tumor, or had disease progression after an initial response to various therapies. Nine patients have died; these patients had a median survival of only 19 months. All cases could be classified within the broad morphologic spectrum previously described for MCL, and no predominant histologic subtype was observed. However, cases could be segregated into two major groups according to tissue architecture: one with a purely diffuse pattern and the other with at least a focal nodular component. Patients with purely diffuse tumors had a lower survival rate (0%) than those with tumors having a nodular component (62% survival rate). In contrast to the morphologic variability, these NHL exhibited a rather homogeneous immunophenotypic pattern. All cases demonstrated intense CD20 expression, with typically intense IgM and light chain expression, and relatively weak IgD expression. In no case was CD10 detected on the neoplastic cells. DNA content analysis showed aneuploidy only in three instances, and two groups of cases could be arbitrarily defined on the basis of their S-phase fraction. A relationship between a purely diffuse growth pattern and a high S-phase fraction (greater than 5%) was observed. As expected from this association, patients with tumors having high S-phase fractions fared worse (14% survival rate) than those patients with tumors showing lower S-phase fractions (57% survival rate). Thirteen NHLs from 12 patients had amplifiable bcl-1 gene rearrangements at the major translocation cluster (MTC). The bcl-1 breakpoints aggregated within a 63-bp region of the MTC, and the amplified tumor DNA from each patient had unique N-nucleotide junctional sequences and Ig joining region breakpoint sites.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534137 TI - Identification of soluble APO-1 in supernatants of human B- and T-cell lines and increased serum levels in B- and T-cell leukemias. AB - The cell-surface protein APO-1 is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 mediates apoptosis in susceptible cells upon stimulation with the monoclonal antibody anti-APO-1 or upon binding of its natural ligand. Soluble receptors had previously been identified for most members of the NGF/TNF receptor superfamily. Recently, a soluble form of APO-1 (sAPO-1) was described. We established a sandwich enzyme linked immunosorbent assay to detect sAPO-1 in culture supernatants of human cell lines and in human sera. sAPO-1 was found in culture supernatants of different human B- and T-cell lines. Molecular weights of sAPO-1 and membrane APO-1 were similar. In addition, in comparison to healthy donors, sera from patients with different high- and low-grade malignant B- and T-cell leukemias and lymphomas contained increased levels of sAPO-1. These findings may have implications for the growth of leukemias and the diagnostic monitoring of individual patients. PMID- 7534139 TI - Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma. AB - From September 1992 to January 1994, we evaluated the use of the CEPRATE SC stem cell concentrator (CellPro, Inc, Bothell, WA) to select CD34+ cells from the bone marrow (BM) of 25 patients with non-Hodgkin's lymphoma in complete remission. This system uses the biotinylated 12.8 IgM MoAb to select CD34+ cells. Cells are retained on an avidin column and detached by agitation. Fifteen patients have been transplanted with the CD34+ purified fraction. The CD34+ purified fraction of the 25 processed BMs contained a median of 0.54% of the original nucleated cells in a volume of 5 to 10 mL. The median concentration of CD34+ cells was 49% (range, 12% to 80%), and the median enrichment of CD34+ cells was 33-fold (range, 9- to 85-fold). This selected CD34+ fraction retained 60% (range, 15% to 95%) of late granulocyte-macrophage colony-forming units (CFU-GM), 55% (range, 12% to 99%) of early CFU-GM, and 31% (range, 2% to 100%) erythroid burst-forming units (BFU-E) corresponding to median enrichments of 22-fold (range, 1- to 71-fold), 19 fold (range, 2- to 58-fold), and 14-fold (range, 2- to 200-fold), respectively. There was a correlation between immune phenotypes and progenitor cells. In the initial buffy-coat fractions, the percentage of CD34+ cells was correlated to the cloning efficiency of both late CFU-GM (P < .05) and early CFU-GM (P < .001). In the final selected fraction, there was a correlation between the percentage of CD34+/CD33- and the cloning efficiency of early CFU-GM (P < .05) and between the percentage of CD34+/CD33+ and the cloning efficiency of late CFU-GM (P < .05). Lymphoma cells positive for t(14; 18) were found by polymerase chain reaction in 9 of 14 buffy coats tested before CD34+ cell purification. In 8 cases, the CD34(+)-selected fraction was found to be negative, and the CD34- fraction was found to be positive. After cryopreservation, the recoveries of progenitor cells in the CD34(+)-purified fraction were 79% for late CFU-GM, 71% for early CFU-GM, and 73% for BFU-E. The 15 patients transplanted with the concentrated CD34+ fraction received a median dose of 1 x 10(6) CD34+ cells/kg (range, 0.3 to 2.96) and 10.62 x 10(4) early CFU-GM/kg (range, 0.92 to 25.55). Median days to recovery to 0.5 x 10(9)/L neutrophils and 50 x 10(9)/L platelets were days 15 (range, 10 to 33) and 23 (range, 11 to 68), respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534140 TI - Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. AB - Peripheral blood stem cells (PBSCs) are widely used in autologous transplantation because of ease of collection and rapid hematopoietic reconstitution. However, PBSCs have rarely been used for allogeneic transplantation because of concerns about donor toxicities from cytokine administration and the theoretical increased risk of graft-versus-host-disease (GVHD) from the large number of T cells infused. Eight patients with advanced malignancies received allogeneic PBSC transplants from genotypically HLA-identical sibling donors. All donors received 5 days of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 16 micrograms/kg/day) subcutaneously and were leukapheresed for 2 days. After treatment of the patient with total body irradiation and cyclophosphamide (n = 7) or etoposide, thiotepa, and cyclophosphamide (n = 1), PBSCs were infused immediately after collection and without modification. All patients received cyclosporine and either methotrexate (n = 6) or prednisone (n = 2) for GVHD prophylaxis, rhG-CSF was well tolerated with mild bone pain requiring acetaminophen occurring in two donors. All patients engrafted and in seven hematopoietic recovery was rapid, with 500 neutrophils/microL achieved by day 18 and 20,000 platelets/microL by day 12. Complete donor engraftment was documented by Y chromosome analysis in all four sex-mismatched donor-recipient pairs tested and by DNA analysis in two sex-matched pairs. One patient died on day 18 of veno occlusive disease of the liver with engraftment but before chromosome analysis could be performed (results are pending in 1 patient). A second patient died of fungal infection 78 days after transplant. Grade 2 acute GVHD occurred in two patients and grade 3 GVHD occurred in one patient. One patient is 301 days from transplant in remission with chronic GVHD; the remaining five patients are alive and disease free 67 to 112 days after transplantation. Preliminary results indicate that allogeneic PBSCs mobilized by rhG-CSF can provide rapid hematologic recovery without an appreciably greater incidence of acute GVHD than would be expected with marrow. Further follow-up is required to determine the incidence of chronic GVHD and any potential beneficial effects on relapse after transplant. PMID- 7534141 TI - Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor) AB - Transplantation of allogeneic peripheral blood progenitor cells (PBPCs) may have advantages over bone marrow transplantation (BMT) with regards to the speed of hematopoietic and immunologic recovery, which may then shorten the time spent in hospital and decrease costs. The recipient might also profit by an enhanced graft versus-leukemia reaction exerted by the high number of natural killer cells contained in such grafts. The donor could be spared the discomfort and risks of general anesthesia and marrow harvesting. Primary transplantation of unmanipulated allogeneic PBPCs has not been reported so far because the vast amount of T cells contained in the collection product was thought to cause severe graft-versus-host disease. We present preliminary data on primary transplantation of allogeneic PBPCs in patients who either suffered from advanced leukemia or had a donor unable to undergo general anesthesia. Eight patients with a median age of 42 years suffering from acute myelogenous leukemia (AML) in first remission (n = 3), AML in third remission, AML in relapse (n = 2), acute lymphoblastic leukemia in second remission, or chronic myelogenous leukemia in accelerated phase received myeloablative therapy followed by transplantation of unmanipulated allogeneic PBPCs mobilized with granulocyte colony-stimulating factor (5 to 10 micrograms/kg of body weight of filgrastim administered for 5 to 6 days) in their HLA-identical donors. Hematopoietic reconstitution was achieved in all patients with a median of 15.5 (16.5) days after transplant needed to surpass an absolute neutrophil count of 0.5 (1.0) x 10(9)/L. The median time to an unsupported platelet count greater than 20 (> 50) x 10(9)/L was 19.5 (41) days after grafting. Three patients did not exhibit signs of acute graft-versus-host disease (GVHD), grade I disease was seen in one patient, and three patients experienced grade II disease limited to the skin. The only patient with severe acute GVHD (grade III) refused to take his oral cyclosporin regularly and had ineffective serum levels for most of the time until relapse. Six of eight patients are currently alive without evidence of disease between 61 and 533 days after grafting; two patients grafted for AML in relapse achieved a complete remission after transplantation but relapsed again and died of leukemia on days +48 and +70, respectively. Primary transplantation of unmanipulated allogeneic PBPCs is feasible and results in long-term engraftment without causing detrimental GVHD. PMID- 7534142 TI - Differential diagnosis of CD3+, CD56+ T-cell leukemias. PMID- 7534143 TI - Granulocyte colony-stimulating factor-mobilized allogeneic peripheral blood stem cells for rescue graft failure after allogeneic bone marrow transplantation in two patients with acute myeloblastic leukemia in first complete remission. PMID- 7534145 TI - Changes in sensory neuropeptides in dorsal root ganglion and spinal cord of spontaneously diabetic BB rats. A quantitative immunohistochemical study. AB - This study examined the expression of the sensory neuropeptides, calcitonin gene related peptide (CGRP) and substance P (SP), in the lumbar 4 and 5 dorsal root ganglion (DRG) and spinal cord of spontaneously diabetic BB rats and non-diabetic controls using quantitative immunohistochemical analysis. In both animal groups immunoreactivities for CGRP and SP were widely distributed within the neurons of DRG and in nerve fibres of the dorsal spinal cord. Image analysis of each neuropeptide subpopulation in the DRG showed that in diabetic rats the cell diameter of immunostained CGRP neurons was significantly decreased compared with controls, while no difference could be found for SP-immunoreactive (IR) neurons. The decrease in the CGRP-IR cell diameter appeared to occur mainly in medium to large neurons (30-50 microns diameter; 2.2% controls, < 1% diabetes), this change being parallel to an increased frequency of small-size neurons (< 20 microns diameter) in diabetic rats (62% controls, 69% diabetes; P < 0.05). However, there was no statistical difference in the total number of cells immunostained for either CGRP or SP between control and diabetic rats. The ratio of CGRP or SP neurons compared to total cells in the ganglion was similar in control and diabetic groups. No difference could be observed for peptide immunoreactivity in the dorsal and ventral horns of either control or diabetic animals. The observed changes of perikaryal size in diabetic rats might relate to the reduced axonal calibre and conduction velocity observed in these animals, and indicate that subpopulations of sensory neurons are affected differently by diabetes. PMID- 7534144 TI - Regulation of insulin-like growth factor binding protein-1 (IGFBP-1) in insulin dependent diabetes mellitus. Effects of hyperglycaemia and insulin. AB - The aim of the present study was to characterize the effect of 44 h of hyperglycaemia on diurnal levels of insulin-like growth factor binding protein-1 (IGFBP-1), insulin-like growth factor-1 (IGF-1), growth hormone (GH) and glucagon in 7 well-controlled subjects with insulin-dependent diabetes mellitus (IDDM). Hyperglycaemia (approximately 15 mmol/l) was induced by a glucose infusion, while the degree of insulinisation was similar to that of a corresponding period with near normoglycaemia (approximately 6.9 mmol/l). Hyperglycaemia for 44 h did not alter the normal diurnal IGFBP-1 levels when the degree of insulinisation was unchanged. The diurnal secretion pattern of IGFBP-1 was preserved in both genders and without any difference between the control and hyperglycaemic periods. However, the IGFBP-1 levels were increased in these IDDM subjects despite a peripheral hyperinsulinemia. An inverse correlation was found between IGFBP-1 and peripheral insulin levels both during periods of rapid changes in IGFBP-1 and insulin concentrations (i.e. morning hours) as well as during the total 24-h sampling period. Total IGF-1 levels were low, but no further decrease was seen after 24 h of hyperglycaemia in the presence of unchanged insulin levels. In conclusion, the present study clearly shows that the increased IGFBP-1 level seen during poor metabolic control in IDDM is not caused by hyperglycaemia. Glucose levels per se do not influence either total IGF-1 or IGFBP-1 concentrations in well-insulinised diabetic patients. PMID- 7534146 TI - Vascular toxicity associated with chemotherapy for testicular cancer. AB - Cisplatin-based chemotherapy considerably improved the outcome of patients with metastatic germ cell tumors. Apart from Raynaud's phenomenon, a frequent side effect, vascular toxicity associated with chemotherapy for testicular cancer, has not been described precisely. Although major vascular complications such as myocardial infarction, stroke and pulmonary embolism seem to occur infrequently, they raise concern with regard to the safety of chemotherapy. Also, potential late vascular toxicity has to be taken into account. Whereas a cause and effect relationship is probable for some vascular events following chemotherapy, some cases may represent coincidence or may be disease related. Presently, the very low incidence of major vascular events should not enter into therapeutic decisions. PMID- 7534147 TI - Modulation of human P-glycoprotein epitope expression by temperature and/or resistance-modulating agents. AB - Three monoclonal antibodies (mAb), MRK16, MM4.17 and MC57, directed against distinct epitopes on the external domain of human P-glycoprotein (Pgp), were used to follow its expression on multidrug resistant (MDR)-cells. The linear MM4.17 epitope and conformational MRK16 epitope showed a 4-fold higher expression at 37 degrees C than at 4 degrees C, while the detection of the conformational MC57 epitope did not change. Inhibition of Pgp function, by a short pretreatment of the MDR-cells with resistance-modulating agents (RMA), such as SDZ PSC 833 and SDZ 280-446, could not be related to depletion of Pgp from the cell surface, since their expression of the MM4.17 and MRK16 epitopes was found unchanged. However, a substantially higher expression of MC57 epitopes was found on RMA treated cells than on untreated ones. Since this effect correlated to the strength of different RMA in reversing the MDR phenotype, MC57 epitopes might be more efficiently expressed on inactivate(d) forms of the Pgp molecules, suggesting that RMA might inhibit Pgp function by disturbing the conformation of individual Pgp molecules, their topographical distribution or polymerization status in the membrane. PMID- 7534148 TI - Synthesis and antigenic properties of reduced peptide bond pseudopeptide analogues of a histone H3 hexapeptide. AB - Pseudopeptide analogues of the C-terminal hexapeptide of histone H3 (-Ile-Arg-Gly Glu-Arg-Ala-OH) were obtained by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond psi (CH2-NH). The resulting analogues were then examined, in ELISA and in the BIAcore system, for their ability to bind polyclonal and monoclonal antibodies generated against the parent natural peptide and the protein. The comparative results show that reduced bond pseudopeptide analogues can mimic the parent peptide. These results present the first unequivocal example for the potent applicability of reduced peptide bond pseudopeptides in the immunological field. PMID- 7534149 TI - A sensitive detection method for peptide using 4-fluoro-7-nitrobenzo-2-oxa-1,3 diazole and its application to measure prolyl endopeptidase activity. AB - A measuring method sensitive to prolyl endopeptidase (EC 3.4.21.26, PEP) activity using native peptides (Arg-vasopressin or substance P) as substrates was established. The investigation of three different derivatization reagents, which had been developed for an amino acid analysis, demonstrated that 4-fluoro-7 nitrobenzo-2-oxa-1,3-diazole (NBDF) was the most suitable for the detection of Arg-Gly-NH2, which was released from Arg-vasopressin by PEP. Arg-Gly-NH2 was reacted with NBDF at 65 degrees C for 5 min at pH 7.6 and the reaction mixture was analysed by HPLC on a reverse-phase column by monitoring the fluorescence intensity. The detection limit was 1 picomol per injection and the linear standard calibration curve could be constructed in the range of 1 to 100 picomol per injection with a 3.0% relative standard deviation. This sensitive detection method for peptide was applied to the measurement of PEP activity using Arg vasopressin as a substrate and 1 x 10(-3) unit of PEP activity was detectable. This method was also applicable to the measurement of PEP activity using substance P as a substrate by detecting the derivative of its fragment peptide (Arg-Pro-Lys-Pro). PMID- 7534150 TI - Influence of genetic and environmental factors on surface expression and occupancy of IgE receptors and on histamine releasability of mast cells. AB - The relationship between the IgE load on mast cells and their secretory capacity when challenged with anti-IgE was studied in peritoneal cells obtained from rats of three different strains, Hooded Lister (HL), Wistar Kyoto (WKY) and Sprague Dawley (SD). IgE was determined cytofluorometrically after labelling with FITC conjugated anti-IgE before and after the saturation of the IgE receptors to provide a measure of the surface expression of IgE receptors (number of receptors available for bindings) as well as the IgE occupancy of the receptors (native IgE content). The secretory capacity of the mast cells was examined in vitro in terms of histamine release as a function of anti-IgE concentration. Mast cells obtained from HL and WKY rats were found to carry significantly higher levels of IgE receptors and IgE than the mast cells of SD rats bred and raised under the same conventional laboratory conditions. The mast cells of SD rats kept under barrier maintained conditions carried significantly less IgE than the mast cells obtained from SD rats kept under conventional conditions, but their IgE receptor levels were similar. The IgE-mediated histamine-releasing capacity of the mast cells, evaluated in terms of maximum release or of the slopes of regression lines (histamine release versus anti-IgE concentration), was positively correlated to the levels of native IgE and IgE receptors in the three strains of rat combined. The mast cells obtained from WKY rats showed the highest secretory capacity in the three strains of rat examined, significantly higher than the mast cells of HL rats, even though the latter displayed similar levels of IgE and IgE receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534151 TI - Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I. AB - The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity. PMID- 7534152 TI - Fetal hemoglobin levels in adults. AB - The synthesis of fetal hemoglobin (HbF) is normally reduced to very low levels of less than 0.6% of the total hemoglobin in adults. The HbF is restricted to a sub population of erythrocytes termed 'F-cells'; 85% of the normal adult population have 0.3% to 4.4% F-cells. The levels of HbF and F-cells vary by more than 10 fold in normal adults; family studies show that these levels are genetically controlled but the number and nature of these genetic factors are still poorly understood. HbF levels may be increased in adults in a number of inherited and acquired disorders, accompanied by an increase in both the number of F-cells and the amount of HbF per F-cell. The clinical significance of these conditions with raised HbF relates to their interaction in disorders such as sickle cell disease and beta thalassaemia in which raised levels of HbF can lead to considerable amelioration of disease severity. Study of the 'natural' mutants primarily associated with increased HbF has provided considerable insight into the understanding of the control of globin gene regulation and hemoglobin switching. Currently considerable effort is being channelled into clinical trials and the search for the 'ideal' therapeutic agents which could increase HbF in adult life with minimal drug toxicity. PMID- 7534153 TI - Mechanisms of cytopenia in human immunodeficiency virus infection. AB - Human immunodeficiency virus (HIV) infection often has effects on the hematopoietic system which can be distinguished from the concurrent effects of medications or opportunistic infections. Exactly how the virus mediates these effects remains uncertain, but both in vivo and in vitro studies have pointed up possible direct and indirect modes of hematopoietic suppression. Whether a significant fraction of CD34+ cells in vivo are infected with HIV remains controversial, but most studies using in situ polymerase chain reaction techniques would suggest not. Other more indirect modes of hematopoietic cell suppression such as production of autoantibodies, production of other humoral inhibitory factors, T-cell mediated suppression of hematopoiesis, or production of inhibitory or stimulatory cytokines may also be contributory. It is probable that several of these mechanisms may occur simultaneously, and an increased understanding of their role may lead to improved strategies to correct the cytopenias which often accompany HIV disease. PMID- 7534154 TI - The inhumanity of medicine. Falling quality of caring mirrors the rise of materialism. PMID- 7534155 TI - Dying for palliative care. Anger is a means of coping with illness. PMID- 7534156 TI - Dying for palliative care. General practitioners have a crucial role. PMID- 7534157 TI - Trials in palliative care. PMID- 7534158 TI - An ethnographic account of opium consumers of Rajasthan (India): socio-medical perspective. AB - This communication gives an ethnographic account of 200 opium users in selected villages of three Western districts in the desert state of Rajasthan. The region is known for its traditional use of raw opium in the form of amal or doda, due to its climate and difficult living conditions. The ethnographic information suggests that opium use is in many ways integrated into the socio-cultural fabric of the local community. Self-medication with opium mitigates various health problems and the drug is also used to relieve mental distress. Besides these uses for relief of distress, the drug is used recreationally and within settings which facilitate social bonding. The traditional roles ascribed to the use of this intoxicant cannot be dismissed when formulating long-term preventive and control measures. PMID- 7534159 TI - Enrichment of peripheral blood stem cells in a primate model following administration of a single dose of rh-IL-1 beta. AB - In a pre-clinical primate model, the effect of a single intravenous injection of rh-GM-CSF (50 micrograms/kg), rh-IL3 (20 micrograms/kg), rh-IL-1 beta (1 micrograms/kg), rh-G-CSF (50 micrograms/kg) and rh-pIXY 321 (50 micrograms/kg) on peripheral blood mononuclear cell and hematopoietic stem cell concentration was determined. The results indicated that administration of a single dose of rh-IL-1 beta increased the concentrations of hematopoietic stem cells, including CFU-GM progenitors and precursors to CFU-GM. No toxicity was noted with this procedure. None of the remaining cytokines tested caused a significant increase in the peripheral blood hematopoietic stem cell concentration when administered in this manner and only G-CSF caused an increase in peripheral blood mononuclear cell concentrations. Autologous transplantation with limiting numbers of peripheral blood mononuclear cells (1 x 10(7) cells/kg) collected either 24 or 72 h following injection of 1 microgram/kg rh-IL-1 beta conferred a survival advantage to recipients compared with controls. These results imply that a single intravenous administration of rh-IL-1 beta may increase the concentration of peripheral blood stem cells to levels sufficient for transplantation. PMID- 7534160 TI - 'Fetal' erythropoiesis following bone marrow transplantation as estimated by the number of F cells in the peripheral blood. AB - The aim of this study was to define factor(s) influencing fetal erythropoiesis following bone marrow transplantation. Thirty-one transplanted patients (14 males, 17 females) were studied. The underlying diseases were chronic myelogenous leukaemia (CML, 18 patients), acute myeloblastic leukaemia (AML, 7 patients) and acute lymphoblastic leukaemia (ALL, 6 patients). Reticulocyte and peripheral F cell estimation was carried out in donors and patients before transplantation and repeatedly during recovery. For F cell estimation, an indirect immunofluorescence assay was utilized. A significant increase above pre-BMT values in the percentage of F cells was observed in all patients from days 11 to 40 after transplantation. The increase of F cells on days 15, 18, 25, 32, 40 and 50 after transplantation was statistically significant in 14 patients who had shown an increase of F cells following chemotherapy (high responders) compared with the remaining 17 patients who did not respond so significantly. This finding supports the influence of the host bone marrow micro environment. The nature of the mechanisms operating remains to determined. PMID- 7534162 TI - Treatment of poor marrow graft function with allogeneic CD34+ cells immunoselected from G-CSF-mobilized peripheral blood progenitor cells of the marrow donor. AB - The treatment effect of immunoselected allogeneic CD34+ blood cells was evaluated in two patients with poor graft function following BMT without evidence for immune-mediated rejection. Patient A had no signs of hematopoietic recovery up to day +34 post-BMT and patient B had normal leukocyte counts only with G-CSF support and remained platelet transfusion-dependent for > 200 days post-BMT. PBPC from the HLA-identical sibling BM donors were mobilized with G-CSF (2 x 5 micrograms/kg sc daily) for 5 days. Aphereses were performed on days 4 and 5 of G CSF administration. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption with the anti-CD34 moAb 12.8 in a biotin-avidin system. Patient A received 0.4 x 10(6) CD34+ and 4.3 x 10(5) CD3+ cells/kg body weight and patient B 3.4 x 10(6) CD34+ and 1.4 x 10(5) CD3+ cells/kg body weight. The trilineage repopulation of BM and the rapid improvement of peripheral blood parameters correlated with CD34+ cell infusion. Patients' blood and BM cell analyses proved the donor origin. Patient A died from CMV pneumonitis and multiorgan failure 27 days after CD34+ cell infusion (day +61 post-BMT). Patient B is still stable and in remission 260 days after CD34+ cell infusion (day +478 post-BMT). Neither patient suffered further exacerbation of GVHD). Thus, immunoselected allogeneic CD34+ blood cells might be appropriate for treatment of post-BMT graft failure. PMID- 7534161 TI - Delayed administration of granulocyte colony-stimulating factor after autologous bone marrow transplantation: effect on granulocyte recovery. AB - Recombinant granulocyte colony-stimulating factor (rhG-CSF) has been shown to hasten granulocyte recovery after autologous BMT. In current protocols, rhG-CSF treatment starts 1 day after BM reinfusion. Our study retrospectively examined the effects on haematological recovery of a day 6 delayed administration. Seventy eight patients receiving autologous BMT for malignant lymphoma (21 non-Hodgkin's lymphoma and 9 Hodgkin's disease) or solid tumors (33 breast carcinoma and 5 ovarian carcinoma) were split up into three study groups. Two groups receiving a 5 micrograms/kg/day of rhG-CSF starting either 1 day (day +1 group, n = 25 patients) or 6 days (day +6 group, n = 24 patients) after BM reinfusion were compared with 29 historical control patients. Granulocyte recovery to 0.5 x 10(9)/l was 12 days in day +6 and day +1 groups versus 16 days in control group (p < 0.005) without any difference in other hematological parameters, infectious complications or length of hospitalisation between the three groups. The day +6 administration allows elimination of a median of 7 days rhG-CSF. It has been concluded that the day +6 administration gives the same clinical benefit as day +1 administration with consequent cost reductions. PMID- 7534164 TI - Impact of liver disease and hepatitis infections on allogeneic bone marrow transplantation in Europe: a survey from the European Bone Marrow Transplantation (EBMT) Group--Infectious Diseases Working Party. AB - This survey investigated allogeneic bone marrow transplantation (BMT) policy in European BMT units by questionnaire, in relation to pre-transplant liver disease. It also assessed diagnostic standards for viral hepatitis infections and their prevalence in BMT candidates. Sixty-three EBMT centers from 22 countries participated in the survey. Median pre-transplant prevalences of HBsAg and anti HCV positivity were 3.5% (range 0-15%) and 5% (range 0-45%), respectively. Forty six (73%) centers adopt the policy of cancelling or postponing BMT in patients with ALT abnormalities but in four of these centers, BMT is not delayed when progressive disease or acute leukemia is present. In 17 institutions (27%) BMT was reported to be carried out irrespective of transaminase values. Data on fatal post-BMT liver disease were provided by 45 centers. The overall mortality rate for liver failure was 4.5% (258 of 5788) with no differences between centers performing or not performing BMT in cases of ALT elevation. These results indicate that there is strong concern in most European BMT units about performing BMT in the presence of ALT elevation and prospective studies on its real impact on fatal post-BMT liver disease should be conducted. PMID- 7534163 TI - Filgrastim post-chemotherapy mobilizes more CD34+ cells with a different antigenic profile compared with use during steady-state hematopoiesis. AB - For the mobilization of CD34+ peripheral blood progenitor cells (PBPC) filgrastim (R-metHuG-CSF) can be administered either during steady-state hematopoiesis or following cytotoxic chemotherapy. We compared both mobilization modalities intra individually in seven patients with breast cancer. The number of circulating CD34+ cells was increased after filgrastim-supported chemotherapy compared with filgrastim administration during steady-state (on average, 129 vs. 19/microliters), resulting in a sevenfold higher yield of CD34+ cells per leukapheresis (5.73 vs. 0.79 x 10(6)/kg bodyweight). CD34+ PBPC harvested post chemotherapy comprised a smaller proportion of early progenitor cells (CD34+/HLA DR- or CD34+/CD38-) compared with filgrastim treatment alone. However, the absolute number of these early progenitor cells harvested was fivefold higher. The filgrastim-supported rebound after chemotherapy was characterized by a greater proportion of CD34+/CD33+ cells. Correspondingly, CD34+ PBPC mobilized post-chemotherapy contained a higher proportion of CFU-GM compared with filgrastim treatment during steady-state, while the cloning efficiency of CD34+ cells for BFU-E tended to be lower. Of note, the proportion of CD34+/CD19+ lymphoid progenitor and B cells was reduced after chemotherapy. In cancer patients, filgrastim mobilizes higher numbers of CD34+ cells when administered post-chemotherapy, compensating for the smaller proportion of early hematopoietic progenitors. PMID- 7534165 TI - [Physiopathology of cervico-urethral obstruction in benign prostatic hypertrophy]. AB - It's known that benign prostatic hypertrophy arise from the glands that surround the proximal prostatic urethra. In these cases voiding disorders may be due by detrusor instability alone or associated with cervical urethral obstruction. At first Griffiths and Schafer and subsequently Sarky have introduced some correlations that are able to differentiate the bladder outlet obstruction from the impaired detrusor contractility. On the contrary with regard to the behaviour of detrusor during obstruction, in connection with histological findings, several interpretations have been expressed. The theory more credited has been expressed by Gosling who has showed a reduction of the parasympathetic innervation of detrusor and consequent hypersensitivity due to denervation. PMID- 7534166 TI - [Assessment of the effects of alfuzosin on miction with dynamic echography]. AB - Alfuzosina has a specific antagonist effect on the alfa-1-adrenergic post junctional receptor posed at urethral laeve musculature, prostatic capsule and vesical trgonum. It's and ideal drug the symptoms of the difficult urinary flow typical of the benign prostatic hypertrophia (IPB) and of some patologies of vesical collum to attenuate. With this work we want to demonstrate the anatomic alterations of vesical collum and prostatic urethra by means of dynamic recording mictional phase with transrectal scan carried out before and after treatment. In this study, 30 patients were divided into three groups of 10 in relation to the pathology: overtone of vesical collum, little benign prostatic ipertrophia and great benign prostatic ipertrophia have included. The patients of each group have also been divided in undergroups of five (selected with the method of double blind) of which the first treated with alfuzosina 7.5 mg/die for 30 days and the second with placebo. Considering the limits of this study, the analysis of the results have permitted to conclude that the drug has determined a subjective improvement in all patients except those with great IPB, while the objective valuation, reached comparing scan dynamic pictures before and after treatment, has made it possible to visualize an improvement on the opening of the vesical collum and the prostatic urethra after therapy with great variations of the size of che mictional funnel and same prostatic urethra. The alfuzosina is efficacious, in the cases under examination, except the third group were the compression on the urethra was of an elevated degree. PMID- 7534167 TI - [Ureteral jet during medical treatment of benign prostatic hypertrophy]. AB - By Color-Doppler ultrasound it's possible to visualize urine flow from ureter into the bladder. Aim of the study was to image these ureteral jets in patients with benign prostatic hyperplasia (BPH) before and after therapy. Seven patients were studied, whose age ranged from 52 to 69 years; they were not affected by methabolic, hepatic, renal diseases and by prostate inflammation. A duplex scanner (Toshiba SSA 270 A) with a convex probe of 3.5 MHz was used. A transabdominal ultrasound study was performed, measuring prostatic volume and imaging ureteral jet before and along a treatment (at four months intervals) with Finasteride 5 mg/die (Finastid, Neopharmed) and at the end of treatment. We found that the mean velocity of jets in patient affected by was higher than in healthy subjects (80 cm/sec vs 60 cm/sec) bur during treatment it decreased along with symptom improvements and decreasing volume at US. We concluded that analysis of ureteral jets with Color-Doppler P.W. may add useful informations to real-time us in the study of patients affected by BPH undergoing treatment with Finasteride. PMID- 7534168 TI - [Usefulness of PSA density (PSAD) in the differential diagnosis between prostatic adenocarcinoma and benign ++ prostatic hypertrophy]. AB - The purpose of this study was to evaluate the usefulness of PSAD in distinguishing benign prostatic hyperplasia (BPH) from prostate cancer. The Authors studied 50 patients, 30 affected with BPH and 20 with prostate cancer. All patients were submitted to: digital rectal examination, trans-rectal ultrasonography and evaluation of serum PSA, before performing any prostatic manipulation. All clinical data were confirmed by histological diagnosis. Although the mean PSAD value was greater significantly in the patients' group affected with prostate cancer, it was impossible to define a PSAD cut off value useful to distinguish malignant from benign prostatic diseases. Similar findings have been observed in a sub-group patients with serum PSA concentration of 3.5-15 ng/ml. The PSAD cut off value of 0.10, proposed from some Authors, showed low specificity. On the contrary a cut off value of 0.15 is not sufficiently sensitive: an elevated number of cancer would have missed using this parameter alone. Therefore, the combined use of clinical findings and serum PSA is recommended to improve the early diagnosis of prostate cancer. PMID- 7534169 TI - [Current role of echography, of PSA and of PSAD in the diagnosis of prostatic carcinoma]. AB - The Authors report their experience in the diagnosis of prostatic carcinoma by means of DRE, TRUS and PSA. They emphasize the improvement of diagnosis given by these exams when used in association, despite a high rate of false positives. In the years 92-93, 182 patients underwent ecoguided prostatic biopsy after DRE and PSA evaluation. PSA density value was calculated as proposed by Benson (PSAD = PSA/V); this parameter should screen between PSA elevation due to BPH and those due to prostatic carcinoma. After their experience, even if limited, they conclude that TRUS should not be used as a "first-line test" but only in patients with abnormal findings in DRE and/or PSA. PSAD may be useful to improve specificity of PSA even if a precise cut-off can not be determined. PMID- 7534170 TI - [ETG follow-up in transurethral incision of the prostate (TUIP)]. AB - In absence of marked benign prostatic hyperplasia, alternative surgical procedure, the transurethral incision of the prostate (TUIP), is available. The Authors report their experience and suggest a comparison between the morphological data obtained with ultrasound and the functional data obtained with the uroflowmetry. PMID- 7534171 TI - [Intraprostatic spiral: our experience]. AB - A spiral urethral prosthesis was inserted with use of local anesthesia in 5 patients with benign prostatic hyperplasia and 2 patients with cancer of the prostate, using ultrasonic guidance. All the patients refused the surgical procedure of had high operative risk. The Authors showed the usefulness of the Ultrasound for insertion of intraprostatic spiral, in selected patients. PMID- 7534172 TI - Ultrastructural identification of entorhinal cortical synapses in CA1 stratum lacunosum-moleculare of the rat. AB - The goal of the present study was to identify and characterize, at the electron microscopic level, the synapses formed by entorhinal cortical (EC) axons in the hippocampal CA1 stratum lacunosum-moleculare of the adult rat. Phaseolus vulgaris leucoagglutinin was ionotophoresed at various loci throughout the mediolateral and dorsoventral extent of the EC to label EC-CA1 synapses. Virtually all labeled synapses were asymmetric and axospinous. EC axons did not preferentially synapse with any particular type of dendritic spine; rather, EC axons formed synapses with the range of dendritic spine morphologies observed in CA1 s. lacunosum moleculare. Spines with either perforated or nonperforated postsynaptic densities were contacted by EC axons. Occasionally both a labeled and an unlabeled axon synapsed on a single dendritic spine head. The data are discussed in relation to the morphology of other afferent systems synapsing in s. lacunosum-moleculare and to the physiology of the EC-CA1 system. PMID- 7534173 TI - Dextran 70 administration after trauma-hemorrhagic shock does not impair cellular immune functions. AB - PURPOSE: Although the effects of the colloid dextran 70 on induction of anaphylactoid reactions or reticuloendothelial phagocytosis have been examined previously, its effects on specific cell-mediated immunity after trauma hemorrhage shock remain unknown. METHODS: Nonheparinized C3H/HeN mice underwent a laparotomy, were bled, and then maintained at a blood pressure of 35 mm Hg for 60 minutes. Then they were resuscitated with either 4 x the shed blood volume as lactated Ringer's solution (LRS) or 2 x LRS + 1 x dextran 70. Control mice underwent all operative protocols but were neither hemorrhaged, nor resuscitated. At 2 or 24 hours posthemorrhage, serum, splenocytes (SPL), and peritoneal macrophages (pM phi, splenic Mo (sM phi) were obtained. Bioassays were used to determine interleukin-2 (IL-2), IL-3, IL-6, and SPL proliferation. RESULTS: Trauma-hemorrhage markedly depressed lymphokine release, splenocyte proliferation, and IL-6 release at 2 hours after the insult. The combination of LRS + dextran did not restore lymphocyte functions, but also did not further suppress them. The release of IL-6 by pM phi and sM phi at 2 and 24 hours after dextran infusion and serum IL-6 remained at the same level as in LRS-treated animals. CONCLUSIONS: The combination of LRS and colloid dextran 70 does not adversely affect ex vivo cell-mediated immune functions during the first 24 hours after its administration after trauma-hemorrhage. Thus, from the immunological standpoint, dextran is a safe resuscitation adjunct. PMID- 7534174 TI - HCV antibodies in Hungarian blood donations. Experiences collected by ELISA tests, immunoblot assays and polymerase chain reaction and protocols for donor management. AB - Routine screening of Hungarian blood donors for anti-HCV commenced in the second half of 1992. Before this, five available anti-HCV ELISA kits were compared in pilot studies. In the first series, 831 random donor samples were tested by one of the tests and the 12 (1.4%) reactives found were retested by the other four. Six of the reactives were positive in all ELISA. In the second series, 325 samples from donors with elevated transaminase levels were tested by all five kits. Forty-four were found to be reactive by one or more of the tests and 32 (10%) were positive in all five assays. Samples concordantly reactive in the ELISA were positive in second or third generation recombinant immunoblot assay (RIBA 2 or RIBA 3); those that gave discordant results were indeterminate or negative. Eleven concordantly reactive samples from the second series were HCV RNA positive by polymerase chain reaction (PCR). In the first period of screening with Abbott ELISA 2 a repeat-reactivity rate of 0.98% was observed in 171,106 samples tested. Reactives were retested for supplementary testing by Wellcozyme anti-HCV. Donors reactive in both tests and strongly reactive (ELISA ratio (ER) = optical density/cut off > or = 2.5) in either of them were permanently deferred. Those negative in the supplementary ELISA or weakly reactive (1.0 < or = ER < 2.5) in both tests were subjected to RIBA 2. On the basis of RIBA, positive donors were permanently deferred, indeterminates were excluded for 1 year and negatives were readmitted. In 1992, 1,347 supplementary tests were completed; 824 (61%) of the respective donors were permanently deferred, 218 (16%) were deferred for 1 year and 305 (23%) were readmitted. PMID- 7534175 TI - [The morphofunctional changes in the alveolar epithelium, macrophages and pulmonary surfactant of rats with bleomycin-induced fibrosis]. AB - Interstitial fibrosis was modelled in 100 male rats by means of a single intratracheal administration of bleomycin (0,5 U/100 g of body mass). The stimulation and activation of secretory activity of large (granular) epitheliocytes (alveolocytes of the 2nd type) and alveolar macrophages with maximum alterations were found 3 weeks later, when in the lungs there were numerous foci of interstitial fibrosis. Biochemically, these phenomena were followed by increasing the amount of phospholipids in surfactant (mainly at the expense of phosphatidylcholine) and in membranes of macrophages, which points to interrelation of metabolic processes in the cells, morphologically detected activation of synthesis of surfactant in alveolocytes of the 2nd type and its utilization by alveolar macrophages at the height of the development of bleomycin induced pneumofibrosis. A supposition has been put forward of a participation of surfactant in the formation of fibrosis. PMID- 7534176 TI - Growth hormone-binding protein-related immunoreactivity in the serum of patients with acromegaly is regulated inversely by growth hormone concentration. AB - In this report we describe a newly developed radioimmunoassay (RIA) for the determination of the high-affinity growth hormone-binding protein (GHBP) in human blood. Using this RIA for the measurement of GHBP in serum of 29 patients with acromegaly, decreased concentrations were found compared to the normal range, depending on the activity of the disease. Growth hormone-binding protein was correlated inversely to log GH (r = -0.7, p < 0.001). A weaker relationship was shown between the GHBP activity determined in a functional assay based on charcoal separation and log GH (r = -0.51, p < 0.01). While insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) were correlated directly to log GH (r = 0.77 and r = 0.66, p < 0.001), an inverse and weaker relationship was evident between GHBP measured by RIA and IGF-I or IGFBP-3 (r = -0.61 and r = 0.57, p < 0.01). In contrast, no correlation could be detected between data of the functional GHBP assay and IGF-I or IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534177 TI - Valid measurements of total IGF concentrations in biological fluids: recommendations from the 3rd International Symposium on Insulin-like Growth Factors. PMID- 7534178 TI - Angiotensin II type 1 receptor-mediated inhibition of cytokine-stimulated inducible nitric oxide synthase expression in vascular smooth muscle cells. AB - In vascular smooth muscle cells (VSMC), inflammatory cytokines such as interleukin 1 (IL1) and tumour necrosis factor alpha (TNF alpha) stimulate nitric oxide (NO) production via the expression of an inducible type of NO synthase (iNOS). This study was performed to determine whether angiotensin II (AII) affected NO production in cultured VSMC. AII by itself did not stimulate the production of nitrite, a stable metabolite of NO, but dose-dependently inhibited IL1 beta-induced nitrite production. This inhibitory effect of AII was blocked by the AII type 1 (AT1) receptor antagonist, 2-ethoxy-1-[[2'-(1H-tetrazol-5 yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974), but not by the AII type 2 (AT2) receptor antagonist, (S)-1-[[4-(di-methylamino)-3 methylphenyl]methyl]-5-(diphenylacetyl++ +- 4,5,6,7-tetrahydro-1H-imidazo[4,5 c]pyridine-6-carboxylic acid (PD-123319). In parallel with the decrease in nitrite production, AII suppressed IL1 beta-induced increases in iNOS mRNA and protein levels, as measured by Northern blotting using an iNOS cDNA probe and by immunoblotting using an anti-iNOS antibody, respectively. AII also inhibited the increases in nitrite production and iNOS mRNA and protein levels caused by TNF alpha. These results indicate that AII inhibits cytokine-induced NO production in VSMC by blocking iNOS expression through the AT1 receptor; this suggests that the AT1 receptor antagonist may modulate the development of atherosclerotic and postangioplastic lesions by blocking this inhibitory effect of AII. PMID- 7534179 TI - Antagonism between the vascular renin-angiotensin and natriuretic peptide systems in vascular remodelling. AB - Vascular remodelling is central to the pathophysiology of hypertension and atherosclerosis. Recent evidence suggests the pivotal role of vasoactive substances occurring in the blood vessel, such as angiotensin II (AII), in the control of vascular growth. We recently discovered that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced by vascular endothelial cells and can act as an endothelium-derived relaxing peptide. We also demonstrated gene expression of CNP and the ANP-B receptor, which is one of the three subtypes of the natriuretic peptide receptor and is specific to CNP in blood vessels in vivo. Thus, we propose the existence of a "vascular natriuretic peptide system (NPS)" similar to the vascular renin angiotensin system (RAS). The present study showed that CNP exerted a growth inhibitory action and antagonised the growth-promoting action of AII, which was mediated through the AII subtype 1 receptor in cultured vascular smooth muscle cells. In neointimal lesions of rat carotid artery, CNP gene transcript was detectable 2 weeks after balloon injury, and ANP-B receptor gene expression was augmented. These findings suggest that the vascular NPS is activated in proliferative vascular lesions, suppressing further proliferation by antagonising the action of the vascular RAS. PMID- 7534180 TI - Effects of RP 67580, a tachykinin NK1 receptor antagonist, on a primary afferent evoked response of ventral roots in the neonatal rat spinal cord. AB - 1. The pharmacological characteristics of RP 67580, a non-peptide tachykinin NK1 receptor antagonist, and its effects on a reflex response evoked by stimulation of primary afferent fibres, were examined in isolated neonatal spinal cord preparations of the rat. Potentials were recorded extracellularly from a lumbar ventral root and drugs were bath-applied in normal artificial cerebrospinal fluid (CSF) or in the presence of tetrodotoxin (TTX). 2. In normal artificial CSF, RP 67580 (0.1-0.3 microM) caused rightward shifts of the concentration-response curves for substance P (SP), neurokinin A (NKA) and substance P methyl ester (SPOMe), an NK1-selective agonist, with pA2 values of 7.25, 7.47 and 7.49, respectively. 3. In the presence of TTX (0.3 microM), RP 67580 also caused rightward shifts of the concentration-response curves for SPOMe and NKA. The pA2 value of RP 67580 against SPOMe (6.75) was significantly lower than that against NKA (7.22). RP 67580 (0.3-1 microM) did not cause a clear parallel shift of the concentration-response curves for SP, and it depressed the depolarizations induced by low concentrations of SP, but slightly potentiated those induced by high concentrations of SP. 4. RP 67580 (1 microM) did not depress the depolarizing responses to bombesin, L--glutamate, gamma-aminobutyric acid (GABA), thyrotropin-releasing hormone and muscarine. RP 67580 (1 microM), however, depressed the response to acetylcholine in the presence of atropine and the response to nicotine. RP 68651 (1 microM), the enantiomer of RP 67580 devoid of activity at tachykinin NK1 receptors, also depressed the response to acetylcholine in the presence of atropine. 5. RP 67580 (1 gAM) did not induce GABA release from the rat spinal cord.6. In the neonatal gerbil spinal cord, the antagonist effects of RP 67580 (0.3-1 JAM) against SPOMe were much less potent than in the neonatal rat spinal cord.7. In the rat spinal cord-saphenous nerve preparation, electrical stimulation of the saphenous nerve atC-fibre strength evoked a prolonged depolarization of the ipsilateral L3 ventral root (slow VRP).RP 67580 (0.1-1 JM) depressed the saphenous nerve-evoked slow VRP. In contrast, RP 68651 (0.3 JAM)had no effect on the slow VRP.8. The results of the present study indicate that RP67580 acts as a high affinity NK, receptor antagonist in the neonatal rat spinal cord, although it also possesses an antinicotinic action. This study further suggests the existence of a subpopulation of tachykinin NK, receptors that are activated by NKA and SPOMe, as well as by low concentrations of SP, and are sensitive to the antagonist action of RP 67580 in the neonatal rat spinal cord. This study also provides further evidence for the involvement of SP and NKA in the slow VRP evoked by C-fibre stimulation in the neonatal rat spinal cord. PMID- 7534181 TI - Attenuation of the antiarrhythmic effects of ischaemic preconditioning by blockade of bradykinin B2 receptors. AB - 1. The possibility that bradykinin is involved in the pronounced antiarrhythmic effects of ischaemic preconditioning in anaesthetized mongrel dogs was examined with the use of the selective B2 antagonist, icatibant (Hoe-140). 2. Preconditioning, achieved by two 5 min occlusions of the left anterior descending coronary artery, followed 20 min later by a 25 min occlusion of the same artery resulted, during this prolonged occlusion, in less severe arrhythmias (VF 0% versus 47% in control non-preconditioned dogs), reductions in the incidence and number of episodes of ventricular tachycardia (VT) and in the number of ventricular premature beats and increased survival following reperfusion (50% versus 0% in the controls). 3. Hoe-140 was given in a dose of 300 micrograms kg-1 either before the preconditioning procedure or after preconditioning but before the prolonged occlusion. This dose of Hoe-140 had insignificant haemodynamic effects and failed to modify the increase in coronary blood flow that occurred in the circumflex coronary artery when the anterior descending branch was occluded. 4. It was difficult to precondition dogs in the presence of Hoe-140. There were more ventricular arrhythmias during the initial 5 min occlusion (44 +/- 12 versus 10 +/- 3) and a higher incidence of ventricular fibrillation (50% versus 21%) during the preconditioning procedure. There was also a more pronounced ST elevation (recorded from epicardial electrodes) during the preconditioning occlusions in those dogs given Hoe-140. 5. In those dogs that survived to the long (25 min) occlusion, Hoe-140 prevented the antiarrhythmic effects of preconditioning (reduction in ventricular premature beats and in VT) although all the dogs survived the occlusion period. However on reperfusion, survival in the preconditioned dogs given Hoe-140 was less than in those dogs preconditioned without the B2 antagonist.6. Changes in the degree of inhomogeneity of conduction within the ischaemic area, which were markedly suppressed by preconditioning, were attenuated in those dogs preconditioned in the presence of Hoe-140.7. These results suggest that bradykinin acts as both a 'trigger' for preconditioning and as one of the mediator protective (antiarrhythmic) substances generated by the myocardium under these conditions.Since the protection afforded both by preconditioning and by local intracoronary infusions of bradykinin is markedly attenuated by an inhibitor of the L-arginine nitric oxide pathway, we suggest that much of the protection afforded by ischaemic preconditioning results from the generation of nitricoxide, and that bradykinin, released early during ischaemia, acts as a stimulant for this generation. PMID- 7534182 TI - Nitric oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced relaxation of the guinea pig stomach. AB - 1. Nitric oxide synthase (NOS) was localized in the guinea pig stomach by immunocytochemistry. In vitro experiments were carried out on the isolated stomach of the guinea pig to study any possible links between nitric oxide (NO) and vasoactive intestinal peptide (VIP) in mediating relaxations induced by vagal stimulation. 2. NOS was localized to nerve cell bodies and nerve fibre varicosities of the myenteric plexus in wholemounts of the longitudinal muscle myenteric plexus of the stomach fundus. The NOS-positive cells had a Dogiel type I morphology characteristic of motor neurones. 3. The cross-sections of the stomach wall showed NOS-positive neurones mainly in the myenteric plexus ganglia and NOS-positive nerve fibre varicosities in the circular muscle layer. 4. Relaxations induced by vagal stimulation were almost completely prevented by L NAME with an IC50 value of 5.5 x 10(-6) M. This inhibition was reversed by L arginine (2 mM). 5. VIP (100 nM) induced reproducible relaxations of the stomach. These were unaffected by tetrodotoxin (2 microM) or N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. Desensitization to the relaxant effect of VIP partially reduced relaxations induced by vagal stimulation, glyceryl trinitrate or sodium nitroprusside but not noradrenaline. 7. These results show that NO has a neuronal origin in the guinea pig stomach, and support NO, and not VIP, as the major neurotransmitter of vagally induced gastric relaxation in the guinea pig. PMID- 7534183 TI - The relative importance of nitric oxide and nitric oxide-independent mechanisms in acetylcholine-evoked dilatation of the rat mesenteric bed. AB - 1. The relative contribution of nitric oxide (NO) to acetylcholine-induced smooth muscle relaxation was investigated in the rat perfused mesenteric vasculature and in isolated segments of second, third and fourth order arterial branches. 2. The EC50 values and maximal relaxation to acetylcholine were not significantly different in the sequential arterial branches, being approximately 0.05 microM and 85%, respectively. 3. The NO synthase inhibitor L-NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) reduced acetylcholine-evoked endothelium-dependent dilatation and relaxation in the perfused mesenteric bed and in isolated arterial segments. The maximum response to acetylcholine in both preparations was reduced by between 35% to 40% while the EC50 values were increased by 5-6 fold. L-NAME had no effect on basal smooth muscle tone in either case. 4. In contrast, endothelium-dependent dilatation of the perfused mesenteric bed evoked by A23187 (0.002-20 nmol), was unaffected by exposure to L-NAME. The EC50 values and maximal responses elicited by A23187 (20 nmol) before and after exposure to L NAME were 0.96 +/- 0.5 nmol and 67.0 +/- 7.0% (n = 4), and 0.7 +/- 0.4 nmol and 70.0 +/- 5.0% (n = 4; P > 0.01), respectively. 5. Perfusion of the isolated mesenteric bed with raised K(+)-Krebs buffer (25 mM) had no effect on basal tone, but reduced the amplitude of both acetylcholine- and A23187-evoked dilatation. The maximum responses to acetylcholine (2 micromol) and A23187 (20 nmol) were reduced from 67.5 +/- 7.3% and 65.4+/-8.2% to 18.9 +/-11.0% (n=5; P<0.01) and 13.5 +/-12.0% (n=4; P<0.01), respectively.6. Exposure of the mesenteric bed to L NAME in the presence of raised K+-Krebs further reduced the maximal response elicited by acetylcholine to only 8.9 +/- 2.8% (n =4; P< 0.01).7. These results indicate that acetylcholine-evoked vasodilatation of the rat mesenteric vasculature is mediated by both NO-dependent and -independent mechanisms. The relative contribution made by these mechanisms does not appear to differ in sequential branches of the mesenteric artery. In contrast,A23187-evoked vasodilatation appears to be mediated predominantly by a NO-independent mechanism which is sensitive to increases in the extracellular potassium concentration and may reflect the action of endothelium-derived hyperpolarizing factor (EDHF). PMID- 7534184 TI - Intracerebral microdialysis combined with recording of extracellular field potential: a novel method for investigation of depolarizing drugs in vivo. AB - 1. The purpose of this study was to examine whether depolarizations evoked by excitatory amino acids can be recorded quantitatively, in vivo, with a microelectrode incorporated within a microdialysis probe. 2. Microdialysis probes incorporating a chlorided silver wire were implanted in the striatum of anaesthetized rats and perfused with artificial cerebrospinal fluid (ACSF). Increasing concentrations of excitatory amino acids were applied for 2 min via the microdialysis probe, and the extracellular direct current (d.c.) potential was recorded between the microdialysis electrode and a reference electrode placed under the scalp. 3. N-methyl-D-aspartate (NMDA, 25-500 microM), alpha-amino-3 hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 5-1000 microM), kainate (5-500 microM), and glutamate (0.25-100 mM) evoked concentration-dependent depolarizations with maxima ranging from 7 to 10 mV, i.e. 3 to 10 times larger than those recorded from brain slices in vitro. Depolarizations evoked by glutamate receptor agonists applied by microdialysis shared several features with those recorded from brain slices. The most characteristic were: steep onset and recovery of NMDA and glutamate responses; marked post-depolarization hyperpolarization with NMDA; and very slow recovery after kainate application. At high concentrations (500 microM), NMDA occasionally initiated spreading depression. The relative potency of glutamate and NMDA was of the same order of magnitude to that obtained with the cortical wedge and hippocampal slices, glutamate being 100 to 400 times less potent than NMDA. 4. Two consecutive series of NMDA-stimuli within the same procedure evoked comparable depolarizations, indicating that reliable quantitative analysis of drug action can be performed, with each animal serving as its own control. This is relevant to the study of drugs acting on glutamate receptors especially antagonists. The remarkable inter animal reproducibility is also a valuable feature.5. Pretreatment with dizocilpine maleate (MK-801, 2mgkg'1, i.p.) reduced by 65% the responses evoked by NMDA (500 fM). The non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX,100 1M) applied via the microdialysis probe reduced by around 78% the responses to AMPA and kainate (250 micro M). The fact that drugs, especially antagonists, can be administered either systemically, or directly through the dialysis probe to by-pass the blood-brain barrier or avoid peripheral effects, is especially relevant for neuropharmacological studies.6. Intracerebral microdialysis combined with in vivo recording of extracellular field potential is a novel and valuable method for the quantitative analysis of the action of drugs acting on glutamate receptors.This method should prove especially useful for comparing the sensitivity of specific brain structures to selective glutamate receptor agonists under normal conditions and when the neuronal micro environment is altered. It should also be useful for investigating the action of other depolarizing agents, such as veratridine, and their antagonists. PMID- 7534186 TI - Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung. AB - 1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi. 6. Similar distributions, densities and characteristics of [I251]-BH-SP binding sites were demonstrated in oval bumin-sensitized and challenged guinea-pig lung and in naive animals.7. Differences in the distribution and characteristics of NKI binding sites labelled with [125I]-BH-SP between guinea pig and human lung suggest limitations in the use of guinea pig models for studying roles of tachykinins in pulmonary disease. However, the similar microvascular distributions of NK,binding sites in human and guinea pig lung suggest that the selective tachykinin receptor antagonistsFK888 and FK224 may be useful in the management of airway inflammation in man. PMID- 7534185 TI - [125I]-PD151242: a selective ligand for endothelin ETA receptors in human kidney which localizes to renal vasculature. AB - 1. The linear tetrapeptide radioligand, [125I]-PD151242 was used to characterize ETA receptors in human kidney which is an ETB-rich tissue. Saturation binding assays with [125I]-PD151242 revealed a single population of high affinity endothelin receptors: KD = 0.75 +/- 0.07 nM and Bmax = 48.4 +/- 1.6 fmol mg-1 protein (n = 3 individuals +/- s.e.mean). Hill slopes were close to unity and a one site fit was preferred to a two site model. 2. ETA-receptor-selective ligands competed for [125I]-PD151242 binding with sub-nanomolar affinity: BQ123 KD = 0.43 +/- 0.10 nM, Bmax = 46.6 +/- 7.9 fmol mg-1 protein; FR139317, KD = 0.37 +/- 0.06 nM, Bmax = 39.5 +/- 6.5 fmol mg-1 protein (n = 3 individuals +/- s.e.mean). In each case, monophasic inhibition curves were obtained and a one site fit was preferred to a two site model. The ETB-selective agonist, BQ3020 at the highest concentration tested (10 microM) inhibited binding by only 50%. The non-selective RO462005 competed for the binding of [125I]-PD151242: KD = 1.31 +/- 1.38 microM, Bmax = 33.0 +/- 9.7 fmol mg-1 protein. Endothelin-2 and sarafotoxin S6B inhibited [125I]-PD151242 binding to renal tissue whereas ET-3 and sarafotoxin S6C were less effective. Non-endothelin and non-sarafotoxin peptides did not compete. 3. No degradation of [125I]-PD151242 was detected following incubation of the ligand with renal tissue under the conditions of the binding assay. 4. Polymerase chain reaction products corresponding to the expected size for mRNA encoding ETA and ETB receptor sub-types were detected in cortex and medulla in each of the five individuals examined.5. Autoradiographical studies showed that ETA receptors visualised with ['25I]-PD151242 were mainly localized to blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins. ETB receptors localized with ['251]-BQ3020 were concentrated in the medulla and the density of binding to vessels was low.6. These data suggest [251I]-PDl51242 is selective for ETA receptors in human kidney and this sub-type is mainly localized to the renal vasculature. The results provide further evidence that the human vasculature mainly expresses the ETA receptor. PMID- 7534188 TI - Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids. AB - 1. These studies examine the effect of retinoids on interleukin 1 beta (IL-1 beta)-induced nitric oxide synthase (NOS) activity in cultured rat aortic vascular smooth muscle (VSM) cells and isolated rat aortic rings. 2. All-trans retinoic acid (all-trans-RA, 0.1-10 microM) and its active analogues produced concentration-dependent inhibition of IL-1 beta (0.1-10 ng ml-1)-induced nitrite production in cultured VSM cells. In contrast, the inactive retinoid, Ro 14-6113 (0.1-10 microM), had no effect on IL-1 beta-induced nitrite production. 3. Since some of the actions of retinoids are mediated by induction of transforming growth factor beta (TGF-beta), its effect on inducible NOS activity in VSM cells was examined. TGF-beta produced concentration-dependent (0.1-10 ng ml-1) inhibition of IL-1 beta-induced nitrite production and the maximum effect (approximately 90% inhibition) was significantly greater than that seen with all-trans-RA (approximately 70% with 10 microM). However, an anti-TGF-beta antibody (50 micrograms ml-1) which blocked the effect of exogenous TGF-beta (5 ng ml-1) did not significantly reverse the inhibitory action of all-trans-RA (10 microM). 4. In addition to inhibiting IL-1 beta-induced nitrite production, all-trans-RA (10 microM) reduced substantially inducible NOS mRNA and protein levels in IL-1 beta induced VSM cells (P < 0.01). 5. Incubation of isolated rat aortic rings with IL 1 beta (10 ng ml-1) caused a progressive resistance of the rings to the vasoconstrictor action of phenylephrine (10 nM to 10 microM). This effect was abolished by the addition of the nitric oxide synthase inhibitor L-NG-monomethyl L-arginine (L-NMMA, 1 mM). All trans-RA (10 micro M) also markedly and significantly reversed this IL-1p-induced vascular hyporeactivity(P<0.01).6. These data show that all-trans-RA and other active retinoids are able to block cytokine-stimulated expression of inducible NOS in cultured VSM cells and isolated aortic rings. PMID- 7534187 TI - Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus. AB - 1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+) dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline 2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro 5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H] NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy 5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX. In contrast, the increase in [Ca2+]i due to stimulation by glutamate was not sensitive to CNQX or MK-801.5. Nitrendipine, at I JAM, did not inhibit the neurotransmitter release induced by AMPA, but, both 0.5 micro M conotoxin GVIA (w-CgTx) and 100 nM w-Aga IVA reduced catecholamine release to 49.03 +/- 3.79% and 46.06 +/- 10.51% of the control, respectively. In the presence of both toxins the release was reduced to 12.58 +/- 4.64% of the control.6. The results indicate that activation of presynaptic glutamate receptors of the NMDA and non-NMDA type induces the release of [3H]-dopamine and [H]-NA from rat hippocampal synaptosomes and that the release induced by AMPA involves the activation of N- and P-type Ca2" channels which allow the influx of Ca2" that triggers the 3H catecholamines release. PMID- 7534190 TI - Ion channel modulation by NS 1619, the putative BKCa channel opener, in vascular smooth muscle. AB - 1. The effects of NS 1619, the putative BKCa channel opener, were investigated on rat intact portal veins and on single smooth muscle cells enzymatically separated from the same tissue. 2. Under whole-cell patch clamp conditions with K-rich pipettes, exposure of single cells held at -10 mV to NS 1619 (10-33 microM) induced a noisy, outward current which reached a maximum (33 microM NS 1619; mean 35.8 +/- 17 pA, n = 8) within about 6 min. 3. On stepping to test potentials (range -50 to +50 mV) from a holding potential of -10 mV, the NS 1619-induced noisy current exhibited time-dependent activation and marked outward rectification. 4. The stimulation of outward currents by NS 1619 at -10 mV was independent of the presence of Ca2+ in the bath or pipette solutions but was antagonized by either charybdotoxin (250 nM) or penitrem A (100 nM) in the bath solution. 5. Stationary fluctuation analysis of the noisy current induced by NS 1619 at -10 mV yielded a value of 70 +/- 8 pS (n = 4) (under the quasi physiological conditions of the experiment) for the unitary conductance of the channel involved. 6. At -10 mV, NS 1619 (10-33 microM) rapidly inhibited spontaneous transient outward currents. 7. With a holding potential of -90 mV, NS 1619 (10-33 microM) produced a reduction of outward currents evoked by depolarizing steps to +50 mV, an effect associated with marked inhibition of the delayed rectifier current, IK(V). 8. NS 1619 (3-100 microM) produced a concentration-dependent inhibition of spontaneous activity in rat portal vein characterized by a reduction in the amplitude and duration of the tension waves. This inhibition was slightly potentiated in the presence of either charybdotoxin (250 nM) or penitrem A (1 microM). NS 1619 also totally inhibited contractions of rat aorta induced by KCl (both 20 mM and 80 mM). 9. Under whole-cell recording conditions and using Cs-rich pipettes, Ca-currents evoked in portal vein cells by stepping from a holding potential of - 90 mV to test potentials in the range - 30 to + 50 mV were totally inhibited in the presence of 33 JAM NS 1619.10. NS 1619 (33 JAM) inhibited the induction of IK(ATP) by levcromakalim (10 JAM).11. It is concluded that NS 1619 activates the large conductance, Ca2+-sensitive channel, BKca and over the same concentration range it inhibits both KV and L-type Ca channels. The observed NS 1619-induced mechanical inhibition in rat portal vein and aorta seems most likely to be due to the observed inhibition of Ca-currents. PMID- 7534192 TI - Is there a role for non-nucleoside reverse transcriptase inhibitors in the treatment of HIV infection? AB - Because the efficacy of currently approved antiretroviral agents used as prolonged monotherapy is limited, there is an urgent need for alternative agents for the treatment of HIV infection. We review the development of a diverse group of new compounds, the non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are potent and specific inhibitors of HIV replication. Early clinical experience with the NNRTIs has demonstrated antiviral activity and a high therapeutic index, but some patients rapidly develop viral strains resistant to these drugs. The contributions of NNRTI studies to the basic science of HIV pathogenesis and the potential clinical role of these compounds, particularly in combination antiretroviral regimens, are discussed. PMID- 7534189 TI - Involvement of tyrosine kinase in the induction of cyclo-oxygenase and nitric oxide synthase by endotoxin in cultured cells. AB - 1. Cyclo-oxygenase (COX) and nitric oxide synthase (NOS) are two enzymes which have distinct cytokine-inducible isoforms (COX-2 and iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used the tyrosine kinase inhibitors, erbstatin and genistein, to investigate the potential role of tyrosine kinase activation in the induction on COX-2 and iNOS caused by endotoxin (lipopolysaccharide; LPS) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages. 2. The main COX metabolites, 6-oxo-prostaglandin F1 alpha (6 oxo-PGF1 alpha) (for BAEC) and PGF2 alpha (for 774.2 macrophages) were measured by radioimmunossay: (i) accumulation of COX metabolites from endogenous arachidonic acid was measured at 24 h after addition of LPS (1 microgram ml-1); (ii) in experiments designed to measure 'COX activity', COX metabolites generated by BAEC or J774.2 macrophages activated with LPS were assayed (at 12 h after LPS administration) after incubation of the washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis with a specific antibody to COX-2 was used to determine the expression of COX-2 protein caused by LPS in cell extracts. Accumulation of nitrite (measured by the Griess reaction) was used as an indicator of NO formation and, hence, iNOS activity. 3. Erbstatin (0.05 to 5 micrograms ml-1) or genistein (0.5 to 50 micrograms ml-1) caused a dose-dependent inhibition of the accumulation of COX metabolites in the supernatant of BAEC or J774.2 macrophages activated with LPS. Erbstatin or genistein also caused a dose dependent inhibition of 'COX activity' in both cell types. Western blot analysis showed that erbstatin (5 ig ml1') or genistein (50gg ml-') inhibited the expression of COX-2 protein in BAEC and J774.2 macrophages activated with LPS (lLgml-' for 24 h).4. Erbstatin or genistein also caused a dose-dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 sg ml-' for 24 h). In contrast to J774.2 macrophages, BAECstimulated with LPS (1 pg ml-' for 24 h) did not produce detectable amounts (<1PiM) of nitrite.5. These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates (i) the induction of COX-2 and iNOS elicited by LPS in J774.2 macrophages, and (ii) the induction of COX-2 by LPS in BAEC. PMID- 7534193 TI - Expression of a multifunctional DNA repair enzyme gene, apurinic/apyrimidinic endonuclease (APE; Ref-1) in the suprachiasmatic, supraoptic and paraventricular nuclei. AB - Apurinic/apyrimidinic endonuclease (APE; also referred to as Ref-1) repairs oxidative damage to DNA and regulates the redox state of DNA binding proteins. This later property influences the ability of DNA binding proteins, which include Fos and Jun, to bind to AP-1 complexes. Since DNA binding proteins may play important roles in regulating neuronal activity in the hypothalamus, we examined the expression of APE in the hypothalami of rats. In situ hybridization studies revealed high levels of APE mRNA expression in the suprachiasmatic nuclei (SCN), supraoptic nuclei (SON) and paraventricular nuclei (PVN). Since the SCN are the site of a biological clock, we examined whether APE gene expression was regulated by the circadian cycle or by light. Quantitative in situ hybridization studies showed that APE mRNA levels remained constant over the circadian cycle and were not increased by light exposure at night. We also tested if APE expression was under osmotic control in the SON and PVN. Hypertonic stimulus, however, did not induce further expression of APE mRNA in either the SON or the PVN. These findings identify the SCN, SON and PVN as sites of high level APE gene expression. These data suggest that APE may play an important role in these structures either to facilitate DNA repair or DNA binding protein action. PMID- 7534191 TI - Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of the GABAB receptor system. AB - 1. Monoarthritis was induced in Lewis rats by interdermal injection in the left hind paw of a suspension of Mycobacterium tubercolusis in mineral oil (500 micrograms 100 microliters-1). Controls were injected with 100 microliters mineral oil. 2. Withdrawal latencies to thermal stimuli of the inflamed paw, the contralateral and both paws of control rats were measured at daily intervals after injection by the plantar test. 3. After detection of the pain threshold, rat spinal cords were removed and horizontal dorsal slices were mounted in a 3 compartment bath to measure electrically-evoked release of substance P-like immunoreactivity (SP-LI). 4. The inflamed paw of monoarthritic rats exhibited a lower pain threshold to thermal stimuli than the contralateral paw of the same animals and both paws of control rats. Inflamed paw hyperalgesia was maximal two days after injection, and declined gradually between 7 to 21 days with no evidence of excitability of withdrawal reflexes after 28 days. 5. During the 28 days study, monoarthritic rats gained less weight than control rats. 6. Electrical stimulation of the dorsal roots attached to rat isolated spinal cord slices induced a significant increase (174 +/- 18% of basal outflow which was 30.3 fmol 8 ml-1, n = 5) in SP-LI release. 7. One-week after induction of inflammation no differences in the amount of SP-LI released from the spinal cord of incomplete Freund's adjuvant-treated rats (IFA) and Freund's adjuvant-treated rats (CFA) were detected. Two weeks after, CFA spinal cord tended to release more SP-LI than IFA cords and, 21 days after injection, the spinal cord of CFA rats released significantly more peptide than IFA rats (17.8 +/- 2.8 fmol ml-1, n = 12 and 6.9 +/- 3.2 fmol ml-1, n = 9, respectively).8. Twenty-one days after treatment, the evoked release from monoarthritic rat spinal cords was increased by 263 + 42% (n = 3) in the presence of the GABAB receptor antagonist, CGP 36742 (100 micro M)which also significantly potentiated monoarthritis-induced hyperalgesia up to 45 min after injection(100 mgkg-1, i.p.).9. These findings may provide a basis for a novel approach to chronic pain therapy but also an explanation for the lack of analgesia produced by the GABAB agonist, baclofen, in chronic as compared to acute pain. PMID- 7534194 TI - Effect of NO synthase inhibition on behavioral changes induced by a single administration of methamphetamine. AB - The present study examined the effects of nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME; 10, 30, 60 mg/kg, i.p.) on behavioral changes induced by a single administration of methamphetamine (MA) (3.22 and 0.805 mg free base/kg, s.c.). MA increased locomotion-stereotypy rating scores and motor activity counts measured by an infrared sensor. L-NAME administered prior to MA significantly decreased the level of locomotion stereotypy rating and motor activity induced by MA. An inactive optical isomer, N omega-nitro-D-arginine methyl ester (D-NAME) had no effects on MA-induced behavioral changes. The results suggest that NO synthesis is involved in the full expression of behavioral effects of MA. PMID- 7534195 TI - A role for non-NMDA excitatory amino acid receptors in regulating the basal activity of rat globus pallidus neurons and their activation by the subthalamic nucleus. AB - We have investigated the hypothesis that excitatory amino acid (EAA) receptors in the globus pallidus (GP) play a significant role in maintaining the firing rates of GP neurons under basal conditions and following activation of the subthalamic nucleus (STN). Drugs were infused directly into the GP and/or STN while the extracellular single unit activity of Type II GP neurons was recorded in ketamine anesthetized rats. Local infusions of the EAA agonists NMDA (30-300 pmol/200 nl) or AMPA (0.1-1 pmol/200 nl) elicited increases in the firing rate of GP neurons in a dose-dependent fashion. Infusion of the GABAA receptor antagonist bicuculline methiodide (1-10 pmol/100 nl) into the STN also elicited dose-related increases in the firing rate of GP neurons. Intrapallidal infusion of the non NMDA (AMPA/kainate) receptor antagonist NBQX (0.1-1.0 nmol) reduced the basal firing rate of GP neurons by 40%. In contrast, the NMDA antagonist MK-801 (0.01 0.1 nmol) produced no significant effect on basal firing rate. Intrapallidal infusion of the non-selective EAA receptor antagonist kynurenic acid or NBQX reversed or blocked the increase in firing rate of GP neurons following bicuculline-induced activation of the STN. Similar treatment with MK-801, however, had no significant effect on this response. These results indicate that tonic stimulation of non-NMDA receptors plays an important role in maintaining the basal activity of GP neurons and in mediating the effects of increased excitatory input from subthalamic afferent neurons. PMID- 7534196 TI - Musculotopic organization in the motor trigeminal nucleus of the reeler mutant mouse. AB - We examined the musculotopic organization in the motor trigeminal nucleus and the somatotopical arrangement in the trigeminal ganglion of the normal and reeler mice. To determine whether or not masticatory motoneurons are malpositioned in the reeler mouse, we injected horseradish peroxidase (HRP) into the masticatory muscles of normal and reeler mice. Injections of HRP into the jaw-closing muscles, i.e., the masseter and temporalis muscles, labeled large multipolar neurons in the dorsolateral division of the motor trigeminal nucleus of both normal and reeler mice. Similar injections into the jaw-opening muscles, i.e., the anterior belly of the digastric muscle and mylohyoid muscle, labeled large multipolar neurons in the ventromedial division of the motor trigeminal nucleus of both mouse strains. Thus, the normal myotopical arrangement of the masticatory muscles on the motor trigeminal nucleus is preserved in the reeler mouse. However, detailed analysis revealed that jaw-opening motoneurons were more widely scattered in the reeler mouse than in the normal control. To examine the somatotopical arrangement of the first-order sensory neurons in the trigeminal ganglion of the normal and reeler mice, we subcutaneously injected Fast blue (FB) into the mental region and Diamidino yellow (DY) into the vibrissal region of the same animals. No differences in distribution patterns of FB-labeled and DY labeled neurons in the whole-mounted trigeminal ganglion could been seen between these two strains, suggesting that migration of trigeminal ganglion cells, which are derived from the neural crest and placode, is not deranged by the reeler genetic locus. PMID- 7534197 TI - Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin. AB - Neutrophil tethering and rolling in shear flow are mediated by selectins and have been thought to be two indistinguishable manifestations of a single molecular interaction between selectin and ligand. However, we report that under physiologic flow conditions, tethering to E-selectin requires a ligand distinct from the one that supports neutrophil rolling. Tethering under shear to E selectin requires a carbohydrate ligand that is closely associated with the lectin domain of L-selectin on the neutrophil surface, as enzymatic removal of L selectin, chemotactic factor-induced shedding of L-selectin, and L-selectin MAbs effectively block tethering. In contrast, this ligand is dispensable for the ability to roll on E-selectin, since rolling adhesions formed after static incubations were not affected by the presence or absence of L-selectin. Thus, E selectin interactions with ligands on neutrophils persist after L-selectin shedding. These findings add an additional step for regulation of leukocyte localization in inflammatory sites. PMID- 7534198 TI - In vitro induction of T cell anergy by blocking B7 and early T cell costimulatory molecule ETC-1/B7-2. AB - APC-associated B7 and ETC-1/B7-2 are two major costimulatory molecules for full activation of T lymphocytes during auto- and allogeneic immune responses. In this report, we further examine the role of the two molecules in murine CD4+ T cell activation and anergy development. As suggested in antibody blocking studies, optimal activation of CD4+ T cells in response to anti-CD3 stimulation requires collaborative signaling through the two molecules. Simultaneous blockade of B7 and ETC-1/B7-2 renders CD4+ T cells unresponsive to anti-CD3 restimulation. PCR analysis and cytokine reconstitution studies show that the observed unresponsiveness is correlated to a significant reduction of Th1-type cytokine production, suggesting B7 and ETC-1/B7-2-mediated costimulatory signaling may be specifically active in regulation of the function of the Th1 subset. PMID- 7534199 TI - The role of the T cell costimulator B7-1 in autoimmunity and the induction and maintenance of tolerance to peripheral antigen. AB - T cell tolerance to peripheral antigens is believed to result mainly from the inability of parenchymal cells to present antigens in an immunogenic form due to the lack of expression of T cell costimulator. We found, however, that transgenic expression of the T cell costimulator B7-1 on the islets of Langerhans is not sufficient to abolish the in vivo tolerance to islets antigen. Here, we present evidence indicating that the level of major histocompatibility complex (MHC) antigen expressed by islet cells plays a critical role. Mice coexpressing the B7 1 transgene and high levels of the class II MHC antigen I-E on the islets develop an autoimmune destruction of the beta cells of the pancreas. By contrast, expression of the I-E molecule by islets, in the absence of T cell costimulator, leads to specific tolerance of these autoreactive T cells that cannot be reversed by costimulation with B7-1. PMID- 7534200 TI - T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice. AB - The identification is made in normal mice of the stages in T cell development at which the rearranged beta chain of the T cell receptor (TCR) is utilized to promote T cell maturation, independent of the TCR alpha chain. In addition, evidence is provided that utilization of beta chains in T cell progenitors does not preclude differentiation to TCR gamma delta + T cells. This is consistent with the view that an initial consequence of beta chain expression by early thymocytes is clonal expansion, increasing the size of the pool of useful precursors. This allows the proposal to be made that allelic exclusion may be a byproduct of cell cycle regulation during early thymocyte differentiation, which may in turn explain why the efficiency of allelic exclusion varies at different TCR or immunoglobulin loci. PMID- 7534201 TI - A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development. AB - RAG-1 and RAG-2 are developmentally regulated genes that are essential for the assembly of antigen receptors in lymphoid cells. Here we describe transgenic mice that carry RAG-1 and RAG-2 under the control of the proximal lck promoter. Persistent expression of RAG-1 and RAG-2 was associated with incomplete thymopoiesis and profoundly compromised cellular immunity. In addition, RAG transgenic mice rapidly developed lymphadenopathy, splenomegaly, and lymphocytic perivascular infiltrates. These effects required both RAG-1 and RAG-2, since mice that carried either gene exclusively were indistinguishable from wild-type controls. We propose that in addition to a previously documented role in V(D)J recombination, RAG-1 and RAG-2 expression must be properly regulated for completion of normal T cell development PMID- 7534202 TI - The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. AB - An engagement of CD40 with CD40 ligand (CD40L) expressed on activated T cells is known to provide an essential costimulatory signal to B cells in vitro. To investigate the role of CD40 in in vivo immune responses, CD40-deficient mice were generated by gene targeting. The significant reduction of CD23 expression on mature B cells and relatively decreased number of IgM bright and IgD dull B cells were observed in the mutant mice. The mutant mice mounted IgM responses but no IgG, IgA, and IgE responses to thymus-dependent (TD) antigens. However, IgG as well as IgM responses to thymus-independent (TI) antigens were normal. Furthermore, the germinal center formation was defective in the mutant mice. These results suggest that CD40 is essential for T cell-dependent immunoglobulin class switching and germinal center formation, but not for in vivo T cell dependent IgM responses and T cell-independent antibody responses. PMID- 7534203 TI - Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin deficient mice. AB - L-selectin, a cell adhesion molecule expressed by leukocytes, mediates the attachment of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes and mediates the earliest interactions between leukocytes and activated vascular endothelium. Mice possessing a mutant L-selectin gene that results in the complete loss of cell surface receptor expression were generated by gene targeting. Lymphocytes from these mice did not bind to peripheral lymph node HEV and these mice had a severe reduction in the number of lymphocytes localized to peripheral lymph nodes. Short-term homing experiments demonstrated that L selectin was also involved in lymphocyte migration to mucosal lymph nodes, Peyer's patches, and spleen. Furthermore, significant defects in leukocyte rolling and neutrophil migration into the peritoneum in response to an inflammatory stimulus were observed. Thus, L-selectin plays an essential role in leukocyte homing to lymphoid tissues and sites of inflammation. PMID- 7534204 TI - The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection. AB - We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo. PMID- 7534205 TI - Role of antibiotic and non-antibiotic therapy in the treatment of sinusitis with an allergic basis. PMID- 7534206 TI - Genomic relatedness among reference strains of different Streptococcus suis serotypes. AB - Hybridization studies using genomic DNA and a rDNA probe revealed genetic relatedness among reference strains of different Streptococcus suis serotypes. Although most serotype 22 isolates are biochemically atypical, the reference strain of capsular type 22 is genetically related to other S. suis serotypes, but not to Streptococcus pneumoniae. Using DNA digested with BamHI and BglII for ribotyping, some S. suis reference strains had common patterns, but this analysis mainly revealed variations in patterns of S. suis strains of different serotypes. PMID- 7534207 TI - Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping. AB - This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds. PMID- 7534209 TI - Influence of divalent cations on androgen and estrogen binding in human prostatic tissue. AB - The influence of divalent cations on estrogen and androgen binding in human benign prostatic hypertrophy (BPH) was investigated. Two tissue types were detected. In type I, important estradiol binding increase in the presence of Mg++ and good evidence of the androgen receptors were noticed. Type II tissues were characterized by weak or inhibitory effect of Mg++ and, poor detection of the androgen receptors in some of the buffers used. Zn++ inhibited estradiol binding. Effects of chelators leaving some endogenous divalent cations in the medium were investigated. For both binding activities no difference could be established between type I and type II tissues. One exception was a somewhat increased androgen binding for type II when endogenous Mg++ was present. Strong inhibition of estradiol binding in the presence of EDTA + Mg++ always occurred. Influence of divalent cations on steroid binding measurements in BPH tissues is discussed. PMID- 7534208 TI - Inhibition of tumor cell growth by low-boiling-point-saturated fatty acids isolated by molecular distillation and reversed phase liquid chromatography of hydrolysates of uncytotoxic wool grease secreted from sheep sebaceous gland. AB - We showed that massive growth of mouse Ehrlich ascites carcinoma (EAC) cells was not inhibited by wool grease secreted from the sheep sebaceous gland, whereas wool fatty acids separated by saponification of wool grease was growth inhibitory. We then fractionated wool fatty acids into 9 fractions using molecular distillation (80-200 degrees C; 1 x 10(-2) mmHg) and found a marked antitumor activity in a low-boiling-point (< 80 degrees C) fraction (MW 200-300; C10-C20), which was further separated by reversed phase liquid chromatography on an octadecylisilica gel column, resulting in 5 fractions. The second most hydrophobic fraction (C8Si-4) obtained was the most growth-inhibitory to EAC cells cultured or implanted into mice, more marked than the antitumor glycopeptide bleomycin. C8Si-4 was suggested to be a mixture of a normal-chain C16-saturated fatty acid and two branched-chain kinds of saturated C16-iso- and C19-anteiso-fatty acids without hydroxyl groups according to gas chromatography mass spectrographic analysis. Thus low-boiling-point saturated fatty acid moieties in some wool grease molecules were shown to become growth-inhibitory in vitro and in vivo only after released in the free acid form by esterolysis. PMID- 7534210 TI - Rat MAbs to the product of the c-erbB-2 proto-oncogene for diagnosis and therapy in breast cancer. AB - The product of the c-erbB-2 protooncogene (p185) is a member of the EGF receptor family of transmembrane tyrosine kinases. Amplification of this gene and overexpression of the product has been observed in adenocarcinomas and has been correlated with poor prognosis in patients with breast and ovarian cancer. The very low levels of expression of p185 by normal adult tissues makes the receptor an almost tumor-specific target. We have prepared rat monoclonal antibodies against five distinct epitopes on the external domain of the c-erbB-2 product overexpressed by the breast cancer line BT474. The antibodies bind to the protein core of p185 and stain specifically the membranes in frozen sections of tumors overexpressing the c-erbB-2 product. Three of the antibodies, ICR12 (epitope A), ICR54, and ICR55 (epitope E), also stain the cell membrane in formalin-fixed, paraffin-embedded sections and bind to p185 in Western blots. An investigation of the stability of the antigen-antibody complexes indicates that the majority are not readily internalized by SKOV3 cells growing in vitro. Antibodies ICR12 (IgG2a) and ICR55 (IgG2a), which are directed against separate epitopes on the c erbB-2 p185, are both of high affinity and immunoreactivity (> 75%) and localize specifically and stably to xenografted breast and ovarian carcinomas growing in athymic mice when labeled with 125I (up to 13% injected dose/g, ICR12 and ICR55) or a range of other radionuclides (up to 20% id/g, ICR12). We conclude that these antibodies may be useful as therapeutic vehicles for targeting radionuclides (imaging and therapy) or enzymes for activating prodrugs (ADEPT). PMID- 7534212 TI - Overexcited or inactive: ion channels in muscle disease. PMID- 7534211 TI - Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor. AB - The receptor (EGFR) for epidermal growth factor (EGF) and transforming growth alpha (TGF alpha) is often overexpressed in certain types of human malignancy and high levels of expression of the receptor and/or coexpression of growth factors. EGF and TGF alpha have also been correlated with poor prognosis in many patients. We have produced a number of rat monoclonal antibodies (MAbs) against four distinct epitopes on the external domain of the EGF receptor and are currently evaluating their potential for therapeutic use. Nine of these of MAbs were found to inhibit the binding of TGF and EGF to the receptor on tumor cells and these MAbs were able to inhibit the growth in vitro and in vivo of tumor cells that overexpress the EGF receptor. Here, we describe the results of experiments to determine the antitumor activity of combination treatment with two antibodies directed against separate epitopes on the external domain of human EGFR. Our results showed that treatment of human tumor xenografts with a combination of two anti-EGFR MAbs that bind to two distinct epitopes on the external domain of EGF receptor was not as effective as treatment with ICR62 alone, which binds to epitope C on the EGFR and is of IgG2b isotype. A phase I clinical trial with antibody ICR62 is currently underway in Royal Marseden Hospital (UK) in patients with head and neck and lung carcinomas. PMID- 7534213 TI - A phosphotyrosine interaction domain. PMID- 7534214 TI - Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. AB - Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class II binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class II binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity. PMID- 7534215 TI - B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. AB - CD4 T helper precursor cells mature along two alternative pathways, Th1 and Th2. Here we show that these pathways are differentially activated by two costimulatory molecules, B7-1 and B7-2. Using anti-B7 antibodies, this developmental step was manipulated both in vitro and in vivo in experimental allergic encephalomyelitis (EAE). Anti-B7-1 reduced the incidence of disease while anti-B7-2 increased disease severity. Neither antibody affected overall T cell induction but rather altered cytokine profile. Administration of anti-B7-1 at immunization resulted in predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease. Since co treatment with anti-IL-4 antibody prevented disease amelioration, costimulatory molecules may directly affect initial cytokine secretion. Thus, interaction of B7 1 and B7-2 with shared counterreceptors CD28 and CTLA-4 results in very different outcomes in clinical disease by influencing commitment of precursors to a Th1 or Th2 lineage. PMID- 7534216 TI - Circulatory responses to laryngeal mask airway insertion or tracheal intubation in normotensive and hypertensive patients. AB - The effects of laryngeal mask airway (LMA) insertion and tracheal intubation on circulatory responses were studied in normotensive (n = 24) and hypertensive (n = 22) patients. In a randomized, double-blind manner, LMA insertion or tracheal intubation was performed after induction of anaesthesia with thiopentone and muscle relaxation with succinylcholine. In both normotensive and hypertensive patients, heart rate (HR), mean arterial pressure (MAP) and rate-pressure product increased after tracheal intubation or LMA insertion compared with baseline (P < 0.05). The haemodynamic changes were greater after intubation than after LMA insertion (P < 0.05). Following intubation of the trachea or insertion of the LMA, HR increased more markedly in hypertensive patients than in normotensive patients (P < 0.05). Plasma adrenaline and noradrenaline concentrations after tracheal intubation or LMA insertion increased compared with baseline values (P < 0.05) in normotensive and hypertensive patients. The increase in noradrenaline concentration after tracheal intubation was greater than that after LMA insertion (P < 0.05). No patient revealed ECG evidence of myocardial ischaemia. We conclude that insertion of LMA is associated with less circulatory responses than tracheal intubation in both normotensive and hypertensive patients. PMID- 7534217 TI - It is not necessary to administer more than 10 micrograms.kg-1 of atropine to older children before succinylcholine. AB - It is common practice at the Hospital for Sick Children, Toronto, to administer atropine 20 micrograms.kg-1 prior to succinylcholine in infants and children. It is unclear whether "prophylactic" administration of this dose of atropine to older children (6-16 yr) is necessary. This study was designed to compare the changes in heart rate, rhythm and mean arterial pressure after administration of either atropine 10 or 20 micrograms.kg-1 with succinylcholine or vecuronium (control group) to older children anaesthetized with thiopentone. Thirty-six ASA I or II patients (6-16 yr) were studied. Anaesthesia was induced with thiopentone 5 mg.kg-1. Patients were randomly assigned to receive: (a) atropine 10 micrograms.kg-1 and succinylcholine 1.5 mg.kg-1 (n = 12), (b) atropine 20 micrograms.kg-1 and succinylcholine 1.5 mg.kg-1 (n = 13) or (c) vecuronium 0.1 mg.kg-1 (n = 11) to facilitate tracheal intubation. Heart rate and rhythm were recorded continuously using a computerised analogue interface whereas blood pressure was monitored non-invasively before induction of anaesthesia, immediately before and at one and three minutes after laryngoscopy. No difference was observed between patients who received atropine 10 or 20 micrograms.kg-1 prior to succinylcholine. No episode of sinus bradycardia occurred. Premature atrial contractions were observed in two patients (one succinylcholine/atropine 20 micrograms.kg-1, one vecuronium). Administration of atropine 20 micrograms.kg 1 prior to succinylcholine provides no advantage over atropine 10 micrograms.kg-1 in older children in terms of cardiovascular stability. PMID- 7534218 TI - Relations between sex hormones, sex hormone binding globulin, insulin-like growth factor-I and insulin-like growth factor binding protein-1 in post-menopausal breast cancer patients. AB - OBJECTIVE: Oestrogens, androgens and anti-endocrine drugs such as tamoxifen and aminoglutethimide influence plasma insulin-like growth factor-I (IGF-I). IGF-I, in turn, has been found to stimulate the peripheral aromatase in vitro. The aim of this study was to examine relations between sex hormones, IGF-I and insulin like growth factor binding protein-1 (IGFBP-1) in post-menopausal women with breast cancer. DESIGN: To measure plasma sex steroids, sex hormone binding globulin (SHBG), IGF-I, IGFBP-1, insulin and urinary oestrogen metabolites in post-menopausal women with breast cancer not receiving any endocrine therapy. PATIENTS: Thirty-two patients had fasting blood samples obtained between 0800 and 1000 h. A sub-group of 10 patients had 24-hour urine oestrogen metabolites determined. MEASUREMENTS: Plasma steroids and proteins were measured by radioimmunoassays. Urinary oestrogens were measured by GC-MS. RESULTS: SHBG correlated negatively with plasma androstenedione (P < 0.001), insulin (P < 0.001), IGF-I, height and plasma oestrone sulphate (P < 0.025 for all), but positively with plasma IGFBP-1 (P < 0.025). IGFBP-1 correlated negatively with IGF-I (P < 0.001) and the testosterone/SHBG ratio (P < 0.05). Neither IGF-I nor IGFBP-1 correlated with any of the plasma or urinary sex hormones or with the oestrone/androstenedione and oestradiol/testosterone ratios. Multivariate analysis revealed plasma SHBG to correlate positively with IGFBP-1 (P = 0.029) and negatively with insulin (P = 0.031). Plasma IGFBP-1 correlated negatively with IGF-I (P < 0.0001) but not with insulin. CONCLUSION: Our results do not suggest any influence of plasma sex steroids in physiological concentrations on IGF-I or IGFBP-1 in post-menopausal breast cancer patients, nor do they indicate IGF-I at physiological concentrations influences the ratios between plasma oestrogens and their androgen precursors. PMID- 7534219 TI - Changes in Lp(a) lipoprotein and other plasma proteins during acute myocardial infarction. AB - The sequential changes of Lp(a) lipoprotein concentrations in patients (n = 59) suffering acute myocardial infarction (AMI) were examined and compared with other plasma proteins. The temporal and quantitative characteristics of the responses in concentration of acute phase reactants (CRP, haptoglobin, alpha 1-antitrypsin, alpha-acid glycoprotein), lipids (total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol) and apolipoproteins AI and B were similar to previous reports. Lp(a) lipoprotein showed transient changes with an initial decrease of 10-25% compared to the 3-month control value, followed by rebound on day 7-11 above admission level, before again declining. We were able to demonstrate a quantitative relationship between infarct size and alterations in plasma levels of acute phase reactants. However, in addition to rather unusual significant fluctuations during AMI, Lp(a) lipoprotein changes seemed unrelated to infarct size. These findings do not support the view that Lp(a) lipoprotein acts as an acute phase reactant. PMID- 7534220 TI - Usefulness of the AMeX method for immunostaining with antikeratin antibodies. AB - We performed an immunohistochemical study with 11 antikeratin antibodies using the newly developed AMeX (acetone-methylbenzoate-xylene) tissue processing method. Specimens processed with this method showed almost as good preservation and morphological detail as routinely processed, formalin-fixed and paraffin embedded tissue specimens, and as good preservation of antigenicity as fresh frozen tissue specimens. Thus, we propose the wide use of this method in dermatology. PMID- 7534221 TI - The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography. AB - In this study radioimmunoassay was used to determine neuropeptide levels in extracts from 17 differing anatomical regions of human skin. Marked regional variations of neuropeptide content for human skin were found and these variations are likely to reflect true physiological functions for the neuropeptides studied. In general the tachykinins, substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were found in highest concentrations in regions of skin with the greatest tactile sensation. By contrast, highest concentrations of vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) were found in axillary skin, where they probably play a part in axillary eccrine sweat production. Neurotensin was not found in any of the skin areas sampled, suggesting that it is relatively unimportant in human physiological skin control. Reverse-phase high-performance liquid chromatography (rpHPLC) was used to verify the results of radioimmunoassay. Both SP and NKA occurred in several regions in both their reduced and oxidized forms, as well as displaying molecular heterogeneity. CGRP occurred as one molecular species, this being alpha-CGRP, suggesting that this is the predominant molecular form in human skin. Likewise, both VIP and PHM displayed molecular homogeneity in the regions investigated by rpHPLC. PMID- 7534223 TI - [Magnetic resonance with intrarectal coil in the study of the prostate]. AB - The objective of this study was to determine the usefulness of the endorectal surface coil in the evaluation of the prostate. The endorectal surface coil provide more detailed visualization of the prostate and periprostatic structures than the body coil. Seminal vesicle architecture and the prostatic capsula are well seen on all of the endorectal coil images thus, disruption of the capsule on the endorectal coil images could be used as a sign of capsular invasion. Furthermore, focal thickening of the tubular walls on the endorectal coil images is considered to be evidence of early seminal vesicle invasion. The limits are represented essentially by the dimension of the endorectal surface coil. PMID- 7534222 TI - [IFN in the treatment of chronic viral hepatitis. Efficacy and tolerance of lymphoblastoid alpha interferon in HIV-negative drug addicts and associated chronic active hepatitis C]. AB - The authors evaluated the efficacy and tolerance of low dose lymphoblastoid alpha interferon in 14 i.v. drug addicts with histologically confirmed chronic active hepatitis C. Patients were treated for 12 months; all completed the whole treatment without complaining of significant side effects. Four months after withdrawal of treatment, 13 patients were found to be in clinical and biohumoral remission with serum transaminases within the normal range already after 12 weeks. PMID- 7534224 TI - [Tumoral vascular density in breast tumors and their effect on recurrence-free survival]. PMID- 7534225 TI - Cytosolic activation of aromatic and heterocyclic amines. Inhibition by dicoumarol and enhancement in viral hepatitis B. AB - The aromatic amines 2-aminofluorene (2AF), 2-acetylaminofluorene, and 2 aminoanthracene, and the heterocyclic amines 2-amino-3-methylimidazo[4,5 f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, and 3-amino-1 methyl-SH-pyrido[4,3-b]indole (Trp-P-2) were activated by rat liver cytosolic fractions to form mutagenic metabolites in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6. In the case of the Trp-P-2, the cytosolic activation was even more potent than the microsomal activation, which is classically ascribed to N-hydroxylation and subsequent esterification. The cytosolic activation was a) NADPH-dependent, b) induced by pretreatment of rats with 3 methylcholanthrene and especially Aroclor 1254 but not by phenobarbital, and c) inhibited by dicoumarol. The hypothesis is that, following a preliminary oxidative step in the cytosol (pure cytosolic activation) or in microsomes via prostaglandin H synthase (mixed microsomal-cytosolic activation), an oxidized intermediate of amino compounds may serve as substrate for DT diaphorase activity and bielectronically reduced to the corresponding N-hydroxyamino derivative. Purified DT diaphorase, in the presence of either NADPH or NADH as electron donor, produced mutagenic derivatives from IQ and Trp-P-2. An NADPH-dependent activation of Trp-P-2 also occurred in the liver cytosol of woodchucks (Marmota monax), but was not inhibited by dicoumarol. As previously demonstrated with liver S-12 fractions in both humans and woodchucks, the cytosolic activation of Trp-P-2 was enhanced in animals affected by hepatitis B virus infection. This enhanced metabolism, which persisted even after appearance of primary hepatocellular carcinoma in virus carriers, is likely to be ascribed to mechanisms other than DT diaphorase induction, such as glutathione depletion. PMID- 7534226 TI - Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency. AB - Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments. PMID- 7534227 TI - Gelatinase A activity directly modulates melanoma cell adhesion and spreading. AB - Interaction of cells with the extracellular matrix (ECM) plays an important role in the regulation of cell behavior. Formation of adhesive contacts leads to transduction of signals into the cell and results in altered gene expression and modulation of the cellular phenotype. Specific adhesive interactions of the fibronectin and vitronectin receptors with their ligands in the matrix modulates expression of ECM-degrading metalloproteases. These proteases are involved in the acquisition of the invasive phenotype by a number of cell types. The activity of matrix metalloproteases (MMPs) is reduced by endogenous inhibitors referred to as tissue inhibitors of metalloproteases (TIMPs). Alterations in the balance between the activity of MMPs and TIMPs alters cellular invasion through effects on matrix degradation. In this study we demonstrate that inhibition of endogenous gelatinase A activity in A2058 human melanoma cells results in enhanced cellular adhesion. To further explore this phenomenon, we have used retroviral infection vectors to control the amount of the MMP inhibitor TIMP-2 in human melanoma A2058 cells. Altering the production of TIMP-2 modulates not only proteolysis of the extracellular matrix, but also the adhesive and spreading properties of the cells and results in altered cell morphology. These effects of TIMP-2 appear to be mediated by inhibition of gelatinase A activity. We conclude that gelatinase A, in addition to contributing to proteolysis of ECM components, also functions to proteolyse cell surface components that mediate attachment of A2058 cells to the ECM. Thus, gelatinase A may function to modulate cell attachment and facilitate cell migration and invasion. PMID- 7534228 TI - Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity. AB - The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition. PMID- 7534229 TI - Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions. AB - The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src. PMID- 7534230 TI - Evaluation of a commercial rRNA target amplification assay for detection of Mycobacterium tuberculosis complex in respiratory specimens. AB - Seventy-one respiratory samples were analyzed in a new commercially available target rRNA amplification assay aimed at detecting Mycobacterium tuberculosis complex directly in patient specimens. The assay (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test) correctly identified 26 of 27 samples positive for Mycobacterium tuberculosis and 43 of 44 samples negative for Mycobacterium tuberculosis in culture. When appropriate controls are performed, the assay also performs well with other specimens, such as tissue biopsies, lymph node or bone marrow aspirates and pleural and ascitic exudates. PMID- 7534231 TI - Development of a ribotyping scheme for Haemophilus influenzae type b. AB - Ribotyping and outer-membrane protein subtyping were used to characterise 283 consecutive isolates of Haemophilus influenzae type b. These isolates were obtained primarily from patients with invasive disease in the UK and were received by the Public Health Laboratory Service Haemophilus Reference Laboratory prior to the implementation of Haemophilus influenzae serotype b vaccine in the UK. A subtyping scheme using the ribotyping method is suggested. Twenty-two ribotypes are described, 14 of which were found amongst the 283 clinical isolates characterised in this study. In contrast, only four outer-membrane protein subtypes were found amongst the 283 isolates. The ribotyping profiles were further used to estimate the relatedness of isolates. The resulting dendrogram suggested a population genetic structure different from that previously described for Haemophilus influenzae type b using multi-locus enzyme electrophoresis. This study shows the value of ribotyping as a subtyping method for epidemiological studies of Haemophilus influenzae type b. However, the further use of ribotyping for population genetic structure analysis of Haemophilus influenzae type b may be misleading and therefore inappropriate. PMID- 7534234 TI - Abnormal subcellular localization of mutated CFTR protein in a cystic fibrosis epithelial cell line. AB - The cystic fibrosis gene product, CFTR, is a Cl- channel that possesses specific binding sites for cytosolic ATP and is activated by cAMP-dependent protein kinase. Most recently, it was reported that CFTR localizes at the surface apical compartment of normal airway epithelial cells, but accumulates in the cytosol of airway cells from CF patients with the delta F508 mutation. In order to explore whether the same difference exists in normal and CF established cell lines that are commonly used in physiological and pharmacological investigations of the CF defect, we employed monoclonal antibodies raised against synthetic peptides corresponding to two different regions of the CFTR protein. One antibody (MATG 1061) was generated against amino acids 503-515 delta 508 in the nucleotide binding domain 1, whereas the other (MATG 1031) was generated against amino acids 107-117 situated in a putative external loop. We used confocal laser scanning microscopy to localize the CFTR protein in T84 (a colonic derived carcinoma), CAPAN-1 (a pancreatic carcinoma), and in CFPAC-1 (a pancreatic carcinoma homozygous for the delta F508 deletion) cell lines. In permeabilized T84 and CAPAN-1 cells, immunolabeling with MATG 1061 predominated at the apical domain. By contrast, CFTR staining with MATG 1061 was homogeneously distributed in the cytoplasm of CFPAC-1 cells. In non-permeabilized non-CF cell lines, MATG 1031 specifically labeled an apical membrane surface epitope. No such labeling was present in CFPAC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534232 TI - Proliferative and metabolic capacity of rat embryo fibroblasts immortalized by c myc depends on cellular age at oncogenic transfection. AB - It is well known that secondary rat embryo fibroblasts are immortalized and transformed with respect to requirements of growth factors by transfection with an overexpressed c-myc protooncogene. On the other hand, c-myc expression of nontransformed cells was shown to be independent of cellular age in vitro. In order to elucidate further the role of the c-myc protooncogene in the process of aging of rat embryo fibroblasts, we have transfected these cells at low (< or = 2) and at high (> or = 16) cumulative population doublings with SV40 promoter/enhancer-driven murine c-myc. These cells transformed young or aged, as well as their nontransformed, young and aged controls, were characterized with respect to their expression of c-myc at the mRNA and the protein level. Furthermore, we have investigated in detail their cell density-dependent growth, dependence of cell proliferation on stimulation by combinations of growth factors, and dependence of entry into cell cycle on cell size. In addition, we have measured rates of synthesis and degradation of cellular RNA and protein. The main result was that cells transformed at old age cannot be distinguished from nontransformed old cells by any of the characteristics investigated except by their immortalization. Thus, the cell lines overexpressing c-myc are immortalized and fixed in the proliferative/metabolic state achieved at the time of transfection. It is concluded that the intracellular effects of c-myc depend on the epigenetic status of the cells. PMID- 7534235 TI - Hepatic extraction of hyaluronic acid in porcine peritonitis. AB - The hepatic extraction of hyaluronic acid (HA) was studied in porcine fecal peritonitis in two groups of animals given various amounts of volume substitution. There was a progressive decrease in hepatic blood flow (QH) and a corresponding increase in the plasma concentration of HA in arterial blood over a 5-hour observation period, less pronounced in animals given more volume substitution. While hepatic clearance of HA decreased, the extraction ratio over the liver was not altered. The extracted amount of HA, which at steady state reflects the turnover of HA, was also unchanged. There was a significant correlation between QH and arterial HA concentration (r = 0.57; p < 0.05). The data suggest that the arterial HA concentration in sepsis reflects QH rather than an altered ability of the liver to eliminate HA. PMID- 7534233 TI - Local increase of beta 1-integrin expression in cocultures of immortalized hepatocytes and sinusoidal endothelial cells. AB - The expression and spatial arrangement of beta 1-integrins was determined on two immortalized liver cell lines held in coculture, namely the epithelial cell line mHepR1 and a sinusoidal endothelial cell line. On mHepR1 cells the distribution of beta 1-integrins was restricted to the basolateral plasma membrane domains, a staining pattern that is typical of polarized epithelial cells. On the endothelial cell line the beta 1-integrins were distributed all over the cell surface. In coculture the endothelial cells tended to cover over the epithelial cells. Epithelial cells located in their vicinity exhibited an increased staining of beta 1-integrins at basolateral plasma membrane domains, which was most prominent with regard to the alpha 2-subunit. When mHepR1 cells were cultivated on various types of extracellular matrix also synthesized by the endothelial cells only collagen IV was found to increase the intensity of beta 1-integrin expression at the cell surface. The results indicate that beta 1-integrin expression in epithelial cell colonies can locally be modulated by interactions with non-parenchymal cells. In addition, the data suggest that mHepR1 cells may be a favorable system for analyzing basic functions of beta 1-integrins in polarized epithelial cells. PMID- 7534237 TI - Chimeric alpha-beta oligonucleotides as antisense inhibitors of reverse transcription. AB - Alpha-beta chimeric 17-mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha-beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell-free system to various extent, via the degradation of the RNA target by RNase H. PMID- 7534238 TI - Internodal cells of the giant green alga Chara as an expression system for ion channels. AB - Internodal cells of the giant green alga Chara corallina were utilized as an expression system for two nicotinic acetylcholine receptor subtypes (nAChR) derived from rat muscle. From Chara internodes that were pressure-injected with the respective cRNA, cytoplasmic droplets were formed, and functional expression of channel proteins in the membrane delineating the droplets was confirmed by patch-clamp techniques. The droplet membrane was recently identified as the original tonoplast, patch-clamp recordings on cytoplasmic droplets were easily performed and single channel activity could be measured with high resolution. The properties of the recombinant nAChR observed in this membrane were similar to those reported for the channel expressed in Xenopus oocytes, and for the native channel recorded in situ. The Chara expression system is, therefore, suitable for functional expression of animal messenger RNAs, without the risk of signal contamination by intrinsic ion channels. It offers a low-cost and practical alternative to the use of Xenopus oocytes for the investigation of heterologously expressed ion channels. PMID- 7534236 TI - Hepatitis C virus infection in non-Hodgkin's B-cell lymphoma complicating mixed cryoglobulinaemia. PMID- 7534239 TI - Alterations in the sensitivity of serum insulin-like growth factor 1 and insulin like growth factor binding protein-3 to octreotide in polycystic ovary syndrome. AB - OBJECTIVE: To determine if the somatostatin analog, octreotide, affects insulin and related peptides and, hence, androgen levels differently between polycystic ovary syndrome (PCOS) patients and controls. DESIGN: Prospective controlled trial. SETTING: Reproductive endocrinology clinic of our medical center. PATIENTS: Eleven women with PCOS and six matched ovulatory controls. INTERVENTIONS: Octreotide (100 micrograms) was administered subcutaneously in the midfollicular phase. Serum was obtained before and at 60, 120, 180, and 240 minutes after octreotide. MAIN OUTCOME MEASURES: Fasting insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), T, androstenedione (A), and LH. RESULTS: In PCOS, baseline levels of T, A, LH, and fasting insulin were significantly higher than in controls. Pretreatment IGF 1 and IGFBP-3 levels were similar in PCOS and controls. Octreotide reduced fasting insulin levels significantly but to a similar degree in control and PCOS patients (77% and 90%, respectively). Both groups also experienced a significant decrease in LH levels after octreotide administration, but no significant changes were demonstrated in serum T or A. However, serum IGF-1 suppression in PCOS was greater (63% versus 8% in controls). Serum IGFBP-3 levels increased after octreotide administration in both groups with a larger increase (40%) occurring in the PCOS patients. CONCLUSIONS: These data suggest that women with PCOS may be more sensitive to the effects of octreotide in decreasing IGF-1 and increasing IGFBP-3. Although no significant changes could be demonstrated in ovarian androgens after a single dose, octreotide effectively reduced serum LH and insulin and, as such, may prove useful in treating some patients with PCOS. PMID- 7534240 TI - Fluorescein isothiocyanate conjugate-wheat germ agglutinin staining of human spermatozoa and fertilization in vitro. AB - OBJECTIVE: To determine the sensitivity and clinical significance of fluorescein isothiocyanate wheat germ agglutinin (FITC-WGA) staining of human spermatozoa. DESIGN: Percentage FITC-WGA staining was assessed in the different morphological zones of human spermatozoa and compared with fertilization rates of metaphase II oocytes in vitro. PATIENTS: Spermatozoa and oocytes were obtained from normospermic (n = 12) and male factor patients (n = 15) attending the Tygerberg Hospital's IVF clinic. MAIN OUTCOME MEASURES: Aliquots of swim-up spermatozoa used in the IVF laboratory were examined for the presence and morphological localization of WGA receptors. Fertilization rates were expressed as the percentage of metaphase II oocytes with successful fertilization. RESULTS: Human spermatozoa capacitated in vitro for use in an IVF program showed variations in the percent positive staining of the acrosomal region which correlated with IVF rates in both patient groups studied. Values of FITC-WGA acrosomal staining of < 35% corresponded with IVF rates of < 50% whereas > 35% WGA acrosomal staining corresponded with IVF rates > or = 50%. Significant differences between acrosomal and equatorial region staining from normozoospermic samples and male factor samples also were observed. CONCLUSION: The results indicate the practical use of the WGA assay for molecular assessment of male fertilizing capacity in conjunction with an assisted reproductive program. PMID- 7534242 TI - Individual and team consensus ratings of child functioning. AB - The authors examined agreement between interdisciplinary teams of professionals in rating the functional abilities of children with disabilities. 129 children were rated on the ABILITIES index by 72 professionals working in nine developmental evaluation centers. Independent individual ratings were made first, followed by a consensus rating. Team members had a high degree of agreement on independent ratings of abilities and limitations, with nearly 90 per cent of the ratings within one point of each other. More disagreement was associated with rating areas of behavior and communication, team experience and increased severity of disability. The team's consensus ratings were more likely to defer to the ratings of the expert in particular areas. Implications for team assessment and team functioning are discussed. PMID- 7534241 TI - Insulin-like growth factor I (IGF-I) and IGF binding proteins in seminal plasma before and after vasectomy in normal men. AB - OBJECTIVE: To examine the levels and origins of insulin-like growth factor I (IGF I) and insulin-like growth factor binding proteins (IGFBPs) in the human male genital tract. DESIGN: Examining seminal plasma before and 3 months after vasectomy. SUBJECTS: Fifteen men who were candidates for vasectomy were included in the study. MAIN OUTCOME MEASURES: Seminal plasma and serum levels of IGF-I, IGFBP-1, and IGFBP-3 were determined by commercially available assays, furthermore, samples were subjected to Western ligand blotting. RESULTS: Seminal plasma concentrations of IGF-I were significantly lower after vasectomy: 18.0 +/- 2.4 micrograms/L (before) and 12.5 +/- 1.2 micrograms/L (after). When the total ejaculate content of IGF-I was calculated, the figures were reduced by 50% after vasectomy: 45.64 +/- 7.8 ng (before) and 23.45 +/- 3.8 ng (after). The patterns observed for seminal plasma IGFBP-3 concentrations were 844.9 +/- 59 micrograms/L (before) and 816.5 +/- 65 micrograms/L (after). When the total ejaculate IGFBP-3 content was calculated there was a 36% reduction after vasectomy: 2,300 +/- 251 ng (before) and 1,474 +/- 217 ng (after). CONCLUSIONS: A considerable amount of seminal plasma IGF-I and IGFBP-3 may be of testicular origin. Although the physiological significance of IGF-I and IGFBPs in the male reproductive system still remains uncertain, the demonstration of their presence in the testes add support to a functional role in the regulation of gonadal function. PMID- 7534243 TI - Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures. AB - Mutations at the Steel (Sl) and dominant white spotting (W) loci affect three embryonic lineages: primordial germ cells, hemopoietic stem cells and neural crest-derived melanocytes. The gene products of these loci are a peptide growth factor, called here stem cell factor (SCF), and its tyrosine kinase receptor, the proto-oncogene c-kit. We have studied how chicken recombinant SCF affects the development of melanocytes from quail neural crest cells in secondary culture under defined conditions. We observed that the total number of neural crest cells, of melanocytes and of their precursors was higher in the presence than in the absence of SCF. Labelling with bromodeoxyuridine showed that SCF had a modest and transient mitogenic effect on the neural crest population. SCF also enhanced the differentiation rate of melanocyte precursors, recognized by the "melanocyte early marker" monoclonal antibody (MelEM MAb), and of melanocytes, since the proportion of both subpopulations significantly increased in the presence of SCF. Finally, SCF increased the survival of the neural crest population since in its presence the total number of cells remained stable while it gradually declined in control cultures. Our results support the notion that SCF sustains the survival of the neural crest population and stimulates the rate of the melanogenic differentiation process. PMID- 7534244 TI - Pyridoindole stobadine is a nonselective inhibitor of voltage-operated ion channels in rat sensory neurons. AB - Pyridoindole stobadine, a compound with known neuroprotective, antioxidant, oxygen-free radical scavenging and antiarrhythmic properties, was analysed as to its effect on inward sodium and calcium currents (INa and ICa), respectively, and on fast inactivating and slow non-inactivating components of potassium outward current (IKf and IKs), respectively, in the neuronal membrane. A voltage-clamp technique was used in internally dialyzed single neurons isolated from young rat sensory ganglia. Stobadine inhibited all currents studied in a concentration dependent manner (0.1-3 mmol/l). Apparent affinity constants pD'2,--(i.e. negative logarithm of IC50) were 3.147 +/- 0.030, 3.112 +/- 0.084, 3.089 +/- 0.062 and 2.918 +/- 0.059 for INa, IKs, IKf, and ICa, respectively. Full current inhibition occurred only in INa at concentrations of > 1 and < or = 3 mmol/l. Saturation of the inhibition of both components of IK at approximately 40% of the control level occurred at concentrations > 1 mmol/l. Stobadine was found to be a nonspecific inhibitor of the currents studied. This property as well as the range of pD'2, values are comparable to those observed in the local anesthetics procaine and trimecaine. The inhibition of voltage-operated ion channels in membranes of excitable cells by stobadine might be responsible for its capability to inhibit synaptic transmission and axonal conduction. It might participate also in the antidysrrhythmic effect of stobadine. PMID- 7534245 TI - [Molecular-genetic analysis of certain mutations of the "cystic fibrosis gene" in Moldavia. Characteristics of molecular markers and their linkage with various mutations]. AB - Sixty-one patients with cystic fibrosis (CF) from Moldova were tested for mutations delta F508, G551D, and R553X. Frequencies of various alleles of the repeated GATT sequence in intron 6B of the CFTR gene, their linkage to other polymorphic markers, and various mutations were determined. The frequency of occurrence of mutation delta F508 was only 25%. An absolute majority of CF patients (80%) had pancreatic insufficiency. Mutations G551D and R553X were not found in our sample. Each of 31 chromosomes with mutation delta F508 carries the 6-GATT allele. Most "non delta F508" (78%) and normal (80%) chromosomes were marked by 7-GATT allele. Twenty-seven delta F508 chromosomes (96.4%) belong to haplotype B6, and only one to D6. Most chromosomes with "non delta F508" mutations are associated with haplotypes D7 (26.3%) and C7 (21%). In addition, a significant portion of chromosomes from this subgroup were associated with haplotypes A7 (23.7%), A6 (10.5%), and C6 (2.7%), which are not yet described for mutant chromosomes. The results obtained demonstrate that CF in Moldova is mainly associated with mutations other than delta F508, G551D, and R553X. Severe forms of the disease, with pancreatic insufficiency, are more frequently caused by these mutations; moreover, our data provides strong evidence about the presence of at least seven additional CF mutations in Moldova, apart from delta F508, G551D, and R553X. Some of these are probably not described. PMID- 7534246 TI - [Use of polymerase chain reaction for determining bcr/abl mRNA in human chronic myeloleukemia]. AB - A modified system with two pairs of primers is suggested for the analysis of different bcr/abl mRNA variants, using the polymerase chain reaction (PCR) method. It was demonstrated that, with the use of this method, mRNA preparations obtained from bone marrow cells are more informative than those obtained from blood cells. Fresh preparations of thermostable Tth DNA polymerase, which exhibited the reverse transcriptase activity, appeared to be optimal for PCR analysis of the bcr/abl mRNA pattern. PMID- 7534247 TI - Y3 is the most conserved small RNA component of Ro ribonucleoprotein complexes in vertebrate species. AB - YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions. PMID- 7534248 TI - Regulation of mouse myelin basic protein gene transcription by a sequence specific single-stranded DNA-binding protein in vitro. AB - Cell- and stage-specific transcription of the myelin basic protein (MBP)-encoding gene (Mbp) in brain is regulated by arrays of cis-acting regulatory elements positioned at the 5'-flanking region of the gene. The proximal element between nucleotides -14 to -50, termed MB1, has previously been shown to have an important role in the cell-type-specific transcription of Mbp in cells derived from the central nervous system (CNS). Here, we utilized band-shift and in vitro transcription assays to examine the ability of MEF-2, an expression factor encoded by a cDNA isolated from mouse brain, in binding to the MB1 element and regulating transcription of the Mbp promoter. Results from the band-shift assays indicated that the bacterially produced MEF-2 recognizes specific nt in the MB1 motif, and its binding to these nt reduces the overall transcriptional activity of Mbp in a cell-free extract. PMID- 7534249 TI - A breakthrough in surgery for age-related macular degeneration? PMID- 7534250 TI - Transplantation of fetal retinal pigment epithelium in age-related macular degeneration with subfoveal neovascularization. AB - BACKGROUND: Age-related macular degeneration (ARMD) is caused by abnormal retinal pigment epithelium (RPE) and may be complicated by choroidal neovascularization. The object of treatment would be to replace the diseased RPE with normal human RPE. METHOD: Five patients with ARMD (preoperative visual acuity 0.08-0.2) underwent removal of subretinal fibrovascular membranes using pars plana vitrectomy techniques. Human fetal RPE (15-17 weeks gestational age) was cultured and transplanted as a monolayer patch into the subretinal space. Transplants were followed by funduscopy and fluorescein angiography. Macular function was assessed using scanning laser ophthalmoscopic (SLO) microperimetry. RESULTS: Three RPE transplants were placed in the fovea; two were placed parafoveally. All transplants have survived for 3 months. They have grown and increased in size covering part of the epithelial defect caused by removal of the fibrovascular membrane. SLO microperimetry indicated that visual function was present in four of the transplants at 1 month but in only two at 3 months after surgery. Function over the transplants, especially those in the fovea, was compromised by cystoidlike macular edema. CONCLUSIONS: Human fetal RPE transplants survive well in the macula for as long as 3 months. They are capable of growing to cover epithelial defects caused by removal of subretinal neovascular membranes. The causes for development of macular edema in transplants directly in the fovea warrant further evaluation. PMID- 7534251 TI - Potential role of superantigen induced activation of cell mediated immune mechanisms in the pathogenesis of Crohn's disease. PMID- 7534252 TI - Tyrosine phosphorylation in the human duodenum. AB - Many growth factor receptors including the epidermal growth factor receptor function through tyrosine kinase activity. The aim of this study was to examine the constitutive level of tyrosine phosphorylation in the normal duodenum and in the hyperproliferative coeliac duodenum. A flow cytometric assay was devised using monoclonal antibody to phosphorylated (but not native) tyrosine residues to determine the levels of tyrosine phosphorylation in both CD3 positive intraepithelial lymphocytes and CD3 negative epithelial cells obtained by EDTA treatment of endoscopically obtained duodenal biopsy specimens. In addition, immunohistochemistry was performed on 18 formalin fixed coeliac duodenal biopsy specimens and eight control specimens. Tyrosine phosphorylation could be detected by flow cytometry on duodenal enterocytes and this expression was up regulated by pretreatment with epidermal growth factor. Tyrosine phosphorylation decreased with progression from the villus to the crypt, however, and was virtually undetectable on crypt enterocytes. Immunohistochemistry of the coeliac duodenum showed virtually absent tyrosine phosphorylation in the crypt. Increased tyrosine phosphorylation was detected in the infiltrating T cells. In conclusion, tyrosine phosphorylation in the duodenum is confined to the non-proliferative villous epithelium and is virtually undetectable in the proliferative crypt compartment. These findings suggest that tyrosine kinase activity is not a significant factor in the regulation of crypt cell proliferation in the human duodenum either in normal subjects or in coeliac disease patients. PMID- 7534253 TI - Expression of mutant p53 protein and CD44 variant proteins in colorectal tumorigenesis. AB - Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukes's A and B carcinomas (n = 21), and Dukes's C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer. PMID- 7534255 TI - In situ mycotoxin production by Candida albicans in women with vaginitis. AB - The virulence attributes of Candida albicans in cases of mucocutaneous disease have not been identified. Based on the recent finding that C. albicans is able to produce an immunosuppressive mycotoxin, gliotoxin, we analyzed vaginal samples of 3 women severely symptomatic for vaginal candidiasis and found that they contained significant levels of gliotoxin. Three control women who were not colonized with C. albicans showed no gliotoxin in vaginal samples. These findings raise the possibility that gliotoxin may play a role in the virulence of C. albicans. PMID- 7534256 TI - [Early diagnosis and therapy of brain metastases. Goal: improved quality of life and perhaps extended survival]. AB - Diagnosis of cerebral metastasis is based on specific anamnestic information, clinical-neurological symptomatology and appropriate imaging. On account of its high level of sensitivity, ready, availability and, in comparison with MRI, relatively low cost, CT scanning remains the most important imaging modality. Curative treatment is not possible. For palliation and, in part, long-term symptom-free survival, radiation is the modality of first choice. On no account must this possibility be neglected by adopting an attitude of therapeutic nihilism. PMID- 7534254 TI - Evaluation of latex agglutination test for alpha-fetoprotein in diagnosing rupture of fetal membranes. AB - To assess the utility of a latex agglutination test for alpha-fetoprotein in vaginal fluid for diagnosing rupture of the fetal membranes, we tested 91 pregnant women, including 32 with an established rupture, 19 with no evidence of rupture and 40 with a suspected rupture. Results were compared with those of the pH indicator and the fern tests. The latex agglutination test had better sensitivity, specificity, or both, than the pH indicator and the fern tests. We conclude that the latex agglutination test is more accurate than either the pH indicator or the fern test for diagnosing premature rupture of fetal membranes. PMID- 7534257 TI - [Age dependence of interleukin 6 and acute phase proteins]. AB - In 59 healthy subjects of different age groups with no signs of inflammatory processes, the basal II-6 levels were measured. The II-6 concentrations were significantly higher in the oldest group (66-90 years) than in the two other age groups (24-40, 41-65 years). A positive correlation was found between age and II 6 concentration (r = 0.470). Otherwise, a statistically detectable relationship was found merely with alpha-2-macroglobulin and with total and LDL cholesterol. PMID- 7534258 TI - [Treatment of benign prostatic hyperplasia. Results of a treatment study with the phytogenic combination of Sabal extract WS 1473 and Urtica extract WS 1031 in urologic specialty practices]. AB - In an open, prospective, multicentric observational study involving 419 specialist urological practices, the efficacy and tolerability of a combination preparation comprising Sabal extract WS 1473 and Urtica extract WS 1031 were investigated in 2080 patients with Alken's stage I or II benign prostatic hyperplasia. A before-and-after comparison revealed an improvement in the pathological findings and in the obstructive and irritative symptoms. For the most part, the efficacy and tolerability of the preparation were assessed by the physician to be "very good" or "good". At the end of treatment, most patients indicated an improvement in their prostatic symptomatology and in general quality of life. Fifteen patients (0.72%) were suspected of having developed mild side effects. PMID- 7534259 TI - Induction of primary antigen-specific immune reponses in SCID-hu-PBL by coupled T B epitopes. AB - Adoptive transfer of human lymphoid cells into immunodeficient (SCID) mice lacking the ability to functionally rearrange T- and B-cell receptor genes constitutes a unique model to study and manipulate human immunocytes. We have investigated this model for the purpose of generating an antigen-specific primary humoral immune response. Peripheral blood lymphocytes (PBL) derived from blood donors were used to repopulate SCID mice, which subsequently were immunized with different B-cell epitopes coupled to either tetanus toxoid (TT), or to a promiscuous helper epitope of TT, or by incorporating the antigens into a liposome construct. By recruiting the necessary T-cell help found in the T-cell memory compartment against TT, primary immune responses were obtained against the hapten dinitrophenyl (DNP), the V3 loop peptide derived from glycoprotein (gp120) (HIV-1), the melanoma-associated GD2 ganglioside and ovine submaxillary mucin. The primary immune response against the GD2 ganglioside was induced by incapsulating TT into GD2-containing liposomes. These liposome constructs also allowed us to induce a high human IgG serotitre (3000-4000) against this normally not very immunogenic ganglioside. PMID- 7534260 TI - Rat, mouse and human neutrophils stimulated by a variety of activating agents produce much less nitrite than rodent macrophages. AB - The role of reactive nitrogen intermediates (RNI) in the antimicrobial activities of neutrophils from various mammalian species is unclear. However, it has been reported that rodent neutrophils possess the inducible form of nitric oxide synthase and that inflammatory neutrophils from rats produce potentially antimicrobial levels of RNI. In the present study, neutrophils from humans, rats and mice were evaluated for production of nitrite, a stable end-product of RNI. Human neutrophil preparations (> 95% neutrophils) isolated from peripheral blood were stimulated for 2-24 hr with agents known to trigger the Ca(2+)-dependent constitutive nitric oxide synthase, or to stimulate synthesis of the inducible nitric oxide synthase. Superoxide dismutase was added to some cultures to decrease the levels of superoxide, a compound reported to react with RNI and yield products other than nitrite. Even though the cells were viable and responsive to stimuli, they did not produce nitrite concentrations indicative of antimicrobial potential. Preparations of inflammatory (casein-elicited) mouse neutrophils also failed to produce high concentrations of nitrite. Inflammatory rat neutrophils (2.5 x 10(6)/ml) produced nitrite concentrations of approximately 40 microM in 24-hr cultures, but plots of nitrite production versus cell number for neutrophil and macrophage preparations indicated that contaminating macrophages could account for all the nitrite production in the neutrophil preparations. Thus, neutrophils from rats, mice and humans seem comparable in their inability to produce high levels of nitrite in response to a variety of stimuli. This suggests that in most circumstances the constitutive nitric oxide synthase known to be present in these cells is limited to the production of low levels of nitric oxide for intercellular signalling. In addition, this raises questions about the presence or functional status of inducible nitric oxide synthase in rodent neutrophils. PMID- 7534261 TI - Alveolar macrophages from humans and rodents selectively inhibit T-cell proliferation but permit T-cell activation and cytokine secretion. AB - Alveolar macrophages (AM) are thought to play a key role in the regulation of immune responses within the lung. While it is well established that AM inhibit T cell proliferation in vitro, it is unclear whether other aspects of the T-cell activation process are also inhibited. The present study demonstrates that AM from rat, mouse and human differ markedly in the potency with which they inhibit mitogen-induced T-cell proliferation, although in humans the degree of inhibition approaches that observed in the animal systems, if antigen (as opposed to mitogen) is employed as the T-cell activating agent. Rodent and human AM also differ in the mechanisms employed to achieve this inhibition; rodent AM appear to utilize reactive nitrogen intermediates, while this does not appear to be the case for human AM. Despite these differences, T cells stimulated in the presence of AM display a similar phenotype in all species examined, i.e. CD3 down modulation, up-regulation of interleukin-2 receptor (IL-2R) expression and IL-2 production, but inability to respond to IL-2. Thus, AM appear to allow T-cell activation and expression of T-cell effector function, while selectively inhibiting T-cell proliferation. PMID- 7534262 TI - Lung eosinophilia is dependent on IL-5 and the adhesion molecules CD18 and VLA-4, in a guinea-pig model. AB - Blood and tissue-eosinophilia is a characteristic feature of a number of disease states. In experimental animals, the intravenous injection of parasitic larvae induces a profound eosinophilia that can be mimicked by the intravenous injection of Sephadex particles. In the present study, this procedure was used to investigate the mechanisms involved in the development of lung eosinophilia in a guinea-pig model. Intravenous administration of Sephadex particles to guinea-pigs resulted in a significant increase in the influx of eosinophils in the airways and in lung tissue eosinophil peroxidase (EPO) activity (at t = 24 hr). An anti interleukin-5 (IL-5) monoclonal antibody (mAb) totally inhibited the eosinophilia in the airways and significantly reduced the lung tissue EPO activity. The concomitant accumulation of neutrophils and mononuclear cells, however, was not affected by this treatment. Monoclonal antibodies to VLA-4 and CD18 caused 58% and 62% suppression of eosinophilia in the bronchoalveolar lavage (BAL), respectively, whilst having no effect on lung tissue EPO activity. Co administration of the two mAb resulted in total inhibition of eosinophil accumulation into BAL and significant suppression of lung tissue EPO activity (55% inhibition). This procedure also resulted in 72% inhibition of mononuclear cell influx and 68% inhibition of neutrophil influx in the BAL, the latter effect being entirely due to the actions of the anti-CD18 mAb. The results of this study indicate for the first time a requirement for IL-5 in the development of lung eosinophilia in this model. Further, it is clear that both the molecules VLA-4 and CD18 contribute to the development of this response and that maximal inhibition of lung eosinophilia is achieved only when the two adhesion pathways are simultaneously blocked. PMID- 7534263 TI - Reversible stimulation of lymphocyte motility by cultured high endothelial cells: mediation by L-selectin. AB - Lymphocyte emigration from blood into peripheral lymph nodes is mediated by specialized high endothelial cells (HEC) lining the postcapillary venules. A current model for this process postulates that it occurs in three steps: weak, selectin-mediated interactions tether lymphocytes to the blood vessel wall; the lymphocytes are activated to increase the affinity of integrin-dependent adhesion and enhance motility; and finally the lymphocytes migrate actively across the endothelial cell layer. Some features of this model are simulated in vitro by cultured HEC, which support the adhesion and transmigration of lymphocytes. In particular, cultured HEC stimulate lymphocytes to change shape from spherical to polar. This shape change provides a convenient assay of the motility activation of lymphocytes. In this paper it is shown that this occurs without the lymphocytes becoming tightly adherent, but depends on contact with the endothelial cell surface. The shape change is labile: non-adherent polar lymphocytes removed from HEC revert to round with a half-time of less than 8 min. Reagents which block the interaction of L-selectin with its ligands inhibit the HEC-induced shape change; these include mannose-6-phosphate, fucoidan, polyphosphomannan ester, treatment of HEC with sialidases and an anti-L-selectin monoclonal antibody known to block its lectin function. The change in shape is partially inhibited by antisera to the L-selectin ligand GlyCAM-1. Thus it is concluded that in this in vitro system, L-selectin-mediated binding of lymphocytes to HEC is essential for optimal induction of the shape change. Lymphocytes change shape in response to cultured HEC without loss of surface L selectin, although activation stimuli are known to promote shedding of neutrophil L-selectin as well as motility and increased adhesiveness. However, the lymphocyte change in shape is a reversible process, and this may have implications for the nature and sequence of the signals transmitted from endothelium to lymphocytes during homing to peripheral lymph nodes. PMID- 7534265 TI - Adhesion molecules on intermediate TCR cells. I. Unique expression of adhesion molecules, CD44+ L-selectin-, on intermediate TCR cells in the liver and the modulation of their adhesion by hyaluronic acid. AB - In addition to thymus-derived T cells, it was demonstrated recently that extrathymically differentiated T cells exist in the liver and other immune organs of mice. Since such extrathymic T cells have T-cell receptors (TCR) of intermediate intensity (i.e. intermediate TCR cells) and constitutively express IL-2 receptor beta-chain (IL-2R beta) similar to natural killer (NK) cells, they are easily distinguished from thymus-derived T cells with a TCR-bright+ IL-2R beta- phenotype (i.e. bright TCR cells). In the present study, the expression of adhesion molecules CD44 and L-selectin was compared between these T-cell subsets. Intermediate TCR cells in the liver and other organs were found to be CD44+ L selectin- and, inversely, bright TCR cells were CD44- L-selectin+. CD3- IL-2R beta+ NK cells were also estimated to be CD44+ L-selectin-. Hyaluronic acid, which is known to adhere to a CD44 ligand, bound to intermediate TCR cells, but not to bright TCR cells. Among many extracellular matrices, hyaluronic acid induced a prominent decrease in the numbers and proportions of intermediate TCR cells and NK cells in the liver from 6 to 24 hr after in vivo administration. The half-life span of injected hyaluronic acid was approximately 7 hr in the plasma. These results suggest that the CD44 molecule, which is uniquely expressed on intermediate TCR cells and NK cells, is eventually associated with their adhesion to the sinusoidal walls in the liver. PMID- 7534264 TI - Differences in E-selectin expression and leucocyte infiltration induced by inflammatory agents in a novel subcutaneous sponge matrix model. AB - We have developed a novel subcutaneous sponge matrix model in major histocompatibility complex (MHC) homozygous SLAb/b inbred pigs to study lymphocyte-endothelial cell interactions during inflammation. Polyether sponges were implanted subcutaneously and left for 12 days before injection of proinflammatory agonists. Implanted sponges became highly vascularized and showed markedly increased uptake of i.v.-injected 51Cr-labelled lymphocytes 5 hr after injection of tumour necrosis factor-alpha (TNF-alpha) (3000 U) or phytohaemagglutinin (PHA) (37 micrograms). Lower levels of traffic were seen in sponges 5 hr after injection with interleukin-1 alpha (IL-1 alpha) (3000 U) and no significant traffic occurred in sponges injected with phorbol 12-myristate 13 acetate (PMA) (15 ng) at 5 hr or PHA at 24 hr (compared to sponges injected with medium alone). Electron microscopy of control sponges revealed low numbers of infiltrating leucocytes and relatively 'flat' endothelium. Many more infiltrating leucocytes were present in PHA-injected sponges. However, no ultrastructural evidence was seen of any significant difference between control and activated endothelium. Immunocytochemistry of frozen sections from sponges showed that E selectin expression was up-regulated markedly by TNF-alpha and PHA at 5 hr, only moderately by IL-1 alpha at 5 hr, and not at all by PMA at 5 hr. By 24 hr in PHA injected sponges E-selectin expression had fallen markedly from the level seen at 5 hr. Flow cytometric analysis of cellular infiltrates dispersed from sponges injected with TNF-alpha, PHA, IL-1 alpha or medium alone, revealed differences in lymphocyte subset populations. The infiltrate in sponges injected with TNF-alpha 5 hr before removal was dominated by high numbers of CD2+ lymphocytes, whereas the infiltrate induced by PHA showed relatively higher levels of CD2- CD4-CD8- gamma delta T-cell receptor+ (TCR+) T cells revealed by population-specific monoclonal antibodies (mAb). This model, which permits harvesting of leucocytes and endothelial cells for manipulation in vitro, will be useful for the study of leucocyte-endothelial cell interactions in subacute and chronic inflammation. PMID- 7534267 TI - The influence of MHC polymorphism on the selection of T-cell determinants of FMDV in cattle. AB - There is a quest for the development of a new generation of vaccines consisting of well-defined subunit antigens. For a number of practical reasons it is attractive to develop vaccines on the basis of synthetic peptides. However, their efficacy may be limited by genetic restrictions imposed on T-cell recognition via major histocompatibility complex (MHC) polymorphism, as shown by many studies using inbred animal species. To study the effect of MHC polymorphism in an outbred species, we selected four cattle homozygous for different A-DR-DQ haplotypes, and another four cattle which shared one haplotype in combination with a haplotype of one of the MHC homozygous animals. We analysed responses to synthetic peptides comprising defined T-cell epitopes of foot-and-mouth disease virus (FMDV) in this selected group of FMDV-vaccinated cattle. This analysis shows that even in outbred animals. MHC polymorphism influences the responses to synthetic peptides. Interestingly, one of the peptides, VP4[20-34], was recognized in association with at least four different MHC haplotypes. Fine specificity analysis of this peptide revealed subtle shifts in the core epitope recognized. All peptides that induced lymphocyte proliferation in vitro were found to induce a T-helper type-1 (Th1) type of response, irrespective of the MHC haplotype involved. Together, these data support the notion that individuals carrying distinct MHC types can be vaccinated successfully by vaccines that include only a limited number of peptides. In the design of a peptide vaccine against FMDV we suggest inclusion of the highly conserved VP4 sequence 20-34. PMID- 7534268 TI - Polyclonal and clonal analysis of human CD4+ T-lymphocyte responses to nut extracts. AB - The induction of IgE antibodies to aeroallergens depends upon antigen-specific CD4+ helper T cells of an 'interleukin-4 (IL-4)-dominant' phenotype. Nuts also drive IgE-mediated hypersensitivity and are the most dangerous of the orally encountered allergens. We have studied the polyclonal T-cell responses of atopic and non-atopic individuals to extracts of peanut, brazilnut and hazelnut. Strong proliferative responses were observed in all patients but specific IgE was only present in the nut-allergic patients suggesting a similar pathogenic mechanism to aeroallergen-mediated hypersensitivity. To investigate this hypothesis a panel of peanut-reactive T-cell clones was raised from a peanut- and brazilnut-allergic individual without hazelnut allergy. The antigen specificity, major histocompatibility complex (MHC) class II restriction and cytokine profiles of the T-cell clones were determined. With the exception of one T-cell clone, which proliferated in response to both peanut and hazelnut extract, the peanut T-cell clones were not cross-reactive with hazelnut or brazilnut. The T-cell clones recognized antigen in association with HLA-DR and HLA-DP but not HLA-DQ class II molecules. The peanut-specific clones produced high levels of IL-4 and low levels of interferon-gamma (IFN-gamma), exhibiting the 'TH2-like' profile which dominates the aeroallergen response. In contrast, the T-cell clone that was cross reactive on both peanut and hazelnut allergen had a Th0-like phenotype, consistent with the lack of specific serum IgE to hazelnut. These results support the importance of functionally distinct T-cell populations that recognize oral allergens. The relative production of IL-4 and IFN-gamma of the cloned T cells in the peanut-allergic patients plays a role in determining whether or not IgE antibody responses are induced with the associated potential to develop anaphylactic reactions. PMID- 7534269 TI - Vitronectin interacts with Candida albicans and augments organism attachment to the NR8383 macrophage cell line. AB - Candida albicans is an increasingly important cause of mucocutaneous and bloodstream infections. The potential role of circulating adhesive glycoproteins such as vitronectin (Vn) in host defense against C. albicans is currently unknown. Accordingly, we investigated the binding of plasma-derived Vn with C. albicans strain 36082. Vn specifically bound to C. albicans in a concentration dependent fashion. Higher affinity binding sites numbered 9.8 x 10(4) sites per organism, with a dissociation constant, Kd of 3.5 x 10(-7) M. Vn binding with C. albicans was significantly inhibited by heparin, suggesting interaction of the organism with Vn's glycosaminoglycan-binding region. To further determine which molecule(s) on the fungus interacted with Vn, C. albicans components were extracted, separated by SDS and blotted with radiolabeled Vn. These studies revealed that Vn binds to a 30 kDa molecule on C. albicans. Finally, we investigated the role of Vn in promoting interaction of C. albicans with phagocytic cells. Incubation of C. albicans in the presence of Vn significantly increased binding of the organism to cultured NR8383 macrophages compared to incubations performed in the absence of Vn. These data demonstrate that C. albicans interacts with the heparin-binding domain Vn and further suggest that Vn augments organism uptake by phagocytic cells. PMID- 7534270 TI - Suppression of phagocytosis by adrenocorticotropic hormone in murine peritoneal macrophages. AB - Phagocytosis of latex beads by peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that adrenocorticotropic hormone (ACTH) suppressed phagocytosis in a dose-dependent manner. ACTH (1-24) was more suppressive than ACTH (1-39). Control phagocytosis was partially suppressed in Ca(2+)-free solution. Phagocytosis was suppressed by ACTH in this solution to the same degree as in the normal solution. Suppression by ACTH was reduced in phosphodiesterase inhibitor-containing solution. These results suggest that (1) ACTH suppresses extracellular Ca(2+)-dependent and -independent phagocytosis, (2) the suppression is not mediated by cAMP and (3) the inhibition of macrophage phagocytosis by ACTH is one of the mechanisms that modulate immune responses in stressful situations. PMID- 7534271 TI - Immunogenic combinations of HIV-1 B- and heterologous T-cell epitopes. AB - Peptides were synthesized which combined HIV-1 B-epitopes from gp41, p34pol and heterologous T-cell epitopes from hepatitis B virus (HBV) core or tetanus toxoid. Mixtures of these composite peptides and peptides representing single HIV-1 B epitopes were used to immunize rabbits in an adjuvant-free immunization regimen. Fusion to T-cell epitopes made the HIV-1 B-epitopes immunogenic and high titers of anti-HIV-1 antibodies were reached. Efficient antibody response against an immunorecessive HIV-1 B-epitope from p34 pol introduced as a B+T-composite also developed in rabbits pre-immunized by composites of the same T-cell epitopes but with a B-epitope from gp41. Fusion changed the fine antigenicity of the epitopes, but at least part of the antibodies against gp41-containing B+T composites recognized whole viral gp160. Composite peptides stimulated T-cells in the majority of the immunized animals. PMID- 7534266 TI - Dexamethasone inhibits the development of mast cells from dispersed human fetal liver cells cultured in the presence of recombinant human stem cell factor. AB - Human fetal liver cells cultured in the presence of recombinant human stem cell factor (rhuSCF) give rise to highly purified mast cell populations. This study examined the effect of steroid hormones on mast cell differentiation. Dispersed fetal liver cells cultured in the presence of rhuSCF at 50 ng/ml and in the presence or absence of various steroid hormones for 4 weeks, were analysed for the presence of mast cells by metachromatic staining with toluidine blue, by immunohistochemistry with a monoclonal antibody against tryptase, and by immunofluorescent flow cytometry with a monoclonal antibody against Kit. Dexamethasone added to the cultures at day 0 resulted in a dose-dependent inhibition of rhuSCF-induced mast cell differentiation with > 85% inhibition seen at a dose of 10(-6) M. A similar effect was seen with hydrocortisone, but not with oestradiol or progesterone. The addition of dexamethasone resulted in decreased DNA synthesis in 14-day-old cultured cells, as assessed by incorporation of bromodeoxyuridine. Addition of dexamethasone to 3-week-old SCF dependent fetal liver mast cells had no significant effect on mast cell survival. Removal of dexamethasone after 3 weeks of culture with SCF did not result in mast cell development. Thus, dexamethasone inhibits SCF-induced development of mast cells from fetal liver cells, but shows no appreciable effect on developed mast cells. PMID- 7534272 TI - Multifocal pattern of postnatal development of the macroglial framework of the rat fimbria. AB - The development of the rat fimbria over the first postnatal month is associated with an approximate doubling of the tract diameter, a large increase in the number of glial cells, and the transformation of the prenatal radial glial skeleton into the adult interfascicular glial rows of solitary astrocytes and contiguous myelinating oligodendrocytes. The ventricular zone is reduced from a heterogeneous germinal layer of three or more cells thick at birth to the mature adult unicellular ependyma of homogeneous pale, mitotically inactive cells by the end of the second postnatal week. Mitoses are present throughout the body of the tract at all times, and persist, at reduced levels, in the adult. At birth the interior of the fimbria has only few scattered glial cell nuclei, largely solitary, or at most in longitudinal pairs. Over the first two postnatal weeks, the numbers and density of the interfascicular glia increase continuously. The scattered cells and cell clusters become progressively transformed into longer unicellular rows, which are aligned along the longitudinal axis of the tract, and which finally coalesce to form the continuous regular astrocyte/oligodendrocyte units that make up the interfascicular glial rows of the adult fimbrial glial skeleton. The increased cell packing density of the developing fimbrial glia is associated with a substantial decrease in nuclear and cytoplasmic size. From the end of the second postnatal week, the characteristic, large pale solitary astrocytes, and the smaller, more numerous, densely stained, closely packed oligodendrocytes are recognisable. Immunostaining for glial fibrillary acidic protein shows that immediately after birth the characteristic embryonic pattern of regular parallel radial glial processes starts to be modified by the progressive accumulation of longitudinal astrocytic processes, so the prenatal radial glial framework is rapidly transformed into the adult type of rectilinear array of radial and longitudinal processes. The development of the oligodendrocytes is shown clearly by immunostaining for myelin basic protein in enlarged, cytoplasm-rich, symmetrically placed cell pairs first seen at around P7. At P8-P10, there is a characteristic pattern of simultaneous multifocal maturation in which a single oligodendrocyte in each cluster develops a full complement of parallel, rather varicose myelinating processes. By P14 myelination is becoming confluent, oligodendrocytes are smaller, darker, with little cytoplasm, and individual myelinating processes cannot be discerned. Even at the end of the first postnatal month there are still many immature glia of indeterminate morphology. Myelination tends at first to be concentrated in the region adjacent to the hippocampus, and only reaches completion by the end of the second month.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534273 TI - Cloning and expression of a neutral phosphatase gene from Treponema denticola. AB - We have isolated and characterized a neutral phosphatase gene, phoN, from Treponema denticola ATCC 35405. The gene was isolated from a T. denticola clone bank constructed in the medium-copy-number plasmid vector pMCL19. Subcloning and nucleotide sequencing of the DNA insert from one phosphatase clone, pTph14, revealed that the activity corresponded to an open reading frame consisting of 1,027 bp coding for a 37.9-kDa protein. Hydrophobicity analysis indicated that the protein exhibits some hydrophobic regions. Indeed, partial purification of the phosphatase suggested that the enzyme was membrane associated both in T. denticola and in the Escherichia coli clone. The pH optimum of the enzyme, approximately pH 6.4, indicated that it corresponded to a neutral phosphatase activity from T. denticola. An examination of possible natural substrates for the enzyme suggested that this enzyme hydrolyzes nucleoside di- and triphosphates. Northern (RNA) blot analysis revealed that this phosphatase gene is not likely to be present in an operon structure. PMID- 7534274 TI - Cryptococcus neoformans fails to induce nitric oxide synthase in primed murine macrophage-like cells. AB - Nitric oxide (NO) is a microbiostatic gas generated by activated murine macrophages. Cytokine signals, gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) act synergistically to induce production of a macrophage nitric oxide synthase (NOS). A variety of intracellular pathogens, when recognized by macrophages primed with IFN-gamma, induce NOS by eliciting TNF alpha secretion, which then functions as a positive autocrine signal. In cell culture assays, a murine macrophage cell line (J774), primed with IFN-gamma, was tested for NOS induction upon challenge with virulent Cryptococcus neoformans. C. neoformans failed to induce macrophage NOS as measured by nitrite production. This was true irrespective of the C. neoformans-to-J774 ratio. Other nonpathogenic Cryptococcus species likewise failed to induce NOS, yet Saccharomyces cerevisiae, Histoplasma capsulatum, and Candida albicans were efficient inducers of NOS. Conditions which promoted attachment and/or phagocytosis of C. neoformans did not lead to NOS induction (including opsonization with specific antibodies against C. neoformans). Assays for transcriptional repressors of NOS were negative. Tests for consumption of nitrite by measurement of additional products of NOS induction were negative. No TNF alpha was detected by enzyme-linked immunosorbent assay in supernatants from C. neoformans-J774 cocultures. A mutant C. neoformans strain with a minimal, but visible, polysaccharide capsule also failed to induce NOS; however, several nonencapsulated mutants of C. neoformans did induce NOS. Failure of C. neoformans to act as an inducer of NOS may be related to the virulence of this pathogen in mice; C. neoformans is a unique example of a facultative intracellular pathogen which fails to induce NOS in primed macrophages. The mechanism appears to involve the failure of TNF-alpha secretion once the macrophage comes in contact with the fungus. The presence of the polysaccharide capsule appears to mask the signal necessary for TNF-alpha secretion and, ultimately, NOS induction. PMID- 7534275 TI - Ability of bacteria associated with chronic inflammatory disease to stimulate E selectin expression and promote neutrophil adhesion. AB - Porphyromonas gingivalis, Pseudomonas aeruginosa, and Helicobacter pylori have been shown to be associated with adult periodontal disease, chronic lung infections, and peptic ulcers, respectively. The ability of these bacteria to stimulate E-selectin expression and promote neutrophil adhesion, two components necessary for the recruitment of leukocytes in response to infection, was investigated. Little or no stimulation of E-selectin expression was observed with either P. gingivalis or H. pylori when whole cells, lipopolysaccharide (LPS), or cell wall preparations added to human umbilical cord vein endothelial cells were examined. P. aeruginosa was able to induce E-selectin to near-maximal levels; however, it required approximately 100 to 1,000 times more whole cells or LPS than that required by E. coli. Neutrophil-binding assays revealed that LPS and cell wall preparations obtained from these bacteria did not promote endothelial cell adhesiveness by E-selectin-independent mechanisms. In addition, P. gingivalis LPS blocked E-selectin expression by LPS obtained from other bacteria. We propose that lack of E-selectin stimulation and the inability to promote endothelial cell adhesiveness are two additional indications of low biologically reactive LPS. We suggest that this property of LPS may contribute to host tissue colonization. In addition, the ability of P. gingivalis to inhibit E-selectin expression may represent a new virulence factor for this organism. PMID- 7534276 TI - A Mycoplasma strain F38 growth-inhibiting monoclonal antibody (WM-25) identifies an epitope on a surface-exposed polysaccharide antigen. AB - Monoclonal antibody (MAb) WM-25 differentiates by in vitro growth inhibition Mycoplasma capricolum subsp. capripneumoniae (Mycoplasma strain F38), which causes contagious carpine pleuropneumonia, from other Mycoplasma spp. (F. R. Rurangirwa, T. C. McGuire, A. J. Musoke, and A. Kibor, Infect. Immun. 55:3219 3220, 1987). The antigen identified by MAb WM-25 was isolated from solubilized Mycoplasma strain F38 organisms by MAb WM-25 affinity chromatography and was stained with Schiff's reagent, but not with Coomassie blue, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of purified F38 polysaccharide with periodate abolished binding with MAb WM-25, and MAb WM-25 binding was blocked with laminarin, a complex oligosaccharide with beta(1-->3) sugar linkages. Purified F38 polysaccharide blocked both growth inhibition and agglutination of live F38 organisms caused by MAb WM-25 and rabbit antiserum to F38 organisms. The results in this paper demonstrate that MAb WM-25 binds a periodate-sensitive epitope on the F38 polysaccharide which is also exposed on the surface of Mycoplasma strain F38. Because MAb WM-25 also causes in vitro growth inhibition of F38, the reactive polysaccharide epitope may induce protective immune responses. PMID- 7534277 TI - Immunogenicity of genetically engineered glutathione S-transferase fusion proteins containing a T-cell epitope from diphtheria toxin. AB - Glutathione S-transferase (GST) has been shown to induce a marginal antibody response in experimental animals as well as partial protection against a number of parasitic worms, including Schistosoma and Fasciola species. The objective of our study was to increase the immunogenicity of GST by adding heterologous T-cell epitopes at the carboxy terminus of the protein. We generated recombinant GST proteins by attaching one or three tandem repeats of a T-cell epitope of CRM197, a nontoxic variant of diphtheria toxin. This T-cell epitope encoding the region of amino acids 366 to 383 of CRM197, when contained in a GST fusion protein and/or after purification as a recombinant peptide, retained the ability to induce a CRM197-specific T-cell response. The fusion protein containing a single T-cell epitope induced a strong T-cell proliferative response to GST and also enhanced anti-GST antibody production in mice. The addition of three repeats of the epitope did not augment the responses when compared with the responses of GST itself. The results suggest that the addition of a single T-cell epitope to a larger protein like GST increases the immunogenicity of the protein. PMID- 7534278 TI - Isolation and characterization of a family of porin proteins from Helicobacter pylori. AB - Two-dimensional gel electrophoresis was used to identify heat-modifiable outer membrane proteins, which were candidates for porins, from Helicobacter pylori membrane preparations. Four such proteins with apparent molecular masses of 48, 49, 50, and 67 kDa were isolated. The four proteins copurified together after selective detergent solubilizations followed by anion-exchange chromatography, and each protein was ultimately purified to homogeneity by gel purification. These proteins were then tested for pore-forming ability with a planar lipid bilayer model membrane system. All four proteins appeared to be present as monomers, and they formed pores with low single-channel conductances in 1.0 M KCl of 0.36, 0.36, 0.30, and 0.25 nS, respectively, for the 48-, 49-, 50-, and 67-kDa proteins which we propose to designate HopA, HopB, HopC, and HopD. N-terminal amino acid sequence analyses showed a high degree of homology among all four proteins, and it appears that these proteins constitute a family of related porins in H. pylori. PMID- 7534279 TI - CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia. AB - This study was carried out to determine the role of CD14 receptors in the uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Treatment of microglial cell cultures with antibodies to CD14 or with soluble CD14 significantly blocked infection by M. tuberculosis H37Rv, suggesting that CD14 receptors could facilitate entry of nonopsonized tubercle bacilli into macrophages within the brain. PMID- 7534280 TI - Preventing the transmission of HCV infection in hemodialysis. PMID- 7534281 TI - Spontaneous fracture of the outlet catheter of a totally implanted catheter system (Port-A-Cath). AB - A case of spontaneous fracture of the outlet catheter of a totally implanted catheter system (Port-A-Cath) is presented. The outlet catheter was fractured at the entrance into the left subclavian vein twenty-one weeks after insertion and the distal part was embolized in the right ventricle. The embolized catheter fragment was retrieved by a 'goose-neck' snare via the right femoral vein. The awareness of a possible spontaneous fracture of the outlet catheter of a totally implanted catheter system (Port-A-Cath) is important to prevent accidental spillage of potent cytotoxic substances. PMID- 7534284 TI - Does Sjogren's syndrome predispose surgical patients to acquired hemophilia? AB - The authors report the first American case of a factor VIII inhibitor found in a patient with Sjogren's syndrome. The inhibitor was isolated from a patient with no known hematologic disorder who was seen with a compartment syndrome of the left thigh after sustaining a fall from bed. The Sjogren's syndrome had been previously diagnosed via lower lip biopsy. After fasciotomy, signs and symptoms of intra-abdominal hemorrhage developed, and the patient was taken to laparotomy, where no hemorrhage was found. A coagulopathy developed, and the patient's hemoglobin continued to fall. A complete factor analysis revealed a factor VIII inhibitor that was quantified at 40 Bethesda units. After vigorous therapy--which included factor concentrates, immunoglobulins, steroids, antifibrinolytic agents, and blood replacement--the patient's bleeding stopped and she continued to convalesce under hospital care until her ultimate death from respiratory problems. Acquired hemophilia with a factor VIII inhibitor may be associated with Sjogren's syndrome. PMID- 7534282 TI - An SV40-immortalized human corneal epithelial cell line and its characterization. AB - PURPOSE: The authors attempted to immortalize human corneal epithelial cells; it is difficult to propagate primary human corneal epithelial cells because of scarcity of available tissue. However, cell immortalization by virus is always accompanied by shedding of free virus. The current study was performed to establish a cell line that produces no free viral particle. METHODS: Primary cultured human corneal epithelial cells were infected with a recombinant sv40 adenovirus vector and were cloned three times to obtain a continuously growing cell line. Morphologic, cytologic, and biochemical characteristics of this cell line were analyzed. RESULTS: This cell line continued to grow for more than 400 generations, exhibiting a cobblestone-like appearance similar to normal corneal epithelial cells in culture. Transmission electron microscopy showed the evidence for the characteristic features of epithelial cells, including desmosome formation and development of microvilli. It expressed cornea-specific, 64-kD cytokeratin in addition to five major insoluble proteins. By enzymatic analysis using NADP as a coenzyme and a gas chromatograph mass spectrometer, this cell line was found to possess 8.71 IU/mg protein of aldehydedehydrogenase activity. When this cell line was grown at air-liquid interface on collagen type I gel, it differentiated in a multilayered fashion. CONCLUSIONS: The authors have established an SV40-immortalized human corneal epithelial cell line with properties similar to normal corneal epithelial cells. PMID- 7534283 TI - Cardiac and hemodynamic tolerability of iopromide with or without sodium or iloprost and of ioversol in the anesthetized rat. AB - RATIONALE AND OBJECTIVES: In this study, the cardiac and hemodynamic effects of iopromide alone were compared with those of two combination preparations (iopromide plus sodium and iopromide plus the prostacyclin analog iloprost) and with ioversol after left ventricular bolus administration in rats. METHODS: The tracheae of anesthetized male Wistar rats were cannulated to facilitate spontaneous respiration. The animals were set up to allow recording or calculation of the following parameters: femoral arterial blood pressure (systolic, mean, and diastolic), left ventricular end-diastolic pressure, heart rate, and contractility. Iopromide (330 mg iodine/mL; 2 g iodine/kg) with or without sodium chloride (20 mmol/L) or iloprost (approximately 50 ng/mL; dose: 300 ng/kg) was injected into the left ventricle within 30 seconds. Ioversol (320 mg iodine/mL) was used at the same dose and injection rate. RESULTS: Iopromide and ioversol induced transient changes in blood pressure (decrease followed by an increase), left ventricular end-diastolic pressure (increase), heart rate (decrease), contractility (increase followed by decrease), and electrocardiogram (extrasystoles, ST depression). Ioversol exhibited more pronounced effects on contractility and ST depression. The differences were statistically significant. The addition of sodium to iopromide resulted in a slight, but not significant influence on cardiac or hemodynamic parameters. The addition of iloprost improved ST depression slightly and hemodynamics significantly resulting in less mean and end-diastolic blood pressure change and less heart rate decrease. Contractility was significantly increased compared with iopromide with or without sodium. CONCLUSIONS: The addition of sodium or iloprost to nonionic contrast media might be useful in the alleviation of cardiac and hemodynamic side-effects. PMID- 7534285 TI - The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl. AB - c-Mpl is a member of the cytokine receptor superfamily, expressed primarily on hematopoietic cells. Recently, the c-Mpl ligand was cloned and found to have thrombopoietic activity. In this paper we report that ligand binding induced tyrosine phosphorylation in BaF3 cells engineered to express the murine Mpl receptor (BaF3/mMpl). Phosphorylation occurred within 1 min at cytokine concentrations sufficient for proliferation of receptor-bearing cells. Using specific antibodies for immunoprecipitation and Western blotting, several of these phosphorylated proteins were identified. Shc and Jak2, known cytokine signaling molecules, and the c-Mpl receptor were shown to be major substrates for tyrosine phosphorylation. In contrast, phospholipase C-gamma and phosphatidylinositol 3-kinase displayed little and no tyrosine phosphorylation, respectively, after thrombopoietin stimulation. Co-immunoprecipitation studies demonstrated that Jak2 became physically associated with c-Mpl relatively late in the observed time course (20-60 min), significantly later than tyrosine phosphorylation of Jak2 (1-5 min). These results suggest that c-Mpl induces signal transduction pathways similar to those of other known cytokines. Additionally, in light of its late physical association with c-Mpl following ligand binding, Jak2 may not be the initiating tyrosine kinase in the thrombopoietin-induced signaling cascade. PMID- 7534286 TI - Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. AB - Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125fak). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210BCR/ABL, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210BCR/ABL and multiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton. PMID- 7534287 TI - Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis. AB - Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation. PMID- 7534288 TI - The rabbit mammary gland prolactin receptor is tyrosine-phosphorylated in response to prolactin in vivo and in vitro. AB - We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor. PMID- 7534289 TI - Insulin-like growth factor-I stimulates tyrosine phosphorylation of endogenous c Crk. AB - Crk, a cellular homolog of v-crk, is an SH2 and SH3 domain-containing adaptor protein related to Grb2 and Nck, two proteins which have been shown to be involved in growth factor signal transduction. Crk proteins have recently been found to associate with two guanine nucleotide releasing proteins, mSos and C3G, and thus appear to lie on the Ras pathway. We investigated whether Crk is a target for the insulin-like growth factor I (IGF-I) receptor tyrosine kinase. We show that IGF-I stimulates tyrosine phosphorylation of Crk II via stimulation of endogenous IGF-I receptors in both 293 cells and NIH-3T3 cells. IGF-I stimulated tyrosine phosphorylation of Crk II in a dose- and time-dependent manner. In 293 cells, which express both IGF-I and insulin receptors, insulin also induced a dose-dependent tyrosine phosphorylation of Crk II, but with somewhat reduced sensitivity, compared to IGF-I. In NIH 3T3 cells, IGF-I also stimulated tyrosine phosphorylation of a 45- kDa protein which co-immunoprecipitated with Crk II. These findings indicate that Crk II is an endogenous substrate of the IGF-I receptor tyrosine kinase and provide the first demonstration that a mitogenic growth factor induces tyrosine phosphorylation of endogenous c-Crk. PMID- 7534290 TI - CD14: physical properties and identification of an exposed site that is protected by lipopolysaccharide. AB - Soluble CD14 (sCD14) is a 55-kDa serum protein that binds lipopolysaccharide (LPS) and mediates LPS-dependent responses in a variety of cells. Using recombinant sCD14 expressed in Chinese hamster ovary (CHO) cells, we examined the structural characteristics of sCD14 and sCD14.LPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial beta-sheet (40%) and a well-defined tertiary structure with the tryptophan residues located in environments with different degrees of hydrophobicity and solvent exposure. The spectra of the sCD14.LPS complex are identical within experimental error to the uncomplexed sCD14. Changes in surface accessibility upon LPS binding were examined using limited proteolysis with endoproteinase Asp-N. This analysis revealed that aspartic acid residues at amino acids 57, 59, and 65 are susceptible to cleavage by Asp-N, while the same residues are protected from proteolytic cleavage in the sCD14.LPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14. PMID- 7534291 TI - Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64. AB - CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Recent limited proteolysis studies have implicated a region in CD14 between amino acids 57 and 64 as being involved in LPS interaction. To specifically assess the importance of this region with respect to LPS binding, we constructed a mutant sCD14 (sCD14 delta 57-64) lacking amino acids 57-64. sCD14 delta 57-64 was isolated from the serum-free conditioned medium of this cell line, and, in all assays, the purified protein failed to recognize LPS or enable LPS-dependent responses in cells. We also demonstrated that the region between amino acids 57 and 64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native polyacrylamide gel electrophoresis assays were used to demonstrate that MEM-18 and LPS compete for the same binding site on CD14. These data strongly suggest that the region spanning amino acids 57-64 binds LPS and that formation of sCD14.LPS complex is required in order for sCD14 mediated responses to occur. PMID- 7534292 TI - Role of p70 S6 protein kinase in angiotensin II-induced protein synthesis in vascular smooth muscle cells. AB - Angiotensin II (AII) is a growth factor which induces cellular hypertrophy in cultured vascular smooth muscle cells (SMC). To understand the molecular basis of this action, we have examined the role of the 70-kDa S6 kinases (p70S6K) in the hypertrophic response to AII in aortic SMC. AII potently stimulated the phosphotransferase activity of p70S6K, which reached a maximal value at 15 min and persisted for at least 4 h. This response was completely abolished when the cells were incubated in the presence of the AT1-selective receptor antagonist losartan. The enzymatic activation of p70S6K was associated with increased phosphorylation of the enzyme on serine and threonine residues. The immunosuppressant drug rapamycin was found to selectively inhibit the activation of p70S6K by AII, but not the activation of mitogen-activated protein kinase or the induction of c-fos mRNA expression. Treatment of aortic SMC with rapamycin also potently inhibited AII-stimulated protein synthesis with a half-maximal concentration similar to that required for inhibition of p70S6K. These results provide strong evidence that p70S6K plays a critical role in the signaling pathways by which AII induces hypertrophy of vascular SMC. PMID- 7534293 TI - E2F-1 and a cyclin-like DNA repair enzyme, uracil-DNA glycosylase, provide evidence for an autoregulatory mechanism for transcription. AB - The cell cycle-dependent transcription factor, E2F-1, regulates the cyclin-like species of the DNA repair enzyme uracil-DNA glycosylase (UDG) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human UDG promoter sequences, that expression of E2F-1 activates the UDG promoter through several E2F sites. The major putative downstream site for E2F, located in the first exon, serves as a target for E2F-1/DP1 complex binding in vitro. We also provide evidence for the functional relationship between the cyclin-like UDG gene product and E2F. High levels of UDG expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of UDG in Saos 2 cells was observed to delay growth late in G1 phase and transiently arrest these cells from progressing into the S phase. This hypothetical model integrates one mechanism of DNA repair with the cell cycle control of gene transcription, likely through E2F. This implicates E2F as a multifunctional target for proteins and enzymes, possibly, responsive to DNA damage through the negative effect of UDG on E2F-mediated transcriptional activity. PMID- 7534295 TI - The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase. AB - We report the first active site substrate specificity analysis of a tyrosine specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well. PMID- 7534294 TI - Lipopolysaccharide (LPS) signal transduction and clearance. Dual roles for LPS binding protein and membrane CD14. AB - Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14. PMID- 7534296 TI - Regulation of the sequence-specific DNA binding function of p53 by protein kinase C and protein phosphatases. AB - The p53 tumor suppressor protein is a transcription factor with sequence-specific DNA binding activity that is thought to be important for the growth-inhibitory function of p53. DNA binding appears to require activation of a cryptic form of p53 by allosteric mechanisms involving a negative regulatory domain at the carboxyl terminus of p53. The latent form of p53, reactive to the carboxyl terminal antibody PAb421, is produced in a variety of eukaryotic cells, suggesting that activation of p53 is an important rate-limiting step in vivo. In this report we provide evidence that phosphorylation of serine 378 within the carboxyl-terminal negative regulatory domain of the human p53 protein by protein kinase C correlates with loss of PAb421 reactivity and a concomitant activation of sequence-specific DNA binding. These effects are reversed by subsequent dephosphorylation of the protein kinase C-reactive site by protein phosphatases 1 (PP1) and 2A (PP2A), which restore the reactivity of p53 to PAb421 and regenerate the latent form of p53 lacking significant DNA binding activity. Thus, p53 is subject to both positive and negative regulation by reversible enzymatic modifications affecting the latent or active state of the protein, suggesting a possible mechanism for the regulation of its tumor suppressor function. PMID- 7534297 TI - Characterization of the promoter region of the human apurinic endonuclease gene (APE). AB - Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region. PMID- 7534298 TI - The MEK kinase activity of the catalytic domain of RAF-1 is regulated independently of Ras binding in T cells. AB - Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain. PMID- 7534299 TI - Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways. AB - Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the tyrosine phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells. PMID- 7534300 TI - Identification of a 190-kDa protein as a novel substrate for the insulin receptor kinase functionally similar to insulin receptor substrate-1. AB - Recently, we generated mice with a targeted disruption of the insulin receptor substrate-1 (IRS-1) gene and demonstrated that they exhibited growth retardation and mild insulin resistance, suggesting the presence of IRS-1-independent pathway that partially substitutes for IRS-1 in IRS-1-deficient mice (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. (1994) Nature 372, 182 186). We have examined the insulin-stimulated tyrosine-phosphorylated proteins in livers of wild type and IRS-1-deficient mice. Tyrosine phosphorylation of an 190 kDa protein (pp190) by insulin was significantly stimulated in livers of IRS-1 deficient mice, which was weakly observed in wild type mice in addition to IRS-1. We also demonstrated that pp190 was immunologically distinct from IRS-1 and was associated with both the 85-kDa subunit of phosphatidylinositol 3-kinase and the Grb2/Ash molecule as IRS-1. We identified pp190 as a novel substrate for insulin receptor kinase (IRS-2), which can bind both PI3-kinase and Ash/Grb2, and whose tyrosine phosphorylation is specifically induced in IRS-1-deficient mice. These data suggested that pp190 may play some physiological roles in insulin's signal transduction; furthermore, induction of tyrosine phosphorylation of pp190 may be one of the compensatory mechanisms that substitute for IRS-1 in IRS-1-deficient mice. PMID- 7534301 TI - Cloning, expression, and chromosome mapping of human galectin-7. AB - The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19. PMID- 7534302 TI - Seven helix cAMP receptors stimulate Ca2+ entry in the absence of functional G proteins in Dictyostelium. AB - Surface cAMP receptors (cARs) in Dictyostelium transmit a variety of signals across the plasma membrane. The best characterized cAR, cAR1, couples to the heterotrimeric guanine nucleotide-binding protein (G protein) alpha-subunit G alpha 2 to mediate activation of adenylyl and guanylyl cyclases and cell aggregation. cAR1 also elicits other cAMP-dependent responses including receptor phosphorylation, loss of ligand binding (LLB), and Ca2+ influx through a G alpha 2-independent pathway that may not involve G proteins. Here, we have expressed cAR1 and a related receptor, cAR3, in a g beta- strain (Lilly, P., Wu. L., Welker, D. L., and Devreotes, P. N. (1993) Genes & Dev. 7,986-995), which lacks G protein activity. Both cell lines failed to aggregate, a process requiring the G alpha 2 and G beta- subunits. In contrast, cAR1 phosphorylation in cAR1/g beta- cells showed a time course and cAMP dose dependence indistinguishable from those of cAR1/G beta+ controls. cAMP-induced LLB was also normal in the cAR1/g beta- cells. Finally, cAR1/g beta- cells and cAR3/g beta- cells showed a Ca2+ response with kinetics, agonist dependence, ion specificity, and sensitivity to depolarization agents that were like those of G beta+ controls, although they accumulated fewer Ca2+ ions per cAMP receptor than the control strains. Together, these results suggest that the G beta-subunit is not required for the activation or attenuation of cAR1 phosphorylation, LLB, or Ca2+ influx. It may, however, serve to amplify the Ca2+ response, possibly by modulating other intracellular Ca2+ signal transduction pathways. PMID- 7534303 TI - v-Abl-mediated apoptotic suppression is associated with SHC phosphorylation without concomitant mitogen-activated protein kinase activation. AB - A temperature-sensitive mutant of the v-Abl protein has previously been shown to exhibit tyrosine protein kinase activity in Interleukin 3 (IL-3)-dependent IC.DP cells grown at the permissive temperature (32 degrees C) but not at the restrictive temperature (39 degrees C). These IC.DP cells are dependent on IL-3 for suppression of apoptosis at 39 degrees C, but at 32 degrees C cells will survive without added growth factor. Both IL-3 and v-Abl stimulated the tyrosine phosphorylation of SHC and GTPase-activating protein. However, while IL-3 stimulated similar levels of tyrosine phosphorylation in p46shc and p52shc, v-Abl preferentially phosphorylated p52shc, an event that occurred within 1 h of temperature switch. v-Abl also differentially associated with p46shc in a temperature-independent manner. In contrast, only IL-3 stimulated detectable increases in both myelin basic protein kinase and mitogen-activated protein (MAP) kinase kinase in in vitro assays, although in more specific MAP kinase activity assays a very slight increase in the activity of this enzyme was observed after 6 h at the permissive temperature. Time course studies suggest that phosphorylation and association of SHC with v-Abl is insufficient to lead to significant activation of MAP kinase and that activation of the MAP kinase kinase/MAP kinase pathway is not required for apoptotic suppression. PMID- 7534304 TI - Differential regulation of alpha 4 integrin-dependent binding to domains 1 and 4 of vascular cell adhesion molecule-1. AB - The vascular cell adhesion molecule-1 (VCAM-1) plays an important role in diverse physiological and pathological processes. The homologous first and fourth immunoglobulin-like domains of the seven domain form of VCAM-1 present binding motifs for alpha 4 beta 1 integrin. Using a panel of VCAM-1 domain deletion mutants we show that alpha 4 beta 7 integrin interacts with both domains 1 and 4. In contrast to their identical domain usage, alpha 4 beta 1 and alpha 4 beta 7 integrins differ in the activation states required for binding to domains 1 and 4 of VCAM-1. We show that integrin alpha 4 beta 1 required significantly higher concentrations of Mn2+ than integrin alpha 4 beta 7 to support half-maximal adhesion to domain 4. Moreover, a clear difference in the capacity of integrins alpha 4 beta 1 and alpha 4 beta 7 to interact with domain 4 was detected in the presence of Ca2+ and Mg2+ cations. Adhesion to domain 1 of VCAM-1, however, was not affected by integrin heterodimer composition. Instead, the activity level of integrin alpha 4 beta 1 for domain 1 binding was regulated by CD24 expression. Binding to seven domain VCAM-1 was not altered significantly by beta 1 and beta 7 subunits or CD24. These data indicate that integrin heterodimer composition and CD24 expression differentially modulate integrin binding to domains 1 and 4 of VCAM-1. Mechanisms that alter integrin binding specificity or monovalent versus divalent interactions may affect the strength of adhesion as well as signal transmission in adherent cells and may therefore be critical to controlling the cellular response to integrin occupancy. PMID- 7534305 TI - Bacterial lipopeptides induce nitric oxide synthase and promote apoptosis through nitric oxide-independent pathways in rat macrophages. AB - Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy) (2R,2S)-propyl]-N-palmytoyl-(R)-C ysSerLys4 or S(-)[2,3-bis(pamitoyloxy)-(2R,2S) propyl]-N-palmytoyl-(R)-++ +CysAlaLys4, two synthetic bacterial lipopeptides, promoted the expression of the inducible form of nitric oxide synthase, exhibiting a temporal pattern of nitric oxide release that was delayed with respect to the induction elicited by bacterial lipopolysaccharide. Treatment of macrophages with genistein blocked the nitric oxide synthesis triggered by the lipopeptides or lipopolysaccharide. Simultaneous incubation with lipopolysaccharide and lipopeptide resulted in an antagonistic effect on nitric oxide synthase mRNA levels and on nitrite plus nitrate release to the medium. Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells. PMID- 7534306 TI - Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA. AB - Analysis of the primary structure of peptide synthetases involved in non ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure. These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation. To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098 amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His6-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATP dependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His). However, it retained the functions of specific amino acid activation and thioester formation. The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP dependent activation as well as in thioester formation and racemization. PMID- 7534307 TI - The alternative splicing pattern of the tenascin-C pre-mRNA is controlled by the extracellular pH. AB - Alternative splicing of primary transcripts is an ubiquitous and reversible mechanism for the generation of multiple protein isoforms from single genes. Here we report that in cultured normal human fibroblasts, small pH variations of the culture medium (from 7.2 to 6.9) strikingly modify the alternative splicing pattern of the tenascin-C primary transcript. Since such extracellular pH variations occur in many normal and pathological conditions, microenvironmental pH may be an important element for the regulation of RNA alternative splicing in vivo. PMID- 7534308 TI - A 40-amino acid segment of the growth hormone receptor cytoplasmic domain is essential for GH-induced tyrosine-phosphorylated cytosolic proteins. AB - It has become evident that intracellular protein phosphorylation plays an important role in mediating signal transduction of hormones and growth factors, including growth hormone (GH). We have previously demonstrated that GH can stimulate tyrosine phosphorylation of cellular proteins with approximate molecular masses of 95,000 daltons (pp95) in GH-treated 3T3-F442A preadipocytes and in mouse L cells that express recombinant porcine or bovine GH receptors. In the present study, a series of GH receptor (GHR) truncation analogs were constructed and examined for their abilities to induce pp95. The results revealed that a region of approximately 40 amino acids in the porcine GHR cytoplasmic domain is essential for induction of pp95. The results also established that the 115 amino acids (517-638) near the C terminus of porcine GHR are not required for pp95 induction. Moreover, the basal levels of GH-induced pp95 in parental mouse L cells was suppressed by expression of these GHR truncation analogs. This suggests that pp95 induced by GH may be mediated by GHR dimerization and can be inhibited by overexpression of truncated porcine GHRs. PMID- 7534309 TI - CD36 induction on human monocytes upon adhesion to tumor necrosis factor activated endothelial cells. AB - Cell adhesion between circulating monocytes and the endothelium is a critical component of vascular thromboregulation and atherogenesis. The biochemical and genetic consequences of adhesion are poorly understood. We have found that monocyte surface expression of CD36, an integral membrane receptor for thrombospondin, collagen, and oxidized low density lipoprotein, increased dramatically upon adhesion to tumor necrosis factor-activated human umbilical vein endothelial cells (HUVEC). Expression was assessed by indirect immunofluorescence microscopy and immunoblotting using monoclonal antibodies to CD36. Steady-state CD36 mRNA levels, detected by RNase protection assay, also showed a similar pattern of up-regulation. To verify the adhesion dependence of the observed phenomenon, monocytes were co-cultured with tumor necrosis factor activated HUVEC in a transwell apparatus that physically separated monocytes from the endothelial cells. Under these conditions, no increase in CD36 expression was detected, demonstrating that the enhanced monocyte CD36 expression observed is not due to soluble factors released by HUVEC. To characterize the specific adhesion molecules involved in the process, co-culture assays were performed on murine L cells transfected with either human E-selectin or intercellular adhesion molecule-1 cDNAs. A dramatic increase in CD36 mRNA was seen upon monocyte adhesion to E-selectin-transfected L cells compared with adhesion to intercellular adhesion molecule-1 or control transfectants. Furthermore, monoclonal antibodies to E-selectin inhibited the adhesion-dependent up regulation of CD36 mRNA induced by transfected L cells or cytokine-activated endothelial cells. These findings demonstrate adhesion-dependent gene regulation of monocyte CD36 and suggest the possible involvement of E-selectin in initiating this process. PMID- 7534310 TI - Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase. AB - More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts. Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end. Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure. Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA. When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process. Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript. Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template. PMID- 7534312 TI - Inhibition of platelet-derived growth factor-BB-induced fibroblast proliferation by plasmin-activated alpha 2-macroglobulin is mediated via an alpha 2 macroglobulin receptor/low density lipoprotein receptor-related protein-dependent mechanism. AB - alpha 2-Macroglobulin (alpha 2M) is a potentially important regulator of platelet derived growth factor-BB (PDGF-BB)-stimulated cell growth due to our previous observation that PDGF-BB binds to alpha 2M noncovalently (Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol. Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor recognized) alpha 2M on the PDGF-BB-induced proliferation of mouse Swiss 3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel electrophoresis showed that plasmin converted alpha 2M to its electrophoretically "fast" form at a 2:1 molar ratio and that 125I-PDGF-BB bound both alpha 2M and alpha 2M-plasmin. PDGF BB-induced growth was not affected by native alpha 2M (0.3 microM) or plasmin (0.6 microM). The combination of plasmin and alpha 2M (2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation 80-90%. Complexes of PDGF-BB.alpha 2M purified by gel filtration chromatography retained growth promoting activity, but the PDGF BB.alpha 2M-plasmin complex did not. Preincubation of fibroblasts (37 degrees C for 24 h) with alpha 2M-plasmin did not change 125I-PDGF-BB binding or affect gene expression of the 6.5-kilobase PDGF-alpha receptor or 5.2-kilobase PDGF-beta receptor mRNA. However, preincubation with alpha 2M-plasmin (0-4 degrees C for 4 h) increased 125I-PDGF-BB binding 2-fold, and this increase was blocked by a receptor-associated protein antagonist of the alpha 2M-receptor/low density lipoprotein receptor-related protein. The receptor-associated protein antagonist blocked 125I-alpha 2M-methylamine binding, inhibited PDGF-BB-alpha 2M-plasmin uptake from fibroblast-cultured supernatants, and abolished the inhibitory effect of alpha 2M-plasmin on PDGF-stimulated growth. These data suggest that inhibition of PDGF-stimulated proliferation by alpha 2M-plasmin is mediated in part by clearance of PDGF-BB-alpha 2M-plasmin through the lipoprotein receptor-related protein. PMID- 7534311 TI - Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine phosphorylation-regulated mechanism. AB - We investigated the mechanism by which ligand-activated epidermal growth factor receptors (EGFR) associate with coated pit adaptor protein (AP) complexes. In vivo association, assayed by coimmunoprecipitation of AP with mutant EGFR, required tyrosine kinase activity, intact autophosphorylation sites, and the regulatory carboxyl terminus of EGFR. The role of autophosphorylation of EGFR in interaction with AP was examined in vitro using a BIAcore instrument. Purified active EGFR, immobilized on the biosensor surface, was reversibly autophosphorylated or dephosphorylated by treatment with ATP or phosphatase. Autophosphorylation of EGFR significantly increased AP binding. Once formed, EGFR AP complexes were resistant to disassembly by dephosphorylation of EGFR or competition with phosphotyrosine, indicating that phosphorylated tyrosine residues do not directly participate in AP binding. Induction of conformational changes in EGFR by treatment with urea increased AP binding up to 10-fold in the absence of EGFR autophosphorylation. A recombinant EGFR carboxyl terminus specifically bound the AP complex and each of the isolated alpha- and beta subunits of AP2. We conclude that tyrosine autophosphorylation of EGFR exposes structural motif(s) in the carboxyl terminus of EGFR that interact specifically with AP2. PMID- 7534313 TI - Identification of a novel heparin binding domain in RHAMM and evidence that it modifies HA mediated locomotion of ras-transformed cells. AB - We have previously reported that the hyaluronan (HA) receptor RHAMM (Receptor for HA Mediated Motility) [Turley et al., 1991] contains two HA binding motifs located within a 35 amino acid region of its C-terminus end [Yang et al., 1993] and that HA stimulation of the motility of ras-transformed fibroblasts is mediated via its interaction with RHAMM. Here we show that RHAMM also contains binding sites for heparin (HP) and that interaction of HP with these sites can regulate the locomotion of ras-transformed fibroblasts. At low concentrations (0.01 mg/ml), HP inhibited HA-induced locomotion of ras-transformed cells in a manner independent of RHAMM. At higher, but still physiological concentrations (0.1 mg/ml), HP alone stimulated cell locomotion and this stimulation appeared to be RHAMM-dependent as it was blocked by anti-RHAMM antibodies. Other related glycosaminoglycans such as chondroitin sulfate and dermatin sulfate had no effect on cell motility. In ligand blotting assays, GST-RHAMM fusion protein was shown to bind biotin-labelled HP and this binding was displaceable with unlabelled HP. In similar ligand binding analyses conducted with truncations of RHAMM fusion protein, the HP binding region was found to be localized in the same 35 amino acid segment of RHAMM that contains the two HA binding domains. Synthetic peptides corresponding to these HA binding domains were retained on and bound effectively to an HP-Sepharose affinity column. Fusion proteins generated by linkage of these peptides to the non-HP binding amino terminus of RHAMM conferred HP binding capacity to the genetically engineered proteins. Conversely, deletion of the HA binding domains of RHAMM resulted in fusion proteins devoid of HP binding activity. The relative affinities of RHAMM for HA and HP, as determined by competition and transblot assays as well as quantification of binding at various salt concentrations, indicated that RHAMM had lower affinity for HP than that for HA. These results demonstrate the existence of a new HP binding motif that has biological relevance to cell locomotion. PMID- 7534314 TI - Increased expression of basic fibroblast growth factor in hyperoxic-injured mouse lung. AB - Basic fibroblast growth factor (bFGF) is a mitogenic polypeptide for a wide variety of cell types and has been immunolocalized in the rodent and human lung. We investigated the mRNA and protein expression of bFGF in hyperoxic-injured adult mouse lungs using northern blot analysis and immunohistochemistry. Mice (6 8 weeks) were continuously exposed to 80% oxygen up to 4 days. Levels of bFGF mRNA were increased from room air control on days 3 and 4 of hyperoxia. mRNA levels of acidic fibroblast growth factor (aFGF), fibronectin, and transin/stromelysin were also examined in this injury model. Similar to bFGF, the fibronectin and transin/stromelysin mRNA levels were increased after 3 days of hyperoxia. In contrast, the aFGF mRNA levels were gradually reduced on each day of hyperoxia. A rabbit polyclonal anti-bFGF antibody was used to determine the distribution and levels of expression in the hyperoxic-injured lungs. The room air control and day 1 hyperoxic-exposed lungs exhibited staining for bFGF in the basement membranes of the blood vessels, airways, and alveoli. Patchy but intense alveolar staining was prominent on day 4 of hyperoxia. The bFGF immunoreactivity of blood vessels and airways was unaffected by the hyperoxia exposure. These results suggest that bFGF may play a role in the alveolar response to hyperoxic induced injury by virtue of the altered mRNA levels and protein distribution in this injury model. PMID- 7534315 TI - Expression of alpha 1-chimaerin (rac-1 GAP) alters the cytoskeletal and adhesive properties of fibroblasts. AB - The small GTP-binding protein rac-1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac-1 protein is regulated by the interplay of several activities: proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac-1/GAP interactions to the overall activity of rac-1 by expressing alpha 1-chimaerin, a rac-1-specific GAP, in fibroblasts. NIH 3T3 cells were transfected with alpha 1-chimaerin cDNA-containing expression vector and stable clones were established. Extracts prepared from alpha 1 chimaerin-expressing cells showed rac-1 GAP activity that was regulated by phosphatidylserine and phorbol ester. The cells expressing alpha 1-chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronectin-coated glass surface, suggesting that the expression of a rac-1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the alpha 1-chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophosphatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac-1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was impaired in the alpha 1 chimaerin expressing cells. These results demonstrate that expression of a GAP for rac-1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac-1 function. PMID- 7534316 TI - NADPH-diaphorase neurons in the retina of the hamster. AB - NADPH-diaphorase-positive neurons have been demonstrated in the inner nuclear layer and ganglion cell layer of the retina of different mammalian species, but so far no experiments have been conducted to identify whether these cells are amacrine cells and/or retinal ganglion cells. We attempted to solve this problem by studying the NADPH-diaphorase-positive neurons in the hamster retina. From the NADPH-diaphorase histochemical reaction, two distinct types of neurons in the hamster retina were identified. They were named ND(g) and ND(i) cells. The ND(g) cells were cells with larger somata, ranging from 10 to 21 microns in diameter with a mean of 15.58 microns (S.D. = 2.59). They were found in the ganglion cell layer only. The ND(i) cells were smaller, with the somata ranging from 7 to 11 microns and having the mean diameter of 8.77 microns (S.D. = 1.24). Most of the ND(i) cells were found in the inner nuclear layer, and only very few could be observed in the inner plexiform layer. On average, there were 8,033 ND(g) and 5,051 ND(i) cells in the ganglion cell layer and inner nuclear layer, respectively. Two experiments were performed to clarify whether any of the NADPH diaphorase neurons were retinal ganglion cells. Following unilateral optic nerve section, which leads to the retrograde degeneration of retinal ganglion cells, the numbers of both ND(g) and ND(i) cells did not change significantly for up to 4 months. In addition, when retinal ganglion cells were prelabeled retrogradely (horseradish peroxidase or fluorescent microspheres) and retinas were then stained for NADPH diaphorase, no double-labeled neurons were detected. These results indicated that the NADPH-diaphorase neurons in the hamster retina were the amacrine cells in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. Dendrites of the ND(g) and ND(i) cells were found to stratify in sublaminae 1, 3, and 5 of the inner plexiform layer, with a prominent staining in the sublamina 5. The possible importance of this arrangement in the rod pathway is also discussed. PMID- 7534317 TI - Corticorubral synaptic organization in Macaca fascicularis: a study utilizing degeneration, anterograde transport of WGA-HRP, and combined immuno-GABA-gold technique and computer-assisted reconstruction. AB - The macaque red nucleus receives afferents from two major sources, the cerebral cortex and the deep cerebellar nuclei. Approximately 90% of the corticorubral afferent axons project to pars parvicellularis of the red nucleus, the neurons of which transmit information to the cerebellum by way of the inferior olivary nucleus. The remaining 10% project to pars magnocellularis of the red nucleus, the major projection of which is to the spinal cord. In this study, corticorubral terminations labeled following lesions or injections of wheatgerm agglutinin conjugated to horseradish-peroxidase into the topographically defined hand area of the primary motor cortex were quantitatively studied via electron microscopy. Cortical afferent terminals within pars parvicellularis and pars magnocellularis synapse upon all regions of the dendritic arbors of rubral projection neurons. However, the majority of these labeled afferents synapse upon thin-diameter shafts or presumed spinous processes of rubral distal dendrites as well as upon vesicle-containing profiles of presynaptic dendrites of local circuit interneurons that are gamma-aminobutyric acid-immunoreactive, as identified by postembedding immunohistochemistry. Synaptic contacts formed by the labeled cortical terminal were large in width and extended through several serial sections. Synaptic contacts formed by the presynaptic dendritic profiles, on the other hand, were more punctate and could be seen in only one or two serial sections. These latter synaptic interactions probably provide a modification of the effects of cortical input to rubral projection neurons as suggested by previous physiological studies that indicated the dominance of cortical input onto distal dendrites as well as involvement with inhibitory circuits. An example of the complexities of these synaptic interactions is further demonstrated by a three-dimensional computer reconstruction. This quantitative study of corticorubral afferents in the macaque monkey provides insight into the interactions of cerebral cortical afferents with rubral projection neurons and their relationship with local circuit inhibitory interneurons to elucidate the role played by the cortex in the activation of rubral neurons. PMID- 7534318 TI - Let me entertain ... er ... teach you: gaining attention through the use of slide shows. AB - The lecture method has been and continues to be the object of criticism. But, perhaps, it is not the lecture which should bear the brunt of the criticism, but rather the lecturer. All too often the audience is bored. This article examines several popular media currently used to enliven the lecture. Instructional design theory is used to discuss their limitations. The use of a self-assembled slide show, featuring novelty and humor, is advocated in order to assist the lecturer in gaining attention and, thereby, avoiding the boredom pitfall. Useful suggestions for developing a slide presentation are provided. PMID- 7534319 TI - Don't leave home (to speak at a conference) without it.... PMID- 7534320 TI - Short-term effect of recombinant human growth hormone in patients with alcoholic cirrhosis. AB - As growth hormone possesses anabolic properties that are active on protein metabolism, and thus of potential benefit to patients with chronic liver disease, we determined the metabolic effects of recombinant human growth hormone on insulin-like growth factor-I (IGF-I) its specific binding proteins, and liver function. Twenty consecutive patients with cirrhosis were randomized to recombinant human growth hormone (Norditropin, 4 I.U. twice daily) subcutaneously for 6 weeks (n = 10) or conventional medical treatment (n = 10). The serum concentrations of insulin-like growth factor-I in the recombinant human growth hormone group increased after 3 (p < 0.01) and 6 weeks (p < 0.02), whereas no significant changes were observed in the control group. The change in insulin like growth factor-I during the treatment period was expressed as area under the curve (AUC). The AUCIGF-I was significantly larger in the recombinant human growth hormone group (median AUCIGF-I: 12.1, range: 0.0-54.7 weeks.nmol/l) than in the control group (median AUCIGF-I: 0.2, range: -10.6-9.9 weeks.nmol/l) (p < 0.007). Insulin-like growth factor binding protein-3 concentrations increased in the recombinant human growth hormone treated patients as well as in controls, whereas no change in insulin-like growth factor binding protein-1 concentrations was found. No significant changes were seen in the area under the curve for biochemical liver function tests. We conclude that administration of recombinant human growth hormone induces an increase in very low levels of insulin-like growth factor-I, even in patients with cirrhosis with advanced disease, but the clinical benefits remain to be demonstrated. PMID- 7534321 TI - Cellular expression of tumour necrosis factor-alpha and interferon-gamma in the liver biopsies of children with chronic liver disease. AB - The liver biopsies of patients with autoimmune liver diseases have a dense portal tract mononuclear cell infiltrate. To investigate whether these cells produce tumour necrosis factor-alpha and interferon-gamma, cytokines which could be involved in the autoimmune attack through a direct cytopathic effect and/or through induction/enhancement of major histocompatibility complex antigen expression, we immunohistochemically stained cryostat liver sections from 21 children with autoimmune liver disease and from 15 children with metabolic liver disorders and histological evidence of portal tract inflammation as controls. Tumour necrosis factor-alpha and interferon-gamma producing cells were detected simultaneously within the inflammatory cell infiltrate in the liver biopsies of 18 patients with autoimmune liver disease, but only one patient with a metabolic disorder was positive for tumour necrosis factor-alpha. There was a significant correlation between frequency of tumour necrosis factor-alpha and interferon gamma producing cells, intensity of inflammatory cell infiltrate (p < 0.03 and p < 0.05, respectively) and transaminase levels (p < 0.008 and p < 0.03, respectively). These results suggest that tumour necrosis factor-alpha and interferon-gamma play a pathogenic role in autoimmune liver cell damage. PMID- 7534322 TI - Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins. AB - The E1 and E2/NS1 genes, encoding the putative hepatitis C virus envelope proteins, show a high rate of sequence variations. We analyzed the degree and distribution of sequence heterogeneity in serum samples from hepatitis C virus infected subjects. The mutations in the E1 region were mainly type-specific and the rate of variability was apparently not linked to the clinical phase of the infection. The sequence evolution of the E1 region during interferon treatment was low, regardless of the response to therapy. In contrast, an increased degree of variation, apparently related to the stage of viral replication, was present in E2 region derived from patients undergoing interferon treatment. These results are consistent with the hypothesis that the E2 protein represents a major target of the immune response. PMID- 7534323 TI - Granulocyte colony stimulating factor (G-CSF) combined with alpha-interferon for the treatment of liver allograft recipients with viral hepatitis. PMID- 7534325 TI - Contaminated collection media as a cause of pseudoinfection. AB - Following the appearance of positive Gram's stains from sterile surgical cases, an investigation was begun. Nonviable but stainable bacteria were found in the gel-based transport media. The use of a cause-and-effect diagram helped to show the numerous items that affected the problem. PMID- 7534324 TI - Hepatitis C virus infection in healthcare workers: risk of exposure and infection. AB - OBJECTIVES: To determine the incidence of hepatitis C virus (HCV) infection among healthcare workers (HCWs) at a university hospital, the proportion of HCWs having non-A, non-B hepatitis (NANBH) who were anti-HCV positive, and the rate of HCV transmission following a HCV-positive needlestick injury. DESIGN: Longitudinal analysis of a dynamic (cohort) population. MEASUREMENTS: From 1980 through 1989, HCWs who had clinical NANBH were identified, and from 1987 through 1989, HCWs who reported a blood or body fluid exposure and the patients who were the source of the exposure were screened for antibodies to HCV. SETTING: A 732-bed, university hospital and outpatient clinics. RESULTS: Over the 10-year period, six cases of occupationally acquired NANBH were observed, for an incidence of 21 cases per 100,000 HCWs per year (standardized incidence ratio, 2.96; 95% confidence interval [CI95], 1.83 to 4.36). Four of the six cases were confirmed to be HCV infection. From 1987 through 1989, 176 (12.7%) of 1,387 patients who were the source of an exposure were anti-HCV positive. Exposures that occurred in the emergency department were more likely to be anti-HCV positive than were exposures from all other locations (relative risk [RR] = 1.7; P = 0.009). Of HCWs who had an HCV-positive needlestick injury and whose serum had been tested for anti-HCV at least 5 months after the exposure, 3 (6.0%) of 50 seroconverted. From 1987 through 1989, the incidence of HCV infection among HCWs was 54 cases per 100,000 HCWs per year. CONCLUSION: The incidence of clinical NANBH among HCWs in this study is approximately three times higher than that of non-HCWs. HCWs are at significant risk for exposure to and acquisition of HCV. PMID- 7534326 TI - Endotoxin administration decreases plasma insulin-like growth factor (IGF)-I and IGF-binding protein-2 in Angus x Hereford steers independent of changes in nutritional intake. AB - Endotoxemia and sepsis cause severe shifts in metabolism towards catabolic events. The objective of the research was to determine whether endotoxin administration changes plasma concentrations of IGF-I and IGF-binding protein-2 (IGFBP-2) in Angus x Hereford steers. In Experiment 1, mean feed intake in endotoxin-treated steers (n = 6) decreased 60% within the first 24 h after endotoxin and averaged, for the duration of the 96 h test period, an intake 35.5% lower than the mean ad libitum intake recorded prior to endotoxin. Plasma concentrations of IGF-I averaged 182 ng/ml in steers before endotoxin (E. coli, 055:B5, 0.2 micrograms/kg, i.v. bolus) and decreased an average of 24.2% at 24 to 96 h after endotoxin. In Experiment 2, a paired feeding strategy was used to determine whether the reduced feed intake was a significant factor in changing plasma concentrations of IGF-I and IGFBP-2 after endotoxin challenge. Steers were divided into endotoxin (0.2 micrograms/kg, i.v., n = 6) or control (saline, i.v., n = 3) treatments. Each of three endotoxin-treated steers was paired to a specific control. In this fashion, the adjusted feed intake of each endotoxin treated steer was fed to it's paired control every 24 h through 96 h after endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534327 TI - Changes induced by non-enzymatic glycosylation of IGF-binding protein-3: effects on its binding properties and on its modulatory effect on IGF-I mitogenic action. AB - The aim of this study was to demonstrate the feasibility of in vitro non enzymatic glycosylation of IGF-binding protein-3 (IGFBP-3) and whether this process affects its binding properties and its modulatory effect on IGF-I mitogenic activity. Swiss 3T3 fibroblasts were cultured and the IGFBP-3 released into the medium (CM) glycated with either labelled or unlabelled glucose. Parallel glycation studies were performed using standard human IGFBP-3. Both species of IGFBP-3 became effectively glycated in a dose-dependent manner. Glycated IGFBP-3 bound larger amounts of 125I-labelled IGF-I than its non glycated form. According to Scatchard analysis this effect might be due to an increase in the number of binding sites of the IGFBP-3 molecule rather than to changes in its affinity constants, which remain unchanged. Preincubation of fibroblasts with CM containing IGFBP-3 for 16 h before the addition of IGF-I enhanced the stimulatory effect of the hormone on thymidine incorporation into cell DNA. This potentiation was blunted when in vitro glycated instead of non glycated IGFBP-3 was employed. These results provide further evidence of the in vitro glycation of IGFBP-3 and demonstrate that this process affects both its binding properties and its enhancing effect on IGF-I mitogenic activity. These changes may explain, at least partially, the development of many alterations observed in poorly controlled diabetic patients. PMID- 7534328 TI - Effects of basic fibroblastic growth factor on the function and proliferation of human clinically non-functional pituitary adenomas which secreted glycoprotein hormones in vitro. AB - The effects of human recombinant basic fibroblastic growth factor (bFGF) on the secretion, viability, proliferation, attachment and morphology of ten dispersed human clinically non-functional (NF) adenomas were examined in vitro. Four clinically NF adenomas secreting FSH and/or LH in vitro were unaffected by 10 nM bFGF over a 4-h period. Over 4 days 10 nM bFGF stimulated LH secretion (66% and 72%, P < 0.01) from two out of seven clinically NF adenomas secreting LH, whilst FSH (three tumours) and alpha-subunit secretion (three tumours) were unaffected. One adenoma co-secreting LH and alpha-subunit and one secreting LH alone were studied over 21 days; LH secretion fell progressively, but the decline was significantly less (P < 0.05) with bFGF (10 nM) treatment after 14 and 21 days in both adenomas, whilst the fall in alpha-subunit secretion was unaffected by bFGF treatment. A 24-h GnRH test performed at the start and end of the 21-day period in one of these tumours showed an increase in both basal and stimulated LH secretion in the bFGF-treated group over control (124%, P < 0.001). There was no effect of bFGF (10 nM) on viability, S-phase proliferation, attachment or morphology of adenoma cells over a 4-day period. These results suggest that bFGF has a role in tumorous LH secretion from these adenomas, but is not mitogenic (at least over 4 days) and is without effect on other parameters of in vitro differentiated function. PMID- 7534329 TI - A homologous radioimmunoassay for ovine insulin-like growth factor-binding protein-2: ontogenesis and the response to growth hormone, placental lactogen and insulin-like growth factor-I treatment in sheep. AB - Although insulin-like growth factor-binding protein-2 (IGFBP-2) is an abundant IGFBP in fetal and postnatal plasma, its regulation is not yet clearly understood. To address this question in sheep, we purified ovine IGFBP-2 and developed a homologous radioimmunoassay. We have studied its ontogenesis and measured serum concentrations of ovine IGFBP-2 after bovine growth hormone (bGH), ovine placental lactogen (oPL) and IGF-I treatment. Concentrations of IGFBP-2 were high at 125 days of gestation (550 +/- 15 micrograms/l) but fell after birth (P < 0.05) and plateaued after 1 year of age (340 +/- 20 micrograms/l). In lactating ewes, bGH treatment for 7 days significantly reduced (21%; P < 0.05) IGFBP-2 relative to the saline-treated group. Similarly, in neonatal lambs, bGH treatment from day 3 to day 23 of life reduced (P < 0.05) IGFBP-2 by 23% relative to the saline-treated group. oPL had no effect on serum levels of IGFBP-2 in the ewe or the neonatal lamb. In well-fed yearling lambs, treatment with IGF-I reduced IGFBP-2 values by 27% (P < 0.05) relative to control animals. In yearling lambs, reduced nutrition increased plasma IGFBP-2 (41%; P < 0.05). However this increase was abolished by IGF-I treatment. The changes in plasma levels of IGFBP 2 were positively related to changes in IGF-II while there was a negative relationship between circulating IGF-I and IGFBP-2 such that both IGF-I and IGF II may play a role in the regulation of IGFBP-2 in serum. PMID- 7534330 TI - Continuous 14 day infusion of IGF-II increases the growth of normal female rats, but exhibits a lower potency than IGF-I. AB - The effects of continuous 14 day infusion of recombinant human IGF-I (104 or 260 micrograms/day) or IGF-II (104, 260 or 650 micrograms/day) via s.c. implanted osmotic pumps were compared in young female rats in order to establish the relative efficacies of these two growth factors. Significant increase in body weight gain and feed conversion efficiency were achieved by 260 micrograms/day of IGF-I or 650 micrograms/day of IGF-II. These treatments were associated with increased nitrogen retention and increases in the fractional weights of kidneys, spleen, total gut and individual gut regions. There was an increase in the size of villi and muscularis lining the jejunum, suggesting an increased absorptive capacity of the gut. However there was no significant change in the amount of faecal nitrogen excretion when expressed as a percentage of nitrogen intake. Interestingly, IGF-II was at least as potent as IGF-I in increasing the depth of jejunal crypts. Infusion of equivalent doses of either IGF-I or IGF-II resulted in similar increases in circulating concentrations of the respective peptides, though IGF-II infusion dose-dependently decreased plasma IGF-I concentrations from those of the controls. Plasma IGF-binding protein levels were increased by both IGF-I and IGF-II treatments, though IGF-I elicited greater responses. In summary, IGF-II can promote the growth of young female rats, although generally less potently than IGF-I. PMID- 7534331 TI - Clinical effects of octreotide compared to placebo in patients with gastrointestinal neuroendocrine tumours. Report on a double-blind, randomized trial. AB - OBJECTIVES: To compare the effect of octreotide with f placebo on symptoms, tumour marker and quality of life in patients with gastrointestinal neuroendocrine tumours and liver metastases. DESIGN: A blinded, placebo controlled, cross-over study was performed. The number of flushing epidodes and diarrhoea episodes were registered for 1 week prior to the study and for the 8 week duration of the study. Quality of life and 24-h urinary 5 hydroxyindoleacetic acid (5-HIAA) excretion were measured before the start, and at 4 and 8 weeks. Quality of life was registered with the Psychosocial Adjustment to Illness Scale (PAIS) and 5-HIAA measured by high-performance chromatography with electrochemical detection. 5-HIAA values exceeding 45 mumol 24 h-1 were considered to be elevated. SETTING: The study was performed in a tertiary referral centre. SUBJECTS: Twelve patients were approached; eleven patients were included, with a mean age of 56.5 (range 30-72) years. The primary tumour originated from the small intestine in nine and from the pancreas in two patients. The main symptoms were diarrhoea, flushing and nausea. The 24-h excretion of 5-HIAA was increased in all patients. INTERVENTIONS: Patients were treated for 4 weeks with octreotide (100 micrograms) subcutaneously, twice daily, and for 4 weeks on placebo (octreotide vehicle) in random starting order. MAIN OUTCOME MEASURES: The main outcome measures were the number of episodes of the main clinical symptom(s) and 24-h 5-HIAA excretion. RESULTS: Octreotide lowered diarrhoea and flushing frequency significantly compared to placebo. 5-HIAA excretion was reduced during treatment with the active drug. Two domains of the PAIS were significantly improved, indicating that the reduction of tumour marker and symptoms were clinically important. CONCLUSIONS: The clinical effect of octreotide on symptoms in patients with neuroendocrine tumours was demonstrated in a controlled, prospective trial. PMID- 7534332 TI - Characterization of neuronal amino acid transporters: uptake of nitric oxide synthase inhibitors and implication for their biological effects. AB - In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors NG-methyl-L-arginine and NG-nitro-L-arginine by the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Uptake of NG-methyl-L arginine was characterized by biphasic kinetics (Km1 = 8 mumol/L, Vmax1 = 0.09 nmol x mg-1 x min-1; Km2 = 229 mumol/L, Vmax2 = 2.9 nmol x mg-1 x min-1) and was inhibited by basic but not by neutral amino acids. Uptake of NG-nitro-L-arginine followed Michaelis-Menten kinetics (Km = 265 mumol/L, Vmax = 12.8 +/- 0.86 nmol x mg-1 x min-1) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of NG-methyl-L-arginine is mediated by system y+, whereas systems L and T account for the transport of NG-nitro-L-arginine. In agreement with these data on uptake of the inhibitors, L-lysine and L-ornithine antagonized the inhibitory effects of NG-methyl-L-arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas L-tryptophan, L-phenylalanine, and L-leucine interfered with the effects of NG-nitro-L-arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors. PMID- 7534333 TI - Interferons suppress nerve growth factor synthesis as a result of interference with cell growth in astrocytes cultured from neonatal mouse brain. AB - Interferon (IFN)-beta and IFN-gamma inhibited the DNA synthesis and nerve growth factor (NGF) synthesis in growing astrocytes cultured from neonatal mouse brain, but they did not affect the NGF synthesis in quiescent astrocytes. IFN-beta and IFN-gamma also inhibited the enhanced DNA synthesis and NGF synthesis in growing astrocytes after the administration of basic fibroblast growth factor. These results indicated that NGF synthesis in astrocytes is regulated by IFNs associated with cell growth. The mechanism of IFN action on NGF synthesis/secretion is unknown, but the results that their effects last long after IFN removal from the cultures present the possibility that IFNs destabilize NGF mRNA. PMID- 7534334 TI - Cystic fibrosis transmembrane conductance regulator is found within brain ventricular epithelium and choroid plexus. AB - The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a Cl- channel that is regulated by cyclic AMP dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR-like immunoreactivity was concentrated in fine puncta localized about 1-2 microns from the CSF-contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP-elevating agents, but the role of this chloride transporter in ependymal function remains to be determined. PMID- 7534335 TI - Regulation of c-fos expression by convulsants and hexachlorocyclohexane isomers in primary cultures of cortical neurons. AB - Primary cortical cultures were used to study the effects of four convulsants on c fos expression. Approximately 30% of the neurons in these cultures displayed c fos nuclear immunostaining under basal conditions. The addition of tetrodotoxin, nifedipine, or delta-hexachlorocyclohexane produced a significant decrease in c fos basal values. Lindane (gamma-hexachlorocyclohexane), Bay K 8644, pentylenetetrazole, and picrotoxinin produced a significant increase in c-fos immunoreactivity and in c-fos mRNA expression. Treatment of cells with tetrodotoxin before administration of the convulsant agents lowered c-fos staining below basal levels. In contrast, delta-hexachlorocyclohexane or nifedipine failed to block only the picrotoxin-induced increase. The differential pattern of expression shown by c-fos after these treatments suggests various mechanisms of action for the compounds studied. The results obtained with delta hexachlorocyclohexane and nifedipine suggest that picrotoxinin activates c-fos expression by calcium-requiring intracellular signaling pathways that are different from those activated by Bay K 8644, pentylenetetrazole, or gamma hexachlorocyclohexane, which, at least in part, act via L-type calcium channels. PMID- 7534336 TI - Corticosterone has a permissive effect on expression of heme oxygenase-1 in CA1 CA3 neurons of hippocampus in thermal-stressed rats. AB - Activity of the stress protein, heme oxygenase-1 (hsp32; HO-1), produces carbon monoxide (CO), the potential messenger molecule for excitatory N-methyl-D aspartate receptor-mediated events, in the hippocampus. Long-term stress caused by elevated adrenocorticoids induces pathological changes in CA1-CA3 neurons of the hippocampus; the adrenal hormones also exacerbate damage from stress. In rats chronically treated with corticosterone, we examined expression of HO-1 and its response to thermal stress in the hippocampus. An unprecedented appearance of scattered immunoreactive astrocytes marked the molecular layer of the hippocampus in corticosterone-treated rats. Steroid treatment showed no discernible effect on whole-brain HO-1 mRNA. When these rats were subjected to hyperthermia, neurons in the CA1-CA3 area, including pyramidal cells, exhibited intense immunoreactivity for the oxygenase and a pronounced increase (approximately 10-fold) in number. HO 1 is essentially undetectable in this area when rats are exposed to chronic corticosterone alone or thermal stress by itself, or in control rats. In contrast, similar analysis of hilar neurons showed no apparent effect on either the number or relative intensity of HO-1-immunostained cells after treatment. Corticosterone treatment also intensified the stress response of cerebellum, including Purkinje cells and Bergmann glia in the molecular layer. In brain, despite a pronounced reduction in NO synthase activity in corticosterone-treated and/or heat-stressed animals, the level of cyclic GMP was not significantly reduced. These observations are consistent with the hypothesis that responsiveness to environmental stress of CA1-CA3 neurons brought about by chronic elevation in circulating adrenocorticoids results in an increased excitatory neuronal activity and eventual hippocampal degeneration. Moreover, these findings yield further support for a role of CO in the production of cyclic GMP in the brain. PMID- 7534337 TI - Enkephalin metabolism by microglial aminopeptidase N (CD13). AB - Rat microglia in culture showed a high capacity to degrade neuropeptides compared with other glial cells. Leu-enkephalin was readily hydrolyzed to free tyrosine and Gly-Gly-Phe-Leu. Inhibition experiments and immunostaining revealed that aminopeptidase N (CD13) on the surface of microglia was responsible for enkephalin cleavage. Endopeptidase-24.11 ("enkephalinase"), angiotensin converting enzyme, or carboxypeptidases could not be detected on microglia. Aminopeptidase N activity in microglia was considerably higher than in rat peripheral monocytes and macrophages, which both also exhibited low endopeptidase 24.11 activities. Activity of aminopeptidase N was upregulated by culture of microglia on astrocytes and down-regulated by exposure of microglia to lipopolysaccharide. The occurrence of aminopeptidase N on microglia is in line with the view that they originate from the monocytic lineage. PMID- 7534338 TI - Striatal met-enkephalin and substance P levels are decreased in mice infected with the LP-BM5 murine leukemia virus. AB - Mice infected with the LP-BM5 murine leukemia virus mixture develop severe immunosuppression and an encephalopathy characterized by spatial learning deficits. Twelve weeks after infection of C57BL/6J mice with LP-BM5, significant (50-60%) reductions in Met-enkephalin and substance P levels were observed in the striatum, whereas somatostatin levels were unchanged. In addition, a 39% decrease in hypothalamic substance P concentrations was observed, with no alteration in Metenkephalin levels. The apparent selectivity of the decrease in neuropeptide concentrations indicates that a functional alteration of the primary striatal efferent neurons occurs in this infection, which may contribute to the impairment of spatial learning observed in these mice. Moreover, this decrease in striatal neuropeptide levels is similar to the neuropathological changes in basal ganglia observed in HIV-infected individuals and is consistent with previous studies suggesting that the LP-BM5-infected mouse may serve as a useful model of AIDS dementia. PMID- 7534340 TI - Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network. AB - Schwann cells polarize their surface membranes into several biochemically and ultrastructurally discrete regions of the myelin internode. To form these membrane domains, Schwann cells must sort, transport, and target membrane proteins appropriately. In this study, microtubule disassembly, confocal microscopy, and electron microscopic immunocytochemistry were used to investigate mechanisms involved in targeting P0 protein (P0), the myelin-associated glycoprotein (MAG), and laminin to different plasma membrane domains in myelinating Schwann cells from 35-d-old rat sciatic nerve. After microtubule disassembly by colchicine, all three proteins accumulated in Schwann cell perinuclear cytoplasm, indicating that microtubules are necessary for their transport. The distributions of Golgi membranes, endoplasmic reticulum, and intermediate filaments were also altered by colchicine treatment. Electron microscopic immunocytochemical studies indicated that P0 and MAG are sorted into separate carrier vesicles as they exit the trans-Golgi network. Following microtubule disassembly, P0-rich carrier vesicles fused and formed myelin-like membrane whorls, whereas MAG-rich carrier vesicles fused and formed mesaxon-like membrane whorls. Microtubule disassembly did not result in mistargeting of either P0 or MAG to surface membranes. These results indicate that following sorting in the trans-Golgi network, certain carrier vesicles are transported along the myelin internode on microtubules; however, microtubules do not appear to target these vesicles selectively to specific sites. The targeting of P0-, MAG-, and laminin-rich carrier vesicles to specific sites most likely occurs by ligand receptor binding mechanisms that permit fusion of carrier vesicles only with the appropriate target membrane. PMID- 7534339 TI - The dopamine transporter: immunochemical characterization and localization in brain. AB - Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C-terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders. PMID- 7534341 TI - Androgen-induced changes in electrocommunicatory behavior are correlated with changes in substance P-like immunoreactivity in the brain of the electric fish Apteronotus leptorhynchus. AB - The hormonal regulation of sex differences in electrocommunicatory behavior and brain substance P-like immunoreactivity (SPI-ir) were examined in the weakly electric fish, Apteronotus leptorhynchus. This animal modulates its electric organ discharge (EOD) to produce discrete electric social signals (chirps), which function in aggressive and reproductive displays. Males readily chirp in response to electrosensory stimuli that mimic the presence of a conspecific; females also chirp in response to such stimuli, but do so at much lower rates than males. We have recently demonstrated that androgen treatment enhances chirping behavior in females and may also lead to a change in chirp quality or structure. In this study, we quantified androgen-induced changes in chirp structure and simultaneously examined whether androgens alter the sexually dimorphic pattern of SPI-ir in a brain region (prepacemaker nucleus, PPn) known to control chirping. Our results demonstrate that, in females, androgens cause both the induction of chirping and an alteration of chirp structure; chirps recorded from androgen implanted females had longer durations and more dramatic frequency and amplitude modulations compared to controls, and appear similar to those reported to be produced during spawning. Moreover, androgen-induced changes in chirping are correlated with increased expression of SPI-ir within specific brain nuclei of females. These changes may underly behavioral changes in chirping, since treated females showed a male-like pattern of SPI-ir in the PPn. However, alterations in SPI-ir were not restricted to the PPn, but also occurred in diencephalic regions related to pituitary function and reproductive behavior. The results suggest that androgens modulate chirping activity and cause both specific and wide-spread changes in SPI-ir that may relate to a functional system that interrelates pituitary function, reproductive behavior, and chirping. PMID- 7534342 TI - Tenascin knockout mice: barrels, boundary molecules, and glial scars. AB - In light of a previous report suggesting that the brains of tenascin-deficient animals are grossly normal, we have studied the somatosensory cortical barrel field and injured cerebral cortex in postnatal homozygous tenascin knockout, heterozygote, and normal wild-type mice. Nissl staining, cytochrome oxidase, and Dil axonal tracing of thalamocortical axonal projections to the somatosensory cortex, all reveal the formation of normal barrels in the first postnatal week in homozygous knockout mice that cannot be distinguished from heterozygote or normal wild-type barrels. In addition to confirming the absence of tenascin in knockout animals, and reporting apparently reduced levels of the glycoprotein in barrel boundaries of heterozygote animals using well-characterized antibodies and immunocytochemistry, we also studied the DSD-1-PG proteoglycan, another developmentally regulated molecule known to be associated with transient glial/glycoconjugate boundaries that surround developing barrels; DSD-1-PG was also found to be expressed in barrel boundaries in apparently normal time frames in tenascin knockout mice. Peanut agglutinin (PNA) binding of galactosyl containing glycoconjugates also revealed barrel boundaries in all three genotypes. We also examined the expression of tenascin-R, a paralog of tenascin-C (referred to here simply as tenascin). As previously reported, tenascin-R is prominently expressed in subcortical white matter, and we found it was not expressed in the barrel boundaries in any of the genotypes. Thus, the absence of tenascin does not result in a compensatory expression of tenascin-R in the barrel boundaries. Finally, we studied wounds of the cerebral cortex in the late postnatal mouse. The astroglial scar formed, for the most part, in the same time course and spatial distribution in the wild-type and tenascin knockout mice. However, there may be some differences in the extent of gliosis between the knockout and the wild type that warrant further study. Roles for boundary molecules like tenascin during brain pattern formation and injury are reconsidered in light of these findings on barrel development and cortical lesions in tenascin-deficient mice. PMID- 7534343 TI - Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. AB - In this study the actions of NGF in regulating peptide expression were examined in vivo in adult rat primary sensory neurons. The hypothesis that NGF might tonically inhibit expression of some peptides was tested specifically. In situ hybridization and immunohistochemistry were used to detect presence or absence of alpha-CGRP, beta-CGRP, SP, SOM, VIP, CCK, NPY, and GAL as well as their mRNAs. In neurons in normal lumbar DRG alpha-CGRP, beta-CGRP, SP, and SOM are abundantly and heterogeneously expressed whereas few neurons have detectable VIP, CCK, NPY, or GAL. Two weeks following sciatic nerve transection, concentrations of alpha CGRP, beta-CGRP, SP, and SOM plus their mRNAs have decreased to background in all but a few neurons. In contrast, VIP, CCK, NPY, and GAL are now synthesized in many neurons. Delayed intrathecal infusion of NGF (125 ng/microliter/hr) for 7 d, starting 2 weeks after injury counteracted the decrease in expression of alpha CGRP, beta-CGRP and SP expression, but not SOM. This lack of influence of NGF on SOM is consistent with the absence of high-affinity NGF receptors and trk mRNA in SOM-positive neurons. Delayed infusion of NGF also reduced the number of neurons expressing VIP, CCK, NPY, and GAL after injury by approximately one-half in each subpopulation. Therefore, we suggest that NGF suppresses expression of these four peptides but only if the neurons also have NGF receptors. The results show that NGF can regulate peptide expression differentially and may also be part of the signal that allows reversion to normal of responses to injury as axons regenerate. PMID- 7534344 TI - Restoration of circadian behavior by anterior hypothalamic heterografts. AB - The suprachiasmatic nucleus (SCN) of the anterior hypothalamus (AH) is a circadian oscillator and an important component of the mammalian circadian system. To determine whether the SCN is the dominant circadian pacemaker responsible for generating a species-typical characteristic of circadian rhythms [i.e., period length (tau)], neural transplantation was conducted using fetal AH donors of different species and SCN-lesioned (SCNx) hosts. The circadian behavior of each of the three donor species is clearly distinguishable by its species typical tau. The extent of SCN pacemaker autonomy was assessed by noting whether the period of the restored circadian rhythm following heterograft transplantation was characteristic of the donor or the host, or whether an atypical circadian period was established. Hamsters rendered arhythmic by SCN ablation were implanted with AH tissue from fetal hamsters (E13-E14, homograft controls) or fetal mice or rats (E15-E17). The AH homografts restored circadian activity rhythms with a tau similar to that of intact hamsters, and fetal mouse AH heterografts restored circadian rhythmicity with a tau similar to that of the donor mouse strain. However, fetal rat AH tissue implanted into SCNx hamsters renewed circadian rhythmicity with a period significantly shorter than either the species-typical tau of the rat donor or the hamster host. In both the mouse and rat AH heterograft experiments, immunocytochemical analysis performed with species-specific monoclonal antibodies revealed extensive fiber outgrowth from the implant into the host hypothalamus, evident up to 7 months postimplantation. The rat implants were consistently larger, more fully vascularized and exhibited less necrosis than the implanted mouse tissue. The histological appearance of the grafts, thus, provides no explantation for the difference in efficacy of the grafts to restore species-typical behavior. However, several interpretations are considered that are consistent with the combined behavioral results observed. PMID- 7534346 TI - Single-channel properties of four calcium channel types in rat motoneurons. AB - Previous studies have demonstrated multiple components of whole-cell calcium currents in hypoglossal motoneurons (HMs); HMs possess a low-voltage-activated (LVA) current and three types of high-voltage-activated (HVA) calcium currents based on sensitivity to omega-Aga IVA, omega-Conotoxin GVIA (omega-CgTx) and dihydropyridine analogs (DHPs). In the present study, we recorded single-calcium channel activities from HMs using a cell-attached patch-clamp method and found four types of channels that could be discriminated based on kinetics, voltage dependency, DHP sensitivity, and single-channel conductances. The average single channel conductances with 110 mM barium as a charge carrier were 7, 14, 20, and 28 pS. T-type channels had a single-channel conductance of 7 pS, activated at the most negative potentials for the calcium channels and inactivated during depolarization. L-type channels (DHP-sensitive channels) did not inactivate during depolarization and had a 28-pS single-channel conductance. Based on kinetics and sensitivity to holding potential, it is likely that the channels with conductances of 14 pS and 20 pS represent N-type and P-type channels, respectively. The N-type channel (14 pS) was sensitive to holding potential, showed modal gating, and inactivated during maintained depolarizations, whereas the P-type channel (20 pS) was rather insensitive to holding potential and did not inactivate during depolarization. PMID- 7534345 TI - Functional interaction of melatonin receptors and D1 dopamine receptors in cultured chick retinal neurons. AB - The possible interaction of melatonin receptors and D1 dopamine receptors was investigated in neural cells prepared from embryonic day 8 chick retinas and cultured for 6 d. Dopamine stimulated cAMP accumulation in cultured retinal cells. This effect of dopamine was antagonized by addition of dopamine receptor antagonists (haloperidol and SCH23390) or melatonin receptor agonists (melatonin, 2-iodomelatonin, and 6-chloromelatonin). The inhibition of dopamine-stimulated cAMP accumulation by melatonin was concentration dependent, with half-maximal inhibition at approximately 160 pM. Melatonin inhibited the effect of dopamine at all dopamine concentrations, suppressing the maximal response to the neurotransmitter by approximately 70%. Melatonin also inhibited the stimulation of cAMP accumulation by SKF 82958, a selective D1 dopamine receptor agonist. Pretreatment of cultures with pertussis toxin had no significant effect on dopamine-stimulated cAMP accumulation, but inhibited the response to melatonin. In contrast to its effect on cAMP accumulation, melatonin had no effect on dopamine-stimulated inositol phosphate accumulation. These results suggest that melatonin receptors are coupled to dopamine receptor-regulated adenylate cyclase via an inhibitory G protein, and demonstrate another mechanism, in addition to inhibition of dopamine release, through which melatonin can modulate dopaminergic neurotransmission. PMID- 7534347 TI - Noradrenergic neurons in the locus coeruleus of birds express TrkA, transport NGF, and respond to NGF. AB - The chicken locus coeruleus contains a population of noradrenergic neurons which express the neurotrophin receptor p75 (von Bartheld and Bothwell, 1992). To determine which neurotrophin may regulate the development of noradrenergic neurons in the chicken locus coeruleus, expression of trk receptors, retrograde transport of neurotrophins, and responses to NGF were examined. P75-expressing noradrenergic neurons were found to project to the basal forebrain. They transport radio-iodinated NGF after injections into this target. The retrograde transport of NGF is specific to the noradrenergic neuronal population as evidenced by double labeling with antibodies against dopamine-beta-hydroxylase. The same neuronal population expresses trkA receptor mRNA. The size of noradrenergic neurons in the locus coeruleus proper, but not in the nucleus subcoeruleus, is significantly increased after injections of NGF into the telencephalon, consistent with the hypothesis that target-derived NGF provides trophic support. Noradrenergic coeruleus neurons are rescued from toxic effects of 6-hydroxydopamine injected into the telencephalon when NGF is injected into the midbrain. NGF has no rescue effect when it is coinjected with 6-hydroxy dopamine into the telencephalon. In explant or dissociated cultures, noradrenergic coeruleus neurons do not respond to elevated levels of NGF with increased neurite outgrowth. Taken together, these results suggest that NGF plays a role in the development and maintenance of noradrenergic coeruleus neurons in the chick brain. The data also support our previous conclusion that major species differences exist between birds (chicken) and mammals with regard to trophic regulation of presumptive homologous neuronal populations. PMID- 7534348 TI - Ductular structures in acute hepatitis with panacinar necrosis. AB - The development of ductular structures in acute hepatitis with panacinar necrosis was studied in 15 cases of fulminant hepatitis with variable clinical duration, using immunohistochemical markers. The immunophenotype of ductular structures was assessed by the expression of two bile duct epithelium determinants, wide spectrum cytokeratin and epithelial membrane antigen (EMA), and by their glycoconjugate expression using the specific binding lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA). Ductular structures showed a predilective, but not a strictly selective location in acinar zone 1 and at the periphery of newly formed parenchymal nodules. All were positive for keratin, while EMA and the lectins were identified less frequently. Cytokeratin expression was additionally observed in hepatic cells with no other phenotypic alteration: this occurred along isolated hepatic cords, within parenchymal remnants, in the spared parenchyma in acinar zone 1 and occasionally at the periphery of parenchymal nodules. The presence of cytokeratin expression in liver cell plates in association with intermediate morphological stages of tubular remodelling speaks in favour of biliary metaplasia of hepatocytes. This process may represent a phenotypic-functional accommodation of hepatocytes to an altered microenvironment, due to loss of parenchymal integrity. During the phenotypic shift, altered cytokeratin expression appears as one of the earliest biliary features, while EMA and the expression of glycoconjugates represent maturation markers. PMID- 7534349 TI - Mechanism of the enhancement effect of n-octyl-beta-D-thioglucoside on the transdermal penetration of fluorescein isothiocyanate-labeled dextrans and the molecular weight dependence of water-soluble penetrants through stripped skin. AB - To clarify the mechanism of the enhancing effect of n-octyl-beta-D-thioglucoside (OTG), which acts as a potent enhancer for skin penetration of peptides and water soluble penetrants, the in vitro penetration of macromolecules [fluorescein isothiocyanate-labeled dextrans (FTIC-dextrans)] was evaluated with hairless rat skin and stripped skin. The FITC-dextrans (MW, 4400, 9600, and 69,000 Da, referred to as FD-4, FD-10, and FD-70, respectively) penetrated more easily in the presence of OTG (1.5%), with high fluxes equivalent to those through stripped skin. This result indicated that the enhancement effect of OTG on the penetration of macromolecules through the stratum corneum was extensive, and the barrier function of the corneum was nearly eliminated by the OTG treatment. OTG significantly solubilized the stratum corneum proteins and ceramides during the initial time stage. Scanning electron microscopic observations demonstrated that OTG treatment dramatically changed the cell membrane (i.e., exfoliation of cell membranes and dissociation of adherent cornified cells), suggesting a significant disturbance of the cohesive laminae and barrier functions. The extent of dissociation of cell membranes increased with treatment time, without significant changes in the cell junctions. These results clarify that the enhancement mechanism of OTG was different from that of laurocapram and other lipophilic enhancers. The permeability of polar solutes with differing molecular sizes (MW, 180-69,000 Da) through stripped skin was size dependent (r = 0.997, p < 0.001). However, the viable epidermis and dermis restricted the penetration of macromolecules, such as FD-70. PMID- 7534351 TI - Nitric oxide synthase inhibitor and lipopolysaccharide effects on reactivity of guinea pig airways. AB - The in vivo and in vitro effects of nitric oxide (NO) synthase inhibitors and lipopolysaccharide (LPS) on reactivity of guinea pig airways were examined. In isolated, perfused tracheas from untreated animals, the NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME; 10(-4)M), NG-methyl-L-arginine (L NMMA; 10(-4) M) and aminoguanidine (10(-4) M) had no effect or inhibited reactivity to extraluminally (EL) or intraluminally (IL) applied methacholine and histamine. L-NMMA (10(-4) M) did not appreciably contract resting or metacholine contracted preparations (+/- 3 x 10(-4) M L-arginine) and L-arginine only weakly relaxed contracted tracheas (+/- L-NMMA). Sodium nitroprusside and S-nitroso-N penicillamine elicited relaxant responses and were more potent extraluminally than intraluminally. Methylene blue (10(-5) M) antagonized relaxation to sodium nitroprusside. Incubation with Escherichia coli LPS (10 micrograms/ml; 30 min incubation) alone in the EL and IL baths depressed methacholine and histamine concentration-response curves. In the presence of LPS, L-NAME potentiated responses to intraluminally applied methacholine but did not affect responses to extraluminally added methacholine. Four days after i.p. injection of animals with LPS (4 mg/kg), L-NAME potentiated responses to IL methacholine, and L-arginine acquired greater relaxant activity. LPS injection increased sensitivity to intraluminally added but not extraluminally added isoproterenol. LPS given by i.p. injection or by inhalation did not affect basal specific airway resistance of conscious animals or reactivity to methacholine aerosol during a postexposure period of 6 to 72 h. NO seems to have little role in regulating reactivity of guinea pig airways to bronchoconstrictor agonists, except after in vitro or in vivo exposure to LPS. After LPS injection the in vitro changes suggestive of NO synthase induction are not associated with altered airway reactivity to inhaled methacholine. PMID- 7534350 TI - Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors. AB - The benefits of nitric oxide synthase (NOS) inhibitors in the treatment of endotoxemia or sepsis presumably arise from inhibition of the type II (inducible) NOS. However, inasmuch as the effect of these inhibitors on NOS function in vivo is rarely assessed, NOS activity was evaluated in rats and mice by measuring changes in plasma nitrite and nitrate concentrations ([NOx]) after administration of lipopolysaccharide (LPS). In both species, [NOx] peaked at 20 hr, returning to base line by 48 to 72 hr. The ED50 values (dose that elicited a 50% inhibition of the LPS-dependent increase in [NOx] 6 hr after LPS administration) for L-NG monomethylarginine acetate, L-NG-nitroarginine methyl ester and aminoguanidine (administered 3 hr after LPS) were 34, 21 and 19 mg/kg in the rat and 32, 5 and 4 mg/kg in the mouse. These compounds also decreased the survival of LPS-challenged animals, which in the case of L-NG-nitroarginine methyl ester was reversed by L arginine. Dexamethasone (which prevents the induction of type II NOS) also inhibited the LPS-dependent increase in [NOx] with ED50 values of 0.05 mg/kg (rat) and 1 mg/kg (mouse), but did not lead to decreased survival. Thus, inhibition of the type I (neuronal) or type III (endothelial) NOS, rather than the type II isoform, may be a possible mechanism for the animal mortality. These models provide a simple and reproducible means for assessing the in vivo inhibition of type II NOS by various compounds. PMID- 7534352 TI - Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5 ethyl-5-phenylhydantoin in the male rat liver or in cultured rat hepatocytes. AB - To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phenylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 microM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 microM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5 ethyl-5-phenylhydantoin. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity. PMID- 7534353 TI - Automated analysis of multiplex microsatellites. AB - The use of automated DNA fragment analysis with the Applied Biosystems 672 Genescanner system was evaluated in a routine diagnostic setting. The aim of the study was to compare automated fragment detection and analysis with conventional methods. For cystic fibrosis analysis the delta F508 mutation in exon 10 of the cystic fibrosis transmembrane regulator (CFTR) gene was multiplexed with two intragenic microsatellites. The analysis of the Prader-Willi/Angelman region of chromosome 15 used a panel of five microsatellites. For dystrophin, seven microsatellites covering the entire dystrophin gene were co-amplified. Automated analysis was faster and more accurate than analysis using radiolabelled products with sequencing gels, although some inconsistencies in the sizing of microsatellite alleles were seen. PMID- 7534354 TI - Assessment of Yqh translocations. AB - Two initially presumed Yqh translocations, one to Xp and another to 21p, were assessed by conventional banding procedures, 5-azacytidine treatment, electron microscopy, and fluorescence in situ hybridisation. While the Yqh nature of the heterochromatic block from Xp was confirmed, this was not the case with the 21ph+ variant. In conclusion, conventional banding techniques are insufficient to diagnose Y;G translocations. PMID- 7534355 TI - Natural history and postmortem anatomy of a patient with tetra-amelia, ectodermal dysplasia, peculiar face, and developmental retardation (MIM 273390) PMID- 7534356 TI - Tau-like immunoreactivity in Alzheimer and control skin fibroblasts. AB - The presence of the microtubule-associated protein tau in skin fibroblasts derived from Alzheimer patients and normal controls was investigated using a panel of well-characterized anti-tau antibodies against epitopes spanning the tau protein from the amino to the carboxyl end. The antibodies immunolabeled a fine, fibrillar cytoplasmic network in all skin fibroblasts. Disruption of the microtubule network with colchicine did not affect the immunolabeling of the fibrillar network nor did treatment with cytochalasin B known to disrupt the microfilament network. Immunoelectron microscopy with the anti-tau antibodies revealed colocalization of the label with the 10 nm intermediate filaments. Furthermore, immunoblots found no reactivity against purified vimentin, suggesting that the antibodies recognize an intermediate filament-associated protein. The findings indicate the presence of tau or a protein with considerable homology to tau in fibroblasts associated with intermediate filaments and not microtubules. PMID- 7534357 TI - Monoclonal antibody Alz-50 reacts with bovine and human serum albumin. AB - Alz-50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF-tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz-50 epitope has been mapped to amino acids 2-10 present in all human tau isoforms, minimal Alz-50 immunoreactivity is present in tissue from control brain, suggesting Alz-50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz-50 binding presents the possibility of Alz-50 immunoreactivity with proteins other than tau. The present study demonstrates Alz-50 cross-reactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA-N-5 neuroblastoma cells, BSA from serum-containing media was present in cell homogenates and was found to be Alz-50-reactive on immunoblots. In fact, Alz-50 (0.1 microgram/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz-50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz-50 reactivity with tau from LA-N-5 cells. On SDS-polyacrylamide gels, HSA (approximately 69 kDa) migrates very closely to the pattern of A68 (PHF-tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using the Alz-50 antibody. PMID- 7534358 TI - Dopamine and serotonin turnover rate in the retina of rabbit, rat, goldfish, and Eugerres plumieri: light effects in goldfish and rat. AB - The concentration of dopamine, and its metabolites 3,4-dihydroxyphenylacetic and homovanillic acids, as well as serotonin and its metabolite 5-hydroxyindoleacetic acid, were determined in the retina of two teleosts, C. auratus (goldfish) and E. plumieri (mojarra), and two mammals, R. norvegicus (rat) and O. cuniculus (rabbit). The turnover rate of these monoamines were investigated in the four species by the calculation of the ratio monoamine/metabolite as an indirect index, and in goldfish and rat by the inhibition of the synthesis with alpha methyl-p-tyrosine or p-chlorophenylalanine, by the increase in dopamine or serotonin by the corresponding precursors, 3,4-dihydroxyphenylalanine or 5 hydroxytryptophan, and by inhibition of monoaminooxidase with pargyline. The modulation by light and dark stimulation was studied in the goldfish and the rat. Differences in the concentration and turnover rate were observed among the species. Serotonin concentration was higher in the teleosts. The administration of inhibitors of dopamine and serotonin synthesis differentially decreased the levels of the monoamines in the retina of goldfish and rat. The rate of formation of dopamine and serotonin by the corresponding precursors was much higher in the goldfish than in the rat. Pargyline administration decreased 3,4 dihydroxyphenylacetic and 5-hydroxyindoleacetic acids at different rates and time dependency in the retina of goldfish and rat. Dopamine and serotonin concentration did not exhibit high modifications by the inhibitor, suggesting the function of regulatory mechanisms or additional effect of pargyline at other sites different from monoaminooxidase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534359 TI - Insertional mutagenesis inducing hypomyelination in transgenic mice. AB - Investigations of myelin disorders, in particular multiple sclerosis (MS), have concentrated on immunemediated damage to formed myelin, while there has been less emphasis on the molecular genetics of myelin formation. We have generated a transgenic mouse mutant (designated 2-50) which carries an insertional mutation in a locus regulating myelination. These mice carry a transgene comprising 1.3 Kb of the mouse myelin basic protein (MBP) promoter conjugated to a fragment containing exons 2 and 3 of the human c-myc gene. Positive mice show a significant reduction in myelin compared to controls and a shivering phenotype. Unlike other myelin mutants, all 2-50 mice lose the shivering phenotype and breed normally. Expression of c-myc is detectable in only 65% of transgene-carrying mice, and when present occurs at extremely low levels. This shows that the phenotype is caused by insertional inactivation of a gene necessary for myelination rather than ectopic expression of the transgene. The transgene was found by in situ hybridization to be inserted into a single site which is very distally located on chromosome 9. The 2-50 mice represent a unique model which will be ideal for investigating the molecular basis of myelin assembly and for developing gene therapy to promote remyelination in conditions such as MS. PMID- 7534360 TI - Tetralogy of Fallot: diagnostic imaging after palliative and corrective surgery. AB - Tetralogy of Fallot was invariably fatal until the development of palliative and later corrective surgical procedures. The prognosis for children with tetralogy of Fallot continues to improve almost a half century after the earliest palliative surgical procedure was performed successfully. Imaging of the child and adult after surgery for tetralogy of Fallot remains an important challenge because surgical complications or limitations frequently require imaging for complete evaluation and further management of the patient. Traditional imaging by chest radiography and arteriography has been largely replaced by echocardiography and ultrafast and conventional CT, as well as magnetic resonance imaging. This article reviews those aspects of diagnostic imaging that are appropriate to study the postoperative chest in the child or adult with tetralogy of Fallot. PMID- 7534361 TI - Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. AB - We have reviewed the basic aspects of history, physicochemical properties, advantages, disadvantages, and applications of nystatin- and amphotericin B perforated patch recording. Recently, we have developed a novel perforated patch technique using gramicidin [49, 132, 133]. Since gramicidin pores are permeable to Na+ and K+ but not to Cl-, it is possible to maintain the physiological concentration of intracellular Cl- and thereby to measure it in real-time from small neurons. Further improvement of the perforated patch techniques would be possible by limiting redistribution of specific intracellular ions. In light of their broad applicability, the perforated patch techniques will prove indispensable for electrophysiologists working on the functions and modulations of receptors, ion channels and transport phenomena. Research with the perforated patch should continue to provide important information relevant to the physiological and pathological conditions of cells. PMID- 7534362 TI - The use of Lucifer Yellow and Mini-Ruby for intracellular staining in fixed brain tissue: methodological considerations evaluated in rat and human autopsy brains. AB - The quality of intracellular filling of Mini-Ruby (MR) is comparable to that of Lucifer Yellow (LY) in both perfusion-fixed rat and immersion-fixed autopsy human tissue. In adult human brain, the use of MR avoids the co-conversion of the typical intracellular lipofuscin deposits as is invariably the case during the photoconversion of LY. In autopsy studies (n = 35), the quality of intracellular staining does not correlate with age (range: 25-91 years) or the postmortem delay (range: 7-50 h). A short agonal status, however, is advantageous. PMID- 7534363 TI - Selective inhibition of neuronal protein synthesis by retrogradely transported ricin. AB - The ability of the lectin, ricinus communis agglutinin I (ricin120), to undergo retrograde axonal transport and cause degeneration of neuronal cell bodies has been frequently exploited to establish the origin of peripheral axons. Since this cytotoxic action of ricin results from its inactivation of ribosomes, the retrogradely transported lectin was employed in the present study to inhibit protein synthesis in dorsal root ganglion (DRG) neurons whose axons project into the lumbar nerve trunk of bullfrog tadpoles. The procedure was developed to examine, during tadpole metamorphosis, the ratio of fast-transported radiolabeled protein accumulating at the proximal side of a nerve trunk ligature to the total newly synthesized protein in the cell bodies of origin. The relatively small diameter and fragility of the developing lumbar nerve trunks necessitated introduction of ricin by bath application to the cut nerve end rather than by intraneural injection. Consistent uptake of ricin was achieved by pretreatment with the phospholipase A2 inhibitor, mepacrine, that blocks resealing of severed nerve fibers. Optimal time and dosage of ricin were established by determining the maximal achievable inhibition of [35S]methionine into DRG protein. In stage XVI tadpoles, maximal inhibition of approximately to 65% was observed after 16 h incubation in 2.5 mg/ml ricin. As evidence that neuronal protein synthesis was effectively suppressed, there was no detectable anterograde axonal transport of [35S]protein subsequent to ricin treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534364 TI - Two models for determining the mechanisms of damage and repair after hypoxic ischaemic injury in the developing rat brain. AB - Hypoxic-ischaemic (HI) brain injury can lead to selective neuronal loss or pannecrosis. Different mechanisms of damage and recovery may be associated with either pattern of cell loss. Two preparations were developed to investigate the role of growth factors and other mechanisms associated with either of these patterns of damage in the developing brain. Twenty-one-day-old Wistar rats underwent permanent unilateral right carotid artery ligation. Following surgery, ligated rats were warmed for 2 h at 34 degrees C with 85 +/- 5% relative humidity and then exposed to either 15 or 60 min of 8% O2. The rats were killed and brains perfusion fixed 5 days post-hypoxia for histological analysis. Rats exposed to 15 min of hypoxia-ischaemia suffered no mortality and 90% developed selective neuronal loss in the frontoparietal cortex and hippocampal CA1 region of the ipsilateral hemisphere. Rats exposed to 60 min of hypoxia consistently developed cortical pannecrosis and cellular loss in the striatum, thalamus, hippocampus and dentate gyrus of the ipsilateral hemisphere. The advantage of these models were ease of preparation, consistent neuronal loss scores and low mortality. The ability to induce either selective neuronal loss or pannecrosis provides an opportunity to investigate the mechanisms of damage or recovery associated with each pattern of cell loss. PMID- 7534365 TI - An alternative method for obtaining high-viability cell suspensions from neonatal mouse brain. AB - Cultured brain cells have contributed greatly to our understanding of a variety of neurobiological processes. The ability to culture brain tissue is important for studying cellular processes underlying unique neural properties. Traditional culturing techniques commonly involve triturating tissue through glass Pastuer pipets, which are inappropriate for use with potentially biohazardous materials. We therefore developed an alternative method for dissociating brain tissue. The protocol combines enzymatic digestion and mechanical dissociation with additives to the dissection medium that protect the cells against other sources of injury, including glutamate neurotoxicity, oxidative damage, and excessively alkaline pH. We find this method works well with post-natal mouse brain, consistently giving cell viabilities in the range of 92-99% and an average yield of 3.1 x 10(6) cells per mouse. PMID- 7534366 TI - [The use of human gamma globulin in the treatment of common variable immunodeficiency]. AB - BACKGROUND: The indication for treatment with human gammaglobulin in patients with primary hypogammaglobulinemia is well established. Nonetheless, there are no uniform criteria with regard to dose, periodicity and route of administration. METHODS: Twenty-seven patients with common variable immunodeficiency (CVI) who received i.m. or i.v. treatment with gammaglobulin were studied, evaluating the secondary effects, stable levels of IgG achieved, control of symptomatology, clinical evolution and the need for adjuvant therapies. RESULTS: Intravenous administration was more effective than intramuscular administration to achieve higher total IgG serum levels (5.2 +/- 1.2 vs 3.5 +/- 1.6 g/l; p = 0.07) in a shorter period of time (2.1 +/- 1.6 months vs 6.3 +/- 2.8 months; p < 0.01) and with new few secondary effects. The dose and periodicity of the treatment was individualized in each patient on the basis of the needs of consumption and the speed of metabolism of the gammaglobulin, with patients with chronic bronchial suppuration and diarrhea being those requiring the greatest doses (p < 0.0001) and a short interdosis time interval (19.2 +/- 3.1 vs 23.6 +/- 3.6 days; p = 0.01). Treatment with human gammaglobulin allowed the control of recurrent bacterial infection; however, adjuvant treatment with respiratory physical measures and antibiotics were required in patients with chronic bronchial suppuration to avoid progressive alteration of respiratory function. CONCLUSIONS: The administration of human gammaglobulin at adequate doses and frequency is effective to control infection, avoid the development of chronic bronchial disease, alteration of pulmonary function and the appearance of other complications. Intravenous route is safer and produces fewer secondary effects than intramuscular administration with the doses and period of the treatment requiring individualization for each patient. PMID- 7534368 TI - [Treatment with human gamma globulin]. PMID- 7534369 TI - Relation of thickening of colon wall to pancreatic-enzyme treatment in cystic fibrosis. AB - Fibrotic strictures of the colon in association with colon-wall thickening on abdominal ultrasound have been described in children with cystic fibrosis. We determined the prevalence of colon-wall thickening in 99 children with cystic fibrosis (aged 6 months to 17 years, 45 male) and 38 healthy controls (aged 1 month to 39 years, 21 male). In controls, peristalsis was uniformly rapid and maximum wall thickness of the fully relaxed colon was 0.8 mm (median 0.6 mm). 24% of the patients had a colon-wall thickness of 1.5 mm or more (maximum 3.4 mm); 48% of children on high-strength pancreatin receiving more than 265 units/kg per day of protease had thickening of more than 1.5 mm compared with less than 12% of children on any other dose. Children on high-strength pancreatin were 5.2 (95% CI 1.2-21) times more likely to have colon-wall thickness of 1.5 mm or more than children taking less than 265 units/kg per day of protease in a standard-strength preparation. This risk rose to 10 (2.5-39) times more likely if laxatives were taken with a high-strength preparation. Lipase or amylase intake, age, sex, genotype, and other medical or drug history were not significantly associated with colon-wall thickening. 1 child required a hemicolectomy for bowel obstruction and another developed haemorrhagic colitis. In 17 children, 2 months after stopping high-strength, high protease, preparations and efforts to reduce enzyme intake, colon-wall thickness had regressed from a median 2.0 mm (range 1.0 3.4) to 1.8 mm (0.9-2.7) (p = 0.005). The use of high-strength pancreatin preparations in conjunction with a high protease intake probably causes thickening of the wall of the colon. PMID- 7534367 TI - [The rapid specific characterization of clinical isolates of the genus Mycobacterium by the polymerase chain reaction and restriction enzyme analysis]. AB - BACKGROUND: Typing at species level of Mycobacterium is usually performed by microbiological and biochemical methods that require a long time and/or sufficient amount of bacteria. Molecular biology can avoid these problems using different techniques. METHODS: A colony growth of the following mycobacteria has been analyzed: M. tuberculosis, M. kansasii, M. avium, M. intracellulare, M. gordonae, M. phlei, M. aurum, M. fortuitum, M. flavescens, M. marinum, M. xenopi, M. nonchromogenicum, M. terrae and M. chelonei. Strains were grown in Lowenstein Jensen medium. DNA was obtained by proteolytic digestion and fenol extraction. The 16S rRNA gen was amplified by polymerase chain reaction (PCR) and the amplification was digested by HaeIII, HpaII, RsaI and AluI restriction enzymes. Restriction fragment patterns were analyzed by agarose gel electrophoresis and UV transillumination. RESULTS: The combination of the patterns obtained with HpaII and RsaI was sufficient to generate 13 different combined ones. The patterns of M. intracellulare and M. avium were the same. CONCLUSIONS: PCR and restriction enzyme analysis is an useful method for typing at species level of clinical isolates of mycobacteria. PMID- 7534371 TI - Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120. AB - The cyanobacterial ntcA gene encodes a DNA-binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen-fixing, heterocyst forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild-type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNAl), that has been shown to be used preferentially after nitrogen step-down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA. PMID- 7534370 TI - mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures. AB - Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro. This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed. PMID- 7534372 TI - Transcriptional attenuation control of the tylosin-resistance gene tlrA in Streptomyces fradiae. AB - The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD. PMID- 7534373 TI - Neoglycolipids: probes in structure/function assignments to oligosaccharides. PMID- 7534374 TI - Sympathomimetic vasoconstrictors as nasal decongestants. PMID- 7534375 TI - End-of-life care in Minnesota. PMID- 7534377 TI - When medicine can't cure. PMID- 7534376 TI - Cancer pain management in the hospice setting. AB - Pain relief for the cancer patient in the hospice setting is almost always achievable. Cancer pain is caused by tumor growth and by psychosocial and spiritual factors. Opioid drugs are the mainstay of effective treatment. Morphine is the opioid drug of choice. Although tolerance to opioids occurs, tumor growth is the usual reason for escalating opioid dose. Addiction almost never occurs in the cancer patient with pain. These patients don't exhibit drug-seeking behavior or experience the psychic high seen in drug addicts. Nonsteroidal anti inflammatory drugs and adjuvant analgesics are synergistic with opioids in providing analgesia and allow lower opioid doses and fewer side effects. Ten to 15 percent of hospice patients will require regional anesthesia for pain relief. The hospice team of physicians, nurses, social workers, chaplains, aides, and volunteers is more effective than any single health care provider in achieving optimal pain relief and comfort. PMID- 7534378 TI - A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution. AB - Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C terminal variants of SRP14 in higher primates. PMID- 7534380 TI - [Transplantation of the small intestine]. PMID- 7534381 TI - Searching for hepatitis C virus antibodies in chronic primary glomerular diseases. PMID- 7534379 TI - Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways. AB - The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5 c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity. PMID- 7534382 TI - Low prevalence of antibodies to hepatitis C virus among adult patients with idiopathic membranoproliferative type I glomerulonephritis in France. PMID- 7534383 TI - Interleukin-6 and selected plasma proteins in healthy persons of different ages. AB - In the present study, interleukin-6 (IL-6) and several acute phase proteins were measured in healthy participants (23-87 years of age). A linear correlation between IL-6 and age was established with an increase of 0.016 pg/ml (0.004) per year of life. Whereas CRP remained below 0.5 mg/dl in all participants, an increase with age for fibrinogen and an inverse relation for albumin as well as transferrin were obtained. However, the increase of IL-6 did not correlate with any of these changes. IL-6 associated diseases may therefore occur more often with advancing age, but in healthy participants IL-6 does not explain the changing plasma protein pattern resembling that of an acute phase reaction. PMID- 7534384 TI - [3H]histamine uptake and release by astrocytes from rat brain: effects of sodium deprivation, high potassium, and potassium channel blockers. AB - Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [3H]-histamine uptake yielded a Km of 0.19 +/- 0.03 microM and a Vmax of 3.12 +/- 0.75 pmol X mg protein-1 X min-1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na+ with choline inhibited the initial rate of uptake showing a strong Na(+)-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K(+)-gradient (in > out) by high K+ or by K(+)-channel blockers, BaCl2, (100 microM), quinine (100 microM) or Sparteine (20 microM) produced also remarkable inhibitions in the uptake of [3H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non competitively in a range of concentrations of 1 to 10 microM with a Ki value of 2.8 microM, indicating the specificity of the uptake. [3H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33 +/- 0.08 microM; and a Vmax of 2.65 +/- 0.35 pmoles x mg protein-1 x min-1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [3H]histamine efflux was sensitive to Na+ omission, ouabain treatment and high K+ or K+ channel blockers, resulting in marked elevations in the efflux.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534385 TI - Enhancement of NMDA-mediated responses by cyanide. AB - The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1-5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion. PMID- 7534386 TI - Effects of hypothyroidism on RNA synthesis in the adult rat brain. AB - In this study we investigated the effects of hypothyroidism on adult brain RNA synthesis. Our data show that in the cerebral hemispheres of hypothyroid rats there is a decrease in microsomal RNA content and microsomal [3H]uridine incorporation. Sucrose gradient analysis revealed that these changes are mainly associated with free ribosomes and subunits and reflect changes in rRNA. The above changes are accompanied by a decrease in RNA polymerase I activity. All of the above mentioned changes returned to normal after thyroxine (T4) treatment. In contrast to RNA polymerase I, RNA polymerase II activity was not affected. However, electrophoretic analysis of the in vitro poly(A)+RNA translation products revealed that hypothyroidism affects a few mRNAs. These results indicate that thyroid hormones have a role in adult brain tissue metabolism. PMID- 7534388 TI - Colocalization of NADPH-diaphorase with neuropeptides in the intrapancreatic neurons of the chicken. AB - Colocalization of nitric oxide with neuropeptides was investigated in the chicken pancreas by use of double staining combined with the indirect immunofluorescence technique and histochemistry for NADPH-diaphorase, a specific marker for neural nitric oxide synthase. NADPH-diaphorase positive ganglia were easily detected in the interlobular connective tissue. Many NADPH-diaphorase positive ganglion cells also showed immunoreactivity for VIP (80.9%) or galanin (76.2%). Some ganglion cells showed enzyme activity only (about 20%). Very few neurons were NADPH diaphorase negative, but immunopositive for VIP (2.0%) or galanin (3.7%). The present study provides evidence that nitric oxide colocates with VIP and galanin in the chicken pancreas. PMID- 7534387 TI - Dopaminergic and muscarinic regulation of striatal enkephalin and substance P messenger RNAs following striatal dopamine denervation: effects of systemic and central administration of quinpirole and scopolamine. AB - Striatal dopamine depletion produces an increase in enkephalin and a decrease in substance P messenger RNAs. Subsequent systemic administration of either the D2 dopamine agonist, quinpirole, or the muscarinic antagonist, scopolamine, results in the reduction of the lesion-induced elevation in striatal enkephalin messenger RNA. These changes in enkephalin messenger RNA levels may be mediated solely within the striatum or through trans-synaptic circuits involving the striatum. To dissociate these possibilities, we have compared the effects of systemic and central administration of quinpirole and scopolamine on striatal enkephalin and substance P messenger RNAs using in situ hybridization histochemistry. Systemic administration of both quinpirole and scopolamine blocked the elevation of striatal enkephalin messenger RNA normally observed in 6-hydroxydopamine-lesioned rats. In addition, high doses of systemic scopolamine (25 and 50 mg/kg per day) prevented the lesion-induced decrease in striatal substance P messenger RNA levels. In order to determine whether the effects of these drugs are mediated directly within the striatum, central administration of quinpirole and scopolamine were compared. In contrast to systemic administration, intraventricular and intrastriatal infusion of quinpirole but not scopolamine prevented the lesion-induced change in striatal enkephalin messenger RNA. However, neither quinpirole nor scopolamine administered centrally affected the level of substance P messenger RNA in the striatum of 6-hydroxydopamine-induced lesioned animals. Together, these data suggest that changes in D2 receptor activation directly in the striatum are responsible for the effects of quinpirole on enkephalin messenger RNA. In contrast, the effect of systemic scopolamine on striatal enkephalin and substance P messenger RNAs may not be mediated within the striatum. PMID- 7534390 TI - Modulation of the nicotinic acetylcholine receptor channels by spermine in Xenopus muscle cell culture. AB - The modulation by endogenous polyamine spermine on acetylcholine (ACh)-induced currents was evaluated using a outside-out patch and whole-cell voltage-clamp recordings from cultured Xenopus muscle cells. Spermine potentiated the ACh induced whole-cell currents at micromolar concentrations but is less effective at millimolar concentrations at a holding potential of -60 mV. It produced a voltage dependent potentiation of ACh-induced whole-cell currents; the degree of potentiation by 100 microM spermine was 18.3 +/- 1.7% at a holding potential of 60 mV while 108.2 +/- 2.9% increase at +60 mV. Arcaine, a putative competitive antagonist at the polyamine-binding site on the N-methyl-D-aspartate (NMDA) receptor-channel complex, reduced the spermine enhancement of ACh-induced whole cell currents. Glycine (10 microM) had no effect on the potentiation of ACh induced whole-cell currents by spermine. In single-channel recordings, spermine (1 microM to 1 mM) increased the opening frequency of both low- and high conductance ACh channels and at higher concentrations (> or = 100 microM) it produced, in addition, a voltage-dependent decrease in channel amplitude and average open time of both types of ACh channels that limits its enhancing action. It is likely that these two actions of spermine, due to differences in concentration- and voltage-dependence, are mediated by different binding sites on the nicotinic ACh receptor-channel complex of the muscle cells. PMID- 7534389 TI - Pb2+ activates potassium currents in bovine adrenal chromaffin cells. AB - Inorganic lead (Pb2+) is a potent environmental toxin which adversely affects several aspects of neuronal and secretory cell function. In this report, we provide evidence that at subnanomolar concentrations, Pb2+ activates the outward K+ currents in bovine adrenal chromaffin cells. Whole-cell patch clamp combined with intracellular perfusion was employed to monitor outward K+ currents in bovine chromaffin cells before and after intracellular application of EGTA-Pb buffers. Intracellular Pb2+ > or = 10(-10) M enhanced the K(+)-currents in a concentration dependent manner, with apparent K0.5 approximately equal to 5 x 10( 10) M. Extracellular application of 40 nM Charybdotoxin (ChTX) blocked the Pb(2+) dependent component of outward currents, suggesting that Pb2+ activates the large conductance Ca(2+)-dependent K+ channels. PMID- 7534391 TI - Fast facts about surgery. PMID- 7534392 TI - Guidelines for the use of expensive medicines. PMID- 7534393 TI - A common enemy in toxicity? PMID- 7534394 TI - The digital indocyanine green videoangiography characteristics of well-defined choroidal neovascularization. AB - PURPOSE: To evaluate the digital indocyanine green (ICG) videoangiography characteristics of well-defined choroidal neovascularization (CNV). METHODS: The authors retrospectively reviewed all ICG angiograms performed at the New England Eye Center over a 2-year period. Included in this study were all patients with the clinical and fluorescein angiographic diagnosis of well-defined CNV according to the Macular Photocoagulation Study Group criteria. RESULTS: Of the 25 eligible patients, 18 (72%) had a well-demarcated area of ICG hyperfluorescence that was observed either both early and late (6 patients = 24%) or only late (12 patients = 48%) on the ICG angiogram. Five patients (20%) showed only poorly demarcated late hyperfluorescence on ICG angiography. Two patients (8%) had type II occult CNV associated with classic CNV as per the Macular Photocoagulation Study Group criteria. Both patients showed a late, well-demarcated area of ICG hyperfluorescence greater than the area imaged with fluorescein angiography. CONCLUSIONS: Choroidal neovascularization which is well-defined on fluorescein angiography has variable ICG appearances. When there is late leakage associated with a well-defined CNV on fluorescein angiography (type II occult CNV), ICG angiography may more completely delineate the extent of the lesion. PMID- 7534395 TI - Cationic amino acid fluxes beyond the proximal convoluted tubule of rat kidney. AB - To investigate the fluxes of cationic amino acids beyond the proximal convolution, we micropunctured and microperfused superficial tubules of male Wistar rats in vivo et situ. In free-flow micropuncture experiments, the concentrations of endogenous L-arginine+, [Arg], and of intravenously infused L homoarginine+, [HoArg], were determined by HPLC. Fluorescein isothiocyanate labeled inulin was detected on-line in the same tubular fluid samples. To determine undirectional fluxes, radiolabeled Arg and inulin were (1) microperfused through short loops of Henle and (2) microinfused into different tubule segments to measure urinary recovery of the radiolabel. At a mean [Arg]plasma of 116 mumol/l, [Arg] was 9.3 mumol/l in the late proximal tubule (LPT), and 35.6 mumol/l in the early distal tubule (EDT) corresponding to fractional deliveries (FD) of 0.055 in LPT and 0.078 in EDT. Fractional urinary excretion (FE) of Arg was 0.00033 (P < 0.05 vs FDEDT). Infusion of HoArg (2.5 or 7.5 mumol/min) led to respective mean [HoArg]plasma values of 1.44 and 3.73 mmol/l, and resulted in respective FDLPT values for HoArg of 0.23 and 0.53, respective FDEDT values of 0.29 and 0.41, and finally, respective FE values for HoArg of 0.25 and 0.58. When short loops of Henle were microperfused with 1 or 50 mmol/l [14C]Arg (+[3H]inulin), fractional recovery (FR) of 14C (relative to inulin) in the EDT was 0.13 and 0.36, respectively. During microinfusion of radiolabeled Arg (1 or 50 mmol/l) and inulin into LPT, the urinary FR of the radiolabel was 0.14, or 0.59, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534396 TI - Neuropeptide Y inhibits Ca(2+)-activated K+ channels in vascular smooth muscle cells from the rat tail artery. AB - The effects of neuropeptide Y (NPY) on the Ca(2+)-activated K+ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single channel patch-clamp recording techniques. In the presence of nifedipine (1 microM), whole-cell outward currents through Ca(2+)-activated K+ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca(2+)-activated K+ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca(2+)-activated K+ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells. PMID- 7534397 TI - [New research findings raises hope for patients with hard-to-treat skin diseases]. PMID- 7534398 TI - Seropositivity to hepatitis C virus in Tunisian haemodialysis patients. AB - The prevalence of anti-hepatitis C virus (anti-HCV) antibodies and of hepatitis B markers (HBs antigen, anti-hepatitis B core antigen) was assessed in 63 haemodialysis patients from the Tunisian Sahel. As measured by second generation ELISA assays (Ortho and Organon), the frequency of anti-HCV antibodies was 42% (27/63), while 4 patients (6.3%) were HBs Ag positive and 30 (47.6%) anti-HBc positive. Anti-HCV seropositivity was significantly correlated with duration of dialysis (p = 0.007) and number of blood transfusions (> 10 units, p = 0.0004). Among 12 subjects with a history of abnormal ALAT levels, 10 were anti-HCV positive (p = 0.0016) and the results suggest hepatitis C viral infection to be the main cause of liver disease in haemodialysis patients in Tunisia. PMID- 7534400 TI - Proliferative cell indices measured by DNA flow cytometry in node-negative adenocarcinomas of breast: accuracy and significance in cytokeratin-stained archival specimens. AB - Proliferative rates of 73 node-negative adenocarcinomas of breast with 5-year or greater follow-up were studied using cytokeratin staining in two-parameter DNA flow cytometry of archival specimens. Quality control data of accuracy of the measurements were determined and all analyses were compared with single-parameter results of the same specimens, using demarcated tumor areas, quadruple analyses, and computerized nonspecific staining subtraction. Mitotic rates of the same samples correlated highly significantly with the S-phase fractions and proliferative index (S + G2 + M phases), especially for the cytokeratin data. The predictive value of mitotic rates was found significant, but that of the DNA flow cytometry-obtained indices was not, probably because of low numbers of deaths in this study. The cytokeratin method identified heteroploid tumors containing a diploid cell population not identifiable by single-parameter analysis. In conclusion, cytokeratin staining can be reliably applied to DNA flow cytometry of archival specimens giving accurate ploidy, S-phase fractions, and proliferative index data limited almost exclusively to neoplastic cell populations. This will permit large-scale retrospective studies aimed at establishing the usefulness of DNA flow cytometry for clinical decisions on therapy of surgically removed node negative adenocarcinomas of breast. PMID- 7534401 TI - Correspondence re: Frierson HF, Bellafiore FJ, Gaffey MJ, McCary WS, Innes DJ Jr, Williams ME. Cytokeratin in anaplastic large cell lymphoma. Mod Pathol 7:317, 1994. PMID- 7534399 TI - Fetal haemoglobin variations following hydroxyurea treatment in patients with cyanotic congenital heart disease. AB - Haematological features of 64 patients suffering from non operable cyanotic congenital heart disease (CCHD) treated with hydroxyurea (HU) were compared with those of 43 patients suffering from the same disorder who had not yet received this drug. Patients with subclinical renal dysfunction were excluded by measuring plasma creatinine levels. MCV and HbF were higher among patients receiving HU, the increase in MCV being cumulative with HU dosage but the rise in HbF dose independent. HbF response to HU was found to be due to the coordinated increase in F-cell and F-reticulocyte production rather than to a selective survival of F cells. Absence of a relationship between plasma erythropoietin and HbF levels excluded a dominant role of the former in increasing F-cell production and results determined after doubling the HU dosage or immediately after initiating therapy suggested genetic differences to be responsible for the individual variations in Hb F response. No irreversible toxic effects or malignancies were noted in this series of patients. HU was administered for a relatively long period of time, the mean duration of treatment exceeding 5 years, while the study also included patients below the age of 10 years. PMID- 7534402 TI - Crystallization and preliminary crystallographic analysis of the N-terminal two domain fragment of vascular cell adhesion molecule-1 (VCAM-1). AB - A molecular fragment comprising the first two domains of the human vascular cell adhesion molecule-1 (VCAM-1) has been crystallized by the vapor diffusion method. Two crystal forms have been examined by X-ray analysis: One crystal form belongs to the space group C2 with two molecules in the asymmetric unit and cell parameters: a = 122.1 A, b = 48.9 A, c = 73.4 A, and beta = 117.4 degrees. The other crystal form belongs to the space group P2(1) with one molecule in the asymmetric unit and cell parameters: a = 40.4 A, b = 45.7 A, c = 54.7 A, and beta = 100.5 degrees. Diffraction data up to 1.9 A resolution have been collected for the C2 crystal form. PMID- 7534403 TI - Polyadenylylation helps regulate mRNA decay in Escherichia coli. AB - As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we examined the effect of the pcnB gene [encoding poly(A) polymerase I] on message stability. Eliminating poly(A) polymerase I (delta pcnB) dramatically stabilized the lpp, ompA, and trxA transcripts. The half-lives of individual mRNAs were increased in both a delta pcnB single mutant and a delta pcnB pnp-7 rnb-500 rne-1 multiple mutant. We also found mRNA decay intermediates in delta pcnB mutants that were not detected in control strains. By end-labeling total E. coli RNA with [32P]pCp and T4 RNA ligase and then digesting the RNA with RNase A and T1, we showed that many RNAs in a wild-type strain contained poly(A) tails ranging from 10 nt to > 50 nt long. When polynucleotide phosphorylase, RNase II, and RNase E were absent, the length (> 100 nt) and number (10- to 20-fold) of the poly(A) tails increased. After transcription initiation was stopped with rifampicin, polyadenylylation apparently continued. Deleting the structural gene for poly(A) polymerase I (pcnB) reduced the amount of 3'-terminal poly(A) sequences by > 90%. We propose a model for the role of polyadenylylation in mRNA decay. PMID- 7534404 TI - Molecular cloning of a ligand for the EPH-related receptor protein-tyrosine kinase Htk. AB - Htk is a receptor protein-tyrosine kinase that is related to the EPH subfamily of tyrosine kinases. The receptor has a wide tissue distribution including expression in several myeloid hematopoietic cell lines. Using an Htk-Fc fusion protein, a protein ligand for this receptor was expression cloned from the murine kidney mesangial cell line SV40MES 13. The Htk ligand cDNA encodes a transmembrane protein of 336 amino acids. Binding competition experiments demonstrated a Kd of 535 pM for binding of Htk-Fc to the Htk ligand. Incubation of 3T3 cells expressing Htk with COS-7 cells expressing the ligand resulted in tyrosine phosphorylation of Htk. The ligand, like its receptor, is widely expressed and may function in a variety of tissues. However, we localized hematopoietic expression of Htk to the monocytic lineage, suggesting that the ligand may play a role in differentiation and/or proliferation of these cells. PMID- 7534405 TI - Rat kidney papilla contains abundant synaptobrevin protein that participates in the fusion of antidiuretic hormone-regulated water channel-containing endosomes in vitro. AB - Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP 2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones. PMID- 7534406 TI - Ethanol enhances growth factor activation of mitogen-activated protein kinases by a protein kinase C-dependent mechanism. AB - Excessive alcohol consumption alters neuronal growth and causes striking elongation of axons and dendrites in several brain regions. This could result from increased sensitivity to neurotrophic factors, since ethanol markedly enhances nerve growth factor (NGF)- and basic fibroblast growth factor (bFGF) stimulated neurite outgrowth in the neural cell line PC12. The mechanism by which ethanol enhances growth factor responses was investigated by examining activation of mitogen-activated protein kinases (MAP kinases), a key event in growth factor signaling. Ethanol (100 mM) increased NGF- and bFGF-induced activation of MAP kinases. This increase, like ethanol-induced increases in neurite outgrowth, was prevented by down regulation of beta, delta, and epsilon protein kinase C (PKC) isozymes. Since chronic ethanol exposure specifically upregulates delta and epsilon PKC, these findings suggest that ethanol promotes neurite growth by enhancing growth factor signal transduction through a delta or epsilon PKC regulated pathway. PMID- 7534407 TI - SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid. AB - Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif. Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC. In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation. These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular. PMID- 7534408 TI - A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli. AB - The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA. The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis. We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter. A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing. DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E. coli. Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation. PMID- 7534409 TI - A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes. AB - Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID- 7534410 TI - The N terminus of phosducin is involved in binding of beta gamma subunits of G protein. AB - Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N terminal half of phosducin showed inhibition, whereas overexpression of the C terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue. PMID- 7534411 TI - Voltage-dependent gating of an asymmetric gramicidin channel. AB - In an effort to understand the molecular mechanisms of voltage activation of ion channels, we have chosen a system of known structure and examined the properties of heterodimeric channels formed between [Val1]gramicidin A ([Val1]gA) and [F6Val1]gramicidin A ([F6Val1]gA). Gramicidin channels are usually not voltage dependent; but the introduction of a single symmetry-breaking dipolar F6Val1 residue into a ([Val1]gA)2 dimer to form the [F6Val1]gA/[Val1]gA heterodimer induces voltage-dependent transitions between two conducting states: a high conductance state and a zero conductance (closed) state. The distribution between these states varies as a function of the applied potential but is not dependent on the nature of the permeant ion (H+ or Cs+). The permeating ions do not seem to contribute to the apparent gating charge. PMID- 7534413 TI - Molecular mimicry: histone H3 and mycobacterial protein epitopes. AB - A 15-kDa protein detected initially in amyloidotic ileum from a transgenic mouse and subsequently in control (nontransgenic) ileum by various polyclonal rabbit antiserums applied to electroblots of extracts derived from these tissues was identified by partial sequence analysis as histone H3. Antiserums were made against immunogens unrelated to the histone, but they recognized calf thymus histone H3 (14.7 kDa) on Western blots. The bacterial component of the Freund's medium used as an adjuvant for the immunogens was either Mycobacterium butyricum or Mycobacterium smegmatis. Absorption tests with histone H3 and sonicated M. butyricum substantiated the presence of anti-histone H3 activity in the antiserums. These findings indicate that the two mycobacterium species make a protein with epitopes perceived as nonself by recipient rabbits but sufficiently similar to epitopes of mammalian histone H3 that the rabbits produced antibodies cross-reactive with the histone. PMID- 7534412 TI - Structural and functional similarities between the promoters for mouse tenascin and chicken cytotactin. AB - Cytotactin/tenascin is an extracellular matrix glycoprotein expressed in a restricted anteroposterior pattern during vertebrate development and is reexpressed in the adult during wound healing, tumorigenesis, and nerve regeneration. Previously, we have characterized the chicken cytotactin promoter and have shown its regulation by homeobox gene products in vitro. We have now isolated the promoter for the mouse tenascin gene in order to determine whether common or different DNA regulatory elements control the expression of this gene in these two species. Like the chicken cytotactin gene, the mouse tenascin gene has a single RNA start site that lies 27 bp downstream of a TATA box. A 4028-bp region of DNA upstream of the mouse tenascin gene was sequenced and examined for regulatory motifs in common with the upstream sequence from the chicken cytotactin promoter. Two hundred thirty base pairs of the proximal promoter regions from both genes had an extended sequence similarity and contained common regulatory motifs such as two tracts of homopolymeric dA.dT sequence, an octamer motif, an ATTA (TAAT) motif which is a common core sequence for binding of homeodomain transcription factors, and a TATA-box/cap-site region. Reporter gene constructs with various 5' deletions of the mouse tenascin upstream sequence were tested in transient transfections of mouse NIH 3T3 and chicken embryo fibroblasts. The conserved proximal promoter region of tenascin was responsible for most of the positive regulatory activity. In addition, an upstream region ( 2478 to -247) repressed proximal promoter activity in mouse fibroblasts and also in chicken embryo fibroblasts. These data indicate that both the structure and function of the cytotactin/tenascin proximal promoters have remained conserved over 250 million years. PMID- 7534414 TI - Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor. AB - In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes. PMID- 7534415 TI - Identification of a major I-Ek-restricted determinant of hen egg lysozyme: limitations of lymph node proliferation studies in defining immunodominance and crypticity. AB - We have chemically analyzed the peptides presented by I-Ek molecules after processing of hen egg lysozyme (HEL) by a murine B-lymphoma line or by splenocytes. In both cases, the identified peptides were derived from a single region of HEL, containing the core residues 85-96 with heterogeneous N and C termini. This was a surprising result because this determinant had previously been described as cryptic--i.e., not presented after processing of intact HEL. Examination of the specificities of T hybridomas isolated after immunization with either HEL or 84-96 peptide (p84-96) provided an explanation for this controversy. Whereas hybridomas induced by immunization with HEL responded equally well to HEL and p84-96, those induced by peptide immunization showed a marked preference for p84-96 over intact HEL. In other words, hybridomas isolated after p84-96 immunization responded poorly to forms of the 84-96 determinant produced by natural processing, leading to the possible erroneous interpretation that 84-96 is a hidden determinant. We conclude that (i) p84-96 is efficiently presented on I-Ek molecules after processing of HEL, (ii) the explanation for the weak lymph node response to this epitope after immunization with HEL lies at the level of the T cell, not the antigen-presenting cell, and (iii) crypticity cannot be defined on the basis of T-cell proliferation studies alone. PMID- 7534416 TI - Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene. AB - There is a need for a large-animal model to investigate the etiology and biology of cystic fibrosis (CF) lung disease and to study potential therapies. The development and electrophysiology of the sheep airway have been shown to exhibit close functional parallels with the human airway, particularly with respect to the respiratory epithelium. We have cloned and sequenced the ovine cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. It shows a high degree of conservation at the DNA coding and predicted polypeptide levels with human CFTR: at the nucleic acid level there is a 90% conservation (compared with 80% between human and mouse CFTR cDNA); at the polypeptide level, the degree of similarity is 95% (compared with 88% between human and mouse). Northern blot analysis and reverse transcription-PCR have shown that the patterns of expression of the ovine CFTR gene are very similar to those seen in humans. Further, the developmental expression of CFTR in the sheep is equivalent to that observed in humans. Thus, overall a CF sheep should show lung pathology similar to that of humans with CF. PMID- 7534418 TI - CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription. AB - CD8+ cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in cultured CD4+ cells by a noncytolytic mechanism that involves a secreted CD8(+)-cell antiviral factor (CAF). The results of this study suggest that CD8+ cells, as well as CAF, arrest HIV replication at the level of viral transcription. Culturing naturally infected CD4+ cells actively producing HIV with autologous CD8+ cells or a 50% dilution of culture fluids from these cells results in a > 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days after exposure to CD8+ cells but required 6 days in the presence of CAF-containing culture fluids to reach the same extent of HIV suppression. Northern blot analysis of CD4+ cell extracts revealed that all viral RNA species (unspliced and single and double spliced) were reduced in quantity to a similar extent. CAF-containing culture fluids also had a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells carrying an LTR-luciferase construct. Suppression of basal levels of LTR-driven transcription was not detected. Thus, the results suggest that the noncytolytic CD8+ cell antiviral activity observed in HIV infection exerts its effects, at least in part, by specifically interrupting HIV transcription. These findings could help in developing therapies for HIV infection. PMID- 7534419 TI - Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry. AB - Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (< 50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (1o(5)) resolving power makes possible detection of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76 mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 50-mer DNA is characterized with < 10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-stranded DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da. PMID- 7534417 TI - Measles virus and C3 binding sites are distinct on membrane cofactor protein (CD46). AB - The human complement regulatory protein membrane cofactor protein (CD46) is the cellular receptor for measles virus (MV), whereas decay accelerating factor (DAF; CD55), a structurally similar complement regulatory protein, does not bind MV. To characterize the interaction between MV and CD46, mutants of the CD46 protein and hybrid molecules between CD46 and DAF were tested for their ability to act as MV receptors. The transmembrane domain and cytoplasmic tail of CD46 were not required for receptor function as cells expressing the CD46 extracellular domain linked to the glycosyl-phosphatidylinositol tail of DAF were rendered susceptible to MV infection. Chimeric proteins exchanging the four extracellular short consensus repeat (SCR) domains between CD46 and DAF indicated that only molecules with both SCR1 and SCR2 from CD46 allowed a productive MV infection. Further, monoclonal antibodies (mAbs) against SCR1 or SCR2 of CD46 blocked MV infection, whereas a mAb against SCR3 and SCR4 did not. The latter mAb blocks C3b/C4b binding (which maps to SCR3 and SCR4) whereas the former mAbs do not. Thus, our data indicate that both SCR1 and SCR2 make up the MV receptor determinant in CD46. These results also suggest avenues for development of therapeutic agents to inhibit MV binding and thus infection and disease. PMID- 7534420 TI - Selection of an RNA molecule that mimics a major autoantigenic epitope of human insulin receptor. AB - Autoimmunity often involves the abnormal targeting of self-antigens by antibodies, leading to tissue destruction and other pathologies. This process could potentially be disrupted by small ligands that bind specifically to autoantibodies and inhibit their interaction with the target antigen. Here we report the identification of an RNA sequence that binds a mouse monoclonal antibody specific for an autoantigenic epitope of human insulin receptor. The RNA ligand binds specifically and with high affinity (apparent Kd congruent to 2 nM) to the anti-insulin receptor antibody and not to other mouse IgGs. The RNA can also act as a decoy, blocking the antibody from binding the insulin receptor. Thus, it probably binds near the combining site on the antibody. Strikingly, the RNA cross-reacts with autoantibodies from patients with extreme insulin resistance. One simple explanation is that the selected RNA may structurally mimic the antigenic epitope on the insulin receptor protein. These results suggest that decoy RNAs may be used in the treatment of autoimmune diseases. PMID- 7534421 TI - Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. AB - A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3' deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template. PMID- 7534422 TI - Methylation profile and amplification of proto-oncogenes in rat pancreas induced with phytoestrogens. AB - Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 micrograms of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras proto-oncogene. No hypermethylation or hypomethylation was observed in the proto-oncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders. PMID- 7534425 TI - [Indications and possibilities of ultrasound diagnosis in skeletal metastases]. AB - Based on the analysis of 921 metastatically affected skeletal regions in 519 tumor patients, the diagnostic relevance of ultrasound for therapy planning and follow-up is shown. As an ideal adjunct to conventional X-ray, it is equivalent to it with respect to involvement of the cortical layer and inferior to infiltration of the medullary space, but superior concerning the periosteum and the paraossal soft tissues. The pain of osteolyses is explained by infiltrating processes into the periosteum and the paraossal space. Ultrasound offers a valuable contribution to tumor characterization by dynamic representation of osseous and paraosseal tumorous processes. The high sensitivity of the method results in effective monitoring of the therapy and indications for its optimization. PMID- 7534424 TI - Facilitated acquisition of a temporal discrimination following destruction of the ascending 5-hydroxytryptaminergic pathways. AB - This experiment examined the effect of destroying the 5-hydroxytryptaminergic (5HTergic) pathways on the acquisition and performance of discrimination between two brief time intervals. Rats that had received injections of 5,7 dihydroxytryptamine into the dorsal and median raphe nuclei, and sham-lesioned control rats were trained in a series of discrete trials to press lever A following a 200-ms presentation of a light stimulus and lever B following an 800 ms presentation of the same stimulus. Both groups gradually acquired accurate performance, attaining 80%-85% accuracy by the end of 40 sessions. The lesioned group learnt the task significantly faster than the control group. When stable performance had been attained, "probe" trials were introduced in which the light was presented for intermediate durations. Both groups showed sigmoid functions relating percent choice of lever B to log stimulus duration. The bisection point (duration corresponding to 50% choice of lever B) did not differ significantly between the two groups; however, the Weber fraction was significantly smaller in the lesioned group than in the control group. The levels of 5HT and 5-hydroxy indole-acetic acid were markedly reduced in the brains of the lesioned rats, but the levels of noradrenaline and dopamine were not altered. The results indicate that destruction of the 5HTergic pathways facilitates acquisition of a temporal discrimination. The lack of an effect of the lesion on the bisection point contrasts with our previous finding using longer stimulus durations; it is suggested that different behavioural processes may underlie millisecond-range and second-range temporal discrimination, and that these may be differently affected by 5HT depletion. PMID- 7534426 TI - [Value of local radiotherapy in treatment of osseous metastases, pathological fractures and spinal cord compression]. AB - Local radiotherapy plays an important and responsible role in the management of bone metastases. The valence will be described according to the different treatment objectives in the sense of pain relief, remineralization and cord decompression. Radiotherapy schedules, aimed at the relief pain, need to take into consideration life expectancy. Patients with a reduced life expectancy could have a good high chance of achieving pain relief with a single dose of 8 Gy. Patients with a solitary metastasis, patients with a longer life expectancy and patients with a pathological fracture should be treated with 'curative' irradiation doses, aimed at killing the maximum number of tumor cells. In addition to pain relief, remineralization is also an important treatment goal. Conventional radiotherapy with doses of 40-50 Gy resulted in pain relief in 70 100% and in remineralization in 60-80% of the patients. Remineralization could not be accelerated by short-course fractionation courses, but resulted in faster pain relief. Short-course fractionation schedules are not indicated as a 'standard' treatment in the vertebral column. Surgery is the treatment of choice for immediate cord decompression and stabilization of a pathological vertebral fracture. Radiotherapy alone could decrease neurological impairment and is suitable for patients with gradual onset and progression of symptoms, no spinal instability and lesions of the cauda equina. PMID- 7534423 TI - Effects of raclopride treatment on plasma and CSF HVA: relationships with clinical improvement in male schizophrenics. AB - Thirty-two acutely psychotic, male schizophrenic patients received raclopride, at 2, 6, or 12 mg/day, or haloperidol, 15 mg/day for 4 weeks after randomized, double-blind assignment. Twenty-six patients, including 19 who had been assigned one of the three doses of raclopride, completed the study. Raclopride, particularly at 12 mg/day, increased CSF homovanillic acid (HVA) at 4 weeks, and plasma HVA at 2 days, of treatment. The clinical response to raclopride was significantly correlated with plasma raclopride concentrations and baseline plasma HVA concentrations. Although raclopride is a substituted benzamide with atypical properties in animals, these results suggest that the doses of raclopride required for clinical efficacy and elevation of clinical indices of brain dopamine turnover are similar. PMID- 7534427 TI - [Embolization of bone metastases]. AB - Eighteen patients (11 renal tumours, 3 bronchogenic carcinomas, 4 others) with 24 different bone metastases were embolized for preoperative devascularization (11 x) or for intractable pain (7 x). Metastases were localized in the spine (17 x), pelvis (5 x), and shoulder girdle (2 x). All metastases were hypervascularized. Post-embolization showed no complications. Intraoperative blood loss was minimized to 2100 ml (600 ml-4200 ml). Pain relief was achieved in 6 out of 7 patients. Eight of 18 patients died as a result of underlying diseases (follow-up 7 months). PMID- 7534428 TI - To color Doppler image the prostate or not: that is the question. PMID- 7534429 TI - Prostate cancer: diagnosis with color Doppler sonography with histologic correlation of each biopsy site. AB - PURPOSE: To correlate the findings at prostate color Doppler sonography (CDS) with those of site-specific transrectal core biopsy. MATERIALS AND METHODS: Forty three patients underwent prostate transrectal ultrasonography (US) and biopsy. CDS was performed at all biopsy sites before US-guided core biopsy. Vascularity at CDS was prospectively graded on a scale of 0-2 (0 = no visible peripheral zone [PZ] flow, 2 = markedly increased PZ vascularity). CDS results were correlated with histologic findings from 220 separate biopsy sites that included 27 focal lesions. RESULTS: Of 34 grade 2 biopsy sites, 21 revealed carcinoma, eight revealed prostatitis, and five were negative. CDS depicted at least one focus of carcinoma in seven patients with no gray-scale abnormality. CDS had a sensitivity of 49%, specificity of 93%, and positive predictive value of 62%. CONCLUSION: Focal PZ hypervascularity at CDS is associated with an increased likelihood of prostate cancer or inflammation at biopsy, often without a focal gray-scale abnormality. CDS may help identify an appropriate site for biopsy. A negative CDS scan, however, should not preclude biopsy, as CDS has a limited sensitivity in the detection of all sites of cancer. PMID- 7534430 TI - Radiation-induced decrease in nitric oxide synthase--containing nerves in the rat penis. AB - PURPOSE: Evaluate effect of prostatic irradiation on erectile function. MATERIALS AND METHODS: Forty-seven male adult rats were divided into three groups according to a single radiation dose to the prostate: control (no irradiation) (n = 15), 1,000 cGy (n = 15), and 2,000 cGy (n = 17). Five months after irradiation, rats underwent evaluation of penile vascularity and of erectile response to central and peripheral stimulation. After the study a proximal-shaft penile segment was obtained for staining. RESULTS: Histologic evaluation demonstrated that, with increasing radiation, the number of nitric oxide synthase-containing nerve fibers per penile segment decreased significantly: control, 225.6 +/- 9.7; 1,000 cGy, 156.3 +/- 12.0; 2,000 cGy, 85.8 +/- 10.1 (standard error of the mean). Maximal intracavernous pressure induced with electrostimulation decreased significantly with increasing radiation dose. After injection of papaverine, maximal intracavernous pressure was significantly decreased in only the 2,000-cGy group. CONCLUSION: A dose of 2,000 cGy over the prostatic bed induces erectile dysfunction by causing defects in the vascular supply of the erectile tissue and in the nerves and smooth muscle. PMID- 7534431 TI - [Flow cytometry identification of CD34-positive cells]. AB - In the present study three clinical applications of flow-cytometric quantification of CD34-positive cells in the field of blood and marrow transplantation are evaluated. By daily determination of CD34-positive cells in the peripheral blood after mobilization of stem cells with chemotherapy or growth factors it is possible to set the optimal time for collection of the transplant material. Thereafter the yield of CD34-positive cells within the harvested material is determined. Finally, the efficacy of positive CD34 selection using a biotin-avidin column is evaluated. Initial experience is presented here. PMID- 7534432 TI - Increased soluble endothelial adhesion molecules in rheumatoid arthritis correlate with circulating cytokines and depletion of CD45RO+ T-lymphocytes from blood stream. AB - Endothelial cells express adhesion molecules and release free forms (e.g., sELAM 1, sGMP-140, sICAM-1 and sVCAM-1). Compared with controls, the serum levels of these soluble adhesion molecules (SAM) were significantly increased in patients with rheumatoid arthritis. We investigated whether this was associated with the circulating cytokines and changes in peripheral blood T-lymphocyte (T-PBL) subsets. In healthy subjects, sELAM-1 correlated with the serum levels of Il-1 beta, Il-1 receptor antagonist (Il-1RA) and Il-6, while sGMP-140 was associated with Il-8, and sVCAM-1 was related to Il-7 and Il-8. Thus, already in controls, relations exist between the levels of SAM and circulating cytokines. The rheumatoid arthritis patients with low and high serum levels of IgA- and/or IgM rheumatoid factors (RF) were separately analyzed. They have different cytokine profiles and showed distinct correlations. In patients with low RF, sGMP-140 and sVCAM-1 correlated with Il-1 beta, while sICAM-1 was associated with Il-7 and TNF alpha. In patients with high RF, sELAM-1 correlated with Il-1RA, and sGMP-140 was associated with many cytokines (e.g., GM-CSF, MIP-1 alpha and TNF-alpha). In addition, lymphopenia (less than 1000 lymphocytes/microliters) was shown in 30% of the patients, and 20% (mostly with low RF levels) had reduced levels of "primed" CD45RO+ cells among T-PBL. In controls, cytokines (Il-7, Il-8 and GM CSF), but not SAM, were associated with less CD45RO+ T-PBL. In patients with low RF only, sGMP-140 and sELAM-1 correlated with the depletion of "primed" CD4+ and CD8+ T-PBL respectively. In such patients, Il-1 beta and GM-CSF also correlated with less CD8+, CD45RO+ T-PBL. Thus, particularly in patients with low RF, increased SAM, possibly released by the endothelial cells, might reflect the cytokine-induced activation of the vascular endothelium and the extravasation of some CD45RO+ T-PBL. PMID- 7534433 TI - [Hydroxyurea, erythrocyte volumes and hemoglobin F]. AB - Hydroxyurea is used in the treatment of sickle cell anemia and beta-thalassemia major to increase the content of hemoglobin F (HbF) and presumably ameliorate clinical symptoms. Under therapy with hydroxyurea an increase of the mean corpuscular volume (MCV) of the erythrocytes can be observed. To evaluate a possible estimation of the content of HbF using the increase of MCV under treatment with hydroxyurea, we measured MCV and HbF during therapy with hydroxyurea. The median MCV before therapy was 87.8 fl (range 74.3-95.7) and under hydroxyurea 104.1 fl (81.0-139.5), and the median HbF 1.8% (0.1-5.4). Although both MCV and HbF increased under treatment with hydroxyurea, a linear correlation between these two parameters was not detectable. Therefore, MCV cannot replace the measurement of HbF. PMID- 7534434 TI - Continuity and discontinuity of psychopathology: a study of patients examined as children and as adults. III--The infancy of "adult personality disorders". AB - We undertook a prospective study of all the children who, having consulted the Child Psychiatry Services (Service Medico-Pedagogique) between 1963 and 1967, later consulted the Adult Public Psychiatry Service (720 cases). We looked for specific clinical clusters which would enable us to differentiate statistically the groups of children according to their adult diagnoses. We present here the results for the group of children who were diagnosed as suffering from "personality disorders" in adulthood. We were able to establish an infantile cluster that significantly differentiated these children. It includes: aggressiveness, oppositional behaviour, unsociability, conflicts with peers. 54.6% had already been diagnosed as suffering from personality disorders in childhood. According to these results, the more a consulting child fits this profile, the greater the risk that he will suffer from a "personality disorder" in adulthood. We discuss the continuity/discontinuity of the symptomatology, and the implications for diagnoses of "personality disorders" and "borderline personalities". PMID- 7534436 TI - [Promoting research in Swiss psychiatry]. AB - There is a lack of junior scientists in the psychiatry of Switzerland since several years. The paper reports about an investigation of 135 residents and fellows of psychiatric university hospitals of the German speaking part of Switzerland. Results indicate that there is no continuous exchange of informations about projected, ongoing and finished research projects between researchers and clinicians. Residents and fellows are interested in being trained in research methodology and in discussions of actual studies carried out by experienced scientists. They appreciate participating in research projects. Finally some recommendations for the improvement of research promotion in the swiss psychiatry are discussed. PMID- 7534435 TI - [Experiences of schizophrenic patients in ambulatory care with oral neuroleptics- baseline for psychoeducational interventions]. AB - In the run-up to a prospective intervention study to assist the preparation of psychoeducational therapeutic measures, schizophrenic out-patients were questioned on their experiences with two oral neuroleptics, Clozapine and Perazine. The two drugs, which are known to have similar side effects, find equal acceptance among patients. Subjective attitudes to medication and general evaluations of drug therapy are extremely positive. Numerous patients had already modified their neuroleptic dosage on their own initiative, in most cases discontinuing their medication but in some cases increasing or reducing the dose. These experiences can be used in designing a psychoeducational intervention directed towards individual needs. PMID- 7534437 TI - [The new role of psychiatry in the management of mentally retarded patients]. AB - The areas of remedial education and special education have profoundly changed the psychiatric care of mentally retarded persons. Desinstitutionalisation, normalisation and integration are widely recognised concepts today and have since gained relatively substantial empirical support. Definition of these concepts has been broadened to include consideration of the optimal management of environmental conditions as an integral part of the comprehensive living environment. The systemic-ecological approach dislodges psychiatry from its central position in the treatment of the mentally ill retarded, and invests a greater degree of trust in remedial educations and special education. Effective treatment thus involves a joint effort on the part of various mental health professionals working towards a common goal, grounded on the coexistence of mental retardation and mental illness. Psychotherapy should be devised so as to address cognitive deficits no longer considered to be contraindicative to the therapy process. The purpose of pharmacotherapy is to finetune psychotropic medication with pedagogical, psychoeducational and psychotherapeutic methods. Although community-based mental health delivery systems and comprehensive (institutional based) services both show promise for the care and treatment of mentally retarded persons there is evidence for the particular viability of the latter model due to prevailing present conditions and to the aspect of cost effectiveness, particularly due to its utility in promoting still acquisition relevant to workers in this field of mental health. PMID- 7534438 TI - [Affective dependency and primary alcoholism]. AB - The relationships between dependency and alcoholism have been studied in two groups of male subjects. In the first group 41 subjects met Spitzer's criteria for primary alcoholism and the 50 subjects of the second group were healthy. All the subjects filled out two rating scales, the Interpersonal Dependency Inventory (IDI) and the Beck Depression Inventory (BDI) (shortened version). The alcoholics were divided into depressives vs non depressives by the means of the score of the BDI. Comparing to controls the alcoholics scored significantly higher on the IDI, but there were no differences for this scale between the two sub-groups of alcoholics (depressed alcoholics, non depressed alcoholics). Interpersonal dependency seems to be a characteristic of the alcoholic personality without influence of secondary depression. PMID- 7534439 TI - [Visions for the development of gerontopsychiatry]. PMID- 7534440 TI - [Functional somatic complaints in the emergency service and 4 year outcome]. AB - The article presents the clinical profile of 72 somatizations taking in charge by a psychiatric team in a emergency room. They represent mood disorders (37.5%), psychotic disorders (11%) and anxiety disorders (20%). The psychiatric antecedents are very poor. Their outcome after 4 years is good on the health sickness rating scale of Luborsky in 35% and very bad in 25%, particularly for symptomatic score. After their taking in charge in the emergency room, these patients go not much in psychiatric hospitals and in psychiatric consultations. This observation gives to this first intervention in the emergency room a very important rule. PMID- 7534442 TI - Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport. AB - Transport of RNAs within nuclei and through nuclear pore complexes (NPCs) are essential, but poorly understood, steps in gene expression. In experiments with mammalian cells, RCC1, the abundant nuclear guanine nucleotide exchange factor for the guanosine triphosphatase Ran/TC4, was shown to be required for nucleocytoplasmic transport of precursors of spliceosomal small nuclear RNAs (snRNAs), intranuclear transport of U3 snRNA, and processing of ribosomal RNAs, but not for export of transfer RNAs. It is proposed that guanosine triphosphate (GTP)-bound Ran/TC4 associates with ribonucleoprotein particles (RNPs) during intranuclear movement, and that GTP hydrolysis promotes deposition of RNPs at targeted sites such as NPCs or nucleoli. PMID- 7534443 TI - Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines. AB - Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells. PMID- 7534441 TI - Air-water gas exchange and evidence for metabolism of hexachlorocyclohexanes in Resolute Bay, N.W.T. AB - Paired air and water samples were collected at Resolute Bay (74 degrees N, 95 degrees W) in summer 1992 to estimate the direction of gas exchange of hexachlorocyclohexanes (HCHs) and investigate possible loss processes in the water column. Average concentrations of alpha-HCH and gamma-HCH in ocean surface water were 4.7 +/- 0.9 and 0.44 +/- 0.11 ng/l, respectively. These alpha- and gamma-HCH levels are approximately 66-104% and 54-72% of values reported for the central Arctic Ocean at the Canadian Ice Island in 1986. Mean atmospheric concentrations of alpha-HCH and gamma-HCH (114 +/- 16 and 9.8 +/- 1.3 pg/m3) were 2-3 times lower than summer Arctic levels in the 1980s. The ocean surface water ( 1.4 degrees C) was approximately within Henry's Law equilibrium with respect to atmospheric gamma-HCH levels. Water/air fugacity ratios were 1.03 for gamma-HCH and 1.57 for alpha-HCH, indicating a slight potential for volatilization of alpha HCH. The two alpha-HCH enantiomers in air and water were separated by chromatography on a gamma-cyclodextrin capillary column. The enantiomeric ratio (ER = ratio of (+)alpha-HCH/(-)alpha-HCH) in air was 1.00 +/- 0.04. This agrees excellently with ER = 1.00 +/- 0.01 found for a racemic alpha-HCH standard. The (+) enantiomer was depleted in seawater, resulting in ER = 0.93 +/- 0.06 in Resolute Bay. ERs of samples from Amituk Lake on Cornwallis Island ranged from 0.65 to 0.99, depending on location, date and relative contributions of fresh snowmelt and older lake water. These results suggest that microbial degradation of HCHs is taking place in Arctic lakes and near-shore marine waters. PMID- 7534444 TI - [The isoantigenic differentiation of human body tissues and excretions by using new immune reagents]. AB - A priority technology has been developed to obtain highly effective group specific reagents for some immunological methods, for the adsorption-elution test and immunofluorescence, first of all. Rapid method making use of these reagents are offered to be used for detection of AB0, MNSs, P, and Le antigens in liquid blood, in trace amounts of tissues, and human excretions in objects of material evidence in forensic medical expert evaluation. These reagents may be used in blood transfusion service as well. PMID- 7534445 TI - [The indications and contraindications for emergency adenomectomy in patients with severe concomitant diseases]. AB - The authors have performed 631 urgent suprapubic transvesical adenomectomies in patients with prostate adenoma complicated by acute urine retention or hemorrhage. Prearranged and urgent interventions had, by the authors' experience, virtually the same rate of postoperative complications and lethal outcomes. The risk in urgent adenomectomy performed in 294 patients was attributed to their concurrent affections: postinfarction cardiosclerosis, myocardial ischemia or hypertensive crisis, hemiparesis after brain apoplexy, bronchial asthma, diabetes mellitus, hepatic cirrhosis, chronic lymphoid leukemia, drug polyallergy, multiple tumors of the urinary bladder, stomach, etc., in stage T1-3NOMO. 80 patients had intermittent chronic renal failure. In compensation of severe concurrent diseases and satisfactory condition of the patients urgent adenomectomy was conducted within 24 hours since hospitalization. Longer interval (within 24-72 hours) was necessary in subcompensation of the concurrent diseases, intermittent chronic renal failure which were intensively treated. The authors achieved uneventful postoperative course for 272 (92.5%) high-risk patients. Postoperative lethality made up 3.06%. According to 1-11-year follow-up 7 patients died, for the most part of blood and respiratory diseases. Functional long-term outcomes were good in 83.5% of the patients. Basing on their experience, the authors specify indications to urgent adenomectomy and optimal time of its conduction. Contraindications to urgent adenomectomy were revised and narrowed. PMID- 7534446 TI - Plasticity of autonomic nerves: differential effects of long-term guanethidine sympathectomy on the sensory innervation of the rat uterus during maturation. AB - The sensory nerves, containing substance P and calcitonin gene-related peptide, and noradrenaline-containing sympathetic nerves of the rat uterus were analyzed following long-term sympathectomy with guanethidine in prepubertal (four weeks), young adult (eight weeks) and fully adult animals (18 weeks). Immunohistochemical and histochemical methods were used in association with nerve density measurements and biochemical assays. The main findings were as follows: (1) long term guanethidine treatment completely abolished the noradrenergic innervation of the uterine horn and parametrial tissue and markedly reduced the tissue levels of noradrenaline in both regions at the three ages analysed; (2) in the uterine horn guanethidine treatment had no effect on the tissue levels of either calcitonin gene-related peptide or substance P or on the density of calcitonin gene-related peptide-containing nerves, at any of the three ages studied; (3) in the parametrial tissue increased levels of calcitonin gene-related peptide were observed at 8 and 18 weeks of age, together with a significant increase in the density of calcitonin gene-related peptide-containing nerves. Substance P levels showed a transient increase in this tissue at eight weeks. In conclusion, long term sympathectomy with guanethidine resulted in an increase in calcitonin gene related peptide and substance P in sensory nerves in the parametrial tissue, but not in the uterine horn. The changes in the parametrial tissue only occurred after puberty. It is suggested that sensory nerves in the uterine horn may be less responsive to sympathetic denervation since loss of sympathetic nerves occurs as part of a normal physiological process during pregnancy in this region. PMID- 7534447 TI - Aplasia cutis congenita: a rare cause of elevated alpha-fetoprotein levels. AB - We present a catastrophic case of aplasia cutis congenita from a pregnancy complicated by elevated maternal serum and amniotic fluid alpha-fetoprotein levels and a positive acetylcholinesterase. Delivery occurred at 27 weeks 1 day after premature rupture of membranes with chorioamnionitis. The neonate lacked > 90% of its skin and died. PMID- 7534448 TI - The advantages of using triple-marker screening for chromosomal abnormalities. AB - OBJECTIVE: Our purpose was to assess the utility of triple-marker serum screening for chromosomal abnormalities. STUDY DESIGN: Our laboratory received 10,605 samples that were between 15 and 22 weeks' gestation for maternal serum screening of chromosomal abnormalities. Triple-marker maternal serum screening consisted of alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol in conjunction with maternal age. Women > or = 35 years old were first offered amniocentesis. If they refused amniocentesis, they were offered the screening test. A second-trimester risk for trisomy 21 > or = 1:270 was considered screen positive. Patients were screen positive for trisomy 18 if all three markers were low: alpha-fetoprotein < or = 0.75 multiples of the median, unconjugated estriol < or = 0.60 multiples of the median, and human chorionic gonadotropin < or = 0.55 multiples of the median. RESULTS: The initial screen-positive rate was 8.3% (880 women); amniocentesis was offered to 766 (7.2%). Twelve of 16 ascertained cases of trisomy 21 (75%), two of three cases of trisomy 18 (67%), five cases of 45,X karyotype, and one case each of 45,X/46,XX, 47,XXY, 47,XYY, 46,XX,ins(2)(q21p13p15)mat, and 69,XXX karyotypes were identified in the screen positive patients. All four known cases of trisomy 21 in the 886 women > or = 35 years old who were screened were detected, with a 21% false-positive rate. Omitting unconjugated estriol from our screening program would have resulted in detecting nine of 16 trisomy 21 and six of 12 other chromosomal abnormalities. The false-positive rate would have remained the same. CONCLUSION: In our sample cohort addition of unconjugated estriol to the screening program resulted in an increased detection rate of chromosomal abnormalities with no change in the false positive rate. Considering the advancement in screening for chromosomal abnormalities, maternal age alone as an indication for amniocentesis should be reevaluated. PMID- 7534449 TI - The relationship between circulating androgens, obesity, and hyperinsulinemia on serum insulin-like growth factor binding protein-1 in the polycystic ovarian syndrome. AB - OBJECTIVE: Our purpose was to evaluate the relationship of obesity, hyperinsulinemia, and hyperandrogenemia on serum insulin-like growth factor binding protein-1 levels in women with the polycystic ovarian syndrome. STUDY DESIGN: Insulin-like growth factor binding protein-1, insulin, and androgen levels were studied during a 3-hour intravenous glucose tolerance test in 16 women with polycystic ovarian syndrome (nine obese and seven nonobese) and 20 healthy control women (10 obese and 10 nonobese). RESULTS: Positive correlations were observed between basal (r = 0.77, p = 0.04) and area under curve (r = 0.86, p < 0.001) insulin-like growth factor binding protein-1 with basal androstenedione in the nonobese women with polycystic ovarian syndrome but not in other groups of women examined or between other androgens and insulin-like growth factor binding protein-1. An inverse relationship was observed between log area under curve insulin-like growth factor binding protein-1 levels and log body mass index in polycystic ovarian syndrome (r = -0.54, p = 0.03) and in normal women (r = -0.43, p = 0.06). The log area under curve insulin-like growth factor binding protein-1 level was approximately an inverse linear function of log area under curve insulin response for both women with polycystic ovarian syndrome (r = 0.70, p < 0.001) and control women (r = -0.72, p < 0.001). Additionally, after the area under curve insulin response during intravenous glucose tolerance testing was controlled for, the decline in area under curve insulin-like growth factor binding protein-1 responses was on average 66% less in both obese and nonobese women with polycystic ovarian syndrome compared with same-weight controls (95% confidence interval 110% to 270%, p = 0.04). CONCLUSION: These data indicate that insulin and body mass index are the major determinants of circulating insulin-like growth factor binding protein-1 and that chronic hyperandrogenemia does not appear to further reduce serum insulin-like growth factor binding protein-1 levels in obese or normal-weight women with polycystic ovarian syndrome. PMID- 7534450 TI - Performance of Israeli versus U.S. preschool children on the Miller Assessment for Preschoolers. AB - OBJECTIVES: The Miller Assessment for Preschoolers (MAP) is a scale that can be used to evaluate preschool children with suspected preacademic problems. Before implementing the MAP in Israel, it was necessary to determine whether the U.S. norms were applicable to the Israeli preschool population. METHOD: In a pilot study carried out in Israel, the Hebrew version of the MAP was administered to 2 age groups of 30 children each. The scores of Israeli children were compared with the U.S. norms on each of the MAP's 27 subtests, the five performance indices, and the total score. RESULTS: There were no significant differences between the Israeli sample and the U.S. standardization sample in either age group on the MAP total score; significant differences were found in both age groups on the Foundations Index and on some specific subtests. Israeli children performed below U.S. norms on the Foundations Index. CONCLUSION: These findings indicate that the performance of Israeli children overall in these two age groups is not significantly different from the performance of U.S. children. If future research demonstrates that these findings are stable across all age groups and for larger samples, the implication is that the MAP can be administered and scored in Israel with the scoring methodology and normative information developed in the United States. However, because of the poorer performance of Israeli children on the Foundations Index, we recommend that specific Israeli norms be developed. PMID- 7534452 TI - Cardiac arrest following Haemaccel--comment. PMID- 7534451 TI - Use of the Bruininks-Oseretsky test of motor proficiency in occupational therapy. AB - OBJECTIVE: In addition to the need for good measurement tools in occupational therapy, there is a need for the tools to be used knowledgeably. The purpose of this article is to investigate the usefulness of the Bruininks-Oseretsky Test of Motor Proficiency (BOTMP) for both descriptive (diagnostic) and evaluative (change over time) purposes. METHOD: The typical profile of subtest scores for children with mild motor problems revealed that certain subtests of the BOTMP may be better indicators of motor problems for these children than others. An analysis also was performed to compare the use of raw (point) scores with standard (age-adjusted) scores in evaluating change. RESULTS: Four subtests that provide a greater degree of discrimination between children with and without motor problems were identified. Raw (points) scores were found to provide a more valid measure of change over time than standard (age-related) scores. CONCLUSION: We recommend that, for clinical use, the BOTMP subtest standard scores be used for diagnostic purposes and that the raw scores be used for evaluative purposes. PMID- 7534453 TI - The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men. AB - Testicular biopsies of infertile patients are often characterized by a mixed atrophy, in which different types of spermatogenic lesions are found in adjacent tubules. In order to evaluate a possible involvement of the state of differentiation of the Sertoli cells, the distribution pattern of cytokeratin and vimentin intermediate filaments within the seminiferous epithelium of 228 biopsy specimens with normal spermatogenesis (n = 10), mixed atrophy (n = 206) or Sertoli Cell Only Syndrome (n = 12) were investigated by means of immunohistochemical techniques. Sertoli cells were regularly found to show vimentin expression in tubules with normal spermatogenesis as well as in tubules with any kind of spermatogenic impairment including SCO. Cytokeratin expression as a marker showing lack of differentiation was common in Sertoli cells of tubules with arrest of spermatogenesis at the level of spermatogonia, and was occasionally associated with arrest at the level of primary spermatocytes or with SCO. Ultrastructural examination of tubules with spermatogonial arrest revealed Sertoli cells with features of typical fetal or prepubertal Sertoli cells, such as round to ovoid nuclei without indentations, stacks of rough ER and spot desmosomes. These data suggest that spermatogenic arrest at the level of spermatogonia might be due to functional impairment of the associated Sertoli cells, which have maintained or regained an undifferentiated state and are not able to initiate or trigger the process of spermatogonial differentiation. PMID- 7534456 TI - Fatal anaphylactic shock after aprotinin reexposure in cardiac surgery. PMID- 7534454 TI - The extravascular contractile system in the human placenta. Morphological and immunocytochemical investigations. AB - In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, alpha-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P. PMID- 7534455 TI - Enhancement of angiogenesis in regenerating gastrocnemius muscle of the toad (Bufo viridis) by low-energy laser irradiation. AB - The effect of low-energy laser (He-Ne) irradiation on the process of neoformation of blood capillaries during regeneration in the toad (Bufo viridis) gastrocnemius muscle was studied using histomorphometric methods. The injured zones of the experimental toads were subjected to four direct He-Ne laser irradiations (632.8 nm wavelength; 6.0 mW for 2.3 min) every alternate day, commencing on the second day after injury. Muscles that were injured as above and subjected to red light irradiation served as control. The volume density (cm3/cm3) of the capillaries in the injured zone at 9 days after injury was significantly (P < 0.01) higher (0.09 +/- 0.006) than in the control muscles (0.048 +/- 0.007). At 14 days after injury, the volume density in the injured zone of the control muscles further increased, while the value in the laser-irradiated muscles remained unchanged. The surface density (m2/cm3) of the capillaries in the injured zone was 2.3-fold higher in the laser-irradiated muscles than in the control muscles at 9 days after injury. The surface density further increased in the control muscles between 9 and 14 days after injury, while in the laser-irradiated muscles there was a decrease in this value during the above period. The surface-to-volume ratios of the capillaries in the injured zone of control and laser-irradiated muscles indicate a straighter, rather than a convoluted appearance between 9 and 14 days after injury. It is concluded that He-Ne laser irradiation during skeletal muscle regeneration in the toad markedly promotes the process of neoformation of blood vessels in the injured zone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534457 TI - [Why use chemotherapy in cancer of the bladder? What are the initial limitations]. AB - Four chemical agents are active, in isolation, against metastatic bladder cancer: cisplatin (nephrotoxicity) (C or Cis), doxorubicin (cardiac toxicity) or adriamycin (A), vinblastine (V) and methotrexate (M). Combinations of these drugs are more effective, with complete response rates of up to 30 to 40%. The current combinations are: CM; Cis cyclophosphamide, A; CMV; MVAC. This last combination appears to be the most effective. Chemotherapy is essentially prescribed for palliative purposes (improvement of symptoms) in metastatic cancers. It may also constitute adjuvant treatment following total cystectomy, in order to decrease the local or distant recurrence rate. Neoadjuvant chemotherapy, before surgery, can reduce the tumour bulk allowing preservation of the bladder. Haematopoietic growth factors are used to reduce the toxicity of chemotherapy and the action of these drugs can also be potentiated by radiotherapy. PMID- 7534458 TI - Dynamic RNA-RNA interactions in the spliceosome. PMID- 7534459 TI - Effect of a major metabolite of the antiallergic drug repirinast on phosphodiesterase and adenylate cyclase activities. AB - MY-1250 (5,6-dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano [3,2-c]quinoline-2 carboxylic acid, CAS 63768-47-8), a major metabolite of the antiallergic drug repirinast (MY-5116, CAS 73080-51-0), inhibits antigen-induced histamine release from rat peritoneal mast cells. MY-1250 causes a rapid increase in cyclic adenosine monophosphate (AMP) levels in rat peritoneal mast cells. MY-1250 inhibited cyclic AMP phosphodiesterase activities from rat peritoneal cells and guinea pig lung tissue in a concentration dependent manner with IC50 values of 2000 mumol/l and 1670 mumol/l, respectively. However, MY-1250 showed no effect on adenylate cyclase activity from rat peritoneal cells. These results suggest that the inhibitory effect of MY-1250 on histamine release may be partly due to the inhibition of cyclic AMP phosphodiesterase activity. PMID- 7534460 TI - Identification and biochemical characterization of the human brain galanin receptor. AB - Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (Kd 0.23 nM, Bmax 44 fmol/mg), the hypothalamus (Kd 0.20 nM, Bmax 25 fmol/mg) and the cortex cerebri (Kd 0.11 nM, Bmax 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax < 3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125-I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3 cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively. PMID- 7534461 TI - Expression of beta 1 integrin receptors in transformed mouse epidermal keratinocytes: upregulation of alpha 5 beta 1 in spindle carcinoma cells. AB - The adhesive properties and the expression of extracellular matrix receptors of the beta 1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two dimensional analyses of the immunoprecipitates obtained with anti-beta 1- and specific anti-alpha-integrin subunits showed qualitative and quantitative changes in the expression of beta 1 integrins by the different cell lines. The polyvalent alpha 3 beta 1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor alpha 5 beta 1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of alpha 5 beta 1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of alpha 5 beta 1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of alpha 5 beta 1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor alpha 2 beta 1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of alpha 2 beta 1 was correlated with the increased adhesion to collagen type I and collagen type IV exhibited by the tumorigenic cell lines. PMID- 7534462 TI - The role of excitatory amino acids in experimental models of Parkinson's disease. AB - The aim of this article was to review the recent literature on the role of excitatory amino acids in Parkinson's disease and in animal equivalents of parkinsonian symptoms. Effects of NMDA and AMPA antagonists on the reserpine induced akinesia, catalepsy and rigidity, on the neuroleptic-induced catalepsy, on the turning behaviour of 6-OHDA-lesioned rats, as well as on the parkinsonian symptoms evoked by MPTP in monkeys were analysed. Moreover, the role of NMDA antagonists in Parkinson's disease was discussed. Data concerning the protective influence of these drugs on degenerative properties of methamphetamine, MPTP and 6-OHDOPA were also presented. On the basis of the above findings, the following conclusions may be drawn: (1) disturbances in the glutamatergic transmission in various brain structures seem to play a significant role in the development of symptoms of Parkinson's disease; (2) the NMDA-receptor blocking component may make a substantial contribution to the therapeutic effect of antiparkinsonian drugs; a similar contribution of AMPA-receptor blocking component has not been sufficiently documented, so far; (3) compounds blocking NMDA receptors may possibly prevent the development of Parkinson's disease; this presumption needs, however further studies; (4) side effects of NMDA receptor antagonists may be a limiting factor in the use of these compounds in humans. PMID- 7534463 TI - Growth hormone-releasing hormone, somatostatin, galanin and beta-endorphin afferents to the hypothalamic periventricular nucleus. AB - A combined retrograde tracing (wheat germ agglutinin-horseradish peroxidase-gold complex)-immunohistochemical technique was used to identify the origin of growth hormone-releasing hormone (GHRH)-immunoreactive (ir), beta-endorphin-ir, galanin (GAL)-ir and somatostatin (SRIH)-ir terminals in the hypothalamic periventricular nucleus, which contains all the hypophysiotrophic SRIH-ir neurons. Retrogradely labeled cells were mostly observed ipsilaterally in the arcuate, dorsomedial (DMH), suprachiasmatic nuclei and the parvocellular part of the paraventricular nucleus. They were less abundant in the ventromedial and periventricular nuclei and in the lateral hypothalamus. The proportion of retrogradely labeled GHRH cells was greater at the outer rim of the ventromedial nucleus (10%) than in the arcuate nucleus proper (3%). In the arcuate nucleus, 14% of the SRIH-ir cells projected to the periventricular nucleus. Of the GAL-ir cells in the arcuate and the DMH 10% were double-labeled. Scattered retrogradely labeled GAL-ir cells were observed in paraventricular and perifornical nuclei and in the lateral hypothalamus. Of the beta-Endorphin-ir cells in the ventral part of the arcuate nucleus 15% were retrogradely labeled. It is concluded that: (1) There is no major direct connection between the hypophysiotropic GHRH and SRIH neurons, respectively, located in the arcuate and periventricular nucleus. (2) GHRH projections to the periventricular nucleus arise mainly from cells located at the outer rim of the ventromedial nucleus. (3) Intrahypothalamic SRIH projections to the periventricular nucleus arise from arcuate SRIH neurons located along the wall of the third ventricle. (4) GAL neurons from the DMH and the arcuate nucleus innervate to the same extent the periventricular nucleus. (5) beta-Endorphin arcuate neurons strongly innervate the periventricular nucleus. PMID- 7534464 TI - Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. AB - In a previous study the authors reported the characterization of some facultatively anaerobic, Gram-positive, non-sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes-like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169T and DSM 9168T, respectively. PMID- 7534466 TI - Simplification of the Bentall procedure with surgical and biological innovations. AB - Most of the technical problem encountered during aortic root replacement may be avoided by the use of fine mattress sutures, gelatin resorcinal formaldehyde (GRF) glue and pre-clotted or bovine collagen grafts in patients whose platelet function is preserved by the intra-operative administration of aprotinin. PMID- 7534467 TI - [Interferons in neurology]. PMID- 7534465 TI - Molecular identities of human sperm proteins that bind human zona pellucida: nature of sperm-zona interaction, tyrosine kinase activity, and involvement of FA 1. AB - The present study was conducted to investigate the molecular identities, nature of interaction, and tyrosine phosphorylation activity of the sperm-zona pellucida binding proteins in humans. Sperm proteins belonging to four major molecular regions, namely 95, 63, 51, and 14-18 kDa, reacted with zona pellucida proteins in the Western blot and immunoprecipitation procedures. In these procedures, zona pellucida protein that reacted strongest with the sperm proteins belonged to the molecular region of 55 kDa (ZP3), besides weakly reacting proteins in the 110-kDa (ZP1/ZP2) and 14-18-kDa molecular regions. The major forces involved in the sperm zona protein interactions were of hydrophobic and ionic in nature. Three (95, 51, and 14-18 kDa) of the four molecular regions of sperm proteins that bound to the zona pellucida proteins also seem to involve o-phospho-L-tyrosine residues in their interaction, and these proteins demonstrated the presence of phosphotyrosine residues, and the 51-kDa protein also showed autophosphorylating activity in the in vitro kinase assay. The sperm binding zona protein of 55 kDa also demonstrated autophosphorylating activity. Using specific monoclonal antibody to the well characterized sperm-specific glycoprotein, designated FA-1, and the competitive inhibition in the immunoprecipitation procedure, it was found that the 51 kDa protein is indeed FA-1 antigen. Besides elucidating the molecular nature of the sperm-zona interaction, these antigens will find application in the development of a multivalent contraceptive vaccine, and may also help in specific diagnosis and treatment of infertility mediated through defective gamete (sperm or oocyte) function. PMID- 7534468 TI - Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system. AB - In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinant Drosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by the Drosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed. PMID- 7534470 TI - Morphology of sweat glands in determining time of death. AB - This study demonstrates post-mortem autolytic alterations in the skin at cellular and subcellular levels and identifies parameters which may assist in determining the time of death in the first few hours post-mortem. Serial skin samples from the ventral surface of the arm were taken at intervals of 3, 6, 9 and 12 h after death in 29 subjects of various ages, with no signs of skin disease; causes of death were various. Three types of tests were performed: cytochemical (hematoxylin-eosin and alcian-PAS), immunohistochemical (S-100, CEA, Cytokeratin, ASM) and ultrastructural (electron microscopy). Electron microscopy proved useful for identifying transformations which were found to be specific for each chronological step considered: reduction of intracellular glycogen in clear cells and reduction of secretory granules in dark cells are typical signs of the first stage (3 h) after death; mitochondrial dilatation and rarefaction of cristae in clear and dark cells are typical of the second stage (6 h); rarefaction of microvilli in dark and clear cells is a sign of the last stage (12 h). Cytochemistry and immunohistochemistry supply useful information--not for all the chronological stage considered here, but for individual phases (3 h for hematoxylin-eosin and 6 h for alcian-PAS). However, it is particularly important to use the results from all such techniques simultaneously, so that the question of the exact time of death within the first 12 h post-mortem may be more accurately answered. PMID- 7534471 TI - Forensic value of the Lugol's staining method: further studies on glycogenated epithelium in the male urinary tract. AB - This study presents findings from a series of investigations on the presence of glycogenated epithelium in the male urinary tract and on the penile surface in order to assess the forensic value of the Lugol's method for the identification of vaginal cells. Direct smears obtained from the urethral opening, glans penis, and penile shaft, along with post-mortem samples of the fossa navicularis, and histological sections of the penis were examined. The presence of polygonal, glycogenated, Lugol-positive epithelium cells in the male urinary tract was found to be common. Our results suggest that these cells originate from the fossa navicularis. Because of the possibility of exfoliation of glycogenated male cells and transfer to the penile surface a Lugol-positive reaction in epithelial cells on penile swabs can no longer be assumed to prove the presence of vaginal cells. PMID- 7534472 TI - Conformation of a T-cell stimulating peptide of interleukin-1 beta protein: circular dichroism studies. AB - A T-cell stimulating peptide Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the 163-171 fragment epitope of interleukin-1 beta (IL-1 beta), has been synthesized in solution phase and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The backbone conformation of the synthetic fragment, investigated in aqueous solution by circular dichroism (CD) spectroscopy, is qualitatively a mixture of beta-turns and random coil. Quantification of the CD spectra revealed the presence of a 9% beta-turn fraction in water at pH 7.0, suggesting the occurrence of the conformation for the epitope fragment in aqueous solution necessary for T-cell stimulation and antigenicity. Concomitant changes in CD spectra were observed with increases in the trifluoroethanol (TFE) concentration in water, and the beta-turn fraction in peptide increased to 28% at a concentration of 90% TFE. This helicogenic solvent, as well as other solvents such as methanol, acetonitrile and dioxane (all favouring an ordered structure in peptides), failed to induce any alpha-helical conformation in the IL-1 beta (163 171) fragment, and CD spectra were attributed to only beta-turn ordered structure. This beta-turn structure has also been found to be a theoretically preferred conformation using Chou-Fasman proclivity data and is in accordance with the presence of an all-beta-globular conformation for its parent molecule IL 1 beta. Thus, the beta-turn conformation is probably involved in retention of T cell stimulation activity in this synthetic epitope. PMID- 7534469 TI - The postmortem activation status of platelets. AB - Platelets are activated by substances from the subendothelial matrix in endothelial lesions or by factors in the plasma coagulation cascade. Conversely, activated platelets are potent activators of this cascade. Only activated platelets express the adhesion molecules Gp53, GMP140 and thrombospondin on the plasma membrane. The postmortem activation status of platelets, therefore, can be determined immunoelectron microscopically by immunogold labeling of antibodies against these glycoproteins. Our studies revealed that the vast majority of these antigens were located within the granules postmortem, hence the platelets had not been activated. Thrombin-induced activation of platelets in vitro was only possible in the early postmortem interval, as demonstrated by labeling of the adhesion molecules on the plasma membrane. Later, such activation was no longer possible even though thrombin-induced fibrin formation gave the appearance of "coagulated blood". In forensic medicine, these findings can possibly be applied to distinguish intravital clotting from the postmortem coagulation phenomena and intravital hematomas from postmortem hematomas. PMID- 7534473 TI - Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies. AB - Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate gamma-poly(glutamate) (gamma PGA) production media containing L-glutamate, citrate and glycerol as carbon sources. A gel permeation chromatography (GPC) method was developed to determine gamma-PGA volumetric yield and molecular weight directly using culture filtrates. For GPC volumetric yield measurements, a calibration curve was generated using purified gamma-PGA to relate the gamma-PGA GPC peak area and polymer weight. Purified gamma-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest gamma-PGA volumetric productivity (approximately 0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 g l 1, 18 to 10 g l-1 and 12 to approximately 1 g l-1, respectively; a decrease in pH from 7.4 to approximately 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of approximately 4.5 g l-1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at approximately 42 h and through a 96 h cultivation period. The presence of 2,3 butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism. When the medium formulation was altered by removal of either citrate, L-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1, respectively, of gamma-PGA were formed. Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of gamma-PGA of relatively higher molecular weight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that gamma-PGA depolymerase is intracellularly located or cell-bound. Culture filtrates showed no significant gamma-PGA depolymerase activity. PMID- 7534475 TI - High-affinity RNA ligands to Escherichia coli ribosomes and ribosomal protein S1: comparison of natural and unnatural binding sites. AB - High-affinity RNA ligands were generated against intact 30S ribosomes, S1 depleted 30S ribosomes, and purified ribosomal protein S1. Sequence analysis indicated two classes of ligand: unstructured RNAs containing a Shine-Dalgarno sequence and structured RNAs containing a pseudoknot. The Shine-Dalgarno containing ligands were generated against S1-depleted 30S ribosomes but, surprisingly, not against intact 30S ribosomes or ribosomal protein S1. In contrast, pseudoknot-containing ligands were generated against intact ribosomes as well as purified S1 protein. The two classes of ligand exhibited specificity for their respective targets, as well as conserved sequence and secondary structure reminiscent of naturally occurring, cis-acting mRNA elements. PMID- 7534474 TI - Secondary structures of Escherichia coli antisense micF RNA, the 5'-end of the target ompF mRNA, and the RNA/RNA duplex. AB - The Escherichia coli micF RNA is a prototype for a class of antisense RNAs encoded by genes at different loci from those that code for their target RNAs. RNAs in this class exhibit only partial complementarity to their targets. micF RNA binds to and regulates the stability of ompF mRNA in response to a variety of environmental stimuli. The secondary structures of micF RNA, ompF-213 mRNA (a segment containing the 213 nucleotides at the 5'-terminus of the target message), and the micF RNA/ompF-213 mRNA duplex were analyzed in vitro by partial digestion with structure-specific ribonucleases and chemical modification. Both micF RNA and ompF mRNA have single-stranded 5'-ends and contain stable stem-loop structures. Strong phylogenetic support for the proposed secondary structure for E. coli micF RNA is provided by a comparison of structural models derived from micF sequences from related bacteria. The micF RNA/ompF-213 mRNA duplex interaction appears to involve only a short segment of micF RNA. Unfolding of only one stem-loop of micF RNA and a minor stem-loop of ompF-213 mRNA appears to be necessary to form the duplex. The probing data suggest that the Shine-Dalgarno sequence and AUG start codon of ompF mRNA, found in single-stranded regions in the free message, are base-paired to micF RNA in the RNA/RNA duplex. PMID- 7534476 TI - Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme binding proximal thiolate ligand as cysteine-415. AB - The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (C1, residues 715 1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The C1 and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of approximately 2-4 mumol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme. PMID- 7534477 TI - Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T hapten. AB - The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3 sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6 sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534478 TI - Permutation of a pair of tertiary nucleotides in a transfer RNA. AB - The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human. PMID- 7534479 TI - Effect of gramicidin A on structure and dynamics of lipid vesicle bilayers. A time-resolved fluorescence depolarization study. AB - We investigated the effects of the hydrophobic small peptide antibiotic gramicidin A (gA) on the properties of vesicle bilayers in the liquid crystalline state. Time-resolved fluorescence anisotropy experiments were performed with unilamellar vesicles of the lipids DMPC, POPC, DOPC, EGGPC, DLPC, DOPG, and SQDG containing various concentrations of gA in two different conformations using TMA DPH and DPHPC as fluorescent probes. These analogues of DPH were taken to study the gA induced change in the structural and dynamical properties of the lipid bilayer in different portions of the hydrophobic region. The time-resolved anisotropy data were analyzed using the recently introduced compound motion model [van der Sijs, D. A., et al. (1993) Chem. Phys. Lett. 216, 559; Muller, J. M., et al. (1994) Chem. Phys. 185, 393]. In general, gA raises the order and reduces the rotational diffusion coefficient for the probes in the bilayer. In DOPC vesicles this ordering effect of gA on the bilayer is found to depend on both the conformation of the peptide and the depth in the bilayer at which the order is probed. This significant effect of gA conformation on the lipid order parameter profile suggests that the shape of the gA dimer in the bilayer, which is determined by its conformation, affects the order of the adjacent DOPC lipid acyl chains. PMID- 7534480 TI - Inhibition of Moloney murine leukemia virus reverse transcriptase by partially 5 thiolated polyuridylic acid. AB - Partially 5-thiolated polyuridylic acid (poly(U60,hs5U40)) is shown to be a potent inhibitor of Moloney murine leukemia virus Reverse Transcriptase (M-MuLV RT). The pattern of this inhibition is competitive, when either poly(A).(dT)16 or poly(C).(dG)16 as template-primer (variable substrate) are used, suggesting that the free enzyme interacts with the modified polynucleotide. Km and Ki values of 25 microM and 11 nM, respectively, were obtained in the presence of poly(A).(dT)16. The Ki value determined in the presence of poly(C).(dG)16 was 31 nM (Km = 22 microM). Inhibitory activities of the 5-thiolated oligouridylic acids, prepared from the polymer, depend on the chain-length. While the 30-mer showed the same activity as the intact polynucleotide, shorter oligonucleotide proved to be less active. PMID- 7534481 TI - Retroviral transfer of the nlsLacZ gene into human CD34+ cell populations and into TF-1 cells: future prospects in gene therapy. AB - Few data are available concerning behavior of reimplanted human hematopoietic cells after autologous stem cell transplantation. This paper reports the possibility to transfer gene markers coding for beta-galactosidase (beta-Gal) activity by retroviral vectors into a human leukemic growth factor-dependent cell line, TF-1, and into human hematopoietic progenitors isolated from peripheral blood or bone marrow. Using various combinations of retroviral vectors and packaging cell lines, we demonstrated high expression of a bacterial beta-Gal activity induced by the LacZ gene, the nlsLacZ gene, or the Sh-ble/LacZ gene, in human hematopoietic cells. The expression of the nlsLacZ construct was stable until the end of the culture in infected CD34+ cell-enriched cell populations, and a slow decrease of transgene expression was observed in a transduced TF-1 cell population during a 1-year long-term culture. Data obtained with the nlsLacZ gene demonstrate that both retroviral transfer and corresponding gene expression were not found to modify the pattern of cell proliferation and differentiation. These results open interesting prospectives for the use of the nlsLacZ gene to mark and follow the fate of progenitor cells isolated from patients with cancers prior to reimplantation. PMID- 7534482 TI - Regulation of the interferon-inducible eIF-2 alpha protein kinase by small RNAs. AB - This review describes the structure and function of the double-stranded RNA dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered. PMID- 7534483 TI - Regulation of thymosin beta 4 mRNA levels during cell proliferation. AB - The levels of thymosin beta 4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum deprived, quiescent cells, the levels of thymosin beta 4 were undetectable; after serum restoration, the cells were induced to proliferate and we found a pronounced increase in thymosin beta 4 mRNA levels at the G1/S transition. Thymosin beta 4 mRNA was induced even in the presence of cycloheximide. On the other hand, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake-off after nocodazole arrest or a double thymidine block did not show any variation in the levels of thymosin beta 4 mRNA when they progressed synchronously through the cycle. In conclusion, the present data indicate that the thymosin beta 4 gene is regulated by cell proliferation but it is not a cell cycle-regulated gene. Finally, we studied thymosin beta 4 mRNA stability by inhibiting thymosin beta 4 gene transcription with actinomycin D. Our results suggest that thymosin beta 4 mRNA has a pronounced stability, a fact that might be relevant to account for the presence of thymosin beta 4 in enucleated cells like platelets. PMID- 7534484 TI - OKT4 epitope deficiency in patients infected with the human immunodeficiency virus: a cause of underestimation of the CD4 lymphocyte count. AB - Two black patients who were infected with the human immunodeficiency virus (HIV) and who had hereditary deficiency of OKT4 epitope were investigated. The patients' lymphocytes lacked the OKT4 surface antigen but reacted with other monoclonal antibodies recognizing the CD4+ helper-inducer T lymphocytes. The CD4 lymphocyte count is a surrogate marker for clinical progression of HIV disease, but it could be unreliable in regard to patients with partial or complete OKT4 epitope deficiency. PMID- 7534485 TI - Effect of surface roughness of hydroxyapatite-coated titanium on the bone-implant interface shear strength. AB - We have investigated the bone-implant interface shear strength of hydroxyapatite (HA)-coated Ti-6Al-4V (HA-coating A) (roughness average, Ra = 3.4 +/- 0.5 microns) and HA-coated Ti-6Al-4V with a rougher surface (HA-coating B) (Ra = 8.4 +/- 1.8 microns). There was no significant difference between HA-coating A and HA coating B implants with respect to the bone-implant interface shear strength as determined in push-out tests using the transcortical model in adult dogs. The bone-implant interface shear strength of bead-coated porous Ti-6Al-4V was significantly greater than that of both HA-coating A and HA-coating B implants. The failure site, as determined by scanning electron microscopy, was the coating substrate interface, not the coating-bone interface. This indicates a need to protect the HA coating from the direct shear forces. HA coating enhances early bone growth into the porous surface of the implant. Long-term fixation should depend on bone anchoring to this porous surface. Hydroxyapatite coatings must be developed which do not obstruct the pores of the surface of the implant. PMID- 7534486 TI - Heparin modulation of the fibrinolytic activity of plasmin, miniplasmin and neutrophil leukocyte elastase in the presence of plasma protease inhibitors. AB - The effect of heparin on the inactivation rates of fibrin-bound plasmin, miniplasmin and neutrophil leukocyte elastase (PMN-elastase) by their plasma inhibitors was studied. While plasmin and miniplasmin bound to fibrin are not inactivated by antithrombin, heparin (800 nM) makes these enzymes available for the inhibitor; the second-order rate constant increases from zero to 1.3 x 10(3) M-1 s-1 and 3.3 x 10(3) M-1 s-1, respectively. Heparin slightly increases the rate of fibrin-bound enzyme inactivation by plasmin inhibitor. alpha 1-Protease inhibitor, on the other hand, is unable to inactivate plasmin or miniplasmin bound to fibrin and heparin has no facilitating effect. In the case of PMN elastase, heparin (300 nM) further increases enzyme protection against alpha 1 protease inhibitor; the rate constant decreases from 41 x 10(3) M-1 s-1 to 23 x 10(3) M-1 s-1. alpha 2-Macroglobulin inhibits fibrin-bound miniplasmin and PMN elastase with a second-order rate constant of 1.8 x 10(4) M-1 s-1 and heparin (300 nM) increases the rate insignificantly for miniplasmin and by a factor of two for PMN-elastase. It is remarkable that plasmin bound to fibrin is not inhibited by alpha 2-macroglobulin independently of the presence of heparin. On the basis of the reported kinetic data a lifespan of 420 s for plasmin, 66 s for miniplasmin and 4 s for PMN-elastase was calculated, when the enzymes are bound to fibrin in the presence of the four protease inhibitors at physiological plasma concentration. If heparin is present (300 nM) these values decrease to 240 s for plasmin and 42 s for miniplasmin, whereas that of PMN-elastase is unchanged. Thus, the present in vitro kinetic model suggests an antifibrinolytic effect of heparin in a plasma milieu. PMID- 7534487 TI - Staurosporine blocks down-regulation of monocyte-associated tissue factor. AB - Inflammatory agents including bacterial endotoxin (LPS) and low concentrations of phorbol myristate acetate (PMA) stimulate human peripheral blood monocytes to transiently express tissue factor procoagulant activity. Concentrations of PMA that cause the cytosol-to-membrane translocation of protein kinase C (PKC) (10( 9)-10(-7) M) induce a rapid decrease in monocyte tissue factor activity. Staurosporine, an inhibitor of protein kinase C, enhances the stimulatory effect of low concentrations of PMA on monocyte expression of tissue factor activity and blocks suppression of tissue factor activity at high PMA concentrations. Furthermore, staurosporine prolongs LPS-induced tissue factor expression in monocytes. These results suggest that protein kinases modulate tissue factor activity in human monocytes by regulating both induction and down-regulation. PMID- 7534488 TI - Nitrite reductase gene cloning of Amycolatopsis mediterranei U-32. AB - Southern blot analysis showed great homology existed between niaD (NR gene) of Aspergillus nidulans and A. mediterranei U-32 chromosome DNA. A 5.0kb PstI fragment from A. mediterranei U-32 complementary to A. nidulans niaD gene was cloned in E. coli NM522 using niaD as a probe. An identical DNA band was observed through back-hybridization of the cloned DNA fragment to PstI digest of A. mediterranei U-32 chromosome DNA. Its 2.1 kb SmaI-EcoRV fragment can only hybridize with total RNA from nitrate-cultured mycelium but not with that from ammonia-cultured mycelium. These data suggested that the cloned DNA fragment contains NR gene of A. mediterranei U-32. This is the first report on NR gene cloning from a aerobic bacterium. It was deduced from the molecular weight of the nitrate reductase that the coding sequence of NR gene is almost 1.5 kb in size. Further hybridization analysis indicated that the cloned DNA fragment covers the full-length NR gene of A. mediterranei U-32. We also constructed the physical map of the recombinant plasmid pJL1 with various restriction endonucleases, among them ten with no restriction site, six with unique site and two with double sites on the insert. PMID- 7534489 TI - Concept and progress in the development of RGD-containing peptide pharmaceuticals. AB - The cell adhesion domain, arginine-glycine-aspartic acid (RGD), has been incorporated into synthetic peptides to perform either of two modes of drug action, antagonist or agonist. Short, conformationally constrained peptides have been developed as antagonists for the platelet membrane glycoprotein complex, the integrin alpha IIb beta 3, using cell-based and integrin-based assays. In combination with a comparative molecular modeling study, these results have helped identify common conformational elements in the pharmacophore of this class of molecules. Peptides are presented that are highly potent, integrin specific, and that possess reduced pharmacological side effects. Also presented is the development of a peptide that modifies, noncovalently, the surfaces of a wide variety of synthetic materials used in medical implants. The agonist activity of [corrected] this molecule is evident from its ability to stimulate cell attachment on these surfaces. This is shown to translate into an in vivo activity of faster and more complete tissue integration, and a reduction in foreign body response. PMID- 7534490 TI - Nitric oxide synthesis in rat mesangial cells induced by cytokines. AB - The production of nitric oxide (NO) is increased in experimental nephritis, with NO thought to be an important mediator of cell damage. The cytokines interleukin 1 beta (IL-1 beta), IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta (TGF-beta) are released from mesangial cells in vitro or are expressed in various forms of glomerulonephritis. We investigated the effects of these cytokines on NO synthesis in cultured rat mesangial cells. Incubation of mesangial cells with IL-1 beta (10 ng/ml) for 24 h increased the accumulation of NO and guanosine 3',5'-cyclic monophosphate (cGMP). IL-6, IL-8, MCP-1 and TGF-beta showed no significant effect on the production of NO or cGMP. Transcripts of the inducible NO synthase (iNOS) gene were not detected in unstimulated mesangial cells. However, exposure of cells to IL-1 beta (10 ng/ml) for 24 h resulted in the appearance of iNos mRNA. IL-1 beta-induced NO synthesis was significantly inhibited by NG-monomethyl-L-arginine, cycloheximide, actinomycin D, dexamethasone, and TGF-beta. These results indicate that, of the various cytokines studied, only IL-1 beta stimulates iNOS mRNA accumulation and NO synthesis in mesangial cells. NO may function in an autocrine manner to modulate the glomerular response to inflammation. PMID- 7534492 TI - Enhanced detection of human IL-5 in biological fluids utilizing murine monoclonal antibodies which delineate distinct neutralizing epitopes. AB - Interleukin 5 (IL-5) is a homodimeric cytokine arranged in a head-to-tail configuration covalently linked by two disulfide bonds. IL-5 has pleiotropic effects on murine and human leukocytes and has been implicated in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor binding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human IL-5 (hIL-5) mAb was generated to baculovirus expressed recombinant hIL-5 (rhIL-5). Fifteen subclones of seven hybridomas were characterized. All mAb bound hIL-5, but not murine IL-5 (mIL-5), and neutralized hIL-5 biological activity in the BCl1 proliferation assay. By competitive ELISA, the mAb were divided into two binding groups. Utilizing comparative analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with hIL-5, at least three hIL-5 neutralizing epitopes were defined. By ELISA and Western analysis, each epitope was shown to be present as a conformationally identical pair on the hIL-5 dimer. Various combinations of mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sensitive and specific sandwich ELISA were developed with a minimum detection limit of 6.25 pg hIL-5/ml (P < 0.05). Quantitation of hIL-5 in both serum and bronchoalveolar lavage (BAL) fluid demonstrated the utility of these anti-hIL-5 mAb for investigating the role of hIL-5 in inflammation. These mAb should also serve as useful reagents for epitope mapping of functional hIL-5 domains. PMID- 7534491 TI - Induction of hepatocyte growth factor in human skin fibroblasts by epidermal growth factor, platelet-derived growth factor and fibroblast growth factor. AB - Hepatocyte growth factor (HGF) is a potent mitogen for rat and human hepatocytes in primary culture and appears to be the physiological hepatotrophic factor that triggers or modulates liver regeneration. Regulation of HGF gene expression and the protein production in human skin fibroblasts was examined. Addition of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) and transforming growth factor-alpha (TGF-alpha) to confluent cultures of the cells markedly stimulated HGF secretion from the cells. The stimulating effect of EGF, PDGF and bFGF was further investigated. The effect of all three growth factors was maximal at 3-30 ng/ml and was accompanied by an increase in HGF mRNA levels. The mRNA levels were not elevated at 5 h but were at 10 h or more after addition of EGF. The levels of HGF mRNA in fibroblasts treated with the optimal doses of EGF, PDGF, bFGF, aFGF and TGF-alpha for 24 h were 6, 4, 5, 4 and 5 times that of control cultures incubated in medium only, respectively. The growth factor induced HGF mRNA expression and HGF secretion was inhibited by addition of TGF beta 1 or dexamethasone. Pretreatment with a high dose of phorbol 12-myristate 13 acetate (PMA), which causes down-regulation in protein kinase C (PKC) activity and PMA-induced HGF secretion, did not reduce the effects of the growth factors on HGF mRNA expression and HGF secretion, but rather enhanced them.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534493 TI - Effects of non-specific immunostimulants (echinacin, isoprinosine and thymus factors) on the infection and antigen expression in herpesvirus-6 exposed human lymphoid cells. AB - Non-specific immunostimulants such as plant extracts and natural and synthetic thymic preparations are widely used for enhancing the reactivity of the human defence system in chronic infections, immunodeficiency, autoimmunity and neoplastic diseases. Considering the high prevalence of latent infections by Lymphotropic herpesviruses and their frequent spontaneous reactivation, one wonders whether the stimulation of lymphoid cells by such immunostimulants may further support virus reactivation. We have performed tissue culture experiments using the well defined infectious system of human herpesvirus-6 (HHV-6) and the immature T cell HSB2 to test the effects of echinacin, isoprinosine and thymus factors on the frequency and extent of virus antigen expression in infected cells. The results show that various viral antigens related to virus replication and to the synthesis of structural components appear earlier in cells stimulated with such substances as echinacin, timunox and TP-1, but not following the stimulation with isoprinosine. Similarly, virus genome containing cells as determined by in situ hybridization techniques increased after stimulation with thymic preparations (thymostimulin and thymopentin), but not with echinacin and isoprinosine. The data suggest that the synthesis of proteins or DNA of lymphotropic viruses may be transiently enhanced when lymphoid cells are stimulated by certain non-specific immunostimulants. There was no evidence, however, of increased virus replication. Since the data presented here are rather preliminary results from tissue culture studies, the use of such substances in patients should include a critical monitoring of the activity of lymphotropic viruses to exclude untoward effects through persistent viral activity and/or autoimmune dysregulations (e.g. secondary to selective expression of viral antigens). More detailed studies are needed to this effect including long-term controls in patients treated by these substances. PMID- 7534494 TI - Expression of CD5 and CD23 on B cells of patients with rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome. Relationship with disease activity and treatment. AB - CD5+ and CD23+ lymphocytes were determined in peripheral blood of patients with rheumatoid arthritis (RA) (n = 56), systemic lupus erythematosus (SLE) (n = 20) and primary Sjogren's syndrome (pSS) (n = 21). No definite correlation between the numbers of CD5+ or CD23+ cells and most of the parameters of disease activity was found. A significant correlation (r = 0.59, p = 0.005) between rheumatoid factor levels and numbers of CD23+ cells was found in RA patients not taking disease modifying agents. For SLE and pSS no signification associations with autoantibody production were found. In conclusion, although CD5+ and CD23+ B cells might be important in the pathogenesis of autoimmune diseases, determination of these subsets in peripheral blood of RA, SLE or pSS patients provides no clear useful clinical information for the individual patient. PMID- 7534495 TI - Prevalence of hepatitis C virus among candidates for blood donation at the Hospital General de Mexico. AB - Among the NonA-nonB hepatitis, only hepatitis C is exclusively of parenteral transmission. The reported seroprevalence among blood donors in the world ranges from 0.5 to 6.0%. In order to discover the seroprevalence of anti-HCV in candidates for blood donation at the General Hospital of Mexico, 330 individuals were studied. Determination of anti-HCV was performed by the UBI HCV EIA diagnostic test. Risk factors such as history of major surgery, transfusions, drug addiction, etc were also assessed. There were only 4 seropositive patients (1.2%), 95% CI = (0, 2.75%). Only major surgery was associated with seropositivity. It was not possible to exclude hemotransfusion during surgery as a risk factor. PMID- 7534496 TI - Contrasting short-term effects of nifedipine on glomerular and tubular functions in glomerulonephritic patients. AB - The short-term effects of nifedipine on glomerular hemodynamics, sieving function, and renal tubular function were assessed in 10 patients suffering from biopsy-verified chronic glomerulonephritis. Three weeks of nifedipine treatment after 2 wk of placebo significantly reduced the blood pressure from 153 +/- 6/90 +/- 3 to 139 +/- 6/84 +/- 4 mm Hg (mean +/- SE; P < 0.05). Renal vascular resistance was reduced from 0.54 +/- 0.10 to 0.46 +/- 0.08 mm Hg/mL per minute. However, GFR (44.3 +/- 7 mL/min), effective RPF (265 +/- 37 mL/min), and filtration fraction (0.17 +/- 0.01) remained unchanged. The excretion of albumin of 1,318 +/- 395 micrograms/min was not affected by nifedipine. The glomerular sieving estimated by use of the fractional dextran clearance technique revealed no significant change by nifedipine compared with placebo in the range of 30 to 60 A of hydrodynamic dextran radius. Fractional proximal reabsorption (lithium clearance method) was reduced by nifedipine from 53 +/- 5 to 46 +/- 4% (P < 0.05). Also, the excretion of beta 2-microglobulin and N-acetyl-beta glucosaminidase increased from 10.98 +/- 4.62 to 11.86 +/- 4.74 mg/24 h (P < 0.05) and from 19.7 +/- 4.2 to 25.3 +/- 7.0 nmol/h per micromoles of creatinine (P = 0.05), respectively. It was concluded that nifedipine treatment acutely represses proximal tubular function but is without significant effect on glomerular sieving and albuminuria in these patients. PMID- 7534497 TI - Overexpression of mitochondrial genes in alloplasmic common wheat with a cytoplasm of wheatgrass (Agropyron trichophorum) showing depressed vigor and male sterility. AB - An alloplasmic hybrid (nucleus-cytoplasm hybrid) of common wheat (Triticum aestivum) with a cytoplasm of wheatgrass (Agropyron trichophorum) shows highly depressed vigor and complete male sterility. The presence of one short-arm telocentric homeologous group 1 chromosome (telosome) of the cytoplasm donor, however, restores normal vigor and male fertility of the hybrid. To study role(s) of the telosome on vigor/fertility restoration, mitochondrial genome organization and gene expression were compared among seedlings of the alloplasmic line showing depressed vigor, the corresponding restored line having a pair of the telosomes, and a euplasmic nuclear donor as control. No differences were detected in the mitochondrial genome structure between the depressed line and the restored line. Northern blot analysis using ten mitochondrial genes as probes showed no differences in transcript size and number between the depressed and restored lines, although clear differences were found in size of the major transcripts of two genes (cob and orf25) between the alloplasmic lines and the euplasmic control. Steady-state transcript levels were higher in the depressed line than in the other lines for all the mitochondrial genes analyzed including rrn18&5 when the same amount of mitochondrial RNA was loaded. The amount of rrn18&5 transcript in the total cellular RNA, however, did not differ among the lines. Run-on transcription analysis demonstrated markedly elevated transcriptional activities of all the mitochondrial genes analyzed in the depressed line based on unit amount of mitochondrial DNA, RNA and protein. The presence of Agropyron telosomes apparently normalized the level of mitochondrial transcription. These observations suggest either direct or indirect association of the observed mitochondrial gene overexpression with the depressed vigor and male sterility of the alloplasmic hybrid. PMID- 7534498 TI - Retrovirus-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human CD34+ bone marrow cells. AB - Retrovirus-mediated gene transfer into human hematopoietic stem cells has been proposed as a means of therapy for various inherited diseases and as a method of gene marking. The transduction efficiency of an amphotropic retroviral vector (PA317/HyTK) containing a hygromycin phosphotransferase-thymidine kinase fusion gene was examined with human CD34+ bone marrow cells in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor. Transduction efficiencies determined from the ability of transduced granulocyte-macrophage colony forming units (CFU-GM) to grow in hygromycin B and from polymerase chain reaction analysis of individual transduced CFU-GM growing in the presence of hygromycin B were 0.3-3.0% (mean +/- S.D., 1.1 +/- 0.9%) and 0.1-1.2% (mean +/- S.D., 0.5 +/- 0.4%), respectively. Ganciclovir at a dose of approximately 1 microM reduced the number of CFU-GM derived from vector-infected CD34+ cells by 50%. These findings demonstrate that human hematopoietic stem cells infected with this retroviral vector are susceptible to ganciclovir, offering the potential to control transduced gene expression in vivo. PMID- 7534499 TI - Increased expression of CD48 on neutrophils activated in childhood patients with aplastic anemia. AB - Activation-associated antigens such as CD11b, CD14 and CD64 of neutrophils have been reported. Although CD48 is an activation antigen of lymphocytes and monocytes, whether it is an activation antigen of neutrophils has not previously been examined. Herein, using FACS analysis, we examined the expression of surface CD48 on neutrophils activated in vivo by G-CSF administered for the treatment of idiopathic aplastic anemia. CD48 expression was increased 24 h after the initial G-CSF infusion and peaked within 1 week. Within a few days after discontinuation of G-CSF administration, it returned to the pretreatment level. This indicates that CD48 is an activation antigen of neutrophils. PMID- 7534500 TI - Action of dopamine on the ionic transport across the isolated skin of Rana esculenta. AB - Dopamine addition to the internal fluid bathing the isolated frog skin results in a strong increase of short circuit current (SCC) across this tissue. The effect is dose-dependent, 10(-4) M being the dose resulting in maximal effect. The measure of transepithelial fluxes of both 22Na+ and 36Cl- across symmetrical parts of skin short-circuited in permanence demonstrates that this effect is due to stimulation of Na+ adsorption and Cl- secretion. The former effect, but not the latter one, is mimicked by both SKF89124A and SKF82525J (D1 and D2 agonists, respectively). Moreover the effect of dopamine on SCC and Na+ net flux is wider than that of its synthetic agonists even when both D1 and D2 agonists were added together. It is suggested that the extraeffect of dopamine on SCC is due to a stimulation of Cl- secretion, probably mediated by dopamine interaction with another receptor. PMID- 7534501 TI - Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels. AB - Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in beta 1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for beta 1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the beta 1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The alpha 5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and alpha 3 continues to be expressed at about the same level as in the epithelium. alpha 6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for alpha 5 beta 1. RGD peptides do not inhibit the transformation but antibodies to beta 1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the beta 1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial-mesenchymal transition. PMID- 7534503 TI - RNA detection using non-radioactive in situ hybridization. AB - Methods of non-radioactive in situ hybridization to RNA now enable the simultaneous detection of two RNAs in the same tissue. Sensitivity has been increased by several modifications, including the use of the polymerase chain reaction. Recently, in situ hybridization has been combined with lineage tracing to determine the origin of cells expressing a specific gene. PMID- 7534502 TI - Fluorescence resonance energy transfer. AB - In the past year, a number of studies have demonstrated the utility of fluorescence resonance energy transfer as a technique for probing complex intermolecular interactions and for determining the spatial extension and geometrical characteristics of multicomponent structures composed of diverse molecular constituents, such as proteins, lipids, carbohydrates, nucleic acids, and even cells with viruses. The benefits of fluorescence resonance energy transfer are becoming increasingly evident to researchers who require measurements with high sensitivity, specificity, non-invasiveness, rapidity, and relative simplicity. PMID- 7534505 TI - Use of reiterative selection for defining protein-nucleic acid interactions. AB - Nucleic acids not only code for proteins, but also play a role in a multitude of biological processes, where they act as structural supports, binding sites, co factors, or catalysts. Recently, an array of techniques has been developed in which molecules that are best fit to perform a given task are selected from a pool of randomized RNA or DNA molecules. These techniques can provide information about the structure/function relationship governing the various biochemical properties of RNA and DNA, including their interaction with proteins. Immediate applications are found not only in the field of transcriptional regulation, but also in the field of RNA-based catalysis. PMID- 7534506 TI - Identification of biologically active peptides using random libraries displayed on phage. AB - The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes. PMID- 7534504 TI - RNA enzymes as tools for gene ablation. AB - Ribozymes have the potential to ablate the expression of any gene in a sequence specific manner and, therefore, may be useful as therapeutic molecules or as tools for the analysis of gene function. Although a number of reports have described ribozymes that are effective in inhibiting gene expression, few studies have attempted, systematically, to analyze the features of ribozymes that affect their potency within cells. Experimental observations suggest that emerging rules governing ribozyme potency in cells can be understood in terms of the competitive interactions between RNA-binding proteins, complementary RNAs and their internal secondary structure. PMID- 7534507 TI - Novel techniques in nuclear magnetic resonance for nucleic acids. AB - Recent techniques for the efficient preparation of isotopically labelled RNA of desired sequence represent a dramatic step forward for NMR of nucleic acids. Three- and four-dimensional NMR experiments greatly facilitate spectral analysis and quantification of structural constraints. Backbone-driven assignment procedures have been introduced to parallel the powerful assignment methods introduced for work with proteins. Additional structural information to complement interproton distances, namely scalar coupling constants defining the backbone conformation, can be obtained using isotopically labelled oligonucleotides. The additional interproton distance and dihedral angle constraints resolved in higher-dimensional spectra will enable the determination of larger DNA and RNA structures and also increase accuracy and precision. PMID- 7534508 TI - Characterization of oligonucleotides and nucleic acids by mass spectrometry. AB - The continued refinement of two recent methods for producing gas-phase ions, electrospray ionization and matrix-assisted laser desorption ionization, has resulted in new techniques for the rapid characterization of oligonucleotides by mass spectrometry. Using commercially available instruments, molecular mass measurements at the 20-mer level, with errors less than 2 Da, can now be made routinely in less than 15 min. Progress has also been achieved in the development of mass spectrometry for rapid sequencing of oligonucleotides smaller than 25 residues. PMID- 7534510 TI - Decreased expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in invasive cell clones derived from human prostate and breast tumor cells. AB - The importance of receptors involved in the clearance of proteases and protease/inhibitor complexes in tumor invasion is unknown. We studied the expression of the low density lipoprotein receptor-related protein/alpha 2 macroglobulin receptor (LRP/alpha 2-MR), the major receptor involved in the clearance of protease/inhibitor complexes, in invasive and noninvasive subclones derived from tumor cells. The receptor activity was 2- to 3-fold lower in invasive subclones compared to noninvasive subclones derived from human prostate PC-3 and DU 145 and melanoma A2058 cells. The receptor activity was decreased in breast cancer (MCF-10A) cells transfected with mutated Ha-ras compared to nontransfected cells. Furthermore, invasive cells derived from ras-transfected MCF-10A cells expressed lower levels of receptor compared to their non-invasive counterparts. These studies indicate a correlation between invasive phenotype and low receptor expression in different tumor cells. Experiments were performed to understand two possible mechanisms (decreased transcription of the receptor and increased transcription of an inhibitory protein) for the decreased cell-surface expression of the receptor in invasive cells. The decreased expression in invasive subclones was due to 2- to 3-fold lower levels of LRP/alpha 2-MR mRNA in all cells. In invasive PC-3 subclones, but not in DU 145, A2058, and MCF-10A subclones, 2-fold higher levels of the 39 kDa receptor-associated protein (an inhibitor of the receptor) mRNA were observed. These studies showed that the decreased expression of LRP/alpha 2-MR activity in invasive subclones was generally correlated with the decreased steady-state mRNA levels of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534509 TI - Spatial and structural segregation of the transcribed and nontranscribed alleles of c-myc in Namalva-S cells. AB - By using various approaches we received evidence that, in Namalva-S cells carrying a t(8;14) translocation and highly expressing c-myc, the two alleles of the gene are spatially and structurally segregated. Spatial segregation of the alleles was observed in all nuclei analyzed by in situ hybridization technique. Their structural segregation, i.e., association with different intranuclear structures, was confirmed in a number of experiments. When high-salt extracted nuclei were digested with EcoRI, which is known to produce fragments containing the entire c-myc locus, the sequences of the gene were found separated between the pellet, containing sequences firmly associated with the heavier matrix structures, and the supernatant, containing sequences from the free length of the DNA loops. Southern hybridization performed with a probe representative for the constant region of the human IgH locus revealed that this fractionation in fact segregates the reorganized from the normal allele of c-myc. Run-on experiments carried out with two fractions, topologically equivalent to the above P and S but isolated as intact chromatin structures, indicated that the allele associated with nuclear matrix is actively transcribed, while that located in the free length of the chromatin loops is practically nontranscribed. Studies on the chromatin organization of transcribed and nontranscribed alleles revealed the existence in them of two alternative chromatin structures. Control experiments with beta-globin gene, performed with cells constitutively nontranscribing or actively transcribing this gene, confirmed our conclusions about the spatial segregation of the two alleles and clarified that their structural segregation occurs when the gene is activated for transcription. PMID- 7534511 TI - Elevated serum levels of soluble adhesion molecules ICAM-1 and ELAM-1 in patients with severe atopic eczema and influence of UVA1 treatment. AB - BACKGROUND: ICAM-1 is known to be strongly expressed on keratinocytes in lesional atopic eczema correlating with the degree of inflammation. ELAM-1 was found to be expressed on dermal vascular endothelium in lesional atopic eczematous skin. OBJECTIVE: The present study was performed to investigate whether elevated serum levels of soluble forms of these molecules are detectable in patients with severe atopic eczema and whether these parameters could be useful markers for disease activity. METHODS: Serum levels of soluble ICAM-1 (sICAM-1) and ELAM-1 (sELAM-1) were measured by ELISA in 18 patients with severe atopic eczema before and after UVA1 therapy. RESULTS: Before onset of treatment, serum sICAM-1 (565 +/- 99 ng/ml) and sELAM-1 (89.7 +/- 29.9 ng/ml) levels were significantly (p < 0.001) elevated compared to 22 healthy control persons (296 +/- 46 and 48.8 +/- 22.7 ng/ml). After achievement of significant clinical improvement after 3 weeks of UVA1 therapy, there was neither a decrease in serum sICAM-1 nor in sELAM-1 levels. The posttherapeutic serum sICAM-1 and sELAM-1 values remained elevated (p < 0.001) above the normal range. CONCLUSION: Based on these data we suggest that (1) serum sICAM-1 and sELAM-1 are elevated in patients with severe atopic eczema, (2) sICAM-1 does not decrease together with reduction of ICAM-1-positive keratinocytes in atopic eczema following clinical improvement and might therefore be mainly of a different origin, i.e. leukocytes/endothelial cells, and that (3) sICAM-1 and sELAM-1 seem not to be suitable markers of actual disease activity in severe atopic eczema. PMID- 7534512 TI - Sulfatide, a specific sugar ligand for L-selectin, blocks CCl4-induced liver inflammation in rats. AB - The effects of sulfatide, which is a specific sugar ligand for L-selectin (LECAM 1), on CCl4-induced liver inflammation was studied in rats. Intramuscular pretreatment with sulfatide suppressed the levels of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) that were increased by CCl4 injection, but galactosylceramide, a desulfated form of sulfatide, did not. A light-microscopic analysis found that the extent and the severity of lesions of the liver cells induced by CCl4 injection were significantly less in the rats treated with sulfatide. These results show that sulfatide suppresses the CCl4 induced liver inflammation by inhibiting the attachment of L-selectin expressing lymphocytes to their native sugar ligands. PMID- 7534514 TI - Angiogenesis: an update. AB - Angiogenesis is the neovascularization or formation of new blood vessels from the established microcirculation. It is particularly important and indispensable in a large number of normal and pathological processes during pre- and post-natal life, including neoplasia, inflammation, wound repair and collaterization in response to ischemic stimuli. The current interest in the role of neovascularization in the transition from hyperplasia to neoplasia, as well as in the tumour growth and metastasis, has brought about a large number of studies on angiogenesis. The complex processes of neovascularization, quiescent in the adult organism, may occur rapidly in several circumstances, with the implication of the following events: a) endothelial cell (EC) and pericyte activation; b) basal lamina degradation; c) migration and proliferation of EC and pericytes; d) formation of a new capillary vessel lumen; e) appearance of pericytes around the new capillaries; f) development of a new basal lamina; g) capillary loop formation; h) persistence or involution, and differentiation of the new vessels; and i) capillary network formation and, eventually, organization into larger microvessels. The use of numerous "in vivo" and "in vitro" systems has facilitated the assessment of angiogenesis control, in which angiogenic (fibroblast growth factors, vascular endothelial growth factor, platelet endothelial growth factor, E series prostaglandin, angiogenin, monobutyrin) and antiangiogenic (cartilage-derived angiogenic inhibitor, thrombospondin, protamine, platelet factor 4, interferon, angiostatic antibiotics, steroids) substances intervene. Heparin and heparin sulphate also play a key role in these mechanisms. A greater knowledge of angiogenesis control may lead to the development of a potential therapy in angiogenesis-related processes. PMID- 7534513 TI - ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia. AB - Cytochemical relationship between Golgi complex and dense-cored granules (DCGs) of small granule-containing (SGC) cells in rat superior cervical ganglia was examined in electron microscopy by zinc-iodide-osmium tetroxide (ZIO) method and by enzyme cytochemistry for thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). After ZIO impregnation, all the saccules of Golgi apparatus and some of tubular rough endoplasmic reticulum (rER) were stained. DCGs in periphery of SGC cells were not stained, but varying degrees of dense deposits occurred in the DCGs in vicinity of Golgi trans-saccules. Both TPPase and ACPase activities were localized in one or two stacked layers of saccules on the trans side of the Golgi complex. No reaction products were demonstrated in the DCGs. From these results, we suggest that the DCGs of SGC cells in rat superior cervical ganglia are derived from the Golgi complex, and that lysosomal cleavage of protein contents in the DCGs may occur in the trans Golgi saccules. PMID- 7534516 TI - Radioimmunoscintigraphy--a new, specific procedure in nuclear oncology. AB - The purpose of this review is to describe in detail a new, specific, nuclear medicine imaging procedure in the field of oncology. Although the history of radioimmunoscintigraphy is not so short, the greatest advances in the realization of this idea have been made during the last two decades. The time has come for radioimmunoscintigraphy to became a standard procedure in the diagnosis of malignant as well as non-malignant diseases. Except some historical facts about the development of radioimmunoscintigraphy, this review also shows all the complexities of the problem which had to be resolved before a good idea was effectively realized. The authors have tried to present, to a reasonable extent, all problems in connection with the selection of radionuclides, antibodies, methods of labeling antibodies, and imaging procedure. In general, what interests medical professionals the most is the clinical application of radioimmunoscintigraphy as well as future development and improvements of this procedure as a step of radioimmunotherapy--a new kind of treatment in oncology. PMID- 7534515 TI - Molecular analysis of four lactate dehydrogenase-A mutants in the mouse. AB - Four electrophoretic and/or enzyme-activity variants of murine LDH-A subunit (Ldhla-m1Neu, Ldhla-m5Neu, Ldhla-m6Neu, Ldhla-m9Neu), induced by procarbazine hydrochloride or ethylnitrosourea (ENU), were analyzed at the DNA level. The exons of the Ldhl gene from homozygous mutants were amplified by PCR and sequenced. Three mutations resulted from nucleotide substitutions in exon 5: the transitions A-->G at codons 216 (Ldhla-m5Neu) and 225 (Ldhla-m6Neu), and the transversion G-->C (Ldhla-m1Neu) at codon 222. The mutations resulted in the replacements of Glu by Gly (Ldhla-m5Neu), Gln by Arg (Ldhla-m6Neu) and Asp by His (Ldhla-m1Neu). The fourth base substitution, the transition T-->C (Ldhla-m9Neu), has been found at the GT donor splice site following the first exon; this mutation affected the efficiency of transcription. All ENU-induced mutations were A/T-->G/C transitions. The mutation events could be correlated with the biochemical and physiological alterations observed in affected mice. PMID- 7534517 TI - Breast cancer prognosis. I. Prognostic factors in patients with node-negative (N0) breast cancer. AB - Predictors of breast cancer survival were investigated among 196 node-negative (N0) breast cancer patients treated at the Department of Surgery of the University Hospital for Tumors in Zagreb between 1969-1988. Selected prognostic factors included patient age, delay in treatment, tumor size, histologic grade of malignancy, estrogen receptor status, progesterone receptor status, tumor site, and type of surgical treatment. Among these predictors, only tumor size (p < 0.001) and pathohistologic grade of malignancy (p < 0.001) caused significant differences in 5-year overall survival rates (T1-92.1%, T2-89.8%, T3-64.1% and T4 45.4%; grade I-95.0%; II-80.6%; III-63.6%). The authors conclude that among eight selected clinical characteristics, only tumor size and pathohistological malignancy grade can serve as helpful predictors in determination of the probability of 5-year overall survival among node-negative breast cancer patients. PMID- 7534518 TI - Breast cancer prognosis. II. Prognostic factors in patients with node-positive (N1-3) breast cancer. AB - Predictors of breast cancer survival were investigated among 282 node-positive (N1-3) breast cancer patients treated at the Department of Surgery of the University Hospital for Tumors in Zagreb between 1969-1988. Selected prognostic factors included patient age, delay in treatment, tumor size, type of lymph-node affection, pathohistological grade of malignancy, estrogen receptor status, progesterone receptor status, tumor site, and type of surgical treatment. Among these predictors, only tumor size (p < 0.001), type of lymph-node affection (p < 0.001), malignancy grade (p < 0.001), and progesterone receptor status (p < 0.001) revealed a significant impact on a 5-year overall survival rates (T1-100%, T2-64.5%, T3-54.5% and T4-23.7%; N1-60.2%, N2-20.8%, N3-30.3%; grade I-85.5%, II 59.4%, III-30.5%; PgR+ 63.8%, PgR--26.4%). The authors conclude that among nine selected characteristics, only tumor size, type of lymph-node affection, pathohistological malignancy grade, and progesterone receptor status can be helpful predictors in the determination of the probability of 5-year overall survival among node-positive breast cancer patients. PMID- 7534519 TI - Breast cancer prognosis. III. Prognostic factors in patients with distant metastases (M1) at the time of diagnosis. AB - Predictors of breast cancer survival were investigated among 66 patients who had distant metastases at the time of diagnosis (M1). All of these patients were treated at the Department of Surgery of the University Hospital for Tumors in Zagreb between 1969-1988. Selected prognostic factors included patient age, delay in treatment, tumor size, type of lymph node affection, response to palliative surgical treatment and administered chemotherapy or hormonal therapy, and site of metastasizing. Among these predictors, several of them revealed a significant impact on a median survival (in months) of these patients: the most important was the response to chemotherapy (p < 0.001), followed by site of metastasizing (p < 0.05) and primary tumor size (p < 0.05). Palliative surgical treatment, apart from improvement of life quality, played no role in determining the survival among breast cancer patients with a distant disease. PMID- 7534520 TI - Clinical and epidemiological characteristics of bleeding duodenal ulcer patients with and without dyspepsia. AB - The most relevant clinical presentations of duodenal ulcer disease are pain and acute bleeding. The purpose of this study was to investigate the relevance of dyspepsia in patients with bleeding duodenal ulcer, and to compare the clinical and epidemiological characteristics of bleeding patients with and without dyspepsia. A total of 82 patients with isolated duodenal ulcer and bleeding were included in this study. There were 48 (58.5%) dyspeptic and 34 (41.5%) nondyspeptic patients. The patients with and without dyspepsia were almost identical with regard to their age (52.9 +/- 11.9 vs 53.4+/- 10.2 years, p > 0.05). In the dyspeptic group, significantly more patients had duodenitis and a deformed bulb (chi 2 = 4.05, p < 0.05 and chi 2 = 3.99, p < 0.05, respectively). Patients with bleeding duodenal ulcers and dyspepsia were more likely to have taken non-steroidal anti-inflammatory drugs (45.8 vs 8.8%; chi 2 = 11.18, p < 0.001), whereas significantly more patients in the nondyspeptic group have taken histamine H2 antagonists (85.3 vs 8.3%; chi 2 = 45.87, p < 0.01). No significant difference was found between the dyspeptic and nondyspeptic groups with regard to the previous diagnosis of peptic ulcer bleeding, the presence of environmental stress, gastritis, and alcohol or tobacco consumption. Furthermore, there was no significant difference in regard to the rebleeding, the need for urgent operation or hospital stay. The results of this study support the evidence that in patients with bleeding duodenal ulcers the dyspeptic symptoms were more often associated with objective signs of duodenal pathology, and the use of non-steroidal anti inflammatory drugs and maintenance treatment with histamine H2 antagonists was associated with silent duodenal ulcer bleeding. PMID- 7534521 TI - Preoperative evaluation of rectal carcinoma by transrectal ultrasonography. AB - The aim of the study was to assess the diagnostic value of transrectal ultrasonography (TRUS) in detecting the depth of tumor infiltration as well as the involvement of lymph nodes in patients with rectal carcinoma diagnosed after rectoscopy and proven by examination of the biopsy specimen. TRUS was performed on thirty-three patients with rectal carcinoma prior to surgery, the first assessment of tumor growth, and extension by pathological examination of the operative specimen. TRUS correctly assessed perirectal growth in 18 out of 22 cases proven by pathology (two understaged, accuracy 86 percent), while in 12 cases with no perirectal growth proven by pathology this method was correct in 9 cases (three overstaged, accuracy 75 percent). The results of this study indicate that TRUS, combined with endoscopy and biopsy, is a useful procedure in the preoperative staging of malignant rectal lesions and provides valuable data for the therapeutic planning of patients with rectal carcinoma. PMID- 7534522 TI - Motivation for the choice of a general practitioner--attitudes of practitioners and patients. AB - The aim of this study was to find out if any significant changes occurred after introducing, the free choice of a general practitioner (GP) into primary health care. The determinants of patients' choice and GP's attitudes regarding this matter were tested. A survey was conducted in 1992 by distributing questionnaires to 71 GP's and 292 patients who chose these GPs. The questionnaires were composed selectively for GPs and patients, and they all answered them anonymously. The group of GPs consisted of 37 (52.1%) GPs with vocational training and of 31 (43.7%) GPs without vocational training. The patients evaluated the GPs' characteristics as very important in choosing a particular GP. Of medium importance were the surgery characteristics (where it was situated, work organization, equipment), and recommendations were the least important. Most frequently, the patients chose a particular GP because he was already the GP of one of the family members. The GPs assessed the family to be the most important determinant for a patient to choose a particular GP. Surgery characteristics (distance and work organization) were evaluated as being of medium importance. The recommendations of a friend were of medium importance, and the recommendations of medical staff were not important, according to the opinion of the surveyed GPs. The only occurrence detected in the survey was that of a "return" phenomenon, in which patients returned to their old GPs, which was a direct consequence of the implementation of the free choice principle into health care practice. The results of this study match the results of similar studies already published, and they are argumented theoretically.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534523 TI - Diagnostic value of growth hormone-releasing hormone tests in short children. AB - The values of GH following GHRH stimulation were determined in 32 short children. Firstly, the secretion of GH was measured by two conventional tests (insulin induced hypoglycemia and arginine tests). According to the response of GH secretion, the children were divided in two groups, the first one with short children suffering from growth hormone deficiency (GHD) (N = 17), and the second with short children without growth hormone deficiency (non-GHD) (N = 15). In the first group (GHD), according to the extent of the GH response to GHRH stimulation, three subgroups were distinguished: in 8 children GH response was < 5 ng/ml, in 5, between 5 and 8 ng/ml, and in 4 the response was > 10 ng/ml. In the second group (non-GHD), the response of GH was between 4.7 and 8.2 ng/ml only in two children, and all other children expressed a response above 10 ng/ml. Comparing the values in both groups by induced hypoglycemia and GHRH test, the response of GH was significantly higher following GHRH stimulation. In the first group the mean value of GH following GHRH stimulation was 10.9 ng/ml (s = 16.35), and in induced hypoglycemic test 1.92 ng/ml (s = 1.86). In the non-GHD group the mean value of GH in the GHRH test was 25.29 ng/ml (s = 14.15), versus GH value in the hypoglycemic test of 11.22 ng/ml (s = 3.045). The authors conclude that the GHRH stimulation test is an acceptable test in differentiation between GH deficiency of pituitary and hypothalamic origin. Still, the GHRH stimulation test alone is not sufficient for the diagnostic procedure of GH deficiency. PMID- 7534524 TI - Alloimmunizations following blood transfusions. AB - A retrospective study was performed to estimate the rate of alloimmunization against red blood cell (RBC) antigens in hospitalized patients and the frequency and specificity of clinically significant alloantibodies (CSA) detected during routine pretransfusion testing of 10,641 blood samples. Clinically significant alloantibodies were found in 116 out of the 10,641 tested serums (1.09%). The incidence of CSA in two surgical groups of patients was 0.18% and 0.68%, respectively. In patients with hematologic diseases the frequency of CSA was 17.6%, in patients with uremic disease it was 14%, and in patients with cirrhosis hepatis it was 6.9%. The incidence of clinically significant alloantibodies in patients who had one to five exposures to RBC antigens was 5.1%; in patients who had six to fifteen exposures, 87.9%; and in patients with more than fifteen exposures, 6.8%. The most frequently found single alloantibodies were anti-Kell (22%), and the following irregular alloantibodies were found with decreasing frequency: anti-E (20%), anti-D (11%), anti-c (10%), anti-C (7%), anti-Kidd (6%). The most frequent alloantibodies found together with other alloantibodies had anti-Duffy specificity (80%), and the following irregular alloantibodies were found with decreased frequency: anti-C (70%), anti-c (57%), and anti-Kidd (50%). PMID- 7534525 TI - A contribution to the knowledge about autoimmune eye diseases. AB - In this paper the autoimmune eye disease complex is presented in detail. The problems of autoagressivity in the light of new conceptions based on modern microchemical, chemical and immunological methods of analysis that allow better insight in the nature of these diseases are discussed. The basic concepts of immunological mechanisms and autoimmunity are explained, with special attention given to those occurring in the eye. Attention is directed towards the importance of various intra- and extraocular factors, as well as to the regulatory mechanisms that influence the autoimmune eye disease's development. In the conclusion the authors analyze the abilities and aims of further investigations of this field of ophthalmoimmunopathology. PMID- 7534526 TI - Spontaneous hypoglycemia associated with chronic renal failure--a preventable life-threatening complication. AB - Hypoglycemia has been reported in three non-diabetic uremic patients undergoing maintenance hemodialysis. There were one female and two males, ranging in age from 26 to 45 years. Only one hypoglycemic episode per patient was documented, and measured blood glucose levels were less than 1.1 mmol/L. Contributing factors of hypoglycemia in CRF patients were hepatic dysfunction, and drug side effects (isoniazid, rifampin and propranolol). The treatment of choice was intravenous hypertonic glucose administration. Proper nutrition, the judicious use of any medication that has the potential for inducing hypoglycemia, the early detection and treatment of associated diseases, and the use of dialysate fluid with glucose in hemodialysis patients can diminish the risks of this potentially lethal complication. PMID- 7534528 TI - Report of the First Croatian Symposium on Hospices and Palliative Care. PMID- 7534527 TI - International cooperation in medicine through the eyes of a physician and scientist. PMID- 7534529 TI - Intermediate filament reactivity in hyperplastic and neoplastic lesions from medaka (Oryzias latipes). AB - To determine if hyperplastic and neoplastic lesions from medaka showed similar immunoreactivity to intermediate filament antibodies as the tissues of origin, two week old medaka were exposed to 10 or 20 mg/L of methylazoxymethanol acetate for two hours and transferred to clean water for up to six months. Using a streptavidin peroxidase method, paraffin embedded Bouins fixed neoplasms were incubated with cytokeratin, vimentin, or neurofilament antibodies. Like their nonneoplastic cellular counterparts, hepatocellular carcinoma, pancreatic acinar carcinoma and mesenchymal neoplasms including hemangioma and hemangiopericytoma reacted negatively to cytokeratin antibodies. Cholangiocarcinoma, mesothelioma, and proliferative lesions containing biliary epithelial cells reacted positively to cytokeratin antibodies. All neoplasms and proliferative lesions were negative with vimentin and neurofilament antibodies. These data indicate that while some epithelial neoplasms showed cytokeratin reactivity similar to the parent tissues, additional markers are needed to identify mesenchymal tissues and neoplasms. PMID- 7534530 TI - [Tetanus vaccination. Evaluation of a program of health promotion in a family medicine unit]. AB - OBJECTIVE: To assess the effectiveness of a tetanus immunization poster at a medical clinic in encouraging patients to request immunization against tetanus with d2T5. DESIGN: Quasi-experimental study. SETTING: The Family Medicine Unit (F.M.U.) at the Centre hospitalier de l'Universite Laval (C.H.U.L.), a tertiary hospital in the region of Quebec City. PARTICIPANTS: All patients over 7 years of age at CHUL's FMU between February 22 and March 19, 1993. INTERVENTIONS: Poster promoting tetanus immunization displayed in waiting room during test weeks 2 and 4 and withdrawn during control weeks 1 and 3. MAIN OUTCOME MEASURE: Proportion of patients vaccinated with d2T5. RESULTS: The proportion of patients vaccinated during the test weeks was the same as the proportion vaccinated during the control weeks. Week 1: 1/507 (0.20); Week 2: 1/394 (0.25); Week 3: 1/441 (0.23); Week 4: 1/486 (0.21). CONCLUSION: The presence of the posters in the waiting room had no impact on tetanus immunization at CHUL's FMU. PMID- 7534531 TI - [Molecular mechanisms underlying the relationship of motivation and supporting stimulation in brain neurons]. AB - The nature of neuronal impulse activities in the sensory area of the brain was studied in rats given ouabain and cycloheximide by electric stimulation of the cold center of the lateral hypothalamus in the presence or absence of readily available feed. Microionophoretic administration of ouabain into the cortical neurons resulted in similar charges in the impulse effects of nerve cells on the stimulation of the lateral hypothalamus both in the presence and in the absence of the feed available to the animals. That of cycloheximide led to significant changes in the frequency of discharge activities of the cells that had initially shown their response to electric irritation of the lateral hypothalamus in the presence of the feed readily available to the animals. PMID- 7534532 TI - [Neurospecific regulatory genome proteins in brain cells and systemic processes of memory]. PMID- 7534533 TI - [Enkephalinase mechanisms of tolerance of analgetic action of opioids. Effects of D-phenylalanine and naloxone]. AB - The paper reviews the data available in the literature and the authors own data demonstrating the differences in the levels of endogenous opioids and in the effects of enkephalinase inhibitor and naloxone in morphine-responsive and morphine-resistant and -tolerant animals. In the morphine-tolerant animals, a single administration of enkephalinase inhibitor or some doses of naloxone was found to produce an analgesic effect leading to a short-term analgesic effect of morphine. Chronic administration of naloxone causing a gradual decrease in and subsequent cessation of its analgesic effect leads to recovery of morphine's analgesic effect in the drug-resistant and -tolerant animals. It is suggested that the morphine-resistant animals have a congenital high activity of enkephalinase, while the drug-tolerant ones have its acquired high activity. Naloxone in certain doses with the high activity of enkephalinase act as its inhibitor, but in high doses acts as its opioid antagonist. PMID- 7534534 TI - [Systemic integration of dominating motivation]. AB - The paper analyzes the investigations into the central architectonics of biological motivations, which have been performed by the P.K. Anokhin scientific school. Each biological motivation is postulated to be a result of specific integration of heterochemical mechanisms of biological motivations. The chemical plasticity of biological motivations is evidenced by the findings of the duplication of different neurotransmitters in cortical-subcortical interrelations, as well as the potentialities of the chemical rearrangement ("chemical compensation") of neurotransmitter mechanisms of biological motivations in case of bilateral failure of hypothalamic motivational centers of the hypothalamus. Some cerebral oligopeptides are found to be able to temporarily and selectively modify the pattern of motivational behavioral responses evoked by electric stimulation of various hypothalamic formations. It is concluded that the biological motivations are a result of the integrative performance of the whole brain wherein hypothalamic pacemakers play the leading but not final role. PMID- 7534535 TI - [Current state of the hypothesis on functional continuum of regulatory peptides]. AB - Further development of the hypothesis of the functional continuum of regulatory peptides is given on the basis of new experimental evidence. Some basic schemes of the regulatory chains and cascades are considered, which explain some long term involved physiological effects of short-living regulatory peptides. PMID- 7534536 TI - [Theoretical physiology: developments in the P. K. Anokhin's scientific school]. PMID- 7534537 TI - [Training of animals with hypoinsulinemia: systems analysis]. AB - Experiments on rabbits and rats with dithizone- and streptozotocin- induced (stz) diabetes were made to study the learning characteristics in animals with hypoinsulinemia. The analysis of operant feeding training of rabbits showed their higher searching behavior and more successful learning in hypoinsulinemic than those in intact animals. The behavior structure differences could be removed by an injection of insulin one hour before the testing. In experiments on rats with electroshock avoidance training some groups with different learning characteristics were distinguished. Searching activity of stz-rats was lower than that of controls, and the most of stz-rats belonged to a lazy group. The results are discussed according to Anokhin's theory of the functional system. PMID- 7534538 TI - [Renin-angiotensin system in the mechanisms of alcohol motivation]. AB - The role of angiotensins in the mechanism of alcohol motivation was studied. Angiotensins were shown to be capable of regulating not only thirst and salt appetite, but also alcohol intake by stimulation of alcohol consumption in non alcohol-dependent rats and suppression of alcohol motivational excitation in alcohol-dependent rats. The mechanism of thirsty stimulation by angiotensins differs from that of their regulation of alcohol uptake. Angiotensins provoke thirst and salt appetite by activation of saralasin-sensitive classical angiotensin-II receptors while the regulation of ethanol intake by angiotensins is not mediated by the same receptors and realized through the participation of the common fragments for all endogenous angiotensins-[3-7]-angiotensin-II. PMID- 7534539 TI - [Systemic activity of taste receptors]. PMID- 7534540 TI - [Change in integrative states of cortical neurons at consecutive stages of systemic behavior pattern]. AB - Cats were trained to press the level after sound conditional stimulus. The spike activity of visual and parietal cortex neurons was registered during this operant feeding behavior, bring activity patterns were individual and their reorganization was observed during interstimulus non-reinforced pressings of the lever and this corresponded to prolongation of a manipulation-reinforcement interval. Thus, the firing pattern reflects the functional state of the neuron and its synaptic activation; the changes in spike activity reflect the alterations in the neuronal integrative state. PMID- 7534541 TI - [Chemical sensitivity of cerebral cortex neurons in systemic pattern of food searching behavior]. PMID- 7534542 TI - [The concept of the nontraditional use of histochemical stains]. AB - A priority concept is produced, according to which stains, which have traditionally been used in pathological anatomy for structural and morphological studies of changes occurring in the cell during pathology, and also for postmortem diagnosis, are suggested for use as medicines. This nontraditional use of strains is based on the results of pathohistologic studies and is regarded as a new pharmacotherapeutic strategy--"address histochemical pharmacotherapy" or "pharmacotopographic therapy". Its main principle is "stopping by staining," i.e., the inhibition of a pathological process by the staining of "address substrates", i.e., the biochemical ingredients of the cell involved in a given pathology. Stain blocking of the "address substrates" of the cells in which pathology originates and develops excludes them from the pathogenetic chain of the disease. Staining biochemically "switches over" the pathology onto the path of normalization and thus softens the course of the disease. "Address histochemical pharmacotherapy" implies the direct effect of the dye on the pathology (the medicating function of the dye) and the delivery of the necessary medicating enzymes, activators, or enzyme inhibitors together with the dye to the site of pathology (the transport function of the dye). A scheme for biological trials of the stains as potential means of "address histochemical pharmacotherapy" is given. The data in the literature on the pathohistology of some diseases is analyzed and various types of pathology control by histochemical stains is suggested. PMID- 7534543 TI - The heme-binding region polymorphism of cytochrome P450IA1 (CypIA1), rather than the RsaI polymorphism of IIE1 (CypIIE1), is associated with lung cancer in Rio de Janeiro. AB - Ile-Val polymorphism in exon 7 of cytochrome P450IA1 (CypIA1) and RsaI polymorphism of cytochrome P450IIE1 (CypIIE1) were examined in a case-control study of lung cancer in Rio de Janeiro, Brazil. The Val-containing genotype in exon 7 of CypIA1 was found to be associated with lung cancer in this population (odds ratio, 2.26; 95% confidence interval, 1.14-4.47 for 99 cases versus 108 controls of 123 matched pairs), whereas RsaI polymorphism in CypIIE1 was not associated with lung cancer susceptibility. In squamous cell carcinoma, the degree of association of Val-containing genotype was greater in those with fewer pack-years of smoking. The RsaI polymorphism of CypIIE1 has a different distribution from the Japanese pattern and is not associated with lung cancer. When we analyzed the association of Ile-Val polymorphism to MspI polymorphism of CypIA1, the Val/Val homozygote was found only in the subpopulation with the MspI site-present homozygote. The apparent lack of association of CypIA1 MspI polymorphism with lung cancer in this area reported in our previous study and the results of the present study indicate that the "true" responsible site for lung cancer susceptibility should be the Ile-Val polymorphism in the catalytic site of CypIA1. PMID- 7534544 TI - Colorimetric assay for the measurement of thymocyte/thymic stromal cell adhesion. AB - 1. We describe a simple and accurate colorimetric adhesion assay (CAA) and illustrate the assay by measuring the adhesion of mouse thymocytes to mouse 2BH4 cells. 2. The assay is based on the crystal violet staining of thymocytes adhered to a subconfluent layer of 2BH4 cells (plated at 2 x 10(4) cells/well for 24 h). The optimal incubation time was shown to be 1 h and washing in PBS of non-bound and non-specifically bound thymocytes is the critical step for the precision and accuracy of the assay. 3. Saturation curves were obtained for thymocytes adhered to plated 2BH4 cells. The blank (only 2BH4 cells) was near 0.200 +/- 0.010 (mean +/- SD) and was quite reproducible. As expected, the extent of adhesion was also dependent on the number of plated 2BH4 cells. Standard curves need to be run with each assay for quantitative measurements. The intra-assay and interassay coefficients of variation were 5% and 20%, respectively. 4. The specificity of the reaction was demonstrated by the reduction of adherence by trypsin pretreatment of thymocytes, and the dose-dependent inhibition of adherence by rabbit anti-mouse thymocyte antisera but not rabbit anti-mouse immunoglobulin antisera. 5. The proposed method is simple and requires less effort than the counting of adhered cells with the light microscope and does not require the use of radioactive material as when labelling with Na2(51)CrO4 is utilized. PMID- 7534545 TI - Immunological induction of flavor aversion in mice. AB - 1. Young adult BALB/c and B6D2F1 mice of both sexes (20 +/- 2 g) immunized ip with 2 doses of 10 micrograms ovalbumin (Ova), but not with 2 doses of 10 micrograms bovine gammaglobulins (BGG), show aversion to the ingestion of sweetened egg white or crystallized Ova solutions which are avidly ingested by normal mice. In 24 h, normal mice or mice immunized with BGG ingested, respectively, 340 +/- 80 and 265 +/- 56 mg of sweetened egg white per gram of body weight (mg/gbw); in the same period, Ova-immunized mice ingested less than one tenth these amounts (18 +/- 5 mg/gbw). ELISA-titers of anti-Ova and anti-BGG antibodies in immune mice were of similar magnitude. 2. Aversion arises coincidentally with the emergence of anti-ovalbumin antibodies in serum in the primary response, 14 days after primary immunization. 3. Previous induction of oral tolerance to ovalbumin by a single gavage with 20 mg Ova 7 days before primary ip immunization, which blocks the increase of specific antibodies in serum, also blocks the development of the aversive phenomenon. 4. Aversion was induced to 1 mg/ml but not 0.1 mg/ml sweetened crystallized ovalbumin solutions and was already noticeable 2 h after exposure of immunized mice to sweetened egg white solutions. 5. We conclude that, at least in experimental situations, immunological factors may be of decisive importance in diet selection. PMID- 7534546 TI - [Diagnostic value of determining specific IgE allergens by the fluoro-FAST method of the 3M firm in atopic dermatitis of children and adults]. PMID- 7534548 TI - [Diagnostic value of allergy-specific IgE determined in dermatitis herpetiformis and atopic dermatitis]. PMID- 7534547 TI - [Allergen specific IgE antibodies in children with atopic dermatitis]. PMID- 7534549 TI - A 170 kDa polypeptide from mung bean shares multiple epitopes with rabbit skeletal myosin and binds ADP-agarose. AB - A 170 kDa polypeptide that has been partially purified from mung beans is retained by ADP-agarose even in the absence of divalent cations when most non myosin ATPases and kinases do not bind. Attempts to demonstrate a myosin-like ATPase activity were inconclusive, however, and the protein accounts at most for only a small part of the total K+ EDTA ATPase activity of mung bean extracts. All four monoclonal antibodies raised to the 170 kDa polypeptide react with rabbit skeletal muscle myosin and localize the 170 kDa polypeptide in mung bean root tip cells to the actin-containing phragmoplast and to sites dispersed throughout the cytoplasm which probably include some but not all actin cables. These 4 monoclonals and 3 commercially available antimyosin monoclonals all recognise rabbit skeletal myosin and 160-170 kDa proteins that are present in two other angiosperms tested. In addition, a 158 kDa protein of mung bean reacts with only one antibody and does not bind ADP-agarose. We conclude that strong but not yet conclusive evidence points to the 160-170 kDa proteins of angiosperms being a widely conserved form of myosin heavy chain. PMID- 7534551 TI - HMO case management and the surgical patient. AB - A nurse serving as a case manager in an HMO describes the major aspects of the role at present and also identifies and explains areas that are expanding. This role is vital to the quality outcomes and cost effectiveness of surgical procedures for HMO members. The case manager is a central member of the health care team of the HMO. PMID- 7534550 TI - Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells. AB - Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25 degrees C and 3 hours recovery at 37 degrees C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-1 fibroblasts. Epithelioid cells survived 3 days at 25 degrees C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25 degrees C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture. PMID- 7534552 TI - Induction of substance P-immunoreactivity by estrogen in neurons containing estrogen receptors in the anterovental periventricular nucleus of female but not male rats. AB - Effects of gonadal steroids on numbers of neurons containing estrogen receptor (ER) and/or substance P (SP) were examined in the anteroventral periventricular nucleus (AVPV) of female and male rats by double-labeling immunohistochemistry employing antibodies specific for ER and SP. Animals were gonadectomized and received subcutaneously either oil alone (Control group), sequential injections of estradiol benzoate and oil (EB + Oil group), or those of EB and progesterone (EB + P group). In the female control rat, a large population of ER immunoreactive (IR) cells were found clustered throughout the AVPV. They were counted more than 2,000 in total of 4 sections in this nucleus. On the contrary, SP-IR neurons were scarcely observed in the same area of this group. Administration of estrogen to female animals decreased the total number of ER-IR cells to 67% of the control group. In contrast to the supressive effect of estrogen to its own receptor, it induced SP-IR neurons in the AVPV of the female. Approximately 50-80 SP-IR neurons were counted in the 4 sections, and 59% of these neurons expressed ER-IR material in their nuclei. In the female EB + P group, the number of ER-IR neurons also decreased to 79% of the control group. Although the number of SP-IR neurons in this group decreased to 32% of that in the EB + Oil group, a ratio of coexistence of ER-IR material in these neurons increased to 75%. The male control group contained a smaller population of ER-IR cells relative to the female control (1497 vs 2143).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534553 TI - Tylosis oesophageal cancer mapped. PMID- 7534554 TI - Arabinogalactan proteins in maize coleoptiles: developmental relationship to cell death during xylem differentiation but not to extension growth. AB - The spatial and temporal expression of arabinogalactan proteins (AGPs) in the coleoptile of maize seedlings was investigated with monoclonal antibodies (MAC207, JIM13, JIM14) raised against particular AGP epitopes in carrot. MAC207 binds to a buffer-soluble AGP fraction of 90-210 kDa that also reacts with beta glucosyl Yariv reagent and the lectin RCA120. Immunogold-labelling showed that the MAC207 epitope is exclusively localized in the plasma membrane. JIM13 binds to a 120 kDa component of the buffer-soluble AGP fraction localized in the plasma membrane of future sclerenchyma cells and secondary-wall thickenings of future tracheids of vascular bundles. JIM14 binds to a 50 kDa component of the salt extractable fraction from cell walls localized in the innermost wall layer of sclerenchyma cells. These AGP epitopes demonstrate different temporal expression patterns which do not correlate with extension growth. Auxin had no effect on the amount of soluble AGP from coleoptile sections, containing the growth-controlling epidermis but no vascular bundles, as measured by crossed electrophoresis. Moreover, incorporation of radioactive arabinose, galactose or proline into this fraction was not stimulated by auxin. These results contradict the hypothesis that AGPs function as wall-loosening agents in auxin-mediated extension growth. The results are compatible with the notion that AGPs serve as developmental markers defining particular features of future cell differentiation. The specific association of the epitopes recognized by JIM13 and JIM14 with disintegrating cells suggests that the related AGPs identify those cells of the coleoptile which are committed to programmed cell death. PMID- 7534555 TI - The blue light-responsive AthH2 gene of Arabidopsis thaliana is primarily expressed in expanding as well as in differentiating cells and encodes a putative channel protein of the plasmalemma. AB - According to our previous studies the Arabidopsis gene AthH2 which is inducible by blue light and phytohormones codes for an intrinsic membrane protein. It bears a resemblance to several distinct channel proteins of plant and animal species classified as the MIP/NOD-26/GlpF family. In the present study biochemical analyses and electron microscopic immunochemistry were used to elucidate the subcellular location of the AthH2 protein. The results clearly demonstrate that it is an exclusive constituent of the plasmalemma. Furthermore, the expression of the AthH2 gene in transgenic Arabidopsis plants containing the promoter region of AthH2 fused to the beta-glucuronidase (gus) reporter gene was studied. The in situ localization of gus activity revealed that the specific promoter is temporally activated by light in expanding and/or differentiating cells comprising newly formed tissues and organs: root elongation zone, guard cells of stomata, vascular bundle sheaths, filaments of stamen and young siliques. Several sites of gus expression coincide spatially with those of in situ hybridization and the immunocytochemical reaction, respectively, suggesting that the AthH2 promoter had correctly responded to light as an important exogenous factor with relevance to the complex pattern of differentiation. Studies with protoplasts from plants transformed with an antisense construct revealed a water transport capacity of the AthH2 protein. PMID- 7534556 TI - [Structural changes in the vascular wall in vasoconstrictor and vasodilator exposures]. AB - Noradrenaline vasoconstriction of the tail artery decreased external, internal diameters and the cross-section area of all the elements of the blood vessel wall. Papaverine vasorelaxation of the same artery increased the diameters but reduced the endothelium and adventitia areas. The data obtained suggest a close relation between the blood vessel tonus and the morphology of all the elements of the vessel wall. PMID- 7534557 TI - [The effect of 4-bromophenacyl bromide on outward-rectifying potassium channels in the membrane of mouse peritoneal macrophages]. PMID- 7534558 TI - [The role of the salivary glands in supporting the relative stability of the blood hydrolytic activity]. AB - Acute and chronic experiments in dogs revealed participation of the salivary glands in ensuring a relative constancy of the blood enzyme activity. The saliva hydrolases reflected the spectrum of blood plasma and its disturbance and extend the possibilities of the sialoenzymodiagnosis. PMID- 7534559 TI - [The species characteristics of the composition of the supraepithelial mucous layer of the digestive tract in rats and mice]. AB - The concentration of protein part of glycoprotein of the adherent mucus gel barrier was found to be the same in respective portions of the rat and mouse digestive tract, whereas the concentration of hexosamines, galactose, fucose was higher in rats rather than in mice. Specific differences in distribution of enzymes, HCO3-, nuclear acids, were shown. PMID- 7534560 TI - [The regulation of the absorptive activity of the intestinal epithelium in lower vertebrates by the enteral portion of the metasympathetic nervous system]. AB - Effects of a few pharmacological agents (ouabain, proserine, atropine) upon absorption velocity in intestinal epithelium, were studied in lower vertebrates. Mechanisms of the intestinal epithelium response to osmotic irritants and regulation of this process by the enteric portion of the metasympathetic nervous system, are discussed. PMID- 7534561 TI - [A comparative evaluation of the importance of hippocampal afferent inputs into the large-cell neurosecretory nuclei of the rat hypothalamus]. PMID- 7534562 TI - [A multichannel microelectrode system for long-term use]. PMID- 7534563 TI - [V. S. Deriabin's memoirs on I. P. Pavlov. An attempt at psychophysiological analysis of the creative personality of a scientist]. PMID- 7534564 TI - [The dynamics of interhemispheric asymmetry during the processing of a transcallosal signal]. AB - Multiple homotopic transcallosal responses were led from the visual, temporal, auditory and motor cortical areas in Wistar rats. Temporal parameters of positive as well as negative components of the responses revealed alteration of the interhemispheric asymmetry by the rule of right-left-right shifting. By the amplitude parameters of negative components the asymmetry was right-left-right whereas by the positive oscillations amplitude it was left-right-left. The data obtained suggest a right-left-right pattern of domineering of the hemisphere cortex in processing of transcallosal signal. PMID- 7534565 TI - [Evoked electrical activity. The methodological problems and outlook]. AB - Basic methodological problems of recording the evoked electrical activity (EEA) are analysed in terms of its genesis. Further progress with non-invasive methods seems to be limited. The proposed technique provides an a priori "space-time" information due to measurement of several classes of the EEA and simultaneous stimulation of two subsystems of the CNS. The problems and possible ways of putting this methodology into practice, are discussed. PMID- 7534566 TI - [Lipid peroxidation in slices of rat olfactory cortex under long-term potentiation]. AB - Long-term potentiation (LTP) was shown to be accompanied by an increase in the lipid peroxidation at the initial stage. Stationary phase resulted in inhibition of the free radical lipid peroxidation. A decline of the LTP is accompanied by normalising of the lipid peroxidation level. The latter's dynamics seems to express the adaptive character of the LTP development in the rat brain slices. PMID- 7534567 TI - [The interaction of vestibular inputs with differing orientations of the otoliths in a gravitational field]. AB - In alert pigeons Columba livia effects of static tilts about fore-aft and binaural axes (angles of tilts: 30-40 deg) on neck and ocular nystagmus elicited by monaural galvanic stimulation of the lateral semicircular canals with sinusoidal currents (peak amplitudes: 20-100 microA; period: 35 sec) were investigated. It was shown that the same angles of the tilts elicited different changes of horizontal nystagmus parameters in different animals. In some of them changes of quantitative characteristics of opposite-directed nystagmus differed with signs. Data support the correctness of recently (Stolbkov, 1989) proposed scheme of forming vestibulomotor reactions, according to which signals from heterolateral vestibular nuclei, during nystagmus, which are integrated, contain both canal and otolith components independent of stimulus mode addressed to canals or otoliths (separately or jointly). Results suggest also that caloric test may be a source of incorrect conclusions concerning state of vestibular system during static tilts the responses of functionally asymmetric semicircular canals may be symmetric ones. Moreover data suggest that sensitivity of convergent vestibular neurons to the canal signals is different depending on separated or combined canal and otolith stimulations. PMID- 7534568 TI - [Linear, circadian and age-related differences in the burrowing reflex in mice and its alteration by ethanol]. AB - The hole reflex (darkness preference) was stable in the C57BL/6 and CC57BR strains by day and in the evening, whereas its was only stable in the evening in the SHR. Intensity of locomotion was the highest in the evening in the C57BL/6 strain. The transition rate between dark and light chambers was the same by day and in the evening in all the strains. The C57BL/6 mice were much more resistant against ethanol-induced alterations of the hole reflex than the SHR. PMID- 7534570 TI - [Circadian fluctuations in the indices of the cardiac intervalogram and their relation to the characteristics of circadian mobility in rats]. AB - Mathematical analysis of the heart rate variability enables one to determine the activity of the sympathetic nervous system at different time of day in rats. A correlation was found between the cardiac intervalogram parameters and the dynamics of rats' circadian motility. Intensification of locomotor activity by day was accompanied by an increase in the sympathetic tone. PMID- 7534569 TI - [The effect of isolation from the mother in the neonatal period on general development, brain formation and behavior]. AB - 24-hr isolation of newborn rats from mother decreases their body weight and growth of the brain mass. The fiber cerebral structures are delayed in their development in deprived newborn rats. Thyroid gland also becomes altered resulting in its hypofunction. The data obtained suggest that the neonatal isolation is a strong stress delaying rather considerably development of a number of cerebral structures, leading to endocrinal disfunction and altering the behaviour. PMID- 7534571 TI - [The transplantation to adult animals of fetal small intestine]. AB - Use of the ectopic growth (culture) of foetal small intestine in vivo as a model for study of normal and pathological development of an organ or its different structures was shown to be possible. Complete structural and functional development of foetal small intestine was reached under certain conditions of its implantation. PMID- 7534572 TI - [The neurophysiological effects of the neuropeptide galanin]. PMID- 7534573 TI - [The thermoregulatory activity of the skeletal muscle motor units in the cat under conditions of sympathetic denervation]. AB - Thermoregulatory activity of motor units was investigated in the cat mm sartorius and semitendinosus on the 7th and 14th days after lumbar sympathectomy. The EMG pattern did not change after gangliotomy but a statistically significant prolongation of the interspike intervals occurred on the 7th day indicating a growing instability of the motoneuronal pool activity. These changes tended to restore on the 14th day. Processes affecting the thermoregulatory activity of the motor units after sympathetic denervation, are discussed. PMID- 7534574 TI - [The noncontractile energy expenditure by an isolated contracting rat heart preparation]. PMID- 7534575 TI - [HCG and its subunits: clinical value]. PMID- 7534576 TI - Expression of keratins K6 and K16 in regenerating mouse epidermis is less restricted by cell replication than the expression of K1 and K10. AB - Biosynthesis of the hyperproliferation-associated keratins K6 and K16 was studied in mouse epidermis following a single topical application of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An epidermal cell cohort was followed by pulse-labelling with the thymidine analogue 5-bromodeoxyuridine (BrdUrd). Mice were injected intraperitoneally with BrdUrd 1 h before a single topical application of TPA. TPA induced regenerative epidermal hyperplasia with hyperproliferation and shortened suprabasal transit time. The BrdUrd-labelled cell cohort was followed for 96 h after TPA-treatment by two-colour immunofluorescence staining with antibodies to BrdUrd, keratin K6 and K16. In control animals, K6 and K16 were found only in the hair follicles. K6 expression was immediately induced in all epidermal cell layers of TPA-treated epidermis, including actively replicating cells and it was expressed during the whole observation period. K16 was only present in post-mitotic cells and was transiently expressed 8-72 h after TPA treatment. Our results suggest that the expression of K6 and K16 is less restricted by cellular replication than the normally occurring K1 and K10 keratins. PMID- 7534577 TI - Radioactive in situ hybridization to replication-banded chromosomes. PMID- 7534579 TI - In situ hybridization to polytene chromosomes of Drosophila melanogaster and other dipteran species. PMID- 7534578 TI - Gene mapping using 3H-labeled heterologous probes. PMID- 7534580 TI - Reverse chromosome painting. PMID- 7534582 TI - Sensitive immunocytochemical detection of viral genomes. PMID- 7534581 TI - Detection of virus nucleic acids by radioactive and nonisotopic in situ hybridization. PMID- 7534583 TI - Primed in situ (PRINS) labeling of RNA. PMID- 7534584 TI - Subcellular location of mRNA by electron microscope hybridization histochemistry. PMID- 7534585 TI - Histamine release from cord blood basophils is influenced by plasma IgE concentration, osmolarity, gestational age at birth and atopic disposition. AB - To characterize the normal values of histamine release from basophil leucocytes in cord blood after stimulation with both IgE-mediating and non-IgE-mediating secretagogues, a population of 1,684 newborn infants was studied using a microfiber-based method for detecting histamine release. A wide variation in anti IgE and Concanavalin A-induced histamine release in the population was found. An increase in osmolarity in the release media selectively enhanced the maximal IgE mediated histamine release, in addition to an increase in sensitivity in the dose response for anti-IgE and Concanavalin A. Both anti-IgE and Concanavalin A mediated histamine release were found to be significantly associated with the concentration of IgE in cord blood. Furthermore, an increased sensitivity to the IgE-mediated response and a close correlation between anti-IgE and Concanavalin A histamine release were found in the group of newborns with high IgE levels. Gestational age at birth and histamine release after stimulation with both IgE mediating, and non-IgE mediating secretagogues were found to be significantly related, thus suggesting a continuing maturation of the human basophil leucocytes in the last weeks of gestation. Parental atopic disposition also affected basophil releasability in cord blood. PMID- 7534586 TI - Histamine blockade after astemizole in children: a single-dose, placebo controlled study. PMID- 7534587 TI - High levels of IgA in HIV-1-perinatally-infected children. Antigen specificity and possible role of increased substance P plasma levels. AB - The specificity of IgA against food, inhalant, bacterial and fungine antigens as well as for HIV-1 proteins was investigated in 14 HIV-1-infected children (CDC stage P-2) and 15 controls. IgA against food- and inhalant antigens as well as against tetanus toxoid were significantly more often present in the HIV positive children than in controls. No difference between the two groups was present for IgA against Candida albicans. A significant increase of substance P, a strong IgA synthesis inducing neuropeptide, was demonstrated in the plasma of HIV-1 infected children. In conclusion, high levels of IgA seem to reflect a complex immune dysfunction in which many factors are involved. The importance of neuroimmune dysregulation is discussed. PMID- 7534588 TI - The Lactococcus lactis triosephosphate isomerase gene, tpi, is monocistronic. AB - Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a lambda GEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit M(r) of 26,802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5' end of the transcript was determined by primer extension analysis to be a G located 65 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DL11 chromosome map was determined to be between map coordinates 1.818 and 1.978. PMID- 7534589 TI - In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. AB - Bacteria of the family Legionellaceae form a monophyletic group within the gamma subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae. PMID- 7534590 TI - Carboxymethyl benzylamide dextrans inhibit breast cell growth. PMID- 7534591 TI - A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig. AB - Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and beta fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor beta 1 (TGF beta 1) at concentrations > or = 1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [> 100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis. PMID- 7534592 TI - Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells. AB - The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species. PMID- 7534594 TI - Acute pancreatitis associated with dual vigabatrin and lamotrigine therapy. PMID- 7534593 TI - Serum-free primary culture of normal mouse mammary epithelial and stromal cells. AB - An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation. PMID- 7534596 TI - Nuclear medicine. PMID- 7534595 TI - [Assessment of blood lymphocyte subpopulations by the immunoenzyme cytochemical PAP method using different fixatives]. AB - The method of fixation of cytologic material was modified for a relatively safe immunocytochemical staining with para-aminophenasine (PAPh) in order to provide the intactness of the principal differentiation antigens of lymphocytic surface membranes of human peripheral blood. The method is recommended for practical studies of immunophenotypic lymphocyte subpopulations in HIV-infected subjects and patients with AIDS and other infectious diseases. PMID- 7534598 TI - Axonal ion channel dysfunction in amyotrophic lateral sclerosis. AB - In amyotrophic lateral sclerosis (ALS) it is not known how or why the motor neurons die, but a clue is provided by observations that the dying cells discharge spontaneously, producing muscle fasciculations. The fasciculations can arise either proximally or distally in the motor unit, suggesting a widespread disturbance of membrane excitability. To test for this, we applied the technique of threshold electrotonus to ulnar motor axons at the wrist, comparing the responses to 100 ms polarizing currents in 11 ALS patients with those from 15 normal controls, six patients with benign fasciculations, 19 with lower motor neuron disorders and six with upper motor neuron disorders. We found that the motor axons of ALS patients, unlike those in the neurological control groups, responded abnormally to subthreshold depolarizing currents, becoming either more (seven cases) or much less excitable (four cases) than normal. Both types of abnormality could be reproduced in rat nerves in vitro, and in a computer model of human motor axons, by reducing voltage dependent potassium conductances. When sufficient potassium channels were blocked, the model axon became unstable and depolarized regeneratively, resulting in an abrupt fall in excitability. We conclude that the fasciculations in ALS are caused by an imbalance between functional sodium and potassium channels, and we propose that this ion channel dysfunction could also be responsible for the motor neuron degeneration in this disease. PMID- 7534597 TI - Biochemical markers of basement membrane disturbances and occupational exposure to hydrocarbons and mixed solvents. AB - To investigate possible mechanisms of hydrocarbon or solvent-induced renal damage, we studied three groups of healthy men employed in a UK manufacturing plant. Group 1 (n = 111) were occupationally exposed to hydrocarbon-based paints, Group 2 (n = 100) were occupationally exposed to petroleum-based mineral oils, and Group 3 (n = 92) had low background occupational exposure to hydrocarbons. Occupational atmospheric exposure levels for toluene, xylene, butanol and oil mist around the time of this study were within UK permissible limits. Group 4 (controls) were males with no known occupational hydrocarbon or solvent exposure (n = 108). Circulating laminin antibodies and the auto-antibody implicated in Goodpasture's syndrome (anti-GBM) were measured, as were serum laminin, a basement membrane turnover marker, and soluble E-selectin, an endothelial activation marker. Group 1 had a significantly greater proportion of subjects with high levels of both anti-laminin antibodies and soluble E-selectin; Group 2 had significantly more subjects with raised anti-GBM antibodies, laminin and soluble E-selectin. Mean levels of soluble E-selectin were increased in Groups 1 and 2. In a small but significant proportion of these workers exposed to hydrocarbons/mixed solvents there are alterations both to basement membranes, resulting in auto-antibody production, and to overlying vascular endothelial cells. PMID- 7534599 TI - Acidic fibroblast growth factor-like immunoreactivity in rat brain following cerebral infarction. AB - Expression of acidic fibroblast growth factor (aFGF) was studied by immunocytochemistry in the rat brain with or without cerebral infarction. In a model of infarction produced by occlusion of the middle cerebral artery, aFGF positive staining that was not seen in controls appeared in both neurons and macrophages. Positive neurons were distributed mainly in the periventricular region of the hypothalamus. The neuronal aFGF began to be detectable about 24 h after the occlusion. The staining intensity for such neuronal aFGF increased until about 14 days after the surgery, but decreased gradually and was undetectable after more than 30 days. Positive macrophages first appeared on the 3rd day after the occlusion and were persistently seen up to the 7th day, but were no longer visible after 14 days. The present results suggest that aFGF may be involved in the repair processes following brain infarction. PMID- 7534600 TI - Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids. AB - The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that 'illness'-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found. PMID- 7534601 TI - Inhibition of nitric oxide synthesis inhibits rat sleep. AB - Previous findings indicate that nitric oxide (NO) may play a role in the regulation of sleep-wake activity. In rabbits, blocking the production of endogenous NO by a nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L NAME) suppresses spontaneous sleep and interferes the somnogenic actions of interleukin 1. In the present experiments we extended our earlier work by studying the long-term effects of L-NAME treatment on sleep-wake activity including power spectra analyses of the electroencephalogram (EEG) in rats. Rats implanted with EEG electrodes, brain thermistor, and intracerebroventricular (i.c.v.) guide cannula were injected i.c.v. with vehicle or 0.2, 1, or 5 mg L NAME at light onset. In separate experiments, rats were injected intraperitoneally (i.p.) with L-NAME three times (50, 50, 100 mg/kg), 12-12 h apart. Both i.c.v. and i.p. injections of L-NAME elicited decreases in time spent in NREMS and REMS. After i.c.v. injection of 5 mg L-NAME the sleep responses were long-lasting; NREMS did not return to baseline even 72 h after injection. EEG delta-wave activity during NREMS (slow wave activity) was also suppressed after 0.2 and 5 mg L-NAME. Brain temperature was slightly increased after the two lower doses of L-NAME, whereas there was a transient decrease in Tbr after 5 mg L-NAME. Acute i.p. injection of 50 mg/kg L-NAME elicited an immediate decrease in NREMS which lasted for approximately 2 h. The second injection of 50 mg/kg L-NAME and the following injection of 100 mg/kg L-NAME induced biphasic decreases in NREMS but not REMS. PMID- 7534602 TI - Regional variations in the pharmacology of AMPA receptors as revealed by receptor autoradiography. AB - Using quantitative autoradiography, we examined the ability of NBQX (2,3-dihydro 6-nitro-7-sulfamoyl-benzo(f)-quinoxaline), S-AMPA (alpha-amino-3-hydroxy-5 methylisoxazole propionic acid), L-glutamate and NS-257 (1,2,3,6,7,8-hexahydro-3 (hydroxyimino)- N,N,7-trimethyl-2-oxobenzo[2,1-b:3,4-c']dipyrrole-5-sulfonam ide) to compete for [3H]AMPA binding sites in several forebrain regions and cerebellar cortex. NBQX had a higher affinity (P < 0.0001) in cerebellar molecular layer than in any forebrain region, whereas the opposite was true for AMPA (P < 0.001). L-Glutamate and NS-257 had different regional patterns of displacement. Consequently, cerebellum and forebrain have distinct rank orders of potency for AMPA receptor ligands. These results suggest that there are regional variations in the pharmacological specificity of AMPA receptors. PMID- 7534603 TI - Release of [3H]GABA evoked by glutamate receptor agonists in cultured chick retina cells: effect of Ca2+. AB - The effect of glutamate receptor agonists (NMDA, kainate, quisqualate and AMPA) on the [Ca2+]i and on [3H]GABA release was studied in cultured chick embryonic retinal cells. The release of [3H]GABA evoked by NMDA, in the absence of Ca2+, was prevented by the NMDA receptor antagonist (+)-methyl-10,11-dihydro-5H dibenzo[a,d]-cyclohepten-5,10-imine (MK-801), and that produced by kainate and quisqualate was prevented by 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX). All the agonists tested increased the [Ca2+]i, and when the GABA transporter was blocked by 1-(2-(((diphenyl-methylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridine-carboxylic acid (NNC-711), NMDA, AMPA or quisqualate, but not kainate, did not induce [3H]GABA release unless Ca2+ (1 mM) was present, showing that exocytotic release of [3H]GABA occurs in retinal cells under these conditions. PMID- 7534605 TI - Histochemical demonstration of membranous localization of endothelial nitric oxide synthase in endothelial cells of the rat brain. AB - We examined cellular and subcellular localization of endothelial nitric oxide synthase by using immunoelectron microscopy. The results showed that immunoreaction products were associated with membranous structure, likely the endoplasmic reticulum and cytoplasmic vesicles. These membranous localization of immunoreactivities at electron microscopic level corresponded to the perinuclear granular structures observed by light microscopic immunohistochemistry for endothelial nitric oxide synthase as well as NADPH diaphorase histochemistry. PMID- 7534604 TI - Causes of calcium accumulation in rat cortical brain slices during hypoxia and ischemia: role of ion channels and membrane damage. AB - To better understand why neurons accumulate calcium during cerebral ischemia, the influence of specific ion channel inhibitors on the rise in cytosolic free calcium ([Ca2+]c) during hypoxia or ischemia was evaluated in rat cerebrocortical brain slices. [Ca2+]c was measured fluorometrically with the dye fura-2 during hypoxia (95% N2/5% CO2 or 100 microM NaCN), simulated ischemia (100 microM NaCN plus 3.5 mM iodoacetate), or 0.5-1.0 mM glutamate. Hypoxia or ischemia increased [Ca+2]c from 100-250 nM to 1,000-2,500 nM within 3-5 min. Greater than 85% of the calcium accumulation was influx from the extracellular medium. The non competitive N-methyl-D-aspartate (NMDA) inhibitor MK-801 reduced [Ca2+]c accumulation during hypoxia, but antagonism of alpha-amino-3-hydroxy-5-methyl-4 isoxazole (AMPA) receptors or voltage-gated sodium or calcium channels or Na+/Ca2+ exchangers had no effect. During ischemia, combined antagonism of NMDA, AMPA and voltage-gated sodium channels slowed the rate of calcium accumulation, but not concentration at 5 min. Membrane damage, as indicated by leakage of lactate dehydrogenase into superfusate, occurred coincidentally with calcium influx and ATP loss during both hypoxia and ischemia. We conclude that cytosolic calcium changes during hypoxia or ischemia in cortical brain slices are due to multiple mechanisms, are incompletely inhibited by combined ion channel blockade, and are associated with disruption of cell membrane integrity. PMID- 7534606 TI - Cellular and extracellular components at nodes of Ranvier in rat white matter. AB - Rat CNS nodes of Ranvier were investigated by electron microscopy and immunohistochemistry. Nodes along thin callosal axons possess tiny node gaps containing few or no astrocytic processes. Nodes along thick spinal axons exhibit spatious node gaps containing relatively few irregularly arranged astrocytic processes. Antibodies against HNK-1, chondroitin sulfate, tenascin or NSP-4 do not label small nodes but stain large nodes. We conclude that rat CNS fibers do not exhibit a strict relation between nodal complexity and fiber size comparable to that found in rat PNS fibers. PMID- 7534607 TI - Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (alpha 4 beta 2) transfected into mouse fibroblasts with ion channel activity. AB - (-)-Nicotine, cytisine and carbachol evoked 86Rb efflux from mouse fibroblasts stably transfected with alpha 4 beta 2 chick brain nicotinic subunits. This response to (-)-nicotine was inhibited by mecamylamine and dihydro-beta erythroidine and was mirrored by a rise in intracellular Ca2+ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant 86Rb from cells and unlike the above agonists displayed significantly different IC50 values for the displacement of [3H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes. PMID- 7534608 TI - Rehydration process from salt-loading: recovery of vasopressin and its coexisting galanin, dynorphin and tyrosine hydroxylase immunoreactivities in the supraoptic and paraventricular nuclei. AB - Salt-loading induces profound metabolic changes in magnocellular vasopressin (AVP)-containing neurons, including changes in levels of coexisting peptides and tyrosine hydroxylase (TH). Although many studies have been conducted on salt loading, little information is available on the recovery processes following its cessation. In the present study, we investigated the changes in AVP, galanin (Gal), dynorphin B (Dyn-B), and TH immunoreactivities in the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN) by immunocytochemistry using specific antisera against these substances. Salt-loading was induced in rats by dissolving 2% NaCl in their drinking water for 7 days. These animals were then allowed free access to fresh water for 2, 4, or 7 days prior to sacrifice. In the SON at the 7th day of salt-loading, AVP, Gal and Dyn-B immunoreactivities decreased in contrast to the marked increase in TH-immunoreactivity compared to those of control rats with free access to water. After a recovery period with free access to water, AVP and Gal immunoreactivities increased with time and returned to the control level at the 7th day. However, Dyn-B immunoreactivity did not recover even at the 7th day. Dehydration-induced TH-immunoreactive neurons almost disappeared at the 7th day. Immunoreactivities for these substances in the PVN showed a similar time course as that in the SON. These findings suggest that AVP and substances coexisting with it change with different time courses in magnocellular neurons following cessation of salt-loading. PMID- 7534609 TI - Invasion of the rat ventral root L5 by putative sympathetic C-fibers after neonatal sciatic nerve crush. AB - The present study examines the occurrence of C-fibers in lumbar ventral roots after sciatic nerve crush in neonatal and adult rats. Electron microscopic analysis showed that the number of C-fibers in the ventral root L5 increased significantly on the lesion side after neonatal but not adult sciatic nerve crush and that the number of C-fibers was higher in the ventral root L5 on the unoperated side compared to this root in normal control rats. In order to determine whether the new C-fibers in the L5 root on the lesion side are sensory or sympathetic we made immunohistochemical studies on roots from neonatally crushed rats. We found that there was no obvious lesion side/contralateral side or operated rat/control rat difference with respect to the occurrence and general configuration of axons with substance P-, calcitonin gene-related peptide- or vasoactive intestinal polypeptide-like immunoreactivity. However, the occurrence of axons with tyrosine hydroxylase-like immunoreactivity appeared clearly higher in the ventral root L5 on the lesion side compared to the unoperated side in neonatally crushed rats. Moreover, these axons seemed to be more numerous also in the ventral root L5 on the unoperated side compared to normal control rats. No lesion side/contralateral side or operated rat/control rat differences were seen in the ventral root L4. We propose that the ventral root L5 is invaded by putative sympathetic C-fibers after sciatic nerve crush lesions in newborn rats. PMID- 7534610 TI - Effects of NMDA receptor blockade and nitric oxide synthase inhibition on the acute and chronic actions of delta 9-tetrahydrocannabinol in mice. AB - The present studies examined the hypothesis that the N-methyl-D-aspartate (NMDA) receptor-nitric oxide (NO) pathway might be involved in the acute and chronic actions of delta 9-tetrahydrocannabinol (THC). The ability of dizocilpine (MK 801), a competitive NMDA receptor antagonist and NG-monomethyl-L-arginine (L NMMA), an inhibitor of NO synthase enzyme to modify the analgesic and hypothermic responses following the acute and chronic treatment of animals with THC was determined in male Swiss-Webster mice. Intraperitoneal administration of THC (5, 10 and 20 mg/kg) produced dose-dependent analgesic and hypothermic effects. MK 801 at 0.1 mg/kg i.p. attenuated the analgesic but not the hypothermic responses to THC (10 and 20 mg/kg, i.p.). The effects of various doses of MK-801 (0.03, 0.1 and 0.3 mg/kg, i.p.) on the analgesic and hypothermic responses to a 10 mg/kg, i.p. dose of THC was also determined. All the doses of MK-801 antagonized the analgesic but not the hypothermic effects of THC. The chronic treatment of animals with THC (10 mg/kg, i.p.) twice daily for 4 days produced tolerance to its analgesic and hypothermic effects. Pretreatment of animals with MK-801 (0.03 0.30 mg/kg, i.p.) did not affect the development of tolerance to the analgesic or the hypothermic action of THC. The pretreatment of animals with L-NMMA (2-8 mg/kg, i.p.), did not alter the analgesic or hypothermic effects of THC. Also, it did not modify the tolerance to its pharmacological actions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534611 TI - Amino acids, serotonin, and 5-hydroxyindoleacetic acid following foot shock in rats. AB - Concentrations of tryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5 HIAA) in brain and plasma, as well as plasma amino acid composition, were measured after 1-h foot shock. Stress induced a rise in both plasma and brain 5 HIAA, whereas 5-HT concentration was found to be increased only in plasma. A prominent rise in brain tryptophan was observed, whereas in plasma, foot shock caused a significant increase only in tryptophan level. Concentrations of other amino acids were found to be either decreased or unchanged. Ratio of tryptophan to the other long-chain neutral amino acids increased significantly following foot shock. It is possible that stress-related changes in 5-HT turnover are due to increased plasma tryptophan, in turn causing a rise in brain tryprophan, necessary to cope with enhanced 5-HT metabolism, reflected as a rise in 5-HIAA levels. PMID- 7534612 TI - Time course effects of MK-801: the relationship between brain neurochemistry and behavior. AB - Separate groups of rats were given saline or MK-801 treatments (0.3 mg/kg) and tested for locomotion activity levels for 10 min at 30, 60, and 120 min postinjection. At each postinjection time interval the MK-801 rats exhibited a marked hyperactivity that was unchanged across the three postinjection intervals. Ex vivo biochemical assays were performed to assess the neurochemical effects of MK-801 at each injection interval. In the striatum, a marked increase in dopamine metabolism was observed in the 120 injection group, but, otherwise, no other changes in striatum were detected. In contrast, a significant increase in dopamine metabolism was observed after 30 min in the medial prefrontal cortex, and this effect persisted across all postinjection intervals. At 120 min, however, the biochemical impact of the MK-801 treatment on medial prefrontal cortex broadened to include a decrease in purine metabolism and norepinephrine. Serotonin metabolism was unaffected in striatum or medial prefrontal cortex across all injection intervals, and there was no effect of MK-801 on plasma corticosterone levels. PMID- 7534613 TI - The psychiatrist in the nursing home, Part I: Collaborative roles. PMID- 7534614 TI - Converting laserdisc video to digital video: a demonstration project using brain animations. AB - Interactive laserdiscs are of limited value in large group learning situations due to the expense of establishing multiple workstations. The authors implemented an alternative to laserdisc video by using indexed digital video combined with an expert system. High-quality video was captured from a laserdisc player and combined with waveform audio into an audio-video-interleave (AVI) file format in the Microsoft Video-for-Windows environment (Microsoft Corp., Seattle, WA). With the use of an expert system, a knowledge-based computer program provided random access to these indexed AVI files. The program can be played on any multimedia computer without the need for laserdiscs. This system offers a high level of interactive video without the overhead and cost of a laserdisc player. PMID- 7534615 TI - The ups and downs of T cell costimulation. PMID- 7534616 TI - Efficiency of MHC class I antigen processing: a quantitative analysis. AB - Listeria monocytogenes is an intracellular pathogen that secretes proteins into host cell cytosol. One such protein, the murein hydrolase p60, is processed by the host cell into the nonamer peptide p60 217-225 and presented to cytotoxic T lymphocytes by the H-2Kd MHC class I molecule. Using strains of L. monocytogenes that secrete different amounts of p60, we show that the rate of p60 217-225 production is proportional to the quantity of intracellular antigen. The appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. By accounting for the rate of intracellular antigen secretion and degradation, we estimate that approximately 35 p60 molecules are degraded to produce one p60 217 225 epitope. These findings provide an estimate of the efficiency of antigen processing and shed light on the capacity of the MHC class I antigen processing pathway to accommodate foreign antigens. PMID- 7534617 TI - Absence of B7-dependent responses in CD28-deficient mice. AB - Costimulation of T cell proliferation can occur through the CD28 signal transduction pathway. In addition, other cell surface receptors, including the CD28 homolog CTLA-4, have been proposed to be capable of providing costimulatory signals. We have examined the response of CD28-deficient T cells to activation by a variety of agonists. We demonstrate that proliferation of CD28-deficient T cells in the presence of antigen-presenting cells or B7-1 transfectants is markedly reduced. Although CTLA-4 can be expressed on CD28-deficient T cells, we observed no B7-dependent costimulation in the absence of CD28. This data demonstrates that CD28 is the major B7-binding costimulatory ligand on T cells. Furthermore, our data suggest that CD28 is the primary, and perhaps exclusive, costimulatory receptor used by traditional antigen-presenting cells to augment the proliferation of antigen-activated T cells. PMID- 7534618 TI - CD14 is a pattern recognition receptor. AB - Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics. PMID- 7534619 TI - Interactions between stem cell factor and c-Kit are required for intestinal immune system homeostasis. AB - Interactions between stem cell factor (SCF) and its receptor, c-Kit, are important for development of hematopoietic, melanocytes, and germ cells. T lymphocytes appeared normal in c-Kit (W/Wv) or SCF (SI/SId) mutant mice, except for those residing within the intestinal epithelium, the intraepithelial lymphocytes (IEL). Normally, IEL are composed of equal numbers of cells with alpha beta or gamma delta T cell receptors. In mutant mice, beginning at 6-8 weeks of age, the number of gamma delta IEL decreased, whereas alpha beta IEL increased. The latter was due largely to an increased CD4+ CD8+ TCR alpha beta subset, suggesting that these cells may be intermediates in the alpha beta IEL lineage. c-Kit or SCF was expressed by IEL or intestinal epithelial cells, respectively, indicating a potential for direct intercellular interaction. This possibility was supported by reconstitution studies that demonstrated that c-Kit mutations directly affected IEL. Thus, SCF-c-Kit interactions are important for homeostasis of the intestinal immune compartment. PMID- 7534620 TI - Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. AB - B7-0 or B7-2 (CD86) is a T cell costimulatory molecule that binds the same receptors (CD28 and CTLA-4) as B7-1 (CD80), but shares with it only approximately 25% sequence identity and is expressed earlier during an immune response. Here we show that human CD86 maintains similar (within approximately 2- to 3-fold) overall receptor binding and T cell costimulatory properties as CD80. However, CD80 and CD86 did not bind equivalently to CTLA-4: CD80 bound Y100A, a form of CTLA4lg with a mutation in the CDR3-like region, > 200-fold better than did CD86; inhibition of CD80-mediated cellular responses required approximately 100-fold lower CTLA4lg concentrations; and CD80-CTLA4lg complexes dissociated 5- to 8-fold more slowly, Thus, CD80 and CD86 utilize different binding determinants and have different kinetics of binding to CD28 and CTLA-4. PMID- 7534621 TI - Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro. AB - The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations. PMID- 7534622 TI - MSAFP levels in aboriginal Canadian women. AB - Maternal serum alpha-fetoprotein (MSAFP) levels in 529 non-diabetic aboriginal Canadian women were compared with levels in 13,285 non-diabetic non-aboriginal women. A woman was considered to be aboriginal if she was listed on the Indian Register of Canada. After controlling for the gestational age on the date at which the sample was drawn and maternal weight, MSAFP levels appeared to be approximately 4 to 5% higher in aboriginal women. The possible implications of this finding on an MSAFP screening program are discussed. PMID- 7534623 TI - Biliary tract cancer. AB - Biliary tract cancer is a rare cancer in most parts of the world, but it is relatively common in some countries and ethnic groups, such as Japan, Central and South America, eastern Europe and in American Indians, and Hispanics. In some countries, such as Japan, Sweden, Finland and Italy, the age adjusted mortality of biliary tract cancer has been increasing, whereas in females in some other countries--Israel, The Netherlands, USA, Canada, Federal Republic of Germany, etc -mortality has been declining. The reasons for these geographical or ethnic variations and time trends for biliary tract cancer are not clear, but some unknown environmental risk factors or a genetic susceptibility are suspected. Not many analytical epidemiological studies on biliary tract cancer have been conducted yet and little is known about its aetiology apart from a close association with gall stones and a female preponderance of gall bladder cancer. Besides gall stones, some other factors such as obesity, pregnancy, female sex hormones, exposure in rubber and some other chemical industries and genetic factors have been suspected of being associated with gall bladder cancer directly or indirectly through cholelithiasis. As an artificial factor, the effect of the prevalence of cholecystectomy on biliary tract cancer must be considered. More studies are needed in the future to elucidate the aetiology of biliary tract cancer and to establish measures to prevent this cancer. PMID- 7534624 TI - Laryngeal cancer. AB - By and large, the incidence of laryngeal cancer is tending to increase over time in much of the world, often in parallel with changes in tobacco and/or alcohol consumption. There are encouraging examples of declining trends that can be attributed to decreasing use of either tobacco or alcohol. These observations are in line with what is known about the multiplicative effects of both exposures. Since it is now well known that the epidemiology of cancer of the epilarynx differs from that of the endolarynx, it is highly desirable that in the future, this should be better reflected in the way laryngeal tumours are classified. There is good reason to believe that a change in alcohol consumption would have an effect on neoplasms of the epilarynx, whereas a change in tobacco consumption would affect incidence of neoplasms of the lower larynx. To examine this hypothesis, combined analyses of both lung and laryngeal cancer trends may have to be carried out together and compared, as in a recent Italian study (Capocaccia et al, in press). Research along these lines should provide a better understanding of how time trends in laryngeal cancer incidence can be related to changes in either tobacco and/or alcohol consumption. We know enough to believe that continuous efforts to reduce the consumption of either factor would result in a decrease in laryngeal cancer and that the biggest effect could be expected from a simultaneous reduction in alcohol and tobacco consumption (Tuyns, 1991). PMID- 7534625 TI - Lung cancer. AB - During the 20th century, a dramatic epidemic of lung cancer deaths has occurred in many countries. The time trends of lung cancer incidence and mortality primarily reflect patterns of cigarette smoking, the predominant risk factor, but inconsistencies are evident in international comparisons. Although the predominant role of tobacco smoking as a cause of lung cancer throughout the world is amply documented, the persistence of tobacco smoking and the continued penetration of manufactured cigarettes into new markets provide a strong reason for continuing to monitor lung cancer trends. Changes in other risk factors for lung cancer and changes in diagnostic and death certificate reporting practices may also affect trends in rates. However, our understanding of the relationship between smoking and lung cancer allows forecasting of lung cancer rates for public health planning. PMID- 7534626 TI - Cancers of the soft tissues. AB - Soft tissue sarcomas are rare and occur more frequently among males (0.9-4.3 per 100,000 age adjusted to the world standard population) than females (0.7-2.6 per 100,000). The international variation in incidence is small, and little change has occurred over time. Mortality rates were half or less than half the incidence and increased slightly in some areas, which is easily explained by changes in classification, coding and diagnosis over time, as well as instability of rates due to small numbers. Survival, in particular comparing data from the 1950s and 1960s with those from the late 1980s, has improved slightly. However, these observations must be interpreted with caution inasmuch as according to the ICD, STS are classified both with specified organs and with the connective tissue. In view of the Danish experience, this fact alone may account for recorded differences in incidence (and thus mortality) of 1 and 4 cases per 100,000 per year. No major risk factor has been clearly identified for soft tissue sarcoma, although genetic traits and certain environmental agents such as ionizing radiation and agricultural chemicals seem to be involved in some cases. The recent rise in AIDS related Kaposi's sarcoma indicates the importance of further studies into the role of infectious agents and immunological mechanisms. Future trends in soft tissue sarcoma should be studied after separating these tumours into Kaposi's sarcoma and other relevant aetiological subgroups. PMID- 7534627 TI - Cutaneous melanoma. AB - Between the early 1960s and the late 1980s, the incidence of melanoma increased at a rate of 3-7% per year in populations of mainly European origin. Corresponding trends were observed in mortality. Higher rates of increase in incidence were observed in a few populations (eg 8.9% per year in Hawaii whites). With the exception of Japan and possibly Puerto Rico, incidence rates of melanoma have remained stable in the few populations of mainly non-European origin for which reliable incidence data were available. A comparison of age specific trends in incidence and mortality in populations of mainly European origin showed two general patterns: a continuous increase in incidence in all age groups but with moderation or cessation of the previous rising trend in mortality in younger people in more recent time periods (eg Canada, continental USA, Denmark and the UK) and recent moderation or cessation of both incidence and mortality trends in younger people (eg New Zealand and, possibly, Hawaii whites). The first of these two patterns appeared to be the most common. Studies of site specific trends in incidence in 13 populations indicate that the highest rates of increase have generally been for melanomas on the trunk and the lowest for those on the head and neck. There is weak evidence to suggest that the rate of increase on the lower limbs has been greater in women than in men. Studies of incidence trends in the 1980s by thickness of melanoma in seven populations show that relative and absolute incidence has increased most for the thinnest melanomas and least for the thickest lesions. Increasing detection, earlier diagnosis and a real rise may together explain the increase in incidence of melanoma. The increases in mortality suggest that incidence has really increased, and the recent moderation in mortality trends may be explained by improved survival from melanoma due, most likely, to increasingly early diagnosis. In some populations, it may also indicate that the incidence increases are coming to an end. The disproportionately increasing incidence of thin melanoma, the divergence between incidence and mortality trends and the recent sharp increases in incidence in some populations suggest that earlier diagnosis or greater detection of less aggressive melanomas may have contributed to the incidence trends. A progressive change from predominantly occupational to predominantly recreational patterns of sun exposure is the most likely cause of increasing real incidence of melanoma in populations of mainly European origin. PMID- 7534628 TI - Oral and pharyngeal cancers. AB - Trends for oral and pharyngeal cancers vary by anatomical subsite. For tumours of the tongue, mouth and pharynx (excluding nasopharynx), rates are rising in many areas of the world, particularly in Europe, at least in part attributable to rising alcohol consumption. Exceptions to this pattern occur in the USA, where incidence and mortality among whites are falling. There are mild downward trends in the incidence of lip, salivary gland and nasopharyngeal cancers, which (except for nasopharyngeal cancer among southern Chinese) occur at much lower frequency. The trends provide interesting clues to the causes of these cancers whose aetiology is partially but not fully understood. PMID- 7534629 TI - Breast cancer. AB - There is a four to fivefold variation in breast cancer incidence rates across different countries. The lowest rates are observed in Asia, and the highest rates are observed in western Europe and North America. The incidence of breast cancer has increased in all countries since 1960. We studied in detail (whenever possible) the changes in incidence and mortality between 1955 and 1990 in four age groups (35-44, 45-54, 55-64 and 65-74) for 11 "representative" countries (USA, England and Wales, Norway, Hungary, Yugoslavia, Spain, Colombia, Singapore, Japan, India and China). The largest increase in incidence took place in Japan and Singapore. The incidence rate for women aged 35-44 in Japan doubled between 1960 and 1985 and by 1985 was roughly two thirds the USA rate. There has been essentially no change in mortality rates in the USA, England and Wales or Norway, whereas there has been a 50-60% increase in Japan, Singapore and Hungary. Most of the observed increase in incidence rates in the USA, England and Wales and Norway may be "artefactual", that is, due to changes in screening patterns. Screening may also have contributed to the rate increase in other countries, but outside western Europe and North America the major part of the increase is likely to be due to changes in known and suspected breast cancer risk factors. PMID- 7534630 TI - Cervical cancer. AB - The overall incidence of and mortality from cervical cancer has declined in western countries and in most developing countries. In women under 40 years of age, however, mortality rates are levelling off or increasing in most countries. The earliest and most marked increases in young women occurred in England and Wales, Scotland, Ireland, New Zealand and Australia. Mortality rates in young women from eastern European countries began to increase later than in the UK, but the increases are of concern because baseline mortality rates are high in these countries. The reasons for the overall decline in cervical cancer are largely unknown but appear to be linked to improvements in the general standard of living. The increases in young women may well be due to the increasing prevalence of HPV infection. Screening for cervical cancer has undoubtedly led to a decline in cervical cancer incidence and mortality in many countries, but its contribution to the trends is difficult to assess without further information. PMID- 7534631 TI - Testicular cancer. AB - Testicular cancer is a disease that predominantly affects young and middle aged men. Our data show that incidence rates have recently increased in men aged 15-54 years in all 13 populations examined, irrespective of whether the populations were at high, moderate or low underlying risk. The annual percentage increase in this age group between 1970 and 1985 varied from 1.9% in the West Midlands, UK, to 6.6% in Miyagi, Japan, with a median of 2.7%. Analysis of the data in two separate age bands, 15-34 and 35-54 years, shows that increases are occurring in both subgroups. This, together with analyses by histological category in Denmark and the West Midlands, UK, indicates that both teratomas and seminomas are increasing in incidence. In contrast to the pattern for incidence rates, testicular cancer mortality rates are now declining in all the nine national populations examined. The time from which mortality rates started to decline varies between populations, and in Poland, a reduction was not observed until the 1980-1985 period. This reflects delay in the uptake of effective chemotherapy for the treatment of teratomas. The decline in mortality, against a background of rapidly increasing incidence in most populations, emphasizes the appreciable improvements in prognosis associated with testicular cancer in recent decades. Although the epidemiology of testicular cancer strongly suggests the presence of environmental risk factors that may be controllable, our ignorance about the nature of these factors precludes any strategy of prevention. Early diagnosis and improved treatment will therefore remain a major focus for the control of this cancer. Our ability to treat testicular cancer is thus a major and necessary achievement given the increase in incidence. PMID- 7534632 TI - Bladder and kidney cancers. AB - The major factor contributing to the increase in incidence of both renal and urothelial cancers has been improved detection rates. To a considerable extent, this accounts for the difference between the trends in incidence and mortality since, on the whole, the efficacy of treatment has improved only slowly. In the case of renal parenchymal cancer, the true burden of disease is rising almost everywhere, probably reflecting changes in standard of living, perhaps chiefly diet, although there will be some contribution from smoking. Where the increase in bladder cancer is not artefactual, it can be attributed in large part to smoking and perhaps to increasing industrial exposure in some localities. Trends in incidence and, to a greater degree, mortality for urothelial cancer are likely to improve substantially with implementation of policies of prevention (reduction in smoking, improvements in industrial hygiene), earlier detection (eg with monoclonal antibodies) and treatment (immunotherapy). No such optimism can be held with respect to renal parenchymal cancer, which is set to become one of the major cancers of affluent societies unless its aetiology becomes known and can be addressed. PMID- 7534633 TI - Brain and other nervous system tumours. AB - Interpretation of time trends in incidence rates for cancers of brain and other and unspecified parts of nervous system is complicated by variation in registration efficiency over time and place, statistical instability of rates, changes in the ICD classification of the tumours, variation in registration practice for the benign and unspecified tumours over time and changes in methods of diagnosis. The reader will undoubtedly have the impression that in this chapter there are too many differences in the content of the data over time and place to have confidence in any of the conclusions drawn. To derive a well documented and sustainable assessment on secular trends will require an international collaborative study collecting incidence data for the malignant, benign, uncertain and unspecified tumours of the brain and other parts of the nervous system by histology and by method of diagnosis. By providing a separate rubric for the meninges, the 10th Revision of the ICD (WHO, 1993) should- combined with the morphology rubrics of the second edition of the International Classification of Diseases-Oncology (Percy et al 1990)--make such an endeavour somewhat easier. The data presented above nevertheless suggest that, as for several other cancer sites, on the whole there has been a slow overall increase in the incidence of brain and other and unspecified nervous system neoplasms on the order of 1-2% per year over the past 30 years. Increases appear to have been greater in older people. The extent to which these increases are due to the unquestioned improvements in methods and precision of diagnosis is not clear. Birth cohort data suggest that in recent years, this influence may have been greatest for those birth cohorts born prior to 1900, and this effect is unlikely to disappear until cohorts born about 1910-1920 have passed on. There is currently little evidence to support the contention that the recent increase in older people cannot be largely explained by improvements in diagnostic methods, wider access to medical care and more intensive investigation. Further work is needed. PMID- 7534635 TI - Hodgkin's and non-Hodgkin's lymphomas. AB - Incidence of HD varies from about 0.5 per 100,000 person-years in parts of Asia to over 3 in parts of North America. In recent decades, many registries have reported slightly declining age adjusted incidence among men and women. Some lymphomas previously diagnosed as HD now would be classified as NHL, but this shift does not explain all of the decline. When analysed by age group, incidence has decreased substantially at older ages, whereas increases have been reported among young adults in some industrial countries. Less developed countries continue to show high rates in childhood. Hodgkin's disease of the nodular sclerosis subtype has increased over time, whereas HD of mixed cellularity has declined. Improved therapy for HD has led to sharply declining mortality rates, but further understanding of the role of EBV and other possible causal agents should afford opportunities for prevention. Non-Hodgkin's lymphoma stands out from most other malignancies because incidence and mortality rates have risen dramatically, steadily and almost universally during the past few decades. Incidence overall has been rising 3-4% per year. No sudden rise has occurred in specific birth cohorts or calendar year of diagnosis, although incidence rates have increased more steeply at older ages. Diagnosis of NHL has improved with time, perhaps beyond the ways considered herein, but has it improved so much more than diagnosis of other malignancies, and roughly simultaneously around the world? Although it appears that diagnostic improvements are partly responsible for the upward trend, it is likely that aetiological factors are playing an important part. Infections with HIV have started to inflate NHL incidence rates further but cannot account for the striking trend already under way for several decades. Clues should be vigorously pursued to determine the role of other known viruses, immunosuppressive states, herbicides and other chemicals in the environment, and commercial products such as hair dyes. To clarify reasons for the upward trends and to take preventive action will require a better understanding of the origins of the lymphomas through epidemiological research, including interdisciplinary approaches that can identify new viruses, host environmental interactions and lifestyle and other exposures that alter susceptibility. PMID- 7534634 TI - Thyroid cancer. AB - Thyroid cancer is one of the rarest forms of cancer, and yet there are wide variations in the degree of malignancy, ranging from the most rapidly fatal to the relatively benign. This difference depends almost entirely on the histological type. A "pool" of individuals with occult thyroid carcinomas (in the vast majority of the papillary type) is probably present in most populations even at a young age. Large differences in the estimated frequency of cancer at this site can therefore be caused by variation in diagnostic intensity. Data on changing trends of incidence and mortality are thus subject to reservation, depending on the degree to which they have been influenced by changing diagnostic criteria and the precision of histopathological description. Nevertheless, there is evidence that mortality is slowly falling, whereas incidence is increasing, in several countries. This chapter considers the upward temporal trends of incidence and substantially stable mortality rates for thyroid carcinoma in the past three decades and attempts to interpret these trends in the light of concurrent changes in diagnostic standards and histological classification. Attention will also be drawn to the public health implications of the recent intensive detection and treatment of occult thyroid carcinomas. PMID- 7534636 TI - Oesophageal cancer. AB - The changing pattern of occurrence of oesophageal cancer is reviewed. Major features include marked decreases in rates among Chinese (in whom most oesophageal cancer is seen globally) as lifestyle changes, on migration or more recently in the People's Republic; steady increases among USA blacks; an indication that traditional extremely high rates in central Asia may be declining; a slow decline in many countries as lifestyles change, and diets presumably improve, seen in such diverse areas as Finland, India and parts of Latin America; a recent upturn in certain male populations, parallel to the increase in alcohol consumption. PMID- 7534637 TI - Multiple myeloma. AB - Trends in mortality from multiple myeloma from 1960 to 1989 are reviewed for 20 countries and examined in greater detail for England and Wales, Sweden and Japan. Percentage increases in mortality were greater in older age groups and were smaller in the most recent decade than in the previous two years. In most countries with relatively high rates, such as Sweden, the rates were stable in the last 10 years considered, whereas in countries with low rates, such as Japan, the rates continued to rise. The male:female ratio is now about 1.45 and is slightly higher in countries with high rates and may be increasing. The British Isles stand out as an area with relatively high rates that are continuing to increase steadily. PMID- 7534638 TI - Leukaemia. AB - The most striking and consistent trend in leukaemia in different countries since 1950 is the recent decline in mortality in childhood, reflecting the advent of effective methods of treatment in the 1960s. Increases at ages 75-84 in several countries since 1950 are consistent with improvements in cancer registration and in the detail of death certification. However, in the main, these have not persisted into the 1980s. Otherwise, there has been no obvious international pattern, and partly for this reason, the rates have been presented in some detail so that readers can make their own judgment. PMID- 7534639 TI - Childhood cancer. AB - Trends in incidence for the main types of childhood cancer are analysed using data from the British National Registry of Childhood Tumours; published reports from other registries are summarized. For registries included in Cancer Incidence in Five Continents, changes in rates reported in successive volumes are calculated and a number of problems in the interpretation of these results are identified. There is some limited evidence for a small increase in the incidence of ALL and more evidence for an increase in brain tumours, although the latter may be at least partly due to changes in diagnostic practice. There is limited, but good, evidence for an increase in the incidence of neuroblastoma. With two exceptions, there is no good evidence for any large increase in incidence for any form of childhood cancer in Britain or other countries over the past 20-30 years. The first exception is Kaposi's sarcoma in Uganda, the cumulative incidence in childhood increasing from fewer than 40 per million in 1968-1982 to more than 500 per million in 1989-1991. The second exception is thyroid carcinoma in Belarus, where the reported incidence probably represents about a 50-fold increase--to about 80 per million per year. This may, however, be at least partly a consequence of improved ascertainment following a screening programme. The results presented here suggest that no widespread important risk factor has been introduced over the past 30 years--unless some other similar factor has been simultaneously removed. Analyses of trends in mortality for childhood cancers are now mainly useful as a means of showing the impact of improved treatment methods. PMID- 7534640 TI - Cancer in developing countries. AB - The developing countries are represented by incidence and mortality datasets from 16 populations. Trends are studied using age specific data from six cancer registries (three in Asia, three in Latin America) and national mortality datasets from Central and South America (three), the Caribbean (two), Asia (two) and Mauritius. In Africa, three cancer registries (in Nigeria, Uganda and Zimbabwe) provide time series of 15 years or more. Systematic examination of time trends is confined to five major sites (stomach, lung, breast, cervix uteri and colon-rectum), with a comment on observed trends in the other important cancers of developing countries (mouth/pharynx, oesophagus and liver). Although uniformity is not to be expected in such diverse material, some overall patterns emerge. Stomach cancer, as in the developed world, appears to be declining in importance. Lung cancer rates are rising, although in males, the increases are most marked in the elderly, with more recent birth cohorts in several populations showing a decline in risk. Breast cancer incidence and mortality rates are rising in most populations, with changes usually more marked in younger women. Conversely, cervix cancer, at present the most common cancer of women in developing countries, shows declines in incidence and mortality in the majority of populations studied, although Africa is clearly an exception. Cancer of the large bowel is becoming more frequent, although there are exceptions (eg Bombay, Mauritius and Trinidad). In the highest risk population (Uruguay), mortality seems to have peaked around 1965 and has since declined. PMID- 7534641 TI - Gastric cancer. AB - Most countries with adequate statistical infrastructure have registered declines in gastric cancer mortality and incidence rates. Such a trend is dominated by the most frequent variant, namely the so-called intestinal type of adenocarcinoma, usually ulcerated and occupying predominantly the antrum and the antrum-corpus junction. This variant is considered the endstage of a prolonged precancerous process with gradual progression from (a) chronic active gastritis to (b) multifocal atrophic gastritis to (c) intestinal metaplasia, first resembling the phenotype of the small intestine and later that of the colon, to (d) dysplasia and (e) finally to invasive carcinoma. Major trends in dietary habits, namely lower intake of salt and increased and more frequent consumption of fresh fruits and vegetables, have been linked to the decline. In parallel with those trends, improved sanitation and more adequate housing may be responsible for the declining rates of infection with Helicobacter pylori, the major cause of chronic active gastritis. A decline in the frequency of papillary adenocarcinoma of the oxyntic mucosa, associated with the pernicious anaemia syndrome, appears to have taken place much earlier. Although the frequency of the pernicious anaemia syndrome seems to have remained at similar levels, its complications in terms of papillary adenocarcinoma have decreased in populations of northern European extraction. This may be related to time trends in dietary habits. The secular decline in diffuse carcinoma has been either of much less magnitude or non existent. Few clues are available on this tumour variant. It is somewhat predominant in women, in subjects of blood group A phenotype, and less frequent in older subjects. Cell lines derived from diffuse carcinomas lack functional calcium dependent adhesion molecules ("cadherins"). Recent increases in incidence rates have been registered for adenocarcinoma of the gastric cardia. This increase parallels that of lower oesophageal adenocarcinoma, frequently linked with Barrett's oesophagus, reflux oesophagitis, a history of duodenal ulcer and gastric hypersecretion. New developments in molecular biology are being used to study the process of gastric carcinogenesis. There is hope that specific molecular alterations may provide better understanding of the different variants of gastric carcinoma and their secular trends. PMID- 7534642 TI - Colorectal cancer. AB - International trends in the incidence, mortality and survival for colorectal cancers are heterogeneous, probably because of interpopulation diversity in trends in risk factors and in systems of medical care and screening programmes. Broadly, colorectal cancer rates correlate with the adoption of a western lifestyle, and, depending on each country's stage of transition, colorectal cancer incidence can be increasing (often rapidly), levelling or declining. In high risk countries, although decreases in incidence might be expected in the longer term in response to current dietary trends, some short term increases in incidence may result from the promotion of screening activities. The growing proportion of early stage tumours detected in screened populations will eventually be reflected in lower mortality rates. PMID- 7534643 TI - Liver cancer. AB - The trends in liver cancer incidence and mortality were investigated by gender in Japan, Norway, Singapore, Spain, Sweden, the UK and the USA. Cancer Incidence in Five Continents, Vols I-VI, were the principal source of the analysis of age specific incidence rates by sex; additional incidence information was obtained from the published data of the Connecticut Tumor Registry and the Surveillance, Epidemiology and End Results Program in the USA. Mortality rates were calculated using the World Health Organization database. The trends observed were generally weak. Increasing occurrence of primary liver cancer was noted in Japan and the Nordic countries, although the rates seemed to be stabilizing or even decreasing in the most recent data from Sweden. By contrast, decreasing trends were apparent in Spain and Singapore. Changes in the rates appeared to be more evident or somewhat stronger for males than for females. Some biological explanations were offered to account for these trends. However, the dominant determinants of the apparent changes in the incidence and mortality of primary liver cancer were felt to be related to classification and coding revisions as well as to variations over time in diagnostic and certification practices. PMID- 7534644 TI - The structure of the O-specific polysaccharide from Escherichia coli O113 lipopolysaccharide. AB - The O-specific polysaccharide from Escherichia coli O113 lipopolysaccharide was separated from the core and lipid A by mild acid hydrolysis and purified by GPC. Methylation analysis and 1H and 13C NMR spectroscopic studies of the O deacetylated polysaccharide allowed the determination of the structure of the pentasaccharide repeating unit of the polysaccharide which can be written as [equation: see text] The position of the O-acetyl groups was not determined. PMID- 7534645 TI - Neutrophil activation in paediatric extracorporeal circuits: effect of circulation and temperature variation. AB - OBJECTIVE: Upregulation of neutrophil adhesion molecules (CD11b and L-selectin) and release of a modulating cytokine (IL8) have been reported in vivo and in vitro in adult cardiopulmonary bypass. The aim of this study was to determine whether paediatric bypass preparations have similar influences and whether neutrophil-endothelium interactions are required for IL8 release. METHODS: In vitro paediatric cardiopulmonary bypass circuits (n = 15) were constructed (identical to those used clinically), as well as static loops (n = 15) using donor blood. The effects of circulation and temperature (17 degrees C, 25 degrees C, 37 degrees C) on the initiation of acute inflammation were examined. Cellular expressions of neutrophil adhesion molecules CD11b and L-selectin were assayed by immunofluorescence technique, and serum IL8, IL6, TNF-alpha, leucocyte elastase, and terminal complement complex were measured by ELISA. RESULTS: In all experiments, an immediate increase in CD11b expression occurred [median values, in relative fluorescence units: 64.9 (range 45.3-212.9) at rest; 365.2 (205 835.4) at 10 min; P < 0.001], along with a decrease in L-selectin expression [153.5 (115.5-220.7) at rest; 42 (12-134) at 10 min; P < 0.01]. Serum concentrations of the following increased gradually and were higher in circulation than in static loops: IL8 [1500 (500-2500) pg.ml-1 in circuit v 600 (180-1500) pg.ml-1 in loop, P < 0.001]; TNF-alpha P < 0.05]; and terminal complement complex [25.9 (6.8-120) v 4.7 (0-21.6) AU.ml-1, P < 0.01]. Cooling decreased and rewarming increased upregulation of CD11b and downregulation of L selectin and release of IL8. IL6 was undetectable. CONCLUSIONS: In the absence of endothelium, in vitro paediatric cardiopulmonary bypass causes profound acute inflammatory changes in donor blood with release of IL8. These changes were greater than in adult cardiopulmonary bypass. Temperature variation and circulation modulate the responses. PMID- 7534646 TI - Hyaline degeneration in heart infarction. PMID- 7534647 TI - Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog, Rana catesbeiana. AB - Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine- and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life. PMID- 7534649 TI - Cytokeratin expression in human thymus: immunohistochemical mapping. AB - Cytokeratin expression in normal postnatal human thymus was studied immunohistochemically by using monoclonal antibodies against various cytokeratin polypeptides. An attempt was made to characterize cell populations giving rise to the cornified structures of Hassal's corpuscles. Monoclonal antibody KB-37, a marker of squamous epithelium basal cells, was applied to distinguish the earliest cells capable of undergoing squamous differentiation. Parts of the subcapsular epithelium were extensively stained with this reagent. This epithelium, like the basal layer of certain squamous epithelia, exhibited a high incidence of cytokeratins 13 and 14, and pronounced expression of cytokeratin 19. Simple epithelium cytokeratins 8, 18, and 19 were present in the cortex. Scattered cells reacted with KB-37 antibody. All stellate epithelial cells in the medulla were positive for cytokeratin 19. Most of the medullar epithelial cells were positive for cytokeratins 13, 14 and 17 of complex epithelium, in contrast to the cortex, where only a few cells were positive for these cytokeratins. A significant proportion of the medullar cells was positive for KB-37 antigen. Cytokeratins 8 and 18 were expressed in single cells and in groups of cells surrounding Hassal's corpuscles. The outermost cells of these corpuscles were positive for cytokeratin 19 and KB-37. In the peripheral parts of Hassal's corpuscles, simple epithelium cytokeratins 7, 8, 18, and cytokeratins 4, 13, 14, and 17, characteristic of stratified nonkeratinizing epithelia, were coexpressed with keratinization-specific cytokeratins 10/11. The inner parts of the swirls were uniformly positive for cytokeratins 10/11. However, the expression of other cytokeratins was reduced. PMID- 7534648 TI - Chemical codes of sensory neurons innervating the guinea-pig adrenal gland. AB - Retrograde neuronal tracing in combination with double-labelling immunofluorescence was applied to distinguish the chemical coding of guinea-pig primary sensory neurons projecting to the adrenal medulla and cortex. Seven subpopulations of retrogradely traced neurons were identified in thoracic spinal ganglia T1-L1. Five subpopulations contained immunolabelling either for calcitonin gene-related peptide (CGRP) alone (I), or for CGRP, together with substance P (II), substance P/dynorphin (III), substance P/cholecystokinin (IV), and substance P/nitric oxide synthase (V), respectively. Two additional subpopulations of retrogradely traced neurons were distinct from these groups: neurofilament-immunoreactive neurons (VI), and cell bodies that were nonreactive to either of the antisera applied (VII). Nerve fibers in the adrenal medulla and cortex were equipped with the mediator combinations I, II, IV and VI. An additional meshwork of fibres solely labelled for nitric oxide synthase was visible in the medulla. Medullary as well as cortical fibres along endocrine tissue apparently lacked the chemical code V, while in the external cortex some fibre exhibited code III. Some intramedullary neuronal cell bodies revealed immunostaining for nitric oxide synthase, CGRP or substance P, providing an additional intrinsic adrenal innervation. Perikarya, immunolabelled for nitric oxide synthase, however, were too few to match with the large number of intramedullary nitric oxide synthase-immunoreactive fibres. A non-sensory participation is also supposed for the particularly dense intramedullary network of solely neurofilament-immunoreactive nerve fibres. The findings give evidence for a differential sensory innervation of the guinea-pig adrenal cortex and medulla. Specific sensory neuron subpopulations suggest that nervous control of adrenal functions is more complex than hitherto believed. PMID- 7534650 TI - Irradiation influences the expression of substance P and enkephalin in the rat larynx. AB - The effects of radiotherapy on neuropeptide expression in the rat larynx were studied. Irradiation was given for five days, 6 or 8 Gray daily. Ten days after the end of irradiation, the larynx, the laryngeal nerves and different ganglia related to the larynx were dissected out from irradiated and control animals and processed for neuropeptide immunohistochemistry. There was an increased immunolabelling for two of the neuropeptides tested, substance P and enkephalin, in the innervation of the subglottic glands and in the acetylcholinesterase positive ganglionic cells of the local ganglia. These cells were interpreted as representing postganglionic parasympathetic ganglionic cells. The changes seen in the subglottic glands were interpreted as most likely being related to the changing pattern of staining seen in the local ganglia. No changes in substance P and enkephalin expression were observed in other laryngeal structures, the nodose ganglia, superior cervical ganglia or laryngeal nerve paraganglia. Thus, in certain respects neuropeptide expression in the larynx is modulated by radiotherapy. Since neuropeptides have both neurotransmitter and/or neuromodulator effects in airway tissue and since they show effects as growth factors, the occurrence of this plasticity in neuropeptide expression should be taken into consideration in future studies examining the effects of irradiation on normal/diseased airway tissues. PMID- 7534651 TI - Localization and quantity of hyaluronan in urogenital organs of male and female rats. AB - The histochemical distribution of hyaluronan was analysed in various urogenital organs of male and female (non-pregnant and pregnant) rats by use of a hyaluronan binding protein and avidin biotin/peroxidase staining. Microwave-aided fixation was used to preserve the extracellular location of hyaluronan. The concentrations of hyaluronan in the different tissues were measured with a highly sensitive radio-assay. Hyaluronan accumulated predominantly in the connective tissue around smooth muscle fibres and in the subepithelial lamina propria. Abundant hyaluronan also occurred in perivascular and perineural connective tissue. In the female urogenital organs, hyaluronan content was high in the vagina and urinary bladder, and highest in the vagina during pregnancy. In the uterus, the surface epithelium of the endometrium stained intensely. In the ovary, the zona pellucida of the oocyte and the theca interna cell layer of the follicles and the follicular fluid of mature follicles exhibited prominent staining. The corpus luteum was devoid of hyaluronan, whereas enlarged corpora lutea of pregnancy exhibited weak, patchy staining. In male urogenital organs, staining for hyaluronan was absent from the testis and epididymis, whereas the erectile connective tissue of the penis stained intensely. The hyaluronan concentrations were high in penile tissue and urinary bladder, while testis, epididymis and the ductus deferens contained only little hyaluronan. PMID- 7534652 TI - Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide. AB - Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO4 fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate. PMID- 7534653 TI - Glycosylation patterns of membrane proteins of the jellyfish Cyanea capillata. AB - Integral and membrane-associated proteins extracted from neuron-enriched perirhopalial tissue of the jellyfish Cyanea capillata were probed with a panel of lectins that recognize sugar epitopes of varying complexity. Of the 13 lectins tested, only concanavalin A, jacalin lectin and tomato lectin stained distinct bands on Western blots, indicating the presence of repeating alpha-1,6-mannoses, terminal Gal-alpha-1,6-GalNAc and repeating beta-1,4-linked GlcNAc, respectively. In whole-mounted perirhopalial tissue, jacalin lectin stained several cell types, including neurons, muscle, cilia and mucus strands. Tomato lectin stained secretory cells intensely, and neurons in a punctate fashion. Concanavalin A stained cytoplasmic epitopes in both ecto- and endodermal cells, and ectodermal secretory cells and the mucus strands emanating from them. With the exception of tomato lectin's sugar epitope, the other sugar epitopes identified in this study are "non-complex". This study suggests that while glycosylation of integral and membrane-associated proteins occurs in Cyanea, the sugars post-translationally linked to these proteins tend to be simple. PMID- 7534654 TI - Nitric oxide nerves in the uterus are parasympathetic, sensory, and contain neuropeptides. AB - Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. The uterus contains abundant NO-synthesizing nerves which could be autonomic and/or sensory. This study was undertaken to determine: 1) the source(s) of NO-synthesizing nerves in the rat uterus and 2) what other neuropeptides or transmitter markers might coexist with NO in these nerves. Retrograde axonal tracing, utilizing Fluorogold injected into the uterine cervix, was employed for identifying sources of uterine-projecting neurons. NO synthesizing nerves were visualized by staining for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and immunostaining with an antibody against neuronal/type I NO synthase (NOS). NADPH-d-positive perikarya and terminal fibers were NOS-immunoreactive (-I). Some NOS-I/NADPH-d-positive nerves in the uterus are parasympathetic and originate from neurons in the pelvic paracervical ganglia (PG) and some are sensory and originate from neurons in thoracic, lumbar, and sacral dorsal root ganglia. No evidence for NOS-I/NADPH-d positive sympathetic nerves in the uterus was obtained. Furthermore, double immunostaining revealed that in parasympathetic neurons, NOS-I/NADPH-d-reactivity coexists with vasoactive intestinal polypeptide, neuropeptide Y, and acetylcholinesterase and in sensory nerves, NOS-I/NADPH-d-reactivity coexists with calcitonin gene-related peptide and substance P. In addition, tyrosine hydroxylase(TH)-I neurons of the PG do not contain NOS-I/NADPH-d-reactivity, but some TH-I neurons are apposed by NOS-I varicosities. These results suggest NO synthesizing nerves in the uterus are autonomic and sensory, and could play significant roles, possibly in conjunction with other putative transmitter agents, in the control of uterine myometrium and vasculature. PMID- 7534655 TI - Ultrastructural localisation of NADPH-diaphorase in the chick thymic medulla. AB - Nicotinamide-adenine-dinucleotide phosphate-diaphorase positive cells in the chick thymus were studied at the electron-microscopic level. The formazan, a marker for the enzyme nitric oxide synthase, labelled cystic, undifferentiated, endocrine-like and myoid cells in the medulla. Some lymphoid and reticulo epithelial cells were also lightly labelled. The reaction product was predominantly bound to the membranes of the endoplasmic reticulum in all the cells labelled and also to the nuclear envelope and outer membrane of mitochondria. The Golgi apparatus and the plasma membrane were free of the reaction product. PMID- 7534656 TI - Porcine aortic smooth muscle cells secrete a serine protease for insulin-like growth factor binding protein-2. AB - Porcine aortic smooth muscle cells secrete two forms of insulin-like growth factor (IGF) binding proteins (IGFBP-2 and -4), and both forms have been shown to modulate IGF-I actions in this cell type. Recently, we showed that IGFBP-4 inhibited IGF-I action and that the cells produced a protease that cleaved IGFBP 4 into non-IGF binding fragments. After the cleavage of IGFBP-4, the cellular DNA synthesis response to IGF-I was enhanced. This study reports that these cells also secrete a protease for IGFBP-2. Like the IGFBP-4 protease, this protease is also secreted constitutively, but unlike the IGFBP-4 protease, its secretion is enhanced if the cells are serum-deprived for 24 hours before the collection of conditioned medium. The protease cleaved IGFBP-2 into 25- and 16-kD fragments, which had reduced IGF-I binding activity. Protease activity was enhanced by coincubation with IGF-I or IGF-II, and IGF-II was more potent than IGF-I. The protease is a serine protease, since its activity can be inhibited by 3,4 dichloroisocoumarin and aprotinin. It is also inhibited by EDTA, and its activity can be restored with calcium but not zinc. The heparin-binding serpins, specifically, heparin cofactor II and antithrombin III, are inhibitory. Heparin alone also had activity, and the combination of antithrombin III plus heparin caused complete inhibition. The conditioned medium also contained proteolytic activities for IGFBP-4 and -5 but it did not cleave IGFBP-1 and -3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534657 TI - Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. AB - In the present study, we examined the hypothesis that dynamic characteristics of flow modulate the production of vasoactive mediators, namely nitric oxide (NO) and endothelin-1 (ET-1), by human umbilical vein endothelial cells (HUVECs). Cells were exposed for 6 hours in a cone-and-plate apparatus to different types of flow: steady laminar, with shear stresses of 2, 8, and 12 dyne/cm2, pulsatile laminar, with shear stress from 8.2 to 16.6 dyne/cm2 and a frequency of 2 Hz; periodic laminar, with square wave cycles of 15 minutes and shear stress from 2 to 8 dyne/cm2, and turbulent, with shear stress of 8 dyne/cm2 on average. A second culture dish was kept in a normal incubator as a static control for each experiment. Laminar flow induced synthesis of NO by HUVECs that was dependent on shear-stress magnitude. Laminar shear stress at 8 dyne/cm2 also upregulated the level of NO synthase mRNA. As observed with steady laminar flow, pulsatile flow also induced an increase in NO release by endothelial cells. When HUVECs were subjected to step-change increases of laminar shear, a further increase of NO synthesis was observed, compared with steady laminar shear of the same magnitude. Turbulent flow did not upregulate NO synthase mRNA or increase NO release. Both laminar and turbulent shear stress reduced, although not significantly, ET-1 mRNA and ET-1 production compared with the static condition. These results indicate that local blood flow conditions modulate the production of vasoactive substances by endothelial cells. This may affect vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone. PMID- 7534658 TI - Exercise training augments flow-dependent dilation in rat skeletal muscle arterioles. Role of endothelial nitric oxide and prostaglandins. AB - We aimed to test the hypothesis that as a consequence of short-term daily exercise, flow (shear stress)-dependent dilation and its mediation by the endothelium are altered in skeletal muscle arterioles. After initial familiarization with the protocol, rats ran on a treadmill once a day (with gradually increasing intensity up to 40 minutes and 28 m/min) for approximately 3 weeks (EX group); a control group remained sedentary (SED group). The active (internal) diameters of isolated gracilis muscle arterioles of SED and EX rats at 80 mm Hg were significantly different (55.2 +/- 2.1 and 49.3 +/- 2.0 microns, P < .05), and their passive diameters (in Ca(2+)-free solution) were 105.3 +/- 3.1 and 111.2 +/- 2.4 microns (not significantly different), respectively. Increases in flow of the perfusion solution from 0 to 12 microL/min elicited a significantly greater increase in diameter of EX arterioles (by 83.5% at maximum flow). This enhanced sensitivity maintained a lower shear stress in EX arterioles (15 to 20 dyne/cm2) compared with SED arterioles (25 to 35 dyne/cm2). In both SED and EX arterioles, flow-dependent dilation was eliminated after removal of the endothelium. Either N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, or indomethacin, an inhibitor of prostaglandin synthesis, shifted the flow diameter and calculated wall shear stress-diameter curves significantly to the right. Each of the inhibitors reduced flow-dependent dilation to a similar degree (approximately 40% to 45%); their combined administration nearly completely eliminated the dilation of arterioles of both SED and EX rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534660 TI - Diminished Ca2+ sensitivity of skinned cardiac muscle contractility coincident with troponin T-band shifts in the diabetic rat. AB - We have measured the apparent Ca2+ sensitivities of force development in skinned cardiac trabeculae at different sarcome lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca(2+)-force relations were measured at a long (2.4-microns) and a short (1.9-microns) sarcomere length. In disease, compared with the control condition, the apparent Ca2+ sensitivity was greatly diminished at a sarcomere length of 1.9 microns but not affected at all at the long length (2.4 microns). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca2+ sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length dependent modifications in Ca2+ sensitivity. The findings raise the possibility that diabetic Ca(2+)-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca2+ sensitivity. PMID- 7534659 TI - Complement-mediated loss of endothelium-dependent relaxation of porcine coronary arteries. Role of the terminal membrane attack complex. AB - Reperfusion of the ischemic myocardium results in the loss of endothelium dependent relaxation. We have shown recently that the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium. We hypothesized that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary arteries. Bradykinin (BK) or substance P concentration-dependently relaxed precontracted (U46619, 50 nmol/L) left anterior descending coronary artery (LAD) rings in vitro. Addition of zymosan to human (10%) or porcine (10%) serum for 30 minutes significantly (P < 0.05) increased the EC50 of BK-induced LAD relaxation from 4 +/- 1 to 418 +/- 159 nmol/L (n = 8) and from 9 +/- 3 to 281 +/- 132 nmol/L (n = 7), respectively. Similarly, addition of zymosan to 10% human serum (HS) for 30 minutes increased the EC50 of substance P-induced LAD relaxation from 0.4 +/- 0.1 to 30 +/- 14 nmol/L (n = 9, P < .05). Basal release of nitric oxide was reduced significantly in LAD rings exposed to zymosan-activated HS compared with HS alone. Addition of soluble CR1 (sCR1, 10 nmol/L) to zymosan-activated HS preserved BK-induced relaxation (EC50) of the LAD rings (control, 4 +/- 1 nmol/L; sCR1 + zymosan+serum, 2 +/- 1 nmol/L; n = 6). Zymosan-activated C8-depleted HS (10%) did not attenuate the EC50 of BK-induced coronary artery relaxation (3 +/- 1 to 3 +/- 1 nmol/L, n = 7, P = NS). Zymosan-activated C8-depleted HS plus C8 (6 micrograms/mL) increased the EC50 of BK-induced coronary artery relaxation from 4 +/- 1 to 423 +/- 141 nmol/L (n = 12, P < .05). We have further demonstrated that C5b-9 complexes can be found on the luminal surface of LAD endothelial cells after 5 minutes of exposure to zymosan-activated HS by using C5b-9 reactive monoclonal antibody fluorescent immunohistochemistry and confocal microscopy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534661 TI - MCI-154 increases Ca2+ sensitivity of reconstituted thin filament. A study using a novel in vitro motility assay technique. AB - MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca2+ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca2+] of the medium (control, pH 7.2 at 25 degrees C). When [Ca2+] was low, the filaments showed only brownian motion. However, above a certain level of [Ca2+] (the threshold [Ca2+]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca2+]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca2+ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca2+] to a lower level (sensitization) in a concentration related manner. And 10(-4) mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15 degrees C), or the addition of inorganic phosphate (10 mmol/L).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534662 TI - Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart. AB - Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT1 through cTnT4, numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30 nt exon in the 5' half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT1 through cTnT4. Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT1 and cTnT2) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT1 and cTnT2, containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT2 being expressed at a very low level. cTnT4, lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT3, containing the 5-residue peptide, is the dominant isoform in the adult heart.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534663 TI - Aspirin inhibits nuclear factor-kappa B mobilization and monocyte adhesion in stimulated human endothelial cells. AB - BACKGROUND: The induction of vascular cell adhesion molecule-1 (VCAM-1) and E selectin by tumor necrosis factor-alpha (TNF) is mediated by mobilization of the transcription factor nuclear factor-kappa B (NF-kappa B). Since salicylates have been reported to inhibit NF-kappa B activation by preventing the degradation of its inhibitor I kappa B, we studied a potential inhibition of this pathway by acetylsalicylate (aspirin) in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Gel-shift analyses demonstrated dose-dependent inhibition of TNF-induced NF-kappa B mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of VCAM-1 and E-selectin surface expression by TNF was dose-dependently reduced by aspirin over the same range, while induction of intercellular adhesion molecule-1 (ICAM-1) was hardly affected. Aspirin appeared to prevent VCAM-1 transcription, since it dose-dependently inhibited induction of VCAM-1 mRNA by TNF. As a functional consequence, adhesion of U937 monocytes to TNF-stimulated HUVECs was markedly reduced by aspirin due to suppression of VCAM 1 and E-selectin upregulation. These effects of aspirin were not related to the inhibition of cyclooxygenase activity, since indomethacin was ineffective. CONCLUSIONS: Our data suggest that aspirin inhibits NF-kappa B mobilization, induction of VCAM-1 and E-selectin, and subsequent monocyte adhesion in endothelial cells stimulated by TNF, thereby providing an additional mechanism for therapeutic effects of aspirin. PMID- 7534664 TI - Blade balloon atrial septostomy in patients with severe primary pulmonary hypertension. AB - BACKGROUND: Patients with severe primary pulmonary hypertension have a poor prognosis, but those with a patent foramen ovale may survive longer. A few reports of clinical improvement after blade balloon atrial septostomy in patients with severe pulmonary vascular disease have appeared. The purpose of this study was to systematically evaluate the effects of blade balloon atrial septostomy on clinical signs and symptoms, hemodynamics, and survival in patients with severe primary pulmonary hypertension. METHODS AND RESULTS: Blade balloon atrial septostomy was performed on 15 children and young adults with severe primary pulmonary hypertension. Despite maximal medical therapy, prior to septostomy all patients had recurrent syncope and 8 had severe right heart failure. Thirteen patients survived the procedure. After blade balloon atrial septostomy, no patient experienced further syncope, and signs and symptoms of right heart failure improved in all New York Heart Association Class IV patients. Within 24 hours after the procedure and at follow-up catheterization 7 to 27 months after septostomy, there was a significant increase in cardiac index, resulting in an increase in systemic oxygen transport. There was improved long-term survival in the 13 patients who survived blade balloon atrial septostomy compared with similar groups of primary pulmonary hypertension patients who received standard therapy (P < .05). CONCLUSIONS: Blade balloon atrial septostomy resulted in clinical and hemodynamic improvement and improved survival in selected patients with severe primary pulmonary hypertension. PMID- 7534665 TI - The nature of the epithelium in acquired cholesteatoma: Part 3--Cytokeratin patterns in aural epithelial and cholesteatoma cells grown in cell culture. AB - The nature and origin of the epithelial layers in acquired cholesteatoma is still unclear. Although previous morphological studies comparing external meatal and cholesteatoma epithelium have shown no significant difference, bone resorption is generally much more severe with cholesteatoma than with chronic otitis media without cholesteatoma. It is possible that cholesteatoma epithelium has undergone transformation leading to its enhanced bone destroying role. In this study the cytokeratin patterns of aural and cholesteatoma epithelia grown in cell culture were compared using monoclonal antibodies. No significant difference in staining patterns were found suggesting that there has been no change in cell phenotype which maintains that of external auditory meatus epithelium. This study therefore supports the immigration theory of cholesteatoma genesis. PMID- 7534666 TI - Pharmacologic activation of expression of immunodominant viral antigens: a new strategy for the treatment of Epstein-Barr-virus-associated malignancies. PMID- 7534667 TI - The blood B-cells and bone marrow plasma cells in patients with multiple myeloma share identical IgH rearrangements. AB - Previous reports have described the phenotypic and functional properties of monotypic late stage B cells in the blood of patients with multiple myeloma and have speculated that these B cells represent a malignant circulating component of myeloma. Here we show that blood B cells have IgH rearrangements identical to those expressed by the bone marrow plasma cells by using Ig Fingerprint and Allele-Specific Oligomer (ASO) polymerase chain reaction (PCR) methods. DNA from purified blood B cells and bone marrow plasma cells taken at the same time, and blood B cells taken at subsequent patient visits was amplified using consensus IgH primers, or ASO primers. In 10/16 patients, a single IgH rearrangement was amplified from the bone marrow plasma cells. In all 10 of those patients the same clonotypic rearrangement was amplified from the purified blood B cells. The relationship of these clonal blood B cells to the malignant bone marrow plasma cells remains undetermined. PMID- 7534668 TI - Circulating B-cells in follicular non-Hodgkin's lymphoma show variant expression of L-selectin epitopes. PMID- 7534669 TI - CD5 transgenic mice. PMID- 7534670 TI - Activation of B-cells by sIgM cross-linking induces accumulation of CD5 mRNA. AB - The surface membrane molecule CD5 is expressed on mature T cells and on the B-1a subpopulation of B cells. These CD5 positive B cells express an antibody repertoire with a relatively high frequency of self-reactivity. There is uncertainty about the origins of CD5 B cells and the reasons for this are reviewed. Recent reports which relate to the lineage/selection debate are discussed. For instance, an increase in the frequency of CD5 B cells is a feature of several genetically determined polysystem autoimmune syndromes. In the case of motheaten (me, mev) the pathogenesis of this increase in CD5 B cells is not yet understood, even though the mutation has been mapped to the Hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Another mutation which affects B cell development, X-linked immunodeficiency (xid), encodes a point mutation in a B cell cytoplasmic tyrosine kinase. Expression of xid in otherwise normal mice causes a lack of CD5 B cells and a shift in the antibody repertoire. Interestingly, expression of both xid and motheaten results in an amelioration of autoantibody production. Evidence is presented that in B cells regulation of expression of CD5 can occur at the level of mRNA and that cross-linking of sIgM can induce the accumulation of CD5 mRNA. The overall concept advanced is that cells expressing natural autoantibodies are triggered via sIgM ligation to become CD5 B cells. PMID- 7534672 TI - Potential role of CD28-B7 interactions in the growth of myeloma plasma cells. PMID- 7534671 TI - Multiple myeloma clones are derived from a cell late in B lymphoid development. AB - We have previously demonstrated that the immunoglobulin (Ig) heavy chain variable region (VH) sequences expressed by the malignant clone in multiple myeloma (MM) contain a high degree of somatic mutation without clonal diversity. This sequence can be used to identify all members of the malignant clone in this B cell malignancy. We sequenced the variable regions expressed by patients with MM and generated primers from the complementarity determining region (CDR) sequences specific for each patient's tumor. Using these primers, we performed PCR amplification on highly purified subpopulations of cells separated by expression of CD10, CD34 and CD38. The results of these experiments demonstrate: 1) there is a small fraction of CD10-expressing tumor cells in MM patients, 2) CD34-bearing malignant cells do not exist in MM, and 3) although the vast amount of tumor is in the CD38-expressing cells, a small amount of tumor is in the CD38-negative population. We also used these primers to determine whether pre-class switch (i.e., Cmu-expressing lymphocytes) clonal cells exist in these patients. After PCR amplification with CDR1 and Cmu primers, colony hybridization was performed using both framework 3 (FR3) and CDR3 probes. Out of > 200 FR3-hybridizing colonies, < or = 5 colonies also hybridized with the CDR3 probe. Colonies which hybridized with both these probes were sequenced, and none of these sequences matched even closely the CDR3 expressed by the malignant clone. These results make the existence of a pre-class switch malignant cell unlikely in MM. Overall, these results suggest that the malignant clone in MM derives from a cell late in B lymphocyte development. PMID- 7534673 TI - B-1 (CD 5+) B-cells as a marker of immune dysregulation in multiple myeloma. PMID- 7534674 TI - Molecular and biological role of CD40 in multiple myeloma. PMID- 7534675 TI - Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis. AB - New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro )myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit). PMID- 7534677 TI - CD3+, CD56+ non-MHC restricted cytotoxic T lymphocytes in two fraternal AIDS patients: a case report. AB - We describe two brothers with hemophilia and AIDS, with an unusually large percentage of CD3+, CD56+ lymphocytes. They experienced no major complications with opportunistic infections (OI) and infrequent secondary infections, even though they had nearly 0% CD4 lymphocytes for 3 years. Both patients died in 1991 of progressive cardiomyopathy. The patients' lymphocytes were immunophenotyped by flow cytometry and analyzed for functional cytolytic activity against K562 and HIV infected HUT 78 cell lines. Single color CD4 counts were 2-9% for 4 years. Dual color CD4 counts at our laboratory demonstrated 0-1% CD4 for the last 6 months. When CD3+ lymphocytes were examined, both patient 1 and patient 2 demonstrated a significantly higher proportion and absolute number of CD3"bright"+, CD56+ double-positive cells, 47% and 22%, respectively, compared to other HIV-positive children with hemophilia (< or = 2%). Functional studies with the K562 target cell line demonstrated the highest natural killer (NK) lymphocyte activity in patient 1 that could not be augmented by in vitro addition of IL-2, whereas patient 2 had no NK activity unless IL-2 was added. Functional studies with HIV-infected HUT-78 cells demonstrated patient 2 had cytolytic activity against HIV-infected cells and patient 1 had high nonspecific cytolytic activity even against uninfected HUT-78 cells, whereas controls had minimal activity to HUT-78 cells or HIV-infected HUT-78 cells. The case report raises a speculative question requiring a larger database, whether the anti-HIV activity and/or unusual clinical course without typical O.I. of some AIDS patients may be related to the presence of CD3"bright"+, CD56+ lymphocytes of their immune system. PMID- 7534676 TI - Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non-lymphoid cells. AB - Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells. PMID- 7534678 TI - Ketotifen ameliorates capsaicin-augmented acetic acid-induced colitis. AB - Capsaicin-sensitive afferent neurons are involved in maintaining the integrity of the gastrointestinal mucosa. These neurons are closely apposed to mast cells and could, therefore, lead to their activation. In the present study, the role of capsaicin-sensitive neurons in the pathogenesis of experimental colitis and the possible involvement of mast cells and nitric oxide were evaluated. Rats were treated with capsaicin subcutaneously, 20, 30, and 50 mg/kg, on three consecutive days, a regimen shown to ablate primary afferent neurons. Colitis was induced two weeks later by flushing 2 ml 5% acetic acid into the proximal colon. Control rats received saline, acetic acid, or capsaicin alone. Another group of rats received ketotifen (100 micrograms/100 g body wt x 2/day) intragastrically 48 hr prior to damage induction and thereafter. Rats were sacrificed 24 hr after damage induction, the colon isolated, damage assessed, explants were organ-cultured for 24 hr for determination of nitric oxide generation, and mucosa extracted for determination of leukotriene B4 generation and nitric oxide synthase activity. Significant increases in colonic weight, nitric oxide synthase activity, and nitric oxide and leukotriene B4 generation accompanied the near tripling of acetic acid-induced damage in capsaicin-pretreated rats. Ketotifen pretreatment significantly decreased the macroscopic damage and the increase in colonic weight. The protection provided by ketotifen was accompanied by a significant decrease in leukotriene B4 generation and nitric oxide synthase activity (80%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534680 TI - Fine-needle aspiration cytology of hepatic leiomyosarcoma. AB - Hepatic leiomyosarcoma is a rare malignancy to involve the liver, occurring as a primary liver sarcoma in patients without an underlying medical disorder, as a metastatic malignancy, and with increasing incidence, as a primary tumor in AIDS patients. A series of hepatic leiomyosarcomas diagnosed by FNA biopsy, including the first reported case in an adult AIDS patient, were reviewed with respect to cytomorphologic features. The series consisted of five men and two women ranging from 24 to 72 years of age. One case was a primary hepatic lesion in a 24-yr-old man with AIDS and six were metastatic from a number of sites (stomach, small bowel, retroperitoneum, vena cava, and seminal vesicle). Two cytologic patterns were identified. Aspirates of spindle-cell leiomyosarcomas (six cases) generally produced hypocellular smears composed primarily of small irregular clusters of cells with blunt-ended, uniform, overlapping nuclei. The differential diagnosis included benign reactive processes and other benign and malignant spindle-cell neoplasms. The aspirate of an epithelioid leiomyosarcoma (one case) revealed a second pattern characterized by highly cellular smears with many single polygonal cells having eccentric, malignant nuclei and a characteristic clear quality to the cytoplasm in Papanicolaou-stained material. This epithelial appearance generated a differential diagnosis that included hepatocellular carcinoma, metastatic carcinoma, and melanoma. Careful integration of clinical information, cytomorphologic features, and ancillary studies will allow specific FNA diagnosis of hepatic leiomyosarcoma in most cases. PMID- 7534679 TI - Nonsurgical management of primary cholangiocarcinoma. Retrospective analysis of 40 cases. AB - Forty patients with cholangiocarcinoma (23 men, 17 women) underwent nonsurgical palliative biliary drainage over a period of 12 years. All were surgically unfit or had unresectable disease. All were jaundiced at presentation with a mean serum bilirubin of 11.5 +/- 1.9 mg/dl. Thirty patients (75%) had hilar obstruction. Twenty-eight were drained percutaneously, three endoscopically and nine by a combined endoscopic and percutaneous procedure. Technical success was 97.5%. Final mean bilirubin was 1.5 +/- 0.4 mg/dl. Minor complications occurred in 10 (25%) patients, and major complications in four (10%). Procedure-related mortality was 2.5% with a 30-day mortality of 7.5%. Mean survival was 8.2 +/- 0.5 months. Stent changes were required in eight patients. In patients with inoperable or unresectable cholangiocarcinoma, percutaneous or endoscopic biliary drainage offers effective palliation. PMID- 7534681 TI - Extraskeletal myxoid chondrosarcoma: fine-needle aspiration biopsy findings. AB - Extraskeletal myxoid chondrosarcoma (EMC) is a malignant soft tissue tumor which typically presents as an enlarging mass in an extremity. In this location it is amenable to sampling and diagnosis by fine-needle aspiration biopsy. We present our experience with three cases of EMC and discuss the differential diagnosis of myxoid soft tissue tumors. PMID- 7534682 TI - Epithelioid sarcoma in FNAB smears. AB - FNAB smears of eleven epithelioid sarcomas were reviewed and analysed. Ten cases had a very similar cytomorphologic picture composed predominantly of dissociated epithelioid-like cells with eccentrically placed nuclei. These tumors were clearly malignant but difficult to differentiate morphologically from melanoma, epithelioid leiomiosarcoma, and Schwannoma or adenocarcinoma. One case was composed of spindle cells and was reminiscent of a fibrohistiocytic tumor. Immunocytochemical reactions to vimentin and cytokeratin were performed in six cases on the Papanicolaou stained smears. The reactions to both antigens were positive in all six cases. Ultrastructural characteristics of eight of the tumors are also described. It seems that epithelioid sarcoma has a rather distinct cytomorphologic picture. Taking into consideration clinical data and using also immunocytochemistry, a definitive diagnosis of epithelioid sarcoma can probably be given from FNAB smears. PMID- 7534683 TI - Nodular lepromatous leprosy: report of a case diagnosed by FNA. AB - Lepromatous leprosy can present with skin nodules which can be misdiagnosed as soft tissue tumors or infected cysts. FNA can be diagnostic if unstained, refractile, intracellular mycobacteria are recognized on Romanowsky stained smears. Fite stain for Mycobacterium leprae confirms the diagnosis. Awareness of the differential diagnosis of skin nodules yielding foamy histiocytes on FNA, briefly discussed, should help avoid error. PMID- 7534684 TI - [A case-control study on relationship between hepatitis C infection and primary liver cancer]. AB - All the cases and controls emerged from a cohort study held in the past years. They were male, more than 20 years of age at the time of their enrollment into the cohort study, with serum specimens taken and kept at -20 degrees C. Every death of primary liver cancer (PLC), occurred since then, as a 'case' (78 in total), was compared with no more than 4 matched controls chosen from cohort members who were still alive until the last follow-up. ELISA technique was used to test anti-HCV antibodies in the stored serum specimens of the cases and controls. The Results showed that anti-HCV prevalence rates were 33.3% (26/78) of the cases and 15.3% (40/262) of their matched controls, respectively (chi 2 = 11.86, P < 0.01, ORMH = 3.0, PAR% = 23.40%). The results suggest that HCV infection is another important factor in the aetiology of PLC in Guangxi, China. Because the serum specimens tested were drawn years before the occurrence of PLC deaths, our conclusion need not worry about the sequential problem, which occurs in most of other kinds of case-control studies. PMID- 7534685 TI - Effects of ivermectin on the activity of enzymes in mammalian cells in vitro. AB - The effects of ivermectin on the activities of lactate dehydrogenase, glucose-6 phosphate dehydrogenase, and glucose-6-phosphatase have been estimated in IB-RS-2 cells in vitro. A 72-hr time course following ivermectin exposure indicated a decrease in the activity of lactate dehydrogenase. The activities of glucose-6 phosphate dehydrogenase and glucose-6-phosphatase remained essentially unchanged. PMID- 7534686 TI - Changes in oxidative metabolism in selected tissues of the crab (Scylla serrata) in response to cadmium toxicity. AB - Changes in oxidative metabolism were studied in hepatopancreas, muscle, and hemolymph of the edible crab Scylla serrata, exposed to a sublethal concentration (2.5 ppm) of cadmium chloride. A significant decrease in glycogen, total carbohydrates, and pyruvate and an increase in lactate levels in hepatopancreas and muscle were observed. Hemolymph sugar levels were increased in experimental crabs. An increase in phosphorylase suggested increased glycogenolysis during cadmium toxicity. The decrease in lactate dehydrogenase activity and the increase in lactate content indicated reduced mobilization of pyruvate into the citric acid cycle. Krebs cycle enzymes such as succinate dehydrogenase and malate dehydrogenase were found to be decreased, suggesting impairment of mitochondrial oxidative metabolism as a consequence of cadmium toxicity. Glucose-6-phosphate dehydrogenase activity was increased, suggesting enhanced oxidation of glucose by the HMP pathway. Cytochrome-c oxidase and Mg2+ ATPase activity levels decreased, indicating impaired energy synthesis during cadmium stress. Acid and alkaline phosphatase activities increased, suggesting enhanced breakdown of phosphates to release energy in view of impaired ATPase system during cadmium exposure. A significant decrease in protein and free amino acid and an increase in ammonia, urea, and glutamine levels were observed in the tissues during exposure. An increase in protease, alanine aminotransaminase, and aspartate aminotransaminase suggested increased proteolysis and transamination of amino acids. The increase in glutamate dehydrogenase, AMP deaminase, and adenosine deaminase indicated increased ammonia production. The increased arginase and glutamine synthetase suggested the detoxification or mobilization of ammonia toward the production of urea and glutamine. These results suggest that cadmium affects oxidative metabolism and induces hyperammonemia, and crabs switch over their metabolic profiles toward compensatory mechanisms for the survivability in cadmium-polluted habitats. PMID- 7534687 TI - Histopathology of kidney of Channa punctatus exposed to chronic nonlethal level of Elsan, mercury, and ammonia. AB - Histopathological changes in the head and trunk kidneys of Channa punctatus induced by chronic nonlethal levels of Elsan (211 ppb), mercuric chloride (16.7 ppb), and aqueous ammonia (15.64 ppm) were studied on 7, 28, 63, and 90 days of exposure. The pathology of the head kidney was characterized by degeneration and dispersion of interrenal and chromaffin tissue and necrosis in the haemopoietic elements. Kidney lesions were observed throughout the entire experimental period in fish exposed to Elsan and mercuric chloride. In contrast, the lesion induced by exposure to aqueous ammonia began to heal during the first phase of treatment. Marked abnormalities in trunk kidney histology were also found. Renal lesions consisted of minimal to mild multifocal, acute tubular epithelial degeneration, karyolysis, and dilation or shrinkage of Bowman's capsule and glomerulus. Elsan treatment resulted in a highly significant decrease in the dimension of Bowman's capsule and glomerulus at all days of sampling, except on Day 28. The response of the fish trunk kidney tissue to mercuric chloride was similar to that observed with Elsan exposure in terms of the alteration in the mean dimensions of Bowman's capsule and glomerulus. The response to ammonia was significant reduction in the size of Bowman's capsule and glomerulus throughout the experimental period except at Day 28. Little dilation of Bowman's capsule and a significant dilation of glomerulus were found at Day 28 of ammonia exposure. This study demonstrated that a chronic nonlethal exposure to Elsan, mercuric chloride affect both endocrine and excretory parts of the kidney while ammonia specifically damages the excretory part of the kidney of C. punctatus. PMID- 7534688 TI - Historical changes in the ecological health of the Newark Bay Estuary, New Jersey. AB - A review of ecological conditions in the Newark Bay estuary over the past century was conducted to characterize chemical, physical, and biological indices of the health of the estuary and to evaluate the relationship of these trends with past and current urban-industrial influences within the watershed. The available data indicate that both the diversity and the abundance of aquatic species within the estuary have been substantially reduced since the late 1800s due to the intense industrialization and urbanization that occurred throughout the region. Water and sediment quality in major rivers and bays linked to the estuary have also been severely impacted. In addition, significant natural habitat has been destroyed as a result of industrial development, urban expansion, and shoreline modifications. Each of these parameters directly influences the status and condition of the ecological community, and their deterioration has likely been the cause of multiple adverse impacts on the overall health of the ecosystem. Review of more recent studies of sediment and water quality indicates that pollution control measures and the reduction or control of other environmental stressors have produced a gradual improvement in the ecosystem over the past 2 decades. PMID- 7534689 TI - An analysis of avian reproduction studies submitted for pesticide registration. AB - Avian reproduction studies are currently part of the regulatory ecotoxicology requirements for pesticides in many countries. In the study, 134 avian reproduction studies were reviewed to determine their ability to identify pesticides that have the potential to affect reproduction in wild birds. A clustering procedure was first used to assign measured variables to parental, developmental, or eggshell effects. This assignation was found to be identical in the two bird species tested. Nineteen of 69 pesticides tested were found to cause developmental effects at levels lower than those giving rise to detectable parental toxicity. At least some of these should not have been registered without some assurance that developmental effects would not occur in the wild. The analysis also found very little similarity in the effects of pesticides on the two bird species commonly used in avian reproduction tests. This casts serious doubts on the ability to extend the results of avian reproduction studies to any potentially affected bird species. Modifications to the avian reproduction test, based on results of the analysis conducted, are suggested. It is recommended that the avian reproduction study be recognized as a rough screening tool only and that efforts not be made to make it more realistic, e.g., such as through a reduced exposure period. PMID- 7534690 TI - Long-term cadmium exposure accelerates oxidant injury: significance of bound/free water states during long-term metal stress. AB - Lepidium sativum (cress) and Lycopersicon esculentum (tomato) plants were grown in peatlite in controlled environments with or without long-term (4 weeks) cadmium stress (Cd) (100 micrograms/ml every fourth day) and with a single exposure (6 hr at 35 parts per hundred million (pphm)) or no exposure to the oxidant ozone (O3). Cress plants which received Cd wilted faster during O3 exposure and became a gray-green color by the end of a 6-hr O3 exposure. Those receiving O3 alone also wilted but were normal in color during wilting. Leaf water content (percentage) significantly declined in both O3 + Cd- and O3-treated plants. However, leaves after Cd + O3 exposure were severely dessicated and necrotic, whereas O3-treated plants recovered their water content completely but had some injury. Increased stomatal aperture in cress but not tomato before O3 exposure and significantly lower water content at 1 and 24 hr after the end of O3 exposure were associated with the higher Cd content of leaves before and subsequent to O3 exposure. These factors contributed to a greater injury and cell death observed in the leaves of combined cadmium-oxidant stress. Dielectric properties of Thlaspi arvense (field penny cress) leaves grown at continuous exposure to Cd and/or nickel (Ni) indicated that there were measurable differences between metal-containing vs control leaves with regard to bound/unbound water status. This indicated that there was more free water under metal stress, and that the bound water content significantly declined in the leaves of these plants. PMID- 7534691 TI - Effect of atrazine herbicide on growth, photosynthesis, protein synthesis, and fatty acid composition in the unicellular green alga Chlorella kessleri. AB - Sublethal atrazine concentrations induced a general inhibition on growth, photosynthesis, and dark respiration in the green alga Chlorella kessleri. 14C protein hydrolysate indicated a maximum incorporation level by 5 microM atrazine after 48 hr. Progressive reduction in protein synthesis was associated with increasing herbicide concentration at all experimental periods. The herbicide had preferential effect on the associated fatty acid composition of total and phospholipids. After 24 and 48 hr the herbicide stimulated fatty acids synthesis at concentrations where photosynthesis was inhibited. Meanwhile, stearic and miristic acids disappeared at 15 microM after 24 hr and the total polyunsaturated fatty acids were not affected after 48 hr. Fatty acid synthesis was sensitive to treatment at 72 hr by 5 and 10 microM atrazine, whereas the total saturated fatty acids were completely inhibited. PMID- 7534693 TI - The effect of dimethoate, dichlorvos, and parathion-methyl on bone marrow cell chromosomes of rats in subchronic experiments in vivo. AB - The three organophosphorous insecticides dimethoate, dichlorvos, and parathion methyl were investigated in subchronic experiments on bone marrow cell chromosomes. In the literature these compounds were reported to exhibit both positive and negative results in mutagenicity tests demanding further investigations in subchronic tests. The treatment of different groups of male Wistar rats lasted for 6 weeks with 5 treatment days per week at doses of 1/100, 1/75, and 1/50 of the LD50. Following the last treatment, bone marrow cell chromosomes were prepared. The frequency of cells revealing any aberrations as well as numeric and structural aberrations were evaluated. In this test both dimethoate and dichlorvos demonstrated mutagenic effects following subchronic treatment of Wistar rats, while parathion-methyl at doses of 1/100, 1/75, and 1/50 of LD50 displayed no significant mutagenicity. PMID- 7534692 TI - Comparative study of plant growth hormone (herbicide) toxicity in various biological subjects. AB - Due to their widespread distribution and toxic nature, herbicides may have a serious impact on the environment and exert adverse effects on associated organisms. The present study was conducted to determine the acute toxicological effects of some plant growth hormones used as herbicides on four biological subjects and compare the subjects' sensitivity to individual testing substances. The herbicides 4-(indol-3-yl)acetic acid C10H9O2N (IAA), N6-(beta 2 isopentenyl)adenosine (pi-indolylpropionic acid) C11H11O2N (IPA), 2,4 dichlorophenoxyacetic acid C8H6O3Cl2 (2,4-D), 4-chloro-2-methylphenoxyacetic acid C9H9O3Cl (MCPA), and 1,napthylacetic acid C12H10O2 (NAA) were tested and the following biological subjects were used: Daphnia magna, Tubifex tubifex, Scenedesmus quadricauda, and seeds of Sinapis alba. For S. alba, the influence of herbicides on seed germination (G) and root growth inhibition (I) was observed. For T. tubifex, the tests lasted 96 hr, for D. magna 48 hr, for S. quadricauda 20 days, and for S. alba 72 hr. The rank order of toxicity of herbicides used for T. tubifex was NAA > IAA > IPA > 2,4-D > MCPA; for D. magna. NAA > IAA > IPA > MCPA > 2,4-D; for S. quadricauda, IAA > IPA > NAA > MCPA > 2,4-D; for S. alba seed germination, NAA > IPA > 2,4-D > MCPA > IAA; and for root growth inhibition. NAA > 2,4-D > MCPA > IAA > IPA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534694 TI - Octamethylcyclotetrasiloxane in aquatic sediments: toxicity and risk assessment. AB - The toxicity of octamethylcyclotetrasiloxane (OMCTS) to the midge Chironomus tentans was examined in a series of aqueous and sediment exposures. Midge larvae were exposed for 14 days to five concentrations of radiolabeled [14C]OMCTS in each of three sediments (low, medium, and high organic carbon content). Additional tests were conducted with a water-only exposure. Results indicate the lowest 14-day no observed effect concentrations (NOEC) to be 65, 120, and 54 mg/kg for the low, medium, and high organic carbon sediments, respectively. For the water-only exposure, the 14-day NOEC was > 15 micrograms/liter. These results were used in the preparation of a risk assessment for OMCTS which took into consideration the physicochemical and environmental fate properties of the compound, as well as actual field monitoring and worst-case modeling results for assessment of exposure. Available evidence suggests that sediment concentrations of OMCTS do not present a risk to sediment-associated organisms. For 95% of sediment samples, data indicate that the margins of safety would be greater than 400 and potentially over 1000. Worst-case margins of safety were greater than 150. PMID- 7534695 TI - The processing of insulin-like growth factor-I (IGF-I) by a cultured kidney cell line is altered by IGF-binding protein-3. AB - The proximal renal tubule is a common site of peptide hormone metabolism, including that of insulin-like growth factor-I (IGF-I). To further explore the renal uptake and processing of IGF-I, a study was carried out with the proximal like cultured opossum kidney (OK) cell line. [125I]IGF-I associated with these cells in a specific manner. Association was competitively inhibited by IGF-I. Des(1-3)-IGF-I was equally effective, insulin had only a small effect, and the unrelated peptides, glucagon and GH, were without effect. Degradation was inhibited in similar manner. Comparisons of [125I]IGF-I with [125I]insulin revealed comparable cell association, but degradation of internalized IGF-I was several-fold slower. Furthermore, IGF-I degradation was less sensitive, by half, to the inhibitory effect of chloroquine. When OK cells were exposed to [125I]IGF I in the presence of IGF-binding protein-3 (IGFBP-3) cell association (binding and internalization) was reduced significantly. Of note, total cell degradation was reduced (P < 0.01), but the IGF-I that was internalized was degraded more rapidly than in control cells. Gel filtration and reverse phase HPLC revealed that the products of IGF-I degradation included large IGF-I-size intermediates in addition to trichloroacetic acid-soluble material. This product profile was not altered by IGFBP-3. Thus, as previously described for insulin, cultured OK cells possess specific IGF-I receptors and degrade internalized IGF-I. However, IGF-I processing differs from that of insulin, in that degradation is slower and relatively insensitive to competition by insulin. This study also shows that IGFBP-3 inhibits the binding and uptake of [125I]IGF-I by these kidney cells. However, once IGF-I is internalized, IGFBP-3 enhances degradation. Although the mechanism of this paradoxical action requires further study, analysis of the products of degradation suggests that the same enzymes are involved in IGF-I degradation regardless of whether IGFBP-3 is present. PMID- 7534696 TI - Quantitative analysis by polymerase chain reaction of growth hormone receptor gene expression in human liver and muscle. AB - A single form of GH receptor (GHR) messenger RNA (mRNA) of 4.5 kilobases, encoding the full-length GHR, has been found in man. To measure the absolute number of mRNA molecules encoding the GHR in human tissues, we developed a quantitative polymerase chain reaction assay. An internal control RNA was constructed by inserting a 50-basepair fragment of the rat PRL receptor complementary DNA into a portion of the human GHR complementary DNA. The internal control RNA and the target mRNA were amplified together with the same set of primers. Twenty-four cycles of amplification were used to satisfy an exponential phase of amplification. It was possible to detect as few as 500 molecules of target mRNA/micrograms total RNA. In 3 liver samples obtained from normal donors at the time of transplant, the amount of GHR mRNA ranged from 0.5 +/- 0.1 to 1.4 +/- 0.4 x 10(6) molecules/micrograms total RNA. These results were confirmed by slot blot analysis of the same samples. The number of receptor transcripts did not appear to be correlated with the receptor-binding capacity found in the 3 liver samples. In 7 muscle biopsies, GH receptor mRNA varied between 4.0 +/- 0.4 and 34.6 +/- 1.4 x 10(4) molecules/micrograms total RNA. This technique allows direct measurement of GHR gene expression in human tissues and represents a valuable tool, particularly for tissues such as muscle, in which the receptor protein cannot be measured using conventional binding assays. PMID- 7534697 TI - Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells. AB - Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106 01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity. PMID- 7534698 TI - Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF binding protein expression in fibroblasts. AB - Insulin-like growth factor (IGF)-binding proteins (IGFBPs) can determine IGF biological competence at the cellular level. IGF-I itself has been shown to be an important peptide regulator of local IGFBP availability. Glucocorticoid also has major effects on IGFBP expression. In the present study, we assessed integrated IGF-I and glucocorticoid regulation of IGFBP messenger RNA (mRNA) and protein expression in two fibroblast model systems. In bovine fibroblasts, IGF-I treatment induced IGFBP-3 and IGFBP-5 mRNA and protein secretion, and had a moderate effect on IGFBP-4 expression. Dexamethasone had little effect on the IGF induced increase in IGFBP-3, but completely blocked the increase in IGFBP-5 expression. Basal IGFBP-4 expression was inhibited by dexamethasone, and this effect was counteracted by IGF-I. IGFBP-2 expression did not vary with IGF-I or dexamethasone treatment in these cells; IGFBP-1 mRNA was not detectable, and IGFBP-6 mRNA was low and inconsistent. In human fibroblasts, IGF-I treatment increased levels of IGFBP-3 and decreased levels of IGFBP-4 without influencing mRNA expression. IGF-I also increased steady state levels of IGFBP-5 mRNA. Dexamethasone alone decreased IGFBP-3, IGFBP-4, and IGFBP-5 mRNA, but it had no significant effect on IGFBP-3, -4, or -5 expression in the presence of IGF-I. Human fibroblast IGFBP-6 expression was stable under the different culture conditions; IGFBP-1 and -2 mRNA were not detected. These data demonstrate that IGF peptide and glucocorticoid individually modulate IGFBP expression and indicate that glucocorticoid has distinct effects on IGF regulation of IGFBP depending upon the particular IGFBP and the underlying mechanism of IGF regulation. Bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3, -4, and -5 gene expression and regulation by IGF I and glucocorticoid, whereas human fibroblasts may be suitable for studying posttranscriptional interactions. PMID- 7534699 TI - Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation. AB - We established a human bone marrow stromal cell line (Saka) by infecting marrow adherent cells from semisolid marrow cultures with a recombinant simian virus-40 (SV40) virus. The cells expressed SV40 large tumor antigen, had a fibroblast-like shape, and expressed fibronectin and vimentin. They did not contain detectable alkaline phosphatase activity; express myeloid, lymphoid, or factor VIII associated antigens; or develop adipocyte-like characteristics with dexamethasone treatment. Polymerase chain reaction analysis of Saka cell RNA detected expression of messenger RNAs for interleukin-6 (IL-6), IL-1 beta, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, and the 1,25-dihydroxyvitamin D3 receptor. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells (MNC) in long term human bone marrow cultures. These MNC expressed calcitonin receptors and formed resorption lacunae on dentine. In contrast, coculture of marrow mononuclear cells with other SV40-transformed human marrow stromal cell lines did not increase MNC formation. Conditioned medium from Saka cells or coculture of bone marrow and Saka cells separated by a Millipore membrane did not enhance MNC formation. Addition of a neutralizing antibody to IL 6 or IL-1 beta blocked the effects of Saka cells on MNC formation. These results suggest that marrow stromal cells enhance osteoclast formation in part through direct cell to cell contact and production of IL-6 and/or IL-1 beta. PMID- 7534700 TI - Rat fibroblast cells overexpressing kinase-inactive human insulin receptors are insulin responsive: influence of growth conditions. AB - The effects of insulin to stimulate metabolic and mitogenic responses were examined in Rat 1 fibroblast cells that overexpressed either normal (HIRc) or kinase-deficient human insulin receptors. When studied at the optimal growth stage for each cell line, insulin-stimulated responses measured in cells containing kinase-defective receptors with a Lys1018-Ala1018 substitution in the ATP-binding site of the kinase domain (A/K1018). Maximal insulin responsiveness for all these effects, measured as fold-increase over basal, was comparable in parental and HIRc cells (1.8- to 2.4-fold increases). Relative insulin responsiveness for all effects was greatest in A/K 1018 cells. One clone (AK-I) expressing a similar number of kinase-inactive receptors as in the HIRc cells displayed maximal responsiveness of 3.6- to 5.5-fold increases. A second A/K cell line containing 1/10 the number of kinase-inactive receptors displayed responsiveness intermediate between AK-I and parental or HIRc cells (1.5- to 4.8 fold increases). Both clones of kinase-deficient A/K1018 cells displayed impaired insulin sensitivity compared with HIRc cells. These findings suggest that expression of insulin receptor kinase activity is a determinant of insulin sensitivity but not necessarily of the final biological responsiveness of cells to insulin. PMID- 7534701 TI - Pituitary cells respond to thyroid hormone by discrete, gene-specific pathways. AB - Type 1 iodothyronine deiodinase (D1) converts T4 to T3, the active thyroid hormone, by removal of the outer ring iodine. Previous studies in liver and thyroid cells have shown that T3 regulates Type 1 deiodinase (dio1) gene expression by a mechanism not requiring ongoing protein synthesis. For certain T3 regulated genes, such as rat GH, T3-induced transcription is blocked by protein synthesis inhibitors. Because the somatotrope tumor cell lines express both dio1 and GH, we compared these two positively T3-regulated genes to establish whether cycloheximide blockade of T3 effects is cell-type or gene specific. In these cells, the T3 stimulation of dio1 messenger RNA (mRNA) is not blocked by cycloheximide, whereas the T3 effect on GH mRNA synthesis is eliminated. Other differences between these two genes were also noted. Retinoic acid does not alter dio1 gene expression or the response to T3 but increases GH and synergizes with T3. Dexamethasone alone had no effect on dio1 mRNA but did enhance the effect of T3 on both dio1 and GH. These results point to distinct pathways for T3 induction of mRNA synthesis from different genes within the same cell. PMID- 7534702 TI - Dihydrotestosterone is the active androgen in the maintenance of nitric oxide mediated penile erection in the rat. AB - Androgens are essential for the expression of normal libido in the male, but their role in the maintenance of the erectile response in humans is controversial. It has been shown previously in the rat that castration induces 1) loss of penile reflexes; and 2) considerable reduction in the erectile response to electric field stimulation (EFS) of the cavernosal nerve. Both of these effects can be reversed by testosterone replacement. The current study was performed to determine whether these testosterone effects are mediated via its conversion to dihydrotestosterone (DHT), and to what extent the synthesis of the mediator of penile erection, nitric oxide, is affected by castration and androgen replacement. Five-month-old rats were either castrated or left intact. The orchiectomized rats were implanted with SILASTIC brand silicon tubing (Dow Corning) containing testosterone or DHT with or without daily injections of the 5 alpha-reductase inhibitor finasteride. After 7 days, rats were submitted to EFS and the intracavernosal pressure was recorded. Castration reduced the EFS-induced erectile response by 50% in comparison with intact rats and testosterone restored this decrease to normal. When finasteride was given to these testosterone-treated castrate rats, erectile response was not restored. DHT was as effective as testosterone in restoring response to EFS in castrates and this effect was not decreased by finasteride. Nitric oxide synthase activity in the penile cytosol was measured by the arginine-citrulline conversion and was found to correlate with the EFS determinations. These results show that DHT is the active androgen in the prevention of erectile failure seen in castrated rats, and suggest that this effect may be mediated, at least partially, by changes in nitric oxide synthase levels in the penis. PMID- 7534703 TI - Does the rat corpus luteum express the progesterone receptor gene? AB - To determine whether the progesterone receptor (PR) gene is expressed in rat corpora lutea (CL), PR messenger RNA (mRNA) and protein levels were examined in CL of both cycling and pregnant rats using in situ hybridization, reverse transcription-polymerase chain reaction, and Western blotting analyses. During the estrous cycle, levels of luteal PR mRNA were below the sensitivity of in situ hybridization. Although no signal could be detected in luteal cells, granulosa cells of preovulatory follicles of the same ovary expressed PR mRNA at high levels during the evening of proestrus. Likewise, CL of pregnant rats expressed undetectable levels of PR mRNA, which remained unchanged throughout pregnancy, as determined by in situ hybridization and reverse transcription-polymerase chain reaction. In addition, no PR protein could be detected in rat CL by Western analysis, indicating that luteal expression of the PR gene, if any, is negligible. To determine whether the lack of PR mRNA in the rat CL is due to progesterone-induced down-regulation of PR mRNA or to low levels of estrogen, aminoglutethimide was used to block the synthesis of progesterone in the presence and absence of exogenous estrogen, and PR mRNA levels were examined in the CL as well as the placenta. Inhibition of progesterone synthesis did increase PR mRNA levels in the placenta, and additional estrogen treatment further increased PR mRNA levels in this tissue. In contrast, neither aminoglutethimide alone nor aminoglutethimide plus estrogen induced PR mRNA expression in CL of the same rats. The temperature-sensitive luteal cell line derived from the rat CL, which produces very low levels of progesterone, also did not express PR mRNA. These results indicate that the rat corpus luteum expresses undetectable levels of PR mRNA and protein, which is not attributable to progesterone-induced down regulation of PR mRNA or to a lack of estrogen-induced up-regulation of PR mRNA in this tissue. PMID- 7534704 TI - Insulin-like growth factor I and its binding proteins in the experimental nephrotic syndrome. AB - Insulin-like growth factor I (IGF-I) is present in serum in association with specific IGF-binding proteins (IGFBPs) primarily in a large (approximately 150K) ternary or a smaller (approximately 50K) binary protein complex or in the free form (< or = 1%). We hypothesized that glomerular proteinuria results in urinary excretion of IGF-I/IGF-binding protein complexes and that the nephrotic syndrome induces abnormal serum distribution and liver synthesis of IGF-binding proteins. In nephrotic rats, serum IGF-I levels are reduced compared with pair-fed control animals. In nephrotic rat serum, binding to IGFBP-3 is reduced and Western immune analysis demonstrates an approximately 27K fragment that does not bind IGF-I, suggesting in vivo proteolysis of IGFBP-3. In contrast, binding and serum levels of IGFBP-2 are increased in nephrotic rats, which results from increased synthesis in the liver. In Nagase analbuminemic rats, the IGF-I levels and IGFBP distribution in serum are normal suggesting that the reduced albumin levels in the nephrotic syndrome do not cause the increased liver synthesis and serum levels of IGFBP-2. Nephrotic rat urine contains IGFBP-3 and IGFBP-2 as well as strong activity of an IGFBP-3 protease. Because the 150K ternary complex in serum but not the smaller binding protein complex is restricted to the intravascular space, the shift of binding from IGFBP-3 (ternary complex) to IGFBP-2 (binary complex) in nephrotic rat serum may help to maintain tissue availability despite the reduction in serum IGF-I levels. PMID- 7534705 TI - Regulation of glucagon and glucagon-like peptide-1 receptor messenger ribonucleic acid expression in cultured rat pancreatic islets by glucose, cyclic adenosine 3',5'-monophosphate, and glucocorticoids. AB - Glucagon and glucagon-like peptide-1 (GLP-1) are important regulators of glucose homeostasis, and both are involved in regulating pancreatic islet hormone secretion. Since the sensitivity of the endocrine pancreas to regulatory hormones can be influenced by their receptor number, we have examined the regulation of glucagon receptor and GLP-1 receptor messenger RNA (mRNA) expression in cultured rat pancreatic islets by various factors, including glucose, cAMP, and glucocorticoids. By ribonuclease protection assay we have demonstrated the expression of both glucagon and GLP-1 receptor mRNA in cultured rat islets. We observed a dose-dependent increase in glucagon receptor mRNA expression with increasing glucose concentrations: an approximately 3-fold increase in glucagon receptor mRNA in islets cultured in 22 mM glucose as compared to 3.5 mM glucose. GLP-1 receptor mRNA levels, on the other hand, were not affected by culturing the islets in low glucose concentrations; however, a small, but significant, decrease in GLP-1 receptor mRNA levels was detected when islets were cultured in 20 mM glucose. Forskolin and 3-isobuty-1-methylxanthine, which increase intracellular cAMP levels, caused a 75% reduction in glucagon receptor mRNA expression. Somatostatin 14 and 28, both of which can inhibit intracellular cAMP production, stimulated glucagon receptor mRNA expression by 40% and 75%, respectively. GLP-1 receptor mRNA levels remained unchanged under all conditions that altered intracellular cAMP levels. Finally, in islets cultured in the presence of 10 nM dexamethasone an approximately 50% decrease in both glucagon and GLP-1 receptor mRNA expression was observed. These results indicate that the expression of glucagon and GLP-1 receptor mRNA is differentially regulated in rat pancreatic islets and suggest that regulation of receptor mRNA expression may be an important mechanism for controlling the sensitivity of the islets to glucagon and GLP-1. PMID- 7534706 TI - Production of thyroid-stimulating antibodies in mice by immunization with T-cell epitopes of human thyrotropin receptor. AB - We examined whether mice, immunized with TSH receptor (TSH-R) peptides, which are known to be T-cell epitopes in patients with Graves' disease, would show thyroid stimulating antibody (TSAb). We immunized DBA/1J mice with TSH-R peptide amino acids 132-150, 145-163, 158-176, and 172-186 and with a pool of these four peptides. The antibodies produced in these mice were evaluated by measurement of TSAb activity using Chinese hamster ovary cells expressing human TSH-R. Seven of 20 mice showed TSAb activity that could be partially blocked with TSH-R peptides. To assess the role of T-cell epitope-specific T-cells in the production of TSAb, we transferred a T-cell line developed from a TSAb-positive mouse to other syngeneic DBA/1J mice. Two of 4 recipient mice showed TSAb activities. These findings suggest that specific T-cell epitopes of TSH-R play a crucial role in the production of TSAb. PMID- 7534707 TI - Anti-neutrophil cytoplasmic antibodies in childhood systemic lupus erythematosus. AB - The prevalence and antigen specificity of anti-neutrophil cytoplasmic antibodies (ANCA) in sera from 23 children with active systemic lupus erythematosus (SLE) were studied utilizing indirect immunofluorescence and IgG and IgM ELISA using crude neutrophil extract and purified proteinase 3, myeloperoxidase, lactoferrin, cathepsin G and elastase. ANCA were present in 69% of SLE children and consisted of IgM and IgG antibodies of variable specificities, but did not correlate with organ involvement or disease activity. It remains unclear whether they have pathogenic significance or are epiphenomena in the category of polyclonal B-cell activation. However, their presence is entirely compatible with SLE even though they have hitherto been commonly associated with other systemic vasculitides. PMID- 7534708 TI - Nitric oxide release from rat aortic smooth muscle cells is not attenuated by angiotensin converting enzyme inhibitors. AB - We investigated the effects of angiotensin converting enzyme inhibitors on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase mRNA by vascular smooth muscle cells. Incubation of the culture with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite levels. The basal and interleukin-1 beta-induced nitrite production by vascular smooth muscle cells were not affected by the presence of angiotensin converting enzyme inhibitors (0.1 approximately 10 microM), enalaprilat, cilazaprilat or captopril. Unstimulated vascular smooth muscle cells expressed no inducible NO synthase transcripts, whereas incubation with interleukin-1 beta for 24 h induced marked inducible NO synthase mRNA expression. The angiotensin converting enzyme inhibitors, however, had no effects on the interleukin-1 beta induced inducible NO synthase mRNA expression. These results indicate that angiotensin converting enzyme inhibitors do not attenuate NO synthesis by vascular smooth muscle cells under basal and interleukin-1 beta-stimulated conditions. PMID- 7534709 TI - The dihydropyridine nitrendipine inhibits [3H]MK 801 binding to mouse brain sections. AB - The L-type Ca2+ channel antagonist nitrendipine inhibits N-methyl-D-aspartate (NMDA)-activated Ca2+ flux into cerebellar granule cells, and [3H]dibenzocyclohepteneimine ([3H]MK 801) binding to mouse cerebral cortical and hippocampal membranes. To further study this interaction between nitrendipine and NMDA-activated channels, the effects of several L-channel active agents on [3H]MK 801 binding to mouse brain were investigated in an autoradiographic assay. Serial slide-mounted sagittal sections of mouse brain were labeled with [3H]MK 801 in the presence of varying concentrations of the L-channel active agents nitrendipine, nimodipine, nifedipine, Bay K 8644, and verapami. Nitrendipine potently displaced 2 nM [3H]MK 801 binding to mouse brain sections (IC50 = 89.8 nM). Dose-dependent inhibition of [3H]MK 801 binding by nitrendipine was demonstrated in most brain regions examined. 10(-5) M and 10(-8) M concentrations of the other dihydropyridines studied, and of verapamil, were without effect. The data supports a unique, direct interaction between nitrendipine and the NMDA activated ion channel. PMID- 7534710 TI - Cytokine-mediated expansion of 5-FU-resistant peripheral blood stem cells. AB - We evaluated the expansion capacity of untreated and 5-fluorouracil (5-FU) resistant peripheral blood stem cells (PBSC) after 7-day incubation with interleukin-1 (IL-1) plus IL-3 plus stem cell factor (SCF) or with medium alone. We found a significant increase in the proportion of CD34+ cells in the PBSC fraction resistant to 25 micrograms/mL 5-FU after 7-day incubation with IL-1 plus IL-3 plus SCF as compared with the untreated fraction (p = 0.011). We also showed that 5-FU-resistant PBSC have a greater capacity for expansion of IL-1/IL-3/SCF responsive immature progenitors (p = 0.05), amplification of IL-3 plus GM-CSF responsive progenitors (p = 0.01), and production of committed single growth factor-responsive (granulocyte-macrophage colony-stimulating factor [GM-CSF]) precursors (p = 0.01) than the untreated PBSC. The expansion of all types of progenitors and CD34+ cells was only observed after 7-day incubation with IL-1 plus IL-3 plus SCF. These results suggest that PBSC contain a primitive stem cell population with an enhanced expansion capacity that is identified by 5-FU resistance. As these cells can be expanded in vitro, they may then be suitable for a number of clinical applications. PMID- 7534712 TI - IL-10 inhibits IL-7-mediated murine pre-B cell growth in vitro. AB - B lymphopoiesis in the bone marrow is mediated by both positive and negative regulatory cytokines. In this report, we demonstrate that interleukin-10 (IL-10) may function to inhibit murine IL-7-dependent pre-B cell growth. Recombinant IL 10 (rmIL-10) inhibited BALB/c bone marrow IL-7 colony-forming unit (CFU) in a concentration-dependent manner, and growth was restored when IL-10 was neutralized with the monoclonal anti-IL-10 antibody, SXC-1. Enriched populations of B220+ bone marrow B lineage cells were also inhibited in their responses to IL 7 by exposure to rmIL-10, suggesting that pre-B cells were directly susceptible to rmIL-10 inhibition. Heterogeneity in the capacity of IL-7 CFU to be inhibited by IL-10 was evident. Although 60% of IL-7 CFU were inhibited by rmIL-10 at 5 U/mL, approximately 20% of IL-7 CFU were not inhibited by rmIL-10 concentrations up to 50 U/mL. Prior incubation of bone marrow cells for 24 hours with IL-7 prevented rmIL-10-mediated growth inhibition, suggesting that prior rIL-7 stimulation of pre-B cells abrogates the inhibitory effects of rmIL-10. These experiments indicate that IL-10, at these concentrations, may function as a potent negative growth regulator for a significant fraction of IL-7-responsive pre-B cells. PMID- 7534713 TI - Development of functional macrophages from embryonal stem cells in vitro. AB - When cultured under appropriate in vitro conditions, embryonal stem cells (ESCs) form embryoid bodies (EBs) that contain mature hematopoietic cells, including cells of the monocyte-macrophage lineage. A two-step in vitro culture system for generation of ESC-derived macrophages has been developed and optimized. Maximum numbers of macrophage-containing colonies developed in secondary hematopoietic cultures of cells from disrupted EBs after 9 to 12 days of differentiation when interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) were included in both primary and secondary cultures. Over 10(5) viable, phagocytically active macrophages were generated from cultures initiated by 7500 ESCs. The inclusion of stem cell factor (SCF) in primary cultures not only increased the frequency of progenitor cells but also the cellular heterogeneity of colonies. SCF in secondary cultures increased the cellularity, but not the frequency, of macrophage-containing colonies; although cellular heterogeneity was also increased, there was still an overall increase in yield of macrophages. PMID- 7534711 TI - Positive selection of CD34+ hematopoietic cells using an immunoaffinity column results in T cell-depletion equivalent to elutriation. AB - Acute graft-vs.-host disease (GVHD) continues to present a barrier to successful allogeneic marrow transplantation. T cell-depletion may prevent severe GVHD but carries an increased risk of graft rejection and relapse posttransplant. Clinical trials have defined the number of lymphocytes associated with sustained engraftment but low risk of significant GVHD (greater than grade I or II skin only) as < or = 10(5)/kg. We examined T cell-depletion resulting from positive selection of CD34+ hematopoietic cells with a biotinylated monoclonal anti-CD34 antibody and an immunoaffinity column. Eleven patients (six myeloma and five breast cancer) underwent both peripheral blood stem cell (PBSC) collection and marrow harvest prior to autologous transplantation. One PBSC collection and one third of each marrow underwent column separation. PBSCs were enriched for CD34+ cells from an initial mean of 1.5 to 53.3%, while marrow went from an initial mean of 2.8 to 65.4%. PBSC were depleted of CD3+ cells from an initial mean of 9.6 x 10(9) to 8.6 x 10(6). Marrow CD3+ lymphocyte content was reduced from an initial mean of 5.6 x 10(9) to 8 x 10(5). Since the column permits quantification and salvage of depleted T cells, its use should allow re-addition of T cell aliquots associated with minimal risk for GVHD and rejection. In addition, since PBSCs were as readily depleted as marrow, allogeneic PBSC transplant may be feasible using this method. PMID- 7534714 TI - Quality of hematologic recovery in patients with aplastic anemia following cyclosporine therapy. AB - To evaluate quality of hematologic recovery in aplastic anemia (AA) patients treated with cyclosporine A (CyA), we examined polymorphonuclear leukocytes (PMNCL) from 25 AA patients for clonality and glycosyl-phosphatidylinositol (GPI) anchored membrane protein expression. Using three different X-linked gene probes, we failed to detect clonal hematopoiesis in seven CyA-responsive female patients. Clonal hematopoiesis was detected in two of six female patients refractory to CyA therapy, although one of these two patients had shown monoclonality before therapy. Flow-cytometric analysis revealed a normal expression of GPI-linked membrane proteins, including CD55, CD59, and CD16 on PMN in all patients treated with CyA, irrespective of response, except for one patient who had a small proportion of GPI-anchored membrane protein-negative cells before therapy. The proportion remained unchanged 41 months after hematologic recovery following CyA therapy. These findings suggest that successful therapy of AA with CyA may not be associated with a significant risk of developing late clonal complications, such as paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplasia. PMID- 7534715 TI - In vitro chronopharmacology of recombinant mouse IL-3, mouse GM-CSF, and human G CSF on murine myeloid progenitor cells. AB - Circadian changes in in vitro pharmacodynamic effects of recombinant mouse interleukin-3 (rmIL-3), rm granulocyte-macrophage colony-stimulating factor (rmGM CSF), and recombinant human G-CSF (rhG-CSF) were investigated in 418 male B6D2F1 mice. Seven distinct experiments were staggered from July to December 1991. All mice were standardized for 3 weeks with a lighting schedule consisting of 12 hours of light and 12 hours of dark (LD12:12). In each experiment, bone marrow was sampled from separate groups of nine to 10 mice each every 4 hours for 24 hours. Data were analyzed with analysis of variance (ANOVA) and Cosinor. This latter method computes the probability of rhythm detection and its parameters. Femoral myeloid progenitors were quantified using the colony-forming units granulocyte/macrophage (CFU-GM) assay in the presence or absence of recombinant CSFs. For each CSF, the number of colonies is a function of circadian time of bone marrow exposure (ANOVA and Cosinor; p < 0.0001) with the values at peak time being double those found at the trough. Peak CSF efficacy occurred at 3 hours after light onset (HALO, early rest span) irrespective of CSF type or dose. Furthermore, in the absence of any added CSF, the number of clusters varied significantly according to sampling time, with a similar peak at 3 HALO (ANOVA and Cosinor; p < 0.001). Further in vivo chronopharmacologic experiments are needed to assess the relevance of these in vitro rhythms in bone marrow responsiveness to hematopoietic growth factors. PMID- 7534716 TI - G-CSF is a major component of colony-stimulating activity (CSA) in the plasma of patients with multiple myeloma after treatment with high-dose melphalan (HDM). AB - Colony-stimulating activity (CSA) was measured by the production of granulocyte macrophage colony-forming units (GM-CFU) from normal donor bone marrow in the plasma of 29 patients with multiple myeloma (MM) after intensive treatment with high-dose melphalan (HDM) with or without autologous bone marrow rescue (ABMR). Although patients who received ABMR had an earlier recovery of circulating neutrophils compared with those who received HDM alone, the time at which CSA reached a maximum was similar in both groups (10 to 11 days) after therapy. The decline in CSA correlated with the recovery of the neutrophil count. In plasma from patients who received recombinant human granulocyte colony-stimulating factor (rhG-CSF), in addition to an autograft, CSA reached a maximum earlier (7 days). Furthermore, neutrophil recovery was earlier in these patients. Platelet recovery was not increased by rhG-CSF. The time at which CSA was maximum in four patients who were undergoing intensive therapy for the second time occurred 9 days after treatment with HDM. Although the period without neutrophils was longer in three (of four) patients who survived long term, one patient who received rhG CSF had a shorter period of neutropenia than the two who had not had the cytokine. G-CSF was detected in plasma from seven of seven patients but not at all times after treatment. In plasma samples that contained G-CSF, colony numbers were increased by recombinant interleukin-4 (rIL-4) in vitro. Neither IL-3 nor GM CSF was detected in plasma; however, antibody to GM-CSF reduced CSA in all samples after intensive therapy. The data suggest that CSA is a consistent physiologic response to intensive therapy, even in previously treated patients, but that hematologic recovery is dependent on the availability of viable progenitor cells. PMID- 7534717 TI - Neurotoxicity induced by prenatal exposure to MPTP on the monoaminergic and peptidergic systems of the marmoset brain. AB - The neurotoxicity induced by incidental prenatal exposure to 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was studied in three marmosets. The baby marmosets exposed in utero to MPTP looked normal in the first few weeks of life but around 8 to 10 weeks of life they started to behave abnormally and were sacrificed when they were 20 weeks old. A marked reduction in DA levels was found in the baby marmosets prenatally exposed to MPTP as compared to the corresponding age-matched controls and this was highly significant in the caudate nucleus, putamen, and nucleus accumbens. Serotonin content was normal in the caudate and putamen and was only significantly reduced in the nucleus accumbens, hypothalamus, and cingulate cortex. Met-enkephalin levels were reduced in the caudate nucleus, putamen, and globus pallidus. Substance P content tended to be lower in all regions examined; however, the decrease was only statistically significant in the substantia nigra. These results indicate that prenatal exposure to MPTP induces a marked and long-lasting alteration in monoaminergic and peptidergic systems of the primate brain. This observation may provide a new insight into the role of toxins in the etiology of Parkinson's disease. PMID- 7534718 TI - Early axonal regeneration: repression by Schwann cells and a protease? AB - We have proposed that mature Schwann cells and an extracellular protease repress the sprouting response of axons. To test this hypothesis, we destroyed all cells by freezing a short span of the rat sciatic nerve or inhibited proteases with subperineurial injections of aprotinin, and a crush was made to induce the sprouting response. In unconditioned or in vehicle-injected nerves, axons began to elongate at a constant rate after a delay of about 1 day. The freezing of the nerve distal to the crush obliterated the delay, but the rate of elongation did not change. A similar pattern was observed when the nerve segment was conditioned with aprotinin for 2 days prior to the crush. These effects were abolished when a short untreated segment was left between crush and conditioned region of the nerve. The electron microscopy of the nerve and the immunolocalization of the growth-associated protein (GAP-43) were consistent with the enhanced regrowth observed in conditioned nerves. Our findings support the notion that Schwann cells repress the onset of regeneration and that a local protease is involved. PMID- 7534719 TI - Neural regeneration and neuronal migration following injury. I. The endocrine hypothalamus and neurohypophyseal system. AB - Central to this investigation are several basic hypotheses that are designed to test the role of nitric oxide (NO) in the complex process of central regeneration and plasticity in a well established model system of the mammalian brain. We have employed histochemical techniques at the light and ultrastructural level coupled with correlative scanning electron microscopy, immunoelectron microscopy, and in situ hybridization in order to determine the functional significance of the increased expression of nitric oxide synthase (NOS) in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei which accompanies regeneration of their axotomized neurites following hypophysectomy. The aim of this investigation was to determine the potential role and temporal up-regulation of NOS in this basic regenerative process and to establish the ultrastructural and neuroanatomical correlates during critical periods of regeneration and regrowth of SON and PVN axons following hypophysectomy in the endocrine hypothalamus of the rat. Our data support the hypothesis that NO may serve as a second messenger molecule that may act in some fashion to govern not only the process of central regeneration and regrowth of magnocellular (SON/PVN) axons into the median eminence, neural stem, and neural lobe (the neurohypophyseal system) but may also influence the regeneration of neurites into new neuroanatomical domains such as the adjacent lumen of the third cerebral ventricle. We have demonstrated a distinct temporal relationship between injury (axotomy) of SON/PVN axons and the establishment of new neurovascular zones following hypophysectomy with the up-regulation of NOS. This up-regulation appears to correlate well with successful regeneration in the mammalian neurohypophyseal system. We have also successfully inhibited axonal regeneration with the use of nitroarginine, a competitive antagonist of NO. NOS up-regulation attendant to regeneration of SON and PVN axons may have inestimable clinical implications, particularly with respect to closed head injury and cerebral contusion that involves the mechanical shearing of the infundibular stalk. In addition, this investigation has reaffirmed that large numbers of bona fide neurons migrate and emerge upon the floor of the adjacent third cerebral ventricle shortly following hypophysectomy (within 2 weeks). The origin and mechanisms of neuronal migration and plasticity following hypophysectomy are the subject of interpretation and discussion in this investigation. PMID- 7534720 TI - Inhibition of tumor necrosis factor is protective against neurologic dysfunction after active immunization of Lewis rats with myelin basic protein. AB - Increasing evidence indicates that the cytokines, tumor necrosis factor (TNF), interleukin-1, and/or interferon-gamma, may play a crucial role in the pathogenesis of multiple sclerosis. Several reports demonstrated that inhibition of TNF is highly protective in experimental allergic encephalomyelitis (EAE) when sensitization is accomplished by the passive transfer of myelin basic protein (MBP) sensitized lymphocytes. However, successful protection has not been reported in EAE that is induced by active immunization with MBP. We examined the effects of a TNF inhibitor, dimeric polyethylene glycol linked form of the type I soluble receptor of TNF, PEG-(rsTNF-RI)2, on actively acquired EAE. Treatment with PEG-(rsTNF-RI)2 at 0.3-3 mg/kg every other day or every third day starting on Day 9 postimmunization with MBP during the effector phase of EAE significantly inhibited clinical signs in a dose-dependent manner. Histological examination of the central nervous system indicated that the administration of PEG-(rsTNF-RI)2 reduced, in part, the cellular infiltrate, particularly in the lumbar and sacral regions of the spinal cord. These studies suggest that TNF is a pivotal mediator of the inflammation resulting from the complete immune response induced by active immunization with MBP. PMID- 7534721 TI - Characteristics of BDNF-induced weight loss. AB - There is evidence that central infusion of brain-derived neurotrophic factor (BDNF) induces weight loss in rats. We have begun to investigate the physiological basis for BDNF-induced weight loss by assessing its relationship to (a) appetite, (b) serum indices of metabolic and renal toxicity, and (c) brain monoamine activity in areas associated with feeding or motor function. BDNF (0-6 microgram/day) was infused into the lateral ventricle (LV) of male Long-Evans rats for 14 days. Body weight and food intake were monitored throughout infusion and recovery periods. BDNF induced severe, dose-dependent appetite suppression and weight loss. Although appetite began to recover after the 10th infusion day, body weight had not returned to control values at the end of the recovery period. The weight loss observed in BDNF-infused rats was related to appetite suppression, since uninfused rats that were pair-fed to high dose BDNF-treated rats showed comparable weight loss. Despite severe weight loss, serum BUN, creatinine, thyroxine, glucose, and total protein were not affected by BDNF infusion. Striatal DO-PAC/DA was similarly unaffected by BDNF. In contrast, BDNF infused rats showed a dose-dependent increase in hypothalamic 5-HIAA/5-HT that was not observed in pair-fed rats, suggesting that the observed increase in hypothalamic 5-HIAA/5-HT was a direct effect of BDNF infusion rather than a secondary effect of food restriction. These data suggest that BDNF may induce appetite suppression and weight loss through a central mechanism. PMID- 7534722 TI - Unique specificity of in vitro inhibition of mosquito midgut trypsin-like activity correlates with in vivo inhibition of malaria parasite infectivity. AB - Synchrony in the egress of Plasmodium ookinetes from the food bolus and enzymatic digestion of the blood meal in the mosquito midgut suggests that digestive enzymes play a role in the successful transmission of malaria parasites. Previously, we found that parasite-produced chitinase is essential for parasite transmission and can be activated by mosquito midgut protease. To determine the suitability of developing a transmission-blocking vaccine directed against mosquito trypsin-like enzyme(s), Aedes aegypti midgut trypsin-like proteases were characterized biochemically and compared to a mammalian trypsin. Mosquito trypsin is more sensitive to inhibition by aprotinin and less sensitive to egg white trypsin inhibitor than is bovine pancreatic trypsin. Soybean trypsin inhibitor and leupeptin inhibit both enzymes to similar extent. Membrane-feeding assays with aprotinin, leupeptin, and egg white trypsin inhibitor revealed a correlation between in vitro inhibition of mosquito trypsin-like activity and transmission blocking activity. The results suggest a role for mosquito midgut trypsin(s) in malaria parasite development and indicate that the protease(s) is a potential target for blocking malaria transmission. PMID- 7534723 TI - Toxoplasma gondii: identification and characterization of a cyst molecule. AB - Monoclonal antibodies were raised against the RRA strain of Toxoplasma gondii, and those with specific reactivity against tissue cysts were selected by differential FAST-ELISA screen. One clone, E7B2, reacted with a novel cyst specific molecule of approximate molecular weight 29,000. The molecule is predominantly found in soluble fractions from intact cysts and bradyzoites, and immunofluorescence studies suggest that it accumulates in the matrix of the cyst. Western blotting experiments show the molecule to be a minor antigen recognized by polyclonal antisera from infected mice. PMID- 7534724 TI - Schistosoma mansoni: molecular cloning and sequencing of the 200-kDa chemotherapeutic target antigen. AB - Praziquantel is the drug of choice for human schistosomiasis. The efficacy of this drug is impaired in immune-deficient mice. However, transfer to B cell depleted mice of a monoclonal antibody that recognizes a 200-kDa GPI-anchored glycoprotein of S. mansoni restores the effectiveness of praziquantel. In order to characterize this target antigen, we have isolated and sequenced cDNA clones encoding the 200-kDa protein. Three overlapping cDNA clones contained the complete nucleotide sequence. The sequences of five tryptic peptides from the native 200-kDa protein could be matched with regions in the amino acid sequence deduced from the nucleotide sequence of the isolated clones. This deduced amino acid sequence differed from sequences available in six databases. Praziquantel exposes epitopes on the worm surface that are normally not exposed, and we have shown by immunofluorescent staining that the fusion protein encoded by one of our cDNA clones expresses epitopes that are exposed on the surface of praziquantel treated worms. PMID- 7534725 TI - Plasmodium yoelii yoelii 17XL MSP-1: fine-specificity mapping of a discontinuous, disulfide-dependent epitope recognized by a protective monoclonal antibody using expression PCR (E-PCR). PMID- 7534726 TI - Does the transcription factor c-ets1 take part in the regulation of angiogenesis and tumor invasion? AB - The c-ets1 proto-oncogene encodes a transcription factor that binds a GGAA/T purine rich core DNA sequence. During normal as well as pathological development, the expression of c-ets1 is associated with the occurrence of invasive processes, either in invading cells or in the invaded tissue. Cellular regulatory sequences responsive to the c-Ets1 proteins include a urokinase-type plasminogen activator (u-PA) gene enhancer, the stromelysin-1 and the collagenase-1 gene promoters. Since invasive processes are thought to require the remodeling of the extra cellular matrix, we investigate the relationships between c-Ets1 and the expression pattern of transcripts encoding these matrix degrading proteases, in embryos and in solid tumors. PMID- 7534728 TI - Role of nitric oxide in the regulation of renin and vasopressin secretion. AB - Research during recent years has established nitric oxide as a unique signaling molecule that plays important roles in the regulation of the cardiovascular, nervous, immune, and other systems. Nitric oxide has also been implicated in the control of the secretion of hormones by the pancreas, hypothalamus, and anterior pituitary gland, and evidence is accumulating that it contributes to the regulation of the secretion of renin and vasopressin, hormones that play key roles in the control of sodium and water balance. Several lines of evidence have implicated nitric oxide in the control of renin secretion. The enzyme nitric oxide synthase is present in vascular and tubular elements of the kidney, particularly in cells of the macula densa, a structure that plays an important role in the control of renin secretion. Guanylyl cyclase, a major target for nitric oxide, is also present in the kidney. Drugs that inhibit nitric oxide synthesis generally suppress renin release in vivo and in vitro, suggesting a stimulatory role for the L-arginine/nitric oxide pathway in the control of renin secretion. Under some conditions, however, blockade of nitric oxide synthesis increases renin secretion. Recent studies indicate that nitric oxide not only contributes to the regulation of basal renin secretion, but also participates in the renin secretory responses to activation of the renal baroreceptor, macula densa, and beta adrenoceptor mechanisms that regulate renin secretion. Histochemical and immunocytochemical studies have revealed the presence of nitric oxide synthase in the supraoptic and paraventricular nuclei of the hypothalamus and in the posterior pituitary gland. Colocalization of nitric oxide synthase and vasopressin has been demonstrated in some hypothalamic neurons. Nitric oxide synthase activity in the hypothalamus and pituitary is increased by maneuvers known to stimulate vasopressin secretion, including salt loading and dehydration. Administration of L-arginine and nitric oxide donors in vitro and in vivo has variable effects on vasopressin secretion, but the most common one is inhibition. Blockade of nitric oxide synthesis has been reported to increase vasopressin secretion, but again variable results have been obtained. An attractive working hypothesis is that nitric oxide serves a neuromodulatory role as an inhibitor of vasopressin secretion. PMID- 7534729 TI - Inhibition of Na/Ca exchange by Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides in intact rat pancreatic B-cells. AB - Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides were recently shown to inhibit Na/Ca exchange in cardiac sarcolemmal vesicles. In the present study, we examined the effects of FMRFa-related peptides on Na/Ca exchange in intact (pancreatic B) cells. At 2.8 mM glucose, FMRFa-related peptides only weakly inhibited Na/Ca exchange although their effect was more marked under depolarizing conditions. The peptides blocked neither the Na/K-ATPase nor Ca2+ channels but slightly reduced membrane K+ permeability. Our data indicate that FMRFa-related peptides are weak and non-specific inhibitors of Na/Ca exchange in intact B cells. The data do not confirm the view that the peptides may exert some of their physiological modulatory role by inhibiting Na/Ca exchange. PMID- 7534727 TI - [The synergistic effect of interferon-gamma on the induction of nitric oxide synthase by lipopolysaccharide in vascular smooth muscle]. AB - Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of nitric oxide synthase (iNOS) in vascular smooth muscle (VSM) which produces large quantities of nitric oxide (NO) and profound vasodilation. This process has been implicated as the cause of gram-negative septic shock. It has been demonstrated that interferon-gamma (IFN) markedly potentiates cytokine-induced NO synthesis in various cell types. However, little is known about the mechanism of this enhancing effect of IFN. The present study was undertaken to investigate the effect of IFN on LPS-induced NO synthesis and iNOS mRNA expression in VSM and the possibility of nuclear factor kB (NFkB) involvement in the effect of IFN. LPS induced NO synthesis is markedly potentiated by IFN in VSM. Expression of iNOS mRNA in VSM costimulated with IFN and LPS was greatly increased compared to that induced by LPS alone. IFN did not alter the lifetime of iNOS mRNA. LPS stimulated translocation into the nuclei of NFkB which is believed to play an important role in the induction of iNOS, but IFN did not enhance NFkB activation by LPS. These results suggest that the enhancing effect of IFN is due to the increased transcription of the iNOS gene rather than a decreased degradation of iNOS mRNA and that the NFkB activation pathway is not involved in this effect of IFN. PMID- 7534730 TI - Bovine activin receptor type II cDNA: cloning and tissue expression. AB - The cDNA encoding the bovine activin type II receptor has been cloned by reverse transcription-polymerase chain reaction (RT-PCR) amplification of a bovine testicular RNA preparation. Sequence comparisons of the bovine activin type II receptor with its human, mouse and rat homologues show strong evolutionary conservation at the nucleotide level of 94.9%, 93.5%, 92.9% and at the amino acid level of 98.6%, 99.0%, 98.8%, respectively. Bovine activin type II receptor mRNA is widely but not strongly expressed in reproductive tissues, with a major RNA band at 6 kb and minor bands at 5 kb and 3 kb. The differential levels of expression observed in these tissues suggest that levels of bActRII gene expression are regulated. Furthermore, we have observed decreasing levels of the bovine activin type II receptor mRNA with testes maturation. PMID- 7534731 TI - Multiple promoter elements in the human chorionic gonadotropin beta subunit genes distinguish their expression from the luteinizing hormone beta gene. AB - The beta subunit of human chorionic gonadotropin (CG beta) is encoded by a cluster of six genes, which have developed through gene duplication from an ancestral LH beta gene. Despite approximately 90% sequence homology between the CG beta and LH beta promoters, the CG beta gene is expressed in the placenta, whereas the LH beta promoter is active only in the pituitary. The CG beta gene uses a TATA-less promoter that is located upstream of the transcriptional start site used by the homologous LH beta gene. The purpose of this study was to use the high degree of homology among members of the CG beta gene cluster and between the CG beta and LH beta promoters to localize regulatory elements that confer CG beta expression in the placenta. The 5'-flanking regions of the different CG beta genes were cloned and expressed in JEG-3 placental cells. Naturally occurring sequence variations were correlated with promoter activity and used to identify candidate regulatory elements. Exchanges of homologous sequences in the CG beta 5 and LH beta proximal identified three separate regions between -362 and +104 that are necessary for full basal expression of the CG beta promoter. Site-directed mutagenesis of four evolutionarily divergent sequences near the CG beta transcription start site confirmed the importance of multiple distinct regulatory elements as each of these mutations resulted in an 80% decrease in promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534732 TI - The nucleotide sequences of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary. AB - Inhibition of the growth of hormone related human tumor cells in vitro by GnRH agonists and antagonists suggests a direct effect on cell growth and proliferation, and this effect may be achieved through its receptors present in tumor cells. However, the nature of the GnRH receptors present in these tumors is controversial. To determine the molecular characteristics of GnRH receptors in such tumors, we used the reverse transcriptase/polymerase chain reaction (RT/PCR) technique to clone these receptors. Primers were selected from the human pituitary GnRH receptor cDNA sequence to amplify the open reading frame and parts of its 5' and 3'-untranslated sequences. Nucleotide sequencing of the GnRH receptor cDNAs from a breast tumor cell line (MCF-7) and from an ovarian tumor showed identity with that of the human pituitary GnRH receptor which binds GnRH with high affinity. GnRH receptor mRNA was found to be expressed in human pituitary, breast, breast tumor, ovary, ovarian tumor, prostate, prostate tumor and in breast tumor cell lines (MCF-7 and MDA-MB 468) and prostate tumor cell lines (PC-3 and LNCaP). These findings demonstrate that a mRNA representing the pituitary form of the GnRH receptor (which shows high affinity binding with GnRH) is also expressed in certain normal tissues and in hormone related human tumors and tumor cell lines derived from them. PMID- 7534733 TI - Studies on the molecular regulation of the inducible form of nitric oxide synthase (iNOS) in insulin-producing cells. AB - Nitric oxide, a radical generated by the enzyme nitric oxide synthase (iNOS), may be an important mediator of beta-cell damage in early insulin-dependent diabetes mellitus. We have investigated the molecular regulation of iNOS in insulin producing RINm5F cells. The data obtained suggest that iNOS is maximally induced in these cells by a 6-h exposure to IL-1 beta or TNF-alpha + IFN-gamma, but not by endotoxin. iNOS mRNA degradation is rapid and it is not affected by IL-1 beta. Interestingly, NO seems to induce a negative feedback on iNOS expression, probably by decreasing iNOS transcription. PMID- 7534734 TI - IGF-binding proteins are differentially regulated in an ovarian granulosa cell line. AB - We have recently established an immortalized granulosa cell line as a model system to investigate ovarian function, with particular emphasis on the insulin like growth factor (IGF) regulatory system. Previous results have shown that these cells express mRNAs for IGF-binding proteins (IGFBPs)-2 to -5. These IGFBPs are also detected by ligand blots. The current work evaluated the regulation by the IGFs and cAMP on the IGFBPs and their mRNAs and compared the findings to that in primary culture. Our results indicate that levels of the IGFBPs are controlled, in part, by expression of the mRNAs. However, evidence for post transcriptional regulation was also discovered. IGFBP-3 was stimulated by IGF-I, IGFBP-4 by forskolin, and IGFBP-5 by IGF-I. IGFBP-2, -3, and -4 are expressed under basal conditions whereas IGFBP-5 is only detectable after IGF-I induction. An alteration in the biphasic actions of cAMP in this cell line, as compared to primary culture, was evident. PMID- 7534735 TI - A CD8+ T-lymphocyte-mediated and CD4+ T-lymphocyte-independent autoimmune diabetes of early onset in transgenic mice. AB - While transgenic mice expressing tumour necrosis factor-alpha under the control of the beta-cell-specific insulin promoter display a marked lymphocytic infiltration of the islets, they never develop insulin-dependent diabetes mellitus (IDDM). In striking contrast, "double" transgenic mice whose beta cells express both tumour necrosis factor-alpha as well as the co-stimulatory B7-1 molecule all develop IDDM at an early age. Furthermore, administration of anti CD8 but not anti-CD4 immunoglobulins prevents diabetes onset. These results indicate that while tumour necrosis factor-alpha induced lymphocytic infiltration is not sufficient to effect beta-cell destruction, locally co-stimulated islet infiltrating CD8+ T lymphocytes could play a critical role in the development of IDDM. PMID- 7534736 TI - Astrocytes in glutamate neurotransmission. AB - Astrocytes maintain ionic, amino acid neurotransmitter, and water homeostasis in the extracellular space of the brain. The anatomy of the cells, with their network formation and their capacity to react to and produce humoral and long distance, slow-speed transfer of information within the syncytium, makes them appear to be a class of cells able to produce integrated responses to multiple stimuli. Impairment of the control by astroglia over the extracellular milieu, e.g., glutamate (Glu) concentration, could lead to disturbances in the neuronal excitability. In this paper we summarize recent evidence of the effects of Glu interactions with astrocytes, i.e., monoamine receptor-mediated regulation of Glu carriers, Glu receptor influences on different ion-channels, and astroglial cell volume. PMID- 7534737 TI - [Effectiveness of blood substitutes based on hydroxyethyl starch in experimental burn shock]. PMID- 7534738 TI - Multidirectional contraction of human hypertrophied prostate. AB - 1. The purpose of the present investigation is to evaluate muscle contraction in two directions (longitudinal and circumferential to urethra) physiologically and morphologically for alpha-adrenoceptor agonists. 2. Norepinephrine (10(-7)-10(-4) M), phenylephrine (10(-7)-10(-4) M) and clonidine (10(-7)-10(-4) M) induced contractions in a dose-dependent manner on human prostate from patients with benign prostatic hypertrophy (BPH). 3. No significant differences were observed between longitudinal and circumferential directions of human prostate in 50% of the maximal muscle contraction (EC50 values) and the maximal muscle contractions caused by any agents used. 4. Morphometric analysis for muscle in prostates was performed using formalin-fixed, paraffin-embedded sections stained by the Mallory Azan method. 5. There was no significant difference in the density of the muscle area between longitudinal and circumferential directions of prostatic strips. 6. These results suggest that there are no significant differences in responsiveness of alpha-adrenoceptor agonists and the smooth muscle contents in longitudinal and circumferential directions to urethra, for human hypertrophied prostate. PMID- 7534741 TI - The effect of naproxen on fever in patients with advanced gynecologic malignancies. AB - The antipyretic action of naproxen has been reported as sufficiently selective for neoplasm-related fever such that the use of this agent has been recommended to distinguish neoplastic from infectious fever. The antipyretic effect of naproxen was evaluated in gynecologic oncology patients with advanced pelvic malignancies and fever without obvious source of infection (suspected neoplastic fever). Naproxen (250 mg orally every 8 hr) was given to 12 patients with (i) a daily temperature greater than 38.3 degrees C, (ii) fever for at least 3 days, (iii) no evidence of infection on physical exam, (iv) negative results of blood and urine cultures, and (v) a chest roentgenogram negative for pneumonia. Ten of the 12 patients initially received a minimum of 3 days of empiric antibiotic therapy without resolution of fever. Within 24 hr of starting naproxen therapy, 10 patients' (83%) fever responded: Eight patients (80%) had a complete lysis of fever and two had partial lysis (20%). Temperature response was accompanied by subjective improvement in patient malaise and fatigue. Naproxen therapy was continued for 5-7 days in these patients, and chemotherapy was administered to those patients scheduled to receive it. Two patients did not respond to naproxen therapy in 24 hr; thus, it was stopped and the fever workup was continued. Of these two patients, one was eventually diagnosed with bacteremia after multiple negative blood cultures and initially no response to antibiotics. Naproxen is clinically useful in the palliation of fever-related symptoms in gynecologic oncology patients with suspected neoplastic fever. Naproxen may also allow the limitation of extensive fever workups and prolonged empiric antibiotic therapy in these patients, and prevent delays in systemic therapy or supportive care. PMID- 7534740 TI - PEBA regimen (cisplatin, etoposide, bleomycin, and adriamycin) in the treatment of drug-resistant choriocarcinoma. AB - Twenty-six drug-resistant choriocarcinoma patients were treated with a PEBA regimen (cisplatin, etoposide, bleomycin, and adriamycin), supplemented by radiotherapy or surgery, from January 1988 to June 1992. A total of 80.8% of the patients had received at least four drugs, and 69.2% had received six or more cycles of combination chemotherapy. All of the patients were primarily treated with PEBA chemotherapy, 5 patients underwent hysterectomy, 9 patients received local lung and/or pelvis cobalt-60 irradiation, and 2 patients with brain metastases received whole brain irradiation. Twenty-five patients achieved a complete remission (CR 96.2%), but 7 patients relapsed. One patient with a relapse reentered sustained CR through surgery and further chemotherapy. Another patient who relapsed lives with tumor after local lung irradiation and chemotherapy. Nineteen patients (73.1%) were in sustained CR lasting at least 1 year. The side effects of the PEBA regimen were moderate and tolerable. We think that PEBA is an optimal regimen for the treatment of drug-resistant choriocarcinoma. PMID- 7534742 TI - Late presentation of an alpha-fetoprotein secreting isolated large upper abdominal retroperitoneal Sertoli-Leydig cell tumor recurrence. AB - The following case reports the surgical and adjuvant treatment of a solitary upper abdominal retroperitoneal recurrence of a poorly differentiated Sertoli Leydig cell tumor (SLCT) which presented 4 years after the original diagnosis. The patient was treated with en bloc resection of the mass, which included her left kidney, adrenal gland, and distal pancreas. She received four courses of adjuvant chemotherapy and remains without evidence of malignancy. Both the primary and recurrent tumor secreted alpha-fetoprotein allowing serologic follow up. The utility of serum alpha-fetoprotein in disease response to treatment and for surveillance of recurrence is also shown. PMID- 7534739 TI - Control of mRNA processing and decay in prokaryotes. AB - Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts. PMID- 7534743 TI - Autoimmunity, autoimmune diseases and lymphoproliferative disorders. PMID- 7534746 TI - [Study of cytochemical properties in the nucleus paragigantocellularis lateralis of rats]. AB - The distribution of cell bodies containing serotonin (5-HT)-, substance-P (SP)-, L-enkephalin (L-ENK)- and neuropeptide Y (NPY)-like immunoreactivity (IR) in the nucleus paragigantocellularis lateralis (PGCL) of the rat was studied immunocytochemically. Perikarya containing 5-HT-, SP- or L-ENK-IR were found throughout the rostro-caudal extent of the PGCL. Large, well-staining 5-HT cells densely clustered rather near the ventrolateral surface of the medulla. SP- and L ENK-IR were distributed more extensively and located more lateraly. A few of perikary on containing NPY-IR were found primarily in the area from the medium to caudal region. The results provided morphological basis for further study of the neurochemical properties of PGCL neurons involved in the regulation of cardiovascular, respiratory and other functions in the rat. PMID- 7534747 TI - Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein. AB - The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR. PMID- 7534745 TI - Cyclophosphamide (3.6 g/m2) therapy with G-CSF support for resistant myeloma. AB - BACKGROUND: In myeloma patients resistance to both melphalan- and doxorubicin containing regimens has been related to very short survival (approximately 6 months). The development of effective regimens combined with a low toxicity rate is mandatory in this patient subgroup. METHODS: Fourteen resistant myeloma patients were treated with cyclophosphamide (a total of 3.6 g/m2 was delivered in 2 doses on days 1 and 3) and prednisone (2 mg/kg, days 1-4) every month for 4 cycles. G-CSF support was administered to reduce myelosuppression. RESULTS: This combination was well tolerated. Granulocyte levels fell below 0.1 x 10(9)/L in all patients after a median of 9 days (range 8-11), followed by recovery to 0.5 x 10(9)/L after a median of 12 days from the start of treatment (range 10-13 days). Platelets never fell below 50 x 10(9)/L. All patients were treated on an outpatient basis and only 2 required hospitalization for major complications (pneumonia and heart failure). Response to cyclophosphamide was observed in 6/14 patients: 2 achieved complete remission, 4 showed a 50% or greater reduction of the M-component. Five patients are still in remission after 2, 6, 7, 9 and 10 months; 1 relapsed after 10 months. All patients except one are alive 4-16 months from the start of treatment. CONCLUSIONS: This schedule may represent a new approach for resistant myeloma, and its very low toxicity allows it to be delivered on an outpatient basis. PMID- 7534744 TI - Ifosfamide, epirubicin and etoposide (IEV) therapy in relapsed and refractory high-grade non-Hodgkin's lymphoma and Hodgkin's disease. AB - BACKGROUND: A fundamental principle in the therapeutic strategy for recurrent lymphomas is the employment of agents that are not part of the usual front line combination regimens. Ideally, the cytotoxic agents should lack complete cross resistance with those utilized up front. PATIENTS AND METHODS: A three-drug combination of ifosfamide, epirubicin and etoposide (IEV) was used to treat 20 patients with relapsing or refractory high-grade non-Hodgkin's lymphoma (HG-NHL) or Hodgkin's disease (HD). RESULTS: Of 14 patients with HG-NHL, 5 (36%) achieved a complete response (CR) and 4 partial remission (PR), giving an overall response rate of 64%. To date, all the complete responders are still in CR at +5, +5, +6, +7, and +9 months, respectively. Of 6 patients with HD, 4 (66%) obtained CR and 2 PR, giving an overall response rate of 100%. The 4 CRs are still in remission after +4, +5, +9, and +13 months, respectively. Clinical and hematologic toxic effects were moderate: neutropenia was responsible for delaying treatment for a week in 6 patients. CONCLUSIONS: These results confirm the efficacy of the IEV regimen in inducing a good remission rate with moderate side effects in relapsing/refractory HG-NHL and HD patients and they show that further investigations with this combination are warranted. PMID- 7534749 TI - Resolution of cyclic neutropenia by intramuscular gamma globulin in a case of common variable immunodeficiency with predominantly antibody deficiency. PMID- 7534748 TI - Identification of a novel missense mutation G239R in exon 6a of the CFTR gene. PMID- 7534750 TI - The capacity of human malignant B-lymphocytes to disseminate in SCID mice is correlated with functional expression of the fibronectin receptor alpha 5 beta 1 (CD49e/CD29). AB - The alpha 5 beta 1 integrin (CD49e/CD29), a heterodimeric membrane protein, is the "classical" fibronectin receptor on many cell types. During B-cell ontogeny, expression of the alpha 5-subunit is developmentally regulated. The alpha 5 beta 1 is decisive for migration on fibronectin substrate and very likely cooperates with other adhesion molecules in transvascular trafficking. To test whether alpha 5 beta 1 influences local growth vs. disseminative spread of neoplastic B-cells in vivo, human B-cell lines mimicking different maturational stages were transferred s.c. into severe combined immunodeficiency (SCID) mice and examined for alpha 5 beta 1 expression and for adherence on fibronectin substrate in vitro and ex vivo. All cell lines were locally tumorigenic. Dissemination was observed in all animals carrying Nalm-6 tumors, in one animal with a BL 60 and in 2 mice carrying a Raji tumor. By contrast, Daudi, BJAB and U266 tumors did not disseminate. As evidenced by immunohistochemistry and flow cytometry, all lines and their tumors were to various extents beta 1-positive but showed considerable differences in alpha 5 expression. The functional surface expression of alpha 5 beta 1 correlated with fibronectin adherence of the lines. Daudi expressed alpha 5 beta 1 in a non-functional configuration which was rendered functional only upon applying high concentrations of Mg++ and Mn++. B-cell lines functionally expressing alpha 5 beta 1 at high or moderate levels disseminated in SCID mice while alpha 5-negative lines and Daudi did not. These results support the conclusion drawn from an earlier in situ analysis of human B-cell lymphomas/leukemias that the alpha 5 beta 1 integrin contributes to the disseminative phenotype of malignant B cells. PMID- 7534751 TI - Circular dichroism (CD) studies on biological activity of mast cell degranulating (MCD) peptide analogs. AB - Analogs of MCD peptide were synthesized by solid-phase methods. Positive charges were deleted at the N-and/or C-terminus, including the helical portion of the molecule. Four peptides were prepared by removing residues 16-18 (Arg-Lys-Ile), 1 2 (Lys), 1-2 and 16-18 and by acetylation of the amino end (Ile). Analogs were tested on mast cells for histamine-releasing activity. Although the helicity of these derivatives, determined by circular dichroism (CD), was not significantly different from the native MCD peptide, two analogs with C-terminal deletions showed a 5- to 10-fold decrease in activity. These findings suggest that the C terminus is more important than the N-terminus in determining bioactivity of MCD peptide. PMID- 7534752 TI - A Southwest Oncology Group phase II evaluation of fluderabine monophosphate in sarcoma. PMID- 7534753 TI - Susceptibility of methicillin-resistant Staphylococcus aureus clinical isolates to various antimicrobial agents. IV. Aminoglycoside-modifying enzyme AAC(6')/APH(2") is responsible for arbekacin-resistance enhanced by bleomycin. AB - Resistance patterns against various antimicrobial agents including beta-lactams, aminoglycosides, tetracyclines, fluoroquinolones, macrolides were examined for 58 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated at Hiroshima University Hospital from April to November 1992. All the MRSA strains produced type II-coagulase but not beta-lactamase. Regarding aminoglycoside modifying enzymes, 7 strains (12%) appeared to be producing aminoglycoside 4',4" adenyltransferase AAD(4',4") encoded by aadD without coproduction of bifunctional aminoglycoside 6'-acetyltransferase/2"-phosphotransferase AAC(6')/APH(2") encoded by aacA-aphD (referred to as tobramycin-resistant type, TOBr). The remaining 51 strains (88%) were phenotypically producers of both enzymes (i.e., mix-resistant type, Mixr). AAD(4',4"), encoded by aadD which was reported to be closely linked with bleomycin (BLM)-resistance determinant, could be seen in 100% MRSA strains and ca. 90% strains expressed AAC(6')/APH(2"). BLM endowed Mixr-type but not TOBr type MRSA strain with enhanced resistance to arbekacin (ABK) dose-dependently, presumably by modifying the production of AAC(6')/APH(2"). The manifestation of ABK-resistant phenotype by Mixr-type MRSA required the coexistence of BLM. Therefore, ABK must be administered carefully to cure MRSA infection in patients who have been treated with BLM. PMID- 7534754 TI - Dextran sulfate activates contact system and mediates arterial hypotension via B2 kinin receptors. AB - To define some of the mechanisms underlying dextran sulfate (DXS)-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor des-Pro2-[Arg15] aprotinin (BAY x 4620) or the specific bradykinin B2 receptor antagonist Hoe-140 on the hypotensive response to DXS. In the first study, anesthetized miniature pigs were given DXS alone, DXS plus BAY x 4620 in various doses, or saline. As expected, DXS alone produced a profound but transient systemic arterial hypotension with a concomitant reduction in kininogen. Circulating kinin levels, complement fragment des-Arg-C3a, and fibrin monomer were all increased. Treatment with BAY x 4620 produced a dose-dependent attenuation of these effects with complete blockade of the hypotension as well as the observed biochemical changes at the highest dose (360 mg). In a second study, two groups of pigs were given either DXS alone or DXS plus Hoe-140. DXS-induced hypotension was completely blocked by Hoe-140 pretreatment; however, kininogen was again depleted. We conclude, therefore, that DXS-induced hypotension is produced by activation of plasma kallikrein that results in the production of bradykinin and that liberation of bradykinin and its action on B2 receptors in the vasculature are both necessary and sufficient to produce the observed effects on circulatory pressure. PMID- 7534755 TI - Cardiac effects of desipramine. PMID- 7534756 TI - Evaluation of diagnostic criteria for autism using latent class models. AB - OBJECTIVE: To illustrate the use of latent class models for comparing alternative diagnostic criteria for autism. The models are based on the notion that the "true" classification of an individual is unknown but does exist at some unobserved, or "latent," level. Estimates of sensitivity and specificity are obtained for each set of diagnostic criteria through maximum likelihood techniques in relation to the latent standard. METHOD: In this paper, latent class models are used to compare DSM-III, DSM-III-R, and ICD-10 criteria for autism in a sample of 342 individuals with autism or other developmental disabilities. The diagnoses were made by one or more child psychiatrists who evaluated each patient and assigned a diagnosis of autism based on their own expert clinical judgment. In addition, the raters also determined whether criteria were met for the various diagnostic systems. RESULTS: The results indicate that the ICD-10 criteria agree best with the latent standard and a diagnosis based on expert opinion. CONCLUSION: It is suggested that latent class models can be usefully applied to the evaluation of other psychiatric disorders as well and represent an important new tool in evaluating diagnostic criteria by providing a way of dealing with data lacking an observable gold standard. PMID- 7534757 TI - Monoclonal antibody HK4013 recognizes an epitope specific for gastric subtype of H+,K(+)-ATPase. AB - Monoclonal antibody HK4013 raised against hog gastric vesicles dose-dependently inhibited gastric H+,K(+)-ATPase activity, formation of phosphoenzyme from ATP, and proton uptake into gastric vesicles. This antibody did not cross-react with related P-type ATPases such as hog kidney Na+,K(+)-ATPase or rabbit sarcoplasmic reticulum Ca(2+)-ATPase. It did not bind to the solubilized gastric H+,K(+) ATPase, indicating that this antibody recognizes a higher-order structural epitope. The epitope is present on the cytosolic surface of H+,K(+)-ATPase. The addition of K+ to a solution containing gastric H+,K(+)-ATPase decreased the fluorescence intensity of the enzyme labeled with fluorescein isothiocyanate (FITC), showing the conformational transition from the E1 to E2K+ form. When H+,K(+)-ATPase was preincubated with HK4013, the addition of K+ did not decrease but increased the FITC fluorescence, indicating that this antibody changed the conformational state, at least near the ATP binding site. This is in contrast with the case of monoclonal antibody HK4001, which inhibited the decrease of the fluorescence. The fact that these two antibodies recognize different epitopes is consistent with previously reported facts that monoclonal antibody HK4001 inhibits the ouabain-insensitive K(+)-ATPase activity of rat distal colon but antibody HK4013 does not, and that the former stains the rabbit distal H+,K(+) ATPase but the latter does not. PMID- 7534759 TI - Purification and properties of L-ornithine delta-aminotransferase from gramicidin S-producing Bacillus brevis. AB - In gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine delta-aminotransferase [EC 2.6.1.13]. The enzyme was purified to homogeneity. The native enzyme had a molecular weight of about 88,000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45,000. The enzyme was specific for L-ornithine (Km = 1.05 mM) as an amino donor and for 2-oxoglutarate (Km = 6.25 mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-gamma-semialdehyde, which is spontaneously cyclized to delta 1-pyrroline-5-carboxylate and L-glutamate. The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit. The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition. The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L ornithine delta-aminotransferases in residues 17-28 of the B. brevis enzyme. PMID- 7534758 TI - Identification of a biochemical lesion, and characteristic response to lipopolysaccharide (LPS) of a cultured macrophage-like cell mutant with defective LPS-binding. AB - We have previously isolated a lipopolysaccharide (LPS)-resistant mutant (named LR 9) of a cultured macrophage-like cell line, J774.1. This mutant had defective LPS binding [Hara-Kuge, S., Amano, F., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 6606-6610]. In this study, we found that: (1) LPS-binding to parental J774.1 cells was dependent on a serum factor with a molecular weight of about 60 kDa, probably LPS binding protein (LBP); (2) LPS-binding to J774.1 cells was markedly reduced by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC); (3) mutant LR-9 cells were defective in LPS-binding even in the presence of serum; (4) LR-9 cells lacked CD14 protein on flow cytometric and immunoblot analyses, but retained normal CD14 mRNA levels on RNA blot analysis; (5) small amounts of LPS (1 to 10 ng/ml) activated J774.1, but not LR-9 cells, to secrete tumor necrosis factor-alpha and to release arachidonate metabolites, whereas both J774.1 and LR-9 were activated by large concentrations of LPS (100 to 1,000 ng/ml). These results provide genetic evidence that CD14 molecules in J774.1 cells play a crucial role in LPS-binding and in LPS-triggered signal transduction, and indicate that large amounts of LPS can activate J774.1 cells without the participation of CD14 molecules. PMID- 7534760 TI - Binding of oligoguanylate to scavenger receptors is required for oligonucleotides to augment NK cell activity and induce IFN. AB - Specific palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity. To study the mechanism of IFN induction by oligonucleotides containing palindromic sequences, we investigated the possible target molecules of the oligonucleotides. Oligo-1, a 30mer single-stranded oligonucleotide with oligoG sequences next to the active palindromic sequence (AACGTT), had more activity than oligonucleotides with oligoA, oligoC, or oligoT sequences. The activity of oligo-1 was inhibited by a guanine homo-oligomer (G30), dextran sulfate, and polyvinyl sulfate. Oligo-1 bound to plastic-adherent mouse splenocytes, and the binding was inhibited by G30, dextran sulfate, and polyvinyl sulfate. Oligo-1 inhibited acetyl-LDL binding to the scavenger receptor on mouse splenocytes. These findings suggest that the binding of an extrapalindromic sequence to the scavenger receptor is required for the immunostimulatory activity of oligo-1. PMID- 7534762 TI - Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. AB - Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during apoptosis may have implications for the membrane blebbing seen during this process. PMID- 7534763 TI - Involvement of N-myristoylation in monoclonal antibody recognition sites on chimeric G protein alpha subunits. AB - Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor. PMID- 7534761 TI - Electrophysiology of the inner mitochondrial membrane. AB - The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K(+)-selective one, a high-conductance "megachannel", a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca2+ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies. PMID- 7534764 TI - The chicken oocyte receptor for lipoprotein deposition recognizes alpha 2 macroglobulin. AB - alpha 2-Macroglobulin (alpha 2M), a major plasma component in all vertebrates, is proposed to function as a broad spectrum protease inhibitor. The alpha 2M proteinase complex (activated alpha 2M; alpha 2M*) is removed rapidly by receptor mediated endocytosis in the liver. Here we demonstrate by Western blotting that alpha 2M is also present in the yolk of chicken oocytes. Plasma levels of alpha 2M are increased by estrogen, and yolk alpha 2M is partially proteolyzed, consistent with the action of cathepsin D on endocytosed alpha 2M. Two known estrogen-induced ligands of the oocyte-specific 95-kDa very low density lipoprotein/vitellogenin receptor (OVR) are also fragmented by yolk cathepsin D (Retzek, H., Steyrer, E., Sanders, E. J., Nimpf, J., and Schneider, W. J. (1992) DNA Cell Biol. 11, 661-672). Since these findings suggested a common uptake mechanism for lipoproteins and alpha 2M by oocytes, we investigated whether OVR, a member of the low density lipoprotein receptor family, functions in the metabolism of alpha 2M. Ligand blotting of oocyte membrane extracts with chicken alpha 2M* revealed that it binds to OVR. Surprisingly, the oocyte receptor also recognizes native alpha 2M, in sharp contrast to the hepatic receptor, which only binds alpha 2M*. Receptor interaction of both forms requires Ca2+; however, competition experiments suggest that alpha 2M and alpha 2M* interact with slightly different, or overlapping, sites on the receptor. Colocalization of alpha 2M and OVR in coated vesicles isolated from growing oocytes, and internalization and degradation of methylamine-activated alpha 2M by COS-7 cells transfected with OVR, strongly suggest that alpha 2M is transported into growing oocytes via OVR. We propose that this multifunctional receptor mediates pathways at the metabolic crossroads of lipoproteins and protease inhibitor complexes. PMID- 7534765 TI - Oral contraceptive-induced expression of prostate-specific antigen in the female breast. AB - Prostate-specific antigen (PSA) is widely used as a tumor marker of prostatic adenocarcinoma. We recently found that 30% of breast tumors produce PSA and that PSA is a favorable prognostic marker in female breast cancer. We measured immunoreactive PSA in cytosolic extracts of normal breast tissue from eight women receiving no medication and one woman who was receiving no medication and one woman who was receiving the progestin-containing oral contraceptive Brevicon. None of the eight cytosolic extracts of normal breast tissue contained appreciable amounts of immunoreactive PSA. However, left and right breast tissues from the woman receiving Brevicon contained high levels of PSA. This immunoreactive species was shown to have a molecular weight identical to that of seminal PSA. Furthermore, reverse transcription of RNA and polymerase chain reaction amplification produced a 571-base pair cDNA that hybridized to a labeled cDNA PSA probe. Upon sequencing, the cDNA polymerase chain reaction product was found to have 100% homology with cDNA from prostatic tissue. PSA production by breast carcinoma cell lines was achieved after in vitro stimulation with norethindrone and ethinylestradiol. Our data suggest that PSA can no longer be regarded as a specific prostatic protein because it is produced by breast tumors with good prognosis and by normal breast tissue after steroid hormone stimulation. PMID- 7534767 TI - Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts. AB - CD31 is a member of the immunoglobulin superfamily consisting of six Ig-related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM 1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31-CD31 interactions are critically important only in the initial phases of intercellular adhesion. PMID- 7534766 TI - Regulation of cell surface beta 1 integrin levels during keratinocyte terminal differentiation. AB - Integrins of the beta 1 family play a central role in controlling adhesion and terminal differentiation within the epidermis. When human epidermal keratinocytes undergo terminal differentiation, intracellular transport of newly synthesized integrins is inhibited, and mature receptors are lost from the cell surface. We have examined the mechanisms underlying these processes, using an experimental model in which keratinocytes are placed in suspension to induce terminal differentiation. The block in intracellular transport was keratinocyte- and integrin-specific since it was not observed when fibroblasts were placed in suspension and did not affect E-cadherin synthesis in suspended keratinocytes. Newly synthesized beta 1 integrins associated with an endoplasmic reticulum resident protein, calnexin; the association was prolonged when keratinocytes were placed in suspension, suggesting a role for calnexin in the inhibition of transport. After 24 h, the level of beta 1 integrin mRNA declines in suspended keratinocytes, reflecting inhibition of gene transcription, but in fibroblasts, the level remained constant. Transport of integrins could be blocked in both adherent keratinocytes and fibroblasts by inhibiting total protein synthesis, raising the possibility that transport is coupled to de novo integrin synthesis. The fate of receptors on the surface of keratinocytes was followed by confocal immunofluorescence microscopy, immunoelectron microscopy, and biochemical analysis: with the onset of terminal differentiation, endocytosed receptors were transported to the lysosomes. These experiments reveal novel mechanisms by which integrin levels can be controlled. Together with our earlier evidence for transcriptional regulation and affinity modulation of integrins, they highlight the complexity of the mechanisms which ensure that the onset of terminal differentiation is linked to detachment of keratinocytes from the underlying basement membrane. PMID- 7534769 TI - Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins. AB - Flow-related shear stress has been shown to modulate endothelial cell structure and function including F-actin microfilament organization. Focal adhesion associated proteins such as vinculin, talin, and specific integrins may play a role in the modulation of these cytoskeletal and morphological changes. Double label immunofluorescence studies indicated that, in static culture, alpha 5 beta 1 fibronectin receptors (alpha 5 beta 1 FNRs) and alpha v beta 3 vitronectin receptors (alpha v beta 3 VNRs) were found predominantly in the peripheral regions of bovine aortic endothelial cells (BAECs) corresponding to the localization of vinculin, talin, and actin microfilament terminations. In response to shear stress, concomitant with cell elongation and the appearance of stress fibers aligned with the direction of flow, there was a prominent localization of vinculin and alpha v beta 3 VNRs as the "upstream" end of the cells. Stress fiber terminations were clearly evident at these concentrations of focal adhesion-associated proteins. These data suggest that the upstream concentration of these proteins may direct shear stress-induced stress fiber formation and may function in the alignment of the fibers in the direction of flow. Levels of surface alpha v beta 3 VNRs were found to decrease in response to flow, possibly reflecting the decrease in numbers of "downstream" receptors. Unlike the arrangement of vinculin and alpha v beta 3 VNRs observed following exposure to flow, talin and alpha 5 beta 1 FNRs, in addition to being localized at the upstream end of the cell, were also evenly distributed throughout the rest of the cell. Surface levels of alpha 5 beta 1 FNRs increased in response to shear stress, perhaps providing an increased adherence of BAECs to the extracellular matrix through these receptors. These data suggest that focal adhesion-associated proteins play specific roles in the response of BAECs to shear stress. PMID- 7534770 TI - Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells. AB - The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE2 stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP 1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. PMID- 7534768 TI - The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. AB - Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an intergrin, VLA-4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin transfected cells can tether in shear flow, and then roll, through binding of the intergrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1. PMID- 7534771 TI - Substance P-related antagonists inhibit vasopressin and bombesin but not 5'-3-O (thio)triphosphate-stimulated inositol phosphate production in Swiss 3T3 cells. AB - The substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP (AntD) and [Arg6, DTrp7,9, MePhe8] SP (6-11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC50 of 0.75 microM and 2 microM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC50 of 1 microM. Strikingly, neither AntD up to 10 microM nor AntG up to 20 microM was able to inhibit GTP gamma S-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 microM AntD or 20 microM AntG. However, neither antagonist affected the dose response of GTP gamma S-stimulated inositol phosphate generation. Furthermore, 20 microM AntD had no effect on AIF-4-induced inositol phosphates in COS-1 cells transfected with G alpha q. AntD inhibited [3H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [3H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca2+ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production. PMID- 7534773 TI - Dynorphin-immunoreactive neurons in the rat nucleus accumbens: ultrastructure and synaptic input from terminals containing substance P and/or dynorphin. AB - The endogenous opioid peptide dynorphin is enriched in neurons in the nucleus accumbens, for which coexistence and synaptic interactions with substance P have been postulated. We examined the immunogold-silver localization of dynorphin and immunoperoxidase labeling for substance P in single coronal sections through the core subregion of the nucleus accumbens of acrolein-fixed rat brain tissue. Dynorphin-immunoreactive somata were more prevalent than substance P-containing neurons throughout the region sampled for ultrastructural analysis. Dynorphin labeled cells were spherical, contained unindented nuclei, and were closely apposed to other somata and dendrites, some of which also contained dynorphin immunoreactivity. The appositions were characterized by the absence of glial processes and contiguous contacts between the plasma membranes. Smooth endoplasmic reticulum and coated vesicles could also be identified in the cytoplasms on either side of the somatic or dendritic appositions. The dynorphin somata and dendrites received synaptic input from numerous unlabeled as well as dynorphin- and/or substance P-labeled axon terminals. Both types of terminals were morphologically similar in their content of small and large dense core vesicles and their formation of mainly symmetric synaptic specializations. In addition to dynorphin-immunoreactive targets, numerous dynorphin- and substance P labeled terminals also formed synapses with unlabeled somata and dendrites. In some cases, terminals separately labeled for dynorphin and substance P converged on common targets with or without detectable dynorphin immunoreactivity. Terminals colocalizing both peptides were also found to synapse on unlabeled or dynorphin-labeled somata and dendrites. Additionally, presynaptic interactions were suggested by close appositions between dynorphin- and/or substance P-labeled terminals and other terminals that were unlabeled, dynorphin labeled, or substance P labeled. These results provide morphological data suggesting nonsynaptic communication between dynorphin-immunoreactive neurons and other neurons possibly mediated through receptive sites or second messengers associated with smooth endoplasmic reticulum in the nucleus accumbens. They also indicate that, in this region, 1) the activity of dynorphin neurons may be dependent on activation of autoreceptors for dynorphin as well as substance P and 2) additional neurons lacking dynorphin immunoreactivity are most likely inhibited (symmetric junctions) by terminals containing either one or both peptides. The findings may have implications for motor and analgesic responses to aversive tonic pain transmitted through dynorphin and substance P pathways within the nucleus accumbens. PMID- 7534772 TI - Distribution and origin of serotoninergic afferents to guinea pig cochlear nucleus. AB - The distribution of serotoninergic fibers in the guinea pig cochlear nucleus was studied with serotonin immunohistochemistry. In addition, the origin of the serotoninergic fibers was determined by combining the retrograde transport of wheat germ agglutinin-apohorseradish peroxidase (gold conjugated) with serotonin immunohistochemistry. Immunoreactivity was present in varicose and nonvaricose fibers that were unevenly distributed throughout the cochlear nucleus. The fibers were most prominent in the superficial layers of the dorsal cochlear nucleus and the anterior spherical cell area of the anteroventral cochlear nucleus. Although less prominent, serotonin-positive fibers were also present in the remaining part of the anteroventral cochlear nucleus and the posteroventral cochlear nucleus. A few positive fibers were present in the auditory nerve root and the dorsal and intermediate acoustic striae. Double-labeled cells were found throughout the rostral-caudal extent of the serotoninergic system from the caudal linear nucleus to the nucleus raphe pallidus. However, most were confined to the dorsal (52%) and median (18%) raphe nuclei. Some serotoninergic cell groups contained retrogradely labeled cells that were not serotonin immunoreactive, indicating nonauditory afferents to cochlear nucleus containing other neurotransmitter substances. Serotonin may tonically modulate auditory processing within the cochlear nucleus as well as influence certain ascending auditory pathways. Most of the serotonin in the cochlear nucleus comes from superior raphe nuclei that also project to basal ganglia motor systems and limbic structures. Therefore, the effect of serotonin on the cochlear nucleus may be related to level of arousal or behavioral state. PMID- 7534774 TI - Substance P phenotype defines specificity of c-fos induction by cocaine in developing rat striatum. AB - Activation of c-fos, a member of the class of immediate early genes that act as transcription factors, may be one of the initial molecular mechanisms underlying plastic changes in gene expression in response to drugs of abuse. By combining c fos (radioactive) in situ hybridization histochemistry with nonradioactive in situ hybridization histochemistry for mRNAs encoding other striatal markers [preprotachykinin (substance P), proenkephalin, and D1 and D2 receptors], we have identified the cellular phenotype of striatal neurons activated by acute administration of cocaine to P8, P15, P28, and adult rats. At each age examined, substance P+, enkephalin- striatal neurons were the predominant class of cells in which cocaine induced c-fos gene expression. In addition, the topography of cellular activation at each age examined was distinct and reflected the topography of distribution of cells expressing high levels of substance P mRNA. We conclude that there is a marked specificity of cellular activation in striatum following acute cocaine administration restricted predominantly to subsets of substance P-expressing cells, with age-specific patterns in their topographic distribution. PMID- 7534775 TI - Medullary visceral reflex circuits: local afferents to nucleus tractus solitarii synthesize catecholamines and project to thoracic spinal cord. AB - Visceral feedback circuits in lower brainstem were elucidated with retrograde tracers by mapping neurons that issue local projections to the general visceral afferent division of the nucleus tractus solitarii (NTS) and dorsomotor vagal nucleus (DMX) in adult male rats. In study 1, spinal and intramedullary afferents to the visceral-sensorimotor complex (NTS-X) were traced to contiguous populations of cell bodies arranged in cylindrical segmental organization. NTS-X afferents derive from curvilinear arrays of neurons that parallel the efferent radiations of the solitariotegmental tract. Newly discovered afferents arise from circumscribed cell groups in the dorsal reticular formation and periventricular zone. Another source was traced to a paraambigual cell column in the apex of the rostral ventrolateral reticular nucleus (n.RVL). In study 2, catecholaminergic afferents were initially defined with combined retrograde transport immunocytochemical methods. Deposits of retrograde tracers into NTS-X transported to neurons containing tyrosine hydroxylase (TH) in the A1, C1, and C3 areas or phenylethanolamine N-methyltransferase (PNMT) in the C1 area of the n.RVL and C3 area. In study 3, it was revealed that NTS-X afferents arise, in part, as collaterals of thoracic reticulospinal neurons. Deposits of the retrograde fluorescent tracer Fluorogold into the upper thoracic cord and rhodamine-labeled microbeads into NTS-X transported to the same neurons within a subambigual locus in n.RVL and parts of nucleus raphe magnus. In study 4, dual retrograde tracer immunocytochemical analysis demonstrated that catecholamines are synthesized by a subset of neurons in the n.RVL that issue collaterals to the NTS-X and thoracic cord. Double retrogradely labeled TH- or PNMT-immunoreactive cell bodies were restricted to the C1 area within a 450-microns column bordered rostrally by the facial nucleus and ventrally by the medullary subpial surface. We conclude that visceral reflex arcs are reciprocally organized. Targets of NTS projection are also sources of local NTS-X afferent innervation. Catecholaminergic and other local afferents from reticular formation, periventricular, and spinal gray may, via collaterals, simultaneously modulate visceral reflex excitability at the level of NTS and the outflow of autonomic and respiratory motoneurons. PMID- 7534776 TI - Acquired cutaneous adherence in patients with androgen-independent prostate cancer receiving ketoconazole and doxorubicin: medication-induced sticky skin. AB - BACKGROUND: The phenomenon of sticky skin has been reported in patients with psoriasis who are receiving retinoids. OBJECTIVE: We describe therapy-induced sticky skin in eight patients receiving ketoconazole and doxorubicin for androgen independent prostate cancer. METHODS: Patients were treated with oral daily ketoconazole (400 mg every 8 hours) and intravenous doxorubicin weekly (20 mg/m2 continuously for 24 hours). Thirty-nine patients were entered into the study; retrospective information regarding sticky skin was obtainable from 28 patients or their spouses. Prostate specific antigen (PSA) values were compared in patients with and without therapy-induced sticky skin. RESULTS: Eight patients (29%) described sticky skin. Six of these patients (75%) had a PSA response. Of the 20 patients without sticky skin a PSA response occurred in 12 (60%). CONCLUSION: Sticky skin occurred in patients with androgen-independent prostate cancer who were simultaneously receiving ketoconazole and doxorubicin. To our knowledge this unique skin manifestation of acquired cutaneous adherence has not been previously described in association with chemotherapeutic or antifungal agents. The pathogenesis of acquired cutaneous adherence in these patients might relate to therapy-induced elevation of endogenous retinoids. The similar PSA response rate in patients with or without sticky skin suggests that retinoids may not play an essential role in the mechanism of antitumor activity of ketoconazole and doxorubicin. PMID- 7534778 TI - Interferon therapy for chronic urticaria. PMID- 7534777 TI - Prolonged occlusion in the treatment of psoriasis: a clinical and immunohistologic study. AB - BACKGROUND: An occlusive dressing that is both cosmetically acceptable and long term is needed for psoriasis treatment. The mechanisms that underlie the efficacy of occlusion in psoriasis are unknown. OBJECTIVE: We performed a clinical and immunohistologic study in patients with psoriasis of the effects of occlusion, topical corticosteroid alone, and occlusion plus corticosteroid, with a new prolonged dressing as the occlusive therapy. METHODS: Nineteen patients completed a 3-week study of efficacy of prolonged occlusion dressing, fluocinonide ointment, or a combination of the two. An immunohistologic study was performed in 10 patients with psoriasis treated for 1 week with prolonged occlusion. RESULTS: The combination of fluocinonide ointment and occlusion produced significantly more improvement than either treatment alone (p < 0.01). There was no significant difference between the efficacy of prolonged occlusion or fluocinonide ointment. On 4-week follow-up plaques treated with occlusion alone or combined fluocinonide and occlusion were still significantly improved (p = 0.05 and p < 0.001, respectively). None of the immunohistologic and proliferation markers assessed in psoriatic plaques was significantly affected by occlusion as compared with untreated plaques. CONCLUSION: Prolonged occlusion is an effective therapy for psoriasis either as monotherapy or in combination with a high-potency topical corticosteroid. However, the mechanism of action of prolonged occlusion alone in the improvement of psoriasis is unknown. PMID- 7534779 TI - Immunosuppression switch in pediatric heart transplant recipients: cyclosporine to FK 506. AB - OBJECTIVES: We studied rejection, allograft function and side effects, such as hypertension, renal dysfunction and hypercholesterolemia, in seven patients switched from cyclosporine-based triple-drug immunosuppression to FK 506. BACKGROUND: A subset of pediatric heart transplant recipients treated with triple drug immunosuppression consisting of cyclosporine, azathioprine and prednisone experience either persistent rejection when attempts are made to taper corticosteroids or morbidity from cyclosporine and corticosteroids. Experience with the new immunosuppressive agent FK 506 has demonstrated its effectiveness as a single agent in heart transplant recipients, and anecdotal evidence has shown that side effects such as hypertension and hypercholesterolemia may be lower. METHODS: Seven patients whom we deemed corticosteroid dependent were switched to FK 506-based therapy. Allograft function, episodes of rejection, need for corticosteroids and incidence of side effects from FK 506 were monitored. The switch to FK 506 was performed using an established protocol. Follow-up time has ranged from 15 to 41 months. Serial right heart catheterizations and endomyocardial biopsies were performed after each reduction of corticosteroid dosing. RESULTS: Catheterization data showed no significant change in pulmonary wedge pressure, mean right atrial pressure or cardiac index, indicating no decline in allograft function. Serial echocardiographic variables of allograft function were also stable. At present, all seven patients are free of the corticosteroid portion of their immune suppression. There have been only two episodes of significant acute rejection requiring treatment with intravenous corticosteroids. Antihypertensive medications have been discontinued in five of six patients previously treated with these drugs. Plasma cholesterol, low density lipoprotein and triglyceride levels were decreased, and renal function was stable. CONCLUSIONS: Preliminary studies suggest that FK 506 may be an alternative immunosuppressive agent for pediatric and adolescent patients experiencing ongoing rejection or significant morbidity from cyclosporine and corticosteroids and in those patients dependent on corticosteroids for immune suppression. PMID- 7534780 TI - Recovery of Pseudomonas cepacia and other Pseudomonas species from the environment. AB - Nine hundred sixteen cultures were obtained from homes of patients with cystic fibrosis, control homes, salad bars, and food markets, and analyzed for the presence of Pseudomonas cepacia and related bacteria. P cepacia was recovered from 5 (18%) of 27 homes, and from 20 (4%) of 509 cultures collected outside of homes. Relative to other pseudomonads, P cepacia is found infrequently in the environment. It is not clear how frequently these sources contribute to acquisition of this bacteria by persons with cystic fibrosis. PMID- 7534782 TI - Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats. AB - We describe a sensitive technique combining dual-label immunocytochemistry (ICC) with isotopic in situ hybridization histochemistry (ISHH). We developed this technique to characterize the receptor and/or peptide content of pheno-typically identified neurons that express cell markers of neuronal activity (immediate early gene products) after physiological or pharmacological perturbation. Tissue was fixed by perfusion with 4% paraformaldehyde in PBS, sucrose-infiltrated, and cryosectioned. Sections were stored in cryoprotectant or immediately hybridized. After stringent hybridization wash procedures, Fos and luteinizing hormone releasing hormone (LHRH) neurons were visualized sequentially using immunocytochemistry. Finally, galanin mRNA was detected autoradiographically. We applied the technique to study of subpopulations of LHRH-containing neurons. Results of this study indicate that a majority of the LHRH neurons activated during the luteinzing hormone (LH) surge (as indicated by presence of nuclear Fos staining) also express mRNA encoding galanin. However, there is not a complete overlap between the subpopulation of LHRH neurons that express Fos and that which expresses galanin mRNA. PMID- 7534783 TI - Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium. AB - Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine secreted by many animal and human tumors, activated macrophages, keratinocytes, rheumatoid synovial cells, embryonic tissues, and by cultured epithelial and mesenchymal cell lines. It acts selectively on vascular endothelial cells to increase their permeability to circulating macromolecules and to stimulate their replication. Although not detectably expressed by vascular cells in the human and animal tumors we have studied, VPF/VEGF accumulates in the microvessels supplying tumors and certain inflammatory reactions in which VPF/VEGF is also overexpressed. Light microscopic immunohistochemistry lacked the resolution necessary to localize VPF/VEGF precisely in such vessels. Therefore, we used a pre-embedding immunocytochemical method to localize VPF/VEGF at the ultrastructural level in the new blood vessels that are elicited in the peritoneal walls of mice bearing a transplantable mouse ascites tumor of ovarian origin. Intense immunostaining for VPF/VEGF was observed on the abluminal plasma membrane of tumor-associated microvascular endothelial cells and in vesiculovacuolar organelles (VVOs) present in these same endothelial cells. (VVOs are recently described cytoplasmic organelles present in tumor vascular endothelium that provide an important pathway for extravasation of circulating macromolecules.) In contrast to labeling of the abluminal plasma membrane and VVO vesicles and vacuoles, endothelial cytoplasmic organelles, such as multivesicular bodies and Weibel-Palade bodies, and the underlying basal lamina, did not stain with antibodies to VPF/VEGF. The distribution of VPF/VEGF here described corresponds to that anticipated for high-affinity VFP/VEGF receptors, although binding of VPF/VEGF to other endothelial cell surface structures, such as plasma membrane proteoglycans, is also a possibility. PMID- 7534781 TI - Localized distribution of alpha 9 integrin in the cornea and changes in expression during corneal epithelial cell differentiation. AB - A recently characterized integrin alpha-chain, alpha 9, forms heterodimers with the integrin beta 1-chain and is present in the skin with a distribution similar to that of alpha 2 and alpha 3, other beta 1 integrins. To determine whether alpha 9 is expressed in the stratified squamous epithelium of the cornea, we used immunohistochemical techniques to compare the distribution of alpha 9 in the adult mouse cornea with that of alpha 3. Abundant alpha 9 was expressed in the lateral and basal membranes of the basal cells of the conjunctiva and corneal limbus, but very little alpha 9 was present in the basal cells of the central corneal epithelium. In contrast, alpha 3 was present in the membranes of basal cells of the conjunctiva, limbus, and central cornea. To determine when during postnatal maturation of the corneal epithelium alpha 9 becomes restricted to the limbus, we looked at the distribution of alpha 9 and alpha 3 in the developing mouse eye from birth to eyelid opening. At birth, the basal cells of the cornea and developing limbal region did not express alpha 9, but there was abundant alpha 9 expressed in suprabasal cells between the fused lids and in the basal cells of the skin and conjunctiva. In contrast, alpha 3, integrin was expressed uniformly in the basal cells across the surface of the conjunctiva, limbus, and cornea and was present only in the basal cells of the epithelium between the fused eyelids. In the central cornea, alpha 9 expression increased in basal cells up until Day 10 after birth. After Day 10, alpha 9 expression in the central cornea began to decrease; after the lids were open, alpha 9 expression in the central cornea became restricted to the limbus. In the basal and suprabasal cells between the fused eyelids expression of alpha 9 became increasingly restricted over time to the basal cells. Recent data suggest that alpha 9 beta 1 can interact with tenascin. Our dual labeling confocal microscopy studies indicate that localization of alpha 9 and tenascin are not coordinated in the developing mouse cornea. Many recent studies have shown an important role for beta 1 integrins in mediating epithelial cell differentiation in vitro; in vivo, changes in integrin expression have been found in wound healing, psoriasis, and in basal and squamous cell carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534785 TI - Immunostaining of chain-specific keratins on formalin-fixed, paraffin-embedded tissues: a comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments. AB - The use of chain-specific monoclonal antibodies (MAbs) against keratins in pathology is hampered by their limited staining on formalin-fixed, paraffin embedded tissue. In the present study, various treatments before immunohistochemistry on paraffin sections were compared, including proteolytic enzymes and microwave antigen retrieval in various solutions. Sections of normal cervical and skin tissue were stained in a three-step immunoperoxidase method, employing a broad panel of MAbs against chain-specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pankeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antigen Retrieval Solution (ARS), a simple detergent solution (DET), PBS, and distilled water (MiQ) were compared. Microwave heating in PBS or MiQ strongly improved staining results. Moreover, microwave pre-treatment in TUF or DET gave excellent and specific staining with the majority of MAbs tested, comparable with or even better than staining obtained on frozen sections. Using microwave antigen retrieval, tissue morphology remained optimal, and only in a very limited number of MAbs did immunoreactivity on paraffin sections fail to be restored. Proteolytic pre-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pre-treatment was able to restore the loss of immunoreactivity, failed to give appropriate staining with proteolytic pre-treatment. Our results show that microwave heating in either TUF or a simple detergent solution before immunohistochemistry is a reliable method for antigen retrieval of chain-specific keratins in formalin-fixed, paraffin-embedded tissues. PMID- 7534784 TI - The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study. AB - We examined the pattern of fibulin-1 mRNA and protein expression in human tissues and cell lines. Fibulin-1 transcripts were found in RNA isolated from most tissues and a variety of cultured cells, including fibroblasts, smooth muscle cells, and several epithelial cell lines, but not endothelial cells, lymphomyloid cells, or a number of carcinoma and melanoma lines. Immunohistochemical analysis showed that fibulin-1 is an intercellular component of connective tissues, predominantly associated with matrix fibers in tissues such as the cervix, dermis, intimal and medial layers of blood vessels, heart valves, meningeal tissue of the brain, Wharton's jelly of the umbilical cord, testis, and lung. Most of the fibers that were immunoreactive with fibulin-1 antibodies also stained with antibodies to the elastic fiber proteins elastin and fibrillin, as well as with Verhoeff's elastin stain. Immunoelectron microscopic analysis of elastin fibers of skin and saphenous vein revealed that fibulin-1 was located within the amorphous core of the fibers, similar to elastin, but it was not in the fibrillin-containing, elastin-associated microfibrils. Our finding that fibulin-1 is an elastic fiber component suggests several possible new functions for fibulin-1, e.g., that it is a structural protein that contributes to the elastic properties of connective tissue fibers or that is involved with the process of fibrogenesis. PMID- 7534786 TI - Phenotypic characterization of CD4+ T cells that exhibit a transendothelial migratory capacity. AB - The phenotype of CD4+ T cells capable of transendothelial migration was determined using an in vitro model system, in which cells migrate through a monolayer of endothelial cells (EC) on collagen gels. A specific subset of resting CD4+ memory T cells was found to migrate. T cells within this subset can be defined by the bright expression of CD11a, CD26, CD44, and CD49d. Additionally, the migratory CD4+ T cell population is largely CD58bright, CD31-, CD62L-, and is also enriched in cells that brightly express CD49c, CD49e, and CD49f. Only a minority of the cells are activated, as indicated by expression of CD69. The EC were found to play a central role in facilitating migration of this subset because selective enrichment of CD11abright, CD26bright, CD44bright, CD4+ T cells was not observed when cells migrated in the absence of EC. Activation of the T cells induced a modest degree of migration of an additional subset of CD45RA+, CD31+ naive T cells. In contrast, TNF-alpha activation of the EC increased the transendothelial migration of an additional subset of activated memory T cells that expressed CD69 and CD62L. Neither activation of the T cells, stimulation of the EC, nor the presence of macrophage inflammatory protein-1 alpha (MIP-1 alpha) or RANTES, however, altered the phenotype of the majority of the migratory CD4+ T cell population, which is characteristic of a particular stage of memory cell differentiation. These results suggest that CD4+ T cells acquire the capacity for transendothelial migration at a specific phase of maturation that is only minimally altered by the activation of either the T cell or the EC, or by the presence of specific chemokines in the subendothelial matrix. PMID- 7534787 TI - B cell antigen receptor-mediated apoptosis. Importance of accessory molecules CD19 and CD22, and of surface IgM cross-linking. AB - Engagement of the B cell Ag receptor can induce a suicide pathway in various B cell types. Earlier studies showed that anti-IgM mAb treatment triggers apoptotic death in the Burkitt lymphoma-derived cell line, Ramos. We show that two B cell surface molecules, CD19 and CD22, which have been reported to interact either functionally or structurally with the B cell Ag receptor, also stimulate cell suicide when sufficiently aggregated, both in the Ramos and EBV-infected Ramos AW cell lines. In conditions of lower cross-linking, both molecules enhance the apoptotic response induced by a suboptimal dose of anti-IgM mAb in Ramos cells, reinforcing the notion that CD19 and CD22 may be involved in the death pathway and modulate Ag-induced B cell apoptosis. Similar outcomes were obtained with human tonsillar B cells, which enter the death program upon treatment with cross linked anti-IgM, -CD19, or -CD22 mAbs. These results indicate that Ag-induced B cell suicide may affect mature B cells in the periphery and may be regulated via the interaction of CD19 and/or CD22 with their respective ligand(s). Early tyrosine phosphorylations were analyzed by Western blotting. The biologic outcome of these various treatments--cell survival or death--could not be related to any detectable new tyrosine-phosphorylated substrate, further questioning the biochemical basis of apoptosis signaling. PMID- 7534788 TI - Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1. AB - Recent evidence indicates that integrins do transduce signals that are involved in the regulation of a number of cellular processes. We have shown previously that human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their adhesion to fibronectin (FN). Here we investigate whether cross linking of beta 1 FN receptors on human NK cells stimulates tyrosine kinase activation. Our results indicate that cross-linking of beta 1 integrins on NK cells, either freshly isolated from PBL or generated from 10-day coculture of nonadherent PBMC with an irradiated EVB+ lymphoblastoid B cell line (long-term activated NK cells), altered the tyrosine phosphorylation pattern. In particular, we found stimulation of tyrosine phosphorylation of two proteins migrating with an apparent mass of 105 and 115 kDa, which was not observed after CD16 engagement. Phosphorylation of pp105-115 was already observed at 1 min, raised maximal values at 3 to 5 min, and persisted until 20 min after stimulation. Tyrosine phosphorylation of pp105-115 was also observed upon engagement of alpha 4 beta 1 and alpha 5 beta 1 FN receptors either with mAb directed against the alpha subunits or after NK cell adhesion to FN or its 120- and 40-kDa fragments. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of phosphorylation stimulated through alpha 4 beta 1 and alpha 5 beta 1 integrins, indicating that ligation of FN receptors on NK cells activates tyrosine kinase(s). Overall our results suggest that beta 1 integrins on NK cells play a major role as signaling molecules in the regulation of NK cell functions. PMID- 7534789 TI - Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage. AB - We have previously reported the identification, using human immune sera, of mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous phage. To test if these mimotopes could be useful in developing a vaccine against the human hepatitis B virus (HBV), we have compared the humoral immune response of animals immunized either with a recombinant HBsAg vaccine, or with mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotopes' amino acid sequences. These immunogens were injected into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific Abs. Our data confirm that mimotopes can induce a humoral immune response resembling that induced by the original Ag, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most reproducible and potent immunization. Mimotopes that react as HBV subtype-specific Ags do not show this specificity as immunogen and induce a nonsubtype-restricted response. Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a strain of mouse reported to show a low response to it. These results indicate that mimotopes identified from random peptide libraries through utilizing human immune sera could be important leads for the derivation of new vaccines. PMID- 7534791 TI - Unique cytokine production profile of anergic human T cells in SCID-hu mice after staphylococcal enterotoxin B administration. AB - Severe combined immunodeficient mice transplanted with human organs (SCID-hu mice), provide a unique in vivo model for studying human intrathymic T cell selection and development of tolerance. In vivo administration of staphylococcal enterotoxin B (SEB) to SCID-hu mice causes intrathymic clonal deletion of SEB specific V beta+ T cells that occurs already at the immature CD4+8+ double positive stage. The expression of activation markers such as CD25, CD71, and HLA DR was specifically increased on V beta+ T cells responding to SEB. The remaining SEB-specific human T cells that had not been deleted in vivo failed to proliferate when rechallenged with SEB in vitro. These SEB-specific T cells that were rendered anergic in vivo had a unique cytokine production profile. They failed to produce IL-2, which correlated with the lack of proliferation of these cells. In addition, they failed to produce TNF-alpha. However, the anergized T cells synthesized considerable amounts of IFN-gamma, granulocyte-macrophage CSF and IL-10 after SEB stimulation. This clonal anergy can be completely reversed in vitro by stimulating the SEB-specific cells in the presence of exogenous IL-2 or by triggering of the CD28/CTLA-4 activation pathway. Under these stimulation conditions, anergic T cells produced levels of IL-2 and TNF-alpha that were comparable to their non-anergized counterparts, whereas the levels of granulocyte macrophage CSF, IL-10 and IFN-gamma production were even higher. Collectively, these data demonstrate that in vivo administration of SEB to SCID-hu mice leads to activation, deletion, and anergy of SEB-specific human thymocytes and that the production of IL-2 and TNF-alpha is selectively switched off in these anergic T cells. PMID- 7534793 TI - Strong similarities in antigen fine specificity among DRB1* 1302-restricted tetanus toxin tt830-843-specific TCRs in spite of highly heterogeneous CDR3. AB - We investigated the Ag fine specificity of four TCRs that shared the same V beta segment but used V alpha s of three different subfamilies and displayed highly heterogeneous alpha and beta CDR3. The TCRs recognized the tetanus toxin tt830 843 (QYIKANSKFIGITE) epitope presented by DRB1*1302. By using a large panel of monosubstituted peptide analogues, we first defined the requirements for tt830 843 binding to DRB1*1302. We found that three residues, I832, N835, and G840, were critical for the interaction with DRB1*1302. Residues potentially contacted by the four TCRs were functionally defined by measuring the IL-2 response to the analogues. Except for the first and the last three residues, as well as I832 and G340, all of the others appeared to provide contacts with the four TCRs, indicating a considerable overlapping in the way these TCRs interact with the peptide. More importantly, and contrary to expectations, the two TCRs expressing the same V alpha/V beta germ-line segments showed a strikingly similar reactivity toward nearly all substitutions; moreover, more pronounced differences were observed when comparing TCRs using different V alpha segments. These results indicate that TCRs with entirely distinct CDR3s in the context of conserved V segments may not differ substantially in the way they recognize the ligand, and may provide new insights into understanding the formation of TCR/peptide/MHC ternary complexes. During these studies, we noticed that analogues with nonconservative substitutions at I832, which bound very unstably to DRB1*1302, could effectively stimulate T cells, suggesting a role of the TCR in contributing toward stabilization of peptide binding. PMID- 7534790 TI - CD28 cross-linking augments TCR-mediated signals and costimulates superantigen responses. AB - The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A sensitive pathway. PMID- 7534792 TI - Expression and induction of nuclear factor-kappa B-related proteins in thymocytes. AB - Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition. PMID- 7534794 TI - Integrin regulation of an inflammatory effector gene. Direct induction of the tissue factor promoter by engagement of beta 1 or alpha 4 integrin chains. AB - Inflammatory genes are regulated in cells of monocyte (Mo) lineage by a variety of cellular encounters, including adhesion mediated by integrins. The role of the beta 1 family of integrins in the direct induction of immediate early gene expression was analyzed by using the tissue factor (TF) gene. Engagement of alpha 4 or beta 1 on Mo, but not members of the beta 2 integrin family, with specific mAbs as surrogate ligands immediately and directly induced high level surface expression of TF, and accumulation of TF mRNA, as well as production of TNF-alpha and HIV-1 virus. The mechanism responsible for induction of TF gene transcription mediated by the engagement of alpha 4 or beta 1 was elucidated by using THP-1 monoblastic leukemia cells. Functional analysis of plasmids containing the TF promoter expressing the luciferase reporter gene identified a cis-acting integrin responsive element (InRE), which contained two AP-1 sites as well as a single kappa B-like site. Mutation of either the AP-1 sites or kappa B-like site greatly diminished responsiveness to integrin engagement. This InRE also conferred responsiveness to a heterologous promoter in the same reporter plasmid. Binding of mAbs to either alpha 4 or beta 1 led to nuclear translocation of the c-Rel/p65 heterodimer that preferentially bound to the TF kappa B-like site. In contrast, constitutive binding of AP-1 proteins to the two AP-1 sites was not increased by alpha 4 or beta 1 integrin engagement. These studies expand knowledge of integrin regulation of immediate early gene expression in Mo and molecular encounters that are inferred to play an active role in Mo effector functions. PMID- 7534795 TI - Receptor-specific induction of individual AP-1 components in B lymphocytes. AB - The inducible nuclear transcription factor complex, AP-1, typically consists of heterodimers between Jun and Fos proteins. Although components are drawn from families of related molecules, little is known about the physiologic regulation of jun- and fos-related gene products. In particular, it is not known whether expression of individual family members is stimulus-specific or whether the same signaling pathways are responsible for induction of all subunits. To clarify these issues, AP-1 components were examined following activation of primary B lymphocytes through two separate receptors, the surface Ig Ag receptor, and the CD40 receptor for T cell influences. Both forms of stimulation led to expression of JunB and JunD mRNA and protein; however, induction of JunB mediated by anti-Ig Ab was protein kinase C (PKC)-dependent, whereas induction mediated by CD40 ligand was resistant to PKC depletion. The two forms of stimulation diverged even further with respect to Fos expression. Although both stimuli induced c-Fos, expression of FosB was stimulus specific at both the mRNA and protein levels, in that this component was induced by anti-Ig but not by CD40 ligand. Stimulated expression of c-Fos and FosB was in all cases PKC-independent. These results provide evidence for receptor-specific differences in the expression of AP-1 components, primarily with respect to FosB. They also indicate that separate intracellular pathways may be used for induction of jun and fos gene products and that the same transcription factor (junB) may be triggered by two surface receptors through separate pathways that differ in PKC dependence. PMID- 7534796 TI - Co-localization of inducible-nitric oxide synthase and Plasmodium berghei in hepatocytes from rats immunized with irradiated sporozoites. AB - Both CD8+ T cells and IFN-gamma (IFN-gamma) are important components in the regulation of inducible-nitric oxide synthase (iNOS) which contribute to liver stage anti-malarial activity in rodents immunized with irradiated sporozoites. IFN-gamma, provided by malaria-specific CD8+ T cells, stimulates liver cells to produce nitric oxide (NO) for the destruction of infected hepatocytes or the parasite within these cells. To identify the cell source of iNOS in livers from Brown Norway rats challenged with Plasmodium berghei sporozoites, we probed tissue sections with antisera that recognize iNOS and the malarial exoerythrocytic stage parasite. Immunofluorescence analysis of parasitized livers demonstrate that 1) iNOS was found in infected hepatocytes, not Kupffer or endothelial cells; and 2) a higher proportion of infected hepatocytes express iNOS in immunized rats compared with naive animals after challenge. There was no immunoreactivity to the iNOS antisera in liver sections of immunized rats 15 h after sporozoite challenge, however, iNOS activity was present in 18% of the infected hepatocytes by 24 h and reached 81% by 31 h. In contrast, < 10% of the infected hepatocytes displayed iNOS activity in naive or immune animals 48 h after challenge. We also found a significant decrease in the ability of the immunized animals to express iNOS in response to sporozoite challenge by accelerating the removal of pre-existing irradiated-attenuated parasites from hepatocytes with the antimalarial drug, primaquine. Therefore, induction and maintenance of iNOS activity were dependent on intrahepatic persistence of the irradiated-attenuated parasite. These results suggest that liver-iNOS expression following sporozoite challenge is restricted to the infected hepatocyte and dependent on the presence of the irradiated-attenuated parasite in immune animals. PMID- 7534798 TI - Crry and CD59 regulate complement in rat glomerular epithelial cells and are inhibited by the nephritogenic antibody of passive Heymann nephritis. AB - Human glomerular epithelial cells (GEC) contain CD59, decay-accelerating factor, and membrane cofactor protein. Crry is the rodent analogue to the latter two proteins. We have previously shown that the nephritogenic Ab of passive Heymann nephritis, anti-Fx1A, impairs C regulation in rat GEC. Here we examined rat GEC C regulation. 125I-labeled GEC membrane proteins were immunoprecipitated with anti Crry, anti-CD59, or anti-Fx1A. Crry and CD59 were present in GEC. Anti-Fx1A reacted with both Crry and CD59 from GEC, as well as with purified rCrry and CD59. The alternative C pathway was studied by incubating GEC in rat serum in Mg(++)-EGTA buffer. To inhibit the function of the C regulators, anti-Crry or anti-CD59 Ab were added to GEC. Inhibition of CD59 function alone had no effect on C regulation, whereas inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. When the classical pathway of C was activated by GEC-bound IgG Ab, inhibition of either Crry or CD59 enhanced cytotoxicity, whereas inhibition of both Crry and CD59 together was additive. Therefore, Crry and CD59 are present and functionally active in GEC. Crry restricts C activation via both alternative and classical pathways. When the classical pathway of C is activated, or when Crry function is inhibited, CD59 limits C5b-9-mediated cytotoxicity. Anti-Fx1A binds to both Crry and CD59, which may account for its ability to activate the alternative pathway in vitro, and for its superior nephritogenicity in vivo. PMID- 7534797 TI - An adenovirus type 5 early region 1B-encoded CTL epitope-mediating tumor eradication by CTL clones is down-modulated by an activated ras oncogene. AB - Mouse embryo cells (C57BL/6, H-2b) transformed by the E1A and E1B genes of adenovirus type 5 (Ad5E1 MEC) are highly immunogenic. Previously, CTL were cloned from mice immunized with Ad5E1 MEC. These CTL clones were capable of tumor eradication in nude mice, and were directed against the Ad5E1A-encoded decapeptide SGPSNTPPEI, presented by the H-2Db MHC molecule. We have now generated Ad5E1 MEC containing a mutated Ad5E1A-encoded epitope. The mutant Ad5E1 MEC induce a strong CTL response when injected into immunocompetent mice. CTL clones generated against mutant Ad5E1-transformed tumor cells recognize an Ad5E1B encoded epitope (VNIRNCCYI) in the context of H-2Db. Because this epitope is also present on wild-type Ad5E1 MEC, it is concluded that Ad5E1-transformed tumor cells express at least two CTL epitopes. Interestingly, the lysis of Ad5E1 MEC by the Ad5E1B-specific, but not by the Ad5E1A-specific, CTL clones was strongly diminished by the action of the activated ras oncogene. CTL directed against the Ad5E1B-encoded epitope were, like Ad5E1A-specific CTL, able to eradicate large established Ad5E1-induced tumors in B6 nude mice, demonstrating that CTL activity directed against different CTL epitopes expressed by the same tumor can be exploited for immunotherapy of cancer. PMID- 7534799 TI - Direct interaction of filamin (ABP-280) with the beta 2-integrin subunit CD18. AB - beta 2-Integrins mediate leukocyte extravasation into inflamed tissues, phagocytosis, and target cell killing, functions that require an intact actin cytoskeleton. Previous studies have focused on elucidating interactions of the cytoplasmic tails of integrins with the cytoskeleton at focal contacts in stationary cells. As integrins are also located at other types of cell-substratum junctions, such as the leading edge of migrating cells, additional cytosolic proteins abundant at these sites may also interact with integrins. In this study, we have identified the actin-binding protein, filamin (ABP-280), as a major cytoskeletal protein that binds to the cytoplasmic tail of the beta 2-integrin subunit CD18. Filamin bound to cytoplasmic CD18 directly and specifically, co immunoprecipitated with beta 2-integrins in detergent cell lysate and co immunolocalized with cross-linked beta 2-integrins in intact cells. The filamin binding site in CD18 was localized to the N-terminal (amino acids (aa) 724 to 747) but not to the C-terminal (aa 743 to 769) half of cytoplasmic CD18. Filamin did not bind to the major alpha-actinin binding site (aa 733 to 742), however, suggesting that these two cytoskeletal proteins bind to distinct but overlapping sites. Given the conservation of the filamin binding region among beta-integrin subunits, these findings suggest the presence of similar associations between filamin and other integrins. These associations may be important in the spreading and extension of lamellipodia at the leading edge during cell movement and, if interrupted, may contribute to the dramatic decrease in cell locomotion observed in genetic deletions involving filamin or integrins. PMID- 7534800 TI - Self-peptides in the initiation of lupus autoimmunity. AB - Systemic lupus erythematosus is characterized by high titers of autoantibodies directed at multiple proteins of the U1/Sm small nuclear ribonucleoproteins (snRNPs). The origin of this type of autoimmunity, that is, whether it is initiated by foreign molecular mimics or by the self-snRNPs, is not known. In this study using normal mice, we investigated the presence of autoreactive B and T cells to the D protein of murine snRNPs. Although neither B nor T cell responses could be detected after immunization with native self-snRNPs, two synthetic self-peptides corresponding to amino acids 26-40 and 56-70 of the snRNP D protein elicited strong autoreactive T cell proliferation as well as a limited Ab response that bound the self-protein in immunoblots. T cells elicited by these peptides did not respond to stimulation with native snRNPs, suggesting that the peptides are cryptic and are not processed from the native protein for presentation by APCs. After priming with either of these cryptic self-peptides, exposure of the immune system to native murine snRNPs resulted in a diversified response with Abs that immunoprecipitated snRNPs and that produced an antinuclear immunofluorescence pattern on murine cell substrates. These studies demonstrate that autoreactive B and T cells specific for self-snRNPs are components of the normal repertoire of mouse lymphocytes; they have been neither deleted nor irreversibly anergized. Furthermore, we show that a diverse autoimmune response to lupus autoantigens, snRNPs, can originate from self-peptides without the influence of foreign Ags or molecular mimics. PMID- 7534801 TI - Design and production of a target-specific monoclonal antibody to parvovirus B19 capsid proteins. AB - Native parvovirus B19 was used as antigen to produce a mouse monoclonal antibody, R92F6, which reacted with B19 VP1 and VP2, neutralised the virus in bone marrow culture, and labelled infected cells in paraffin-embedded tissues from cases of B19-related fetal hydrops. The B19 epitope recognised by R92F6 (amino acids 328 344 from the amino terminal region of B19 VP2) appears to be highly conserved, since these tissue specimens were obtained over a 13 year period from widely spaced locations in the UK. This epitope was synthesised as a peptide (S7b) which was used as antigen to produce a mouse monoclonal antibody, 3H8, which specifically reacted with the B19 capsid proteins VP1 and VP2 in immunofluorescence and immunoblot assays. 3H8 was also capable of labelling formalin-fixed, paraffin-embedded, B19-infected fetal tissue and was shown to be of the same isotype as R92F6 (IgG1). Highly conserved epitopes derived from conserved amino acid sequences are valuable in the diagnosis of infectious disease. If these can be recognised and accurately synthesised, the production of specific mouse monoclonal antibodies may be possible for many human pathogens. Considering the vast amount of sequence data available in the literature, this approach seems to be both feasible and of wide potential. PMID- 7534802 TI - Monoclonal antibodies differentially reactive with native and reductively modified Bowman-Birk protease inhibitor. AB - Bowman-Birk protease inhibitor (BBI) is a potent anticarcinogen that suppresses malignant transformation at nanomolar concentrations. Small amounts of BBI in its native form can be measured by immunoassay using specific monoclonal antibodies (MAbs); however, the MAbs currently available are not capable of detecting BBI metabolites in human body fluids. To develop new reagents for the study of BBI exposure and pharmacokinetics, we produced four MAbs, designated 3B6, 3E3, 4H8 and 5G2, from hybridomas derived from a mouse immunized with reductively modified BBI. The epitopes recognized by the four MAbs were characterized using BBI in its native form or modified by different methods. MAb 3B6 reacted with native BBI. Partial reduction of BBI with 720 Gy of gamma radiation in an oxygen-free solution of 100 mM formate increased the reactivity of BBI with 3B6; however, extensive reduction of BBI with 100 mM DL-dithiothreitol (DTT) completely abolished this antigenic reactivity. In contrast, the other three MAbs reacted with BBI molecules that had been reduced either with 720 Gy of radiation in formate solution or with DTT. Alkylation of the radiochemically reduced BBI with N-ethylmaleimide further increased the reactivity of BBI with 3E3, 4H8 and 5G2, possibly by preventing the formation of new disulfide bonds within the BBI molecules. The binding of 4H8 and 5G2 to BBI antigen was inhibited by the binding of 3E3, and vice versa. Thus, the epitopes recognized by 3E3, 4H8 and 5G2 are probably located close to one another on the reduced BBI molecules. These three MAbs were able to react with BBI metabolites in urine samples collected from volunteers after oral administration of BBI. The ability of these MAbs to detect BBI metabolites indicates that BBI may be reductively modified in vivo and these MAbs may be useful reagents for monitoring the uptake of BBI into human tissues in cancer chemoprevention studies with BBI. PMID- 7534803 TI - Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein. AB - The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass--i.e., approximate sequence positions--that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1 177 and 94-323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94-177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping. PMID- 7534804 TI - Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC restricted cytotoxicity. AB - Using colorimetric MTT assay the susceptibility of a newly established bladder epithelial cell line, Fen cells was compared with conventional target cells, i.e., K562 and Daudi and other epithelial lines for investigation of non-specific killing activity (NK/LAK) of effector cells previously activated with interleukin 2 (IL-2). The results showed that Fen cell line was more sensitive than K562 and Daudi targets and this was seen whether the effector cells were IL-2-activated or not. The percent killing of effector cells from nine normal donors against Fen, K562 and Daudi targets at effector/target (E/T) ratio of 10/1 after IL-2 activation were 63.4 +/- 7.3, 42.6 +/- 4.3 (p = 0.0001) and 38.6 +/- 5.1 (p = 0.0001) respectively. The corresponding values for inactivated effector cells at 50/1, E/T ratio were 44.8 +/- 9.0, 25.1 +/- 8.3 (p = 0.0001) and 24.4 +/- 9.4 (p = 0.0001) indicating exquisite sensitivity of Fen cells to NK/LAK killing. The susceptibility of Fen cells was found to increase by pre-treatment of target cells with interferons (IFN). Thus the percent killing of untreated, IFN-alpha (1000 U/ml), beta (2000 U/ml) and gamma (100 U/ml) treated cells were 52%, 64% (p = 0.005), 70% (p = 0.001) and 67% (p = 0.001) respectively. These results indicated that Fen cells were more susceptible to NK/LAK killing than the conventional K562 and Daudi target cells. These results and the epithelial origin of Fen cells indicate that this cell line might prove to be a more realistic system to replace the conventional approach for assessment of NK/LAK activity in patients with cancer of epithelial origin. PMID- 7534805 TI - Immunochemical analyses of human plasma fibronectin-cytosolic transglutaminase interactions. AB - Fibronectin is a glycoprotein involved in cell adhesion, tissue organization and wound healing. Transglutaminase binding and covalent cross-linking of fibronectin are physiologically important reactions. We describe microtiter plate-based immunochemical methods to analyze cytosolic transglutaminase-human plasma fibronectin interactions. The method was sensitive, specific, species-independent and capable of simultaneously analyzing 96 samples for binding. Binding was time , temperature- and concentration-dependent and demonstrable with either protein immobilized to the plastic. The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked immunosorbent assays. CaCl2 (8 mM) enhanced transglutaminase binding by two-fold. Molar concentrations of NaCl or millimolar concentrations of chloride salts of barium, copper or zinc inhibited binding by 50-60%. The binding was also competitively blocked by soluble fibronectin (IC50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 microM). Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a concentration-dependent inhibition of transglutaminase-fibronectin interactions (IC50 = 1.5 mM and 20 mM, respectively). A complex of [anti transglutaminase IgG-transglutaminase-fibronectin-anti- fibronectin IgG] suggested that the binding sites and antibody epitopes could overlap, but are distinct and surface-exposed in the two proteins. Liver transglutaminase bound fibronectin 30-50% less compared to erythrocyte transglutaminase. Fibronectin transglutaminase affinity was adequate for quantitating either antigen in lysates of lung fibroblasts, breast carcinomas or Escherichia coli. These immunochemical analyses will be useful for determining the affinity and mapping the domains involved in antibody recognition or protein-protein interactions using recombinant molecules of transglutaminase and fibronectin. PMID- 7534806 TI - Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures. AB - We have investigated the effect of different cell preparation procedures on the surface expression of CD11b/CD18 and CD62L on human monocytes. Both EDTA and heparin anticoagulated blood were used as sources for leukocytes. The monocytes were analysed by flow cytometry in a mixed leukocyte suspensions obtained after ammonium chloride mediated lysis and in mononuclear cell suspension prepared by density gradient centrifugation (Ficoll-Paque) performed both at 4 degrees C and at 20 degrees C. Monocytes from heparinized blood had a higher expression of CD11b/CD18 and a more pronounced inter-individual variation than monocytes from EDTA blood. Monocytes isolated by Ficoll-Paque had a higher degree of ex vivo activation by means of altered expression of CD11b/CD18 and CD62L compared to monocytes prepared by ammonium chloride mediated lysis. This was more pronounced when the isolation procedure was performed at 20 degrees C. Our findings indicate that monocytes prepared by ammonium chloride mediated lysis of EDTA blood and with the cell handling temperature kept at 4 degrees C are exposed to the smallest ex vivo modulation by means of receptor alteration. An awareness of ex vivo modulation by different cell preparation procedures is of importance especially when comparing the expression of functional receptors on leukocytes of disparate origin. PMID- 7534808 TI - Spatial distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA in the ocular lens: relationship to cholesterologenesis. AB - This study probes the regulation of cholesterol biosynthesis in the ocular lens by estimating the concentration and distribution of the messenger RNA for the rate-controlling enzyme for sterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Because the lens is dependent on biosynthesis for cholesterol, HMGR activity is crucial for the life-long growth of this organ. Young rat lenses were serially divided into several fractions by dissolution in an SDS-containing buffer and each fraction was equated to a percent of the lens radius based upon its protein content. HMGR enzyme activity and cholesterol synthesis has been shown to disappear from the lens cortex over a narrow arc of radius due to loss of enzyme protein. Using a published competitive reverse transcriptase-polymerase chain reaction method for amplifying HMGR mRNA (Powell, E. E., and P. A. Kroon. 1992. J. Lipid Res. 33: 609-614), an average of about 46,000 copies of this mRNA was estimated per lens at all rat ages examined (5-day old to adult). However, copies/microgram total RNA decreased with aging. The distribution of HMGR mRNA across 95-60% of the lens radius was essentially uniform at 2000-3000 copies/mm3 tissue. But the very superficial cortex contained 5- to 7-times this concentration and accounted for about 35% of the total copies/lens. We estimated that cells in this region each contained 1 to 2 copies of message, a value similar to the estimated copy number of HMGR message in human lymphocytes (Powell and Kroon, ibid). This suggests that the translational efficiency and stability of lens HMGR mRNA must be very high.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534809 TI - Paroxysmal supraventricular tachycardia initiated by ventricular couplets in concealed ventricular trigeminy. AB - A case report of a man with ventricular concealed trigeminy in whom ventricular couplets initiating paroxysmal supraventricular tachycardia were occasionally seen is reported. This is the first report on concealed trigeminy with couplets. This strengthens the authors' previous suggestion that supernormal conduction may occur in the reentrant pathway of extrasystoles, as a mechanism of ventricular couplets. PMID- 7534807 TI - Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. AB - A number of cell types possess an L-arginine-nitric oxide (NO) pathway. We studied the presence of constitutive and inducible forms of NO synthase in human platelets. N omega-nitro-L-arginine, an inhibitor of NO synthase, potentiated thrombin-induced aggregation of washed human platelets, whereas L-arginine inhibited it. The direct evidence for the presence of constitutive form of NO synthase came from the observation of conversion of tritium-labeled L-arginine to tritium-labeled L-citrulline by washed platelets suspended in Ca(++)-rich but not in Ca(++)-free buffer. Incubation of washed platelets in Ca(++)-free buffer with cytokines (tumor necrosis factor-alpha and interferon-gamma) or cytokines plus lipopolysaccharide caused a marked increase in the conversion of [3H]L-arginine to [3H]L-citrulline, suggesting the presence of inducible form of NO synthase. Gel electrophoresis identified an approximately 130 kd protein band with NO synthase in the platelet cytosol, which on isolation converted [3H]L-arginine to [3H]L-citrulline. This 130 kd protein required the presence of Ca++, reduced nicotinamide adenine dinucleotide phosphate tetrahydro-L-biopterin, and flavin adenine dinucleotide for expression of NO synthase activity. Platelet sonicates demonstrated presence of nitrite, and its concentrations were lowered by preincubation of platelets with NG-nitro-L-arginine methyl ester and enhanced in cytokine-treated platelets. Reverse-transcription polymerase chain reaction demonstrated messenger RNA expression of the constitutive endothelial (but not brain) and inducible isoforms of NO synthase in platelets. These observations indicate that human platelet cytosol possesses both constitutive and inducible forms of NO synthase. PMID- 7534810 TI - Lymphocyte recognition of picornaviruses. PMID- 7534811 TI - Antibodies to parvovirus B19 NS-1 protein in infected individuals. AB - Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease). The infection is usually benign and self limiting, but in adults cases of severe arthritis which may persist for years have been reported. Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection. The immune response against the third major protein, the non-structural protein NS-1, of parvovirus B19 has not been characterized so far. We cloned and expressed the full-length NS 1 protein and fragments thereof in Escherichia coli. The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection. Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA. Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1. The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein. PMID- 7534812 TI - Evidence of a major neutralizable conformational epitope region on VP2 of infectious pancreatic necrosis virus. AB - A collection of neutralizing monoclonal antibodies (MAbs) produced against the LWVRT 60.1, Jasper and N1 strains of infectious pancreatic necrosis virus (IPNV) were selected for the analysis of VP2 epitopes. Previous characterization of the LW and JA MAbs allowed the identification of continuous and discontinuous epitopes but the topological localization of these sites remained obscure. The ability of these MAbs to differentiate individual epitopes was evaluated by additivity and competition assays using antigen-coated plates and by surface plasmon resonance (SPR), an automated biosensor system that is able to retain the conformation integrity of proteins. IPNV-neutralizing MAbs defined a major, conformational-dependent and immunodominant area where continuous epitopes represent portions of a larger discontinuous epitope. Moreover, weakly neutralizing MAbs could interact with internal sites, located within the foldings of the polypeptide chain. Anti-VP2 MAbs prepared against the European serotype N1 recognized and competed for epitopes present on the North American strain LWVRT 60.1 and Jasper. Attempts to establish the proximity of VP2 and VP3 epitopes have been made by SPR. Results indicate that these major structural proteins do not overlap in the virion. PMID- 7534813 TI - The role of template-primer interactions in cleavage and initiation by the influenza virus polymerase. AB - An in vitro cleavage/initiation assay was used to analyse cleavage site choice and transcription initiation by the influenza virus polymerase. A synthetic mRNA which is cleaved by the polymerase to produce a single 11 base primer fragment was altered around this cleavage site. Depending upon the mutations made, alternative cleavage sites were used. This system was then used in extracts from recombinant vaccinia virus infected cells which express the polymerase. These extracts require the addition of a synthetic vRNA in order to induce cleavage and initiation activity. The data show that the choice of cleavage site is wholely controlled by the mRNA and does not depend upon interactions with the vRNA template. However, the site of initiation of the cleaved primer on the template is influenced by template-primer interactions. PMID- 7534814 TI - Recognition of endogenous ecotropic murine leukaemia viruses by anti-AKR/Gross virus cytotoxic T lymphocytes (CTL): epitope variation in a CTL-resistant virus. AB - AKR/Gross virus-specific cytotoxic T lymphocytes (CTL) from C57BL/6 (B6) mice are H-2Kb-restricted and recognize epitopes encoded by the prototype endogenous ecotropic murine leukaemia virus (Emv) AKR623. Four CTL epitopes have been identified by the use of synthetic peptides corresponding to AKR623-encoded amino acid sequences. Here we present both functional and nucleotide sequence data indicating that three closely related Emv share all of these CTL epitopes. We also found that one other murine leukaemia virus (MuLV) was not susceptible to lysis by these CTL. This is the ecotropic component of the LP-BM5 virus complex that causes murine AIDS. Nucleotide sequencing revealed that three of the four epitopes, including the immunodominant peptide, are altered in this virus. The other epitope was unchanged. These data implied that the inability of anti AKR/Gross virus CTL to lyse cells infected with the LP-BM5 ecotropic (BM5eco) MuLV was due to the functional loss of three of the four CTL epitopes. Using recombinant vaccinia and Sindbis virus vectors, we have shown that the BM5eco encoded form of the immunodominant epitope, which differs only by an arginine for lysine substitution at the N-terminal residue, fails to induce a CTL response in B6 mice. Immunization with BM5eco-infected cells also failed to induce MuLV specific CTL. In light of the long in vivo passage history of the LP-BM5 complex in B6 mice, our results are consistent with a contribution of CTL-mediated immune selection to the evolution of the BM5eco MuLV. PMID- 7534815 TI - Human foamy virus infection activates class I major histocompatibility complex antigen expression. AB - We examined the effect of human foamy virus (HFV) infection on the expression of human major histocompatibility complex molecules. Our data show that in vitro HFV infection of U373-MG glioblastoma cells results in increased expression of class I human leukocyte antigen (HLA) and transcripts. Transient transfection assays of plasmids containing the reporter gene chloramphenicol acetyl transferase driven by different 5' deletions of the HLA-A11 class I promoter allowed identification of cis-acting elements involved in this regulation. HFV infection has two opposite effects on the HLA class I promoter: transactivation of the HLA-A11 promoter through a positive regulatory element located in the -525 to -335 region upstream of exon 1 and down-regulation of transcriptional activity driven by the 335 to -205 class I promoter region. Additional experimental data indicate that the effect of HFV on HLA class I expression is not mediated by the interferon pathway. PMID- 7534816 TI - Diversity of genotypes of hepatitis C virus in southern India. AB - A second generation assay for antibody to hepatitis C virus (anti-HCV) was used to screen 78 southern Indian individuals with a high risk of infection. RT-PCR targeted at the 5' end untranslated region (5'UTR) of the HCV genome was used to evaluate evidence of viraemia in 32 anti-HCV positive sera. The PCR products amplified from the 5'UTR of the HCV genome from 24 patients were sequenced, revealing the existence of two distinct groups of sequences: 21 corresponded to HCV type 1 while the other three sequences had 95% to 99% identity to HCV type 3. Two of these three isolates had more than 90% nucleotide identity in the NS5 region to established 3b sequences whereas the other had less than 74% nucleotide identity to any of the published genotype 3 (3a, 3b, 3c, 3d, 3e and 3f) sequences. However, a search of the EMBL nucleotide database revealed 91% identity to the unpublished sequence of an isolate of HCV from Indonesia. We provide evidence that these two isolates may represent a novel subtype within genotype 3. Our data also suggest that HCV genotype 1 predominates over HCV genotype 3 in southern India. PMID- 7534817 TI - Molecular epidemiology of enterovirus outbreaks in Canada during 1991-1992: identification of echovirus 30 and coxsackievirus B1 strains by amplicon sequencing. AB - The relatedness of enteroviral isolates associated with two recent outbreaks in Canada was assessed using direct sequencing of amplicons derived from a large portion of the 5' nontranslated region (NTR) of the viral genome. The amplicons of 60 echovirus 30 isolates originating from seven different provinces in 1991 were found to share 99% or greater sequence identity. Recent coxsackievirus B1 isolates characterised in the same manner were identical to each other. When the 5' NTR sequence of these isolates was compared to prototype strains a difference of 11-15% in nucleotide composition was observed. These results indicate that the variability of nucleotide sequence found in 5' NTRs can be utilized to identify rapidly enteroviral strains associated with particular outbreaks and distinguish them from other strains and serotypes. PMID- 7534818 TI - Development and preliminary evaluation of an enzyme immunosorbent assay for the detection of adenovirus type 8. AB - Epidemic keratoconjunctivitis (EKC) is a highly contagious adenoviral ocular infection which can occur in outbreaks and is predominantly caused by adenovirus type 8 (Ad8). Detection and typing of this virus after isolation is generally by serum neutralisation or more complex molecular techniques. These methods can be time consuming particularly during outbreaks. Therefore, an Ad8 specific antigen capture enzyme immunosorbent assay (EIA) was developed as a rapid and cost effective diagnostic method for laboratories. An Ad8 type-specific monoclonal antibody was used in a direct antigen capture method and an anti-hapten fluorescein isothiocyanate (FITC)-anti-FITC system. Assay configuration studies indicate that the system in which a type specific capture antibody and a group specific detector antibody are used provides greater sensitivity to the assay than a system using a group specific monoclonal or polyclonal capture antibody. Also, this allows the use of the same detector antibody with varying type specific capture antibodies on the solid phase. In a preliminary evaluation of the two formats, the direct antigen capture assay and the FITC-anti-FITC system had a sensitivity of 98.75% and 100%, respectively, with a specificity of 100%. However, these results are not statistically significant due to the low numbers of Ad8 and other viral isolates obtained from eye swabs. The direct EIA format has been shown to be able to detect different Ad8 genome types including four isolated from four epidemics of EKC in Brest, France, the Ad8 prototype strain, and some DNA-variant isolates which had not been typed by restriction endonuclease analysis (REA). PMID- 7534820 TI - Hepatitis C and arboviral antibodies in the island populations of Mauritius and Rodrigues. AB - A serological survey for antibodies to hepatitis C virus (HCV), dengue viruses (DEN), West Nile virus (WN), and sindbis virus (SIN) was carried out in sera of selected groups of the population of the Islands of Mauritius (n = 449) and Rodrigues (n = 115), Indian Ocean. 8.3% of 564 sera were positive for anti-HCV. In Mauritius, 2.1% of sera of healthy individuals were found with anti-HCV. The highest prevalence was found in sexually transmitted disease (STD) patients and prison inmates with 46.2% and 43.8%, respectively. None of the sera from blood donors sampled from Rodrigues Island had anti-HCV. Antibodies to arboviruses were detected in sera of individuals from both islands. Anti-DEN IgG was detected in 3.8% of sera from Mauritius and 0.9% from Rodrigues. Anti-WN IgG was detected in 2.2% of sera from Mauritius and 0.9% from Rodrigues. All sera from Rodrigues were without anti-SIN IgG, 1.1% of those from Mauritius were positive. This suggests that arboviruses occur on these islands. PMID- 7534819 TI - Nucleotide sequence of the core region of hepatitis C virus in Pakistan and Bangladesh and the geographic characterisation of hepatitis C virus in south Asia. AB - A large number of complete and partial hepatitis C virus (HCV) sequences have been reported and classified into several genotypes, although none have been reported from South Asia. We have determined and evaluated partial sequences in the core region of HCV obtained from patients with chronic hepatitis in Pakistan and Bangladesh. Nucleotide sequences from these viruses show significant homology with the Japanese HCV-TR isolate (91.7%-97.9%) and low homology with other Japanese, American, and UK isolates including HCV-1, HC-J4, HC-J6, HC-J8, and E b1 (79.3%-86.2%). The homologies of their deduced amino acids sequence with HCV 1, HC-J4, HC-J6, HC-J8, E-b1, and HCV-TR were 84.3%-89.8%, 85.0-87.9%, 84.1% 86.9%, 84.3%-87.0%, 90.2%-93.1%, and 89.8%-93.5%, respectively. These results suggest that our clones might be classified into the same genotype as HCV-TR. Further analysis using molecular evolutionary methods strongly supported the classification of these sequences with the HCV-TR genotype. Moreover, we could not detect any isolates which were closely related to our clones or HCV-TR in countries outside the South Asian area. These data further support the association of HCV genotypes with distinct geographic regions. PMID- 7534821 TI - E2 and NS5: new antigens for detection of hepatitis C virus antibodies. AB - The value of two new hepatitis C virus (HCV) antigens for detection of HCV antibodies was studied. These two recombinant antigens were derived from the nonstructural-5 (NS5) and envelope-2 (E2) region of the HCV genome. In a panel of 33 HCV-RNA positive samples with indeterminate Riba-2 confirmatory test results, 29 samples (88%) showed additional antibody reactivity against E2 and 12 samples (36%) showed additional reactivity against NS5. Among 39 HCV-RNA negative, Riba-2 indeterminate donor samples, no additional E2 or NS5 reactivity was found in 34 samples (87%); while 5 samples (13%) showed additional reactivity against NS5 and/or E2. E2 reactivity thus resolved the majority of hitherto indeterminate samples. In serial samples from nine posttransfusion hepatitis C patients, NS5 and E2 antibodies did not appear earlier than classical HCV antibodies. However, E2 antibodies eventually appeared in all nine patients. The recombinant E2 might be a candidate antigen for future HCV antibody assays. PMID- 7534822 TI - Transganglionic labelling of primary sensory afferents in the rat lumbar spinal cord: comparison between wheatgerm agglutinin and the I-B4 isolectin from Bandeiraea simplicifolia. AB - We recently reported that the I-B4 isolectin from Bandeiraea simplicifolia could be used as a transganglionic neuronal tracer which appears to be selective for unmyelinated cutaneous afferents (C fibres) and their terminals in the superficial dorsal horn. As terminals in the superficial dorsal horn are also labelled by wheatgerm agglutinin, we sought to compare these two neuronal tracers. Three days after the injection of 1% wheatgerm agglutinin-HRP or 1% BSI B4-HRP into the sciatic nerve of adult rats the lumbar spinal cord was processed for HRP reactivity. The majority of labelled structures was found in the superficial dorsal horn, with fewer labelled structures seen in the overlying white matter (including Lissauer's tract). In wheatgerm agglutinin-HRP experiments most labelled structures were synaptic terminals (63%) and unmyelinated axons (32%). About 3% of wheatgerm agglutinin-HRP-labelled structures were fine myelinated fibres (which were found only in lamina I and outer lamina II) and about 2% of label was located in neuronal somata. In contrast, label from BSI-B4-HRP experiments was found only in synaptic terminals (37%) and unmyelinated axons (63%). Previous studies have shown that small diameter dorsal root ganglion neurons and their terminals in the superficial dorsal horn express a range of structurally related carbohydrates that contain binding sites for BSI-B4 or wheatgerm agglutinin or both. Comparison of the labelling patterns produced by the two transganglionic tracers in the present study suggests that unmyelinated sciatic afferents express wheatgerm agglutinin and BSI-B4 binding sites, but some thin myelinated afferents, and a distinct form of synaptic terminal in lamina I/II outer, express the wheatgerm agglutinin binding site and not the BSI-B4 binding site. PMID- 7534823 TI - Morphological analyses of NADPH-diaphorase/nitric oxide synthase positive structures in human visual cortex. AB - Human visual cortex was studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. Large, strongly stained, sparsely spined non pyramidal cells (average soma diameter: 16 x 16 microns) occur in layers II-VI, but are commonest in layers II-III. Small weakly stained multipolar cells (average soma diameter 3.6 x 4 microns, stellate like cells) in layers II-VI are concentrated in layer IV of areas 17 and 18. The density of these cells, measured with a computer assisted microscopy system is less in area 18 than 17. Large, strongly stained, predominantly horizontal cells (average soma diameter 12 x 19 microns) are localized in the underlying white matter. Axons of the large, strongly NADPH-diaphorase positive cells are thin and unbranched with fine boutons. These axons ascend to layer I. The large, strongly stained cells in layers II-VI we identify as Martinotti neurons. In layer I parallel unbranched positive fibres with some fine boutons run horizontally and build dense axonal plexuses together with the axons of Martinotti neurons. Axons of presumed extrinsic origin are morphologically different from NADPH-diaphorase positive intrinsic fibres. They show thick varicosities running in different directions and forming a network in layers III-VI. Basket like formations of these fibres were frequently observed in layers IV, V and VI. Other fibres seem to innervate blood vessels. Nitric oxide synthase was also demonstrated immunohistochemically by a polyclonal rabbit nitric oxide synthase antiserum. The morphology and distribution of the immunostained cells correspond with those seen with NADPH diaphorase histochemistry. Double labelling experiments confirm the colocalization of NADPH-diaphorase and nitric oxide synthase in all demonstrated cells. Immunohistochemical demonstration of glial fibrillary acidic protein has shown that astrocytes are not involved in the NADPH-diaphorase/NOS system in the human visual cortex. PMID- 7534824 TI - Subcortical contributions to head movements in macaques. II. Connections of a medial pontomedullary head-movement region. AB - 1. In the companion article, a variety of head movements were elicited by stimulation in, and adjacent to, the gigantocellular reticular nucleus (Cowie and Robinson 1994). We refer to this area, caudal to the abducens nucleus, as the gigantocellular head movement region. In the present paper, the anatomical connections of this region, as determined by injections of wheat-germ agglutinin conjugated horseradish peroxidase (WGA-HRP), are reported. The majority of efferent and afferent connections were with areas related to head movements. 2. Efferent fibers from the region projected via two paths to the caudal medulla and upper cervical spinal cord. Labeled fibers descended in the anterolateral funiculus of the ipsilateral spinal cord to terminate in lateral parts of the ventral horn. A second pathway descended bilaterally in the medial longitudinal fasciculus to the anterior funiculi and medial portions of the ventral gray. These efferents paralleled the head-movement topography demonstrated physiologically. Other projections included efferents to the interstitial nucleus of Cajal, caudal field H of Forel, paramedian pontine reticular formation, and caudal vestibular nuclei. Other efferent fibers projected to the trigeminal, facial, and hypoglossal nuclei, as well as to the parvocellular reticular field, which contains interneurons for these motor groups. However, no efferent or afferent labeling involved the ocular motor nuclei. 3. Afferents to the gigantocellular head movement region arose mainly from head-movement areas. In all animals, labeled cells were found in the intermediate and deep layers of the caudal superior colliculus. Labeled neurons also were found in the caudal field H of Forel, interstitial nucleus of Cajal, pontine medial tegmentum including the pontine paramedian reticular formation, nucleus subcoeruleus, and vestibular nuclear complex. Caudally, filled cells were located in the parvocellular, magnocellular, dorsal, and ventral reticular nuclei, the supraspinal nucleus, and the upper cervical ventral horn. 4. In one animal, the ipsilateral frontal cortex contained retrogradely labeled neurons. These cells were found in layer V of cortical areas 4 and 6. Other afferent cells were found consistently in the periventricular and periaqueductal gray matter. 5. A control injection into the caudal vestibular nuclear complex showed projections to the gigantocellular reticular formation and labeled cells in the vestibular and parvocellular reticular nuclei. These observations show that the connections of the gigantocellular region are not typical of all head movement sites. 6. These data indicate that the gigantocellular head-movement region has the requisite efferent and afferent connections to function in the subcortical control of head, but not eye, movements.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534825 TI - Substance P potentiates calcium channel modulation by somatostatin in chick sympathetic ganglia. AB - 1. The whole cell patch clamp was used to measure calcium current in isolated chick sympathetic ganglion neurons. Previous results showed that somatostatin inhibits calcium currents (ICa) in a voltage-dependent manner. The effect of somatostatin rapidly desensitizes. In addition, the action of somatostatin on the calcium current is inhibited by activation of protein kinase C (PKC). Because substance P (SP) has been shown to activate PKC in the chick sympathetic neurons, we here test the effects of SP on the calcium current and on the modulatory action of somatostatin. 2. At a concentration of 1 microM, SP had small, variable effects on ICa. 3. SP in the presence of guanosine 5'-triphosphate-gamma-S, or at higher concentrations (10 microM), inhibited ICa in a voltage-dependent manner, similar to the action of somatostatin. 4. Rather than inhibiting the action of somatostatin, SP (1 microM) potentiated the response to somatostatin. This effect of SP was only observed after the response to somatostatin had partially desensitized. SP had no effect on nondesensitized responses to somatostatin. 5. Desensitization of the somatostatin response involved a shift in its dose response curve toward higher somatostatin concentrations as well as a decrease in the maximal response. SP appears to counteract the shift of the dose-response curve selectively. 6. The potentiation of the somatostatin response by SP is blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), but not by Calphostin C, Compound 5, k252a, protein kinase C (PKC)19-36, or adenylyl imidodiphosphate (AMP-PNP), suggesting that phosphorylation is not involved and that the H-7 action does not depend on kinase inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534826 TI - Hyperpolarization-activated Na(+)-K+ current (Ih) in neocortical neurons is blocked by external proteolysis and internal TEA. AB - 1. After 1 day in culture, neurons derived from the neonatal cerebral cortex of the rat exhibited a slowly activating current gated by hyperpolarizing voltage clamp pulses. The current was blocked by extracellular Cs+ and unaffected by extracellular Ba2+, and was permeable to both Na+ and K+ (PNa/PK = 0.29). Its form and pharmacology are consistent with a current termed Ih in other preparations. 2. Ih was absent from cells acutely dissociated from both the neonatal and mature cerebral cortex, despite the use of low enzyme concentrations. The sensitivity of Ih to extracellular proteolysis was demonstrated by superfusing the cells with trypsin (1 mg/ml) while monitoring the presence of Ih in the whole cell mode of recording. Ih was rapidly abolished (t1/2 approximately 5 min at 22 degrees C) by proteolysis and exhibited no shifts in its range of activation or changes in its activation kinetics during the course of the digestion. 3. Intracellular tetraethylammonium (TEA), at a concentration of < 15 mM was shown to block Ih completely, while extracellular TEA had no effect on the current. This suggests that the inner vestibule of Ih may be structurally related to that of potassium channels. PMID- 7534827 TI - Physiology of single putative cochlear efferents in the chicken. AB - 1. An experimental approach was developed that allowed recording of neurophysiological activity from single putative cochlear efferents in the auditory brain stem of anesthetized chickens with the use of glass micropipettes. The aim of this study was to study spontaneous and tone-evoked activity from single efferent neurons in the chick and to compare their properties with those of other vertebrate hair cell organs. Because the birds, like mammals, have a complex hearing organ with different hair cell types and different afferent and efferent innervation, the purpose of this study was also to determine whether different types of efferents exist. 2. In the same electrode penetrations, putative trapezoid fibers were also isolated. In addition, the penetration angle permitted recordings from units in both cochlear nuclei, nucleus magnocellularis and nucleus angularis (probably mostly cochlear afferents), in the same animal. This allowed monitoring of the auditory sensitivity of the individual animal during the experiment. With the use of physiological criteria, it was possible to distinguish between trapezoid fibers and putative cochlear efferents. Possible alternative origins of the responses described are discussed. 3. Tuning curve characteristics of putative efferents were determined. They were as sensitive as ascending auditory neurons. Q10 dB values of efferent tuning curves were < 2.5 and thus showed poorer frequency selectivity than ascending fibers; in some cases they covered the entire hearing range of the chicken. 4. Latencies to tone pips were different for ascending neurons and putative efferent units. For trapezoid fibers and neurons from the cochlear nuclei, latencies usually did not exceed 5 ms, whereas latencies of efferents were always longer. 5. Because of the interaural canal that connects both middle ear cavities in birds, the measurement of the lateralization of the efferents was difficult. In any case, the majority of putative cochlear efferents responded more sensitively to sound stimulation of the contralateral side. 6. Of the efferent units, 28% showed no spontaneous activity. The others either showed regular spontaneous activity, or their time interval histograms showed longer modes than ascending fibers. In general, mean spontaneous activity was lower than in ascending fibers, being < 30 spikes/s. 7. In contrast to reports from mammalian studies, in which efferents only showed on peristimulus time (PST) response pattern to tonal stimuli (chopper), two different response types were found in this study: two excitation types (chopper and primary-like) and one suppression type.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534828 TI - Inhibition of simian immunodeficiency virus (SIV) replication by CD8+ cells of SIV-infected rhesus macaques: implications for immunopathogenesis. AB - The ability of the CD8+ cells from simian immunodeficiency virus (SIV)-infected rhesus macaques to inhibit SIV replication was investigated. Inhibition was produced by a heat-stable soluble factor of molecular size greater than 10kDa. CD8+ supernatants from some macaques were found not only to suppress SIV growth but also to be cytolytic toward both infected and uninfected CD4+ cells. Such indiscriminate CD8+ cell-mediated cell killing may therefore account for DC4+ cell depletion in certain SIV-infected macaques. PMID- 7534829 TI - Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes. AB - In the present paper we describe the characterization of a Trypanosoma cruzi cDNA (L1Tc) corresponding to a transcript from a new long terminal repeat (LTR) retrotransposon. This element is present in a high-copy number, and is found dispersed throughout the T. cruzi genome. Northern analysis shows an abundant expression of L1Tc-related sequences with a major band of about 5 kb. The transcript has at its 3' end a fragment of a highly repetitive DNA sequence (E12A), at its 5' end a ribosomal mobile element-like sequence and three putative open reading frames (ORF) in different frames. The ORF2 codes for a protein which has significant homology with the retrotranscriptase-related sequences from non LTR retrotransposons containing the seven domains present in all the retrotranscriptase and retrotranscriptase-related proteins. The ORF3 codes for a gag-like protein showing unusual cysteine motifs present in all non-LTR trypanosomatid elements, similar to the C2H2 zinc finger family of transcription factors. Interestingly, ORF1 codes for a protein with significant homology to the major human AP endonuclease protein, and maintains in similar positions most of the amino acid domains described for all the Ape family of proteins. The presence of Ape-related sequences, described for the first time in a non-LTR retrotransposon (L1Tc), may have functional relevance for these types of elements. PMID- 7534830 TI - Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. AB - We had previously used in vitro RNA selection techniques to describe a consensus RNA pseudoknot that binds and inhibits HIV-1 reverse transcriptase (HIV-RT). In this work we constructed variants of this consensus pseudoknot in order to evaluate the contributions of individual nucleotide identities and secondary structure to affinity for HIV-RT. We have also used chemical modification of ligand RNAs to corroborate the predicted structure of the pseudoknot, to discover which modifiable groups are protected from chemical attack when bound to HIV-RT, and to find which modifications interfere with binding to HIV-RT. A novel interference study is presented which involves selection of ligands from a pool created by mixed reagent oligonucleotide synthesis in order to rapidly determine allowed substitutions of 2'-OCH3 groups for the usual 2'-OH group in such RNA ligands. PMID- 7534831 TI - Endotoxin-induced nonendothelial nitric oxide activates the Ca(2+)-activated K+ channel in cultured vascular smooth muscle cells. AB - Endotoxin induces an enzyme that synthesizes nitric oxide (NO) from L-arginine (NO synthase) in vascular smooth muscle cells, resulting in nonendothelial NO release. In this study, we measured the NO release and its intracellular action on the Ca(2+)-activated K+ channel (KCa channel) in cultured smooth muscle cells of porcine coronary artery using a newly-developed porphyrinic-based microsensor and the patch-clamp technique. In smooth muscle cells pretreated with endotoxin, extracellular application of 10(-4) M L-arginine increased NO release, which induced rapid and prolonged activation of the KCa channel. This activation was only partially blocked by application of 10(-5) M 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-oxyl 3-oxide, which neutralizes NO. NO formation and activation of the KCa channel were suppressed by pretreatment with 10(-3) M NG methyl-L-arginine or 10(-3) M N omega-nitro-L-arginine methyl ester, each of which is a specific antagonist of the L-arginine-NO pathway. One micromolar methylene blue, a blocker of guanylate cyclase, inhibited L-arginine-induced activation of the KCa channel. The effect of nitroprusside in opening the KCa channel was transient, although it induced production of larger amounts of NO in the bath. These results suggest that the endotoxin-induced and L-arginine pathway generates NO and directly modulates the KCa channel intracellularly in an autocrine manner. PMID- 7534832 TI - Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: effects of NGF or prior peripheral axotomy. AB - Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increases in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. PMID- 7534833 TI - Lindane cytotoxicity in cultured neocortical neurons is ameliorated by GABA and flunitrazepam. AB - The effect of lindane (gamma-hexachlorocyclohexane) on [35S]t butylbicyclophosphorothionate ([35S]TBPS) binding and GABA-stimulated 36Cl- influx was investigated in cultured cerebral cortical neurons. In addition, the cytotoxic action of lindane as well as a protection by GABA and flunitrazepam were studied together with the ability of lindane to increase the intracellular concentration of free Ca2+. Lindane was found to be toxic to the neurons, an effect that could be completely prevented by the simultaneous presence of GABA (0.1 microM) and flunitrazepam (100 microM) and reduced by GABA alone. An interaction with the GABA receptor-gated chloride channel was demonstrated by an inhibitory action of lindane on [35S]TBPS binding (IC50 188 +/- 51 nM) and on GABA-stimulated 36Cl- influx in the neurons. Lindane only marginally increased the intracellular Ca2+ concentration in the neurons. It is concluded that the cytotoxic action of lindane is mediated through interaction with GABA receptors in a manner essentially independent of changes in intracellular Ca2+ homeostasis. PMID- 7534834 TI - Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404. AB - The microtubule-associated protein tau is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic tau peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF-1. The present study extends this observation by showing that PHF-1 recognizes tau peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a > 10-fold increase in the sensitivity of detection of tau peptides by PHF-1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in tau by PHF-1 can also be demonstrated in Chinese hamster ovary cells transfected with full-length wild type tau constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF-1 epitope contains both phosphorylated Ser396 and Ser404. PMID- 7534836 TI - [Water channel family proteins]. AB - 17 members of MIP family from bacteria, yeast, plants and animals are compared in this review. These proteins appear to function in (1) water channels (CHIP, WCH CD, MIWC, AQP3, gTIP, RD28, TobRB7), (2) neurogenesis (Bib), (3) small-molecule permeating channels (MIP, AQP3, NOD, Glpf), (4) unknown function (WCH-3, AtRB7, Pea R7A, FPS1). However, the biological functions are not well established. The most conserved residues in the first and the second halves of all MIP family proteins are asparagine-proline-alanine (NPA) sequences in the loops (NPA boxes). This structural similarity may lead to functional similarity (water and/or small molecule permeation). This signature sequence for the MIP family will facilitate the identification of new protein members of this family. PMID- 7534837 TI - [Utility of QRST isointegral map]. AB - The clinical usefulness of QRST isointegral map (IQRST map) was evaluated in many disease entities, i.e., 1) detection of myocardial infarction with and without bundle branch block, 2) identification of the site of ventricular premature beat and ventricular tachycardia, 3) left ventricular hypertrophy, 4) diagnosis of the location and size of myocardial infarction. PMID- 7534838 TI - [Effects of combined aprotinin and prostagrandin E1 therapy on aortic arch replacement]. AB - This study was undertaken to compare the effects of combined aprotinin and prostaglandin E1 therapy on aortic arch replacement. Twenty patients were divided into 2 groups with (group A; n = 10) or without (group B; n = 10) the treatment (200 KIU of aprotinin and 0.01-0.02 microgram/kg/min of prostaglandin E1 during cardiopulmonary bypass (CPB) and the first postoperative day. Preoperative evaluation of respiratory function and all parameters related to CPB procedure were revealed to be equal between the groups. Postoperative A-a DO2 and respiratory index (RI) as functional parameters of oxygenation capacity, dosage of dopamine were monitored at 0, 3, 6, 12, 18 and 24 hr after termination of CPB and at extubation period. Serum creatinine, platelet numbers and blood coagulation function (PT, APTT) were also assayed postoperatively. The recovery of respiratory and cardiac function were superior in group A with treatment, but renal and blood coagulation function showed no difference in the groups. We suggest the combined therapy with aprotinin and prostaglandin E1 for aortic arch replacement may emerge as a valuable treatment to save postoperative respiratory and cardiac function. PMID- 7534839 TI - [A case of primary mediastinal germ cell tumor, successfully treated with chemotherapy and curative resection]. AB - A 20-year-old man was admitted to our hospital because of an abnormal shadow on chest X-ray Laboratory data revealed a high serum alpha-fetoprotein (AFP) and LDH level. Percutaneous needle biopsy of the tumor suggested primary mediastinal germ cell tumor. Curative resection was performed after three courses of combination chemotherapy (cis-platinum, VP-16, bleomycin and adriamycin). A post-operative histological examination of the mass revealed total necrosis. Post-operative course was uneventful, and he has been free of recurrence for the last six months. PMID- 7534835 TI - [Diagnostic significance of QRST isointegral map in patients with premature ventricular contraction]. AB - Identification of the focus of premature ventricular contraction (PVC) has been reported by various body surface mapping methods: i.e. PVC isopotential map, ventricular activation time map, QRS isointegral map, PVC T wave map, etc. For evaluating the clinical usefulness and diagnostic significance of QRST isointegral map in patients with PVC, 23 patients without apparent underlying heart disease (group N), and 44 patients with dilated cardiomyopathy (group D), were studied. The significant changes in QRST values at maximal points during two activation sequences (basic rhythm and PVC) were detected in group D (N:D = 26.6 +/- 17.6:52.6 +/- 27.0%, p < 0.01), which were frequently directed to the PVC focus and influenced by the short coupling interval (Q-Q interval: 300-390 ms). In conclusion, QRST isointegral map reflecting ventricular gradient may be useful for identifying the presence of underlying heart disease and may correlate with the focus and coupling interval of PVC. PMID- 7534840 TI - ["Can't piss", "can't hold"]. PMID- 7534841 TI - Turapy--transurethral ablation prostatectomy by high temperature radiofrequency- preliminary results of a multicenter study. AB - Thermal treatment of BPH has been introduced several years ago as an alternative treatment modality for patients suffering from symptomatic BPH. The initial temperatures ranged from 44.5 to 48 degrees C. With these temperatures, about 60 70% of the patients showed significant improvement of their subjective, mainly irritative symptoms with only mild changes in their obstructive parameters. In order to improve the therapeutic effects, increase in energy delivered allowing higher temperatures in the order to 70-100 degrees C was investigated regarding the feasibility, tolerance, pathological lesions and clinical effectiveness. A new radiofrequency technique, TURAPY--TransURethral Ablation Prostatectomy, an outpatient ablative BPH treatment has been introduced recently. The treatment is applied transurethrally with a helicoidal antenna without any cooling system. Temperature delivery in the order of medium 75 degrees C was applied during a medium time of 45 minutes. An ongoing multicenter study included 38 patients. 15 patients were operated by retropubic prostatectomy after 6-30 days, for a histopathology study and 23 eligible patients were entered in a clinical study and analyzed to determine the efficacy of the TURAPY treatment. The tolerance was excellent and although patients experienced initial heating sensation at the onset of treatment, as the temperature rose to 40-50 degrees C (as with previous modalities of thermotherapy), they did not experience further pain and the procedure was never stopped. Urinary retention was observed in about 80% of the patients, due to the extensive tissular destruction. These patients had suprapubic catheter inserted one day to a maximum of one week (medium 3 days).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534842 TI - [Molecular aspects of invasion and metastasis of bladder cancer]. PMID- 7534843 TI - [Usefulness of hyper sensitive PSA assay kits for determination on low range of prostate specific antigen in prostate cancer]. AB - Prostate specific antigen (PSA) levels after total prostatectomy or radiation therapy to localized prostate cancer and also during endocrine therapy are within normal range. Therefore, it is necessary to use hyper sensitive PSA assay kits for early detection of relapse. The present study was undertaken to evaluate two hyper sensitive assay kits (Delfia kit, lower limit 0.1 ng/ml, Kabi Pharmacia Diagnostics Co. and Markit M kit, 0.5 ng/ml, Dainippon Pharmaceutical Co.) and to compare them with conventional PSA kit (Eiken Chemical Co., 1.0 ng/ml). Total of 291 sera were examined: patients consisted of 10 total prostatectomy+endocrine therapy, 9 radiation therapy+endocrine therapy, 5 radiation therapy alone and 44 endocrine therapy alone. Values of endocrine therapy alone were divided into two groups according to duration after start of treatment; more or less than 5 years. The following results were obtained. 1. In non-relapse patients after total prostatectomy+endocrine therapy and radiation+endocrine therapy, PSA showed under lower limit with hyper sensitive kit. On the contrary, conventional kit indicated more than 1.0 ng/ml. 2. Radiation therapy alone kept PSA in detectable range with hyper sensitive kit in spite of no sign of relapse. 3. In non-relapsed patients under endocrine therapy alone, long duration (more than 5 years after start of treatment) decreased PSA in non detectable values with hyper sensitive kits. In this case, conventional kit still showed PSA as more than 1 ng/ml. 4. Doubling time at relapse was estimated similar with Delfia kit and Markit M kit, and much longer with conventional kit. It is concluded that hyper sensitive kit is more useful to manage patients after therapy than conventional kit. PMID- 7534844 TI - [Laser-TURP with a lateral firing fiber for contact irradiation]. AB - We have operated 25 BPH patients with transurethral Nd-YAG laser ablation for prostate from June of 1993 till December of 1993. We used UltraLine fiber for contact laser irradiation of the prostate. This is a lateral firing quartz fiber with high density laser energy output, and so made it possible for us to vaporize prostatic tissue rapidly and deeply. We used 60 Watts of power for all cases. For the prostates larger than 50 cm3, limited resection after laser ablation allowed early relief of patients' symptoms. Two months after the treatment, average flow rate improved from 3.7 ml/sec to 7.8 ml/sec. Volume of residual urine decreased from 48.4 ml to 17.0 ml. Twenty-four percent decrease of prostatic volume was also recognized with transrectal sonography. Only 4 patients complained of transient urinary retention, although another 3 or 5 days catheter placement relieved their symptoms. Bleeding was negligible and the other postoperative complications were not recognized at all. Laser-TURP is a new treatment that is effective, less morbid and economical, compared with the ordinary TUR-P. So it will soon become an alternative method to electrocautery TUR-P. PMID- 7534845 TI - [Prostate specific antigen (PSA)/gamma-seminoprotein ratio in the cases with PSA levels less than or equal to 10.0 ng/ml]. AB - Prostate specific antigen (PSA) and gamma-seminoprotein (gamma-Sm) have been revealed to be the same protein and used as tumor markers for prostate cancer (CaP). However, it seems impossible to detect prostate cancer in the cases with PSA levels of 10.0 ng/ml or less. We now report on PSA/gamma-Sm ratio in the cases with PSA levels of 10.0 ng/ml or less, and on the relation between PSA and gamma-Sm in those cases. Serum samples were obtained from the patients with no cancer (NC) (n = 118) and CaP (n = 39). In the cases with PSA ranging from 4.1 to 10.0 ng/ml, gamma-Sm levels in the patient with CaP were significantly lower than in those with NC (3.744 +/- 2.481 (mean +/- SD, n = 27) VS. 7.573 +/- 4.182 (n = 41), p < 0.0001) though PSA levels in both groups were not significantly different, and consequently, PSA/gamma-Sm ratio in the patients with CaP were significantly higher than in those with NC (2.181 +/- 0.802 VS. 1.095 +/- 0.804, p < 0.0001). In the cases with PSA levels of 4.0 ng/ml or less, gamma-Sm levels in the patient with CaP were significantly lower than in those with NC (1.600 +/- 0.705 (n = 12) VS. 3.243 +/- 2.456 (n = 77), p = 0.0064), while PSA levels in the patients with CaP were not significantly different from those in the patients with NC, and consequently, PSA/gamma-Sm ratio in the patients with CaP were significantly higher than in those with NC (1.762 +/- 0.544 VS. 0.808 +/- 0.330, p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534846 TI - [Soft roentgen therapy in cutaneous metastases of the eyelids]. AB - HISTORY: Within 6 months a 88-year-old woman developed cutaneous metastases with infiltration of the eye-lid so that an opening of the eye-lids became impossible. The primary tumor histologically resembled a mamma carcinoma. THERAPY: Within a few days the cutaneous metastases of the eye-lids decreased by using x-ray therapy with the Dermopan II with a single dosage of 5 Gy and a total dosage of 35 Gy on the eye-lids. Even 12 months after palliative therapy the opening of the eye-lids was possible without any problems. CONCLUSIONS: By using the x-ray therapy immediate and gentle help could be given to the terminally ill patients so that eye-lid surgery could be avoided. PMID- 7534847 TI - [Heart rupture in the acute period of myocardial infarction: prognostication and ways of prevention]. AB - Clinical, instrumental, and biochemical examinations were carried out in 587 patients with transmural myocardial infarctions (381 patients with the first infarction and 206 ones with the repeated disease). Heart ruptures occurred in 5.7% of cases. Clinical forms and risk factors of heart rupture are described. Another group consisted of 815 patients with the first transmural infarction; in 44 patients at a high risk of heart ruptures preventive therapy with pantripin and contrykal was carried out. The incidence of heart ruptures was reduced twofold in this group as against the controls. Heart rupture risk factors in patients with myocardial infarction are described. The syndrome of excessive resorption in the pre-rupture period of myocardial infarction was singled out, manifesting by increased activities of lactate dehydrogenase, lysosomal enzymes, and elevated level of cardiac antigen. Lysosomal enzyme levels were measured in the myocardium in heart ruptures. The authors have defined the notion of 'mechanical incompetence' in myocardial infarction, consisting in inability of the involved myocardium to resist the intraventricular blood pressure. The program of heart rupture prevention in myocardial infarction is presented. PMID- 7534848 TI - Ribosomal RNA patterns identify additional strains of Campylobacter jejuni and C. coli among isolates serotyped by heat-stable and heat-labile antigens. AB - Heat-stable (HS, O-antigen) and heat-labile (HL) serotyping are the most common methods used to type Campylobacter jejuni and C. coli for epidemiologic purposes. In this study, we conducted RRNA analysis to differentiate strains of C. jejuni and C. coli that had been serotyped by use of the passive hemagglutination (heat stable) and slide agglutination (heat-labile) methods. Ribotyping of isolates within HS and HL serotypes revealed further discrimination of strains. Four ribotypes were identified by Pvu II and Pst I digests of eight HS serotype-34 isolates. Ribotyping also differentiated strains within HL serotypes. Ribotyping also was conducted on 10 representative isolates of C. jejuni and C. coli isolated from an infant macaque. The eight ribotypes confirmed previous results of serotyping and other phenotypic analyses, which indicated that the infant was repeatedly reinfected with different strains of C. jejuni and C. coli. Results of the study indicated that ribotyping is a sensitive molecular marker for distinguishing strains of C. jejuni and C. coli. Furthermore, some isolates with similar ribotype patterns had variability in their HS and HL serotypes. PMID- 7534849 TI - Changes in serum protein fractions and specific alpha and beta concentrations during lactation in Macaca fascicularis. AB - Physiologic variations are known to develop in milk and serum proteins of women during lactation. This study was intended to evaluate whether modifications of the major serum proteins and alpha, beta, and gamma fractions occur during lactation in Macaca fascicularis. The alpha 1 and gamma globulins decreased and the alpha 2 and beta globulins increased; results of specific protein determinations indicated a decrease in almost all globulins. The protein trends during lactation in M. fascicularis are probably modified by estrogen and progesterone variations after delivery. Estrogen trends are similar in women and cynomolgus monkey females, whereas progesterone patterns between women and female macaques are different during the postpartum period. PMID- 7534850 TI - Transgenic mice carrying a PSArasT24 hybrid gene develop salivary gland and gastrointestinal tract neoplasms. AB - BACKGROUND: Although prostate cancer is one of the most prevalent tumors in men, knowledge of its biology has been hindered by lack of animal models. We have attempted to develop a prostate cancer model utilizing transgenic mouse technology. EXPERIMENTAL DESIGN: Two lines of transgenic mice were derived from one cell stage embryos injected with a fusion gene consisting of a mutated (codon 12) ras gene driven by the human prostate specific antigen (PSA) promoter in an attempt to target the oncogene specifically to the mouse prostate gland. Nontransgenic FVB/N mice were used as controls. The animals were sacrificed for study between 4 and 55 weeks of age. RESULTS: All organs were normal except the salivary glands and gastrointestinal tracts, both of which developed carcinomas in animals older than 44 weeks. The salivary gland tumors were of ductal origin, exhibited a variable degree of differentiation, and were shown to contain abundant PSAras mRNA by in situ hybridization. The gastrointestinal tract tumors were undifferentiated but appeared to be of stromal origin. Both salivary gland and gastrointestinal tumors occasionally metastasized. No transgene expression could be demonstrated in the prostate gland by either reverse transcription polymerase chain reaction or in situ hybridization. CONCLUSIONS: Lack of transgene expression by the prostate can be explained on the basis of the apparent species specificity previously observed for PSA. Expression in salivary gland is best attributed to identity between the nucleotide sequences of the PSA promoter and of a mouse glandular kallikrein normally secreted by the salivary gland. PMID- 7534852 TI - Inducible nitric oxide synthase modulates fibronectin production in the EA.hy926 cell line and cultured human umbilical vein endothelial cells. AB - We wished to test the hypothesis of a connection existing between inducible nitric oxide (NO) synthesis and production of extracellular matrix proteins in endothelial cells (EC). We recently reported that the inducible-NO pathway contributes to cytokine-induced enhancement of tumor cell (TC) adhesion to cultured vascular endothelium, independent of changes in E-selectin expression on endothelial cells (EC). We now show that inducible NO-synthase is involved in enhancing fibronectin production by EC. Indeed, fibronectin synthesis and secretion increased both in the EA.hy926 EC line and in human umbilical vein EC (HUVEC) after prolonged exposure to tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). This effect was reversed by the reported inhibitor of NO synthase N omega-nitro-L-arginine methyl ester (L-NAME 10(-5) M). The two cytokines exerted no additive effect, suggesting that they trigger a common metabolic pathway. NO production by cytokine-stimulated EC was dependent on the inducible NO-pathway, as demonstrated by studies of EC-dependent inhibition of platelet aggregation. This inhibition was also evident in calcium-free medium and was reversed by L-NAME and by two inhibitors of protein synthesis that are reported to block the inducible-NO synthase, such as dexamethasone (Dex 10(-7) M) and cycloheximide (Chx 10(-6) M). We conclude that modulation of the inducible NO synthase may regulate matrix protein production by vascular endothelium during inflammation. PMID- 7534851 TI - Development of the conduction system in the rat heart as determined by Leu-7 (HNK 1) immunohistochemistry and computer graphics reconstruction. AB - BACKGROUND: The development of the heart conduction system is controversial. Our previous study demonstrated anti-Leu-7 antibody to cross-react with the cells of the conduction system in the embryonic rat heart. Thus, detailed analysis of the development of the conduction system was performed by immunohistochemical reaction with the use of the antibody. DESIGN: Horizontal serial sections of hearts obtained from embryonic and neonatal rats were treated for immunohistochemical reaction with anti-Leu-7 antibody, and three-dimensional images were reconstructed by computer graphics. Images were analyzed by superimposing the results on scanning electron micrographs. RESULTS: The development of the rat heart conduction system starts at 10 days and is completed by 18 days of gestation. The presence of three internodal tracts (INT1, -2, and 3) and two primordia of the atrioventricular node were confirmed in this process. INT1 and -3 pass through the septum spurium. INT2 follows the same route as the posterior internodal tract of James, but the other two INTs showed no correlation to previously described tracts. The sinuatrial node and INTs were found to be derivatives of the so-called S-A ring, and the bundle of His and bundle branches were found to be derived from the B-V ring. In this study, no immunoreactivity was observed in cells of the truncobulbar portion. CONCLUSIONS: Three internodal tracts and two atrioventricular node primordia were observed in the developing heart. The paths of the three internodal tracts showed intimate relationships with the internal structures of the heart. The developmental significance of the septum spurium, sinus septum, and venous valve was discussed. PMID- 7534853 TI - Ovarian mucinous cystadenocarcinoma with sarcoma-like mural nodules. AB - A rare case of ovarian mucinous cystadenocarcinoma with sarcoma-like mural nodules is reported. Macroscopically, nodular or granular lesions were scattered over the inner surface of a huge, multilocular tumor in the left ovary. The histopathological features were consistent with those of previously documented cases, except significant squamous metaplasia in the present case. The pleomorphic sarcoma-like cells showed a positive reaction for vimentin and alpha antichymotrypsin but were negative for carcinoembryonic antigen and common epithelial membrane antigen by immunohistochemistry. Electron microscopically, the tumor cells had neither desmosomes nor secretory granules, but they possessed abundant intermediate filaments. Both immunostaining and ultrastructure suggested that the sarcoma-like nodules were derived from reactive proliferation of fibrohistiocytic cells. The fibrohistiocytic reaction seemed to occur in response to hemorrhage, because hemorrhage was reported to be associated with the mural nodules in most of the previous cases as well as ours. PMID- 7534854 TI - [Granulocyte colony-stimulating factor and HIV infection]. PMID- 7534855 TI - CD44 variant exon epitopes in primary breast cancer and length of survival. AB - CD44 designates a group of closely related cell-surface proteins generated by alternative splicing. We have previously shown that splice variants carrying sequences encoded by exon v6 are preferentially expressed in metastatic animal cancer cell lines and that they confer metastatic behaviour on non-metastatic animal tumour cell lines. In this study we set out to assess the expression of CD44 epitopes specific for variant exon sequences in human breast cancer and their potential for determining prognosis. We used affinity-purified polyclonal sera and four monoclonal antibodies raised against the human homologues of CD44 variant exon sequences to investigate the presence of CD44 on 100 primary invasive breast tumours, 12 local recurrences, 18 lymph node metastases, and normal tissue controls. Whereas normal mammary ductal epithelial cells and cells derived from hyperplastic lesions do not express CD44 variant exons, expression of v3, v5, and v6 epitopes was found in most tumour samples. The DIII (exon v6) epitope was present in 84% of the primary tumours and in 100% of axillary lymph node metastases and local recurrences. The presence of these CD44 epitopes is correlated with poor overall survival. 15 patients with exon-v6-negative tumours had good survival compared with 76 patients with exon-v6-positive tumours (p = 0.005; log rank test). Multivariate analysis showed that the CD44 epitope encoded by exon v6 was a good marker for prognosis independent of progesterone receptor, lymph node status, tumour size, and grade. PMID- 7534856 TI - Tacrine. PMID- 7534857 TI - Measles virus in Crohn's disease. PMID- 7534858 TI - HIV-1 RNA serum-load and resistant viral genotypes during early zidovudine therapy. AB - The response of HIV-1 to initial zidovudine (ZDV) treatment was assessed in 11 patients with severe HIV disease. We quantified serum HIV-1 concentrations and mutations associated with ZDV resistance by culture-independent methods. There was a prompt fall in serum HIV-1 RNA within 1-2 days of treatment with maximum suppression by seven days, which was paralleled by changes in serum p24 antigen (p24 Ag). Serum RNA started to return to pretreatment levels within weeks. The HIV reverse transcriptase (RT) gene in most patients developed mutations associated with drug resistance within months and as early as 25 days on therapy in one patient. The codon changes were not sufficient to explain the early return of serum HIV-1 RNA levels and their patterns continued to evolve after patients stopped taking ZDV. The significance of these findings is discussed in relation to the limited long-term efficacy of ZDV. The dynamic time course of viral load and RT responses to ZDV is of particular importance in short-term interventions such as pregnancy. PMID- 7534859 TI - Patient preference for extended palliative chemotherapy for non-small cell lung cancer. PMID- 7534860 TI - Controlled delivery of Gd-containing liposomes to lymph nodes: surface modification may enhance MRI contrast properties. AB - Surface modification of liposomes containing Gd-diethylenetriaminepentaacetyl phosphatidylethanolamine (Gd-DTPA-PE) as an amphiphilic paramagnetic label influences their contrast properties as MRI agents. Liposomes with the mean diameter ca. 220 nm and 10% mol content of Gd-DTPA-PE were modified with dextran (molecular weight 6 kDa) or polyethyleneglycol (PEG, molecular weight 5 kDa). The preparations obtained were used for MR visualization of axillary and subscapular lymph nodes after SC injection into rabbit's paw. Dextran-modified liposomes were found to have limited or no effect on lymph node/muscle MR signal intensity ratio during the first 2 h after the administration when compared to nonmodified plain liposomes, whereas the liposomes modified with PEG demonstrated a 3-3.5-fold enhancement of lymph node signal. However, the partial biodistribution studies with the 111In-labeled liposomes revealed that PEG-modified vesicles accumulated in the axillary lymph node at half the amount comparing with nonmodified and dextran-modified liposomes. The phenomenon found might be explained by noticeably increased relaxivity of PEG-modified Gd-liposomes. PMID- 7534861 TI - Stabilization of neurofilament transcripts during postnatal development. AB - Neurofilament (NF) mRNAs in primary sensory neurons are long-lived transcripts that undergo transcription-dependent destabilization when placed in primary culture [32]. Destabilization of NF transcripts implies that the transcripts are stabilized in high-expressing neurons and that stabilization may coordinate and increase levels of NF expression. The present study examines the stabilities of the three NF subunit mRNAs in postnatal cultures of dorsal root ganglia (DRG) to determine whether increased stability of NF mRNAs could be responsible for the coordinate postnatal upregulation of the three NF subunits [29]. The studies show that the light (NF-L), mid-sized (NF-M) and heavy (NF-H) NF mRNAs are lost at 8 and 16 h in primary cultures from postnatal day 2 (P2) rats, but much less so in cultures from postnatal day 16 (P16) and day 30 (P30) rats. Losses of each NF mRNAs in P2 cultures occurs simultaneously in the presence or absence of actinomycin. The findings support the view that stabilization of NF transcripts contribute to the high and coordinate level NF expression and that components of the stabilizing process are acquired during postnatal development. PMID- 7534863 TI - Preparation of fluorescent glycoconjugates for energy transfer studies. PMID- 7534864 TI - Mapping of hydrogen bonding between saccharides and proteins in solution. PMID- 7534862 TI - Polysaccharide affinity columns for purification of lipopolysaccharide-specific murine monoclonal antibodies. PMID- 7534865 TI - Preparation of tyrosinamide-oligosaccharides as iodinatable glycoconjugates. PMID- 7534867 TI - [The change of lymphocyte subset and effect of FK506 in experimental small bowel transplantation of rats]. AB - I analyzed the relationship of the change of lymphocyte subsets in the blood and acute allograft rejection of heterotopic small bowel transplantation in the rats. The effect of immunosuppressive agent, FK506 was monitored by lymphocyte subsets and effective dose in small bowel transplantation was assessed. Four different combinations were investigated: (A) syngenic graft; (B) allogenic graft without FK506; (C) allogenic graft with FK 0.135mg/kg/day; and (C-2) allogenic graft with FK 0.270mg/kg/day. Lymphocytes obtained from the caudal artery blood at 1, 3, 5 and 7 days after transplantation, were stained by monoclonal antibodies (W3/13, W3/25, OX-8, 68-IB3) and then enumerated by flow cytometry. In group A, lymphocyte subsets showed no significant change after transplantation. Group B showed remarkable decrease of W3/13 and OX-8 positive cells at 3 and 5 days after transplantation. In group C-1, the change of lymphocyte subsets was similar to group B. In group C-2, the lymphocyte subsets revealed almost same change as group A. The change of lymphocyte subsets, especially W3/13 and OX-8 positive cells, may be useful marker for early diagnosis of acute rejection and for determination of the effective dose of FK506 in the small bowel transplantation. PMID- 7534866 TI - Glioma-stimulated chemoattraction of endothelial cells and fibroblasts in vitro: a model for the study of glioma-induced angiogenesis. AB - Induction of angiogenesis is essential for the continued growth of solid tumors, and one critical component of tumor-induced angiogenesis involves the stimulation of microvascular cells to migrate into the growing mass. We have developed a convenient model system utilizing dual co-culture chambers to study cellular chemotaxis induced by glioma cells in vitro. In this system, rat C6 glioma cells induced migration of fibroblasts and brain capillary endothelial cells. The migratory response was directly related to the number of C6 cells serving as stimulus in the lower chamber. Similar migratory responses were induced by C6 cell conditioned medium in a concentration dependent fashion. Medium conditioned by cultured human anaplastic astrocytoma cells was also found to contain potent chemotactic factor(s). This system may ultimately be employed in the identification of particular glioma cell population(s) and secreted factor(s) responsible for the chemoattraction of microvascular cells. PMID- 7534869 TI - Hydrophobic, hydrophilic and other interactions in epitope-paratope binding. AB - Macroscopic, non-covalent, aspecific interactions between hydrophilic biopolymers, particles and cells in aqueous media tend to be repulsive; they are caused by Lifshitz-van der Waals (LW), Lewis acid-base (AB) and electrostatic (EL) forces. Microscopic scale specific interactions, e.g. between epitopes and paratopes, are also non-covalent and caused by attractive LW, AB and EL forces, which locally must be able to overcome the long- to medium-range macroscopic aspecific repulsive forces. Thus epitopes and paratopes need to be able to attract each other over a distance of at least 3 nm. The medium- and long-range specific attractive forces are mainly of hydrophobic (AB) and of EL origin; in aqueous media the medium- and long-range LW attractions are usually much weaker. It has been shown that hydrophobic (AB) interactions are as often enthalpic as entropic. Upon expulsion of interstitial water of hydration between epitope and paratope, a strong interfacial bond ultimately arises which is mainly caused by LW forces. PMID- 7534870 TI - Beneficial effects of iloprost on acute myocardial ischemia in dogs. AB - The effects of intravenous administration of iloprost, a prostacyclin analogue, on myocardial energy metabolism and myocardial blood flow (MBF) were examined in anesthetized open-chest dogs subjected to 60 min of myocardial ischemia by coronary ligation. Iloprost administration at levels of 0.1 or 0.2 micrograms/kg/min was started 30 min before the commencement of ischemia and continued throughout the 90 min observation period. Since systolic aortic pressure in the iloprost 0.2 micrograms/kg/min group showed significantly lower values than that in the control group, whereas no clear effect was observed with the lower concentration (0.1 micrograms/kg/min), this latter group was further investigated. This 0.1 microgram/kg/min dose of iloprost lacked influence on MBF in both ischemic and nonischemic areas but did result in a significantly higher value for high energy phosphate contents in the ischemic myocardium. Moreover, myocardial mitochondrial respiratory function in the ischemic area was significantly improved. These results indicate that iloprost brought about preservation of myocardial energy metabolism without alteration of coronary perfusion, suggesting that it may exert a direct cardioprotective effect. PMID- 7534871 TI - Special issue dedicated to Dr. Sidney Ochs. PMID- 7534868 TI - Are most transporters and channels beta barrels? AB - Given the sequence of transporters or channels of unknown secondary structure, it is usual to predict their putative transmembrane regions as alpha-helical. However, recent evidence for a facilitative glucose transporter (GLUT1) appears inconsistent with such predictions, which has led us to propose an alternative folding model for GLUTs based on the 16-stranded antiparallel beta-barrel of porins. Here we apply the same predictive algorithms we used for GLUTs to several other membrane proteins. For some of them, a high-resolution structure has been derived (beta-barrels: Rhodobacter capsulatus and Escherichia coli porins; multihelical: colicin A, bacteriorhodopsin, and reaction center L chain); we use them to test the prediction procedures. The other proteins we analyze (GLUT1, CHIP28, acetylcholine receptor alpha subunit, lac permease, Na(+)-glucose cotransporter, shaker K+ channel, sarcoplasmic reticulum Ca(2+)-ATPase) are representative of classes of similar membrane proteins. As with GLUTs, we find that the predicted transmembrane segments of these proteins are consistently shorter than expected for transmembrane spanning alpha-helices, but are of the correct length and number for the proteins to fold instead as porin-like beta barrels. PMID- 7534872 TI - Depression of fast axonal transport in axons demyelinated by intraneural injection of a neurotoxin from K. humboldtiana. AB - Tullidinol, a neurotoxin extracted from the Karwinskia humboldtiana fruit, dissolved in peanut oil was injected into the right sciatic nerve of adult cats. The contralateral sciatic nerve received an equivalent volume of peanut oil alone. The fast axonal transport of labeled ([3H]Leucine) protein was studied in sensory and motor axons of both sciatic nerves. The radioactive label was pressure injected either into the L7 dorsal root ganglion or the ventral region of the same spinal cord segment. Several days after the toxin injection, the cat limped and the Achilles tendon reflex was nearly absent in the right hind limb. The amount of transported label was decreased distal to the site of toxin injection. Proximal to this site, the transported material was damned. Sensory and motor axons showed similar changes. In addition, the toxin produced demyelination and axonal degeneration. Axonal transport and the structure of the axons were normal in the contralateral nerve. Both, Schwann cells and axons of the right sciatic nerve showed globular inclusions, presumably oil droplets containing the toxin. We conclude that Schwann cells and axons as well are tullidinol targets. PMID- 7534874 TI - Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as a function of age. AB - Calcitonin gene-related peptide (CGRP) has been implicated in the trophic regulation of acetyl-choline receptors and G4 acetylcholinesterase at the rat neuromuscular junction. Since these latter molecules exhibit significant changes with advancing age, we examined the possibility that certain aspects of CGRP transport are also influenced by aging. Double nerve ligations and CGRP radio immunoassay of 3-mm nerve segments permitted the assessment of the peptide's apparent transport rates in sciatic nerves from 3-, 12-, and 24-month-old Fischer 344 rats. Results confirm that CGRP is conveyed by anterograde axoplasmic transport; more importantly, they suggest that CGRP is also transported retrogradely, but in smaller amounts and at slower rates. In addition, our findings indicate that the apparent rates of CGRP transport in both directions significantly decline with advancing age. These data are consistent with the notion that changes in CGRP delivery may contribute to age-related changes in junctional acetylcholine receptors and acetylcholinesterase. PMID- 7534873 TI - Insulin-like growth factor binding protein-1 is pre-synaptic at mouse neuromuscular synapses and is transported in nerve. AB - In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were detected by Western ligand blot (WLB) with 125I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse. PMID- 7534875 TI - Cholinergic innervation of the retrosplenial cortex via the fornix pathway as determined by high affinity choline uptake, choline acetyltransferase activity, and muscarinic receptor binding in the rat. AB - The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC. PMID- 7534876 TI - Fast and slow axonal transport-different methodological approaches give complementary information: contributions of the stop-flow/crush approach. AB - This 'minireview' describes experiments in short term crush operated rat nerves, to study endogenous substances in anterograde and retrograde fast axonal transport. Immunofluorescence was used to recognize transported antigens, and cytofluorimetric scanning was employed to quantitate different antigens which had accumulated proximal and distal to the crushes. Vesicle membrane components p38 (synaptophysin) and SV2 accumulated on both sides of a crush. This was expected from a number of studies from different laboratories. Surface associated molecules, however, like synapsins and rab3a, have been studied by other groups with biochemical methods, and suggested to be transported with slow transport. The crush method, however, revealed that a considerable fraction of these two substances are transported with the fast transport system, and, thus, associated with fast transported organelles in the living neuron. Evidently, more than one technique is required to give a more complete picture of intraneuronal transport related events. PMID- 7534877 TI - Is the intrasomal phase of fast axonal transport driven by oscillations of intracellular calcium? AB - An hypothesis is presented suggesting that the delivery of vesicle-packaged protein from the neuronal soma to the axonal transport system is physiologically coupled to spontaneous fluctuations of intracellular calcium (Cai). Evidence is reviewed that oscillations of Cai, commonly detected as agonist- or voltage triggered waves and spikes propagating through the cytosol, also occur as spontaneous events. Endogenously-generated oscillations are examined since intrasomal transport persists in the absence of extracellular signals or nerve impulse activity. Vesicle budding from the endoplasmic reticulum (ER) may be a key step at which anterograde transport is regulated by events related to the release and reuptake of ER stores of Ca2+. PMID- 7534880 TI - Histaminergic and non-histamine-immunoreactive mast cells within the cat lateral geniculate complex examined with light and electron microscopy. AB - Mast cells and their location in the cat lateral geniculate complex of the thalamus were examined by means of histamine immunohistochemistry and the mast cell stain pinacyanol erythrosinate. Brain sections from seven normal adult pigmented cats were processed for light or electron microscopy. Histamine containing and pinacyanol erythrosinate-stained mast cells were widespread throughout the dorsal and ventral lateral geniculate nuclei and the surrounding regions. Mast cells were especially numerous rostrally in the complex and in the geniculate C laminae. The cells were found consistently in association with blood vessels, ranging from capillary size to vessels c. 150 microns diameter, and twice as often with arterioles as with venules. Large clusters of many mast cells associated with single blood vessels were seen. Individual mast cells were typically 8 microns in diameter and somewhat oval, although multipolar and crescent-shaped cells were also seen, up to twice as long. The amount of histamine labeling varied across cells. When histamine-labeled material was secondarily stained with pinacyanol erythrosinate, many mast cells were double labeled. In addition, there was a small population of mast cells that stained only with pinacyanol erythrosinate, but was otherwise identical to the histamine immunoreactive mast cells. Electron microscopic examination showed that the mast cells lie on the brain side of the blood-brain barrier. Mast cells were found in close proximity to the thalamic neuropil, primarily apposed to the processes of astrocytes, but also apposed to neural elements. The distinctive electron-dense cytoplasmic granules in the fully granulated, mature state were largely amorphous in appearance and as large as 700 nm in diameter. Histamine was dispersed throughout some granules and contained within restricted areas of other granules. In degranulated mast cells, large, irregularly shaped, electron-lucent granules were seen fused with the cell membrane on the neuropil side, as well as the lumen side of the mast cell. More mast cells were observed at the electron microscopic level than were expected from the light level observations, which suggests that, despite the numbers of mast cells labeled, these results may still underestimate the total mast cell population present in this region of the thalamus. Mast cells, by their numbers, their distribution and the potent chemical substances they contain, may significantly influence vascular and neural function, directly and indirectly, in the cat lateral geniculate complex. PMID- 7534879 TI - GR138676, a novel peptidic tachykinin antagonist which is potent at NK3 receptors. AB - GR138676, a conformationally constrained analogue of neurokinin B, is a novel, potent NK3 receptor antagonist. GR138676 was a competitive antagonist of neurokinin B-dependent arachidonic acid mobilization from prelabelled Chinese hamster ovary cells stably transfected with a human NK3 receptor gene (pKB 8.3) and of contractions induced by senktide in rat portal vein (pKB 8.2). However, GR138676 was also a competitive antagonist of the increase in intracellular calcium evoked by the selective NK1 agonist, GR73632, in the human astrocytoma U373MG cell-line (pKB 8.3). GR138676 had little activity at NK2 receptors, inhibiting binding of the NK2 antagonist radioligand [3H]-GR100679 to Chinese hamster ovary cells transfected with the human ileum NK2 receptor with a pKi of 6.0. In summary, despite its activity at NK1 receptors, GR138676 will be a useful tool for characterizing NK3 receptors as well as defining the physiological and pathophysiological function of this receptor subtype. PMID- 7534878 TI - [32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo. AB - Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [32P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble 32P-carrier that was axonally transported faster than neurofilaments. 32P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons. 32P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total 32P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [35S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons. PMID- 7534881 TI - Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons. AB - Two isoforms of the enzyme heme oxygenase are expressed in distinct populations of neurons in the brain. These enzymes catalyse the oxidative cleavage of heme to the cellular antioxidant biliverdin resulting in the release of carbon monoxide in the process. Both heme and carbon monoxide may play important roles in regulating the nitric oxide-cyclic guanosine monophosphate signal transduction system. Thus we have examined the distributions of both isoforms of heme oxygenase in the rat brain, and compared their localizations with that of nitric oxide synthase determined with the NADPH-diaphorase histochemical technique. Heme oxygenase-1 is highly expressed in a few select populations of neurons including cells in the hilus of the dentate gyrus, in the hypothalamus, cerebellum and brainstem. This enzyme appears to be coexpressed with nitric oxide synthase only in a few cells in the dentate gyrus. Heme oxygenase-2 is much more widely expressed. It is present in mitral cells in the olfactory bulb, pyramidal cells in the cortex and hippocampus, granule cells in the dentate gyrus, many neurons in the thalamus, hypothalamus, cerebellum and caudal brainstem. However, only some of these labelled neurons also displayed nitric oxide synthase. Instead, many neurons expressing heme oxygenase-2 correspond to those known to express high levels of the hemoprotein soluble guanylyl cyclase. These results suggest that heme oxygenase may play a role in modulating guanylyl cyclase independent of nitric oxide synthase. This may result from regulation of intracellular heme and carbon monoxide levels by the heme oxygenase system. PMID- 7534882 TI - Distribution, origin and projections of nitric oxide synthase-containing neurons in gut and pancreas. AB - Nitric oxide has been put forward as an important inhibitory neurotransmitter in the gut. Nitric oxide synthase-containing neurons were visualized by immunocytochemistry using antibodies against neuronal nitric oxide synthase or by beta-nicotinamide adenine dinucleotide phosphate diaphorase staining in whole mounts and cryostat sections from the gastrointestinal tract and pancreas of several mammals (mouse, rat, hamster, guinea-pig, cat and man). Nitric oxide synthase-containing neuronal cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut of all species. Varicose nerve terminals formed extensive networks in the circular smooth muscle and the myenteric ganglia. Nitric oxide synthase-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals were also found in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. In the rat small and large intestine nitric oxide synthase-containing submucous neurons terminated within the mucosa/submucosa and nitric oxide synthase-containing myenteric neurons issued short descending projections, approximately 3 mm, to the smooth muscle and other myenteric ganglia. In the pancreas of all species nitric oxide synthase containing nerve cell bodies were regularly seen in intrapancreatic ganglia. Positive nerve fibers were mainly found within nerve trunks in interlobular spaces and as delicate fibers within the islets. Double staining for nitric oxide synthase and neuropeptides in intestine and pancreas of rat, guinea-pig and man revealed that only occasionally the nitric oxide synthase-containing nerve cell bodies stored in addition vasoactive intestinal peptide and neuropeptide Y, or enkephalin. However, nitric oxide synthase-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained vasoactive intestinal peptide/neuropeptide Y (rat and man) or vasoactive intestinal peptide/enkephalin (guinea-pig). In intrapancreatic ganglia few nitric oxide synthase-containing nerve cell bodies were also vasoactive intestinal peptide-immunoreactive. Coexistence of nitric oxide synthase and vasoactive intestinal peptide in nerve terminals could here be detected around blood vessels and interlobular ducts. The distribution of nitric oxide synthase indicates a major role of nitric oxide in the regulation of gut motility; a role in the regulation of blood flow and secretion in both gut and pancreas is also likely. PMID- 7534884 TI - The effects of intrathecal neuropeptide Y on the spinal nociceptive flexor reflex in rats with intact sciatic nerves and after peripheral axotomy. AB - We examined the effects of intrathecally administered neuropeptide Y on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats with intact sciatic nerves, or 11-39 days after unilateral transection of the sciatic nerve. In rats with intact sciatic nerve, intrathecal neuropeptide Y at low doses (10 and 100 ng) caused a brief facilitation of the flexor reflex. At a dose of 300 ng, the effect of neuropeptide Y on the flexor reflex was biphasic, i.e. a brief facilitation followed by slight depression. At higher doses (1 and 10 micrograms), the effect of neuropeptide Y was mainly inhibitory, causing substantial and usually prolonged depression of the flexor reflex magnitude. The reflex depression caused by intrathecal neuropeptide Y was not reversed by the opioid antagonist naloxone or the alpha 2 adrenoceptor antagonist atipamezole. Intrathecal neuropeptide Y at doses up to 1 and 10 micrograms had no effect on reflex facilitation caused by conditioning stimulation of C-fibers, intrathecal substance P or neurokinin A. Topical application of neuropeptide Y (1 microgram/microliter) failed to influence the monosynaptic reflex in normal rats. Eleven to 16 days after peripheral axotomy, the initial excitation of the flexor reflex to intrathecal neuropeptide Y was significantly enhanced in axotomized compared with normal rats. However, the depressive effect of neuropeptide Y on the flexor reflex was unchanged. Neuropeptide Y did not influence the monosynaptic reflex in axotomized rats at this period. In experiments performed on rats in which the sciatic nerve had been transected 31-39 days previously, the facilitatory effect of neuropeptide Y on the flexor reflex remained enhanced compared with normal rats. Furthermore, the inhibitory effect of neuropeptide Y also increased as 100 ng intrathecal neuropeptide Y was able to produce reflex depression in a similar fashion as 300 ng neuropeptide Y normally and the reflex depression caused by 1 microgram neuropeptide Y was stronger and longer lasting than in normal rats. Intrathecal neuropeptide Y (100 ng-10 micrograms) in rats with intact sciatic nerves caused a moderate decrease in spinal cord dorsal surface blood flow as measured with a laser Doppler flowmeter. This effect of neuropeptide Y was unchanged in axotomized rats. The present results support previous observations that spinal application of neuropeptide Y in normal rats caused antinociception. As the depressive effect of neuropeptide Y is independent of spinal opioid and alpha 2-adrenergic systems, it may be mediated by its own receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7534883 TI - Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons. AB - Nitric oxide can act as a neurotransmitter and a retrograde modulator of synaptic transmission, but uncontrolled nitric oxide synthase activity has been associated with neural degeneration. Although earlier studies using immunohistochemistry, in situ hybridization, and NADPH-diaphorase staining had suggested that nitric oxide synthase is not expressed in the CA1 neurons of the hippocampus, we have recently demonstrated that NADPH-diaphorase activity can be detected in CA1 neurons of the hippocampus. To confirm that this diaphorase activity reflects nitric oxide synthase, we have developed a more sensitive in situ hybridization procedure, and an RNase protection assay to detect message for constitutive nitric oxide synthase, the form constitutively expressed in many neurons. Message for constitutive nitric oxide synthase is expressed in the hippocampus, and it is localized to neural cell layers CA1, CA3, the dentate gyrus and some displaced neurons, but not to CA2. Expression of constitutive nitric oxide synthase message in the CA1 region was lost when pyramidal neurons died due to transient forebrain ischemia, supporting the conclusion that CA1 pyramidal cells express constitutive nitric oxide synthase. Although constitutive nitric oxide synthase message is strongly expressed in CA3 and the dentate gyrus, there is little diaphorase activity in these cells, suggesting that there may be post-transcriptional controls that limit constitutive nitric oxide synthase expression in some cells. Message for constitutive nitric oxide synthase is also present in a number of other regions, including the amygdala, several hypothalamic nuclei, the cerebellum, the olfactory bulb, two distinct regions of the perirhinal cortex, the subthalamic nuclei, a neuronal layer in the retrosplenial granular cortex, the lateral geniculate nucleus, the presubiculum, the inferior colliculus, the superior colliculus, the pedunculopontine tegmental nucleus, and scattered individual neurons in the cortex, hippocampus and brainstem. These studies support a role for nitric oxide in multiple regions of the central nervous system. In particular, nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide, is expressed in the CA1 region of the hippocampus, where there is evidence that nitric oxide may play a major role in long-term potentiation. CA1 hippocampal neurons are an example of a population of neurons that express constitutive nitric oxide synthase but are very sensitive to excitotoxicity and ischemic insults. PMID- 7534885 TI - Synaptotagmin I is present mainly in autonomic and sensory neurons of the rat peripheral nervous system. AB - The distribution of synaptotagmin I in the peripheral nervous system of the rat was investigated by immunofluorescence and confocal laser scanning microscopy. After crushing of the sciatic nerve, synaptotagmin I-like immunoreactivity accumulated proximally as well as distally to the crushes in thin and medium sized axons. Double labelling studies revealed that synaptotagmin I co-localized with tyrosine hydroxylase, a marker of sympathetic adrenergic neurons, and with substance P, a marker for sensory neurons. No synaptotagmin I-like immunoreactivity was found in large axons, while accumulations of the synaptic vesicle proteins synaptophysin and synapsin I were found in all types of axons. Furthermore, no synaptotagmin I-like immunoreactivity was detected in motor endplates. In contrast, the protein was found in muscle spindles of young rats and in perivascular terminals, where it co-localized with synaptophysin and synapsin I. Lumbar sympathectomy resulted in a marked reduction of the amount and intensity of synaptotagmin I-like immunoreactivity in sciatic nerve. High magnification revealed that synaptotagmin I-like immunoreactivity was mainly distributed in a fine granular pattern, but large, brightly fluorescent granules which were not labelled by anti-synaptophysin or anti-synapsin I were occasionally observed. We conclude that synaptotagmin I is mainly expressed in adrenergic and sensory neurons and is absent from, or below detection levels, in motoneurons. PMID- 7534886 TI - Activation of galanin pathways across puberty in the male rat: galanin gene expression in the bed nucleus of the stria terminalis and medial amygdala. AB - Galanin and vasopressin are coexpressed in the bed nucleus of the stria terminalis and medial amygdala of the male rat. In adult males, the level of gene expression for both peptides in these regions is dependent on circulating levels of testosterone. We hypothesized that galanin messenger RNA levels would be enhanced in adult males compared with prepubertal males due to the rise in plasma testosterone levels. We used in situ hybridization and quantitative autoradiography to measure galanin messenger RNA in cells of the bed nucleus of the stria terminalis and medial amygdala of prepubertal and adult male rats. Our results show that significantly (P < or = 0.05) more galanin messenger RNA expressing neurons are detectable in the bed nucleus of the stria terminalis of adult compared with prepubertal male rats. In contrast, no differences were observed between the groups in the number of labeled neurons detected within the medial amygdala. However, the average labeling intensity was significantly enhanced in both the bed nucleus of the stria terminalis (P < or = 0.001) and medial amygdala (P < or = 0.001) of adult compared with prepubertal animals. The present findings are consistent with the hypothesis that gonadal hormones regulate galanin gene expression in some brain regions and suggest that the activation of the hypothalamic-pituitary-gonadal axis which occurs naturally with puberty is associated with activation of galanin pathways in the bed nucleus of the stria terminalis and medial amygdala. PMID- 7534888 TI - Amino acids differentially inhibit the L-[3H]arginine transport and nitric oxide synthase in rat brain synaptosomes. AB - The nitric oxide synthase (NOS) present in the cytosol obtained from rat cerebral cortex synaptosomes was inhibited by NG-nitro-L-arginine (L-NOArg) with an IC50 value of approximately 0.06 microM. This compound did not affect the transport of L-arginine (L-Arg) into synaptosomes at concentrations up to 100 microM but other potential inhibitors of NOS (NG-monomethyl-L-arginine, NG-amino-L-arginine and L arginine methyl ester) inhibited L-Arg transport at a concentration < 5 microM. We showed that concentrations of L-NOArg (0.001-3 microM) that did not block the uptake of tritiated arginine (L-[3H]Arg) inhibited the catalytic activity of NOS in intact synaptosomes. L-NOArg at a concentration of 1 microM inhibited the cytosolic enzyme by 98.0 +/- 2.0% of the total NOS activity whereas the enzyme studied in the intact synaptosomes was only inhibited by 75 +/- 5% which suggested that the NOS in synaptosomes is not fully accessible to the external L NOArg. On the other hand, L-Lysine did not inhibit the cytosolic NOS activity of ruptured synaptosomes but at a concentration that blocked 50.0 +/- 4.5% of L [3H]Arg uptake it inhibited the NOS activity in intact synaptosomes by 12.6 +/- 3.6%, suggesting that the transport of L-Arg may be an important regulatory step in the pathway for nitric oxide generation. PMID- 7534887 TI - Activation of galanin pathways across puberty in the male rat: assessment of regional densities of galanin binding sites. AB - Galanin-like immunoreactivity and galanin messenger RNA levels increase across puberty in neurons of gonadal steroid-dependent brain nuclei. We hypothesized that this activation and the associated increase in endogenous galanin release would result in changes across puberty in both galanin binding density and the level of receptor occupancy. Here we have assessed the density of galanin binding sites in several brain regions of prepubertal and adult male rats with or without GTP to induce dissociation of endogenous galanin from its binding sites. The developmental changes in the level of receptor occupancy were used as an indirect measure of changes in neuropeptide release from galanin expressing neurons. In standard binding conditions (buffer preincubation), 125I-labeled galanin binding showed a generalized decline in adult brains (34-68%) compared with prepubertal levels in most regions of the telencephalon and diencephalon. Following preincubation with 10(-5) M GTP, galanin binding showed a dramatic increase in most regions of the adult (152-504%) and several regions of the prepubertal brain (132-245%) over their standard binding levels. However, this increase was greatest in adult animals. Finally, although preincubation of brain slices with GTP eliminated most of the apparent age-related differences observed in standard binding conditions, several brain regions of the adult brain continued to show a significant reduction (38-76%) in 125I-labeled galanin binding compared with prepubertal animals. Only one region, the lateral preoptic area, exhibited enhanced 125I-labeled galanin binding in adult (160%) compared with prepubertal brain after GTP preincubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534889 TI - Monoclonal IgM reactive with several gangliosides in a chronic relapsing polyneuropathy. AB - A possibly pathogenic serum monoclonal IgM lambda from a patient with chronic relapsing polyneuropathy was shown to react with the disialosyl-lactosyl residue (NeuAc alpha 2-8NeuAc alpha 2-3) -Gal beta 1-4Glc expressed by GD1b, GT1b, GQ1b, GD2 and GD3. A part of this epitope in terminal position in GM3, GD1a and LM1 was also recognized by the IgM lambda. PMID- 7534890 TI - Detection of a 23 kDa human brain protein that shares epitope(s) with the 17 kDa core protein of human immunodeficiency virus (HIV). AB - To search for brain molecules that share epitopes with HIV (human immunodeficiency virus) proteins, we investigated non-AIDS human brains by immunohistochemical and Western blotting methods, and detected a 23 kDa protein which was immunoreactive with antibodies against HIVp17, a core protein of HIV with a molecular weight of 17 kDa. The 23 kDa protein was localized mostly in reactive astrocytes in and around pathologic areas of the brain, but some neurons also had it. We propose that any HIV vaccine must be checked up if it induces a cross-reacting antibody to a human brain or other organs. PMID- 7534891 TI - Decrease and increase of responses to glutamate receptor agonists in RNA-injected Xenopus oocytes by the epileptogenic agent pentylenetetrazol: dependence on the agonist concentration. AB - The effects of the epileptogenic agent pentylenetetrazol (PTZ) on current responses to glutamate (Glu) and to the Glu receptor agonist N-methyl-D-aspartate (NMDA), kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QA) were studied on RNA-injected Xenopus oocytes. PTZ antagonized the reactions to Glu as well as to NMDA, KA, AMPA and QA. With increasing concentration of AMPA, however, the depressive effect of PTZ turned to an augmentation. The complete change from a decreasing to an increasing effect of PTZ with elevated agonist concentration was unique for current responses by AMPA. PMID- 7534892 TI - Effect of opioid receptor agonists on nitric oxide synthase activity in rat cerebral cortex homogenate. AB - The effect of drugs acting at mu-, delta-, or kappa-opioid receptors on the activity of nitric oxide synthase (NOS) was determined in the cerebral cortex of the rat. The drugs included morphine and D-Ala2,Met,Phe4, Gly-ol5-enkephalin (DAMGO) (mu receptor), D-Ser2,Thr6-Leucine-enkephalin (DSTLE) and D-Pen2,D-Pen5 enkephalin (DPDPE) (delta receptor) and U-50, 488H (kappa receptor). As controls, two known inhibitors of NOS, NG-monomethyl-L-arginine (NMMA) and NG-nitro-L arginine (NNA) were also included. The activity of NOS was determined by the rate of conversion of [3H]arginine into [3H]citrulline. NMMA and NNA inhibited the activity of NOS with IC50 values of 3.28 +/- 0.10 and 0.79 +/- 0.20 microM, respectively. DAMGO, DSTLE and DPDPE had no effect on the NOS activity. Morphine inhibited NOS activity by 25% at 10 mM concentration whereas the U-50,488H inhibited the NOS activity with an IC50 value of 107 microM. It is conclude that NNA is four time more potent than NMMA in inhibiting NOS activity whereas drugs acting at mu-, delta- and kappa- receptors have no direct action on central NOS activity in vitro. PMID- 7534893 TI - Interleukin-1 beta induces histamine release in the rat hypothalamus in vivo. AB - We have previously demonstrated the increase of histidine decarboxylase activity and histamine content in the murine hypothalamus after intracerebroventricular injection of lipopolysaccharide possibly due to inducible interleukin-1 beta (IL 1 beta). Therefore, we investigated the effects of IL-1 beta on brain histamine dynamics by directly injecting it into the tuberomammillary nucleus of the rat hypothalamus (TM) using an in vivo microdialysis method. Injection of artificial cerebrospinal fluid or recombinant murine IL-1 beta at 0.1 ng into the TM did not evoke a significant change in core temperature, however, a significant monophasic febrile response was observed following injection of IL-beta at more than 1 ng per animal. Histamine release in the anterior hypothalamic area in vivo was significantly augmented from 140 min to 360 min following injection of IL-1 beta at 10 ng dose. These results suggest the possibility that interrelationship between histamine and IL-1 beta may modulate the acute phase reaction in the central nervous system. PMID- 7534894 TI - Exposure of rat spinal neurones to NMDA, AMPA and kainate produces only short term enhancements of responses to noxious and non-noxious stimuli. AB - The ability of excitatory amino acids (EAAs) to modulate nociceptive and non nociceptive responses was tested on spinal neurones of the anaesthetized rat. NMDA (N-methyl-D-aspartate), AMPA ((RS)-alpha-amino-3-hydroxy-5-methyl-4 isoxazole-propionate) and kainate were applied by iontophoretic ejection to increase the background firing rate of each cell to approximately 25 spikes/s. Responses to noxious heat and pinch and innocuous tap stimuli were enhanced to similar degrees by all three EAAs and returned to control immediately following termination of EAA ejection. This result shows that, whilst NMDA does enhance synaptic responses of spinal neurones, this effect is little or no greater than for AMPA or kainate. Furthermore, the rapid recovery of nociceptive responses indicates that more than NMDA receptor activation alone is required to induce longer-term enhancement of nociceptive responses (hyperalgesia). PMID- 7534895 TI - Focal epithelial hyperplasia. PMID- 7534896 TI - Histopathologic evaluation of proliferating cell nuclear antigen (PC10) in oral epithelial hyperplasias and premalignant lesions. AB - As the therapeutic options for malignant lesions expand, early accurate diagnosis of premalignancy is becoming increasingly important in the concept of cancer prevention. Because it has been hypothesized that abnormal cell proliferation is related to subsequent malignant transformation, many proliferation markers such as proliferating cell nuclear antigen have been studied in a variety of malignant tumors. In oral surface epithelium, proliferating cell nuclear antigen activity is restricted to basal layers of normal squamous mucosa. In this preliminary study, 169 formalin-fixed, paraffin-embedded oral epithelial lesions, including 28 carcinomas in situ, 82 epithelial dysplasias, 21 epithelial atypia, and 38 typical epithelial hyperplasias, were studied with a monoclonal antibody, PC10, to determine whether proliferating cell nuclear antigen suprabasal expression correlated with premalignancy. The findings revealed that with progression of lesions toward malignancy, there was a significant predilection for basal/suprabasal staining pattern for proliferating cell nuclear antigen as compared with the strictly basal staining pattern seen in normal and benign epithelial conditions. One unexpected staining pattern, suprabasal positive stain only, was also noted mostly in reactive hyperplasia and dysplasia. The data suggested that a positive basal/suprabasal staining pattern for proliferating cell nuclear antigen is indicative of premalignancy in oral epithelial lesions. PMID- 7534897 TI - Identification and semiquantification of estrogen and progesterone receptors in pyogenic granulomas of pregnancy. AB - Cases of pyogenic granuloma in pregnant women, nonpregnant women, and men were evaluated for the detection of estrogen and progesterone receptor proteins by immunoperoxidase staining. Immunostaining for estrogen receptors revealed a marked immunoreactivity of the endothelium within lesional tissue and in the overlying mucosal epithelium in many cases. Progesterone receptor immunoreactivity was only present within the epithelium, where it was much less than that of estrogen receptor immunoreactivity in both quantity (proportion of positive cells) and intensity. No characteristic staining pattern or significant quantitative difference among the three study groups could be discerned. These findings suggest that the quantity of estrogen or progesterone receptors in pyogenic granuloma is not the determining factor in the pathogenesis of this lesion. Rather, such a role may be attributed to the levels of circulating hormones. The levels of estrogen and progesterone are markedly increased in pregnancy and could therefore exert a greater effect on the endothelium of the pyogenic granuloma. PMID- 7534898 TI - Immunohistochemical analysis of salivary gland canalicular adenoma. AB - Canalicular adenoma is a newly recognized salivary gland adenoma that may be confused with malignant salivary gland tumors. To better characterize this neoplasm, six examples were investigated with a panel of immunohistochemistry antibodies including anti-keratin (AE1/AE3), anti-epithelial membrane antigen, anti-carcinoembryonic antigen, anti-vimentin, anti-S-100, anti-muscle specific actin, and anti-glial fibrillary acid protein. All canalicular adenomas stained in a similar fashion showing positive staining with anti-keratin, anti-vimentin, and anti-S-100 (6 of 6 cases each). Rare focal staining with anti-epithelial membrane antigen and anti-glial fibrillary acid protein was noted (1 of 6 cases each). This immunohistochemistry staining pattern was compared with those of ameloblastoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma. Immunohistochemistry may be useful in the distinction of canalicular adenoma from other salivary gland tumors. PMID- 7534899 TI - Loss of p53 function leads to metastasis in ras+myc-initiated mouse prostate cancer. AB - To study the interactions between dominantly acting oncogenes and tumor suppressor genes we used p53 'knockout' mouse urogenital sinus tissue for retroviral transduction of ras and myc in the mouse prostate reconstitution (MPR) model system. Epithelial hyperplasia was observed in all wild-type p53 MPRs with one small focal cancer and no evidence of metastasis. Prostatic cancer was found in 100% of the heterozygous and homozygous p53 mutant MPRs with metastatic deposits in 95% of the mice. The pattern of metastasis was remarkably similar to that in human prostate cancer with gross metastatic deposits in the lung, lymph nodes, bone and liver of many animals. Progression of carcinomas in the ras+myc initiated heterozygous p53 mutant MPRs was invariably associated with either complete loss, partial deletion or loss of expression of the wild-type p53 allele. Southern blotting analysis of proviral-cellular DNA junction fragments in primary carcinomas and cell lines derived from metastatic deposits revealed that metastases do not necessarily seed out from the most abundant clone in the primary carcinoma. PMID- 7534900 TI - Hyperactive autocrine loop mediated by a NDF-related factor in neoplastic hamster embryo fibroblasts expressing an activated cph oncogene. AB - We described previously the characterization of a novel oncogene, cph, activated in primary Syrian hamster embryo fibroblasts by exposure to 3-methylcholanthrene (Velasco et al., Oncogene 9:2065-2069, 1994). The present report describes the participation in the neoplastic conversion of cph-expressing (81C39) hamster fibroblasts of a hyperactive autocrine loop involving a neu differentiation factor [NDF]-like protein. The tyrosine phosphorylation of the p185erbB-2 receptor in the human breast carcinoma MDA-MB-453 cells was stimulated by conditioned medium from neoplastic 81C39 cells. The extent of this stimulatory effect was much greater than that induced by conditioned medium from normal 84-3 hamster cells. The p185erbB-2 tyrosine phosphorylation-stimulating activity was partially blocked by the heparin analogue pentosan polysulfate [PPS], a known antagonist of p185erbB-2 ligands, and was partially purified from 81C39 conditioned medium by heparin-Sepharose chromatography. The level of p185erbB-2 tyrosine phosphorylation-stimulating activity in the heparin-Sepharose fractions correlated directly with their content in NDF-like protein as immunodetected with an anti-rat NDF antibody. Consistently, the steady-state level of NDF-related mRNA was found to be four times greater in neoplastic 81C39 cells than in normal 84-3 cells. However, the levels of erbB-2 mRNA were similar in both cell types, while the expression of erbB-4 mRNA was upregulated in the neoplastic fibroblasts. The ability of 81C39 conditioned medium to stimulate protein tyrosine phosphorylation and to induce other PPS-sensitive growth responses on 81C39 cells themselves suggested the involvement of an autocrine loop in their neoplastic conversion. The participation of a NDF-related factor in this autocrine loop was confirmed by the ability of an anti-NDF antibody to block the mitogenic activity present in their own conditioned medium. The involvement of the cph oncogene in the upregulation of NDF-related expression was evidenced when cph-transformed NIH3T3 fibroblasts showed elevated levels of NDF-related mRNA, and their conditioned medium induced tyrosine phosphorylation on MDA-MB-453 cells, reproducing the effect of the medium from 81C39 hamster cells. PMID- 7534901 TI - Insulin-like growth factor I and insulin-like growth factor binding protein 3 as determinants of blood hemoglobin concentration in healthy subjects. AB - We studied the serum concentrations of IGF-I, IGF-binding protein 3 (IGFBP-3), and testosterone in relation to blood Hb in 60 healthy prepubertal or early pubertal boys twice, with a 9-mo interval. Serum IGF-I and testosterone levels were measured by RIA, and serum IGFBP-3 was measured by monoclonal immunofluorometric assay. Positive correlations were observed between the concentrations of blood Hb and serum IGF-I at the first examination (r = 0.36, p = 0.008) and Hb and IGFBP-3 at both examinations (r = 0.53, p < 0.001, and r = 0.39, p = 0.003). No association between Hb and testosterone concentrations was found. Our results show that blood Hb is positively correlated to serum IGF-I and IGFBP-3 levels, indicating indirectly the involvement of growth hormone in the regulation of physiologic Hb concentration. Because no association was found between Hb and testosterone concentrations, this may indicate that the role of androgens in erythropoiesis may be different at different stages of puberty. It is concluded that the IGF system may be involved in the rise of Hb level during early puberty. PMID- 7534902 TI - Effect of intravenous insulin-like growth factor I in two patients with leprechaunism. AB - Leprechaunism (Donohue syndrome) is an autosomal recessive disorder characterized by hyperglycemia, extreme insulin resistance, dysmorphic features, failure to thrive, and early death. In this study, recombinant IGF-I, which has both insulin like and anabolic effects, was administered to two infants with leprechaunism in an attempt to reduce hyperglycemia and improve nutritional status. IGF-I was infused for 66 h in patient FL-1 and 62 h in patient NC-2, with maximal infusion rates of 110 and 40 micrograms/kg/h, respectively. Although supraphysiologic concentrations of IGF-I were achieved (459 and 1583 micrograms/L in FL-1 and NC 2, respectively), there were no apparent glucose-lowering or nitrogen-sparing effects. Insulin concentrations decreased from extremely high values (16804 and 10224 pmol/L) but remained elevated (611 pmol/L in FL-1 and 5869 pmol/L in NC-2). No changes in serum and urinary urea nitrogen or electrolytes occurred. IGF binding protein-2, which was the predominant IGF binding protein in serum by ligand blot and immunoblot, did not change with IGF-I infusion. IGF binding protein-3 levels were low at baseline and increased slightly during the infusion. We hypothesize that the lack of significant glucose-lowering and anabolic responses to IGF-I could be secondary to a postreceptor defect in IGF-I signaling resulting from the absence of functional insulin receptors. PMID- 7534903 TI - Effect of nitric oxide synthase inhibition during group B streptococcal sepsis in neonatal piglets. AB - Nitric oxide (NO), an important vasodilatory modulator of systemic and pulmonary vascular tone, is synthesized from L-arginine by the enzyme NO synthase in vascular endothelial and smooth muscle cells. L-Arginine analogs, such as N omega nitro-L-arginine methyl ester (L-NAME), are competitive antagonists of NO synthase and inhibit NO synthesis. Group B streptococcus (GBS) causes pulmonary hypertension, hypoxemia, lung vascular injury, and reduced cardiac output in both human newborns and neonatal piglets. Lung vascular injury associated with prolonged GBS infusion in piglets may attenuate NO production and thus promote severe pulmonary hypertension. We studied the effect of the NOS inhibitor, L-NAME and the precursor of NO, L-arginine, on pulmonary and systemic hemodynamics during late-phase GBS sepsis in the piglet model. Neonatal piglets were anesthetized, ventilated with room air, and randomized to receive a continuous infusion of saline (n = 5) or GBS (n = 5) for 4 h. After 3 h of infusion, both groups received a bolus of L-NAME (3 mg/kg). Hemodynamic and gas exchange indices were measured at baseline, 30 min, and 3 h of infusion, and 30 min and 1 h after L-NAME treatment. L-NAME treatment caused 1) significant increases in mean pulmonary arterial pressure, pulmonary vascular resistance, mean systemic arterial pressure, and systemic vascular resistance for both groups; 2) a similar percentage of increase in pulmonary vascular resistance for the two groups; 3) greater reduction in cardiac output and SV in the GBS compared with the control group; and 4) no significant alterations in arterial partial pressure of oxygen or the difference between alveolar and arterial partial pressure of oxygen for either group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534904 TI - L-selectin is down-regulated in umbilical cord blood granulocytes and monocytes of newborn infants with acute bacterial infection. AB - The leukocyte glycoprotein L-selectin mediates an early step in the recruitment of leukocytes to sites of inflammation. L-Selectin surface expression is rapidly down-regulated by inflammatory signals in vitro. In a prospective study, we found L-selectin expression on umbilical cord blood granulocytes and monocytes to be significantly decreased in newborn infants with acute bacterial infection compared with controls (p < 0.01). A significantly reduced L-selectin expression of both granulocytes and monocytes was also found to be associated with an increased neutrophil immature/total ratio (p < 0.01) but not with other laboratory markers of neonatal sepsis. There was no apparent impact of prematurity, low birth weight, gestational hypertension, or gestational diabetes on L-selectin expression. Although the mode of delivery did not affect granulocyte L-selectin expression, umbilical cord blood monocytes showed an increased L-selectin expression after emergency cesarean delivery compared with samples obtained after elective cesarean or vaginal delivery (p < 0.01). We conclude that acute systemic inflammation results in down-regulation of granulocyte and monocyte L-selectin expression in vivo similar to that observed in vitro. PMID- 7534905 TI - Concurrent validity of the Kaufman Hand Movement Test as a measure of limb apraxia. AB - The concurrent validity of the Kaufman Hand Movement Test as a measure of limb apraxia was investigated. For 23 adult aphasic subjects, a Pearson r of 0.84 was obtained between scores on this test and on the Limb Apraxia Test, a battery of tasks often used in the assessment of limb apraxia. The Kaufman test is a shorter and simpler test to administer than the Limb Apraxia Test. The concurrent validity of the Kaufman test encourages its use in the assessment of limb apraxia. PMID- 7534906 TI - Bulged-out nucleotides protect an antisense RNA from RNase III cleavage. AB - Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo. PMID- 7534908 TI - Detection of point mutations with a modified ligase chain reaction (Gap-LCR). AB - DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA. PMID- 7534907 TI - Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo. AB - Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage. PMID- 7534909 TI - Decoding with the A:I wobble pair is inefficient. AB - tRNAs with inosine (I) in the first position read three codons ending in U, C and A. However, A-ending codons read with I are rarely used. In Escherichia coli, CGA/U/C are all read solely by tRNAICGArg. CGU and CGC are very common codons, but CGA is very rare. Three independent in vivo assays show that translation of CGA is relatively inefficient. In the first, nine tandem CGA cause a strong rho mediated polar effect on expression of a lacZ reporter gene. The inhibition is made more extreme by a mutation in ribosomal protein S12 (rpsL), which indicates that ribosomal binding by tRNAICGArg is slow and/or unstable in the CGA cluster. The second assay, in which codons are substituted for the regulatory UGA of the RF2 frameshift, confirms that aa-tRNA selection is slow and/or unstable at CGA. In the third assay, CGA is found to be a poor 5' context for amber suppression, which suggests that an A:I base pair in the P site can interfere with translation of a codon in the A site. Two possible errors, frameshifting and premature termination by RF2, are not significant causes for inefficiency at CGA. It is concluded that the A:I pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare usage of codons read with the A:I base pair. PMID- 7534911 TI - Modified Apt test. PMID- 7534910 TI - Quantitation of RNA editing substrates, products and potential intermediates: implications for developmental regulation. AB - Kinetoplast mitochondrial RNA editing is the developmentally regulated post transcriptional process of uridine insertion and deletion in mRNAs directed by short guide RNAs (gRNAs), which creates functional mRNAs. Two mechanisms are proposed: transesterification which predicts gRNA/mRNA chimeric intermediates, and enzymatic steps which allow but do not require chimeric intermediates. We quantitated the copy number of apocytochrome b (CYb) gRNAs, edited/unedited mRNAs and gRNA/mRNA chimeras in bloodstream and procyclic form cells of Trypanosoma brucei. Both forms have 35 copies/cell of two gRNAs. Bloodstream forms contain 15 unedited and edited CYb mRNA molecules/cell while procyclic forms have four times as much unedited and over 10 times as much edited mRNA. Chimera levels are very low, 350-5000-fold lower than unedited mRNA or gRNAs, but are over 10 times more abundant in procyclic than bloodstream forms. These results are consistent with chimeras being editing intermediates if their resolution is rapid in respect to their formation, although they could be non-productive byproducts of the editing reaction. Bloodstream chimera sequences differ from procyclic chimeras. These results indicate that developmental regulation is not by gRNA abundance and suggest that it occurs at the level of gRNA utilization possibly by changing abundance of unedited CYb mRNA. PMID- 7534912 TI - Actions of molecules which regulate hemopoiesis on endothelial cells: memoirs of common ancestors? AB - The proliferation and differentiation of hematopoietic stem cells (hematopoiesis) takes place in close contact with stromal cells and matrix in bone marrow. Hematopoiesis requires cytokines, collectively termed colony stimulating factors (CSFs), which act on progenitor cell populations and induce their commitment to a specific lineage. For instance, leukemia, inhibitor factor and stem cell factor act on pluripotent cells and immature progenitors, granulocyte-macrophage colony stimulating factor (GM-CSF) acts at early stages of the development of myelomonocytic lineage, whereas granulocyte-colony stimulating factor (G-CSF) and macrophage-colony stimulating factor (M-CSF) act on more mature cells of the same lineage and are only required later during the differentiation of this cell lineage. A second important element for the hematopoietic process is the presence of extracellular matrix proteins, which bind CSFs and correctly present the molecules to specific receptors present on the surface of the progenitor cells. Finally, stromal cells (i.e. fibroblasts, endothelial cells and adipocytes) which support the growth of hematopoietic stem cells in vitro, are crucial for the production of CSFs and protein matrix and regulate the passage of mature cells from bone marrow to bloodstream. Idiopathic myelofibrosis is an example of the relevance of microenvironment in hematopoiesis. This disease is characterized by fibroblast and basement membrane accumulation, appearance of myofibroblasts and modification of the capillary network and provokes a bone marrow aplasia. In this article we review recent studies on the role of hemopoietic cytokines on stromal cells, in particular on endothelial cells, and propose a double role for CSFs in hematopoiesis: to induce the commitment of progenitor cells and to maintain the behavior of bone marrow endothelial cells. PMID- 7534913 TI - The role of basement membrane in angiogenesis and tumor growth. AB - Expansion of the tumor-cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Blood vessel growth is controlled, in part, by the matrix surrounding it, in particular, the basement membrane underlying the endothelium. Here we illustrate that laminin, a major component of basement membrane, has several biologically active sites that can bind to endothelial and tumor cells, and have the ability to regulate angiogenesis and tumor growth. We show that synthetic peptides at two sites in the laminin B1 chain (the RGD and YIGSR sequences) inhibit angiogenesis, whereas a third site in the A chain, designated SIK-VAV, stimulates vessel and tumor cell growth. By developing strategies that promote or inhibit the activities of these sites in laminin, we may obtain methods to inhibit angiogenesis and subsequent tumor growth. PMID- 7534915 TI - Serum levels of integrins in chronic liver diseases. AB - The serum levels of beta 1 integrin (microgram/ml) were significantly higher in the patients with chronic persistent hepatitis (2.59 +/- 0.04), chronic active hepatitis (3.45 +/- 0.13), cirrhosis (4.77 +/- 0.30) and hepatocellular carcinoma (4.71 +/- 0.49) than in normal subjects (2.11 +/- 0.08). Serum levels of beta 3 integrin (microgram/ml) were significantly higher in the patients with chronic active hepatitis (10.48 +/- 1.22), liver cirrhosis (13.55 +/- 1.54) and hepatocellular carcinoma (14.1 +/- 1.77) when compared with normal subjects (5.51 +/- 0.52). A positive correlation was found between serum levels of beta 1 and beta 3 integrins (p < 0.001). A strong positive correlation was observed between serum levels of beta 1 integrin and histologic features, particularly in the degree of hepatic fibrosis, while no correlation was found between serum levels of beta 3 integrin and hepatic fibrosis. Immunohistochemical studies revealed that the beta 1 integrin was present on the plasma membranes of hepatocytes and sinusoidal lining cells in the normal liver, and was increased in fibrotic areas, and on the plasma membranes of hepatocytes and sinusoidal lining cells of the chronic liver disease. However, no positive staining for beta 3 integrin was observed in fibrotic area. The serum level of beta 1 integrin in patients with chronic liver diseases may therefore be a useful marker of hepatic fibrosis. PMID- 7534914 TI - New approaches to tissue regeneration and repair. AB - Several Heparin Binding Growth Factors (HBGFs) are thought to play a key role in the natural processes of tissue regeneration or repair after being released by neighbouring, inflammatory or circulating cells as well as from extracellular matrix associated heparan sulfate proteoglycosaminoglycans. In order to better understand how the bioavailability of these HBGFs can take part in the regulation of the wound healing processes, we have studied the healing effect of various chemically substituted dextrans (CMDBS) selected for their affinity for HBGFs, alone and in association with HBGFs. The CMDBS was obtained by substitution of methylcarboxylic (CM), benzylamide (B) and benzylamine sulfonate (S) groups in proportion of 83%, 23% and 13% respectively for CMDBS K that we have further used (Mauzac et al., 1985 Biomaterials. 6: 61-63). CMDBS K could 1: potentiate the biological activity of 1 or 2 FGFs, 2: protect 1 and 2 FGFs against thermal or pH inactivation, 3: protect a and b FGFs against proteolytic degradation (Tardieu et al., 1992 J. Cell. Physiol. 150: 194-203). CMDBS K was tested alone in cutaneous and bone wound healing models and for its ability to stabilize FGFs. Rats were punched and skin regeneration was studied by morphometric and histological analysis. The wounds (6 mm diameter) were filled with collagen plaster alone or soaked with CMDBS. CMDBS K in collagen plaster was able to induce a remarkable effect both on the kinetics and on the quality of the restored skin. These results suggest that endogenous growth factors naturally released during the regeneration process could be trapped, protected and released by CMDBS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534916 TI - Microvascularity in benign prostatic hyperplasia. AB - The vascular density of benign prostatic hyperplasia (BPH) has not been characterized. Previously we reported vessel density (vv/mm2) in prostatic carcinoma was twice that of normal prostate [Bigler et al.: Hum Pathol 24:220-226 1993]. To further characterize vessel density in benign prostate tissue we examined 15 cases of BPH obtained by open prostatectomy. Vessels were stained with antibodies to Factor VIII-related antigen, and vessel density was measured using computer-assisted image analysis. Vessel density was analyzed between various histologic tissue types. Mean vessel density in all transition zone tissue was 70.2 vv/mm2. Vessel density in epithelial hyperplastic nodules (mean 99.3, SD 40.7) exhibited density levels similar to those found in prostatic carcinoma (mean 101.4, SD 35.6). Vessel density in epithelial nodules was significantly higher than in non-nodular epithelial tissue (mean 76.7, SD 23.1; P < 0.001, ratio = 1.3). Higher vessel densities were found in hypercellular stromal nodules (mean 64.7, SD 19.1) than in adjacent stromal areas (mean 36.5, SD 15.3; P < 0.001, ratio = 1.8). Overall, vessel density in BPH was higher than previously found in benign tissue measured in radical prostatectomy specimens, especially in areas of nodular morphology. PMID- 7534918 TI - Synergistic effects of estrogen and androgen on the prostate: effects of estrogen on androgen- and estrogen-receptors, BrdU uptake, immunohistochemical study of AR, and responses to antiandrogens. AB - To investigate the synergistic effects of estradiol (E2) and testosterone (T) on prostate growth, castrated Wistar rats were treated with 1 mg/day of T or with 1 mg/day of T and 0.01 mg/day of E2 for 6 weeks. The weight of the prostate in T + E2-treated rats was significantly higher than that in T-treated and normal rats, parallel with the bromodeoxy-uridine (BrdU) labeling index (LI). Nuclear androgen receptor (AR) content in the T + E2 group was significantly higher than that in the T group. But they were lower than that in normal rats. And there were no significant differences between the groups in nuclear estrogen receptor (ER) content. Immunohistochemical studies with the AR antibody revealed positive staining in the prostatic epithelium and stromal cells in the normal, T-treated, and T + E2-treated animals. However, castration led to loss of staining. Response to steroidal antiandrogens was also determined. Antiandrogen treatment abrogated the increases in nuclear AR content and BrdU LI, and prevented immunohistochemical staining. These results suggest that AR and ER, which were measured in this study, were not indicators of prostatic proliferation. We further need to investigate other factors, including other types of receptors, growth factors involved in epithelial-stromal interaction, and so on. PMID- 7534917 TI - Inhibition of androgen synthesis by 22-hydroximino-23,24-bisnor-4-cholen-3-one. AB - The 22-hydroximino-23,24-bisnor-4-cholen-3-one (22-oxime) was synthesized and evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and the 5 alpha-reducatase of human prostatic microsomes from patients with benign prostatic hypertrophy. The 22-oxime demonstrated moderate inhibition for the 17 alpha-hydroxylase (Ki 74 nM vs. Km 29 nM) with progesterone as substrate and potent inhibition (Ki 18 nM vs. Km 76 nM) for the C17,20-lyase activity with 17 alpha-hydroxyprogesterone as substrate. Further investigation of this enzyme with progesterone as substrate demonstrated the inhibition occurred mainly at the 17 alpha-hydroxylation step of the progesterone substrate. The 22 oxime also demonstrated potent and competitive inhibition of 5 alpha-reductase in human prostatic microsomes (Ki 1.4 nM vs. Km 14 nM). When adult male rats were injected subcutaneously (sc) daily with 22-oxime (50 mg/kg/day) for 21 days, the concentrations of serum and testicular testosterone were significantly reduced by 65% and 59%, respectively, in comparison to vehicle-treated controls. Furthermore, both testosterone and DHT concentrations in rat prostatic tissue were significantly decreased by 60% and 44% compared to control tissue. Serum LH concentrations were unchanged in the 22-oxime-treated group compared to the control group. This indicates that the reduction in androgen concentrations in animals treated with this compound is not due to its influence on pituitary feedback mechanisms which result in reduced LH secretion. These results suggest that 22-oxime is effective in reducing androgen synthesis through the inhibition of 17 alpha-hydroxylase, C17,20-lyase, and 5 alpha-reductase both in vitro and in vivo. PMID- 7534919 TI - Comparison of prostate specific antigen, prostate specific membrane antigen, and LNCaP-based enzyme-linked immunosorbent assays in prostatic cancer patients and patients with benign prostatic enlargement. AB - Serum assays for prostate specific antigen (PSA; monoclonal), for prostate specific membrane antigen (PSM; Western blot), and a LNCaP/7E11.C5-based competitive enzyme-linked immunosorbent assay (ELISA) were evaluated in a small number of prostate cancer patients with localized or disseminated disease, and judged to be in clinical progression or remission based on National Prostate Cancer Project (NPCP) criteria. PSA values recognized the presence of clinical progression in localized disease (B1-C) and to a lesser degree disseminated disease (D1-D2). In contrast, to a limited degree the ELISA test recognized clinical progression mainly in disseminated disease and chiefly in stage D2. PSM values were elevated in both D1 and D2 but not in a linear fashion as observed with PSA. The ELISA and PSM results may be assessing a different clinical response to prostatic cancer than that recognized by PSA. This could reflect a developing clone of resistant prostatic cells as previously postulated. To further pursue this possibility a secondary generation of monoclonal antibodies to PSM is being developed. The ELISA levels for benign prostatic enlargement were not elevated above normal. In contrast both with PSA and PSM the assays reflected levels significantly above the normal range in benign prostatic hypertrophy (BPH). PMID- 7534920 TI - Formation and characterization of cisplatin-loaded poly(benzyl l-glutamate) microspheres for chemoembolization. AB - Chemoembolization using microspheres of 100- to 200-microns is a useful way to treat primary and secondary hepatic tumors. In a search for a better embolic material, we described in detail the preparation and characterization of a poly(benzyl l-glutamate) (PBLG) microspheres containing cisplatin (CDDP). We determined the optimal experimental conditions to produce spherical free-flowing microspheres that were able to release drug content (44% [w/w] CDDP) in a sustained manner. We found that solvent viscosity played a key role in determining the resulting microsphere characteristics. Microscopic studies showed that increasing the polymer concentration (to 10% [w/v]) and the viscosity of the organic phase produced microspheres with uniform drug distribution. Increasing polymer concentration also markedly improved drug incorporation efficiency. In vitro release studies revealed that the release of CDDP was a function of drug loading; microspheres with a higher amount of entrapped CDDP had a slower release rate. This observation and the fact that CDDP/PBLG microspheres did not show "burst effect" at higher loading is ascribed to the formation of uniformly distributed drug crystal networks within the polymer matrix. The favorable properties of the CDDP/PBLG system warrants its further evaluation on experimental animal models for the treatment of hepatic tumors. PMID- 7534921 TI - Infusion of nicotine in the ventral tegmental area or the nucleus accumbens of the rat differentially affects accumbal dopamine release. AB - The present study examined the effects of acute, continuous infusion of nicotine in either the ventral tegmental area or the nucleus accumbens on extracellular concentrations of dopamine in the nucleus accumbens by applying in vivo microdialysis in freely moving rats. Nicotine (1000 microM) infusion for 80 min. in the ventral tegmental area produced a long-lasting increase in accumbal dopamine, whereas similar nicotine infusion in the nucleus accumbens increased dopamine levels only within the first 20 min. of administration. This effect was blocked by systemic pretreatment with the nicotinic receptor antagonist mecamylamine (1 mg/kg, subcutaneously). In contrast to the effects of nicotine, N methyl-D-aspartate infusion in the ventral tegmental area as well as in the nucleus accumbens produced a long-lasting increase in accumbal dopamine levels. The more procounced effect of infusion of nicotine in the ventral tegmental area as compared to the nucleus accumbens on accumbal dopamine release may be due to a lower rate or even lack of tolerance to nicotine's stimulatory action in the ventral tegmental area. These results support the notion that nicotinic receptors in the ventral tegmental area may be of greater importance than those located in the nucleus accumbens for mediating some of the stimulatory effects of nicotine on the reward-related mesoaccumbens dopamine system. PMID- 7534922 TI - Risk assessment of amniocentesis between 11 and 15 weeks: comparison to later amniocentesis controls. AB - We studied 693 consecutive early amniocenteses (prior to 15 weeks) and found a spontaneous abortion rate to 28 weeks' gestation of 1.5 per cent. A control group of women having standard amniocentesis (15-20 weeks) experienced a 0.6 per cent fetal loss in the same period. There were no other apparent differences between the two groups. Early amniocentesis results are generally available 4-6 weeks before standard amniocentesis and 1-3 weeks after chorionic villus sampling (CVS). Alpha-fetoprotein (AFP) can be accurately assayed in 11- to 15-week amniotic fluid samples but additional studies are necessary to determine the accuracy of neural tube defect (NTD) detection. Including the present study, over 5800 early amniocenteses have been reported and the results suggest that this is a relatively safe prenatal diagnostic test and an alternative to CVS and later amniocentesis. PMID- 7534924 TI - Second-trimester diurnal variation of maternal serum alpha-fetoprotein, human chorionic gonadotropin, and unconjugated oestriol: is it present and does it affect the prediction of a patient's risk for fetal Down syndrome? AB - Both a cross-sectional and a longitudinal study were performed to investigate whether or not the collection time should be taken into consideration when generating a patient's risk for fetal Down syndrome with multiple marker screening. Diurnal variations of third-trimester alpha-fetoprotein (AFP) levels and first-trimester human chorionic gonadotropin (hCG) levels have been previously reported. In addition, large episodic fluctuations of conjugated and unconjugated oestriol (uE3) as well as a diurnal variation have also been reported in the third trimester. If the levels of these analytes routinely fluctuate during the day, they could affect a patient's risk calculation for fetal Down syndrome. The longitudinal study evaluated ten non-diabetic women who underwent sequential sampling for AFP, hCG, and uE3. The cross-sectional study evaluated 1953 patients for these three markers whose time of sampling was recorded between 8.00 a.m. and 5.59 p.m. Using either study design, no significant effect was seen in the median MOM levels of the screening analytes as a function of the time of day. PMID- 7534923 TI - Prenatal diagnosis of xeroderma pigmentosum and Cockayne syndrome. AB - In a study of fetal cells from a series of 12 pregnancies in ten families at risk for the ultraviolet light-sensitive, DNA repair-deficient diseases xeroderma pigmentosum (XP) and Cockayne syndrome (CS), we detected one XP and two CS homozygote fetuses. The diagnoses were confirmed by analysis of fetal skin fibroblasts or second amniotic samples after termination of the pregnancies. The measurement of ultraviolet light sensitivity and DNA repair depended on properties common to the seven excision repair-deficient XP complementation groups (A-G) and the two CS complementation groups (A, B). No XP variant families were included in the study, because the variant requires different testing techniques. Reliable and rapid diagnosis proved possible in all but one of the 12 pregnancies, supporting the use of these methods until the spectrum of mutations in the various XP and CS genes of the U.S. population is fully characterized and a DNA sequence-based diagnostic procedure becomes available. PMID- 7534926 TI - First-trimester maternal serum alpha-fetoprotein as a marker for fetal chromosomal disorders. Dutch Working Party on Prenatal Diagnosis. AB - We evaluated first-trimester maternal serum alpha-fetoprotein (MS-AFP) as a marker for fetal chromosomal disorders. The multicentre study was performed under the auspices of the Dutch Working Party on Prenatal Diagnosis. MS-AFP was measured in 2404 normal pregnancies and 72 chromosomally abnormal pregnancies. The median multiple of the normal median (MOM) in 32 Down's syndrome pregnancies was 0.83 with a 95 per cent confidence interval ranging from 0.60 to 1.04. The difference between the distributions of first-trimester MS-AFP in normal and Down's syndrome pregnancies was statistically significant (t-test: t = 2.34, P < 0.05). Thirty-one per cent of the Down's syndrome pregnancies were found below the tenth percentile. We found no difference between normal pregnancies and pregnancies with other chromosomal disorders (eight cases with trisomy 18, MOM = 1.26; seven cases with sex chromosome abnormalities, MOM = 1.07; 22 cases with a chromosomal mosaic pattern in chorionic villi, MOM = 1.08). We conclude that first-trimester MS-AFP can discriminate between normal and Down's syndrome pregnancies, but is not an effective marker. First-trimester MS-AFP has no value as a marker for other fetal chromosomal disorders. PMID- 7534925 TI - Urinary beta-core human chorionic gonadotrophin: a new approach to Down's syndrome screening. AB - Human chorionic gonadotrophin (hCG) is the most discriminatory maternal serum marker of Down's syndrome. We have carried out a study to establish whether urinary beta-core-hCG, a major metabolic product of hCG, might be an even better marker. Urine samples were available from seven singleton pregnancies with Down's syndrome, and one each of Edwards' syndrome, triploidy, and twins discordant for Down's syndrome. beta-Core-hCG levels were corrected for creatinine and expressed as multiples of the normal gestation-specific median (MOM) level derived from 67 singleton controls. There was a highly statistically significant elevation in level among the singleton Down's syndrome cases (P < 0.0005; Wilcoxon rank sum test). All had levels exceeding 2 MOM with a median of 6.11 MOM (95 per cent confidence interval 3.7-10.0). The levels were extremely low in Edwards' syndrome (0.08 MOM) and triploidy (0.02 MOM), but the twin pregnancy discordant for Down's syndrome did not have a raised beta-core-hCG level (0.64 MOM). The findings are sufficiently encouraging to investigate the possibility of urinalysis as a routine modality in the prenatal screening for Down's syndrome and other common serious aneuploidies. PMID- 7534927 TI - Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis. AB - For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This double-MACS' procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40-55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45-/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45-/CD14- cells, paternal alleles could be detected. In the CD45-/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non invasive prenatal diagnosis. PMID- 7534928 TI - Early detection of abdominal pregnancy by maternal serum AFP+ screening. PMID- 7534929 TI - Prenatal diagnosis of exencephaly associated with high levels of maternal serum human chorionic gonadotropin and urinary beta-core fragment of hCG/creatinine ratio. PMID- 7534930 TI - [Whipple disease: a single or multiple origin?]. AB - After having been considered as an essentially digestive disease, Whipple's disease has appeared more and more to be a multivisceral disease with two main characteristics: on one hand Whipple's disease yields a diffuse infiltration of tissues by abnormal macrophages without any other inflammatory reaction; on the other hand, aspects of microbial invasion by intra or extracellular unique rod shaped Gram+bacteria are found. This unusual pathological complex has alternatively been considered as suggestive of an immunological defect or as a very unusual type of bacterial infection. Though recent studies support the hypothesis of a primary microbial infection due to a hitherto undescribed bacterium (Tropheryma whippelii) or more or less related bacteria belonging to the actinomycetes family, they do not totally exclude a primary or acquired impairment of antigen processing by macrophages. Speculations about this fascinating pathophysiological model and about its optimal therapeutic modalities are not likely to reach a conclusion in the near future. PMID- 7534931 TI - [Cat-scratch disease and disease caused by Bartonella (Rochalimaea)]. AB - The aetiology of cat scratch disease remains controversial since both Afipia felis and Bartonella (Rochalimaea) henselae have been isolated from diseased lymph nodes. Bartonella henselae, Bartonella (Rochalimaea) quintana and Bartonella (Rochalimaea) elizabethae cause endocarditis and Bartonella bacilliformis cause septicemia (Oroya's fever) in non-immunocompromized patients, and Bartonella henselae and Bartonella quintana cause fever, bacillary angiomatosis, and visceral peliosis in human immunodeficiency virus-infected patients. Bartonella quintana is the historical agent of trench fever and we recently isolated it from chronic adenopathy. The diagnosis of Afipia felis and Bartonella infections relies upon the isolation of the bacterium from blood, node tissue after inoculation of cell cultures systems and molecular identification, and upon the serology. In vitro both species are sensitive to aminoglycosides, and we recommend aminoglycosides be included in antibiotic regimens for treating cat scratch disease and Bartonella infections. PMID- 7534932 TI - Receptors and antagonists for substance P and related peptides. PMID- 7534934 TI - Stem cell factor enhances the survival of murine intestinal stem cells after photon irradiation. AB - Recombinant rat stem cell factor (SCF) has been shown to decrease lethality in mice exposed to total-body irradiation (TBI) in the lower range of lethality through radioprotection of hematopoietic stem cells and acceleration of bone marrow repopulation. This study evaluates the effect of SCF on the survival of the intestinal mucosal stem cell after TBI. This non-hematopoietic stem cell is clinically relevant. Gastrointestinal toxicity is common during and after abdominal and pelvic radiation therapy and limits the radiation dose in these regions. As observed with bone marrow, the administration of SCF to mice prior to TBI enhanced the survival of mouse duodenal crypt stem cells. The maximum enhancement of survival was seen when 100 micrograms/kg of SCF was given intraperitoneally 8 h before irradiation. This regimen increased the survival of duodenal crypt stem cells after 12.0 Gy TBI from 22.5 +/- 0.7 per duodenal cross section for controls to 30.0 +/- 1.7 after treatment with SCF (P = 0.03). The TBI dose producing 50% mortality at 6 days (LD50/6) was increased from 14.9 Gy for control mice to 19.0 Gy for mice treated with SCF (dose modification factor = 1.28). These findings demonstrate that SCF has radioprotective effects on a non hematopoietic stem cell population and suggest that SCF may be of clinical value in preventing radiation injury to the intestine. PMID- 7534933 TI - IS1237, a repetitive chromosomal element from Clavibacter xyli subsp. cynodontis, is related to insertion sequences from gram-negative and gram-positive bacteria. AB - We describe here a repetitive chromosomal element, which appears to be an insertion sequence, isolated from Clavibacter xyli subsp. cynodontis, a gram positive plant-associated bacterium. The element, IS1237, is 905 bp in size, is bounded by 19-bp perfect inverted repeats and 3-bp direct repeats, and appears at least 16 times in the genome. It contains three open reading frames which show similarity to open reading frames from various other insertion sequences. We have found that there are two groups of related mobile elements: one in which two open reading frames are read separately and the other in which these two open reading frames are fused together to give one predicted protein product. Using one of these open reading frames to search amino acid sequence databases, we found two instances in which similar reading frames flank genes carried on plasmids. We believe therefore that these plasmid-borne genes may be parts of previously unidentified mobile elements. For IS1237, a frameshift in two of the open reading frames and a stop codon in the third may indicate that this particular copy of the element is no longer active in transposition. The similarity of IS1237 to other elements from both gram-negative and gram-positive bacteria provides further evidence that mobile elements have been transferred between these two bacterial groups. PMID- 7534935 TI - Radiotherapy in the treatment of metastases of soft tissue sarcomas. PMID- 7534936 TI - Malaria-specific memory T cells: putative roles of different types of memory responses in immunity and disease. PMID- 7534937 TI - An HLA-based approach to the design of a CTL-inducing vaccine against Plasmodium falciparum. PMID- 7534939 TI - Evolving peripapillary choroidal neovascular membrane demonstrated by indocyanine green choroidal angiography. PMID- 7534940 TI - [How I treat.... Benign prostatic hypertrophy. Part 1: restatement and nonmedical treatment]. PMID- 7534938 TI - Effect of polymorphism of sporozoite antigens on T-cell activation. PMID- 7534941 TI - The influence of tumour necrosis factor-alpha, interleukin-1 beta and interferon gamma on the expression and function of the complement regulatory protein CD59 on the human colonic adenocarcinoma cell line HT29. AB - CD59 is a 18-25 kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up-regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose-dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-alpha and interleukin-1 beta. A dose-dependent increase in the level of CD59 expression was also seen using low concentrations of interferon gamma (IFN-gamma), while CD59 expression on cells cultured with high IFN-gamma concentrations was comparable to non-stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non-stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape. PMID- 7534943 TI - Alternative therapy for benign prostatic hypertrophy. PMID- 7534942 TI - Reiter's syndrome and reactive arthritis: a current view. AB - This paper reviews advances in the understanding of the pathogenesis of reactive arthritis that have occurred over the last decade. Inflammatory aseptic joint disease has been linked with prior infection initiated by many different species of microorganisms. The presence of intra-articular bacterial antigens has now been firmly established with the demonstration of bacteria, bacterial fragments, DNA, RNA, and bacterial lipopolysaccharide in joints of patients with reactive arthritis. Chlamydia trachomatis, Salmonella enteritidis, and Shigella flexneri have all been detected in the joint by immunological techniques, although there is still some doubt as to the form in which they reach the joint and whether or not they persist. A number of phlogistic bacterial components could be acting as arthritogens. Negative joint culture results from patients with reactive arthritis make it unlikely that bacteria in the joint are viable, although chlamydial DNA has been shown in the joints of patients with sexually acquired reactive arthritis using the polymerase chain reaction. The use of antimicrobial therapy in the treatment of reactive arthritis is under review; data suggests that long-term antibiotic treatment warrants further study. The role of HLA-B27 in disease pathogenesis is discussed as are possible mechanisms of interplay between germ and gene. HLA-B27 might confer disease susceptibility by affecting immune mechanisms other than classical antigen presentation. The immunopathogenesis of joint inflammation in reactive arthritis is explored with reference to studies of humoral and cellular immune responses. Serological evidence to support the concept of molecular mimicry is far from conclusive; the results of relevant studies are summarized. Lymphocyte proliferation experiments suggest that antigen presenting cells play an important role. Finally, our views on reactive arthritis in the 1990s, and areas of new and potentially fruitful future research are presented. PMID- 7534944 TI - Transurethral resection of the prostate for benign prostatic hyperplasia--a local review. AB - There has been a recent resurgence of interest in the role of transurethral resection of the prostate for benign prostatic hyperplasia in view of the introduction of new modalities. We have conducted a retrospective analysis of 175 cases operated from October 1988 to June 1989 with an aim to ascertain the present mortality and morbidity rates associated with this procedure. The main presenting symptoms were acute retention of urine (54%) and bladder outlet obstruction (33%). The average weight of the prostate resected was 24.2 gm and 3% of specimens revealed malignant changes on histology. Seventy-five percent of the patients have post-operative stay of 5 days or less. Urinary tract infection was the commonest complication (16%). Clot retention requiring re-scope occurred in 2% of our patients. Twelve percent (12%) of our patients developed acute retention post-operatively but only 2% required re-scope as the rest resolved conservatively. We had one mortality in our series as a result of post-operative pneumonia. After 6 months follow-up, 4% complained of mild urinary incontinence and another 4% noted retrograde ejaculation. Six percent developed urethral strictures which required surgical treatment. Three years after the procedure, we retrieved the case notes of our cohort to analyse long-term results. We note that ninety-eight percent of our patients were discharged after nine months follow-up. This includes the 4% who complained of mild stress incontinence at 6 months follow-up. The remaining 2% was discharged after 24 months because of recurrent urethral stricture.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534945 TI - Study of the interaction of dextran and enoxaparin on haemostasis in humans. AB - The effect on haemostatic variables by dextran 70, enoxaparin and their combinations, given in doses of 500 ml i.v. and 40 mg s.c. respectively, was studied in a randomised cross-over fashion in twelve healthy male volunteers. Antifactor-IIa activity, antifactor-Xa activity, APTT, factor VIII, vWF, bleeding time and blood counts were analysed over a 24-h period. Dextran alone did not affect antifactor-IIa activity and antifactor-Xa activity. No difference in antifactor-IIa and antifactor-Xa activity was found for Amax, tmax, AUC0-8 h and AUC0-24 h in the groups treated with enoxaparin or the combination of enoxaparin and dextran. Only minor changes in APTT were observed without statistical significance between the treatment groups. Factor VIII did not change significantly in the three treatment groups. However, vWF was significantly reduced in the dextran and the dextran/enoxaparin group (p = 0.046 and 0.01 respectively) but no difference was found between the two groups. Bleeding time was not significantly increased four hours after administration of the test substances and no difference was found between the individual treatment groups. Our findings indicate that dextran can be combined with enoxaparin, when used in thromboprophylactic doses, without increased risk for bleeding. PMID- 7534947 TI - A P-selectin/CD62P monoclonal antibody (LYP-20), in tandem with flow cytometry, detects in vivo activated circulating rat platelets in severe vascular trauma. AB - P-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet alpha granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% +/- 3.44) or in peripheral blood (78.5% +/- 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% +/- 3.34) or in peripheral blood (18.0% +/- 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 +/- 15 x 10(3) platelets versus 48 +/- 31 x 10(3) platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% +/- 1.84) or in the peripheral blood (11.2% +/- 3.30) showed little activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534946 TI - Pharmacokinetics and pharmacodynamics of saruplase, an unglycosylated single chain urokinase-type plasminogen activator, in patients with acute myocardial infarction. AB - We examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active two-chain urokinase-type plasminogen activator (tcu-PA) and evaluated its effect on haemostatic parameters. Twelve patients were studied during and after administration of 20 mg bolus plus 60 mg continuous 1 h i.v. infusion of saruplase. For u-PA antigen and total u-PA activity (expressed as protein equivalents), where 234 U corresponds to 1 microgram, respectively, steady state plasma concentrations were 2.75 +/- 8.3 and 2.50 +/- 7.0 micrograms/ml (mean +/- standard deviation) and were reached within 20 min, t1/2 lambda 1 was 9.1 +/- 1.8 and 7.8 +/- 1.3 min, t1/2 lambda 2 1.2 +/- 0.2 and 1.9 +/- 0.5 h, and the total clearance was 393 +/- 110 and 427 +/- 113 ml/min. Inactivation of saruplase in plasma was negligible. After 15 min, tcu-PA was detected in plasma. From the ratio of the areas under the curve of tcu-PA and total u-PA activities it was calculated that 28 +/- 9.3% of the saruplase dose is converted into active tcu PA. Systemic plasminaemia occurs as shown by a decrease in alpha 2-antiplasmin and fibrinogen and an increase in fibrinogen degradation products. Thrombin antithrombin complex formation indicated activation of the clotting system. Saruplase is eliminated rapidly from plasma in AMI patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534949 TI - Aortic valve replacement for end-stage aortic valve disease. AB - To evaluate the outcome of surgical intervention for end-stage aortic valve disease, we carried out a retrospective, longitudinal survey of 85 patients (65 males, 20 females; mean age 53 period. All the patients presented in New York Heart Association (NYHA) class IV in cardiac failure (3 had cardiogenic shock and 27 had bacterial endocarditis). In-hospital mortality was 9.4% (8/85) overall. Those with endocarditis had a significantly higher mortality, 6/27 (22%) vs 2/58 (3.4%), p < 0.01. In-hospital mortality was not significantly increased in those with renal failure, reoperation, simultaneous coronary artery surgery, age > 65 years nor was it related to the predominance of aortic regurgitation or stenosis. After a mean follow-up period of 5.9 years (range 0 to 12.5 years), the overall actuarial survival was 82% and 74% at 5 and 10 years respectively. For 66 late survivors, the NYHA status improved to class I in 51, to II in 10, to III in 4 patients, and one patient remained in class IV. The incidence of paraprosthetic leak, reoperation, thromboembolism, anticoagulant-related haemorrhage, and endocarditis were respectively 0.8, 0.8, 1.6, 1.4, and 0.2 per 100 patient-years. Aortic valve replacement in the patient with end-stage aortic valve disease is a high-risk procedure, the risk being higher in the presence of endocarditis. The favourable long-term survival, long-term improvement in functional class and the relatively low incidence of valve-related complications justify surgical intervention in such patients, who would otherwise have a very poor prognosis. PMID- 7534948 TI - Neutrophil but not monocyte activation inhibits P-selectin-mediated platelet adhesion. AB - Selectins are Ca(2+)-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534950 TI - Valve reoperations--identification of risk factors and comparison with first-time operations. AB - Fourty-seven patients with a least one heart valve operation each who underwent reoperations (Gr. I) were analyzed with special regard to risk factors influencing the perioperative mortality and compared to 203 patients operated for the first time (Gr. II) during the same time period. Mean age was 57.1 years in Gr. I and 64.1 years in Gr. II (p < 0.05). There were no differences between the groups with regard to sex, smoking, obesity, or concomitant peripheral vascular disease. Hypertension, hyperlipidemia, and diabetes were more frequently seen in Gr. I, p < 0.05. A significantly higher number of patients in the redo group (Gr. I) belonged preoperatively to NYHA class III or IV, p < 0.001 and needed emergency surgery more often, p < 0.01, but left-ventricular function did not differ between the groups. There was no significant difference in the position of valves operated or the number of multiple valve replacements/repairs between the groups, and no difference in aortic cross-clamping or cardiopulmonary bypass time. Most patients were referred from other hospitals. Overall perioperative mortality for Gr. I was 6.4% and Gr. II 4.4% (n.s.). Mortality after first reoperation was 5.0%, after second or more 14.3%. Perioperative mortality was related to age, preoperative NYHA class, and urgency of operation in both groups, and to multiple valve replacement/repair in Gr. I. Elective reoperation carried a mortality of 4.8% but emergency reoperation 20%; reoperation mortality was 2.6% for single valves and 25% for multiple valves.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534951 TI - Coronary sinus retroperfusion combined with intraaortic balloon pumping to perfuse the acutely ischemic myocardium. AB - This study was planned to show the effect of retroperfusion and intraaortic balloon pumping (IABP) on myocardial hemodynamic recovery. Twelve dogs entered this study. Half of them received IABP and coronary sinus retroperfusion (CSPR) combination (Group II) and the remaining received IABP alone (Group I). Left anterior descending artery was occluded for a period of three hours. 15 minutes after occlusion IABP and IABP + CSRP were initiated. The average cardiac output was 1.41 +/- 0.18 L/min in the group I and 1.72 +/- 0.24 L/min in the group II (p < 0.03) after 3 hours of occlusion. Mean arterial pressure was 82.1 +/- 4.8 mmHg in the group I and 89.7 +/- 2.6 mmHg in the group II (p < 0.03). On the basis of this study it was concluded that CSRP + IABP could be an alternative treatment to IABP alone during the acutely developing ischemia. PMID- 7534952 TI - Laser welding--suitable for vascular anastomosis? AB - Carotid arteries of 21 piglets were transsected and reanastomosed either by laser welding (Neodym:YAG laser) or by conventional suture anastomosis. Histological specimens of the anastomoses obtained 2 to 32 days after the operation showed less foreign body reaction and intimal hyperplasia after laser welding than after suturing. There was, however, no significant difference when comparing occurrence of thrombosis, patency rate, or growth of the anastomosis in growing animals. Neither our study nor a review of the literature of laser-assisted vascular anastomosis in microvessels and large arteries up to 5 mm diameter could establish a definite clinical application for laser welding in vascular anastomosis. PMID- 7534953 TI - Long-term results of the gamma-irradiation-preserved homograft monocusp for transannular reconstruction of the right-ventricular outflow tract in tetralogy of Fallot. AB - The long-term results of a consecutive series of transannular gamma-irradiated homograft monocusp patches in tetralogy of Fallot were studied. Seven survivors out of 8 patients are doing clinically well after a mean of 22.4 years of follow up, 5 of them being in New York Heart Association class I and 2 in class II. In one patient the completely calcified gamma-irradiated homograft monocusp patch was replaced 15 years after intracardiac repair. Echocardiographic investigation at the end of follow-up of the remaining 6 patients with a gamma-irradiated homograft monocusp patch in situ showed mild pulmonary regurgitation in 1, moderate regurgitation in 3, and severe regurgitation in 2. Residual pulmonary stenosis was present in 4 patients (gradients ranging from 10 to 40 mmHg). Right ventricular dilatation was present in all patients. In the long-term the gamma irradiated transannular homograft monocusp patch behaves like a simple transannular patch. PMID- 7534955 TI - Costotomy and "hand inside": a useful adjunct to video-assisted thoracic surgery or just a silly idea? AB - We report on our first experiences with an access in video-assisted thoracoscopic surgery (VATS) which to the best of our knowledge has not yet been reported. One of the main problems in VATS is the absence of palpation which evidently plays an important role in tumor surgery. In three cases we therefore performed dorsolateral 8 cm incisions just beneath the scapula and resected a corresponding length on 6th or 7th rib to be able to insert one hand into the thorax. No rib spreader was used in either case, the idea being that a small costotomy would be less painful and traumatic than a standard posterolateral thoracotomy. Visualization of the operative field was obtained by inserting a camera through the 9th intercostal space, one or two additional small incisions were made for the other instruments. PMID- 7534954 TI - Long-term experience with pericardiectomy: analysis of 105 consecutive patients. AB - A retrospective analysis of the records of 105 patients who underwent pericardiectomy from 1983 to 1993 was performed. Primarily, 40 patients (38%) had tuberculosis, 16 patients (15%) a malignancy, 12 patients (11.4%) uremia, and 11 patients (10.5%) had rheumatic disorders. Pericardiectomy was performed through midline sternotomy in all cases, 9 of them required cardiopulmonary bypass. On operation, the anterior pericardium was excised parallel to the phrenic nerves on both side. The early mortality rate was 10.5% (11 patients). Mean follow-up time was 5.8 +/- 2.1 years, ranging from 1 year to 11 years. Actuarial survival of the patients at 1 year and 5 years were 81.1% +/- 6.8% and 63.5% +/- 8.2%, respectively. We conclude that pericardiectomy using midline sternotomy with or without cardiopulmonary bypass can be performed safely and can lead to good functional results and long-term survival. PMID- 7534956 TI - The coronary subclavian steal syndrome: an uncommon sequel to internal mammary coronary artery bypass surgery. AB - Coronary subclavian steal syndrome is a possible sequel in patients who have undergone myocardial revascularization with an internal mammary artery. We report a case of this syndrome in a 67-years-old man. In 1990 he underwent a quadruple bypass: aorta-obtuse margin, aorta-right coronary (two sequential), internal artery mammary-descending coronary artery. Three months later he started to have angina pectoris. In April 1992 an aortic arch angiography and a coronary angiography were performed. The examination showed an occlusion of the left subclavian artery at its origin. The artery was opacified countercurrently by the left vertebral artery but the left mammary artery was not opacified. Left coronary angiography showed a very severe disease of left anterior descending coronary artery and retrograde flow through the anastomosis in the left mammary artery. The patient underwent a left common carotid-subclavian artery bypass operation using a 6 mm vascutex graft. Eighteen months later the patient is doing well without angina pectoris and with very little alteration of the perfusion in the left frontal lobe observed by SPECT neuroimaging with a lipophilic tracer (99mTc-HMPAO). We think that the coronary-subclavian steal syndrome can be treated successfully with low risk by means of common carotid-subclavian artery bypass. PMID- 7534957 TI - Spontaneous non-traumatic rupture of the thoracic aorta. AB - Spontaneous non-traumatic rupture of the aorta is a very rare condition that requires immediate surgery. However, correct preoperative diagnosis is difficult or impossible to establish. We report a case of spontaneous aortic rupture which was successfully treated by emergency surgical exploration following a transesophageal echographic diagnosis of hemopericardium. This case, as well as other reports, suggests that hemopericardium along with symptoms which suggest aortic dissection or rupture (e.g., acute chest or back pain) should raise a strong suspicion of spontaneous aortic rupture even in the absence of intimal flap or aortic dilatation, and the emergency surgery may be life-saving. PMID- 7534958 TI - Reversed flow in the internal carotid artery after occlusion of the common carotid artery. AB - Patency of the internal carotid artery in case of common carotid artery occlusion is usually maintained by a flow of blood from the external carotid into the internal carotid artery. A case where a reversed flow of blood was established from the cerebral circulation into the internal carotid artery and eventually into the external carotid artery is presented. Careful reading of the cerebral views of the standard carotid angiogram allowed detection of a retrograde flow into the internal carotid artery. Surgical exploration confirmed the patency of the internal carotid artery; the occluded common carotid artery was bypassed by a prosthesis, which restored an antegrade flow into the internal carotid artery. PMID- 7534959 TI - Intravenous leiomyomatosis extending into the right ventricle: one-stage radical excision during hypothermic circulatory arrest. AB - A case of intravenous leiomyomatosis with extension into the right ventricle is described. A tumor in the inferior vena cava was detected 5 years after a hysterectomy had been performed for a myomatous uterus, but was misdiagnosed as an intracaval thrombus. Three years later this tumor was discovered after it had enlarged and intruded into the right ventricle. The correct diagnosis was made after a complete evaluation. The patient underwent surgery employing simultaneous sternotomy and laparotomy. Radical excision was achieved using cardiopulmonary bypass with hypothermic circulatory arrest. The diagnostic and surgical approaches are reviewed and discussed. PMID- 7534960 TI - Left-ventricular aneurysm in a young male due to spontaneous coronary artery dissection. AB - A case of spontaneous coronary artery dissection in a 26-year-old male with subsequent left-ventricular aneurysm is reported. A coronary bypass operation using left internal mammary artery combined with left-ventricular aneurysmectomy was performed successfully. This is the youngest case in the literature which describes a successful surgical treatment for a spontaneous coronary artery dissection with left-ventricular aneurysm. PMID- 7534961 TI - Bifurcate tracheal stent with foam cuff for tracheo-esophageal fistula: utilization of reconstruction modes of spiral computed tomography. AB - Malignant tracheo-esophageal fistulae can be palliated with bifurcate tracheal stents. However, stents must be manufactured according to the individual anatomy. In these instances a foam cuff can result in better sealing of the fistulized area. We present a successful implantation of a bifurcate tracheal stent with foam cuff. To facilitate preoperative planning reconstruction possibilities offered by spiral computed tomography were used. PMID- 7534962 TI - Effects of substance P on gastric motility differ depending on the sites and vagal innervation in conscious dogs. AB - The effect of substance P on gastric motility was studied in conscious dogs by means of strain gauge force transducers chronically implanted on the gastric body, antrum, and a vagally-denervated fundic pouch. Intravenous infusion of substance P in the interdigestive state induced phasic contractions in the pouch and antrum. Atropine inhibited these contractions in the pouch and antrum. Hexamethonium enhanced substance P-induced contractions in the gastric antrum, but reduced those in the pouch. Pretreatment with phentolamine, propranolol, or naloxone did not affect substance P-induced contractions in the pouch and antrum. The intact gastric body scarcely reacted to substance P. Mean systemic blood pressure was lowered by substance P-infusion, but there was no dose-dependency in the reduction of the blood pressure, nor was it affected by the pretreatment with atropine or hexamethonium. These results suggest that 1) the vagal innervation influences the effect of substance P on motility in the gastric body, and that 2) substance P may stimulate postsynaptic excitatory cholinergic and presynaptic inhibitory neurons simultaneously in the gastric antrum. PMID- 7534963 TI - The role of granulocyte colony-stimulating factor (filgrastim) in maintaining dose intensity during conventional-dose chemotherapy with ABVD in Hodgkin's disease. AB - BACKGROUND: The aim of the study was to evaluate the role and potential benefit of granulocyte colony-stimulating factor (G-CSF, Filgrastim), administered following cytotoxic chemotherapy with the ABVD regimen in Hodgkin's disease, in maintaining cycle schedule and dose intensity and in decreasing neutropenia and number of infections. PATIENTS AND METHODS: Twenty-two patients affected by high risk Hodgkin's disease (14 localized and 8 diffuse), aged 15 to 69 years (median, 34), were given ABVD chemotherapy for a total of 6 courses (for the purpose of this study, each single course of chemotherapy was considered as two 15-day periods). No patient was given G-CSF after the first cycle. After each cycle, G CSF was administered only for: 1)absolute neutrophil count < 1 x 10(9)/L between cycles; 2) delay in cycle schedule due to an absolute neutrophil count < 1 x 10(9)/L on the planned day of treatment; or 3) fever or a documented infection, regardless the absolute neutrophil count. Once administered, G-CSF was maintained in the subsequent cycles. RESULTS: Seventeen of 22 patients (77%) required the administration of G-CSF (5 micrograms/kg b.w.; a median of 5 doses/cycle); most of them (13/17) before the 5th dose of chemotherapy. The main reason for introducing G-CSF into therapy was neutropenia during the interval between courses (n = 4) or on the planned day of treatment (n = 11). Comparing 112 courses where G-CSF was not administered with 124 where it was, in the latter group we observed: 1) a significantly lower (P = 0.0002) incidence of cycle delays (0 vs 13), with a median delay of 7 days (5 to 11). The main reason for cycle delay was neutropenia (n = 13); 2) a greater dose intensity delivered to the patients while on G-CSF (100% vs 95.2 +/- 8.8%; P = 0.0001); 3) an absolute neutrophil count significantly higher at day 8 (P < 0.0001) and day 15 (P < 0.0001); 4) a significantly lower (P = 0.0003) incidence of neutropenia (2 vs. 17). No difference in the incidence of infections was observed between the two groups of cycles (P = 0.5889), but the duration and severity of the same were greater during chemotherapy without G-CSF, requiring antibiotic therapy and causing cycle delay. CONCLUSIONS: In conclusion, our data suggest the use of Filgrastim in Hodgkin's disease also during conventional-dose chemotherapy with ABVD. It is not required from the first dose of therapy, but as soon as neutropenia appears between cycles or on the planned day of treatment. Then, its use allows maintenance of the chemotherapy schedule and dose intensity. It also decreases frequency, duration and severity of neutropenia and its sequelae. PMID- 7534964 TI - Histopathologic investigations of acute and subchronic toxicities of some organotin compounds in chickens. AB - The acute and subchronic oral toxicities of 4 organotin derivatives were investigated in chickens. The LD50 values oscillated between 200 and 400 mg/kg bw and revealed a moderate or weak toxicity. Subchronic dosing with 2 doses from each compound at 2 and 10 mg/kg bw induced dose-dependent lesions seen at necropsy and histologic examinations. Involution of the thymus and bursa Fabricius, fatty degeneration of the liver, and nephrotic syndromes were the most evident effects. The butyltin derivatives induced more drastic lesions than did the phenyltins. PMID- 7534965 TI - [Concentration of acute phase proteins in serum of children with rheumatoid arthritis]. AB - An attempts was made at evaluation of the changes of acute phase proteins seromucoid (BRS), alpha 1-acid glycoprotein (alpha 1-AGP), alpha 1-antitrypsin (alpha 1-AT), haptoglobin (Hp), C3 protein of complement system (C3), C1 esterase inhibitor (C1-INH), and transferrin (Tf) in the serum of children with rheumatoid arthritis. The patients were divided into clinical and age groups. The studies carried out have shown in ill children increase of BRS, alpha 1-AGP, alpha 1-AT, Hp, and C1-INH levels, During remission increased levels of alpha 1-AGP and Hp persisted. In the studied clinical groups a positive correlation was found between the intensity of changes of the studied indices and the degree of disease activity. In the age groups greater increase of the levels of the studied acute phase proteins was found in the group of preschool children and the group of puberty spurt. The obtained results suggest that the determination of acute phase proteins may be useful in laboratory investigation of children with rheumatoid arthritis. PMID- 7534966 TI - Comparative study of three amniotic fluid markers in premature rupture of membranes: fetal fibronectin, alpha-fetoprotein, diamino-oxydase. AB - BACKGROUND: To study the comparative diagnostic value in premature rupture of membranes (PRM) of three amniotic components: fetal fibronectin (fFN), alphafetoprotein (AFP), diamino-oxydase (DAO). METHODS: 131 pregnant women took part in our prospective study. Three samples were obtained successively for each patient in a random order. Two clinical situations were studied: group A of 68 women with clinical certain PRM and group B of 63 women with a highly unlikely PRM. RESULTS: fFN is the best marker for diagnosis of PRM (sensitivity of 94% and specificity of 97%). AFP and DAO are complementary: the NPV is greater for AFP (87% against 84%) whereas the PPV is greater for DAO (95% against 86%). CONCLUSION: Apart from its value in predicting premature labor, vaginal fFN represents a diagnostic test of PRM with good specificity and sensitivity. PMID- 7534967 TI - Assessment of estrogen receptor distribution in human endometrium by direct immunofluorescence. AB - OBJECTIVE: To use a direct immunofluorescence technique employing a fluorescein labeled anti-idiotypic antibody that recognizes the estrogen receptor (ER) order to assess the distribution of ER in the uteri of normal women throughout the normal menstrual cycle and of a woman exposed prenatally to diethylstilbestrol (DES). SUBJECTS: Included in the study were 25 women aged between 35 and 50 years and an amenorrheic patient diagnosed as "DES Syndrome". LOCALIZATION: Localization of ER expression in frozen sections of uterine tissue was achieved by direct immunofluorescence using a fluorescein labeled anti-idiotypic antibody that interacts with ER. RESULTS: Analysis of the immunofluorescence staining indicated that in the normal human endometrium the intensity of ER staining varied according to the phase of the cycle as well as according to the cell type. On the other hand, endometrial ER evaluation of the patient with DES syndrome showed minimal expression of ER and after treatment with conjugated estrogens, endometrial biopsy revealed a significant increase in ER expression. CONCLUSIONS: These findings indicate that the fluorescein labeled anti-idiotypic antibody can be used to detect ER in normal and pathological human endometrium and to monitor changes in ER expression in the endometrium during hormonal therapy. PMID- 7534968 TI - Distribution of NADPH diaphorase-reactive nerves in the human female genital organ. AB - OBJECTIVE: This study was designed to histochemically clarify the presence of nerves containing NADPH diaphorase, representing the catalytic activity of nitric oxide synthase, in the human female genital organ. STUDY DESIGN: Female genital organs were isolated for NADPH diaphorase staining by extensive or simple hysterectomy from ten patients. RESULTS: Paracervical ganglia contained many nerve cells intensely stained. The nerve from the ganglia contained many axons intensely stained, which selectively innervated blood vessels distributing to the whole genital organ. NADPH diaphorase-reactive nerve fibers around the vascular wall distributed in the adventitia, closely contacting to the medial layer. The distribution of the axons was much denser in the arterial wall than in the venous wall. CONCLUSION: The NADPH diaphorase-reactive nerve fibers, possibly originated from paracervical ganglia, appear to play an important role in the regulation of uterine vascular tone by liberating nitric oxide, and such a neurogenic control may be predominant in the artery over the vein. PMID- 7534969 TI - Effects of labor on serum levels of insulin and insulin-like growth factor binding proteins at the time of delivery. AB - BACKGROUND: The purposes of this study were to explore whether serum levels of insulin, insulin-like growth factor-I (IGF-I), insulin-like growth factor-binding protein-1 (IGFBP-1) and IGFBP-3 in both maternal and fetal compartments were affected by the stress of labor, and to investigate the relationship between the fetal birthweight and serum levels of insulin, IGF-I and IGFBPs. METHODS: Blood samples were collected at the time of delivery from 147 parturients with vaginal delivery and 128 cases of Cesarean section (112 cases without labor and 16 cases with arrest of cervical dilatation during the active phase of labor). Serum concentrations of insulin, IGF-I, IGFBP-1 and IGFBP-3 were determined by radioimmunoassays (insulin, IGFBP-1 and IGFBP-3) and immunoradiometric assay (IGF I). RESULTS: Maternal circulating IGFBP-1 levels in parturients with normal spontaneous delivery (NSD) and in subjects receiving Cesarean section (CS) due to arrest of cervical dilatation during active phase of labor were higher than those undergoing scheduled CS without labor. By contrast, insulin levels in both maternal and umbilical cord serum were higher in parturients with CS without labor than those with NSD. No difference in maternal serum IGFBP-3 levels was observed between NSD and CS at the time of delivery. As for all measurements (insulin, IGF-I, IGFBP-1 and IGFBP-3), serum levels in pregnant women (from both NSD and CS) were strikingly higher than those in the fetus. Serum levels of IGFBP 1 in umbilical cords from both groups of NSD (p < 0.02) and scheduled CS (p < 0.01) were inversely correlated with birthweight (BW). By contrast, serum concentration of insulin and IGF-I in umbilical cords from NSD (p < 0.005 and p < 0.01; respectively) and scheduled CS (p < 0.01 and p < 0.05; respectively) were positively related to BW. CONCLUSIONS: From the present results, we conclude that insulin appears to be a regulator for circulating IGFBP-1 during pregnancy. The fetal growth may not be well reflected by maternal serum IGFBP-1 levels, nor by IGFBP-3. By contrast, cord serum IGFBP-1 from CS group without labor may preeminently reflect fetal weight. In additional, serum concentration of insulin and IGF-I in umbilical cord may also be good indicators to reflect the result of neonatal birthweight. PMID- 7534971 TI - Beta-HCG concentration in peritoneal fluid and serum in ectopic and intrauterine pregnancy. AB - OBJECTIVE: To evaluate the significance of beta-HCG levels in peritoneal fluid and serum in the diagnosis of ectopic pregnancy. STUDY DESIGN SETTING: Obstetrics and Gynecology department of a regional general hospital. SUBJECTS: Sixty-two women who presented with a differential diagnosis of ectopic pregnancy vs. nonviable intrauterine pregnancy. INTERVENTIONS: All patients underwent D & C and culdocentesis. beta-HCG was measured in simultaneously obtained peritoneal fluid (PF) and serum (S), and the PF/S ratio calculated. RESULTS: Twenty-three patients had an ectopic pregnancy (Group I). All 23 had higher beta-HCG concentrations in the PF than in the serum, with a mean PF/S ratio of 19.1 +/- 16.9. Twenty-four patients had an intrauterine pregnancy (Group II). The beta-HCG levels in the PF and serum were similar (mean PF/S 1.1 +/- 0.2). The difference in PF/S ratio between groups I and II was statistically significant (p < 0.001). CONCLUSION: The measurement of beta-HCG in peritoneal fluid and serum is a useful diagnostic tool in differentiating ectopic from intrauterine pregnancy. PMID- 7534970 TI - Serum tetranectin in patients with acute pelvic inflammatory disease (PID). Correlation to clinical and laboratory findings. AB - AIM OF STUDY: To clarify the impact of the presence of pelvic inflammatory disease (PID) and activation of the acute phase response on the serum level of tetranectin (Se-TN), a potential new tumor marker for ovarian cancer. MATERIALS AND METHODS: The study group consisted of 70 patients with a laparoscopically verified PID and 47 healthy female controls. RESULTS: Lower Se-TN levels were found for the PID group compared to the control group (p < 0.0001). It was not possible to relate the decreases in Se-TN levels to any distinct bacterial strain. Neither was it possible to find any correlation between Se-TN and severity of PID (p = 0.5). A significant positive correlation was found between Se-TN and ALB (p < 0.001). A just significant negative correlation was found between Se-TN and C-reactive protein (CRP) (p = 0.04), while no correlation was found with any of the other acute phase reactants. Highly significant correlations were found between all the acute phase reactants and grade of PID. CONCLUSIONS: A slight, but significant reduction in Se-TN was found in PID patients. The decrease was minor compared to the reported findings of very low Se TN levels for ovarian cancer patients. However, the finding is important in the assessment of TN used as a potential screening marker for ovarian cancer, or as a diagnostic tool for pelvic tumors. Furthermore, Se-TN does not seem to behave as a negative acute phase reactant. PMID- 7534972 TI - [Clinical evaluation of TANDEM PSA in Japanese cases and comparison with other methods]. AB - Clinical evaluation of TANDEM PSA which is the most frequently used prostate specific antigen (PSA) assay method in the world and a comparison with other methods were performed in Japanese cases in a cooperative research fashion. The minimum detectable level of the method was found to be 0.50 ng of PSA in one ml of serum and 1.9 ng/ml was regarded as the upper normal value in Japanese males. The distribution of serum PSA showed a significant difference between the benign prostate hypertrophy (BPH) cases and patients with stage C or D prostate cancer. The sero-diagnosis prostate cancer at an early stage with the TANDEM PSA was difficult. The correlation to other methods of PSA detection was very high. Furthermore, the clinical use of the method in following-up the clinical course of prostate cancer patients was very useful. These findings suggested that the PSA detection using TANDEM PSA is applicable even in Japanese cases although the upper cut-off level is decreased. PMID- 7534973 TI - Characteristic MR imaging of the trichilemmal cyst. PMID- 7534974 TI - Interventional cardiac catheterization under transesophageal echocardiographic guidance. PMID- 7534976 TI - Neoadjuvant chemotherapy for advanced nasopharyngeal carcinoma. AB - Between October 1990 and November 1991, a total of 16 male patients with advanced nasopharyngeal carcinoma were treated by neoadjuvant chemotherapy before conventional radiotherapy. They belonged to the AJCC stage IV with multiple bulky neck nodes metastases. The chemotherapy consisted of bleomycin, epirubicin, and cisplatin. Six patients completed 3 cycles, 9 patients finished 2 cycles, and 1 patient received 1 cycle of chemotherapy. Seven of the 16 patients (44%) were in complete response, and 50% (8/16) achieved partial response. The overall response rate was 94%. The major toxicities consisted of leucopenia (12/37 cycles had grade III-IV), nausea/vomiting, alopecia. Aplastic marrow developed in 1 patient, and one died of bleomycin-induced pneumonitis. Subsequent radiation therapy was well tolerated. After a minimal follow-up time of 24 months, the 2-year actuarial survival rate was 56%. Although we confirmed the impressively high response rate of this regimen, the toxicities were high and most patients failed at distant site(s). The efficacy of neoadjuvant chemotherapy for advanced nasopharyngeal carcinoma is doubtful and should be further studied in prospective randomized trials. PMID- 7534975 TI - Neuroendocrine primary small cell carcinoma of the breast. Report of a case and review of the literature. AB - One case of breast neuroendocrine primary small cell carcinoma with light microscopic and immunohistochemical findings is reported. The patient died of unrelated disease 21 months after diagnosis and treatment by modified radical mastectomy, radiotherapy and subsequent chemotherapy. Immunohistochemical studies revealed cytokeratin and neuroendocrine markers (chromogranin, neuron-specific enolase) immunostaining on tumoral cells. Expression for neuropeptides (met enkephalin, leu-enkephalin, beta-endorphin) and CALLA antigen was found. Based on this case report and six other previously reported cases, breast neuroendocrine primary small cell carcinoma appears to be a very aggressive tumor for which no firm conclusions regarding treatment can be drawn. PMID- 7534977 TI - Oral etoposide and carboplatin. Effective therapy for elderly patients with small cell lung cancer. AB - PURPOSE: Elderly patients with small cell lung cancer (SCLC) and/or those with comorbid conditions are frequently not considered candidates for standard combination chemotherapy. An active, but less toxic regimen is needed for this group of patients. PATIENTS AND METHODS: Forty-seven elderly (> 65 years) or medically unfit patients with SCLC were treated with oral etoposide 100 mg/m2 x 7 days and carboplatin 150 mg/m2 day 1. Treatment was given every 3-4 weeks for six cycles in responding patients. Patients responding to the chemotherapy regimen were also treated with prophylactic cranial irradiation, and limited-stage patients received thoracic radiotherapy. The study population included 36 extensive-stage patients and 11 limited-disease patients with renal or cardiac disease that precluded standard chemotherapy. The median age of the study population was 69 years (range: 47-84). RESULTS: Nine of 47 patients were inevaluable for response, including four patients who succumbed to sepsis. Of the 38 patients evaluable for response, 71% responded (95% CI: 56-86%) (88% LD; 67% ED) with a complete response in 29% of patients (50% LD; 23% ED). Based on an analysis of intent to treat, the overall response rate was 60% and the median survival time of the whole group was 46 weeks (LD, 59 weeks; ED, 45 weeks). Treatment was generally well tolerated. Neutropenia was the dose-limiting toxicity; the median nadir granulocyte count was 1.04 x 10(9)/L (range: 0-8.2). CONCLUSION: We conclude that this regimen can provide palliation to SCLC patients who might not otherwise be considered for systemic chemotherapy. PMID- 7534978 TI - A phase II study of fazarabine in patients with advanced ovarian cancer. A Gynecologic Oncology Group study. AB - BACKGROUND: A total of 22 patients with recurrent ovarian cancer previously treated with cisplatin-containing chemotherapy were treated with Fazarabine. METHODS: The drug was administered at an initial dose of 30 mg/m2/day for 5 consecutive days. Cycles were repeated every 28 days. There were 19 evaluable patients. RESULTS: No complete or partial responders were observed in this study; 48% of patients were deemed to have stable disease. The major toxicity was hematologic, with four patients exhibiting grade 4 neutropenia. CONCLUSIONS: Fazarabine shows no useful activity as a single agent when given at this dose and schedule in the management of previously treated patients with cancer of the ovary. PMID- 7534980 TI - PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln. AB - Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor. PMID- 7534981 TI - Role of Ca(2+)-activated K+ channels in electrical activity of longitudinal and circular muscle layers of canine colon. AB - The role of Ca(2+)-activated K+ channels (BK channels) in the canine colon was evaluated by testing the effects of charybdotoxin (ChTX) and tetraethylammonium on K+ currents of isolated myocytes and on electrical and mechanical activity of tissue strips. ChTX blocked Ca(2+)-activated outward current [IK(Ca)] in a dose- and voltage-dependent manner. No significant differences in IK(Ca) density, ChTX block, or Ca2+ sensitivity of BK channels were observed between circular and longitudinal myocytes. ChTX (100 nM) blocked 60% of current at +80 mV. Delayed rectifier current was not inhibited by 100 nM ChTX. In the absence of agonists, ChTX did not affect electrical or mechanical activity of circular muscle strips. In the presence of 10(-6) M BAY K 8644 or 10(-6) M acetylcholine, ChTX increased slow-wave duration and amplitude, induced membrane potential oscillations, and potentiated contraction. In unstimulated longitudinal muscle strips, ChTX depolarized the tissue, increased burst duration and spiking frequency, and resulted in an increase in contractions. These results indicate that BK channels are important regulators of colonic motility. In the longitudinal layer, BK channels are involved in setting membrane potential and determine excitability. In the circular layer, ChTX-sensitive channels do not participate in the in vitro basal electrical activity but limit the responses to excitatory agonists. PMID- 7534982 TI - Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current. AB - Whole cell epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents exhibited a linear current-voltage (I-V) relationship with high symmetrical transmembrane Cl- concentrations. However, when intracellular Cl- (Cli-) was reduced by replacement with glutamate, I-V relationships were outwardly rectifying. Rectification was not affected by reducing extracellular Cl to eliminate or reverse the gradient, indicating that rectification is not a function of the Cl- gradient. Rectification was affected by Cli- in a concentration-dependent manner, and it was weaker when Cli- was reduced by replacement with sucrose. These characteristics are identical to those of the cardiac isoform of CFTR, and the experimental data could be simulated by an Eyring rate theory model assuming that permeating anions interact at a single binding site within the channel pore. No evidence was found for multiple binding sites. These results indicate that rectification is a function of the concentration and permeability of the anions inside the cell. It is concluded that rectification of CFTR Cl- current is a property of ion channel permeation that would occur under physiological conditions and that permeation of the epithelial and cardiac isoforms of CFTR is identical. PMID- 7534983 TI - Impact of nitric oxide on macrophage glucose metabolism and glyceraldehyde-3 phosphate dehydrogenase activity. AB - Conflicting evidence has been presented regarding the role of nitric oxide (NO) in the regulation of cellular glucose metabolism. While it enhances glucose uptake and utilization through glycolysis and the hexose monophosphate shunt in macrophages and other cells, NO also inhibits glyceraldehyde-3-phosphate dehydrogenase, an enzyme catalyzing the metabolism of intermediates generated by both pathways. Indeed, it has been proposed that NO modulates glycolytic flux by suppressing glyceraldehyde-3-phosphate dehydrogenase activity. To establish the relative impact of these apparently incompatible actions, the effects of exogenous or endogenous NO on different aspects of glucose metabolism in macrophages were investigated. Cell activation increased NO production, maximal glyceraldehyde-3-phosphate dehydrogenase activity, and glucose metabolism through glycolysis and the hexose monophosphate shunt. NO generated endogenously or from S-nitroso-N-acetylpenicillamine (> 500 microM) reduced maximal glyceraldehyde-3 phosphate dehydrogenase activity in culture. The suppression of maximal glyceraldehyde-3-phosphate dehydrogenase coincided with decreased lactate accumulation only in concert with a marked loss of viable cells in the cultures. The maximal glyceraldehyde-3-phosphate dehydrogenase activity did not appear to be rate limiting for glucose metabolism when moderately inhibited by NO. A potential causal relationship between profound glyceraldehyde-3-phosphate dehydrogenase inhibition and cell death remains to be established. PMID- 7534979 TI - Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction. AB - Reorganization of cytoskeletal-membrane interactions during contractile stimulation may contribute to the regulation of airway smooth muscle contraction. We investigated the effect of contractile stimulation on the phosphorylation of the actin-membrane attachment proteins talin, vinculin, and paxillin. Stimulation of 32P-labeled canine tracheal smooth muscle strips with acetylcholine (ACh; 10( 3) M) resulted in a rapid 2.6-fold increase in phosphorylation of serine and/or threonine residues, compared with resting levels of 0.22 mol PO4(3-)/mol talin. After stimulation with ACh, phosphorylation of tyrosine residues on paxillin increased approximately threefold. Two-dimensional phosphopeptide mapping of in vivo labeled talin and paxillin indicated phosphorylation on a limited number of sites. Vinculin phosphorylation was undetectable in either resting or ACh stimulated muscle. We conclude that phosphorylation of talin and paxillin occurs during ACh-stimulated contraction of tracheal smooth muscle and that distinct signaling pathways activate a serine/threonine kinase that phosphorylates talin and a tyrosine kinase that phosphorylates paxillin. The pharmacological activation of airway smooth muscle cells might involve the anchoring of contractile filaments to the membrane. PMID- 7534984 TI - Angiotensin II decreases inducible nitric oxide synthase expression in rat astroglial cultures. AB - Consistent with stimulation of expression of an inducible form of nitric oxide synthase (iNOS), exposure of rat astroglial cultures to lipopolysaccharide (LPS) caused a time-dependent increase in the accumulation of nitrite in the culture media. Addition of the peptide angiotensin II (ANG II) with LPS decreased subsequent formation of nitrite in a concentration-dependent manner (concentration inhibiting 50% of maximal response approximately 1 nM). The ANG II effect could be blocked by the ANG II type 1 (AT1 receptor antagonist losartan but not by the ANG II type 2 (AT2) receptor antagonist PD-123177. ANG II had no effect on nitrite formation stimulated by a combination of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma). A brief 10-min exposure to ANG II was sufficient to cause an approximately 30% inhibition of the LPS response, with maximal inhibition of approximately 65% after 3 h, and occurred only when ANG II was added during the iNOS induction phase. Consistent with partial inhibition of LPS-stimulated expression of iNOS, ANG II reduced the levels of both iNOS mRNA and iNOS protein. These results demonstrate that ANG II can decrease LPS-stimulated NO production in astroglia by inhibiting induction of iNOS expression. PMID- 7534985 TI - cAMP- but not Ca(2+)-regulated Cl- conductance in the oviduct is defective in mouse model of cystic fibrosis. AB - Defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- transport in cystic fibrosis (CF) reflects defects in the cystic fibrosis transmembrane conductance regulator (CFTR). A moderate level of CFTR mRNA expression has been found in rodent and human oviductal epithelium, but unlike other CFTR-expressing tissues, the oviduct in CF patients is apparently normal. The present study was carried out to investigate the relative magnitude of the cAMP- and intracellular Ca2+ (Cai2+)-regulated Cl- secretion in primary cultures of the oviduct from normal and CF mice generated by targeted disruption of the murine CF gene. Normal oviductal epithelium exhibited a basal equivalent short-circuit current (Ieq) of 20.3 +/- 1.7 muA/cm2. CF oviduct exhibited a lower basal Ieq of 4.5 +/- 1.9 muA/cm2. In normal mice, forskolin (10(-5) M, apical) elicited a slowly developing sustained rise in Ieq, whereas ionomycin (5 x 10(-6) M, apical) and ATP (10(-4) M, apical) induced larger increases in Ieq consisting of a prompt, transient response followed by a slowly decreasing component. The Ieq response to forskolin was totally abolished in CF mouse oviducts, but the magnitudes of the peak Ieq responses to ionomycin and ATP were not different from normal. The time courses of the ionomycin- and ATP-evoked responses, however, were significantly more transient in CF than in normal oviducts. These results demonstrate that CF mouse oviduct exhibits defective cAMP- but not Cai(2+)-mediated Cl- secretion. The relatively high level of functional expression of the alternative Cai(2+) activated Cl- secretory pathway in the mouse oviduct may contribute to the absence of major pathology in the CF oviduct. PMID- 7534986 TI - Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells. AB - To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl(-)-dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3 phenylpropylamino)benzoic acid (100 microM) or Ca2+ chelators such as ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) of approximately 200 nM. The swelling-induced rise in [Ca2+]i and RVD were diminished in the presence of 10 microM La3+, 50 nM 12-O tetradecanoylphorbol 13-acetate, and nominally Ca(2+)-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca(2+)-free EGTA 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation. PMID- 7534987 TI - Lysine uptake by human placental microvillous membrane: comparison of system y+ with basal membrane. AB - Transport of lysine by microvillous membranes was investigated by characterization of L-[3H]lysine uptake in membrane vesicles isolated from human placentas. At least one Na(+)-independent system was observed at 22 degrees C and two systems at 37 degrees C. Lysine concentration dependence data were fit by a one- or two-system model with a Michaelis-Menten constant (Km) of 124 +/- 28 microM and a maximum velocity (Vmax) of 33.1 +/- 7.7 pmol.mg protein-1.min-1 at 22 degrees C and with Km values of 1 +/- 0.6 and 245 +/- 51 microM and Vmax values of 0.14 +/- 0.07 and 45.8 +/- 8.7 pmol.mg protein-1.30 s-1 at 37 degrees C. In the presence of N-ethylmaleimide, the uptake (37 degrees C) data were fit by a one-system model with kinetic parameters similar to the lower Km system. Uptake of L-lysine in the absence of Na+ was inhibited completely by L-arginine, L-histidine, and L-homoarginine. In the presence of Na+, uptake was inhibited completely by these same three amino acids and L-leucine but only partially by other neutral amino acids. To compare directly microvillous and basal membrane from the same placenta, we examined the inhibition of 20 microM lysine uptake in the presence of Na+. Inhibition by L-leucine was similar in the two membranes. However, L-homoserine, L-alanine, and L-phenylalanine over a wide concentration range inhibited substantially less in microvillous (at both temperatures) than in basal membrane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534988 TI - Amiloride-sensitive and HCO3(-)-dependent ion transport activated by aldosterone and vasotocin in A6 cells. AB - We studied the effects of aldosterone (Aldo) and arginine vasotocin (AVT) on ion transport of renal epithelial cell line (A6) by measuring short-circuit current (Isc). AVT induced a rapid, transient increase in Isc, followed by a decrease toward the baseline in cells untreated with Aldo. In cells treated with Aldo, Isc showed a biphasic response to AVT, i.e., both transient and sustained increases over 40 min after addition of AVT. The transient increase was composed only of amiloride-insensitive Isc regardless of Aldo treatment, whereas the sustained increase contained both amiloride-sensitive and amiloride-insensitive components. The main part of the amiloride-insensitive, sustained Isc depended on HCO3(-). In cells treated with Aldo for 1 day, removal of HCO3(-) in the bathing solution enhanced the amiloride-sensitive component and decreased the amiloride insensitive one. These data suggest that 1) Aldo treatment is necessary for an AVT-induced sustained increase of Isc and 2) a HCO3(-)-dependent Isc mainly contributes to the sustained increase in amiloride-insensitive Isc. PMID- 7534990 TI - Cation channel mechanisms in ET-3-induced vasopressin secretion by rat hypothalamo-neurohypophysial explants. AB - Endothelins modulate not only vasoregulation but also neurotransmission and hormone secretion, specifically vasopressin (AVP) secretion. The present studies were designed to ascertain the site of action and the participation of membrane cation channels mediating endothelin-3-induced AVP release. Experiments were performed using standard and compartmentalized hypothalamo-neurohypophysial explants. The stimulatory action of endothelin-3 on AVP release occurred at the neural lobe, consistent with the failure of sodium channel blockade to decrease AVP secretion. Calcium channel antagonism or chelation of extracellular calcium inhibited neurohormone release, but blockade of calcium mobilization from intracellular stores with 8-(diethyl-amino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) did not. Inhibition of the calcium-activated potassium channel with charybdotoxin increased AVP levels dose dependently. Potassium ionophore abolished this response, as did TMB-8, but inhibition of calcium entry failed to do so. A subthreshold dose of charybdotoxin potentiated AVP secretion to submaximal stimulation with endothelin-3 that was prevented only by concomitant blockade of calcium influx and intracellular mobilization. The data support interaction between calcium and potassium channels at the secretory terminal. Collectively, these data are consistent with endothelin-3 receptor activation at the secretory terminal initiating calcium entry, thereby leading to depolarization independent of sodium conductances. This mechanism is opposed by hyperpolarizing forces linked to calcium accumulation, namely, the charybdotoxin sensitive calcium-activate potassium channel. Interaction of the depolarizing and repolarizing systems enables grade AVP secretion from the neural lobe. These findings do not preclude the participation of other systems as well. PMID- 7534989 TI - Phosphatase inhibitors potentiate adrenergic-stimulated cAMP and cGMP production in rat pinealocytes. AB - The role of phosphoprotein phosphatase in the regulation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat pinealocytes was investigated using the three phosphatase inhibitors calyculin A, tautomycin, and okadaic acid. Calyculin A (0.1 microM) was found to enhance the isoproterenol- and norepinephrine-stimulated cAMP accumulation six- and threefold, respectively, whereas tautomycin and okadaic acid were less effective. The effect of calyculin A was rapid (within 5 min) and persisted in the presence of phosphodiesterase inhibition. However, in contrast to protein kinase C activation or intracellular calcium elevation, the phosphatase inhibitors were less effective in potentiating the cAMP response stimulated by forskolin or cholera toxin, and their effects were not blocked by calphostin C or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. The adrenergic-stimulated cGMP response was also less sensitive to the phosphatase inhibition. Therefore, our results suggest that 1) the adrenergic-stimulated cAMP signal is subjected to the tonic inhibition by phosphoprotein phosphatase; 2) phosphatase inhibitors enhance cAMP synthesis through their actions at the receptor level; and 3) the cAMP signal is more sensitive to the regulation by phosphorylation than cGMP in rat pinealocytes. PMID- 7534992 TI - Loss of sensitivity to cholecystokinin stimulation of isolated pancreatic acini from genetically diabetic rats. AB - Pancreatic exocrine function of a new inbred strain Otsuka Long-Evans Tokushima Fatty (OLETF) rat that develops spontaneous persistent hyperglycemia was evaluated in in vitro isolated pancreatic acini and compared with that in the control Long-Evans Tokushima Otsuka (LETO) rat. Serum glucose and insulin concentrations in the OLETF rats were significantly high (glucose: 270 +/- 12 vs. 208 +/- 10 mg/100 ml, P < 0.01; insulin: 12.4 +/- 1.7 vs. 4.9 +/- 0.6 ng/ml, P) < 0.01), whereas pancreatic wet weight was significantly low (803 +/- 20 vs. 1,138 +/- 17 mg, P < 0.01) compared with those in the LETO rat. Pancreatic acini isolated from the OLETF rat were totally insensitive to cholecystokinin (CCK)-8 stimulation at concentrations of up to 100 nM. However, neither the responsiveness nor the sensitivity to carbamylcholine, bombesin, and secretin of the acini from the OLETF rat was altered or even increased, probably due to the larger amylase content in the OLETF rat acini compared with those of the LETO rat acini (31.5 +/- 2.0 vs. 13.0 +/- 1.1 Somogyi units/micrograms DNA, P < 0.01). The responsiveness to fluoride, a direct activator of guanine nucleotide-binding protein, in the OLETF rat acini was similar to that in the LETO rat, suggesting that the transmembrane signaling and effectors and subsequent intracellular signal transduction molecules in the OLETF rat acini are normal. Moreover, 125I CCK binding to the acini prepared from the OLETF rat was totally absent. These present results indicate that the OLETF rat has a selective defect in the binding of CCK to its receptors on the acinar cell surface. PMID- 7534991 TI - Amrinone prevents muscle protein wasting during chronic sepsis. AB - The time course for the effects of sepsis on rates of protein synthesis, RNA contents, and translational efficiencies was measured in mixed muscles of rat hindlimb perfused in vitro 3, 5, and 10 days after induction of sepsis. Furthermore, the effect of daily injections of amrinone (5 mg.kg-1.day-1) on muscle protein synthesis was investigated. On day 3 of sepsis, decreased rates of protein synthesis in muscle from untreated septic animals or septic rats treated with amrinone resulted from a reduced food intake. When food intake became normalized to control after 5 days, rates of protein synthesis in untreated septic rats remained depressed. Treatment of septic animals with amrinone for 5 days prevented the sepsis-induced inhibition of protein synthesis by abolishing the inhibition of peptide-chain initiation and restoring translational efficiency to control values. In contrast, amrinone treatment of control rats for 5 days did not cause an accretion of muscle protein or augment protein synthesis. Ten days after induction of sepsis, there were no differences in rates of protein synthesis, RNA content, or translational efficiency in septic animals compared with control or amrinone-treated septic rats. Thus, amrinone prevented the sepsis induced abnormalities in skeletal muscle protein synthesis. PMID- 7534993 TI - Expression of prolactin and growth hormone receptor genes and their isoforms in the gastrointestinal tract. AB - Distribution of transcripts for prolactin and growth hormone receptors (PRLR and GHR) and their isoforms was characterized in the gastrointestinal (GI) tract from several species by reverse transcription-polymerase chain reaction combined with Southern analysis. Human, rabbit, and fetal and adult rat PRLR and GHR transcripts were detected in isolated gastric glands, gastric cell fractions, and intestinal mucosa lineages. Human PRLR and GHR transcripts were also observed throughout the cancerous progression of the colonic and gastric mucosa from adenomas to colonic liver metastasis and gastrointestinal cancer cell lines at various stages of growth and differentiation. Prolactin (PRL) produced no detectable effect on M1 gastric mucin secretion in HT-29 cells adapted to methotrexate (HT-29-MTX) or on acid secretion in isolated rabbit parietal cells. GHRd3, an isoform of human GHR transcript missing exon 3, was also broadly expressed and was the only form found in gastric and colorectal adenocarcinomas. Interestingly, several extra bands of polymerase chain reaction products of the human PRLR, which were smaller than the expected size, were observed not only in the GI tract but also in liver and T-47D breast cancer cells. These products from human intestinal and breast cancer cell lines were subsequently subcloned and sequenced, and we isolated six isoforms of the receptor transcripts. One of these clones encodes a putative human PRL binding protein. The expression of PRL and PRLR transcripts was also clearly observed in intraepithelial lymphocytes purified from the mouse intestine. The widespread expression of the PRL and GH receptor transcripts in gastric and intestinal mucosal lineages, particularly in epithelia, suggests regulatory roles of these hormones on digestive and immune functions, including metabolism, growth, or differentiation. PMID- 7534994 TI - Cholinergic and VIP-ergic pathways mediate histamine H2 receptor-induced cyclical secretion in the guinea pig colon. AB - Previous studies demonstrated neurally mediated recurrent increases in short circuit current (Isc) suggestive of anion secretion in guinea pig distal colon. To determine the neural pathways involved, segments of distal colon from guinea pigs were mounted in flux chambers. In muscle-stripped or whole thickness preparations, serosal addition of the histamine H2 receptor agonist, dimaprit, caused cyclical increases in Isc, which were reduced by the chloride channel blocker, N-phenylanthranilic acid, but not by the sodium channel blocker amiloride. Dimaprit stimulated release of [3H]acetylcholine and vasoactive intestinal polypeptide (VIP) from submucosal/mucosal sheets. Dimaprit caused recurrent increases in Isc, which were significantly decreased by mecamylamine, a nicotinic receptor antagonist, and nearly abolished by the muscarinic antagonist, atropine (M3 > M1 = M2). The muscarinic antagonist, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP, M3 > M1), was more potent than pirenzepine (M1 > M3) in reducing recurrent increases in Isc. Dimaprit- and electrically evoked secretion were inhibited by the VIP antagonists [4Cl-D-Phe6, Leu17]VIP and VIP hybrid. The results suggest the involvement of VIP-ergic and cholinergic neurons utilizing nicotinic and muscarinic synapses in mediating secretion. PMID- 7534995 TI - Ion transport across the jejunum in normal and cystic fibrosis mice. AB - Cystic fibrosis (CF) mice created by targeted disruption of the murine cystic fibrosis transmembrane conductance regulator gene lack adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and exhibit marked intestinal complications secondary to inadequate fluid secretion. The basal short-circuit current (Isc) in the normal murine jejuna [43.2 +/- 5.9 microA.cm-2, n = 10 (mean +/- SE)] exhibits marked spontaneous n = 10 (mean +/- SE)] exhibits marked spontaneous oscillations (amplitude = 47.9 microA.cm-2, n = 18), which were completely absent in the CF jejunum. Treatment of normal jejuna with the neuronal blocker tetrodotoxin completely eliminated the oscillations and decreased the Isc to levels not significantly different from the low basal Isc (5.4 +/- 2.8 microA.cm-2, n = 16) exhibited by CF tissue. Ion substitution studies revealed basal Isc in normal jejuna to be due primarily to Cl- secretion but these tissues appeared to be capable of HCO3- secretion as well. In contrast, CF jejuna spontaneously secreted neither Cl- nor HCO3-, which may indicate that CF jejuna have a defect in the ability to secrete both of these anions. Apical glucose elicited an electrogenic absorption of Na+ of identical magnitude in normal and CF jejuna. Without apical glucose, CF jejuna exhibited a very small Isc response to forskolin (delta 2.2 +/- 0.67 microA.cm-2, n = 10). However, in the presence of apical glucose, forskolin elicited an eightfold greater Isc response in the CF tissue (delta 17.2 +/- 4.8 microA.cm-2, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7534996 TI - Modulation of acetylcholine release in rabbit airways in vitro. AB - We investigated the effects of substance P (SP) and vasoactive intestinal peptide (VIP) on acetylcholine (ACh) released from nerve endings by electrical field stimulation (EFS) in rabbit airways in vitro. ACh release was directly measured using high-performance liquid chromatography with electrochemical detection. Airway smooth muscle (ASM) segments, dissected from the midtrachea down to the left mainstem bronchus, were obtained from New Zealand White rabbits and mounted in organ baths containing modified Krebs-Henseleit solution, physostigmine, and choline. EFS at 20 Hz was delivered for 15 min to define baseline ACh release (pmol per gram of tissue per minute). There were no significant regional differences in ACh release during these baseline studies. A second stimulation was then performed in the absence (control) and presence of one or more of the following substances: SP (10(-7) M), a nonpeptide antagonist of the NK1 receptor (10(-7) M CP-96,345; Pfizer), and VIP (10(-7) M). Results for ACh release are expressed as a percentage of the first stimulation (means +/- SE). SP significantly increased ACh release in all ASM segments. This effect was abolished by CP-96,345. VIP alone did not affect ACh release. However, it significantly decreased SP-induced ACh release in all ASM segments. We conclude that SP significantly increases ACh release, thus facilitating cholinergic neurotransmission; its effect is abolished by CP-96,345. VIP decreases SP-induced ACh release, indicating a modulatory effect on cholinergic neurotransmission. PMID- 7534997 TI - Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators. AB - Nitric oxide (.NO) is a short-lived mediator that can be induced by different cytokines and lipopolysaccharide (LPS) in a variety of cell types and produces many physiological and metabolic changes in target cells. In the current study, we show that a combination of cytokines, LPS, and zymosan-activated serum (ZAS; called for convenience cytomix Z) induces production of high concentrations of the NO oxidation products nitrite (NO2-) and nitrate (NO3-) by cultured rat fetal lung epithelial type II cells in a time-dependent fashion. Interferon-gamma and tumor necrosis factor-alpha alone did not significantly affect .NO synthesis, whereas ZAS, LPS, and interleukin-1 beta caused only a modest increase in formation of .NO oxidation products. Production of NO2- and NO3- was inhibited by NG-monomethyl-L-arginine and cyclohexmide. After exposure of these cells to a combination of the above cytokines, Escherichia coli LPS, and ZAS (cytomix Z), enhanced inducible nitric oxide synthase (iNOS) expression was indicated by an elevation in steady-state mRNA specific for iNOS (via Northern blot analysis) and increased immunofluorescence for iNOS after cell permeabilization, incubation with anti-iNOS antibody, and treatment with Cy3.18-conjugated rabbit-specific antibody. The extent of inflammatory mediator-induced.NO production by alveolar epithelium, which exceeds that of other lung cell types, reveals new insight into mechanisms of pulmonary host defense and pathways of free radical-mediated lung injury. PMID- 7534998 TI - In vivo treatment with endotoxin induces nitric oxide synthase in rat main pulmonary artery. AB - Our aim was to demonstrate increased NO activity from inducible NO synthase (iNOS) in pulmonary arteries (PA) from rats treated with endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip]. LPS treatment diminished the contractile response of PA to potassium chloride (KCl) and phenylephrine (PE) and increased levels of guanosine 3',5'-cyclic monophosphate (cGMP) in endothelium-denuded vessels. Both the NO synthase (NOS) antagonists NG-monomethyl-L-arginine (L-NMMA; nonselective) and aminoguanidine (selective for iNOS) enhanced PE-induced contraction in endothelium-denuded vessels from LPS-treated rats. Furthermore, L NMMA-induced contraction of endothelium-denuded vessels from LPS-treated rats was stereospecifically antagonized by L-arginine and associated with decreased cGMP levels. These data suggest that NO is produced in increased amounts from PA smooth muscle after LPS treatment. LPS treatment caused increased expression of mRNA for iNOS in PA. This effect of LPS was attenuated by pretreatment with dexamethasone, suggesting that induction of NOS in PA smooth muscle underlies the increased NO activity associated with LPS administration. PMID- 7534999 TI - Influence of protein kinase C inhibitors on vasoconstrictor responses in the pulmonary vascular bed of cat and rat. AB - The effects of staurosporine and calphostin C, two different protein kinase C (PKC) inhibitors, on pressor responses were studied in the pulmonary vascular bed of the intact chest anesthetized cat and the isolated rat lung. Under conditions of constant lobar blood flow in the cat, injections of the angiotensin peptides, norepinephrine (NE), serotonin, and U-46619 into the lobar arterial perfusion circuit caused dose-related increases in lobar arterial pressure and responses were reproducible with respect to time. Infusion of staurosporine into the perfused lobar artery at 1-2 micrograms/kg for 10 min reduced the pressor response to the angiotensin peptides and to NE; however, staurosporine did not alter pressor responses to serotonin or to the thromboxane mimic U-46619. In a separate series of experiments, the effects of calphostin C were investigated and infusion of the PKC inhibitor into the perfused lobar artery at 1-5 micrograms/kg for 10 min also reduced pressor responses to the angiotensin peptides and to NE and did not alter pressor responses to serotonin or to U-46619. In the isolated rat lung, the inhibitory effects of staurosporine on pulmonary pressor responses were investigated and injections of angiotensin II, NE, and serotonin produced dose-related increases in pulmonary arterial perfusion pressure that were decreased after administration of 20 micrograms ia of the PKC inhibitor staurosporine. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535001 TI - Critical role of endothelium in sustained arterial contraction during prolonged hypoxia. AB - Acute anoxia or severe hypoxia causes an initial transient contraction followed by marked relaxation of vascular tissues. We observed a spontaneous gradual sustained contraction of rat aortic rings following relaxation when hypoxia was prolonged. Deendothelialization as well as treatment of the endothelium-intact rings with nitric oxide synthase inhibitors or oxyhemoglobin abolished the late hypoxic contraction despite prolonged hypoxia. The prolonged hypoxia-induced sustained contraction was not affected by adenosine receptor blockade, cyclooxygenase inhibition, free radical scavengers, or the endothelin receptor antagonists. The ATP-sensitive K+ channel blocker glibenclamide abbreviated the duration of hypoxic relaxation and potentiated the magnitude of late hypoxic contraction. These data suggest that the late-sustained hypoxic contraction of arterial tissues is dependent on the presence of intact functional endothelium. Activation of ATP-sensitive K+ channels may participate in the genesis of hypoxic relaxation. However, cyclooxygenase products, free oxygen radicals, adenosine, and endothelin are not involved in the regulation of hypoxia-mediated events in rat aortic rings. PMID- 7535000 TI - Water channel expression in human ADPKD kidneys. AB - Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) results in part from the transport of solute and fluid into the lumen of the cyst. In proximal tubules and thin descending limbs of normal kidneys, the high transepithelial water permeability of these segments is due to the presence of the water channel protein, aquaporin-CHIP (AQP-CHIP, i.e., AQP-1). The collecting ducts of normal kidneys express another member of this gene family, the aquaporin collecting duct (AQP-CD, i.e., AQP-2). The expression and distribution of these two members of the aquaporin gene family were examined in ADPKD and normal human kidneys. In both tissues, Western blotting with the anti-AQP-CHIP antibody revealed a major 28-kDa band. By immunofluorescence, AQP-CHIP was present in proximal tubules and thin descending limbs of Henle of both normal and ADPKD kidneys. In the latter, AQP-CHIP was detected in the epithelia lining 71% of cysts. Many cysts were positive for the proximal tubule marker gp330 (44%). Some cysts expressing AQP-CHIP did not stain for gp330, suggesting a descending thin limb origin, and a few cysts were negative for both markers. In normal human kidney, Western blotting with the anti-AQP-CD antibody revealed a band at 28 kDa. AQP-CD was localized to collecting ducts and did not show colocalization with gp330 in normal human kidney. In ADPKD kidney, AQP-CD was expressed by only 8% of cysts. In summary, water channels, primarily AQP-CHIP, are expressed in epithelial cells lining cysts in approximately 80% of cysts in ADPKD kidneys.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535003 TI - Role of nitric oxide and cAMP in beta-adrenoceptor-induced pial artery vasodilation. AB - The present study was designed to investigate the role of nitric oxide (NO), guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3',5'-cyclic monophosphate (cAMP) in the vasodilator response to beta-adrenoceptor agonists in newborn pigs equipped with a closed cranial window. Dobutamine (10(-8) and 10(-6) M), a beta 1-agonist, produced pial artery dilation that was blunted by NG-nitro L-arginine (L-NNA; 10(-6) M), a NO synthase inhibitor (12 +/- 1 vs. 0 +/- 2% and 24 +/- 3 vs. 4 +/- 1% for 10(-8) and 10(-6) M dobutamine, respectively). Dobutamine-induced vasodilation was associated with increased cortical periarachnoid cerebrospinal fluid (CSF) cGMP, and these changes in CSF cGMP were blocked by L-NNA (391 +/- 10 and 675 +/- 36 fmol/ml vs. 307 +/- 3 and 346 +/- 37 fmol/ml for control and 10(-6) M dobutamine before and after L-NNA, respectively). In contrast, dobutamine-associated changes in CSF cAMP were unchanged by L-NNA (1,108 +/- 56 and 2,623 +/- 139 fmol/ml vs. 1,059 +/- 24 and 2,500 +/- 61 fmol/ml for control and 10(-6) M dobutamine before and after L-NNA, respectively). Salbutamol, a beta 2-agonist, and isoproterenol, a nonselective beta-agonist, elicited similar changes in pial diameter and cyclic nucleotides; vasodilation and changes in CSF cGMP also were similarly inhibited by L NNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535004 TI - Differential regulation of L-arginine transport and inducible NOS in cultured vascular smooth muscle cells. AB - Experiments were performed to characterize the uptake of L-arginine in rat aortic smooth muscle cells (SMC) and to examine whether inducers of nitric oxide synthase (NOS) could regulate the transport of L-arginine into these cells. L Arginine transport by SMC was saturable, Na+ independent, strongly inhibited in the presence of other cationic amino acids, and could be stimulated by preloading the cells with cationic amino acids. Kinetic studies revealed the presence of both a high [Michaelis constant (Km) approximately 125 microM] and low (Km approximately 1.4 mM) affinity L-arginine transporter. Treatment of vascular SMC with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) resulted in parallel increases in L-arginine transport and nitric oxide (NO) synthesis, as measured by nitrite production. Increasing the concentration of IL 1 beta and TNF-alpha caused a progressive elevation in nitrite production but did not further stimulate L-arginine uptake. Treatment of SMC with the combination of TNF-alpha and interferon-gamma (IFN-gamma) synergistically enhanced nitrite release without having any additional effect on L-arginine transport. Both induction of L-arginine transport and NOS activity by these cytokines were blocked by cycloheximide. Finally, treatment of SMC with interferon-gamma and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate selectively stimulated the formation of nitrite by SMC but had no effect on L-arginine transport. These results demonstrate that L-arginine transport by cultured vascular SMC is mediated by the system y+ carrier and that inducers of NOS differentially regulate the activity of this transporter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535005 TI - A model study of changes in excitability of ventricular muscle cells: inhibition, facilitation, and hysteresis. AB - A model study was carried out to investigate the mechanism of changes in excitability at long cycle lengths (i.e., > 1,000 ms), which are responsible for various phenomena, including electrotonic inhibition, active facilitation, and hysteresis of excitability in ventricular muscle at slow frequencies of stimulation. Experimental studies suggested that with repetitive activity the inward rectifier potassium current (IK1) is not a passive component of membrane response and that the dynamics of IK1 are responsible for the changes in excitability at long cycle lengths. In the present study, we have used new experimental data as the basis to modify the equations for IK1 in the ionic model for ventricular muscle of the Luo and Rudy (LR) model. The modified equations for IK1 incorporate an additional slow gate (s-gate), which governs the transition from a high steady-state conductance at rest to a lower conductance with repetitive stimulation. In simulation studies, electronic inhibition was seen in the original and the modified LR model and was shown to depend on changes in the delayed rectifier current (IK). However, addition of the s-gate to IK1 of the LR model extended the frequency dependence of excitability to longer cycle lengths and allowed for the demonstration of active facilitation and hysteresis. These results support the hypothesis that the inward rectifier is involved in the dynamic control of membrane excitability. The overall results provide mechanistic explanations for heart rate-dependent excitation abnormalities that may be involved in the genesis of cardiac arrhythmias. PMID- 7535002 TI - Loss of adenosine-induced negative inotropic effect in hyperexcited rabbit hearts: relationship to PKC. AB - Adenosine produced a negative inotropic effect in hearts isolated from calm rabbits but not from those exhibiting alarm behavior during handling. This study was conducted to determine whether protein kinase C (PKC) activation is responsible for the loss of adenosine-induced negative inotropism in the hearts of hyperexcited rabbits. Adenosine (10 microM) decreased myocardial contractility (dP/dtmax) in the hearts of calm, but not hyperexcited, rabbits but decreased heart rate (HR) and coronary perfusion pressure (PP) in the hearts of both calm and hyperexcited animals. During infusion of calphostin C (200 nM), a PKC inhibitor, adenosine also decreased dP/dtmax in the hearts of hyperexcited rabbits. Calphostin C did not alter the actions of adenosine in the hearts of calm rabbits. Agents that stimulate PKC directly [phorbol 12,13-dibutyrate (PDBu), 1 nM] or indirectly [norepinephrine (NE), 3 nM; angiotensin II (ANG II), 5 nM] abolished the adenosine-induced decrease in dP/dtmax but not HR or PP in the hearts of calm rabbits. During calphostin C, infusion of PDBu, NE, and ANG II failed to prevent the adenosine-induced decrease in dP/dtmax. These data suggest that the lack of a negative inotropic effect of adenosine in hyperexcited rabbits is due to an increase in PKC activity. PMID- 7535006 TI - Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma. AB - There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs. In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines. To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA. By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC. Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells. iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535007 TI - Acetylcholine activates a glibenclamide-sensitive K+ current in cat atrial myocytes. AB - The preceding paper [Y. G. Wang and S. L. Lipsius, Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1313-H1321, 1995.] showed that when an atrial myocyte is treated with two consecutive exposures to acetylcholine (ACh) separated by a recovery interval, the second ACh exposure elicits a larger increase in K+ conductance than the first ACh exposure. In the present study a nystatin perforated patch whole cell recording method was used to determine the mechanisms underlying the potentiating effect of ACh on ACh-induced K+ currents and the nature of the potentiated K+ current. The K+ current potentiated by the second ACh exposure was selectively abolished by 1) M1 muscarinic receptor block by 0.1 microM pirenzepine, 2) depletion of sarcoplasmic reticulum (SR) Ca2+ stores by 1 microM ryanodine or 10 mM caffeine, 3) intracellular dialysis with 10 mM ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 4) omitting external Ca2+, 5) 50% external Na+, 6) inhibition of protein kinase C by 0.01 microM staurosporine or 0.1 microM calphostin C, or 7) inhibition of ATP sensitive K+ channels with 10 microM glibenclamide (Glib). AFDX-116 (100 microM), an M2 muscarinic receptor antagonist, selectively abolished the conventional ACh activated K+ current and revealed an ACh-activated Glib-sensitive K+ current. In addition, with K+ conductances blocked and zero external Ca2+, 10 microM ACh induced a small nonselective inward current carried by Na+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535008 TI - Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation. AB - Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during simulated extracorporeal circulation. After 60 min of recirculation in the presence of the slow tight-binding boronic acid inhibitor, Bz-Pro-Phe-boroArg-OH (10.7 microM), complete inhibition of kallikrein-C1-inhibitor complex formation and marked inhibition of C1-C1-inhibitor complex formation and the release of human neutrophil elastase were observed. Arg15-aprotinin (3.1 microM), Ala357,Arg358 alpha 1-antitrypsin (2.6 microM), and soybean trypsin inhibitor (48.0 microM) either completely or partially inhibited the generation of kallikrein-C1-inhibitor complexes but were less effective inhibitors of human neutrophil elastase release. The second-order rate constants for the inhibition of kallikrein in purified systems are consistent with the order of effectiveness of the inhibitors in blocking human neutrophil elastase release in heparinized blood. Our results suggest that low-molecular-weight selective inhibitors of kallikrein may be effective agents in the attenuation of the contact-mediated inflammatory response in cardiopulmonary bypass. PMID- 7535009 TI - Release of EDRF and NO in ex vivo perfused aorta: inhibition by in vivo E. coli endotoxemia. AB - Previous studies have yielded contradictory results about interrelations between endotoxin and endothelium-derived relaxing factor (EDRF). We tested the hypothesis that in vivo endotoxemia inhibits basal and/or agonist-mediated release of EDRF and nitric oxide (NO). EDRF bioactivity, NO production, and NO synthase (NOS) activity were measured in aorta from guinea pigs following 16 h of Escherichia coli endotoxemia (4 mg/kg endotoxin i.p.). Endothelium-dependent relaxation of aortic rings was studied under standard isometric conditions. Endotoxemia resulted in an 89% reduction in basal EDRF bioactivity and a 62% reduction in basal NO production in perfused aorta. EDRF bioactivity and NO production in response to the receptor-dependent agonists acetylcholine and ADP were significantly reduced in perfused aorta from endotoxemic animals. In contrast, endotoxin did not significantly inhibit EDRF bioactivity and NO production by the receptor-independent agonist A-23187. Aortic rings from endotoxemic animals likewise showed decreased vasodilator responses to acetylcholine and ADP but not to A-23187. Inducible (Ca2+ independent) NOS activity was not significantly different in control and endotoxin-treated animals. These findings indicate that prolonged endotoxemia resulted in diminution of release of EDRF, consistent with the interpretation that endotoxemia decreases basal and agonist-stimulated EDRF bioactivity and NO production with loss of endothelium-dependent vasodilator reserves during gram negative sepsis. PMID- 7535010 TI - Substance P-induced calcium entry in endothelial cells is secondary to depletion of intracellular stores. AB - Substance P (SP) induces an elevation in cytosolic Ca2+ concentration ([Ca2+]i) in porcine coronary artery endothelial cells by way of Ca2+ influx and release from intracellular stores. We tested the hypothesis that SP-induced Ca2+ influx occurs due to activation of a Ca(2+)-permeable influx pathway coupled to depletion of intracellular stores. With the use of the perforated patch technique and fura 2 microfluorimetry, a fivefold greater increase in [Ca2+]i per unit decrease in membrane potential was obtained in the presence of SP (10 nM) compared with resting state, implying that SP increased Ca2+ conductance. When K+ channels were blocked, SP activated a net inward current with a reversal potential (2.5 +/- 1 mV) not significantly different from that (2 +/- 1 mV) for inward current recorded in response to store depletion by (2,5-di-tert butylhydroquinone) (BHQ, 10 microM). Increasing bath [Ca2+] induced a similar shift in reversal potential for SP- and BHQ-induced currents. Inositol 1,4,5 trisphosphate (20 microM), applied through the patch pipette, activated an inward current with Ca2+ selectivity similar to SP- and BHQ-activated currents. Dialysis of cells with heparin (5 mg/ml) completely blocked SP-induced inward current but not BHQ-induced current. These results suggest that the SP-induced increase in Ca2+ conductance can be completely explained by activation of a Ca(2+)-permeable influx pathway coupled to depletion of intracellular stores. PMID- 7535011 TI - Interaction of the renal nerves and prostaglandins on the phosphaturic response to PTH in phosphate-deprived rats. AB - Previous studies demonstrated that catecholamines modulate the phosphaturic response to parathyroid hormone (PTH) in normal rats. The present study was performed to determine the effect of unilateral renal denervation (DNX) and the interaction with prostaglandin synthesis on the blunted phosphaturic response to PTH in phosphate-deprived rats. One week before the acute experiment, rats were anesthetized, and the left kidney was denervated or sham surgery was performed. Rats were fed either a low-phosphate diet (LPD, 0.07%) or a normal-phosphate diet (NPD, 0.7%) for 2 days before the experiment. All rats were thyroparathyroidectomized (TPTX). Control clearances were taken from the left kidney 2 h after TPTX. PTH (33 U/kg + 1 U.kg-1.min-1) was infused for 45 min, and then the urine collections were repeated. In phosphate-deprived rats with an innervated kidney, PTH infusion resulted in a blunted phosphaturic response [changed fractional excretion of phosphate (delta FEPi) of 9.2 +/- 3.7%, n = 9] compared with rats fed NPD (delta FEPi 45.7 +/- 9.3%, n = 6) or those in the phosphate-deprived group with renal DNX (delta FEPi 23.6 +/- 5.0%, n = 12). Indomethacin pretreatment (3 mg/kg) markedly attenuated the phosphaturic response to PTH in phosphate-deprived rats with a denervated kidney (delta FEPi, 3.2 +/- 1.3%, n = 7) but not in animals fed an LPD with innervated kidneys or in rats fed an NPD. Infusion of Iloprost (2 ng.kg-1.min-1), a stable prostaglandin I2 analogue, in indomethacin-treated phosphate-deprived rats enhanced the phosphaturic response to PTH in rats with a denervated kidney (delta FEPi 17.3 +/ 3.5%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535012 TI - Neurochemical interactions in the parabrachial nucleus mediating visceral inputs to visceral thalamic neurons. AB - Previously we demonstrated that glutamatergic and noradrenergic receptors mediate the relay of visceral information through the parabrachial nucleus (PBN) and that calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), neurotensin (NT), and cholecystokinin (CCK) may modulate these responses. The interactions of these neurotransmitters and neuropeptides were examined in male Wistar rats (17) that were anesthetized with chloral hydrate and ventilated and in which blood pressure and heart rate were continuously monitored. The left cervical vagus nerve was stimulated at submaximal current intensities to elicit changes in single and multiunit activity of visceral thalamic neurons (VTNs). Peristimulus-time and continuous-time histograms of VTN activity were made before and after 200-nl injections of peptides, neurotransmitter agonists or antagonists, or artificial cerebrospinal fluid into the PBN. Combined injection of CGRP and SP into the PBN produced a synergistic inhibition of spontaneous VTN activity and the vagally evoked VTN response. Combined injection of NT and phenylephrine (PE) into the PBN produced only an additive increase in the spontaneous activity of VTNs. Prior administration of SOM in the PBN blocked the excitatory action of an alpha-adrenergic agonist (phenylephrine) injection on the spontaneous activity of VTNs, whereas CGRP, SP, or CCK had no effect on the alpha agonist-induced response. Prior injection of an alpha-adrenergic antagonist (phentolamine) prevented the excitatory effect of NT in the PBN. Injection of CGRP, SP, NT, or CCK into the PBN did not change the response of VTNs to application of glutamate. These results suggest mechanisms for peptide interaction with primary neurotransmitters in the PBN and indicate whether the neuropeptides are acting before the primary neurotransmitter synapse or postsynaptically. PMID- 7535014 TI - Monoclonal antibodies to Pseudomonas pseudomallei and their potential for diagnosis of melioidosis. AB - Monoclonal antibodies (MAbs) specific for Pseudomonas pseudomallei antigens were produced by immunizing BALB/c mice with a crude whole cell extract. Hybrids secreting MAbs specific for P. pseudomallei antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of crude whole cell extracts from P. pseudomallei, P. cepacia, P. aeruginosa, P. putida, P. alcaligenes, Xanthomonas maltophilia, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Salmonella typhi, S. krefeld, S. enteritidis, Proteus mirabilis, and Staphylococcus aureus. Of the six specific clones, clone 5F8, which was IgM-producing and which reacted with all 56 P. pseudomallei isolates, was selected for further characterization and evaluation of its possible diagnostic potential. Results obtained from the indirect ELISA against various P. pseudomallei antigens, from direct bacterial agglutination, and from immunofluorescence tests suggested that 5F8 reacted with the surface envelope, and probably specifically with an epitope of the lipopolysaccharide. The antibody could be readily used to identify P. pseudomallei in primary culture or in a simulated hemoculture. The antibody was also used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in patients with acute septicemic melioidosis. PMID- 7535013 TI - Nitric oxide responses of air-breathing and water-breathing fish. AB - Nitric oxide (NO), exogenously administered or endogenously produced by NO synthase (NOS), is an important regulator of lung ventilation and perfusion in mammals. This study attempts to investigate the evolutionary history of this system in fish and its possible relationship to air breathing. The gas bladder of Hoplerythrinus unitaeniatus (air-breathing teleost) and Oncorhynchus mykiss (non air-breathing teleost) and the lung of Lepidosiren paradoxa (air-breathing dipnoan) all exhibited elevated guanosine 3',5'-cyclic monophosphate (cGMP) levels in response to 1 microM sodium nitroprusside. Only the H. unitaeniatus gas bladder responded to 10 microM acetylcholine chloride (ACh) with increased cGMP levels. The ACh response was inhibited by N omega-nitro-L-arginine methyl ester, which inhibits NOS. These data suggest that although tissues from each species may respond to exogenous NO, only the gas bladder of H. unitaeniatus appears to synthesize NO through NOS. This is the first report of constitutive NOS outside of the central nervous system in a teleost. These results also imply that NOS did not necessarily coevolve with air breathing in fish. PMID- 7535015 TI - Lack of correlation between HLA class II alleles and immune responses to Pf155/ring-infected erythrocyte surface antigen (RESA) from Plasmodium falciparum in Madagascar. AB - To investigate the relationships between predominant HLA class II alleles and immune responses to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), 50 individuals from the highlands of Madagascar were followed-up from 1988 to 1991. The T cell reactivity and antibody responses to synthetic peptides (EENV)4, (EENVEHDA)4, and (DDEHVEEPTVA)3, representing major T and B epitopes of Pf155/RESA antigen, were assessed with an average of five determinations per individual over the four-year follow-up period. The T cell reactivity was investigated by lymphocyte proliferation and assays for interferon gamma and interleukin-2 release. Antipeptide antibodies were measured using the Falcon assay screening test-enzyme-linked immunosorbent assay. The cumulative prevalence rates of cellular (range for the three peptides = 64-68%) and antibody responders (range = 70-74%) were similar for each peptide. The HLA class II typing was performed using polymerase chain reaction-restriction fragment length polymorphisms. The prevalent alleles or groups of alleles (frequency > 20%) were similar in responders and nonresponders, both for cellular and antibody responses to each peptide. These were HLA-DR 5 group and HLA-DQA1 *0601, *0101-0102-0104, HLA-DQB1 *0301, and HLA-DPB1 *0101-2601 alleles. Allelic distribution was similar in individuals presenting with (74%) or without (26%) a malaria attack during a 20-week follow-up conducted when malaria was hyperendemic (P > 0.05, by Fisher's exact test). Despite repeated immunologic measures that better identify the responders, no relationship was found between HLA class II alleles and the cellular or antibody responses to Pf155/RESA epitopes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535016 TI - Surgical treatment of carcinoma of the proximal esophagus. AB - BACKGROUND: Proximal esophageal cancer is usually diagnosed at an advanced stage, and the treatment is often limited to palliation. Surgery offers the best relief of dysphagia but it remains controversial, because a cure is unlikely even at the price of laryngeal mutilation. PATIENTS AND METHODS: We treated 40 patients with transhiatal esophagectomy for cancer of the proximal esophagus. The esophageal substitute was a stomach tube in 37 patients and colon in 3 patients. The larynx was preserved in 27 patients whose tumors did not extend to cricopharyngeus. Adjuvant treatment consisted of postoperative radiotherapy for 22 patients, chemotherapy for 1 patient, and a multimodality regimen for 4 patients. RESULTS: The postoperative complication and hospital mortality rates with gastric tube transpositions were 22% and 8%, respectively, with a 3% fistula rate. The 1- and 3-year overall survival rate was 53% and 21%, respectively. The unfavorable prognostic factors were tumor wall penetration, lymph nodal involvement, and cricopharyngeal involvement. Local recurrence of cancer was the major cause of failure. CONCLUSIONS: These results indicate that transhiatal esophagectomy with gastric tube transposition offers good palliation of dysphagia with low morbidity and mortality for proximal esophageal cancer. The laryngeal preservation can be attempted for tumors located close to, but not involving, the cricopharyngeus in order to retain speech in patients with a limited life expectancy. PMID- 7535017 TI - Bile and pancreatic juice replacement ameliorates early ligation-induced acute pancreatitis in rats. AB - BACKGROUND: In healthy rats, combined bile and pancreatic juice diversion from gut has a synergistic rather than additive effect on stimulation of exocrine pancreatic protein secretion. We hypothesized that exclusion of combined bile and pancreatic juice from gut exacerbates bile and pancreatic-duct ligation-induced acute pancreatitis in rats to a greater extent than exclusion of either bile or pancreatic juice alone. METHODS: Bile and pancreatic juice (obtained fresh from donor rats) were replaced, separately or together, via a duodenal fistula beginning immediately before 6 hours of duct ligation. Pancreatic morphologic changes were evaluated with an acute pancreatitis histology score and morphometric quantitation of acinar-cell necrosis. Plasma amylase and cholecystokinin concentrations and pancreatic subcellular distribution of cathepsin B activity were determined. Characteristics of bile and pancreatic juice obtained from donor rats were also studied. RESULTS: Combined bile and pancreatic juice replacement limited the increase in acute pancreatitis histology score by 77%, acinar cell necrosis by 95%, hyperamylasemia by 77%, and hypercholecystokininemia by 99%, while preventing subcellular redistribution of cathepsin B. Amelioration of pancreatic morphologic changes was significantly greater with combined bile and pancreatic juice replacement than with replacement of either bile or pancreatic juice alone. CONCLUSION: In this experimental corollary of early gallstone-induced acute pancreatitis, combined bile and pancreatic juice exclusion from gut contributes to disease pathogenesis to a greater extent than exclusion of either bile or pancreatic juice alone. PMID- 7535020 TI - Quantification of a major bovine allergen by a two-site immunometric assay based on monoclonal antibodies. AB - A two-site immunometric assay based on monoclonal antibodies (mAb) was developed for the measurement of a 20-kDa major allergen of cow. The sensitivity of this assay was (BDA20) 1 ng/ml. It was used to measure airborne allergen concentrations in 10 Finnish cowsheds. The mean concentration of the BDA20 measured at two stationary sites was 280 ng/m3. Concentrations varied more than 10-fold among cowsheds (54-804 ng/m3). The mean intertest coefficient of variation was 8.2%, and the mean intratest variation 4.1%. PMID- 7535018 TI - Immunolocalization of extracellular matrix components in mixed tumors of the skin. AB - Mixed tumors of the skin are characterized by a proliferation of epithelial cells embedded in a mesenchymal matrix with a wide spectrum of histologic appearances. The characterization of the extracellular matrix components of mixed tumors of the skin has so far received little attention. We performed an immunohistochemical study of type IV collagen, laminin, fibronectin, and tenascin distribution in a series of 10 mixed tumors of the skin. Laminin localized at the basement membrane around solid epithelial nests and tubulo-alveolar structures, whereas type IV collagen, fibronectin, and tenascin were also expressed in the myxoid stroma. Tenascin and fibronectin localized in the chondroid matrix. The extracellular matrix components were prominently expressed in mixed tumors of the skin, suggesting that they could play an important role in the formation and organization of myxoid and chondroid matrices and in the epithelial-mesenchymal interactions of these tumors. PMID- 7535019 TI - Type II collagen in mixed tumors of the skin. An immunohistochemical study and new speculations concerning histogenesis. AB - We studied 11 cases of mixed tumor of the skin with a polyclonal antibody against human type II collagen. Two of the cases were of the eccrine type, six were apocine, and three were malignant. In all of the benign cases of mixed tumor and in some of the malignant type, "chondroid" and mucinous portions stained. The clearly epithelial portions of the tumor stained as well, and staining was most intense in the eccrine types and least intense in the malignant types. The positive staining in mixed tumors may help elucidate the histogenesis of these unusual tumors as well as help differentiate among the various subtypes. PMID- 7535021 TI - Identification of protein kinases that modify specific epitopes. PMID- 7535022 TI - A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). AB - The isolation of biologically active RNA from cotton (Gossypium hirsutum L.) is difficult due to interference by high levels of endogenous phenolics, polysaccharides, and secondary metabolites. A modified hot borate procedure was developed to combat these cellular constituents during tissue homogenization, resulting in the quantitative recovery of RNA suitable for hybridization analysis, in vitro translation, and cDNA synthesis. The efficacy of several hot borate buffer adjuvants for the qualitative and quantitative recovery of leaf RNA was monitored by absorbance spectra, gel electrophoresis, protein, and cDNA synthesis. Of the buffer adjuvants evaluated, polyvinylpyrrolidone-40 (PVP-40) exhibited the single, most significant impact on the yield and quality of RNA isolated from cotton leaves, although inclusion of deoxycholate and/or Nonident 40 (NP-40) further enhanced the quality of the RNA. The unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot borate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other recalcitrant plant species as well as for especially difficult tissue types. PMID- 7535023 TI - Investigation of pseudorabies virus DNA and RNA in trigeminal ganglia and tonsil tissues of latently infected swine. AB - Pseudorabies virus (PRV) nucleic acids in the trigeminal ganglia and tonsils of swine latently infected with the virus were analyzed. By use of DNA-polymerase chain reaction (PCR), 14 of 14 trigeminal ganglia and 12 of 14 tonsils were positive for PRV genomes. By use of RNA-PCR, RNA containing the large latency transcript splice junction were detected in 4 of 4 trigeminal ganglia and 4 of 5 tonsils. In general, results of both PCR procedures indicated that the amounts of PRV DNA and RNA per microgram of cellular nucleic acids were higher in trigeminal ganglia than in tonsils. Identification of peripheral tissues that harbor latent PRV is an important asset for PRV research. The presence of large latency transcript in tonsil tissues, in the absence of virus replication, is a critical characteristic, which indicates that the tonsil is a site of PRV latency. For diagnostic purposes, animals need not be euthanatized to obtain their nervous tissue to determine latency; instead, tonsil biopsy specimens could be obtained from live animals for analysis. For pathogenesis, studies, multiple specimens obtained sequentially from the same animal would be available for examination for the duration of the experiment. PMID- 7535024 TI - Endothelial heterogeneity and intimal blood-borne cells. Relation to human atherosclerosis. PMID- 7535025 TI - Vascular endothelium. An integrator of pathophysiological stimuli in atherogenesis. PMID- 7535026 TI - Down-regulation of vascular adhesion molecule-1 by fluid shear stress in cultured mouse endothelial cells. AB - This study was undertaken to determine whether blood flow modulates the adhesive property of vascular endothelial cells to lymphocytes and, if it does, what adhesion molecules are involved. Cultured mouse endothelial cells were exposed to medium flow in a parallel plate chamber, and binding assay using fluorescence labeled lymphocytes was carried out. The adhesion rate of endothelial cells to lymphocytes, which was high in the static control state, decreased when exposed to shear stress (1.5 dynes/cm2) for 6 h. The treatment of static endothelial cells with a monoclonal antibody of vascular cell adhesion molecule-1 (VCAM-1) depressed the adhesion rate to the same extent as that caused by flow, while monoclonal antibodies of CD44 and intercellular adhesion molecule-1 had no effect on it. Flow cytometric analysis revealed that the application of flow decreased markedly the amount of VCAM-1 expressed on the cell surface. A reverse transcriptase-polymerase chain reaction of mRNA showed that flow depressed VCAM-1 mRNA levels. These results suggest that blood flow can modulate the adhesive property of endothelial cells to lymphocytes via affecting the surface expression of adhesion molecules, e.g., down-regulation of VCAM-1. PMID- 7535027 TI - Abnormalities in the fibrinolytic system of the vascular wall associated with atherosclerosis. AB - In summary, studies of the expression of fibrinolytic genes in the vessel wall suggest an active, ongoing proteolytic process, the activity of which is dependent on the relative amounts of tPA, uPA, and PAI-1 secreted and locally deposited. Disturbances in the balance of pro- and antifibrinolytic activity in atherosclerotic vessels have considerable potential for influencing both intra- and extravascular fibrinolytic events and may be causally related to the development of vascular disease. PMID- 7535028 TI - Fish oil, atherogenesis, and thrombogenesis. AB - Marine fish consumption is known to reduce mortality from ischemic heart disease. The use of fish oil as a dietary supplement, however, is not universally recommended. In large doses, fish oil reduces plasma cholesterol and triacylglycerol but increases low density lipoprotein (LDL) levels and the potential for free radical generation and bleeding. Moderate marine fish consumption is known to reduce mortality without altering commonly measured variables, i.e., plasma cholesterol levels, in vitro platelet aggregation, and bleeding times. In swine, we observed that monocyte adhesions and platelet clumps over the lesion surface of proximal left anterior descending (LAD) coronary arteries are markedly reduced when an atherogenic diet was supplemented with cod liver oil, even when the cholesterol levels were equalized with the untreated group. These findings suggest that fish oil is hypothrombogenic. We developed an in vitro assay to delineate the mechanism whereby fish oil reduced monocyte endothelial cell interactions in vivo. The effects of supplementing the culture medium with different fatty acids on adhesions between lipopolysaccharide (LPS) stimulated swine aortic endothelial cells (SAEC) and the human monocyte-like cell line, U937, was investigated in a 10 minute adhesion assay at 37 degrees C. Exposure of SAEC for 6 hours to media containing 50-200 microMs eicosapentaenoic (EPA), stearic, oleic, linoleic, and arachidonic acid, respectively, revealed that only EPA reduced U937-SAEC adhesion. Exposure of U937 to EPA also reduced adhesions. EPA was not effective when added to the SAEC more than 2 hours after they were stimulated with LPS. Exposure of human umbilical vein endothelial cells (HUVEC) to EPA reduced the expression of VCAM-1, ELAM-1, and ICAM-1 after 5 hours of stimulation with LPS. These results suggest that EPA may functionally impair the induction/expression of adhesion molecules. PMID- 7535029 TI - Endothelial cell adhesion molecules. Immunohistological examination on acetone fixed and paraffin-embedded human aortas. PMID- 7535030 TI - The 66, 56, 50, and 47 kDa vitronectins in atherosclerotic lesions and in serum. PMID- 7535031 TI - Activation of smooth muscle and endothelial cells following balloon injury. PMID- 7535032 TI - Activation and inhibition of nitric oxide synthase from cultured bovine aortic endothelial cells by phospholipids and arachidonic acid. PMID- 7535033 TI - Cyclic AMP elevating agents synergize with inflammatory cytokines to induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. PMID- 7535034 TI - Episodic ataxias as channelopathies. PMID- 7535035 TI - Experimental pneumococcal meningitis: cerebrovascular alterations, brain edema, and meningeal inflammation are linked to the production of nitric oxide. AB - We investigated whether treatment with the nitric oxide synthase inhibitor N nitro-L-arginine (L-NA) and the free radical scavenger superoxide dismutase influences cerebral blood flow changes, brain edema, and cerebrospinal fluid pleocytosis in early experimental pneumococcal meningitis. Compared to untreated infected rats, superoxide dismutase given 3 hours after infection significantly attenuated the increase of brain water content, intracranial pressure, and cerebrospinal fluid white blood cell count, but did not modulate the increase in regional cerebral blood flow. N-Nitro-L-arginine treatment (5 mg/kg intravenously, followed by 5 mg/kg/hour) reversed the increase in regional cerebral blood flow; prevented an increase in brain water content, intracranial pressure, and cerebrospinal fluid nitrite concentrations; and reduced cerebrospinal fluid white blood cell count. With a closed cranial window preparation, N-nitro-L-arginine prevented pneumococci-induced dilatation of pial arterioles. When the effective dose was increased twofold, the effects of N-nitro L-arginine became more pronounced but resulted in the death of 4 of 5 rats, probably due to hemodynamic side effects. In primary cultures of rat cerebral endothelial cells, nitrite concentrations increased after pneumococcal stimulation, which could be prevented by N-nitro-L-arginine and cycloheximide. These data suggest that (a) nitric oxide accounts for regional cerebral blood flow changes and pial arteriolar dilatation in the early phase of experimental pneumococcal meningitis; (b) both superoxide radical and nitric oxide are involved as mediators of brain edema and meningeal inflammation; and (c) cerebral endothelial cells can be stimulated by pneumococci to release nitric oxide presumably via the inducible nitric oxide synthase. PMID- 7535036 TI - Pharmacokinetics, toxicity, and efficacy of liposomal capreomycin in disseminated Mycobacterium avium beige mouse model. AB - Capreomycin was incorporated into multilamellar vesicles of pure dipalmitoylphosphatidylcholine. The pharmacokinetics and nephrotoxicity of capreomycin in the free and liposomal forms were studied in normal mice. The efficacies of the two forms were evaluated by using the Mycobacterium avium complex beige mouse model. Approximately 10(7) viable M. avium cells were injected intravenously. Seven days later, treatment with either liposomal or free capreomycin at 60 or 120 mg/kg of body weight was administered daily for 5 days. Mice were sacrificed 5 days after the end of treatment, and the viable bacteria in liver, spleen, lungs, and blood were counted. After 5 days of treatment with dosages of 60 or 120 mg/kg/day, the level of blood urea nitrogen increased in the group treated with free capreomycin but not in the group treated with liposomal capreomycin. After intravenous injection of 120 mg/kg, liposomes enhanced the diffusion of capreomycin in the spleen, lungs, and kidneys and increased the half life in serum. The 120-mg/kg dose of liposomal capreomycin significantly reduced the number of viable mycobacteria in the liver, spleen, and blood compared with those in the controls. Although these results are promising, further studies are needed to assess the efficacy of liposomal capreomycin for the treatment of M. avium complex infections. PMID- 7535037 TI - New tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione derivatives are potent inhibitors of human immunodeficiency virus type 1 replication and are synergistic with 2',3'-dideoxynucleoside analogs. AB - Tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives were shown to specifically block human immunodeficiency virus type 1 (HIV-1) replication through a unique interaction with the HIV-1 reverse transcriptase (RT). Through further modification of the lead compounds and structure-activity relationship analysis several new TIBO derivatives that show high potency, selectivity, and specificity against HIV-1 have been obtained. A new TIBO derivative, R86183, inhibits the replication of HIV-1, but not HIV-2, in a variety of CD4+ T-cell lines and peripheral blood lymphocytes, at a concentration of 0.3 to 30 nM, which is at least 4 orders of magnitude lower than the 50% cytotoxic concentration. Whereas an HIV-1 strain containing the Leu-100- >Ile mutation in the RT gene is about 400-fold less susceptible, R86183 still inhibits the replication of an HIV-1 strain containing the Tyr-181-->Cys RT mutation by 50% at a concentration of 130 nM. R86183 inhibits the poly(C).oligo(dG)12-18-directed HIV-1 RT reaction by 50% at a concentration of 57 nM. The antiviral activity of 22 TIBO derivatives in cell culture correlated well with their activity against HIV-1 RT. No such correlation was found for their cytotoxicity. The combination of R86183 with either zidovudine or didanosine resulted in a synergistic inhibition of HIV-1 (strain IIIB) replication. Combination of R86183 with the protease inhibitor Ro31-8959 was found to be additive. Also described is a dilution protocol circumventing overestimation and underestimation of antiviral activity due to adherence to plastic surfaces. PMID- 7535038 TI - Cloning, sequencing, and site-directed mutagenesis of beta-lactamase gene from Streptomyces fradiae Y59. AB - The beta-lactamase gene from Streptomyces fradiae Y59 was cloned and sequenced. To determine which amino acid residues are critical in binding activity to blue dextran, chimera beta-lactamases were constructed and their binding abilities were determined. The results suggested that blue dextran binding may depend more on overall conformation of about two-thirds of the beta-lactamase molecule from the N terminus than on the primary structure. PMID- 7535040 TI - Aprotinin improves hemostasis after cardiopulmonary bypass better than single donor platelet concentrate. AB - Platelet transfusion and aprotinin administration improve platelet function and clinical hemostasis after extracorporeal circulation. To compare two methods of improving postoperative hemostasis, we preoperatively randomized 40 patients undergoing various open heart procedures into two groups. Group A included 20 patients who, immediately after bypass, received single-donor plateletpheresis concentrates collected from ABO-compatible donors (Baxter Autopheresis-C System). They were compared with 20 patients who received high-dose aprotinin (6 x 10(6) KIU) before and during cardiopulmonary bypass (group B). Group A patients showed significantly higher platelet count after single-donor plateletpheresis concentrate transfusion (157 +/- 36 x 10(9)/L compared with 118 +/- 42 x 10(9)/L (p < 0.05). However, platelet aggregation on extracellular matrix was better in group B (3.4 +/- 0.7 versus 2.8 +/- 0.9; p < 0.05). Total 24-hour blood loss and exposure to homologous blood products were significantly less in group B (396 +/- 125 mL and 1.1 +/- 1.6 units compared with 617 +/- 233 mL and 5.4 +/- 3.4 units; p < 0.01). Despite higher platelet count in patients after single-donor plateletpheresis concentrates transfusion, hemostasis in patients receiving aprotinin is better due to improved platelet function. PMID- 7535041 TI - Aprotinin reduces blood loss in lung transplant recipients. AB - After early experience with perioperative bleeding in sequential single-lung transplant recipients, aprotinin was introduced in an attempt to reduce this complication and the attendant morbidity. Records of sequential single-lung transplantations (n = 33) performed between January 1989 and November 1991 were reviewed to assess the impact of aprotinin on perioperative blood loss and blood product requirements. Recipients were divided according to whether or not they required cardiopulmonary bypass. In patients requiring cardiopulmonary bypass (n = 15), mean estimated postoperative blood loss was 3,000 +/- 500 mL in those who did not receive aprotinin (n = 4) compared with 1,177 +/- 253 mL in those who received aprotinin (n = 11) (p < 0.05). An average of 8.0 +/- 0.7 units of packed red blood cells were administered to patients not receiving aprotinin compared with 3.1 +/- 0.7 units to those who received aprotinin (p < 0.05). Requirements for fresh frozen plasma were similar in each group. There were no differences in blood loss or blood product replacement in the group not undergoing cardiopulmonary bypass (n = 18). Therefore, we conclude that aprotinin decreases postoperative blood loss and blood product requirements in patients undergoing sequential single-lung transplantation under cardiopulmonary bypass. PMID- 7535039 TI - Macromolecular mechanisms of sputum inhibition of tobramycin activity. AB - Tobramycin, an aminoglycoside antibiotic, is used in the treatment of Pseudomonas aeruginosa infections in cystic fibrosis patients. Tobramycin bioactivity, however, is antagonized by sputum. Glycoproteins (mucins) and high-molecular weight DNA make up 2 to 3% (P. L. Masson and J. F. Heremans, p. 412-475, In M. J. Dulfano, ed., Sputum: Fundamentals and Clinical Pathology, 1973) and 3 to 10% (W. S. Chernick and G. J. Barbero, Pediatrics 24:739-745, 1959, and R. Picot, I. Das, and L. Reid, Thorax 33:235-242, 1978) of the dry weight of sputum, respectively. tobramycin binds to both mucins and DNA obtained from sputum (R. Ramphal, M. Lhermitte, M. Filliat, and P. Roussel, J. Antimicrob. Chemother. 22:483-490, 1988). In vitro, recombinant human DNase (rhDNase) hydrolyzes high-molecular weight DNA of > 50 kb within sputum to fragments of 2 to 4 kb. Studying dialyzable tobramycin, we examined drug binding to whole sputum and to "mock sputum," which consisted of porcine gastric mucin and calf thymus DNA. We also studied the effects of rhDNase treatments of sputum, mock sputum, and calf thymus DNA on tobramycin binding. We found that treatments of sputum, mock sputum, and calf thymus DNA with rhDNase did not significantly increase the tobramycin bioactivity within the dialysates; surprisingly, sputum binding of tobramycin was increased by rhDNase. We conclude that rhDNase does not increase the bioactivity of tobramycin in sputum. PMID- 7535042 TI - [An in vivo experiment on the antitumor action of bleomycin bound to an ion exchange carrier]. AB - Bleomycetin, an antitumor antibiotic of the domestic origin, is component A5 of the bleomycin complex. Design of new drug delivery systems especially for local application is an important problem of antitumor chemotherapy. Bleomycetin immobilized on ion exchange medical gauze was studied with this purpose in an in vivo experimental model of murine lympholeukemia NK/Ly. Subcutaneous implantation of the new dosage form of bleomycetin with prolonged action provided a significant antitumor systemic effect evident from an increase in the mouse life span. In vitro determination of the bleomycetin contents in the gauze revealed that the antibiotic was completely absorbed from the carrier within 72 hours. The bleomycetin half-life in the plasma after the antibiotic administration in the bound form on the ion exchange gauze was twice as long as that after the antibiotic injection with NaCl isotonic solution. PMID- 7535043 TI - [Aprotinin antiviral aerosol: study of the local irritating and allergenic action after inhalation]. AB - The aerosol of aprotinin, a natural low molecular weight polypeptide (m.w. about 160 kD) inhibiting a wide range of serine proteases may be used as an antiviral drug. The animal studies showed that it had no local irritating action on the mucosa. A long-term use of aprotinin in the form of a fine aerosol practically induced no allergenic side effects. The results of the study indicative of the absence of the allergic complications made it possible to recommend the aprotinin inhalations as a safe means in the treatment and prophylaxis of viral infections of the respiratory tract. PMID- 7535046 TI - [The location of keratins 17 and 8 in the metaplastic proliferates of the endocervix]. AB - Monoclonal antibodies to keratin 8 which is characteristic for glandular epithelium and those to keratin 17 characteristic of basal cells of complex epithelium were used. Material from 14 females after surgery because of ovarian tumours and uterine carcinoma and from 10 females with dysplasias of various degree and uterine carcinoma was investigated. In addition, ectocervix of vaginal portion at a distance from the point of two epithelia junction was studied in 42 samples. It is shown that keratins 8 and 17 are expressed in the ectocervix sporadically and have a basal location. Keratin 8 is expressed in column epithelium of the areas having normal structure of endocervix. Proliferation of subcolumn reserve cells is followed by an active expression of keratin 17. Proliferative multilayer areas with foci of the immature metaplasia, dysplasias and carcinoma in situ are characterized by cells with coexpression of both keratins, i.e. cells with double differentiation (glandular and squamous). PMID- 7535047 TI - [The Guillain-Barre syndrome]. AB - A clinicomorphological study was conducted in 11 patients with severe forms of Guillain-Barre syndrome (GBS) at different periods of the disease. Five postmortem cases of GBS were investigated. In all the cases there was a multifocal loss of myelin in the peripheral nervous system with axon degeneration of various degree. Macrophages always took part in demyelination sometimes followed by lymphocytic infiltration. Ultrastructural investigation of nerve biopsies from 6 patients with GBS showed macrophage-associated demyelination with little or no lymphocytic infiltration. It is likely that axonal damage revealed in the biopsies and at the autopsies occurs as secondary consequence of demyelination. PMID- 7535048 TI - Fluid and protein secretion by the submandibular glands of weanling rats in response to various agonists. AB - Secretion of fluid and protein by the submandibular glands of 25-day-old rats was investigated by stimulation with 22 sialogogues classified into five categories, four cholinergic, five beta 1-, seven alpha 1- and three alpha 2-adrenergic, and three peptidergic, at optimal doses. For fluid secretion, cholinergic and peptidergic agonists were the most powerful, whereas the beta 1- and alpha 1 adrenoceptor agonists were the most effective for the concentration of protein among the five categories, except for methoxamine. For total output of protein, the beta 1- and alpha 1-adrenoceptor agonists and pilocarpine were the most powerful among the 22 agonists, except for methoxamine and norephedrine. Cholinergic, peptidergic and alpha 2-adrenergic agonists among the five categories were less effective for protein secretion, except for pilocarpine. For the specific activity of esteroprotease, methoxamine and oxymetazoline, as alpha adrenoceptor agonists, were the most powerful among the 22 agonists. Thus fluid and protein secretion evoked from the submandibular glands of weanling rats in response to a wide variety of agonists are similar to those of adult rats. PMID- 7535049 TI - Patterns of fractionation for palliation of bone metastases. AB - The patterns of fractionation used to treat bone metastases in a single centre in two time periods were determined. Clinical audit was carried out, for two periods of 6 months each, in 1988 and 1993. Data recorded included patient, tumour and treatment variables. Palliation of bone metastases represented 40% and 44% of palliative treatment courses, and 20% and 21% of total treatment courses, respectively, in both time periods. Shorter treatment schedules were used in 1993 compared to 1988, with mean fraction numbers of 6.4 versus 8.5, respectively (P < 0.001). This reduction in fraction numbers occurred for the common primary tumour sites. In 1993, the mean numbers of fractions used were related to the primary tumour site (7.8 fractions for breast, 5.0 for lung, 6.7 for melanoma, 6.4 for other primary sites, P = 0.05), the treatment site (7.3 fractions for weight bearing bones, 4.0 for non-weight-bearing bones, P < 0.001) and patient address (6.0 fractions for city postcode vs 7.8 for country postcode, P = 0.02). Treating radiation oncologist (5.7-7.8 mean fractions, P = 0.06) and patient age (P = 0.56) were not significant factors for the number of fractions used. It is likely that there were multiple causes for a reduction in the number of fractions used to treat bone metastases, including the results of clinical trials and increasing pressure to optimize the use of scarce resources. However, patients who may have a better prognosis (breast primary) and those with metastases in weight-bearing bones continued to receive longer treatment schedules. PMID- 7535044 TI - [Suppression of conjunctival provocation by 0.1% pemirolast potassium ophthalmic solution in VKC]. PMID- 7535045 TI - [An electron radioautographic study of the dynamic decrease in the level of RNA synthesis in human brain cells at different times after death]. AB - The above study is performed in neurons, oligodendrocytes and capillary endotheliocytes of human brain 4-13 hours after death. Brain biopsies after the surgery and early necropsies served as the material. RNA synthesis in some neurons and glial cells is retained to some degree 8-13 hours after death. Oligodendrocytes are more and neurons less resistant. Grave disturbances of the CNS microcirculation is the obstacle to reversibility of destructive processes. PMID- 7535050 TI - Intracranial meningioma with hepatic metastases and hypoglycaemia treated by selective hepatic arterial chemo-embolization. AB - Meningiomas rarely give rise to metastases despite frequent dural invasion. A rare case of symptomatic hypoglycaemia due to extensive liver metastases from an angioblastic meningioma is described along with the use of hepatic arterial chemo embolization to effect palliation by reduction in tumour bulk. PMID- 7535051 TI - ATP-activated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II. AB - Previous studies in freshly isolated rat biliary epithelial cells and in the human cholangiocarcinoma cell line Mz-ChA-1 have demonstrated that ATP activates a calcium-dependent chloride conductance. The coupling between the rise in intracellular calcium and activation of chloride channels has not previously been investigated. In the present study, we evaluated the potential role of calmodulin dependent protein kinase II (CaMKII) in ATP-activated chloride permeability in Mz ChA-1 cells. ATP stimulated [125I] efflux, a marker for Cl- movement. Peak efflux rates were inhibited by approximately 60% in cells pretreated with the calmodulin antagonist, W-7. In whole-cell patch clamp recordings, ATP and ionomycin activated calcium-dependent Cl- currents. Pretreatment of cells with the CaMKII inhibitor KN-62 blocked activation by either agent. It is concluded that calcium dependent activation of chloride currents in Mz-ChA-1 cells is coupled to a CaMKII-dependent process. PMID- 7535053 TI - Substance P and bradykinin are natural inhibitors of CD13/aminopeptidase N. AB - Aminopeptidase N (EC 3.4.11.2) is an important enzyme that is involved in the degradation of regulatory peptides including enkephalins. We report here that purified and native membrane-bound aminopeptidase N will sequentially and completely hydrolyze both Leu-enkephalin and Met-enkephalin from the amino terminus. Both purified pig aminopeptidase N and the enzyme on live HL60 cells displayed similar Km values for enkephalin. The naturally occurring neuropeptides substance P and bradykinin, and the morphine agonist, morphiceptin, were not hydrolyzed by aminopeptidase N and each inhibited the enzymatic activity. Each of these peptides contains a proline at the second residue. The Ki values for substance P (0.44 microM), bradykinin (9.4 microM), and morphiceptin (169 microM) were obtained with the enzyme on live HL60 cells. The values for the purified enzyme from pig were similar. The potent inhibition of aminopeptidase N by substance P and bradykinin suggests that these peptides may be natural inhibitors of the enzyme. PMID- 7535052 TI - Pharmacological study of stem-cell-factor-induced mast cell histamine release with kinase inhibitors. AB - Stem cell factor (SCF) is a ligand for c-kit receptor and has a critical role in the development of mast cells. In this study, we investigated the effect of a panel of kinase inhibitors on SCF-induced histamine release from rat peritoneal mast cells. Genistein, an inhibitor of tyrosine kinases, inhibited SCF-induced histamine release with IC50 of 1.6 x 10(-5) M. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), inhibited histamine release stimulated with SCF dose-dependently with IC50 of 4 x 10(-9) M. KT5926, an inhibitor of myosin light chain (MLC) kinase, reduced histamine release with IC50 of 1.8 x 10(-7) M. Staurosporine, an inhibitor of protein kinases, also inhibited SCF-induced histamine release with IC50 of 6.5 x 10(-8) M. These results show the early involvement of tyrosine kinase and PI3 kinase and the possible role of MLC kinase in the late secretory phase in the signaling pathway used by SCF. PMID- 7535054 TI - Dual modulation of phosphorylation of a 60 kDa nuclear protein in interferon alpha treated Daudi cells. AB - To explore the modulation of phosphorylation and/or kinase activity of cellular proteins in interferon-alpha (IFN-alpha) treated Daudi cells, experiments were performed to determine the effect of IFN-alpha on the ability of cellular proteins to undergo autophosphorylation reaction in-vitro. Treatment of cells with IFN-alpha significantly inhibited the amount of phosphorylation of a nuclear protein of about 60 kDa [+/- 3000 KDa, (P60)]. Results of phosphoamino acids analysis indicated that the nuclear P60 was a phosphotyrosine containing protein and was predominantly phosphorylated on threonine and serine in the absence of IFN-alpha. Treatment with IFN-alpha had a dual effect on relative phosphoamino acids content of P60: inhibited the phosphorylation on threonine residue, and enhanced the phosphorylation on serine and tyrosine residues. The nuclear P60 is not one of earlier described IFN-responsive signaling protein as it was not translocated from the cytosol. PMID- 7535055 TI - Tyrosine kinases but not cAMP-dependent protein kinase mediate the induction of leukocyte alkaline phosphatase by granulocyte-colony-stimulating factor and retinoic acid in acute promyelocytic leukemia cells. AB - Leukocyte alkaline phosphatase (LAP) is synergistically induced by the combination of all-trans retinoic acid (ATRA) and granulocyte-colony-stimulating factor (G-CSF) in acute promyelocytic leukemia (APL) cells (Gianni' M. et al., Blood 83: 1909-1921, 1994). The role of cAMP and tyrosine kinases in the induction of LAP was investigated. In the APL cell line NB4, adenosine-3': 5' monophosphothioate, cyclic, Rp isomer, a reversible inhibitor of cAMP-dependent protein kinase (PKA), has no effect on the induction of LAP enzymatic activity and mRNA triggered by ATRA+G-CSF, in conditions where this compound completely blocks the upregulation of LAP transcript caused by the combination of the PKA agonist, dibutyryl-cAMP (db-cAMP), and ATRA. Challenge of NB4 cells with G-CSF, dbcAMP and ATRA causes a much higher induction of LAP relative to that observed in the presence of ATRA+G-CSF or ATRA+dbcAMP. Treatment of NB4 with ATRA and G CSF results in increases in the tyrosine phosphorylation of several proteins. In the presence of the cytokine and the retinoid, tyrosine kinase inhibitors decrease LAP enzymatic activity and mRNA. PMID- 7535056 TI - Design, synthesis, DNA sequence preferential alkylation and biological evaluation of N-mustard derivatives of Hoechst 33258 analogues. AB - The design, synthesis and biological evaluation of a series of bis-benzimidazole analogues of Hoechst 33258 bearing nitrogen mustard moieties is described. The novel compounds show clear evidence of interstrand cross-linking of linear lambda DNA, in contrast to their distamycin nitrogen mustard counterparts. Interference of the cross-linking reaction by the minor groove-selective distamycin suggests that this process takes place in the minor groove of DNA. Sequence preferential alkylation is revealed by high-resolution polyacrylamide gel electrophoresis and autoradiography. Alkylation occurs predominantly at the 5'-A or 5'-G termini of mixed sequences, determined largely by the sequence-recognizing properties of the bis-benzimidazole carrier moiety. An analysis of the frequency of bases around the alkylation sites reveals marked individual base preferences, especially for A at -3 and +3 positions. All of the compounds tested showed cytotoxic properties against the human tumor cell line KB in culture. PMID- 7535057 TI - Skeletal metastases in advanced prostate cancer: cell biology and therapy. PMID- 7535058 TI - Alternative processing pathways for MHC class I-restricted epitope presentation to CD8+ cytotoxic T lymphocytes. AB - CD8+ cytotoxic T lymphocytes (CTL) mediate protective immunity against many intracellular pathogens. New generation vaccines that exploit the potential of recombinant protein technology have to meet the challenge to identify immunodominant antigen systems of the pathogen of interest, to produce these antigens in recombinant form, and to find ways to selectively deliver them to the compartments of the specific immune system that mediate the protective responses. Immunization with soluble protein antigens usually primers CD4+ T cells but not CD8+ T cells because of the stringent requirements for 'endogenous processing' for major histocompatibility complex (MHC) class I-restricted epitope presentation to CD8+ CTL. This rule is not absolute because priming of class I restricted CTL by soluble, recombinant viral protein antigens injected without adjuvants has been reported in various antigen systems. An understanding of the cell biology of alternative pathways of protein antigen processing for MHC class I-restricted epitope presentation is emerging. We describe subcellular sites, alternative peptide transport mechanisms, and alternative modes of MHC class I molecule recycling that allow different modes of peptide loading of class I molecules. Experimental evidence for some of these novel pathways of 'endogenous' processing for class I-restricted epitope presentation is discussed. Some pathways are still hypothetical. The issue of alternative modes of 'endogenous' processing for class I-restricted antigen presentation is of theoretical and practical relevance. Immunologists are interested in the question from which source protein antigens are derived that are potentially accessible to specific recognition by different T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535059 TI - Antisense principle or ribozyme action? AB - When incorporated into long stretches of antisense RNA, the catalytic domain of the hammerhead type ribozyme significantly increases the extent of antisense mediated inhibition in living cells. A kinetic model based on the rate constants determined for duplex formation and cleavage of target RNA in vitro is not compatible with the observed biological effects and indicates that further assumptions have to be included to explain the action of ribozymes in vivo. PMID- 7535060 TI - Inhibition of nitric oxide synthase--potential for a novel class of therapeutic agent? AB - The free-radical gas nitric oxide (NO) plays an important role in a wide and diverse range of physiological processes. As progress is made in understanding the biological function of NO, there is growing interest in the possibility that inhibitors of NO synthase (NOS) may be of clinical use in the therapy of certain disease states. The search for novel and clinically relevant inhibitors of this enzyme represents a truly multidisciplinary approach to drug screening and will no doubt benefit from the application of recombinant DNA (rDNA) technology. PMID- 7535062 TI - MUM-4, a monoclonal antibody reacting with resident peritoneal mouse macrophages. AB - A new rat monoclonal antibody, MUM-4, which recognizes a murine antigen/epitope that is absent on monocytes, strongly expressed on resident peritoneal macrophages and almost completely absent from peritoneal macrophages 4 days after an intraperitoneal injection of thioglycollate or heat-killed Propionibacterium acnes organisms, is described. Immunocytochemistry and flow cytometry have been used to characterize the specificity of the antibody. MUM-4 did not react with blood granulocytes, peritoneal exudate granulocytes, lymphocytes from blood or peritoneum, isolated spleen dendritic cells, or veiled cells from the thoracic duct of mesenteric lymphadenectomized mice. The MUM-4 antibody reacted with resident peritoneal macrophages from all the mouse strains studied. MUM-4 appears to represent a new specificity. The MUM-4 antibody is of the rat IgG2c isotype and exhibits complement-mediated cytotoxicity with rabbit complement. The staining achieved with MUM-4 by FACS or immunocytochemical methods is intense on most resident peritoneal macrophages and the antibody should be a valuable addition to the panel of monoclonal antibodies available for studies on mouse macrophages. PMID- 7535061 TI - Fathers helping out: shared child care and marital satisfaction of parents of children with disabilities. AB - The relation between division of child care and marital satisfaction in couples rearing children with disabilities was explored. Greater father participation in child care was related to higher marital satisfaction for both parents. Higher family income was also related to higher marital satisfaction for fathers only. Results were discussed in the framework of recent adaptations to Hill's (1949) ABCX Model of Family Adaptation. PMID- 7535064 TI - Influence of aprotinin on early graft thrombosis in patients undergoing myocardial revascularization. AB - One hundred sixty-five patients undergoing primary myocardial revascularization were prospectively entered into a randomized, double-blind, placebo-controlled study, in a single institution, in order to determine the influence of high- and low-dose aprotinin application on early coronary artery bypass graft patency. All patients were operated on by the same team and the three treatment groups were comparable in all demographic data and surgical variables. Postoperative chest tube drainage and transfusion requirements were significantly reduced in patients receiving high or low doses of aprotinin. In all patients vein and internal mammary artery graft patency was assessed by control coronary angiograms 4 to 15 days (median 8.2 days) postoperatively. In the high-dose aprotinin group, 140 of 142 vein grafts and in the low-dose aprotinin group all of the 128 vein grafts were patent compared with 138 of 139 in the placebo group. The difference was not statistically significant (P > 0.05). All pedicled internal mammary artery grafts were patent in the three treatment groups. The prevalence of perioperative myocardial infarction was evaluated by serial creatine kinase-myocardial band (CK MB) isoenzyme measurements and by electrocardiographic recordings. No additional changes that could be attributed to aprotinin were observed. In conclusion, these results suggest that perioperative myocardial infraction secondary to aprotinin induced native coronary artery or conduit thrombosis is not increased by aprotinin in patients undergoing primary myocardial revascularization. PMID- 7535063 TI - Phenotype and differentiation potential of a novel rat tracheal epithelial cell line. AB - In this report we described the establishment and characterization of a continuous rat tracheal epithelial (RTE) cell line spontaneously derived from secondary RTE cell cultures. Designated SPOC1, this cell line is nontumorigenic and maintains a diploid karyotype with specific, nonrandom chromosomal alterations involving chromosomes 1, 3, and 6. SPOC1 cells demonstrate decreased requirements for peptide growth factors, compared with primary RTE cells. Upon inoculation into denuded rat tracheas, which are then implanted into syngeneic hosts, SPOC1 cells initially form a stratified squamous epithelium, which becomes less stratified with time and forms glandlike invaginations into the surrounding lamina propria. No evidence of ciliated cell differentiation is detected. The epithelium formed by SPOC1 cells in tracheal grafts reacts with antibodies specific for keratin 14, 13, and 19 (but not keratin 18) at both early and late time points, although the localization of antibody staining changes as the epithelium becomes less stratified with time. The suprabasal epithelial cells become positive for alcian blue-periodic acid-Schiff staining at later time points. The near-normal karyotype and differentiation potential of SPOC1 cells make this cell line a unique window into early changes occurring during immortalization of airway epithelial cells and will allow studies of relationships between differentiation state and neoplastic transformation. PMID- 7535065 TI - Glycosylation of recombinant human granulocyte colony stimulating factor: implications for stability and potency. AB - The production of recombinant human granulocyte colony stimulating factor (HuG CSF) by gene cloning has made this growth factor available in large quantities for clinical application. There is accumulating evidence to suggest that the glycosylation of HuG-CSF confers advantages in terms of in vitro stability to temperature, pH and degradation by proteases, and a recent report attributes a greater biological potency, in the absence of larger biological mass, to the property of glycosylation. In this study, the biological potency of glycosylated rHuG-CSF (lenograstim) was compared with that of non-glycosylated rmetHuG-CSF (filgrastim) and a non-glycosylated rHuG-CSF (non-commercial preparation), using duplicate assays of neutrophil and erythroid colony formation in three human bone marrows. Serial doubling dilutions of each rHuG-CSF resulted in a concentration range of 0.008-128 ng/ml. Qualitative (number) and quantitative (size) assessments of colonies were performed at day 14 of culture. Lenograstim proved twice as potent as filgrastim (and non-commercial rHuG-CSF) at maximal colony stimulation, and 20 times more potent than both at half-maximal colony stimulation (P = 0.0001). Incubation with lenograstim also produced a higher proportion of colonies with > 200 cells than either of the other preparations. In conclusion, glycosylated rHuG-CSF (lenograstim) had a greater qualitative and quantitative potency than the non-glycosylated rHuG-CSFs (filgrastim and non commercial rHuG-CSF), indicating that glycosylation confers a potency advantage on lenograstim. PMID- 7535066 TI - Granulocyte colony stimulating factor: from laboratory bench to clinical use. AB - A great deal has been learned about haematopoiesis since the discovery, purification and cloning of haemopoietic growth factors. The function of these growth factors is to promote survival and enhance mitosis (at higher concentrations), and influence differentiation and development of stem cells and progenitor cells. The type of mature cells produced reflects the combinations of growth factors to which the stem cells are exposed. Growth factors may also control the functional activity of mature cells. Treatment with granulocyte colony stimulating factor (G-CSF) produces neutrophils that are at least equivalent to those produced during normal haematopoiesis and does not appear to deplete the haematopoietic stem or progenitor cell populations. PMID- 7535067 TI - Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor. AB - Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture. It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line. In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures. Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor. Purification of lenograstim involves a four-step chromatographic process. The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk. The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin. PMID- 7535068 TI - The optimal dose of glycosylated recombinant human granulocyte colony stimulating factor for use in clinical practice: a review. AB - In pharmacokinetic studies, higher maximum plasma concentrations of granulocyte colony stimulating factor (G-CSF) were achieved after intravenous administration of lenograstim than after subcutaneous administration of the same dose. However, subcutaneous administration resulted in a longer elimination half life, and a greater and more sustained neutrophil response. The safety of lenograstim at doses up to 40 micrograms/kg/day has been demonstrated in Phase I studies involving 21 healthy volunteers and 37 cancer patients. In controlled Phase II dose-ranging studies which included 66 solid tumour and 121 bone marrow transplantation (BMT) patients, doses from 2 micrograms/kg/day were shown to effectively restore neutrophil counts. A dose of 5 micrograms/kg/day was chosen for further clinical trials. Phase III double-blind randomised studies in a further 439 patients with solid tumours or postBMT confirmed the clinical efficacy of the recommended therapeutic dose of lenograstim of 150 micrograms/m2/day which is equivalent in efficacy to 5 micrograms/kg/day. At this unique dose, one 263-microgram vial of lenograstim is sufficient to effectively treat the average adult patient in solid tumour or bone marrow transplantation settings. PMID- 7535069 TI - The clinical benefits of recombinant human granulocyte colony stimulating factor in the treatment of cancer patients. AB - Recombinant human granulocyte colony stimulating factor (rHuG-CSF) has been used for several years in clinical haematology and it is now routinely employed to prevent or treat chemotherapy-induced neutropenia. rHuG-CSF is also administered after autologous or allogeneic bone marrow transplantation (BMT), since it can significantly shorten the duration of neutropenia. However, probably its main use at the moment is to facilitate the collection of peripheral blood stem cells (PBSC) from patients with lymphoma, myeloma and breast cancer. Within controlled trials, it is also used as an adjunct to immunosuppression for patients with aplastic anaemia. rHuG-CSF has a number of other potential uses such as increasing the numbers of progenitor cells for transplantation by in vivo and/or ex vivo amplification; treatment of non-neutropenic infections post transplant, and prophylaxis and treatment of cytomegalovirus infections. In the future, autologous stem cell transplants may be performed in the outpatient department thus expanding the use of PBSC transplantation to disease areas not previously considered suitable for such myelosuppressive treatment. PMID- 7535070 TI - Chemotherapy dose intensity facilitated by use of lenograstim--implications for quality of life and survival. AB - Increasingly more aggressive chemotherapeutic regimens are being used in pursuit of better tumour response and patient survival. Studies in advanced breast cancer, small cell lung cancer (SCLC), urothelial cancer and sarcoma indicate that recombinant human granulocyte colony stimulating factor (rHuG-CSF) (lenograstim) facilitates modest dose intensification (20-30%), but at high intensity doses, chemotherapy-related toxicity remains a problem. Survival outcome in SCLC patients is still uncertain although in one randomised trial lenograstim facilitated a 20% increase in chemotherapy dosage which resulted in a 25% increase in complete remissions. In studies of breast cancer, an initially improved response rate was not maintained. However, increased dose intensity of chemotherapeutic agents in aggressive lymphoma was possible using lenograstim. Studies to date with haematopoietic growth factors have not been designed to specifically address the issue of survival and/or quality of life, although they have inferred that this is possible through dose intensification. The use of haematopoietic growth factors will allow the design of such studies, but a modest chemotherapy dose increase may be insufficient to improve survival. The future for dose intensification in cancer therapy may lie in the use of lenograstim primed peripheral blood progenitor cells (PBPC) transplants to enhance haematopoietic recovery after high-dose sequential or myeloablative chemotherapy. PMID- 7535071 TI - The changing face of stem cell transplantation by the use of recombinant human granulocyte colony stimulating factor. AB - The use of peripheral blood progenitor cells (PBPC) for autografting following high-dose chemotherapy in a variety of malignancies has increased markedly with the advent of efficacious and safe myeloid growth factors, which have dramatically improved collection yields. There is still controversy as to whether PBPC should be harvested after a combination of chemotherapy and growth factors, or after growth factors alone. The use of powerful combinations of newer growth factors, such as interleukin-3 with granulocyte-macrophage colony stimulating factor (GM-CSF), or granulocyte colony stimulating factor (G-CSF) with stem cell factor, may obviate the need for chemotherapy. The problem of providing sufficient numbers of PBPC for successful transplantation and sustained engraftment has led to the development of a variety of in vitro expansion systems for stem cells, using cocktails of growth factors. Single blood collections may contain sufficient CD34+ cells to be expanded on a large scale for clinical transplantation, eliminating costly and labour intensive apheresis procedures. PBPC transplants may carry less risk for contamination with malignant cells; however, sensitive detection techniques have revealed that tumour cell contamination of PBPC harvests may be much more prevalent than was previously suspected and both positive and negative selection of CD34+ cells for clinical transplantation is being investigated. PBPC transplantation generally results in more rapid engraftment, with faster recovery of neutrophils and platelets, compared with autologous bone marrow transplants. In most studies, PBPC transplants also resulted in fewer septic episodes, fewer intensive care admissions, fewer transfusions of red blood cells and platelets, reduced antibacterial and parenteral nutrition use, and reduced hospital costs. Dose optimisation and intensification of chemotherapy, relying on repeated administration of growth factor-mobilised PBPC, offers interesting prospects in the treatment of a variety of tumours. However, well-controlled, randomised prospective trials will be needed to prove the real value of PBPC transplants with regard to survival and cure. In the clinical setting, PBPC transplantations are likely to replace autologous marrow transplants in the near future. Manipulation of PBPC in vitro, i.e. expansion or gene transfer, may prove to be the most exciting perspective in the treatment of both malignant and non malignant haematological conditions. PMID- 7535072 TI - Future strategy for cancer treatment using recombinant human granulocyte colony stimulating factor. AB - Recombinant human granulocyte colony stimulating factor (rHuG-CSF) has an accepted use in reducing the severe neutropenia and associated complications in patients who undergo chemotherapy or bone marrow transplantation (BMT). It is valuable in mobilising peripheral blood progenitor cells (PBPCs) for stem cell rescue after myeloablative chemotherapy as an alternative to BMT. rHuG-CSF also allows chemotherapy dose/schedule optimisation and intensification, although a clear survival advantage has yet to be demonstrated. Recommendations for the focus and methodology of future studies with rHuG-CSF have been made, incorporating the endpoints of survival, quality of life and cost/benefit. Future studies need to address the questions of optimal dosing level and duration, cytokine combinations, the factors influencing patient survival, and the most cost-effective use of therapy. rHuG-CSF has potential for extended use in cancer therapy and in a variety of non-malignant indications. PMID- 7535073 TI - Physicochemical and biochemical characteristics of glycosylated recombinant human granulocyte colony stimulating factor (lenograstim). AB - Lenograstim, glycosylated recombinant human granulocyte colony stimulating factor (rHuG-CSF), is a glycoprotein that contains 4% O-glycosides with an approximate molecular weight of 20 kDa. The carbohydrate chain contributes to stabilisation of the protein by its ability to suppress polymerisation due to pH changes and to prevent denaturation at elevated temperatures. The carbohydrate chain also has an important role in maintaining biological stability in normal human serum. Furthermore, it is shown that glycosylated rHuG-CSF is more stable and resistant to degradation than non-glycosylated rG-CSF in vitro. In addition, we show the remarkable effects of combined therapy of lenograstim with an antibiotic in severe neutropenic infection models. These data demonstrate the possibility that such combination therapy may be applied to neutropenic patients with serious infections and that there is increased efficacy of antibiotic treatment when administered concurrently with lenograstim. PMID- 7535074 TI - Volume adjustment for intermediate prostate-specific antigen values in a screening population. AB - In a screening population of 812 men, between the ages of 55 and 77 years, and prostate-specific antigen (PSA) below 10.0 ng/ml (Hybritech), digital rectal examination (DRE) and transrectal ultrasonography of the prostate (TRUS) were performed. Seventeen prostate carcinomas were detected. Four methods of prostate volumetry were used to determine volume-adjusted PSA levels. These were PSA density for the total gland volume (PSAD), for the inner zone volume (PSAT) and population-specific excess PSA values. There was a significant difference between the benign and the malignant population for age, PSA, PSAD, PSAT and excess PSA values. The maximal discriminatory potential, analysed by the area under receiver ROC curve, was 0.90, reached for prolate spheroid determined excess PSA. For PSA alone this was 0.86. Volume-adjusted PSA values have no additional benefit beyond unadjusted values in screening for prostate carcinoma in this study. PMID- 7535076 TI - A recent increase in the incidence of prostatic carcinoma in a French population: role of ultrasonography and prostatic specific antigen. AB - Between 1979 and 1990, the incidence rate (World Standard) for cancer of the prostate in the region of Isere (France) increased from 22.1 to 45.0 cases per 100,000 men, although there was no concurrent increase in mortality (16.0 to 17.6 cases per 100,000 men). This represents a mean increase per year of 6.3% for incidence, compared with 1.3% (NS) for mortality. Incidence of cases with metastases at diagnosis also remained stable with time. In this area, Prostatic Specific Antigen assays began in 1987, and rectal ultrasonography was implemented in 1984, but activity peaked only in 1988. Thus, during 1986-1988, there was both an implementation of new diagnostic procedures and an increase in the incidence of prostatic carcinoma, which suggests that the latter was the result of increased detection of small latent carcinomas. This has implications for public health since apart from increasing costs, it might unduly disturb the life of otherwise healthy people. PMID- 7535075 TI - Adjuvant radiotherapy following radical prostatectomy--results of 56 patients. AB - Patients with adenocarcinoma of the prostate with positive surgical margins and/or seminal vesicle invasion after radical prostatectomy (RP) have a high risk of local recurrence or distant spread of disease. Several investigators reported increased local control rates following adjuvant radiotherapy (RT). However, it is unclear whether this procedure, with or without hormonal therapy (HT), improves the outcome. From 1975 to 1987, 56 patients with adenocarcinoma of the prostate underwent adjuvant RT following RP (pathological stage C1, n = 19; stage C2, n = 17; stage D1, n = 20). In 27 of 56 patients an additional immediate orchiectomy was performed. 48 patients received 4000-5000 cGy to the pelvic lymphatics, including the prostatic fossa, followed by a boost to the prostatic fossa to complete 6400-7000 cGy, whereas 8 patients were treated to the prostatic fossa only. With a median follow-up of 89 months, the overall survival rate of patients with stages C1, C2 and D1 did not differ significantly (10-year overall survival rate 84, 74 and 71, respectively). The local control rate for 5- and 10 years was 96 and 90%, respectively. A significant advantage in overall survival (5- and 10-year rate: 92 versus 93% and 92 versus 63%; P < 0.05, respectively) and clinical disease-free survival (5- and 10-year rate: 92 versus 72% and 92 versus 49%; P < 0.05, respectively) was seen in 27 patients with orchiectomy compared with 29 patients without HT. A total of 15 patients (26%) developed at least one form of late toxicity, in most cases a mild proctitis, cystitis, or penile or leg oedema. However, 6 patients (11%) had severe grade 3 or 4 side effects that necessitated a cystectomy in 2 cases as well as a colostomy in 2 cases. In all patients with grade 3 or 4 side-effects, 70 Gy as a tumour encompassing isodose were applied. Adjuvant RT, following RP in stage C and D1 prostate cancer with positive surgical margins and/or seminal vesicle invasion increases local control. Whether immediate HT influences the outcome, as seen in this study, should be proven in prospective clinical trials. PMID- 7535077 TI - Insulin sensitivity, hormonal levels and skeletal muscle protein metabolism in tumour-bearing exercising rats. AB - We have previously shown that spontaneous physical exercise can delay onset of experimental anorexia and cachexia, and retard tumour growth; we now report the effects on insulin sensitivity, hormonal levels and skeletal muscle protein metabolism. Insulin sensitivity determined with a euglycaemic hyperinsulinaemic clamp revealed a normalised glucose disposal rate in tumour-bearing exercising (TBE) versus sedentary (TBS) animals (TBE 15.55 +/- 2.71 versus TBS 2.47 +/- 2.12 mg/kg/min; P < 0.05). Both TBE and TBS animals had decreased levels of corticosterone during the clamp. Serum levels of insulin during tumour progression were unaffected by exercise, but the insulin: glucagon ratio increased and the progressive decrease in rT3 was attenuated. The concentration of glucagon decreased in both tumour-bearing groups during the experiment, while TBE animals showed a relative reduction in corticosterone. Capacity for skeletal muscle protein synthesis, expressed as RNA: protein ratio, was normalised in TBE animals in two tumour protocols (TBE 5.9 +/- 0.6 versus TBS 4.7 +/- 0.3; TBE 2.9 +/- 0.4 versus TBS 1.8 +/- 0.2; P < 0.05, respectively). Incorporation rate of 14C-phenylalanine into skeletal muscle protein was increased in the TBE group in vitro and in vivo. In the postexercise period, protein degradation evaluated by tyrosine release in vitro was increased, but decreased over time. This study has confirmed a positive skeletal muscle protein balance in exercising tumour-bearing animals, partly explained by the increased insulin sensitivity. This conclusion was further supported by the less catabolic pattern indicated by hormonal levels. PMID- 7535079 TI - Aprotinin in knee replacement surgery. PMID- 7535078 TI - Histaminoid reactions in anaesthesia. PMID- 7535080 TI - P-VEBEC: a new 8-weekly schedule with or without rG-CSF for elderly patients with aggressive non-Hodgkin's lymphoma (NHL). AB - BACKGROUND: Chemotherapy regimens devised for elderly patients with intermediate high grade NHL are a matter of discussion. The aim is to reduce general toxicity without loosing an antilymphoma effect. The most important limiting factor of chemotherapy is myelotoxicity; for this reason the use of growth factor may be useful in these patients. PATIENTS AND METHODS: From November '91 to November '92, 67 pts older than 65 years with intermediate-and high-grade advanced-stage NHL were treated with the P-VEBEC regimen, an original scheme with epirubicin 50 mg/m2, cyclophosphamide 350 mg/m2 and etoposide 100 mg/m2 on weeks 1, 3, 5, 7; vinblastine 5 mg/m2 and bleomycin 5 mg/m2 on weeks 2, 4, 6, 8, prednisone 50 mg/m2/day p. os in the first 2 weeks and thereafter every other day. Twenty-eight pts received r-GSF 5 micrograms/kg/day throughout the treatment starting on day 2 of every week for 4 consecutive days. Their median age was 71 years (65-80), 31 pts were male and 36 female, histology according W.F. was D 6; E 17; F 16; G 19; H 9. Twenty-five percent of pts had B symptoms, 35% had bulky disease, 41% LDH level > normal, 44% stage IV and 26% had B.M. involvement. RESULTS: C.R. was achieved by 66% of pts. Adverse prognostic factors for CR were E histology, stage IV, bone marrow infiltration and LDH above normal. Severe toxicity was never recorded, no toxic death was observed. With a median follow-up of 24 months OS, DFS and EFS were 55%, 52%, and 33%, respectively. EFS was influenced by stage, BM involvement and level of LDH. The relative dose intensity (RDI) was calculated by the method of Hryniuk and Bush. Patients who received rG-CSF had a significantly higher median RDI (94% vs 79%) and lower myelotoxicity (neutrophil nadir < 500 18% vs 56%). The rate of CR was influenced by RDI > 80% (89% vs 56%). EFS was also better in pts who received a RDI higher than 80% (50% vs 18% p = 0.05). CONCLUSION: P-VEBEC is a feasible cycle in elderly patients; the use of rG-CSF improves RDI. In patients with adverse prognostic factors (BM involvement, poor performance status) a RDI > 0.80 could play a role in improving the outcome. PMID- 7535081 TI - The management of primary mediastinal B-cell lymphoma with sclerosis. PMID- 7535083 TI - N- and KRAS mutations in primary testicular germ cell tumors: incidence and possible biological implications. AB - Recently, conflicting results have been reported on the incidence of RAS mutations in primary testicular germ cell tumors of adults (TGCTs). In four studies a low incidence of mutations (less than 15%) in a variety of TGCTs or derived cell lines was found, whereas in two other studies a high incidence of N- or KRAS mutations (over 40%) was shown. A total of 62 testicular seminomas (SE) and 34 nonseminomatous TGCTs (NS) were studied thus far. The largest series consisted of 42 TGCTs, studied on paraffin embedded tissue. We present the results of analysis for the presence of N- and KRAS mutations, in codons 12, 13, and 61, in snap frozen samples of 100 primary TGCTs, comprising 40 SE and 60 NS. Using the polymerase chain reaction (PCR) and allele specific oligonucleotide hybridization (ASO), mutations were found in five SE (three in NRAS and two in KRAS, all codon 12), and in one NS (KRAS, codon 12). To exclude underestimation of the incidence of RAS mutations in TGCTs due to the presence of an excess of wild type alleles in the analyzed sample, a PCR technique preferentially amplifying KRAS alleles with a mutation in codon 12 was applied to all SE. This approach, allowing a 250 times more sensitive assay, resulted in the detection of only one additional SE with a mutation. Based on a critical analysis of published data and on our results from the largest series of frozen samples investigated thus far, we conclude that N- or KRAS mutations are rare and apparently not essential for initiation or progression of TGCTs. PMID- 7535082 TI - Keratinocyte growth factor receptor ligands induce transforming growth factor alpha expression and activate the epidermal growth factor receptor signaling pathway in cultured epidermal keratinocytes. AB - Epidermal growth factor receptor (EGFR) ligands are fundamental regulators of epithelial growth, differentiation, and neoplastic transformation. In addition to being potent mitogens for murine epidermal keratinocytes in vitro, transforming growth factor alpha (TGF alpha) and EGF elicit distinctive changes in keratin expression: Ca(2+)-mediated induction of the differentiation-specific keratins K1 and K10 is blocked, while simple epithelial keratins K8 and K18 are expressed aberrantly (C. Cheng et al., Cell Growth, & Differ., 4: 317-327, 1993). We have evaluated several additional growth factors to determine the specificity of this response for EGFR ligands. TGF alpha, keratinocyte growth factor (KGF), and acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF) or insulin-like growth factor type I, block Ca(2+)-mediated expression of K1 while inducing K8. Since KGF and aFGF (but not bFGF) are ligands for the KGF receptor (KGFR), we explored the possibility that the TGF alpha/EGFR pathway is an intermediary in signaling through the KGFR. TGF alpha mRNA was increased in cells treated with KGF, aFGF, or TGF alpha but not bFGF or insulin-like growth factor type I. Similar changes were detected at the protein level; TGF alpha in conditioned medium (CM) from control, KGF-, TGF alpha-, and aFGF-treated cultures was 54 (+/- 8, SEM), 365 (+/- 50), 146 (+/- 20), and 120 (+/- 50) pg/ml, respectively. KGF and TGF alpha also increased expression of cell-associated TGF alpha measured in keratinocyte lysates. KGF increased TGF alpha secretion and mRNA levels in human as well as mouse keratinocytes. CM from KGF-treated cultures stimulated cell growth when added to cultures of normal keratinocytes. Preincubation with neutralizing antibodies to both TGF alpha and KGF, but not KGF antibody alone, blocked cell growth in cultures treated with KGF CM, suggesting that the predominant keratinocyte mitogen in KGF CM is TGF alpha. In support of this hypothesis, treatment of keratinocytes for 5 min with either KGF CM or purified TGF alpha resulted in EGFR autophosphorylation. Furthermore, after approximately 24 h, KGF as well as TGF alpha induced EGFR down-regulation based on Western blot analysis and 125I-EGF binding. Induction of TGF alpha in KGF treated keratinocytes, coupled to activation and down-modulation of the EGFR, suggests that TGF alpha may be a proximal effector of KGF action for at least certain aspects of epidermal growth and differentiation. PMID- 7535084 TI - Screening for germ-line mutations in the NF2 gene. AB - Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease that predisposes to the development of tumors of the nervous system, particularly meningiomas and schwannomas. The gene which, when altered, causes NF2, is localized on chromosome 22 and has recently been identified. The NF2 gene is also the site of somatic mutation in tumors, suggesting that it might have a tumor suppressor activity. We here report a screening method for the detection of point mutations in NF2 which takes advantage of denaturing gradient gel electrophoresis (DGGE). This method efficiently screens 95% of the coding sequence and 90% of intron/exon junctions. When applied to 91 unrelated NF2 patients, it enabled the identification of 32 germ-line mutations. Since mutations are found in only one third of the patients, it is expected that mutations or deletions affecting the promoter and/or intronic regions of the NF2 gene occur frequently. The characterized mutations are preferentially located within the 5' half of the gene. Most of them are predicted to lead to the synthesis of a truncated protein. A search for genotype/phenotype correlations showed that, at least in this series of patients, mild manifestations of the disease were associated with mutations which preserve the C-terminal end of the protein. PMID- 7535086 TI - A genetic model of melanoma tumorigenesis based on allelic losses. AB - Previous karyotypic studies have indicated a possible series of non-random chromosomal events involved in progression of melanoma. We sought to verify and augment this model of melanocyte tumorigenesis by studying allelic deletions of markers mapping to these regions in 30 matched pairs of melanoma and constitutional DNA samples. Polymorphic loci on chromosomes 1, 7, 10, 11, 17, and 21 were analyzed and data combined with those previously obtained for chromosome arms 6q and 9p in the same series of tumours. The most frequent and earliest deletions were found on 9p (57%) and 10q (32%). With the exception of one case, no sample had loss of markers on another chromosome without concomitant loss of markers on 9p or 10q. Losses on 6q were also a frequent (31%) and early event whereas losses of loci on distal 1p (22%) or 11q (26%) occurred only in metastatic melanomas. A "background" rate (0-17%) of allele loss was seen on chromosomes 7, 17, and 21. These data strongly support the previous model based on karyotypic findings in melanocytic lesions. However, we have been able to further, augment that model by delimiting the regions of loss on 10q, to that distal to D10S254, and on 1p, to between D1S243 and D1S160. PMID- 7535087 TI - Atypical cytogenic aberrations in two childhood peripheral primitive neuroectodermal tumors. AB - Atypical cytogenetic abnormalities were detected in peripheral primitive neuroectodermal tumors (PPNET) of the extremity in two children. One had an osseous tumor with a balanced reciprocal translocation, t(5;9)(q22;q32), and had a complete response to therapy. The other had a non-osseous tumor with an interstitial deletion, del(18)(q12.2q21.2), was resistant to combination therapy, and at autopsy had evidence of possible clonal evolution with the karyotype 46,XX der(8)t(8;8)(p11.2;q13), inv(16)(p13.2q12),del(18)(q12.2q21.2). Neither tumor demonstrated the t(11;22)(q24;q12) typically found in Ewing's sarcoma and PPNET, suggesting heterogeneity of the cytogenetic aberrations seen in this rare childhood malignancy. PMID- 7535085 TI - 11q13 amplification in local recurrence of human primary breast cancer. AB - Breast cancer can relapse both locally and at distant metastatic sites. The mechanism of local recurrence is unknown, but seems to be due not only to the number of residual cancer cells (inadequate irradiation or surgery), but also to their genetically determined malignant potential. To identify genetic alterations associated with local recurrence risk in breast carcinoma, we analyzed 28 local recurrences and 173 primary breast tumors for the ten most frequently altered genetic regions in breast carcinomas, i.e., loss of heterozygosity on chromosomal arms 1p, 3p, 7q, 11p, 17p, 17q, and 18q, and amplification of the MYC and ERBB2 protooncogenes and of genes in 11q13. Only INT2/FGF3 and CCNDI, located in 11q13, were more frequently amplified in local recurrences than in primary tumors (39% vs. 17%; P < 0.01). Moreover, recurrence-free survival was shorter when the 11q13 region was amplified. These results suggest that one or more genes located in 11q13 play an important role in local relapses of breast cancer. PMID- 7535088 TI - Translocation t(8;13)(p11;q11-12) in stem cell leukemia/lymphoma of T-cell and myeloid lineages. AB - An unusual hematologic neoplasia has been described recently in which the predominant clinical features include T-cell lymphoma, myeloid hyperplasia, and eosinophilia. The multilineage involvement in this disorder suggests transformation of a primitive stem cell. Abnormal karyotypes have been described in three such cases, including one case with t(8;13)(p11.2;q12) and a second case with t(8;13)(p23;q14). We report translocation of chromosomes 8 and 13 in lymph node karyotypes from two patients with this syndrome. Fluorescence in situ hybridization confirmed an identical translocation, t(8;13)(p11;q11-12), in lymphoma cells from each patient. The translocation breakpoints are of particular interest because the FLT3 receptor tyrosine kinase gene has been mapped 13q12. FLT3 is expressed highly in hematopoietic progenitor cells and in myeloid and lymphoid acute leukemias. PMID- 7535089 TI - Loss of heterozygosity in sporadic primary cutaneous melanoma. AB - Difficulties in obtaining clinical samples from primary melanomas have meant that most genetic analyses of melanoma have concentrated on cell lines and metastases. Because the Breslow thickness of the primary tumour is the single best prognostic indicator, it is important to identify genetic abnormalities in primary melanomas and relate these changes to the thickness of the lesion. We have investigated 47 sporadic melanomas, of which 41 were primary lesions, for loss of heterozygosity (LOH) on several chromosomal arms, including areas where genes involved in familial melanoma and other relevant hereditary syndromes map, and where LOH has previously been reported in cell lines, or metastatic lesions. LOH was identified at 66 (18%) of 358 informative loci in primary melanomas, and there was a significant relationship between the overall frequency of LOH and Breslow thickness (P < 0.0005). Loss of chromosome arm 9p was most frequent, occurring in 15 (47%) of 32 informative primary tumours, and was observed in 3 of 11 informative lesions < or = 1.5 mm in depth. LOH on chromosome arms 3p, 6q, 10q, 11q, and 17p was also relatively frequent, with loss of 3p and 10q heterozygosity in lesions < or = 1.5 mm in depth, while LOH on 6q, 11q, and 17p was only detected in more invasive tumours. The results suggest that loss of these chromosome regions are important in sporadic cutaneous melanoma, and are consistent with chromosome arm 9p loss occurring before loss of other chromosome arms. PMID- 7535091 TI - Detection of hidden structural rearrangements by FISH in pleomorphic adenomas. AB - Previous cytogenetic analysis of pleomorphic adenomas of the salivary glands has revealed a subgroup of tumors with interstitial deletions of 8q with breakpoints at q12 and q21-23. In this paper we have re-examined four adenomas with confirmed or suspected deletions of 8q by FISH. Painting of interphase nuclei and metaphase chromosomes with whole chromosome libraries revealed that in each of the four cases the deleted chromosome 8 material was found inserted or translocated onto other chromosomes. In two of the cases we were also able to resolve several other complex rearrangements. Our FISH data thus suggest that gross deletions of 8q do not occur in pleomorphic adenomas. These observations are of crucial importance for the molecular interpretations of the 8q12 rearrangements. The fact that all rearrangements with breakpoints at 8q12 seem to be translocations or insertions strongly indicates that they involve a similar molecular mechanism. Our findings illustrate that chromosome painting is a valuable and useful adjunct to conventional cytogenetic analysis of solid tumors. PMID- 7535090 TI - The detection of E2A rearrangement or E2A-PBX1 fusion transcript in t(1;19) leukemia. PMID- 7535092 TI - Expression of the Wilms' tumor gene WT1 in human malignant mesothelioma cell lines and relationship to platelet-derived growth factor A and insulin-like growth factor 2 expression. AB - Mutations in the WT1 tumor suppressor gene are known to contribute to the development of Wilms' tumor (WT) and associated gonadal abnormalities. WT1 is expressed principally in the fetal kidney, developing gonads, and spleen and also in the mesothelium, which lines the coelomic cavities. These tissues develop from mesenchymal components that have subsequently become epithelialized, and it has therefore been proposed that WT1 may play a role in this transition of cell types. To test the possible involvement of this gene in malignant mesothelioma, we have first studied its expression in a panel of human normal and malignant mesothelial cell lines. WT1 mRNA expression levels varied greatly between the cell lines and no specific chromosomal aberration on 11p, which could be related to the variation in WT1 expression in these cell lines, was observed. Furthermore, no gross deletions rearrangements, or functionally inactivating point mutations in the WT1 coding region were identified. All four WT1 splice variants were observed at similar levels in these cell lines. The WT1 gene encodes a zinc-finger transcription factor and the four protein isoforms are each believed to act as transcriptional repressors of certain growth factor genes. Lack of WTI expression in thus predicted to result in growth stimulation of tumor cells. Binding of one particular WT1 isoform construct to the insulin-like growth factor 2 (IGF2) and platelet-derived growth factor A (PDGFA) gene promoters has been demonstrated to result in repression of these genes in transient transfection studies. Analysis of IGF2 and PDGFA mRNA expression levels compared with WTI mRNA expression levels failed to demonstrate an inverse correlation in the mesothelial cell lines, which endogenously express these genes. Finally, the putative role of WT1 in the transition of cell types was investigated. No obvious correlation between WT1 expression levels and cell morphology of the malignant mesothelial cell lines was evident from this study. Moreover, no change in WT1 expression was observed in normal mesothelial cells which were, by alteration of culture conditions, manipulated to switch from the mesenchymal to epithelial morphology. PMID- 7535093 TI - Karyotypic characterization of colorectal adenocarcinomas. AB - Cytogenetic analysis of short-term cultures from 52 primary colorectal adenocarcinomas revealed clonal chromosome aberrations in 45 tumors, whereas the remaining 7 had a normal karyotype. More than 1 abnormal clone was detected in 26 tumors; in 18 of them, the clones were cytogenetically unrelated. The modal chromosome number was near-diploid in 32 tumors and near-triploid to near tetraploid in 13. Only numerical aberrations were identified in 13 carcinomas, only structural aberrations in 3, and 29 had both numerical and structural changes. The most common numerical abnormalities were, in order of decreasing frequency, gains of chromosomes 7, 13, 20, and Y and losses of chromosomes 18, Y, 14, and 15. The structural changes most often affected chromosomes 1, 17, 8, 7, and 13. The most frequently rearranged chromosome bands were, in order of decreasing frequency, 13q10, 17p10, 1p22, 8q10, 17p11, 7q11, 1p33, 7p22, 7q32, 12q24, 16p13, and 19p13. Frequently recurring aberrations affecting these bands were del(1)(p22), i(8)(q10), i(13)(q10), and add(17)(p11-13). The most common partial gains were from chromosome arms 8q, 13q, and 17q and the most common partial losses from chromosome arms 1p, 8p, 13p, and 17p. A correlation analysis between the karyotype and the clinicopathologic features in our total material, which consists of altogether 153 colorectal carcinomas, including 116 with an abnormal karyotype, showed a statistically significant association (P < 0.05) between the karyotype and tumor grade and site. Carcinomas with structural chromosome rearrangements were often poorly differentiated; well and moderately differentiated tumors often had only numerical aberrations or normal karyotypes. Abnormal karyotypes were more common in rectal carcinomas than in carcinomas situated higher up. Near-triploid to near-tetraploid karyotypes were more than twice as frequent in tumors of the distal colon as in those of the proximal colon and rectum. The cytogenetic data indicate that carcinomas located in the proximal colon and rectum, which often are near-diploid with simple numerical changes and cytogenetically unrelated clones, probably arise through different mechanisms than do tumors located in the distal colon, which more often have complex near triploid to near-tetraploid karyotypes. PMID- 7535095 TI - Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes. AB - Apoptosis appears to play a major role in the differentiation and selection of T and B lymphocytes, but the mechanisms of clonal deletion of T cells in thymus are not well understood. We have prepared an anti-human Fas IgM mAb with associated apoptosis-inducing activity in Fas antigen-positive target cells including human T cells. We analyzed the expression of apoptosis antigen Fas on human thymocytes by cytofluorometry showing low, but significant amounts of Fas antigen on double negative and double-positive undifferentiated thymocytes. On the contrary, most of the differentiated thymocytes (single-positive or CD3-brightest) expressed undetectable levels of Fas antigen. About 1-2% of thymocytes expressed high amounts of Fas antigen, and these cells, which were CD3-bright, were CD4-bright and CD8-low at the stage of late double-positive lineage. Immunohistological analysis shows these Fas-bright cells on the edge of the medulla. Stimulation through the TCR complex was shown to induce the expression of Fas antigen on thymocytes at the late double-positive stage and prolonged stimulation through the TCR complex rendered the Fas-bright thymocytes sensitive to apoptosis inducing activity of anti-Fas. To show the involvement of the Fas system in the negative selection/clonal deletion of thymocytes, we organ-cultured human thymus in the presence of the superantigen, staphylococcus enterotoxin B (SEB), and new antagonistic anti-Fas mAb, which can inhibit the apoptosis-inducing activity of the original anti-Fas mAb. The SEB-reactive TCR complex on thymocytes was at first down-regulated by SEB, then the SEB-reactive clone was deleted by apoptosis, which was inhibited by an antagonistic anti-Fas mAb. Thus, Fas antigen is shown to be involved in the negative selection/clonal deletion of superantigen reactive thymocytes. PMID- 7535096 TI - Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation. AB - Antibodies recognising the products of alternatively spliced exons near the N terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone. PMID- 7535094 TI - Antibody response elicited by T-dependent and T-independent antigens in gene targeted kappa-deficient mice. AB - Animal models substantially contribute to the understanding of the pathogenesis of various human diseases, including those associated with genetic defects. Our study investigated the characteristics of antibody responses elicited by T dependent and T-independent antigens in mice rendered kappa-deficient by targeted deletion of the J kappa C kappa gene segments. It is known that in normal murine species the kappa repertoire dominates the antibody repertoire (kappa/lambda ratio = 95:5). Our results indicate that the kappa gene deletion causes the alternative usage of lambda 1 (93%) and lambda 2 (7%) light chains, confirming previous studies demonstrating that in kappa-deficient mice all B cells express Ig lambda receptors. The anti-trinitrophenylbenzene (TNP) response in K-/- mice was compensated for by lambda 1 and lambda 2 bearing Igs. However, isoelectric focusing analysis of anti-TNP antibodies showed a considerably more restricted pattern of lambda anti-TNP antibodies in K-/- as compared with kappa antibodies in normal mice. No major differences were observed in the affinity for the hapten of kappa or lambda 1 or lambda 2 mAbs obtained from 129/Sv and K-/- mice. Furthermore, lambda 1 and lambda 2 chains can reconstitute the expression of an idiotype (460Id) borne on kappa anti-TNP antibodies. The 460Id was detected both in polyclonal and monoclonal anti-TNP antibodies obtained from K-/- mice. Our results clearly showed that the kappa anti-TNP repertoire is compensated by the lambda repertoire even though the latter is clonally restricted in K-/- mice. PMID- 7535097 TI - CD40 ligation induces lymphotoxin alpha gene expression in human B cells. AB - CD40 plays an important role in T cell mediated B cell proliferation and isotype switching. The cytokines tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) alpha are expressed by B cells, and are known to play a role in B cell activation. We have studied TNF-alpha and LT-alpha expression in human tonsillar B cells following stimulation with anti-CD40 mAb. Anti-CD40 induced weak TNF alpha mRNA expression but strong LT-alpha mRNA expression and had little effect on the constitutive expression of LT-beta mRNA in B cells. Induction of TNF-alpha mRNA was inhibited by actinomycin D suggesting that CD40 ligation results in transcriptional activation of the TNF-alpha and LT-alpha genes. Anti-CD40 caused minimal increase in the expression of TNF-alpha on the B cell membrane and no detectable secretion of TNF-alpha. Anti-CD40 as well as soluble CD40 ligand caused sustained induction of LT-alpha on the membrane of the B cells lasting up to 120 h but induced no detectable secretion of LT-alpha. IL-4, a cytokine known to synergize with anti-CD40 in inducing B cell proliferation and isotype switching, augmented the induction of LT-alpha mRNA and of mLT-alpha expression by anti-CD40. These results indicate that CD40 ligation vigorously induces expression of membrane LT-alpha in B cells and that membrane LT-alpha may play a role in CD40 mediated B cell activation. PMID- 7535098 TI - Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries. AB - The integrin alpha v beta 3 binds promiscuously to cell-adhesive proteins: vitronectin, fibronectin, and several others containing the RGD motif. We have explored molecular recognition by alpha v beta 3 through selection of ligands from large random libraries of peptides displayed on phage. Ligands bound by alpha beta 3 consisted primarily of RGD peptides; however, these peptides showed considerable heterogeneity with respect to the identities of amino acids flanking RGD. The tolerance of alpha v beta 3 for RGD peptides of diverse composition is consistent with its role in vivo as a versatile receptor for RGD-containing extracellular matrix proteins. Peptide ligands for alpha v beta 3 also included a novel binding sequence, identical to a tetrapeptide found in vitronectin, which is a candidate for a synergistic site in this adhesive protein that may act in concert with RGD to promote molecular recognition. PMID- 7535099 TI - Purine functional groups in essential residues of the hairpin ribozyme required for catalytic cleavage of RNA. AB - Synthetic chemistry techniques have been used to study the functional group requirements of the essential purine residues in hairpin ribozyme cleavage. Three stranded ribozymes were prepared that had functional group deletions or alterations at single purine sites within loops A and B of the hairpin, and the kinetics of cleavage were compared to those of the unmodified ribozyme. Adenosine analogues used were purine riboside and N7-deazaadenosine, and guanosine analogues used were inosine, N7-deazaguanosine, and O6-methylguanosine. In many cases, introduction of one of these analogues caused substantial loss of ribozyme cleavage activity. Most of the impairments of activity were found to be due to changes in kcat rather than in KM. The losses corresponded in magnitude to loss of at least one hydrogen bond, and the results were rationalized in terms of removal of potential cross-strand hydrogen bonds as well as potential hydrogen bonds between loops A and B. A new secondary structure model for loop B was proposed. Finally, the magnesium ion dependence of cleavage was studied for the modified ribozymes and compared to that of the unmodified ribozyme. It is proposed that magnesium binds in the ground state to the N7-positions of G + 1 and A43 and in the transition state to the N7-position at A9. The results provide further evidence for the folding of the two arms of the hairpin so that in the active conformation loops A and B approach closely to form a specific three dimensional structure with a magnesium ion (or ions) placed between the loops, making contacts in the ground state and in the transition state. PMID- 7535102 TI - Activation and deactivation of membrane currents in human fibroblasts following infection with human cytomegalovirus. AB - The whole cell patch clamp technique was used to study the effects on membrane currents of infection of cultured human embryonic lung (HEL) fibroblasts with human cytomegalovirus (CMV). Four types of membrane currents were found in uninfected HEL cells, namely: Ca(2+)-activated potassium current, inward rectifier potassium current, delayed rectifier potassium current and voltage dependent CMV. Voltage-dependent sodium current was detected in 30% of uninfected HEL cells whenever they were examined up to 72 h after seeding; however this current had completely disappeared by 18 h after infection with CMV. The delayed rectifier potassium current was detectable in 8% of uninfected HEL cells but, after infection, the proportion of cells expressing this current gradually increased from 20% at 18-24 h post-infection to 100% at 48 h and 72 h. Pharmacological agents known to regulate the activity of ion channels, via cellular secondary messengers, did not alter the frequency at which either current was detected in uninfected and infected cells. Phosphonoformate, an inhibitor of CMV DNA polymerase, caused 95% block of expression of CMV 'late' proteins in infected cells but did not prevent the switching off of the sodium current or the increased expression of the potassium current. The results indicate an association between the expression of CMV 'immediate-early' or 'early' proteins and the down-regulation of the sodium current and up-regulation of the potassium current. PMID- 7535100 TI - Origins of the large differences in stability of DNA and RNA helices: C-5 methyl and 2'-hydroxyl effects. AB - Recent studies have shown that there can be large differences in the stability of double and triple helical nucleic acid complexes, depending on whether RNA or DNA strands are involved. These differences have been attributed to structural differences in the sugar-phosphate backbone of these two polymers. However, since there are in fact two structural features which distinguish DNA from RNA (the 2' hydroxyl and C-5 methyl groups), the stability differences may arise from either or both of these factors. We have separated effects of the 2'-hydroxyl and C-5 methyl groups by synthesizing nucleic acid strands which contain all possible combinations with and without these groups. Studies of the stabilities of double and triple helices involving these strands show that in fact the C-5 methyl group of thymine and the 2'-OH group of ribose have equally large effects on stability. The two effects vary with secondary structure and can be reinforcing or even opposing in their influence on stability. Three types of complexes are specifically examined: bimolecular pyrimidine.purine duplexes, termolecular pyrimidine.purine.pyrimidine triplexes, and bimolecular triplexes formed from circular pyrimidine oligonucleotides with purine target strands. It is found in general that the two types of substitutional effects are independent of one another and that C-5 methyl groups are in all cases stabilizing, while 2'-OH groups can be stabilizing or destabilizing, depending on the type of complex. In addition, studies with partially methylated duplexes lend evidence that the largest contribution to stabilization by the methyl group arises from increased base stacking ability rather than from a favorable hydrophobic methyl-methyl contact.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535101 TI - Differential electrophoretic behavior in aqueous polymer solutions of red blood cells from Alzheimer patients and from normal individuals. AB - The recently reported phenomenon that red blood cells (RBC) from Alzheimer disease (AD) patients and normal individuals, which have identical electrophoretic mobilities (EPM) in phosphate-buffered saline (PBS), have different EPM in appropriately selected polymer solutions, has been further explored. Of a number of in vitro treatments to which AD and normal RBC were subjected prior to EPM measurements in bottom phase (from a dextran-poly(ethylene glycol) (PEG) aqueous phase system) only trypsin eliminated the difference. Thus, the differential polymer interaction between AD and normal RBC, thought to be the basis for their dissimilar EPM, can be abolished by appropriate proteolytic modification of the cell surfaces and suggests protein as a source of difference. Because young and old RBC from normal individuals, which have the same EPM in PBS, have different EPM in certain polymer solutions, and the RBC from AD patients have been reported to age abnormally, we also compared the young and old RBC subpopulations from these two sources. By the criterion of cell electrophoresis in polymer solutions the differences between AD and normal RBC and between young and old RBC are distinct. The EPM of AD and normal RBC differ in bottom phase or PEG but not in dextran solution; while the EPM of young and old RBC differ predominantly in dextran. We speculate that since the observed difference in EPM of RBC from AD patients and normals depends on protein(s) yet is anticoagulant-related (being obtained only when blood is collected in citrate or oxalate) it might be the result of an interaction (Ca(2+)-mediated?) between the surfaces of these cells and protein component(s) of their respective, compositionally differing sera. PMID- 7535103 TI - Clinical trials of antiangiogenic agents. AB - Complete inhibition of angiogenesis should be well tolerated in most adults because under physiologic conditions angiogenesis is required only for wound healing and reproduction. However, angiogenesis is required for malignant solid tumor growth beyond 1 to 2 cubic millimeters, and microvessel counts in tumor specimens have been correlated with prognosis in patients with malignancies of the breast, prostate, and central nervous system. Antiangiogenic agents that bind to heparin-binding growth factors, inactivate matrix metalloproteinases, or inhibit endothelial cell proliferation are currently being tested as single agents in clinical trials. Recombinant proteins such as interferon alfa and platelet factor 4 may also have antiangiogenic activity through mechanisms that are not yet completely defined. Because they act through diverse mechanisms of action, antiangiogenic agents may achieve maximum biologic effect when administered together. These agents are particularly attractive in the surgical adjuvant setting because the risk for development of drug resistance and induction of second malignancies is low. PMID- 7535104 TI - Isolation of biologically functional RNA during programmed death of a colonial ascidian. AB - The blastogenic (asexual) cycle of the colonial ascidian Botryllus schlosseri (Tunicata, Ascidiaceae) concludes in a cyclical phase of programmed cell and zooid death called takeover, in which all asexually derived adults die synchronously by apoptosis. The characterization of developmentally regulated genes whose expression patterns are selectively modulated during this process could pave the way to understand how this model organism dies. However, isolation of biologically functional RNA in this and other colonial ascidians with conventional phenol/chloroform-based procedures is hampered by extensive contamination of RNA preparations by pigments. Upon cell lysis, pigments that normally reside within specialized cells in the mantle wall of the adult are released and tightly associate with nucleic acids. Here, we report on the usefulness of a single-step RNA isolation method in which acid guanidinium isothiocyanate is used as an extraction medium, followed by preparative cesium chloride ultracentrifugation. This procedure successfully isolated biologically active, high-purity total RNA (OD260/OD280 = 1.9-2.1) from Botryllus colonies during takeover, as well as other species of colonial ascidians (Diplosoma macdonaldii, Botrylloides diegense) irrespective of pigmentation. Northern blot analysis performed with a 32P-labeled tunicate actin probe detected two polyadenylated transcripts of 1.5 and 1.7 kilobases in length from both growth phase and takeover colonies. Two-dimensional protein gel assays from in vitro translated mRNA preparations further revealed that specific transcripts were up regulated during takeover, while others were repressed or down-regulated. Growth phase and takeover-specific cDNA libraries were constructed from pooled poly(A)+ RNA with a complexity of 1.0 x 10(7) and 1.2 x 10(7) recombinants respectively per 100 ng of cDNA before amplification.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535105 TI - Analysis of multiple antigenic determinants of the bovine conglutinin molecule using different synthetic peptides. AB - Twenty-three overlapping peptides corresponding to the complete amino acid sequence of the bovine conglutinin molecule were synthesized on the basis of the sequence data to analyze antigenic determinants. Of the synthetic peptides tested, peptides 31-50, 166-185, and 241-260 were highly reactive with rabbit antibodies against native conglutinin, whereas peptides 1-20, 16-35, 31-50, 76 95, 136-155, 151-170, 181-200, 271-290, 301-320, and 316-335 were less reactive with the antibodies. Peptides 16-35, 31-50, 46-65, 76-95, 136-155, 151-170, 181 200, 271-290, 301-320, and 316-335 were less reactive with the antibodies. Peptides 16-35, 31-50, 46-65, 76-95, 91-110, 106-125, 151-170, 166-185, 211-230, 226-245, and 286-305 inhibited the binding of native conglutinin to mannan. On the other hand, all of these synthetic peptides elicited rabbit antibodies reactive with native conglutinin. Antibodies to peptide 241-260 showed the highest reactivity with native conglutinin. Of these anti-peptide antibodies, however, only antibodies to synthetic peptide 46-65 inhibited effectively the binding of conglutinin to mannan. Antibodies to the remaining peptides failed to block the binding at the highest concentrations tested. These results suggest that the multiple antigenic determinants associated with both ligand binding and immunogenicity may be located on the conglutinin molecule and that the predominant antigenic determinant responsible for binding saccharides may be located in the vicinity of the region 46-65. PMID- 7535106 TI - Analysis of multiple antigenic determinants of Clostridium perfringens enterotoxin as revealed by use of different synthetic peptides. AB - To identify the antigenic determinants of Clostridium perfringens enterotoxin (CPE), overlapping peptides corresponding to the entire amino acid sequence of CPE were synthesized and their antigenicity and immunogenicity were analyzed. Of the 21 synthetic peptides (C-1 to C-21) tested, peptide C-7 (amino acid residues 91-110) showed the highest reactivity with rabbit antibodies against CPE. Peptides C-4 (46-65), C-9 (121-140), and C-11 (151-170) were reactive with the antibodies, whereas peptides C-2 (16-35), C-3 (31-50), and C-20 (286-305) were less reactive. Other peptides were much less reactive. On the other hand, all of the synthetic peptides reactive with the anti-CPE antibodies were found to elicit rabbit antibodies reactive with both their respective antigens and native CPE molecule. These findings demonstrate that CPE molecule contains at least 10 different antigenic determinants which are all immunogenic and supposed to be responsible for functionally and biologically active sites. PMID- 7535107 TI - [Preparation of monoclonal antibodies to E1-component and their use for studying the pyruvate dehydrogenase complex]. AB - A monoclonal antibody against the E1-component of pigeon breast muscle has been prepared. The dissociation constant of the E1-mAb F7F10 complex was determined to be equal to 5.93.10(-8) M. The cross-reaction between mAb F7F10 and the E1 component of the pyruvate dehydrogenase complex from various species (including human beings) was established. The F7F10 antibody was shown to interact with both alpha- and beta-subunits of E1, which suggests that the amino acid residues in the both subunits are constituents of the antigenic determinant. Binding of the F7F10 antibody to the antigen had no effect on the enzymatic activity of E1 but induced rapid inactivation of the pyruvate dehydrogenase complex in the pyruvate:NAD oxidoreductase reaction. The competition between the F7F10 antibody and the E2-component of the pyruvate dehydrogenase complex for the binding to E1 was revealed by immunoenzymatic analysis. It was concluded that the antigenic determinant and the E1 site responsible for the E1-E2 interaction within the pyruvate dehydrogenase complex may overlap. PMID- 7535108 TI - All cytokeratin assays are not the same. PMID- 7535109 TI - Theoretical models of the ion channel structure of amyloid beta-protein. AB - Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs. PMID- 7535110 TI - Hofmeister effect in ion transport: reversible binding of halide anions to the roflamycoin channel. AB - We have studied the anion-dependent gating of roflamycoin ion channels using spectral analysis of noise in currents through multichannel planar lipid bilayers. We have found that in addition to low frequency current fluctuations that may be attributed to channel switching between open and closed conformations, roflamycoin channels exhibit a pronounced higher frequency noise indicating that the open channel conductance has substates with short lifetimes. This noise is well described by a Lorentzian spectrum component with a characteristic cutoff frequency that depends on the type of halide anions according to their position in the Hofmeister series. It is suggested that transitions between the substates correspond to a reversible ionization of the channel by a penetrating anion that binds to the channel structure, more chaotropic anions being bound for longer times. Within a framework of a two substate model, the duration of the substate with reduced electrostatic barrier for cation current varies exponentially with anion electron polarizability. This explains two features of the roflamycoin channel reported earlier: the increase in apparent single-channel conductance along the series F- < Cl- < Br- < I- and the reverse of channel selectivity from anionic for KF to cationic for KI. PMID- 7535111 TI - Potentiation of large conductance KCa channels by niflumic, flufenamic, and mefenamic acids. AB - Large conductance calcium-activated K+ (KCa) channels are rapidly activated by niflumic acid dose-dependently and reversibly. External niflumic acid was about 5 times more potent than internal niflumic acid, and its action was characterized by an increase in the channel affinity for [Ca2+], a parallel left shift of the voltage-activation curve, and a decrease of the channel long-closed states. Niflumic acid applied from the external side did not interfere with channel block by charybdotoxin, suggesting that its site of action is not at or near the charybdotoxin receptor. Accordingly, partial tetraethylammonium blockade did not interfere with channel activation by niflumic acid. Flufenamic acid and mefenamic acid also stimulated KCa channel activity and, as niflumic acid, they were more potent from the external than from the internal side. Fenamates applied from the external side displayed the following potency sequence: flufenamic acid approximately niflumic acid >> mefenamic acid. These results indicate that KCa channels possess at least one fenamatereceptor whose occupancy leads to channel opening. PMID- 7535113 TI - Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2 stretching region. AB - Vibrational circular dichroism (VCD) spectra were measured for H2O solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC)-poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the PO2-stretching region. Only the symmetric PO2 stretch at approximately 1075 cm-1 yields a significant intensity VCD signal. Differences of the PO2-stretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO2- VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO2- VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO2- stretching mode VCD spectra to the base sequence as opposed to that found for the C = O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense. PMID- 7535112 TI - ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions. AB - Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer. PMID- 7535114 TI - Extended dipolar chain model for ion channels: electrostriction effects and the translocational energy barrier. AB - We reinvestigate the dipolar chain model for an ion channel. Our goal is to account for the influence that ion-induced electrostriction of channel water has on the translocational energy barriers experienced by different ions in the channel. For this purpose, we refine our former model by relaxing the positional constraint on the ion and the water dipoles and by including Lennard-Jones contributions in addition to the electrostatic interactions. The positions of the ion and the waters are established by minimization of the free energy. As before, interaction with the external medium is described via the image forces. Application to alkali cations show that the short range interactions modulate the free energy profiles leading to a selectivity sequence for translocation. We study the influence of some structural parameters on this sequence and compare our theoretical predictions with observed results for gramicidin. PMID- 7535115 TI - Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy. AB - A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation. PMID- 7535117 TI - Immunocytochemical localization of nitric oxide synthase in the brain of the chicken. AB - The distribution of neuronal nitric oxide synthase (NOS) in the chicken brain was investigated by immunocytochemistry. Strongly stained neurones were concentrated in the paleostriatum augmentatum, lobus parolfactorius, ventral pallidum, olfactory tubercle, parts of the neostriatum, mesencephalic reticular formation and locus coeruleus. Cells in these areas have previously been shown to stain for NADPH-diaphorase, a histochemical activity associated with NOS. However, various structures which NADPH-diaphorase staining has suggested to contain NOS were not immunoreactive: these included the glomeruli of the olfactory bulb, magnocellular preoptic neurones, the median eminence, subcommissural organ and mesencephalic trigeminal neurones. NOS was sparsely present in the hyperstriatum ventrale, providing evidence against the involvement of nitric oxide in certain forms of learning and memory processes known to occur in this region. PMID- 7535116 TI - Viscosity dependence of O2 escape from respiratory proteins as a function of cosolvent molecular weight. AB - Laser photodissociation of respiratory proteins is followed by fast geminate recombination competing with escape of the oxygen molecule into the solvent. The escape rate from myoglobin or hemerythrin has been shown previously to exhibit a reciprocal power-law dependence on viscosity. We have reinvestigated oxygen escape from hemerythrin using a number of viscous cosolvents of varying molecular weight, from glycerol to dextrans up to 500 kDa. In isoviscous solutions, the strong viscosity dependence observed with small cosolvents is progressively reduced upon increasing the cosolvent's molecular weight and disappears at molecular weights greater than about 100 kDa. Thus, viscosity is not a suitable independent parameter to describe the data. The power of the viscosity dependence of the rate coefficient is shown here to be a function of the cosolvent's molecular weight, suggesting that local protein-solvent interactions rather than bulky viscosity are affecting protein dynamics. PMID- 7535118 TI - Calcium dependence of glutamate receptor-evoked alkaline shifts in hippocampus. AB - Glutamate receptor activation induces an extracellular alkalinization in rodent hippocampus. We studied its Ca2+ dependence and pharmacology in hippocampal slices. Glutamate-evoked alkaline shifts were blocked by 0 Ca2+ saline. Alkalinizations induced by AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid) and NMDA (N-methyl-D-aspartate) were abolished by 20 microM CNQX (6-cyano-7-nitro-nitroquinoxaline-2,3-dione) and 50 microM APV (DL-2-amino-5 phosphonovalerate), respectively. The AMPA- and NMDA-evoked alkaline shifts were blocked by 0 Ca2+, however, AMPA-induced [K+]o elevation was unaffected. These data suggest that the glutamate receptor-channel does not mediate H+ influx, and support a role for Ca(2+)-H+ exchange. PMID- 7535119 TI - Ca2+ channel inhibition by kappa opioid receptors expressed in Xenopus oocytes. AB - Functional coupling between kappa opioid receptors and voltage-dependent Ca2+ channels was studied in the Xenopus oocyte translation system, in which specific RNAs encoding rat kappa opioid receptor, rabbit BI-2 alpha 1 subunit, and human beta subunit were co-injected. Perfusion of the oocytes with U50488H inhibited depolarization-evoked Ba2+ current (IBa) in a reversible manner, showing maximal inhibition of 25% at 1 microM (IC50 = 31 nM). The inhibitory effect of U50488H was desensitized by pre-exposure of the oocytes to U50488H and abolished by the kappa opioid antagonist nor-binaltorphimine and by overnight pretreatment with pertussis toxin. Agents affecting the activity of protein kinase A or C did not affect the U50488H-induced inhibition of IBa. These findings suggest that kappa opioid receptors inhibit the activity of neuronal Ca2+ channels via GTP-binding proteins, without the participation of protein kinase A or C. PMID- 7535120 TI - Neurone-specific enolase persists in some hippocampal cells 24 hours after an NMDA injection. AB - Neurone-specific enolase immunoreactivity (NSE-IR) was examined in the hippocampal dentate gyrus 5 h, 24 h and 6 days following local injection of N methyl-D-aspartate (NMDA), and was compared with Nissl substance and acid fuchsin staining. Five and 24 hours post-injection extracellular NSE-IR was increased, and the number of NSE-IR neurones in the granule cell layer was decreased. However, at 24 h post-injection only, 10-15% of the granule cells were strongly NSE-IR, in contrast to the Nissl/acid fuchsin staining which showed a homogeneous population of degenerating neurones. By 6 days there were no viable neurones in the lesion area stained by either method. These results show that neuronal degeneration after an excitotoxic insult occurs in a non-uniform way in an apparently homogeneous group of central neurones. PMID- 7535121 TI - Reduction of MPP(+)-induced hydroxyl radical formation and nigrostriatal MPTP toxicity by inhibiting nitric oxide synthase. AB - N-Methyl, 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces experimental parkinsonism after oxidation to N-methylpyridinium ion (MPP+), accumulation in dopamine neurons and concentration in mitochondria. Inhibition by MPP+ of mitochondrial electron transport impairs respiratory function, but the molecular mechanisms of cell death are not clear. We tested the hypothesis that locally produced nitric oxide is a key component in MPTP toxicity by providing a necessary intermediate in the production of hydroxyl free radicals. Inhibition of nitric oxide synthase reduced MPP(+)-induced hydroxyl radical formation in striatum and MPTP toxicity to nigrostriatal dopamine terminals, but did not interfere with inhibition of complex-I activity. Nitric oxide appears to be necessary for hydroxyl free radical generation in MPP+ toxicity and may play a role in neuronal degeneration in Parkinson's disease. PMID- 7535122 TI - Cross-modal neuroplasticity in the blind mole rat Spalax ehrenbergi: a WGA-HRP tracing study. AB - Spalax ehrenbergi is a subterranean rodent possessing extremely degenerated eyes but a distinguishable dorsal lateral geniculate nucleus (dLGN) which projects ipsilaterally to a fair sized striate cortex. 2-Deoxyglucose and electrophysiological studies have shown that these areas are vigorously activated by auditory input. We show here, using the neuroanatomical tracer WGA-HRP, that the major source of the auditory input to the visual system is the inferior colliculus that, in addition to all its typical auditory targets, also projects to the dLGN. The visual thalamo-cortical pathway thereupon processes auditory information. This is the first naturally blind mammal in which the origin of auditory compensatory projections have been traced and described. PMID- 7535123 TI - CHARGE association. AB - CHARGE association is a heterogeneous condition that affects several organ systems, including the eye, ear, nose, heart, and genitals. Hearing loss is a common finding in CHARGE association; therefore, audiologists and otolaryngologists need to be familiar with this relatively common entity. PMID- 7535125 TI - Early progression stage of malignancy as revealed by immunohistochemical demonstration of DNA instability; I, Human gastric adenomas. AB - The degree of DNA-instability as revealed by the immunohistochemical staining with monoclonal anti-single-stranded DNA antibody after acid hydrolysis (DNA instability test) was used as the marker of malignancy. This was applied to human gastric regenerative epithelium in chronic peptic ulcer (5 cases), adenoma (35 cases), and well differentiated tubular adenocarcinoma (5 cases). Proliferative activity was evaluated by proliferating cell nuclear antigen (PCNA) immunohistochemistry, and the quantitative analyses of the mean number and mean area of silver-stained nucleolar organizer regions (AgNORs) per one nucleus were performed for all these cases. All cancers and adenomas were positively stained by the DNA-instability test diffusely, indicating the malignant character of the latter from the view point of DNA-instability, in contrast to the negative stainability of all regenerative epithelium. The percent number of PCNA-positive cells and mean number and mean area of AgNORs tended to be larger in adenoma and cancer than in regenerative epithelium, although the differences were not usually statistically significant. Supporting the malignant character of adenoma, single cell necroses and abnormal mitoses were almost always present in the lesion. In conclusion, all adenoma lesions were regarded as malignant in nature, namely, in situ carcinoma, existing at an early stage of progression of malignancy. PMID- 7535124 TI - An investigation into the use of protein cross-linking agents as cell fixatives for confocal microscopy. AB - A variety of compounds which are known to cross-link proteins have been tested as possible cell fixatives for confocal microscopy. The criteria for good fixation that were used for this work were that the fixative should make the cells resistant to lysis and the loss of protein from the cell under hypotonic conditions. In addition, fixation should not change the shape nor the volume of the cell. Of the compounds tested, one compound, EDC, appeared to have an excellent potential as a cell fixative and has the additional advantage that it does not require amino groups for its crosslinking activity. Another compound, SPDP, appeared to have some potential as a readily reversible fixative by virtue of its disulfide bridge. PMID- 7535126 TI - Early progression stage of malignancy as revealed by immunohistochemical demonstration of DNA instability; II, Otorhinolaryngeal border-line neoplastic lesions. AB - The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to benign, border line, and malignant neoplastic lesions found in the otorhinolaryngeal regions (31 cancer, 22 leukoplakia, 10 nasal inverted papilloma, 33 salivary gland pleomorphic adenoma, and 7 Warthin's tumor cases). Proliferative activity and polarity of the proliferative cell distribution were evaluated by PCNA immunohistochemistry, and the quantitative analyses of the number, mean size, largest size, and maximum shape-irregularity of AgNORs in a nucleus were performed for all these cases. As the results, 31 cancer (100%), 20 leukoplakia (90.1%), 10 nasal inverted papilloma (100%), and 21 pleomorphic adenoma (63.6%) cases were positively stained by the DNA-instability test diffusely or sporadically, indicating their malignancy. Reflecting the malignant character, these cases showed a remarkable increase in the PCNA-index with the loss of polarity of PCNA-positive cell distribution, and also increased number, mean and largest sizes, and maximum shape-irregularity of AgNORs. These results indicate that all nasal inverted papillomas are malignant in nature, namely, in situ carcinoma, and the majority of leukoplakia is also regarded as in situ cancer, although a certain percentage of simple hyperplasia may be included. Furthermore, the pleomorphic adenoma of the salivary gland can be regarded as an "unstable tumor" which often contains or predisposes to bear malignant subclones with occasional capsular or extracapsular invasion, reflecting the progression of malignancy. In the present study, no sign of malignancy was detected in Warthin's tumor. PMID- 7535127 TI - Langerhans cells in the immunopathology of contact allergic dermatitis. AB - The functional morphology, topography and frequency of Langerhans cells (LCs), which are significant factors in the pathogenesis of contact allergic dermatitis (CAD), were studied by histoenzymatic methods (adenosine triphosphatase (ATP ase), acid phosphatase (AF) and alpha naphtylacetate esterase (ANAE), immunohistochemical methods (indirect immunoperoxidase (IPO) with the monoclonal antibody OKT 6), and the peroxidase-antiperoxidase (PAP) method with the polyclonal S-100 antibody in skin biopsies of 24 patients with CAD, as well in skin biopsies in experimental models in guinea pigs. The results confirmed the significant role of LCs in the pathogenesis of contact allergic dermatitis. PMID- 7535128 TI - Histochemical and ultrastructural changes induced by zidovudine in mitochondria of rat cardiac muscle. AB - Zidovudine (azidothymidine, AZT), a drug used in acquired immune deficiency syndrome (AIDS), blocks reverse transcriptase and therefore inhibits human immunodeficiency virus (HIV) replication. We carried out an ultrastructural and histoenzymatic study in rat cardiac muscle. Groups of animals (3 rats per group) were given drinking water with or without AZT (1 or 2 mg AZT/ml). After 30, 60 and 120 days, the hearts were studied by light and electron microscopy. Histochemical analysis of isocitrate, succinic, malic, NADH and NADPH dehydrogenase activities revealed no changes in AZT-treated rats compared with control rats. The ultrastructural study showed a disruption of cristae and an increased size of mitochondria in rats treated with AZT for 30- and 60-days. No alterations were observed in rats that received the 120-day treatment. A statistical analysis based on electron micrographs demonstrated a time-dependent ratio between intact and disrupted mitochondria. Rats that received AZT for 30 days showed a higher number of abnormal mitochondria than rats that received the 60 day treatment. No differences with respect to rat controls were observed in the rats that received AZT for 120 days. We conclude that AZT-induced ultrastructural alterations in cardiac muscle did not modify the histochemical activity of several mitochondrial enzymes. PMID- 7535129 TI - Ontogenesis of some endocrine cells in the duck gastrointestinal tract. AB - The time of appearance, morphology, and topographic distribution of somatostatin, neurotensin, bombesin, gastrin/CCK and serotonin immunoreactive cells during embryonic development were studied in the duck gastrointestinal tract by immunohistochemical methods. Somatostatin immunoreactive cells first appeared in the duodenum of duck embryos at 9 days of incubation (d.i.). They progressively appeared in the other segments at 19 d.i., and at hatching they were present in all gastrointestinal segments except for the caecum. At hatching, the antrum was the richest region in somatostatin endocrine cells, and the gizzard the poorest. Neurotensin immunoreactive cells were detected at 21 d.i. in the proventriculus, antrum, duodenum, and rectum; at 23 d.i. they were present in all the other segments. Bombesin immunoreactive cells were observed in the proventriculus at 17 d.i., and in the gizzard and antrum at 23 d.i. No cells were detected in the intestinal segments. Gastrin/CCK immunoreactive cells first appeared at 17 d.i. in the antrum region; at 21 d.i. they appeared in the small intestine and around hatching they were found in the other intestinal segments except for the proventriculus and gizzard. Serotonin immunoreactive cells appeared at 21 d.i. in the proventriculus, duodenum, and jejunum-ileum. At 23 d.i., they were present in all other segments. Our results show that the time of appearance of immunoreactive cells may be related to the general development of the gut wall in the duck and may reflect cell differentiation in the mucosa. PMID- 7535130 TI - Electron spectroscopic imaging of secretory granules in human eccrine sweat glands. AB - A study of human eccrine sweat glands by electron spectroscopic imaging has been performed. A high nitrogen content in secretory granules of the dark mucous cells has been found while mucous cells in the digestive or respiratory systems did not contain nitrogen detectable in our experimental conditions. High concentrations of nitrogen were found in zymogenic granules and in the core of the granules in the eosinophilic leucocytes. Our findings seem to suggest that the granules of the dark secretory cells are composed not only of mucoid substances but contain also high concentrations of proteins. The descriptive term "granulated cells" seems to be more correct than "mucous cells" to indicate these elements. PMID- 7535131 TI - Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry. AB - The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations. PMID- 7535132 TI - The T-cell repertoire contains cells reactive with hormones of the hypothalamic pituitary-adrenal axis: recognition of synthetic peptide fragments of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) in the Lewis rat. AB - This report characterizes T-cell lines developed against peptide fragments of the neuroendocrine hormones, corticotropin-releasing hormone (CRH) and pro opiomelanocortin (POMC). A MHC Class II binding motif containing a serine (S) and glutamic acid (E) residue separated by five intervening amino acids was used as a template for synthesizing peptides that may serve as T-cell epitopes. T-cell lines were generated specifically against a 17-amino-acid peptide of POMC or CRH peptide. These T-cell lines were predominantly CD4+ T cells and proliferated in an antigen-specific fashion. Furthermore, proliferation of T-cell lines specific for peptide-hormones could be inhibited by anti-MHC Class II antibody. In vitro the whole CRH protein could be processed and recognized as antigenic by CRH peptide-specific T cells. In addition, POMC-specific T cells can recognize POMC peptide presented on the membrane of MHC Class II+ POMC T cells. These results indicate that the normal T-cell repertoire of the rat contains elements which can recognize and specifically proliferate to self-proteins of the hypothalamic hypophyseal axis. Moreover, it seems that T lymphocytes themselves may present antigens which they synthesize. The relationship of these observations to autoimmune reactions affecting the hypothalamus and/or pituitary gland, or T-cell regulation, is the subject of ongoing investigation. PMID- 7535133 TI - Predicting antigenic determinants in proteins: looking for unidimensional solutions to a three-dimensional problem? AB - In a recent review, Hopp (Peptide Research 6:183-190, 1993) claimed that the Hopp and Woods hydrophilicity method for locating antigenic determinants is superior to all other existing methods for predicting the B cell epitopes of proteins but that it is not useful to aid the investigator in producing peptide-protein cross reactive antisera. In this article, we challenge both these assertions. Most investigators utilize antigenicity prediction algorithms because they wish to produce anti-peptide antibodies capable of cross-reacting with the intact protein. All prediction methods are based on propensity scales for the 20 amino acids, which describe the tendency of each residue to be associated with properties such as hydrophilicity, surface accessibility or segmental mobility. When we compared the prediction efficacy of 22 different scales, taking into account both correct and incorrect predictions, we found that none of the scales gave a level of correct prediction higher than about 50%-60%. If no antigenicity was found in a particular region of the protein, we took the view that hydrophilicity peaks located in that region amounted to wrong predictions. The much higher success rate reported by Hopp for this method stems from the way he assesses prediction efficacy, i.e., by counting the number of known epitopes located inside and outside hydrophilicity peaks. Reasons for the low success rate of antigenicity prediction are discussed. In most cases, it is unrealistic to try to reduce the complexity of discontinuous, conformational epitopes to simple, linear peptide models. PMID- 7535134 TI - Different views of protein antigenicity. PMID- 7535135 TI - Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1. AB - To identify critical amino acid residues for carbohydrate binding of galectins (soluble beta-galactoside-binding lectins found in the animal kingdom). site directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992) J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate binding site of galectin. PMID- 7535136 TI - The recognition of three different epitopes for the H-type 2 human blood group determinant by lectins of Ulex europaeus, Galactia tenuiflora and Psophocarpus tetragonolobus (winged bean). AB - The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc alpha (1-2)Gal beta (1-4)GlcNAc beta Me) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc alpha (1-2)Gal beta (1-4)Xyl beta Me was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction. PMID- 7535139 TI - Surgical management of cancer pain. AB - Although the technology has improved rapidly and provided many different techniques, information is lacking concerning the application of the technology. These techniques are applied, for the most part, only to patients with severe pain, because many of the nonsurgical options adequately manage pain that is minimal or mild in severity. Decisions for selection of a specific technique can be based on expected survival time, need for focal or diffuse pain control, or preference for ablative or augmentative options. For patients with good prognosis, augmentative techniques or simple ablative techniques are used more frequently, whereas ablative procedures with higher risk or destruction of more tissue are used later. Patients with severe pain and a poor prognosis represent the most difficult treatment group for three reasons: (1) the severity of the pain and the tendency of the pain to worsen significantly during the patient's remaining time, (2) the limited expected survival time of the patient, which some what limits the use of an expensive, complicated intervention or operation, and (3) the considerable controversy that has been generated by the large number of different treatment options for this group. Because of these issues, there is not a general consensus concerning a treatment algorithm for this group of patients. This emphasizes the need for controlled, comparative studies of systemic treatments and interventional treatments as well as side-by-side comparisons of the efficacy and complications of the different types of surgical techniques. In light of the recently released cancer pain guidelines from the United States Agency for Health Care Policy Research, one would hope that the research of the next 5 years would focus on providing many of the answers to these problems. PMID- 7535137 TI - Does an animal peptide: N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein? AB - Recently, we have reported purification and characterization of a de-N glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L 929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L 929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively. PMID- 7535140 TI - Polyostotic heterogeneity of the spine in osteoporosis. Quantitative analysis and three-dimensional morphology. AB - It was the aim of this study to record quantitatively and qualitatively the distribution of the three-dimensional microarchitecture throughout the human spine in osteoporosis. Bone biopsies of the iliac crest and the complete spine of 26 autopsy cases without skeletal disease and 11 female patients with proven osteoporosis were removed. Grindings of all vertebrae by a technique which we developed allowed two- and three-dimensional measurements simultaneously. The analysis included an evaluation of trabecular bone volume, trabecular interconnection, and trabecular thickness, as well as a qualitative investigation of the structure of cancellous bone. The bone loss in osteoporosis is a loss of structure. The relative loss of the trabecular microarchitecture is greater in the iliac crest than in the lumbar spine. It is a gradual change from normal bone to osteoporosis. Transformation from plates to rods and the loss of whole trabeculae are caused by perforations. The polyostotic heterogeneity in osteoporosis is remarkable. Adjacent vertebrae may show differences of up to 100% in bone structure and bone volume. This explains the difficulties in early diagnosis of osteoporosis. Due to the polyostotic heterogeneity it is impossible to define a threshold mineral content for osteoporotic fractures. PMID- 7535138 TI - Lectin domains in the toxin of Bordetella pertussis: selectin mimicry linked to microbial pathogenesis. AB - The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells. Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs. This is illustrated by the toxin of Bordetella pertussis. Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages. These activities are mediated by the lectin-like properties of the binding oligomer of the toxin. A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities. Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis. PMID- 7535141 TI - [The polyethylene oxide correction of the hemodynamic sequelae in the infusion therapy of massive blood loss]. AB - High molecular weight polyethylene oxides (2 x 10(6), 4 x 10(6), and 6 x 10(6)) were tested for their effects on blood pressure, cerebral circulation in rats with massive hemorrhage and turbulent blood flow velocity in the pre- and posthemorrhagic and polyglucine posttreatment. Polyethylene oxide (4 x 10(6) and 6 x 10(6)) alleviated hypotension and cerebral hypoperfusion, which appeared 0.5 1 hour after polyglucine therapy and increased turbulent blood flow velocity. Polyethylene oxide failed to affect spontaneous and alcian blue-induced red blood cell aggregation. PMID- 7535142 TI - Delayed commencement of granulocyte colony-stimulating factor following autologous bone marrow transplantation accelerates neutrophil recovery and is cost-effective. AB - It is known that Granulocyte colony-stimulating factor (G-CSF) accelerates neutrophil recovery following bone marrow transplantation (BMT), though the optimal timing is not clear. We have undertaken a pilot study in 19 recipients of autologous BMT for non-myeloid malignancy, in which G-CSF was commenced 10 (13 cases) or 7 (6 cases) days after BM infusion. These patients were compared with 18 historical controls, who did not receive G-CSF. The median time to achieve both 0.5 and 1.0 x 10(9) neutrophils/Litre was significantly shorter in the treated group (18 and 21 days respectively) than the control group (20.5 and 29 days; p = 0.03 and 0.02 respectively). No differences between the two groups were seen for the number of febrile days, days on antibiotics or the cost of the antibiotics. G-CSF-treated patients remained in hospital for significantly less time after marrow infusion (21 days compared to 29 days; p = 0.007). The cost of the G-CSF therapy was offset by the decreased bed utilisation, so that the median combined antibiotic, G-CSF and hospitalisation cost was 754 pounds less for G-CSF treated patients. It is concluded that delaying the commencement of G-CSF after autologous BMT accelerates neutrophil recovery, and may allow earlier discharge from hospital, whilst not adversely affecting the cost of the procedure. PMID- 7535144 TI - TNF-alpha, the great imitator: role of p55 and p75 TNF receptors in hematopoiesis. AB - The clinical application of tumor necrosis factor-alpha (TNF-alpha) has so far been limited due to the severe adverse effects associated with its systemic use. Recently, two distinct TNF receptors with molecular weights of 55 kDa (TNFR55) and 75 kDa (TNFR75) have been cloned and characterized. The subsequent development of TNF-alpha mutants with selective activity on either TNFR55 or TNFR75 suggest that such mutants might maintain the therapeutic (anti-tumor) potential of wild type TNF-alpha, but exhibit reduced toxicity (proinflammatory effects). In the present article we discuss previous studies on the effects of TNF-alpha in in vitro and in vivo hematopoiesis. In addition, we summarize more recent data from our laboratory as well as others, elucidating the role of TNF alpha as a direct bifunctional regulator of in vitro hematopoiesis. Specifically, TNF-alpha is a potent inhibitor of the clonal growth of primitive and committed murine and human bone marrow progenitors in combination with multiple cytokines, including granulocyte colony-stimulating factor (G-CSF), CSF-1, erythropoietin (Epo), stem cell factor (SCF), and flt3 ligand (FL). In contrast, TNF-alpha at low concentrations can synergistically and directly enhance the clonal growth of primitive and more mature human CD34+ bone marrow progenitors when combined with GM-CSF or interleukin (IL)-3. Thus, a critical determinant of whether TNF-alpha elicits a stimulatory or inhibitory effect on the in vitro growth of hematopoietic progenitors appears to be the specific growth factors with which it interacts, rather than the maturity of the targeted progenitor. Furthermore, we describe the involvement of the two TNF receptors in signaling in vitro hematopoietic effects of TNF-alpha. Whereas TNFR55 is involved in most observed responses to TNF-alpha, signaling of TNFR75 appears to be restricted to inhibitory effects on primitive progenitors. Finally, we discuss the complexity of direct and indirect actions of TNF-alpha in in vivo hematopoiesis, and the potential clinical applications of TNF-alpha or TNF mutants. PMID- 7535143 TI - Granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, PIXY-321, stem cell factor, interleukin-3, and interleukin-7: receptor binding and effects on clonogenic proliferation in acute lymphoblastic leukemia. AB - Cytokines are frequently used after chemotherapy of leukemias and solid tumors to augment recovery of normal hematopoiesis. While the regulation of normal and leukemic myelopoiesis is well investigated, little is known about effects of cytokines on growth and differentiation of lymphoblastic leukemia. In this study, we investigated the expression of receptors for G-CSF, GM-CSF, SCF, IL-3, and IL 7 on acute lymphoblastic leukemia (ALL) blasts and the effects of these growth factors (GF) on ALL blast colony formation. The binding of fluorescence-tagged cytokines to receptors on ALL blasts was studied by flow-cytometry in 27 cases of ALL (24 precursor B-ALL, 3 T-ALL). Receptor-binding for myeloid-associated GF was observed in the majority of precursor B-ALL (G-CSF = 100%, GM-CSF = 65%, IL-3 = 83%, SCF = 74%), but not in T-ALL. Binding of labelled IL-7 was detected in both precursor B- (92%) and T-ALL (100%). The presence of receptors for SCF in ALL was confirmed by polymerase chain reaction for c-kit mRNA in 19/21 cases tested. Expression of receptors for G-CSF, GM-CSF, IL-3, and SCF was not associated with expression of myeloid antigens, or with specific cytogenetic abnormalities. The effects of these GF on clonogenic cells were tested in the ALL blast colony assay and varied between samples, but all cytokines were able to increase clonogenic growth. The GM-CSF/IL-3 fusion molecule PIXY-321 was most effective in promoting colony growth. In some cases inhibition of colony formation was found. We conclude that ALL blast cells have receptors not only for IL-7, but also for G CSF, GM-CSF, SCF, and IL-3. ALL precursors can respond to these GF with changes in their clonogenic growth indicating the presence of functional receptors. Results may have implications for therapeutic approaches combining cytokines and chemotherapy. PMID- 7535146 TI - Peripheral blood stem cells: in vivo biology and therapeutic potential. AB - Peripheral blood stem cells (PBSC) have been studied for their use after high dose chemotherapy. The combination of a standard-dose chemotherapy [VIP: VP16 (etoposide), ifosfamide, cisplatin] in combination with hematopoietic growth factors was shown to provide effective anti-cancer activity as well as to enable sufficient stem cell mobilization for clinical use. Different growth factor regimens [granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, interleukin (IL)-3/GM-CSF] resulted in a differential induction of high levels of circulating PBSC after VIP chemotherapy, with the sequential combination of IL-3 and GM-CSF inducing maximal numbers of CD34+ cells as well as clonogenic progenitors. Our studies revealed a correlation between prior treatment and PBSC recruitment: the highest numbers of PBSC were mobilized in untreated patients whereas stem cell harvest was considerably impeded in heavily pretreated patients. Phase I/II trials demonstrated that transplantation of PBSC collected after VIP plus growth factor mobilization was safe, and engraftment was rapid and sustained. However, PBSC mobilization carried the risk of concomitant tumor cell recruitment in patients with detectable levels of tumor cells prior to therapy and in 21% of patients without circulating tumor cells. Positive selection of CD34+ cells by immunoadsorption that leads to an approximately three log depletion of contaminating tumor cells therefore was investigated with regard to feasibility and capability as a source for PBSC transplantation. Twenty-one patients with advanced malignancies received autologous CD34+ cell transplantation after high-dose chemotherapy. Hematological recovery was as rapid as recorded for unseparated PBSC preparations, indicating that CD34+ cells can be safely used for autologous PBSC transplantation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535145 TI - The mobilization of primitive hemopoietic progenitors into the peripheral blood. AB - There is considerable interest in the use of peripheral blood progenitor cells (PBPC) for hemopoietic rescue following high dose chemotherapy. Current regimens mobilize CD34+ with variable efficacy and there remains considerable empiricism in the design of these regimens. Some involve myelosuppression, some the administration of various cytokines alone or in combination, while a combination of chemotherapy and cytokines is employed in others. Certain protocols result in mobilization within one week while in others, maximal PBPC levels occur only after several weeks. Thus, procedures required for optimal mobilization of PBPC remain to be defined. An understanding of the mechanisms responsible for mobilization may lead to the development of improved mobilization strategies. Herein we review data that explore the mechanisms involved in the mobilization of PBPC in man. These data demonstrate that mobilization is associated with marked changes in the expression and function of cell adhesion molecules (CAMs) on hemopoietic progenitor cells (HPC), suggesting that the release of HPC into the blood involves a perturbation of the adhesive interactions between these cells and the marrow stroma that, in steady-state conditions, serve to restrict HPC to the bone marrow. Downregulation of c-kit is invariably associated with successful mobilization which, when combined with data from in vitro studies, implies a key role for stem cell factor (SCF) as an orchestrator of mobilization. PMID- 7535148 TI - Analysis of steel factor (stem cell factor) isoforms in the hematopoietic microenvironment. AB - Hematopoietic cell proliferation and differentiation is dependent in part on the interaction of hematopoietic stem and progenitor cells with cells making up the hematopoietic microenvironment (HM). Direct cell-cell interactions appear to be important in the hematopoietic microenvironment. One mechanism to accomplish such interactions is the expression of membrane-associated growth factors. Stem cell factor (SCF), the product of the steel gene in mice (also termed mast cell growth factor, c-kit ligand, or Steel factor), is a hematopoietic growth factor demonstrating substantial synergistic activity with a number of other cytokines on primitive hematopoietic stem and progenitor cells. Cloned SCF cDNA encode both a membrane-associated and a secreted growth factor. The physiologic relevance of these isoforms is unknown at present. In order to better understand the physiologic role of these SCF isoforms in normal hematopoiesis, we have established multiple stromal cell lines expressing each isoform. We have used these cell lines to study protein sequences that are required for appropriate post-translational processing of SCF protein in HM-derived stromal cell lines. These lines have also been used to study the interaction of membrane-associated and secreted SCF with murine and human hematopoietic cells. In addition, we have generated transgenic mice expressing each isoform of murine and human SCF. These transgenic mice will be used to study the function of each isoform in hematopoiesis in vivo. PMID- 7535147 TI - The clinical utility of granulocyte colony-stimulating factor: early achievements and future promise. AB - Recombinant granulocyte colony-stimulating factor (rHuG-CSF) is a hematopoietic growth factor that acts selectively on the neutrophil lineage, and has had a major impact on clinical practice. Two forms are in clinical use: filgrastim has been approved for use in more than 45 countries for the amelioration of chemotherapy-induced neutropenia and restoration of granulopoiesis following bone marrow transplantation and lenograstim has been approved in Europe and Japan. In some countries, rHuG-CSF is also approved for various other indications, such as severe chronic neutropenia. Infection and neutropenia are a major cause of morbidity and mortality following cytotoxic chemotherapy, and there is a known correlation between neutropenia and the risk of infection. Hematopoietic growth factors have been used successfully in the prevention and treatment of neutropenia. There is evidence to suggest that use of rHuG-CSF before the onset of neutropenia allows patients to receive the maximum benefit; however, patients who do not receive rHuG-CSF prophylactically still benefit from the use of rHuG CSF for the treatment of febrile neutropenia. These patients have an accelerated neutrophil recovery and a shorter duration of febrile neutropenia. These effects seem to translate into a significant reduction in the number of patients requiring prolonged hospitalization. This paper reviews the use of rHuG-CSF in the treatment of febrile neutropenia and describes how it is routinely used by hematologists and oncologists in non-clinical trial settings. PMID- 7535149 TI - The flt3 ligand: a hematopoietic stem cell factor whose activities are distinct from steel factor. AB - A number of growth factors have been described that affect the hematopoietic system. Among this group are Steel factor (also known as mast cell growth factor, stem cell factor and kit ligand), and the more recently described flt3 ligand. These factors have been shown to function by binding to and activating the c-kit and flt3 tyrosine kinase receptors, respectively. Both of these factors stimulate the growth of mouse and human hematopoietic progenitor cells. These factors therefore differ from such later acting hematopoietic factors as colony stimulating factor (CSF)-1, which regulates the growth, survival and differentiation of monocytic cells through the c-fms tyrosine kinase receptor. Like Steel factor, the flt3 ligand has little biological activity on its own, but synergizes well with a number of other colony stimulating factors and interleukins. One major difference between the two factors appears to be their effect on mast cells. Steel factor stimulates both the proliferation and activation of mast cells, while preliminary data with the flt3 ligand suggests that it has no effect on mast cells. Although the flt3 ligand and Steel factor each act on early hematopoietic cells, differences in their activities suggest that they are not redundant and are both required for normal hematopoiesis. PMID- 7535150 TI - Recognition and elimination of senescent erythrocytes: implication of antibodies specific for malonic dialdehyde-protein adducts, as demonstrated by flow cytometry. AB - Many different hypotheses have been formulated about the mechanisms of specific recognition of senescent red blood cells (RBC). It is usually assumed that novel epitopes appear on RBC membranes during ageing and are responsible for recognition of aged RBC by antibodies, which is followed by binding to mononuclear phagocytes and then phagocytosis. But these age-related epitopes have not so far been identified. Lipoperoxidation is known to produce aldehydes, among which malonic dialdehyde (MDA). This dialdehyde reacts with primary amino groups of biological molecules, producing 1-amino-3-imino propene (AIP) bridges, and we had previously shown that sera of healthy mammals contain antibodies recognizing epitopes containing AIP bridges (AbAIP). Lipoperoxidation is responsible for many age-related damages in RBC membrane, and we tried in the present work to determine whether age-related epitopes responsible for recognition of aged RBC were not derived from lipoperoxidation. Using flow cytometry techniques, we demonstrated that some of the epitopes recognized by immunoglobulins which bind to aged RBC contain AIP bridges, and that some of these RBC-bound immunoglobulins are AbAIP. Consequently, AbAIP/AIP bridges interactions appear to play a role in recognition and elimination of senescent RBC. PMID- 7535151 TI - Biological characterization of stem cell present in mobilized peripheral blood of CML patients. AB - Peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood and whether stem cells isolated from CML patients are depleted of Philadelphia chromosome positive cells. The presence of stem cells in mobilized peripheral blood (MPB) was examined by staining the CD34+Lineage- (Lin-) population for the expression of the Thy-1 antigen. The extent and kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied. In our analysis the percentage of these cells was found to vary widely depending on the day of leukapheresis and the individual patient. Fluorescence activated cell sorting (FACS) was used to isolate Thy-1 subsets from MPB which were analyzed for activity in in vitro long term cultures and in two in vivo models in which SCID-hu mice were implanted with human fetal bone or thymus grafts. The long term culture assay shows that the cells which express Thy-1 are enriched for cobblestone area forming activity (CAFC), an assay which can be used as a measure of detecting PBSC's in vitro. This CD34+Thy-1+ Lin cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy 1+Lin- cell subset possessed a higher ability to engraft and to produce multilineage hematopoietic lineages in the SCID-hu bone assay and T cells in the SCID-hu thymus assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535152 TI - Ph-negative blood progenitor cells (BPCs) can be recruited after chemotherapy and G-CSF during early hemopoietic recovery in patients at diagnosis of CML or pretreated only with hydroxyurea. PMID- 7535154 TI - Intensive treatment in order to minimize the Ph-positive clone in chronic myelogenic leukemia (CML). Swedish CML-group. PMID- 7535156 TI - An improvement of the endocrine diagnosis by high-performance liquid chromatography. AB - The assessment of catecholamines (NE, E), essential metabolites (VMA, HVA) and serotonergic metabolite 5HIIA in urine by HPLC with UV detection is of interest for clinical diagnosis concerning HTA, pheochromocytoma, neuroblastoma, carcinoid syndrome, hypotension, etc. In the same urine sample one can detect (following preliminary steps) by HPLC the above--mentioned biochemical parameters with good reproducibility and sensitivity. PMID- 7535153 TI - Selection of Philadelphia-negative progenitors from chronic myelogenous leukemia. PMID- 7535157 TI - Is non-invasive diagnosis possible in Down's syndrome? PMID- 7535155 TI - Vitamin D3, a possible nuclear protector in normal hepatic and medullar tissue in white Wistar rats treated with cytostatics. Radioisotopic and cytogenetic aspects. AB - Vitamin D3 was given to male Wistar rats in 3 doses totalling 10,000 IU/kg b.w. then, within 24 hrs, girostan (10 mg/kg b.w.), lomustin or CCNU (10 mg/kg b.w.), all cytostatic substances, were injected in a single dose. Association of vitamin D3 with these cytostatics was found to reduce incorporation of 3H thymidine and a return to normal of RNA synthesis. This suggests that vitamin D3 has a protective effect by maintaining the stability of the double helix. Cytogenetically, the level of the recombinations and of the lesions themselves changes in relation to the structure of the alkylant used, and the damage is smaller if vitamin D3 is given before cytostatics. PMID- 7535158 TI - Gel-filtration chromatography. PMID- 7535159 TI - Ion-exchange chromatography. PMID- 7535160 TI - Epitope prediction methods. PMID- 7535162 TI - Multiple and combinatorial peptide synthesis. Chemical development and biological applications. PMID- 7535161 TI - Epitope mapping of a protein using the Geysen (PEPSCAN) procedure. PMID- 7535163 TI - Reversed-phase high-performance liquid chromatography. A semipreparative methodology. PMID- 7535164 TI - Characterization of proteasomes isolated from rat liver. AB - Proteasomes are cylindrical particles which have a pseudohelical arrangement of subunits. On 2D-PAGE gels, rat liver proteasome preparations give rise to up to 25 proteins which are encoded by at least 16 different genes that are all members of the same family. Proteasomes are able to degrade protein substrates to acid soluble peptides. They have at least five different catalytic components which can be distinguished by the use of synthetic peptide substrates and inhibitors which have very different reactivity at the different sites. Proteasomes can undergo conformational changes when treated with various effectors of their multiple peptidase activities. They are found in the nucleus and in the cytoplasm and, in cultured cells, show changes in localization during the course of the cell cycle. PMID- 7535166 TI - Increased numbers of cytokeratin-positive interstitial reticulum cells (CIRC) in reactive, inflammatory and neoplastic lymphadenopathies: hyperplasia or induced expression? AB - A total of 291 enlarged lymph nodes showing a range of reactive-inflammatory processes, primary and metastatic neoplasms were studied to determine the distribution and immunoprofile of their cytokeratin-positive interstitial reticulum cells (CIRC) in comparison with normal nodes. In 258/291 nodes (89%), CIRC numbers were distinctly increased in the subcapsular, paracortical and, occasionally, in the medullary zones; often, these increased CIRC formed networks around follicles, sinuses and vessels. CIRC had comparatively small, irregularly shaped bodies and dendritic processes; occasionally, giant forms were noted. CIRC contained cytokeratins (CK) 8 and 18 but not 19, as shown by immunohistochemistry, and by gel electrophoresis with subsequent immunoblotting. They co-expressed vimentin consistently, alpha-smooth-muscle actin frequently, and desmin less frequently. They did not contain desmoplakins, Factor VIII, S 100, LCA, B and T lymphocyte- and macrophage-associated antigens, chromogranin A, synaptophysin or the A-80 glycoprotein. We found no clear correlation between the increased CIRC and given nodal disease processes. However, CIRC were most abundant in nodes free of but draining malignant tumours; bizarre CIRC assemblies were noted in HIV lymphadenopathy. CIRC appear to represent a subset of the so called "fibroblastic reticulum cells" of lymph nodes. Their function remains undetermined; their increase in diverse lymphadenopathies suggests that they partake in nodal reactions to injury. It remains unclear whether the increase in CIRC relative number is due to proliferation or to CK gene induction processes but their presence and potential capability to undergo hyperplasia with dysplastic forms should alert pathologists to possible diagnostic pitfalls. In addition, we discuss that CIRC may undergo transformation and represent the "cell of origin" of certain CK-positive tumours restricted to lymph nodes. PMID- 7535168 TI - The Palliative Care Centre of Hotel-Dieu Hospital. AB - In 1989, two affiliations of Centre de Soins Palliatifs were created by the Assistance Publique-Hopitaux de Paris, the largest medical complex in Europe. At Hotel-Dieu de Paris, a mobile team from Soins Palliatifs was formed. The members were recruited from hospital services in order to help the team in the care and support of patients with advanced diseases. A description of the service, team activities (care, formation, teaching and research) is proposed. PMID- 7535167 TI - Ethical dilemmas in hospice and palliative care. AB - In order to understand some of the ethical dilemmas that face hospice programs in the United States, one must understand the Medicare Hospice Benefit, which is the model by which hospice programs provide palliative care to terminally ill patients in the United States. Unlike palliative care programs outside the United States, patients must have a prognosis of 6 months or less to receive hospice care under the Medicare Hospice Benefit. Care is reimbursed on a per diem basis, and inpatient care is restricted to pain and symptom management that cannot be managed in another setting. Ethical dilemmas that face physicians referring patients to hospice programs include the ability of clinicians to predict accurately a patient prognosis of 6 months or less, and to what extent hospice programs and clinicians are obligated to provide patients with full information about their illness, as the Medicare Hospice Benefit requires that patients sign an informed consent in order to elect the hospice benefit. There are ethical dilemmas that affect day-to-day patient management in palliative care programs including physician concern over the use of morphine because of possible respiratory depression in the advanced cancer patient, the question of providing enteral or parenteral nutritional support to patients who refuse to eat near the end of life, and the question of providing parenteral fluids to patients who are unable to take fluids during the terminal phases of illness. A final ethical dilemma concerns the methodology for quality of life research in palliative care. By following current research dogma, and only considering patient-generated data as valid, the patient population that most needs to be studied is excluded. A new methodology specifically for palliative care research is needed to provide information on the patients who are cognitively or physically impaired and unable to provide input regarding their needs near the end of life. PMID- 7535169 TI - Colony-stimulating factors for adjunctive therapy of infections in neutropenic cancer patients. PMID- 7535165 TI - Conditioned media of carcinoma cells cultured in hypoxic microenvironment stimulate angiogenesis in vitro; relationship to basic fibroblast growth factor. AB - Conditioned media (CM) harvested from human pulmonary squamous cell carcinoma (QG56), pulmonary small cell carcinoma (QG90) and gastric adenocarcinoma (MKN28) cultivated under hypoxic conditions (3% oxygen), enhanced the angiogenic activity in vitro more than those obtained under normoxic cultivation (20% oxygen). The total length of the tube structures formed by bovine capillary endothelial cells (BCEs) in the CM cultured at 3% oxygen was about 1.5 (QG56 and MKN28) or 1.9 (QG90) times longer than that at 20% oxygen. Tube formation was diminished by the preincubation of CM with anti-basic fibroblast growth factor (bFGF) IgG. After performing the fractionations of the CM and the crude extracts of cell lysates cultured using a heparin-Sepharose column, the mitogenic activity in the CM from all cancer cells at 3% oxygen was about twice that of CM at 20% oxygen, while it decreased in the cell lysates at 3% oxygen to about 40% of those at 20% oxygen. This mitogenic activity of BCEs in the CM from all cancer cells was almost totally suppressed by anti-bFGF IgG, but not with anti-vascular endothelial growth factor IgG. Hypoxia is an important factor in tumor angiogenesis by bFGF or bFGF-like molecule(s) derived from tumour cells. PMID- 7535171 TI - Activation of cAMP-dependent protein kinase triggers a glial-to-neuronal cell fate switch in an insect neuroblast lineage. AB - BACKGROUND: The grasshopper median neuroblast (MNB) is a multipotent progenitor cell that produces neurons and midline glia in distinct temporal phases. The MNB generates pioneer neurons during its first few divisions, and then switches to production of midline glial precursors. After the glia have been produced, the MNB reverts to generating neurons. We have investigated the molecular mechanism underlying the transition from glia production back to neuron production in the MNB lineage. RESULTS: We report evidence that this second transition in the MNB lineage is triggered by the activation of cAMP-dependent protein kinase (PKA). PKA is a heterodimer of a catalytic (PKA-C) and a cAMP-binding regulatory (R) subunit. The R subunit dissociates from PKA-C on binding cAMP, and free PKA-C than translocates into the nucleus. Nuclear localization of PKA-C can thus be used as an indicator of PKA activation within a cell. We have found that PKA-C is translocated into the nucleus at the time of the second switch in the MNB lineage. When PKA is prematurely activated in the MNB by microinjection of purified PKA-C, or by pharmacological agents that elevate intracellular cAMP levels, the glial-to-neuronal cell-fate switch takes place prematurely. Inhibition of PKA activity by microinjection of a peptide inhibitor, or by a non hydrolyzable cAMP analog, blocks the glial-to-neuronal switch. CONCLUSIONS: Our results imply that elevation of cAMP in the MNB, and the resultant activation of PKA, is likely to be a trigger for the glial-to-neuronal cell-fate transition within the MNB lineage. PMID- 7535170 TI - Cancer therapy. Less blood means more sanguinity. AB - Angiostatin, a recently discovered anti-angiogenic factor, offers the hope of long-term control of metastatic cancers following surgery. PMID- 7535172 TI - Mesoderm formation in response to Brachyury requires FGF signalling. AB - BACKGROUND: The Brachyury (T) gene is required for the formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In these species, and in Xenopus, the gene is expressed transiently throughout the presumptive mesoderm, and transcripts then persiste in notochord and posterior tissues. In Xenopus embryos, expression of the Xenopus homologue of Brachyury, Xbra, can be induced in presumptive ectoderm by basic fibroblast growth factor (FGF) and activin; in the absence of functional FGF or activin signalling pathways, expression of the gene is severely reduced. Ectopic expression of Xbra in presumptive ectoderm causes mesoderm to be formed. As Brachyury and its homologues encode sequence-specific DNA-binding proteins, it is likely that each functions by directly activating downstream mesoderm-specific genes. RESULTS: We show that expression in Xenopus embryos of RNA encoding a dominant-negative FGF receptor inhibits the mesoderm-inducing activity of Xbra. We demonstrate that ectopic expression of Xbra activates transcription of the embryonic FGF gene, and that embryonic FGF can induce expression of Xbra. This suggests that the two genes are components of a regulatory loop. Consistent with this idea, dissociation of Xbra-expressing cells causes a dramatic and rapid reduction in levels of Xbra, but the reduction can be inhibited by addition of FGF. CONCLUSION: Formation of mesoderm tissue requires an intact FGF signalling pathway downstream of Brachyury. This requirement is due to a regulatory loop, in which Brachyury activates expression of a member of the FGF family, and FGF maintains expression of Brachyury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535173 TI - Protein kinase C inhibitors, H7 and calphostin C, inhibit induction of DNA synthesis by cytosolic extracts of exponentially growing neuroblastoma cells in isolated nuclei. AB - Cytoplasmic extracts from proliferating Neuro-2a cells contain a protein factor, ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. Cytoplasmic extracts derived from quiescent-made Neuro-2a cells contain none or very little ADR activity, but this activity can be generated after a brief exposure of cytosolic extracts to a membrane-enriched fraction derived from exponentially growing Neuro-2a cells. ADR activity appears at the beginning of the S phase of the cell cycle. Moreover it appears to be a protease, because aprotinin inhibits ADR activity. ADR activity can be also inhibited by the protein kinase C inhibitors, 1-(5-isoquinoline-sulfonyl)-2- methylpiperazine (H7) and calphostin C. PMID- 7535174 TI - Words as symbols. PMID- 7535176 TI - Topographic relationship of the ventromedial lymphatic bundle and the superficial inguinal nodes to the subcutaneous veins. AB - The relationships between lymph vessels and veins are of clinical importance for the prevention of injury to collectors and resulting edema after removal of veins. Injection preparations of the lymph vessels and veins showed that on the dorsum of the foot the collectors overcross the veins; however, in all remaining areas they undercross them. Only the great saphenous vein is overcrossed by lymph collectors. It also has accompanying lymph vessels but its vascular sheath, however, does not contain any lymph vessels. The position of the collectors depends on the thickness of the subcutaneous adipose tissue. On the dorsum of the foot the lymphatics are often closely bound up with the corium; on the lower leg, however, they are embedded in the adipose tissue. Some collectors are closely related to perforator veins. In the knee region the lymph bundle ascends dorsomedially to the medial condylus of the femur. On the thigh, where the subcutaneous fatty tissue is thick, the collectors from three layers. The superficial inguinal lymph nodes draining the leg and the external genitalia are situated around the saphenous opening and are closely related to the subinguinal venous star, so that in surgical intervention the collectors of this region are more exposed to danger than in other regions. PMID- 7535177 TI - Monoclonal antibodies to Legionella Mip proteins recognize genus- and species specific epitopes. AB - Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains. PMID- 7535175 TI - Evidence that TRH controls prolactin release from rat lactotrophs by stimulating a calcium influx. AB - Prolactin (PRL) release and intracellular free calcium concentration [Ca2+]i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd2+ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca2+]i was reversibly inhibited by Cd2+. In a Ca(2+)-free EGTA-containing medium, TRH did not modify [Ca2+]i. PMID- 7535178 TI - Preparation and characterization of antibodies against mouse prion protein (PrP) peptides. AB - Antisera were raised in rabbits against three peptides, representing amino acid sequences 150 to 159, 165 to 174, and 213 to 226 of mouse prion (PrP), which were synthesized by using a multiple antigenic peptide (MAP) system. The reactivities of these sera to PrP were examined by an enzyme-linked immunosorbent assay (ELISA), Western immunoblotting (WB), and immunohistochemical procedures. The results of both ELISA and WB showed that antisera to peptide sequence 150 to 159 (Ab150-159) did not react with purified mouse PrP. On the other hand, sera to the sequence 165 to 174 (Ab165-174) reacted weakly with purified mouse PrP, as detected by WB but not by ELISA. However, antiserum to peptide sequence 213 to 226 (Ab213-226) reacted strongly with mouse, Syrian hamster, and sheep PrP by WB and with mouse PrP as shown by the results of ELISA. Moreover, Ab213-226 clearly detected PrP immunohistochemically in mouse, Syrian hamster, and sheep brains affected with scrapie as well as in the brain of a cow with bovine spongiform encephalopathy. From these data, we conclude that rabbit antiserum against the MAP representing amino acid sequence 213 to 226 of mouse PrP is useful as a diagnostic tool for prion disease of animals. PMID- 7535181 TI - Control of memory CD4 T cell activation: MHC class II molecules on APCs and CD4 ligation inhibit memory but not naive CD4 T cells. AB - Memory or antigen-experienced CD4 T cells differ from naive CD4 T cells both phenotypically by cell surface marker expression, and functionally by their dissimilar pattern of cytokine secretion and activation requirements through their T cell receptor (TCR). We show here that activation of memory CD4 T cells (CD45RBlo subset), but not naive CD4 T cells (CD45RBhi subset), is inhibited by MHC class II molecules on antigen-presenting cells and by CD4 ligation. We propose that the selective negative signal in memory cells is a direct result of the differences in signaling via CD4 and CD3, exemplified in the disparate pattern of tyrosine-phosphorylated proteins visible after activation of the two subsets. In vivo, this inhibitory signal may serve to prevent irrelevant interactions between memory CD4 T cells and bystander MHC class II+ cells, and may also be responsible for the defective functioning of memory CD4 T cells in AIDS. PMID- 7535180 TI - Memory B cells from human tonsils colonize mucosal epithelium and directly present antigen to T cells by rapid up-regulation of B7-1 and B7-2. AB - Human memory B cells that carry mutated IgV region genes were isolated from tonsils by negative selection of IgD+ naive B cells and CD38+ germinal center B cells and plasma cells. They were mainly found within the intraepithelial areas, but not in the B cell follicles of human tonsils. Memory B cells but not naive B cells have the capacity to present antigen directly to T cells, owing to the constitutive expression of the accessory molecules B7-1/CD80 and B7-2/CD86. Signals through antigen receptors and CD40 antigen result in these two molecules being further up-regulated more rapidly and strongly on memory B cells than on naive B cells. The unique anatomical localization of memory B cells beneath the surface of mucosa, together with their strong APC capacity, may explain the well known prompt and robust secondary antibody responses. PMID- 7535182 TI - Lipopolysaccharide interferes with the induction of peripheral T cell death. AB - In mice injected with superantigens, T cells specific for that antigen proliferate and then die. It has been suggested that the target cells die because they encounter superantigen on the surfaces of nonprofessional presenting cells, such as B cells, which cannot deliver costimulatory signals to T cells. A number of reagents that induce costimulatory molecules on B cells were tested. Lipopolysaccharide very effectively prevented T cell death driven by superantigen. Perhaps surprisingly, the action of lipopolysaccharide was not mediated through the expected costimulatory molecule, B7. Rather, the effects of lipopolysaccharide involved the production of inflammatory cytokines, in particular TNF alpha. The rescued cells survived in vitro culture and were resistant to Fas-induced killing. These data demonstrate that LPS can block antigen-induced T cell death perhaps by interfering with Fas signaling. PMID- 7535183 TI - Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. AB - Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation. PMID- 7535179 TI - Humoral immune response against human cytomegalovirus (HCMV)-specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occurring proteins. AB - The humoral immune response to four intracellularly located cytomegalovirus (CMV) proteins was studied in 15 lung transplant recipients experiencing active CMV infections. Five patients had primary infections, and 10 had secondary infections. Antibodies of the immunoglobulin M (IgM) and IgG classes were measured in an enzyme-linked immunosorbent assay (ELISA) system in which procaryotically expressed recombinant proteins were used as a substrate and also in a monoclonal antibody-based capture ELISA which uses naturally occurring proteins as a substrate. The proteins investigated were the lower matrix protein pp65 (ppUL83), the major DNA-binding protein p52 (ppUL44), and the two immediate early proteins IE1 and IE2 (different splicing products of UL123). Higher levels of antibodies were found to pp65 and especially to p52 than to the immediate early antigens. Antibody levels detected in the recombinant protein-based ELISAs were generally lower than antibody responses detected with the matching antigen capture ELISA. Moreover, some patients appeared to have antibodies mainly to epitopes present on naturally occurring proteins. The antibody responses detected in both assays were related to the viral load during infection as assessed by the CMV antigenemia test, which is a quantitative marker for CMV load. It was found that although epitopes on naturally occurring proteins induce higher antibody responses and responses in more patients, antibodies directed to epitopes present on the recombinant proteins were inversely related to the viral load during a CMV infection. Therefore, antibodies to epitopes on the recombinant proteins might be more clinically relevant in this group of lung transplant recipients. PMID- 7535184 TI - Acute-phase protein response and survival duration of patients with pancreatic cancer. AB - BACKGROUND: Current methods to predict survival duration of patients with pancreatic cancer are limited. The aim of this study was to determine whether certain nutritional indices and the acute-phase protein response are prognostic factors independent of disease stage for patients with unresectable pancreatic cancer. METHODS: Variables at the time of diagnosis of 102 patients with unresectable pancreatic cancer were entered into a Cox's proportional hazards model. Included in the analysis were the serum concentration of C-reactive protein (CRP) and albumin, the extent of weight loss, age, sex, and disease stage (International Union Against Cancer criteria). RESULTS: A multivariate analysis in which each factor was adjusted for the influence of the other factors revealed the patient age, disease stage, serum albumin, and serum CRP to be independent predictors of survival. The presence of an acute-phase protein response was the most significant independent predictors of survival duration. The median survival of those with an acute-phase protein response (CRP > 10 mg/L, n = 45) was 66 days compared with 222 days for those with no acute-phase protein response (n = 57, P = 0.001, Mann-Whitney U test). CONCLUSION: The acute-phase protein response is a useful prognostic indicator for patients with unresectable pancreatic cancer. Moreover, the metabolic disturbances associated with an acute-phase protein response of patients with pancreatic cancer may be a worthwhile therapeutic target. PMID- 7535185 TI - Detection of residual prostate cancer after external radiotherapy. Role of prostate specific antigen and transrectal ultrasonography. AB - BACKGROUND: Although the incidence of positive post-radiotherapy biopsies has been reported, the range is considerable (19-93%) and depends partly on patient selection criteria. In addition, the prognostic significance of transrectal ultrasonography (TRUS) and prostate specific antigen (PSA) used alone and in combination in predicting residual cancer after radiotherapy has yet to be determined. METHODS: Transrectal ultrasonography-guided prostate biopsies were performed on 31 unselected patients with prostate cancer 34-77 months (mean, 51 months) after definitive external beam radiotherapy was completed. Biopsy results were compared for pre- and posttreatment parameters (clinical stage, grade, PSA, and TRUS). RESULTS: In 22 patients (71%), residual cancer was detected histologically by biopsy. All but one patient (12/13, 92%) with a pretherapy PSA value greater than 10 ng/mL had a positive biopsy compared with 50% of those (7/14) with a pretherapy PSA value less than or equal to 10 (P < 0.05). After radiotherapy, a positive biopsy was noted for 15 of 16 patients (94%) with a PSA value greater than 2 and in 20 of 23 (87%) of those with abnormal TRUS findings (P < 0.01 and P < 0.005, respectively). CONCLUSIONS: The most clinically useful model for predicting histologically identifiable residual cancer was either a serum PSA value greater than 2 or a PSA value less than or equal to 2 and abnormal TRUS findings. The positive and negative predictive values of the model were 84% and 83%, respectively. The model predicted biopsy results correctly in 26 of 31 patients (84%). Transrectal ultrasonography is recommended as a diagnostic tool for patients whose PSA level ranges from a detectable level to less than or equal to 2 ng/mL. PMID- 7535186 TI - A phase I study of the somatostatin analogue somatuline in patients with metastatic hormone-refractory prostate cancer. AB - BACKGROUND: Somatuline, a somatostatin analogue, has proven to be effective in several animal models of prostate cancer. Preliminary clinical studies also have suggested antitumor activity in patients with prostate cancer. The authors conducted a dose-escalation trial of 25 patients with metastatic hormone refractory prostate cancer. METHODS: Dosages of 4, 7, 10, 13, 18, and 24 mg/day were administered by continuous intravenous infusion for at least 28 days. RESULTS: Plasma levels of insulin-like growth factor-I (IGF-I), but not those of IGF-II, declined modestly during therapy. Toxicities included grade I diarrhea, bloating, infection, nausea, and flatus. The gastrointestinal side effects were typically self-limiting and occurred during the initial portion of treatment cycles. In addition, three patients experienced grade II catheter-related infections. No clinical response was noted by either radiographic or tumor marker criteria. The maximally tolerated dose of somatuline was not determined. CONCLUSION: A continuous intravenous infusion of 24 mg/day of somatuline is well tolerated and could be evaluated in other types of cancer or possibly in less advanced prostate cancer, but no clinical activity was noted at this dose in patients with advanced metastatic hormone-refractory prostate cancer. PMID- 7535188 TI - Lipopolysaccharide core region of Hafnia alvei: structure elucidation using chemical methods, gas chromatography-mass spectrometry, and NMR spectroscopy. AB - Sugar and methylation analysis with the use of gas chromatography-mass spectrometry and 1H NMR spectroscopy proved that the core oligosaccharides isolated from lipopolysaccharides of eight Hafnia alvei strains have the identical hexasaccharide skeleton. However, 1H, 31P heterocorrelated spectra showed that the phosphorylation pattern is not the same. The branched heptose for the ATCC 13337, 1187, 2, 1191, 1196, 1220, and 481L strains is phosphorylated as in the following formula, where P = -O-P(O)(O-)2 and P-PEtN = [-O-P(O)(O-)]2 O(CH2)2NH3+ [formula: see text] A different phosphorylation pattern was found for the 1211 strain, where the branched heptose residue is 6-substituted by a monophosphorylethanolamine group, ...-->3(-->7)(PEtN-->6)-alpha-LD-Hepp-(1- >3)..., where PEtN = -O-P(O)(O-)-O(CH2)2NH3+. PMID- 7535187 TI - Analysis of chromosomal abnormalities in human sperm after chemotherapy by karyotyping and fluorescence in situ hybridization (FISH). AB - The frequency of numerical and structural chromosomal abnormalities was studied in the sperm of a lymphoma patient 3 years after MACOP-B chemotherapy (CT). Sperm karyotyping was performed by fusion of human sperm with hamster oocytes and analysis of 193 Q-banded sperm chromosomes. Multicolor fluorescence in situ hybridization (FISH) was performed on 10,228 sperm for analysis of disomy frequencies for chromosomes 1 and 12 and on 10,664 sperm for chromosomes X and Y. Sperm karyotyping demonstrated numerical abnormalities in 7.3% of the spreads, 6.7% hypohaploid and 0.5% hyperhaploid, giving a conservative estimate of aneuploidy of 1%. Structural chromosomal abnormalities were present in 7.3% of the karyotypes and 0.5% had both numerical and structural abnormalities. Results of FISH analyses yielded disomy frequencies of 0.10%, 0.11%, 0.04%, 0.05%, and 0.18% for chromosomes 1, 12, X, Y, and XY, respectively. The frequency of diploid sperm was 0.09%. The frequency of abnormalities was not significantly increased compared to control donors for any of the studies. Also, the frequencies of X- and Y-bearing sperm did not differ significantly from 50% in the sperm karyotyping or FISH studies. PMID- 7535189 TI - Structural studies of the O-antigen oligosaccharides from two strains of Moraxella catarrhalis serotype C. AB - The oligosaccharide parts from Moraxella (Branhamella) catarrhalis serotype C lipooligosaccharides were isolated by mild acid hydrolysis followed by gel permeation chromatography. Four different oligosaccharides could be identified from strain RS26 and two from strain RS10. The structures of the O oligosaccharides were established by methylation analyses, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide O-antigens from RS26 are a mixture of octa-, deca-, and undeca-saccharides, and most likely a heptasaccharide. Strain RS10 contains the deca- and the undeca-saccharide only. The structures for the oligosaccharides are shown below. [formula: see text] OS(7) [formula: see text] OS(8) [formula: see text] OS(10) [formula: see text] OS(11) Methylation analysis of the intact lipooligosaccharides showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. It also showed that they consisted of a lipid A portion with 6-substituted glucosamine residues. PMID- 7535190 TI - Structure of the O10 antigen of Stenotrophomonas (Xanthomonas) maltophilia. AB - A polysaccharide containing L-rhamnose and L-xylose was isolated from the lipopolysaccharide extracted from the cell walls of the reference strain for Stenotrophomonas (Xanthomonas) maltophilia serogroup O10. By means of NMR studies and methylation analysis, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. [formula: see text] PMID- 7535191 TI - Effect of arotinolol on insulin sensitivity in obese hypertensive patients. AB - Insulin sensitivity in terms of glucose disposal rate was determined by using the hyperinsulinemic euglycemic clamp technique in seven obese hypertensive patients and nine normotensive control subjects. The technique was used on all study subjects at baseline and on the hypertensive patients after 12 weeks of arotinolol treatment. Mean supine systolic blood pressure decreased from 155 +/- 3 mm Hg before treatment to 133 +/- 6 mm Hg after treatment (P < 0.02), and diastolic blood pressure decreased from 100 +/- 3 mm Hg before treatment to 86 +/ 3 mm Hg after treatment (P < 0.05). The mean dose of arotinolol was 10 mg/d, except for one patient who received 20 mg/d. The glucose disposal rate during the last 30 minutes of the glucose clamp procedure was not significantly affected by treatment with arotinolol. Compared with the hypertensive patients, the glucose disposal rate in the normotensive subjects was significantly increased, both before and after treatment (P < 0.01). The study results show that the obese hypertensive patients were insulin resistant and that treatment with arotinolol did not significantly affect insulin sensitivity. PMID- 7535193 TI - Surprises at the 3' end of prokaryotic RNA. PMID- 7535194 TI - Changes in CD44 expression during B cell differentiation in the human tonsil. AB - CD44 is a widely distributed cell surface glycoprotein that has been implicated in a number of cellular adhesion processes and signal transduction events. These functional capabilities qualify CD44 as a potential mediator of contact-signaling events underlying the process of antigen-dependent B cell differentiation in secondary lymphoid tissues. We postulated that changes in the expression of CD44 during B cell differentiation reflect the cells' changing requirements for this receptor. It has been reported that germinal center B cells are low to negative for CD44 expression, implying that the receptor is lost upon activation. Correlation of the expression of CD44 with surface immunoglobulin and a number of B cell differentiation markers revealed a trimodal expression pattern. High levels of CD44 are expressed on resting IgD+/IgM+ cells. The receptor is still expressed at the early activation stage defined by the expression of CD23. At the early blast stage, when the blast marker CD38 appears on the cell surface and IgD and CD23 disappear, CD44 is downregulated. The majority of CD38+/IgM+ blasts and CD38+/Ig- centroblasts are CD44 low/negative. The receptor is re-upregulated at the point of transition from the centroblast to the centrocyte level. Centrocytes expressing IgG or IgA comprise CD44high and CD44low fractions. IgG+ or IgA+ cells at the postgerminal center stage express high levels of CD44. The functional implications of this expression pattern are discussed. PMID- 7535192 TI - Nitroglycerin-induced headache is not dependent on histamine release: support for a direct nociceptive action of nitric oxide. AB - The molecular mechanisms of migraine pain have not yet been clarified. Monoamine and the peptide neurotransmitters involved in neurogenic inflammation do not cause significant head pain. Our previous studies of glyceryl trinitrate (GTN) and histamine-induced headaches have suggested that nitric oxide (NO) is the causative molecule in migraine pain. We furthermore suggest that substances capable of inducing experimental vascular headache do so via a common mediator which is NO. Finally, it is suggested that drugs exert their antimigraine activity by inhibiting NO or subsequent steps in the cascade of intracellular reactions triggered by NO. These novel observations change current views on vascular headache mechanisms and the importance of NO as an initiator of the migraine attacks dictates new approaches to the pharmacological treatment of migraine and other vascular headaches. PMID- 7535195 TI - Superantigens induce primary T cell responses to soluble autoantigens by a non-V beta-specific mechanism of bystander activation. AB - Superantigens have been suggested to act as powerful TCR V beta-specific inducers of T cell reactivity in autoimmune diseases. We have investigated the capacity of staphylococcal enterotoxins (SE) to prime autoreactive T cell responses in naive animals in the Lewis rat model of experimental autoimmune encephalomyelitis (EAE), where myelin basic protein (MBP)-specific CD4+ effector T cells express almost exclusively V beta 8.2 TCR elements. By taking advantage of the reactivity of V beta 8.2+ MBP-specific T cells to SEE but not to other SEs in vitro, we estimated the potential of different SEs (SEA, SEB, and SEE) to induce a primary T cell response to soluble MBP in vivo. Upon immunization of naive rats with soluble MBP alone or MBP and SEB (which is only a very weak superantigen for rat T cells), no MBP-responses could be retrieved. Similarly, when coimmunizing naive rats with MBP and V beta 8.2-activating SEE, no autoreactivity was inducible. By contrast, coimmunization of animals with soluble MBP and the superantigen SEA that is strongly activating various T cell subpopulations in Lewis rats but not V beta 8.2+ (i.e., potentially MBP reactive) T cells led to a significant primary MBP-specific T cell autoreactivity. These SEA-induced MBP-reactive T cells expressed V beta 8.2 TCRs at levels similar to those seen in autoreactive T cells conventionally induced by immunization with MBP administered in complete Freund's adjuvant (CFA) and could induce disease in a transfer model of EAE. Thus, our results are consistent with the notion that superantigens are able to induce primary T cell responses to soluble autoantigens by a non-V beta specific mechanism of bystander priming. PMID- 7535196 TI - Tumor necrosis factor-alpha facilitates induction of CD80 (B7-1) and CD54 on human B cells by activated T cells: complex regulation by IL-4, IL-10, and CD40L. AB - Expression of immune accessory molecules, such as CD80 (B7-1), on antigen presenting cells governs whether such cells can activate antigen-specific T cells. As such, the factors that regulate the expression of these accessory molecules may determine whether presentation of antigen leads to immune activation or anergy. We previously reported that anti-CD3-activated T cells (Ta) can induce expression of CD80 and CD54 (ICAM-1) on human B cells through a contact-dependent signal delivered to the CD40 molecule via the CD40 ligand. Here, we demonstrate that another molecule in the CD40-ligand family, namely tumor necrosis factor-alpha (TNF-alpha), also plays a role in the Ta-mediated induction of CD80 or CD54 on human B cells. Neutralizing mAbs specific for TNF alpha can inhibit B cell expression of CD80 or CD54 that is induced when B cells are cultured with Ta cells or in Ta-cell conditioned media. Moreover, soluble, recombinant TNF-alpha or TNF-beta can induce significant increases in B cell expression of CD80 and CD54. The phenotypic changes effected by TNF-alpha can be recapitulated by crosslinking CD120b (p75 TNF-receptor), but not CD120a (p55 TNF receptor), with mAbs presented on Fc gamma RII (CD32)-expressing L cells. IL-4 augments the expression of CD80 induced by crosslinking either CD40 or CD120b. However, although IL-10 augments CD40-induced expression of CD80, this cytokine inhibits the expression of CD80 that is induced by crosslinking CD120b. Further regulation of TNF-mediated CD80 expression may occur at the level of CD120b expression itself. We find that stimulation with exogenous IL-4 or CD40-cross linking induces B cell expression of CD120b, but not CD120a. This study identifies an ancillary, TNF-mediated pathway, whereby activated T cells can induce B cells to express enhanced levels of the important costimulatory molecules, CD80 and CD54. PMID- 7535197 TI - Synthesis and antiallergic activity of novel azaazulene derivatives. AB - Various azaazulene derivatives were synthesized and their antiallergic activity was examined. The structure-activity relationship among various derivatives modified by introducing substituents at the 1-,2-, or 3-position of the azaazulene ring was investigated. The inhibitory activities on allergic histamine release of the compounds bearing a 5-tetrazolyl group at the 3-position were more potent than those of the corresponding compounds with other groups (CN, COOH, and CHO). The compounds substituted with amino, azide and carboxymethylamino groups at the 2-position showed strong inhibitory activity. The compounds with various phenylalkyl groups at the 1-position showed a greater activity than those with other substituents. Among the compounds with substituents at the 1-,2-, or 3 position of the azaazulene ring, 1-benzyl-7-isopropyl-3-(5-tetrazolyl)-1 azaazulen-2-one (18f) and 1-(4-fluorobenzyl)-7-isopropyl-3-(5-tetrazolyl)-1 azaazulen- 2-one (19c) had the most potent inhibitory activities on histamine release from mast cells and on passive cutaneous anaphylaxis (PCA) in rats after oral administration (ED50 = 0.56 and 0.58 mg/kg, respectively). PMID- 7535198 TI - The effect of child hyperactivity on mothers' expectations for development. AB - Mothers of hyperactive (M-Hyp) and control children (M-NHyp) estimated the age at which their child and children normally would reach a number of significant developmental milestones. M-Hyp and M-NHyp had similar timetables for normal development while their judgements about their own child's development were different. M-Hyp saw their children as delayed relative to normal development and M-NHyp saw theirs as advanced. This group differences spanned the social, communicative, cognitive and self-care domains. The generality of this effect was particularly surprising as the two groups of children had similar IQ scores. A number of explanations for this are discussed, including the possibility that this finding represents an important generalization of low expectations from the purely social to the communicative/cognitive domains. The implications of this possibility for hyperactive children's development are discussed. PMID- 7535199 TI - Effect of neonatal hypoxia-ischemia on nigro-striatal dopamine receptors and on striatal neuropeptide Y, dynorphin A and substance P concentrations in rats. AB - Perinatal hypoxic-ischemic brain injury was induced in 7- to 8-day-old rats by ligating the left carotid artery with subsequent exposure to 9% oxygen atmosphere for 2.5 h. The animals were killed 7 days later and grouped according to the degree of brain injury sustained after hypoxia-ischemia. Total protein content measured in striatum ipsilateral to the ligation, and dissected from brains showing extensive damage, was reduced to 64% of contralateral tissue. The protein content was not altered in other groups including control animals exposed to air and in sham-operated animals exposed to hypoxic conditions. The concentration of (pg/mg protein) and total (pg/striatum) striatal dynorphin A-like immunoreactivity (DLI) from brains with extensive damage were increased to 481% and 285% of the contralateral side, respectively. Hypoxia-ischemia increased striatal neuropeptide Y-like immunoreactivity (NPYLI) concentration from brains with extensive damage to 157% of contralateral side, but when the results were expressed as total NPYLI content per striatum, NPYLI content in striatum with extensive damage remained unaltered. Substance P-like immunoreactivity (SPLI) concentration and total content per striatum from brains with extensive damage were reduced to 66% and 43% of the contralateral side, respectively. D1 and D2 receptor density in animals killed 10 days after injury was reduced by 24% and 22% of control, respectively, in striatum from brains with extensive damage. These results indicate complex changes in brain neuropeptides following neonatal hypoxia-ischemia. Damage in the substance P system could have functional effects on dopaminergic transmission while the increase in NPYLI and in DLI concentrations may respectively reflect the relative preservation from neuronal damage and possibly an increase in neuropeptide synthesis or decrease in release. The decrease in SPLI concentration and the increase DLI concentration induced by hypoxia-ischemia suggests that these peptides may be present in separate neurons. PMID- 7535200 TI - Expression of the ceramide galactosyltransferase gene during myelination of the mouse nervous system. Comparison with the genes encoding myelin basic proteins, choline kinase and CTP:phosphocholine cytidylyltransferase. AB - The present study documents the patterns of mRNA expression for the ceramide galactosyltransferase (CGT), the CTP:phosphocholine cytidylyltransferase (CT), and the choline kinase (CK) during the myelination period of the mouse central nervous system (CNS) and peripheral nervous system (PNS). Using the Northern blot technique with densitometric analyses, we show that the CK gene is not developmentally regulated during the period studied, whereas a peak of expression of the CT gene is observed around day 10. On the other hand, the expression of the CGT gene is similar to that of the MBP gene in the CNS and the PNS. Therefore, the synthesis of the galactosylceramides during the myelination period seems to be controlled at the level of the expression of the CGT gene. These results were compared to those of a neurological mutant, the trembler mouse, whose PNS myelination is deficient. Our results clearly indicate that the deficit in the accumulation of the galactosylceramides documented for this mutant is well correlated to a reduced CGT gene expression. PMID- 7535201 TI - Acidic and basic fibroblast growth factors delay the maturation of neural crest derived neurons. AB - Neural crest (NC) cultures were prepared from lumbosacral segments of 12 day rat embryos and maintained in a defined medium. Post-mitotic, flat, neurofilament+ neurons with broad neuritic processes ('nascent neurons') appeared within 24 h. Timing of the next stage in neuronal differentiation, the formation of bipolar, phase-bright cells that bound tetanus toxin with long, slender neurites ('bipolar neurons'), was markedly influenced by acidic or basic fibroblast growth factor (FGF). The transition from nascent to bipolar neuron occurred several days prematurely in medium without added FGF, but took place with a time-course like that in vivo when 10 ng/ml of acidic or basic FGF was added. PMID- 7535202 TI - Neuronal markers, peptides and enzymes in nerves and chromaffin cells in the rat adrenal medulla during postnatal development. AB - Neuronal markers, peptides and enzymes were analyzed in the rat adrenal medulla during the postnatal period, i.e., when the 'functional' splanchnic innervation is assumed to 'mature'. Nerve fibers were present on day 2 as indicated by neurofilament 10 (NF10)- and growth associated protein 43 (GAP43)-like immunoreactivities (LIs). Acetylcholinesterase (AChE)- and enkephalin (ENK) immunoreactive (IR) fibers, presumably of preganglionic nature, increased in number and intensity during the postnatal period. In contrast, calcitonin gene related peptide (CGRP)- and galanin (GAL)-IR fibers were almost fully developed on day 2. Thus, the presumably sensory innervation of the adrenal gland seems to precede the development of the autonomic nerves. The AChE- and ENK-IR fibers may exert a suppressive effect on ENK-, CGRP- and neurotensin (NT)-LIs in chromaffin cells, since the levels of these peptides were high in the early postnatal period and then decreased. On the other hand, GAL-LI in chromaffin cells was low also in young rats, while GAP43-IR cells were observed at all stages. Neuropeptide tyrosine (NPY) was expressed in many chromaffin cells at all stages and its turnover rate seemed to decrease towards the adult stage. The expression of the catecholamine synthezising enzymes changed only marginally during development. These results indicate that the preganglionic fibers, but not the sensory axons, in the splanchnic nerve are involved in the developmental control of expression of some, but not all, peptides in the chromaffin cells and that these changes thus may reflect the maturation of a 'functional' transmission. PMID- 7535204 TI - The development of enkephalin and substance P neurons in the basal ganglia: insights into neostriatal compartments and the extended amygdala. AB - To study the comparative development of the two major neuropeptide genes of the striatum, we used immunocytochemistry to detect immunoreactivity (ir) for substance P and synenkephalin (the N terminus of proenkephalin), and in situ hybridization to detect proenkephalin mRNA. Earliest detection of substance P-ir was in the anlage of the bed nucleus of the stria terminalis (BST, at E15) and in the rostral-lateral caudate-putamen (CPu), at E16. Substance P in the BST was immediately subjacent to the medial ganglionic eminence, while immunoreactivity in the CPu was associated with the lateral ganglionic eminence. Earliest detection of synenkephalin-ir or proenkephalin mRNA was in the caudal-lateral CPu and the adjacent central nucleus of the amygdala (Ce), at E16. Over the next several days, expression of each neuropeptide spread toward the region of first expression of the other neuropeptide. The first overlap of expression of the two neuropeptides was at E18, at the level of the septum. Despite correspondence of substance P-ir and proenkephalin mRNA in patches at P0, very little co-expression of the two neuropeptides was evident in individual neurons. We propose a model in which the CPu develops primarily from the lateral ganglionic eminence, and the extended amygdala develops primarily from the medial ganglionic eminence. Within each structure, two poles of neuropeptide gene expression are established initially: substance P-ir in the rostral CPu and in the rostral-medial pole of the extended amygdala (represented by the BST), and synenkephalin/proenkephalin in the caudal CPu and in the caudal-lateral pole of the extended amygdala (represented by the Ce). A stream of substance P-ir cells connects the two poles of the extended amygdala, in the sublenticular substantia innominata. PMID- 7535203 TI - Genesis and migration patterns of neurons forming the patch and matrix compartments of the rat striatum. AB - The mammalian striatum is divided into two compartments, the patch (or striosome) and the matrix, which differ on the basis of several cytochemical markers, connection patterns, and time of neurogenesis. In the rat, the patch compartment consists of clusters of neurons isolated by matrix neurons; included in the patch compartment is a rim of neurons subjacent to the corpus callosum and external capsule, called the subcallosal streak. To study the genesis and migration patterns of striatal neurons forming these compartments, we injected pregnant rats with 5-bromo-2'-deoxyuridine (BrdU, which is incorporated into DNA during S phase mitosis) on embryonic (E) day 14, to label patch neurons, or on E19, to label matrix neurons. Embryos were sacrificed at intervals after injection, for detection of BrdU by immunocytochemistry. Cells labeled at E14 were distributed fairly uniformly in the differentiated portion of the caudate-putamen through E19. However, by the day of birth (P0), E14-labeled cells were clustered into patches and the subcallosal streak. Using double immunocytochemistry for BrdU and for the patch marker substance P, we demonstrated a caudal-rostral gradient in the birth dates of neurons in the patch compartment; E14-labeled cells occupied substance P-labeled patches at the level of the posterior limb of the anterior commissure, but patches further rostral were nearly devoid of E14-labeled cells. The distance between the lateral ventricle and the nearest E14-labeled cells was greater on E19 than on E16 or on P0, suggesting secondary movement of early-born neurons during the process of cluster formation. Neurons labeled at E19 formed the matrix surrounding clusters of unlabeled cells, except in the nucleus accumbens (ventral striatum), where E19-labeled cells formed clusters. The data suggest that the uniformly-distributed population of early-born neurons is disrupted by the invasion of later-born (matrix) neurons, forcing the early-born neurons into clusters which are displaced toward the ventricular surface to form the patch compartment. Early-born neurons adjacent to the external capsule are not displaced, forming the subcallosal streak. PMID- 7535205 TI - EX-1, a surface antigen of mouse neuronal progenitor cells and mature neurons. AB - Using membrane fragments of PCC7-Mz1 embryonal carcinoma cells, an established in vitro model of neural differentiation (Lang et al., J. Cell Biol., 109 (1989) 2481-2493), we have raised monoclonal antibodies (mAb) against developmental stage-specific cell surface antigens. As shown by double-immunofluorescence labeling studies, employing differentiating PCC7-Mz1 cells, primary cultures of mouse cerebellum cells and cryosections of mouse brain and other tissues, rat mAb anti-mouse EX-1 recognizes a membrane protein which is exclusively expressed by cells of the neuronal cell lineage. EX-1-expressing neuronal precursor cells were identified by double labeling with antibodies directed against stem cell markers or BrdU, EX-1-expressing postmitotic neurons by labeling with antibodies directed against phenotypic markers. In the developing mouse brain, the EX-1 antigen is expressed in the neuroepithelium already at prenatal day 8, i.e. clearly before the onset of mature neuron-specific marker expression. Increasing co-expression with the latter is observed from embryonic day E10 throughout neuronal maturation, but not with markers of other cell types tested. From these studies, the EX-1 antigen is the earliest marker for the mouse brain neuronal cell lineage so far discovered. PMID- 7535206 TI - Expression of P0 protein mRNA along rat sciatic nerve during development. AB - The temporal and spatial expression of P0 protein mRNA in the spinal nerve roots and in distal (popliteal trifurcation) segments of the developing rat sciatic nerve was examined using in situ hybridization. P0 mRNA was undetectable in the PNS portions at day 16 post-fertilization (E16) but present in the roots as well as in the sciatic nerve by E18. While Schwann cells were labelled by in situ hybridization at E18, it was not until birth (P1) that cells revealed the typical clusters of silver grains in a perinuclear distribution. Hybridization signal rose between P5 and P10, than decreased at P40, with no significant difference of P0 mRNA levels between the different PNS portions at each age studied. These findings demonstrate the lack of proximo-distal gradient in abundance of P0 mRNA during rat PNS development and add further insight in understanding the dynamics of axonal regulation of myelination in the PNS. PMID- 7535207 TI - Serum amyloid A protein in patients with non-insulin-dependent diabetes mellitus. AB - We determined the serum amyloid A protein (SAA) levels in patients with non insulin-dependent diabetes mellitus (NIDDM), and investigated the possible association between SAA and the complications of NIDDM. The concentrations of SAA were measured in the plasma of 105 patients with NIDDM (52 men and 53 women, age mean +/- SD, 61 +/- 13 years) and 91 healthy subjects (37 men and 54 women, aged 57 +/- 11 years). SAA concentrations were assayed by enzyme-linked immunosorbent assay. SAA concentrations in the patients with NIDDM were significantly higher than those in healthy subjects (2.1 +/- 1.3 vs. 1.2 +/- 0.5 mg/L). There were no obvious relationships between SAA levels and duration of diabetes, type of therapy, or control of blood sugar in the patients with NIDDM. However, SAA levels in patients with NIDDM increased significantly, with increase of urinary albumin excretion (p = 0.027). The increase of SAA in the patients with NIDDM did not influence the serum concentrations of lipid or lipoprotein. The SAA concentration in NIDDM was unrelated to the type of treatment, but seemed to be related to the development of diabetic nephropathy. PMID- 7535209 TI - Tyrosine phosphorylation pathway is involved in interferon-gamma (IFN-gamma) production; effect of sodium ortho vanadate. AB - The molecular mechanisms regulating IFN-gamma production have yet to be well characterized. We describe here how treatment of activated cultures of peripheral blood mononuclear cells (PBMC) with the phosphotyrosine phosphatases (PTP) inhibitor sodium ortho vanadate results in greatly enhanced IFN-gamma production. Conversely, cellular proliferation of the same cultures is profoundly inhibited by treatment with vanadate, while the expression of IL-2R and DR molecules on activated lymphocytes remains substantially unmodified. Increased IFN-gamma production, but not inhibition of cellular proliferation, was also observed in mitogen-activated vanadate-treated Jurkat cells. On the other hand, IFN-gamma production induced in cultures of PBMC treated or not with vanadate, was strongly inhibited by incubation with the protein tyrosine kinase (PTK) inhibitor herbimycin A. As a result of the inhibited phosphatase activity, substrates for PTK become hyperphosphorylated on tyrosine residues, as shown by Western blot analysis of cell lysates from cultures of PBMC treated with vanadate. We suggest that the tyrosine phosphorylation pathway plays a role in regulating IFN-gamma production. PMID- 7535208 TI - Combined effects of FK506 (tacrolimus) and cyclophosphamide on atypical B220+ T cells, cytokine gene expression and disease activity in MRL/MpJ-lpr/lpr mice. AB - Groups of female MRL/MpJ-lpr/lpr mice received either saline or FK506 (tacrolimus; 2 mg/kg intraperitoneally) three times weekly, cyclophosphamide (CY; 20 mg/kg) once monthly, or both drugs from 8 weeks of age. Median survival for untreated and CY-treated mice was 26 weeks, and for FK506- and FK506 + CY-treated groups was > or = 44 weeks. Severity of skin lesions and lymph node hyperplasia was markedly reduced by the drug combination, whereas either drug alone was less effective. FK506 or CY alone delayed the onset of proteinuria, but by 24 weeks all of these animals were positive. In contrast, drug combination reduced the prevalence of proteinuria to < or = 60% throughout the 44 weeks of study. Sequential monitoring of peripheral blood lymphocytes revealed that combination therapy but not monotherapy markedly reduced the proportion of atypical CD3+ B220+ and CD3+CD4-CD8- T cells. Neither FK506 nor CY affected the reduction in IL 2 and IL-4 mRNA levels observed in lymph nodes of diseased animals compared with normals. Although the drug combination also did not affect IL-2 mRNA levels, IL-4 mRNA transcripts were increased six-fold compared with saline-treated controls. IL-10 and interferon-gamma (IFN-gamma) mRNAs were induced by FK506, CY and by the drug combination. Serum levels of anti-dsDNA antibodies were reduced in all treatment groups. These data demonstrate improved efficacy of combined T and B cell-directed immunosuppression in murine lupus, associated with marked inhibition of atypical T cells and selective augmentation of IL-4 within the affected lymphoid tissue. PMID- 7535210 TI - Human neutrophil Fc receptor-mediated adhesion under flow: a hollow fibre model of intravascular arrest. AB - Human polymorphonuclear cells (PMN) were found to adhere to a novel model of blood vessel wall-associated IgG. The internal surfaces of cellulose acetate hollow fibres, of comparable internal diameter to small blood vessels, were coated with normal serum human IgG, heat-aggregated IgG (HAIgG), laminin or fibrinogen. Under conditions of flow mimicking those in a small vessel, PMN were found to adhere markedly only to immunoglobulin-coated fibres. Arrest on HAIgG was inhibited by excess soluble IgG but not by bovine serum albumin (BSA), demonstrating that the adhesion was IgG-specific and presumably mediated by Fc gamma R on the PMN surface. Pre-adsorption of serum components onto HAIgG-coated fibres enhanced PMN arrest, due most probably to fixation of complement components by immobilized HAIgG, resulting in additional potential to entrap PMN via complement receptors such as CR3. Treatment of PMN with the regulatory neuropeptide substance P also enhanced adhesion to HAIgG-coated fibres and caused increased surface expression of Fc gamma RI, Fc gamma RII and Fc gamma RIII. A mouse cell line derived from L cells, hR4C6, stably transfected with human Fc gamma RII, was found to adhere under flow to HAIgG-coated fibres, whilst untransfected parent L cells did not. This adhesion was similarly inhibited by excess soluble IgG, confirming the capability of Fc gamma R to mediate cell arrest. The study strongly suggests that Fc gamma R may play an important role in intravascular PMN arrest and we speculate that in inflammatory diseases PMN may adhere via Fc gamma R to immobilized immunoglobulin on the vascular endothelium, with subsequent degranulation and tissue damage. PMID- 7535211 TI - Dendritic cells in synovial fluid of chronic inflammatory arthritis lack CD80 surface expression. AB - Dendritic cells (DC) act as potent primary antigen-presenting cells in many immune responses and therefore may have a role in the initiation and perpetuation of the synovial inflammation in chronic inflammatory arthritis. To examine their function, it is important to isolate fresh DC from arthritic joints without aberrant activation. We have developed a technique using minimal cell manipulation to isolate DC from the synovial fluid of chronic arthritic patients. Using this method, DC were shown to be potent allostimulatory cells, with 63-90% of cells lacking lineage-specific markers (lin-), but positive for MHC class II molecules. Two morphologically distinct populations of these cells were identified in 10 out of 13 DC preparations. Both populations expressed CD40, intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3 and leucocyte function associated antigen-3 (LFA-3), but the predominant population, which was larger and more typical of cultured blood DC, had a higher density of these antigens compared with the minor population, which were smaller and morphologically similar to lymphocytes. Two new MoAbs which label activated human blood DC, HB15 (CD83) and CMRF-44, were tested. CD83 labelled very weakly or not at all, whereas CMRF-44 was positive on the larger cells only. Likewise, the costimulator molecule, B7/BB1 (CD80), was not detected on the surface of either synovial lin- cell population, reverse transcriptase polymerase chain reaction (RT-PCR) showed little or no CD80 mRNA, and no binding of the CTLA-4Ig fusion protein was found. These results suggest that synovial DC are not, despite the inflammatory environment, in a fully activated state. PMID- 7535213 TI - Pharmacokinetics of tacrolimus in liver transplant patients. AB - OBJECTIVE: To characterize the pharmacokinetics of the immunosuppressive agent tacrolimus (FK 506) in liver transplant patients. METHODS: Patients (n = 16) were assessed during and after 1- to 3-day intravenous infusions followed by a 2-week course of oral dose therapy. Plasma and whole blood data were fitted simultaneously with equations accounting for nonlinear drug binding by red blood cells to generate clearance (CL) and volume of distribution (V). RESULTS: The maximum blood/plasma ratio of tacrolimus was 55.5 +/- 26.8 (SD) and half-life averaged 12.1 +/- 4.7 hours. The CL and V were relatively high based on plasma concentrations (CL, 1.7 L/hr/kg; V, 30 L/kg) and low based on whole blood (CL, 54 ml/hr/kg; V, 0.9 L/kg), with moderate variability (coefficient of variation, 34% to 49%) among the patients. Correlations of plasma CL and V with maximum blood/plasma ratios (ranging from 13 to 114) were strong (r = 0.65 and r = 0.73). Blood binding affects the disposition of tacrolimus, and plasma concentrations are indirectly and inversely related to red cell binding. The oral dose data for tacrolimus yielded a brief absorption lag time (tlag, 0.39 hour), a variable first-order absorption rate constant (ka, 4.5 +/- 3.0 hr-1), and consistent bioavailability (F, 25% +/- 10%). The area under the concentration-time curve versus 12-hour minimum concentration relationships for both whole blood and plasma were nearly linear, confirming the utility of trough values for monitoring drug exposure. CONCLUSION: This study provides pharmacokinetic guidelines for the use of tacrolimus in patients undergoing hepatic transplantation. Nonlinear blood binding is a major source of interpatient variation in the disposition of tacrolimus. PMID- 7535214 TI - An analysis of spontaneous conversational speech fluency in children with acquired aphasia. AB - We report on an instrumental analysis of spontaneous conversational speech (SCS) fluency in acquired childhood aphasia (ACA). Tape-recorded SCS samples of 25 children with ACA (clinical judgment: 12 nonfluent and 13 fluent), and of 12 dysarthric and 12 nonaphasic and nondysarthric right hemisphere injured children were analysed in order to: (1) investigate whether a more refined analysis can objectively contribute to the differentiation of patients who were labelled as fluent or nonfluent on the basis of a clinical judgment: (2) verify whether an instrumental analysis of phonation duration does confirm the subjective estimation of verbal rate (i.e. the number of words produced in a unit of time) in groups of children with acquired neurogenic speech/language disorders frequently met in clinical practice. The results are: (1) phonation rate (i.e. the vocalization percentage) seems to represent an adequate variable to distinguish clinically diagnosed nonfluent aphasic children from speech/language impaired children belonging to other clinical groups of acquired neurogenic speech/language disorders; (2) the verbal rate is highly correlated to the phonation rate in all investigated groups except the dysarthric one. We suggest the instrumental method discussed here might contribute to the differential diagnosis between dysarthric and aphasic disturbances in the acute stage of the disease. Concerning the study of ACA, the main issue of the present investigation is that an objective fluency measurement has succeeded in identifying aphasic children who obviously do not fit in with the standard doctrine on ACA, which claims that ACA is invariably nonfluent irrespective of lesion location. PMID- 7535215 TI - A test of the immunoreactive theory for the origin of neurodevelopmental disorders in the offspring of women with immune disorder. AB - Gualtieri and Hicks (1985) proposed that male vulnerability for neurodevelopmental disorders (NDs) was partially due to intrauterine immune attack of the fetus. One group of mothers with heightened immunoreactivity might be women with immune disorder. This was tested within an epidemiological sample of 17,283 mother/child pairs. Maternal immune disorders considered were ulcerative colitis or asthma. NDs in the child included: cerebral palsy, mental retardation, seizures, articulation disorder, reading, or arithmetic disability, verbal or performance aptitude deficits, and attention deficit disorder. Unlike prior studies, we controlled for demographic perinatal variables that might confound interpretation of the data. Results indicated that immune dysfunction in the mother, be it autoimmune (ulcerative colitis) or defensive (asthma) was not associated with an increased incidence of any NDs in the offspring, but mothers with ulcerative colitis did have a disproportionate number of offspring who were non-right handed. Few variables discriminated between the children of ulcerative colitis mothers who became right handed when compared to those who did not. We suggest that a) only certain maternal autoimmune disorders such as systemic lupus erythematosus (but not ulcerative colitis or asthma) elevate the risk of intrauterine immune attack and b) the elevated rate of non-right handed offspring among ulcerative colitis mothers was not an instance of immune attack but instead represents some kind of genetic association. PMID- 7535212 TI - The rheumatoid arthritis-associated autoantibodies to filaggrin label the fibrous matrix of the cornified cells but not the profilaggrin-containing keratohyalin granules in human epidermis. AB - Since they were first described, serum IgG antibodies to the stratum corneum of rat oesophagus epithelium, highly specific for rheumatoid arthritis (RA), have been consensually called antikeratin antibodies (AKA). However, we recently demonstrated that they actually recognize three new proteins of rat oesophagus epithelium distinct from cytokeratins, and also human epidermal filaggrin. In this work we provided further evidence that AKA and RA-associated anti-filaggrin autoantibodies are the same antibodies. Moreover, analysing by indirect immunofluorescence on human skin a large series of 212 well characterized RA sera and anti-filaggrin autoantibodies purified from RA sera by affinity chromatography, we demonstrated the specific binding of AKA to the stratum corneum of human epidermis and the absence of any staining of the granular keratinocytes. This binding was confirmed and the AKA antigen precisely localized in human epidermis by immunoelectron microscopy. The antigen was found to be restricted to the filaggrin-containing intracellular fibrous matrix of the corneocytes, up to the desquamating cells. In contrast, MoAbs directed to human filaggrin and to profilaggrin, its precursor, not only stained the intracellular matrix of the lower corneocytes but also the keratohyalin granules of the granular cells, where profilaggrin is stored. These results reinforced by the absence of immunoblotting reactivity of RA sera to profilaggrin suggest that the epitopes recognized by AKA are absent from profilaggrin. Their identification may provide more insight into the pathogenesis of RA. PMID- 7535216 TI - Cervical and scalp recorded short latency somatosensory evoked potentials in response to epidural spinal cord stimulation in patients with peripheral vascular disease. AB - Somatosensory evoked potential (SEP) studies were performed in 14 patients with peripheral vascular disease who received epidural spinal cord stimulation (SCS) for chronic pain relief of the lower limbs. Signals were amplified and filtered between 20-2000 Hz and 200-2000 Hz to better identify activities in the high frequency range. In 7 patients bit-colour maps were also computed. In all the patients a homogeneous short-latency scalp evoked potential with a prevalent diphasic shape (P1-N1) was recorded. In all our scalp records, even with the wide bandpass, small short-latency positive deflections were observed on the descending front of the first major positive wave and they were better defined as a series of up to 6 wavelets, preceding the major negative scalp wave in the tracings filtered through the narrow bandpass. They appeared in an interval ranging from 5.5 to 15.6 msec. Bit-colour maps showed consistent positive fields, with a maximum at the vertex, starting mainly at about 5.5 msec; in 3 patients, a prominent positivity between 8.5 and 10.5 msec was recorded followed by smaller components preceding the major positive-negative (P1-N1) complex. More synchronous volleys during direct SCS produced clear short-latency SEPs. Although they were of larger amplitude, we regarded them as corresponding to those described by previous authors obtained by stimulation of nerves of the lower limbs, and probably arising from subcortical structures. PMID- 7535218 TI - Somatotopy of human hand somatosensory cortex revealed by dipole source analysis of early somatosensory evoked potentials and 3D-NMR tomography. AB - Somatosensory evoked potentials (SEPs) to median nerve and finger stimulation were analyzed by means of spatio-temporal dipole modelling combined with 3D-NMR tomography in 8 normal subjects. The early SEPs were modelled by 3 equivalent dipoles located in the region of the brain-stem (B) and in the region of the contralateral somatosensory cortex (T and R). Dipole B explained peaks P14 and N18 at the scalp. Dipole T was tangentially oriented and explained the N20-P20, dipole R was radially oriented and modelled the P22. The tangential dipole sources T were located within a distance of 6 mm on the average and all were less than 9 mm from the posterior bank of the central sulcus. In 6 subjects the tangential sources related to finger stimulation arranged along the central sulcus according to the known somatotopy. The radial sources did not show a consistent somatotopic alignment across subjects. We conclude that the combination of dipole source analysis and 3D-NMR tomography is a useful tool for functional localization within the human hand somatosensory cortex. PMID- 7535217 TI - Two distinct cervical N13 potentials are evoked by ulnar nerve stimulation. AB - To investigate the dual nature of the posterior neck N13 potential, we attempted to establish the presence of a latency dissociation between caudal (cN13) and rostral (rN13) potentials on stimulating the ulnar nerve, in view of its lower radicular entry compared to the median nerve. SEPs were evaluated in 24 normal subjects after both median and ulnar nerve stimulation. cN13 was prominent in the lower cervical segments, and rN13 was localized mainly in the upper ones using anteroposterior and longitudinal bipolar montage, respectively. The N9-cN13 interpeak latency did not differ significantly from N9-rN13 when stimulating the median nerve. On the other hand, the N9-rN13 interpeak was significantly longer than the N9-cN13 interpeak when the ulnar nerve was stimulated. The rN13 presented the same latency as P13-P14 far-field potentials in 17 out of 24 ulnar nerves tested. Therefore, the ulnar nerve stimulation evokes two distinct posterior neck N13 potentials. It is widely accepted that the caudal N13 is a postsynaptic potential reflecting the activity of the dorsal horn interneurons in the lower cervical cord. We suggest that the rostral N13 is probably generated close to the cuneate nucleus, which partly contributes to the genesis of P13-P14 far-field potentials. PMID- 7535220 TI - Tonotopic organization of the human auditory cortex: N100 topography and multiple dipole model analysis. AB - The tonotopic organization of the human auditory cortex has been investigated by means of scalp potential mapping and dipole modelling of the evoked response occurring around 100 msec after the stimulus onset. The major characteristics of the topographical changes observed with increasing stimulus frequency were statistically demonstrated. Using a 3-concentric sphere head model, the scalp potential distributions can be explained in first approximation by two equivalent current dipoles, located in the supratemporal plane and mimicking the activity of both auditory cortices. To take into account the temporal aspects of the brain activities, 3 time-varying dipole strategies were tested. Frequency dependence of the dipole orientation has been evidenced in both hemispheres with the 3 models, whereas no significant change in dipole position was found. The tilt in dipole orientation could be related to the folding geometry of Heschl's gyrus, which varies with depth. In agreement with previous MEG findings, this brings new evidence for a tonotopic organization of the auditory cortical area involved in the N100 wave generation. Moreover, distinct frequency dependences of the equivalent current dipoles were observed in the early and the late parts of the N100. This study demonstrates that simple dipolar models, applied on electrical data, make it possible to reveal functionally distinct cortical areas. PMID- 7535219 TI - The interaction of the somatosensory evoked potentials to simultaneous finger stimuli in the human central nervous system. A study using direct recordings. AB - In order to investigate the interaction of sensory electrophysiologic fields arising from the adjacent second (II) and third (III) fingers and the distant second and fifth (V) fingers, direct recordings of somatosensory evoked potentials (SEPs) were performed from the sensory and motor cortices, the sensory thalamic nucleus (nucleus ventralis caudalis, VC) and the cuneate nucleus in humans during neurosurgical operations. Electrical stimulation was given to the II, III or V fingers individually, and also to pairs of either the II and III fingers or the II and V fingers simultaneously. The interaction ratio (IR) was devised as the ratio of amplitude attenuation caused by the simultaneous stimulation to two fingers compared with the amplitude of the arithmetically summed SEPs to the individual stimulation of two fingers. The IRs were calculated on N20 and P25 from the sensory cortex, P22 from the motor cortex, P17thal from the VC, and N16cune and P35cune from the cuneate nucleus. With both stimulations to the II and III fingers and the II and V fingers, P25 showed the greatest IR, followed by P22, then by P17thal, with N16cune exhibited the smallest IR. N20 and P35cune showed similar IRs and significantly greater IRs with II and III finger stimulation compared with II and V finger stimulation. These results thus indicate that the interaction of somatosensory impulses occurs in several structures along the sensory pathway in CNS, including the cuneate nucleus, the sensory thalamic nucleus, as well as sensory and motor cortices, with the greatest IRs in the cerebral cortices and the weakest ones in the brain stem.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535221 TI - Auditory event-related potentials dissociate early and late memory processes. AB - Event-related potentials (ERPs) to environmental sounds were recorded from 15 young control subjects in an auditory recognition memory task. Subjects listened to a continuous string of binaurally presented sounds, 20% of which were presented once and 80% were repeated. Of the repeated sounds, some repeated immediately after the initial presentation (2 sec; short delay repetition) while others repeated after 2-6 intervening sounds (4-12 sec; long delay repetition). Subjects were instructed to indicate whether they had heard the sounds before by pressing a "yes" or "no" button. The initial stimulus presentation and long delay repetition stimuli generated both an N4 component and a prolonged latency P3 component while the short delay repetition stimuli elicited no N4 component and an earlier latency P3 component. Subjects' responses were faster and more accurate for short delay repetition. All stimuli generated a sustained frontal negative component (SFN). These data indicate that auditory recognition memory for environmental sounds may involve two processes. The P3 generated by both short and long delay repetition stimuli may index activation of a neocortical template matching system. The N4 generated by initial stimulus presentations and long delay repetition is proposed to measure additional activation of limbic memory systems at long retention intervals. PMID- 7535222 TI - Event-related desynchronization (ERD) patterns during memory processes: effects of aging and task difficulty. AB - Event-related desynchronization (ERD) was studied in 10 young (mean age = 19.1) and 10 older (mean age = 62.8) subjects during two recognition tasks: verbal and visuo-spatial. The difficulty of these tasks varied according to the difficulty to distinguish between targets and distractors. EEGs recorded from 29 electrodes were used to compute ERDs from 14 source derivations in 125 msec intervals. Thereafter, they were displayed as spatio-temporal maps. The results show that desynchronization was more widespread in the visuo-spatial compared to the verbal task. This was observed in the two age groups, although it was more pronounced in the young subjects. The effect of task complexity was also influenced by the kind of material to be remembered: more differences between the two levels of difficulty were observed during the verbal task. The results revealed significant influences of the task and time variables on the ERD patterns. A distinct time course of the desynchronization phenomenon was observed to be related to the kind of recognition task. Age and task complexity interacted with the other variables. PMID- 7535223 TI - Mental representations of movements. Brain potentials associated with imagination of hand movements. AB - The present study was designed in order to contribute towards the understanding of the physiology of motor imagery. DC potentials were recorded when subjects either imagined or executed a sequence of unilateral or bilateral hand movements. The sequence consisted of hand movements in 4 directions, forwards, backwards, to the right and to the left, and varied from trial to trial. The sequence had been cued by visual targets on a computer screen and had to be memorized before the trial was initiated. Changes of DC potentials between task execution and imagination were localized in central recordings (C3, Cz, C4) with larger amplitudes when executing the task than when imagining to do so. Stimulation of peripheral receptors associated with task execution or a different level of activation of the cortico-motoneural system could account for this finding. The main result of the present study was that with unilateral performance, the side of the performing hand (right, left) had localized effects in recordings over the sensorimotor hand area (C3, C4) which were qualitatively the same with imagination and execution and quantitatively similar (i.e., without significant difference). Performance of the right hand augmented negative DC potentials in C3, performance of the left hand augmented amplitudes in C4. This result is consistent with the assumption that the primary motor cortex is active with motor imagery. Finally, the question has been addressed whether motor imagery may involve the left hemisphere to a larger extent than the execution of the movement. It is shown that a particular contribution of the left hemisphere associated with motor imagery may only show up under strictly controlled conditions. PMID- 7535224 TI - Catecholaminergic function and P300 amplitude in major depressive disorder (P300 and catecholamines). AB - The neurobiology of P300 is still a subject of controversy. P300 amplitude appears to be modulated by multiple neurotransmitter systems, especially dopaminergic, noradrenergic as well as cholinergic and GABAergic. In this study, we investigated the relationship between P300 amplitude and catecholaminergic neurotransmission as assessed by the growth hormone (GH) response to clonidine and apomorphine challenges in 20 major depressive patients. Results showed a correlation of P300 amplitude with the apomorphine test (r = 0.54; P = 0.01), but not with the clonidine test (r = 0.22; NS). This study supports a role for dopamine in the neurobiological modulation of P300 amplitude. PMID- 7535225 TI - Comparison of the pattern reversal visual evoked potential mediated by separate cone systems. AB - With the purpose of recording responses mediated by the 3 cone systems visual evoked potentials (VEPs) were elicited by the reversal of monochromatic checkerboards superimposed upon strong monochromatic backgrounds (yellow, purple and blue-green). The sensitivity to light of various wave lengths were measured as the reciprocal of the intensity necessary to elicit a VEP amplitude of 3 microV. The spectral sensitivity curves based on this VEP amplitude criterion in the presence of blue-green, purple and yellow adaptation showed peak sensitivities in the red, the green and the blue part of the spectrum, respectively. This indicates that the responses reflect separate modulation of the 3 different cone mechanisms. The potentials obtained with yellow adaptation differed from those obtained with purple and blue-green adaptation. The amplitude versus log intensity function was flatter and the latency of the major positive peak was increased by 20-25 msec. Repeated examinations of 4 subjects suggest that the method yields reliable latency measurements of responses mediated by separate cone mechanisms. PMID- 7535227 TI - 1-Nitropyrene as a marker for the mutagenicity of diesel exhaust-derived particulate matter in workplace atmospheres. AB - The use of 1-nitropyrene (1-NP) as a marker for the occupational exposure to diesel exhaust (DE) mutagens was investigated in workplace atmospheres contaminated with DE from a variety of emission sources, such as power supplies, forklifts, trucks, caterpillar vehicles, trains, ships' engines, and vehicles in city traffic. Total suspended particulate matter was collected by area sampling. The 1-NP content of acetone extracts of these samples as determined by gas chromatography-high resolution mass spectrometry varied from 0.080 to 17 micrograms/g acetone extractable matter, corresponding to air concentrations of 0.012 to 1.2 ng/m3. A sample collected in a rural area contained 0.0017 ng/m3 1 NP. The mutagenicity of the extracts was tested in the Salmonella typhimurium strains TA98 and TA1538, using the microsuspension assay with and without metabolic activation by an exogeneous metabolizing system (rat liver S9 fraction). In addition, the S. typhimurium strains YG1021 and YG1024 were used because of their high sensitivity towards the mutagenicity of nitro polycyclic aromatic hydrocarbons. When plotting the mutagenic potency of the air sample extracts as determined in the absence of liver S9 versus the particle-associated 1-NP level, a relatively high correlation (r = 0.80-0.91) was observed in all of the S. typhimurium strains. High correlations (r = 0.80-0.93) were also observed when plotting the results of mutagenicity testing after activation by S9 versus the outcome of chemical analysis. These results show that the 1-NP content of workplace air samples is associated with their mutagenic potency, suggesting that 1-NP may be used as a marker for occupational exposure to DE-derived particle associated mutagens. PMID- 7535226 TI - Amplified interactive toxicity of chemicals at nontoxic levels: mechanistic considerations and implications to public health. AB - It is widely recognized that exposure to combinations or mixtures of chemicals may result in highly exaggerated toxicity even though the individual chemicals might not be toxic. Assessment of risk from exposure to combinations of chemicals requires the knowledge of the underlying mechanism(s). Dietary exposure to a nontoxic dose of chlordecone (CD; 10 ppm, 15 days) results in a 67-fold increase in lethality of an ordinarily inconsequential dose of CCl4 (100 microliters/kg, ip). Toxicity of closely related CHCl3 and BrCCl3 is also enhanced. Phenobarbital (PB, 225 ppm, 15 days) and mirex (10 ppm, 15 days) do not share the propensity of CD in this regard. Exposure to PB + CCl4 results in enhanced liver injury similar to that observed with CD, but the animals recover and survive in contrast to the greatly amplified lethality of CD + CCl4. Investigations have revealed that neither enhanced bioactivation of CCl4 nor increased lipid peroxidation offers a satisfactory explanation of these findings. Additional studies indicate that exposure to a low dose of CCl4 (100 microliters/kg, ip) results in limited injury, which is accompanied by a biphasic response of hepatocellular regeneration (6 and 36 hr) and tissue repair, which enables the animals to recover from injury. Exposure to CD + CCl4 results in suppressed tissue repair owing to an energy deficit in hepatocytes as a consequence of excessive intracellular influx of Ca2+ leading initially to a precipitous decline in glycogen and ultimately to hypoglycemia. Supplementation of cellular energy results in restoration of the tissue repair and complete recovery from the toxicity of CD + CCl4 combination. In contrast, only the early-phase hepatic tissue repair (6 hr) is affected in PB + CCl4 treatment, but this is adequately compensated for by a greater stimulation of tissue repair at 24 and 48 hr resulting in recovery from liver injury and animal survival. A wide variety of additional experimental evidence confirms the central role of stimulated tissue repair as a decisive determinant of the final outcome of liver injury inflicted by CCl4. For instance, a 35-fold greater CCl4 sensitivity of gerbils compared to rats is correlated with the very sluggish tissue repair in gerbils. These findings are consistent with a two-stage model of toxicity, where tissue injury is inflicted by the well described "mechanisms of toxicity," but the outcome of this injury is determined by whether or not sustainable tissue repair response accompanies this injury.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7535229 TI - Effect of substance P and capsaicin on urinary bladder of diabetic rats and the role of the epithelium. AB - The in vitro responses of rat urinary bladder, to substance P and capsaicin were studied at 1, 4, 16, and 26 weeks of diabetes induction by streptozotocin. We also studied the role of epithelium in these responses. The results were compared with those obtained in age-matched control rats. The bladder contractile response to exogenous substance P was similar in both groups at all stages (1-26 weeks) studied, whereas the bladder response to capsaicin gradually decreased with the progression of diabetes. Atropine did not inhibit these responses whereas indomethacin slightly reduced substance P- but not capsaicin-induced responses in control and diabetic rats. The removal of epithelium slightly increased the substance P- and capsaicin-induced responses in control tissue; these responses were significantly reduced in tissue excised from diabetic rats. Our results indicate that, in rat urinary bladder, diabetes (1) provokes an impairment of capsaicin-sensitive sensory fibers but not of the cholinergic system even at an early stage (4 weeks) of the disease, (2) has no effect on the sensitivity of smooth muscle cells to substance P, (3) stimulates the release of epithelial contracting factors, partially non-prostanoic. Furthermore epithelium removal impairs acetylcholine-induced contraction in bladder excised from diabetic rats but not in controls. PMID- 7535228 TI - Effect of nonapeptide fragments of uteroglobin and lipocortin I on oedema and mast cell degranulation. AB - The anti-inflammatory action of nonapeptide fragments of uteroglobin or lipocortin I known as antiflammins, was tested in the carrageenan or phospholipase A2 rat paw oedema model. The development of carrageenan-induced oedema in rats was significantly inhibited during the early and late phases of the oedema by the local administration of antiflammins 1 and 2. However, the peptides were not able to inhibit phospholipase A2-induced oedema. The time course of the anti-oedematous activity of nonapeptides after intradermal carrageenan injection may be attributed to their effect on mast cell degranulation and accumulation and activation of leukocytes. Naja naja phospholipase A2 exhibited strong histamine release-inducing activity, which may have contributed to the rat paw oedema induction. Surprisingly, antiflammins had a limited but significant inhibitory effect on histamine secretion. PMID- 7535230 TI - Heterogenous effects of histamine on isolated rat coronary arteries. AB - The influence of increasing concentrations of histamine (0.1 microM-1 mM) was studied on proximal and distal ring segments of left anterior descendens coronary arteries isolated from rats. Addition of histamine to prostaglandin F2 alpha (10 microM)-precontracted proximal segments elicited a further contraction. This effect was endothelium-independent and mediated by a histamine H1 receptor mechanism since it was blocked by the histamine H1 receptor antagonist, mepyramine (10 microM), and not by the histamine H2 receptor antagonist, cimetidine (100 microM), and since it was mimicked by the histamine H1 receptor agonist, 2-pyridylethylamine, and not by the histamine H2 receptor agonist, dimaprit. Addition of histamine to prostaglandin F2 alpha-precontracted distal segments elicited concentration-dependent relaxation. This relaxation is histamine H2 receptor-mediated since it was blocked by cimetidine (100 microM) and not by mepyramine (10 microM) and since dimaprit but not 2-pyridylethylamine elicited relaxation. The relaxation was not due to the release of endothelial NO, prostaglandins or activation of ATP-regulated K+ channels since it was not inhibited by NG-nitro-L-arginine methyl ester (100 microM) or NG-nitro-L-arginine (100 microM), indomethacin (10 microM) or glibenclamide (10 microM). Our results show that the effects of histamine on rat left anterior descendens coronary arteries are heterogenous, depending on the relative location within the coronary vasculature and possibly, the relative preponderance of histamine H1 or H2 receptors on the smooth muscle cells. PMID- 7535232 TI - NG-nitro-L-arginine methyl ester modulates intestinal secretion and motility produced by carbachol. AB - The effects of the nitric oxide (NO) synthesis inhibitor, NG-nitro-L-arginine methyl ester, on carbachol-induced diarrhoea, fluid accumulation and motility changes were studied. Pretreatment of mice with NG-nitro-L-arginine methyl ester (1-25 mg/kg i.p.) and NG-nitro-L-arginine (2.5-50 mg/kg i.p.) but not NG-nitro-D arginine methyl ester (25 mg/kg i.p.) prevented in a dose-related manner the carbachol (0.5 mg/kg i.p.)-induced diarrhoea in mice. L-Arginine (150-1500 mg/kg i.p.) administered to mice pretreated with NG-nitro-L-arginine methyl ester counteracted the antidiarrhoeal activity of NG-nitro-L-arginine methyl ester in a dose-related manner. Pretreatment of rats with NG-nitro-L-arginine methyl ester (2.5-25 mg/kg i.p.) decreased the intestinal fluid accumulation induced by carbachol in rats. NG-Nitro-D-arginine methyl ester was without effect. Intraperitoneal pretreatment of rats with NG-nitro-L-arginine methyl ester (2.5 25 mg/kg) reduced the increase in small intestinal transit induced by carbachol. NG-nitro-L-arginine methyl ester had no effect. These results provide evidence that nitric oxide may play a role in diarrhoea, intraluminal fluid accumulation and motility changes induced by carbachol. PMID- 7535231 TI - Modulation of neuropeptide FF release from rat spinal cord slices by glutamate. Involvement of NMDA receptors. AB - This study examined the effects of glutamate receptor agonists on the release of neuropeptide FF-like immunoreactivity from rat spinal dorsal half slices. Glutamate (10 microM) only induced release in Mg(2+)-free medium enriched with glycine (1 microM) and with slight depolarization (15 mM K+). This effect was abolished by the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (100 microM), suggesting major participation of NMDA receptors. The quisqualate and metabotropic receptor agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4 propionate (AMPA) and trans-1-hydroxy-5-methylisoxazole-4-propionate (t-ACPD) respectively, had no effect at 10 microM. In contrast, NMDA dose dependently stimulated neuropeptide FF release, even in the presence of the Na+ channel blocker, tetrodotoxin (1 microM), suggesting that NMDA receptors involved in the release of neuropeptide FF are mainly located on nerve terminals. The NMDA receptor antagonists, 2-amino-5-phosphonovalerate or (+)-5-methyl-10-11-dihydro 5H-dibenzo [a,d]cyclohepten-5,10-imine (MK-801) (100 microM), blocked the 10 microM NMDA effect. Furthermore, neuropeptide FF-like material inhibited binding of [125I]Y8Fa, a radioiodinated analog of neuropeptide FF, to spinal membranes, suggesting physiological relevance of NMDA-induced release. Taken together, these results suggest a relationship between neuropeptide FF and NMDA receptors in the spinal cord. PMID- 7535236 TI - Tenascin-C induction in Whitlock-Witte culture: a relevant role of the thiol moiety in lymphoid-lineage differentiation. AB - The extracellular matrix (ECM) glycoprotein tenascin-C is expressed in a temporally and spatially restricted pattern during embryogenesis and carcinogenesis in association with stromal-epithelial interactions. First, we investigated the production of tenascin-C and other ECM glycoproteins in the established in vitro model system specific for the lymphoid-lineage hemopoiesis, i.e., the Whitlock-Witte (W-W) culture system. In murine primary long-term bone marrow cultures, tenascin-C was produced constitutively and was expressed significantly in higher amounts in this system than in the other established in vitro model system specific for the myeloid-lineage hemopoiesis, i.e., the Dexter culture system. 2-Mercaptoethanol (2-ME), a component of the W-W system, induced the secretion of tenascin-C and upregulated the expression of its mRNA. Furthermore, the reduced glutathione, which, like 2-ME, contains a thiol moiety, induced tenascin-C glycoprotein and its mRNA. By contrast, hydrocortisone (HC), a component of the Dexter system, inhibited the secretion of ECM glycoproteins. 2 ME and TGF-beta 1, the latter of which is known as an inducer of ECM glycoproteins, had an additive effect on the induction of tenascin-C when they were simultaneously added to the W-W system. The TGF-beta receptor binding analysis demonstrated that this induction by 2-ME was not mediated by the cell surface TGF-beta receptors, suggesting that it was regulated independently of TGF beta 1. Then, the role of thiol compounds in the lymphoid-lineage differentiation was examined. The omission of 2-ME from the W-W system completely eliminated its ability to support the lymphoid-lineage differentiation. Glutathione, which, unlike 2-ME, does not passively permeate through the plasma membrane, did not support the development of a lymphoid lineage. These results indicate that 2-ME, essential for the lymphoid-lineage differentiation in the W-W culture system, is a potent inducer of tenascin-C expression in vitro. PMID- 7535234 TI - L-canavanine restores blood pressure in a rat model of endotoxic shock. AB - Administration of lipopolysaccharide to anaesthetised rats produced a reduction in mean arterial pressure, an increase in heart rate, and death at 4-6 h. Intravenous infusion of NG-nitro-L-arginine methyl ester (50 mg/kg), an inhibitor of constitutive and inducible nitric oxide (NO) synthase, 60 min after challenge with lipopolysaccharide, caused an immediate increase in blood pressure followed by a precipitous fall in pressure, and death. In contrast, intravenous infusion of L-canavanine (100 mg/kg), reported to be a selective inhibitor of inducible NO synthase in vitro, 60 min and 180 min after lipopolysaccharide challenge, produced an increase in mean arterial pressure and reversed the lipopolysaccharide induced hypotension. However, in lipopolysaccharide challenged animals protected from hypotension by administration of L-canavanine (60 min post challenge), intravenous infusion of NG-nitro-L-arginine methyl ester at 180 min post challenge caused an immediate rise in mean arterial pressure, followed by a rapid fall in blood pressure and heart rate, and sudden death. In contrast, a second dose of L-canavanine at 180 min post challenge maintained blood pressure for the duration of the experiment. These findings indicate that inhibition of both constitutive and inducible NO synthase during endotoxaemia is lethal. However, the use of a selective inhibitor of inducible NO synthase restores mean arterial pressure to baseline, and offers a therapeutic approach to managing hypotension in shock. PMID- 7535233 TI - Tachykinins contract trachea from Fischer 344 rats by interaction with a tachykinin NK1 receptor. AB - The contractile effect of substance P, neurokinin A, carbachol and serotonin (5 HT) on isolated Fischer 344 rat trachea was studied. Contractions of two distal tracheal rings were measured isometrically in a 2-ml organ bath. Cumulative concentration-response curves were obtained for carbachol (EC50 ring 1: 1.6 x 10( 7) M and ring 2: 2.2 x 10(-7) M) and for 5-HT (EC50 ring 1: 10.2 x 10(-7) M and ring 2: 10.5 x 10(-7) M). Non-cumulative administration of substance P and neurokinin A (10(-8) to 10(-5) M) caused a concentration-dependent contraction with an EC50 (x 10(-7) M) of 1.10 +/- 0.27 and 1.97 +/- 0.45 respectively. The maximal contraction was 32.6 +/- 2.5% (substance P) and 32.6 +/- 1.5% (neurokinin A) of the maximal contraction with carbachol. In contrast, neither substance P nor neurokinin A caused contraction of trachea from BDE rats. The tachykinin NK1 receptor agonist, Ac[Arg6, Sar9, Met(O2)11] substance P-(6-11), caused a concentration-dependent contraction with an EC50 (x 10-(-9) M) of 1.38 +/- 0.09 and a maximal effect of 25.5 +/- 2.1% of the maximal contraction with carbachol. The tachykinin NK2 receptor agonist, [beta-Ala8]neurokinin A-(4-10), had a small contractile effect at 10(-6) M (8.4 +/- 0.8% of the maximal contraction with carbachol) while the tachykinin NK3 receptor agonist, senktide, had no effect up to 3.3 x 10(-6) M.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535235 TI - Expression of beta 1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage. AB - Expression of beta 1 integrins was studied in vitro as articular chondrocytes reestablished a matrix in culture and in situ in a nonhuman primate model of osteoarthritis in order to investigate a potential role for integrins in mediating cell-extracellular matrix interactions in cartilage. Chondrocytes were found to express alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins both in vitro and in situ. Cell surface expression of beta 1 integrins increased as chondrocytes were maintained in cultured from 3 to 7 days. Increased beta 1 integrin expression was also observed in osteoarthritic cartilage compared with normal cartilage. The greatest relative increase in both systems was noted for the alpha 1 beta 1 integrin. The increase in chondrocyte beta 1 integrin expression in vitro was noted in both monolayer and alginate cultures and occurred prior to detectable changes in the differentiated phenotype of the chondrocyte. Disruption of the cytoskeleton with the drug dihydrocytochalasin B inhibited the cell culture induced increase in integrin expression, while treatment of cultured cells with TGF-beta resulted in increased expression of the alpha 5 beta 1 integrin. The modulation of beta 1 integrin expression noted in vitro and in situ indicates that chondrocytes are capable of regulated expression of beta 1 integrins and suggests that beta 1 integrins may play an important role in mediating chondrocyte-extracellular matrix interactions in cartilage. PMID- 7535238 TI - Effects of expressing human Hsp70 and its deletion derivatives on heat killing and on RNA and protein synthesis. AB - We have established rat cell lines stably and constitutively expressing intact human heat shock protein (hsp) 70 and its deletion mutant derivatives. Using these stable cell lines, the functions of the various domains of human hsp70 were studied. An elevated level of human hsp70 or fragments containing the C-terminal portion of the protein, but not fragments containing the amino-terminal portion of the protein, is found to protect cells against thermal killing. Only the expression of intact human hsp70, however, can reduce the degree of heat-induced inhibition of translation and most effectively accelerate the recovery from heat induced inhibition of transcription and translation. The peptide-binding domain in the carboxyl portion of hsp70 is of primary importance in protecting cells from thermal stress, probably through its binding to unfolded or partially folded polypeptides; this interaction, in turn, retards detrimental thermal denaturation or aggregation of key cellular proteins and protects cells from thermal stress. ATP binding and hydrolysis, on the other hand, are presumably necessary in the refolding and reassembly of thermally damaged proteins through multiple cycles of interactions between hsp70 and its targets. PMID- 7535237 TI - Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-beta, IGF-II, and IGFBP-1. AB - Trophoblast invasion of the human uterus is stringently controlled by the microenvironment. Invasive extravillous trophoblast cells in situ as well as in culture express a selective repertoire of cell surface integrins. Since migration is a necessary step in the invasion cascade, we tested whether certain integrins or invasion-regulating molecules, i.e., TGF-beta, IGF-II, and IGFBP-1 produced at the fetomaternal interface had a functional role on trophoblast migration. Flow cytometric analysis of integrin expression and the use of an in vitro cell migration assay revealed that exogenous TGF-beta upregulates integrin expression and reduces migratory ability to the invasive trophoblast, whereas IGF-II has no effect on integrin expression but stimulates migration. Trophoblast migration was inhibited in the presence of alpha 5 and beta 1 integrin blocking antibodies, indicating its dependence on the expression of these subunits. Furthermore, IGFBP 1, which contains an RGD sequence recognizing certain integrins, stimulated migration, an effect that was blocked by pretreatment with anti-alpha 5 or -beta 1 blocking Abs. These studies demonstrate that the migration of first trimester invasive trophoblast in vitro (1) requires the expression of alpha 5 and beta 1 integrin subunits, (2) is inhibited by TGF-beta, possibly due to increased cell adhesiveness to the extracellular matrix, (3) is stimulated by IGF-II by an as yet undetermined mechanism, and (4) is stimulated by IGFBP-1, likely by interaction with the RGD binding site of the alpha 5 beta 1 integrin. The invasion-regulating effects of TGF-beta, IGF-II, and IGFBP-1 may thus, at least in part, be due to their migration-regulating effects on the invasive trophoblast. PMID- 7535239 TI - Relationships between DNA fragmentation, chromatin condensation, and changes in flow cytometry profiles detected during apoptosis. AB - The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G0/G1 cell cycle region (the sub-G1 region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G1 region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies. PMID- 7535240 TI - Comparative study of glycosphingolipid composition in mammalian lenses. AB - The carbohydrate epitope Gal alpha 1-3Gal-R (alpha-galactosyl epitope), which is detectable by its binding with Bandeiraea simplicifolia-IB4 lectin, was found in glycosphingolipids (GSLs), both neutral and acidic (gangliosides), from lens tissues of non-primate mammals, but not in those of human senile cataracts and Old World monkeys. Instead, human cataractous and Old World monkey non cataractous lenses expressed Lewisx (Le(x)) epitopes (Gal beta 1-4(Fuc alpha 1 3)GlcNAc-R) in neutral GSLs. Sialylated Le(x) epitopes were found in rat and pig lenses as well as in human and Old World monkey lenses. Ganglio-series gangliosides, consisting mainly of GM3, GM1, GD1a and GD3, were detected in a species-specific fashion. On the other hand, alpha-galactosyl epitopes were expressed in lens tissues only in water-insoluble proteins of non-primate mammals, but Le(x) and sialylated Le(x) epitopes were not detectable in lens proteins. Among the several mammalian lenses examined, humans and Old World monkeys showed similar GSL compositions, in particular the presence of Le(x) and sialylated Le(x) epitopes and the absence of alpha-galactosyl epitopes, in lens tissue. PMID- 7535241 TI - Receptors for hyaluronan on corneal endothelial cells. AB - Previous investigations suggest that the corneal endothelium has specific binding sites for hyaluronan (HYA). In the present study, biochemical and immunological techniques were used to characterize these binding sites and to compare them with the liver endothelial cell (LEC) HYA receptor. Affinity chromatography of solubilised, 125I-labelled corneal endothelial cell surface proteins on immobilised HYA proved that there were molecules that were strongly bound to the polysaccharide. A part of these molecules formed a 100-kDa band when analysed by autoradiography after SDS polyacrylamide electrophoresis (PAGE). A specific antibody against the rat LEC HYA receptor was used for immunohistochemical studies of monkey and human corneas. There was a specific staining of the corneal endothelium of both species, and hyaluronan treatment before isolation of the human eyes reduced the staining intensity. Hyaluronidase treatment of the tissue sections before receptor staining strikingly increased the specific staining of the corneal endothelial cells (CEC). Immunoblotting of human corneal proteins, separated by SDS-PAGE, showed staining at 200, 150-160 and 55 kDa. Uptake experiments of tritiated HYA in cultured monkey CEC showed only a slight increase in cell associated radioactivity over 2-6 hr. The results make it unlikely that the corneal endothelial receptor, like its liver endothelial counterpart, is actively involved in receptor-mediated endocytosis. Our studies suggest that CEC carry receptors for HYA that are immunologically similar to the LEC receptors. CEC receptors might act as binding structures increasing the concentration of HYA close to the CEC as a protection of these vulnerable cells from physicochemical damage. PMID- 7535243 TI - Gamma-chain heterogeneity in Greek (delta beta)zero-thalassemia. AB - A molecular and biochemical population study of (delta beta)zero thalassemia in central Greece is described. The molecular study was focused on the type of the deletion and the status of G gamma-XmnI polymorphism, whereas the biochemical approach was centered on the G gamma/A gamma ratio as well as the frequency of the A gamma T chain in the fetal hemoglobin of 19 delta beta-thalassemia heterozygotes and 3 homozygotes. This study includes individuals from the mountainous district of Epirus (northwestern Greece) where the trait was found to be concentrated along the river Arachthos. The Sicilian (delta beta)zero thalassemia deletion was found in all subjects tested by direct PCR. The levels for the G gamma-chain presented values ranging from 29 to 83% of the total gamma chain content. Thirteen heterozygotes had the adult G gamma/A gamma ratio (mean G gamma: 35% +/- 10) of whom 10 were XmnI-negative (- / -), 6 had the newborn ratio (mean G gamma: 70% +/- 9) and were XmnI-positive, while homozygotes had equal amounts of G gamma and A gamma. Five of the 19 heterozygotes were A gamma T positive with low levels of this A gamma-chain variant, suggesting an in-trans to the delta beta-thalassemia determinant production. PMID- 7535245 TI - Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors. AB - To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum-free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin-3 (rIL-3), granulocyte/macrophage-CSF (rGM-CSF), granulocyte-CSF (rG-CSF), macrophage-CSF (rM-CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM-CSF, rG CSF, rGM-CSF and rIL-3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non-erythroid MDS progenitor cells. An increase of undifferentiated "blast" cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high-risk group. In the presence of these three CSFs, rIL-3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL-3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM-CSF or rIL-3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M-CSF is present in normal human serum at a concentration of 550 +/- 110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G-CSF combined with GM-CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non-erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high-risk group. PMID- 7535244 TI - Intensive treatment of AIDS-related non-Hodgkin's lymphomas with the MACOP-B protocol. AB - The usefulness of intensive chemotherapy with the MACOP-B protocol was evaluated in 8 patients with AIDS-related non-Hodgkin's lymphoma (NHL). Four patients had a prior AIDS diagnosis. The median CD4+ lymphocyte count was 0.079 cells x 10(9)/l (range 0.016-0.330). All patients responded to treatment. Four patients finished chemotherapy, all with complete remission, while another 3 patients deteriorated prior to finishing treatment and died. The median survival was 4 months (range 1 to 86 months). Major causes of the poor outcome were AIDS-related opportunistic infections and meningeal CNS involvement by NHL developing during or after chemotherapy. Patients with AIDS-related NHL usually do not appear to benefit from treatment with MACOP-B protocol. Advanced immunodeficiency is associated with poor tolerance to treatment and inability to finish this chemotherapy protocol. MACOP-B chemotherapy does not prevent meningeal spread of lymphoma in spite of using repeatedly systemic methotrexate crossing the blood-brain barrier. CNS prophylaxis with repeated application of intrathecal methotrexate may lower the risk of meningeal spread of lymphoma, which developed in 1 of 5 patients given CNS prophylaxis as compared to 2 of 3 patients without CNS prophylaxis. PMID- 7535246 TI - Enhancing peptide antigenicity by helix stabilization. AB - The engineering of antigenic determinants on super secondary structures using de novo design approaches often involves synthesis of long peptide chains (35-80 residues long). This communication illustrates that the stabilization of secondary structure by rational design can also greatly enhance immunogenicity and antigenicity, but in much shorter peptide sequences (21 residues long). A peptide epitope the sequence of which has been derived from the C-terminus of the chicken riboflavin carrier protein (cRCP), H2N-Tyr-His-Ala-Cys-Gln-Lys-Lys-Leu Leu- Lys-Phe-Glu-Ala-Leu-Gln-Gln-Glu-Glu-Gly-Glu-Glu-OH, has been chosen for analysis. Helical conformations were induced in the peptide in aqueous trifluoroethanol. Analogs were designed to stabilize this conformation in water by either the introduction of appropriately spaced ion pairs or the strongly helix nucleating residue alpha-aminoisobutyric acid (Aib), substituted for Ala/Gly, thus affording a comparison of the helix stabilization strategies. Circular dichroism (CD) results demonstrate that all the designed analogs are appreciably more helical than the parent peptide in 50% aqueous trifluoroethanol. Peptide antisera were raised for all analogs in rabbits. The affinities of these antisera for the native protein antigen, determined using a chaotrope disrupted binding assay, correlated very well with the helix content determined by CD. PMID- 7535247 TI - The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils. AB - Azide and hydroxylamine release nitric oxide (NO) enzymatically in biological conditions. We observed that both compounds were able to inhibit in vitro the programmed cell death of human eosinophils from peripheral blood. This protective effect could be mimicked by permeable cGMP analogs and by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Moreover, the soluble guanylate cyclase inhibitor LY-83583 inhibited in a dose-response manner the effects of the NO donors. Consequently, via the increase of eosinophil survival, NO could contribute to the amplification of inflammatory and allergic processes. This effect appears to be mediated, at least in part, by the soluble guanylate pathway. PMID- 7535248 TI - Interaction of primer tRNA(Lys3) with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation. AB - In the interaction between HIV-1 RT and tRNA(Lys3) each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV 1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed. PMID- 7535242 TI - Calcium activity and post-ischemic suppression of protein synthesis. AB - Increase in intracellular calcium concentration is a prominent feature of ischemia and has been considered a major factor in the initiation of ischemic pathology, which involves inhibition of protein synthesis. A reduction of calcium ion activity during and immediately after in vitro ischemia did not prevent inhibition of protein synthesis in hippocampae slices. When slices were overloaded with calcium by NMDA receptor activation or by the calcium ionophore A23187, no significant inhibition of protein synthesis was observed. We conclude that calcium overload plays only a limited role in ischemic inhibition of protein synthesis. PMID- 7535250 TI - Primary sequence and post-transcriptional modification pattern of an unusual mitochondrial tRNA(Met) from Tetrahymena pyriformis. AB - In a previous investigation of the rDNA region in Tetrahymena pyriformis mitochondrial DNA, we identified a putative tRNA(Met) gene [Heinonen et al. (1987) J. Biol. Chem. 262, 2879-2887]. On the basis of Northern hybridization analyses, we suggested that this gene is expressed, even though the resulting tRNA would be unusually small and have an atypical dihydrouridine stem-loop domain. We report here the complete nucleotide sequence and post-transcriptional modification pattern of this tRNA(Met), confirming its predicted primary structure and supporting the view that this structurally aberrant species functions in translation in T. pyriformis mitochondria. PMID- 7535249 TI - Differential codon usage: a safeguard against inappropriate expression of specialized genes? AB - Recent work has suggested that rare codons are sometimes used for the regulation of specialized gene expression in bacteria. Moreover, the cellular levels of certain tRNAs may fluctuate with growth conditions. Evidence implicating such mechanisms in the control of photosynthesis in Rhodobacter, solventogenesis in Clostridium, sporulation in Streptomyces, and fimbrial phase variation in E. coli is summarized. It is suggested that such mechanisms will prove applicable to the control of numerous additional specialized functions, and that the empirical tools for testing this possibility are currently available. PMID- 7535251 TI - Formation of the alpha 1-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism? AB - alpha 1-Microglobulin is an immunosuppressive plasma protein synthesized by the liver. The isolated protein is yellow-brown, but the hypothetical chromophore has not yet been identified. In this work, it is shown that a human liver cell line, HepG2, grown in a completely synthetic and serum-free medium, secretes alpha 1 microglobulin which is also yellow-brown, suggesting a de novo synthesis of the chromophore by the cells. alpha 1-Microglobulin isolated from the culture medium of insect cells transfected with the gene for rat alpha 1-microglobulin is also yellow-brown, suggesting that the gene carries information about the chromophore. Reduction and alkylation or removal of N- or O-linked carbohydrates by glycosidase treatment did not reduce the colour intensity of the protein. An internal dodecapeptide (amino acid positions 70-81 in human alpha 1 microglobulin) was also yellow-brown. The latter results indicate that the chromophore is linked to the polypeptide. In conclusion, the results suggest that the alpha 1-microglobulin gene carries information activating a post translational protein modification mechanism which is present in mammalian and insect cells. PMID- 7535252 TI - Overproduction of the bleomycin-binding proteins from bleomycin-producing Streptomyces verticillus and a methicillin-resistant Staphylococcus aureus in Escherichia coli and their immunological characterisation. AB - The bleomycin-binding proteins designated BLMA and BLMS, which confer resistance to bleomycin (Bm), from Bm-producing Streptomyces verticillus ATCC15003 and a methicillin-resistant Staphylococcus aureus B-26, respectively, were overexpressed in Escherichia coli. The present study showed that both BLMA and BLMS quench the antibacterial activity of Bm by the binding to the drug. To immuno-characterize the Bm-binding proteins, we constructed a monoclonal antibody against BLMA. The antibody, designated 893-12, did not cross react to BLMS and another Bm-binding protein from tallysomycin-producing Streptoalloteichus hindustanus. Although the ability of Bm to cleavage DNA was eliminated by a binding of BLMA to Bm, as shown by Sugiyama et al. [Gene 151 (1994) 11-15], the Bm-induced DNA degradation was restored by pre-incubation of BLMA with the anti BLMA monoclonal antibody. PMID- 7535254 TI - Immune privilege in the eye: an evolutionary adaptation. AB - The purpose of this study was to determine whether immune privilege exists in the eye of goldfish and to explore from an evolutionary point of view the relationship between the immunological and neurobiological microenvironments in the eye. Neural retinal or scale allografts and autografts were implanted into the vitreous cavity or the anterior chamber of goldfish eyes. Histological examinations were conducted to determine the fate of these grafts. In order to detect donor-specific immune suppression induced by intraocular retinal allografts, scale allografts obtained from the same donors were subsequently implanted orthotopically and evaluated. Neural retinal allografts implanted intraocularly were rapidly rejected by postimplantation day 8. In contrast, neural retinal autografts survived well within the eye with no inflammation. Prior intraocular allografts, either scale or retinal grafts, did not prevent rejection of subsequent scale allografts nor did they induce down-regulation of systemic immunity. Thus, immune privilege does not exist in the goldfish eye, implying that immune privilege in the eye (or central nervous system) may be an evolutionary adaptation acquired by higher vertebrates. Considering that the capacity for neural regeneration in the central nervous system diminishes during evolution, the hypothesis that immune privilege and neural regeneration may be mutually exclusive properties is addressed. PMID- 7535253 TI - Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads. AB - A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and destroys malaria parasites in the midgut. Normal development of parasites is observed in a closely related susceptible strain. To examine the basis for the difference in response, the two strains were compared for responses to inoculated Sephadex beads of varying charge. Negatively charged C-25 beads elicited a much stronger reaction in the refractory strain where 49% of the beads were strongly melanized by 24 h, compared with only 5% in the susceptible strain. Male mosquitoes of each strain responded similarly, with 100% of the beads strongly melanized by 24 h in the refractory strain compared with only 5% in the susceptible strain males. A time course revealed that the melanization in refractory but not susceptible mosquitoes increases substantially over time; 91% of C-25 beads were melanized in refractory females at 72 h compared with 9% in the susceptible sample. Neutral G-25 beads and positively charged A-25 beads were melanized in both strains, demonstrating that the capacity to melanize foreign particles is present in susceptible mosquitoes. PMID- 7535256 TI - Caesium-binding sites in the gramicidin pore. PMID- 7535255 TI - Identification, characterisation, and measurement of immunoglobulin concentrations in grey (Haliocherus grypus) and common (Phoca vitulina) seals. AB - Concentrations of macroglobulin, total gammaglobulin, and a gammaglobulin subclass were measured in grey and common seals. In pups, immunoglobulin M (IgM) was found to rise rapidly, concentrations reaching adult values by approximately 14 days postpartum. Total IgG concentrations increased more slowly, only approaching 50% of juvenile and adult male values by 30 days after birth. Concentrations of the IgG subclass did not change significantly postpartum. Total IgG concentrations measured in adult female grey seals sampled during lactation were found to be lower than in males and juveniles. This apparent immunosuppression may be associated with pregnancy. PMID- 7535257 TI - Histamine release from mast cells by polyamines: an NMDA receptor-mediated event? PMID- 7535258 TI - Immunochemical analysis of FK506 binding proteins in neuronal cell lines and rat brain. PMID- 7535259 TI - The subcellular distribution of FK506 binding proteins in rat brain. PMID- 7535260 TI - Body composition in immature rats with D-galactosamine induced hepatitis and bile duct ligation. PMID- 7535262 TI - The acute effects of a single dose of ethanol on biochemical indices of skeletal muscle composition: time course changes and comparison with the effects of endotoxin. PMID- 7535263 TI - Simple and efficient cDNA capture utilising a short gene-specific probe attached to magnetic beads. PMID- 7535261 TI - Insulin-like growth factor binding proteins in fed and nutritionally restricted rats: interaction with acute ethanol dosage. PMID- 7535265 TI - Polyamines and polyamine amides from wasps and spiders. PMID- 7535264 TI - Ornithine decarboxylase-induced cellular transformation: the involvement of protein tyrosine kinase(s) and pp130. PMID- 7535266 TI - Gi2 and Gi3 proteins mediate the inhibition of adenylyl cyclase by galanin in the RINm5F cell. AB - Inhibition of adenylyl cyclase activity is one of at least four mechanisms by which the neuropeptide galanin inhibits insulin secretion from pancreatic beta cells. In a membrane preparation of the insulin-secreting cell line RINm5F, a maximally effective concentration of galanin inhibited forskolin-stimulated adenylyl cyclase activity by 30%. Pretreatment of the cells with pertussis toxin abolished the inhibitory effect of galanin, indicating the involvement of Gi or Go guanine nucleotide binding proteins (G-proteins). Because galanin receptors interact with four G-proteins (Gi1, Gi2, Gi3, and Go1), any or all of these may inhibit adenylyl cyclase. Therefore, to identify the G-protein(s) involved, antibodies raised against various G-protein alpha-subunits were used to block the inhibition of forskolin-stimulated adenylyl cyclase activity by galanin in RINm5F membrane preparations. Antisera AS/7 and EC/2, specific for G alpha i1/alpha i2 and G alpha i3, respectively, were able to significantly attenuate the inhibitory effect of galanin, whereas antisera specific for Go proteins were not. The use of additional antisera specific for the various subtypes of Gi proteins indicated that Gi2 and Gi3, but not Gi1, are involved. Simultaneous application of antisera AS/7 and EC/2 resulted in a greater attenuation of the effect of galanin than application of either antiserum alone. Thus, galanin inhibition of adenylyl cyclase activity in these cells is selectively mediated by two inhibitory G proteins, Gi2 and Gi3. PMID- 7535267 TI - A clinical trial of the process-oriented treatment approach for children with developmental co-ordination disorder. AB - The process-oriented treatment (POR chi) approach is a time-limited programme aimed at increasing the kinaesthetic performance of children with mild motor problems in order to improve their motor performance. The approach was compared with a traditional or general motor approach and with no treatment in a randomized clinical trial of 75 children with developmental co-ordination disorder. The children were assessed before and after treatment and after a six week follow-up period. The results were mixed. The study provides evidence of the severity of so-called 'mild' motor problems of children referred to occupational therapy. The data suggest that these children do not improve spontaneously, and that their motor problems are very resistant to treatment. The data also suggest that an appropriate treatment strategy might be one that involves direct, repetitive training of a specific skill. PMID- 7535268 TI - A case of paroxysmal tonic upward gaze associated with psychomotor retardation. AB - The authors report a female with paroxysmal tonic upward gaze similar to the cases reported by Ouvrier and Billson (1988). She developed abnormal eye movements at the age of six months. Several anticonvulsants and levodopa were not effective in the control of the episodes; however, her upward eye-deviations spontaneously decreased during the one-year observation. Her clinical features were almost identical to those reported by Ouvrier and Billson, except that she had neurodevelopmental disorder and abnormal brain MRI. More data are required to clarify this unique phenomenon. PMID- 7535269 TI - The mastomys gastric carcinoid: aspects of enterochromaffin-like cell function. AB - The mastomys rodent exhibits a genetic propensity to develop gastric carcinoid tumors. Utilizing acid inhibitory pharmacotherapy (histamine-2 receptor antagonists and proton pump inhibitors), we have demonstrated transformation from normal to neoplastic enterochromaffin-like (ECL) cells in a well-defined fashion over a period of 4 months. In addition, we have demonstrated inhibition of tumor growth with either somatostatin or histamine-1 receptor antagonists (terfenadine and cyproheptadine). In order to define the regulation of growth and secretion of transformed ECL cells, we developed an isolated pure ECL cell system. ECL cells secrete histamine in response to gastrinergic (gastrin), muscarinic (carbachol), and beta-adrenergic (isoproterenol) stimulation. Both cAMP and intracellular calcium-dependent mechanisms are involved in the process of histamine secretion. PMID- 7535270 TI - Surgical management of carcinoid tumors: role of debulking and surgery for patients with advanced disease. AB - Surgery is the only potentially curative treatment for patients with carcinoid tumors. Patients with localized disease even with lymph node metastases can be resected for potential cure. Patients with distant metastatic disease have been reported to be cured by resection of all tumor. However, long-term follow-up of these individuals suggests that these patients probably will recur. Debulking surgery, that is removal of part but not all disease, has been advocated by some to decrease symptoms secondary to hormone secretion, relieve intestinal obstruction and ischemia, and prolong survival. Certainly, the first and second indications have been demonstrated by retrospective analysis of patient records. The final indication is less substantiated. It is my opinion that surgery to prolong survival will be beneficial if all gross tumor can be removed. Debulking procedures may improve quality but not quantity of life. Because of the potential benefits of surgery in the management of all patients with carcinoid tumors, a surgeon should be part of the team of physicians who manage these complex patients. PMID- 7535271 TI - [Effect of the neuropeptide substance P on the activity of the brain dopaminergic system in experimental parkinsonism]. PMID- 7535273 TI - Role of nitric oxide in the relationship of pancreatic blood flow and exocrine secretion in cats. AB - BACKGROUND/AIMS: Recent studies have suggested that, in the gastrointestinal tract, nitric oxide is an important mediator of alterations in blood flow and, in some organs, a second messenger involved in secretion. This study examined the role of NO in changes in pancreatic blood flow associated with basal and stimulated pancreatic exocrine secretion. METHODS: In anesthetized cats, we determined the effects of the NO synthase inhibitor NG-monomethyl-L-arginine (10 mg/kg) and the NO donor sodium nitroprusside (10 micrograms.kg-1.min-1) on pancreatic secretion and blood flow (hydrogen gas clearance). RESULTS: NG monomethyl-L-arginine had no effect on the increase in blood flow associated with secretin stimulation (271 +/- 52 vs. 290 +/- 50 mL.min-1.100 g-1) but reduced that associated with cholecystokinin stimulation (189 +/- 17 vs. 53 +/- 15 mL.min 1.100 g-1; P < 0.001). In contrast, NG-monomethyl-L-arginine significantly reduced both secretin- and cholecystokinin-stimulated secretion. Sodium nitroprusside had no effect on basal blood flow but significantly increased secretion. CONCLUSIONS: NO has a selective role in mediating changes in pancreatic perfusion and secretion. It seems to be important in stimulus secretion coupling with both secretin and cholecystokinin but is only responsible for the accompanying increase in pancreatic blood flow with cholecystokinin. PMID- 7535272 TI - A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator. AB - BACKGROUND/AIMS: In the intestine, the cystic fibrosis transmembrane conductance regulator (CFTR) has been localized to the apical pole of crypt epithelial cells. Recent data indicate that some villus cells may also express CFTR, although the identity of these cells has not been established. The aim of the current study was to characterize the distribution, morphology, and surface marker expression of CFTR-expressing villus cells. METHODS: Immunofluorescence and immunoelectron microscopy was performed using anti-CFTR and enzyme marker antibodies. RESULTS: In the rat and human proximal small intestine, a subpopulation of scattered villus and superficial crypt epithelial cells label brightly with anti-CFTR antibodies. The fluorescent signal is detected throughout the cells with its greatest concentration apically. At the ultrastructural level, labeling involves the brush border and a prominent subapical vesicular compartment. The cells resemble adjacent villus enterocytes in their abundance of mitochondria and expression of basolateral Na(+)-K(+)-adenosine triphosphatase yet differ in their absence of brush-border sucrase and lactase expression. CONCLUSIONS: A previously uncharacterized subpopulation of villus cells with high levels of intracellular CFTR expression exists in the proximal small intestine. Morphological and cytochemical studies suggest that this subset of villus cells has a unique transport function. PMID- 7535274 TI - Effect of a wheat amylase inhibitor on canine carbohydrate digestion, gastrointestinal function, and pancreatic growth. AB - BACKGROUND/AIMS: Chronic amylase inhibition might be useful to treat diabetes mellitus and obesity. Duodenal and ileal cannulas were placed in 8 dogs to determine if long-term ingestion of a wheat amylase inhibitor maintained amylase inhibition or affected gastrointestinal or metabolic function or pancreatic growth. METHODS: Five dogs were fed and 3 were not fed 1.5 g of the inhibitor with meals for 9 weeks. Postprandial and cholecystokinin octapeptide stimulated pancreatic secretion, and fecal balance studies were performed at intervals. After the experiment, the pancreas was analyzed. RESULTS: Weight loss was similar in both groups. Amylase inhibition persisted throughout the 9 weeks; it declined from 91% to 37% from the first to the sixth postprandial hour. Amylase inhibition decreased plasma glucose levels during the first hour (P < 0.05), increased carbohydrate delivery to the ileum (315 vs. 555 mg/h; P = 0.002), and increased cholecystokinin octapeptide-stimulated amylase secretion. However, amylase inhibition did not significantly change plasma concentrations of insulin, peptide YY or neurotensin, postprandial pancreatic secretion, gastrointestinal transit or pancreatic weight, and protein or DNA content. CONCLUSIONS: Prandial ingestion of 1.5 g of the inhibitor for 9 weeks reduces postprandial amylase levels enough to delay carbohydrate digestion and absorption and lower plasma glucose levels without altering pancreatic growth. This dose may be effective to treat diabetes mellitus but not obesity. PMID- 7535275 TI - Preoperative cyst fluid analysis is useful for the differential diagnosis of cystic lesions of the pancreas. AB - BACKGROUND/AIMS: It has been suggested that activity of pancreatic enzymes and concentrations of tumoral markers in cyst fluid may help to distinguish pseudocyst, serous, and mucinous cystadenomas. The aim of this study was to prospectively assess the reliability of preoperative biochemical and tumor marker analysis in cyst fluids obtained by fine-needle aspiration for pathological diagnosis. METHODS: Cyst fluid was obtained preoperatively by fine-needle aspiration, and biochemical and tumoral marker values were measured. The diagnosis of cystic tumors (7 serous cystadenomas and 12 mucinous tumors) was established by surgical specimen analysis. Thirty-one pancreatic pseudocysts complicating well-documented chronic pancreatitis were also studied. RESULTS: Carbohydrate antigen 19.9 levels of > 50,000 U/mL had a 75% sensitivity and a 90% specificity for distinguishing mucinous tumors from other cystic lesions. Carcinoembryonic antigen levels of < 5 ng/mL had a 100% sensitivity and an 86% specificity for distinguishing serous cystadenomas from other cystic lesions. Amylase levels of > 5000 U/mL had a 94% sensitivity and a 74% specificity for distinguishing pseudocysts from other cystic lesions. CONCLUSIONS: High carbohydrate antigen 19.9, low carcinoembryonic antigen, and high amylase levels in cyst fluid are very indicative of mucinous tumors, serous cystadenomas, and pseudocysts, respectively. PMID- 7535277 TI - Self-expanding metal stents for palliation of esophagocardial malignancies. PMID- 7535276 TI - Expression of pancreatic enzymes (alpha-amylase, trypsinogen, and lipase) during human liver development and maturation. AB - BACKGROUND/AIMS: Although pancreatic enzymes have been found in hilar intrahepatic bile ducts in adult humans, their expression during human liver development is unclear. The aim of this study was to clarify the temporal expression of pancreatic enzymes at various stages of human liver development. METHODS: We immunohistochemically investigated pancreatic alpha-amylase, trypsinogen, and lipase expression in fetal, neonatal, juvenile, and adult human livers. RESULTS: In hilar duct development, alpha-amylase but not trypsinogen or lipase was expressed in the ductal plate. These three enzymes were expressed in biliary cells migrating into the mesenchyma, in immature ducts in fetal livers, and in maturing and mature ducts in postnatal livers. Their expression was weak and diffusely cytoplasmic in fetal livers, whereas in postnatal livers their expression was strong, granular, and located in the supranuclear cytoplasm. Expression was not found in developing peripheral ducts. These enzymes were expressed in immature hepatocytes (9-25 weeks' gestation) but disappeared thereafter. Enzyme expression was mild in fetal pancreata and strong in adult pancreata. CONCLUSIONS: Pancreatic enzymes may be present in primitive hilar bile ducts and hepatocytes in fetal livers; hilar ducts, hepatocytes, and pancreas may have similar fetal enzymatic profiles. Intrahepatic hilar bile ducts, hepatocytes, and exocrine pancreas may have a common cell lineage. PMID- 7535278 TI - Use of a very flexible guide wire to permit dilation of complex malignant strictures of the esophagus. AB - Risk of perforation is a major impediment to the use of polyvinyl bougies in palliative dilation of cancerous strictures of the esophagus. We encountered 23 patients with complex malignant strictures in whom initial dilation with Savary Gilliard bougies was thwarted because attempts to pass a conventional Eder Puestow guide wire were unsuccessful. As a recourse, we probed these strictures with a very flexible guide wire of the type used to implant prostheses in the biliary tract. The purpose was to establish a passage through which a standard guide wire could then be inserted. The procedure was successful in all but 4 of the 23 patients. We conclude that in such cases the preliminary use of the very flexible guide, even though time-consuming, improves the chance of effective dilation with minimal added risk. PMID- 7535279 TI - enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties. AB - Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development. PMID- 7535281 TI - Characterization of a putative 23S-5S rRNA operon of Buchnera aphidicola (endosymbiont of aphids) unlinked to the 16S rRNA-encoding gene. AB - Buchnera aphidicola (Ba) is an endosymbiont of the aphid Schizaphis graminum. In order to obtain information on highly expressed genes, we have chosen to study Ba genes coding for rRNAs. Previously, the single-copy rrs gene was cloned and sequenced [Munson et al., Gene 137 (1993) 171-178], and found to constitute a single transcription unit unlinked to rrl and rrf. In the present study, a 6.1-kb Ba DNA fragment containing rrl was cloned into Escherichia coli (Ec) and sequenced. Based on sequence similarity to Ec, the following genes were identified: aroE-tRNA(Glu)-rrl-rrf-cysS. AroE and CysS had 48 and 54% amino acid (aa) identity, respectively, to the corresponding Ec proteins; tRNA(Glu), rrl and rrf had 80-90% nucleotide (nt) identity with the corresponding genes of Ec rrnB. Ba tRNA(Glu)-rrl-rrs appears to be part of a single transcriptional unit; a putative promoter and a Rho-independent terminator were identified. Comparisons of sequences of aroE-rrl from endosymbionts of seven additional species of aphids indicated conservation of the -35 (TTGACT) and -10 (TGTAA/TT) promoter regions, and boxA, tRNA(Glu) and boxC. Secondary structure analysis indicated that the Ba tRNA(Glu)-rrl-rrf operon resembled the homologous region of Ec rrnB. The results of this and previous studies indicate that Ba differs from most bacteria in having the single-copy rRNA genes organized into two transcription units. PMID- 7535280 TI - Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA. AB - A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA. RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures. Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant. The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation. PMID- 7535283 TI - Expression of binding of plasminogen, thrombospondin, vitronectin, and fibrinogen, and adhesive properties by Escherichia coli strains isolated from patients with colonic diseases. AB - Escherichia coli strains isolated from patients with colonic disorders (n = 27) and strains isolated from the rectal mucosa of healthy subjects (n = 24) were compared with respect to expression of cell surface hydrophobicity, carriage of intestinal virulence factors, adhesion to tissue culture cells, and expression of binding of extracellular matrix proteins and plasma proteins. Strains isolated from patients with colonic disease did not express a more hydrophobic cell surface than strains from healthy subjects. Few strains from both groups carried genes encoding for recognised virulence factors of E coli. Only one strain, carrying the eae gene induced actin polymerisation in tissue culture cells. Strains from patients with colonic diseases adhered to HT29 cells, which are of intestinal origin, to a higher extent than E coli from healthy subjects. Significantly more strains from patients with colonic disorders than E coli from healthy subjects expressed binding of fibronectin, collagens, laminin, vitronectin, plasminogen, throbospondin, and fibrinogen. Expression of binding of these proteins may influence the pathogenesis of colonic disease by mediating binding to ulcerated tissue, preventing complement induced lysis of bacteria and by exerting proteolytic activity. There was no correlation between serotype, expression of cell surface hydrophobicity, and binding of extracellular matrix and plasma proteins. PMID- 7535282 TI - Pharmacological manipulation of gastric juice: thrombelastographic assessment and implications for treatment of gastrointestinal haemorrhage. AB - The impairment of formation and maintenance of a formed fibrin clot contributes to the prolonged bleeding and high incidence of rebleeding in upper gastrointestinal haemorrhage. To investigate the basis for the use of drug therapy in gastric bleeding, this study used thrombelastography to determine the effects of pharmacological manipulation of gastric juice on coagulation and fibrinolysis. The thrombelastograph is a mechanical device that provides a visual assessment of all stages of coagulation and fibrinolysis. The effects of fresh and pharmacologically changed gastric juice was assessed after its addition to fresh whole blood in the thrombelastograph cuvette. Pharmacological manipulation was achieved through alkalisation or through addition of tranexamic acid, aprotinin, or sucralfate. Fresh gastric juice delayed clot formation, decreased maximum clot amplitude, and stimulated clot lysis. Alkalisation inhibited the lytic effects of fresh gastric juice and improved the induced abnormalities in coagulation. Tranexamic acid partially inhibited gastric juice induced clot lysis but did not exhibit a beneficial effect on coagulation. Sucralfate, and to a lesser extent aprotinin significantly inhibited fibrinolysis but exacerbated the detrimental effect of gastric juice on the parameters of coagulation. Alkalisation of gastric juice reduces the adverse effect on coagulation and fibrinolysis. Tranexamic acid, aprotinin, and sucralfate can all reduce or inhibit clot lysis, but the adverse effects on clot formation may outweigh any potential benefit in the treatment of gastrointestinal bleeding. PMID- 7535285 TI - Adenine arabinoside 5'-monophosphate in patients with chronic hepatitis B: comparison of the efficacy in patients with high and low viral replication. AB - This study compared the response to adenine arabinoside 5'-monophosphate (ARA AMP) in 60 patients with chronic hepatitis B according to the pretreatment serum hepatitis B virus DNA concentration. The level of hepatitis B virus replication was defined as low (30 patients) or high (30 patients) when serum hepatitis B virus DNA concentration was below or above 100 pg/ml, respectively. Patients received a 28 day course of ARA AMP and a second course of ARA AMP was given six months later to patients with persistent hepatitis B virus replication. At the end of the first course of ARA AMP, 11 of the patients (37%) with low replication and one of the patients (3%) with high replication became negative for hepatitis B virus DNA (p = 0.0012); five of the patients (17%) with low replication and none of the patients with high replication had HBe seroconversion (p = 0.06). Two of these five patients lost HBsAg. Kinetics of serum hepatitis B virus DNA during treatment showed a considerable but transient antiviral effect of ARA AMP. Three of 32 retreated patients became negative for hepatitis B virus DNA and one patient had HBe seroconversion. In conclusion, ARA AMP exerts a considerable but transient antiviral effect on hepatitis B virus. Complete and sustained inhibition of hepatitis B virus replication was only obtained in the patients with low hepatitis B virus replication. PMID- 7535284 TI - Increased expression of cell adhesion molecule P-selectin in active inflammatory bowel disease. AB - The pathogenic changes of inflammatory bowel disease (IBD) depend on migration of circulating leucocytes into intestinal tissues. Although leucocyte rolling and tenuous adhesion are probably regulated by inducible selectins on vascular endothelia, little is known about the expression of these molecules in Crohn's disease and ulcerative colitis. Using immunohistochemistry on surgically resected specimens, this study investigated endothelial P-selectin (CD62, granular membrane protein-140) in frozen sections of histologically uninvolved tissues adjacent to inflammation (Crohn's disease = 10; ulcerative colitis = 10), from highly inflamed areas (Crohn's disease = 20; ulcerative colitis = 13), and from normal bowel (n = 20). By light microscopy, two forms of P-selectin immunoreactivity were detected that apparently corresponded ultrastructurally to stored and released distributions. Compared with the normal gut, there was a 3.7 fold increase of P-selectin immunoreactivity on veins (p < 0.0001), venules (p < 0.0001), and capillaries (p < 0.05) in the highly inflamed gut, without differences between Crohn's disease and ulcerative colitis. In the uninvolved gut, P-selectin expression was similar to that seen in normal controls, except for a focal increase of P-selectin in the vicinity of small lymphocyte aggregates. The dramatic upregulation of P-selectin in the inflamed tissue and its potential role in leucocyte trafficking support the concept of P-selectin blocking therapy for the control of active IBD. PMID- 7535286 TI - Mapping a putative tumor suppressor gene on chromosome 9 bands p21-p22 with microdissection probes. AB - Deletions of the short arm of chromosome 9 have been observed in a number of malignant cell lines and primary tumor samples using cytogenetic and molecular techniques. These tumors include acute lymphoblastic leukemias, lymphomas, gliomas, melanomas, mesotheliomas, bladder cancer, and lung cancer. The smallest region of overlap (SRO) of these deletions is thought to contain a tumor suppressor gene. A microdissection library was constructed from bands 9p21-p23 to obtain DNA probes that would be useful in further defining the limits of the deletions. Eight single-copy probes were found to be homozygously deleted in at least 1 of the 10 cell lines examined. The mapping of these 8 clones using a panel of cell lines with deletions revealed that 3 probes mapped telomeric to the SRO and 5 clones mapped centromeric to the SRO. PMID- 7535287 TI - Arrangement of a cluster of three mouse type I keratin genes expressed sequentially during esophageal-type epithelial cell differentiation. AB - Keratins are intermediate filament proteins expressed in epithelial cells. They are divided into two groups, type I and type II, that must associate to form filaments. The genes encoding these proteins are clustered in two type-specific loci. In stratified epithelia, differentiation of the basal cells is accompanied by a switch in the expression of keratin genes. However, how this switch is controlled is not yet understood. We report here the cloning and mapping of a 55 kb region surrounding the keratin 19 (K19) gene in the mouse genome. This gene encodes a type I subunit expressed in simple and complex epithelia, notably in nonkeratinizing stratified epithelia of internal organs. In these tissues, it is expressed in basal cells and not in suprabasal cells, where the main type I subunit is keratin 13. Using probes corresponding to highly conserved sequences in intermediate filament proteins, we mapped two other genes downstream from the K19 gene. Restriction mapping and sequencing data indicate that they encode the mouse K15 and K13. The three genes are separated by about 5-6 kb, and they are in the same transcriptional orientation. Because the three genes are expressed together in stratified epithelia and because their order of expression during differentiation is the same as their order on the chromosome, we suggest that there is a relationship between their genomic organization and the control of their expression. PMID- 7535288 TI - Localization of the alpha 2-macroglobulin receptor-associated protein 1 gene (LRPAP1) and other gene fragments to human chromosome 4p16.3 by direct cDNA selection. PMID- 7535289 TI - Fas antigen and sphingomyelin-ceramide turnover-mediated signaling: role in life and death of T lymphocytes. PMID- 7535290 TI - The role of APO-1-mediated apoptosis in the immune system. PMID- 7535291 TI - From apoptosis to autoimmunity: insights from the signaling pathways leading to proliferation or to programmed cell death. PMID- 7535292 TI - Regulation of C3 deposition on gp120 coated CD4 positive cells by decay accelerating factor and factor H. AB - We investigated complement activation by recombinant gp120 (rgp120) treated CD4 cells and the role host complement regulatory proteins play in controlling C3 deposition. Complement activation was determined by detection of C3 on rgp120 coated cells in the presence and absence of HIV seropositive sera using flow cytometry. Treatment of rgp120 coated cells with complement resulted in C3 deposition only if HIV positive sera was included. Examination of C3 fragments on these cells demonstrated rapid cleavage of C3b to iC3b. The role of the regulatory proteins was examined by pretreating cells with mAb to block decay accelerating factor (DAF) or membrane cofactor protein (MCP) or by using factor H depleted sera as a complement source. Inhibition of DAF or use of factor H depleted sera significantly increased C3 deposition on rgp120 coated cells. In contrast, C3 deposition on rgp120 coated cells was not increased after blocking MCP. The sensitivity of rgp120 coated cells to complement lysis was unchanged after inhibition of the regulatory proteins, despite the increase in C3 deposited. These results indicate that in a model of virus infected cells, C3 deposition is regulated by DAF and factor H but MCP appears to have no role. PMID- 7535293 TI - Lack of correlation between early intracellular calcium ion rises and the onset of apoptosis in thymocytes. AB - Apoptosis, a well-recognized process of cell death, is usually defined by chromatin condensation, plasma membrane blebbing, reduction in cell volume, and in many cell types the cleavage of DNA into nucleosomal multiples, and finally the formation of apoptotic bodies. We have characterized the time of onset and the range of concentrations at which the toxins gliotoxin and thapsigargin induce apoptosis in thymocytes. We also looked for early changes in cytosolic calcium ion concentration ([Ca2+]i). Three methods were used to detect apoptosis: cellular morphology, DNA fragmentation and a flow cytometric method using ethidium bromide. Calcium fluxes were measured using both flow cytometry and bulk cell fluorimetry. Gliotoxin concentrations of 50 nmol/L to 10 mumol/L induced significant numbers of cells to become apoptotic in a dose dependent manner. At these concentrations there was no observable increase in [Ca2+]i as determined by flow cytometry or in bulk cells. However, when thymocytes were treated with gliotoxin at concentrations greater than 500 mumol/L, rises in [Ca2+]i were apparent, but these cells died by necrosis. Thapsigargin induced low levels of apoptosis in thymocytes; the maximum effect observable after a 10 nmol/L treatment. Thapsigargin is known to inhibit the Ca(2+)-ATPase in the endoplasmic reticulum thereby causing a sustained increase in [Ca2+]i in thymocytes. The rise in [Ca2+]i observed was quantitatively similar when thymocytes were treated with thapsigargin concentrations ranging between 10 and 100 nmol/L. These results led us to investigate the effect of dexamethasone on [Ca2+]i. In these experiments thymocytes showed no rises in [Ca2+]i above the control over 85 min following treatment with 10 mumol/L dexamethasone. PMID- 7535295 TI - Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment. AB - Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI. distamycin A (DA) and 4',6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level. PMID- 7535294 TI - Hepatitis C virus and Sjogren's syndrome. AB - Viral infection has been suggested as a possible cause of Sjogren's syndrome. After we had noted Sjogren's syndrome in several patients infected with hepatitis C virus (HCV), we set up a prospective study to investigate the association of Sjogren's syndrome and HCV liver disease. We studied 10 patients with primary Sjogren's syndrome and observed four with HCV infection. A striking association between HCV infection and Sjogren's syndrome was found; however, a direct link could not be proved. PMID- 7535297 TI - Retention of glycogen in cryosubstituted mouse liver. AB - A periodic acid-Schiff (PAS)-type reaction in which osmium-ammine was used as the reagent was carried out on ultrathin sections of mouse liver in order to study the extent to which glycogen is preserved. Comparisons were made between tissues that were, on the one hand, conventionally fixed and dehydrated and, on the other, those that were high-pressure frozen and cryosubstituted in acetone. A control was carried out for both groups using a routine uranyl acetate-lead citrate staining procedure. In the latter case, glycogen could be identified as electron-clear patches in the cytoplasm whereas after a PAS-type reaction, glycogen became darkly contrasted. In the case of conventionally fixed samples, glycogen appeared to display a certain amount of clumping separated by gaps whereas in cryosubstituted specimens it was denser and often showed elongated interconnecting structures. These results suggest that cryofixation and cryosubstitution provide better preservation of glycogen in mouse liver tissue compared with chemically fixed specimens. In addition, the fine structure of glycogen appears more homogeneous, showing less aggregation in cryo-treated liver samples. PMID- 7535296 TI - Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland. AB - The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed "ringlet like" structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9 +/- 4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7 +/- 2.0 pmol/g).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535298 TI - Expression and distribution of fetuin in the developing sheep fetus. AB - Tissue distribution and developmental expression of fetuin were studied in the sheep fetus from embryonic day (E) 30 to adult (gestational period is 150 days). The presence of fetuin was demonstrated immunocytochemically using anti-fetuin antibodies; in situ hybridisation using short anti-sense oligonucleotide probes labelled with digoxigenin was used to study the ability of the developing tissue to synthesise fetuin, and reverse transcription-polymerase chain reaction (RT PCR) was used to estimate the level of fetuin mRNA in selected tissues. Tissue distribution of fetuin was widespread in the younger fetuses (E30 to E40). The most prominent presence due to in situ synthesis was demonstrated in the liver, central nervous system (CNS) including anterior horn cells, dorsal root ganglia and in skeletal muscle cells. Other developing tissues and organs that showed evidence of fetuin synthesis and presence of the protein included mesenchyme, kidney, adrenal, developing bone, gut, lung and heart. In the immature liver (E30 40) there was a strong signal for fetuin mRNA in hepatocytes and also in numerous haemopoietic cells; the proportion of these latter cells that was positive for fetuin mRNA increased between E30 and E40. Only some hepatocytes and a proportion of the haemopoietic stem cells were immunoreactive for fetuin itself at E30-40; immunoreactive hepatocytes were more frequently observed in the more mature outer regions of the developing liver. Lung and gut contained scattered fetuin-positive epithelial cells, especially at E30; a weak fetuin mRNA signal could be detected above background in many of these cells up to E40, but not at E60-E115 or in the adult. Particularly at E30 to E40, mesenchymal tissue both within organs such as the gut and lung and around forming bone and skeletal muscle contained cells that were positive for fetuin mRNA. Mesenchyme at these ages was also very strongly stained for fetuin protein, much of which may reflect fetuin in tissue extracellular spaces and be derived from the high concentration in plasma. By E80 fetuin mRNA was mainly present in the liver and the CNS; staining of the muscle tissue was becoming less pronounced. However in developing bone tissue, staining of chondrocytes for fetuin mRNA was still prominent in older (E80) fetuses; there was also fetuin protein staining of chondrocytes at the growing surfaces of bones and in bone marrow at this age. In the adult, weak immunocytochemical staining for fetuin itself was present in hepatocytes, but the mRNA signal was barely above the threshold limit of detection.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7535299 TI - Improved in situ beta-galactosidase staining for histological analysis of transgenic mice. AB - The present study describes a novel method for the histochemical demonstration of beta-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro 3-indolyl-beta-D-galactoside (X-Gal) with 5-bromo-indolyl-beta-o galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial beta galactosidase (lacZ). After beta-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo Gal results in greatly enhanced sensitivity of the staining method for beta galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of beta-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter. PMID- 7535300 TI - Lectin staining of sheep microglia. AB - The B4-isolectin from Griffonia simplicifolia is known to stain microglial cells in a variety of species. The present report describes a lectin staining method that has been modified to facilitate staining of resting microglia, as well as perivascular cells, in vibratome sections of normal sheep brain. This modified method employs tissue fixed in formaldehyde or paraformaldehyde and requires incubating sections with Triton X-100 prior to staining. PMID- 7535301 TI - Fractional clearances of low molecular weight proteins in lead workers. AB - Urinary alpha 1-microglobulin (alpha 1-m) and beta 2-microglobulin (beta 2-m) can be used as early indicators of renal tubular dysfunction. However, low levels of lead exposure cause an increase in urinary alpha 1-m, but not in urinary beta 2 m. In order to clarify the level of tubular dysfunction in early lead nephropathy, fractional clearances of alpha 1-m (FC-alpha 1-m) and beta 2-m (FC beta 2-m), i.e., the ratios of these clearances to the creatinine clearance, were measured in 99 male lead workers. Blood urea nitrogen, serum creatinine, uric acid, and urinary creatinine and N-acetyl-beta-D-glucosaminidase activity were also measured to diagnose the presence of other renal dysfunction. The median of FC-alpha 1-m was 0.13% in the control group. The FC-alpha 1-m increased in lead workers with blood lead (B-Pb) levels above 20 micrograms/dl. The correlation of FC-alpha 1-m with urinary alpha 1-m was highly significant, but there was no correlation with serum alpha 1-m. The median of FC-beta 2-m was 0.065% in the control group. There was a correlation of FC-beta 2-m with FC-alpha 1-m, but there was no correlation with B-Pb, or with serum beta 2-m. These results suggest the following: There was a very low excretion rate of alpha 1-m and beta 2-m in both the control group and the lead exposed groups. The excretion rate of alpha 1 m was higher than that of beta 2-m.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535302 TI - Quality improvement in the biomedical communications audio visual unit. AB - The audio visual unit is a highly visible and most frequently requested service provided by a biomedical communications department. To continuously improve service, delivery, and operation of an audio visual communications function is a challenge. This article presents a field-tested audit tool for describing audio visual unit strengths and limitations. The tool suggests four assessment areas: the service plan, the client communication network, training and managing staff, and standard operating policy and procedures. PMID- 7535303 TI - KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B. AB - Kinesin is known as a representative cytoskeletal motor protein that is engaged in cell division and axonal transport. In addition to the mutant assay, recent advances using the PCR cloning technique have elucidated the existence of many kinds of kinesin-related proteins in yeast, Drosophila, and mice. We previously cloned five different members of kinesin superfamily proteins (KIFs) in mouse brain (Aizawa, H., Y. Sekine, R. Takemura, Z. Zhang, M. Nangaku, and N. Hirokawa. 1992. J. Cell Biol. 119:1287-1296) and demonstrated that one of them, KIF3A, is an anterograde motor (Kondo, S., R. Sato-Yashitake, Y. Noda, H. Aizawa, T. Nakata, Y. Matsuura, and N. Hirokawa. J. Cell Biol. 1994. 125:1095-1107). We have now characterized another axonal transport motor, KIF2. Different from other KIFs, KIF2 is a central type motor, since its motor domain is located in the center of the molecule. Recombinant KIF2 exists as a dimer with a bigger head and plus-end directionally moves microtubules at a velocity of 0.47 +/- 0.11 microns/s, which is two thirds that of kinesin's. Immunocytological examination showed that native KIF2 is abundant in developing axons and that it accumulates in the proximal region of the ligated nerves after a 20-h ligation. Soluble KIF2 exists without a light chain, and KIF2's associated-vesicles, immunoprecipitated by anti-KIF2 antibody, are different from those carried by existing motors such as kinesin and KIF3A. They are also distinct from synaptic vesicles, although KIF2 is accumulated in so-called synaptic vesicle fractions and embryonal growth cone particles. Our results strongly suggest that KIF2 functions as a new anterograde motor, being specialized for a particular group of membranous organelles involved in fast axonal transport. PMID- 7535306 TI - Dissociation of minor satellite from the centromere in mouse. AB - The minor satellite DNA of mouse is believed to constitute the centromere. We report that centromeres of some chromosomes in the Cl1D cells of mouse are not associated with this DNA even though the latter is present on these chromosomes. The satellite DNA was detected distally from the centromere and could not be mistaken as a component of the centromere. We also report that the site of the primary constriction may not always coincide with the site of the anti kinetochore antibody reaction. Whereas the regions containing the major satellite decondense upon treatment with bisbenzimidazole (Hoechst 33258), the sites carrying minor satellite resist decondensing. PMID- 7535305 TI - The exocytotic fusion pore of small granules has a conductance similar to an ion channel. AB - We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex. PMID- 7535307 TI - An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti). AB - Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer ('thread,' approximately 60 cm length, approximately 3 microns width). In view of the unusual ultrastructure of the thread, its extracellular role in modulation of the viscoelastic properties of mucus, and the ancient lineage of this primitive vertebrate, we report the nucleotide and deduced amino acid sequences of one major thread IF subunit, alpha (pI 7.5), which is coexpressed with a second polypeptide, gamma (pI 5.3). These two polypeptides coassemble in vitro into approximately 10 nm filaments. The alpha-thread chain, a 66.6 kDa polypeptide, has an unusual central rod domain containing 318 residues flanked by N- and C terminal domains of 192 and 133 residues, respectively. Each peripheral region exhibits some epidermal keratin-like features including peptide repeats and a high total content of glycine and serine residues. The terminal domains, however, lack the H1 and H2 subdomains characteristic of known keratins. Moreover, when the central rod is aligned either in relation to established homology profiles (J. F. Conway and D. A. D. Parry (1988) Int. J. Biol. Macromol. 10, 79-98) of other IF subunits (types I-V, nestin, non-neuronal invertebrate), or by computer based homology searches of the GenBank/EMBL Data Bank, a low identity (< 30%) is evident, with no preferred identity to keratins or other known IF types. Although the central rod of 318 residues consists of the canonical apolar heptad repeats interspersed with three linker regions, a discontinuity in phasing of the heptad substructure in rod 2B, and conserved sequences at either end of the rod domain, other collective characteristics are atypical: overall high threonine content (13.2% vs 2.3-5.4% for other IFs), high threonine content in rod 1B (18.8% vs 1 6%), five Thr-Thr repeats in coiled coil segments, L12 of length greater than in keratins, substitution of phenylalanine for a highly conserved glutamate in the sixth position of L2, and a glycine-proline sequence in segment 2B. Possibly as a result of the high threonine content, the percentage of both acidic and basic residues in most helical subdomains is reduced relative to type I and II chains. Fast Fourier transform analyses show that only the acidic residues in segment 1B and basic residues in segment 2 have near typical IF periods.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7535308 TI - A specifically apical sub-membrane intermediate filament cytoskeleton in non brush-border epithelial cells. AB - Although many pieces of evidence support the notion of a role for the cytoskeleton in epithelial polarization, no cytoskeletal component has been found to be specifically apical, except for some actin-binding proteins. Here we report the apical distribution of a 53 kDa cytokeratin. Furthermore, this cytokeratin co purified with biotinylated apical plasma membrane proteins in high density complexes. Differential biotinylation of the basolateral domain showed that the 53 kDa protein is mainly attached to the apical membrane, although a companion 58 kDa protein attaches to both apical and basolateral membrane proteins. Immunoprecipitation experiments indicated that a number of apical components are directly or indirectly linked to the 53 kDa protein. These results indicate the existence of a terminal web-like structure in non-brush-border cells, which attaches to the apical domain and may play a role in apical polarization, especially during the acquisition of polarity from non-polarized cellular stages. PMID- 7535304 TI - PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra-peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes. AB - We have previously described mutant S. cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. J. Cell Biol. 123:1133-1147.). In some mutants, peroxisomes are undetectable. Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359.). In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence). These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information. Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment. The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions. The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant. The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies. The PEB1 gene product does not cleave the thiolase-targeting sequence. It may function to draw thiolase into peroxisomes. PMID- 7535309 TI - New variant of Vibrio cholerae O1 from clinical isolates in Amazonia. AB - A survey of pathogenic Vibrio cholerae O1 strains from the north of Brazil by using arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vivo and in vitro techniques and the group has been denominated the Amazonian variant of V. cholerae O1. PMID- 7535310 TI - Detection and quantitation of the glucuronoxylomannan-like polysaccharide antigen from clinical and nonclinical isolates of Trichosporon beigelii and implications for pathogenicity. AB - Sera from patients with systemic infections caused by the opportunistic fungus Trichosporon beigelii have been shown to cross-react with anticryptococcal antibodies. We quantitatively compared the amounts of antigen produced and examined the expression of O-acetyl epitopes from 35 strains of T. beigelii isolated from deep and superficial infections. By counterimmunoelectrophoresis, 10 of 10 isolates from deep infections were positive for polysaccharide, compared with 7 of 13 isolates from superficial infections (P = 0.02). All 23 strains tested were positive for polysaccharide when screened by immunodot. By enzyme immunoassay, the cross-reactive antigen produced by deep isolates (n = 9) had a mean titer of 1:5,500. In contrast, superficial isolates (n = 22) produced significantly less antigen than the deep isolates (P < 0.001), with a mean titer of 1:700. Isolates from environmental sources (n = 3) were similar to the superficial isolates, with a mean titer of 1:600. The mean concentrations +/- standard errors of cross-reactive polysaccharide released by deep isolates and superficial isolates were 3.09 +/- 0.44 and 1.74 +/- 0.30 micrograms/ml, respectively, when measured by rocket immunoelectrophoresis (P = 0.02). O-Acetyl epitopes were detected on polysaccharide from 8 of 9 strains of T. beigelii isolated from deep sources, while only 2 of 12 superficial isolates expressed detectable O-acetyl epitopes (P = 0.01). Thus, while all isolates of T. beigelii tested were capable of producing glucuronoxylomannan-like cross-reactive antigen, pathogenic isolates produced significantly more antigen than superficial or environmental isolates. Furthermore, significantly more pathogenic isolates than superficial or environmental isolates expressed antigen that was O acetylated. PMID- 7535312 TI - Serological reactivity and viral genotypes in hepatitis C virus infection. AB - Patients infected with hepatitis C virus (HCV) were examined with four commercial HCV immunoblotting assays and for anti-GOR antibody to ascertain whether serological findings varied with the genotype of the infecting virus. The results indicate that patients infected with different HCV genotypes tend to show different immunoblotting profiles, mainly due to a low prevalence of antibodies to the viral region NS4 in patients infected with genotypes III and IV. Differences were more evident with second- than with third-generation assays. Patients infected with genotype IV exhibited a lower prevalence of anti-GOR antibody than patients infected with other genotypes. PMID- 7535311 TI - Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers. AB - We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37 kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% (kappa = 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions. PMID- 7535313 TI - Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens. AB - An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens. PMID- 7535314 TI - Use of UV ParaLens adapter for detection of acid-fast organisms. AB - Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears. PMID- 7535315 TI - Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. AB - Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains. PMID- 7535317 TI - Comparison of cerebral blood flow velocity estimation with cranial ultrasound imaging for early prediction of outcome in preterm infants. AB - Seventy-four low birth weight infants underwent serial measurements of cerebral blood flow velocity (CBFV) using Doppler ultrasound together with ultrasound imaging of the brain. Surviving infants were examined and assessed using the Bayley scales of mental and motor development at 18 months post-term. There were no significant differences in mean CBFV between normal and impaired infants. Infants with abnormal neurological signs (n = 6) or developmental delay (n = 3) did not show the usual steady rise in CBFV during the first few days of life. Four of nine had a rise, then a fall in CBFV compared with 4 of 31 with complete data in the normal group. This difference is significant (chi 2 = 4.3, p = 0.03). The magnitude of the percentage increase between day 1 and day 3 was also smaller in the abnormal group (median 0% vs. 39%, Mann-Whitney p = 0.03). A structural abnormality seen on the cranial ultrasound image was a better predictor of adverse outcome than an abnormal CBFV pattern, with a better sensitivity and specificity (66% and 97% for imaging compared to 44% and 87% for CBFV). The addition of serial ultrasound Doppler measurements of CBFV did not improve the prediction of outcome obtained using ultrasound imaging alone. PMID- 7535316 TI - Clinical evaluation of a Mycobacterium tuberculosis PCR assay. AB - On the basis of previously published PCR primer sequences, we have designed a sensitive system for detecting DNA of the Mycobacterium tuberculosis complex (MTB) in patient sputum samples which employs a fast and simplified sample preparation method appropriate for routine diagnostic testing. In order to evaluate the accuracy of the PCR assay, we performed a prospective study with 103 patients, comparing PCR results with culture results of samples obtained from a parallel culture assay as well as with subsequent culture results. Using two MTB specific PCR primer systems, we found 48 of 49 tuberculosis (Tb) patients to be PCR positive (PCR sensitivity, 0.98). Sixteen of 54 presumably non-Tb patients showed amplifiable MTB DNA (specificity, 0.7). The study demonstrates that for diagnostic applications of MTB PCR two MTB-specific primer pairs should be used. MTB infection is extremely unlikely in cases of MTB PCR-negative samples: with our method for the exclusion of active Tb, the validity of one PCR assay seems to be equivalent to those of at least three culturing procedures. Positive PCR results do not necessarily reflect active MTB infection. It remains to be shown whether positive PCR results in Tb-negative patients mean false-positivity, an early laboratory finding which predicts a subsequent reactivation of a prior Tb infection, or whether asymptomatic patients may carry PCR-amplifiable MTB DNA without any clinical relevance. It is important to point out that the validity of PCR results in clinical studies depends on the use of contamination controls parallel to all PCR steps and the simplicity of the DNA extraction method as well as on the specificity of the PCR results. PMID- 7535318 TI - Dendritic reorganisation in the basal forebrain under degenerative conditions and its defects in Alzheimer's disease. II. Ageing, Korsakoff's disease, Parkinson's disease, and Alzheimer's disease. AB - Changes in the dendritic arborisation of Golgi-impregnated basal forebrain neurones with respect to size, shape, orientation, and topology of branching were quantitatively investigated in ageing, Alzheimer's disease (AD), Korsakoff's disease (KD), and Parkinson's disease (PD). A reorganisation of the whole dendritic tree characterized by an increase in both the total dendritic length and the degree of dendritic arborisation as well as by changes in the shape of the dendritic field was found during ageing, in KD, PD, and AD. Dendritic growth under these conditions was related to the extent of cell loss in basal forebrain nuclei. There appeared to be major differences, however, with respect to the overall pattern of dendritic reorganisation between AD on one side and ageing, KD, and PD on the other side. In both ageing and KD, dendritic growth was largely restricted to the terminal dendritic segments, resulting in an increase of the size of the dendritic field (pattern of "extensive growth") In AD, however, dendritic growth mainly resulted in an increase of the dendritic density within the dendritic field without being accompanied by an increase in the size of the volume occupied by the dendritic tree (pattern of "intensive growth"). In AD, aberrant growth processes were frequently observed in the perisomatic area or on distal dendritic segments of basal forebrain neurones of the reticular type. Neurones with aberrant growth profiles were typically located in the direct vicinity of deposits of beta/A4 amyloid. Perisomatic growth profiles were covered by the low-affinity receptor of nerve growth factor p75NGFR. Aberrant growth processes were not present in ageing, KD, and PD. On the basis of the present study, it is concluded that under certain degenerative conditions, reticular basal forebrain neurones undergo a compensatory reorganisation of their dendritic arborisation, a process that has become defective in AD, thereby converting a physiological signal into a cascade of events contributing to the pathology of the disease. PMID- 7535319 TI - Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron. AB - Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed. PMID- 7535321 TI - Sclerosing B-cell lymphoma involving the skin. AB - We report a case of sclerosing B-cell lymphoma involving the skin. The patient is a 43-year-old man who came to our institution with a 4-year history of multinodular masses in the back. A thoracic CT scan demonstrated subcutaneous masses with extension to the posterior parietal pleura and compression of several epidural spaces. An incisional biopsy was performed and demonstrated an infiltrative process in the lower dermis composed of interconnected thick sclerosing bands forming compartments around groups of large neoplastic cells. These neoplastic cells were shown to be B lymphocytes using immunohistochemical stains. A diagnosis of diffuse sclerosing B-cell lymphoma, large cell type, was made. Our case is reported to alert dermatologists and dermatopathologists to the occurrence of this neoplasm in the skin which could be confused with a deep inflammatory process or other neoplastic conditions. PMID- 7535320 TI - NADPH-diaphorase active and calbindin D-28k-immunoreactive neurons and fibers in the olfactory bulb of the hedgehog (Erinaceus europaeus). AB - The hedgehog, a macrosomatic insectivore with an extraordinary development of the olfactory structures, has a crucial value for any phylogenetic or comparative study in mammals. The distribution pattern and morphology of NADPH-diaphorase active and calbindin D-28k-immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. NADPH-diaphorase (ND) staining was carried out by a direct histochemical method, and the calbindin D-28k (CaBP) immunoreaction by using a monoclonal antibody and the avidin-biotin immunoperoxidase method. The possible coexistence of both markers was determined by sequential histochemical-immunohistochemical double labeling of the same sections. Specific neuronal populations were positive for both ND and CaBP markers. No cell colocalized both stains in the hedgehog olfactory bulb. A subpopulation of olfactory fibers, and a subpopulation of olfactory glomeruli, located on the medial side, were positive for ND. Surrounding both the ND positive and ND-negative glomeruli, there were ND- and CaBP-positive periglomerular cells, the latter group being much more abundant. A subpopulation of superficial short-axon cells was CaBP positive but, contrary to what is observed in rodents, this neuronal type was always ND negative. In addition, three neuronal types were observed in the GL-EPL border after CaBP immunostaining. These neuronal types have not been previously described either in the hedgehog or in the rodent olfactory bulb. Horizontal cells and vertical cells of Cajal were also observed after both ND and CaBP labeling. Distinct groups of ND- and CaBP-positive cells, differing in size, shape, dendritic branching pattern, and staining intensity, were distinguished in the granule cell layer and in the white matter. The large and medium-sized cells were identified as a very heterogeneous population of deep short-axon cells, whereas a subpopulation of granule cells was ND positive. The accessory olfactory bulb showed ND staining in all vomeronasal fibers and glomeruli, and in subpopulations of periglomerular cells, granule cells, and deep short-axon cells. The CaBP immunolabeling was more restricted and located in subpopulations of periglomerular cells and in deep short-axon cells. These results indicate different and more complex ND and CaBP staining patterns in the hedgehog olfactory bulb than those previously described in rodents, including the presence of specific, chemically and morphologically defined new neuronal types. PMID- 7535323 TI - Screening for hepatocellular carcinoma in patients with Child's A cirrhosis: an 8 year prospective study by ultrasound and alphafetoprotein. AB - One hundred and forty-seven patients with Child's A cirrhosis and no evidence of hepatocellular carcinoma were followed up in an 8-year prospective surveillance program with testing by ultrasound and alphafetoprotein every 6 months. Eighteen of 147 patients were HBsAg positive. Anti-hepatitis C virus antibodies were found in 103 out of 133 cases tested. Sixteen patients had a history of heavy drinking. Thirty hepatocellular carcinomas were detected during follow up. At the time of diagnosis, ultrasound detected focal lesions in all the patients whereas alphafetoprotein was below diagnostic levels. The hepatocellular carcinoma was single in 26 patients and multiple in four. The overall 8-year cumulative tumor free rate was 69% (95% confidence interval = 58-73). The yearly hepatocellular carcinoma incidence from 1985 to 1992 was respectively 2%, 1.5%, 2%, 3%, 5%, 4.8%, 7% and 10%. The initial value of AFP > 50 ng/ml and < 400 ng/ml was significantly related to the development of hepatocellular carcinoma. This series shows that the cumulative incidence of hepatocellular carcinoma in cirrhosis in Italy is higher than previously reported, but lower than that observed in Asiatic areas. A 6-month interval for ultrasound is reasonable to detect treatable tumors. Alphafetoprotein has no value for early diagnosis, although its intermediate values (> 50 and < 400 ng/ml) may indicate the presence of undetectable cancer which will appear during the follow up, and suggests that ultrasound should be employed more frequently in patients with these values. PMID- 7535322 TI - Glucoseoxidase from Aspergillus niger in reverse micelles: pH and wo dependence. AB - The well known enzymatic method for the determination of glucose in aqueous solution with the system glucose oxidase/peroxidase/o-dianisidine was modified in a way that enables the quantitative determination of glucose in various reverse micellar systems. For instance in the 0.1 M sodium-bis-(2-ethylhexyl) sulfosuccinate/n-octane system the useable glucose measuring range is between 10 and 70 micrograms glucose per 3 ml micellar solution. Investigations concerning the influence of the pH-value and the wo-value on the enzyme activity of glucose oxidase in the system 0.1 M sodium-bis-(2-ethylhexyl) sulfosuccinate/n-octane led to a shift of the pH-optimum from pH 5.5-6.0 in aqueous solution, to pH 4.0, in reverse micellar solution. The bell-shaped wo-dependence of the enzyme activity, typical for many other enzymes, was not found for glucose oxidase at pH 4.0, and was less clearly visible at the other investigated pH-values with an optimum at wo = 5.6. PMID- 7535324 TI - Activation of ras oncogene in livers with cirrhosis. AB - Activation of cellular oncogenes and inactivation of anti-oncogenes have been postulated as important mechanisms during hepatocarcinogenesis. This study was conducted to detect abnormal levels of several proto-oncogenes (c-jun, c-fos, c-H ras) and of the p53 and the alpha-fetoprotein gene in the liver during cirrhosis, a pathological process which predisposes to the development of hepatocarcinoma. Liver tissue from 11 patients with cirrhosis of different etiologies, and seven histologically normal liver fragments taken at the periphery of benign liver tumors of metastases were studied. Transcripts of the various oncogenes and of the alpha-fetoprotein gene were detected by in situ hybridization, and the p53 protein was revealed by immunocytochemistry. No overexpression of any of the mRNA tested or of the p53 protein was found in histologically normal liver in contact with benign or metastatic tumors. In contrast, 10 of the 11 specimens with cirrhosis (90.9%) displayed abnormally high levels of c-H-ras transcripts. Five samples with cirrhosis revealed a moderate increase in the level of c-fos mRNA. Only one case and two cases, respectively, exhibited increased levels of c-jun and alpha-fetoprotein mRNA. No cases were positive for the p53 antigen. Liver cell proliferation, as assessed by immunocytochemistry with the Ki 67 monoclonal antibody, was low in both the group with cirrhosis and the control groups (0.49% and 0.55% positive cells, respectively). These data demonstrate that activation of c-H-ras mRNA is an almost constant finding in hepatocytes of livers with cirrhosis. This gene overexpression is not linked to hepatocellular proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535325 TI - Exacerbation of lichen planus but not of acute intermittent hepatic porphyria during interferon therapy in a patient with chronic hepatitis C. PMID- 7535326 TI - Retreatment with interferon in chronic hepatitis C. PMID- 7535327 TI - Clinical relevance of the detection of hepatitis delta virus RNA in serum by RNA hybridization and polymerase chain reaction. AB - Hepatitis delta virus nucleic acid was detected by dot-blot hybridization using RNA probe and reverse transcription/polymerase chain reaction amplification in 223 serum samples from 66 patients with hepatitis D virus infection. Seven cases with chronic hepatitis D virus infection were treated with interferon: six for 3 months and one for 7.5 years. By using the primers located in the putative conserved regions, the technique of reverse transcription/polymerase chain reaction amplification was 10(3) to 10(4) times more sensitive than that of dot blot hybridization. The main findings of this study are: (i) HDV RNA could be detected in the absence of any other serological hepatitis D virus marker in serum from acute hepatitis patients with IgM anti-HBc; (ii) high titer anti-HD antibodies (IgM and total anti-HD) persisted in patients during short-term interferon treatment, and in one patient during long-term interferon treatment, despite clearance of serum HDV RNA even after 3 years; (iii) total anti-HD alone was detected in the absence of IgM anti-HD and serum HDV RNA. These observations indicate that the detection of HDV RNA by molecular techniques in serum is a useful, sensitive and non-invasive technique for the early diagnosis and follow up of hepatitis D virus infection, as well as for the monitoring of antiviral therapy. In addition, total anti-HD antibody in the absence of HDV RNA may be the only residual marker of past infection. Finally, the choice of the technique for hepatitis D virus detection is important for the optimal assessment of the clinical stage and monitoring of antiviral therapy in hepatitis D virus-infected patients. PMID- 7535328 TI - [Maternal serum alpha-fetoprotein as a screening test for fetal Down syndrome]. AB - An association between a low maternal serum alpha-fetoprotein (MSAFP) level and an increased incidence of Down syndrome is well documented. This study was undertaken to elucidate the diagnostic efficacy of MSAFP as a screening test for Down syndrome in 711 Japanese women who underwent genetic amniocentesis between 14 and 20 gestational weeks of age in Hyogo Medical College Hospital. The mean value for the multiple of the median (MoM) of the MSAFP level in 11 pregnancies associated with Down syndrome was 0.78 +/- 0.47 and 1.12 +/- 0.67, respectively. A cut-off value of < 0.45, < 0.50, < 0.55 and < 0.60 MoM yielded negative predictive values of 98.6%, 98.5%, 99.0% and 99.2%, positive predictive values of 6.3%, 4.0%, 9.1% and 8.6%, a sensitivity of 9.1%, 9.1%, 36.4% and 54.5%, and false positive rates of 2.1%, 3.4%, 5.7% and 9.1%, respectively. The combining of MSAFP and maternal age improves the expected results of a strategy for Down syndrome. PMID- 7535330 TI - [The expression of Fas antigen in human endometrium]. PMID- 7535329 TI - [Study on peripheral blood stem cells mobilized by different chemotherapies with granulocyte-colony stimulating factor in ovarian cancer]. AB - A new therapeutic strategy for advanced ovarian cancer is to administer ultra high doses of anticancerous drugs, followed by peripheral blood stem cell transplantation (PBSCT) to recover normal marrow functions. There are, however, no clear regimens to induce mobilization of peripheral blood stem cells (PBSCs). We therefore used three different chemotherapies and granulocyte colony stimulating factor (G-CSF) to produce a rebound increase in PBSCs during myelosuppression by apheresis. Eleven patients with advanced ovarian cancer (FIGO Stage; IIc-1, IIIc-5, IV-4, Recurrence-1) received chemotherapy with CEP (cyclophosphamide: 500-750mg/m2, epirubicin: 50-70mg/m2, cisplatin: 70mg/m2), CEE (cyclophosphamide: 2,000mg/m2, epirubicin: 50mg/m2, etoposide: 300mg/m2, and PEC salvage (cisplatin: 75mg/m2, etoposide: 300mg/m2, cyclophosphamide: 1,000mg/m2) followed by lenograstim (G-CSF; 2 micrograms/kg subcutaneous injection daily for 14-18 days) to mobilize PBSCs. A range of 0-38.2 x 10(4) (mean +/- S.D.; 11.1 +/- 5.0 x 10(4) colony forming unit granulocyte-macrophage (CFU-GM)/kg in the CEP regimen (n = 15), 1.6-40.8 x 10(4) (mean +/- S.D.; 12.3 +/- 3.6 x 10(4) CFU-GM/kg in the CEE regimen (n = 11), and 8.6-11.4 x 10(4) (mean +/- S.D.; 10.0 +/- 2.0 x 10(4) CFU-GM/kg in the PEC salvage regimen (n = 2) were recruited by a single apheresis. Although the CEE regimen to mobilize PBSCs was much more efficient than the CEP regimen, a large number of PBSCs showing 38.2 x 10(4) CFU-GM/kg were mobilized by the CEP regimen with a dose-escalation of cyclophosphamide 750mg/m2 and epirubicin 70mg/m2. The number of CFU-GM/kg correlated well with that of CD34+ (r = 0.693).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535331 TI - Redistribution of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) to the surface membrane of human platelets during the release reaction. AB - Treatment of human washed platelets with 5 mM EDTA at 37 degrees for 60 min irreversibly dissociated glycoprotein (GP) IIb/IIIa complex (alpha IIb beta 3 integrin) on the surface membrane, since transmission immunoelectron microscopy studies demonstrated that these EDTA-pretreated platelets in the presence of added Ca2+ ion could not bind P2, an anti-GPIIb/IIIa complex-specific monoclonal antibody, to their surface membrane. The treatment, however, had no effect on the GPIIb/IIIa complex on the alpha-granule membrane. At 30 sec after the EDTA pretreated platelets were activated with 0.1 U/ml of thrombin, alpha-granules fused with each other or with the surface-connected canalicular system (SCCS) to form swollen SCCS, the membrane of which was found to have the intact GPIIb/IIIa complex detectable by P2. In addition, at this time the intact GPIIb/IIIa complex reappeared on the surface membrane. At 5 min, the intact GPIIb/IIIa complex increased on the surface membrane with a reciprocal decrease or disappearance on the membrane of the swollen SCCS. The observation under scanning immunoelectron microscopy also confirmed the same translocation of the intact GPIIb/IIIa complex. These results indicate that alpha-granule membrane GPIIb/IIIa is redistributed to the surface membrane via the membrane of SCCS during the release reaction. PMID- 7535332 TI - Facultative role of germinal centers and T cells in the somatic diversification of IgVH genes. AB - The development of memory B cells takes place in germinal centers (GC) of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. However, the mechanisms that drive this complex response are not well understood. We explored the relationship between GC formation and the onset of hypermutation in response to the hapten phosphorylcholine (PC) coupled to antigenic proteins in mice bearing different frequencies of CD4+ T cells. PC-reactive GC were identified by staining frozen splenic sections with peanut agglutinin (PNA) and with monoclonal Abs against AB1-2, a dominant idiotope of T15+ anti-PC antibody. The nucleotide sequences of rearranged T15 VH1 genes were determined from polymerase chain reaction amplifications of genomic DNA from microdissected GC B cells. T15+ GC became fully developed by day 6-7 after primary immunization of euthymic mice with either PC-keyhole limpet hemocyanin (KLH) or PC-chicken gamma globulin (CGG). Yet the VH1 gene segments recovered from the primary GC as late as day 10-14 had low numbers of mutations, in contrast to responses to the haptens nitrophenyl or oxazolone that sustain high levels of hypermutation after GC formation. PC-reactive B cells proliferate in histologically typical GC for considerable periods with no or little somatic hypermutation; the signals for GC formation are independent of those for the activation of hypermutation. We then examined GC 7 d after secondary immunization with PC-KLH in euthymic mice, in nu/nu mice reconstituted with limited numbers of normal CD4+ cells before priming (CD4(+)-nu/nu) and in nu/nu mice. All of these animals develop T15+ GC after antigen priming, however, the patterns of V gene mutations in the secondary GC reflected the levels of CD4+ cells present during the primary response. VDJ sequences from secondary GC of euthymic mice were heavily mutated, but most of these mutations were shared among all related (identical VDJ joints) sequences suggesting the proliferation of mutated, memory B cells, with little de novo somatic hypermutation. In contrast, the patterns of V gene diversity in secondary GC from CD4(+)-nu/nu mice suggested that there was ongoing mutation and clonal diversification during the first week after rechallenge. The secondary GC from T cell-deficient, nu/nu mice showed little evidence for mutational and/or recombinational diversity of T15+ B cells. We conclude that the participation of CD4+ helper cells is required for full activation of the mutator in GC and takes place in a dose-dependent fashion. PMID- 7535333 TI - Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. AB - Previous studies from our laboratory demonstrated an inverse relationship between the expression level of inducible nitric oxide synthase (iNOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the expression of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NG-methyl-L-arginine (NMA). Positive transfectants were identified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogeneic BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. Experiments with [125I]dUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4.L8.5 cells died by 48-72 h after injection. Enhanced survival of all K-1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis. PMID- 7535335 TI - The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors. AB - The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells. PMID- 7535334 TI - Mutational analysis and an alternatively spliced product of B7 defines its CD28/CTLA4-binding site on immunoglobulin C-like domain. AB - Costimulatory molecules B7 and B7-2 interact with T cell surface receptors CD28/CTLA4 and deliver a costimulatory signal essential for T cell growth. However, the structure basis of this interaction is not known. B7 and B7-2 are members of immunoglobulin (Ig) superfamily and their extracellular portion consists of an IgV- and IgC-like domain. Here we report that a naturally occurring, alternatively spliced form of B7 reveals that exon 3-encoded IgC domain is essential for CD28/CTLA4 binding. Mutational analysis of B7 demonstrates a critical role of several amino acids around loops between strands B and C and D and E, for binding CTLA4/CD28. These amino acids are clustered to form a single binding site centered at 201Y. A comparison of the effects of mutations on the binding of CD28 and CTLA4 reveals that CD28 and CTLA4 binds to the same site on B7. These results have important implications on the role of CTLA4 and CD28 in T cell costimulation. The structure of the CD28/CTLA4-binding site also provides valuable information for immune intervention targeted at the B7/B7-2-CD28/CTLA4 interactions. PMID- 7535336 TI - Activated mast cells produce interleukin 13. AB - When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1 2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy. PMID- 7535337 TI - Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes. AB - The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration. PMID- 7535338 TI - Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7. AB - The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL-4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor. PMID- 7535340 TI - Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells. AB - Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes. Most studies have found that these leukemic CD5+ B cells, like their normal counterparts, use immunoglobulin (Ig) variable (V) region genes that exhibit minimal, if any, somatic diversity. These and other observations have suggested that CD5+ B cells may be incapable of generating Ig V gene diversity, and therefore may not be able to develop higher affinity binding sites that could be selected by antigen. However, most of the studies of CLL and normal CD5+ B cells have focused on IgM-producing cells. Since somatic mutations are most often seen in B cells that have undergone an isotype class switch, we analyzed the Ig heavy (H) and light (L) chain variable region genes of seven IgG+CD5+ CLL B cells to determine if somatic diversification and antigen selection had occurred. The data derived provide evidence for skewed use, somatic diversification, and antigenic selection of the Ig V region genes. Nonrandom use of both H and L chain V region genes was manifested by an overrepresentation of VH4 and VKI family genes and the underrepresentation of the JH4 gene segment. Furthermore, VH4 gene use was restricted to only two family members (4.21 and 4.18). In four of the seven cases, the VH and VL genes displayed > or = 5% difference from the most homologous known germline counterparts. Polymerase chain reaction and Southern blot analyses performed in two of these patients demonstrated that their unique VH CDR2 and adjacent sequences were not present in their germline DNA. In addition, a significant level of diversity was seen in the rearranged DJH segments and at the VL-JL junctions of every patient that occurred both at the time of recombination and subsequently. The localization of replacement changes to complementarity determining regions of some patients suggested that antigen selection had occurred. Furthermore, the mutations identified in the VH and VL genes of each individual patient were strikingly similar, both in number and location. Collectively, the data indicate that a subset of CD5+ CLL B cells can display Ig V region gene mutations. In addition, they are consistent with the notions that in some cases antigen selection of these mutations may have occurred, and that antigen stimulation may be a promoting factor in the evolution of certain CLL clones. PMID- 7535339 TI - CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. AB - CD11c/CD18 is a member of the leukocyte integrin family, heterodimeric adhesion molecules that interact with a diverse repertoire of ligands, including bacterial lipopolysaccharide (LPS). Their role as signal transducing receptors remains uncertain. We used a heterologous expression system to determine if CD11c/CD18 was capable of initiating signal transduction in response to LPS-binding, as assessed by the induced translocation of nuclear factor-kappa B. We have previously reported that Chinese hamster ovary (CHO)-K1 fibroblasts, normally unresponsive to LPS, acquire serum-dependent macrophage-like responses to LPS when transfected with CD14 (Golenbock, D.T., Y. Liu, F. Millham, M. Freeman, and R. Zoeller. 1993. J. Biol. Chem. 268:22055-22059), a known LPS receptor. In contrast, CHO cells acquired serum-independent responses to Gram-negative bacteria and LPS when transfected with CD11c/CD18 (CHO/CD11c). In comparison to CHO cells transfected with CD14 (CHO/CD14), responses in CHO/CD11c cells were slower, required higher endotoxin concentrations for maximal response, and were not inhibited by the presence of antibodies to CD14. CD11c/CD18 is, thus, the second phagocyte receptor, in addition to CD14, which has been shown to have the capacity to activate cells after binding to LPS. The function of this receptor in normal phagocytes may be limited to the recognition of LPS in infected tissues, where LPS-CD14 interactions are not favored because of the absence of serum proteins. PMID- 7535342 TI - Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells. AB - CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100 kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6. PMID- 7535344 TI - A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina. AB - The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage-dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels. PMID- 7535341 TI - Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice. AB - Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes. Results indicate that antibody to IgM is sufficient to induce a state of dormancy. Antibodies against other cell surface molecules including IgD and CD44 (Pgp1) had no effect on tumor growth. Id-immune T cells by themselves also had no effect on tumor growth in SCID mice. However, simultaneous transfer of anti-Id and Id-immune T cells enhanced both the induction and duration of the dormant state. In vitro studies indicated that antibody to IgM induced apoptosis within several hours and cell cycle arrest by 24 h. Hyper cross-linking increased apoptosis. The Fc gamma RII receptor played little or no role in the negative signaling. Antibodies that did not negatively signal in vitro did not induce dormancy in vivo. The results suggest that anti-IgM plays a decisive role in inducing tumor dormancy to BCL1 by acting as an agonist of IgM-mediated signal transduction pathways. PMID- 7535345 TI - Inhibitory molecules in development and regeneration. AB - In addition to chemotrophic and contact guidance theories that explain how long projection neurons weave intricate patterns of connectivity within developing or regenerating neuronal networks, there has been recent interest in mechanisms that guide axons by actively constraining, inhibiting or repelling axon growth cones. Developmental boundaries are especially important in regions where large numbers of growing axons must change direction in order to remain on course towards their potential targets. Regenerative boundaries can also have severe pathological consequences since they limit the potential for axon regrowth following injury or diseases. Some of the molecular mechanisms that generate repulsive environments in the embryo are re-expressed in the adult following injury. In the developing retina, a chondroitin sulfate-proteoglycan appears to play an essential role in controlling the sequence of ganglion cell differentiation and initial direction of axons. In several lesion models, re-expression of a chondroitin sulfate proteoglycan by reactive astrocytes limits regeneration through glial scars; conversely, in experiments where boundary molecules have been manipulated by chondroitinase digestion, axons are stimulated to regrow or re-route to inappropriate pathways. PMID- 7535343 TI - Identification of the ligand-binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand. AB - CD22 is a B cell-restricted member of the immunoglobulin (Ig) superfamily that functions as an adhesion receptor for leukocytes and erythrocytes. CD22 is unique among members of the Ig superfamily in that it has been suggested to bind a series of sialic acid-dependent ligands, potentially through different functional domains expressed by different splice variants of CD22. In this study, the epitopes identified by a large panel of function-blocking and non-function blocking CD22 monoclonal antibodies were localized to specific Ig-like domains, revealing that all function-blocking monoclonal antibodies bound to the first and/or second Ig-like domains. Consistent with a single ligand-binding region, the two amino-terminal domains were the functional unit that mediated CD22 adhesion with lymphocytes, neutrophils, monocytes, and erythrocytes. The predominant cell surface species of CD22 was a full length 140,000 relative molecular mass seven Ig-like domain glycoprotein and a minor 130,000 relative molecular mass form lacking the fourth domain. While the two amino-terminal Ig like domains of CD22 are structurally similar to those found in other members of the Ig superfamily involved in cell adhesion and containing an amino acid sequence motif associated with integrin recognition, site-directed mutagenesis of critical residues surrounding this motif did not disrupt CD22-mediated adhesion. These results demonstrate that the unique ligand-binding properties of CD22 are distinct from those of other members of the Ig superfamily involved in integrin mediated cell adhesion. PMID- 7535346 TI - Combined antitumor effects of TNF and G-CSF on a human medulloblastoma xenograft line. AB - The antitumor effects of TNF and G-CSF on a xenograft line of human medulloblastoma were examined. (Method):1) A human medulloblastoma xenograft line was transplanted into nude mice. Tumor bearing nude mice were divided into the following eight groups: untreated controls (C); those receiving a subcutaneous injection of G-CSF for one week (G1); for four weeks (G2); those receiving an intratumoral injection of TNF for four weeks (Tit); an intravenous injection of TNF (Tiv); those receiving a combination of G1 and Tit (G1 + Tit); a combination of G2 and Tit (G2 + Tit); and a combination of G2 and Tiv (T2 + Tiv). The relative tumor weight in each group was calculated and any antitumor effects were examined by calculating a tumor growth inhibition ratio. 2) Tumor bearing nude mice were divided into the following two groups: those receiving a subcutaneous injection of G-CSF and an intravenous injection of TNF (G+T); and only an intravenous injection of TNF (T). We evaluated the pathological findings from the tumors at 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h and 48 h after the TNF injection. Routine H.E. staining and immunostaining using antigranulocyte and antimacrophage antibodies were performed. (Results): 1) The tumor growth inhibition ratio was 0.112, 0.190, 0.287, 0.451, 0.347, 0.635, and 0.622 at G1, G2, Tit, Tiv, G1 + Tit, G2 + Tit, G2 + Tiv group. A combined antitumor effect was clearly seen in the G2 + Tit and the G2 + Tiv groups. 2) The tumor was fragmented by the infiltration of many inflammatory cells 24 hours after TNF injection. Many more macrophages were observed in the tumors of G+T mice than in the T mice. Granulocytes were observed only in the tumors of the G+T mice. PMID- 7535349 TI - Guidelines in palliative care for voluntary service in Italy. Italian League against Cancer. PMID- 7535347 TI - Osteosarcoma following radiation treatment for meningioma: report of a case and effective treatment with chemotherapy. AB - A case of effective chemotherapy in postirradiation sarcoma is reported. A 30 year-old woman underwent a subtotal resection of a benign mixed meningioma followed by a course of radiotherapy. Five years later she developed a skull tumor which was resected. Histological study showed sarcomatous change corresponding most likely to a postirradiation osteosarcoma. Adjuvant chemotherapy (methotrexate, carboplatin, and VP16) was given. A progression occurred 8 months later and the patient was treated unsuccessfully with tamoxifen, LHRH, and later with high dose methotrexate and carboplatin. Six months later a lung metastasis was discovered and she received four cycles of IVP (ifosfamide, vepeside, cisplatin) alternating with IVA (ifosfamide, vepeside, adriamycin). Within 4 months the primary and lung tumours had decreased substantially without evidence of progression at follow-up 5 months later. We conclude that IVP/IVA regimen is a potentially useful therapy when an osteosarcoma of the skull is in progression. PMID- 7535348 TI - Intrapleural bleomycin in the treatment of chylothorax. AB - Chylothorax is an accumulation of thoracic lymph or chyle in the pleural cavity. It is a rare condition and is usually caused by trauma or malignant disease. We present three cases with chylothorax due to malignant non-Hodgkin's lymphoma [high grade malignant (1 case) and low grade malignant (2 cases)] treated with pleurodesis with bleomycin and systemic chemotherapy (CHOP, CNOP, trofosfamide). Complete remissions (CR) were achieved in all three cases. Two patients had a recurrent chylothorax 3 and 12 months after initial treatment. They were treated with a second intrapleural installation of bleomycin and continuing systemic chemotherapy (CNOP, trofosfamide) and are still alive in CR with a follow-up period of 28 and 30 months respectively. One patient died of relapsing non Hodgkin's lymphoma after 23 months of follow-up. There was no sign of recurrent chylothorax. We conclude that chylothorax caused by lymphoma can be satisfactorily controlled by pleurodesis with bleomycin combined with systemic chemotherapy. Immediate action is necessary to prevent great loss of lipids and proteins. The underlying malignancy must be controlled to achieve a good prognosis. PMID- 7535350 TI - Management of nosocomial respiratory tract infections in terminally ill cancer patients. PMID- 7535351 TI - Providing palliative care to older adults: context and challenges. AB - This paper presents the findings of a study aimed at understanding more fully the work of nurses who provide care to older adults who are dying at home. The method employed was qualitative in nature and involved the use of focus groups for data collection. Data were gathered from a total of 40 community-based nurses during four sessions lasting approximately two hours each. Analysis revealed that the provision of care occurred within a context of aging and dying characterized by clients' awareness of impending death, the presence of multiple pathologies, diminishing social support, and a lack of control. Challenges to providing care stemmed from an ethic of high expectation and a health care system experienced as fragmented, bureaucratic, and driven by cost efficiency. Challenges included working in isolation, achieving closure, securing personal support, working collaboratively with others, and keeping up to date. Findings from this study have implications for both education and practice. PMID- 7535352 TI - Cell-mediated immune status of children with recurrent infection. AB - OBJECTIVE: To evaluate the cell-mediated immune status of children with recurrent respiratory tract infections. DESIGN: We evaluated the cell-mediated immune status of 76 patients referred because of recurrent infection. Patients were divided into those with serologic abnormalities and those without such findings. Twenty-three healthy children served as control subjects. Studies of lymphocyte phenotype included CD4+ CD29+ cells (an immunologically mature phenotype), lymphocyte proliferation studies, cytokine production including interleukin-2 (IL 2), IL-4, IL-6, and interferon gamma), and measurement of in vitro IgM and IgG synthesis. RESULTS: Lymphocyte proliferation and T-cell phenotype were similar in both patient groups as well as in control subjects. The proportions of CD4+ CD29+ cells at different ages were similar in all groups. Patients with serologic abnormalities (e.g., partial IgA deficiency, partial IgG subclass deficiency) produced more IL-2 and IL-4 than did other patients. The control population had greater spontaneous IgM and IgG synthesis than the patient groups. CONCLUSION: Routine studies of T-cell function of patients with recurrent infection provide little information useful in making clinical decisions. PMID- 7535353 TI - Absence of infection in breast-fed infants born to hepatitis C virus-infected mothers. AB - The role of breast-feeding in perinatal transmission of hepatitis C virus (HCV) was explored in 15 HCV-infected mothers and their infants. The 15 carrier mothers had anti-HCV titers ranging from 1:80 to 1:40,000 and also had HCV-ribonucleic acid with concentrations ranging from 10(4) to 2.5 x 10(8) copies/ml. Both anti HCV antibody and HCV-ribonucleic acid were present in colostral samples in much lower levels, but none of the 11 breast-fed infants had evidence of HCV infection for up to 1 year of age. Thus breast-feeding seems safe for these infants. PMID- 7535354 TI - Control of severe pain in children with terminal malignancy. AB - OBJECTIVE: To identify the characteristics of the subset of children with malignancy in whom massive opioid infusions are needed during the terminal phase. DESIGN: Retrospective review of the records of the 199 patients who died of malignancy after treatment at Children's Hospital, Boston, from March 1989 to July 1993, identifying characteristics of patients who required massive opioid infusions (operationally defined as infusion of > 3 mg/kg per hour of morphine dose equivalent) during the terminal phase. RESULTS: Twelve patients (6%) required massive opioid infusions, and eight of these patients required extraordinary measures (epidural or subarachnoid infusion and/or sedation) to achieve adequate analgesia. The duration of epidural or subarachnoid infusions in three patients ranged from 3 to 9 days, and minimal complications occurred. The duration of sedation ranged from 1 to 15 days. Maximal intravenous opioid dosing ranged from 3.8 to 518 mg/kg per hour of morphine equivalent. The maximal infusion rate (exceeding all previous published reports) occurred in an infant with an isolated metastasis in the periaqueductal gray matter, a brain-stem site linked to mediating analgesia and defense reactions. The need for massive opioid dosing in 11 of 12 patients was associated with tumor spread to the spinal nerve roots, nerve plexus, large peripheral nerve, or spinal cord compression. CONCLUSIONS: Standard dosing of opioids adequately treats most cancer pain in children; however, a significant group requires more extensive management. These problems occur more commonly among patients with solid tumors metastatic to spine and major nerves. PMID- 7535355 TI - Lack of evidence for human T cell lymphotrophic virus type I or II infection in patients with systemic lupus erythematosus or rheumatoid arthritis. AB - OBJECTIVE: Human retroviruses including human immunodeficiency virus (HIV) and human T cell lymphotrophic virus Types I and II (HTLV-I/II) have been associated with forms of connective tissue autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We looked for evidence of HTLV I/II infection in a large population of SLE, RA, and control patients. METHODS: One hundred fifteen patients with connective tissue autoimmune disease and other rheumatological disorders were screened for antibodies to HTLV-I/II by Western immunoblots (WIB). Due to the transforming characteristic of these retroviruses, the patients' peripheral blood mononuclear cells (PBMNC) were cultured in attempts to establish continuous cell lines. Furthermore, PBMNC culture supernatants were analyzed for reverse transcriptase activity and/or HTLV-I/II gag antigen production. The presence of HTLV-I/II proviral sequences in short term culture and fresh PBMNC was determined by Southern blot analysis and polymerase chain reaction (PCR), respectively. respectively. RESULTS: All 115 patients were HTLV-I/II and HIV seronegative. Seventy-four attempts to establish PBMNC cell lines from 65 patients were unsuccessful with a mean culture survival time of 3.6 (+/- 1.4) months. Reverse transcriptase activity and HTLV-I/II gag antigen production were not detected in 51 and 16 culture supernatants tested, respectively. Cells from 11 patients tested by Southern blot analysis and from 57 patients tested by PCR were negative for HTLV-I/II related sequences. CONCLUSION: Our results failed to establish an association between human retroviruses (HTLV I/II and HIV) and SLE, RA, or other rheumatological disorders. However, these results do not rule out other exogenous or endogenous retroviruses that may play a role in the initiation and/or promotion of these diseases. PMID- 7535356 TI - Peripheral blood CD5+ B cell subset in the remission phase of systemic connective tissue diseases. AB - OBJECTIVE: To get a better insight into the level of circulating CD5+ B cells as related to the systemic connective tissue disease activity. METHODS: Peripheral blood CD5+CD19+ cells of patients in the remission phase of systemic lupus erythematosus (SLE) (n = 28), Sjogren's syndrome (SS) (n = 20), rheumatoid arthritis (RA) (n = 26), and 19 control healthy subjects were analyzed by 2-color flow cytometry. RESULTS: In comparison to control group, the patients with SLE had a significant increase in the relative CD19+CD5+ blood cell count (p < 0.0005); this count was also different from the finding in both RA (p < 0.005) and patients with SS (p < 0.05). In contrast, the proportion of B cells expressing CD5 (within an individual B cell population) was significantly increased in all the 3 diseases compared to healthy subjects (SLE, p < 0.0001; SS, p < 0.05; and RA, p < 0.01). In the multivariate discriminant analysis, a discriminant function defined by the CD19+CD5+ subset strongly discriminated SLE, SS and RA from the control, but also SLE from both SS and RA. CONCLUSION: Our findings demonstrated that, in relation to healthy control subjects, the blood CD5+ B subset tended to be elevated in the patients in the remission phase of systemic connective tissue diseases, particularly in SLE. PMID- 7535357 TI - Natural killer cell function and expression of beta 7 integrin in psoriatic arthritis. AB - OBJECTIVE: To examine the cytotoxic activity of natural killer (NK) cells from peripheral blood (PB) and synovial fluid (SF) of patients with psoriatic arthritis (PsA). The influence of selected inflammatory mediators on the cytolytic function and integrin expression of NK cells was also studied. METHODS: Paired samples of PB and SF lymphocytes (PBL and SFL) were obtained from 8 patients with PsA for comparison of NK activity between PBL and SFL. In 6 patients the phenotype of NK cells was determined by flow cytometry using monoclonal antibodies (Mab) to natural killer associated antigen (NKH-1) and the beta 7 integrin, HML-1 (human mucosal lymphocyte adhesion molecule). RESULTS: NK activity of PB samples was significantly greater than paired SF (p = 0.015). SF NK activity was enhanced by overnight culture with interleukin 2 (IL-2) (p < 0.05). A trend towards reduction of NK activity by prostaglandin E2 (PGE2) was noted (p = 0.06) whereas interleukin 6 (IL-6) and indomethacin had no significant effect. NK activity did not correlate with the percentage of NK cells in PB or SF. However, all SF samples contained a greater proportion of monocytes than PB samples. The expression of HML-1 on NK cells correlated with expression HML-1 on CD3+ cells (r = 0.82) and was greater in SF than PB in PsA and RA patients. Effects of IL-2 on HML-1 expression by NK cells were variable in the 3 patients studied. CONCLUSION: In PsA, HML-1 is an activation marker on NK cells. IL-2 expands or maintains the population of HML-1/NKH1 positive cells and increases NK cytolytic activity. However, cytolytic activity of activated NK cells may be inhibited by monocyte derived PGE2. PMID- 7535358 TI - Immunohistochemical changes in synovial tissue during the course of adjuvant arthritis. AB - OBJECTIVE: To study immunohistochemical changes occurring in knee synovial membranes of rats during the time course of adjuvant arthritis (AA). The effect of treatment with dexamethasone after establishment of arthritis was also studied. METHODS: AA was induced in Wistar rats by means of a single injection of a suspension of Mycobacterium butyricum. On Days 7, 14, 17, 21, 28 and 42 after induction, synovial membranes were obtained, frozen and sectioned on a cryostat. Tissue sections were tested by peroxidase-antiperoxidase method, using the following monoclonal antibodies: OX19 (CD5), W3/25 (CD4), OX8 (CD8), OX6 (Ia), OX33 (LCA) and OX39 (CD25). RESULTS: Knee synovial membranes obtained from arthritic rats on days of maximum inflammation showed few or no CD5+ cells, a higher proportion of CD8+ cells, and a higher number of CD4+ and Ia+ cells than healthy synovial tissues. CD25+ cells were observed from Day 7 postinduction and remained numerous throughout the study. CONCLUSION: Few T lymphocytes (CD5+) were found in arthritic synovial membrane, whereas an increase of cells expressing CD8, CD4, CD25, Ia, and LCA was found. These increases (except CD8) are not evident in dexamethasone treated rats. PMID- 7535359 TI - Localization of the alpha v subfamily of integrins and their putative ligands in synovial lining cell layer. AB - OBJECTIVE: The lining cell layer of the synovium proliferates strongly in rheumatoid arthritis. It has been suggested that it has a central role in the destruction of cartilage. We have analyzed the structure of the extracellular matrix and the adhesion molecules of normal, osteoarthritic and rheumatoid lining cell layer. METHODS: We localized the alpha v integrin subunit and its 4 putative partner beta subunits in synovial samples by using indirect immunofluorescence. The specimens were also analyzed by confocal microscopy. Indirect immunofluorescence was also used to analyze the ligands of alpha v integrins, namely fibronectin and vitronectin. RESULTS: The alpha v integrin was abundant in the lining cell layer of normal and osteoarthritic synovia, whereas it was not expressed in the proliferating rheumatoid lining cell layers. A similar expression pattern was found for beta 5 subunit, suggesting that it is the major partner for alpha v. However, also some alpha v beta 1 and alpha v beta 3 heterodimers may be present. The confocal microscopy revealed the presence of both alpha v beta 5 positive and negative lining cells. The putative ligands for alpha v integrins, namely fibronectin and vitronectin were found in the lining cell layer of all the synovial specimens. CONCLUSION: In spite of the proliferation of the lining cell layer in rheumatoid inflammation, the extracellular matrix stays very similar to that in normal and osteoarthritic synovium, whereas the pattern of the adhesion receptors is completely altered. PMID- 7535360 TI - Terminal complement pathway activation and low lysis inhibitors in rheumatoid arthritis synovial fluid. AB - OBJECTIVE: To investigate terminal complement activation and lysis inhibitors in rheumatoid arthritis (RA). METHODS: C5a, vitronectin and clusterin were quantitated by enzyme immunoassays in plasma and synovial fluid (SF) in RA (n = 30) and osteoarthritis (OA) (n = 11). RESULTS: In RA the concentration of C5a was 3-fold increased in plasma (21.9 vs 7.2 micrograms/l) and 5-fold increased in SF (7.8 vs 1.7 micrograms/l) compared to OA. The SF/plasma ratios for C5a, vitronectin and clusterin were 0.35, 0.36 and 0.23, respectively, not significantly different in the 2 diseases. CONCLUSION: SF terminal pathway activation in RA combined with low local levels of lysis inhibitors might allow lytic or sublytic attacks on local cells, resulting in inflammation and cell damage. PMID- 7535361 TI - A pilot study of the effect of oral 8-methoxypsoralen and intraarticular ultraviolet light on rheumatoid synovitis. AB - OBJECTIVE: To determine the feasibility and safety of combining oral 8 methoxypsoralen (8-MOP) and intraarticular ultraviolet A band light (UVA) to treat rheumatoid synovitis, and to demonstrate a favorable biological effect. METHODS: Six patients with rheumatoid arthritis (RA) and clinically evident knee synovitis were given a single oral dose of 8-MOP (0.6 mg/kg) followed by arthroscopy with a UVA laser equipped small arthroscope. Nine tissue samples treated with UVA doses ranging from 4 to 52 J/cm2 were examined by light microscopy and by immunohistochemistry for vascular cell adhesion molecule 1 (VACM-1), intracellular adhesion molecule 1 (ICAM-1), E-selectin and HLA-DR expression. RESULTS: No reduction in inflammation was evident on light microscopy, nor was there any evidence of tissue injury on gross inspection or light microscopy. At 28 and 52 J/cm2, VCAM-1, ICAM-1 and E-selectin staining were reduced in the posttreatment synovial biopsies. No local or systemic complications were observed by Day 30 in any patient. CONCLUSION: This treatment modality appears to be feasible and safe and may potentially be useful in the treatment of the synovitis associated with RA. PMID- 7535364 TI - [A study on frequency characteristics of filter for detecting high frequency components of QRS complex and its application to the intraventricular conduction system]. AB - A method to detect the high frequency component of QRS complex was developed and the mode of intraventricular conduction was analyzed from its sequential changes. An originally developed subtraction method was used to diminish the transient phenomenon related to phase shift and to obtain easily changeable filtering characteristics. The subjects included normal healthy persons (N), persons having right or left bundle branch block (R and L) and ventricular premature contraction of R or L type (VR and VL). Three components, initial low, mid high and terminal low amplitude were obtained in the N group. In the R group, mid component was lower and duration of the terminal component was longer than in the N group. In the L group, mid and terminal components were not clearly discriminated. Further, their amplitudes were low and duration of each component was markedly prolonged. In the VR and VL groups, the amplitude of the initial component was low and its duration was prolonged. From these findings, the excitation wave through the normal conduction system was short in duration and relatively high in amplitude and includes high frequency component. As contrasted, the duration was prolonged and the amplitude was low in conduction disturbance, seen in bundle branch block or ventricular premature contraction. Thus, the mode of intraventricular conduction could be defined by this newly developed method. PMID- 7535362 TI - Synthesis and biological evaluation of NK1 antagonists derived from L-tryptophan. AB - The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan (3), which was derived from the screening lead N-ethyl-L-tryptophan benzyl ester, has been used as a starting point to identify high-affinity substance P receptor antagonists with improved in vivo activity. Altering the ester moiety to an amide or ether led to a substantial loss in binding affinity, but conversion to a ketone provided compounds with affinity comparable to the equivalent esters. A homochiral synthesis of the key intermediate amino ketone 15 was developed which allows its preparation on a large scale. From this intermediate a range of amine containing acylamino derivatives were prepared with affinity optimized in the morpholinylbutyramide 161 which has an IC50 of 0.17 nM at the human NK1 receptor. In addition to improving affinity, the amino group also provided aqueous solubility for a number of these derivatives. When tested in vivo the quinuclidine derivative L-737,488 (16i) was found to be an orally active (ID50 = 1.8 mg/kg) inhibitor of substance P-induced dermal extravasation in the guinea pig. PMID- 7535363 TI - Phylogeny of African monkeys based upon mitochondrial 12S rRNA sequences. AB - The suborder Anthropoidea of the primates has traditionally been divided in three superfamilies: the Hominoidea (apes and humans) and the Cercopithecoidea (Old World monkeys), together comprising the infraorder Catarrhini, and the Ceboidea (New World monkeys) belonging to the infraorder Platyrrhini. We have sequenced an approximately 390-base-pair part of the mitochondrial 12S rRNA gene for 26 species of the major groups of African monkeys and apes and constructed an extensive phylogeny based upon DNA evidence. Not only is this phylogeny of great importance in classification of African guenons, but it also suggests rearrangements in traditional monkey taxonomy and evolution. Baboons and mandrills were found to be not directly related, while we could confirm that the known four superspecies of mangabeys do not form a monophyletic group, but should be separated into two genera, one clustering with baboons and the other with mandrills. Patas monkeys are clearly related to members of the genus Cercopithecus despite their divergence in build and habitat, while the talapoin falls outside the Cercopithecus clade (including the patas monkey). PMID- 7535365 TI - [Evaluation of late potential by using--event-related signal-averaging technique]. AB - Event-related (ER) signal-averaging technique was used to evaluate whether beat to-beat changes of filtered QRS complex possibly relate to the appearance of ventricular arrhythmias (VA) or not. 82 patients with premature ventricular contractions (PVC) were studied. ANALYSIS-I:56 pts (16 with VT): a conventional method, in which all sinus beats were averaged (ALL), was compared with the new ER methods, in which only sinus beats immediately before (B) or after (A) each PVC can be separately averaged. ANALYSIS-II: 26 pts (10 with VT): five modes of ER averaging, 2nd preceding (2-B), just preceding (1-B), immediately after (1-A), 2 beat after (2-A), and isolated (ISO) from each PVC, were compared. RESULT: I: Significantly longer F-QRS and smaller RMS40 than ALL were observed at B both in VT+ and VT- pts. These transient changes tended to remain even at A only in VT+ not in VT+ pts.II: No significant changes compared with ISO were seen at 2-B and 2-A at all. The widening of F-QRS and the decrease of RMS40 occurred only at 1-B in VT- and unti 1-A in VT+ pts. In conclusion, pre- and post- extra-systolic changes of filtered QRS complex were very transient and event-related, suggesting the close relationship to the mechanism of VA. PMID- 7535368 TI - [Simultaneous production of parathyroid hormone-related protein (PTHrP) and granulocyte colony-stimulating factor (G-CSF) in lung cancer patients with hypercalcemia and leukocytosis]. AB - Hypercalcemia and leukocytosis are often associated with primary lung cancer as a paraneoplastic syndrome. Recently, parathyroid hormone-related protein (PTHrP) and granulocyte colony stimulating factor (G-CSF) have been identified as major causative peptides for hypercalcemia and leukocytosis, respectively. We studied four men with advanced primary lung cancer (stages from IIIA to IV) who presented with hypercalcemia (corrected serum calcium levels: 10.5 mg/dl) and leukocytosis (WBC > 10,000 per mm3). The age of the patients ranged from 59 to 79 years old. The pathological subtypes were squamous cell carcinoma in three and adenocarcinoma in one. The mean serum calcium levels and leukocyte counts were 15.8 +/- 1.4 mg/dl (mean +/- SE) and 24,800 +/- 3,253 cells/mm3 (mean +/- SE), respectively. Abnormally high serum levels of PTHrP and G-CSF were found in three patients (mean +/- SE: 137 +/- 68 pg/ml; normal range in human serum, < 16 pg/ml), and in all four (mean +/- SE: 72 +/- 7.7 pg/ml; normal range in human serum, < 20 pg/ml), respectively. Immuno-histochemical examination of cancerous tissue obtained from these patients showed positive staining for both PTHrP and G CSF within the cytoplasm of all the cancerous tissue. These results suggest that the association of hypercalcemia and leukocytosis in patients with advanced primary lung cancer is caused by production of both PTHrP and G-CSF by cancerous tissue. PMID- 7535367 TI - [Inhibition of gene expression by antisense DNA]. AB - We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV, MMLV, or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment, bound to the antisense oligonucleotide, by reverse transcriptase associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization, by binding to the AMV- and MMLV-RTs, rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription. Furthermore, the RNase H activity of HIV-RT was only slightly affected by the phosphorothioate oligonucleotide. The anti-HIV activities of phosphorothioate- or 5'-linked lipid-oligonucleotides are also described and some of the problems that still need to be solved are pointed out. PMID- 7535366 TI - [The expression pattern of adhesion molecules and their role of the tumor cells in patients with multiple myeloma]. AB - The expression pattern of beta 1 integrin on the surface of tumor cells in patients with multiple myeloma (MM) is reviewed and compared with that of other B cell malignancies. The expression pattern of beta 1 integrin of plasma cells of healthy individuals was also compared with that of malignancies of plasma cells. Normal immature CD10+ B cell precursors in bone marrow are alpha 4+ and alpha 5+, while mature peripheral B cells are alpha 4+ and alpha 5+. In contrast to mature peripheral B cells, it is reported that plasma cells are alpha 4+ and alpha 5+. There are three points following in the phenotype characteristic of MM cells; (i) MM cells are alpha 4 strong positive, (ii) MM cells are beta 1 strong positive and (iii) MM cells are alpha 6strong positive. Because alpha 6+ cell lines of malignant plasma cells showed spread and chemotaxis on stimulation with laminin, alpha 6 beta 1 integrin might contribute of MM cells to transit laminin rich basement membrane of blood vessel to exit to the extravascular space. The difference of the phenotype between plasma cells and MM cells is the expression of alpha 5; plasma cells express alpha 5, but MM cells are lost in one half of the cases. The functional role of VLA-5 in MM cells is reviewed. PMID- 7535369 TI - Hepatitis C virus-associated glomerulonephritis. Effect of alpha-interferon therapy. AB - Hepatitis C virus (HCV) infection may present as a primary glomerular disease. We report 34 adult patients who presented with proteinuria and had circulating anti HCV antibodies. Primary risk factors included a history of intravenous drug abuse (56%) or blood transfusion (18%). Patients presented with nephrotic syndrome (71%) or with non-nephrotic proteinuria (29%) and had membranoproliferative or acute proliferative glomerulonephritis on renal biopsy. Signs of clinical liver disease were infrequent (18%), though elevated liver function tests were common (66%) and liver biopsy in 16 of 18 patients showed chronic active hepatitis. Cryoglobulinemia was frequent (59%), but only 44% had extrarenal manifestations. In 100% of cases tested, HCV RNA could be found in the serum or cryoprecipitates. Fourteen patients received interferon alpha for 6 to 12 months with a significant reduction in proteinuria but no improvement in renal function. A good clinical response correlated with disappearance of HCV RNA from the serum during treatment; however, relapse of viremia and renal disease was common after completing therapy. Evidence for HCV infection should be sought in all patients with primary glomerular disease. The optimal treatment strategy, however, remains to be defined. PMID- 7535370 TI - Growth inhibition of malignant CD5+B (B-1) cells by antisense IL-10 oligonucleotide. AB - Malignant B-1 cells derived from NZB mice, a murine model of chronic lymphocytic leukemia, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for malignant B-1 cells. By addition of antisense oligodeoxynucleotides specific for IL-10 mRNA, we were able to dramatically inhibit the growth of leukemic B-1 cells in a time and dose dependent manner. Control cell lines which do not depend on IL-10 for growth were not affected. Antisense therapy targeted at the 5' region of the IL-10 mRNA not only resulted in inhibition of malignant B-1 cell proliferation but also inhibited IL-10 production by malignant B-1 cells. Because endogenous IL-10 gene activation is critical for B-1 cell expansion, inactivation of the endogenous IL 10 gene by IL-10 antisense rather than extracellular regulation of the IL-10 gene product should be successful in controlling the malignant growth. PMID- 7535371 TI - Isolation of nucleic acid from paraffin embedded tissue for PCR amplification and sequencing of TcR V beta genes. AB - PCR analysis of DNA and RNA from fresh and frozen tissue is now a routine practice in most laboratories. Furthermore DNA extracted from paraffin embedded tissue is routinely used for clonality studies and for HLA typing. However the use of RNA from paraffin embedded tissue is more problematical due to degradation and only short sequences are suitable for amplification. In this report we examine the suitability of extracted nucleic acid from paraffin embedded tissue for the PCR amplification of TcR V beta genes and sequencing of resultant PCR products. PMID- 7535372 TI - Relationship between responsiveness to colony stimulating factors (CSFs) and surface phenotype of leukemic blasts. AB - We examined the responsiveness of leukemic cells to colony stimulating factors (CSFs) as determined by 3H-TdR incorporation and surface phenotypes of leukemic blasts. In acute myeloid leukemia (AML), CD13 and/or CD33 positive and HLA-DR negative M1 and M3 cases tended to show high response to G-CSF, GM-CSFs and IL-3, however, all HLA-DR positive M1, M2, M4 and M5 cases were unresponsive to CSFs but showed high autonomous growth. In acute lymphocytic leukemia (ALL), no response was observed to any CSFs but high autonomous growth was found in mixed leukemia cases. Sole T or B lineage cases showed low autonomous growth. These results suggest the varied nature of the proliferative state in leukemia and the existence of a subgroup in M1. PMID- 7535373 TI - Chlamydia trachomatis does not bind to alpha beta 1 integrins to colonize a human endometrial epithelial cell line cultured in vitro. AB - Chlamydia trachomatis is the leading cause of bacterially acquired sexually transmitted diseases in the United States and Europe. As an obligate intracellular pathogen, this bacterium must invade epithelial cells in order to survive and grow. Thus, multiple strategies probably exist for initial binding of chlamydiae to their target cells. Since a variety of bacteria have exploited integrins to colonize tissues, and a precedent existed for the involvement of extracellular matrix components in chlamydial attachment, this study first analyzed, by flow cytometry, integrins expressed by the human endometrial epithelial cell line HEC-1B. The genital cells were then exposed to monoclonal antibodies directed against those integrins and assayed for chlamydial attachment and inclusion development. Monoclonal antibodies bound to the alpha and/or beta 1 subunit of classic integrin receptors displayed by HEC-1B cells were not able to prevent colonization and infection of the epithelial cells by a genital isolate of C. trachomatis. PMID- 7535374 TI - Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus. AB - When applied at low concentrations (< 10 micrograms/ml), staphylococcal alpha toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the presence of serum in the medium and is very sensitive towards pH. Decreases of pH in the medium to < or = 7.0 as well as increases to > or = 7.8 causes the repair mechanism to fail. The fate of cell-bound toxin molecules was investigated by using a radiolabelled tracer and by immunological detection of toxin exposed at the cell surface. The results indicated that 50-70% of the toxin was shed from cell membranes. However, there was no clear correlation between shedding and recovery, and shedding was also observed in cells that died at pH 7.8. Shedding was not decisive for repair, since cells that had recovered from toxin attack continued to carry 30-40% of initially bound toxin on their cell surface. Blockade of Na+/K(+)-ATPases with ouabain evoked similar kinetics of K(+)-depletion in control cells, compared with cells that had just recuperated from toxin attack and that still carried 30-40% alpha-toxin on their surface. We therefore tentatively concluded that repair of alpha-toxin lesions was due to closure of small pores, rather than from compensation of membrane leaks by up-regulation of Na+/K(+)-ATPase activity. We speculate that repair of small membrane lesions may extend to other agents that produce channels of similar nature in nucleated cells. Larger pores created by E. coli hemolysin or streptolysin O, both of which form larger functional transmembrane lesions, could not be repaired by fibroblasts. PMID- 7535375 TI - Phylogeny, rates of evolution, and patterns of codon usage among sea urchin retroviral-like elements, with implications for the recognition of horizontal transfer. AB - Phylogenetic relationships, rates of evolution, and codon usage were investigated in a family of retrotransposons (SURL elements) found in echinoids. The phylogeny of SURL element reverse transcriptase sequences from 10 echinoid species clearly shows the phylogenetic signature of the host taxa as well as paralogous sequences that diverged prior to speciation events. Two subfamilies (1 and 5) of SURL element reverse transcriptase sequences are recognized that diverged prior to the radiation of the Echinometridae. Comparisons of synonymous versus nonsynonymous substitutions indicate that SURL elements have been active in echinoid genomes and have evolved under purifying selection for millions of years. Rates of synonymous substitution for reverse transcriptase are similar to rates of single copy DNA evolution and to rates of synonymous substitution for the H3 and H4 histone genes, contradicting the assumption that rates of evolution are accelerated in retrotransposons. Finally, codon usage in SURL elements is biased for codons ending in A or U relative to 42 sea urchin nuclear genes. Biased codon usage is sometimes cited as evidence for horizontal transfer, but in the case of SURL elements this bias occurs in spite of a long history of vertical transmission rather than because of horizontal transfer. PMID- 7535376 TI - Identification of a membrane protein and a truncated LysR-type regulator associated with the toluene degradation pathway in Pseudomonas putida F1. AB - A 3 kb DNA region upstream of the toluene degradation (tod) genes, todFC1C2BADEGIH, in Pseudomonas putida F1 (PpF1) was sequenced. Two divergently arranged open reading frames, todR and todX, were identified. A toluene-inducible promoter was localized in front of todX, and the transcription start point was mapped. This promoter is probably responsible for the expression of all tod structural genes. TodX was found to be a membrane protein. Its predicted amino acid sequence (453 residues; M(r) 48,265) exhibits considerable similarity with the FadL protein of Escherichia coli, an outer membrane protein required for binding and transport of long-chain fatty acids. An apparent function of TodX is likely to be involved in facilitating the delivery of exogenous toluene inside the PpF1 cells. The sequence of TodR (100 residues) exhibits extensive homology with the DNA-binding domain of transcriptional activators of the LysR family, but todR was found to have a negligible role in tod gene regulation. PMID- 7535377 TI - Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression. AB - The transport of citrate in Lactococcus lactis biovar diacetylactis is mediated by the citrate permease P. This polypeptide is encoded by the citP gene carried by plasmid pCIT264. In this report, we characterize the citP transcript, identify a cluster of two genes cotranscribed with citP and describe their post transcriptional regulation. The transcriptional promoter is located 1500 nucleotides upstream of the citP gene and the transcriptional terminator is positioned next to the 3'-end of this gene. The DNA sequence was determined of the region upstream of the citP gene, including the promoter. Two partially overlapping open reading frames, citQ and citR were identified, which could encode polypeptides of 3.9 and 13 kDa respectively. These two genes, together with citP, constitute the cit cluster. Moreover, an IS-like element located between the cit promoter and the citQ open reading frame was identified. This element includes an open reading frame ORF1, which could encode a 33 kDa polypeptide. A translational fusion between the citP and a cat reporter gene showed that translation of citR and citP is coupled, and regulated by CitR. The cit mRNA was subjected to specific cleavage after addition of rifampicin to the bacterial cultures. We propose that expression of the cit cluster is controlled at the post-transcriptional level by mRNA processing at a putative complex secondary structure and by translational repression mediated by CitR. PMID- 7535378 TI - Nonopioid mechanism of morphine modulation of the activation of 5 hydroxytryptamine type 3 receptors. AB - The effect of morphine on the ion current mediated by 5-hydroxytryptamine (5-HT3) receptors was investigated in rat nodose ganglion neurons and in Xenopus oocytes expressing the cloned 5-HT3 receptor. Morphine reversibly inhibited the 5-HT induced current and shifted the 5-HT concentration-response curve to the right in a parallel fashion, without reducing the maximal 5-HT response. IC50 values for morphine were 0.3 microM in nodose neurons and 0.32 microM in oocytes. The apparent Kd of morphine in nodose neurons was 0.903 microM. This effect of morphine was immediate not dependent on membrane potential, and not prevented by the opioid receptor antagonists naltrexone and beta-chlornaltrexamine. It is concluded that opioid receptors were not involved in the present study and that morphine acted at the agonist recognition site located on the 5-HT3 receptor. PMID- 7535379 TI - Nitric oxide modulation of agonist-evoked intracellular Ca2+ release in neurosecretory PC-12 cells: inhibition of phospholipase C activity via cyclic GMP dependent protein kinase I. AB - Nitric oxide is a signaling molecule involved in events crucial to neuronal cell function, such as neurotransmitter release, gene transcription, and neurotoxicity, i.e., a number of processes in which a key role appears to be played by increases in intracellular Ca2+ concentration. In the neurosecretory/neuronal cell line PC-12, we have investigated the role of nitric oxide in the modulation of Ca2+ release from intracellular stores elicited by activation of three different receptors coupled to phosphatidyl-inositol-4,5 bisphosphate hydrolysis, i.e., the purinergic P2U, muscarinic M3, and bradykinin B2 receptors. The results obtained show that nitric oxide donors have an inhibitory effect on agonist-evoked Ca2+ release. This effect is not due to nitric oxide-induced modifications of Ca2+ storage, because the total releasable Ca2+ pool, measured as the radioactivity released by thapsigargin and ionomycin in cells loaded at equilibrium with 45Ca2+, was unchanged. In contrast, nitric oxide donors decreased agonist-evoked inositol-1,4,5-trisphosphate generation and total inositol phosphate accumulation. Similarly, nitric oxide inhibited total inositol phosphate accumulation stimulated by either aluminium fluoride or Ca2+. All of these effects were mimicked by the cGMP analogue 8-bromo-cGMP. When cells were incubated with nitric oxide synthase inhibitors, the results observed were opposite those produced by nitric oxide donors. All of the effects of nitric oxide were abolished when cells were treated with the cGMP-dependent protein kinase I inhibitor KT5823. Furthermore, KT5823 mimicked the effects of nitric oxide synthase inhibitors. We conclude that nitric oxide and Ca2+ signaling pathways are interconnected in PC-12 cells. Modulation of inositol phosphate generation and Ca2+ release by nitric oxide appears to be exerted primarily at the level of phospholipase C functioning and to be mediated by the activation of cGMP-dependent protein kinase I. PMID- 7535381 TI - Zinc is a mixed antagonist of homomeric rho 1 gamma-aminobutyric acid-activated channels. AB - The transition metal Zn2+ is differentially distributed in the central nervous system, where it is proposed to be a neuromodulator. One of the documented effects of Zn2+ is the antagonism of gamma-aminobutyric acid (GABA)-mediated synaptic inhibition. This antagonism is presumed to result from a direct interaction of Zn2+ with the GABA receptor/ionophore complex, although the characteristics of Zn2+ sensitivity are dependent on the particular GABA subunit combination. In this study, we examined the effects of Zn2+ on homomeric rho 1 GABA-activated channels expressed in Xenopus oocytes. Zn2+ was found to be a mixed antagonist of these recombinant rho 1 GABA receptors. The antagonism was predominantly competitive at low Zn2+ concentrations (< or = 100 microM), whereas at high Zn2+ concentrations (> 100 microM) a noncompetitive antagonism was apparent. Evidence is presented showing that the antagonism was not due to an interaction of GABA and Zn2+ in solution but, rather, resulted from interactions of these two ligands with the GABA-activated channel. A mechanism is proposed for Zn(2+)-mediated antagonism in which GABA and Zn2+ bind to distinct sites on the GABA complex. The apparent mixed antagonism may arise from different Ki values for the binding of Zn2+ to non-agonist-bound or agonist-bound receptors. However, two distinct Zn2+ binding sites, one competitive and one noncompetitive, could also give rise to the dual antagonism. PMID- 7535382 TI - The effect of fuel composition on the mutagenicity of diesel engine exhaust. AB - The effect of fuel composition on the mutagenicity of diesel engine emission was investigated. To this end, a fuel matrix comprising fuels with different contents of aromatic and naphthenic compounds was used. Extracts of the organic phase of raw exhausts obtained with different fuels were tested for mutagenicity in bacterial reversion assays. The results obtained demonstrate that the mutagenicity of diesel exhaust is largely dependent on the aromatic content of the fuel. In fact, mutagenicity was greatly reduced when the aromatic content of the fuel was lowered by hydrogen treatment. Conversely, mutagenicity was enhanced when the fuel was enriched with fractions of di- or triaromatic compounds. The addition of di- and trinaphthenic compounds only produced borderline mutagenicity. No clear relationship was observed between sulfur content of the fuel and mutagenicity of the exhaust. Assays in bacterial strains with different sensitivity to nitroaromatic compounds suggest a low contribution of the highly mutagenic dinitropyrenes to the responses observed, and a relatively greater contribution of 1-nitropyrene or other nitroaromatics processed by the same bacterial nitroreductase. PMID- 7535383 TI - NO in hydra feeding response. PMID- 7535380 TI - Novel adamantane derivatives act as blockers of open ligand-gated channels and as anticonvulsants. AB - We examined the influence of the molecular structure of four novel adamantane derivatives on their ability to block the channels of nicotinic acetylcholine (ACh) and N-methyl-D-aspartate (NMDA) receptors. The structure of the drugs is Ad CH2-N+H2-(CH2)5-R, where Ad is adamantane and R was varied from ammonium (IEM 1754) to tert-butyldimethylammonium (IEM-1857) radical. The compounds induced double-exponential decays of postsynaptic currents in frog muscles and flickering of NMDA-activated channels, suggesting that each drug acts as a fast open-channel blocker at both types of receptors. The equilibrium dissociation constants (Kd) of the drugs for ACh-activated channels at -80 mV were similar, whereas the Kd values for NMDA-activated channels at -80 mV were 2-10 times lower. Several observations suggested that occupation of either type of channel by these compounds inhibited channel closure; the time constant (tau) of the slow component of the decay of postsynaptic currents in the presence of each compound was greater than the control tau, the IC50 of IEM-1754 for inhibition of NMDA activated whole-cell currents was > 20 times larger than its Kd for the open channel, and a transient increase in NMDA-activated whole-cell currents was observed after washout of IEM-1754. Thus, these drugs appear to act on nicotinic ACh and NMDA receptors via similar mechanisms, although the voltage dependence of block suggested that the drugs bind at a more superficial site in the ACh activated channel. All compounds also potently prevented NMDA-induced convulsions in mice. The ED50 of IEM-1754 was 4 times lower than the ED50 of MK-801. PMID- 7535384 TI - MHC evolution. PMID- 7535385 TI - Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow. AB - Selectins tether to the blood vessel wall leukocytes that are flowing in the bloodstream and support subsequent labile rolling interactions as the leukocytes are subjected to hydrodynamic drag forces. To support this rolling, selectins have been proposed to have rapid bond association and dissociation rate constants, and special mechanical properties linking tensile forces and bond dissociation. We have visualized transient tethering and release of neutrophils in hydrodynamic flow on lipid bilayers containing densities of P-selectin below those required to support rolling. We report here that transient tethers had first-order kinetics and other characteristics suggesting a unimolecular interaction between P-selectin and its glycoprotein ligand (PSGL-1). The unstressed dissociation constant (off rate) was 1 s-1. Hydrodynamic shear stresses of up to 1.1 dyn cm-2, corresponding to a force on the bond of up to 110 pN, increased the off rate only modestly, to 3.5 s-1. The data was adequately matched by a proposed equation relating off rate to the exponential of tensile force on the bond and the bond interaction distance, and gave a bond interaction distance of 0.5 A. This distance is compatible with hydrogen and metal coordination bonds between P-selectin and PSGL-1. Fast on and off rates, together with the high tensile strength of the selectin bond, appear necessary to support rolling at physiological shear stresses. PMID- 7535386 TI - [Kawasaki disease; what to look for and what to do?]. PMID- 7535388 TI - Somatotropes and thyrotropes in the rat anterior pituitary gland cosecrete substance P: analysis by the sandwich cell immunoblot assay. AB - Substance P (SP) which is synthesized and secreted by anterior pituitary cells has been suggested to alter pituitary functions as a local modulator. We determined which cell type(s) of rat anterior pituitary gland secretes SP. A new technique, named the sandwich cell immunoblot assay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior pituitary cells were sandwiched with pairs of transfer membranes and incubated to absorb secretory substances on the membranes. Small reference points for identifying the location of cell blots were simultaneously put on the pairs of transfer membranes after the incubation. The validity of the sandwich CIBA was revealed by the following: (1) cell blots were detected at the same locations on the pairs of transfer membranes that were immunostained with the same antisera against the anterior pituitary hormones, and (2) the areas of these cell blots detected at the same locations on different membranes correlated well. When one of the pair of transfer membranes was immunostained for SP and the remaining immunostained for one of the anterior pituitary hormones, it was found that 78 and 27% of SP-immunoreactive cell blots showed also GH and TSH immunoreactivity, respectively. None of the SP immunoreactive cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These results suggest that the sandwich CIBA is useful to identify two substances cosecreted from a cell and that a subpopulation of rat somatotropes or thyrotropes cosecretes SP. PMID- 7535387 TI - Endothelin-1 stimulates the in vitro release of neurohypophyseal hormones, but not corticotropin-releasing hormone, via ETA receptors. AB - The endothelins consist of a family of vasoconstrictor peptides originally isolated from endothelial tissue which are now known to be involved in neuroendocrine regulation. However, while there are data indicating the involvement of endothelins in the modulation of the hypothalamo-pituitary-adrenal (HPA) axis, the precise mechanisms involved have been unclear. We have therefore used a previously validated rat hypothalamic explant system in order to investigate the possible modulation of the neurohypophyseal hormones vasopressin and oxytocin, and corticotropin-releasing hormone (CRH), by endothelin-1 (ET-1) and endothelin-3 (ET-3). Following a period of stabilisation, the release of vasopressin, oxytocin and CRH remained approximately constant in successive 20 min incubations. Addition of ET-1 stimulated the release of vasopressin at a dose of 0.1 nmol/l (p < 0.05), and both vasopressin and oxytocin at 10 nmol/l (p < 0.01 and 0.05, respectively). The release of vasopressin and oxytocin induced by 10 nmol/l ET-1 were both totally blocked by co-incubation with either 1 or 10 mumol/l of the specific ETA receptor subtype antagonist cyclo (D-Trp-D-Asp-Pro-D Val-Leu) (BQ-123). ET-1 had no effect on CRH release in the dose range of 0.1 1,000 nmol/l. In case any possible stimulation of CRH might be masked by simultaneous generation of nitric oxide (NO), an inhibitor of CRH secretion, addition of ET-1 was also carried out in the presence of the NO synthase inhibitor, L-NO-Arg: ET-1 was again without effect in this dose range.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535389 TI - Concentrations of corticotropin-releasing hormone, norepinephrine, MHPG, 5 hydroxyindoleacetic acid, and tryptophan in the cerebrospinal fluid of alcoholic patients: serial sampling studies. AB - Abnormalities in corticotropin-releasing hormone (CRH) secretion, noradrenergic neurotransmission, and serotonergic activity in the central nervous system (CNS) have all been hypothesized to exist in alcoholic patients, as have abnormalities in hypothalamic-pituitary adrenal function. To test these hypotheses, we continuously sampled cerebrospinal fluid (CSF) from alcoholic patients after 38 124 days of abstinence and from normal volunteers via a flexible, indwelling lumbar subarachnoid catheter and measured CRH, norepinephrine (NE), 3-methoxy-4 hydroxyphenylglycol (MHPG), tryptophan, and 5-hydroxyindoleacetic acid (5-HIAA) concentrations at 10-min intervals, from 11:00 through 17:00 h. The spinal canal catheter was inserted at approximately 08:00 h. Serial plasma ACTH, cortisol, and NE concentrations were also measured. A mixed liquid meal was consumed at 13:00 h. CSF CRH concentrations were lower in alcoholic patients than in normal volunteers (26 +/- 15 vs. 60 +/- 30 pg/ml, respectively, p < 0.05 by ANOVA), as were CSF NE levels (0.33 +/- 0.09 vs. 1.15 +/- 0.51 pmol/ml, respectively, p < 0.01). Plasma NE and CSF MHPG levels were normal in the alcoholic patients. CSF tryptophan and 5-HIAA and plasma ACTH and cortisol concentrations did not differ between the groups. These studies extend our finding of reduced spinal canal CSF CRH concentrations in depressed patients to abstinent chronic alcoholics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535390 TI - Chronic treatment with a classical neuroleptic alters excitatory amino acid and GABAergic neurotransmission in specific regions of the rat brain. AB - The purpose of the following experiments was to describe some of the neurochemical changes that occur in the basal ganglia of rats exposed chronically to a classical neuroleptic, fluphenazine, and to relate these changes to extrapyramidal motor dysfunction. For these studies a combination of behavioural, receptor autoradiographic and in situ hybridization methods were employed. Preliminary pharmacological studies on GABA receptors showed that incubation in Tris-acetate rather than Tris-citrate buffer increased the number of binding sites labelled by [3H]muscimol by over 120% without affecting binding affinity or selectivity. The results of experiments with fluphenazine showed that treatment for six months increased the frequency of vacuous chewing movements compared to controls. In the striatum, changes in GABA transmission were observed in fluphenazine-treated rats with increases in glutamate decarboxylase mRNA levels in the caudate nucleus, dorsal shell and core of the accumbens and decreases in [3H]muscimol binding in the caudate and dorsal shell regions. These data suggest that fluphenazine treatment increased GABA transmission in specific subregions of the caudate and accumbens nuclei. In addition, glutamate decarboxylase mRNA levels were elevated in the entopeduncular nucleus of fluphenazine-treated animals. Autoradiographic analysis of excitatory amino acid binding showed that fluphenazine exposure decreased [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid binding in entopeduncular nucleus and in the ventrolateral thalamic nucleus and decreased [3H]dizocilpine maleate binding in the medial geniculate nucleus. These experiments show that in addition to altering GABA transmission, chronic neuroleptic exposure alters excitatory amino acid transmission in specific regions of the basal ganglia-thalamocortical motor system. The neuroleptic dependent increases in glutamate decarboxylase mRNA levels in the entopeduncular nucleus may reflect changes in neurotransmission in the indirect pathway connecting the major input and output nuclei of the basal ganglia. Changes in some of these brain regions may be related to the occurrence of extrapyramidal motor disturbances. PMID- 7535391 TI - Differential expression of membrane currents in dissociated mouse primary sensory neurons. AB - The whole cell configuration of the patch clamp technique has been applied to identify the membrane currents expressed by populations of dissociated mouse primary sensory neurons. Three discrete populations of cells were distinguished on the basis of cell size and the array of currents expressed. Group 1 cells (capacitance 10-30 pF) expressed a Na+ current resistant to tetrodotoxin (1 microM) and a prominent, low threshold, inactivating, K+ current sensitive to 4 aminopyridine (IA). A population (53%) of these small cells responded to capsaicin (10 microM) with an inward current, suggesting a functional correlate with nociceptive "C"-cells. The cells of Group 2 (capacitance 55-85 pF) were characterized by the expression of a Na+ current sensitive to tetrodotoxin and a prominent inward current activated by hyperpolarization (IH). They also showed a variant of the A-type K+ current, which was a low threshold, but sustained K+ current, sensitive to dendrotoxin (30 nM). Group 3 cells, of intermediate size (capacitance 30-55 pF) were similar to Group 2 cells, in that they expressed a tetrodotoxin-sensitive Na+ current and (through reduced in amplitude), IH. The most notable feature of Group 3 cells was the expression of a transient, low threshold Ca2+ current. The differential expression of these conductances was reflected in the behaviour of cells under current clamp control. Each group of cells could thus be distinguished by the selective expression of specific ionic conductances which correlated clearly with cell size, suggesting a correlation with well recognised functional differentiation of sensory neurons. The selective expression of specific subsets of membrane channels may provide valuable markers in studying the developmental regulation of phenotype in this population of cells. PMID- 7535393 TI - Specialization of tachykinin NK1 and NK2 receptors in producing fast and slow atropine-resistant neurotransmission to the circular muscle of the guinea-pig colon. AB - We studied the relative contribution of tachykinin NK1 and NK2 receptors in producing nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig proximal colon in response to electrical field stimulation. All experiments were performed in the presence of atropine, guanethidine, indomethacin, apamin and L-nitroarginine. In organ bath experiments, electrical stimulation produced a tetrodotoxin-sensitive frequency-dependent contraction. The NK1 receptor antagonists, FK 888 (1-10 microM) and GR 82,334 (0.3-3 microM) markedly reduced but did not abolish the nonadrenergic noncholinergic response. The NK2 receptor antagonist, GR 94,800 (0.3-3 microM) was partly effective at 3 microM. The combined administration of FK 888 (10 microM) and GR 94,800 (3 microM) or GR 82,334 and GR 94,800 abolished the nonadrenergic noncholinergic contraction. The response to a prolonged period of stimulation (3 Hz for 5 min) was evenly depressed by FK 888 or GR 82,334, while GR 94,800 was more effective in inhibiting the late (87% inhibition) than the peak response (25% inhibition). In the presence of nifedipine (1 microM) a marked inhibition of the nonadrenergic noncholinergic contraction was observed and a time lag was evident between stimulus application and onset of contraction, which showed slow onset and offset kinetics. The nifedipine-resistant nonadrenergic noncholinergic contraction was unaffected by FK 888 or GR 82,334 but was suppressed by GR 94,800. Submaximally effective (1-3 nM) concentrations of substance P and neurokinin A produced distinct patterns of contraction: the response to substance P was fast and declined rapidly toward baseline; the response to neurokinin A was slow and sustained. In the presence of nifedipine, the response to substance P was greatly depressed and became slower in onset; nifedipine did not affect the contraction to neurokinin A but slowed its time-course. In sucrose gap experiments, either a short (10 Hz for 1 s) or a prolonged period of electrical stimulation (3 Hz for 3 min) evoked membrane depolarization, action potentials and contraction: in response to the "prolonged" stimulation, distinct phasic and tonic component of contraction were observed. Nifedipine abolished action potentials and the phasic contraction produced by a short period of stimulation, reduced by about 50% the maximal contraction developed during the prolonged stimulation without affecting the amplitude of the tonic response. In the presence of nifedipine, GR 82,334 (3 microM) blocked the membrane depolarization but did not affect contraction; GR 94,800 (0.1 microM) did not affect depolarization but abolished contraction.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7535392 TI - Glutamate-induced swelling of single astroglial cells in primary culture. AB - Glutamate induced an increase in cell volume within one minute and evoked cytosolic Ca2+ transients in type 1 astroglial cells in primary culture obtained from the cerebral cortex of newborn rat. Even the metabotropic glutamate receptor agonists (1S,3R)-1-aminocyclopentane- 1,3-dicarboxylic acid (1S-3R-ACPD) and L(+) 2-amino-4 phosphonobutyric acid (L-AP4) induced a cell swelling with ACPD inducing a parallel Ca2+ transient while L-AP4 did not. A new method was used where rapid changes in relative cell volume could be followed at the single cell level. Relative volume changes in cultured single astroglial cells were examined by microspectrofluorimetry after loading the cells with the highly fluorescent intracellular probe fura-2/AM. At its isosbestic point, 358 nm, fura-2 is ion insensitive and the fluorescent signals emitted are related only to the intracellular dye concentration. By varying the excitation wavelengths, changes in intracellular Ca2+ transients could be recorded simultaneously with the relative volume variations of the individual cells. Thus, as rapid changes in cell volume were followed, the results from this method could be of physiological significance. Glutamate-induced cell swelling was blocked by BaCl2 and by tetraethylammonium, suggesting that K+ channels are operative in glutamate induced cell swelling. Furthermore, the glutamate-induced swelling was blocked by the Na+; K+, and 2Cl- co-transport inhibitor furosemide. The glutamate-induced swelling was partially blocked by pertussis toxin and partially blocked also by the glutamate carrier-blocker dihydroaspartate. When the ionotropic glutamate receptor alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid was blocked with the antagonist 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo(F)quinoxaline, glutamate still induced a swelling, suggesting that this receptor was not directly involved in the glutamate-induced volume increase. Even in situations of blocked or partially blocked swelling, intracellular Ca2+ transients could be obtained. Furthermore, the glutamate-induced swelling was evoked even in low extracellular Ca2+ concentrations. Our data suggest that glutamate-induced rapid swelling is a complex process at the molecular level. One hypothetical mechanism might be that glutamate interacts with metabotropic glutamate receptors and induces a release of Ca2+ from internal stores. Furthermore glutamate interacts with K+ channels, and probably at least one co-transporter and the sodium dependent high-affinity uptake glutamate carrier, resulting in cell swelling. PMID- 7535394 TI - Characterization of alkaline phosphatase-reactive neurons in the guinea-pig small intestine. AB - Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons. PMID- 7535395 TI - Molecular characterization of immunoreactivities of peptides derived from chromogranin A (GE-25) and from secretogranin II (secretoneurin) in human and bovine cerebrospinal fluid. AB - Chromogranin A and secretogranin II are members of the so-called chromogranins, the acidic proteins stored in neuroendocrine large dense-core vesicles. We characterized chromogranin A and secretogranin II immunoreactivities in cerebrospinal fluid by radioimmunoassays using synthetic peptides derived from these components (GE-25 for chromogranin A and secretoneurin for secretogranin II). In lumbar cerebrospinal fluid, high levels (more than 1000 fmol/ml) of these two components were found, whereas in ventricular cerebrospinal fluid the secretoneurin levels were relatively low. The cerebrospinal fluid/serum ratio for secretoneurin was close to 170. High-performance liquid chromatography revealed that in both cerebrospinal fluid and extracts from human brain secretoneurin was the predominant immunoreactive component. In cerebrospinal fluid chromogranin A immunoreactivity was present as intermediate-sized peptides with little intact chromogranin A and free GE-25 peptide. In human brain samples smaller peptides including GE-25 were more predominant. Analogous findings for secretoneurin and chromogranin A were obtained for bovine brain samples. We can conclude that chromogranins are present in cerebrospinal fluid in concentrations much higher than those of classical neuropeptides also stored in large dense-core vesicles. Therefore, their degree of proteolytic processing can be analysed with small samples of cerebrospinal fluid. A possible disturbance of proteolytic processing in large dense-core vesicles in various pathological conditions can now be discovered. PMID- 7535396 TI - Synaptic activation of metabotropic glutamate receptors in the parallel fibre Purkinje cell pathway in rat cerebellar slices. AB - Glutamate, the major excitatory neurotransmitter in the central nervous system, acts through two broad classes of receptors: ion channel-linked (ionotropic) receptors, which include N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl 4-isoxazolepropionic acid receptors, and metabotropic receptors which couple via G-proteins to intracellular messenger cascades. Seven subtypes of mGluR are known to exist but their roles in synaptic physiology are poorly understood. In cerebellar Purkinje cells, application of the mGluR agonist, trans-1 aminocyclopentane-1,3-dicarboxylic acid, or the active enantiomer, 1S,3R-ACPD, results in a depolarization associated with an inward current and an elevation of intracellular Ca2+ (for review see Ref. 29). Moreover, using an extracellular (grease-gap) technique that monitors population responses, we have previously discovered that, in Purkinje cells of adult rat cerebellum, brief tetanic stimulation of the glutamatergic parallel fibre input gives rise to a slow depolarising synaptic potential that is resistant to ionotropic glutamate receptor blockers and to antagonists acting at GABA receptors. It was suggested that this novel potential is mediated by metabotropic receptors. The advent of antagonists for metabotropic receptors has allowed us to test this hypothesis. We find that the S-enantiomer of alpha-methyl-4-carboxyphenylglycine stereoselectively antagonizes the slow synaptic potential recorded using the grease-gap method. The results were confirmed by intracellular recording from Purkinje cells. To our knowledge this is the first direct evidence of an mGluR mediated EPSP in intact brain tissue. PMID- 7535397 TI - N-methyl-D-aspartate receptor-mediated changes in thermal nociception: allosteric modulation at glycine and polyamine recognition sites. AB - The effects of allosteric modulators of the N-methyl-D-aspartic acid receptor ion channel complex on the nociceptive tail-flick reflex were studied in awake rats. Intrathecal administration of D-serine (100 fmol-1 mumol) but not L-serine or glycine to the lumbar spinal cord produced a facilitation of the tail-flick reflex at doses > or = 1 pmol (maximum at 0.5-1 min). Intrathecal pretreatment with the glycine modulatory site antagonist 7-chlorokynurenic acid (3 pmol) blocked both D-serine-produced and N-methyl-D-aspartate-produced facilitation of the tail-flick reflex. D-serine-produced facilitation was also blocked by intrathecal pretreatment with a N-methyl-D-aspartate receptor ion-channel blocker, MK 801 (100 fmol), or with an alternate substrate for nitric oxide synthase, NG-nitro-L-arginine-methyl ester (100 nmol). Intrathecal administration of spermine (0.01 nmol-3 mumol) produced biphasic effects on tail-flick latency accompanied by mechanical hyperesthesia and vocalization at greater doses. Spermine-produced facilitation (maximum with 0.01 nmol to 1 nmol at 1 min) was blocked by intrathecal pretreatment with MK 801 (100 fmol), NG-nitro-L-arginine methyl ester (100 nmol) or the polyamine modulatory site antagonist, arcaine (10 nmol). Spermine-produced inhibition (maximum with 300 nmol at 2 min) was blocked by intrathecal administration of MK 801 (1 nmol). Intrathecal administration of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoic acid (1 nmol), blocked inhibition and uncovered a facilitation produced by 1 mumol spermine. In addition, spermine produced multi-stage motor effects (immediate- and late-onset). Intrathecal pretreatment with MK 801 (1 nmol) blocked only the immediate-onset motor effects while the late-onset motor effects were selectively blocked by pretreatment with the kappa opioid receptor antagonist, nor binaltorphamine (200 nmol). Taken together, these data suggest that D-serine and spermine facilitate nociceptive transmission by positive allosteric modulation of the N-methyl-D-aspartate receptor ion-channel. Furthermore, activation of the N methyl-D-aspartate receptor is also necessary to elicit the immediate-onset motor effects and inhibition of the tail-flick reflex produced by greater doses of spermine. Because kappa opioid receptors appear to be involved, the spermine produced late-onset motor effects may involve endogenous dynorphin release. PMID- 7535398 TI - Properties of isolated GABAB-mediated inhibitory postsynaptic currents in hippocampal pyramidal cells. AB - Whole-cell recording techniques were used to record isolated slow inhibitory postsynaptic currents in CA1 pyramidal neurons from rat hippocampal slices. Application of 6-cyano-7-nitroquinoxaline-2,3-dione and 3-(2-carboxypiperazin-4 yl)propyl-1-phosphonic acid eliminated excitatory synaptic transmission, resulting in a 38% reduction in slow inhibitory postsynaptic current magnitude. Subsequent addition of the GABAA antagonist picrotoxin caused a further decrease in slow inhibitory postsynaptic current amplitude. The remaining, isolated slow inhibitory postsynaptic current was blocked by the GABAB antagonist 2 hydroxysaclofen and when cesium was substituted for intracellular potassium. The kinetics of isolated slow inhibitory postsynaptic currents were characterized by single exponential, fourth power activation, and double exponential inactivation. These slow inhibitory postsynaptic currents had a reversal potential of -85.7 +/- 1.6 mV, and a slope conductance of 935 +/- 277 pS. Single slow inhibitory postsynaptic currents carried a total charge flux of 13.4 +/- 7.6 pC. Repetitive stimulation up to 1 Hz progressively reduced steady-state slow inhibitory postsynaptic current amplitude. This attenuation was characterized by a decrease in slope conductance, but slow inhibitory postsynaptic current reversal potential remained unchanged, as did slow inhibitory postsynaptic current kinetics. These results indicate that, under physiological conditions, both ionotropic glutamate- and GABAA-mediated transmission contribute to slow inhibitory postsynaptic current recruitment. Given this finding, activity-dependent decreases in GABAA transmission could contribute to slow inhibitory postsynaptic current depression, though not exclusively, since isolated slow inhibitory postsynaptic currents also demonstrated this property. The use-dependent depression of isolated slow inhibitory postsynaptic currents may be a consequence of a reduction in transmitter release. PMID- 7535400 TI - Nerve growth factor reduces apoptosis of axotomized retinal ganglion cells in the neonatal rat. AB - It has recently been reported that the degeneration of retinal ganglion cells induced by transection of the optic nerve in the neonatal rat is due to an active process of apoptosis, as opposed to passive necrosis. Here we tested whether the administration of the trophic factor nerve growth factor could prevent the apoptotic death of the axotomized cells. We administered nerve growth factor by two intraocular injections, one immediately after the lesion and the second 12 h later. The retinas were taken at 24 h post-lesion and stained as whole mounts with Cresyl Violet. Pyknotic as well as surviving cells were counted in the retinal ganglion cell layer. In this layer at least 95% of the total cell population is composed by ganglion cells, as revealed by retrogradely labelling these cells with horseradish peroxidase injected in the superior colliculi. We found that intraocular administration of nerve growth factor diminishes the degeneration induced by optic nerve transection in the neonatal rat. After nerve growth factor injection, in fact, the number of pyknotic cells is reduced by 39% compared with controls (lesioned, injected with saline); in addition, nerve growth factor also increases the survival of retinal ganglion cells by 30% at 24 h post-lesion. PMID- 7535399 TI - Tonic desensitization of hippocampal alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid receptors regulates 5-hydroxytryptamine release in vivo. AB - Strong evidence implicates glutamate as an excitatory neurotransmitter in the central nervous system. In the present study we have investigated the effects of different concentrations of the excitatory amino acid agonist alpha-amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid on release of 5-hydroxytryptamine in rat hippocampus using in vivo microdialysis. Infusion of alpha-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid at 1 microM led to an increase in dialysate 5 hydroxytryptamine. In contrast 100 microM alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid decreased extracellular 5-hydroxytryptamine, collected in 30 min samples, and this decrease was sustained for several hours. alpha-Amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization is well documented in vitro, and is reversed by the drug diazoxide. We therefore studied the possibility that this was occurring in hippocampus in vivo. Collection of dialysates at 5 min time intervals revealed a brief increase in dialysate 5 hydroxytryptamine in response to 100 microM alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid, although basal level of 5-hydroxytryptamine was below the level of detection. When 100 microM agonist was co-infused with 500 microM diazoxide, a substantial and prolonged increase in dialysate 5-hydroxytryptamine was seen. Diazoxide alone was observed to cause an increase in extracellular 5 hydroxytryptamine. Diazoxide is known to active ATP-dependent K+ channels, however, cromakalim (100 microM), an activator of ATP-dependent K+ channels, reduced hippocampal 5-hydroxytryptamine release, suggesting that the effect of diazoxide is not the result of such an action.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535401 TI - Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis. AB - Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell "suicide" whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6 hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricarboxylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations. PMID- 7535403 TI - Quantitation, cellular localization and regulation of neurokinin receptor gene expression within the rat substantia nigra. AB - The diverse biological effects of substance P and related peptides are mediated by multiple neurokinin receptors. The CNS sites of neurokinin receptor biosynthesis have not been fully elucidated and little is known about the regulation of neurokinin receptor gene expression. In the present study, the abundance of neurokinin-1, neurokinin-2 and neurokinin-3 receptor messenger RNAs in various rat brain regions was quantitated using a sensitive solution hybridization assay. Midbrain neurokinin receptor gene expression was then examined in detail. In situ hybridization experiments localized high levels of neurokinin-3 receptor messenger RNA to presumptive dopamine neurons, as evidenced by sensitivity to 6-hydroxydopamine lesions and the presence of tyrosine hydroxylase messenger RNA in serial sections. Lesions of nigral afferent (including substance P-containing) pathways from the caudate-putamen increased both nigral neurokinin-3 and neurokinin-1 receptor messenger RNA levels two- to three-fold. These data provide the anatomical substrate for physiological data suggesting that substance P (released from striatonigral neurons) may act on nigral cells through neurokinin-1 receptors, while the substance P co-transmitter neurokinin A may act preferentially on dopamine neurons through neurokinin-3 receptors. The magnitude of denervation-induced changes in neurokinin receptor messenger RNAs suggests significant plasticity of neurokinin receptor gene expression. PMID- 7535402 TI - Evidence for a preferential loss of enkephalin immunoreactivity in the external globus pallidus in low grade Huntington's disease using high resolution image analysis. AB - Previous studies have shown that in advanced cases of Huntington's disease, enkephalin-immunoreactive striatal projections to the external globus pallidus may be more affected than substance P-containing striatal projections to the inner segment of the pallidum [Reiner A. et al. (1988) Proc. natn. Acad. Sci. U.S.A. 85, 5733-5737]. Other immunohistochemical [Ferrante R. J. et al. (1990) Soc. Neurosci. Abstr. 16, 1120] and neurochemical observations [Storey E. and Beal M.F. (1993) Brain 116, 1201-1222] suggest no difference in the loss of these peptide-containing pathways in Huntington's disease. In view of the potential significance of this issue for understanding the neuropathological process in Huntington's disease, we examined the globus pallidus in control and Huntington's disease brains, using a quantitative approach which involved high resolution image analysis of 7 microns frozen sections to determine the overall density of peptide-immunoreactive terminals. Results showed that in the controls there was no significant difference between the density of enkephalin- and substance P immunoreactive terminals in the external and internal globus pallidus, respectively. In all Huntington's disease brains, including grade 1 cases, enkephalin-immunoreactive terminals in the external globus pallidus were significantly reduced compared to substance P-positive boutons in the internal segment of the adjacent section. In comparison to controls, enkephalin immunoreactivity in all Huntington's disease cases was significantly lower; substance P-immunoreactive terminals in the internal globus pallidus were significantly lower than controls in some of the grade 2 cases and in the grade 3 cases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535404 TI - Thalamocortical projections activated by phrenic nerve afferents in the cat. AB - This study identified thalamocortical projections activated by respiratory afferents. Cortical evoked potentials were recorded in the right primary somatosensory cortex of the cat following electrical stimulation of the left C5 root of the phrenic nerve. The majority of primary sites were located in the vicinity of the postcruciate dimple, in area 3a near the 3a/3b border, corresponding to the trunk region of the cortical body map. Retrograde fluorescent tracers injected at the sites of primary activation produced labeled cells in the oralis nucleus of the ventroposterior complex [4]. Control injections made in adjacent cortical areas not activated by phrenic stimulation resulted in labeling in the ventroposterior complex which did not overlap that seen with injections of primary activation sites. We conclude that respiratory muscle afferents in the phrenic nerve elicit activity in the trunk region of primary somatosensory cortex via specific thalamocortical projections originating in the oralis portion of the thalamic ventroposterior complex. PMID- 7535405 TI - Projections from the globus pallidus to the thalamic areas projecting to the dorsal area 6 of the macaque monkey: a multiple tracing study. AB - In the brains of 3 Japanese monkeys (Macaca fuscata), a combined injection of the tract-tracers Fast Blue (FB), Diamidino Yellow (DY), and horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) was performed: the retrograde tracers, FB and DY, were injected into a rostral and a caudal sectors of the dorsal part in area 6, and the anterograde tracer, WGA-HRP, was injected into the internal segment of the globus pallidus (GPi). In the thalamus, FB- and DY labeled neurons were distributed in a segregated manner. A substantial number of the FB-labeled and DY-labeled thalamic neurons were located within the thalamic areas containing fine axons labeled with WGA-HRP. The results suggest that the GPi may project to both the rostral and caudal sectors of the dorsal part of area 6 through the thalamus. PMID- 7535406 TI - Postnatal development of nitric oxide synthase type 1 expression in the lumbar spinal cord of the rat: a comparison with the induction of c-fos in response to peripheral application of mustard oil. AB - The distribution of the neuronal isoform of the enzyme nitric oxide synthase (type 1) has been investigated in the lumbar spinal cords of neonatal rats (2-20 days old). Large multipolar neurones were present from day 2 around the central canal, in a band across the neck of the dorsal horn and at the thoracic level in the intermediolateral cell column, whereas staining was absent from laminae II. By 20 days the laminae II staining was similar to that found in the adult. NOS expression in lamina II paralleled the development of c-fos expression in this lamina in response to peripheral application of mustard oil. PMID- 7535407 TI - All calbindin-immunoreactive myenteric neurons project to the mucosa of the guinea-pig small intestine. AB - The projections of Dogiel type II myenteric neurons to the mucosa of the guinea pig ileum were quantified by combining retrograde transport of DiI, in vitro, with immunohistochemistry. After DiI application to the mucosa over an area of 1.5 x 10 mm2, virtually all (> 97%) calbindin-immunoreactive Dogiel type II neurons in the myenteric plexus underneath the mucosal DiI application site were labelled, indicating that essentially all of these neurons project to the mucosa. From cell counts, on average 5 calbindin-immunoreactive neurons project to each villus, and each calbindin-immunoreactive neuron supplies on average 10 villi. Since Dogiel type II neurons that were not immunoreactive for calbindin (19% of all labelled nerve cells) also projected to the mucosa, it is likely that all Dogiel type II neurons, which are putative sensory neurons of the gut, project to the mucosa. PMID- 7535408 TI - Modulation by nitric oxide of rat brain GABAA receptors. AB - The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells. PMID- 7535411 TI - Intracellular study of substance P in human Scarpa's ganglion cells. AB - The effect of substance P on surgically removed human Scarpa's ganglion cells was investigated by intracellular recordings and bath application under in vitro conditions. The neuropeptide produced a slow depolarization of the membrane potential which was accompanied by an increase in membrane resistance. Furthermore, an enhanced firing in response to depolarization occurred. The results strongly support a modulatory action of the neuroactive peptide substance P on human Scarpa's ganglion cells. PMID- 7535412 TI - [Interferon therapy of hepatitis B and C]. PMID- 7535410 TI - GABAergic modulation of striatal peptide expression in rats and the alterations induced by dopamine antagonist treatment. AB - GABAergic modulation of enkephalin, substance P and glutamic acid decarboxylase (GAD67) gene expression and the alterations induced by dopamine receptor blockade were studied in the rat striatum. Following subchronic treatment with the GABA-A agonist muscimol, the GABA-B agonist baclofen or the GABA transaminase inhibitor gamma-vinyl GABA there were no significant changes in striatal peptide and GAD67 gene expression. Following repeated administration of the D-2 antagonists, eticlopride and haloperidol, there was an increase in enkephalin and GAD67 mRNA levels and parallel decrease in that of substance P. These were unaffected by co administration of gamma-vinyl GABA. The D-1 antagonist, SCH 23390 administered alone or together with gamma-vinyl GABA did not alter peptide or GAD67 mRNA levels. It seems that pharmacological stimulation of GABA receptors has little effect on enkephalin, substance P or GAD67 mRNA expression in striatal output neurons. PMID- 7535409 TI - Modulation of the dihydropyridine-insensitive Ca2+ influx by 8-bromo-guanosine 3':5'-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells. AB - Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 microM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca2+]i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Combined incubations with nifedipine 10 microM and 8-Br-cGMP reduced KCl-evoked intracellular Ca2+ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) under conditions of low depolarization. Neomycin (0.2 mM) and omega-AgaToxin-IVA (AgTx) (1 microM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca2+ influx pathway in the chromaffin cell. PMID- 7535413 TI - [Screening for congenital anomalies in mid-term pregnancy. Prospective epidemiologic study. Fetal trisomy]. AB - The authors have presented their experiences on prenatal screening of fetal trisomies in this second part of a prospective study between 1988 and 1990. They gained their conclusions by processing 63,496 pregnancies during three years. The results show that maternal age plays the most important role in the prenatal screening of fetal trisomies in Hungary. They recommend fetal karyotyping for every pregnant woman aged 35 years or more. They emphasized that using of a combined screening method (i.e. maternal age, serum alpha-fetoprotein, human chorionic gonadotropin, oestriol) is only permissible if the hormonal and cytogenetic laboratory background are provided under standard circumstances. Since these are not available for the vast majority of pregnant women in Hungary they concluded that, at least for the time being, the main criteria for prenatal screening of fetal trisomies is the maternal age. By applying this recommendation 25-30 percentage of Down syndrome fetuses can be detected. PMID- 7535414 TI - [Distribution, structure and transmitter content of nerve elements affecting the function of Oddi's sphincter]. AB - Nerve elements containing neuropeptides were observed by using different antisera and Avidin-Biotin-Peroxidase technique and the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a marker for nitric oxide (NO) synthase were studied in the ampulla hepatopancreatica (sphincter of Oddi) in the cat. A large amount of NPY, VIP, Substance P, somatostatin immunoreactive nerve fibers were found in all layers. Some immunoreactive nerve cell bodies (NPY, VIP, SP), were also observed in the wall. The NADPH-d stained cell bodies could be distinguished according to their size and the number of processes into two neuronal subtypes: large neurons with many dendrites and smaller, round cells with one or two processes. 99% of the cell bodies showed pozitive reactions for NADPH-d. The nerve fibers with NADPH-d activity were found in all layers, chiefly in the muscle layers. According to the distribution of the nerve fibers and the relationship to the effector cells, it is suggested, that these neuropeptides might have an important role in the function, and the NO containing nerve fibers are responsible for the nonadrenergic and noncholinergic inhibitory function. PMID- 7535415 TI - Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of src. AB - To search for the biochemical properties of cells relevant to transformation by p60v-src, we examined the activities and amounts of metalloproteinase (gelatinase) released from chicken embryonic fibroblasts infected with various mutants of Rous sarcoma virus by zymography and immunoblotting. While nontransforming Src proteins, including cellular p60c-src and its nonmyristylated form, had no stimulatory effect, wild-type p60v-src and the transforming mutant of cellular p60c-src stimulated the secretion and proteolytic activation of metalloproteinases. Moreover, the activation of metalloproteinase secretion strongly correlated with the invasiveness of cells assayed by the modified Boyden Chamber method. Chimeric mutants between v-src and c-src, which are transforming but produce less distinct morphological changes in infected cells, also stimulated the secretion of metalloproteinases as well as wild-type p60v-src. Deletion mutants of v-Src in which varying portions of the NH2-terminal half of p60v-src are deleted stimulated secretion to a level similar to that of wild-type regardless of the degree of morphological change they induce. Together with Src protein, other oncogene products including Yes, Fps, ErbB and Crk were also found to stimulate the secretion of metalloproteinases. Thus, these results suggest that transformation of cells with src and other oncogenes is closely associated with the enhanced secretion of metalloproteinases that may play an important role in tumor invasion and metastasis. PMID- 7535417 TI - The DNA binding activity of wild type p53 is modulated by blocking its various antigenic epitopes. AB - Interaction of wild type p53 with specific DNA target sequences, which is dictated by several structural domains, can be modified by blocking the different antigenic epitopes of the protein. Comparison of p53 protein expressed by recombinant bacteria (wtp53-Bac) to that produced in an eukaryotic system by a vaccinia expression vector (wtp53-Vac), indicated that only the later exhibited spontaneous DNA-binding activity. Furthermore, DNA-binding patterns of these wild type p53 proteins were affected differently by their interactions with monoclonal anti-p53 antibodies recognizing individual antigenic epitopes of the molecule. While the vaccinia derived p53 that spontaneously bound DNA is supershifted by the N'-terminal specific antibodies PAb-248, the bacterial derived p53 protein that retains this antigenic epitope but does not bind DNA spontaneously, is not affected. The C'-terminal specific PAb-421 antibodies accelerated binding of the bacterial p53 protein and modified the pattern of the interaction of the vaccinia derived p53 DNA. DNA-binding patterns generated by PAb-421 and PAb-248, suggest that either interaction of wild type p53 is dependent on modification of the p53 protein or that it interacts with cellular factors which their activity can be mimicked by PAb-421. Saturation of both types of wild type p53 with several anti p53 monoclonal antibodies directed against the wild type p53 specific epitope that maps to the N'-terminal border of the DNA-binding region, blocked specific DNA-binding. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA-binding activity. The wild type specific epitope which maps to the N'-terminal border of the DNA-binding region is neighboring the first beta-strand detected by the recent crystallographic analysis. PMID- 7535416 TI - Expression and activation of B-Raf kinase isoforms in human and murine leukemia cell lines. AB - The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms previously shown to be expressed predominantly in neural tissues. We report here that B-Raf proteins of 95 and 72 kDa are also expressed in various human and murine hematopoietic cell lines. Their relative level of expression is variable depending on the cell line examined. The highest level of expression of p95B-raf was found in UT-7 cells, a human pluripotent cell line established from a patient with a megakaryoblastic leukemia. These cells are able to differentiate toward erythroid or myeloid lineage phenotypes in presence of erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. We show that treatment of UT-7 cells with EPO, GM-CSF or stem cell factor (SCF) rapidly induces phosphorylation of p95B-raf as indicated by a shift of electrophoretic mobility. This increase in phosphorylation is correlated with a three-fold increase of B-Raf kinase activity. B-Raf activation also increases in a dose-dependent manner in response to EPO and GM-CSF. We also show that both p95B-raf and p72B-raf can be activated by IL-3 in murine BAF-3 pro-B cells and by anti-CD3 in human Jurkat cells, respectively. These observations provide the first evidence that the B-Raf kinase is involved in signal transduction pathways regulating proliferation and differentiation of hematopoietic cells of both myeloid and lymphoid lineages. PMID- 7535418 TI - [Biopolymer metabolism in connective tissue of aorta and myocardium in chronic emotional stress and stress-limiting effect of substance P]. PMID- 7535419 TI - Plasma growth hormone-binding protein activity, insulin-like growth factor I, and its binding protein levels in patients with Turner's syndrome: effect of short- and long-term recombinant human growth hormone administration. AB - Plasma growth hormone-binding protein (GH-BP) activity and the levels of IGF-I and its binding proteins (IGFBP) were studied in eight girls with Turner's syndrome before and during recombinant-hGH (r-hGH) administration. Growth hormone and GH-BP activity were assayed at baseline and hourly, over a 12-h period, after an intramuscular bolus of 0.09 mg/kg of the hormone. After 7 d, each patient received r-hGH at 0.33 mg/kg/weekly s.c. every day at nighttime; plasma growth hormone-binding protein activity, blood IGF-I, and IGFBP were evaluated before and on d 7, 30, 180, and 360. Baseline reference values were obtained from 10 bone age-matched healthy girls. Basal GH-BP activity, IGF-I, and IGFBP levels were similar in patients and controls. Four h after the intramuscular injection, GH-BP activity maximally increased and returned to baseline 6-7 h later; during long-term r-hGH administration GH-BP activity peaked at +180 d but declined to pretreatment at +360 d. IGF-I, IGFBP-3, and IGFBP-4 increased under r-hGH and, in contrast to GH-BP activity, remained high throughout the study. In conclusion, in girls with Turner's syndrome, GH-BP activity, IGF-I, IGFBP-3, and IGFBP-4 are induced by r-hGH. However, the increase of IGF-I and IGFBP-3 does not require an increased level of the cellular growth hormone receptors, as suggested by the unchanged +360 d values of plasma GH-BP activity compared with baseline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535421 TI - Ventricular- versus atrial-based lower rate timing in dual chamber pacemakers: does it really matter? PMID- 7535420 TI - Chronic neutropenia and defect in superoxide generation of granulocytes in two patients: enhancement of bactericidal capacity and respiratory burst activity by treatment with recombinant human granulocyte colony-stimulating factor. AB - We have identified two unrelated girls with chronic neutropenia [absolute neutrophil counts (ANC) 10-870 and 10-940/microL in patients 1 and 2, respectively] and severe defect in superoxide anion generation by granulocytes. Formyl-methionyl-leucyl-phenylalanine-induced superoxide release was 1.2 +/- 0.9 and 1.9 +/- 1.9% (mean +/- SEM, n = 3) of normal controls', mean value in patients 1 and 2, respectively. However, granulocytes from both patients released a normal amount of superoxide upon stimulation with phorbol myristate acetate. Patient 2 exhibited characteristic features of Duane syndrome, a rare disorder of eye movement. Treatment of the patients with recombinant granulocyte colony stimulating factor led to significant clinical improvements and reduction of infectious complications and to increases in the ANC, to 400-2100/microL in patient 1 and to 500-3000/microL in patient 2. Treatment with 5 micrograms/kg/d resulted in increased intracellular killing of opsonized Staphylococcus aureus by granulocytes and an enhancement of superoxide release upon stimulation with formyl-methionyl-leucyl-phenylalanine in both patients up to 11.1 +/- 6.0 and 13.5 +/- 7.0% (mean +/- SEM, n = 5) of normal controls', mean value in patient 1 and patient 2, respectively. These data suggested that recombinant human granulocyte colony-stimulating factor treatment enhanced resistance to bacterial infection by stimulation of superoxide generation and increasing the bactericidal capacity of peripheral blood granulocytes. PMID- 7535422 TI - Evidence for cholecystokinin receptor subtype in endocrine pancreas. AB - Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCKA receptors. CCK4 (Trp-Met-Asp-Phe-NH2) has an insulinotropic effect, but is 1000-fold less potent than CCK8. The in vitro potencies and selectivity of newly synthesized CCK4 analogs were investigated. Exchanging various a amino acids, for example Met by Nle and modifying Phe and/or Trp, led to compounds that were much more effective than CCK4 itself and show insulinotropic effects comparable with those of CCK8. Compounds that possess electron withdrawing groups on the C-terminal phenylalanine were especially effective; compounds with electron-donating groups had no effect. In contrast to CCK8 the synthetic CCK4 compounds were selective for the endocrine pancreas: they had no agonistic or antagonistic effect on the contraction of the guinea pig ileum, amylase release from isolated acini, and no major effect on the feeding behavior of mice being supplied with either compound by an implantable AlzetR pump for 8 days. The data indicate that some of the synthetic tetrapeptides exhibit a high affinity for the CCK receptor of the endocrine pancreas and that they are highly selective for this (peripheral) CCKA receptor subtype. The beta cell CCKA receptors are different from those in exocrine pancreas, smooth muscle, and those for regulating appetite; these peripheral receptor subtypes can be discriminated for the first time. PMID- 7535423 TI - Vasoactive intestinal polypeptide stimulates cyclic AMP production in mouse N1E 115 neuroblastoma cells: modulation by a protein kinase C activator and ionomycin. AB - In this study, we investigated the vasoactive intestinal polypeptide (VIP) stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca2+ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4 beta-phorbol 12 myristate 13-acetate reduced VIP- but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E 115 cells, and that although activation of protein kinase C inhibits the VIP stimulated cAMP response, elevation of intracellular Ca2+ potentiates this signaling pathway. PMID- 7535425 TI - Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP. AB - Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 +/- 2 microns and, with carbachol treatment, contracted to 89 +/- 2 microns. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC50 of 3-7 nM. Galanin (1 microM) and VIP (1 microM) increased cellular cAMP from 118 +/- 10 pmol/10(6) cells in control to 212 +/- 14 and 214 +/- 12 pmol/10(6) cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 microM, completely inhibited the relaxant effect of an EC50 concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, L-NNA (NG-nitro-L-arginine), at 100 microM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, L NNA (100 microM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535424 TI - Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release. AB - An examination of the binding characteristics of a large number of somatostatin analogues with respect to the five known somatostatin receptor subtypes has recently resulted in the discovery of several peptides with some selectivity for types 2, 3, and 4 and little affinity for type 1 or 5 receptor. A panel of these peptides has thus far implicated type 2 receptors in the inhibition of release of pituitary growth hormone and type 4 receptors in inhibiting pancreatic insulin release. In the present article, we have examined the inhibitory effects of the same group of peptides on in vivo rat gastric acid and pancreatic amylase release and binding to rat pancreatic acinar cells. The type 2-selective ligand NC-8-12 was a potent inhibitor of gastric acid release (EC50s in the 1.5 nM region) whereas the type 4-selective ligand, DC-23-99, elicited little response. However, some involvement of type 3 receptors could not be ruled out because the type 3 selective analogue, DC-25-20, exhibited inhibitory effects at higher dose levels (EC50 > 10 nM). Conversely, the type 4 analogue was a potent inhibitor of amylase release (EC50 1.1 nM) whereas the type 3 analogue had no significant effects at doses tested. DC-23-99 also bound with high affinity to rat acinar cells (EC50 3.8 nM), whereas DC-25-20 exhibited more than 10-fold less affinity. Thus, these two major biological functions of somatostatin appear to be controlled by different receptors and, furthermore, effects on both endocrine and exocrine pancreas appear to be type 4 receptor mediated. PMID- 7535426 TI - Effects of galanin on short circuit current and electrolyte transport in rabbit ileum. AB - Galanin decreased short circuit current (Isc) and increased active Na+ and Cl- absorption in rabbit ileum. In the absence of calcium, the galanin-induced decrease in Isc was inhibited by approximately 60%. Tetrodotoxin significantly reduced the effect of galanin on Isc, and tetrodotoxin and EGTA totally blocked the effect, indicating that the nonneuronal mediator of the effect is Ca2+ dependent. Galanin binding to basolateral membranes prepared from ileal epithelial cells was specific and of high affinity. These results suggest the involvement of this peptide in the regulation of intestinal epithelial cell function. PMID- 7535427 TI - Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas. AB - The peptide galanin (GAL), when injected into the rat hypothalamus, is known to stimulate feeding behavior and affect the secretion of various hormones, including insulin and the adrenal steroid, corticosterone. To determine whether endogenous peptide levels shift in relation to natural rhythms of feeding and circulating hormone levels, rats were sacrificed at different times of the light/dark cycle, and their GAL levels were measured, via radioimmunoassay, in medial hypothalamic dissections and micropunched hypothalamic areas. The results suggest the existence of two distinct diurnal rhythms for hypothalamic GAL. One rhythm, detected exclusively in the area of the SCN, is characterized by bimodal peaks of GAL, threefold higher than basal peptide levels, around the onset of the dark and light periods. The second rhythm shows a single peak of GAL towards the middle of the nocturnal feeding cycle, specifically between the third and sixth hour. This latter rhythm is evident in the dorsal region of the medial hypothalamus, localized specifically to the lateral portion of the PVN. Moreover, it is inversely related to circulating insulin but unrelated to the adrenal steroids, suggesting a possible association between this pancreatic hormone and GAL in the PVN. PMID- 7535428 TI - Distribution and relative proportions of neuropeptide Y- and proenkephalin containing noradrenergic neurones in rat superior cervical ganglion: separate projections to submaxillary lymph nodes. AB - The distribution and relative proportions of neuropeptide Y (NPY)- and [Met]enkephalyl-Arg-Gly-Leu (ME-RGL)-containing sympathetic neurones in the rat superior cervical ganglion (SCG) and their projections to submaxillary lymph nodes (SLN) were determined by retrograde tracing and immunocytochemistry. Three subpopulations of neurones were detected in the SCG: 64% contained NPY, 30% contained ME-RGL, and 6% were immunonegative for both. Immunoreactive neurones were also present inside the external carotid nerve of the SCG. An injection of Fluoro-Gold (FG) into the left SLN retrogradely labeled a few neurones in the ipsilateral SCG. FG-labeled neurones contained tyrosine hydroxylase (TH) and were either positive for ME-RGL or for NPY. FG-labeled neurones immunostained for ME RGL out-numbered by 4:1 FG-labeled neurones immunopositive for NPY. It is suggested that the sympathetic/peptidergic innervation to SLN may have distinct vasoregulatory and immunomodulatory functions. PMID- 7535429 TI - On the structure and function of platelet integrin alpha IIb beta 3, the fibrinogen receptor. AB - Platelet membrane glycoprotein (GP) IIb/IIIa (alpha IIb beta 3), a Ca(2+) dependent heterodimer, serves as an inducible receptor for fibrinogen and other adhesive plasma proteins, and is the most thoroughly studied integrin receptor. Intensive research during the past several years has elucidated the major features of its biosynthetic pathway, covalent structure, domain organization, and topography, and we are beginning to get an insight into the cellular mechanisms controlling integrin function. The emerging picture indicates that platelet-specific elements initiate at the cytoplasmic domains of GPIIb/IIIa a signal that leads to conformational changes within the integrin's extracellular domains and expression of the fibrinogen receptor. The simultaneous occupancy on adjacent platelets of receptors with dimeric fibrinogen molecules leads to platelet aggregation. Further structural alterations promote clustering of occupied GPIIb/IIIa complexes and their attachment to the remodelling cytoskeletal network. This interaction provides the physical link for clot retraction to occur and appears to regulate the compartmentalization, and local activation, of a multienzymatic complex which translates the ligand-binding information into time-dependent irreversibility of the fibrinogen-GPIIb/IIIa interaction. Platelet GPIIb/IIIa plays, thus, a central role in thrombus formation both in health and disease: abnormalities in the platelet adhesive mechanisms responsible for the formation of the hemostatic plug, lead to major pathophysiologic disorders, ranging from severe bleeding to thrombosis. It is, therefore, not surprising that GPIIb/IIIa has been the subject of intensive research during the last decades, since a detailed knowledge of the molecular biology and the mechanism underlying the platelet activation and aggregation processes may aid in the rational design of both an effective gene replacement therapy, and of potent and specific anti-thrombotic drugs. The aim of this minireview is to summarize many functional and structural data from different laboratories in the perspective of an emerging model that may help us to understand structure-function relationships of GPIIb/IIIa and of other members of the integrin family. PMID- 7535430 TI - A two-phase approach to B lymphocyte purging of autologous bone marrow grafts for patients with malignant lymphoma contaminated bone marrow. PMID- 7535431 TI - Specific capture of targeted hematopoietic cells by high gradient magnetic separation by the use of ordered wire array filters and tetrameric antibody complexes linked to a dextran iron particle. PMID- 7535432 TI - Ex vivo expansion of CD34+ hematopoietic progenitors. PMID- 7535434 TI - Separation of clinical quantities of CD34+ cells from human marrow using immunomagnetic procedures. PMID- 7535433 TI - Measurement of marrow repopulating potential of human hematopoietic progenitor and stem cells using a fetal sheep model. PMID- 7535436 TI - Selection and purification of CD34+ cells using monoclonal antibody and ferrofluids. PMID- 7535435 TI - CD34 may be used to determine the adequacy of a stem cell harvest for hematologic recovery following high dose chemotherapy. PMID- 7535437 TI - The clinical use of elutriation and positive stem cell selection columns to engineer the lymphocyte and stem cell composition of the allograft. PMID- 7535438 TI - Single and multicolor flow cytometric CD34 analysis in different cell sources. PMID- 7535440 TI - T-cell depletion from allogeneic bone marrow by counterflow centrifugation is not associated with a substantial loss of CD34-positive cells. PMID- 7535439 TI - Expression of differentiation-associated antigens and adhesion molecules on CD34 positive cells harvested from peripheral blood and bone marrow. PMID- 7535441 TI - Impact of the degree of maturation and differentiation of CD34+ cells in grafts of different origin on the duration of aplasia. PMID- 7535442 TI - Cultivation and ex vivo expansion of human CD34+ progenitor cells under serum free culture conditions. PMID- 7535444 TI - CD34 analysis in erythropoietin mobilized peripheral blood stem cells. PMID- 7535443 TI - Administration of GM-/G-CSF prior to bone marrow harvest increases collection of CD34+ cells. AB - This study compares differences in the cellularity and levels of CD34 positive cells in bone marrows from patients treated with G-/GM-CSF prior to harvest and marrows from untreated patients. The average volume of marrow aspirated was 1302mL in the untreated group containing an average of 2.6 x 10(10) nucleated cells, while an average volume of 1147mL of marrow was aspirated from patients treated with GM-/G-CSF prior to harvest which contained an average of 5.6 x 10(10) nucleated cells. Analysis of these marrows by flow cytometry revealed a higher percentage of CD34 positive cells within the lymphoid gate of marrow specimens from patients receiving GM-/G-CSF as compared with their untreated counterparts (21.4% vs. 9.1%). All patients receiving GM-/G-CSF prior to harvest were also given G-CSF subcutaneously (5 micrograms/kg/day) following the infusion of autologous marrow after high dose myelosuppressive chemotherapy and the duration of neutropenia (AGC < 500/mm3) in this group was shortened, an average of 12 days as compared to 24 days in untreated patients. This decreased duration of neutropenia is similar to that reported in patients receiving GM-/G-CSF only after transplantation (Lieschke & Burgess, 1992). Further studies are needed to determine whether the administration of GM-/G-CSF prior to bone marrow harvest is clinically beneficial. PMID- 7535445 TI - Is CD33 a differentiation marker? PMID- 7535446 TI - Ex vivo expansion of CD34+ cells from purified adult human bone marrow and umbilical cord blood hematopoietic progenitor cells. PMID- 7535447 TI - Characterization of hematopoietic stem cells in human cord blood. PMID- 7535448 TI - Expansion of human umbilical cord blood CD34+ hematopoietic progenitor cells with recombinant growth factors. PMID- 7535449 TI - Enrichment of G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation by negative selection. PMID- 7535450 TI - Comparative assays of megakaryocyte progenitors in CD34+ peripheral blood progenitor cell fractions. PMID- 7535453 TI - Yield of primed progenitor cells as determined by days of priming with recombinant human granulocyte-colony stimulating factor. PMID- 7535452 TI - A retrospective comparison of three different methods for mobilization of peripheral blood stem cells. PMID- 7535451 TI - Comparison of three mobilization regimens for peripheral blood progenitor cell rescue after high dose chemotherapy. PMID- 7535454 TI - Peripheral blood mononuclear mobilization with sargramostim (GM-CSF). PMID- 7535455 TI - Combined therapy with rG-CSF and GM-CSF for the mobilization of hematopoietic progenitor cells. PMID- 7535456 TI - Kinetics of low and high dose granulocyte/macrophage colony stimulating factor mobilization of peripheral blood stem cells. PMID- 7535458 TI - High-dose therapy and growth factor mobilized peripheral blood progenitor cell support for responding patients with metastatic breast cancer. PMID- 7535457 TI - Comparative analysis of peripheral stem cells collected and transplanted in combination with granulocyte-macrophage and granulocyte colony-stimulating factors. PMID- 7535459 TI - Quantitative assessment of colony growth and hematologic engraftment in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation for breast cancer. PMID- 7535460 TI - High-dose chemotherapy for peripheral stem cell procurement in leukemia, lymphoma and multiple myeloma. PMID- 7535461 TI - Peripheral blood stem cell rescue in children with advanced stage neuroblastoma. PMID- 7535462 TI - Selective separation of cells using magnetic colloids. AB - We have developed a reliable, purely immunological means of quantitatively labeling cells with magnetic colloidal dextran iron. Labeled cells can be efficiently separated with a high gradient magnetic filter inside a magnetic field. Separation conditions and filter design can be changed to accommodate large and small scale positive or negative selection. This separation technique has been applied to tumor cell depletions, stem cell enrichment, and large scale preclinical T-cell depletions. PMID- 7535464 TI - Quality assurance in flow cytometry: comparison of three analytical methods for quantifying CD34+ cells in marrow. PMID- 7535463 TI - A semi-automated process for antibody sensitization and washing of mononuclear cells prior to CD34+ cell selection. PMID- 7535465 TI - Quality control for peripheral stem cell collection: the Ottawa experience. PMID- 7535466 TI - 4-Hydroperoxycyclophosphamide purged autologous bone marrow transplantation of relapsed, responding non-Hodgkin's lymphoma with granulocyte colony stimulating factor support results in reliable hematopoietic recovery. PMID- 7535467 TI - Purging tumor cells from bone marrow or peripheral blood using avidin-biotin immunoadsorption. PMID- 7535468 TI - Reinforcing properties of the neuropeptide substance P in Carassius auratus: evidence of dopaminergic system involvement. AB - The aim of the present study was to investigate whether neuropeptide substance P (SP) has reinforcing effects in Carassius auratus and whether this effect could be related to dopaminergic neurotransmission. For this purpose fishes were put in a three-compartment box in which one compartment gave access to two others that did not directly link. The time spent in each compartment was registered for 10 min to determine a possible preferred compartment. Twenty-four hours later, the fish were given one of the following intraperitoneal treatments: a) Group VEH, injected with the vehicle of substance P; b) Group SP25, injected with SP, 25 micrograms/kg body wt.; c) Group SP50, injected with SP, 50 micrograms/kg; or d) Group HALO, injected with haloperidol (2 mg/kg) 30 min before an injection of SP (50 micrograms/kg). Immediately after the treatment the fish were kept for 30 min in the compartment preferred least the day before. On the next day the fish were retested for 10 min to verify the time spent in each compartment. The results indicate that SP at the dose of 50 micrograms/kg enhanced the time spent on the paired compartment, and that the pretreatment with haloperidol abolished this enhancement. It is suggested that SP has reinforcing effects in C. auratus that may be mediated by the dopaminergic system. PMID- 7535470 TI - A new conductivity staining method for tissue analysis under scanning electron microscope. AB - A conductivity staining method for tissue samples were developed using a solution of 10 ml of 5% carbolic acid solution, 2 g of tannic acid, and 10 ml of saturated aluminium sulfate 12-hydrated after fixation with osmium. Specimens of kidney (mouse) with the above treatment can be analyzed under a scanning electron microscopy without the charging phenomenon. PMID- 7535471 TI - Allergen-induced changes of B-cell phenotypes in patients with allergic rhinitis. AB - We investigated sub-populations of B-lymphocytes in nasal mucosa and peripheral blood of 17 patients with seasonal allergic rhinitis (birch pollen) and 10 controls. The study included provocation with allergen during the non-pollen season, during which no participant used medication. Samples were also taken during the pollen season. Subsets of B-cells as expressed by different CD antigens were investigated by immunohistochemistry on frozen sections and by flow cytometry of peripheral blood. Nasal CD23+ B-cells decreased in allergic patients during provocation, indicating that mature virgin CD23+ B-cells switch into a memory B-cell phenotype with loss of CD23 expression. This indicates differentiation towards cells that can represent a local source for IgE synthesis. No decrease was observed during the pollen season when the patients used medication. Serum IgE was significantly higher in allergic patients on all occasions. The observed up-regulation of CD40 expression on peripheral blood B cells in allergic patients during the pollen season clearly indicate B-cell activation. Furthermore, a relative increase of CD19+ B-cells was observed in peripheral blood during provocation. Upregulation (by IL-4) of CD40 on B-cells which then may be stimulated by gp39 (CD40 ligand) can constitute an early and important event in the IgE-mediated allergic reaction. PMID- 7535469 TI - p-Chloroamphetamine (PCA), 3,4-methylenedioxy-methamphetamine (MDMA) and d fenfluramine pretreatment attenuates d-fenfluramine-evoked release of 5-HT in vivo. AB - Previous work has suggested that repeated treatment with substituted amphetamines including PCA, MDMA and d-fenfluramine produces a persistent neurodegeneration which is relatively selective for the fine serotoninergic terminals arising from the dorsal raphe nucleus. The aim of the present study was to investigate whether the acute releasing effect of d-fenfluramine might also be sensitive to lesions produced by PCA, MDMA and d-fenfluramine itself. Basal and 5-HT release evoked by d-fenfluramine or 100 mM KCl was measured by microdialysis in frontal or parietal cortex of rats 2 weeks after they had been treated with a neurodegenerative regime of PCA, MDMA, d-fenfluramine, or vehicle. In frontal cortex of vehicle controls, d-fenfluramine (10 mg/kg IP) and KCl (100 mM via microdialysis probe) evoked an increase in 5-HT of 1740% and 779% of basal, respectively. PCA pretreatment reduced d-fenfluramine-evoked 5-HT release by 90.9% while potassium evoked release was reduced by only 66.8%. Similar results were obtained in parietal cortex. MDMA (20 mg/kg x 8) and d-fenfluramine (1.25 mg/kg x 8) pretreatment reduced d-fenfluramine-evoked release of 5-HT in frontal cortex by 45.2% and 72.0%, respectively. Overall, the present data are consistent with the hypothesis that the acute release of 5-HT evoked by d-fenfluramine occurs via those terminals destroyed by pretreatment with PCA, MDMA and d-fenfluramine, while KCl evokes release from both PCA-sensitive and PCA-insensitive terminals. The significance of these results for the interpretation of neuroendocrine data from d-fenfluramine challenge tests is discussed. PMID- 7535472 TI - The interiorized environment. AB - Biological reactions occur regularly at the right time, on the right place and in the right quantity. How does this biological order work? Mechanicistics and vitalists fail to explain it and holism is an alternative that misses the very point. Our investigation is restricted to the relation between the organism and its specific environment, stressing their mutual penetration, common to twins. The twin organisms/environment in organisms as a whole, in the cellular environment of DNA and around the biological clock. PMID- 7535473 TI - Intercellular interactions in PC12 cells overexpressing beta/A4 amyloid. AB - The amyloid precursor protein (APP) is an integral membrane component of eukaryotic cells. A variety of research approaches have addressed the contribution of the beta amyloid peptide region of the APP to neuritic plaque structure and formation in the Alzheimer disease brain as well as the relationship between beta amyloid accumulation and the occurrence of dementia. However, there is limited information available concerning the cellular consequences of amyloid deposition. The present studies were undertaken to investigate the relationship between beta amyloid and intercellular junctions. Transfected PC12 cell lines, that overexpress the beta amyloid peptide, exhibit structural and functional alterations at the cell surface and tend to form aggregates more readily than normal control cells. Intermediate junctions were the most common intercellular interactions of both normal and transfected cells. However, the control and transfected cells differed since areas of continuous and extensive junctions were readily seen in transfected cells and infrequently seen in control cells. The data suggest that excess accumulation of beta amyloid is associated with the junctional apparatus and may be related to increased intercellular adhesion. PMID- 7535474 TI - Glycol methacrylate embedding and microwave staining for light microscopy of the mouse cochlea. AB - This study examined the utility of a methacrylate-based embedding medium and microwave staining for light microscopic quantification of hair cells and spiral ganglion cells in the mouse cochlea. The most important phase of the preparation process involved slowing down the polymerization process. The tissue molecules so locked within the plastic matrix produced excellent preservation of the organ of Corti and adjacent structures including the spiral ganglion, as well as tissue ionic charges. Excitable by microwaves, these ionic charges accelerated the movement of the basic dye (hematoxylin) into the tissue, reducing the time for this segment of the staining process from approximately 45 minutes to 1-2 minutes. When embedded in glycol methacrylate (GMA), acidic dyes show less stain cell affinity so that staining intensity and time cannot be improved significantly. However, addition of color extenders to the counterstain eosin produced distinguished staining of all tissue constituents. Thus, a combination of GMA embedding medium, use of the microwave for staining and addition of color extenders to the counterstain generated excellent structural resolution and contrast. This made both hair cell and spiral ganglion cell counts possible from within the same specimen and provided an opportunity for qualitative evaluation as well. PMID- 7535476 TI - Antigen-specific development of primary and memory T cells in vivo. AB - The expansion and contraction of specific helper T cells in the draining lymph nodes of normal mice after injection with antigen was followed. T cell receptors from purified primary and memory responder cells had highly restricted junctional regions, indicating antigen-driven selection. Selection for homogeneity in the length of the third complementarity-determining region (CDR3) occurs before selection for some of the characteristic amino acids, indicating the importance of this parameter in T cell receptor recognition. Ultimately, particular T cell receptor sequences come to predominate in the secondary response and others disappear, showing the selective preservation or expansion of specific T cell clones. PMID- 7535477 TI - Digestion in the premature infant: the effects of human milk. PMID- 7535475 TI - Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. AB - The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development. PMID- 7535478 TI - Fatal sepsis due to Pseudomonas cepacia. AB - Pseudomonas cepacia is known as an opportunistic pathogen in the patients with altered host defence. We report a case of hospital-acquired fatal sepsis due to P. cepacia in a child with no known defect in immune system. Virulence of the organism was indicated by the high magnitude of septicaemia. The organism was successfully eliminated by ceftriaxone but the patient died of renal failure. PMID- 7535479 TI - Reinforcement of cyclosporin A-induced red cell destruction by superoxide. AB - To determine the mechanism of hemolysis following organ transplantation, we studied the effect of immunosuppressants and/or superoxide (SO) on the in vitro destruction of red cells. The immunosuppressants tested included cyclosporin A (CyA), deoxyspagarine (DSG), and FK506. SO was obtained from the hypoxanthine xanthine oxidase reaction. Of the three immunosuppressants studied, only CyA affected the size of red cells and directly produced hemolysis in an isotonic buffer without the involvement of an immune mechanism. In addition, SO and CyA showed a synergistic effect on hemolysis during prolonged incubation. Catalase and allopurinol prevented hemolysis by counteracting the activity of SO. In that SO is produced in excess during the recovery of blood flow after organ transplantation, the prolonged contact of red cells with CyA and SO may be involved in the development and reinforcement of hemolysis in vivo. PMID- 7535480 TI - 5 alpha-reductase inhibition by finasteride (Proscar) in epithelium and stroma of human benign prostatic hyperplasia. AB - Finasteride is a specific 5 alpha-reductase inhibitor that has been shown to reduce the size of human benign prostatic hyperplasia (BPH) by inhibiting the intraprostatic conversion of testosterone to 5 alpha-dihydrotestosterone. The aim of the present in vitro study was to describe in more detail the inhibitory effect of finasteride on 5 alpha-reductase in epithelium and stroma of human BPH. 5 alpha-Reductase activity in epithelium and stroma was inhibited dose dependently by finasteride. The mean IC50 (50% inhibitory concentration) values, determined in the presence of various testosterone concentrations, were generally 2- to 4-fold lower in epithelium than in stroma. With finasteride concentrations greater than 5 nM, competitive inhibition of 5 alpha-reductase occurred both in epithelium and stroma. The mean inhibition constant Ki[nM +/- SEM] was 7 +/- 3 and 31 +/- 3 in epithelium and stroma, respectively. In the presence of finasteride concentrations < or = 5 nM, the epithelial 5 alpha-reductase seems to be inhibited in an uncompetitive manner, whereas such low finasteride concentrations cause either no inhibition (1-2 nM) or competitive inhibition (5 nM) in stroma. Our present study provides evidence that the inhibitory effect of finasteride on 5 alpha-reductase is much stronger in epithelium than in stroma. Therefore, it is conceivable that the global size-reduction of BPH under finasteride treatment is primarily due to the regression of BPH epithelium. PMID- 7535481 TI - Quantitation of CD62, soluble CD62, and lysosome-associated membrane proteins 1 and 2 for evaluation of the quality of stored platelet concentrates. AB - BACKGROUND: Platelets become activated during storage, which results in secretion of granules, vesiculation of microparticles, secretion of protein, and a number of other biochemical and morphologic processes that decrease the utility of platelet concentrates stored for transfusion. STUDY DESIGN AND METHODS: To evaluate the quality of stored platelet concentrates, the cell surface expression of specific activation-dependent antigens (CD62 and lysosome-associated membrane proteins 1 and 2 [LAMP-1, LAMP-2]) on platelets stored in a hospital blood bank over a 7-day period was examined. Relative microparticle counts and the expression of CD62 by microparticles, as well as platelet concentrate supernatant levels of soluble CD62, were determined. RESULTS: The percentage of platelets expressing CD62 increased significantly from Day 1 to Day 5 (p < 0.05) of storage; the mean fluorescence values for CD62 did not. In contrast, the mean fluorescence values of LAMP-1 and LAMP-2 rose significantly (p < 0.01 and p < 0.05, respectively) between Days 1 and 5. Significant declines in CD62, LAMP-1, and LAMP-2 percent expression and mean fluorescence were seen on Day 6 of storage (p < 0.001). Microparticle numbers increased significantly during storage and correlated with levels of CD62 protein (free and membrane-bound) (r = 0.95 vs. Day 2, p < 0.05; r = 0.88 vs. Day 5, p < 0.05). CONCLUSION: Flow cytometric evaluations of the expression of cell surface CD62, LAMP-1, and LAMP-2 are complementary tests that, especially when used in conjunction with the quantitation of CD62 protein, provided a simple and effective means of evaluating the quality of platelet concentrates stored for transfusion. PMID- 7535482 TI - Major infectious complications after orthotopic liver transplantation and comparison of outcomes in patients receiving cyclosporine or FK506 as primary immunosuppression. AB - A retrospective cohort study was conducted to determine the incidence of major infectious complications after orthotopic liver transplantation and to compare outcomes in patients receiving either cyclosporine (CsA) or FK506 (tacrolimus) as primary immunosuppression. Of 133 transplants performed in 118 patients, 124 transplant episodes were evaluated. Cytomegalovirus (CMV) infection (INF) and disease (DIS), deep fungal infection (DFI), and intraabdominal bacterial infections (IAI) were catalogued. The overall incidences of major infectious outcomes were: CMV INF = 33%; CMV DIS = 19%; DFI = 15%; and IAI = 25%. Cox proportional hazard analysis identified donor seropositivity, OKT3 as secondary immunosuppression and initial intensive care unit (ICU) duration as risk factors for CMV INF and DIS in the overall population. Fungal colonization was the dominant risk factor associated with deep fungal infection. A choledochojejunostomy anastomosis, the number of cellular blood products transfused at the time of transplantation surgery, and prior CMV INF were independent risk factors for both fungal colonization and deep infection. The single risk factor identified for intraabdominal bacterial infections was the number of cellular blood products transfused at the time of surgery. In the Cox proportional hazards model the relative risk (RR) for each category of infection was lower in the FK506 group (CMV: RR = .87, 95% confidence interval [C.I.] = [.32-2.4]; DFI: .58 [.13-2.6]; IAI: .51 [.15-1.7]), but the effect was not statistically significant. Survival was similar in patients receiving FK506 or CsA. CMV INF and DFI were independent predictors of death for all patients. Risk factors identified for CMV INF and DIS support the findings of others. Higher intraoperative blood product requirements and complicated intraoperative or postoperative courses increase the risk for IAI or DFI. The development of effective strategies to prevent CMV and fungal infections in liver transplant recipients remains a priority for future endeavors. PMID- 7535483 TI - Genetics and molecular biology of Huntington's disease. AB - In 1993, the genetic abnormality responsible for Huntington's disease was identified as a trinucleotide-repeat expansion in a novel gene. Much has been learned about the molecular genetics of Huntington's disease and the possible effects of the trinucleotide expansion in the development of this disease and other neurological disorders. The Huntington's disease locus is widely expressed throughout the brain and in many non-neural tissues. Current speculation about the pathogenesis of neuronal death concentrates on a 'gain of function' effect in which the abnormal protein has acquired a new and lethal property. Future research will define the normal function of the Huntington's disease locus, test hypotheses regarding the putative gain of function, and explore the factors that determine neuronal susceptibility to the effects of the abnormal allele. PMID- 7535485 TI - Visual ecology and voltage-gated ion channels in insect photoreceptors. AB - That particular membrane conductances are selected for expression to enable the efficient coding of biologically relevant signals is illustrated by recent work on insect photoreceptors. These studies exploit the richness of insect vision and the accessibility of insect photoreceptors to cellular analysis in both intact animal and isolated cell preparations. The distribution of voltage-gated conductances among photoreceptors of different species correlates with visual ecology. Delayed-rectifier K+ channels are found in the rapidly responding photoreceptors of fast-flying flies. The conductance's activation range and dynamics match light-induced signals, and enable a rapid response by reducing the membrane time constant. Slow-moving flies have slowly responding photoreceptors that lack the delayed rectifier, but express an inactivating K+ conductance that is metabolically less demanding. Complementing these findings, locust photoreceptor membranes are modulated diurnally. The delayed rectifier is exhibited during the day and the inactivating K+ current is exhibited at night. Insect photoreceptors also demonstrate the amplification of signals by voltage gated Na+ channels. In drone-bee photoreceptors, voltage-gated Na+ channels combine with K+ channels to enhance the small transient signals produced by the image of a queen bee passing over the retina. This subthreshold amplifier operates most effectively over the range of light intensities at which drones pursue queens. PMID- 7535486 TI - Monotremunculi and brain evolution. PMID- 7535484 TI - Neurotrophic factors in diabetic neuropathy. PMID- 7535487 TI - Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. AB - The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization. PMID- 7535489 TI - Compensatory plasticity and sensory substitution in the cerebral cortex. AB - Cats deprived visually from birth show few overt impairments in their natural behavior. Therefore, they seem well suited as an animal model for the study of compensatory plasticity after early vision loss. It can be demonstrated that binocularly deprived cats show improved abilities of auditory localization, and at least equal tactile behavior compared to normal controls. Within the anterior ectosylvian cortex of binocularly deprived cats, where different sensory modalities come together, the anterior ectosylvian visual area is completely taken over by auditory and somatosensory inputs. Furthermore, the auditory spatial tuning of single units in this cortical region is sharpened significantly as a result of visual deprivation. Somatosensory compensation for early loss of vision can be demonstrated by a hypertrophy of the facial vibrissae, and a corresponding expansion of their central representation in the somatosensory cortex of binocularly deprived animals. The compensatory changes in the cortex can be explained by a reorganization of sensory representations under the guidance of sensorimotor feedback rather than by instruction through an extraneous 'supervisory' signal. These processes might form the neural basis of sensory substitution in blind humans. PMID- 7535488 TI - The cellular basis of classical conditioning in Aplysia californica--it's less simple than you think. AB - Classical conditioning of the withdrawal reflex of the marine snail Aplysia californica can be used as an important model system for investigating the neurobiology of associative learning. It results when weak tactile stimulation of the snail's mantle shelf or siphon is repeatedly paired with strong electrical shocks to the animal's tail. This learned behavioral change is thought to be mediated by a presynaptic neuronal mechanism-activity-dependent presynaptic facilitation of the connections between sensory and motor neurons in the CNS of Aplysia. Recent evidence suggests, however, that another type of synaptic plasticity-Hebbian potentiation of the sensorimotor connections-might contribute to classical conditioning in Aplysia. Additional evidence indicates that this relatively simple form of learning is likely to be mediated by multiple neuronal mechanisms. PMID- 7535490 TI - Do antidepressants stabilize mood through actions on the hypothalamic-pituitary adrenocortical system? AB - Patients suffering from severe depression often show an increased activity of the hypothalamic-pituitary-adrenocortical (HPA) system, a premature escape from the cortisol suppressant action of dexamethasone, and a number of other neuroendocrine changes. This might be explained by defective glucocorticoid feedback inhibition. Normalization of the hyperactive HPA system occurs during successful antidepressant pharmacotherapy of depressive illness, and this could be achieved by antidepressant-induced increases in the cellular corticosteroid receptors, rendering the HPA system more susceptible to feedback inhibition by cortisol. Both mineralocorticoid- and glucocorticoid-receptor mRNA levels and hormone-binding activities are found to be increased following treatment of different cell lines or animals with antidepressants. Since the timecourse of antidepressant actions on corticosteroid receptors follows more closely that of clinical improvement of depression, antidepressants might elevate mood in depressives through their long-term effects on HPA regulation. PMID- 7535491 TI - [Centrioles can replicate in cultured cells in the absence of a nucleus]. AB - Cytoplasts of cultured L fibroblasts, obtained by enucleation at the end of G1 of synchronized cells collected in mitosis, have usually two centrioles. Starting from 8 h after enucleation, the centrioles in all cytoplasts were seen replicating. The formed procentrioles had an average length of 0.2 micron and did not grow within the next 16 h. When the same synchronized L cells were treated from the end of G1 with actinomycin D, inhibiting more than 96% of RNA synthesis, procentrioles were not observed for about 16 h of incubation. It is concluded that centrioles in cultured cells could replicate in the absence of RNA synthesis, and that the nucleus may not only regulate the centriole replication, but also suppress this process. PMID- 7535492 TI - [Monoclonal antibodies to the HNK-1 antigen of human natural killers]. AB - The ability of monoclonal antibodies (MAb) to HNK1 antigen of natural killers (NK), previously purified by affinity chromatography, to react with human blood lymphocytes has been studied by flow cytometry. The results obtained point to some cells, among blood lymphocytes, which have antigenic determinants to these MAbs. A decrease in the NK cell cytotoxic activity was shown after addition of MAb in comparison with the control. This enables us to suggest a reactivity of these MABs to the CD57 leucocyte differential antigens. PMID- 7535493 TI - [Future treatment of prostatic hypertrophy]. PMID- 7535494 TI - [The conservative and surgical treatments of patients with Leriche's syndrome]. PMID- 7535495 TI - [A possible role of the family of macroglobulins in cancer]. PMID- 7535497 TI - Preparation and properties of a therapeutic inter-alpha-trypsin inhibitor concentrate from human plasma. AB - Inter-alpha-trypsin inhibitor (ITI) is a serine protease inhibitor found in human plasma. Its antiprotease activity is due to bikunin which is effective in various types of experimental shock and pancreatitis. Therefore ITI, which releases bikunin by proteolytic cleavage, could be of therapeutic interest. A method for the large-scale isolation of ITI from human plasma is described. ITI was purified from the prothrombin complex concentrate (PCC) by diethylaminoethyl-Sepharose fast-flow chromatography followed by a chromatographic step on immobilized heparin designed to remove C4, factor X and protein C. With this procedure, which was performed under mild conditions, a homogeneous preparation of native ITI was obtained, as demonstrated by electrophoretic and chromatographic analyses. ITI maintained its biological activity, as exhibited by its specific antitryptic activity of 420 +/- 65 IU/g. In order to decrease or eliminate the risk of transmission of viral disease due to lipid-enveloped viruses, the process incorporated a solvent-detergent treatment. Animal studies on the final product revealed no adverse side-effects in terms of toxicity, thrombogenicity or hypotension. This preparation appears suitable for therapeutic evaluation in animal experimental models. PMID- 7535496 TI - [Effects of infusion therapy on immunity and calcium contents in patients with stomach cancer]. AB - The humoral factor of non-specific immunity was shown to be inhibited significantly in gastric cancer patients. According to an analysis of immunologic status, phagocytic activity and blood plasma level of lysozymes increased during treatment of stomach cancer, thus pointing to favorable course of postoperative period. PMID- 7535498 TI - Circulating stem cell collection in lymphoma and myeloma after mobilization with cyclophosphamide and granulocyte colony-stimulating factor for autologous transplantation. AB - We report the results of 72 leukapheresis procedures performed for autologous peripheral blood stem cell collection in 18 patients with lymphoma and myeloma, after combined mobilization with cyclophosphamide and granulocyte colony stimulating factor (G-CSF). The numbers of mononuclear cells (MNCs), CD34+ cells and granulocyte-macrophage colony-forming units (CFU-GM) either in the peripheral circulation (preleukapheresis sample) or in the product obtained from leukapheresis (leukapheresis sample) were evaluated. A highly superior proportion of CD34+ cells (14-fold) and CFU-GM (5-fold) resulted from the mobilization therapy. CFU-GM and CD34+ cells were highly enriched with respect to all MNCs (relative recoveries: 2.13, range 0.3-41, and 1.08, range 0.2-8.5, respectively) due to an additional mobilization effect by the leukapheresis procedure. Also, a relatively strong linear correlation between the three different parameters was found in the leukapheresis product (CD34+:CFU-GM, r = 0.81; MNCs:CD34, r = 0.69; MNCs:CFU-GM, r = 0.75; CFU-GM:CD34+, and MNCs, r = 0.85). Our data suggest that the number of MNCs and CD34+ cells obtained after combined mobilization with cyclophosphamide and G-CSF can be used as predictor of the number of granulomonocytic progenitors. PMID- 7535499 TI - Detection of HIV-1, HBV and HCV antibodies in blood donors from Surat, western India. PMID- 7535500 TI - Effect of dry-cow therapy on subclinical mastitis--an evaluation of long-acting and short-acting intramammaria. AB - This field study was conducted to evaluate the effect of selective dry-cow therapy with long-acting and short-acting antibiotics, respectively, and also in comparison to control groups without antibiotic treatment. A total of 684 cows from 288 different herds in three Norwegian regions fulfilled the criteria of the study design. There were 104 cows in control group A (sampling only), 115 cows in control group B (placebo), 221 cows treated with long-acting intramammaria Benestermycin vet. 'Leo' for 1 day at drying off in group C, and 244 cows treated with four short-acting intramammaria Leocillin with Dihydrostreptomycin vet. 'Leo' every second day before drying off in group D. The overall effect, measured as the cow being healthy after therapy, was 14.2% in control groups and 33.7% in therapy groups 30 +/- 17 days into the next lactation. Of quarters infected with S. aureus both in late lactation (45 +/- 32 days before drying off) and at drying off, 38.4% in the control group were bacteriologically negative 30 +/- 17 days into the next lactation, compared with 49.5% in the long-acting group and 68.6% in the short-acting group. Of quarters infected with Str. dysgalactiae both in late lactation (45 +/- 32 days before drying off) and at drying off, 10 out of 27 were still infected with Str. dysgalactiae in the control group 30 +/- 17 days into next lactation, compared with 0 out of 31 in the therapy groups. Dry-cow therapy in coagulase-negative Staphylococcus spp. (CNS)-infected quarters led to a 5.2 odds ratio of being healthy quarters 30 +/- 17 days into the next lactation, compared with control groups. Despite this, the overall frequency of CNS in the material was unchanged after therapy compared with controls. Short acting compared to long-acting preparations had a significantly better effect in preventing new infection with S. aureus or Str. dysgalactiae in untreated healthy quarters in cows with fewer than three infected quarters. This difference in preventive effect was greater in cows with one infected quarter during previous lactation (the new infection rates being 0.078 for short-acting and 0.149 for long-acting) than in those with two infected quarters (the new infection rates being 0.042 and 0.063, respectively). PMID- 7535501 TI - Immunohistochemical demonstration of the expression of neurofilament proteins in Merkel cells. AB - The presence of immunoreactive neurofilament proteins has previously been reported in Merkel cell carcinomas but not in normal human epidermal and dermal Merkel cells. We have studied the immunoreactivity of epidermal Merkel cells for neurofilament triplet proteins (68 KD, 70 KD, 160 KD, 200 KD), using epidermal sheets prepared from the plantar skin of human adults, which enabled us to survey large numbers of Merkel cells. Neurofilament protein 200 KD-positive cells were readily identified, while neurofilament protein 68 KD-, 70 KD- and 160 KD positive cells were largely absent. 200 KD-positive cells in the epidermis were confirmed to represent Merkel cells by a sequential immunoenzyme labeling for the simple epithelial type cytokeratin (No. 8). 200 KD-positive cells were 5.9% of the total number of epidermal Merkel cells. Despite a heterogeneous expression of neurofilament protein subspecies between the normal and transformed Merkel cells, the presence of neurofilament proteins in epidermal Merkel cells may link them to Merkel cell carcinomas. PMID- 7535502 TI - The effect of a hydrocolloid occlusive dressing (DuoDERM E) on keratinization in psoriasis vulgaris. PMID- 7535504 TI - Changes in Timm staining of the dentate molecular layer. PMID- 7535503 TI - Reinnervation by axon collaterals from single facial motoneurons to multiple muscle targets following axotomy in the adult guinea pig. AB - To study the process of recovery from facial palsy experimentally, the location of cranial motoneurons supplying the posterior belly of the digastric muscle (PDG) and the extratemporal portion of the facial nerve trunk was examined in a double-labeling paradigm using two retrograde tracers in the adult guinea pig of which the facial nerve had been surgically injured. In different stages after the induced facial palsy had recovered functionally (4-13 weeks after the surgical operation), wheat germ-agglutinated horseradish peroxidase (WGA-HRP) was injected into the PDG and Fluoro-Ruby (FR) was applied to the proximal cut end of the extratemporal portion of the facial nerve trunk. Distribution of neurons retrogradely labeled with WGA-HRP and/or FR was plotted in the brainstem and compared with that of the controls. In the intact cases, HRP-labeled neurons were restrictedly seen in the accessory facial nucleus (Acs7), while FR-labeled neurons were found within the main facial nucleus (FMN). In the axotomized cases: (1) HRP-labeled neurons were seen diffusely in the Acs7 as well as in the FMN, where normal myotopical representation no longer seemed to be maintained. (2) FR labeled neurons were also observed diffusely in the FMN and the Acs7. (3) A considerable number of neurons were doubly labeled with WGA-HRP and FR in both the Acs and the FMN in cases with shorter survival periods (4-7 weeks), but not in cases with longer survival periods (12-13 weeks). Thus, new findings show that connections are temporarily maintained by single, facial motoneurons with axon collaterals to multiple muscle targets in adult mammals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535505 TI - Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production. AB - The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of L-NOARG was completely reversed by L-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. L-NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 M HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO2. In vessel segments in which the endothelium had been removed beforehand 15% CO2 induced relaxation that was not different from that seen in vessels with intact endothelium. L-NOARG had no affect in endothelium denuded vessels. The results suggest that high CO2 elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by L-NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall. PMID- 7535507 TI - [Histological study of allergic conjunctivitis--1. Study on the adhesion molecules to allergic conjunctivitis]. AB - We performed an immunohistochemical study on the relationship between inflammatory cells and adhesion molecules to investigate inflammatory reaction in allergic conjunctivitis. Guinea pigs were sensitized with an intraperitoneal injection of ovalbumin with hydroxyalminium. Ovalbumin was applied topically to the conjunctiva 3 weeks after the initial treatment. Then the cornea, conjunctiva and lacrymal gland were taken and processed for frozen sections. The specimens were stained with acid Giemsa for inflammatory cells and enzyme-labelled antibody for ICAM-1 and VCAM-1. Neutrophils were dominantly observed 6 hours after the challenge with the antigen, eosinophils at 12 hours, and eosinophils and lymphocytes at 24 hours, respectively. ICAM-1 and VCAM-1 were detected at 6 and 24 hours in the vascular tissue in the subconjunctival tissue and lacrymal gland. There results suggest that adhesion molecules play a role in the infiltration of inflammatory cells in allergic conjunctivitis. PMID- 7535506 TI - The comparative estimation of proteolytic and amylolytic activity in pancreatic preparations. PMID- 7535509 TI - [Protective effect of alpha-tocopherol in retinal light damage of pigmented rabbits--evaluation by nitro blue tetrazolium staining]. AB - Free radicals are thought to be one of the causes of retinal light damage. Experiments were performed to identify the production of free radicals in light exposed retinas of rabbits using nitro blue tetrazolium (NBT) staining. In addition, a free radical scavenger, alpha-tocopherol, was administered systemically before light exposure to evaluate the protective effect against the retinal light damage. Pigmented rabbits were exposed to xenon light without long waves for 60 minutes. The eyes were enucleated immediately after light exposure, and incubated in the equilibrated media at 37 degrees C with 0.1 mM NBT solution for 60 minutes. After incubation, the eyes were embedded in paraffin, sectioned, and mounted without further staining. The same procedure was done to the tocopherol treated retinas. In the light exposed eyes, the NBT staining was markedly observed at the inner retina, and the staining was more remarkable than that of the non-exposed eyes. The tocopherol treated eyes showed weaker NBT staining in the inner retina than the non-treated eyes. The results suggest that free radicals are increased in the inner retina by light exposure and alpha tocopherol suppresses production of free radicals. PMID- 7535508 TI - [Observation of retinal vasculature in experimental renal hypertension rat using bis-benzimide]. AB - The retinal vessels of experimental renal hypertensive rats (RHRS), spontaneously hypertensive rats (SHRS), and normotensive Wistar-Kyoto rats (WKYS) were studied using bis-benzimide vital staining. In the bis-benzimide stained flat retinal preparations (B-FRP) of SHRS, it was only observed that the endothelial nuclei were elongated and their arrangement was irregular. In B-FRPS of RHRS, the arrangement was more irregular and the nuclei disappeared at the point where the caliber irregularity was marked. The nuclei of smooth muscle cells, which were arranged circumferentially, and the perivascular tissues were clearly identified. This finding suggests that hyperpermeability or disruption of the blood-retinal barrier did not occur in the mild hypertension but in the severe hypertension. The retinal vessels of RHRS in the regressive stage also showed seved the same kinds of findings in electron microscopy. PMID- 7535511 TI - Mutations in the sequence flanking the microsatellite at the KAP8 locus prevent the amplification of some alleles. AB - The microsatellite described for the glycine- and tyrosine-rich keratin locus (KAP8) in sheep was used to type nine large three-generation families comprising the AgResearch International Mapping Flock. The apparent non-Mendelian inheritance in these families suggested that some of the microsatellite alleles were not being amplified. Sequence analysis showed that a deletion within one of the published primer sites was the likely cause of the 'null' alleles. By redesigning the primer all alleles could be amplified. PMID- 7535510 TI - Mediastinal teratoma and precocious puberty in a boy with mosaic Klinefelter syndrome. AB - We describe a boy who developed precocious puberty resulting from chorionic gonadotropin produced by a mediastinal germ cell tumor. Following tumor removal he began spontaneous precocious sexual development which was treated and then arrested spontaneously. Investigation of this arrested puberty established that he had Klinefelter syndrome (KS) mosaicism. He represents the first instance of KS mosaicism reported with a mediastinal germ cell tumor, a neoplasm commonly reported in males with a 47,XXY karyotype. We recommend that all males with KS and early sexual development or with "normal" testicular growth be screened with measurement of germ cell tumor markers including beta-subunit of human chorionic gonadotropin and alpha-fetoprotein. PMID- 7535512 TI - [Topical tretinoin in the treatment of lichen planus and leukoplakia of the oral mucosa. A biochemical evaluation of the keratinization]. AB - In earlier work, we demonstrated that 0.1 p. 100 topical tretinoin is clinically effective and well tolerated compared with placebo for the treatment of oral leukoplakia and oral keratosic or erythematous lichen planus. Here we aimed to complete this clinical protocol with histological and biochemical analyses comparing the biopsy specimens collected at inclusion and those collected after 4 months of treatment. Histological results were based on changes in keratinization observed between onset of treatment and 4 months treatment. Biochemical studies included the use of antibodies (anti-cytokeratins 10-11, anti-filaggrine) for the immunohistochemical evaluation of keratinization and 2-dimensional gel electrophoresis for measuring cytokeratins. In patients with lichen planus, histological changes during treatment showed that, in the 10 patients in the tretinoin group, keratinization disappeared in 6 and decreased significantly in 3. Immunohistochemistry revealed that cytokeratins 10-11 and filaggrin disappeared in 57 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the patients given placebo. Bidimensional gel electrophoresis showed that cytokeratins 1, 2, 10 and 11 disappeared only in the tretinoin group (60 p. 100 of the cases). In patients with leukoplakia, histological changes during treatment showed that, in the tretinoin group, keratinization disappeared in 5 cases and decreased in 5 others. Immunohistochemistry revealed that cytokeratins 10-11 disappeared in 30 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the placebo group. Bidimensional electrophoresis demonstrated that cytokeratins 1, 2, 10 and 11 disappeared in 43 p. 100 of the patients treated with tretinoin. PMID- 7535513 TI - [Carcinoid tumor with revealed by skin manifestation]. AB - INTRODUCTION: Carcinoid tumours are often diagnosed late because a large quantity of vasoactive peptides must be accumulated to express the carcinoid syndrome. OBSERVATION: A 54-year-old male was seen for rosacea and intense episodes of vasomotor flush. The rosacea was associated with episodes of diarrhoea. The carcinoid tumour was diagnosed on the basis of raised levels of 5-hydroxyindol acetic acid. Radiology and surgery demonstrated liver, lymph node and mesenteric metastases of the primative carcinoid tumour which could not be located. Treatment combined chemotherapy (5-fluorouracil-streptozotocine) and a somatostatin analogue (octreotide). A good clinical response was obtained with a 1 year follow-up. COMMENTS: This case was discovered in misleading conditions since the rosacea overshadowed the other clinical manifestations of the carcinoid syndrome. Symptomatology resulted from the metastasic carcinoid tumour. Surgery should always be proposed. It is rarely very extensive but does allow reduction of tumour size. The role of medical treatment with a somatostatin analogue should be emphasized. This treatment can be combined with more classical chemotherapy (5 fluorouracil, streptozotocine). PMID- 7535514 TI - A method of enhancing regeneration of conventionally repaired peripheral nerves. AB - The effect of axoplasmic fluid injection on regeneration of conventionally repaired nerve was studied. Twenty New Zealand rabbits were studied in two experimental groups: immediate nerve repair (Group 1A, no axoplasm; Group 1B, axoplasm injection) and delayed nerve repair (Group 2A, no axoplasm; Group 2B, axoplasm injection group). A total of 40 sciatic nerve repairs were performed. After epineurial nerve repair, axoplasmic fluid collected from the proximal and distal nerve stumps was injected under epineurium proximally and distally to the anastomotic site. In the two control groups (immediate nerve repair and delayed nerve repair, both without axoplasm), 0.9% saline was injected in a similar way. Nerve regeneration was assessed by measuring hair and nail growth, plantar and dorsal foot temperature, muscle atrophy, and the toe spread test. In the 3- and 6 month follow-up periods, nerve samples were taken for histological evaluation. Specimens were stained with toluidine blue, and 50% of the nerve cross-section area was counted from microphotographic reconstructions. After immediate repair in Group 1, axoplasmic fluid-treated animals showed a 27% increase in the number of myelinated axons at 3 months when compared with saline-treated controls (3,768 vs. 2,962 +/- 1,921 SD). After delayed repair in Group 2, a 32% increase in myelination axon counts was detected at 3 months in axoplasm-treated animals compared with saline-injected controls (1,040 +/- 684 SD vs. 786 +/- 389 SD). In addition, a positive toe spread test result was seen at 6 months in the axoplasmic fluid-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535515 TI - Triglyceride hydrolysis and stability of a recombinant cutinase from Fusarium solani in AOT-iso-octane reversed micelles. AB - A recombinant cutinase from Fusarium solani was encapsulated in AOT reversed micelles. Physicochemical parameters of the system were optimized relative to triolein hydrolysis. Kinetic studies of triglyceride hydrolysis showed a decrease in specificity with increase of the acyl chain length. Stability of cutinase in the system under study is lower than in aqueous solution and decreases with increase in the water content in the system (W0 = [H2O]/[AOT]). The products of triolein hydrolysis had little effect on the cutinase stability. Although glycerol did not alter the stability, oleic acid decreases the enzyme stability. The increase in log P of solvent (from iso-octane to n-dodecane) decreased the stability. Deactivation profiles were fitted with the Henley and Sadana model (1). PMID- 7535517 TI - Endothelial cell adhesion molecules in psoriasis. AB - Skin biopsies from patients with psoriasis and normal controls were examined for the expression of cell adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), HECA-452 and 4D10, using an immunoperoxidase techique. This study demonstrates that psoriatic skin exhibits a wide variety of markers of endothelial cell activation which are either induced or increased in expression (ICAM-1, ELAM-1 & 4D10). Moreover, ICAM-1 & HECA-452 are also expressed on leukocytes. These antigens may facilitate the adhesion of inflammatory cells to endothelium and antigen presenting cells in psoriatic skin. Thus, they may play a role in faciliating the infiltration of leukocytes into psoriatic skin. PMID- 7535516 TI - A monoclonal antibody against blood forms of Trypanosoma cruzi lyses the parasite in vitro and inhibits host cell invasion. AB - Monoclonal antibodies (mAbs) of the IgM isotypes were produced from mice immunized with blood forms of Trypansoma cruzi Y strain. Characterization of the epitope recognized by one of the mAbs, 164C11, as well as the effects of this mAb on complement-mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the mAb was reactive with various strains of T. cruzi (Y, WSL, and Colombiana) as well as other trypanosomatids. The mAb 164C11 demonstrated a high complement-mediated lytic activity against bloodstream trypomastigotes, being more effective than chronic mouse serum. A protein with an apparent molecular weight of 72 kDa was detected by this mAb on all developmental stages of T. cruzi. Studies using periodate and endoglycosidase treatments suggested that the epitope is not a carbohydrate and seems to be located on the parasite membrane. In addition, preliminary results are presented, suggesting that the 72-kDa protein is involved in adhesion/or internalization of bloodstream trypomastigotes. PMID- 7535518 TI - Analysis of humoral immune response in chickens after inoculation with Cryptosporidium baileyi or Cryptosporidium parvum. AB - White leghorn chickens aged 14 days were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium baileyi or C. parvum to compare the specific immune responses. Cross-reactions were evaluated using homologous or heterologous antigens in enzyme-linked immunosorbent assay (ELISA) and Western blot to determine the occurrence of an antigenic homology between these two species. Blood, bile, whole intestine, bursa of Fabricius, and feces were collected daily from the day of inoculation (day 0) to day 22 postinoculation (PI). Eight control chickens remained negative up to day 22 PI. Chickens inoculated with C. baileyi did not express clinical symptoms but shed oocysts from days 6 to 21 PI. Chickens inoculated with C. parvum exhibited no clinical signs, no oocysts in feces, and no developmental stages of the parasite. However, a specific immune response to both antigens appeared on day 9 PI. ELISA using homologous or heterologous antigens showed that anti-C. baileyi and anti-C. parvum antibodies in serum or bile were detectable using C. baileyi or C. parvum oocysts as antigen, but the intensity of the response was significantly higher when C. baileyi was used. Cross-reactions in immunoblot analysis confirmed ELISA results, revealing a greater number of bands using C. baileyi as antigen but showing that epitopes recognized on the protein with a molecular weight of 15,000-17,000 were different. PMID- 7535519 TI - Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. AB - Collagen plays a major role in the structural organization of the heart and therefore direct visualization of collagen fibers is a crucial component of cardiac analysis. Although linearly polarized light has proven an effective tool for the examination of myocardial collagen in histologic sections, the use of circularly polarized light may offer advantages and additional possibilities. We examined the potential enhancement of collagen analysis using circularly polarized light in two ways. We first measured the brightness, and hence indirectly assessed the birefringence, of collagen fibers in scars examined at different times after myocardial infarction. Secondly, we measured collagen content in myocardial tissue and compared results obtained from brightfield analysis of trichrome stained sections with those obtained from circularly polarized light analysis of picrosirius red stained sections. We observed a progressive increase in the maximum brightness of collagen fibers in the scar with time, and a time-dependent shift in the relative distribution of collagen fiber brightness from lower to higher levels. We found consistently lower values of collagen content in trichrome stained versus picrosirius red stained tissue, and concluded that trichrome staining underestimated collagen content. The information provided by these studies could not be obtained by brightfield analysis and could be only partially obtained from linearly polarized light analysis. Thus, analysis using circularly polarized light has the ability to enhance histologic assessment of tissue and can provide additional insights into the composition and structure of myocardial collagen. PMID- 7535521 TI - Cholinergic regulation of amylase gene expression in the rat parotid gland. Inhibition by two distinct post-transcriptional mechanisms. AB - Stimulation of the beta-adrenergic or cholinergic muscarinic receptors are the principal mechanisms by which parotid salivary secretion is regulated in vivo. In this study we have examined the effects of cholinergic stimulation on amylase gene expression in dispersed rat parotid cells. [3H]Leucine incorporation into amylase and total protein was inhibited by carbamylcholine. Within 5 min of its addition, 10 microM carbamylcholine induced a 50-60% reduction in the rate of amylase synthesis which was sustained for more than 2 h. Blockade of the muscarinic receptor with atropine 8 min after addition of 10 microM carbamylcholine reversed the carbamylcholine-induced inhibition of amylase synthesis. When cells were exposed to carbamylcholine for 2 h before addition of atropine, there was only a slight reversal of inhibition. Carbamylcholine had no significant effect on the rate of total RNA synthesis but caused a progressive loss of amylase mRNA. After 2 h, amylase mRNA in cells treated with 10 microM carbamylcholine was 46% of control levels. Actinomycin D (5 micrograms/ml) lowered amylase mRNA by 8%; cycloheximide and phorbol 12-myristate 13-acetate had no effect. Isoprenaline (isoproterenol; at a concentration of 10 microM), which is an inducer of amylase gene transcription, elevated the amylase mRNA content by 30% after 2h. The calcium ionophore A23187 mimicked the effect of carbamylcholine by inhibiting [3H]leucine incorporation into amylase and lowering amylase mRNA content. The results suggest that acute stimulation of the muscarinic cholinergic receptor inhibits amylase biosynthesis in parotid cells not only by rapid attenuation of translation but also by causing a gradual loss of amylase mRNA, apparently by a Ca(2+)-dependent destabilization of the mRNA. PMID- 7535520 TI - Plasma exudation in conscious dogs with experimental heart failure. AB - The purpose of this study was to determine the effects of bradykinin (BK), substance P (SP) and histamine on plasma exudation in the skin of conscious dogs with and without pacing-induced heart failure. We also determined the role tissue angiotensin I-converting enzyme (ACE) and neutral endopeptidase (NEP) play in modulating these responses. We found that intradermal injection of BK, SP and histamine induced a significant, concentration-dependent Evans blue exudation in normal dogs (p < 0.05). Bradykinin-induced responses were significantly potentiated by captopril (p < 0.05). In contrast, phosphoramidon potentiated BK induced responses only at low concentrations of BK. Both captopril and phosphoramidon had no significant effects on SP- and histamine-induced Evans blue exudation. BK- and SP-induced responses were significantly attenuated, whereas histamine-induced Evans blue exudation was significantly potentiated in dogs with heart failure. We conclude that heart failure is associated with attenuation of BK- and SP-, but not histamine-induced plasma exudation in the peripheral microcirculation and that these responses are not modulated by tissue ACE and NEP. PMID- 7535522 TI - CD44 isoforms during differentiation and development. AB - During mouse early development cell adhesion molecules are indispensable for the embryo organisation. A family of molecules probably involved in development is the transmembrane glycoprotein CD44 family, which exists in multiple isoforms. These are generated by alternative splicing of the pre-mRNA, resulting in the enlargement of the extracellular part of the molecule. The standard form of CD44 is widely expressed in adult tissues and in embryos from day 9.5 post coitum onwards, while the numerous variant isoforms exhibit highly specialised patterns of expression that are already in the egg cylinder at day 6.5 of development. In lymphohemopoiesis, specific variant isoforms also emerge at decisive differentiation stages. Although specific ligands for the variant region still await isolation, the highly organised expression of CD44 variant isoforms suggests they have a pivotal role in cellular interactions during early development, pattern formation and hemopoiesis. PMID- 7535523 TI - Degradation of DNA in dried tissues by atmospheric oxygen. AB - Recently, DNA of extinct creatures have been brought into analyses. The difficulty in this field of research is DNA degradation. Oxidative damage is considered to be one of the causes of the DNA degradation. We studied how DNA in dried tissue was affected by atmospheric oxygen using freeze-dried rat liver as a model. In tissues exposed to oxygen, DNA degradation occurred within several months and the amount of 8-hydroxy-2'-deoxyguanosine in DNA rapidly increased. The DNA degradation was inhibited by lipid extraction prior to the exposure to atmospheric oxygen. Purified lambda phage DNA was not affected by oxygen. Cellular DNA and RNA were degraded slowly in nitrogen air. These results suggest that both atmospheric and endogenous oxygens play a role in DNA degradation in dried tissues. PMID- 7535525 TI - Erythropoietin induces tyrosine phosphorylation of the beta chain of the GM-CSF receptor. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) induce tyrosine phosphorylation of the GM-CSF receptor beta chain and the EPO receptor, respectively, although their receptors lack the tyrosine kinase activity. We have shown that EPO as well as GM-CSF induces tyrosine phosphorylation of the beta chain. Conversely, GM-CSF does not induce tyrosine phosphorylation of the EPO receptor. Tyrosine phosphorylation of the beta chain by stimulation with EPO is rapid and transient. EPO may trans-modulate a signaling pathway of GM-CSF by phosphorylating the beta chain of the GM-CSF receptor. PMID- 7535524 TI - Nonsteroidal anti-inflammatory drugs inhibit expression of the inducible nitric oxide synthase gene. AB - Increased nitric oxide production is associated with acute and chronic inflammatory processes. Accordingly, we tested the hypothesis that the therapeutic action of nonsteroidal anti-inflammatory drugs could be attributed at least in part to inhibition of excess nitric oxide production. We report here that sodium salicylate, aspirin, ibuprofen, and indomethacin markedly inhibited the appearance of the inducible inflammatory nitric oxide synthase in rat alveolar macrophages activated with lipopolysaccharide and interferon gamma. We attribute the mechanism of nitric oxide synthase inhibition by nonsteroidal anti inflammatory drugs to pretranslational control of enzyme expression and not to direct inhibition of enzymatic activity. These observations indicate that the chronic anti-inflammatory action of nonsteroidal anti-inflammatory drugs may be due not only to inhibition of prostaglandin synthesis but also to inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis. PMID- 7535526 TI - Active transport by the CscB permease in Escherichia coli K-12. AB - The cscB gene encoding a putative sucrose permease from Escherichia coli EC3132 [Bockmann, J., Heuel, H. & Lengeler, J.W. (1992) Mol. Gen. Genet. 235, 22-32] was placed under control of the lacZ promoter/operator and overexpressed. Cells of E. coli K-12 expressing cscB gene product from a high copy number plasmid accumulate sucrose against a concentration gradient with an apparent Km of about 1 mM and a Vmax of approximately 23 nmol/min/mg protein. In contrast, transport of lactose or melibiose is not observed. Epitope-tagging and immunological analysis reveal that concomitant with increased transport activity, a protein with an apparent M(r) of 33-34 kDa appears in the cytoplasmic membrane. The results provide direct support for the contention that the cscB gene encodes a sucrose permease and provide the basis for further molecular analysis. PMID- 7535527 TI - Molecular cloning and characterization of mouse HS1. AB - Here we report the cloning of a cDNA and a genomic DNA encoding the mouse homolog of human HS1, a hematopoietic cell-specific protein-substrate of non-receptor type protein-tyrosine kinase(s). Sequence analysis of the mouse HS1 cDNA revealed that it is highly homologous to human HS1 (total percent match = 84%) especially in the amino-terminal half, which contains unique repeating motifs, and in the carboxyl-terminal Src-homology 3 domain. As is the human counterpart, the mouse HS1 gene is expressed exclusively in hematopoietic cells. Genomic fragments covering most of the HS1 gene were isolated and used to map this gene to mouse chromosome 16. PMID- 7535528 TI - Integrin-linked tyrosine phosphorylation increases membrane association of protein kinase C alpha in pancreatic acinar cells. AB - Ligation of beta 1 integrin receptors resulted in increased tyrosine phosphorylation of at least five proteins (Mrest = 110, 85, 55, 30 and 24 kD) from rat pancreatic acinar cells. Increased protein kinase C (PKC) activity and elevated amounts of immunoreactive PKC alpha were demonstrated in membrane fractions from integrin-ligated acinar cells. Membrane translocation of PKC alpha was confirmed using scanning confocal laser microscopy in immunocytochemical preparations of acinar cells following beta 1 integrin ligation. These studies establish the presence of a beta 1 integrin-linked protein tyrosine phosphorylation system in exocrine pancreatic cells and provide evidence for integrative activity of this system with PKC, a primary signalling pathway of central importance in these cells. PMID- 7535530 TI - Derivatized dextrans modulate collagen synthesis in aortic smooth muscle cells. AB - The effect of specifically derivatized dextrans, with or without antiproliferative activity on smooth muscle cells (SMC), was investigated on type I and type III collagen biosynthesis and mRNA levels in post-confluent SMC cultures. Our results indicate that dextran derivatives decreased total protein and collagen synthesis independently of their antiproliferative activities. However, the most substituted dextran, the one exhibiting the strongest antiproliferative activity towards SMC, was the most active in modulating type III collagen expression. In addition, only the two dextran derivatives bearing benzylamide groups inhibited collagen excretion. PMID- 7535531 TI - The glutamate uptake inhibitor L-trans-2,4-pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain. AB - High-affinity glutamate uptake (HAGU) transporters rapidly remove released glutamate from the synaptic cleft. If HAGU is suppressed, neurotoxic concentrations of excitatory amino acids may accumulate. To seek further evidence in support of the neurotoxicity of endogenous glutamate in the developing brain, we assessed the neurotoxicity of the selective HAGU inhibitor L-trans-2,4 pyrrolidine dicarboxylate (L-PDC) in postnatal day 7 (PND 7) rats. The hippocampus of PND 7 rats is susceptible to EAA agonist-mediated injury; features of injury include atrophy and neuronal loss. Since HAGU is energy-dependent, we hypothesized that moderate hypoxia would increase L-PDC-mediated injury by further suppressing HAGU. L-PDC was stereotaxically injected into dorsolateral hippocampus of PND 7 rats (568 nmol, n = 20). Prior to return to the dam, rats were divided into two groups, one of which was subjected to moderate hypoxia (3 h, FiO2 = 0.08) (n = 11; 2 died acutely). On PND 12, hippocampal neuropathology was assessed by a blinded observer using a five-point scale and also by measuring hippocampal cross-sectional areas with computerized image analysis. Three brains were excluded from analysis, since markedly asymmetric tissue sectioning precluded valid side-to-side comparison of hippocampal areas. Injection of L-PDC alone elicited focal pyramidal cell loss (6/7); in the (L-PDC + hypoxia) group, injury was significantly increased (median scores: L-PDC = 2; [L-PDC + hypoxia] = 3.5; p < 0.005). Hippocampal atrophy was noted only after L-PDC + hypoxia (4/8) (percent right-left difference in mean hippocampal area [+/- SE]: L-PDC = 2.5% [+/- 2.6]; [L-PDC + hypoxia] = 8.9% [+/- 3.2]; p < 0.02). In tissue from PND 7 rats, L-PDC (10 microM) inhibited hippocampal synaptosomal HAGU by > 85%; at the same concentration, L-PDC did not displace [3H]glutamate from NMDA- or AMPA sensitive hippocampal binding sites. These results support the hypothesis that increased synaptic accumulation of endogenous excitatory amino acid neurotransmitters may produce hippocampal injury in perinatal rodents. PMID- 7535529 TI - Inhibition of ADP-ribosyltransferase increases synthesis of Gs alpha in neuroblastoma x glioma hybrid cells and reverses iloprost-dependent heterologous loss of fluoride-sensitive adenylate cyclase. AB - Exposure of NG108-15 cells to 50 mM nicotinamide [an inhibitor of mono(ADP ribosyl)transferase] for 18 hr led to an increase in membrane associated Gs alpha measured either as cholera toxin substrate or by immunoblotting with a specific antiserum. Prolonged exposure of NG108-15 cells to iloprost is followed by homologous loss of iloprost sensitivity, and heterologous loss of fluoride dependent activation of adenylate cyclase. Nicotinamide reversed the loss of fluoride sensitivity, but failed to restore iloprost-dependent activation of adenylate cyclase. These results with nicotinamide in NG108-15 cells contrasted with those from platelets, which also exhibit heterologous desensitization of fluoride sensitivity following prolonged exposure to iloprost. Treatment of platelets with 50 mM nicotinamide for 18 hr led to an increase of 75.0 +/- 19.4% in the amount of membrane associated cholera toxin substrate. However, there was no associated increase in the abundance of Gs alpha as determined by immunoblotting. Furthermore, in platelets there was no restoration by nicotinamide of the iloprost-dependent loss of fluoride-sensitive adenylate cyclase activity. It follows that heterologous desensitization in platelets is accompanied by inactivation of Gs alpha, which is retained within the plasma membrane in its inactive state. The nicotinamide-dependent increase in the abundance of membrane associated cholera toxin substrate and immunoreactive Gs alpha in NG108-15 cells is associated with an increase of 72.0 +/- 20.3% in the levels of mRNA encoding Gs alpha. The capacity of nicotinamide to increase the abundance of membrane associated Gs alpha was reversed when the cells were cultured in the presence of 20 micrograms/mL cycloheximide. These results suggest that the ability of nicotinamide to increase the abundance of Gs alpha in NG108 15 cells is mediated by de novo protein synthesis. PMID- 7535533 TI - The effect of gamma-hexachlorocyclohexane (lindane) on the activities of liver lipogenic enzymes and on serum lipids in rats. AB - The effect of dietary gamma-hexachlorocyclohexane (lindane) (50-350 ppm, 0.17 1.19 mumol/kg chow) on the activity of enzymes of lipogenesis, viz., fatty acid synthase (FAS; EC 2.3.1.85), citrate cleavage enzyme (CCE; EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44), and on serum lipid levels, was investigated in livers of 35-day-old male Wistar rats. Lindane (150 ppm) caused a substantial decline of enzyme activities within the first 24 h of treatment. The decrease was transient, however, and enzyme activities subsequently recovered despite continuation of lindane feeding. The recovery of enzyme activities was comparatively fast in the case of ME, G6PDH and PGDH, but very slow with FAS and CCE. Activities of lipogenic enzymes decrease when animals are starved, and increase much beyond prestarvation levels upon subsequent refeeding. Lindane in the refeeding diet blunted this overshoot of FAS and CCE activities in a dose-dependent manner. In contrast, activities of Me, G6PDH and PGDH responded to low dietary lindane concentrations with a substantial stimulation of the increase of activity, whereas at high lindane concentrations the overshoot was inhibited. According to their responses to lindane exposure, liver lipogenic enzymes could be grouped into 2 categories with FAS and CCE representing one and ME, G6PDH and PGDH representing the other group. Polychlorinated biphenyls (PCBs) in the diet caused basically opposite changes of the activities of the lipogenic enzymes. Co-administration of lindane and PCBs resulted in an apparent cancellation of effects, suggesting that lindane and PCBs affect fatty acid synthesis at opposite points.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535532 TI - Kuehneromycins A and B, two new biological active compounds from a Tasmanian Kuehneromyces sp. (Strophariaceae, Basidiomycetes). AB - In a search for new inhibitors of RNA-directed DNA-polymerases kuehneromycin A (1) was isolated from fermentations of a Tasmanian Kuehneromyces species. Its structure was elucidated by spectroscopic methods. Kuehneromycin A (1) is a non competitive inhibitor of avian myeloblastosis virus (Ki 200 microM) and moloney murine leukemia virus (Ki 40 microM) reverse transcriptases. The second compound, kuehneromycin B (2) is a strong inhibitor of platelet aggregation stimulated with different inducers. In addition, both compounds exhibit cytotoxic and antimicrobial activities. PMID- 7535534 TI - Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, the in vivo site in brain myelin basic protein. AB - In a previous report [Yang et al., (1987a), J. Biol Chem. 262, 7034-7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C18-reversed phase high-performance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio approximately 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio approximately 0.9) and TT(p)HYGSLPQK (molar ratio approximately 0.8) as the major phosphorylation site sequences in 32P-MBP phosphorylated by autokinase, further indicating that -Arg-X-Ser/Thr (neutral amino acid)3-(amino acid-containing hydroxyl group such as Ser/Glu/Asp) (neutral amino acid)2-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/multifunctional protein kinase in the brain. PMID- 7535535 TI - Autoimmunity against thyroid hormones. AB - The presence of thyroid hormone autoantibodies (THAA) is a common phenomenon. More than 270 cases have been reported by the end of 1993 involving not only thyroidal but also nonthyroidal disorders. Clinically, THAA in a patient's serum produces variation in thyroid hormone metabolism and, in particular, may interfere with the radioimmunoassay (RIA) results of total or free thyroid hormone measurements, which can cause unusually high or low values of the hormones depending on the B/F separation method used. This in vitro interference can give clinicians confusing information about the patient's thyroid state. As a result, the patient may receive inappropriate treatment from physicians who are unaware of this disorder. The presence of THAA has been reported not only in humans but also in dogs, chickens, and rats. In this review article, clinical features of THAA and the mechanism of autoantibody production are discussed. PMID- 7535537 TI - Snail hosts of Paragonimus in Asia and the Americas. AB - We have undertaken a comprehensive review of snail hosts of Paragonimus world wide exclusive of Africa based on modern malacological data, where available, and with consideration of the phylogeny of the snail groups involved. This is the first comprehensive review since those made by Chen (1979) and Chen et al. (1983), and there have been considerable taxonomic changes over the past decade. A number of names and concepts found in the medical malacological and parasitological literature up to the present time require revision or correction. There are vast radiations of snails of the superfamilies Cerithiacea and Rissoacea involved in the transmission of Paragonimus in China. We list 54 species world-wide of which 35(65%) occur in China. Revisions and corrections pertaining to China include: (i) The family Hydrobiidae does not occur in China or S.E. Asia, and the Pomatiopsidae should be used. (ii) The genus Bythinella is entirely European (Hydrobiidae: Amnicolinae). The so-called Bythinella of China belongs to the genus Erhaia (Pomatiopsidae). (iii) The generic name Pseudobythinella described from China is preoccupied, a junior synonym of Pseudobythinella Melville 1956, a fossil from England. All Chinese Pseudobythinella are now classified as Erhaia. (iv) Akiyoshia has been used as a generic name for some snails in Hunan transmitting Paragonimus. Akiyoshia is from Japan and biological/ecological data indicate that the Chinese taxon is not Akiyoshia. (v) The genus Tricula in recent Chinese literature is comprised of four genera determined by detailed comparative anatomical data: Tricula, Neotricula, Gammatricula, and Jinhongia. Shells cannot be used to discriminate among them. (vi) Tricula cristella has been consistently misidentified in collections in China and literature. However, genuine T. cristella does transmit Paragonimus skrjabini. (vii) Tricula minutoides in the Chinese literature has been misidentified, and specimens are T. cristella. (viii) The genus Melania and the family Melaniidae are used in the Chinese literature. The so-called melaniidae snails belong to the families Thiaridae, Pleuroceridae, and Melanopsidae. Asian Semisulcospira is pleurocerid while Melanoides and Tarebia are thiarid (ix) Semisulcospira libertina is correctly identified for populations ranging throughout Japan to Taiwan. This species is most probably not distributed throughout all south China, and intense systematic research on Semisulcospira is needed. A modern classification is given for snails transmitting Paragonimus. PMID- 7535538 TI - Use of the minimal function for partial structure development in direct methods. AB - The shake-and-bake procedure, which is based on the minimal function, has been tested and shown to be extremely effective in molecular-fragment recycling applications. Correctly positioned fragments as small as 5% of the scattering power of the structure typically have a 50% chance of producing a solution in a single recycling trial. While starting models for tangent-formula recycling methods normally require an average r.m.s. displacement error of less than approximately 0.25 A from the refined structure to ensure an adequate chance of success, the shake-and-bake method often tolerates r.m.s. model errors well in excess of 0.5 A. Tests indicate that the new method can outperform traditional tangent-formula procedures in difficult structural applications involving multiple copies of pseudosymmetrically related molecules or low-resolution data. PMID- 7535536 TI - Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction. AB - The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage. PMID- 7535539 TI - Lymphoepithelial cysts of salivary glands in HIV-infected patients: clinico pathological and immunohistochemical features. AB - The parotidectomy specimens of 3 HIV-infected patients with lymphoepithelial cysts (CLL) have been used to study, with an immunohistochemical method, the expression of the following antigens in histopathological sections: Cytokeratins, CD3, CD20, CD35, CD68, MAC387, Kappa and Lambda light chains. The purpose of the study was to ascertain if CLL of salivary glands parallel the immunophenotype of extrasalivary systemic lymphadenopathy (LAS). The results of this study show that the epithelial lining of CLL does not derive from preexisting ducts and that the immunophenotype of CLL very closely resembles that of LAS. These features seem to suggest that CLL and LAS are tightly related diseases and that probably surgery is not the elective treatment for this lesion. PMID- 7535540 TI - Recovery of tissue plasminogen activator enzymatic activity following RNA extraction. PMID- 7535541 TI - Mitochondrial biogenesis in early mouse embryos: expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H(+)-ATP synthase. AB - The mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two-cell stage onward (Piko and Taylor, 1987: Dev Biol 123:364-374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H(+)-ATPase subunit 9 (P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos. All four mRNAs showed a similar developmental pattern of prevalence, characterized by a steady decline in mRNA copy numbers from the late growth-phase oocyte through the two-cell embryo, and an about 30-fold rise during cleavage through the blastocyst stage. However, the ATPase subunit 9 (P1) mRNA was about three times more prevalent in cleavage-stage embryos than the COX mRNAs. A similar pattern was obtained previously for the mitochondrial-encoded COX I and II mRNAs, but the latter accumulate at a 30-50-fold excess over the nuclear-encoded COX subunit mRNAs during the cleavage stages. The results suggest a coordinated activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus beginning with the two-cell stage. It is estimated that new respiratory chains are produced at a rate of 50 100 chains hr-1/mitochondrion in the early blastocyst, accounting for 3.5-7% of the total protein synthetic activity at this stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535542 TI - Extra-articular fractures of the distal radius in young adults. A technique of closed reposition and stabilisation by mono-segmental, radio-radial external fixator. AB - Some fractures of the distal radius need an operative treatment in order to restore and maintain both length and anatomical angles of the distal joint surfaces. We present a technique of closed reposition and stabilisation by external fixator on the distal radius, without any bridging of the wrist joint, allowing for active and passive motion of the wrist during bone healing. Two cases are illustrated as examples. PMID- 7535545 TI - [Kirner's deformity. 4 case reports and review of the literature]. AB - Kirner's disease is a rare deformity of the distal phalanx of the little finger, with a combination of camptodactyly and clinodactyly and clinodactyly. A review of the literature and analysis of our cases show that this anomaly appears to result from a unusual insertion of the deep flexor over the growth cartilage. No treatment is required in view of the absence of any clinical and functional symptoms. PMID- 7535543 TI - [Reconstruction with free vascularized or island flaps of soft tissue loss in the upper limb after tumor resection. 16 cases]. AB - We have reviewed 16 cases of soft tissue sarcomas of the upper limb treated with wide excision of the tumour and immediate soft tissue reconstruction by means of a microvascular flap. 9 patients were males, 7 females; age ranged from 12 to 74 years. All the patients had been referred to us after previous failed or inadequate excisions. All the tumours were high-grade malignancies in an extracompartmental location (stade IIB according to Enneking). Histopathologic diagnosis was: synovial-sarcoma in 11 cases; fibrosarcoma in 2 cases; malignant fibrous histiocytoma in 1 case, soft tissue osteosarcoma in 1 case, epitheloid sarcoma in 1 case. The site of the tumour was the hand in 2 cases, the wrist in 6 cases, the forearm in 5 cases and the elbow in 3 cases. The free flaps used for soft tissue reconstructions were the latissimus dorsi in 6 cases, the dorsalis pedis in 2 cases, the lateral arm flap in 2 cases, the serratus in 1 case, the gracilis in 1 case. The island flaps were the radial forearm flap in 1 case, the posterior interosseous flap in 1 case, the cubital flap in 1 case. 14 cases required immediate associated reconstructive procedures such as tendon transfers or grafts (5 cases), vascularized or non vascularized bone grafts (4 cases), articular allografts (1 case), ligament reconstruction (1 case), nerve grafts (2 cases). Brachytherapy was associated to microsurgical reconstruction in the last 6 cases of this group. The catheters were charged with the radioisotope 5 days after surgery. No complications were observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535544 TI - [Elongation and coverage of an inguinal flap over a short forearm traumatic amputation stump]. AB - A particularly short traumatic amputated forearm finally led to a simplified under-elbow prosthesis after a long surgical program including: 1) A progressive 8 cm ulnar lengthening with a Wagner device; 2) and a secondary groin flap resurfacing the distal stump. Complications essentially concerned the ulnar lengthening particularly because of a weak osseous callus. Final aesthetic and functional results are satisfying with a 6 years follow-up. PMID- 7535546 TI - [Fluctuating phlegmons. Seven cases]. PMID- 7535547 TI - [Fibro-osseous pseudo-tumor of the fingers. A little known entity. A case of an 11 year-old girl]. AB - The authors report a case of fibro-osseous pseudo-tumour affecting a long finger. This is a rare tumour difficult to diagnose. Complete excision of the tumour was delayed till two years after its appearance as it's true nature had not been recognised at the time of the initial histopathologic examination. It is a benign tumour with a good prognosis as it does not undergo malignant degeneration and does not recur locally if excision has been complete. Errors in interpretation of the histopathologic picture have, in the past, led to unwarranted amputations of the involved fingers. PMID- 7535548 TI - The treatment of chronic thumb metacarpophalangeal joint instability in an adolescent secondary to thumb hypoplasia. AB - A fourteen-year-old female presented with untreated thumb hypoplasia consisting only of chronic instability of the thumb metacarpophalangeal joint. Successful treatment consisted of metacarpophalangeal joint fusion. PMID- 7535549 TI - [Treatment of comminuted fractures of the lower end of the radius with internal osteosynthesis, traction and early mobilization]. AB - Goals for treatment of comminutive fractures of the distal radius include restoration of the articular profile of the proximal part of the joint, while axial loading forces must be avoided as much as possible to prevent secondary displacement. The choice of an internal fixation protected by an external wrist distractor-fixator, with early activo-passive mobilisation, seems to achieve the goal. Twelve patients with a comminuted fracture of the distal radius, including axial articular impigment displacement were reviewed for this study. All fractures were Frykman's type III, IV, VII or VIII. Distraction was done with a specific external apparatus, allowing an internal fixation, using an anterior plate and posterior Kirschner wires for the more complex cases. Distraction was released at the end of the surgical procedure, while the distractor was left in place. The wrist was mobilised early in the post-operative period, and the distractor was removed two months later. At a mean follow-up of 8.5 months, two patients were still painful. Mean motion of the wrist joint was 115 degrees for flexion-extension and 35 degrees for radio-ulnar deviation. Radiological results were good (10 cases), in both planes sagittal and frontal, and stable with time. The radio-ulnar index was correct in 11 cases. Only two cases of Sudeck's atrophy were noted. Authors use a specific external wrist distractor to obtain and maintain reduction in comminuted fractures of the distal end of the radius, using internal fixation in combination. Early motion of the wrist, protected by the wrist distractor seems to lower rates of Sudeck's atrophy. PMID- 7535551 TI - Reflex sympathetic dystrophy syndrome secondary to organophosphate intoxication induced neuropathy. AB - Reflex sympathetic dystrophy or Sudeck's atrophy is a syndrome that can usually be followed by a traumatic insult. This disorder is accompanied by signs and symptoms of vasomotor instability, trophic skin changes, and rapid development of bony demineralization. This report presents a case with reflex sympathetic dystrophy syndrome secondary to organophosphate intoxication induced neuropathy. The patient was threated with calcitonin well. PMID- 7535550 TI - Scintigraphic changes in bone metastasis from prostate cancer after hormonal therapy--comparison with tumor markers and bone X-ray. AB - Bone scintigraphy is often performed to assess the response to systemic therapy of bone metastasis from prostate cancer. We examined the changes in bone scintigraphic findings and the agreement with AIP, AcP, or other tumor markers measured in the follow-up of patients with known bone metastasis after hormonal therapy. Out of 32 patients, 22 (69%) showed improved scintigraphic findings on the first follow-up bone scintigraphy after hormonal therapy. However, 7 out of 22 patients who showed improvement on the first follow-up scintigraphy, deteriorated thereafter. Changes in the scintigraphic findings were closely correlated with those in the measured tumor markers except for patients with small bone metastasis. Though there were no significant differences in the agreement ratios of the 6 tumor markers evaluated, AIP might be a practical and acceptable indicator. Bone X-ray findings did not change at all in almost half of the cases though the scintigraphic findings showed improvement or deterioration. PMID- 7535553 TI - Low-level X-ray exposures on rat skin. Hyperkeratinization and concomitant changes in biometal concentration. AB - Integumentary structures naturally are more exposed to solar radiation than other body tissues, so there is a big question regarding the biological threshold of skin in respect to low-dose ionizing radiation. In this study, adult male albino rats were exposed chronically to low-dose (0.015 cGy/sec) X-rays for 9 and 18 mo, with total X-ray dose of 2.025 and 4.050 cGy, respectively. In both the dose groups, hyperkeratinization was noted in skin by transmission electron microscopic (TEM) study. Atomic absorption spectrometric (AAS) study revealed decreased zinc concentration (p < 0.01), increased iron concentration (p < 0.001), and status quo cadmium concentration. Moreover, the ratio between zinc and iron became highly depleted in both the irradiated groups. Hence it may be stated that chronic low-level X-rays induce redistribution of biometals in the skin. Nevertheless, specific concentrations of biometals indicate the risk-prone status of irradiated skin. PMID- 7535552 TI - CYFRA 21-1 as a tumor marker used in measuring the serum fragment of cytokeratin subunit 19 by immunoradiometric assay. AB - Serum levels of cytokeratin subunit 19 (CYFRA 21-1) were measured in 42 healthy volunteers, 104 cases of malignant diseases, 30 patients with chronic renal failure and 13 patients with nonmalignant and infectious diseases. The reliability of the method was demonstrated after dilution of serum samples and intra- and inter-assay reproducibility. Serum CYFRA-21-1 concentrations were less than 2.00 ng/ml in all healthy controls and 86% of the malignant cases had high serum CYFRA 21-1 levels. However slightly elevated values of CYFRA 21-1 were observed in most chronic renal failure patients. High correlation was observed between serum CYFRA 21-1 and Tissue Polypeptide Antigen (TPA) values (r = 0.90, n = 10) but not with serum alpha-feto protein (AFP) concentrations. Furthermore, cross binding tests with the CYFRA 21-1 tracer/CYFRA 21-1 antibody-coated beads and CYFRA 21-1 tracer/TPA antibody-coated beads also gave an almost linear graph. These results indicate that CYFRA 21-1 and TPA share similar type of antigens. PMID- 7535554 TI - In vivo models for testing of cytostatic agents in non-small cell lung cancer. PMID- 7535557 TI - The certification of wild poliovirus eradication from the Western Hemisphere. PMID- 7535556 TI - Interferon and lung cancer. PMID- 7535555 TI - The role of hematopoietic growth factors in small cell lung cancer: a review. PMID- 7535558 TI - First inter-American conference on society, violence, and health. AB - As a part of the strategy to promote the Regional Plan of Action on Violence and Health, the First Conference on Society, Violence, and Health was held at the headquarters of the Pan American Health Organization in Washington 16-17 November 1994. The objectives of the Conference were to: 1) Draft a declaration calling upon the heads of state of the American States to mobilize resources in order to prevent violence: 2) Create a consensus among the national and international cooperation organizations on methods and procedures to achieve effective support for democratic values and the respect of the human rights in societies living in peaceful coexistence: 3) Promote a regional movement of nongovernmental and other organizations in order to create nonviolent communities; 4) Urge the governments of the Hemisphere to commit themselves individually and through international organizations to allocate financial resources capable of reversing the trend toward violence. The Conference had five panels: 1) Violence as a Public Health Issue; 2) Towards a Democracy without Violence; 3) The Political Economy of Non Violence; 4) a Culture of Peace for the XXI Century; and 5) Building Non-Violent Social Relations. Distinguished speakers representing various institutions related to the subject area participated in each. As part of the objectives, the participants approved a Declaration that considered the nature of violence, examined its multiple causes and different expressions, and specified the structures of the most vulnerable social groups. Explicit reference was made to the need for carrying out efforts in order to strengthen democracy, seek healthier forms of life, and strive for a culture of peace and coexistence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535559 TI - Preparation of cell-affinity adsorbents by immobilization of peptides to Sephadex G-10. AB - In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10(DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/- 300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method' is a good alternative for polystyrene and other solid phase materials. PMID- 7535560 TI - Adhesion of human peripheral lymphocytes on biomaterials preadsorbed with fibronectin and vitronectin. AB - The adhesion of human peripheral lymphocytes (HPL) was studied after preadsorption of fibronectin (FN) and vitronectin (VN) on hydrophilic glass and hydrophobic octadecyl glass. The adhesion of HPL was shown to be dependent not only on the wettability but also on the protein preadsorbed. Vitronectin expressed not only a higher extent of adhesion under static conditions but also a stronger interaction with HPL, indicated by the low detachment under shear stress. The flow experiments also demonstrated that FN adsorbed on octadecyl glass may undergo conformational changes because HPL could be easily removed. Scanning electron microscopy revealed that HPL on both FN- and VN-coated glass spread well whereas particularly on FN-coated octadecyl glass less cell spreading was observed; moreover, some round cells were detected. The typing of adherent HPL by immunofluorescence microscopy showed that on FN- and VN-coated glass about 70% of all HPL were T-cells (CD 3+). However, on octadecyl glass, particularly on VN, a smaller percentage of CD 3+ cell was observed. The testing for the beta 1 integrin--the receptor for FN and the alpha v integrin--the receptor for VN demonstrated that about 70% of all cells on FN-coated glass were positive for the beta 1 integrin. On VN-coated glass, however, only 5% of HPL were positive for the beta 1 integrin. Although on VN a high adhesion and strong binding of HPL was observed, no presence of the alpha v integrin was detected. PMID- 7535561 TI - Carboxanilide derivative non-nucleoside inhibitors of HIV-1 reverse transcriptase interact with different mechanistic forms of the enzyme. AB - Researchers at the National Cancer Institute first recognized the anti-HIV potential of the carboxanilide compound oxathiin carboxanilide (UC84) [Bader, J. P., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6740-6744]. We have compared the inhibitory effect of UC84 and a second-generation thiocarboxanilide derivative, UC38, on HIV-1 reverse transcriptase (RT) RNA-dependent DNA polymerase activity. UC38 was a much better inhibitor (IC50 = 0.8 microM) than UC84 (IC50 = 4.3 microM). Inhibition by UC84 was competitive with respect to primer/template (P/T), whereas that by UC38 was uncompetitive. Both compounds were mixed noncompetitive inhibitors with respect to deoxynucleoside triphosphate (dNTP). Both compounds protected RT from irreversible photoinactivation by an azido derivative of nevirapine, implying that UC84 and UC38 bind to the same region of RT as nevirapine. UC84 photoprotected both free RT and the RT-P/T binary complex, but did not protect the RT-P/T-dNTP ternary complex. In contrast, UC38 completely photoprotected the RT-P/T-dNTP ternary complex, but not free RT or the RT-P/T binary complex. UC84 and UC38 thus appear to bind to different mechanistic forms of RT in the polymerase reaction sequence. PMID- 7535562 TI - Persistence length of RNA. AB - A set of 10 double-stranded RNAs ranging in length from 92 to 317 base pairs has been synthesized, and their rotational diffusion times were measured by transient electric birefringence. A hydrodynamic analysis of the birefringence decay data yields a helix persistence length of 720 +/- 70 A, based on a helix with an effective hydrodynamic diameter of 26 A and a rise per base pair of 2.7 A in the presence of Mg2+ ions. Thus, duplex RNA is somewhat stiffer than DNA, for which the persistence length is 450-500 A. The measurements also suggest that (i) current hydrodynamic theory for the rotational decay times is applicable to RNA molecules that are longer than 100 base pairs and (ii) weak Mg(2+)-RNA association decreases the rise per base pair slightly, by no more than 0.1 A. PMID- 7535563 TI - Membrane topology of P-glycoprotein as determined by epitope insertion: transmembrane organization of the N-terminal domain of mdr3. AB - P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR). Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain. To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376). Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells. Intracellular and extracellular localization of the tag in the full length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope. While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance. The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane. These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions. These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6). Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane. PMID- 7535564 TI - Conformational transitions in the cell binding domain of fibronectin. AB - Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin. PMID- 7535566 TI - [A new phenomenon induced by ion conformational interaction in biomembrane channels]. AB - In several experimental studies it has been observed that when the ion flow approaches zero one of the conduction sublevels disappears irrespective of how the controlling parameters are changed. It is explained by the fact that the bistable regime in the ion channel appears only when the difference of the electrochemical potentials exceeds the threshold level, and such systems can function only in strongly now-equilibrium conditions. We describe the conformational mobility of the ion channel structure in terms of stochastic differential equations. It allows us to study behaviour of the ion channel, that is, volt-ampere characteristics, and kinetic parameters such as the lifetime and probability of the open state, ect. Ion-conformational interaction is taken into account based on experimental data. It is demonstrated that in some cases this interaction plays a decisive role in functioning of ion channels, since it is what causes emergence of discrete conduction levels. Such channels are described in the frame of the synergistic approach, which is characterised by the monostable regime of functioning near the equilibrium state, whereas bistability is possible only when the ion flow exceeds the threshold value. PMID- 7535565 TI - Nonlocal interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. AB - A search for the topology of the chain folding of reduced bovine pancreatic trypsin inhibitor (BPTI), in unfolded and partially folded states, was done by means of time resolved dynamic nonradiative excitation energy transfer (ET) measurements. Four double labeled BPTI derivatives were used in which the donor was attached to the N-terminal arginine residue and the acceptor was specifically attached to one of the lysine residues. The four derivatives form a series of labeled backbone segments of increasing length spanning the full lengths of the BPTI chain: 15, 26, 41, and 46 residues. The intramolecular segmental end-to-end distance (EED) distributions were determined for all the derivatives by global analysis of the decay curves of both the donor and the acceptor in the reduced state, in low (0.5 M) guanidinium chloride (GuHCl) concentrations at pH 3.6 and 2.1 (R and A states, respectively). The results show that, in the partial folding conditions of low GuHCl concentration, reduced BPTI is in a compact state, but in this state the polypeptide chain is not in a condensed statistical coil conformation. Two distinct subpopulations were found for the four intramolecular EED distributions. One subpopulation was compact, with native-like EED distribution, while the second was unfolded. The pairs of sites, residues 1 and 26 and residues 1 and 46, showed close proximity in the dominant subpopulation. These contacts form two loops (probably collapsed): one consists of the first 26 residues, and the second comprises the full length of the chain from the N- to the C-terminal segments, which is in fact made up to two shorter loops (1-26 and 27-46). The N-terminal 15 residue segment was relaxed into statistical coil-like non-native conformation, in contrast to its extended conformation in the native state. The effect of temperature in the range of 2-60 degrees C was small; the folded subpopulations were stable over this range. These results show that in BPTI the compact conformations found under unfolding and partially folding conditions have native-like chain topology. Under the conditions of transition to partially folding conditions the compact conformation is stabilized, not only by the hydrophobic collapse and the local interaction but also by nonlocal interactions (NLIs). Few specific, very stable NLIs between the three segments which form the main structural elements of the native conformation direct the formation of native-like topology of the chain in the transition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535567 TI - Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor. AB - The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy. PMID- 7535568 TI - A new histochemical double-stain method using three-dimensional analysis with confocal laser scanning microscopy. AB - We describe a new technique for immunohistochemical and enzyme-histochemical double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for Ca(2+) ATPase in the rat kidney. The lead precipitation method for Ca(2+)-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35 calbindin-D28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca(2+) ATPase, were distributed deep in the section. The most intense signals from the silver particles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution. PMID- 7535569 TI - Interleukin-7 selectively enhances natural kill cytotoxicity mediated by the CD56bright natural killer subpopulation. AB - Both the CD56bright and CD56dim NK cell subpopulation mediate non-major histocompatibility complex-restricted cytolysis of NK-sensitive tumor cell lines, and IL-2-dependent augmentation of cytolysis and proliferation of CD56bright and CD56dim NK cells was recently reported. We investigated the effects of IL-7 and IL-6 on the killing mediated by these cells to determine whether other cytokines besides IL-2 regulate their activity. IL-7 increased the cytotoxicity in only the CD56bright NK cell population. The effect of IL-7 varied from donor to donor but was comparable to that of IL-2. Furthermore, IL-7 was found to induce lymphokine activated killer (LAK) cell generation primarily in the CD56bright cells. CD56bright NK cells also proliferated in response to IL-7, but only weakly in comparison with IL-2. In contrast to the results with CD56bright NK cells, IL-7 had little effect on the CD56dim subset. However, IL-2 enhanced NK cytotoxicity, induced LAK activity, and caused proliferation of these cells. An anti-IL-2 antibody did not inhibit the IL-7-induced increase in CD56bright cytotoxicity, suggesting that IL-7 acted independently of IL-2. However, the IL-7 effect on CD56bright NK cell cytotoxicity was partially inhibited by anti-CD2, anti-CD11a, and anti-CD18 antibodies and almost completely abrogated by a combination of anti CD2 and anti-CD11a. These data suggest that cell adhesion molecules (CAM) play a role in the regulation of IL-7-induced CD56bright NK cell cytolysis. In contrast to IL-7-mediated effects, IL-6 alone had no effect on CD56+ NK cell cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535570 TI - Alterations in L-selectin expression and elastase activity in neutrophils from patients receiving granulocyte colony-stimulating factor alone or in conjunction with high-dose chemotherapy with autologous bone marrow transplantation. AB - We determined L-selectin expression and elastase levels in neutrophils obtained from patients receiving granulocyte colony-stimulating factor (G-CSF) either alone (given for increasing peripheral progenitor cells for harvest) or in combination with high-dose chemotherapy with autologous bone transplantation support (BMT). Administration of G-CSF alone for 3-5 days produced a decrease in L-selectin expression in neutrophils (25 +/- 4 versus 7 +/- 1, mean +/- SEM; mean channel fluorescence, n = 10) with no effect on neutrophil elastase activity (3.1 +/- 0.3 versus 3.4 +/- 0.6; micrograms elastase/million cells; n = 9). In contrast, in patients in the BMT group the L-selectin expression was increased (26 +/- 2 versus 38 +/- 3; n = 20) and elastase activity was markedly decreased (2.9 +/- 0.2 versus 1.4 +/- 0.2, n = 12) compared with values before BMT. The changes in L-selectin expression correlated with the ability of neutrophils to adhere to human umbilical vein endothelial cells. The decrease in the neutrophil elastase activity was not associated with an increase in the plasma elastase/alpha 1-antitrypsin complex levels, indicating that the decrease in the neutrophil elastase activity is not caused by activation of neutrophils and release of the enzyme into the plasma. Administration of G-CSF alone did not cause a decrease in the neutrophil elastase activity but increased plasma elastase/alpha 1-antitrypsin complex levels. There was no change in CR3 expression on neutrophils under any of these conditions. These observations suggest that the changes seen in neutrophils during BMT are influenced by various factors associated with BMT other than the administered cytokine alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535571 TI - Cytokines and T cell switching. AB - In recent years, the phenotypic characterization of T cell subsets has given way to a functional dichotomy based essentially on their cytokine profiles. In this context, the CD4+ helper T cell subset has been shown to consist of two types, termed Th1 and Th2. In general, Th1 cells produce interleukin (IL)-2 and interferon (IFN)-gamma, while Th2 cells characteristically produce IL-4, IL-5, and IL-6. The major function of the Th1 subset is to mediate delayed-type hypersensitivity reactions and their secondary function is suppression of B cell activity. In contrast, the major function of the Th2 subset is to provide B cell help, while their secondary function is cell-mediated immune suppression. A similar dichotomy has also been described for CD8+ T cells. The role that these functional T cell subsets and their cytokines play in terms of their protective and nonprotective outcomes in a variety of infectious and oral diseases is reviewed. PMID- 7535572 TI - Chronic nitric oxide synthase inhibition aggravates glomerular injury in rats with subtotal nephrectomy. AB - Besides its glomerular hemodynamic effects, nitric oxide (NO) inhibits platelet aggregation and mesangial cell proliferation, two mechanisms possibly involved in the pathogenesis of glomerulosclerosis (GS). Chronic NO synthase inhibition in the rat leads to marked arterial hypertension and promotes glomerular and interstitial injury, but only mild GS. In this study, NO synthase blockade by nitro-L-arginine methyl ester (L-NAME) was associated with 5/6 nephrectomy, a well-known model of GS. Sixty-eight adult male Munich-Wistar rats were distributed among four groups: SHAM (no renal ablation or drug treatment), NX (5/6 nephrectomy), NX+NAME (5/6 nephrectomy and chronic treatment with L-NAME, 5 mg/dL in drinking water) and NX+NAME+L (as in group NX+NAME but also receiving the angiotensin II receptor inhibitor Losartan potassium (L), 25 mg/dL in drinking water). One week after ablation, rats of Group NX showed moderate glomerular hypertension and hypertrophy. Although glomerular enlargement was also modest in Group NX+NAME, glomerular hypertension was particularly severe in this group. Both alterations were absent in Group NX+NAME+L. Only incipient glomerular and interstitial injury occurred at this phase. Three weeks after ablation, renal structural injury was still modest in Group NX. By contrast, Group NX+NAME exhibited marked GS, glomerular ischemic injury, interstitial expansion, and creatinine retention. Renal injury was largely prevented in Group NX+NAME+L. Tuft enlargement occurred in all groups but was most prominent in Group NX. NO synthase inhibition aggravates parenchymal injury and functional impairment in the remanent kidney by mechanisms that may involve glomerular hypertension and renin-angiotensin activation but that appear to be unrelated to glomerular enlargement. PMID- 7535573 TI - Neuronal localization of glutamate receptor subunits in the basolateral amygdala. AB - Antibodies to the NMDAR1 glutamate receptor subunit and the GluR1 and GluR2/3 subunits of the AMPA glutamate receptor were used to localize these receptor components in the basolateral amygdala (ABL) of the rat and monkey. A similar localization pattern was observed in both species. Pyramidal neurons exhibited high levels of NMDAR1 and GluR2/3 immunoreactivity (ir), but low levels of GluR1 ir. Some non-pyramidal cells exhibited high levels of NMDAR1-ir or GluR1-ir, but none exhibited significant levels of GluR2/3-ir. This differential localization of receptor subunits suggests that glutamate receptors will exhibit specific functional properties in distinct subpopulations of ABL neurons. PMID- 7535574 TI - Differential subcellular distribution of glutamate and taurine in primary olfactory neurones. AB - Taurine and glutamate are widely distributed amino acids in the mammalian brain, including the olfactory bulb where their functions have not been fully elucidated. This study investigated the precise cellular distribution of taurine and glutamate in the nerve layer and the glomeruli of the olfactory bulb, using semiquantitative immunocytochemistry at the electron microscopic level. The results show that both amino acids are present at higher concentrations in primary olfactory neurones than in the postsynaptic dendrites. Glutamate is also enriched in terminals vs axons of the primary olfactory neurones. This suggests that glutamate acts as a neurotransmitter at the primary synapse in the olfactory system. An opposite concentration gradient is found for taurine, with a higher level of taurine-like immunoreactivity in axons than in terminals of primary olfactory neurones. Further studies are required before conclusions can be drawn about the function of taurine in these neurones. PMID- 7535575 TI - NADPH-diaphorase localization in the olfactory system. AB - NADPH-diaphorase staining was studied in the intact olfactory system and following transections of the olfactory nerve. Intense staining was found in the olfactory epithelia of rat and salamander, especially at the mucosal surface. The olfactory bulb nerve and glomerular layers were also stained. Transection of the olfactory nerve in salamander, or bulbectomy in rat, resulted in decreased staining at the mucosal surface, although staining increased in cells deeper in the epithelium. Deafferentation also caused the disappearance of staining in the olfactory nerve and glomerular layers of the bulb. These results support the notion of a role for NADPH in the olfactory system, perhaps as a biosynthesis substrate for NO modulation of cGMP in the developing epithelium and a complementary role for CO in the adult. PMID- 7535577 TI - Ultrastructural study on NOS-immunoreactive nerve terminals in the rat coeliac ganglion. AB - Nerve terminals immunoreactive for nitric oxide synthase (NOS) were studied in the rat coeliac-superior mesenteric ganglion by electron microscopy using a pre embedding immunostaining method. The immunoreactive material was distributed in the axoplasmic matrix and was not specifically associated with any subcellular organelle. In most NOS-immunoreactive axon terminals numerous small clear vesicles (35-50 nm) were seen, and in some terminals a small number of large granular vesicles (70-120 nm) were intermingled with small clear vesicles. Most NOS-immunoreactive axon terminals formed axodendritic as well as axo-somatic synapses with non-immunoreactive ganglion neurones, and axo-axonic contacts were very scarce. These results suggest that NO may be released at the synaptic sites from the axon terminals and may affect ganglion neurones. PMID- 7535576 TI - Myelin degrading activity in the CSF of HIV-1-infected patients with neurological diseases. AB - Degradation of purified myelin basic protein (MBP) was studied by SDS gel electrophoresis after addition of CSF samples obtained from HIV-1-infected patients. An increase in MBP degradation was detected in patients with neurological complications, such as AIDS dementia complex (ADC) or progressive multifocal leukoencephalopathy (PML), when compared with patients with no neurological symptoms (NA) or with other neurological opportunistic infections (OI). In the ADC and PML patients, in addition to CSF proteolytic activity, an increase in CSF-MBP levels and presence of white matter lesions were also observed by neuroimaging (MRI). In other opportunistic infections of the brain, MBP levels but not anti-MBP proteolytic activity increased. Results suggest the involvement of proteases in the virus-induced demyelination. PMID- 7535578 TI - NOS is present in the brain of Triatoma infestans and is colocalized with CCK. AB - Immunohistochemistry was used to establish the presence of nitric oxide synthase (NOS) in the central nervous system of Triatoma infestans which is the main vector for Chagas' disease in Argentina and neighbouring countries. In addition, we have investigated the presence of cholecystokinin (CCK) and studied the possible coexistence of these molecules. The results show NOS-like immunoreactivity (LI) in neurones of the soma rind of the protocerebrum, the optic lobe and in the lateral part of the sensory deutocerebrum with a few cells in the suboesophageal and the prothoracic ganglia. The distribution of CCK-LI was similar to that of NOS-LI and in several areas both molecules coexisted in neurones and fibres. The results suggest that nitric oxide may act as a neurotransmitter in the brain of insects. PMID- 7535579 TI - NO synthase and free radical generation in brain regions of old rats: correlations with individual behaviour. AB - We have investigated the activity of NO synthase (NOS) and generation of free radicals (FRG) in selected brain regions of old male Wistar rats. Using the emotional resonance test, two groups of rats were selected for the experiment: passive, preferring dark space and active, preferring light space. Highest NOS activity and FRG were seen in cerebellum. As a rule, NOS activity was lower and FRG higher in the respective brain regions of active rats than in passive rats. Positive linear inter-regional cross-correlations of NOS activity as well as of FRG were found. When all rats were assessed as one group, negative correlations between NOS and FRG in cerebral cortex were revealed. PMID- 7535581 TI - [Blast transformation of lymphocytes and natural killer cell activity in the presence of gamma-globulin in vitro]. PMID- 7535580 TI - [Circulating serum interferon and its effect on human natural killer cell activity]. PMID- 7535583 TI - Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells. AB - We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM-1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow-conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury. PMID- 7535582 TI - To C or not to C: these are the questions. PMID- 7535584 TI - Metabolic persistence of fetal hemoglobin. AB - Hereditary persistence of fetal hemoglobin (HPFH) has typically been ascribed to mutations in the beta-globin gene cluster. Pharmacologic agents, including the short-chain fatty acid butyrate, have been shown to upregulate fetal and embryonic globin gene expression. In this report we investigate the possibility that metabolic derangements characterized by an inability to metabolize another short-chain fatty acid, propionate, could be associated with a persistence of fetal hemoglobin unrelated to alterations in the beta-globin cluster. Embryonic globin gene upregulation in a murine adult erythroid cell culture was shown by RNase protection after induction with three short-chain fatty acids (C2-C5). Chart reviews and measurement of fetal hemoglobin in five patients with abnormalities in propionate (C3) metabolism were undertaken; SSCP/dideoxy fingerprint analysis of the gamma-globin gene promoters was done in three of these five patients. Twelve patients with other metabolic derangements served as controls. Only the four patients with clinically severe abnormalities in propionate metabolism (ages 2 to 11), but without anemia, showed a sustained elevation in fetal hemoglobin (3% to 10%). The level of elevation of fetal hemoglobin in these patients, who lack erythropoietic stress, suggests that propionic acid and/or its metabolites are potent stimulators of fetal hemoglobin expression. Study of this group of patients should allow unique insights into the long-term effects of sustained exposure to elevations of short-chain fatty acid levels. PMID- 7535587 TI - Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors. AB - Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL 6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353 fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-). PMID- 7535585 TI - Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. AB - Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU-Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg. PMID- 7535586 TI - Influence of molecular characteristics on clinical outcome in human immunodeficiency virus-associated non-Hodgkin's lymphoma: identification of a subgroup with favorable clinical outcome. AB - The relationship between clinical and molecular characteristics of 45 treated individuals with histologically-documented human immunodeficiency virus (HIV) associated B-cell non-Hodgkin's lymphoma was examined to determine whether differences in molecular features of lymphoma were associated with differences in clinical outcome. Tissue specimens from these tumors were evaluated for evidence of Ig heavy-chain gene rearrangements using both Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Lymphomas were also evaluated for the presence of Epstein-Barr virus (EBV) DNA sequences and c-myc gene rearrangements. Twenty-five lymphomas were characterized as polyclonal and 20 as monoclonal. PCR amplification of expressed Ig variable (V)-region genes confirmed polyclonality in three extensively studied polyclonal lymphomas. The median CD4 count was significantly higher in the group with polyclonal disease (277/microL) than in the group with monoclonal disease (123/microL), P = .04. The complete response rate to therapy was significantly higher in patients with polyclonal disease (78%) and CD4 greater than 200/microL (81%) than in those with monoclonal disease (31%) and CD4 less than 200/microL (33%). CD4 count, clonality, and presence of EBV DNA sequences were the most important predictors of survival. Both Kaplan-Meier and Cox proportional hazards analyses showed a markedly prolonged survival in those patients with both CD4 > or = 200/microL and polyclonal disease. Histologically the polyclonal lymphomas were high grade in appearance and contained prominent macrophages. All seven surviving patients were in this group. Median survival for those individuals whose tumors contained EBV sequences was only 3.2 months (range, 0.4 to 19.5), whereas those with EBV- tumors survived for a median of 9.0 months (range, 0.7 to 65.2), P = .0007. These data indicate that molecular features of HIV-associated lymphomas may be important predictors of clinical outcome. These characteristics define a distinct subset of patients with polyclonal EBV- tumors and CD4 counts greater than 200/microL that appear to have a less aggressive clinical course. PMID- 7535589 TI - Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old. AB - Approximately 6% of dog platelets are positive for staining with thiazole orange, a dye frequently used to stain ribonucleic acid. In this report, thiazole-orange positivity is shown to mark platelets that are less than 24 hours old. Dog platelets were derivatized in vivo with N-hydroxysuccinimido biotin such that greater than 95% of all platelets were biotinylated. Newly synthesized, nonbiotinylated platelets were then monitored by flow cytometry for their ability to bind thiazole orange. After biotinylation, the percentage of biotin-negative, thiazole-orange-positive platelets increased gradually from 0.72% at 30 minutes to 5.44% at 24 hours. These data indicate that thiazole-orange staining does label newly synthesized platelets. PMID- 7535588 TI - Transforming growth factor beta 1 inhibits expression of the gene products for steel factor and its receptor (c-kit). AB - Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF-beta 1 has been shown to repress human and murine leukemic cell and murine lin- bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin- hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo. PMID- 7535590 TI - B220- bone marrow progenitor cells from New Zealand black autoimmune mice exhibit an age-associated decline in Pre-B and B-cell generation. AB - New Zealand Black (NZB) autoimmune mice exhibit progressive, age-dependent reduction in bone marrow pre-B cells. To ascertain the capacity of NZB bone marrow B220- cells to generate pre-B cells in a supportive environment, B-lineage (B220+) cell-depleted and T-cell-depleted bone marrow cells from NZB mice at 1 to 3, 6, and 10 to 11 months of age were adoptively transferred into irradiated (200R) C.B17 severe combined immunodeficient (SCID) mice. Bone marrow pre-B cells (sIgM- CD43[S7]- B220+) were assessed 3 and 10 weeks posttransfer. Pre-B cells and B cells were reconstituted in SCID recipients of older NZB progenitor cells by 10 weeks posttransplant, in contrast to the very low numbers of pre-B cells present in the donor bone marrow. However, B220- bone marrow progenitor cells from greater than 10-month-old NZB donors were deficient in the reconstitution of both pre-B and B cells in SCID recipients at 3 weeks post-transfer. This reflected a slower kinetics of repopulation, because older NZB-->SCID recipients had numbers of both pre-B and B cells similar to recipients of young NZB progenitor cells by 10 weeks posttransplant. Adoptive transfer of equal mixtures of BALB/c and older NZB bone marrow B220- progenitor cells into irradiated C.B17 SCID recipients failed to demonstrate active suppression. These results suggest that, with age, NZB bone marrow has reduced numbers and/or function of early B220 B-lineage progenitors. Consistent with this hypothesis, B220- bone marrow cells from older NZB mice were deficient in progenitors capable of yielding interleukin 7 (IL-7) responsive pre-B cells in vitro on stimulation with the pre-B-cell potentiating factor, insulin-like growth factor 1 (IGF-1). PMID- 7535591 TI - Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via alpha 4 beta 1 or alpha 5 beta 1 integrins. AB - Recent studies have shown that cell-surface integrins expressed on platelets, fibroblasts, or carcinoma cell lines serve not only as adhesion receptors that connect the extracellular matrix to the cytoskeleton, but also as signal transducing molecules involved in altering cellular patterns of tyrosine phosphorylation. In this present report we provide evidence that adhesion of freshly purified human natural killer (NK) cells to fibronectin (FN) induces tyrosine phosphorylation of intracellular proteins of approximate molecular mass of 60, 70, and 120 kD. Increases in phosphorylation induced by NK cell binding to immobilized FN were partially blocked by EILDV- (CS-1) or RGD-containing peptides, which compete specifically for a distinct binding site for either alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, within the FN molecule. The presence of either one of the inhibitory peptides alone inhibited tyrosine phosphorylation primarily during short-term (30 minutes) and, to a lesser extent, during long-term (2 to 3 hours) periods of adhesion. These observations indicate that triggering either via alpha 4 beta 1 or alpha 5 beta 1 integrins, which are constitutively expressed on NK cells, induces protein tyrosine phosphorylation. Moreover, FN fragments of 40 or 120 kD, known to contain the binding sites for alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, used as immobilized substrates for NK cell adhesion, were able to initiate tyrosine kinase activity. The induced tyrosine phosphorylation was observed mainly on intracellular proteins of greater than 50 kD molecular weight. We have identified a 70-kD tyrosine phosphoprotein as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions. Thus, interaction of NK cells with immobilized extracellular matrix glycoproteins required for migration and extravasation of these cells involves activation of intracellular protein tyrosine kinases and tyrosine phosphorylation of cytoskeleton-associated protein, paxillin, which may play a role in signaling between beta 1 integrins and the underlying cytoskeleton. PMID- 7535592 TI - Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia. AB - PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1). PMID- 7535593 TI - Alternative splicing restricts translation of rearrangements at the T-cell receptor delta/alpha locus. AB - Lymphocytes expressing alpha beta or gamma delta T-cell receptors (TCR) represent distinct T-cell populations. Because TCR delta genes lie within the TCR alpha locus, the rearrangement processes, transcription, and translation of TCR delta or TCR alpha variable domain exons require tight regulation. Human precursor B cell leukemias (eg, the REH cell line) constitute an interesting model to study TCR delta/alpha recombination because they rearrange TCR delta/alpha loci along a hierarchically ordered pathway in which V delta 2D delta 3 segments are joined to the J alpha cluster. We now show for REH cells that chimeric TCR delta/alpha variable domain exons are posttranscriptionally modified by alternative splicing resulting in truncated V delta 2C alpha transcripts. This process also takes place during thymic differentiation. CD7+/CD3- T-cell precursors exhibit V delta 2D delta 3 rearrangements. Further differentiation into CD7+/CD3+ thymocytes is associated with the expression of a truncated V delta 2C alpha RNA species. In contrast, chimeric TCR delta/alpha rearrangements containing a V delta 1 segment (but no D delta sequences) are predominantly expressed as full-length V delta 1J alpha C alpha transcripts. These data suggest that alternative splicing constitutes a mechanism that restricts the production of distinct chimeric TCR alpha chains. PMID- 7535594 TI - CD40 ligand triggered interleukin-6 secretion in multiple myeloma. AB - Previous studies have suggested that interleukin-6 (IL-6) may mediate growth of multiple myeloma (MM) in either an autocrine or paracrine growth mechanism. However, those molecules which can trigger IL-6 secretion either by tumor cells or non-MM marrow cells are not well characterized. In the present study, we have examined the expression and functional significance of CD40 on MM and plasma cell leukemia (PCL) cells and derived cell lines, as well as long-term bone marrow stromal cells (BMSCs) and derived cell lines. CD40 was expressed on the majority of MM cells (> 90%) and BMSCs (> 70%). Triggering via CD40 using NIH3T3 CD40 ligand transfectant (CD40LT) cells increased (> 30%) cell surface CD80, CD18, CD11a, CD11b, and CD11c expression on MM cell lines. Culture with either fresh or paraformaldehyde fixed NIH3T3 CD40LT cells upregulates IL-6 secretion in MM cells and MM-derived cell lines, as well as normal and MM bone marrow mononuclear cells (BMMCs), BMSCs, and BMSC lines; this effect can be specifically blocked by anti CD40 monoclonal antibody (MoAb). BMMCs and BMSCs from patients with MM secreted significantly more IL-6 than those from healthy donors (n = 3, P < .001); moreover, after stimulation using CD40L, IL-6 secretion was fourfold greater (n = 3, P < .001) from MM BMMCs and BMSCs than from normal BMMCs and BMSCs. Myeloma (CD38+CD45RA-) cells and non-MM (CD38+CD45RA+, CD38-CD45RA+, and CD38-CD45RA-) BMMCs were separated by dual fluorescence cell sorting. The latter secreted fourfold more IL-6 than the former (n = 2, P < .001). Increased IL-6 secretion (up to 28-fold) and proliferation (Stimulation index 10) by CD38+CD45RA-MM cells was triggered by culture with NIH3T3 CD40LT cells. Finally, anti-CD40MoAb partially (30%) blocked tumor cell to BMSC adhesion-induced IL-6 secretion. These studies support the view that CD40L may trigger IL-6 secretion by both MM cells and BMSCs and that IL-6-mediated autocrine and paracrine growth mechanisms may be possible in MM. PMID- 7535595 TI - Blood-derived autografts collected during granulocyte colony-stimulating factor enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells. AB - It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA DR- cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy related cytotoxic damage. PMID- 7535596 TI - Classification of lymphoid neoplasms between the hematopathologists and the "common person". PMID- 7535597 TI - Histiocytes and histiocytosis. PMID- 7535598 TI - Amino acid sequence of hen ovomacroglobulin (ovostatin) deduced from cloned cDNA. AB - The complete amino acid sequence of the hen ovomacroglobulin (ovostatin) subunit has been determined from cDNA and partial peptide sequence analysis. Ovostatin is a tetrameric member of the alpha-macroglobulin (alpha M) family of proteins. The 4715 nt ovostatin cDNA encodes a 36- or a 16-residue signal peptide and a 1437 residue mature protein (162.2 kDa). At the protein level the overall score of sequence identity between ovostatin and mammalian alpha Ms is 39-44%, indicating an early divergence from the line leading to the mammalian alpha Ms. Ovostatin contains 56 mol glucosamine per mol subunit, and 12 of its Asn-residues are likely to be N-glycosylated. Including carbohydrate, the size of the ovostatin subunit is approx. 185 kDa. The ovostatin subunit is predicted to contain 12 intrachain disulfide bridges, and two subunits are predicted to be disulfide bound by two interchain bridges. One Cys residue may be unpaired or participate in dimer formation as a third interchain disulfide bridge. Ovostatin contains a unique 40-residue bait region. In contrast to other alpha Ms, ovostatin contains no internal beta-Cys-gamma-Glu thiol ester, as a result of a Cys-to-Asn replacement (TGC or TGT to AAT), but the Gln-moiety of the thiol ester is preserved. By comparing the sequences of the receptor binding domain in alpha Ms with the corresponding region of ovostatin possible determinants for receptor recognition of mammalian alpha Ms are proposed. PMID- 7535601 TI - Studies on non-covalent associations of immunosuppressive drugs with serum albumin using pneumatically assisted electrospray ionization mass spectrometry. AB - Pneumatically assisted electrospray ionization (ion-spray) mass spectrometry was employed in the detection of non-covalent interactions of immunosuppressive drugs with proteins. Non-covalent complexes of albumin (rat and bovine) with FK506, dihydro-FK506, and FK520 were observed. No complexation was detected between albumin and cyclosporin A (CsA), even at a protein-ligand molar ratio of 1:20. It is suggested that the lack of detection of an albumin-CsA non-covalent complex may be attributed to dissociation of the weakly associated species during ion vaporization. Nonetheless, preliminary results indicate that ion-spray mass spectrometry has potential in qualitative evaluation of drug-plasma protein interaction. PMID- 7535599 TI - Antibody pattern's lack of predictivity in determining the response of viral hepatitis C to interferon therapy. AB - The aim of the study was to investigate whether the immunoblot pattern for HCV is a predictor of the response to interferon treatment. In a group of 60 patients with persistent rise of aminotransferase, all were treated with 3-6MU of Alfa-IFN from normal leucocytes every other day for 6 months, followed by one weekly dose of 1-3 MU for 3 months. HCV serum markers were detected before treatment and every three months thereafter. In 22 out of 60 (36.6%) patients aminotransferase normalized and remained so for 3 months after therapy; 12 patients (54.5%) relapsed during a follow-up of 9-12 months. The most frequent pattern in responders and non responders was the positivity to four antibodies (55%). The pattern did not change during or after IFN therapy, nor was it related to the variation of aminotransferases. Three patients lost antibodies linked to viral replication (c100-3, 5-1-1) and 3 others became positive to the same antigens. No changes were observed during the follow-up of patients who had an initial normalization of ALT/AST levels and who then relapsed (either during the maintenance dose or during the whole follow-up:n = 19 pts). Therefore neither the antibody clearance of viral replication (c100-3 and 5-1-1) nor the antibody pattern is a valid predictor as to the efficacy of interferon therapy. PMID- 7535600 TI - Whipple's disease: a continuous challenge. AB - Whipple's disease is a rare disease with protean clinical manifestations, often mimicking those of other pathological conditions. We describe two new cases, one admitted to hospital only after Giardia lamblia infestation had drawn attention to gut symptoms, and the other who was treated for a long time with steroids for suspected Horton's arteritis. Once again, we stress the importance of bearing this polymorph disease in mind, especially in older people. PMID- 7535602 TI - [Results of chemotherapy in cancers of the colon]. AB - With 25,700 new cases a year, colorectal cancer is the most common cancer in France. Until the late 80s, the only effective treatment was surgical excision. However, chemotherapy (palliative and adjuvant) has made great progress and interesting results have begun to be published, especially since 1990. Palliative treatment: the rate of tumor reduction is two times higher when using a combination of 5FU and folinic acid rather than 5FU alone and, according to some studies, this combination might significantly improve the duration and quality of survival. Two recent controlled studies, comparing systemic chemotherapy with symptomatic treatment, showed a significant improvement in the duration of survival and a study in asymptomatic patients indicated a significant extension of the duration of the symptom-free period. Intra-arterial hepatic chemotherapy significantly improves the survival but toxicity is high. Further clinical testing is needed to evaluate new agents and to improve response rates and tolerance to treatment. More study will also be required to evaluate chemotherapy as debulking of initially inoperable metastasis. Adjuvant treatment: in Dukes stage C colonic cancer a combined regimen using 5FU and levamisole is now the standard treatment and reduces the relative risk of recurrence and death by 41 and 33% respectively. A combination of 5FU and folinic acid has been shown at least as effective in term of 3 year outcome in three studies. Current studies have not demonstrated the efficacy of chemotherapy in patients with Dukes B colonic cancer. Efficacy is probably marginal and clearly lower than for stage C. USe of new prognostic factors such as ploidy which appears to be an independent and reliable predictor of the risk of recurrence in stage B patients, should allow accurate identification of patients able to benefit from adjuvant treatment. Some findings support early postoperative use of intraportal chemotherapy. Present trials in stage B and C patients which no longer include untreated control groups are under way to compare systemic treatments (5FU levamisole, 5FU-AF-levamisole) or combination of locoregional and systemic treatments (intraperitoneal or intraportal 5FU and systemic treatment vs systemic treatment alone). One such trial is the recently begun European study that is to include 2000 patients upon completion. Development of large-scale controlled trials will be necessary to build on the major gains that have been made in adjuvant treatment. PMID- 7535603 TI - [Antitumor electro-chemotherapy]. AB - We summarize the way which, starting at the electropermeabilization of cultured cells, a technique currently used in molecular and cellular biology, had led up to the electrochemotherapy, the new antitumor treatment that we have designed at the Institut Gustave-Roussy. Electrochemotherapy consists in intravenous administration of a low dose of bleomycin followed by local delivery of electric pulses on tumor nodules by means of two electrodes located at each side of the nodule. We describe the basis of the electrochemotherapy and we report the results obtained in mice and in clinical trials. Lastly we discuss the reasons for which electrochemotherapy, a treatment relevant to some precise clinical situations, might be widely instrumental in the treatment of cancer. PMID- 7535604 TI - When the nurse wields the scalpel... PMID- 7535605 TI - How does the law view patient consent? PMID- 7535606 TI - Alteration of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands. AB - Nontoxic doses of the histamine H2 antagonists ranitidine, cimetidine, lamtidine and mifentidine rapidly and reversibly increased tyrosinase activity in an amelanotic human melanoma cell line (MM96L) with low constitutive activity. The H2 antagonists, famotidine and MGTI, and the imidazol(in)e receptor ligand clonidine had no effect either alone or in competition with ranitidine, whilst metiamide decreased tyrosinase activity. Lysosomotropic amines had a similar effect to ranitidine, except that induction reached a plateau at 6 h and was insensitive to amiloride. Human melanocytes and pigmented human melanoma cell lines exhibited minimal levels of tyrosinase induction, which was dependent on protein synthesis but not on RNA or DNA synthesis. Constitutive tyrosinase activity in MM96L cells was much less stable than in melanocytes and pigmented melanoma cells. No change was observed in expression of gp75, neural specific octamer binding proteins, or in mRNA levels of tyrosinase, Pmel-17 and gp75 (TRP 1). Tyrosinase was inhibited by the H3 agonist imetit but not by alpha methylhistamine or the H3 antagonist thioperamide. Overall, this work showed that certain H2 antagonists activate an unstable form of tyrosinase in amelanotic melanoma cells by a post-transcriptional mechanism dependent on protein synthesis. An imidazoline/guanidinium receptor site rather than the H2 receptor appeared to be involved. PMID- 7535607 TI - Posters for accident departments: simple method of sustaining reduction in x ray examinations. AB - OBJECTIVE: To assess whether a simple strategy would sustain a reduction in the number of unnecessary x ray examinations. DESIGN: Use of posters to display guidelines encouraging the more effective use of radiology in patients with head injuries, twisted ankles, neck injuries, and abdominal pain. SETTING: Accident department of a large metropolitan district general hospital. PATIENTS: 15,875 patients attending the accident department over two years. MAIN OUTCOME MEASURE: Proportion of patients having radiography. RESULTS: Referrals for skull radiography fell from 56% to 20% and those for abdominal radiography fell from 31% to 7%. Referral patterns for adults attending with twisted ankles and cervical spine injuries did not change. Reductions were sustained over two years. CONCLUSION: Carefully designed posters provide a simple method of reducing unnecessary x ray examinations. PMID- 7535608 TI - Chemotherapy in head and neck cancer. AB - Chemotherapy has been used for many years as a palliative approach to advanced squamous cell carcinoma of the head and neck. Regimens employed have slowly evolved during this time, and the combination of cisplatin and 5-fluorouracil is still standard chemotherapy for such a tumour. However, clinical approaches to advanced squamous cell carcinoma of the head and neck are changing dramatically as physicians become increasingly familiar with multidisciplinary treatments. Integrating chemotherapy and radiotherapy, neo-adjuvant or adjuvant treatments and organ preservation are stimulating fields of investigation involving chemotherapy which definitely warrant further investigation. PMID- 7535609 TI - Combined therapy for angioimmunoproliferative lesions. AB - 43 patients with a diagnosis of angioimmunoproliferative lesions (AIL) entered onto a prospective clinical trial to evaluate the use of combined therapy as a primary therapeutic approach. Patients were treated initially with involved field radiotherapy 40-55 Gy (40 patients received 45 Gy) followed by six cycles of chemotherapy which consisted of CEOP-Bleo (cyclophosphamide, epirubin, vincristine, prednisone and bleomycin). Complete response was achieved in 41 cases (95%). At a median follow-up of 40 months, 40 patients (91%) remain in first complete remission. 2 patients died during radiotherapy secondary to sepsis and tumour progression. Treatment was well tolerated. The treatment of AIL remains controversial. Our results show that combined therapy appears to be the best therapeutic approach in patients with this type of malignant lymphoma. More studies are necessary to define the role of combined therapy in patients with AIL. PMID- 7535610 TI - Expression of human cytokeratin 14 in normal, premalignant and malignant oral tissue following isolation by plaque differential hybridisation. AB - Differences in gene transcription between RNA samples extracted from oral normal and squamous cell carcinoma (SCC) tissue were examined using the technique of cDNA library differential plaque screening. A differentially expressed transcript was selected on the basis of it being under-expressed in the cancer tissue and was identified, using DNA sequencing, as cytokeratin 14. The level of cytokeratin 14 transcription in RNA samples extracted from a range of oral SCC and normal tissue, as well as "white patch" lesions, was then investigated. Cytokeratin 14 appeared to be significantly under-expressed in oral cancer specimens studied compared to normal and white-patch tissue (P < 0.01). The trend for higher levels of cytokeratin 14 transcription in the dysplastic "white patch" samples compared to that observed for the malignant tissue (P < 0.05) suggests that the decrease in cytokeratin 14 transcription is a late event in the carcinogenic pathway. PMID- 7535611 TI - Response of mouse oral mucosa to repeated doses of bleomycin. AB - Bleomycin (BLM) applied at systemically tolerable doses induces denudation of tongue mucosa in the C3H-Neuherberg mouse strain. The dose-incidence curve after single injections has a sigmoid shape with an ED50 of 17.5 mg/kg. In contrast, the dose-response curves to repeated (two, five and 10) drug injections follow triphasic shapes and show dose-effect inversions. The effect initially increases with dose to a maximum of 70-100% at 2 x 7, 5 x 2, and 10 x 0.9 mg/kg. A marked decrease in response is observed at higher doses with a nadir of 10-30% after 2 x 11 mg/kg, 5 x 4 to 5 x 5 mg/kg and 10 x 2 mg/kg, followed by a second rise when dose is further increased. These clinical results were confirmed in a histological study. Variation of the time interval between two drug injections caused marked fluctuations in the treatment efficacy. A clear increase in drug response was induced by splitting total drug doses of 6, 14 or 22 mg/kg, the maximum effect (100%) was seen at intervals of 2 h, 0.5-1 h and 0.25 h between two injections of 3, 7 or 11 mg/kg, respectively. At longer intervals of up to 6 h, a dose-dependent decrease in drug efficacy resulted in an inverse dose-effect. Original tissue tolerance to BLM was restored only in the 2 x 3 mg/kg arm but was still elevated in the other arms after 96 h. The results can be plausibly explained by the dose-dependent induction of detoxifying processes. PMID- 7535612 TI - Redesigning the hydrophobic core of a four-helix-bundle protein. AB - Rationally redesigned variants of the 4-helix-bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native-like physical properties of the wild-type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild-type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild-type protein. The rate of chemically induced folding and unfolding of wild-type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type. PMID- 7535615 TI - [Modeling the effect of structural reorganization during formation of the secondary structure of RNA]. PMID- 7535614 TI - Immunoglobulin fold characteristics of B7-1 (CD80) and B7-2 (CD86). AB - B7-1 and B7-2 are expressed on antigen-presenting cells and bind to the CD28 and CTLA-4 receptors on T cells. These interactions trigger a costimulatory pathway that is essential for T-cell activation. B7-1 and B7-2 are members of the immunoglobulin superfamily (IgSF) and, despite sharing common function, have only limited sequence similarity. The B7-1 extracellular region was previously subdivided into 2 IgSF domains, an N-terminal V(ariable)-like domain, followed by a C(onstant)-like domain. We recently reported that the V-like domains of B7-1 and B7-2 share some significant sequence similarities with 3 major histocompatibility complex (MHC)-encoded members of the IgSF. We have now applied inverse folding methodology to assess the compatibility of the B7-1 and B7-2 extracellular region sequences with currently available 3-dimensional structures. In these calculations, the sequences of the N-terminal (V-like) domains in B7-1 and B7-2 were not compatible with known structures, including the IgSF V-set. In contrast, the sequences of the C-like domains were compatible with IgSF C-set structures and were best recognized by the beta 2-microglobulin (beta 2m) domain of MHC Class I. A sequence comparison of the C-like domains in the B7 molecules showed that 11 of 17 rigorously conserved residues in B7-1 and B7-2 are not IgSF C-1 set consensus residues. When mapped onto the corresponding positions of the beta 2m structure, the conserved residues in B7 cluster on the surface, where they may interact with the B7 V-like domain or other molecules. PMID- 7535616 TI - Amino acid functional groups involved in the binding of Escherichia coli ribosomal protein S1 to ribosomes and nucleic acids. AB - Histidine, arginine, tyrosine, lysine and cysteine residues of protein S1 were modified with diethyl pyrocarbonate & rose bengal, 2,3-butanedione (diacetyl), tetranitromethane, pyridoxal 5-phosphate, and N-ethyl-maleimide, respectively. Modification of the residues and the number of modified residues were determined by either fluorescence or UV spectroscopy. The effect of chemical modification on the function of protein S1 was studied with respect to nucleic acid (poly U and M13 ssDNA) binding and ribosome binding properties of the protein. We tested S1 binding to these two types of polynucleotides because of their reported (Draper et al. (1977) PNAS 74, 4786-4790) binding to two different sites on S1. The results indicate that histidine and lysine residues of S1 play an important role in the binding of S1 to both types of nucleic acids and that histidine and to some extent tyrosine residues are involved in the binding of S1 to ribosomes. The Data indicate the need for a re-evaluation of the two nucleic acid binding-site model. PMID- 7535613 TI - A structural model for the prostate disease marker, human prostate-specific antigen. AB - Prostate-specific antigen (PSA) provides an excellent serum marker for prostate cancer, the most frequent form of cancer in American males. PSA is a 237-residue protease based on sequence homology to kallikrein-like enzymes. To predict the 3 dimensional structure of PSA, homology modeling studies were performed based on sequence and structural alignments with tonin, pancreatic kallikrein, chymotrypsin, and trypsin. The structurally conserved regions of the 4 reference X-ray proteins provided the core structure of PSA, whereas the loop structures were modeled on the loops of tonin and kallikrein. The unique "kallikrein loop" insert, between Ser 95b and Pro 95k of kallikrein, was constructed using molecular mechanics, dynamics, and electrostatics calculations. In the resulting PSA structure, the catalytic triad, involving residues His 57, Asp 102, and Ser 195, and hydrophobic and electrostatic interactions typical of serine proteases were extremely well conserved. Similarly, the 5-disulfide bonds of kallikrein were also conserved in PSA. These results, together with the fact that no major steric clashes arose during the modeling process, provide strong evidence for the validity of the PSA model. Calculation of the electrostatic potential contours of kallikrein and PSA was carried out using the finite difference Poisson-Boltzmann method. The calculations revealed matching areas of negative potential near the catalytic triad, but differences in the positive potential surrounding the active site. The PSA glycosylation site, Asn 61, is fully accessible to the solvent and is enclosed in a positive region of the isopotential map. The bottom of the substrate specificity pocket, residue S1, is a serine (Ser 189) as in chymotrypsin, rather than aspartate (Asp 189) as in tonin, kallikrein, and trypsin. This fact, plus other features of the S1 binding-pocket region, suggest that PSA would prefer substrates with hydrophobic residues at the P1 position. The location of a potential zinc ion binding site involving the side chain of histidines 91, 101, and 233 is also suggested. This PSA model should facilitate the understanding and prediction of structural and functional properties of this important cancer marker. PMID- 7535617 TI - Helicobacter pylori aggregating activity of gastric mucin with ulcer healing by ebrotidine. AB - The mucin isolated from gastric secretion of duodenal ulcer patients before and after therapy with a new antiulcer agent, ebrotidine, was assessed for H. pylori aggregating activity and macromolecular organization. Analyses of mucin molecular forms revealed that successful therapy with ebrotidine was accompanied by a 2.6 2.9-fold increase in the high molecular weight mucin form. The H. pylori aggregation inhibition assays showed that therapy with ebrotidine evoked a 4-fold increase in mucin anti-H. pylori titer. The changes in the functional properties of mucin following ebrotidine therapy were also accompanied by a 36% increase in the content of sulfomucin. The results demonstrate that ulcer therapy with ebrotidine lead to a marked enhancement in mucin's qualities associated with maintenance of gastric mucosal integrity and strengthening the indigenous defenses against H. pylori. PMID- 7535618 TI - Secretory proteins and growth factors of the baboon (Papio anubis) uterus: potential roles in pregnancy. AB - The primate endometrium undergoes distinct morphological changes during the menstrual cycle. These alterations are regulated by the steroid hormones, estrogen and progesterone. Several lines of evidence suggest that some of these hormonally induced changes may be modulated by growth factors. Our studies have focused on characterizing the secretory activity of the uterine endometrium associated with these hormonally regulated morphological changes during the menstrual cycle and pregnancy in the baboon. Additionally, we have also attempted to study the regulation of specific growth factors and their receptors. In this review we present evidence to indicate that growth factor receptors for insulin like growth factor-I (IGF-I) and epidermal growth factor (EGF), and secretory proteins, insulin-like growth factor binding protein-1 (IGFBP-1) and retinol binding protein (RBP), which are present in the glandular epithelium during the menstrual cycle, undergo cell-specific changes in gene expression at the implantation site during pregnancy. We postulate that these alterations in growth factor receptor and secretory protein expression are conceptus modulated and may play important regulatory roles during trophoblast invasion and decidualization. PMID- 7535619 TI - Identification of the cDNA, gene and promoter for a major protein from flexible cuticles of the giant silkmoth Hyalophora cecropia. AB - We have isolated and sequenced a cDNA and region of genomic DNA that encode HCCP12, a major protein found in flexible cuticles of all three metamorphic stages of the giant silkmoth, Hyalophora cecropia. The gene for HCCP12 contains two introns with the first intron interrupting the signal peptide. The 5' flanking region contains 478 base pairs (bp) with about 95% identity to the prototype of a common Cecropia insertion element. The RACE procedure was used to produce cDNAs corresponding to the 5' ends of mRNAs for HCCP12 isolated from larval, pupal and pharate adult epidermal cells. Sequences from 21 cloned cDNAs were almost identical. It was thus concluded that a single gene and a single promoter (as evidenced by a single transcriptional start site) for HCCP12 are used in all three metamorphic stages. Hence, neither stage-specific gene sets nor stage-specific promoters are essential for metamorphic transitions of cuticular protein gene expression. PMID- 7535622 TI - Objective noninvasive evaluation of benign prostatic hypertrophy. AB - An objective noninvasive procedure has been developed to evaluate the urodynamics of benign prostatic hypertrophy. The test uses pneumatic occlusive cuffs similar to those used for blood pressure measurements and the electrical engineering concepts of open-circuit, short-circuit, and transient response measurements. The cuff is first inflated to measure pressure, then rapidly released, yielding the transient response and subsequent unimpeded flow. From the pressure and flowrate recordings as functions of time, objective evaluations of bladder strength and urethral obstruction are extracted. PMID- 7535621 TI - Role of T cell DNA methylation in lupus syndromes. AB - Current theories postulate that exposure to certain environmental agents will induce lupus in genetically predisposed individuals. However, the mechanisms by which environmental agents interact with the immune system to trigger lupus is unclear. Recent work has shown that some environmental agents associated with lupus, such as procainamide, hydralazine and ultraviolet light, will inhibit T cell DNA methylation, increase LFA-1 expression and induce autoreactivity. In addition, T cells isolated from patients with active lupus have hypomethlated DNA, diminished DNA methyltransferase activity and overexpress LFA-1 on an autoreactive subset of cells which spontaneously lyses autologous macrophages. More recent work has shown that the adoptive transfer of murine T cells made autoreactive with DNA methylation inhibitors is sufficient to cause a lupus-like disease in otherwise healthy syngeneic recipients. Together, these results support a new model of autoimmunity, in which certain environmental agents modify T cells by inhibiting DNA methylation and altering expression of certain genes, thereby inducing autoreactivity. The autoreactive cells then interact with the host to produce a lupus-like disease. PMID- 7535620 TI - Laparoscopic bilateral cutaneous ureterostomy for palliation of ureteral obstruction caused by advanced pelvic cancer. AB - In cases of advanced urologic malignancies with impairment of renal function secondary to tumor infiltration in high-risk patients, the possibility of performing a laparoscopic instead of an open cutaneous ureterostomy should be considered. We performed laparoscopic cutaneous ureterostomy in three male patients, two with prostate cancer and one with bladder cancer, and in one female patient with uterine cancer. Five operative ports were used. The ureters were identified, dissected, severed, and passed through two 10-mm ports; and cutaneous ureterostomies were performed in the usual manner. The mean operative time was 96 minutes. Patients were discharged after 5 to 7 (mean 6) days. The two patients with prostate cancer are now in treatment with GnRH analogues with a follow-up of 3 and 7 months. The patient with bladder cancer underwent palliative radiotherapy and is well after 6 months. The patient with uterine cancer has stable disease after 3 months. Laparoscopic urinary diversion causes less discomfort and has a low complication rate and may be the first-choice diversion in patients with advanced cancer who have a life expectancy longer than 6 months. PMID- 7535623 TI - Four patients with polyendocrinopathy with associated pituitary hormone deficiency. AB - Four cases of polyglandular endocrine disorders associated with pituitary hormone secretion failure are reported. Three of them had both insulin dependent diabetes mellitus (IDDM) and Hashimoto's disease. Each of these patients (cases 1-3) showed isolated deficiency of ACTH, TSH or gonadotropin, respectively. Another patient (case 4) had both Hashimoto's disease and isolated ACTH deficiency. Anti pituitary antibody to AtT-20 cells was detected in case 1. Serum gamma-globulins from patients 1 and 4 attenuated corticotropin releasing hormone-induced ACTH release in monolayer cultured rat anterior pituitary cells. Gamma-globulins from patients 1 and 2 decreased baseline TSH release but stimulated baseline prolactin release in pituitary cell cultures. It is possible that pituitary hormone deficiency in these patients may be caused by autoimmune disorders. PMID- 7535624 TI - The role of cAMP- and Ca(2+)-dependent intracellular mechanisms in the control of oxytocin and vasopressin secretion by bovine ovarian granulosa cells in vitro. AB - The secretion of oxytocin and arginine-vasopressin was demonstrated in bovine granulosa cell culture. It was found that dbcAMP or 3-isobutyl-1-methyl-xanthine (an inhibitor of intracellular cAMP metabolization) additions increased both oxytocin and vasopressin release. The Ca2+ ionophore A23187 also stimulated, while the Ca2+ channel blocker verapamil inhibited the secretion of both nonapeptide hormones. These results suggest the involvement of cAMP- and Ca(2+) dependent intracellular mechanisms in the stimulation of both oxytocin and vasopressin secretion by bovine granulosa cells. PMID- 7535625 TI - Surveillance of measles since the vaccination campaign. PMID- 7535626 TI - An in vivo screen for the luciferase transgene in zebrafish. AB - A simple and economical large-scale in vivo screen for firefly luciferase expression in transgenic zebrafish is described. The screen is a film assay of luminescence during embryogenesis. Either luciferin substrate can be microinjected into the embryo, or the embryo can be raised in a luciferin solution. In a test of transient expression in the G0 (microinjected) generation, a construct with the human cytomegalovirus (CMV) promoter gave higher levels of expression than three other constructs. Using the CMV promoter, injection of supercoiled or linear DNA led to approximately equivalent amounts of expression. Although G0 transient luciferase expression is high enough to be reliably screened, G1 integrated expression is either low or nonexistent, and therefore unscreenable. In the G1 and G2 generations, low-level expression was increased with application of 5-azacytidine. The fact that both transgene methylation and 5 azacytidine activation of expression occurred suggests that methylation is involved in either reducing or eliminating integrated luciferase expression. This in vivo luciferase screen may be useful for insertional mutagenesis, promoter, gene, or enhancer traps, promoter analysis, and optimization of conditions for gene transfer. PMID- 7535627 TI - Arsenic speciation by micellar liquid chromatography with inductively coupled plasma mass spectrometric detection. AB - Four environmentally and biologically important arsenic species, dimethylarsenic acid (DMA), monomethylarsonic acid (MMA), As(III) and As(V) are separated by micellar liquid chromatography. Linear dynamic ranges for the four species are three orders of magnitude and detection limits are in the picogram range with inductively coupled plasma mass spectrometric (ICP-MS) detection. This paper discussed in detail the development of the chromatographic conditions. The micellar mobile phase, which consisted of 0.05 M cetyltrimethylammonium bromide, 10% propanol and 0.02 M borate buffer, showed good compatibility with ICP-MS. This method allowed direct injection of urine samples onto the chromatographic system without extensive pretreatment and presented no interference from chlorine in the matrix. Detection limits are comparable with other LC-ICP-MS studies. An SRM urine sample was used to demonstrate the applicability of this technique to "real-life" situations. Results indicated that DMA, MMA and As(V) were present in the urine sample. PMID- 7535629 TI - Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells. AB - To better understand the role of insulin-related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)--an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at 10(-8) M, increased the proportion of PM1-positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted-local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF's role, local IGFBPs expression can contribute to the fine modulation. PMID- 7535628 TI - The L2/HNK-1 carbohydrate is carried by the myelin associated glycoprotein and sulphated glucuronyl glycolipids in muscle but not cutaneous nerves of adult mice. AB - We have previously shown that myelinating Schwann cells associated with motor, but not sensory, axons in peripheral nerves of adult mice express the L2/HNK-1 carbohydrate epitope. This carbohydrate structure carried by glycolipids and neural cell adhesion molecules has been suggested to specifically foster regrowth of motor as opposed to sensory axons after infliction of a lesion. To determine which molecular components may be the carriers of the L2 carbohydrate in motor axon-associated myelinating Schwann cells, we have isolated the purely sensory, cutaneous branch and the mixed sensory and motor muscle branch of the femoral nerve of adult mice, isolated the myelin fraction thereof and analysed the molecules expressing the L2 carbohydrate by several immunochemical methods. L2 immunoreactivity in myelin of the muscle branch was four to five times higher than that of the cutaneous branch. The 110 kDa L2-immunoreactive glycoprotein in myelin of the muscle branch, which is not L2-immunoreactive in the cutaneous branch, was identified as the myelin-associated glycoprotein by a combination of immunoprecipitation and Western blot analysis. Myelin extraction with organic solvents additionally revealed the two L2-carrying glycolipids, which amounted to approximately 40 ng glycolipid/mg dry weight in myelin of the muscle branch, whereas no significant amounts of the L2 glycolipids were found in myelin of the cutaneous branch. These observations suggest an astonishing degree of differential regulation of carbohydratesynthesizing activities in myelinating Schwann cells. PMID- 7535630 TI - Location and anatomical connections of a paradoxical sleep induction site in the cat ventral pontine tegmentum. AB - The brainstem mechanisms for the generation of paradoxical sleep are under considerable debate. Previous experiments in cats have demonstrated that injections of the cholinergic agonist carbachol into the oral pontine tegmentum elicit paradoxical sleep behaviour and its polygraphic correlates. The different results on the pontine structures that mediate this effect do not agree. We report here that limited microinjections of a carbachol solution into the ventral part of the oral pontine reticular nucleus in the cat induce, with a short latency, a dramatic, long-lasting increase in paradoxical sleep. Moreover, neuronal tracing experiments show that this pontine site is connected with brain structures responsible for the different bioelectric events of paradoxical sleep. These two facts suggest that the ventral part of the oral pontine reticular nucleus is a nodal link in the neuronal network underlying paradoxical sleep mechanisms. PMID- 7535631 TI - Detection of Buchnera, the primary prokaryotic endosymbiont of aphids, using the polymerase chain reaction. AB - Members of the genus Buchnera constitute a distinct prokaryotic lineage containing the primary endosymbionts of aphids (Homoptera: Aphidoidea). Using synthetic oligonucleotides in conjunction with the polymerase chain reaction, we propose three approaches for the identification of members of this genus. The first is based on unique sequences within rrs (gene coding for 16S ribosomal RNA). The second is based on a different and unique organization of the ribosomal RNA operons of Buchnera and the close proximity of aroE upstream of rrl (gene coding for 23S rRNA). The third is based on the linkage relationship of argS which is upstream of rrs. Validation of these three approaches requires their more extensive application. PMID- 7535632 TI - Differential expression of beta 1, beta 3 and beta 4 integrins in sarcomas of the small, round, blue cell category. AB - Integrins are a large and complex family of membrane spanning alpha beta heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of beta 1, beta 3 and beta 4 integrins in situ. We found complex and to some extent differential patterns of beta 1, beta 3 and beta 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently beta 1+, beta 4-, alpha 2-. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was alpha 5+ throughout and three RMS were focally alpha 5+. Three RMS expressed the alpha 6 and alpha v chains. In contrast to RMS, pPNET and ES, all of which were alpha 1-, alpha 3-, (G)NB were alpha 3+ and frequently co-expressed alpha 1. The eight pPNET and seven ES studied showed a similarly restricted integrin profile that was limited to the expression of beta 1 and alpha 5 in nearly all cases. In summary, RMS were beta 1+, alpha 1-, alpha 3- and heterogeneously expressed alpha 5 and alpha 6. (G)NB were generally beta 1+, alpha 1+, alpha 3+, alpha 5-, alpha 6-. pPNET and ES were beta 1+, alpha 1-, alpha 3-, alpha 5+, alpha 6-. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES. PMID- 7535633 TI - Expression of myosin heavy chain isoforms in mammary epithelial cells and in myofibroblasts from different fibrotic settings during neoplasia. AB - The expression of smooth muscle (SM) and non-muscle (NM) myosin heavy chain (MyHC) isoforms has been studied in fibroblastic cells of different fibrotic lesions (hypertrophic scars, Dupuytren's nodules and stromal reaction to mammary carcinoma) and in epithelial cells of non-neoplastic and neoplastic mammary glands, using anti-myosin antibodies in immunofluorescence and Western blotting. Two antibodies were specific for SM-MyHC isoforms (SM1 and SM2) and three antibodies were directed against different sequences of NM-MyHC isoforms. Myofibroblasts containing SM-MyHC were present in a variable number of cases of the different lesions: 1 of 11 hypertrophic scars, 3 of 9 Dupuytren's nodules and 20 of 25 breast cancers. The distribution of NM-MyHC sequences recognized by our antibodies was heterogeneous in fibroblasts from normal dermis and mammary stroma, but became homogeneous in myofibroblasts from all the pathological conditions examined. Moreover, the expression of these MyHC sequences differed in normal mammary epithelium when compared with invasive carcinoma. These results show that cellular modulation from fibroblast to myofibroblast may be accompanied by the appearance of SM-MyHC and is characterized by a uniform expression of MyHC of NM type, and that tumour progression in mammary epithelial cells may be paralleled by the disappearance of a specific NM-MyHC sequence. This suggests that MyHC modulation participates in the process of fibrosis as well as in the process of malignant epithelial transformation. PMID- 7535634 TI - Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. AB - This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79, n = 28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF beta 1, and TGF-beta 2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5 alpha-Reductase, 3 alpha-hydroxysteroid oxidoreductase, and 17 beta-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis. PMID- 7535638 TI - A study of acute side-effects related to palliative radiotherapy treatment of lung cancer. AB - Two recent studies carried out by the Medical Research Council Lung Cancer Working Party have suggested that large fraction radiotherapy to the chest in either 10-Gy single fraction or 17-Gy two-fraction doses, 1 week apart, is safe and effective for patients who require palliation for bronchogenic cancer. The Beatson Oncology Centre, Glasgow, participated in the original MRC trial and anecdotal reports of acute chest pains, fevers, sweats and rigors in some patients during the first 24-hour period after radiotherapy treatment were noted. These acute side-effects were not monitored during the Medical Research Council trials. It was felt that this area warranted further evaluation in order to identify the incidence of such acute side-effects and to what extent they caused a reduction in the patients' remaining quality of life. A pilot study of 10 patients confirmed the manifestation of the side-effects reported anecdotally in the MRC trial. It was on this basis that the study was extended, with a further 51 patients being invited to participate over a 4-month period. The findings indicate a significant incidence of adverse side-effects in patients receiving large fraction radiotherapy to the chest in either 10-Gy single fraction or 17-Gy two-fraction doses, but that these are transient and do not cause unacceptable disruption to the patients over an extended period. PMID- 7535635 TI - HH2A, an immortalized bovine mammary epithelial cell line, expresses the gene encoding mammary derived growth inhibitor (MDGI). AB - We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression. PMID- 7535637 TI - Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium. AB - The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins. PMID- 7535636 TI - An immortal cell line to study the role of endogenous CFTR in electrolyte absorption. AB - The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from 15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535639 TI - Prostanoid modulation of synovial antigen-specific CD4+ T-cell cytotoxic function in rheumatoid arthritis. AB - The recent demonstration of cytolytic mediators within synovial CD4+ T-cells of patients with rheumatoid arthritis (RA) has suggested an additional role for these cells in the pathogenesis of the disease. In this study we have investigated the function and regulation of antigen-specific class II-restricted cytotoxic T-cells from the synovial fluid (SFMNC) and peripheral blood (PBMNC) of 20 seropositive RA patients, and correlated in vitro findings with clinical data. Regulatory factors including prostaglandin E2 (PGE2), interferon-gamma (IFN gamma) and interleukin-4 (IL-4) were measured in cell supernatants. A diversity in SFMNC antigen-specific cytotoxicity that correlated with therapy and PGE2 production was found, and shown to be mediated by synovial prostanoid (products of cyclooxygenase metabolism) inhibition of effector function. Our findings indicate that SFMNC cytotoxicity may be important in the pathogenesis and treatment of RA. Cyclooxygenase inhibition as the sole treatment early in RA may reduce the potentially beneficial inhibitory effect of synovial prostanoids on antigen-specific SFMNC cytotoxicity. PMID- 7535640 TI - The value of neuroendocrine markers in non-small cell lung cancer: a comparative immunohistopathologic study. AB - In order to estimate the value of immunohistochemical identification of neuroendocrine (NE) differentiation markers in non-small cell lung carcinomas (NSCLCs), we investigated the expression of five neuroendocrine and neural differentiation-related antigens in 51 NSCLCs. Additionally, 20 epithelial lung tumors with NE differentiation [15 carcinoids and five small cell lung carcinomas (SCLCs)] and 61 epithelial tumors of various other origin (breast, prostate, colon and head-neck carcinomas) were studied. An indirect two-stage immunoperoxidase method was performed in formalin-fixed and paraffin-embedded tissue specimens, by using commercially available monoclonal antibodies. These antibodies are directed against neuron-specific enolase (NSE), chromogranin-A and Leu-7 which are general markers of NE differentiation, bombesin, which is a specific NE secretory product and neurofilament triplet protein (NFTP), an intermediate filament protein of neuronal differentiation. All five markers demonstrated a positive immunoreactivity in NSCLCs, equally distributed to all three histologic subtypes, ranging from 16 to 47% of the cases (NSE 47%, bombesin 21.5%, Leu-7 21.5%, chromogranin-A 18% and NFTP 16%). Most of the carcinoids and SCLCs expressed multiple or all NE markers. The other four epithelial tumors showed a positive immunoreactivity for bombesin, Leu-7 and NFTP, ranging from 11 to 40% of the cases. Chromogranin-A was not expressed in any of these tumors, whereas NSE was demonstrated only in 17% of breast carcinomas. The following remarks can be drawn from this study: (1) some NSCLCs showed immunophenotypic NE differentiation; (2) among all the markers used, NSE was the most sensitive (sensitivity, 100%) and chromogranin-A the most specific (specificity, 100%); and (3) NSE and chromogranin-A appear to be the most valuable and useful indicators of probable neuroendocrine differentiation in lung epithelial tumors. PMID- 7535641 TI - Immunosuppressive drugs and the induction of transplantation tolerance. PMID- 7535642 TI - Serum soluble adhesion molecules and cytokines in cardiac allograft rejection. The Manchester Transplant Cytokine Group. AB - AIMS: To examine the relationship between soluble adhesion molecules ICAM-1, E selectin and VCAM-1, serum cytokines TNF alpha, IL6 and IL2, the IL2 soluble receptor p55 and cardiac rejection in cardiac allograft recipients. METHODS: Seventy-six serum samples from 56 patients were examined. Samples were taken on the day of biopsy. No patient was experiencing concurrent infection. All the samples were examined for ICAM-1, TNF alpha, IL6 and IL2R p55. A smaller number were examined for E-selectin, VCAM-1 and IL2. Specific enzyme-linked immunosorbent assays were used. RESULTS: When grade 0 and grade 3a rejection groups were compared a significant difference was seen between IL6 levels (means 32 pg/ml vs 51 pg/ml, medians both 32 pg/ml, p = 0.007), and a significant difference between ICAM-1 levels (medians 207 ng/ml vs 250 ng/ml versus 303 ng/ml, p = 0.045). No patient without rejection had detectable levels of IL6. There was a correlation between ICAM-1 and E-selectin levels (R = 0.6, p = 0.003). There was no correlation between the other parameters and rejection or each other. CONCLUSIONS: Cytokines and adhesion molecules are of great importance in the mechanisms of transplant rejection and this, in some cases, is reflected in the serum. However, this is not sufficiently consistent to be of diagnostic value. PMID- 7535643 TI - The importance of E-selectin as a marker for renal transplant rejection. AB - Vascular endothelial cells express membrane bound adhesion molecules which play a direct role in the localization and subsequent movement of leucocytes from the blood into sites of inflammation. E-Selectin is a cytokine induced adhesion molecule, known to be expressed by endothelial cells in inflammatory conditions, which binds to various leucocyte subpopulations. In a prospective study we have investigated the expression and distribution of E-selectin on renal allograft needle biopsies taken from 16 pretransplant kidneys and 119 post-transplant kidneys. Post-transplant biopsies were taken at times of graft dysfunction and at times of normal graft function. Formal histology was also performed and assessed independently. E-Selectin was found predominantly on the intertubular endothelium and on the endothelium of larger vessels. E-Selectin was present, at low intensity, in some pretransplant biopsies and also some post-transplant biopsies which were reported histologically as normal. In post-transplant biopsies taken for dysfunction E-selectin was present in the majority of cases. Expression was strong in biopsies showing acute cellular rejection and this was associated with a CD4 positive cellular infiltrate. Biopsies showing other causes of dysfunction, in particular acute tubular necrosis, also were E-selectin and CD4 positive with lower intensity than those with acute cellular rejection. These results suggest that E-selectin is a good marker for endothelial activation in renal transplant biopsies. Its presence in histologically apparently normal biopsies suggests that its in vivo kinetics may differ from previously reported in vitro kinetics. E Selectin may be a potential target for therapeutic intervention. PMID- 7535644 TI - Human endothelial stimulation of allogeneic T cells via a CTLA-4 independent pathway. AB - It is accepted that T cells require at least two signals to undergo proliferation and cytokine release: an antigen dependent signal mediated via the the TCR (T cell receptor) and an antigen independent signal mediated via one or more accessory or adhesion molecules. Interaction between CD28 or CTLA-4 and the B7 co receptors found on many antigen presenting cells (APC) is known to be essential for antigen specific (including alloantigen) expansion of T cells in vitro and in vivo. CTLA-4-Ig is a fusion protein with very high affinity for B7. It has been used in vivo to block both allograft and xenograft rejection. Most of the work investigating second signal requirement has used 'professional' APC. In view of the observations that class II positive human endothelial cells can cause direct allostimulation of resting CD4+ and CD8+ T cells, we have investigated the requirement of CTLA-4 in this response. The current studies show that the proliferative response of allogeneic CD4+ and CD8+ T cells to interferon-gamma treated HUVEC (human umbilical vein endothelial cells) is inhibited by monoclonal antibodies (mAbs) against MHC class II and class I antigens, respectively, but not by CTLA-4-Ig. In contrast, lymphocytes proliferating in response to allogeneic splenocytes are inhibited by CTLA-4-Ig. Cell surface binding studies using flow cytometry demonstrated failure of endothelial cells to bind either CTLA-4-Ig or mAbs against B7 receptors. In conclusion, different APC use different co-stimulatory signals. The possibility that this leads to different cytokine profiles needs to be investigated to further understand the role of endothelial cells in transplant rejection. PMID- 7535645 TI - Lack of correlation between proliferative and colony-forming assays and the true regenerative potential of transplanted bone marrow. PMID- 7535647 TI - Use of nitric oxide synthase inhibitors in animal models of sepsis. AB - Sepsis, as a general inflammatory process, affects the whole organism, mainly because of the intense vasodilation and reduced perfusion pressures associated with it. The high mortality rates seen with sepsis are correlated with a reduction in mean arterial pressure. Therefore, the restoration of adequate arterial pressures is imperative. Nitric oxide (NO.) is at least partly responsible for the vasodilation. Inhibition of nitric oxide synthase (NOS) is, therefore, a logical approach for the treatment of sepsis. As with any other vasoconstrictive drug, NOS inhibitors are clinically indicated only in hyperdynamic sepsis. In animal models, their administration leads to an immediate restoration of blood pressure, accompanied by improved myocardial, pulmonary, and renal function. An increase in oxygen extraction prevents oxygen consumption from decreasing, despite a marked reduction in cardiac output to normal concentrations. In sepsis, virtually all regional blood flows are increased. In our experiments, no organ systems showed a reduction below preseptic baseline values when NOS inhibitors were administered. Furthermore, NOS inhibition did not cause an increase in lactate concentrations, indicating adequate nutritive organ blood flow. Consequently, NOS inhibitors seem to be beneficial and safe when administered under the right circumstances and in a controlled fashion. PMID- 7535646 TI - Reconstitution of the DNA base excision-repair pathway. AB - BACKGROUND: The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes. RESULTS: We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I. CONCLUSIONS: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches. PMID- 7535648 TI - Alterations in nitric oxide production in various forms of circulatory shock. AB - The free radical nitric oxide (NO.) is synthesized from the guanidino group of L arginine by a family of enzymes termed NO. synthase (NOS). In the earlier phases of shock, activation of the endothelial, constitutive NOS (ecNOS) occurs, which, in the case of endotoxic shock, is triggered by endotoxin-induced, acute release of platelet-activating factor (PAF) and also other potential mediators. This early overproduction of NO. results in reduced contractile responsiveness to norepinephrine and contributes to the acute decrease in blood pressure afforded by endotoxin. In the delayed phase of endotoxic shock, a distinct isoform of NOS (iNOS) is induced in various organs and in the vessel wall. The induction of iNOS is mediated by the release of endogenous tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and PAF by endotoxin. These mediators, in turn, act in parallel, or in synergy to induce iNOS. Induction of iNOS contributes to delayed vascular hyporeactivity in vivo and ex vivo, and to the delayed decrease in blood pressure in rats with endotoxic shock. As endotoxic shock, hemorrhagic shock also leads to an early activation of ecNOS, which is responsible for the early vascular hyporeactivity, and a delayed induction of iNOS that contributes to delayed circulatory failure (vascular decompensation and hyporeactivity). The induction of iNOS in hemorrhagic shock is unlikely to be mediated by endogenous release of endotoxin, e.g., due to intestinal ischemia. Endogenous circulating glucocorticoids exert a tonic suppression of the induction of iNOS, as well as the cardiovascular failure in response to endotoxin. Endotoxin tolerance is associated with increased plasma levels of glucocorticoids, which may account for the blunted cardiovascular response and reduced induction of iNOS in these animals. A wide variety of drugs that exert protective effects in various models of circulatory shock also inhibit the induction of iNOS, and this effect is likely to contribute to their protective actions. These drugs include glucocorticoids, TNF-alpha antibodies, IL-1 receptor blockers/antibodies, PAF antagonists, dihydropyridine calcium-channel antagonists, tyrosine kinase inhibitors, and the experimental drug cloricromene. Various forms of shock can also lead to an inhibition of NO. production by the calcium-dependent ecNOS.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7535649 TI - Nitric oxide in inflammation and immunity. AB - Few discoveries have had as comprehensive an impact on the understanding of cellular physiology as the production of nitric oxide (NO.) from the terminal guanido amino group of L-arginine through nitric oxide synthases. The sheer volume of data presently coming forth on the physiology and pathophysiology of NO. ensures that any attempt at a comprehensive review will result in a simple snapshot of the event, soon to be outdated. PMID- 7535650 TI - Regulation and functions of nitric oxide in the liver in sepsis and inflammation. AB - The liver plays important roles in metabolic and immune responses during sepsis. It is the major site of acute-phase protein synthesis and is responsible for the clearance of circulating pathogens. In addition to mediators such as cytokines and eicosanoids, numerous studies have emphasized the role of nitric oxide (NO.) in influencing hepatic function during sepsis. The induction and the distribution of inducible nitric oxide synthase in the liver, the regulation of the enzyme, and the functions of NO. in the liver are the subject of this review. PMID- 7535651 TI - Picolinic acid protects against quinolinic acid-induced depletion of NADPH diaphorase containing neurons in the rat striatum. AB - Previous studies in our laboratory have demonstrated that focal injections of picolinic acid (PIC) protect the cholinergic neurons of the nucleus basalis magnocellularis (nbm) against quinolinic acid (QUIN)-induced neurotoxicity. The present study was designed to examine the effects of chronic infusions of QUIN and PIC on nicotinamide adenine dinucleotide (NADPH) diaphorase containing neurons of the rat striatum. Using osmotic minipumps, QUIN (6 nmol/h) and PIC (18 nmol/h) were infused alone or in combination to examine the neurotoxic effects of QUIN and the potential anti-neurotoxic action of PIC. Exposure to QUIN for 7 days severely depleted NADPH diaphorase-positive neurons. When co-infused with this neurotoxic dose of QUIN, PIC attenuated the depletion of NADPH diaphorase neurons induced by QUIN. The infusion of PIC alone did not affect the number of these neurons. These results indicate that PIC itself is not neurotoxic and effectively prevents chronic QUIN-induced neurotoxicity in the rat striatum. Since PIC and QUIN are derived from the same metabolic pathway, a balance between endogenous compounds that produce neurotoxicity and those antagonizing these effects may be important in normal neuronal function. PMID- 7535652 TI - Further localization of cardiovascular and behavioral actions of substance P in the rat brain. AB - Cardiovascular and behavioral actions of substance P (SP) were examined after microinjection into the medial preoptic area (MPO), anterior hypothalamic area (AH), and ventral tegmental area (VTA) in conscious unrestrained rats. SP elicited marked increases in mean arterial pressure and heart rate as well as stereotyped behaviors of excessive grooming and exploring when injected into the MPO or AH. In the MPO, the latencies to the cardiovascular responses were observed after SP injection into the VTA. These results, together with our previous results, suggest that SP acts as transmitter or modulator in the rostral hypothalamic areas to elicit cardiovascular defense responses. In contrast, SP may not be involved in causing a defense reaction in the more caudal areas of the defense center. PMID- 7535653 TI - Efferent connections from the external cuneate nucleus to the medulla oblongata in the gerbil. AB - The present study revealed the efferent projections from the external cuneate nucleus (ECN) to various medullary nuclei in the gerbil as demonstrated in fresh living brainstem slices by using in vitro anterogradely tracing with the dextran tetramethyl-rhodamine-biotin. The tracer-labelled ECN axon terminals were observed (1) in most of the vital autonomic-related nuclei: the nucleus solitary tractus, nucleus ambiguus, rostroventrolateral reticular nucleus and C2 adrenergic area, (2) in the reticular formation: the medullary, parvocellular, intermediate, gigantocellular, dorsal paragigantocellular and lateral paragigantocellular reticular nuclei and medullary linear nucleus, and (3) in sensory nuclei: the cuneate nucleus, spinal trigeminal nuclei caudalis and interpolaris, paratrigeminal nucleus, medial and spinal vestibular nuclei, inferior olive and prepositus hypoglossal nucleus. These new findings are discussed in relation to possible roles of the ECN in cardiovascular, respiratory and sensorimotor controls. PMID- 7535654 TI - Spinal cord NADPH-diaphorase histochemical staining but not nitric oxide synthase immunoreactivity increases following carrageenan-produced hindpaw inflammation in the rat. AB - Recent reports suggest that NADPH-diaphorase (NADPH-d) may be a histochemical marker for neuronal nitric oxide synthase (nNOS) in the central nervous system. Carrageenan-produced unilateral hindpaw inflammation in the rat results in a bilateral increase in NADPH-d in spinal cord neurons. This suggests there would be a bilateral increase in NO, which mediates thermal hyperalgesia. However, carrageenan-produced unilateral hindpaw inflammation results in hyperalgesia of the inflamed hindpaw only. This study determined (1) if neurons that labeled for NADPH-d following carrageenan-produced unilateral hindpaw inflammation colocalized nNOS, and (2) whether there was an increase in nNOS-ir neurons following inflammation. Following unilateral hindpaw inflammation, double labeling of tissue sections and single labeling of alternate serial sections revealed a lack of colocalization or mismatch between NADPH-d histochemical activity and nNOS-like immunoreactivity in neurons in lamina I, the dorsolateral funiculus and lamina X. Quantitative analysis showed no difference in the number of nNOS-ir neurons and NADPH-d labeled neurons in the superficial laminae of the spinal cord in non-inflamed animals. Following unilateral hindpaw inflammation, there was a 34% increase in the number of NADPH-d labeled neurons but no increase in the number of nNOS-ir neurons. These results indicate that nNOS-immunoreactive neurons and NADPH-diaphorase stained neurons are not identical and that nNOS does not increase as a result of hindpaw inflammation, leaving the source of NO involved in thermal hyperalgesia following injury in question. PMID- 7535656 TI - A major role for calcium-dependent potassium current in action potential repolarization in adrenal chromaffin cells. AB - To determine the extent which Ca dependent K current (IKCa) contributes during an action potential (AP), bovine chromaffin cells were voltage-clamped using a pre recorded AP as the command voltage waveform. Based on (1) differential sensitivity of IKCa and Ca-independent K current (IK) to tetraethylammonium; (2) measurements of AP currents under conditions where Ca activation of IKCa had been abolished; and (3) blockade by charybdotoxin, IKCa comprised 70-90% of the outward K current during AP repolarization. In addition, observations are made concerning the form of AP-evoked Ca current. PMID- 7535657 TI - Dose-dependent effect of nitric oxide synthase inhibition following transient forebrain ischemia in gerbils. AB - The extensive research concerning the interaction between nitric oxide (NO) and ischemic brain tissue has yielded contradictory results. The present study was designed to explore the effect of gradual inhibition of NO production on brain ischemia. Gerbils were administered (i.p.) either saline (control-ischemia), or 5, 10, 25 or 50 mg/kg of NG-nitro-L-arginine (NARG), a specific inhibitor of NO synthase (NOS), and 4 h later were subjected to 5 min of forebrain ischemia. A group receiving 50 mg/kg NARG with sham operation served as a second control (control-NARG) group. Body weights and spontaneous activity were monitored daily until day 6, when the gerbils were sacrificed and their brains processed for histologic-morphometric evaluation. All ischemia groups displayed significant decreases in body weights starting on day 1, as compared to control-NARG (non ischemic) gerbils. At 24 h post-ischemia spontaneous activity was increased in all ischemia groups in a dose-dependent manner, reaching a peak at 25 mg/kg. Typical ischemia-induced neuronal cell degeneration was observed at the hippocampal CA1 layer in control-ischemia and in each of the dose-groups of 10 mg/kg NARG and above. The 5 mg/kg group displayed damage which was not different from control-NARG, and was milder (P < 0.01) than control-ischemia gerbils and each of the other dose-groups. It is suggested that during ischemia, NO activates a series of processes which are beneficial to brain tissue, whereas an excess amount of NO causes neurotoxic effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535655 TI - Trolox protects mitochondrial complex IV from nitric oxide-mediated damage in astrocytes. AB - The efficacy of cystine, ascorbate and trolox, a vitamin E analogue, at protecting against nitric oxide-mediated mitochondrial complex IV damage has been investigated in cultured astrocytes. Of these compounds, only trolox afforded protection. It is suggested that lipid peroxidation is responsible for nitric oxide-mediated mitochondrial damage and that inhibitors of this process may be of therapeutic benefit in conditions where excessive nitric oxide production is implicated. PMID- 7535659 TI - Day care admissions. PMID- 7535660 TI - Hemangiomas and vascular malformations. PMID- 7535658 TI - Detection and characterization of nitric oxide synthase in the mammalian cochlea. AB - The messenger molecule nitric oxide (NO) is involved in blood flow regulation, cytotoxicity, and neural signalling, processes that are important in the physiology and pathophysiology of the mammalian cochlea. However, neither the presence of NO nor its synthetic enzyme, NO synthase, has been established in the peripheral auditory system. NO synthase activity, measured as the enzymatic conversion of radioactive arginine to citrulline, was predominantly soluble in the auditory nerve, lateral wall, vestibule and cochlear neuroepithelium. N methyl-L-arginine and trifluoperazine inhibited NO synthase activity in the lateral wall and auditory nerve. Histochemical staining by NADPH-diaphorase localized NOS activity to the lateral wall and the neuronal elements of the organ of Corti. Based on these results, the predominant NO synthase isoform in the cochlea is the neuronal type-I isoform. PMID- 7535661 TI - Germ cell tumors. PMID- 7535662 TI - Parathyroid carcinoma. PMID- 7535663 TI - Prostate cancer. PMID- 7535664 TI - Benign prostatic hyperplasia. PMID- 7535665 TI - Graves' disease in children. PMID- 7535666 TI - Thyroid storm. PMID- 7535667 TI - Perspectives on reality. PMID- 7535668 TI - Lack of T cell tolerance in mice exposed to a protein antigen through lactation. AB - Offspring of mother mice treated immediately after delivery with deaggregated human gamma-globulins (dHGG) are unable to produce HGG-specific antibodies when challenged with immunogenic forms of HGG (HGG/CFA) in adulthood. Despite a defective antibody response, animals rendered tolerant to HGG as neonates retain tolerogen-specific T cells able to proliferate and secrete lymphokines. The pattern of IL-2 and IL-4 secretion by T cells isolated from tolerant animals could not be distinguished from the corresponding cells in control mice, suggesting that neonatal exposure to dHGG did not affect T cell reactivity or Th1/Th2 in vivo balance. Moreover, immunization of tolerant animals with haptenated HGG confirmed the presence of tolerogen-specific helper T cells in vivo. Functional T cell depletion by anti-CD3 mAbs during lactation failed to modify induction of B cell tolerance, suggesting that T cells are neither affected nor required to induce the selective tolerance status observed in this model. Based on the finding that antigen-presenting cell functions in secondary organs (spleen, peritoneal cavity) are a late acquisition during ontogeny and reach adult-like levels at weaning, we propose that most soluble proteins elude T cell recognition during lactation due to defective antigen presentation. PMID- 7535669 TI - Effects of cholinergic modulation on serum insulin-like growth factor-I and its binding proteins in normal and diabetic subjects. AB - OBJECTIVE: We wished to study alterations in serum insulin-like growth factor-I (IGF-I) and its binding proteins in subjects with insulin dependent diabetes mellitus (IDDM) and possible relations with metabolic and GH secretory status, before and after cholinergic modulation. In addition, we have investigated whether cholinergic modulation exerts any effects on IGF-I secretion, independently of any actions on GH secretory status. DESIGN: All subjects received GH releasing hormone (GHRH) 1-44; 80 micrograms i.v.) alone and 60 minutes following 120 mg of pyridostigmine orally or 200 mg of pierenzepine orally. The three tests were carried out in random order at least one week apart. Blood was sampled at 15-minute intervals over 120 minutes. PATIENTS: Twelve male subjects with IDDM and no clinical evidence of complications were selected on the basis of HbA1 levels to provide a wide range of metabolic control. Six normal male subjects were also studied. MEASUREMENTS: Serum IGF-I, IGF-binding protein 1 (IGFBP-1) and IGFBP-3 were measured at regular intervals throughout the study. Fasting plasma glucose and HbA1 were measured before each study to provide measures of metabolic control. RESULTS: Serum IGF-I and IGFBP-3 levels were significantly lower while serum IGFBP-I levels were significantly higher in the diabetic subjects. Pirenzepine had no effect on serum IGF-I, IGFBP-1 or IGFBP-3 in diabetic subjects but caused a significant increase in serum IGF-I and IGFBP-3 levels in normal subjects. Pyridostigmine had no effect on IGF-I, IGFBP-1 or IGFBP-3 in either diabetic or normal subjects. IGFBP-1 levels were significantly correlated with fasting plasma glucose but no correlation was demonstrated between measures of diabetic control and serum IGF-I or IGFBP-3 levels in diabetic subjects, nor was there any correlation between GH responses to GHRH alone or after pirenzepine or pyridostigmine pretreatment and serum levels of IGF I, IGFBP-1 or IGFBP-3. CONCLUSION: These data confirm that subjects with IDDM have reduced serum IGF-I and IGFBP-3 and increased IGFBP-1 levels, the latter being directly related to the fasting plasma glucose concentrations. The absence of any relation between changes in the IGF-I system and altered GH neuroregulation after cholinergic modulation suggests that changes in IGF-I are not the sole contributors to the altered GH neuroregulation which occurs in IDDM. We have also shown an acute stimulatory effect of pirenzepine on serum IGF-I and IGFBP-3 in normal subjects which is not present in IDDM although the underlying mechanisms is unknown. PMID- 7535670 TI - Effect of octreotide on circulating IGF-I chromatographic profile: evidence for an inhibitory action on the formation of the 150-kDa ternary complex. AB - OBJECTIVE: The increasing use in clinical practice of octreotide (a somatostatin analogue which inhibits the secretion of GH and other peptide hormones) led us to study the effects of this treatment on GH, insulin-like growth factors (IGF)-I and II and IGF-binding protein (IGFBP)-3, as well as on circulating IGFBP complexes in acromegalic patients. DESIGN: The circulating concentrations of GH, IGF-I, IGF-II and IGFBP-3 were measured in acromegalic patients before and after 3, 6, 9, and 12 months of treatment with octreotide (group I: n = 5), and compared with those found in a group of patients (group II) treated with bromocriptine (n = 3), cabergoline (n = 7) radiotherapy (n = 3) or surgical therapy (n = 2). In pools of serum obtained from patients treated with octreotide, dopaminergic drugs, surgery and radiation, before and after therapy, immunoreactive IGF-I and IGFBP-3 were also evaluated after Superdex 200 gel filtration in neutral conditions. RESULTS: Before treatment, the concentration of IGF-I and IGFBP-3 were above the normal range in all patients, while IGF-II levels were slightly reduced. After treatment with octreotide, IGF-I (P = 0.004), IGF-II (P = 0.02) and IGFBP-3 (P < 0.001) were significantly reduced as compared to basal levels. In subjects of group II, only IGF-I concentration was significantly reduced by the treatment (P = 0.02), and a negative correlation between IGF-I and IGF-II concentrations was found (r = -0.58, P < 0.0001). After gel filtration immunoreactive IGF-I and IGFBP-3 were found in the 150-kDa mol.wt. region in serum obtained from untreated patients and from treated patients of group II, while in the serum of octreotide-treated patients the IGF-I and IGFBP-3 peaks were shifted to the 60-kDa mol.wt. region, thus suggesting that the acid labile subunit of the 150-kDa complex was drastically reduced. Since the GH concentrations in groups I and II were similar (M +/- SEM; 13.8 +/- 7.4 and 21.2 +/- 10.6 mU/l respectively), the marked reduction in acid-labile subunit in the octreotide treated patients can be explained by a direct inhibitory effect of somatostatin on the subunit. CONCLUSIONS: Octreotide exerts an inhibitory effect not only on IGF-I but also on IGF-II. The reduced formation of the 150-kDa complex probably causes an increased metabolic clearance rate of IGF peptides which can account for the reduced concentration of both IGFs after treatment with octreotide. PMID- 7535672 TI - Potential roles of the B7 and CD28 receptor families in autoimmunity and immune evasion. AB - Recognition of self major histocompatability complex (MHC)-presented antigen (MHC:Ag) by the T cell antigen receptor (TCR) is by itself not sufficient to induce T cell proliferation. Rather, to be fully activated T cells require both a TCR-generated signal and a "costimulatory" signal. This important costimulatory signal is not completely understood. Recent evidence suggests that this costimulatory signal is generated by the interaction of the T cell CD28 receptor with the B7 counterreceptor found on antigen-presenting cells. Regulation of costimulation may well prove to be more complex than was previously imagined based on the discovery that CD28 and B7 are each members of larger gene families. The present review highlights recent advances in the understanding of the CD28 and B7 receptor families with an emphasis on controversies in the field. Certain forms of immunopathology that might result from the aberrant regulation of CD28 and/or B7 expression are also discussed. PMID- 7535671 TI - Thyrotropin receptor T cell epitopes in autoimmune thyroid disease. AB - The human TSH receptor represents the primary target of thyroid-stimulating immunoglobulins responsible for the hyperthyroidism of Graves' disease. In the present series of investigations, the distribution of T cell epitopes has been mapped using synthetic peptides spanning the entire extracellular region of the human TSH receptor. In vitro proliferative responses of the mononuclear cells were measured using flow cytometric analysis of bromodeoxyuridine incorporation into nuclei. In 8 of 11 samples from patients with Graves' disease, at least one (and up to 9) regions of the human TSH receptor induced proliferation, with the mean stimulation index being 39.8 +/- 47.3. No single universal stimulatory peptide was identified. In contrast, stimulation was not observed in three control subjects, while one control subject showed minimal stimulation (index of 5.7) to peptides encompassing a limited area (amino acids 31-65). The immunodominant epitope of patients with recent-onset Graves' disease was localized between amino acids 271 and 365, whereas the immunodominant epitope of patients with disease duration greater than 1 year localized between amino acids 91 and 215. We conclude that the bromodeoxyuridine incorporation method is a useful and important tool for detecting antigen-induced lymphocyte proliferation. The TSH receptor-specific T cells from different Graves' disease patients recognize variable distinct sites within the extracellular region of the TSH receptor, and the immunodominant epitope apparently shifts toward the N-terminus of the receptor protein during the course of treated Graves' disease. PMID- 7535673 TI - Comparison by extended ribotyping of Pseudomonas cepacia isolated from cystic fibrosis patients with acute and chronic infections. AB - Multiple isolates of Pseudomonas cepacia, from two cystic fibrosis (CF) patients who were chronically infected and two others who suffered acute fatal lung infections, were examined by multilocus enzyme electrophoresis and four-enzyme ribotyping. The strains isolated from the fatalities belonged to a clone, electropherotype 12 (ET12) that is endemic in the Ontario patients' province of origin. ET12 strains have also been isolated from outbreaks in CF patients in the United Kingdom, where they are considered to be strains of high virulence and transmissibility and epidemiologically related to Ontario strains. Four-enzyme ribotyping (EcoRI, Xho, PstI, and ClaI) established the close genetic relationship of the Ontario ET12 isolates and those from the United Kingdom, particularly an isolate from Manchester. In addition, four enzyme ribotypes of the sequential isolates taken during life and at autopsy of the ET12 clone were highly variable in comparison with the stability of the ribotypes of clone ET16 isolated sequentially from living chronic carriers. This extreme ribotype variability may be indicative of a highly virulent strain and poor prognosis. Isolates from our chronically infected CF patients belonged to a different clone, ET16, and it is also endemic in its region, 1000 miles east of ET12, in Nova Scotia. In both endemic circumstances, person-to-person transmission was easily demonstrated by four-enzyme ribotyping. The ET12 clone was found to be transmitted among summer campers and during a nosocomial outbreak, whereas an E16 strain was found to infect a sibling of a chronically infected patient; both infections were of the same ribotype. PMID- 7535674 TI - Analyzing correlations of three types in selected lines of Drosophila melanogaster that have evolved stable extreme geotactic performance. AB - The behavior-genetic analysis of Drosophila melanogaster with geotactic performance as the phenotype is an ideal model system with which to investigate the complex relations between heredity and behavior. As part of a long-term, 38 year study, we report 4 experiments that identify and analyze trait correlations in the selected high- and low-geotaxis lines. We performed F2 correlational analyses and backcrosses to examine 3 types of correlations: (a) genotype genotype (alcohol dehydrogenase [Adh]-amylase [Amy]), (b) genotype-phenotype (Adh and Amy-geotaxis), and (c) phenotype-phenotype (mate preference-geotaxis). Only the Adh-geotaxis correlation survived meiosis and reappeared in the F2 generation, which indicates a genotype-phenotype correlation, whereas the others did not. The importance of hybrid correlational analysis to the behavior-genetic analysis of a species is discussed. PMID- 7535675 TI - Serum deprivation and the turnover of short-, medium- and long-lived proteins in 3T3 and HeLa S-3 cells. AB - Protein turnover in serum-deprived Swiss 3T3(U) untransformed and 3T3(T) transformed cells was comprehensively studied. When 3T3(U) cells were cultured in 0.2% serum medium, protein synthesis fell to 28% of the control value by 48 h, but took at least 12 h to become manifest. Meanwhile the rates of degradation of medium- and long-lived proteins were increasing, with a 1.65-fold increase in degradation of the former within 2 h, and the turnover of long-lived proteins doubling in 24 h. A significant increase in the degradation of truly short-lived proteins was not apparent until 24 h of deprivation. After serum restoration in cultures deprived of serum for 24 h, protein synthesis increased from 25 to 40% of the control values within 1 h, reaching 93% by 24 h. The rate of medium-lived protein degradation in 3T3(U) cells quickly returned to control levels, and that of long-lived proteins fell to less than control levels by 3 h, decreasing to 67% by 24 h. This contrasted with short-lived protein turnover, which remained at the same level as the cells kept in 0.2% serum for a further 24 h. The turnover of medium- and long-lived proteins is initially more important than short-lived proteins in growth regulation following the removal of growth factors. PMID- 7535677 TI - Signal transduction pathways in GnRH- and dopamine D1-stimulated growth hormone secretion in the goldfish. AB - In goldfish, growth hormone (GH) secretion is regulated by multiple neuroendocrine factors. Among these regulators, gonadotropin-releasing hormone (GnRH) and dopamine (DA) are effective stimulators of GH release. The stimulatory actions of GnRH and DA are mediated by GnRH and DA D1 receptors on somatotropes, respectively. In this article, results from recent in vitro pharmacological and electrophysiological studies examining the possible involvement of extracellular Ca2+, protein kinase C, voltage-sensitive Ca2+ channels (VSCC) and phospholipase A2 in mediating GnRH-induced GH release are presented. Results from experiments investigating the possible interactions of cyclic adenosine 3',5'-monophosphate (cAMP), and extracellular Ca2+ entry through VSCC in mediating the DA D1-elicited GH response are also reported. These data were discussed in conjunction with other information available in the literature on the signal transduction mechanisms mediating GH secretion in goldfish. Based on these findings, a model for the transduction pathways integrating the initiation and maintenance of the distinct GnRH-induced and DA D1-elicited GH responses was proposed. GnRH and DA stimulate GH release via separate PKC- and cAMP-dependent mechanisms, respectively. These signalling mechanisms appear to act on distinct GH pools. PKC and cAMP subsequently activate VSCC. Ca2+ entry through VSCC plays a role in the sustained GH release response by enhancing the PKC- and cAMP-induced GH release. PMID- 7535676 TI - Effects of colchicine on IgE-mediated early and late airway reactions. AB - BACKGROUND: The pathogenesis of bronchial asthma is thought to involve elements of both acute and chronic inflammation. Hence, there is growing interest in the potential of immunomodulatory drugs in asthma therapy. This study examines the effects of the anti-inflammatory compound colchicine on early and late allergen induced, IgE-mediated airway reactions. METHODS: Nine mildly allergic asthmatic subjects were evaluated in a single-blind, two-way crossover study designed to examine the effects of colchicine and placebo on early and late airway reactions to ragweed allergen and related changes in nonspecific responsiveness to methacholine. RESULTS: Compared with placebo, colchicine provided 19% (p = 0.036) and 40% (p = 0.004) inhibition of early and late airway reactions to allergen, respectively. Allergen-induced increases in methacholine responsiveness were observed with both types of treatment, although there was a trend toward a smaller increase after administration of colchicine (p = 0.13). We also found that methacholine responsiveness per se was not directly altered by colchicine (n = 7). In 6 subjects, we found suppression of neutrophil leukotriene B4 generation after colchicine treatment, suggesting that the colchicine dose (0.6 mg twice daily) was sufficient to produce an anti-inflammatory effect. Further in vitro studies using purified human lung tissue mast cells failed to demonstrate inhibition of mediator release at concentrations corresponding to achievable tissue or blood levels during the in vivo trial. CONCLUSION: Colchicine partially inhibits IgE-mediated early and late airway reactions at conventional clinical doses. This inhibitory effect may be mediated via suppression of some cell species other than the lung tissue mast cell. Controlled studies to examine the benefits of colchicine in clinically evidenced asthma are warranted. PMID- 7535678 TI - Islet amyloid polypeptide and its N-terminal and C-terminal flanking peptides' immunoreactivity in islet amyloid of diabetic patients. AB - We determined immunohistochemically whether the islet amyloid polypeptide (IAPP)/amylin precursor is one component of islet amyloid, using polyclonal antibodies specific for human IAPP8-17 and amino (N)-terminal and carboxy (C) terminal flanking peptides. To enhance immunostaining of the amyloid, we pretreated the pancreatic tissue sections with 100% formic acid. In three non diabetic subjects, pancreatic islet cells were immunoreactive to anti-IAPP8-17 and anti-N-terminal and C-terminal flanking peptide antibodies and the reactivity was enhanced with formic acid pretreatment. In six type 2 diabetic subjects and a subject with type A insulin resistance, islet amyloid deposits were reactive to anti-IAPP8-17 antibody, but not to anti-N-terminal and C-terminal flanking peptide antibodies. Formic acid pretreatment markedly enhanced the reactivity to anti-IAPP8-17 antibody; however, it failed to show the reactivity to anti-N terminal and C-terminal flanking peptide antibodies. Formic acid pretreatment of pancreatic tissue sections prepared for immunostaining is useful for visualization of buried epitopes of mature IAPP and its precursor molecules, either in islet amyloid deposits or in the islet cells. We conclude that the IAPP precursor and N-terminal and C-terminal flanking peptides are not constituents of human islet amyloid. PMID- 7535679 TI - The impact of microglia-derived cytokines upon gliosis in the CNS. AB - Injury to the CNS elicits a complex cellular response involving both astrocytes and microglia. Reactive glial populations make up the so-called 'glial scar' that has long been implicated as a barrier to axonal regeneration or as a causal factor in the genesis of epilepsy. Using in vitro models involving highly enriched populations of brain cells we have observed that astroglial growth is regulated in part by an immunomodulatory growth factor, or cytokine, called interleukin-1 (IL-1). A second cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF) serves as a potent microglial mitogen and regulator of the microglial component of the glial scar. Employing cytokines as tools to manipulate reactive gliosis, we found that IL-1 supported neuronal growth by action upon astroglia, while GM-CSF initiated epileptic-like discharges through mechanisms involving reactive microglia. We propose that a 'cytokine network' involving IL-1 and GM-CSF mediates the composition of glial scars at sites of CNS injury; these reactive glia, in turn, influence the survival and function of neighboring neurons. PMID- 7535680 TI - Reactive nitrogen intermediates in human neuropathology: an overview. AB - Nitric oxide (NO) is a recently recognized messenger molecule that has been shown to possess pleiotropic properties, including vasodilation, neurotransmission, cytotoxicity and antimicrobial activity. Constitutive and inducible forms of NO synthase (NOS) have been identified. Activation of cNOS releases relatively low levels of NO for short periods of time whereas induction of iNOS releases high levels of NO for extended periods of time. In rodents, iNOS is predominantly found in cells of the monocyte/macrophage series, including microglia, where it is induced by a combination of bacterial products and cytokines. cNOS and iNOS have also been reported in rodent astrocytes. Activation of iNOS in the CNS could be toxic to many different cell types, including neurons and oligodendrocytes. iNOS, however, has been difficult to demonstrate in human peripheral blood cells, suggesting that the regulation of expression of this enzyme in humans is different from that found in rodents. In this overview, we show that in human glial cells cultured in vitro, astrocytes, but not microglia, can be induced by cytokines to express NO-like activity. Bacterial products are without effect, but a combination of IL-1 and TNF alpha or IFN gamma is a potent stimulus. NO production by astrocytes inhibits Cryptococcus neoformans growth in vitro. In vivo, we show in acute multiple sclerosis lesions, intense NADPH-diaphorase activity is present in hypertrophic astrocytes in the lesion center and at the lesion edge, whereas microglia are nonreactive. Increased NADPH-diaphorase activity colocalizes with immunoreactivity for IL-1 and TNF. These results suggests that the induction of reactive nitrogen intermediates in humans differs from that found in rodents, and supports the conclusion that hypertrophic astrocytes are the major source of NO-like activity in the inflamed CNS. PMID- 7535681 TI - Differential sensitivity to nitric oxide in immortalized, cloned murine oligodendrocyte cell lines. AB - Using five different immortalized, cloned murine oligodendrocyte cells lines, we have assessed their sensitivity to the nitric oxide donor chemical S-nitroso, N acetyl-DL-penicillamine (SNAP). The five lines were quite different in their sensitivity to SNAP as determined by assessment of viability, mitochondrial function, single-stranded DNA breaks, DNA/RNA/protein synthesis, morphology, and myelin gene expression. Single-stranded DNA breaks as well as mitochondrial and DNA damage occurred in viable cells. Thus, cell damage was independent of cell death. In the most mature oligodendrocyte cell line, cell morphology was altered and the expression of myelin basic protein mRNA was inhibited by nitric oxide in a time- and dose-dependent fashion. These findings suggest two things. First, oligodendrocytes at different states of differentiation are differentially sensitive to nitric oxide. Second, while not all oligodendrocytes may be destroyed in multiple sclerosis plaques, many could be significantly damaged and thereby nonfunctional in repair processes triggered during the pathology of this disease. PMID- 7535682 TI - Is p75NGFR involved in developmental neural cell death? AB - The tumor necrosis factor receptor superfamily includes twelve members, at least two of which--tumor necrosis factor receptor I and FAS/Apo-1--induce cell death following ligand binding. This review summarizes data suggesting that two other members of the family--p75NGFR and CD40--achieve a similar effect in the inverse fashion; they induce apoptosis constitutively when unbound by their respective ligands, with the induction of apoptosis being inhibited by the binding of their respective ligands. The potential roles that such receptors may play in development and pathological processes are discussed. PMID- 7535683 TI - Reactive gliosis as a consequence of interleukin-6 expression in the brain: studies in transgenic mice. AB - Gliosis is a characteristic pathologic state in many CNS disorders. Cytokines are considered to be effectors of gliosis. In order to explore the role of IL-6 in gliosis, the temporal and spatial expression of the IL-6 gene and its consequent effects on the brain were studied in a GFAP-IL6 transgenic mouse model. In GFAP IL6 mice, IL-6 transgene expression was detectable in the brain at 1 week postnatally and increased to maximal levels by 3 months of age before declining at 8 and 12 months. Enhanced glial fibrillary acidic protein (GFAP) (marker for astrocytes) and Mac-I (marker for microglia) mRNA expression were first prominent at 1 month, increased to maximum levels by 3 months and remained significantly elevated through 12 months of age. Western blot analysis revealed that the enhanced GFAP mRNA expression in these transgenic mice was accompanied by increased GFAP protein levels. Immunostaining for Mac-I demonstrated that in addition to an increased staining intensity, the number of cells expressing the microglial/macrophage marker was also apparently increased, particularly in the cerebellum and brain stem. Concurrent with IL-6 transgene mRNA expression and reactive gliosis, upregulation of IL-1 alpha/beta, TNF alpha, ICAM-1 and EB22/5.3 (acute-phase reactant) but not inducible nitric oxide synthase gene expression was also observed. EB22/5.3 mRNA expression was most prominent and increased progressively with age. Expression of the IL-6, GFAP and EB22/5.3 RNAs was found to have similar distribution in the brain being found predominantly in the cerebellum, brain stem and sub-cortical regions. In conclusion, the constitutive expression of IL-6 in the brain induced the development of a pronounced and lifelong reactive gliosis affecting both astrocytes and microglia. The altered state of these cells may contribute to the functional and structural CNS impairment exhibited by the GFAP-IL6 mice. Finally, in these mice, expression of the EB22/5.3 gene correlated closely with the progression of neuropathy indicating that this acute-phase response gene was a good marker for and may be involved in the pathogenesis of CNS injury mediated by the expression of IL-6. PMID- 7535684 TI - Serum alphafoetoprotein, hepatitis B virus infection and primary hepatocellular carcinoma in Nigerians. AB - Serum alphafoetoprotein and hepatitis B antigen were estimated by radioimmuno assay and haemagglutination methods respectively in 42 Nigerian adults comprising 14 subjects in each of three groups, viz, controls, liver cirrhosis and primary hepatocellular carcinoma. At an abnormal concentration of serum alphafoetoprotein greater than 200 micrograms/L, a correct diagnosis of primary liver cancer was made in 64.3% of the patients at a specificity of 100%. However, no correlation was found between serum concentrations of alphafoeto-protein and status of hepatitis B surface antigen in the patients with primary liver cancer. It may be concluded that serum alphafoeto-protein is useful in the diagnosis of primary hepatocellular carcinoma in Nigerians and secretion of the onco-foetal protein by neoplastic hepatocytes is unlikely to be influenced by hepatitis B virus infection. PMID- 7535685 TI - Amelioration of bleomycin-induced lung fibrosis in hamsters by dietary supplementation with taurine and niacin: biochemical mechanisms. AB - Interstitial pulmonary fibrosis induced by intratracheal instillation of bleomycin (BL) involves an excess production of reactive oxygen species, unavailability of adequate levels of NAD and ATP to repair the injured pulmonary epithelium, and an overexuberant lung collagen reactivity followed by deposition of highly cross-linked mature collagen fibrils resistant to enzymatic degradation. In the present study, we have demonstrated that dietary supplementation with taurine and niacin offered almost complete protection against the lung fibrosis in a multidose BL hamster model. The mechanisms for the protective effect of taurine and niacin are multifaceted. These include the ability of taurine to scavenge HOCl and stabilize the biomembrane; niacin's ability to replenish the BL-induced depletion of NAD and ATP; and the combined effect of taurine and niacin to suppress all aspects of BL-induced increases in the lung collagen reactivity, a hallmark of interstitial pulmonary fibrosis. It was concluded from the data presented at this Conference that the combined treatment with taurine and niacin, which offers a multipronged approach, will have great therapeutic potential in the intervention of the development of chemically induced interstitial lung fibrosis in animals and humans. PMID- 7535688 TI - Benign prostatic hyperplasia. AB - Benign prostatic hyperplasia (BPH) is the most common cause of bladder outlet obstruction and voiding symptoms in elderly men. The pathogenesis is not fully determined but a combination of androgens and age are needed for development of BPH. Symptoms of BPH are divided into obstructive and irritative symptoms but large interpersonal variability is found and no specific BPH symptom exists. Treatment modalities include surgery (TURP, TUIP, open prostatectomy, laser ablation, balloon dilatation, hyperthermia and thermotherapy, and urethral stents) and medical therapy. TURP is the gold standard treatment and TUIP is a safe and effective alternative to TURP in patients with smaller prostates. Laser ablation, hyperthermia and thermotherapy, and urethral stents are at the present time under investigation. Balloon dilatation is FDA-approved but not often used because of low efficacy and poor long-term results. Medical treatment includes alpha-blocker or finasteride treatment and is indicated in patients with moderate to severe symptoms of BPH without a strong indication for surgery. PMID- 7535689 TI - Order despite a multitude of molecular anaesthetic actions. PMID- 7535687 TI - Fluoromicroscopic studies of bleomycin-induced intracellular oxidation in alveolar macrophages and its inhibition by taurine. AB - The mechanism of bleomycin-induced pulmonary fibrosis is not yet clear. Recent studies have shown that alveolar macrophages (AM) can be stimulated by bleomycin in vitro releasing inflammatory cytokines, suggesting that the interaction of bleomycin with AM is an important step in the drug-induced fibrotic process. Bleomycin is known to bind DNA and generate oxygen radicals through complexation with Fe2+ and oxygen. To provide more insight into the cellular oxidative property of bleomycin, we have developed a fluoromicroscopic method using 2',7' dichlorofluorescin diacetate (DCFHDA) as an oxidative fluorescence probe to study the bleomycin-induced intracellular oxidation in rat AM and the inhibition of the oxidation by taurine, a compound known to inhibit the bleomycin-induced fibrosis. Bleomycin at 5 to 20 micrograms/ml has a moderate stimulatory effect (1.87- to 2.66-fold) on the secretion of superoxide anion. A high concentration of bleomycin (20 micrograms/ml), however, inhibits cell response to zymosan-induced secretion of superoxide anion. At 4 micrograms/ml, bleomycin has no effect on cell membrane integrity or morphology but results in a significant increase in intracellular oxidation. This oxidative process is Fe(2+)-dependent and is accompanied by an increase in intracellular calcium (35 nM). Both the intracellular oxidation and calcium rise induced by internalized bleomycin are inhibited by pretreatment of cells with varying concentrations of taurine (25, 125, and 187.5 microM). The inhibitory effect on intracellular oxidation was found to be 36, 57, and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535686 TI - Attenuation of oxidant-induced lung injury by 21-aminosteroids (lazaroids): correlation with the mRNA expression for E-selectin, P-selectin, ICAM-1, and VCAM 1. AB - We compared the effects of treatment with methylprednisolone or the 21 aminosteroids, U-74389 and U-74006F (Tirilizad mesylate), on hyperoxic lung injury and the associated expression of mRNA for several adhesion molecules in rats. Inhalation of > 95% oxygen for up to 72 hr in Sprague-Dawley rats produced a marked increase in lung weight and an accumulation of fluid in the thorax when compared with air-breathing controls. Hyperoxia also induced a marked neutrophil rich influx of inflammatory cells into the bronchial lumen as measured by bronchoalveolar lavage. Neutrophil numbers in bronchoalveolar lavage fluid peaked after 60 hr of exposure to s 95% oxygen; this was associated with a marked upregulation of mRNA for the adhesion molecules P-selectin and E-selectin but not VCAM-1. mRNA for ICAM-1 was constitutively expressed at high levels in both air breathing controls and in the lungs of rats exposed to high concentrations of oxygen. Pretreatment with the 21-aminosteroids reduced hyperoxic lung damage and improved survival times in animals exposed to > 95% oxygen. However, treatment with methylprednisolone significantly decreased survival times. Treatment with U 74389 did not significantly (p > 0.05) inhibit the BAL neutrophilia and did not significantly (p > 0.05) reduce hyperoxia-induced increases in mRNA expression for P-selectin and E-selectin. The inhibition of hyperoxic lung damage coupled with improved survival seen in treated animals suggests that 21-aminosteroids may provide valuable treatments for pulmonary disorders in which oxidant damage has been implicated. PMID- 7535690 TI - Molecular sites of general anaesthetic action on acetylcholine receptors. AB - Mechanisms of anaesthetics still remain unclear. However, various attempts have been made to elucidate the effects of anaesthetics at the molecular level. The nicotinic acetylcholine receptor has proved to be a good model of membrane-bound ligand-gated ion channels. It is available in abundance from Torpedo electroplaques, thus enabling multiple experimental approaches. The receptor exists in different states: the resting state, the open state and the desensitized state, amongst others. For each of these states, effects of general anaesthetics at the receptor molecule have been shown. Displacement studies show barbiturates to bind to a site on the resting state of the nicotinic acetylcholine receptor; cation flux studies suggest barbiturates may also bind to the same, or a similar, site on the open state of the receptor when inhibiting its function. Long-chain alcohols inhibit the open receptor, perhaps by binding to such a site or sites. However, short-chain alcohols do not inhibit and do not share this long-chain alcohol binding site; instead they nonspecifically enhance the agonist's apparent affinity. All the alcohols also cause desensitization by a non-specific mechanism possibly involving perturbation of the lipid bilayer. Thus, general anaesthetics exert both specific and non-specific actions on the acetylcholine receptor. PMID- 7535691 TI - Interactions of general anaesthetics with single acetylcholine receptor channels. AB - We used single-channel recording techniques to study the effects of general anaesthetics on nicotinic acetylcholine receptor channels. Normally, these channels remain open for a few milliseconds. Anaesthetics induce three different patterns of channel activity. Ether causes the channel amplitude to be smaller and noisier than normal; isoflurane induces a flickery pattern in which openings occur in bursts of brief openings; propofol causes the channels to appear as isolated brief openings. These patterns can all be understood in terms of a model in which the anaesthetics bind directly to the channel protein and interrupt the flow of ions through the channel. The difference in pattern is determined by the duration of anaesthetic binding. Ether remains bound for the shortest period (< or = 0.01 ms), followed by isoflurane (0.5 ms) and propofol (> or = 2 ms). The anaesthetics may either be physically obstructing the pore of the channel or acting allosterically by inducing a new, non-conducting conformation of the channel. PMID- 7535692 TI - Integrative effects of general anaesthetics: why nerve axons should not be ignored. AB - The effects of low and clinically relevant concentrations of inhalation anaesthetics and related compounds on the firing behaviour of nerve axons from a number of species are described. The observation of substantial, if sometimes transient, changes in excitability is contrasted with the view that axonal ion channels are insensitive to many general anaesthetics. Taking the squid giant axon as a well-studied example, we show that a full understanding of these effects can only be achieved through a detailed investigation of the actions of a range of compounds on a number of ion channels. Small alterations in the properties of individual channel types can in combination result in major changes in the behaviour of a multi-channel system such as an axon. Finally, as an example of the spectrum of activity of structurally related compounds, we compare the effects of inhalation anaesthetics with those of volatile convulsants. PMID- 7535693 TI - The synaptic basis of general anaesthesia. AB - The cellular and synaptic mechanisms that underpin the state of general anaesthesia are discussed. Anaesthetics act principally on synaptic processes and this provides a satisfactory basis for understanding their effects on neural networks. Although anaesthetics affect both the release of neurotransmitters and post-synaptic receptor function, the effects on post-synaptic receptors are always of importance in the modulation of synaptic transmission. Effects on action potential firing patterns also play a role in anaesthetic modulation of neuronal signalling. Many of these complex data can be explained in terms of altered ion channel function. PMID- 7535694 TI - Volatile anaesthetics: cellular mechanisms of action. AB - The mechanisms by which volatile anaesthetics act in the central nervous system are reviewed. The main cellular targets are excitatory synapses and the cell membrane of the neuronal cell body/dendrite, although there also appears to be a significant effect on thin unmyelinated fibres. Experiments quantifying these effects have shown that 1 MAC isoflurane reduces the activity in afferent fibres by 18%, excitatory synapses by 27% and postsynaptic neurones by 24%. Two important effects observed when recording from individual neurones are hyperpolarization of the cell membrane by an enhanced potassium conductance and increased threshold for initiation of action potentials. Excitatory synaptic transmission is most probably reduced due to a decreased release of transmitter from presynaptic terminals. Antagonistic and biphasic effects are often seen, for instance depression of both inhibitory and excitatory transmission and depolarization-hyperpolarization of the postsynaptic membrane. PMID- 7535695 TI - The filamentous brush border glycocalyx, a mucin-like marker of enterocyte hyper polarization. AB - The probably sole constituent of the filamentous brush border glycocalyx, which has been defined on the basis of electron microscopic data as a set of filaments radiating from the tip of rabbit intestinal brush border microvilli, has been purified. It consists of a mucin-type glycoprotein that can be solubilized by either Triton extraction or papain treatment of the brush border membrane vesicles but is insensitive to phosphatidylinositol phospholipase C. The detergent- and papain-solubilized forms both have the same apparent molecular mass of 400 kDa (SDS/PAGE). This suggests that the filamentous brush border glycocalyx may be anchored to the membrane by a small hydrophobic peptidic tail. Ser, Thr, Pro and Ala amount to 65% of the protein core amino acid residues. The glycosidic moiety, which amounts to 73% of the molecular mass, has high O acetylated sialic acid contents. A monoclonal antibody (3A4) raised against the purified material was produced which specifically recognized the 400-kDa band by immunoprecipitation and immunoblotting, and the filamentous brush border glycocalyx of villus enterocytes when jejunum sections were immunolabelled. The 3A4 determinant was identified with a filamentous brush border glycocalyx specific carbohydrate structure containing an O-acetylated sialic acid. The fact that the labeled glycocalyx was anchored entirely in a membrane microdomain at the tip of the microvilli shows that mature enterocytes are hyper-polarized epithelial cells. PMID- 7535696 TI - The pathophysiology of the insulin-like growth factor axis in fetal growth failure: a basis for programming by undernutrition? AB - Recent evidence suggests that a number of adulthood conditions, including non insulin dependent diabetes mellitus (NIDDM) and lipid and cardiovascular abnormalities are associated with intra-uterine growth retardation (IUGR). It is possible that this arises from programming of endocrine axes during development as a result of an adverse intra-uterine environment. Insulin-like growth factors (IGFs) are mitogenic polypeptides which stimulate cellular proliferation and differentiation and are important in human fetal development. The functions of IGFs are modulated by specific high affinity binding proteins (IGFBPs). IGFBP-1 is antagonistic to the insulin-like and growth promoting effects of IGF-I, and IGFBP-3 holds IGFs in the circulation by associating with IGFs and an acid labile subunit to form a ternary complex. Using specific radioimmunoassays and fetal serum obtained during diagnostic cordocentesis we have investigated the role of the IGF/IGFBP axis in human fetal development. In a study of 130 singleton pregnancies we have examined levels of immunoreactive IGFs and IGFBPs in normally grown fetuses (AGA), starved small fetuses affected by uteroplacental insufficiency (UPI), and non-starved small fetuses (SGA). IGF-I was significantly lower in the UPI group (n = 14, 7.8 +/- 0.6 micrograms l-1), than in either the SGA group (n = 22, 31.4 +/- 3.5 micrograms l-1, P = 0.0001) or the AGA group (n = 94, 36.3 +/- 1.9 micrograms l-1, P = 0.0001). IGFBP-3 showed similar changes (UPI: 682.6 +/- 50.0 micrograms l-1; SGA: 831.9 +/- 55.5 micrograms l-1; AGA: 847.7 +/- 19.8 micrograms l-1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535697 TI - Nitric oxide and the pathogenesis of heart muscle disease. PMID- 7535698 TI - Antibodies that activate beta 2 integrins can generate different ligand binding states. AB - A human erythroleukemic cell line (K562) that does not normally express beta 2 integrins has been transfected with the genes encoding these integrins. The resulting cell lines show minimal background adhesion but can be stimulated to bind to appropriate substrates when activated with either of two different antibodies to CD18. The two antibodies appear to generate different ligand binding states in LFA-1 such that different members of the ICAM family are recognized. Antibody-activated complement receptor type 3 and p150,95-transfected cells bind protein-coated surfaces, although they require slightly different activation conditions for optimal binding. PMID- 7535699 TI - Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors. AB - Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4+ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 microgram/ml. PMID- 7535700 TI - Role of the thymus in the generation of skin-homing alpha beta and gamma delta virgin T cells. AB - Current models of lymphocyte traffic suggest that homing specificities of T cells to tissues such as skin are generated outside the thymus as a result of activation of naive T cells by antigen in lymph nodes. Virgin T cells are thought to home to high endothelial venules in lymph nodes, but are thought to be unable to home to extra-lymphoid tissues such as skin. We used the technique of in situ labeling of the thymus with fluorescein isothiocyanate to examine the homing specificities of authentically naive T cells in vivo, immediately after their export from the thymus. We report that homing specificities for skin as well as lymph node are imprinted on T cells inside the thymus, independent of antigen. We also show that both alpha beta and gamma delta emigrant T cells exhibit homing patterns to skin and lymph nodes which are identical to those of mature T cells. Our findings demonstrate a key role for the thymus in the induction of skin homing specificities on T cells indicating that skin-homing specificities of T cells are not generated solely outside the thymus as a result of the activation of virgin T cells by antigen. The migration of thymic emigrants to extra-lymphoid tissues within a few hours of leaving the thymus may have implications for mechanisms of peripheral self-tolerance. This pathway provides an opportunity for direct virgin T cell interactions with self components only expressed in the periphery at a time when emigrants may be more susceptible to tolerance induction than mature circulating T cells. PMID- 7535701 TI - Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability. AB - Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase. PMID- 7535702 TI - CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1 alpha, interleukin-8 and RANTES. AB - The chemokines macrophage inflammatory protein 1 alpha (MIP 1 alpha), interleukin 8 (IL-8) and RANTES are potent regulators of leukocyte trafficking. Examination of chemokine secretion by human peripheral blood lymphocytes after stimulation with anti-CD3 or phorbol 12, 13 myristate acetate and ionomycin showed CD8+ cells were the dominant source of MIP 1 alpha and RANTES. Although production of MIP 1 alpha and IL-8 were similar in pharmacologically stimulated CD4+ CD45RA+, CD4+ CD45RO+, and CD8+ CD45RA+ cells, the largest amounts of MIP 1 alpha and RANTES were secreted by CD8+ CD45RO+ lymphocytes. A parallel pattern of prolonged chemokine mRNA expression for at least 18 h after activation was observed in the T cells subsets. These results confirm that human T lymphocytes have a unique capacity for secretion of these three chemokines. In addition, CD8+ cells have an unrecognized role in recruiting cells to sites of inflammation, and adult human CD45RA+ cells have a physiologically significant secretory capacity. PMID- 7535703 TI - Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity. AB - Crude preparations of chicken interferon (ChIFN) from various sources contain both antiviral and macrophage-activating factor (MAF) activity. Previous serological data indicated that unlike mammals, birds might express only a single type of IFN in response to viruses and mitogens that exhibits both activities. We have now expressed a complementary DNA for virus-induced ChIFN in transfected COS cells and in Escherichia coli. Purified recombinant ChIFN is a powerful antiviral agent and has high Mx promoter-inducing activity. However, as the sole agent, recombinant ChIFN lacks MAF activity: it does not induce the secretion of nitric oxide in primary monocyte-derived chicken macrophages. A neutralizing antiserum prepared against cloned ChIFN blocks most of the antiviral and Mx promoter inducing activity present in preparations of natural ChIFN, but does not inhibit the MAF activity. These results demonstrate that chicken cells can be induced to secrete a novel cytokine which probably represents the avian homolog of mammalian IFN-gamma. PMID- 7535704 TI - The action of pyrantel as an agonist and an open channel blocker at acetylcholine receptors in isolated Ascaris suum muscle vesicles. AB - The anthelmintic pyrantel is believed to act as an agonist at acetylcholine receptors on somatic muscle from the parasite Ascaris suum. This study aimed to confirm this mode of action of pyrantel. Single-channel recordings from muscle vesicles formed from the extrasynaptic region of the bag of somatic muscle cells of Ascaris suum were made using the patch-clamp technique. Pyrantel (0.03-100 microM) activated cation-selective channels with at least 2 conductance levels: main conductance 41 +/- 2.04 pS (mean +/- S.E., n = 28), smaller conductance 22.4 +/- 0.34 pS (mean +/- S.E., n = 8). The current/voltage plots showed a linear relationship. Detailed kinetic analysis revealed that activation of the receptor by pyrantel resulted in at least 2 distinct open and burst states and at least 3 distinct closed states. The mean open time of the channel, with 0.1 microM pyrantel, was 1.53 +/- 0.22 ms (mean +/- S.E., n = 7) at -75 mV. We have previously shown that acetylcholine activated channels with similar properties to the pyrantel-activated channels (Pennington, A.J. and R.J. Martin, 1990, J. Exp. Biol. 154, 201) confirming that pyrantel is an acetylcholine agonist. With high concentrations (100 microM) of pyrantel a sequence of rapid openings and closings of the channel was observed, indicating the presence of an open channel block. Previous experiments have shown that the anthelmintic levamisole, which also acts as an acetylcholine agonist on this preparation, induced channel block at hyperpolarised potentials with high concentrations (Robertson, S.J. and R.J. Martin, 1993, Br. J. Pharmacol. 108, 170). A comparison is made of the actions of the 3 agonists pyrantel, levamisole and acetylcholine at the nicotinic receptor in Ascaris muscle, and implications for the therapeutic use of the compounds are discussed. PMID- 7535705 TI - Endogenous nitric oxide modulates behavioural effects elicited by substance P in rat. AB - Several studies have shown that the undecapeptide, substance P, alters behaviour following central or peripheral administration in the rat. Here we report that L arginine administration increases substance P-induced locomotion and changes in food intake in rats. NG-Nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, reduces substance P-induced effects. These results suggest that endogenous nitric oxide plays a role in the modulation of the catecholaminergic effect of substance P on motor behaviour. They also clarify the mechanism underlying food intake induced by substance P. PMID- 7535706 TI - Selective inhibition of sympathetic nerve-mediated contraction by L-arginine in lipopolysaccharide-treated tail artery of rats. AB - The effects of L-arginine on the adrenergic responses to either electrical transmural stimulation or phenylephrine were studied in isolated endothelium denuded strips of rat tail arteries treated with lipopolysaccharide for 6 h in vitro. L-arginine did not relax the strips precontracted by phenylephrine. However, the adrenergic contractions induced by electrical transmural stimulation were significantly inhibited by the addition of L-arginine. This inhibitory effect was reversed by NG-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor) but was not affected by hemoglobin (a scavenger of nitric oxide). These results indicate that the adrenergic neurogenic contractions may be directly modulated by nitric oxide derived from the sympathetic nerves and/or neighboring cells in the lipopolysaccharide-treated rat tail arteries, and the nitric oxide production may be associated with the reduction of sympathetic tone in sepsis. PMID- 7535709 TI - Regulation of ileal electrolyte transport by K+ channel activators. AB - Pinacidil (N"-cyano-N-4-pyridyl-N'-1,2,2-trimethylpropylguanidine monohydrate) and BRL 38227, a benzopyran derivative, two K+ channel activators, were found to decrease short-circuit current (ISC), a measure for ion movement across the intestinal tissue. This decrease in ISC was correlated with an increase in NaCl absorption. These results suggest the possibility of new forms of drug therapy for diarrheal diseases. The effects of pinacidil were compared to galanin which also increased NaCl absorption. Galanin increased potassium currents in whole cell patch clamp studies. The effects of galanin and pinacidil on Isc were not additive suggesting a common pathway in their mechanism of action. PMID- 7535708 TI - Augmented antinociception following 7-nitro indazole and flurbiprofen in the conscious mouse. AB - Co-administration of the nitric oxide synthase inhibitor, 7-nitro indazole (1 mg/kg i.p.), with the cyclooxygenase inhibitor, flurbiprofen (5-75 mg/kg i.p.), resulted in significantly enhanced antinociceptive activity in mice (formalin induced hindpaw licking assay) without affecting hindpaw inflammation. No antinociception was observed in animals pretreated with 7-nitro indazole (1 mg/kg i.p.) and flurbiprofen (100 micrograms subplantar). Flurbiprofen (50 mg/kg i.p.) pretreatment did not influence the inhibition of cerebellar or spinal cord nitric oxide synthase activity observed after 7-nitro indazole (1 or 25 mg/kg i.p.) administration and did not alter blood pressure in anaesthetised animals. Thus, flurbiprofen acts by a mechanism unrelated to a local anti-inflammatory effect in the hindpaw. Since nitric oxide synthase inhibitors are antinociceptive by an effect in the spinal cord (dorsal horn) this would appear to be a likely location for the nitric oxide synthase and cyclooxygenase enzymes targetted by 7-nitro indazole and flurbiprofen respectively. PMID- 7535707 TI - Functional evidence for a glibenclamide-sensitive K+ channel in rat ileal smooth muscle. AB - The motor activity of gastrointestinal smooth muscle is closely related to the membrane potential. Controlling the membrane potential via modulation of K+ channels is essential for the action of neurotransmitters on smooth muscle. In the present study the effect of the K+ channel activator, lemakalim, on longitudinal smooth muscle of the rat ileum was investigated. Segments of rat ileum were stimulated by the muscarinic receptor agonist, carbachol (10(-6) M). Lemakalim (10(-10) to 3 x 10(-5) M) induced a dose-dependent inhibition of the carbachol-induced contraction. This inhibitory effect of lemakalim was not modified by neural blockade with tetrodotoxin (10(-6) M, n = 9). Glibenclamide (10(-7) to 10(-5) M), a specific blocker of ATP-dependent K+ channels antagonized dose dependently the relaxant effect of lemakalim (IC50: 3.4 x 10(-6) M, n = 11, P < 0.001). In contrast, apamin (10(-7) M, n = 9, n.s.) and charybdotoxin (10(-7) M, n = 9, n.s.), specific blockers of Ca2+-dependent K+ channels and the non specific K+ channel blocker, tetraethylammonium (10(-4) to 10(-1) M), had no influence on the inhibitory effect of lemakalim. Contractions induced by the Ca2+ channel activator, Bay-K-8644, were completely inhibited by lemakalim (10(-5) M, n = 12). This inhibitory effect was also selectively antagonized by glibenclamide (10(-5) M). Potential non-adrenergic non-cholinergic (NANC) inhibitory mediators like ATP, nitric oxide (NO) or neurotensin showed no sensitivity to glibenclamide. These functional data indicate that the relaxant effect of lemakalim is due to a specific activation of glibenclamide-sensitive K+ channels, which in turn can modulate the activity of dihydropyridine-sensitive (voltage dependent) Ca2+ channels. A physiological or pathophysiological role of the glibenclamide-sensitive K+ channels in intestinal smooth muscle is discussed; however, they seem not to be involved in the effect of the NANC inhibitory mediators tested. PMID- 7535710 TI - Differential pharmacology of GABAA and GABAC receptors on rat retinal bipolar cells. AB - GABAA and GABAC receptors were studied on cultured or freshly isolated rat retinal bipolar cells. The cells displayed GABA-induced whole-cell currents, which were only partially blocked by high concentrations (100 microM) of the GABAA receptor antagonist bicuculline. The bicuculline-resistant (GABAC) component was insensitive to the GABAA receptor modulators flunitrazepam (1 microM) and pentobarbital (50 microM). The bicuculline-sensitive portion of the current was strongly augmented by both drugs, indicating that it was mediated by conventional GABAA receptors. The GABAC and GABAA receptor subtypes displayed a 7 fold difference in their binding affinity for GABA, the EC50 values being 4.2 microM and 27.1 microM, respectively. The Hill coefficient was approximately 2 for both receptors. The bicuculline-insensitive GABAC receptors were markedly blocked by 100 microM picrotoxinin, 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide (SR-95531) and gamma-hexachlorocyclohexane, drugs known to be antagonists of GABAA receptors. Examination of single-channel currents indicated main-state conductances of 7.9 pS and 29.6 pS for GABAC and GABAA receptors, respectively. The pore diameter of open GABAC receptor channels was 5.1 A, i.e. close to the value of 5.6 A reported for the GABAA receptor. These results demonstrate that rod bipolar cells possess two populations of pharmacologically distinct GABA receptors, GABAA and novel-type GABAC receptors, which might subserve different physiological functions in controlling visual transduction in the retina. PMID- 7535711 TI - Cyclosporine A inhibits protein-kinase-C-mediated amylase release from isolated rat pancreatic acini. AB - The present study was performed to characterize a direct influence of cyclosporine A (CsA) on exocrine pancreatic enzyme secretion. CsA inhibited dose dependently amylase release from isolated rat pancreatic acini in response to carbachol or cholecystokinin octapeptide (CCK-8). A significant reduction in amylase release by 17% was observed at 0.2 mumol/l CsA (p < 0.001) when compared to controls. At 0.2 mmol/l, CsA reduced amylase release in response to both secretagogues by maximally 45%, whereas basal secretion was not affected. CsA had no influence on CCK-8-stimulated increase in intracellular Ca2+ concentrations or amylase release in response to the Ca2+ ionophore A 23187. In contrast, the dose response curve for amylase secretion induced by the phorbol ester phorbolmyristate-13-acetate was shifted to the right without a reduction of the maximal secretory response. Unexpectedly, vasoactive-intestinal-polypeptide- and secretin-stimulated acinar secretion was not diminished by CsA. In isolated pancreatic lobules exposed to 0.1 mmol/l CsA, amylase release stimulated by cerulein or veratridine was reduced by 26.7 +/- 2 or 28.3 +/- 4%, respectively. CsA had no influence on the displacement of 125I-Bolton-Hunter-labeled CCK-8 from acinar CCK receptors. The ultrastructure of cellular organelles in isolated lobules was not altered after incubation with 0.1 mmol/l CsA for 60 min. Our data suggest that CsA interferes with protein-kinase-C-mediated signal transduction in isolated rat pancreatic acini, without affecting cAMP-dependent signalling. PMID- 7535712 TI - Influence of chronic nicotine intake and acute ethanol challenge on gastric mucus level and blood flow in rabbits. AB - The effects of nicotine pretreatment on ethanol-induced gastric mucosal lesions and changes of gastric mucosal mucus levels and blood flow (GBF) were studied in anaesthetized rabbits. Nicotine treatment 25 or 50 micrograms/ml drinking water did not affect the volume of water consumption during the 10-day experimental period. It did not produce gastric mucosal lesions or affect the superficial adherent mucus content. The length of mucus-containing cells and the basal GBF were also unaffected. Intragastric administration of absolute ethanol reduced GBF, this effect was not altered by nicotine. However, the alkaloid potentiated the ulcerogenic actions of ethanol both on lesion formation and mucus depletion evoked by graded oral doses of ethanol (50 or 100%, v/v). Ultrastructurally, the mucous cells were more degenerated in the animals co-treated with nicotine and ethanol. It is concluded that reductions of mucus-containing cells and adherent mucus on the gastric mucosa are likely to be the contributory factors involved in the aggravating action of nicotine on ethanol-induced gastric mucosal lesions in rabbits. PMID- 7535714 TI - [Follow-up of beta-hCG after pelviscopic linear salpingotomy for therapy of tubal pregnancy]. AB - From 1987 until 1993 a total of 337 patients underwent a therapy for tubal pregnancy at the Department of Gynaecology and Obstetrics of the University of Kiel. The percentage of cases treated by tubal-preserving pelviscopic linear salpingotomy and subsequent extraction of the conceptus, increased from 59% in 1987 to 88% in 1993. In 6.5% a second look pelviscopy was performed for suspected remnants of trophoblastic tissue. Remaining trophoblastic cells can be recognised by serial determination of serum beta-hCG titers postoperatively. However, the interval of the postoperative beta-hCG decline reveals considerable interindividual variation. A regression following the "Cox Proportional Hazard" model shows that the period of the beta-hCG decline depends on the initial titer. The Kaplan Meier curve obtained via beta-hCG determination in 98 patients following pelviscopic linear salpingotomy until values reached 20 mIU/ml or less, shows that 50% of the patients had to be controlled for more than 7 days, 10% for more than 28 days and individual patients even for more than 70 days. The relative beta-hCG titers differ significantly from the unresolved group compared to the group with resolved ectopic pregnancy starting at postoperative day 2 (p < 0.01). At postoperative day 3/4 10% of the initial beta-hCG value is attained in 50% of cases. Even then a serial beta-hCG follow-up should be obtained down to the detection limit to ensure complete resolution of viable trophoblastic cells. PMID- 7535713 TI - [Intra-arterial hepatic chemotherapy. Experience of 200 cases]. AB - OBJECTIVES: This retrospective study had three aims: a) to investigate whether one catheter, whatever the number of hepatic arteries, associated with miscellaneous arterial ligations, produced the same results as the implantation of multiple catheters, b) to study the survival rate after intra-arterial chemotherapy, and c) based on a multifactorial study of prognostic factors, to define the best indications of this treatment in the future. METHODS: Two hundred catheters were surgically implanted to perform intrahepatic arterial chemotherapy in patients with multiple unresectable isolated liver tumors. The origins of these tumours were colorectal cancer in 152 cases, neuroendocrine tumour in 13 cases, hepatocellular carcinoma in 9 cases, and miscellaneous tumours in 22 cases. The hepatic arteries were ligated in 32% of the cases. Resection of the primary or local recurrence was performed at the same time in respectively 41% and 16% of the cases. RESULTS: The use of only one catheter was always possible, and the ligations of miscellaneous accessory hepatic arteries did not modify the therapeutic results. There were no postoperative deaths. Numerous complications occurred during local chemotherapy, mainly digestive and hepatobiliary complications (38.5% of the cases). Early discontinuation of local chemotherapy was due to the occurrence of thrombosis of the hepatic artery in 23% of the cases (after a mean delay of 5.2 months) and to the occurrence of digestive or hepato biliary complications in 11% of the cases. The rate of objective response was 44%, with 22 complete and 62 partial responses on morphologic examination. The crude survival rates were 65 +/- 3% at 1 year, 33 +/- 4% at 2 years and 5 +/- 2% at 5 years, with a median survival of 16 months. The median survival was different according to the primary tumour (34 months for neuroendocrine tumours, 17 months for colorectal tumours and 12 months for hepatocellular and miscellaneous carcinomas). For the 152 colorectal cancers, a multifactorial study identified 2 main independent adverse prognostic factors: an involvement of the liver greater than 50% (P = 0.006) and circulating carcinoembryonic antigen > 100 ng/L (P = 0.001). PMID- 7535715 TI - [The so-called adenomatoid tumor of the uterus: a case report of evaluation of primary sterility]. AB - A description of the clinical diagnosis, localisation and the histological structure of an adenomatoid tumour of the uterus is presented. The tumour was diagnosed during investigation of sterility and localised in the uterus wall. Fallopian tubes and ovaries can also be affected. Adenomatoid tumours are small, slow growing and of benign origin, and hence organ-preserving tumourectomy is an adequate therapy. Histogenesis of this kind of tumour has been established by means of immunological techniques as being of mesothelial origin. PMID- 7535716 TI - Mutations in the unc-52 gene responsible for body wall muscle defects in adult Caenorhabditis elegans are located in alternatively spliced exons. AB - The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52 encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping. PMID- 7535719 TI - Tumor angiogenesis as a prognostic factor in cervical carcinoma. AB - Angiogenesis, the induction of new capillaries and venules, has been associated with tumor growth. Increased tumor size and new vessel growth may further the opportunity for tumor cells to enter the circulation and potentiate metastatic disease. To investigate if tumor angiogenesis could serve as a prognostic factor in cervical carcinoma, we counted microvessels (capillaries and venules) in 29 patients with squamous cell carcinoma of the cervix. Surgical specimens were stained for endothelial cells specifically with Factor VIII to identify all vessels. The microvessels were counted by light microscopy (per 200 x field) in tumor sections with the highest population of microvessels. This was performed by two investigators without knowledge of patient outcome or extent of disease. Microvessel counts in patients with squamous cell carcinoma were significantly different from those of control subjects: 56 +/- 28.9 and 16.3 +/- 3.3 (P = 0.013). There was no correlation between microvessel count and node status, parametrial involvement, depth of invasion, or gross disease. Microvessel count was significantly correlated with vascular space involvement (P = 0.017). Four patients who developed recurrent disease within 1 year had high microvessel counts and yet were node negative and VSI negative at surgery. As shown by Folkman in breast cancer, angiogenesis may also be an independent predictor for recurrent disease in squamous cell carcinoma of the cervix. Microvessel counts could be of prognostic value in patients who do not have other risk factors for disease recurrence. PMID- 7535717 TI - Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila. AB - Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium does seem to be present in the 5' half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. PMID- 7535720 TI - Cytokeratin subunit 19 measured by CYFRA 21-1 assay in follow-up of cervical cancer. AB - The purpose of this study was to evaluate the serum tumor markers CYFRA 21-1 and squamous-cell carcinoma antigen (SCC) in the follow-up of squamous-cell cervical cancer patients. One hundred-ninety-three serum samples, which were collected pretherapeutically and during follow-up of 30 patients suffering from squamous cell cervical cancer FIGO stage III, were analyzed for SCC and CYFRA 21-1. Cutoff values for SCC and CYFRA 21-1 were 3 and 3.3 micrograms/liter, respectively. Fifteen cases were keratinizing and 15 nonkeratinizing squamous-cell carcinomas. Serum tumor marker results were correlated to the results of the clinical and radiologic examinations. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of serum SCC and serum CYFRA 21-1 for the whole study group and separately for the keratinizing and nonkeratinizing squamous-cell carcinoma group. Sensitivity, specificity, PPV, and NPV of serum SCC were 66, 91, 93, and 60%, respectively. Serum CYFRA 21-1 showed a sensitivity of 63%, specificity of 96%, PPV of 96%, and NPV of 59%. The combination of SCC and CYFRA 21-1 increased the sensitivity to 78%, with a specificity, PPV, and NPV of 87, 91, and 69%, respectively. In keratinizing squamous-cell carcinoma serum SCC, CYFRA 21-1 and the combination of both had a sensitivity of 76, 64, and 85%, respectively. In nonkeratinizing squamous-cell carcinoma sensitivity was 58, 61, and 72%, respectively. The detection of cervical cancer recurrences with SCC is improved by the combination with CYFRA 21 1. Especially in nonkeratinizing squamous-cell carcinoma CYFRA 21-1 showed promising results. PMID- 7535718 TI - A novel role for a U5 snRNP protein in 3' splice site selection. AB - The choice of a 3' splice site in Saccharomyces cerevisiae introns involves recognition of a uridine-rich tract upstream of the AG dinucleotide splice junction. By isolating mutants that eliminate the normal preference for uridine containing 3' splice sites in a cis-competition, we identified a mutation that is an allele of PRP8, prp8-101. This was unexpected because previous analysis has demonstrated that the U5 snRNP protein encoded by PRP8 is required for spliceosome assembly prior to the first catalytic step of splicing. In contrast, the uridine recognition defect caused by the prp8-101 mutation selectively inhibits the second catalytic step of splicing. This defect is seen not only in 3' splice site cis-competitions but also in the splicing of an unusual intron in the TUB3 gene and in the ACT1 intron when utilization of its 3' splice site is rate limiting for splicing. Consistent with a direct role in 3' splice site selection, Prp8 can be cross-linked to the 3' splice site during the splicing reaction. These data demonstrate a novel function for Prp8 in 3' splice site recognition and utilization. PMID- 7535721 TI - Mucinous adenocarcinomas of the vulva. AB - An 80-year-old nullipara had a 2.0-cm cystic tumor of the right labium majus. Histologic diagnosis was mucinous eccrine carcinoma. Seventy-five percent of these rare skin adnexal tumors arise on the face, eyelid, or scalp; but none has been reported on the vulva. Indolent localized growth is usual with regional nodal spread in 11% and distant metastases in 3%. A 67-year-old multipara had a 1.2-cm polypoidal nodule of the posterior fourchette. Histologically, a colonic type mucinous carcinoma was arising within a villous adenoma. Mucicarmine and CEA stains were positive. Extensive workup failed to reveal other primary cancers in either patient. Both patients are well 19 and 17 months after radical vulvectomies and node-negative groin dissections. These cases illustrate further the diversity in cell type and biologic behavior of vulvar adenocarcinomas. PMID- 7535722 TI - Conus-cauda syndrome as a presenting symptom of endodermal sinus tumor of the ovary. AB - We report on a case of a 46-year-old woman with a conus-cauda syndrome due to an endodermal sinus tumor of the right ovary with multiple metastases in the spine and pelvic bone. Before removing the tumor surgically, combination chemotherapy was given to treat the metastases, which threatened to compromise the spinal cord. PMID- 7535724 TI - Roots of interferon research. PMID- 7535723 TI - Establishment and characterization of two human ovarian clear cell adenocarcinoma lines from metastatic lesions with different properties. AB - Two permanent human ovarian clear cell adenocarcinoma lines (OVISE and OVTOKO) were established from metastatic tumors of two patients who were treated with five to six courses of CAP chemotherapy. The two cell lines grow on monolayers and showed a variety in both size and shape: small or moderately sized cuboidal cells, columnar cells, spindle-shaped cells, and malignant tumor giant cells. The cell lines have been in culture for 4 to 6 years, the passage number varying from 160 to 220. The mean population-doubling time of the two cells was 60 to 70 hr. The OVISE cells shed tumor-associated antigens CA19-9, CA125, and TPA in the culture medium, whereas the OVTOKO cells did not secrete them at detectable levels. Immunohistochemical analysis showed that coexpression of cytokeratins and vimentin was preserved in the two cell lines, which is a feature of cultured epithelial origin. Cytokeratin polypeptides 7, 8, 18, and 19 were expressed in both cell lines. The EGF receptor was more intensely expressed in the OVTOKO cells than in the OVISE cells. The estrogen and progesterone receptors were negative in both cell lines. The two cell lines showed no chemosensitivity to anticancer drugs including cisplatin, doxorubicin, cyclophosphamide, and etoposide. Heterotransplantation of the two cell lines reflected the origin of cells. Intraperitoneal transplantation of the OVTOKO cells yielded peritoneal implantation and distant metastasis, whereas that of the OVISE cells showed no dissemination and metastasis. These new ovarian clear cell adenocarcinoma lines will provide a relevant experimental system for further investigations into the intrinsic alterations responsible for malignant progression and chemoresistance. PMID- 7535725 TI - The dream for interferon. PMID- 7535726 TI - Interferon induction: regulation by both virus and cell. AB - Both the virus and its host cell play significant roles in the regulation of interferon (IFN) induction and production. The virus exerts a regulatory effect through variation in its capacity to deliver an IFN inducer molecule in the form of dsRNA, and by suppressing activation of the IFN gene(s) or their subsequent expression. Regulation of IFN induction by genetically closely related viruses may encompass a 10,000-fold range of IFN yields. Viruses that fail to induce IFN express a dominant phenotype in that they suppress IFN induction in cells otherwise programed to produce IFN. The loss of the IFN induction-suppressing phenotype through non-temperature-sensitive mutations is accompanied by the acquisition of IFN inducibility from an otherwise latent state. The cell regulates production of IFN through genetically programed events as part of the developmental process in embryos, and through physiological changes that occur during incubation in vitro. In the developing chicken embryo, the acquisition of IFN inducibility may span a 1,000-fold range of IFN yields, and appears as a time dependent process proceeding uninterrupted from in ovo to in vitro culture. Populations of established cell lines contain subpopulations (clones) that can differ 10-fold from the mean IFN yielding capacity of the parent, and may undergo physiological changes upon "aging" in vitro that also span a 10-fold range of IFN yields. During development, and most likely as a result of physiological changes brought on by aging in vitro, IFN regulatory factors themselves are regulated. This report reviews the regulation of IFN induction and production with vesicular stomatitis virus (VSV) as a model virus, and primary chick embryo cells and an established line of mouse L cells as model host cells to define systematically the extent and manner in which the virus and cell regulate IFN induction and production. PMID- 7535727 TI - Interferon-induced proteins and their mechanisms of action. AB - Interferons modulate a number of biological functions including cell growth and differentiation, the immune response, and virus replication. The antiviral activity of interferons (IFNs), the property that led to their discovery, is mediated by multiple cellular proteins. Among the IFN-induced cellular proteins with antiviral activity are the protein kinase PKR, the enzymes of the 2', 5' oligoadenylate pathway, and the Mx proteins. The antiviral activities of these proteins differ significantly against viruses of the myxoviridae, rhabdoviridae, and picornaviridae virus families. PMID- 7535728 TI - G-CSF mobilization in steady state for peripheral blood stem cell transplantation. AB - Peripheral blood stem cells were collected by granulocyte-colony stimulating factor (G-CSF) mobilization in normal volunteers and patients with hematological malignancy in complete remission without anti-cancer drug synchronization. The yields of PBSC and the possibility of G-CSF mobilization in steady state for PBSC transplantation (PBSCT) were studied. For collecting PBSC, G-CSF was subcutaneously injected at the dose of 100 micrograms/m2 on 5 consecutive days. PBSC collection was performed on day 4 and/or 5 by using cell separator, CS-3000. In normal volunteers, the yields of colony forming unit in granulocyte and macrophage (CFU-GM) was 1.9 x 10(4) kg by processing the plasma of 1.5L, and CD34 positive cell was 1.4% on the average. In patients with hematological malignancy in complete remission, the processing volume ranges from 10 to 18L/1-2 cycles, the average CFU-GM number was 2.2 x 10(5)/kg, and the average CD34 positive cell was 2.2%. In acute leukemia case, PBSCT was performed by using G-CSF mobilized PBSC, engraftment was achieved earlier. In conclusion, the yields of G-CSF mobilized PBSC from normal volunteers suggested the possibility of PBSCT and the yields of PBSC from patients in complete remission proved that G-CSF mobilization method in complete remission status could take the place of drug synchronized G CSF mobilization for which the timing of collection and the drug choice for synchronization are intricate. PMID- 7535729 TI - Formation of anti-sense RNA to IFN mRNA in poly (rI):(rC) induced HEL cells. AB - To elucidate the mechanism of super induction of IFN, mRNA and anti-sense RNA were analyzed by using reverse transcription-polymerase chain reaction (RT-PCR) method, and following results were obtained. (1) When human embryonic lung fibroblast cells (HEL cells) were exposed to poly (rI):(rC) for 1 hr, treated with cycloheximide for 3 hr, treated with actinomycin-D for 30 min at the final period of cycloheximide treatment, then washed and replenished with maintenance medium, (HEL-I:C/CH/Act. D), they produced much higher amount of IFN and IFN production continued for longer period when compared to the HEL cells which were not treated with actinomycin-D (HEL-I:C/CH). In agreement with these IFN production kinetics, HuIFN-beta mRNA was stabilized in HEL-I:C/CH/Act. D. In HEL I:C/CH/Act.D, the mRNA was detected at the end of cloheximide treatment (4 hr after induction) and 6 hr after induction, however, it was not detected at 6 hr after induction in HEL-I:C/CH. (2) Anti-sense RNA was detected in HEL I:C/CH/Act.D at 4 hr and 6 hr after induction, and also in HEL-I:C/CH at 4 hr after induction, however, it was not detected at 6 hr after induction in HEL I:C/CH. These results indicated that an anti-sense RNA to HuIFN-beta mRNA was formed in poly (rI):(rC) induced HEL cells, and that aRNA had the same fate as mRNA, suggesting it's role in the regulation of IFN production. Possible role of aRNA in the regulation mechanism of IFN production was discussed. PMID- 7535730 TI - [Clinical studies on the detection of hepatitis C virus genome in the serum and the liver]. AB - Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Since the HCV viral genome was molecularly cloned and antibody detection systems were established, a considerable amount of information on HCV has been accumulated. In the present study, I have examined the sera and liver tissues of the patients of type C chronic liver disease using molecular biological methods and tried to correlate the data obtained with the clinical, pathological and therapeutic outcomes. A reverse transcription-polymerase chain reaction (RT-PCR) method using radiolabeled nucleotides provided constant results in genome amplification, and could be shown to be a reliable method for quantifying the levels of HCV genome. The amount of HCV RNA in the serum tended to increase as a function of the duration of the disease and the progression of histopathologic changes of the liver. In addition, the serum HCV RNA titers correlated well with those of liver tissues, which showed that the amount of circulating HCV genome reflected the intrahepatic amount of HCV. HCV, a positive-stranded RNA virus, is likely to make a negative strand during viral replication. In studies to detect the positive and negative strands of HCV RNA separately using strand specific primers, the existence of both strands in the liver was demonstrated, confirming viral replication in the liver. The ratio of negative to positive strands ranged from 0.03% to 30%, being 3.7% in chronic persistent hepatitis, 4.8-6.0% in chronic aggressive hepatitis and 6.7% in liver cirrhosis. The viral factors influencing the interferon (IFN) response were also examined. Individuals who had low titers of HCV genome tended to respond well to IFN. Moreover, IFN was more effective for patients with HCV type III than for those with HCV type II. Thus, IFN response was shown to depend both on virus titers and genotypes. However, mean levels of HCV RNA were almost the same between the patients with HCV type II and those with HCV type III before the treatment, there were significant differences in the disappearance of HCV and the effects of IFN therapy. It is suggested that the susceptibility to IFN is different for each genotype. Hokkaido J Med Sci 69 (6), 1382-1398, 1994. PMID- 7535731 TI - [Fas fever among researchers]. PMID- 7535732 TI - Alternative splicing of the insulin receptor isoforms is altered in patients with leprechaunism. AB - Leprechaunism is a syndrome of severe insulin resistance. In general, mutations in both alleles of the insulin receptor are required for developing the leprechaun phenotype. Recently, we described a leprechaun patient having an Arg for Gly substitution in one allele of the insulin receptor whereas the other allele has the normal sequence. To explain the leprechaun phenotype, we searched for additional defects at the receptor level. The insulin receptor exists as two splice variants, either with (B form) or without exon 11 (A form). It has been suggested that a decrease in the relative amount of the A isoform can contribute to the development of an insulin resistant state. Fibroblasts from the leprechaun patient show levels of the A isoform of less than 20% whereas in control fibroblasts approximately 50% of the A isoform is present. However, in fibroblasts from two other patients with severe insulin resistance where the disease has been demonstrated to result from two mutated insulin receptor alleles, the same decline of the A isoform was seen. We conclude that the decrease in the relative amount of the A isoform in these patients is probably an epiphenomenon due to impaired insulin action. PMID- 7535733 TI - Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression. AB - Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized histologically by nonsuppurative destructive cholangitis. Sera from patients with PBC react with a series of intramitochondrial enzymes with the immunodominant response directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Recently, using tissue sections of late-stage PBC, we showed that there is increased expression in biliary epithelial cells of patients with PDC-E2 or a molecule cross-reactive with PDC-E2. Previous work has shown that biliary epithelial cells of patients with PBC express an increased amount of class II. To address the sequence of events in the evolution of PBC, we have focused our attention in this study on early biliary epithelial lesions. In particular, we have studied the liver of 22 female patients with PBC that was diagnosed as either stage I or stage II using both a mouse monoclonal antibody that has reactivity similar to human autoantibodies as well as a human Fab combinatorial prepared from the lymph node of a PBC patient. Tissues were simultaneously stained using antibodies to PDC-E2, class II, and BB1/B7. As a positive control, tissues from late-stage PBC were studied concurrently. By determining the order of expression among the three molecules, PDC-E2, class II, and BB1/B7, we report that the expression of PDC-E2 or a PDC-E2-like molecule on biliary duct epithelium of patients with PBC precedes the expression of BB1/B7 and major histocompatibility complex (MHC) class II molecules. The alteration of an autoantigen in biliary duct epithelium may be the earliest lesion in PBC. PMID- 7535734 TI - In vitro tumor necrosis factor cytotoxicity in Hep G2 liver cells. AB - Tumor necrosis factor-alpha (TNF-alpha) is a mediator of liver injury. The objective of this study was to develop an in vitro model of TNF-mediated liver cell injury using the Hep G2 cell line. Hep G2 cells normally are insensitive to TNF cytotoxicity, but they were rendered susceptible, or sensitized, to TNF cytotoxicity by inhibitors of RNA and protein synthesis. The concentration of TNF required to kill 50% of Hep G2 cells sensitized with 0.8 mumol/L actinomycin D (Act D) was 35 pmol/L compared with 5 pmol/L for LM fibroblasts, a classic target cell used in TNF cytotoxicity bioassays. Similarly, TNF cytotoxicity occurred in Hep G2 cells sensitized with cycloheximide (CHX), and cytotoxicity to both inhibitors was dose dependent. Both protein and RNA synthesis were inhibited in Hep G2 cells by the concentrations of CHX and Act D associated with TNF cytotoxicity. Hep G2 cells pretreated with TNF alone and later exposed to normally toxic concentrations of TNF with DACT did not develop cytotoxicity. Thus, in vitro tolerance to TNF was induced. Cytotoxicity also was more severe at modestly increased temperatures (39 degrees C versus 37 degrees C), which may have clinical relevance to hepatic decompensation during febrile episodes. We suggest that the Hep G2 cell line sensitized by inhibiting RNA and protein synthesis is a useful in vitro model for evaluating mechanism(s) of TNF-mediated liver cell injury. PMID- 7535735 TI - Cyclosporin A and FK-506 in inhibition of rat Ito cell activation in vitro. AB - Ito cells are the primary matrix-producing cells in the liver. In hepatic fibrosis in vivo or culture on plastic, these cells undergo activation, a process characterized by cell proliferation, fibrogenesis, and smooth muscle alpha-actin expression. The cytosolic-binding proteins of cyclosporin A (CsA) and FK506 accelerate folding of various proteins including collagen and become inactivated by binding to those agents. CsA is shown to inhibit collagen synthesis in cultured fibroblasts. These findings prompted us to examine the effect of cyclosporin A and FK506 on Ito cell activation. CsA and FK506 reduced DNA synthesis in a dose-related manner, to 26% and 45% of controls at 5 mumol/L, respectively, without affecting total protein synthesis. CsA reduced collagen synthesis in a dose-related manner, to 70% of controls at 5 mumol/L without affecting noncollagenous protein synthesis, whereas FK506 changed neither collagen synthesis nor noncollagenous protein synthesis. Moreover, smooth muscle alpha-actin expression was reduced by 0.5 mumol/L CsA, but not by FK506. CsA merits consideration for the therapy of hepatic fibrosis. FK506 may also be a candidate for such therapy through inhibitory action on Ito cell proliferation. PMID- 7535737 TI - Hepatitis C virus RNA and liver histology in blood donors reactive to a single antigen by second-generation recombinant immunoblot assay. AB - The clinical significance of single band reactivity (indeterminate pattern) at anti-hepatitis C virus (HCV) second-generation recombinant immunoblot assay (RIBA 2) was investigated in symptomless subjects with normal liver function tests to obtain data for their counseling and clinical management. Serum and hepatic HCV RNA were determined by the nested polymerase chain reaction, and liver histology was evaluated in 40 symptomless blood donors with stable indeterminate RIBA-2 pattern, including 38 reactive to c22-3. All but one had normal alanine aminotransferase (ALT) levels. Two new immunoblot tests, RIBA-3 and INNO-LIA HCV Ab III (LIA-III), which incorporate additional HCV antigens, were also done to assess whether they could identify the viremic subjects. Ten cases (25%, confidence interval 12 to 38) were HCV RNA positive. Three of the HCV RNA positive and none of the HCV RNA-negative subjects had chronic hepatitis. RIBA-2 strong intensity of reaction (score > 2+) was observed in all the HCV RNA positive and in 12 HCV RNA-negative subjects. RIBA-3 and LIA-III gave positive results in 9 of 10 and 10 of 10 HCV RNA-positive, but also in 8 of 30 and 24 of 30 HCV RNA-negative subjects. A c-22-3 reactivity score of 4+ by RIBA-3 and E2/NS1 reactivity by LIA-III were both strongly associated with HCV RNA (P < .001). Based on relatively high prevalence of chronic hepatitis in our series (30%), apparently healthy subjects with stable indeterminate RIBA-2 pattern and HCV RNA positivity should be considered for liver biopsy independently of ALT profile.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535736 TI - Cellular immune responses in patients with dual infection of hepatitis B and C viruses: dominant role of hepatitis C virus. AB - Several lines of evidence have suggested that immune mechanisms are involved in the pathogenesis of hepatitis B virus (HBV)- and hepatitis C virus (HCV)-related hepatitis. Study of patients with dual HBV and HCV infection raises the question of which is etiologically more relevant in determining the liver cell damage. To address this issue, proliferation of peripheral blood mononuclear cells (PBMCs) in response to a panel of HBV and HCV antigens was assayed in 13 patients with chronic dual hepatitis B and C, 7 patients with chronic hepatitis B, 7 patients with chronic hepatitis C, and 6 patients with hepatitis B surface antigen (HBsAg) carrier state. Although HBV or HCV hepatitis patients had a significant response to HBV or HCV antigens, respectively, the patients with dual hepatitis B and C exclusively responded to HCV antigens, but not to HBV antigens. One patients who was seropositive for both HBV-DNA and HCV-RNA showed a low response to HBV antigens initially but lost the response 3 months later and became responsive to more HCV antigens. These findings suggest that HCV has a dominant role in the immune response of the patients with dual HBV and HCV infection. PMID- 7535738 TI - Hepatobiliary disease in cystic fibrosis patients with pancreatic sufficiency. AB - Focal and multilobular biliary cirrhosis are considered pathognomonic of cystic fibrosis (CF) and almost invariably have been reported in patients with steatorrhea. In contrast, patients with pancreatic sufficiency and normal absorption are considered less likely to develop liver or biliary tract problems. The authors report three patients with CF and pancreatic sufficiency, presenting with recurrent abdominal pain (unrelated to pancreatitis). All had common bile duct disease, one with multilobular cirrhosis and portal hypertension. Pancreatic sufficiency was proven by quantitative pancreatic stimulation tests, 3-day fecal fat analyses, and serum pancreatic isoamylases. All three patients had mild lung disease. Two were homozygous for the common delta F508 mutation, and the other, a delta F508 compound heterozygote. Hepatobiliary structure and function were determined by serial hepatobiliary scintigraphy, percutaneous transhepatic cholecystography, and biochemical liver function tests. Patients 1 and 3 had mild hepatomegaly, normal liver biochemistry, and distal common bile duct strictures. Patient 2 had a firm nodular liver with splenomegaly, abnormal liver biochemistry, and a cholangiographic appearance of sclerosing cholangitis. All have undergone operative treatment for persistent abdominal pain. These cases confirm the occurrence of common bile duct pathology and liver disease in patients with CF and pancreatic sufficiency. They demonstrate that liver and biliary tract disease can occur independently of the underlying disease severity and the presence of steatorrhea. Further, they suggest that obstruction of the biliary tract may be an additional factor in the evolution of liver disease in CF. PMID- 7535739 TI - Expression of the retinoblastoma gene product in human hepatocellular carcinoma. AB - Using the mouse anti-human retinoblastoma gene product (pRB) monoclonal antibody, PMG3-245, pRB was detected immunohistocytochemically in human hepatocellular carcinoma (HCC) tissues and a human HCC cell line, designated OCUH-16, recently established in our laboratory. This antibody reacted with human pRB and yielded a single band of approximately 110 kd from cultured OCUH-16 cells. The granules that stained for pRB were found mostly in the HCC cell nuclei, with a few granules observed in the rough endoplasmic reticulum by electron microscopy. Most of the stained granules were located in the euchromatin-rich areas. The percentage of OCUH-16 cells that expressed pRB or DNA polymerase alpha (DNA-PA) decreased over time as the number of OCUH-16 cells increased. The number of HCC cells that stained for pRB in the biopsy specimens from 11 patients varied and pRB expression was well maintained in early and advanced HCC. The level of pRB expression did not correlate with the differentiation of HCC cells or the clinical prognosis. The expression of pRB statistically correlated with that of DNA-PA (P < .01; r = .92). Some sinusoidal cells also stained for pRB. These findings imply that large deletions in the pRB gene are rare in the initiation or promotion of HCC. The correlation between pRB and DNA-PA may suggest that stained pRB participates in the proliferation of both HCC and non-HCC cells. PMID- 7535740 TI - CD-34 and keratin expression distinguishes solitary fibrous tumor (fibrous mesothelioma) of pleura from desmoplastic mesothelioma. AB - Solitary fibrous tumors (SFTs) often involve the pleura and also may encompass the peritoneum and nonserosal sites. On occasion SFTs mimics other neoplasms, including desmoplastic mesothelioma. CD-34, initially characterized as a hematopoietic progenitor cell antigen, recently has been identified in a small number of SFTs. Based on this observation, we compared the keratin, vimentin, and CD-34 expression of 19 SFTs and eight desmoplastic mesotheliomas. Fifteen of 19 SFTs (78.9%) expressed CD-34, whereas keratin expression was absent in all SFTs. In contrast, none of the desmoplastic mesotheliomas expressed CD-34 and keratin expression was found in seven of eight (87.5%). Vimentin expression was noted in 18 of 19 SFTs and in seven of eight desmoplastic mesotheliomas. We conclude that CD-34 expression distinguishes SFT from desmoplastic mesothelioma. Additionally, the results of our study support the idea that SFT is not derived from or related to conventional mesothelium. PMID- 7535741 TI - Intimal neovascularization in human coronary atherosclerosis: its origin and pathophysiological significance. AB - To investigate the histopathological characteristics of the newly formed vessels in the atherosclerotic intima of human coronary arteries, we conducted postmortem angiography in 31 cases, including 11 with myocardial infarction. Vessels were examined three-dimensionally under the stereoscope. In addition, we evaluated 25 anterior descending coronary arteries unrelated to the occurrence of myocardial infarction by light microscopy using 3-mm stepwise sections and 5-microns serial sections. Histological alterations were analyzed morphometrically to determine the correlation between the degree of intimal neovascularization and the growth of the endothelium into the atherosclerotic intima from the adventitia or lumen. There was a significant positive correlation between the density of new vessels in the intima and the incidence of luminal stenosis, the extent of chronic inflammatory infiltrate, the formation of granulation tissue, or the atheromatous changes, whereas the vascular density decreased in the extensively hyalinized and calcified intima. The newly formed intimal vessels originated mainly from the adventitial vasa vasorum and also partly from the proper coronary lumen. The intimal vessels that originated from the adventitia occurred approximately 28 times more frequently than those that originated from the luminal side. The frequency of former vessels increased as the luminal stenosis became more severe, whereas the latter vessels were found most frequently in the presence of 40% and 50% stenosis. Vessels originating from the proper lumen were more often associated with fresh or old hemorrhage. We conclude that intimal neovascularization largely originates from the adventitia and is closely associated with the extent of coronary stenosis and the histological inflammatory reaction. PMID- 7535743 TI - Molecular basis of inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) polymorphism. AB - A genetic polymorphism of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) was analyzed at the nucleic acid level. Three common alleles, ITIH1*1, ITIH1*2 and ITIH1*3, were characterized by mutations at codons 551 and 561 in exon 14. ITIH1*1 was characterized by GAG (Glu) at codon 551 and CAG (Gln) at codon 561, ITIH1*2, by GTG (Val) and CGG (Arg), and ITIH1*3, by GAG (Glu) and CGG (Arg). PMID- 7535744 TI - Exclusion of the 13-kDa rapamycin binding protein gene (FKBP2) as a candidate gene for multiple endocrine neoplasia type 1. AB - The MEN1 gene is considered to be a tumour suppressor gene and has been localised to a 1-Mb region of 11q13.1. In this study, we report the physical localisation of the 13-kDa FK506 and rapamycin binding protein gene (FKBP2) to the cosmid marker D11S750, which is located inside the MEN1 region of non-recombination. The product of this gene is involved in signal transduction and is thus a candidate cell growth regulator or tumour suppressor gene. Northern studies have revealed that FKBP2 is expressed in those tissues predisposed to hyperplasia in MEN1; however, single-strand conformation polymorphism analysis and direct sequencing of DNAs from affected members of MEN1 kindreds and sporadic tumour DNAs have been performed and no mutations have been found. These studies exclude FKBP2 as a candidate gene for MEN1. PMID- 7535742 TI - Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations. AB - A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1 8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype. PMID- 7535748 TI - Immunocytochemical distribution of gamma isoform of protein kinase C (PKC-gamma) following incomplete ischaemia. AB - Immunocytochemical distribution of PKC-gamma was examined in rat brain in relation to molecular mechanisms of post-ischaemic neuronal modulation following incomplete ischaemia. Incomplete ischaemia was developed by either permanent occlusion of one common carotid artery (CA) or permanent occlusion of one CA with temporary occlusion of opposite CA. Unilateral CA (UCA) occlusion resulted in a pronounced increase in the intensity of staining and number of PKC-gamma positive neurons in the neocortex ipsilateral to the insult after 3 h. The effect was maximum at 6-12 h and was undetectable after 7 days. CA1 neurons showed an increase immunoreactivity (IR) after 1 day, reached to a peak by 3 days, then reduced to basal levels after 7 days. Bilateral CA (BCA) occlusion showed almost similar changes in the neocortex, but on both sides and short durated. The altered patterns of PKC-gamma IR in the neocortex and hippocampus following CA occlusion may reflect activation and/or down-regulation of PKC-gamma in ischaemic neurons. PKC-gamma may, therefore, potentially play an important role in the post ischaemic modulation of synaptic efficacy in these neurons and in the neuronal damage following incomplete ischaemia. PMID- 7535747 TI - C to T and/or G to A transitions are responsible for loss of a MspI restriction site at the 5'-end of the human apolipoprotein AI gene. AB - We detected the loss of a MspI restriction site by a C to T transition at +83 bp and a G to A transition at +84 bp of the 5'-end non-coding region of the human apolipoprotein AI gene. This base change occurred at the "hot spot" (CCGG) for methylation, which may be important in the regulation of gene expression. The population frequency for the loss of the MspI site is 6.1%. PMID- 7535746 TI - A new polymorphism in exon 7 of the cystic fibrosis transmembrane regulator (CFTR) gene. AB - Here we describe a new polymorphism, located in exon 7 of the cystic fibrosis transmembrane regulator (CFTR) gene at nucleotide position 1104 (C-->G), detected by a single-strand conformational polymorphism (SSCP) analysis. PMID- 7535745 TI - Fluorescent multiplex microsatellites used to identify haplotype associations with 15 CFTR mutations in 124 Northern Irish CF families. AB - The cystic fibrosis transmembrane conductance regulator gene contains three highly informative microsatellites; IVS8CA, IVS17BTA and IVS17BCA. Fluorescent multiplexes of these microsatellites were assayed in 124 CF families carrying 15 different CFTR mutations, from N. Ireland. PMID- 7535749 TI - Functional characterization of rat chemokine macrophage inflammatory protein-2. AB - Expression of mRNA for the C-X-C chemokine, macrophage inflammatory protein-2 (MIP-2), is induced during acute inflammation in rat models of disease. We have characterized the phlogistic potential of rat recombinant MIP-2 (rMIP-2) protein in vitro and in vivo. Recombinant MIP-2 caused marked PMN chemotaxis in vitro, with peak chemotactic activity at 10 nM. Incubation of whole blood with rMIP-2 caused a significant loss of L-selectin and a significant increase in Mac-1 expression on the PMN surface. Under similar conditions rMIP-2 also caused a modest respiratory burst in PMNs. The intratracheal instillation of 10 and 50 micrograms of rMIP-2 caused a significant influx of PMNs into the airspace of the lungs. Rat MIP-2 is a potent neutrophil chemotactic factor capable of causing neutrophil activation and is likely to function in PMN recruitment during acute inflammation in rat disease models. PMID- 7535752 TI - Suppression of pulmonary metastasis by angiogenesis inhibitor TNP-470 in murine osteosarcoma. AB - We treated a murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs, with a new angiogenesis inhibitor, TNP-470, to evaluate the efficacy of this compound in the suppression of pulmonary metastasis of osteosarcoma. In an in vivo experiment, tumor cells were inoculated i.v. into C3H mice, and TNP-470 or vehicle alone (control group) was administered s.c. every day for 3 weeks. In the TNP-470-treated groups, both the number of pulmonary metastatic nodules and the lung wet weight were significantly reduced in a dose-dependent manner. Similarly, vascular density in the metastatic tumors estimated by immunohistochemical staining with anti-von-Willebrand factor antibody as an endothelial marker were significantly reduced. No severe side effects were found. In an in vitro experiment, viable tumor cells were counted after 3 days' treatment with TNP-470. The 50% inhibitory concentration was 0.6 ng/ml for LM8, which was more sensitive than other tumor cells previously reported. Our results show that TNP-470 suppresses the pulmonary metastasis of LM8 and suggest that both its anti-angiogenic activity and cytostatic activity towards LM8 are responsible for the antitumor effect. PMID- 7535751 TI - MAPAG: a computer program to construct 2- and 3-dimensional antigenic maps. AB - The contact area between an antibody (Ab) and the antigen (Ag) is called antigenic determinant or epitope. The first step in the characterization of an Ag by using monoclonal antibodies (MAb) is to map the relative distribution of the corresponding epitopes on the Ag surface. The computer program MAPAG has been devised to automatically construct antigenic maps. MAPAG is fed with a binary matrix of experimental data indicating the ability of paired MAb to bind or not simultaneously to the Ag. The program is interactive menu-driven and allows the user an easy data handling. MAPAG utilizes iterative processes to construct and to adjust the final map, which is graphically shown as a 2- or a 3-dimensional model. Additionally, the antigenic map obtained can be optionally modified by the user or readjusted by the program. The suitability of MAPAG was illustrated by running experimental data from literature and comparing antigenic maps constructed by the program with those elaborated by the investigators without the assistance of a computer. Furthermore, since some MAb could present negative allosteric effects leading to misinterpretation of data, MAPAG has been provided with an approximate reasoning module to solve such anomalous situations. Results indicated that the program can be successfully employed as a simple, fast and reliable antigenic model-builder. PMID- 7535750 TI - Protein kinase C inhibitor calphostin C prevents cytokine-induced angiogenesis in the rat. AB - A rat sponge implant model was used to examine the role of protein kinase C (PKC) in angiogenesis. Neovascular response was determined by measurements of relative sponge blood flow by a 133Xe clearance technique and confirmed histologically. Morphometric analysis was used to quantitate the amount of fibrovascular growth in the sponges. Daily doses of recombinant human basic fibroblast growth factor (bFGF, 100 ng), tumor necrosis factor-alpha (TNF-alpha, 50 ng), or interleukin-1 alpha (IL-1 alpha, 50 ng) caused neovascular responses that were blocked by daily coadministration of the selective PKC inhibitor, calphostin C (4 micrograms). To confirm that calphostin C was able to inhibit PKC in vivo, its effect on the angiogenic response elicited by the PKC activator, phorbol 12-myristate 13 acetate (PMA, 30 micrograms) was examined. The blood flow and morphometric data clearly showed that the intense neovascularization induced by PMA was totally suppressed by coadministration of calphostin C (4 micrograms). Thus, these results suggest that cytokine-induced angiogenesis may be mediated in part through the activation of PKC and that selective inhibition of this enzyme could have therapeutic benefit in angiogenic diseases. PMID- 7535754 TI - Characterization of mature and immature RadLV-induced thymic T-cell lines for tumorigenesis and MHC-class-I gene expression. AB - Class-I-MHC molecules are divided into class-Ia molecules, which play major roles in recognition of virus-infected cells, graft rejection and immune responses against tumors, and class-Ib molecules, which are less polymorphic and may be responsible for presenting unique classes of peptides. Our report characterizes RadLV-induced thymic T-cell lines that differ both in their tumorigenic potential and in the level of protein for class-Ia and TL genes. The PD1.1 cell line is CD4 CD8+ and expresses relatively high levels of class-I as compared with the CD4+CD8+ PD1.2 cell line. These class-I-expression levels correlate with thymocytes and splenic T cells of the same phenotype, except that normal cells fail to express TL3b. Interferon-treated PD1.2 cells demonstrate significantly lower levels of class-I expression than do interferon-treated PD1.1 cells, and were shown to contain large amounts of degraded class-I mRNA, at least some of which was TL in origin. These RNA products were not detected in PD1.1 cells, suggesting the existence of a mechanism controlling cell-specific and gene specific mRNA stability. Such RadLV-induced cell lines provide a means for obtaining stage-specific T cells, which can be used for studying the regulation of class-I gene expression during T-cell differentiation, as well as factors that differentially regulate class-Ia and class-Ib expression and are potentially useful for studying T-cell differentiation in general. PMID- 7535753 TI - Regulation of cytoskeletal organization in tumor cells by protein phosphatases-1 and -2A. AB - Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-1 and -2A) are inhibited by okadaic acid, attaining the same level of motility as metastatic LLC (LLC-LN7) variants. This stimulation of LLC-C8 motility was tempered when protein kinase A activity was inhibited. We examined whether the okadaic acid-stimulated LLC-C8 motility was associated with alterations in the cytoskeletal organization so that these non-metastatic cells acquire the rounded morphology and diffuse cytoskeletal organization previously described for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 are typically adherent during culture, achieving a spread morphology. Treatment of non metastatic LLC-C8 cells with okadaic acid resulted in a contraction of most of their extended processes, formation of spikes and membrane blebs within 10 min, and complete cell rounding within 20 min for most of the cells. While the overall level of F-actin was minimally affected by the okadaic acid, its uniform distribution shifted to localization toward the periphery of the rounded cells, often concentrating at a single focus. Immunofluorescent staining for vimentin showed a similar shift to the cell periphery and similar capping. After okadaic acid treatment, the filamentous network of microtubules in non-metastatic LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta tubulin. These results show that PP-1 and -2A maintain cytoskeletal organization and that inhibition of this control reduces cytoskeletal organization and increases tumor cell motility. PMID- 7535755 TI - Glycosylation of a novel member of the immunoglobulin gene superfamily expressed in rat carcinoma cell lines. AB - MAb E4 recognizes a 66-kDa glycoprotein, pE4, which is a member of the immunoglobulin gene superfamily. This protein is expressed at the cell surface in rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. Since expression of aberrant carbohydrate structures is often associated with malignant transformation, glycosylation of pE4 was analyzed. Reactivity of lectins with pE4 suggested the absence of N-acetylneuraminic acid, terminal galactose and O-linked glycan, and the presence of N-linked glycans. Tunicamycin treatment reduced the binding of MAb E4 to cancer cells suggesting that the E4 epitope is at least partially glycosylated. Digestions with neuraminidases, O glycosidase and peptide-N-glycosidase F confirmed these results. Pronase treatment abolished the binding of MAb E4, indicating that E4 epitope involves not only a carbohydrate determinant but also a peptide moiety. Mild periodate oxidation abolished the binding of MAb E4, indicating that non-reducing terminus carbohydrates are part of the E4 epitope. Neutral sugar analysis revealed the absence of galactose and the presence of fucose. Since fucose is sensitive to periodate oxidation, this sugar could be the carbohydrate part of the determinant recognized by MAb E4. Reactivity of lectins specific for fucose indicated the presence of alpha(1-6)-fucose on pE4. PMID- 7535756 TI - Glycoconjugate profile and CD44 expression in human melanoma cell lines with different metastatic capacity. AB - Changes in glycoconjugate production have been reported for tumor cells. In this study, we investigated the glycoconjugate expression pattern in normal human melanocytes and in a panel of 6 human melanoma cell lines with different metastatic capacity after s.c. inoculation into nude mice. Glycoconjugates were labeled in vitro with [35S] sulphate and [3H] glucosamine, purified from cells and culture medium by column chromatography and identified by treatment with specific glycosidases. Characterization of the purified glycoconjugate fractions as well as alcian-blue staining of xenograft lesions revealed that hyaluronic acid (HA) is the main glycoconjugate produced by all cell lines. Highly metastatic cell lines expressed higher levels of HA than melanocytes and than weakly metastatic or non-metastatic cell lines. In addition, a shift in dominance from chondroitin-sulphate proteoglycan to heparan-sulphate proteoglycan was observed with increasing metastatic capacity. We also studied the expression and binding activity of the HA receptor CD44. Immunoprecipitation experiments indicated high CD44 synthesis only in highly metastatic cell lines, but FACS analysis demonstrated approximately the same surface expression in melanocytes as in all cell lines. Adhesion assays to immobilized HA showed that CD44 can be present in an inactive or an active conformation. Our data suggest that a combination of increased HA production and the expression of CD44 on the cell surface may be associated with high metastatic potential of human melanoma cell lines in nude mice. PMID- 7535758 TI - A rapidly emerging field: water channel proteins in the eye. PMID- 7535757 TI - Comparison of the inhibitory effect of the angiogenesis inhibitor, TNP-470, and mitomycin C on the growth and liver metastasis of human colon cancer. AB - Angiogenesis inhibitors have attracted considerable interest. The anti-tumor and anti-metastatic effects of TNP-470, an angiogenesis inhibitor, and mitomycin C (MMC), a representative anti-neoplastic agent, were investigated using a xenotransplanted human colon cancer, TK-4. Suturing of small pieces of TK-4 tumors to the cecal wall in nude mice (orthotopic transplantation) induced liver metastasis. Mice were randomly divided into 3 groups; a control group given saline solution, a group receiving TNP-470 and a group receiving MMC. TNP-470 was given s.c. on alternate days for 5 weeks from day 10 after cecal transplantation and MMC was administered intraperitoneally (i.p.) once a week from day 10 after cecal transplantation. MMC significantly inhibited cecal tumor growth. In the control group, liver metastases developed in 9 out of 10 mice, including 3 with more than 20 metastatic foci. Liver metastasis also developed in 8 out of 10 mice receiving MMC, 2 of which had many metastases. In contrast, liver metastasis developed in only 2 out of 8 mice in the TNP-470 group and neither of these animals had numerous metastases. PMID- 7535759 TI - [Substance P and vasoactive intestinal peptide in patients with progressive scleroderma. Determination of plasma level before and after autogenic training]. AB - In 12 patients suffering from systemic sclerosis (SSc) the influence of autogenic training on the plasma level of the neuropeptides substance P and vasoactive intestinal peptide (VIP) was studied. Compared with healthy controls the SSc patients exhibited significantly elevated levels of substance P (mean +/- SD: 7.1 +/- 3.2 pmol/l vs 1.6 +/- 1.6 pmol/l). Apart from variations the VIP plasma concentration did not significantly differ from that in healthy controls (mean +/ SD 10.7 +/- 7.1 pmol/l versus 12.0 +/- 5.3 pmol/l). Autogenic training did not bring about any significant changes in the plasma levels of neuropeptides. PMID- 7535761 TI - Simple quantitative reverse transcribed-polymerase chain reaction (RT-PCR) method involving recombinant RNA generated by a false-priming PCR product. AB - A simple and efficient method for preparing rcRNA through competitive RT-PCR has been developed. The basis of this method is the use of a false-priming PCR product including the same primers as a specific product. A 290 bp fragment obtained by two-step PCR was subcloned into a plasmid vector and then the cloned DNA was transcribed into rcRNA. After competitive RT-PCR using sample RNA and rcRNA had been carried out, Southern blot hybridization was performed. The method was applied to determine the amounts of PIMT mRNAs in the rat pituitary. The quantitative analysis indicated that an at least 2-fold difference in PIMT mRNA level can be accurately determined with our method. PMID- 7535760 TI - Sequential transplants using mobilized peripheral blood progenitor cells. AB - Modest success has been achieved with the use of high-dose cytotoxic therapy and bone marrow transplantation in solid tumors. Patient outcome can potentially be improved with further intensification of the therapy. The rapid hematologic recovery achieved with mobilized peripheral blood progenitor cells (PBPC) may reduce the toxicity of transplantation enabling the use of sequential courses of myeloablative therapy. We report on 42 patients with solid tumors enrolled in a tandem transplant protocol involving the use of PBPC mobilized with cyclophosphamide (4 g/m2), etoposide (1 g/m2), and granulocyte-colony-stimulating factor (G-CSF: 10 micrograms/kg/day). This regimen significantly increased the number of circulating progenitor cells; only 1-2 aphereses were sufficient to collect 2.5 x 10(8)/kg mononuclear cells, our goal for each transplant course. The median number of circulating colony-forming units (CFU) and CD34+ cells obtained for each transplant course were 70.3 x 10(4)/kg, and 11.7 x 10(6)/kg, respectively. There was a significant correlation between the numbers of CD34+ cells and CFU measured in the apheresis product (r = 0.49, P = .003). The first transplant regimen given to 38 patients consisted of thiotepa, carboplatin, and cyclophosphamide. The second transplant regimen given to 29 patients consisted of busulfan and etoposide. Hematologic recovery was comparable after each of the two transplant courses. The median time to neutrophil recovery over 0.5 x 10(9)/L and to platelet transfusion independence was 9 and 8 days, respectively. There was no difference in engraftment rates after transplant with PBPC only (n = 28 courses) compared to transplant with PBPC plus bone marrow (n = 39 courses).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535762 TI - Human laminin M chain: epitope analysis of its monoclonal antibodies by immunoscreening of cDNA clones and tissue expression. AB - We have produced four monoclonal antibodies (mAbs) against human placenta laminin purified by immunoaffinity chromatography. Three clones, 2D9, 3DM, and 4F1, recognized 320 kDa (M) chain of the laminin and the other one, 3DB, recognized B2 chain. One cDNA clone (HLM-1, 3.5 kb) was immunoscreened from human placenta cDNA library using 2D9, and three further overlapping cDNA clones (HLM-2, HLM-3, and HLM-4) covering 2.0 kb were isolated. Nucleotide sequencing and fusion protein analysis demonstrated that the amino acid sequence deduced from HLM-1 coincided with that of the G-domain of human merosin chain, and HLM-2, HLM-3, and HLM-4 encoded the long-arm and EGF-like domain of M chain. The binding regions of 2D9 and 3DM to M chain were defined as homologous repeating sequences of G2-G3 region of G-domain and the carboxy-terminal region of the long-arm, respectively. The extents of identity of amino acid sequences of the long-arm and EGF-like domains between human M chain and A chain were about 37% and 52%, respectively, which were lower than between mouse and human A chains. Northern blot analysis revealed that M chain mRNA, 8.6 kb, was highly expressed in heart and placenta, but less highly expressed in skeletal muscle, brain, and lung. Immunostaining showed selective distributions of M chain in nerve fibers in the dermis and mesangial matrix of the kidney, and B2 chain in subepidermal and kidney glomerular basement membranes. PMID- 7535763 TI - Highly efficient transfection into primary cultured mouse hepatocytes by use of cation-liposomes: an application for immunization. AB - Transfection methods for primary cultured mouse hepatocytes were examined. Of four conventional transfection methods examined, involving use of DEAE-dextran, calcium phosphate, cation-liposomes (lipofection), and cation-multilamellar liposomes, only cation-liposomes induced highly efficient transfection into primary cultured mouse hepatocytes. The highest transfection rate reached more than 60% of the total cells. Three other commonly used cell types (CHO-K1, COS-1, 3T3-L1) were also tested as target cells, but highly efficient transfection was observed specifically in primary cultured mouse hepatocytes. The transfection remained at a high level from 6 to 48 h after the start of incubation with the cation-liposome-DNA complex in the absence of serum, and the transfection rate decreased in inverse relation to the increase in cell density. The transfection was inhibited by free low density lipoprotein (LDL), EDTA, and an endocytosis inhibitor, cytochalasin B. These data suggest that the transfection is mediated not only by membrane fusion, as is generally accepted, but also by endocytosis. This information should be useful for research in hepatocyte biology and the development of gene therapy. As one of the applications, simple and successful immunization was achieved by administration of hepatocytes transfected with murine adhesion molecule, integrin VLA beta 1 subunit, genes into a Syrian hamster. PMID- 7535764 TI - Intracellular tyrosine residues of the human growth hormone receptor are not required for the signaling of proliferation or Jak-STAT activation. AB - Ligand binding and dimerization of the growth hormone (GH) receptor leads to the rapid tyrosine phosphorylation of the intracellular kinase, Jak2, to the tyrosine phosphorylation and activation of STAT protein(s) and to the tyrosine phosphorylation of the receptor itself. Expression of the human GH receptor in the mouse promyeloid, interleukin-3-dependent cell line, FDC-P1, shows that this receptor can signal ligand-dependent proliferation in these cells as well as induce the tyrosine phosphorylation of Jak2 and the activation of transcription factors. We now examine the requirement for tyrosine phosphorylation of the GH receptor for these three events by expression of a receptor without tyrosine residues in the intracellular domain. Six of the seven intracellular tyrosine residues were removed by a carboxyl-terminal truncation, and the remaining tyrosine was changed to phenylalanine to yield the GH receptor D351Stop/Y314F. When expressed in FDC-P1 cells, this receptor retained its ability to induce the tyrosine phosphorylation of Jak2, to induce the activation of transcription factors, and to signal ligand-dependent cell proliferation. Thus, tyrosine phosphorylation of the GH receptor is not essential for the signaling of these three events at least in this system. This finding contrasts with that for the interferon-gamma receptor system where data indicate that the specific tyrosine phosphorylation of the interferon-gamma receptor leads to an association with the STAT protein, p91, that is the mechanism by which ligand couples the receptor to the signal transduction system. PMID- 7535765 TI - Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer. AB - The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication. PMID- 7535766 TI - Evidence that the bradykinin-induced activation of phospholipase D and of the mitogen-activated protein kinase cascade involve different protein kinase C isoforms. AB - The effect of alkylglycerol supplementation on protein kinase C (PKC)-mediated signaling events has been studied in fibroblasts from Zellweger patients (SF 3271 cells). Western blotting analysis established that Zellweger fibroblasts express PKC alpha, epsilon, and zeta. Incubation with bradykinin induced a rapid transient translocation of PKC alpha and a more sustained translocation of PKC epsilon to the particulate fraction; translocation of PKC zeta was unaffected. Bradykinin-induced translocation and activation of PKC alpha, but not translocation of PKC epsilon, was blocked in SF 3271 cells which had been incubated with 1-O-hexadecylglycerol (1-O-HDG; 20 micrograms/ml) for 24 h and then incubated in the absence of 1-O-HDG and serum for a further 24 h. Supplementation with 1-O-HDG increased the mass of ether-linked phospholipid. Bradykinin initiated a transient increase in cytosolic Ca2+ concentration in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplementation. Bradykinin also caused a rapid activation of phospholipase D (PLD), measured by phosphatidylbutanol accumulation, and mitogen-activated protein kinase (MAPK) determined by myelin basic protein phosphorylation of Mono Q fractions. Both events were blocked by preincubation of the cells with 12-O-tetradecanoylphorbol 13-acetate for 24 h to deplete PKC protein. 1-O-HDG supplementation prevented the bradykinin-induced activation of PLD, but had no effect on the stimulation of MAPK activity. These results establish that modulation of the ether lipid composition of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC alpha, most likely PKC epsilon, is involved in MAPK activation. PMID- 7535767 TI - Heregulin stimulates mitogenesis and phosphatidylinositol 3-kinase in mouse fibroblasts transfected with erbB2/neu and erbB3. AB - Heregulin (HRG) is a pluripotent growth factor that can stimulate the growth of some human mammary tumor cells and the differentiation of others. Two members of the epidermal growth factor receptor family of receptor/tyrosine kinases, p180erbB3 and p180erbB4, serve as receptors for the HRG ligand. While HRG appears to be capable of stimulating the autophosphorylation activity of p180erbB4, the co-expression of p185erbB2/neu with p180erbB3 is necessary for the HRG-stimulated tyrosine phosphorylation of both of these receptors. On the basis of the sequences surrounding their putative tyrosine phosphorylation sites, we predict that the different HRG-responsive receptors couple to different intracellular SH2 domain-containing proteins. Hence, the different receptors may mediate different cellular responses to the HRG ligand. In the present study we show that HRG beta 1 is mitogenic for erbB3-transfected DHFR/G8 cells, an NIH3T3 mouse fibroblast derivative that over-expresses p185erbB2/neu. HRG stimulated the incorporation of [3H]thymidine into the DNA of these cells with an EC50 of 70 +/- 7 pM. HRG was not mitogenic for parental DHFR/G8 cells that do not express the ErbB3 protein. Phosphatidylinositol (PI) 3-kinase, an enzyme believed to be important in cellular growth regulation by growth factors and oncogenes, is predicted to couple to tyrosine-phosphorylated ErbB3. We observed that HRG stimulated the association of PI 3-kinase with both p185erbB2/neu and ErbB3 in transfected DHFR/G8 cells, but not in the parental cell line. We conclude that the ErbB3 protein is capable of mediating a proliferative response of fibroblasts to HRG, and that the activation of PI 3-kinase is an integral part of the growth signaling mechanism. PMID- 7535768 TI - Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase. AB - The mitogen-activated protein kinase (MAPK) also known as extracellular signal regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation. PMID- 7535769 TI - RNA ligase and its involvement in guide RNA/mRNA chimera formation. Evidence for a cleavage-ligation mechanism of Trypanosoma brucei mRNA editing. AB - RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is thought to be directed by guide RNAs and may proceed via a chimeric guide RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprotein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylylated proteins of 50 and 57 kDa co-sediment on glycerol gradients with RNA ligase activity as components of the ribonucleoprotein particles. The two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addition of ligatable RNA substrate with the concomitant release of AMP and by the addition of pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate results in an accumulation of the adenylylated RNA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable analogs indicates that both RNA ligase and chimera-forming activities require alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera formation. These results strongly suggest the involvement of RNA ligase in in vitro chimera formation and support the cleavage-ligation mechanism for kinetoplastid RNA editing. PMID- 7535770 TI - Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. AB - Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group. PMID- 7535771 TI - A novel ligand for CD44 is serglycin, a hematopoietic cell lineage-specific proteoglycan. Possible involvement in lymphoid cell adherence and activation. AB - The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation. PMID- 7535772 TI - Staurosporine causes epidermal growth factor to induce differentiation in PC12 cells via receptor up-regulation. AB - Although they all utilize tyrosine kinase receptors and activate signaling pathways characterized by a similar set of phosphoproteins, epidermal growth factor (EGF) promotes only cell division while fibroblast growth factor (FGF) and nerve growth factor (NGF) can induce division followed by differentiation in PC12 cells. EGF, in contrast to NGF and FGF, cannot maintain the sustained phosphorylation and activation of mitogen-activated protein (MAP) kinase kinase and MAP kinases, which may account for the difference in phenotypic response. The pretreatment of PC12 cells with staurosporine, a protein kinase inhibitor, causes a substantial increase in both receptor and MAP kinase phosphorylation that results in a differentiative response (neurite proliferation). However, neurites begin to disappear after 3 days, despite the continual presence of EGF, and are largely gone after 5 days, which is not the case with NGF and FGF. Thus, the effect of staurosporine is not permanent. Northern and Western blots indicate that the staurosporine response mainly results from a substantial up-regulation in EGF receptor synthesis, thus providing a much stronger cell surface signal and supporting the view that quantitative rather than qualitative differences distinguish the EGF versus NGF/FGF signaling pathways in these cells. PMID- 7535773 TI - Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC association with Grb2. AB - Growth hormone (GH) has been shown to stimulate the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2 are activated by tyrosine kinases involves the Src homology (SH)-2 containing proteins SHC and Grb2. To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound to SHC SH2-GST fusion protein, demonstrating that the SHC SH2 domain can bind tyrosyl-phosphorylated JAK2 in the absence of GHR. Regions of GHR required for GH-dependent tyrosyl phosphorylation of SHC were examined using Chinese hamster ovary cells expressing mutated rat GHR. In cells expressing GHR1-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1 454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHR delta P, the latter lacking amino acids 297-311 containing the proline-rich motif required for JAK2 activation by GH. In contrast to SHC, Grb2 appeared not to interact directly with GHR or JAK2. However, Grb2 was shown to associate rapidly with SHC proteins in a GH-dependent manner. These findings suggest that GH stimulates: 1) the association of SHC proteins with JAK2.GHR complexes via the SHC-SH2 domain, 2) tyrosyl phosphorylation of SHC proteins, and 3) subsequent Grb2 association with SHC proteins. These events are likely to be early events in GH activation of MAP kinases and possibly of other responses to GH. PMID- 7535774 TI - HER4 expression correlates with cytotoxicity directed by a heregulin-toxin fusion protein. AB - We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells. PMID- 7535775 TI - Insulin-dependent tyrosine phosphorylation of the vav protooncogene product in cells of hematopoietic origin. AB - Insulin activates the ras signaling pathway and promotes hematopoietic cell proliferation. One possible mediator in such signaling is the vav proto-oncogene product (p95vav), which is specifically expressed in cells of hematopoietic origin and contains domains typical of guanine nucleotide exchange factors as well as Src homology 2 and Src homology 3 domains. We studied the tyrosine phosphorylation of p95vav in hematopoietic cells expressing insulin receptors. Immunoblotting experiments with an antiphosphotyrosine monoclonal antibody disclosed that insulin induces rapid and transient tyrosine phosphorylation of p95vav in the human U-266 myeloma cell line. These findings were confirmed by immunoprecipitation experiments performed with 32P-labeled cells and phosphoamino acid analysis of the bands corresponding to p95vav. Similarly, insulin-dependent tyrosine phosphorylation of p95vav was observed in the human IM-9 and mouse J558L hematopoietic cell lines. Furthermore, insulin treatment of cells led to the association of the Src homology 2 domain of p95vav with the activated beta subunit of the insulin receptor in vitro. Altogether, these data suggest that p95vav is a substrate for the insulin receptor tyrosine kinase and may be involved in an insulin signaling pathway linking receptor-generated signals to Ras or other GTP-binding proteins in cells of hematopoietic origin. PMID- 7535776 TI - Thiazolidine derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein-tyrosine phosphatase activities. AB - The mechanisms for the insulin resistance induced by hyperglycemia were investigated by studying the effect of high glucose concentration (HG) and its modulation by thiazolidine derivatives, on insulin signaling using Rat 1 fibroblasts expressing human insulin receptors (HIRc). Incubating HIRc cells in 27 mM D-glucose for 4 days impaired the insulin-stimulated phosphorylation of pp185 and receptor beta-subunits. Both protein kinase C activities and phorbol dibutyrate binding to intact cells were unchanged; however, cytosolic protein tyrosine phosphatase (PTPase) activity increased within 1 h prior to the impairment of insulin receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S., Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi, A. (1993) Biochem. Biophys. Res. Commun. 197, 1078-1082). Increased PTPase activity was consistent with a 2-fold increase in the amount of PTP1B, and anti-PTP1B antibody inhibited this increment of cytosolic PTPase activity in HG cells. Co incubating cells with pioglitazone prevented these abnormalities in cytosolic PTPase, the PTP1B content and the impaired phosphorylation of pp185 and receptor beta subunits in HG cells. Finally, HG cells had impaired insulin-stimulated alpha-amino-isobutyric acid uptake, which was ameliorated by exposure to thiazolidine derivatives. In conclusion, exposing cells to high glucose levels desensitizes insulin receptor function, and thiazolidine derivatives can reverse the process via the normalization of cytosolic PTPase, but not of protein kinase C. PMID- 7535777 TI - Transforming growth factor-beta 1 induction of novel extracellular matrix proteins that trigger resistance to tumor necrosis factor cytotoxicity in murine L929 fibroblasts. AB - The molecular basis by which transforming growth factor (TGF)-beta 1 protects certain tumor cells from tumor necrosis factor (TNF) cytotoxicity was investigated. When pretreated, with TGF-beta 1, -beta 2, and -beta 3, murine L929S fibroblasts developed resistance to TNF cytotoxicity. Time course experiments revealed that TGF-beta 1 initially induced both cellular protein tyrosine phosphorylation and simultaneous secretion of a novel extracellular matrix TNF-resistance triggering (TRT) protein(s), which closely preceded the acquisition of TNF-resistance. TGF-beta 2 and -beta 3 also increased tyrosine phosphorylation. However, both molecules failed to stimulate TRT secretion. The increased levels of phosphorylation, particularly to 9 specific protein tyrosine kinase inhibitor-sensitive cellular proteins, appeared to alter the TNF killing pathway. TGF-beta 1-induced TRT secretion required participation of unknown serum factors. TRT adhered strongly to polystyrene plates and resisted treatment with heat (60 degrees C, 30 min), collagenase, alpha 2-macroglobulin, heparin, antibodies against TGF-beta s, and limited trypsin digestion. Notably, TRT promoted TNF-resistance via activation of tyrosine and serine/threonine kinase functions in L929S. Thus, the molecular pathway involves TGF-beta 1-mediated initiation of a rapid tyrosine phosphorylation of cellular protein substrates (which alters TNF cytotoxic pathway), and a simultaneous secretion of TRT, which in turn signals the cells to maintain the levels of phosphorylation, thereby sustaining the TNF-resistance. PMID- 7535778 TI - Demonstration of functionally different interactions between phospholipase C gamma and the two types of platelet-derived growth factor receptors. AB - Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr 1018 are able to compete with the intact receptor for binding to immobilized PLC gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma. PMID- 7535779 TI - Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells. AB - The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl and hemopoietic factors. PMID- 7535780 TI - Regulation of myelin basic protein gene transcription: identification of a distal cis-acting regulatory element. AB - The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial-specific transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis-acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated MB3, which is located between -93 to -130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the MB3 regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double-stranded MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G-residues within nucleotides -100 to -108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 DNA with the nuclear proteins from various cells of glial origin. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells. PMID- 7535781 TI - Identification of insulin-like growth factor binding proteins from cultured human epidermal keratinocytes. AB - The role and mechanisms of action of insulin-like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF-I receptors and are responsive to IGF-I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF-I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37 42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP-2. Radioimmunoassay of IGFBP-1, -3, and -6 revealed detectable levels of IGFBP-3 and significant levels of IGFBP-6, but not IGFBP-1. Northern analysis following culture in complete medium revealed that at early passage IGFBP-1, -2, -4, and -6 mRNAs were detectable. IGFBP-3 and -5 mRNAs were not detectable. Following culture in growth factor-free medium a 37-42 kD band, consistent with IGFBP-3, was predominant and a 24 kD band consistent with IGFBP-4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte-derived IGFBPs are likely to play a role in the transport and targeting of IGF-I from dermally derived fibroblasts to the epidermis. PMID- 7535782 TI - Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. AB - Vascular endothelial cells (ECs) are constantly subjected to mechanical strain due to relaxation and contraction of vessel walls. The effects of cyclical strain on endothelin-1 (Et-1) secretion and Et-1 mRNA levels in human umbilical vein ECs were examined. Cultured ECs grown on a flexible membrane base were deformed by negative pressure (16 kPa at 60 cycles/min). Cells subjected to strain showed increased Et-1 secretion (0.54 ng/hr/10(6) cells) compared with unstrained control cells (0.22 ng/hr/10(6) cells). Northern blot analysis of cells strained for 2 hours or longer demonstrated a sustained elevated Et-1 mRNA level at more than double the level in unstrained controls. This strain-induced ET-1 mRNA level returned to its basal level 2 hours after the release of strain. Cells treated with actinomycin D before or during strain treatment showed no strain-induced gene expression. Pretreatment of ECs with a protein kinase C (PKC) inhibitor, Calphostin C, strongly inhibited the strain-induced Et-1 gene expression. Pretreatment of ECs with cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 or KT5823) only partially inhibited the increased Et-1 mRNA levels in strain-treated cells. EGTA strongly inhibited the Et-1 gene expression. The intracellular calcium chelator BAPTA/AM also showed an inhibitory effect on Et-1 mRNA levels. We conclude that mechanical strain can stimulate the secretion of Et 1 from ECs by increasing Et-1 mRNA levels via transcription, and that this gene induction is mediated predominantly via the PKC pathway and requires extracellular Ca2+. This strain-induced Et-1 gene expression in ECs may contribute to the regulation of vascular tone and structure in normal and pathological states of the cardiovascular system. PMID- 7535783 TI - The crypt cycle in mouse small intestinal epithelium. AB - We have used a mutation-induced marker system in the intestine of mice heterozygous at the Dlb-1 locus, which determines the expression of binding sites for the lectin Dolichos biflorus agglutinin, and the frequency of clustering of mutated crypts with time as a means of investigating the frequency of the crypt fission process and the crypt cycle. Whole-mount preparations from heterozygous Dlb-1b/Dlb-1a mice were stained with a peroxidase conjugate of Dolichos biflorus agglutinin. Mutations at the Dlb-1b locus in crypt stem cells result in loss of DBA-Px binding in these cells and subsequently their progeny, which eventually results in a rare isolated single, unstained crypt. The subsequent development of pairs, triplets and clusters of negative staining crypts has been assumed to be the result of crypt fission. The frequency of these fission events has been measured in control untreated mice. These negative crypts are the result of spontaneous mutations. We have also looked at mutated crypts after treatment with N-nitroso-N-ethylurea or N-methyl-N'-nitro-N-nitrosoguanidine of young adult mice, which elevates the number of mutations. Our results suggest that the crypt cycle in control animals is very long, 187 +/- 44 weeks (3.6 years, i.e. essentially the life of a laboratory mouse). This implies that about a third of the crypts may divide once in the life of a mouse. After sufficient time for conversion of mixed crypts to monophenotypic crypts after mutagen treatment several clusters of negative crypts were seen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535784 TI - Selective increase in the binding of the alpha 1 beta 1 integrin for collagen type IV during neurite outgrowth of human neuroblastoma TR 14 cells. AB - Regulation of beta 1 integrins in neurite outgrowth following N6,2'-O dibutyryladenosine 3':5'-cyclic monophosphate (dBcAMP) treatment was investigated using the human neuroblastoma cell line TR 14. Three beta 1 integrins were identified: the alpha 1 beta 1 receptor bound collagen type I, collagen type IV and probably laminin; the alpha 2 beta 1 integrin bound collagen type I; and the alpha v beta i receptor bound fibronectin. Neurite extension was detectable as early as 30 minutes following dBcAMP treatment, was maximal after 24 hours and remained constant during treatment for 4 days. Adhesion-perturbing beta 1 subunit specific antibodies, added together with dBcAMP, prevented the outgrowth of new neurites. During the first 24 hours of neurite outgrowth, no change was observed in the amount of beta 1 integrins nor in their topographic distribution. However, dBcAMP treatment increased the binding of alpha 1 beta 1 receptors to collagen type IV-Sepharose by a factor 2.3 +/- 0.6 (P < 0.02), while no alteration in the binding to collagen type I was detected. Moreover, neurites and growth cones were immunoreactive for collagen type IV but not for collagen type I. Consistently dBcAMP-induced neurite outgrowth was inhibited by adhesion-perturbing alpha 1 subunit-specific antibodies. Following maximal neurite outgrowth, the amount of beta 1 integrins determined by immunoprecipitation and by confocal microscopy decreased to 58.3 +/- 11.2% (P < 0.001) and to 55.4 +/- 17.5% (P < 0.001) of untreated levels, respectively, without any change in the level of beta 1 mRNA or de novo synthesized beta 1 precursor. However, pulse-chase experiments showed an increased turnover of the beta 1 subunit: the amount of beta 1 precursor that was degraded after 1 hour chase was 50.5 +/- 8.4% in cells treated for 4 days and 34.2 +/- 3.9% in untreated cells (P < 0.02); the amount of mature beta 1 after 24 hours chase was smaller in cells treated for 4 days compared to untreated cells. In conclusion, during neurite outgrowth, alpha 1 beta 1 integrins are required and acquire an enhanced binding activity for collagen type IV; but following maximal neurite outgrowth, expression of beta 1 integrins is reduced. PMID- 7535785 TI - Isolated plasma membranes induce the loss of oriented detyrosinated microtubules and other contact inhibition-like responses in migrating NRK cells. AB - We have previously shown that detyrosinated microtubules (Glu MTs), which are oriented toward the direction of locomotion in motile fibroblasts, disappear from the area adjacent to cell-cell contact soon after a cell-cell collision. To identify cell surface molecules that trigger this phenomenon, we have established a system in which this and other cellular reactions to cell-cell contact can be reproduced by the addition of isolated plasma membranes. Experimental wounds were made in confluent monolayers of NRK cells, and cells at the wound margin were allowed to develop oriented Glu MTs. Test samples were added to these cells and after a 1 hour incubation the distributions of Glu MTs, tyrosinated MTs (Tyr MTs) and microfilaments were determined by immunofluorescence. When plasma membranes isolated from NRK cells were added, oriented Glu MTs disappeared from the leading lamella of target cells and instead a small number of Glu MTs were found clustered around the nucleus. As observed for cell-cell contact, plasma membranes did not significantly affect the distribution of Tyr MTs. We also found that both cell-cell contact and membrane treatment caused the collapse of lamellipodia and loss of associated staining with antiactin antibody. Time-lapse recordings of directed locomotion of NRK cells showed that membranes suppressed the forward movement of cells. The loss of Glu MTs from the leading lamella was the most amenable response for quantification and we used it to examine the biochemical properties of the membrane activity. The ability of membranes to induce the loss of oriented Glu MTs was observed at as low as 4 micrograms/ml of membrane protein and was detectable 10 minutes after membrane addition. The loss of oriented Glu MTs was reversible upon removal of membranes, demonstrating that the membranes were not toxic to the cells. The oriented Glu MT reducing activity could be solubilized from the membranes by detergent, was enriched in a plasma membrane fraction, and was labile to heat and acid treatment. In summary, we have successfully reconstituted a number of responses of contact inhibition using solubilized preparations of membranes. Our preliminary results suggest that there is a specific factor in plasma membranes that is capable of triggering contact inhibition. With the assay we have developed, it should now be possible to dissect contact inhibition of motility at the molecular level. PMID- 7535786 TI - A group of integral membrane proteins of the rat liver Golgi contains a conserved protein of 100 kDa. AB - Rat liver Golgi membranes were washed with KCl and urea, and a polyclonal antiserum that stained the Golgi complex by immunofluorescence microscopy was raised. A group of proteins of apparent molecular mass 500 kDa, 200 kDa and 100 kDa were identified by immunoblotting with the antiserum, and were enriched in the Golgi membrane fraction. These proteins were also localised to the Golgi by immunofluorescence microscopy with affinity-purified antibodies. They are integral membrane proteins, and protease digestion experiments suggested that they are not exposed on the cytoplasmic face of the Golgi membrane. Immunofluorescence microscopy showed that staining of the Golgi complex by antibodies to the 100 kDa Golgi protein can be demonstrated among a wide range of mammalian species. This conservation may point to an important structural or functional role for the molecule. When the 100 kDa protein was reduced with dithiothreitol it was no longer recognised by the anti-Golgi antiserum. During phase separation in Triton X-114 the 100 kDa protein partitioned into the aqueous phase, rather than into the detergent phase, suggesting that it has a large luminal domain of hydrophilic amino acids. PMID- 7535787 TI - Branching morphogenesis of human mammary epithelial cells in collagen gels. AB - To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1-7 HB2. In standard medium 1-7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblast-conditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentration dependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the alpha 2- and beta 1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the alpha 2 beta 1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the alpha 3-integrin subunit (P1B5) mimicked the effect of soluble 'morphogens'. Our results suggest that the modulation of alpha 3 beta 1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells. PMID- 7535789 TI - Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immunodeficient (SCID) mice. AB - Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2s) strain mice into severe combined immunodeficient (SCID) C.B-17scid/scid (H-2d) mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139-151-specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139 151. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4+, TCR+ T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse SJL mouse chimeras. PMID- 7535788 TI - Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue. AB - We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express alpha 1 beta 1, intercellular CAM-1 (ICAM-1), and sialylated Lewisx, slightly expressed alpha 2, alpha 4, alpha 5, alpha 6 beta 1, alpha v beta 3, lymphocyte function-associated antigen-3 (LFA-3), Lewisx, sialylated LewisLewisx, had a good expression of alpha 3 beta 1 and CD44, and strongly expressed neural CAM (NCAM). GBM expressed alpha 2, alpha 3, alpha 5, alpha 6 beta 1, alpha v beta 3, ICAM-1, LFA-3, CD44, Lewisx, sialylated Lewisx, and sialylated LewisLewisx significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of alpha 2 beta 1, alpha 4 beta 1, and LFA-3 RIU appeared twice higher than in normal endothelium and alpha 6 beta 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue. PMID- 7535790 TI - Neonatal tolerance to an immunodominant T cell reactivity does not confer resistance to EAMG induction in Lewis rats. AB - The overall goal of this study was to determine, during induction of experimental autoimmune myasthenia gravis (EAMG) in Lewis rats, the relative importance of acetylcholine receptor (AChR)-reactive helper T cells associated with one particular immunodominant fine specificity. Thus, experiments presented below were designed to evaluate the immunopathological role played by helper T cells with reactivity against the AChR alpha subunit region associated with amino acid residues 100-116 (i.e., alpha 100-116); in particular, the relationship between T cell reactivity with this specificity and disease induction was assessed. In order to examine the importance of this T cell reactivity, Lewis rat neonates were made T cell tolerant to a synthetic peptide alpha 100-116 and subsequently evaluated for anti-AChR antibody production and resulting neuromuscular dysfunction. Results indicated that although T cell reactivity against the alpha 100-116 peptide could be effectively removed from the Lewis T cell repertoire, tolerized Lewis rats immunized with AChR could undergo an active anti-AChR antibody response that produced symptoms of EAMG. Thus, other AChR T cell reactivities appeared capable of providing adequate help to B cells leading to production of anti-AChR antibodies with pathogenic potential. PMID- 7535791 TI - Increased serum alpha 1-antichymotrypsin in patients with probable Alzheimer's disease: an acute phase reactant without the peripheral acute phase response. AB - Serum levels of alpha 1-antichymotrypsin (alpha 1-ACT) were measured in patients with early and late onset Alzheimer's disease (e-AD, 1-AD), patients with vascular dementia (VD) and healthy elderly. Patients with 1-AD were divided into two groups, one had normal alpha 1-ACT values and one had increased serum levels of alpha 1-ACT. Other acute phase proteins were also measured. The serum levels of alpha 2-macroglobulin (alpha 2-MG), alpha 1-antitrypsin (alpha 1-AT), ceruloplasmin (CER), transferrin (TRSF) and alpha 1-acid glycoprotein (alpha 1 ac.GL) were within the normal range. The C reactive protein (CRP) was occasionally detectable at low concentrations in e-AD, in both groups of 1-AD patients and in VD patients. Low serum concentrations of interleukin-6 (IL-6) were found in a higher proportion of 1-AD than in patients with e-AD or VD. These results indicated that increased levels of alpha 1-ACT along with occasional detection of IL-6 might be peripheral markers of the 'acute reaction' in the brain. PMID- 7535792 TI - Synergy between antibody and P2-reactive T cells in experimental allergic neuritis. AB - Studies were conducted in experimental allergic neuritis (EAN) to evaluate the possible interaction of cellular and humoral immune mechanisms in the demyelinating process. EAN was induced in Lewis rats by passive transfer of T cells reactive to P2 myelin protein or by active immunisation with whole myelin. Animals were then given systemic antimyelin antibody or control serum and assessed clinically, electrophysiologically and with semiquantitative histological studies. Animals given intraperitoneal (i.p.) P2-reactive T cells and systemic antimyelin antibody developed much more severe disease than those given i.p. T cells alone (P < 0.001). In actively immunised animals, the addition of systemic antimyelin antibody did not significantly alter disease severity. We believe the more severe disease in animals receiving T cells and antimyelin antibody reflects synergy between cellular and humoral immune mechanisms whereby neural antigen-specific T cells breach the blood-nerve barrier, allowing demyelinating antibody access to the endoneurium. In EAN induced by active immunisation with whole myelin it is likely that both B and T cell activation occurs and that the more severe demyelination characteristic of this disease reflects the involvement of both humoral and cellular immunity. PMID- 7535793 TI - Thymic expression of the golli-myelin basic protein gene in the SJL/J mouse. AB - The T cell antigen-specific repertoire is thought to be shaped by thymic expression of self molecules. Since a myelin basic protein (MBP)-like gene (golli MBP) has been reported to be expressed by cells of the immune system, the present study was undertaken to determine whether the golli-MBP gene was expressed in the mouse thymus and, if so, to characterize transcripts of this gene in this organ. Using exon-specific primers for MBP and golli-MBP, cDNA from thymus and other tissues was amplified, and the amplified products analyzed by Southern blotting with exon-specific oligonucleotide probes. The amplified products were subcloned, and the inserts characterized by DNA sequencing. The thymic transcripts were found to contain golli-MBP exons 1, 2, 3, 5A, 5B, 5C, 6, 7, 8, and 11. PMID- 7535794 TI - Adhesion molecule knockouts: one step forward and one step backward. PMID- 7535795 TI - Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats. AB - Hypertension is a multigene and multifactorial disorder affecting approximately 25% of the population. To demonstrate potential therapeutic effects of human tissue kallikrein in hypertension, spontaneously hypertensive rats were subjected to somatic gene therapy. Two human tissue kallikrein DNA constructs, one under the promoter control of the metallothionein metal response element and the other under the control of the Rous sarcoma virus 3'-LTR, were generated. We delivered naked DNA constructs into spontaneously hypertensive rats via intravenous injection. The expression of human tissue kallikrein in rats was identified in the heart, lung, and kidney by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. A single injection of both human kallikrein plasmid DNA constructs caused a sustained reduction of blood pressure which began 1 wk after injection and continued for 6 wk. A maximal effect of blood pressure reduction of 46 mmHg in rats was observed 2-3 wk after injection with kallikrein DNA as compared to rats with vector DNA (n = 6, P < 0.05). The hypotensive effect caused by somatic gene delivery of human tissue kallikrein in hypertensive rats is reversed by subcutaneous injection of aprotinin, a potent tissue kallikrein inhibitor. No antibodies to either human tissue kallikrein or kallikrein DNA were detected in rat sera after injection of the human kallikrein gene. These results show that direct gene delivery of human tissue kallikrein causes a sustained reduction in systolic blood pressure in genetically hypertensive rats and indicate that the feasibility of kallikrein gene therapy for treating human hypertension should be studied. PMID- 7535796 TI - Cytokines from activated T cells induce normal endothelial cells to acquire the phenotypic and functional features of AIDS-Kaposi's sarcoma spindle cells. AB - Kaposi's sarcoma (KS) is a proliferative disease of vascular origin particularly frequent in HIV-1-infected homosexual men (AIDS-KS) and characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Previous work has suggested that KS spindle cells are of endothelial cell origin and that chronic immune activation via the release of inflammatory cytokines may cooperate with basic fibroblast growth factor (bFGF) and the HIV-1 Tat protein in the induction and progression of AIDS-KS. Here we show that KS spindle cells have features of activated endothelial cells, and that conditioned media from activated T cells, rich in the same inflammatory cytokines increased in HIV-1-infected individuals, induce normal endothelial cells to acquire the phenotypic and functional features of KS cells. These include (a) acquisition of a similar pattern of cell surface antigen expression; (b) similar proliferative response to bFGF; (c) induction of the responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein that is now able to promote the G1-S transition of endothelial cell cycle; and (d) induction in nude mice of vascular lesions closely resembling early KS as well as the lesions induced by inoculation of KS cells. These results suggest that chronic immune activation, via release of inflammatory cytokines, may play a role in the induction of KS. PMID- 7535797 TI - Prevention of in vitro neutrophil-endothelial attachment through shedding of L selectin by nonsteroidal antiinflammatory drugs. AB - The activation of the endothelial cells by extravascular stimuli is the key event in the extravasation of circulating leukocytes to target tissues. L-selectin, a member of the selectin family, is constitutively expressed by white cells, and is the molecule involved in the initial binding of leukocytes to activated endothelium. After activation, leukocytes rapidly release L-selectin from the cell surface, suggesting that the functional activity of this molecule is controlled in large part by its appearance and disappearance from cell surface. We have studied in a neutrophil-activated endothelial cell binding assay, the effect of different antiinflammatory drugs (steroidal and nonsteroidal) in the L selectin-mediated interaction of neutrophils with activated endothelial cells. Some nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, diclofenac, ketoprofen, and aspirin, but not steroids, strongly inhibited the neutrophil-endothelial cell attachment. Furthermore, we also investigated the underlying mechanism of this functional effect. The expression of L-selectin on the neutrophil surface rapidly decreased in the presence of different NSAIDs, in a dose- and time-dependent manner, whereas no changes in the expression of other adhesion molecules such as CD11a, CD11b, CD31, or ICAM-3 (CD50) were observed. Interestingly, studies in vivo on healthy volunteers treated with physiological doses of indomethacin showed a significant decrease of L-selectin neutrophil expression. Only diclofenac induced an upregulation of CD11b expression, suggesting an activating effect on neutrophils. No enzyme release was observed upon treatment of neutrophils with different NSAIDs, indicating a lack of degranulatory activity of NSAIDs, with the exception of diclofenac. The downregulation of L-selectin expression was due to the rapid cleavage and shedding of the membrane L-selectin, as determined by both immunoprecipitation from 125I-labeled neutrophils, and quantitative estimation in cell-free supernatants. These results suggest that NSAIDs exert a specific action on adhesion receptor expression in neutrophils, which might account, at least in part, for the antiinflammatory activities of NSAIDs. PMID- 7535798 TI - P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli. AB - Neutrophil emigration during an inflammatory response is mediated through interactions between adhesion molecules on endothelial cells and neutrophils. P Selectin mediates rolling or slowing of neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Removing the function of either molecule partially prevents neutrophil emigration. To analyze further the role of P-selectin and ICAM-1, we have generated a line of mice with mutations in both of these molecules. While mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum during Streptococcus pneumoniae-induced peritonitis, double mutant mice show a complete loss of neutrophil emigration. In contrast, neutrophil emigration into the alveolar spaces during acute S. pneumoniae-induced pneumonia is normal in double mutant mice. These data demonstrate organ-specific differences, since emigration into the peritoneum requires both adhesion molecules while emigration into the lung requires neither. In the peritoneum, P-selectin-independent and ICAM-1-independent adhesive mechanisms permit reduced emigration when one of these molecules is deficient, but P-selectin-independent mechanisms cannot lead to ICAM-1-independent firm adhesion and emigration. PMID- 7535800 TI - Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. AB - Lithium, a widely used treatment for bipolar affective disorders, often causes nephrogenic diabetes insipidus. The effect of chronic lithium therapy on the expression of the vasopressin-regulated water channel Aquaporin-2 (AQP2) in rat kidney was examined. Membranes were prepared from inner medulla of one kidney from each rat, while the contralateral one was fixed for immunofluorescence and immunoelectronmicroscopy. Immunoblotting revealed that lithium treatment reduced AQP2 expression dramatically, to 31 +/- 8% after 10 d and to 4 +/- 1% after 25 d, coincident with development of severe polyuria. Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2). The downregulation was only partly reversed by return to lithium-free diet for 1 wk (40 +/- 8% of control). Furthermore, immunoblotting and immunogold quantitation revealed that 2 d of thirsting or 7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by six- and threefold, respectively, coincident with increased urinary osmolality. Thirsting increased AQP2 immunolabeling mainly of vesicles, whereas dDAVP caused accumulation of AQP2 predominantly in the subapical region and plasma membrane. Thus, lithium causes marked downregulation of AQP2 expression, only partially reversed by cessation of therapy, thirsting or dDAVP treatment, consistent with clinical observations of slow recovery from lithium-induced urinary concentrating defects. PMID- 7535799 TI - Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. AB - To investigate the relationship between angiogenesis and hepatic tumorigenesis, we examined the expression of vascular endothelial growth factor (VEGF) in 8 human colon carcinoma cell lines and in 30 human colorectal cancer liver metastases. Abundant message for VEGF was found in all tumors, localized to the malignant cells within each neoplasm. Two receptors for VEGF, KDR and flt1, were also demonstrated in most of the tumors examined. KDR and flt1 mRNA were limited to tumor endothelial cells and were more strongly expressed in the hepatic metastases than in the sinusoidal endothelium of the surrounding liver parenchyma. VEGF monoclonal antibody administration in tumor-bearing athymic mice led to a dose- and time-dependent inhibition of growth of subcutaneous xenografts and to a marked reduction in the number and size of experimental liver metastases. In hepatic metastases of VEGF antibody-treated mice, neither blood vessels nor expression of the mouse KDR homologue flk-1 could be demonstrated. These data indicate that VEGF is a commonly expressed angiogenic factor in human colorectal cancer metastases, that VEGF receptors are up-regulated as a concomitant of hepatic tumorigenesis, and that modulation of VEGF gene expression or activity may represent a potentially effective antineoplastic therapy in colorectal cancer. PMID- 7535801 TI - Molecular basis of subtotal complement C6 deficiency. A carboxy-terminally truncated but functionally active C6. AB - Individuals with subtotal complement C6 deficiency possess a C6 molecule that is 14% shorter than normal C6 and present in low but detectable concentrations (1-2% of the normal mean). We now show that this dysmorphic C6 is bactericidally active and lacks an epitope that was mapped to the most carboxy-terminal part of C6 using C6 cDNA fragments expressed as fusion proteins in the pUEX expression system. We thus predicted that the abnormal C6 molecule might be carboxy terminally truncated and sought a mutation in an area approximately 14% from the carboxy-terminal end of the coding sequence. By sequencing PCR-amplified products from this region, we found, in three individuals from two families, a mutation that might plausibly be responsible for the defect. All three have an abnormal 5' splice donor site of intron 15, which would probably prevent splicing. An in frame stop codon is found 17 codons downstream from the intron boundary, which would lead to a truncated polypeptide 13.5% smaller than normal C6. This result was unexpected, as earlier studies mapped the C5b binding site, or a putative enzymatic region, to this part of C6. Interestingly, all three subjects were probably heterozygous for both subtotal C6 and complete C6 deficiency. PMID- 7535802 TI - Amplification of nitric oxide synthase expression by nitric oxide in interleukin 1 beta-stimulated rat mesangial cells. AB - Nitric oxide (NO) plays an important role in immunological reactions as a host defense mechanism against tumor cells and invasive microorganisms, but it may also damage healthy tissue. The excessive formation of NO in IL-1 beta-stimulated renal mesangial cells not only alters glomerular filtration, but it may also cause tissue injury and thus contribute to the pathogenesis of certain forms of glomerulonephritis. We report here that, although NO alone has no evident effect on NO synthase expression, it potently augments IL-1 beta-stimulated NO synthase expression in mesangial cells. NO donors such as sodium nitroprusside and S nitroso-N-acetyl-D,L-penicillamine markedly increase IL-1 beta-induced NO synthase mRNA and protein levels as well as enzyme activity. Nuclear run-on experiments suggest that NO acts to increase IL-1 beta-induced NO synthase gene expression at the transcriptional level. Furthermore, inhibition of NO synthesis by different pharmacological approaches reduces IL-1 beta-induced NO synthase expression, thus suggesting that NO functions in a positive feedback loop that speeds up and strengthens its own biosynthesis. We suggest that this potent amplification mechanism forms the basis for the excessive formation of NO in acute and chronic inflammatory diseases. PMID- 7535803 TI - Immunophenotypic analysis of childhood Burkitt's lymphoma in the West Midlands 1957-1986. AB - AIMS: To analyse the immunophenotype of a large number of non-endemic Burkitt's lymphomas to determine whether a B cell phenotype is consistently recognisable using formalin fixed, paraffin wax embedded archival material and a standard panel of commercially available antibodies. METHODS: Archival material was obtained from 30 cases of childhood Burkitt's lymphoma registered with the West Midlands Regional Children's Tumour Research Group. These were analysed by a standard avidin biotin complex immunoperoxidase method using antibodies to CD45, CD43, CD30, CD20, CD15, and immunoglobulin heavy and light chains. RESULTS: There was a high incidence of the CD45RB and CD20 immunophenotypes, with a clearly recognisable B cell lineage even in archival material. IgM was identifiable in 13 of the 23 (56.5%) cases tested. Only three of 17 (18%) cases expressed CD30. Positive membrane staining with CD45RO was observed in two (6.7%) cases. CONCLUSIONS: A B cell lineage can be identified in Burkitt's lymphoma in formalin fixed, paraffin wax embedded material, even in archival tissue. There was a low incidence of membrane staining with CD45RO which is a potential source of diagnostic confusion. PMID- 7535805 TI - Contamination of crystal violet in the gram stain method. PMID- 7535804 TI - Cytokeratin intermediate filament expression in benign and malignant breast disease. AB - AIM: To carry out a comprehensive study of cytokeratin expression in benign and malignant breast epithelium and breast myoepithelial cells; to examine changes in the cytokeratin profile in malignant and benign epithelium and in carcinomas of increasing histological grade. METHODS: Frozen sections from fibroadenomas (19 cases), fibrocystic disease (19 cases), and infiltrating ductal (68 cases), lobular (seven cases), and mucinous carcinomas (three cases) were examined using a panel of monoclonal antibodies. RESULTS: The luminal epithelium in all fibroadenomas and all cases of fibrocystic disease, as well as tumour cells in most carcinomas, reacted with the specific antibodies to cytokeratins 7, 8, 18, and 19 and to antibodies which included these cytokeratins in their specificities (Cam 5.2, AE1, AE3, RCK102, and LP34). In a few ductal carcinomas none of the tumour cells reacted for cytokeratins 7, 8, or 18. Three ductal carcinomas expressed cytokeratin 14. Only occasional cases expressed cytokeratins 3, 4, 10, and 13. Antibodies which included cytokeratins 5 and 14 in their specificities detected myoepithelial cells less efficiently than antiactin antibodies. CONCLUSION: The cytokeratin profiles in the luminal epithelium in benign breast disease and in tumour cells in most carcinomas are similar in most cases. Some carcinomas, however, are negative for cytokeratins 7, 8, or 18. This may provide a means of predicting the biological behaviour of a histologically borderline lesion. PMID- 7535808 TI - Organization of the colliculo-suprageniculate pathway in the cat: a wheat germ agglutinin-horseradish peroxidase study. AB - A wheat germ-agglutinated horseradish peroxidase (WGA-HRP) tracing technique was used to label the cell bodies of neurons in the superior colliculus that send projections into the visually sensitive region of the suprageniculate nucleus (Sg) in the feline thalamus. After determination of the position of the Sg by detecting characteristic single-unit responses to moving visual stimuli, WGA-HRP was injected into the Sg in five pentobarbital-anesthetized cats. The animals were than sacrificed, and serial frozen sections of the midbrain were processed for the demonstration of peroxidase activity. A total of 2,736 WGA-HRP-stained neurons were located within the ipsilateral superior colliculus (SC), and a few labeled cells were consistently found bilaterally in the external nuclei of the inferior colliculus. In each cat, a small but significant fraction of the labeled cells were encountered contralateral to the injection. Medial SC neurons tended to project to the posterior Sg, and lateral SC neurons tended to project to more rostral Sg. However, labeled cells were distributed homogeneously along the rostrocaudal extent of the SC, indicating the absence of a well defined topographic relationship. Nor was the Sg injection site location related to the laminar distribution of SC projection neurons. In all cases, the majority of the labeled cells were found in layer IV (49.0%), with fewer cells in layers III (17.5%) layer V (20.0%), and layer VI (11.8%). No labeled cells were located in layer I, although a few were located in the deep part of layer II. Five types of SC projection cells were distinguished morphologically. Of the 258 labeled cells that could be characterized, 25% were stellate cells, 25% vertical cells, 20% granular cells, 17% triangular cells, and 12% horizontal cells. The average diameter of 226 cells ranged between 8 and 47 microns. We conclude that a mixed population of SC cells projects to the Sg; the morphological heterogeneity and the distribution of these cells suggests that several functionally different pathways may be involved in the colliculothalamic pathway and in the processing of visual input in the SC. PMID- 7535806 TI - Lack of topography in the spinal cord projection to the rabbit soleus muscle. AB - Several mammalian muscles in the limb and trunk receive topographically organized innervation from spinal cord motor neurons. Some muscles in which topographic innervation has been demonstrated have a sheet-like architecture; others are compartmentalized and/or have more than one origin. An interesting question is whether topography is related to these anatomical features, or whether it occurs as a general consequence of the development of innervation. To address this question, we examined the pattern of projections to the soleus muscle, which lacks these anatomical features. Intracellular recordings of endplate potentials in early and intermediate age rabbits were used to assess the spinal origin of inputs to two distinct regions of the muscle. Both regions were innervated by both rostral and caudal portions of the motor pool. These experiments also showed that individual muscle fibers frequently receive separate inputs arising from widely separated regions of the motor pool. In another set of experiments, physiological measurements of tension overlap in young, polyinnervated muscles showed that the relative positions of motor neurons in the spinal cord do not correlate with the extent to which motor units share muscle fibers. In a third set of experiments, motor neurons were retrogradely labeled following local injections of tracer into muscle. Small and large local injections resulted in comparably dispersed labeling of motor neurons within the motor pool. Moreover, the rostrocaudal position of labeled neurons was not correlated with the position of the injection site within the muscle. Together, these results provide evidence that the soleus muscle is not topographically innervated. Furthermore, an examination of several age groups suggests that the innervation pattern in this muscle is not altered by postnatal synapse elimination. PMID- 7535809 TI - NADPH-diaphorase in the central nervous system of the tench (Tinca tinca L., 1758). AB - The distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (ND)-positive cells and fibers in the tench central nervous system was mapped by using a direct histochemical method. This enzyme was observed in specific cell populations throughout all main divisions of the tench brain. In the telencephalon, we found strongly labeled olfactory fibers, as well as positive cells and fibers in the area ventralis of the telencephalic lobes. Positive staining was observed in the following diencephalic nuclei: nucleus preopticus magnocellularis pars magnocellularis, nucleus recessus lateralis, nucleus recessus posterioris, nucleus posterior tuberis, and nucleus diffusus torus lateralis, as well as small cells with a diffuse distribution surrounding the diencephalic ventricle. In the mesencephalon, heavily stained ND positive neurons were observed in the nucleus fasciculi longitudinalis medialis, nucleus nervi oculomotorius, and nucleus nervi trochlearis. In the hindbrain the most evident staining was observed as large neurons located in the nuclei of the cranial nerves, scattered positive cells located between the negative fibers of the cranial nerves, and in the nucleus fasciculi solitari. Finally, in the spinal cord, ND-positive cells and fibers were mainly located in the ventral horn. This distribution of ND labeling in the brain of the tench is significantly different from previous data on ND activity in the brain of terrestrial vertebrates and does not correlate with the presence and distribution patterns of several neurotransmitters and neuroactive substances in the teleost brain. PMID- 7535810 TI - Efferent connections of the lateral cortex of the lizard Gekko gecko: evidence for separate origins of medial and lateral pathways from the lateral cortex to the hypothalamus. AB - The lateral cortex of the lizard Gekko gecko is composed of three parts: a dorsal and ventral part located rostrally and a posterior part located caudally. In order to obtain detailed information about the efferent connections of these lateral cortex subdivisions, iontophoretic injections of the anterograde tracers Phaseolus vulgaris leucoagglutinin and biotinylated dextran were made in the various parts. The main projection from the dorsal part terminates in the caudal part of the medial cortex. Other cortical projections were noted to the ipsi- and contralateral lateral cortex, the large-celled part of the medial cortex, and the dorsal cortex. Additional fibers were found bilaterally in the anterior olfactory nucleus and the external amygdaloid nucleus. The ventral part of the lateral cortex projects mainly to the ipsilateral, posterior part of the dorsal ventricular ridge and the external amygdaloid nucleus. Minor contralateral projections to these nuclei were also found. Other projections were observed to travel to the caudal part of the medial cortex, to the nucleus sphericus, and bilaterally to the lateral cortex and the anterior olfactory nucleus. The posterior part of the lateral cortex has similar efferent connections as the dorsal part and should be regarded as the caudal continuation of the dorsal part. Because previous studies have shown that the medial cortex and the amygdaloid complex project to different hypothalamic areas, we conclude that the dorsal and ventral parts of the lateral cortex transmit olfactory information to separate hypothalamic areas that are probably involved with different types of behavior. PMID- 7535807 TI - Enhanced but delayed axonal sprouting of the commissural/associational pathway following a combined entorhinal cortex/fimbria fornix lesion. AB - From previous lesion studies of the hippocampus it has been reported that axons of the commissural/associational pathway expand their termination zone in the molecular layer of the dentate gyrus by 20-25% in response to loss of input from the entorhinal cortex. However, although much is known about the response of the commissural/associational pathway with regard to extent, latency, and speed of the reinnervation response following an entorhinal cortex lesion, little is known about how the loss of additional afferent systems might modulate this response. To address this issue, we examined at 14, 30, and 45 days postlesion, the sprouting of commissural/associational afferents following either a unilateral fimbria fornix transection, a unilateral entorhinal cortex lesion, or combined lesions of both the entorhinal cortex and the fimbria fornix. Loss of septal innervation to the hippocampus was assessed using the cholinesterase stain, whereas sprouting from the commissural/associational pathway was determined from Holmes fiber-stained sections. In addition, the Timms stain was used to examine the time course of the loss of terminal fields of the various zinc-containing afferent systems within the hippocampus. Following the removal of input to the hippocampus via the fimbria fornix transection, there was no evidence of sprouting of the commissural/associational fibers into the deafferented portion of the dentate gyrus. In contrast, rats receiving an entorhinal cortex lesion showed a significant increase (28%) in the width of the commissural/associational fiber plexus that was present by 14 days postlesion. By comparison, the magnitude of the expansion of the commissural/associational fiber plexus was significantly larger after lesioning both the entorhinal cortex and the fimbria than after the entorhinal cortex lesion alone (45% vs. 28%). In addition, the expansion of the commissural/associational fiber plexus was not increased at 14 days postlesion but was significantly increased at 30 days postlesion. The delay in the sprouting of the commissural/associational pathway coincided with the time course of loss of zinc-containing fibers in the outer molecular layer of the dentate gyrus as assessed with the Timms stain. These results suggest that the magnitude and time course for the sprouting of axons from the commissural/associational pathway into the partially deafferented hippocampus of the adult rat is lesion dependent and that the effect of the loss of input from the entorhinal cortex can be modulated and enhanced by the concomitant depletion of input from the fimbria fornix. PMID- 7535811 TI - pp60c-src expression is induced by activation of normal human T lymphocytes. AB - We have re-examined whether pp60c-src, the normal cellular homologue of the transforming protein of Rous sarcoma virus, is present in human T cells. By in vitro immune-complex kinase assay or Western blotting with the anti-pp60c-src mAbs 327 or GD11, pp60c-src was found to be present in lysates of T cell lines, including the Jurkat T cell line. The 327 and GD11 mAbs have been reported to be specific for pp60c-src and not to cross-react with other src family members or other kinases. Furthermore, the size of the pp60c-src bands present on Western blotting and in vitro kinase assay were clearly different from those of p56lck or p59fyn. In addition, pp60c-src is detected in the HTLV-I-derived T cell lines S1T and C8, which lack expression of p56lck and p59fyn. RNase protection assays confirmed that pp60c-src mRNA is present in Jurkat T cells. We also found pp60c src protein to be constitutively present in freshly isolated thymocytes. In contrast, pp60c-src was absent, or present at extremely low levels, in normal, resting peripheral blood T lymphocytes, which is in agreement with previous findings. However, after stimulation of resting T cells with the mitogenic lectin PHA or with Ab to the TCR complex, pp60c-src expression is induced in both CD4+ and CD8+ T cell subsets, with peak expression detectable 12 to 24 h after T cell activation. The levels of pp60c-src are low in all T cells except Jurkat, where levels of pp60c-src are comparable to levels found in a glioblastoma cell line (T98G). Nevertheless, significant levels of pp60c-src kinase activity are readily detectable in thymocytes and activated normal T cells as well as in T cell lines. The finding that pp60c-src is inducible following activation through the TCR suggests that pp60c-src may play a specific role in the normal T cell activation pathway. PMID- 7535812 TI - Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression. AB - TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated. PMID- 7535814 TI - Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. AB - Chromatin condensation and nuclear envelope breakdown are characteristic features of apoptotic cell death, but the mechanisms underlying these phenomena have not been identified. Solubilization of nuclear lamin is responsible for both events in mitosis. In this work, we report that glucocorticoids stimulate rapid degradation of lamin B1 that occurs before oligonucleosomal DNA fragmentation in apoptotic thymocytes. Protease inhibitors and the Ca2+ buffering agent BAPTA-AM block lamin degradation and DNA fragmentation, indicating that the processes are regulated by similar or identical mechanisms. Incubation of isolated thymocyte nuclei with Ca2+ stimulates lamin degradation before the detection of oligonucleosomal DNA fragments. However, in contrast to lamin dissolution during mitosis and some other forms of apoptosis, glucocorticoid-induced degradation of lamin B1 in thymocytes is not accompanied by dephosphorylation-mediated activation of cdc2. Our results demonstrate that lamin degradation is an early feature of apoptosis in thymocytes and suggest that chromatin condensation and breakdown of the nuclear envelope may occur as a result of disruption of nuclear lamina architecture. PMID- 7535813 TI - Regulation of human T lymphocyte chemotaxis in vitro by T cell-derived cytokines IL-2, IFN-gamma, IL-4, IL-10, and IL-13. AB - There has been a number of conflicting reports regarding the T lymphocyte chemotactic activities of several cytokines. IL-2 and IFN-gamma are known to promote augmentation of immune inflammation, whereas IL-4, IL-10, and IL-13 display immunomodulatory effects on inflammatory cells including inhibition of cytokine production. Their effects on chemotaxis of inflammatory cells are unknown. We observed that IL-1 alpha could induce chemotaxis both in overnight cultured and anti-CD3 mAb-activated T lymphocytes and that overnight culture and anti-CD3 activation increase the number of IL-1R on T lymphocytes. In contrast, IL-8 selectively attracts freshly isolated T lymphocytes. Staurosporine inhibits freshly isolated T lymphocyte chemotaxis toward IL-8, whereas tyrphostin 23 inhibits chemotaxis of overnight cultured and anti-CD3-activated T lymphocytes toward IL-1 alpha. We have found that IL-2 and IL-13 inhibit the chemotactic migration of both CD4+ and CD8+ T lymphocytes toward IL-8, and RANTES. IL-4 inhibits only CD8+ T lymphocyte chemotaxis toward RANTES, IL-8 and IL-10. IL-10 inhibits only CD4+ T lymphocytes in their chemotactic response toward RANTES and IL-8. IFN-gamma does on the other hand augment the sensitivity of human T lymphocytes to chemotactic stimuli. Thus, our results demonstrate that different proinflammatory cytokines will induce chemotactic migration of T lymphocytes under different circumstances acting through different signaling pathways. The T cell-derived cytokines IL-2, IL-4, IL-10, and IL-13 are able to block further T lymphocyte chemotaxis, thus leading to a focusing of T lymphocytes in an area of T lymphocyte activation. These mechanisms seem relevant in our understanding of the specific and continuous localization of T lymphocytes in allergic and autoimmune disorders. PMID- 7535815 TI - Self-reactive T cell hybridomas and tolerance. Same range of antigen dose dependence but higher numbers of self-reactive T cell hybridomas from mice in which self-tolerance has been broken by antiserum treatment. AB - Mechanisms of self-tolerance of 4-hydroxyphenylpyruvate dioxygenase (HPPD) are explored. It is well established that negative selection based on TCR affinity occurs in the thymus. We have investigated the frequency with which self-reactive T cell hybridomas can be obtained in relation to self-tolerance. Mice immunized with the self-form of HPPD gave rise to T cell hybridomas that were able to recognize self-protein and a synthetic peptide representing the T cell epitope, at higher Ag concentration than was necessary for recognition of allo-protein. The efficiency of negative selection was then reduced by treating neonatal mice with anti-HPPD antiserum. This reduced T cell tolerance of the self-protein, as judged by in vitro proliferation, and enabled self-reactive T cell hybridomas to be generated at a higher frequency. However, the Ag concentration requirements of these hybridomas for the self-protein and the self-peptide remained unaltered. The possibility that these findings reflect an auxiliary mechanism of self tolerance based on frequencies of self-reactive T cells is discussed. PMID- 7535816 TI - Isolation of a kidney-specific peptide recognized by alloreactive HLA-A3 restricted human CTL. AB - The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of these peptides, their function, and their possible relationship to allograft rejection, we have utilized human kidney-specific CD8+ CTL clones to screen reversed-phase HPLC (RP HPLC)-separated self peptides presented by allo-class I molecules. One of these clones is HLA-A3-restricted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropriate HLA molecules. We have identified a biologically active RP-HPLC fraction containing self peptides eluted from affinity-purified MHC molecules from HLA-A3+ kidney. This peptide is not expressed in HLA-A3+ spleen. Similarly, a HLA-B62-associated peptide fraction was identified in kidney but not in spleen using the HLA-B62-restricted CTL clone. Sequence analysis of the biologically active fraction from HLA-A3 kidney revealed multiple peptides. Because of the ambiguity of the peptide sequence, a mixed peptide library corresponding to this sequence was synthesized that included the HLA-A3 binding motif. The biologically active peptide library was RP-HPLC fractionated and the fraction containing HLA A3-restricted CTL activity was sequenced. The resulting sequence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVTIVK. By using this unique strategy, we describe the successful isolation and sequencing of an antigenic peptide that is recognized by a human alloreactive kidney-specific CTL clone. PMID- 7535817 TI - Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum. AB - Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2 microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation. PMID- 7535818 TI - Differentiation-dependent effects of IL-1 and TGF-beta on human articular chondrocyte proliferation are related to inducible nitric oxide synthase expression. AB - This study analyzed the effect of chondrocyte differentiation on iNOS expression and responses to IL-1 and TGF-beta. During subculturing of chondrocytes, the growth-stimulatory effects of TGF-beta decreased, and cells in later passages even were growth inhibited by TGF-beta. IL-1 beta responses showed an inverse pattern. The antiproliferative effects of IL-1 beta decreased, and, after passage 6, IL-1 beta became a growth stimulator for chondrocytes. This change in growth factor response pattern was associated with a decrease in type II collagen expression. To determine whether these changes in the growth regulatory effects of IL-1 beta and TGF-beta were related to nitric oxide (NO), inducible nitric oxide synthase (iNOS) expression and NO release were analyzed. In primary chondrocytes, TGF-beta did not stimulate iNOS mRNA expression or NO release, and, during co-incubation, it did not detectably alter the IL-1 beta effect. Preincubation with TGF-beta resulted in a time-dependent increase in IL-1-induced NO. With increasing passage number, the IL-1 beta effects decreased, and, after passage 6, IL-1 beta no longer detectably stimulated iNOS expression or NO release. However, TGF-beta increased NO production synergistically with IL-1 beta during the same culture period when it lost its growth-stimulatory effects. The antiproliferative effects of TGF-beta in late passage chondrocytes were reversed by the NO synthase inhibitor NG-monomethylarginine. These results suggest a novel pattern of iNOS regulation by IL-1 and TGF-beta and show that the factors that modulate iNOS expression and proliferation are dependent on the differentiation status of the cells. PMID- 7535819 TI - The Fc gamma RI receptor signals through the activation of hck and MAP kinase. AB - U937 cells differentiated with IFN-gamma (termed U937IF cells) were used to study Fc gamma RI signaling. IFN induces a functional Fc gamma RI receptor signaling pathway in U937 cells, leading to the activation of the respiratory burst. IFN induces the expression of the nonreceptor protein tyrosine kinase, hck, and cross linking the Fc gamma RI receptor in U937IF cells results in the activation of hck kinase as evidenced by the three- to fivefold increased tyrosine phosphorylation of hck. In vitro kinase assays demonstrate that the specific kinase activity of hck is increased 10-fold after Fc gamma RI stimulation. hck is observed to associate with two prominent tyrosine-phosphorylated proteins, p72 and p95, after Fc gamma RI-activation. Fc gamma RI cross-linking also results in mobility shift in MAP kinase in U937IF cells, suggesting that the Fc gamma RI receptor signals through the activation of MAP kinase. The data suggest that hck, p72, p95, and MAP kinase are involved in signal transduction through the Fc gamma RI receptor. PMID- 7535820 TI - Hyaluronic acid enhances cell proliferation during eosinopoiesis through the CD44 surface antigen. AB - We examined the effect of hyaluronic acid in promoting proliferation of undifferentiated progenitor cells through the CD44 receptor during eosinopoiesis in vitro. Undifferentiated umbilical cord blood cells were purified on the first day to isolate primitive progenitor cells expressing the CD34 hemopoietic surface marker. Culture in wells coated with 100 micrograms/ml hyaluronic acid caused a 198 +/- 28.7% augmentation of proliferation of CD34+ progenitor cells at 3 wk (p < 0.01). By contrast, concentrations of hyaluronic acid > 10 micrograms/ml inhibited proliferation of unfractionated cord blood mononuclear cells. The augmented proliferation of precursor cells caused by hyaluronic acid was associated with complete (93.0 +/- 5.12%) differentiation to eosinophil morphology. By contrast, concentrations of hyaluronic acid > or = 10 micrograms/ml inhibited eosinophilic differentiation of unfractionated mononuclear cells. Wright-Giemsa staining demonstrated 95.4 +/- 2.92% eosinophils for CD34+ cells cultured for 3 wk without hyaluronic acid (control) and 93.8 +/- 5.11% for CD34+ cells cultured in hyaluronic acid-coated wells (100 micrograms/ml); for unfractionated cells, 94.0 +/- 3.02% demonstrated eosinophilic morphology in control wells at 3 wk vs 55.4 +/- 8.34% in hyaluronic acid-coated (100 micrograms/ml) wells (p < 0.05). Augmented proliferation caused by hyaluronic acid was attenuated completely by the anti-CD44 mAbs, 212.3 and IM7.8.1. Pretreatment of CD34+ cells with 5 micrograms/ml 212.3 inhibited the augmented proliferation caused by the optimal concentration of hyaluronic acid (100 micrograms/ml) from 260 +/- 39.2% of control growth to 114 +/- 16.4% of control growth (p = 0.02). Inhibition was comparable for IM7.8.1. Control mAb (LM2) to the beta 2 integrin subunit CD11b had no effect on proliferation induced by hyaluronic acid. We demonstrate that hyaluronic acid stimulates the growth of CD34+ selected umbilical cord blood cells into specifically differentiated mature eosinophils. This process is modulated by the CD44 receptor on the progenitor cell population. PMID- 7535821 TI - The adhesion molecules used by monocytes for migration across endothelium include CD11a/CD18, CD11b/CD18, and VLA-4 on monocytes and ICAM-1, VCAM-1, and other ligands on endothelium. AB - CD11/CD18 and VLA-4 integrins mediate interactions of monocytes with HUVEC cultured on human amniotic tissue. In the present study, the roles of individual CD11/CD18 integrins and endothelial adhesion molecules were examined using blocking mAbs and peptides. After 20 min of incubation, monocyte adhesion to and migration across unstimulated endothelium was dependent primarily on CD11a/CD18. When incubation was extended to 2 h to allow for completion of migration, either CD11a/CD18 or CD11b/CD18 could be used. Similarly, either CD11a/CD18 or CD11b/CD18 could be used by monocytes to bind to and traverse IL-1 beta stimulated endothelium. Although both CD11a/CD18 and CD11b/CD18 are known to bind to ICAM-1, results of Ab-mixing experiments suggest that alternative ligands on HUVEC for CD11/CD18 integrins also may be used during transendothelial migration of monocytes. Our previous studies indicate that VLA-4 on monocytes interacts primarily with VCAM-1 on unstimulated endothelium. In contrast, migration of monocytes across IL-1 beta-stimulated endothelium was less dependent on VCAM-1. mAbs directed against binding sites for VLA-4 in domain 1 and domain 4 of VCAM-1 did not, by themselves, inhibit interactions of monocytes with stimulated HUVEC. VLA-4-dependent migration across IL-1 beta-stimulated endothelium was markedly inhibited only when mAbs to VCAM-1 were added in combination with peptides of fibronectin. Therefore, VLA-4 can interact with either VCAM-1 or alternative ligands on IL-1 beta-stimulated HUVEC-amnion cultures to mediate transendothelial migration of monocytes. PMID- 7535822 TI - Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. AB - The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h. PMID- 7535823 TI - Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3. AB - It has been shown that CC chemokines activate basophil and eosinophil leukocytes with different selectivities and patterns of activity. The most effective are monocyte chemotactic protein-1 (MCP-1), a potent stimulus of mediator release in basophils without effects on eosinophils, RANTES, a weak stimulus of release and strong chemoattractant for basophils and eosinophils, and MCP-3, which combines the activities of MCP-1 and RANTES. We have now compared MCP-2, which has 62 and 60% of sequence identity with MCP-1 and MCP-3, respectively, with the other CC chemokines. MCP-2 induced mediator release by human basophils with lower efficacy and potency than MCP-1 and MCP-3. It promoted transient changes of cytosolic-free calcium concentration ([Ca2+]i) and chemotactic responses in both basophils and eosinophils, however somewhat less efficiently than MCP-3 and RANTES. Desensitization studies indicate that MCP-2 interacts with receptors recognizing MCP-1 as well as RANTES. These results demonstrate that MCP-2 and MCP-3 exert qualitatively similar biologic activities on basophils and eosinophils. In basophils that had not been treated with IL-3, MCP-2 induced minimal exocytosis only, but desensitized the cells toward MCP-1 and MCP-3, suggesting that MCP-2 may act as a functional inhibitor of CC chemokine actions. The results of this study further indicate that MCP analogues display partially distinct, partially overlapping bioactivities toward eosinophils and basophils, and may thus regulate inflammatory processes involving these effector cell types. PMID- 7535825 TI - Induction of anagen in telogen mouse skin by topical application of FK506, a potent immunosuppressant. AB - The effect of topical application of FK506 on the normal hair cycle of C57BL/6J mice was investigated. When telogen mice (7 weeks of age) were treated topically with 1 mumol FK506 on days 0 and 3, 50% of the tested mice entered anagen by day 9 and 100% by day 16. With 0.1 mumol of FK506, 50% of the tested mice entered anagen by day 13 and 80% by day 19, indicating that the effect of FK506 is dose dependent. In control mice, a spontaneous shift from telogen to anagen started on day 14, and 30% of the control animals were in anagen at day 19. Histologic studies revealed that FK506 markedly stimulated the skin and thickened it. The depth and size of hair follicles were also markedly increased in FK506-treated skin compared to control skin. The data on hair growth also support the contention that FK506 induces early onset of anagen and stimulates hair growth. The hair growth stimulated by FK506 looked normal and the hairs were of normal length. The hair growth was restricted to the site of application. These results clearly demonstrate that topical application of FK506 induces anagen hair growth in telogen mouse skin and indicate that the hair-growth-stimulating effect of FK506 is due at least in part to its promoting effect on the hair cycle. PMID- 7535824 TI - CTLs from lymphoid organs recognize an optimal HLA-A2-restricted and HLA-B52 restricted nonapeptide and several epitopes in the C-terminal region of HIV-1 Nef. AB - In a previous analysis of HIV-1-specific CTLs in lymphoid organs from HIV seropositive patients, we reported high frequencies of in vivo differentiated CTLs directed against two immunodominant regions in the central and in the C terminal part of the HIV-1 Nef protein. The present study analyzes the epitopes recognized by CTLs in the carboxyl terminus of Nef (amino acids 182-205). In addition to several epitopes that are recognized in association with different HLA molecules (A1, A2, A25(10), B35, B52), we defined an optimal nonapeptide (190 198). This nonapeptide was recognized by CTLs down to nanomolar concentrations in the context of at least two HLA molecules, HLA-B52 and HLA-A2, including three HLA-A2 subtypes: HLA-A2.1, -A2.2, and -A2.4. We also determined the relative frequencies of effector CTLs directed against peptide 190-198 to be as high as 10(-4), as opposed to lower frequencies ranging between 5 x 10(-5) and 5 x 10(-6) observed for the other peptides recognized in the same region, thus confirming the optimal presentation of this nonapeptide in vivo. Molecular modeling of the interactions between HLA-A2.1 and Nef peptide 190-198 suggests the formation of a stable complex and allowed us to study sequence motifs that are important for the binding of the HIV-1 peptide in the pockets of the HLA-A2.1 molecule. PMID- 7535827 TI - Sexual transmission of hepatitis C virus among patients attending sexually transmitted diseases clinics in Baltimore--an analysis of 309 sex partnerships. AB - The prevalence of antibodies to hepatitis C virus (anti-HCV), the behavioral and laboratory-derived risk factors for anti-HCV, and the quantity and homology of HCV RNA were assessed among 1039 non-injection drug-using sexually transmitted disease (STD) patients representing 309 sex partnerships. Thirty-seven (7%) of 555 males and 19 (4%) of 484 females had anti-HCV. In logistic regression analyses, factors associated with anti-HCV included age (P < .001), greater numbers of lifetime sex partners (P = .023), human immunodeficiency virus infection (P < .001), Trichomonas infection (P < .001), cigarette smoking (P < .001), and male homosexual exposure (P = .012). Among couples, females whose sex partners were anti-HCV positive were 3.7 times more likely to have anti-HCV than females whose sex partners were anti-HCV negative (P = .039). The proportion of RNA homology between anti-HCV positive females and their male partners (94%) was higher than among randomly selected patients (82%). Sexual transmission of HCV may contribute to the high prevalence of anti-HCV reported in urban settings. PMID- 7535826 TI - Circulating L-selectin is elevated in patients with Plasmodium falciparum malaria. PMID- 7535828 TI - Multivariate models for predicting progression to AIDS and survival in human immunodeficiency virus-infected persons. AB - Nine hundred thirty persons enrolled in the US Air Force Human Immunodeficiency Virus (HIV) Natural History Study were evaluated with a standard battery of 30 potential surrogate markers of disease progression. A risk score for predicting progression to AIDS was then calculated for each patient in the cohort by using the four highest-ranking variables from multivariate analysis: percentage of CD4 CD29 cells, anergy status, age, and hemoglobin. For predicting survival, beta 2 microglobulin replaced age in the Cox model. Stratification according to the risk score demonstrated that rates of progression to AIDS and survival were significantly different between risk groups (P < .0001). The novel combination of these markers results in extremely accurate risk scores, which may serve as the basis for the development of true surrogate markers of disease progression. PMID- 7535829 TI - Antibodies against viral nonstructural proteins in response to infection with poliovirus. AB - Sera from recent and past cases of poliomyelitis as well as from healthy subjects vaccinated with live attenuated poliovirus, all contain antibodies to poliovirus nonstructural proteins. Reactivity of these antisera with nonstructural protein antigens present in virus-infected HeLa cells or translated from cDNA transcripts in rabbit reticulocyte lysates was sometimes sensitive to denaturation of the antigens. The finding of antibodies to nonstructural proteins of poliovirus and other viruses should have applications in the development of diagnostic assays for virus infection and may have implications for future vaccine design. PMID- 7535830 TI - Etiology of cat scratch disease: comparison of polymerase chain reaction detection of Bartonella (formerly Rochalimaea) and Afipia felis DNA with serology and skin tests. AB - To determine the role of Bartonella (formerly Rochalimaea) species and Afipia felis in cat scratch disease (CSD), two polymerase chain reaction (PCR) hybridization assays were developed to detect DNA from these organisms. These assays were applied on 89 pus aspirates from skin test-positive CSD patients (group 1) and on 137 pus and lymph node specimens from CSD suspects (group 2). Bartonella DNA was detected in 96% of the samples from group 1 patients and in 60% of group 2 samples; however, A. felis DNA could not be detected in any clinical samples. These results suggest that CSD is caused by bartonellae and that A. felis does not play a significant role in this zoonosis. A strong correlation between Bartonella PCR positivity and Bartonella henselae antibody titer was found. Comparison of CSD skin test results with those obtained by Bartonella PCR suggests a low sensitivity of the skin test. PMID- 7535831 TI - [Angiogenic factor in the ovaries]. PMID- 7535836 TI - The effects of ovine placental lactogen infusion on metabolites, insulin-like growth factors and binding proteins in the fetal sheep. AB - It has been suggested, but not shown, that in the fetus placental lactogen (PL) may affect the regulation of the IGFs and fetal metabolism. To examine the effects of PL on the circulating concentrations of the IGFs, IGF-binding proteins (IGFBPs), glucose, free fatty acids (FFAs) and amino nitrogen (AN), we infused late gestation sheep fetuses with recombinant ovine PL (roPL). Five chronically catheterised sheep fetuses were infused intravenously with three 24 h infusions of saline, roPL (100 micrograms bolus then 500 micrograms over 24 h) and then saline again. Fetal roPL infusion increased plasma oPL from 0.4 +/- 0.1 to 3.3 +/ 0.5 nM (mean +/- S.E.M.; P < 0.05; factorial analysis of variance and Scheffe's test). Fetal plasma IGF-I, IGF-II, insulin, FFAs and blood glucose were unaffected by the roPL infusion. Fetal plasma IGFBP-3, as measured by Western ligand blotting, decreased by 30% during fetal roPL infusion while other fetal plasma IGFBPs were unaffected. Fetal roPL infusion decreased fetal blood AN from 7.3 +/- 0.5 to 6.6 +/- 0.2 mM (P < 0.05). Maternal plasma IGF-I, IGF-II, IGFBPs, insulin, FFAs, blood glucose and AN were unaffected by the fetal roPL infusion. Saline infusion had no effect on any parameter. The data suggest that PL is not a significant determinant of plasma IGFs in the late gestation sheep fetus although there may be an indirect effect via alterations in levels of IGFBP-3. The effect of fetal roPL infusion on fetal blood AN concentrations may suggest some role for PL in the regulation of fetal amino acid metabolism. PMID- 7535835 TI - Changes in IGF-I and -II expression and secretion during the proliferation and differentiation of normal rat osteoblasts. AB - IGF-I and -II have potent effects on proliferation and differentiation of osteoblasts in vitro. These cells secrete both IGFs and expression of these peptides is regulated by several of the hormones and growth factors that promote bone resorption and/or formation. However, the physiological role(s) of IGFs in the remodelling process of adult bone is still unclear. Some confusion may arise from results influenced, in part, by differences in the state of osteoblast development of in vitro cultures. Several laboratories have demonstrated that murine osteoblast cultures progress from proliferating preosteoblasts, to mature differentiated osteoblasts that form an extracellular matrix, to cultures that form a mineralized matrix. We have recently documented changes in IGF-binding protein expression and secretion in these cultures. To complement and extend this work, we have examined IGF-I expression and secretion and IGF-II expression during in vitro osteoblast development. Steady-state mRNA levels of both IGF-I and -II increased from the earliest time examined, day 5 in culture, to a maximum at day 11 and, thereafter, declined. IGF-I secreted into the medium also changed in a biphasic manner, but IGF-II could not be quantitated due to the sensitivity of our assay. Secretion of IGF-I was lowest between days 8 and 14. IGF-I secretion on day 5 was significantly greater than day 8. Similarly, IGF-1 secretion from day 17 to 26 was also greater than observed for days 8 to 14. If differentiation of the cells was inhibited, this late rise in IGF-I secretion was abolished.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535833 TI - [Expression and subtype analysis of vascular endothelial growth factor (VEGF) and its receptor (flt-1) in human ovarian tumors]. AB - Angiogenesis is very important not only for embryogenesis and wound healing but also for tumor growth in vivo because vessels supply oxygen and nutrition to the tumor mass. In this study, we focused on Vascular Endothelial Growth Factor (VEGF), a newly characterized endothel-specific growth factor and investigated the expression of VEGF in 13 ovarian tumors and 3 normal ovaries by using polymerase chain reaction (PCR) analysis and Northern blot analysis. Further, we examined the expression pattern of 4 alternatively spliced forms of VEGF in these tissues. The level of VEGF mRNA was higher in 77% of ovarian tumors when compared with that in normal ovaries. Among subtypes of VEGF, 121-, 165- and 189-amino acid types were detected but 206-amino acid type was not observed in ovarian tumors. The most abundant form of VEGF was 121-amino acid type and the relative amounts of the various forms of VEGF were 121-amino acid type > 165-amino acid type >> 189-amino acid type. Expression of flt-1, a receptor for VEGF was detectable by PCR but not by Northern blot analysis. These results suggest that like other epithelial cell-derived carcinomas, ovarian tumors use the VEGF/flt-1 system for tumor angiogenesis. PMID- 7535832 TI - [B lymphocyte antigen CD40 and its ligand, CD40L]. PMID- 7535837 TI - Treatment of cutaneous nodules using electrochemotherapy. AB - The ability to increase the amount of chemotherapeutic agents absorbed by tumor cells can be accomplished using electrochemotherapy. A preclinical study demonstrated the potential of this treatment; therefore, a clinical trial was initiated to determine if it would be effective in cutaneous and subcutaneous malignancies. Three melanoma, one adenocarcinoma and two basal cell carcinoma patients were treated. Each received a 10 unit/m2 dose of bleomycin intravenously. Eight 99 microseconds pulses at an amplitude of 1.3 kV/cm were administered directly to the tumors approximately 10-12 minutes after initiating the bleomycin injection and following the injection of 1% lidocaine solution around the treatment site. Two of three melanoma patients have responded positively to the treatment. Both basal cell carcinoma patients responded. The patient with adenocarcinoma responded as well. All patients tolerated the treatment well with no residual effects from the electric pulses. Although conclusions cannot be made, the results thus far are very encouraging. PMID- 7535838 TI - Development of wing sensory axons in the central nervous system of Drosophila during metamorphosis. AB - The development of new, adult-specific axonal pathways in the central nervous system (CNS) of insects during metamorphosis is still largely uncharacterized. Here we used axonal labeling with DiI to describe the timing and pattern of growth of sensory axons originating in the wing of Drosophila as they establish their adult projection pattern in the CNS during pupal life. The wing of Drosophila carries a small number of readily identifiable sensory organs (sensilla) whose neurons are located in the periphery and whose axons travel along specific routes within the adult CNS. The neurons are born and undergo axonogenesis in a characteristic order. The order of axon arrival in the CNS appears to be the same as that of their development in the periphery. Within the CNS, the formation of four prominent axon bundles leading to distant termination sites is followed by the formation of a compact axon termination site near the point of wing nerve entry into the CNS. This sensillum-specific pattern persists into adulthood without discernible modification. We also find a small number of axons filled with DiI prior to the formation of the four permanent bundles. We have only been able to fill them for a few hours in early pupal life and therefore consider them to be transient. The bundles of wing sensory axons travel within tracts that contain other axons as well. Using immunocytochemistry, the tracts start to be histologically identifiable at around 12 h after pupariation (AP), and grow substantially as metamorphosis proceeds. Wing sensory neurons are found in the tracts by 18-20 h AP and the full adult pattern is established by 48 h AP. When sensory axons first enter the CNS, they fan out in the region where their appropriate tracts are located, but they do not wander extensively. They quickly form bundles that become increasingly compact over time. Calculations show that the rate of axon extension within the CNS varies from bundle to bundle and is equal to or greater than that of the same axons growing through wing tissue. PMID- 7535834 TI - An alternative to regular dressings for otitis externa and chronic supperative otitis media? AB - Otitis externa and chronic otitis media often present to the otolaryngologist with a discharging ear. The conventional method of treatment is to perform regular aural toilet and insert medicated dressings into the external auditory canal. This treatment is either performed by trained nurses or medical staff, but in either case is time consuming. This study compares the efficacy of the above standard regimen with a novel treatment where a single aural toilet is carried out and medicated ointment instilled into the ear. Both regimens were evaluated at three weeks. Our results shows that there was no significant difference between the two treatment regimens with regard to the resolution of either of the conditions studied or the improvement in the symptom status of the patients. On the basis of this it would seem that a single aural toilet and instillation of medicated ointment is a valid treatment option, cuts down hospital attendance and could be performed in the community by general medical practitioners or trained practice nurses. PMID- 7535839 TI - Lectin binding distinguishes between neuroendocrine and neuronal derivatives of the sympathoadrenal neural crest. AB - Lectin cytochemistry was used to identify surface epitopes selectively expressed by chromaffin cell chemoreceptors (glomus cells) in the rat carotid body. Unexpectedly, these studies revealed that binding sites for peanut agglutinin (PNA; Arachis hypogea) were highly expressed by all neuroendocrine-derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, and adrenal chromaffin cells in situ. In contrast, principal sympathetic neurons did not express PNA receptors. PNA binding was inhibited by 2% galactose. To determine whether expression of PNA receptors was selectively induced by neuroendocrine differentiation of sympathoadrenal precursors, we compared PNA labeling of embryonic sympathoblasts in the presence of either nerve growth factor (NGF) or the synthetic glucocorticoid dexamethasone (DEX). DEX treated cells, which expressed several neuroendocrine traits, bound PNA, whereas NGF-treated neuronal derivatives did not. In addition, to examine whether expression of existing PNA receptors was down-regulated by neuronal differentiation of chromaffin cells, we compared labeling of PC12 cells, which normally bind PNA, in the presence and absence of NGF. Although PC12 cells acquired characteristic neuronal morphologies in the presence of NGF, they did not lose PNA labeling, even after 8 days of NGF treatment. These findings indicate that neuronal and neuroendocrine derivatives of the sympathoadrenal lineage can be distinguished by differential expression of carbohydrate epitopes and suggest that PNA receptors are induced by neuroendocrine differentiation. PMID- 7535840 TI - Herpes simplex virus encephalitis in a mouse model: PCR evidence for CNS latency following acute infection. AB - We have used a mouse model of herpes simplex encephalitis produced by intranasal inoculation of virus to study the expression of viral immediate early, early and late genes and latency associated transcript (LAT) in trigeminal ganglia and brain at various times after inoculation. A PCR technique was used to detect the viral gene transcripts. All viral genes were expressed between post-inoculation days 1 and 13. On post-inoculation day 42 when the acute infection had subsided only the LAT could be detected, most commonly (70%) in the trigeminal ganglion but also, in 50% of mice, in the brain stem, in 40% in olfactory bulbs and in 20% in cerebrum and cerebellum. These findings suggest that latent infection by HSV-1 may be relatively readily established in the CNS as well as in sensory ganglia. The frequency of establishment of latency appears to be related to the neuroanatomical accessibility of each brain region to the site of entry of the virus. PMID- 7535841 TI - Alterations in serum thrombospondin in patients with amyotrophic lateral sclerosis. AB - Thrombospondin (TSP), an endogenous extracellular matrix (ECM) glycoprotein, is secreted from platelet alpha-granules after thrombin stimulation. Alterations in blood TSP levels occur in different pathologic conditions, suggesting it is a marker for certain disorders. We previously found a marked increase in TSP deposition in the muscle ECM of patients with amyotrophic lateral sclerosis (ALS) in comparison with controls. Because the mechanism for this increase is unknown, we compared serum TSP levels in 11 patients to 15 controls using three different site-specific monoclonal antibodies (MA-I, MA-II and A6.1). We found mean serum TSP concentrations by indirect ELISA to be significantly decreased in the ALS patients. Using laser densitometry we calculated the ratio of fragmented to native TSP from Western immunoblots probed with A6.1, where a higher ratio corresponds to increased fragments. Mean values for this ratio were 6.3 +/- 4.9 and 18.3 +/- 8.2 for controls and patients, respectively. Thus significant decrease in native TSP and increase in its proteolytic fragments in ALS is consistent with increased proteolytic enzyme activity. Dysregulation of the protease: inhibitor balance in this degenerative condition may be reflected in the quantitative and qualitative changes in TSP. PMID- 7535842 TI - Clinical applications of hematopoietic growth factors. AB - PURPOSE AND DESIGN: To review the current clinical uses, ongoing investigations, and future applications of hematopoietic growth factors. Approved cytokines, as well as cytokines not yet released for general use, are included in this review. RESULTS: Clinical applications of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin, the three recombinant hematopoietic growth factors currently commercially available for clinical use in the United States, are discussed. Macrophage colony stimulating factor (M-CSF), interleukin-3 (IL-3), PIXY321, stem-cell factor (SCF), IL-1, IL-6, and IL-11 represent cytokines not yet approved; the majority of these newer agents have their principal action at an earlier time point in the hematopoietic cascade than the currently approved cytokines. Current clinical uses of hematopoietic growth factors include decreasing cytopenias associated with chemotherapy, those due to congenital or acquired bone marrow failure states, those that occur after high-dose chemotherapy and bone marrow transplantation, peripheral-blood progenitor mobilization, and supportive care of leukemia patients. CONCLUSION: Hematopoietic growth factors have made a significant impact on the prevention of infections associated with chemotherapy induced neutropenia, shortening of neutropenia following high-dose chemotherapy and progenitor-cell transplantation, and chemotherapy-associated anemia. Cost effectiveness and cost-benefit analyses in future phase III and pharmacologic studies will aid in the assessment of these agents. PMID- 7535846 TI - State Board Connection. PMID- 7535845 TI - [Arterial vascularization of the lenticular nucleus]. AB - The arterial vasculature of the lenticular nucleus was studied on 30 human brains the vascular system of which was injected with Indian ink mixed with gelose. The arteries of the lenticular nucleus belong to the group of anterior central arteries issued from the internal carotid artery and its terminal branches, except for the posterior cerebral artery. Examination of serial sections made translucent by the Spalteholtz method enabled us to follow the courses of these vessels within the lenticular nucleus. In this nucleus two vascular networks were present, with different vascular organization and density. These differences were due to the histological structure and specific embryonic origin of each part of the lenticular nucleus. PMID- 7535843 TI - Granulocyte colony-stimulating factor following peripheral-blood progenitor-cell transplant in non-Hodgkin's lymphoma. AB - PURPOSE: To compare the hematologic recovery after high-dose chemotherapy and circulating peripheral-blood progenitor-cell (PBPC) transplant between patients who received recombinant human granulocyte colony-stimulating factor (G-CSF) (treated group) and those who did not (control group). PATIENTS AND METHODS: From December 1992 through June 1994, two sequential and consecutive cohorts of 20 patients each with histologically proven non-Hodgkin's lymphoma (NHL) received high-dose chemotherapy (carmustine [BCNU], cytarabine [Ara-C], etoposide and melphalan [BEAM]) followed by PBPC transplant. The first 20 patients were treated with G-CSF (5 micrograms/kg/d) after PBPC administration. Since the time of platelet and leukocyte recovery in this group was short (< 15 days), with a narrow standard deviation from the mean value, the last 20 patients were not given G-CSF. Hematologic recovery, number of febrile days, rate of documented infections, number of hospital days, duration of gastrointestinal complications, platelet and RBC transfusions, and antibiotic requirements were compared in the two groups. RESULTS: The two groups of patients were comparable according to disease status, histology, stage, bulky disease bone marrow involvement, elevated lactate dehydrogenase (LDH) level, and median number of infused CD34+ cells and colony-forming units granulocyte-macrophage (CFU-GM). The median time to reach 0.5 x 10(9)/L and 1.0 x 10(9)/L neutrophils was 2 days shorter in G-CSF group, but this difference was not statistically significant. The median times to reach 20 x 10(9)/L and 50 x 10(9)/L platelets were, respectively, 10 and 14 days in the G-CSF group and 11 and 16 days in the control group, but again this was not statistically significant. Moreover, when considering clinically relevant end points including the number of documented infections and antibiotic requirements, platelet transfusions, gastrointestinal toxicity, and days of hospitalization, no differences were demonstrated between the two groups. CONCLUSIONS: Provided an optimal dose of circulating progenitors is infused, NHL patients transplanted with PBPC do not benefit by the administration of hematopoietic growth factors. PMID- 7535847 TI - [Immunohistochemical distribution of laminin in squamous cell carcinoma of the head and neck--correlations between laminin staining and clinical and histological features]. AB - The distribution of the basement membrane glycoprotein laminin was investigated immunohistochemically in 66 patients with squamous cell carcinoma of the head and neck region. There were 18 patients with laryngeal cancer, 23 with pharyngeal cancer, 11 with maxillary cancer, and 14 with oral cancer. The specimens were taken from untreated primary lesions, and laminin was detected using the avidin biotin-peroxidase complex method. In carcinoma tissue, staining was seen in border zone between the cancer cells and surrounding connective tissue, but discontinuity or disappearance of the staining was commonly observed. The patients were classified on the basis of laminin distribution into the following three groups: a normal group, in which staining was almost continuous around the cancer cells, a discontinuous group, in which staining was discontinuous but present in up to 50% of the cancer cells, and an absent group, in which staining was seen in 49% or less of cancer cells. These groups were matched against clinical and histological features. Laminin distribution was significantly correlated with metastasis, survival rate and keratinization. In the absent group, 12 (92%) of the 13 patients had lymph node metastasis, and 6 (46%) had distant metastasis. In the discontinuous group, 17 (94%) of the 18 patients had lymph node metastasis and 3 (17%) had distant metastasis. In contrast, in the normal group, 13 (37%) of the 25 patients had lymph node metastasis and 2 (6%) had distant metastasis. The 5 year survival rate was 31%, 52%, and 67% in the absent, discontinuous and normal group, respectively. Histologically, the ratios of poorly keratinized to highly keratinized carcinomas were 62%, 67%, and 34% in the absent, discontinuous, and normal group, respectively. These findings suggested that the absent and the discontinuous, group required intensive neck dissection and systemic anticancer therapy. PMID- 7535844 TI - CEOP-B alternated with VIMB in intermediate-grade and high-grade non-Hodgkin's lymphoma: a pilot study. AB - PURPOSE: To improve response and toxicity in treatment of non-Hodgkin's lymphomas (NHLs), a prospective single-arm trial was initiated using cyclophosphamide, epirubicin, vincristine, prednisone, and bleomycin (CEOP-B) alternated with etoposide (VP-16), ifosfamide, mitoxantrone, and bleomycin (VIMB). PATIENTS AND METHODS: From December 1988 to April 1992, 60 consecutive previously untreated patients with intermediate- or high-grade NHL were admitted to the study and were assessable. Patient characteristics were as follows: 32% greater than 60 years of age, 63% with stage III to IV disease, 42% with a performance status (PS) of 2 or 3, 23% with high lactate dehydrogenase (LDH) levels, and 22% with two or more extranodal disease sites. Stage I and II patients received three cycles of CEOP B/VIMB plus radiotherapy (RT) to involved fields; stage III and IV patients received four cycles of chemotherapy alone. RESULTS: The complete remission (CR) rate was 77%; actuarial 48-month overall survival (OS) and time to treatment failure (TTF) rates were 70% and 59%, respectively. With univariate analysis, CR, OS, and TTF rates were significantly influenced by serum LDH levels (P = .0485, P = .0017, and P = .0064, respectively) and performance status (P = .0005, P < .00005, and P = .0001, respectively). The actuarial 48-month disease-free survival (DFS) rate was 83% and was negatively influenced only by high-grade histology (P < .004). Toxicity was mild. A lower epirubicin dose-intensity (DI) was found in patients older than 60 years of age, with a borderline P value. Patients were divided into four groups according to the International Prognostic Factor Project; low-risk and low-intermediate-risk groups had similar OS and TTF rates; when considered together, they showed superior, but not statistically significant, OS and TTF rates as compared with the high-intermediate-risk group, which in turn had significantly superior OS and TTF rates when compared with the high-risk group. CONCLUSION: CEOP-B/VIMB compares favorably with third-generation regimens and results in lower toxicity. PMID- 7535848 TI - Hemocyte alterations during melanotic encapsulation of Brugia malayi in the mosquito Armigeres subalbatus. AB - The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms. PMID- 7535849 TI - Pharmacological characterization in vitro of prostanoid receptors in the myometrium of nonpregnant ewes. AB - Prostanoid receptors regulating the contractility of strips of myometrium obtained from nonpregnant ewes during the breeding season were classified pharmacologically. Natural prostanoids, receptor-type selective synthetic analogues, and selective antagonists were used where available. The natural prostanoids PGD2, PGE2, and PGF2 alpha were equipotent in causing contractions (pD2 values of 6.9, 6.7, and 6.9, respectively) but were 100 times less potent than oxytocin (pD2 = 9.2). The synthetic prostanoids iloprost (pD2 = 8.3), GR63799x (pD2 = 7.0), cloprostenol (pD2 = 6.8), and U46619 (pD2 = 6.2) also caused contractions. The effects of iloprost, but not of GR63799x, were blocked by the selective EP1 antagonist AH 6809. This suggests the presence of both EP1 and EP3 receptors. The similar potencies of cloprostenol and PGF2 alpha suggest the presence of FP receptors. Although the potency of the TP agonist U46619 was relatively low, its effects were blocked by the selective TP antagonist L 670596 (pKB = 8.4), an observation consistent with the presence of TP receptors. Thus, all currently recognized excitatory prostanoid receptors (EP1, EP3, FP and TP) appeared to be present. Contractions induced by cloprostenol and KCl could be inhibited by the beta-adrenoceptor agonist isoprenaline (pD2 = 8.8 against cloprostenol) and the Ca(2+)-channel blocker, D600 (pD2 = 6.3 against cloprostenol), but a number of relaxant prostanoids, BW245c, ZK110841, AH13205 and cicaprost, could not produce inhibition. These results suggest that DP, EP2 and IP receptors do not regulate contractility under these conditions. PMID- 7535850 TI - Expression of carbohydrate antigens in the rat uterus during early pregnancy and after ovariectomy and steroid replacement. AB - Monoclonal antibodies were used to examine the expression of a number of carbohydrate antigens in the rat endometrial epithelium from day 1 to day 8 of pregnancy and in ovariectomized rats supplemented with ovarian steroids. Carbohydrate antigens based on the Gal beta 1-GlcNAc backbone structure were expressed and some of these (Le(y), Le(x), B antigen) were present at all stages of pregnancy and independent of ovarian steroids. The H-type-2 antigen was stimulated by progesterone and expressed in the sensitized and receptive uterus, but was not detected after implantation or, in ovariectomized rats, in the refractory phase. The H-type-1 antigen, which is stimulated by oestrogen in the mouse, appeared to be stimulated by progesterone in rats. It was expressed throughout the period of pregnancy but maximal expression was found on days 4-5. The histo-blood group A antigen appeared in ovariectomized rats only after treatment with progesterone followed by three daily injections of progesterone and oestrogen, and in the corresponding postimplantation period of pregnant rats. Its appearance corresponded to the loss of detectable H-type-2 antigen. This study shows that the rat endometrial epithelium expresses some carbohydrate antigens not expressed in mice (A and B antigen) or under completely different steroidal regulation (H-type-1). Moreover, the T antigen was expressed on the endometrial epithelium adjacent to decidua on days 7 and 8 of pregnancy, but not in rats given ovarian steroids to mimic the sensitized, receptive or refractory phase. Differences in expression between glandular and luminal epithelial indicated differences in steroidal regulation, as found in mice. PMID- 7535852 TI - D2O-induced ion channel activation in Characeae at low ionic strength. AB - Effects of D2O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca2+ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D2O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (Rm = 0.12 +/- 0.03 k omega.cm2), thus preventing further voltage clamping. APW with 25% D2O caused a two-step reduction of Rm: first, down to 2.0 +/- 0.8 k omega.cm2, and then further to 200 omega.cm2, in 2 min. It was shown that in the first stage, Ca2+ channels are activated, and then, Ca2+ ions entering through them activate the Cl- channels. The Ca2+ channels are activated irreversibly. If 100 mM CsCl was substituted for 200 mM sucrose (introduced for iso-osmoticity), no effect of D2O on Rm was observed. Intracellular H2O/D2O substitution also did not change Rm. In experiments on single Ca2+ channels in BLM H2O/D2O substitution in a solution containing 100 mM KCl (trans side) produced no effect on channel activity, while in 10 mM KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H2O/D2O substitution. The discussion of the possible mechanism of D2O action on Ca2+ and Cl- channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond. PMID- 7535853 TI - Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells. AB - The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl- but inhibited by addition of MK196 (anion channel blocker) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl-, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a "Mini Cl- channel." The Cl- efflux via this Cl- channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated "Mini Cl- channel" and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels. PMID- 7535854 TI - The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line, CRI-G1. AB - The activity of a calcium-activated nonselective (Ca-NS+) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, beta-NAD+, beta NADH, beta-NADP+ and beta-NADPH were very similar when tested at 0.1 mM, and at 1 mM the phosphorylated forms, beta-NADP+ and beta-NADPH, appeared to be slightly more potent than beta-NAD+ and beta-NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch. The application of beta-NAD+, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the beta-NAD(+)-NS+ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP. PMID- 7535856 TI - Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri. AB - Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halphytic angiosperm Zostera muelleri. In wholecell preparations, both outward and inward rectifying currents that activate in a time- and voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K+. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K+ than to Na+. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K+. In addition, the plasma membrane contains a much larger K+ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl- channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd3+, an effect that is reversible upon washout. PMID- 7535851 TI - Reconstitution of the influenza virus M2 ion channel in lipid bilayers. AB - M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle. PMID- 7535855 TI - Role of F-actin in the activation of Na(+)-K(+)-Cl- cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells. AB - The influence of microtubules and F-actin on Na(+)-K(+)-Cl- cotransport was investigated in cultured cells derived from outer-medullary thick ascending limb tubules microdissected from the mouse kidney. The cultured cells contained Tamm Horsfall protein, produced cAMP in response to dD-arginine vasopressin (dD-AVP), isoproterenol, prostaglandin E2 and forskolin (FK), and exhibited an ouabain resistant furosemide-sensitive (Or-Fs) component of 86Rb+ influx mediated by the Na(+)-K(+)-Cl- cotransporter. Both FK and dD-AVP stimulated the Or-Fs component of Rb+ influx. Neither agent altered the tubulin and cytokeratin networks nor the shape of the tight junction using a specific anti-ZO-1 antibody. In contrast, they did induce a marked redistribution of F-actin to the periphery of the cells delineating the tight junctions. Preincubation of the cells with nocodazole, to disrupt microtubules, did not alter the FK- or dD-AVP-elicited Or-Fs Rb+ influx. In contrast, phalloidin and NBD-phallicidin, which stabilize F-actin, markedly impaired the stimulation of Na(+)-K(+)-Cl- cotransport by FK or dD-AVP, without affecting the Na(+)-K+ ATPase pumps and the rate constant of 36Cl- and 86Rb+ efflux. These results strongly suggested that cAMP-stimulated Na(+)-K(+)-Cl- cotransport is linked to F-actin in renal TAL cells. PMID- 7535858 TI - Simulation of human B-lymphocyte proliferation by AGM-1470, a potent inhibitor of angiogenesis. PMID- 7535857 TI - Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants. AB - We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3' end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex. PMID- 7535859 TI - Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells. AB - BACKGROUND: Vascular endothelial growth factor (VEGF) is a secreted endothelial specific growth factor that is angiogenic in vivo. It is commonly expressed in a range of carcinomas. PURPOSE: The study was designed to investigate the effect of constitutive expression of VEGF on tumor formation by estrogen-dependent human MCF-7 breast carcinoma cells. METHODS: A full-length complementary DNA encoding the shortest isoform of VEGF (VEGF121) was stably transfected into MCF-7 cells. Transfected clones were screened for VEGF121 messenger RNA (mRNA) expression by ribonuclease protection analysis and for secretion of VEGF121 protein by Western blot analysis. Secretion of biologically active VEGF121 by transfectants was confirmed by 1) a competitive radioreceptor-binding assay, 2) stimulation of the growth of microvascular endothelial cells in vitro, and 3) potent angiogenic activity in the rabbit corneal assay. Tumor models were then established by subcutaneously implanting wild-type or VEGF121-transfected MCF-7 cells, together with either mouse BALB/3T3 clone A31 fibroblasts or human MDA-435S breast carcinoma cells, into ovariectomized nude mice either with or without a separately implanted slow-release estrogen pellet. Tumor vascularity was quantitatively assessed by capillary vessel counting after staining with the pan endothelial marker CD31. RESULTS: Stable VEGF121-overexpressing MCF-7 cells were isolated and designated V12 cells. When implanted into the rabbit cornea, V12 cells elicited a strong directional outgrowth of capillaries. The growth rate of V12 cells in vitro was indistinguishable from that of MCF-7 wild-type cells. V12 cells formed faster growing tumors than did wild-type cells (P < .01) when xenografted subcutaneously into nude mice with either 3T3 fibroblasts or MDA-435S cells. Tumors formed from V12 cells were more vascular (P < .01) and showed a heterogeneous distribution of vessels when compared with the homogeneous distribution seen in tumors formed from wild-type cells. VEGF121 overexpression had no effect on hormone dependence or tamoxifen sensitivity of tumor formation by MCF-7 cells in mice. No macroscopic evidence for metastasis from subcutaneous implants was obtained. CONCLUSIONS: VEGF121 expression by breast carcinoma cells confers a growth advantage in vivo but not in vitro. Tumors formed by V12 transfectants were more vascular than those formed by wild-type MCF-7 cells, and we surmise that the growth advantage arises from increased tumor vascularization induced by VEGF121. IMPLICATIONS: Tumor formation by V12 cells could provide a useful model for the assessment of anti-angiogenic drugs. PMID- 7535860 TI - Response and resistance to interferons and interacting cytokines. PMID- 7535861 TI - Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases. AB - BACKGROUND: The importance of matrix metalloproteinases in angiogenesis, tumor growth, and metastasis is well known. However, little is known about the role of matrix metalloproteinases in the formation of hemangiomas and about the possible therapeutic use of matrix metalloproteinase inhibitors in aggressive vascular tumors. PURPOSE: To study the role of matrix metalloproteinase in vascular tumors, we tested the antineoplastic activity of a synthetic inhibitor of matrix metalloproteinases, batimastat, on an experimental model of hemangioma, formed by murine endothelioma cells transformed by polyoma middle-T oncogene (eEnd.1). METHODS: The effect of batimastat was studied in vivo on the formation of hemorrhaging, cavernous hemangiomas by eEnd.1 endothelioma cells injected subcutaneously in nude mice and on the angiogenic response induced by an endothelioma cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel). The effect of batimastat was investigated in vitro on endothelial cell proliferation, motility, and invasion of a layer of Matrigel. RESULTS: Daily treatment with batimastat (30, 3, and 0.3 mg/kg at the site of eEnd.1 cell injection) inhibited tumor growth, with increased doubling time. The carboxamide derivative of batimastat, BB-374, a poor inhibitor of matrix metalloproteinase activity, was less active in reducing hemangioma growth. Histologic analysis of treated tumors indicated a reduction in the size of blood filled spaces and in hemorrhage. Batimastat also inhibited the angiogenic response induced by cultured eEnd.1 endothelioma cell supernatant embedded in a pellet of Matrigel. Batimastat significantly inhibited endothelial cell invasion in vitro through a layer of Matrigel, but it showed no direct cytotoxic activity. CONCLUSIONS: Batimastat reduces in vivo growth of experimental hemangiomas, most probably by blocking endothelial cell recruitment by the transformed cells or by interfering with cell organization in vascular structures. IMPLICATIONS: These results confirm the importance of matrix metalloproteinase in endothelial cell recruitment that occurs in angiogenesis and in the formation of vascular tumors and suggest a therapeutic potential for synthetic matrix metalloproteinase inhibitors. PMID- 7535862 TI - Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein. AB - We established a reverse genetics system for the M gene of influenza A virus, using amantadine resistance as a selection criterion. Transfection of an artificial M ribonucleoprotein complex of A/Puerto Rico/8/34 (H1N1), a naturally occurring amantadine-resistant virus, and superinfection with amantadine sensitive A/equine/Miami/1/63 (H3N8), followed by cultivation in the presence of the drug, led to the generation of a transfectant virus with the A/Puerto Rico/8/34 (H1N1) M gene. With this system, we attempted to generate a virus containing a deletion in an M-gene product (M2 protein). Viruses lacking the carboxyl-terminal Glu of M2, but not those lacking 5 or 10 carboxyl-terminal residues, were rescued in the presence of amantadine. These findings indicate that carboxyl-terminal residues of the M2 protein play an important role in influenza virus replication. The M-gene-based reverse genetics system will allow the study of different M-gene mutations to achieve a balance between attenuation and virus replication, thus facilitating the production of live vaccine strains. PMID- 7535863 TI - Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. AB - The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes. PMID- 7535864 TI - Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. AB - The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles. PMID- 7535866 TI - Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein. AB - Bluetongue virus produces large numbers of tubules during infection. The tubules are formed from a 552-amino-acid, 64-kDa NS1 protein encoded by the viral double stranded RNA segment M6. A series of deletion and extension mutants of bluetongue virus serotype 10 NS1 has been generated and expressed in insect cells in order to identify the carboxy-terminal components of the protein which are important for tubule formation. The mutants AcCT5 and AcCT10, lacking 5 and 10 of the carboxy-terminal residues, respectively, were prepared. By analyzing their abilities to form tubules, it was shown that AcCT5 was capable of this function whereas AcCT10 was not, indicating that the last five amino acids are not strongly involved in NS1 tubule formation. Extension mutants including foreign antigenic sequences involving up to 16 amino acids added to the C terminus of NS1 were shown to form tubules, although an extension of 19 amino acids inhibited tubule formation. Analysis of a panel of monoclonal antibodies has established that an NS1 antigenic site is located near the carboxy terminus of the protein. It appears to be exposed on the surface of tubules. The opportunities to develop new vaccines using recombinant NS1 to deliver foreign epitopes are discussed. PMID- 7535865 TI - Preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells is epitope and major histocompatibility complex dependent. AB - Regulatory T-helper (Th) cells have been categorized into two functional subsets, Th1 and Th2 cells, which produce distinct lymphokines. In general, Th1 cells mediate cellular immune responses and Th2 cells mediate humoral immunity. Recent serological studies suggest that the Th1-Th2 balance may be relevant in acute and chronic hepatitis B virus (HBV) infections. The purpose of this study was to determine the potential of the nucleocapsid antigens (Ags) (hepatitis B core and e Ags [HBc/eAg]) of HBV to preferentially elicit either a Th1 or a Th2 dominant response. For this purpose, H-2 congenic B10.S and B10 mice were immunized with HBc/eAg, and Ag-specific T-cell proliferative responses, T-cell helper function, and T-cell cytokine production were analyzed. The results indicated that B10.S mice preferentially develop a Th1-like response whereas B10 mice preferentially develop a Th2-like response after immunization with HBc/eAg. Furthermore, the preferential Th1 and Th2 response patterns were reproduced when 12-residue peptides representing the dominant HBc/eAg-specific T-cell sites for B10.S (peptide 120-131) and B10 (peptide 129-140) mice were used as immunogens. Therefore, the combination of the T-cell site recognized and the major histocompatibility complex restricting element can in large part determine the Th phenotype of the HBc/eAg-specific T-cell response. Other factors that influenced Th phenotype were the presence of exogenous cytokines, Ag structure, and tissue distribution. PMID- 7535868 TI - Echovirus 1 interaction with the isolated VLA-2 I domain. AB - The isolated I domain of the integrin VLA-2, produced as a bacterial fusion protein, specifically bound echovirus 1 and prevented virus attachment to cells. These results demonstrate that the receptor structures critical for virus attachment are contained solely within the VLA-2 I domain and that soluble receptor fragments are capable of preventing infection. PMID- 7535870 TI - From the Centers for Disease Control and Prevention. Update: Vibrio cholerae O1- Western Hemisphere, 1991-1994, and V. cholerae O139--Asia, 1994. PMID- 7535867 TI - Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen. AB - Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association. PMID- 7535869 TI - Human transluminally placed endovascular stented grafts: preliminary histopathologic analysis of healing grafts in aortoiliac and femoral artery occlusive disease. AB - PURPOSE: The purpose of this study was to perform a preliminary histopathologic analysis of explanted human endovascular stented grafts from patients treated for occlusive disease. METHODS: Over a 16-month period, 26 endovascular stented grafts were placed in 21 patients with limb-threatening ischemia caused by aortoiliac or femoral artery occlusive disease. All grafts were inserted through open arteriotomies remote from the region of primary disease. During the follow up period, two patients died of preexisting heart disease 2 weeks and 7 months after grafting, and a portion of their endovascular grafts were the surrounding artery was explanted. Specimens from five other endovascular grafts were obtained during surgical revision for graft stenosis after 3 and 6 weeks and for outflow artery stenosis after 3, 5, and 6 months. All specimens were formalin fixed and studied with hematoxylin and eosin and trichrome staining and immunohistochemically for factor VIII-related antigen, alpha actin smooth muscle, macrophage antigen (MAC-387) and PC-10 (a mouse monoclonal antibody which specifically recognizes proliferating cell nuclear antigen in paraffin sections). RESULTS: Three weeks after placement of the stented grafts, organizing thrombus was present on both surfaces of the expanded polytetrafluoroethylene (PTFE) grafts. At 6 weeks, evidence of a neointima with overlying endothelium was seen in the perianastomotic region, and 3 months after grafting it was seen 1 to 3 cm from the anastomosis. The specimen explanted at 5 months demonstrated factor VIII positive cells 8 cm from the anastomosis. The histopathologic condition of the external capsule appeared to vary, depending on the presence or absence of an external wrap on the PTFE graft and on which layer in the arterial wall the graft was inserted. A foreign body reaction characterized by multinucleated giant cells was seen adjacent to wrapped grafts or around those placed in an intraadventitial plane. Grafts inserted within the media were surrounded by orderly, arranged, smooth muscle cells and few mononuclear cells. Extensive smooth muscle cell proliferation (PC-10 activity) was not seen within native artery atherosclerotic plaques peripherally displaced and external to prosthetic endovascular grafts. CONCLUSIONS: These preliminary observations on the healing of PTFE endovascular stented grafts in human beings demonstrate limited plaque hyperplasia and the presence of endothelial cells on the luminal surface remote from the graft-artery anastomosis. It is unclear whether this is a unique manifestation of healing in prosthetic grafts inserted within the walls of arteries. PMID- 7535872 TI - [A call for attention to the organization of palliative care in oncology]. PMID- 7535871 TI - [Pancreatoduodenal reconstruction after previous palliative surgery]. AB - The performing peculiarities of reconstructive stage of pancreatoduodenal resection after previously conducted palliative operation in 212 patients were analyzed. The drainage operation was previously conducted to 89 of them. PMID- 7535873 TI - [Serum analysis in pregnant women. Mass screening is an effective way to discover chromosome aberrations--arguments for and against]. PMID- 7535874 TI - [Quality assurance of pain relief in terminal care. Diagnose the pain and evaluate therapeutic results!]. PMID- 7535875 TI - Hypertrophic cardiomyopathy associated with tacrolimus in paediatric transplant patients. AB - Reported side-effects of tacrolimus, a potent immunosuppressive agent, have not included cardiotoxicity. We describe 5 consecutive paediatric transplant recipients (3 small bowel with or without liver and 2 liver) who received tacrolimus. 2 developed congestive heart failure and hypertrophic obstructive cardiomyopathy which resolved after changing to cyclosporin. In the other 3 patients the cardiomyopathy regressed or improved with a lower dose of tacrolimus or after stopping the drug. PMID- 7535877 TI - Absence of sex-hormone receptors in Kaposi's sarcoma. PMID- 7535876 TI - Polonium-210 and vehicle exhaust pollution. PMID- 7535879 TI - Hydration and alimentation in terminal patients: a hospice approach. PMID- 7535878 TI - The effect of temporary hepatic ischemia on liver surface pH and potassium ion activity in the rat. AB - The effects of hepatic ischemia can be analyzed with a wide variety of functional and morphological methods. In this study we used a new organ monitoring device that accurately determines pH and K+ activities on the liver surface with ion selective electrodes. Four groups of rats (n = 6 each) were subjected to complete, arterial or portal warm ischemia for 15 min (achieved by clamping of the hepatoduodenal ligament, the hepatic artery or the portal vein) and subsequent reperfusion. One group was sham-operated. Complete and portal ischemia were characterized by an immediate decline of pH and a more retarded rise of K+ activity on the liver surface. Arterial ischemia had almost no effect on these two parameters when compared with the sham group. Upon reperfusion the shifts of pH and K+ activity reversed towards initial baseline values. The organ monitoring system offers the option to assess ional shifts non-invasively during organ procurement, ischemia and reperfusion and may be used as an additional criterion for the estimation of organ viability. PMID- 7535880 TI - The nucleotide and deduced amino acid sequence of a rat cysteine string protein. AB - Cysteine string proteins are novel, heavily lipidated components of synaptic vesicles. They have previously been studied in Drosophila (insect) and Torpedo (fish). To facilitate further investigation of the structure and function of these proteins in mammals, we isolated and sequenced the cDNA and conducted an initial characterization of a rat cysteine string protein. Nucleotide sequencing reveals that this rat protein is highly homologous to the insect and fish cysteine string proteins. At the amino acid level, the fish and rat proteins are 82% identical. The rat cysteine string protein is encoded by an approximately 5 kb mRNA that is ubiquitously expressed in rat brain. Using antibodies that cross react with the rat protein, we find that the rat cysteine string protein is predominantly associated with nerve endings and synaptic vesicles. Moreover, like its Torpedo (fish) counterpart, it is extensively fatty acylated. It will be of considerable interest to ascertain the functional correlates of these cross species similarities of cysteine string proteins. PMID- 7535881 TI - Prefabrication of a vascularized nerve graft by vessel implantation: preliminary report of an experimental model. AB - Regeneration through vascularized nerve grafts (VNG) seems to be better than nonvascularized nerve grafts (NVNG), especially in hostile beds. We report on an experimental technique of prefabrication of VNG by direct vessel implantation. An arteriovenous fistula was created in the groin region with autologous vein grafts in the Wistar rat model, and implanted into the sciatic nerve. Five weeks later the sciatic VNG was elevated on the prefabricated pedicle. The flap was free transferred orthotopically over a silicone sheet to impede plasmatic imbibition. Flap viability at 3 days was complete. India ink injection of the AV fistula resulted in capillary ink filling within the nerve and surrounding tissues. Histologic sections of the flap were examined, revealing its neovascularity. In an ongoing study, the regeneration through this prefabricated VNG is being compared to native VNG. PMID- 7535882 TI - Effects of child age and level of developmental delay on family practice physicians' diagnostic impressions. AB - A 2 x 2 factorial design was used to examine the effects of child age (20 and 40 months) and level of developmental delay (mild and severe) on identification of developmental disorders by 155 family practice physicians. Results provide preliminary evidence that level of delay influences identification of developmental delay, with mild delay less likely to be detected. Implications in light of federal legislation as well as directions for future research were discussed. PMID- 7535883 TI - The use of photoactivatable reagents for the study of cell lineage in Drosophila embryogenesis. AB - Photoactivatable lineage tracers represent a major advance for clonal analysis in the early embryo and the study of cell movements. Any cell in the blastoderm can be marked, and the nuclear localization of the signal allows excellent resolution in identifying the daughters of individual cells. Although the technique is limited by the availability of the water-soluble caged fluorescein and its derivatives for synthesis of the complete tracer, these may become commercially available in the future. The use of caged rhodamine derivatives or antibody amplification of the signal may greatly extend the developmental period over which marked clones can be identified. PMID- 7535884 TI - Methods for quantitative analysis of transcription in larvae and prepupae. PMID- 7535886 TI - [Metal stent implantation for reopening of a gastroenterostomy in incurable stomach carcinoma]. PMID- 7535885 TI - In situ hybridization to RNA. PMID- 7535887 TI - [Successful therapy of cerebral Whipple disease with rifampicin]. PMID- 7535889 TI - Thallium increases monoamine oxidase activity and serotonin turnover rate in rat brain regions. AB - The effect of thallium acetate administration on monoaminergic pathways was studied in male Wistar rats using 30 mg/kg and 50 mg/kg acute IP doses. We found that thallium activated both monoamine oxidase (MAO) activity and serotonin turnover rate in rat brain regions, that may contribute to the neuronal damage mechanism of the agent. MAO activity in midbrain and pons was increased at both doses (at 30 mg/kg dose by 27.7% and 37%; at 50 mg/kg dose by 48% and 47%, respectively vs. control group). Serotonin turnover rate in pons was also increased at the 30 mg/kg dose (172%) while midbrain and pons serotonin turnover was increased only at the 50 mg/kg dose (56% and 166%, respectively vs. control group). Dopamine turnover rate was not significantly changed. The results indicate that thallium induced a significant increase in pons and midbrain MAO activity and also in serotonin turnover rate as compared with control animals, and this could led to behavioral and toxic alterations in the rats intoxicated with thallium. PMID- 7535891 TI - Progress toward global poliomyelitis eradication, 1985-1994. AB - In 1985, the Pan American Health Organization (PAHO) established as a goal the elimination of poliomyelitis from the Western Hemisphere by 1990; the last confirmed case of paralytic polio caused by wild poliovirus occurred in 1991 in Peru. In 1988, the World Health Assembly established the objective of global polio eradication by the year 2000. Substantial progress toward this goal has resulted from the use of four strategies recommended by the World Health Organization (WHO): 1) maintenance of high vaccination coverage levels among children with at least three doses of oral poliovirus vaccine (OPV); 2) development of sensitive systems of epidemiologic and laboratory surveillance, including use of the standard WHO case definition; 3) administration of supplementary doses of OPV to all young children (usually those aged < 5 years) during National Immunization Days (NIDs) to rapidly interrupt poliovirus transmission; and 4) "mopping-up" vaccination campaigns--localized campaigns targeted at high-risk areas where wild poliovirus transmission is most likely to persist at low levels (3). This report summarizes progress toward global polio eradication from 1985 through 1994 based on data submitted to WHO as of March 20, 1995. PMID- 7535890 TI - Neuroglial responses to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine in mouse striatum. AB - We have studied the reactive responses of both astrocytes and microglia to dopaminergic denervation of the striatum by MPTP. Following MPTP treatment, increased GFAP immunoreactivity reached a peak at 2 days and persisted for at least 6 weeks. Immunoreactivity to vimentin was also markedly increased in astrocytes 48 h after MPTP treatment. Striatal laminin immunoreactivity, however, appeared to be unaffected by drug treatment. GFAP protein levels increased to 196% and 321% of control 24 and 48 hours after MPTP treatment, respectively. Concomitantly, GFAP mRNA levels increased to 560% and 1620% of control, respectively. These reactive changes in striatal astrocytes in response to MPTP treatment were also accompanied by a reactive microglial response as evidenced by increased immunohistochemical visualization of striatal microglia using antibodies to Mac-1. Our results and those reported previously by O'Callaghan et al., strongly suggest that MPTP-induced reactive gliosis in mouse striatum is associated with reactive microglia, albeit without increased interleukin-1 beta. PMID- 7535888 TI - Phylogenetic identification and in situ detection of individual microbial cells without cultivation. AB - The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail. PMID- 7535892 TI - The calcitonin exon and its flanking intronic sequences are sufficient for the regulation of human calcitonin/calcitonin gene-related peptide alternative RNA splicing. AB - The primary transcript of the calcitonin (CT)/calcitonin gene-related peptide (CGRP) is alternatively spliced in a cell-specific fashion to produce CT in thyroid C cells and CGRP in neuronal cells. The key step in this regulatory process is the recognition and inclusion of exon 4 to produce CT mRNA or nonrecognition and exclusion of exon 4 to produce CGRP mRNA. To determine whether inclusion/exclusion of CT exon is regulated independently of its position, we created a series of minigene constructs containing decreasing amounts of CT gene sequence. A human glioblastoma cell line, T98G, was tested and used as a CT exon exclusion cell line, while HeLa cells were used as a CT exon inclusion cell line. CT exon inclusion/exclusion was regulated when either the relative position of exon 4 within the CT gene was changed or when the exon with flanking sequence was inserted into a completely heterologous gene. Our results demonstrate that CT exon functions as a unit in a position-independent fashion in regulating its own inclusion/exclusion. We believe that the heterologous fusion gene containing only exon 4 and part of its flanking intron sequences will be useful for further defining the sequence elements involved in the regulation of CT/CGRP splicing. PMID- 7535893 TI - Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. AB - The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the direct induction of proglucagon gene transcription by a cAMP-dependent pathway and suggest that the GLUTag cell line represents a useful model for the analysis of the molecular determinants of enteroendocrine gene expression. PMID- 7535895 TI - Secretion of lutropin and follitropin from transfected GH3 cells: evidence for separate secretory pathways. AB - Although lutropin (LH) and follitropin (FSH) are synthesized in the same pituitary gonadotropes, their secretion patterns in response to several experimental paradigms are not the same. Previous studies showing differences in secretion kinetics and the magnitude of hormone release by secretagogues imply differences in mechanisms for the storage and release of these hormones. To examine the secretory fate of LH and FSH, the genes encoding the common alpha subunit and the corresponding beta-subunits were transfected in rat somatotrope derived GH3 cells, which contain regulated and constitutive secretory pathways. The use of a gene transfer/heterologous cell system avoids physiological variations and functional heterogeneity of gonadotropes. Pulse-labeling and subsequent chase experiments demonstrated that although one third of newly synthesized FSH enters a regulated pathway, the majority is released constitutively. This contrasts with LH, which is mainly secreted through a regulated pathway. Although stored LH and FSH are released from GH3 cells in response to both KCl and forskolin, the magnitude of FSH release by secretagogues is smaller than that of LH. In Chinese hamster ovary cells, which are devoid of regulated secretory pathway and lack secretory granules, the mature forms of LH and FSH are neither stored nor released by secretagogues. These observations indicate that the intracellular mechanisms for the storage and release of LH and FSH differ and suggest that primary secretion of LH and FSH is via the regulated and constitutive pathways, respectively. PMID- 7535894 TI - Binding of a growth hormone-inducible nuclear factor is mediated by tyrosine phosphorylation. AB - The nuclear mechanism by which GH acts to induce gene expression after binding to its receptor on the cell surface is not defined. We have characterized an element in the 5'-flanking region of the rat GH-responsive serine protease inhibitor (Spi) 2.1 gene responsible for its induction by GH. This element binds a hepatic nuclear protein(s) in a GH state-specific manner. Activation of binding by GH does not require de novo protein synthesis, suggesting that a reversible posttranslational process is required for binding to the element. To define the mechanism of this process, hepatic nuclear extracts were analyzed by electrophoretic mobility shift assays using a DNA fragment (-147 to -103) of the Spi 2.1 gene. Treatment of extracts with phosphatases resulted in a marked reduction of GH state-specific binding. Addition of phosphatase inhibitors antagonized the reduction in binding after phosphatase treatment. The specific nature of the phosphorylation event involved in binding was explored using phosphotyrosine antibodies and a protein tyrosine phosphatase. Treatment of nuclear extracts with either of these reagents ablated binding to the response element. Because the tyrosine-phosphorylated transcription factor protein p91 has recently been implicated in cytokine signal transduction mediated by JAK2, we sought evidence that p91 was part of the GH-responsive binding complex. Analysis of an enriched preparation of GH-inducible binding complexes by Western blots using anti-p91 demonstrated no immunoreactivity. We conclude that tyrosine phosphorylation of a nuclear factor is required for GH state-specific binding to this GH response element in vivo, but that p91 is not present in the binding complex. PMID- 7535897 TI - In vivo modulation of histamine release by autoreceptors and muscarinic acetylcholine receptors in the rat anterior hypothalamus. AB - The modulation of histamine release by histamine and muscarinic acetylcholine receptors was investigated by using the push-pull technique. The anterior hypothalamic area of the conscious, freely moving rat was superfused through the push-pull cannula with CSF or with CSF containing drugs and the release of endogenous histamine was determined in the superfusate. Hypothalamic superfusion with tetrodotoxin (10 mumol/l) led to a pronounced and sustained decrease in the histamine release rate. Superfusion with compound 48/80 (100 mg/l) was ineffective. Hypothalamic superfusion with the H3 agonist (R)-alpha methylhistamine inhibited, while superfusion with the H3 antagonist thioperamide enhanced the release of histamine. The release of histamine was inhibited on hypothalamic superfusion with the muscarinic receptor agonists carbachol or oxotremorine. Histamine release was enhanced by atropine, and this release enhancing effect was abolished by oxotremorine. The selective M1 antagonist pirenzepine (100 mumol/l) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10 mumol/l), which blocks M1 and M3 receptors, also enhanced the release rate of histamine. On the other hand, 50 and 100 mumol/l methoctramine (M2 receptor antagonist) 10 and 100 mumol/l p-fluoro-hexahydro-siladifenidol (p-F-HHSiD, a M3 receptor antagonist) were ineffective. It is concluded tht histamine released in the hypothalamus originates predominantly from neurons. The release of histamine is modulated by H3 autoreceptors. The histamine release is also modulated by cholinergic neurons which modify histamine release from histaminergic neurons by stimulating M1 muscarinic acetylcholine heteroreceptors probably located on histaminergic neurons. PMID- 7535896 TI - Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle. AB - The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [3H]inositol phosphate ([3H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [3H]Ins labelled BTSM slices from 37 degrees C to 27 degrees C for 20 min prior to the addition of agonist caused a substantial (73.0 +/- 2.5%) inhibition of carbachol (100 microM, 30 min)-stimulated [3H]InsP accumulation compared to values measured at 37 degrees C. The degree of inhibition of [3H]InsP accumulation was similar at all agonist time points (2-30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37 degrees C to 27 degrees C resulted in a 34% reduction in basal Ins(1,4,5)P3 mass (37 degrees C, 13.1 +/- 0.6 pmol mg-1 protein; 27 degrees C, 8.9 +/- 0.9 pmol mg 1 protein; P < 0.02) and markedly attenuated carbachol (100 microM)-stimulated increases in Ins(1,4,5)P3 accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [3H]phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) /deoxycholate assay system, was also significantly lower at 27 degrees C compared to 37 degrees C (initial velocities of PtdIns(4,5)P2 hydrolysis of 853 +/- 167 (37 degrees C) and 418 +/- 119 (27 degrees C) pmol min-1 ml-1 (1/400 diluted) BTSM cytosol; p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535898 TI - Inhibition by ethanol of excitatory amino acid receptors and nicotinic acetylcholine receptors at rat locus coeruleus neurons. AB - The frequency of spontaneous action potentials of locus coeruleus (LC) neurons was recorded extracellularly in pontine slices of the rat brain. Ethanol (1-100 mM) elevated the firing rate in most neurons; this effect was concentration dependent. (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 0.03-1 microM), kainate (0.1-3 microM), N-methyl-D-aspartate (NMDA; 1-30 microM), substance P (0.01-1 microM), nicotine (0.1-10 microM) and alpha,beta-methylene ATP (alpha,beta-meATP; 0.3-30 microM), all increased the firing. Application of ethanol (10-100 mM) to the superfusion medium for 10 min, reproducibly and concentration-dependently inhibited the facilitatory effect of NMDA (10 microM). However, the inhibitory effect of ethanol (100 mM) decreased during a 30-min superfusion period and after the wash-out of ethanol the sensitivity of LC neurons to NMDA (10 microM) tended to overshoot above their initial level. Although NMDA was more potent in the absence than in the presence of external Mg2+, ethanol (100 mM) continued to depress the facilitatory effect of a low concentration of NMDA (3 microM) in a Mg(2+)-free medium. By contrast, in a medium containing normal Mg2+, ethanol (100 mM) failed to significantly interfere with the increase in firing rate induced by a high concentration of NMDA (30 microM). The effects of kainate (0.5 microM), AMPA (0.3 microM) and nicotine (1 microM) were also depressed by ethanol (100 mM), while the effects of substance P (0.03 microM) and alpha,beta-meATP (30 microM) were not changed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535900 TI - The association of urinary 5-hydroxyindoleacetic acid and vanillylmandelic acid in patients with generalized anxiety. AB - There is evidence that serotonin and norepinephrine are in some way involved in the pathophysiology of anxiety disorders. Urinary levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the norepinephrine metabolite vanillylmandelic acid (VMA) were measured in 46 patients with generalized anxiety disorder. There was a significant association between urinary levels of 5-HIAA and VMA: r = 0.79; p = 0.0001. Possible implications of this finding are discussed. PMID- 7535899 TI - Ramipril prevents the detrimental sequels of chronic NO synthase inhibition in rats: hypertension, cardiac hypertrophy and renal insufficiency. AB - Inhibition of the angiotensin converting enzyme (ACE) with ramipril was studied in male Wistar rats during long-term inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME). Chronic treatment with L-NAME in a dose of 25 mg/kg per day over 6 weeks caused myocardial hypertrophy and a significant increase in systolic blood pressure (245 +/- 16 mmHg) as compared to controls (155 +/- 4 mmHg). Animals receiving simultaneously L-NAME and ramipril were protected against blood pressure increase and partially against myocardial hypertrophy. L-NAME caused a significant reduction in glomerular filtration rate (GFR: 2.56 +/- 0.73 ml.kg-1.min-1) and renal plasma flow (RPF: 6.93 +/- 1.70 ml.kg-1.min-1) as compared to control (GFR: 7.29 +/- 0.69, RPF: 21.36 +/- 2.33 ml.kg-1.min-1). Addition of ramipril prevented L-NAME-induced reduction in GFR and renal plasma flow. L-NAME produced an elevation in urinary protein excretion and serum creatinine and a decrease in potassium excretion which was antagonised by ramipril. L-NAME-induced increase in plasma renin activity (PRA) was further elevated with ramipril treatment. Isolated hearts from rats treated with L-NAME showed increased post-ischaemic reperfusion injuries. Compared to controls duration of ventricular fibrillation was increased and coronary flow reduced. During ischemia the cytosolic enzymes lactate dehydrogenase and creatine kinase, as well as lactate in the venous effluent were increased. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535901 TI - Calcium loading of secretory granules in stimulated neurohypophysial nerve endings. AB - The total calcium content of secretory granules, Cag, was evaluated in isolated neurohypophysial nerve endings. The Cag in the resting state, as measured by X ray microanalysis, is relatively high with an average of 7.4 +/- 0.6 mmol/kg wet weight. Following a depolarizing potassium challenge, a subpopulation of granules with even higher Cag could be detected, dispersed over a wider range of concentrations (up to 70 mmol/kg wet weight). After subsequent rinsing in physiological saline, Cag decreased to control values. This could have resulted from Ca2+ extrusion, or from preferential secretion of calcium-enriched granules. Our data can be interpreted in favor of the second explanation since no decrease in Cag was observed when secretion was blocked by a hyperosmotic saline. The effect of hyperosmotic conditions on isolated nerve endings was further studied by monitoring free cytoplasmic Ca2+ with the calcium-sensitive dye Fura-2 and by conventional electron microscopy. It was demonstrated that hyperosmotic treatment alone did not increase basal cytosolic Ca2+ concentrations but did significantly reduce the potassium-induced cytosolic rise in Ca2+. Electron microscopy of nerve endings in hyperosmotic conditions showed numerous exocytotic figures at various stages. The observed changes in Cag are in accord with a published hypothesis which proposes that intragranular calcium is a significant variable in regulated secretion. PMID- 7535903 TI - Granulocyte-colony-stimulating factor in patients treated by continuous ambulatory peritoneal dialysis. PMID- 7535902 TI - Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states. AB - Microglia were enriched in brain cell cultures from newborn mice as a result of supplementation with the growth factors macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor. When separately administered these two cytokines promote the outgrowth of loosely adherent cells with similar morphology which stained positive for CD11b and nonspecific esterase. Microglial cells isolated from both types of culture were electrophysiologically characterized using the whole cell configuration of the patch-clamp technique. Different resting membrane potentials were measured. In response to hyperpolarizing and depolarizing voltage commands 68 of 91 macrophage colony stimulating factor-cultured microglial cells exhibited only an inward rectifying potassium current. By contrast, an outward potassium current was observed on 71 of 95 granulocyte/macrophage colony-stimulating factor-grown cells. Parallel testing of their capability for antigen presentation proved the activated functional state of these microglial cells. They induce antigen-specific T cell response without prior stimulus. In comparison, cells developed with macrophage colony-stimulating factor failed to present antigen. In such resting microglia a short-term treatment with granulocyte/macrophage colony-stimulating factor or interferon-gamma provoked a strong appearance of outward potassium currents, however, only the interferon-gamma-trigger resulted in efficient antigen presentation. The differential induction of both functional parameters suggests the detection of outward potassium currents to provide an electrophysiological activation marker of microglia which is subjected to cytokine regulation but not compellingly linked to antigen presentation. PMID- 7535904 TI - Ipsilateral connections of the anterior cingulate cortex with the frontal and medial temporal cortices in the macaque monkey. AB - The present study was attempted to study ipsilateral corticocortical connections of the anterior part (area 24) of the cingulate cortex of the macaque monkey by means of wheat germ agglutinin-conjugated peroxidase (WGA-HRP) method. In 2 out of 4 Japanese monkeys (Macaca fuscata) that were injected with WGA-HRP into the anterior part of the cingulate cortex, the sites of injection were successfully localized within the cortical regions corresponding to areas 24a and 24b. The results obtained from these monkeys indicate that areas 24a and 24b in the anterior part of the cingulate cortex are reciprocally connected with the prefrontal, premotor, and motor cortical regions, and also with the medial temporal cortical regions. Areas 24a and 24b were strongly connected with the lateral and medial prefrontal cortices and area 6a beta of the premotor cortex, moderately with the remaining premotor cortex, and weakly with the motor cortex. In the medial temporal cortex, areas 24a and 24b were strongly connected with the prosubiculum, entorhinal cortex (area 28), and perirhinal cortex (areas 35 and 36), and weakly with areas TF and TH of the parahippocampal gyrus, throughout their rostrocaudal extent. In addition, areas 24a and 24b projected to the molecular layer of the CAI subfield of Ammon's horn and the external pyramidal layer of the presubiculum. Our findings suggest that areas 24a and 24b of the anterior cingulate cortex may constitute relays in the reciprocal pathways between the prefrontal cortex and the hippocampal, entorhinal and/or perirhinal cortical regions. PMID- 7535907 TI - [Epidermal keratinocyte proliferation, differentiation and apoptosis in rats in postnatal ontogeny]. AB - Quantitative ratios between reproduction of the keratinocytes and their apoptotic death vary in different periods of postnatal ontogenesis. During the first month of life of the rats the mitotic activity of keratinocytes and thickness of the epidermis gradually decrease. The epidermis undergoes further development, which terminates by the 30th day after birth. The number of apoptotic cells in the epidermis increases from the 1st until 16th day and that of intraepidermal lymphocytes from the 1st until 30th day. In 3-month old animals the peak of mitotic activity coincided with the enhanced apoptotic death of the keratinocytes. The anagen signs were noted in such animals. In the old rats the mitotic activity of the epidermal cells was markedly reduced with the increased rate of apoptosis of the keratinocytes and the decreased thickness of epidermis. Single Merkel cells were visualized in the epidermis using the silver plating technique. Hence, their role in apoptosis of the keratinocytes cannot be significant. The possibility of lymphocyte-associated apoptosis of the keratinocytes in the early postnatal ontogenesis of rats is assumed. PMID- 7535905 TI - Site of origin of projections from the thalamus to dorsal versus ventral aspects of the premotor cortex of monkeys. AB - Retrograde tracers were injected into the forelimb regions of three cortical motor areas: (1) a dorsal aspect of the premotor cortex (PMd) immediately lateral to the superior precentral sulcus; (2) a ventral aspect of the premotor cortex (PMv) immediately caudal to the genu of the arcuate sulcus and lateral to the arcuate spur; and (3) the primary motor cortex (MI). Before tracer injection, single-unit recordings were made to select injection sites in the forelimb regions where neurons with set- and/or movement-related activity before forelimb movements were densely located. Following the PMd injections, labeled cells were found mainly in rostral portion of VLc and VLo. Cells projecting to PMv were found mainly in X and VPLo. Projection cells to the MI were found in VPLo, VLc, and VLo. Locations of neurons projecting to different motor areas were not overlapped in the thalamic nuclei. Combining available reports, the results suggest that major inputs to PMd come from globus pallidus and that, in contrast, cerebellum is a main source to the PMv. The differential inputs to PMd and PMv may contribute to their functional specialization. PMID- 7535906 TI - Presynaptic ionotropic glutamate receptors modulate in vivo release and metabolism of striatal dopamine, noradrenaline, and 5-hydroxytryptamine: involvement of both NMDA and AMPA/kainate subtypes. AB - In order to explore further the presynaptic modulation of monoamine release by glutamatergic nerve fibers, we investigated the effects of selective agonists for ionotropic glutamate (GLU) receptors on striatal release of dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). In the striatum of anesthetized Sprague-Dawley rats, in vivo microdialysis was performed to measure the release of monoamines and metabolities, and also to administer GLU agonists locally in the tissue. L-GLU and its selective agonists (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate (KA)) evoked simultaneous release of striatal DA, NA and 5-HT in a dose dependent manner. Pretreatment with MK-801 (5 mg/kg i.p.), a noncompetitive NMDA receptor antagonist, selectively suppressed NMDA-evoked monoamine release. The rank order of GLU agonist efficacy in releasing monoamines was different among DA, NA, and 5-HTergic terminals: AMPA = KA > NMDA for DA release, AMPA > NMDA = KA for NA release, and NMDA = AMPA = KA for 5-HT release. In conclusion, presynaptic ionotropic GLU receptors exist extensively on monoaminergic terminals including not only catecholaminergic (DA and NA) but also indoleaminergic (5-HT) terminals in the rat striatum. Their subtypes include both NMDA subtype and AMPA/KA subtype, and show a differential distribution among these three monoaminergic terminals and a differential contribution to facilitating monoamine release. PMID- 7535908 TI - [Local transcatheter arterial chemoembolization in the palliative treatment of inoperable hepatocellular carcinoma]. AB - Hepatocellular carcinoma is a tumor with high mortality. Adequate oncological therapy is essential to modify the poor prognosis. Transcatheter arterial chemoembolisation has been proposed as a useful and well-tolerated treatment for unresectable carcinoma. In the study 51 patients with unresectable carcinoma (mean age 61.6, range 45-81, Child-Pugh A = 34 patients, Child-B = 13, Child-C = 4; Okuda I = 33 patients, Okuda II = 18) underwent chemoembolisation. A total of 122 procedure were performed, with a median number of 2.4 (range 1-6) per patient. One and two year survivals are 91% and 74% respectively (Child-A: 100% and 82%; Child-B: 100% and 63%; Child-C 0% at 1 year). The difference among the 3 groups is statistically significant (p = 0.001). Median overall survival is 20 months, with 22, 20 and 6 month in Child-A, B and C patients respectively (p = 0.006). Commonly reported side effects and biochemical changes included: fever, pain and increased serum amylase, transaminase levels. One patient developed a liver abscess and died of liver failure. In addition, in 18 patients (35%) mild to severe changes in glucose metabolism were also observed. Mild hyperglycemia was observed in 14 patients, with severe derangement in 4 patients (8%). It is suggested that careful evaluation of glucose metabolism is advisable in patients being considered for chemoembolisation. Their results confirm the usefulness of chemoembolisation in Child-A and B patients with unresectable hepatocellular carcinoma. PMID- 7535909 TI - Comparative DNA analysis of breast cancer by flow cytometry and image analysis. AB - Measurement of DNA ploidy can be performed either with Flow Cytometry (FCM) and Image-Analysis (IA); both methods provide prognostic information in primary breast cancer. We compared the results of quantitative DNA analysis of formalin fixed, paraffin embedded tissue from 62 invasive ductal breast cancers. For FCM nuclear suspensions from disaggregated tumor were stained with Propidium Iodide and analyzed by means of Ortho Cytoron Absolute. For IA nuclear suspensions were stained by the Feulgen method and analyzed by means of Vidas system. We found a good correlation between flow cytometry DNA Index and Histogram Type, according to Auer classification (rs = 0.65, p < 0.001) and between DNA Index and Grading of Malignancy (MG) which had been measured by Bocking's algorithm (rs = 0.38, p < 0.05). Concerning to disease free survival (DFS), flow cytometric DNA Index showed a better correlation (rs = 0.56, p < 0.001). We concluded that the two methods provide comparable results, but offer individual advantages and are complementary for analyzing DNA ploidy in breast cancer. PMID- 7535910 TI - [Cytometry of ependymomas using Feulgen-positive DNA analysis]. AB - Ependymomas are neoplasms which, on the one hand, supply useful morphological findings that help to make the diagnosis, and, on the other, do not provide certain elements for a reliable prognosis. The difficulties related to the neoplasm topography, the type of surgery and other clinical-operational problems can be overcome, whereas those arising from the limitations of descriptive morphology remain insurmountable since the latter does not supply precise predictive information concerning such a histotype. The Authors have re-examined the issue by studying 31 cases of benign ependymomas and by applying to them static cytometry on Feulgen stained samples. The results obtained (see the histograms enclosed) have revealed the presence of heterogeneous cell clones with polyploid and hyperploid cell populations. These cell populations, though a minority and often also marginal, represent the starting point not only of real recurrence, but also of malignant transformations. The cytometric method has analytically revealed a composite cytologic reality of which the common histological methods can only provide approximate, unreliable and subjective evaluations. PMID- 7535911 TI - Frequency of Candida sp infection in tuberculous patients with acquired immunodeficiency syndrome: morphological and immunocytochemical study in sputum. AB - Sputum samples from AIDS patients with and without pulmonary tuberculosis were analyzed morphologically and immunocytochemically to determine the frequency of occurrence of Candida sp. Mycobacterial infection was detected by bacterioscopy and/or culture and cytological evaluation was performed using Papanicolaou and Toluidine Blue staining. Immunoreaction for Candida sp was performed using polyclonal antibody in selected cases with fungal structures in smears stained by the Papanicolaou or Toluidine Blue method. An increased frequency of Candida sp (2.5 times) was observed in the tuberculous group compared to the group of AIDS patients without tuberculosis. The Toluidine Blue stain showed good results for the detection of Candida sp in sputum. Due to the increased risk of this opportunistic infection among more severely immunocompromised patients. Toluidine Blue staining of sputum samples submitted to analysis seems to be a reliable screening method. PMID- 7535912 TI - [Bone marrow granular-cell tumor with histological and immunohistochemical analysis: 1st case in the literature]. AB - Here is described a rare case of a tumour with granular cells, placed in the bone marrow (radius bone) which caused a spontaneous fracture. The tumour has answered positivity to the immuno-histochemical SP 100 and NSE tests. Besides, wide focusing of moderate dysplasia of the granular cells were found, which answered partly in histochemical tests NSE and PS 100. There has been analysed the literature on multi-visceral incidence of this tumoral histotype and on its histogenesis. On the basis of the heterogeneous nature of this tumor, it was thought the name of tumour with granular cells to be more appropriate than myoblastoma. PMID- 7535913 TI - [Technico-scientific bases of the cardiac nervous and conduction system in relation to the problem of sudden cardiac death]. PMID- 7535914 TI - [Histological and histochemical techniques for serial sectioning and staining the cardiac nervous and conduction system]. PMID- 7535915 TI - Pancreatic enzyme therapy in patients with cystic fibrosis: the high dose lipase issue. PMID- 7535917 TI - Differential anesthetic-induced opening of calcium-dependent large conductance channels in isolated ventricular myocytes. AB - Under conditions of low Ca buffering of the pipette intracellular dialyzing solution, the opening of Ca dependent large conductance (approximately 310 pS) channels (LCCs) was observed in isolated ventricular myocytes using the whole cell patch clamp technique. With Na-Ca exchange current (INaCa) suppressed by elimination of intracellular and extracellular Na, sustained LCC activity, which is markedly enhanced by caffeine-stimulated Ca release from the sarcoplasmic reticulum (SR), was increased by application of the inhalational anesthetic halothane, but not isoflurane. Halothane (0.90 mM in solution) reversibly increased the frequency of LCC openings, fo, by a factor of approximately 30 with a concurrent rise in the observed probability of opening, NPo, by a factor of approximately 50. The effect of halothane on LCC activation was suppressed by either strong Ca buffering in the whole-cell pipette solution or pretreatment of the myocytes with ryanodine (10 microM) to decrease SR Ca release. In the presence of intracellular and extracellular Na, a transient inward current was evoked by application of caffeine (5 mM) or halothane (1.80 mM) suggesting that Ca release from the SR by either agent can activate INaCa. Our findings are consistent with the notion that halothane, in contrast to isoflurane, causes SR release of Ca. The eventual depletion of SR activator Ca may account, at least in part, for the differential effects of these anesthetics on myocardial tension development. PMID- 7535916 TI - cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus. AB - Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage clamp method, in vitro. Inward rectifier current (IH) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately -60 mV in nominally zero-Ca2+ Krebs solution containing tetrodotoxin (1 microM), tetraethylammonium (40 mM), Cd2+ (500 microM) and 4 aminopyridine (1 mM). IH developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time. IH was selectively blocked by extracellular Cs+ (1 mM). The activation of the H-channel conductance (GH) ranged between -55 and -120 mV. The GH was 80-150 pS (n = 4) at the half-activation voltage of -84 +/- 7 mV (n = 4). The reversal potential of IH obtained by instantaneous current voltage (I/V) relations was -41 +/- 6 mV (n = 4); it shifted to -51 +/- 8 mV (n = 3) in low-Na+ (20 mM) solution and to -24 +/- 4 mV (n = 4) in high-K+ (20 mM) solution. Forskolin (1-10 microM) produced an inward current and increased the amplitude of IH. Forskolin did not change the half-activation voltage of GH. 8-Bromo-adenosine 3',5'-cyclic monophosphate (8-Br cAMP, 0.1-1 mM) and dibutyryl-cAMP (0.1-1 mM) enhanced IH. 3-Isobutyl-1 methylxanthine (IBMX, 1 mM) also enhanced IH. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN. PMID- 7535918 TI - Characterization of a transient outward current in a rapidly adapting insect mechanosensory neuron. AB - This paper describes the first voltage-clamp recordings from an arthropod cuticular sensory neuron. In the femoral tactile spine neuron of the cockroach Periplaneta americana, a rapidly activating and inactivating outward current, IA, appeared when the neuron was hyperpolarized for a short period before a depolarizing test pulse. IA could be separated from the other outward currents using 5 mM 4-aminopyridine (4-AP), which specifically blocked it. Tetraethylammonium (TEA), (50 mM) did not remove IA, but decreased the steady state outward current by about 50%. The threshold for IA activation was about -75 mV. The minimum activation and inactivation time constants were approximately 0.2 ms and 15 ms, respectively. The voltage dependencies of activation and inactivation were well fit-ted by Boltzmann distributions, giving values of membrane potential at half-maximal activation (V50) equal to -56.5 mV and an equivalent gating charge of n = 3.9 for activation and V50 = -86.7 mV and n = 3.4 for inactivation. In current-clamp recordings, 4-AP reversibly reduced the cell's normal adaptation by lowering the threshold for action potentials, but did not affect the amplitude or duration of single action potentials. These results indicate that IA plays a role in short-term adaptation by opposing membrane depolarization and reducing the spike frequency during maintained stimulation. PMID- 7535919 TI - A ubiquitous non-selective cation channel in the mouse renal tubule with variable sensitivity to calcium. AB - Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K(+)-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5 +/- 3.1 pS and 17.3 +/- 1.8 pS, respectively (n = 4). In contrast, cell attached patches were usually inactive when a Na(+)-rich solution filled the patch pipette. However, another type of channel with a conductance of 20-30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1 +/- 1.2 pS (n = 17), excluded anions (PCl/PNa = 0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa = 1.5; PK/PNa = 0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa = 0.09; PBa/PNa approximately 0). The cation channel was moderately voltage-dependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 mumol/l-0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition of Po at 1.2 mumol/l AMP. As in other cell models, 3',5' dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another. PMID- 7535920 TI - Volume-activated, gadolinium-sensitive whole-cell currents in single proximal cells of frog kidney. AB - Stretch-activated channels (SACs) have been implicated in the control of epithelial cell volume. Such channels are generally sensitive to the trivalent lanthanide, gadolinium (Gd3+). In this study, using Gd3+ sensitivity and volume activation as indices, we have looked for ionic currents attributable to SACs using the whole-cell-patch clamp technique in freshly isolated proximal tubule cells of the frog. Hypotonic shock caused a reversible increase in whole-cell conductance, which was inhibited by Gd3+. In conjunction with this increase in conductance, cell length (measured using an optical technique) also increased. We observed two types of volume- and Gd(3+)-sensitive currents: voltage-dependent IVD and voltage-independent IVI. IVD was found in all cells, activated by depolarisation and hypotonic shock, and was inhibited reversibly by 10 microM Gd3+. The conductance did not discriminate between Na+ and K+ but was slightly anion-selective and was Ca(2+)-permeable. IVI was observed in only 50% of cells and was also inhibited by Gd3+. Although the inhibition was irreversible, it was dose-dependent, suggesting a specific effect of Gd3+ on IVI. Cells that showed IVI had a significantly higher conductance than those that did not (38.7 +/- 4.4, n = 20, and 20.5 +/- 0.7, n = 15, microS.cm-2 respectively). In contrast to IVD, IVI was mildly cation-selective, Ca(2+)-permeable, and also selective for Na+ over K+. As with IVD, volume-induced increases in IVI were inhibited by Gd3+. Both of these currents are activated during hypotonic shock and may be involved in volume-regulatory processes in frog proximal cells. PMID- 7535921 TI - Multiple RNA binding domains (RBDs) just don't add up. AB - One of the most common motifs for binding RNA in eukaryotes is the RNA binding domain (RBD) or RNA Recognition Motif (RRM). One of the more intriguing aspects of these proteins is their modular nature. Proteins have been found containing from one to four RRMs. In most instances, these domains have some basal level of non-sequence specific RNA binding affinity. In addition, many also have a higher affinity for a specific structure or sequence of RNA. In the cases of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), yeast poly-A binding protein and splicing factor U2AF65, the individual free energy of binding of the RBDs for RNA are not strictly additive. By invoking a model in which the amino acids connecting adjoining RBDs are considered to be flexible linkers with an interresidue spacing of about 3.5 A, it is possible to predict the apparent association constants for at least some multi-RBD proteins to single-stranded RNA. We have surveyed the literature and found that individual RBDs are separated by 'linker' sequences of highly variable length. These linkers provide a critical determinant of binding affinity and may modulate cis versus trans binding. A clearer understanding of multi-RBD binding is essential to critically evaluating the role of these proteins in RNA splicing, packaging and transport. PMID- 7535922 TI - The large subunit of HIV-1 reverse transcriptase interacts with beta-actin. AB - HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV 1 virions. PMID- 7535924 TI - Surgical clinical nurse specialist facilitates discharge of patients undergoing breast surgery. PMID- 7535923 TI - Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1. AB - We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function. PMID- 7535925 TI - Pancreatic digestive enzyme secretion in the rabbit: rapid cyclic variations in enzyme composition. AB - The role and mechanism of nonparallel pancreatic secretion of digestive enzymes, in which enzyme proportions change in rapidly regulated fashion, remain controversial. Secretion was collected from male 2.2-kg New Zealand rabbits in 5 min intervals for 3 h under basal conditions or constant stimulation with cholecystokinin (CCK; 0.1 microgram per kg per h i.v.) or methacholine chloride (MCh; 40 micrograms per kg per h i.v.). Both CCK and MCh produced an 8-fold stimulation of protein output. Enzymes were separated by SDS/PAGE and quantitated by densitometry of Coomassie blue-stained gels. Under both basal conditions and constant MCh infusion, rapid neurosecretory-like 12-min cyclic changes occurred in the proportions of amylase, lipase I, chymotrypsinogen, and trypsinogen. During constant infusion their percentages changed as much as 10-fold, and their ratios cycled by as much as 30-fold. The mean percentage for the entire infusion period for lipase I declined > 25% with CCK or MCh, for amylase it rose approximately 30%, and for chymotrypsinogen and trypsinogen it doubled (for all, P < 0.05). CCK and MCh elicited subtly but significantly different mean enzyme percentages and enzyme ratios (P < 0.05) for amylase, chymotrypsinogen, and trypsinogen; these differences were also confirmed by regression and correlation analyses. The changes in enzyme percentages and ratios were explicitly consistent with secretagogue-caused shifts in the intrapancreatic enzyme secretory sources. Nonparallel secretion of digestive enzymes occurs routinely, even during constant stimulation, and is due to cyclic neurosecretory-like secretion from heterogeneous intrapancreatic sources. PMID- 7535926 TI - Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways. AB - We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C. PMID- 7535927 TI - Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression. AB - Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression. PMID- 7535930 TI - Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3' dideoxycytidine 5'-triphosphate and other drugs. AB - A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the pp6/p51 heterodimer of wild-type RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays. PMID- 7535929 TI - Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity. AB - Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain. PMID- 7535931 TI - Opposite effects of transforming growth factor alpha and epidermal growth factor on mouse placental lactogen I secretion. AB - This study was undertaken to determine whether transforming growth factor alpha (TGF-alpha) regulates the production of mouse placental lactogen I (mPL-I) and mPL-II in a manner that is similar to that of epidermal growth factor (EGF), which was previously shown to stimulate mPL-I secretion and inhibit mPL-II secretion. In contrast to the activity of EGF, human (h) and rat (r) TGF-alpha (each at 100 ng/ml) inhibited secretion of mPL-I by placental cells isolated from mice on day 7 of pregnancy. Maximum inhibition of mPL-I secretion occurred on the third day of a 5-day culture period and ranged between 37% and 56% in multiple trials. Incubation of cells with hTGF-alpha and EGF was not followed by a change in the mPL-I concentration of the medium, suggesting the peptides antagonized each other's effects. hTGF-alpha and rTGF-alpha inhibited secretion of mPL-II; maximum inhibition ranged between 62% and 84% in multiple trials. The lowest concentrations of hTGF-alpha that affected mPL-I and mPL-II secretion were 10 ng/ml and 1 ng/ml, respectively. EGF and hTGF-alpha bound to the same receptors on placental cells, as assessed by cross-linking, and both peptides stimulated receptor phosphorylation, as assessed by Western blot analysis. There are three types of mPL-containing cells in placental cultures: cells that contain only mPL I, cells that contain only mPL-II, and cells that contain both mPLs. The percentage of each type of mPL-containing cell in the culture was determined by immunostaining. hTGF-alpha affected the differentiation of the subpopulations of PL-containing cells in a manner that differed from that of EGF. The data suggest that TGF-alpha and EGF do not regulate the production of mPL-I and mPL-II in a similar manner. PMID- 7535928 TI - Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo. AB - Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR- i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo. PMID- 7535932 TI - gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells. AB - gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation. PMID- 7535933 TI - Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms. AB - Nitric oxide (NO) produces rapid osteoclast detachment and contraction in vitro, and this effect is accompanied by a profound inhibition of bone resorption. Work by others has confirmed these findings in vivo: inhibition of NO synthase [NOS; L arginine, NADPH: oxygen oxidoreductase (NO-forming), EC 1.14.13.39] in normal rats is followed by increased bone resorption reflected by a marked loss in bone mineral density. In our present study, immunocytochemistry and Northern blotting show the presence of the constitutive calcium-sensitive NOS isoform (cNOS) in normal rat osteoclasts and in the human preosteoclast cell line (FLG 29.1). The inducible NOS isoform (iNOS) was also clearly demonstrable in the rat cells especially after treatment with gamma interferon (IFN-gamma) and bacterial wall products [lipopolysaccharide (LPS)], while a basal level of transcript was detected in the untreated human preosteoclast line. However NADPH-diaphorase activity was intense only in neonatal rat osteoclasts attached to bone, perhaps reflecting either enhancement of cNOS activity by calcium or increased amounts of the inducible isoform in activated osteoclasts in situ compared with isolated neonatal rat osteoclasts. These actively resorb devitalized bone but the untreated cells contain relatively low levels of NOS; they are extremely sensitive to inhibition by NO. The iNOS inhibitor aminoguanidine markedly enhances in vitro resorption by activated NOS-rich chick osteoclasts and by normal rat osteoclasts treated with LPS or IFN-gamma. In contrast, the nonselective NOS inhibitor NG-monomethyl-L-arginine inhibits resorption by untreated neonatal rat osteoclasts. Thus, osteoclast function may require intermittent calcium-stimulated increases in NO production by cNOS against a basal inhibitory background activity of the iNOS isoform. However, bone resorption depends on precursor replication and on the activity of the mature cells, and we found that the NO donor 3-morpholinosydnonimine (SIN-1) (50 microM) profoundly depressed replication in the human preosteoclast line. Taken together, these results strongly suggest that NO maintains a central control of bone resorption in both avian and mammalian species by exerting a powerful tonic restraint of osteoclast numbers and activity. The presence of NOS in human cells implies a similar function in man and that conventional views of calcium homoeostasis and skeletal metabolism will need substantial revision. Since NO also influences behavior of the osteoblast, the bone-forming cell, in vitro, a similar effect in vivo might imply a general influence on bone remodeling. PMID- 7535934 TI - The effects and molar potency of iloprost, U46619 and sodium nitroprusside on capsular and vascular smooth muscle of the isolated perfused canine spleen. AB - The isolated canine spleen was perfused at constant flow with continuous recording of splenic arterial perfusion pressure (SAPP) and spleen weight. Intra arterial injections of the thromboxane A2 (TXA2) mimetic U46619 caused dose related increases in splenic arterial perfusion pressure (SAPP) of short duration (ED50 0.31 nmol). There were very small changes in spleen weight accompanying any of the vasoconstrictor responses to U46619. The stable analogue of prostacyclin, iloprost, caused dose-dependent reductions in SAPP (ED50 1.3 nmol) indicating vasodilation. There were no changes in spleen weight to any doses of iloprost indicating a lack of action on capsular smooth muscle. Similarly, the nitric oxide (NO) mimetic sodium nitroprusside caused dose-related reductions in SAPP of short duration (ED50 5.8 nmol). No changes in spleen weight accompanied splenic vasodilator responses to any dose of sodium nitroprusside (SNP). The results indicate the potential actions and intrinsic potency of three endogenous vasoactive substances and provide information about their relative roles in the control of the splenic microcirculation in situations when they are released. PMID- 7535936 TI - Iloprost enhances survival of axial-pattern skin flaps in an ischemia-reperfusion model. AB - Iloprost, a stable prostacyclin analogue, significantly enhances the survival of an axial-pattern groin flap in rats after ischemia-reperfusion. Treated flaps showed viability over 90 percent of their area 7 days after an 8-hour ischemic episode, whereas the saline-treated controls were viable over only 20 percent of their area (p = 0.002). This effect was seen at a low (10 ng/kg) dosage, and no apparent side effects were noted. PMID- 7535935 TI - Omega-3 and omega-6 fatty acids and PGE2 stimulate the growth of normal but not tumor mouse mammary epithelial cells: evidence for alterations in the signaling pathways in tumor cells. AB - The direct effect of omega-3 and omega-6 fatty acids on the proliferation of mouse mammary tumor cells (MTC) was examined in a serum-free cell culture system. While the EGF-induced proliferation of normal mammary epithelial cells was shown to be enhanced by omega-3 and omega-6 fatty acids and prostaglandins (PGs), a majority (75-80%) of primary mammary tumors were not stimulated by these agents. Compared to normal cells, some MTC cultures showed a higher susceptibility to inhibition by omega-3 fatty acids. The general lack of response of MTC cultures to PGE2 and cyclic adenosine monophosphate (cAMP) suggests some alterations in the cAMP-mediated pathway. However, the PGE2-induced cAMP levels and cAMP dependent protein kinase (PKA) activities in the tumor cells were comparable to normal cells. We conclude that the proliferation of mammary tumor cells either follow a cAMP-PKA-independent pathway or have some alterations in the serine/threonine kinase mediated signaling pathway. PMID- 7535937 TI - Comparison of desmethylsertraline with sertraline as a monoamine uptake inhibitor in vivo. AB - 1. Desmethylsertraline, a metabolite of the antidepressant drug sertraline, was compared with sertraline for its ability to produce effects characteristic of inhibitors of the serotonin transporter in vivo. Desmethylsertraline antagonized brain serotonin depletion by p-chloroamphetamine, a depletion dependent upon the serotonin transporter, being less potent than sertraline in rats but almost as potent as sertraline in mice. Desmethylsertraline was a weak antagonist of 6 hydroxydopamine-induced depletion of heart norepinephrine in mice; sertraline had no effect at the doses studied. 2. Desmethylsertraline decreased brain concentrations of 5-hydroxyindoleacetic acid (5HIAA) in rats as did sertraline, the duration of the effect after both drugs being at least 24 hrs but less than 48 hrs. 3. After sertraline injection, desmethylsertraline was present in rat brain at higher concentrations than the parent drug at 8 hrs and thereafter. 4. In rats, repeated injections of sertraline, at doses previously shown to diminish beta-adrenergic receptor-mediated responses, led to marked accumulation of desmethylsertraline in brain and to inhibition of the catecholamine transporters. 5. In mice, brain concentrations of desmethylsertraline were higher than those of parent drug within 7 hrs after sertraline injection and probably contributed importantly to the antagonism of p-chloroamphetamine effects. 6. These data show that desmethylsertraline is less potent than sertraline as a serotonin uptake inhibitor in vivo, as the in vitro data would have predicted, but that desmethylsertraline may nonetheless contribute to the prolonged inhibition of the serotonin transporter after sertraline administration, perhaps more in mice than in rats. PMID- 7535941 TI - Guidelines for the presentation of numerical tables. PMID- 7535938 TI - The utility of salivary amylase as an evaluation of M3 muscarinic agonist activity in Alzheimer's disease. AB - 1. In this double-blind, placebo-controlled phase I study of the safety/tolerance of two doses of xanomeline tartrate (100 mg and 115 mg tid) given to 12 AD patients, the authors measured serum amylase, fractionated into pancreatic and salivary isoenzymes, as a potential marker for M3 activity associated with maximally tolerated dose (MTD). 2. MTD of xanomeline was determined to be 100 mg tid based on intolerable adverse events at 115 mg tid. One patient at the 115 mg tid level presented with moderate hypersalivation and salivary amylase levels 400% of baseline. 3. Overall amylase results were not significant, however a trend in the results for salivary amylase in the 115 mg panel suggests that salivary amylase may be a useful marker for M3 activity. PMID- 7535940 TI - [Distributors of poisons]. PMID- 7535939 TI - Hypofractionated radiotherapy as palliative treatment in poor prognosis patients with high grade glioma. AB - We report the palliative effectiveness of a hypofractionated radiotherapy regimen in patients with poor prognosis high grade glioma. Thirty-eight elderly, and/or disabled patients received radiotherapy to a dose of 30 Gy in 6 fractions over 2 weeks to a planning target volume defined by the enhancing tumour and a 2-cm margin. The median survival was 6 months with a 1-year survival rate of 23%. Treatment was without acute toxicity. One month after radiotherapy, functional status, assessed using a verbally administered Barthel index, improved in 38% and remained stable in a further 39% of surviving patients. At 3 months 39% of surviving patients had improved and a further 12% remained stable. We conclude that in the poor prognostic group of patients with high grade glioma hypofractionated partial brain radiotherapy is well tolerated, convenient and provides effective palliation in a proportion of patients. Comparison with conventional radiotherapy or symptomatic care alone require further evaluation in randomised studies. PMID- 7535942 TI - Ability of linear and cyclic peptides of neutralization antigenic site 1 of poliovirus type 1 to induce virus cross-reactive and neutralizing antibodies. AB - Eight peptides encompassing neutralization antigenic site 1 of poliovirus type 1 (residues 93-103 of VP1) were synthesized in linear or cyclized form and used to immunize rabbits. The resulting anti-peptide antibodies were tested for their ability to react with linear peptide 95-104, with infectious virus D-particles and heated C-particles and for their capacity to neutralize poliovirus infectivity. A good correlation was observed between the ability of different peptide antisera to immunoprecipitate D-particles and neutralize virus infectivity. The peptides that induced a neutralizing antibody response in the highest number of immunized animals contained flanking residues 104-115 in addition to the 93-103 residues of the epitope. However, a high neutralizing antibody titre was also obtained in two of ten animals immunized with peptide 93 104 cyclized via an amide bond between Asp93 and Lys103. It seems, therefore, that, at least in rabbits, the T-cell epitope recently identified in residues 103 115 of VP1 need not be present in the peptide immunogen in order to obtain poliovirus-specific neutralizing antibodies. PMID- 7535943 TI - Retinal vasculitis. PMID- 7535945 TI - Serum lipids, acute phase proteins and serum cholinesterase in normal subjects. AB - Although serum cholinesterase (CHE) is elevated in some hyperlipidaemic subjects, the relationship between serum CHE and lipids in normolipidaemic subjects is scanty. Furthermore, serum CHE is reduced in conditions in which there is an acute phase response. Serum CHE activity was measured in 46 normal individuals (22 males and 24 females). There was no significant difference between the activity of serum CHE in males or females being 6.2 +/- 1.8 U1(-1) vs. 6.4 +/- 1.5 U1(-1) respectively (mean +/- SD). There was, however, a significant correlation between serum CHE and subject age (Spearman rho 0.35, p < 0.05). There was also a significant correlation between serum CHE and serum nonfasting triglyceride concentration (rho 0.34, p < 0.05) and also apolipoprotein B (rho 0.38, p < 0.05) but not serum cholesterol or HDL-cholesterol. Five serum acute phase proteins were measured namely serum alpha-1 antichymotrypsin (ACT), alpha-1 acid-glycoprotein (AGP), alpha-2-macroglobulin (AMG), C-reactive protein (CRP), haptoglobin (HAP). Only serum AGP showed a significant negative correlation with serum CHE (rho - 0.43, p < 0.02). PMID- 7535944 TI - [Are there alternative forms of therapy in breast carcinoma? Status and perspectives for the treatment of metastasized breast carcinoma]. AB - The emergence of new cytotoxic agents and techniques for treatment of systemic disease as single modalities or in combination with irradiation and surgery will impact on the use of such agents in the management of systemic breast cancer. Metastatic breast carcinoma, unlike other solid tumors, is highly responsive to chemotherapy, response rates of 50 to 70% have been reported consistently, although there has not been a significant improvement on long-term survival of these patients in the last ten years. New therapeutic approaches include cytotoxic and hormonal agents, growth and differentiation factors, monoclonal antibodies, hematopoietic stem cell support, conquest of tumor cell resistance by MDR-modulation, genetic manipulation, identification of new targets on the tumor surface, synthesis of target-oriented designer-drugs and inhibition of tumor angiogenesis. In breast cancer the tumor growth correlates with vascularization and angiogenesis. Tumor angiogenesis is stimulated by the vascular endothelial growth factor (VEGF). Microvessel density is a significant predictor of survival among node-negative women, who are at risk for having occult metastases at presentation. These patients could then be given systemic adjuvant therapy. Animal experiments show promising inhibition of tumor growth in nude mice after application of antibodies against VEGF. Other methods of manipulation of molecular mechanisms of angiogenesis are under investigation. PMID- 7535946 TI - Interactions in vitro and in vivo between porcine tissue kallikrein and porcine plasma proteinase inhibitors. AB - The elimination of radio-iodinated porcine tissue kallikrein, after intravenous injection in the pig, showed a rapid initial clearance from plasma (T1/2 approximately 10 min), followed by a phase of slower elimination (T1/2 approximately 100 min). Gel filtration of plasma samples showed complexes with alpha 1-alpha 2-macroglobulin (A1a2-M) and alpha 1-proteinase inhibitor (A1-PI), which decreased with time. The urinary excretion of undegraded tissue kallikrein was about 1.8%. Gel filtration of urine showed a minor peak representing free tissue kallikrein and a major peak representing degradation products. On average, 8.3% was found in the liver and 1.3% in the kidneys post mortem, indicating that these are the primary organs for the elimination of tissue kallikrein. The in vivo findings were supported by in vitro experiments. A1a2-M were found to be the major inhibitors of tissue kallikrein, when a mixture of the enzyme and porcine plasma was analysed by gel filtration, immunoelectrophoresis, crossed immunoelectrophoresis and autoradiography. A1-PI was only a minor inhibitor of tissue kallikrein. Both the A1a2-M and A1-PI complex formation was found to be time-dependent and slow; unbound glandular kallikrein was still detected after 12 h, even when there was a molar surplus of A1a2-M and A1-PI. The complexes with A1a2-M and the unbound tissue kallikrein were found to be enzymatically active against low-molecular-weight chromogenic substrate. The total tissue kallikrein inhibiting capacity of plasma seemed to be exceeded at a concentration of 800 K U/l when analysed using the rat uterus bioassay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535947 TI - [Neural regeneration: problems and prospects]. PMID- 7535948 TI - [Nitric oxide and neural injury]. PMID- 7535949 TI - [The function of pancreatic exocrine in diabetic rats]. PMID- 7535950 TI - The release of the clinical practice guideline: benign prostatic hyperplasia: diagnosis and treatment. PMID- 7535951 TI - Laser ablation of the prostate: nursing management. PMID- 7535953 TI - [Clinical and histopathological characteristics of nonpalpable prostatic carcinoma]. PMID- 7535952 TI - [Advances in the treatment of head and neck tumors. 2. Radiochemotherapy]. AB - PURPOSE: Local recurrences are one of the main reasons of failure of therapy for locally advanced cancer of the head and neck. Concomitant chemoradiotherapy and accelerated radiation techniques are supposed to improve the locoregional control rates. METHODS: We review the theoretical background and the most important larger clinical trials using concomitant or alternating chemoradiotherapy. The data of alternative fractionation schedules are also briefly reviewed. RESULTS: For many chemotherapy agents a radiosensitizing effect has been shown. Significantly improved locoregional control has been shown for mitomycin C, cisplatin and 5-fluorouracil. Most studies failed to show improved survival. The major factor for this negative result is the common multimorbidity of patients leading to death of other causes then cancer. CONCLUSION: Concomitant, alternating or split course chemoradiotherapy can improve locoregional control. Improved survival has been shown rarely. Confirmatory studies are necessary before adapting this approach in daily practice outside clinical trials. Accelerated hyperfractionation with or without chemotherapy may further improve the control rates. Reirradiation with concomitant chemotherapy in recurrent tumors achieves complete response rates of over 40%. PMID- 7535954 TI - Effect of nitric oxide synthase inhibition on the cerebral vascular response to hypercapnia in primates. AB - BACKGROUND AND PURPOSE: The role of nitric oxide in cerebrovascular response to changes in PCO2 is unclear. In the present study, we assessed responses at two levels of hypercapnia in a primate model before and after blockade of nitric oxide synthesis. METHODS: We compared the effects of two levels of hypercapnia, defined as PCO2 of approximately 70 mm Hg (high-CO2 group, n = 5) and PCO2 of approximately 50 mm Hg (moderate-CO2 group, n = 6), on increases in regional cerebral blood flow (microspheres) before and after inhibition of nitric oxide synthase with N omega-nitro-L-arginine methyl ester (L-NAME; 60 mg.kg-1) in isoflurane-anesthetized cynomolgus monkeys (1.0% end-tidal concentration). RESULTS: Before L-NAME administration, hypercapnia increased flow in all regions (eg, forebrain, high-CO2 group 69 +/- 10 to 166 +/- 15 mL.min-1.100 g-1; moderate CO2 group, 49 +/- 7 to 93 +/- 15 mL.min-1.100 g-1) and decreased cerebral vascular resistance (high-CO2, 1.1 +/- 0.1 to 0.4 +/- 0.1 mm Hg.mL-1.min.100 g; moderate-CO2, 1.4 +/- 0.1 to 0.7 +/- 0.1 mm Hg.mL-1.min.100 g). During normocapnia, L-NAME decreased cerebral blood flow (high-CO2, 37 +/- 9%; moderate CO2, 40 +/- 6%) and increased cerebral vascular resistance (high-CO2, 93 +/- 33%; moderate-CO2, 88 +/- 20%). After L-NAME, hypercapnia still increased blood flow in all regions (eg, forebrain: high-CO2, 56 +/- 7 to 128 +/- 3 mL.min-1.100 g-1, moderate-CO2, 36 +/- 5 to 57 +/- 8 mL.min-1.100 g-1) and decreased vascular resistance (high-CO2, 1.5 +/- 0.1 to 0.6 +/- 0.1 mm Hg.mL-1.min.100 g; moderate CO2, 2.0 +/- 0.3 to 1.2 +/- 0.1 mm Hg.mL-1.min.100 g). In both groups L-NAME attenuated hypercapnia hyperemia by approximately 30% in cortex but not in other regions. CONCLUSIONS: Nitric oxide contributes to basal vascular tone but is not a major contributor to the mechanism of hypercapnia-induced cerebral vasodilation, except in cortex, in primates. PMID- 7535955 TI - DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins. PMID- 7535956 TI - Association of acidic fibroblast growth factor and untreated low grade rejection with cardiac allograft vasculopathy. AB - Acidic fibroblast growth factor (aFGF) is a potent growth factor for vascular smooth muscle cells and may mediate vasculopathy in cardiac allografts subjected to chronic immunological injury. Therefore, we examined cardiac expression of aFGF, the number of rejection episodes, and other potential risk factors in 32 heart transplant patients who underwent intravascular ultrasound (IVUS) for detection of cardiac allograft vasculopathy (CAV). As defined by IVUS, CAV was present in 21 patients and absent in 11 patients (follow-up time: 52 +/- 21 vs. 51 +/- 12 months, respectively, P = NS). The level of aFGF in myocardial biopsies obtained at the time of IVUS was measured by semiquantitative reverse transcriptase polymerase chain reaction and expressed as the aFGF:GAPDH ratio. Higher level of aFGF were associated with CAV (mean aFGF:GAPDH ratio was 1.45 +/- 0.99 in patients with vs. 0.18 +/- 0.12 in patients without CAV [P < 0.001]). A strong association was found between high levels of cardiac aFGF and CAV, as 18 of 19 patients (95%) with high levels of aFGF (aFGF:GAPDH > 1) but only 3 of 13 patients with low levels of aFGF had CAV (P < 0.001). The relative risk of high level of aFGF for CAV was 4.1. Untreated low grade rejection (ISHLT I), but not treated high grade rejection (ISHLT > 2), was also associated with CAV (average number of untreated low grade rejection episodes was 3.5 +/- 1.8 in patients with vs. 2.1 +/- 1.0 in patients without CAV [P = 0.04]). Among other risk factors examined (age, sex, serum cholesterol, blood pressure, CMV infection, dose of immunosuppressants, and ischemic time), only triglycerides were higher in patients with than those without CAV (P = 0.003). We conclude that increased cardiac production of aFGF is significantly associated with CAV, which suggests that aFGF may serve as an important mediator in CAV. Untreated low grade rejection also poses an increased risk for CAV. PMID- 7535958 TI - Normal pancreas allograft function following simultaneous pancreas kidney transplantation after rescue therapy with tacrolimus (FK506). PMID- 7535959 TI - Demyelinating sensorimotor polyneuropathy after administration of FK506. PMID- 7535957 TI - Formation of a regular neo-epidermis by cultured human outer root sheath cells grafted on nude mice. AB - The outer root sheath of hair follicles mainly consists of basal-like keratinocytes which can substitute for interfollicular epidermal keratinocytes, as during healing of skin wounds when outer root sheath cells migrate onto the denuded area, thus contributing to epidermal regeneration. Human outer root sheath cells represent a repeatedly available source of keratinocytes which can be easily and extensively expanded in culture. Close comparison of organotypic cultures of either outer root sheath cells or epidermal keratinocytes grafted onto nude mice demonstrated that outer root sheath cells formed a stratified epithelium resembling normal epidermis that is virtually indistinguishable from that developed by epidermal keratinocytes. Typical epidermal differentiation markers, such as the suprabasal keratins 1 and 10, involucrin, filaggrin, the basement membrane components collagen type IV and laminin, and the integrin chains alpha 2, alpha 3, alpha 6, and beta 1, were readily expressed in a mostly regular localization. These data suggest that outer root sheath cells, bearing essential advantages as compared with interfollicular keratinocytes, are suitable for skin replacement. PMID- 7535961 TI - Prevalence of hepatitis C infection in health care workers affiliated with a liver transplant center. AB - The risk of hepatitis C to liver transplantation health care workers has not been identified. We compared the occupational risk of hepatitis C in health care workers associated with liver transplantation with risk of health care workers affiliated with the same institutions but not involved in transplantations. Health care workers were recruited from 2 transplant centers. Participation was voluntary; results were confidential. An occupational and health history questionnaire was completed and blood was donated for testing. Health care workers were categorized into 3 groups according to risk for hepatitis C infection: very high, high, and low risk. A total of 241 health care workers were recruited from 2 transplant centers. Fifty-nine percent (142/241) were female; mean age was 38.7 years. Health care workers included: 48.5% (117/241) nurses, 24.9% (60/241) physicians, and 17% (42/241) laboratory personnel. The mean number of years in their occupation was 13.5 years (range < 1 year to 38 years). Twenty four percent (57/241) were categorized in a very high risk occupation for hepatitis C, 66% (158/241) in a high risk occupation, and 10.8% (26/241) in a low risk occupation. A total of 2.1% (5/241) of health care workers were reactive to hepatitis C by enzyme immunosorbent assay; three of these were positive by polymerase chain reaction testing. Of the 3, none had a history of hepatitis or transfusion. However, 5.3% (3/57) of health care workers involved with liver transplantation were infected, as compared with 0% (0/184) who were not (P = 0.013). We conclude that health care workers associated with liver transplantation may be at a higher risk for hepatitis C when compared with health care workers not associated with transplantation. PMID- 7535962 TI - [The comparative characteristics of the immunogenic and immunomodulating activity of different antigenic preparations of the causative agent of Q fever Coxiella burnetii]. AB - The immunogenicity of the Coxiella burnetii corpuscle antigen and two its components, obtained after the treatment with chloroform-methanol or extraction by trichloroacetic acid, was investigated. All the studied antigens were highly immunogenic. A strong correlation was discovered between the immunogenic and immunomodulating properties of the C. burnetii antigens. Cells of the mononuclear phagocytic system are the targets for stimulating action of the studied antigens. A cyclic increase of antibacterial activity and oxygen-dependent metabolism of the peritoneal cells was noted, in addition to the increase of the natural splenocyte cytotoxicity caused by the action of these antigens. The role of cytokines in rising the activity of peritoneal cells and splenocytes, caused by immunization of mice with the above antigenic substances, is discussed. PMID- 7535960 TI - Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. AB - We examined the cytokine profiles associated with tolerance and rejection using the mouse model of neonatal tolerance. BALB/c mice primed with CAF1 splenocytes during the neonatal stage showed increased A/J skin graft survival of > 60 days and failed to develop anti-A/J cytotoxic responses, but rejected third-party C57BL/6 grafts. Lymph node cells that drained A/J grafts on neonatal-primed mice produced allospecific immune cytokine responses characterized by high IL-4 and low IFN-gamma levels. In contrast, lymph node cells that drained either rejected third-party grafts or rejected A/J grafts placed on adult controls produced less IL-4 and more IFN-gamma. Tolerogen-specific immune responses from neonatal-primed mice made up to 100 times higher IL-4 to IFN-gamma ratios than did controls. Alloantigen priming during the immediate neonatal stage induced constitutive expression of IL-4 mRNA in the spleen without IFN-gamma mRNA, whereas alloantigen stimulation during adulthood induced the opposite pattern. IL-4 production from neonatal primed mice was confined to the CD4 population. The altered cytokine profile of enhanced IL-4/IFN-gamma in neonatal primed mice persisted for up to 12 weeks after priming in in vitro secondary MLR assays, which suggests that the initial timing of antigen stimulation critically influenced CD4 maturation. The results support a model of immunoredirection as a mechanism of tolerance and provide rationale for examining the therapeutic use of cytokines in transplantation. PMID- 7535963 TI - [The treatment of the Budd-Chiari syndrome (a review of the foreign literature)]. PMID- 7535964 TI - Comparative toxicological studies of chlorpyrifos in rats and chickens. AB - Rats and chickens were given single oral doses of 50 mg chlorpyrifos/kg to compare toxic effects in these 2 species. Oral administration resulted in decreased cytochrome P-450 and aminopyrine N-demethylase activities and increased cytosolic glutathione S-transferase activity in rats. On the contrary, there was increased cytochrome P-450 and aminopyrine N-demethylase activities in chickens. A significantly higher inhibition of serum cholinesterase (82%) was noted in rats than in chickens (55%). Serum gamma-glutamyl transferase, a marker of hepatotoxicity, remained unchanged in both species, indicating the absence of hepatotoxicity. These studies project chlorpyrifos to be an inhibitor of hepatic microsomal drug-metabolizing enzymes in rats and an inducer in chickens, and a non-hepatotoxic organophosphate insecticide in both species when given at the dosage of 50 mg/kg. PMID- 7535965 TI - [Occlusive jaundiced caused by liver metastases. Results of endoscopic surgical treatment]. AB - The results of endoscopic drainage treatment in 69 patients with metastases of the liver have been analyzed for the period from 1982 to 1992. Fifty-five patients had follow-up. Criteria for inclusion were: positive diagnosis of liver metastases, jaundice, and primary origin of tumors distant from the pancreaticobiliary system ("distant" primary). Localisations of metastic obstructions were: hilum of the liver (n = 24), commonbile duct (n = 16), and prepapillary region (n = 15). Treatment was performed by transpapillary applications of one or more (n = 3) pig-tail or Tannenbaum stents. Concentrations of mean serum bilirubin could be reduced from 14.5% to 8.8 mg%, 16 patients reached a normal level (less than 1.0 mg%). The median survival time was 76 days ranging vom 4 to 299 days. The most frequent complication was cholangitis in 29% of the cases with mortality in 6 patients. Patients less than 60 years of age and with obstructions in the mid commonbile duct gained most by this kind of treatment. Results were poor in elderly patients with hilar stenosis. Selected patients with known liver metastases may benefit from endoscopic treatment of jaundice. PMID- 7535966 TI - [Initial experiences with a new color technique: ultrasound angiography]. AB - The sonographic diagnosis can be expanded by Color Doppler. Nevertheless something is missing, especially concerning the demonstration of the very slow velocities as it can be found in neovascularized malignant tumors. A recently developed new color technique--the Angio-Color of the Diasonics Corporation, Sonotron (other companies have prototypes of this color)--promises to improve the detection of very low flow velocities. Due to a method very different to the conventional Doppler technique the registered signal is coded in the color image of the blood flow: that means that the amplitude and not the frequency shift is coded in color. Therefore there is less noise in the color mode with the possibility of showing the lower flow in comparison to the conventional Doppler. In Gynaecology and Obstetrics the advantages in the demonstration of the placental blood flow were obvious. In eutrophic fetuses the blood flow could be registered over the whole breadth of placenta, while in dystrophic fetuses this was possible only at the margin of the placenta with some color pixels in the middle of the organ. The conventional Color Doppler was not able to show the flow in the placenta even in eutrophic fetuses. Also the flow in fetal organs produced different results using both methods. So the angio-technique showed more color pixels in the periphery. In 8 malignant breast tumors both methods were able to show blood flow, but the Angio-Color showed more color pixels as the conventional color did.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535968 TI - A novel liposome immune lysis assay (LILA) for determination of CRP antigen using two monoclonal antibodies recognizing different antigenic determinants. AB - We have already developed the liposome immune lysis assay (LILA) for the determination of C-reactive protein (CRP) by employing an inhibition method and a sandwich method. We herein report a new LILA system involving the use of monoclonal antibodies-bearing liposomes. We established five monoclonal antibodies to CRP antigen, AC-1, -2, -3, -4, -5 which had the capacity to activate complement and form antigen-antibody complex. Each of these antibodies was covalently coupled to carboxyfluorescein-entrapped multilamellar liposomes. When the liposomes were incubated with CRP antigen in the presence of guinea pig complement, CRP antigen-dependent liposome lysis was observed but the sensitivity was not great enough for practical use. On the other hand, when liposomes coupling two monoclonal antibodies (AC-1, AC-2) which recognized distinct CRP antigenic determinants were employed in the assay, the sensitivity increased compared with that using only one monoclonal antibody, and the detectable concentration range was 5-300 ng/ml. These results indicated that the combination of two or more monoclonal antibodies which recognize distinct CRP antigenic determinants is effective for increasing the sensitivity of the assay. PMID- 7535967 TI - Intrafamilial clustering of genotypes of hepatitis C virus RNA. AB - Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family. The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household. PMID- 7535969 TI - Chronic administration of citalopram inhibited El mouse convulsions and decreased monoamine oxidase-A activity. AB - Serotonin (5-HT) is thought to play an important role in the seizures of El mice because the seizure threshold of El mice correlates with the 5-HT concentration in the central nervous system. In this study, the anticonvulsant effect of a 5-HT reuptake blocker, citalopram, was evaluated behaviorally and biochemically. El mouse convulsions were inhibited by chronic administration of citalopram (80 mg/kg/day, p.o. for 2 weeks), but were not inhibited by acute administration of citalopram (80 mg/kg, i.p., 2 h after single injection). Both chronic and acute administration of citalopram decreased the concentration of 5-hydroxyindolacetic acid in the brain, whereas the concentration of 5-HT was not changed by treatment with citalopram. Tryptophan hydroxylase activity was not different between the citalopram and control groups, although the monoamine oxydase-A activity was lowered by chronic administration of citalopram. These findings suggest that both acute and chronic administration of citalopram depresses the 5-HT turnover rate, however chronic administration is necessary to inhibit El mouse convulsions. PMID- 7535970 TI - Long-term follow-up results of 175 patients with malignant glioma. PMID- 7535972 TI - Complement and antibody enhance binding and uptake of HIV-1 by bone marrow cells. PMID- 7535973 TI - The differences in survival and phenotype between centroblasts and centrocytes. PMID- 7535971 TI - Autoimmune lpr and gld mice: models of abnormal adhesion molecule regulation and defective lymphocyte traffic. PMID- 7535974 TI - Expression and function of DRC-1 antigen. PMID- 7535976 TI - In vivo gp39-CD40 interactions occur in the non-follicular compartments of the spleen and are essential for thymus dependent antibody responses and germinal center formation. PMID- 7535975 TI - Selection of anti-arsonate idiotype (CRIA) in A/J mice by the immune network. PMID- 7535977 TI - Family history and prostate cancer risk in black, white, and Asian men in the United States and Canada. AB - Increased risk of prostate cancer in men with a family history of the disease has been observed consistently in epidemiologic studies. However, most studies have been confined to white men; little is known about familial aggregation of prostate cancer in populations with unusually high incidence, such as African Americans, or in populations with low incidence, such as Asian-Americans. The authors report results from a population-based case-control study of prostate cancer among blacks, whites, and Asian-Americans in the United States and Canada. Controls were matched to cases on age (5-year groups), ethnicity (black, white, Chinese-American, Japanese-American), and region of residence (Los Angeles, San Francisco, Hawaii, Vancouver, Toronto). In the combined group of participants, 5% of controls and 13% of cases reported a father, brother, or son with prostate cancer. These prevalences were somewhat lower among Asian-Americans than among blacks or whites. A positive family history was associated with a statistically significant two- to threefold increase in risk in each of the three ethnic groups. The overall odds ratio associated with such a family history, adjusted for age and ethnicity, was 2.5 (95% confidence interval 1.9-3.3). This odds ratio varied by neither ethnicity nor age of the participants. Sera from 1,087 controls were used to examine the relations between family history and serum concentrations of androgens and prostate-specific antigen. The concentrations of sex hormone-binding globulin were slightly higher in men with than without a positive family history. Prostate-specific antigen concentrations were unrelated to family history. PMID- 7535978 TI - Prostate specific antigen decline following the discontinuation of flutamide in patients with stage D2 prostate cancer. PMID- 7535979 TI - Gadolinium-enhanced magnetic resonance imaging assessment of hydroxyapatite orbital implants. AB - PURPOSE: Successful prosthesis attachment depends on complete vascularization of porous coralline hydroxyapatite when it is used as an orbital implant. We retrospectively assessed the utility of gadolinium-enhanced magnetic resonance imaging to evaluate and characterize the temporal progression of this fibrovascular process, which has been histologically documented elsewhere. METHODS: Serial T1-weighted gadolinium-enhanced orbital magnetic resonance examinations were performed in five patients receiving hydroxyapatite orbital implants. Retrospective evaluation of the enhancement patterns was performed. Magnetic resonance imaging enhancement patterns guided timing of final drilling for prosthesis fixation. RESULTS: Serial gadolinium-enhanced T1-weighted sequences consistently demonstrated centrally advancing, peripheral enhancement centered on the drilled access channels. Progression over time varied, with the following two patterns demonstrated: (1) rapid peripheral enhancement, which led to diffuse enhancement (three patients); and (2) enhancement limited to the periphery, which failed to advance centrally. CONCLUSIONS: The temporal enhancement seen on magnetic resonance imaging is identical to the histologically proven fibrovascular ingrowth pattern and most likely reflects this process. Magnetic resonance imaging can identify progression of fibrovascular ingrowth into the hydroxyapatite orbital implants and guide surgical planning. It may also identify implants that fail to vascularize, thereby preventing the morbidity encountered by drilling into an avascular hydroxyapatite implant. PMID- 7535980 TI - Naturally occurring human IgA autoantibodies against IgE-DES myeloma protein. Prevalence and specificity. AB - The prevalence and specificity of naturally occurring human IgA anti-IgE autoantibodies (a-E Ab) were studied by ELISA with anti-IgA monoclonal antibodies (mAb) and a purified myeloma IgE as solid-phase protein, i.e., IgE-DES(kappa). Such detected IgA a-E Ab were common among adults, and significantly increased geometric means (GM) were found in patients with atopy (P = 0.006; n = 41; GM = 79.3 arbitrary units (AU)/ml) and filariasis (P = 0.02; n = 41; GM = 75.9 AU/ml), as compared with nonatopic controls (n = 42; GM = 48.8 AU/ml). No such difference was observed between age-matched nonatopic (n = 22; GM = 36.7 AU/ml) and atopic (n = 22; GM = 38.6 AU/ml) children. Children had significantly (P < 0.001) lower IgA a-E Ab concentrations than adults, probably as a result of age, because IgA a E Ab concentrations and age of children were significantly correlated (n = 44; P < 0.05; r = 0.30). IgA a-E Ab concentrations were very low in cord serum (n = 32; median < 0.1 AU/ml). Sex did not influence IgA a-E Ab concentrations in any study group. The specificity of IgA a-E Ab in nine sera was studied by ELISA inhibition assay using IgE-DES myeloma as solid-phase protein and inhibitory proteins of the IgG, IgM, IgD, and IgE classes, including five different IgE myeloma proteins, as well as three enzymatic fragments of IgE-DES. The inhibitions indicated that all IgA a-E Ab tested reacted in a low-affinity reaction with determinants restricted to IgE-DES, i.e., the solid-phase protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535982 TI - Allergy to ingested cereals in atopic children. AB - Clinical features, hypersensitivity mechanisms, and differential diagnosis of cereal allergy or intolerance were investigated in children with atopic dermatitis (AD). On oral provocation, 18 children exhibited a positive response to wheat, three to rye, one to barley, and one to oats. Cereal-induced symptoms were dermatologic, gastrointestinal, or oropharyngeal, and their onset after provocation was immediate (eight cases), delayed (14 cases), or both immediate and delayed (one case). A combination of type I allergy tests (prick test, RAST, and histamine-release test) detected all immediate reactors and 9/14 delayed reactors. Of the five subjects remaining negative in these tests, three were positive in the patch or lymphocyte-proliferation tests. Subjects with cereal allergy or intolerance frequently possessed IgE, IgA, and IgG antibodies against gliadin, but only one of these children was HLA-DR3-positive, and none had reticulin antibodies typical of celiac disease. Combining tests of immediate and delayed hypersensitivity can confirm allergy to cereals in a more reliable way. The coexistence of cereal allergy and celiac disease seems to be rare. PMID- 7535983 TI - A specific colorimetric staining method for gamma-carboxyglutamic acid-containing proteins in polyacrylamide gels. AB - In this paper we describe a specific staining method for gamma-carboxyglutamic acid (Gla)-containing proteins in polyacrylamide gels. The procedure is based on the colorimetric detection of Gla using 4-diazobenzenesulfonic acid and has the advantage of being simple and fast (1 h). The detection limit is 9.4 pmol for prothrombin and 150 pmol for osteocalcin. It is demonstrated that Gla-proteins can be visualized independent of the presence of an excess of contaminating non Gla-proteins. The technique may be used for screening of large number of fractions during the purification of Gla-proteins from complex protein mixtures. PMID- 7535981 TI - Effect of cytokines on mediator release from human dispersed lung mast cells. AB - To evaluate the contribution of human lung mast cells (HLMC) to allergic inflammation, we investigated whether or not cytokines, including stem-cell factor (SCF), monocyte chemotactic and activating factor (MCAF), and RANTES, activate HLMC. SCF induced histamine release from dispersed HLMC in a dose dependent fashion (P < 0.01). The release was 7.8 +/- 1.0% at 500 ng/ml SCF (n = 9). This response was also observed in chopped lung tissue. HLMC from which surface IgE molecules had been removed by treatment with lactic acid responded to SCF, while these cells lost their response to anti-IgE. The process was relatively rapid and reached a maximum in 5 min. This response required extracellular calcium, and it was observed at 37 degrees C, but not at 4 degrees C or 20 degrees. A brief preincubation (10 min) with lower concentrations of SCF, which were ineffective in releasing histamine, enhanced anti-IgE-induced histamine release (P < 0.05), while its enhancing effect was lost by the longer preincubation (30 min). SCF did not prime basophils to enhance stimulated histamine release. Interleukin (IL)-1 alpha, IL-1 beta, IL-3, IL-4, IL-5, granulocyte/macrophage-colony stimulating factor (GM-CSF), MCAF, and RANTES neither induced histamine release nor enhanced the release stimulated by anti-IgE after a 10- or 30-min preincubation. The combination of IL-3 and IL-4 showed no effect on histamine release from HLMC. Leukotriene (LT)C4/D4/E4 production by SCF was negligible, as compared with anti-IgE-induced LT production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535984 TI - Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. AB - A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring single protein bands from gel onto small-sized (13 x 5 mm2) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for the protein electroblotting in such minisandwiches. Microsequence analysis of the electroblotted proteins showed initial yields in the range of those achieved when the transfer was done from unstained control gels. Protein bands kept in the reverse-stained gel for prolonged time periods (even for as long as 2 years) could be similarly analyzed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535985 TI - Double staining of coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl sulfate-zinc reverse staining: sensitive detection of coomassie blue undetected proteins. AB - The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water (15 min, two times) and then subjected to imidazole zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-staining gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinase, 47 kDa) over a wide range of protein molecular weights (10 to 100 kDa) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed. PMID- 7535986 TI - Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard. AB - Advances in our understanding of molecular and cellular physiology necessitate that mRNA levels for specific growth factors and other rare transcripts be measured quantitatively in small samples. Conventional methods such as Northern blot analysis and solution hybridization/ribonuclease protection are not sufficiently sensitive. We now report the theory, development, and validation of a rapid and highly sensitive assay, the RNA/DNA quantitative polymerase chain reaction (RD-PCR), which uses a competitive PCR approach to measure the number of copies of a specific mRNA per cell. Total nucleic acid (RNA and genomic DNA) is isolated from cells in culture. The mRNA of interest is first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3' end, and a sequence complementary to an intron of the desired gene at the 5'-end. Competitive PCR is then performed in the presence of the cDNA product and endogenous genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest, and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The cell's own genomic DNA is thereby used as the internal standard. To control for the efficiency of reverse transcription, a standard curve is used in each assay. The technique was validated by comparing the quantitation of insulin-like growth factor I (IGF-I) mRNA in two human cell lines by RD-PCR and by RNase protection analysis. Both methods gave similar numbers of copies of IGF-I mRNA per cell. For accurate analysis, RNase protection required at least 10(7) cells; RD-PCR required as little as 10(2) cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7535987 TI - Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay. AB - A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use. PMID- 7535988 TI - Nonradioactive 3'-end-labeling of RNA molecules of different lengths by terminal deoxynucleotidyltransferase. AB - Terminal deoxynucleotidyltransferase is well known for its ability to add nucleotides to the 3'-ends of DNA; here we demonstrate a corresponding activity with RNA as acceptor molecule. Using DIG- or biotin-dUTP as substrate, a tailing reaction is used for nonradioactive 3'-end-labeling of RNA oligonucleotides, isolated RNA molecules such as tRNA from E. coli, and MS2 RNA as well as in vitro transcripts. The tail length and associated sensitivity of detection can be varied using DIG-ddUTP, DIG-dUTP, or a nucleotide mixture of DIG-dUTP and dATP. PMID- 7535990 TI - Effect of dietary supplementation of red or white wine on human blood chemistry, hematology and coagulation: favorable effect of red wine on plasma high-density lipoprotein. AB - Twenty healthy males were divided into two groups: 10 subjects were supplemented for 2 weeks with 400 ml of red wine (11% alcohol) per day and the other 10 subjects were given 400 ml of white wine (11% alcohol) per day for a similar period. Blood samples were drawn prior to wine supplementation, after 1 week and at the end of the study. No significant effects were found on plasma concentrations of urea, creatinine, bilirubin, creatine kinase, amylase, blood cell counts, platelet counts and platelet aggregation. Both red- and white-wine supplementation resulted in a transient minor reduction in plasma glucose concentration and in a minor elevation in blood coagulation properties such as prothrombin time and partial thromboplastin time. Red (but not white) wine resulted in an 11 and 26% increment in plasma triglyceride concentrations after 1 and 2 weeks of supplementation, respectively. Plasma cholesterol, as well as very low- and low-density-lipoprotein levels did not change during the 2 weeks of red- or white-wine supplementation. The most impressive effect of red-wine intake was a significant (p < 0.01) increase in plasma high-density lipoprotein (HDL) cholesterol and in plasma apolipoprotein A-I concentrations by up to 26 and 12%, respectively. These effects were not observed after the intake of white wine. We conclude that the major effect of red-wine supplementation (about 40 g of alcohol per day for a period of 2 weeks) was a significant increase in plasma HDL concentration which may contribute to the reduced risk for cardiovascular diseases observed in red-wine drinkers. PMID- 7535989 TI - Comparative evaluation of the effects of two different forms of dietary fibre (rice bran vs. wheat bran) on rat colonic mucosa and faecal microflora. AB - The aim of the study was to compare the physiological consequences of two dietary fibre sources on the faecal microflora and colonic mucosal growth in rats. The studied sources, a moderately well-soluble fibre (rice bran, RB) and a less well soluble fibre (wheat bran, WB), were included in diets of rats at a level of 10% for 3 weeks and compared with a totally fibre-deprived diet. RB significantly increased faecal water compared to the control diet (p < 0.05). Faecal nitrogen content and bacterial mass, as estimated from the 2,6-diaminopimelic acid (DAPA) output, were greatly and significantly increased by RB, and to a lesser extent by WB, compared to the control diet. Total bile acid excretion was significantly higher by rats fed RB than by those fed WB. Faecal bacterial enzyme activities tested (beta-glucuronidase, mucinase and nitroreductase) were significantly reduced by the two different fibre sources, but RB was more effective than WB, except for nitroreductase activity which was reduced at the same level for each fibre source. Although measurements of mucosal colonic weight and RNA content were significantly different between groups fed RB and WB (p < 0.05), DNA content and the ratio RNA/DNA did not significantly differ between these groups. Our results indicate that the differential changes observed in beta-glucuronidase and mucinase activities and DAPA and bile acid excretion may depend on the nature of the fibre consumed. They also suggest that RB, which had similar effects, sometimes more marked than WB, on the studied parameters, may be a new valuable fibre source. PMID- 7535991 TI - In vitro protein-binding characteristics of atevirdine and its N-dealkylated metabolite. AB - The in vitro protein-binding characteristics of atevirdine (ATV), a non nucleoside reverse transcriptase inhibitor with activity against HIV-1, and its N dealkylated metabolite (N-ATV) were studied using equilibrium dialysis. ATV and N ATV were studied at concentrations of 5, 10, 20, and 30 microM in five protein containing solutions [albumin 4%, plasma, serum, immune globulin (IgG) 1.5%, alpha 1-acid glycoprotein (AAG)] for 5 h at 37 degrees C. All samples were analyzed by high-performance liquid chromatography. The free fraction of atevirdine in plasma, albumin, and serum was 0.01-0.02 over the range of drug concentrations studied. The fraction unbound (fu) in these protein solutions statistically differed from IgG and AAG (P < 0.05), where the fraction unbound averaged 0.96 and 0.53, respectively. N-ATV had a similar binding profile as ATV with a fraction unbound of 0.04, 0.03, 0.03 in albumin, plasma and serum, respectively. A difference existed in N-ATV binding when compared to IgG and AAG with an average fu of 0.87 and 0.59 (P < 0.05 vs. plasma). The potential clinical implications of the high degree of protein binding for ATV and N-ATV are discussed. PMID- 7535992 TI - Antiviral activity of natural and semi-synthetic chromone alkaloids. AB - The activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV), of the non-polar fraction of a methanolic extract of the rootbark of Schumanniophyton magnificum was found to be present in a fraction containing the chromone secondary amine schumannificine 1. Other chromone alkaloids present in the plant were isolated and tested for inhibition of HIV and HSV infections in C8166 and Vero cells, respectively. Acyl and methyl derivatives were prepared and tested. Of all the compounds tested, schumannificine 1 displayed the greatest activity against HIV, whereas potent anti-HSV activity was observed for a number of its derivatives. The presence of a piperidine ring and unsubstituted hydroxy groups on the molecules seems to favour the anti-HIV activity. The anti-HIV activity is considered to be due to irreversible binding to gp120 rather than inhibition of reverse transcriptase or protease. PMID- 7535993 TI - Effects of nefiracetam (DM-9384), a pyrrolidone derivative, on brain monoamine systems. AB - The pyrrolidone cyclic gamma-aminobutyric acid (GABA) derivative nefiracetam [DM 9384; N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide] has been shown to enhance acquisition and ameliorate amnesia in different learning tasks in rodents. In the present study, the effects of nefiracetam on the brain monoamine systems were studied. Male, adult Sprague-Dawley rats were treated with nefiracetam in single doses (0, 1, 3, 10, 30 or 100 mg/kg, p.o.) and analyzed after 1 and 4 hr, or treated by daily doses (0, 1, 3, 10 or 30 mg/kg, p.o.) for 2 weeks. In general, no or only weak effects were observed on tissue monoamine levels, either following acute or 14 days treatment with nefiracetam. Acute administration of intermediate doses of nefiracetam induced minor increases in dopamine (DA) and homovanillic acid (HVA) tissue levels in the striatum, while hypothalamic 3,4-dihydroxyphenylacetic acid (DOPAC) decreased at 1 hr. Noradrenaline (NA) and serotonin (5-HT) levels increased in some regions after higher doses of nefiracetam. Increases in 5-hydroxyindoleacetic acid (5-HIAA) were also seen at 4 hr, but only after the 3 mg/kg dose. Minor decreases of HVA and DOPAC levels were seen in some regions after treatment with various doses of nefiracetam for 14 days, while an increase in 5-HT levels was observed occasionally. Using in vivo microdialysis in freely moving animals, no significant effects on extracellular levels of HVA, DOPAC and 5-HIAA in the striatum or of HVA, DOPAC and NA levels in the dorsal hippocampus were seen after acute administration of nefiracetam. On the other hand, extracellular hippocampal 5-HIAA levels decreased by 20% after the 1 and 3 mg/kg doses. Nefiracetam, in a concentration range of 1 nM to 10 microM, did not affect the in vitro synaptosomal uptake of [3H]NA and [3H]5-HT in the cortex or of [3H]DA in the striatum. Taken together, nefiracetam appears to exert minor, regionally restricted and not dose-dependent effects on the monoamine systems following either acute or repeated administrations in normal rats. A direct or indirect, possible GABA-mediated, influence of nefiracetam may underlie the modest changes seen on monoamines. The cognitive-enhancing action of nefiracetam does not seem to be related to effects on presynaptic monoamine functions. PMID- 7535994 TI - Influence of nifedipine, verapamil and diltiazem on pulmonary vascular resistance and vasoconstrictors in cats. AB - In the present study, the effects of three classes of L-type calcium channel blocking agents, nifedipine, verapamil and diltiazem, on the lobar arterial pressure and the vasoconstrictor responses in the pulmonary vascular bed were compared to those of cromakalim, a KATP channel activator, in the anaesthetized cat under controlled pulmonary blood flow and constant left atrial pressure. These drugs were infused intralobarly in doses selected which did not raise left atrial pressure, change cardiac output or alter systemic arterial pressure. Intralobar bolus injections of calcium channel-blocking agents and of the K+ channel activator decreased the lobar arterial pressure in a dose-related manner when pulmonary vasomotor tone was actively elevated by intralobar arterial infusion of U46619. The pulmonary vasodilator response to these agents was accompanied by a dose-related decrease of systemic arterial pressure. In decreasing lobar arterial pressure at elevated pulmonary vasomotor tone, the order of potency was nifedipine > verapamil > diltiazem, whereas in reducing systemic arterial pressure, the order of potency was nifedipine > diltiazem > verapamil. The calcium channel-blocking agents were less active than the reference drug, cromakalim, in both vascular beds. Intralobar arterial infusions of nifedipine, verapamil and diltiazem, at the rates of 0.03 mumol/min, 0.2 mumol/min and 0.1 mumol/min, respectively, caused no changes in cardiac output and in systemic and pulmonary arterial pressure. Infusion of all three calcium channel-blocking agents blocked the pulmonary vasoconstrictor responses to BAY K 8644 (calcium entry promoter) and U46619 (thromboxane A2 mimic). Nifedipine infusion also reduced the pulmonary vasoconstrictor responses to methoxamine and BHT933 (alpha 1- and alpha 2-adrenoceptor agonists, respectively), whereas verapamil infusion reduced the responses only to methoxamine. Infusion of diltiazem caused no significant decrease of responses to either alpha adrenoceptor agonist. The results of the present study suggest that the dihydropyridine, nifedipine, is more potent than the non-dihydropyridines, verapamil and diltiazem, in reducing the pulmonary vascular resistance and more effective in inhibiting the vasoconstrictor responses to the alpha-adrenoceptor agonists, to U46619 and to BAY K 8644 in the feline pulmonary circulation at the infusion rates which cause no or little hemodynamic changes. PMID- 7535995 TI - Cloning and sequencing of the genes encoding the light-harvesting B806-866 polypeptides and initial studies on the transcriptional organization of puf2B, puf2A and puf2C in Chloroflexus aurantiacus. AB - The genes encoding the alpha- and beta-polypeptide subunits of the B806-866 membrane-bound light-harvesting complex of Chloroflexus aurantiacus have been cloned and the nucleotide sequences determined. The gene puf2A, which encodes the B806-866 alpha-polypeptide, began 28 bases downstream of the stop codon of puf2B, which encodes the B806-866 beta gene. The gene-encoding cytochrome c-554, puf2C, was found about 250 bp downstream of puf2A. puf2A encoded a 13 amino acid extension at the C-terminus of the B806-866 alpha-polypeptide that was not present in the mature protein. These genes, unlike those of purple nonsulfur bacteria, did not form a contiguous operon with puf1L or puf1M, the genes encoding the L and M subunits of the photochemical reaction center. The occurrence of the two latter genes and of puf2B and puf2A in two separate operons has not been observed in purple bacteria. Under photoheterotrophic growth conditions, puf2B and puf2A were encoded on an abundant mRNA that was 0.5 kb long. Two monocistronic transcripts for puf2C were observed that had different 5' ends. One transcript encoding all three genes was also detected. Nucleotide sequences very similar to the consensus promoter sequence of the Escherichia coli RNA polymerase sigma 70 subunit were found seven and eight bases upstream of the 5'-end of mRNA encoding puf2B and for one of the monocistronic mRNA encoding puf2C, respectively. PMID- 7535996 TI - Perioperative plasma volume expansion reduces the incidence of gut mucosal hypoperfusion during cardiac surgery. AB - OBJECTIVE: To test the hypothesis that perioperative plasma volume expansion would preserve gut mucosal perfusion during elective cardiac surgery. DESIGN: Prospective randomized open study. SETTING: Teaching hospital. PATIENTS: Sixty American Society of Anesthesiology grade III patients with a preoperative left ventricular ejection fraction of 50% or greater undergoing elective cardiac surgery. INTERVENTIONS: Patients were allocated randomly to a control or protocol group. The control group was treated according to standard practices. After induction of general anesthesia, the protocol group received, in addition, 200-mL boluses of a 6% hydroxyethyl starch solution to obtain a maximum stroke volume. This procedure was repeated every 15 minutes until the end of surgery, except when the patient underwent cardiopulmonary bypass. MEASUREMENTS AND RESULTS: Cardiac stroke volume was estimated by an esophageal Doppler system, and gastric mucosal perfusion was measured by tonometric assessment of gastric intramucosal pH in all patients. Patients were followed up postoperatively until discharge from the hospital or death. The incidence of gut mucosal hypoperfusion (gastric intramucosal pH < 7.32) at the end of surgery was reduced in the protocol group (7% vs 56%) (P < .001), as were the number of patients in whom major complications developed (0 vs 6) (P = .01), mean number of days spent in the hospital (6.4 [range, 5 to 9] vs 10.1 [range, 5 to 48]) (P = .011), and mean number of days spent in the intensive care unit (1 [range, 1 to 1] vs 1.7 [range 1 to 11] days) (P = .023). CONCLUSIONS: Perioperative plasma volume expansion with colloid during cardiac surgery, guided by esophageal Doppler measurement of cardiac stroke volume, reduced the incidence of gut mucosal hypoperfusion. This group of patients also had an improved outcome when compared with controls. PMID- 7535997 TI - Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions. AB - A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants. PMID- 7535999 TI - Long-term results of laser treatment in the ocular histoplasmosis syndrome. AB - OBJECTIVE: To determine the long-term visual outcome, rate of persistent choroidal neovascularization, and rate of recurrent choroidal neovascularization in eyes undergoing laser photocoagulation for choroidal neovascularization secondary to ocular histoplasmosis syndrome. DESIGN AND PATIENTS: One hundred one eyes with 5 to 16 years of follow-up that presented with choroidal neovascularization secondary to ocular histoplasmosis were retrospectively evaluated. Patients were grouped according to location of choroidal neovascularization and assignment to observation or laser photocoagulation. MAIN OUTCOME MEASURES: Visual acuity outcome and loss for all groups were compared. The rates of persistent and recurrent choroidal neovascularization for the treated eyes were also evaluated. RESULTS: Visual acuity of 20/40 or better was obtained in 71% of eyes with treated extrafoveal choroidal neovascularization and 68% with treated juxtafoveal choroidal neovascularization. Recurrent choroidal neovascularization was observed in 23% of treated eyes during a mean follow-up of 9.6 years. CONCLUSION: Results support the long-term benefit of photocoagulation and need for careful follow-up. PMID- 7535998 TI - Immune recognition of genetically diverse simian T-cell lymphotropic virus type I isolates. AB - Nucleotide sequence analysis of selected regions of the gag, pol, env and pX genes of simian T-cell lymphotropic virus type I (STLV-I) strains indicated that African isolates were more closely related to human T-cell lymphotropic virus type I (HTLV-I) than Asian isolates. Despite these recent comparative studies on nucleotide sequence homology between HTLV-I and STLV-I isolates, only limited information is available regarding the influence of genetic differences on antigen-antibody recognition of distinct STLV-I strains. In this study, we demonstrated that sera from STLV-I-infected yellow baboons (Papio cynocephalus) reacted strongly with env gp62/68 from HTLV-I-infected cell lines MT-2 and C10/MJ. In contrast, sera from Japanese macaques (Macaca fuscata) naturally infected with Asian STLV-I had weak reactivity to env gp62/68 of these prototypic HTLV-I strains. Pst-1 restriction enzyme analysis of proviral DNA indicated that the baboon virus isolates were more closely related to HTLV-I than the Japanese isolates. These results indicate that nucleotide sequence diversity, correlates with variations in proviral restriction enzyme sites and antibody recognition of viral envelope proteins. These differences in immunoreactivity may have important implications for serologic diagnosis, as well as epidemiological and vaccine studies of STLV-I infection. PMID- 7536000 TI - Vessel formation by choroidal endothelial cells in vitro is modulated by retinal pigment epithelial cells. AB - OBJECTIVE: To elucidate the mechanism of bovine choroidal endothelial (BCE) cell angiogenesis and, in particular, the role of retinal pigment epithelial (RPE) cells by use of an in vitro coculture assay system. METHODS: The BCE cells were isolated from choroidal tissues and cultured. They were embedded in type I collagen gel and incubated. The gel-embedded BCE cells were then covered with a monolayer of RPE cells, pericytes, choroidal fibroblasts, or additional BCE cells on culture day 0 or day 14. The BCE cells in culture formed a meshwork of tubelike structures. The length of the tubelike structures (micrometers per field) was quantified by image analysis, as an indicator of angiogenesis. The effect of RPE cells on normal and growth-arrested BCE cell tube formation was evaluated in this assay system. The mechanism of RPE-induced angiogenesis was studied by parallel experiments with the use of neutralizing antibodies against specific growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor beta). RESULTS: Ultrastructural analysis revealed that the tubelike structures had features typical of choroidal endothelial cells. Cocultures initiated on day 0 revealed that BCE cell angiogenesis was promoted by overlying RPE cells and, to a lesser extent, by pericytes, choroidal fibroblasts, and additional BCE cells when compared with BCE cells without covering cells. In cocultures initiated after BCE tube formation (day 14), there was inhibition of BCE angiogenesis by overlying RPE cells when compared with cultures without overlay or with an overlay of BCE cells. The RPE cells stimulated tube formation of growth-arrested BCE cells less effectively than did normal BCE cells. Neutralizing antibody for basic fibroblast growth factor and vascular endothelial growth factor, but not transforming growth factor beta, inhibited control and RPE-induced tube formation by BCE cells. CONCLUSIONS: Overlying RPE cells stimulate the formation of tubelike structures by choroidal endothelial cells more effectively than do fibroblasts or pericytes but inhibit BCE tube formation in older cultures. The effect involves endothelial proliferation and differentiation. The stimulatory effect of overlying RPE cells can be inhibited by neutralizing antibodies to vascular endothelial growth factor and basic fibroblast growth factor, which suggests that these growth factors play an important role in this phenomenon. PMID- 7536001 TI - Comparison of silk sutures and n-butyl-2-cyanoacrylate on the healing of skin wounds. A pilot study. AB - The synthetic tissue adhesive n-butyl-2-cyanoacrylate (Histoacryl Blue) and silk sutures were compared on the backs of guinea-pigs as the means for closing skin incisions. On the post-operative 3rd, 7th, 14th and 21st days, specimens from each group were obtained and evaluated under light microscopy. Sections were analysed for the degree of inflammation, giant cell reaction, fibroblastic and capillary cell activity and were ranked statistically. The results showed that the silk sutures caused severe inflammatory and giant cell reaction when compared with Histoacryl and the healing process was slower than with n-butyl-2 cyanoacrylate. PMID- 7536002 TI - Quantitation of immunogold with an interactive image analysis system. A new, practical approach to antibody-induced modulation, internalization and intracellular transport of surface antigens in viable hematopoietic cells. AB - This work aimed to evaluate the value of computerized quantitative analysis in ultrastructural studies on internalization and intracellular transport of leukocyte antigens traced with immunogold in viable lymphoid cells. The time of analysis and the results were compared with those obtained with a standard method based on counting gold particles on electron micrographs. A commercially available, personal computer-based, single-screen system was used for capturing and processing the ultrastructural images, counting gold particles and presenting data. The viable lymphoblasts of RAJI and NALM-6 cell lines were labeled with B4 (anti-CD19) murine monoclonal antibody followed by rabbit antimouse immunoglobulins coupled to 12.8-nm colloidal gold, cultured for six hours to allow internalization of CD19 antigen, collected after various periods of time and processed for electron microscopy. Each sample was analyzed by counting the gold particles on 30 randomly chosen, equatorial cell sections. Surface-bound particles and those located within certain intracellular compartments (that is, cytoplasmic vesicles, multivesicular bodies and lysosomes) were counted separately. No significant differences were found between the results obtained using electron micrographs and those yielded by on-screen analysis of digitized images. However, the time of analysis varied considerably--approximately eight and two hours, respectively. Also, using computerized analysis saved the cost of photographic processing. In conclusion, the system provided a reliable, rapid and inexpensive alternative for quantitation of immunogold-labeled leukocyte antigens at the ultrastructural level. PMID- 7536003 TI - Semiautomated nuclear shape analysis of prostatic carcinoma and benign prostatic hyperplasia. AB - Nuclear shape analysis has predicted outcome better than histologic grading in patients with clinically localized prostatic carcinoma. However, the requirement for manual nuclear contour tracing makes the method tedious and slow. Currently available image analysis systems for nuclear shape analysis using light absorption microscopy provide nuclear boundaries of insufficient clarity for automatic segmentation. We improved image resolution using confocal laser scanning microscopy, automatically detected nuclear boundaries by a multiscale segmentation algorithm and discriminated artifacts in a semiautomated way. A manual quantitative morphometry system and our semiautomated system distinguished eight cases of prostatic carcinoma from seven cases of benign prostatic hyperplasia by nuclear roundness factor, ellipticity, nuclear area and perimeter. The ease of semiautomated nuclear shape analysis should allow evaluation of large numbers of patients with known outcomes after treatment for clinically localized prostatic carcinoma to determine whether nuclear shape analysis can be extended from research to clinical usage. PMID- 7536004 TI - NADPH-diaphorase/nitric oxide synthase containing neurons in normal and Parkinson's disease putamen. AB - Nitric oxide (NO) is thought to be involved in neurodegenerative processes. Concerning Parkinson's disease (PD) it remains to be elucidated, if NO contributes to pathological alterations in the striatum. The present study evaluates the post-mortem putamen of PD patients and control subjects for distribution patterns of NO-synthase containing neurons, using the NADPH diaphorase technique. The ratio of positively stained neurons and the total number of cells (control: 1,120 +/- 69 per mm2, n = 5; PD: 575 +/- 164mm2, n = 5) shows striking differences between controls and PD patients. Our findings give reason to conclude that NADPH-diaphorase positive structures may have pathogenetic importance in degenerative processes in PD putamen. PMID- 7536005 TI - Markedly elevated alpha-fetoprotein and positive acetylcholinesterase in amniotic fluid from a pregnancy affected with dystrophic epidermolysis bullosa. AB - Prenatal diagnosis of dystrophic epidermolysis bullosa (DEB) has been achieved in the past by fetal skin sampling. However, this invasive procedure is associated with a relatively high rate of pregnancy loss. We present a consanguineous Arab family ascertained by 2 affected offspring to be at risk for DEB. In a previous gestation, fetoscopic skin sampling for prenatal diagnosis yielded a false positive result. In the index pregnancy, abnormally elevated amniotic fluid alpha fetoprotein (13.7 MOM) and positive acetylcholinesterase were highly suggestive of an affected fetus. Fetal skin biopsy was declined. At term, the patient delivered a male infant with DEB that expired on the 3rd day of life. It is apparent from our experience and from review of the literature that in some genodermatoses, markedly elevated alpha-fetoprotein and positive acetylcholinesterase in amniotic fluid are highly suggestive of an affected fetus and may obviate the need for fetal skin sampling in the prenatal diagnosis of these disorders. PMID- 7536006 TI - Fetomaternal hemorrhage: diagnostic problems. Three case reports. AB - This report describes 3 successive cases in which fetomaternal hemorrhage was suspected but confirmed in only 2. The manifestations of fetomaternal hemorrhage are often nonspecific and diagnosis can be difficult. We discuss diagnostic methods, especially the value of the Kleihauer-Betke test and maternal serum alpha-fetoprotein measurement, and approach. PMID- 7536007 TI - Characterization of the 5'-flanking region of the human preprogalanin gene. AB - Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and 122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor. PMID- 7536008 TI - Long-term maintenance of liver-specific functions in three-dimensional culture of adult rat hepatocytes with a porous gelatin sponge support. AB - The three-dimensional culture of adult rat hepatocytes with a porous gelatin sponge (gelfoam) support was investigated. Hepatocytes were immobilized on the surface as well as within the pores of the support. The morphology of the hepatocytes immobilized on the support was close to that observed in vivo. In some parts of the support the spheroids of hepatocytes could be observed. To examine the liver-specific functions of the hepatocytes in the culture, the levels of serum albumin and bile acids secreted into the medium were assessed. The secretion of albumin and bile acids was stable over the course of 12 days, longer than that in collagen-gel culture. To elucidate further the function of hepatocytes immobilized on gelfoam, the metabolic activities of the hepatocytes, as measured by the competency of removal of NH4+ and the synthesis of urea nitrogen, were determined. The rates of ammonium removal and urea nitrogen synthesis were comparable with those in conventional monolayer culture. Albumin secretion was enhanced by the treatment of gelfoam with either heparin or acidic fibroblast growth factor (aFGF), the gelfoam having a high affinity for these substances. DNA synthesis was also enhanced by aFGF. These results demonstrate that gelfoam is a suitable support for the in vitro culture of hepatocytes. Combined with its easy manipulation, it is suggested that the culture system described could be used for both basic and applied studies. PMID- 7536009 TI - The expression of self antigenic determinants: implications for tolerance and autoimmunity. AB - T lymphocytes respond to small peptides in the context of major histocompatibility molecules and a host of other cell-surface proteins on antigen presenting cells. By design, therefore, T-cell responses are dependent on the efficient and accurate processing of both foreign and self peptides by antigen presenting cells. This review examines the functions of T cells that may be specific for self peptides processed and presented under less than ideal conditions or outside the normal pathways of antigen processing. Do these T cells survive selection events and remain in the repertoire of normal lymphocytes? Moreover, can these cells become activated and are they important in the pathogenesis of autoimmunity? PMID- 7536010 TI - Self-determinants in autoimmune demyelinating disease: changes in T- cell response specificity. AB - Recent research developments support the following views regarding antigen recognition in autoimmune demyelinating disease: there may be no single autoimmune target protein; diverse peptide self-determinants from multiple myelin proteins can be recognized; target determinant epitopes may differ among individuals; and target epitope recognition can change with time during the course of disease. PMID- 7536011 TI - TCR V gene usage in autoimmunity. AB - With the use of polymerase chain reaction technology, investigators now have the ability to assess, in a comprehensive and rapid manner, the entire repertoire of T cell antigen receptors expressed by pathogenic cells present in virtually any disease site. A summary of studies of T cell receptor variable gene usage suggests that preferential expression can be identified most reproducibly when cells are isolated directly from pathogenic lesions. This provides a framework for future investigations in other autoimmune settings. PMID- 7536012 TI - Apoptosis, Fas and systemic autoimmunity: the MRL-lpr/lpr model. AB - Proteins encoded by the fas and fas ligand (fasL) genes are involved in apoptotic cell death in lymphocytes. In this article we review the recent elucidation of the role of the Fas-FasL interactions in the maintenance of tolerance to self antigens and in the homeostatic regulation of lymphocyte clonal expansion, and discuss the mechanisms of autoimmunity in Fas- and FasL-deficient mutant mouse strains. PMID- 7536013 TI - The subthalamo-nigral pathway regulates movement and concomitant acetylcholinesterase release from the substantia nigra. AB - Within the substantia nigra acetylcholinesterase is released independently of cholinergic transmission: this release could be related to some aspects of motor control. To investigate this possibility, acetylcholinesterase release was continuously monitored in relation to specific movements evoked by central electrical stimulation. Increased intensities of stimulation of the subthalamic nucleus in awake guinea-pigs produced a behavioural response, ranging from a decrease in spontaneous movement, to chewing, to both chewing and circling movements. Enhancement of acetylcholinesterase release occurred only when large scale movements (circling as well as chewing) were evoked by subthalamic stimulation: however, a similar protocol of stimulation during ketamine-induced anaesthesia did not produce any comparable movements nor any concomitant change in the release of acetylcholinesterase. Perfusion of the glutamate agonist N methyl-D-aspartate (NMDA) into the substantia nigra also induced an increase in release of acetylcholinesterase from the substantia nigra of conscious animals, whereas (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) did not significantly enhance acetylcholinesterase levels. It is concluded that AChE release in the substantia nigra can occur as a result of activation of glutamatergic subthalamic afferents, and that this activation may also be associated with changes in movement. PMID- 7536014 TI - Contrast-enhanced dynamic MR imaging of postmolar gestational trophoblastic disease. AB - Conventional spin-echo (SE) and contrast-enhanced dynamic MR imaging were performed on a 1.5 T superconductive unit for evaluation of myometrial lesions in postmolar gestational trophoblastic disease (GTD) in 10 women. MR imaging was done at the time of the initial examination (n = 10), during (n = 6), and after repeated courses of chemotherapy (n = 10). The T2-weighted SE image revealed an enlarged uterus (n = 7), disappearance of zonal anatomy (n = 6), and heterogeneous signal intensities (n = 8) with prominent flow voids (n = 7). However, these abnormalities remained after repeated courses of chemotherapy, when the S-beta-HCG level returned to the normal range. Myometrial lesions characteristically had marked enhancement with areas of unenhancement on dynamic MR images in patients with highly elevated S-beta-HCG. Areas of contrast enhancement correlated with changes in S-beta-HCG level. The enhancement was reduced with decrease in S-beta-HCG level after repeated courses of chemotherapy. Six of 8 masses seen on T2-weighted images proved to be active trophoblastic lesions and 2 masses proved to be hematoma or necrosis. In 2 patients, abnormal myometrial lesions were detected only on contrast-enhanced dynamic MR imaging. These preliminary data indicate that contrast-enhanced dynamic MR imaging more clearly demonstrates myometrial involvement of postmolar GTD than conventional SE imaging. PMID- 7536015 TI - Recognition of surface antigens on spermatozoa of the common carp (Cyprinus carpio L., Teleostei) using monoclonal antibodies and scanning electron microscopy. AB - The distribution of antigenic determinants, recognized by seven anti-carp spermatozoa monoclonal antibodies (MAbs) and two anti-carp spermatogonia MAbs were studied using fresh, unfixed carp spermatozoa. The location of the antigenic determinants was analysed in light- and in scanning electron microscopy, in the latter with the backscattered imaging mode. With all seven anti-carp spermatozoa MAbs a similar regular distribution of the immuno-gold labeling was present on heads, midpieces and tails of carp spermatozoa, whereas with the anti-carp spermatogonia MAbs only low or no labeling was observed. The regular distribution of antigenic determinants, as observed with the anti-spermatozoa MAbs in carp, is in agreement with data on agglutination of spermatozoa by autoantibodies in Salmo gairdneri and data on distribution of antigenic sites on spermatozoa in Xenopus laevis. It differs from data in mammals in which the presence of specialized domains on spermatozoa was shown. These results are discussed. PMID- 7536016 TI - Light microscopic studies of the stomach of the lesser mouse deer (Tragulus javanicus). AB - The stomach of the lesser mouse deer was studied at the light microscopic level using histological and immunohistochemical methods. The stomach was clearly differentiated into rumen, reticulum including reticular groove, a small transition zone and abomasum. The mucosal surface of the rumen, reticulum and transition zone was lined with a stratified squamous epithelium and that of the abomasum with a simple columnar type. The epithelial keratinization was weak in the rumen, floor of the reticular groove and transition zone, while it was strong in the reticulum, especially on the tip of the reticulum papillae. Large sinusoidal capillaries were often present in the ruminal papillae. In the ruminal mucosa, a thin layer of alpha-smooth muscle actin immunoreactive cells was demonstrated by immunohistochemistry. The muscularis mucosae of the reticulum was continuous and well-developed. The transition zone appeared as a nonglandular area having many low mucosal folds and two layers of tunica muscularis. The abomasal mucosa consisted of cardiac, proper gastric and pyloric glands. Cells immunoreactive for bovine pepsinogen and bovine prochymosin antisera were demonstrated in the abomasum. It is suggested that the characteristic features observed might be adaptations to a relatively rapid passage and rapid absorption of the fermentation products. There is some evidence that the transition zone is not a part of either the floor of the reticular groove or the abomasum, suggesting a possible reevaluation of the term used for the reticulo-abomasal orifice in the mouse deer. PMID- 7536017 TI - P-glycoprotein homologues. PMID- 7536018 TI - Biochemical modulation as an approach to reversal of antimetabolite resistance. PMID- 7536019 TI - Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands. AB - The corticotropin releasing factor (CRF) receptor is known to be coupled to Gs and transduces its signal through stimulation of cyclic AMP (cAMP) production. Here we describe the characterization of several stable CRF receptor-expressing LVIP2.0Zc cell lines that also contain an exogenous cAMP-responsive beta galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of beta-galactosidase in response to CRF. Rat/human and bovine CRF stimulated beta-galactosidase activity in a dose-dependent manner with EC50 values of approximately 0.1 nM; the biologically weak deamidated analog of bovine CRF was approximately 500-fold less potent. The CRF receptor antagonist, [d-Phe12,Nle21,38,Ala32]r/hCRF(12-41) produced a dose-dependent inhibition of CRF stimulated beta-galactosidase activity, further demonstrating the pharmacological specificity of the interaction. The magnitude of the maximal response to CRF varied among individual cell lines. This variation was independent of the level of CRF receptor expression, but reflected differences in the intrinsic activity of adenylate cyclase. In contrast to most cAMP assay systems, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine decreased the CRF-induced beta-galactosidase activity when used in the context of the assay regimen described here. Since the assay can be easily performed in a high-throughput 96 well plate format, these cell lines provide an efficient way for the identification of CRF receptor agonists and antagonists. PMID- 7536020 TI - Rapid isolation of tissue-specific and developmentally regulated brain cDNAs using RNA arbitrarily primed PCR (RAP-PCR). AB - RNA arbitrarily primed PCR (RAP-PCR) was used to isolate cDNAs that represent developmentally regulated brain-specific genes. Five clones with a restricted pattern of expression were identified and sequenced. Four cDNAs had no obvious homology to the sequences in GenBank. One clone had over 95% homology to a Ca2+/calmodulin-insensitive adenylyl cyclase, a recently cloned gene that was isolated from rat brain and was shown to be expressed only in adult brain and lung. Two novel cDNAs were investigated further by Northern blot analysis and were found to be expressed differentially during development; their expression was confined to the forebrain in the adult mouse. Further characterization by in situ hybridization showed that the mRNA corresponding to one clone was localized to a limited number of differentiating functional structures in the developing nervous system. In the adult brain, this message is confined to the forebrain with the highest level of expression in the cortex. These data suggest that the product of this gene is involved in the establishment of neuronal networks during brain development and in synaptic plasticity in the mature cortex. This work demonstrates that RAP-PCR is a powerful method for the simultaneous detection of differences between multiple RNA populations and, as such, can be used to study differential gene expression in the brain. PMID- 7536021 TI - Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay. AB - We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro. PMID- 7536022 TI - Transient perfusion in human melanoma xenografts. AB - Studies of transplantable rodent tumours have suggested that malignant tissue might experience transient perfusion at the microvascular level. The purpose of the work reported here was to investigate whether transient perfusion can be demonstrated in xenografted human tumours. Tumours of four melanoma lines (A-07, D-12, R-18, U-25), grown orthotopically in Balb/c nu/nu mice, were included in the study. Transient perfusion was studied by using the double-fluorescent staining technique. Hoechst 33342 and DiOC7(3) were either administered simultaneously or Hoechst 33342 was administered 20 min before DiOC7(3). Detection of transient perfusion by this method requires that vessels are non functional for at least 5 min owing to the distribution half-lives of the dyes in the blood. Usable combinations of dye concentrations were found by varying the concentrations of Hoechst 33342 and DiOC7(3) systematically. The level of perfusion mismatch following simultaneous administration of the dyes ranged from approximately 1.5% for U-25 tumours to approximately 3.0% for R-18 tumours at these combinations. Moreover, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly different whether the dyes were administered simultaneously or sequentially. Transient perfusion could not be demonstrated in any of the tumour lines. Thus, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly higher after sequential than after simultaneous administration of the dyes. Moreover, the vessels stained only with Hoechst 33342 and the vessels stained only with DiOC7(3) were randomly distributed within the tumours whether the dyes were administered simultaneously or sequentially. Consequently, acute hypoxia caused by transient perfusion is probably a less pronounced phenomenon in malignant tissue than previous studies of rodent tumours have suggested. PMID- 7536025 TI - High-dose carboplatin, thiotepa and cyclophosphamide (CTC) with peripheral blood stem cell support in the adjuvant therapy of high-risk breast cancer: a practical approach. AB - In 29 chemotherapy-naive patients with stage II-III breast cancer, peripheral blood stem cells (PBSCs) were mobilised following fluorouracil 500 mg m-2, epirubicin 90-120 mg m-2 and cyclophosphamide 500 mg m-2 (FEC) and granulocyte colony-stimulating factor (G-CSF; Filgrastim) 300 microgram s.c. daily. In all but one patient, mobilisation was successful, requiring three or fewer leucocytopheresis sessions in 26 patients; 28 patients subsequently underwent high-dose chemotherapy consisting of carboplatin 1600 mg m-2, thiotepa 480 mg m-2 and cyclophosphamide 6 g m-2 (CTC) followed by PBSC transplantation. Haemopoietic engraftment was rapid with a median time to neutrophils of 500 x 10(6) l(-1) of 9 days (range 8-10) in patients who received G-CSF after PBSC-transplantation; platelet transfusion independence was reached within a median of 10 days (range 7 16). Neutropenic fever occurred in 96% of patients. Gastrointestinal toxicity was substantial but reversible. Renal, neural or ototoxicity was not observed. Complications related to the central venous catheter were encountered in 64% of patients, with major vein thrombosis occurring in 18%. High-dose CTC-chemotherapy with PBSC-transplantation, harvested after mobilisation with FEC and G-CSF, is reasonably well tolerated without life-threatening toxicity and is a suitable high-dose strategy for the adjuvant treatment of breast cancer. PMID- 7536023 TI - Immortalisation of human oesophageal epithelial cells by a recombinant SV40 adenovirus vector. AB - We introduced the origin-defective SV40 early gene into cultured human oesophageal epithelial cells by infection of a recombinant SV40 adenovirus vector. The virus-infected cells formed colonies 3-4 weeks after infection in medium containing fetal calf serum. When the cells derived from 'serum-resistant' colonies were then maintained in the serum-free medium with a low calcium ion concentration, some of them passed the cell crisis and kept growing for over 12 months. These cells, regarded as immortalised cells, resembled the primarily cultured oesophageal epithelial cells in morphology and had some of their original characteristics. Treatment of the cells with a high calcium concentration induced phenotypic changes. These cells still responded to transforming growth factor beta. When the immortalised cells were injected into severe combined immunodeficient mice, they transiently formed epithelial cysts, although the typical differentiation pattern of the oesophageal epithelium was not observed. These cysts regressed within 2 months without development into tumours. The results indicated that human oesophageal epithelial cells were reproducibly immortalised by infection with a recombinant SV40 adenovirus vector at relatively high efficiency. The immortalised cells should be useful in studies on oesophageal carcinogenesis and in assessing the cooperative effects with other oncogene products or carcinogens. PMID- 7536024 TI - Indoloquinone EO9: DNA interstrand cross-linking upon reduction by DT-diaphorase or xanthine oxidase. AB - We report DNA interstrand cross-linking caused by the anti-tumour indoloquinone EO9 following reductive activation with purified rat liver DT-diaphorase or xanthine oxidase. Reduction was a necessary event for cross-linking to occur. DNA cross-link formation by EO9 following DT-diaphorase reduction was completely inhibited by addition 10 microM dicoumarol, whereas only a minor effect of dicoumarol on xanthine oxidase-mediated DNA cross-linking by EO9 was observed. DNA cross-linking was pH dependent, with increasing cross-link formation from pH 5.5 to 7.0 for both DT-diaphorase and xanthine oxidase mediated reactions. Also, conversion of EO9 upon reduction was pH dependent. However, in contrast to DNA cross-linking, conversion rates of EO9 decreased at higher pH. EO9 was shown to be more efficient in DNA cross-linking than mitomycin C under identical conditions, using both DT-diaphorase and xanthine oxidase reductive activation at pH 5.5 and 7.0. This study indicates that the anti-tumour activity of EO9 may be at least partly mediated by interstrand DNA cross-link formation, and that various reducing enzymes may be important for activation of EO9 in vitro and in vivo. PMID- 7536026 TI - Pancreatic carcinoma. AB - The management of patients with pancreatic carcinoma poses many problems. The diagnosis is usually made late, generally because the patients present late, but it is not unusual to find patients who have had many negative investigations for vague upper abdominal symptoms only to be diagnosed as having pancreatic carcinoma many months later. Staging the disease is equally difficult and often inaccurate. The results of treatment are to date discouraging even in those patients diagnosed early. But the outlook is not totally dismal; in recent years the results for surgical resection of pancreatic lesions have improved; adjuvant treatment may finally be having an effect, although small, on this relentless disease. The most notable inroad made in the management of pancreatic cancer in the last 10 years is the improvement in palliation due to the use of the endoprosthesis. In spite of the poor results we must continue to search actively for more accurate methods of diagnosis and better methods of treatment. PMID- 7536027 TI - Secondary tumours following etoposide containing therapy for germ cell cancer. AB - BACKGROUND: Reports have implied etoposide as the cause of secondary leukaemia in patients treated for germ cell cancer. PATIENTS AND METHODS: Between 1979 and 1992, 679 male patients with germ cell cancer received etoposide containing chemotherapy. RESULTS: Six of 679 patients developed acute myeloid leukaemia (relative risk 150; CI: 55-326). None of these patients had a primary mediastinal germ cell tumour and only 1 patient received previous radiotherapy. The median interval between the onset of cytotoxic treatment and the development of leukaemia was 27 months. The FAB M4 morphology was seen in 4 of 6 cases. CONCLUSION: The benefit of etoposide containing protocols outweigh the risk of leukaemia in patients with intermediate or high risk disease, however in patients with good risk disease non-etoposide containing protocols should be explored. PMID- 7536028 TI - Quality versus quantity of life in the treatment of patients with advanced small cell lung cancer? A randomized phase III comparison of weekly carboplatin and teniposide versus cisplatin, adriamycin, etoposide alternating with cyclophosphamide, methotrexate, vincristine and lomustine. Swiss Group for Clinical Cancer Research (SAKK). AB - BACKGROUND: Based on a promising pilot study with weekly carboplatin and teniposide (CBDCA/VM) the Swiss Group for Clinical Cancer Research (SAKK) performed a randomised phase III trial in patients with extensive-disease small cell lung cancer aimed at the development of an effective palliative treatment with low subjective toxicity. PATIENTS AND METHODS: From September 1989 to September 1991 patients were randomised to a weekly regimen of CBDCA/VM or to our 'standard chemotherapy' of cisplatin, adriamycin and etoposide alternating with cyclophosphamide, methotrexate, vincristine and lomustine (PAV-CyMOC). RESULTS: The trial was closed before the planned accrual of 140 evaluable patients due to a significant survival difference shown by an interim analysis. Of the 61 patients 59 were eligible and included in the final analysis. The results achieved with the PAV-CyMOC regimen were significantly better than those observed in patients treated with weekly CBDCA/VM (remission rate of 65% vs. 29%; p = 0.006). The median survival of patients treated with the PAV-CyMOC combination was significantly longer than that of patients receiving weekly CBDCA/VM (260 days vs. 147 days; p = 0.0035). The 1-year survival rate was 30% in the PAV-CyMOC arm compared to 4% in the CBDCA/VM-treated patients. As expected, side effects including myelosuppression, alopecia and mucositis were significantly more pronounced in patients treated with the PAV-CyMOC regimen. No significant difference was found in patient-rated tumor symptoms and general quality-of-life categories. CONCLUSION: Contrary to our initial expectation that we would achieve similar therapeutic results with less subjective toxicity, in this randomised prospective trial the results achieved by weekly carboplatin and teniposide were significantly inferior in terms of remission rate and survival to those of our 'standard regimen' of cisplatin, adriamycin and etoposide alternating with cyclophosphamide, methotrexate, vincristine and lomustine. The weekly regimen was less toxic than the standard chemotherapy. Whether patients are willing to accept a significant trade-off between quantity and quality of life remains to be evaluated. PMID- 7536029 TI - Myelo-ablative therapy with peripheral blood progenitor cell (PBPC) support in patients with haematological malignancy. AB - BACKGROUND: Myelo-ablative therapy with peripheral blood progenitor cell (PBPC) support is increasingly being used in patients with haematological malignancy considered to be at high risk for recurrence. The results of this approach, in comparison with the previous experience at St. Bartholomew's Hospital (SBH) using autologous bone marrow transplantation form the basis of this report. PATIENTS AND METHODS: 42 patients (age range 18-63 years, median 42 years), deemed to have a poor prognosis with conventional therapy received myelo-ablative therapy with PBPC support. Diagnoses comprised: non-Hodgkin's lymphoma (NHL): 16 patients, Hodgkin's disease (HD): 9, Multiple Myeloma (MM): 12, and solid tumours (ST): 5. PBPC were mobilised using adriamycin: 35 mg/m2 i.v. on day 1 and etoposide 100 mg/m2 orally, days 1-5, followed by G-CSF: 5 micrograms/kg, subcutaneously, for a median of 7 days (range 6-9 days). RESULTS: A total of 67 PBPC collections were performed, 1 being 'sufficient' (i.e. mononuclear cells > or = 1.5 x 10(8)/kg and CD34+ cells > or = 1 x 10(6)/kg) in 21 of the 42 patients. The median time to haematological recovery following reinfusion of PBPC was 13 days for both neutrophils > 0.5 x 10(9)/l and platelets > 20 x 10(9)/l (ranges: 8-27, and 8-48 days, respectively) which is significantly shorter than for patients in the historical control group. Supportive care requirements were also significantly reduced, as was the duration of hospital stay i.e., median 19 days (range 12-73 days) compared with 29 days (range 9-180 days). CONCLUSION: These results confirm rapid blood count recovery following myelo-ablative therapy with PBPC support and the feasibility of this approach. PMID- 7536030 TI - Microvessel density, endothelial cell proliferation and tumour cell proliferation in human colorectal adenocarcinomas. AB - BACKGROUND: Thymidine incorporation studies performed in animal tumour models, revealed major differences in endothelial cell proliferation when tumour tissue was compared with normal tissue. The fraction of proliferating endothelial cells is reported to be increased by a factor of 30 to 40 in tumour tissue. PATIENTS AND METHODS: To make it possible to analyze the endothelial cell proliferation in human tumours, an immunohistochemical double staining technique comprising CD31, an endothelial cell marker, and Ki-67, a proliferation marker, was developed. Endothelial cell proliferation was analysed in 21 primary human colorectal adenocarcinomas and in the adjacent mucosa. RESULTS: Proliferating endothelial cells were found throughout the entire carcinoma. The mean overall endothelial cell labeling index (ECLI) was 9.9% (range, 5.4-18.0), and the labeling index of endothelial cells in areas of intense neovascularisation was even higher. Mean ECLI in the vascular hot spots was 21.0% (range, 6.8-35.0), and the mean tumour cell labeling index (TCLI) in the maximally Ki-67 immunostained areas was 78.3% (range 47.0-89.7). In 14 of 21 carcinomas, these areas were predominantly found at the luminal margin of the tumour, as were the vascular hot spots. A significant positive correlation was found between tumour vascularity, measured in the vascular hot spots, and tumour cell proliferation, measured in the maximally Ki-67 immunostained areas (p < 0.05). To analyse this relation in more detail, microvessel density (MVD), TCLI and ECLI were determined per x400 microscopic field by scanning in sequence from the luminal tumour margin to the invasive tumour base. In all tumours, the pattern of the MVD per x400 field, from the luminal margin to the tumour base, was similar to that of the TCLI and ECLI. CONCLUSIONS: These findings confirm that the fraction of cycling endothelial cells is higher in human colorectal carcinoma than in the adjacent mucosa which suggests that endothelial cells are proliferating in most of the individual capillaries in tumour tissue. Regional differences in MVD correlate with differences in tumour cell proliferation in these tumours. PMID- 7536032 TI - Detection at diagnosis of tumor cells in bone marrow aspirates of patients with small-cell lung cancer (SCLC) and clinical correlations. AB - BACKGROUND: Immunocytochemistry has often been used to identify tumor cells in bone marrow aspirate (BMA) of SCLC patients in order to improve the results of conventional histomorphology. However, whether the detection of bone marrow microlocalisation at diagnosis had implications for prognosis has not been clear. PATIENTS AND METHODS: Eighty-four slides (44 patients) and 66 bone marrow biopsies (from 42/44 patients) were evaluated. Cytospins of BMA were incubated with the monoclonal antibody (MAb) NCC-LU-243, recognising the cluster 1 antigen (NCAM) and then stained by the APAAP (alkaline phosphatase-anti-alkaline phosphatase) method. The relationship among BMA and PS (performance status), NSE (neuron-specific enolase), stage, survival was also studied. RESULTS: 33/84 (39%) BMA were positive for NCAM, compared with 8/66 (12%) bone marrow biopsies (p = 0.009), (17/44 and 6/42 patients, respectively). Moreover, BMA was positive for NCAM in 6/19 patients with limited disease. The presence of positive BMA did not correlate with PS, NSE or stage, but patients with positive BMA had shorter survivals than those with negative BMA (median survival: 7 and 12 months, respectively, p = 0.007). CONCLUSIONS: Bone marrow involvement detected by immunocytochemistry appears to be related to survival but not to parameters of tumor burden (NSE, stage), suggesting that this technique might help to select patients with better prognoses for new therapeutic strategies. PMID- 7536031 TI - Phase II study of carboplatin and continuous infusion bleomycin followed by cisplatin and 5-fluorouracil in recurrent head and neck cancer. AB - BACKGROUND: Recurrent squamous cell carcinoma of the head and neck is poorly responsive to most chemotherapy regimens. Carboplatin and bleomycin are effective single agents with non-overlapping toxicity; therefore, we sought to explore the efficacy of this regimen in a phase II study. In the second stage of the study, patients who did not respond to carboplatin and bleomycin were given treatment with cisplatin and 5-fluorouracil (5-FU). PATIENTS AND METHODS: Patients with recurrent squamous cell carcinoma of the head and neck were treated with carboplatin 400 mg/m2 followed by bleomycin 15 units intravenously as a continuous infusion for 4 days. Patients with no tumor response after 3 cycles of carboplatin and bleomycin were crossed-over to receive cisplatin 100 mg/m2 and 5 FU 1000 mg/m2/day continuous infusion for 5 days. RESULTS: Among the 20 carboplatin-bleomycin patients evaluable for toxicity, no cases of grade 4 granulocytopenia were reported and grade 3 or 4 thrombocytopenia developed in only three patients. Three partial responses occurred among the 19 patients (16%) [95% C.I. 0% to 32%] evaluable for response to carboplatin-bleomycin. None of the 11 patients crossed-over to cisplatin and 5-FU had a major response. CONCLUSION: The combination of carboplatin and bleomycin is well tolerated in patients with recurrent head and neck cancer, but the activity does not appear to be superior to the activity of either agent alone. Patients who did not respond to carboplatin and bleomycin were also resistant to the cisplatin and 5-FU regimen. PMID- 7536033 TI - Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H. AB - The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template. PMID- 7536034 TI - Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides. AB - Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus. PMID- 7536035 TI - Structural and functional characterization of DsbC, a protein involved in disulfide bond formation in Escherichia coli. AB - DsbC is a soluble protein of the bacterial periplasm that was identified genetically as being involved in protein disulfide formation. The gene sequence was corrected to include an additional proline residue and was then consistent with the molecular weight of the purified protein. Gel filtration and subunit hybridization indicate that DsbC is a stable dimer of identical subunits. Each subunit has a -Cys-Gly-Tyr-Cys- segment that forms an unstable and reactive disulfide bond; only the first cysteine residue is accessible, similar to thioredoxin and DsbA. The other two cysteine residues of DsbC form a buried, structural disulfide bond. The reactivities and stabilities of the active site disulfide bond of DsbC have been characterized and compared to that of DsbA. Both are very unstable and can be transferred rapidly to reduced proteins and peptides, although they differ somewhat in their kinetic reactivities. The two active sites of the DsbC dimer appear to function independently. DsbC is much more active than DsbA in catalyzing protein disulfide rearrangements, and this may be its main function in vivo. PMID- 7536036 TI - Control of the alpha 5 beta 1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin. AB - Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector. PMID- 7536037 TI - Synthesis and interaction of fluorescent thapsigargin derivatives with the sarcoplasmic reticulum ATPase membrane-bound region. AB - Fluorescent derivatives of thapsigargin (TG) were synthesized by replacing the C8 butanoyl chain with a dansyl (DTG) or eosin (ETG) moiety. DTG and ETG retain the inhibitory effect of TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 and 10 microM Ki, respectively. Steady state and lifetime fluorescence measurements are consistent with energy transfer between tryptophanyl residues assigned to the ATPase membrane-bound region and DTG. This phenomenon exhibits saturation behavior, occurs in the presence of DTG concentrations producing ATPase inhibition, and is partially prevented by inhibitory concentrations of TG. Although long range conformational effects of TG binding affect the fluorescence properties of endogenous tryptophans as well as of a fluorescein 5' isothiocyanate (FITC) label of the ATPase extramembranous region, no significant energy transfer was detected between DTG and the FITC label. It is concluded that the inhibitors partition within the membrane and the binding domain resides within or near the membrane-bound region of the ATPase. PMID- 7536038 TI - Structural specificity of substrate for S-adenosylmethionine:protein arginine N methyltransferases. AB - The enzymatic methylation of polypeptides on the guanidino group of internal arginine residues by S-adenosylmethionine:protein arginine N-methyltransferase (protein methylase I) yields NG-monomethylarginine, NG,NG-dimethylarginine and NG,NG-dimethylarginine. It has commonly been observed that these arginine residues are present in glycine-and-arginine rich motifs. To understand structural features which are essential for serving as the methyl acceptor for protein methylase I, we have investigated substrate capacities of several synthetic oligopeptides whose sequences are homologous and/or analogous to the methyl acceptor region of the naturally occurring arginine-methylated proteins. These studies have led to the following conclusions. (i) The preferred amino-acid sequence of methyl-accepting peptides was shown to be an arginine-containing peptide with glycine in both the N- and C-flanking positions. While a tetrapeptide with such a sequence (residues 106-109 of bovine myelin basic protein) exhibited almost negligible substrate activity, an overlapping hexapeptide was a moderate substrate. (ii) Substitution of the C-flanking glycine in GKGRGL (residues 104-109 of myelin basic protein) with histidine, phenylalanine, lysine or aspartic acid completely abolished the ability of these hexapeptides to serve as substrates. (iii) A heptapeptide with a repeated glycine arginine motif (GRGRGRG) was an excellent substrate for the enzyme. (iv) A cyclic octapeptide (CGKGRGLC), which was formed by cyclization of GKGRGL by introduction of disulfide bridge to cross-link N- and C-terminus of the hexapeptide, was an even better substrate than the hexapeptide. (v) Upon HPLC amino-acid analysis, all enzymatically methyl-14C-labeled oligopeptides were found to yield predominantly NG-monomethylarginine with a minor fraction of NG,NG dimethylarginine in certain peptide samples. However, no NG,NG-dimethylarginine formation was detectable. (vi) The recombinant hnRNP protein A1 (residues 1-320) is known to be methylated at arginine-194 by nuclear-protein/histone protein methylase I (Rajpurohit et al. (1994) J. Biol. Chem. 269, 1079-1082). However, the hexapeptide (SSSQRG) which corresponds to residues 189-194 of protein A1 containing the methylatable arginine residue was relatively inert as a substrate. Furthermore, the N-terminal fragment of protein A1 (residues 1-196) generated by controlled trypsin digestion was also completely inactive as a substrate for the enzyme. These results indicate that the remainder of the A1 protein molecule plays an important though not yet understood role in enzymatic methylation of the arginine-194. PMID- 7536039 TI - Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src. AB - The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values. PMID- 7536040 TI - Transcriptional regulation of the mts1 gene in human lymphoma cells: the role of DNA-methylation. AB - The transcription of the mts1 gene putatively involved in the control of tumor metastasis was studied in three human lymphoma cell lines: MOLT-4, CEM and Jurkat. The level of the mts1 gene transcription is high in MOLT-4 cells, lower in CEM cells and hardly detectable in Jurkat cells. This correlates with the hypomethylation of DNA in the first exon and the first intron of the mts1 gene in the analyzed culture cells. This area was also found to be undermethylated in human peripheral blood cells--macrophages, neutrophils and lymphocytes where the mts 1 gene is highly expressed. 5-Azadeoxycytidine (AzadC)--an inhibitor of the eukaryotic DNA-methylase--significantly induces the expression of the mts1 gene in CEM and Jurkat cells and has little effect on mts1 gene transcription in MOLT 4 cells. The drug does not influence mts1 transcription in cultivated peripheral blood lymphocytes. These data indicate the possible involvement of the methylation of the first exon/first intron sequences in the transcriptional repression of the mts1 gene. The finding of two DNAaseI hypersensitivity sites (DHSs) mapped in the first intron of the mts1 gene supports this suggestion. PMID- 7536041 TI - Design, synthesis, and sequence selective DNA cleavage of functional models of bleomycin. 1. Hybrids incorporating a simple offal-complexing moiety of bleomycin and lexitropsin carriers. AB - The syntheses of functional models for bleomycin, which are composed of a simple analog of the metal-complexing moiety of bleomycin and oligo-N-methylpyrrole peptide DNA-binding moieties, are described. The extent and the relative rate of their cleavage of DNA in the presence of reductants were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The rate of DNA cleavage increases with the number of N-methylpyrrole units in the carrier moiety. The sequence selectivity of DNA cleavage was demonstrated by polyacrylamide sequencing gel electrophoresis of cleavage reactions on two 5'-32P labeled restriction fragments: a 158 bp GC-rich fragment from pBR322 and a 241 bp AT-rich fragment fragment from SV40. Comparison of the sequence selectivity with that of bleomycin A2 indicates that the poly-N-methylpyrrole moiety directs the hybrid compounds to AT-rich sequences of DNA. High-resolution denaturing gel electrophoresis of DNA cleaved by the model compounds reveals that 3' phosphate is produced exclusively, indicating that the chemistry of DNA cleavage differs from that of bleomycin. PMID- 7536042 TI - Comparison of a protein model with its X-ray structure: the ligand binding domain of E-selectin. AB - E- and P-selectin are cell adhesion molecules implicated in the early events of inflammation. Three-dimensional models of the lectin domains have been reported by us and others prior to the availability of X-ray structural information. The models have been used to outline the ligand binding site in the selectins and to identify residues critical for function. Recently, the crystal structure of E selectin has been reported, and thus, comparison of our E-selectin model with the X-ray data is now possible. The comparison shows that the assumptions on which the modeling was based were generally correct and provides an instructive example for the opportunities and the limitations of comparative modeling. PMID- 7536043 TI - Localization of nitric oxide synthase in the reproductive organs of the male rat. AB - Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536044 TI - Rapid cDNA sequencing in combination with RNA expression studies in mice identifies a large number of male germ cell-specific sequence tags. AB - A large number of cDNA clones were isolated from an adult mouse testis cDNA library and partially sequenced. Sequence comparisons revealed that many of them displayed similarities to genes previously identified only in invertebrates and lower eukaryotes, including, e.g., one cDNA clone related to the Drosophila melanogaster suppressor of forked gene. Other cDNA clones were found to be related, but not identical, to rodent genes involved in a variety of cellular activities, e.g., signal transduction and tumor development. The RNA expression patterns of 258 cDNA clones were analyzed through use of Northern blot methodology. Forty-two novel cDNA clones were found to be expressed only in male germ cells, the majority of them during spermiogenesis. One male germ cell specific mouse cDNA clone was found to be similar to tektin A1, a protein known to interact with the flagellar microtubules of sea urchin sperm. PMID- 7536045 TI - Interleukin-1 beta and the endometrium: an inhibitor of stromal cell differentiation and possible autoregulator of decidualization in humans. AB - Interleukin-1 beta (IL-1 beta), which modulates cell proliferation and differentiation in a number of cell types, is present in human endometrial stromal cells. However, both the function of IL-1 beta in endometrium and the factors that modulate its expression in endometrial stromal cells are unknown. To examine the effects of IL-1 beta on decidualization, human proliferative endometrial stromal cells were cultured for 12 days in medium (Dulbecco's Modified Eagle's medium with 2% fetal bovine serum) containing 1 microM medroxyprogesterone acetate, 10 nM estradiol, and 1 microM prostaglandin E2 (PGE2) with and without IL-1 beta (17 pg/ml). Morphologic changes as well as release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) were used as markers of decidualization. Morphologic analysis of cells exposed to IL-1 beta revealed incomplete decidualization. In addition, cells exposed to IL-1 beta released 40% less PRL and 85% less IGFBP-1 than cells cultured in the absence of IL-1 beta, and the PRL mRNA content of the IL-1 beta exposed cells was decreased by 68%. A possible role for ovarian steroids in the modulation of IL-1 beta expression in the endometrium is suggested by the increase in IL-1 beta mRNA that occurs in late secretory endometrium and by the induction of IL-1 by estrogen and progesterone in the mouse uterus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536046 TI - Expression of c-kit receptor and its autophosphorylation in immature rat type A spermatogonia. AB - The objective of this study was to examine the expression and activation of the c kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation. PMID- 7536047 TI - FLB1, a human protein of epididymal origin that is involved in the sperm-oocyte recognition process. AB - CA6 antibody was selected out of a monoclonal antibody library raised against human sperm proteins primarily for its ability to recognize an epididymal antigen and to modify sperm adhesion to zona-free hamster oocytes. In the present study, CA6 was shown to decrease sperm binding to zona-free hamster and human oocytes by 40-92% and 38-48%, respectively. The corresponding protein, which was referred to as FLB1, was found to be secreted by the epididymis and to bind specifically to a human, macaque, and rodent subacrosomal sperm region. Western blotting revealed a molecular mass of 94 kDa in human epididymal extracts and of 100 kDa in human, macaque, mouse, rat, and hamster sperm, suggesting further modifications after its binding to sperm. An equivalent protein was not observed in human liver, ovary, testis, plasma, or epidermis. Two-dimensional electrophoresis showed that FLB1 is formed of two subunits with the same 47-kDa molecular mass and slightly different pI (5.8, 5.9). Microsequencing of the protein revealed a partial homology with human cytokeratins 1 and 10. These results suggest that FLB1 is an epididymis-specific cytokeratin-like protein that is involved in the sperm-oocyte recognition process. PMID- 7536049 TI - Increasing concentrations of 17 beta-estradiol has differential effects on secretion of luteinizing hormone and follicle-stimulating hormone and amounts of mRNA for gonadotropin subunits during the follicular phase of the bovine estrous cycle. AB - The hypothesis tested was that 17 beta-estradiol (E2) would increase amounts of mRNA for alpha, LH beta, and FSH beta subunits during the follicular phase of the estrous cycle prior to the preovulatory surge of gonadotropins in cows. On Day 16 (Day 0 = estrus) of the estrous cycle, all cows were treated with prostaglandin F2 alpha (PGF2 alpha). Cows served as intact controls (CONT, n = 4) were ovariectomized (OVX, n = 5), or were ovariectomized and administered E2 (OVXE, n = 6) in increasing doses starting at the time of treatment with PGF2 alpha. Cows were bled for 6 h before and for 40 h after PGF2 alpha treatment to characterize pulsatile secretion of LH and FSH. Forty hours after PGF2 alpha treatment, pituitaries were collected for evaluation of amounts of mRNA for alpha, LH beta, and FSH beta subunits. Amplitude of LH pulses was greater (p < 0.05) in cows from the OVXE than from the CONT group. Concentrations of FSH were greater in cows from both the OVXE and OVX (p < 0.01) groups than from the CONT group. Amounts of mRNA for alpha and FSH beta subunits were greater (p < 0.01) in pituitaries of cows from the OVX than from the CONT or OVXE groups. Amounts of mRNA for LH beta subunit in pituitaries of cows from the OVX group tended to be greater (p < 0.08) than from the CONT group. Cows in the OVXE group tended (p < 0.08) to have greater amounts of mRNA for FSH beta subunit than did CONT cows.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536050 TI - Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase. AB - In previous experiments, the sperm-specific isozyme of lactate dehydrogenase (LDH C) had been purified from mouse testes and shown to suppress the fertility of female baboons by 70% compared to controls. Although these results demonstrated the feasibility of this approach for contraceptive vaccine development, it is not practical to purify enough of the protein from natural sources for human use. Therefore, a need exists to develop a contraceptive vaccine based on synthetic peptides. In the current study, baboon LDH-C cDNA was amplified by the reverse transcriptase-polymerase chain reaction technique. The amino acid sequences of human and baboon LDH-C were 99.3% identical, indicating that the human LDH-C would be an effective antigen in nonhuman primates. The immunodominant epitope of human LDH-C was identified, synthesized, and conjugated to diphtheria toxoid (DT). This construct was used to immunize 15 female baboons; 15 control animals were immunized with DT alone. The fertility of the experimental group was reduced by 75% as compared to the controls (p < 0.02). One year after the last immunization, the contraceptive effect was completely eliminated (no statistical difference between the groups). These results show that a synthetic peptide based on the sequence of human LDH-C is effective in preventing pregnancy in nonhuman primates. The effect is completely reversed 1 yr after the last immunization. The contraceptive effect is not related to serum antibody titers, and human LDH-C is only slightly more effective than mouse LDH-C in female baboons. PMID- 7536048 TI - Fas antigen-mediated apoptosis in human granulosa/luteal cells. AB - The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of tumor and hematopoietic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. Using reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry, we demonstrated that human granulosa/luteal cells express the Fas antigen. An anti-human Fas antigen monoclonal antibody (Fas mAb; clone CH-11), which induces apoptosis in other cell types by binding to the Fas antigen, induced significant cell death (30%) in cultures pretreated with interferon gamma (IFN gamma). This agrees with studies on tumor cell lines showing that IFN gamma enhances cytotoxic effects of Fas mAb. Granulosa/luteal cells exhibited morphological characteristics typical of apoptosis, including membrane blebbing and condensed chromatin. DNA fragmentation into oligonucleosomal units of approximately 180 bp, typical of apoptosis, was detected at elevated levels in Fas mAb-treated cultures via 3' end-labeling and gel electrophoresis. Examination of cultured cells in situ for apoptotic DNA cleavage by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) indicated that more apoptotic death occurred in Fas mAb treated cultures than in control cultures. Effects of hCG-induced luteinization of cultures on Fas mAb-induced cytotoxicity was examined: combined pretreatment with IFN gamma and hCG induced a synergistic increase in Fas mAb-induced cytotoxicity (40%) over that obtained with IFN gamma-pretreatment alone (15%). In summary, granulosa/luteal cells express the Fas antigen and are sensitive to Fas mAb-induced apoptosis. Human CG synergized with IFN gamma to increase Fas mAb induced death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536051 TI - Evidence that a GABAA-like receptor is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa. AB - We have investigated whether progesterone-triggered acrosomal exocytosis involves the activation of a gamma-aminobutyric acid (GABA) receptor, and whether activation of this receptor is linked to Ca2+ entry via Ca2+ channels. Mouse spermatozoa preincubated in a modified Tyrode's medium underwent exocytosis when stimulated with progesterone, as revealed by an increase in the number of cells exhibiting an "AR" pattern after staining with chlortetracycline; the effect was concentration dependent. Only capacitated spermatozoa underwent exocytosis in response to progesterone: cells preincubated for 15 min (uncapacitated) did not exocytose in response to this agonist, whereas cells preincubated for 120 min did. Stimulation of capacitated spermatozoa with GABA or muscimol, two GABAA receptor agonists, resulted in acrosomal exocytosis; this response was enhanced by half-maximal concentrations of progesterone. Bicuculline, a GABAA receptor antagonist, inhibited exocytosis stimulated by progesterone or GABA. Picrotoxin, another GABAA receptor antagonist, inhibited only GABA-stimulated exocytosis. These results suggest that progesterone effects are mediated by a GABAA receptor but that such receptor may not be identical to the neuronal GABAA receptor. The ability of progesterone, GABA, or muscimol to stimulate exocytosis was blocked by the Ca2+ channel antagonists verapamil or nifedipine. The Ca2+ channel agonist Bay K 8644, on the other hand, stimulated exocytosis in capacitated sperm cells. The stimulatory ability of progesterone and Bay K 8644 was additive. These results indicate that acrosomal exocytosis involves activation of a GABAA receptor apparently linked to Ca2+ channels. PMID- 7536052 TI - Insulin-like growth factor (IGF) binding protein 3 in the rat testis: follicle stimulating hormone dependence of mRNA expression and inhibition of IGF-I action on cultured Sertoli cells. AB - Insulin-like growth factor binding protein 3 (IGFBP-3) is the predominant IGF binding protein produced by cultured rat Sertoli cells. Previous studies have shown that FSH lowers the abundance of IGFBP-3 protein in Sertoli cell culture medium, suggesting that this binding protein has a physiological role in modulating insulin-like growth factor-I (IGF-I) activity in the testis. To characterize the physiological relevance of the FSH regulation of IGFBP-3, in this study we examined effects of FSH on IGFBP-3 mRNA expression in testes from hypophysectomized rats and in Sertoli cell culture. We then examined whether or not IGFBP-3 could alter the effects of IGF-I on cultured Sertoli cells. FSH (1 300 ng/ml) treatment of rat Sertoli cells dose-dependently decreased IGFBP-3 mRNA, with near-complete inhibition by 12 h of treatment. To evaluate this effect in the whole animal, male rats were hypophysectomized at 20 days of age and injected with FSH 10 days later. Testis IGFBP-3 mRNA increased following hypophysectomy relative to the value in sham animals, while FSH treatment decreased IGFBP-3 mRNA to sham levels within 6 h. To determine whether or not IGFBP-3 modulates IGF-I activity in the Sertoli cell, recombinant human non glycosylated rIGFBP-3 was added to Sertoli cell cultures in the presence or absence of IGF-I. The rIGFBP-3 dose-dependently inhibited IGF-I-stimulated lactate production with a half-maximal dose of 100 ng/ml. Neither basal lactate production nor FSH-stimulated lactate was altered by addition of rIGFBP 3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536053 TI - Changes in equine endometrial retinol-binding protein RNA during the estrous cycle and early pregnancy and with exogenous steroids. AB - A cDNA library was constructed from poly(A) RNA obtained from Day 14 nonbred equine endometrium. A cDNA probe for porcine retinol-binding protein (RBP) was used to screen the library, and a complete cDNA sequence (1133 bp, excluding the poly(A) tail) was obtained. Endometrial biopsies were obtained from cycling, nonbred mares at Days 0, 1, 4, 8, 10, 11, 13, and 15 and from pregnant mares at Days 11, 13, 15, and 17 after ovulation (n = 2 mares each day). Endometrial biopsies were also taken from 18 noncycling anestrous mares after the following treatments: C (vehicle control for 1 day, n = 3), E (estradiol-17 valerate, 5 mg/day for 6 days, n = 3), P (progesterone, 250 mg/day for 6 days, n = 4), ESP (E for 6 days followed by, P for 6 days, n = 4), and ELP (E for 6 days followed by P for 12 days, n = 4). Northern blot analyses were performed on total RNA (30 micrograms) using cDNA probes to equine (e) RBP and human glyceraldehyde-3 phosphate dehydrogenase (G3PDH). The RBP RNA levels (normalized to G3PDH) from nonbred mares were low during early diestrus and increased after Day 10, and RBP RNA levels from pregnant mares were similar to those of nonbred mares for corresponding days. E tended to decrease endometrial RBP RNA; and P, ESP, and ELP increased it compared to C. There were no significant differences among P, ESP, and ELP RBP RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536054 TI - Measuring the length of the pore of the sheep cardiac sarcoplasmic reticulum calcium-release channel using related trimethylammonium ions as molecular calipers. AB - After incorporation of purified sheep cardiac Ca(2+)-release channels into planar phospholipid bilayers, we have investigated the blocking effects of a series of monovalent (CH3-(CH2)n-1-N+(CH3)3) and divalent ((CH3)3N(+)-(CH2)n-N+(CH3)3) trimethylammonium derivatives under voltage clamp conditions. All the compounds tested produce voltage-dependent block from the cytoplasmic face of the channel. With divalent (Qn) derivatives the effective valence of block decreases with increasing chain length, reaching a plateau with a chain length of n > or = 7. No decline in effective valence is observed with the monovalent (Un) derivatives. A plausible interpretation of this phenomena suggests that for the 90% of the voltage drop measured, the increase in length following the addition of a CH2 in the chain spans 12.7% of the electrical field. Extrapolating this distance to include the remaining 10% suggests that the applied holding potential falls over a total distance of 10.4 A. In addition, at high positive holding potentials there is evidence for permeation of the trimethylammonium ions and a valency specific relief of block. PMID- 7536056 TI - Use of nitric oxide synthase inhibitors as a novel treatment for septic shock. AB - OBJECTIVE: To review the current literature regarding the role of nitric oxide (NO) in the pathogenesis of septic shock and to describe the potential role of NO synthase (NOS) inhibitors in the treatment of septic shock. DATA SOURCES: A MEDLINE, Cancerlit, Biosis, Scisearch, CBAC, bibliography, and current journal search of applicable articles on the involvement of NO in mediating septic shock and the use of NOS inhibitors in septic shock was conducted. Articles that were searched included animal and human studies from January 1990 to August 1994. STUDY SELECTION: Because of the preliminary nature of the research involving NOS inhibitors in septic shock, all available studies were evaluated. DATA SYNTHESIS: NO appears to have a role in the mediation of the hemodynamic instability associated with septic shock. Cytokines and endotoxin stimulate synthesis of the inducible NOS, which produces large amounts of NO over an extended period of time. NO may be the key mediator in the pathogenesis of septic shock. Derivatives of the precursor to NO, L-arginine, have been used to investigate the role of NO in septic shock and as possible therapeutic agents. Comparison of study results among animal studies shows much variability. This variability may be attributable to differences in dosing regimens and models of septic shock. Data obtained from human studies are more consistent, but are limited to a few case series. Results indicate that NOS inhibitors increase blood pressure and systemic vascular resistance and decrease cardiac output. The effects of NOS inhibitors on morbidity and mortality have not been assessed because of the lack of an appropriate sample size. CONCLUSIONS: NO appears to play a role in septic shock; however, the use of NOS inhibitors to treat septic shock requires further studies to determine an appropriate dosing regimen and to determine the effects of these agents on morbidity and mortality. PMID- 7536055 TI - Cyclic GMP diffusion coefficient in rod photoreceptor outer segments. AB - Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10( 8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges. PMID- 7536057 TI - Clinical and ethical perspectives on rationing of high-cost drugs. AB - OBJECTIVE: To analyze the use of high-cost drugs from a clinical decision-making approach and ethical perspectives on rationing. CASE: The case of a 26-year-old intravenous drug user with AIDS raises issues of how to ration high-cost drugs such as foscarnet, monoclonal antibodies (MAbs) for septic shock, and granulocyte colony-stimulating factor. ASSESSMENT: Should a patient with a terminal illness receive high-cost drugs given limited healthcare resources? Necessary clinical information including treatment algorithms, risk to benefit ratios, and cost effectiveness data are evaluated. Rationing, especially bedside rationing, by the clinician is rejected because it is contrary to the clinician's ethical obligation of beneficence and nonmaleficence. Patient autonomy and desires may also conflict with society's interest in equitably distributing resources. Treatment could be denied if costs exceed benefits for the outcomes and thus deny resources to others who have more basic healthcare needs. There is no obligation to offer medically futile care or for the patient to accept extraordinary medical care. CONCLUSIONS: An ethical argument for rationing cannot be made because of the lack of a clear clinical and societal consensus on specific criteria for rationing of healthcare dollars. The decision to use high-cost drugs in the case presented is made using a clinical decision-making approach based on available treatment guidelines. This assumes that the patient continues to consent to therapy and that there is continued benefit. PMID- 7536058 TI - Expression and role of c-myc protooncogene in murine preimplantation embryonic development. AB - PURPOSE: The present study was conducted to investigate the expression and possible role of the c-myc protooncogene in preimplantation embryos by using reverse-transcriptase/polymerase chain reaction (RT-PCR) technique and microinjection of synthetic antisense c-myc oligonucleotide probe, respectively. Total RNA was extracted from oocytes and two cell-, four cell-, early morula-, late morula-, early blastocyst-, and late blastocyst-stage embryos, and cDNA was constructed using MMLV reverse transcriptase. Sense primer (P1) and antisense primer (P2) used were based on the c-myc gene sequence bp 1609-1629 and bp 3279 3299, respectively, that span a 1.37-kb intron. PCR-amplified products of cDNA from oocyte-, two cell-, four cell-, early and late morula-, and blastocyst-stage embryos demonstrated the expected 313-bp product in Southern blot hybridization using a c-myc specific DNA probe, with an indication of lower levels in oocytes and early morulae. RESULTS: Cytoplasmic injection of the antisense c-myc oligonucleotide probe (P2) and not the sense probe (P1) into pronuclear-stage zygotes caused a significant (P = 0.02 to 0.0001) inhibition of development to blastocysts in a concentration-dependent manner, with a maximal inhibition at the first cleavage of zygotes to two cell-stage embryos. There was no effect on the P2 antisense injection on pronucleus formation. CONCLUSION: These results indicate that the c-myc protooncogene is expressed in preimplantation embryos and may have an essential role in normal embryogenesis in mice. PMID- 7536059 TI - Modulatory effects of calcitonin gene-related peptide and substance P on human cholinergic sweat secretion. AB - Immunoreactivity to various peptides has been demonstrated in nerve terminals around the sweat glands, suggesting a regulatory function for these peptides on sweating. The present study evaluated the calcitonin-gene related peptide and substance P related regulation of sweating in man. Both calcitonin-gene related peptide and substance P, when administered alone, failed to cause sweat secretion, whereas sweating induced by methacholine chloride alone was four times greater when administered with calcitonin-gene related peptide and suppressed by 70% when administered with substance P. The degree of calcitonin-gene related peptide dependent augmentation and substance P dependent suppression of the methacholine chloride induced sweating was dependent on the concentration of calcitonin-gene related peptide and substance P. These findings suggest that calcitonin-gene related peptide enhances cholinergic sweating and substance P inhibits it. PMID- 7536061 TI - Immunomodulatory effects of antithyroid drugs. PMID- 7536060 TI - Localization of NADPH-diaphorase reactivity in the chick and mouse thyroid gland. AB - In the present study, nicotinamide adenine dinucleotide hydrogen phosphate diaphorase (NADPH-d) histochemistry has been used as a marker for nitric oxide synthase (NOS). The colored reaction product, formazan, was localized in neuronal cell bodies, nerve fibers, and vascular endothelium in the thyroid of chick and mouse. In these two animal species, most of the NADPH-d-labeled neuronal cell bodies were found in the thyroid capsule and interfollicular connective tissue while some were associated with blood vessels. Most nerve fibers travelled with blood vessels supplying the thyroid gland, while a few of them were intimately associated with the thyroid follicular cells. Control sections not incubated with beta-NADPH failed to show labeling of the above structures. It is concluded that nitric oxide may play an important role in endocrine secretion by controlling the regional blood flow in the thyroid gland and by directly acting on the thyroid follicular cells. PMID- 7536062 TI - The expression of osteoclast markers on foreign body giant cells. AB - The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (M phi Ag), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50-200 mM and expressed VNR and a restricted range of M phi Ag. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express M phi Ag such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies. PMID- 7536063 TI - Interleukin-1 beta induces cyclic AMP formation in isolated human osteoblasts: a signalling mechanism that is not related to enhanced prostaglandin formation. AB - Interleukin-1 (IL-1) is a potent stimulator of bone resorption. Induction of osteoclastic bone resorption by various endocrine or paracrine factors is mediated via the osteoblasts. We have therefore investigated the effects of IL-1 beta on cell signalling in isolated human osteoblasts. Special interest was focused on prostaglandin synthesis, since indomethacin, an inhibitor of prostaglandin synthesis, partly inhibits IL-1-induced bone resorption. IL-1 beta, at and above 0.3 pM, dose dependently stimulated PGE2 formation in isolated human osteoblasts, with half maximal stimulation, EC50, at 3 pM. Treatment with the calcium ionophore A23187 (1 microM), or with forskolin (30 microM), also stimulated PGE2 formation in human osteoblasts. The time-course for IL-1 beta induced PGE2 formation was similar to that of forskolin, with a significant increase in the formation of PGE2 seen after 1 h. In contrast, A23187-induced PGE2 formation was seen within minutes. IL-1 beta stimulated the accumulation of cyclic AMP in isolated human osteoblasts incubated for 15 min. This increase in cyclic AMP formation was not secondary to PGE2 formation since it was not blocked by the addition of indomethacin (1 microM). Pretreatment with the phosphodiesterase inhibitor IBMX did not augment IL-1 beta-induced PGE2 formation, nor did the protein kinase A inhibitor Rp-cAMPs inhibit IL-1 beta induced PGE2 formation, suggesting that cyclic AMP does not mediate the stimulatory effect of IL-1 on PGE2 formation. We conclude that IL-1 beta enhances the formation of cyclic AMP as well as PGE2 in primary cultures of isolated human osteoblasts. The IL-1 beta-induced cyclic AMP formation is, however, not related to the enhanced prostaglandin formation. The findings implicate that both cyclic AMP- and PGE2-formation in osteoblasts might be involved as independent mediators of IL-1 beta-induced bone resorption. PMID- 7536064 TI - Assessing blood glucose control in diabetes mellitus. Variant haemoglobin may affect measurements. PMID- 7536065 TI - Palliative care in terminal cardiac failure. PMID- 7536066 TI - Abscopal depression of G-CSF mobilized peripheral stem cells. AB - Transient suppression of hematopoietic colony formation from cytokine mobilized PBSC was observed in a patient following local radiation therapy for lymphoma. This effect was seen 2 weeks after completion of radiation and reversed by 6 weeks. The patient successfully engrafted after myeloablative chemotherapy with harvested PBSC. This report documents the first reported case of suppression of PBSC colony formation following radiation and we speculate that this finding may be due to an abscopal effect. PMID- 7536067 TI - Blood stem cell collection with G-CSF. PMID- 7536068 TI - Large-scale enrichment of mobilized CD34+ peripheral blood hematopoietic progenitors by removal of nylon wool-adherent mature cells. AB - With the aim of facilitating the ex vivo manipulation of peripheral blood hematopoietic progenitors (CPCs = circulating progenitor cells) collected by leukapheresis, we removed polymorphonuclear cells and monocytes that naturally adhere to nylon wool fibers. Leukapheresed cells harvested at the time of hematopoietic recovery after cancer therapy with high-dose cyclophosphamide plus hematopoietic growth factors were incubated with nylon wool fibers for 1 h at 37 degrees C. Evaluation of the cells non-adherent to the nylon wool in all experiments (n = 14) showed that the median recovery of nucleated cells and CPCs detected as CD34+ cells, CFU-GM and BFU-E was 16.4% (range 4.8%-34.0%), 60.0% (range 30.8-80.8%), 60.9% (range 33.4-74.5%) and 65.5% (range 30.8-69.2%), respectively. Therefore exposure to the nylon wool determined a selective removal of mature cells and a complementary enrichment of CPCs. The wide range of results depended on the significantly different cell compositions of the unmanipulated leukaphereses. The latter from patients receiving rhG-CSF (n = 10) comprised a median of 88.5% (range 77.8-93.8%) and 11.5% (range 6.2-22.2%) polymorphonuclear and mononuclear cells, respectively. In contrast, leukaphereses from patients receiving rhGM-CSF or PIXY321 (n = 4) comprised a median of 71.1% (range 55.4 85.0%) and 28.9% (range 15.0-44.6%) polymorphonuclear and mononuclear cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536070 TI - Comparison of G-CSF-primed peripheral blood progenitor cells and bone marrow auto transplantation: clinical assessment and cost-effectiveness. AB - The introduction of hematopoietic growth factors (HGFs) offers new opportunities for autologous transplantation by facilitating and enriching collection of circulating progenitor cells from peripheral blood as a source of stem cell rescue. Substitution of peripheral blood progenitor cells (PBPC) from bone marrow in autologous transplantation for therapy in advanced cancers requires clinical and economic assessment. We carried out the first clinical and cost-effectiveness study in an experimental group of 16 patients autografted with PBPC primed by G CSF alone and with G-CSF stimulation post-transplantation, comparing these with two other groups of 17 and 21 patients who received autologous bone marrow transplantation with and without G-CSF stimulation, respectively, post transplantation. We confirmed the ability of primed PBPC to achieve durable engraftment in a shorter time than classical BMT (median number of days to reach 0.5 x 10(9)/l neutrophils = 10.5 versus 12 and 16, respectively) to improve overall hematological recovery (median number of days to recover a platelet count > or = 25 x 10(9)/l, independent of platelet transfusion = 14.5 vs 23 and 20) and to shorten length of hospitalization. Total costs of PBPC autografting remain lower than those of autologous BMT either with or without G-CSF, and cost effectiveness ratios using hematological recovery end points are in favour of PBPC. Finally, PBPC is a safe and effective way of performing dose intensification in cancer patients, although further improvements are required to optimize the procedure and so further decrease the costs. PMID- 7536071 TI - Low-dose cyclophosphamide in combination with cisplatin or epirubicin plus rhG CSF allows adequate collection of PBSC for autotransplantation during adjuvant therapy for high-risk cancer. AB - Six patients with advanced ovarian carcinoma (OvCa), and six patients with stage II or III resectable breast cancer (BrCa) were treated with low-dose CY (LD-CY, 1500 mg/m2) and cisplatin (CDDP) 100 mg/m2 (OvCa) or epirubicin (EPR) 120 mg/m2 (BrCa) plus recombinant human G-CSF (rhG-CSF). Twelve days after chemotherapy, all patients underwent PBSC collection on an outpatient basis. Following the completion of the induction programme, all patients underwent high-dose chemotherapy (HDC) with carboplatin 1200 mg/m2, etoposide 900 mg/m2 and melphalan 100 mg/m2 with the reinfusion of PBSC. LD-CY plus rhG-CSF in combination with CDDP or EPR mobilised a very large number of PBSC. After a median of 13 days from chemotherapy, the concentration of PBSC in the peripheral blood was 40-fold higher than the same patient's baseline value. Each collection yielded a median of 10.8 x 10(4)/kg colony-forming unit granulocyte-macrophage. Severe myelosuppression occurred in all patients following HDC, but the infusion of PBSC produced a rapid and sustained haemopoietic recovery. After a median of 11 days from reinfusion, haemopoietic engraftment was complete and 80% of the patients had platelets > 100 x 10(9)/l and PMN > 1 x 10(9)/l within 14 days after reinfusion. We can conclude that the present therapeutic approach is an excellent option for mobilisation, collection and transplantation of PBSC during intensive dose adjuvant polychemotherapy of high-risk cancer. PMID- 7536072 TI - Comparison of G-CSF with GM-CSF for mobilizing peripheral blood progenitor cells and for enhancing marrow recovery after autologous bone marrow transplant. AB - Primed peripheral blood progenitor cells (PBPC) with hematopoietic growth factors enhance marrow engraftment after autologous bone marrow transplantation (BMT). G CSF and GM-CSF stimulate the production of PBPC; both cytokines alone also stimulate neutrophil recovery after autologous BMT. Little data exist comparing these two cytokines. We prospectively studied G-CSF and GM-CSF in autologous BMT. Forty-four consecutive patients with either Hodgkin's disease or non-Hodgkin's lymphoma underwent autologous BMT using both PBPC and autologous marrow. The autologous BMT preparative regimen was CBV (VP-16 2400 mg/m2, CY 1800 mg/m2 i.v. four times daily for 4 days, BCNU 600 mg/m2). Sixteen patients received G-CSF 5 micrograms/kg sc daily for 8 days for mobilization of PBPC and received G-CSF 16 micrograms/kg i.v. four times daily after autologous BMT. Twenty-eight patients received GM-CSF to mobilize PBPC (14 patients received 250 micrograms/m2 sc daily for 8 days; 14 patients received 125 micrograms/m2 sc twice daily for 8 days) and GM-CSF (250 micrograms/m2 i.v. four times daily) after autologous BMT. Patients underwent three to five pheresis procedures to harvest at least 3 x 10(8) nucleated cells/kg. Patients receiving G-CSF had higher peripheral WBC counts than did those receiving GM-CSF. Total numbers of mononuclear cells, total CD34+ cells and total CD34+/33-negative cells were similar in the two treatment groups. The patients receiving G-CSF after autologous BMT experienced a more rapid engraftment of both neutrophils (9 days vs 13 days, p = 0.0001) and platelets (14 days vs 18 days, p = 0.027) than did patients receiving GM-CSF after transplant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536073 TI - Pilot trial of combined administration of erythropoietin and granulocyte colony stimulating factor to children undergoing allogeneic bone marrow transplantation. AB - We carried out a pilot study to evaluate the combined use of recombinant human erythropoietin (rhEpo) and granulocyte colony-stimulating factor (G-CSF) for accelerating marrow engraftment in children given allogeneic bone marrow transplantation (BMT). Fifteen consecutive children were enrolled in this study; 13 completed it and were evaluable. Using analysis of variance, laboratory and clinical data referring to these children were compared with those of 15 patients previously treated with rhEpo alone and with those of 16 historical controls. Erythroid repopulation, evaluated sequentially through serum transferrin receptor and reticulocyte count, was similarly accelerated in children receiving rhEpo alone and in those receiving combined treatment. These latter, however, showed a further reduction in the total number of red blood cell units required to reach transfusion independence (1.1 +/- 0.7 in the study population vs 2.7 +/- 1.2 in rhEpo group vs 4.2 +/- 2.3 in historical controls; values are mean +/- 1 SD; p < 0.001). Neutrophil engraftment, i.e. time for neutrophils to reach 0.5 x 10(9)/l, was 11 +/- 3 days in children receiving combined treatment, significantly shorter than that of the control groups (16 +/- 3 and 18 +/- 5, respectively; p < 0.001). Acceleration of neutrophil recovery translated into fewer infections: days of fever were significantly reduced in the study population (4 +/- 2 vs 11 +/- 8 vs 15 +/- 6, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536074 TI - Liquid storage of peripheral blood progenitor cells for transplantation. AB - Peripheral blood progenitor cells (PBPC) were mobilised by recombinant human G CSF (rhG-CSF) and cyclophosphamide, harvested by apheresis from 9 patients with NHL and stored at 4 degrees C without further manipulation. They were then reinfused after high-dose chemotherapy. We monitored the change in colony-forming units-granulocyte, macrophage (CFU-GM) proliferation as well as plasma glucose, lactate and pH levels over the period of the study. In an attempt to maintain CFU GM numbers, rhG-CSF was added to storage bags at collection and 48 h later. Our observations suggest that cell viability is well maintained and that CFU-GM numbers rise over the first 48 h of storage before falling rapidly. Metabolic changes cause a fall in the pH and glucose levels with a reciprocal rise in plasma lactate. The addition of rhG-CSF at a concentration of 10 ng/ml to cells in storage showed no detectable benefit. Following storage for 96 h, 77% (SEM +/- 8%) of the initial CFU-GM remained. PMID- 7536075 TI - The challenge of change. PMID- 7536076 TI - Avidin-biotin labeling of cellular antigens in cryostat-sectioned tissue. PMID- 7536069 TI - Factors affecting the mobilization of primitive and committed hematopoietic progenitors into the peripheral blood of cancer patients. AB - Rapid hematopoietic reconstitution following peripheral blood progenitor cell (PBPC) autotransplantation is thought to result from reinfusion of committed progenitor cells. This has raised concern that PBPC autografts might be rich in committed hematopoietic progentors responsible for early engraftment, but deficient in more primitive progenitors required for long-term hematopoietic reconstitution. The granulomonocytic colony-forming unit (CFU-GM) assay measures committed progenitors responsive to a single species of colony-stimulating activity such as granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas the pre-CFU assay identifies more primitive progenitors by measuring interleukin-3 (IL-3) and kit ligand (KL) induced generation of secondary CFU-GM from CD34+, 4-hydroperoxycyclophosphamide resistant progenitors that require multiple cytokine stimuli. Paired bone marrow (BM) and PBPC samples from 17 breast and ovarian cancer patients participating in four separate clinical trials were compared in these assay systems. In seven of nine patients, PBPC autografts mobilized with cyclophosphamide rebound and G-CSF compared favorably with paired BM autografts in both committed and primitive progenitor capacity. Failure to mobilize substantial primitive progenitor cell numbers occurred in two of nine patients undergoing this mobilization regimen and could not have been predicted by either circulating CFU-GM or CD34+ cell number. Prior myelosuppressive treatment experiences reduced peripheral progenitor yields somewhat, but still allowed for the collection of PBPC autografts which compared favorably with BM autografts in total CFU-GM and Pre-CFU. Mobilization of PBPC with G-CSF or GM-CSF alone in patients who had received prior myelosuppressive therapies produced autografts which were relatively deficient in committed progenitors, but absolutely deficient in primitive progenitors. We conclude that optimization of patient characteristics and mobilization parameters can achieve PBPC autografts rich in both the primitive and committed hematopoietic progenitor cells. PMID- 7536077 TI - Overview of flow cytometry and fluorescent probes for cytometry. PMID- 7536079 TI - Fluorescent labeling of DNA. PMID- 7536078 TI - Indirect immunofluorescent labeling of fixed cells. PMID- 7536080 TI - Pre-embedding labeling methods. PMID- 7536081 TI - Postembedding labeling methods. PMID- 7536082 TI - Overview of fluorescence photomicrography. PMID- 7536083 TI - Overview of automated immunostainers. PMID- 7536084 TI - Conjugation of fluorochromes to antibodies. PMID- 7536085 TI - Preparation of frozen sections for analysis. PMID- 7536086 TI - Processing of cell-touch preparations. PMID- 7536087 TI - Processing of cytological specimens. PMID- 7536088 TI - Processing of tissue-culture cells. PMID- 7536089 TI - Processing of tissue specimens. PMID- 7536090 TI - Quantification of urinary insulin-like growth factors (IGFs) and IGF binding protein 3 in healthy volunteers before and after stimulation with recombinant human growth hormone. AB - We examined excretion of urinary insulin-like growth factors I and II (IGF-I and IGF-II) and their major binding protein IGFBP-3 in comparison to their respective serum concentration in nine healthy female volunteers (median age 25 years, range 22-27) under baseline conditions and after stimulation with recombinant human growth hormone (rhGH), 4.5 IU twice daily subcutaneously for a period of 3 days. The IGFs were measured in unconcentrated urine by use of recently developed, highly sensitive radioimmunoassays. The IGFBP-3 was measured by a specific radioimmunoassay. The mean (+/- SD) urinary concentrations of IGF-I (0.08 +/- 0.07 micrograms/l), IGF-II (1.02 +/- 0.47 micrograms/l) and IGFBP-3 (19.1 +/- 6.9 micrograms/l) were two to three orders of magnitude lower than in serum. The ratio of IGF-II over IGF-I concentration in urine (13:1) was five times higher than in serum (2.5:1), and the ratio of IGFBP-3 over the sum of IGF-I and IGF-II in urine (17:1) was four times higher than in serum (4:1). Urinary excretion was 63.3 +/- 46.6 ng.m-2.24h-1 for IGF-I, 1002 +/- 598 ng.m-2.24h-1 for IGF-II and 18039 +/- 4983 ng.m-2.24h-1 for IGFBP-3. Using fast protein liquid exclusion chromatography, only immunoreactive IGFBP-3 components of less than 60 kD were detected in urine, with a major peak at 20 kD. Urinary IGFBP-3 excretion correlated with serum IGFBP-3 (r = 0.61, p < 0.01) and the glomerular filtration rate (r = 0.56, p < 0.05) measured by steady-state inulin infusion clearances.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536091 TI - Use of prostate-specific antigen for prostate cancer screening in primary care practice. AB - OBJECTIVE: To examine prostate-specific antigen (PSA) as a screening test in randomly selected primary care practices. DESIGN: Chart abstractions of a random selection of 552 men (age, > or = 50 years) and physician and practice-level surveys. SETTING: Fifty-eight randomly selected, nonteaching, non-governmental, primary care practices in a 43-county area in Virginia. MAIN OUTCOME MEASURES: Documented evidence of PSA screening for asymptomatic male patients and physician self-report on regular (annual or biannual) use of PSA screening. RESULTS: Of 496 asymptomatic men, 123 (25%) seen in a 1-year period had documentation of PSA screening. Sixty (50%) of 120 asymptomatic men with documentation of a health maintenance examination had a PSA screening, whereas only 63 (17%) of 376 men without health maintenance examination had a PSA screening (chi 2, P < .001), after adjusting for race, insurance status, and age. Comparison of PSA screening use before and after the November 1992 publication of the American Cancer Society guidelines on PSA screening revealed that PSA screening after the publication date was 26%, compared with 19% before the data (P = .045), adjusting for health maintenance examination and the length of time for which the patient was eligible for screening. Logistic regression on physician self-report of regular vs not regular PSA use found an odds ratio of 6.12 (95% confidence interval, 1.28 to 29.30) for influence of the guidelines and 0.96 (95% confidence interval, 0.93 to 0.99) for the proportion receiving Medicaid or uninsured in the practice. CONCLUSIONS: Despite controversy over PSA as a screening test, PSA screening has now spread substantially in primary care practice. PMID- 7536092 TI - Distinct populations of identified glial cells in the developing rat spinal cord slice: ion channel properties and cell morphology. AB - Four types of glial cells could be distinguished in the grey matter of rat spinal cord slices at postnatal days 1-19 (P1-P19), based on their pattern of membrane currents as revealed by the whole cell patch clamp technique, and by their morphological and immunocytochemical features. The recorded cells were labelled with Lucifer Yellow, which allowed the subsequent identification of cells using cell-type-specific markers. Astrocytes were identified by positive staining for glial fibrillary acidic protein (GFAP). These were morphologically characterized by multiple, very fine and short processes and electrophysiologically by symmetrical, non-decaying K+ selective currents. Oligodendrocytes were identified by a typical oligodendrocyte-like morphology, lack of GFAP staining and positive labelling with a combination of O1 and O4 antibodies (markers of the oligodendrocyte lineage), and their membrane was dominated by symmetrical, passive, decaying K+ currents. The third population of glial cells was also characterized by positive staining for O1/O4 or only for O4 antigens, lack of GFAP staining and, in some cells, oligodendrocyte-like morphology. However, these cells could be distinguished by the presence of inwardly rectifying (KIR), delayed outwardly rectifying (KDR) and A-type K+ currents (KA), representing the most likely glial precursor cells of the oligodendrocyte lineage. The fourth population of glial cells had small somata and a widespread network of long processes with no apparent orientation preference. In one case, processes were positively labelled with GFAP, while 30% were characterized by faint, diffuse staining. These cells expressed a complex pattern of voltage-gated channels, namely Na+, KDR, KA and KIR channels. In contrast to neurons, the amplitude of Na+ currents was at least one order of magnitude smaller than the K+ currents, and none of these cells showed the ability to generate action potentials in the current clamp mode. Since none of these cells could be labelled by oligodendrocyte markers we assume that they were either astrocytes or glial precursor cells of the astrocyte lineage. The four cell types were found in all regions of the grey matter. When randomly accessing the glial cells, the probability of recording from the oligodendrocyte precursor cells and the glial cells with Na+ currents decreased during development. At P1-P3, 50% of the cells revealed the Na+ current, while at P13-P15 only 18% did. Concomitantly, the number of glial cells with astrocyte- and oligodendrocyte-like membrane currents increased from 19 and 12% to 41 and 35.5% respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536093 TI - Mitosis of Schwann cells and demyelination are induced by the amyloid precursor protein and other protease inhibitors in the rat sciatic nerve. AB - We studied the cytological alterations produced in the rat sciatic nerve by the amyloid precursor protein (APP) containing the Kunitz insert (APP K+) and other protease inhibitors. Conditioning of nerve segments with APP K+, aprotinin or leupeptin for 5 days or more resulted in mitosis of Schwann cells, demyelination of fibres, and a < 10-fold increase in Schwann cells, associated with demyelinated fibres. Altered fibres nevertheless involved a small part of the population. Nerve segments proximal and distal to the conditioned region showed almost no alteration. Conditioning with saline, heated APP K+, or APP without the Kunitz insert was not effective. We conclude that APP K+ and other protease inhibitors induce Schwann cells to enter the cell cycle, and once committed to proliferate they resorb their myelin. These functional properties of APP may be relevant to the pathogenesis of Alzheimer's disease. PMID- 7536094 TI - A phylogenetic analysis of micro-organisms isolated from subsurface environments. AB - Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram positive bacteria and appeared to be most closely related to Arthrobacter globiformis--a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria-including the pseudomonads and Agrobacterium tumefaciens--or the low-G+C Gram-positive bacteria. PMID- 7536095 TI - Effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, on electromechanical coupling in the guinea-pig ureter. AB - 1. We have investigated the effect of the sarcoplasmic reticulum (SR) Ca(2+) ATPase inhibitor, cyclopiazonic acid (CPA), on electromechanical coupling in the guinea-pig ureter. All experiments were performed in capsaicin-pretreated (10 microM for 15 min) ureters to prevent the release of sensory neuropeptides from afferent nerves. 2. In organ bath experiments, electrical field stimulation (EFS, 10 Hz for 1 s, 5 ms pulse width, 60 V) produced tetrodotoxin- (1 microM) resistant phasic contractions which were enhanced by Bay K 8644 (1 microM) and abolished by nifedipine (10-30 microM). 3. CPA (10 microM) enhanced the EFS evoked contractions both in the absence and presence of Bay K 8644. The effect of CPA was concentration-dependent between 1 and 30 microM. The response to 10 microM CPA was biphasic: the maximal enhancement (58 +/- 3% increase) was observed within 10-20 min from CPA administration, followed by a decline to a new steady state (25 +/- 5% increase over baseline) at 50-60 min. The effect of CPA was reversed by washout. 4. Ryanodine (100 microM) produced a prompt enhancement of the EFS-evoked contractions of the guinea-pig ureter, which peaked at 42 +/- 3% increase over baseline; the co-administration of CPA (10 microM) and ryanodine (100 microM) produced a peak effect (60 +/- 8% enhancement) which was not different from that produced by CPA alone. With either ryanodine alone or ryanodine plus CPA, the enhancement of the EFS-induced contractions was biphasic, showing a time-course similar to that observed with CPA alone. Tetraethylammonium (10 mM) produced a significantly larger effect (93 +/- 13% increase over baseline) and its effect was sustained throughout the 60 min observation period. 5. In the presence of Bay K 8644, superfusion for 30 min with a low Na+ medium (60% of extracellular Na+ replaced by Li+ or choline) reduced the amplitude of EFS-evoked contractions by 20-35%. In both Li(+)- and choline-substituted media, spontaneous activity developed during superfusion with low Na+ Krebs solution which was suppressed by 10 microM nifedipine. CPA (10 microM) produced a marked enhancement of the EFS-evoked contractions in low-Na+ medium (both Li(+)- and choline-substituted) and this effect was sustained throughout the 60 min observation period. 6. In the absence of Bay K 8644, the response of the ureter smooth muscle to EFS is characterized by a refractory period: an interval of about 30 s was required between two applied stimuli to produce a second response comparable in size to that elicited by the first stimulus. CPA (10 micro M, 10-20 min before)markedly reduced the refractory period of the guinea-pig ureter to EFS.7. CPA (10 micro M, 30-60 min before) increased the phasic component of contraction produced by 80 m MKCl. The tonic component of the response to KCl was slightly but not significantly reduced by CPA,and a 'hump' in the tonic contraction was observed at 1-2 min from addition of KCl.8. In sucrose gap experiments, 10 micro M CPA produced a sustained depolarization of the membrane and reduced the latency between application of electrical stimuli and onset of the action potential; these effects were maintained throughout the 60 min superfusion with CPA. CPA also transiently prolonged the plateau phase of the action potential and increased the peak amplitude of contraction: these effects peaked at about 10-20 min from start of superfusion with CPA and then declined. At the peak of its enhancing effect on contraction amplitude, CPA prolonged the contractile phase of the contraction relaxation cycle.9.Superfusion with a low Na, choline-substituted Krebs solution produced a reversible membrane depolarization. In the presence of Bay K 8644 (1 micro M), action potentials and phasic contractions were superimposed on this depolarization which were abolished by nifedipine (1O micro M).10. These findings indicate that CPA augments the excitability and affects the contraction-relaxation cycle of the smooth muscle of the guinea-pig ureter, implying a role for sarcoplasmic reticulum Ca2+-ATPase in the regulation of electromechanical coupling. The effects of CPA resemble those produced by ryanodine and the effect of the two agents on the amplitude of contractions is non-additive.It appears that following blockade of the CPA sensitive SR Ca2+ pump, other mechanism(s) may come into action to reduce intracellular Ca2+. The Na+/Ca2+ exchanger could be involved in the compensatory changes responsible for the fading of the response to CPA. PMID- 7536096 TI - The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha. AB - 1. Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha). These cytokines depress cardiac mechanical function by a mechanism which is not well defined. 2. Bacterial endotoxin or cytokines cause the expression of Ca(2+)-independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL-1 beta plus TNF-alpha in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation. 3. Rat isolated working hearts perfused with IL-1 beta plus TNF-alpha showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time-matched control hearts. The depressant action of IL-1 beta plus TNF-alpha was first apparent after 1 h of perfusion; no early (15 min) cardiac depressant actions were seen. 4. The competitive inhibitor of Ca(2+)-dependent and Ca(2+) independent NO synthases, NG-nitro-L-arginine methyl ester (L-NAME, 3 microM) when given concurrently with IL-1 beta plus TNF-alpha prevented the loss in contractile function such that these hearts after 2 h of perfusion had similar function to time-matched controls. L-NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL-1 beta plus TNF-alpha. The protective action of L-NAME in the presence of cytokines was concentration dependent and was not seen at a higher concentration (10 micro M) due to the significant reduction in coronary flow observed at this concentration.5. In contrast, when L-NAME (3 micro M) was given in the absence of IL-l beta plus TNF alpha it depressed contractile function over the 2 h perfusion period by significantly reducing coronary flow.6. Inhibition of protein synthesis with cycloheximide (Cx) abolished the loss in function that occurred over 2h in both control and IL-1 beta plus TNF-a-treated hearts.7. Inducible, Ca2+-independent NO synthase activity was not observed in freshly isolated hearts but was observed in control hearts perfused for 2 h in vitro and was doubled in hearts perfused with IL-1 beta plus TNF-a. Cx prevented the expression of Ca2+-independent NO synthase in both control and cytokine-treated hearts.8. In summary, these results suggest that the depression of myocardial function by IL-l beta plus TNF-alpha is mediated, at least in part, by induction of Ca2+-independent NO synthase activity in the heart. PMID- 7536097 TI - Reduction of carrageenin oedema and the associated c-Fos expression in the rat lumbar spinal cord by nitric oxide synthase inhibitor. AB - 1. Three hours after intraplantar carrageenin (6 mg/150 microliters of saline) Fos-like immunoreactivity (Fos-LI) was mainly observed in L4 and L5 segments of the dorsal horn. Both superficial (I-II) and deep laminae (V-VI) neurones were labelled. 2. We have studied the effect of systemic administration of a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on carrageenin evoked c-Fos expression and thus the contribution of nitric oxide to this expression. 3. Pre-administration of L-NAME (10, 25, 50, 100 mg kg-1, i.v.) dose-dependently reduced the number of superificial and deep laminae Fos-LI neurones, 100 mg kg-1 produced a 63 +/- 2% and 72 +/- 4% reduction of Fos-LI neurones respectively, P < 0.0001 for both superficial and deep neurones. 4. Pre administered L-NAME dose-relatedly reduced the carrageenin-evoked paw and ankle oedema, with 100 mg kg-1 of L-NAME resulting in a 74 +/- 2% and 103 +/- 2% reduction respectively. 5. Post-administration of L-NAME (10 mg kg-1, i.v.) reduced the number of superficial and deep laminae Fos-LI neurones (65 +/- 7% and 53 +/- 8% reduction respectively, P < 0.01 for both superficial and deep neurones). 6. Post-administered L-NAME reduced both the paw and ankle oedema (52 +/- 8% and 62 +/- 10% reduction respectively, P < 0.0001 for both paw and ankle). 7. Pre-administered D-NAME (100 mg kg-1, i.v.), the inactive isomer of L-NAME, produced a weak reduction of the number of superficial laminae Fos-LI neurones (26 +/- 8% reduction, P<0.05), without influencing the deep Fos-LI neurones (5 +/ 8% enhancement) or the oedema.8. Systemic L-arginine (1200 mg kg-1) did not reverse the reduction of the total number of Fos-LI neurones induced by 100mg kg 1 of L-NAME, or the effect of L-NAME on the paw and ankle oedema.9. Intraplantar L-arginine (30 mg) did not reverse the effect of L-NAME (100 mg kg-1) on the total number of Fos-LI neurones. However, the inhibitory effects of L-NAME on the paw and ankle oedema were partially reversed by intraplantar L-Arginine (34 +/- 9% and 45 +/- 11% reduction of carrageenin oedema respectively) with these effects being significant as compared to the effect of L-NAME alone(P<0.05 for both).10. There is a strong correlation between the reduction of the number of Fos-LI neurones and the oedema by L-NAME, clearly demonstrating a predominant role of peripheral NO in the development of one of the signs of carrageenin inflammation. PMID- 7536098 TI - Acute versus chronic administration of phosphodiesterase inhibitors on allergen induced pulmonary cell influx in sensitized guinea-pigs. AB - 1. The aims of this study were to determine which phosphodiesterase (PDE) isoenzymes are involved in the control of eosinophil accumulation in the airways of ovalbumin (OVA)-immunized guinea-pigs by the use of isoenzyme selective inhibitors and to compare the effects of acute versus chronic administration of PDE isozyme inhibitors on pulmonary cell influx in ovalbumin-immunized guinea pigs. 2. Guinea-pigs were sensitized and subsequently challenged with aerosolized OVA. Twenty four hours later bronchoalveolar lavage (BAL) was performed to permit assessment of inflammatory cell accumulation. A significant increase in the number of eosinophils was observed in the lavage fluid from OVA-immunized (13.6 +/- 1.4 x 10(4) ml-1 in acute experiments and 10.1 +/- 1.4 x 10(4) ml-1 in chronic experiments) animals compared with sham-treated controls (5.6 +/- 0.6 x 10(4) ml-1 in acute experiments and 5.1 +/- 0.6 x 10(4) ml-1 in chronic experiments). There was no difference in neutrophil, mononuclear cell or total cell numbers between the two groups. 3. Acute administration of a high dose of selective and non-selective PDE inhibitors by the i.p. route had no significant effect on eosinophil accumulation in the airways. 4. Chronic administration of a low dose (3 mg kg-1, i.p., twice daily for 7 days) of the type IV PDE inhibitor, RO 20-1724, and the PDE III/IV inhibitor, zardaverine, produced a significant inhibition of eosinophil accumulation (46% and 59% respectively). 5. These results suggest that the type IV PDE isoenzyme plays a role in the control of allergen-induced eosinophil infiltration into the airways, but indicate that a period of low dose chronic treatment with a type IV or mixed type III/IV PDE inhibitor is necessary for eosinophil accumulation in the airways to be reduced. PMID- 7536099 TI - Epitopes and radicals: early events in glomerular injury in membranous nephropathy. PMID- 7536100 TI - IGF-I stimulates renal function in the isolated rat kidney: inhibition by aminoguanidine and nitroarginine methyl ester. AB - IGF-I is known to increase renal function when administered in vivo. To establish whether IGF-I has a direct effect on renal function, the present experiments were performed to measure the effects of IGF-I in the isolated perfused rat kidney (IPRK). We have examined the influence of l-nitroarginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS) and aminoguanidine (AG), a selective inhibitor of the inducible isoform of NOS on the direct effects of recombinant human IGF-I (rhIGF-I) on renal function in the IPRK. rhIGF-I (100 nM) increased both glomerular filtration rate (GFR) (+109 +/- 17%, n = 6, p < 0.01) and renal perfusate flow (RPF) (+100 +/- 15%, n = 6, p < 0.01), effects which were completely blocked by l-NAME (10 microM). In the presence of the enantiomer d-NAME (10 microM, n = 6) which is not an inhibitor of NOS, rhIGF-I (100 nM) still produced a significant increase in both GFR (+175 +/- 15%, n = 6, p < 0.01) and RPF (+94 +/- 7%, n = 6, p < 0.01). AG blunted the renal haemodynamic response to rhIGF-I (GFR +49 +/- 10, RPF + 31 +/- 7, n = 6, p < 0.05), but unlike l-NAME, did not abolish it. The haemodynamic responses of the IPRK to rhIGF-I in the presence of AG were significantly greater than those of rhIGF-I in the presence of l-NAME (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536101 TI - Muscarinic agonists modulate calcium-dependent outward currents in an identified insect motoneurone. AB - The soma membrane of the fast coxal depressor (Df) motoneurone of the cockroach, Periplaneta americana possesses a population of acetylcholine receptors which respond to both nicotinic and muscarinic ligands. Activation of these 'mixed' acetylcholine receptors by McN-A-343 or oxotremorine results in the generation of an inward current at potentials positive to - 40 mV. A large proportion of the outward current induced by membrane depolarization in Df somata is due to a calcium-activated potassium conductance (IKCa) which shows a characteristic N shape voltage-dependence. This N-shape of the membrane current-voltage relationship is suppressed by McN-A-343 or oxotremorine indicating that the agonist-induced inward current is due to a reduction in IKCa. PMID- 7536103 TI - Trigeminal ganglion stimulation increases facial skin blood flow in the rat: a major role for calcitonin gene-related peptide. AB - Activation of the trigeminovascular system leads to neurogenic inflammation within the dura mater and cerebral vasodilatation. These processes have been implicated in the pathogenesis of migraine headache. Neurogenic vasodilator responses to trigeminal ganglion stimulation were investigated in rat facial skin, an area innervated by the trigeminal nerve. Microvascular blood flow changes in the facial skin were measured in anaesthetised rats, using laser Doppler flowmetry. Electrical stimulation of the trigeminal ganglion caused an ipsilateral increase in facial skin blood flow which was found to be frequency dependent (0.5-10 Hz). The role of several neuropeptides in these blood flow responses was studied using selective receptor antagonists. The calcitonin gene related peptide antagonist, CGRP8-37 (400 nmol.kg-1, i.v.) had no effect on resting levels of facial skin blood flow, but markedly inhibited responses induced by trigeminal ganglion stimulation (5 Hz, 10 V, 1 ms for 30 s). However, neither the neurokinin-1 (NK1) receptor antagonist, RP67580 (0.23 or 2.3 mumol.kg 1, i.v.) nor the vasoactive intestinal peptide (VIP) antagonist, [p-Cl-D Phe6,Leu17]-VIP (15 or 30 nmol.kg-1, i.v.) had any effect on these responses. These results suggest that CGRP is the major neuropeptide involved in the vasodilator response to trigeminal ganglion stimulation in rat facial skin. Clarification of the mechanisms involved in this neurogenic vasodilator response may aid the development of drugs that target the trigeminovascular system during migraine headache. PMID- 7536102 TI - Pharmacology of nicotine-induced increase in cytosolic Ca2+ concentrations in chick embryo ciliary ganglion cells. AB - Chick embryo ciliary ganglion cells were acutely isolated, and the mechanism(s) underlying the increase in the cytosolic Ca2+ concentration ([Ca]in) induced by high concentrations of nicotine examined using fura-2 microfluorometry. The order of potencies of nicotinic receptor agonists in increasing [Ca]in was ACh > nicotine = dimethylphenylpiperazinium > cytisine. The nicotine-induced increase in [Ca]in was inhibited not only by nicotinic antagonists but also by muscarinic antagonists, while the muscarine-induced [Ca]in increase was little affected by nicotinic antagonists. The nicotine-induced [Ca]in increase was inhibited by both L- and N-type Ca2+ channel blockers and potentiated by an L-type Ca2+ channel agonist, Bay-K-8644. Nicotine also increased the cytosolic Na+ concentration ([Na]in) as measured by sodium binding benzofuranisophthalate microfluorometry, and this [Na]in increase was inhibited by various agents which reportedly affected nicotinic receptor channels in adrenal chromaffin cells. These results suggest that nicotine increased Na+ influx through nicotinic receptor channels resulting in membrane depolarization, which in turn increased Ca2+ influx through voltage-dependent Ca2+ channels. However, nicotine still increased influxes of Ca2+ and Mn2+ in the absence of external Na+, suggesting that nicotinic receptor channels in these cells are permeable not only to monovalent cations but also to Ca2+ and Mn2+. PMID- 7536104 TI - Bilateral projections from the superior colliculus to the suprageniculate nucleus in the cat: a WGA-HRP/double fluorescent tracing study. AB - Following WGA-HRP injection into the right suprageniculate nucleus of the cat brain, retrogradely-labeled neurons were found not only in the ipsilateral, but also in the contralateral superior colliculi. After WGA-HRP injection into the unilateral superior colliculus, anterogradely-labeled axon terminals were observed in the bilateral suprageniculate nuclei, and electron microscopic examination revealed that these were large terminals which made asymmetric synaptic contacts with dendrites. When a different kind of fluorescent tracer was injected into each suprageniculate nucleus (Fast blue and Nuclear yellow), double labeled neurons were observed in the rostral and middle portions of both superior colliculi. These results suggest that there are direct bilateral projections from the superior colliculus to the suprageniculate nuclei, and that some of these projections originate from branching colliculo-suprageniculate axons. PMID- 7536105 TI - The trochlear nucleus of the frog Rana ridibunda: localization, morphology and ultrastructure of identified motoneurons. AB - The organization of the trochlear nucleus (N IV) was investigated in the frog Rana ridibunda. Retrograde tracing with horseradish peroxidase and biotinylated dextran amines resulted in labeling on the contralateral N IV of motoneurons with diverse morphologies and in direct caudal continuation with the oculomotor nucleus. Their dendritic arborizations extend profusely in the ipsilateral tegmentum and reach the oculomotor nucleus, the fasciculus longitudinalis medialis and also small processes branch towards the ventricle. Occasionally, one to three cells are labeled in the ipsilateral N IV, whereas mesencephalic trigeminal cells that would send their peripheral branch in the IVth nerve are never observed. The course of the labeled trochlear axons varies depending on the localization within the N IV of their cells of origin and different points of decussation are present above the midbrain ventricle. The ultrastructural analysis of identified trochlear motoneurons shows numerous axo-somatic synaptic contacts and six types of terminals with variable morphologies have been observed. Among them, a peculiar type of axon terminal forms mixed junctions with synaptic specializations and gap junctions together in the membrane interfaces that could represent the simultaneous presence of a chemical as well as an electrical component. The present data give more insights into the organization of the N IV and demonstrate that, although the organization of the trochlear nucleus is highly conservative in gnathostome vertebrates, it shows specific features for each species studied, as demonstrated for amphibians. PMID- 7536106 TI - Sympathetic preganglionic neurons contain nitric oxide synthase and project to the superior cervical ganglion: combined application of retrograde neuronal tracer and NADPH-diaphorase histochemistry. AB - Nitric Oxide (NO), which was initially identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in teh intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS) immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1-Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG. PMID- 7536107 TI - Thymic immunopathology and progression of SIVsm infection in cynomolgus monkeys. AB - Thymuses from 22 cynomolgus monkeys infected with simian immunodeficiency virus (SIVsm) developed characteristic cortical and medullary changes including formation of B-cell follicles (8/21) and accumulation of virus immune complexes. Advanced thymic histopathology was correlated with more pronounced immunodeficiency. SIVsm provirus was detected by polymerase chain reaction (PCR) in most (16/18) thymuses and spliced viral env mRNA in 3 (3/7) thymuses with advanced histopathologic changes indicative of thymic SIVsm replication. By combined in situ hybridization (ISH) and immunohistochemistry, viral RNA was localized mainly to the follicular dendritic network, macrophages, multinucleated giant cells, and lymphocytes of the medullary regions. Latent infection by an Epstein-Barr-related herpesvirus (HVMF1) was also found by PCR and by ISH in medullary regions of three (3 of 8) thymuses with B-cell follicles, suggestive of an inductive role for B-cell proliferation in these thymuses. In a control group of HIV-2-infected nonimmunosuppressed monkeys, no comparable thymic changes were observed. Our results indicate that SIV, and probably by analogy HIV, can have direct and diverse pathogenic effects on the thymus that are important in the development of simian (human) AIDS. PMID- 7536109 TI - High specificity of hepatitis C second-generation enzyme immunoassay in HIV infected patients. PMID- 7536108 TI - Immunodominant epitope regions of HIV-1 reverse transcriptase: correlations with HIV-1+ serum IgG inhibitory to polymerase activity and with disease progression. AB - Anti-HIV-1 polymerase antibodies are present in high titer throughout the course of HIV disease. The possibility that a subset of these immunoglobulins might represent an adaptive immune response was suggested by several studies, from this laboratory and others, identifying IgGs capable of inhibiting the catalytic activity of reverse transcriptase (RT) in asymptomatic HIV+ individuals, which were absent from most AIDS patients. We have now determined the dominant B-cell epitope segments of RT recognized during natural HIV-1 infection, with one epitope region, amino acids 244-269, recognized by the majority of HIV-1 positive sera. We have also correlated IgG RT inhibitor activity with amino acids 157-178, corresponding to sequences previously identified by monoclonal antibody mapping and site-directed mutagenesis as necessary for the maintenance of polymerase function. Absence of reactivity to synthetic peptides prepared from this latter region correlated with both advanced HIV disease in a cross-sectional survey of 57 HIV-1+ individuals, and progression of clinical symptoms in a small cohort followed longitudinally. Given current interest in examining diverse components of HIV in development of peptide-based protective vaccines and specific active immunotherapies, these segments of RT might be considered as potential candidates. PMID- 7536110 TI - Determinants of fetal haemoglobin in newborn infants. AB - Percentage fetal haemoglobins (% HbF) were measured in 7081 newborns at the University Hospital, Nottingham, between 1990 and 1992 to assess the effects of a range of factors, and to determine if there was an association between % HbF at birth and risk of SIDS (sudden infant death syndrome). Data from the obstetric clinical records were analysed by multiple linear regression to establish the major determinants of % HbF. New findings were the identification of maternal smoking, twin pregnancy, ethnic origin, and season of birth as significant determinants. Alcohol use, drug and vitamin intake, and duration of labour were not found to be important. The previously reported correlations between % HbF and sex, gestational age, and birthweight were confirmed, and the relative effects quantified. Overall, the major determinants of an increase in % HbF in neonates are similar to the known antenatal risk factors for sudden infant death syndrome (SIDS). PMID- 7536111 TI - Antibody-antigen interactions: new structures and new conformational changes. AB - During the past year, many new antibody structures have been determined, increasing our understanding of these immunologically important molecules. Of special interest are new catalytic antibodies, antibody-peptide and antibody virus complexes, NMR structures, and structures illustrating conformational changes and antibody cross-reactivity. PMID- 7536112 TI - Fibrinolytic abnormalities following liver transplantation in patients with fulminant hepatic failure. AB - OBJECTIVE: To evaluate the fibrinolytic system after liver transplantation in patients with fulminant hepatic failure. DESIGN: Seven patients were studied prior to, and for 4 days after, liver transplantation. METHODS: Both activators and inhibitors of the fibrinolytic system were investigated in seven patients with fulminant hepatic failure who underwent liver transplantation. RESULTS: alpha 2-antiplasmin and C1-inhibitor levels increased rapidly after transplantation (81 and 53% of normal on day 1; 106 and 99% on day 2, respectively). Plasminogen levels remained low throughout the 4-day study period. Plasminogen activator inhibitor-1 was higher than normal before transplantation (21.0 compared with 7.4 U/ml) and increased further on the first day after operation (37.5 U/ml; P < 0.05 versus pre-transplantation). Tissue plasminogen activator levels remained normal (pre-operative, 7.0 IU/ml; Day 4, 0.2 IU/ml). D dimer remained elevated during the postoperative period showing increased fibrinolytic activity. Thrombin-antithrombin III complex was also elevated during the study period. Antithrombin III was greatly reduced prior to transplantation (13.7% of normal) and plasma levels were less than 50% of normal values during the study. CONCLUSIONS: Measures of fibrinolytic activity are raised after liver transplantation in patients with fulminant hepatic failure. This is probably due to increased fibrin formation caused by a coexisting hypercoagulable state. PMID- 7536113 TI - Does hepatitis C contribute to liver injury in alcohol abusers in the west of Scotland? AB - OBJECTIVE: To test the hypothesis that many patients with alcoholic liver disease have coexisting hepatitis C virus (HCV) infection which promotes the development of cirrhosis. DESIGN: Prospective, two-centre study comparing patients with alcoholic liver disease with HCV-positive blood donors identified by the Regional Blood Transfusion Service. SETTING: Two teaching hospitals in Glasgow, UK. PATIENTS: Sixty patients admitted to hospital with a diagnosis of alcoholic liver disease on the basis of clinical and histological tests. For comparison, a group of 50 anti-HCV-positive subjects identified from 305,012 blood donors during the same period (1991-1993) were questioned about their alcohol consumption and liver biopsy specimens are taken. MAIN OUTCOME MEASURES: The prevalence of HCV infection was determined by a second generation enzyme-linked immunosorbent assay (ELISA) for anti-HCV and by liver histology. RESULTS: No patients with alcoholic liver disease were anti-HCV-positive. Of the blood donors with chronic HCV infection, 11 (22%) reported previous or continuing consumption of more than 80 g alcohol daily for at least 2 consecutive years but liver histology in all 50 cases showed features characteristic of chronic HCV. There was no difference in liver histology between donors with a history of high alcohol consumption [mean grade 2.6 (range, 1-5), stage 0.4 (range, 0-2)] and abstinent, anti-HCV-positive donors [grade 2.8 (0-5), stage 0.5 (range 0-1)]. CONCLUSIONS: The absence of anti HCV in this population of patients with alcoholic liver disease shows that HCV is not a necessary or a common cofactor in the development of alcoholic liver disease in the west of Scotland. PMID- 7536114 TI - Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium. AB - Increasing evidence indicates that cationic liposomes are capable of safely transferring foreign genes to pulmonary epithelium in vitro and in vivo. To transfer reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) to mammalian respiratory epithelium we used two cationic lipid formulations: N-[1-(2,3-dioleoyloxy)propyl] N,N,N-triethylammonium chloride (DOTMA), and 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). Lipid DNA conjugates containing either CFTR or LacZ were instilled directly into the airways of Sprague-Dawley rats. Rats treated with LacZ cDNA in vivo demonstrated expression in 30-50% of the large and medium-sized airways, with some airways showing high efficiency gene transfer and expression (in the most proximal airways, 70-80% of surface epithelial cells were positive for expression of a nuclear targeted LacZ). While control and LacZ treated tracheas mounted in Ussing chambers showed minimal stimulation of transepithelial chloride (Cl)-currents by cAMP (suggesting low levels of endogenous rat CFTR activity), tracheas taken from animals receiving CFTR exhibited significant forskolin-stimulated currents at 72 h after gene transfer. Human CFTR gene expression was also detected by polymerase chain reaction (PCR) analysis of reverse transcribed lung RNA. These results, together with previous studies using lipid-mediated gene transfer in mice, help confirm the potential for cationic lipid-mediated gene transfer in the gene therapy of cystic fibrosis in humans. PMID- 7536115 TI - In vitro rat pancreatic digestive enzyme activities and raw and heated glandless cottonseed and soybean flours. AB - Higher nitrogen and lipid digestibilities have been obtained with diets containing cottonseed flour rather than soybean flour. To explain these results, in vitro studies were carried out to compare the effects of raw and heated glandless (without gossypol) cottonseed flours versus soybean flours on pancreatic digestive enzyme activities. These effects were compared with those obtained without addition of flour in standard assays. Apparent lipase (lipase colipase dependent) and potential lipase (lipase with saturating amounts of colipase), colipase, phospholipase A2, amylase, trypsin and chymotrypsin activities were measured on specific substrates. Phospholipase A2 and amylase activities were enhanced, while chymotrypsin activity was diminished with both raw and heated flours. Compared with raw and heated soybean flours, raw and heated cottonseed flours promoted higher potential lipase, chymotrypsin, trypsin and lipase activities. Heat treatment of cottonseed flour enhanced apparent lipase, colipase, chymotrypsin, trypsin activities and diminished potential lipase, phospholipase A2 and amylase activities. When soybean flour was heated, apparent lipase, phospholipase A2, chymotrypsin, trypsin and amylase activities were raised while those of potential lipase were decreased. Our findings show that in vitro raw or heated cottonseed flours affect less digestive enzymes than raw or heated soybean flours, apparent lipase activity excepted. Moreover, only chymotrypsin activities were seriously lowered with both flours, especially with raw soybean flour. Hypotheses are suggested to account for the differences in alterations. PMID- 7536116 TI - The art of lecturing. How to become a scientific entertainer. AB - It is known to all nutritionists and food scientists that the presentation of papers at (inter)national meetings is not always optimal. In this paper on effective lecturing some very practical advice is presented on how to entertain the audience scientifically. With the prerequisite of a clear scientific message, the basic elements for an optimal presentation are discussed with emphasis on the proper use of audiovisual means, including slides and overhead transparencies. Some additional comments are made about posters, chairpersons of sessions, and audiences. PMID- 7536117 TI - [Antigenic changes in skin after cryopreservation]. AB - It is well accepted impression that antigenic properties of skin are lowered after cryopreservation. In order to prove it, this experiment was done. Fresh guinea pig and cadaveric skin homogenates with appropriate adjuvants were injected hypodermically separately to rabbits once a week. Five week later, blood was withdrawn from rabbits, thus anti-guinea pig skin serum (AGS) and anti cadaveric skin serum (ACS) were obtained. Homogenates of fresh guinea pig skin (FG) and guinea pig skin stored at -196 degrees C (SG), homogenates of fresh cadaveric skin (FC) and cadaveric skin stored at -196 degrees C (SC) were used as antigens. Microimmunoelectrophoresis and rocket immunoelectrophoresis were performed between related antigens and antibodies. The results of microimmunoelectrophoresis showed that there were four dense and wide precipitated are lines in AGS vs FG and ACS vs FC groups, while only two light and thin precipitated are lines in AGS vs SG and ACS vs SC groups. Rocket immunoelectrophoresis showed that the rocket precipitated area in AGS vs FG and ACS vs FC groups are larger (longer in axis, and broader in width) than in AGS vs SG and ACS vs SC groups. The antigenicity of skin is lowered after being stored at -196 degrees C as compared with that of the fresh one as shown by electrophoresis method. PMID- 7536118 TI - Determination of human plasma biogenic amines and their metabolites using liquid chromatography with a dual-channel electrochemical detector. AB - BACKGROUND: Measurement of biogenic amines and their metabolites in plasma has been used for decades to explore the pathophysiology of neuropsychiatric disorders or to examine the mode of action of psychotropic medications. Many advantages of ultrafiltration and high-performance liquid chromatography (HPLC) in assaying human plasma content have been described in our previous study. METHODS: The present HPLC assay, applying a dual-channel electrochemical detector, provides an additional reliable measurement or confirmation of peaks by identifying each peak retention capacity factor and its current ratio in the dual channel detector. Simultaneous measurements of plasma serotonin, catecholamines and their metabolites by ultrafiltration and microbore HPLC technique is applied to evaluate moclobemide and fluoxetine therapy in patients with major depression. RESULTS: This present HPLC method provides highly specific detection, free from interference by the large offscale peak of plasma ultrafiltrates. Isocratic separation of all analytes by a microbore column is achieved within 15 mins. The detection limit (signal-to-noise ratio = 3) of this method is about 0.2-0.5 pg per injection for all analytes. In addition, the required volume of plasma samples is only 100 microliters. CONCLUSIONS: This rapid, simple and sensitive method for the measurement of human plasma serotonin, catecholamines and their metabolites can be used as a clinical or biomedical research tool. Blood loss is minimal in the present assay, especially in repeated blood sampling for pharmacokinetic studies. PMID- 7536120 TI - Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood. AB - BACKGROUND: This study was performed to develop a sensitive method for the detection of circulating hepatocellular carcinoma (HCC) cells in peripheral blood, in advance of the diagnosis of distant metastasis of HCC by conventional means. METHODS: Peripheral blood (5 ml) samples were obtained from 64 patients with HCC and from 48 control subjects (31 patients with benign liver disease, 8 with metastatic liver cancer, and 9 with normal liver function). To identify HCC cells in peripheral blood, liver-specific human alpha-fetoprotein (hAFP) mRNA was amplified from total RNA extracted from whole blood by reverse transcriptase polymerase chain reaction. RESULTS: Human alpha-fetoprotein mRNA was detected in 23 blood samples from the HCC patients (23/64, 36%), in 17 patients in whom there was no clinical evidence of distant metastasis. In contrast, there were no control patients whose samples showed detectable hAFP mRNA in the peripheral blood. The presence of hAFP mRNA in blood seemed to be correlated with the stage (by TNM classification) of HCC, the serum hAFP value, and the presence of intrahepatic metastasis, portal vein thrombosis, and/or distant metastasis. CONCLUSIONS: Reverse-transcriptase polymerase chain reaction is a very sensitive method for detecting circulating HCC cells. With this technique, important information for the management of HCC can be acquired, such as the indications for orthotopic liver transplantation in HCC patients. Moreover, use of this detection method may encourage investigation of the mechanism of metastasis in HCC. PMID- 7536119 TI - Detection of Helicobacter pylori infection in early stage gastric cancer. A comparison between intestinal- and diffuse-type gastric adenocarcinomas. AB - BACKGROUND: Helicobacter pylori (H pylori) infection has been suggested to be a risk factor for gastric carcinogenesis. However, those previous studies have been concerned with advanced cancer cases. To the authors' knowledge, no detailed investigation on the prevalence of H pylori in early stage gastric cancer tissue has been performed. The relationship between early stage gastric cancer and the prevalence of H pylori was studied by a immunohistochemical staining analysis. METHODS: Sixty-eight patients who were endoscopically and surgically diagnosed as having early stage gastric cancer were enrolled in this study. All tissue specimens were obtained from patients by endoscopic biopsy, and were classified histopathologically as the intestinal-type of early stage gastric cancer in 34 patients (male-to-female ratio, 28:6; age, 64 +/- 11 years) and the diffuse-type of early stage gastric cancer (male-to-female ratio, 23:11; age, 57 +/- 14 years) in the other 34 patients. The amount of H pylori in tissue samples was graded from 0 (no characteristic bacteria) to 3 (numerous bacteria) using the fluorescent microscopic and an immunohistochemical technique. RESULTS: Twenty nine of the 34 cases of the intestinal-type of gastric cancer had H pylori infection, as compared with 11 of the 34 cases of diffuse-type early stage gastric cancer. A significantly higher incidence (85%; P < 0.001) of H pylori infection and, thus, higher grading scores of the number of H pylori were found in the intestinal-type early stage gastric cancer. CONCLUSIONS: These findings suggest that the infection of H pylori may have a crucial relationship to the early stages of carcinogenesis of intestinal-type gastric cancer. PMID- 7536121 TI - Etiologic factors and clinical presentation of hepatocellular carcinoma. Differences between cirrhotic and noncirrhotic Italian patients. AB - BACKGROUND: It is not known whether the prevalence of hepatocarcinogenic factors differs between cirrhotic and noncirrhotic patients with hepatocellular carcinoma (HCC) or whether the clinical presentation of HCC in these two groups differs. METHODS: The prevalence of the putative etiologic factors of HCC and its clinical presentation in 373 patients with cirrhosis and 102 without cirrhosis seen from 1981 to 1992 were evaluated. RESULTS: Hepatitis C virus infection (76 vs. 48%, P = 0.003) and both current (22.5 vs. 10%, P = 0.007) and past (50.5 vs. 34.5%, P = 0.045) hepatitis B virus infections were more common in cirrhotic than in noncirrhotic patients with HCC. The absence of exposure to both viruses was much less frequent in the former (7 vs. 40%, P < 0.001). Heavy alcohol intake prevailed in patients with cirrhosis (30 vs. 16.5%, P = 0.01). Alpha-fetoprotein elevation was more common in cirrhotic patients (63% vs. 31%, P < 0.001); however, the prevalence of diagnostic (> 400 ng/ml) levels did not differ significantly (24 vs. 17%) between the two groups. Extrahepatic extension of HCC was more common in noncirrhotic patients (20.5 vs. 6.5%, P < 0.001) and its independent predictors were poor cancer differentiation and absence of cirrhosis. "Asymptomatic" cancers were more frequently encountered with cirrhosis. Abdominal pain was the most common presenting symptom in both groups. Signs of hepatic decompensation prevailed in cirrhotic patients, whereas a "toxic syndrome" dominated the clinical picture of the noncirrhotic patients. CONCLUSIONS: Hepatitis viruses are associated more with carcinogenesis of the cirrhotic than of the noncirrhotic liver. Alpha-fetoprotein is not a sensitive neoplastic marker, particularly in noncirrhotic patients. In the latter, HCC appears more advanced at diagnosis and symptoms of neoplastic toxicity are prominent. PMID- 7536123 TI - Evaluation of hyperbaric oxygen as a chemosensitizer in the treatment of epithelial ovarian cancer in xenografts in mice. AB - BACKGROUND: Resistance to chemotherapy is common in bulky hypoxic tumors such as epithelial ovarian cancer. Hyperbaric oxygen (HBO) oxygenates hypoxic tissues and promotes neovascularization. These unique properties of HBO may help overcome chemotherapy resistance by increasing both tumor perfusion and cellular sensitivity. This study was undertaken to determine if HBO increases the response of epithelial ovarian cancer to cisplatin chemotherapy. METHODS: In Phase I, 64 nu/nu mice were divided into four groups and subcutaneously inoculated with cells from the A2780 human epithelial ovarian cancer cell line. Group 1 served as controls. Group 2 received weekly intraperitoneal cisplatin (3.15 mg/kg). Group 3 was exposed to HBO (dives) at 2.4 atmospheres absolute pressure for 90 minutes, 7 days a week. Group 4 received both cisplatin and HBO. In Phase II, 72 mice were divided into two groups and similarly inoculated. Both groups received weekly intraperitoneal cisplatin (2.5 mg/kg). Group 1 was not exposed to HBO. Group 2 was exposed to HBO for 5 days a week. RESULTS: Dramatic tumor neovascularization was found in tumors of mice exposed to HBO (P = 0.0001). There was significant (P = 0.014) tumor growth retardation in Phase I for mice receiving both cisplatin and HBO compared with those treated with cisplatin alone. This significance was noted after just two doses of cisplatin but subsequently lost due to reduced numbers of mice. In Phase II, neovascularization was detectable after 10 HBO treatments (2 weeks) and was maximal after 15 treatments (3 weeks). CONCLUSIONS: Hyperbaric oxygen increases vascularity in bulky tumors such as epithelial ovarian cancer. There appears to be a relationship between increased vascularity and enhanced response to chemotherapy that merits further investigation. PMID- 7536122 TI - A phase I study of anti-GD3 ganglioside monoclonal antibody R24 and recombinant human macrophage-colony stimulating factor in patients with metastatic melanoma. AB - BACKGROUND: Macrophages activated by macrophage-colony stimulating factor (M-CSF) are potent immune effector cells and can mediate both in vitro cytotoxicity and antitumor effects in vivo. A Phase I trial combining M-CSF with R24, a mouse monoclonal antibody against GD3 ganglioside that has been shown to localize to melanoma tumors, induce inflammation at tumor sites, and result in major tumor responses in some patients with melanoma was performed. METHODS: Nineteen patients with metastatic melanoma received a 14-day continuous intravenous infusion of 80 micrograms/kg/day of recombinant human M-CSF. R24 was administered daily by intravenous infusion on days 6-10 at doses of 1, 3, 10, 30, and 50 micrograms/m2/day. RESULTS: All patients developed pruritus and urticaria; 13 patients developed transient thrombocytopenia less than 100,000/mm3. The maximum tolerated dose was not reached. All patients developed a monocytosis characterized by increased expression of the antigen HLA-DR and decreased expression of CD14, a phenotype reported to represent a subpopulation of monocytes active in mediating antibody-directed cellular cytotoxicity. Other biologic effects of treatment included marked but transient decreases in total cholesterol, low density lipoprotein, and high density lipoprotein. Three patients experienced tumor regression in breast, liver, and lymph node metastases and received a second course of therapy. Six of the 19 patients, one of whom received no further therapy, survived more than 2 years and 4 of these patients remain alive 24 to 37 months after treatment. Of the six patients with liver metastases, three (50%) survived more than 2.5 years and one remains alive at 37+ months. CONCLUSIONS: Combination therapy with R24 and M-CSF resulted in both clinical and biologic effects that warrant further investigation of this combination. PMID- 7536127 TI - The gold standard of pain control. PMID- 7536125 TI - Potential benefit of improved local tumor control in patients with prostate carcinoma. AB - BACKGROUND: In the case of prostate carcinoma, radiation therapy is a locally applied treatment modality in a malignancy known for systemic dissemination. Because significant efforts and resources currently are being consumed to improve local tumor control, failure patterns and potential curative gain deserve appropriate assessment. METHODS: From 1975-1989, 647 patients with clinically localized prostate carcinoma were definitively irradiated for biopsy-proven adenocarcinoma of the prostate. Failure patterns were examined, and survival advantage based on improvement in either local or distant disease control was calculated. Distant metastatic rate and cause-specific survival analyses were used as parameters by which to compare the outcome for patients in whom local tumor control was achieved with patients who experienced local failure, thereby assessing further the importance of the effectiveness of locally applied therapy. RESULTS: Three hundred ninety-two (61%) patients at the time of this writing were clinically disease free. Sixty-two (10%) patients failed locally only, 133 (20%) distantly only, and 60 (9%) developed local and distant recurrent disease. Both local and distant failure rates were higher in patients with more advanced stage lesions at presentation, and distant failure rates significantly increased in patients with less differentiated tumors. Pretreatment prostate-specific antigen was found to be useful in predicting recurrence patterns. Overall, there appeared to be more potential for improvement in survival secondary to reducing distant metastasis. The distant survival advantage (DSA) of reducing distant metastases, compared with the local survival advantage (LSA) of improving local tumor control, was 26 versus 14%. Although DSA was greater than LSA within each stage category, the potential to improve survival was most significant in the Stage C group, where DSA was 35% and LSA 16%. Although LSA varied little according to tumor grade, DSA was dependent on tumor grade and varied from 13% for well differentiated lesions to 38% for poorly differentiated lesions. Distant failure free survival at 10 years was 63% for patients with local control and 45% for those with local failure (P = 0.01). Similarly, 10-year cause-specific survival was 75% in locally controlled patients compared with 48% for those with local recurrence (P < 0.001). CONCLUSIONS: Although better local tumor control should translate into at least modest survival gain for patients with prostate carcinoma, additional advantage may be seen with improved systemic therapy or perhaps earlier diagnosis to reduce further the distant metastasis rate. PMID- 7536124 TI - The impact of the inclusion of endorectal coil magnetic resonance imaging in a multivariate analysis to predict clinically unsuspected extraprostatic cancer. AB - BACKGROUND: The introduction of the endorectal coil magnetic resonance imaging (MRI) technique has improved the accuracy of preoperative staging for prostate cancer. This study quantifies the improvement in the ability to identify clinically unsuspected extraprostatic disease with the use of the endorectal coil MRI. METHODS: A retrospective review of the pathologic findings of 347 patients with prostate cancer treated with a radical retropubic prostatectomy was performed. The preoperative clinical indicators including prostate specific antigen (PSA), clinical stage, Gleason score, and endorectal coil MRI data were employed in a multivariate analysis to identify patients who were at high risk for seminal vesicle invasion (SVI) or extracapsular extension (ECE). The sensitivity, specificity, and positive and negative predictive values for predicting SVI and ECE were calculated using the significant clinical indicators found on the multivariate analysis. RESULTS: The clinical factors identified on multivariate analysis as significant predictors of SVI include the endorectal coil MRI data (P < 0.0001), PSA (P = 0.0096), and the Gleason score (P = 0.012). Endorectal coil MRI data (P < 0.0001), PSA (P = .0001), and Gleason score (P < .0001) were significant predictors of ECE. In the patient subgroup with PSA (> 10 20 ng/ml) and Gleason score of 5 to 7, the addition of the endorectal coil MRI data enabled an additional 71 and 27% of patients with SVI and ECE, respectively, to be correctly identified. These patients would have been missed based on the prediction obtained from the PSA and Gleason score alone. CONCLUSIONS: The use of the endorectal coil magnetic resonance imaging data, in addition to prostate specific antigen and Gleason score, provides a more accurate prediction of the pathologic outcome of seminal vesicle invasion and extracapsular extension than the PSA and Gleason score alone for the patient subgroup with a PSA of greater than 10 to 20 ng/ml and Gleason score of 5 to 7. PMID- 7536126 TI - Prostate specific antigen-monitored combination radiotherapy for patients with prostate cancer. I-125 implant followed by external-beam radiation. AB - BACKGROUND: Because results of retropubic I-125 implantation for prostate cancer have been poor, external beam radiation was added postimplant. Serum prostate specific antigen (PSA) was used to assess this approach of combined irradiation. METHODS: Two hundred and thirty-nine patients with clinical Stage T1 or T2 but surgically node-negative prostate cancer were treated. Results were monitored by serial serum PSA evaluation. Positive clinical findings or a rising PSA level defined recurrent disease. RESULTS: With a median follow-up of 45 months (range, 24-120 months), the Kaplan-Meier projected disease free survival rate was 74% at 5 years and 66% at 10 years. Overall at a 60-month minimum follow-up, 77% of patients had a PSA of 0.5 ng/ml or less. This nadir level is highly predictive of a long term disease free survival, significantly better than pretreatment PSA, grade, or clinical Stage T1 or T2. CONCLUSIONS: The PSA-monitored disease free survival rate after combination irradiation appears comparable with that after radical prostatectomy and perhaps superior to that after external-beam radiation. This effect was achieved even though lower-than-usual doses of radiation from both sources were administered. The combination of an I-125 implant followed by external-beam radiation produces high doses within the prostate and, to a lesser extent, in the periprostatic tissue. Additive and synergistic effects from simultaneous irradiation may be responsible for these results. PMID- 7536128 TI - Expression of the prostate-specific antigen gene by a primary ovarian carcinoma. AB - We describe a patient with primary ovarian carcinoma that developed after liver transplantation whose tumor was highly positive for prostate-specific antigen (PSA). PSA in tumor tissue was characterized by two immunoassays, HPLC, immunohistochemistry, reverse transcription-PCR, Southern blotting, and DNA sequencing. PSA in the ovarian tumor was present as free, M(r) 33,000 protein (> 90%) and as PSA bound to alpha 1-antichymotrypsin (M(r) 100,000; < 10%). Immunohistochemistry localized PSA in the cytoplasm of epithelial cells of the tumor. Two separate reverse transcription-PCRs for PSA amplified the expected products which hybridized specifically to a PSA cDNA probe on Southern blots. Sequencing of the PCR products, representing the whole coding sequence of the PSA gene, revealed identity with the sequence of PSA cDNA from prostate tissue. These data suggest that the PSA produced by the ovarian tumor was identical in molecular weight and sequence to prostatic PSA. Based on data of tissue culture experiments with breast carcinoma cell lines, we speculate that the PSA gene in the tumor of this patient was up-regulated by the therapeutically administered glucocorticoids after liver transplantation. PMID- 7536129 TI - Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase. AB - Human thymidine phosphorylase (dThdPase) has been reported to be identical with the platelet-derived endothelial cell growth factor (PD-ECGF). To investigate whether the dThdPase activity of PD-ECGF/dThdPase is indispensable to its angiogenic activity, three PD-ECGF/dThdPase mutants, K115E (Lys-115-->Glu), L148R (Leu-148-->Arg) and R202S (Arg-202-->Ser) were made by site-directed mutagenesis. Although the expression level of the three mutant PD-ECGF/dThdPases in the COS-7 cells was similar to that of wild-type PD-ECGF/dThdPase, dThdPase activity was not detected in the COS-7 cells transfected with the mutant PD-ECGF/dThdPase cDNA. The lysates of COS-7 cells transfected with the wild-type PD-ECGF/dThdPase cDNA had angiogenic activity, but those transfected with the mutant PD ECGF/dThdPase cDNAs did not. An inhibitor of dThdPase, 6-amino-5-chlorouracil, inhibited the angiogenic activity of purified dThdPase. These findings indicate that dThdPase activity is indispensable to the angiogenic activity of PD ECGF/dThdPase. PMID- 7536131 TI - Transformation of thyroid epithelium is associated with loss of c-kit receptor. AB - The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation. PMID- 7536130 TI - Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic antitumor cytolytic T lymphocyte responses. AB - Anticancer vaccine strategies can now target intracellular antigens that are involved in the process of malignant transformation, such as oncogene products or mutated tumor suppressor genes. Fragments of these antigens, generally 8-10 amino acids in length and complexed with MHC class I molecules, can be recognized by CD8+ T lymphocytes (TCD8+). To explore the possibility of using a genetically encoded, minimally sized fragment of an intracellular antigen as an immunogen, we constructed a recombinant vaccinia virus encoding an 8-residue peptide derived from chicken ovalbumin that is known to associate with the mouse H-2Kb molecule. Compared to standard methods of immunization, recombinant molecule. Compared to standard methods of immunization, recombinant vaccinia virus expressing the minimal determinant as well as full length ovalbumin were the only approaches that elicited specific primary lytic responses in C57BL/6 mice against E.G7OVA, a transfectant of the murine thymoma EL4 containing the ovalbumin gene. Stimulating these effectors in vitro with OVA257-264 peptide induced H-2Kb-restricted TCD8+ that not only lysed but also specifically secreted IFN-gamma in response to an antigen. Furthermore, when transferred adoptively, these anti-OVA257-264 TCD8+ cells significantly reduced the growth of established ovalbumin-transfected tumors in a pulmonary metastasis model system. Synthetic transfected tumors in a pulmonary metastasis model system. Synthetic oligonucleotides encoding minimal antigenic determinants within expression constructs may be a useful approach for treatment of neoplastic disease, thus avoiding the potential hazards of immunizing with full-length cDNAs that are potentially oncogenic. PMID- 7536134 TI - [The role of growth factor binding proteins (insulin-like growth factor binding proteins) in humans]. PMID- 7536135 TI - [Analysis of insulin-like growth factor binding proteins]. PMID- 7536132 TI - Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat. AB - 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing. 2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzyme Bbv1. Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G). 3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated with Bbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts. 4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62 +/- 6.0% of total (cortex), 43 +/- 5.3% (striatum), 52 +/- 5.3% (hippocampus), 91 +/- 6.3% (thalamus), 85 +/- 10.2% (hypothalamus), 82 +/- 6.5% (cerebellum), 88 +/- 6.8% (pons/medulla oblongata), and 41 +/- 2.7% (white matter). 5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain. PMID- 7536133 TI - Erythropoietin modulation of intracellular calcium: a role for tyrosine phosphorylation. AB - We have reported that erythropoietin induces a dose-dependent increase in cytosolic calcium ([Cai]) in single human peripheral blood BFU-E derived erythroblasts which is specific for stage of differentiation and that this increase is modulated by erythropoietin through an ion channel permeable to Ca2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Cai] stimulated by erythropoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosine kinases, staurosporine (100 nM), blocked the increase in [Cai] over 20 min following erythropoietin stimulation. However, erythropoietin-induced calcium influx was unaffected by preincubation of cells with specific inhibitions of protein kinase C (calphostin C) or the cAMP- or cGMP dependent kinases (KT 5720, HA 1004), and [Cai] did not increase following stimulation with phorbol 12-myristate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C nor protein kinase A mediate the erythropoietin-induced [Cai] increase. In contrast, preincubation with genistein, a tyrosine kinase inhibitor, blocked the erythropoietin induced increase in [Cai]. To further study calcium entry in erythroblasts, we determined mastoparan, a peptide from wasp venom, induced a dose-dependent rise in [Cai] in erythroblasts which required external calcium. Stimulation of erythroid precursors with 10 microM mastoparan resulted in an increase in [Cai] from 52 +/- 3 nM to 214 +/- 36 nM which peaked at 20 min. The mastoparan-induced [Cai] increase was also dependent on tyrosine phosphorylation since it was blocked by preincubation with genistein. These results demonstrate that both erythropoietin and mastoparan stimulate calcium entry by a mechanism which has a genistein sensitive step and suggest that tyrosine kinase activation is required for the rise in [Cai] to occur. PMID- 7536136 TI - [Effects of tanshionone to bleomycin induced pulmonary fibrosis of rats on histological changes and production of lipid peroxides and hydroxyproline]. AB - Study was designed to explore the effect of sodium tanshionone IIA sulfonate on bleomycin-induced pulmonary fibrosis in rat. The amount of lipid peroxides (LPO), hydroxyproline in rat lung tissue homogenate was determined at 3, 7, 14, 28 day after administration respectively. The results suggest that the amount of LPO, hydroxyproline of rat lung homogenate were significantly decreased in sodium tanshionone IIA sulfonate group. In addition, histological changes and width of alveolar septa and percentage area of pulmonary fibrosis scars was measured. The results show that sodium tanshionone IIA sulfonate could ameliorate bleomycin induced pulmonary fibrosis in rat. The mechanism that sodium tanshionone IIA sulfonate could prevent and treat bleomycin-induced pulmonary fibrosis in rat was discussed. PMID- 7536137 TI - Urinary laminin P1 as an index of glycemic control in children with insulin dependent diabetes mellitus. AB - OBJECTIVE: To evaluate the clinical meanings of urinary laminin P1, urinary laminin P1 concentrations in children with insulin-dependent diabetes mellitus (IDDM) were measured and compared with urinary levels of albumin, kappa light chain (kappa-LC), alpha 1-microglobulin (alpha 1-MG), beta 2-microglobulin (beta 2-MG), and n-acetyl-b-D-glucosaminidase (NAG). RESEARCH DESIGN AND METHODS: Forty six children with IDDM and 31 age-matched controls were studied. The first urine in the morning was collected for three days and stored at -20 degrees C until assayed. The mean values were used for the study. Radioimmunoassay (RIA) was used to measure of laminin P1, kappa-LC, alpha 1-MG, and beta 2-MG. NAG and haemoglobin Aic (HbA1c) concentrations were measured by a colorimetric method and high-performance liquid chromatography, respectively. RESULTS: Urinary laminin P1 levels in IDDM were 54.2 + 3.4 (SE) mU/microM.Cr, significantly higher than those in control subjects (40.7 +/- 2.3 mU/microM.Cr, p < 0.01). In 10 out of 46 patients (21.7 percent), the values were higher than the mean +/- 2 SD of controls. Urinary albumin, kappa-LC, alpha 1-MG, beta 2-MG, and NAG in IDDM were higher than those in control subjects (p < 0.01). There were significant correlations between urinary laminin P1 levels and urinary albumin, kappa-LC, NAG, alpha 1-MG, and beta 2-MG. A significant correlation was also shown between urinary laminin P1 and HbA1c concentrations (p < 0.01). CONCLUSIONS: From the above results, we conclude that urinary laminin P1 concentrations could be one of the clinical indexes for glycemic control and damage of the glomerulus in IDDM. PMID- 7536139 TI - Effects of L-arginine and L-nitro-arginine treatment on blood pressure and cardiac output in a rabbit endotoxin shock model. PMID- 7536138 TI - Early postoperative enteral nutrition with arginine-omega-3 fatty acids and ribonucleic acid-supplemented diet versus placebo in cancer patients: an immunologic evaluation of Impact. AB - OBJECTIVE: To evaluate the effect of early postoperative feeding with a nutritionally complete enteral diet supplemented with the nutrients arginine, ribonucleic acid (RNA), and omega-3 fatty acids on the immune function in patients undergoing surgery for upper gastrointestinal (GI) malignancies. DESIGN: Prospective, randomized, placebo-controlled, double-blind study. SETTING: Surgical intensive care unit (ICU) in a German university hospital. PATIENTS: Forty-two consecutive patients receiving an enteral diet via needle catheter jejunostomy after GI surgery for cancer. INTERVENTIONS: Patients were randomized to receive either the arginine, RNA, and omega-3 fatty acids supplemented diet or an isocaloric and isonitrogenous placebo diet. Early enteral nutrition was started on postoperative day 1 in the surgical ICU with 20 mL/hr and progressed to the optimal goal of 80 mL/hr by postoperative day 5. MEASUREMENTS AND MAIN RESULTS: Clinical examination and adverse GI symptoms were recorded on a daily basis. Body weight was determined twice weekly. Immunoglobulin concentrations were determined by laser nephelometry. Interferon-gamma concentrations were measured with a modified enzyme-linked immunosorbent assay method. Fluorescence activated cell scan flow cytometry was performed to analyze B cells, T lymphocytes and their subsets. Clinical patient characteristics and mean caloric intake were similar between the two groups and both formulas were well tolerated. The number of T lymphocytes and their subsets, helper T cells (CD4) and activated T cells (CD3, HLA-DR), were significantly higher in the supplemented diet group on postoperative days 10 and 16 (p < .05). Mean interferon-gamma concentration after phytohemagglutinin stimulation was higher in the supplemented diet group on postoperative day 16. In the supplemented diet group, mean immunoglobulin M concentrations were significantly higher on postoperative day 10 and mean immunoglobulin G concentrations were higher on postoperative day 16 (p < .05) compared with the results in the placebo group. B-lymphocyte indices were significantly higher in the supplemented vs. the placebo diet group on postoperative days 7 and 10 (p < .05). CONCLUSIONS: Supplementation of enteral diet with arginine, RNA, and omega-3 fatty acids in the early postoperative time period improves postoperative immunologic responses and helps to overcome more rapidly the immunologic depression after surgical trauma. PMID- 7536140 TI - Symbolic meanings of the body in Chinese culture and "somatization". AB - It is proposed in this paper that the term "somatization" as applied to the Chinese requires further exploration. By way of a study of Chinese language usage employing body-related expressions, an indirect method of finding the "equivalence in meaning" is demonstrated. Results of the study reveal the scope and depth of the symbolic meanings of the body in Chinese culture. PMID- 7536142 TI - Absorptive function of segmental allotransplanted small intestines in pigs. AB - Ten outbred pigs were each operated on for three times. First, a 130 cm length of terminal ileum of each pig was isolated on its vascular pedicle as a Thirty-Vella loop. One week later, the solitary ileal segments were transplanted heterotopically in two pigs. And after 28 days of heterotopic transplantation, the transplanted intestine was interposed into continuity of host intestine as orthotopic transplant. During the experiment, tests were made on 6th day after the first operation (period I), the 14th (II), 28th (III) day after heterotopic transplantation, and 3 weeks after interposition (IV) respectively for the levels of glucose, palmitate and leucine. Additionally, at period I, III, and IV, a 3 cm length of intestinal mucosa was excised for morphologic observation and determination of DNA, RNA and protein contents. After heterotopic transplantation, the absorptive function of transplanted intestine was severely impaired for two weeks. The absorption of glucose and palmitate was partially recovered by period III, at which time leucine level had return to normal. At period IV, the absorptive function of glucose and leucine had surpassed normal levels, while palmitate had risen to the level of pretransplantation. After transplantation, at period III, DNA, RNA and protein contents were well below normal. Three weeks after orthotopic transplantation, RNA and protein had risen to normal level, while DNA content remained below normal. The morphologic changes during the experiment were correlated with the changes of contents in RNA, protein and DNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536141 TI - Prostate cancer. AB - Prostate cancer is the most common noncutaneous malignancy diagnosed in American men, and in 1994 it will pass lung cancer as the most common cancer diagnosed in the United States, with an estimated 200,000 new cases. The molecular biology of prostate carcinogenesis is rapidly advancing, and it is clear that, to a degree, prostate cancer is a heritable disease. The use of serum prostate-specific antigen (PSA) as a screening tool has been widely accepted by the medical community, although the evidence to support the efficacy of screening is not yet available. The curative approaches to organ-confined, clinically localized prostate cancer include radiation therapy, radical prostatectomy, and close observation in selected patients. The absence of well-designed clinical trials contributes to the confusion surrounding which curative treatment is the best option in individual patients. The standard approach to patients with evidence of extracapsular spread without distant metastases has been external-beam radiotherapy, although the results with radiation therapy alone in these patients has left considerable room for improvement. Innovative combined-modality approaches are currently being investigated at a number of institutions for these poor-prognosis patients. Three-dimensional conformal radiation therapy is currently being investigated at multiple institutions and offers some hope for improved results. The treatment of metastatic disease remains hormonal manipulation, although the exact nature of optimal androgen deprivation is currently a matter of considerable debate. In patients with hormone-refractory disease newer regimens using novel chemotherapy regimens offer some promise. PMID- 7536143 TI - Double cortical line in the acetabular roof of paraplegic patients. AB - The pelvic radiographs of 60 patients with paraplegia or quadriplegia were reviewed and double cortical line in the acetabular roofs on both sides was found in 35 patients. It was concluded that the double cortical line was a radiologic sign of disuse osteoporosis after spinal cord injury. It was noticed that the acetabular double cortical line in incomplete paralysis was not so prominent as that in complete paralysis (P < 0.05), suggesting that the degree of acetabular osteoporosis may be related to the severity of spinal cord injuries. The etiology of double cortical line and related factors were discussed. PMID- 7536144 TI - [Simplified combination bypass or single biliodigestive anastomosis in nonresectable pancreatic carcinoma?]. PMID- 7536145 TI - [Treatment of advanced and recurrent gynecologic cancer with chemotherapy by cannula perfusion into both internal iliac arteries]. AB - This paper reports fourteen cases of advanced and recurrent gynecologic cancers treated with prolonged arterial perfusion chemotherapy by catheterization of both internal iliac arteries through femoral arteriopuncture under X-ray. The immediate effective rate was 78.5%, follow-up rate 100%. By this technique anticancer drugs can be selectively injected into vessels supplying the tumors. The drug is concentrated within the tumor resulting in longterm better local effects, and fewer general side reactions, and a much higher dose than that which could be given conventionally. This technique is considered as a new route of treatment in gynecologic tumors. PMID- 7536146 TI - [Adenomatoid tumors in the uterus: an immunohistochemical and ultrastructural study]. AB - In 2 cases of adenomatoid tumors of the uterus FVIII, keratin, vimentin and carcinoembryonic antigen (CEA) were observed by immunohistochemical method. No staining for FVIII and CEA were seen in the tumor cells but staining for keratin and vimentin were seen coexistent in the tumor cells. Electron microscopic finding showed tumor cells attached to one another by numerous long slender microvill. Adenomatoid tumors had typical mesothelial features. It suggests that adenomatoid tumor may be a mesothelioma. PMID- 7536147 TI - [CD5+B cells and autoimmune diseases]. AB - By using two colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 surface antigen (CD5+B cell). The level of CD5+B cells was markedly increased in active chronic ITP and active hyperthyroid patients with Graves' disease. There was an inverse correlation between the percentage of circulating CD5+B cells and platelet counts, and a positive correlation between the level of CD5+B cells in peripheral blood and serum T3 level. These results suggested that the level of CD5+B cells might play a role in producing autoantibodies in some autoimmune diseases. PMID- 7536148 TI - [A study on the patterns of early specific antibody response in patients with posttransfusion hepatitis C]. AB - Techniques of enzyme-linked immunosorbent assay based on synthetic multiple peptide fragments and second generation recombinant immunoblot assay (RIBA) were used to study the patterns of specific antibody response in 10 cases of posttransfusion hepatitis (PTH) during a period of 36-38 weeks after blood transfusion. Nine cases were positive with serum anti-HCV, including 8 cases positive with serum HCV-RNA. Antibodies to core protein of HCV showed a higher positive rate and were detected 1-3 weeks earlier than those to the putative nonstructural (NS) protein. Anti-HCV IgM to core protein were detected 1-4 weeks earlier than anti-HCV IgG and the detective absorbent values of anti-HCV IgM were positively correlated with the levels of serum ALT (P < 0.01). "Passive transfer" of anti-HCV were found in 3 cases. These facts suggest that HCV is the major causative agent of PTH cases in our district and anti-HCV IgM to core protein is a putative serological marker not only for early diagnosis of HCV infection but also for demonstration of active HCV infection. PMID- 7536149 TI - [Adjuvant and palliative chemotherapy for stomach carcinoma]. PMID- 7536150 TI - Heart period variability in sleep. AB - Analysis of heart period variability is a dynamic noninvasive technique to quantify the autonomic control over the heart period. We recorded electroencephalographic, electro-oculographic, electromyographic and electrocardiographic data from 10 normal subjects during sleep using an ambulatory polysomnographic monitor. R-R intervals were determined for 10 min segments of electrocardiographic data from wakefulness, stage 2 sleep, slow wave sleep and REM sleep. Average heart period, instantaneous changes greater than 50 msec and fractal dimension were calculated and the time domain and phase plots were depicted. The R-R interval time domain plots were subsequently analyzed using the discrete Fourier transform. We found sleep stage specific, time domain and frequency domain changes in heart period variability, particularly using spectral analysis of heart period. Increased power in the 0.2-0.4 Hz band was associated with stage 2 sleep when compared to awake and slow wave sleep states. Power in the 0.0-0.04 and 0.04-0.12 Hz bands was increased in association with REM sleep when compared to non-REM sleep, and slow wave sleep had diminished power in all frequency bands. Our results support other investigations demonstrating stage 2 sleep is associated with increased parasympathetic influences and REM sleep is associated with increased sympathetic and neurohumoral influences. We feel that spectral analysis of heart period variability is an effective noninvasive method to quantify changes in the autonomic influences over the heart during sleep. PMID- 7536152 TI - EEG activation patterns during the performance of tasks involving different components of mental calculation. AB - In this study we demonstrate the existence of different patterns of EEG activation during the performance of 4 different tasks involving different components of mental calculation in normal subjects. The EEG was recorded in all monopolar leads of the 10/20 system using linked ear lobes as reference. Absolute and relative power were calculated in the delta (1.5-3.5 Hz), theta (3.5-7.5 Hz), alpha (7.5-12.5 Hz) and beta (12.5-19 Hz) bands. The tasks were presented randomly and the EEG segments preceding presentation of the stimulus were considered as the rest corresponding to the task requested by the stimulus. Tasks were of 4 different types, involving number comprehension, recognition of mathematical symbols, the calculation process and the spatial component. ANOVAs between the rest periods showed no differences in any band. Neither did ANOVAs between tasks. However, other variables (task minus rest), which were calculated as the differences in power between task and rest respectively, showed significant differences between tasks in the delta and beta bands in the frontal lobes. In addition, new variables were calculated as the difference between tasks, since many factors were common across several tasks. These variables correspond to the EEG change due to a specific component of mental calculation. Significant differences were obtained in delta and theta bands in right posterior areas and in the beta band in frontal areas. We concluded that the EEG differences observed during different components of mental calculation suggest the participation of different networks. PMID- 7536151 TI - Symptomatic and asymptomatic high-grade unilateral internal carotid artery stenosis: scalp topography of event-related potentials (P300) and psychometric testing. AB - Unilateral internal carotid artery (ICA) stenosis may be accompanied by widespread atherosclerosis of extra- and intracranial vessels leading to subtle cognitive disorders. We applied multichannel recording of P300 in 28 patients (68.3 +/- 8.1 years; 15 asymptomatic, 13 with a history of transient ischemic attack (TIA)) and compared them with an age- and sex-matched control group. All underwent a visual "odd-ball paradigm" as well as a psychometric test, the Cognitive Performance Test (CPT), testing mainly visual attention and memory. The potentials were derived from 16 electrodes according to the 10/20 system against linked mastoids. The latencies and amplitudes of N250 and P300 were measured and their amplitudes additionally mapped. Furthermore, the early sensory exogenous potentials, P1 and N1, within the P300 potentials as well as conventional pattern reversal visual evoked potentials (PVEPs) were evaluated. (1) Both the early exogenous potentials and the conventional PVEPs showed no significant differences among all groups. (2) There were no significant differences between asymptomatic patients and those with a TIA history in all parameters of the P300 complex so that one total patient group was constructed and compared to the controls. (3) Patients' P300 amplitudes showed significant reductions over hemispheres ipsilateral (P < or = 0.014) and contralateral (P < or = 0.044) to the stenosis. (4) The N250 amplitudes were reduced only in the central leads (P < or = 0.05). (5) The latencies of N250 potentials were significantly prolonged at many electrodes, not only ipsi-(P < or = 0.0007) but also contralateral (P < or = 0.022) to the stenosis. (6) The patients' P300 latencies showed significant lengthening only at occipital sites (P < or = 0.05) compared to controls. (7) In all measured parameters, within the patient group, the differences between hemispheres ipsilateral versus contralateral to the ICA stenoses did not reach statistical significance. (8) The CPT values detected slight cognitive disorders for both patient groups and they correlated significantly with the latencies in many leads. (9) The highest test sensitivity to classify patients versus controls (z score > 2) was reached in P300 maps of TIA patients (77%). An altered P300 indicates electrophysiologically, and CPT behaviorally, subclinical cognitive deficits even in asymptomatic patients with unilateral tight ICA stenoses. Interestingly, no differences between asymptomatic and TIA patients with a high grade unilateral ICA stenosis could be found. PMID- 7536153 TI - Functional localization of bilateral auditory cortices using an MRI-linked whole head magnetoencephalography (MEG) system. AB - In 20 healthy subjects, auditory evoked magnetic fields were measured over the entire head, using a helmet-shaped 66-channel MEG system linked to MRI. When the left or right ear was stimulated by 60 msec 2 kHz tones, the prominent 100 msec response (N100m) appeared significantly earlier in the contralateral hemisphere than in the ipsilateral one. In 16 cases, the N100m dipolar field patterns were clear in both hemispheres, overlapping each other across the midline. The N100m sources were estimated using a 2-dipole model in a spherical conducting medium with the size and location of the sphere determined individually according to the MRI images. No differences were found between the contralateral and ipsilateral N100m dipole positions in one hemisphere. When superimposed on MRI, the N100m dipoles were located precisely on the upper surface of bilateral temporal lobes with a standard deviation of 2.2 mm in the superior-inferior direction. In 16 right handed males, the right hemispheric N100m dipoles were 6 mm anterior to the left hemispheric dipoles. The whole head MEG is suitable to see small but significant differences of bilateral cerebral function, with exceptionally high spatial resolution, confirmed by the MRI-linked system. PMID- 7536154 TI - Intracerebral potentials to rare target and distractor auditory and visual stimuli. I. Superior temporal plane and parietal lobe. AB - Event-related potentials were recorded from 537 sites in the superior temporal plane and parietal lobe of 41 patients. Depth electrodes were implanted to localize seizure origin prior to surgical treatment. Subjects received an auditory discrimination task with target and non-target rare stimuli ("standard oddball paradigm"). In some cases, the target, distracting and frequent tones were completely balanced across blocks for pitch and volume. Variants included an analogous visual discrimination task, or auditory tasks where the rare target event was the omission of a tone, or the repetition of a tone within a series of alternating tones. In some subjects, the same auditory stimuli were delivered but the patient ignored them while reading. Three general response patterns could be distinguished on the basis of their wave forms, latencies and task correlates. First, potentials apparently related to rarity per se, as opposed to differences in sensory characteristics, or in habituation, were observed in the posterior superior temporal plane, beginning with a large positivity superimposed on early components. This positivity peaked at 150 msec after stimulus onset and inverted in sites superior to the Sylvian fissure. Subsequent components could be large, focal and/or inverting in polarity, and usually included a positivity at 230 msec and a negativity at 330 msec. All components in this area were specific to the auditory modality. Second, in the posterior cingulate and supramarginal gyri, a sharp triphasic negative-positive-negative wave form with peaks at about 210-300 400 msec was observed. This wave form was of relatively small amplitude and diffuse, and seldom inverted in polarity. It was multimodal but most prominent to auditory stimuli, appeared to remain when the stimuli were ignored, and was not apparent to repeated words and faces. Third, a broad, often monophasic, wave form peaking at about 380 msec was observed in the superior parietal lobe, similar to that which has been recorded in the hippocampus. This wave form could be of large amplitude, often highly focal, and could invert over short distances. It was equal to visual and auditory stimuli and was also evoked by repeating words and faces. The early endogenous activity in auditory cortex may embody activity that is antecedent to the other patterns in multimodal association cortex. The "triphasic" pattern may embody a diffuse non-specific orienting response that is also reflected in the scalp P3a. The later broad pattern may embody the cognitive closure that is also reflected in the scalp P3b or late positive component. PMID- 7536155 TI - The use of a long-acting somatostatin analogue (octreotide) for prophylaxis of acute pancreatitis after endoscopic sphincterotomy. AB - Acute pancreatitis is a serious complication of endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy (EST). In addition, serum pancreatic enzymes increase without clinical symptoms in about 40-50% of patients undergoing these endoscopic procedures. We evaluated the potential of octreotide, a long-acting somatostatin analogue, to prevent these complications in patients who underwent EST for choledocholithiasis. 151 patients were randomly allocated to two groups (A and B). Group A was given 0.1 mg of octreotide subcutaneously 120 and 30 min before EST and four hours after; group B was given a placebo. Serum amylases (normal range 20-220 IU/l) were measured before premedication and 4, 24, and 48 hours after the end of endoscopy. After EST, the increase in the mean serum amylase was greater in the control group, but the difference was statistically significant only at the 48-hour measurement. There were five cases of acute pancreatitis in each group, with a trend (but not statistically significant) toward less severe pancreatitis in the treated group. In the control group, one patient with acute pancreatitis died. In conclusion, octreotide does not seem to prevent acute post-EST pancreatitis. PMID- 7536157 TI - Transurethral microwave therapy: patient selection and outcome. AB - Forty-one patients with benign prostatic disease awaiting transurethral resection of the prostate were offered transurethral microwave therapy as an alternative. Pre-operative assessment consisted of symptom scores, prostate-specific antigen levels, flow rates and urinary tract ultrasound with residual urine estimation. Patients were reassessed 6 weeks, 3 months and 6 months after microwave treatment. Twenty-three patients had a successful outcome and 18 an unsuccessful outcome to treatment. Fifteen of the 18 with an unsuccessful outcome could have been predicted by the presence of one or more of the following pretreatment features: glands over 50 g (10 patients), the presence of a median lobe (5 patients), high residual urine (6 patients), a history of recurrent urinary infection (2 patients) and coexisting neurological disorders such as parkinsonism (1 patient) and CVA (1 patient). Three failures had none of these criteria present and could not have been predicted from their pretreatment assessment. Transurethral microwave therapy produces subjective and objective improvements in appropriately selected patients. Patients with large glands or decompensated bladders fail to benefit and should continue to be treated by conventional surgery. PMID- 7536158 TI - Urodynamic changes in benign prostatic hyperplasia patients treated by transurethral microwave thermotherapy. AB - Our study describes the results obtained in 44 patients with benign prostatic hyperplasia (BPH) who underwent transurethral microwave thermotherapy (TUMT) and their follow-up at 12 months. Prostatron, a prostatic TUMT device which comprised a microwave heat generator and a cooling system, was used at our centre. Forty four out of the 60 patients given the treatment had a 12-month follow-up to be evaluated. The evaluation of subjective symptoms, according to the Boyarsky symptom score, demonstrated a significant rate of response (p < 0.0005). The analysis of pressure-flow recordings demonstrated a reduction of mean detrusor opening pressure (p < 0.0005), a reduction of detrusor pressure at maximum flow 12 months after treatment (p < 0.0005) and an improvement of mean values of maximum flow after treatment (p < 0.0005). This study shows that TUMT, though it cannot be considered an alternative to surgical or endoscopic therapy of BPH for severely obstructed patients, can produce improvement of both subjective and objective parameters of mild prostatic bladder outflow obstruction. PMID- 7536159 TI - Endothelin-induced facilitation of sympathetic neurotransmission to the rat vas deferens: effects of suramin. AB - Experiments were conducted to elucidate the mechanisms of action of endothelins in facilitating neurotransmission to the rat isolated vas deferens. Endothelin-1 and endothelin-3 potentiated field stimulation-induced contractions and those evoked by ATP and alpha, beta-methylene ATP. Responses to noradrenaline were unaffected. The C-terminal hexapeptide, endothelin-(16-21) was without effect on neurotransmission. The facilitation by endothelin-1 of responses to trains of stimulation (10 Hz for 10 s) was absent in the presence of the P2-purinoceptor antagonist, suramin, in concentrations which antagonised the contractile effects of alpha, beta-methylene ATP, but not those of noradrenaline. Suramin did not affect 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)- phenyl]-3 pyridine carboxylic acid methyl ester (Bay K 8644)-induced potentiation of contractions in response to field stimulation. These results support the hypothesis that endothelin-induced facilitation of sympathetic neurotransmission to the vas deferens is due to potentiation of the postjunctional effects of the co-transmitter, ATP, acting at P2X-purinoceptors, and indicate that this effect is mediated through actions at endothelin receptors that are not of the ETB subtype. PMID- 7536156 TI - Lead alters the immunogenicity of two neural proteins: a potential mechanism for the progression of lead-induced neurotoxicity. AB - Some heavy metals have been suspected of playing a role in the pathogenesis of nervous system diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. In these disorders, autoantibodies against neural proteins are evident at some stage of the disease. Lead is known to affect both the immune and nervous systems. Work in our laboratory has shown that lead exposure leads to the production of autoantibodies against neural proteins, including myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). We hypothesize that lead aggravates neurological disease by enhancing the immunogenicity of nervous system proteins, including MBP and GFAP. To test this hypothesis, lead-altered protein was prepared by incubating MBP or GFAP with lead acetate for 24 hr. On days 0, 14, and 28, mice received inoculations with either saline, native protein, or lead-altered protein. Anti-MBP and anti-GFAP, isotypes IgM and IgG, were measured in sera by ELISA on day 38. Sera of mice treated with lead-altered MBP had statistically higher anti-MBP IgG titers than both control and native MBP-immunized mice. An analogous response was seen in mice immunized with lead-altered GFAP. Supernatants from lectin-stimulated splenocytes were also examined for antibody titers and for interleukin 2 (IL-2) and interleukin 6 (IL 6) levels. A significant increase in IL-6 production was seen in mice immunized with lead-altered MBP but not with lead-altered GFAP. No changes were observed in the IL-2 levels of mice immunized with either lead-altered protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536160 TI - Inhibition of hind-paw edema and cutaneous vascular plasma extravasation in mice by acetylshikonin. AB - Acetylshikonin, a naphthoquinone isolated from the Chinese herb medicine, tzu ts'ao, was demonstrated to inhibit the polymyxin B-induced hind-paw edema in normal as well as in adrenalectomized mice. Liver glycogen content was increased in adrenalectomized mice pretreated with dexamethasone, but not with acetylshikonin. Like diphenhydramine, methysergide and isoproterenol, acetylshikonin reduced the plasma exudation evoked in dorsal hind-paw skin by antidromic stimulation of the saphenous nerve, and in passive cutaneous anaphylactic reaction, bradykinin-, substance P-, compound 48/80-, histamine- and serotonin-induced ear edema. Indomethacin was ineffective in these respects. Bradykinin- and substance P-induced plasma exudation were also significantly reduced when [Thi5,8,D-Phe7]bradykinin and [D-Pro2,D-Trp7,9]substance P were coinjected with bradykinin and substance P, respectively. In isolated rat peritoneal mast cell preparation, acetylshikonin produced a concentration dependent inhibition of histamine and beta-glucuronidase release from mast cells challenged by compound 48/80. In compound 48/80-pretreated mice, acetylshikonin and isoproterenol produced significantly more inhibitory effect on bradykinin- and substance P-induced plasma exudation than did diphenhydramine in combination with methysergide. Pretreatment with diphenhydramine/methysergide in compound 48/80-pretreated mice significantly further reduced the bradykinin- and substance P-induced plasma exudation if [Thi5,8,D-Phe7]bradykinin and [D-Pro2,D Trp7,9]substance P were coinjected with bradykinin or substance P, respectively. The results suggest that the inhibitory effect of acetylshikonin on the edematous response is due neither to the release of steroid hormones from the adrenal gland nor to the glucocorticoid activity, but probably partly to the suppression of mast cell degranulation and partly to protection of the vasculature from mediator challenge. PMID- 7536161 TI - Human and rat cutaneous mast cells: involvement of a G protein in the response to peptidergic stimuli. AB - Recent evidence suggests that peptides induce the release of mediators from rat peritoneal mast cell by means of a receptor-independent mechanism, possibly involving an interaction with sialic acid residues at the cell surface followed by the activation of a guanine nucleotide binding protein (G protein). We have now examined the potential involvement of sialic acid residues and of G protein stimulation in the activation of both human and rat cutaneous mast cells by neuropeptide Y, its C-terminal fragments and the wasp venom peptide, mastoparan. Neuropeptide Y-(18-36) was the most effective histamine releaser of the fragments tested, the order of potency being neuropeptide Y-(18-36) > neuropeptide Y-(22 36) > neuropeptide Y-(1-36). This order of potency suggests that the effects of the peptides are not mediated through classical NPY receptors. The hydrolysis of sialic acid residues by neuraminidase and the inhibition of G proteins by benzalkonium chloride or pertussis toxin significantly inhibited the secretory response of cutaneous mast cells to neuropeptide Y-(18-36) and mastoparan. These results demonstrate that the peptidergic pathway described for the activation of peritoneal rat mast cells is also involved in the response of cutaneous human and rat mast cells to peptides. PMID- 7536163 TI - E-selectin involvement in the pathogenesis of splanchnic artery occlusion shock. AB - We investigated the involvement of E-selectin in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham-operated animals were used as controls. Survival time, serum tumor necrosis factor-alpha, while blood cell count, mean arterial blood pressure and myeloperoxidase activity were determined. Splanchnic artery occlusion-shocked rats had a decreased survival time (85 +/- 8 min, while sham-shocked rats survived more than 4 h), reduced mean arterial blood pressure, increased serum levels of tumor necrosis factor-alpha (186 +/- 9 U/ml) and myeloperoxidase activity in the ileum (0.10 +/- 0.04 U x 10(-3)/g tissue) and in the lung (1.5 +/- 0.06 U x 10(-3)/g tissue). Shocked rats showed histological alterations in the ileum and in the lung. Administration of a hyperimmune serum containing specific antibodies raised against E-selectin significantly increased survival time (225 +/- 10 min), reduced leukopenia and myeloperoxidase activity both in the ileum (0.035 +/- 0.001 U x 10(-3)/g tissue) and in the lung (0.3 +/- 0.005 U x 10(-3)/g tissue), improved the cardiovascular changes and reduced the histological alterations in the ileum and lung. Our data are consistent with an involvement of E-selectin in the pathogenesis of splanchnic artery occlusion shock. PMID- 7536162 TI - Aminoguanidine inhibits both constitutive and inducible nitric oxide synthase isoforms in rat intestinal microvasculature in vivo. AB - The effects of aminoguanine on the intestinal vascular permeability following endotoxin administration in vivo has been compared to those of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) in the rat. Concurrent administration of aminoguanidine. (12.5-50 mg/kg, s.c.) with endotoxin (E. coli lipopolysaccharide, 3 mg/kg, i.v.), dose dependently increased vascular leakage of radiolabelled albumin in the ileum and colon after 1 h, an effect reversed by the pretreatment with L-arginine (300 mg/kg, s.c.). Aminoguanidine (50 mg/kg, s.c.) also elevated arterial blood pressure over the 1 h investigation period. Similar acute potentiation of endotoxin-provoked vascular injury was observed 1 h following L-NMMA (50 mg/kg s.c.) which also increased blood pressure, indicating the inhibition of constitutive NO synthase. By contrast, administration of aminoguanidine (12.5-50 mg/kg, s.c.) 3 h after endotoxin, at the time of the expression of the inducible NO synthase, reduced the subsequent endotoxin-induced vascular leakage, as did L-NMMA (50 mg/kg). In homogenates of rat ileal or colonic tissue, aminoguanidine inhibited both the constitutive and inducible NO synthase activity showing only 2-fold selectivity for the inducible isoform. Thus, although aminoguanidine inhibits these isoforms of NO synthase, it is not a selective inhibitor of the inducible isoform in the intestinal microvasculature in vivo. PMID- 7536164 TI - Inhibition of nitric oxide synthase partially attenuates alterations in the blood cerebrospinal fluid barrier during experimental meningitis in the rat. AB - The permeability of the blood-cerebrospinal fluid (blood-CSF) barrier is increased upon exposure to lipopolysaccharides during bacterial meningitis. Lipopolysaccharides induce nitric oxide (NO) synthase in a variety of cells. Increased meningeal NO production and blood-CSF barrier permeability were observed in a rat model of meningitis. Administration of aminoguanidine, an inhibitor of NO synthase, blocked meningeal NO production and significantly attenuated permeability changes in the blood-CSF barrier. It is hypothesized that pathological production of NO may contribute to the disruption of the blood-CSF barrier during meningitis. PMID- 7536165 TI - Thy1 (+) and (-) lung fibrosis subpopulations in LEW and F344 rats. AB - Appreciation of the potential of fibroblasts as effector cells in inflammation has led to the recognition of fibroblast subpopulations, the most stable of which are the Thy1 (+) and Thy1 (-) subpopulations in mouse lung fibroblasts. We investigated the presence of Thy1 (+) and (-) fibroblasts in rats, comparing the percentage in primary cultures from rats with different susceptibility to fibrosis, and whether the characteristics were similar in mice and rats, and between normal and fibrotic rats lungs. Using primary cultures of rat fibroblasts obtained both from normal and fibrotic lungs, we analysed the percentage of Thy1 (+) and (-) fibroblasts by fluorescence-activated cell sorter (FACS) analysis. We sorted the fibroblasts to evaluate immune region associated antigen (Ia) expression, which tends to be raised in tissues involved in inflammation, and other characteristics. We found that Thy1 (+) and (-) fibroblasts: 1) are distinct subpopulations in rat lungs; 2) are found in different proportions in rat strains with different propensity towards lung fibrosis; and 3) have similar but not identical characteristics in mice and rats. We also found that bleomycin induced fibrosis increases the percentage of Ia expression in Thy1 (-), but not Thy1 (+) fibroblasts. The presence of these stable fibroblast supopulations in multiple species, and the fact that these fibroblasts differ in their response to a fibrosing agent, suggests the importance of considering fibroblast subpopulations in development and disease. PMID- 7536166 TI - [Acute phase blood proteins: role in the homeostasis and their synthesis induction in the liver]. AB - Role of acute phase proteins (APP) and mechanisms that regulate their synthesis in the liver are considered. It is known that acute infection, trauma and neoplastic growth cause the acute phase response characterized by a complex of local and systemic reactions of the organism. Production of some acute phase plasma proteins increases (positive APP) while concentration of the others decreases (negative APP). In this review the role of the most important positive APP, such as C-reactive protein, the third component of the complement, alpha 1 antitrypsin, serum amyloid haptoglobin, hemopexin and some others and of negative APP such as albumin, transferrin, apoliprotein A-1 is discussed. Synthesis of APP in hepatocytes is regulated by three main inflammatory cytokines, such as IL-1, IL-6, TNF, produced mainly by immunocompetent cells, and by glucocorticoids. Peculiarities of each cytokine effect, their interaction during APP synthesis as well as their regulation by receptors-antagonists and by autoantibodies are discussed. The data described provide evidence for the close relation which exists between the immune system and the liver and is required to maintain homeostasis. PMID- 7536167 TI - [Effect of cultured mice hepatocytes on the proliferation of immunocompetent cells]. AB - Mice hepatocytes in primary cultures were treated during 2 h with normal rabbit antibodies (0.5 mg/ml), antiliver antibodies (0.5 mg/ml) and CCl4 (5 mM). Functional state of cells was studied according to DNA, RNA and protein synthesis. Cell-free supernatants were collected during 24 h after cells treatment. Spontaneous proliferation in vitro of syngeneic cells from the spleen, thymus, lymph node and bone marrow cultured with hepatocyte supernatants was investigated. Cultured intact hepatocytes were shown to produce factors that changed proliferation of the thymus and spleen cells. Hepatocyte supernatants demonstrated different effect on cells from different immune organs. Hepatocyte function alteration (minimal under normal antibodies treatment and maximal under CCl4 treatment) changed liver cells humoral influence on the immune cells. PMID- 7536168 TI - A new method of image analysis of fluorescein angiography applied to age-related macular degeneration. AB - Quantitative analysis of retinal and choroidal abnormalities using current photographic techniques is complex and laborious. Digital image analysis techniques using the scanning laser ophthalmoscope overcome many of the problems with present techniques and allow reliable quantitation. A prerequisite of quantitation is accurate image acquisition and registration. The authors describe a reliable method of image analysis and apply it to the quantitation of hyperfluorescence in scanning laser fluorescein angiograms of different forms of age-related macular degeneration. Retrospective analysis of scanning laser fluorescein angiograms obtained using a standardised technique was undertaken. Eighty-six angiograms from patients with age-related macular degeneration were analysed and categorised as dry maculopathy, geographic atrophy of the retinal pigment epithelium, retinal pigment epithelial detachment (PED) or subretinal neovascularisation (SRNV). Fluorescein characteristics of both SRNV and PED showed a characteristic pattern of fluorescence. The advantages and disadvantages of the technique are discussed. PMID- 7536169 TI - The effect of compounds altering the cAMP level on reversing the 2-cell block induced by hypoxanthine in mouse embryos in vitro. AB - The possibility of reversing the hypoxanthine induced 2-cell block in mouse embryos when cultured in conditions supplemented with compounds that increase (FSH, hMG, IBMX, hCG) or inhibit (GnRH-analogue) cAMP was assessed. When embryos were cultured in Ham's F-10 without hypoxanthine supplemented with each of the above compounds, no inhibition of blastocyst development was observed. Embryos were then cultured in Ham's F-10 with hypoxanthine supplemented again with each compound. For the addition of GnRH-analogue or FSH, the rate of blastocyst formation was comparable with that of the control medium with hypoxanthine alone. Instead, the addition of IBMX or hMG reversed the induced block. There was no reversible effect for the addition of 2 micrograms/ml hCG while the latter was observed with higher doses. The results from GnRH-analogue and IBMX addition show that, contrary to what was found for oocytes, stimulation of cAMP reverses the hypoxanthine-induced block in mouse embryos. FSH and hCG also had effects opposite to those observed for oocytes. It is unknown why hMG (FSH + LH) reverses the block. A lower cAMP degradation rate resulting in a higher cAMP level is a possible explanation. Our results provide further evidence that cleavage arrest by hypoxanthine has a different mechanism than the hypoxanthine-induced arrest of meiosis. PMID- 7536172 TI - Characterization of a gene encoding a cysteine-rich keratin associated protein synthesized late in rabbit hair follicle differentiation. AB - Many different cysteine-rich proteins are synthesized during hair follicle differentiation, forming part of the interstitial matrix between bundles of intermediate filaments. We have isolated a rabbit gene (rKAP4L), a member of a multigene family that encodes a small cysteine-rich hair keratin associated protein. This is the first complete gene sequence for this family. The rKAP4L gene is expressed in the cortex of rabbit pelage hair follicles at a late stage of hair follicle differentiation, well after the synthesis of the other major hair proteins, the intermediate filament and glycine/tyrosine-rich keratin associated proteins, has commenced. The protein contains 36 mol % cysteine, with a molecular size of 13593 daltons, and its sequence appears to be based on a pentapeptide repeat. It is predicted to adopt a folded conformation characterized by beta-turns interspersed with short stretches of beta-sheet or random coil. PMID- 7536170 TI - Effect of octreotide and insulin on manifest renal and glomerular hypertrophy and urinary albumin excretion in long-term experimental diabetes in rats. AB - Treatment of diabetic rats with octreotide can inhibit early diabetic renal hypertrophy. Octreotide administration for 6 months from the day of diabetes induction inhibits renal hypertrophy and diminishes increase in urinary albumin excretion. To investigate the effect of octreotide on manifest diabetic renal changes, octreotide treatment was given for 3 weeks after an untreated diabetic period of 3 or 6 months. In addition, following 6 months of diabetes, a group of diabetic rats was treated with insulin for 3 weeks. Renal and glomerular hypertrophy, and increased urinary albumin excretion were observed in diabetic rats compared to non-diabetic control rats from 3 months and throughout the study period. Octreotide treatment did not affect body weight, food intake, blood glucose or serum fructosamine levels. We observed no effect of octreotide treatment on renal and glomerular hypertrophy or urinary albumin excretion compared to placebo-treated diabetic rats. Insulin treatment for 3 weeks after 6 months of untreated diabetes normalized blood glucose and serum fructosamine levels, and furthermore renal hypertrophy was significantly diminished compared to the placebo-treated diabetic rats. However, insulin treatment had no effect on glomerular hypertrophy or urinary albumin excretion. In conclusion, octreotide treatment for 3 weeks following an untreated diabetic period of 3 or 6 months is unable to reduce the increased renal and glomerular volume or urinary albumin excretion. However, insulin treatment for 3 weeks with induction of euglycaemia diminishes the renal hypertrophy but has no effect on glomerular volume or urinary albumin excretion. PMID- 7536173 TI - Interaction of amiloride with rat parotid muscarinic and alpha-adrenergic receptors. AB - 1. In rat parotid acini, amiloride inhibited the secretion of amylase and the efflux of calcium and rubidium in response to carbamylcholine and to norepinephrine. 2. Amiloride competitively inhibited the binding of [3H]N methylscopolamine and [3H] is thus a competitive antagonist of muscarinic and norepinephrine alpha-adrenergic receptors. 3. Amiloride did not affect the response to substance P with respect to secretion or ion movements. 4. Thus the Na+/H+ antiporter is not involved in the short-term regulation of amylase secretion and calcium and potassium movements in rat parotid gland function. PMID- 7536174 TI - Non-genomic effects of catecholestrogens in the in vitro rat uterine contraction. AB - 1. The effects of catecholestrogens 2-hydroxyestradiol (2-OH E2, 0.6-30 microM), 4-hydroxyestradiol (4-OH E2, 1-30 microM) and 2-methoxyestradiol (2-MeO E2, 0.6 30 microM) on rat uterine contraction induced by KCl (60 mM), have been assayed. 2. All drugs assayed relaxed the tonic-contraction induced by KCl in a concentration-dependent way. The EC50s were: 4.4 +/- 0.5, 4.2 +/- 0.3 and 8.5 +/- 0.7 microM for 2-MeO E2, 2-OH E2 and 4-OH E2, respectively. This relaxing effect was counteracted by CaCl2 (1-10 mM) but not by the calcium channel agonist Bay k 8644 (1 nM-1 microM). 3. The effect of 2-MeO E2 is not modified by propranolol (1 microM), cycloheximide (35 microM), actinomycin D (4 microM), alpha difluoromethyl-ornithine (10 mM) or genistein (10 microM). Nor did cycloheximide (35 microM) or actinomycin D (4 microM) modify the relaxing effect of 2-OH E2 and 4-OH E2. Propranolol (1 microM) significantly increased the effect of 4-OH E2 but not the effect of 2-OH E2. 4. Our results suggest that the relaxing effect of catecholestrogens in the rat uterus is a non-genomic effect and could be related to inhibition of extracellular calcium entry. PMID- 7536171 TI - Comparison of mRNA contents of interleukin-1 beta and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice. AB - Interleukin-1 beta (IL-1 beta) has been suggested to mediate beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) by inducing nitric oxide production. In this study, we assessed the levels of IL-1 beta and the inducible form of nitric oxide synthase (iNOS), using a semi-quantitative polymerase chain reaction assay, and performed determinations of nitrite accumulation and IL-1 beta bioactivity, on pancreatic islets isolated from 5- and 16-week-old female and male nonobese diabetic (NOD) mice and from nondiabetes prone NMRI mice. NOD mouse islets contained notable amounts of IL-1 beta mRNA. At 5 weeks of age, but not at 16 weeks, the values were higher in islets isolated from NOD females compared to males. The IL-1 beta bioactivity showed differences roughly reflecting the mRNA levels in the NOD mouse islets. In the NMRI mouse islets the IL-1 beta bioactivity was very low. The expression of iNOS mRNA increased in both male and female islets between 5 and 16 weeks of age. Immunocytochemistry of pancreatic sections indicated the presence of macrophages especially in the peri-insular area of the NOD mice which suggests that IL-1 beta was produced by macrophages. The levels of IL-1 beta activity and mRNA in freshly isolated islets from NOD 5-weeks-old females did not correlate to the iNOS mRNA content or to the nitrite production. However, after incubation with IL-1 beta in vitro, both NOD and NMRI islets responded with a marked increase in nitric oxide production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536175 TI - Myocardial action potential prolongation by calcium channel activation under calcium free-EGTA condition in rats: developmental and regional variations. AB - 1. Prolongation of action potentials upon the addition of isoproterenol, forskolin, isobutylmethyl-xanthine (IBMX) and dibutyril cAMP (dbcAMP) under Ca free EGTA condition was examined in isolated myocardial preparations from neonatal and adult rats, whose action potential configuration greatly differ. 2. The prolongation of the action potential was previously suggested to be produced by persistent sodium influx through calcium channel due to the lack of calcium mediated inactivation of calcium channels under such experimental condition. 3. Preparations used were papillary muscles and free walls of the right and left ventricles from neonatal and adult rats. 4. In adult preparations, the prolongation produced by isoproterenol, forskolin and IBMX in the right free wall was smaller than those in the other three regions, while no regional difference was observed with dbcAMP. 5. The degree of prolongation by all of the four drugs were smaller in the neonate than in the adult. No regional difference was observed with any of the drugs in the neonate. 6. Our present results suggest that contribution of calcium-mediated inactivation of calcium channels to the repolarization of rat myocardium may increase postnatally to produce the developmental shortening of its action potential. Also, regional difference in the cAMP related mechanisms may appear postnatally. PMID- 7536176 TI - [Differential diagnosis of hepatitis A, B, C, D and E. Clinical, biochemical and virus serologic criteria. 5: Hepatitis C serology]. PMID- 7536177 TI - [New substance against AIDS in clinical trial]. PMID- 7536178 TI - Keratin expression in the normal anal canal. AB - The pattern of epithelial keratin expression in the normal anal canal has not been extensively defined and is a necessary prerequisite to the interpretation of alterations in these intermediate filaments in pathological anal epithelial lesions. Thirty-five frozen tissue specimens of resected haemorrhoids were investigated immunohistologically for expression of 14 individual keratins (K) using a panel of 17 monoclonal antibodies. Perianal skin showed basal expression of keratinocyte Ks 5, 14 and 17, and suprabasal expression of keratinocyte Ks 14, 10, 1 and 16. Anal squamous epithelium showed persistent basal K5 and 17, basal and suprabasal K4, 13 and 16 positivity, with sporadic expression of K1 and 10. The expression of simple epithelial keratins in squamous epithelium adjacent to the anal transitional zone varied with basal expression of K7, K8, K18 and K19 and sporadic suprabasal expression of K7 and K19. The anal transitional zone (ATZ) expressed K19, as found in transitional epithelia elsewhere. The full thickness of epithelium was positive for the simple epithelial Ks 7, 8 18 and 19. Marked heterogeneity of keratinocyte keratin expression was seen. Basal layers expressed Ks 4, 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks 4 and 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks 4 and 13 and variably K14, 16 and 17. This anomalous expression of keratinocyte K4 and 13 has also been documented in transitional epithelium of the bladder. The anal glands and ducts showed a keratin distribution similar to the transition zone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536179 TI - Immunohistochemical staining patterns of keratins in normal oesophageal epithelium and carcinoma of the oesophagus. AB - To clarify the keratin staining patterns of invasive carcinoma of the oesophagus, 22 cases of formalin-fixed paraffin-embedded surgical specimens were examined immunohistochemically with the labelled streptavidin biotin method using a panel of six different monoclonal anti-keratin antibodies. The antibody reacted adequately when antigen was retrieved in a microwave oven, and the relationship between morphological characteristics and keratin reaction patterns was analyzed in carcinomas and compared with adjacent histologically normal epithelium. In the normal oesophageal epithelium, AE3 and CK8.12 labelled all layer of cells, KS 1A3, E3 and KL1 labelled suprabasal cells, and LL002 selectively labelled the basal cells. In squamous cell carcinomas, AE3, CK8.12, KL1 and LL002 labelled almost all the tumour cells regardless of their differentiation, E3 only labelled keratinized cells, while marked decrease or loss of KS-1A3 staining was seen in all cases examined. Therefore, the characteristic profile of squamous cell carcinoma was a strong and diffuse expression of keratin 14 and 16, strong but localized expression of keratin 17, and loss of keratin 13 expression. Undifferentiated carcinoma totally lacked all keratin reactivity. The findings suggested that the neoplastic epithelial cells showed different keratin reactivity and distribution compared to normal oesophageal epithelium. In addition, histologically normal epithelium, dysplasia and carcinoma-in-situ adjacent to or overlying carcinoma expressed keratin 14. PMID- 7536180 TI - Female adnexal tumour of probable Wolffian origin: a clinicopathological and immunohistochemical study of three cases. AB - The clinical and pathological features of three adnexal tumours of probable Wolffian origin are reported. One case was an incidental finding in a patient who died from ovarian carcinoma; in the other two cases the patients presented with lower abdominal pain. The three tumours were well-circumscribed, solid masses arising in the leaves of the broad ligament and histological examination showed bland epithelial cells forming tubular, solid and microcystic patterns. The immunohistochemical profile of the tumours was similar to that of Wolffian duct remnants. They co-expressed cytokeratin and vimentin and lacked epithelial membrane antigen (EMA) reactivity, in contrast to tumours of Mullerian origin which usually express EMA. The differential diagnosis of female adnexal tumours is discussed. PMID- 7536181 TI - Carcinosarcoma arising in a duct papilloma. PMID- 7536182 TI - Regional assignment of conserved reference loci anchors unassigned linkage and syntenic groups to ovine chromosomes. AB - Seven loci that have been previously mapped to human and mouse chromosomes have now been regionally assigned to six sheep chromosomes. Nerve growth factor beta (NGFB), antigen CD3 zeta polypeptide (CD3Z), inhibin beta A (INHBA), estrogen receptor (ESR), rhodopsin (RHO), insulin-like growth factor 2 (IGF2), and myelin basic protein (MBP) were mapped by in situ hybridization to sheep chromosomes 1p24-p21, 1p14-p11, 4q26-q31, 8q25-q27, 19q23-qter, 21q21-qter, and 23q11-q12.3, respectively. ESR, RHO, IGF2, and MBP are the first markers to be assigned to their respective sheep chromosomes. These new data allow the previously unassigned sheep linkage groups H, J, K, and S to be provisionally assigned to chromosomes 21, 19, 4, and 8, respectively. The unassigned sheep syntenic groups U8 and U13 are provisionally assigned to sheep chromosomes 8 and 21, respectively. The new assignments support the emerging picture that there is extensive conservation of human chromosomal segments in the sheep and cattle genomes. The position of another evolutionary breakpoint on human chromosome 1q is suggested. PMID- 7536183 TI - Organization of the human keratin type II gene cluster at 12q13. AB - Keratin proteins constitute intermediate filaments and are the major differentiation products of mammalian epithelial cells. The epithelial keratins are classified into two groups, type I and type II, and one member of each group is expressed in a given epithelial cell differentiation stage. Mutations in type I and type II keratin genes have now been implicated in three different human genetic disorders, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, and epidermolytic palmoplantar keratoderma. Members of the type I keratins are mapped to human chromosome 17, and the type II keratin genes are mapped to chromosome 12. To understand the organization of the type II keratin genes on chromosome 12, we isolated several yeast artificial chromosomes carrying these keratin genes and examined them in detail. We show that eight already known type II keratin genes are located in a cluster at 12q13, and their relative organization reflects their evolutionary relationship. We also determined that a type I keratin gene, KRT18, is located next to its partner, KRT8, in this cluster. Careful examination of the cluster also revealed that there may be a number of additional keratin genes at this locus that have not been described previously. PMID- 7536184 TI - Detection of Mycobacterium leprae by three-primer PCR. AB - Recently, polymerase chain reaction has been introduced for the species-specific assessment of Mycobacterium leprae (1). To avoid Southern blotting techniques using radioactively labelled oligonucleotide probes, the aim of this study was to establish a three primer-based single-step PCR technique. Using primers designed for this purpose we amplified a part of the gene encoding for the 16S ribosomal RNA of slowly growing mycobacteria. Due to the species-specific antisense primer a second, smaller fragment specific for M. leprae was amplified. Our results show that the employment of a second antisense primer in the PCR may be a substitution for Southern blot hybridization. PMID- 7536185 TI - Validation of the psychoanalytic clinical process: the role of dreams. AB - This paper explores the place of dreams in the validation of the actuality of an evolving psychoanalytic clinical process. The specificity of psychoanalysis, in the author's perspective, consists basically in its being an intersubjective clinical practice; because of this, positivistic notions of validation, such as verification as a search for constancies and regularities, have no proper place here. The shared emotional experience--the intersubjectivity--of the psychoanalytic pair is the place where our method must be validated. Dreams, not only as a solitary intrapsychic production during sleep, but also as narratives made by the analysand within the setting, are an essential part of working through and provide a feasible way to attain some degree of validation of the process. The analysand's narrative of his dreams leads to a 'shared dreaming' in session of the analysand-analyst pair; leaving aside their role as resistance, dreams emerge from emotional experiences as a 'construction of knowledge', as an attempt at symbolic elaboration. PMID- 7536186 TI - Localization of nuclear RNA by pre- and post-embedding in situ hybridization using different gold probes. AB - Pre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. Pre-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments. PMID- 7536187 TI - The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study. AB - In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 micrograms ml-1 proteinase K for 12 min at 37 degrees C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 microns before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for 10 min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml-1. Hybridization temperature (37-42 degrees C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536188 TI - Expression of cytokeratin-mRNAs in squamous-cell carcinoma and balloon-cell formation of human oesophageal epithelium. AB - Using digoxigenin-labelled cRNA probes, relationships between morphological characteristics and in situ hybridization for cytokeratin (CK)-mRNAs were analysed in cases of squamous-cell carcinoma of variable differentiation and in balloon-cell formation within the oesophageal mucosa. The present results were correlated to our previous findings on normal oesophageal epithelium. Our results from in situ hybridization study on oesophageal squamous-cell carcinoma provide strong evidence that changes in CK expression occur with differences in malignant potential. Cells of poorly differentiated carcinoma lose an ability to produce CK mRNAs characteristic of their normal progenitor cells. Moderately differentiated and, still more pronounced, well differentiated carcinoma cells retain an ability to produce CKs characteristic of their tissue of origin (CK 6, CK 14, CK 15 and CK 19). Furthermore, well differentiated carcinoma cells may also gain an ability to synthesize new types of CKs that are not characteristic of the normal oesophageal epithelium (CK 8 and CK 18 characteristic of most simple epithelia, and CK 10 characteristic of keratinizing epithelia). Moreover, some oesophageal CK-genes are expressed in an obviously higher amount (CK 6, CK 14, and CK 19), but the expression of genes coding for the oesophageal differentiation-related CKs (CK 4 and CK 13) is obviously decreased or apparently lost. At the interface zone, observed in sections of well differentiated carcinomas, CK 8 and CK 18 mRNA were expressed in intermediate cell layers, and the centrally located cell layers were found positive for CK 10 mRNA. These findings largely extend the existing results from immunoblotting and immunohistochemical studies. The reduced or non detectable expression of oesophageal differentiation-related CK-mRNAs (CK 4 and CK 13) on the appearance of balloon cells, suggests molecular changes that may be a marker for pathological progression. In addition, the abundant expression of CK 6 and CK 14 mRNA within areas of balloon-cell formation showing basal hyperplasia, and the higher expression of CK 19 in comparison with normal epithelium, points rather to de-differentiation than to normal vertical differentiation of the oesophageal epithelium. Whether CK-mRNAs can be used as biomarkers for evaluation of oesophageal pathologies remains to be further elucidated. PMID- 7536189 TI - Lung growth in hypobaric normoxia, normobaric hypoxia, and hypobaric hypoxia in growing rats. I. Biochemistry. AB - Adaptive changes in cellular and connective tissue components of the lung after chronic exposure to reduced ambient oxygen and/or pressure were studied. Four week-old male Sprague-Dawley rats were randomly divided into five groups (n = 12 each): 1) general control, room air (GC); 2) hypobaric normoxic; 3) normobaric hypoxic; 4) hypobaric hypoxic; and 5) weight-matched control, restricted food intake (WMC; weight matched to hypobaric hypoxic animals). Lung growth (lung weight and DNA, RNA, protein, hydroxyproline, and desmosine contents) diminished in WMC compared with GC. Somatic growth decreased in hypobaric and normobaric hypoxic rats compared with GC. Lung weight; DNA, RNA, protein, hydroxyproline, and desmosine contents; and RNA/DNA, protein/DNA, and desmosine/DNA ratios increased in both hypobaric and normobaric hypoxic rats compared with WMC. Hydroxyproline and desmosine contents and the hydroxyproline/DNA ratio were significantly higher in hypobaric than normobaric hypoxic rats. Hypobaric normoxia caused a slight somatic growth reduction, but biochemical parameters of lung growth remained unaffected. In conclusion, in growing animals, despite inhibition of lung growth due to reduced food consumption, accelerated lung growth in hypobaric or normobaric hypoxia occurs by hyperplastic and hypertrophic changes. Hypobaric normoxia does not affect lung growth, but connective tissue proteins accumulate slightly more in hypobaric hypoxia than in hypoxia alone. PMID- 7536191 TI - Further characterization of HeLa DNA polymerase epsilon. AB - DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7 kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon. PMID- 7536190 TI - A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. AB - Signaling for cell death by Fas/APO1 occurs via a distinct region in its intracellular domain. This region contains a conserved sequence motif, the death domain motif, that is also found in the intracellular domains of the p55 tumor necrosis factor receptor and the low affinity nerve growth factor receptor, as well as in the regulatory domain of the ankyrins. A novel protein that specifically binds to the death domain of Fas/APO1 but not to Fas/APO1 molecules with a loss of function point mutation occurring in lprcg mice was cloned by a two-hybrid screen of a HeLa cells' cDNA library. The cloned protein itself contains a death domain motif, and this region binds to the death domain of Fas/APO1, while the region upstream to the death domain prompts self-association of the protein. Induced expression of the protein results in ligand-independent triggering of cytotoxicity, suggesting that it is involved in cell death induction by Fas/APO1. PMID- 7536192 TI - Selective induction of prostaglandin G/H synthase I by stem cell factor and dexamethasone in mast cells. AB - This study examines the regulatory effects of two cytokines, stem cell factor (SCF) and interleukin-3, and a glucocorticoid, dexamethasone, on lipid mediator generation in mouse bone marrow-derived mast cells (BMMC). Treatment of BMMC with SCF induced a modest, dose-dependent increase in three eicosanoids, thromboxane B2, prostaglandin D2, and leukotriene B4. These increases were accompanied by a marked elevation in cytosolic PLA2 (cPLA2). Dexamethasone blocked the induction of cPLA2 levels and the elevation in leukotriene B4 induced by SCF. By contrast, the combination of SCF and dexamethasone dramatically increased (5-8-fold) the capacity by BMMC to produce prostanoid products. This increase in prostanoid products was mirrored by an increase in prostaglandin G/H synthase I (PGHS-I) levels. Dexamethasone, alone, had no effect on PGHS-I, cPLA2, or prostanoid levels. Moreover, neither SCF or dexamethasone, alone or in combination, influenced prostaglandin G/H synthase II (PGHS-II) levels. In contrast to SCF, interleukin-3 alone or in combination with dexamethasone had no effect on prostanoid synthesis or PGHS-I or II levels. To better understand the SCF and dexamethasone effect, PGHS-I and PGHS-II mRNA expression were examined by Northern analysis. PGHS-I mRNA was markedly induced (maximal levels at 5 h) by the combination of SCF and dexamethasone. PGHS-II mRNA was undetectable in either control or SCF/dexamethasone-treated BMMC. Neither SCF or dexamethasone, alone, altered mRNA for either PGHS isotype. Taken together, these studies reveal that PGHS-I may be critical to prostanoid formation in mast cells exposed to cytokines and glucocorticoids. Moreover, they suggest that synergistic induction of PGHS-I could represent a novel mechanism for the anti-inflammatory action of glucocorticoids. PMID- 7536193 TI - Identification of two 17-kDa rat parotid gland phosphoproteins, subjects for dephosphorylation upon beta-adrenergic stimulation, as destrin- and cofilin-like proteins. AB - We previously reported that when 32Pi-loaded rat parotid slices are incubated with the beta-adrenergic agonist isoproterenol, the level of a soluble 32P labeled 17-kDa protein (pp17) decreases rapidly (Kanamori, T., and Hayakawa, T. (1982) Biochem. Int. 4, 517-523). Here we show that pp17 consists of two distinct phosphoproteins (pp17a and pp17b), identify their unphosphorylated forms (p17a and p17b, respectively), and provide evidence for their beta-adrenergic stimulation-induced dephosphorylation. Since p17a and p17b were predominant forms even in nonstimulated cells, peptides were generated from them with Staphylococcus aureus V8 protease or cyanogen bromide; subsequent sequencing of these peptides and homology search allowed identification of p17a and p17b as destrin- and cofilin-like proteins, respectively. Interestingly, they were also dephosphorylated in response to cholinergic stimulation. Because destrin and cofilin are actin-depolymerizing proteins whose activities are possibly regulated by their phosphorylation/dephosphorylation, the two parotid proteins reported here might be involved in cortical F-actin disruption observed in parallel with exocytotic amylase secretion. PMID- 7536194 TI - Modulation of E-selectin structure/function by metal ions. Studies on limited proteolysis and metal ion regeneration. AB - E-selectin is a member of the selectin family of proteins that recognize carbohydrate ligands in a Ca(2+)-dependent manner. In order to better understand the role of Ca2+ in E-selectin-ligand interactions, we examined the E-selectin structure by limited proteolysis. Apo-Lec-EGF-CR6, a Ca(2+)-free form of soluble E-selectin containing the entire extracellular domain, was sensitive to limited proteolysis by Glu-C endoproteinase. Amino-terminal sequencing analysis of the proteolytic fragments revealed that the major cleavage site is at Glu98 which is in the loop (residues 94-103) adjacent to the Ca2+ binding region of the lectin domain. Upon Ca2+ binding, Lec-EGF-CR6 was protected from proteolysis. This Ca(2+)-dependent protection was further augmented upon sialyl Lewis x (sLex) ligand binding. These results implied that Ca2+ binding to E-selectin induces a conformational change and perhaps facilitates ligand binding. The sLex-bound complex in turn stabilizes Ca2+ binding. Lec-EGF-CR6 contains only one high affinity Ca2+ site (Kd = approximately 3.5 microM) as determined by equilibrium dialysis. In addition, we found that Ba2+ was a potent antagonist in blocking Lec EGF-CR6-mediated HL-60 cell adhesion. By competitive equilibrium dialysis and proteolysis analysis, we demonstrated that Ba2+ bound to apo-Lec-EGF-CR6 5-fold tighter than Ca2+ and abolished ligand binding activity. Sr2+ also bound to apo Lec-EGF-CR6 tighter than Ca2+. However, Sr(2+)-regenerated Lec-EGF-CR6 showed 50% ligand binding activity. Mg2+ bound to apo-Lec-EGF-CR6 with much weaker affinity than Ca2+ and did not show any activity. Thus, E-selectin function can be modulated by different metal ions. PMID- 7536198 TI - Evidence that acetylcholine mediates increased cerebral blood flow velocity in crucian carp through a nitric oxide-dependent mechanism. AB - Nitric oxide (NO)-dependent regulation of brain blood flow has not been proved to exist in fish or other ectothermic vertebrates. Using epi-illumination microscopy on the brain surface (optic lobes) of crucian carp (Carassius carassius), we show that superfusing the brain with acetylcholine (ACh) induces an increase in cerebral blood flow velocity that can be completely blocked by the NO synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) and NG-nitro-L-arginine. Also, sodium nitroprusside, which decomposes to liberate NO, causes an increase in cerebral blood flow velocity. By contrast, L-NAME does not block the increase in blood flow velocity caused by anoxia. The results suggest that NO is an endogenous vasodilator in crucian carp brain that mediates the effects of ACh. Because teleost fish deviated from other vertebrates 400 million years ago, these results suggest that NO-dependent brain blood flow regulation was an early event in vertebrate evolution. PMID- 7536196 TI - Intermittent interferon and polychemotherapy in metastatic melanoma. AB - This study was conducted to evaluate the efficacy and the tolerability of a four drug chemotherapy regimen combined with interferon alpha (IFN) in metastatic melanoma. Between March 1991 and August 1993, 55 patients with advanced melanoma were enrolled for the present multicentre phase II study. Forty-nine patients were eligible and evaluable for toxicity; 48 patients were evaluable for response. The treatment schedule consisted of a 5-day regimen of dacarbazine, vincristine, bleomycin and lomustine, plus 6 x 10(6) IU IFN alpha three times weekly subcutaneously for 2 weeks starting on day 8. The cycle was repeated on day 29. Among the 48 assessable patients, 16 objective responses were seen, yielding a response rate of 33% (95% confidence interval 20%-46%). Seven patients achieved a complete response (CR) of a median of 6+ months (range 1+ to 21+ months) and 9 patients achieved a partial response (PR) of a median of 9 months (range 4-13 months). The median overall survival was 12+ months (range 6+ to 23+ months) for the patients with CR and 15+ months (range 8-20 months) for the patients with PR. Even the survival of the 7 patients with stable disease was fairly long (median 12, range 7-17 months), appearing to be significantly longer than the survival of the 25 patients with progressive disease (median 5, range 1 24+ months). The treatment was moderately well tolerated, although all patients experienced some mild form of toxicity, mostly gastrointestinal symptoms, neurotoxicity and haematotoxicity. Grade 3-4 adverse effects were noted in 39% of the patients. No toxic deaths occurred. It can be concluded that the present regimen produces meaningful responses for patients with metastatic melanoma. A randomised study is needed to determine the effect on survival. PMID- 7536197 TI - Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. AB - Cerebral ischemia is followed by a local inflammatory response that is thought to participate in the extension of the tissue damage occurring in the postischemic period. However, the mechanisms whereby the inflammation contributes to the progression of the damage have not been fully elucidated. In models of inflammation, expression of the inducible isoform of nitric oxide synthase (iNOS) is responsible for cytotoxicity through the production of large amounts of nitric oxide (NO). In this study, therefore, we sought to establish whether iNOS is expressed in the ischemic brain. Rats were killed 6 h to 7 days after occlusion of the middle cerebral artery. iNOS expression in the ischemic area was determined by reverse-transcription polymerase chain reaction. Porphobilinogen deaminase mRNA was detected in the same sample and used for normalization. In the ischemic brain, there was expression of iNOS mRNA that began at 12 h, peaked at 48 h, and returned to baseline at 7 days (n = 3/time point). iNOS mRNA expression paralleled the time course of induction of iNOS catalytic activity, determined by the citrulline assay (17.4 +/- 4.4 pmol citrulline/micrograms protein/min at 48 h; mean +/- SD; n = 5 per time point). iNOS immunoreactivity was seen in neutrophils at 48-96 h after ischemia. The data provide molecular, biochemical, and immunocytochemical evidence of iNOS induction following focal cerebral ischemia. These findings, in concert with our recent demonstration that inhibition of iNOS reduces infarct volume in the same stroke model, indicate that NO production may play an important pathogenic role in the progression of the tissue damage that follows cerebral ischemia. PMID- 7536195 TI - Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of alpha v integrins. AB - We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3), poorly differentiated type; OSC-19, well differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-alpha v antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-beta 1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-beta 5 and anti-alpha v antibodies, and only HSC-3 cells were positive for anti-beta 3 antibody. alpha 5 beta 1 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects alpha v beta 3- and/or alpha v beta 5-mediated carcinoma cell invasion. PMID- 7536199 TI - A sensitive assay for the IFN-regulated 2-5A synthetase enzyme. AB - 2-5A synthetase is the central enzyme of the 2-5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2-5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2-5A. Quantitation of labeled 2-5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2-5A. The combination of these procedures accounts for a 3-4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2-5A synthetase activity in vivo leading to a better understanding of the role of the 2-5A system in virus infection and other cellular processes. PMID- 7536200 TI - Concentrations, release, and disposal of insulin-like growth factor (IGF)-binding proteins (IGFBP), IGF-I, and growth hormone in different vascular beds in patients with cirrhosis. AB - The liver is thought to be the major source of circulating insulin-like growth factor (IGF-I) and IGF-binding protein-1 (IGFBP-1), whereas the primary production site of circulating IGFBP-3 remains unknown. As other tissues may contribute to the circulating pool of IGF-I and IGFBP, the aim of the present study was to assess the hepatic and renal arterio-venous difference and production rates of IGF-I, IGFBP-1, IGFBP-3, and GH in cirrhotic patients (n = 22) and matched control subjects (n = 27). IGFBP-1 and -3, IGF-I, and GH levels were measured by RIA in hepatic, renal, and peripheral veins and in the femoral artery. Levels of IGFBP-1 to -4 were additionally determined by Western ligand blotting. Hepatic venous IGFBP-1 was significantly increased in the cirrhotic patients (mean +/- SEM, 33.6 +/- 9.1 vs. 10.4 +/- 1.9 micrograms/L; P < 0.001), and arterio-renal-venous extraction was significant in both patients (6 +/- 2%; P < 0.01) and controls (11 +/- 1%; P < 0.001). Conversely, IGFBP-3 was decreased in the cirrhotic patients (1265 +/- 149 vs. 2712 +/- 137 micrograms/L; P < 0.001). IGFBP-3 correlated significantly with the wedged hepatic venous pressure (r = 0.49; P < 0.05), serum aspartate aminotransferase (r = -0.66; P < 0.01), serum bilirubin (r = -0.65; P < 0.01), serum albumin (r = 0.64; P < 0.01), and the Child score (r = -0.57; P < 0.01). IGF-I was significantly lower in the cirrhotics (57 +/- 10 vs. 143 +/- 11 micrograms/L; P < 0.001). No significant IGFBP-3 proteolysis was demonstrated in cirrhotics or controls. No significant differences were found in the values obtained simultaneously from hepatic, renal, and brachial veins or femoral artery, which suggests that no major net production or release of IGFBP-3 or IGF-I occurs in these tissues. No differences in IGFBP-2 or IGFBP-4 determined by Western ligan blot were found between patients and controls. The IGF-I concentrations correlated significantly with parameters of biochemical liver function. Basal GH concentrations were significantly higher in the cirrhotics (1.19 +/- 0.13 vs. 0.58 +/- 0.08 micrograms/L; P < 0.001). A significant hepatic disposal of GH was found in the patients (P < 0.05) and controls (P < 0.001).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536201 TI - N-terminal truncated insulin-like growth factor-I in human urine. AB - Urinary insulin-like growth factor-I (IGF-I) from healthy human subjects was examined using two antisera directed toward the whole molecule (WM) and the N terminal of IGF-I. Pooled urine samples from normal adults were dialyzed, lyophilized, then subjected to Sephacryl S-200 chromatography. The gel filtration profile of immunoreactive IGF-I measured by RIA using WM antiserum showed two peaks. Of the total IGF-I, approximately 40% was free, and the rest was present as a 50-kilodalton complex. To characterize the IGF-I forms present in those two peaks, antibody capture enzyme-linked immunoassays (EIA) using the two antisera were established for detection of intact IGF-I and N-terminal-truncated IGF-I variants. The WM antibody recognizes intact IGF-I and des(1-3)-IGF-I, an N terminal-truncated variant, equally well, whereas the N-terminal IGF-I antibody recognizes intact IGF-I, but not des(1-3)-IGF-I (< 1% cross-reactivity). As both antibodies show similar cross-reactions with IGF-II, the difference between IGF-I levels recognized by the two antisera was considered to indicate the presence of N-terminal-truncated IGF-I variants. Of the free immunoreactive IGF-I in the urine, 64% was not recognized by N-terminal IGF-I antiserum and was considered to represent N-terminal-truncated IGF-I. In contrast, only 6% of the IGF-I present in the 50-kilodalton fraction was truncated. Urine samples from normal human subjects were analyzed by RIA with WM antiserum and EIA with both WM and N terminal IGF-I antisera after extraction of IGF-I from binding proteins. IGF-I values measured by EIA with the WM antiserum correlated well with those values obtained by RIA using WM antiserum (r = 0.98; P < 0.001). The total urinary IGF-I level measured by EIA with the WM antiserum was 216.0 +/- 41.1 ng/L (mean +/- SEM), and 35.2 +/- 6.1% of this was considered to represent N-terminal-truncated IGF-I. Using an immobilized biotinylated peptide corresponding to the N-terminal six amino acids of IGF-I, we detected proteolytic activity toward the N-terminal of IGF-I in all four human serum samples tested. In contrast, only two of seven urine samples had detectable protease activity, and in these samples, activity was very low compared to that in serum.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536202 TI - Insulin-like growth factor (IGF)-I and -II and IGF-binding proteins-1, -2, and -3 in children and adolescents with diabetes mellitus: correlation with metabolic control and height attainment. AB - The putative effects of diabetes and metabolic control on circulating levels of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) remain controversial. In the present study, serum levels of IGF-I and IGF-II and IGFBP 1, -2, and -3 were measured in 58 patients (age, 0.8-17 yr) with treated (51 subjects) or untreated (7 subjects) insulin-dependent diabetes mellitus (IDDM) and were compared with the levels in normal subjects. In the untreated patients IGF-I and IGF-II were decreased as compared with the healthy controls. In the treated diabetics IGF-I and IGF-II were reduced; IGFBP-2 (only in prepubertal subjects) and IGFBP-3 were increased. Furthermore, age-adjusted values of IGF-I, IGF-II, and IGFBP-3 were lower in prepubertal than in pubertal patients. Regression analysis revealed a negative correlation between hemoglobin (Hb)A1c and standard deviation scores (SDS) of IGF-I and a positive association between HbA1c and IGFBP-1 SDS or IGFBP-2 SDS. In the treated patients HbA1c was positively related to IGFBP-1 SDS and IGFBP-2 SDS when applying simple regression analysis and to IGFBP-2 SDS when using a multiple regression model. Strong correlations were observed between height SDS and IGF-I SDS, IGF-II SDS, and IGFBP-3 SDS in prepubertal subjects who had had IDDM for at least 2 yr, but not in adolescents. Such correlations have also been found in healthy children and adolescents. In conclusion; 1) IDDM is associated with alterations of the IGF IGFBP system, which are partially accounted for by differences in metabolic control and pubertal status; 2) the lower plasma concentrations of serum IGF-I may play a role in the pathogenesis of growth impairment of poorly controlled prepubertal, but not pubertal, children and adolescents with IDDM; and 3) in addition, a potential role of the altered IGF-IGFBP system for the development of diabetic late complications is hypothesized. PMID- 7536203 TI - Lack of pineal growth during childhood. AB - During childhood, serum melatonin concentrations drop by approximately 80%, but the 24-h melatonin excretion is stable. Arrest of pineal growth after the end of infancy has been proposed as one possible mechanism underlying that phenomenon. To test this hypothesis, we reviewed 332 magnetic resonance imaging brain studies, classified as normal, of endocrine-normal children, aged 1 day to 15 yr, and estimated the pineal and pituitary sizes. The pineal was identified in 277 of 332 magnetic resonance imaging studies (83%). The average size (mean +/- SEM) of the pineal gland (transaxial diameter, 5.6 +/- 2.1; midsagittal diameter, 5.0 +/- 2.4; planimetric area, 28.5 +/- 17.8) did not differ with age. A total of 74 of 277 pineals with cysts (26.7%) were found. The occurrence of pineal cysts was equally distributed among the different age groups (chi 2 = 11.6; df = 14; P = 0.7). Ten pineals showed more than 1 cyst (3.6%). The pituitary was identified in 325 of 332 brain images (97.9%). The average pituitary size increased by some 100% from 1 to 15 yr of age [transaxial diameter: F = 2.2; P = 0.005 (by two-way analysis of variance); midsagittal diameter: F = 3.7; P = 0.0001; planimetric area: F = 7.1; P = 0.0001]. The pituitary was slightly larger in females than in males [midsagittal diameter: F = 8.8; P = 0.003 (by two-way analysis of variance); planimetric area: F = 7.9, P = 0.005]. The data presented indicate a lack of a discernible pineal growth after age 1 yr, which contrasts with pituitary development in the same individuals. The data are in agreement with a hypothesis suggesting a growth arrest of the pineal after infancy. PMID- 7536204 TI - Differential effects of insulin and insulin-like growth factor I on the production of plasma steroid-binding globulins by human hepatoblastoma-derived (Hep G2) cells. AB - Changes in the plasma levels of corticosteroid-binding globulin (CBG) and sex hormone-binding globulin (SHBG) from birth to adulthood suggest that growth factors might influence clearance and/or hepatic secretion of CBG and SHBG in humans. The effects of insulin-like growth factor I (IGF-I) and insulin on CBG and SHBG synthesis by a clone of human hepatoblastoma-derived (Hep G2) cell lines were therefore investigated. The results showed that the immunoconcentrations of CBG and SHBG, as well as total protein concentration in culture medium from Hep G2 cells, were decreased by IGF-I and insulin. However, although the CBG-to-total protein ratio was decreased dose dependently by IGF-I and insulin, IGF-I and insulin did not dose-dependently decrease the SHBG-to-total protein ratio. The steady state levels of CBG and SHBG messenger RNAs (mRNAs) were reduced dose dependently by IGF-I with a half-effect at 5.4 +/- 1.9 and 4.6 +/- 1.6 nmol/L, respectively, and by insulin with a half-effect at 4.3 +/- 1.1 and 4.3 +/- 1.4 nmol/L, respectively. The maximum inhibitory effect of IGF-I on CBG mRNA level was 48 +/- 17% of control values and 60 +/- 13% for SHBG mRNA level. The changes in CBG mRNA levels were quantitatively similar to the changes in CBG immunoconcentration in the Hep G2 medium. In contrast, the inhibitory effects of insulin were only 17 +/- 8% and 31 +/- 12% of control values on CBG and SHBG mRNAs and 37 +/- 4% and 43 +/- 4% on CBG and SHBG concentrations, respectively. These results demonstrate that IGF-I reduces CBG and SHBG production by Hep G2 cells by decreasing mRNA steady state levels. The discrepancy between the inhibitory effects of insulin on CBG and SHBG mRNAs and protein secretion suggests that insulin exercises its inhibitory effects mainly on the mechanism(s) of translation and/or excretion of CBG and SHBG. The respective effects of IGF-I and insulin in the regulation of CBG and SHBG levels during fetal life and pubertal development in humans merit further study. PMID- 7536205 TI - Effect of insulin on the insulin-like growth factor system in children with new onset insulin-dependent diabetes mellitus. AB - To further characterize the mechanism of impaired growth in children with insulin dependent diabetes mellitus, we examined the serum components of the insulin-like growth factor (IGF) system in 11 children with new-onset insulin-dependent diabetes mellitus and followed the effect of insulinization on the IGF system longitudinally 1 day, 1 week, and 1 month after starting insulin treatment. Before insulin therapy, serum IGF-I, IGF-II, IGF-binding protein-3 (IGFBP-3), and GH-binding protein (GHBP) levels were significantly decreased, whereas IGFBP-1 and cortisol were significantly increased in diabetic children compared to those in an age-, sex-, and stage of puberty-matched control group. Random serum GH concentrations did not differ significantly. The alterations in the IGF system reversed with insulin therapy in a sequential manner. IGFBP-1 fell rapidly and was comparable to control values within 24 h after insulin treatment. IGF-I rose 1 week after treatment, reaching levels comparable to those in controls and continued to rise through 1 month of treatment. IGF-II, IGFBP-3, and GHBP showed a slower pattern of change, with their levels reaching control values only 1 month after the start of insulin treatment. Improvement in glycemic control, as determined by a change in hemoglobin-A1c, correlated positively with improvement in IGF-I, IGF-II, IGFBP-3, GHBP, and weight gain after 1 month of insulin therapy. These data are consistent with the hypothesis that changes in the IGF system in the insulinopenic state are similar to those during nutritional deprivation and may serve to minimize IGF's anabolic actions. The decreases in IGF-I, IGF-II, and IGFBP-3 may in part be due to a decrease in the GHBP/receptor. However, the observation that an increase in serum IGF-I was observed earlier than an increase in GHBP and without a significant change in serum GH suggests a direct stimulatory effect of insulin on liver IGF-I production or reversal by insulin of some postreceptor defect in GH action independent of GHBP. PMID- 7536206 TI - Effect of the acid-labile subunit on the binding of insulin-like growth factor (IGF)-binding protein-3 to [125I]IGF-I. AB - In normal subjects, the major form of circulating insulin-like growth factor (IGF) is the GH-dependent 150K complex. This complex is formed by the IGF peptide, the acid-stable binding protein IG-FBP-3, and the acid-labile subunit (ALS), which, although not binding IGF, appears to be necessary to reconstitute the complex in its natural form. The ALS was purified in our laboratory from human serum by ammonium sulfate precipitation, ion exchange chromatography using DEAE-Sephadex A-50, Concanavalin-A-Sepharose-4B chromatography, and two sequential gel filtrations by fast performance liquid chromatography. As demonstrated by gel permeation chromatography on fast performance liquid chromatography, incubation for 2 h at 20 C of this preparation with [125I]IGF-I and recombinant IGFBP-3 (rIGFBP-3) allows the reconstitution of a complex of about 150 kilodaltons. In these experimental conditions, the ALS is not only able to increase the mol wt of the complex, but also to greatly increase the amount of IGF-I bound; in the absence of ALS, radioactivity in the mol wt volume of the complexed forms was lower than that in the mol wt volume of the free form (percentage of total [125I]IGF-I: rIGFBP-3 alone, 15% and 44%; in the presence of ALS, 41% and 24%, respectively). In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [125I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP 3-binding activity was dose dependent. These data show that the non-IGF-binding ALS subunit of the 150-kilodalton complex can play an important role in the regulation of IGF-I or IGF-II binding to rIGFBP-3 and, therefore, on the levels of free IGF peptide, possibly by inducing conformational changes in rIGFBP-3. In addition, ligand and immunoblot reveal that ALS and rIGFBP-3 are able to form a high mol wt complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol wt of the IGF-IGFBP-3 complex. PMID- 7536207 TI - Characterization of the paradoxical growth hormone inhibitory effect of galanin in acromegaly. AB - Galanin is a 29-amino acid straight-chain biologically active peptide which has been found to decrease circulating GH levels in some acromegalic patients, whereas is able to increase GH secretion in normal subjects. The aim of our study was to ascertain the incidence, entity and mechanism of the paradoxical GH inhibitory effect of galanin in acromegaly also looking at possible correlations between the GH responses to galanin and the main clinical and biochemical features of the patients. Finally, the effects of either successful or unsuccessful neurosurgical intervention on the GH inhibitory effect of galanin in acromegaly were investigated. A series of 23 consecutive patients with active acromegaly seen at the Endocrine Section of the Department of Internal Medicine of the University of Brescia (Italy) between 1991 and 1994 was examined. The acromegalic patients were subdivided in group 1 (i.e. patients who were 1) untreated, 2) evaluated before surgery, and 3) not cured after surgery and radiotherapy) and group 2 (i.e. surgically cured). All patients were submitted at least once to the following biochemical and radiological evaluations: 1) baseline serum insulin-like growth factor-I and PRL samples, 2) iv infusion of synthetic porcine galanin (500 micrograms in 100 mL saline) from -10 to 30 min, 3) iv bolus injection of TRH (200 micrograms) at time zero, 4) oral glucose tolerance test (75 g glucose, orally) at time zero, and 5) magnetic resonance of the pituitary sella. Adenomatous tissue obtained during neurosurgery in four patients was cultured in vitro, and the effect of the addition of galanin in the culture medium on GH secretion was tested. During galanin infusion in 19 of 21 group 1 patients, serum GH levels were lower with respect to baseline (range of GH decrease, -6.2 to -85.4% with respect to basal levels). During galanin infusion, no reductions in GH levels were observed in the acromegalic patients cured after neurosurgery (group 2); on the contrary, 6 of 7 patients displayed a normal stimulatory response to galanin (range of GH increase, +120-1533.3% of the basal level). A significant correlation between the percent decrease in GH levels after galanin treatment and the percent increase after TRH was found in group 1 patients (r = -0.783; P < 0.05). In three of the four adenomas examined, galanin determined a clear decrease in GH secretion (mean nadir, 63.3 +/- 12% of the baseline secretion rate). In conclusion, we demonstrated that the large majority of numerous patients with active acromegaly show a decrease in serum GH levels after galanin administration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536208 TI - Choice of treatment affects plasma levels of insulin-like growth factor-binding protein-1 in noninsulin-dependent diabetes mellitus. AB - Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) modulates the metabolic and mitogenic effects of IGFs. Although IGFBP-1 levels are abnormally high in insulin-dependent diabetes (IDDM), relatively little is known in NIDDM; conflicting data have suggested both high and low levels. We investigated whether treatment modifies IGFBP-1 levels in two groups of NIDDM patients. Study 1 examined fasting concentrations in groups of patients with NIDDM, comparable except for treatment type (sulfonylurea, n = 23; once daily insulin, n = 15; sulfonylurea plus once daily insulin, n = 14; multiple insulin injections, n = 9) and 25 nondiabetic subjects. In sulfonylurea-treated patients there were markedly reduced plasma IGFBP-1 concentrations (median, interquartile range in parentheses): control, 61.0 (36-96) micrograms/L; sulfonylureas alone, 31.5 (21 61) micrograms/L (P < 0.01); and sulfonylureas plus insulin, 31.5 (9-53) micrograms/L (P < 0.01). Once daily insulin was associated with values similar to those in the control group [62.0 (27-103) micrograms/L; P = NS], whereas IGFBP-1 levels were higher with multiple insulin injection therapy [156.0 (71-184) micrograms/L; P < 0.05]. Proinsulin levels were higher in sulfonylurea-treated patients, but there was no significant correlation between IGFBP-1 and proinsulin within any individual group. Study 2 examined the effects of treatment on the dynamics of IGFBP-1 levels between 0800-1900 h. In control subjects (n = 8), levels fell from 0800 h (mean +/- SEM, 22.4 +/- 5.2 micrograms/L) to 1000 h (14 +/- 5.2 micrograms/L), followed by a rise, more rapid after food, to a peak at 1240 h (20.6 +/- 3.7 micrograms/L). Levels then declined until 1500 h (10.7 +/- 2.9 micrograms/L), with a further postprandial peak at 1840 h (23.1 +/- 3.2 micrograms/L). Sulfonylurea therapy (n = 6) resulted in a complete loss of this pattern, with a marked fall in IGFBP-1 from 0800 h (22 +/- 2.7 micrograms/L) to less than 7 micrograms/L for the remainder of the study (area under the curve, 1150-1400 h, P < 0.001 vs. control). By contrast, in metformin-treated patients (n = 7), neither IGFBP-1 levels nor postprandial peaks were significantly different from those in the control group. Our findings suggest that in patients with NIDDM, the regulation of IGFBP-1 is markedly influenced by the choice of treatment. PMID- 7536209 TI - A randomized, double blind, placebo-controlled trial on safety and efficacy of recombinant human insulin-like growth factor-I in children with growth hormone receptor deficiency. AB - GH insensitivity due to GH receptor deficiency is a rare autosomal recessive condition, characterized by deletions or mutations of the GH receptor gene. Patients are refractory to both endogenous and exogenous GH, resulting in severe growth retardation. Therapy with recombinant human insulin-like growth factor-I (rhIGF-I) can bypass the defect in the GH receptor and potentially stimulate growth. We previously identified a genetically homogeneous group of patients in southern Ecuador, thus providing a patient base for a controlled clinical trial of rhIGF-I therapy. Seventeen prepubertal patients were entered in a randomized, double blind, placebo-controlled trial. Subjects received either a 12-month course of rhIGF-I (120 micrograms/kg, sc, daily) or 6 months of placebo followed by 6 months of rhIGF-I. Subjects receiving rhIGF-I showed a significant increase in growth rate, which was sustained over the 1-yr course of therapy (from 2.9 +/- 0.6 to 8.6 +/- 0.4 cm/yr). Incidents of hypoglycemia were equal in frequency in the placebo and rhIGF-I groups. One recipient of rhIGF-I developed papilledema, which resolved spontaneously. rhIGF-I therapy did not alter serum IGF-binding protein-3 concentrations. rhIGF-I treatment is effective in stimulating skeletal growth in GH receptor deficiency. Although the therapy proved to be safe, the potent metabolic actions of rhIGF-I and the persistently low levels of serum IGF carrier protein necessitate continued careful observation for side-effects. PMID- 7536210 TI - Massive weight loss restores 24-hour growth hormone release profiles and serum insulin-like growth factor-I levels in obese subjects. AB - In the present study, we 1) determined whether the impaired spontaneous 24-h GH secretion as well as the blunted GH response to provocative testing in obese subjects are persistent disorders or transient defects reversed with weight loss and 2) investigated 24-h urinary GH excretion and basal levels of insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP-3), as well as insulin in obese subjects before and after a massive weight loss. We studied 18 obese subjects (age, 26 +/- 1 yr; body mass index, 40.9 +/- 1.1 kg/m2); 18 normal age-, and sex-matched control subjects; and 9 reduced weight obese subjects after a diet-induced average weight loss of 30.3 +/- 4.6 kg. Twenty-four-hour spontaneous GH secretion was estimated by obtaining 3240 integrated 20-min blood samples using a constant blood withdrawal technique and computerized algorithms. Body composition was determined using anthropometric measurements and dual energy x ray absorptiometry scanning (DXA). In the obese subjects, 24-h spontaneous GH release profiles and the GH responses to insulin-induced hypoglycemia and L arginine as well as basal IGF-I levels and the IGF-I/IGFBP-3 molar ratio were decreased, whereas insulin levels were elevated compared to those in normal subjects. In obese subjects, 24-h spontaneous GH secretion and serum IGF-I levels were inversely related to abdominal fat (r = -0.67; P < 0.01) and percent body fat (r = -0.69; P < 0.01), respectively. The decreased 24-h spontaneous GH release profiles, the decreased GH responses to insulin-induced hypoglycemia and L-arginine, the decreased basal IGF-I levels and IGF-I/IGFBP-3 molar ratio, as well as the elevated insulin levels were returned to normal after a massive weight loss in the obese subjects. In conclusion, the present study has shown reversible defects in 24-h spontaneous GH release profiles, basal IGF-I levels, and the IGF-I/IGFBP-3 molar ratio in obese subjects. The recovery of the 24-h GH release points to an acquired transient defect rather than a persistent preexisting disorder. PMID- 7536211 TI - CD56+ lymphoid cells in human first trimester pregnancy decidua as a source of novel transforming growth factor-beta 2-related immunosuppressive factors. AB - The lymphomyeloid cells isolated from normal first trimester pregnancy decidua may be separated into a CD56+ population of natural killer (NK)-lineage cells with the morphology of granulated lymphocytes, and a CD56- population which includes other cell types. Unlike CD56+ NK cells in peripheral blood, decidual CD56+ cells lack type III Fc receptors (CD16) and did not express significant levels of either type I FcR (CD64) or type II FcR (CDw32). By contrast to the decidual CD56- cells, CD56+ cells could release biologically active transforming growth factor (TGF)-beta in vitro, detectable using an normal rat kidney fibroblast colony-forming assay. The CD56+ cells could be stained using an antibody specific for TGF-beta 2, and similarly staining cells could be detected in intact biopsies of normal pregnancy decidua. Bioactive TGF-beta is known to suppress the generation of cytotoxic cells in vitro, and high performance liquid chromatography fractionation of supernatants conditioned by CD56+ but not CD56- cells contained reproducible peaks of immunosuppressive activity at 40-45 and 15 20 kDa, similar to the TGF-beta 2 immunosuppressive activity in supernatants conditioned by unfractionated decidua. PMID- 7536212 TI - Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction. AB - This study was designed to test the effects of pentoxifylline and progesterone upon capacitation of fresh human spermatozoa. Capacitation and acrosomal integrity were assessed using the fluorescent probe chlortetracycline on spermatozoa co-stained with a supravital fluorescent dye, Hoechst 33258. Hyperactivated motility was measured using computer-assisted movement analysis. After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent 'B' pattern, characteristic of capacitated, acrosome-intact cells, increased significantly (P < 0.01), though no increase in 'AR' pattern, characteristic of acrosome-reacted cells, was detected. There was a significant increase in hyperactive motility (P < 0.001). Exposure to progesterone (1 microgram/ml; 60 min) resulted in a significant increase in 'B' pattern (P < 0.05) and 'AR' pattern (P < 0.005), though no effect on the expression of hyperactivation was detected. No effect upon hyperactivation was detected on exposure of fresh or cryopreserved spermatozoa to a physiological range of progesterone concentrations (0.1-1000 ng/ml). Sequential exposure to pentoxifylline then progesterone resulted in a significant increase in 'B' pattern, acrosome loss and hyperactivation. Sperm viability was not affected in any treatment group. These observations suggest that pentoxifylline and progesterone affect capacitation through independent mechanisms. Stimulation of both capacitation and acrosome reaction resulted from sequential exposure to pentoxifylline and progesterone. This may have implications for sperm handling for assisted reproductive techniques. PMID- 7536213 TI - Detection of Mycobacterium tuberculosis directly from spiked human sputum by Q beta replicase-amplified assay. AB - We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic acid hybridization assay for the detection of Mycobacterium tuberculosis directly from spiked human sputum. Specimens were processed by either an N-acetyl-L-cysteine NaOH or a 2% NaOH digestion-decontamination method and then washed to neutralize the pH of the cell pellet. The washed sputum pellets were heated at 100 degrees C to inactivate the M. tuberculosis organisms. The heat-inactivated samples were mechanically lysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrument in the presence of zirconium oxide beads and a buffer containing guanidine thiocyanate. The released nucleic acid was subjected to the GENE-TRAK Q Beta replicase-amplified, dual-capture assay. The assay sensitivity was 10(3) purified rRNA targets or 1 CFU of M. tuberculosis spiked into M. tuberculosis negative human sputum. There was a low level of noise because of the limitations of performing a signal amplification assay in an open system. High levels of other mycobacterial rRNA (approximately 10(7) organisms), including rRNAs of Mycobacterium avium and Mycobacterium gordonae, did not interfere with the sensitivity of the assay. PMID- 7536214 TI - Reverse transcriptase sequence of paired isolates of cerebrospinal fluid and blood from patients infected with human immunodeficiency virus type 1 during zidovudine treatment. AB - Human immunodeficiency virus type 1 (HIV-1) isolates obtained from the blood of patients undergoing treatment with 3'-azido-3'-deoxythymidine (zidovudine [AZT]) show a decreased sensitivity to the drug in vitro. The aim of the present study was to determine if HIV-1 variants resistant to AZT are present also in the brain compartment. We selected sequential HIV-1 isolates from the blood and the cerebrospinal fluid (CSF) of six patients with HIV-1 infection undergoing AZT therapy for a time varying between 1 and 3 years. The isolates were used to infect peripheral blood mononuclear cell cultures which were used to prepare viral DNA. The viral DNA was amplified by PCR and then directly sequenced. Analysis of the reverse transcriptase (RT) sequence of the isolates from the CSF during therapy demonstrated that CSF-resistant isolates are characterized by the same mutations documented in resistant isolates from the blood compartment. Isolates obtained from one patient (patient 3) showed the same two mutations (codons 70 and 215) in blood and CSF, whereas isolates obtained from an additional four patients presented a different pattern of mutations in the two compartments. We also analyzed the degree of amino acid homology between RT sequences from blood and CSF isolates in patients before and during AZT treatment. The percentages of amino acid variations were approximately equal when isolates from the same or different compartments were considered. Excluding the codons involved in AZT resistance, the time point of sampling did not affect RT variations during therapy significantly. In conclusion, our studies show that AZT resistant HIV-1 can be found in the CSF of patients undergoing treatment. The mutations linked to AZT resistance in the CSF isolates are the same as those identified in AZT-resistant isolates from blood. PMID- 7536216 TI - Comparison of four different methods for detection of Cryptosporidium species. AB - Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost. We conclude that all methods tested were equally sensitive and specific for the detection of Cryptosporidium spp. Ease of use, adaptability to batch testing, and cost are important criteria in determining the method of choice. PMID- 7536215 TI - Restriction fragment length polymorphism of rRNA genes for molecular typing of members of the family Legionellaceae. AB - Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested. PMID- 7536218 TI - Mixed infection of different Borrelia species among Apodemus speciosus mice in Hokkaido, Japan. AB - The wood mouse (Apodemus speciosus) serves as a wildlife reservoir for Lyme disease spirochetes in Hokkaido, Japan. To isolate Borrelia species, we captured 34 wood mice in an area where Borrelia species are endemic during October 1993. The earlobes (right and left), heart, spleen, and urinary bladder from each mouse were used as culture sources. As a result of culture 73 isolates from 21 mice were classified by rRNA gene restriction fragment length polymorphism analysis. Ribotype groups III (Borrelia afzelii) and IV (unknown species) were detected among those isolates. Thirty-one (77.5%) of 40 earlobe isolates were classified as group IV. In contrast, 6 (40.0%) of 15 heart isolates, 5 (50.0%) of 10 spleen isolates, and 7 (87.5%) of 8 urinary bladder isolates were B. afzelii. Seven mice showed mixed infection with B. afzelii and group IV. The data indicate that different Borrelia species can coexist in a reservoir host that is suitable for them. PMID- 7536217 TI - Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. AB - A fusiform bacterium with 3 to 14 multiple bipolar sheathed flagella and periplasmic fibers wrapped around the cell was isolated from the liver, bile, and lower intestine of aged, inbred mice. The bacteria grew at 37 and 42 degrees C under microaerophilic conditions, rapidly hydrolyzed urea, were catalase and oxidase positive, reduced nitrate to nitrite, did not hydrolyze indoxyl acetate or hippurate, and were resistant to both cephalothin and nalidixic acid but sensitive to metronidazole. On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel helicobacter, Helicobacter bilis. This new helicobacter, like Helicobacter hepaticus, colonizes the bile, liver, and intestine of mice. Although the organism is associated with multifocal chronic hepatitis, further studies are required to ascertain whether H. bilis is responsible for causing chronic hepatitis and/or hepatocellular tumors in mice. PMID- 7536219 TI - Immunohistochemical identification of neurons in ganglia of the guinea pig sphincter of Oddi. AB - The sphincter of Oddi is a smooth muscle sphincter that regulates the flow of bile into the duodenum. To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry were employed to assay for putative neurotransmitters and related synthetic enzymes in wholemount preparations, with and without colchicine treatment. Immunoreactivities for enkephalin-endorphin (ENK-END), substance P (SP), nitric oxide synthase, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene related peptide (CGRP) were demonstrated within the ganglionated plexus. Roughly half of the neurons in the sphincter of Oddi expressed immunoreactivity for both SP and ENK-END, but not for nitric oxide synthase. About 25% of the neurons expressed nitric oxide synthase immunoreactivity as well as NADPH-diaphorase activity. This contingent of neurons was made up of two subgroups: one that expressed immunoreactivity for VIP, the other for NPY. Neurons that expressed CGRP immunoreactivity were sparse in sphincter of Oddi ganglia; however, many axons immunoreactive for both CGRP and SP were present in the ganglionated plexus. The CGRP/SP fibers are probably visceral afferents that may influence ganglionic output through axon reflex circuits. These results, along with studies of the actions of these neuroactive compounds on sphincter tone, support the view that ganglia of the sphincter of Oddi are largely comprised of excitatory (SP/ENK END-immunoreactive) and inhibitory (nitric oxide synthase/VIP- or NPY immunoreactive) neurons, and that sphincter of Oddi tone is controlled by the regulation of the outputs of these two groups of cells. PMID- 7536220 TI - Distribution of spinal cord projections from the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: a Phaseolus vulgaris-leucoagglutinin study in the rat. AB - The distribution and organization of descending spinal projections from the dorsal part of the caudal medulla were studied in the rat following injections of Phaseolus vulgaris-leucoagglutinin into small areas of the subnucleus reticularis dorsalis (SRD) and the adjacent cuneate nucleus (Cu). The caudal aspect of the Cu projected only to the dorsal horn of the ipsilateral cervical cord via the dorsal funiculus. These projections were mainly to laminae I, IV, and V. More ventrally located reticular structures projected to the full length of the cord. Fibers originating from the SRD travelled through the ipsilateral dorsolateral funiculus and terminated within the deep dorsal horn and upper layers of the ventral horn, mainly in laminae V-VII. Fibers originating from subnucleus reticularis ventralis (SRV) travelled ipsilaterally through the lateral and ventrolateral funiculi and bilaterally through the ventromedial funiculus. These fibers terminated within the ventral horn. The density of labeling within the gray matter varied at different levels of the cord was as follows: cervical > sacral > thoracic > lumbar. The reciprocal connections between the caudal medulla and the spinal cord suggest that the former is an important link in feedback loops that regulate spinal outflow. PMID- 7536221 TI - Heterogeneous topographical distribution of the striatonigral and striatopallidal neurons in the matrix compartment of the cat caudate nucleus. AB - The topographical organization of the striatonigral projection was investigated in the cat by comparing the localization and the intensity of labelling of retrogradely labelled cells in the caudate nucleus following one or multiple injections of horseradish peroxidase-wheat germ agglutinin into the center or along the rostrocaudal axis of the substantia nigra pars reticulata. Second, the localizations of retrogradely labelled striatopallidal neurons and of clusters of aggregated striatonigral neurons (as outlined by the transport of 14C-material) were compared in cats that received four horseradish peroxidase-wheat germ agglutinin injections into the internal segment of the globus pallidus and three nigral injections of 14C-amino acids into the substantia nigra pars reticulata. Two types of striatonigral neurons located predominantly within the matrix compartment were identified: poorly collateralized aggregated cells distributed in clusters and more numerous collateralized cells distributed outside the clusters. In addition, two cell types were distinguished within each cluster of aggregated neurons. Those innervating the center of the substantia nigra pars reticulata were observed after a single nigral injection of the tracer, whereas those projecting to distinct sites of the substantia nigra pars reticulata along a rostrocaudal axis were observed only after multiple injections. Striatal neurons innervating the internal segment of the globus pallidus were heterogeneously distributed predominantly within the matrix but outside the clusters of aggregated striatonigral neurons. Together, these results provide further evidence for the heterogeneity of the matrix and for the complexity of matrix striatonigral connections that send both diverging and converging signals to the substantia nigra pars reticulata. PMID- 7536222 TI - Embryonic development of the antennal lobes of a hemimetabolous insect, the cockroach Periplaneta americana: light and electron microscopic observations. AB - In the hemimetabolous insect Periplaneta americana, the adult-like organization of the primary olfactory centers, the antennal lobes, is established during the approximately 31 days of embryogenesis. This report describes the temporal sequence of developmental events as viewed in the light and electron microscope by means of histological stains and by DiI labeling of antennal receptor axons with subsequent photoconversion. Glomeruli, characteristic differentiations of the antennal lobe neuropil, are first observed on day 19; their development, which is not synchronous in the various parts of the antennal lobe, lasts until about day 22. From day 10 on, glial cells begin to form a narrow boundary layer between the soma cortex and the central neuropil. They exhibit a lengthening of their processes in parallel with the formation of glomeruli. Marked proliferation or migration of these glial cells into the neuropil between glomeruli has not been observed. Antennal receptor axons could be labeled from stage 15 on. They terminate in an elongated growth cone with numerous filopodia. From day 18 on, some of these become bent or show an initial bifurcation. From day 22 on, the first afferent axons develop an adult-like arborization pattern. Synaptic contacts between receptor axons and unidentified neurons were observed as early as stages 16 and 19, in which the axons still have a growth cone-like form. In stage 27, in which the fibers have adult-like arborizations, many output contacts and few input contacts were found. PMID- 7536223 TI - Spinal projection neurons to the laterodorsal pontine tegmental nucleus: relationship to preganglionic neurons and nitric oxide synthase. AB - The region of the rat sacral parasympathetic nucleus (SPN) contains distinct subpopulations of neurons that project supraspinally or are preganglionic neurons. Some preganglionic neurons in the SPN serve as the motor outflow for urinary bladder contraction; other neurons in the SPN project to regions of the rostral pons that subserve micturition reflexes. Previous studies utilizing immunohistochemistry or staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) have demonstrated that numerous neurons in the SPN contain nitric oxide synthase (NOS), the enzyme for nitric oxide synthesis. Thus, the objectives of this study were to determine 1) the distribution of neurons in the region of the SPN that project to the laterodorsal tegmentum (LDT) of the pons, 2) whether spinal neurons projecting to a peripheral autonomic ganglion also project to the LDT, and 3) whether NOS or NADPH-d is present in LDT projection neurons. Preganglionic neurons were identified by injecting the retrograde tracer fluorogold (FG) into the major pelvic ganglion (MPG). Supraspinally projecting neurons were identified by injecting the retrograde tracer fast blue (FB) into the LDT. Numerous FB-labeled neurons were present in the ipsi- and contralateral SPN and were immediately dorsal to FG-labeled preganglionic neurons. Neurons containing both tracers were not observed. Approximately 20% of preganglionic neurons, but no LDT projection neurons, were reactive for NOS and NADPH-d. These data suggest that the region of the SPN is a site for distinct subpopulations of neurons that project to the LDT and to the MPG and that NOS is contained in some preganglionic neurons, but is not a marker for LDT projection neurons. PMID- 7536224 TI - Perceived occurrence and importance of caring behaviours among patients and staff in psychiatric, medical and surgical care. AB - The present study identified, within psychiatric, medical and surgical care, patient and staff perceptions of the occurrence and importance of caring behaviours. A Swedish version of the 'CARE-Q' instrument, including 50 caring behaviours, was used for the assessment of importance, and the 'CARE-How often' questionnaire (containing the same behaviours) was used for the determination of occurrence. In psychiatric and medical care, but not in surgical care, staff considered several behaviours to occur more frequently than did patients. However, in each type of care, the groups agreed fairly well with respect to rankings of behaviours. 'Explains and facilitates' occurred rarely, and 'Monitors and follows through' occurred often, according to both patients and staff. Overall, patients and staff differed both with regard to perceived levels and rankings of the importance of behaviours. Psychiatric patients perceived 'Explains and facilitates' as most important, and somatic patients perceived 'Monitors and follows through' as most important, while staff in both somatic and psychiatric care considered 'Comforts' as the most important subscale. Neither patient nor staff perceptions of the occurrence of caring behaviours were well matched with their perceptions of the importance of these. Implications for nursing practice and for studying patient satisfaction with care are given. PMID- 7536226 TI - The localization of neurons innervating the carotid sinus in the dog. AB - The localization of neurons innervating the carotid sinus of the dog was studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Labeled cell bodies were found in the petrosal (198 +/- 108; mean +/- S.D.) and superior cervical ganglia (SCG) (890 +/- 354 mean +/- S.D.) supporting the existence of both a sensory afferent and a sympathetic efferent innervation of the carotid sinus. Labeled neurons in the petrosal ganglion were round pseudounipolar neurons of variable size. Labeled neurons in the SCG were multipolar and appeared distributed over the whole ganglion, but with a higher density toward its caudal half. No labeled perikarya appeared either in the brainstem or in the nodose or jugular ganglia, suggesting that in the dog a vagal pathway for carotid sinus baroreceptor afferents does not exist. PMID- 7536225 TI - Immunopositive GABAergic neural sites display nitric oxide synthase-related NADPH diaphorase activity in the human colon. AB - In the enteric nervous system, gamma-aminobutyric acid (GABA) is a transmitter of interneurons which are proposed to innervate excitatory and inhibitory motor neurons. Nitric oxide (NO) is a putative transmitter of enteric inhibitory motor nerves targeted by GABA. In addition, NO is synthesized by a variety of enteric nerves throughout the gut wall indicative of its potential to be a transmitter of other nerve types, including interneurons. We sought to determine if some populations of nitrergic neurons are interneurons in human infant colon. As enteric neural GABA is exclusive to interneurons, colocalization with NO synthase related NADPH diaphorase was examined. GABA-transaminase (GABA-T) immunohistochemistry was used to identify GABAergic neurons and a histochemical protocol was used as a marker of neuronal NO synthase-related NADPH diaphorase activity in enteric layers. GABA-T immunoreactive neurons were seen in the ganglionated nerve networks of the myenteric and submucosal layers. GABA-T immunoreactive fibres were also present in the longitudinal and circular muscle layers. A subpopulation of GABA-T immunoreactive neurons within both the myenteric and submucosal ganglia express NO synthase-related activity. This colocalization extends further to a subpopulation of fibers within the muscle layers. These findings strongly suggest that in addition to its role in inhibitory motor neurons, NO may also be a transmitter of enteric interneurons. PMID- 7536228 TI - Shaping attitudes to postoperative pain relief: the role of the acute pain team. AB - Postoperative pain relief is often inadequate. Ignorance and misconceptions about opioids by ward staff contribute to this poor management. The introduction of acute pain teams has done much to improve pain relief for patients. It may also have contributed to changes in attitudes and knowledge of medical and nursing staff. We questioned 48 doctors and nurses on their knowledge and beliefs about postoperative pain relief. Staff members were questioned on two units, one with access to an acute pain team and one without. Over half those on the unit using traditional postoperative care thought patients did not receive adequate pain relief (58%). In comparison, only one respondent from the unit with the pain team thought this was the case (P < 0.001). More staff members that had experience of patient-controlled analgesia (PCA) were optimistic about its benefits than those in the unit with no experience; they were also less concerned about possible side effects. Only one respondent on the unit using PCA thought it carried a risk of drug dependence, compared to over half (55%) of those on the unit with no experience in this technique (P < 0.001). Over two-thirds of staff familiar with PCA thought nursing workload had decreased. Acute pain teams have an important role in educating ward staff. The impact of establishing such teams on staff knowledge and attitudes needs further study to ensure that they can carry out this role most effectively. PMID- 7536227 TI - Intravenous regional sympathetic blockade for pain relief in reflex sympathetic dystrophy: a systematic review and a randomized, double-blind crossover study. AB - The first aim was a systematic review of intravenous regional sympathetic blocks (IRSBs) in patients with reflex sympathetic dystrophy (RSD). Randomized controlled trials (RCTs) of IRSBs in patients with RSD were identified by MEDLINE search (1966 to May 1993) and by hand search of 30 journals (1950 to May 1993). Authors of eligible trials were asked for information on additional trials and for unpublished data. Seven RCTs of IRSBs in RSD were found. Four used guanethidine; none showed significant analgesic effect in IRSBs to relieve pain due to RSD. Two reports, one using ketanserin and one bretylium, with 17 patients in total, showed some advantage of IRSBs over control. RCT results were not combined because of the variety of different drugs and outcome measures and because of methodological deficiencies in most of the reports. The second aim was a randomized, double-blind, crossover study to assess the effectiveness of IRSBs with guanethidine. Patients fulfilling diagnostic criteria for RSD and who had reported pain relief after an open IRSB with guanethidine received IRSBs with guanethidine high dose, guanethidine low dose, and normal saline. Pain intensity and relief, adverse effects, mood, duration of analgesia, and global scores were recorded. Sixteen patients with diagnosis of RSD were recruited, but only nine entered the double-blind phase. The trial was stopped prematurely because of the severity of the adverse effects. No significant difference was found between guanethidine and placebo on any of the outcome measures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536229 TI - Detection of hepatitis C virus-RNA by polymerase chain reaction in dental surgeries. AB - The mean prevalence of anti-hepatitis C virus (HCV) in Italy is 0.87%. It reaches 2% in Campania, Southern Italy. Approximately 50% of community acquired non-A, non-B (NANB) hepatitis cannot be associated with known parenteral exposure. A recent Italian study has shown that the only demonstrable risk factor in 9% of acute C/NANB hepatitis is dental treatment. There are no data on direct contamination by HCV of dental surgeries. Possible environmental contamination by HCV-RNA was investigated in dental surgeries after treatment of anti-HCV and HCV RNA positive patients. Thirty-five anti-HCV and HCV-RNA positive patients with chronic hepatitis underwent dental treatment and were enrolled in this study. Eight had chronic persistent hepatitis (CPH), 23 chronic active hepatitis (CAH), and 4 cirrhosis. A total of 328 samples collected from instruments and surfaces were tested after dental treatment of 35 anti-HCV positive patients. The presence of HCV-RNA was determined by polymerase chain reaction (PCR) to evaluate contamination of instruments and surfaces in dental surgeries. Twenty (6.1%) out of 328 collected samples were positive for HCV-RNA. The positive samples were from work benches (two), air turbine handpieces (one), holders (four), suction units (one), forceps (four), dental mirrors (two), and burs (six). Our data indicate that there is extensive contamination by HCV of dental surgeries after treatment of anti-HCV patients and that if sterilisation and disinfection are inadequate there is the possible risk of transmission to susceptible individuals. PMID- 7536230 TI - Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes. AB - The effect of interferon-gamma (IFN-gamma) on dengue virus multiplication in human peripheral blood monocytes was investigated. Enriched monocytes were treated with IFN-gamma and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-gamma caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-gamma also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-gamma-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-gamma in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937. PMID- 7536232 TI - Is the detection of anti-hepatitis C virus core IgM influenced by the presence of serum rheumatoid factor? AB - Rheumatoid factor (RF) induces false-positive results in the detection of serum antibodies, especially of the IgM type. About 70% of the patients with chronic hepatitis C have abnormal levels of serum RF. The aim of this study was to determine whether the presence of serum RF could influence the detection of anti HCV core IgM, using an assay designed not to pick up RFs by the addition of goat antibodies directed against human IgG in the sample diluent. Serum anti-HCV core IgM antibodies and RF were sought in 60 patients with chronic hepatitis C. Serum anti-HCV IgG antibodies and anti-HCV core IgM antibodies were also sought in 101 patients with high levels of RF. Anti-HCV core IgM antibodies were found in 45% and serum RF in 72% of the patients with chronic hepatitis C. Neither the prevalence nor the levels of RF differed significantly between IgM positive and negative patients. Eight percent of the 101 patients with raised RF had anti-HCV antibodies and two of them had anti-HCV core IgM antibodies. No patient without anti-HCV antibodies had anti-HCV core IgM antibodies. These results show that: a) the detection of anti-HCV core IgM in patients with chronic hepatitis C is independent of the presence of serum RF; b) high titers of serum RF are not responsible for false-positive results of anti-HCV IgM tests. The study suggests that the test used could be a confident tool for studies on the significance of anti-HCV core IgM antibodies in chronic hepatitis C. PMID- 7536233 TI - Obsessive-compulsive symptom clusters and urinary amine correlates in Tourette syndrome. AB - This study was conducted to identify clusters of obsessive-compulsive characteristics in Tourette syndrome subjects and to explore their neurochemical correlates. Patients completed a 40-item questionnaire assessing obsessive compulsive symptoms. Each subject had a 24-hour urine specimen collected and analyzed for a variety of biogenic amines and their metabolites. Factor analysis identified eight symptom clusters, the majority of which appeared to reflect obsessive symptoms. Consistent relationships were observed between symptom clusters and levels of catecholamine and indolamine amines and metabolites. Overall, the primary metabolite of serotonin, 5-hydroxyindoleacetic acid, appeared to be the most highly correlated with the individual obsessive compulsive symptoms. PMID- 7536231 TI - Evaluation of a multiple peptide assay for typing of antibodies to the hepatitis C virus: relation to genomic typing by the polymerase chain reaction. AB - A panel of 16 type-specific synthetic peptides corresponding to variable antigenic regions within the hepatitis C virus (HCV) core, nonstructural 4 (NS4), and NS5 proteins was synthesised. The peptide panel was used to develop an enzyme immunoassay (EIA) for the detection of antibodies directed to HCV type 1 (genotypes I/1a and II/1b), type 2 (genotypes III/2a and IV/2b), and type 3 (genotype V/3). The peptides corresponded to residues 68-81 of the HCV core (types 1, 2, and 3), residues 1692-1705 and 1710-1728 of HCV NS4 (types 1a, 1b, 2a, 2b, and 3), and residues 2303-2319 of HCV NS5 (types 1a, 1b, 2a, and 2b). The 16-peptide panel was evaluated using human sera from 46 carriers of HCV, which were genotyped in parallel by the polymerase chain reaction (PCR) using primers specific for types I, II, III, IV, and V of HCV core. Of the 46 carriers, 14 (30%) were infected by HCV genotype I, 7 (15%) by genotype II, 16 (35%) by HCV genotype IV, and 6 (13%) by HCV of genotype V. Two carriers had double infections of types I and II, and the HCV strain of one carrier could not be genotyped. Using the serotyping system, 40 (89%) out of the 45 genotyped carriers were found to contain type-specific antibodies corresponding to the genotypes identified by PCR. In 5 of the 23 carriers infected by genotypes I and/or II, antibodies specific for HCV type 1 could not be detected, whereas all 16 carriers infected by genotype IV were serologically typed as type 2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536235 TI - Unilateral posterolateral decompression without stabilization for neurological palliation of symptomatic spinal metastasis in debilitated patients. AB - Patients with symptomatic spinal metastases and limited life expectancy are often too debilitated to withstand anterior or posterolateral spinal cord decompression and segmental stabilization. More limited surgery aiming solely at preservation or restoration of neurological function and relief from pain offers the potential for significant improvement in the quality of remaining life without incurring undue perioperative morbidity and mortality. Eight patients with spinal metastases and limited life expectancy underwent a unilateral transpedicular decompression procedure on their most symptomatic side and/or the side of maximum tumor involvement. All patients were neurologically improved within the 1st postoperative week; all were ambulatory and continent postoperatively. Postoperatively, all five patients with preoperative motor deficits demonstrated increased motor strength, and the three patients with predominant radicular pain reported marked improvement. There were no perioperative deaths and two transient perioperative complications. The average length of hospitalization was 6 days for patients without complications and 10 days for the entire group. Unilateral transpedicular decompression without stabilization is an effective and safe method for palliating symptomatic spinal metastases in debilitated patients with widespread malignancy and limited life expectancy. This therapeutic option should be considered in select cases as an alternative to either nonoperative management or anterior or posterolateral decompression and segmental stabilization. PMID- 7536234 TI - Differential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole-induced depolarization. AB - ZENK is an immediate early gene (IEG) that encodes a transcription factor protein, and its induction has been proposed as a necessary step in the cellular process underlying long-term memory formation. We have previously shown that ZENK is induced in adult songbird brain by the sound of birdsong, but, interestingly, induction did not occur in several areas known to respond to song stimuli. Conceivably, the ZENK gene may be repressed in these areas in adult birds. As a further test of this hypothesis, we administered metrazole, a strong GABAergic antagonist that leads to widespread excitation in the brain. Following metrazole, ZENK mRNA increases more than 10-fold throughout most of the telencephalon in both canaries and zebra finches, and primarily in neurons. In contrast, ZENK induction is much lower or absent in the archistriatum, the primary telencephalic sensory-recipient areas (including auditory field L), and the three telencephalic androgen receptor-containing song nuclei (high vocal center, lateral magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum). We did not observe any differences in ZENK induction patterns in juvenile versus adult zebra finches, or fall versus spring male canaries. Together with our previous studies of induction by song, these results suggest that in specific parts of the forebrain, including most of the song control system, IEG expression is subject to different constraints than in the rest of the forebrain. Understanding the molecular basis for this differential gene regulation may prove invaluable in understanding the organization of the song control circuit and the avian telencephalon. PMID- 7536237 TI - Problems of preterm infants after discharge. AB - Preterm infants often experience continuing health problems after discharge and have a higher rate of readmittance to the hospital during the first year. These infants also are at risk for neurodevelopmental problems, such as language, learning, and school difficulties. The continuing health and developmental problems of preterm infants creates stress for the family. Nurses caring for these infants in hospital settings must understand the problems for which preterm infants are at risk after discharge as a basis for appropriate discharge planning and teaching. A similar knowledge base will help nurses working in community settings in assessment and interventions with infants and their families after discharge. PMID- 7536236 TI - Alternative RNA splicing of the hyaluronic acid receptor CD44 in the normal human brain and in brain tumors. AB - The cell-surface receptor for hyaluronic acid, CD44, is expressed by both normal and malignant cells. Numerous CD44 isoforms have recently been identified that are derived by alternative ribonucleic acid splicing. The expression of some CD44 isoforms has been shown to be involved in tumor progression and metastatic spread in a rat carcinoma model and in human carcinomas. In the present study, CD44 isoform expression was evaluated by reverse transcriptase-polymerase chain reaction (PCR) analysis in frozen sections derived from three samples of normal brain tissue and from 40 brain tumors, including samples of glioblastoma multiforme, anaplastic astrocytoma, low-grade astrocytoma, cerebral primitive neuroectodermal tumor, medulloblastoma, metastatic colon carcinoma, and metastatic melanoma. Normal brain tissue adjacent to the tumors was also examined in 14 of 18 glioblastomas. In all normal brain and tumor samples, with the exception of metastases from colon carcinoma, PCR analysis demonstrated one prominent product that corresponded to the CD44H hematopoietic form of CD44. Metastases from colon carcinoma demonstrated two prominent PCR amplification products corresponding to CD44H and CD44R1. These results suggest that CD44H is the predominant isoform of this protein in normal human brain tissue and in human neuroectodermal tumors of varying degrees of malignancy. The ability of CD44H to mediate tumor cell motility and invasiveness (in contrast to CD44R1) suggests that the CD44 alternative splicing pattern of neuroectoderm-derived tumors may enhance their local biological aggressiveness and intracerebral spread. The lack of expression of larger molecular weight CD44 variants by primary brain tumors may also partially explain why these tumors rarely metastasize to distant sites. PMID- 7536238 TI - Prostate-specific antigen expression by various tumors. AB - There is a growing body of evidence indicating that prostate-specific antigen (PSA) may be present in many steroid hormone-stimulated epithelial tissues other than that of the prostate. In particular, breast tumor cell lines treated with steroid hormone receptor agonists, breast tumors, and normal human breast have recently been found by our group to contain PSA. To investigate whether PSA may also be present in other human tumors, we employed a highly sensitive immunofluorometric assay technique to quantify PSA immunoreactivity in tumor extracts. Using a PSA-positivity cutoff value of 0.005 ng per mg of protein, 23 of 43 diverse tumors tested positive for PSA protein. Confirmatory analyses for PSA by a commercially available method (IMx) on six samples demonstrated a high degree of concordance between the two methods. To establish the molecular weight of the immunoreactive species, the most highly positive tumor extracts of each tumor type were fractionated by high performance liquid chromatography. Whereas the majority of tumors had immunoreactivity eluting at both 100 KDa and 33 KDa, corresponding to PSA bound to alpha 1-antichymotrypsin and free PSA, respectively, the colon and parotid tumors displayed immunoreactivity only at the 33 KDa fraction. We conclude that in addition to breast tumors and normal breast, colon, ovarian, liver, kidney, adrenal, and parotid tumors can also produce PSA. The physiological role of PSA in these tumors is currently under investigation. PMID- 7536239 TI - Exaggerated effect of bilateral medial rectus recession in developmentally delayed children. AB - Many have suggested that the esotropia associated with developmental delay should be considered separately. However, the esotropia surgery recommended for developmentally delayed children has been similar to that performed in normal children. We have noticed a tendency for developmentally delayed children to develop consecutive exotropia following bilateral medial rectus recessions. Of 94 children undergoing such surgery between 1981 and 1991, 31 were developmentally delayed. Follow up ranged from 7 months to 202 months (mean 24 months). Surgical effect, defined as the change in alignment following each amount of surgery, was greater in the developmentally delayed group than in control subjects (P = .002). The increase in effect of the same amount of surgery in a developmentally delayed patient averaged 5.28 prism diopters, but was much larger in specific instances. Variability of effect was more marked among developmentally delayed children. We conclude that bilateral medial rectus recessions in developmentally delayed children may be better postponed in some cases, deferred for smaller angles, or decreased in amount. PMID- 7536240 TI - Wide-angle X-ray diffraction of human stratum corneum: effects of hydration and terpene enhancer treatment. AB - Wide-angle X-ray-diffraction experiments were used to investigate the molecular organization of barrier components of human stratum corneum. Diffraction lines related to the side-by-side lipid packing arrangements in the intercellular bilayers were identified as were patterns arising from secondary protein structures in intracellular keratin. Reflections were also identified which may be produced by proteins in the corneocyte envelopes. The effects of hydration on stratum corneum structure were monitored using 0, 20-40, 40-60, 60-80 and approximately 300% hydrated samples. The packing arrangements in the intercellular lipid bilayers remained the same over the entire hydration range, as did keratin structures. A new diffraction ring, attributable to liquid water, was produced by 300% hydrated samples with a repeat spacing of 0.35 to 0.30-0.29 nm. The effects of three terpene enhancers, (+)-limonene, nerolidol and 1,8 cineole, on stratum corneum structure were monitored. Treatment with each of the terpenes produced additional reflections which were attributed to the presence of the respective liquid enhancers within the stratum corneum. (+)-Limonene produced an additional reflection at 0.503-0.489 nm, nerolidol, an additional reflection at 0.486-0.471 nm and 1,8-cineole, an intense reflection at 0.583-0.578 nm. Reflections characteristic of gel-phase lipids and crystalline lipids also remained after all terpene treatments. These results provide no clear evidence of lipid bilayer disruption by the terpenes and suggest that areas of liquid terpene exist within the stratum corneum. The mechanisms underlying propylene glycol synergy with terpene enhancers were investigated. Treatment of stratum corneum with each terpene mixed with propylene glycol gave rise to two additional reflections. One reflection, always positioned at 0.452-0.448 nm, had been observed in control studies following propylene glycol treatment and may have been associated with bilayer structures disrupted by propylene glycol or altered keratin structures. The second reflection was developed by the respective terpene enhancer. For example, treatment with a 1,8-cineole/propylene glycol mixture produced reflections at 0.457-0.451 nm (propylene glycol-disrupted lipids or altered keratin) and 0.591-0.578 nm (liquid 1,8-cineole). Since the reflection at 0.452-0.448 nm was unaffected by co-application of propylene glycol with terpene enhancers, this study offers no evidence to support the theory that propylene glycol synergy with the terpenes occurs through enhanced lipid disruption. PMID- 7536241 TI - Deamidation of polyanion-stabilized acidic fibroblast growth factor. AB - The deamidation of polyanion-stabilized acidic fibroblast growth factor (aFGF; FGF-1) can be induced by prolonged storage under accelerated conditions of elevated pH and temperature. A urea-isoelectric focusing (urea-IEF) method has been developed to monitor aFGF deamidation in the presence of highly negatively charged polyanions which are required to maintain the conformational stability of the protein. The kinetics of aFGF deamidation have been established by a combination of urea-IEF and an enzymatic ammonia assay. Native, non-deamidated aFGF (complexed with heparin) has a half-life of 16 weeks at pH 7, 30 degrees C, and 4 weeks at pH 8, 40 degrees C. The mitogenic activity and biophysical properties of deamidated aFGF were compared to the non-deamidated protein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction with heparin, or bioactivity. At longer times, however, limited aggregation of the protein was observed after prolonged storage under some conditions. N-terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexible, peptide-like region of the protein (Asn8-Tyr9). PMID- 7536242 TI - Secretion of cyclic GMP by cultured epithelial and fibroblast cell lines in response to nitric oxide. AB - LLC-PK1 epithelial cells and RFL-6 fibroblasts secreted both cyclic AMP (cAMP) and cyclic GMP (cGMP) when costimulated with forskolin and 3 morpholinosydnonimine (a chemical nitric oxide generator). Intracellular cAMP levels as high as 1100 and 12,000 pmol/10(6) cells were achieved for the two cell types, respectively. These levels were high enough to reach approximately 50% saturation of the cAMP transporter and inhibited transport of cGMP to an equal extent, suggesting that the two cyclic nucleotides compete for a common transport system. The rates of secretion of cGMP and cAMP from LLC-PK1 cells increased in proportion to their rates of synthesis as concentrations of stimulant were varied, but increased only 25% relative to intracellular concentrations in response to inhibition of phosphodiesterases by 3-isobutylmethylxanthine. It is proposed that secretion of cyclic nucleotides is not simply proportional to the total intracellular pool in these cells, but rather is coupled to synthesis. In support of this model, oxyhemoglobin was used to trap nitric oxide and block activity of guanylate cyclase in cells treated with 3-morpholinosydnonimine. As a result, secretion of cGMP ceased within 1 min, whereas intracellular levels decreased slowly over 60 min. Probenecid [p-(dipropylsulfamoyl)benzoic acid] is a nonselective antagonist of anion transport that inhibited secretion of cAMP in both cell types but, unexpectedly, blocked synthesis of cGMP, and this was reflected in direct inhibition of soluble guanylate cyclase in cell lysates. Two heat-stable, high molecular weight factors that confer sensitivity to probenecid were identified, and these factors increased the sensitivity of guanylate cyclase to nitric acid by an order of magnitude. PMID- 7536244 TI - The discordant influences of infarct healing on the electrophysiologic effects of procainamide and N-acetylprocainamide. AB - Ischemic zone refractoriness and conduction delay respond differently to infarct healing and, hypothetically, may exert discordant influences on the electrophysiologic action of different classes of antiarrhythmic drugs. This study evaluated the influence of infarct healing on the electrophysiologic effects of procainamide (PA) and N-acetylprocainamide (NAPA) in a sedated, closed chest canine model with a healing anterior wall myocardial infarction, indwelling myocardial electrodes and inducible sustained ventricular tachyarrhythmias (VT). Infarct zone refractory periods, conduction times and the inducibility of VT were tested at base line and during infusion of PA or NAPA in a crossover study design at 1, 4 and 8 weeks of infarct healing. Data were presented as the percent magnitude of change from base line induced by drug. The magnitude of change during PA infusion in infarct zone refractory periods, but not conduction times, decreased during infarct healing (P < .001). The magnitude of change in refractory period and conduction time during NAPA was not significantly altered by the stage of myocardial infarction healing. At week 1, PA prevented inducible VT in 9 of 14 animals vs. 3 of 15 during NAPA infusion (P < .05). At weeks 4 and 8 there was no significant difference in VT suppression between PA and NAPA. We conclude that the stage of infarct healing can selectively influence the response of the infarct zone to the effects of PA, but not NAPA. This discordant effect may be class-specific. These data may have important implications for the management of lethal ventricular arrhythmias soon after myocardial infarction. PMID- 7536243 TI - Gramicidin as a potential immunosuppressant for organ transplantation: suppression of human lymphocyte blastogenesis in vitro and prolongation of heart allograft survival in the rat. AB - Linear polypeptide antibiotic gramicidin is known to interact with the cell membrane and deregulate cation exchange. Because perturbation of cell membrane function may suppress the immune cell network, the authors investigated the effects of gramicidin on lymphocyte blastogenesis in vitro and allograft survival in vivo. Gramicidin blocked blastogenesis of human peripheral blood lymphocytes that responded to mitogens (IC50 = 0.2-10.1 ng/ml) or allogeneic lymphocytes (IC50 = 4.9 ng/ml). The extent of these suppressive effects was equal to or superior to that of cyclosporine or prednisolone. The antibiotic caused no apparent cytotoxicity at a dose of 10,000 ng/ml. The suppression of lymphocyte blastogenesis in vitro by cyclosporine or prednisolone was restored by addition of interleukin (IL)-1, IL-2, IL-4, IL-5 or IL-6. In contrast, none of these cytokines affected the suppressive activity of gramicidin on lymphocyte blastogenesis. The immunosuppressive efficacy of gramicidin was further examined in vivo in heterotopically heart-transplanted rats. A heart graft from (Lewis x BN) F1 donor was implanted in the neck of allogeneic Lewis rats. In this model, beating of the graft in control recipients (placebo group) stopped as a result of acute allograft rejection at 5.4 +/- 0.4 days after transplantation (n = 38), whereas beating of the graft in recipients that received 2.0 or 4.0 mg kg-1 day-1 of gramicidin i.p. for 6 days was significantly prolonged to 15.4 +/- 4.3 (n = 5) or 18.4 +/- 3.9 (n = 5) days after transplantation, respectively (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536245 TI - Muscarinic regulation of the cardiac CFTR Cl- current by quaternary ammonium compounds. AB - In guinea pig ventricle, the protein kinase A-regulated Cl- current (ICl) is conducted by an alternatively spliced isoform of the cystic fibrosis transmembrane conductance regulator. We studied muscarinic regulation of this current using the whole-cell configuration of the patch-clamp technique. Acetylcholine (ACh) antagonized activation of ICl activated by 1 microM isoproterenol (ISO) in a concentration-dependent manner. The concentration of ACh that produced a half-maximal effect (K1/2) was 36 nM, the slope factor was 1.1, and the relative magnitude of the Cl- conductance at maximally effective concentrations of ACh (Gmin) was 21% of that observed in the presence of ISO alone. In the presence of 100 nM atropine, a competitive antagonist at the muscarinic receptor, the K1/2 value for ACh inhibition of ICl was increased to 4.3 microM, but the slope factor and Gmin were not affected, which indicated that the dissociation constant (KB) for atropine was < 1 nM. ACh-induced inhibition of the ISO-activated ICl was also blocked by the quaternary ammonium compound tetraethylammonium (TEA). Like atropine, TEA increased the K1/2 value for ACh inhibition of ICl without affecting the slope factor or Gmin. Schild analysis confirmed that TEA is also a competitive antagonist at the muscarinic receptor, with a KB value of 137 microM. However, tetramethylammonium (TMA), a structurally related compound, acted as an agonist at the muscarinic receptor. TMA inhibited ICl activated by 1 microM ISO with a K1/2 value of 342 microM, a slope factor of 0.87 and a Gmin value of 17%. Increasing the concentration of ISO shifted the K1/2 value for both ACh and TMA inhibition of ICl to higher concentrations and increased Gmin, without significantly affecting the slope factor. These results indicate that muscarinic regulation of ICl depends on the level of beta adrenergic stimulation in a functionally uncompetitive manner. They also suggest that TMA acts like ACh, a full agonist at the muscarinic receptor. Furthermore, we conclude that quaternary ammonium compounds, which are often used as ion substitutes and direct ion channel blockers, should be used with caution because of the significant and diverse effects they exert at muscarinic receptors. PMID- 7536246 TI - Agonist-independent, muscle-type-specific signal transduction pathways in cat esophageal and lower esophageal sphincter circular smooth muscle. AB - Smooth muscle cells isolated from the circular muscle layer of cat esophagus and lower esophageal sphincter (LES) exhibit distinct contractile intracellular signal transduction pathways in response to acetylcholine. To determine whether these contractile pathways are muscle type dependent, the authors examined the signal transduction pathways utilized by substance P and bombesin, which in other tissues, use different signal transduction pathways, and by the GTP analog, guanosine 5'-O-3-thiotriphosphate (GTP gamma S), which activates all available G proteins. Western blot analysis of esophageal and LES circular muscle revealed the presence of Gq-G11 (42 kD), Gi1-Gi2 (40 kD) and Go-Gi3 (40 kD) types of G proteins. The responses of esophageal cells to bombesin and substance P were blocked by 1) a Gi3 protein antibody, 2) the inhibitor of specific phosphatidylcholine-phospholipase C (PLC) D609 potassium tricyclo-[5.2.1.0(2.6)] decyl-(9[8])-xanthogenate, 3) inhibition of phosphatidic acid phosphohydrolase by propranolol, 4) the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2 methylpiperazine dihydrochloride (H7) and 5) incubation in Ca(++)-free medium. Conversely, the responses of LES muscle cells to bombesin and substance P were blocked by 1) a Gq-G11 antibody, 2) a phosphatidylinositol-specific PLC antagonist U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), 3) the calmodulin inhibitor CGS9343B (1,3 Dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin++ +-4 - yl)methyl 4-piperindinyl]-2H-benzimidazol-2-one maleate) and 4) incubation in Sr++. After permeabilization by saponin, inositol 1,4,5-trisphosphate contracted LES but not esophageal cells. The inositol 1,4,5-trisphosphate receptor antagonist heparin and depletion of intracellular Ca++ stores by thapsigargin or A23187 4 Benzoxazolecarboxylic acid, 5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2 oxo-2-(1H-pyrrol- 2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-, [6s [6.alpha. (2S*,3S*),8.beta. (R*), 9.beta., 11. alpha.]]-(9Cl), blocked bombesin- and substance P-induced contraction of LES but not of esophageal muscle. In addition, contraction in response to GTP gamma S, which activates all G proteins, was blocked in esophageal cells by a Gi3-protein antibody, propranolol, D609 and H7. In LES muscle cells, the response to GTP gamma S was blocked by a Gq protein antibody, U-73122 and CGS934B. These data demonstrate that, in esophageal muscle, different agonists activate the same Gi3 protein, phosphatidylcholine-specific phospholipases and protein kinase C-dependent pathway.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536247 TI - Cytochrome P450 mono-oxygenase-regulated signalling of Ca2+ entry in human and bovine endothelial cells. AB - 1. We tested the hypothesis that agonist-stimulated Ca2+ entry, and thus formation of endothelium-derived nitric oxide (EDNO) in vascular endothelial cells, is related to activation of microsomal P450 mono-oxygenase (P450 MO) and the biosynthesis of 5,6-epoxyeicosatrienoic acid (5,6-EET). 2. Several P450 inhibitors diminished the sustained [Ca2+]i plateau response to agonist or intracellular Ca2+ store depletion with ATPase inhibitors by 31-69% (fura-2 technique). Mn2+ influx stimulated by agonists or ATPase inhibitors was prevented by P450 inhibitors. 3. Histamine- or ATPase inhibitor-stimulated formation of EDNO was strongly attenuated (50-83%) by P450 inhibitors, without any effect on EDNO formation by the Ca2+ ionophore A23187, indicating that decreased EDNO synthesis is due specifically to the inhibition of Ca2+ entry by these compounds. 4. Induction of P450 MO by beta-naphthoflavone potentiated agonist-induced Ca2+ and Mn2+ influx by 60 and 53%, respectively. Intracellular Ca2+ release remained unchanged. 5. The P450 MO product, 5,6-EET (< 156 nmol l-1), activated Ca2+/Mn2+ entry without any depletion of intracellular Ca2+ stores. The 5,6-EET-stimulated Ca2+/Mn2+ entry was not affected by P450 inhibitors. 6. As with the bradykinin stimulated Ca2+ entry pathway, the 5,6-EET-activated Ca2+ entry pathway was permeable to Mn2+ and Ba2+, sensitive to Ni2+, La3+ and membrane depolarization, and insensitive to the removal of extracellular Na+ or the organic Ca2+ antagonist, nitrendipine. 7. In the presence of 5,6-EET, stimulation with bradykinin only transiently increased [Ca2+]i. Vice versa, 5,6-EET failed to increase [Ca2+]i further in bradykinin-stimulated cells. The sustained [Ca2+]i plateau phase induced by a co-stimulation with bradykinin and 5,6-EET was identical to that observed with bradykinin or 5,6-EET alone. 8. These results demonstrate that Ca2+ entry induced by the P450 MO product, 5,6-EET, is indistinguishable to that observed by stimulation with bradykinin. 9. All data support our hypothesis that depletion of endothelial Ca2+ stores activates microsomal P450 MO which in turn synthesizes 5,6-EET. We propose that the arachidonic acid metabolite 5,6-EET or one of its metabolites is a second messenger for activation of endothelial Ca2+ entry. PMID- 7536250 TI - Bulge-induced bends in RNA: quantification by transient electric birefringence. AB - Bulges represent one of the most common non-helical elements in RNA, often displaying a strong degree of phylogenetic conservation, both in location and sequence, within larger RNA molecules. Thus, knowledge of the conformation and flexibility of RNA bulges is an important prerequisite for understanding the rules governing the formation of tertiary structure within the larger molecules. In the current investigation, the magnitudes of the bends induced in a 148 base pair duplex RNA molecule by single, centrally located bulges of varying size (n = 1 to 6) and base composition (An and Un series) have been determined through the use of transient electric birefringence (TEB). The TEB approach is highly sensitive to the changes in the global shape of RNA (or DNA) helices that accompany the introduction of points of bending or flexibility near the center of the helix. In the current instance, bulge angles deduced from TEB measurements ranged from approximately 7 degrees to approximately 93 degrees, with the angle increasing with increasing n for both An and Un series. For both An and Un series in the absence of Mg2+, the angle increment per added nucleotide varied from approximately 20 degrees to approximately 8 degrees as n increased from 1 to 6. These angle increments remained unchanged for the An series in the presence of Mg2+; however, the angle increments for the Un series were reduced by a factor of 2 for all values of n. Thus the current observations have identified structural transitions in one of the simplest non-helical elements in RNA, transitions that are dependent on both sequence and counterion valence. Finally, the measured bend angles are strongly correlated with the degree of reduction in electrophoretic mobility of bulge-containing RNA helices. The observed correlation was used to obtain a semi-empirical relationship between bend angle and mobility in order that additional angles might be assigned, by interpolation, through the use of gel data alone. PMID- 7536249 TI - A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope. AB - A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry. PMID- 7536251 TI - Genetic analysis of helper virus-specific selective amplification of cucumber mosaic virus satellite RNAs. AB - Satellite RNAs (sat-RNAs) are small molecular parasites associated with a number of plant RNA viruses. The cucumber mosaic virus (CMV) sat-RNAs are ca. 335 nucleotides and have evolved to produce a large number of closely related sat RNAs. Different cucumoviruses can act as helper viruses in the amplification of CMV sat-RNAs. We have found that different helper viruses show a preference for a particular sat-RNA in a mixed infection. In this study the specificity of WL47 sat-RNA amplification by LS-CMV and of D4 sat-RNA amplification by tomato aspermy virus in mixed infections was examined. Recombinant cDNA clones of D4 sat-RNA and WL47 sat-RNA were used to map the sat-RNA sequences responsible for the helper virus selection of a specific sat-RNA for amplification. PMID- 7536248 TI - Dendritic glutamate receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons. AB - 1. Properties of dendritic glutamate receptor (GluR) channels were investigated using fast application of glutamate to outside-out membrane patches isolated from the apical dendrites of CA3 and CA1 pyramidal neurons in rat hippocampal slices. CA3 patches were formed (15-76 microns from the soma) in the region of mossy fibre (MF) synapses, and CA1 patches (25-174 microns from the soma) in the region of Schaffer collateral (SC) innervation. 2. Dual-component responses consisting of a rapidly rising and decaying component followed by a second, substantially slower, component were elicited by 1 ms pulses of 1 mM glutamate in the presence of 10 microM glycine and absence of external Mg2+. The fast component was selectively blocked by 2-5 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the slow component by 30 microM D-2-amino-5-phosphonopentanoic acid (D-AP5), suggesting that the fast and slow components were mediated by the GluR channels of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and NMDA type, respectively. The peak amplitude ratio of the NMDA to AMPA receptor mediated components varied between 0.03 and 0.62 in patches from both CA3 and CA1 dendrites. Patches lacking either component were rarely observed. 3. The peak current-voltage (I-V) relationship of the fast component was almost linear, whereas the I-V relationship of the slow component showed a region of negative slope in the presence of 1 mM external Mg2+. The reversal potential for both components was close to 0 mV. 4. Kainate-preferring GluR channels did not contribute appreciably to the response to glutamate. The responses to 100 ms pulses of 1 mM glutamate were mimicked by application of 1 mM AMPA, whereas 1 mM kainate produced much smaller, weakly desensitizing currents. This suggests that the fast component is primarily mediated by the action of glutamate on AMPA preferring receptors. 5. The mean elementary conductance of AMPA receptor channels was about 10 pS, as estimated by non-stationary fluctuation analysis. The permeability of these channels to Ca2+ was low (approximately 5% of the permeability to Cs+). 6. The elementary conductance of NMDA receptor channels was larger, with a main conductance state of about 45 pS. These channels were 3.6 times more permeable to Ca2+ than to Cs+.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536253 TI - Incidence of erectile impotence secondary to transurethral resection of benign prostatic hyperplasia, assessed by preoperative and postoperative Snap Gauge tests. AB - In an attempt to assess the impotence rate secondary to transurethral resection of the prostate more objectively than by merely interviewing patients, potency was evaluated with the Snap-Gauge test. The test was used preoperatively to recruit patients with intact potency. The 98 patients studied underwent transurethral resection of the prostate and were retested during postoperative night 4. Of the 98 patients 64 remained potent while 34 did not. These 34 men were retested 3 months later, and 26 were potent and 8 were impotent. Therefore, 8 of 98 patients (8.3%) became impotent as a consequence of transurethral resection of the prostate. The risk specific to subgroups in cases of small (less than 10 gm. resectable tissue) and larger adenomas is 11.1% and 7.7%, respectively, for men older than 65 years, and 7.1% and 0%, respectively, for men younger than 65 years. A selective indication taking into account patient age and prostatic size might further lower the already low impotence risk of transurethral resection of the prostate. PMID- 7536252 TI - Molecular staging of prostate cancer. II. A comparison of the application of an enhanced reverse transcriptase polymerase chain reaction assay for prostate specific antigen versus prostate specific membrane antigen. AB - Current imaging modalities used to stage prostate cancer clinically fail to detect extracapsular disease in a significant subset of patients. A molecular based peripheral blood assay using the reverse transcriptase polymerase chain reaction has recently been shown to be a highly sensitive staging modality for detecting extraprostatic disease preoperatively. The assay uses primers that are specific for prostate specific antigen (PSA). We compare the application of the reverse transcriptase polymerase chain reaction assay using primers specific for the human prostate specific membrane antigen with results obtained from the same specimens by reverse transcriptase polymerase chain reaction for PSA. Prostate specific membrane antigen, a recently cloned prostatic antigen, is a transmembrane glycoprotein that has been described as prostate specific. These assays were applied to ribonucleic acids extracted from the peripheral blood lymphocyte fraction of 80 patients with clinically localized prostate cancer. In addition, blood specimens from 20 female patients, 20 young male patients, 25 age matched control men under treatment for benign prostatic hypertrophy and 20 men with established, untreated metastatic prostate cancer were tested. All 3 groups of noncancer patients had negative polymerase chain reactions for PSA as well as prostate specific membrane antigen. Of 20 metastatic prostate cancer patients 16 (80%) had positive polymerase chain reactions for PSA, while only 10 (50%) had positive results for prostate specific membrane antigen. Among the 80 patients with clinically localized disease (stages T1 to T2cN0M0), 27 and 19 had positive polymerase chain reaction for PSA and prostate specific membrane antigen, respectively, from blood specimens obtained preoperatively. Analyzing the final pathology in each patient with the reverse transcriptase polymerase chain reaction assay identified a significantly stronger correlation with tumor invasion using the results of the PSA test rather than the results of the prostate specific membrane antigen reverse transcriptase polymerase chain reaction test (67% versus 34% sensitivity for detecting capsular penetration, 87% versus 46% sensitivity for detecting disease to the surgical margin and 83% versus 16% sensitivity for detecting seminal vesicle invasion). In contrast to the reverse transcriptase polymerase chain reaction assay for PSA, a similar assay done for prostate specific membrane antigen did not correlate with pathological stage of prostate cancer. PMID- 7536254 TI - Repeat prostate needle biopsy: who needs it? AB - The indications for repeat prostate needle biopsy after a transrectal ultrasound guided sextant biopsy are not defined. We examined 100 sextant prostate needle biopsies without a diagnosis of malignancy, which were repeated. Carcinoma was detected in 20 repeat biopsies (20%). Stratification based on initial biopsy result revealed carcinoma in 10 of 69 cases (14.5%) without prostatic intraepithelial neoplasia or atypia, 5 of 17 (29.4%) with atypia, 5 of 5 (100%) with grade II or III prostatic intraepithelial neoplasia and 0 of 9 with grade I prostatic intraepithelial neoplasia. Examination of prostate specific antigen (PSA) levels and PSA velocity did not provide statistically significant stratification, perhaps due to the wide variance in these parameters and the small sample size. We conclude that patients with a diagnosis of glandular atypia, or grade II or III prostatic intraepithelial neoplasia on initial biopsy are at high risk for invasive carcinoma and should undergo repeat prostate needle biopsy. A rapidly increasing serum PSA level or grossly abnormal digital rectal examination may also indicate carcinoma not discovered on initial biopsy. PMID- 7536255 TI - Transurethral incision of the prostate following renal transplantation. AB - Urinary retention in male patients after renal transplantation may cause serious complications in terms of graft function and even patient survival. Only few data concerning the management and outcome of these patients are reported in the literature. Therefore, we retrospectively analyzed the outcome of patients who underwent transurethral incision of the prostate immediately after renal transplantation. Between 1990 and 1993, we performed 259 renal transplantations and 15 patients had symptoms of urinary retention postoperatively. These patients underwent a midline transurethral incision of the prostate from the bladder neck to the verumontanum. Median peak flow preoperatively was 7 ml. per second (range 0 to 11.4) and median residual urine volume was 100 ml. (range 30 to 500). Median prostate volume was 28 ml. (range 12 to 45). After transurethral incision of the prostate a significant improvement (p < 0.001) in peak flow rates (19.6 ml. per second, range 8 to 49) as well as a significant decrease in residual urine volumes (40 ml., range 20 to 80) could be achieved. After a median followup of 19 months the effect was still present. PMID- 7536256 TI - Laser prostatectomy performed with a right-angle firing neodymium:YAG laser fiber at 60 watts power setting. AB - A total of 50 patients with symptomatic bladder outlet obstruction due to benign prostatic hyperplasia was entered into a prospective trial of laser prostatectomy performed with the right-angle firing neodymium:YAG laser fiber at 60 watts power setting. Mean estimated resectable prostatic weight was 30 gm. with a mean prostatic urethral length of 3.3 cm. Nine patients (18%) were in urinary retention requiring catheterization before treatment. Mean energy delivery was 30,834 joules (range 14,400 to 127,800) with a mean operative time of 31 minutes. Efficacy of treatment was assessed at 3, 6 and 12 months postoperatively by standardized American Urological Association symptom scores, peak urinary flow rates and post-void residual urine volumes. At preoperative baseline the mean symptom score was 20.8, mean peak urinary flow rate 7.6 cc per second, and mean post-void residual urine volume 353 cc. At 1 year postoperatively mean symptom score was 8.4, mean peak urinary flow rate was 18.7 cc per second and mean post void residual urine volume was 175 cc. Two patients required retreatment for residual tissue. There were 6 complications including 5 bladder neck contractures requiring incision (10%) and 1 case of postoperative prostatitis requiring antibiotic therapy (2%). Of 37 evaluable sexually active patients 31 (84%) reported preservation of antegrade ejaculation postoperatively. PMID- 7536257 TI - Does transurethral resection of the prostate pose a risk to life? 22-year outcome. AB - The clinical outcomes of 717 patients who underwent transurethral resection for benign prostatic hyperplasia between 1971 and 1981, and of 48 who underwent open prostatectomy during the same period were evaluated. All living patients could be followed for a minimum of 12 years postoperatively. The cumulative percentage of patients undergoing a secondary operation was substantially greater after transurethral resection of the prostate than after open prostatectomy. The volume of resected tissue, operating time, requirement for blood transfusion and hyponatremia during or after the procedure did not affect long-term outcome of patients in the transurethral resection group. Abnormal preoperative electrocardiography and azotemia appeared to be associated with increased risk of postoperative mortality after controlling for other variables (p < 0.05). Actuarial survival rates did not differ substantially for patients who underwent transurethral resection and open prostatectomy, and exceeded the expected survival rates in the general male population in the same age group in Japan. Both procedures are safe for the symptomatic relief of urinary obstruction due to benign prostatic hyperplasia. Prostatectomy does not jeopardize long-term survival of the patients. PMID- 7536259 TI - International prostate symptom score and quality of life assessment versus urodynamic parameters in men with benign prostatic hyperplasia symptoms. AB - In 207 men with symptomatic benign prostatic hyperplasia the international prostate symptom score and score on quality of life were collected. Cystometric tests with pressure-flow studies were performed in these patients with special attention to the assessment of obstruction grade. Of the men 24% appeared not to have obstruction. A significant correlation was found between size of the prostate and grade of obstruction. No correlation was found between obstruction grade, and any symptom score or quality of life score, while symptom score and quality of life correlated well. In 41 patients the outcome of laser prostatectomy was evaluated 6 months after the procedure. Mean symptom score decreased from 19.1 to 5.4 and mean quality of life score decreased from 3.7 to 1.3 in the 31 obstruction patients, compared to decreases of 19.6 to 10.1 and 3.6 to 2.6, respectively, in 10 without obstruction. The improvement in the obstruction patients was significantly better but nonobstruction patients will benefit from prostatectomy as well. PMID- 7536260 TI - Variability of pressure-flow analysis parameters in repeated cystometry in patients with benign prostatic hyperplasia. AB - Urodynamic investigation becomes increasingly important in the diagnosis of bladder outflow obstruction in patients with benign prostatic hyperplasia. To date, different methods for evaluation of the pressure-flow relationship and quantification of the grade of obstruction are available. Models for pressure flow analysis are briefly explained. The variability of the parameters is investigated by evaluation of 75 patients in whom 2 sequential voidings during urodynamic investigation were analyzed. The results showed that in 87% of these patients individual maximum flow differences of first and second voidings were less than 2 ml. per second. Individual detrusor pressure at maximum flow differences were less than 15 cm. water in 80% of these patients, while in 80% the intra-individual variation of the pressure-flow results was less than 15 cm. water for the minimal voiding pressure parameters (minimal urethral opening detrusor pressure and urethral resistance factor). For the pressure-flow parameter that defines the theoretical urethral lumen during voiding, the variation was less than 1.5 mm.2 in 84% of the patients. Patients with larger intra-individual differences are discussed. We concluded that the observed, aforementioned differences can be regarded as an indication of normal intra individual variability of voiding during urodynamic investigation. This intra individual variability, however, seldom leads to a change in the clinical grade of bladder outflow obstruction. We conclude that investigators involved in therapeutic trials of benign prostatic hyperplasia must be aware of this intra individual variability of micturition, since this variability is greater than the refined scale of the pressure-flow analysis models. PMID- 7536258 TI - Natural history of prostatism: relationship among symptoms, prostate volume and peak urinary flow rate. AB - We describe relationships among symptoms, prostate volume and peak urinary flow rate in an age stratified, community based random sample of white men 40 to 79 years old with no prior prostate surgery, prostate cancer or other conditions known to interfere with voiding. Symptoms were assessed with an instrument comparable to the American Urological Association symptom index. Prostate volume was estimated by transrectal ultrasonography and peak urinary flow rate was measured by a portable device. Subject age was significantly associated with symptom score but accounted for only 3% of its variation, while prostate volume and peak urinary flow rate explained only an additional 10% of the symptom variability. The odds (95% confidence interval) of moderate to severe symptoms increased with age from 1.9 (1.1 to 3.1), 2.9 (1.7 to 5.0) and 3.4 (1.8 to 6.1) for men 50 to 59, 60 to 69 and 70 to 79 years old, respectively, relative to men 40 to 49 years old. Adjusting for age, the odds of moderate to severe symptoms were 3.5 times greater for men with prostatic enlargement (more than 50 ml.) than for men with smaller prostates, while the odds were similarly increased (2.4 fold) for men not achieving a peak urinary flow rate of 10 ml. per second. Estimated odds changed little when other cutoff points were considered for peak urinary flow rate (15 ml. per second) or prostate volume (40 ml.). These results, based on randomly selected white men, suggest a somewhat stronger, albeit modest, relationship among symptoms, prostate size and urinary flow rate than previously reported in clinic based studies. The strength of these relationships is comparable to that found with other diseases. PMID- 7536262 TI - Histopathological evaluation of laser thermocoagulation in the human prostate: optimization of laser irradiation for benign prostatic hyperplasia. AB - We have previously shown in a canine prostate model that a noncontact low power neodymium:YAG laser regimen of 15 watts for 180 seconds yields a larger volume of coagulation necrosis than the currently recommended high power regimen of 50 watts for 60 seconds. These 2 regimens have not yet been compared in humans. The objective of this study was to evaluate histopathologically the thermocoagulation effect of these 2 laser regimens in the human prostate and the effect of the spatial distribution of the laser lesions on the extent of coagulation necrosis. The study was conducted in 10 patients undergoing radical prostatectomy or cystoprostatectomy. The laser treatment was given transurethrally 1 hour before removal of the specimen. There were no fractures of the prostate. The coagulation necrosis did not reach the peripheral zone and it was minimal in areas rich in fibromuscular tissue, such as the bladder neck. At times, nodular benign prostatic hyperplasia was unaffected. A 4-quadrant treatment in the same plane often yielded small nonconfluent lesions. Confluent lesions in the same plane yielded approximately 30% greater depth of coagulation necrosis, which was achieved when 3 or 4 lesions were created on each side of the prostate (per single transverse plane). Likewise, coagulation necrosis observed with 15 watts for 180 seconds was approximately 40% greater than that noted with the 50 watts for 60 seconds regimen. Our findings suggest that noncontact laser prostatectomy is a safe procedure that can be improved by modifying the laser regimen and the spatial distribution of lesions. PMID- 7536261 TI - The prediction of clinical outcome from transurethral microwave thermotherapy by pressure-flow analysis: a European multicenter study. AB - A total of 100 patients treated with a single session of microwave thermotherapy at 4 European centers was stratified according to 2 different types of obstruction (constrictive and compressive) and compared to clinical outcome at 6 months. Patients had a Madsen-Iversen score of 8 or more, maximum flow rate of 15 ml. per second or less and residual urine volume of 300 ml. or less at entry. The change in Madsen-Iversen score was the same in the 2 groups. Maximum flow rate increased from 8.71 +/- 2.62 to 14.73 +/- 4.04 ml. per second in the constrictive group, and from 8.54 +/- 2.26 to 10.41 +/- 4.52 in the compressive group (p < or = 0.0001). Residual urine decreased from 96.00 +/- 72.85 to 40.34 +/- 56.33 ml. in the constrictive group and from 109.86 +/- 67.09 to 84.65 +/- 81.45 ml. in the compressive group (p < or = 0.0001). Success, as defined by an increase of 50% or more in maximum flow rate and Madsen-Iversen score, was noted in 68% of the constrictive but only 15% of the compressive groups (p < or = 0.0001 chi-square test for trend). Selection by pressure-flow criteria for patients being considered for thermotherapy should improve the overall clinical results. PMID- 7536263 TI - Systematic removal of catheter 48 hours following transurethral resection and 24 hours following transurethral incision of prostate: a prospective randomized analysis of 213 patients. AB - The interval before removal of the catheter used in prostatic transurethral surgery depends to a great extent on the surgeon, with a frequently empirical orientation. We conducted a prospective, randomized and controlled study of 213 patients who underwent transurethral surgery for benign prostatic hyperplasia. The catheter was removed systematically 24 hours after transurethral incision and 48 hours after transurethral resection of the prostate (group 1-52 and 54 patients, respectively) or the catheterization interval was determined by each surgeon in accordance with the usual criteria (group 2-52 and 55 patients, respectively). No statistically significant differences were noted between these 2 groups in regard to complications. We conclude that systematic removal of the catheter at the aforementioned periods is cost-effective, safe and comfortable for the patient. PMID- 7536264 TI - Benign prostatic hyperplasia. PMID- 7536265 TI - Automated prostate volume determination with ultrasonographic imaging. AB - A method for automated determination of the prostate volume based on planimetric volumetry was developed to overcome subjectivity in prostate volume measurements using ultrasonography. An edge detection method is applied to locate the prostate boundary in sequential ultrasonographic images. During regular examinations, the volume was assessed automatically in 55 patients. The Pearson correlation coefficient of the automated volume compared to the reference prostate volume was 0.93. The estimated ratio of the automated volume to the reference volume was 0.92. This method overcomes the individual subjectivity when interpreting ultrasonographic images, and provides accurate and objective results for followup of benign prostatic hyperplasia treatments and for comparison of multicenter trials. Also, this method is a useful tool for objective determination of prostate specific antigen in proportion to the volume (prostate specific antigen density). PMID- 7536267 TI - Prostate specific antigen following radiotherapy for local prostate cancer. AB - The clinical recurrence-free survival rates were compared to the combined clinical and biochemical recurrence-free survival rates in 285 patients with stages A2 (T1b), B (T2) and C (T3) prostate cancer treated with radiation therapy. For stages A2 (T1b) and B (T2) disease the 10-year clinical recurrence free survival rate was 48% and the corresponding 10-year clinical and biochemical recurrence-free survival rate was 33%. For stage C (T3) disease these rates were 33% and 22%, respectively. These results demonstrate that approximately a third of the patients who are considered recurrence-free by standard clinical criteria at 10 years after radiation therapy have elevated prostate specific antigen values and are at significant risk for later clinical failure. PMID- 7536268 TI - Patterns of positive specimen margins and detectable prostate specific antigen after radical perineal prostatectomy. AB - Positive specimen margins and detectable postoperative prostate specific antigen (PSA) levels were analyzed after 200 consecutive radical perineal prostatectomies for clinical stages T1 and T2 adenocarcinoma of the prostate. Clinical parameters that correlated with lymph node metastases in concomitant pelvic lymphadenectomies were also noted. At a mean followup of 35 months 79% of the patients had undetectable PSA levels. Step-section pathological analysis of specimens obtained by either nerve sparing or extended radical modifications revealed that 41% of the tumors were organ confined and 56% had negative margins. Selective sacrifice of the posterolateral periprostatic fascia and the enclosed neurovascular bundle achieved negative margins and undetectable PSA levels despite capsular penetration in 15% of all patients. Of all positive margins with the perineal approach, solitary positive apical and posterolateral margins were infrequent (7% and 16%, respectively) but solitary positive anterior margins were more so (25%). Of those positive anterior margins 41% appeared to be artifactual and 45% might have been eliminated by avoiding avulsion of the puboprostatic ligaments. Pelvic lymphadenectomy could have been eliminated in 58% of the patients (clinical stage T2b or less, biopsy Gleason score 6 or less and PSA level 11 or less, for a node negative predictive value of 99%). PMID- 7536266 TI - Laparoscopic pelvic lymph node dissection combined with real-time interactive transrectal ultrasound guided transperineal radioactive seed implantation of the prostate. AB - Laparoscopic pelvic lymph node dissection with real-time interactive transrectal ultrasound guided transperineal radioactive seed implantation is a new method of treatment for localized carcinoma of the prostate. A total of 58 patients with clinically confined prostate cancer and negative seminal vesicle biopsies underwent staging laparoscopic pelvic lymph node dissection immediately followed by prostate implantation: 50 had 125iodine and 8 had 103palladium implants. Mean operating time was 226 minutes (range 120 to 475), mean blood loss was 57 cc (range 5 to 400) and average hospital stay was 2.2 days (range 0.5 to 8). At a mean followup of 12 months (range 6 to 24), complications included proctitis in 1.7% of the cases, dysuria in 24%, nocturia in 21% and outlet obstruction in 17%. Erectile function remained unchanged. Prostate volume decreased to 58.9% of the pretreatment value by 12 months and to 44.3% by 24 months. Mean prostate specific antigen level was 18.4 +/- 26.3 ng./ml. before treatment, 3.4 +/- 3.9 ng./ml. at 6 months, 2.3 +/- 2.3 ng./ml. at 12 months and 4.9 +/- 6.0 ng./ml. at 24 months (1.2 +/- 1.0 ng./ml. for patients with no evidence of disease). Of the patients 15.8% had local failure at 18 to 24 months as determined by positive transrectal ultrasound guided biopsy. Five of 58 patients (8.6%) had persistently elevated prostate specific antigen levels, only 1 of whom had a positive biopsy. Laparoscopic pelvic lymph node dissection with transrectal ultrasound guided implantation is a safe and promising mode of therapy for patients with localized prostate cancer. PMID- 7536269 TI - Prostate cancer diagnosis. PMID- 7536270 TI - TAG-72 expression in primary, metastatic and hormonally treated prostate cancer as defined by monoclonal antibody CC49. AB - Monoclonal antibodies CC49 and B72.3, which recognize a tumor associated glycoprotein (TAG-72) related to sialyted Tn antigen, have been used in clinical trials for radionuclide imaging, and treatment of colon, breast and ovarian carcinoma. In addition, studies with CC49 in patients with metastatic hormone refractory prostate cancer have been initiated based on the observed expression of TAG-72 in primary prostate cancer. We examined whether TAG-72 expression is a common feature of primary, metastatic and hormonally treated prostatic carcinoma. Immunohistochemical analysis of 25 primary prostatic carcinomas confirmed previous data that 21 of 25 specimens (80%) were immunoreactive with CC49. CC49 staining was noted in all 6 well (Gleason score 2 to 4), 8 of 10 moderately (Gleason score 5 to 6) and 7 of 9 poorly (Gleason score 7 to 9) differentiated tumors. CC49 immunoreactivity was noted in 10 of 20 hormonally treated prostate cancers and in 21 of 25 tumors without hormonal therapy. Intense CC49 staining of prostatic intraepithelial neoplasia was present in all 5 specimens examined. In contrast to the primary lesion, many metastatic prostate cancers lacked detectable CC49 immunoreactivity. Of 24 pelvic lymph node metastases from different patients only 4 (17%) had significant CC49 staining and 5 others had rare CC49 positive cells. However, 6 of 12 bone metastases showed CC49 immune staining. One specimen from an anaplastic locally recurrent tumor showed no reactivity. To our knowledge we present the first analysis of TAG-72 expression in a large series of patients with hormonally treated and metastatic prostate cancer, the most likely candidates for CC49 immunotherapy. Our findings that lymph node and bone metastases from prostate cancer are less likely to express significant amounts of TAG-72 than primary prostate cancer suggest that pretreatment biopsy typing for TAG-72 may be necessary to optimize the results of ongoing CC49 imaging and therapy studies. PMID- 7536272 TI - Anticoagulant associated hematuria: a prospective study. AB - We studied prospectively 32 consecutive patients with new onset of gross or microscopic hematuria while on anticoagulant therapy. Of the patients 19 men and 11 women (mean age 65 years) underwent urological evaluation. Of 6 patients with microscopic hematuria 3 with nephrolithiasis subsequently underwent extracorporeal shock wave lithotripsy. Two of 24 patients (7%) with gross hematuria had neoplastic disease invading the bladder, 1 had benign prostatic hyperplasia requiring resection, 1 had urethral stricture, 1 had ureteropelvic junction obstruction and 1 had nephrolithiasis. Thus, significant urinary tract disease was present in 9 patients (30%). Hematuria resolved in more than 90% of the patients after treatment. Based upon these observations, we believe that gross or microscopic anticoagulant associated hematuria is frequently precipitated by a significant genitourinary pathological condition and its prompt evaluation is recommended. PMID- 7536273 TI - Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. AB - In patients who recover erectile function after radical prostatectomy (with preservation of at least 1 neurovascular bundle), a recovery time of 6 to 18 months is not uncommon. As this is also the usual time required for regeneration of spinal nerves, we believe that regeneration of cavernous nerves, partially damaged inadvertently, may be responsible. In a rat model, we examined the long term effect of unilateral and bilateral cavernous nerve transection on the nonadrenergic/noncholinergic (NANC) nervous system and erectile function. In 31 rats, nitric oxide synthase (NOS), the enzyme that catalyzes nitric oxide production, was identified in penile nerve fibers from a mid-shaft segment with nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining and antibody to neuronal NOS. Animals were divided into three groups: 5 rats underwent pelvic exploration without transection of cavernous nerves (sham group); 13 rats underwent unilateral neurotomy of a 5-mm. segment of the cavernous nerve; and 13 rats underwent bilateral neurotomy. After bilateral ablation, the NOS-positive nerve fibers were significantly decreased at 3 weeks and remained so at 6 months; no erectile response could be elicited by pelvic nerve stimulation. After unilateral ablation, the NOS-positive nerve fibers were similarly decreased on the side of the neurotomy at 3 weeks, but by 6 months the number had increased significantly and approximated the level on the contralateral side. Furthermore, electrostimulation of the intact side induced a greater intracavernous pressure response at 6 months than at 3 weeks (N.B. the rat has an incomplete septum). Fibers positive for NOS were also identified in the dorsal nerve. The staining pattern diminished as rapidly and significantly on the side of neurotomy as in tissue from the corpus cavernosum. However, regeneration was not seen. To our knowledge, this is the first demonstration of regeneration of NOS-containing nerves after cavernous nerve neurotomy. Our findings support the reports by others that unilateral nerve-sparing is sufficient to preserve erectile function. PMID- 7536271 TI - The results of a phase II randomized trial comparing 5-fluorouracil and 5 fluorouracil plus alpha-interferon: observations on the design of clinical trials for androgen-independent prostate cancer. AB - The therapeutic benefit of chemotherapy in androgen independent prostate cancer is limited. 5-Fluorouracil has been reported to have modest antitumor activity in androgen independent prostate cancer. Although alpha-interferon is inactive as a single agent in prostate cancer, preclinical data indicate that it increases the in vitro cytotoxicity of 5-fluorouracil against a variety of malignant cells. We evaluated the relative antitumor activity and tolerance of 5-fluorouracil versus 5-fluorouracil plus alpha-interferon in 50 patients with histologically confirmed metastatic adenocarcinoma of the prostate. These patients had progressive disease in the presence of castrate levels of testosterone. A prospective randomized phase II open labeled trial was performed because of the difficulty in measuring responses in patients with metastatic prostate cancer. Of 23 patients treated with 5-fluorouracil alone and 28 treated with 5-fluorouracil plus alpha interferon 17 and 23, respectively, were evaluable for response and toxicity, and 5 and 5, respectively, were evaluable for toxicity only. Only 2 of 17 (11.7%) and 4 of 23 (17%) patients, respectively, showed a greater than 50% decrease in serum prostate specific antigen (no significant difference). There was no difference in duration of response or duration of survival between the 2 groups (mean duration of response 8.64 and 6.17 weeks, respectively, and mean duration of survival 33.70 and 38.65 weeks, respectively). Both regimens caused significant morbidity (mucositis and neurotoxicity) and 3 treatment related deaths at the high 5 fluorouracil doses. 5-Fluorouracil alone and with alpha-interferon at the doses used have minimal antitumor activity against androgen independent prostate cancer and, therefore, should not be tested further in these patients. Androgen independent prostate cancer selected using our criteria is a rapidly progressive disease, and these patients are an ideal target population for phase II studies. PMID- 7536275 TI - [Continuation of complete remission by oral administration of cytarabine ocfosfate in a patient with M0, who achieved remission by small doses of cytosine arabinoside with G-CSF]. AB - A 70-year-old male was admitted to our hospital because of leukocytosis. The laboratory examination revealed leukocytosis (44,500/microliters) with blasts (99%) in the peripheral blood. Myeloperoxidase staining of the leukemia cells was negative, but the surface phenotype was CD13- and CD33-positive, and negative for all lymphoid antigens. Peroxidase staining using the electron microscope was positive. Thus, the patient was diagnosed as acute myeloblastic leukemia (M0), according to the FAB classification. Although the therapy regimens commonly used for ANLL were effective for some cases with M0, the regimens mainly for ALL were more effective for the others. Thus we cannot determine what is the most effective regimen for M0. Since the patient had many complications, he was treated with low-dose AraC instead of combination chemotherapy. After the beginning of treatment, he became febrile and we added G-CSF to Ara-C. One month later, the patient achieved complete remission without severe infection. After four courses of consolidation therapy, the patient was discharged. He has been maintained in remission for more than 3 years and 8 months with only 5-day oral administration of cytarabine ocfosfate every four weeks. PMID- 7536276 TI - [Complete remission in acute myeloblastic leukemia (M0) after treatment with rhG CSF]. AB - A 57-year-old man was admitted because of fever and night sweat. The bone marrow was hypercellular with 86.4% blast cells. The diagnosis of AML (M0) was made, because the blast cells were negative for peroxidase stain and had CD13 and no lymphoid antigens in marker analysis. The patient was treated with BH-AC.TMP, BH AC.MVP and low dose Ara-C without any hematological improvement, and even additional treatment with medium dose Ara-C resulted in 66.4% blast cells in the bone marrow. Subsequent administration of rhG-CSF (150 micrograms/day) by continuous intravenous infusion resulted in the decrease of the blast cells in the bone marrow to a level that was evaluated as complete remission. He remains in complete hematological remission at present. As shown in this case, rhG-CSF might be an effective agent for the treatment of AML, even if the mechanism of its effectiveness is unclear at present. Further clinical studies should will supply useful information to analyze the pathophysiology of AML. PMID- 7536274 TI - [Primary nasopharyngeal lymphoma with CD3- and CD56+ phenotype]. AB - A 73-year-old man was admitted to our hospital with nasal hemorrhage and high grade fever on Aug, 1992. Physical examination revealed a tumor in the nasopharyngeal cavity, generalized skin eruptions and three tumors on different subcutaneous lesions, splenomegaly 2 cm below the costal margin, and the enlargement of the right cervical and axillary lymph nodes. Biopsy of the nasopharyngeal and cutaneous tumor disclosed non-Hodgkin's lymphoma (WF: Diffuse small cleaved). Peripheral blood examination showed a WBC of 4,800/microliters with 10% blastoid cells. Bone marrow examination showed 60% blastoid cells which frequently appeared a hand mirror configuration had no azurophilic granules in the cytoplasm. Flow cytometic analysis of these cells in the bone marrow showed that they expressed CD56 (NKH-1) and Ia but not expressed T-cell antigens as well as B-cell antigens and myeloid cell antigens. Phenotype of subcutaneous tumor biopsy cells was similar to that of blastoid cells in the bone marrow. T-cell receptor gene (TCR beta and gamma) rearrangements in blastoid cells were not found. The patient was treated with local radiotherapy to nasopharyngeal and skin tumors, followed by chemotherapy. The patient died of complication with pulmonary bleeding due to DIC. These results suggested that this nasopharyngeal lymphoma derived from NK cell. PMID- 7536278 TI - [Characterization of peripheral blood natural killer (NK) cells in two patients with CD16+ CD56- NK cell-lineage granular lymphocyte-proliferative disorder]. AB - In normal peripheral blood natural killer (NK) cells, the subset of CD16+ CD56+ cells is predominant, and that of CD16+ CD56- cells is rarely present. Because we have found the expansion of CD16+ CD56- NK cells in the peripheral blood of two patients with NK cell-lineage granular lymphocyte-proliferative disorders (NK GLPD), the clinical findings and cellular characteristics of these patients were compared with those of CD16+ CD56+ NK-GLPD patients. Although CD16+ CD56- and CD16+ CD56+ NK-GLPD cells were morphologically different, clinical findings and courses, and NK activity did not differ significantly. Because strong NK activity was demonstrated in CD16+ CD56- NK-GLPD cells, the CD56 antigen, one of the adhesion molecules, did not seem to play a major role in NK cell-mediated cytotoxicity. The CD56 antigen is known to be more strongly expressed by immature NK cells than by mature NK cells. However, because interleukin 2-activated CD16+ CD56- NK-GLPD cells rapidly expressed the CD56 antigen, the degree of CD56 antigen expression did not always correlate with the maturity of NK cells. PMID- 7536277 TI - [Complete remission achieved by low-dose Ara-C, aclarubicin and rhG-CSF (CAG) therapy in acute non-lymphocytic leukemia with monosomy 7 occurring after severe aplastic anemia]. AB - We report a case of acute myelogenous leukemia (AML), which developed from severe aplastic anemia (SAA) and was successfully treated by low-dose Ara-C and aclarubicin with concomitant use of G-CSF (CAG therapy). A 37-year-old male was admitted for scrutiny of pancytopenia and diagnosed as SAA because of hypocellular bone marrow without abnormal or dysplastic cells. Although hematopoiesis recovered with steroid pulse therapy followed by administration of anabolic steroids, 29 months after initial onset of SAA, he presented as AML (FAB M6), as his bone marrow Contained 21.6% leukemic myeloblasts and 56% of erythroblasts. Chromosome study revealed 45, XY, -7 in 14 of 20 cells analyzed. Complete remission was achieved by administration of low-dose Ara-C (20 mg/m2 for 7 days) and aclarubicin (14 mg/m2 for 4 days) along with G-CSF (200 micrograms/m2 for 7 days), without any severe complications. In the previous reports in Japan since 1982, 7 out of 8 cases with AML developing from SAA died within a year. Our results indicate that CAG therapy is useful for treatment for this subset of AML with poor prognosis. PMID- 7536280 TI - Differential sensitivity of thoracic malignant tumors to adenovirus-mediated drug sensitization gene therapy. AB - Malignant mesothelioma may prove to be an attractive candidate for somatic gene therapy with replication-deficient recombinant adenovirus transfer of a toxic, or drug sensitization gene. Transfer of the herpes simplex thymidine kinase type I gene (HSVtk), followed by exposure to the acyclic nucleoside drug ganciclovir, has been shown to be an effective tumor cell killing system. To study generalized applicability, we tested a number of thoracic malignant cell lines for their sensitivity to gancyclovir after infection with an adenoviral vector containing the HSVtk gene (Ad.RSVtk). Using the concentration of gancyclovir required to kill 50% of the cells (IC50) as a measure of sensitivity, we detected variable sensitivity among cell lines, with mesothelioma most sensitive (IC50 = 0.075 to 2.8 mumol/L gancyclovir), and non-small-cell carcinoma lines having an intermediate sensitivity (IC50 = 1.5 to 100 mumol/L). In contrast, an ovarian carcinoma line was extremely resistant (IC50 > 2000 mumol/L). To study the possible mechanisms for these differences, we studied cell lines with regard to their ability to be infected with an adenoviral vector containing a marker gene (Ad.CMVlacZ) and expression of the vitronectin receptor alpha v (an integrin cell adhesion molecule shown to be required for adenovirus internalization after initial binding). We found that the degree of lacZ transduction correlated with HSVtk sensitivity, whereas vitronectin receptor expression did not, suggesting that differences in initial viral binding ability, rather than internalization, may explain the sensitivity differences seen in vitro. PMID- 7536279 TI - [The effects of COP-BLAM regimen with G-CSF for intermediate and high grade non Hodgkin's lymphoma]. AB - G-CSF was used concomitantly with the COP-BLAM regimen, and its therapeutic results and adverse effects were evaluated. A total of 104 patients with untreated non-Hodgkin's lymphoma (NHL), including 22 in stage II, 52 in stage III and 30 in stage IV. Seventy five patients had diffuse large cell type, 18 diffuse medium cell type, and 11 diffuse mixed cell type. The treatment consisted of the COP-BLAM regimen based on the method of Laurence et al., was performed every 3 weeks. Complete remission was achieved in 98 out of 104 patients (94.2%), and the 4-year survival rate was 82.4%, while at the time of evaluation the median observation period was 26 months. The survival time was significantly prolonged in patients with low LDH values, B-cells, stage II or low CRP values. The COP BLAM regimen with concomitant G-CSF administration achieved a high remission rate and reduced the frequency of infections. Almost all of the patients could be treated in 21-day cycle and this appeared to be effective for treatment with increased dose intensity. PMID- 7536282 TI - Vertical transmission of hepatitis C. PMID- 7536281 TI - Immunocytochemical characterization of malignant mesothelioma and carcinoma metastatic to the pleura: IOB3--a new tumor marker. AB - We have tried to find a reliable panel of markers that would allow distinction between mesotheliomas and carcinomas metastatic to the pleura. In a prospective study, we evaluated 54 pleural effusions: In 27 of the patients, a diagnosis of histologically proven metastatic carcinoma was subsequently established, 7 patients had biopsy-proven malignant mesotheliomas and 20 had benign, reactive effusions whose benign etiologies were established by more than 2 years clinical follow-up. The MAb (monoclonal antibody) IOB3 proved to be diagnostic for carcinomas in all 27 cases (100%), whereas CEA (carcinoembryonic antigen) expression was found in only 22 out of 27 (81%). None of the malignant mesotheliomas, nor benign reactive mesothelial cells reacted with these two markers. All carcinomas, as well as one malignant mesothelioma, reacted with the MAb HEA125. Antibodies against 12 single cytokeratins, vimentin, and EMA (epithelial membrane antigen) were not helpful in the differentiation between malignant mesotheliomas and malignant carcinomatous pleural effusions. We conclude that adding the antibody IOB3 to the CEA assay should allow a reliable differentiation between these two entities. PMID- 7536283 TI - Vertical transmission of hepatitis C. PMID- 7536284 TI - Vertical transmission of hepatitis C. PMID- 7536285 TI - Determination of nonendemic nasopharyngeal carcinoma by in situ hybridization for Epstein-Barr virus EBER1 RNA: sensitivity and specificity in cervical node metastases. AB - After time-consuming and costly investigations, patients with neck metastases from an occult primary often receive unnecessarily large radiation volumes to treat a possible origin in the nasopharynx. In this study a colorimetric antisense Epstein-Barr early ribonucleoprotein 1 (EBER1) oligonucleotide probe specific for Epstein-Barr virus RNA was hybridized in situ to metastatic tissue obtained from 18 nasopharyngeal, 54 oral and pharyngeal, and 12 occult carcinomas derived from an unselected population. All 16 nonkeratinizing nasopharyngeal carcinomas (NPCs) were positive for EBER1. Both cases of keratinizing NPC and all 54 other metastases were negative. A single positive case of occult carcinoma indicated its origin from NPC. In retrospect, 7 patients with occult carcinoma had received unnecessary treatment with irradiation to the nasopharynx. Nasopharyngeal carcinoma appears to be a less common origin of occult carcinoma than previously considered. In the proper clinicopathologic context EBER1 in situ hybridization (EBER1-ISH) allows exclusion of NPC with a high degree of accuracy. Thus unnecessarily large radiation volumes and their adverse sequelae may be reduced in the treatment of occult carcinoma. Conversely, a positive result of ISH allows exclusion of further extensive diagnostic procedures. PMID- 7536286 TI - Evaluating the role of photodynamic therapy in the management of pancreatic cancer. AB - BACKGROUND AND OBJECTIVE: Cancer of the pancreas constitutes one of the major causes of cancer related death throughout the world. A 5-year survival rate of only 2% and a maximum of 20 months median survival in multi modality treatment studies dealing with the most favorable patients only, has been demonstrated. This review analyzes the principal treatments and available experimental data in view of a clinical application of photodynamic therapy (PDT) for the treatment of pancreatic cancer. STUDY DESIGN/MATERIALS AND METHODS: On the basis of published results, we examined the palliation of pancreatic cancer by chemotherapy alone; radiation alone and multimodality schedules (radiation and chemotherapy). Radical tumor resection was examined as attempted curative treatment. RESULTS: In reported therapeutic procedures, palliative or potentially curative, median survival was below 2 years. The GTSG reported survival time increases from 10.9 to 21.0 months when surgery is followed by adjuvant chemotherapy and radiation. This combination postoperatively does not increase mortality, but adds 30% morbidity. Photodynamic therapy has been demonstrated in preclinical studies to have a selective effect on malignant versus the normal pancreas. CONCLUSION: PDT is highly effective in eliciting the destruction of experimental pancreatic tumors with the lack of significant effect on the normal pancreas. The poor prognosis for patients with this disease, especially those patients with advanced disease, warrants closer examination of PDT for the treatment of pancreatic cancer. PMID- 7536287 TI - [Interferon in the treatment of solid tumors]. AB - Discovered almost four decades ago, interferons were being investigated as potential anticancer agents from early sixties, because of their antiviral antiproliferative and immunomodulating properties and their relatively modest toxicity. Unlike very good effects on some lymphoproliferative disorders where complete durable remissions were observed, results of their application in solid tumors are not so unambiguous. Among large number of tumors investigated so far, the best, reproducible, although modest, activity was found against melanoma, renal cell carcinoma and Kaposi's sarcoma. There are still many doubts concerning their optimal doses, sequencing and schedules, most probably in combination with other biological response modifiers and/or cytotoxic agents, which have to be clarified in further clinical trials, together with investigation of more reliable biological indicators of response to interferons in vivo. PMID- 7536288 TI - Elucidation of linear epitopes of pertussis toxin using overlapping synthetic decapeptides: identification of a human B-cell determinant in the S1 subunit indicative of acute infections. AB - To identify relevant linear epitopes within the immunodominant ADP-ribosyl transferase (S1 subunit) of pertussis toxin (PT), its complete amino acid sequence was synthesized as consecutive, overlapping decapeptides on solid phase and probed for seroreactivity with pertussis specific human antisera in 'peptide scans'. Comparison of the resulting antigenic profiles revealed two distinct types of human antisera, though amino acids 140-200 could not be assessed as the corresponding peptides reacted non-specifically with the detection system. Human anti-pertussis sera predominantly recognized linear immunodominant epitopes located in three separated segments spanning amino acids 3-16, 21-30, and 211 222. Antisera originating from infants with acute B. pertussis infections (type I) identified determinants in all three segments, while type-II antisera from convalescent patients only recognized epitopes in the N-terminal regions. The binding of pertussis specific antisera--both type I and type II--to the holotoxin was inhibited by preincubation of antibodies with synthetic peptides corresponding to two linear determinants located at the N-terminus of S1:R 3-16 and R 21-30. However, competitive binding of antibodies to PT and to synthetic peptides equivalent to the third epitope (R 211-222) was only observed with type I antisera. Thus, the linear immunogenic determinant identified at the C-terminus of the A-protomer represents a human epitope which is apparently specific for antisera from pertussis patients with acute infections. The possible application of this determinant in serologic diagnosis will be a valuable tool to detect and distinguish acute Bordetella pertussis infections. PMID- 7536289 TI - Mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the P2 porin protein of non-typeable Haemophilus influenzae. AB - The P2 protein is the major outer-membrane protein of non-typeable Haemophilus influenzae (NTHI) and shows extreme heterogeneity among strains. Based on the analysis of antigenic structure, the P2 protein consists of eight potentially surface-exposed loops. Previous studies of monoclonal antibodies (mABs) to a single strain of NTHI showed that P2 contains potentially immunodominant epitopes in loop 5 of the molecule. The goal of the current work is to test the hypothesis that strain-specific and potentially immunodominant epitopes are located in loop 5 of P2 in other strains of NTHI as well. Gene fragments which encode peptides of loop 5 of strains 2019 and 5657 were cloned into an expression vector and subjected to immunoassays with mABs which recognize surface-exposed, bactericidal, strain-specific epitopes. Each mAB recognized loop 5 of the P2 protein of the homologous strain. Analysis of mutant clones with minor amino acid changes showed a loss of reactivity with the mABs. These observations indicate that loop 5 of the P2 molecule contains strain-specific, abundantly expressed surface-exposed epitopes. This further supports the hypothesis that loop 5 is an immunodominant region of the P2 molecule. PMID- 7536290 TI - Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation. AB - RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosome binding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The significance of these observations for the RNA folding problem in general is discussed. PMID- 7536291 TI - The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth-phase-dependent expression. AB - The dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L-amino acids. The periplasmic dipeptide-binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem-binding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential -35 and -10 regions of a sigma 70 promoter. During exponential growth in Luria-Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF-binding palindromic) element, whose deletion from a multi-copy plasmid causes fivefold and 10-fold reductions in the levels of upstream and downstream dpp mRNA, respectively. PMID- 7536292 TI - Partial purification of the rat erythrocyte receptor for the channel-forming toxin aerolysin and reconstitution into planar lipid bilayers. AB - The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N-glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte. PMID- 7536294 TI - Combination chemotherapy with vinblastine, bleomycin, cisplatin, and etoposide (VBPE) in children with primary intracranial germ cell tumors. AB - We have treated 13 children with primary intracranial germ cell tumors with the combination of vinblastine, bleomycin, cisplatin, and etoposide, the so-called VBPE regimen in the past seven years at one institution. The clinical diagnosis of these children were: three germinomas, seven non-germinomatous germ cell tumors, and three recurrent non-germinomatous germ cell tumors without previous chemotherapy. Of this group, three germinomas and three non-germinomatous germ cell tumors were treated primarily with VBPE plus radiotherapy. All of them responded completely, and were free of tumor. Primary VBPE regimen without radiotherapy was used in another four non-germinomatous germ cell tumors. After six courses of VBPE regimen, only one of the four patients achieved complete response for 30 months. VBPE regimen was used as salvage chemotherapy in three recurrent non-germinomatous germ cell tumors without previous chemotherapy. After six courses of VBPE regimen, two had complete response for 58+ months and 78+ months respectively. We concluded that in newly-diagnosed intracranial non germinomatous germ cell tumors, the combination of chemotherapy VBPE regimen and radiotherapy led to good results. Primary chemotherapy with VBPE alone was not adequate for the treatment of non-germinomatous germ cell tumors. However, VBPE regimen might be one of the salvage therapies for recurrent germ cell tumors without chemotherapy before relapse. PMID- 7536293 TI - The characterization of ion channels formed by Pasteurella multocida dermonecrotic toxin. AB - The influence of the dermonecrotic lethal toxin (approximately 120 kDa) produced by Pasteurella multocida serovarian D on planar phospholipid bilayers was studied. It was found that the toxin is able to increase the conductance of the bilayers by formation of low-conductive and cation-selective ion channels [27 pS at 4.0 M KCl, pH 7.5; zero current potential equals to -14.5 +/- 0.5 mV at threefold transmembrane gradient KCl (120 mM/40 mM)]. In biionic conditions the channels displayed weak selectivity between Na, K and Ca ions. The shapes of current-voltage characteristics (which were measured at different pH and salt concentrations) indicate that an energetic barrier for passing ions is situated near the center of the water pore of the ion channels. The effective diameter of the ion channel's water pore was established to be equal to 2.1 +/- 0.3 nm. PMID- 7536295 TI - [The laboratory diagnosis of Pneumocystis infection (an evaluation of existing methods for staining the causative agent and the use of crystal violet for demonstrating P. carinii in pathological material)]. PMID- 7536297 TI - The specificity of disease-associated anti-fibrillarin autoantibodies compared with that of HgCl2-induced autoantibodies. AB - Autoantibodies against nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. An important target of these autoantibodies is a protein with an apparent molecular weight of 36 kDa and a pI value of 8.5, located in the dense fibrillar component of the nucleolus and therefore termed fibrillarin. Animal models in which abundant anti-nucleolar antibodies appear spontaneously have not yet been described; however, high levels of anti-fibrillarin antibodies can be induced by treating susceptible strains of mice with sub-toxic amounts of mercuric chloride. In this study, we have analysed the specificity of anti-fibrillarin autoantibodies of human and murine origin. Our results suggest that both species have similar, if not identical conformational epitopes that are the target of anti-fibrillarin autoantibodies; these epitopes require the presence of a 30-kDa fragment of the fibrillarin molecule. Post-translational modifications such as the dimethylation of arginines in the N terminus of the protein are not essential for antibody recognition. PMID- 7536296 TI - Evidence for post-transcriptional regulation of the synthesis of the Escherichia coli HlyB haemolysin translocator and production of polyclonal anti-HlyB antibody. AB - Extensive attempts were made to overexpress the Escherichia coli haemolysin translocator protein HlyB, and HlyB fragments, utilising high copy number plasmids or hlyB expressed from strong promoters including lambda PR, ptrp and the T7 promoter. Analysis of both cytoplasmic and membrane fractions failed to detect any overexpression of the protein, although all the constructs showed biological activity and there was no evidence of HlyB-induced toxicity. In some constructs, the effect of removing a stem-loop structure, immediately upstream of the start codon and implicated in rho-independent termination of transcription, was tested but this did not lead to over-expression. Nevertheless, analysis of hlyB specific mRNA synthesis revealed that some constructs showed at least a 50 fold increase in mRNA levels, indicating that expression of HlyB may be limited at the translational level. When HlyB was expressed as a hybrid, downstream of LacZ, extremely high level overproduction was then detected in total cell extracts. When the expression of HlyB or HlyB fragments expressed from a T7 promoter was examined, the C-terminal ATPase domain was dramatically overexpressed but the production of fragments encompassing the N-terminal membrane domain, was reduced at least 1000-fold. These results indicate that mRNA structures corresponding to the membrane domain of HlyB greatly limit the post transcriptional expression of HlyB. When such structures are deleted, or disrupted when part of a larger mRNA, HlyB or the HlyB ATPase domain can be overproduced in milligram quantities and this has facilitated the production of high titre antibodies to HlyB. PMID- 7536298 TI - Carbohydrate specificity of IgM autoantibodies to CD45 in systemic lupus erythematosus. AB - Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinant E. coli fusion proteins encoded by exons 3-7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45. PMID- 7536300 TI - Sickle cell anemia--basic research reaches the clinic. PMID- 7536301 TI - Idiopathic dilated cardiomyopathy. PMID- 7536299 TI - All you wanted to know about SELEX. AB - In vitro selection, or SELEX, is a technique that allows the simultaneous screening of highly diverse pools of different RNA or DNA (dsDNA or ssDNA) molecules for a particular feature. Different examples from a great variety of applications of in vitro selection experiments are described and a detailed overview of the method and its variations will be given. Some especially conclusive in vitro selection experiments are discussed in detail to illustrate the potential power and diversity of this method. Potential restrictions of the methods and possible ways to overcome them are pointed out. PMID- 7536302 TI - Origins of homochirality. PMID- 7536303 TI - Molecular basis of antigen mimicry by an anti-idiotope. AB - Idiotopes are antigenic determinants, unique to an antibody or group of antibodies, defined by the reaction of anti-idiotopic antibodies with the antibodies bearing the idiotopes. The ensemble of idiotopes of an antibody constitutes its idiotype. Idiotypes are useful as markers to follow specific antibodies and clones of cells in immune responses and the inheritance of immunoglobulin genes. As external antigens and anti-idiotypic antibodies can competitively bind the combining site of specific antibodies, some anti-idiotypic antibodies may resemble the external antigen, thus mimicking its structure. It has been proposed that an anti-idiotypic antibody, anti-anti-X, may resemble the external antigen X and thus carry its 'internal image', but this idea is not unequivocally supported by the three-dimensional structures of anti-idiotopic antibodies, either because the structures of the external antigen or of the anti idiotopic antibody were unknown, or because the anti-idiotopic antibodies showed no resemblance to the external antigens (reviewed in ref. 10). Functional mimicry of ligands of biological receptors by anti-idiotypic antibodies has been described in several systems (reviewed in ref. 11). But how closely can antibodies mimic antigens at the molecular level? Here we present the crystal structure of an idiotope-anti-idiotope complex between the Fv fragments of the anti-lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2. D1.3 contacts the antigen, lysozyme and the anti-idiotopic E5.2 through essentially the same combining-site residues. In addition, E5.2 interacts with D1.3, making contacts similar to those between lysozyme and D1.3. Thus, the anti-idiotopic antibody E5.2 mimics lysozyme in its binding interactions with D1.3. Validating these observations, E5.2, used as an immunogen, induces an anti-lysozyme response. PMID- 7536305 TI - Influence of posttetanic potentiation of slices of the cerebral cortex of rats on the content of proteins and RNA in neurons and gliocytes. PMID- 7536304 TI - Propagation of impulses in the guinea-pig ureter and its blockade by calcitonin gene-related peptide (CGRP). AB - The guinea-pig ureter was placed in a three-compartment organ bath to enable the application of electrical stimuli or drugs to its renal end (R-site), the middle region (M-site) or the bladder end (B-site) while recording mechanical activity at the R- and B-sites. All experiments were performed in ureters pre-exposed to capsaicin (10 microM for 15 min) to prevent the release of sensory neuropeptides from afferent nerves. Electrical field stimulation (EFS, 5-25 ms pulse width, 20 V) produced a phasic contraction at the site of stimulation ('direct' response to EFS) which propagated to the other end of the ureter. Section of the ureter at the M-site abolished the propagated response to EFS; after section, EFS applied at the M-site induced a phasic contraction at both the R- and B-sites. Likewise, the application of KCl at the M-site produced phasic contractions at both the R- and B-sites. Tetrodotoxin (1 microM), nifedipine (1 microM) or Bay K 8644 (1 microM) applied at the M-site had no influence on the direct or propagated responses to EFS; nifedipine (10 microM) applied at the M-site abolished the propagated responses without affecting the direct responses to EFS. Bay K 8644 (1 microM) applied at the R-site produced a marked enhancement of the direct response (EFS applied at R-site) while having no effect on the amplitude of the propagated response to EFS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536306 TI - Amiridin and tacrine modulation of the activity and plasticity of the cholinoreceptors of neurons of the common snail: phenomenology and mechanisms. AB - The influence of amiridin and tacrine on the membrane potential, activity, and plasticity of cholinoreceptors was investigated using the methods of recording of intracellular potentials and transmembrane currents in identified RPa3 and LPa3 neurons of the common snail. Amiridin and tacrine (1-100 mumole/liter) do not exert appreciable influences on the membrane potential of the cells. Both compounds modulate the activity of cholinoreceptors, judging from their influence on the inward current induced by local application of the acetylcholine: the duration of the ACh current increases and its amplitude varies biphasically (a short-latency increase is succeeded by a decrease). Amiridin and tacrine intensify the extinction of the ACh current induced by repeated applications of ACh to the soma. The acetylcholinesterase inhibitor, physostigmine, exerts a similar modulatory influence on the ACh current and on its extinction, and also prevents the manifestation of the effects of amiridin and tacrine. Amiridin and physostigmine directly influence the cholinoreceptors and the ionic membrane channels controlled by them by similarly altering the voltage-current characteristics (VAC) of the ACh current, and by shifting the reversal potential of the ACh current in the negative direction, bringing it closer to the equilibrium potential for chloride ions; this may be interpreted as a result of the nonidentical action of these compounds on the various ionic currents controlled by cholinoreceptors. The hypothesis is advanced that the modulatory influence of amiridin, tacrine, and physostigmine on the activity and plasticity of the cholinoreceptors is governed by their direct membrane-cytoplasmic action. PMID- 7536307 TI - The human neostriatum shows compartmentalization of neuropeptide gene expression in dorsal and ventral regions: an in situ hybridization histochemical analysis. AB - Expression of neuropeptide messenger RNAs in striatal neurons was studied in post mortem human brain tissue by the use of in situ hybridization histochemistry. Clusters of cells expressing high levels of prodynorphin messenger RNA, and less strikingly, preprotachykinin messenger RNA, were prominent in the caudate nucleus and were present but less pronounced in the putamen. Proenkephalin and prosomatostatin messenger RNA-containing cells were more homogeneously distributed throughout the striatum, though the latter were much sparser. The four neuropeptide messenger RNA patterns in the nucleus accumbens were rather homogeneous compared with the dorsal striatum. Of these, prodynorphin messenger RNA showed a higher level of expression per cell in the nucleus accumbens relative to the dorsal striatum. The relationship of neuropeptide-containing cell clusters to the striosomal organization was characterized by looking at the register of these markers with patterns of low acetylcholinesterase activity and dense mu opiate receptor binding. In the caudate and putamen, clusters of cells expressing high levels of dynorphin and preprotachykinin messenger RNAs were clearly in register with the striosomes. The accumbens was defined by high prodynorphin messenger RNA levels, both low and high levels of acetylcholinesterase staining, and very low to absent mu opiate receptor binding. The distribution of high-expressing prodynorphin messenger RNA-containing cells- to the patch compartment and throughout the entire ventral striatum/nucleus accumbens region--defines the limbic domain of the neostriatum and suggests particular relevance to human striatal organization and function, because the distribution of this opioid neuropeptide is considerably more compartmentalized in human than in non-human species. PMID- 7536309 TI - The non-peptide neurokinin-1 antagonist, RPR 100893, decreases c-fos expression in trigeminal nucleus caudalis following noxious chemical meningeal stimulation. AB - The effect of RPR 100893, a selective and specific neurokinin-1 antagonist, or its enantiomer RPR 103253 was examined on c-fos antigen expression in brain stem and upper cervical cord 2 h after intracisternal capsaicin injection (30.5 micrograms/ml) in pentobarbital-anesthetized Hartley guinea-pigs. Positive cells were counted at three levels corresponding to obex, -2.25 mm and -6.75 mm in 18 sections (50 microns). Immunoreactivity was strongly expressed within laminae I and IIo of trigeminal nucleus caudalis, area postrema and the leptomeninges. Moderate labeling was present in the nucleus of the solitary tract and the medullary lateral reticular nucleus, whereas few positive cells were found in the ventral portion of the medullary reticular nucleus and Rexed laminae III-V and X. The distribution of labeled cells was consistent with previously reported results following subarachnoid placement of the noxious agents, blood or carrageenin. Pretreatment with RPR 100893 (1, 10 and 100 micrograms/kg, i.v.) but not its enantiomer (100 micrograms/kg, i.v.) 30 min prior to capsaicin injection significantly reduced the number of positive cells in the trigeminal nucleus caudalis (P < 0.01) in a dose-dependent manner, but not within area postrema or nucleus of the solitary tract. These results indicate that (i) the instillation of capsaicin into the subarachnoid space is an effective stimulus for the induction of c-fos antigen within trigeminal nucleus caudalis, presumably through activation of trigeminovascular afferents, and (ii) the neurokinin-1 antagonist RPR 100893 reduces the number of positive cells selectively within this nucleus. The findings are significant because drugs which alleviate vascular headaches decrease the number of c-fos-positive cells within trigeminal nucleus caudalis following noxious meningeal stimulation. Hence, strategies aimed at blocking the neurokinin-1 receptor may be useful for treating migraine and cluster headache. PMID- 7536308 TI - The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration. AB - Tachykinin peptides such as substance P and neurokinin B have been widely studied as mediators of pain transmission. The expression of neurokinin-1 and neurokinin 3 receptor messenger RNAs in the spinal cord is increased following intense nociception. The opiate ligands morphine and naltrexone alter behavioral responses to formalin-induced pain and alter evoked substance P release. This study investigated whether these opiates similarly alter the expression of substance P-, neurokinin B-, neurokinin-1 receptor- and neurokinin-3 receptor encoding messenger RNAs in spinal systems following formalin-induced nociception. Expression levels of various messenger RNAs were quantitated using solution hybridization-nuclease protection assays. Six hours after hindpaw treatment, the levels of substance P-encoding preprotachykinin messenger RNA in the lumbar dorsal root ganglia and neurokinin B, neurokinin-1 receptor and neurokinin-3 receptor messenger RNAs in the lumbar dorsal horn were increased by approximately two-fold as compared to sham-treated controls. Pretreatment with naltrexone resulted in a further increase in the nociception-induced substance P messenger RNA expression in the dorsal root ganglia; preprotachykinin messenger RNA expression was not affected by morphine. Nociception-induced neurokinin-1 receptor messenger RNA expression in the dorsal horn was blocked by morphine, but was not affected by naltrexone. Both morphine and naltrexone blocked the formalin induced increases in neurokinin B and neurokinin-3 receptor messenger RNA levels. Increased neurokinin B messenger RNA expression may reflect increased neurokinin B turnover in spinal interneurons activated by nociception. Neurokinin-3 receptor messenger RNA expression levels varied closely with, and thus may be regulated by, the levels of neurokinin B messenger RNA in the same regions. The results of this study indicate that pretreatment with opiate ligands modulates the expression of tachykinin peptide and neurokinin receptor encoding mRNAs in spinal systems following a peripheral chemogenic inflammatory stimulus. Thus, endogenous opioid systems may be involved in activity-induced changes in the expression of spinal tachykinin peptides and neurokinin receptors. PMID- 7536311 TI - Capsaicin-sensitive area in the ventral surface of the rat medulla. AB - In acute experiments on nembutal-urethan-anesthetized rats, structures selectively sensitive to capsaicin were found near the ventral surface of the medulla at the exit of hypoglossal nerve roots. Microinjection of 5-50 nl 0.01% capsaicin to the rostral region of the capsaicin-sensitive area mostly activated respiration, arterial pressure and heart rate (HR) while that to the caudal region inhibited arterial pressure and HR. In chronic experiments on rats, injection of 25 nl 1% capsaicin to the caudal capsaicin-sensitive area led to a decrease in arterial pressure by 35-45% and in HR by 10-15% within a week after operation. Arterial pressure and HR virtually reached the control level and the rostral and caudal ventral medulla showed asymmetric distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive cells by the end of the 2nd week. It is suggested that nitric oxide may be involved in the mechanisms of neurochemical rearrangements in the brainstem after application of capsaicin to the caudal ventrolateral medulla (CVLM). PMID- 7536310 TI - Differential contribution of L- and N-type calcium channels on rat hippocampal acetylcholine release. AB - Bay K 8644, nimodipine and omega-conotoxin GVIA (omega-CgTx) were used to study the different contribution of voltage-sensitive calcium channels (VSCC) to [3H]acetylcholine ([[3H]ACh) release in rat hippocampal synaptosomes. In our experimental conditions, the percentage of calcium-dependent ACh release was approximately 80%. Nimodipine (0.01-10 microM) and Bay 8644 (0.01-10 microM) were not able to modify the [3H]ACh release under stimulating conditions (15 mM K+). Nevertheless, when K+ concentration was reduced to 8 mM, a significant increase in [3H]ACh release was observed at 1 and 10 microM of Bay K 8644. Nimodipine (0.01-10 microM) failed to reverse the effect of Bay K 8644 on [3H]ACh release. Finally, omega-CgTx (0.001-1 microM) caused a concentration-dependent reduction of [3H]ACh release in K+ (15 mM)-stimulating conditions. These results suggest that the N-type VSCC probably play a predominant role in regulating the [3H]ACh release in synaptosomes from rat hippocampus. PMID- 7536312 TI - Neuroprotective effect of free radical scavengers on beta-N-oxalylamino-L-alanine (BOAA)-induced neuronal damage in rat hippocampus. AB - The neurotoxin beta-N-oxalylamino-L-alanine (BOAA), found in Lathyrus sativus seeds, is thought to be the causative agent of neurolathyrism. We have investigated the neuroprotective effects of free radical scavengers on BOAA induced toxicity following focal injection (1 microliter) of BOAA and comparing the pathological outcome with the effects of injections of alpha-amino-3-hydroxy 5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) or N-methyl-D-aspartate (NMDA) into the dorsal hippocampus of male Wistar rats. Cellular damage was assessed histologically. BOAA (50 nmol) induced a highly selective pattern of hippocampal damage identical with that seen with AMPA (1 nmol). BOAA-induced neurotoxicity, but not AMPA, KA (0.5 nmol) or NMDA (25 nmol)-induced neurotoxicity, was prevented in a dose-dependent manner by focal co-injection of four potential free radical scavengers; dimethyl sulphoxide (DMSO) (1750-7000 nmol), dimethylthiourea (DMTU) (8000 nmol), dimethylformamide (DMF) (7000 nmol) and mannitol (1000 nmol). These findings suggest that hippocampal damage induced by BOAA involves an interaction between AMPA receptors and free radicals. PMID- 7536313 TI - Preprotachykinin-A and substance P receptor (NK1) gene expression in rat astrocytes in vitro. AB - The presence of mRNA transcripts coding for preprotachykinin-A and the substance P receptor in cultured astrocytes is demonstrated by a combination of reverse transcription/polymerase chain reaction (PCR) and Southern blotting. These findings showed that astrocytes in culture are capable of synthesing both the precursor of substance P (preprotachykinin-A) and the cognate receptor, substance P receptor (NK1). The simultaneous presence of both the ligand (substance P) and the receptor (NK1) may indicate an autocrine nature of astrocyte communication. PMID- 7536314 TI - Acetylcholinesterase staining in the auditory brainstem nuclei of the leopard frog, Rana pipiens. AB - We examined the distribution of acetycholinesterase (AChE) in the brainstem auditory nuclei of leopard frogs (Rana pipiens). Numerous AChE-labeled somata were observed in caudal brainstem nuclei including the dorsolateral nucleus, superior olivary nucleus, and superficial reticular nucleus. At midbrain levels, AChE-labeled somata were observed in the magnocellular, principal and laminar nuclei of the torus semicircularis, though, most were located in the magnocellular nucleus. Neuropil labeling, while present throughout the auditory brainstem, was particularly pronounced in the principal nucleus of the torus. These findings suggest that the auditory system of anurans, like that of birds and mammals, comprises, in part, distinct cholinergic pathways. PMID- 7536315 TI - Histochemical localization of nitric oxide synthase in the bullfrog intracardiac ganglion. AB - Histochemical and immunohistological methods have been used to demonstrate the presence of nitric oxide synthase (NOS) in the intracardiac parasympathetic ganglion of bullfrog heart. Both NADPH-diaphorase staining and indirect immunofluorescence with polyclonal antibodies raised against the C-terminal regions of a cloned NOS from rat cerebellum show that the postganglionic, parasympathetic neurons in the intracardiac ganglion contain NOS. This suggests that nitric oxide may have a role in neuronal activity in frog heart. PMID- 7536316 TI - Carrier-mediated uptake and release of histamine by cultured rat cerebral endothelial cells. AB - The present study demonstrates that histamine could be taken up by and released from endothelial cells of brain capillaries. Incubation of cultured endothelial cells, with low (0.01-0.50 microM) concentrations of [3H]histamine, resulted in a rapid uptake of the amine. The uptake was saturable, Na(+)-dependent and yielded an apparent Km 0.3 +/- 0.02 microM and a Vmax 4.6 +/- 0.04 pmol/mg protein per min. After a 10-min incubation in a histamine-free medium, about 65% of [3H]histamine was released from the cells. Na(+)-deprivation and high K+, as well as the treatment of the cells with ouabain affected the release, resulting in significantly higher rates of the efflux. The ability of cerebral endothelial cells to take up histamine from both luminal and abluminal sides but to release it mainly luminally, may function as an important mechanism to protect the neural tissue from the harmful effects of this endogenous mediator of inflammation. PMID- 7536317 TI - Clinical evaluation of CYFRA 21-1 in malignant pleural fluids. AB - The diagnostic value of the novel tumor marker CYFRA 21-1 in malignant pleural fluid was assessed in comparison to carcinoembryonic antigen (CEA). CYFRA 21-1 and CEA were measured in pleural fluid obtained from patients with 108 malignant and benign diseases. The levels of pleural fluid CYFRA 21-1 in malignant diseases (median: 84.5 ng/ml) were statistically higher than those in benign diseases (median: 13.9 ng/ml; p < 0.01). The CYFRA 21-1 test was able to discriminate significantly between squamous cell lung cancer and pneumonia (p < 0.01), while pleural fluid CEA levels could not. Receiver operating characteristic (ROC) curve analysis showed that the CYFRA 21-1 test has an advantage over CEA because of its higher specificity. These results indicate that measurement of CYFRA 21-1 in pleural fluid is a new tool, in addition to cytologic examination, to discriminate between malignant and benign diseases. PMID- 7536318 TI - Recombinant human granulocyte colony-stimulating factor does not influence distribution or sister chromatid exchange frequency of proliferating mononuclear cells of the peripheral blood. AB - Phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) were treated with increasing concentrations of filgrastim, the unglycosylated methionine granulocyte colony-stimulating factor of man (rhG-CSF), and cultured for 72 h. There were no impaired proliferation or differentiation of proliferating PBMC, no impaired expression of activation markers such as the low affinity interleukin 2 receptor and transferrin receptor, and no induction of sister chromatid exchanges. Under these conditions, no effects of a general DNA destabilization of peripheral blood leukocytes was observed. Thus, longterm administration of therapeutical concentrations of rhG-CSF should not produce severe mutagenic effects. PMID- 7536319 TI - Lymphocyte-induced angiogenesis: effect of different antigenic stimuli. AB - It is known that tumor cells activate spleen cells to induce an angiogenic response. In this report we studied whether different antigenic stimuli, other than tumor cells, were able to activate spleen lymphocytes to induce angiogenesis in syngeneic combination (SLIA). For this purpose, mice were inoculated with sheep red blood cells (SRBC), allogeneic kidney and syngeneic fetal tissues. The effect of pregnancy (syngeneic or allogeneic) on the ability of spleen cells to induce a neovascular response was also assessed. None of the different stimuli were able to induce spleen lymphocytes to evoke angiogenesis. Although allogeneic lymphocytes from virgin females induced a strong neovascular response, the same population, but from allogeneic pregnant mice, did not evoke this response. We conclude that tumor cells seem to be the only antigenic stimuli able to activate spleen lymphocytes to induce SLIA. PMID- 7536320 TI - CMDBS, functional analogue of heparin sulfate as a new class of corneal ulcer healing agents. AB - Soluble dextran polymer derivatives (CMDBSs) are originally synthesized as heparin-like plasma substitutes. Some of them mimic heparin in its interactions and stabilize, protect and facilitate actions of heparin binding growth factors. The wound healing activity of one specific CMDBS was studied in a model of corneal ulcer on the rabbit eye and compared with the activity of basic fibroblast growth factors (bFGF) added alone or in association with CMDBS. Total reepithelization was observed with bFGF + CMDBS, bFGF alone and CMDBS alone after, respectively, 3.8 +/- 0.78, 4.3 +/- 0.67 and 4.4 +/- 0.51 days. All treatments were efficient if compared with eyes treated with saline (p < 0.0001). The grade of significance of the applied treatments was as follows: bFGF + CMDBS > bFGF > CMDBS > saline. Our study pinpoints that some specific CMDBS are as potent agents as bFGF for corneal ulcer healing, and can therefore be proposed for therapeutic use. PMID- 7536321 TI - Investigations on the role of flagella in adhesion of Pseudomonas aeruginosa to mouse and human corneal epithelial proteins. AB - Antiflagellar monoclonal antibodies (MAbs) were used in an overlay assay to determine whether flagella bind to blots of mouse and human corneal epithelial proteins (CEPs). The role of carbohydrates and surface charge was also explored by preincubation of blots with periodate or neuraminidase, or flagella with monosaccharides or charged compounds, respectively. Periodate slightly decreased binding of flagella, while sialic acid inhibited binding, and the effect was dose dependent. Neuraminidase treatment of blots or incubation of flagella with a negatively, but not a positively charged compound, also blocked binding. The data suggest that flagella interact with mouse and human CEPs by electrostatic mechanisms. PMID- 7536322 TI - Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini. AB - Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism. PMID- 7536324 TI - Simultaneous extraction of total RNA and peptides from tissues: application to tachykinins. AB - Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use. PMID- 7536323 TI - Effect of neurotensin and substance P on adrenal cortex regeneration. AB - The influence of neurotensin (NT) and substance P (SP) on the early stage of adrenal regeneration was investigated. Neurotensin (5 and 50 micrograms/kg) and SP (10 and 100 micrograms/kg) were given to rats subjected to adrenal enucleation combined with contralateral adrenalectomy. The mitotic index was employed to assess cell growth, and plasma corticosterone was determined by a radioimmunological assay. The animals were killed 4 and 8 days after operation. Neurotensin did not evoke any significant changes either in the proliferation ratio or corticosterone production of regenerating adrenal cortex. However, it was found that SP (100 micrograms/kg) stimulated both the proliferation and steroidogenesis on the eighth day after operation. PMID- 7536325 TI - Distribution and chromatographic analysis of galanin immunoreactivity in the heart. AB - The presence and distribution of the biologically active neuropeptide galanin (GAL), in the rat heart as well as in mouse, guinea pig, rabbit, cat, and dog heart, were analyzed. With some minor variations in the overall distribution, extractable GAL-like immunoreactivity (-LI) was present in all major portions of the heart. In the rat heart, GAL-immunoreactive (GAL-IR) nerve fibers were present in the atria as well as in the ventricles; thin GAL-IR fibers were present in the myocardium as well as around some cardiac blood vessels. A few larger GAL-IR nerve fiber bundles were also present on the surface of the heart. Characterization of extractable GAL-LI in the rat heart, using HPLC, revealed one GAL-IR form, coeluting with synthetic rat GAL. Our findings suggest that galanin is of importance in the control of certain cardiac functions and/or of circulation. PMID- 7536326 TI - Expression of cellular adhesion molecules on human prostate tumor cell lines. AB - By flow cytometric analysis we have examined the expression of cellular adhesion molecules (CAMs) on the surface of four different human prostate tumor cell lines: DU 145, from a brain metastasis; PC 3, from a bone metastasis; LNCaP.FGC, from a lymph node metastasis; and a primary tumor cell line, ND 1. The corresponding ligands for the expressed CAMs were, by and large, extracellular matrix proteins. We detected high-level expression of ICAM-1 on three of the four prostate cell lines, whereas LNCaP cells were negative. We observed unstable, heterogeneous expression of E-cadherin in the cell lines DU 145, PC 3, and ND 1. Flow cytometric cell sorting enabled us to divide PC 3 cells into negative and bright positive subpopulations but, after several cell divisions in culture, sorted cells returned to the original heterogeneous phenotype. The laminin specific alpha 6 beta 4 integrin was not expressed by LNCaP, and was expressed at a low level and heterogeneously on DU 145 and PC 3 cell lines. In contrast, ND 1 cells, derived from a primary tumor, showed homogeneous and high-level expression of the alpha 6 beta 4 integrin. All of the prostate cell lines expressed the RGD dependent binding of alpha 3 beta 1 and alpha 5 beta 1 integrins and did not reveal non-RGD-dependent alpha 4 beta 1 integrin expression. This finding provides a stimulus to investigate the inhibition capacity of RGD-containing peptides on the metastatic behavior of prostate tumor cells. PMID- 7536327 TI - Isolation of cDNAs that are differentially expressed between androgen-dependent and androgen-independent prostate carcinoma cells using differential display PCR. AB - In the development of prostate cancer there is an important transition from androgen-dependent growth (which can be treated) to androgen-independent growth (which is beyond medical control). This transition is probably accompanied by genetic changes, resulting in the activation of oncogenes or the inactivation of tumor suppressor genes. In the present manuscript, the isolation of genes that may be involved in advanced, androgen-independent prostate cancer growth is described. Using differential display PCR, 13 cDNAs were isolated representing genes that are differentially expressed between the androgen-dependent prostate carcinoma cell line LN-CaP and the androgen-independent prostate carcinoma cell lines PC-3 and DU 145. These clones were divided into four groups: androgen responsive genes (TL5, TL25, TL32, and TL35); genes with a marked decreased expression in one of the prostate cancer cell lines (TL27); genes with a marked, increased expression in one or more of the prostate cancer cell lines (TL4, TL16, TL21, and TL22); and genes with minor (but repeatable) changes in expression between prostate cancer cell lines (TL7, TL15, TL18, and TL33). The 13 genes were analyzed for their sequence information, tissue specificity, and androgen responsiveness in order to identify genes of interest. In summary, differential display PCR appears to provide an attractive alternative to existing molecular techniques to screen for differentially expressed genes in prostate cancer cells. PMID- 7536328 TI - The effect of helodermin in rat dispersed pancreatic acini. AB - Helodermin is a 35 amino acid-residue peptide of the vasoactive intestinal polypeptide (VIP) family, which was originally isolated from the venom of Heloderma suspectum on the basis of its capacity to stimulate adenylate cyclase in the rat pancreas. In the present study, using rat dispersed pancreatic acini, we examined the binding characteristics of helodermin, its action on amylase secretion, and the production of intracellular cyclic AMP (cAMP). Helodermin stimulated intracellular cAMP production dose dependently in a manner that was nearly identical to that of VIP. Helodermin stimulated amylase secretion dose dependently, showing an efficacy similar to that of VIP but with 100 times less potency than VIP. Adding 0.5 mM 3-isobutyl-1-methylxanthine increased the potency of helodermin's action on amylase secretion but did not change the efficacy. The binding study showed that the order of binding affinity to VIP receptors was VIP = helodermin > secretin, while the order of that to secretin receptors was secretin > helodermin > VIP. These results suggest that helodermin stimulated amylase secretion from rat dispersed pancreatic acini via VIP-preferring receptors that are coupled to the production of intracellular cAMP, but a part of the postreceptor mechanism for enzyme secretion is different from that of VIP. PMID- 7536330 TI - Lipase/amylase ratio: a review. PMID- 7536329 TI - Peptide YY inhibits exocrine pancreatic secretion in isolated perfused rat pancreas by Y1 receptors. AB - Peptide YY (PYY) inhibits exocrine pancreatic secretion in several species. Two receptors, Y1 and Y2, are known to mediate PYY actions. While PYY 1-36 binds equally to both receptor subtypes, a second endogenous form of PYY, PYY 3-36, selectively activates Y2 receptors. The importance of Y receptor subtypes for inhibition of exocrine pancreatic secretion by PYY is unknown. We studied the effects of PYY 1-36 on cholecystokinin octapeptide (CCK-8)-stimulated amylase secretion in an isolated perfused rat pancreas model. To characterize functionally the receptors involved we determined the effects of a Y1-selective agonist, [Pro34]PYY; a Y2 selective agonist, PYY 3-36; and neuropeptide Y (NPY) in this model. PYY 1-36 significantly inhibited stimulated amylase secretion in the denervated rat pancreas. [Pro34]PYY and NPY both inhibited exocrine pancreatic secretion as potently as PYY 1-36. Contrary to that, the Y2 selective agonist, PYY 3-36, was inactive. We conclude that PYY inhibits exocrine pancreatic secretion in this extrinsically denervated rat pancreas model by Y1 receptors. PMID- 7536331 TI - Change in vascular cAMP and cGMP contents in portal hypertensive rats. AB - The purpose of this study was to investigate the possible changes of cyclic nucleotide contents in portal hypertensive rats. Portal hypertension was induced by partial portal vein ligation (PVL) in Sprague-Dawley rats. Sham-operated rats served as controls. Hemodynamic and cyclic nucleotide measurements were performed at 14 days after surgery. The portal venous pressure was significantly higher, while systemic arterial pressure and heart rate were lower in PVL rats than those in controls. Basal cAMP (PVL, 10.91 +/- 0.98, vs. sham, 8.08 +/- 0.81 pmol/mg protein) and cGMP (PVL, 0.91 +/- 0.12, vs. sham, 0.59 +/- 0.05 pmol/mg protein) contents in the tail artery were significantly higher in PVL rats. Isobutyryl methylxanthine (10(-5) M), a nonspecific phosphodiesterase inhibitor, exerted similarly stimulating effects on the tissue cGMP (PVL, 158 +/- 10, vs. sham, 178 +/- 20%) and cGMP (295 +/- 28 vs. 316 +/- 71%) levels in both PVL and control rats; so did forskolin (10(-6) M) on the cAMP (184 +/- 20 vs. 197 +/- 66%) content in both groups. Our results showed that the arterial cAMP and cGMP contents were higher in PVL rats, which may contribute to the reduction of peripheral resistance in portal hypertension. PMID- 7536332 TI - The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes. AB - 5-Azacytidine (5-aza-C) analogs demonstrate a remarkable ability to induce heritable changes in gene and phenotypic expression. These cellular processes are associated with the demethylation of specific DNA sequences. On the other hand, 5 aza-C analogs have dramatic effects on chromosomes, leading to decondensation of chromatin structure, chromosomal instability and an advance in replication timing. Condensation inhibition of genetically inactive chromatin occurs when the DNA is still hemimethylated or fully methylated. In cell cultures prolonged for several replication cycles, chromosomal rearrangements and instability affect the 5-aza-C-sensitive regions. Moreover, the normally late-replicating inactive chromatin undergoes a transient temporal shift to an earlier DNA replication, characteristic of activatable chromatin. zThe induced alterations of chromosome structure and behavior may trigger the 5-aza-C-dependent process of cellular reprogramming. Apart from their differentiating and gene-modifying effects, 5-aza C analogs can tumorigenically transform cells and modulate their metastatic potential. High doses of 5-aza-C analogs have cytotoxic and antineoplastic activities. PMID- 7536333 TI - Primary carcinoma of the gall-bladder: potential for external radiation therapy. AB - Nineteen patients (14 women, 5 men) received external radiation therapy (ERT) between 1980 and 1988 for gall-bladder carcinoma. Eleven patients had complete resection (cholecystectomy in eight cases), six incomplete gross resection and two only percutaneous transhepatic biliary drainage (PTBD). The modalities of ERT were variable and doses ranged from 30 Gy/10 fractions to 50 Gy/25 fractions. Among 11 patients with complete resection (9/11 with T1 or T2 stages), overall survival was 55% at 48 months and 36% at 60 months, median survival was 48 months and at the time of this report 3/11 patients were alive with no evidence of disease, 54, 65, 76 months after surgery, and eight dead of cancer 8-114 months. Local control was achieved in 66 patients with T1 or T2 stages. All eight patients who had palliative surgery or PTBD died of cancer after 4-20 months with median survival of 6 months. Three complications were noted: one gastric ulcer in the course of ERT (surgical treatment), one duodenal ulcer which occurred 6 months after completion of ERT (medical treatment) and one regressive radiation hepatitis. From this experience it appears that ERT in gall-bladder carcinoma is well tolerated, can obtain local control and prolonged survival after complete resection and good palliation in non-resectable tumors. PMID- 7536334 TI - Immunohistochemical localization of peptidergic nerve fibers and neuropeptide receptors in Peyer's patches of the cat ileum. AB - This study examined the distribution of peptidergic nerve fibers in Peyer's patches to determine whether appropriate receptors were present. Vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and receptors for VIP and SP were localized in lymphoid follicles of the cat ileum using a combined indirect horseradish peroxidase and streptavidin biotin method. The margins of follicles were innervated by nerve fibers containing VIP, SP and CGRP. Nerve fibers were predominantly around lymphatics and high endothelial venules at the edges of follicles. Specific receptors for VIP and SP were present at the margins of follicles and in the lamina propria around crypts. VIP receptors were numerous on T cells within and around high endothelial venules and lymphatic vessels and at the margins of follicles. SP receptors were identified on a small number of T and B cells, granulocytes and macrophages, restricted to the margins of follicles. The defined distribution in ileal lymphoid tissue of nerve fibers containing VIP and SP and the corresponding localization of their appropriate receptors support immunoregulatory roles for neuropeptides in mucosal immunity. PMID- 7536335 TI - Inhibitory effect of vasoactive intestinal peptide (VIP) on phagocytosis in mouse peritoneal macrophages. AB - The effect of VIP on phagocytosis in peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that VIP suppressed phagocytosis in a dose-dependent manner. VIP(1-12) did not suppress phagocytosis. VIP(10-28) was more suppressive than VIP(1-28). A known VIP-antagonist (N-Ac-Tyr1,D-Phe2)-growth hormone-releasing factor (GRF) (1-29)-NH2 suppressed phagocytosis less than VIP. Control phagocytosis was partially suppressed in Ca(2+)-free solution. Phagocytosis was suppressed by VIP further in Ca(2+)-free solution than in the normal solution. Phagocytosis was suppressed in a known phosphodiesterase inhibitor IBMX-containing solution. The degree of suppression by VIP was the same in the presence or the absence of IBMX. These results suggest that VIP suppresses extracellular Ca(2+)-dependent and -independent phagocytosis, that the C-terminal fragment of VIP is essential for VIP action, that the suppression is mediated by cAMP and that the inhibition of macrophage phagocytosis by VIP is one of the mechanisms which modulates immune responses by the nervous system. PMID- 7536336 TI - Pharmacological study of gastrin-mediated amylase release in pancreatic acinar cells (AR4-2J). AB - In rat pancreatic acinar cells, amylase release and Ca2+ mobilization are related to the occupancy of CCKA receptor. The rat pancreatic acinar cell line (AR4-2J) possesses both CCKA (CCKA R) and CCKB (CCKB R) sub-type receptors. Using this cell line we attempted to determine the relative involvement of each sub-type in both amylase release and Ca2+ mobilization. For this purpose we used L 364718 a selective antagonist for CCKA R and PD 135158 a selective antagonist for CCKB R. We showed on AR4-2J cells that: a minority of CCKA R (Kd = 0.7 nM), a classical CCKB R (Kd = 0.93 nM) and a new high affinity gastrin binding site (Kd = 2.1 pM) coexisted; CCK through CCKA R and CCKB R, was more potent to stimulate amylase secretion (EC50 = 34 pM) and Ca2+ mobilization (EC50 = 30 pM) than to occupy its receptor. Gastrin induced a biphasic stimulation of amylase release. Gastrin through CCKB R was equally potent to stimulate amylase release (EC50 = 1.72 nM) and Ca2+ mobilization (EC50 = 3.1 nM), whereas through the high affinity gastrin binding site, gastrin-induced amylase release (EC50 = 0.73 pM) did not correlate with the Ca2+ mobilization (EC50 = 3.1 nM). These results demonstrated for the first time the existence, on AR4-2J cells, of a high affinity gastrin receptor whose occupation by gastrin induces amylase release. PMID- 7536337 TI - Captopril increases endothelin serum concentrations and preserves intestinal mucosa after mesenteric ischemia-reperfusion injury. AB - Endothelial cells modulate the tone of the underlying smooth muscle by generating endothelium-derived relaxing and constricting factors. Captopril (CPT), unlike other angiotensin-converting enzyme (ACE) inhibitors, contains a sulfhydryl (-SH) group and can act as a free radical scavenger. Iloprost (ILO) is a synthetic analogue of prostacyclin and mimics the effects of this compound. This study was designed to investigate the effect of ILO and CPT on the mechanism of endothelin (ET) release after mesenteric ischemia-reperfusion (I/R) injury in the rat. Sprague-Dawley rats were divided into five groups: sham-operated, control, ILO (25 micrograms/kg), CPT (10 micrograms/kg), and ILO + CPT. The superior mesenteric artery was occluded for 30 min and then allowed 90 min of reperfusion, except in the sham-operated group, and the corresponding agents were given to the treated groups prior to I/R injury. After I/R injury, portal venous blood was obtained for ET assay, and ileal tissue samples were also obtained for the determination of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) and for histopathological examination. MDA levels were significantly lower in the CPT, ILO and, ILO + CPT groups than in the control group, indicating the inhibition of lipid peroxidation in all groups. ET levels increased in the control group, and this increase was reversed with ILO. In the CPT group, ET levels were significantly increased, and the addition of ILO did not affect this increase. Significant cytopreservative effect was achieved with ILO and CPT, the latter being more prominent histopathologically. CPT exerts a significant protective effect on the intestinal mucosa after I/R injury. This protection is accomplished by increased ET levels and seems to be unrelated to its inhibitory effect on lipid peroxidation and also unrelated to the arachidonic acid cascade. PMID- 7536339 TI - [Sociomedical syndrome. Intervention strategies to manage a new public health problem]. AB - Medical and social services are confronted with the increasing demands that our ageing society presents. Some patients (specially geriatric, chronically disabled and oncologic patients) pose multiple needs of social and medical care that very often are not met in a coordinated and comprehensive way. In this work authors present case management as a tool to achieve a better organization of social and medical resources in accordance with the demands of this growing part of the population. PMID- 7536338 TI - [Simplified model of induction of experimental acute pancreatitis with a supra maximal dose of cerulein]. AB - The study was performed to compare an usual method of induction of acute experimental pancreatitis with a simplified, easier and faster induction through a subcutaneous and intravenous injection of cerulein, with good reproducibility as compared to the literature. Four groups were studied. In the group I, continuous three hour intravenous injection of 15 micrograms/Kg of cerulein, was given. Group II was a control group with saline infusion. Group III received a subcutaneous injection of 20 micrograms/Kg and an intravenous injection of 20 micrograms/Kg of cerulein one hour later. Group IV was the control group with saline. The results of biochemical measurements, such as tecidual trypsinogen, chimotrypsinogen, proelastase, cathepsin and serum amylase, showed no difference between the two methods. Histologic study revealed edematous pancreatitis in group I and III, with moderate acinar necrose in group III. These results suggest that the proposed simplified method induces enough acute and edematous pancreatitis to allow studies in physiopathology and therapeutics. PMID- 7536340 TI - Fighting all the time. PMID- 7536341 TI - Dendrites shed their dull image. PMID- 7536342 TI - Specific DNA-RNA hybrid binding by zinc finger proteins. AB - Zinc finger proteins of the Cys2His2 type represent a large class of proteins that have been assumed to function by means of specific interactions with DNA. Experiments motivated by structural characteristics of zinc finger protein-DNA complexes revealed that certain zinc finger proteins bound DNA-RNA hybrids with affinities comparable to or greater than those for DNA duplexes. The interactions between the zinc finger proteins and the DNA-RNA hybrids were dependent on which strand was RNA and were sequence-specific. Thus, interactions with DNA-RNA hybrids should be considered with regard to the biological roles of zinc finger proteins. PMID- 7536343 TI - FAP-1: a protein tyrosine phosphatase that associates with Fas. AB - Fas is a cell surface receptor that controls a poorly understood signal transduction pathway that leads to cell death by means of apoptosis. A protein tyrosine phosphatase, FAP-1, capable of interacting with the cytosolic domain of Fas, was identified. The carboxyl terminal 15 amino acids of Fas are necessary and sufficient for interaction with FAP-1. FAP-1 expression is highest in tissues and cell lines that are relatively resistant to Fas-mediated cytotoxicity. Gene transfer-mediated elevations in FAP-1 partially abolished Fas-induced apoptosis in a T cell line. These findings are consistent with an inhibitory effect of FAP 1 on Fas signal transduction. PMID- 7536344 TI - Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. AB - The ribosome scanning model predicts that eukaryotic ribosomal 40S subunits enter all messenger RNAs at their 5' ends. Here, it is reported that eukaryotic ribosomes can initiate translation on circular RNAs, but only if the RNAs contain internal ribosome entry site elements. Long-repeating polypeptide chains were synthesized from RNA circles with continuous open reading frames. These results indicate that ribosomes can translate such RNA circles for multiple consecutive rounds and that the free 5' end of a messenger RNA is not necessarily the entry point for 40S subunits. PMID- 7536345 TI - FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides. AB - Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity. PMID- 7536347 TI - Pseudoleukemia after granulocyte colony-stimulating factor therapy. AB - Therapy with myeloid colony-stimulating factors has been safely and effectively used in a wide variety of situations associated with neutropenia. We present a case of pseudoleukemia occurring in a patient with lymphoma and pancytopenia after 2 days of treatment with granulocyte colony-stimulating factor (G-CSF). Bone marrow aspirate and flow cytometry study results were consistent with acute myelomonocytic leukemia but were normal after G-CSF was discontinued for 4 days. As previous phase I studies of bone marrow morphology after G-CSF use have not described the extreme myeloid immaturity seen in this patient, it seems likely that the action of G-CSF was enhanced by factors associated with the patient's illness. We emphasize the clinical importance of this case in light of the widespread use of G-CSF. PMID- 7536348 TI - Screening program for prostate cancer. PMID- 7536346 TI - Biliary sludging in critically ill trauma patients. AB - We used serial weekly ultrasonography to prospectively screen 19 critically ill trauma victims for the development of biliary sludge. Fourteen patients had sludge formation during their hospitalization. Sludge development was positively associated with increased transfusion requirements, but not with any other laboratory or clinical findings, including injury severity scores. The enteral feedings administered to most patients did not prevent sludge formation in the majority of cases; all five patients receiving total parenteral nutrition had sludge. Three patients had complications that could possibly be attributed to the sludge (one case of acalculous cholecystitis and two cases of mild pancreatitis). No such problems occurred in the five patients who did not have sludge. No long term clinical problems related to sludge have occurred. We conclude that gallbladder sludge frequently develops in critically ill trauma patients and that sludge may be associated with pancreatobiliary complications. PMID- 7536350 TI - [Prostate-specific antigen in patients with benign prostatic hyperplasia]. AB - The authors examined 119 patients with the histological finding of benign prostatic hyperplasia (BPH). Before the planned operation of the prostate (transurethral resection or open prostatectomy) the authors examined in all patients the level of the prostate specific antigen (PSA) and the prostate volume by sonography. Of 119 patients 70 (58.8%) had elevated serum levels of PSA and in 35 (29.4%) the PSA level was higher than 10 ng/ml. The authors demonstrated a highly significant (p < 0.0001) linear relationship of the PSA serum level and volume of prostate in patients with BPH. Based on the results of simple regression analysis they elaborate an equation for the prostate volume: PSA ng/ml = (3.26 + 0.12 x volume of prostate (ml) +/- 8.44). By adding the value of the standard deviation of PSA the authors created an interval in which are most of the patients with BPH. The authors recommend supplementary examinations (transrectal sonography and biopsy of the prostate) in patients where there is a disproportion between the PSA level and the prostate volume. PMID- 7536349 TI - [Viral hepatitis C--a problem in the treatment of children with renal insufficiency on hemodialysis]. AB - The aim of the study was to assess the frequency of positive anti HCV in the haemodialysis unit (staff and patients) of the University Children's Hospital in Belgrade. Twenty four patients, 14 girls and 10 boys, on chronic haemodialysis from 1 to 164 months, and ten staff members who worked for an average of 9.3 years in the haemodialysis unit, were examined. Patients were screened for HCV antibody in November 1990 (15 pts), July 1992 (14 pts) and September 1992 (13 pts) using the Hepatitis C virus (HCV) 1st generation test for the first check up, and 2nd generation tests for the following ones. Five patients were tested 3 times and 8 twice. Staff members were tested in September 1992 by the HCV 2nd generation test. Complete screening for Hepatitis B virus (HBV) was done by ELISA at the same time as the tests for HCV antibody. All patients and staff members were Hbs Ag negative. Eleven patients and 9 staff members had one or more HBV antibodies due to HBV infection or HBV vaccine. None of staff members had HCV antibody while overall prevalence in patients was high, reaching 50%. It raised with duration of dialysis, and was more frequent in polytransfused and/or patients with positive HBV infection anamnesis. PMID- 7536351 TI - [Levels of prostate-specific antigen, acid phosphatase, alkaline phosphatase and pain in the prediction of bone metastases in patients with newly diagnosed prostatic carcinoma]. AB - In a retrospective analytical study the authors evaluated in 86 patients, mean age 69 years (range 55-85 years), with a newly diagnosed untreated prostate carcinoma the sensitivity, specificity, positive and negative predictive value of specific prostatic antigen (PSA), acid phosphatase (AP), alkaline phosphatase (AP') and pain in relation to possible affection of bones by secondaries. The authors found a highly negative predictive value for assessment of bone metastases when PSA values were lower than 10 ng/ml (96%), at levels under 20 ng/ml (94%) and a highly positive predictive value at levels higher than 50 ng/ml (97%). When AP and AP' are negative and there is no pain the occurrence of secondaries is of low probability. These results make it possible to differentiate some patients where scintigraphy of the skeleton is not inevitable. This procedure can be applied above all in patients where radical prostatectomy is not indicated. PMID- 7536352 TI - [Electrophoresis of 1,149 samples of umbilical cord blood and assessment of the heterogeneity of the relationship between gamma G and gamma A chains of fetal hemoglobin]. AB - A total number of 1,149 specimens of cord-blood were subjected to cellulose acetate gel electrophoresis in order to estimate the incidence of haemoglobinopathies in newborn infants (NBI). It was found that 4.37% of black NBI and 0.54% of white NBI were AS carriers. Those samples with HbS or HbC were verified by means of agar-citrate electrophoresis at pH 6.2. Haemoglobin Bart was commonest in black (3.1%) than in white (0.4%) NBI. A slow haemoglobin variant was found with an alpha-chain mutation. The concentration of gamma G was estimated by separation of gamma G and gamma A chains with polyacrylamide gel electrophoresis, and it ranged between 52% and 94%, which could be attributed to the fact that the shift from HbF to HbA is not simultaneous in all the red-cell population. PMID- 7536353 TI - Antigenic specificities of "antiphospholipid" autoantibodies. PMID- 7536354 TI - A P-selectin-immunoglobulin G chimera is protective in a rabbit ear model of ischemia-reperfusion. AB - BACKGROUND: Neutrophils have been shown to play a role in ischemia-reperfusion injury, and the initial interaction of neutrophils with the endothelium is mediated through the selectin family of adhesion molecules. Thus the purpose of these studies was to determine whether a P-selectin-IgG chimera was protective in a model of ischemia-reperfusion injury. METHODS: The model used was a rabbit ear model of ischemia-reperfusion. Selectin-IgG chimeras were given at the time of reperfusion of the tissue, and their efficacy was compared with an anti-CD18 antibody (MHM23). RESULTS: The P-selectin-IgG was as protective in this model as an anti-CD18 antibody. The chimera did not mediate its effect by causing the animals to become neutropenic. CONCLUSIONS: P-selectin plays a role in ischemia reperfusion injury. This is in agreement with data from other groups. The fact that the chimera was effective in this model suggests that carbohydrates or small molecule mimics of carbohydrates would be effective in this model. Such antiinflammatory agents may have fewer side effects in terms of increased risk of sepsis. PMID- 7536355 TI - Cytokeratin staining reveals micrometastasis in lymph nodes of early gastric cancer. PMID- 7536356 TI - Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'-deoxycytidine. AB - DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5-aza-2'-deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose-related accumulation of cells in the S and G2/M phases was observed at 4 and 8 hr after maternal dosing. S phase accumulation was the most sensitive indicator of effect; a dose-related increase in the percentage of hindlimb bud cells in S-phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication-associated cytotoxicity, at doses of > or = 0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of > or = 0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536357 TI - 24th Annual meeting of the German Society for Thoracic and Cardiovascular Surgery. Bonn, Germany, February 15-18, 1995. Abstracts. PMID- 7536358 TI - German Society for Thoracic and Cardiovascular Surgery, membership list. PMID- 7536359 TI - [Lymph node cytology]. AB - The cytological preparation of a lymph node is a relatively simple technique. A fine needle aspirate is smeared as a blood sample and stained with a hematological method, Wright-stain or Diff-Quik. The microscopical examination can be completed in a few minutes. The cytology of a lymph node is a valuable diagnostical step for the classification of a lymphadenopathy. Reactive hyperplasia, forms of lymphadenitis, primary or metastatic neoplasias, microorganisms and parasites can be diagnosed. PMID- 7536360 TI - Role of tumor necrosis factor-alpha in cadmium-induced hepatotoxicity. AB - Liver and kidney injury following acute or chronic exposure to cadmium is well characterized. While hepatocytes and endothelial cells of the sinusoids are thought to be the primary cellular targets in the liver, ultrastructural changes may vary depending upon the exposure regimen and the time following administration. Since acute and chronic liver disease is often associated with the presence of cytokines, we investigated the role of proinflammatory cytokines in cadmium-induced hepatotoxicity. Supernatants from cultured liver slices obtained from acute or subchronic cadmium-exposed rats and mice were collected and cytokine secretion was examined. In addition, mRNA transcripts for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, MIP-2, IFN-gamma, and ICAM-1 from livers of treated mice were quantitated by reverse transcription-polymerase chain reaction. Modest increases in secretion of TNF-alpha, IL-1 alpha, and IL-6 were observed in response to cadmium which were enhanced in LPS-primed mice. Additionally, cadmium exposure increased IL-1 alpha, IL-1 beta, TNF-alpha, MIP-2, IL-6, and ICAM-1 mRNA transcripts in the liver. Immunohistochemical analysis revealed that TNF-alpha was associated with nonparenchymal cells in livers of cadmium-treated mice. Cadmium exposure produced a marked increase in plasma hepatocellular enzyme levels (i.e., AST, LDH, SDH), acute phase proteins (i.e., serum amyloid A), and foci formation in the liver, while focal inflammation and serum amyloid A (SAA) secretion, but not plasma enzymes, were further increased in cadmium-exposed mice primed with LPS. SAA secretion and focal inflammation were prevented by pretreatment with antibodies to TNF-alpha, indicating that these pathological manifestations are cytokine dependent. These data indicate that TNF-alpha, released from nonparenchymal cells as well as associated cytokines, are responsible for certain manifestations observed with cadmium-induced hepatotoxicity. PMID- 7536361 TI - Sidestream smoke effects on lung morphology and C-fibers in young guinea pigs. AB - Children raised in homes with smokers have more frequent respiratory symptoms, decreased lung function, and increased airway reactivity. This study was designed to evaluate whether chronic exposure of developing guinea pigs to sidestream smoke (SS) would impair lung function and morphology and/or change the activity of a pulmonary defense mechanism, the local bronchopulmonary C-fiber system. Duncan-Hartley guinea pigs (n = 29) were exposed to filtered air (FA) or to SS for 6 h/day, 5 days/week from 8 to about 43 days of life. Their lungs were then studied in an isolated buffer perfused system where increasing doses of capsaicin (a C-fiber stimulant) or substance P (SP, a C-fiber neurotransmitter) were injected into the pulmonary artery. SS exposure significantly increased baseline dynamic compliance (Cdyn) by 17% but did not change baseline pulmonary resistance (RL). SS exposure reduced the capsaicin-induced change in RL and Cdyn but did not change lung responsiveness to SP. SS exposure did not change fixed lung volume, surface area, mean linear intercept length, or elastin deposition. We conclude that SS exposure to developing guinea pigs (1) increased lung compliance without affecting alveolar size or elastin deposition and (2) decreased the airway reactivity of the C-fiber system without changing reactivity to one of its neurotransmitters, SP. If humans are similarly affected, children raised in the homes of smokers may have a diminished pulmonary defense mechanism. PMID- 7536362 TI - Documentation of coomassie-stained protein gels using a UV transilluminator. PMID- 7536363 TI - In situ hybridization and beta-galactosidase: a powerful combination for analysing transgenic mice. PMID- 7536366 TI - Prostate-specific antigen variability in men without prostate cancer: effect of sampling interval on prostate-specific antigen velocity. AB - OBJECTIVES: To evaluate short-term and long-term variability between prostate specific antigen (PSA) measurements to determine the most appropriate PSA sampling interval and rate of PSA change (PSA velocity) to distinguish between men with and without prostate cancer. METHODS: Retrospective study of PSA variability and PSA velocity in three groups of men without a diagnosis of prostate cancer and PSA levels less than 10 ng/mL: 56 men with a histologic diagnosis of benign prostatic hyperplasia (BPH; histologic BPH group) and 527 men with no history of cancer (noncancer group) who were part of the Baltimore Longitudinal Study of Aging and had PSA sampled at 2-year intervals (long-term), and 223 men with a clinical diagnosis of BPH (clinical BPH group) who had PSA sampled at 3-month intervals (short-term). PSA variability (deviation between consecutive measurements) and PSA velocity based on both two consecutive measurements and three consecutive measurements (average velocity) were calculated for each study group. RESULTS: PSA velocity is the deviation in PSA measurements relative to the elapsed time between the measurements. Because the variability in PSA between measurements was similar for the groups, the major factors that influenced PSA velocity were the sampling interval between PSA measurements, and to a lesser extent, the number of repeat PSA measurements. The 99th percentile for PSA velocity was 0.7 (histologic BPH group) and 0.75 ng/mL per year for the noncancer group when three measurements with a 24-month PSA sampling interval were used. However, the 99th percentile for PSA velocity was 5.8 and 2.4 ng/mL per year when three measurements with 3-month and 6-month PSA sampling intervals were used. Using three measurements, the percentage of subjects with a PSA velocity more than 0.75 ng/mL per year was 1% for the groups with a 24-month PSA sampling interval and 28% and 17% for 3-month and 6-month PSA sampling intervals, respectively. The 99th percentile for PSA velocity and the percentage of subjects with a PSA velocity more than 0.75 ng/mL per year was higher using two measurements compared to three measurements regardless of PSA sampling interval. CONCLUSIONS: PSA velocity is inversely related to the interval between PSA measurements. A PSA velocity more than 0.75 ng/mL per year is useful in distinguishing between men with and without prostate cancer when: (1) velocity is based on three consecutive measurements; and (2) PSA is sampled long-term (2 years) but not short-term (3 to 6 months). PMID- 7536364 TI - Finding the hairpin in the haystack: searching for RNA motifs. AB - A growing list of examples underscores the roles that regulatory RNA motifs play in controlling the genetic repertoire of cells and developing organisms. Once either an RNA-processing signal, a ribozyme, an element that controls translational or mRNA stability or an RNA localization signal has been identified, it is important to search for other RNA sequences that bear similar regulatory signals. While DNA regulatory elements can often be described by a consensus sequence, RNA signals are frequently composed of a combination of sequence and structure motifs. Here, we discuss the approaches that can be used to identify RNA motifs by searching databases. PMID- 7536367 TI - A rapid and simple method for the detection of prostate-specific antigen mRNA in archival tissue specimens using a reverse transcription-polymerase chain reaction assay. AB - OBJECTIVES: Polymerase chain reaction (PCR) amplification of DNA from archival, fixed tissue sources is now performed routinely. In this report, we describe a reproducible technique for mRNA amplification from archival tissues. METHODS: Archival, fixed tissue was treated with a modification of a rapid, acid guanidinium technique. The RNA yielded was reverse transcribed and subjected to 40 cycles of two-step PCR, using a panel of primers designed to encompass relatively short (less than 250 bp) cDNA fragments. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. RESULTS: Using fixed, paraffin-embedded specimens of human prostate, we have demonstrated a reproducible pattern of reverse transcription (RT)-PCR products using several different primer sets. Our data suggest that RNA degradation proceeds to fragments approximately 250 bp or shorter in length but that many of these fragments survive intact over extended periods of time and are of suitable quality to serve as a template for RT and subsequent PCR amplification. CONCLUSIONS: We describe a technique that will allow the retrospective analysis of archival tissues for gene expression. These methods are generally applicable to a wide variety of systems. Such a method will make it possible to perform retrospective studies of gene expression in the archival tissues of patients whose eventual clinical course is already known, greatly shortening the time needed for genetic outcome studies in slowly growing tumors, such as prostate cancer. PMID- 7536365 TI - Transurethral electrovaporization of the prostate: a novel method for treating men with benign prostatic hyperplasia. AB - OBJECTIVES: To determine the preliminary safety and efficacy of transurethral vaporization of the prostate (TVP) using the VaporTrode as a therapeutic alternative in the management of men with bladder outlet obstruction. METHODS: Twenty-five men (mean age, 63.5 +/- 3.1 years) with mild to moderate symptoms of prostatism underwent TVP since August 1994. Patients were assessed at baseline for both safety and efficacy and in follow-up at 1 week and 1 and 3 months. Efficacy parameters evaluated included operative time (in minutes), change in hematocrit and serum sodium, postoperative catheterization time, American Urological Association symptom score, peak uroflow (Qmax) and postvoid residual urine. Safety parameters evaluated included incidence of side effects, changes in serum sodium and hematocrit, and evaluation of sexual function. RESULTS: Symptoms decreased from 17.8 to 5.9 and 4.2 at 1 and 3 months, respectively (P < 0.01). Qmax increased from 7.4 to 15.3 and 17.3 mL/s at 1 and 3 months, respectively (P < 0.02). Mean operative time was 40.3 minutes; mean interval to catheter removal was 14.6 hours. Changes in serum parameters included a 0.9 mL/dL decrease in hematocrit and a 1.1 mEq/L change in sodium. Complications of the procedure included mild hematuria (n = 3) and distal bulbar urethral stricture (n = 1). There were no associated significant postprocedure irritative symptoms and no patient required recatheterization. CONCLUSIONS: TVP is a new and potentially useful modification of performing transurethral resection of the prostate. In this preliminary study, there has been significant clinical improvement maintained with minimal morbidity. This early clinical experience highlights several potential advantages of TVP, including significantly lower cost and minimal postoperative irritative symptom score. Currently, a multicenter clinical trial is under way to determine the long-term efficacy and safety of TVP. PMID- 7536369 TI - Antiandrogen withdrawal syndrome in prostate cancer after treatment with steroidal antiandrogen chlormadinone acetate. AB - OBJECTIVES: A case report is presented of 2 patients whose levels of serum prostate-specific antigen (PSA) improved after the withdrawal of a steroidal antiandrogen. METHODS: Two cases with prostate cancer had been treated with surgical castration and the steroidal antiandrogen chlormadinone acetate (CMA), and, on disease progression, the administration of CMA was terminated. RESULTS: Following withdrawal of CMA, a fall in PSA levels and remarkable clinical improvement were observed in both cases. One patient revealed a decrease and the other an increase in serum prostate acid phosphatase after the discontinuation of CMA. Serum levels of testosterone, prolactin, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione were not significantly elevated after CMA withdrawal. CONCLUSIONS: Withdrawal of the steroidal antiandrogen CMA resulted in a decline in PSA levels and clinical improvement in prostate cancer patients with disease progression. Changes in testosterone, prolactin, or adrenal androgens were not a cause of the antiandrogen withdrawal syndrome. PMID- 7536368 TI - Clear cell adenocarcinoma of the female urethra showing positive staining with antibodies to prostate-specific antigen and prostatic acid phosphatase. AB - A case of clear cell adenocarcinoma arising from the female urethra is described. Histologically, solid and glandular areas consisted of clear cells. The tumor cells stained positively with antibodies to prostate-specific antigen and prostatic acid phosphatase, suggesting that the clear cell adenocarcinoma arises from the female paraurethral duct, rather than embryonic remnants. PMID- 7536371 TI - [Treatment of adult acute lymphatic leukemia with polychemotherapy supported by the leukocyte growth factor G-CSF]. AB - The treatment results in 13 patients with acute lymphatic leukemia are reported. All patients were treated according to the Hoelzer protocol. Granulocyte colony stimulating factor (G-CSF) was administered till the onset of neutropenia and was continued as long as the leukocyte number increased above 3 x 10(9)/l. In phase I of Hoelzer's protocol G-CSF was administered on the average for 12 days, in phase II for 15 days. The chemotherapy of phase I was administered in scheduled time to 11 patients. In phase II the average delay against the scheduled time was 14 days. Reductions of dose were made only in 2 person. After finishing phase II 9 patients were in complete remission. Three patients died during therapy, one patient with small response died one month after completing phase II therapy. We believe that the administration of G-CSF only at the onset of leukopenia also abbreviate its duration as if the application were started immediately with the chemotherapy. PMID- 7536370 TI - Comparison of an antigen capture enzyme-linked assay with reverse transcription- polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections. AB - A study to compare the merits of three different tests for the diagnosis of ruminant pestivirus infections was carried out. Sensitivity studies using reference strains of bovine viral diarrhoea virus (BVDV) and buffy coat samples from persistently infected (PI) carriers showed the reverse transcription polymerase chain reaction (RT-PCR) had a greater sensitivity than the other tests. The antigen capture enzyme-linked immunosorbent assay (ELISA) was least sensitive and could only be used on samples containing cells (tissue or blood). When 169 clinical samples were examined, the RT-PCR detected the most positives (42) compared to the ELISA (32) and the immunoperoxidase test (IPT) (20). The RT PCR was more successful when specific antibody was also present in the sample. The lower sensitivity of the IPT was related to the use of a 1 passage (4-day) test and the testing of toxic or contaminated samples. The ELISA was found to be most suitable for large-scale testing for the diagnosis and control of pestivirus infections. PMID- 7536372 TI - [Biliary-duodenal prosthesis in the palliative treatment of tumors in the subhepatic region]. AB - In the group of 347 patients (pts) with biliary cancer (218 females and 129 males, age range 42-88 years), the large size bilio-duodenal endoprostheses (BDE) were inserted. The success rate in BDE implantation varied from 68 to 92% according to the localisation of the common bile duct stenosis and its anatomy. The BDE introduction was followed by step-by-step normalisation in laboratory markers of biliary obstruction in 76% of pts, the full functional capacity of BDE varied from 26 to 311 days (median 168 days). In pts treated by BDE the survival median was 210 days. Early complications did not exceed 2.2%, late complications were observed in 8% out of cases. The 30-day mortality was 1.7%. PMID- 7536374 TI - [Change in interferon status in patients with acute viral hepatitis during intramuscular and rectal administration of genetically engineered alpha-2 interferon]. AB - Viferon500 suppositories representing a combination of recombinant alpha 2 interferon and membrane-stabilizing agents were used in the treatment of adult patients with acute viral hepatitis B. Changes in interferon status of the blood under the effect of reaferon and viferon500 were studied. Both reaferon and viferon500 suppositories were found to stimulate interferon synthesis in patients with acute viral hepatitis B, their stimulating effect correlating with the positive time course of clinical manifestations of the disease. The results permit us recommend viferon500 for the treatment of viral hepatitis in the early period of the icterus in order to prevent the disease progress. Parameters of the interferon system may serve as an objective criterion for assessment of the treatment efficacy. PMID- 7536375 TI - [Experiences in the treatment of far advanced primary breast cancers, extensive tumor recurrences and radiation damage (reflections on on the value of surgical therapy)]. AB - Ulcerating primary breast cancer, local recurrence with extension to the thoracic wall and skin and ulceration after radiation therapy, sometimes with malignant degeneration, call for interdisciplinary evaluation and treatment. For the benefit of the patient an effective cooperation of the involved specialties seems desirable at an early stage of management. We performed breast tumor resection and immediate coverage of the defect in 25 patients (median age 54 years). In choosing the reconstruction procedure great attention was paid to short hospitalization and the possibility of commencing cytostatic or radiation therapy as soon as possible. We are able in all cases to resect the open and partly infected, ulcerated and bleeding tumors and to close the defects completely by means of various flaps. Among the many flaps at our disposal to reconstruct the thoracic well defects, we prefer the cranial based, ipsi- or contralateral extended rectus abdominis flap. PMID- 7536373 TI - [Study of certain indicators of immunity upon infecting CBA/Calac line mice with Lassa virus]. AB - Some immunity parameters (interferon, tumor necrosis factor, interleukin 1, etc.) were studied in CBA/Calac mice infected with Lassa virus. The results permit a hypothesis that a pathologic inflammatory reaction is responsible for the death of animals in experimental Lassa fever. One of the components of this reaction is endogenous shock involving a manifest production of immune response mediators, such as interferon, interleukin 1. and tumor necrosis factor. PMID- 7536376 TI - Fixation-dependent vimentin immunoreactivity of mono- and polyclonal antibodies in brain tissue of cattle, rabbits, rats and mice. AB - The immunohistochemical staining of vimentin in paraffin-embedded sections from adult cattle, rabbit, rat and mouse brain fixed in different fixatives (formaldehyde, methacarn, ethanol) was examined using two monoclonal antibodies and a polyclonal antiserum. In non-trypsinized formaldehyde-fixed tissue sections both monoclonal antibodies and the polyclonal antibodies failed to stain vimentin. Following trypsinization of formaldehyde-fixed sections of the four species the meninges, endothelial cells of blood vessels, ependymal cells and the stroma of the choroid plexus were labelled by the monoclonal and polyclonal antibodies used. Astrocytes and Bergmann glial fibers in pretrypsinized formaldehyde-fixed sections from cattle, rabbit and rat brain, however, showed only weak staining. Fixation of cattle and rat brain in methacarn markedly improved the vimentin immunoreactivity of astrocytes and Bergmann glial fibers. The best fixative for the preservation of immunoreactive determinants of vimentin in astrocytes and Bergmann glial fibers in cattle, rabbit and rat brain was ethanol. In brain tissue from mice both monoclonal antibodies labelled only mesoderm-derived tissue components, but did not recognize vimentin in astrocytes and Bergmann glial fibers. Pre-heating formaldehyde-fixed sections from cattle, rabbit and rat brain in a microwave oven prior to the immunohistochemical reaction resulted in an enormous enhancement of vimentin staining of mesoderm derived tissues, of astrocytes and bergmann cell fibers. PMID- 7536377 TI - Peptidergic innervation of the human testis and epididymis. AB - Neuropeptidergic innervation of the human testis and epididymis was investigated by immunohistochemical methods. The innervation of the epididymis was more dense than that of testis. In the testis only tyrosine hydroxylase- and neuropeptide Y positive nerves could be found between seminiferous tubules and around blood vessels. In the connective tissue capsule of the testis also small calcitonin gene-related peptide- and metenkephalin-containing nerve fibres were seen. The epididymis was densely innervated by nerve fibres immunoreactive to tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, calcitonin gene related peptide, galanin, peptide histidine isoleusine and substance P. PMID- 7536378 TI - Modulation of pan-cadherin expression in alveolar epithelial cells of mini pigs with pulmonary fibrosis. AB - The expression of pan-cadherin was investigated immunohistochemically in normal and irradiated mini pig lungs. Using a double labelling immunofluorescence technique, pan-cadherin expression was also compared with that of cytokeratin 18, which is selectively present in type II cells. In untreated animals pan-cadherin was detected in type I pneumocytes, in alveolar macrophages and in endothelial cells of larger blood vessels. Radiation caused increased pan-cadherin immunoreactivity of bronchial epithelial cells and interstitial cells. In addition, pan-cadherin expression was induced in type II pneumocytes. These changes in the distribution of epithelial cadherin molecules may implicate a role of cadherins for epithelial remodeling in response to radiation-induced lung injury. PMID- 7536379 TI - Mediator difference in contractions between trachea and bronchus of guinea pig induced by stimulation of C-fibers in vitro. AB - In trachea and bronchus of guinea pig in vitro, electric field stimulation (EFS) induced a rapid contraction (phase I) followed by a long-lasting contraction (phase II). The pretreatments of chlorphenamine (Chl) 1 mumol.L-1 and disodium cromoglycate (Cro) 10 mumol.L-1 reduced the tracheal contraction of phase II from 49 +/- 23 mg and 34 +/- 18 mg to 27 +/- 21 and 18 +/- 12 mg, respectively. The contractile responses of the tracheae to increasing concentrations of substance P (SP) 0.1-3.0 mumol.L-1 were reduced by the pretreatment of Cro 10 mumol.L-1 (P < 0.01). On the contrary, the contractile responses of the bronchi were not inhibited by Cro or Chl but were inhibited by pretreatment of atropine 1 mumol.L 1 from 61 +/- 36 mg to 36 +/- 15 mg. These results show that there are different mechanisms in the EFS-induced contractions between the trachea and bronchus; that different mediators amplify the phase II contractions. PMID- 7536380 TI - [Effect of digital rectal examination on PSA]. AB - A prospective randomized study on 100 patients was conducted to determine the effect of the rectal digital examination on prostate specific antigen (PSA) serum concentration. The control group consisted of 50 patient, all with two PSA determinations with no urological manipulation between them. The study group had 50 patient, who underwent digital examination 24 hours before the second PSA determination. Mean difference in PSA concentration in the study group is 0.84 ng/ml, compared to 0.0048 ng/ml in the control group (p << 0.05). Only 7 patients with initial PSA within the normal range (0.0-0.4 ng/ml) had a second PSA higher than 4.0 ng/ml. Only a patient with initial PSA lower than 4.1 ng/ml, had a second PSA higher than 10.1 ng/ml. This minimal change is independent of the initial concentration and the diagnosis (benign prostate hyperplasia, prostate adenocarcinoma or chronic prostatitis). Though changes are statistically significant they are not clinically so because the 0.84 increase is irrelevant. Based on these data we believe rectal digital examination has no effect on PSA serum levels. PMID- 7536381 TI - Cicaprost inhibits collagen-induced platelet accumulation in rat lungs for some hours. AB - A new method is introduced admitting of direct quantification of collagen-induced platelet trapping in the rat lung. The synthetic PG1(2)-mimetics, Iloprost and Cicaprost, are capable of inhibiting the trapping of platelets induced by collagen. The described method has proved to be suited for performing both pharmacodynamic and effectkinetic investigations with inhibitors of collagen induced platelet trapping. PMID- 7536382 TI - The endothelial cell nitric oxide synthase: is it really constitutively expressed? AB - During the past two years, the enzyme responsible for production of endothelium derived nitric oxide, the endothelial cell NO synthase (ecNOS) has been cloned and the gene encoding this enzyme isolated, cloned and its structure characterized. This research has provided direction for a variety of studies of regulation of the ecNOS. Several features of the ecNOS are compatible with a constitutively expressed, poorly regulated gene, including absence of a TATA box and numerous SP-1 sites. The promoter also contains a number of putative binding domains which suggest that it may be regulated by a variety of transcription factor mediated signals. In this review we will discuss evidence to support the concept that the ecNOS is a constitutively expressed gene subject to a modest degree of regulation by important physiological influences. PMID- 7536383 TI - A neutrophil-derived NO-synthase (NOS) inhibitor. AB - In the present work we have demonstrated that the low NOS activity of circulating blood neutrophils is related to an endogenous inhibitory factor. This factor inhibits constitutive NOS (cNOS) of cerebellum and inducible NOS (iNOS) of macrophages in a concentration-dependent manner. Boiling only partially diminished its activity. The inhibition of cNOS was specific, since to some degree NADPH (0.5-4 mM) and more effectively L-arginine (0.1-1 mM), but not D arginine, reversed the inhibition. PMID- 7536384 TI - Vasodilator effects of PGE1 in the coronary and systemic circulation of the rat are mediated by ATP-sensitive potassium (K+) channels. AB - This study was undertaken to investigate the possible involvement of K+ channels in PGE1-mediated vasodilatation. The increase in coronary flow elicited by PGE1 in isolated working rat hearts was attenuated by phentolamine and glibenclamide, inhibitors of ATP-regulated K+ channels, whereas apamin and charybdotoxin, inhibitors of calcium-activated K+ channels, were ineffective. In the anaesthetized rat, the duration of the hypotensive action of PGE1 was markedly attenuated by glibenclamide. It is concluded that the vasodilatory action of PGE1 in the coronary and systemic circulation of the rat is, at least in part, mediated via an opening of ATP-sensitive K+ channels. PMID- 7536385 TI - Diminished inhibition of adhesion molecule expression in prostacyclin receptor desensitized human platelets. AB - Long-term exposure of platelets to prostacyclin or iloprost (100nM, 3hr) results in receptor desensitization measured as decrease in 3H-iloprost binding sites by 47 +/- 14%. Desensitized platelets respond with an increased adhesion to endothelial cells. The mechanism of increased adhesiveness was studied by measuring the expression of the adhesion molecule CD62p (p-selectin; GMP140) on washed human platelets by flowcytometry. In thrombin stimulated platelets CD62p expression was dose-dependently reduced by iloprost. In receptor desensitized platelets IC50 for iloprost inhibition of thrombin-induced CD62p expression increased from 0.48 +/- 0.10 to 2.4 +/- 0.7 nM. PMID- 7536386 TI - Iloprost-induced inhibition of proliferation of coronary artery smooth muscle cells is abolished by homologous desensitization. AB - In addition to inhibition of platelet function, prostacyclin and its stable analogues are reported to attenuate vascular smooth muscle cell proliferation. However, desensitization of prostacyclin responsiveness is a known phenomenon in platelets. In this study we investigated the time-dependent effects of the prostacyclin-mimetic iloprost and of PGE1, respectively, on PDGF-induced proliferation of cultured coronary artery smooth muscle cells. Proliferation, assessed by [3H]thymidine incorporation was markedly inhibited by coincubation with iloprost (100 nM) and PGE1 (100 nM) for 4 h. In contrast, addition of iloprost (100 nM) for 24 h did not decrease smooth muscle cell proliferation, whereas inhibition by PGE1 or by forskolin was not diminished. These results suggest a homologous desensitization of anti-mitogenic effects of iloprost in coronary artery smooth muscle cells, probably at receptor-level. PMID- 7536387 TI - The cardioprotective actions of iloprost in myocardial ischemia of the rabbit can be separated from its vasodilatory effects mediated by KATP(+)-channel opening. AB - The modification of cardioprotective actions of iloprost by K(+)-channel blockade was studied in ischemic rabbit hearts. Glibenclamide, a blocker of ATP-dependent K(+)-channels, prevented coronary vasodilation mediated by the prostacyclin mimetic iloprost. In contrast, the cardioprotective effects of iloprost which were determined from prevention of ischemia induced rise in left ventricular enddiastolic pressure and loss of cytosolic troponin T in hearts made globally ischemic for two hours were not affected by glibenclamide. It is concluded that the cardioprotective action of iloprost can be separated from ist coronary vasodilator effect mediated by opening KATP(+)-channels. PMID- 7536388 TI - Erythromycin-induced cardiac toxicity. PMID- 7536390 TI - Interferon treatment of cirrhotic patients with chronic hepatitis C. PMID- 7536389 TI - Treatment of metastasized midgut carcinoids with dacarbazine. AB - Seven patients with metastasized midgut carcinoids were treated with intravenous infusion of dacarbazine [dimethyltriazenoimidazole carboxamide (DTIC)] (650 mg/m2) every 4 wk. After 2 wk, white blood cell counts decreased transiently in three patients. No other DTIC-associated side effects occurred. Biochemical markers of disease activity decreased significantly in four patients for 4-20 months (mean duration, 12 months). Size of hepatic metastases was reduced or remained unchanged in six patients for 6-20 months (mean duration, 10 months). Clinical symptoms such as cutaneous flush, diarrhea, abdominal pain, constipation, night sweat, or weight loss improved in six of seven patients. We conclude that DTIC represents a useful therapeutic option in the treatment of advanced and metastasized carcinoid tumors. PMID- 7536391 TI - Hemostatic complications of cardiopulmonary bypass. PMID- 7536392 TI - Physiological neutrophilia of pregnancy is not associated with a rise in plasma granulocyte colony-stimulating factor (G-CSF) PMID- 7536393 TI - Overlapping submicroscopic deletions in Xq28 in two unrelated boys with developmental disorders: identification of a gene near FRAXE. AB - Two unrelated boys are described with delay in development and submicroscopic deletions in Xq28, near FRAXE. Molecular diagnosis to exclude the fragile X (FRAXA) syndrome used the direct probe pfxa3, together with a control probe pS8 (DXS296), against PstI restriction digests of DNA. Deletions were detected initially by the control probe pS8, which is an anonymous fragment subcloned from YAC 539, within 1 Mb distal to FRAXA. Further molecular analyses determined that the maximum size of the deletion is < 100 kb in one boy (MK) and is wholly overlapped by the deletion of up to approximately 200 kb in the other (CB). These deletions lie between the sequences detected by the probe VK21C (DXS296) and a dinucleotide repeat VK18AC (DXS295). The patient MK had only speech delay with otherwise normal development, while patient CB had global developmental delay that included speech delay. Detection of overlapping deletions in these two cases led to speculation that coding sequences of a gene(s) important in language development may be affected. Hybridization of the pS8 and VK21A probes to zooblots revealed cross-species homology. This conservation during evolution suggested that this region contains sequences with functional significance in normal development. The VK21A probe detected a 9.5-kb transcript in placenta and brain and a smaller, 2.5-kb, transcript in other tissues analyzed. PMID- 7536394 TI - Dubowitz syndrome: long-term follow-up of an original patient. AB - Dubowitz syndrome is an autosomal recessive disorder of growth retardation, characteristic face, mild mental retardation, and eczema originally described by Dubowitz [1965]. Little information is available on natural history and adulthood in this disorder. We report on a 30-year-old woman who was one of the first patients to be diagnosed with the condition [Grosse et al., 1971, Z Kinderheilkd 110:175-187]. Microcephaly, short stature, leg length discrepancy, hyperextensible joints, spina bifida occulta, and absence of anterior cruciate ligaments were present. Her facial appearance had been modified by several plastic surgery procedures. Eczema resolved with age, with occasional flareups. Asthma, headaches, and seizures were additional medical findings. Speech delays, an unusually soft, high-pitched voice, submucous cleft palate, and velopharyngeal insufficiency were noted in childhood. Mild mental retardation was present. At age 30 years she is living independently in her own apartment and working full time in a nearby sheltered workshop. PMID- 7536395 TI - Heterogeneity in Roberts syndrome. AB - Roberts syndrome (RS) is a rare, autosomal recessive condition characterized primarily by growth retardation, developmental delay, and limb anomalies. Some RS patients (RS+), but not others (RS-), have an abnormality of their constitutive heterochromatin (the "RS effect"). RS+ patients also show a cellular hypersensitivity to DNA damaging agents such as mitomycin C (MMC). Lymphoblastoid cell lines from 2 unrelated RS+ patients were fused and hybrid cells examined for correction of the RS effect and MMC hypersensitivity. Neither cellular defect was corrected in the 2 hybrid cell lines examined, suggesting that these 2 patients represent a single complementation group. Fusions were also performed between one RS+ cell line and 2 different RS- cell lines. In both fusions, the hybrids demonstrated correction of both the heterochromatin abnormality and MMC hypersensitivity. These observations suggest that RS+ and RS- patients belong to different complementation groups and do not arise from the same single gene mutation. PMID- 7536396 TI - 'ACT': taking a positive approach to end-of-life care. PMID- 7536397 TI - How cyclosporine modifies histological and molecular events in the vascular wall during chronic rejection of rat cardiac allografts. AB - Accelerated allograft arteriosclerosis (chronic rejection) has emerged as a major factor affecting long-term survival of human cardiac allografts. The underlying mechanism of this disorder remains unclear. The purpose of this study was to investigate the effect of cyclosporine on the development of cardiac allograft arteriosclerosis at the cellular and molecular level. Heterotopic rat cardiac allografts from DA donors to WF recipients, with a strong genetic disparity in major histocompatibility complex and non-major histocompatibility complex loci, were used. The allograft recipients received triple-drug immunosuppression consisting of methylprednisolone (0.5 mg/kg/day), azathioprine (2 mg/kg/day), and three different doses of cyclosporine A (CsA; 5, 10, and 20 mg/kg/day). The grafts were removed 3 months after transplantation and processed for histology and immunohistochemistry. Low dose CsA (5 mg/kg/day) was associated with a severe form of intimal cell accumulation and intimal thickening in epicardial arteries and in smaller intramyocardial arterioles with nearly occluded vessel lumens 3 months after transplantation. The intermediate dose CsA (10 mg/kg/day) significantly inhibited arterial intimal thickening but was not efficient in reducing intimal cell accumulation. Instead, high dose CsA (20 mg/kg/day) significantly inhibited all arteriosclerotic vascular wall changes in the allografts. Immunohistochemistry revealed that the occluded epicardial arteries of cardiac allografts with low dose CsA expressed VCAM-1 on the endothelium. Higher CsA doses significantly reduced the expression of endothelial VCAM-1. Neither ICAM-1 nor major histocompatibility complex class II were expressed. Perivascular arterial infiltrates consisting of T helper cells and monocytes/macrophages were a characteristic finding in the allograft with low dose CsA. In the allografts treated with higher doses of CsA, arterial perivascular infiltrates were seldom seen. Our results conclusively demonstrate that sufficient immunosuppression with CsA inhibits intimal thickening and intimal cell accumulation of long-surviving rat cardiac allografts in a dose dependent fashion. Arteriosclerotic alterations associated with increased expression of arterial endothelial VCAM-1 were totally down-regulated by high doses of CsA. PMID- 7536398 TI - Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Roles for TNF and IL4. AB - The in vivo mechanisms of vascular endothelial activation and VCAM-1 expression were studied in murine heterotopic cardiac grafts. Preliminary studies demonstrated that cardiac allograft endothelia develop reactivity with MECA-32 monoclonal antibody (MAb) and M/K-2 (anti-VCAM-1) MAb within 3 days of transplantation, whereas cardiac isografts develop MECA-32 reactivity but no M/K 2 reactivity. Additional studies demonstrated that a single treatment of cardiac isograft recipients with the anti-CD3 MAb 145-2C11 induces VCAM-1 expression on isograft microvascular endothelia within 24 hours. We have used this experimental system to identify the cytokines responsible for expression of VCAM-1 and MECA-32 MAb reactivity on graft vascular endothelia. We report that the expression of VCAM-1 on isograft endothelia that was induced with anti-CD3 MAb was blocked by simultaneous treatment with either pentoxifylline, soluble tumor necrosis factor (TNF) receptor (TNFR-Fc), anti-IL4 MAb, or soluble IL4R, but not by anti-IFN gamma MAb. Alternatively, a similar pattern of isograft endothelial VCAM-1 expression was stimulated in the absence of anti-CD3 MAbs with a single injection of human recombinant TNF-alpha, or with recombinant murine IL4 provided as IL4/anti-IL4 MAb complexes. In addition, the IL4-induced VCAM-1 expression was completely blocked by a single intravenous treatment of the isograft recipients with TNFR:Fc. This suggests that high concentrations of TNF-alpha can stimulate endothelial VCAM-1 expression, but these concentrations are apparently not achieved in cardiac isografts. In the absence of an inducing agent such as anti CD3 MAb, the stimulation of VCAM-1 expression with exogenous IL4 may reflect functional interaction between endogenous TNF and exogenous IL4, as suggested by the blocking experiments with TNFR:Fc. Although cardiac isograft endothelia normally develop reactivity with MECA-32 MAb within 3 days of transplantation, treatment of cardiac isograft recipients with anti-CD3 MAb accelerated MECA-32 reactivity to within 24 hours of transplantation. This accelerated expression can be experimentally manipulated in the same way as M/K-2 reactivity, suggesting that similar mechanisms may control the development of these two inflammatory endothelial phenotypical markers, despite their differential expression in cardiac isografts and allografts. PMID- 7536399 TI - Surface-induced dissociation of multiply-protonated proteins. AB - A novel surface design compatible with the open cell geometry allows nonglancing angle collisions of selected ions stored in a Fourier transform mass spectrometer. Dissociation efficiencies of 36%, 22%, and 14% are achieved for gramicidin S, melittin, and carbonic anhydrase (29 kDa), respectively. Ion neutralization by the surface, which is highly competitive for many singly charged ions, is minimal, and dissociation products of hypervalent neutral species are not detected. Instead, the spectra are similar to those from collisionally activated and infrared multiphoton dissociation; the fragmentation pathways are relatively independent of the method of energy deposition. For carbonic anhydrase, however, the single event excitation inherent to surface induced dissociation appears to minimize secondary fragmentation, a critical advantage for tandem mass spectrometry of such large ions. Electrically floating the open cell below ground greatly enhances the collection efficiency. PMID- 7536400 TI - The efferent connections of the nucleus accumbens in the lizard Gekko gecko. A combined tract-tracing/transmitter-immunohistochemical study. AB - The present investigation of connections in the basal ganglia of the lizard Gekko gecko has revealed that the efferent projections of the nucleus accumbens are far more elaborate than previously thought. The projections reach not only the ventral pallidum, the preoptic area, the lateral hypothalamic area and the ventral tegmental area, but also the bed nucleus of the stria terminalis, the dorsomedial thalamic nucleus, the substantia nigra and the retrorubral dopaminergic cell group. Additional projections were observed within the nucleus accumbens and, caudally, to the peribrachial region and the superior raphe nucleus. Moreover, the results of the present study indicate that the accumbal projections to both the ventral pallidum and midbrain dopaminergic cell groups have a medial-to-lateral topographical organization. This expanded view of the efferent projections of the nucleus accumbens of Gekko gecko suggests a great resemblance in accumbal output systems between reptiles and mammals. A notable difference, however, is observed in the "limbic loop", which in reptiles, in contrast to mammals, seems to lack a cortical involvement. PMID- 7536401 TI - An outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection associated with nebulized albuterol therapy. AB - OBJECTIVE: To investigate an outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection in mechanically ventilated patients. DESIGN: A retrospective case-control and bacteriologic study. SETTING: Veterans Affairs medical center. PATIENTS: 42 mechanically ventilated patients who developed respiratory tract colonization or infection with B. cepacia and 135 ventilator-dependent controls who were not colonized and did not develop infections. MEASUREMENTS: Clinical and demographic data; benzalkonium chloride concentrations and pH levels in albuterol sulfate solutions; repetitive-element polymerase chain reaction (PCR)-mediated molecular fingerprinting on eight patient isolates and three environmental B. cepacia isolates that were available for study. RESULTS: 42 patients had B. cepacia respiratory tract colonization or infection. Observation of intensive care unit and respiratory care personnel showed faulty infection control procedures (for example, the same multiple-dose bottle of albuterol was used for many mechanically ventilated patients). More case patients (39 [92.9%]) than controls (95 [70.4%]; P = 0.006) received nebulized albuterol, and case patients (67.5 treatments) received more treatments than controls (18 treatments; P < 0.001). In-use albuterol solutions had pH values that were unstable, and benzalkonium chloride concentrations declined over time to levels capable of supporting bacterial growth. Medication nebulizers and in-use bottles of albuterol harbored B. cepacia. Molecular fingerprints of patient isolates and environmental B. cepacia isolates were identical using repetitive-element PCR. No further isolates of B. cepacia were identified after institution of appropriate infection control procedures. CONCLUSIONS: Multiple dose medications and reliance on benzalkonium chloride as a medication preservative provide a mechanism for nosocomial spread of microorganisms, particularly if infection control procedures are not carefully followed. Repetitive-element PCR is a useful fingerprinting technique for molecular epidemiologic studies of B. cepacia. PMID- 7536402 TI - The adhesion molecule and cytokine profile of multiple sclerosis lesions. AB - The expression of the adhesion molecules, vascular cell adhesion molecule-1 (VCAM 1) and intercellular adhesion molecule-1 (ICAM-1), and their respective receptors on leukocytes, very late activation antigen-4 (VLA-4) and lymphocyte function associated antigen-1 (LFA-1), together with a selection of proinflammatory and immunomodulatory cytokines (interleukin [IL]-1, IL-2, IL-4, IL-10, tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta [TGF-beta], and interferon-gamma [IFN-gamma] was examined by immunocytochemistry in multiple sclerosis (MS) lesions of different ages and compared with central nervous system (CNS) tissue from other neurological diseases, both inflammatory and noninflammatory, and normal CNS tissue. These molecules play key roles in lymphocytic infiltration and interactions during tissue inflammation and are in large part normally not expressed by CNS cells. High levels of expression of all the molecules tested were found in MS, particularly in chronic active lesions. Positivity for all molecules was also seen in other neurological diseases, even in noninflammatory conditions. There was some suggestion that the VCAM-1/VLA-4 adhesion pathway was expressed at higher levels in chronic MS lesions, while ICAM 1/LEA-1 was used more uniformly in lesions of all ages. Of the cytokines examined, there was increased expression of TNF-alpha and IL-4 in MS; this was found to be statistically significant when compared with noninflammatory neurological diseases. The expression of most adhesion molecules and some cytokines was negligible in normal CNS tissue although low-level reactivity for ICAM-1 TGF-beta, IL-4, TNF-alpha, and IL-10 was detected, perhaps indicative of immunoregulatory mechanisms. Microglial cells and astrocytes were the major CNS cell types expressing cytokines. The results indicate a potential in the CNS for widespread induced expression of molecules involved in the inflammatory cascade. No adhesion or cytokine molecule or pattern of expression unusual for MS was apparent. PMID- 7536404 TI - Making your point: principles of visual design for computer aided slide and poster production. PMID- 7536403 TI - [Recent progress in the development of anti-tumor metastatic drugs]. AB - Metastasis is often a terminal stage of cancer when tumor fragments lodge and grow in different parts of the body. There is currently no way to prevent metastasis, and there are few effective treatments. We believe that it is very important to develop an anti-tumor metastatic drug. Recent studies on the mechanism of tumor metastasis reveal that this multi-step process requires complex interaction between the tumor cells and their environment. In vitro experiments and animal cancer model studies have shown that the inhibitors of invasion (including adhesion, proteolysis, and migration) and neovascularization may suppress tumor metastasis. The author show a the mechanism of tumor metastasis, an in vitro screening method, and the recent progress of the development of various candidate anti-tumor metastatic drugs, including invasion inhibiting factor-2. Finally, the importance of the development of an anti-tumor metastatic drug in Japan is underscored. PMID- 7536407 TI - [Properties of white sorghum diastatic malt]. AB - Diastatic malt enzymes have potential to hydrolize pregelatinized starches releasing soluble sugars, lowering viscosity of slurries and allowing the use of high nutrient densities for preparation of cereal-based creams, baby food and drinks. Determination of the extent in which sorghum malt is able to develop desirable functional properties such as viscoty, water solubility and nutritional quality is fundamental. In this work the characteristics of a white sorghum during germination and the resulting malt were evaluated. "Dorado" white sorghum was germinated at 28 degrees C and 95% RH during 6 days in complete darkness, dried at 55 degrees C and ground to produce diastatic malt flour. Physicochemical, chemical, diastatic and nutritional characteristics of malt and its ability to liquify precooked flour slurries were determined. Maximum diastatic activity occurred at 3-4 days germination. Dry matter loss increased during germination at a rate of 2.7 percent units per day. Longer germination resulted in increased dry matter loss and decreased diastatic activity. Water solubility index of sorghum increased linearly during the first 5 days of germination probably caused by the production of soluble sugars and free amino acids. In vitro protein digestibility, lysine content and C-PER increased during germination. Sorghum malt was able to liquify precooked rice, wheat, oats or millet pastes (20% solids) in 5 min mixing at 30 degrees C. Utilization of malts with maximum diastatic activity are useful to liquify precooked cereal pastes or drinks and to increase the total solid contents and nutrient density while keeping the liquid properties of the product.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536406 TI - Age-related, different clinicopathologic features of hepatocellular carcinoma patients. AB - OBJECTIVE: The authors attempted to clarify the clinicopathologic differences of hepatocellular carcinoma (HCC) patients, according to age distribution, and to investigate whether these differences contribute a certain hepatocarcinogenesis. SUMMARY BACKGROUND DATA: Hepatitis-associated viruses causing HCC have been investigated, and the infection of the viruses and etiologically, the peak age of the disease vary according to geographic barriers. However, a correlation between clinicopathologic differences and the age distribution of the patients is not well understood. METHODS: The authors reviewed their institutional experience from 1978 to 1990 in treating 428 patients with HCC. The carrier rate for hepatitis B surface antigen (HBsAg), the frequency of occurrence of high serum alpha-fetoprotein (AFP) of 2000 ng/mL, the degree of liver damage represented by the retention rate of indocyanine green dye at 15 minutes (ICGR15), and the incidence of accompanying liver cirrhosis were investigated and compared in each decade of age. RESULTS: The HBsAg carrier rate and the frequency of high serum AFP values were significantly prominent in the younger patients (20-49 yrs). The degree of liver damage and the incidence of liver cirrhosis were prominent in the elderly patients (older than 70 yrs) or the middle-aged patients (50-69 yrs); however, these four values in the middle-aged patients were intermediate with respect to those observed in the other two age groups. In addition, there was a positive correlation between the HBsAg carrier rate and the frequency of high serum AFP values or between the degree of liver damage represented by ICGR15 and the incidence of liver cirrhosis, showing that the former correlation was inversely related to the latter. CONCLUSIONS: The authors' study indicates that there are age-related differences of clinicopathologic features in HCC patients, suggesting that there are different steps or mechanisms of hepatocarcinogenesis according to the patient's age-distribution. PMID- 7536405 TI - Cystic duct patency in malignant obstructive jaundice. An ERCP-based study relevant to the role of laparoscopic cholecystojejunostomy. AB - OBJECTIVE: This endoscopic retrograde cholangiopancreatography-(ERCP)based study estimates the potential role of laparoscopic cholecystojejunostomy for palliation of patients with malignant obstructive jaundice. SUMMARY BACKGROUND DATA: Traditional treatment of malignant obstructive jaundice has used a standard bilioenteric anastomosis. Laparoscopic biliary bypass via a gallbladder conduit currently is an established technique; it provides a low initial morbidity alternative to open procedures, similar to endoscopic stenting. No study has specifically addressed anatomic factors relevant to cholecystojejunostomy, such as prior cholecystectomy, stricture location in reference to the hepatocystic junction, and cystic duct patency in patients with malignant obstructive jaundice. METHODS: Retrograde cholangiograms were reviewed from consecutive patients with malignant obstructive jaundice and a control group without biliary disease who underwent ERCP during a 2-year period. Patients with either prior biliary surgery or hilar tumors were excluded. The presence of gallbladder or cystic duct filling was assessed. In patients with patent cystic ducts, the distance from obstruction to the cystic duct takeoff was classified as either greater or less than 1 cm. RESULTS: Nearly half the patients with malignant obstructive jaundice were ineligible for cholecystojejunostomies because of prior biliary surgery (29%) or hilar tumors (17%). Half (50 of 101) of the remaining potential candidates had patent hepatocystic junctions. Patients with ampullary carcinoma and patent hepatocystic junctions (5 of 9) were all ideal candidates for cholecystojejunostomies, having biliary obstruction more than 1 cm from the cystic duct takeoff. Two thirds of the remaining eligible patients (28 of 45) had obstructions less than 1 cm from patent hepatocystic junctions. CONCLUSIONS: Palliation of malignant obstructive jaundice by laparoscopic cholecystojejunostomy should only be attempted after direct cholangiography demonstrates a patent hepatocystic junction that is well separated from the malignant stricture. The majority of patients with malignant obstructive jaundice are ineligible for cholecystojejunostomies because of prior cholecystectomies, hilar obstructions, or tumor involvement of the hepatocystic junction. Nonoperative treatments will continue to be indicated for the majority of patients with malignant obstructive jaundice. PMID- 7536408 TI - Effect of various antidotes on the biliary and intestinal excretion of arsenic in situ and into the feces in vivo in guinea-pigs after injection of As2O3. AB - The effect of various antidotes on the excretion of arsenic into the feces in vivo and on the biliary and enteric excretion in situ was investigated on segments of jejunum and colon in anesthetized guinea-pigs using the pendular perfusion technique, according to Henning and Forth (1982). In the in situ experiments guinea-pigs received As2O3 (0.02 mmol As(III)/kg) and 30 min later, British-Anti-Lewisite (BAL), dimercaptopropanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA) or 2,3-bis-(acetylthio)propanesulfonamide (BAPSA) (0.1 or 0.7 mmol/kg each) into the jugular vein. In the in vivo experiments guinea-pigs received As2O3 s.c. (same dose as above) and 30 min later the same antidotes (0.1 mmol/kg i.p.). The feces were collected for 24 h and the arsenic content measured. During the 60-min perfusion period the amount of arsenic excreted into the jejunum or colon was only 3% or 0.4% of the dose administered, respectively. Of the arsenic dose, 8% was found in the bile. None of the antidotes had an effect on the arsenic excretion into the jejunum or colon. No change in biliary excretion was found in animals treated with BAL, 0.1 or 0.7 mmol/kg, respectively. DMSA, BAPSA or DMPS, 0.1 mmol/kg, increased the biliary excretion of arsenic to 14, 33, or 43% of the dose administered and after 0.7 mmol/kg to 29, 37, or 42%, respectively. Furthermore, a significant increase (P > 0.05) was found for the bile/blood concentration ratio in the following order: control < BAL < DMSA < BAPSA approximately DMPS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536410 TI - Indications for laparoscopic pelvic lymph node dissection in the staging of prostate cancer. AB - Laparoscopic pelvic lymph node dissection is increasing in popularity, in the absence of validated surgical indications. This retrospective study was performed to identify those patients who would benefit from laparoscopic pelvic lymphadenectomy. The case notes of 50 patients who underwent bilateral open pelvic lymphadenectomy with frozen section examination were reviewed. The patients were staged clinically as having A2 (16), B1 (20) and B2 (14) prostate cancer. Except for the two patients who had macroscopically involved pelvic lymph nodes at operation, all proceeded to total prostatectomy. Six patients were found to have positive pelvic nodes. Of the six, four patients were clinical stage B2, one was B1 and one was A2. The Gleason score of two of these six patients was greater than seven. The prostate specific antigen (PSA) levels correlated more with high tumour volume (i.e. grade C) rather than with D1 disease. Frozen section at the time of lymphadenectomy had a false negative rate of 2 of 6. We conclude that laparoscopic pelvic lymph node dissections (LPLND) should be performed where there is a high likelihood of nodal disease, viz. bulky tumours, poorly differentiated histology and a high PSA reading. We consider that such selective use of LPLND optimizes its diagnostic utility while minimizing its unnecessary application. PMID- 7536409 TI - Failure of engraftment of lenograstim mobilised allogeneic peripheral blood progenitor cells in aplastic anaemia. PMID- 7536411 TI - Anti-(human immunodeficiency virus) activity of polyoxotungstates and their inhibition of human immunodeficiency virus reverse transcriptase. AB - Heteropolyoxotungstates of the Keggin class containing different heteroatoms were tested for inhibition of two strains of human immunodeficiency virus 1 (HIV-1); they exhibited varying antiviral activity. Compounds containing boron were inactive, only one of those containing phosphorus showed selective anti-viral activity, whereas all silicon-containing compounds exhibited significant anti viral activity in C8166 cells infected with the IIIB strain. Their effectiveness was some 10-fold higher in JM cells with selectivity indices of about 2000. The silicotungstates were effective inhibitors of HIV reverse transcriptase, showing greater inhibition with RNA/DNA template primers than with DNA/DNA template.primer. Kinetic analysis demonstrated that they inhibit the enzyme by different mechanisms, as, of the four compounds examined, two competed with template.primer and two competed with deoxynucleoside triphosphate. Inhibition of DNA polymerase activity by these compounds was compared using polymerases from different sources, including human; although not necessarily most specific for HIV-1 reverse transcriptase, they did not inhibit all DNA polymerases to a similar degree. PMID- 7536414 TI - The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by 5 azacytosine is likely to involve methyl transfer to the inhibitor. AB - The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by the mechanism-based inhibitor 5-azacytosine has remained unclear, mainly because of the unavailability of a substrate in which the inhibitor, but not normal cytosine, is present at the target site. We synthesized an oligonucleotide duplex containing a single target site for the EcoRII methyltransferase, in which the target base is 5-azacytosine. This substrate formed a stable covalent complex with EcoRII methyltransferase in the absence and in the presence of the cofactor S-adenosylmethionine. The complex formed in the presence of the cofactor was resistant to SDS and moderate heat treatment, and a methyl group was incorporated into the complex. Enzyme titration and kinetic studies of inhibition suggest that methyl transfer to the complex occurred only during the first turnover of the reaction. These results suggest that, when the enzyme binds to 5-azacytosine in the presence of the cofactor, a methyl group is transferred to the N-5 position of the base, resulting in the inactivation of the enzyme. PMID- 7536412 TI - Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle. AB - 1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented. PMID- 7536413 TI - A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes. AB - An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself. PMID- 7536415 TI - Demonstration of granzyme A and perforin messenger RNA in the synovium of patients with rheumatoid arthritis. AB - OBJECTIVE: To examine the gene expression of 2 highly specific markers of cytotoxic T lymphocyte (CTL) activation, the serine protease granzyme A and the pore-forming protein perforin, in synovial tissue of patients with rheumatoid arthritis (RA), and to compare the findings with those in osteoarthritis (OA) synovial tissue. METHODS: Snap-frozen synovial tissue specimens from 9 patients with RA and 5 patients with OA were examined. The number of CTL that expressed granzyme A or perforin messenger RNA was determined by in situ hybridization using nonradioactive riboprobes for granzyme A and perforin, and by a novel in situ reverse transcriptase technique. The signals were visualized by an immunogold-silver immunohistochemistry technique and compared with immunohistochemical labeling of T and B cells. Additional double-labeling was achieved using anti-type IV collagen, anti-macrophage (anti-CD68), anti-T lymphocyte (anti-CD45RO), anti-B lymphocyte (anti-CD20), and anti-natural killer cell (anti-CD56) antibodies in an alkaline phosphatase-anti-alkaline phosphatase assay. RESULTS: Granzyme A and perforin messenger RNA (mRNA) was observed in CTL in synovial specimens from all of the RA patients, whereas in specimens from OA patients only a few, single cells with a positive mRNA signal for these molecules could be detected. In the RA specimens, the number of lymphocytes showing a positive mRNA signal for granzyme A or perforin varied from 10% to 50%, reflecting the recent findings of other investigators studying synovial fluid. CONCLUSION: Our results demonstrate that gene expression of at least 2 CTL products, granzyme A and perforin, is up-regulated in the synovium of patients with RA compared with that in the synovium of patients with OA. These molecules presumably play an important role not only in lymphocyte-mediated cytotoxicity, but also in facilitating the migration of blood-borne mononuclear cells through the vascular basement membrane into the rheumatoid synovium. PMID- 7536416 TI - Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes. AB - OBJECTIVE: To determine whether apoptosis occurs in rheumatoid arthritis (RA) synoviocytes, and if this phenomenon is dependent on the Fas/Apo-1 pathway. METHODS: Apoptotic change in vivo was examined in RA synovial cells by several standard methods. The ability of cells to undergo Fas-induced apoptosis was determined in vitro. RESULTS: Typical apoptotic change was demonstrated in RA synovial cells by each method. Anti-Fas antibody induced apoptotic synovial cell death in vitro. CONCLUSION: This is the first reported study to demonstrate apoptosis in RA synovial cells. The findings indicate that rheumatoid synoviocytes undergo Fas-mediated apoptosis. PMID- 7536417 TI - Medical education in palliative care. PMID- 7536418 TI - Nitric oxide and asthmatic inflammation. AB - Asthmatic patients show an increased expression of inducible nitric oxide synthase (iNOS) in airway epithelial cells and an increased level of nitric oxide (NO) in exhaled air. The NO derived from airway epithelial cells may be a mechanism for amplifying and perpetuating asthmatic inflammation, through inhibition of T helper 1 (Th1) cells and their production of interferon gamma (IFN-gamma). This would result in an increase in the number of Th2 cells and the cytokines interleukin 4 (IL-4) (which is important for IgE expression) and IL-5 (which plays a critical role in the recruitment of eosinophils into the airways). Although this mechanism may be part of our nonspecific airway defence against parasite invasion, it appears to have been activated inappropriately in asthma. Here, Peter Barnes and Eddy Liew argue that the development of specific iNOS inhibitors may represent a novel therapeutic approach for asthma and other allergic diseases. PMID- 7536419 TI - Cyclodextrin glycosyltransferase may be the only starch-degrading enzyme in Bacillus macerans. AB - Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source. In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch. At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells. Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B. macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid. PMID- 7536420 TI - Toxic effects of bleomycin on the hypothalamus following its administration into a cystic craniopharyngioma. AB - A 48-year-old man with a cystic craniopharyngioma developed hypersomnia, personality changes, memory impairment and thermal dysfunction following intracystic administration of bleomycin through an Ommaya reservoir after partial removal of the tumour. The patient's neurological deterioration was thought to be related to the toxic effects of bleomycin on the hypothalamus. PMID- 7536421 TI - Induction of a primary human cytotoxic T-lymphocyte response against a novel conserved epitope in a functional sequence of HIV-1 reverse transcriptase. AB - OBJECTIVE: To identify novel major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) epitopes conserved in HIV-1. METHODS: Potential conserved CTL epitopes were selected using a predictive computer algorithm based on a human leukocyte antigen (HLA)-A*0201 peptide-binding motif and tested for actual binding to the human processing defective cell line 174.CEM T2 (T2). Hence, the amino-acid sequences of 14 full-length sequenced HIV-1 strains were analysed. An in vitro primary peptide-specific human CTL response was induced with responding lymphocytes of an HIV-1-seronegative donor. Responding T cells were cloned by limiting dilution and tested for their ability to recognize naturally processed antigen in a 51Cr-release assay using recombinant vaccinia-HIV protein-infected B-lymphoblastoid cells (B-LCL) as target cells. RESULTS: The analysis of peptides bearing the HLA-A*0201 motif for conservation resulted in one peptide of Env, three of Gag and 12 of Pol. Only Gag340-348, Pol83-92, Pol267-277 and Pol960-968 showed binding properties to T2 comparable with those of known CTL epitopes Gag76-84 SLYNTVATL and Pol468-476 ILKEPVHGV. A successful primary MHC class I-restricted CTL response was induced against Pol468-476 and Pol267-277 VLDVGDAYFSV, a peptide in a functional sequence of reverse transcriptase (RT). The resulting CD8+ CTL clones were peptide specific and able to specifically lyse recombinant vaccinia-HIV-1 RT-infected HLA A*0201-matched B-LCL. CONCLUSION: The method used to screen proteins sequences for potential CTL epitopes, test selected peptides for binding to MHC class I and induction of an in vitro primary response against optimal binding peptides resulted in the identification of at least one novel conserved CTL epitope. The novel epitope is located in an area crucial for RT activity. This study demonstrates the feasibility of identifying highly conserved HIV-1-derived peptides capable of eliciting novel anti-HIV-1 CTL responses. PMID- 7536423 TI - Constitutive overexpression of secreted heterologous proteins decreases extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae. AB - High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast. The rate-limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER-resident proteins involved in folding--heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)--are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse--chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast. PMID- 7536422 TI - The HIV envelope protein gp120 is toxic to human brain-cell cultures through the induction of interleukin-6 and tumor necrosis factor-alpha. AB - OBJECTIVE: To investigate the induction of cytokines as a possible mechanism for the neurotoxicity of the HIV-1 envelope protein gp120. DESIGN: The gp120 protein was tested directly on primary human brain cultures to examine its ability to induce cytokines and its neurotoxicity on human neural cells because gp120 is known to be toxic to rodent ganglion cultures, and neural cells such as astrocytes and microglia produce cytokines when stimulated. METHODS: Primary cultures of human brain cell aggregates, astrocytes and macrophages were exposed to HIV-1 recombinant (r) gp120SF2. Induction of cytokines was assayed by enzyme linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR); neurotoxicity of rgp120SF2 and interleukin (IL)-6 on human brain cultures was examined by electron microscopy. RESULTS: ELISA and RT-PCR studies revealed that rgp120SF2 induced IL-6 and tumor necrosis factor (TNF) alpha in brain cultures; IL-6 could also be induced by TNF-alpha added to brain cultures. Both IL-6 and TNF-alpha were upregulated in astrocytes and macrophage cultures on rgp120SF2 treatment. Ultrastructural studies demonstrated that IL-6 treatment for 72 h induced large cytoplasmic vacuoles in neural cells with morphology consistent with neurons; rgp120SF2 treatment for 7 days resulted in chromatin condensation along the inner margins of nuclear envelopes of neural cells. CONCLUSIONS: Our results demonstrated that HIV-1 rgp120SF2 can upregulate at least two known neurotoxic cytokines, IL-6 and TNF-alpha, which may injure neural cells and contribute to the neuropathology observed in AIDS dementia patients. PMID- 7536424 TI - The action of substance P methyl ester on cochlear potentials in the guinea pig. AB - The action of the substance P agonist, substance P methyl ester (SPME) on cochlear potentials was examined in the guinea pig. Previous studies have shown that SPME is a selective agonist for neurokinin 1 (NK1) receptor. Perfusion with SPME at a concentration of more than 10(-6)M produced an increase in the amplitudes of the compound action potential and negative summating potential in a dose-dependent manner. N1 latency showed a tendency to be shortened, but this change was not significant. Amplitudes of the cochlear microphonics and endocochlear potential remained unchanged. Substance P fragment 7-11, an inactive analogue, produced no changes in the cochlear potentials. In contrast, the substance P antagonist [D-Pro2, D-Trp7,9]-SP blocked the action of SPME on the cochlear potentials. These results suggest that substance P may modulate neurotransmission through NK1 receptors in the cochlea. PMID- 7536425 TI - Recurrent amyloid tumor of the parotid gland. AB - A case of an organ-limited amyloid tumor of the left parotid gland is described with a history of recurrence. A slowly growing parotid mass was the only symptom. After 5.5 years following local excision, the patient was readmitted with a slowly growing recurrence in the superficial lobe of the previously treated gland. Lateral parotidectomy was performed with wide excision of the infiltrated tissue and preservation of the facial nerve. Primary amyloidosis of the AL type was confirmed with immunohistochemical studies revealing staining for lambda but not kappa light chains of immunoglobulins. There has been no clinical or laboratory evidence of systemic amyloidosis or recurrence after 2 years. To the best of our knowledge, this is the first report of a recurrent amyloid tumor of the parotid gland. PMID- 7536427 TI - Localization of regions affecting an allosteric transition in cyclic nucleotide activated channels. AB - Sensory transduction in olfactory receptors and photoreceptors is mediated by cyclic nucleotide-activated ion channels. We have studied the gating mechanism in olfactory and rod channels expressed in Xenopus oocytes. We report that the differences in cyclic nucleotide affinity and efficacy between these channels resulted from sequence differences outside the cyclic nucleotide-binding domain, especially in the amino-terminal domain, influencing the free energy of the closed to open allosteric conformational change. In addition, Ni2+ inhibited activation of the olfactory channel, decreasing both the maximum current and the apparent affinity for cyclic nucleotides. Ni2+ exerted its effect by binding preferentially to the closed configuration of the channel, thereby destabilizing the opening conformational change. We have localized this inhibition to a single histidine (H396) following the last transmembrane segment, suggesting a role for this region in channel gating. PMID- 7536426 TI - Growth cone advance is inversely proportional to retrograde F-actin flow. AB - In a previous study, F-actin appeared to play a key role in guiding microtubules during growth cone-target interactions. Here, F-actin flow patterns were assessed to investigate the relationship among F-actin flow, microtubule/organelle protrusion, and rates of outgrowth. We first demonstrated conditions in which surface markers (beads) moved at the same rate as underlying F-actin. These beads were then positioned, using laser tweezers, to assess F-actin movements during target interactions. We found retrograde F-actin flow was attenuated specifically along the target interaction axis in direct proportion to the rate of growth cone advance. Retrograde actin flow adjacent to the interaction axis was unperturbed. Our results suggest that growth cones transduce retrograde F-actin flux into forward movement by modulating F-actin-substrate coupling efficiency. PMID- 7536428 TI - Primary radiation therapy in the treatment of localized prostatic cancer. AB - Prostatic carcinoma is one of the leading causes of male cancer deaths. However, the routine diagnostic and therapeutic strategies have not yet been established. Although the outcome of surgical and radiotherapeutical approaches has frequently been reported to be comparable, the profile of side effects is different. This could offer the basis for selecting the treatment of choice in individual cases. During the last decade the radiotherapeutical technique has markedly improved, in part due to the achievements in the field of computer assisted tomography planning and conformal technique; the outcome of side-effects has decreased with concurrent increase in the rate of local control. The prescribing, recording and reporting of irradiation have also recently developed, as well as the staging of the disease. Therefore we consider it timely to review progress in this subject and to emphasize the role of radiotherapy in the treatment of localized prostatic cancer. PMID- 7536429 TI - Elevated alpha-feto protein in a 51-year-old male with relapsing seminoma, previous hepatitis B and acute hepatitis C. PMID- 7536430 TI - Palliative radiotherapy of mucocutaneous lesions in malignant acanthosis nigricans. PMID- 7536431 TI - [Experimental study on blocking agent of substance P nerves in the treatment of allergic rhinitis]. AB - Experimental guinea pigs with allergic rhinitis were treated with capsaicin (CAP). During the whole course of treatment, nasal secretions, sneezing and nasal scratching were observed and recorded, as compared quantitatively with those of the control group. By means of specific radioimmunoassay, determination of the amount of substance P (SP) in the nasal mucosa was done in the treated group, untreated group and normal control group. By using the filter paper technique, the functional status of the nasal secretion was observed. The experimental group was given CAP by dripping into the nasal cavities for 15 days. The results indicated that the various symptoms of allergic rhinitis were obviously relieved. The nasal secretions were decreased by 64% as compared with those before the treatment; and SP content in the nasal mucosa was remarkably reduced in comparison with untreated group. The results of the experiment indicated: the repeated use of CAP might effectively deplete SP content in the nasal mucosa and prove the new theory of the effects of CAP on the desensitization of the SP nerves. CAP as a blocking agent of SP nerves blocked the axon reflex and exerted curative effect on allergic rhinitis. PMID- 7536432 TI - An evaluation of four types of freshwater model ecosystem for assessing the hazard of pesticides. AB - Experimental results are reported on four types of freshwater model ecosystem after administration of a single dose of chlorpyrifos. The fate, and primary and secondary effects of chlorpyrifos were compared between the model ecosystems, and were evaluated in the light of the predictive value of the current ecotoxicological hazard assessment procedure for pesticides. 'Slootbox', a fate model used in the ecotoxicological risk assessment of pesticides in the Netherlands, overestimated chlorpyrifos concentrations. The primary effects of chlorpyrifos can be predicted accurately on the basis of single species laboratory toxicity data. The population effects observed in the microecosystems, microcosms, and mesocosms were consistent between all experiments and with the single species tests. Community metabolism, as a functional endpoint, was less sensitive than the structural parameters measured. Secondary effects, both for structural and functional endpoints, varied between the micro- and mesocosm experiments. At the present 'state of the art' in ecotoxicology, no a priori prediction of secondary effects in natural ecosystems will generally be possible. 0.1 times the lowest acute L(E)C50 for chlorpyrifos, as measured in the current ecotoxicological hazard assessment procedure, matched the NOECmesocosm for a single dose of chlorpyrifos. Recovery of populations affected by insecticide stress was found to depend on factors such as life cycle characteristics and ecological infrastructure, in addition to the toxicant concentration. The onset of (potential) recovery is likely to start at an approximate concentration of the EC10(48 h). PMID- 7536433 TI - The feasibility of using glycosylated recombinant human granulocyte colony stimulating factor (G-CSF) to increase the planned dose intensity of doxorubicin, cyclophosphamide and etoposide (ACE) in the treatment of small cell lung cancer. Medical Research Council Lung Cancer Working Party. AB - This study was conducted to test the feasibility of reducing the interval between cycles of doxorubicin, cyclophosphamide, etoposide (ACE) chemotherapy to 2 weeks, thereby increasing dose intensity, by adding granulocyte colony-stimulating factor (G-CSF) to reduce the duration of neutropenia following a cycle. 20 patients with small cell lung cancer (SCLC) were prescribed six cycles of 2 weekly ACE, with G-CSF on the intermediate days. 3 patients died during the treatment period and a further 5 had ACE terminated, 3 for toxicity and 2 for progressive disease. Of the 71 intervals between cycles, 42 (59%) were of the prescribed 14 days, 9 (13%) of 15-20 days, 15 (21%) of 21 days and five (7%) longer, but during the first four cycles, 36 (77%) of 47 intervals were of 14 days. The main reason for delay was haematological toxicity. All 20 patients experienced WHO grade 3 or 4 neutropenia, but at 2 weeks after a cycle only 3 had grade 4 and 1 grade 3. 17 patients required blood transfusion and 12 platelet transfusion. The only potentially serious adverse reaction to G-CSF was an episode of rash with facial oedema. Adding G-CSF allows ACE chemotherapy to be intensified by reducing the interval between cycles. PMID- 7536434 TI - Treatment of poor prognosis epidemic Kaposi's sarcoma with doxorubicin, bleomycin, vindesine and recombinant human granulocyte-monocyte colony stimulating factor (rh GM-CSF). AB - The efficacy and toxicity of doxorubicin, bleomycin and vindesine in epidemic Kaposi's sarcoma, and the role of rh GM-CSF in chemotherapy-induced neutropenia were evaluated in this Phase II study. Patients with progressive Kaposi's sarcoma were eligible, and were staged according to ACTG criteria. Treatment consisted of 20 mg/m2 doxorubicin, and a fixed dose of 15 mg bleomycin and 4 mg vindesine every 2 weeks. All patients continued antiretroviral medication with severe myelosuppression, patients received subcutaneous 5 micrograms/kg rh GM-CSF (Leucomax) from days 2-12. Response and toxicity were measured according to ACTG and WHO criteria. 27 patients were evaluable, 25 patients classified as having a poor prognosis. The response rate was 70% (3 CR, 16 PR), the duration of response was 18 weeks (range 8-25) and the median survival 30 weeks (range 4-63+). The cause of death was mostly opportunistic infection. 4 patients died of pulmonary Kaposi's sarcoma. The toxicity of this regimen was mainly myelosuppression and 13 patients were treated with rh GM-CSF. Complete recovery of the white blood cells occurred in seven of the 27 courses of rh GM-CSF (26%). No bacterial infections were recorded, but 5 patients (19%) developed an opportunistic infection during treatment. Peripheral neuropathy occurred in 16% of patients. Combination chemotherapy is effective in poor prognosis Kaposi's sarcoma but has a shortlasting effect. The main toxicity of this treatment is severe myelosuppression which can be ameliorated by rh GM-CSF. It remains to be established whether rh GM-CSF is also able to reduce the incidence of opportunistic infections. PMID- 7536435 TI - Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid. AB - Recombinant proteins containing a short stretch of contiguous histidine residues (approximately 6) ("a His-tag") can be specifically bound to N-nitrilotriacetic acid-chelated nickel ions, providing a convenient general method for their purification. A lipid derivatized with a nickel-chelating head group may provide a general approach to two-dimensional crystallization of the His-tagged proteins, using the lipid layer technique. We have designed a synthetic phospholipid that carries a chelated nickel ion (Ni-NTA-DOPE). His-tagged recombinant HIV-1 reverse transcriptase (HIV-RT) bound specifically to lipid layers containing Ni-NTA-DOPE and formed crystals within minutes from a dilute protein solution. Two dimensional crystals preserved in negative stain diffracted strongly to approximately 21 A. The projection map computed from averaged Fourier transforms revealed a structure similar in size and shape to a selected projection view of the 3-D structure that was previously determined for HIV-RT by X-ray crystallography. PMID- 7536436 TI - Increased intracellular glycerophosphoinositol and arachidonic acid are biochemical markers for lindane toxicity. AB - Lindane stimulates the release of both glycerophosphoinositol and arachidonic acid from phospholipids in rat renal proximal tubular cell cultures. When lindane was added to the culture medium, a correlation between the time-course profiles of glycerophosphoinositol and arachidonate release was found. This suggests a pathway in which phosphatidylinositol is not directly broken down by phospholipase C, but can instead be broken down to glycerophosphoinositol and arachidonic acid by phospholipase A enzymes. Therefore, a mechanism of action of lindane is through its effect on glycerophosphoinositol and arachidonic acid metabolism. PMID- 7536437 TI - Immunosuppressive therapy for vasculitis. AB - There are few controlled trials of immunosuppressive therapy for vasculitis, making the further study of long-term outcome of these diseases with contemporary modes of management necessary. Relapse is frequent in many forms of vasculitis. The consequences of immunosuppressive therapy, including opportunistic infection, have been emphasized. Novel forms of therapy have been described in small series and case reports, although the precise role of such therapies in the treatment of vasculitis in general is far from certain in the absence of collaborative, multicenter controlled studies. The relationship between vasculitis and hepatitis C virus has prompted the use of interferon therapy in the treatment of vasculitic complications resulting from this infection. PMID- 7536438 TI - Tumour necrosis factor alpha is pro-inflammatory in normal human skin and modulates cutaneous adhesion molecule expression. AB - Tumour necrosis factor alpha (TNF-alpha) is a potent immunoregulatory cytokine produced by many cutaneous cells, including keratinocytes, mast cells and Langerhans cells. To explore its potential role in inflammatory skin disease, we have studied immunohistochemically the effects of intradermal recombinant human TNF-alpha (rHuTNF-alpha) on cutaneous inflammatory cells, adhesion molecules and Langerhans cells in normal human skin. Volunteers receive rHuTNF-alpha 100 U (group A), 5000 U (group B), or 100 U daily for 5 days (group C), and biopsies were taken at 6 h (groups A and B), or 6 h after the final injection (group C). An inflammatory cell infiltrate developed in all cases: following single injections of either 100 or 5000 U rHuTNF-alpha this was predominantly neutrophilic, whereas following multiple injections of 100 U few neutrophils were seen, although many lymphocytes (CD3+, CD4+) were present. In all groups there was an increase in cells of monocyte/macrophage lineage (CD36+). TNF-alpha induced a dose- and time-dependent decrease in CD1a+ epidermal Langerhans cell numbers and an increase in dermal CD1a+ cells, suggesting migration of Langerhans cells away from the epidermis. TNF-alpha induced endothelial E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in all groups, and adhesion molecule expression by interstitial dermal dendritic cells (ICAM-1 and VCAM-1) and keratinocytes (ICAM-1) was observed. These findings indicate that TNF-alpha is a potent modulator of cutaneous immune function in vivo, and this central role in the cutaneous immune response suggests that TNF-alpha may be an attractive target for therapeutic inhibition. PMID- 7536439 TI - In vivo and in vitro serine/threonine phosphorylations of epidermal growth factor receptor upon entry into the cell cycle. AB - Protein phosphorylation and dephosphorylation is one of the main mechanisms of cell cycle regulation. This study examines the modulation of epidermal growth factor receptor phosphorylation as cells emerge from quiescence and enter the S phase of the cell cycle. The epidermal growth factor receptor is phosphorylated primarily on serine and threonine, but not on tyrosine residues, in an S phase dependent fashion, as determined by phosphoamino acid analysis and anti phosphotyrosine immunoblotting. These phosphorylations occur both in vitro and in vivo and are ligand independent. Some of the sites that are phosphorylated in vitro also appear to be phosphorylated in vivo, as determined by two-dimensional tryptic phosphopeptide analysis. At least one of the in vivo phosphorylation sites is phosphorylated by mitogen-activated protein kinase. Although the mechanism for this ligand-independent phosphorylation is not known, its correlation with emergence from quiescence and entry into the cell cycle suggests that the phosphorylation of epidermal growth factor receptor on serine and threonine residues may have heretofore unknown role(s) in cell cycle entry and progression. PMID- 7536440 TI - Only late, nonmitotic stages of granulocyte differentiation in 32Dcl3 cells are blocked by ectopic expression of murine c-myb and its truncated forms. AB - In murine leukemia virus-induced myeloid leukemias, insertional mutagenesis of the c-myb locus has been shown to occur frequently. Proto-oncogene activation is achieved in most leukemias by integration of murine leukemia virus upstream of exons 3 or 4 or by integration into exon 9 with consequent truncation of the protein. The present study investigates the effect of ectopic expression of full length c-myb or c-myb containing amino- or carboxyl-terminal truncations (minus 47 and 248 amino acids, respectively) on granulocyte differentiation in vitro. Recombinant myb retroviruses were used to infect an interleukin 3-dependent progenitor cell line, 32Dcl3, which undergoes terminal differentiation to mature neutrophilic granulocytes in the presence of granulocyte colony-stimulating factor. Overexpression of c-myb did not abrogate the interleukin 3 dependency of the parental cell line. However, cells expressing all forms of c-myb were blocked at an intermediate stage of granulocyte differentiation and continued to proliferate in the presence of granulocyte colony-stimulating factor. After 14 days in medium with granulocyte colony-stimulating factor, myb-expressing cultures predominantly consisted of promyelocytes with some myelocytes and almost undetectable numbers of neutrophilic granulocytes. This suggested that early stages of granulocyte differentiation were not inhibited, a finding that was further supported by the induction of myeloperoxidase, a biochemical marker of promyelocytes. Interestingly, the expression of lactoferrin, known to be a marker of late stages of granulocyte differentiation, was completely inhibited in the cells infected with myb viruses. It was concluded that c-myb expression blocked granulocyte differentiation to the terminal mitotic stages and that deletion of the NH2-terminal 47 amino acids and/or the COOH-terminal 248 amino acids of c-myb neither enhanced nor diminished this effect. PMID- 7536441 TI - Organization of the hamster intergeniculate leaflet: NPY and ENK projections to the suprachiasmatic nucleus, intergeniculate leaflet and posterior limitans nucleus. AB - The intergeniculate leaflet (IGL) is an integral part of the circadian visual system. It receives direct retinal input and relays photic information to the circadian clock in the suprachiasmatic nucleus (SCN) through a geniculohypothalamic tract (GHT). In both rat and hamster, neuropeptide Y immunoreactive (NPY-IR) IGL cells project through the GHT to the SCN. However, the hamster GHT also contains enkephalin-IR (ENK-IR) fibers, presumably of IGL origin. In the present investigations, the IGL was examined for NPY-, ENK-, or dual-IR cells. Their projections to the SCN, contralateral IGL and pretectum were also studied. The results show that the hamster IGL contains both NPY- and ENK-IR neurons and that about 50% of these are immunoreactive to both peptides. Double label retrograde analysis indicates that cells of each peptide class project to the SCN. Similarly, IGL neurons, many of which are NPY- and ENK-IR, project to the pretectum, particularly the posterior limitans nucleus. While numerous IGL neurons project contralaterally, very few are NPY- or ENK-IR. The distribution of SCN- and pretectum-projecting cells, in conjunction with the distribution of peptide-IR neurons, allows expansion of the IGL definition to include the region medial to the ventral lateral geniculate nucleus (VLG). The VLG is ventrolateral to the IGL and does not contain either neurons projecting to the SCN nor NPY- or ENK-IR cells, but does have numerous neurons projecting to the pretectum. The results substantiate and expand the previous definition of the hamster IGL, elaborate the species difference in IGL organization, and demonstrate the increased breadth of the circadian visual system. PMID- 7536442 TI - The value of coarse needle biopsy in evaluating thyroid nodules. AB - At Columbia Presbyterian Medical Center, CPMC, the incidence of thyroid cancer found at surgery (CI) has recently changed. CI prior to 1950 was 3-4%. The CI gradually increased to 29% in 1975 without the use of needle biopsy. Fine needle biopsy (FNB) was introduced in 1976. The CI did not change during the next 7 years but remained stable at 27% in patients who did not undergo coarse needle biopsy (CNB). The overall CI using both FNB and CNB in 1982 was 42% and with CNB alone, 47%. Since that time, extensive experience using both CNB and FNB has led to a CI of 51%. The CI in patients who only received FNB remains at 28%. The increase in CI seen in patients undergoing CNB are a result of better distinction between hyperplastic lesions and microfollicular neoplasms. 78 patients, referred for surgery because of suspicion of a follicular neoplasm determined on FNB alone, underwent CNB. 35 of these patients were shown to have benign macro-micro follicular lesions (hyperplastic). In a review of 1,625 patients who have undergone CNB there were 3 complications which required surgical intervention (.018%). All of these were for bleeding 6-72 hours after CNB. These 3 patients underwent total thyroidectomy for follicular cancer without complications. There were no other significant complications. These experiences demonstrate that CNB is extremely useful in evaluating thyroid nodules. The complication rate is low and is offset by a large decrease (40%) in the number of patients referred for operation. The primary value of CNB is in differentiating between true microfollicular neoplasms and hyperplastic ones. PMID- 7536443 TI - Aetiology of thyroid cancer: an epidemiological overview. AB - Thyroid cancer is a relatively rare cancer (5 new cases/y/10(5) inhabitants). An excess of thyroid carcinoma has been found in some but not in all goiter endemic areas. Follicular and anaplastic carcinomas have been found particularly frequent in regions of goitre endemia. A significant increase of thyroid carcinoma has also been found in iodine sufficient areas (Norway, Iceland, Hawaii). In several surveys a positive correlation has been found between parity and incidence of differentiated thyroid carcinoma. Natural goitrogens and chemotherapeutic agents have been proved to induce hyperplasia but their role in carcinogenesis of exposed populations is not yet definitely ascertained. Exposure to external radiation is carcinogenic for the thyroid both in human and in experimental animals. Patients treated for hyperthyroidism or thyroid cancer or given diagnostic doses of 131-I (0.5 Gy/test) indicate that under these conditions 131 I is not carcinogenic. Findings on population exposed to radioactive fallout showed an increased incidence of thyroid carcinomas compared to unexposed populations. After the Chernobyl accident (1986) particular attention was given to calculate the risk of thyroid cancer caused by the fallout of 131-I. Up to now a considerable increase of thyroid carcinoma has been reported in children of a region near Chernobyl (Belarus). PMID- 7536444 TI - Diagnosis of thyroid carcinoma by ultrasonic examination: comparison with diagnosis by fine needle aspiration cytology. AB - Ultrasonic examination was performed on 120 patients who later underwent surgery. Ultrasonically, among the 64 cases of benign nodules and the 56 of carcinomas, 50 and 46 cases respectively were diagnosed correctly. In making the ultrasonic diagnosis, the following findings were considered; shape, margin, boundary, internal echoes, hyperechoic spots, the echo level of the nodule and the cystic pattern. The sensitivity for carcinoma diagnosis was 0.82, specificity was 0.78 and accuracy was 0.80. The diagnostic accuracy for the same patients using a newly devised diagnostic system that utilized a fuzzy inference was almost as high as that of the conventional method. Furthermore, the diagnostic accuracy of both of these methods did not differ significantly from that obtained by fine needle aspiration cytology. Therefore, non invasive ultrasonic examination is considered to be very useful for the detection and diagnosis of thyroid carcinoma. PMID- 7536445 TI - Bone metabolism in premenopausal women with nontoxic goiter and reduced serum thyrotropin levels. AB - OBJECTIVE: To study whether premenopausal women with nontoxic goiter, but reduced serum TSH as a sign of spontaneous subclinical hyperthyroidism have decreased bone mass or other indications of increased bone turnover. DESIGN AND SUBJECTS: In a cross-sectional study, bone mass measurements were performed in the distal forearm and the lumbar spine in 11 premenopausal women with nontoxic goiter and a stable reduction in serum TSH (median (range) 8 months (6-108 months)), and in 22 matched controls. In a longitudinal study, measurements were repeated every 3 months for a maximum period of 2 years in 9 of the patients (6 completed 9 months, 4 completed 2 years follow-up). RESULTS: Serum TSH in the patients were (median (range)) 0.025 mU/l (< 0.005-0.256 mU/l). Patients had a bone mineral content of the distal forearm and a bone mineral density of the lumbar spine similar to those of controls, medians of controls: 98.4% and 93.8%, respectively. The risk of a type 2 error for over-looking a 10% difference was 2% for the distal forearm and 20% for the spine. TSH correlated negatively with marginal significance (p < 0.10) with markers of bone turnover: plasma osteocalcin, serum alkaline phosphatase, and fasting urinary hydroxyproline corrected for creatinine, although all patients had absolute values of these parameters within normal range. During follow-up bone mass measurements did not tend to decrease, but serum alkaline phosphatase and fasting urinary hydroxyproline showed increasing trends (p < 0.05). CONCLUSIONS: Premenopausal women with nontoxic goiter and reduced serum TSH do not seem to have reduced bone mass but seem to demonstrate signs of increased bone turnover. PMID- 7536446 TI - Polymyositis-like syndrome in hypothyroidism: report of two cases. AB - An increase of muscle enzymes in hypothyroidism has sometimes been correlated with a polymyositis-like syndrome and hypothyroid patients have been misdiagnosed and mismanaged as suffering from polymyositis. Actually, muscle symptoms, such as aches and pain, stiffness, weakness and cramps or, more rarely, hypertrophy, are observed in hypothyroidism and increased serum muscle enzyme values, particularly the level of creatine phosphokinase (CPK), indeed seem to suggest polymyositis. The muscular groups most commonly affected by the above mentioned symptomatology are those of the shoulder and pelvic girdles. In this report two hypothyroid patients complaining of muscle symptoms, whose serum muscle enzymes were particularly elevated, are described. In the first case the patient had been suffering from pain and weakness of the thenar eminence for about 4 months. The clinical features suggested a diagnosis of Carpal Tunnel Syndrome, but thyroid function tests revealed primary hypothyroidism. In the second case the patient had been afflicted with muscular weakness of the shoulder girdle for 2 months and was unable to keep his arms raised over his head. A study of thyroid function demonstrated a picture of hypothyroidism. Both patients were treated with L thyroxine and in a relatively short time biochemical parameters improved remarkably, and the symptoms disappeared. The hypothesis that a muscular effort of long duration by hypothyroid patients may have been responsible for the muscular damage and the symptoms may explain why only a few hypothyroid patients develop a clinical picture similar to polymyositis. PMID- 7536447 TI - The value of aspiration needle biopsy in evaluating thyroid nodules. AB - From 1980 to 1990 4,229 consecutive euthyroid patients with thyroid nodule (73% with single and 27% with multiple nodules) were examined by FNA cytology for preoperative selection. One thousand four hundred and eight of these patients (33%) had nodules also suitable for evaluation by large needle biopsy histology (Aspiration Needle Biopsy, ANB). No significant complications occurred following ANB. The proportion of inadequate specimens was 25% for ANB and 15% for FNA, however a definite diagnosis was obtained with ANB in 62 patients with inadequate FNA finding. Diagnostic sensitivity was higher (93%) for FNA than for ANB whereas specificity was better (82%) for ANB diagnoses. Nonetheless ANB contributed to the increase of overall sensitivity as four of all the malignant nodules diagnosed as benign by FNA were correctly identified by ANB. Analysis of the postoperative results of 102 nodules with FNA and ANB finding of benign nodule or of suspected cancer showed that the addition to the same FNA finding (benign nodule or suspected cancer) of a different ANB diagnosis (suspected cancer or benign nodule) greatly changed the probability of finding a malignant nodule at postoperative histology. ANB was also useful in showing a macrofollicular component in 52% of 150 nodules diagnosed by FNA as pure microfollicular nodules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536448 TI - Thyroid diseases in cohort studies of A-bomb survivors. AB - A fixed population of about 120,000 subjects was selected from those A-bomb survivors and unexposed controls for the purpose of follow-up studies on their late health effects. One hundred twelve cases of thyroid cancer were found in the study period between 1958 to 1979. The relative risk increased with thyroid dose: the excess relative risk per Gray was 1.1. The prevalence of thyroid diseases was evaluated in 2,587 subjects in Nagasaki: one or more diagnoses for thyroid diseases were made in 447. PMID- 7536449 TI - The incidence of ground glass nuclei in thyroid diseases. AB - Ground glass nuclei are an important finding in the diagnosis of papillary carcinoma of the thyroid. However, they appear only in specimens in paraffin embedded histological sections, and not in frozen sections or cytological specimens. This has led to the conclusion that they are an artifact of fixation and/or embedding. However, if this is the case, a question arises as to whether or not such an artifact can be of value as a definitive sign for establishing a diagnosis of papillary carcinoma. This study was undertaken to try to answer that question. 1) To confirm whether or not the author's microscopic observations were correct, we carried out automatic imaging analysis with the Interaktives Bild Analysen System (IBAS). The author's microscopic observations were consistent with the data obtained by the IBAS. 2) Among 20 cases of papillary carcinoma, clear nuclei were observed in 11 cases (55%), and pseudoclear nuclei were noted in 20 cases (100%). When 1,000 papillary cancer cells were counted in 10 cases, the average incidences of clear nuclei and pseudoclear nuclei were found to be 0.51% and 18.3% respectively. Clear nuclei were seen in papillary carcinomas in all kinds of fixatives except one but were not seen in benign lesions. Even though ground glass nuclei are a type of artifact, we believe that their presence may be considered to be a characteristic feature of papillary carcinomas of the thyroid. PMID- 7536451 TI - Neuroelectrophysiological evaluation of untreated hyperthyroid patients. AB - Hyperthyroidism is a common endocrinologic disorder affecting many organ systems. Musculoskeletal and neurological involvement present themselves as fatigue, muscle weakness and paralysis. Electromyography (EMG) is essential for differential diagnosis of muscle weakness. Well defined neuropathy and myopathy have been described in these patients. In the present study 17 hyperthyroid patients were evaluated with electrophysiological tests in addition to physical and neurological examinations and biochemical laboratory studies. Needle EMG, motor and sensory conduction velocities, median and tibial somatosensory evoked potentials (SEP) were studied. For assessment of the activity of disease clinical status, neurological symptom and disability scores and serum T3, T4 and TSH levels were examined. Statistical analysis of neuroelectrophysiological findings of the patient and the control groups yielded meaningful difference in the needle EMG, sensory conduction velocity and evoked potential findings. Abnormalities were observed in 80% of the proximal muscles besides polyphasic potentials that were seen in 20% of the extensor digitorum brevis muscle. Median, ulnar and sural nerve sensory action potential amplitudes were found to be lower than that of the control group. Sural sensory nerve conduction velocity of patients was decreased in 35.5%, prolongation of median SEP latencies and increase in the amplitudes were not however statistically significant. Prolongation of Tibial SEP N1, P2 latencies were seen in 47%, amplitudes of N1 were increased in 88.2%, P2 in 58.8%, N2 in 47%. The thyroid clinical status score was correlated with Tibial SEPs amplitude. These findings suggest the presence of an initial axonal type of mild polyneuropathy. As a conclusion electrophysiological studies can be useful in the diagnosis of asymptomatic polyneuropathy in hyperthyroid patients. PMID- 7536450 TI - Incidence of different forms of thyroid dysfunction and its degrees in an iodine sufficient area. AB - STUDY OBJECTIVE: The aim of the study was to determine the incidence of thyroid dysfunction in an iodine sufficient area (Vigo city, Galicia, North-West of Spain). DESIGN: Case-finding study during a 3-year (1990-1992) period. SUBJECTS: Subjects from a random sample of the population with abnormal results on FT4, sensitive-TSH or antithyroid autoantibodies. MEASUREMENTS: Thyroid size by ultrasound study. FT4 by RIA; TSA Ab by radio receptor assays; TSH, Tg Ab and TMS Ab by IRMA. MAJOR RESULTS: Overall incidence of thyroid dysfunction was 97.96 per 100,000 per year (CI 95% 78.86-117.06); female 162.45, male 17.44. Incidence rate of hyperthyroidism was 52.37 per 100,000 per year (CI 95% 38.41-66.36); 24.24 for Graves' disease, 11.63 for nodular hyperthyroidism, 13.57 for iatrogenic hyperthyroidism and 2.90 for others causes. Incidence rate of hypothyroidism was 45.58 per 100,000 per year (CI 95% 32.55-58.620 27.15 for hypothyroid autoimmune thyroiditis, 8.72 for postoperative hypothyroidism, 4.89 for miscellaneous hypothyroidism, 1.93 for amiodarone induced hypothyroidism and 2.90 for secondary hypothyroidism. CONCLUSIONS: This investigation provides extensive data on incidence of clinical and subclinical thyroid dysfunction and its different forms in an iodine sufficient area. PMID- 7536453 TI - Occurrence of papillary carcinoma in a hyperfunctioning thyroid nodule: report of a case and diagnostic considerations. AB - Papillary carcinoma of the thyroid occurring within a hyperfunctioning nodule is an extremely rare disorder. A case in a woman is reported. We believe that diagnostic and therapeutic procedures to be used in the case of a hot thyroid nodule must take this possible occurrence into account. PMID- 7536454 TI - Consistent presence of isochromosome 7q in hepatosplenic T gamma/delta lymphoma: a new cytogenetic-clinicopathologic entity. AB - Peripheral T-cell lymphoma (PTL), which is characterized by hepatosplenic presentation and the gamma/delta T-cell receptor (TCR) phenotype on the malignant cells, is a rare but distinct subtype of non-Hodgkin's lymphomas. Little is known about the chromosomal changes in these lymphomas. We report the cytogenetic analysis of three patients who had neoplastic proliferation of T gamma/delta cells in the spleen, bone marrow, and liver, but not in lymph nodes or skin. Isochromosome 7q and trisomy 8 were observed in all three patients. Isochromosome 7q as the sole abnormality has been previously reported in one patient with similar clinicopathologic features. It is suggested that i(7q) is a primary, nonrandom chromosomal abnormality in hepatosplenic T gamma/delta PTL. PMID- 7536452 TI - Puzzling biochemical thyroid profile in patients with multinodular goitre originating from a mild iodine deficient area in Tunisia. AB - Thirty Tunisian patients with a voluminous multinodular goiter with cold nodules and inhabiting a region of mild iodine deficiency, have been studied. The 131 I fixation by the thyroid, three hours after ingestion was significatively higher than in normal population. Fourty six per cent of the patients have stable iodine urine contents less or equal to 0.39 mumol/L. Three serum concentrations were paradoxically abnormal: thyrotropin (TSH), thyroglobulin (TG) and gammaglobulin concentrations. Fourty seven per cent of the serum thyrotropin (TSH) levels were less than the normal values though the serum total triiodotyronine (TT3), tetraiodotyronine (TT4), free triiodotyronine (FT3), free tetraiodotyronine (FT4), antithyroglobulin antibodies, total protein, triglycerides and cholesterol concentrations were normal. Serum thyroglobulin (TG) rates were elevated, ranged from 2 to 10,180 ng/ml and twenty five per cent of our patients showed levels greater than 500 ng/ml. The gammaglobulin levels were greater than normal in sixty eight per cent of our patients though only 10% of our patients manifest antithyroglobulin antibodies. PMID- 7536455 TI - Deletion of chromosome arm 17p DNA sequences in pediatric high-grade and juvenile pilocytic astrocytomas. AB - In adults, loss of heterozygosity for DNA on 17p has been shown in high-grade anaplastic astrocytomas (AAs) and glioblastomas multiforme (GMs), and mutation of the TP53 tumor suppressor gene has been reported in all grades of astrocytomas. Little is known, however, about 17p deletion and TP53 mutation in juvenile pilocytic astrocytomas (JPAs), the most common low-grade tumors seen in children. To elucidate the genetic characteristics of pediatric high-grade astrocytomas and JPAs, we performed restriction fragment length polymorphism analysis with probes derived from 17p and TP53 mutational studies in 28 tumor specimens. Telomeric chromosome arm 17p markers 144-D6 and ABR were lost in 6 (75%) of 8 informative tumors classified as high-grade (7 AAs, 1 GM) and in 2 (10%) of 20 informative JPAs. Loss of 17p probes centromeric to the TP53 gene were also detected in 3 AAs and 5 JPAs. Four of the 6 (66%) JPAs with losses of 17p DNA sequences recurred rapidly despite aggressive therapy, whereas only 5 of the other 14 (36%) recurred. Mutation of the TP53 gene was detected by polymerase chain reaction and denaturing gradient gel electrophoresis in only 1 JPA and 1 AA. These tumors were also examined for MDM2 gene amplification as an alternate inactivation mechanism for TP53 gene function: no instances of alteration were identified. These results suggest that a gene or genes in addition to TP53 on 17p may be involved in the etiology or progression of high-grade astrocytomas and aggressive JPAs in children.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536456 TI - Chromosome analysis of 97 primary breast carcinomas: identification of eight karyotypic subgroups. AB - Chromosome banding analysis of 97 short-term cultured primary breast carcinomas revealed clonal aberrations in 79 tumors, whereas 18 were karyotypically normal. In 34 of the 79 tumors with abnormalities, two to eight clones per case were detected; unrelated clones were present in 27 (34%) cases, whereas only related clones were found in seven. These findings indicate that a substantial proportion of breast carcinomas are of polyclonal origin. Altogether eight abnormalities were repeatedly identified both as sole chromosomal anomalies and as part of more complex karyotypes: the structural rearrangements i(1)(q10), der(1:16)(q10;p10), del(1)(q11-12), del(3)(p12-13p14-21), and del(6)(q21-22) and the numerical aberrations +7, +18, and +20. At least one of these changes was found in 41 (52%) of the karyotypically abnormal tumors. They identify a minimum number of cytogenetic subgroups in breast cancer and are likely to represent primary chromosome anomalies in this type of neoplasia. Other candidates for such a role are translocations of 3p12-13 and 4q21 with various partner chromosomes and inversions of chromosome 7, which also were seen repeatedly. Additional chromosomal aberrations that give the impression of occurring nonrandomly in breast carcinomas include structural rearrangements leading to partial monosomies for 1p, 8p, 11p, 11q, 15p, 17p, 19p, and 19q and losses of one copy of chromosomes X, 8, 9, 13, 14, 17, and 22. The latter changes were seen consistently only in complex karyotypes, however, and we therefore interpret them as being secondary anomalies acquired during clonal evolution. PMID- 7536458 TI - Comparative genomic hybridization in the investigation of myeloid leukemias. AB - Comparative genomic hybridization (CGH) was used for the examination of ten cases of myeloid leukemia (eight acute myeloid leukemias and two myelodysplastic syndromes). In five cases, genomic gains or losses were identified, which mapped to chromosomal regions known to be involved in this group of malignancies. In comparison to the results obtained by banding analysis, discrepancies were found in three of the ten cases; in two cases, chromosomal imbalances were not identified by CGH because they were present only in small subclones. In the other case, there were no evaluable metaphase cells for banding analysis; CGH revealed an overrepresentation of chromosome 8, which was confirmed by interphase cytogenetics with a chromosome 8-specific alphoid probe. All abnormalities revealed by CGH were confirmed by G-banding or subsequent interphase cytogenetic analysis, which demonstrates the high specificity of the method. Furthermore, in all cases, CGH identified the chromosomal imbalances present in the major clone as detected by banding analysis. The good correlation between CGH and chromosome banding results in myeloid leukemias makes this tumor a good model for the assessment of tools that are developed for automated and quantitative CGH analysis. PMID- 7536457 TI - Detection of the t(2;13)(q35;q14) and PAX3-FKHR fusion in alveolar rhabdomyosarcoma by fluorescence in situ hybridization. AB - Cytogenetic studies of the pediatric solid tumor alveolar rhabdomyosarcoma have demonstrated the presence of a consistent chromosomal translocation, t(2;13)(q35;q14). We recently identified PAX3 and FKHR as the genes on chromosomes 2 and 13, respectively, that are juxtaposed by this translocation. As one means of detecting the t(2;13) translocation in clinical specimens, we have developed a fluorescence in situ hybridization (FISH) assay that may be used for both interphase and metaphase cells. Translocation of the 5' region of the FKHR gene to the derivative chromosome 2, and retention of the 3' region of FKHR on the derivative chromosome 13 [(der(13)], were demonstrated in metaphase cells from a rhabdomyosarcoma cell line with a previously identified t(2;13) translocation. A 5' PAX3 cosmid probe was shown to localize to 2q35 in normal cells, and to translocate to the der(13) in the rhabdomyosarcoma cell line. In order to detect the der(13) in interphase nuclei, we labeled the 3'FKHR and the 5'PAX3 cosmid probes with digoxigenin and biotin, respectively, and used these in a two-color FISH assay. The presence of the der(13) was visualized as juxtaposed or overlapping red and green signals in metaphase and interphase tumor cells. The PAX3-FKHR FISH assay was then applied to a series of cytogenetically characterized pediatric sarcoma cell lines. The presence of the der(13) was demonstrated by FISH in all cases containing a cytogenetically detectable t(2;13). The FISH assay was then applied to a series of 20 embryonal and alveolar rhabdomyosarcoma samples. All 10 of the alveolar rhabdomyosarcoma specimens demonstrated a der(13) with the FISH assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536459 TI - New translocation t(2;13)(p12;q34) and rearrangement of the MLL gene in a childhood leukemia cell line. AB - Here we report the case of a 7-month-old boy who presented with biphenotypic acute leukemia, but with leukemia cells of B-cell phenotype present at the time of relapse. Two cell lines were derived from bone marrow specimens obtained at relapse, and immunophenotyping and analysis of antigen receptor gene configuration revealed concordance between the patient's leukemic cells and the cell lines. Cell line PER-377 shows a new chromosomal abnormality, t(2;13)(p12;q34), a molecular rearrangement at chromosome band 11q23 in the absence of a cytogenetically detectable abnormality of this band, and deletion of the genes for IGK. PMID- 7536460 TI - Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma. AB - Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome. PMID- 7536461 TI - Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization. AB - Comparative genomic hybridization (CGH) makes it possible to detect losses and gains of DNA sequences along all chromosomes in a tumor specimen based on the hybridization of differentially labeled tumor and normal DNA to normal human metaphase chromosomes. In this study, CGH analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy. Losses affecting 11p, 11q, 8p, 9, 17p, 3p, and 12q were all seen in more than 20% of the tumors. The minimal common region of loss in each chromosome was identified based on the analysis of overlapping deletions in different tumors. Gains of DNA sequences were most often found at chromosomal regions distinct from the locations of currently known oncogenes. The bands involved in more than 10% of the tumors were 8q21, 13q21-q34, 1q31, 3q24-q26, and 1p22. In conclusion, these CGH data highlight several previously unreported genetic alterations in bladder cancer. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of tumor suppressor genes and oncogenes that play an important role in bladder tumorigenesis. PMID- 7536462 TI - Cytogenetic and histologic findings in 17 pulmonary chondroid hamartomas: evidence for a pathogenetic relationship with lipomas and leiomyomas. AB - Pulmonary chondroid hamartomas (PCH) are benign tumors that contain mesenchymal and epithelial components. In this series, we identified clonal chromosome aberrations in mesenchymal cells from 10 of 17 PCH. Chromosome band 12q15 was rearranged most frequently (N = 4), and one case had a t(12;14)(q15;q24) that was identical cytogenetically to the characteristic translocation in uterine leiomyomas. Histologic review revealed diverse mesenchymal populations, including undifferentiated cells, cartilage, adipose tissue, and smooth muscle, in most of the PCH. These findings suggest that PCH result from neoplastic transformation of a primitive mesenchymal cell that differentiates along chondroid, adipose, and smooth muscle pathways. PMID- 7536463 TI - Defining the position of the breakpoint of the constitutional t(3;6) occurring in a family with renal cell carcinoma. AB - In a family with a constitutional translocation t(3;6), the oldest member carrying the translocation had developed multiple nonpapillary renal cell carcinomas (RCCs). The translocation breakpoint was positioned between 3p13 and 3p14.1. This is close to the region in which a t(3;8) breakpoint has been reported in a family with hereditary RCC. We defined the location of the t(3;6) and t(3;8) breakpoints by fluorescence in situ hybridization (FISH) analysis with yeast artificial chromosomes (YACs) from the 3p14-13 region. Both interphase nuclei and metaphase cells from translocation-carrying members of both families have been used, allowing the definition of flanking YACs for each breakpoint. We could thereby clearly confirm that the breakpoints are different, the t(3;8) breakpoint being most distal. In addition, we have shown that both translocation breakpoints are located distal to the homozygously deleted region in the U2020 lung cancer cell line. PMID- 7536464 TI - Concurrent presence of inv(14)(q11q32) and t(4;11)(q21;q23) in pre-B acute lymphoblastic leukemia. AB - The inv(14)(q11q32) is a non-random chromosomal aberration which has been associated with a variety of T-cell malignancies. We have studied a case of inv(14)(q11q32) that is unique in several respects. First, the inversion, which is expressed at the mRNA level, occurred in the context of a pre-B acute lymphoblastic leukemia (ALL) as opposed to a T-cell malignancy. Second, cloning and sequencing of the inversion revealed that it resulted from a fusion between an immunoglobulin heavy chain variable (V) segment and a T-cell receptor delta diversity (D) segment. In addition, the patient had a second chromosomal abnormality at diagnosis, a t(4;11)(q21;q23) which disrupted the MLL gene. The fact that there were two distinct chromosomal abnormalities at diagnosis enabled us to address the question of leukemic clonal evolution during the course of this patient's disease. We present evidence suggesting that the t(4;11)(q21;q23) occurred first, with the inv(14)(q11q32) occurring as a second event. PMID- 7536465 TI - Spontaneous loss of Ph chromosome with maintenance of clonal hemopoiesis in an untreated patient with myeloproliferative disease and a long survival. AB - The unusual case of myeloproliferative disease described here is characterized by the following features: (1) a clinically completely silent course for 11 years without splenomegaly, marrow fibrosis, or cellular morphologic alterations; (2) the presence, at the onset, of a Philadelphia (Ph) chromosome without DNA breakpoints in the M-bcr region; (3) the spontaneous loss of detectable Ph positive cells, 5 years after the first finding of leukocytosis, in the absence of any therapy; (4) the maintenance of the clonal nature of hematopoiesis, as revealed by the PGK X-linked inactivation pattern, in the absence of the Ph chromosome; and (5) a biphasic trend in the levels of leukocytes, red cells, and platelets during the years of observation. PMID- 7536466 TI - Isolation and characteristics of tonsil centroblasts with reference to Ig class switching. AB - Most tonsil B cells have high levels of surface CD44 but this molecule is either expressed at low levels or is absent from germinal centre B cells (GCB). On average 62% of isolated GCB were found to be CD44- and the remainder CD44low. Most CD44- GCB were in cell cycle, indicating that they were centroblasts, while centrocytes, non-dividing GCB, were mainly CD44low. Immunohistological analysis confirms that centrocytes, which are located in the light zone of germinal centres, express low levels of CD44, while centroblasts, cells of the dark zone, are CD44-. While most CD77high GCB are centroblasts and CD77low GCB centrocytes, many centroblasts and centrocytes express intermediate levels of CD77, making this less reliable than CD44 for discriminating between these cells. Most CD44low and CD44- GCB were shown to have undergone Ig switch recombination in vivo. This indicates that switch recombination is independent of the maturation of centroblasts to centrocytes and precedes the signals that induce GCB to differentiate to plasma cells or memory B cells. The average rate of entry of the CD44- GCB fraction to apoptosis on culture at 37 degrees C was faster than that of the total GCB preparation. It is suggested that this may reflect strict stromal-dependence of centroblasts while centrocytes have to survive for long enough to have the chance of receiving antigen-specific selection signals. Inhibition of apoptosis by CD40 mAb with IL-4 or phorbol myristate acetate with ionomycin was similar in the CD44- and CD44low preparations. PMID- 7536467 TI - De novo development and self-replenishment of B cells. AB - Previous studies distinguished two murine B cell lineages: the conventional lineage, which comprises the majority of B cells, and the Ly-1 B lineage (B-1a), which represents a small percentage of total adult B cells. A third subset, B-1b cells, shares many properties with B-1a cells, including the characteristic ability to self-replenish, but does not express Ly-1 (CD5). Reconstitution studies presented here show that (i) although the B220- population in adult spleen and bone marrow contains very little progenitor activity for B-1a cells, it can reconstitute roughly half the normal number of B-1b cells; (ii) B-1 progenitors present in adult bone marrow and spleen function at low levels in adult animals; (iii) peritoneal B-1 cells (principally B-1b) that develop following bone marrow transfer, like B-1 cells from normal animals, are capable of substantial self-replenishment; and (iv) conventional B cells do not expand (self-replenish) in adoptive recipients, although they can persist for long periods. Collectively, these progenitor and self-replenishment characteristics provide a developmental base for distinguishing B-1a, B-1b and conventional B cells. PMID- 7536468 TI - Post-irradiation changes in the blood vessels of the adult human mandible. AB - Changes in the inferior alveolar artery and periosteal vessels of the post irradiation mandible have been investigated. The inferior alveolar artery underwent comparatively little change, stenosis being noted in just over half the specimens examined. The number of periosteal arteries and veins increased, the changes being most marked on the body where the effective tissue dose of radiation was highest. The increase in periosteal vessels was independent of stenosis of the inferior alveolar artery. PMID- 7536470 TI - Evidence that eosin-5-maleimide binds close to the anion transport site of human erythrocyte band 3: a fluorescence quenching study. AB - The interaction between eosin-5-maleimide (EMA), an inhibitor of the anion transport protein, band 3, and I-, a transportable substrate, was investigated by fluorescence quenching. The Stern-Volmer plot for the quenching reaction between EMA-labeled band 3 and I- exhibits downward curvature both in human erythrocyte ghosts and in purified band 3. The quenching reaction is insensitive to the viscosity of the bulk phase. The shape of the Stern-Volmer plot becomes more linear with increasing temperature. Following the approach of Blatt et al. [(1986) Biophys. J. 50, 349-356], we have developed a binding-diffusion model which is in good agreement with the quenching data. The model supposes that EMA is located in a compartment or "pocket" in band 3 which is separate from the bulk phase and contains a binding site or sites for the quencher. Quenching of band 3 bound EMA fluorescence by I- is inhibited by DIDS and by the transportable anions Cl-, HCO3-, and Br-. Analysis of these experiments yields dissociation constants for the anions which are in reasonable agreement with those determined from transport kinetics and by NMR. We thus deduce that the quencher binding site is the anion binding/transport site on band 3. We propose that EMA is located in the wall of the anion access channel such that it does not inhibit anion binding. The methods described in this report should facilitate detailed studies of anion binding to the transport site on band 3 under a variety of experimental conditions. PMID- 7536469 TI - Structural and biochemical studies of human galanin: NMR evidence for nascent helical structures in aqueous solution. AB - The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3 11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536471 TI - Site-specific conjugation of diethylenetriaminepentaacetic acid to recombinant human granulocyte-colony-stimulating factor: preservation of protein structure and function. AB - The chelating agent diethylenetriaminepentaacetic acid (DTPA) was conjugated site specifically to the N-terminus of recombinant human granulocyte-colony stimulating factor (rhG-CSF) by reaction of the protein with DTPA dianhydride at an initial pH of 6.0. The reaction was efficient in that 84% of the starting rhG CSF was N-terminally modified and could be purified to homogeneity by cation exchange chromatography. Chelation of 111In by the DTPA-rhG-CSF conjugate was demonstrated by cation-exchange HPLC and thin-layer chromatography. Metal contamination of conjugate preparations, as well as metal-loading onto the conjugate, could be monitored by either cation-exchange HPLC or isoelectric focusing. The 1:1 stoichiometric molar ratio of DTPA to protein for the DTPA-rhG CSF conjugate was determined by thin-layer chromatography and mass spectrometry, and the localization of the conjugated DTPA moiety was resolved using a peptide mapping procedure. The secondary structure (i.e., alpha-helicity) of the protein was unmodified following conjugation as revealed by circular dichroism. Furthermore, the conjugate induced a similar induction of peripheral WBC counts as unmodified rhG-CSF when injected subcutaneously into hamsters, demonstrating preservation of protein bioactivity. These results reveal a simple and efficient method for conjugating DTPA to protein, via reaction with the dianhydride, to yield a homogeneous and well-defined product. The procedure may prove to be a useful method of labeling growth factors and related proteins while preserving structural and functional integrity. PMID- 7536472 TI - The high-conductance channel of porin-less yeast mitochondria. AB - Patch-clamp and planar bilayer experiments on porin-less yeast mitochondria have allowed the characterization of a cationic channel activated at matrix-side positive (unphysiological) potentials. In voltage-pulse experiments, inactivation was a faster process than activation and the time constant for inactivation was more steeply dependent on voltage than the one for activation. The channel exhibited various conductance states whose occupancy depended on the applied transmembrane potential. In bilayer experiments, the presence of the pCOx-IV leader peptide induced fast gating in a voltage-dependent manner. A comparison with previously described activities suggests that the pore may coincide with the peptide-sensitive channel (PSC) (Thieffry et al. (1988) EMBO J. 7, 1449-1454) as well as with two other activities (Dihanich et al. (1989) Eur. J. Biochem. 181, 703-708; Tedeschi et al. (1987) J. Membr. Biol. 97, 21-29) assigned to the mitochondrial outer membrane. The possible relationship of this channel to the mitochondrial megachannel is discussed. PMID- 7536474 TI - In vitro bioassay with enhanced sensitivity for human granulocyte colony stimulating factor. AB - A method for the determination of human granulocyte colony-stimulating factor (hG CSF) activity, based on stimulation of cellular proliferation, was developed using a subclone of the murine myeloid leukemia cell line NFS-60, with an improved sensitivity for hG-CSF, as indicator. The optimal range for quantitative analysis of hG-CSF was about 4-60 pg ml-1. The stimulatory effect was measured by a colorimetric microassay: the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was obtained by reading plates in a multi-channel photometer. The assay was designed as a five-dose parallel line test, employing three or four doses for potency determinations, which fulfil pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation (RSD) varied between 5.2 and 12.0%. Most assay experiments revealed potencies within limits of error of 90 110% and the mean index of precision value was 0.057. The recently developed yeast cell-derived International Standard (88/502) served as a reference for activity of rhG-CSF. Specificity of the assay was demonstrated by absence of response upon exposure to a panel of biomolecules, including recombinant human interleukin-3, and by the suppression of growth stimulation in the presence of neutralizing anti hG-CSF antibodies. Potency readings of unglycosylated rhG-CSF were dependent on pH of assay medium with higher relative activities observed at pH 6.6 than at 7.4. Moreover, SDS-PAGE analysis of the carbohydrate-deficient preparation, following incubation at physiological pH, revealed several high molecular weight rhG-CSF bands and decreased monomeric form. The method described was found suitable for potency assessments of pharmaceutical formulations of hG CSF. PMID- 7536473 TI - Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils. AB - In human neutrophils, alkaline phosphatase (AlkPase), a low-affinity receptor for IgG (FcRIIIB), and complement decay accelerating factor (DAF) are glycosyl phosphatidylinositol (GPI)-anchored proteins. Varying greatly in biological function these three integral membrane proteins exhibit regulated cell surface expression in neutrophils. Defined by their common membrane-linkage motif, AlkPase, FcRIIIB, and DAF can be released from the lipid bilayer by the action of phosphatidylinositol-specific phospholipase C and are relatively resistant to low temperature extraction with Triton X-100 (TX-100). In this study we show that neutrophil AlkPase, FcRIII, and DAF display differential extractibility; they are relatively insensitive to TX-100 solubilization at 4 degrees C, but are readily extracted with TX-100 at 37 degrees C or by the detergent octyl glucoside at 4 degrees C. The differential extractibility of these GPI-anchored proteins is the same in unstimulated cells, where these proteins exist primarily in an intracellular pool, and stimulated cells, where they are expressed principally at the cell surface. However, no differential extraction effect is observed with two neutrophil transmembrane proteins, complement receptor 1 (CD35, CR1) and MHC Class I in either stimulated or unstimulated cells. PMID- 7536476 TI - Risks of rhG-CSF treatment in drug-induced agranulocytosis. AB - Nine patients with drug-induced agranulocytosis received recombinant human granulocyte colony-stimulating factor (rhG-CSF) to accelerate myeloid recovery because of life-threatening infections related to neutropenia. All patients showed a quick recovery of their granulocyte counts. Side effects were substantial, however. Three patients, two with a severe infection and one with preexisting pulmonary infiltrates, developed worsening of their respiratory status during neutrophil recovery, resulting in clinical manifestations of the adult respiratory distress syndrome (ARDS). In view of these major complications, the exact place of hematopoietic growth factors in the treatment of drug-induced agranulocytosis remains to be determined. PMID- 7536475 TI - The impact of interferon versus busulfan therapy on the reticulin stain-measured fibrosis in CML--a comparative morphometric study on sequential trephine biopsies. AB - To evaluate treatment-related changes of the reticulin stain-measured fibrosis in Ph(1+)-CML, a clinicopathological study was performed on sequential trephine biopsies of the bone marrow following either interferon (IFN) or busulfan (BU) monotherapy. Using the monoclonal antibody CD61 for the identification of megakaryopoiesis and Gomori's silver impregnation method, number of megakaryocytes and density of argyrophilic (reticulin and collagen) fibers were determined by morphometry. We studied specimens from 26 patients with IFN-alpha 2b (including nine patients with additional IFN gamma) therapy and from 23 patients who had received BU. In both groups, repeated bone marrow biopsies (total 125) revealed a significant increase in the fiber content, as well as in the number of megakaryocytes during treatment. To assess the dynamics of myelofibrosis more precisely, computation of differences in the degree of fiber density between the first and last examination was carried out. Regarding the considerable variations in the biopsy intervals, a so-called myelofibrosis progression index (MPI) was calculated. Following this rationale, we were able to demonstrate that, in comparison to the BU-group, speed of progression of bone marrow fibrosis was significantly increased in CML patients treated with IFN. Preliminary statistical analysis indicated a relationship between myelofibrosis on admission, which was always associated with increased growth of megakaryocytes, and the MPI with survival. Even when these parameters were regarded, prognosis was significantly more favorable in the IFN-treated patients. The failure of IFN and BU to inhibit the evolution of myelofibrosis may be related to several conversely acting pathomechanisms. Among others, the inability of both therapeutic agents to reduce the number of megakaryocytes more effectively should be taken into consideration. PMID- 7536477 TI - Molecular identification of hereditary persistence of fetal hemoglobin type 2 (HPFH type 2) in patients from Brazil. AB - The HPFH deletion type 2 was first described in a patient from Ghana and is characterized by a large deletion of approximately 105 kb extension. We report here the results obtained in studying a black Brazilian patient who presented an association of beta-thalassemia and HPFH type 2, using a PCR strategy for detection of the breakpoint region. This procedure allows a rapid molecular identification of this condition and is a reliable procedure for screening patients with a hematological picture of HPFH deletion types. PMID- 7536478 TI - Lineage-unrestricted hematologic response to granulocyte colony-stimulating factor in a patient with refractory anemia with excess blasts. AB - We report a patient with refractory anemia with excess blasts who showed a lineage-unrestricted hematologic response to granulocyte colony-stimulating factor (G-CSF). After 17 months of a stable disease state, the patient developed pneumonia, progression of cytopenia, and reduced cellularity and blast mass in the bone marrow. He was given G-CSF to overcome the pneumonia. Not only the neutrophil count, but also the platelet count increased soon after initiation of the G-CSF therapy; both counts became normal on the fifth day of the G-CSF therapy. Additionally, the anemia improved gradually. The neutrophil and platelet counts were maintained in the normal range for 3 months after cessation of the G CSF. In vitro studies showed that G-CSF alone stimulated megakaryocyte colony formation from bone marrow mononuclear cells (BMMNC), and accessory cells in the BMMNC were necessary for expression of this G-CSF-induced in vitro megakaryocytopoiesis. These results suggest that, in coordination with accessory cells, G-CSF stimulated megakaryocytopoiesis in the patient. This case provides valuable information for understanding the mechanisms of a lineage-unrestricted hematologic response to G-CSF, which is very rarely observed in MDS. PMID- 7536479 TI - Diagnostic testing during pregnancy: a descriptive analysis of utilisation data. AB - To describe patterns of diagnostic testing during the antenatal period and to assess the potential benefit of using Medicare claims data in monitoring testing practice, we examined the matched claims data (with identifying details removed) on approximately 10,000 women having a confinement for which a Medicare benefit was claimed between 1 July and 30 September 1990. The results showed that almost all the women included in the study sample had an ultrasound and blood group and antibody examination. A smaller proportion had serological tests for syphilis (77 per cent), rubella (51 per cent) and hepatitis B carriage (73 per cent). Two thirds had urine microscopy and culture, and under half (40 per cent) had serum alpha-fetoprotein estimation. Few (18 per cent) had a claim processed for microscopy and culture of a genital swab and fewer than 8 per cent claimed for any other pathology tests. There were differences in the proportions having tests, depending on whether the clinician managing the confinement was a specialist obstetrician or a general practitioner, and depending on geographic area and age group. While the data do not represent all women having a confinement in New South Wales, the selective use of antenatal diagnostic tests found in this study is of considerable public health importance and analysis of claims data can provide useful information for health professionals. PMID- 7536480 TI - Altered cerebral blood flow during hysterical paresthesia. PMID- 7536483 TI - [Cascade aftereffects of administering delta-sleep inducing peptide]. PMID- 7536481 TI - The use of ultra-low-dose aprotinin to reduce blood loss in cardiac surgery. AB - One hundred patients due to undergo primary cardiac surgery were prospectively randomized to receive aprotinin or placebo. In the aprotinin group, 250,000 kallikrein inhibitory units (KIU) of aprotinin were added to the cardiopulmonary bypass prime solution. A further 250,000 KIU of aprotinin were infused intravenously over 30 minutes immediately before the start of cardiopulmonary bypass. The control group received 0.9% saline in equal volumes at identical times. The study was designed to have a 90% chance of demonstrating a 30% reduction in blood loss. No significant differences were found between the two groups. The median blood loss in the aprotinin group was 750 mL (interquartile range 556 to 1025 mL, 95% confidence interval 600 to 800 mL). In the control group, the median blood loss was also 750 mL (interquartile range 500 to 988 mL, 95% confidence interval 625 to 925 mL). In the aprotinin group, 12 patients received postoperative autotransfusion of shed mediastinal blood of median volume of 665 mL (interquartile range 500 to 925 mL, 95% confidence interval 450 to 1000 mL). In the control group, 14 patients received postoperative autotransfusion of mediastinal blood of median volume of 663 mL (interquartile range 600 to 800 mL, 95% confidence interval 600 to 700 mL). Five patients in the aprotinin group and seven patients in the control group required postoperative homologous blood transfusion. Reassessment of inclusion criteria showed a 19% reduction in blood loss in patients undergoing only aortocoronary bypass receiving aprotinin compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536482 TI - Blood loss and transfusion requirements in cystic fibrosis patients undergoing heart-lung or lung transplantation. PMID- 7536484 TI - [Synthesis and properties of CD5-ricin immunotoxin]. PMID- 7536485 TI - [Morphogenesis of a canalicular-hypertensive model of pancreatitis]. PMID- 7536486 TI - [Pathomorphology of radiation-induced changes in the bronchi at a distant period after exposure]. PMID- 7536487 TI - Androgen repression of cytokeratin gene expression during rat prostate differentiation: evidence for an epithelial stem cell-associated marker. AB - Cytokeratin (CK) 8 mRNA expression in developing and degenerating rat prostate was studied using in situ hybridization with an antisense RNA-probe. It was found that: 1) the CK 8 antisense probe was accumulated only within prostatic epithelial cells; 2) after castration, CK 8 mRNA signals in ventral prostate (VP) sections were significantly increased, and elevated CK 8 mRNA expression persisted even long after prostate involution was complete; and 3) during prostate development, the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, a shift of CK 8 mRNA staining to peripheral regions and decreased overall CK 8 mRNA levels were noted. These data indicate that excessive expression of CK 8 mRNA is a characteristic of prostatic stem cells, and CK molecules are excellent markers for determining the hierarchical pathway of cell differentiation in prostate epithelium. PMID- 7536488 TI - Interleukin-3 and interleukin-7 are alternative growth factors for the same B cell precursors in the mouse. AB - Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain- B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell. PMID- 7536489 TI - Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines. AB - Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo. PMID- 7536491 TI - Induction of IgA1 and IgA2 production in immature human fetal B cells and pre-B cells by vasoactive intestinal peptide. AB - We studied the effects of vasoactive intestinal peptide (VIP) on IgA1 and IgA2 production in human fetal B cells and pre-B cells derived from bone marrow. VIP induced IgA1, IgA2, and IgM production in sIgM+, CD19+ fetal B cells stimulated with anti-CD40 monoclonal antibody (MoAb) without inducing the production of IgG1, IgG2, IgG3, IgG4, or IgE. The anti-CD40 MoAb plus VIP also induced IgA1, IgA2, and IgM production in sIgM-, CD19+ pre-B cells, which was enhanced by the addition of interleukin-7 (IL-7). This induction by VIP was specific, as the anti CD40 MoAb plus other neuropeptides [ie, somatostatin (SOM) or substance P (SP)] had no effect, and moreover, the induction was specifically blocked by a VIP antagonist. Furthermore, the anti-CD40 MoAb plus various cytokines, including IL 1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, transforming growth factor beta (TGF beta), low-molecular-weight B-cell growth factor (BCGF), and interferon-gamma (IFN-gamma), did not induce IgA1 and IgA2 production in fetal B cells or pre-B cells. These findings indicate that, in the presence of costimulators, VIP may induce IgA1 and IgA2 production by isotype switching. PMID- 7536490 TI - Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis. AB - In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7536492 TI - Predictive value for treatment outcome in acute myeloid leukemia of cellular daunorubicin accumulation and P-glycoprotein expression simultaneously determined by flow cytometry. AB - To evaluate the clinical relevance of multidrug resistance (MDR) phenotype, the intracellular daunorubicin accumulation (IDA) and P-glycoprotein (P-gp) expression were investigated in 87 adult patients with acute leukemia: 69 patients with de novo acute myeloid leukemia (AML), 10 with AML at relapse, and eight with secondary leukemia to myelodysplastic syndromes (MDS-AML). IDA and P gp expression were determined by double-labeling flow cytometry analysis. Of 87 patients, 36 expressed P-gp (41%). P-gp expression was more frequently observed in AML at relapse and MDS-AML as compared with de novo AML (P = .0001). P-gp expression was significantly associated with CD34 expression (P = .0003) and chromosome 7 abnormalities (P = .027). A significantly reduced IDA was observed in P-gp+ as compared with P-gp- patients (P = .0007). Of the 87 patients, 51 achieved complete remission (CR). A reduced IDA was observed in patients in failure as compared with patients in CR (22% +/- 17% v 42% +/- 21%; P = 10(-4). Twelve of 36 P-gp+ patients as compared with 40 of 51 P-gp- patients achieved CR (33% v 78%; P = 10(-4). The prognostic value of IDA and P-gp expression was confirmed in multivariate analysis. These data suggest that the determination of IDA and P-gp expression may be useful in designing therapy for patients with AML. PMID- 7536494 TI - Markedly high population of affected reticulocytes negative for decay accelerating factor and CD59 in paroxysmal nocturnal hemoglobinuria. AB - Paroxysmal nocturnal hemoglobinuria (PNH) blood cells lack glycosylphosphatidylinositol-anchored membrane proteins such as decay accelerating factor (DAF) and CD59. This lack is of diagnostic value in PNH. Because reticulocytes in PNH are not yet well characterized, we analyzed reticulocytes obtained from 12 patients with PNH and from 5 healthy volunteers by two-color flow cytometry with a membrane-permeable fluorescent dye, thiazole orange, to identify reticulocytes and monoclonal antibodies to DAF and CD59. Healthy individuals had no affected cells. In all patients, the population of affected reticulocytes negative for DAF and CD59 was markedly higher than the population of affected erythrocytes. Moreover, the population of affected erythrocytes became obviously low in patients who received transfusions and suffered from hemolytic precipitation, whereas the population of affected reticulocytes was unchanged. The persistently high population of affected reticulocytes, despite cytolytic exclusion and an inherently short lifetime, might possibly be explained by relative reticulocytosis caused by an anemia induced feedback stimulation of erythropoiesis in PNH. Thus, affected reticulocytes could be a reliable marker for the diagnosis of PNH and for the evaluation of erythropoiesis by PNH stem cell. PMID- 7536493 TI - Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia. AB - The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38- subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte-macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients. PMID- 7536496 TI - Localization of the neuronal form of nitric oxide synthase to mouse chromosome 5. PMID- 7536495 TI - Antigenic specificity of the "anticardiolipin" antibodies. PMID- 7536499 TI - [Effect of promoting blood circulation to remove stasis and supplementing qi and nourishing yin on the lipid peroxide and superoxide dismutase during experimental pulmonary fibrosis]. AB - The preventive and therapeutic effect of the traditional Chinese compound medicine Feixiankang (FXK) on the mice pulmonary fibrosis formation have been studied with the mice model. The level of LPO and the activity of SOD in the mice lung showed that LPO increased remarkably (P < 0.01), while the activity of SOD decreased significantly (P < 0.01) during the alveolar pneumonia period, although the LPO content had no significant change (P < 0.01) during experimental pulmonary fibrosis formation. In addition, the effect of FXK on the reducing of LPO was much stronger than that of the antioxidative agent Vitamin E. Also, FXK could improve the activity of SOD. PMID- 7536500 TI - The effect of peroxides on the vascular endothelium of isolated pig aorta in vitro. AB - The effect of peroxide on endothelial cells (perfused pig aorta) was examined using an in vitro perfusion model. Hydrogen peroxide was added to the perfusion medium (pig serum together with a buffer solution) which was expected to lead to an increased oxidation of lipids and lipoproteins. Oxidation processes of this type play a decisive role in the pathogenesis and progression of arteriosclerosis. The aim of the present investigation was to demonstrate by introducing hydrogen peroxide (H2O2) in varying concentrations (0.5-1.5%), the destructive impact of peroxides on the endothelium, while these cells are believed to play a key role in the pathogenesis of arteriosclerosis. The extent of endothelial cell impairment was assessed by means of silver staining visualisation of endothelial cell borders as well as light- and scanning electronmicroscopic investigation. It was discovered that the endothelial cells show increasing impairment after 10 h of perfusion due to the effect of peroxide (hydrogen peroxide). PMID- 7536498 TI - Interferons for viral hepatitis. PMID- 7536497 TI - Blood group antigens on complement receptor/regulatory proteins. PMID- 7536501 TI - Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3. AB - The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P 815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells. PMID- 7536503 TI - Experimental study for the development of an in vitro test for contact allergens. 1. Primary activation of hapten-specific T cells by hapten-conjugated epidermal cells. AB - We conducted a study on the primary in vitro activation of T cells from non sensitized mice by using hapten-conjugated Pam 212 cells (keratinocyte cell line). Furthermore, we attempted to develop a simple, quantitative in vitro test to assess the sensitizing potency of contact allergens and applied it to determine the stimulation index (SI) of various chemicals with known degrees of sensitizing potency. Monolayered Pam 212 cells were incubated with a variety of chemicals exhibiting allergenic potential. Washed and fixed T cells depleted of autoreactive T cells and macrophages from spleens of nonsensitized Balb/c mice were cocultured for 5 days with those monolayered Pam cells conjugated with chemicals. They were then harvested and restimulated with mitomycin-C-treated spleen cells conjugated with chemicals in 96-well culture plates to inhibit the proliferation of stimulator cells. We evaluated the sensitizing potency of the following chemicals: oxazolone, TNBS, DNFB and FITC (strong sensitizers); p phenylendiamine (p-PD), nickel chloride and potassium dichromate (potent sensitizers); betamethasone and budesonide (corticosteroids), and methyl salicylate (MS) as an irritant. T cells sensitized in vitro with TNP-Pam cells and macrophages demonstrated antigen-specific proliferation when restimulated in vitro with mitomycin-C-treated TNP-spleen cells. Subcutaneous injection of these T cells induced contact sensitivity in vivo in an antigen-specific fashion. While T cells cocultured with TNP-3T3 could not be activated even in the presence of macrophages. The SI of strong sensitizers was about 4.00 and that of p-PD was 2.36. The SIs of budesonide, betamethasone and MS were 1.62, 0.98 and 1.21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536502 TI - Human heart mast cells: a definitive case of mast cell heterogeneity. AB - Mast cells and their chemical mediators play a role in cardiac and systemic anaphilaxis. Perivascular and cardiac mast cells have been implicated in the pathogenesis of coronary artery spasm, atherosclerosis, myocardial ischemia, and cardiomyopathy. Despite this, nothing is known about the immunological and biochemical characteristics of the human heart mast cell (HHMC). We have isolated and partially purified HHMC and compared them with mast cells isolated from lung (HLMC) and skin (HSMC) tissues. Cross-linking of the high-affinity receptor for IgE (Fc epsilon RI) by a polyclonal anti-Fc epsilon antibody caused the release of preformed (histamine and tryptase) and de novo synthesized mediators [peptide leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)]. The tryptase content of HHMC (19.4 +/- 1.5 micrograms/10(6) cells) was lower than HSMC (33.4 +/- 2.5 micrograms/10(6) cells) and higher than HLMC (10.6 +/- 1.9 micrograms/10(6) cells). Maximal stimulation of HHMC with anti-IgE led to the release of LTC4 (17.5 +/- 5.1 ng/10(6) mast cells) and PGD2 (17.8 +/- 5.0 ng/10(6) mast cells, whereas HSMC synthesized more PGD2 (65.0 +/- 6.8 ng/10(6) mast cells) and much less LTC4 (< 5 ng/10(6) cells). Recombinant human C5a anaphylatoxin and protamine induced histamine release from HHMC and HSMC, but not from HLMC. Substance P and morphine selectively induced the release of histamine from HSMC, but not from HHMC and HLMC. Compound 48/80 caused histamine release from HSMC and HHMC, but not from HLMC. The pattern of mediators synthesized and the responsiveness of HHMC to different secretagogues appear unique providing strong evidence of human mast cell heterogeneity. PMID- 7536504 TI - Possible role of thromboxane A2 in hyperresponsiveness of isolated rat lung tissue in a Sephadex-induced eosinophilia model. AB - Antigen-stimulated contraction and release of chemical mediators were examined in saline- or Sephadex-treated rat lung parenchymal strips. Sephadex treatment caused eosinophilia in the blood and the lung tissue. Antigen challenge of the isolated parenchymal strips in Sephadex-treated rat was followed by passive sensitization, resulted in an augmented contraction and elevated releases of thromboxane (TX) B2 and peptide-leukotrienes (p-LTs) in bath fluid compared with those of saline-treated control. Although 5-hydroxytryptamine (5-HT) and histamine were significantly released after antigen challenge, the levels were not different between saline- and Sephadex-treated groups. DP-1904, a selective thromboxane synthetase inhibitor, and methysergide but not atropine significantly reduced the augmented contraction and inhibited the elevated TXB2 release in the Sephadex-treated group. Similar increased contraction and the elevated TXB2 release above were observed when Sephadex-treated rat lung strips were stimulated by exogenous 5-HT and LTD4. These augmented contractions were closely correlated with the increase in TXB2 level (r = 0.83; p < 0.01). In addition, contraction to U-46619, a thromboxane mimetic, was significantly greater in Sephadex-treated rat lung strips. Our results indicate that the ability of Sephadex-treated rat lung tissue to synthesize newly generated mediators such as TXA2 and p-LTs is increased, and the spasmogenic susceptibility of the lung tissue to TXA2 itself is modified by Sephadex treatment, suggesting these are due to the augmented contraction in an established hyperresponsiveness state induced by Sephadex. PMID- 7536505 TI - [Continuing hospice care of cancer--a three-year experience]. AB - The hospice at Mackay Memorial Hospital was established in February 1990. A group of team workers including physicians, nurses, social workers and the clergy were involved in this holistic care program for terminal cancer patients. Four hundred and seventy-nine patients were eligible for the program up to February 1993. Regarding duration of stay, 62.5% of patients resided for 14 days. Those surviving under 90 days constituted 75.5% of patients. Fifty-one point eight percent of patients died in the hospice and 18.2% died at home soon after being discharged from the hospice. Pain is the most common symptom among the patients. Treatment strategies vary according to the three-step-ladder protocol designed by WHO. Total pain relief was achieved in 80% of patients. Opportune private talking and family conferences formed the basis of the "peer model". Through this model, treatment decisions including physical, psychosocial and spiritual issues were made. Before the peer model, only 36 (10.3%) patients agreed with the idea of hospice care, while 257 (73.6%) patients agreed after the model was established. Awareness of dying was evident in 412 (86%) patients. Two hundred and eighty (68%) patients became aware of the prospect of death through guessing, while the other 132 (32%) patients were informed by medical staff. Problems encountered by the team workers included 1) needs in education and training, 2) psychological pressure, 3) management of loss and grief, 4) needs in supportive system and 5) troubles caused by families' lying to patients. The team workers were satisfied with the quality of care in 38.4% of patients and fairly satisfied with 30.7% of patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536506 TI - Odontogenic myxoma--characterisation of the extracellular matrix (ECM) of the tumour stroma. AB - In order to elucidate the origin of the odontogenic myxoma, the composition and the structural organisation of the extracellular matrix (ECM) of this tumour were characterised. Collagen type I, VI, procollagen type III, undulin, tenascin and fibronectin were demonstrated in biopsy material of 4 cases by polyclonal antibodies. The tumour stroma showed a pronounced reaction for collagen type I. Fibroblasts displayed an intense intracytoplasmatic reaction for procollagen type III, and collagen type I was not found in the fibroblasts of the adjacent normal oral mucosa. In contrast to the surrounding connective tissue, label for collagen type VI was weak, as was the reaction for fibronectin and tenascin. Undulin was almost undetectable. The immunohistochemical results suggest that the odontogenic myxoma is characterised by an as yet unobserved structural organisation of ECM proteins, a secretion defect of fibrillar collagens type I and III with no resemblance to physiological tooth development. PMID- 7536507 TI - Immunoreactive tenascin in tumours of salivary glands: evidence for enhanced expression in tumour stroma and production by tumour cells. AB - Tenascin, a large molecular weight extracellular glycoprotein expressed at the epithelial-mesenchymal interface during morphogenesis in embryo, wound healing and in the stroma of various benign and malignant tumours was evaluated in a series of primary epithelial tumours of salivary glands using a monoclonal antibody. Normal salivary glands (n = 5) had linear delicate band-like immunoreactive tenascin in relatively large excretory or intralobular ducts. Pleomorphic adenomas (n = 40) had heterogeneity of expression in modified myoepithelial cell-associated myxoid, hyaline and chondroid areas. Warthin's tumours (n = 10) had a linear immunoreactivity profile of tenascin just adjacent to the basal cells of the epithelial tumour component. A heterogeneity of expression with intense to low or negative stromal immunoreactivity was observed in adenoid cystic carcinomas (n = 8), mucoepidermoid carcinomas (n = 8), epithelial-myoepithelial carcinomas (n = 4), polymorphous low-grade carcinomas (n = 3), papillary cystadenocarcinomas (n = 15) and undifferentiated carcinomas (n = 3). In addition, small cystic spaces or lumens of epithelial-lined tubulo-ductal structures in numerous salivary tumours had positive immunoreactivity for tenascin, suggesting its production by the epithelial tumour component. An enhanced expression of tenascin in salivary tumours suggests a role of this protein in the stromal remodelling and tumour growth. PMID- 7536508 TI - DNA and keratin analysis of oral exfoliative cytology in the detection of oral cancer. AB - Refinements in oral exfoliative cytology may make it a suitable screening technique for the early diagnosis of oral cancer. In this study DNA range profiles were combined with keratin expression in an attempt to improve the diagnostic accuracy of oral exfoliative cytology. Smears were taken from 33 biopsy-proven oral cancers and the contralateral normal site. For DNA range profiles the smears underwent Feulgen hydrolysis, with DNA distribution being assessed using the Vickers M85 microdensitometer. For keratin expression a panel of antikeratin antibodies were applied. The smears for keratin expression were then graded on a three-point scale. Abnormal DNA range profiles were observed in 23 of 33 smears taken from oral cancers and in two smears from normal oral mucosa (sensitivity 70%, specificity 90%, positive predictive value 90%). The simple epithelial keratins 8 and 19 were identified in the majority of oral cancer smears. The sensitivity of keratin 19 was greater (90%). However, keratin 8 was the most useful keratin marker associated with malignancy (sensitivity 62%, specificity 100%, positive predictive value 100%). The combination of simple keratin expression and DNA content improved the cancer detection rate beyond that obtainable with DNA range profile alone. PMID- 7536509 TI - Induction of bleomycin resistance in a human oral squamous carcinoma cell line and characterisation of bleomycin-resistant and -sensitive clones. AB - We examined the change of sensitivity to antitumour agents by repeated treatment with bleomycin (BLM) using two oral squamous carcinoma cell lines, SCCTF and SCCKN. SCCTF exhibited minimal sensitivity to BLM and strong heterogeneity in BLM sensitivity, whereas SCCKN was highly sensitive to BLM and showed weak heterogeneity. When SCCTF was treated continuously with low-dose BLM (0.1 microgram/ml) but not intermittently with high-dose BLM (1 microgram/ml), the BLM sensitivity was rapidly decreased to acquire drug resistance. On the other hand, SCCKN was completely killed by the same treatments. To investigate the mechanism of induction of resistance in SCCTF, BLM-sensitive and -resistant clones, TF-S and TF-R, were isolated and analysed. Consequently, TF-R showed decreased cellular accumulation and retention of BLM, increased BLM hydrolase activity and elevated DNA repair activity concomitant with increased poly(ADP-ribose) polymerase activity as compared with TF-S. Therefore, it was suggested that antitumour drug-resistant clones were selectively grown from heterogeneous tumour cell population. PMID- 7536510 TI - Expression of c-kit receptor (CD117) and CD34 in leukemic cells. AB - The expression of c-kit receptor (c-kit R; CD117) and CD34 was examined in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) in blastic transformation (BT), and myelofibrosis (MF) in myeloid BT. In myeloid leukemia including AML, CML-myeloid BT and MF-myeloid BT, both c-kit R and CD34 were expressed synchronously, while in lymphoid leukemia including ALL and CML-lymphoid BT, only CD34 was highly expressed. A close correlation between c-kit R and CD33 expression and an inverse correlation between c-kit R and CD19 expression were observed when all of the myeloid plus lymphoid leukemia cells were analysed. There was a close correlation between c-kit R and CD34 expression in the myeloid leukemia cells. c-kit R expression may be associated with myeloid phenotypes of leukemic cells and may be useful for the diagnosis of myeloid leukemia. The literature of c-kit R expression in leukemic cells is reviewed here and the comparison of c-kit R and CD34 expression in normal hematopoietic progenitor cells with those on the leukemic counterparts was discussed. PMID- 7536511 TI - Effect of recombinant human interferons in inducing differentiation of acute megakaryoblastic leukaemia blast cells. AB - The effects of interferons (IFN)-alpha, beta 2 and -gamma in inducing megakaryocytic differentiation of blast cells from a patient with acute megakaryoblastic leukaemia (AMegL) was investigated in liquid suspension culture by the increase in CD41 and CD42b expressions using monoclonal antibodies in the APAAP technique. After six days of culture, the percentage of CD41 and CD42b positive cells increased in control cultures from 15.2% and 10.6% on day 0 to 32.0 +/- 4.3% and 22.1 +/- 2.5%, respectively. The addition of IFN-alpha significantly increased the number of CD41 and CD42b positive cells by about two to three fold compared to control cultures (p < 0.01) and by about four to six fold compared to day 0 (p < 0.001) Similarly, IFN-beta 2 induced a significant increase in CD41 and CD42b positive cells. On the other hand, IFN-gamma failed to increase the number of CD41 and CD42b positive cells in comparison to control cultures on day 6 and instead stimulated a significant increase in the number of monocytes/macrophages by about ten fold compared to control cultures in IFN-gamma treated cultures (p < 0.001). The present results suggest that megakaryocytic differentiation of blast cells in AMegL could be induced by IFN-alpha and beta 2 and support a clinical role for them in the treatment of AMegL patients. Also, the present results showed that monocytic differentiation of blast cells in AMegL could be stimulated by IFN-gamma, supporting the multipotent stem or progenitor cell origin of the AMegL subtype of acute myelogenous leukaemia. PMID- 7536512 TI - Synergism of interleukin 12, interleukin 3 and serum factor on primitive human hematopoietic progenitor cells. AB - Interleukin 12 (IL-12), or natural killer cell stimulatory factor (NKSF), has multiple biologic effects on T lymphocytes and natural killer cells. In this study, we evaluated the effect of IL-12 on human hematopoiesis by analyzing the growth of CD34+33- cells in methylcellulose culture with or without serum. Blood cells were collected by apheresis from an expanded stem cell pool during the rebound phase of hematopoiesis after chemotherapy and purified by combined purification procedures to yield target CD34+33- cells. IL-12, by itself or in combination with IL-3 and erythropoietin (Epo), had no effect on the formation of colonies from the unfractionated blood mononuclear cells. In the presence of fetal bovine serum (FBS), IL-12 increased the number of colony forming units for granulocyte-macrophage (CFU-GM) from CD34+33- cells which responded to IL-3, but not the number of CFU-GM from those which responded to stem cell factor (SCF) or granulocyte colony stimulating factor (G-CSF). This effect was not abolished by the addition of monoclonal antibodies against tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN-gamma), IL-1 alpha, IL-6, G-CSF, or granulocyte/macrophage CSF (GM-CSF). However, the stimulatory effect of IL-12 was abolished in secondary plating in serum-containing methylcellulose culture after short-term (48 h) liquid incubation with IL-3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536513 TI - Effects of continuous stem cell factor administration on normal and erythropoietin-stimulated murine hemopoiesis: experimental results and model analysis. AB - The aim of this study was to determine how stem cell factor (SCF) modifies hemopoietic cell production. First we determined the effects of a prolonged SCF administration on murine hemopoiesis and analyzed the results by a mathematical simulation model of hemopoiesis in order to explain the data. Subsequently we investigated the effects of simultaneous coadministration of SCF+erythropoietin (Epo), to see how effects of early and late cytokines superimpose. SCF administration during 14 days induced a proliferative wave through the hemopoietic system; colony forming units-granulocyte macrophage (CFU-GM), burst forming units-erythroid (BFU-E) and colony forming units erythroid (CFU-E) were the first to be augmented, followed by their respective progeny, ultimately leading to increased blood cell numbers. Despite continued treatment most cell numbers returned to normal values in 14 days, colony forming units-spleen (CFU S), however, remained elevated. This wave pattern could be explained within the framework of a previously established mathematical model of hemopoiesis, if it was assumed that SCF affected the cycling status of primitive cells and if regulatory feedback loops of erythroid and myeloid progenitors on these cells were also allowed. Simultaneous SCF and Epo administration led to synergistic effects on CFU-E numbers and hematocrit values at moderate Epo doses. At high Epo doses, however, this was less pronounced. We conclude that SCF increases the input into committed hemopoietic lineages, where late acting cytokines can induce further amplification. PMID- 7536514 TI - [Tissue-specific expression of the estS gene in transgenic Drosophila]. PMID- 7536515 TI - [The effect of loratadine on skin and bronchial reactivity and histamine liberation from basophils in patients with atopic asthma and allergic rhinitis]. AB - The effect of the therapy with Loratadine on skin and bronchial reactivity to histamine and on a specific histamine release from isolated basophils of allergic patients was assessed. The studies were carried out on a 34 patients (23 with pollen rhinitis and 11 with a mild bronchial asthma). The effect loratadine versus placebo on the skin reactivity to histamine was measured in patients suffering from pollen rhinitis. Outside the pollen season in 12 patients from the above group a histamine release from isolated basophils was evaluated by Shore method, using a-IgE and pollen allergen as liberators, before and after 4 days therapy with loratadine. In the group of the 11 asthmatic patients the effect of the single dose of 10 mg Loratadine on spirometric parameters and on a bronchial response to histamine was studied. The Bronchial Provocation Test with histamine was performed by the Ryan's method. The results were expressed as PC20 FEV1. It was shown that loratadine statistically significant limited of the skin reactivity to histamine and specific histamine release from basophils. The one tablet of Loratadine have not changed of spirometric parameters in the patients with mild asthma, but statistically significant reduced their bronchial response to histamine. PMID- 7536516 TI - [Spontaneous and allergen induced basophil histamine release and bronchial hyperreactivity]. AB - In this study we have measured spontaneous and allergen induced basophil histamine release before and 24 hours after specific allergen bronchoprovocation challenge. Non-specific bronchial hyperreactivity was estimated twice: before and after allergen challenge. 15 atopic asthmatic entered the study. 8 of them developed only early asthmatic reaction and 7 demonstrated dual asthmatic reaction. In dual responders group higher values of spontaneous basophil histamine release before and increase of allergen induced basophil histamine release after specific allergen bronchoprovocation challenge were observed. There was an inverse correlation between values of allergen induced basophil histamine release and values of PC20 for histamine. PMID- 7536517 TI - Defining potential targets for immunotherapy in Burkholderia cepacia infection. AB - Burkholderia cepacia has become a serious source of infection in patients with cystic fibrosis. Antibiotic therapy is difficult as the bacteria are intrinsically resistant to most antibiotics. The present study compared the antibody response by immunoblot of 50 negative control sera, 22 patients with cystic fibrosis and no evidence of B. cepacia, 9 clinically well patients with cystic fibrosis colonised by B. cepacia and 5 patients with cystic fibrosis and deteriorating or fatal B. cepacia infection. Nineteen antigenic bands varying in apparent molecular weights from 19 to 170 kDa were identified. Two bands at 19 and 21 kDa were only present when the organism was grown in an iron-deficient medium. The band at 30 kDa was identified as a porin and the possession of IgG antibody carried a statistically significant (P = 0.00003) better prognosis. This antigen was thus a potential target for immunotherapy. PMID- 7536518 TI - The effect of the inhibition of the nitric oxide synthase by NG-nitro-L-arginine on the erection of the rabbit. PMID- 7536519 TI - Rabbit penile corpus cavernosum: electrical and mechanical activity of isolated strips. PMID- 7536520 TI - Proliferation control in individual cell cultures from prostatic carcinomas: characterization by using a multiparameter system. PMID- 7536522 TI - E-cadherin: expression of the epithelial cell-cell-adhesion molecule in prostatic carcinoma and normal prostate. PMID- 7536521 TI - Prevention of colon tumors following urinary diversion by antagonizing of nitrosamines or separation of feces and urine? PMID- 7536523 TI - Establishment and characterization of a seminoma cell-line (S2). PMID- 7536525 TI - Effect of local pentosan polysulphate pretreatment on the immune response to intravesical BCG. PMID- 7536524 TI - In-vitro cell growth of human urothelial tumor cell lines under exposure with IL 3, GM-CSF, G-CSF and by combined administration with methotrexate, vinblastine, adriamycin and cisplatin. PMID- 7536527 TI - [The role of the laser in urology]. AB - There is currently a renewed interest in laser in the field of urology, essentially for the treatment of benign prostatic hypertrophy, as a result of recent developments in the field of fibres. Laser is light coherent in time and space emitted continuously or in pulses. Only its thermal and photochemical properties are used in urology. Endoscopic coagulation of superficial bladder tumours by Nd YAG laser does not seem to be better than classical endoscopic resection, at the present time, as although it is less haemorrhagic, it does not decrease the recurrence rate. Photochemotherapy of bladder carcinoma in situ still constitutes a complex treatment protocol reserved for specialized centres. Lastly, upper urinary tract tumours can be treated by Nd YAG laser coagulation, in rare indications which are the same as those of ureteroscopic or percutaneous resection. The recent development of lateral firing laser fibres and contact tip fibres has led to a renewed interest in laser in benign prostatic hypertrophy. Two different techniques have been proposed: Nd YAG laser coagulation under direct visual or ultrasound control (TULIP), which gives delayed objective results (two or three months) and contact tissue vaporization (Nd YAG, diode), whose effects are more immediate. In both cases, intraoperative bleeding is minimal and the length of hospital stay is decreased, but the duration of urine drainage remains to be defined. Endoscopic pulsed laser urinary lithotripsy (dye, Ho YAG), although effective and atraumatic, is not justified at the present time because of its high cost compared to mechanical percussion lithotripters. Lastly, laser treatment for urethral stricture has not been found to be superior to classical scalpel urethrotomy and laser tissue welding is still in the experimental stages. In conclusion, laser technology, especially fibers, has currently reached an important phase of development with applications for urological disease, essentially in the treatment of benign prostatic hypertrophy in order to reduce the morbidity of classical endoscopic resection. However, other urological applications of laser could be validated in the near future due to the development of less expensive lasers (diode) and/or with multiple functions (Ho YAG). PMID- 7536528 TI - [Cancer of the prostate: the experiences and opinions of T. A. Stamey (Stanford) on the natural history, positive resection margins and the role of radiotherapy. (I). The conference of 20 November 1993, CNIT, Paris]. PMID- 7536529 TI - [The value of PSA density for the diagnosis of prostatic cancer and for the indication of radical prostatectomy]. AB - We evaluated the diagnostic and prognostic value of PSA density (PSAD) in a prospective study of 100 men consulting for various urinary tract disorders. PSAD was calculated by the ratio of serum PSA over the ultrasonographic volume of the prostate gland, and was expressed in ng/ml/ml. The mean PSAD of 31 patients with prostate cancer was significantly higher than that of the 69 patients without prostate cancer (p = 0.0003). The patients were classified into two groups according to the serum PSA value. Forty nine patients had a serum PSA < or = 10 ng/ml, 31 of whom had a PSAD < or = 0.15 ng/ml/ml and 29 of these 31 patients (94%) did not have prostate cancer; 18 patients had a PSAD > 0.15 ng/ml/ml and six of them (33%) had prostate cancer (p = 0.04). Fifty one patients had a serum PSA > 10 ng/ml and 23 of them (45%) has prostate cancer; 14 of these 51 patients had a PSAD < or = 0.30 ng/ml/ml and two of them (14% a had prostate cancer; 37 patients had a PSAD > 0.30 ng/ml/ml, 21 of whom (57%) had prostate cancer (p = 0.016). Twenty one of these 31 patients with prostate cancer were candidates for radical prostatectomy. Four patients were N+ and 17 underwent radical prostatectomy. Capsular invasion was significantly correlated with PSAD. None of the four patients with PSAD < or = 0.30 ng/ml/ml showed capsular invasion, while 11 of the 13 patients (85%) with PSAD > 0.30 ng/ml/ml had capsular invasion. In conclusion, in patients with PSA < or = 10 ng/ml, calculation of the PSAD avoids approximately two-thirds of prostatic biopsies, with a very low risk of missing a clinically significant cancer. On the other hand, PSAD is of more limited value in patients with PSA > 10 ng/ml. In cancers considered to be operable clinically, the cut-off value of 0.30 ng/ml/ml for PSAD is predictive of capsular or lymph node invasion with a diagnostic accuracy of 94%. PMID- 7536526 TI - [Cancer of the prostate. 1. Epidemiology]. AB - Prostatic cancer is the second most frequent cancer in men in France. It is a serious disease with a relative 5-year survival of 42%. Although the incidence of latent forms appears to be constant throughout the world, the incidence of clinical forms varies from country to country and according to race. These aspects are in favour of a dual mechanism of prostatic carcinogenesis: initiation of a cellular modification, which may be transmitted genetically according to an autosomal dominant mode, but whose expression may be influenced by the environment, and successive steps of transformation (epigenetic factors) which are essentially environment-dependent. The main identified risk factors essentially consist of a direct family history, age and a diet rich in animal fats. In contrast, neither the presence of benign prostatic hypertrophy, nor the characteristics of the sex life or a history of vasectomy appear to influence the incidence of prostatic cancer. The main epidemiological data currently available are presented. PMID- 7536530 TI - [Treatment of benign prostate hypertrophy using Prostatron: a study of the effects of treatment temperature elevation]. AB - Transurethral microwave thermotherapy was administered to 91 consecutive men with moderate to severe benign prostatic hyperplasia. Mean patient age was 68 years, with a range of 55 to 87 years. Treatment was given in one 60-minute session, on an outpatient basis and without anesthesia. Of the 91 original patients 67 (73.6%) were followed from 3 to 18 months, mean 6.1 months. The microwave emission was continuously monitored to permit the control of the urethral temperature level. Patients were prospective and randomly assessed into 2 groups: group 1 (34 patients) who underwent treatment with urethral temperature between 42 and 43 degrees C and group 2 (33 patients) treated with higher temperature ranging between 43.1 and 45 degrees C. There was improvement of the irritative and obstructive symptoms, prostate weight and residual urine, in both groups after treatment (p < 0.001). PSA did not show a statistically significant difference after TUMT in both groups. Uroflow improved in both groups after the treatment; however, only the group treated with higher temperature showed statistically significant difference (p < 0.001). In the lower temperature group there were 2/34 (5.8%) failures; both patients were still obstructed and underwent transurethral resection. A total of 2/33 (6%) failures were observed in the higher temperature group. Patients showed no general conditions which might allow the use of anesthesia. Therefore, they underwent a second postoperative microwave treatment after 2 and 3 months with uneventful clinical recovery at an early follow-up. No systemic complications were encountered. There were minor complications such as epididymitis, urethral bleeding and severe micturition discomfort within the first 30 days postoperative.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536531 TI - [Intracranial pressure in monkeys under microgravity]. AB - The results of measurement of the intracranial pressure (ICP) in monkeys on board of the biosatellite "Cosmos-2229" are presented. The ICP grew insignificantly in microgravity. The ICP pulse waveform changed significantly in the first day of the flight but returned to normalcy later. PMID- 7536532 TI - [The role of the beta-adrenoreceptors in the manifestation of the peristaltic reflex]. AB - Distension of the ileal segment was shown to induce its contraction in isolated portion of the cat ileum, the response being abolished by M-cholinergic blockade and preserved after N-cholinergic blockade. Blockade of the beta-adrenoreceptors or their stimulation did not affect the peristaltic reflex. The findings suggest that the excitatory beta-adrenoreceptors are not directly involved in the first phase of peristaltic reflex. PMID- 7536533 TI - [The transplantation to adult animals of the pancreas from fetuses]. AB - The possibility of using the syngeneic and allogeneic foetal pancreas implantation was shown for therapy of diabetes mellitus. Cases of the implantation with no immunosuppression were studied in 57 patients. PMID- 7536534 TI - [The dynamics of milk secretion in white mice and its consumption by progeny]. AB - Only about 72 per cent of the litter manifested milk ingestion during every next nursing in white mice. The correlation between the dynamics of the milk ejection from the mammary gland and individual real milk intake by the litter, is discussed. PMID- 7536535 TI - [A method for the microdialysis of the cerebral cortex in research on the neurophysiological correlates of cognitive processes in monkeys]. PMID- 7536536 TI - [The computerized identification of the phases of the migrating myoelectric complex]. PMID- 7536537 TI - [The level of cyclic nucleotides during spontaneous smooth-muscle contractions in the guinea pig]. AB - The relationships between isotonic tension and changes in cAMP and cGMP levels was studied in isolated preparations of the guinea-pig taenia coli and myometrium during spontaneous contractions. No significant changes in cyclic nucleotide levels were detected at any stage of the spontaneous contraction-relaxation cycle. The finding suggests that these changes play no significant role in the regulation of spontaneous contractions of the taenia coli and myometrium. However, other data obtained suggests the possibility of combined actions of the cGMP and phosphatidylinositols in control of smooth muscle motility during muscarine stimulation. PMID- 7536539 TI - [The prolactin level in the cerebrospinal fluid and blood plasma of cattle at different stages of ontogeny]. AB - The prolactine level in the CSF was shown to be higher than in the blood plasma in 5-8-month-old and 1-2-month-old calf. On the 9th month, the prolactine level in the blood becomes higher than in the CSF. No sexual difference was found in the prolactine levels. The data obtained suggest an extrahypophyseal origin of the CSF prolactine and its important role in early stages of the ontogeny. PMID- 7536538 TI - [The lateralized effects of the melanostatin analog alaptide in rats raised in isolation and in a community]. AB - Effects of alaptide on self-stimulation of the lateral hypothalamus, rotation behaviour and learning ability were studied in rats growing either in social adaptation or in groups. The effects on self-stimulation were opposite in these rats. Alaptide facilitated the learning process in grouped rats and potentiated the indirect dophamine-mimetic action of amphetamine in isolated rats. PMID- 7536540 TI - [The role of amplitude modulation in forming the auditory images of speech signals]. AB - The identification of stationary vowels in influenced by the change in relations of formant amplitudes as if their "centre gravity" is used in phoneme decision. That is valid for formants spaced in critical frequency band equal approximately 3.5 Bark. The hypothesis about large-scale spectral integration in auditory system is based on this effect. The data obtained supports the hypothesis in respect to non-stationary sounds, too. Imitation of the large-scale spectral integration on the model of auditory processing suggests that the frequency band of the integration may be equal to 3.5 Bark. PMID- 7536541 TI - [The influence of baroreceptor reflexes on the effects of rapid changes in blood volume in rats]. AB - The effect of suppression of the sinocarotid and cardioaortal areas on systemic pressor responses were studied during fast administration of fluid. The data obtained does not corroborate the traditional ideas of the universal buffer role of the baroreflexes in regulation of arterial pressure under live conditions of formation of systemic pressor responses as the suppression of the baroreceptor areas tells not on the increment of the pressor responses but either suppresses them or preserves them intact. PMID- 7536542 TI - [The reactions of the cardiorespiratory system to exposure to a hyperbaric environment in the simulation of free-ascent conditions]. AB - In 44 adult rabbits simulating 360 to 700-m ascent, bradyarrhythmia, auricular extrasystoles, partial antegrade block, dyspnea and transitory apnoea were found. Changes in the R-R histograms as well as autocorrelation function and spectrum of the cardiointervalogram showed overstrain and exhaustion of the cardiac rhythm control system at the end of decompression. Mechanisms of the cardiorespiratory system's responses to hyperbaric ambient conditions are discussed. PMID- 7536543 TI - [The reactivity of the mouse aorta during multiple prolonged hyperbarism]. PMID- 7536544 TI - [The contractile activity of the lymphatic vessels in rats after head-down tilt exposure]. AB - After antiorthostatic attitude of rats during 12 days, the contractile activity of their isolated lymphangions of the thoracic duct was studied. The amplitude of contractions was found to be decreased as well as the effects of serotonine, histamine, and acetylcholine, whereas that of adrenaline increased. The latter finding suggests enhancing of the activity of lymphatic vessels' adrenergic structures in antiorthostatic. PMID- 7536545 TI - [Metabolic acidosis under an acid load and the role of the respiratory system in its compensation in middle-aged and elderly persons]. AB - 30 healthy 60- to 89-year-old subjects and 20 young ones (20 to 31 years) were given orally ammonium chloride 125 mg/kg. The signs of subsequent metabolic acidosis were more obvious in older subjects and they lasted longer. In young subjects, the intake of ammonium chloride induced a considerable hyperventilation. PMID- 7536546 TI - [The effect of immobilization during adrenoreceptor blockade on the neutrophil lysosomal apparatus in rabbits]. AB - Peripheral adrenoreceptors were shown to take part in responses of the neutrophil lysosomal apparatus in formation of the stress syndrome. Different mechanisms of neutrophilic leucocytosis and degranulation were suggested. PMID- 7536547 TI - [The asymmetry of parietal-visual and parietal-auditory transcallosal potentials and their sex dimorphism]. AB - "Reverse" transcallosal responses (TCR) occurring in the visual and auditory cortex during stimulation of the temporal associative cortex of opposite hemisphere, were studied. The right hemisphere was shown to domineer in females, whereas a greater latency of the temporal-visual positive-negative TCRs was found in the males' right hemisphere and the females' left one. These and other data obtained suggest a greater lateralisation of the brain in males. PMID- 7536548 TI - [The role of adrenoreceptor mechanisms in the feedback inhibition of pancreatic secretion]. PMID- 7536549 TI - [The reactivity of the mesenteric arteries and vein to exposure to endogenous noradrenaline in normal rats and rats with DOCA-salt hypertension]. AB - The DOCA-salt hypertension was shown to induce changes of different types in reactivity of the mesenteric artery and vein, acting on the contractility of the vessels rather than on the sensitivity of their receptor apparatus. The observed heterogeneity of the alpha-adrenoreceptors in the artery and the vein suggests that the artery's reactivity is under the control of both humoral and neurogenic factors, whereas the vein's reactivity is neurogenically controlled rather. PMID- 7536550 TI - [The characteristics of the neuronal organization of the sympathetic-activating and sympathetic-inhibiting mechanisms of the visceral area of the limbic cortex]. AB - Effects of high frequency stimulation of dorsal and ventral regions of anterior area 25 of the limbic cortex on the tonic electrical activity of two postganglionic sympathetic nerves of stellate ganglion-inferior cardiac and vertebral nerves and arterial blood pressure were studied in anesthetized cats. It was revealed three types of medullary influences: generalized activation of both sympathetic nerves and pressor responses mainly in case of stimulation of ventral structures, generalized inhibition of electrical activity of both sympathetic nerves and depressor responses in case of stimulation of dorsal region of area 25 and selective modulation of activity, namely increase in activity of inferior cardiac nerve and simultaneous decrease of activity of vertebral nerve. According to data of our previous comparative studies of sympathetic responses of inferior and vertebral postganglionic nerves to stimulation of anterior and posterior hypothalamus as a working hypothesis it is supposed that the dorsal region of area 25 of limbic cortex is the analogy of trophotropic system of anterior hypothalamus, the ventral region--of ergotrophic system of posterior hypothalamus. PMID- 7536551 TI - [A device for the digital recording, synthesis and playback of spatial acoustic signals]. PMID- 7536552 TI - [At the sources of the physiological direction in Russian psychoneurology]. PMID- 7536553 TI - [The dopaminergic and serotoninergic components of the self-stimulation reaction of the lateral hypothalamus in rats with disruption of the medial prefrontal cortex]. AB - Amphetamine (1 mg/kg) promoted a pedal self-stimulation of lateral hypothalamus of Wistar rats in the Skinner box. An unipolar lesion of medical prefrontal cortex with kainic acid 10-14 days prior to an experiment did not prevent the facilitating effect of amphetamine on self-stimulation, though the levels of hypothalamic noradrenaline and dopamine are 43% and 4.5-fold decreased respectively, the hypothalamic serotonin concentration not changing. Lysergic acid diethylamid (10 mkg/kg) did not influence on self-stimulation response in rats with damaged medial prefrontal cortex, but after preliminary administration prevented the stimulating effect of amphetamine on self-stimulation of lateral hypothalamus. The data are discussed from the point of view of a hypothesis that the phenomenon obtained is associated with the presence of a hypothalamic autoregulatory dopaminergic system providing the realization of self-stimulation. However, it does not exclude that the modulating influence of medial prefrontal cortex on lateral hypothalamus is connected with not only dopaminergic but with serotoninergic axons too. PMID- 7536554 TI - [The effect of the prolonged experience of aggression and subordination on the adrenal cortical and androgenic functions of inbred male mice]. AB - Repeated experience of aggression and submission in daily agonistic confrontations differently changed stress response in winners and losers of CBA/Lac and C57BL/6J strains. Experience of defeats was accompanied by expressed decrease of testosterone level in losers. Number of hemorrhages and erosions was significantly more in losers when compared to winners. Some strain differences in stress reaction were found in winners and losers as compared to controls. PMID- 7536555 TI - [The glycosaminoglycans of the bone marrow erythroblast islands in the rat]. AB - The glycosaminoglycans (GAG) types of normal rat bone marrow's erythroblastic islands were found to be similar to that of human macrophages. Induced anaemia increased the sulphated GAG. PMID- 7536556 TI - [The effect of short-term anoxia on the mechanisms of intracellular signal transduction in the cat cerebral cortex]. AB - Changes in the intracellular bound calcium, polyphosphoinositides and cAMP were studied during 60 min. of recovery after 50-90 s of anoxia in the cat cerebral cortex. Different changes of the intracellular regulatory system's components were revealed in the reoxygenation period. PMID- 7536557 TI - [Muramyl peptides and sleep]. AB - Effects of muramyl peptides from bacterial cell walls (MDP and GMDP), their fragments, steric isomers and structural analogues were studied on sleep in rabbits. An increase in the SWS, decrease in PS, rise in body temperature were found following minimal doses, whereas pathological responses in the EEG and sleep as well as pyrogenic effects occurred after higher doses. The GMDP analogues affected sleep weakly, and isomers and fragments were inactive. Possible role of muramyl peptides in normal and pathological regulation of sleep is discussed. PMID- 7536559 TI - [Steroidogenesis in the adrenals and gonads of rats following the neonatal administration of hydrocortisone]. AB - Neonatal administration of hydrocortisone decrease steroidogenesis in the rat adrenals in immature and mature animals production of progesterone and androstenedione reducing, at that. Primary long-term disturbance of central regulatory influences controlling steroidogenesis in adrenals, was proposed. The steroid production disturbance in the gonads was lesser than in the adrenals and was found only in mature rats. PMID- 7536562 TI - [Optimization of the electrostimulation parameters for cochlear implantation]. PMID- 7536558 TI - [The glucocorticoid receptors in the hippocampus of rats with different excitabilities of the nervous system]. PMID- 7536563 TI - [The temperature of different body organs in white rats being cooled in water and in the recovery period]. AB - In water cooling of white rats down to rectal temperature 17.37 +/- 0.03 degrees C, the temperature of the liver decreased by 1.13 +/- 0.03 degrees C more than in the brain. However, during independent recovery from the hypothermia, the liver temperature reached a higher value earlier than in other organs. This can be of importance for heating the organism. PMID- 7536560 TI - [The participation of serotonin S2 receptors in realizing the effects of stress on ulcer formation in the gastric mucosa of inbred mice]. AB - Significant interstrain differences in intensity and character of changes in gastric mucosa induced by affecting the serotoninergic system, were found. The main mechanism of stress-induced stomach ulceration was found to involve brain serotonin in central regulation of the stomach blood flow. Use of exogenic amine and its precursor corroborated the serotonin-dependent ulceration of the stomach in mice. PMID- 7536561 TI - [The principles of oxygen standardization in artificial hyperbaric gas mixtures during the prolonged stay of aquanauts at depths down to 500 m]. AB - In simulation of saturation dives at 3.6-5.1 MPa, physiologically optimal limits of oxygen partial pressure were determined in breathing gas mixtures. The parameters of oxygen-dependent carbohydrate and lipid metabolism as well as the blood oxygen transport function were found to be the most important criteria for determination of the optimal concentration of oxygen in hyperbaric breathing gas mixtures. PMID- 7536564 TI - [The neuronal reaction of the rabbit thalamus to thermal stimulation of the skin]. AB - Skin temperature of the rabbit back and face was increased to 38-40 degrees C or decreased to 22-26 degrees C. Out of 48 units under study 10 neurons responded to the cold skin stimulation, the latency of the responses varying from 10 to 50 s. The thalamic neurons responded also to the light, sound and mechanical stimuli. PMID- 7536565 TI - [Protein starvation and the trophic barrier functions of the enzymatic and transport systems of the digestive and nondigestive organs in adult and growing rats]. AB - A reduced activity of cytosol dipeptidases was revealed both in epithelial and subepithelial layers of the small intestine against the background of protein deprivation in adult and maturing rats. The latters' enzyme systems of the small intestine and kidneys' layers proved to be particularly sensitive to protein deprivation. The disturbance of the digestive-barrier functions provides favourable conditions for penetration of foreign protein and peptide substances into the organism's inner milieu which can be dangerous for a maturing organism. PMID- 7536566 TI - [Cortical mechanisms in the sensory organization of goal-directed movements]. PMID- 7536567 TI - [Brain activity induced by changes in acoustic stimuli]. AB - The averaged variants of the EPs to deviant stimuli had more obvious components N100, N250 and P300 as compared with the EPs to standard stimuli. The data obtained show occurrence of a negative shift during presentation of a rare stimulus within the sequence of standard ones. This negativity seems to reflect the processes of comparison of acting excitations with the traces of previous stimuli. PMID- 7536569 TI - [The neuroendocrine aspects of the participation of the epiphysis in the regulation of lactation]. AB - Pinealectomy was shown to suppress the milk ejection reflex and increase the luteinizing hormone secretion in lactating rats. Suckling caused the same type changes of prolactin though at a lower level, in these rats. The data obtained suggest that the pineal gland together with the oxytocinergic system are involved in complex mechanisms of neurohumoral regulation of the lactogenic and gonadotropic hormones secretion during lactation. PMID- 7536568 TI - [Cardiovascular system reactions and bioenergy metabolism in relation to adaptation to apnea]. AB - Responses of the cardio-vascular system and bioenergetic metabolism were studied in the heart tissue in apnoea in naturally adapted to diving musk-rats and unadapted rats and mice. A sharp bradycardia was shown to develop in former animals in arresting a breathing as well an increase in the neutrophils contents in the blood. The data obtained suggest that release of the necessary oxygen and antiradical defence functions can be related to the catalase activity. PMID- 7536570 TI - [The comparative characteristics of the electroretinograms of vertebrates evoked by light and x-ray stimulation under different conditions of adaptation]. AB - Existence of two electrical responses specific for the X-rays and differing from the responses to red or blue light, was confirmed in the electroretinogram of the frog eye during darkness adaptation, monochromatic illumination as well as in response to paired stimuli in different combinations of red and blue light and the X-rays. The data obtained suggest the possibility of using roentgenophosphen as a test for determining of individual radiation sensitivity in humans. PMID- 7536571 TI - [Bioelectrical reactions on the surface of the mamma in the goat]. AB - Spontaneous and evoked biopotentials were recorded on the mammary gland surface in goats. The biopotentials appear in the course of secretion during milk ejection as well as under electrical, mechanical and hormonal actions. They might be considered as adequate indices of secretion and ejection processes in the mammary gland. PMID- 7536572 TI - [The action of tetraethylammonium on the electrical responses of the neurons of the stretch receptors in the crayfish]. AB - The effects of intracellular pressure injection of tetraethylammonium and potassium salt solution has been studied for its effect on the receptor potential of the slowly adapting stretch receptor of crayfish. It was found that the pressure injection caused an increase in the static phase of the receptor potential. Intracellular microiontophoretic injection and extracellular application of tetraethylammonium have not exert an effect on the adaptation of receptor potential. The results suggest that tetraethylammonium-sensitive potassium channels are not involved in adaptation of the receptor potential of mechanoreceptors. PMID- 7536573 TI - [The mechanisms of the regulation of receptor-dependent calcium ion influx into the cells of Ehrlich ascites carcinoma]. PMID- 7536574 TI - [Creatine kinase as an index of the body functional state]. AB - Factors affecting activity and isoenzyme pattern of creatinine kinase in animal tissues, were analysed. Based on the analysis and study of creatine kinase systems at the molecular, cellular, tissue and organism levels, the activity and isoenzyme pattern of creatinine kinase was shown to be able to serve as an indicator of the organism functional state. PMID- 7536575 TI - [Fibroblast growth factors (a short review)]. AB - Some features of structure and expression of bFGF gene and FGF receptors genes, unique secretion property of bFGF, as well as FGF's interactions with heparin containing components of the cell and extracellular matrix are briefly observed. These special features may underlie the multifunctionality of members of ubiquitously distributed fibroblast growth factor family which recently have been filled up with two new members: androgen-induced growth factor--FGF-8 (Tanaka A., Miyamoto K., Minamino N. at al., 1992) and glia-activating factor--FGF-9 (Naruo K., Seco C., Kuroshima K. at al. 1993). PMID- 7536576 TI - [The A. A. Ukhtomskii Physiological Institute of Saint Petersburg University in the history of its development (on the 60th anniversary of its founding)]. PMID- 7536577 TI - [The control of milk consumption in white mouse pups]. PMID- 7536579 TI - [Seasonal changes in adrenoreactivity and a modification in the fractional phospholipid composition of the frog bladder]. AB - The study has shown seasonal changes in the responses of the frog bladder isolated preparation to exogenous adrenalin. The finding is explained by heterogeneity of the smooth muscle adrenoreceptors and alteration of functionally domineering populations during the winter-spring cycle. The changes corresponded to modifications of the fraction composition of phospholipids, the modifications being, probably, one of the ways of regulation of the smooth muscle adrenoreceptor apparatus' activity. PMID- 7536581 TI - [The modulating effect of an optical sensory input on the excitability of the motor center in insects]. AB - Changes in slow and fast motoneurone unit activity of the cockroach in response to light stimulation of the eye revealed a modulating effect of the sensory input on launching and frequency of the unit discharges. The effect was shown to depend on initial state of the excitability which can be changed by either activating or blockade of the GABA-ergic receptors and extracellular anode polarisation of the segmental motor centre. PMID- 7536578 TI - [The metasympathetic mechanisms supporting the constancy and stability of the internal environment in vertebrates]. AB - The data were obtained with the aid of extracellular recording of unit activity in the cardial, enteral and respiratory parts of the metasympathetic nervous system, staining of the neural tissue preparations of atrial walls, intestine and lower part of the guinea pig trachea. The neurons were shown to be very sensitive to the main homeostatic contants: pH of the perfusion solution, its temperature and the grade of oxygenation. These structures were shown to take a direct part in maintenance of the constancy and stability of the organism inner milieu. Sympathetic nervous system differs from the above system in that the former is the system of transmission, defence, mobilisation of resources which is necessary for active interaction of the organism with ambient milieu. PMID- 7536580 TI - [Heart-stimulating neurons in the subesophageal ganglia of the African snail Achatina fulica Ferussac]. AB - 5 cardiostimulating neurons belonging to 3 different functional groups were studied in visceral and right parietal ganglia of the African snail. The cell VG 1 formerly believed to be an interneuron, was shown to be a motoneuron of the heart producing the EPSPs in the myocardium. The data obtained show a considerable similarity in organization of the cardioregulating neurons system in different species of the gastropods. PMID- 7536582 TI - [Experimental and theoretical research on the phenomena of the Vvedenskii Ukhtomskii school]. PMID- 7536583 TI - [The effect of muscle metabolites on neuromuscular transmission and muscle fatigue]. PMID- 7536584 TI - [The restoration of the work capacity of the fatigued rat diaphragm after the use of exogenous acetylcholine]. PMID- 7536586 TI - [The inhibitory neuronal reactions of the hypothalamic supraoptic nucleus in rats]. AB - Experiments with electric stimulation of the hypophyseal stalk and hippocampal formation revealed a great significance of the direct inhibitory control of the neurosecretory unit activity. Various types and mechanisms of primary inhibitory responses and postactivation [correction of pastactivafahory] inhibitory processes, as well as their role in the activity of neurosecretory cells and other neurons of the supraoptic area, are discussed. PMID- 7536585 TI - [The role of vacuum and tactile stimuli during milk ejection from the human breast]. AB - Milk ejection in lactating women was performed by breast pump with vacuum and tactile (touch-compression) components. It was found that milk flow rate changed in a pulsatile manner at about 1-2 min interval and peaks milk flow coincided with peaks intramammary. Switch off the tactile component increased the latency of milk ejection 1.5-2 fold and decreased total flow rate 2 fold. It was concluded that touch-compressions stimuli necessary for generation milk ejection reflex and milk ejection from the women mammary gland. PMID- 7536587 TI - [The electrophysiological characteristics of long-term homo- and heterotopic cortical transplants in the rat motor cortex]. AB - Microelectrode experiments were performed on the transplants (TPs) in the motor cortex, MI, of adult rats 2 to 6 mo and 8 to 11 mo after grafting TPs taken from one of hemispheres of newborn rat. Within TPs, latencies of neuronal responses evoked by means of electrocutaneous stimulation of forelimbs, hindlegs and vibrissal pads were ranged, from 13 to 30 ms, 17 to 38 ms and 10 to 15 ms, respectively. The values are similar with those in intact MI in both experimental and normal rats. A trend to widening receptive fields was observed in many neurons recorded in long-surviving TPs. In some units, a predominance of ipsilateral somesthetic inputs over contralateral ones was demonstrated which is more usual phenomenon for immature neurons in MI during early normal development. It indicates on the deficiency of maturation's mechanisms for synaptic inputs into the TPs. No motor responses were induced by low-threshold microstimulation in TPs and only on the depths lower than 2000 microns motor responses were obtained in a few cases, presumably due to the activation of white matter axons. PMID- 7536588 TI - [The principle of the dominant in the organization of goal-directed behavior]. PMID- 7536589 TI - Tuberculosis in England and Wales 1982-1993: notifications exceeded predictions. AB - About 8000 more cases of tuberculosis were notified in the years 1988 to 1993 than would have been expected if previous trends had continued (95% confidence interval (CI) 3000-12,000). Tuberculosis notifications were analysed to determine the population groups and geographical areas in which increases occurred. Increases were greatest in regional health authorities (RHAs) with large conurbations, while rates generally continued to decline in RHAs that serve more rural areas. The rate of notification rose significantly in males aged 15 to 34 years and fell in men over 35. In females, the rate increased only in the 35 to 64 year age group. Non-respiratory tuberculosis accounted for 21% of notifications in 1987 and 27% in 1993. In most age, sex, and geographical groups significant increases were confined to non-respiratory tuberculosis, but the rate of both respiratory and non-respiratory tuberculosis rose in males aged 15 to 34 years in North Thames RHAs. A number of factors may have contributed to the observed increase. Reporting of cases may have improved following the appointment of consultants in communicable disease control in 1988, whose role includes improving notification practices. Such improvements might explain the increased proportion of notifications of non-respiratory tuberculosis, as this may have been undernotified to a greater extent than respiratory tuberculosis in the past. Other factors associated with recent increases are discussed. PMID- 7536591 TI - How many people in England and Wales risk infection from injecting drug use? AB - Injecting drug users who share equipment may transmit and acquire bloodborne virus infections, including HIV, hepatitis B virus, and hepatitis C virus. Even without sharing, injection with non-sterile equipment, drugs, or mixing agents may result in infection due to bacteria or fungi. Estimates of the number of people who are currently at risk of infection from injecting drug use are needed in order to plan services and care, and to interpret surveillance data. This paper examines the data from registries of drug use and two recent surveys of the general population from which estimates of the number of current injecting drug users in England and Wales have been derived. Drug registries include only those whose drug use is identified during contact with drug or medical services, so these sources provide minimum estimates but may be used to monitor trends: 25,706 drug users in England and Wales were notified to the Home Office in 1993, 12,253 of whom were current injectors. Estimates derived from surveys of the general population suggest, however, that between 51,900 (95% confidence interval (CI): 33,000-71,600) and 77,700 (95% CI: 4100-151,200) people in England and Wales are at risk of infection from current injecting drug use, of whom between 10,400 (95% CI: 7200-13,800) and 15,500 (95% CI: 800-30,200) are at risk of bloodborne virus infections as a result of sharing injecting equipment. In the 16 to 34 year age group about one in 200 men, and one in 400 to 500 women may be current injectors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536590 TI - A collaborative case control study of sporadic hepatitis A in England. AB - A case control study of sporadic hepatitis A was carried out in 201 districts in England from July 1990 to June 1991. The aims were to determine the risk factors associated with the infection and to identify individuals or groups who might benefit from prophylactic measures, such as human normal immunoglobulin or hepatitis A vaccine. Factors associated with risk of hepatitis A included travel abroad (odds ratio (OR) 19.8; 95% confidence interval (CI) 4.87-80.6), a household contact with hepatitis A (OR 13.5; 95% CI 6.49-28.0) and sharing a household with a child aged 3 to 10 years (OR 1.57; 95% CI 1.1-2.22). This study provided no clear evidence of increased risk in health care workers, teachers, or other occupational groups. A non-significant trend towards an increased risk in nursery nurses and child minders aged 20 to 29 years was observed. Pre-exposure prophylaxis with hepatitis A vaccine may be considered for people who travel frequently to areas where hepatitis A is highly or moderately endemic. Post exposure prophylaxis with human normal immunoglobulin should be given to contacts of known cases in accordance with national guidelines. Immunoglobulin alone has been recommended in outbreaks, but the use of vaccine alone or combined with immunoglobulin should be evaluated. PMID- 7536592 TI - Chromatographic separation of low-molecular-mass recombinant proteins and peptides on Superdex 30 prep grade. AB - The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30 60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown. PMID- 7536593 TI - The need for inpatient palliative care facilities for noncancer patients in the Thames Valley. AB - Inpatient facilities in palliative care units are generally considered to be mainly for cancer patients. We present and discuss the results of a survey that attempted to estimate the number of noncancer patients requiring inpatient palliative care. Questionnaires sent to all general practices in the Thames Valley area asked about the diagnosis and the number of bed-days that would have been required for each noncancer patient in the practice dying in the last year or still in their care. The replies suggest that about 11 noncancer patients per practice per year were in need of respite or continuing care. For the Thames Valley area this would amount to at least 66,000 bed-days per year for noncancer patients, compared with the current provision, mainly for cancer patients, of about 40,000 bed-days per year. The diagnoses involved and the reasons why our figures may overestimate need, are discussed. There can be no doubt that, if the need is to be met, current facilities will be inadequate and additional beds and services will be required. PMID- 7536594 TI - Palliation in chronic respiratory disease. AB - Prognosis in severe chronic obstructive pulmonary disease is poor, and it is increasingly accepted that such patients need good palliative care. This paper reviews the medical management of chronic obstructive pulmonary disease, and also discusses the place of long-term oxygen therapy. A multidisciplinary programme termed 'pulmonary rehabilitation' is being used increasingly, and, although this probably does not improve survival, there is evidence that it increases quality of life. The drug treatment of dyspnoea has been disappointing, but close attention to psychosocial aspects can improve mobility and control. The place of palliation in a number of other chronic lung conditions is also mentioned. PMID- 7536596 TI - Palliative Care Research Forum. Dublin, 9-10 November 1994. Abstracts. PMID- 7536595 TI - Professional needs of palliative care nurses in New South Wales. AB - A survey of 108 palliative care nurses practising in New South Wales, Australia, was undertaken to explore their professional needs and clinical knowledge. Opportunity for improved training was the most frequently nominated professional need. Only 12% of the sample had postgraduate qualifications in palliative care and fewer than 20% were currently undertaking postgraduate training. Sixty-three per cent of nurses indicated that a lack of opportunity for formal study was a problem for them. The results of the knowledge survey revealed a need for additional training. Many nurses did not have the clinical knowledge identified as minimal by an expert committee. Those nurses who had a postgraduate qualification in oncology scored more highly on the knowledge questionnaire than did those whose general nursing training was undertaken outside Australia. The implications of these findings for training and other professional support are discussed. PMID- 7536597 TI - Subcutaneous ketorolac. PMID- 7536598 TI - Peritoneovenous shunt for palliation of gynecologic malignant ascites. AB - BACKGROUND: Ascites is a common sequela of advanced or recurrent gynecologic malignancies, such as carcinoma of the ovary, fallopian tube, or endometrium. Symptomatic treatment with repeated paracentesis is the initial management after failure of chemotherapy. STUDY DESIGN: This study was done to evaluate the safety and effectiveness of a peritoneovenous shunt (PVS) in the palliation of these patients with recurrent ascites. A retrospective review of 25 patients having a PVS between 1982 and 1992 was performed. RESULTS: The 25 patients consisted of 21 patients with carcinoma of the ovary, two with primary carcinoma of the peritoneum, one with carcinoma of the endometrium, and one patient with carcinoma of the fallopian tube. The mean weight and abdominal girth decreased after shunt insertion (p < 0.001). Gastrointestinal dysfunction and dyspnea also improved with PVS insertion. There was no change in mean Karnofsky score after placement of a PVS. Two patients died within ten days postoperatively. The median survival period was 80 days and shunt occlusion occurred in four patients. CONCLUSIONS: The insertion of a PVS is effective in relieving refractory malignant ascites in gynecologic malignancies. The impact on quality of life requires further study. PMID- 7536599 TI - [Screening for Down's syndrome: results at the Klimentska Polyclinic Screening Center 1992-1994]. AB - Between January 1992 and March 1994 in Klimentska Screening Centre 635 second trimester amniocenteses were performed because of the risk of a chromosomal aberration. 416 (66 per cent) procedures were recommended because of the mother's age over 35 years and 219 ones (34 per cent) because of a positive screening test. Maternal serum alpha-fetoprotein and human chorionic gonadotrophin were used as screening markers in most cases. Five trisomy 21 cases in the advanced age group were detected. Serum markers were assessed in three older mothers with an affected foetus after amniocentesis. All were ex post screen positive. Three trisomy 21 foetuses of mothers younger than 35 years were detected in the screen positive group. Recent advances in screening for Down's syndrome and other congenital defects are discussed. PMID- 7536600 TI - [Anticardiolipin antibodies and oncofetal antigens in pregnant women]. AB - The presence of antiphospholipid antibodies (APA) in the blood stream is associated with some reproductive disorders in women. In the serum of 84 pregnant women, using the Elisa method, anticardiolipin antibodies (ACA) were examined. The first group of 53 women was formed by women with pathological levels of at least one of the oncofoetal antigens (alpha-1-fetoprotein-AFP, human chorionic gonadotropin-HCG and trophoblast-specific beta-1-glycoprotein- SP1). The second group of 31 pregnant women comprised women hospitalized on account of a risk pregnancy. In the first group women with reduced HCG were most frequently represented, where in 8.8% positive ACA were recorded. Of 11 patients with elevated HCG only in one the presence of ACA was detected. In nine pregnant women with reduced HCG and SP1 or AFP ACA positivity was found in 44.4%. In the second group ACA were detected in the serum of six pregnant women who had also pathological SP1 or HCG levels. From the results ensues that ACA could interfere with transduction signalizing processes in the cell and thus influence the synthesis of some proteins. PMID- 7536601 TI - The role of the thymus in myasthenia gravis. AB - The experimental work discussed here supports the hypothesis that in the pathogenesis of MG the initial and essential steps take place within the thymus. Most if not all thymuses of MG patients contain B cells capable of producing AChR specific autoantibody along with appropriate stroma elements. Hyperplastic thymuses characteristically contain germinal centers with cellular complexes of AChR-producing MC and surrounding interdigitating dendritic cells. In thymomas, the source of the myasthenogenic autoantigen is less obvious. There are data suggesting that thymoma epithelium expresses a protein sharing certain peptide epitopes with the AChR alpha chain, although there is no further molecular similarity. A unique type of 'molecular self-mimicry' cold be involved in the initiation of thymoma-associated MG. PMID- 7536602 TI - The role of major histocompatibility complex genes in myasthenia gravis and experimental autoimmune myasthenia gravis pathogenesis. PMID- 7536603 TI - Molecular recognition of acetylcholine receptor. Recognition by alpha-neurotoxins and by immune and autoimmune responses and manipulation of the responses. PMID- 7536605 TI - An improved NOESY simulation program for partially relaxed spectra: BIRDER. AB - An improved 2D NOESY simulation program, BIRDER (back-calculation considering incomplete recovery and differential external relaxation), has been developed. BIRDER has the following features: (1) It allows for differential external relaxation rates for protons with different chemical environments, (2) it automatically accounts for incomplete recovery of z magnetization between scans, and (3) it incorporates anisotropic tumbling motional modes for nonspherical molecules. It was found that a lower R factor is obtained if differential external relaxation rates are used in the NOESY spectrum simulation. The method for experimentally determining these external relaxation rates is discussed. In practice, the ability to account for incomplete recovery of z magnetization is very useful in the quantitative analysis of 2D NOESY spectra of spin systems with long longitudinal relaxation times (T1). For example, the T1's of some protons in RNA duplexes are approximately 4-6 s. Thus, a more than 15 s relaxation delay is needed to allow the z magnetization to return to thermal equilibrium between scans, requiring more than a week to obtain a single NOESY spectrum. Unless incomplete recovery of z magnetization is explicitly considered, distance errors may arise when shorter relaxation delays are used. The situation is further complicated in studies of DNA:RNA hybrid or DNA-RNA chimeric duplexes in which the DNA protons relax much faster than the RNA protons. It is shown that BIRDER successfully corrects for the incomplete recovery of z magnetization and makes it possible to save spectrometer time by using shorter relaxation delays without introducing serious errors. The nonsymmetrical nature of NOESY spectra when the spin system is not fully relaxed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536604 TI - T cell receptor gene regulation of experimental autoimmune myasthenia gravis. PMID- 7536607 TI - A reappraisal of serial isotope bone scans in prostate cancer. AB - Carcinoma of the prostate is the commonest malignancy of the genitourinary tract in the male and is frequently associated with metastatic bone disease. Serial isotope bone scans for screening secondary deposits are not cost-effective. We have evaluated the serum prostate markers prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) as an alternative to conventional serial bone scanning in 129 patients with newly diagnosed prostate cancer over a period of 3 years. Although serum PSA did not reflect local tumour burden at presentation, it was significantly elevated in those who presented with stage D disease (p < 0.01). 45 patients presented de novo with metastatic bone deposits and a further 18 patients developed metastases during the study period. The sensitivity of PSA in detecting secondary deposits at presentation for levels in excess of 100 micrograms/l was 93.75%, the positive predictive value 95.7% and the negative predictive value for levels less than 5 micrograms/l was 90.6%. During the follow up period the sensitivity was 94.4%, the positive predictive value 100% and the negative predictive value 100%, with a median lead time of 3 months in predicting metastases in the 18 patients with progressive disease. When compared with PAP, PSA was found to be a statistically superior marker of bone metastases both at presentation and follow-up (p < 0.05). We recommend that PAP measurements are no longer necessary and should be replaced by PSA, and that serial serum PSA estimations should determine the need for future isotope bone scans in the patient with established prostate cancer. PMID- 7536606 TI - Comparative biochemical study of coated vesicles purified from cock brains. AB - Cock brain coated vesicles (CBCVs) were purified and compared with porcine brain coated vesicles (PBCVs) from several biochemical aspects. Clathrin heavy and light chains of CBCVs were immunologically similar to those of PBCVs. Coat proteins (CPs) of CBCVs behaved in almost the same manner as those of PBCVs for limited proteolysis. Dissociation of CPs from CBCVs by treatment with 10 mM Tris Cl, pH 8.5, or 2M urea resembled that of CPs from PBCVs. pH dependency of dissociation of CPs from CBCVs was slightly different from those of PBCVs. PMID- 7536608 TI - The efficacy of radiotherapy in the treatment of intraocular metastases. AB - From January 1980 to May 1991, 28 patients with intraocular metastases were seen at our Institute. Three presented with bilateral metastases and two developed contralateral involvement. Out of the 33 ocular metastases 27 were managed by radiotherapy. The most common primary tumour sites were breast (18/28 patients) and lung (3/28). 22 patients were treated with an 8 MV linear accelerator, using a 4 x 4 cm anterior direct field. The median dose was 40 Gy/20 fractions (range 28 Gy/14 fractions to 50 Gy/25 fractions). Of the 27 treatments reported, 16 resulted in a complete response (59%), six in a partial response (22%) and five resulted in no change (19%). Complete and partial responses lasted for a median time of 13 months (range of 3-89+ months). The median survival time from the start of ocular treatment was 13 months. The aim of radiation treatment is either to prevent or to postpone the visual loss caused by intraocular metastases. PMID- 7536610 TI - Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3. AB - Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel. Channel activity was transient; inactivation of ensemble currents, like that of whole-cell currents, occurred as a single exponential process. Both single-channel and macroscopic currents exhibited use-dependent inactivation in response to repetitive depolarizations. Two scorpion toxins, margatoxin and charybdotoxin, blocked this transient K+ channel, with half-maximal inhibition at 200 pM and 5 nM, respectively. In contrast, dendrotoxin (500 nM) had little effect. In summary, the outwardly rectifying K+ channel in osteoclasts resembles the Shaker-related K+ channel, Kv1.3. When membrane potential was recorded in whole-cell configuration, charybdotoxin (50 nM) caused a depolarization of 5 to 10 mV from resting levels of -50 mV or more positive; therefore this K+ channel contributes to the membrane potential of osteoclasts under some conditions. To investigate the molecular nature of osteoclast K+ channels, we performed RT-PCR on osteoclast RNA using primers for Kv1.3 and the inward rectifier, IRK1. mRNA encoded by Kv1.3 and IRK1 was detected and message identity confirmed by restriction enzyme digestion and sequence analysis. We conclude that osteoclasts exhibit, in addition to the previously described inward rectifier, an outwardly rectifying K+ conductance with properties of the Kv1.3. channel. PMID- 7536609 TI - Molecular analysis and functional expression of the human type E neuronal Ca2+ channel alpha 1 subunit. AB - A human brain alpha 1 Ca2+ channel subunit was cloned and expressed in Xenopus laevis oocytes. The open reading frame, encoding 2,312 amino acids, has high homology to the marine ray doe-1, the rat E-type, and the rabbit brain BII alpha 1 subunits. The amino and carboxy termini of this human.E-type alpha 1 subunit (alpha 1E) are most similar to the rabbit BII-1 splice variant, the remainder being colinear with the BII alpha 1 with the exception of two insertions, one of 43 amino acids in the C-terminus and another of 7 amino acids, found also in the rat alpha 1E, between domains II and III. Two potential Ca2+ binding sites are predicted from its primary structure. The expression of inward Ba2+ currents reveals voltage-dependent activation and inactivation measured by the cut-open oocyte vaseline-gap technique, with kinetics that correspond to that of a high voltage-activated neuronal Ca2+ channel, and pharmacologic properties that resemble those of some low-voltage-activated neuronal Ca2+ currents. The human alpha 1E currents are insensitive to omega-conotoxin-GVIA (1 microM), omega agatoxin-IVA (200 nM), a synthetic funnel web spider toxin (FTX, 20 microM), and Bay-K8644 (0.5 microM); they are inhibited 20% by high concentrations of methoxyverapamil and diltiazem, 65% by 0.1% crude funnel web spider venom and 100% by Ni2+ (IC50 = 30 nM). Single-channel records show a complex activity pattern with several apparent conductance states, the largest having a conductance of 14 pS. PMID- 7536611 TI - Functional expression and pharmacological characterization of the human EAA4 (GluR6) glutamate receptor: a kainate selective channel subunit. AB - A cDNA encoding an ionotropic glutamate receptor subunit protein humEAA4 (GluR6), has been cloned from a human fetal brain library. This cDNA when expressed in COS or HEK-293 cells is associated with high-affinity kainate receptor binding and ion channel formation. We have successfully established cell lines stably expressing humEAA4 in HEK-293 cells This is the first report of the establishment of stable cell lines expressing a glutamate receptor channel. The relative potency of compounds for displacing [3H]-kainate binding to humEAA4 receptors expressed in COS or HEK-293 cells is domoate > kainate > quisqualate > 6-cyano-7 nitroquinoxaline-2,3-dione > L-glutamate = 6,7- dinitroquinoxaline-2,3-dione > dihydrokainate. Applications of kainate, glutamate, and domoate but not AMPA evoked rapidly desensitizing currents in cells expressing homo-oligomeric humEAA4 in a concentration dependent manner. The order of potency was: domoate > kainate > L-glutamate. Although AMPA did not itself activate humEAA4 receptors it did reduce, to a limited extent, kainate-evoked responses. AMPA may therefore be a weak partial agonist for this receptor. To date this effect has not been demonstrated with rat GluR6. It is possible that subtle species differences may exist in the nature of agonist receptor interaction. Kainate evoked currents were attenuated by the quinoxalinediones CNQX and DNQX but not by DAP5. The receptor desensitization was attenuated on application of concanavalin A. Ion-permeability studies indicated that the receptor-linked ion channel is permeable to both Na+ and Ca2+ ions. PMID- 7536613 TI - An immunohistochemical and in situ hybridization study of insulin-like growth factor I within fetal neuron cell cultures. AB - Fetal neuron cell cultures (NCC) from 22 day gestation and 18 day gestation fetal rabbit brain were studied for the presence of insulin-like growth factor I (IGF I). The 22 day gestation NCC were incubated in an IGF I free/insulin free/serum free medium. The 18 day gestation NCC were incubated in: (1) IGF I free/insulin free/serum free medium, (2) IGF I containing medium (100 ng)/serum free medium, and (3) serum containing medium. The 22 day gestation NCC survived in the IGF I free/insulin free/serum free medium. Furthermore, IGF I was detected in the medium by RIA from day one to day ten of incubation. In contrast, the 18 day gestation NCC did not survive in the IGF I free/insulin free/serum medium, but survived in the serum medium. When the 18 day gestation NCC were incubated in the serum free medium containing 100 ng IGF I the cells survived for a period of 2-3 days. Immunoreactive IGF I was found within the 22 day gestation NCC incubated in the IGF I free/insulin free/serum free medium and 18 day gestation NCC in serum medium. Likewise, IGF I mRNA was found only within the 22 day gestation NCC. Internalization studies of IGF I have shown that the peptide was internalized from the medium by the two different gestational age NCC's studied. IGF I receptors were found in both 22 day gestation and 18 day gestation NCC. In conclusion IGF I may promote cell survival in early stages of brain development, and may be of exogenous origin. In contrast the 22 day gestation NCC are capable of producing and secreting IGF I, and indeed appear to respond to this growth factor in an autocrine fashion. PMID- 7536612 TI - Beta-adrenergic modulation of currents produced by rat cardiac Na+ channels expressed in Xenopus laevis oocytes. AB - In Xenopus oocytes coexpressing beta 2-adrenergic receptors and the rat cardiac alpha SkM2 Na+ channel, superfusion with 10 microM isoproterenol led to modest (approximately 30%) increases in peak Na+ inward current. Intracellular injection of cAMP and of protein kinase A (PKA) catalytic subunit reproduced this increase, showing that the second messenger pathway involves PKA dependent phosphorylation. Coexpression of the Na+ channel beta 1 subunit had no influence on the modulation. The modulation had little or no effect upon Na+ current waveforms, steady-state activation, steady-state activation, steady-state inactivation, or recovery from both fast and slow inactivation; but maximum Na+ conductance was increased. Mutation of the five major consensus PKA phosphorylation sites on alpha SkM2 did not abolish the observed effect. In parallel experiments, beta adrenergic stimulation of the neuronal alpha IIA Na+ channel subunit led to an attenuation of Na+ current. It is concluded that (i) the alpha SkM2 subunit might be directly phosphorylated by PKA, but at serine/threonine residue(s) in a cryptic phosphorylation site(s); or that (ii) the modulation might also be mediated by phosphorylation of another, as yet unknown protein(s). The divergent modulation of neuronal and cardiac Na+ channel alpha-subunits suggests that differential physiological modulation by identical second messenger pathways is the evolutionary basis for the isoform diversity within this protein family. PMID- 7536615 TI - Adverse reactions to antihistamine-decongestant. PMID- 7536614 TI - Effects of strongly anisosmotic and NaCl deficient solutions on muscimol- and glutamate evoked whole-cell currents in freshly dissociated hippocampal neurons. AB - Sudden exposure of dissociated hippocampal neurons to strongly hypo- or hyperosmotic solutions suppresses voltage gated Na+, K+ and Ca2+ currents. We investigated whether ligand gated ion currents were similarly shut down by exposure to anisosmotic solutions. The effect of hypo-osmotic, NaCl deficient (mannitol-substituted), or hyper-osmotic test solutions delivered from a flow pipette was tested on voltage gated Ca2+ currents and on currents and conductance changes evoked by brief administration of either the GABAA-agonist muscimol or glutamate. Hyper-osmotic solution caused cells to shrink, but cell membrane capacitance did not change. Muscimol-induced conductance increases were depressed by hypo-osmotic and by NaCl deficient solutions and often by hyper-osmotic solution. Voltage gated Ca2+ currents were depressed by anisosmotic, but not by NaCl deficient isosmotic solution. NMDA- and non-NMDA evoked conductance increases were depressed by hyperosmotic solution; hypo-osmotic and NaCl deficient solutions were not tested on glutamate induced currents. Ligand gated currents are suppressed by anisosmotic solutions more slowly than are voltage gated channels. The changes caused by anisosmotic and NaCl deficient solutions were much greater then expected from calculated electrochemical effects and are probably the result of change in receptor controlled channels. PMID- 7536616 TI - Mapping of B-cell determinants in the nucleocapsid protein of Puumala virus: definition of epitopes specific for acute immunoglobulin G recognition in humans. AB - The complete amino acid sequence of the Puumala (PUU) virus nucleocapsid protein (N), deduced from the genome of the prototype strain Sotkamo, was synthesized as decapeptides with 5-amino-acid overlaps. By use of the PEPSCAN method, 86 peptides were examined for reactivity with sera from serologically confirmed nephropathia epidemica (NE) patients and 11 PUU virus N-specific bank vole monoclonal antibodies. The human sera showed reactivity with several different regions, while only one of the monoclonal antibodies reacted with one single peptide. Sequences were selected by this PEPSCAN analysis of human antibody reactivities, and five 15-amino-acid peptides were synthesized and evaluated as antigens by an enzyme-linked immunosorbent assay (ELISA). Peptide-reactive antibodies of the immunoglobulin M (IgM) class were measured in serum samples drawn from patients with acute NE. In comparison with the results of a mu-capture IgM ELISA using native PUU virus antigen, only a few serum samples were found positive (sensitivity, 2 to 10%). Interestingly, when antibodies of the IgG class were measured, the sensitivities of the five peptide ELISAs were found to be 79, 46, 2, 100, and 40%, respectively, as compared with the sensitivity of an IgG ELISA based on native viral antigen. The IgG reactivities of sequentially drawn sera from NE patients with the two peptides giving the highest assay sensitivities were analyzed and compared with their reactivities with native viral antigen. All patients had detectable anti-peptide IgG in the acute-phase sample, which, however, had totally declined in samples drawn after 2 years. The opposite pattern was seen with native viral antigen, in which case all patients showed the highest levels of specific IgG after 2 years. The results suggest the presence of epitopes specific for the acute IgG response. PMID- 7536617 TI - CNS gene delivery by retrograde transport of recombinant replication-defective adenoviruses. AB - The ability to program recombinant gene expression in specific sets of motor and sensory neurons would facilitate the treatment of a number of acquired and inherited central nervous system (CNS) diseases. In this report, we demonstrate that intramuscular injection of replication-defective recombinant adenovirus results in high-level recombinant gene expression, specifically in the CNS motor and sensory neurons that innervate the inoculated muscles. Neural expression of the recombinant genes results from virus transport into the CNS, presumably by retrograde axonal transport. This novel method of neural gene delivery may be of value in studies designed to improve understanding and treatment of inherited and acquired neurological diseases. PMID- 7536618 TI - Expression and localization of CFTR in the rhesus monkey surface airway epithelium. AB - The Rhesus monkey has been used as a model for evaluating the possibility of introducing the CFTR gene into the airway epithelium in vivo. We addressed the question of whether the simian airway surface epithelium exhibits a CFTR distribution and functional activity (ciliary beating frequency) similar to that of human airway surface epithelium. Expression of CFTR mRNA was demonstrated on Rhesus monkey tracheobronchial tissue by reverse transcription polymerase chain reaction (RT-PCR) analysis. By immunofluorescent light microscopy, CFTR was localized on the apical plasma membrane of ciliated cells as we previously described for human tracheobronchial surface epithelium. The ciliary beat frequency (CBF) measured on the explant of Rhesus monkey tracheobronchial tissue appeared to be similar to that of the CBF of human tracheobronchial ciliated cells. To compare the Rhesus monkey CFTR gene with that of the human, we sequenced parts of exon 13 (encoding the R domain) and exon 24 (encoding the C terminal part of the protein) of the Rhesus monkey CFTR gene. The nucleotide sequence identity with the human counterpart was found to be 98% and 94% respectively, although restriction enzyme differences allow discrimination between Rhesus monkey and human CFTR cDNA. Taken together, these results suggest that the airway epithelium of the Rhesus monkey is a suitable model of human respiratory epithelium for analyzing the effect of human CFTR gene transfer. PMID- 7536619 TI - Gene therapy for cystic fibrosis: a clinical perspective. AB - Since the isolation of the cystic fibrosis (CF) gene in 1989, the potential for gene therapy for this disease has existed. Although current treatments have resulted in a mean life expectancy of approximately 30 years, there is clearly a need for more effective therapy. A large number of studies have now assessed both in vitro and in vivo CFTR gene transfer into cell lines, animal models and most recently in CF subjects. Despite this rapid progress several difficulties remain including efficient in vivo gene transfer and the measurement of end-points to assess such gene transfer. This article reviews some of the clinically related aspects of gene therapy for CF. PMID- 7536620 TI - FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway. AB - Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis. PMID- 7536621 TI - Defective expression of hematopoietic cell protein tyrosine phosphatase (HCP) in lymphoid cells blocks Fas-mediated apoptosis. AB - Protein tyrosine dephosphorylation after Fas cross-linking occurred in Fas apoptosis-sensitive CEM-6 cells but not in Fas apoptosis-resistant MOLT-4 cells, and apoptosis in the CEM-6 cells could be inhibited by the protein tyrosine phosphatase inhibitor, pervanadate. The time course and level of dephosphorylation were correlated with increased hematopoietic cell protein tyrosine phosphatase (HCP) activity, but not with the activity of two other tyrosine phosphatases. The level of expression of HCP was correlated with Fas apoptosis function in eleven human and murine Fas-positive lymphoid cell lines. Expression of recombinant HCP in the MOLT-4 cell line converted this Fas apoptosis-resistant cell line to Fas apoptosis sensitive. HCP-mutant mev/mev mice exhibited increased expression of Fas but decreased Fas-mediated apoptosis function in lymphoid organs after anti-mouse Fas antibody treatment in vivo. Thus, HCP-mediated protein dephosphorylation is involved in the delivery of the Fas apoptosis signal in lymphoid cells. PMID- 7536622 TI - Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand. AB - We demonstrate that cognate peptide ligands altered at T cell receptor (TCR) contact residues and bound to class II major histocompatability complex can change the cytokine pattern of mature T cell clones. Myelin basic protein peptide 85-99-reactive Th0 T cell clones were stimulated with altered peptide ligands, which acted both as TCR antagonist and induced new mRNA synthesis and protein secretion of TGF-beta 1, while no longer inducing mRNA synthesis or protein secretion of IL-2, IL-4, IL-10, and IFN gamma. The modified peptides failed to induce a detectable calcium flux, p56lck activation, or thymidine incorporation, yet triggered nearly equal amounts of IL-4 secretion in the presence of ionomycin. Antigen-induced modulation of T cell cytokine secretion may be important in regulating the immune response. PMID- 7536623 TI - Glycosaminoglycan polysulfuric acid (GAGPS) in osteoarthritis of the knee. AB - We studied the efficacy and tolerability of glycosaminoglycan polysulfuric acid (GAGPS) in 80 patients with osteoarthritis (OA) of the knee. Patients received two series of five intra-articular injections, at 1-week intervals, of 25 mg (0.5 ml) GAGPS into the knee in a double-blind, parallel, randomized, placebo controlled trial. There was an immediate decrease in pain after the injections of 43% with GAGPS and 33% with placebo (P = 0.047) (Jezek pain index). Pain relief of GAGPS vs placebo was not different at other intervals (10, 14, 22, 26 weeks after start of treatment). At 6 weeks the Lequesne index decreased 20% after GAGPS and 9% after placebo (P = 0.17). At 10 weeks the Lequesne index decreased 24% after GAGPS and 13% after placebo (P = 0.20). The decrease in Lequesne index at 14 weeks was 31% after GAGPS and 15% after placebo (P = 0.06). The other measured parameters tended to be more favorably influenced by GAGPS than placebo. GAGPS was well tolerated, with associated mild adverse reactions in 8% of cases. GAGPS may have a role as a symptomatic slow acting drug for OA. Further study appears appropriate. PMID- 7536625 TI - Contractile-tailed bacteriophages adsorb to Escherichia coli O128ab lipopolysaccharide that is altered by large plasmids to provide receptors and lipopolysaccharide heterogeneity within the serogroup. AB - The verotoxigenic Escherichia coli strain H.I.8 (originally O128:B12, now not typeable) contained a ColB+M plasmid and two morphologically identical temperate bacteriophages (H18A and H18B). Both phages were O128ab specific, using the lipopolysaccharide O side chains of susceptible clinical isolates as receptors. SDS polyacrylamide gel electrophoresis with silver staining of O128ab lipopolysaccharide revealed four distinct types of ladder with different interband spacings. No specificity was found between ladder type and sensitivity to either phage. One of the numerous large plasmids present in O128ab isolates was found to modify the structure of the lipopolysaccharide O side chains to provide phage receptors. PMID- 7536624 TI - Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects. AB - Chondrocyte-collagen composites were evaluated for resurfacing of large articular defects. Isolated chondrocytes were cultured in expanded collagen scaffolds for 7 10 days to provide a composite containing 3.6 x 10(4) cells/mm3. The graft was transplanted into 15 mm full thickness articular defects in the femoropatellar joint of 12 horses using arthroscopic techniques. Ungrafted defects in the opposite femoropatellar joint served as controls. Synovial fluid, clinical progress and pain responses were evaluated in groups of 6 horses over 4-month and 8-month periods. Following termination, gross, histochemical and histologic evaluations of the repair tissues and synovial membrane were performed. Arthroscopic defect debridement and chondrocyte implantation resulted in minimal post-operative effusion or pain, and synovial fluid constituents were not significantly different in grafted and ungrafted joints. Gross differences in grafted defects were not apparent. Increased chondrocyte numbers and cartilage histochemical staining were evident in the deeper layers of grafted defects, whereas ungrafted defects were almost entirely fibrous tissue. The surface layers of grafted defects were fibrous tissue. There were no synovial fluid cellular responses, synovial membrane histiocytic reaction or subchondral bone infiltrates to suggest immune-related reaction to the allograft cells. Chondrocyte-collagen grafts were arthroscopically implanted and resulted in improved cartilage healing in extensive defects. However, the structural organization of the surface layers was inadequate and suggested poor long-term durability. PMID- 7536626 TI - Tenascin in breast cancer development--is epithelial tenascin a marker for poor prognosis? AB - (1) In mouse mammary gland development, immunoreactive tenascin (TN) is expressed in the dense mesenchyme surrounding the epithelial component of 14-day embryos, endbuds at puberty, and tumors. (2) Cells that produce TN are myofibroblastic and are characterized by nuclear invaginations, rough endoplasmic reticulum, and pinocytotic vesicles. These cells are not normally present in the stroma of mammary glands but present in cancer stroma, originating probably from fibroblasts differentiated under the influence of TGF-beta 1 stimulation. (3) Breast cancer cells are capable of synthesing TN under certain conditions. TN-non producing MCF7 cells can produce TN when co-cultured with embryonic fibroblasts or with their conditioned medium. (4) Nine primary human breast cancers were examined for TN expression by in situ hybridization. TN mRNA was expressed in all nine cases in the stroma and in four cases in carcinoma cells as well. (5) Immunohistochemistry for TN was performed in human breast cancers, and it was found that the five-year survival after surgery was markedly lower in the group whose cancer cells were positive [corrected] for TN. TN expression in cancer cells appears to indicate poor prognosis. PMID- 7536627 TI - MHC class I-peptide interactions and TCR recognition. AB - The recent crystal structure determinations of MHC class I molecules with single bound peptides have allowed us to understand the guidelines that govern peptide binding in a given MHC allele. Evolution has provided for MHC class I molecules whose antigen binding clefts possess distinct physical and chemical properties as a result of the particular arrangement of variable residues lining the binding cleft. As a result, a given molecule binds a unique set of peptides, the position and identity of whose anchor residues are dictated by the features of the cleft. In addition to the interactions between the anchor residues and the cleft, the peptide is held in the groove by a highly conserved array of hydrogen bonds. A tantalizing application of our newfound understanding of peptide binding will be in the design of model peptides that either block or enhance immune response, in order to achieve effective treatments for a variety of disorders of the immune system. PMID- 7536628 TI - Class I MHC-peptide interactions: structural requirements and functional implications. AB - In this chapter, we have defined the structural motifs that dictate the capacity of peptides to bind to five different HLA-A alleles that represent some of the most common alleles found in different ethnic populations. In general, these peptide motifs were very specific for the individual HLA-A alleles, with the exception of HLA-A degree 0301 and HLA-A degree 1101, for which the motifs were very similar. When these motifs were tested against an unbiased and complete set of nonamer peptides derived from human papillomavirus E6 and E7 proteins, it was found that the vast majority of high and intermediate binding peptides contained the appropriate motif. Furthermore, using the dominant anchor residues, together with the amino acid positions that interact with secondary anchor residues, it was possible to predict high and intermediate binders. Finally, the finding that there is a direct correlation between binding affinity for MHC and immunogenicity suggests a practical application of being able to predict those peptides that have a high affinity binding for a particular MHC allele--that is, in the design of peptide based vaccines for prophylactic or therapeutic use. PMID- 7536629 TI - Recognition of viral antigens at the cell surface. AB - There is now a very good understanding of the way in which epitope peptides are generated from virus proteins and how the peptides bind to the class I MHC molecules. This gives a framework in which to analyse the potentially protective cytotoxic T lymphocyte response in human immunodeficiency virus infection. A detailed understanding of the specificity of the responding T cells, the clonality of these T cells and the effects of virus variation on the CTL gives a possible explanation for the ultimate failure of the CTL response to control this virus infection. PMID- 7536630 TI - Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. AB - Rho and rac, two members of the ras-related superfamily of small GTPases, regulate the polymerization of actin to produce stress fibers and lamellipodia, respectively. We report here that cdc42, another member of the rho family, triggers the formation of a third type of actin-based structure found at the cell periphery, filopodia. In addition to stress fibers, rho controls the assembly of focal adhesion complexes. We now show that rac and cdc42 also stimulate the assembly of multimolecular focal complexes at the plasma membrane. These complexes, which are associated with lamellipodia and filopodia, contain vinculin, paxillin, and focal adhesion kinase, but are distinct from and formed independently of rho-induced focal adhesions. Activation of cdc42 in Swiss 3T3 cells leads to the sequential activation of rac and then rho, suggesting a molecular model for the coordinated control of cell motility by members of the rho family of GTPases. PMID- 7536631 TI - The CTL's kiss of death. AB - The potent and specific lytic activity of CTLs can occur by at least two distinct pathways. In the secretion and perforin-mediated pathway, the direct effect(s) on the target cell membrane of the pore-forming agent perforin, probably in conjunction with granzymes, also secreted from the CTLs, causes the target's demise. Intercytoplasmic transfer of granzymes is believed to be involved in inducing target apoptosis. In the Fas-mediated pathway, engagement of a CTL membrane ligand with an apoptosis-inducing target cell surface receptor, such as the FasL with Fas, triggers programmed disintegration of the CTL-bound target; secretion of granzymes and pore formation by perforin are not involved in this receptor-mediated mechanism. Despite the fundamental differences in their onset for both pathways, the downstream sequence of events that culminate in target cell apoptosis appears to be similar. Further studies will resolve this enigma. PMID- 7536632 TI - Constitutive alpha V beta 3 integrin-mediated adhesion of human lymphoid B cells to vitronectin substrate. AB - Adherence to cells and matrices participates in lymphocyte migration and tissue localization and contributes to the regulation of growth and differentiation of the lymphoid cells. The adherence is mainly mediated by three families of cell surface proteins: integrins, immunoglobulin (Ig)-related molecules, and selectins. Integrins recognize Ig-related molecules such as ICAMs as well as fibronectin (FN), vitronectin (VN), and other matrix proteins. In this study, the in vitro adhesive properties of two Epstein-Barr virus-carrying B lymphoblastoid cell lines, IB-4 and NAD-20, were compared. IB-4 cells grow as a monolayer in contrast to NAD-20 cells, which grow as cell clusters. IB-4 cells were found to adhere to the tissue culture vessel through a component of the fetal bovine serum. By using blocking monoclonal antibodies to cell-surface molecules and serum proteins, IB-4 cells were found to use alpha V beta 3 integrin (CD51/CD61) and serum VN as the adhesive molecules. alpha V beta 3 integrin also mediated adhesion of IB-4 cells to human serum VN and to purified VN and FN. This constitutive adherence was not enhanced by phorbol ester treatment and was inhibited by RGD-containing peptides, in contrast to the homotypic adhesion of NAD-20 cells, which was mediated by beta 2 integrin CD11a/CD18 and its ligand ICAM-1 (CD54). Since VN is a component of both lymphoid tissue matrix and plasma, adhesion to this protein may affect functions and activities of B lymphocytes. PMID- 7536633 TI - Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella-zoster virus antigen. AB - Fas antigen and bcl-2 expression on peripheral blood mononuclear cells (PBMC) cultured with cytomegalovirus (CMV) and varicella-zoster virus (VZV) antigen was analyzed by three-color flow cytometry. Expression of Fas antigen increased significantly in CD45RO+ populations of both CD4+ and CD8+ cells obtained from CMV and VZV seropositive donors after culture with CMV and VZV antigen for 6 days. Fas antigen expression did not increase in PBMC cultured with control antigen. In contrast, bcl-2 expression decreased both in CD4+CD45RO+ and CD8+CD45RO+ cells from the same donors. Fas antigen and bcl-2 expression in CD45RA+ populations did not change. Cell viability of cultured cells with CMV and VZV antigen decreased after treatment with anti-Fas antibody and DNA fragments indicating apoptosis were detected in the cell lysate of these cultured cells after treatment with anti-Fas antibody. These data suggest that Fas antigen and bcl-2 protein may interact in regulating the cell death process of activated memory lymphocytes and eliminating lymphocytes activated by viral infection. PMID- 7536634 TI - A novel angiogenesis inhibitor suppresses rat adjuvant arthritis. AB - Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470 treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases. PMID- 7536637 TI - [The biological significance of endocrine cells in pancreatic carcinoma]. AB - Endocrine cells (EC) were found in 19 out of 42 cases of the pancreas carcinoma (42.5%). Among them, 4 cases had a positive rate of EC more than 50%. The positive rate of EC in the well differentiated carcinomas (5/20) was lower than that of the poorly-differentiated ones (12/19) or mucinous carcinoma (2/2), and the positive rate in histologic grade I cases (5/18) was significantly lower than that of the grade III cases (7/8). The number of mast cells infiltrating in the matrix in EC positive cases was significantly higher than that of the negative ones. The positive rate of EC in the cases with metastasis (8/14) was higher than that of the non-metastasis cases (7/21). Immunocytochemical staining showed that GN (8), SS(4), HCG(5), CK(12), EMA(13) and CEA(9) were positive in 19 EC positive cases. PMID- 7536638 TI - [Preparation and application of ELISA kit for detection of G-CSF]. AB - By adding monoclonal antibodies from BALB/c mice to the granulocyte colony stimulating factor (G-CSF), we established a sandwich ELISA for detecting G-CSF levels in human serum samples. G-CSF was measured in 157 normal serum samples and in 153 serum samples from patients with no clinical manifestation of bacterial infection and in 269 serum samples from patients with clinical diagnosis of bacterial infection and in 31 serum samples from patients with positive bacterial infection according to cell culture. It was found that the positive rates of G CSF were zero in normal serum samples, only 6.5% in patients with no clinical manifestation of bacterial infection, 89.2% in patients with clinical diagnosis of bacterial infection and 100% in patients with bacterial culture positive for infection. Our results reveal that the sandwich ELISA for detection of G-CSF levels in human serum samples may be useful for diagnosing patients with bacterial infection and for clinically guiding the rational use of antibiotics. PMID- 7536636 TI - Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and alpha CD3-induced CD3-AK cell responses. AB - This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL 2R to generate LAK cells and through TCR-CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the alpha CD3-induced CD3-AK cell response. First, the alpha CD3 induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2 benzyoxycarbonyl-L-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR-CD3. The former pathway is primarily PTK-dependent. Activation through TCR-CD3 is a more complex event.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536635 TI - Transient expression of human interleukin-2 and interferon-gamma genes is regulated by interaction between distinct cell subsets. AB - The level of transient expression of human IL-2 and IFN-gamma genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-gamma gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-gamma was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-gamma gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-gamma mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition. PMID- 7536640 TI - [Reconfirmation of golgiphobic dendrites of the motoneurons in rat spinal cord]. AB - With CB-HRP method (injections into 5 muscles of anterior and posterior extremities) and Golgi technique, the corresponding sections of spinal cords were observed on same aged rats of the identical parent rats. Comparing the cell numbers of the lateral groups of anterior horns, the former (CB-HRP) revealed twice as many as the cell numbers of the latter (Golgi). As to the surface densities of the white matter dendrites (WMD) in the lateral funiculi from the neurons of the lateral groups of the anterior horns, the density revealed by injection of CB-HRP to the tibialis anterior is 2-9 times more than that in Golgi sections. All of WMD revealed in CB-HRP sections could extend into the peripheral portions of the lateral funiculi, and quite a few of them even form a subpial marginal plexus, that is one example of Golgiphobic dendrites (GBD). For labeled medial cell groups of the anterior horn with CB-HRP, their dendrites could reach to the ependymal layer of the central canal (another GBD). These two types of GBD were not present on the Golgi material. The significance of GBD was also discussed. PMID- 7536639 TI - [Bleomycin-induced fibroblast proliferation is inhibited by IH764-3]. AB - It reports that IH764-3 has both therapeutical and prophylactic effect on bleomycin-induced pulmonary fibrosis in rats and mice. The data presented indicate that IH764-3 is a potent inhibitor of the two important ways commonly thought to be the main causes of pulmonary fibrosis induced by bleomycin (BLM): 1) BLM stimulates the alveolar macrophages to secrete fibroblast growth factor (FGF); and 2) BLM increases the proliferation rate of the fibroblast directly. This demonstration of suppression of the stimulative effect of BLM on the fibroblast proliferation by IH764-3 provides direct evidence that IH764-3 could be a promising anti-fibrogenetic drug candidate. PMID- 7536642 TI - [Snail hosts of Paragonimus in Asia and America]. AB - This is a comprehensive review of snail hosts of Paragonimus distributed worldwide exclusive of Africa. Fifty-eight species of snails are listed, of which 67% occur in China. Most of the Chinese snails are classified as Pomatiopsinae Erhaiini or Triculinae of the family Pomatiopsidae. Problems associated with a number of names and concepts have been clarified. PMID- 7536641 TI - Antitumor effects of oral administration of an interferon-inducing pyrimidinone, Bropirimine, on murine renal-cell carcinoma. AB - Bropirimine [2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone] is a low-molecular weight compound that acts as an inducer of interferon in several animal species. Experiments were designed to explore the possibility of using this drug for the treatment of renal-cell carcinoma (RCC). Euthymic BALB/c mice were inoculated with murine RCC (Renca) cells and given graded doses of Bropirimine p.o. for 5 consecutive days beginning on day 1 following tumor inoculation. These mice were killed and tumors were excised on day 21. Bropirimine significantly (P < 0.01) inhibited the tumor growth at a daily dose of 1,000 or 2,000 mg/kg. No adverse effect or toxicity was noted at 1,000 mg/kg, and at 2,000 mg/kg there was only a marginal body-weight reduction without any other appreciable side effect. In addition to the inhibition of tumor growth, there was a small yet significant (P < 0.05) increase in the duration of survival (in days) in the Bropirimine-treated animals. When the treatment was delayed to begin on day 6 following tumor inoculation, Bropirimine did not suppress tumor growth in euthymic mice, pointing to the importance of the timing of the treatment. In athymic nude BALB/c mice lacking T-cells or T-cell function, Bropirimine also inhibited tumor growth (P < 0.01). The antitumor effect of this drug was abolished by pretreatment with anti asialo GM1 serum, which eliminated natural killer (NK) activity in euthymic mice. In vivo treatment with Bropirimine augmented the cytotoxicity of lymphocytes isolated from the spleens or lungs of the tumor-bearing mice, which were active against Renca and YAC-1 cells in vitro. This activity was NK-cell-dependent as judged on the basis of the results of the in vitro complement-dependent cytotoxicity assay. Since Bropirimine induced interferon (IFN)-alpha/beta production, significantly (P < 0.05) elevating its serum concentration, and since this drug mimics the effects of IFN-alpha/beta, it seemed likely that the Bropirimine-induced NK cell augmentation we found was mediated by IFN-alpha/beta. These results suggest that Bropirimine, a booster of NK activity, may have potential as an adjunct to other therapeutic modalities in the treatment of human RCC. PMID- 7536643 TI - Intracellular injections of lucifer yellow into lightly fixed mitral cells reveal neuronal dye-coupling in the developing rat olfactory bulb. AB - We report that intracellular injections of Lucifer Yellow into lightly fixed mitral cells revealed dye-coupling between mitral cells and between mitral and granule cells in the form of discrete, radially oriented cell clusters. Dye coupling was observed in animals as early as postnatal day 10 (P10) and at least until P30. In P10 rats, a mean of 2.5 dye-coupled mitral cells and 27 granule cells were observed per column. Mean column depth and width were 169 microns and 86 microns respectively. Most of the dye-filled granule cells were found within 150 microns of the mitral cell layer. No significant changes were found at P20. By P30, the mean number of granule cells per column increased to 42 and the addition of granule cells occurred in areas proximal to the mitral cell layer. Immunocytochemical results indicate that the developing bulb contains a large concentration of the gap junction protein Connexin 43 (Cx43). Cx43-like immunoreactivity was found at all ages examined, with the most intense staining in the nerve and glomerular layers. Less intense Cx43-like immunoreactivity was found in both the mitral and granule cell layers, with Cx43-like immunoreactive puncta observed between and around cell body profiles. Freeze-fracture analysis revealed the presence of gap junction-like plaques on mitral cells, further suggesting that the dye-coupling occurred across interneuronal gap junctions. Neuronal coupling during development could provide an inter-cellular pathway for the passage of relevant developmental signals which could influence the formation and/or strengthening of synaptic contacts. The coupling could also be involved in the synchronization of neuronal activity, which may be important for olfactory coding. PMID- 7536644 TI - Enzymatic markers of gallstone-induced pancreatitis identified by ROC curve analysis, discriminant analysis, logistic regression, likelihood ratios, and information theory. AB - We investigated the diagnostic utility of frequent serial determinations of aspartate aminotransferase, alanine aminotransferase (ALT), lipase, amylase, and the lipase/amylase (L/A) ratio for distinguishing patients with acute pancreatitis due to biliary obstruction from those with acute pancreatitis due to other pathogenesis. Analyzed were enzyme activities obtained at admission and peak enzyme activities identified retrospectively from serial measurements in 53 patients with acute pancreatitis due to various causes. We evaluated the data with multiple statistical tools. Discriminant analysis and logistic regression revealed the diagnostic significance of ALT at initial and peak values, and the maximum information provided by peak ALT was confirmed by both logistic regression and stratum-specific likelihood ratios. Stratum-specific likelihood ratios showed peak ALT > 150 U/L was highly diagnostic of biliary pancreatitis. The L/A ratio, either at admission or at peak, was the only other significant variable for identifying patients with acute pancreatitis due to biliary obstruction. A multivariate logistic discriminant function including ALT and the L/A ratio significantly discriminated biliary acute pancreatitis from pancreatitis due to other causes. Evaluation of initial and peak enzyme data by information theory revealed that the optimal test depended on disease prevalence. Initial ALT activities were the test of choice for identifying biliary pancreatitis, up to a disease prevalence of approximately 0.75. At disease prevalence > 0.75, the initial L/A ratio provided the greatest amount of diagnostic information. PMID- 7536645 TI - Serum concentrations of 10 acute-phase proteins in healthy term and preterm infants from birth to age 6 months. AB - Aiming to define the evolution pattern of 10 acute-phase proteins in early infancy, we measured nephelometrically the serum concentrations of albumin, prealbumin, retinol-binding protein, transferrin, ceruloplasmin, hemopexin, haptoglobin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, and alpha 1 antitrypsin in 395 term and preterm infants (gestational ages 26-41 weeks). Measurements were performed within 24 h after birth and then at the end of 1 (n = 171), 3 (n = 155), and 6 (n = 90) months afterwards. Data obtained from 250 healthy adults were used as adult reference values. All proteins increased progressively with postnatal age, except for alpha 1-antitrypsin, which remained stable from birth to the 6th month. Concentrations of almost all measured proteins were significantly lower in preterm than in term infants in the first 3 months. Compared with adult values, alpha 2-macroglobulin and alpha 1-antitrypsin were higher in infants throughout the 6 months. The other proteins were significantly lower at birth than adult values but after 6 months, only albumin, prealbumin, retinol-binding protein, and alpha 1-acid glycoprotein still remained lower in infants. Thus both gestational and postnatal age should be considered when interpreting concentrations of these proteins in early infancy. PMID- 7536646 TI - Nitric oxide synthase activity is increased in patients with sepsis syndrome. AB - 1. We measured nitric oxide synthase activity in peripheral blood polymorphonuclear leucocytes from 10 patients with sepsis syndrome and 10 healthy subjects. 2. Synthase activity was significantly higher in patients with sepsis than in control subjects (1202 +/- 579 compared with 595 +/- 544 pmol of nitric oxide min-1 mg-1 of cell protein, P < 0.05). 3. Activity was greatest in those patients with the larger number of organ failures, although this failed to reach significance (1489 +/- 560 in patients with three or more organ failures and 843 +/- 404 pmol of nitric oxide min-1 mg-1 of cell protein in those with less than three, P = 0.11). 4. This study provides evidence for the role of overproduction of the vasodilator nitric oxide in sepsis syndrome. PMID- 7536647 TI - Effects of dietary polyunsaturated fatty acids on small intestinal secretory and absorptive function: studies in rat jejunum in vitro. AB - 1. Recent initiatives which advocate an increase in dietary polyunsaturated fat intake have led to the study of the effects of this upon gastrointestinal function. 2. Weanling rats were for 21 weeks fed diets containing 10% fat that were either high or low in polyunsaturated fats. Jejunal function was studied in vitro in an Ussing chamber. 3. Basal intestinal short-circuit was similar in both groups. 4. A decreased EC50 for the non-neural electrogenic secretory responses to acetylcholine, bethanecol and isobutylmethylxanthine was apparent in the jejuna of rats fed a diet high in polyunsaturated fatty acids. 5. Submaximal electrogenic galactose absorption was increased in the rats fed a diet high in polyunsaturated fatty acids. 6. Changing the composition of dietary lipid resulted in a change in the fatty composition of the apical enterocyte membrane. 7. Diets high in polyunsaturated fatty acids may be both prosecretory and proabsorptive in the small intestine. PMID- 7536648 TI - [Treatment of chronic atherosclerotic obliterative arteriopathy in the II and IV Fontaine stage. Personal observations using iloprost]. AB - The traditional medical treatment of atherosclerotic artery disease of the lower limbs with vasodilators, antiplatelet agents, hemorheologic agents and heparin has so far yielded only partly satisfactory results. In view of this, we have treated ten patients with lower limb arterial disease, Fontain stages III and IV, with a stable prostacyclin analogue, iloprost, with antiaggregant, vasodilating, and cytoprotective activity. Patients' mean age 73.6 +/- 9.9 yrs., M/F ratio 9/1. The drug was infused by peristaltic pump at dosages varying from 1.5 to 2 ng/kg body weight/min for 6 hours daily and for an average of 25 days. Our observations, albeit on a limited number of subjects, appear to confirm the good tolerability, and above all the efficacy of iloprost (relative number of patients improved: 0.70). Nevertheless, in our view, a hypertensive diabetic woman who under treatment developed a lower myocardial infarction deserves special attention. Data found in the literature do not allow us to hold iloprost responsible for this effect with any degree of certainty. However, the doubt remains that as a result of its vasodilating action the drug may have caused steal phenomena from the subendocardial to subepicardial districts, thus provoking the ischemic event. PMID- 7536649 TI - Localization of the complement membrane attack complex inhibitor (CD59) in human conjunctiva and lacrimal gland. AB - Recent studies have established that complement is present in the eye and participates in ocular defense. The mechanisms by which ocular tissues are protected from bystander injury arising from local activation of the cascade, however, have not been characterized. Decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59) are cell surface regulatory proteins that protect blood cells from uptake of autologous C3b and polymerization of autologous C9 on their surfaces. In previous studies, we found that DAF is expressed in high levels on corneal, conjunctival, and lacrimal gland acinar surfaces. In this study we assayed ocular and lacrimal gland tissues for CD59. Immunohistochemical analyses demonstrated large amounts of the protein the same locations. The presence of CD59 in these sites is consistent with the proposal that CD59 functions together with DAF in protecting ocular tissues from autologous complement-mediated injury. PMID- 7536650 TI - Increased catalase levels and hypoxanthine-enhanced nitro-blue tetrazolium staining in rat retina after ischemia followed by recirculation. AB - In the present study, using retinal ischemia as a model, we examined if different periods of ischemia and recirculation influenced the generation of reactive oxygen species, i.e. in hydrogen peroxide generation and nitroblue tetrazolium (NBT) reduction. Ischemia was induced for 30 and 90 min by ligation of the optic nerve with the vessels and recirculation was established by removing the ligature. The rats were sacrificed after 15 min or 3 days of recirculation. The retinas were separated from the pigment epithelium for measurements of catalase activity and examination of NBT staining. Compared to controls, the catalase activity was increased after 30 and 90 min of ischemia followed by 15 min of recirculation, and after 90 min of ischemia followed by 3 days of recirculation. As in controls, NBT staining was observed, both after 30 and 90 min of ischemia followed by 15 min of recirculation, in photoreceptors, in both plexiform layers, in some ganglion and glial cells, and, occasionally, in cells in the inner nuclear layer. Opposite to controls, addition of hypoxanthine to the NBT solution resulted in an increased staining in vessels in the inner nuclear layer in retinas subjected to 30 min of ischemia followed by 3 days of recirculation. The increased catalase activity suggests an increased amount of this free radical scavenger after ischemia followed by short-term and long-term recirculation. The hypoxanthine-enhanced NBT staining of blood vessel walls after ischemia followed by long-term recirculation indicates an activation of xanthine oxidase and an increased production of NBT reductants, some of which may represent oxygen free radicals. PMID- 7536652 TI - Further metabolism of FK506 (tacrolimus). Identification and biological activities of the metabolites oxidized at multiple sites of FK506. AB - To characterize the metabolic pathway of FK506 (tacrolimus), FK506 or its 31-O desmethyl metabolite was incubated with liver microsomes prepared from dexamethasone-treated rats in the presence of a NADPH-generating system under aerobic conditions. Besides the four oxidized metabolites already reported, four new metabolites were isolated and identified by HPLC, mass spectrometry, and NMR spectroscopy, and their biological activities were examined. The di-demethylated metabolites at the 15- and 31-, 13- and 31-, and 13- and 15-methoxy groups of FK506, were designated respectively as M-V, M-VI, and M-VII. The fourth, M-VIII, was the metabolite produced after O-demethylation at the 31-methoxy group and formation of a fused 10-membered ring structure through the 19- to 22-carbon of the macrolide ring after oxidation of the 19-methyl group, and of the 36- and 37 vinyl group of FK506. The immunosuppressive activity of the isolated metabolites was estimated in a mouse mixed lymphocyte reaction system and the IC50 values for M-V, M-VI, M-VII, M-VIII, and FK506 were > 1000, 8.78, > 1000, 15.27, and 0.11 ng/ml, respectively. Reactivity of the metabolites with mouse anti-FK506 monoclonal antibody was studied and immunocrossreactivity of M-V was 92.3% of FK506, but no reactivity was observed for M-VI, M-VII, and M-VIII. FK506 thus was metabolized at multiple sites by rat hepatic microsomes and the metabolites formed (M-V) - (M-VIII) exhibited weak or negligible immunosuppressive activity. PMID- 7536653 TI - [Intracardial thrombus formation in heparin-associated thrombocytopenia type II]. AB - Deep vein thrombosis of the right leg occurred in a 77-year-old woman after percutaneous cardiac catheterization via the right femoral vein, performed to assess mitral valve disease with atrial fibrillation. She thereupon received intravenous heparin (1,000 IU/h; partial thromboplastin time 60-70s). 13 days later she developed a transient incomplete right brachiofacial hemiparesis with motor aphasia. Transthoracic echocardiography revealed a fresh left atrial thrombus. Platelet count fell from initially normal levels to 20 x 10(9)/l. Because type II heparin-associated thrombocytopenia was suspected heparin administration was discontinued and phenprocoumon administered. Heparin-dependent antibodies were demonstrated with the heparin-induced platelet activation test. Cross reactions occurred in vitro against all low-molecular heparins and heparinoid ORG 10172. The platelet count had become normal 17 days later, the leg veins had recanalized and the intraatrial thrombus had become much smaller. The patient declined cardiac surgery and was discharged on the 41st hospital day in satisfactory general condition on maintenance anticoagulant dosage. PMID- 7536651 TI - Colocalization of NADPH-diaphorase staining and VIP immunoreactivity in neurons in opossum internal anal sphincter. AB - Nitric oxide and vasoactive intestinal polypeptide (VIP) are important inhibitory neurotransmitters mediating relaxation of the internal anal sphincter. The location and coexistence of these two neurotransmitters in the internal anal sphincter has not been examined. We performed a double-labeling study to examine the coexistence of nitric oxide synthase and VIP in the opossum internal anal sphincter using the NADPH-diaphorase technique which is a histochemical stain for nitric oxide synthase. In perfusion-fixed, frozen-sectioned tissue, VIP immunoreactive neurons were labeled using immunofluorescence histochemistry. After photographing the VIP-immunoreactive neurons, nitric oxide synthase was labeled using the NADPH-diaphorase technique. Ganglia containing neuronal cell bodies were present in the myenteric plexus for the entire extent of the internal anal sphincter. VIP-immunoreactive and NADPH-diaphorase-positive neurons were present in ganglia in the myenteric as well as the submucosal plexuses. Most of the VIP-immunoreactive neurons were also NADPH-diaphorase positive. VIP and nitric oxide synthase are present and frequently coexist in neurons in the internal anal sphincter of the opossum. These neurons may be an important source of inhibitory innervation mediating the rectoanal reflex-induced relaxation of the sphincter. The demonstration of the coexistence of these two neurotransmitters will be of fundamental importance in unraveling their relationship and interaction in the internal anal sphincter as well as other systems. PMID- 7536654 TI - Discrete endogenous signals mediate neural competence and induction in P19 embryonal carcinoma stem cells. AB - Endogenous signals capable of inducing neuroectodermal differentiation are expressed by differentiating P19 EC cells in vitro. The present study demonstrates that at least two discrete signals are required. One is expressed by isolated primitive streak mesoderm-like cell lines and has the capacity to induce the expression of Pax-3 but, alone, induces neural differentiation inefficiently. The second signal is not expressed by the primitive streak mesoderm-like cell line but is present in conditioned media from differentiating P19 EC cells following DMSO treatment. This signal does not induce either Pax-3 expression or morphological differentiation and does not commit stem cells to a neuroectodermal fate. Rather, it acts synergistically with the signal derived from the primitive streak mesoderm-like cells to increase the efficiency with which stem cells respond initially by Pax-3 expression and subsequently by differentiation towards neural lineages. The activity of this second signal can be replaced by forskolin and 3-isobutyl-1-methyl-xanthine suggesting that its effects are transduced by a cyclic nucleotide-dependent pathway. PMID- 7536655 TI - Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway. AB - Trunk neural crest cells segregate from the neuroepithelium and enter a 'migration staging area' lateral to the embryonic neural tube. After some crest cells in the migration staging area have begun to migrate on a medial pathway, a subpopulation of crest-derived cells remaining in the migration staging area expresses mRNAs for the receptor tyrosine kinase, c-kit, and tyrosinase-related protein-2, both of which are characteristic of melanocyte precursors. These putative melanocyte precursors are subsequently observed on the lateral crest migration pathway between the dermatome and overlying epithelium, and then dispersed in nascent dermal mesenchyme. Melanocyte precursors transiently require the c-kit ligand, Steel factor for survival. Although Steel factor mRNA is transiently expressed in the dorsal dermatome before the onset of trunk neural crest cell dispersal on the lateral pathway, it is no longer produced by dermatomal cells when melanocyte precursors have dispersed in the dermal mesenchyme. To assess the role of Steel factor in migration of melanocyte precursors on the lateral pathway, we analyzed melanocyte precursor dispersal and fate on the lateral pathway of two different Sl mutants, Sl, a null allele, and Sld, which lacks cell surface-associated Steel factor but produces a soluble form. No melanocyte precursors were detected in the dermatome of embryos homozygous for the Sl allele or in W mutants that lack functional c-kit. In contrast, in embryos homozygous for the Sld allele, melanocyte precursors appeared on the lateral pathway, but subsequently disappear from the dermis. These results suggest that soluble Steel factor is required for melanocyte precursor dispersal on the lateral pathway, or for their initial survival in the migration staging area. In contrast, membrane-bound Steel factor appears to promote melanocyte precursor survival in the dermis. PMID- 7536656 TI - Regulation of desmocollin transcription in mouse preimplantation embryos. AB - The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527 539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo. PMID- 7536657 TI - [Nuclear pathway of interferons]. PMID- 7536658 TI - Expression of insulin-like growth factor binding proteins in the rat kidney: effects of long-term diabetes. AB - Recent studies have shown that the renal synthesis of insulin-like growth factor binding proteins (IGFBPs) is altered in insulin-deficient diabetes mellitus, suggesting that these changes may be implicated in the alterations in renal function and morphology that accompany diabetes. To investigate the time course and the precise cellular distribution of changes in IGFBP expression, we used quantitative in situ hybridization to analyze renal IGF-I and IGFBP-1 to -5 messenger RNA (mRNA) localization and levels from 2 days to 6 months after the onset of streptozotocin-induced diabetes. There was an immediate sharp decline in IGF-I mRNA levels in the outer medulla that persisted for up to 3 months and a much smaller reduction in IGF-I mRNA levels in the medullary thick ascending limbs (MTALs). In nondiabetic animals, IGFBP-1 mRNA is most abundant in the MTALs. Immediately after the induction of diabetes, however, there was a greater than 2-fold increase in cortical IGFBP-1 mRNA and a 75% decrease in IGFBP-1 mRNA in MTALs. These changes persisted for up to 6 months in the diabetic animals. In contrast, IGFBP-5 mRNA levels were increased in the outer medulla and decreased in the cortex of diabetic kidneys. No significant changes in renal IG-FBP-2 mRNA levels or distribution were noted, and changes in IG-FBP-3 and -4 mRNA levels were subtle. In summary, streptozotocin-induced diabetes is associated with very prominent and complex alterations in renal IGF system gene expression, including robust increases in cortical IGFBP-1 and profound decreases in cortical IG-FBP-5 mRNA and medullary IGF-I mRNA levels. The divergent changes in IGFBP-1 and -5 mRNA levels in cortex vs. outer medulla indicate that regulation of IGFBP mRNA levels is quite complex. PMID- 7536659 TI - Glucocorticoid regulation of insulin-like growth factor-binding protein-3. AB - Short stature and decreased growth velocity are prominent features of endogenous and pharmacological glucocorticoid excess in children. Underlying processes may involve direct cellular effects or defective generation of insulin-like growth factors (IGFs) and/or IGF-binding proteins (IGFBPs), which modulate IGF stimulated events and regulate growth. To evaluate potential mechanisms, we investigated the impact of dexamethasone (dex) on hepatic expression of IGFBP-3, the major carrier protein for IGFs. Using cocultured hepatic parenchymal and nonparenchymal cells, dex at 10(-8) and 10(-6) M decreased IGFBP-3 secretion by 67 +/- 9% and 73 +/- 9%, respectively (both P < 0.05 vs. no dex). In a separate experiment, IGFBP-3 messenger RNA (mRNA) expression was decreased by 84 +/- 2% and 75 +/- 2% (both P < 0.05 vs. no dex). In combined studies, levels of IGFBP-3 protein in conditioned medium were strongly correlated with the abundance of IGFBP-3 mRNA (r = 0.75; P < 0.01), consistent with regulation at a pretranslational level. After inhibition of transcription, levels of IGFBP-3 mRNA decreased 85% and 86% over 24 h in cells cultured in 10(-6) M and no dex, respectively; the t1/2 was 13.6 h at 10(-6) M and 12.6 h with no dex, indicating that dex had no effect on IGFBP-3 mRNA stability. To evaluate transcriptional effects, the rate of IGFBP-3 gene transcription was measured by incorporation of [alpha-32P]UTP into preinitiated message in isolated nuclei and fell 78% after the addition of 10(-6) M dex for 48 h (compared to cells cultured in 10(-9) M dex), an inhibition of a magnitude similar to the effects on protein release and mRNA abundance. We conclude that dex may reduce the production of IGFBP-3 by inhibiting IGFBP-3 gene transcription. PMID- 7536660 TI - Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotrophic neurons: estrogen dependency. AB - Hypophysiotrophic neurons projecting to hypophyseal portal vessels in the median eminence of the hypothalamus maintain the operation of the master gland, the pituitary, by secreting releasing and release-inhibiting hormones into the bloodstream. LHRH, synthesized in neurons of the rat prosencephalon, is one of the key substances that governs the anterior pituitary-gonadal axis. Recently, it has been shown that the peptide galanin (GAL) is coproduced in a subpopulation of LHRH neurons and is a potent modulator of central processes regulating reproduction. A better understanding of the secretory mechanisms involved in pulsatile hormone release from LHRH axons of the median eminence requires exploration of the organelle domain that displays the cosynthesized peptides in terminal boutons. This study shows that LHRH- and GAL-immunoreactive axons overlap heavily in the lateral part of the median eminence. Double fluorescent labeling revealed colocalization of the peptides at the level of single axon terminals. By means of dual colloidal gold immunolabeling, LHRH and GAL were detected in the same secretory vesicles at the ultrastructural level. The incidence of colocalizing vesicles was high in the female (45%) and low in the male (3%) rat. Ovariectomy resulted in a dramatic decline in the number of LHRH/GAL-coexpressing vesicles (23%), which was reversed (55%) by the administration of estradiol. The observations indicate a sex-related difference in the packaging of LHRH and GAL and suggest that the events are estrogen dependent. Furthermore, the simultaneous release of GAL and LHRH from the colocalizing vesicles provides a mechanism that might ensure the potentiating effect of GAL on LHRH by synchronizing events at the receptor sites in the anterior pituitary. PMID- 7536662 TI - Oxytocin stimulates mitogen-activated protein kinase activity in cultured human puerperal uterine myometrial cells. AB - The regulation of mitogen-activated protein (MAP) kinase by oxytocin in cultured human uterine myometrial cells was investigated. Oxytocin caused the rapid stimulation of MAP kinase activity detected in 32P incorporation of MAP-2. Oxytocin also stimulated the phosphorylation of MAP kinase detected in incorporation of [32P]orthophosphate into MAP kinase. Furthermore, oxytocin induced the tyrosine phosphorylation of MAP kinase. The oxytocin-dependent increase in the tyrosine phosphorylation of MAP kinase displayed a transient time course and was dependent on the concentration of oxytocin applied to the cells. Furthermore, we examined the mechanism by which oxytocin induced MAP kinase phosphorylation. Islet-activating protein (100 ng/ml), which inactivates Gi/Go proteins, blocked the oxytocin-induced phosphorylation of MAP kinase. Moreover, 1 microM ritodrine, which is known to relax uterine muscle contraction, attenuated oxytocin-induced MAP kinase activity and phosphorylation. These results provide evidence that oxytocin acutely activates MAP kinase through an islet-activating protein-sensitive G-protein in human uterine myometrial cells, suggesting that this new pathway may play an important role in the biological action of oxytocin on these cells. PMID- 7536661 TI - Insulin-like growth factor (IGF) I and retinoic acid induce the synthesis of IGF binding protein 5 in rat osteoblastic cells. AB - The insulin-like growth factor (IGF) regulatory system has a major impact on bone physiology. Among the modulators of IGFs, a family of structurally related proteins, the IGF-binding proteins (IGFBPs), have been shown to either potentiate or inhibit IGF actions on bone growth. However, the regulation of IGFBP expression in bone cells is not completely understood. In the present study, the expression of IGFBP-5 was analyzed in primary osteoblastic cells (Ob cells) isolated from 22-day-old fetal rat calvariae. Treatment of Ob cells with either IGF-I or all-trans-retinoic acid (RA) caused a time- and dose-dependent increase in IGFBP-5 messenger RNA (mRNA) levels, as determined by Northern blot analysis. Stimulation of IGFBP-5 mRNA was obtained at 100 nM IGF-I between 6 and 16 h (2- to 2.5-fold) and 100 nM RA between 16 and 24 h (3- to 4-fold). Concomitant treatment of Ob cells with IGF-I and RA revealed an additive effect and a 5- to 7 fold increase in IGFBP-5 mRNA levels after 16-24 h. The effect of IGF-I and RA and their combination on IGFBP-5 transcripts was similar in confluent and subconfluent cultures of Ob cells. IGF-I and RA did not change IGFBP-5 mRNA stability in Ob cells after transcription arrest with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole. IGF-I and RA at 100 nM elevated the levels of IGFBP-5 heterogenous nuclear RNA, measured by reverse transcription-polymerase chain reaction. The effect was similar to that observed on mRNA levels. IGFBP-5 from rat Ob cells appeared as a single band of 31 kilodaltons in both the conditioned medium and the extracellular matrix as determined by Western immunoblots. IGF-I and RA, both at 100 nM, increased IGFBP 5 by 2- to 3-fold after 24 h. In conclusion, IGF-I and RA modify the synthesis and secretion of IGFBP-5 in rat Ob cells through pathways that may involve increased transcription and elongation and/or altered processing of heterogenous nuclear RNA. Our data suggest that IGFBP-5 may play a role in the osteoblastic differentiated function regulated by IGF-I and RA. PMID- 7536663 TI - Natriuretic peptide-augmented induction of nitric oxide synthase through cyclic guanosine 3',5'-monophosphate elevation in vascular smooth muscle cells. AB - To elucidate the role of natriuretic peptides in vascular remodeling, the effects of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide (CNP) on the induction of inducible nitric oxide (NO) synthase (iNOS) in rat aortic smooth muscle cells were examined. Although none of the peptides when applied alone induced the production of nitrite, a stable end product of NO, each peptide dramatically enhanced nitrite production induced by a cytokine combination of interleukin-1 alpha and tumor necrosis factor-alpha. Each natriuretic peptide stimulated intracellular cGMP accumulation in a dose dependent manner. Time-dependent nitrite production by the cytokines was increased by CNP cotreatment and inhibited by NG-methyl-L-arginine, indicating involvement of the L-arginine-NO pathway. Northern blot analysis showed that the augmented nitrite production was accompanied by an increase in iNOS messenger RNA. A cGMP analog, 8-bromo-cGMP, completely mimicked all of the effects of CNP described above. A cGMP-dependent protein kinase inhibitor, KT5823, paradoxically increased nitrite production and iNOS messenger RNA levels induced by the combination of 8-bromo-cGMP and both cytokines or by the two cytokines only. These data demonstrate the stimulatory effect of cGMP on cytokine-induced iNOS and imply that natriuretic peptides may play a regulatory role in vascular remodeling via the production of large amounts of NO. PMID- 7536664 TI - Androgen and fibroblast growth factor (FGF) regulation of FGF receptors in S115 mouse mammary tumor cells. AB - We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent. PMID- 7536665 TI - Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation. AB - Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat. PMID- 7536666 TI - Coordinate regulation of nitric oxide and 1,25-dihydroxyvitamin D production in the avian myelomonocytic cell line HD-11. AB - Cells of the monocyte/macrophage lineage are capable of both nitric oxide (NO) and 1,25-dihydroxyvitamin D [1, 25-(OH)2D] production through expression of inducible nitric oxide synthase (iNOS) and a putative 25-hydroxyvitamin D (25 OHD)-1-hydroxylase, respectively. We have recently reported that 1,25-(OH)2D synthesis in the chick myelomonocytic cell line HD-11 is restricted by inhibition of iNOS. In the current set of experiments, measuring nitrite, a stable water soluble secreted metabolite of NO as an index of iNOS activity and 1,25-(OH)2D3 in lipid extracts of cells incubated with 200 nM 25-OHD3 as an index of 1 hydroxylase activity, we demonstrate that NO and 1,25-(OH)2D production by HD-11 cells are temporally related, induced by the same kinds of activating agents, and coordinately regulated. NO and 1,25-(OH)2D3 production by HD-11 cells was stimulated severalfold by the macrophage stimulators interferon-gamma and lipopolysaccharide and by an autologous, nonlipid, heat-labile factor with an apparent molecular mass approximately 10,000 daltons. As expected NO synthesis was 1) dependent upon the presence of L-arginine in the extracellular medium, 2) subject to significant stimulation by Nw-hydroxy-L-arginine, an L-arginine derived intermediate in NO biosynthesis, and by sodium nitroprusside, a non-L arginine-dependent source of intracellular NO, and 3) inhibited by Nw-nitro-L arginine methyl ester, a competitive inhibitor of iNOS. At high NO production rates, induced either by high-dose lipopolysaccharide or by sodium nitroprusside exposure, there was an apparent downturn in 1,25-(OH)2D3 synthesis, suggesting functional dependence of the 1-hydroxylase on NO but ultimate inhibition of 1,25 (OH)2D3 synthetic capacity at high levels of intracellular NO production. On the basis of these results we postulate that the macrophage 25-OHD-1-hydroxylation reaction may be dependent on iNOS-generated NO as a soluble source of electrons and regulated in an autocrine mode by a macrophage-derived NO stimulatory factor and NO itself. PMID- 7536667 TI - Role of the Pro-Leu-Arg motif in glycosylation of human gonadotropin alpha subunit. AB - CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta subunit. Processing of the N-linked oligosaccharide of the glycoprotein family is both tissue and dimer specific. LH, TSH, and free alpha synthesized in pituitary bear oligosaccharide terminating with sulfate (SO4) and N-acetylgalactosamine (GalNAc), whereas the termination of oligosaccharide in CG synthesized in placenta and FSH is sialic acid and galactose (Gal). Using site-directed mutagenesis and gene transfer, we studied the role of the Pro-Leu-Arg motif, which has been shown to be a recognition marker of glycoprotein hormone-specific GalNAc transferase, in sulfation of N-linked oligosaccharide in alpha-subunit. The wild-type or mutated alpha gene was transfected into GH3 cells. Our data revealed that substitution of the Pro-Leu-Arg motif by Ala-Leu-Ala did not affect the sulfation of N-linked oligosaccharide, but generated the attachment of O linked oligosaccharide. alpha-Subunit containing either of the two N-linked glycosylations is also sulfated. We conclude that in GH3 cells, the Pro-Leu-Arg motif plays no role in the sulfation of oligosaccharide in alpha-subunit, and both N-glycosylations are terminated with SO4. PMID- 7536668 TI - Growth of LNCaP human prostate cancer cells is stimulated by estradiol via its own receptor. AB - We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor. PMID- 7536669 TI - Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients. AB - A new and fast method is described for detection of point mutations in polymerase chain reaction (PCR)-amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant-temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6 degrees C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo- and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four-band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exon 17b (R1066H, R 1066C, F1052V) and of two polymorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. PMID- 7536670 TI - Size-dependent separation of proteins in the presence of sodium dodecyl sulfate and dextran in capillary electrophoresis: effect of molecular weight of dextran. AB - Dextran solutions are widely used as sieving medium in protein analysis by capillary electrophoresis in the presence of sodium dodecyl sulfate. We studied the effect of dextran molecular weight on the separation efficiency using different dextran preparations with wide and very narrow molecular weight distributions, in the range between 1270 and 2,000,000. Migration times and band broadening of proteins were significantly affected by the molecular weight of dextran. Migration times of proteins decreased as molecular weights of the dextrans decreased. Satisfactory separation of all the proteins was possible with all dextrans except those with molecular weights of 70,000 and 23,800 where bovine serum albumin and phosphorylase b failed to be separated. Unexpectedly rapid separation of all the proteins with enhanced resolution could be observed using two dextrans with a narrow molecular weight distribution, with molecular weights of 1270 and 5220. Clearly the use of dextran with higher molecular weight is not the only way to achieve efficient separation of proteins. The separation mechanism in the presence of the low molecular weight dextrans remains to be made clear in a future study. PMID- 7536671 TI - Sample application by in-gel rehydration improves the resolution of two dimensional electrophoresis with immobilized pH gradients in the first dimension. AB - We describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5% T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 microL for a standard 3 x 160 x 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design. PMID- 7536673 TI - Antibodies to an epitope on synapsin I detect a protein associated with the endocytic compartment in non-neuronal cells. AB - To detect potential substrate proteins for Ca2+/calmodulin-dependent protein kinase II outside the central nervous system, antibodies were made to a synthetic peptide corresponding to a sequence within synapsin I which is phosphorylated by this enzyme. In neural tissues, this antibody (212) identified an 86/80 kDa doublet corresponding to synapsin I. In rat liver, intestinal enterocytes and the clone 9 cell line this antibody identified two proteins of 170 and 85 kDa. These proteins were present in the particulate fraction of liver postnuclear supernatant, and were released into the soluble fraction when extracted with 100 mM NaCl. In liver, enterocytes, and clone 9 cells, these antigens were localized by immunocytochemical techniques to small intracellular vesicles. The endocytic compartment of clone 9 cells was labeled by continuous uptake of horseradish peroxidase; antibody 212-labeled vesicles exhibited overlap with the compartment. To confirm the identity of this compartment as endosomal, rat liver endosomes were labeled in vivo by intravenous injection of horseradish peroxidase. Horseradish peroxidase-containing endosomes of approximately 80 nm were recognized by antibody 212. Occasionally, larger endosomes (approximately 300-500 nm) were also labeled. In clone 9 cells, partial overlap was observed between the 212 antigen and a transferrin receptor-positive, brefeldin A-sensitive compartment. In clone 9 cells double-labeled with anti-tubulin and antibody 212, then imaged using confocal microscopy, these vesicles appeared to be associated with microtubules. This antigen has properties similar to that of CLIP-170, a membrane-associated endosomal phosphoprotein. These findings demonstrate that a 170/85 kDa antigen containing an epitope for the Ca2+/calmodulin-dependent protein kinase II phosphorylation sequence is associated with an endocytic compartment. PMID- 7536672 TI - Expression of the functional soluble form of human fas ligand in activated lymphocytes. AB - Fas is a type I membrane protein which mediates apoptosis. Fas ligand (FasL) is a 40 kDa type II membrane protein expressed in cytotoxic T cells upon activation that belongs to the tumor necrosis factor (TNF) family. Here, we found abundant cytotoxic activity against Fas-expressing cells in the supernatant of COS cells transfected with human FasL cDNA but not with murine FasL cDNA. Using a specific polyclonal antibody against a peptide in the extracellular region of human FasL, a protein of 26 kDa was detected in the supernatant of the COS cells. The signal sequence of granulocyte colony-stimulating factor was attached to the extracellular region of human FasL. COS cells transfected with the cDNA coding for the chimeric protein efficiently secreted the active soluble form of human FasL (sFasL). Chemical crosslinking and gel filtration analysis suggested that human sFasL exists as a trimer. Human peripheral T cells activated with phorbol myristic acetate and ionomycin also produced functional sFasL, suggesting that human sFasL works as a pathological agent in systemic tissue injury. PMID- 7536674 TI - Apical membrane trafficking during regulated pancreatic exocrine secretion--role of alkaline pH in the acinar lumen and enzymatic cleavage of GP2, a GPI-linked protein. AB - The GP2/THP family of glycosyl phosphatidylinositol (GPI)-anchored proteins is targeted to apical secretory compartments in polarized epithelial cells. We demonstrate in the rat exocrine pancreas that enzyme-mediated release of GP2 from acinar cell membranes represents a pH-dependent process regulated by bicarbonate secreted from ductular cells. Release of GP2 from secretin-stimulated pancreatic lobules, which retain intralobular ducts, was inhibited by (i) bicarbonate substitution, (ii) chloride substitution, and (iii) DIDS, a potent inhibitor of chloride-bicarbonate exchange. These inhibitory effects were not observed in preparations of pancreatic acini devoid of ductal elements. Enzymatic cleavage of GP2 and amylase release from pancreatic acini varied directly as a function of pH of the acinar human. Alkali-induced GP2 release could be correlated with ultrastructural and biochemical evidence for stimulated retrieval (endocytosis) of exocytic membranes at the acinar lumen. Our study defines functional roles for ductal bicarbonate in acinar cell and lumen physiology and provides a potential explanation for the biological significance of enzyme-mediated cleavage of GP2 from the apical plasma membrane. PMID- 7536675 TI - Identification of beta-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans. AB - Several cell wall proteins released from yeast and hyphal cells of Candida albicans by laminarinase reacted with an affinity-purified antiserum raised against beta-1,6-glucan. Binding of the antiserum was competitively inhibited by beta-1,6-glucan, but not by beta-1,3-glucan or isolated N-chains. Immunodetection was completely abolished when the proteins were treated with periodate. These results demonstrate that the laminarinase-released wall proteins of C. albicans possess an epitope consisting of beta-1,6-linked glucose residues. The yeast form of C. albicans contained four beta-1,6-glucosylated wall proteins, an Endo H resistant protein of 125 kDa and three glycoproteins which became only detectable after Endo H digestion and had a molecular mass of 320, 170 and 44 kDa, respectively. As for the hyphal form, a different set of beta-1,6-glucosylated wall proteins was found consisting of two Endo H-resistant glycoproteins of 125 and 80 kDa, respectively, and two glycoproteins that after Endo H digestion had a molecular mass of 320 and 38 kDa, respectively. Sodium dodecyl sulfate extractable wall proteins and medium proteins did not react with the beta-1,6 glucan antiserum. The beta-1,6-glucan epitope could be removed by aqueous hydrofluoric acid indicating that the epitope is phosphodiester-linked to the cell wall proteins. It is speculated that the epitope forms part of a GPI-anchor and might be involved in the anchoring of mannoproteins into the cell wall. PMID- 7536676 TI - Prevalence of antibody to Rochalimaea henselae among Austrian cats. PMID- 7536677 TI - Use of recombinant alpha 1-adrenoceptors to characterize subtype selectivity of drugs for the treatment of prostatic hypertrophy. AB - Several alpha 1-adrenoceptor antagonists have recently been developed for the treatment of benign prostatic hypertrophy because of their less frequent systemic side-effects compared to conventional alpha 1-adrenoceptor blockers. One potential explanation for their good tolerability would be the selectivity for a certain subtype of alpha 1-adrenoceptor. Utilizing COS-7 cells expressing the rat alpha 1A, the hamster alpha 1B and the human alpha 1C-adrenoceptors, we investigated affinities of alfuzosin, doxazosin, terazosin, indoramin and (+)- and (-)-5-[2-[[2-(o-ethoxyphenoxy)ethyl] amino]propyl]-2-methoxybenzesulfonamide HCl (YM 617) compared to prazosin. Radioligand binding studies showed that the affinities of alpha 1-adrenoceptor subtypes for alfuzosin (Ki value; alpha 1A: 2.4 nM, alpha 1B:1.4 nM, alpha 1C:4.2 nM), doxazosin (Ki value; alpha 1A:2.7 nM, alpha 1B:3.2 nM, alpha 1C:7.5 nM), terazosin (Ki value; alpha 1A:2.5 nM, alpha 1B:2.7 nM, alpha 1C:7.1 nM), indoramin (Ki value; alpha 1A:69 nM, alpha 1B:21 nM, alpha 1C:13 nM) and prazosin (Ki value; alpha 1A:0.16 nM, alpha 1B:0.19 nM, alpha 1C:0.2 nM) were equipotent to the three receptor subtypes. Unlike these antagonists, both (+)- and (-)-YM617 had relatively lower affinity for alpha 1B receptors compared to the other subtypes (Ki value; for (+)-YM617, alpha 1A:22 nM, alpha 1B:96 nM, alpha 1C:4.3 nM; for (-)-YM617, alpha 1A:0.11 nM, alpha 1B:0.7 nM, alpha 1C:0.035 nM). The data suggest that alpha 1-adrenoceptor antagonists currently used for the treatment of the benign prostatic hyperplasia do not show substantial subtype selectivity. PMID- 7536678 TI - Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. AB - The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [125I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [125I] angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of NG-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8 bromo-cGMP, NaNO2 or NaNO3 failed to significantly alter the affinity or number of [125I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism. PMID- 7536679 TI - The effects of ethanol in combination with the alpha 2-adrenoceptor agonist dexmedetomidine and the alpha 2-adrenoceptor antagonist atipamezole on brain monoamine metabolites and motor performance of mice. AB - The time course of the effects of ethanol alone and in combination with the selective alpha 2-adrenoceptor agonist dexmedetomidine and the alpha-adrenoceptor antagonist atipamezole was studied in NIH-Swiss mice. Core body temperature, rotarod performance, motility and changes in the noradrenaline, dopamine, and 5 hydroxytryptamine (5-HT) metabolite contents of different brain parts (limbic forebrain, striatum, lower brainstem, the rest of the forebrain + midbrain and hypothalamus) were measured. Atipamezole (3 mg/kg) attenuated the hypothermia induced by either ethanol (3 g/kg) alone or ethanol in combination with dexmedetomidine (0.3 mg/kg). Atipamezole shortened the duration of the ethanol impaired and ethanol + dexmedetomidine-impaired rotarod performance. Further, atipamezole prevented the decreased motility due to the combined treatment with ethanol and dexmedetomidine. Ethanol increased 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) values. Dexmedetomidine alone decreased MHPG and 5-hydroxyindoleacetic acid (5 HIAA) concentrations and increased DOPAC and HVA values. Dexmedetomidine combined with ethanol resulted in a further increase in DOPAC and HVA values. Pharmacokinetic parameters did not contribute to this antagonism of ethanol's effects by atipamezole, nor did the antagonism observed in rotarod performance or hypothermia seem to correlate with the changes seen in the brain noradrenaline and dopamine or 5-HT metabolism. In conclusion, these findings suggest that several ethanol effects are not mediated via direct activation of alpha 2 adrenoceptors, even though some of ethanol's behavioral and physiological effects may be antagonized by coadministration of alpha 2-adrenoceptor antagonists. PMID- 7536680 TI - Fibronectin, laminin, vitronectin and their receptors at newly-formed capillaries in proliferative diabetic retinopathy. AB - Proliferative diabetic retinopathy (PDR) is characterized by intraocular formation of fibroglial vascularized tissue by active vasoproliferative mechanisms. Using immunocytochemistry, we have studied changes in the distribution pattern of fibronectin (FN), laminin (LN), vitronectin (VN) and their receptors in the newly-formed capillaries of PDR. In intraocular vascularized tissue of PDR patients, FN was present on both luminal and basal surfaces of endothelial cells, and was diffusely distributed in the interpericyte space. LN was also associated with the interpericyte space. VN was occasionally detected on the luminal capillary side, but was frequent in the basal aspect of the endothelium in the interpericyte space, where it was colocalized with FN. Beta-1 subunit complex receptors were detected on the luminal side, while alpha v beta 3 integrin was identified on both sides, more so in the luminal than in the basal endothelial domain. By slot-blotting techniques and densitometric analysis, increased concentrations of intravitreous FN and VN were found in PDR in comparison with normal samples. These results suggest that FN, VN and LN have a key role in the structural arrangement of newly formed capillaries in PDR, and that receptor expression could be involved in events of endothelial cell adhesion and proliferation. PMID- 7536681 TI - Influence of in vivo administration of GM-CSF and G-CSF on monocyte cytotoxicity. AB - The influence of colony-stimulating factors (CSFs) on the monocyte functions of 32 patients with refractory testicular cancer receiving high-dose chemotherapy followed by autologous bone marrow transplantation (ABMT) was tested. Eight patients were treated as a control group without CSF therapy, 12 patients received recombinant human granulocyte-macrophage-CSF (rhGM-CSF), and 12 patients received recombinant human granulocyte-CSF (rhG-CSF). For the assessment of monocyte activation induced by CSF expression of major histocompatibility complex (MHC) class I and II antigens, production of tumor necrosis factor (TNF) and monocyte-mediated cytotoxicity against tumor cell targets were chosen. Monocytes from patients with GM-CSF therapy showed a significant increase in MHC class I and II antigen expression as compared to patients without CSF treatment (p < 0.001). A significant increase in the expression of MHC class I was seen in monocytes from patients under G-CSF treatment, whereas no change of class II antigens was noticed. Production of TNF and monocyte-mediated cytotoxicity against U937 tumor cells was significantly increased in monocytes derived from patients receiving GM-CSF, as compared to those from the control group, while no effect was detectable in monocytes from patients with G-CSF therapy. However, after in vitro stimulation with interferon-gamma (IFN-gamma), monocytes derived from GM-CSF as well as from G-CSF treated patients responded with a significantly higher TNF-production and cytotoxicity than monocytes from control patients. PMID- 7536682 TI - An improved panning technique for the selection of CD34+ human bone marrow hematopoietic cells with high recovery of early progenitors. AB - The human hematopoietic pluripotent repopulating "stem cell" is thought to be present within a minor subpopulation of CD34+ cells. This has not been definitively shown, although the more primitive CD34+ cell subset contains precursors for all lymphoid and nonlymphoid cell lineages. When purifying CD34+ cells, it is important to recover these early progenitors, which are more strongly immunoadsorbent to the separation devices. Using a commercialized panning system (AIS CELLector flasks), we observed that a high degree of purity requires a thorough washing procedure so that cells not binding or weakly binding to CD34 antibodies are removed. High recoveries can be obtained if the adherent cells are then efficiently detached by a 2-hour incubation in culture medium without added cytokines. In this way, we can routinely obtain 93.5 +/- 3.4% purity of CD34+ cells with a 74% yield of the multipotent colony-forming units (CFU-GEMM). Complete recovery of the putative "stem cell," or at least the early progenitor cell compartment (CD34+ CD38low/- CD34+ Thy-1+ cells), is also obtained. More than 30% of these cells can generate day-14 colonies in vitro. Comparable results were obtained when the separation was scaled up for clinical application using appropriate large-scale devices. The various incubation times of the procedure can be easily adjusted to the work schedule. This renders the procedure easy to handle, efficient, safe, and, because the cells can be observed under light microscopy, easy to control. PMID- 7536683 TI - Steel factor supports the cycling of isolated human CD34+ cells in the absence of other growth factors. AB - Steel factor (SF) acts synergistically with other hematopoietic growth factors to support the proliferation of progenitor cells in a variety of culture systems. To determine whether SF alone could directly stimulate proliferation of early hematopoietic progenitor cells, isolated CD34+ cells from adult bone marrow and umbilical cord blood were studied in a short-term suspension culture in serum free medium. Numbers of CD34+ and proliferating cells were quantified by flow cytometry; proliferation was assessed by simultaneous measurement of expression of the nuclear antigen Ki67 and DNA content (propidium iodide [PI]). In the absence of growth factors, the numbers of CD34+ and cycling cells declined over 2 days. Cells cultured in the presence of SF alone maintained the number of CD34+ and cycling cells at levels equal to the starting population. Withdrawal of growth factors led to a dramatic decrease in the number of cells in G1. Compared to cells grown in the absence of growth factors, cells grown in the presence of SF had significantly higher numbers of CD34+ and cycling cells (mean fold increase = 1.3 and 2; p < 0.05 and 0.01, respectively). SF increased the numbers of cells in both G1 and later phases of the cell cycle. Addition of interleukin-3 (IL-3) to SF led to further significant increases in CD34+ and cycling cells. The effects of SF could not be attributed to inhibition of apoptosis. CD34+ cells isolated from peripheral blood (PB) from patients with chronic myelogenous leukemia (CML) displayed similar characteristics. As assessed by binding of phycoerythrin (PE)-labeled ligand and flow cytometry, c-kit was expressed on 65 +/- 6% of isolated CD34+ cells and was detected on HLA-DRlow, CD38low, and Thy1+ subsets, as well as on more mature progenitor cells. Thus, while the effects of SF are most marked in combination with other growth factors, SF appears to bind to and directly maintain the active cell-cycle characteristics of isolated CD34+ cells. PMID- 7536684 TI - Differential effects of the hematopoietic inhibitors MIP-1 alpha, TGF-beta, and TNF-alpha on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver. AB - We have previously characterized the proliferative response of primitive CD34+ cells, purified from adult bone marrow, umbilical cord blood, and fetal liver, to a mixture of hematopoietic stimulators (steel factor [SF], interleukin-3 [IL-3], IL-6, and erythropoietin [Epo]) in serum-free liquid cultures. In the present study, we assessed the effects of the hematopoietic inhibitors, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor-beta (TGF beta), and tumor necrosis factor-alpha (TNF-alpha), on the cytokine-induced proliferation of three different CD34+ cell subpopulations derived from cord blood and on total CD34+ cells derived from fetal liver. In cultures of cord blood cells, addition of MIP-1 alpha inhibited the numerical expansion of primitive CD34+ cells (CD34+ CD45RAlow CD71low cells) without inhibiting the proliferation of more mature subpopulations enriched for myeloid (CD34+ CD45RA+ CD71low cells) or erythroid (CD34+ CD45RAlow CD71+ cells) progenitors. TGF-beta significantly reduced the proliferation of all three subpopulations, although its effects were more pronounced on cells of the erythroid lineage, particularly immature erythroid progenitors. Similarly, TNF-alpha preferentially inhibited total nucleated and CD34+ cell production in the subpopulation enriched for erythroid cells. However, in contrast to TGF-beta, TNF-alpha preferentially inhibited the proliferation of more mature erythroid progenitors. In a separate set of experiments, MIP-1 alpha, TGF-beta, and TNF-alpha were added to cultures of total CD34+ cells purified from fetal liver. In keeping with the fact that the majority of the progenitors contained in these cells were erythroid progenitors, the inhibitory effects of the three cytokines were similar to those observed in cultures of CD34+ CD45RAlow CD71+ cord blood cells. The results of the present study demonstrate that MIP-1 alpha, TGF-beta, and TNF-alpha have the capacity to modulate cytokine-induced proliferation of cord blood and fetal liver progenitors. The differential effects of these three cytokines confirm their pleiotropic nature as regulators of hematopoiesis. PMID- 7536685 TI - Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. AB - Stimulatory cytokines may induce murine hematopoietic progenitor cells (HPCs) to survive, self-renew, proliferate, or differentiate. We studied the role of active cell cycling induced by the cytokines interleukin-3 (IL-3), IL-6, IL-11, and Steel factor (SF) on murine progenitor cell frequency and cell cycle status in an in vitro system and on engraftment potential in nonmyeloablated mice. Marrow exposure to IL-3, IL-6, IL-11, and SF in in vitro liquid culture maintained or expanded seven factor-responsive high and low proliferative potential colony forming cells (HPP-CFC and LPP-CFC). The HPP-CFC and LPP-CFC were dormant at the initiation of culture, as determined by 3H-thymidine suicide. There was an increase in the number and proliferation of HPP-CFC and LPP-CFC at 48 hours; by 48 hours, 62% of HPP-CFC and 56% of LPP-CFC were killed by 3H-TdR exposure. In engraftment studies of cytokine-stimulated marrow cells into normal hosts, female BALB/c mice received the equivalent of 40 x 10(6) starting male marrow cells exposed to cytokines in vitro for 48 hours for 3 consecutive days and were sacrificed 8 weeks after transplantation. Control groups received either 40 x 10(6) male uncultured marrow cells, 40 x 10(6) starting marrow cells cultured in medium without growth factors for 48 hours, or phosphate-buffered saline (PBS) for 3 days. Engraftment of male cytokine-treated cells was analyzed by Southern blot analysis using the Y-chromosome-specific pY2-cDNA probe. There was minimal engraftment (approaching background levels) in marrow, spleen, and thymus of nonmyeloablated female recipients. Transplant recipients that had received uncultured marrow directly after sacrifice showed engraftment levels of 21% (11 mice; range = 8 to 44%) into marrow, of 9% (range = 0 to 22%) into spleen, and 13% (range = 2 to 43%) into thymus. We conclude that active cell cycling of marrow cells induced by cytokine stimulation is associated with an engraftment defect in the normal host. PMID- 7536686 TI - Effect of nerve growth factor on peptide neurons in dorsal root ganglia after taxol or cisplatin treatment and in diabetic (db/db) mice. AB - In our study we have used morphological and radio-immunological methods for the investigation of calcitonin gene-related peptide (CGRP) and substance P in cervical dorsal root ganglia (DRGs) in mice after administration of taxol or cisplatin and in spontaneously diabetic animals (db/db mice). The results were compared to findings in animals receiving recombinant human nerve growth factor (rhNGF). Morphometric analysis did not reveal any significant changes of cell size distribution in diabetic and taxol-treated mice, whereas cisplatin induced a significant decrease in the number of large- and medium-sized neurons, indicating neuronal atrophy. This finding correlated with a highly significant loss of neuropeptides after cisplatin-application. Measurement of peptide levels in the taxol-treated groups and in diabetic mice demonstrated a decrease predominantly for CGRP. Application of 10 mg/kg NGF caused a significant elevation in peptide immunoreactivity in control animals and in taxol-treated mice, i.e., statistically significant increase in peptide concentrations and in the number of substance P- and CGRP-immunoreactive DRG-neurons, suggesting a recruitment of additional peptide cells. In diabetic animals a restoration in CGRP-content was observed under NGF-treatment; however, in this model the quantitative parameters did not demonstrate further elevation above control levels. Our data support the hypothesis that NGF exerts a major effect on the metabolism of transmitters associated with nociception and sensation in "healthy" controls and in various models of toxic and metabolic neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536687 TI - HIV Tat protein potentiates in vitro granulomonocytic progenitor cell growth. AB - The direct involvement of hematopoietic progenitor cells in AIDS which results in dysregulated hematopoiesis, probably due to regulatory factors yet to be determined, has been reported by us and others. In this study we demonstrate that the HIV-2 Tat gene product is released in supernatant of HIV-2-infected long-term bone marrow cultures (LTBMC). These Tat-containing supernatants specifically enhanced growth of CFU-GM in agar culture system and this promoting activity was specifically neutralized with anti-Tat antibodies. The use of different recombinant Tat proteins confirmed that Tat has been responsible for enhanced in vitro growth of CFU-GM after HIV-2 infection of LTBMC. Moreover, limiting dilution analysis showed that Tat acts directly on the CD34+ cell population in which it increases the frequency of IL3-responding cells. PMID- 7536688 TI - Differential inhibition of cytosolic and membrane-derived protein kinase C activity by staurosporine and other kinase inhibitors. AB - The hypothesis was tested that 9 kinase inhibitors with diverse specificities for protein kinase C (PKC), including staurosporine and four of its analogues, interfere differently with PKC derived from either the cytosolic or particulate fractions of MCF-7 breast carcinoma cells. GF 109203X inhibited the enzyme identically in either preparation. CGP 41251 and calphostin C inhibited cytosolic PKC more effectively than membrane-derived PKC with ratios of IC50 (cytosolic PKC) over IC50 (membrane-derived PKC) of 0.07 and 0.04, respectively. The other six agents inhibited membrane-derived PKC more potently than cytosolic enzyme. Staurosporine and RO 31 8220 exhibited IC50 ratios of 12.3 and 21.6, respectively. The results suggest that there are dramatic differences between kinase inhibitors in their divergent effects on cytosolic and membrane-derived PKC which should be borne in mind in the interpretation of their pharmacological properties. PMID- 7536689 TI - Nerve growth factor stimulates tyrosine phosphorylation of paxillin in PC12h cells. AB - Nerve growth factor (NGF) induces tyrosine phosphorylation of various cellular proteins to activate multiple signal transduction pathways. We show that one of these proteins is paxillin, a cytoskeletal component associated with adhesion plaques. Phospho-amino acid analysis showed that NGF stimulated phosphorylation of its serine in addition to tyrosine residues. Tyrosine phosphorylation of paxillin by NGF was blocked by the pretreatment of the cells with cytochalasin D, an inhibitor of actin polymerization. These results suggest that phosphorylation of paxillin is involved in the signaling pathway of NGF in PC12 cells. PMID- 7536690 TI - Selective inhibition of the mitochondrial permeability transition pore at the oxidation-reduction sensitive dithiol by monobromobimane. AB - In this paper we introduce monobromobimane, a thiol reagent, as a selective blocker of the recently identified dithiol whose oxidation-reduction status modifies voltage sensing by the mitochondrial permeability transition pore, a cyclosporin A-sensitive channel. Monobromobimane does not inhibit the phosphate carrier, nor does it interfere with Ca2+ transport, energy coupling or ATP production and transport. We show that monobromobimane selectively prevents the shift in pore gating potential caused by some dithiol oxidants or crosslinkers but not by increasing [Ca2+], allowing a clear distinction of the pore agonists which act at this site. PMID- 7536691 TI - Palliation of pancreatic cancer. A prospective multicentre study. AB - The aim of this study was to compare palliation of patients with verified pancreatic carcinoma after radical surgery (n = 84), bypass surgery, i.e. biliary (n = 106), biliary and duodenal (n = 85), and for patients with advanced disease, i.e. laparotomy alone (n = 56), not operated (n = 106), 3 months after discharge from hospital. 128 of 233 patients (55%) still alive were followed up. Radically resected patients had the best palliation and clinical performance; on average they could perform normal activities if helped, 82% were mobile at home and 62% were pain-free. Bypassed patients can on average look after themselves but normal activities are not possible, 31% were in hospital, 26% were pain-free and 39% suffered from nausea and vomiting. Patients with advanced disease were on average incapable and required special help and care, about 70% were in hospital, 8-17% were pain-free. 'Good risk' bypassed patients, i.e. in stage I-III, used stronger analgesics (29 vs 3%) (P = 0.006) and more frequently complained of nausea and vomiting (38 vs 12%) (P = 0.02) than radically resected patients. Palliation was comparable for the two bypassed groups. PMID- 7536693 TI - Suppression of follicular phase pituitary-gonadal function by a potent new gonadotropin-releasing hormone antagonist with reduced histamine-releasing properties (ganirelix). AB - OBJECTIVE: To determine if daily subcutaneous doses of ganirelix will suppress and maintain E2 < or = 30 pg/mL (conversion factor to SI unit, 3.671), the serum profiles of LH and FSH during and after cessation of treatment, the time-course of the resumption of normal ovarian function after ganirelix cessation, and to identify side effects of daily treatment. DESIGN: Open-label nonrandomized clinical study. SETTING: Normal human volunteers in an academic research center. PATIENTS: Women 21 to 45 years of age, with documented ovulatory menstrual cycles. INTERVENTIONS: Ganirelix was administered subcutaneously daily for 8 days. Blood samples were obtained during dosing as well as before and after cessation of dosing. MAIN OUTCOME MEASURES: Changes in serum E2, LH, FSH, P, and ganirelix. RESULTS: Ganirelix treatment rapidly decreased serum levels of gonadotropins and E2 after both 1 and 2 mg administration. Twenty-four hours after the first dose of ganirelix, E2 decreased from a mean +/- SEM of 50 +/- 8 and 67 +/- 11 pg/mL at baseline to 25 +/- 4 and 20 +/- 3 in the 1 mg and 2 mg groups, respectively. Estradiol remained suppressed (mean levels < 26 pg/mL) on all subsequent 7 days of ganirelix dosing in both groups. After the final dose of ganirelix, there was a rapid return of ovarian function in all volunteers. All women had P levels indicative of ovulation in the subsequent cycle, and the mean number of days from the final ganirelix dose to the next menses was 25.8 +/- 2.1 and 27.3 +/- 1.6 in the 1 and 2 mg groups, respectively. CONCLUSIONS: Daily ganirelix administration is effective in suppressing the pituitary-gonadal axis and has a side effect profile that should be well tolerated. PMID- 7536692 TI - The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. AB - OBJECTIVE: To determine if nitric oxide synthase activity within the male reproductive tract is regulated by androgen. DESIGN: Nitric oxide synthase activity was measured in the reproductive organs of three groups of mature rats: unoperated controls, 1-week castrates, and 1-week castrates given T capsules at the time of surgery. The presence of nitric oxide synthase activity was confirmed by using the nitric oxide synthase-specific inhibitor N-nitro-L-arginine methyl ester (L-NAME). RESULTS: After castration, nitric oxide synthase activity was significantly reduced by 88%, 73%, and 54% in the caput, corpus, and cauda epididymidis, respectively. In the penis, nitric oxide synthase activity decreased 45% and nitric oxide synthase protein decreased 57% after castration. In the seminal vesicle and lateral prostate, nitric oxide synthase activity increased significantly after castration from nondetectable levels in controls. Nitric oxide synthase activity in the coagulating gland and ventral and dorsal prostate did not change after castration. The changes in nitric oxide synthase activity in all organs after castration were prevented by T replacement. Additionally, the activity measured in every organ in all three treatment groups was > 90% inhibited by L-NAME. CONCLUSION: These data demonstrate that androgen differentially affects nitric oxide synthase activity in the male reproductive tract. To the best of our knowledge this is the first time that nitric oxide synthase activity has been shown to be influenced by androgen in any tissue. PMID- 7536695 TI - Characterization and quantification of full-length and truncated Na,K-ATPase alpha 1 and beta 1 RNA transcripts expressed in human retinal pigment epithelium. AB - We have characterized cDNA clones encoding the alpha 1 and beta 1 subunits of Na,K-ATPase produced in the human retinal pigment epithelium (hRPE). In addition to isolating clones corresponding to known sequences of Na,K-ATPase subunits, we report hitherto unknown forms of Na,K-ATPase with unique deduced amino acid (aa) sequences in their C-termini. Truncated cDNA sequences were found for both the beta 1 and alpha 1 subunits. While the beta 1 sequence is truncated by two aa residues at the C terminus, in the alpha 1 sequence 342 aa have been replaced by a unique sequence containing only 44 aa. Interestingly, this new C-terminal polypeptide shows sequence similarities to the Ca(2+)-ATPase and contains consensus sequence elements for phosphorylation and cell adhesion, suggesting expression of Na,K-ATPase subunits with unique functions. Using reverse transcription-polymerase chain reaction, RNA sequences for alpha 1, beta 1 and their corresponding truncated isoforms were quantified. 4.0 x 10(5) alpha 1 and 2.3 x 10(5) beta 1 molecules were found per ng of mRNA from hRPE. Much lower levels were detected for truncated alpha 1 and beta 1 (3.6 x 10(3) and 2.7 x 10(3) molecules/ng, respectively). These data corroborate the expression of truncated transcripts coding for unique aa sequences in hRPE, and suggest that factors other than alpha 1 and beta 1 mRNA levels regulate the equimolar accumulation of alpha and beta subunits in the plasma membrane. PMID- 7536694 TI - Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells. AB - The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4 1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3 7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized. PMID- 7536697 TI - Spindle cell carcinoma of the conjunctiva. PMID- 7536698 TI - [Hysteroscopy in the diagnosis and treatment of residua after evacuation of hydatidiform mole]. AB - The authors showed the case of 23 years old multipara treated in Department of Reproduction, Institute of Gynecology and Obstetrics, Academy of Medicine, Poznan because of hydatidiform mole using the exhaustion of uteri. After 8 weeks the hysteroscopy was done in order to explain the reasons of elevated serum beta hCG levels. During the hysteroscopy the polypoid lesion was recognized and removed using the electric resectoscope. After this endoscopic operation the normalization of serum beta hCG levels was achieved. PMID- 7536696 TI - Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats. AB - We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest. PMID- 7536699 TI - [Biophysical and pharmacological characterization of receptor-operated nonselective cation channels (ROCC) and their regulatory mechanisms in smooth muscle]. AB - Stimulation of excitatory receptors in smooth muscle often leads to the opening of ROCC. These channels exhibit considerable permeability to Ca2+, and they have been regarded as the most probable candidate for the "receptor-operated Ca2+ entry" pathway. The muscarinic receptor ROCC in guinea pig ileum (mROCC) have a unitary conductance of -25pS and are activated through a pertussis toxin sensitive G protein. mROCC permeate Ca2+ and Ba2+ several fold more preferably than monovalent cations, and they are inhibited by various types of K channel blockers, diphenylamine-2-carboxylate derivatives and even by nicardipine and D 600 at high concentrations. mROCC are efficiently regulated by various physiological factors including the membrane potential, intracellular Ca2+ concentration, external pH and osmolarity. The effective ranges of these factors span their dynamic ranges under physiological conditions. In addition to these properties, mROCC have several sites sensitive to external polyvalent cations. The alpha 1-adrenergic receptor ROCC in rabbit portal vein resemble mROCC in many respects, e.g., the unitary conductance, ionic selectivity, activation kinetics, sensitivity to polyvalent cations and voltage-dependence. These complex characteristics of ROCC suggest that they play other roles in addition to being just a passive cation permeable pore in agonist-mediated Ca2+ mobilization in smooth muscle. PMID- 7536700 TI - [Effect of fluvoxamine, a new antidepressant drug, on monoamine dynamics in the rat cerebral limbic system and its pharmacological characteristic]. AB - The effect of fluvoxamine (FLU) on the amine dynamics in serotonergic, noradrenergic or dopaminergic nervous systems in the rat cerebral limbic system was investigated, with reference to the pharmacological characteristics of FLU. Following single oral administration of 30 and/or 60 mg/kg FLU, significant decreases in the 5-hydroxyindole-3-acetic acid (5-HIAA)/serotonin (5-HT) ratios in the amygdala and hippocampus and the 4-hydroxy-3-methoxyphenylglycol (MHPG)/norepinephrine (NE) ratio in the amygdala were produced. FLU at 15 mg/kg, p.o. also caused a significant increase in MHPG/NE ratio in the hippocampus. On the other hand, imipramine (IMI) at 30 mg/kg, p.o. induced significant decreases in 5-HIAA/5-HT ratio in the hippocampus, MHPG/NE ratios in the amygdala and hippocampus, and 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) ratios in the amygdala, hippocampus and septum. The 5-HIAA/5-HT ratios in the amygdala and hippocampus and the MHPG/NE ratio in the septum were significantly decreased after repeated oral administration of 30 mg/kg FLU twice daily for 14 days, but the MHPG/NE ratio in the amygdala was increased. In the septum, DOPAC/DA ratio was significantly decreased following repeated administration of IMI at 30 mg/kg, p.o. These findings suggest that FLU inhibits 5-HT turnover in the amygdala and hippocampus as a result of the interference with the neuronal re-uptake mechanism for 5-HT. Also, FLU has an effect on NE turnover in the cerebral limbic system, and the effect may appear in connection with a change in the interaction of nervous systems. PMID- 7536701 TI - [An elderly patient with arthralgia, fever, weakness and weight loss. Diagnosis of Whipple disease by PCR detection of Tropheryma whippelii]. PMID- 7536702 TI - Histamine and serotonin released from the rat perfused heart by compound 48/80 or by allergen challenge influence noradrenaline or acetylcholine exocytotic release. AB - Terminal nerve fibres of the autonomic nervous system closely approach mast cells in peripheral organs, and mutual influences between release of neurotransmitters or mast cell mediators may cause neuro-immunological interactions. We have studied the influence of mast cell degranulation on the release of endogenous noradrenaline and newly incorporated acetylcholine (such as 14C choline/acetylcholine overflow) evoked by stimulation of extrinsic postganglionic sympathetic or preganglionic vagal nerves in the rat Langendorff heart perfused with Tyrode solution. Compound 48/80 perfused in normal hearts, or ovalbumin infused into hearts from rats sensitized to ovalbumin, enhanced the overflow of endogenous histamine and serotonin. Both stimuli increased the release of mediators to a similar extent and with fast kinetics. Maximum average concentrations in the perfusate of histamine were about 800 nmol/l, and of serotonin 40 nmol/l, in a sample collected within 4 min after mast cell degranulation. Stimulation of autonomic nerves did not affect basal histamine or serotonin overflow. Whereas basal overflows were unaffected, the stimulation evoked releases of both noradrenaline and acetylcholine, were facilitated when compound 48/80 was perfused before and during nerve stimulation. The facilitation of noradrenaline overflow was more pronounced (by 60%) when compound 48/80 induced mediator overflow started 4 min before nerve stimulation as compared to 30 s (15%), and was reduced by cocaine (by 50%), and, in the presence of cocaine, abolished by cimetidine (but was unaffected by mepyramine and thioperamide) and NG-nitro-(L)-(-)-arginine. In the presence of cimetidine and cocaine, when the facilitatory components were abolished, the evoked noradrenaline overflow observed 30 s after the start of infusion of compound 48/80 was inhibited, and the inhibition was partly reduced by methiotepin and ketanserin. Ovalbumin infusion in hearts from sensitized animals caused an inhibition of evoked noradrenaline overflow sensitive to methiotepin and also partly to ketanserin, and no facilitation was observed. The facilitation (> 100%) of evoked overflow of acetylcholine observed at 4 min after the start of perfusion with compound 48/80 was partly reduced by thioperamide (but not mepyramine or cimetidine) and to a comparable extent either by tropisetron (3 mumol/l) alone or by tropisetron plus methiotepin. In conclusion, degranulation of immunological cells is followed by histamine and serotonin release in the rat heart and may affect the release of autonomic neurotransmitters in rather unusual ways, by i) an uptake1-dependent and ii) an H2-mediated facilitation which probably involves nitric oxide as a permissive mediator, and iii) a serotonergic inhibition, of noradrenaline release, and iv) an H3- and serotonergic facilitation of acetylcholine release. PMID- 7536703 TI - Should patients over 80 years old be operated on for colorectal or gastric cancer? AB - One hundred and three patients aged 80 years or more were operated on for colorectal cancer (n = 80) or gastric cancer (n = 23). The postoperative mortality rate was 8%. Hospital mortality (12%) was not influenced by the factors age or emergency procedure, but by the factors ASA score (P = 0.06), palliative procedure (p = 0.08), and gastric surgery (P = 0.05). The overall 5-year survival rate was 23% after colorectal surgery, and 10% after gastric surgery (P = 0.001). After colorectal surgery, long-term survivals were observed, even in presence of serosal and/or lymph nodes involvement. Thirteen patients were alive more than five years after surgery. In contrast, after gastric surgery, all patients, except those with early carcinoma, died within one year. Indications for operation must be wide in colorectal cancers, and more restrictive in gastric cancers. Long-term survivals were observed after colorectal surgery in the elderly, even in the presence of lymph node involvement. In contrast, after gastric surgery, all patients, except those with early carcinoma, died within one year. PMID- 7536704 TI - End-to-end duodenojejunal bypass for unresectable periampullary carcinoma. AB - Palliative operation plays an important part in the treatment of periampullary carcinoma. However, gastric bypass such as the widely practiced side-to-side gastrojejunostomy frequently fails to provide adequate drainage. Here we attempted to fashion an end-to-end duodenojejunostomy in the hope of establishing physiological continuity of the stomach and duodenum. Biliary bypass with side-to side choledochojejunostomy is performed simultaneously. Eight patients underwent this surgery. In seven of these, radical resection proved to be impossible, and in one the duodeno-biliary decompression was attempted before the radical operation. Early results were satisfactory in all patients. They began to eat liquid meals within a week, and were discharged uneventfully within the third postoperative week, when they were able to eat a regular diet. No ulcer developed in any of the patients. Plasma gastrin levels following a test meal was significantly lower after the operation, but plasma CCK-N and GIP levels showed no statistical difference prior to and after surgery. This duodenojejunal bypass is recommended as a means of improving the quality of the remaining life of the patients. PMID- 7536705 TI - The significance of mucosal staining for the endoscopic diagnosis of chronic esophagitis as assessed in biopsy findings. AB - A total of 115 patients with clinical signs of esophagitis were examined by endoscopy and mucosal biopsy combined with Lugol solution. Overall, 175 mucosal areas (105 stained and 70 unstained) were subjected to histopathological examination. The correlation between the staining properties of the esophageal mucosa and the histopathological diagnosis resulted in a diagnostic sensitivity of 51.9%, a specificity of 70.2%, a positive predictive value of 60.0% and a negative predictive value of 62.9% for Lugol combined endoscopy. Lugol combined endoscopy did not appear to result in any improvement in the diagnosis or staging of reflux esophagitis in our study. PMID- 7536706 TI - The inhibitory effect of FK506 on cytotoxic T-lymphocyte killing. AB - The immunosuppressant FK506 inhibits N-alpha-benzyloxylcarbonyl-L-lysine thiobenzyl ester (BLT) esterase release from cytotoxic T lymphocytes (CTL). In addition, serine esterase has been demonstrated to be strongly associated with CTL killing. In the present study, the effect of FK506 on the activity of CTL killing against target cells was examined. FK506 inhibited lysis of antigen (Ag) treated CTL target cells by auto-CTL, but failed to inhibit lysis of conventional P815 target cells by CTL. Moreover, FK506 inhibited DNA fragmentation of CTL target cells lysed by auto-CTL. Killing of CTL target cells by FK506-pretreated auto-CTL was inhibited, but FK506-pretreated CTL target cells were killed by auto CTL. Incubation of both FK506 and Ag-pretreated CTL target cells with untreated auto-CTL induced DNA fragmentation. These indicated that FK506 inhibited CTL killing by influencing effector, but not CTL target cells. These results suggest that FK506 may function by inhibiting alloreactive CTL killing in organ transplantation in addition to the interruption of the T-cell receptor signal transduction leading to cytokine production. PMID- 7536707 TI - In vitro activation and detection of antibody-secreting cells from Trypanosoma congolense-infected cattle. AB - B cells from the peripheral blood and spleen of Trypanosoma congolense-infected cattle and from the peripheral blood of an uninfected cohort were analysed for ability to secrete antibody and for expression of surface antigens before and after in vitro culture with interleukin-2, lipopolysaccharide and pokeweed mitogen. Antibody-secreting cells (ASC) were only detected in lymphocytes from peripheral blood after in vitro stimulation. The frequency of ASC was greater in cultures of lymphocytes from infected cattle than from the uninfected cohort. The frequency of ASC was positively correlated with the number of B cells expressing the transferring receptor but not with the expression of the CD5 antigen. PMID- 7536709 TI - Self peptides bound by HLA class I molecules are derived from highly conserved regions of a set of evolutionarily conserved proteins. AB - An evolutionary analysis of self peptides reported to be bound by HLA class I molecules showed that these peptides are largely derived from proteins that have been highly conserved in the history of mammals. These proteins also often have universal tissue expression and have a higher than average frequency of highly hydrophilic residues. The peptides themselves are generally still more highly conserved than the source proteins and have a higher frequency of highly hydrophobic residues, evidently often being derived from conserved hydrophobic cores of the source proteins. These results suggest that the mechanism by which peptides are derived for MHC presentation may preferentially select peptides from conserved protein regions. In the case of parasite-derived peptides, such a mechanism would be adaptive in that it would reduce the likelihood of escape mutants. PMID- 7536708 TI - Anti-HIV effect of beta subunit of human chorionic gonadotropin (beta hCG) in vitro. AB - Human chorionic gonadotropin (hCG)--a pregnancy-associated immunomodulating hormone--has been recently shown in vitro to suppress reverse transcriptase activity in chronically HIV-infected lymphocytes and monocytes and to block viral transmission resulting from cell-cell contact between virus-carrying lymphocytes and placental trophoblasts. In further pursuit of the query into the mechanism of action, purified alpha and beta subunits of hCG were tested for the inhibition of p24 gag protein synthesis in virus-producing ACH-2 lymphocytes and U1 monocytes. Unlike the alpha subunit, beta-hCG displayed a distinct U-shaped dose response, characteristic of the effect of dimer hCG. Maximum inhibition of viral expression has been achieved at 10-100 ng/ml, the concentration corresponding to blood levels of beta-hCG in pregnant women. The doses that were several logs higher of normal levels seemed to increase viral production in monocytes. The data presented supports our original observations regarding the effect of intact hCG on HIV replication. While the mechanism of action remains to be established, the results suggest that the virus-interfering activity of hCG is determined by hormone-specific beta chain but not by the alpha subunit--shared with the family of glycoprotein hormones from the pituitary--follicle-stimulating hormone, luteinizing hormone and thyrotropin. PMID- 7536710 TI - Structural analysis of the RH-like blood group gene products in nonhuman primates. AB - Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M(r), immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene. PMID- 7536711 TI - Low-density lipoprotein induces vascular adhesion molecule expression on human endothelial cells. AB - We tested the hypothesis that low-density lipoprotein (LDL) and its acetylated form influence surface expression of vascular adhesion molecules on human endothelial cells. Vascular adhesion molecule surface expression was assessed with flow cytometry on cultured endothelial cells with a modified enzyme-linked immunosorbent assay. LDL acetylation was determined by chromatography. Monocyte adhesion to endothelial cells was assessed with U937 cells by direct counting. Tumor necrosis factor-alpha (10 ng/mL), a positive control, induced a time dependent expression of vascular adhesion molecules (P < .05), which peaked at 5 hours. Incubation of endothelial cells with LDL (1.3 to 26.0 mmol/L) led to an increase in expression at 2 and 5 hours (P < .05). Prolonged (24-hour) exposure to LDL resulted in a second peak. The effect of acetylated LDL on expression was not different from that of native LDL. Incubation with the protein kinase C inhibitor staurosporine (5 x 10(-8) mol/L) blocked the effects of both native and acetylated LDL completely (P < .05). The calcium channel blocker nitrendipine (10(-7) mol/L) did not influence the expression of vascular adhesion molecule at 2 and 5 hours but did reduce the effect of LDL on expression at 24 hours. LDL (2.6 mmol/L) also induced a significant increase in the surface expression of intercellular adhesion molecule-1 but did not affect the expression of endothelial adhesion molecules. LDL (2.6 mmol/L) induced a significant increase in monocyte binding. We conclude that LDL can induce the expression of vascular adhesion molecules on endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536712 TI - cGMP upregulates nitric oxide synthase expression in vascular smooth muscle cells. AB - 8-Bromo-guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), an analogue of cyclic guanosine monophosphate (cGMP), induced a time- and dose-dependent enhancement of interleukin-1-induced nitric oxide production in vascular smooth muscle cells. Human atrial natriuretic polypeptide, which stimulates cGMP accumulation in vascular smooth muscle cells, also enhanced interleukin-1-induced nitric oxide release at a concentration of 100 nmol/L. In contrast, coincubation with 10 mumol/L methylene blue, an inhibitor of soluble guanylate cyclase, inhibited interleukin-1-induced nitric oxide release from vascular smooth muscle cells. Furthermore, coincubation with 8-Br-cGMP also enhanced the interleukin-1-induced increase in inducible nitric oxide synthase messenger RNA in vascular smooth muscle cells. However, the enhancement of nitric oxide production induced by 8-Br cGMP was significantly prevented by coincubation with neutralizing antibody against tumor necrosis factor-alpha. Furthermore, 8-Br-cGMP enhanced the interleukin-1-induced increase in tumor necrosis factor-alpha messenger RNA level in vascular smooth muscle cells. These findings indicate that cGMP may upregulate inducible nitric oxide synthase gene expression through the stimulation of tumor necrosis factor-alpha production in vascular smooth muscle cells. Thus, there may be a positive feedback mechanism between nitric oxide and the cGMP system in vascular smooth muscle cells. PMID- 7536714 TI - Cyclosporin A inhibits nitric oxide synthase induction in vascular smooth muscle cells. AB - The effect of cyclosporin A on induction of nitric oxide synthase in rat aortic smooth muscle cells was examined. A combination of interleukin-1 alpha (100 U/mL) and tumor necrosis factor--alpha (5000 U/mL) induced accumulation of nitrite/nitrate, the stable end products of nitric oxide, in culture media within 48 hours. Cyclosporin A inhibited this nitrite/nitrate accumulation in a concentration-dependent manner with an IC50 of 4 x 10(-7) mol/L when applied simultaneously with the cytokines. The expression of inducible nitric oxide synthase messenger RNA (mRNA) induced by the combination of interleukin-1 alpha and tumor necrosis factor-alpha was inhibited by the cyclosporin A cotreatment. Cyclosporin A did not decrease inducible nitric oxide synthase mRNA stability in the presence of transcription inhibitor actinomycin D (5 micrograms/mL). Induction of nitrite/nitrate production by the combination of tumor necrosis factor-alpha and bacterial lipopolysaccharide or that of interleukin-1 alpha and interferon gamma (100 U/mL) was also inhibited by cyclosporin A cotreatment. Another inhibitor of calcineurin, FK506 (up to 10(-6) mol/L), had no effect on the induction of nitrite/nitrate production, suggesting the possibility that the inhibitory effect of cyclosporin A may be exerted by means of a novel pathway other than inhibition of calcineurin. These results indicate that cyclosporin A inhibits inducible nitric oxide synthase induction at the mRNA level and that inducible nitric oxide synthase in vascular smooth muscle cells can be a target for cyclosporin A, providing a possible mechanism for the interference of the drug with the balance of vasoactive substances. PMID- 7536713 TI - Relaxation of the aorta during hypoxia is impaired in chronically hypertensive rats. AB - We investigated mechanisms by which hypoxia produces relaxation of the aorta and tested the hypothesis that these mechanisms are altered during chronic hypertension. Tension of thoracic aortae from normotensive Wistar-Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) was measured in an organ bath under control conditions and at two levels of hypoxia. In WKY rats, mild and severe hypoxia produced relaxation of the aortae (precontracted with phenylephrine) by 33 +/- 4% and 82 +/- 3%, respectively (mean +/- SEM). Removal of endothelium or administration of NG-nitro-L-arginine (10(-4) mol/L), an inhibitor of nitric oxide synthase, abolished relaxation of the aortae in response to mild hypoxia but did not affect relaxation during severe hypoxia. Glibenclamide (10(-6) mol/L), an inhibitor of potassium channels, attenuated relaxation of the aortae during mild and severe hypoxia by 49 +/- 16% and 74 +/- 4%, respectively. In SHRSP, mild hypoxia produced little relaxation of the aortae (3 +/- 4%, P < .05 compared with WKY). Indomethacin did not increase relaxation to mild hypoxia in SHRSP, which suggests that a cyclooxygenase-derived contracting factor does not contribute to impaired relaxation. Severe hypoxia relaxed the aortae by 86 +/- 4% in SHRSP, and glibenclamide inhibited this response by 60 +/- 9%. These findings suggest that relaxation of the aorta in response to mild hypoxia in WKY rats is mediated primarily by endothelium-derived relaxing factor, and the response to mild hypoxia is markedly impaired in SHRSP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536715 TI - Angiotensin-(1-7) and nitric oxide interaction in renovascular hypertension. AB - New studies suggest that vasodilator systems may play an important role in restraining the rise in peripheral vascular resistance associated with the evolution of arterial hypertension. We characterized in conscious dogs the hemodynamic and hormonal effects of 4 weeks of feeding either the nitric oxide synthase inhibitor N omega-nitro-L-arginine (3 mg.kg-1.d-1) or the nitric oxide precursor L-arginine (0.3 mg.kg-1.d-1) during the evolution of two-kidney, one clip hypertension. Inhibition of nitric oxide production elicited a form of hypertension more severe than that produced in placebo-fed two-kidney, one clip dogs. The higher levels of blood pressure were accompanied by lower levels of plasma renin activity and lower angiotensin II concentrations. During the chronic phase of renovascular hypertension, the fall in blood pressure produced by acute systemic injections of lisinopril or losartan was significantly reduced in dogs given the nitric oxide inhibitor. In contrast, chronic administration of L arginine had no effect on the magnitude of hypertension or on the increases in renin activity and hyperangiotensinemia associated with the evolution of renal hypertension. Likewise, the fall in blood pressure produced by pharmacological blockade of angiotensin II was not different from that recorded in untreated renal hypertensive dogs. The vasodilator component of the blood pressure response due to intravenous injections of angiotensin-(1-7) (1 to 100 nmol/kg) was augmented in both untreated and L-arginine-treated two-kidney, one clip hypertensive dogs, but was significantly attenuated in hypertensive dogs fed the nitric oxide synthase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536717 TI - Relation between pressure natriuresis and urinary excretion of nitrate/nitrite in anesthetized dogs. AB - Alterations in intrarenal nitric oxide (NO) formation during changes in renal arterial pressure (RAP) have been suggested as a mechanism mediating pressure natriuresis. To test this hypothesis further, we examined the relation between RAP and the urinary excretion rate of nitrate/nitrite (NO3-/NO2-; NO metabolites) in anesthetized sodium-replete dogs before (n = 9) and during (n = 6) intrarenal infusion of the NO synthesis inhibitor nitro-L-arginine (NLA; 50 micrograms.kg 1.min-1). Urinary NO3-/NO2- concentrations were measured with the Griess reaction and spectrophotometry methods after enzymatic reduction of NO3- to NO2- in the samples. During control conditions, there were decreases in the urinary NO3-/NO2- excretion rate in response to reductions in RAP (150 to 75 mm Hg; slope, 0.04 +/- 0.01 nmol.min-1.g-1.mm Hg-1) in association with decreases in urinary sodium excretion (UNaV). There was a positive correlation between changes in NO3-/NO2- excretion rate and changes in RAP (r = .48; P < .005) or UNaV (r = .59; P < .001). NLA infusion resulted in decreases in NO3-/NO2- excretion rate (4.8 +/- 1.4 to 1.0 +/- 0.3 nmol.min-1.g-1) in association with reductions in UNaV (4.3 +/ 0.3 to 0.7 +/- 0.2 microL.min-1.g-1), fractional excretion of sodium (2.9 +/- 0.2% to 0.5 +/- 0.1%), and renal blood flow (4.8 +/- 0.3 to 3.3 +/- 0.2 mL.min 1.g-1), without changes in glomerular filtration rate. Furthermore, there was a marked attenuation of the NO3-/NO2- and sodium excretory responses to alterations in RAP during NO synthesis inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536716 TI - Effect of angiotensin II blockade on the fibroproliferative response to phenylephrine in the rat heart. AB - In this study we infused phenylephrine into adult Wistar rats and used losartan to test for a possible role of angiotensin II in the phenylephrine-induced fibrosis. Phenylephrine, given by Alzet minipumps at a rate of 25 mg.kg-1.d-1, produced a rapid and striking fibrotic response that was obvious after 1 day and progressed throughout a 3-day infusion period. Northern and Western blot analyses showed large increases in cardiac fibronectin expression and atrial natriuretic peptide mRNA, corresponding to fibroblast proliferation and myocyte hypertrophy, respectively. Cardiac fibrosis, fibronectin mRNA, and atrial natriuretic peptide mRNA were blocked by prazosin (7 mg.kg-1.d-1). Administration of losartan (10 mg.kg-1.d-1) resulted in a threefold decrease in interstitial and perivascular fibroblast proliferation, as measured by proliferating cell nuclear antigen immunoreactivity (p < .05), a marked reduction of fibronectin mRNA in the heart, and a moderate reduction of cardiac atrial natriuretic peptide mRNA. The data suggest that effects mediated by a1-adrenergic and angiotensin type 1 receptors may promote cardiac fibrosis. PMID- 7536719 TI - Correlation between contrast enhancement in dynamic magnetic resonance imaging of the breast and tumor angiogenesis. AB - RATIONALE AND OBJECTIVE: The authors determined the relation between tumor angiogenesis in small invasive breast carcinoma and contrast enhancement on magnetic resonance imaging (MRI) after gadolinium injection. MATERIALS AND METHODS: Magnetic resonance imaging was performed before surgery in a prospective study of 20 patients who had a small palpable lump. Spin-echo sequences after injection of gadolinium were studied by factor analysis of medical image sequences and were compared with a histologic quantification of tumor angiogenesis after immunocytochemical staining. RESULTS: In nine cases, there was good correlation between the MRI and the histologic plane. In four patients, an early factor was found on MRI. This factor was related to a high concentration of arterioles located in the stroma or, in one patient, to the intratumor repair process. CONCLUSION: Early enhancement correlated well with the number of vessels determined histologically. PMID- 7536718 TI - The natural history of prostatism: the effects of non-response bias. AB - BACKGROUND: In epidemiological studies, non-response may raise the question of generalizability to the target population. Most investigations have not been able to access data that could provide information about the potential impact of non response bias. METHODS: A 55% response rate was realized at baseline for a prospective cohort investigation of the natural history of benign prostatic hyperplasia in Olmsted County, Minnesota, during 1989-1991 (the Olmsted County Study of Urinary Symptoms and Health Status Among Men). This prompted a preliminary study of potential non-response bias among full participants, partial participants and complete non-responders. The medical diagnostic index maintained by the Rochester Epidemiology Project was used to ascertain the prevalence of specific conditions in the 9 years prior to study inception. RESULTS: The age adjusted period prevalence rate for benign prostatic hyperplasia (%) was 9.6 (95% confidence interval [CI]: 8.1-11.0) for full participants, 8.2 (95% CI: 5.8-10.6) for partial participants and 5.3 (95% CI: 3.6-6.9) for complete non-responders. Other urologic diagnoses followed the same pattern. However, age-adjusted prevalence rates for general medical examination history and major non-urologic morbidities were decidedly similar across response groups. CONCLUSIONS: These data suggest response may have been driven, in part, by concerns about urologic disease. However, the similarity in non-urologic diagnoses and general medical examinations provide some preliminary reassurance that the 55% response rate did not necessarily compromise generalizability. PMID- 7536720 TI - Radiotherapy and androgen ablation for clinically localized high-risk prostate cancer. AB - PURPOSE: The response of patients with clinical stages T1-4 prostate cancer to radiotherapy is variable. A particularly poor prognostic group has been found to be comprised of those with pretreatment prostate specific antigen (PSA) levels above 30 ng/ml with any tumor grade, or PSA levels > 10 and < or = 30 with tumors grade 3 or 4. These patients have over an 80% actuarial risk of biochemical failure 3 years after definitive external beam radiotherapy. Thus, patients with these high-risk features require more aggressive therapy. During the last 3-4 years, the policy to treat such patients with radiotherapy and androgen ablation (XRT/HORM) was instituted. A retrospective comparison was made between high-risk patients treated with radiotherapy alone (XRT) vs. XRT/HORM. METHODS AND MATERIALS: Between 1987 and 1991, there were 81 high-risk patients treated with XRT. There were 38 high-risk patients treated with XRT/HORM between 1990 and 1992. The median follow-up was 37 months for the XRT group and 22 months for the XRT/HORM group. No patient had clinical, radiographic, or pathologic evidence of lymph node involvement. The median dose to the prostate was 66 Gy for the XRT group and 68 Gy for the XRT/HORM group. RESULTS: The distributions of several potential prognostic factors were analyzed. Significant differences between the groups were observed for tumor grade, pretreatment prostatic acid phosphatase, and age. The XRT/HORM group was composed of patients with worse features, including a greater proportion of patients with grade 4 tumors, more with abnormal acid phosphatase levels, and more under 60 years of age. The actuarial incidence of a rising PSA at 3 years for the XRT group was 81% vs. 15% for the XRT/HORM group (p < 0.0001). In addition, local relapse at 3 years was 34% for the XRT group and 15% for the XRT/HORM group (p < 0.02). There was no difference between the groups in terms of survival. Cox proportional hazards analyses were performed using several disease end points, including a rising PSA, a rising PSA or disease relapse, any disease relapse, and local relapse, and the only prognostic factor of independent predictive value was treatment group, i.e., XRT vs. XRT/HORM. CONCLUSIONS: Based on biochemical and disease relapse end points, definitive radiotherapy is insufficient treatment for high-risk prostate cancer patients. The addition of androgen ablation significantly reduces the recurrence rates, although longer follow-up is needed to determine if the combined treatment impacts significantly on survival. PMID- 7536721 TI - Measurement of weekly prostate specific antigen levels in patients receiving pelvic radiotherapy for nonprostatic malignancies. AB - PURPOSE: To study the response of nonmalignant prostatic tissue to ionizing irradiation in terms of the resultant changes in serum prostate specific antigen (PSA) levels. METHODS AND MATERIALS: Weekly serum PSA values were determined during radiotherapy (RT) in nine patients ("treatment group") without clinical evidence of prostate cancer (PC), and who received pelvic RT for other indications. Slopes for the rate of change in PSA was determined using model: log PSA = beta 0 + beta 1*week + beta 2*week2 + error. These results are compared with 17 normal volunteers ("control group") who were not exposed to ionizing irradiation. An attempt is made to compare any similarities and differences in subsets of 64 T1-T4N0M0 PC patients who received pelvic RT. RESULTS: An elevation in the serum PSA levels were noted in eight of nine patients in the "treatment group" with a median time of 4.2 weeks to reach the maximum serum PSA values. After an initial increase, PSA values declined. In some patients, manifold increase in PSA was noted, for example, from 1.8 to 13.5 ng/ml and 3.3 to 9.8 ng/ml in two patients. The PSA increase ranged from 50-650%. The median slope was 0.601 week-1 (range 0.192-3.045 week-1). No such increases were seen in the "control group" (median slope = 0.03 week-1; range, 0.18-0.13 week-1). When differences between the mean increase/decrease for each week compared to pretreatment values were analyzed, the irradiated group had statistically significant elevations in the PSA for weeks 3 (p = 0.034), 4 (p = 0.035), and 5 (p = 0.024). A similar trend of increasing PSA levels during radiotherapy was noted in prostate cancer patients whose initial PSA values were < or = 20 ng/ml: whereas positive slopes (i.e., increasing PSA levels during radiotherapy course) was seen in 7.1% of those with > 20 ng/ml preradiotherapy PSA values, such trends were seen in 52.7% of those with < or = 20 ng/ml preradiotherapy PSA values. CONCLUSIONS: (a) Incidental exposure of noncancerous prostate to ionizing irradiation appears to lead to an initial elevation followed by a decline in serum PSA. (b) Similar elevations in serum PSA levels are seen in over 50% of prostate cancer patients with < or = 20 ng/ml pretreatment PSA. (c) Acinal cell death and sudden release of PSA into the circulation is the most likely explanation for our observations, although other mechanism cannot be excluded. (d) Our observations have to be considered in modeling PSA kinetics induced by RT and in correlating such kinetics to long-term outcomes. (e) Our findings in the control group indicate that there appears to be no significant variation in serum PSA over many weeks under physiological conditions in normal healthy ambulatory men. PMID- 7536722 TI - The source of pretreatment serum prostate-specific antigen in clinically localized prostate cancer--T, N, or M? AB - PURPOSE: Prostate-specific antigen (PSA) is an important marker for prostate cancer and has been shown to be secreted from the primary tumor and from metastases. However, the relative contribution of the primary and micrometastatic disease to the serum level of PSA in patients with clinically localized disease has not been delineated. This study addresses the source of pretreatment serum PSA in patients with clinically localized disease. METHODS AND MATERIALS: The fall in serum PSA level following radical prostatectomy (280 patients; 105 T1, 165 T2, 10 T3) or definitive radiotherapy (427 patients; 122 T1, 147 T2, 158 T3/T4) was analyzed with the assumption that any fall in PSA following local treatment reflects the fraction of PSA produced in the prostate and its primary tumor. RESULTS: Serum PSA level became undetectable in 277 of the 280 (99%) patients within 6 months of radical prostatectomy. The three patients who did not achieve undetectable levels had postsurgical values < or = 0.9 ng/ml. Following definitive radiotherapy, nadir serum PSA values were between < or = 0.3 and 20.3 ng/ml, with mean and median values of 1.9 and 1.2 ng/ml, respectively. Nadir PSA was undetectable in 52 patients (12%). Four patients' PSA did not fall, but rose from the start, and each developed metastatic disease within 9 months, and in each metastases appeared to contribute to pretreatment serum PSA. In the remaining patients, the maximal factor by which PSA fell to its nadir was higher the higher the pretreatment PSA level. We present arguments that this is most consistent with the hypothesis that virtually all detectable pretreatment serum PSA derives from the primary tumor. Confirmatory evidence that little of the pretreatment serum PSA came from metastases was obtained by extrapolating the rising PSA profile in 97 patients back to pretreatment time. Back-extrapolated PSA contributed a mean of 7% and a median of 5% to the pretreatment serum value. Because such back-extrapolated values estimate the potentially maximal micrometastatic contribution, metastatic disease at diagnosis contributes little to pretreatment serum PSA. CONCLUSION: In patients with clinically localized prostate cancer, putative micrometastatic disease contributes negligibly to the pretreatment serum PSA level even when the latter is high. Most likely such patients, when they have metastases, have a very low metastatic burden. PMID- 7536723 TI - Pulmonary complications of bone marrow transplantation: a comparison of total body irradiation and cyclophosphamide to busulfan and cyclophosphamide. AB - PURPOSE: To retrospectively compare the acute and long-term pulmonary toxicities of total body irradiation and busulfan in bone marrow transplantation. METHODS AND MATERIALS: From March 1984 through February 1991, 144 patients received high dose therapy with cyclophosphamide plus either total body irradiation (TBI-CY) or busulfan (BU-CY) followed by bone marrow rescue. Treatment protocols were based on disease type. Cyclophosphamide dose was 120-200 mg/kg, given in 2-4 days. Total body irradiation was given as 12 Gy in four fractions over 4 days, or 14.4 Gy in eight fractions over 4 days. Busulfan dose was 16 mg/kg given over 4 days. RESULTS: Seventy-nine patients were treated with TBI-CY and 65 patients with BU CY. More patients in the TBI group had allogeneic transplants (40 vs. 18). Pulmonary events occurred in 48 patients, 19 in BU-CY and 29 in TBI-CY. Of the 58 patients with allogeneic transplants, 21 (36%) developed chronic graft-vs.-host disease (GVHD), and 10 of those patients developed pulmonary complications (including 2 with obliterative bronchitis and 1 with asthma). Interstitial pneumonitis (IP) occurred in 14 patients, 12 in the TBI-CY group and 2 in the BU CY group. Cytomegalovirus and pneumocystis infections were associated with IP in 11 of those patients. Fatal idiopathic IP occurred in one patient in each of the TBI-CY and BU-CY groups. Multivariate analysis showed that only chronic GVHD and prior bleomycin use were significant predictors of interstitial pneumonitis; no difference was seen between TBI-CY and BU-CY. CONCLUSIONS: Pulmonary complications were most commonly associated with GVHD and prior bleomycin use. The incidence of cytomegalovirus or pneumocystis carinii pneumonitis was greater in the patients receiving the TBI regimen; fatal pulmonary complications were not significantly different between TBI and nonTBI regimens. PMID- 7536724 TI - Neuropeptides in the human penis: an immunohistochemical study. AB - In the present study, the distribution of neuropeptides in the human penis is demonstrated by immunohistochemistry (IHC). IHC screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine-methionine (PHM), prepro-VIP (111-122), neuropeptide Y (NPY), C flanking peptide of NPY (C-PON), calcitonin gene-related peptide, substance P, and galanin immunoreactivities. Special attention was also given to the recently isolated, VIP-related lizard peptide helospectin, which could also be detected in neuronal elements in the penis. Colocalization studies showed the coexistence of VIP, PHM, and partly helospectin, and of NPY with C-PON within nerve fibers in the cavernous and spongious body, the glans penis, and the urethra. PMID- 7536725 TI - Estrogen receptor messenger RNA expression in human benign prostatic hyperplasia: detection, localization, and modulation with a long-acting gonadotropin-releasing hormone agonist. AB - We have analyzed human benign prostatic hyperplastic (BPH) tissue derived from eight radical prostatectomy specimens from patients with prostate cancer for the expression of the estrogen receptor (ER) messenger RNA. Four of the eight patients received a long-acting gonadotropin-releasing hormone agonist (GnRHa) for 4 months prior to surgery. An RNase protection assay utilizing six riboprobes spanning most of the ER protein-coding sequences demonstrated expression of the ER mRNA in human BPH tissue. A comparison of ER mRNA expression in four patients who had received 4 months pretreatment with the GnRHa vs. the four untreated patients suggested that there is upregulation of ER mRNA expression with the GnRHa treatment. The combined techniques of in situ hybridization and immunocytochemistry localized the ER mRNA expression to the prostatic basal epithelial cells and stroma. We conclude that ER mRNA is expressed in human BPH tissue and that this expression is modulated by treatment with a long-acting GnRH agonist. PMID- 7536726 TI - The immunohistochemical localization of alpha 2-macroglobulin in rat testes is consistent with its role in germ cell movement and spermiation. AB - alpha 2-Macroglobulin (alpha 2-MG) is a nonspecific protease inhibitor and binding protein for peptide hormones that was recently isolated from Sertoli cell enriched culture medium and shown to be the same protein as alpha 2-MG in serum. The present study was conducted to determine the localization of alpha 2-MG in the seminiferous epithelium in order to gain insight into its possible site(s) of action. Immunostainable alpha 2-MG was present in the lumen of the tubules consistent with its proposed role as a protease inhibitor needed to inactivate the protease released from defective spermatozoa in the male reproductive tract. Immunoreactive alpha 2-MG was also localized adjacent to the heads of elongated spermatids, the most mobile cells in the seminiferous epithelium; immunostainable alpha 2-MG was not observed adjacent to round spermatids and spermatocytes, which are relatively less mobile. The intensity of the staining around the elongated spermatids was dependent on the stage of the spermatogenic cycle. Stainable alpha 2-MG was present adjacent to the spermatids in stage XI soon after the elongation process began. Immunoreactive product was in stages XI-XIV but only faintly visible. The most intense staining reaction for alpha 2-MG was in stages I-VI; it was reduced in stage VII; and virtually no alpha 2-MG was detectable in stages VIII-X at and just after spermiation. The postnatal changes of alpha 2-MG in the testis was also examined. During the first 2 weeks after birth, alpha 2-MG was not detected in the seminiferous epithelium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536727 TI - Antegrade ejaculation following transurethral laser ablation of the prostate. AB - A major drawback for many sexually active men who undergo a prostatectomy for benign prostatic hyperplasia, either via a transurethral resection or an open procedure, is the high incidence of retrograde ejaculation. Transurethral laser ablation of the prostate (TULAP) is a minimally invasive alternative to these procedures for prostatic outlet obstruction. We retrospectively reviewed 37 patients who underwent the TULAP procedure. Our data revealed that in addition to a significant improvement in voiding symptoms, 92% of sexually active patients retained both potency and antegrade ejaculation postoperatively. We conclude that this procedure is a viable alternative to transurethral prostatectomy, especially for the men who wish to remain sexually potent with antegrade ejaculation. A rationale for the mechanism of preservation of ejaculatory function is discussed. PMID- 7536728 TI - Evolutionary perspective on the structure and function of ribonuclease P, a ribozyme. PMID- 7536731 TI - Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria. AB - To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium. PMID- 7536730 TI - Sequence, regulation, and functions of fis in Salmonella typhimurium. AB - The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located. PMID- 7536732 TI - Transcriptional analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions. AB - The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Qs RNA and QL RNA), much larger than the prgQ gene, were expressed in the prgQ region. QS RNA was produced constitutively, whereas QL RNA was produced inducibly by pheromone. The lack of any other open reading frame in QL RNA and significant sequence complementarity between the 3' end of QL RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of QL RNA. A model for transcriptional activation of prgB is presented. PMID- 7536729 TI - The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA. AB - A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4 hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37- >ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone. PMID- 7536733 TI - Differential expression in Escherichia coli of the Vibrio sp. strain ABE-1 icdI and icdII genes encoding structurally different isocitrate dehydrogenase isozymes. AB - The expression of two structurally different isocitrate dehydrogenase isozymes of Vibrio sp. strain ABE-1 in Escherichia coli was examined. At a low temperature (15 degrees C), a thermolabile and monomeric type isozyme (IDH-II), which is quite different in amino acid sequence from the E. coli isocitrate dehydrogenase, was expressed and conferred glutamate prototrophic ability on an E. coli mutant defective in isocitrate dehydrogenase. The ability of IDH-II to confer restoration of the E. coli mutant to glutamate prototrophy was similar to that of IDH-I, which is a dimeric enzyme homologous to the E. coli isocitrate dehydrogenase. At a high temperature (37 degrees C), no functional IDH-II was expressed. Transcription of icdI and icdII genes, which encode IDH-I and IDH-II, respectively, was regulated differently by different environmental conditions. The level of icdII mRNA was increased by lowering the growth temperature for E. coli transformants, while the level of icdI mRNA was increased when E. coli transformants were cultured in acetate minimal medium. Similar patterns of transcriptional regulation of the two icd gene were observed also in Vibrio sp. strain ABE-1. However, activity of isocitrate dehydrogenase kinase, which can phosphorylate IDH-I and consequently inactivate the enzymatic activity, was detected in cell lysates of E. coli but not of Vibrio sp. strain ABE-1. PMID- 7536734 TI - Genes encoded on a cyanobacterial plasmid are transcriptionally regulated by sulfur availability and CysR. AB - A cyanobacterial sulfur-regulated gene (cysR), which encodes a protein with similarity to the Crp family of prokaryotic regulatory proteins, has recently been isolated and characterized. Polyacrylamide gel electrophoresis of periplasmic protein extracts reveals that a cysR mutant fails to synthesize a 36 kDa polypeptide that is normally induced in wild-type cells that have been grown under sulfur-deficient conditions. The amino-terminal sequence of this protein was obtained, and a synthetic oligonucleotide was used to isolated a clone containing a 1.9-kb NruI-KpnI fragment from a Synechococcus sp. strain PCC 7942 genomic library. RNA blot analysis indicates that this fragment encodes a transcript that is detectable in wild-type but not cysR mutant cells that have been starved for sulfur. DNA blot analysis revealed that the 1.9-kb NruI-KpnI fragment is contained within the Ba4 BamHI fragment of the endogenous 50-kb plasmid pANL. RNA blot studies indicate that the accumulation of a large number of pANL transcripts is regulated by sulfur levels and CysR. DNA sequence analysis confirmed that the gene encoding the sulfur-regulated 36-kDa periplasmic protein is encoded on the Ba4 fragment of pANL. The sequence of the 36-kDa protein displays sequence similarity to the enzyme catalase, and two downstream proteins exhibit 25 and 62% identity to a subunit of a P-type ATPase complex involved in Mg2+ transport and a chromate resistance determinant, respectively. Surprisingly, a strain in which the putative chromate resistance gene was interrupted by a drug resistance marker exhibited increased resistance to chromate when grown in media containing low sulfate concentrations. The possible role of this protein in the acclimation of cyanobacteria to conditions of low sulfur availability is discussed. PMID- 7536736 TI - Evidence for the binding of a biologically active interleukin-2 to human alpha 2 macroglobulin. AB - Human alpha 2-macroglobulin (alpha 2M), which irreversibly entraps proteinases through a drastic conformational change, has also been reported to bind various cytokines. The meaning of cytokine binding to native and/or transformed alpha 2M molecules is, however, not understood. In an attempt to elucidate this question, we have studied the interaction of radioiodinated recombinant human interleukin-2 (125I-rhIL-2) with native and chymotrypsin (alpha 2M-C)- or methylamine transformed (alpha 2M-MA) alpha 2M. Our results show that native and alpha 2M-MA are able to bind 125I-rhIL-2, with binding occurring only with the latter in a covalent manner, whereas the labeled cytokine is proteolyzed when incubated with alpha 2M-entrapped chymotrypsin. The degradation of uncomplexed 125I-rhIL-2 has also been observed in the presence of trypsin, whereas 125I-rhIL-2 bound to alpha 2M-MA is protected. Moreover, the proliferative activity of this cytokine on responsive cells is still maintained either with native alpha 2M- or alpha 2M-MA complexed rhIL-2 in comparison with that observed with the cytokine alone. Our results, which lead us to consider alpha 2M molecules as IL-2-binding proteins, emphasize the possible role of these molecules as immune response regulators. PMID- 7536735 TI - Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system. AB - The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9 specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man (1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed. PMID- 7536737 TI - Generation of monoclonal antibodies against autologous proteins in gene inactivated mice. AB - Induction of an immune response is strongly dependent on the phylogenetic distance between antigen and recipient. In general, antibodies will not be raised against self-antigens nor against highly conserved domains. In the present study we describe the production and characterization of murine monoclonal "auto antibodies" against murine tissue-type plasminogen activator (t-PA) raised in "knock-out" mice, homozygously deficient of the functional gene. 203 stable hybridomas were obtained producing murine monoclonal antibodies against murine t PA. Analysis of the species reactivity revealed that 182 cross-reacted with one or more (t-)PAs originating from other species including rat t-PA, human t-PA, and vampire bat-PA. 121 reacted with epitopes conserved among murine, rat, and human t-PA. In addition, 31 of the monoclonal antibodies were directed against domains present in all four species. Epitope mapping indicated a high frequency of specificity toward diverse epitopes that are highly conserved across species. Comparative analysis of their influence on the enzymatic activity of t-PA and their species cross-reactivity clearly demonstrated that the domains required for the biological activity of plasminogen activators are more conserved (p < 0.02) than non-functional domains. The availability of such unique antibodies against a wide variety of conserved epitopes may facilitate studies on the structural homologies between (t-)PAs isolated from various species. The present approach should also apply to various other classes of proteins, allowing the generation of monoclonal antibodies, against conserved epitopes, which could not be raised in wild-type animals because of their "self-antigen" nature. PMID- 7536738 TI - Differential modulation of bombesin-stimulated phospholipase C beta and mitogen activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P. AB - Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor mediated activation of a complex array of effectors, including phospholipase C beta and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C beta activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C beta by a GTPase-deficient form of the Gq-like protein, G16, indicating that the peptide does not inhibit phospholipase C beta and is acting at a point upstream of the activated form of the G protein alpha subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C beta, this inhibition could be overcome with 30 nM bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, 50 nM bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C beta and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells. PMID- 7536739 TI - Different susceptibility of small and large human tenascin-C isoforms to degradation by matrix metalloproteinases. AB - Two major tenascin-C (TN-C) isoforms are generated by the alternative splicing of the pre-mRNA. The large isoform contains seven extra type three repeats that, by contrast, are omitted in the small TN-C isoform. The large TN-C isoform is mainly expressed at the onset of cellular processes that entail active cell migration, proliferation, or tissue remodeling such as occur in neoplasia, wound healing, and during development. Thus, the large TN-C isoform seems to be a specific component of the provisional extracellular matrix. Here we have studied the degradation of the large and small TN-C isoforms by matrix metalloproteinases (MMPs) 2, 3, 7, and 9. Among these proteolytic enzymes only MMP-7 can degrade the small TN-C isoform removing the NH2-terminal knob. The large TN-C isoform shows the same MMP-7-sensitive site adjacent to the NH2-terminal sequence, but is further degraded in the splicing area where three fibronectin-like type III repeats are completely digested. Moreover, the large TN-C isoform is degraded by MMP-2 and MMP-3 which completely digest a single type III repeat inside the splicing area. By contrast, the large TN-C isoform is resistant to MMP-9 digestion. The results show that the presence of the spliced sequence introduces new protease-sensitive sites in the large TN-C isoform. PMID- 7536740 TI - Involvement of the Ca(2+)-dependent phosphatase calcineurin in gene transcription that is stimulated by cAMP through cAMP response elements. AB - Gene transcription can be induced by cAMP and Ca2+ through distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Ca2+ has been shown recently to depend on the Ca2+/calmodulin-dependent protein phosphatase calcineurin in pancreatic islet cells. This study investigates the role of calcineurin in CRE-directed gene transcription after stimulation by cAMP. Reporter fusion genes under the transcriptional control of CREs were transiently transfected into the cell line HIT. Pharmacological evidence suggests that cAMP stimulates CRE-mediated transcription through a Ca(2+)-dependent mechanism. The immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by cAMP. At the same concentrations they also inhibited calcineurin phosphatase activity. Reversal of calcineurin inhibition by rapamycin or overexpression of calcineurin led to disinhibition of CRE-mediated gene transcription. Immunoblots with a phosphoCREB-specific antibody showed that cyclosporin A and FK506 do not interfere with CREB phosphorylation at serine 119 stimulated with cAMP or membrane depolarization. These results indicate that in HIT cells stimulation of CRE-mediated transcription depends not only on the activity of protein kinases phosphorylating CREB but also on the Ca2+/calmodulin dependent protein phosphatase calcineurin that is necessary for the transcriptional competence of phosphorylated CREB. PMID- 7536743 TI - C-terminal extension of truncated recombinant proteins in Escherichia coli with a 10Sa RNA decapeptide. AB - When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene. PMID- 7536741 TI - Cell type-specific transactivation of the VCAM-1 promoter through an NF-kappa B enhancer motif. AB - Cytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-kappa B enhancer motifs, kappa L-kappa R (positions -77 and -63). However, cytokine activation of the dimeric NF-kappa B transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor-alpha markedly transactivated a transiently transfected minimal kappa L-kappa R motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the kappa L-kappa R motif. Both cell types exhibited similar inductions of NF-kappa DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-kappa B(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-kappa B may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression. PMID- 7536742 TI - Water channel properties of major intrinsic protein of lens. AB - The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions. PMID- 7536744 TI - The MATK tyrosine kinase interacts in a specific and SH2-dependent manner with c Kit. AB - We have cloned a protein tyrosine kinase, MATK, which is expressed abundantly in megakaryocytes and the brain. We investigated whether MATK participates in the c Kit ligand/stem cell factor (KL/SCF) signaling pathway in the megakaryocytic cell line CMK. After KL/SCF stimulation, five major proteins of molecular masses of 145, 113, 92, 76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in a time-dependent manner, peaking within 5 min, and returning to basal levels within 60 min. To study the role of MATK in the KL/SCF signaling pathway, glutathione S-transferase (GST) fusion proteins containing SH2 and SH3 domains of MATK were cloned, expressed in Escherichia coli, and purified. MATK-SH2, but not MATK-SH3, precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145 kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins containing the SH2 domain of p85 of phosphatidylinositol 3-kinase, phospholipase C gamma-1, and ras GAP also precipitated c-Kit. The tyrosine-phosphorylated c-Kit was co immunoprecipitated with anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but not in granulocyte-macrophage colony stimulating factor or interleukin-6-stimulated cells, suggesting receptor specificity. These results indicate that MATK associates with the c-Kit receptor following specific stimulation by KL/SCF via its SH2 domain and likely participates in transduction of growth signals induced by this cytokine in megakaryocytes. PMID- 7536745 TI - Fatty acylation of alpha z. Effects of palmitoylation and myristoylation on alpha z signaling. AB - As the first step in an investigation of roles played by fatty acylation of G protein alpha chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three alpha subunits. At site I, only HA-tagged alpha q and alpha z functioned normally. alpha s, alpha q, and alpha z subunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged alpha z, we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly2 (G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys3 (C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Gly2 and Cys3 (G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of alpha z with the particulate fraction. Gz-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by alpha z was unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated alpha z mutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated alpha z. From these findings we conclude: (a) fatty acylations of alpha z increase its association with membranes; (b) myristoylation is not required for palmitoylation of alpha z or for its productive interactions with adenylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by alpha z. PMID- 7536746 TI - Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. AB - A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus. PMID- 7536747 TI - Transforming growth factor-beta 1 regulates axon/Schwann cell interactions. AB - We have investigated the potential regulatory role of TGF-beta in the interactions of neurons and Schwann cells using an in vitro myelinating system. Purified populations of neurons and Schwann cells, grown alone or in coculture, secrete readily detectable levels of the three mammalian isoforms of TGF-beta; in each case, virtually all of the TGF-beta activity detected is latent. Expression of TGF-beta 1, a major isoform produced by Schwann cells, is specifically and significantly downregulated as a result of axon/Schwann cell interactions. Treatment of Schwann cells or Schwann cell/neuron cocultures with TGF-beta 1, in turn, has dramatic effects on proliferation and differentiation. In the case of purified Schwann cells, treatment with TGF-beta 1 increases their proliferation, and it promotes a pre- or nonmyelinating Schwann cell phenotype characterized by increased NCAM expression, decreased NGF receptor expression, inhibition of the forskolin-mediated induction of the myelin protein P0, and induction of the Schwann cell transcription factor suppressed cAMP-inducible POU protein. Addition of TGF-beta 1 to the cocultures inhibits many of the effects of the axon on Schwann cells, antagonizing the proliferation induced by contact with neurons, and, strikingly, blocking myelination. Ultrastructural analysis of the treated cultures confirmed the complete inhibition of myelination and revealed only rudimentary ensheathment of axons. Associated defects of the Schwann cell basal lamina and reduced expression of laminin were also detected. These effects of TGF beta 1 on Schwann cell differentiation are likely to be direct effects on the Schwann cells themselves which express high levels of TGF-beta 1 receptors when cocultured with neurons. The regulated expression of TGF-beta 1 and its effects on Schwann cells suggest that it may be an important autocrine and paracrine mediator of neuron/Schwann cell interactions. During development, TGF-beta 1 could serve as an inhibitor of Schwann cell proliferation and myelination, whereas after peripheral nerve injury, it may promote the transition of Schwann cells to a proliferating, nonmyelinating phenotype, and thereby enhance the regenerative response. PMID- 7536750 TI - Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and human colon cancer cell lines. AB - Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R) and B (Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1 4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4-21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer. PMID- 7536749 TI - Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes. AB - Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells. PMID- 7536748 TI - Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules. AB - A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+) ATPase is localized to both apical and basal-lateral membranes. Expression of E cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+) ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types. PMID- 7536751 TI - Protein kinase A inhibitors enhance radiation-induced apoptosis. AB - In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149-1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 microM) gave rise to significantly increased levels of apoptosis at 2-6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 microM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis. PMID- 7536753 TI - The self-renewal process of murine hemopoietic stem cells supported by interleukin-3 and the synergistic factors and the probability of its occurrence. AB - By use of the in vitro murine blast cell colony (Bl) assay system, Bl constituting cells supported by interleukin-3 (IL-3), IL-3 + interleukin-6 (IL 6), IL-3 + granulocyte colony-stimulating factor (G-CSF) and IL-3 + interleukin-1 (IL-1) were replated and the frequencies of secondary (2nd) granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colony and Bl progenitors were examined. According to the statistical method reported by Nakahata et al, the p values for the hemopoietic stem cells to self-renew were calculated and all cytokine groups produced similar p values ranging between 0.576 and 0.596. Further studies using IL-3-supported and IL-3 + G-CSF-supported Bl showed that the 2nd Bl progenitors could be produced even when there were more than 150 primary Bl-constituting cells per colony in the case of IL-3 + G-CSF, but no 2nd Bl progenitors were found in the case of IL-3. Their appearance was limited within the smaller primary Bl when supported by IL-3. Again there was a difference in the net product number of 2nd Bl progenitors, that is, the addition of G-CSF to the primary culture could produce around double the number of 2nd Bl progenitors. These data led us to hypothesize that synergistic factors could not modify the self-renewal probability, but maintained the self-renewal process for a longer period, in other words, for several cellular divisions. PMID- 7536752 TI - IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. AB - We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-gamma, IFN-alpha, and IFN-beta 2 (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased approximately 25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-gamma. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-gamma inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-alpha, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D3, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85-95 kDa in the nuclear extracts of IFN-gamma-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-gamma, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, 124PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents. PMID- 7536754 TI - Innervation of the hard palate in the rat studied by anterograde transport of horseradish peroxidase conjugates. AB - The innervation of the rat hard palate and the bordering part of the soft palate was studied after anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) and to choleragenoid (B-HRP) in separate experiments. WGA-HRP labeling showed leakage from several types of nerve endings, whereas B-HRP did not. Both conjugates gave rise to heavy labeling of a variety of nerve endings. Intragemmal and, especially, perigemmal fibers were labeled in chemosensory corpuscles, which were most common in the medial wall of the incisive canal and in the most anterior part of the soft palate. Ruffini endings of different sizes were labeled in the incisive papilla. Other subepithelial endings forming elongated expanded profiles with medium- to large-caliber source fibers were most common in protruding parts of the palate. Labeled intraepithelial endings included Merkel endings, which were most frequent in the incisive papilla and the rugae. Other labeled profiles were medium-caliber afferents giving rise to irregular, beaded, and sometimes branched endings often located far superficially in the epithelium. Such endings were present both within and between protruding parts of the palate. Fine-caliber intraepithelial endings were labeled almost exclusively in WGA-HRP experiments. PMID- 7536755 TI - Convergence and divergence of ascending binaural and monaural pathways from the superior olives of the mustached bat. AB - The lateral superior olive and medial superior olive give rise to pathways that terminate in the dorsal nucleus of the lateral lemniscus and central nucleus of the inferior colliculus. In most mammals, neurons in both the medial and lateral superior olives are binaural, but in the mustached bat most neurons in the medial superior olive are monaural. The aims of this study were to determine how the inputs to the medial superior olive contribute to its monaurality and to determine whether the ascending projections from the lateral and medial superior olives overlap or remain segregated at their targets. Injections of two different tracers were placed in tonotopically matched areas of the lateral and medial superior olives in the same animal. Retrograde transport from injections in the medial superior olive labeled spherical cells in the contralateral anteroventral cochlear nucleus and principal cells in the ipsilateral medial nucleus of the trapezoid body. Few cells were labeled in ipsilateral cochlear nucleus. Anterograde transport resulted in tonotopically specific distributions of label with the same laterality as in nonecholocating mammals. In the dorsal nucleus of the lateral lemniscus, label from the lateral and medial superior olives largely overlapped. In the inferior colliculus, label from the lateral and medial superior olives largely overlapped. In the inferior colliculus, label from the two sources overlapped in the high and low frequency ranges, but in the frequency range around 60 kHz, label from the medial superior olive extended more dorsally than that from the lateral superior olive. These results indicate that projections of the lateral and medial superior olives overlap extensively at their targets. PMID- 7536756 TI - Transient subcortical connections of inferior temporal areas TE and TEO in infant macaque monkeys. AB - As part of a long-term study designed to examine the ontogeny of visual memory in monkeys and its underlying neural circuitry, we have examined the subcortical connections of the inferior temporal cortex in infant monkeys and compared them to those previously described in adult monkeys (Webster et al. [1993] J. Comp. Neurol. 335:73-91). Inferior temporal areas TEO and TE were injected with wheat germ agglutinin conjugated to horseradish peroxidase and tritiated amino acids, respectively, or vice versa, in 1-week-old (N = 6) and 3-4-year-old (N = 6) Macaca mulatta, and the distributions of labeled cells and terminals were examined in subcortical structures. Although the connections of inferior temporal cortex with subcortical structures were found to be similar in infant and adult monkeys, several projections appear to undergo refinement during development. Quantitative analysis showed that 1) whereas the projection from TE to the superior colliculus is consistent (5 of 5 cases) and widespread in infants, it is less reliable (2 of 7 cases) and limited in areal extent in adults; 2) although the projections from TE to nucleus medialis dorsalis and the tail of the caudate are present in infants and adults, they are reduced in adults; and 3) TEO receives input from the dorsal lateral geniculate nucleus in both infants and adults, but the number of cells giving rise to this projection is lower in adults. There was also a suggestion that TE projects to nucleus paracentralis in infants (2 of 5 cases) but not in adults (0 of 7 cases). No differences between infants and adults were apparent in other subcortical connections, including those with the pulvinar, reticular nucleus, claustrum, and putamen. PMID- 7536757 TI - Birth dates and survival after axotomy of neurochemically defined subsets of trigeminal ganglion cells. AB - Trigeminal (V) ganglion cells with different neurochemical phenotypes or different birth dates are affected differently by neonatal axonal transection. The aim of the present study was to determine if V ganglion cell birth date and neurochemical phenotype were correlated and if these two variables could be related to responses to neonatal axonal transection. Immunocytochemistry, histochemistry, and [3H]thymidine labelling were used to determine the birth dates of V ganglion cells recognized by antibodies directed against neurofilament protein (NF), calcitonin gene-related peptide (CGRP), and substance P (SP) and those that bound the lectin Bandierea simplicifolia-I (BS-I). All V ganglion cells were born between embryonic days (E-) 9.5 and 14.5. All ganglion cells were born between E-9.5 and E-14.5. In a normalized population (percentages normalized to equal 100%), over 90% of NF-positive V ganglion cells were born between E-10.5 and E-12.5. The majority of CGRP-positive and SP-positive ganglion cells (> 90%) were generated from E-13.5 to E-14.5 and E-12.5 through E-14.5, respectively. Almost 85% of BS-I-positive ganglion cells were generated on E-12.5 through E 14.5. Previous results and additional data from this study indicated that NF- and BS-I-positive ganglion cells are proportionally more likely to be lost after neonatal axotomy and that SP-positive cells are more likely to remain. The percentage of CGRP-positive cells in the V ganglion was not significantly altered by neonatal infraorbital nerve transection. Overall, these findings do not indicate a strong relationship between cell birth date and the probability of survival after neonatal axonal damage for all V ganglion cell phenotypes. PMID- 7536758 TI - Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy. AB - Using immunocytochemistry combined with confocal and electron microscopy, the secretory pathways related to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), and neuropeptide Y (NPY) were investigated in neurons in rat lumbar (L) 4 and L5 dorsal root ganglia (DRGs) before and after peripheral axotomy. All four peptides were processed through the regulated secretory pathway in many small neurons in normal DRGs, and CGRP through this pathway also in some large neurons. In many small neurons, two neuropeptides could be sorted into the same or separate large dense-core vesicles (LDCVs). The LDCVs had a significantly larger diameter in small as compared to large DRG neurons. Fourteen days after sciatic nerve cut, the levels of SP- and CGRP-like immunoreactivities (-LIs) and the number of LDCVs containing these peptides were markedly reduced, but SP- and CGRP-LIs were still seen in the regulated pathway. GAL-LI was markedly increased in many small neurons and some large neurons and NPY-LI mainly in large neurons. Both peptides were particularly abundant in the Golgi region. In small neurons, the number of LDCVs containing GAL- or NPY-LI was increased, but did not appear to reach the numbers containing SP- or CGRP-LI in normal DRG neurons. After axotomy, CGRP-LI and GAL-LI were often in separate LDCVs. One type of NPY positive large neurons showed budding off of LDCVs after axotomy, but also some "scattered" labeling in the cytoplasm. In the second type, NPY-LI was mainly found in multivesicular bodies. In several myelinated nerve fibers a "diffuse" distribution of NPY was seen together with some LDCVs containing NPY-LI. In contrast, in unmyelinated nerve fibers, NPY-, GAL-, SP-, and CGRP-LIs were always observed in LDCVs. Thus, both in normal and axotomized DRG neurons, peptides are processed through the regulated pathway. However, in some large neurons, NPY is, in addition, secreted through the constitutive pathway, perhaps as a consequence of limited sorting mechanisms for NPY, i.e., the plasticity of the secretory mechanisms does not match the rate of peptide synthesis after axotomy. PMID- 7536759 TI - Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cis diamminedichloroplatinum. AB - It has been speculated elsewhere that growth factors produced by macrophages in response to tissue damage induce a modulation of pre-existing fibroblasts into myofibroblasts, leading to fibrosis. The development of these cells in cis diamminedichloroplatinum (CDDP)-induced rat renal interstitial fibrosis was observed immunohistochemically. In the cortico-medullary junction, nuclear changes and epithelial necrosis of the proximal renal tubule (mainly the P3 segment) were seen on days 1 and 4 after a single dose (6 mg/kg body weight) of CDDP, and regenerating epithelium appeared on day 7. Gradually developing fibrosis was observed around the affected tubules on days 14 and 28. The increase in fibrosis was confirmed by histometrical analysis. The number of ED-1 (primary antibody) positive macrophages reached a peak in the affected cortico-medullary junction on day 7 and this was accompanied by an increase in muscle actin positive myofibroblasts. On days 14 and 28, macrophages had declined in number, but the number of muscle actin-positive myofibroblasts in the fibrotic area was still high as compared with control values. Cytoplasmic myofilaments were observed in myofibroblasts by electron microscopy. These findings suggest that the myofibroblasts participate in renal interstitial fibrosis in the rat, and that their appearance may be related to macrophage infiltration in response to tubular injury, at least in the early stages of fibrosis. PMID- 7536760 TI - Bacillary angiomatosis and related diseases caused by Rochalimaea. PMID- 7536761 TI - Lymphoepithelioma-like carcinoma of the skin treated with Mohs micrographic surgery in combination with immune staining for cytokeratins. AB - Lymphoepithelioma-like carcinoma of the skin (LLCS) is a rare cutaneous neoplasm that histologically resembles nasopharyngeal lymphoepithelioma. Conventional surgical excision carries a considerable rate of recurrence (three of 11 reported cases with such treatment, with one patient dying of metastatic disease). We report the first case of lymphoepithelioma-like carcinoma of the skin treated with Mohs micrographic surgery. Because of its tendency to occur on the face and its potential for recurrence after incomplete removal, this tumor is a good candidate for treatment with Mohs micrographic surgery. Immunohistochemical staining of frozen sections for cytokeratins may help to detect neoplastic cells that may be obscured by the dense lymphoplasmacytic infiltrate associated with this tumor. PMID- 7536762 TI - Localization of active and inactive elastase, alpha-1-proteinase inhibitor, and alpha-2-macroglobulin in human gingiva. AB - Biochemically, there is usually much less elastase activity in gingival tissue than in crevicular fluid. The tissue distributions of active and inactive elastase and the endogenous inhibitors alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) were therefore compared. Inflamed tissue was obtained from chronic periodontitis patients, and cryostat sections were incubated with the histochemical elastase substrate MeOSuc-Ala-Ala-Pro-Val-MNA. Adjacent sections were examined immunocytochemically with antibodies to neutrophil elastase, alpha 1PI, alpha 2M, and leukocyte differentiation antigens. Antigenic elastase was widely distributed in CD15-positive granulocytes in both the epithelium and lamina propria as well as in granulomatous tissue from infrabony defects. However, there was very limited histochemical staining of these cells, and biochemical activity against the equivalent substrate MeOSuc-Ala Ala-Pro-Val-AFC could be extracted only from sections with such staining. The pH optimum and effector response of the activity in the extracts were, nevertheless, consistent with those of leukocyte elastase. The large difference between the total elastase content of the tissue, as determined immunocytochemically, and the limited amount of active enzyme, as demonstrated histochemically, indicated that the majority was in an inactive form. The involvement of tissue inhibitors was suggested by the fact that extracts from sections with no histochemical staining reduced biochemical elastase activity in crevicular fluid. alpha 2M was found in many fibroblasts and also some CD68-positive macrophages, which additionally contained alpha 1PI.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536763 TI - Treatment of cutaneous sarcoidosis with tranilast. AB - Tranilast is an anti-allergic drug clinically used for the treatment of atopy or urticaria. The drug has been shown to have an anti-fibrotic effect as well. We treated two cutaneous sarcoidosis patients with tranilast, resulting in remission within three months of administration. This drug should be an excellent tool for the treatment of other granulomatous diseases. PMID- 7536764 TI - CD44 expression in normal human skin and skin tumors. AB - CD44 is thought to be a principal cell surface receptor for hyaluronic acid. Although the distribution of hyalulonic acid has been studied, little is known about the distribution of the CD44 molecule in the human skin and skin tumors. This study was undertaken to investigate the distribution of the CD44 molecule in normal human skin as well as in benign and malignant skin tumors. In normal skin, CD44 was expressed on 1) keratinocyte cell surfaces throughout the epidermis except for the granular and horny layers, 2) hair follicular cells, 3) eccrine sweat gland cells, and 4) cell surfaces of dendritic cells in the dermis. In skin tumors, although CD44 was expressed on the tumor cell surface of seborreic keratosis, Bowen's disease, and squamous cell carcinoma as in normal skin, we could not detect any CD44 expression on the cell surface of the tumor cells of basal cell carcinoma. However, CD44 positive dendritic cells were observed in the tumor islands of basal cell carcinoma. Phenotypic analysis suggested that these CD44 positive cells were melanocytes. PMID- 7536765 TI - Presidential address. Fiftieth annual meeting of the American Academy of Allergy and Immunology: Anaheim, California, March 1994. PMID- 7536766 TI - Limb transplantation in rats: immunosuppression with FK-506. AB - The efficacy of a new immunosuppressive agent, FK-506, to prevent the rejection of limb transplants was investigated across a major histocompatibility barrier in rats and compared with Cyclosporine. We performed 129 limb transplants between donor ACI rats and recipient Lewis rats. The mean rejection time of the skin component of limb allografts without immunosuppression was 7 days. Animals receiving a 14-day course of Cyclosporine 25 mg/kg had a mean rejection time of 30 days. In contrast, animals receiving a 14-day course of FK506 1 mg/kg had a mean rejection time of 54 days and those receiving a 14-day course of FK-506 2 mg/kg had a mean rejection time of 122 days. Intermittent administration of FK 506 twice weekly further prolonged the mean rejection times to 149 days in animals receiving 1 mg/kg and to 296 days in animals receiving 2 mg/kg. PMID- 7536767 TI - Recently activated naive CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2 type cytokines. AB - Development of T cells during primary responses was investigated using pigeon cytochrome C-specific naive Th from TCR transgenic mice. Naive CD4 cells did not activate and help resting B cells. This failure was found to be primarily because the resting B cells were incapable of stimulating the naive Th. Provision of a costimulatory signal such as anti-CD28, or addition of APCs that express costimulatory molecules, such as dendritic cells, activated B cells, and B7+ and B7+ICAM(+)-expressing fibroblasts, induced naive Th activation and promoted T cell-dependent help for IgM secretion. T cell activation for as little as 24 h promoted helper activity, and Ig secretion required production of small amounts of IL-4 by the activated naive Th. On initial stimulation, naive Th secrete only IL-2. By mRNA analysis, activated naive Th were also shown to produce IL-4, however induction of IL-4 message only occurred 24 h after initial activation and required additional stimulation with Ag. A single exposure of naive CD4 to Ag/APC followed by 4 to 12 days in culture led to generation of effector Th which secreted IL-2 and some IFN-gamma, and no detectable IL-4 or IL-5, and which could only help B cells to IgM secretion. In contrast, similar cultures that received Ag/APC one or more times during this period generated effector cells capable of secreting easily detectable titers of IL-4 and IL-5, as well as IL-2 and IFN gamma, and able to now promote IgG1 and IgE responses. Generation of these Th0 like effectors was accompanied by increasing amounts of IL-4 secreted during the culture period after each restimulation, and addition of anti-IL-4 in culture inhibited development of the capacity to produce Th2 cytokines. These studies reinforce the notion that naive CD4 must interact with a costimulatory professional APC, rather than a resting B cell, for initiation of the primary response, but show that such an interaction can result in rapid development of the ability to interact with and provide cognate help to B cells. They also suggest that if activated naive CD4 cells receive multiple stimulations from Ag/APC, enough endogenous IL-4 can be produced to drive differentiation into effectors secreting type 2 cytokines. The existence of such an autocrine feedback mechanism suggests that the amount and availability of Ag could influence the nature and polarization of the Th response. PMID- 7536768 TI - Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4+ T cells responding to a bacterial superantigen. AB - Exposure of naive CD4+ T lymphocytes to superantigens such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell population with SEB leads to the apoptotic events of activation-induced cell death (AICD). However, T cells derived from either Fas-deficient lpr or Fas ligand-deficient gld autoimmune mouse strains, fail to undergo AICD under these conditions. Instead, these autoimmune T cells mount a vigorous proliferative response, suggesting a critical role for Fas/FasL interactions in this form of autoapoptosis. In the current study, we found that SEB-induced AICD was tied to the rapid induction of FasL expression in cells constitutively expressing high levels of Fas. Furthermore, the addition of soluble Fas-IgG fusion protein to the SEB-restimulated cultures blocked AICD and resulted in a 2 degrees proliferative response that was comparable in magnitude and kinetics to that of the lpr and gld T cells. The rapid onset of apoptosis in normal T cells subsequent to restimulation with SEB was in direct contrast to the proliferative response of the initial cultures, even though comparable levels of Fas and FasL RNA were found in T cells after 1 degree and 2 degrees challenge. The clonal expansion of the normal T cells responding to the initial SEB stimulation was, however, dramatically compromised when the normal cells were cocultured with an MRL-lpr responder population; addition of soluble Fas-IgG rescued the normal component of the response. Together, these data demonstrate first, that Fas/FasL interactions are intimately tied to superantigen-induced AICD, a form of autocrine cell death, and second, that FasL-mediated cytotoxicity is responsible for the disappearance of normal CD4+ T cells in lpr cocultures. PMID- 7536769 TI - Regulation of L-selectin mRNA in Jurkat cells. Opposing influences of calcium- and protein kinase C-dependent signaling pathways. AB - L-Selectin initiates leukocyte attachment to venular endothelium during lymphocyte recirculation through lymph nodes, leukocyte recruitment into sites of inflammation, and the hematogenous spread of lymphoid malignancies. The density of L-selectin at the cell surface is a major determinant of binding activity and entry into tissues. Post-transcriptional shedding is one control mechanism; however, the extent and physiologic relevance of pre-translational regulation has not been defined. The current study shows that mitogen-/IL-2-driven proliferation of human T cells first increased then markedly decreased the expression of L selectin on the blast population. The prevalence of specific mRNA showed parallel changes, implying that receptor density is controlled, in part, at the pretranslational level. We used the IL-2-independent Jurkat cell line to determine whether signaling through C-type protein kinases and intracellular calcium regulated L-selectin mRNA directly. Selective pharmacologic activation of these pathways with phorbol esters and calcium ionophore, respectively, resulted in opposite effects on both L-selectin density and mRNA levels. Phorbol esters induced receptor shedding followed by progressive increases in L-selectin density and steady state levels of mRNA. Addition of a calcium ionophore with the phorbol ester blocked both the reexpression of surface receptor and the increase in mRNA. Treatment with ionophore alone resulted in a steady decline in L-selectin expression and mRNA levels. Cyclosporin A, a specific inhibitor of calcineurin, blocked the impact of ionophore on both basal and phorbol-induced levels of L selectin mRNA. Ionophore alone did not induce apoptosis, significantly alter cell cycle kinetics, or increase transcription of the IL-2 gene under conditions that suppressed L-selectin. Thus, calcineurin seems to be a proximal enzyme in a novel regulatory cascade that suppresses L-selectin expression independent of its known effects on proliferating T cells. In light of the findings in Jurkat, we propose that the protein kinase pathway up-regulates L-selectin mRNA and surface expression early in mitogen-driven T cell proliferation. Chronic elevation of intracellular calcium in repeatedly stimulated T cells then down-regulates expression at the pretranslational level through prolonged activation of calcineurin. PMID- 7536770 TI - Expression and function of mouse Fas antigen on immature and mature T cells. AB - We prepared mAbs specific for the mouse Fas Ag (CD95) and used them to analyze the expression and apoptosis-inducing activity of the Fas Ag on murine immunocytes. Cytofluorometry of mouse bone marrow, thymus, and splenocytes using the mAbs indicated that cells of the T lineage, except for bone marrow cells, expressed Fas Ag on the surface. CD4-CD8- undifferentiated thymocytes expressed low levels of Fas Ag. Immature CD4+CD8+ thymocytes and mature CD4+CD8- and CD4 CD8+ thymocytes were highly positive for Fas Ag. CD4+CD8+ thymocytes were specifically sensitive to the apoptosis-inducing activity of anti-Fas, although CD4-CD8-, CD4+CD8-, and CD4-CD8+ thymocytes were resistant. Spleen T cells were resistant to anti-Fas, whereas they expressed Fas Ag. The superantigen, staphylococcal enterotoxin B (SEB) administered to BALB/c mice, induced clonal expansion and successive clonal deletion of spleen T cells bearing the V beta 8 TCR, which specifically reacts to SEB. Such clonal deletion of V beta 8 T cells was highly suppressed in lpr mice, which have defects in the Fas Ag gene. In SEB administrated BALB/c mice, expression of Fas Ag was significantly enhanced on V beta 8, but not on V beta 6 T cells, which cannot react to SEB. Moreover, V beta 8 T cells in SEB-primed mice were sensitive to the cell-killing activity of anti Fas, although V beta 6 T cells were resistant. These findings show that the expression level and apoptosis-inducing activity of Fas Ag on peripheral T cells are directly up-regulated by stimulation through the TCR in vivo. PMID- 7536771 TI - Tissue distribution of natural peptides derived from a ubiquitous dehydrogenase, including a novel liver-specific peptide that demonstrates the pronounced specificity of low affinity T cell reactions. AB - The peptides recognized by CD8+ CTL normally arise by proteolysis of intracellular proteins. To learn whether these peptides are generated similarly in diverse cell types, we examined the variety and abundance of naturally processed peptides that derive from a ubiquitous enzyme, alpha-ketoglutarate dehydrogenase, and are recognized in association with the class I MHC protein, Ld, by a CTL clone (2C). A characteristic set of three peptides was found in diverse tissues, but their abundance varied greatly, apparently unrelated to differences in class I MHC expression, e.g., they were surprisingly abundant in liver. We also found in liver a fourth naturally processed peptide (p2Ca-Y4, LSPYPFDL) that differs by one oxygen atom from a previously characterized natural peptide (p2Ca, LSPFPFDL). CTL discrimination between these peptides in association with the same class I MHC protein, Kb, demonstrates the striking specificity that can be exhibited by low affinity T cell reactions. PMID- 7536772 TI - Identification of a unique costimulatory activity for murine T helper 1 T cell clones. AB - We have examined the ability of several class II-positive tumor cell transfectants to stimulate murine Th1 clones. Most of the transfectants failed to activate the Th1 clones and, in fact, induced Ag-specific anergy. However, we found that one tumor, a UV-induced fibrosarcoma (6130-VAR1), was capable of stimulating both cytokine production and proliferation in Th1 clones. We believe that 6130-VAR1 cells possess a unique costimulatory activity for the following reasons. First, these cells fail to express known costimulatory molecules including B7-1 and B7-2. Second, 6130-VAR1-mediated stimulation of Th1 clones was not blocked by anti-CD28 Fab or by CTLA4Ig, which suggests that members of the B7 family were not up-regulated during the course of stimulation and that activation does not occur via a CD28-dependent pathway. Third, 6130-VAR1 could provide costimulation when presented on a different surface than the class II/peptide ligand for the TCR. This last finding suggested that the activity on these cells was not simply an adhesion molecule that facilitated increased efficiency of T cell:MHC interactions. Finally, like B7-1 transfectants, stimulation by class II positive 6130-VAR1 cells prevented the induction of anergy in the Th1 clones. Taken together, these results strongly suggest that 6130-VAR1 expresses a unique costimulatory activity (VAM-1) that, like B7-1, can promote T cell activation and prevent anergy induction. PMID- 7536773 TI - Characterization of T cell epitopes restricted by HLA-DP9 in streptococcal M12 protein. AB - Interaction of the HLA-DP9 (DPA1*0201/DPB1*0901) molecule and M protein of serotype 12 (SS95/12) streptococci, a main component of the streptococcal cell wall Ag, has been investigated to decipher peptide-binding capacity and T cell activation in the context of the HLA-DP molecule. Seven antigenic peptides (amino acids 19-25) restricted by the HLA-DP9 molecule were identified in M12 protein, using M12 protein- or peptide-specific T cell lines from naturally exposed individuals. The binding affinity of each peptide to the HLA-DP9 molecule was measured by fluorescence intensity of biotinylated peptides bound to L cell transfectants expressing HLA-DP9, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to the HLA-DP9 molecule was inhibited by an excess amount of corresponding nonbiotinylated peptides and other nonbiotinylated peptides, indicating that the peptides were bound to the HLA-DP9 molecule at a single binding site. Seven synthetic peptides containing the T cell epitopes restricted by the HLA-DP9 molecule had high binding affinity to the HLA-DP9 molecule. Comparison of the amino acid sequences of truncated analogues that could bind to the HLA-DP9 molecule and/or activate T cells suggested an HLA-DP9 specific binding motif, composed of a positively charged residue (R or K) at position 1, a hydrophobic residue (A, G, or L) at position 6, and another hydrophobic residue (L or V) at position 9. Analysis of single amino acid substituted analogues suggested that the positively charged amino acid in the motif served as a key anchor residue for binding to the HLA-DP9 molecule, which differs from the binding motif to the HLA-DR molecules. PMID- 7536774 TI - Retargeting of CTL by an efficiently refolded bispecific single-chain Fv dimer produced in bacteria. AB - A single-chain bispecific Fv dimer (bs(sFv)2) having specificity for mouse CD3 epsilon chain and human transferrin receptor was produced in bacterial inclusion bodies. To overcome difficulties associated with in vitro protein folding, we used a novel renaturation approach to obtain active bs(sFv)2. The protein was dissolved in the weak ionic detergent sodium lauroylsarcosine, and disulfides were formed by oxidation in air. After oxidation, the bs(sFv)2 exhibited very little covalent aggregation and migrated as a single species in nonreducing SDS PAGE, suggesting that disulfides were correctly paired. The detergent was removed using an ion exchange resin and the protein fractionated by size exclusion chromatography. The recovered 65-kDa protein was monomeric in non-denaturing solvent, homogeneous by SDS-PAGE, and comprised 15 to 20% of material applied to the gel filtration column. This protein bound specifically to both mouse CD3 epsilon chain and human transferrin receptor with affinities indistinguishable from those of the parental Fabs or single-chain Fvs. The bs(sFv)2 specifically redirected mouse cytotoxic T cells to lyse target cells expressing human transferrin receptor at picomolar concentrations. Bacterially produced and detergent oxidized bs(sFv)2 molecules may therefore provide the abundant amounts of homogeneous active material required to redirect cytotoxic cells against tumors and other unwanted cells in animal models and in patients. PMID- 7536775 TI - Cloning of the murine CD5 promoter and its tissue-specific regulation. AB - A genomic clone containing 1700 bp of the 5'-flanking region and first exon of the murine CD5 gene was isolated by screening a NIH 3T3 fibroblast genomic library with the previously characterized murine CD5 cDNA. The CD5 5'-flanking region lacks a consensus TATA box but contains Sp1, AP-1, IgH muE2, SV40 enhancer core, CCAAT, TCF2 alpha/PEA3/ets, and C/EBP motifs. Transcription is initiated from multiple sites upstream of an Inr-like sequence. When linked to the CAT reporter gene, the CD5 5'-flanking region was an active promoter in transient transfection assays using the EL4 (T), PD36 (pre-B) and M12 (B) cell lines, but was inactive in NIH 3T3 fibroblasts. This pattern of lymphoid-specific expression reflects the pattern of in vivo CD5 expression. Successive 5' to 3' deletions of the CD5 promoter/CAT reporter construct were transfected into T and B cells revealing a succession of positive and negative regulatory elements until promoter activity was eliminated at position -27. This result identified sequences from -125 to -27 (which contain the Inr, IgH muE2, SV40 enhancer core, and AP-1 sites) as sufficient for tissue-specific promotion. Isolation and characterization of the CD5 promoter represents an initial step in elucidating the control over tissue-specific expression of the murine CD5 gene. PMID- 7536776 TI - Inducible nitric oxide synthase in cattle. Differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages. AB - We assessed bovine bone marrow-derived macrophages and monocyte-derived macrophages for expression of inducible nitric oxide synthase (iNOS) activity. Both cell types expressed iNOS activity upon stimulation with Salmonella dublin (S. dublin) or with LPS. A 372-bp fragment of the bovine iNOS mRNA could be amplified by reverse transcription-PCR from mRNA of stimulated macrophages. Cloning and sequencing of the fragment revealed a high degree of homology to human hepatocyte, rat vascular smooth muscle cell, and mouse macrophage iNOS both at the nucleotide (87 to 92%) and amino acid levels (94 to 97%). iNOS mRNA was expressed maximally 6 h after stimulation with S. dublin, whereas maximal nitrite accumulation in supernatants was measured at 24 to 48 h. Significant differences with regard to cytokine regulation of iNOS were observed between murine and bovine macrophages cultured under identical conditions. The most striking difference was the inability of homologous IFN-gamma to induce iNOS both at the level of nitrite production and of mRNA expression in bovine macrophages. TNF alpha, IL-2, and IL-1 alone or together with IFN-gamma neither induced iNOS nor primed bovine macrophages for enhanced iNOS expression or activity upon stimulation with S. dublin. RhuIL-4, but not rhuTGF-beta, down-regulated S. dublin-induced iNOS activity and mRNA expression in bovine macrophages. Thus, an enzyme with a high degree of homology to rodent iNOS is inducible by stimulation of bovine macrophages with bacteria, but induction and regulation by cytokines occur under more restricted conditions than in rodent macrophages. PMID- 7536777 TI - Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines. AB - C biosynthesis at extrahepatic sites remote from plasma C may be important in the protection of tissues against inflammation and infection but may also contribute to tissue injury. This latter possibility is particularly relevant in the central nervous system (CNS), where several cell types are susceptible to damage by C. We have previously shown that human astrocyte-derived tumor cell lines synthesize and secrete all of the components of the activation pathways of C. In this study, we demonstrate that these cells also produce the components (C6, C7, C8, and C9) and regulators (S-protein and clusterin) of the lytic terminal C pathway. The terminal components produced are hemolytically active, and secretion is markedly up-regulated by the inflammatory cytokine IFN-gamma. Primary human fetal astrocytes also expressed C6, C7, S-protein, and clusterin. The human monocyte/macrophage cell line, used here as a model for microglia, also produced all terminal components and regulators when appropriately stimulated. These studies raise the prospect of the intrathecal synthesis of a complete, functional C system and its regulators in the inflamed CNS. Intrathecal C synthesis may be important in the resolution of infection and inflammation but, given the C susceptibility of some CNS cell types, may also exacerbate damage in demyelination and neurodegeneration. PMID- 7536778 TI - Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B. AB - N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation. PMID- 7536779 TI - Prostanoid enhancement of interleukin-6 production by rat peritoneal mast cells. AB - Mast cells are traditionally associated with an acute response involving the short-term release of mediators such as histamine. We have shown previously that mast cells can produce IL-6 without prior histamine release. In this study we examined the hypothesis that mast cell IL-6 production can be selectively regulated by PGs. Highly purified rat peritoneal mast cells were cultured in the presence of PGE1, PGE2, or PGD2 alone or in combination with anti-IgE or bacterial LPS. Histamine release was assessed after 10 min; IL-6 and TNF-alpha production was measured in supernatants after 18 h. Mast cell IL-6 production was induced by PGE1 and PGE2 to a similar level to that observed in anti-IgE activated cells. In contrast, constitutive production of TNF-alpha was inhibited by PGE1 and PGE2, but not by PGD2. PGE2 had a synergistic effect, inducing IL-6 in the presence of LPS, whereas an additive effect was observed in the presence of anti-IgE. None of the prostanoids alone induced significant histamine release at the 10-min time point. However, PGE2 significantly increased histamine release when added concurrently with anti-IgE. Flurbiprofen in the context of anti-IgE or LPS activation did not alter mast cell IL-6 or TNF-alpha production. IL-6 production in response to each of the stimuli was significantly inhibited by the corticosteroid dexamethasone. These observations of selective modulation of mast cell cytokine production are important to understand the mechanisms by which mast cells interact with other cells during an inflammatory process involving prostanoid synthesis. PMID- 7536780 TI - Induction of tissue factor on monocytes by adhesion to endothelial cells. AB - Activated monocytes express tissue factor (TF), a protein that is important in the pathogenesis of thrombotic disorders. We sought to characterize an adhesion dependent pathway for monocyte activation. In this study, we showed that adhesion of monocytes to cytokine-activated endothelial cells (EC) increased (approximately 5- to 10-fold) monocyte procoagulant activity (PCA). The PCA was attributed to TF because it was dependent on the coagulation factors VII and X, but not VIII, and was completely blocked by an anti-TF mAb. Direct cell-cell contact between monocytes and EC was required. The induction of TF was rapid, peaked at 30 min, and persisted for 4 h. Northern analysis revealed a rapid (approximately 30 min) increase in TF mRNA following adhesion, distinct from that induced by LPS (approximately 2-4 h). Four-hour TNF-treated EC supported TF expression, but 0.5- and 24-h TNF-treated EC had no effect. An anti-E-selectin mAb (H18/7) exerted partial inhibition, whereas anti-VCAM-1, ICAM-1, and CD11/CD18 mAbs had no inhibition. Synthetic Lewis X (Le(x)) oligosaccharide partially blocked TF induction, whereas sialyl-Le(x) (sLex) oligosaccharide had no effect. Cross-linking Le(x) on monocytes, but not sLex, significantly increased (approximately 10-fold) the TF expression. Le(x) cross-linking induced TF in 30 min and lasted for 4 h. All seventeen anti-Le(x) mAbs induced significant amount of TF generation, and epitope mapping revealed a single binding epitope on Le(x). These findings indicate that adhesion of monocytes to activated EC induces TF generation, and also suggest that Le(x) may represent an important signaling molecule on monocytes. PMID- 7536781 TI - Taurine chloramine inhibits production of nitric oxide and TNF-alpha in activated RAW 264.7 cells by mechanisms that involve transcriptional and translational events. AB - We previously reported that taurine chloramine (Tau-Cl) inhibits the production of both nitric oxide and TNF-alpha by activated RAW 264.7 cells. The current studies were conducted to gain insight into the mechanisms through which Tau-Cl exerts these effects. RAW 264.7 cells were activated by LPS (10 micrograms/ml) and rIFN-gamma (50 U/ml) in the absence or presence of either 0.8 mM Tau-Cl or taurine. Production of NO and TNF-alpha by RAW 264.7 cells was monitored: NO was measured spectrophotometrically as nitrite and TNF-alpha was measured by ELISA. Cell lysates were analyzed for the inducible form of nitric oxide synthase (iNOS) by Western blot analyses, and TNF-alpha and iNOS mRNAs were assessed by northern blot analyses. Tau-Cl inhibited transcription of the iNOS gene, or some earlier event in the signal transduction pathway, because iNOS protein and iNOS mRNA were undetected in lysates of cells activated in the continuous presence of Tau-Cl. In contrast, steady-state levels of TNF-alpha mRNA increased in the presence of Tau Cl to at least the same extent as that in untreated activated cells and persisted for a longer period of time. Metabolic labeling experiments demonstrated that Tau Cl inhibited translation of TNF-alpha mRNA because the presence of the presecretory 26-kDa form and the secreted 17-kDa form of TNF-alpha were greatly reduced in lysates and culture media, respectively, of cells activated in the presence of Tau-Cl. Inhibition of TNF-alpha synthesis by Tau-Cl is not the result of a generalized effect on protein synthesis because the amount of radiolabeled protein precipitated from metabolically labeled cells by TCA was unaffected by Tau-Cl, and cell viability was unaffected. The results of these studies demonstrate that Tau-Cl decreases production of tissue-damaging inflammatory mediators and thus may act as a physiologic modulator of macrophage function. PMID- 7536782 TI - Suppression of TNF-alpha mRNA expression in LPS-primed macrophages occurs at the level of nuclear factor-kappa B activation, but not at the level of protein kinase C or CD14 expression. AB - Previously, we reported that preexposure of proteose peptone-elicited murine peritoneal exudate macrophages (P-PEM) to a low dose of LPS suppressed the expression of TNF-alpha mRNA, but not of IL-1 beta mRNA, induced by a second round of LPS exposure. To elucidate the mechanisms underlying this hyporesponsiveness to LPS, we focused on two molecules: nuclear factor (NF)-kappa B and CD14. Activation of NF-kappa B induced by a second round of LPS was suppressed in LPS-primed P-PEM much like the suppression of TNF-alpha mRNA expression. However, protein kinase C (PKC), a candidate as an activator of NF kappa B, was not desensitized by LPS priming. LPS-induced TNF-alpha production was not affected by depletion of PKC, and LPS could not induce translocation of PKC. CD14 expression showed no significant difference between control and primed P-PEM. In contrast with J774.1 cells and thioglycolate medium-elicited macrophages (T-PEM), P-PEM exhibited serum-independent TNF-alpha production, and a polyclonal Ab to murine CD14 had no inhibitory effect on the LPS-induced TNF alpha production by P-PEM. These results suggest that priming by LPS causes blockage at an early step, at least before the activation of NF-kappa B, in the LPS signal transduction pathway, but not at the expression of CD14. Our results also suggest that, in P-PEM, in contrast to J774.1 cells and T-PEM, neither PKC nor CD14 is involved in the LPS-induced activation and suppression of TNF-alpha gene expression. PMID- 7536784 TI - [Hypertension and endogenous nitric oxide]. PMID- 7536783 TI - [A case of cryoglobulinemia with decreased C4 and HCV antibody positivity]. PMID- 7536785 TI - Serologic diagnosis of hepatitis C infection. Review of available tests. AB - Several tests are currently available to assist in the diagnosis of the hepatitis C virus. These tests can be classified as antibody tests (ELISA, RIBA, and MATRIX HCV) or tests that actually detect or quantify the hepatitis C virus (PCR techniques). These tests vary in cost, technical complexity, specimen handling requirements, and diagnostic information achieved. Those tests currently available to the clinician are reviewed along with their associated benefits and disadvantages. PMID- 7536787 TI - Selective responses (actin polymerization, shape changes, locomotion, pinocytosis) to the PKC inhibitor Ro 31-8220 suggest that PKC discriminately regulates functions of human blood lymphocytes. AB - The results suggest that protein kinase C (PKC) plays a pivotal role in the control of F-actin levels, locomotion, pinocytosis, and cell shape in lymphocytes. The PKC inhibitor Ro 31-8220 elicits a high proportion of polarized (ED50 = 1.5 x 10(-6) M) and locomoting cells and reduces the relative amount of F actin (by 29% at 10(-5) M) in initially resting cells. Phorbol myristate acetate (PMA) counterbalances the polarizing effect of Ro 31-8220. This indicates that the spherical shape and the F-actin content of resting cells are maintained by constitutive PKC activity. PMA-induced increases in fluid pinocytosis, F-actin content, and formation of nonpolar cells with surface protrusion are suppressed by Ro 31-8220 (IC50 = 2-4 x 10(-7) M). Spherical cells and, at higher concentrations (ED50 = 3.3 x 10(-6) M), polarized cells are formed instead. As a result, lymphocyte function switches from fluid pinocytosis to cell polarity and locomotion. The data indicate that PKC is instrumental in selectively switching lymphocyte function between resting state, locomotor activity, and fluid pinocytosis. Ro 31-8220 is extremely potent in stimulating lymphocyte polarity and locomotion (B and T cells). It acts faster and/or produces a higher proportion of polarized lymphocytes than other available agonists. It may thus be used as a tool in further experiments requiring locomoting lymphocytes. PMID- 7536786 TI - Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages. AB - Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS. PMID- 7536791 TI - Effects of granulocyte colony-stimulating factor and interferon-gamma on antifungal activity of human polymorphonuclear neutrophils against pseudohyphae of different medically important Candida species. AB - Polymorphonuclear neutrophils (PMNs) are the major host defense against pseudohyphae, the invasive form of Candida species. We studied the effects of granulocyte colony-stimulating factor (G-CSF) and interferon-gamma (IFN-gamma) on the PMN-induced damage of pseudo-hyphae of Candida albicans, Candida tropicalis, and Candida parapsilosis in vitro by using two antifungal assays: a modified limiting dilution assay and a colorimetric metabolic 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay. PMNs from healthy volunteers were incubated with either G-CSF (100-10,000 U/ml) or IFN-gamma (10-5000 U/ml) or buffer at 37 degrees C for 90 min and their capacity to damage nonopsonized pseudohyphae was then measured. C. tropicalis appeared to be the most susceptible species, whereas C. parapsilosis showed the highest rate of resistance to PMN damage. G-CSF (500-10,000 U/ml) and IFN-gamma (100-1000 U/ml) enhanced the antifungal activity of PMNs against C. albicans pseudo-hyphae (P < .01 and P < .05). Among the others, G-CSF enhanced PMN-induced damage of C. parapsilosis at concentrations 500-10,000 U/ml (P < .05), whereas it enhanced damage of C. tropicalis only at 10,000 U/ml (P < .01). IFN-gamma (100-1000 U/ml)-primed PMNs also caused augmented damage of C. parapsilosis (P < .05) but not of C. tropicalis at the same concentrations. Species-dependent differences exist in the responses of PMNs to Candida pseudohyphae and G-CSF as well as IFN-gamma are important immunomodulators of phagocytic host defenses against them. PMID- 7536790 TI - Selective sensitivity of macrophages to cytotoxicity by inhibitors of macromolecular synthesis: induction of apoptosis. AB - Initial studies designed to measure the effect of inhibiting RNA synthesis by dactinomycin on macrophage functions revealed that the cells were uniformly killed at concentrations that have been routinely used to inhibit RNA synthesis in other cell types. We, thus, determined the dose curve for the cytotoxicity of dactinomycin for macrophages and two other cell types, L929 cells and splenic lymphocytes. Macrophages were extremely sensitive to the cytotoxicity of dactinomycin compared to the other cell types. Submicromolar concentrations that induced 100% cytotoxicity in macrophages caused little death in L929 cells or lymphocytes. Concentrations of dactinomycin that inhibited RNA synthesis by 40% in macrophages induced almost complete cell death but inhibition of over 80% of RNA synthesis in L929 cells or lymphocytes induced no measurable cytotoxicity. Macrophages did take up more dactinomycin than other cells but the amount was not sufficient to account for the large differences in cytotoxicity. We next tested the effects of doxorubicin and cycloheximide and found that macrophages were also extremely sensitive to killing by these compounds, and there was a very close association between the amount of inhibition of protein synthesis and the amount of toxicity. The morphology of macrophages exposed to these agents was consistent with death by apoptosis. This was further supported by assays measuring membrane integrity and DNA fragmentation. These data demonstrate that inhibition of macromolecular synthesis in macrophages, by different mechanisms, causes macrophages to undergo apoptosis. They further suggest that, in contrast to other cell types that require protein synthesis for apoptosis, macrophages require the synthesis of certain proteins to avoid apoptosis. PMID- 7536789 TI - Tumoricidal response of liver macrophages isolated from rats bearing liver metastases of colon adenocarcinoma. AB - Intraportal inoculation of CC531 adenocarcinoma cells into syngeneic rats causes an increase of liver macrophage cell number but not of major histocompatibility complex class II antigen expression. On day 1 after inoculation of 10(5) CC531 cells, a fixed number of isolated liver macrophages lysed significantly more target cells in vitro than did control cells. This effect was still present after 4 weeks. A 10-fold higher initial tumor dose significantly suppressed the macrophage response during the first 2 weeks. In contrast to tumoricidal activity induced by lipopolysaccharide in vitro, the tumoricidal response following in vivo challenge with tumor cells appeared not closely related to the production of reactive nitrogen intermediates, as in the latter case it was not abrogated in the presence of nitric oxide synthase inhibitor. Furthermore, the liver macrophage population appeared not fully activated after tumor inoculation as lipopolysaccharide further increased tumoricidal activity in vitro. The observed numerical and functional response of liver macrophages to intraportally inoculated tumor cells points at an important role of these cells in aspecific immune reactivity aimed at the reduction of local tumor growth. Results suggest that mechanistic differences exist between macrophage tumoricidal activity induced by tumor cells as compared with lipopolysaccharide. PMID- 7536788 TI - Mechanisms of beta 1 integrin-dependent adherence of granulocytic HL60 to fibronectin. AB - We investigated the mechanism of beta 1 integrin-mediated adherence of stimulated granulocytic HL60 cells to fibronectin using a monoclonal antibody (15/7) that recognizes beta 1 integrins only when the receptors are active for ligand binding. Phorbol myristate acetate (PMA) stimulated expression of the 15/7 epitope on granulocytic HL60 by nearly fivefold but had an insignificant effect on the expression of the epitope on undifferentiated HL60 cells. These results paralleled the effect of PMA on HL60 and granulocytic HL60 adhesion to fibronectin, indicating that activation of beta 1 integrins is important for beta 1-mediated adherence of granulocytic HL60 cells to fibronectin. Agonists that stimulate alpha 5 beta 1-dependent human polymorphonuclear leukocyte (PMN) adhesion to fibronectin (C5a and PMA) also upregulated the 15/7 epitope on purified human PMNs. Although PMA rapidly induces increased levels of filamentous actin (F-actin) in granulocytic HL60 cells and a decrease in F-actin levels in undifferentiated HL60 cells, depolymerization of the actin cytoskeleton with cytochalasin B did not affect increased expression of the 15/7 epitope on granulocytic HL60 cells. Cytochalasin B did, however, inhibit granulocytic HL60 adherence to fibronectin by 50%, demonstrating that actin polymerization is important for optimal beta 1-dependent granulocytic adherence. PMID- 7536792 TI - Contribution of mannose receptor to signal transduction in Fc gamma receptor mediated phagocytosis of mouse peritoneal macrophages induced by liposomes. AB - The contribution of mannose receptors on the cell surface of mouse peritoneal macrophages to the process of liposome-induced phagocytosis of immunoglobulin G opsonized sheep red blood cells (SRBCs) through Fc gamma receptor has been investigated. Fc gamma receptor-mediated phagocytosis of opsonized SRBCs was activated by modified alpha 2-macroglobulin, which was produced in the incubation mixture of alpha 2-macroglobulin and liposome-treated splenic B cells. The phagocytosis was specifically inhibited by the addition of D-mannose, and the inhibition was dependent on the D-mannose concentration. The binding of modified alpha 2-macroglobulin to macrophages was also reduced by the addition of D mannose. The activation effect of modified alpha 2-macroglobulin was not inhibited when in the presence of alpha 2-macroglobulin-trypsin and -methylamine complexes. In the presence of cycloheximide, activated phagocytosis was reduced to the control level. By Scatchard plot analysis of IgG binding studies, the number of Fc gamma receptors of a macrophage had been increased to 4.6-fold that of a control macrophage by treatment with modified alpha 2-macroglobulin. These findings suggest that macrophage mannose receptors are involved in activating the process of Fc gamma receptor-mediated phagocytosis of opsonized SRBCs induced by modified alpha 2-macroglobulin. Lectins may participate in a signal transduction in macrophage activation by liposomes. PMID- 7536793 TI - Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids. AB - Activation of T cell hybridomas induces a G1/S cell cycle block and apoptosis. We isolated a variant of the 2B4.11 T cell hybridoma that, when activated via the TCR, produced IL-2 and underwent growth inhibition but did not die. Analysis of a variety of cell surface molecules revealed that the variant cell line, termed VD1, expressed very low levels of Fas compared to the wild type cells. Unlike 2B4.11 cells, VD1 cells were not killed by Fas ligand (FasL)-bearing effector cells. To determine if Fas is involved in activation-induced apoptosis, two different reagents that specifically bind Fas without killing the T cell hybridomas, a monoclonal antibody and a soluble Fas:Fc chimeric molecule, were added to activated T cell hybridomas. Both treatments prevented activation induced apoptosis in a dose-dependent manner, but had no effect on IL-2 production or growth inhibition. Northern blot analysis revealed that unactivated 2B4.11 cells expressed negligible levels of FasL mRNA, but transcripts were detectable as early as 2 h after activation and continued to increase up to 4-6 h after activation. Anti-TCR induced activation of 2B4.11 cells in the presence of a TCR- 2B4.11 variant resulted in death of the unactivated "bystander" cells, which was inhibited by anti-Fas antibodies. Finally, treatment of T hybridoma cells with 9-cis retinoic acid or glucocorticoids, which are known to prevent activation-induced T cell apoptosis, inhibited the up-regulation of FasL. We conclude that up-regulated expression of FasL and its subsequent interaction with Fas accounts for the apoptotic response of T cell hybridomas to activation, and that retinoic acid and corticosteroids inhibit activation-induced apoptosis by preventing up-regulation of FasL. PMID- 7536794 TI - Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein. AB - We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP. PMID- 7536795 TI - Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. AB - Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL 3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors. PMID- 7536796 TI - HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases. AB - We established six T cell clones specific for pyruvate dehydrogenase complex (PDC)-E2 peptides from four different patients with primary biliary cirrhosis using 33 different peptides of 17-20 amino acid residues corresponding to human PDC-E2 as stimulating antigens. The minimal T cell epitopes of these six T cell clones were all mapped to the same region of the PDC-E2 peptide 163-176 (GDLLAEIETDKATI), which corresponds to the inner lipoyl domain of PDC-E2. The HLA restriction molecules for this epitope were all identified as HLA DRB4 0101. The common essential amino acids of this epitope for these T cell clones were E, D, and K at positions 170, 172, and 173, respectively; other crucial amino acids for this epitope differed in each T cell clone. In addition, the alanine-substituted peptides at positions 170 and 173, but not 172, inhibited the proliferation of all T cell clones induced by the original peptide of human PDC-E2 163-176, indicating that amino acid D at position 172 is a critical MHC-binding site for all T cell clones tested. Interestingly, all T cell clones reacted to PDC-E2 peptide 36-49 (GDLIAEVETDKATV), which corresponds to the outer lipoyl domain of human PDC-E2. Furthermore, one T cell clone cross-reacted with exogenous antigens such as Escherichia coli PDC-E2 peptide 31-44/134-147/235-248 (EQSLITVEGDKASM), which has an EXDK sequence. This is a definite demonstration of the presence of molecular mimicry at the T cell clonal level in human autoimmune diseases. It is also considered possible to design peptide-specific immunotherapy based on the findings of T cell autoepitopes in primary biliary cirrhosis. PMID- 7536797 TI - CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis. AB - Phagocyte recognition and ingestion of intact cells undergoing apoptosis are key events in this generally important program of cell death. Insufficient phagocyte capacity for apoptotic cells can result in failure to clear dying cells before membrane integrity is lost, resulting in leakage of noxious cell contents and severe tissue damage. However, no means has been available to increase phagocytic clearance of apoptotic cells. We now report that transfection of the macrophage adhesion molecule CD36 into human Bowes melanoma cells specifically conferred greatly increased capacity to ingest apoptotic neutrophils, lymphocytes, and fibroblasts, comparable to that exhibited by macrophages. Furthermore, when CD36 was transfected into another cell type with limited capacity to take up apoptotic bodies, the monkey COS-7 cell, similar effects were observed. Therefore, CD36 gene transfer can confer "professional" capacity to ingest apoptotic cells upon "amateur" phagocytes. PMID- 7536799 TI - The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity. AB - The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se. PMID- 7536798 TI - CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2. AB - Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo. PMID- 7536801 TI - Cytosine arabinoside induces apoptosis in cerebellar neurons in culture. AB - Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC50 of approximately 60 microM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 microM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells. PMID- 7536800 TI - Induction of functional interleukin-2 receptor in mouse microglia. AB - Interleukin (IL)-2, initially discovered for its mitogenic activity on T cells, also acts on monocytes, resulting in the activation of cytokine production, superoxide production, and tumoricidal activity. Because severe brain damage was observed in IL-2-transgenic mice, this cytokine may have some influence(s) on the cells of the CNS. We investigated IL-2 receptor-bearing cells in the CNS and found that activated microglia expressed alpha-chain mRNA and immunoreactive IL-2 receptor beta-chain protein in culture. Although microglia did not express IL-2 receptors under normal culture conditions, they were induced to express these receptors by lipopolysaccharide (LPS) in a time-dependent manner. The IL-2 receptors were found to be functional because the viability and growth activity of LPS-treated microglia, but not untreated controls, increased in response to recombinant mouse IL-2 as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide colorimetric assay and bromodeoxyuridine uptake experiment, respectively. These effects of recombinant IL-2 were blocked by pretreatment with anti-mouse IL-2 receptor beta-chain antibody. Our findings suggest that activated microglia in the CNS can respond to this T cell-derived factor regulating their growth, which may be an important mechanism of communication between nervous and immune systems in physiological and pathological conditions. PMID- 7536802 TI - Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5. AB - Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components. PMID- 7536803 TI - Adenosine A1 agonists and the Ca2+ channel agonist bay K 8644 produce a synergistic stimulation of the GTPase activity of Go in rat frontal cortical membranes. AB - The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10(-5) M, the increase above basal GTPase in frontal cortex was 25 +/- 4, 20 +/- 3, and 8 +/- 1%, respectively, and in the cerebellum 55 +/- 2, 41 +/- 4, and 22 +/- 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2 chloroadenosine were inhibited by (1) A1 antagonists (76-96% reduction), (2) pretreatment with pertussis toxin (90-100% reduction), and (3) antibodies raised against the alpha-subunit of Gi and G(o) (55-57% reduction by each), suggesting that A1 receptors interact equally with Gi and G(o). (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of Gi or G(o). Previously, (+/-)-Bay K 8644 enhanced GTP hydrolysis by G(o) but not Gi. Now we report a profound synergistic stimulation of GTPase in the presence of (R) phenylisopropyladenosine and (+/-)-Bay K 8644 (10(-7) to 10(-5) M). (+/-)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G(o). It appears that a positive cooperative stimulation of G(o) occurs when it is first activated by A1 receptors and subsequently interacts with the L-type Ca2+ channel. PMID- 7536805 TI - Roles for nitric oxide as an intra- and interneuronal messenger at NMDA release regulating receptors: evidence from studies of the NMDA-evoked release of [3H]noradrenaline and D-[3H]aspartate from rat hippocampal slices. AB - N-Methyl-D-aspartate (NMDA) receptors regulating the release of [3H]noradrenaline ([3H]NA) and D-[3H]aspartate (D-[3H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg(2+)-free Krebs Henseleit buffer, the NMDA-evoked release of [3H]NA and D-[3H]Asp was Ca2+ dependent and inhibited by the NMDA antagonist (+/-)-3-(2-carboxypiperazin-4 yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [3H]NA was tetrodotoxin (TTX; 0.1-2 microM) sensitive, whereas that for D-[3H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for D-[3H]Asp appear to be presynaptic, whereas those for [3H]NA are extrasynaptic in location. L-Arginine (100 microM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [3H]NA (100%) and D-[3H]Asp (700%). Exogenous NO donors--sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso N-acetylpenicillamine (all 100 microM)--stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [3H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (NG-nitro-, NG-methyl-, and NG-amino-L-arginine, all 100 microM) attenuated (50-80%) the NMDA-stimulated release of [3H]NA and D-[3H]Asp, as did KN-62 (10 microM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger. PMID- 7536804 TI - Cyclothiazide modulates AMPA receptor-mediated increases in intracellular free Ca2+ and Mg2+ in cultured neurons from rat brain. AB - We investigated the modulation of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)-induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca2+]i were increased by 0.3-100 microM cyclothiazide, with an EC50 value of 2.40 microM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N-methyl-D-aspartate (NMDA) receptor antagonists were also potentiated by 10 microM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na(+)-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 microM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide. PMID- 7536807 TI - Modulation of ion gradients and glutamate release in cultured cerebellar granule cells by ouabain. AB - Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L-type Ca2+ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6-10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 mM KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl. PMID- 7536806 TI - Protein kinase C modulates calcium channels in isolated presynaptic nerve terminals of rat hippocampus. AB - Nerve terminals ("synaptosomes") isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura-2 and were subjected to depolarization with an elevated K+ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca2+ channels with different inactivation kinetics (tau of approximately 60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the "reverse" mode. The activity of both classes of voltage-dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13 acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca2+ channel activity, but the effect of DHS could be slowed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals. PMID- 7536808 TI - Phosphatase resistance of ERK2 brain kinase PK40erk2. AB - We have previously shown that a brain protein kinase, termed PK40, catalyzes the multiple phosphorylation of the KSP-repeat site of neurofilaments (NFs) and also can transform tau proteins into the paired helical filament-like state as found in Alzheimer's disease (AD) brains. Protein sequence analysis suggests that PK40 is a form of the extracellular signal-regulated kinase ERK2. A subpopulation of ERK2 species in soluble brain fractions can be efficiently phosphorylated and activated in cell-free systems, simply by adding Mg(2+)-ATP. Two phosphoisoforms of PK40erk2 are formed in this process, which have a reduced gel mobility, very much like the ERK2 form obtained in cell culture by stimulation with growth factors. One of these low-mobility forms cannot be inactivated with protein phosphatase 2A (PP2A) or with tyrosine phosphatases. The second form can be slowly inactivated by PP2A. In this case two Ser/Thr phosphates are removed at different rates during inactivation: One phosphate is very quickly removed to result in the formation of a high-mobility 39-kDa ERK2 species without consequence for activity; the other, slowly removed Ser/Thr phosphate controls the activity but has no effect on the gel mobility of ERK2. These results show that forms of ERK2 exist with properties different from the previously characterized ERK2 (p42mapk) from stimulated cell cultures. The active ERK2 forms produced in the presence of Mg(2+)-ATP alone could provide an explanation for the existence of constitutive ERK2-like NF phosphorylation in vivo. Excessive formation of an ERK2 species resistant to inactivation by PP2A might be relevant to the persistent pathological tau hyperphosphorylation in AD. PMID- 7536809 TI - Involvement of free radicals in excitotoxicity in vivo. AB - Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N methyl-D-aspartate (NMDA), kainic acid, and alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA). In a similar manner, striatal lesions produced by 1-methyl-4-phenylpyridinium (MPP+), malonate, and 3-acetylpyridine were significantly attenuated by either S-PBN or alpha-phenyl-N-tert-butylnitrone (PBN) treatment. Administration of S-PBN in combination with the NMDA antagonist MK-801 produced additive effects against malonate and 3-acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (.OH) as assessed by the conversion of salicylate to 2,3- and 2,5 dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S PBN, consistent with a free radical scavenging effect. S-PBN had no effects on malonate-induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated. PMID- 7536810 TI - Exacerbation of NMDA, AMPA, and L-glutamate excitotoxicity by the succinate dehydrogenase inhibitor malonate. AB - We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-D-aspartate (NMDA), S-alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (S-AMPA), and L-glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 mumol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 mumol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 +/- 1.4 mm3), as did coinjection of malonate with S-AMPA (11.0 +/- 1.0 mm3) or glutamate (12.8 +/- 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 +/- 0.3 mm3). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 +/- 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by approximately 40% (7.5 +/- 0.9 mm3). Coinjection of the AMPA antagonist 2,3 dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 +/- 1.8 and 14.0 +/- 0.9 mm3, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 +/- 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536811 TI - The beta-subunit of Caenorhabditis elegans avermectin receptor responds to glycine and is encoded by chromosome 1. AB - A full-length cDNA encoding the beta subunit of the recently described avermectin receptor was amplified from Caenorhabditis elegans mRNA. When this cDNA was injected into Xenopus oocytes a dose-dependent response to glycine was observed, together with a smaller response to 1 mM GABA. The EC50 of the glycine response was similar to that described previously for glutamate (0.38 mM). Hybridisation of the cDNA to polytene filters identified three yeast artificial chromosome clones that gave a positive signal, Y37B3, Y38E5, and Y24C9, all of which are mapped to chromosome 1. Hybridisation to a series of cosmid clones covering this area further mapped the gene encoding this subunit to the region -2,818 to 2,824. PMID- 7536812 TI - Inhibition of nitric oxide synthase activity attenuates striatal malonate lesions in rats. AB - Mitochondrial inhibitors such as malonate are potent neurotoxins in vivo. Intrastriatal injections of malonate result in neuronal damage reminiscent of "excitotoxic" lesions produced by compounds that activate NMDA receptors. Although the mechanism of cell death produced by malonate is uncertain, overactivation of NMDA receptors may be involved; pretreatment of animals with NMDA antagonists provides neuroprotection against malonate lesions. NMDA receptor activation stimulates the enzyme nitric oxide (NO) synthase (NOS). Elevated tissue levels of NO may generate highly reactive intermediates that impair mitochondrial function. We hypothesized that NO may be a mediator of malonate toxicity. We investigated whether in vivo inhibition of NO production by the NOS inhibitor N omega-nitro-L-arginine (NLA) would attenuate lesions produced by intrastriatal injections of malonate. We found that systemic injections of 3 mg/kg of NLA significantly reduced the extent of histologic damage elicited by intrastriatal injections of 1.5 mumol of malonate in adult rats. PMID- 7536813 TI - Inhibition of kinesin synthesis in vivo inhibits the rapid transport of representative proteins for three transport vesicle classes into the axon. AB - We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign to specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide containing synaptic vesicles, the amyloid protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations. PMID- 7536814 TI - B cell responses to the PNS protein P0 in experimental autoimmune neuritis. PMID- 7536816 TI - Treatment with basic fibroblastic growth factor following focal cerebral ischemia does not prevent neuronal injury. AB - This experiment determined if postischemic administration of basic fibroblast growth factor (bFGF) would result in neovascularization to minimize neuronal injury following a focal cerebral ischemia insult. Fifty-eight Sprague-Dawley rats underwent middle cerebral artery (MCA) occlusion and were divided into three groups receiving either vehicle, serum, or 50 ng bFGF biweekly through an indwelling ventricular cannula. At variable time intervals, the animals underwent carbon black perfusion of capillary beds and histological staining for assessment of neuronal injury. Following MCA occlusion, there was a significant decrease in capillary bed density in peri-infarction cortex which normalized by two weeks. The number of alive neurons in the peri-infarction cortex was also significantly decreased compared to contralateral control cortex. The chronic administration of bFGF commencing two days after MCA occlusion did not result in either a significant increase in capillary bed density or the number of alive neurons in the peri-infarction cortex. PMID- 7536815 TI - Nitric oxide changes in the rat brain after transient middle cerebral artery occlusion. AB - Using a porphyritic microsensor, we measured the cortical NO concentration within ischemic tissue during 2 h of middle cerebral artery (MCA) occlusion and 1 h of reperfusion in the rat (n = 36). Local cerebral blood flow was simultaneously measured by laser Doppler flowmetry to verify MCA occlusion and reperfusion. Baseline concentration of NO was < 10(-8) M. The maximum concentrations of NO during MCA occlusion and reperfusion were, respectively, 1.47 +/- 0.45 microM and 0.54 +/- 0.24 microM. Administration of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, prior to ischemia, significantly (p < 0.05) reduced NO release to 0.04 +/- 0.02 microM during MCA occlusion and completely inhibited NO release during 1 h of reperfusion. Administration of L-arginine 30 min after administration of L-NAME restored NO release (3.45 +/- 1.14 microM) during MCA occlusion; however, administration of L-arginine did not overcome the effect of L NAME on mean arterial blood pressure. Our data indicate that NO is released in the brain after the onset of ischemia and NO levels can be modulated by administration of NO substrate and NO antagonists. PMID- 7536817 TI - Functional identification of central afferent projections conveying information of acute "stress" to the hypothalamic paraventricular nucleus. AB - Intraperitoneal administration of hypertonic saline is a potent stimulus to the "stress" responsive hypophysiotrophic parvicellular neurons of the hypothalamic paraventricular nucleus (PVN), as well as to magnocellular neurons of the hypothalamo-neurohypophysial system. Precise identification of the pathways gating information about the "stress" of intraperitoneal hypertonic saline to the PVN has not been ascertained earlier. In this study we demonstrate that intraperitoneal injection of hypertonic saline induces increased expression of c fos immunoreactivity within neurons of the medial parvicellular division of the PVN, within the circumventricular organs surrounding the anteroventral tip of the third ventricle, and within the magnocellular neurons of the PVN and supraoptic nucleus. Also, neurons involved in conveying visceral information to the PVN, including the parabrachial nucleus, the nucleus of the solitary tract and the ventrolateral medulla responded with increased c-fos expression to the "stress" paradigm. Combined c-fos immunocytochemistry and retrograde tracing experiments with cholera toxin B (ChB) was used to identify neurons projecting to the PVN activated by the applied "stressor." Neither the mere intracerebral presence of ChB nor intraperitoneal administration of isotonic saline influenced the number of c-fos immunoreactive nuclei in the brain. Dual immunocytochemistry revealed that intraperitoneal administration of hypertonic saline induced expression of c fos immunoreactive nuclei in approximately half of the retrogradely labeled neurons projecting to the PVN from the anteroventral tip of the third ventricle (AV3v), including the subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT). In the brainstem, the "stressor" induced expression of c-fos IR nuclei in almost all of the retrogradely labeled cells of the ventrolateral part of the medulla oblongata (A1 and C1), while only about 25% of the ChB labeled cells of the caudal part of the nucleus of the solitary tract (A2) were concomitantly immunoreactive to c-fos. Within the parabrachial nucleus, only 20% of the ChB-labeled cells were also immunoreactive for c-fos. The present results provide evidence that information about the "stress" of intraperitoneal hypertonic saline is conveyed to both magnocellular neurons projecting to the neurohypophysis and hypophysiotrophic parvicellular neurons the PVN via afferent projections from a variety of neurons in the osmosensitive anteroventral tip of the third ventricle and visceromotor neurons of the parabrachial nucleus, the ventrolateral medulla and the nucleus of the solitary tract. PMID- 7536818 TI - Complementary distribution of receptors for neurotensin and NPY in small neurons in rat lumbar DRGs and regulation of the receptors and peptides after peripheral axotomy. AB - Neurotensin (NT) has been reported to have antinociceptive effects at the spinal level. In situ hybridization, electrophysiology, immunohistochemistry, and electronmicroscopy were used to investigate the distribution of NT receptors, possible effects of NT on primary sensory neurons, and the effect of nerve injury on the expression of NT receptors and NT. NT receptor (R) mRNA was observed in more than 25% of the small dorsal root ganglion (DRG) neurons, which lacked neuropeptide Y NPY-R mRNA and essentially other neuropeptide mRNAs. Intracellular recording using voltage-clamp mode showed that NT evokes an outward current in NPY-insensitive small neurons, and NPY an outward current in NT-insensitive small neurons. Both peptides lacked effect on several small DRG neurons. In the superficial dorsal horn NT immunoreactive (IR) terminals directly contacted primary afferent terminals without synaptic specializations. This new category (> 25%) of the small DRG neurons expressing NT-R mRNA was complementary to the around 60% of small neurons expressing NPY-R mRNA (and also substance P and calcitonin gene-related peptide mRNAs) and to the rest exhibiting somatostatin mRNA expression. The electrophysiological results support this classification, showing that NT and NPY have inhibitory effects on separate subpopulations of small DRG neurons. After sciatic nerve transection, a marked decrease was observed in (1) the number of NT-R mRNA-positive neurons in DRGs, (2) NT mRNA positive neurons in the dorsal horn, and (3) NT-IR cell bodies and fibers in laminae I-II. Thus, axotomy causes downregulation of several NT systems at the spinal level, suggesting that the possible effects of NT on primary sensory neurons is attenuated after peripheral axotomy. PMID- 7536819 TI - Increased collateral sprouting of primary afferents in the myelin-free spinal cord. AB - After partial lesions, uninjured nerve fibers have been shown to sprout and expand their connections within the CNS of adult mammals. The extent of this anatomical plasticity in adults is rather limited in comparison to embryonic or neonatal animals. Factors that might limit sprouting of nerve fibers and suppress anatomical plasticity in the CNS include myelin-associated neurite growth inhibitory molecules present in the CNS of adult mammals. To examine further the role of these neurite growth inhibitors, we have studied the ability of primary afferent fibers to sprout in the absence of myelin within a partially deafferented spinal cord. Myelination was suppressed in the lower thoracic and lumbar spinal cord of rats using neonatal x-irradiation. Dorsal roots of lumbar segments L2-L4 were cut in myelin-free and normal spinal cords of 8- or 15-d-old rats. Sprouting of primary afferents was measured after 20 d using thiamine monophosphatase (TMP) histochemistry. TMP is a specific marker enzyme for small diameter primary afferents that terminate in the substantia gelatinosa (lamina II) of the spinal cord. When compared to the control groups, collateral sprouting of TMP-positive afferents was significantly enhanced in the myelin-free spinal cords: in animals deafferented at postnatal day P8, the average volume occupied by sprouting fibers in the upper dorsal horn was 0.103 mm3 +/- 0.008 (mean +/- SEM) in myelin-free spinal cords and 0.044 mm3 +/- 0.011 in control spinal cords. In spinal cords deafferented at P15, this difference was even larger, with 0.106 mm3 +/- 0.010 in the absence of myelin and 0.031 mm3 +/- 0.010 in controls. Our results indicate that myelin and its associated neurite growth inhibitors restrict collateral sprouting. These data provide further evidence that CNS myelin and its associated neurite growth inhibitors are involved in the regulation of anatomical plasticity in the normal CNS. PMID- 7536820 TI - Synaptic NMDA receptor channels have a low open probability. AB - Realistic estimates of channel-gating parameters of synaptic receptors are essential to an understanding of synaptic transmission and modulation. However, the gating of N-methyl-D-aspartate (NMDA) channels appears to differ, depending on recording conditions; thus, it remains unclear what measurements are most relevant to synaptic receptors. To further explore this discrepancy, we examined the open probability (Po) of NMDA channels in whole-cell and outside-out patch recording from cultured hippocampal neurons. Currents were evoked by rapid application of saturating concentrations of NMDA in the presence or absence of the "irreversible" open channel blocker, MK-801 ((+)-5-methyl-10,11-dihydro-5H dibenzo[a,d]cyclohepten-5,10-imine) . The reduction of the peak amplitude and the acceleration of the decay of the current in MK-801 were used to derive Po by fitting the current traces to a multistate kinetic model. The Po in whole cell was low (0.04), similar to that previously measured for synaptically activated NMDA channels. In contrast, ensemble average currents from outside-out patches were much more rapidly blocked in the presence of MK-801, indicative of a significantly higher Po. The Po also gradually increased with the duration of recording in both whole-cell and outside-out configurations, suggesting that channel gating is sensitive to mechanical alterations of the patch or that washout of cytoplasmic factors leads to an increase in channel open probability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536821 TI - Pharmacological types of calcium channels and their modulation by baclofen in cerebellar granules. AB - Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the dihydropyridine nimodipine reversibly inhibited the maximum current by 25% and the dose dependence showed IC50 close to 50 nM. omega-Conotoxin GVIA (cgtx, 5 microM) and omega-agatoxin IVA (agatx, 200 nM) irreversibly inhibited the current by 17% and by 47%, respectively. The effect of nimodipine was additive with that of the toxins. The GABAB agonist (+/-)baclofen, or (-)baclofen (100 microM), reduced the calcium current by 30 +/- 5%, with a IC50 4 microM. The effect was mediated by a pertussis toxin-sensitive G-protein. In cells treated with cgtx during the experiment or preincubated with the toxin for 30 min, the effect of baclofen was significantly reduced. However, the action of baclofen was not confined to cgtx-sensitive channels: application of nimodipine or agatx resulted in a 50% reduction of the baclofen effect as well. In contrast, baclofen inhibited approximately the same amount of current both before and after the increase caused by the dihydropyridine agonist BayK 8644 and did not modify the slow BayK-induced tail current. These results indicate (1) the modulation through GABAB receptors does not clearly discriminate between pharmacologically distinct calcium channels and (2) L-type calcium channels represent an heterogeneous population in these neurons. PMID- 7536822 TI - Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons. AB - TrkA is a receptor tyrosine kinase whose activation transduces NGF signaling. TrkA expression has been demonstrated in NGF-responsive adult basal forebrain cholinergic neurons (BFCNs). Several lines of evidence have suggested that endogenous NGF plays a role in the development and differentiation of these neurons. We examined TrkA expression during development. TrkA mRNA and protein were present in basal forebrain neurons during the entire postnatal period; the distribution of neurons bearing these markers was identical to that for those containing choline acetyltransferase (ChAT) mRNA, suggesting that, as in the adult, TrkA gene expression is localized to BFCNs. The expression of TrkA and ChAT followed a very similar temporal pattern, suggesting regulation by the same factor(s). We discovered that NGF administration in vivo activated TrkA receptors, and increased both TrkA and ChAT mRNA; conversely, anti-NGF infusions suppressed expression of both genes. These results suggest that endogenous NGF regulates expression of TrkA and ChAT. Finally, while NGF infusion increased the size of developing BFCNs, NGF antibodies inhibited the normal developmental increase. The results are evidence that endogenous NGF acts on developing BFCNs to enhance gene expression and cellular differentiation. PMID- 7536824 TI - Anterograde tracing of trigeminal afferent pathways from the murine tooth pulp to cortex using herpes simplex virus type 1. AB - Due to its predominantly nociceptive innervation, viral tracing from the tooth pulp provides a potential means for tracing central pain pathways. The neural pathways from the tooth pulp to cortex were determined using in situ hybridization to detect the anterograde transneuronal spread of herpes simplex virus type 1 strain H129 following inoculation into the murine mandibular incisor pulp. Virus first appeared in the brain at day 3 in the dorsomedial region of all three subnuclei of the spinal trigeminal nucleus and the principal sensory nucleus. By days 5-6 virus had spread to the contralateral medial nucleus of the medial geniculate complex, posterior thalamus, and ventroposteromedial thalamus. At days 7-8 virus was detected in laminae IV and Va of the primary somatosensory cortex and lamina IV of the secondary somatosensory cortex in regions previously shown to receive input from the lower jaw. Several mice also showed infection of laminae II/III of the ipsilateral dysgranular insular cortex, along with labeling for virus in the ipsilateral external lateral parabrachial nucleus, posterior thalamus, and posterior basolateral amygdala. Our results are highly consistent with previous tracing and electrophysiological studies utilizing the tooth pulp and with studies implicating the infected structures in nociception. Viral spread appeared to define two separate afferent systems with infection of structures which have been implicated in the sensory-discriminative aspects of pain, such as the ventroposteromedial thalamus and somatosensory cortex, as well as in the dysgranular insular cortex and related subcortical nuclei which may have a role in the affective-motivational aspects of pain. PMID- 7536826 TI - The activation of protein kinase A pathway selectively inhibits anterograde axonal transport of vesicles but not mitochondria transport or retrograde transport in vivo. AB - To shed light on how axonal transport is regulated, we examined the possible roles of protein kinase A (PKA) in vivo suggested by our previous work (Sato Yoshitake et al., 1992). Pharmacological probes or the purified catalytic subunit of PKA were applied to the permeabilized-reactivated model of crayfish walking leg giant axon, and the effect was monitored by the quantitative video-enhanced light microscopy and the quantitative electron microscopy. Dibutyryl cyclic AMP caused concentration-dependent transient reduction in the number of anterogradely transported small vesicles, while the retrogradely transported organelles and anterogradely transported mitochondria showed no decrease. This transient selective inhibition of anterograde vesicle transport was reversed by the application of a specific inhibitor of PKA (KT5720) in a concentration-dependent manner, and was reproduced by the application of the purified catalytic subunit of PKA and augmented by the application of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). Corresponding biochemical study showed that several axoplasmic proteins including kinesin were specifically phosphorylated by the activation of the PKA pathway. These findings suggest the possible roles of PKA in the regulation of the direction of the axonal transport in vivo. The finding that only vesicle transport but not mitochondria transport was inhibited also suggests that the transport of vesicles and that of mitochondria are differently regulated and might be supported by different motors. PMID- 7536825 TI - Neuronal regeneration enhances the expression of the immunophilin FKBP-12. AB - Immunophilins are a group of proteins that serve as receptors for the immunosuppressant drugs cyclosporin A and FK506. The immunophilin designated FK 506 binding protein-12 (FKBP-12) is concentrated more than 10 times higher in the brain than in immune tissues. The complex of FK506 and FKBP-12 inhibits the calcium activated phosphatase, calcineurin, increasing phosphorylated levels of calcineurin substrates with growth associated protein-43 (GAP-43), being most prominent in the brain. We now demonstrate an association of FKBP-12 with neuronal regeneration and GAP-43 disposition. Facial nerve crush markedly augments expression of FKBP-12 mRNA in the facial nucleus with a time course paralleling changes in GAP-43 mRNA. Following sciatic nerve lesions, similar increases in FKBP-12 mRNA occur in lumbar motor neurons and dorsal root ganglia neuronal cells. Increased FKBP-12 expression appears linked to regeneration rather than degeneration as facial nerve lesions elicited by ricin injection, which produce neuronal death without regeneration, fail to augment FKBP-12 expression in the facial nucleus. The time course for accumulation of FKBP-12 in sciatic nerve segments following nerve crush indicates rapid axonal transport at a rate similar to GAP-43. PMID- 7536823 TI - Effect of bacterial endotoxin and interleukin-1 beta on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. AB - In this study the effect of immune system stimulation and intracerebroventricular (i.c.v.) administration of interleukin-1 beta (IL-1 beta) on hippocampal serotonergic neurotransmission, behavioral activity, and the hypothalamic pituitary-adrenocortical (HPA) axis is described. An in vivo microdialysis method was used to measure hippocampal extracellular concentrations of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in conscious, freely moving rats. In addition, we established a method to continuously monitor free corticosterone levels in dialysates. Behavioral activity was scored by measuring the time during which rats were active (locomotion, grooming, eating, drinking). We found a significant, positive relationship between behavioral activity and hippocampal extracellular concentrations of 5-HT. Intraperitoneal (i.p.) administration of the bacterial endotoxin lipopolysaccharide (LPS; 30, 100, and 300 micrograms/kg body weight) produced an increase in the extracellular concentrations of 5-HT and 5-HIAA in the hippocampus, which was paralleled by a significant decline in behavioral activity and a marked increase in extracellular corticosterone levels. Thus, the close correlation between hippocampal extracellular 5-HT levels and behavioral activity observed in control rats was disrupted in the LPS-treated animals. The effects of i.p. LPS could be mimicked by i.c.v. application of recombinant human IL-1 beta (hIL-1 beta; 100 ng). i.c.v. pretreatment with the IL-1 receptor antagonist (IL-1ra; 10 micrograms) antagonized the hIL-1 beta-induced effects. IL-1ra showed no intrinsic effects. Furthermore, it was found that i.c.v. pretreatment with IL-1ra (10 micrograms) significantly attenuated the i.p. LPS-induced (100 micrograms/kg body weight) rise in hippocampal extracellular 5-HT levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536827 TI - cGMP inhibits L-type Ca2+ channel currents through protein phosphorylation in rat pinealocytes. AB - In this study, the effect of cGMP on the dihydropyridine-sensitive (L-type) Ca2+ current was investigated using the whole cell version of the patch-clamp technique in rat pinealocytes. Dibutyryl-cGMP (1 x 10(-4) M) induced a pronounced inhibition of the L-type Ca2+ channel current. The dibutyryl-cGMP effect was concentration dependent. Elevation of cGMP by nitroprusside had a similar inhibitory action on the L-type Ca2+ channel current. Norepinephrine, which increased cGMP in rat pinealocytes, also inhibited this current. The action of cGMP was independent of cAMP elevation since the cAMP antagonist, Rp-cAMPs, had no effect on the inhibitory action of dibutyryl-cGMP. The involvement of cyclic GMP-dependent protein kinase was suggested by the blocking action of two protein kinase inhibitors, (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and N-(2 guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), on the dibutyryl-cGMP effect on the L-type Ca2+ channel current. Taken together, these results suggest that (1) cGMP modulates L-type Ca2+ channel currents in rat pinealocytes, causing inhibition of this current; (2) the action of cGMP appears to be independent of cAMP elevation; and (3) phosphorylation by cGMP-dependent protein kinase may be involved. PMID- 7536829 TI - The differences in intestinal viscosity produced by barley and beta-glucanase alter digesta enzyme activities and ileal nutrient digestibilities more in broiler chicks than in cocks. AB - The objectives of this study were to determine whether intestinal viscosity caused by mixed linked barley beta-glucan depresses ileal nutrient digestibility and digestive enzyme activities and to determine the interaction of intestinal viscosity, digestive enzyme activities and ileal nutrient digestibility in different ages of poultry. In Experiments 1 and 2, 1-d-old broiler chicks and 1-y old cocks, respectively, were fed diets with 60% corn, low and high viscosity barley with or without beta-glucanase, for 3 wk. A 3 x 2 factorial design was used. Comparisons were made only within the same age group. In Experiment 3, 1-d old broiler chicks were fed high viscosity barley with and without beta-glucanase to measure fecal nutrient and ileal and fecal amino acid digestibility. Broiler chicks fed barley ate less and gained less weight than those fed corn; added beta glucanase resulted in increases in both food consumption and weight gain for the barley-fed chicks (P < 0.05). Relative pancreas weight was higher (P < 0.05) in chicks fed barley than in those fed corn, and lower with beta-glucanase (P < 0.05). Digesta from barley-fed birds had the highest viscosities, which were decreased (P < 0.05) by beta-glucanase. Amylase and lipase were lower in broiler chicks fed high viscosity barley compared with corn (P < 0.05), and beta glucanase increased both activities and that of trypsin as well (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536828 TI - Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C. AB - Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl-currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses. PMID- 7536830 TI - History and design of the INCAP longitudinal study (1969-77) and its follow-up (1988-89). AB - This is an overview of the design and methods of the INCAP longitudinal study (1969-77) and its follow-up study (1988-89). The first study had the objective of assessing the effects of intrauterine and preschool malnutrition on growth and mental development. To achieve this, food supplements were provided to pregnant women and young children residing in four Gautemalan villages. Two villages were given a high-protein, high-energy drink and two were provided a no-protein, low energy drink. Both supplements contained vitamins and minerals. Longitudinal information was collected during the first seven years of life on physical growth, mental development, attendance and consumption of supplement, home diet, morbidity and on characteristics of the family. Health and nutrition data on mothers also were collected. The INCAP follow-up study was a cross-sectional evaluation of former participants of the first study and was carried out when the subjects were 11-27 y old. The hypothesis of the INCAP follow-up study was that improved nutrition in early childhood leads to enhanced human capital formation in adolescents and adults. Data were collected on physical growth and body composition, maturation, work capacity, intellectual performance and school achievement. PMID- 7536831 TI - Nutrition in early life and the fulfillment of intellectual potential. AB - The effects of early supplementary feeding on cognition are investigated using data collected during two periods in four Guatemalan villages. The first was the Institute of Nutrition of Central America and Panama (INCAP) longitudinal study from 1969 to 1977 and the second was a cross-sectional follow-up of former participants carried out in 1988-1989. The principal objective of these studies was to assess the differential effect of two dietary supplements, Atole containing 163 kcal/682 kJ and 11.5 g protein per cup or 180 mL and Fresco containing 59 kcal/247 kJ and 0 g protein per cup, that were given to mothers, infants and young children. Performance was assessed on a battery of psychoeducational and information processing tests that were administered during adolescence. Consistent differences between groups were observed on psychoeducational tests. Subjects receiving Atole scored significantly higher on tests of knowledge, numeracy, reading and vocabulary than those given Fresco. Atole ingestion also was associated with faster reaction time in information processing tasks. In addition, there were significant interactions between type of dietary supplement and socioeconomic status (SES) of subjects. In Atole villages, there were no differences in performance between subjects in the lowest and highest SES categories. On the other hand, performance in Fresco villages was best in the highest compared with the lowest SES group. After close scrutiny of alternative hypotheses, it is concluded that dietary changes produced by supplementation provide the strongest explanation for the test performance differences observed in the follow-up between subjects exposed to Atole and those exposed to Fresco supplementation. PMID- 7536833 TI - Results and implications of the INCAP follow-up study. AB - This article is a critical synthesis of 12 papers included in this supplement. The set deals with the short- and long-term effects of improving nutrition in Guatemalan villages characterized by deficient diets, high rates of infection and pronounced growth retardation in the first 3 y of life. The data reviewed come from two studies carried out over two decades: the Institute of Nutrition of Central America and Panama (INCAP) longitudinal study (1966-1977) and its follow up (1988-1989). The longitudinal study included a nutrition intervention that improved the energy and nutrient intakes of women and preschool children. Its effects included improved birthweights, reduced infant mortality rates and improved growth rates in children < 3 y of age. Growth rates from 3 to 7 y of age, similar to those of well-nourished children, were not affected by the intervention. The follow-up study was conducted when the subjects were 11-27 y old. Among the long-term effects found were greater stature and fat-free mass, particularly in females, improved work capacity in males and enhanced intellectual performance in both genders. The nutrition intervention did not, on the other hand, accelerate maturation during adolescence, as measured by skeletal age or age at menarche. It is concluded that improved nutrition in early childhood has important long-term effects in the adolescent and adult. PMID- 7536832 TI - Malnutrition and human function: a comparison of conclusions from the INCAP and nutrition CRSP studies. AB - The overall objective of both the Institute of Nutrition of Central America and Panama (INCAP) studies and the Nutrition Collaborative Research Support Program (CRSP) was to determine if marginal malnutrition affects human function. The conclusions from the two studies were consistent, notably that growth stunting occurs early in life and is accompanied by functional impairments. These consequences of early malnutrition persist later in life. The comparison of INCAP and Nutrition CRSP results also illustrates that the Guatemalan children were more malnourished and stunted than those in Kenya, Mexico or Egypt and that this greater degree of stunting occurs before 18 months of age. Even the Atole did not bring the average size of supplemented children in Guatemala up to those of the unsupplemented children in the CRSP populations. It is also likely that their functional potential was not realized fully by supplementation. The CRSP studies provide evidence of multiple micronutrient deficiencies associated with poor growth and function and it is probable that these associations exist in Guatemala as well. The overall conclusion is that our attention should be directed to determining the adequacy of micronutrient status in the perinatal period and to the development of approaches that prevent early growth failure. PMID- 7536834 TI - Evaluation of free PSA isoforms, PSA complex formation, and specificity of anti PSA antibodies by HPLC and PAGE-immunoblotting techniques. AB - Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14 fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values. PMID- 7536836 TI - Purification of PSA-ACT complex: characterization of PSA-ACT complex by various chromatographic procedures. AB - We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536835 TI - Correlation of serum concentrations of PSA-ACT complex with total PSA in random and serial specimens from patients with BPH and prostate cancer. AB - The current assays for serum prostate specific antigen (PSA) have failed to produce the same PSA values on the same specimens because of problems with antibody specificity and calibrator preparation. To eliminate these problems, we proposed to replace the current serum PSA assay with an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex in the serum. An assay specific for the PSA-ACT complex was established using the anti-PSA antibody to coat the microplate for capturing the PSA complex and anti-ACT polyclonal antibodies for quantification. There was an excellent correlation between serum concentrations of PSA-ACT and total PSA, using either the Hybritech calibrator (gamma = 0.996) or a serum calibrator prepared in house (gamma = 0.993), in random as well as in serial specimens from 14 individual patients. Even though we did not find a gradual increase in the percentage of PSA-ACT with the increase of total PSA in cancer patients, a slightly higher percentage of free PSA was measured in pooled normal sera (18%) and in pooled sera containing only 12 ng/ml of total PSA (12%), compared to serum pools containing elevated PSA (> 100 ng/ml) level, in which most PSA was in the complex form (95%). Therefore, using an assay that specifically measures the PSA-ACT complex in the serum not only simplifies the preparation of calibrator but eliminates the difficulty of antibody selection, it also allows various assay kits to produce identical PSA values and also improve the test specificity for prostate cancer. PMID- 7536837 TI - New strategies in vaccination against fungal infections. PMID- 7536839 TI - Current priorities for the clinical mycology laboratory. PMID- 7536838 TI - Advances in veterinary mycology. PMID- 7536840 TI - Fungal polysaccharides. AB - Fungal polysaccharides are cell wall components which may act as antigens or as structural substrates. As antigens, the role of mannans in Saccharomyces cerevisiae and Candida albicans, and of glycoproteins in Aspergillus fumigatus are discussed. Analyses on beta-glucan synthetase in Paracoccidioides brasiliensis and the inhibitory effect of Hansenula mrakii killer toxin on beta glucan biosynthesis are also considered. PMID- 7536841 TI - Pathophysiology of congenital diaphragmatic hernia. X: Localization of nitric oxide synthase in the intima of pulmonary artery trunks of lambs with surgically created congenital diaphragmatic hernia. AB - The pathophysiology of congenital diaphragmatic hernia (CDH) results from a combination of pulmonary hypoplasia, pulmonary hypertension, and surfactant deficiency. Previously we demonstrated that inhaled nitric oxide (NO), a known vasodilator, only improves oxygenation and decreases pulmonary artery pressures when the lamb model of CDH is pretreated with exogenous surfactant. Nitric oxide synthase (NOS) in endothelial cells is responsible for the production of NO, a mediator of smooth muscle cell relaxation. Pulmonary hypertension in CDH may result from a defect in the endogenous production of NO. Our aim was to determine whether the main pulmonary artery trunks in CDH lambs have NOS immunoreactivity. Cryostat sections of paraformaldehyde-fixed specimens of pulmonary artery and aortic rings from 10 CDH lambs and five control lambs were processed for NADPH diaphorase activity. Immunolocalization of NOS was studied in paraformaldehyde fixed sections and compared with serially cut specimens from identical rings that were tested for NADPH-diaphorase activity. Intense NADPH-diaphorase staining was present in the intimal layer (endothelial lining) of the pulmonary artery and aortic rings of both the CDH and control lambs. This activity colocalized with NOS immunoreactivity in all specimens. Both NOS immunoreactivity and NADPH diaphorase staining were lacking in cartilage, which were used as negative controls. NOS is present in the main pulmonary artery trunks of CDH lambs. To our knowledge, this is the first report of NOS immunoreactivity in CDH. We can only speculate whether this activity is preserved in other areas of the vascular tree in CDH, ie, pulmonary capillaries and veins. Perhaps the pulmonary hypertension in CDH is not caused by an NOS deficiency. PMID- 7536843 TI - Effects of single doses and daily melatonin treatments on serotonin metabolism in rat brain regions. AB - The acute effects of two doses (0.5 and 1 mg/kg) of melatonin on the levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid in several rat brain regions were studied. Tryptophan content in the brain regions was unchanged by the treatments. Melatonin at a dosage of 0.5 mg/kg increased medial hypothalamic serotonin levels at 60 and 90 min after the injection. However, the dose of 1 mg/kg increased the levels of this amine or its metabolite in the preoptic area anterior hypothalamus, medial and posterior hypothalamus, amygdala, and midbrain. These results suggest a specific regional sensitivity to melatonin as well as a dose-dependent response. The stimulatory melatonin effect on the serotoninergic system was also observed after a daily treatment with this hormone (0.5 mg/kg, twice daily during 10 days) in both intact or pinealectomized rats. In intact rats, melatonin treatment increased the levels of 5-hydroxyindole-3-acetic acid in the preoptic area-anterior hypothalamus and medial hypothalamus, while in pinealectomized rats melatonin increased the serotonin content in the medial hypothalamic region. The data support the idea that melatonin has a selective action on serotonin metabolism in regions that contain serotoninergic terminals, especially at medial hypothalamic level. PMID- 7536842 TI - Osteopontin adhesion receptors on gingival fibroblasts. AB - Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM-609, a monoclonal antibody to the alpha v beta 3 integrin (a vitronectin receptor), inhibited OPN-mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN-sepharose affinity chromatography using detergent extracts of 35S methionine or 125I-surface labeled gingival fibroblasts. Proteins bound to the OPN-matrix were eluted with EDTA and subjected to SDS-PAGE under reducing conditions. EDTA eluates from both 125I-surface labeled and 35S-methionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to alpha v beta 3. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than alpha v beta 3. PMID- 7536846 TI - Inflammatory cell infiltrate associated with primary and transplanted tumours in an inbred model of oral carcinogenesis. AB - This study characterised the nature of the local cellular immune responses associated with an inbred animal model of oral carcinogenesis. Inbred F344 rats developed moderately- to well-differentiated primary oral squamous cell carcinomas (SCC) after treatment with the carcinogen 4-nitroquinoline N-oxide (4 NQO) in vivo for 5-6 months. The inflammatory cell infiltrate associated with the primary tumours was predominantly of the macrophage lineage (CD45+, Ia+) and contained smaller numbers of CD8+ cells (NK cells, cytotoxic/suppressor T cells), CD5+ cells (T cells) and CD25+ cells (activated cells; T and NK cells). Keratinocyte cell lines were established from three lingual and one palatal SCC. By contrast to normal keratinocytes, tumour-derived cell lines were immortal and independent of 3T3 fibroblast support. All of the tumour-derived cell lines were tumorigenic in athymic (nu/nu) mice and showed contrasting latent periods of tumour development and histological differentiation; normal keratinocyte grafts were non-tumorigenic in athymic mice. Three of four malignant cell lines formed well-differentiated tumours in syngeneic F344 rats; the tumours regressed after 10-14 days. Regressing grafts contained significantly larger numbers of NK cells (CD5-, CD8+) in the inflammatory cell infiltrate compared with that associated with primary tumours (p < 0.04). One malignant cell line and normal keratinocytes were non-tumorigenic in syngeneic hosts. The results demonstrate phenotypic variation in the cell-mediated immune responses associated with the actively growing primary SCC and the regressing tumours in syngeneic hosts and suggest that NK cells, possibly activated by local T cell responses, are important for tumour rejection in this model. PMID- 7536845 TI - Effect of orpiment (As2S3) on cytochrome P-450, glutathione and lipid peroxide levels of rat liver. AB - Oral administration of orpiment (50 mg/kg b. wt.) caused significant decreases of cytochrome P-450 (44%) and glutathione levels (47%) of rat liver microsomes. Concomitantly, there was an increase in the lipid peroxidation (78%). Orpiment caused significant decreases in the activities of antioxidant enzymes. The glutathione-S-transferase activity was, however, lowered by 42%. The cytochrome P 450-dependent mixed function oxygenase system was also affected. The results show the hepatotoxic nature of orpiment. PMID- 7536847 TI - Laparoscopic diagnosis and treatment of cornual pregnancy. A case report. AB - An early diagnosis of interstitial pregnancy was made by serial quantitative human chorionic gonadotropin beta-subunit evaluation, transvaginal ultrasound findings and laparoscopy. Cornual resection was performed using operative endoscopic techniques; the postoperative course was unremarkable. PMID- 7536844 TI - [Recent advances in studies of GABA receptors: neurochemical aspects]. AB - gamma-Aminobutyric acid (GABA) has been established as a major inhibitory neurotransmitter in the brain. The GABA-induced inhibitory transmission is mediated by two distinct types of GABA receptors which are termed as GABAA and GABAA receptors. The GABAA receptor forms a Cl- channel that consists of several subunits. The recent development of molecular cloning clarified the presence of multiple and heterogeneous molecules in the GABAA receptor subunits. Therefore, it is suggested that multiple GABAB receptors generate various functions in the brain. In contrast, the GABAB receptor exhibits various metabotropic actions for the inhibitory neurotransmission, since it is coupled with GTP-binding proteins. The GABAB receptor of approximately 80 kDa protein in its molecular weight was purified from the bovine cerebral cortex and the presence of another molecular species of the GABAB receptor was also suspected. These results indicate that studies on the molecular diversity of the GABA receptors is important for elucidating the functional roles of GABAergic neurons in the brain. PMID- 7536848 TI - NusA contacts nascent RNA in Escherichia coli transcription complexes. AB - We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template. PMID- 7536849 TI - Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding. AB - The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells. PMID- 7536852 TI - From the Centers for Disease Control and Prevention. Progress toward global poliomyelitis eradication, 1985-1994. PMID- 7536851 TI - Hypertonic acetate dextran achieves high-flow-low-pressure resuscitation of hemorrhagic shock. AB - OBJECTIVE: For resuscitation of hemorrhagic hypovolemia, we compared the effectiveness of (1) isotonic lactated Ringer's solution (LRS), (2) 2400 mOsm of 7.5% NaCl:6% dextran 70 (HSD), and (3) 2400 mOsm of 7.9% sodium acetate:1.9% NaCl:6% dextran 70 (HAD). DESIGN: In six randomized, blinded experiments for each solution, conscious instrumented adult sheep were hemorrhaged by removing approximately 1.8 L (42 +/- 3 mL/kg) of blood, while maintaining the mean arterial pressure (MAP) at 50 mm Hg for 2 hours. METHODS: Test solutions were infused as needed to restore the cardiac index to baseline. RESULTS: Volume requirements with HAD (236 +/- 29 mL) and HSD (244 +/- 39 mL) were significantly less (p < 0.05) than LRS (3463 +/- 234 mL). Mean arterial pressure was normalized with HSD and LRS, but not with HAD, which resulted in MAPs of 20 to 25 mm Hg less than baseline resulting from a reduced peripheral resistance. Oxygen delivery, however, was significantly higher with HAD during the resuscitation period. Acid base balance (pH) and oxygen consumption were normalized within 5 minutes of infusion only with HAD. CONCLUSIONS: Small-volume infusion with HAD resulting in "high-flow-low-pressure" resuscitation may offer unique hemodynamic and metabolic advantages for the initial treatment of hemorrhage from trauma. PMID- 7536850 TI - Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes. AB - Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity. PMID- 7536853 TI - The role of ions on histamine-induced depolarization in isolated guinea pig adipocytes. AB - Effects of ions on histamine (Hi)-induced depolarization were studied in guinea pig adipocytes. Depolarization induced by Hi in guinea pig adipocytes was decreased by removal of K+ from the medium or pretreatment with ouabain at concentrations that showed no significant effect themselves. The decrease in membrane potentials induced by Hi was also abolished potently by replacement of Na+ by choline or pretreatment with tetrodotoxin at a concentration that caused no significant action alone. Pretreatment with monensin at a concentration lower than that eliciting the action resulted in a potentiation of Hi-induced depolarization. The depolarization induced by Hi was not affected by the presence of Ca2+ in the medium or pretreatment of the cells by diltiazem. PMID- 7536854 TI - Study of EHS type IV collagen lacking Goodpasture's epitope in glomerulonephritis in rats. AB - The Goodpasture's epitope has been mapped to the alpha 3 non-collagenous chain (NC1) of type (IV) collagen [alpha 3col(IV)]. We have developed a model of experimental autoimmune glomerulonephritis (EAG) in rats immunized once with collagenase solubilized GBM (csGBM). Engelbreth-Holm-Swarm (EHS) tumor contains abundant col(IV) with little or no alpha 3col(IV). To test the hypothesis that antigens related to Goodpasture epitope are required to produce EAG in our model, we immunized rats once with 40 micrograms csEHS. Positive controls immunized with csGBM developed typical EAG with GBM bound antibody, proteinuria, and glomerulonephritis. EHS rats developed circulating and bound antibody to mesangium and tubular basement membrane with minimal GBM deposits, but did not develop proteinuria or glomerulonephritis. Although circulating antibody in EHS rats bound to csGBM by ELISA, there was no binding in ELISA to M2 antigen containing the Goodpasture epitope while EAG rat's serum did bind. By Western blot with antisera to Goodpasture epitope, EHS antigen was less complex than GBM in the monomer/dimer regions and appeared to lack NC1 corresponding to alpha 3col(IV). Blotting with sera from EHS rats demonstrated reactivity to various components of GBM but not to alpha 3col(IV). EAG sera and renal eluates bound to alpha 3col(IV). EAG rats evidenced cell mediated immunity while EHS rats did not (stimulation index EHS 1.1, EAG rats 8.0).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536855 TI - Intracellular apoprotein B degradation is suppressed by decreased albumin concentration in Hep G2 cells. AB - It is generally accepted that hepatic secretion of apoprotein (apo) B-containing lipoproteins is substantially increased in nephrosis. To elucidate the mechanisms for the oversecretion of apo B, we investigated the effect of a various concentration of albumin on apo B kinetics in the absence or presence of oleate in Hep G2 cells. Hep G2 cells were labeled with [3H]-leucine in leucine-free medium containing 0, 1.5, 3.0 or 4.5% BSA for 180 minutes, and the secreted radiolabeled apo B, apo A1 and albumin were isolated by immunoprecipitation and counted. The secretions of apo B and albumin were suppressed by BSA (bovine serum albumin) in a dose-dependent manner, but the secretion of apo A1 was not suppressed significantly. Oleate (0.4 mM) increased the rate of apo B secretion by 2.5-fold when oleate was bound to 1.5% BSA, but at higher concentrations of BSA (3.0 or 4.5%), apo B secretion was less responsive to oleate. A pulse-chase study indicated that early apo B degradation was significantly suppressed in cells incubated with lower concentrations of BSA (0 or 1.5% BSA), thereby rapidly stimulating apo B secretion. Oleate (0.4 mM) potently inhibited apo B degradation when oleate was bound to 1.5% BSA, whereas the inhibition was not observed when oleate was bound to 4.5% BSA. Intracellular albumin synthesis was stimulated in BSA-free medium, but intracellular decay of albumin was essentially unaffected by concentration of BSA. Similar to BSA, a higher concentration of dextran (3.0 or 4.5%) reduced apo B secretion, and this was the result of increased early apo B degradation in the cells. These results indicate that reduced albumin suppresses intracellular apo B degradation, and the inhibition of apo B degradation by oleate is manifested only at a low concentration of albumin. Therefore, the present study suggests that free fatty acids bound to low concentration of albumin in the circulating plasma play an important role on hepatic oversecretion of apo B-containing lipoprotein in hypoalbuminemic state, such as nephrotic syndrome. PMID- 7536856 TI - Guanidino compound metabolism in rats subjected to 20% to 90% nephrectomy. AB - In mammalian kidney, the proximal convoluted tubule (PCT) is the main site of arginine (Arg) production. Arginine can be used in the biosynthesis of guanidino compounds (GC). Since uremic rats have a lower functional mass of PCT, GC synthesis might be modified, especially that of guanidinoacetic acid (GAA) which occurs in PCT. In order to study GC metabolism at different steps of uremia, rats were subjected to either 42% or 80% nephrectomy (NX); the experiment lasted for three weeks. Results show that: (1) in plasma, the pattern of GC levels in 42% NX rats was similar to that of controls except for a clear increase of beta guanidinopropionic acid (beta-GPA), whereas in 80% NX rats, all GC levels sharply increased except that of creatine which decreased. (2) Urinary excretion of GC in control and 42% NX rats is quite similar except for GAA which strongly decreased, and for homoarginine (HArg) and argininic acid (ArgA) which increased. In rats with 80% NX, the principal modification in GC excretion was a four- to five-fold reduction in GAA output. (3) After induction of renal failure, Arg, creatine and guanidinosuccinic acid reabsorption remained unchanged, and that of HArg decreased. For guanidine and methylguanidine the negative renal balance remained unchanged, and that of gamma-guanidinobutyric acid, GAA and alpha-keto-delta guanidinovaleric acid became smaller, suggesting a better reabsorption. In conclusion, uremia strongly modified GC metabolism involving mainly those synthesized from Arg; both GAA and creatine synthesis were strongly decreased probably because of the loss of renal tissue, mainly PCT. PMID- 7536857 TI - Na+/myo-inositol cotransport is regulated by tonicity in cultured rat mesangial cells. AB - Mesangial cells are considered to be faced with osmotic stress under physiological (such as extraglomerular mesangial cells) and pathophysiological (for example, diabetes mellitus) conditions. To see if mesangial cells have an osmoregulatory mechanism, like renal medullary cells, we measured the intracellular contents of organic osmolytes in isotonic and hypertonic conditions. Cultured rat mesangial cells are well tolerant of acute increase in osmolality up to 500 mOsm/kg. The myo-inositol content increased in hypertonic cells more than six-fold the value in isotonic cells. The contents of glycerophosphorylcholine and sorbitol also increased but were less than that of myo-inositol. The Na(+)-dependent myo-inositol uptake in hypertonic cells was a 12-fold uptake in isotonic cells, reaching a maximum 24 hours after the switch to a hypertonic medium. The uptake rate increased as medium osmolality increased from 300 to 500 mOsm/kg. Raffinose is the most effective solute to increase the myo-inositol uptake. NaCl, glucose and mannitol also increased the uptake rate (NaCl > glucose > mannitol). The increased uptake by hypertonicity was the result of an increase in Vmax without change in Km and was dependent on RNA and protein synthesis. These results indicate that mesangial cells respond to extracellular hypertonicity by increasing myo-inositol transport activity and accumulating myo inositol into the cells, suggesting that myo-inositol functions as an organic osmolyte in mesangial cells. PMID- 7536859 TI - Strategic locus for the activation of the superoxide dismutase gene in the nephron. AB - Upon exposure to a transient ischemia, the distal tubule of the kidney often escapes the severe damage which afflicts the proximal tubule. To ascertain whether this feature of the distal tubule is attributable to its intrinsic cellular properties, we focused on two pairs of unique tubule segments; distal versus proximal convoluted tubules in the superficial cortex and distal versus proximal straight tubules in the outer stripe of the outer medulla. These tubules were chosen because, firstly, they can be identified by morphology and immunostaining, and secondly, each pair has the same anatomical relationship to the circulation. Detailed morphometric analyses were performed six hours following unilateral transient ischemia in adult rats to semiquantitate the local tissue damage in these specific nephron segments. The architecture of the distal convoluted and straight tubules was remarkably well preserved, contrasting to the moderate to extensive necrotic changes seen in the proximal tubules. In search of the potential intrinsic cellular mechanism that underlies the observed difference, we examined the segmental distribution along the nephron of manganese superoxide dismutase gene transcripts by in situ hybridization. This antioxidant enzyme gene was expressed primarily in the distal tubules with contrastingly low levels of expression in the proximal tubules. Moreover, following ischemia reperfusion, this distal tubule-dominant pattern was further accentuated immediately following reperfusion. The study indicates that the marked difference between the proximal and distal tubules in their susceptibility to injury in vivo is attributable to their intrinsic cellular properties, which include the local level of antioxidants. PMID- 7536860 TI - Modifications in cytokeratin and actin in cultured liver cells derived from griseofulvin-fed mice. AB - BACKGROUND: Hepatocytes from mice fed griseofulvin (GF) for 8 months form Mallory bodies (MBs), which represent a pathologic state of intermediate filaments (IFs). The cellular mechanisms that lead to MB formation are not known. EXPERIMENTAL DESIGN: This study was aimed to investigate if MB formation could be related to modification in cytokeratin (CK) metabolism. Primary cultures of hepatocytes from control and GF livers were studied. Immunofluorescence microscopy was used to study the organization of the cytoskeleton in these cells. The hepatocytes were labeled with [35S]methionine or [32P]orthophosphate to study, respectively, the level of amino acid incorporation into IF proteins (CK 8 and CK 18) and their phosphorylation levels. The response to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate stimulation of the phosphorylation of CK 8 and CK 18 was also elicited in contrast to control hepatocytes. RESULTS: We found that there was a change in the organization of actin and the IF network in the hepatocytes from GF treated animals. This was associated with an increase in labeled amino acid incorporation into CK 8 and CK 18 as well as in actin. Although there was no significant difference in the absolute level of CK phosphorylation, we found modifications in the phosphorylated isomers of CK 8, the more phosphorylated isomers becoming more prominent. The treatment of the hepatocytes with 12-O tetradecanoyl-phorbol-13-acetate did not induce changes in the level of CK phosphorylation in GF-pretreated hepatocytes. CONCLUSIONS: These results suggest that the modification of the IF network and MB formation are the consequences of increased CK synthesis and the modification of phosphorylation. They could alter the normal interaction of the IFs with different cellular components, which results in conformational changes of CKs and the reorganization of the IF network to the form of MBs. PMID- 7536858 TI - Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli. AB - The presence of the inducible isoform of nitric oxide synthase (iNOS) in glomerular mesangial cells facilitates the synthesis of nitric oxide (NO) after stimulation with cytokines or lipopolysaccharide (LPS). As the role of NO within the glomerulus may be important in conditions such as glomerulonephritis, we have studied the effect of dexamethasone (DX) and pirrolidine dithiocarbamate (PDTC), an inhibitor of the nuclear transcription factor, NF-kappa B activation on the induced synthesis of NO in rat mesangial cells (RMC). LPS, tumor necrosis factor alpha (TNF-alpha) and the combination of both were able to induce NO synthesis in a dose-dependent manner as measured with the determination of NO2- levels. Treatment with LPS (10 micrograms/ml) + TNF-alpha (100 ng/ml) for eight hours was the most potent stimulus for iNOS activity. DX (1 microM) had an inhibitory effect on LPS-, TNF-alpha- and LPS + TNF-alpha-induced NO synthesis (51.2, 42.5 and 68% of inhibition, respectively). The inhibitory effect of DX was confirmed using a reporter cell bioassay, whereas cGMP was measured as a reflection of bioactive NO. DX inhibited induced NO synthesis when RMC were exposed to this agent before (16 hr of pretreatment, 75.7% inhibition) or at the same time (8 hr of cotreatment, 61.2% inhibition) as TNF-alpha + LPS but not four hours after the stimuli. Northern blot analysis showed marked blunting of mRNA expression in RMC treated with DX, in concordance with functional studies. Both actinomycin D and cycloheximide significantly inhibited NO synthesis and iNOS mRNA expression. PDTC (100 microM) was able to inhibit the iNOS activity induced by LPS and TNF-alpha independently (56.8 and 49.9% inhibition, respectively), and in combination (79.1% inhibition). PDTC (1 to 100 microM) inhibited LPS + TNF-alpha-induced NO synthesis and iNOS mRNA expression in a concentration-dependent fashion (69 to 86% inhibition of NO synthesis and 50 to 100% inhibition of mRNA expression). Addition of PDTC four hours after exposure to TNF-alpha + LPS was still able to markedly inhibit NO synthesis. The effects of DX and PDTC were also demonstrated in isolated glomeruli, where two different combinations of inductive stimuli for NO synthesis were employed. Our results establish DX and PDTC as useful tools to study the regulation of NO synthesis in the mesangial cell and glomerulus, and suggest that NF-kappa B is involved in the transcriptional regulation of iNOS in RMC. PMID- 7536861 TI - Forensic drug testing for opiates. VI. Urine testing for hydromorphone, hydrocodone, oxymorphone, and oxycodone with commercial opiate immunoassays and gas chromatography-mass spectrometry. AB - Opiate testing for morphine and codeine is performed routinely in forensic urine drug-testing laboratories in an effort to identify illicit opiate abusers. In addition to heroin, the 6-keto-opioids, including hydromorphone, hydrocodone, oxymorphone, and oxycodone, have high abuse liability and are self-administered by opiate abusers, but only limited information is available on detection of these compounds by current immunoassay and gas chromatographic-mass spectrometric (GC-MS) methods. In this study, single doses of hydromorphone, hydrocodone, oxymorphone, and oxycodone were administered to human subjects, and urine samples were collected before and periodically after dosing. Opiate levels were determined in a quantitative mode with four commercial immunoassays, TDx opiates (TDx), Abuscreen radioimmunoassay (ABUS), Coat-A-Count morphine in urine (CAC), and EMIT d.a.u. opiate assay (EMIT), and by GC-MS. GC-MS assay results indicated that hydromorphone, hydrocodone, oxymorphone, and oxycodone administration resulted in rapid excretion of parent drug and O-demethylated metabolites in urine. Peak concentrations occurred within 8 h after drug administration and declined below 300 ng/mL within 24-48 h. Immunoassay testing indicated that hydromorphone, hydrocodone, and oxycodone, but not oxymorphone, were detectable in urine by TDx and EMIT (300-ng/mL cutoff) for 6-24 h. ABUS detected only hydrocodone, and CAC failed to detect any of the four 6-keto-opioid analgesics. Generally, immunoassays for opiates in urine displayed substantially lower sensitivities for 6-keto-opioids compared with GC-MS. Consequently, urine samples containing low to moderate concentrations of hydromorphone, hydrocodone, oxymorphone, and oxycodone will likely go undetected when tested by conventional immunoassays. PMID- 7536863 TI - Microwave fixation and localization of calcium in synaptic vesicles. AB - The distribution of Ca2+ ions is demonstrated in the synaptic terminals by means of a 2-step chemical precipitation of Ca2+ ions in nervous tissue. K-oxalate/K antimonate chemical replacement with simultaneous computerized microwave irradiation is used. This precipitate in cell structures was investigated by computerized electron probe X-ray micro-analysis. The calculated values (from the theoretical, standards and sections), elemental binding ratios and elemental molecular weight ratios were compared. Each calculated value coincided with the theoretical value. This method can reliably detect Ca2+ ions at the micromolar level. Ca2+ ions were distributed in the synaptic vesicles and surrounding membranes. Further progress is expected in freeze-substitution and in the application and propagation of the EELS-Imaging system in calcium determinations. PMID- 7536864 TI - In vitro purging with BCR-ABL antisense oligodeoxynucleotides does not prevent haematologic reconstitution after autologous bone marrow transplantation. AB - We treated a patient with chronic myeloid leukaemia in accelerated phase with autologous bone marrow transplantation. Before reinfusion, cells were purged in vitro with a 26-mer phosphorothioate antisense oligodeoxynucleotide specific for the B2A2 junction. Incubation with antisense oligodeoxynucleotides produced a 24 and 41% reduction of CFU-GM and CD34+ cells, respectively. However, an in vitro test previously performed as a screening for patient inclusion in this procedure, revealed a 38 and 75% reduction of colony formation after 24-h and 168-h incubation, respectively. The patient showed bone marrow engraftment 15 days after reinfusion and haematological reconstitution after 17 and 25 days for platelets and neutrophils, respectively. Using fluorescence in situ hybridization in interphase nuclei, we demonstrated the presence of a proportion of Ph-negative cells in repeated controls after the autograft. The patient is now in unmaintained complete haematological remission 9 months after the autograft. PMID- 7536862 TI - Responses to endothelins-1, -2, and -3 and sarafotoxin 6c after ischemia/reperfusion in isolated perfused rat heart: role of vasodilator loss. AB - Coronary constrictor actions of endothelin-1 (ET-1) are enhanced after myocardial ischemia/reperfusion (I/R), possibly owing to enhanced ETA-receptor-mediated constriction and/or loss of the opposing ETB-receptor-mediated vasodilatation. We examined the actions of ET-1, ET-2, and ET-3 and the selective ETB-receptor agonist sarafotoxin 6c (Sx6c) after I/R in perfused rat heart. To examine the effects of a loss of ETB-receptor-mediated vasodilatation on coronary constrictor responses to ET-1, we used repeated doses of Sx6c to desensitize ETB receptors. After I/R, the coronary constrictor effects of all three ETs were enhanced, whereas their initial vasodilator effects were inhibited. The pure coronary dilator effect of Sx6c observed in control hearts was also inhibited after I/R. After desensitization of ETB receptors, the coronary constrictor action of ET-1 was enhanced by an amount equivalent to the vasodilatation that had been lost. This enhancement of constriction was not as marked as that noted after I/R, suggesting that the enhanced coronary constrictor action of ET-1 after I/R is not simply due to loss of opposing ETB-receptor-mediated vasodilatation and that other mechanisms are involved. The most likely explanation is upregulation of functional ETA receptors after I/R because ETB-receptor stimulation did not cause coronary constriction in this preparation. The vasoconstrictor enhancement therefore is likely to be the combined effect of receptor upregulation and vasodilator loss. PMID- 7536866 TI - Measuring disease-specific health status in men with benign prostatic hyperplasia. Measurement Committee of The American Urological Association. AB - In preparation for an outcomes study of benign prostatic hyperplasia (BPH), two measures of disease-specific health status were developed to supplement a symptom score and overall health status measures. The symptom problem index (SPI) captures how troublesome patients find their urinary symptoms. The BPH impact index (BII) measures how much their urinary problems affect various domains of health. A prospective revalidation of the refined instruments (N = 108 BPH patients and 50 controls) documented that both indices had good internal consistency (Cronbach's alpha = 0.88 and 0.79, respectively) and test-retest (r = 0.88 for both) reliabilities, correlated strongly with symptom scores (r = 0.86 and 0.77), and discriminated between BPH and control subjects (receiver-operating characteristic areas = 0.87 and 0.85, respectively). These indices were nearly as responsive as symptom scores in 50 men actively treated for BPH, and much more responsive than a non-disease-specific General Health Index (GHI), a Mental Health Index (MHI), and an Activity Index (AI). Finally, these measures capture most of the health status significance of BPH symptoms. In linear regression models constructed to predict scores on the GHI, MHI, and AI, symptom scores added little explanatory power to the SPI and, particularly, to the BII. These measures help clarify how BPH affects overall health status and function. Such measures have an important role to play in studies of the outcomes of treatment for BPH, and probably for other conditions that interfere with health status and function. PMID- 7536865 TI - Production of transforming growth factor alpha by human leukemia cells (HL-60 and U-937) during monocytic differentiation. AB - We have previously demonstrated that human promyelocytic HL-60 cells express transforming growth factor-alpha (TGF-alpha) during granulocytic differentiation. The present experiments were carried out in order to determine whether cells differentiated towards monocytes/macrophages will analogously express the TGF alpha proto-oncogene product. HL-60 cells were induced to differentiate with 1 microM 1,alpha 25-dihydroxycholecalciferol (vitamin D3), and the human monocytoid cell line, U-937, was induced with 1 microM retinoic acid (RA), 0.1 microM vitamin D3, or 0.16 microM phorbol-12-myristate-13-acetate (PMA), ie experimental protocols known to induce monocyte/macrophage differentiation in these cells. In HL-60 cells, lacking constitutive TGF-alpha mRNA, vitamin D3 caused expression of the TGF-alpha gene and protein as demonstrated by Northern blot analysis and enzyme-linked immunoabsorbant assay (ELISA). In U-937 cells, showing constitutive TGF-alpha expression, RA but not vitamin D3 or PMA, caused marked increase in TGF alpha mRNA (approximately 5-fold) and protein (approximately 3-fold) levels. In both cell lines the increase in TGF-alpha mRNA was evident within 24 h and continued throughout the observation period. Thus, it is established that differentiation of human leukemia cells towards monocytes/macrophages may be accompanied by TGF-alpha gene and protein expression in vitro. This is in conformity with the observed ability of mature activated macrophages to produce TGF-alpha. PMID- 7536867 TI - The role of community-based longitudinal studies in evaluating treatment effects. Example: benign prostatic hyperplasia. AB - To determine the appropriateness and effectiveness of medical interventions, it is necessary to understand both the natural history of the conditions the interventions are intended to treat or prevent, and the normal reference ranges of tests used in diagnosis and management. The acquisition of this information through clinic-based studies can yield misleading conclusions owing to selection bias. What appears to be intervention-related variation in outcomes may be variation in the magnitude and form of selection bias. To minimize selection bias, population-based studies are required. Results from the Olmsted County Study of Urinary Symptoms and Health Status Among Men were used to show how a population-based longitudinal study of the natural history of benign prostatic hyperplasia (BPH) can complement research programs of the BPH Patient Outcomes Research Team. Population-based studies of disease natural history are a necessary part of medical outcomes research, and deserve greater emphasis in the medical treatment effectiveness research initiative of the Agency for Health Care Policy and Research. PMID- 7536868 TI - Comparing prospective and retrospective measures of treatment outcomes. AB - This study examines the correspondence between retrospective and prospective assessments of treatment outcomes among female patients treated for gynecologic symptoms (n = 800) and male patients having surgery for benign prostatic hyperplasia (n = 434). The overall health and symptom status of patients in both samples was assessed at enrollment and again 3 months after treatment; at the 3 month follow-up, patients also were asked to compare retrospectively their current health and how they were feeling with their condition before treatment. Findings indicate that prospective and retrospective measures of change do not yield the same results. Retrospective assessments consistently produce higher estimates of the benefits of treatment, although that pattern was clearer for overall health status than for measures of symptoms. Patients' posttreatment health and symptom status contributes as much to retrospective assessments of change as does prospectively measured change, although the retrospective assessments of female patients whose gynecologic symptoms were medically managed were more strongly related to prospective change than those of hysterectomy patients or prostate surgery patients. Overall, we conclude that these alternative measurement strategies yield divergent assessments of change, depending on the type of treatment a patient receives and, to some extent, what is being measured. PMID- 7536869 TI - [Angiogenesis is induced by growth factors. The molecular mechanisms are not yet known]. AB - Angiogenesis, the formation of new vessels from the vessel bed, is crucially involved in physiological and pathological processes. Angiogenesis may be regarded as a series of cellular events--e g, basement membrane degradation, and the migration, proliferation and differentiation of endothelial cells. Several different growth factors trigger one or more of these events both in vivo and in vitro, though the underlying mechanisms are still largely unknown. Elucidation of the function of growth factor receptors in angiogenesis, and their propagation of intracellular signals eventually resulting in angiogenesis is an important task for research. In all likelihood, such studies will give rise to clinically useful tools for the regulation of angiogenesis, which might be of importance in such disease as diabetes and cancer. PMID- 7536870 TI - Identification of conserved antigenic components for a cytotoxic T lymphocyte inducing vaccine against malaria. AB - Several cellular and humoral mechanisms probably play a role in natural immunity to Plasmodium falciparum malaria, but the development of an effective vaccine has been impeded by uncertainty as to which antigens are targeted by protective immune responses. Experimental models of malaria have shown that cytotoxic T lymphocytes (CTL) which kill parasite-infected hepatocytes can provide complete protective immunity against certain species of Plasmodium in mice, and studies in The Gambia have provided indirect evidence that CTL play a protective role against P falciparum in humans. By using an HLA-based approach, termed reverse immunogenetics, we have previously identified peptide epitopes for CTL in liver stage antigen-1 and the circumsporozoite protein of P falciparum. We have extended this work to identify CTL epitopes for HLA class I antigens that are found in most individuals from Caucasian and African populations. Most of these epitopes are in conserved regions of P falciparum. CTL peptide epitopes were found in a further two antigens, thrombospondin-related anonymous protein and sporozoite threonine and asparagine rich protein, indicating that a subunit vaccine designed to induce a protective CTL response may need to include parts of several parasite antigens. However, CTL levels in both children with malaria and in semi-immune adults from an endemic area were low suggesting that boosting these low levels by immunisation might provide substantial or even complete protection against infection and disease. PMID- 7536871 TI - Transient hypoxaemia during neutrophil recovery in febrile patients. AB - In previous anecdotal reports, treatment with granulocyte colony-stimulating factor has been associated with pulmonary toxicity. In 35 consecutive admissions for chemotherapy-induced febrile neutropenia, transient hypoxia occurred in 12. 10 of the 12 followed treatment with filgrastim to induce neutrophil recovery. There was no consistent association with cytotoxic regimen. PMID- 7536872 TI - Harnessing cytotoxic T lymphocytes for vaccine design. PMID- 7536873 TI - Beta-hCG inhibits Kaposi's sarcoma. PMID- 7536874 TI - Hepatitis C transmission associated with intravenous immunoglobulins. PMID- 7536875 TI - Hepatitis C transmission associated with intravenous immunoglobulins. PMID- 7536876 TI - Screening for biliary atresia. PMID- 7536877 TI - Opioid analgesia in uraemic patients. PMID- 7536878 TI - Prostate-specific antigen and lack of specificity for prostate cells. PMID- 7536879 TI - Substance P modulating effect on the binding capacity of hamster Leydig cell LH receptors. AB - The influence of Substance P was studied on the binding characteristics of LH receptors in purified Leydig cells collected from golden hamsters kept under natural long or short days. Substance P exerted a differential effect on the binding capacity of LH receptors. A significant increase in Bmax was estimated in Leydig cells obtained from young hamsters living under long days. In contrast, Substance P reduced the number of the LH binding sites in Leydig cell cultures prepared from adult hamsters housed under short-day conditions. PMID- 7536881 TI - Contribution of bradykinin to heat-induced substance P release in the hind instep of rats. AB - To investigate the possible contribution of the bradykinin (BK) system to heat induced substance P (SP) release from the peripheral endings of primary afferent nerves, we used the high molecular weight (HMW) and low molecular weight (LMW) kininogen-deficient rat strain (Brown Norway-Katholiek, B/N-Ka) and the normal rat strain (Brown Norway-Kitasato, B/N-Ki). We found that immersion of the paw of B/N-Ki rats in water of 47 degrees C for 20 min led to significant increases of BK, SP and Evans blue extravasation in the s.c. perfusate, and that similar treatment resulted in significantly lower levels in B/N-Ka rats. Local application of BK (10(-4) M) to the s.c. perfusate and intra-arterial infusion of BK 10(-5) mol/kg) increased Evans blue extravasation and SP release evoked by heat stimulation, respectively, in B/N-Ka rats to similar levels to those in B/N Ki rats after heat-stimulation without BK treatment. These results indicate that BK released into the extravascular space by noxious stimulation is involved in SP release from the peripheral endings of capsaicin-sensitive primary sensory neurons. PMID- 7536880 TI - Phosphorylation and adrenergic chronotropism and inotropism in guinea pig cardiac muscles. AB - A question whether phosphorylation is involved in adrenergic effects on cardiac tissues has long been a matter of dispute. We examined whether phosphorylation is involved in adrenergic chronotropism and inotropism in excised cardiac muscles from guinea pigs. KT5720, a specific inhibitor of A-kinase, abolished the late phase of adrenergic chronotropism. Okadaic acid, an inhibitor of phosphoprotein phosphatases, and IBMX (1-methyl, 3-isobutylxanthine), a phosphodiesterase inhibitor, potentiated the chronotropism. The adrenergic inotropism was influenced neither by KT5720, okadaic acid, nor by IBMX. The specific beta 1 adrenergic agonist, denopamine, showed actions similar to adrenaline and susceptibility to the inhibitors. Adrenaline of 10 microM showed a chronotropic time course consisting of early and late components. We concluded that only the late component results from phosphorylation, and its early one and the inotropism is entirely independent of phosphorylation. PMID- 7536882 TI - Differences in eucaryotic cell binding of Pseudomonas. AB - The lungs of cystic fibrosis (CF) patients are frequently chronically colonized by Pseudomonas aeruginosa. Recently there has been an increase in colonization by another pathogen Pseudomonas cepacia, which can cause a rapid decline in clinical condition or death of the patient. The nature of the factor(s) which predispose CF patients to colonization by one or both of these opportunistic pathogens is unknown. It has been suggested that the genetic defect in CF patients results in an increase in the number of epithelial cell receptors available to P. aeruginosa in the lung, thus rendering CF patients more susceptible to bacterial colonization than non-CF individuals. In this study, we have examined adherence of several strains of P. aeruginosa and P. cepacia to a variety of continuous cell lines, as well as primary cultures of CF and non-CF nasal polyp cells. The results suggested that there may be a decrease in the number of receptors available to both strains of Pseudomonas on cells of canine origin compared to human cells. Both strains appear to use pili as the primary adhesin, but there is also evidence that non-pilus adhesins contribute significantly to eucaryotic cell binding. P. cepacia exhibited microcolony formation on all cell types, which is typical of the localized adherence pattern characteristic of the enteropathogenic Escherichia coli. However, we were unable to demonstrate, with either P. cepacia or P. aeruginosa, a significant increase in adherence to CF compared to non-CF nasal polyp cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536883 TI - Amplification and analysis of promoter region of insulin receptor gene in a patient with leprechaunism associated with severe insulin resistance. AB - A patient with leprechaunism associated with severe insulin resistance was studied to identify the molecular and genetic basis for insulin resistance. Insulin binding and surface labeling of transformed lymphocytes prepared from the patient showed a significantly decreased insulin receptor number on the cell surface. Southern blot analysis of the insulin receptor gene showed no evidence of large insertions or deletions. Furthermore, direct sequencing of all 22 exons and exon-intron junctions of the insulin receptor gene failed to show any missense mutations, nonsense mutations, or mutations at exon-intron junctions. However, Northern blot analysis indicated significantly decreased insulin receptor mRNA expression in the patient's cells. Moreover, restriction endonuclease digestion of the amplified cDNA suggested that the expression levels of one allele were less efficient than the other. These findings suggested that the regulatory region of the insulin receptor gene might have abnormalities. Therefore, we examined the 5' flanking region of the insulin receptor gene. Southern blot analysis showed no major deletions or insertions between positions 1,823 and -2 relative to the translation initiation site. A 5' flanking region of the insulin receptor gene spanning positions -881 approximately +7 was amplified by polymerase chain reaction (PCR) and introduced into a reporter plasmid carrying the human growth hormone (hGH) gene. The nucleotide sequence of the amplified fragment showed two polymorphic sites at positions -603 and -500 in the patient, as well as in normal subjects. No other abnormal sequence was found in the patient. Promoter activity measured by hGH expression in transfected mouse L cells was not influenced by the polymorphism at position -603 located in a cluster of GC boxes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7536884 TI - Effect of substance P on basal and thyrotropin-releasing hormone-stimulated thyrotropin release in humans. AB - To test the possible effects of intravenous administration of substance P (SP) on basal and thyrotropin-releasing hormone (TRH)-stimulated thyrotropin (TSH) release, SP was infused alone (0.5 or 1.5 pmol/kg-1/min-1 for 60 minutes) or after TRH (20 or 400 micrograms in an intravenous bolus) in 21 normal male subjects (aged 26 to 36 years) and in 18 normal women (aged 25 to 32 years). Women were studied during follicular (day 6 to 8) and luteal (day 21 to 23) phases of following regular menstrual cycles. In addition, plasma cortisol levels during SP infusion were measured. In agreement with previous findings, significant increments in plasma cortisol levels were observed in men and women when the higher (1.5 pmol/kg-1/min-1) but not the lower (0.5 pmol/kg-1/min-1) amount of SP was administered. In contrast, in both men and women basal and TRH (20 or 400 mg)-induced TSH releases were not modified by SP at any tested amount. Results in the follicular and luteal phase were similar. These data suggest that in normal men and women plasma SP is not involved in the control of TSH release, at least not outside the blood-brain barrier. PMID- 7536885 TI - Recombinant endothelial nitric oxide synthase: post-translational modifications in a baculovirus expression system. AB - Nitric oxide synthesized by the endothelial isoform of nitric oxide synthase (ecNOS) is importantly involved in the homeostatic control of blood pressure and platelet aggregation. The different members of the nitric oxide synthase protein family have several biochemical features in common but serve distinct physiological functions and are the products of distinct genes. The ecNOS is further distinguished by its subcellular distribution in the endothelial cell membrane, and the enzyme undergoes several post-translational modifications, including myristoylation, palmitoylation, and phosphorylation. Overall, however, the ecNOS has remained less well characterized because of the challenges involved in isolating sufficient quantities of this membrane-associated protein from native or cultured endothelial cells. In this report, we describe the purification and characterization of ecNOS expressed in a heterologous system in recombinant baculovirus-infected insect Sf9 cells. Recombinant ecNOS is targeted to the Sf9 cell membrane and comprises approximately 10% of the total cellular protein, allowing purification to homogeneity in a single-step procedure to yield a stable protein that retains the essential features of the native enzyme. Using biosynthetic labeling and immunoprecipitation, we show that recombinant ecNOS is myristoylated, palmitoylated, and phosphorylated when expressed in insect Sf9 cells. The interpretation of structural and enzymological studies of recombinant ecNOS will be facilitated by the apparent fidelity of its biosynthesis and post translational modification in insect Sf9 cells. PMID- 7536886 TI - Characterization of the interaction of diacylpiperazine antagonists with the human neurokinin-1 receptor: identification of a common binding site for structurally dissimilar antagonists. AB - We recently described a novel series of diacylpiperazine antagonists of the human neurokinin (NK)-1 receptor. The diacylpiperazine compounds are structurally dissimilar from previously described NK-1 antagonists. L-161,664 [1-(N,N diphenylaminocarbonyl)-4-(N',N'-di-n-pentylaminocarbony l) piperazine-2 diethylaminopropylcarboxamide] inhibits 125I-substance P binding to the human NK 1 receptor with an IC50 of 43 +/- 21 nM but has 50-fold and 200-fold lower affinity for the human NK-2 and NK-3 receptors, respectively. L-161,664 inhibits substance P-stimulated inositol monophosphate accumulation in Chinese hamster ovary cells expressing the human NK-1 receptor by increasing the EC50 for substance P but not its maximal effect. The compound decreases the apparent affinity of the NK-1 receptor for 125I-substance P and does not alter the rate of dissociation of 125I-substance P from the receptor. These data indicate that L 161,664 is a potent and selective competitive antagonist of the human NK-1 receptor. L-161,664 has reduced affinity for mutants of the NK-1 receptor in which alanine has replaced Gln-165 in transmembrane helix 4, His-197 in helix 5, His-265 in helix 6, or Tyr-287 in helix 7. Similarly, a novel series of acyclic 2 benzhydryl-2-aminoethyl ethers that we have recently shown to be competitive NK-1 receptor antagonists have reduced affinity for the Q165A. H197A, and H265A mutant receptors. These residues have been shown to be important for binding of quinuclidine, tryptophan benzyl ester, and perhydroisoindole antagonists to the receptor. Analysis of the interaction of structural analogs of L-161,664 with the Q165A mutant receptor suggests that this residue interacts with the 2 diethylaminopropylcarboxamide side chain of L-161,664. Thus, even though the diacylpiperazine antagonists are structurally dissimilar from other classes of antagonists described to date, these data suggest that a common antagonist binding site that accomodates much structural diversity is present in the human NK-1 receptor. Furthermore, these data, combined with those obtained from medicinal chemistry approaches, suggest a minimum pharmacophore map for the interaction of these diverse ligands with the NK-1 binding site. PMID- 7536887 TI - Effects of chromium on basal and insulin-induced tyrosine phosphorylation in H4 hepatoma cells: comparison with phorbol-12-myristate-13-acetate and sodium orthovanadate. AB - Chromium, in its various forms, is recognized both as a human carcinogen and as a nutrient essential in glucose homeostasis. Although the genotoxicity of this element is associated with its carcinogenic properties, the manner in which chromium mediates its epigenetic effects on cells, including its ability to potentiate insulin action, is not known. In the current studies, Western blotting with antiphosphotyrosine antibodies was used to study the effects of chromium on protein tyrosine phosphorylation in intact H4 rat hepatoma cells. Treatment of cells with hexavalent chromium [Cr(VI)] was found to induce the tyrosine phosphorylation of three prominent sets of proteins, having median molecular masses of 210, 125, and 87 kDa. Cr(VI) pretreatment also inhibited the insulin induced tyrosine phosphorylation of the major substrate of the insulin receptor kinase, insulin receptor substrate-1, and its subsequent association with the 85 kDa regulatory subunit (p85) of phosphatidylinositol 3'-kinase. Furthermore, Cr(VI) was found to alter the pattern of other p85-binding (insulin-induced) phosphoproteins that were distributed throughout the soluble and particulate fractions of cells. Virtually all of the alterations in basal and insulin-induced phosphorylations associated with Cr(VI) treatment were also observed in cells treated with the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate. However, the effects of Cr(VI) were determined to be independent of PKC activity, because they were sustained in PKC-depleted cells. The pattern of phosphoproteins induced by Cr(VI) also had similarities to the pattern generated in response to the phosphatase inhibitor sodium orthovanadate. However, several specific differences, including the ability of vanadate to increase insulin receptor beta subunit autophosphorylation [i.e., an effect not observed with Cr(VI)], indicated that these agents modulate phosphorylation by distinct mechanisms. The ability of Cr(VI) to alter the phosphorylation state of key regulatory proteins in a manner similar to that of other biologically active agents suggests a mechanism by which this element can modulate the growth and metabolism of cells. PMID- 7536889 TI - Novel potent and selective inhibitors of inducible nitric oxide synthase. AB - We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6 dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases. PMID- 7536888 TI - Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction. AB - Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction. PMID- 7536890 TI - [Spectrophotometric complexes of nucleic acids with pyronine G dyes as a test for radiation damage of DNA]. AB - The spectrophotometric titration curves of the complexes of polynucleotide matrix with pyronine G are highly specific depending on the type and conformation of polyelectrolyte (native and denatured DNA, polyribonucleotides, tRNA). It is found that DNA from the liver of experimental animals of different age, permanent inhabitants of the Chernobyl zone, forms complexes with RNA. A method for testing the defects in a DNA secondary structure have been developed based on comparative analysis of the spectra for the complexes of different nucleic acids with dyes and relative curves of spectrophotometric titration. PMID- 7536891 TI - [An important role for weak interactions during the recognition of long DNA and RNA molecules by enzymes]. AB - Recognition of small ligands by the enzymes occurs usually due to the strong specific contacts, such as hydrogen bonds, electrostatic and stacking interactions. A complex formed by long DNA or RNA molecules and site-specific enzymes includes, as a rule, not all mononucleotide units of an enzyme binding cleft. Recognition is based preferentially on the strong interaction of the enzymes with certain structural elements or specific nucleotides of the sequences. The weak hydrophobic of van-der-Waals (electrostatic) interactions usually are not very important in enzyme interaction with small ligands or definite units of long nucleic acid molecules and do not exceed the Kd estimation limits. However, some enzymes catalyzing reactions of nucleic acid conversion independently of their structures, most likely, interact with all mononucleotide units within the protein molecules. In this case, due to the additivity of the free energies, when the length of the recognized substrate is about 10-30 nucleotides, the contribution of the weak interactions to the substrate affinity may be about 4-7 orders of magnitude. These interactions become stronger than any other. New approaches for studying the weak interactions were analyzed. An attempt is made to prove the extremely important role of DNA/RNA weak interactions with the enzymes in their recognition and conversion with reference to some important enzymes participating in replication and repair processes. PMID- 7536892 TI - Determination of the active site of CD59 with synthetic peptides. AB - CD59 inhibits the formation of membrane attack complex (MAC) of human complement by binding to C8 and C9 in the nascent membrane attack complex and inhibiting C9 binding to C8 in C5b-8 and C9 polymerization. Considering five disulfide bridges of CD59, we divided the molecule into two portions and synthesized the two peptides. One represented an amino-terminal half, P1-41, consisting of residues 1 41, while another represented a carboxyl-terminal half, P42-77, consisting of residues 42-77. P1-41 inhibited the MAC formation much more strongly than P42-77, indicating that the amino-terminal half contained the active site. We further synthesized P4-18 that consisted of residues 4-18 and P19-41 that consisted of residues 19-41. The activity of P4-18 was less than that of P19-41. Surprisingly, P19-41 showed higher activity than P1-41 and was comparable to urine CD59. Residues 19-41 were further divided into two portions: P20-25 which consisted of residues 20-25 and P27-38 which consisted of residues 27-38. Although their activities were significantly less than the activity of P19-41, P27-38 showed higher activity than P20-25. Residues 27-38 were further divided into three portions: P27-32 which consisted of residues 27-32, P30-34 which consisted of residues 30-34 and P33-38 which consisted of residues 33-38. When these peptides were assayed for the activities, all of them showed significant activities, even though they needed 10-fold more concentrations than P19-41. These data suggest that the portion made up of residues 27-38 is the active site constituting the binding site to C8 and C9. PMID- 7536893 TI - Is hydropathic complementarity involved in antigen-antibody binding? PMID- 7536894 TI - Ribozymes. Exploration by lamp light. PMID- 7536895 TI - Programmed cell death and Bcl-2 protection in very low oxygen. AB - Programmed cell death (PCD) is a fundamental feature of animal cells, but the mechanism remains unknown. Similarly, the Bcl-2 oncoprotein can suppress PCD in a variety of cell types and circumstances, but it is not known how it does so. It has been suggested that PCD involves the generation of reactive oxygen species (ROS) and that Bcl-2 protects against PCD by inhibiting the generation or action of ROS. To determine whether ROS are required for PCD, we cultured cells in a near-anaerobic atmosphere where the generation of ROS would be expected not to occur, or at least to be greatly reduced. We find that these conditions inhibit PCD induced by ROS-generating agents but do not inhibit PCD induced by other means. Furthermore, we show that Bcl-2 can protect cells from PCD in these anaerobic conditions. These results suggest that ROS are not required for PCD, and that Bcl-2 protects against PCD in ways that do not depend on the inhibition of ROS production or activity. PMID- 7536896 TI - Motor-neuron diseases. Interfering with the runners. PMID- 7536898 TI - Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. AB - Amyotrophic lateral sclerosis (ALS) is a degenerative disease of motor neurons, characterized by depositions of neurofilaments in the perikarya and proximal axons. The pathogenesis of ALS remains poorly understood, but two lines of evidence suggest that neurofilament accumulation may play a causal role. First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration and, second, variant alleles of the neurofilament heavy-subunit gene (NF-H) have been found in some human ALS patients. To investigate how disorganized neurofilaments might cause neurodegeneration, we examined axonal transport of newly synthesized proteins in mice that overexpress the human NF-H gene. We observed dramatic defects of axonal transport, not only of neurofilament proteins but also of other proteins, including tubulin and actin. Ultrastructural analysis revealed a paucity of cytoskeletal elements, smooth endoplasmic reticulum and especially mitochondria in the degenerating axons. We therefore propose that the neurofilament accumulations observed in these mice cause axonal degeneration by impeding the transport of components required for axonal maintenance, and that a similar mechanism may account for the pathogenesis of ALS in human patients. PMID- 7536899 TI - Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. AB - Arrest of cell division is a prerequisite for cells to enter a program of terminal differentiation. Mitogenesis and cytostasis of neuronal cell precursors can be induced by the same or by different growth or trophic factors. Response of PC12 cells to nerve growth factor (NGF) involves a proliferative phase that is followed by growth arrest and differentiation. Here we present evidence that the cytostatic effect of NGF is mediated by nitric oxide (NO), a second messenger molecule with both para- and autocrine properties that can diffuse freely and act within a restricted volume. We show that NGF induces different forms of nitric oxide synthase (NOS) in neuronal cells, that nitric oxide (NO) acts as a cytostatic agent in these cells, that inhibition of NOS leads to reversal of NGF induced cytostasis and thereby prevents full differentiation, and that capacity of a mutant cell line to differentiate can be rescued by exogenous NO. We suggest that induction of NOS is an important step in the commitment of neuronal precursors and that NOS serves as a growth arrest gene, initiating the switch to cytostasis during differentiation. PMID- 7536900 TI - Involvement of an ICE-like protease in Fas-mediated apoptosis. AB - Fas is a type-I membrane protein that transduces an apoptotic signal. Binding of Fas ligand or agonistic anti-Fas antibody to Fas kills the cells by apoptosis. Studies in the nematode Caenorhabditis elegans have suggested that proteases such as interleukin-1 beta-converting enzyme (ICE) or the product of the C. elegans cell-death gene ced-3 are involved in apoptotic signal transduction. The activity of ICE can be inhibited by the product of crmA, a cytokine-response modifier gene encoded by cowpox virus. We report here that expression of crmA inhibits cytotoxicity induced by anti-Fas antibody or tumour necrosis factor (TNF). We have found a specific ICE inhibitor tetrapeptide (acetyl-Tyr-Val-Ala-Asp chloromethylketone) that also prevents apoptosis induced by anti-Fas antibody. These results suggest an involvement of an ICE-like protease in Fas-mediated apoptosis and TNF-induced cytotoxicity. PMID- 7536897 TI - Disruption of imprinting caused by deletion of the H19 gene region in mice. AB - The imprinted H19 gene, which encodes an untranslated RNA, lies at the end of a cluster of imprinted genes in the mouse. Imprinting of the insulin-2 and insulin like growth factor 2 genes, which lie about 100 kilobases upstream of H19, can be disrupted by maternal inheritance of a targeted deletion of the H19 gene and its flanking sequence. Animals inheriting the H19 mutation from their mothers are 27% heavier than those inheriting it from their fathers. Paternal inheritance of the disruption has no effect, which presumably reflects the normally silent state of the paternal gene. The somatic overgrowth of heterozygotes for the maternal deletion is attributed to a gain of function of insulin-like growth factor 2, rather than a loss of function of H19. PMID- 7536901 TI - Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. AB - The Fas/APO-1 receptor is one of the major regulators of apoptosis. We report here that Fas/APO-1-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-1 beta-converting enzyme (ICE), which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/APO-1 rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/APO-1-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor CrmA or an antisense ICE construct, substantially suppressed Fas/APO-1-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/APO 1-mediated cell death. PMID- 7536902 TI - Cerebral ocular Whipple's disease: a 62-year odyssey from death to diagnosis. AB - A 47-year-old white man with dementia, supranuclear ophthalmoplegia, and myoclonic ocular and facial jerks died in 1931. The case report in 1936 by Ford and Walsh diagnosed encephalitis. In 1993, we made a clinical diagnosis of Whipple's disease on the basis of the 1936 publication. We restudied the pathologic material and found, in addition to extensive encephalitis, PAS positive material in only the eye, brain, spinal cord, and pituitary. Electron microscopy demonstrated free and intracytoplasmic microorganisms in the eye and brain. We review the history of cerebral ocular Whipple's disease and the implications from this case, which occurred before the development of antibiotics. PMID- 7536903 TI - Cascade filtration in Refsum's disease. PMID- 7536904 TI - Characterisation of potential regulatory elements within the rat preprotachykinin A promoter. AB - The rat preprotachykinin-A (rPPT-A) gene encodes the precursor of several tachykinin neuropeptides including substance P. Previous studies have demonstrated the presence of multiple DNA sequences important for directing expression of the rPPT-A gene in dorsal root ganglion neurons within a region of the promoter spanning nucleotides -865 and -47. In order to identify potential cis acting elements, we have carried out DNase 1 footprinting analysis using a series of constructs containing fragments from this region of the promoter. This study has defined three potential AP-1 complex interactions, two E box binding protein interactions and two dG rich elements, which are potentially bound by complexes related to AP-2 or Sp1 in this region of the promoter. PMID- 7536905 TI - Induction of calcium-independent nitric oxide synthase activity in cultured cerebellar granule neurons. AB - Cultured cerebellar granule neurons were assayed for nitric oxide synthase (NOS) activity by measuring the conversion of L-arginine to L-citrulline. Granule neurons expressed constitutive NOS activity which was calcium-dependent. Treatment of neuron cultures for 24 h with the combined stimulation of IFN-gamma plus IL-1 beta, TNF-alpha and LPS induced NOS activity by 87-fold which was calcium-independent. We conclude that cultured cerebellar granule neurons can express both the constitutive and inducible forms of NOS activity. PMID- 7536906 TI - Currents caused by the spontaneous release of quanta of acetylcholine onto chromaffin cells in guinea-pig adrenal medulla. AB - Membrane potentials were recorded from chromaffin cells in isolated bisected adrenal glands from guinea-pigs. Spontaneous excitatory synaptic potentials (SESPs) were recorded whose frequency was increased following brief (up to 10 s) periods of presynaptic nerve stimulation at 10-30 Hz. The single electrode voltage-clamp method was used to record the currents underlying all but the largest SESPs. Spontaneous excitatory synaptic currents (SESCs) decayed according to a single exponential with a time constant of about 8 ms at 30 degrees C. Thus the neuronal nicotinic receptor-channels giving rise to SESCs in chromaffin cells are probably very similar to those that are opened by quanta of acetylcholine in sympathetic ganglion cells. PMID- 7536907 TI - Catalase activity as a predictor of amniotic fluid culture results in preterm labor or premature rupture of membranes. AB - OBJECTIVE: To evaluate catalase activity as a rapid predictor of microbial invasion of amniotic fluid (AF). METHODS: The study population consisted of 74 patients before 36 weeks' gestation with preterm labor or premature rupture of membranes (PROM). Subjects were excluded if there was evidence of clinical chorioamnionitis or fetal distress at admission. Amniocentesis was done within 24 hours of admission, and the AF was cultured for aerobic and anaerobic bacteria and for Mycoplasma species. All AF samples were Gram stained, and slides were examined by microbiology technologists. Amniotic fluid catalase activity was measured immediately after amniocentesis using a commercially available kit. The sensitivity of the Gram stain and catalase activity were compared using McNemar exact test. RESULTS: Amniotic fluid cultures were positive in 12 of 37 (32%) patients presenting with preterm labor and in 21 of 37 (56%) patients with PROM. Catalase activity was significantly more sensitive than Gram stain in detecting positive AF cultures in cases of PROM (P < .001) and preterm labor (P < .04). CONCLUSION: Catalase activity is a simple, rapid test that is useful in identifying subclinical intra-amniotic infection in patients with preterm labor or PROM. PMID- 7536908 TI - Prevention of carcinoma in situ of human papillomavirus type 16-immortalized human endocervical cells by retinoic acid in organotypic raft culture. AB - OBJECTIVE: To determine the effect of retinoic acid on the development of severe dysplasia or carcinoma in situ from endocervical cells containing human papillomavirus (HPV) type 16. METHODS: Two independent lines of HPV 16 immortalized endocervical cells were reconstructed into two squamous epithelial tissues using the organotypic raft culture system to examine the differentiated phenotype. The effect of retinoic acid on dysplastic morphology of differentiation of the epithelia was examined by light microscopy of stained sections and electron microscopy. The endocervical cell type cytokeratin expression pattern was determined by indirect immunofluorescence using specific monoclonal antibodies. Ribonucleic acid expression of the HPV 16 E7 oncogene was examined by in situ hybridization. RESULTS: Untreated HPV 16-immortalized endocervical cells were reconstructed into squamous dysplastic lesions resembling carcinoma in situ observed in women. Retinoic acid-treated rafts formed epithelia composed of two to three cell layers of columnar-like cells resembling simple epithelium of the endocervix. Electron microscopy and cytokeratin expression patterns confirmed the histology of a differentiated endocervical phenotype after treatment with retinoic acid. Expression of HPV 16 E7 was modestly lower in treated epithelia, preferentially in basal cells. CONCLUSION: Retinoic acid prevents the histology and cytokeratin differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells. Because the epithelia closely mimic HPV 16-containing severe dysplasias and native endocervical epithelium in women, this immortalized endocervical cell-raft system may be useful as a model to assess the efficacy of agents such as retinoic acid for preventing progression of these lesions to malignant cervical carcinoma. PMID- 7536909 TI - Complications of porous spherical orbital implants. AB - PURPOSE: To determine the complications observed with using porous spherical orbital implants (hydroxyapatite and porous polyethylene) and the factors leading to their occurrence. METHODS: A total of 101 cases of porous spherical orbital implantation by five ophthalmic surgeons were reviewed retrospectively. The demographic data, diagnosis, prior surgery, type and technique of surgery, implant characteristics and prosthesis fitting were described in patients with complications. RESULTS: Eleven of the 101 patients had implant exposure. There were six male (1 with bilateral involvement) and four female patients, ranging in age from 2 to 71 years. Preoperative diagnosis included trauma in five patients, nontrauma in five, and tumor in one. Seven had prior eye surgeries. Three patients underwent evisceration, whereas eight underwent enucleation. Eight hydroxyapatite and three porous polyethylene implants were used with diameters of 16 to 20 mm. Three were unwrapped, six were wrapped in sclera, and two were wrapped in preserved fascia. Exposures, which generally occurred within 1 year, were grouped into small (1-5 mm), medium (6-10 mm), and large (> 10 mm). One delayed case occurred after drilling. Small stable exposures were managed conservatively. Larger exposures were managed either by implant revision or replacement. All patients were fit ultimately with a prosthesis. Histopathologic findings of explanted spheres showed fibrovascularization limited to the periphery with moderate inflammatory reaction. CONCLUSION: Complications were significantly higher in cases of eviscerations than enucleations. Complications occurred in implants either unwrapped or wrapped in homologous grafts. None of the autologous wrapping had exposure. Secondary procedures may initiate exposure when fibrovascular status of implant is inadequate. PMID- 7536910 TI - Indocyanine green videoangiographic findings in detachment of the retinal pigment epithelium. AB - BACKGROUND: Several forms of retinal pigment epithelial detachment have been reported. The authors used indocyanine green (ICG) videoangiography, which is useful to study the choroidal vasculature and Bruch membrane, to study pigment epithelial detachments. METHODS: Ninety-eight pigment epithelial detachments in 75 eyes were classified based on the appearance of choroidal neovascular membranes or late phase findings of ICG videoangiography done at the initial examination. The authors also followed the evolution of 51 such detachments not associated with choroidal neovascularization (CNV). RESULTS: Sixty-four pigment epithelial detachments without CNV were divided into five groups. Among eyes with pigment epithelial detachments that showed intense hyperfluorescence, all except one of the patients had both eyes involved and had several pigment epithelial detachments, sometimes with exudative retinal detachments. Weak hyperfluorescence was observed more often in younger patients. During follow-up of eyes with pigment epithelial detachments that showed irregular hypofluorescence, a neovascular membrane developed in one eye, microrips developed in four eyes and retinochoroidal folds in one eye. Most eyes that showed irregular hyperfluorescence developed atrophy of the retinal pigment epithelium. In 34 pigment epithelial detachments with CNV, either irregular hypofluorescence or absence of fluorescence was observed in areas that corresponded to the pigment epithelial detachment. CONCLUSION: The intense hyperfluorescence is thought to be due to the accumulation of protein-rich fluid within the pigment epithelial detachment. Most pigment epithelial detachments that showed weak fluorescence probably represent variants of central serous choroidopathy. Pigment epithelial detachments that showed irregular hypofluorescence or hyperfluorescence were associated with age-related macular degeneration, and the former was correlated closely with CNV. Close follow-up therefore is recommended for eyes with pigment epithelial detachments that show irregular hypofluorescence. PMID- 7536911 TI - Retinal distortion and cotton-wool spots associated with epiretinal membrane contraction. AB - PURPOSE AND BACKGROUND: Several articles on surgery for macular pucker have noted the presence of intraretinal white spots, presumably cotton-wool spots, associated with epiretinal membranes. It was proposed that membrane contraction and resultant nerve fiber layer distortion might lead to blockage of axoplasmic flow and thus a cotton-wool spot. To see whether the observations supported this concept, the authors studied patients with epiretinal membranes associated with intraretinal white spots involving the center of the fovea, where there are normally no retinal vessels and the only axons are in Henle fiber layer. METHODS: Four patients with epiretinal membranes associated with prominent areas of intraretinal whitening involving the center of the fovea were examined before and after surgery. Observations and measurements were made from projected fundus photographs. RESULTS: The intraretinal white spots, which appeared preoperatively to lie in the center of the fovea, proved to have been pulled in from more peripheral retina by the epiretinal membrane. These white areas were associated with surprisingly great distortion of the inner retina. Stretching of the inner retina seemed to play a larger role in the retinal distortion than full-thickness retinal folding. CONCLUSION: The appearance and time course of these intraretinal white lesions were consistent with cotton-wool spots caused by mechanical disruption of axonal flow. The marked retinal stretching and distortion of the innermost layers of the retina were the most striking findings in this study and also seemed consistent with the concept that membrane contraction and resultant nerve fiber layer distortion might lead to blockage of axoplasmic flow and thus a cotton-wool spot. PMID- 7536912 TI - Localization of parasite antigens in Cryptosporidium parvum-infected epithelial cells using monoclonal antibodies. AB - An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immunofluorescence antibody test monoclonal antibody (MAb) 1B5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite. PMID- 7536913 TI - [Epitopes of the envelope of the hepatitis B virus: a structural approach]. AB - Hepatitis B is a major public health problem. More than 300 million people are chronically infected by the virus. During infection very large quantities of complete virions and empty envelopes, consisting of spherical or filamentous lipoprotein particles, are present in the blood. DNA genome coding for envelopes is divided into three domains, preS1, preS2 and S. All available data suggest that the preS1 and preS2 products are exposed at the surface of the virions. These proteins are more immunogenic than S in terms of in vivo antibody response and the number of epitopes identified. The three dimensional mapping of antigenic sites of the HBV will provide important strategic information for vaccine development and identification of targets for immunorecognition or immunoregulation of the disease. PMID- 7536914 TI - Perinatal and neonatal issues of substance abuse. AB - Substance abuse during pregnancy can be teratogenic for the fetus and can cause decreased growth parameters in the newborn infant. Short-term and long-term neurobehavioral problems have been documented also in babies born to substance abusing mothers. The problem of substance abuse during pregnancy and its effects on the fetus is unique in medicine in that it is 100% preventable. Physicians will need to take an active role in leading society to take action in preventing substance abuse during pregnancy, and emphasis on this action should occur long before the woman becomes pregnant. Until medical schools and residency programs take responsibility for teaching the importance of preventing substance abuse and of identifying the substance-abuser, drug and alcohol use will continue to exact its tragic toll on future generations. PMID- 7536915 TI - Adverse outcomes of bacterial meningitis in school-age survivors. AB - OBJECTIVE: To determine the outcomes of bacterial meningitis in school-age survivors. DESIGN: Prospective cohort study. SETTING: Teaching pediatric hospital. CHILDREN: During 1983 through 1986, 158 meningitis survivors, ages 3 months to 14 years, treated at a single center were enrolled. Between 1991 and 1993, 130 children, 82% of the original cohort, were evaluated at a mean age of 8.4 years and a mean of 6.7 years after their meningitis. OUTCOME MEASURES: Blinded neurologic, neuropsychologic, audiologic, behavior, and socio-demographic assessments were compared with those from grade- and sex-matched control children. Multivariate analyses adjusted for age at testing and socio-demographic variables. RESULTS: There was a systematic increase in risk of abnormality or poorer functioning for children with meningitis, compared with control children, across all categories tested, which was significant for fine motor function, Intelligence quotient (IQ) scores, and tests of school behavior, neuropsychologic function, and auditory figure-ground differentiation. Eleven children who had experienced meningitis (8.5%) had major deficits (IQ < 70, seizures, hydrocephalus, spasticity, blindness, or severe to profound hearing loss); a further 24 (18.5%) cases and 14 (10.8%) control children had minor deficits (IQ 70 to 80, inability to read, mild to moderate hearing loss, abnormalities in speech discrimination, or school behavior problems). Overall, children who had meningitis were at greater risk (26.9%) for disability. Children with acute neurologic complications had more adverse outcomes than those with uncomplicated meningitis and control children (39% vs 18% vs 11%, respectively). CONCLUSIONS: One in four school-age meningitis survivors has either serious and disabling sequelae or a functionally important behavior disorder, neuropsychologic or auditory dysfunction adversely affecting academic performance. As a group, survivors function less well than their classroom peers, and risk is greatest for, but not confined to, those who had acute neurologic complications. All survivors require careful follow-up, at least until school age. PMID- 7536917 TI - Ribavirin for hepatitis C infection. PMID- 7536916 TI - Cat scratch disease in two children presenting with fever of unknown origin: imaging features and association with a new causative agent, Rochalimaea henselae. AB - OBJECTIVE: To report the clinical course, imaging findings, and method of diagnosis of two patients with systemic manifestations of cat scratch disease, presenting with fever of unknown origin. DESIGN: Case study. PATIENTS: Two children with fever of unknown origin who had multiple lesions in the liver and spleen, shown on ultrasound, computed tomography, and magnetic resonance imaging. Initial diagnoses were Kawasaki disease (case 1) and metastatic neuroblastoma (case 2). RESULTS: Biopsy material showed granulomatous hepatitis in both patients. The diagnoses were confirmed by positive assays for Rochalimaea henselae, currently thought to be the causative agent of cat scratch disease. CONCLUSION: Cat scratch disease presenting as fever of unknown origin is now well described and can be more readily diagnosed because of the availability of new serologic assays, as well as polymerase chain reaction assays for R henselae DNA in tissue specimens. PMID- 7536918 TI - Are blood tests of value in the primary assessment and resuscitation of patients in the A&E department? AB - Over a three month period an audit of the use of emergency blood tests was carried out in the accident and emergency (A&E) department at Southampton General Hospital. Few blood tests were found to be useful in the primary assessment and treatment of patients by the attending doctors. Only cross-matching of blood, an amylase level and an arterial blood gases were felt to be consistently of clinical benefit in the acute management of the patient. The reasons for inappropriate investigations and possible solutions are discussed. Significant cost savings can be made, allowing for better targeting of resources. PMID- 7536919 TI - Cultured human sweat gland epithelia: isolation of glands using neutral red. PMID- 7536920 TI - Stage-dependent effects of analogs of gramicidin A on the growth of Plasmodium falciparum in vitro. AB - Tryptophan-N-formylated gramicidin A, a nonhemolytic derivative of the toxic peptide antibiotic gramicidin A, has previously been shown to induce potassium leakage from Plasmodium falciparum-infected erythrocytes in vitro and to inhibit the growth of the parasite. In the present study the antimalarial activities of two other nonhemolytic derivatives of gramicidin A, viz., acylated gramicidin A and desformylated gramicidin A, were tested and compared with those of gramicidin A and tryptophan-N-formylated gramicidin A. The 50% growth-inhibitory concentrations (IC50 values) of the four compounds varied from 0.3 to 18.3 nM, and complete growth inhibition was detected within one parasitic growth cycle. Using highly synchronized cultures of P. falciparum, it was furthermore shown that the gramicidin analogs are inhibitory to all developmental stages of the parasite, although their efficiency in accomplishing growth inhibition was found, as expected, to be clearly stage-dependent and to increase with the age of the parasite. PMID- 7536921 TI - Developmental changes in the circumsporozoite proteins of Plasmodium berghei and P. gallinaceum in their mosquito vectors. AB - The circumsporozoite (CS) protein covers the surface of the sporozoite of plasmodia. Its role in the development of the malaria parasite in mosquito vectors remains unknown. CS-epitope-containing proteins appear on undifferentiated oocysts on day 7 in Plasmodium berghei and on day 5 in P. gallinaceum as demonstrated by indirect fluorescence antibody tests using monoclonal antibodies directed against the CS-protein repeats. The three dimensional distribution of the CS-epitope-containing proteins on oocysts was analyzed by confocal scanning laser microscopy. A strong antibody binding was found in patches around the oocysts of P. berghei and P. gallinaceum, and an accumulation of labeled proteins was found at the base of the oocysts of both species. In Western blots of infected midguts and salivary glands the antibodies recognized two peptides in the salivary glands but up to ten peptides in midgut extracts. The larger number of peptides recognized in midgut preparations might indicate breakdown products during the escape of the sporozoites from the oocyst and their migration on the midgut in the mosquito vector. The data indicate a possible involvement of the CS protein in an active migration process of the sporozoites in the mosquito vector. PMID- 7536923 TI - Passive immunization during experimental amebic liver-abscess development. PMID- 7536922 TI - Detection of Theileria parva in the salivary glands of Rhipicephalus appendiculatus: evaluation of staining methods. AB - A comparison of ten methods for staining tick salivary glands for detection of Theileria parva infection from ticks fed on rabbits for various periods was undertaken. Staining with azure without hydrochloric acid hydrolysis was found to be the most reliable method for detection of the presporozoite stages (sporoblasts) of T. parva in the salivary gland acini of unfed Rhipicephalus appendiculatus and could be applied to field ticks. All the stains proved suitable for the detection and quantitation of sporozoites in ticks fed for 4 days on rabbits. The capacity of the stains to allow detection of early stages of T. parva differed, but it became more reliable during tick feeding as sporoblasts developed and matured. Giemsa's stain and Feulgen's stain followed by superimposition of Giemsa's stain were superior to other stains for the detection and quantitation of immature salivary gland stages in feeding ticks. PMID- 7536924 TI - Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family. AB - Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs. PMID- 7536925 TI - Proline-rich sequences that bind to Src homology 3 domains with individual specificities. AB - To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another. PMID- 7536926 TI - Nitric oxide mediates the increase in local cerebral blood flow during focal seizures. AB - The role of nitric oxide (NO) in the increase in local cerebral blood flow (LCBF) elicited by focal cortical epileptic seizures was investigated in anesthetized adult rats. Seizures were induced by topical bicuculline methiodide applied through two cranial windows drilled over homotopic sites of the frontal cortex, and LCBF was measured by quantitative autoradiography by using 4-iodo[N-methyl 14C]antipyrine. Superfusion of an inhibitor of NO synthase, N omega-nitro-L arginine (NA; 1 mM), for 45 min abolished the increase of LCBF induced by topical bicuculline methiodide (10 mM) [164 +/- 18 ml/100 g per min in the artificial cerebrospinal fluid (aCSF)-superfused side and 104 +/- 12 ml/100 g per ml in the NA-superfused side; P < 0.005]. This effect was reversed by coapplication of an excess of L-arginine substrate (10 mM) (218 +/- 22 ml/100 g per min in the aCSF superfused side and 183 +/- 31 ml/100 g per min in the NA + L-Arg-superfused side) but not by 10 mM D-arginine, a stereoisomer with poor affinity for NO synthase (193 +/- 17 ml/100 g per min in the aCSF-superfused side and 139 +/- 21 ml/100 g per min in the NA + D-Arg-superfused side; P < 0.005). Superfusion of the guanylyl cyclase inhibitor methylene blue attenuated the LCBF increase elicited by topical bicuculline methiodide by 25% +/- 16% (P < 0.05). The present findings suggest that NO is the mediator of the vasodilation in response to focal epileptic seizures. PMID- 7536928 TI - Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development. AB - The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production. PMID- 7536927 TI - Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal. AB - Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance. PMID- 7536929 TI - Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans. AB - Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors. PMID- 7536931 TI - Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor mediated channel current and neurotransmitter release. AB - The effect of the two metal-ion chelators EDTA and citrate on the action of N methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity. PMID- 7536930 TI - B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes. AB - Treatment of small resting B cells with soluble F(ab')2 fragments of anti-IgM, an analogue of T-independent type 2 antigens, induced activation characterized by proliferation and the expression of surface CD5. In contrast, B cells induced to proliferate in response to thymus-dependent inductive signals provided by either fixed activated T-helper 2 cells or soluble CD40 ligand-CD8 (CD40L) recombinant protein displayed elevated levels of CD23 (Fc epsilon II receptor) and no surface CD5. Treatment with anti-IgM and CD40L induced higher levels of proliferation and generated a single population of B cells coexpressing minimal amounts of CD5 and only a slight elevation of CD23. Anti-IgM- but not CD40L-mediated activation was highly sensitive to inhibition by cyclosporin A and FK520. Sp-cAMPS, an analogue of cAMP, augmented CD40L and suppressed surface IgM-mediated activation. Taken together these results are interpreted to mean that there is a single population of small resting B cells that can respond to either T-independent type 2 (surface IgM)- or T-dependent (CD40)-mediated activation. In response to different intracellular signals these cells are induced to enter alternative differentiation pathways. PMID- 7536933 TI - Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells. AB - To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases. PMID- 7536932 TI - Activation of Ca2+ signaling in neutrophils by the mast cell-released immunophilin FKBP12. AB - The immunophilins of the FK506-binding protein (FKBP) family are intracellular proteins that bind the immunosuppresants FK506 and rapamycin. In this study we show that HMC-1 mast cells sensitized with IgE release FKBP12 upon stimulation with anti-IgE. The release is rapid and not affected by actinomycin D or cycloheximide, suggesting that it is due to exocytosis from a storage compartment. FKBP12 from HMC-1 mast cells exhibits biological activity. When applied extracellularly to human neutrophils, it induces transient changes in the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ release from intracellular stores. Inhibition of [Ca2+]i changes by ruthenium red and ryanodine indicates that ryanodine receptor/Ca2+ release channels are involved in FKBP12-induced Ca2+ signaling. Neutrophil activation by mast cell-derived FKBP12 is prevented by complexing FKBP12 with FK506 or rapamycin. These results demonstrate that extracellular FKBP12 functions as a cytokine in cell-to-cell communication. They further suggest a pathophysiological role for FKBP12 as a mediator in immediate or type I hypersensitivity and may have implications for novel therapeutic strategies in the treatment of allergic disorders with FK506 and rapamycin. PMID- 7536934 TI - Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat. AB - Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock. PMID- 7536935 TI - Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. AB - Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal. PMID- 7536936 TI - Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist. AB - During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated. PMID- 7536937 TI - Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen. AB - Heart tissue destruction in chronic Chagas disease cardiopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests that there is antigenic crossreactivity between T. cruzi and heart tissue. As there is evidence for immune recognition of cardiac myosin in CCC, we searched for a putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate immunoblots were probed with anti-cardiac myosin heavy chain IgG antibodies (AMA) affinity purified from CCC or asymptomatic Chagas disease patient-seropositive sera. A 140/116-kDa doublet was predominantly recognized by AMA from CCC sera. Further, recombinant T. cruzi protein B13--whose native protein is also a 140- and 116-kDa double band--was identified by crossreactive AMA. Among 28 sera tested in a dot blot assay, AMA from 100% of CCC sera but only 14% of the asymptomatic Chagas disease sera recognized B13 protein (P = 2.3 x 10(-6)). Sequence homology to B13 protein was found at positions 8-13 and 1442-1447 of human cardiac myosin heavy chain. Competitive ELISA assays that used the correspondent myosin synthetic peptides to inhibit serum antibody binding to B13 protein identified the heart specific AAALDK (1442-1447) sequence of human cardiac myosin heavy chain and the homologous AAAGDK B13 sequence as the respective crossreactive epitopes. The recognition of a heart-specific T. cruzi crossreactive epitope, in strong association with the presence of chronic heart lesions, suggests the involvement of crossreactivity between cardiac myosin and B13 in the pathogenesis of CCC. PMID- 7536939 TI - Histamine effect on ornithine decarboxylase of rat intestine in cases of ischemia reperfusion compared with refeeding. AB - Our previous study suggested that histamine might enhance the increase of ornithine decarboxylase activity in injured intestinal mucosa. To test this hypothesis, we measured histamine content in mesenteric lymph and ornithine decarboxylase activity in intestinal mucosa after ischemia-reperfusion in the rat. We examined the effect of alpha-fluoromethylhistidine, a suicide inhibitor of histidine decarboxylase, on ornithine decarboxylase activity after ischemia reperfusion and compared this with its effect on the rat after refeeding. Ischemia-reperfusion was performed by 15-min occlusion of the superior mesenteric artery. After ischemia-reperfusion, histamine content in mesenteric lymph increased, and this increase was completely suppressed by alpha fluoromethylhistidine pretreatment. In contrast to ischemia-reperfusion, histamine content in mesenteric lymph did not change after refeeding. Ornithine decarboxylase activity increased markedly 3 and 6 hr after ischemia-reperfusion and refeeding, whereas alpha-fluoromethylhistidine attenuated the increase in ornithine decarboxylase activity only in the ischemia-reperfusion group. These results indicate that increase in histamine synthesis in the intestinal mucosa plays an important role in the increase of ornithine decarboxylase activity after ischemia-reperfusion but that histamine is not related to the increase in ornithine decarboxylase activity after refeeding. PMID- 7536940 TI - Rapid induction of mRNA for nitric oxide synthase II in rat alveolar macrophages by intratracheal administration of Mycobacterium tuberculosis and Mycobacterium avium. AB - Mycobacterium avium complex (MAC) organisms are among the most common bacterial cause of disseminated infection in patients with acquired immune deficiency syndrome (AIDS). An increase in the incidence of virulent Mycobacterium tuberculosis (MTB) is also occurring throughout the world. In vitro data suggest that nitric oxide (NO) may be important in restricting the growth of MAC. However, the ability of MTB to stimulate NO production and the susceptibility of MTB to the bactericidal activity of NO produced by murine alveolar macrophages (AM) is controversial. This study tested the hypothesis that in vivo administration of heat-killed MAC (strain 100 and 101) and human virulent MTB (strain F1) to rats stimulated NO production by rat AM, ex vivo. We show that heat-killed MTB instilled into rat lungs rapidly induced mRNA for NO synthase (iNOS) II in AM obtained by bronchoalveolar lavage (BAL). In contrast, expression of AM iNOS mRNA was only found in 40% of the rats given MAC. Moreover, the change in iNOS mRNA in the AM obtained from rats given MTB and MAC correlated with the production of the reactive nitrogen intermediates (RNI) NO2- and NO3- in BAL fluid, lung homogenate, and the spontaneous generation of RNI by isolated AM ex vivo and occurred without measurable increases in BAL fluid tumor necrosis factor alpha (TNF-alpha). L-NG-monomethylarginine (50 mg/kg, ip) given 30 min before MAC or MTB attenuated the increase in RNI in lung homogenates and BAL fluid. This is the first demonstration that in vivo exposure to MTB results in rapid upregulation of gene expression for iNOS which is associated with functional RNI production by rat AM. These results show that MTB human virulent strain 1 has the ability to rapidly upregulate iNOS mRNA in AM. If human AM generate NO from L arginine by either iNOS or other NADPH oxidases then NO may play a role in the overall host-defense response of the lung to MAC and MTB. PMID- 7536941 TI - Inhibition of endothelial nitric oxide synthase by cytochrome P-450 reductase inhibitors. AB - Nitric oxide synthase (NOS) shows similarities to cytochrome P-450 reductase. The two enzymes catalyze the oxidation of N-omega-hydroxy-L-arginine by NADPH and oxygen to nitric oxide (NO) and citrulline. Nitric oxide synthase activity is inhibited by L-arginine analogs like N-omega-nitro-L-arginine, which does not affect cytochrome P-450 reductase. Dihydroergotamine, miconazole, and troleandomycin are classical inhibitors of cytochrome. The present study shows the concentration-dependent inhibitory effect of these compounds and of L- but not D-N-omega-nitro-arginine on the activity of constitutive nitric oxide synthase from bovine aortic endothelial cells. Activity of nitric oxide synthase was estimated by measurement of conversion of [3H]arginine to [3H]citrulline. The tested cytochrome P-450 inhibitors are likely to interfere with heme of nitric oxide synthase. The data confirms a similarity as well as functional differences between the enzymes. PMID- 7536938 TI - Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor. AB - Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. We evaluated rolipram, a type IV phosphodiesterase inhibitor, for its efficacy in preventing EAE in the common marmoset Callithrix jacchus. In a blinded experimental design, clinical signs of EAE developed within 17 days of immunization with human white matter in two placebo-treated animals but in none of three monkeys that received rolipram (10 mg/kg s.c. every other day) beginning 1 week after immunization. In controls, signs of EAE were associated with development of cerebrospinal fluid pleocytosis and cerebral MRI abnormalities. In the treatment group, there was sustained protection from clinical EAE, transient cerebrospinal fluid pleocytosis in only one of three animals, no MRI abnormality, and marked reduction in histopathologic findings. Rolipram-treated and control animals equally developed circulating antibodies to myelin basic protein. Thus, inhibition of type IV phosphodiesterase, initiated after sensitization to central nervous system antigens, protected against autoimmune demyelinating disease. PMID- 7536942 TI - The effect of DHEA given chronically to Zucker rats. AB - Dehydroepiandrosterone (DHEA) has been reported to exert antiglucocorticoid activity. When administered to obese, hypercorticosteronemic Zucker rats, it causes a diminution of food intake and a reduction in their rate of weight gain. This experiment was conducted to evaluate whether this biologic effect could be ascribed to chronic adrenal insufficiency. Obese and lean Zucker rats were treated with DHEA as a food supplement for 28 days. Upon sacrifice, organ weights and serum chemistries were measured, along with neurotransmitter levels in regions of the hypothalamus. Results showed that although the obese animals gained weight more slowly, had lower insulin levels, and ate less, their serum glucose, corticosterone, and ACTH levels were not different from control. Hypothalamic neurotransmitters in the obese rat were unaffected by chronic DHEA treatment. We concluded that, although DHEA clearly affects Zucker weight gain, it does not induce chronic adrenal insufficiency. PMID- 7536943 TI - The neuronal cytoskeleton: roles in neuronal morphogenesis and organelle transport. PMID- 7536944 TI - Biology of cyclosporin A and FK506. PMID- 7536946 TI - Therapeutic effect of percutaneous ethanol injection on small hepatocellular carcinoma: evaluation with CT. AB - PURPOSE: To evaluate the therapeutic effect of percutaneous ethanol injection (PEI) on small hepatocellular carcinoma (HCC) with computed tomography (CT). MATERIALS AND METHODS: Sixty-seven patients with histologically proved HCC 3 cm or less in diameter underwent PEI. The patients were regularly followed up with sonography and contrast material-enhanced CT for more than 1 year (range, 12-96 months). The CT findings were evaluated for three tumor types distinguished on the basis of their appearance relative to that of the surrounding liver parenchyma: type 1 = hyperattenuating at the early phase (n = 39), type 2 = iso- or hypoattenuating at the early phase and hypoattenuating at the late phase (n = 18), and type 3 = isoattenuating (not detected) at both the early and late phases (n = 10). RESULTS: After PEI, a necrotic area of HCC and the surrounding liver parenchyma was characterized as hypoattenuating at both early and late phases of contrast-enhanced CT, regardless of the type. When an HCC appeared to be completely necrotic within 3 months after PEI, this status was retained until the latest observation in all but three cases. CONCLUSION: Contrast-enhanced CT can correctly depict PEI-induced necrosis in HCC and is reliable for evaluating the therapeutic effect of PEI. PMID- 7536947 TI - Neonates treated with ECMO: predictive value of early CT and US neuroimaging findings on short-term neurodevelopmental outcome. AB - PURPOSE: To determine if neuroimaging findings in infants who undergo extracorporeal membrane oxygenation (ECMO) are predictive of developmental outcome. MATERIALS AND METHODS: At 1-2 years of age, 183 ECMO survivors (69 female, 114 male) underwent developmental examination. Neuroimaging studies obtained at time of ECMO were assigned a neuroimaging score. Neuroimaging findings were correlated with developmental outcome. RESULTS: Eighty-five infants had neuroimaging abnormalities. Development was normal in 105 infants, suspect in 37, and delayed in 41. Mean neuroimaging scores were significantly worse in survivors with delayed development (P < or = .0001). The sensitivity and specificity of normal neuroimaging findings in prediction of normal outcome were 65% and 63%, respectively. Survivors with nonhemorrhagic abnormalities had a higher risk of delayed development than did those with isolated hemorrhagic abnormalities (39% vs 21%). CONCLUSION: Although they cannot be used alone to predict outcome, early neuroimaging scores can be used to assign risk categories for developmental outcome. PMID- 7536945 TI - Dopamine D2 receptor blockade in vivo with the novel antipsychotics risperidone and remoxipride--an 123I-IBZM single photon emission tomography (SPET) study. AB - Risperidone and remoxipride are recently introduced atypical antipsychotics, with clinical efficacy comparable to that of classical antipsychotics but lower propensity to induce extrapyramidal side effects (EPS). It is unclear whether these properties relate to weak dopamine D2 receptor blockade in vivo, as has been suggested for the archetypal atypical antipsychotic clozapine. We have used 123I-IBZM single photon emission tomography (SPET) to characterize the patterns of striatal D2 receptor binding in vivo in DSMIII-R-diagnosed schizophrenic and schizo-affective patients treated with either risperidone (n = 6) or remoxipride (n = 4) but predominantly EPS free. These groups were compared to age- and BPRS- matched subjects from a previously reported D2 receptor binding database of patients treated with clozapine (n = 10) and classical antipsychotics (n = 10). Patients on risperidone and remoxipride had high levels of D2 receptor blockade, comparable to those of patients on classical antipsychotics, and significantly greater than those obtained with clozapine-treated patients (risperidone versus clozapine, P < 0.005; remoxipride versus clozapine, P < 0.025). These results suggest high levels of striatal D2 receptor occupancy in association with remoxipride and risperidone treatment and argue against modest D2 antagonism as the explanation for the low incidence of EPS associated with these drugs. PMID- 7536948 TI - [LPS receptor of macrophages]. PMID- 7536949 TI - Neuropeptide levels in discrete brain regions in the iminodipropionitrile-induced persistent dyskinesia rat model. AB - To clarify the role of neuropeptides in dyskinesia induced by iminodipropionitrile (IDPN), the levels of five representative neuropeptides were examined in discrete regions of the rat brain 4 weeks after intraperitoneal injection of IDPN. The five neuropeptides examined were methionine-enkephalin (Met-Enk), substance P (SP) and somatostatin, which are closely related to extrapyramidal function, and thyrotropin-releasing hormone (TRH) and cholecystokinin octapeptide (CCK-8), which are closely related to the neural mechanism of the dopamine system. IDPN pretreatment significantly increased Met Enk in the basal ganglia but not SP or somatostatin; however, all three neuropeptide levels were increased in the hindbrain. In IDPN-treated rats, TRH and CCK-8 levels were increased in the nucleus accumbens, and the frontal cortical CCK-8 level was extremely increased. These findings, together with previous reports, suggest that neuropeptides in the basal ganglia, hindbrain and cerebral cortex play important roles in the manifestation of dyskinetic symptoms. PMID- 7536950 TI - Capsaicin effects on substance P and CGRP in rat adjuvant arthritis. AB - The effects of capsaicin on the sensory neuropeptides substance P and calcitonin gene-related peptide were analyzed in the ankle joints and dorsal root ganglia (L2-L6) of adult female Lewis rats. The study included 23 normal rats and 23 arthritic rats, all injected subcutaneously with capsaicin (total dose 200 mg/kg bw). Another two groups of animals from a previous study, i.e., 23 normal rats and 23 arthritic rats not given capsaicin served as controls. Adjuvant arthritis was induced by inoculation with heat-killed mycobacteria. The morphological distribution of sensory neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay. In normal rats, capsaicin significantly reduced the concentrations of substance P and calcitonin gene-related peptide in ankle joints (54 and 36%, respectively) as well as dorsal root ganglia (40 and 54%, respectively). In arthritic rats those pretreated with capsaicin had significantly lower concentrations of substance P and calcitonin gene-related peptide in dorsal root ganglia (19 and 42%, respectively) compared to the arthritic controls. In the ankle joints, however, only the SP concentration was reduced (42%). Notably, this was accompanied by a 40% reduction in inflammatory response as assessed by comparing the ankle joint weights of the experimental groups. In general, there was a good correlation between the neuropeptide concentrations in ipsilateral ankle joints and the corresponding dorsal root ganglia as assessed in individual rats. The present study of adjuvant induced arthritis shows that capsaicin administration reduces the otherwise up regulated levels of sensory neuropeptides in dorsal root ganglia and ankle joints. However, capsaicin at the dose given can only mitigate, not completely prevent the development of joint inflammation. Nonetheless, the findings suggest that antineuronal therapy targeted against specific neurotransmitters may prove useful in inflammatory joint disease. PMID- 7536951 TI - [How I treat... benign prostatic hypertrophy. 2. Medical treatment and proposed strategy]. PMID- 7536952 TI - [Biological diagnosis of hepatitis C virus infection]. PMID- 7536954 TI - [Medical treatment of chronic viral hepatitis]. AB - alpha-interferon and vidarabine monophosphate are currently available for the treatment of chronic active hepatitis B. Treatment is indicated in patients with high transaminase levels, evidence of replicating virus and histological inflammation. Interferon or vidarabine suppress viral replication and normalize transaminase activity in 30 to 40% of the patients. The main predictive factors of response to therapy are a high transaminase level, a low replication level, and the absence of immunodepression. The response is usually sustained and the Ag HBs to anti-HBs seroconversion occurs a few years after treatment. No efficacious treatment is still available in patients with chronic hepatitis D. Standard treatment of chronic hepatitis C with alpha-interferon (3 millions units 3 times a week during 6 months) induces a complete response in only 15% of the patients. New modalities of interferon therapy and associated treatment are currently evaluated. PMID- 7536953 TI - Measurement of colony-stimulating factors in synovial fluid: potential clinical value. AB - In this study, 100 synovial fluid (SF) samples from patients with a variety of arthritides were assayed for levels of colony-stimulating factors (CSFs) using a human bone-marrow bioassay and enzyme immunoassays for granulocyte (G-) and granulocyte-macrophage (GM-) CSFs. GM-CSF was found more frequently in samples from rheumatoid arthritis (RA) subjects (49%) than in non-RA samples (29%). Absence of GM- but not G- or bioassay CSFs characterised samples from subjects with psoriatic arthritis and ankylosing spondylitis (n = 14). There was strong evidence of an antagonistic relationship between levels of G- and GM-CSFs in samples from RA patients, an effect independent of drug treatment. However, treatment with non-steroidal anti-inflammatory agents (NSAIDs) may affect reported CSF concentrations: G-CSF levels were significantly lower in samples from subjects not taking NSAIDs. These results suggest that SF-CSF estimations using commercially available assays could provide useful diagnostic clues for clinicians, but careful interpretation is warranted particularly in patients on long-term NSAID treatment. PMID- 7536955 TI - [Semiology of urination disorders in men]. AB - With the ageing of population, the mictional disorders affect 35 at 47% of men 50 years old in many studies. The origins are numerous but especially dominated by prostate adenoma. For this reason, the semiology of mictional disorders of men is necessary at diagnosis orientation and for specific complementary examination. A meticulous interrogation is necessary because these disorders are ignored by the patients or under-estimated and on specific clinical examination. Here, we look at the functional signs and the clinical of prostatic adenoma, prostatisis, prostate cancer and uretral stenosis. Also practical conducting in urine blockage is reviewed. PMID- 7536956 TI - The importance of non-charged amino acids in antibody binding to Humicola lanuginosa lipase. AB - The antigenicity of 36 Humicola lanuginosa lipase (HL) variants, generated by site directed mutagenesis, was compared with that of the unchanged enzyme. Polyclonal antibodies raised against variant lipases were investigated and compared with the antibodies raised against the wild type lipase in an ELISA competition assay. The results showed that exchange of charged amino acids with polar residues in surface epitopes of HL, results in a tighter binding of the antibody to the epitope. Four amino acids (Trp at position 89, Asp at positions 96 and 254 and Phe at position 211) were found to be essential for antibody binding in each their epitope of the wild type enzyme. PMID- 7536957 TI - Anti-GD3 antibodies are potent activators of human gamma/delta and alpha/beta positive T cells. AB - The ganglioside GD3 has a variety of biological functions. These include stimulatory effects on proliferation, natural killer activity and cytokine production by freshly isolated peripheral T cells. In this study we have characterized anti-GD3 antibody (MoAb Z21) mediated effects on T cell clones. Our data indicate that alpha/beta TCR CD4+ and CD8+ as well as gamma/delta TCR positive T cells can be stimulated resulting in proliferation and cytokine production. This effect could be blocked by cyclosporin A and did not involve the LFA-3 or CD4 molecule. Apart from IFN-gamma and IL-2 production by T helper 1 and T helper 0 cells we have observed production of IL-4 and IL-10 by T helper 2 cells indicating that the GD3 molecule is not a marker for a certain functional T cell subset. In contrast to anti-CD3 mediated activation, the responsiveness of T cells to stimulation via GD3 was dependent on the cell surface expression of the molecule and could be enhanced by costimulation via CD2, CD3, CD26 or CD28. In addition, anti-GD3 antibodies delivered a potent costimulatory signal for antigen induced proliferation of CD4+ T lymphocytes. In summary, our experiments illuminate the mechanisms of anti-GD3 antibody induced T cell activation. PMID- 7536958 TI - Nucleic acid amplification mediated microbial identification. AB - Nucleic acid amplification techniques have and are revolutionising the way research, academia, medicine, environmental and quality control issues are dealt with. In particular, the polymerase chain reaction, (PCR), originally mentioned in 1985 has greatly modified the ways in which scientists and clinicians go about nucleic acid sequence analysis and manipulation and has great implications for molecular biology in general and diagnostics in particular. This report attempts to review the use of PCR and isothermal nucleic acid amplification techniques in the rapid identification of microorganisms. Key methodologies have been noted, their strengths and weaknesses commented upon and comparisons have been drawn between isothermal and thermally cycled nucleic amplification techniques in rapid microbial identification. PMID- 7536960 TI - [The effect of hemosorption on the glycoprotein concentration of the blood serum in inflammatory diseases of the large intestine]. AB - 71 patients with nonspecific diseases of the colon were examined before and after hemosorption for concentrations of seromucoid, orosomucoid, immunoglobulins G,M,A, C3 and C4 complement component fractions, haptoglobin, ceruloplasmin, alpha 1-antitrypsin, C-reactive protein, properdin, transferrin. It was found that hemosorption results in lowering of all the above concentrations in nonspecific ulcerative colitis, except seromucoid and orosomucoid in moderate and acute colitis. In response to hemosorption serum glycoproteins levels varied with the disease severity, its duration and metabolic activity of the liver. Concentrations of seromucoids, orosomucoid, alpha 1-antitrypsin changed in correlation with clinical manifestations of colon inflammation and may serve as criteria of hemosorption efficacy. PMID- 7536959 TI - Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha induced angiogenesis. AB - B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation. PMID- 7536961 TI - [The antiviral therapy of chronic hepatitis B]. PMID- 7536962 TI - High-resolution two-part basic urea gels for analysis of venom phospholipase A2 isoforms. AB - The performance of acidic and basic urea polyacrylamide gels has been improved by adopting a two-part gel system with a concentration discontinuity to act as a stacking boundary and by increasing the urea concentration to 8 M. The contributions of primary amino-group and guanidino-group ionizations to mobility have been evaluated by acetylation and phenyl glyoxal treatment respectively. The chromogenic PLA2 detection method of Shier and Trotter (Analyt. Biochem. 87, 604, 1978) has been modified for use with basic urea PAGE. The results have confirmed the major findings of other workers, but have demonstrated the presence of many hitherto uncharacterised isoforms of PLA2 in a variety of whole snake venoms. The basic urea PAGE (BG) method is proposed as the basis of a simple and rapid method for the classification of PLA2 isoforms which should allow unambiguous identification of isoforms by referring bands for purified material to the isoform content of whole venoms. PMID- 7536963 TI - Direct toxic effect of bleomycin on alveolar type 2 cells. AB - We studied the in vitro toxicity of bleomycin (BLM) on primary cultures of rat alveolar type 2 cells (T2 cells). It was shown that BLM was directly toxic for T2 cells in a dose- and time-dependent manner. Lung fibroblasts (LF) appear to be more resistant than T2 cells. Modulation of intracellular glutathione concentration was associated with changes in cytotoxicity. Furthermore, the addition of O-phenanthroline to the cellular medium reduced significantly BLM toxicity, suggesting the involvement of intra-cellular ferric ion. We also found that BLM toxicity was associated with a decreased release of phosphatidylcholine by T2 cells, the main component of surfactant. Protective effect of O phenanthroline and the involvement of glutathione may be an alternative approach to the protection of BLM-induced damage. PMID- 7536964 TI - Interaction of tumour cells with cultured stromal cells from human bone marrow. AB - Adhesion of tumour cells to cultured bone marrow stromal cells has been studied in an in vitro model system. Stromal cells were isolated from bone marrow aspirates. Immunohistochemical and electron microscopical analysis revealed a uniform cell monolayer of myofibroblastic cells, expressing fibroblast antigens and smooth muscle actin. Cell interactions with tumour cells lines showed different patterns. The K562 cells bound in low numbers to stromal cells. HEL-DR- and HL60 cells adhered to stromal cells showing an enlarged cell contact area (spreading) attenuated by distinct contact sites and they invaded the monolayer. Adhesion molecules, important for cell contacts, were detected on tumor cells. Different VLA antigens were detected on tumour cells, but on stromal cells only VLA-5 and CD29 were found. In vitro inhibition studies with mAbs against adhesion molecules indicated two major pathways for binding of tumour cells to stromal cells: VCAM-1/VLA-4 and fibronectin/VLA-5. Variation in inhibition of mAbs to VLA 4 and VCAM-1 indicated the existence of critical epitopes in the adhesion of tumour cells. PMID- 7536967 TI - The determinant of R73 MAb exists on the constant domain of rat T-cell receptor beta-chain. PMID- 7536965 TI - An adhesion-promoting anti-rat CD18 monoclonal antibody differentially alters thymocyte responses to various mitogens. AB - Leukocyte function-associated antigen (LFA)-1 represents one of the major lymphocyte adhesion molecules being capable of both strengthening cell-cell contacts and cooperating with other relevant surface molecules in signal transduction. We have studied the effects of an adhesion-promoting anti-CD18 monoclonal antibody (mAb) (NG2B12) on thymocyte proliferation induced by various mitogens. As we have recently described, the adhesion-promoting function of this mAb strictly depends upon the activation of several intracellular protein kinases and phosphatases. In the present work we have found that NG2B12 inhibits concanavalin (Con) A-induced thymocyte proliferation suppressing IL-2 production by these cells. Conversely, this mAb enhances proliferative responses of thymocytes to phytohemagglutinin (PHA) and interleukin (IL)2 alone or in combination with the phorbol ester PMA. The effects of this mAb were compared with those of another anti-rat CD18 mAb which potently blocks the LFA 1/Intercellular adhesion molecules (ICAMs) interactions. PMID- 7536966 TI - A monoclonal antibody reacts with T cells and novel subpopulation of rat bone marrow cells. PMID- 7536969 TI - Engraftment of rat bone marrow cells in mice transgenic for granulocyte-colony stimulating factor. PMID- 7536968 TI - Vaccination with a synthetic T-cell receptor V-region peptide: immunomodulation or vaccination. PMID- 7536970 TI - Target antigen specificity of a monoclonal antibody (HAR-1) that causes the rejection of hamster heart xenografts in rats. PMID- 7536971 TI - Cellular basis of acquired immunologic tolerance following total bowel transplantation in rats. I. Relation between donor lymphocyte chimerism and host cell-mediated immunity. PMID- 7536972 TI - Cellular basis of immunologic tolerance following total bowel transplantation in rats. II. Dominant role of suppressor T cells in adoptive transfer of acquired tolerance to vascularized allografts. PMID- 7536973 TI - Spleen grafting might induce transplantation tolerance in rats. PMID- 7536974 TI - Induction of tolerance in rat cardiac allograft model by intrathymic injection of donor bone marrow cells. PMID- 7536976 TI - Adhesion molecule expression in acute humoral and acute cellular rejection of renal grafts. PMID- 7536975 TI - Outcome of lymphocyte subsets and cytokines during combined CD4/anti-IL-2R monoclonal antibody therapy in kidney transplantation. PMID- 7536977 TI - Expansion of CD4+CD7- T helper cells with a TH0-TH2 function in kidney transplant recipients. PMID- 7536979 TI - Peripheral nerve allotransplant immunosuppressed with FK 506: preliminary results. PMID- 7536978 TI - Attempted prevention of perioperative cytomegalovirus infection after liver transplantation. PMID- 7536980 TI - Effect of FK 506 in experimental pancreas transplantation. PMID- 7536981 TI - Minimal threshold of FK 506 for enhancing liver regeneration in thymectomized rats. PMID- 7536982 TI - Odontoameloblastoma in a Japanese monkey (Macaca fuscata). PMID- 7536983 TI - Immunohistochemical and morphologic features of a cecal adenocarcinoma in a white handed gibbon (Hylobates lar). PMID- 7536984 TI - Cloning bovine cytokine cDNA fragments and measuring bovine cytokine mRNA using the reverse transcription-polymerase chain reaction. AB - Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-gamma activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression. PMID- 7536985 TI - Newcastle disease virus-induced apoptosis in chicken peripheral blood lymphocytes. AB - Chicken peripheral blood lymphocytes (PBL) were infected with the GB strain of Newcastle disease virus (NDV). At 0, 1.5 and 3 h postinfection, PBL were stained with acridine orange-ethidium bromide and examined for apoptotic and necrotic indices under a UV microscope. At the end of 3 h, the cells were pelleted and were either fixed for electron microscopy or used for DNA extraction. Infected cells showed a significant amount of apoptosis and necrosis when compared with controls. Electron microscopy of the infected cells also showed characteristics of apoptosis, i.e. apoptotic bodies, margination of chromatin with crescent formation and fusion of cells. DNA extracted from virus-infected cells showed extensive fragmentation. Based on these findings it is concluded that NDV, in addition to causing necrosis in chicken lymphocytes, can induce apoptosis. PMID- 7536986 TI - Chicken interferon-mediated induction of major histocompatibility complex class II antigens on peripheral blood monocytes. AB - Conditioned medium containing immune interferon (IFN) activity was prepared by stimulating spleen lymphocytes obtained from inbred SC chickens with 10 micrograms concanavalin A (Con A) for 48 h. Pretreatment of spleen cells with monoclonal antibody against CD4, but not CD8, abrogated IFN production suggesting that CD4+ lymphocytes are responsible for immune IFN production. Immune IFN was purified 25-fold from Con A conditioned medium using controlled-pore glass column chromatography resulting in an increase in specific antiviral activity from 7 to 3290 units mg-1. Partially purified immune IFN retained antiviral and macrophage activating factor (MAF)-like activities. Normal peripheral blood macrophages, when cultured in the presence of partially purified immune IFN, showed a dose dependent increase in cell surface major histocompatibility complex Class II antigen expression by flow cytometry. Northern blot analysis of mRNA obtained from IFN-treated macrophages showed a concomitant increase in Class II gene expression. This effect was more obvious in cells induced for 48 h than in those induced for 24 h. These results strongly suggest that existence of an avian homologue of the MAF-like activity. PMID- 7536987 TI - Improved detection of anti-HCV in post-transfusion hepatitis by a third generation ELISA. AB - The sensitivity of ORTHO HCV 3.0 ELISA Test System (ELISA 3) for the detection of anti-HCV was compared with the second-generation ELISA, OR-THO HCV 2.0 ELISA Test System (ELISA 2). ELISA 3 differs from ELISA 2 in that it incorporates the HCV recombinant antigen NS5, in addition to recombinant antigens derived from the NS3, NS4 and core regions of the HCV genome. Specimens tested consisted of serial bleeds obtained from 21 individuals undergoing seroconversion following acquisition of post-transfusion HCV infection. ELISA 3 demonstrated significantly greater sensitivity than ELISA 2, detecting seroconversion earlier in 24% (5/21) of cases. Although one of these cases appeared to represent early seroconversion to NS5, most of the improved sensitivity of ELISA 3 appeared to derive from increased detectability of anti-c33c. PMID- 7536989 TI - Evidence that the granulocyte-specific antigen NC1 is identical with NA2. AB - The neutrophil-specific antigen NC1 is defined by an antibody in the serum of a mother who gave birth to a child with alloimmune neonatal neutropenia. NC1 has been reported to be associated with the neutrophil-specific antigen NA2, but the precise relation of NC1 and NA2 remained unclear. Therefore, we investigated the serum using the antigen capture assay MAIGA and the granulocyte (GIFT) and lymphocyte (LIFT) immunofluorescence tests. In GIFT, no NA association was observed. In LIFT, serum antibodies bound preferably to lymphocytes with the HLA antigens HLA-B7 and cross-reacting antigens. In MAIGA, an antibody specific for the NA2 variant of the granulocyte Fc gamma-receptor III was observed. The NA2 specificity was confirmed by testing granulocytes from 40 further different donors. This indicates that the NC1 and NA2 antigens are identical. A positive GIFT result but a negative one in LIFT using cells of an NA2-negative typed individual suggest the presence of an additional, non-NA2-specific granulocyte antibody. PMID- 7536988 TI - Monitoring anti-HPA-1a platelet antibody levels during pregnancy using the MAIPA test. AB - Anti-HPA-1a platelet antibody levels in pregnant women with a history of fetomaternal alloimmune thrombocytopenia (FMAIT) were monitored longitudinally using the monoclonal antibody immobilisation of platelet antigens (MAIPA) assay, in order to examine any variation in optical density (OD) readings obtained over the course of pregnancy and after delivery. Seven women were selected; 4 were studied retrospectively and 3 prospectively (the latter being treated with intravenous gammaglobulin; IVGG). Levels of anti-HPA-1a were measured at various intervals after delivery of the first affected infant, to post delivery of the following affected infant. A decrease in MAIPA OD was demonstrated in all patients during the course of these pregnancies. This assay is a useful tool for monitoring anti-HPA-1a in women with a history of infants affected with FMAIT. A maternal antibody 'resting' level prior to, or early in the first trimester, must be established for comparison. PMID- 7536990 TI - Transmission of hepatitis C virus by anti-HCV-negative blood transfusion. Case report. AB - Acute posttransfusion hepatitis C was reported in a recipient of 3 units of red cells. The recipient became acutely icteric 6 weeks after transfusion, and HCV infection was diagnosed. Stored serum samples of the 3 implicated donations, which were negative with ELISA-2, were retested by PCR and 3rd-generation antibody tests. One implicated donation was PCR positive, but anti-HCV negative. Both other donations were negative in all tests. The donor was recalled to the Blood Bank 13 weeks after the implicated donation and was found to be ELISA-3 plus RIBA-3 positive. Eight months after the implicated donation, the donor is still PCR and RIBA-3 positive, whereas the recipient became PCR negative but remained anti-HCV RIBA-3 positive. The case shows that blood products from donors collected during the open window period of an HCV infection can transmit HCV to recipients. PMID- 7536991 TI - HCV core IgM and IgG antibodies in blood donors. PMID- 7536992 TI - Anti-HCV antibodies and HCV RNA in Libyan blood donors. PMID- 7536993 TI - Screening for prostate cancer. AB - Prostate cancer is a serious health care problem in the United States. Whether or not to screen for it has become a timely issue. Although a large number of men have clinically important, asymptomatic, undetected prostate cancer, an even larger number have clinically unimportant cancer. To justify screening programs, not only must we avoid detecting biologically unimportant cancers, we must also detect and effectively treat that subset of tumors that, if undiagnosed, would progress, produce symptoms, and reduce life expectancy. Serum prostate-specific antigen (PSA) assay, or its variations such as PSA density, PSA velocity, and age specific reference ranges, and the digital rectal examination are the best tests for detecting clinically important, asymptomatic, curable tumors. Recent data suggest that using serum PSA levels does not result in an overdetection of unimportant tumors. Highly effective, curative treatment of localized prostate cancer is available. These factors promote optimism that screening for prostate cancer will ultimately prove beneficial. Nonetheless, men should be informed regarding the benefits and possible risks before being screened for prostate cancer. PMID- 7536994 TI - Screening for prostate cancer today. PMID- 7536997 TI - The effect of the long-acting somatostatin analogue octreotide on caerulein induced pancreatic injuries in rats. AB - The efficacy of long acting somatostatin analogue, octreotide acetate (SMS 201 995) on the caerulein-induced acute pancreatitis and on the regeneration of the gland was examined. The effect of the drug on the acute injury was examined at 6 and 24 hours following the intervention, while the regeneration was examined on Day 3 and Day 5 in all cases by determination of plasma amylase levels and by analysis of the pancreatic tissue. The use of octreotide could not counteract the occurrence of acute pancreatitis, however, it has some benefit as seen by it's ability to moderate the increases of serum amylase levels. During the examination of pancreatic regeneration it was found that the weight of the pancrease decreased and this was not affected by octreotide. As a matter of fact, the octreotide coadministered with caerulein counteracted the caerulein-induced increase of pancreatic DNA content and therefore acted against the reactive pancreatic hyperplasia. Thus long term administration of octreotide in acute pancreatic injury may not be rational. PMID- 7536995 TI - Insulin-like growth factor I receptors and insulin-like growth factor-binding proteins in human parathyroid tumors. AB - Studies have demonstrated the presence of epidermal growth factor receptors in human parathyroid tumors. However, there is little information on the effect of other peptide growth factors on parathyroid cell growth. We therefore studied the interaction of insulin-like growth factor I (IGF-I) with human parathyroid tumor cells. Parathyroid tissues were obtained from 24 patients with primary or secondary hyperparathyroidism. There were 15 solitary adenomas, 5 carcinomas, and 4 hyperplastic tissues. First, the binding of [125I]IGF-I to the crude membrane fractions was studied by competitive inhibition with unlabeled IGF-I. Second, isolated parathyroid cells were cultured with IGF-I and examined for DNA synthesis. The IGF-binding protein (IGFBP) content of tissue homogenates was determined by ligand blot analysis. The binding of [125I]IGF-I to parathyroid membranes was dependent on time, temperature, and pH of the medium. Maximum binding was obtained after incubation for 18 hours at 4 degrees C. Specific binding to parathyroid cancer membranes (mean +/- SE, 10.75 +/- 10.55%/mg protein) was significantly (p < 0.05) greater than that in adenoma tissues (3.71 +/- 2.11%/mg). The value in hyperplastic tissues (4.78 +/- 2.97%/mg) was not different from that in adenomas. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Cultured parathyroid cells responded to IGF-I with increased DNA synthesis. The parathyroid tumor tissues expressed IGFBPs. These results suggest that IGF-I and IGFBPs are involved in the growth regulation of parathyroid tumor cells. PMID- 7536996 TI - [Viral hepatitis C]. AB - Soon after the isolation of the hepatitis C virus (HCV) genome in 1988 it became evident that HCV is the most important cause of non-A, non-B-hepatitis. In recent years the structure of this (+)-stranded RNA-virus, the different genotypes of HCV and the replication in hepatic and extrahepatic sites have been investigated. HCV has a remarkable degree of genetic heterogeneity, mutates rapidly, leading to the simultaneous coexistence of different genoms in the same individual (quasispecies) and most likely to the generation of neutralization escape mutants. The cytotoxicity of the hepatitis C virus appears to be mainly immune mediated. This review article summarizes basic, diagnostic and clinical aspects of acute and chronic HCV-infection, association with other diseases and complications such as liver cirrhosis and hepatocellular carcinoma. Interferon alpha has been shown useful in normalizing serum aminotransferases and decreasing liver inflammatory lesions in about half of the patients with chronic hepatitis C. However, relapses after the cessation of interferon-alpha are frequent, leading to a sustained response in less than 30% of treated patients. Several clinical and biochemical parameters for response to interferon-alpha have been proposed. In patients with orthotopic liver transplantation due to progressive chronic hepatitis C and decompensated liver cirrhosis, reinfection of the donor organ frequently occurs. However, in transplanted patients under immunosuppression the course of hepatitis C reinfection is usually mild. Due to screening programs of blood and blood products the incidence of posttransfusion acquired hepatitis C has declined. However, further efforts in understanding the transmission of community-acquired hepatitis C are necessary. The development of a hepatitis C vaccine will be difficult due to the high degree of viral genetic heterogeneity. PMID- 7536998 TI - [Doppler ultrasound studies in placental insufficiency as an indication of the effectiveness of hemodilution therapy]. AB - Early diagnosis of intrauterine growth retardation is important to ensure optimal obstetric monitoring. Current methods of diagnosing and assessing the growth retardation include clinical evaluation, various ultrasonic parameters and Doppler ultrasound. In this study Dopplersonographic measurements of the umbilical artery, fetal aorta and the middle cerebral artery were performed on 19 patients having been diagnosed with a placental insufficiency and maternal haemoconcentration. The patients were treated 10 days by a daily hypervolemic haemodilution with 500 ml 10% hydroxyethylstarch and 1000 ml ringersolution. The blood flow velocimetry showed changes in the fetal circulation characterized by a redistribution of the fetal blood flow. The aortic and umbilical resistance indices decreased. The resistance index of the middle cerebral artery demonstrated a significant rise during the haemodilution. The fetal circulatory changes represents a better oxygenation during the therapy with hydroxyethylstarch. The intravenous haemodilution can be seen as an effective and easy treatment of fetal growth retardation by increasing the placenta perfusion. PMID- 7536999 TI - [Prostacyclin (iloprost) as an adjuvant in local surgical therapy of stage IV arterial occlusive disease--is quantification of the therapeutic effect possible with tcPO2 measurements?]. AB - The effect of intravenous therapy with iloprost (average duration 6 weeks) on the prognosis of leg-preserving local surgical treatment and on the transcutaneously measured oxygen pressure (tcPO2) in patients with ischaemic lesions was investigated in a prospective, open, controlled, randomized study. All of the 30 patients recruited to the study suffered from a peripheral arterial occlusive disease Fontaine stage IV with progressive symptoms and arterial occlusions below the knee. 15 patients received iloprost intravenously for 6 hours per day in an individual tolerable dose (mean dose: 1.7 ng/kg/min). 15 patients in the control group received no trial therapy. During the treatment period each patient underwent minor amputation or skin grafting. Wound healing after surgery and tcPO2 were assessed. The long-term efficacy of iloprost was assessed by following the patients for one year. The iloprost treatment was in general well tolerated and all patients completed the study in accordance with the trial protocol. Primary postoperative wound healing as the responder-criterion was observed in 9 patients (60%) of the iloprost group and in 4 patients (27%) of the control group. Patients with iloprost therapy were especially likely to show wound healing if they received the operation approximately in the mid of the treatment phase. The effect of therapy lasted for one year after the end of the treatment in 7 of 9 classified responders of the iloprost group. After one year the responder rate of 47% in the iloprost group was still 20% higher than in the control group. Surgical revisions were necessary in 7 patients (47%) of each group. 2 patients (13%) treated with iloprost and 4 control patients (27%) underwent major amputation. The mortality was 13% = 2 of 15 patients (iloprost) and 7% = 1 of 15 patients (control). Transcutaneous PO2-measurements were very reproducible over a long-term period under standardized conditions. A significant effect of iloprost on the tcPO2 was shown. There was a mean increase of 8.1 mmHg +/- 11.2 (mean +/- SD) in the foot of the affected limb after an average 6-week therapy (p < 0.05). At the same time a significant difference in the tcPO2 changes between responders and nonresponders was demonstrated (p < 0.05). Patients with primary wound healing had a greater increase in tcPO2 than patients whose wounds did not heal. An increase of at least 10 mmHg (6 patients) in tcPO2 during the infusion after 3 weeks of therapy predicted primary post-operative wound healing. PMID- 7537001 TI - Galanin immunoreactive neurons in the medulla oblongata of rats. AB - The distribution of galanin-immunoreactive (-ir) neurons in the medulla oblongata was mapped with light microscopic immunohistochemistry. No immunopositive perikarya were seen in untreated rats. Two days after colchicine treatment, galanin immuno-positive neurons were localized in the following areas: 1) raphe nuclei (magnus, pallidus and obscurus); 2) in various parts of the reticular formation, mainly in the territory of the catecholaminergic groups and in the peritrigeminal subdivision of the lateral reticular nucleus; 3) vagal nuclei (nucleus of the solitary tract, nucleus ambiguous); 4) two cell groups at the ventral surface of the rostro-caudal middle portion of the medulla oblongata (they do not correspond to any known demarkated anatomical nuclei, but related to the chemosensitive medullary area); 5) in the gelatinous part of the spinal trigeminal nucleus. The wide distribution of galanin neurons in the medulla support data that had been reported on the role of this peptide in various viscerosensory and autonomic mechanisms. In addition to these, galanin seems to be an important factor in the restoration of lesioned neurons (nerve growth factor-like activity). An increased galanin mRNA expression can be seen in dorsal vagal or hypoglossal motor neurons after intracranial transections of vagal or hypoglossal nerves, respectively. Transections of the olivocerebellar tract induced galanin gene expression in neurons of the contralateral inferior olive. After brainstem hemisection, galanin immunopositivity was seen in cells of the nucleus of the solitary tract due to the transection of ascending projections of this primary autonomic center in the medulla oblongata. PMID- 7537000 TI - Ontogeny of galanin-immunoreactive neurons in the central nervous system of the chicken. AB - 1. The ontogenic development and differentiation of the galanin-immunoreactive (GA-IR) neuron systems in the avian brain was studied by the aid of immunocytochemistry. 2. The first traces of GA-immunoreactivity can be detected already on day 2 of embryonic life within the neural tube, and the system appears to be fully developed around day 16. 3. GA-IR neuron system develops parallel in the hypothalamus and in extrahypothalamic sites. 4. The first well-defined groups of GA-IR perikarya are visible within the diencephalon and myelencephalon as early as day 6. 5. In the embryonic hypothalamus, the periventricular and the tuberal area are especially rich in GA-IR neurons, while in extrahypothalamic regions, the mesencephalon and lower brain stem is prominent in this respect. 6. Since similarities have been observed in developmental patterns of the GA-IR system and the releasing-hormonal systems, it might be assumed that the galaninergic system might have functional relationship with these latter neuronal structures during development. PMID- 7537003 TI - Gamma globulin administration in relapsing Clostridium difficile-induced pseudomembranous colitis with a defective antibody response to toxin A. AB - A 53-year-old woman suffered six episodes of Clostridium difficile pseudomembranous colitis. The serological follow-up demonstrated the absence of a rise in IgG and IgA to toxin A. Human pooled gamma globulin was administered during the fifth relapse and raised IgG levels to toxin A. Normal stools reappeared a week later. The role of the antibodies to toxin A and gamma globulin in C. difficile colitis are discussed. PMID- 7537002 TI - Long-term effect of perinatal neurochemical lesions of brain monoaminergic neurons on body growth, growth hormone secretion and thyroid activity. AB - In the present experiments, extended studies were performed on long-term effect of perinatal neurotoxic damage of brain monoaminergic neurons exerted on Grown Hormone (GH) and on Thyrotrophic Hormone (TSH) secretion. Neurotoxins were applied for selective destruction of the different components of the monoaminergic neuron system. Deficient GH secretion and reduced TSH-mobilizing capacity were observed in consequence of perinatal injury of dopaminergic neurons, meanwhile in perinatal serotonergic lesion bearing rats, reduced GH secretion was associated with increased reactivity of the TSH-mobilizing mechanism. The results show that perinatal damage of monoaminergic neurons induce long-lasting alteration of the neural mechanisms regulating GH and TSH secretion. PMID- 7537004 TI - Pityrosporum ovale in healthy children, infantile seborrhoeic dermatitis and atopic dermatitis. AB - The occurrence of Pityrosporum ovale was studied in healthy children, children with infantile seborrhoeic dermatitis (ISD) and in patients with atopic dermatitis (AD). Twenty children with ISD and twenty healthy infants were subjected to culture for P. ovale. Positive cultures were found in 18 of 20 infants with ISD, compared with 4 of 20 controls. The same culture medium containing olive oil as one of the lipids was used to evaluate the frequency of positive P. ovale cultures in 60 patients with AD, 40 patients with rhinoconjunctivitis and/or asthma (RA) and 40 children and young adults with no atopic history (HC). The results of the quantitative cultures from the forehead did not differ between the groups. P. ovale cultures were positive in 0-20% of children aged 0-10 years and in 60-90% of the 11-20-year-old subjects. Positive P. ovale cultures were found in 87% of 138 healthy children aged 2 months to 15 years when cultures were performed on a medium containing whole fat cows' milk as one lipid source. The largest number of colonies was found among children aged 2 23 months and among children older than 9 years. The occurrence of specific IgE antibodies to P. ovale was evaluated with a skin prick test (SPT) and RAST and compared in 3 groups (AD, RA, HC) of patients aged 0-20 years. Specific IgE were found most often in patients with AD. In patients with AD on different parts of the body, 15% had a positive SPT to P. ovale. In another group of patients, aged 14-53 years, with AD localised mainly to the head and neck area, the SPT was positive in 55% of the patients. Sera from 13 patients with positive SPT to P. ovale were further analysed with IgE immunoblotting using both P. ovale and C. albicans antigens. Simultaneous IgE-binding to both these yeasts was found in 5 sera and these were analysed with RAST-inhibition. Cross-reacting IgE antibodies to P. ovale and C. albicans were found in two of these sera. Cross-reacting sera were pooled and used as an IgE probe in crossed radioimmunoelectrophoresis and Tandem-crossed immunoelectrophoresis. Cross-reacting epitopes were suggested to be located in the mannan polysaccharide of C. albicans and in a high molecular weight fraction of P. ovale.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7537005 TI - Oxygen metabolism changes and outcome in response to immediate colloid treatment in the endotoxaemic rat. AB - A colloid (Hespan) fluid regimen in a 4 h rat model of endotoxaemia was used to prevent the development of the early hypodynamic phase of shock. Groups (N = 10 each) of isoflurane-anaesthetized, male Sprague-Dawley rats received either 1) E. coli endotoxin (E, 20 mg.kg-1 BW, i.v.), 2) 0.9% saline (S), 3) endotoxin + Hespan (E + H), or 4) saline + Hespan (S + H). After a 30 min baseline, 15 ml of 6% hetastarch (Hespan) were infused over 1 h beginning 1 min after endotoxin or saline. Pulmonary artery wedge pressures suggested no fluid overload in the E + H or S + H groups. By the end of the study, there were six spontaneous deaths in the E group vs. no deaths in the other groups. However, despite successful prevention of the early hypodynamic response together with increased cardiac output, increased oxygen delivery, decreased oxygen extraction, and sustained normal oxygen consumption in the E + H group, this fluid regimen failed to prevent significant and progressive acidaemia and hyperlactataemia. Also, by 4 h the E + H group exhibited declining blood pressures, marked hypoglycaemia, and significant small intestinal damage. Our results indicate that the early hypotensive, hypodynamic period is not crucial for the development of significant pathology in endotoxaemia, and that early flow-dependency of whole body oxygen uptake is not inherent to the early response to endotoxin in this model. PMID- 7537010 TI - [Role of intracellular calcium in the programmed cell death of prostatic cancer cells]. AB - The growth of any tissue depends on the quantitative relationship between the rate of cell proliferation and cell death. In normal adults tissues, this steady state balance is regulated by a series of both systemic hormones and local growth factors. Programmed cell death, also called apoptosis, is one of the two pathways for cell death. In programmed cell death, specific intracellular signals induce the cell to undergo an active, energy-dependent process leading to a cascade of biochemical and morphologic events that result in the irreversible fragmentation of genomic DNA and then of the cell itself. Biochemical and morphological studies have demonstrated that the involution of the normal, hyperplastic and cancerous prostate after castration is the result of programmed cell death. Numerous group have studied the role of calcium in programmed cell death's triggering. In androgen-independent prostatic cell it had been proved that it was possible to trigger programmed cell death by inducing a sustained elevation of the intracellular calcium. Actually, some features tend to explain more precisely the exact role of calcium in apoptosis of prostatic cells. PMID- 7537006 TI - Changes in end-tidal carbon dioxide tension following sodium bicarbonate administration: correlation with cardiac output and haemoglobin concentration. AB - An intravenous administration of sodium bicarbonate (NaHCO3) forms excess CO2, resulting in an immediate increase in end-tidal carbon dioxide tension (PETCO2). We hypothesized that the time until PETCO2 reached a maximum, and the magnitude of the increase in PETCO2 are influenced by cardiac output and haemoglobin concentration, respectively. To test this hypothesis, we examined changes in PETCO2 following an intravenous administration of NaHCO3 at different levels of cardiac output and haemoglobin concentration. We administered 0.2 mmol.kg-1 of 8.4% NaHCO3 into the vena cava in 15 anesthetized dogs under mechanical ventilation of 20 breaths per min. Cardiac output was increased by dopamine infusion, and decreased by blood withdrawal under halothane anaesthesia. Haemoglobin concentrations were changed by haemodilution with hydroxyethyl starch. When control measurements were taken, time-max (the time until the increase in PETCO2 reached a maximum) was 4 +/- 0.2 breaths-time, and delta CO2 max (the magnitude of the increase in PETCO2) was 0.90 +/- 0.04 kPa (6.6 +/- 0.3 mmHg). Cardiac output was inversely correlated with time-max (r = 0.94, P < 0.0001), while it revealed a poor correlation with delta CO2-max. Haemoglobin concentration showed a significant correlation with delta CO2-max (r = 0.736, P < 0.005), but not with time-max. We concluded that the time course and the magnitude of changes in PETCO2 following intravenous administration of NaHCO3 reflect changes in cardiac output and haemoglobin concentration, respectively. PMID- 7537008 TI - Histamine release in man. 1975. PMID- 7537009 TI - Role of intra- and extracellular calcium in histamine release from rat peritoneal mast cells. 1981. PMID- 7537011 TI - [Organ culture of human tissue as study model of hormonal and pharmacological regulation of benign prostatic hyperplasia and of prostatic cancer]. AB - We have established organ cultures of human prostate in vitro analysis of the hormone responsiveness of prostatic carcinoma. The influence of dihydrotestosterone (DHT), the epidermal growth factor (EGF) and prolactin (PRL) was assessed on the proliferation rate of 30 benign human prostatic hyperplasias (BPH) maintained for 48 hours as short-term primary cultures. The influence exerted by the EGF, the DHT and the PRL on the BPH cell proliferation was characterized by adding these compounds alone or in combination to the culture media. The proliferation rate was assessed by means of histo-autoradiographical nuclear labeling with titriated thymidine. The results show that of the DHT, EGF and PRL, the first two induced a statistically significant increase in the cell proliferation rate in a proportion of BPH significantly higher than the proportion of BPHs whose cell proliferation was increased significantly by the PRL. Furthermore, the PRL was able to antagonize the EGF-induced stimulatory effect on the BPH glandular cell proliferation rate. Lastly, the EGF- and PRL mediated effects on the BPH glandular cell proliferation correlated with each other, while the DHT-mediated did not correlate with either the PRL- or the EGF mediated effects. This model of organ, culture provide a basis for developing a method for in vitro testing of the individual hormone and drug responsiveness of a prostatic carcinoma. PMID- 7537012 TI - [Predictive value of the serum PSA level in patients having undergone neo adjuvant hormone treatment before radical prostatectomy]. AB - Great interest has been generated recently in preoperative androgen deprivation for clinical stage B ou C (T2 ou T3) prostate cancer. The influence of neoadjuvant hormonal therapy on down-staging and down-grading is still controversial. To assess the influence of preoperative androgen deprivation on serum PSA levels, we compared pre- and post-treatment serum PSA levels in 54 patients who received complete pre-operative androgen blockade (LHRH agonist + flutamide) 3 months prior to surgery. All patients with a pretreatment PSA > 20 ng/ml had extra-prostatic disease excepted two patients who presented lesions of acute prostatitis with adenocarcinoma. After hormonal deprivation, 51/54 patients experienced a return of PSA to normal values (< 4 ng/ml). Among this patient, 33 had undetectable PSA levels (< 0.25 ng/ml). 90% of the patients with undetectable had tumor confined to the gland (pT2/B). On the other hand, patients who still have PSA > 4 ng/ml after hormonal deprivation, had extra-prostatic cancer (pT3 pT4). Thus, PSA levels after 3 months neo-adjuvant hormonal treatment might have a useful predictive value in patients selection for radical surgery. PMID- 7537007 TI - Treatment of cerebral arteriovenous malformations by neuroradiological intervention and surgical resection. AB - We present the results of a series of 105 patients with cerebral arteriovenous malformations (AVMs) who were treated by a combined protocol including endovascular occlusive measures and open surgical resection. 25 patients were treated only by surgical intervention, 72 patients underwent preoperative endovascular embolization and 8 patients were treated only by embolization, seven of which were only treated for palliation. After superselective angiography the vascular territories suitable for endovascular or microsurgical approach were defined, and in most cases these territories were complementary to each other. In 56 cases, only one embolization was necessary and due to an advantageous co localization of the departments the whole combined endovascular/neurosurgical procedure was done in one anesthesia. If several endovascular sessions were necessary (16 patients), the resection was mostly carried out immediately after the last neuroradiological session in the same anesthesia with total time of such combined procedure now averaging about 7 hours. According to the proposed grading system by Spetzler we treated 25 grade 1, 24 grade 2, 40 grade 3, 11 grade 4, and 5 grade 5 lesions. The overall success rate defined as complete resection without additional permanent neurological deficit was 89.6% (87 out of 97 surgical cases). The benefits of such combined approach to cerebral AVMs become apparent in shortened and safer surgical procedures as well as in a low complication rate. PMID- 7537014 TI - [PSA and anatomopathological stage of prostatic cancer]. AB - Accurate preoperative staging is important for adequate selection of patients for radical prostatectomy. We reviewed the preoperative PSA levels of 214 patients who underwent radical retropubic prostatectomy and analysed the results in relation to pathological stage. 32 (15%) patients had a PSA level below 4 ng/ml; nevertheless 8 of them had already a extracapsular disease and one present with positive lymph nodes. Thus, although a specificity of 90%, the sensitivity is only 18%. 98 (46%) patients had PSA levels beyond 10 ng/ml of which 47 (48%) had extracapsular tumor; this gives a specificity of 62% and a sensitivity of 59%. Finally 35 (16%) patients had a PSA over 20 ng/ml of which 18 (51%) present a extracapsular disease thus the specificity is 87% but the sensibility remains low at 23%. We concluded that although PSA levels are grossly related to the pathological stage, that relation is not reliable to distinguish patients with organ-confined from those who have extracapsular tumor extension and for either definition of a positive PSA level many patients would be denied a curative operation. Therefore, PSA value cannot be used to decide whether to recommend radical prostatectomy for potential cure. PMID- 7537013 TI - [Is a single prostatic biopsy sufficient for selection of medical treatment in benign prostatic hypertrophy?]. AB - Medical treatments in BPH (5-alpha reductase inhibitors and alpha-blockers) have been extensively studied in the last years, but their clinical outcome is unpredictible. Therefore, a better selection of the patients before prescribing these medical treatment of BPH is certainly needed. The variability in tissue composition in BPH may partially explain the differences observed in clinical outcome after medical therapy. In the present study, we have quantified the percentage of the different tissue components (smooth muscle, epithelial cells) in a prostatic biopsy and in the corresponding whole-adenoma in 14 patients operated on of a retropubic adenomectomy. Immunostaining with actin anti-actin was used to stain the smooth muscle cells and with anti-PSA for the epithelial cells components. Quantification was made using a computerized image analysis system. Our results indicates that a statistically significative difference in the percentage of smooth muscle cells (p < 0.02) exists when comparing a single biopsy and the corresponding adenomas. In contrast, no difference was found in the percentage of epithelial cells, nor for the glandular acini between a single biopsy and the entire adenoma (paired t-test). Using a multivariate matrix, a statistically significative correlation was found between the percentage of smooth muscle cells in the biopsies and the prostatic adenomas in toto. In conclusion, our study demonstrates that major differences in the percentage of the different tissue components exist between different prostatic adenomas. A single prostatic biopsy could help in selecting patients prior to medical therapy for BPH. PMID- 7537015 TI - Cyst fluid analysis in the differential diagnosis of pancreatic cysts: a new approach to the preoperative assessment of pancreatic cystic lesions. AB - Pancreatic cysts include pseudocysts, serous tumors, and mucinous tumors, some of which are malignant. The clinical and radiologic features of these lesions may not permit a preoperative diagnosis. Analysis of percutaneous cyst fluid aspirates for cytologic findings, tumor markers, enzymes, and viscosity has been proposed as an aid to differential diagnosis. The combination of cytologic findings, carcinoembryonic antigen level, and viscosity will usually differentiate mucinous from nonmucinous cysts, whereas cytologic findings and levels of tumor markers CA 15-3 and CA 72-4 are useful to predict malignancy. Pseudocysts can be identified by a combination of cytologic features; levels of tumor markers, including NB/70K; and levels of enzymes, including amylase isoenzymes and leukocyte esterase. Serous tumors are a diagnosis of exclusion, although in 50% of cases the cytologic findings will indicate a serous tumor. When compared with conventional methods of differentiating pancreatic cysts, cyst fluid analysis is a promising new approach to the evaluation of these diagnostically difficult lesions. PMID- 7537016 TI - Biliary sludge after liver transplantation: 2. Treatment with interventional techniques versus surgery and/or oral chemolysis. AB - OBJECTIVE: Interventional treatment of biliary sludge in liver transplant recipients includes transhepatic biliary drainage and saline irrigation, catheter chemolysis and/or basket extraction, and endoscopic intervention. The purpose of this study was to compare these interventional procedures with oral chemolysis and with surgical treatment of biliary sludge in order to evaluate the effectiveness of interventional procedures as an alternative to surgery in the treatment of this complication. MATERIALS AND METHODS: We retrospectively evaluated the outcome of several forms of treatment for biliary sludge occurring after liver transplantation in 49 cases. Treatments included oral chemolysis with chenodeoxycholic acid (n = 35), percutaneous transhepatic biliary drainage (n = 13) followed by irrigation with heparinized saline solution (n = 4), intraluminal chemolysis with glycero-octanoate-carnosine and bile salts-EDTA (n = 3) and/or basket extraction (n = 5), and endoscopic intervention (n = 2) or surgery (n = 26). Oral chemolysis was attempted in all cases of biliary sludge if no other complications were present. If this conservative treatment failed and the sludge was limited to the main bile ducts, interventional procedures were attempted. Surgical removal of the sludge (n = 15) or retransplantation (n = 5) without any attempt at prior nonsurgical treatment was performed if concomitant complications were present (n = 14) or if the extent of the sludge was considered too time consuming for an interventional attempt (n = 6). The six patients in whom nonsurgical treatment failed underwent surgery. Treatment was considered successful if cholangiograms obtained after therapy showed no more evidence of sludge. Treatment was considered a failure if biliary sludge was shown after therapy by means of cholangiography, surgery, or autopsy. RESULTS: Complete disappearance of biliary sludge as a result of oral chemolysis was achieved in 14 (40%) of 35 cases. Interventional procedures were performed in 15 of the patients in whom oral treatment failed. After percutaneous transhepatic biliary drainage, the sludge was successfully removed by chemolysis with glycero-octanoate carnosine in three cases, by basket extraction in one case, and by a combination of chemolysis and basket extraction in three cases. In two other cases, underlying recurrent tumor was treated palliatively with percutaneous transhepatic biliary drainage or endoscopic stenting. Irrigation with heparinized saline solution failed in four cases, and percutaneous or endoscopic basket extraction failed in one case each. Surgical treatment was successful in 18 (86%) of 21 cases, and retransplantation was successfully done in five patients. In all, interventional techniques were used in 43% of the patients with biliary sludge who could not be treated successfully with oral chemolysis, and the overall success rate was 60%. CONCLUSION: Interventional techniques are effective therapeutic alternatives for treating biliary sludge occurring after liver transplantation and should be considered before surgical procedures. An indication for interventional procedures in biliary sludge is lack of success of oral chemolysis and an absence of other complications that require surgery or retransplantation. PMID- 7537017 TI - Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy. AB - Recent evidence suggests that patients with chronic hepatitis C virus (CHCV) who respond to interferon-alpha (IFN) therapy have a lower hepatic iron concentration than those who do not. The object of this study was to assess the concentration and distribution of iron in liver biopsies from 15 patients with CHCV seen at the authors' medical center between June 1992 and March 1993. Patients with complete response to IFN were compared to those with non-complete response with respect to quantitative hepatic iron concentration, serum iron indices, and a detailed analysis of histologic features of hematoxylin-and-eosin and iron-stained pre-IFN biopsies. Patients with non-complete response had significantly higher scores for stainable iron in sinusoidal cells (P = .02) and portal tracts (P = .05) than did patients with complete response. Total hepatic iron scores, mean quantitative hepatic iron, and mean serum ferritin were higher in patients with noncomplete response, but the differences were not significant. In conclusion, iron deposition in sinusoidal cells and portal tracts is significantly less frequent in patients with complete response to IFN than in those with poor or no response, and may be a useful, objective predictor of response to IFN therapy. PMID- 7537019 TI - New syndrome of macrocephaly, hypertelorism, short limbs, hearing loss, and developmental delay. AB - We describe a boy with an apparently unique constellation of anomalies, including macrocephaly, short stature, relatively short limbs, hearing loss, developmental delay, sparse anterior scalp hair, hypertelorism, downslanting palpebral fissures, and a short nose with a broad, flat nasal bridge and anteverted nares. Chromosomes were normal and radiographs failed to show a bone dysplasia. We conclude that this represents a new syndrome. PMID- 7537018 TI - Genetic linkage for Darier disease (keratosis follicularis). AB - Darier disease is an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion. Recent data have provided evidence for linkage of the Darier disease locus to 12q23-24.1 in British families. We have carried out linkage analysis using the 12q markers D12S58, D12S84, D12S79, D12S86, PLA2, and D12S63 in 6 Canadian families. Pairwise linkage analysis generated positive lod scores at all 6 markers at various recombination fractions, and each family showed positive lod scores with more than one marker. The peak lod score in the multipoint analysis (Zmax) was 5.5 in the interval between markers D12S58 and D12S84. These positive lod scores in North American families of varied European ancestry confirm the location of the Darier disease gene, and suggest genetic homogeneity. The future identification and sequencing of the gene responsible for Darier disease should lead to improved understanding of the disease and of keratinocyte adhesion in general. PMID- 7537021 TI - Clinical appearance and treatment strategies for human papillomavirus: a gynecologic perspective. PMID- 7537020 TI - MOMs (multiples of the median) and DADs (discriminant aneuploidy detection): improved specificity and cost-effectiveness of biochemical screening for aneuploidy with DADs. AB - OBJECTIVE: Our purpose was to assess the efficacy of double- and triple-screening paradigms for Down syndrome and to develop a more logical, statistical approach to risk prediction that will decrease the cost of screening and allow the incorporation of new parameters appropriately weighted for their contribution. STUDY DESIGN: Data from 24,504 patients who had biochemical screening for Down syndrome by single (alpha-fetoprotein), double (alpha-fetoprotein, beta-human chorionic gonadotropin), or triple screening (alpha-fetoprotein, beta-human chorionic gonadotropin, unconjugated estriol) who had complete outcome information were analyzed by (1) existing gaussian-based methods, (2) the Glasgow ratio method, and (3) a new statistical approach (i.e., directly adjusted data sets for discriminant aneuploidy detection [DADs]) RESULTS: By use of individual risk-based thresholds for "at risk" status, both double and triple screening performed far better than single screening, but the percentages of patients at risk varied widely. When the percentages at risk were held constant, the sensitivity of double and triple screenings was similar, suggesting that there are no benefits of using estriol as a third marker. For 25,000 patients the use of only alpha-fetoprotein and beta-human chorionic gonadotropin would save about $500,000, with no decrease in sensitivity. With the DADs approach a statistically sound model giving more stable estimates was developed that permits each factor to be analyzed for its own explained proportion of variance and allows each parameter to have different weighting. For this data set the same sensitivity was seen with, conservatively, a 1% reduction in the percentage of patients at risk, which would reduce by 250 the number of amniocenteses, at a further savings of about $400,000. CONCLUSIONS: By use of existing methods, double screening is equally as effective as triple screening, so that the expense of estriol is unnecessary. The DADs approach, by allowing for variable weighting of parameters, lowers the at risk percentage and will permit a much more flexible approach as new parameters become available. Changing to DADs and eliminating estriol should achieve higher specificity for the same sensitivity and save, conservatively, about $900,000 in this series. Extrapolated nationally, if confirmed, the annual savings could approach $72,000,000. PMID- 7537022 TI - Interferons: biochemistry and mechanisms of action. PMID- 7537023 TI - Human papillomavirus infection and therapy with interferon. AB - The studies summarized have shown that therapy of condylomata acuminata with interferon is effective. The route of administration does not appear to influence the results; the intralesional, intramuscular, and subcutaneous routes were effective. Additional research is required to determine whether the natural interferon or recombinant product is superior. The appropriate administration schedule may also not have been attained. PMID- 7537024 TI - Atypical adenomatous hyperplasia of the prostate. Relationship with carcinoma in 217 whole-mount radical prostatectomies. AB - Atypical adenomatous hyperplasia (AAH) of the prostate is a microscopic proliferation of small glands that may be mistaken for adenocarcinoma. The extent and multicentricity of this histopathologic lesion have not been fully defined, and the spatial relationship with carcinoma has not been described in whole-mount surgical specimens. We sought to determine whether the extent and zonal location of AAH is related to prostate cancer by evaluating 217 totally embedded radical prostatectomy specimens with cancer. All but 17 patients had clinically localized cancer, and none had received preoperative therapy. The number of foci and volume of AAH were measured using a grid-counting method; proximity to cancer was recorded as either less than or equal to 2 mm from cancer or greater than 2 mm from cancer. AAH was identified in 23.0% of cases and was more frequent in the transition zone (19.8% of cases) than in the nontransition (peripheral and central) zone (6.0%). It was found within 2 mm of cancer in 34% of cases of AAH, including 30% of cases in the transition zone and 31% cases in the nontrasition zone. The number of foci of AAH in the transition zone was always greater than that in the nontransition zone, regardless of whether it was within 2 mm of cancer or more than 2 mm from cancer. AAH was frequently multicentric (46% of cases), especially in the transition zone (47% of transition zone cases) compared with the nontransition zone (23% of nontransition zone cases). The mean volume of AAH was 0.029 cc (range, 0-1.29 cc) and was much higher in the transition zone than the nontransition zone, regardless of whether it was within 2 mm of cancer or more than 2 mm from cancer. In cases of AAH within 2 mm of cancer, the volume was lower than in cases more than 2 mm from cancer; this was true regardless of zonal location. AAH was more common in older patients and in those with greater prostatic weight, higher prostatic volume, greater percentage of nodular hyperplasia, greater volume of cancer, greater percent of Gleason patterns 4 and 5 cancer, higher volume of prostatic intraepithelial neoplasia, and higher serum prostate-specific antigen level. There was no correlation of number of foci of AAH or volume of AAH with pathologic stage, seminal vesicle invasion, Gleason primary pattern or score, nuclear grade, perineural invasion by tumor, or DNA ploidy. Our results indicate that AAH is usually found iin the transition zone in association with nodular hyperplasia and is often multicentric. The extent and zonal distribution of AAH and carcinoma show a weak but significant association. PMID- 7537025 TI - Malonic aciduria. AB - Three infants with malonic aciduria are reported, one of whom could be studied in detail. All children had severe and progressive encephalopathy with intermittent ketoacidosis and hypoglycemia. One infant died of cardiomyopathy. Biochemical studies revealed that one patient had neither malonyl-CoA decarboxylase nor glutaryl-CoA dehydrogenase deficiencies. This variant of malonic aciduria is different from that of four patients previously reported, both in its clinical and biochemical presentations. The biochemical pathology of this variant malonic aciduria is unknown. PMID- 7537026 TI - Plasma N-methylhistamine concentration as an indicator of histamine release by intravenous d-tubocurarine in humans: preliminary study in five patients by radioimmunoassay kits. AB - Histamine disappears rapidly from plasma because of its short half-life. Because a metabolite of histamine, N-methylhistamine, is stable and has a longer half life, determination of its plasma concentration can be useful in retrospective determination of histamine release. In this study, we measured plasma histamine and N-methylhistamine concentrations after d-tubocurarine (dTc) administration to evaluate the use of plasma N-methylhistamine measurement for confirming histamine release. After the induction of anesthesia, five patients received dTc, 0.8 mg/kg intravenously. A radioimmunoassay kit was used to determine plasma histamine and N-methylhistamine before and 1, 3, 5, and 10 min after administration of dTc. Histamine released by the injection of dTc reached a maximum level at 1 min, but decreased rapidly, whereas N-methylhistamine increased at 1 min and remained increased for at least 10 min. Good correlations were found between histamine concentration at 1 min and N-methylhistamine concentrations at 1, 3, 5, and 10 min, especially r = 0.999 (n = 5) at 10 min. N-Methylhistamine measurement with this kit can ascertain histamine release retrospectively in a semiquantitative manner. PMID- 7537027 TI - The effect of graded hemorrhage and intravascular volume replacement on systolic pressure variation in humans during mechanical and spontaneous ventilation. AB - In dogs and humans, the magnitude of the variation in systolic pressure (SPV) over the respiratory cycle during mechanical ventilation appears to be inversely related to intravascular volume. Also reported to correlate with changes in volume status is delta down, the difference between systolic pressure at end expiration and the lowest value during the respiratory cycle. These variables were examined during graded hemorrhage in eight anesthetized, mechanically ventilated subjects, and seven awake, spontaneously breathing subjects. SPV and delta down were measured in sequence at baseline, after 500 mL blood removal, after 1000 mL (total) blood removal, after 500 mL hetastarch replacement, after 1000 mL (total) hetastarch replacement, and after 500 mL normal saline (NS). Repeated-measures analysis of variance was used to test the significance of the change in SPV and delta down among the interventions. During mechanical ventilation, each 500-mL hemorrhage significantly increased SPV and delta down, and each 500-mL hetastarch infusion significantly decreased SPV and delta down. After hetastarch, both SPV and delta down were smaller than at baseline and may explain why the infusion of NS caused nonsignificant reductions in SPV and delta down. A SPV of 5 mm Hg or less, or a delta down of 2 mm Hg or less appeared to indicate minimal intravascular volume depletion. During spontaneous ventilation, delta down could not be determined accurately in several subjects, and SPV did not change in the appropriate direction in all cases of hemorrhage and volume infusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537028 TI - Hydroxyethyl starch versus lactated Ringer's solution in the chronic maternal fetal sheep preparation: a pharmacodynamic and pharmacokinetic study. AB - Administration of fluids intravenously prior to spinal and epidural analgesia in obstetrics is required to prevent maternal hypotension and fetal hypoxia. A colloid solution, such as hydroxyethyl starch (HES), might be preferable considering the capacity to stay intravascularly for a longer period. In this study the placental transfer of HES and the hemodynamic effects after infusion were investigated using a chronic maternal-fetal sheep preparation. Either 500 mL HES 10% or 750 mL lactated Ringer's solution (RL) was infused intravenously into the ewe over 30 min. Fetal and maternal blood were assayed for HES, blood gases, and acid-base status before and at regular intervals after infusion. Maternal and fetal cardiovascular variables were recorded continuously for 90 min. After HES infusion, maternal HES levels peaked at 30 min ranging from 6.9 mg/mL to 12.1 mg/mL and declined at 24 h to levels between 0.3 mg/mL and 2.8 mg/mL. Mean fetal HES concentrations remained below 0.3 mg/mL. Infusion of HES decreased hemoglobin (Hb) and plasma viscosity (PV) in the mother. Infusion of RL decreased Hb, but did not change PV. Infusion of HES significantly increased uterine blood flow (UBF), cardiac output (CO), total oxygen-delivery capacity, and uterine artery oxygen delivery. In contrast, infusion of RL did not significantly change these variables. Infusion of HES increases UBF, CO, and uterine and total oxygen carrying capacity in the pregnant ewe. No significant transplacental transfer of HES was shown. PMID- 7537030 TI - Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase. AB - Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses. PMID- 7537031 TI - Dietary carbohydrates: effects on self-selection, plasma glucose and insulin, and brain indoleaminergic systems in rat. AB - The aim of the study was to investigate the effect of different dietary carbohydrates such as corn starch, sucrose, fructose and glucose on carbohydrate and protein self-selection and on arterial and venous concentrations of glucose and insulin, and brain indoleamines in rats. Fructose and sucrose feeding induced the lowest food intakes which were due respectively to a lower carbohydrate and protein selection. The present data showed that feeding with dietary glucose as the main carbohydrate source gave the highest glycemic response, the lowest one being found with fructose and corn starch, and an intermediate one with sucrose feeding. The insulin response to the dietary carbohydrates followed a somewhat different pattern with the highest insulin secretion observed after fructose feeding whereas highly variable and inconsistent results were obtained following corn starch, sucrose and glucose feeding. Feeding chemically different sugars was also characterized by decreased serotonin synthesis in the raphe nuclei, brainstem and thalamus, and increased 5-HT synthesis in the hypothalamus of rats fed fructose when compared to glucose fed animals. The present results highlight the importance of considering the nature of dietary carbohydrates in the regulation of feeding. PMID- 7537029 TI - Michellamine B, a novel plant alkaloid, inhibits human immunodeficiency virus induced cell killing by at least two distinct mechanisms. AB - Studies of the mechanism of action of michellamine B, a novel anti-human immunodeficiency virus (HIV) alkaloid from the tropical plant Ancistrocladus korupensis, have revealed that the compound acts at two distinct stages of the HIV life cycle. The compound had no direct effect on HIV virions and did not block the initial binding of HIV to target cells. Postinfection time course studies revealed that the agent partially inhibited HIV-induced cell killing and syncytium formation when added up to 48 h following acute infection; however, viral reproduction was fully inhibited only when the compound was added immediately after infection. Time-limited treatments of HIV-infected cells revealed that michellamine B had to be present continuously to provide maximum antiviral protection. HIV replication in cells in which infection was already fully established or in chronically infected cells was unaffected by michellamine B. Biochemical studies showed that michellamine B inhibited the enzymatic activities of reverse transcriptases (RTs) from both HIV type 1 and HIV type 2 as well as two different nonnucleoside drug-resistant RTs with specific amino acid substitutions. In addition, human DNA polymerases alpha and beta were inhibited by the alkaloid. Michellamine B exerted a potent dose-dependent inhibition of cell fusion in two independent cell-based fusion assays. Thus, michellamine B acts both at an early stage of the HIV life cycle by inhibiting RT as well as at later stages by inhibiting cellular fusion and syncytium formation. PMID- 7537032 TI - Epidermolysis bullosa: hereditary skin fragility diseases as paradigms in cell biology. AB - Recent research into the molecular basis of epidermolysis bullosa has provided a unique insight into a variety of mechanisms in normal cell biology, such as cell matrix interactions, and has uncovered an excellent model for studies on keratin intermediate filaments. The simplex forms of epidermolysis bullosa are caused by mutations in the genes for the basal epidermal keratins, K5 and K14. Most mutations affect highly conserved parts of the molecules, illustrating their importance in normal keratin filament assembly and integrity. Mutations in corresponding regions of the differentiation-associated keratins, K1 and K10 can also occur in epidermolytic ichthyosis. Both recessive and dominant forms of dystrophic epidermolysis bullosa result from mutations in an anchoring fibril collagen gene, COL7A1. Junctional epidermolysis bullosa is caused by mutations in the genes encoding different chains of the novel laminin isoform, nicein/kalinin, also known as laminin 5, which is associated with the anchoring filament hemidesmosome complex of the basement membrane zone. These recent findings strengthen the evidence for the role of nicein/kalinin and type VII collagen in adherence and stabilization of the dermo-epidermal junction. PMID- 7537033 TI - Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. AB - Mechanisms affecting mast cell and melanocyte growth and function are still poorly understood. This report summarizes the current state of knowledge on a recently described growth factor for both these cell types and for primitive haematopoietic stem cells. Stem cell factor (SCF), also named mast cell growth factor or kit-ligand, has only recently been cloned and has been shown to be encoded on human chromosome 12. It may be of specific importance in cutaneous physiology and pathology since it is produced by several cell types in the skin (e.g. fibroblasts, keratinocytes, endothelial cells) and since it affects melanocyte and mast cell growth, survival, secretion and adhesion as well as migration into tissues. Defects in the genes encoding for the SCF receptor (c-kit protein) have been shown to be responsible for human piebaldism. A pathogenetic role in mastocytosis has recently been proposed, but remains to be proven. SCF receptor expression is decreased on cells of some malignant cell lines compared to their physiological counterparts, making it unlikely that SCF is a key factor in malignant transformation and cellular hyperproliferation. In haematopoiesis, SCF acts primarily in concert with other growth factors, and we show here that alone in serum-free culture it has no effect on mast cell growth. Furthermore, there is evidence that besides SCF, additional mast cell growth factors are secreted by fibroblasts and keratinocytes, suggesting a complex orchestration of several growth factors in the regulation of cutaneous growth and differentiation in which SCF plays only one part. PMID- 7537034 TI - Enhancement of fibroblast-dependent mast cell growth in mice by a conditioned medium of keratinocyte-derived squamous cell carcinoma cells. AB - We investigated the increase in mast cell numbers at the sites of inoculation of keratinocyte-derived squamous cell carcinoma cell line (KCMH-1) cells in mice. A significant increase in the number of mast cells was observed at the sites of tumours developed at the sites of inoculation of the KCMH-1 cells. Enhancement of mast cell growth was observed by culturing bone marrow-derived mast cells (BMMC) on NIH/3T3 fibroblast monolayers in the presence of conditioned medium (TCM) obtained from KCMH-1. Activities of known factors for mast cell growth, such as interleukin-3 (IL-3), IL-4, IL-9, IL-10 and stem cell factor (SCF), were not detected in the TCM. Nerve growth factor (NGF) did not induce mast cell growth. Mast cell growth induced by the TCM needed 3T3 fibroblasts. These results suggest that KCMH-1 cells may produce a factor which induces mast cell growth with 3T3 fibroblasts, other than the already known mast cell growth factors. This may be the mechanism of mast cell accumulation at sites of tumours. PMID- 7537036 TI - Increased intrapancreatic trypsinogen activation in ischemia-induced experimental pancreatitis. AB - OBJECTIVE: The potential of pancreatic ischemia to cause acute pancreatitis as indicated by morphologic changes and ectopic trypsinogen activation was investigated. BACKGROUND: Experimental evidence has shown that pancreatic ischemia is important in the evolution of severe pancreatitis, but whether ischemia can initiate pancreatitis has been disputed. METHODS: Pancreatic ischemia was induced in rats by hemorrhagic hypotension (30 mm Hg for 30 min; n = 64). Changes of pancreatic microcirculatory perfusion were studied using diffuse reflectance spectroscopy. Serum amylase, trypsinogen activation peptide (TAP) in serum and pancreatic tissue, wet/dry weight ratio, and histology were determined over 24 hours and compared with sham-operated control subjects (n = 35). RESULTS: In control animals, serum amylase (47.9 +/- 2.1 units/L), serum (7.9 +/- 0.7 nmol/L) and tissue TAP (63.0 +/- 5.4 nmol/L x g), wet/dry weight ratio (2.8 +/- 0.1), and histology remained unchanged. Temporary hypotension markedly decreased pancreatic perfusion with incomplete recovery after reperfusion. Pancreatic isoamylase activity increased within 1 hour (110 +/- 5 units/L, p < 0.05) and further to 151 +/- 18 units/L at 24 hours. Tissue TAP was elevated at 1 hour (134 +/- 16 nmol/L x g, p < 0.05) and increased to 341 +/- 43 nmol/L x g (p < 0.001) after 24 hours, whereas serum TAP remained unchanged (8.3 +/- 0.5 nmol/L). Morphologic alterations included elevated wet/dry weight ratio (4.1 +/- 0.3, p < 0.01) and increased histologic scores for edema (p < 0.05) and acinar necrosis (p < 0.05) at 24 hours. Trypsinogen activation preceded the development of pancreatic necrosis. CONCLUSIONS: In addition to its potentiating role, severe pancreatic ischemia can play a pathogenetic role in the initiation of acute pancreatitis. PMID- 7537037 TI - Generation of monoclonal antibodies that distinguish between mesotheliomas and other tumor of the lung. AB - The accurate diagnosis of mesothelioma remains difficult despite advances of diagnostic technique. And specific monoclonal antibody (McAb) against mesothelioma have not been reported. In an attempt to develop mesothelioma specific McAb(s), spleen cells from a mouse immunized with isolated tumor cells were fused to a drug resistant mouse myeloma cell lines. Over 200 hybridomas were assayed for their preferential reactivity with mesothelioma cell lines or mesothelioma tumor biopsy tissues. Two monoclonal antibodies 2A3 and 4E1 were identified that bound 6/7 of the mesotheliomas, tested, but did not bind to the majority, 11/13 (for 2A3) and 12/13 (for 4E1), of other lung tumor types. Based upon western blot analysis of one and two-dimensional gels and upon the distribution pattern of the antibody recognized molecule in mesotheliomas and non mesothelioma lung tumors, 2A3 binds to the cell adhesion molecule CD44. While the specificity of 4E1 has not yet been unequivocally established it appears to recognize a variant form of the CD44 molecule. PMID- 7537038 TI - Effects of H2O2 on membrane potential and [Ca2+]i of cultured rat arterial smooth muscle cells. AB - The effect of 1 mmol/l H2O2 was studied on the membrane potential and [Ca2+]i with microelectrodes and the fura-2 technique, respectively. H2O2 induced a biphasic increase in [Ca2+]i with a fast transient peak and a subsequent plateau. H2O2 also led to a biphasic hyperpolarization of the cells with a similar time course. This was followed by a slight depolarization after wash-out of H2O2. External Ca2+ free solutions and treatment with the Ca2+ ionophore A23187 (1 mumol/l) abolished the effect of H2O2 on [Ca2+]i and almost entirely reduced the effect on the membrane potential. Phenylephrine (10 mumol/l) or A23187 also induced very similar biphasic hyperpolarizations of the membrane as H2O2 which were fully reversible after wash-out. It is concluded that H2O2 hyperpolarizes the membrane by opening of Ca2+ dependent K+ channels. PMID- 7537039 TI - Mitochondrial VDAC can be phosphorylated by cyclic AMP-dependent protein kinase. AB - The voltage dependent anion channel (VDAC) of the outer membrane of mitochondria is thought to play a role in transport of metabolites including ATP across mitochondrial membrane and modulate mitochondrial functions such as respiration. However, regulation of this anion channel is only poorly understood. In this paper we demonstrate that VDAC purified from rat liver mitochondria can be phosphorylated by the catalytic subunit of cAMP dependent protein kinase (PKA). PKA phosphorylates VDAC linearly up to fifteenfold in sixty minutes. The level of VDAC phosphorylation increases to twofold and sevenfold of control value after ten and thirty minutes of reaction, respectively. Data presented here suggest the possibility that voltage dependent anion channel of the outer membrane of mitochondria may be a target of PKA in vivo. PMID- 7537040 TI - Mast cell chemotactic activity of RANTES. AB - RANTES is a cytokine produced by activated T-lymphocytes that has been shown to exert chemotactic activity for memory-type CD4 T-lymphocytes and eosinophils. In this study, RANTES caused directional migration of human mast cells. When compared to other potential chemoattractants of the same cells, RANTES was found to be more potent than fibronectin and the c-kit receptor ligand, on a molar basis. This cytokine may be a common mechanism in allergic reactions which culminate in the selective migration of memory CD4 T-lymphocytes, eosinophils and mast cells at the tissue site. Asthma and allergic rhinitis may represent possible clinical examples. PMID- 7537041 TI - Tumor necrosis factor alpha and interleukin-1 beta but not interferon gamma induce vascular cell adhesion molecule-1 expression on primary cultured murine hepatocytes. AB - Inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-1 beta (IL-1 beta) play important roles in the mechanisms of hepatitis. The effects of these cytokines on the expression of vascular cell adhesion molecule-1 (VCAM-1) in hepatocytes were examined. TNF alpha and IL-1 beta but not IFN gamma or IL-6 induced VCAM-1 expression on primary cultured murine hepatocytes in a dose- and a time-dependent fashion. TNF alpha is significantly more effective than IL-1 beta on the induction of VCAM-1 expression. The results of RT-PCR demonstrate that these cytokines regulate VCAM-1 expression at mRNA level. These results suggest that TNF alpha and IL-1 beta participate in the pathogenesis of hepatitis via induction of VCAM-1 molecules on hepatocytes. PMID- 7537042 TI - Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-kappa B/Rel proteins. AB - The activity of the inducible nitric oxide synthase enzyme (iNOS) is tightly controlled, partly at the transcriptional level. We find NF-kappa B/Rel activation (p50-p50 and p50-p65) in RAW 264.7 macrophages after lipopolysaccharide treatment and binding to both NF-kappa B sites in the mouse iNOS promoter. To delineate the importance of NF-kappa B/Rel in iNOS gene transcription, we used an unusually direct approach to try to improve on the antioxidant-treatment or reporter techniques, namely the depletion of NF-kappa B/Rel activity through the use of a phosphorothioate-modified oligonucleotide containing three copies of the NF-kappa B consensus sequence. The reduction in NF kappa B/Rel activity (particularly that binding to the downstream of the two sites) was associated with a 50% reduction in NO output and a reduction in the quantity of the iNOS protein expressed. These results point to the probability that physiologically relevant NF-kappa B/Rel activators or repressors other than lipopolysaccharide might crucially affect the macrophage NO response. PMID- 7537035 TI - Nitric oxide. Novel biology with clinical relevance. AB - OBJECTIVE: The author provides the reader with a view of the regulation and function of nitric oxide (NO), based on the three distinct enzyme isoforms that synthesize NO. SUMMARY BACKGROUND DATA: Nitric oxide is a short-lived molecule exhibiting functions as diverse as neurotransmission and microbial killing. Recent advances in the characterization of the enzymes responsible for NO synthesis and in the understanding of how NO interacts with targets have led to new insights into the many facets of this diverse molecule. METHODS: Nitric oxide is produced by one of three enzyme isoforms of NO synthesis. These enzymes vary considerably in their distribution, regulation, and function. Accordingly, the NO synthesis or lack of NO production will have consequences unique to that isoform. Therefore, this review summarizes the regulation and function of NO generated by each of the three isoforms. RESULTS: Nitric oxide exhibits many unique characteristics that allow this molecule to perform so many functions. The amount, duration, and location of the NO synthesis will depend on the isoform of NO synthase expressed. For each isoform, there probably are disease processes in which deficiency states exist. For induced NO synthesis, states of overexpression exist. CONCLUSIONS: Understanding the regulation and function of the enzymes that produce NO and the unique characteristics of each enzyme isoform is likely to lead to therapeutic approaches to prevent or treat a number of diseases. PMID- 7537045 TI - Detection of dairy Leuconostoc strains using the polymerase chain reaction. AB - This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe. PMID- 7537043 TI - [Benzene emissions in the neighbourhood of filling stations]. AB - BTXE-Immissions (benzene, toluene, ethylbenzene, xylene) were measured in 32 flats in the neighbourhood of 12 filling stations in Frankfurt on the Main. Mean benzene-concentration in the indoor air nearby filling stations was 10.2 micrograms/m3, whereas in reference flats 5.6 micrograms/m3 were measured. Maximum levels were 22.4 micrograms/m3 and 8.0 micrograms/m3. Indoor air levels were slightly higher than outdoor air contaminations. High Toluene-concentrations were not caused by the filling stations but by other commercial enterprises, such as paint shops. Mean ethylbenzene or xylene-concentrations in the neighbourhood of filling stations did not differ from the concentrations measured in the reference flats. Because of the cancerogenicity of benzene the benzene contaminations in the neighbourhood of filling stations have to be lowered. The newly established German law to prevent gas leakage in filling stations will lead to some improvement in the future. PMID- 7537046 TI - Counting total cell numbers microscopically on stained films. AB - Two main sources of error in the conventional stained film cell count method, i.e. errors from imperfect spreading of the suspension and uneven distribution of cells in the film, have been eliminated by counting all the cells in the film. Staining solution and sodium alginate were added to the suspension before spreading over the slide. With a microsyringe, 20-microliters suspension was spread in two bands of ca 1.7 mm x 60 mm each. Then total cells could be counted easily in the x 100 magnification field by moving the slide in one direction. Results were satisfactory. Coefficients of variation were 2.6 and 1.1% in two separate yeast determinations each with five replicates. For counting higher magnifications, a suitable image analyser for automating the method is believed worthy of study. PMID- 7537044 TI - Tyrosine- versus serine-phosphorylation leads to conformational changes in a synthetic tau peptide. AB - One of the major immunodominant epitopes of the paired helical filaments (PHF) of Alzheimer's disease is the peptide sequence GAEIVYKSPVVSGD (T3), comprising amino acids 389-402 of the microtubule-associated protein, tau, when it is phosphorylated at the first serine residue. While the corresponding anti-PHF monoclonal antibody recognizes the peptide phosphorylated at either serine, it does not recognize the tyrosine-phosphorylated peptide. Here we describe the effect of serine- versus tyrosine-phosphorylation on the conformation of a synthetic tau peptide. While adding a phosphate to the serine residue has practically no impact on the structure of the non-phosphorylated peptide, phosphorylation of the tyrosine results in considerable conformational changes. PMID- 7537047 TI - Molecular cloning and expression in Escherichia coli of bleomycin-resistance gene from a methicillin-resistant Staphylococcus aureus and its association with IS431 mec. AB - A gene that confers bleomycin resistance was cloned from the chromosomal DNA of methicillin-resistant Staphylococcus aureus (MRSA) B-26 into the plasmid pUC18. It is of chromosomal origin rather than plasmid and exists in the chromosome making a cluster with the kanamycin-resistance gene. We found that the nucleotide sequence of the bleomycin-resistance gene from the chromosome of MRSA B-26 is identical to that from a staphylococcal plasmid, pUB110. The partial sequence of IS431mec was also found upstream from the DNA fragment containing the bleomycin- and kanamycin-resistance genes. PMID- 7537048 TI - Improved direct PCR screen for bacterial colonies: wooden toothpicks inhibit PCR amplification. PMID- 7537049 TI - Efficient method for preparing normal and tumor tissue for RNA extraction. PMID- 7537050 TI - [Oxidative stress in the treatment of tumors and AIDS. Iatrogenic disease caused by antineoplastic agents. Preventive action of antioxidant agents]. PMID- 7537051 TI - [Self-expandable prosthesis for palliative treatment of non-functioning gastrojejunostomy]. PMID- 7537053 TI - [Sialomucins in the follow-up of patients surgically treated for colorectal cancer: preliminary results of a prospective study]. AB - Preliminary results of a prospective study on sialomucin content found either at resection margins of colo-rectal cancer after curative surgery or in endoscopic biopsy specimens of colonic mucosa in thirteen patients selected for the follow up are reported. Though not statistically significant because of the small number of cases enrolled, preliminary data indicate that clinical application of differential mucin staining in colo-rectal neoplasms is potentially useful to predict subgroups of patients at risk for local recurrence as well as metachronous colo-rectal cancers. PMID- 7537054 TI - MPs back cannabis motion. PMID- 7537052 TI - Myelin basic protein-reactive T cells in multiple sclerosis: pathologic relevance and therapeutic targeting. AB - Autoreactive T cells specific for myelin proteins, such as myelin basic protein (MBP), are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In MS, these MBP-reactive T cells are activated and clonally expanded in vivo and found to accumulate in the brain compartment, suggesting their pathologic role in the disease. There is experimental evidence supporting the beliefs that MBP-reactive T cells are regulated in vivo by the clonotypic regulatory network. This concept has led to the paradigm of T cell vaccination where attenuated MBP-reactive T cells are used as vaccines to effectively prevent and treat experimental autoimmune encephalomyelitis, an animal model for MS. In this paper, the recent evidence regarding the pathologic relevance of MBP reactive T cell in MS is reviewed. In particular, we discuss our recent clinical trial in which patients with MS were vaccinated with inactivated autologous MBP reactive T cell clones to investigate the nature of clonotypic responses in vivo, and whether the responses are effective in depleting circulating MBP-reactive T cells in patients with MS. Our study presented in this paper demonstrated the successful depletion of MBP-reactive T cells by T cell vaccination and touched upon important issues related to the clinical application of T cell vaccination in humans. This review provides new insights into the current development in designing effective therapeutic strategies, such as T cell vaccination, to treat patients with MS and other autoimmune diseases. PMID- 7537055 TI - 16S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus Ehrlichia. AB - Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, > 95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia. PMID- 7537056 TI - A new genus of marine budding phototrophic bacteria, Rhodobium gen. nov., which includes Rhodobium orientis sp. nov. and Rhodobium marinum comb. nov. AB - Strains of a previously undescribed species of purple nonsulfur phototrophic bacteria were isolated from coastal seawater in Japan. These new isolates were gram-negative, motile, budding rods that contained lamellar intracytoplasmic membranes and produced pink to red cultures. Cell extracts of photosynthetic cultures exhibited absorption maxima at 377, 468, 500, 530, 591, 802, and 870 nm, indicating that bacterio-chlorophyll a and carotenoids of the spirilloxanthin series were present. The new isolates were halophilic, facultatively aerobic photoheterotrophs that grew anaerobically in the light or aerobically in the dark. Maximum growth occurred in the presence of 4 to 5% NaCl. Anaerobic growth in the dark with nitrate as a terminal electron acceptor also occurred. Various organic compounds were used as photosynthetic electron donors and carbon sources. Sulfate was used as a sulfur source. Both menaquinone 10 and ubiquinone 10 were produced; these quinones were the major quinones. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MB312T (T = type strain), a representative of the new phototrophs, was a member of a lineage that was distinct from members of the genus Rhodopseudomonas; Rhodopseudomonas marina was the closest relative. On the basis of the data described above, we propose the name Rhodobium orientis gen. nov., sp. nov. for the new isolates. We also propose that Rhodopseudomonas marina Imhoff 1983 should be transferred to the genus Rhodobium as Rhodobium marinum comb. nov. PMID- 7537057 TI - "Streptococcus milleri" strains displaying a gliding type of motility. AB - Isolates belonging to the "Streptococcus milleri" species group that appear to exhibit a gliding type of motility, which is expressed as spreading growth on certain types of agar media, are described. These strains resembled a biotype of "S. milleri" that is usually isolated from genitourinary sources and is notable for its ability to ferment a wide array of carbohydrates. This biotype, which is currently included in the species Streptococcus anginosus, has been implicated in cases of neonatal infection. The "S. milleri" isolates which we studied lacked any observable organelles of motility and gave negative results when they were tested in conventional motility test medium stab cultures. Colonies growing on certain agar media, however, spread over the surfaces of plates and increased in area with increasing time of incubation. Chocolate agar supported maximum spreading, while this characteristic was barely discernible on blood agar. Electron microscopy studies revealed that there was more production of extracellular glycocalyx by motile strains than by a nonmotile isolate having a similar biotype. The results of an analysis of 16S rRNA gene sequences suggested that the motile strains are closely related to S. anginosus and represent a distinct rRNA population within the "S. milleri" species complex. PMID- 7537058 TI - A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. AB - Partial sequences of the 16S rRNA genes of the type strains of nine species of the genus Nocardia were determined following the isolation and cloning of the amplified genes. These sequences were aligned with the sequences of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and Tsukamurella, and phylogenetic trees were inferred by using the Fitch-Margoliash and neighbor-joining methods. The genus Nocardia formed a distinct clade that was most closely associated with the genus Rhodococcus. The average level of sequence similarity found among the type strains of the Nocardia species was 97.2 +/- 0.7%. Two sublines were recognized within the Nocardia clade; one encompassed Nocardia asteroides and related species, and the other encompassed Nocardia otitidiscaviarum and allied taxa. Separation of the two sublines is based on differences in helix 37-1. The results of isoprenoid quinone analyses provided evidence that nocardiae can be distinguished from all other actinomycete taxa on the basis of their characteristic menaquinone profiles. Nocardiae typically contain hexahydrogenated menaquinones with eight isoprene units in which the two end units are cyclized. PMID- 7537059 TI - Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics. AB - The 16S rRNA gene of a new infectious agent, strain AS145T (T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris. PMID- 7537060 TI - Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov. AB - The Mycobacterium chelonae-like organism (MCLO) is a recently described member of the Mycobacterium fortuitum complex which causes posttraumatic skin infections and catheter sepsis. This taxon is a distinct group biochemically and has a unique mycolic acid profile as determined by high-performance liquid chromatography. Its phylogenetic relationships to other mycobacteria, however, have not been studied previously. We sequenced 1,062 bp of the 16S rRNA genes from three MCLO strains obtained from the American Type Culture Collection and compared our results with the sequences of previously described taxa of rapidly growing and slowly growing mycobacteria. Two biochemically typical strains (ATCC 49650T [T = type strain] and ATCC 49651) had identical sequences, while the sequence of a biochemically atypical strain (ATCC 49649) differed by 4 bp from the sequence of the two typical strains. The Hamming distances between these MCLO strains and related rapidly growing mycobacteria are comparable to the Hamming distances among taxa of rapidly growing mycobacteria established as species by DNA-DNA hybridization. We propose the name Mycobacterium mucogenicum sp. nov. for this new taxon because of the highly mucoid nature of most isolates on solid media. PMID- 7537061 TI - Roseobacter algicola sp. nov., a new marine bacterium isolated from the phycosphere of the toxin-producing dinoflagellate Prorocentrum lima. AB - We describe a new species on the basis of phenotypic characteristics and the results of an analysis of small-subunit rRNA sequences. Three strains of this organism were isolated from a culture of the toxin-producing dinoflagellate Prorocentrum lima. These bacteria are gram-negative, strictly aerobic, ovoid organisms that are motile by means of one or two subpolar flagella. They grow at temperatures ranging from 10 to 37 degrees C and in the presence of NaCl concentrations ranging from 0.1 to 2 M and have an absolute requirement for sodium ions. They are strictly aerobic with a nonfermentative type of metabolism and are not able to grow anaerobically in presence or absence of nitrate. They do not denitrify. They exhibit oxidase, catalase, gelatinase, esculinase, beta galactosidase, and (to a lesser extent) amylase activities. The three strains which we examined require thiamine and biotin for growth. They grow only when glucose, trehalose, saccharose, fructose, maltose, pyruvate, malate, citrate, esculin, 2-ketoglutarate, 5-ketogluconate, glutamate, or shikimate is present as a sole carbon source. The three strains have identical small-subunit rRNA sequences. A phylogenetic analysis of these sequences revealed that these bacteria belong to the alpha subdivision of the Proteobacteria and that they form a distinct and robust monophyletic group with Roseobacter denitrificans and Roseobacter litoralis. This result and the general phenotypic characteristics of the organisms place them in the genus Roseobacter, although they do not produce bacteriochlorophyll a, in contrast to previously described Roseobacter species.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537062 TI - Succiniclasticum ruminis gen. nov., sp. nov., a ruminal bacterium converting succinate to propionate as the sole energy-yielding mechanism. AB - A gram-negative, anaerobic, nonmotile, non-spore-forming, rod-shaped bacterium that fermented succinate quantitatively to propionate was isolated from a high dilution of rumen ingesta obtained from a dairy cow fed a production diet containing grass silage as the main roughage source. This organism did not grow on any of the following energy sources: 12 carbohydrates, pyruvate, lactate, 7 dicarboxylic acids, aspartate, citrate, and trans-aconitate. Both rumen fluid and yeast extract were necessary for good growth on succinate. The organism was negative for the following characteristics: production of propionate from threonine, protein digestion, sulfide production, nitrate reduction, catalase activity, and urease activity. There was no growth at 22 degrees C and reduced growth at 45 degrees C compared with growth at 39 degrees C. The DNA base composition was 52 mol% G + C. The complete 16S rRNA sequence (EMBL accession number, X81137) was obtained, and the phylogenetic relationships of the organism were determined. The most closely related genera were the genera Acidaminococcus and Phascolarctobacterium. The name proposed for this bacterium is Succiniclasticum ruminis gen. nov., sp. nov.; the type strain is strain SE10 (= DSM 9236). Additional isolation attempts revealed that S. ruminis is a common inhabitant of the rumina of cows that are fed production diets and of cows on pasture. PMID- 7537063 TI - Haloanaerobium alcaliphilum sp. nov., an anaerobic moderate halophile from the sediments of Great Salt Lake, Utah. AB - A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST (T = type strain). Strain GSLST grew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0). The optimum temperature for growth was 37 degrees C, and no growth occurred at 15 or 55 degrees C. The optimum salt concentration for growth was 10%. Strain GSLST required yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine. Strain GSLST was resistant to penicillin, D-cycloserine, tetracycline, and streptomycin. The G + C content of this isolate was 31 mol%. The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLST fermented glycine betaine to acetate and trimethylamine. All of these traits distinguish this organism from all previously described halophilic anaerobes. The 16S rRNA gene sequence of strain GSLST was found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens. Therefore, strain GSLST (= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum. PMID- 7537065 TI - Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever. AB - Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species. PMID- 7537064 TI - Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil producing well. AB - A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T (T = type strain), was isolated from an African oil-producing well. This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose). Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66 degrees C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459T is not similar to the three previously described Thermotoga species. Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described. On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii. The type strain of T. elfii is SEBR 6459 (= DSM 9442). PMID- 7537066 TI - Isolation and characterization of Rhodovulum strictum sp. nov. and some other purple nonsulfur bacteria from colored blooms in tidal and seawater pools. AB - Several strains of phototrophic purple nonsulfur bacteria were isolated from colored blooms occurring in tidal and seawater pools in Japan. All of these isolates had ovoid to rod-shaped cells that were motile by means of single polar flagella and contained vesicular intracytoplasmic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series. They produced ubiquinone 10 as the major quinone and contained straight-chain fatty acids, with C18:1 predominating. They were mesophilic, halophilic, and photoheterotrophic, utilized sulfide and thiosulfate as electron donors for phototrophic growth, and photoassimilated a wide variety of organic compounds as carbon sources. Our results suggested that all of these isolates are members of the recently described genus Rhodovulum. The isolates were classified into four groups (designated groups I through IV) on the basis of phenotypic and genotypic data. The group I isolates, which were the most abundant purple nonsulfur bacteria recovered from the blooms, grew in the presence of NaCl concentrations ranging from 0.5 to 3.0% (optimum NaCl concentration, 0.8%) and at pH values ranging from 7.5 to 9.0 (optimum pH, 8.0 to 8.5). On the basis of these unique physiological traits, together with genotypic and phylogenetic data, we propose that the group I isolates should be classified as members of a new species, Rhodovulum strictum. The group II isolates were identified definitely as Rhodovulum sulfidophilum, and the group III and IV isolates were phenotypically most similar to R. sulfidophilum and Rhodovulum adriaticum, respectively, but could be differentiated from these species by DNA-DNA pairing data. PMID- 7537067 TI - Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria. AB - In the past workers have isolated several pseudomonad strains which have been used for studies of anaerobic aromatic metabolism. The best studied of these strains are strains KB 740T (T = type strain) and K 172T. The taxonomic positions of these two organisms were determined by classical methods, including experiments to determine substrate spectrum, quinone type, and total fatty acid composition. Our results clearly excluded these strains from the authentic genus Pseudomonas, which belongs to the gamma subclass of the Proteobacteria. Instead, the properties of these organisms indicated that they belong to the beta subclass of the Proteobacteria. The sequences of the 16S ribosomal DNA genes confirmed this conclusion and indicated that strain K 172T represents a new species of the genus Thauera, Thauera aromatica, and that strain KB 740T represents a new species of the genus Azoarcus, Azoarcus evansii. PMID- 7537068 TI - Taxonomic study of bacteria isolated from plants: proposal of Sphingomonas rosa sp. nov., Sphingomonas pruni sp. nov., Sphingomonas asaccharolytica sp. nov., and Sphingomonas mali sp. nov. AB - The taxonomic positions of 10 strains of 3-ketolactose-forming bacteria which were isolated from the roots of plants (Rosa sp., Psychotria nairobiensis, Ardisia crispa, Prunus persica, and apple trees) were investigated. The DNA base compositions of these strains ranged from 64.0 to 65.7 mol%, the isoprenoid quinone of each strain was ubiquinone 10, 3-hydroxy fatty acids were lacking in the cellular fatty acids of these organisms, and all of the strains contained a sphingolipid with the long-chain base dihydrosphingosin. These are characteristics of the genus Sphingomonas. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with DNA-DNA hybridization and 16S ribosomal DNA sequence comparison data, we propose the following four new species of the genus Sphingomonas: Sphingomonas rosa (type strain, IFO 15208) for the strains isolated from rose plants and formerly named [Agrobacterium rhizogenes]; Sphingomonas pruni (type strain, IFO 15498) for the strains isolated from Prunus persica; and Sphingomonas asaccharolytica (type strain, IFO 15499) and Sphingomonas mali (type strain, IFO 15500) for the strains isolated from apple trees. Two strains which were isolated from Psychotria nairobiensis and formerly named [Chromobacterium lividum] were identified as Sphingomonas yanoikuyae strains. PMID- 7537069 TI - Taxonomic analysis of the tortoise mycoplasmas Mycoplasma agassizii and Mycoplasma testudinis by 16S rRNA gene sequence comparison. AB - The nucleotide sequences of the 16S rRNA genes of two mycoplasmas, Mycoplasma agassizii (proposed sp. nov.) and Mycoplasma testudinis, isolated from tortoises were determined and used for taxonomic comparisons. Signature nucleotide sequence motifs and overall sequence similarities to other mollicutes positioned these mycoplasmas in the M. hyorhinis and M. pneumoniae phylogenetic groups, respectively. A third, previously unrecognized tortoise mycoplasma was detected by 16S rRNA gene amplification and sequence analysis and was positioned in the M. fermentans phylogenetic group. The 16S rRNA gene of Acholeplasma laidlawii was similarly detected in a tortoise isolate, showing that diverse mollicutes can share the same family of reptilian host. PMID- 7537070 TI - A phylogenetic analysis of the genus Saccharomonospora conducted with 16S rRNA gene sequences. AB - Nearly complete sequences of 16S rRNA genes of representative strains of the genus Saccharomonospora were determined following the isolation and cloning of the amplified genes. The sequences were aligned with those of representatives of the family Pseudonocardiaceae, and a phylogenetic tree was inferred by the neighbor-joining method. The genus Saccharomonospora formed a distinct clade within the evolutionary radiation encompassed by the family Pseudonocardiaceae. The average nucleotide similarity value found between the type strains of the four validly described Saccharomonospora species was 97.5% +/- 1.0%. The most distant relationship was found between Saccharomonospora azurea and Saccharomonospora viridis K73 (96.3% similarity). In contrast, Saccharomonospora azurea K161 and "Saccharomonospora caesia" K163 had identical 16S rRNA gene sequences. The nucleotide sequence data suggest that the genus Saccharomonospora contains several new centers of variation. PMID- 7537071 TI - Lentzea gen. nov., a new genus of the order Actinomycetales. AB - We describe a new genus of mesophilic actinomycetes, for which we propose the name Lentzea. The strains of this genus form abundant aerial hyphae that fragment into rod-shaped elements. Whole-cell hydrolysates contain the meso isomer of diaminopimelic acid and no characteristic sugar (wall chemotype III). The phospholipid pattern type is type PII (phosphatidylethanolamine is the characteristic phospholipid); the major menaquinone is MK-9. The fatty acid profile comprises saturated, unsaturated, and branched-chain fatty acids of the iso and anteiso types in addition to tuberculostearic acid (10Me-C18:0). A 16S ribosomal DNA sequence analysis revealed that the genus Lentzea is phylogenically related to the genera Actinosynnema, Saccharothrix, and Kutzneria. The type species of this genus is Lentzea albidocapillata sp. nov.; the type strain of this species is strain IMMIB D-958 (= DSM 44073). PMID- 7537072 TI - Inability of the polyphasic approach to systematics to determine the relatedness of the genera Xenorhabdus and Photorhabdus. AB - Comparative analysis of the genes coding for 16S rRNA of the type strains of Xenorhabdus and Photorhabdus species indicates the close phylogenetic relationship of these two genera. However, distance matrix analyses do not unambiguously separate the symbionts of entomopathogenic nematodes according to their assignment into different genera. When various 16S rRNA gene sequences from a selection of members of the gamma subclass of Proteobacteria and outgroup taxa were used, the intrageneric relationships of Xenorhabdus species and the positions of Photorhabdus luminescens and related species changed significantly. PMID- 7537074 TI - Proposal to reclassify Leuconostoc oenos as Oenococcus oeni [corrig.] gen. nov., comb. nov.. AB - Wine strains belonging to the genus Leuconostoc were classified as Leuconostoc oenos by Garvie in 1967, and this name was confirmed on the Approved Lists of Bacterial Names in 1980. L. oenos is distinguished from other Leuconostoc spp. by its growth in acidic media, by its requirement for a growth factor in tomato juice, and by a number of carbohydrate fermentation characteristics. In addition, the results of a total soluble cell protein analysis, an electrophoretic analysis of NAD-dependent D-(-)-lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and alcohol dehydrogenase, and an analysis of cross-reactivity with anti-glucose 6-phosphate dehydrogenase and anti-NAD-dependent D-(-)-lactate dehydrogenase performed with other Leuconostoc spp. clearly indicated that L. oenos should be distinguished from the other Leuconostoc species. Phylogenetic studies, in particular 16S and 23S rRNA sequencing studies, have revealed that L. oenos represents a distinct subline that is separate from other Leuconostoc spp. and lactic acid bacteria. In view of the phenotypic and phylogenetic distinctiveness of L. oenos, we propose that this species should be assigned to a new genus as Oenococcus oeni [corrig.] gen. nov., comb. nov. The type strain of O. oeni is NCDO 1674 (= ATCC 23179). PMID- 7537073 TI - Classification of Erysipelothrix strains on the basis of restriction fragment length polymorphisms. AB - Restriction fragment length polymorphisms of the 16S rRNA genes of Erysipelothrix strains were studied by cleavage of the chromosomal DNA with restriction endonuclease EcoRI, followed by hybridization to a 420-bp internal fragment of the 16S rRNA gene. Thirty-two Erysipelothrix type and reference strains were classified, together with seven field strains. Reference strains of all serotypes and the type strains of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum were included. Nine ribopatterns were observed. Pattern A was represented by 16 strains and included strains of serotypes 1b, 2 to 8, 11 to 13, 15 to 17, 19, and 23. Pattern B was represented by two strains (serotypes 1a and 9). Pattern C was represented by five strains (serotypes 5, 6, and 21). Pattern D was represented by one strain of serotype 4. Pattern E was represented by 11 strains of serotypes 2, 7, 10, 20, 22, 24, and 25. Patterns F, G, H, and I were each represented by a single strain of serotypes 26, 2, 18, and 3, respectively. All the different ribopatterns had some bands in common. Patterns B, C, and D were most similar to pattern A, while patterns F, G, H, and I resembled pattern E. Partial sequencing of the 16S rRNA gene of nine selected strains resulted in three different sequences, i.e., the typical E. rhusiopathiae sequence, the E. tonsillarum sequence, and a third sequence found for two strains. Strains of the same serotype were found to have different ribopatterns as well as different partial 16S rDNA sequences. PMID- 7537075 TI - Phylogenetic placement of Dialister pneumosintes (formerly Bacteroides pneumosintes) within the Sporomusa subbranch of the Clostridium subphylum of the gram-positive bacteria. AB - The nucleotide sequence of the 16S rRNA gene of the type strain of Dialister pneumosintes was determined. Phylogenetic analysis revealed that this species belongs to the Sporomusa branch of the Clostridium subphylum of the gram-positive bacteria and should therefore be excluded from the family Bacteroidaceae. Within this branch, which encompasses several other gram-negative taxa, such as Acidaminococcus, Pectinatus, Phascolarcobacterium, Quinella, Selenomonas, and Zymophilus, Dialister showed a specific, albeit distant, affinity with the genera Megasphaera and Veillonella. PMID- 7537076 TI - Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. AB - We determined the 16S rRNA sequences of the type strains of Streptococcus mitis and Streptococcus gordonii and calculated the phylogenetic distances between those organisms and other members of the genus Streptococcus. The viridans group streptococci were separated into five phylogenetic groups; we named these groups the anginosus group, the mitis group, the salivarius group, the bovis group, and the mutans group. S. mitis and S. gordonii clustered in the mitis group together with Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus parasanguis at levels of sequence homology of more than 96%. Within this group, S. mitis, S. oralis, and S. pneumoniae exhibited more than 99% sequence homology with each other, although the DNA-DNA similarity values for their total chromosome DNAs were less than 60%. PMID- 7537077 TI - Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis. AB - Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions. PMID- 7537078 TI - Partition of polysaccharide-coated liposomes in aqueous two-phase systems. AB - Hydrophobized polysaccharides such as cholesterol-bearing pullulan (CHP), dextran (CHD) and mannan (CHM) effectively coat the liposomal surface. Partition of the hydrophobized polysaccharide-coated liposomes in an aqueous two-phase system (PEO (top)/pullulan (bottom) or PEO (top)/dextran (bottom)) was investigated (PEO = poly(ethylene oxide)). Conventional liposomes without a polysaccharide coat mostly locate at the interface between the two polymer phases. The polysaccharide coated liposomes, on the other hand, were partly partitioned to the bottom polysaccharide phase depending on the structure of the hydrophobized polysaccharide on the liposomal surface. The affinity between the polysaccharide on the liposomal surface and that in the bulk bottom phase controls the efficiency of partition. The sequence of interaction strength between the two carbohydrates was the following: for the PEO/dextran two-phase system, dextran(liposome)-dextran(bulk) > mannan(liposome)- dextran(bulk) > pullulan(liposome)-dextran(bulk); while for the PEO/pullulan system, the sequence of interaction strength was pullulan(liposome)- pullulan(bulk) > dextran(liposome)-pullulan(bulk) approximately mannan(liposome)-pullulan(bulk). PMID- 7537079 TI - Retinoic acid upregulates c-kit ligand production by murine keratinocyte in vitro and increases cutaneous mast cell in vivo. AB - Mouse-transformed epidermal cell line (Pam 212) generated the soluble mediators for promoting the growth of a mast cell line (MC9) in the presence of retinoic acid at a concentration of 10(-6)-10(-7) M. The effective molecule of MC9 cell growth promoting factor (MC9-GF) was non-dialyzable and eluted between the molecular weight of 45 K and 68 K on a TSK 2000 G column. Chromatofocusing analysis revealed that this factor had a pI range between 7.0 and 7.5. Anti-c-kit ligand antibody abrogated MC9-GF activity and RT-PCR analysis demonstrated that retinoic acid upregulates c-kit ligand mRNA expression by Pam cells. Several recombinant cytokines including IL1-alpha, IL-1 beta, IL-2, IL-3 or IL-4 did not promote MC9 cell growth at a concentration of 100 U/ml. The presence of anti-IL-1 alpha, -IL-1 beta, -IL-2, -IL-3 or -IL-4 antibodies did not abrogate the MC9-GF activity except for anti-c-kit ligand antibody. PMID- 7537080 TI - Keratinocyte growth factor and keratin gene regulation. AB - Keratinocyte growth factor (KGF) is a stromally derived paracrine mitogen that belongs to the fibroblast growth factor (FGF) family. It is secreted by dermal fibroblasts and specifically promotes keratinocyte proliferation. We have recently shown that epidermal growth factor (EGF) and transforming growth factor beta (TGF beta), modulators of keratinocyte proliferation, regulate expression of specific keratin genes. However KGF, unlike EGF and TGF beta, allows keratinocytes to differentiate normally. With this in mind, we sought to determine whether KGF may be involved in keratinocyte differentiation through a mechanism that does not involve regulation of keratin gene expression. We transfected human epidermal keratinocytes with ten different keratin gene promoters linked to a reporter gene, and grew the transfected cells in the presence or absence of KGF. Interestingly, no significant change in keratin gene regulation was observed in the presence of KGF relative to control. The possibility that KGF influences the induction of keratin gene expression by other keratinocyte modulators, such as EGF, TGF beta and gamma interferon (IFN gamma), was also explored. In these experiments, the transformed keratinocytes were exposed simultaneously to KGF and another modulator. KGF did not significantly change the effects of EGF, TGF beta or IFN gamma on keratin gene expression. KGF's lack of ability to directly regulate keratin gene expression suggests that KGF affects keratinocyte growth and differentiation through a pathway independent of keratin gene regulation. These results illustrate that regulation of keratinocyte proliferation can be separated from the regulation of keratin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537081 TI - Tranilast inhibits collagen synthesis in normal, scleroderma and keloid fibroblasts at a late passage culture but not at an early passage culture. AB - We have previously reported that tranilast, an anti-allergic agent, specifically suppresses collagen synthesis in normal skin fibroblasts and to a greater extent in keloid fibroblasts. We found in this study that the specific suppression of collagen synthesis by tranilast was limited to the fibroblasts with a high passage number (passage 8-10). In normal skin fibroblasts with a low passage number (passage 1-2), tranilast exerted no significant effect on collagen synthesis. This was also observed with scleroderma and keloid fibroblasts. This result suggests that inhibition of collagen by tranilast will be dependent on in vitro cellular aging and that serial cell passages result in the loss of the cell phenotype resistant to tranilast effect. PMID- 7537082 TI - Effects of immunosuppressive peptidyl-prolyl cis-trans isomerase (PPIase) inhibitors, cyclosporin A, FK506, ascomycin and rapamycin, on hair growth initiation in mouse: immunosuppression is not required for new hair growth. AB - The effects of immunosuppressive peptidyl-prolyl cis-trans isomerase (PPIase) inhibitors, cyclosporin A, FK506, ascomycin and rapamycin, on hair growth initiation (anagen hair induction) in mouse were studied by topical application on the dorsal skin surface during the telogen phase of the hair cycle. Single applications of cyclosporin A and FK506 (10 to 100 nmol in 5 microliters of ethanol) induced new hair growth in 12 days within the restricted area where the compounds were applied. On the other hand, ascomycin and rapamycin did not initiate new anagen hairs even at higher doses (1 mumol in 5 to 10 microliters of ethanol). The effects of simultaneous application of the immunosuppressants were also tested by a single topical application. Ascomycin did not inhibit the anagen hair induction by cyclosporin A, but inhibited hair induction by FK506. Rapamycin inhibited new hair growth induced by cyclosporin A and FK506. These results suggest that the inhibition of PPIase is not required for the initiation of a new hair cycle in mice, and that anagen hair induction caused by cyclosporin A and FK506 is not a result of immunosuppression. The present results also indicate that a single application of an adequate quantity of cyclosporin A and FK506 is sufficient to initiate new hair growth. PMID- 7537083 TI - Structural study of a DNA.RNA hybrid duplex with a chiral phosphorothioate moiety by NMR: extraction of distance and torsion angle constraints and imino proton exchange rates. AB - The solution structure of the thiophosphate-modified DNA.RNA hybrid duplex d(GCTATAApsTGG).r(CCAUUAUAGC) has been studied by NMR. Two samples with pure stereochemistry in the modified phosphate have been investigated. Two-dimensional NMR (2D NMR) methods have been applied to assign nearly all the resonances in both duplexes. Scalar coupling constants have been determined by comparing quantitative simulations with experimental double-quantum filtered COSY (DQF COSY) cross-peaks. More than 300 distance constraints have been obtained from two dimensional nuclear Overhauser spectroscopy (2D NOE) spectra recorded in D2O and H2O by using a complete relaxation matrix analysis as implemented in the program MARDIGRAS. This hybrid duplex presents a heteronomous structure. Riboses in the RNA strand are found in a N-type conformation typical of the A-form family as shown by the lack of H1'-H2' cross-peaks in DQF-COSY spectra and confirmed by the measured interproton distances. In contrast, the DNA strand adopts a different conformation with sugar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses are not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-state dynamic equilibrium between N- and S-type conformers. In general, the population of major S conformer is lower than in double-stranded DNA duplexes, indicating that hybrid duplexes may be more flexible than pure DNA or RNA. The only differences observed in the spectra between the two stereoisomers studied originate from resonances of protons located near the modified phosphate. No significant differences in interproton distance have been detected, and only a slight difference of sugar pucker in the 5' neighbor has been found. The sulfur atom appears to be well accommodated without further changes in the structure of the hybrid. PMID- 7537084 TI - Crystal structure of bovine heart phosphotyrosyl phosphatase at 2.2-A resolution. AB - The first X-ray crystallographic structure of a member of the class of low molecular weight (M(r) 18,000) phosphotyrosyl phosphatases is presented. Bovine heart phosphotyrosyl phosphatase (BHPTP) exemplifies this class and is highly homologous (94% sequence identity) to an isoenzyme known as red cell acid phosphatase that is present throughout human tissues. The high-resolution (2.2-A) crystal structure of BHPTP shows that the enzyme consists of a four-strand central parallel beta sheet with alpha helices packed on both sides in a manner characteristic of a Rossmann fold. A bound phosphate ion defines the active site location in a loop of the first beta alpha beta motif at the C-terminus of the beta sheet. The location and enzymatic significance of the residues in the characteristic low molecular weight PTPase active site motif, including the essential arginine (Arg 18) and nucleophilic cysteine (Cys 12), are described. The functional role of a histidine (His 72) suggested previously to be near the active site is defined in the structure, as well as a potential proton donor for the leaving group in the tyrosyl phosphate cleavage. Surface maps of BHPTP define a hydrophobic crevice suitable for phosphotyrosyl peptide binding. Comparison of the BHPTP structure to the related, but structurally distinct enzyme PTP1B is made, illustrating the unique way this smallest of these phosphatases has formed the phosphotyrosine active site. PMID- 7537085 TI - Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies. AB - Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane. The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer. The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions. PMID- 7537086 TI - Neurotrophin binding to human alpha 2-macroglobulin under apparent equilibrium conditions. AB - alpha 2-Macroglobulin (alpha 2M) is a broad-spectrum proteinase inhibitor and a carrier of certain growth factors. The purpose of this investigation was to characterize the interaction of alpha 2M with nerve growth factor-beta (NGF beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ciliary neurotrophic factor (CNTF) under apparent equilibrium conditions. Binding in solution was assessed using the cross-linking agent bis(sulfosuccinimidyl) suberate (BS3). Noncovalent binding of NGF-beta, NT 3, NT-4, and BDNF to native alpha 2M and alpha 2M-methylamine (a conformationally modified form of alpha 2M that is recognized by the alpha 2M receptor) reached apparent equilibrium in less than 20 min at 37 degrees C. Apparent KD values for the binding of NT-4, NGF-beta, NT-3, and BDNF to alpha 2M-methylamine were 61, 110, 120, and 150 nM, respectively. Native alpha 2M bound all four neurotrophins with decreased affinity. Unlabeled NGF-beta competed with the radioiodinated neurotrophins for binding to immobilized alpha 2M-methylamine. The K1 for unlabeled NGF-beta was 120 nM, in good agreement with the apparent KD determined by the BS3 method. The number of NGF-beta binding sites per immobilized alpha 2M methylamine was 1.0. CNTF bound minimally, if at all, to native alpha 2M and alpha 2M-methylamine as determined using a number of techniques. The extent of binding was insufficient for the determination of an affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537087 TI - The stability and structure of tandem GA mismatches in RNA depend on closing base pairs. AB - UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A. PMID- 7537088 TI - Proton NMR assignments and solution conformation of RANTES, a chemokine of the C C type. AB - 1H NMR has been used to investigate the structural properties of RANTES, a protein from the C-C branch of the chemotactic cytokine family that has a strong chemoattractive effect on monocytes, lymphocytes, and eosinophils. Titration of pH from 5.0 to 2.5 indicates that RANTES is extensively aggregated in solution above pH 4.0. At pH 3.7 the protein is mostly dimeric, although this species does dissociate to the monomer with a Kd of 35 microM. NMR data have been acquired and resonance assignments made for the dimeric species. Structures of the dimer have been generated by distance geometry and simulated annealing calculations that utilized 1956 intramolecular distance restraints, 120 intermolecular distance restraints, 164 dihedral angle restraints, and 68 restraints enforcing 34 hydrogen bonds (17.0 restraints per residue). The structure is well-defined (average root mean square deviation from the average structure of 0.38 +/- 0.06 and 0.53 +/- 0.12 A for backbone heavy atoms of residues 4-66 of the monomer and dimer, respectively). Each monomer consists of a C-terminal alpha-helix packing against a three-stranded antiparallel beta-sheet and two short N-terminal beta strands; dimerization occurs between the N-terminal regions of each monomer. This quaternary structure is very different from that of the C-X-C chemokines such as interleukin-8 and melanoma growth stimulatory activity but similar to that found for the C-C chemokine macrophage inflammatory factor 1 beta. Distinct structural differences between RANTES and other chemokines at both the tertiary and quaternary level are discussed with regard to the distinct biological functions of the C-C and C-X-C members of this protein family. PMID- 7537089 TI - An expanded model of replicating human immunodeficiency virus reverse transcriptase. AB - Replication complexes containing wild-type and RNase H-deficient p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) were analyzed by DNase I and S1 footprinting. While crystallography and chemical footprinting data demonstrate that 15-18 bases of primer and template occupy the DNA polymerase and RNase H active centers, enzymatic footprinting suggests that a larger portion of substrate is encompassed by the replicating enzyme. Independent of the position of DNA synthesis arrest, template nucleotides +7 to -23 and primer nucleotides -1 to -25 are nuclease resistant. On both DNA strands, position -20 remains accessible to DNase I cleavage, suggestive of an alteration in nucleic acid structure between exiting the RNase H catalytic center and leaving the C-terminal p66 domain. A model of HIV-1 RT containing an extended single-stranded template and duplex region was constructed on the basis of the structure of an RT/DNA complex. Mapping of footprint data onto this model shows consistency between biochemical and structural data, implicating a contribution from domains proximal to the catalytic centers. PMID- 7537091 TI - Retardation of a surface protein chimera at the cis Golgi. AB - Dipeptidyl peptidase IV (D4) and the alpha subunit of human chorionic gonadotrophin (alpha hcg) are plasma membrane and secretory proteins, respectively. In the course of studies to understand mechanisms involved in transport along the exocytotic pathway, the ectoplasmic domain of D4 was replaced by the mature polypeptide of alpha hcg, resulting in the membrane anchored chimera, D4 alpha hcg. Surprisingly, when transfected into Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells, strong perinuclear Golgi staining was predominant, in addition to the expected surface staining. By following the biogenesis and transport of the molecule, it was established that newly synthesized D4 alpha hdg is eventually transported to the cell surface but only after a significant retardation in the Golgi apparatus. The compartment of retardation was identified as the early or cis Golgi, before the medial Golgi, where resistance to endoglycosidase (endo) H is conferred. As a result of the transport retardation of the chimera, we were able to document the appearance of an endo D sensitive intermediate, which is usually too transient to be apparent in normal cells. The retardation of this chimera in the cis Golgi complements our previous report in which the D4 molecule with its transmembrane domain replaced by that of aminopeptidase N resulted in retardation in the trans Golgi/trans Golgi network in MDCK and CHO cells [Low, S. H., Tang, B. L., Wong, S. H., & Hong, W. (1994) J. Biol. Chem. 269, 1985-1994).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537090 TI - Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. AB - When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively. PMID- 7537092 TI - Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity. AB - Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (.NO). NOS is a hemoprotein containing a cytochrome P-450-type heme that has been shown to be involved in catalysis. It has been suggested that .NO is able to bind tightly to the heme of NOS and may in this way serve to regulate enzymatic activity. We report here the formation of both ferric and ferrous heme nitrosyl complexes with the inducible NOS from murine macrophages. The ferric nitrosyl complex is characterized by a Soret peak at 443 nm and two distinct peaks in the alpha/beta region at 549 and 585 nm. The ferrous nitrosyl complex has absorbance maxima at 436 and 566 nm. A transient spectral intermediate is observed under conditions of NOS turnover. This intermediate appears to be a mixture of ferric and ferrous nitrosyl complexes and is unstable in the presence of oxygen. Binding of L-arginine decreases the affinity of .NO for the ferric heme but does not appear to decrease the affinity of .NO for the ferrous heme. Addition of either oxyhemoglobin or methemoglobin to NOS assays results in a nearly 2-fold increase in enzymatic activity. This result is attributed to the ability of both forms of hemoglobin to decrease the concentration of .NO in solution and is consistent with .NO inhibition of NOS under assay conditions. Our results show that NOS nitrosyl complexes form under certain conditions but suggest that the relevance of such complexes to activity in vivo may be limited by their instability in an aerobic environment. PMID- 7537094 TI - Effects of chemical modification on the binding activities of P-selectin mutants. AB - P-Selectin (GMP140, CD62P, PADGEM), a 140-kDa glycoprotein found on activated platelets and endothelial cells, is involved in one of the early events in the inflammatory response due to its role in initiating the recruitment of circulating leukocytes. From a three-dimensional model of P-selectin and site specific mutagenesis studies, a number of residues were previously identified as critical for the binding of P-selectin to HL-60 cells, a human myeloid cell line. Included among them were lysines 111 and 113 (K111 and K113). In this study, the roles of K111 and K113 were further characterized by the generation and specific chemical modification of two cysteine mutants, K111C and K113C, of a P-selectin immunoglobulin fusion protein (P-selectin-Rg). Both K111C and K113C displayed significantly reduced binding activity compared to the wild-type P-selectin from which they were derived, further illustrating the importance of these particular lysines for ligand binding. Reaction of K111C with aziridine or nipsylcysteamine resulted in the formation of K111C-AZ and K111C-CY, both of which displayed significant increases in HL-60 binding activity. No such increase took place upon reaction of K111C with N-ethylmaleimide, indicating that a free amine at position 111 is important for binding. Residue length at position 111 is not critical, since the synthetic side chains are 0.5-2.0 A longer than lysine yet still impart binding activity. Similar modification studies of K111A and K113C did not lead to any detectable increase in binding of these proteins to HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537093 TI - In vitro selection of RNA ligands to substance P. AB - RNA ligands to the tachykinin substance P have been selected from a large pool of random sequence RNA molecules. Substance P is an undecapeptide that plays a variety of roles as a neurotransmitter and neuromodulator in the central and peripheral nervous system of mammals. A systematic evolution of ligands by exponential enrichment (SELEX) procedure was used to isolate RNAs that bind substance P immobilized on a solid support. RNAs that also bind substance P in solution were identified, and the tightest binder was subjected to mutagenesis in a second SELEX procedure to evolve ligands with a higher affinity for the peptide. A comparative analysis of 36 ligands isolated from the second SELEX experiment revealed two main sequence classes with highly conserved secondary structures within each class. Dissociation constants for the interaction of these ligands with substance P in solution were determined by equilibrium dialysis. The amino acid residues involved in the interaction with the highest affinity ligand (190 nM Kd) were mapped by determining which of a set of overlapping fragments of substance P can compete with the intact peptide for binding. A binding competition experiment also demonstrated the ability of the same ligand to discriminate between substance P and the reverse orientation of the same amino acid sequence. The results from this study demonstrate that SELEX can yield high affinity RNA ligands to small nonconstrained peptides. PMID- 7537095 TI - Correlations between function and dynamics: time scale coincidence for ion translocation and molecular dynamics in the gramicidin channel backbone. AB - The polypeptide backbone of gramicidin lines the channel lumen, which maintains a single file string of water molecules and the occasional cation. Whether or not local motions of the backbone are important for facilitating cation transport has been the subject of debate. Here it is demonstrated that motions of the backbone occur on the same time scale as cation translation through this transmembrane channel. To characterize the frequency of the local motions, field-dependent 15N T1 relaxation times were interpreted in light of an independently determined motional model that defines the motions as occurring about the C alpha-C alpha axis. To overdamp these local motions, several models that involve extensive correlations of the molecular motions were considered. One of these schemes for correlated motions suggests a unidirectional reaction path that would minimize the time necessary for the cation to pass through the low-dielectric center of the bilayer. PMID- 7537096 TI - Formation of signal transfer complexes between stem cell and platelet-derived growth factor receptors and SH2 domain proteins in vitro. AB - Cellular growth and differentiation signals are generated and defined by the interaction of specific phosphotyrosine residues of activated receptor tyrosine kinases (RTKs) and src homology-2 (SH2) domain-containing intracellular signal transducers. This appears to involve for both the p145c-kit and beta platelet derived growth factor receptor (PDGF-R) cytoplasmic domains the formation of multiprotein signal transfer complexes, which include combinations of noncatalytic and enzymatically active subunits of phosphatidylinositol 3'-kinase (PI3'-K), phospholipase C-gamma (PLC gamma), and guanosine trisphosphatase activating protein (GAP). In vitro association experiments indicate that PLC gamma and PI3'-K bind the beta PDGF-R simultaneously, while these two SH2 proteins compete for association to p145c-kit binding sites, with p85/PI3'-K exhibiting higher affinity. Interestingly, GAP and p85/PI3'-K binding to distinct p145c-kit phosphotyrosines is cooperative, enhancing formation of a heterotetrameric signaling complex, which may include different combinations of p85 alpha and p85 beta with p110, p112, and p116 by interaction with the same tyrosine 721 docking site. The diversity of molecular interactions observed for PDGF-R and p145c-kit suggests a new mode of signal definition and modulation. PMID- 7537097 TI - Bulge-specific cleavage in transactivation response region RNA and its DNA analogue by neocarzinostatin chromophore. AB - On the basis of the finding that in the absence of thiol the nonprotein chromophore of the antitumor drug neocarzinostatin (NCS-chrom) induces highly efficient site-specific cleavage at a single site on the 3' side of a bulge in single-stranded DNA involving entirely 5' chemistry [Kappen, L. S., & Goldberg, I. H. (1993) Science, 261, 1319-1321], transactivation response region (TAR) RNA (29-mer) and its DNA analogue which presumably contain bulge structures were tested as potential substrates for NCS-chrom. In TAR RNA NCS-chrom generates a distinct but weak band due to cleavage at U24 in the bulge. Cleavage at U24 has a pH dependence and time course similar to those for previously studied DNA bulges. This band is not produced in drug reactions containing glutathione, by the protein component of native NCS, or by inactivated NCS-chrom. Cleavage at U24, albeit weak, occurs in an RNA substrate made up of two linear RNA oligomers which presumably can form a bulge akin to that in TAR RNA. In the DNA analogue of TAR RNA, as well as in a DNA duplex made of two linear oligomers that can form a similar bulge, NCS-chrom causes strand cleavage at the T residues in the bulge and at the bases flanking the bulge. Cleavage at T25 in the bulge involves, in addition to 5' chemistry, 4' attack which results in a fragment with mobility characteristic of 3'-phosphoglycolate-ended fragments. Experiments using DNA substrate having deuterium selectively at the 4' or 5' positions of T25 confirm 4' attack and show kinetic shuttling between the two positions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537098 TI - Effect of dose of cyclosporine or FK506 and antithrombotic agents on cardiac allograft vascular disease in heterotopically transplanted hearts in rats. AB - BACKGROUND: We compared the severity of cardiac allograft vascular disease in rats treated with cyclosporine or FK506 and studied the effect of antithrombotic agents on cardiac allograft vascular disease. METHODS: One group each was treated with 2 and 5 mg/kg/day of cyclosporine. Two other groups were injected with heparin and dipyridamole, respectively, in addition to cyclosporine. Four other groups were similarly divided by dose of FK506 (0.1 or 0.25 mg/kg/day) and concomitant anticoagulant treatment. RESULTS: Grade of rejection and percentage stenosis of coronary arteries were lower in groups with high doses of immunosuppressive agents or with heparin. Major histocompatibility class II antigens were expressed by the endothelium of grafted hearts, and IgM and C3 were deposited in the intimal and medial layers in all groups except those administered the higher doses of immunosuppressive drugs. However, no remarkable differences in density of major histocompatibility class II antigens were found between groups demonstrating expression of these antigens. On the other hand, the intensity of IgM or C3 expression grew significantly as coronary stenosis increased in severity. CONCLUSIONS: A significant difference in severity of cardiac allograft vascular disease was not found between the groups treated with cyclosporine and FK506, and cardiac allograft vascular disease was almost entirely suppressed when doses of cyclosporine and FK506 sufficient to suppress graft rejection were administered. Our findings also showed that concomitant heparin administration reduced the extent of allograft rejection and the incidence of cardiac allograft vascular disease. PMID- 7537099 TI - Preservation of competent rabbit lung function after 30 hours of storage with a low-potassium dextran solution. AB - BACKGROUND: The goal of organ preservation is maintenance of physiologic functions during extended extracorporeal storage. METHODS: This study was designed to evaluate the efficacy of using low-potassium (4 mmol/L) dextran (1%) solution on lung function after 30 hours hypothermic (10 degrees C) storage and to compare this with lung function after no storage. With low-potassium dextran solution rabbit lungs were flushed (10 degrees C, 40 ml/kg, 60 cm H2O), excised, inflated (with room air), and either not stored (control; no preservation; n = 9) or stored in low-potassium dextran solution (10 degrees C) 30 hours (experimental group; n = 9). RESULTS: During the flush the infusion pressure and pulmonary vascular resistance for the two groups did not differ (17.56 +/- 1.3 versus 16.74 +/- 1.5 mm Hg/ml/sec). After either no preservation or after 30 hours of storage, the lungs were first reperfused with low-potassium dextran solution (37 degrees C) for 4 minutes and then with blood (37 degrees C) for 30 minutes at 100 ml/min. During the reperfusion period the mean pulmonary artery pressure and end inspiratory airway pressure for the control and experimental groups did not differ. After reperfusion the wet and dry weights of the left lung were determined. The wet/dry ratio for the two groups did not differ (5.32 +/- 2.20 versus 4.70 +/- 2.70, respectively). CONCLUSIONS: These data suggest that cold flush, cold storage, and initial warm perfusion with low-potassium dextran solution crystalloid preserve lung function after 30 hours of storage. PMID- 7537101 TI - Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds. AB - The stabilities of the SH-reagent eosin-5-maleimide (EMA) and its adducts with the SH-compounds L-cysteine, N-acetyl-L-cysteine and glutathione (reduced form) were studied under various conditions in comparison with those of the adducts of N-ethylmaleimide (NEM). Studies by reversed-phase high performance liquid chromatography and mass spectrometry showed that EMA was less stable than NEM at neutral and moderately alkaline pH values. EMA formed a succinimide-type adduct with SH-compounds, and then underwent further modification by nucleophilic attack of OH- or an amino group. The succinimide-type adducts with acetylcysteine and glutathione were converted to open-type adducts, in which the succinimide ring was cleaved, whereas the adduct with cysteine was modified to a thiazine-type adduct. Kinetic analyses showed that these open-type and thiazine-type adducts were readily formed and were stable at moderately alkaline pH values such as pH 8.0 or 9.0. PMID- 7537102 TI - Endocytosis of poly(ethylene oxide)-modified liposome by human lymphoblastoid cells. AB - Egg PC liposome as reconstituted with poly(ethylene oxide)-bearing lipid (coded as PEO-lipid (n = 15)) was remarkably endocytosed by Jurkat cell, which was a lymphoblastoma derived from human T cell. To confirm the endocytosis, two kinds of fluorescent probes (FITC-dextran and octadecyl rhodamine B) were employed. The former was loaded in the aqueous phase of the liposome, while the latter was embedded in the liposomal membrane. Both probes were found coincidentally at the same site in the cytosol, clearly suggesting that whole liposome entered the cell. The endocytosis was most obvious when PEO-lipid (n = 15) was employed above 50 mol%. FITC-Dextran entered the cell was found small dots in the cell, not dispersive. Even when octadecyl rhodamine B was used, no membrane fluorescence was observed at all. The uptake closely related to the cell metabolism as affected by the culture temperature and serum in the incubation medium. Furthermore, the addition of cytochalasin B completely prohibited the cell uptake of liposomes. PMID- 7537100 TI - Effect of a chemical or proteolytic modification on the biological activity of guinea-pig cationic peptide. AB - Guinea-pig neutrophil cationic peptides (GNCPs) are single polypeptides containing 31 amino acid residues and three intramolecular disulfide bonds, which show both antibacterial and histamine-releasing activities. Reduction and alkylation of the disulfide bonds of GNCP did not reduce both biological activities. When pyridylethylated GNCP was digested with trypsin, the biological activities were almost lost, whereas the chymotryptic digest retained the biological activities. Chymotrypsin digested fragments were purified by RP-HPLC, and three active peptide fragments containing two Arg residues at the N-terminal sequence were isolated. When the biological activities were examined using synthetic peptides containing various numbers of Arg residue at the N-terminus, the omission of the Arg residues was found to reduce remarkably the antibacterial and histamine-releasing activities. Together these observations indicate that the primary structures containing Arg residues at the N-terminus but not the intramolecular disulfide cross-linking are important for the expression of the biological activities of GNCP. PMID- 7537104 TI - Receptor-ligand interactions measured by an improved spun column chromatography technique. A high efficiency and high throughput size separation method. AB - Size exclusion chromatography may under the right circumstances be an easy and powerful way to measure in solution the interaction between a receptor an dits ligand. Spun column chromatography is a fast size exclusion technique of increasing popularity, however, little information exists on the method development essential to obtain efficient separation in particular when used for analytical purposes. In this paper we describe a systematic approach to select the optimal parameters for spun column separation including a simple modification of the technique whereby the spun columns are eluted by high-speed gradient centrifugation. This modification is easy to implement and it considerably improves spun column performance. We hypothesize that the high-speed centrifugation step leads to the release of additional buffer which assists in the complete elution of excluded molecules while the gradient centrifugation helps to achieve equilibrium across the gel matrix during the elution. The new method has been used successfully for several different receptor-ligand interactions, and this paper describes a general approach on how to develop new applications of the technique. PMID- 7537103 TI - Localization and interaction of bovine pancreatic trypsin inhibitor and tryptase in the granules of bovine mast cells. AB - The interaction of bovine pancreatic trypsin inhibitor and bovine tryptase, isolated from liver capsule mast cells, was investigated. They form a complex in vitro with a Ki of 5.6 nM at pH 8.0 and are localized within the mast cell granules, as shown by immunogold staining at the electron microscope level. In addition, double immunogold electron microscopy revealed that the inhibitor and the enzyme are present in the same granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiological role for bovine pancreatic trypsin inhibitor in the regulation of tryptase proteolytic activity. PMID- 7537105 TI - Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor. AB - The interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) induced by ristocetin or botrocetin resulted in associated platelet aggregation, protein tyrosine phosphorylation (PTP) of a 64 kDa protein, as detected by a monoclonal antibody against phosphotyrosine (PY-20), and intracellular Ca2+ elevation that is largely dependent upon Ca2+ influx in human platelets. It is of interest that 75-80, 97 and 125 kDa proteins which are strongly tyrosine phosphorylated in platelet activation induced by thrombin and other agonists were not detected. Neither vWF nor a coaggregating agent (ristocetin or botrocetin) alone induced aggregation, [Ca2+]i elevation or the 64 kDa PTP. NMC-4, an antibody which inhibits both ristocetin- or botrocetin-induced vWF binding to GPIb, abolished the appearance of the 64 kDa PTP as well as other responses, suggesting that it is specifically induced by the GPIb-vWF interaction. Aspirin, or ONO-3708, a competitive inhibitor of thromboxane A2, did not modify the 64 kDa PTP, while [Ca2+]i elevation was moderately suppressed. Depletion of extracellular Ca2+ or RGD peptides suppressed neither the 64 kDa PTP nor aggregation. H-7, a protein kinase C inhibitor, did not inhibit the 64 kDa PTP, while staurosporine, a potent protein kinase inhibitor, inhibited the 64 kDa PTP and Ca2+ influx, but not aggregation, in a dose-dependent manner. It is suggested that the 64 kDa PTP is associated with platelet aggregation induced by the interaction between GPIb and vWF. PMID- 7537106 TI - Location of an epitope shared by Alzheimer's amyloid peptide and brain creatine kinase using a newly developed monoclonal antibody. AB - Amyloid plaques, composed mainly by a peptide termed A4-amyloid, derived by proteolytic processing from the amyloid precursor protein (APP), are a hallmark in the brain of Alzheimer's disease patients. We have prepared a collection of monoclonal antibodies as tools to study APP expression and proteolysis in different systems. One of these, 5AH10, raised against residues 9-22 of A4 peptide, was selected for its ability to recognize only A4 subpeptides having the intact APP-secretase target sequence, as well as whole recombinant APP. By using synthetic subpeptides, we have located 5AH10 epitope between amino acids 15 and 22 of A4. In addition, 5AH10 showed a strong immunoreactivity to a 47 kDa protein present in rat brain extracts, that was identified as the B (brain specific) subunit of creatine kinase by immunochemical data and direct N-terminal sequencing. The cross-reaction observed is most probably due to a high degree of sequence identity between amino acids 15 to 22 of A4 peptide and amino acids 9 to 16 of rat B creatine kinase. 5AH10 did not recognize the muscle specific isoform (M subunit) of rat creatine kinase, nor the B subunit of human and rabbit creatine kinase, suggesting that glutamine at first position of the epitope is essential for antigen recognition by 5AH10. PMID- 7537107 TI - IL-6-induced changes in synthesis of alpha 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80). AB - The synthesis of the human acute-phase alpha 1-acid glycoprotein (AGP) is primarily controlled by IL-6 and IL-1 in liver cells. In the present study, monoclonal antibodies against human gp80 interleukin-6 receptor (IL-6R) were utilized to study the role of the IL-6R in the control of the IL-6-induced AGP synthesis in the human hepatoma Hep3B cell line. Two of the 4 MAbs used in this study, M164 and M195, identified 2 different epitopes involved in IL-6 binding and two others, M91 and M182, recognized epitopes not involved in IL-6 binding. Dose-response experiments indicated that up to 55% of AGP synthesis was inhibited by 10(5) ng/ml of MAbs 164 or 195 when Hep3B cells were treated by IL-6 for 48h. Kinetics of the inhibition of AGP synthesis after addition of anti-IL-6R indicated that the decrease of the IL-6-induced AGP synthesis by Hep3B cells was obtained immediately after the addition of the anti-IL-6R MAbs. Of the two MAbs not involved in IL-6 binding, M91 was unable to interfere with the IL-6-induced AGP synthesis whereas, surprisingly, M182 decreased it by about 25%. Since M182 was also able to interfere with the proliferative response of an IL-6 dependent plasma cell line, our results suggested that M182 may be directed to a structure involved in the IL-6/IL-6R gp130 complex formation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537108 TI - Cancer biotherapy with interferon, interleukin-2 and tumor-derived activated cells (TDAC). AB - Interleukin-2 (Il-2) allows for the therapeutic augmentation of immunity in vivo and/or in vitro. In extensive studies in nearly 1000 patients with advanced cancer, we have demonstrated that Il-2 by continuous infusion, with cellular therapy (LAK or TDAC), is feasible, cost effective and useful in selected patients with melanoma and kidney cancer. Studies in patients with other tumor types are underway. PMID- 7537109 TI - Peripheral blood progenitor cells mobilized by recombinant human granulocyte colony-stimulating factor plus recombinant rat stem cell factor contain long-term engrafting cells capable of cellular proliferation for more than two years as shown by serial transplantation in mice. AB - Mobilized peripheral blood progenitor cells (PBPC) have been shown to provide rapid engraftment in patients given high-dose chemotherapy. PBPC contain cells with long-term engraftment potential as shown in animal models. In this study we have further analyzed mobilized PBPC for their ability to support serial transplantation of irradiated mice. Transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) plus recombinant rat stem cell factor (rrSCF) mobilized PBPC resulted in 98% donor engraftment of primary recipients at 12 to 14 months post-transplantation. Bone marrow (BM) cells from these primary recipients were harvested and transplanted into secondary recipients. At 6 months posttransplantation, all surviving secondary recipients had donor engraftment. Polymerase chain reaction (PCR) analysis showed greater than 90% male cells in spleens, thymuses, and lymph nodes. Myeloid colonies from BM cells of secondary recipients demonstrated granulocyte/macrophage colony forming cells (GM-CFC) of male origin in all animals. In comparison, transplantation of rhG-CSF mobilized PBPC resulted in decreased male engraftment in secondary recipients. BM cells from secondary recipients, who originally received PBPC mobilized by the combination of rrSCF and rhG-CSF, were further passaged to tertiary female recipients. At 6 months posttransplantation, 90% of animals had male-derived hematopoiesis by whole-blood PCR analysis. These data showed that PBPC mobilized with rhG-CSF plus rrSCF contained cells that are transplantable and able to maintain hematopoiesis for more than 26 months, suggesting that the mobilized long-term reconstituting stem cells (LTRC) have extensive proliferative potential and resemble those that reside in the BM. In addition, the data demonstrated increased mobilization of LTRC with rhG-CSF plus rrSCF compared to rhG-CSF alone. PMID- 7537111 TI - c-kit ligand gene expression in normal and sublethally irradiated mice. AB - The c-kit ligand (KL; Steel factor, mast cell growth factor, stem cell factor) is a hematopoietic factor that has been shown to act as a potent cofactor for hematopoietic growth and differentiation in vitro. The in vivo effects of KL, however, have been variable. To study the hematopoietic role of KL in vivo, we evaluated KL gene expression in both normal mice and mice recovering from myelosuppressive radiation exposure using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. In a single RNA sample, we found that the RT PCR technique has high precision (co-efficient of variation, 15.7%). Amplifications of serial 1:2 dilutions of template RNA precisely correlated with starting RNA concentrations at 20 cycles or at 25 cycles, depending on the level of expression. Amplification of individual normal bone marrow and spleen cell RNA showed basal expression in all normal bone marrows but irregular expression in normal spleens. On day 2 after a sublethal 7.75-Gy (0.4 Gy/min) 60Co irradiation, splenic KL gene expression increased approximately 2.5-fold (P = .011), and bone marrow expression increased 15-fold (P = .004). During a 28-day postirradiation recovery period, KL expression increased in bone marrow on days 2 through 7. Splenic expression during the same period was more variable. In conclusion, the KL gene is invariably expressed in normal murine spleens. Postirradiation, recovering bone marrow and spleen both express increased levels of KL mRNA at day 2 and continue to express increased levels for several days postexposure. These data support a role for KL in the endogenous recovery of hematopoiesis after hypoplastic injury. PMID- 7537112 TI - CD64/Fc gamma RI is a granulo-monocytic lineage marker on CD34+ hematopoietic progenitor cells. AB - The aim of this study was to identify markers specific for granulo-monocytic commitment of progenitor cells. Large panels of antibodies were screened for selective staining of subsets of CD34+ cells from fetal and adult bone marrow. Flow cytometric analysis showed that CD64/fc gamma RI was undetectable on noncommitted progenitor cells (CD34++, CD38-/lo, HLA-DR+) and expressed on a subset of lineage-committed progenitors (CD34+, CD38+) with higher mean orthogonal light scatter than the remaining CD34+ cells. The CD34+, CD64+ cells were CD19- and the majority were CD45RA+, CD71lo, suggesting that CD64 recognized granulomonocytic progenitor cells. Specificity of CD64 for the granulo-monocytic lineage was shown by demonstrating that colonies arising from CD34+, CD64+ cells consisted of 98% +/- 2% colony-forming unit-granulocyte-macrophage (CFU-GM) in semisolid medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). In contrast, 63% +/- 15% of the colonies from the CD34+, CD64- cells were burst-forming unit-erythroid/colony-forming unit-erythroid (BFU-E/CFU-E). Furthermore, four-color immunofluourescence and cell sorting was used to analyze the progeny of cells cultured in liquid medium containing identical cytokines as used in the semisolid medium. This analysis showed that CD34+, CD64+ cells gave rise to 83% +/- 10% granulo-monocytic cells whereas progeny of the CD34+, CD64- cells contained 81% +/- 11% erythroid cells. Neutrophils as well as basophils and monocytes/macrophages were present in the cultures from CD34+, CD64+ cells, showing that this population contains progenitors of most types of granulo monocytic cells. Two widely used myeloid markers, CD13 and CD33, were not myeloid specific, because both were clearly positive on noncommitted progenitor cells. Of 40 antigens tested, CD15 was the only other marker fulfilling the criteria of a myeloid-specific marker. However, at concentrations of CD15 that did not induce aggregation, CD15+ cells constituted less than 50% of the CD34+, CD64+ cells. Furthermore, the CD34+, CD15- cells showed more than 50% higher CD34 mean fluorescence intensity than the CD64+, CD15+ cells, indicating that CD64 appears earlier than CD15 during differentiation. Thus, among a large number of antigens screened, CD64 was the most useful for the identification and purification of granulo-monocytic progenitor cells. PMID- 7537110 TI - Thrombopoietic effects and toxicity of interleukin-6 in patients with ovarian cancer before and after chemotherapy: a multicentric placebo-controlled, randomized phase Ib study. AB - Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in normal and sublethally irradiated mice, dogs, and primates. To assess its tolerance and efficacy in clinical use, we performed a randomized phase Ib study in patients with ovarian carcinoma. IL-6 was administered during an initial 7-day cycle before any chemotherapy. Beginning 7 days later, six cycles of chemotherapy containing carboplatin were administered every 3 weeks. During chemotherapy cycles 2 to 6, IL-6 was administered from day 4 through day 17 at escalating dose levels from 0.5 to 10 micrograms/kg/d. At each level, three patients received IL-6 and one patient received a placebo. During the prechemotherapy cycle of IL-6, a dose-dependent increase in platelet count was observed from day 12 to 15 and was maximal on day 15 (r = .77; P < .01). The median ploidy of bone marrow megakaryocytes shifted from 16 N to 32 N after 7 days of the initial prechemotherapy IL-6 administration. Dose-dependent increases in C-reactive protein (CRP) and fibrinogen levels were observed on day 8 (P < .0001 for both). A significant decrease in hemoglobin level occured rapidly after initiation of IL-6 therapy and was maximal on day 8 (P < .001). When given after chemotherapy, IL-6 accelerated platelet recovery after chemotherapy cycles 2 to 6. Postponements of scheduled chemotherapy due to thrombocytopenia were less frequent in patients treated with IL-6. No difference in either neutrophils or peripheral blood progenitor assays was observed during or after IL-6 treatment. Toxicity of IL-6 appeared mild and was not dose-limiting up to 10 micrograms/kg/d. Systemic symptoms such as fever, headache, and myalgia were the main side effects and were easily relieved by acetaminophen administration. No biologic toxicity was observed. The data indicate that IL-6 is a well-tolerated cytokine and capable of accelerating platelet recovery in patients receiving chemotherapy. PMID- 7537113 TI - Contact- and growth factor-dependent survival in a canine marrow-derived stromal cell line. AB - Cell-cell interactions and the presence of growth factors such as stem cell factor (SCF; or c-kit ligand) or interleukin-6 (IL-6) are involved in the proliferation and differentiation of the canine marrow-derived stromal cell line DO64. In the presence of SCF, stromal cells are induced to differentiate, but not to proliferate. In contrast, in the presence of IL-6, stromal cells are induced to proliferate rather than to differentiate in culture. Both SCF and IL-6 are produced by the stromal cells themselves and, thus, act as autocrine factors. In addition, DO64 cells also interact physically with each other in culture when grown under optimal culture conditions (70% to 90% cell confluence and in the presence of serum), thereby supporting proliferation and maintaining viability. Under conditions of lower cell density or low serum or growth factor concentrations in culture, DO64 cells tend to aggregate and form clusters. This increase in local cell concentration is associated with preservation of viability, presumably because of the accumulation of autocrine factors. If no signal, neither intercellular nor soluble, is provided, and DO64 cells are not able to reach a critical cell density or to produce sufficient factors in an autocrine fashion, the cells cease to proliferate and eventually die. PMID- 7537114 TI - The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors. AB - There is a long-standing controversy as to whether a single bone marrow (BM) derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence-activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38-, HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM. PMID- 7537115 TI - Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome. AB - The Wiskott-Aldrich syndrome (WAS) is an X-linked (Xp11.22) recessive immunodeficiency syndrome characterized by susceptibility to opportunistic and pyogenic infections, thrombocytopenia, and eczema. Previous studies of obligate carriers of WAS documented that nonrandom inactivation of the X chromosome carrying the defective gene is observed in all peripheral blood cells. The existence of both abnormal platelets and lymphocytes is consistent with a defect that affects early hematopoietic precursors. We isolated CD34+ hematopoietic progenitor cells collected from obligate carriers of WAS by apheresis and used polymerase chain reaction analysis of a polymorphic variable number of repeats (VNTR) within the X-linked androgen receptor to document nonrandom inactivation. These data show that nonrandom inactivation of the X-chromosome in WAS-obligate carriers occurs early during hematopoietic differentiation. PMID- 7537116 TI - Effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers. AB - We explored the ex vivo alteration in the cytokine release of stimulated blood taken from healthy volunteers treated subcutaneously with 480 micrograms granulocyte colony-stimulating factor (G-CSF). In a double-blind, controlled, randomized study with 21 volunteers who received G-CSF once or twice 24 hours apart, we measured lipopolysaccharide (LPS)-inducible release of various cytokines and soluble receptors at different times after treatment. At day 1 after a single dose of G-CSF, mediator release was also initiated with muramyl dipeptide, Staphylococcus aureus enterotoxin A, lipoteichoic acid, streptolysin O, complement factor C5a, phytohemagglutinin, or phorbol myristate acetate. In blood from G-CSF-treated subjects, our major findings were (1) a maximal 12-fold increase in interleukin-1 receptor antagonist (IL-1ra) release and an increase of both the p55 and p75 soluble tumor necrosis factor (TNF) receptors; (2) a reduction in TNF release when using all the various stimuli described except LPS; (3) an increase in G-CSF and, to lesser extent, in IL-6, IL-8, and IL-10 release; and (4) an attenuation of interferon-gamma (IFN-gamma) and granulocyte-macrophage (GM)-CSF release. Our findings demonstrate that the major effect of G-CSF treatment is a change in the responsiveness of blood towards a variety of stimuli, which we interpret as a shift toward an antiinflammatory cytokine response. PMID- 7537117 TI - Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis. AB - Between December 1990 and January 1994, bone marrow (BM) samples from 151 patients with multiple myeloma (MM), including 117 patients evaluated at diagnosis, were collected for cytogenetic analysis. A total of 129 patients had assessable metaphases (100 patients at diagnosis). Cytogenetic studies were performed on BM cells after longterm cultures (6 days) with stimulation of cultures by granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF plus interleukin (IL)-6, IL-3 plus IL-6, or GM-CSF plus IL-3 plus IL-6 to improve myeloma cell growth, and 91 patients had an additional unstimulated culture. Sixty-six patients (51%) had cytogenetic abnormalities, including 47 of 100 patients at diagnosis (47%) and 17 of 24 patients at relapse (71%; P = .04). The aberration rate increased with stage (P = .007), BM plasmacytosis (P = .003), beta 2 microglobulin level (P = .001), C-reactive protein (CRP) level (P = .001), and Ki-67 (P = .007). The abnormality detection rate was higher in stimulated than unstimulated cultures, and the difference was statistically significant (P < .01). Hyperdiploidy was observed in 39 patients (30% of patients with an assessable karyotype) and hypodiploidy in 19 patients (15%). Among numeric changes, gains predominantly involved chromosomes 3, 5, 7, 9, 11, 15, 19 and losses, chromosomes 8, 13, 14, and X. The most frequent loss was loss of chromosome 13, observed in 22 patients (15%), including 18 patients at diagnosis (12%). We observed frequent structural changes of chromosomes 1 (15%) and 14 (10%) but also a 5% incidence of 19q13 abnormality and two patients with translocation t(1;16)(p11;p11). By using the proportional hazard univariate model, patients with abnormal karyotypes were demonstrated to have 2.5-fold greater chance of death than patients with normal karyotypes (P < .014). Despite a multivariate approach with the same model, the respective roles of karyotype abnormality, age, stage, and beta 2 microglobulin level could not be clearly ascertained. From these results we conclude that cytogenetic analysis using stimulation of cultures by cytokine(s) may be a promising method to identify about 50% of cytogenetic abnormalities in patients with newly diagnosed MM. Cytogenetic analysis may help to define a high-risk population that would benefit from intensive therapeutic approaches. PMID- 7537118 TI - Role of B7-1 in mediating an immune response to myeloid leukemia cells. AB - A costimulatory signal from B7-1 (CD80) to its counter-receptor CD28 is required for T-cell activation. Many tumors, including most human leukemias, lack expression of B7-1, and this has been suggested to contribute to the failure of immune recognition of these diseases. A murine leukemia model system was developed to assess the potential role of B7-1 in the induction immunity to leukemia cells. The nonleukemic 32Dc13 myeloid cell line was transformed by transfection of the BCR/ABL gene, generating a subline (32Dp210/clone 26) that was leukemic and rapidly lethal to syngeneic, immunocompetent C3H/HeJ mice or T cell-deficient nude mice. B7-1-modified leukemic cells remained lethal in nude mice, but caused only a transient, nonlethal leukemia in C3H/HeJ mice. After a single exposure to live, nonirradiated B7-1-modified leukemic cells, C3H/HeJ mice developed protective immunity against subsequent challenge with B7-1(-) leukemic cells. Further, hyperimmunization with B7-1(+) leukemic cells prolonged the survival of mice previously injected with a lethal number of B7-1(-) leukemic cells. These results indicate that myeloid leukemic cells may be attractive candidates for B7-1 gene transfer. PMID- 7537119 TI - Serum levels of interleukin-10 in patients with diffuse large cell lymphoma: lack of correlation with prognosis. AB - Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor, has multiple effects on lymphoid development. In addition, it has been previously reported that serum levels of IL-10 correlate with failure-free and overall survival in patients with non-Hodgkin's lymphoma. In this study, we used a sensitive enzyme-linked immunosorbent assay specific for human IL-10 (lower limit of sensitivity, 5 pg/mL) to measure serum levels in 52 newly diagnosed patients with diffuse large cell lymphoma and at least one adverse prognostic feature who were subsequently treated in a uniform way. Lymphoma patients had significantly higher serum levels of IL-10 (median, 7.98 pg/mL; range, < or = 5 to 27,143 pg/mL) than healthy volunteers (N = 50; median, < or = 5 pg/mL; range, < or = 5 to 19.21 pg/mL) (P = .0000012). Individuals with B symptoms had significantly higher serum levels of IL-10 than those without them (P = .03), but there was no correlation between IL-10 levels and any of the other prognostic variables analyzed, including age, lactic dehydrogenase, beta 2-microglobulin levels, performance status, bulky disease, Ann Arbor stage, or International Index score. More importantly, we found no correlation between IL-10 levels and the achievement of complete remission, nor with failure-free survival or overall survival. We conclude that in a uniform population of untreated patients with diffuse large cell lymphoma, serum levels of IL-10 do not appear to have any prognostic value. PMID- 7537120 TI - Biotherapy for xenografted human central nervous system leukemia in mice with severe combined immunodeficiency using B43 (anti-CD19)-pokeweed antiviral protein immunotoxin. AB - The study of central nervous system (CNS) leukemia has been hampered by the lack of a suitable animal model. We report that severe combined immunodeficiency (SCID) mice invariably develop rapidly progressive fatal CNS leukemia within 3 weeks after intravenous injection of NALM-6 pre-B acute lymphoblastic leukemia (ALL) cells. Colonization of the dura mater and subarachnoid space, usually of the distal spinal cord with occasional extension into the Virchow-Robin spaces of blood vessels subjacent to the meninges, followed involvement of bone marrow in the skull, vertebrae, and, occasionally, the appendicular skeleton. Occult CNS leukemia was detectable by polymerase chain reaction amplification of human DNA as early as 8 days postinoculation of leukemia cells. We used this in vivo model of human CNS leukemia to examine the therapeutic efficacy and toxicity of intrathecally administered B43 (anti-CD19)-pokeweed antiviral protein (PAP), an anti-B-lineage ALL immunotoxin directed against the pan-B-cell antigen CD19/Bp95. Intrathecal therapy with B43 (anti-CD19)-PAP immunotoxin at nontoxic dose levels significantly improved survival of SCID mice and was superior to intrathecal methotrexate therapy. PMID- 7537121 TI - Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors. AB - The cobblestone-area forming cell (CAFC) assay permits a direct measurement of the seeding of primitive and more mature murine hematopoietic stem cell subsets by comparing the number of CAFC in the original transplant with the number of CAFC retrieved from bone marrow (BM) and spleen after transplantation. We found no differences in seeding efficiency between the more mature and primitive CAFC subsets, nor between seeding efficiencies of stem cells from low-density (LD) fractions of normal and day-6 post-5-fluorouracil BM. The data show that 18% to 20% of all intravenously transplanted stem cell subsets seed to the BM, whereas 8% to 10% seed to the spleen. In addition, similar seeding efficiencies were found for day-12 spleen colony-forming unit (CFU-S-12) as was determined by retransplantation. Previously, it has been reported that a 2- to 3-hour preincubation of BM with interleukin-3 (IL-3) enhances the in vivo repopulating ability of a graft. To test whether hematopoietic growth factors affected this increased engraftment by enhancing the seeding of the transplanted marrow, we assessed the 16- to 18-hour seeding efficiency of short- and long-term in vivo repopulating stem cell subsets to BM and spleen using the CAFC assay, after preincubation with or without hematopoietic growth factors. A 2- to 3-hour preincubation with IL-3, or a combination of IL-3, IL-12, and steel factor, at 37 degrees C, led to a substantial decrease in seeding compared with control (which was kept on ice) of all hematopoietic subsets measured, both in spleen and BM. In concert with these data, the long-term in vivo repopulating ability of growth factor incubated BM was also decreased when compared with control. In conclusion, we have been unable to observe a beneficial effect of growth factor preincubation on the repopulating ability of a graft. PMID- 7537123 TI - Peripheral blood progenitor cell (PBPC) counts during steady-state hematopoiesis allow to estimate the yield of mobilized PBPC after filgrastim (R-metHuG-CSF) supported cytotoxic chemotherapy. AB - Peripheral blood progenitor cells (PBPC) can be mobilized using cytotoxic chemotherapy and cytokines. There is a substantial variability in the yield of hematopoietic progenitor cells between patients. We were looking for predictive parameters indicating a patient's response to a given mobilization regimen. Multiparameter flow-cytometry analysis and clonogenic assays were used to examine the hematopoietic progenitor cells in bone marrow (BM) and peripheral blood (PB) before filgrastim (R-metHuG-CSF; Amgen, Thousand Oaks, CA)-supported chemotherapy and in PB and leukapheresis products (LPs) in the recovery phase. Fifteen patients (four with high-grade non-Hodgkin's lymphoma [NHL], two with low-grade NHL, two with Hodgkin's disease, two with multiple myeloma, three with breast cancer, one with ovarian cancer, and one with germ cell tumor) were included in this study. The comparison of immunofluorescence plots showed a homogenous population of strongly CD34+ cells in steady-state and mobilized PB whereas in steady-state BM, the CD34+ cells ranged from strongly positive with continuous transition to the CD34- population. Consistent with the similarity in CD34 antigen expression, a correlation analysis showed steady-state PB CD34+ cells (r = .81, P < .001) and colony-forming cells (CFCs; r = .69, P < .01) to be a measure of a patient's mobilizable CD34+ cell pool. Individual estimates of progenitor cell yields could be calculated. With a probability of 95%, eg, 0.4 steady-state PB CD34+ cells x 10(6)/L allowed to collect in six LPs 2.5 x 10(6) CD34+ cells/kg, the reported threshold-dose of progenitor cells required for rapid and sustained engraftment after high-dose therapy. For the total steady state BM CD34+ cell population, a weak correlation (r = .57, P < .05) with the mobilized CD34+ cells only became apparent when an outlier was removed from the analysis. Neither the CD34+ immunologic subgroups defined by the coexpression of the myeloid lineage-associated antigens CD33 or CD45-RA or the phenotypically primitive CD34+/HLA-DR- subset nor the BM CFC count had a predictive value for the mobilization outcome. This may be caused by the additional presence of maturing progenitor cells in BM, which express lower levels of the CD34 antigen and do not circulate. Our results permit us to recognize patients who are at risk to collect low numbers of progenitor cells and those who are likely to achieve sufficient or high progenitor cell yields even before mobilization chemotherapy is administered. PMID- 7537124 TI - Molecular mechanism of histamine release: the role of intermediate filaments and membrane skeletons. AB - It has been recognized that cytoskeletons play some important roles in the histamine release from mast cells. We previously reported the role of microfilaments and microtubules in the histamine release from mast cells, and in the present study, the roles of intermediate filaments and membrane skeletons were investigated. When permeabilized mast cells were stimulated with Ca2+, a translocation of protein kinase C from cytosol to the membrane fraction was observed. This lead to the phosphorylation of vimentin, one of the component proteins of the intermediate filaments. Phosphorylation of vimentin induced disruption of intermediate filaments and resulted in an increase in the mobility of granules. This may be favorable for the initiation of degranulation. In the membrane skeletons of rat mast cells, alpha- and beta-fodrin, ankyrin and actin were found. Changes in the distribution of the fodrin network were elicited by antigen-antibody reaction. It is suggested that membrane skeletons may act as a barrier between the plasma membrane and the granule membrane and that the changes in the distribution of membrane skeletons may facilitate the initiation of the fusion of the plasma membrane and granular membrane. PMID- 7537122 TI - Coblockade of the LFA1:ICAM and CD28/CTLA4:B7 pathways is a highly effective means of preventing acute lethal graft-versus-host disease induced by fully major histocompatibility complex-disparate donor grafts. AB - We have developed an in vitro system in which C57BL/6 donor splenocytes are exposed to B10.BR host alloantigens in the context of deficient CD28:B7 signaling as a means of preventing graft-versus-host disease (GVHD). Although 54% to 82% of MLR alloresponse was inhibited by cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig treatment of host stimulator cells, treated splenocytes were still capable of causing GVHD when infused in vivo. By adding anti-leukocyte function antigen 1 (anti-LFA1) antibody to hCTLA4-Ig in vitro to coblock the LFA1:intercellular adhesion molecule (ICAM) signaling, splenic alloresponse was inhibited by > or = 89%, yet GVHD induction capabilities were retained. Because antigen-primed cells might be more susceptible to CD28:B7 blockade, we investigated whether hCTLA4-Ig alone, anti-LFA1 antibody alone, or the combination of both added to donor antihost in vitro primed cells could reduce GVHD. To facilitate hyporesponsiveness induction and to block B7 and ICAM ligands that are upregulated during GVHD, these reagents were also administered to recipients post BMT. We have shown that hCTLA4-Ig plus anti-LFA1 antibody is highly effective in preventing GVHD-induced lethality (88% to 100% of treated mice surviving versus 0% to 28% of controls surviving). For optimal prevention, both hCTLA4-Ig and anti LFA1 must be used in vitro in the context of donor-antihost primed splenocytes and continued in vivo. This in vitro-in vivo combined approach was associated with donor engraftment, and recipients were not globally immunosuppressed. We conclude that blocking both the CD28/B7 and the LFA1:ICAM pathways are critical to effective GVHD prevention and may offer advantages to in vitro donor T-cell removal. PMID- 7537125 TI - Relations between Fc epsilon RI crosslinking-induced mast cell activation and adhesion to fibronectin. AB - Demonstration of murine mast cell adhesion to fibronectin (FN) following PMA mediated cell activation raised the question whether crosslinking of high affinity IgE receptors on mouse mast cells might induce changes in adhesiveness of these cells to FN. Murine mast cells of line MCP5/L were used to investigate the effect of antigenic stimulation on cell adhesion to fN and mediator secretion. effect of antigenic stimulation on cell adhesion to FN and mediator secretion. Adhesion assays were performed using sensitized radiolabeled cells and FN- or BSA-coated 96-well plates. The presence of antigen in the concentrations up to 10 ng/ml resulted in concentration-dependent adhesion potentiation, which was detectable after 5 min, reached maximum at 30 min and persisted or decreased over the next 30 min. Adhesion potentiation decreased at antigen excess and was abolished by heat inactivation of IgE in the antiserum prior to cell treatment. External calcium ion and temperature dependence of adhesion together with the observation that RGD (Arg, Gly, Asp)--containing peptide blocked cell binding to FN suggests that FC epsilon RI crosslinking-induced adhesion potentiation involves an integrin type receptor on cell surface. Sensitized mast cells allowed to adhere spontaneously to FN released more histamine and beta-hexosaminidase upon antigen challenge. Hence, the results show the relations between IgE-induced mast cell activation, adhesion to FN and mediator secretion. PMID- 7537126 TI - Gastric cytoprotective activity of endogenous 5-HT. AB - The role of endogenous serotinin in the formation of gastric damage was studied in rats. Stress ulcers were induced by ultrasounds, immobilization and immobilization plus cold. The damage of gastric mucosa was estimated (arbitrary scale) and serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in this tissue measured. In all examined groups of animals with gastric mucosal damages the lower levels of 5-HT and 5-HIAA in gastric mucosa were observed. In some experimental groups animals were treated with serotonergic receptor antagonists 30 min. before stress. The administration of ICS 205-930 (80 micrograms/kg), 5-HT3 receptor antagonist, and DAU-62855 (80 micrograms/kg), 5 HT4/5-HT3 receptors antagonist, reduced the intensity of stress gastric injuries. In contrast the administration of methysergide (8 mg/kg), 5-HT1/5-HT2 receptors antagonist, enhanced the stress gastric mucosa damage. 16, 16 dimethyl PGE2 (10 micrograms/kg) protected stomach against stress stimuli and accompanied increase of serotonin and 5-HIAA concentration in gastric mucosa was observed. Both 5 HT3/5-HT4 receptor antagonist had an additive cytoprotective effect when given in combination with PGE2 analog. In the presence of methysergide gastroprotective effect of PGE2 was abolished. The present studies demonstrate that cytoprotective effect of endogenous serotonin depends on 5-HT1 and 5-HT2 receptors stimulation in the gastric mucosa and the protective effect of prostaglandins depends partly on the regulation of serotonin metabolism. PMID- 7537127 TI - A comparison of the effects of isoproterenol and forskolin on immunologic and nonimmunologic release of histamine from guinea-pig superfused trachea and dispersed tracheal cells. AB - This study has compared the abilities of isoproterenol and forskolin to inhibit immunologic- and nonimmunologic-induced histamine release from guinea-pig superfused trachea and enzymatically dispersed tracheal cells. Contraction was also measured in the former preparation. The potency of isoproterenol was similar for inhibition of all parameters associated with immunologic (ovalbumin) challenge in the two preparations. In contrast, forskolin appeared less potent in inhibiting ovalbumin-induced histamine release from dispersed tracheal cells. Histamine release by the nonimmunologic secretagogues d-tubocurarine and compound 48/80 was not altered by either substance. However, inhibition by isoproterenol and forskolin of tracheal contraction was evident when challenge was conducted with d-tubocurarine and compound 48/80. Inhibition of contraction appears to be a result of functional antagonism at the level of the smooth muscle. The superfused trachea is a useful preparation in which to explore the effects of substances that modulate mast cell mediator release. PMID- 7537128 TI - Idiopathic atrophy of the nails: clinical and pathological study of 2 cases. AB - BACKGROUND: Whether idiopathic atrophy of the nails (IAN) should be considered a separate entity or a clinical variant of nail lichen planus is still controversial. OBJECTIVE: We report here the pathological study of 2 patients with IAN. METHODS: Our patients had similar clinical features consisting of severe nail atrophy with and without pterygium. RESULTS: The nail matrix architecture was markedly deformed with complete disappearance of the keratogenous zone that was replaced by a 3- to 10-cell-thick granular layer. CONCLUSION: The hypothesis that IAN is an acute and self-limited variety of lichen planus is still the most presumable. Even though this hypothesis can not be definitely proven, it is nevertheless not excluded by the clinical and pathological findings of our cases. PMID- 7537129 TI - Soluble E-selectin and soluble tumour necrosis factor receptor (60 kD) serum levels in patients with psoriasis. AB - BACKGROUND: Increased tumour necrosis factor alpha has been found in psoriatic skin. This cytokine activates endothelial cells and induces the membrane E selectin molecule (E-selectin or endothelial leucocyte adhesion molecule 1); the same cytokine is able to induce its own receptors. Since the soluble forms of E selectin and tumour necrosis factor receptor (TNF-R, 60 kD) may be reliably measured in body fluids, these determinations have been performed in the sera of psoriatic patients. OBJECTIVE: To evaluate endothelial activation in psoriatic patients, sE-selectin has been determined in patient sera and compared with those of a control group. sTNF-R (60 kD) was also measured in the same samples. METHODS: Two commercially available enzyme immunoassay methods have been used to determine sE-selectin and sTNF-R (60 kD) in the sera of 19 patients with plaque type psoriasis; 22 healthy subjects were used as controls. CONCLUSIONS: Significantly increased amounts of sE-selectin serum levels were found in psoriatic patients as compared to healthy controls. Moreover, a direct correlation between sE-selectin and PASI scores was observed. On the contrary, sTNF-R (60 kD) serum levels presented no increases. These data suggest that sE selectin serum levels are a reliable marker of disease activity in psoriatic patients. PMID- 7537130 TI - Trichorhinophalangeal syndrome. AB - Trichorhinophalangeal syndrome (TRPS) comprises a distinctive combination of hair, facial and bony abnormalities with variable expression. A 20-year-old man with TRPS was seen because of marked androgenetic alopecia. Scanning electron microscopic studies of the hair revealed flattened hair with an elliptoid transverse section pattern. Mechanical behavior of the hair was abnormal with a significant increase in the viscous parameter, indicating a decreased intermolecular bridging within the keratin matrix. The dermatologist confronted with premature or marked alopecia in young adults should always consider the possibility of an underlying congenital syndrome involving the hair and prompt further investigation. PMID- 7537131 TI - [New molecules with potential antitumor effect]. PMID- 7537132 TI - Bacteriophages specifically recognizing the lipopolysaccharide antigens O4, O5, O6, and O7 of Escherichia coli. AB - Four bacteriophages recognizing the Escherichia coli lipopolysaccharide (LPS) antigens O4, O5, O6, and O7, respectively, were isolated from pooled sewage samples. Electron microscopic investigations revealed icosahedral phage structures. Phages phi O4, phi O5, and phi O7 belonged to Bradley's morphology group C, while phi O6 had a tail and resembles phages of group A of Bradley. The nucleic acid of the phages was identified as double-stranded DNA of different genomic sizes. Host range studies showed that only E. coli strains with homologous O antigens were attacked. No lysis of encapsulated and rough E. coli strains was observed. The phages specifically depolymerized the homologous LPS of their host strains; they may be useful for detecting respective non-capsulated E. coli strains in epidemiological studies as simple alternative to the laborious serological typing. Diagnostic application is restricted, however, as strains carrying K antigens have not been detected. The high specificity of the phage associated enzymes provides a mild method for the preparation of oligosaccharides from the LPS for structural studies. PMID- 7537133 TI - rRNA gene restriction patterns as possible taxonomic tools for the genus actinomyces. AB - Species delineation in the genus Actinomyces remains unclear, particularly regarding the two taxa, A. naeslundii and A. viscosus. The ribotyping patterns of 64 strains of Actinomyces, representing 8 species and comprising different serotypes, were studied as possible taxonomic tools, using an acetyl aminofluorene (AAF)-labelled E. coli 16S + 23S rRNA probe. Similarities between patterns were assessed using Jaccard's coefficient and clustering achieved using the unweighted pair-group method with average linkage (UPGMA) on a Macintosh II (Apple, Cupertino, USA) computer. The dendrogram obtained from the ribotypes gave results which were in reasonable agreement with many previous reports: A. bovis, A. gerensceriae, A. israelii, A. meyerii, A. odontolyticus and A. pyogenes were found to be distinct species but the two taxa A. naeslundii and A. viscosus remained unclear. Further investigations, using a larger number of A. naeslundii and A. viscosus strains and other endonucleases, need to be carried out to provide more information concerning the relatedness of these two taxa. Nevertheless, these preliminary results suggested that the Actinomyces chromosome contains multiple rRNA operons which may be used as an epidemiological and taxonomical tool. PMID- 7537134 TI - Partitioning of streptokinase in aqueous two-phase systems. AB - The partitioning of streptokinase in aqueous two-phase systems, containing poly(ethylene glycol) and dextran or poly(ethylene glycol) and salt was investigated. The protein partitions in favour of the upper, PEG-rich phase, if PEG of low molecular weight and potassium phosphate or ammonium sulphate were used. This property was found to be independent of the degree of purity and was exploited for the partial purification of streptokinase from crude material. The protein was shown to exhibit negligible affinity to diverse triazine dyes applied in affinity partitioning experiments. PMID- 7537135 TI - Experimental and theoretical analysis of the chromatographic behaviour of protein purification fusions carrying charged tails. AB - Poly(glutamic acid) tail consisting of 6 glutamate residues was fused to the N terminus of Escherichia coli beta-galactosidase (beta-gal), by genetic engineering techniques. The wild-type and modified genes were expressed intracellularly and in soluble state in Escherichia coli, leading to the proteins respectively designated beta-gal2 and E6-beta-gal. Both enzymes were purified by affinity chromatography. The specific activity of purified E6-beta-gal was found to be comparable to the wild-type enzyme and its increased net charge was indicated by lon-Exchange Chromatography (IEC). The use of such a charged fusion for selective recovery of beta-gal from cell extract using IEC and Ion-Exchange Membrane Chromatography (IEMC) was explored. The additional charges enabled the separation factor to be increased about two-fold on both IEC and IEMC, but the IEMC step achieved a better throughput than the IEC step. The selectivity of recovery promoted by the charged tail was further analysed by processing the experimental data obtained in IEC with the Stoichiometric Displacement Model, a recent model very appropriate for the understanding of the retention of polymeric biomolecules on ion-exchangers. It was shown that E6-beta-gal had the same characteristic charge as beta-gal2 but that the binding constant to the ion exchanger of the tagged beta-gal was 6 times greater than for the wild-type enzyme. PMID- 7537137 TI - Dying for palliative care. PMID- 7537136 TI - [CD44 splice variants as prognostic factors in invasive cervix carcinoma]. AB - OBJECTIVE: Aberrant expression of specific isoforms of the cell adhesion molecule CD44 has been detected in various malignant human tumors. We wanted to verify whether the expression of these splice variants was associated with a higher incidence of metastasis and poor prognosis. METHODS: We studied the expression of CD44 splice variants v5, v6 and v7-8 by immunohistochemistry in human cervical cancer. 105 surgically treated patients with cervical cancer of stages IB-IIB were included in the study. RESULTS: Tumors expressing exon v6 had significantly more often metastasized to the pelvic nodes (p = 0.04), and patients suffering from tumors expressing CD44v6 showed poorer overall survival. Even in cases with negative pelvic lymph nodes, we found a significantly poorer prognosis when tumors expressed CD44v6. CONCLUSIONS: Among the investigated CD44 splice variants, expression of exons v6 and v7-8 is the most promising prognostic marker in cervical cancer. They can possibly identify patients with an increased risk earlier than morphologic prognostic parameters. PMID- 7537138 TI - Effect of the local anesthetics phenethyl alcohol and procaine on hns mutants of the acid-induced biodegradative arginine (adi) and lysine (cad) decarboxylases of Escherichia coli. AB - The environmentally responsive biodegradative arginine (adi) and lysine (cad) decarboxylases are maximally induced when Escherichia coli is cultured under acidic, anaerobic conditions in rich medium. Previously, transposon mutagenesis led to the identification of hns (encoding H-NS, a histone-like DNA binding protein) as being a trans-acting regulatory factor of both systems. The hns mutants show depressed expression of adi or cad (i.e., their expression is increased). The effects of the local anesthetics phenethyl alcohol (PEA) and procaine (both environmental perturbants) were investigated with lacZ operon fusions to either adi or cad and their respective hns mutants. These results indicate that wild-type fusion strains are insensitive to either PEA or procaine, but that hns mutants show decreased beta-galactosidase synthesis in the presence of one or both of the local anesthetics. This is the first report of the effect of local anesthetics on hns mutants in this or any other environmentally responsive system. PMID- 7537139 TI - Epidemiology of breast cancer. PMID- 7537140 TI - Levels of selenium in plasma and glutathione peroxidase in erythrocytes in patients with prostate cancer or benign hyperplasia. AB - Plasma selenium and glutathione peroxidase in erythrocytes were analysed in a case-control study encompassing 164 cases with prostate cancer and 152 controls with benign prostate hyperplasia. Plasma selenium levels were divided into three groups; I > 1.17, II 1.00-1.17 and III < 1.00 mumol/l. For the 124 cases with no supplementary intake of selenium pills, the mean plasma selenium level was 0.99 (range 0.27-1.47) and for the corresponding 121 controls 1.08 (range 0.52-1.50) mumol/l, a difference which was significant (P = 0.0007). The three categories of selenium levels had odds ratios (OR) of 0.3 and a 95% confidence interval (CI) of 0.1-0.7 for group I, an OR of 0.6 and a CI of 0.3-1.1 for group II, and group III was used as the reference entity. No significant differences in levels of glutathione peroxidase in erythrocytes were found between cases and controls. PMID- 7537142 TI - Ask the experts. What is a multiple marker (triple screen) test? PMID- 7537141 TI - Mathematical models for tumour angiogenesis: numerical simulations and nonlinear wave solutions. AB - To ensure its sustained growth, a tumour may secrete chemical compounds which cause neighbouring capillaries to form sprouts which then migrate towards it, furnishing the tumour with an increased supply of nutrients. In this paper a mathematical model is presented which describes the migration of capillary sprouts in response to a chemoattractant field set up by a tumour-released angiogenic factor, sometimes termed a tumour angiogenesis factor (TAF). The resulting model admits travelling wave solutions which correspond either to successful neovascularization of the tumour or failure of the tumour to secure a vascular network, and which exhibit many of the characteristic features of angiogenesis. For example, the increasing speed of the vascular front, and the evolution of an increasingly developed vascular network behind the leading capillary tip front (the brush-border effect) are both discernible from the numerical simulations. Through the development and analysis of a simplified caricature model, valuable insight is gained into how the balance between chemotaxis, tip proliferation and tip death affects the tumour's ability to induce a vascular response from neighbouring blood vessels. In particular, it is possible to define the success of angiogenesis in terms of known parameters, thereby providing a potential framework for assessing the viability of tumour neovascularization in terms of measurable quantities. PMID- 7537144 TI - Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats. AB - Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation. PMID- 7537143 TI - Attenuation of parasite cAMP levels in T. cruzi-host cell membrane interactions in vitro. AB - Previous investigations have shown that the adhesion of T. cruzi plasma membrane vesicles (PMV) to monolayers of host cell myoblasts and to immobilized heart muscle sarcolemma membranes (PAM) on polyacrylamide beads is mediated by the interaction of T. cruzi attachment sites with the muscarinic cholinergic and beta adrenergic receptors of the host cell membrane. It has also been shown that this interaction is blunted by the specific antagonists of the mammalian receptors atropine and propranol, respectively. In the studies reported here, PAM also rapidly attached to swimming T. cruzi trypomastigotes in a complex, concentration dependent fashion and binding isotherms showed that the equilibrium between free and bound PAM is rapidly reached within 2 minutes of incubation in physiologically balanced salt solutions. In this time frame, trypomastigote cAMP levels are significantly reduced from steady state values within 30 seconds of the addition of PAM in a buffer system containing a diesterase inhibitor. Maximal attenuation of cAMP levels was measured between 1 and 2 minutes of the addition of PAM to T. cruzi trypomastigotes. The degree of cAMP level attenuation was reduced by blocking PAM attachment with either atropine or propranol. On the basis of these results we propose that a likely pathway for the negative parasite signal generated upon adhesion of host muscle cell membranes to the surface of the flagellates is from the parasite's surface attachment sites directly to a Pertussis toxin sensitive inhibitory protein Gi, thereby blunting adenyl cyclase activity and cAMP formation. PMID- 7537145 TI - Histochemical detection of carbohydrates of Blastocystis hominis. AB - The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole. PMID- 7537146 TI - Chemorepellents in Paramecium and Tetrahymena. AB - Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels. PMID- 7537148 TI - Analysis of linkage disequilibrium between different cystic fibrosis mutations and three intragenic microsatellites in the Italian population. AB - Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVS17bTA and IVS17bCA, located in intron 17b and a CA repeat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and difference CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all delta F508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1G-->A, which might have been originated more recently than delta F508. PMID- 7537149 TI - Leu-676-Pro mutation of the androgen receptor causes complete androgen insensitivity syndrome in a large Hutterite kindred. AB - A large Manitoba Hutterite kindred with X-linked receptor negative complete androgen insensitivity syndrome (CAIS) was studied. In attempts to identify all carriers of the syndrome in this kindred, using the androgen receptor (AR) cDNA, we have found a novel diagnostic MspI polymorphic pattern, which cosegregates with the disease. This polymorphism was not detected in 79 unrelated X chromosomes of which 22 were from Hutterite controls. We were able to localize the polymorphism to exon 4, which is known to encode part of the androgen receptor hormone binding domain. A single base substitution (T-->C) was detected, which creates a new MspI site. This novel transition mutation replaces Leu-676 with Pro at a site which is conserved in numerous members of the steroid receptor gene family. Sequencing all 8 exons of the AR revealed the Leu-676-->Pro mutation as the only change in the primary structure of the receptor. Transfection of COS 1 cells with an expression vector of the mutant AR demonstrates that this point mutation of nucleotide 2558 abolishes receptor binding activity. The mutation can easily be detected by MspI digestion of the polymerase chain reaction (PCR) amplified exon 4 product. PMID- 7537150 TI - Identification of six mutations (R31L, 441delA, 681delC, 1461ins4, W1089R, E1104X) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - Six new mutations have been identified in the CFTR gene. These mutations, representing three different categories--missense (R31L, W1098R), nonsense (E1104X), and frameshift (441delA, 681delC, 1461ins4)--are located in exons 2, 4, 5, 9, and 17b of the gene and presumed to cause cystic fibrosis (CF) in patients. All these mutations are probably rare in the population, as no additional examples were found for any of them in a cohort of 545 CF patients. Our study also revealed a benign sequence variation (3499 + 45T-->C) in intron 17b. PMID- 7537147 TI - A splicing mutation (1898 + 1G-->T) in the CFTR gene causing cystic fibrosis. PMID- 7537152 TI - The role of air pollutants. PMID- 7537154 TI - Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2. AB - Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2 producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2 secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers. PMID- 7537151 TI - [Ectopic pregnancy: criteria to choose therapy]. AB - The ectopic pregnancy (EP) could be treated by spontaneous resolution, or medical treatment or laparoscopic treatment. Spontaneous resolution of EP with beta hCG < 1,000 mUl/ml, plasmatic progesterone < 5 ng/ml had 74% success rate. Methotrexate (MTX) injection is the common Medical treatment of EP with beta hCG < or = 5,000 mUl/ml and mean diameter of EP < or = 3 cm: a single dose of 50 mg/m2 or 1 mg/kg intramuscular injection had 93.4% success rate; local injection under sonographic control (1 mg/kg) had 80.2% success rate. The surgical conservative treatment by laparoscopy had 94% success rate. PMID- 7537153 TI - Management perspectives. Surgical unit nursing assignments. PMID- 7537156 TI - Surgery for pancreas divisum. AB - Recurrent acute pancreatitis has a documented association with pancreas divisum. The intermittent inflammation of the gland is caused by episodic obstruction of pancreatic secretions at the minute dorsal duct orifice. Selection of appropriate patients for operative treatment is paramount if long-term success is to be achieved. Dorsal duct sphincterotomy alone, performed by a transduodenal approach, requires precise surgical technique. Long-term freedom from recurrent attacks of pancreatitis can be achieved by operative treatment. PMID- 7537155 TI - Hepatocellular carcinoma. AB - Hepatocellular carcinoma is regarded as an aggressive tumour with a poor short term prognosis. Recent research has suggested that additional aetiological factors may be involved in hepatocarcinogenesis. The development of orthotopic liver transplantation has offered renewed hope for disease-free survival. PMID- 7537158 TI - Macroamylasemia: a possible cause of unexplained hyperamylasemia in rheumatoid arthritis. AB - Macroamylasemia is a benign acquired condition, characterized by a serum amylase unusually large in molecular size that has been found to occur in apparently healthy humans as well as in a variety of diseases including liver disease, diabetes, cancer malabsorption and autoimmune disorders. Most commonly macroamylasemia results from the formation of immune complexes between amylase and immunoglobulins. We describe the first case of an association between macroamylasemia/hyperamylasemia and rheumatoid arthritis characterized by the absence of immunoglobulins, as amylase binding globulins, within the macroamylase complex. Failure to identify macroamylase as the cause of unexplained but benign hyperamylasemia correctly, can lead to costly studies (e.g. ultrasonography, computerized tomography) to rule out pancreatic disease, and could induce prescription of unnecessary elemental diets and replacement therapies, as reported in our patient. PMID- 7537157 TI - Regulation of the interferon-induced PKR: can viruses cope? AB - Viruses that fail to block the lethal effects of the double-stranded-RNA activated protein kinase (PKR) may be doomed; why do so many viruses go to so much trouble to downregulate this interferon-induced protein kinase? PKR may regulate cell growth and proliferation in uninfected cells, suggesting that it also participates in the antiproliferative arm of the interferon response. PMID- 7537159 TI - Effect of PAF antagonists on cerulein-induced pancreatitis. AB - The present study was undertaken to investigate the involvement of PAF in acute pancreatitis induced by cerulein in rats. Cerulein (two doses of 20 micrograms/rat, the first s.c. and the second i.v., 1 h apart) induced a significant increase in vascular permeability in the pancreas, evaluated by the Evans blue (EB) extravasation method. Plasma amylase levels were also significantly increased in this group. The PAF antagonists, BN-52021 (5 mg/kg) and WEB-2170 (1 and 10 mg/kg), both significantly reduced the extravasation of EB in the pancrease induced by i.v. injection of PAF (1 microgram/kg). At these concentrations, BN-52021 was effective at inhibiting cerulein-induced pancreatitis (60-70% of inhibition) whereas WEB-2170 had no significant effect. Although the inhibition induced by BN-52021 suggests the involvement of PAF in cerulein-pancreatitis, the lack of effect of WEB-2170 reported here does not allow a definite conclusion. Further studies are needed to elucidate the differential effect of the PAF antagonists. PMID- 7537160 TI - Eicosanoid release in allergen-induced bronchoconstriction in dogs. Its relationship to airways hyperreactivity and pulmonary inflammation. AB - In vitro studies have demonstrated the release of histamine, PGD2, TXB2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha and the peptidoleukotrienes from sensitized lung. We have assessed the release of these mediators following antigen challenge in vivo. Antigen challenge of allergic dogs resulted in an acute bronchoconstriction and an increase in bronchial reactivity to histamine and an increase in neutrophil and eosinophil numbers recovered by bronchoalveolar lavage (BAL), 2 and 24 h later. The acute response was associated with histamine and PGD2 release but there was no increase in the levels of other eicosanoids in recovered BAL. Bronchial hyperreactivity and pulmonary inflammation, 2 and 24 h post-challenge was not associated with a concommitant (antigen specific) increase in BAL fluid eicosanoid levels. We conclude that although PGD2 is released during the acute response to antigen the subsequent changes (hyperreactivity and inflammation) are not characterised by a maintained elevation in eicosanoid levels detectable by BAL. PMID- 7537161 TI - Cloning and characterization of pentylenetetrazol-related cDNA, PTZ-17. AB - cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol. PMID- 7537163 TI - Serotonergic and non-serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study. AB - Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat. After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD). A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups. Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups. In the RD, 30% of CTb stained cells were immunoreactive to 5-HT. After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70%. These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment. To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed. Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero posterior differences. PMID- 7537162 TI - Different mechanisms of L-arginine induced dilation of brain arterioles in normotensive and hypertensive rats. AB - We evaluated the response of pial arterioles to L-arginine in anesthetized normotensive rats and spontaneously hypertensive rats equipped with a closed cranial window. Topical application of 10(-6)-10(-4) mol/l L-arginine, which is known to be the endogenous substrate for the synthesis of nitric oxide, induced dose-dependent arteriolar vasodilation. The response was more pronounced in hypertensive than in normotensive rats (at the concentration of 10(-4) mol/l L arginine: 18.3 +/- 3.3% vs. 6.7 +/- 1.7%, respectively, means +/- S.E.). The stereoisomer D-arginine had no effect in hypertensive rats. Topical application of the nitric oxide synthase inhibitor N-nitro-L-arginine converted L-arginine induced dilation to constriction in normotensive and hypertensive rats. The cyclooxygenase inhibitor indomethacin (5 micrograms/ml cerebrospinal fluid) also blocked the dilation in both strains. Photochemical endothelial injury blocked L arginine-induced dilation in normotensive rats, but only partly antagonized the response in hypertensive animals. Intravenous or topical pretreatment with the free radical scavenger superoxide dismutase significantly reduced the dilating response to 10(-4) mol/l L-arginine in hypertensive rats. Superoxide dismutase did not significantly change the response to L-arginine in normotensive animals. It is concluded that nitric oxid formation in the endothelium and liberation of cyclooxygenase products cause L-arginine-induced dilation in normotensive rats. While nitric oxide and cyclooxygenase products are also involved in L-arginine induced dilation in spontaneously hypertensive rats, superoxide radicals contribute to the enhanced response in this strain. This mechanism appears to be specific for the hypertensive animals and is only partly dependent on an intact endothelium. PMID- 7537165 TI - Role of mucin, mannose, and beta-1 integrin receptors in Escherichia coli translocation across Caco-2 cell monolayers. AB - Our previous work suggests that Caco-2 cells play an active role in bacterial translocation (BT). Since bacterial enterocyte interactions may be receptor mediated, the current study was performed to investigate the role of beta 1 integrin and mannose receptors as well as the general protective effect of the mucous layer in this process. Caco-2 cells grown to confluence on semipermeable membranes contained in the upper compartment of a two compartment system were utilized. BT was assessed by quantitating the number of Escherichia coli crossing the monolayers after challenge with 10(8) E. coli C25. Pretreatment of the Caco-2 cells with the beta 1 integrin receptor competitive inhibitors fibronectin or RGD did not inhibit BT; while pretreatment of Caco-2 cells with the LFA-1 (lectin) receptor competitive inhibitor mannose (12 mg/ml) or purified mucin (8 mg/ml) decreased BT compared to control membranes (p < .001). Transepithelial resistance was similar among all the groups indicating maintenance of tight junction integrity. These studies suggest that E. coli BT in the Caco-2 system can be reduced by mannose and that intestinal mucin contributes to the barrier function of the monolayer. PMID- 7537166 TI - Effect of resuscitation with hydroxyethyl starch and lactated Ringers on macrophage activity after hemorrhagic shock and sepsis. AB - Hemorrhagic shock appears to predispose patients to subsequent sepsis. This study examined the effect of different resuscitation fluids on macrophage function following hemorrhagic shock. Male Sprague-Dawley rats were bled to a blood pressure of 50 mmHg for 60 min and then resuscitated with 6% hydroxyethyl starch (HES) or Lactated Ringers (LR). Phagocytic function was assessed by clearance of IV colloidal carbon (C). Carbon clearance was not statistically different between control (154.89), shock LR (169.16), and shock HES (144.60). Computerized image analysis of C distribution in sections of liver and spleen taken 4 h after C infusion exhibits a significant decrease in C distribution after resuscitation with HES compared to control and animals resuscitated with LR (Student's t test, p < .05). Male CBA/J mice were bled to a mean blood pressure of 50 mmHg for 60 min and then resuscitated with either LR (N = 18) or HES (N = 17). In separate experiments CBA/J mice had no shock, but were given LR or HES followed by cecal ligation and puncture and later excision (CLPE). A final group had shock with either LR or HES resuscitation and then CLPE as above. Splenocytes were harvested 48 h after shock for mixed lymphocyte culture (MLC). Animals undergoing shock with subsequent septic challenge (Shock/CLPE) showed significantly increased mortality 40 vs. 0% (chi square, p < .05) and immunosuppression on MLC 2,088(LR)/3,300 (HES) vs. 18,570 (LR)/17,705 (HES) compared to CLPE alone (Student's t test, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537164 TI - Hepatic artery dexamethasone infusion inhibits colorectal hepatic metastases: a regional antiangiogenic therapy. AB - BACKGROUND: A randomized trial treating colorectal hepatic metastases demonstrated that hepatic arterial floxuridine (FUdR) with dexamethasone increased tumor response compared with hepatic arterial FUdR alone (Cancer 1992;69:327-34). The mechanism of this improvement is unclear. METHODS: We investigated the effect of hepatic arterial dexamethasone with or without FUdR on the growth of colorectal hepatic metastases in an animal model. BD-IX rats were inoculated intrasplenically with 10(7) K12/TRb colon cancer cells on day 0. On day 14, the hepatic metastases were counted and hepatic arterial catheters placed for chemotherapy. Forty-eight animals were randomized to 4 groups for 14 days of infusion with heparinized saline alone (group A), heparinized saline with dexamethasone 0.03 mg/kg/d (group B), heparinized saline with FUdR 2 mg/kg/d (group C), or heparinized saline with dexamethasone 0.03 mg/kg/d plus FUdR 2 mg/kg/d (group D). The hepatic metastases were recounted by laparotomy on day 28. Response in each rat was expressed in terms of percentage change in number of hepatic nodules between the number of hepatic nodules seen on days 14 and 28. In vitro chemosensitivity of K12/TRb to dexamethasone with or without FUdR was examined using an MTT (3-(4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, U.S.A.) assay. The effect of dexamethasone on tumor-induced angiogenesis was tested using an in vivo assay. RESULTS: The mean percentage change in tumor nodules was +129% in group A, +17% in group B, -4% in group C, and -29% in group D (p = 0.002 A vs. B, p = 0.04 C vs. D). The MTT assay showed that dexamethasone had no direct effect on K12/TRb growth or on tumor FUdR sensitivity. Dexamethasone inhibited K12/TRb-induced angiogenesis in vivo. CONCLUSIONS: Hepatic arterial dexamethasone is effective in treating colorectal hepatic metastases and is more effective when combined with hepatic arterial FUdR. The antiangiogenic activity of dexamethasone may partially contribute to its efficacy. PMID- 7537167 TI - Invited opinion: role of nitric oxide in hemorrhagic, traumatic, and anaphylactic shock and thermal injury. AB - The free radical nitric oxide (NO) is synthesized from the guanidino group of L arginine by a family of enzymes termed NO synthase (NOS). Hemorrhagic shock leads to an inhibition of NO production by the calcium-dependent, endothelial NOS (ecNOS), which may lead to maldistribution of blood flow leading to, e.g., coronary, renal, and cerebral ischemia and may enhance the adhesion of neutrophil granulocytes and platelets to the endothelial surface. Prolonged periods of hemorrhagic shock are associated with the induction of a calcium-independent isoform of NOS in a variety of organs and in the vascular smooth muscle. The formation of large quantities of NO by inducible isoform of NOS (iNOS) contributes to the delayed vascular decompensation and to the hyporeactivity of the vasculature to vasoconstrictor agents. An impairment of NO formation by the ecNOS has been demonstrated in various models of traumatic shock, whereas there is good experimental evidence supporting the hypothesis that an enhanced formation of NO contributes to the pathophysiology of experimental thermal injury and anaphylactic shock. We speculate that a pharmacological modulation of NO biosynthesis which either enhances NO concentration in the vicinity of endothelium (i.e., NO donors) or inhibits NO overproduction following iNOS expression (i.e., iNOS-selective NOS inhibitors) may become novel therapies to improve the outcome of patients with circulatory shock of various etiologies. PMID- 7537169 TI - Allergens in rhinitis. B- and T-cell epitopes of allergen molecules. PMID- 7537170 TI - Oral antihistamine/decongestant treatment compared with intranasal corticosteroids in seasonal allergic rhinitis. AB - This international, multicentre, randomized, double-blind, double-dummy, parallel group trial was undertaken to compare the efficacy and tolerability of once-daily astemizole-D (10 mg astemizole plus 240 mg pseudoephedrine) with beclomethasone nasal spray (0.05 mg/ml) two puffs/nostril administered twice daily in a total of 204 patients with seasonal allergic rhinitis. Treatment duration was 4 weeks. Although investigator assessments of symptom severity were generally comparable in the two treatment groups throughout the trial, statistically significant differences in favour of astemizole-D for sneezing and ocular symptoms were apparent at the end of the 4-week treatment period (P < 0.05). Patient diary data support these findings, with significant differences in favour of the antihistamine/decongestant combination reported for ocular symptoms after 2 weeks of treatment (P < 0.05) and non-significant trends for sneezing after 2 weeks and ocular symptoms over the entire treatment period (P = 0.07). Use of rescue medication for ocular symptoms was also significantly lower in the astemizole-D treatment group (P < 0.05). A wide range of adverse experiences were reported, however, there were no statistically significant differences in the type or incidence of those between the two treatment groups. In conclusion, astemizole-D appears to be at least as effective and well tolerated as intranasal beclomethasone in the treatment of seasonal allergic rhinitis, providing at least comparable relief from all nasal symptoms including congestion and significantly greater relief from ocular symptoms than the topical steroid. PMID- 7537171 TI - Pollen allergen homologues in barley and other crop species. AB - Pollen from 10 agricultural plant species was surveyed for the presence of proteins crossreactive with group I, group IV and group IX allergens. Barley (Hordeum vulgare), maize (Zea mays), rye (Secale cerale), triticale (xTriticosecale cereale), oats (Avena sativa), Canola (Brassica napus) and sunflower (Helianthus annus) pollens contained numerous allergen cognate proteins. Northern blot analysis of barley pollen RNA revealed the presence of group I and group IX allergen transcripts. The barley pollen cDNA hvp9742, and three other cloned allergens: phlenum protense (Phl p) V, Phl p Va and Lolium perenne (Lol p) 1b, were demonstrated to have extensive nucleotide and amino acid sequence similarity to the Poa p IX isoallergens. It was concluded that hvp9742 represents a Poa p IX isoallergen homologue expressed by barley pollen, and was therefore designated Hor v IX. It is further shown that the most highly conserved domains of all seven proteins, including Hor v IX, map to previously defined Poa p IX antibody binding epitopes. PMID- 7537168 TI - Protein kinase C is a mediator of lipopolysaccharide-induced vascular suppression in the rat aorta. AB - Treatment of vascular tissue with lipopolysaccharide (LPS) in vitro induces hyporesponsiveness to contractile agonists. We investigated whether protein kinase C (PKC) transduces the LPS signal into contractile dysfunction. Rat aortic tissue was incubated .5-18 h with LPS (10 or 30 ng/mL) or alpha- and beta-phorbol 12,13-dibutyrate (PDB, .1 or 1 microM), either alone or combined with cycloheximide (50 microM) or the kinase inhibitors sphingosine (20 microM), H7 (1 (5-isoquinolinylsulfonyl)-2-methyl piperazine, 25 microM), and HA1004 (N-(2 guanidinoethyl)-5-isoquinolinesulfonamide, 25 microM). LPS and beta-PDB induced a sustained translocation of PKC activity from the cytosol to the membrane, an increased protein synthesis-dependent expression of nitric oxide synthase (NOS) activity, and an impaired contractility that could be partially reversed by treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester. Incubation with alpha-PDB, an inactive isomer of beta-PDB, did not alter any of the tissue functions. Sphingosine blocked LPS- and beta-PDB-induced NOS activity and LPS-induced impairments in tissue contractility and PKC translocation. Incubation with H7 also protected against LPS-induced vasoplegia, while HA1004, used as a negative control for H7, provided little protection against LPS. These data indicate that PKC plays a role as an intracellular mediator of LPS-induced NOS activity and vascular suppression. PMID- 7537172 TI - BC network to improve palliative care. PMID- 7537173 TI - Activity of pp60c-src in 60 different cell lines derived from human tumors. AB - The activity of the protein tyrosine kinase pp60c-src was determined for each of the 60 human cell lines in the panel used by the National Cancer Institute for the random screening of potential anticancer drugs. The leukemia, lymphoma, melanoma, and small-cell lung cancer derived cell lines had low pp60c-src activity. Surprisingly, non-small-cell lung and ovarian cell lines had a median pp60c-src activity which was greater than that of the panel of cells representing colon cancer, which is most often associated with elevated pp60c-src activity. This data defines homologous cell lines which contain low and high pp60c-src activity which will aid attempts to understand the role of this enzyme in human cancer. PMID- 7537174 TI - CD44 isoform expression in primary and metastatic pancreatic adenocarcinoma. AB - CD44 is the transmembrane adhesion molecule which binds hyaluronate. The gene encoding CD44 is found on chromosome 11p and comprises 20 exons. Differential splicing of the 10 extracellular juxtamembranous exons (v1-10) generates the major isoforms of CD44. The major CD44 isoform found on hematopoetic cells (CD44s) contains none of the variably expressed exons, while the major isoform expressed on epithelial cells [CD44(v8-10)] contains exons v8-10. Metastasis specific isoforms of CD44 were first documented in a model of rat pancreatic adenocarcinoma [CD44(v4-7), CD44(v6-7)] and subsequently in other cancers. This study is the first characterization of CD44 isoforms in primary and metastatic human pancreatic adenocarcinomas. CD44 isoforms were analyzed in specimens of 15 primary and 6 metastatic pancreatic adenocarcinomas as well as in 6 specimens of control pancreata by two different methods. Radiolabeled reverse transcriptase PCR coupled with 8% PAGE allowed analysis of the major isoforms of CD44, while Southern blot hybridization with [alpha-32P]dCTP-labeled probes permitted analysis for metastasis-specific CD44 isoforms containing CD44(v6) or CD44(v8 10). No differences in the expression of CD44(v8-10) and CD44s were found among the primary and metastatic pancreatic adenocarcinomas, and control specimens of pancreata. However, a novel CD44(v6) isoform was found in metastatic lesions and may represent the human homologue of the rat pancreatic adenocarcinoma metastasis associated CD44 isoform. PMID- 7537176 TI - Neovascularization induced growth of implanted C6 glioma multicellular spheroids: magnetic resonance microimaging. AB - Magnetic resonance imaging has been used to follow noninvasively tumor neovascularization and tumor growth in a model system of multicellular C6 rat glioma spheroids implanted s.c. in nude mice. By positioning a single spheroid approximately 1 cm from the site of incision both the vascularization of the tumor and the wound healing processes were spatially separated and could be simultaneously followed. The model proposed here provides defined initial conditions of tumor geometry and cell proliferative status and separation of initial tumor growth from neovascularization. Magnetic susceptibility relaxation provided an intrinsic marker for blood containing vessels. The implanted spheroid induced vessel growth within 4 days after implantation that was geometrically oriented toward the spheroid and distinct from wound healing at the site of incision. Volume measurements showed a corresponding 4-day lag in growth followed by Gompertz progression. Sham implantation of agarose beads of similar diameter showed no induction of vessel growth, ruling out a direct effect of wound healing. The new vessels penetrating the tumor were highly permeable to the contrast reagent gadolinium-diethylenetriaminepentaacetic acid. This permeability may be due to the action of vascular endothelial growth factor, a major angiogenic growth factor in this system, and a potent permeability factor. PMID- 7537175 TI - Tumor-associated sialylated antigens are constitutively expressed in normal human colonic mucosa. AB - Immunohistochemical studies have indicated that sialylated carbohydrate antigens such as sialyl-Tn, sialyl-Le(a), and sialyl-Le(x) are expressed in a tumor associated fashion in human colon. Since sialic acid residues are O-acetylated more extensively in normal colonic epithelium than in colon cancer cells, we examined whether deacetylation of colonic tissues might enable monoclonal antibodies to recognize these tumor-associated sialylated antigens. In normal colon, deacetylation turned most cases (82%) positive with anti-sialyl-Tn mAb TKH2; and in colon cancers, it increased the number of TKH2-positive cells. Sialyl-Le(a) and sialyl-Le(x) detection was also increased after deacetylation of normal and malignant colonic tissues so that the frequency of positive cases in normal tissues was similar to that in the cancers. However, in the stomach and pancreas, the same treatment rarely increased the detection of the sialylated epitopes in normal or cancerous tissues. Thus, the same sialylated epitopes can be expressed in a tumor-associated fashion by different mechanisms in different gastrointestinal organs; in the colon, these antigens are constitutively expressed and O-acetylated, whereas in the upper gastrointestinal tract, they are rarely O-acetylated, suggesting that other mechanisms such as differences in glycosylation account for the cancer-associated expression. PMID- 7537177 TI - Release of iron from ferritin by 1,2,4-benzenetriol. AB - Release of iron from ferritin in the presence of polyhydroxy metabolites of benzene i.e., hydroquinone (HQ) or 1,2,4-benzenetriol (BT) was studied in acetate buffer, pH 5.6. The release of iron from ferritin was quantitated by monitoring the formation of iron-ferrozine complex. The presence of hydroquinone (330 microM) did not result in the release of iron from ferritin, whereas the same concentration of BT resulted in the release of significant amounts of iron (3.2 microM/min) from ferritin. BT concentration-dependent increase in iron release from ferritin was observed although the increase was not linear with the concentration of BT. Under a N2 atmosphere the presence of BT resulted in the release of iron (2.1 microM/min) from ferritin. The presence of oxyradical scavengers i.e., albumin, catalase or superoxide dismutase significantly inhibited iron release from ferritin by BT. The iron released from ferritin by BT enhanced lipid peroxidation in rat brain homogenate and released aldehydic products from bleomycin-dependent degradation of DNA. Addition of BT to bone marrow lysate resulted in an increase of iron release as a function of time. These studies indicate that BT is a potent reductant of ferric iron of ferritin and also mobilizes and releases iron from ferritin core. The release of iron from bone marrow lysate by BT may be of toxicological significance as this could lead to disruption of intracellular iron homeostasis in bone marrow cells. PMID- 7537178 TI - Synthesis and structure-activity relationships of 2-(4-benzhydryl-1-piperazinyl) 1-phenylethanols as new calcium blockers. AB - A series of 2-(4-benzhydryl-1-piperazinyl)-1-phenylethanols (4) was synthesized and evaluated for calcium entry-blocking activity, assessed as inhibitory activity on calcium current in rat hippocampal pyramidal neurons by using a patch clamp technique (10(-5) M), and cerebral vasodilating activity, assessed in terms of increase of vertebral blood flow after intravenous administration (1 mg/kg) in anesthetized dogs. Alkoxy substituents on the phenyl ring of the phenylethanol moiety conferred potent calcium entry-blocking activity and potent cerebral vasodilating activity. Among these compounds, 4i (NC-1100) was selected as the best analog. Some pharmacological properties of 4i are presented. PMID- 7537179 TI - Novel fluoroprostacyclin analogs with modified cycloalkylenyl chains. Highly potent and orally active anti-anginal agents. AB - Novel fluoroprostacyclin analogs (1a-f) have been synthesized and pharmacologically evaluated. Compounds 1a-c given intravenously or orally showed potent and long-lasting anti-anginal activities in an animal model. PMID- 7537180 TI - Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor receptor and c-erbB-2 protein in hyperplastic human prostates. AB - Transforming growth factor-alpha (TGF-alpha) is a potent mitogenic factor which acts by binding to the epidermal growth factor (EGF). c-erbB-2 is a member of the EGF receptor family and is known to be associated with cellular growth and differentiation. The roles played by these factors in benign prostatic hyperplasia (BPH) are not clearly known. In the present study, expression of these factors was investigated immunohistochemically in frozen and formalin fixed prostate tissues from subjects suffering from BPH. Intracytoplasmic localization of TGF-alpha was observed in the epithelium of nine out of 39 (23.07%) cases. Twenty-six out of 36 (72.22%) frozen BPH tissues exhibited moderate to strong staining for immunoreactive EGF-receptor in the cell membrane. c-erbB-2 oncoprotein was localized in 35 out of 39 cases (89.74%) with the intensity of staining being variable. All nine cases positive for TGF-alpha were also positive for both EGF-receptor and c-erbB-2 protein. Staining reaction had no correlation with the serum testosterone, prostate specific acid phosphatase and prostate specific antigen levels. Immunohistochemical studies indicate the expression of TGF-alpha, EGF receptor and c-erbB-2 protein in BPH tissues. Further study is required to elucidate the precise roles played by these factors in benign growth of prostates. PMID- 7537181 TI - Selective inhibition of repair of active genes by hyperthermia is due to inhibition of global and transcription coupled repair pathways. AB - Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes. To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts. The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia. PMID- 7537182 TI - Direct evidence for hydroxyl radical-induced damage to nucleic acids by chromium(VI)-derived species: implications for chromium carcinogenesis. AB - Reduction of Cr(VI) by NADH and NADPH has been shown to yield Cr(V) species, which have been detected by electron paramagnetic resonance (EPR) spectroscopy. The fine structure on the EPR signal of the Cr(V) species is consistent with the presence of two NAD(P)H ligands in a square-pyramidal arrangement with a single oxygen (oxo) group at the apex. Neither this species nor the initial Cr(VI) complex damage DNA components as evidenced by the lack of effect of these compounds on the optical and EPR signals of the Cr(VI) and Cr(V) species respectively. Addition of hydrogen peroxide to the Cr(V) species is shown to result in the formation of a further transient EPR signal, the parameters of which are consistent with an assignment to a Cr(V)-peroxide complex. Inclusion of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide in this system demonstrates that hydroxyl radicals are also generated, possibly via the decomposition of the peroxide complex. Inclusion of DNA components in this system together with the spin trap 2-methyl-2-nitrosopropane results in the detection of base- and sugar derived radicals; the characteristic EPR signals of these species have allowed both the identification of these species and their mechanism of formation to be determined. The signals from the former species are consistent with radical addition to the base, whereas the sugar-derived species are believed to be formed via hydrogen atom abstraction. In each case, this behaviour is consistent with hydroxyl radicals being the damaging species in systems where Cr(V) is generated in the presence of hydrogen peroxide. These results therefore suggest that it may be the hydroxyl radical that is the ultimate carcinogenic species in cells and systems exposed to Cr(VI). PMID- 7537183 TI - Induction of CYP1A and glutathione S-transferase activities by 2,3,7,8 tetrachlorodibenzo-p-dioxin in human hepatocyte cultures. AB - Induction of CYP1A and glutathione S-transferase activities with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) was studied in human hepatocytes in primary culture to investigate the variability of inducibility and the potency of TCDD. Determining induction of 7-ethoxyresorufin O-deethylase activity, preferentially catalyzed by CYP1A isozymes, we obtained concentration-response diagrams in TCDD treated hepatocyte cultures from transplant donors and patients undergoing hepatic surgery. At a concentration of 10(-10) M TCDD approximately half-maximal induction of CYP1A was observed. Northern analysis of CYP1A gene expression showed a similar concentration-response relationship. In comparison with rat hepatocytes, human hepatocytes were about 10-fold less sensitive towards the CYP1A-inducing effect of TCDD. No pronounced interindividual differences in the inducing potency of TCDD (concentration which leads to half-maximal induction) were obvious in the six human individuals studied, whereas the efficacy of CYP1A induction was highly variable. In addition, inducibility of glutathione S transferase (GST) activity also revealed a considerable degree of interindividual variation, i.e. a complete lack of induction in three out of six hepatocyte preparations and a highly variable efficacy of GST induction among responders which was not related to CYP1A inducibility. PMID- 7537184 TI - Localization of porcine cardiac myosin epitopes that induce experimental autoimmune myocarditis. AB - Porcine cardiac myosin induced severe myocarditis in Lewis rats, similar to that seen after immunization with human or rat cardiac myosin. We investigated the localization of pathogenic epitopes by using porcine cardiac myosin. Subfragment 1 (S-1) and the rod were obtained by alpha-chymotryptic digestion. The rod was further fragmented by using cyanogen bromide cleavage. Three subfragments of S-1 were prepared by tryptic digestion. All Lewis rats immunized with the rod exhibited severe myocarditis, and the immunization of the cyanogen bromide cleaved peptide equivalent to human beta-cardiac myosin heavy chain RDCB9 (residues 1070 to 1165) induced moderate myocarditis. Although none of the rats immunized with the whole S-1 exhibited any myocardial lesions, moderate inflammatory cardiac lesions were detectable in rats immunized with the tryptic digests of S-1. Our results indicate that the myocardiogenic epitopes in Lewis rats are located in the RDCB9 (residues 1070 to 1165) of the cardiac myosin rod and that a cryptic minor epitope may reside in S-1. PMID- 7537185 TI - Increased activity of constitutive nitric oxide synthase in cardiac endothelium in spontaneous hypertension. AB - BACKGROUND: We analyzed the activity of nitric oxide synthase in the rat heart to study whether this activity is affected by high blood pressure. Hearts from young (3 to 4 weeks old) and adult (15 to 25 weeks old) Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were excised and frozen in liquid nitrogen. The activities of Ca(2+)-dependent (cNOS) and Ca(2+)-independent (iNOS) were determined in homogenized tissues by measuring the conversion of [14C]-L-arginine to [14C]-L-citrulline in the presence or absence of either EGTA (1 mmol/L) or EGTA plus NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L each). METHODS AND RESULTS: Arterial pressure was higher in adult SHR than in young SHR and WKY rats of both ages (P < .01). The cNOS activity was two to three times higher in hypertensive than in normotensive hearts (P = .01 to P = .04). No significant activity of iNOS was detected in any tissue. Studies of the right and left ventricles demonstrated a higher cNOS activity in the left sides of the hearts of adult SHR (P < .05). No differences were found in hearts from WKY rats. Selective removal of endocardial or coronary endothelial cells in hearts of SHR and WKY rats substantially reduced cNOS activity (P < .01). CONCLUSIONS: The cNOS activity is upregulated in cardiac endothelial cells of genetically hypertensive rats. The high activity of cardiac cNOS is related to increased arterial pressure of these animals. We propose that in the heart, endothelial cells respond with a higher production of nitric oxide as a compensatory mechanism against high blood pressure and its damaging effects in this organ. PMID- 7537186 TI - Biological variation and the effect of fasting and halothane anesthesia on plasma glutathione S-transferase concentrations. AB - Using a specific RIA, we have investigated in patients and volunteers whether fasting, diminished hepatic clearance, hemoconcentration, or within-day biological variation might be responsible for the transient increases in plasma glutathione S-transferase (GST) concentration observed after anesthesia. GST concentration was measured in 44 healthy volunteers after an overnight fast and at 3, 6, and 24 h after the fasting sample. The concentration was significantly lower at 3 and 6 h after than in the fasting sample (P = 0.0019 and P = 0.015, respectively). The change in GST concentration caused by fasting was examined in 30 subjects by comparing pre- and postfasting values. Fasting had no significant effect on GST concentration overall (P = 0.4721), but two individuals showed a marked increase in GST concentration after fasting overnight. In a separate study of 10 patients, plasma amylase activity and plasma concentrations of GST and albumin were measured immediately before and 3 h after induction of halothane anesthesia. Although GST concentration was increased at 3 h in each of the 10 patients, plasma amylase activity and plasma albumin concentration were significantly decreased in all patients (P = 0.002). Apparently, increases in GST concentration after anesthesia do not result from incidental factors. PMID- 7537188 TI - Sulfasalazine interference with HPLC assay of 5-hydroxyindole-3-acetic acid. PMID- 7537187 TI - Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol. AB - Lipoprotein(a) [(Lp(a)], a low-density lipoprotein (LDL)-like particle, contains in addition to LDL a specific protein component, apolipoprotein(a) [apo(a)]. Conventionally, Lp(a) has been measured by immunological methods that distinguish between Lp(a) and LDL by dealing with apo(a) as an antigen. We describe a new method to determine Lp(a) on the basis of its cholesterol content. Very-low density lipoproteins were removed from serum by preparative ultracentrifugation at a density of 1.006 kg/L. The infranate was subjected to agarose gel electrophoresis to separate Lp(a) and LDL. Lp(a) cholesterol was then determined by direct enzymatic staining for cholesterol. On electrophoresis of the > 1.006 kg/L (bottom) fraction, Lp(a) migrates to the pre-beta position, regardless of the genetic apo(a) isoform. The interassay CVs of Lp(a) cholesterol determinations ranged from 6.9% to 11.5%, and the results correlated well with the Lp(a) concentrations measured by immunonephelometry (r = 0.937). There was an inverse relation between the molecular mass of the genetically determined apo(a) isoforms and Lp(a) cholesterol concentrations. Patients with angiographically proven coronary artery disease (CAD) had significantly more Lp(a) cholesterol than healthy controls did. The ratio of Lp(a) cholesterol to immunologically determined Lp(a) tended to be lower in CAD patients, suggesting that Lp(a) particles contained less cholesterol than apo(a). In addition, the new method allows determination of LDL cholesterol without contamination by Lp(a). PMID- 7537189 TI - [A familial Charcot-Marie-Tooth disease type 1B (CMTD1B) manifesting a new mutation of myelin P0 gene]. AB - A 15-year-old girl (case 1) was admitted to our hospital because of progressive muscle weakness of the lower limbs and numbness of the upper limbs. She noted these symptoms beginning at 13 years of age. Neurological examination revealed that deep tendon reflexes were absent and hypesthesia of touch and pain sensation were distributed in a glove-and-stocking pattern. Muscle weakness and atrophy were predominantly present in the distal portions of the extremities. There were obvious pes cavus and champagne-bottle shape deformities. The motor conduction velocity of the median nerve was markedly delayed and sensory potentials were not evoked in any nerves examined. Lumbar MRI showed thickening of the nerve radices. Cranial MRI showed thickening of the acoustic nerves, as well. Histological studies of a biopsied sural nerve revealed a marked decrease in the number of myelinated and unmyelinated fibers and remarkable onion bulb formation. The patient's clinical manifestations and histological findings were more severe than that seen in the usual case of CMT1A. Her mother (case 2, 39 years old) had similar neurological and electrophysiological findings. DNA duplication encoding peripheral myelin protein 22, was not detected in either case 1 or 2. Sequencing of DNA from these patients revealed the presence of a mutant allele containing an A- to G-substitution of nucleotide 245, which replaced tyrosine with cysteine in the extracellular Ig-domain of the P0 protein. PMID- 7537190 TI - User acceptability of an alpha-fetoprotein screening programme. AB - The objective of the study was to determine user acceptability among women who were classified as false positives or test negatives in an alpha-fetoprotein screening programme. The study was performed as a questionnaire study over a one year period from October 1, 1988 to September 30, 1989 at Hvidovre University Hospital, Copenhagen, and the county hospitals of Sonderjylland, Denmark. The participating subjects were 4104 pregnant women who had had an alpha-fetoprotein test and had completed 30 weeks of gestation, when the questionnaire was delivered. Main outcome measures were degree and duration of anxiety, influence on daily life and whether the woman wanted the alpha-fetoprotein test again in a new pregnancy. Three thousand, three hundred and thirty-one questionnaires were analyzed. The participation rate was 81.2%. For 219 women (6.6%), the first alpha fetoprotein test was abnormal (high or low) and the tests were later found to be false positives. There was a strong association between anxiety experienced in conjunction with the alpha-fetoprotein screening programme and the alpha fetoprotein test result. Two percent of the women with a normal test result reported severe anxiety, compared to 36% of the women who had an abnormal test result followed by amniocentesis (p < 0.001). Two percent of the women with a normal alpha-fetoprotein result reported that they were anxious one month or more after testing, in contrast to 39% of those who had had an amniocentesis (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537191 TI - [Action of granulocyte-colony stimulating factor and cisplatin on two models of human digestive, esophageal and colonic, cancers]. AB - A combined therapy using a colony-stimulating factor and a chemotherapeutic agent has been designed, in vitro and in vivo, on digestive tumor cells to assess the effects of these agents administered alone or in combination, on the cells' or the tumor's growth rate. A recombinant human granulocyte colony-stimulating factor (rh G-CSF) and Cisplatin (CDDP) has been administered at various concentrations in vitro on oesophageal (ECYO) or on colonic (COLO 205) cell lines and in vivo on oesophagal tumor transplanted in Nude mice. In vitro, the oesophagal model is sensitive to G-CSF. The administration of G-CSF induces a stimulation of the cell proliferation. The DNA synthesis is stimulated or inhibited by low or high concentrations of G-CSF without any dose-response relationship. CDDP inhibits the DNA synthesis in ECYO cells. The association of the two agents leads to an activation of the DNA synthesis but only for some concentrations. On the contrary, the colonic model treated or not by CDDP is not sensitive to G-CSF. In vivo, G-CSF does not allow any inhibition or activation of oesophagal tumor's growth rate. CDDP alone is also inefficient. When G-CSF is administered before CDDP, there is no effect on the tumors. On the contrary, when G-CSF is administered with or after the chemotherapeutic agent, there is a significant inhibition of the tumor's growth rate. PMID- 7537193 TI - Low-dose iloprost infusion improves insulin action in aged healthy subjects and NIDDM patients. AB - OBJECTIVE: To investigate the effect of iloprost infusion on insulin action. RESEARCH DESIGN AND METHODS: Thirteen healthy subjects and 13 non-insulin dependent diabetes mellitus (NIDDM) patients matched for age (68.2 +/- 0.5 vs. 67.9 +/- 0.5 years, NS), gender ratio (7 men:6 women vs. 6 men:7 women), body weight, body fat distribution, arterial blood pressure, and plasma triglyceride levels (1.89 +/- 0.09 vs. 1.87 +/- 0.08 mmol/l, NS) were studied. In eight healthy subjects and eight NIDDM patients, we studied insulin action by euglycemic glucose clamp (insulin infusion rate 2 mU.kg-1.min-1) along with saline and iloprost delivery (0.7 ng.kg-1.min-1). In the other five subjects of each group, forearm blood flow and insulin-mediated glucose uptake during saline and iloprost infusion (0.7 ng.kg-1.min-1) were investigated. RESULTS: Iloprost infusion improved insulin-stimulated whole-body glucose uptake and oxidative and nonoxidative glucose metabolism in both study groups. Forearm blood flow under basal conditions and with insulin infusion (2 mU.kg-1.min-1) did not show any significant difference from that during saline and iloprost infusion (0.7 ng.kg 1.min-1) in healthy subjects and diabetic patients. CONCLUSIONS: Iloprost infusion improves insulin action in healthy subjects and NIDDM patients. PMID- 7537192 TI - Role of SPan-1 antigen in adhesion of human colon cancer cells to vascular endothelium. AB - Recently E-selectin (ELAM-1, endothelial leukocyte adhesion molecule-1) was shown to recognize not only sialyl Lewis X but also sialyl Lewis A, and these carbohydrate antigens may be involved in the process of the adhesion between cancer cells and endothelial cells in cancer metastasis. To investigate the contribution of sialylated carbohydrate antigen, SPan-1, and sialic acid to the adhesion of human colon cancer cells to endothelial cells, adhesion assay using HUVECs (human umbilical vein endothelial cells) was performed. The adhesion was significantly inhibited by pretreatment with anti-E-selectin antibody, indicating that this adhesion was thought to be mediated by E-selectin. When these cancer cells were pretreated with SPan-1 antibody, the adhesion was significantly inhibited in a concentration-dependent manner. The adhesion was also inhibited by pretreatment with neuraminidase. These findings suggest that the SPan-1 antigen plays a significant role in the adhesion of human colon cancer cells to endothelial cells, and sialylation of the terminal structure of carbohydrate antigens is important in this adhesion. PMID- 7537195 TI - Displaying electrocorticographic findings on gyral anatomy. AB - Human electrocorticographic findings recorded from subdural arrays of electrodes were topographically mapped directly onto magnetic resonance images of gyral anatomy. With this technique gyri involved in generating somatosensory evoked potentials and epileptic phenomena are easily identified. Regions of the cortex which exhibit local spectral changes associated with cognitive tasks can also be visualized. These composite images of structure and function can provide insight regarding the functional organization of human cortex in relation to gyral anatomy and localized pathologic rhythms. PMID- 7537194 TI - Doxazosin. An update of its clinical pharmacology and therapeutic applications in hypertension and benign prostatic hyperplasia. AB - Doxazosin is a long-acting alpha 1-adrenoceptor antagonist structurally related to prazosin and terazosin. Its antihypertensive effect is produced by a reduction in the smooth muscle tone of peripheral vascular beds resulting in a decrease in total peripheral resistance without significant effect on cardiac output or heart rate. In benign prostatic hyperplasia, doxazosin's effect of relieving bladder outflow obstruction is produced through a reduction in prostatic tone mediated via alpha 1-adrenoceptor blockade. In most comparative trials doxazosin has proven to be equally effective as the comparator drug in the treatment of mild to moderate hypertension. It has been used in a variety of patient populations including the elderly, Blacks, smokers, and patients with concomitant disease states such as renal dysfunction, hypercholesterolaemia, non-insulin dependent diabetes mellitus (NIDDM) and respiratory disease. Doxazosin has also been used successfully in combination with beta-adrenoceptor antagonists, diuretics, calcium channel antagonists, and angiotensin-converting enzyme inhibitors in patients with hypertension that is uncontrolled with monotherapy. Doxazosin has a beneficial effect on some of the risk factors associated with coronary heart disease including elevated serum lipid levels, impaired glucose metabolism, insulin resistance and left ventricular hypertrophy. Modest decreases in total cholesterol, low density lipoprotein cholesterol and triglycerides are seen with doxazosin therapy while small increases in high density lipoprotein cholesterol and the high density lipoprotein cholesterol/total cholesterol ratio are consistently reported. Some studies have reported an improvement in glucose tolerance although this effect has been more consistently seen in nondiabetic patients than in patients with NIDDM. Additionally, doxazosin produces a similar reduction in left ventricular hypertrophy to other antihypertensive agents. Modelling-based calculations suggest that doxazosin significantly reduces the risk of developing coronary heart disease in patients with mild to moderate hypertension, although this remains to be confirmed in long term prospective studies. Doxazosin appears to be a promising agent in the treatment of urinary symptoms associated with benign prostatic hyperplasia. Similar to other alpha 1 adrenoceptor antagonists, doxazosin treatment produces increases in peak and mean urinary flow rates and improves other objective and symptomatic measures.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7537196 TI - Intracerebral potentials to rare target and distractor auditory and visual stimuli. II. Medial, lateral and posterior temporal lobe. AB - Event-related potentials were recorded from 1221 sites in the medial, lateral and posterior aspects of the temporal lobe in 39 patients. Depth electrodes were implanted for about 4 days in order to localize seizure origin prior to surgical treatment. Subjects received an auditory discrimination task with target and non target rare stimuli. In some cases, the target, distracting and frequent tones were completely balanced across blocks for pitch and volume. Some subjects also received an analogous visual discrimination task, or auditory tasks in which the rare target event was the omission of a tone, or the repetition of a tone within a series of alternating tones. In some subjects, the same auditory stimuli were delivered but the patient ignored them while reading. A complex field was recorded, indicating multiple components with overlapping time-courses, task correlates and generators. Two general patterns could be distinguished on the basis of their waveforms, latencies and task correlates. In the temporal pole and some middle temporal, posterior parahippocampal and fusiform gyrus sites, a sharp triphasic negative-positive-negative waveform with peaks at about 220-320-420 msec was usually observed. This wave was of relatively small amplitude and diffuse, and seldom inverted in polarity. It was multimodal but most prominent to auditory stimuli, appeared to remain when the stimuli were ignored, and was not apparent to repeated words and faces. A second broad, often monophasic, waveform peaking at about 380 msec was generated in the hippocampus, a limited region of the superior temporal sulcus, and (by inference) in the anterobasal temporal lobe (possible rhinal cortex). This waveform was of large amplitude, often highly focal, and could invert over short distances. It was equal to visual and auditory stimuli, was greatly diminished when the stimuli were ignored, and was also evoked by repeating words and faces. Preceding this waveform was a non-modality specific negativity, possibly generated in rhinal cortex, and a visual-specific negativity in inferotemporal cortex. The early triphasic pattern may embody a diffuse non-specific orienting response that is also reflected in the scalp P3a. The late monophasic pattern may embody the cognitive closure that is also reflected in the scalp P3b or late positive component. PMID- 7537197 TI - Intracerebral potentials to rare target and distractor auditory and visual stimuli. III. Frontal cortex. AB - Evoked potentials (EPs) were recorded from 991 frontal and peri-rolandic sites (106 electrodes) in 36 patients during an auditory discrimination task with target and non-target (distractor) rare stimuli. Variants of this task explored the effects of attention, dishabituation and stimulus characteristics (including modality). Rare stimuli evoked a widespread triphasic waveform with negative, positive and negative peaks at about 210, 280 and 390 msec, respectively. This waveform was identified with the scalp EP complex termed the N2a/P3a/slow wave and associated with orienting. It was evoked by rare target and distractor auditory and visual stimuli, as well as by rare stimulus repetitions or omissions. Across most frontal trajectories, N2a/P3a/SW amplitudes changed only slowly with distance. However, large (120 microV) P3as with steep voltage gradients were observed laterally, especially near the inferior frontal sulcus, and clear inversions of the P3a were noted in the orbito-frontal and the anterior cingulate cortices. The frontal P3a was earlier to distractor than to target stimuli, but only in some sites and with a latency difference much smaller than that observed at the scalp. Frontal P3a latencies were significantly shorter than those recorded simultaneously at the scalp and often were also shorter than P3a latency in the parietal or temporal lobes. In summary, this study demonstrates an early P3a-like activity that polarity inverts over short distances in the medial frontal lobe, and that it has a significantly shorter latency than similar potentials recorded in the temporal and parietal cortices. PMID- 7537198 TI - EEG prior to hemispherectomy: correlation with outcome and pathology. AB - Hemispherectomy, for the treatment of seizures, is highly successful but has a significant morbidity rate. The procedure is usually restricted to patients with an intractable seizure disorder and hemiparesis. Because of the inherent risk of surgery, patient selection is a critical issue. This report describes the evaluation of background activity and ictal patterns on surface and invasive EEG in 12 children who underwent both anatomical (7) and functional (5) hemispherectomy in order to determine the role of electroencephalography in the selection of patients for hemispherectomy, and to correlate EEG findings with underlying pathology and outcome. A favorable outcome was predicted by an interictal EEG with two or more of the following: suppression over the abnormal hemisphere, absence of contralateral slowing, absence of generalized discharges and absence of bilateral independent spiking; or by unilateral onset of ictal discharges on invasive intracerebral EEG recording. Outcome did not correlate with the underlying pathology. Hemispherectomy can be successful in patients with a variety of predominantly unilateral pathologic entities. PMID- 7537199 TI - Interictal EEG changes in eclampsia. AB - The pathogenesis of seizures and encephalopathy in eclampsia remains obscure even today. There are only a few reports on EEG findings in eclampsia. This is a report of the interictal EEG changes in 8 patients with eclampsia who were evaluated prospectively. The major EEG changes observed were (1) slowing of the background activity (generalized slowing in 4 patients and focal slowing in 3 patients) and (2) intermittent spike and sharp wave transients (5 patients). These abnormalities had disappeared in all except 2 patients when the EEG was repeated on the seventh day. A comparison is drawn between eclampsia and hypertensive encephalopathy for the clinical, EEG and CT/MRI findings. The pathogenesis of seizures and encephalopathy could be similar in these two conditions. It is worth while considering these EEG changes while planning the treatment of eclampsia. PMID- 7537200 TI - The effects of phonological and semantic features of sentence-ending words on visual event-related brain potentials. AB - Event-related brain potentials (ERPs) were recorded to terminal words of visually presented sentences that were identical to those used in an auditory modality study examining the effects of phonological and semantic characteristics of words on ERPs (Connolly and Phillips, 1994). Phonological and/or semantic characteristics of terminal words were manipulated in 4 conditions in which: (1) the terminal word was the highest cloze probability ending for the sentence and was thus phonologically and semantically appropriate to the sentence context (e.g., Ray fell down and skinned his knee(s).; (2) the terminal word had the initial phonemic sound of the highest cloze probability sentence-ending word but was semantically anomalous to the context of the sentence (e.g., They sat together without saying a single worm. [word]); (3) the initial phoneme of the terminal word was phonologically unexpected but the word was semantically correct (e.g., The dough was put in the hot pan. [oven]); or (4) the initial phoneme of the terminal word was phonologically unexpected and the word was semantically inappropriate to the sentence context (e.g., Bill jumped into the lake and made a big farm. [splash]). A left fronto-temporally distributed negative peak was observed in the 250-300 msec range to varying degrees in all conditions but was largest in the condition in which terminal words were both phonologically unexpected and semantically inappropriate. A second, parietally distributed, symmetrical negativity (the N400) was found which peaked around 365 msec and was largest in the two conditions involving semantically anomalous terminal words. Results are discussed in terms of modality factors in ERP manifestations of receptive language functions and the implications of ERP modality differences for theories of word recognition. PMID- 7537201 TI - Classification of single-trial ERP sub-types: application of globally optimal vector quantization using simulated annealing. AB - Examination of the single trials which are traditionally averaged to form late component ERPs reveals a number of different sub-types of response. This study introduces an automated and robust approach to objectively classify these ERP sub types. Auditory oddball ERP (target tones) data were examined in 25 normal subjects. Globally optimal vector quantization using simulated annealing (the "Metropolis algorithm") was employed to determine the natural groupings of the single-trial responses that constitute the average ERP. No prior assumptions about the ERP patterns were imposed. This is the first study to employ a cluster analysis technique with globally optimal properties in ERP research. We demonstrate that, due to the presence of many different undesirable local minima, a globally optimal solution is crucial if the classification of the single-trial ERPs is to reflect their real structure. The results of this study showed that only around 40% of single trials had a morphology which resembled the averaged ERP wave form. The remaining single trials had a response morphology which was different from the average, in terms of the amplitude and latency of the components. Single-trial ERP response sub-types may provide fundamental complementary functional information to the ERP average. PMID- 7537202 TI - Multichannel detection of magnetic compound action fields with stimulation of the index and little fingers. AB - Magnetic compound action fields (CAFs) over the right arm were measured from 63 sensor positions with two 7-channel SQUID gradiometer systems following electrical stimulation of the index and little fingers as well as the ring finger separately. The wave forms of the CAFs were primarily biphasic, corresponding to the depolarization and repolarization currents of the stimulated nerves. Maximum amplitudes of the CAFs were 60-140 fT for the index finger stimulation and 40-90 fT for the little finger stimulation. The field mapping of the CAFs revealed a propagating quadrupolar pattern with different distributions for the index and little fingers. The results agree with the anatomical location of the median and ulnar nerves for the index and little finger stimulation respectively. The isofield maps, due to ring finger stimulation, showed complex patterns as a result of simultaneous activation of the median and ulnar nerves. By comparing the amplitudes of the maxima of the CAFs due to index finger stimulation with those after median nerve stimulation at the wrist, the numerical ratios of the constituent digital nerve fibers for the index finger within the median nerve at the wrist were estimated. The ratios of 0.14-0.41 (mean 0.27), determined with measurement of the CAFs, are fairly consistent with those calculated from the reported histological data. PMID- 7537203 TI - Modulation of motor activity by cutaneous input: inhibition of the magnetic motor evoked potential by digital electrical stimulation. AB - We examined the inhibitory effect of a brief train of digital (D2) electrical stimuli at 4 times perception threshold on transcranial magnetic motor evoked potentials (MEPs) recorded from abductor pollicis brevis (APB) and flexor carpi radialis (FCR) muscles ipsilateral to the side of D2 stimulation. We compared this to the inhibitory effect of ipsilateral D2 stimulation on averaged rectified EMG recorded at 10% maximum voluntary contraction and on F-responses and H reflexes recorded from these same muscles. We also compared MEPs recorded following D2 stimulation just above perception threshold to MEPs following higher intensity D2 stimulation. As well, we assessed the effect of preceding D2 stimulation on MEPs recorded from a relaxed versus tonically contracted hand muscle. D2 stimulation elicited a triphasic response of modest MEP facilitation followed by inhibition and further facilitation. The duration and onset of MEP inhibition correlated with those of the initial period of rectified EMG inhibition, however, the magnitude of MEP inhibition was generally less than the magnitude of EMG inhibition, consistent with a greater inhibitory effect of digital afferents on smaller motor neurons. MEPs were not facilitated during the rebound of EMG activity (the E2 period) that usually followed the initial period of EMG inhibition (I1 period). The behavior of H-reflexes and F-responses following ipsilateral D2 stimulation suggested that inhibition of both EMG and MEPs is not mediated via presynaptic inhibition of Ia afferents, and that inhibition is augmented by descending rather than segmental input to spinal motor neurons. Tonic contraction of the target muscle during D2 stimulation decreased the inhibitory effect of the preceding digital stimulus possibly due to recruitment of larger spinal motor neurons less likely to be inhibited by cutaneous input. PMID- 7537204 TI - Late facilitations of motor evoked potentials by contralateral mixed nerve stimulation. AB - The effect on motor evoked potentials (MEPs) of a peripheral afferent volley (a single square pulse delivered to the contralateral median nerve at the wrist) was studied in abductor digiti minimi muscle of 9 normal patients following magnetic stimulation of the motor cortex. Long intervals were used (200-700 msec) between the electrical conditioning stimulus and magnetic test stimulation. An MEP amplitude increase, with no change in latency, was observed when the C-T interval was about 500 msec, and the motor threshold was decreased by the conditioning stimulation. The observed long latency and duration of the facilitation effect would seem consistent with activation of suprasegmental areas. PMID- 7537205 TI - Effect of transcranial magnetic stimulation on cerebral function in a monkey model. AB - The effect of transcranial magnetic stimulation on higher cerebral function was studied using 3 monkeys. They were trained in a delayed response task which required spatial short-term memory. The task was presented by a computer on a cathode-ray tube and results of the delayed response task, which consisted of the percentage of correct choices, reaction time and trial number, were analyzed. For stimulation, small and large round coils were used as well as a figure 8 configuration. Their maximal B-fields were 3.3 T, 1.9 T and 2.4 T, respectively. A total of more than 7000 stimuli were given to each monkey in various patterns. There was no deficit in the delayed response. Further complications such as epileptic seizures were not observed either. In conclusion, transcranial magnetic stimulation does not appear to have any effect on higher cerebral functions in monkeys. PMID- 7537206 TI - Central motor conduction in Hirayama disease. AB - The pathogenesis of Hirayama disease is usually attributed to microcirculatory disturbances in the anterior spinal artery territory, leading to segmental anterior horn cell loss and occasional lower limb hyperreflexia. In 7 patients with Hirayama disease, central motor conduction to upper (CMCT-ADM) and lower limbs (CMCT-TA) was evaluated. CMCT-TA was normal in all, but CMCT-ADM was marginally prolonged (8.4 msec, amplitude 0.8 mV) on one side only. Peripheral delay in the upper limbs was found in 2 patients (1 side each) which might be due to fall-out of anterior horn cells. In 2 patients with lower limb hyperreflexia, HM ratio, vibratory inhibition and reciprocal inhibition of soleus H reflex were also normal, suggesting lack of pyramidal dysfunction. Our results do not suggest any pyramidal dysfunction as a cause of lower limb hyperreflexia in Hirayama disease. PMID- 7537207 TI - Feedforward postural adjustments in a simple two-joint synergy in patients with Parkinson's disease. AB - Patients with Parkinson's disease, age-matched controls and young control subjects performed discrete elbow or wrist movements in a sagittal plane under the instruction to move one of the joints "as fast as possible." Relative stability of the other, postural joint was comparable in all 3 groups, while movement time was the highest in the patients and the lowest in young controls. Typically, EMG patterns in both muscle pairs acting at the joints demonstrated a commonly observed "tri-phasic" pattern. A cross-correlation analysis of the EMGs confirmed virtually simultaneous bursts in the wrist and elbow flexors and in the wrist and elbow extensors. In all 3 groups, there were no signs of anticipatory activation of postural muscles in about 90% of movements. We consider postural anticipation not a separate process, but a separate peripheral pattern of a single control process that may involve a number of joints and muscles. We conclude that the postural deficits in Parkinson's disease are not related to a basic deficit in the ability to generate feedforward postural adjustments but to other factors that may include the specificity of maintaining the vertical posture in the field of gravity. PMID- 7537208 TI - Comparison of single motor unit responses to transcranial magnetic and peroneal nerve stimulation in the tibialis anterior muscle of patients with amyotrophic lateral sclerosis. AB - Responses of single tibialis anterior motor units to transcranial magnetic stimulation and to a synchronized Ia volley evoked by peripheral nerve electrical stimulation were obtained in amyotrophic lateral sclerosis (ALS) patients and normal controls. Whereas the units of normal subjects exhibited rather stereotyped short-latency spike density peaks in response to both types of stimulus, the responses of ALS patient units were much less uniform. All ALS patient units exhibited a response to the synchronized Ia volley indistinguishable from that of normal subjects, indicating that the investigated spinal motoneurons are capable of normal excitatory responses in ALS patients. More than half of the ALS patient units responded to the transcranial magnetic stimulus with prolonged spike-density peaks appearing at a latency consistent with the notion that these pathological peaks are evoked by some relatively hyperexcitable structures presynaptic to the corticomotoneurons. PMID- 7537209 TI - Repetitive magnetic stimulation and motor evoked potentials. AB - The aim of the study was to evaluate the effect of varying stimulus rates of repetitive transcranial magnetic stimulation (RTMS) on the motor evoked potentials (MEPs) recorded from the right abductor pollicis brevis muscle (APB). Thirteen normals were included. Stimuli were applied to the cortex and to the median nerve at the wrist. The cortical stimuli were applied without and with facilitation. Stimulus intensity was 1.2 times the motor threshold (TmAPB) for cortical stimulation and supramaximal for peripheral stimulation. Stimulus rates were 1, 2, 3, 5, 10 and 20 Hz. Nine pulses were applied in each stimulus series. At stimulus rates between 1 and 3 Hz no amplitude changes were observed throughout the stimulation. At 5 Hz stimulation inhibition of some of the MEPs was observed followed by MEPs with increased amplitude. At 10 Hz stimulation some MEPs were totally inhibited, interrupted by an MEP with increased amplitude, resembling clonic contraction. At 20 Hz the inhibition of the MEPs decreased (P < 0.05) compared to 10 Hz stimulation. Facilitation decreased the inhibition at 5 and 10 Hz stimulation (P < 0.01). On peripheral stimulation a decrement was observed with stimulus rates of 10 and 20 Hz. The study shows that RTMS exerts a complex influence on the MEPs depending upon stimulus rates. The pronounced inhibition and excitation of the MEPs at 5 and 10 Hz stimulation possibly reflects inhibition and excitation in the cortico-spinal neurons. PMID- 7537210 TI - Advantages of Sirius Red staining for quantitative morphometric collagen measurements in lungs. AB - Sirius Red staining is presented as a method for collagen determination, enabling quantitative morphometric measurements to be performed in locally defined tissue areas. The advantage of this method is especially shown for alveolar lung tissue. By excluding the bronchial areas in the tissue sections, the differences in the degree of fibrosis proved to be more discrete after different loads of quartz dust than by any other method. The difference of 12 micrograms collagen measured colorimetrically represented a 1.2-fold increase. The collagen measured in the alveolar tissue by the morphometric method rose from 9.8 to 28.6%. This is a 2.9 fold increase, underlining the vast improvement in sensitivity. Thus, this method is specifically suitable for the evaluation of very small fibrotic lesions. The quartz doses given are particularly low compared to most other investigations. Histologic lung and lymph node sections from female Wistar rats injected intratracheally with differing quantities of quartz dust (0.03, 0.1, 0.5, 1.75 mg) were stained with Sirius Red, and the collagen fibers measured with a quantitative image analysis. The results for lymph nodes using different methods (wet weight determination, quantitative measurement of quartz typical areas, colorimetric and morphometric collagen determination) showed a high correlation at the different doses. This showed that the morphometric method is suitable for the quantitative measurement of collagen. Corresponding results were also found in the comparative lung tissue measurements (colorimetric and morphometric collagen determination). However, the morphometric method has the decisive advantage that measurements can be restricted to defined tissue areas and do not destroy the section. PMID- 7537211 TI - Effect of arsenic exposure on alveolar macrophage function. II. Effect of slightly soluble forms of As(III) and As(V). AB - The pulmonary toxicity of a substance depends on a number of chemical and physical characteristics, including the solubility of the compounds. In the lung, insoluble forms of metals may be more tumorigenic than soluble forms despite the fact that this effect has not been quantitated and the mechanism of action has not been elucidated. The toxic effects of slightly soluble forms of As(III) and As(V) were evaluated by determining alteration in function of pulmonary alveolar macrophages (PAM) following in vivo and in vitro exposure. Male Sprague-Dawley rats were used throughout. Twenty-four hours following intratracheal instillation of 1 mg/kg (as arsenic) of either arsenic trisulfide (As(III)) or calcium arsenate (As(V)), PAM were lavaged and analyzed for alterations in superoxide (O2 ), and tumor necrosis factor (TNF-alpha) production. There were no differences in bronchoalveolar lavage fluid TNF-alpha. PAM lavaged from As(V)-exposed animals showed significant increases in O2- production and in basal release of TNF-alpha. PAM lavaged from animals receiving As(III) did not show significant alterations. To test the direct effects of arsenic, PAM were lavaged from control animals and exposed to concentrations of 0.1 to 300 micrograms/ml arsenic in vitro for up to 24 hr. Doses used were not cytotoxic to PAM, since LDH release was not significantly increased. Significant dose-dependent inhibition of O2- production was only evident after 24 hr exposure to arsenicals. Both As(III) and As(V) produced inhibition at concentrations of 10 micrograms/ml. Suppression of LPS induced release of TNF-alpha also occurred at similar concentrations for both arsenicals (4-5 micrograms/ml). Neither arsenical inhibited prostaglandin E2 production. Measurement of soluble arsenic concentrations indicated dissolution of the compounds could not account for all of the effects seen. Arsenic-induced alteration in PAM function may compromise host defense. PMID- 7537212 TI - Dynamin GTPase is stimulated by crosslinking through the C-terminal proline-rich domain. AB - Dynamin is a 100 kDa GTPase required for endocytic-coated vesicle formation. Recombinant human neuronal dynamin (dynamin-1) was used for monoclonal antibody (mAb) production. Two mAbs, designated hudy-2 (for human dynamin) and hudy-4, were chosen for further study based on their differential ability to recognize dynamin-1 and its non-neuronal isoform, dynamin-2. Both bind to the proline-rich C-terminal domain (PRD) of dynamin and inhibit the ability of microtubules and grb2 to stimulate GTPase activity. Hudy-4 binds to an epitope within the last 20 amino acids of dynamin-1 and has no effect on its intrinsic GTPase activity. Hudy 2 binds to an epitope within amino acids 822-838 that is common to dynamin-1 and dynamin-2. Hudy-2 stimulates dynamin's intrinsic GTPase activity in a manner proportional to the valency of the immunoglobulin (Ig) G. Crosslinking IgGs with secondary antibodies caused a 2-fold increase in GTPase activity, while F(ab)s were inactive. Importantly, our findings suggest that the stimulation of dynamin GTPase activity by multivalent proteins which bind in vitro to the PRD may not be a valid criterion on its own for assessing the in vivo functional significance of these interactions. PMID- 7537214 TI - A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. AB - The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin 6 (IL-6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three-kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL-6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL-6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase-negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL-6 response pathway are, nevertheless, clearly established for these cells. PMID- 7537213 TI - Interleukin-3 signals through multiple isoforms of Stat5. AB - The interleukin (IL)-3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL-3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL-3 activates STF-IL-3a, which comprises two tyrosine-phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL-3 and granulocyte-macrophage colony-stimulating factor activate STF-IL-3b, which consists of a 94 and 96 kDa tyrosine-phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF-IL-3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL-3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5. PMID- 7537215 TI - FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter. AB - The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase. PMID- 7537217 TI - Chronic diarrhea due to a single strain of Aeromonas caviae. AB - Over a period of 17 months, Aeromonas caviae was cultured 15 times as the sole enteropathogen from the feces of a man who had developed chronic diarrhea after traveling to Turkey. Determination of rRNA gene restriction patterns confirmed that the seven isolates of Aeromonas caviae studied were identical (hybridization group [HG]4). After therapy with ciprofloxacin for four weeks, the patient was culture negative for the original isolate, and six months later a novel strain of Aeromonas media with a different ribopattern (HG 5A) was isolated from the feces of the patient. The patient responded again, both clinically and bacteriologically, to a four-week course of ciprofloxacin and has remained asymptomatic since then. PMID- 7537218 TI - Trypanosoma cruzi: transfer of protection by lymph node cells obtained from mice immunized with exoantigens of pI 4.5. AB - Exoantigens of pI 4.5 (Ea 4.5) of T. cruzi released to the circulation of infected mice are able to induce partial protective immune response in mice (F. Cerban et al. 1991, International Archives of Allergy and Applied Immunology 96, 35-40). In order to analyze the participation of cellular immunity in the parasitemia control, we i.d. immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The role of immune cells in protective immunity was examined by adoptive transfer experiments. The immune lymph node cells (LNC) transferred the capacity to control the parasitemia, since it was observed that the normal recipients of immune LNC, which were afterward infected, presented a significant decrease in parasite levels with respect to the animals receiving LNC from control mice. This capacity was absent in the spleen cells. In addition, polystyrene nonadherent cells from immune LNC transferred the capacity to control T. cruzi infection. It was observed that Ig+ cells and enriched T cells from immunized mice are able to control the parasitemia. To define epitopes of Ea 4.5 able to stimulate protective immunity, the levels of parasitemia were examined in mice immunized with Ea 4.5 untreated or treated with sodium metaperiodate. These animals presented similar levels of parasitemia and in both cases they are significantly lower than the parasitemias of the control animals, suggesting that the most relevant epitopes for the protective immune response that control the beginning of the infection are not carbohydrates. Later, on Day 30 postinfection only the animals immunized with untreated Ea 4.5 maintained a significant decrease in parasite levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537216 TI - Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti Tat single chain intrabodies. AB - Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS. PMID- 7537220 TI - A rapid method for protein localization in trypanosomes. PMID- 7537219 TI - Schistosoma mansoni: characterization of p50, an immunophilin. AB - A 1.4-kb cDNA clone encoding a 50-kDa homologue of p59, a heat shock binding immunophilin, has been isolated from a Schistosoma mansoni cercariae cDNA library. We have designated this clone S. mansoni p50 (Smp50). From the sequence comparison, we speculate that Smp50 is similar in structural organization to other p59 proteins. As with other p59s, Smp50 also shows homology to various FK binding proteins (FKBPs). The amino acids in human FKBP12 which are proposed to be important for FK506 interaction are conserved in the schistosome protein. We have expressed and purified the recombinant protein (rSmp50) from Escherichia coli. rSmp50 demonstrated peptidyl-prolyl cis-transisomerase (PPIase) activity, typical of immunophilins. Western blot analysis of extracts from S. mansoni adult worms probed with rabbit polyclonal antisera, generated against the recombinant polypeptide, has indicated the presence of Smp50 in the parasite. The antisera did not cross-react with proteins from E. coli or HeLa cell extracts. All of the p59s characterized to date have been from vertebrate species. Therefore, our finding represents the first identification of the protein in an invertebrate system. PMID- 7537221 TI - Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the beta 1 subunit. AB - Integrins are the major family of receptors involved in the adhesive interactions of cells with extracellular matrix macromolecules. Although it is known that integrins can exist in active or inactive states, the molecular mechanisms by which integrin activity is modulated are poorly understood. A novel anti-integrin monoclonal antibody, 12G10, that enhances alpha 5 beta 1-fibronectin interactions has been identified. 12G10 binds to the beta 1 subunit and appears to recognise a region of the subunit that contains the epitopes of several previously described activating or inhibitory monoclonal antibodies. However, unlike other activating anti-beta 1 antibodies, the binding of 12G10 to alpha 5 beta 1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the beta 1 integrin family of an antibody that recognises a ligand induced binding site, and further emphasises the functional importance of a specific region of the beta 1 subunit in regulating integrin-ligand interactions. PMID- 7537222 TI - The convergence-divergence duality in lectin domains of selectin family and its implications. AB - A comparison of the three-dimensional structures of P-, L-, and E-selectin lectin domains reveals that there is a convergence-divergence duality for the 77-107 polypeptide in the three domains; i.e. part of the peptide is folded into a closely similar conformation, and part of it into a highly different one. Since the 77-107 residues are associated with the putative binding sites of the selectin family for ligands, this kind of duality might well reflect the common character of ligands to the selectin family as well as the specificity to each of their respective receptors. The finding may be of use for rationally designing selectin inhibitors with a given specificity and possible antiadhesion drugs. PMID- 7537223 TI - Protein kinase C and phospholipase D activation in rat parotid glands. AB - We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12 myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism. PMID- 7537224 TI - Phosphorylation of human plasminogen activators and plasminogen. AB - Plasminogen (PG), urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) are the main molecules involved in fibrinolysis and in many other physiological and pathological processes. In the present study we report that human t-PA, purified from human melanoma cells, and PG, purified from human plasma, both contain P-Tyr residues, as revealed by immunoblotting analyses with monoclonal anti-P-Tyr antibodies. In addition HPLC amino acid analysis of acid hydrolyzed t-PA, PG and u-PA, shows that: (i) P-Ser and P-Tyr residues are present in t-PA; (ii) P-Thr and P-Tyr are present in PG; (iii) P-Ser, P-Thr and P Tyr are present in u-PA. The utilization of monoclonal anti-P-Ser and anti-P-Thr antibodies in immunoblotting experiments has confirmed these data which indicate that phosphorylation is a common feature of PAs and of PG. PMID- 7537225 TI - The importance of two specific domains in ligand binding to the AMPA/kainate glutamate receptors GluR2 and GluR6. AB - Chimeric receptor subunits of the AMPA receptor subunit GluR2 and the kainate receptor subunit GluR6 were constructed and stably expressed in baby hamster kidney cells. By using Ca2+ imaging and radioligand binding, we demonstrated that substitution of a specific domain showing homology to a bacterial leucine isoleucine-valine binding protein (LIVBP) had no effect on the affinities of the tested agonists, but decreased the affinities of the antagonists CNQX, DNQX, and NBQX. On the other hand, when the first of two domains showing homology to a bacterial glutamine binding protein (QBP) in GluR2 was substituted with the corresponding region from GluR6, the affinity of AMPA decreased sevenfold and the affinity of kainate increased fourfold, indicating the importance of this domain in binding of these agonists. In contrast to this, the affinities of quisqualate and domoate, two other agonists, were unchanged, indicating that a region located C-terminal to the QBP domain is also involved in agonist binding. PMID- 7537226 TI - Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to glutathione-S-transferase. AB - CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S transferase, the soluble portion was purified and identified as a structured protein by its CD spectrum. Association reactions of the recombinant NBF-2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP-, ADP- and AMP-affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled adenine nucleotides occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s-1). Our findings support the concept of a central role of NBF-2 in CFTR activity regulation acting as an allosteric switch between channel opening and closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2. PMID- 7537227 TI - Monoclonal antibodies against the acetylcholine receptor gamma-subunit as site specific probes for receptor tyrosine phosphorylation. AB - Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) may be involved in AChR desensitization and clustering. Torpedo AChR gamma-subunit is phosphorylated at Tyr365. Using overlapping synthetic peptides, we have precisely mapped the epitopes of five anti-gamma-subunit monoclonal antibodies (mAbs) and found that the epitope(s) for the mAbs 154, 165 and 168 (gamma 365-370) all contain Tyr365. mAb 168 is a known blocker of AChR channel function. Using peptide analogues, Tyr365 was found to be indispensable for mAb165 binding; furthermore its binding was selectively inhibited by in vitro AChR tyrosine phosphorylation. The possible connection between gamma-subunit phosphorylation and regulation of AChR function and the proven usefulness of these mAbs as tools should facilitate functional studies of AChR gamma-subunit phosphorylation. PMID- 7537228 TI - 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis, downregulates the expression of iNOS protein and mRNA in primary murine macrophages. AB - 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, blocks the synthesis of tetrahydrobiopterin (BH4), which is a known cofactor of inducible nitric oxide synthase (iNOS). Previously, DAHP was shown to suppress the production of nitric oxide by cytokine-activated fibroblasts, smooth muscle cells or endothelial cells which could be attributed to its function as a cofactor antagonist. Here, we demonstrate that in interferon-gamma-activated murine peritoneal macrophages DAHP suppresses the expression of iNOS mRNA and protein in a BH4-independent manner and, thus, acts by a novel mechanism. PMID- 7537229 TI - ADP-ribosylation of Rho enhances adhesion of U937 cells to fibronectin via the alpha 5 beta 1 integrin receptor. AB - To examine the role of Rho GTP binding proteins in the adhesion of monocytic cells to fibronectin we used the C3 exoenzyme of Clostridium botulinum which ADP ribosylates and inactivates Rho proteins in situ. Treatment of human monocytic U937 cells with C3 exoenzyme (10 micrograms/ml, 24 h) increased adhesion to fibronectin 2-fold but had no effect on adhesion to collagen or huamn serum albumin. The increase in fibronectin adhesion was prevented by antibodies against the alpha 5 and beta 1 integrin subunits, but surface expression of beta 1 and alpha 5 was not altered. These results suggest that Rho proteins regulate the interaction of the monocyte alpha 5 beta 1 integrin receptor with fibronectin by post receptor mechanisms. PMID- 7537230 TI - Evidence for multiple interacting binding sites in bovine tryptase. AB - The interaction of bovine pancreatic trypsin inhibitor (BPTI) and bovine tryptase, which are co-localized in the same granules of bovine mast cells, has been analyzed at 30 degrees C in 0.1 M Tris-HCl, pH 8.0. The analysis has unravelled that the functional unit of bovine tryptase is formed of (at least) four binding sites for this inhibitor. These interaction sites display a simple binding behaviour for small inhibitors (and substrates), whereas heterogeneous properties have been observed in the binding of BPTI. Furthermore, in the presence of BPTI, a positive functional interaction can be detected among the binding sites also for a small synthetic inhibitor, like benzamidine. Such features indicate the existence of a complex functional interplay among the sites of the functional unit which is transmitted through the secondary specificity sites. PMID- 7537231 TI - Lethality of Rw/Rw mouse embryos during early postimplantation development. AB - Three mutations in the mouse, white spotting (W), rump white (Rw), and patch (Ph), are described as a "gene triplet" on the basis of their close genetic linkage and similar mutant phenotypes. The finding that the W phenotype results from mutations altering the c-kit protooncogene, and that Ph is associated with the deletion of Pdgfra, suggested specific molecular reagents which could be used for the analysis of the chromosomal structure of the third mutation, rump white. Such studies indicated that Rw is associated with a large chromosomal inversion. In this study, we showed that it is possible to generate molecular markers specific for the Rw chromosome, as recombination is suppressed between the inverted portion of the Rw chromosome and the wild-type homologue. Using one such marker, we were able to genotype the offspring of Rw/+ intercrosses. This enabled us to show that Rw homozygote embryos die around 9.5 days of gestation. Histological analysis revealed that the embryos undergo gastrulation, forming three germ layers, and in some cases, exhibit a defined axial midline with an apparent notochordal plate. However, mutant embryos are significantly smaller than the wild-type, with the size difference evident from Day 7.5 and becoming more disparate as development progresses. These morphological data further support the genetic evidence indicating that the developmental lethality of the Rw mutation is not caused by the disruption of a gene within the cluster of RTKs in the central portion of mouse chromosome 5. Furthermore, we present evidence that both Kit and Pdgfra are expressed from the Rw chromosome in several adult tissues. The results of these studies suggest that the identification of the sequence(s) disrupted by the Rw mutation will provide further insight into the regulation of early postimplantation development. PMID- 7537233 TI - Developmental regulation of alpha-fetoprotein expression in intestinal epithelial cells of transgenic mice. AB - The alpha-fetoprotein (AFP) gene is transcribed in most epithelial cells lining the fetal mouse small intestine, but transcription persists in only a subset of enteroendocrine cells representing less than 1% of the total intestinal epithelial cells in the adult. The decrease in AFP expression after birth is mediated in part by a repressor element lying between -838 and -250 bp of the AFP gene. Deletion of this element from AFP minigene constructs results in high-level minigene expression in the intestines of adult transgenic mice. Although high levels of AFP minigene RNA are expressed, the fetal pattern of expression is not maintained upon deletion of the repressor element. Instead, the number of cells in which the minigene is expressed increases from less than 1% to approximately 10% of the epithelial cells in the adult small intestine, and includes the majority of the goblet cells in addition to the enteroendocrine cells. In contrast, the pattern of AFP minigene expression in the enterocytes is unaffected by deletion of the repressor element and continues to decrease in the neonate. These studies indicate that the identified AFP repressor is active specifically in goblet cells. The decrease in AFP expression in the enterocytes may be mediated by a separate cis-acting element that is contained in the AFP minigene construct. Alternatively, it is possible that mature enterocytes lack some of the positive factors required for initiation and maintenance of minigene transcription in the absence of the identified negative element. PMID- 7537232 TI - Ectoderm-mesenchyme and mesenchyme-mesenchyme interactions regulate Msx-1 expression and cellular differentiation in the murine limb bud. AB - The apical ectodermal ridge (AER) is a specialized thickening of the distal limb mesenchyme that has been demonstrated to support limb outgrowth and proper limb development. The homeobox gene, Msx-1, is associated with the distal limb mesenchyme (progress zone) and its expression depends upon the presence of the AER in chick limbs. We demonstrate here that the expression of Msx-1 is dependent upon the limb ectoderm in the mouse, but that the inductive capacity of murine limb ectoderm is not restricted to the AER. Msx-1 can also be maintained in limb mesenchyme by the substitution of FGF 4 for the ectoderm; however, we see that local cell-cell interactions are required for high levels of expression. Disruption of cell-cell interactions in the limb mesenchyme results in a dramatic decrease in Msx-1 levels and a precocious expression of MyoD1, suggesting that the limb environment represses differentiation and promotes cell proliferation during early development. BMP 4 and FGF 2 can also maintain Msx-1 expression in limb mesenchyme as well as retinoic acid which is usually associated with polarizing activity in the early limb. Msx-2 expression does not appear to be dependent upon cell-cell interactions as measured in these experiments. Taken together, our data suggest that the expression of Msx-1, but not Msx-2, not only requires factors from the limb ectoderm, but also relies upon cues from local cell interactions and that the spatial distribution of inductive capacities in limb ectoderm differs between the avian and murine systems. PMID- 7537234 TI - Structure, expression, and extracellular targeting of PM27, a skeletal protein associated specifically with growth of the sea urchin larval spicule. AB - The PM27 gene encodes a sea urchin skeletal protein. Both the transcript and encoded protein appear at the mesenchyme blastula stage and are restricted to the primary mesenchyme cell (PMC) lineage throughout development. Transgenic expression of PM27 promoter constructs demonstrates that this cell specificity is regulated at the level of transcription. The PM27 sequence predicts a nonglycosylated secretory product of 27 kDa in mature form. The N-terminal "repeat" domain of the deduced amino acid sequence consists of a series of tandem repeats and shares both sequence and predicted structural similarities with several fiber-forming proteins. The C-terminal "lectin" domain is similar to the C-class lectins. Antisera against either domain of PM27 detect two major proteins in embryo extracts, with apparent molecular weights of 27 and 30 kDa. Immunolocalization in whole embryos demonstrates that PM27 antigen is produced uniformly and exclusively by PMCs through the early prism stage and that this specificity is further restricted during skeletogenesis to a subpopulation of PMCs associated with the growing tips of the spicules. It is secreted to the skeletal compartment and accumulates predominantly at the advancing mineralizing surface of the spicule tips. As the spicules elongate PM27 protein disappears from the more mature mid-shaft regions. The observed characteristics of PM27 are consistent with a role in the regulation or execution of skeletal growth, as opposed to maintenance or structural integrity of the spicules. PMID- 7537235 TI - Evidence against a role for inducible nitric oxide synthase in the hyperdynamic circulation of portal-hypertensive rats. AB - BACKGROUND/AIMS: Excessive nitric oxide biosynthesis caused by expression of inducible NO synthase has been implicated in the hyperdynamic circulation of portal hypertension. The aim of the study was to investigate whether inducible NO synthase is expressed in portal hypertension an accounts for the hyperdynamic circulation. METHODS: In study 1, NO synthase activities were measured by the conversion of L-arginine to citrulline in tissues from portal-hypertensive, cirrhotic, and sham-operated rats and from normal rats pretreated with endotoxin and after long-term administration of dexamethasone, which inhibits the expression of inducible NO synthase. In study 2, systemic and splanchnic hemodynamics (radiolabeled microspheres) and gastric blood flow (hydrogen gas clearance and reflectance spectrophotometry) were measured in portal-hypertensive rats after long-term administration of dexamethasone (0.25 mg.kg-1.day-1) or vehicle. RESULTS: In study 1, constitutive and inducible NO synthase activities in portal-hypertensive or cirrhotic rats were similar to those observed in sham operated rats. The significant increase in the inducible activity observed after endotoxin injection was prevented when rats received long-term treatment with dexamethasone. In study 2, cardiac index, portal-pressure, portal venous inflow, and gastric blood flow were similar in dexamethasone-or vehicle-treated portal hypertensive rats. CONCLUSIONS: These results to not support a role for an increased expression of the inducible NO synthase in the hyperdynamic circulation of portal hypertension. PMID- 7537237 TI - Phylogenetic analysis of a novel sulfate-reducing magnetic bacterium, RS-1, demonstrates its membership of the delta-Proteobacteria. AB - Most of the 16S ribosomal RNA gene of a sulfate-reducing magnetic bacterium, RS 1, was sequenced, and phylogenetic analysis was carried out. The results suggest that RS-1 is a member of the delta-Proteobacteria, and it appears to represent a new genus. RS-1 is the first bacterium reported outside the alpha-Proteobacteria that contains magnetite inclusions. RS-1 therefore disrupts the correlation between the alpha-Proteobacteria and possession of magnetite inclusions, and that between the delta-Proteobacteria and possession of greigite inclusions. The existence of RS-1 also suggests that intracellular magnetite biomineralization is of multiple evolutionary origins. PMID- 7537236 TI - The role of angiogenesis in the tumor growth of Syrian hamster pancreatic cancer cell line HPD-NR. AB - BACKGROUND/AIMS: New therapeutic approach is required for pancreatic cancer, one of the most intractable malignancies. The role of angiogenesis in the tumor growth of a Syrian hamster pancreatic cancer cell line HPD-NR, which closely resembles its human counterpart, was investigated. METHODS: Angiogenic activity was measured as stimulation of growth of human umbilical vein endothelial cells (HUVEC), and angiogenic factors produced by HPD-NR cells were identified by reverse-transcription polymerase chain reaction and Northern blot analysis. Then in vitro and in vivo antitumor effects of a potent angiogenesis inhibitor, O (chloroacetylcarbamoyl)fumagillol (AGM-1470), were examined. RESULTS: The conditioned medium of HPD-NR cells stimulated the growth of HUVEC, and four hamster angiogenic factors were detected with an overexpression of transforming growth factor alpha and vascular endothelial growth factor messenger RNAs. AGM 1470 specifically inhibited the growth of HUVEC and that of HPD-NR tumors in vivo with decreased vascularity of the tumors but not the growth of HPD-NR cells in vitro. CONCLUSIONS: The results suggest that angiogenesis plays an important role in tumor growth of HPD-NR cells and can be a new target of medical therapy for pancreatic cancer. PMID- 7537239 TI - Immunological disorder against the Epstein-Barr virus infection and prognosis in patients with cervical carcinoma. AB - Epstein-Barr virus antigen-specific killer T cell (EBV-KT) activity and Epstein Barr virus (EBV) shedding at uterine cervix and oropharynx were examined in patients with cervical carcinoma. EBV-KT activity was determined by a modified EBV-induced B cell focus regression assay. EBV shedding was examined by infectivity assay. EBV-KT activity in the patients was decreased significantly compared with that of controls (P < 0.001) and became increasingly lower as the clinical stage of the disease advanced. EBV-KT activity was changed by treatment, and decreased EBV-KT activity after treatment was clearly related to patient survival. EBV shedding at oropharynx and uterine cervix were also higher in the patients than in the controls (P < 0.01 and < 0.05). These results suggest that EBV-KT activity is closely associated with the prognosis in patients with cervical carcinoma and that it may be a useful parameter in monitoring the cell mediated immunity of patients with cervical carcinoma. PMID- 7537238 TI - Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. AB - T cell-specific transcription factor (TCF-1) and lymphoid enhancer factor 1 (LEF 1) have been implicated exclusively in the regulation of T cell-specific genes. The only adult tissue other than thymus known to express these factors is spleen and lymph node, which contain low levels of LEF-1 and no TCF-1. We noticed that genes involved in hair-specific gene expression possess LEF-1/TCF-1 consensus motifs located in similar positions relative to their TATA box. We show that of the two factors only LEF-1 is expressed in hair follicles; it can be cloned in both splice forms from human skin keratinocytes and it can bind to these sites in the hair promoters. We show that LEF-1 mRNA is present in pluripotent ectoderm, and it is up-regulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and commitment of overlying ectodermal cells to invaginate and become hair follicles. New waves of ectodermal LEF-1 spots appear concomitant with new waves of follicle morphogenesis. To test whether LEF-1 patterning might be functionally important for hair patterning and morphogenesis, we used transgenic technology to alter the patterning and timing of LEF-1 over the surface ectoderm. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. This provides the first direct evidence that ectodermal cues are critical in establishing these developmental processes, which at later stages are known to be influenced by underlying mesenchyme. Remarkably, elevated LEF-1 in the lip furrow epithelium of developing transgenic animals triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates. Collectively, our findings demonstrate that ectodermal expression of LEF-1 plays a central role in gene expression, pattern formation, and other developmental processes involving epithelial-mesenchymal associations. PMID- 7537240 TI - Based on biochemical and physiological behavior, where is Aspergillus egyptiacus better placed? AB - Physiological and biochemical properties were tested in 45 isolates of Aspergillus egyptiacus (16 isolates), Emericella nidulans (16) and Aspergillus versicolor (13). The three fungal species exhibited common and similar features. The big similarity between A. egyptiacus and E. nidulans was greater than between A. egyptiacus and A. versicolor. It included the inability to produce base either from sodium citrate or lactic acid media, growth at 45 degrees C (thermophilicity), and production of very similar pigmentations on Aspergillus flavus and parasiticus agar. A. egyptiacus is therefore better placed in the Aspergillus nidulans-Emericella assemblage. PMID- 7537241 TI - Chronic toxicity of tar from heavy-duty diesel exhaust following intratracheal instillations to the lungs of hamsters. AB - Chronic toxicity of tar from heavy-duty diesel exhaust (HD tar) was studied in male Syrian golden hamsters which received 15 mg, 7.5 mg or 1.5 mg of HD tar as the total dosages by intratracheal instillations once a week for 15 weeks. As a control group, hamsters were treated with the 0.1 ml of Tween 60: ethanol: phosphate buffer (pH 6.88, 0.25 M) solution (5.8: 8.7: 100 by volume) once a week in the same manner. The survival rate during the instillation period in the group given 15 mg of HD tar, the high-dose group of HD tar, was the lowest, and the effect was dose-dependent. However, the survival rates during the subsequent observation period showed no marked differences among HD tar treated groups. During their total life span, one papilloma in the larynx was seen in the 44 hamsters in the group given 1.5 mg of HD tar, one papilloma in the larynx was appeared in the 59 hamsters in the group given 1.5 mg of HD tar and one lung adenoma was developed in the 58 hamsters in the control group. There were no tumors in the respiratory tract in the group given 7.5 mg of HD tar. Concerning the histopathological findings of the lung, the incidence of alveolar cell or bronchiolar cell hyperplasia in the group given 1.5 mg of HD tar was significantly higher than that in the control group. From these results, although we could not observe any tumorigenicity or carcinogenic effect of HD tar, it would seem that HD tar caused weak but positive damage to the lungs of hamsters. PMID- 7537242 TI - Human uterine amylase in relation to infertility. AB - In the uterine fluid of infertile women the amylase activity is significantly lower (90.00 to 357.75 mg/hr/100 ml) than that in parous women (172.14 to 536.99/hr/100 ml) during the different phases of menstrual cycle. Possibly the low enzyme activity in infertile women can impair the sperm capacitation and thus adversely affect the fertility of these infertile women. PMID- 7537245 TI - Human genetic diversity (immunoglobulin GM allotypes), linguistic data, and migrations of Amerindian tribes. AB - GM haplotype frequencies were examined in 49 Amerindian tribes (from North, Central, and South America) to investigate the congruence of genetic variation with that observed in language and geography. We used two approaches: (1) the mobile site method, which allows a two-dimensional representation of genetic variation where the distances between reference points (i.e., the locations of the populations in the geographic map after displacements) are close to the genetic distances, and (2) a multivariate analysis (factorial correspondence analysis), which permits a visual interpretation of the geographic distribution of GM haplotypes on a map, completed by a cluster analysis. The results show a strong gradient from the Bering Strait to South America. The Eskimo and Na-Dene are genetically different from all other Amerindians, reflecting their more recent migrations. The orientation of most trajectories of the tribes from Central and South America can be interpreted as earlier migrations along the Pacific and Atlantic coasts. We conclude that geographic and linguistic factors played a part in the genetic diversity of Amerindian tribes. PMID- 7537243 TI - In vitro development of placode-derived LHRH neurons: possible involvement of alpha-fetoprotein. AB - Using the olfactory placode (NAP) of 12.5-day-old and vomeronasal organ (VNO) of 14.5-day-old (E12.5, E14.5) rat embryos, we examined in vitro the roles of brain tissues in the development of LHRH neurons. The culture was performed singly or in combination with various brain tissues of E12.5 and E14.5 embryos. Furthermore, a serum-free basal culture medium was supplemented with a water extract of cerebral cortex or septopreoptic-medial basal hypothalamus (S-MBH) of E18.5 embryos and 2-day-old (N2) newborns. In cocultures, many LHRH cells derived from the NAP or VNO migrated into the collocated tissues of the forebrain vesicles and medial basal hypothalamus, especially into the latter, along neural cell adhesion molecule (NCAM)-positive fibers projecting from the NAP and/or the brain tissues, especially in the case of the medial basal hypothalamus. The effects of the brain extracts were evaluated by differentiation, migration, neurite extension, and survival of LHRH neurons. E18.5 S-MBH extract was most effective. In the analysis of the protein composition of the extracts, alpha fetoprotein (AFP) was determined only in the E18.5 S-MBH extract. When anti-AFP IgG was added to the cultures containing E18.5 S-MBH extract, the stimulative effects of the extract were reduced to the level of the N2 S-MBH extract. When we cultured the NAP by adding AFP in the basal culture medium, the development of LHRH cells was somewhat promoted. The actual roles of AFP thus remain to be further elucidated. PMID- 7537244 TI - Regulation of galanin and gonadotropin-releasing hormone gene expression in the hypothalamus and basal forebrain of the rat. AB - Galanin is a cotransmitter in GnRH neurons and is thought to play a role in the control of gonadotropin secretion. The aim of our research has been to learn how galanin mRNA is regulated in GnRH neurons with the goal of understanding galanin's physiological significance. We have used double-label in situ hybridization and computerized image analysis to identify GnRH neurons coexpressing galanin mRNA and to estimate cellular levels of galanin message in these cells under different physiological conditions in the rat. In adult females, levels of galanin mRNA in GnRH neurons increase two- to fourfold with the onset of the proestrous and steroid-induced LH surges. Pharmacological blockade of synaptic transmission with either a general anesthetic (pentobarbital) or an alpha-adrenergic receptor antagonist (phenoxybenzamine) inhibits both the steroid-induced LH surge and the associated induction of galanin expression in GnRH neurons. Compared with the day of diestrus of the estrous cycle, during lactation cellular levels of galanin mRNA in GnRH neurons are profoundly reduced. In contrast to galanin mRNA in GnRH neurons, we could adduce no evidence for changes in cellular levels of GnRH mRNA under any physiological conditions or with any pharmacological manipulations. We conclude that alterations in galanin gene expression play a fundamental role in governing the functional activity of GnRH neurons, possibly by acting presynaptically to shape GnRH pulses, thereby determining the biological efficacy of GnRH action at its target cells in the pituitary. PMID- 7537246 TI - dra-related X adhesins of gestational pyelonephritis-associated Escherichia coli recognize SCR-3 and SCR-4 domains of recombinant decay-accelerating factor. AB - Bacterial adhesins are important virulence factors that allow colonization of the human urogenital tract by Escherichia coli. Adhesins of the Dr family have been found to be more frequently expressed in strains associated with symptomatic urinary tract infections. Because of the high frequency of symptomatic urinary tract infections during pregnancy, we screened E. coli isolates from 64 gestational pyelonephritis patients for the expression of Dr and X adhesins to address their potential virulence roles in this population. Using PCR and primers for the afaB gene, we detected dra-related operons in 17 isolates (27%). On the basis of the lack of hemagglutination of Dr(a-) erythrocytes containing a point mutation in the decay-accelerating factor (DAF) short consensus repeat-3 (SCR-3) domain, 12 of these strains were categorized as classical Dr adhesins. The hemagglutination of O erythrocytes by Dr+ strains was blocked or reduced by a monoclonal antibody to the DAF SCR-3 domain. The remaining five dra-positive strains agglutinated Dr(a-) erythrocytes. Monoclonal antibody to the DAF SCR-3 domain failed to block O-erythrocyte hemagglutination. Adhesins in these strains did not fulfill criteria for Dr hemagglutinins because of the undefined receptor specificities and were categorized as X. E. coli strains bearing dra-related X adhesins bound to DAF cDNA-transfected Chinese hamster ovary cells. Three of these dra-related X-adhesin-bearing E. coli strains failed to attach to the SCR-3 delta deletion transfectant, which suggested that binding sites were located in the SCR-3 domain but outside the region blocked by the monoclonal anti-SCR-3 immunoglobulin G. The binding sites of the remaining two dra-related X adhesin strains were localized to the SCR-4 domain, as the attachment was shown to be abolished on an SCR-4 delta mutant but unaffected by an SCR-3 delta deletion. The heterogeneity in the binding sites of E. coli DAF (Dr) family adhesins from gestational pyelonephritis isolates may reflect the ability of the adhesins to evolve to recognize alternate peptide epitopes for efficient colonization. PMID- 7537248 TI - Pseudomonas aeruginosa exoenzyme S induces proliferation of human T lymphocytes. AB - Pseudomonas aeruginosa is a gram-negative bacterium that is responsible for devastating acute and chronic infections, which include bronchiectasis in cystic fibrosis, nosocomial pneumonia, and infection of burn wounds. Previous studies have demonstrated that these patients have impaired host responses, including cell-mediated immune responses, which are important in anti-Pseudomonas host defense. The P. aeruginosa exoproduct, exoenzyme S, has a number of characteristics which suggest that it might be important in cell-mediated immunity. To determine whether exoenzyme S activates lymphocytes to proliferate, peripheral blood mononuclear cells (PBMC) from normal volunteers were stimulated with purified exoenzyme S, and the lymphocyte response was assessed by measuring [3H]thymidine uptake and by counting the number of cells after various times in culture. Ninety-five percent of healthy adult donors had a lymphocyte response to exoenzyme S. The optimal lymphocyte response occurred on day 7, with 4 x 10(5) PBMC per microtiter well when cells were stimulated with 10 micrograms exoenzyme S per ml. [3H]thymidine uptake correlated with an increase in the number of mononuclear cells, indicating that proliferation occurred. In unseparated PBMC, T cells, and to a lesser extent B cells, proliferated. Purified T cells proliferated, while purified B cells proliferated only after the addition of irradiated T cells. Thus, T lymphocytes are necessary and sufficient for the proliferative response to exoenzyme S. We speculate that exoenzyme S from P. aeruginosa is important in T-lymphocyte-mediated host defense to P. aeruginosa. In strategies to enhance impaired cell-mediated immunity, exoenzyme S should be considered as a potential stimulant. PMID- 7537249 TI - Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay. AB - The competitive binding specificities of glucuronoxylomannan (GXM) and its derivatives to factor sera of Cryptococcus neoformans were studied by enzyme linked immunosorbent assay. An effort was made to determine the epitope specificity of each factor serum. Despite the presence of antigenic factor 1 on all serotypes of C. neoformans, variations in inhibition ability were observed with different GXMs. The panspecific component of factor serum 1 (antibody 1) appeared to be due to the presence of more than one antibody component. The activity was dependent on the 6-O-acetyl substituent. GXMs of serotypes A and D inhibited factor serum 2 equally well, indicating a low titer for the antibody 7 component. Serotype B GXM was a poor inhibitor, and serotype C GXM did not inhibit factor serum 2. The activity of factor serum 2 was 6-O-acetyl dependent. GXMs from typical serotype A and serotype D isolates were excellent inhibitors of factor serum 3. GXMs from serotype B were poorly inhibitory and serotype C did not inhibit factor serum 3. The activity of factor serum 3 was 6-O-acetyl dependent. The activity of factor serum 4 was due predominantly to antibody component 6. The activity of factor 4 was directed mainly against serotype C, and it was independent of 6-O-acetyl substitution Factor serum 5 was specific for serotype B GXMs. The inhibitory effect was independent of 6-O-acetyl substitution, but the effect was diminished by reduction of the glucuronic acid. The GXMs with a typical serotype C structure inhibited antibody 6. O deacetylation of the GXMs did not affect their inhibitory activity. However, reduction of glucuronic acid reduced factor serum 6 binding. Factor serum 8 was specific to serotype D; native GXMs of serotype A were slightly inhibitory. O deacetylation of the serotype D GXMs abrogated the inhibitory effect. O deacetylation alone abrogates the activity of antibody components 1, 2, 3, and 8. Reduction of glucuronic acid reduces the inhibitory activity of the GXM to antibody components 4, 5, and 6. Partial GXM structures and methyl glycosides did not effectively inhibit the activity of any of the factor sera. PMID- 7537247 TI - Identification of rfbA, involved in B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O5. AB - Previous work from this laboratory has shown that a 26-kb insert in cosmid clone pFV100, isolated from a Pseudomonas aeruginosa gene library, contained genes that could restore serotype-specific B-band lipopolysaccharide (LPS) expression in rough mutant ge6. In this study, subclones from pFV100 were made to identify genes responsible for B-band LPS synthesis. Transformation of Escherichia coli HB101 with cosmid clone pFV100 resulted in expression of P. aeruginosa serotype O5 B-band LPS, indicating the presence of an rfb cluster in pFV100. Expression of P. aeruginosa LPS could not be achieved in E. coli HB101 transformed with any of the subclones. Complementation studies of well-characterized rough mutants of P. aeruginosa PAO1 deficient in B-band LPS biosynthesis were performed with the various subclones. Subclone pFV110, containing a 1.4-kb XbaI-HindIII insert, restored B-band LPS biosynthesis in mutant AK44 (A+B-; complete core). Probing chromosomal DNA from the 20 International Antigenic Typing Scheme serotypes with the 1.4-kb insert from pFV110 in Southern hybridizations revealed a positive reaction to restriction fragments in serotypes O2, O5, O16, O20, and O18. LPS of serotypes O2, O5, O16, and O20 were shown earlier to have a similar backbone structure in their O antigen. The insert in pFV110 was sequenced, and the deduced amino acid sequence was compared with sequences of protein databases. No significant homology could be detected with any sequences in the database. Open reading frame analysis identified one region, ORF303, which could encode a 33-kDa protein. Using E. coli maxicells for protein expression, orf303 mediated the expression of a unique polypeptide with an apparent molecular mass of 32.5 kDa. The deficiency in the synthesis of B-band LPS biosynthesis in mutant AK44 is apparently complemented by the 33-kDa protein encoded by orf303. We have designated this ORF rfbA. This investigation is the first report on cloning and sequencing of an rfb gene involved specifically in O-antigen biosynthesis in P. aeruginosa PAO1. PMID- 7537251 TI - Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein. AB - Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans. PMID- 7537250 TI - Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures. AB - One mechanism by which interferons (IFNs) can inhibit chlamydial infection is by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO), which restricts the availability of tryptophan, which is required for chlamydial growth. Other immunomodulating agents, including interleukin-1 (IL-1), can interact synergistically with IFNs, resulting in increased IDO activity in macrophages. The objectives of this study were to establish that IL-1 can enhance IFN-mediated inhibition of chlamydial growth by increasing the amount of IDO activity induced by IFNs and to identify immunomodulatory agents in culture supernatants from chlamydia-infected macrophages that interact synergistically with IFNs in restricting chlamydial growth. Monocyte-derived macrophages were treated with IL 1 combined with gamma IFN (IFN-gamma) or IFN-beta. The ability of treated cells to support the growth of Chlamydia psittaci was directly related to the amount of IDO activity induced; as IDO activity increased, so did inhibition of chlamydial growth. Furthermore, concentrations of IFNs were identified at which little IDO activity was induced and chlamydial growth was permitted yet which in the presence of IL-1 resulted in increased IDO activity and restriction of chlamydial growth. The addition of exogenous tryptophan reversed the effect of combined IFN and IL-1 treatment, indicating that IDO activity induced by combined cytokine treatment was responsible for chlamydial inhibition. Supernatants from chlamydia infected macrophages were capable of potentiating IDO induction by IFN-gamma and of restricting the growth of C. psittaci. Antibody to IL-1 beta neutralized the potentiating effects of supernatants from chlamydia-infected cells on both IDO induction and chlamydial inhibition. Thus, IL-1 produced in response to chlamydial infection may contribute to the elimination of the infection. PMID- 7537252 TI - Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates. AB - This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations. Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB. Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations. Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested. Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung. Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations. Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations. This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide protein conjugate. The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization. PMID- 7537253 TI - Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guerin. AB - C57BL/6 and BALB/c mice were vaccinated with either live or heat-killed Mycobacterium bovis bacillus Calmette-Guerin (BCG) organisms, and splenic T cells were used to screen the stimulatory potential of fractionated somatic and secreted mycobacterial proteins by production of gamma interferon (IFN-gamma). Maximum responses were obtained with fractionated secreted proteins of Mycobacterium tuberculosis. There was no single dominant antigen, but five regions of mycobacterial proteins induced high concentrations of IFN-gamma. However, only two of the five regions stimulated T cells from both mouse strains: two were exclusively recognized by T cells from BALB/c mice, and one was exclusively recognized by T cells from C57BL/6 mice. T cells from mice vaccinated with heat-killed M. bovis BCG organisms failed to respond to fractionated secreted proteins but recognized several somatic antigen fractions. As late as 1 year after primary vaccination, memory T cells responded to similar protein regions, and IFN-gamma production was intensified by secondary infection. Our data confirm a central role for secreted proteins in immunity to mycobacteria. Moreover, we demonstrate that a major set of mycobacterium-reactive T cells is stimulated only by vaccination with live but not with heat-killed M. bovis BCG organisms. Because a major impact of genetic host factors on antigen recognition was observed, we favor the use of live carrier organisms which secrete mycobacterial proteins over subunit vaccines as an improved antituberculosis vaccine. PMID- 7537254 TI - Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli. AB - In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined. EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam. The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E. coli strains but not K-12 strains. The log jam phenotype may represent a basal means by which E. coli bacteria attach to the human intestine. PMID- 7537256 TI - Glycosylation of beta-1 integrins in B16-F10 mouse melanoma cells as determinant of differential binding and acquisition of biological activity. AB - Studying B16-F10 cells we could identify beta-1 integrins as laminin, fibronectin and collagen receptors. Gradient ionic strength elution analysis of affinity chromatography showed differential interactions between laminin-binding beta-1 integrins (two beta-1 polypeptides of 105 and 120 kDa) and fibronectin and collagen-binding beta-1 integrins (elution of one major beta-1 polypeptide of 120 kDa) and their respective ligands. To evaluate this diversity we submitted B16 F10 extracts to IEF and SDS-PAGE and found that one beta-1 integrin formed acidic and larger isoforms, while another formed basic and smaller isoforms. To study this difference we also submitted material eluted from WGA-Sepharose columns to IEF but now only the acidic beta-1 isoform was found. Extracts of B16-F10 treated with neuraminidase showed only the basic beta-1 isoform, suggesting that terminal sialic acid residues may be responsible for this acidic pattern, an interpretation supported by the fact that MAA (Maackia ammurensis agglutinin) reacts only with the acidic isoform. Differential glycosylation of beta-1 integrin isoforms in B16-F10 was also demonstrated since the smaller laminin binding beta-1 integrin isoform reacted only with GNA (Galanthus nivalis agglutinin), whereas the mature larger form reacted with DSA (Datura stramonium agglutinin) and MAA; thus this heterogeneity of beta-1 chains is essentially due to variable glycosylation. Autoradiography and immunoblotting analysis of material separated by 2-dimensional electrophoresis show that only the processed forms of beta-1 integrins are expressed at the cell surface. PMID- 7537255 TI - Induction of CD44 expression by the Epstein-Barr virus latent membrane protein LMP1 is associated with lymphoma dissemination. AB - Epstein-Barr Virus (EBV) is implicated in the pathogenesis of endemic Burkitt's lymphoma (BL), B-cell lymphomas occurring under immunosuppression, nasopharyngeal carcinoma and Hodgkin's disease. Two distinct patterns of latent EBV gene expression occur in EBV-associated lymphomas. BLs typically display expression of the nuclear antigen EBNAI only, whereas EBV-associated, non-Burkitt B-cell lymphomas express at least 9 latent viral genes (6 EBNAs and 3 latent membrane proteins), reminiscent of in vitro EBV-immortalized lymphoblastoid cell lines (LCL). BLs are characterized by local, extra-nodal growth, whereas EBV-associated B-cell lymphomas often disseminate to peripheral lymphoid tissue. We show here that BL cells forming local tumors after xenotransplantation into SCID mice disseminate to lymphoid tissue following introduction of the latent membrane protein I (LMP 1) gene. Introduction of LMP 1 into BL cells induced expression of CD44 on the cell surface, a molecule implicated in enhanced lymphoid tumor growth and dissemination. Introduction of CD44 into LMP 1-/CD44-BL cells was observed to confer the disseminated tumor growth pattern associated with LMP 1 expression. Taken together our results show that expression of LMP 1 may regulate expression of CD44 and play an important role in the behavior of EBV-based lymphomas. PMID- 7537257 TI - Investigating the validity of the Clinical Linguistic Auditory Milestone Scale. AB - The purpose of this investigation was to determine the validity of the Clinical Linguistic Auditory Milestone Scale (CLAMS) as a screening test to help physicians detect delays in language development. Language delays in children are often not diagnosed until about 3 years of age because (1) parents may not be aware of what constitutes normal language development, (2) diagnosis of a language problem is difficult for physicians to make because they see the child for brief periods of time, (3) there is a wide range of what constitutes normal language so professionals are reluctant to classify a child as delayed without thorough testing. The hypothesis for this investigation was that children who have language delays can be identified quickly and easily through the administration of the CLAMS. Subjects included 99 children between the ages of 1 and 3 years. The procedure had 3 parts: (1) parents completed a form consisting of questions such as parent occupation, number of children in the family, birth history, etc., (2) the CLAMS, a parent report that can be given while the child is seen by the physician, was administered, (3) the Sequenced Inventory of Communication Development (SICD), was administered to assess receptive and expressive language. The purpose for administering the SICD was to obtain a more in-depth picture of the child's ability to comprehend and use language, thereby providing data to determine the validity of the CLAMS as a screening device. Results indicated that the CLAMS is a valid instrument for identifying language delayed children. PMID- 7537260 TI - Lens-specific expression of a major histocompatibility complex class I molecule disrupts normal lens development and induces cataracts in transgenic mice. AB - PURPOSE: Lens epithelial tissue does not normally express major histocompatibility complex (MHC) class I molecules. In addition, the mechanism of self-tolerance to intraocular antigens is unknown. To study the effect of class I expression in the lens, transgenic mice were produced that express an allo-MHC class I molecule under the alpha A-crystallin proximal promoter. METHODS: p alpha Dd was generated by fusion of the H-2Dd structural gene to the alpha A-crystallin proximal promoter. Transgenic mice were produced, and founder lines were identified by Southern blot hybridization. Eyes from transgenic mice were cryostat sectioned and stained for Dd expression or fixed in paraformaldehyde and stained for histologic analysis. Lens RNA was isolated by acid phenol extraction, and transgene expression was analyzed by nuclease protection. RESULTS: The transgenic mice demonstrated dose-dependent, nonimmunologic lens defects consistent within a given line. In the highest expressing lines, ocular defects, including microphthalmia and cataract formation, were observed. Many adult mice from these lines demonstrated lens capsule rupture and a Dd-specific inflammatory response. Inflammation did not occur in mice with intact lens capsules. CONCLUSIONS: Overexpression of H-2Dd in the lens had serious nonimmunologic consequences on lens development and cataract formation. In addition, the high copy number mice revealed at least a partial loss of immunologic tolerance on lens capsule rupture. The lack of an inflammatory response in transgenic mice with intact lens capsules suggests that the physical barrier of the lens capsule is one mechanism of maintaining immune privilege. PMID- 7537261 TI - Symbolic issues in embryo research. PMID- 7537258 TI - Effect of tecogalan sodium on angiogenesis in vitro by choroidal endothelial cells. AB - PURPOSE: To examine the possible inhibitory effect of tecogalan sodium, derived from bacteria, on three important components of in vitro angiogenesis (endothelial proliferation, migration, and tube formation in a collagen gel) using bovine choroidal endothelial cells (CECs). METHODS: The effects of tecogalan sodium (1, 5, 25, 125, and 250 micrograms/ml) on cultured CECs were examined when basic fibroblast growth factor (bFGF, 10 ng/ml), vascular endothelial growth factor (VEGF, 50 ng/ml), a combination of bFGF (10 ng/ml) and VEGF (50 ng/ml) (bFGF/VEGF) and 10% fetal calf serum (FCS) were used as angiogenic stimulants. For the proliferation assay, CECs were cultured and the cell numbers counted on days 1, 3, and 5. For migration assay, CECs were seeded in the upper half of a Boyden chamber while an angiogenic growth factor was loaded in the lower half. After 6 hours of incubation, cell migration was evaluated by counting the numbers of migrated cells per microscopic field on the lower side of the filter. For the tube-forming assay, CECs were seeded in a type I collagen gel, and the length of the tube-like structures (an indicator of angiogenesis) formed by CECs per microscopic field was quantified by image analysis. The effect of neutralizing antibody for bFGF also was tested in these three assays. RESULTS: All tested angiogenic stimulants induced CEC proliferation. The stimulatory effect of bFGF and bFGF/VEGF was reduced by tecogalan sodium (IC50 for bFGF effect, 26.1 micrograms/ml). However, the effect of VEGF and of 10% FCS was not altered by low doses of tecogalan sodium (< 25 micrograms/ml). Chemotaxis of CECs was stimulated by bFGF alone and by bFGF/VEGF, and this effect was inhibited by tecogalan sodium (IC50 for bFGF, 3.2 micrograms/ml). Stimulation of chemotaxis by VEGF alone and by 10% FCS was not affected by tecogalan sodium in low doses but was inhibited by high doses. Tube formation was stimulated by administration of each of the factors. Stimulation of tube formation by bFGF and by bFGF/VEGF was inhibited by tecogalan sodium (IC50 for bFGF, 18.2 micrograms/ml). High doses of tecogalan sodium (125 and 250 micrograms/ml) also inhibited 10% FCS-induced proliferation, migration, and tube formation. CONCLUSION: bFGF, VEGF, and a combination of bFGF and VEGF stimulated proliferation, migration, and tube formation by CECs in vitro. These stimulatory effects, but especially those of bFGF, were inhibited by tecogalan sodium. If tecogalan sodium can be shown to have a similar effect in vivo, it might have the potential for pharmacologic control of subretinal neovascularization. PMID- 7537259 TI - The effect of diabetes on neuropeptide content in the rat cornea and iris. AB - PURPOSE: To determine the effect of diabetes mellitus on the levels of substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP) in the rat cornea and iris. METHODS: Corneas and irides from control and diabetic rats were processed for neuropeptide radioimmunoassay 3 months after induction of diabetes with streptozotocin. Corneas and irides also were processed for SP and CGRP immunohistochemistry and were evaluated qualitatively. RESULTS: The radioimmunoassay data revealed no significant differences in either the content or concentration of SP, CGRP, and VIP between control and diabetic corneas. In contrast, iridial levels of CGRP and SP were significantly increased by 38% and 256%, respectively, in the diabetic animals. Iridial VIP levels were unchanged in the diabetic versus control groups. Immunohistochemical demonstrations of corneal and iridial SP- and CGRP immunoreactive fiber plexuses were indistinguishable on the basis of purely qualitative criteria. CONCLUSIONS: The results of this study have demonstrated a target- and peptide-specific effect of short-term diabetes on SP and CGRP expression in ocular nerves of the anterior eye segment. The absence of demonstrable changes in corneal neuropeptide levels argue against the theory that corneal abnormalities seen in clinical diabetes are caused, in part, by deficits in synthesis or axonal transport of "trophic" peptides in corneal sensory nerves. In contrast, elevated iridial SP and CGRP levels may be responsible for reported clinical deficits in pupillary diameter regulation. PMID- 7537262 TI - The ability of membrane potential dyes and calcafluor white to distinguish between viable and non-viable bacteria. AB - Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed: by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3 dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful. PMID- 7537263 TI - Effects of bidisomide (SC-40230), a new class I antiarrhythmic agent, on ventricular arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized rats; comparison with mexiletine and disopyramide. AB - We investigated the antiarrhythmic effects of bidisomide (SC-40230), a new class I antiarrhythmic drug, in early-phase ventricular arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized rats. The effects of bidisomide were compared with those of mexiletine (MXT) and disopyramide (DSP), established class I antiarrhythmic drugs. Drugs were administered intravenously, 5 min before induction of coronary occlusion. Bidisomide (5 mg/kg) reduced the number of premature ventricular complexes and the incidence of ventricular tachycardia and ventricular fibrillation similarly to MXT and DSP in rats with ventricular arrhythmias induced by coronary artery occlusion. In rats with ventricular arrhythmias induced by coronary artery reperfusion following a 5-min coronary occlusion, the antiarrhythmic effects of 5 mg/kg of bidisomide were similar to those of the same doses of MXT and DSP. All three drugs significantly slowed the heart rate. Our results suggest that bidisomide may effectively reduce the severity of life-threatening ventricular arrhythmias that occur during acute coronary syndrome. PMID- 7537264 TI - Cooperation of calcineurin and vacuolar H(+)-ATPase in intracellular Ca2+ homeostasis of yeast cells. AB - Saccharomyces cerevisiae VMA genes, encoding essential components for the expression of vacuolar membrane H(+)-ATPase activity, are involved in intracellular ionic homeostasis and vacuolar biogenesis. We report here that the immunosuppressants FK506 and cyclosporin A cause general growth inhibition of the vma3 mutant. Upon addition of the drugs, the mutant grew neither in the presence of more than 5 mM Ca2+ nor above pH 6.0. The action of the immunosuppressants is dependent on their binding proteins and ascribable to inhibition of calcineurin activity; a mutation of a calcineurin subunit (cnb1) shows synthetic lethal interaction with the vma mutation. The addition of FK506 decreases the cytosolic free concentration of Ca2+ in the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of a nonexchangeable Ca2+ pool. These results suggest that calcineurin controls calcium homeostasis by repression of Ca2+ flux into a cellular compartment(s) and that the vacuolar H(+)-ATPase is essential for cell growth cooperating with calcineurin to regulate the cytosolic free concentration of Ca2+. PMID- 7537265 TI - Functional interaction between c-Src and its mitotic target, Sam 68. AB - The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src approximately Ras GTPase activating protein > p85 alpha (amino-terminal) > Grb2 >> p85 alpha (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 alpha SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68. PMID- 7537266 TI - The c-jun proto-oncogene down-regulates the rat alpha-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA. AB - The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis. PMID- 7537267 TI - Sequences located 3' to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice. AB - Expression of fetal gamma-globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3' regulatory elements juxtaposed to gamma-globin genes or to deletion of gamma-gene silencers normally residing within the beta-globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of beta-globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A gamma-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A gamma-mRNA using RNase protection assays. Three mice lacked A gamma expression in the yolk sac indicating non-optimal integration site. Four expressed A gamma-mRNA at the embryonic stage only, while two expressed A gamma mRNA in both embryonic and fetal liver erythroid cells. Since the A gamma-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal gamma-globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the gamma-globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3' to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic epsilon-globin gene promoter by 2-fold but not the adult beta- or delta-globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3' A gamma- and the chicken 3' beta-globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3' to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal gamma-globin genes, resulting in their continued expression during adult life. PMID- 7537268 TI - Inter-alpha-inhibitor is required for the formation of the hyaluronan-containing coat on fibroblasts and mesothelial cells. AB - Cultured cells of various origins have been shown to be surrounded by a hyaluronan-containing coat, a structure that can be visualized by its ability to exclude large particles such as erythrocytes. When cultured in medium with no or low concentrations of serum, the cells lose their coats, although they still produce hyaluronan; upon the addition of serum, the coats are formed again. Here, we show that the serum protein inter-alpha-inhibitor can replace whole serum as an inducer of the formation of the coats on fibroblasts and mesothelial cells. The physiological role of inter-alpha-inhibitor has so far been unclear; our findings, together with those obtained with cumulus cell-oocyte complexes (Chen, L., Mao, S.J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), suggest that inter-alpha-inhibitor and related proteins have a general function as stabilizers of hyaluronan-containing pericellular coats. PMID- 7537272 TI - A voltage-dependent porin-like channel in the inner envelope membrane of plant chloroplasts. AB - The electrical activity of a single channel of 525 +/- 12 picosiemens in 150 mM KCl was measured after fusion of the inner envelope membrane of the chloroplast with planar lipid bilayers. The reversal potentials measured in KCl gradients indicate that this channel is weakly anion selective (PCl/PK = 1.6 +/- 0.2). The gating mechanism of the pore is voltage dependent. The channel shifts from a fully open state to a substrate at positive electrical potentials and remains closed at negative electrical potentials. Succinylation of the protein increases the open probability of the fully open state and reverses the channel selectivity. Analysis of the single-channel conductance as a function of the salt concentration and of the open probability at various voltages suggests that this channel is a new membrane porin not previously identified. PMID- 7537269 TI - Human immunodeficiency virus type 1 reverse transcriptase. 3'-Azidodeoxythymidine 5'-triphosphate inhibition indicates two-step binding for template-primer. AB - Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T.P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T.P binary complex (RTT.P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T.P (for single nucleotide incorporation). To explore cycling through these alternate rate limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3' azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T.P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Km should be equivalent to Ki, and since Km for AZTTP should be influenced by the dissociation rate constant for RTT.P, we examined the effect of altering RTT.P dissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T.P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T.P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower steady-state phase representing kcat (0.42 min-1) which was similar to the rate constant for RTT.P dissociation. Additionally, Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT.P dissociation rate constant and Ki,AZTTP was not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT.P exist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T.P where the binary RTT.P complex undergoes an isomerization before binding of the deoxynucleotide substrate. PMID- 7537270 TI - Enzymatically deacylated lipopolysaccharide (LPS) can antagonize LPS at multiple sites in the LPS recognition pathway. AB - Like other tetraacyl partial structures of lipopolysaccharide (LPS) and lipid A, LPS that has been partially deacylated by acyloxyacyl hydrolase can inhibit LPS induced responses in human cells. To identify the site(s) of inhibition in the LPS recognition pathway, we analyzed the apparent binding affinities and interactions of 3H-labeled enzymatically deacylated LPS (dLPS) and [3H]LPS with CD14, the LPS receptor, on THP-1 cells. Using (i) incubation conditions that prevented ligand internalization and (ii) defined concentrations of LPS binding protein (LBP), which facilitates LPS and dLPS binding to CD14, we found that dLPS can antagonize LPS in at least three ways. 1) When the concentration of LBP in the medium was suboptimal for promoting LPS-CD14 binding, low concentrations of dLPS were able to compete with LPS for binding CD14, suggesting competition between LPS and dLPS for engaging LBP. 2) When LBP was present in excess, dLPS could compete with LPS for binding CD14, but only at dLPS concentrations that were at or above its KD for binding CD14 (100 ng/ml). 3) In contrast, substoichiometric concentrations of dLPS (1 ng/ml) inhibited LPS-induced (3 ng/ml) interleukin-8 release without blocking LPS binding to CD14. Functional antagonism was possible without competition for cell-surface binding because both LPS-induced interleukin-8 release and dLPS inhibition occurred at concentrations that were far below their respective CD14 binding KD values. In addition to its expected ability to compete with LPS for binding LBP and CD14, dLPS thus potently antagonizes LPS at an undiscovered site that is distal to LPS-CD14 binding in the LPS recognition pathway. PMID- 7537271 TI - Ca2+ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. AB - Osteopontin (OPN) is an extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin alpha v beta 3. We measured the binding between OPN and integrin alpha v beta 3 with recombinant human OPN and the urinary form of human OPN, uropontin. Recombinant OPN was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and cleaved from glutathione S-transferase with Factor Xa. The mass of this form of OPN (rOP27) is 27,046 Da. rOP27 is truncated at arginine residue 228, 69 amino acids short of the native carboxyl terminus. Uropontin and rOP27 support RGD-dependent cell adhesion and to bind purified integrin alpha v beta 3 with similar affinities. Further study showed that OPN is the only known naturally occurring RGD-containing protein with a much greater affinity for alpha v beta 3 than for the platelet integrin alpha IIb beta 3. Most importantly, we find that physiologic levels of Ca2+ block cell adhesion to OPN. Measurement of binding constants between rOPN and purified integrin alpha v beta 3 with surface plasmon resonance showed that the affinity between rOPN and alpha v beta 3 is 26-fold lower in Ca2+ (Kd = 1.1 x 10(-8) M) than in Mn2+ (Kd = 4.3 x 10(-10) M) and 9 fold lower than in Mg2+ (Kd = 1.3 x 10(-9) M). In bone, the resorbing osteoclast generates elevated levels of extracellular Ca2+, therefore the findings presented here suggest a previously unappreciated mechanism for the modulation of bone resorption by extracellular Ca2+. PMID- 7537273 TI - Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. AB - It has been known for a number of years that glycosyl-phosphatidylinositol (GPI) anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes. PMID- 7537274 TI - Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. AB - DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells. PMID- 7537275 TI - Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase. AB - Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin. PMID- 7537276 TI - Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing. AB - The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. PMID- 7537278 TI - Transforming growth factor-beta 1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis. AB - Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon gamma (IFN-gamma) that makes MS worse and transforming growth factor-beta (TGF beta), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-beta in MS, we examined the effects of recombinant TGF-beta 1 (rTGF-beta 1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-beta 1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-beta 1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-gamma, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha), TNF-beta and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-beta itself. rTGF-beta 1 also suppressed numbers of myelin antigen reactive IFN-gamma- and IL-4-secreting cells in MS and AChR-reactive IFN-gamma and IL-4 secreting cells in MG. The selective suppressive effects of TGF-beta 1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-beta 1 attractive as a treatment alternative in MS and MG. PMID- 7537279 TI - Suppression of experimental allergic encephalomyelitis in offspring of DA and Wistar rats following immunization of mother with encephalitogen. AB - Immunization of female rats with encephalitogen before gestation, during gestation, and during lactation differentially decreased susceptibility to experimental allergic encephalomyelitis (EAE) in their offspring. The most pronounced suppression, revealed by lowered incidence and weaker clinical signs of the disease, was observed in offspring of mothers immunized before gestation and during lactation in both Dark August (EAE-susceptible), and Wistar (EAE relatively resistant) rat strains. Induction of EAE in mothers during pregnancy only delayed the onset of the disease in DA progeny. The overall effect on EAE in offspring did not depend on the disease intensity in mothers. Our results suggest that anti-myelin basic protein (MBP) antibodies passively transferred from mothers are not responsible for the observed protection in offspring. PMID- 7537280 TI - Suppression of experimental autoimmune myasthenia gravis by nasal administration of acetylcholine receptor. AB - Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freund's adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-gamma-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR. PMID- 7537277 TI - Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. AB - Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors. PMID- 7537281 TI - Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis. AB - Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-alpha), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-alpha suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN gamma secretion in both donors and recipients. IFN-alpha acts orally by modifying the encephalitogenicity of donor spleen T cells. PMID- 7537282 TI - The beta-endorphin inhibition of mitogen-induced splenocytes proliferation is mediated by central and peripheral paracrine/autocrine effects of the opioid. AB - In this study we show that the opioid peptide beta-endorphin exerts a tonic inhibitory effect on the proliferative response of splenocytes to the polyclonal mitogen phytohemoagglutinin throughout two separate sites of action: one central and one peripheral. The intracerebroventricular administration of beta-endorphin, in fact, induces a significant inhibition of splenocyte proliferation. In contrast, both the intracerebroventricular and the peripheral administration of anti-beta-endorphin gamma globulins induce a significant increase in proliferation. Moreover, an increase of splenocyte proliferation was observed also after the intravenous administration of gamma globulins and intraperitoneal naloxone, and this effect was still present in hypophysectomized rats. The data reported suggest that beta-endorphin exerts a tonic inhibitory effect on proliferation, acting centrally, and peripherally throughout a paracrine/autocrine mechanism. FACS experiments show that the effect observed is not the consequence of an alteration of lymphocyte trafficking induced by the opioid. PMID- 7537283 TI - Administration of nitric oxide synthase inhibitors in experimental autoimmune neuritis and experimental autoimmune encephalomyelitis. AB - The nitric oxide (NO) synthase pathway is activated during experimental autoimmune inflammation of the central nervous system, and administration of aminoguanidine, an inhibitor of the cytokine-inducible NO synthase (NOS), ameliorated the disease course of autoimmune encephalomyelitis in the SJL mouse. We studied the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune neuritis (EAN) and experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. NG-L-monomethyl-arginine (L-NMMA), a competitive inhibitor of NOS, partially suppressed T cell line-mediated EAN, but not myelin-induced EAN, myelin basic protein (MBP)-induced EAE, or T cell line mediated EAE. Aminoguanidine (AG), a selective inhibitor of the cytokine inducible NOS, enhanced MBP-induced EAE, but had no significant effects on myelin induced EAN. Two other NOS inhibitors, nitro-arginine methyl-ester and N-nitro arginine, had only little or no effects in EAN and EAE. The administration of NOS inhibitors showed some striking effects in EAN and EAE, but the observed diversity of actions points to a much more complex role of the NO pathway than previously suggested. PMID- 7537284 TI - Positron emission tomography studies on D2 and 5-HT2 receptor binding in risperidone-treated schizophrenic patients. AB - By the use of positron emission tomography (PET), high central dopamine D2 receptor occupancy (70 to 90%) has been demonstrated in patients treated with conventional neuroleptics. In patients treated with the atypical antipsychotic clozapine, the D2 occupancy was low (20 to 67%). The effects of clozapine may thus be mediated by a mechanism distinct from D2 occupancy. The observation that low doses of clozapine (125 to 175 mg daily) induced more than 80% (5 hydroxytryptamine) 5-HT2 occupancy supports the view that 5-HT2 antagonism may be related to the atypical effects of clozapine. Risperidone is a new antipsychotic drug with high affinity in vitro for both central 5-HT2 and D2 receptors. In this study, we determined the D2 and 5-HT2 occupancy induced by clinical treatment with risperidone. Four patients with acute exacerbation of schizophrenia were examined by PET after 4 weeks of treatment with risperidone, 6 mg daily. The D2 occupancy in the striatum was 75 to 80%. The 5-HT2 occupancy in the neocortex was 78 to 88%. This study confirms that, in patients with schizophrenia, treatment with risperidone induces a high D2 and 5-HT2 occupancy. Risperidone is, accordingly, a suitable drug for the examination of the clinical benefit of combined serotonin and dopamine antagonism. PMID- 7537285 TI - Clinical efficacy of serotonin-dopamine antagonists relative to classic neuroleptics. AB - Serotonin-dopamine antagonists (SDAs) offer the possibility of improved treatment of schizophrenia compared with conventional neuroleptics and have superior safety profiles. Clinical trial data have so far been published for only three SDAs to date, namely, risperidone, sertindole, and olanzapine. Of these, extensive data are available only for risperidone, showing that at doses of 4 to 16 mg/day, it is superior to haloperidol at 10 to 20 mg/day. Furthermore, risperidone, 6 and 16 mg/day, significantly improved negative/symptoms, whereas 20 mg/day of haloperidol was ineffective. Risperidone also appears to cause fewer extrapyrimidal symptoms (EPS) than haloperidol, 10 or 20 mg/day. Similar advantages of risperidone over perphenazine have also been found. A clinical trial of sertindole showed that, at 20 mg/day, it was equivalent to haloperidol, 16 mg/day, and caused fewer EPS. Olanzapine, a chemical derivative of clozapine, has also been shown to be superior to haloperidol (10 to 20 mg/day) at doses of 7.5 to 17.5 mg/day. In addition, at doses of 12.5 to 17.5 mg/day, olanzapine was found to have a significantly superior effect on negative symptoms over haloperidol, 10 to 20 mg/day. Doses of up to 17.5 mg/day of olanzapine also caused fewer EPS than haloperidol, 10 to 20 mg/day. There was no evidence of any leukopenia in patients treated with olanzapine in this small study (N = 335). The low EPS liability of these SDAs, combined with their efficacy, suggests that SDAs should become the mainstay of treatment for schizophrenia. PMID- 7537286 TI - Effects of risperidone in tardive dyskinesia: an analysis of the Canadian multicenter risperidone study. AB - In the Canadian multicenter, double-blind clinical trial of risperidone, 135 hospitalized chronic schizophrenic patients were randomly assigned to one of six parallel treatment groups for 8 weeks: risperidone, 2, 6, 10, or 16 mg/day; haloperidol, 20 mg/day; or placebo. Risperidone (6 to 16 mg)-treated patients showed significantly (p < 0.05) lower dyskinetic scores than those receiving placebo, whereas in haloperidol- and placebo-treated patients, no significant differences for dyskinetic symptoms were noted. To explore the antidyskinetic effect of risperidone, a post hoc analysis was performed on two selected patient samples: (1) patients meeting Research Diagnosis Criteria (RDC) for tardive dyskinesia (TD) at baseline or during double-blind treatment (N = 49) and (2) patients with RDC TD and with a Clinical Global Impression (CGI) Severity of dyskinesia score > or = 5 (at least moderately severe) (N = 48). The composition of the two subsamples was found to be almost identical because all but one of the patients with RDC TD (N = 49) were members of the group with at least moderately severe TD (N = 48). Analysis of four parameters (Extrapyramidal Symptom Rating Scale-dyskinesia total score, CGI severity of dyskinesia, buccolinguomasticatory [BLM] factor score, and extremities [choreoathetoid factor] score confirmed the antidyskinetic effect that was noted in the intent-to-treat analysis, which included all patients, whether they had RDC TD or not. Results indicated that risperidone at 6 mg/day had the most beneficial effect on TD, especially on the BLM syndrome, without inducing significant parkinsonism while treating psychotic symptoms. This antidyskinetic effect was greater than with either placebo or haloperidol. PMID- 7537287 TI - Randomized, double-blind, controlled trial of risperidone versus clozapine in patients with chronic schizophrenia. AB - This study compares the antipsychotic efficacy and the tolerability of risperidone and clozapine in patients with schizophrenia. Patients were randomized to double-blind treatment with risperidone, 4 mg (N = 20), risperidone, 8 mg (N = 19), or clozapine, 400 mg (N = 20), daily for 28 days. Efficacy was assessed by improvement of psychotic symptoms, measured on the Brief Psychiatric Rating Scale, and Clinical Global Impression. The tolerability was assessed by the Simpson and Angus scale for extrapyramidal side effects (EPS), the Association for Methodology and Documentation in Psychiatry (AMDP) scale for somatic side effects, spontaneous reports of adverse events, clinical laboratory assessments, and vital signs. All treatments reduced psychotic symptoms. The global tolerability was significantly better in the risperidone than in the clozapine-treated patients (p < 0.01). There were no differences between treatments on the AMDP scale. The most frequent spontaneously reported adverse effects were dizziness, fatigue, accommodation disturbance, and EPS in all treatment groups and increased salivation, mainly in the clozapine-treated patients. Although there were no changes in vital signs during risperidone treatment, clozapine was associated with a mean reduction in heart rate of 10 beats/minute. Risperidone tolerability at endpoint was classified as "very good" by 60 and 47% of patients treated with risperidone, 4 and 8 mg daily, respectively; the corresponding figure in clozapine-treated patients was 30%. The results suggest that risperidone is at least as effective as an antipsychotic as clozapine, providing a valuable new approach for the treatment of schizophrenia. PMID- 7537288 TI - Skilled nurses' experiences of caring. AB - The content and the meaning of nurses' caring in practice remain poorly illuminated. To contribute to a better understanding, the meaning of caring, abilities needed for caring, experiences that make caring worthwhile, and the source of the strength needed to continue caring were focused on in interviews with experienced nurses in surgical (n = 15), medical (n = 15), and long-term (n = 15) care. The analysis showed both similarities and differences between the three groups of nurses. One similarity lay in the ability of the nurses within their caring role to understand the situation, to establish contact with the patient, and to act in the patient's best interest especially regarding the relief of pain and anxiety. On the other hand, one of the differences was that the nurses engaged in long-term care emphasized support of the patients' psychosocial needs more than nurses engaged in surgical and medical care, who emphasized support of the patients' weakened vital functions. The analysis also disclosed that almost all of the nurses had been troubled by problems of patient care when they were new to their jobs, but experience and maturity had given them strength. Care in itself gave the nurses satisfaction and pleasure and made it worthwhile. Positive cooperation with coworkers and patients' families were the main sources of their strength to continue caring. PMID- 7537290 TI - The care of dying patients: a position statement from the American Geriatrics Society. AGS Ethics Committee. PMID- 7537292 TI - Ultrastructural localization of ras-related proteins using epitope-tagged plasmids. AB - To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy. For ultrastructural localization of these tagged proteins, a method was devised to process microinjected cells in situ into low-temperature resin. The proteins were localized on the sections using 9E10 detected with colloidal gold conjugates. Ha ras protein was localized almost exclusively on the cell membranes. Rho-A and rho C were predominantly associated with the submembraneous actin network, and rho-B was found in association with multivesicular bodies. Rac1 protein induces the formation of large pinocytotic vesicles and was detected on the cytoplasmic face of these vacuoles. These experiments demonstrate the successful use of this approach for detection of de novo synthesized proteins from microinjected plasmids by both light and electron microscopy on a small (< 50 cells) sample size. PMID- 7537293 TI - In vivo tolerance induction and associated cytokine production by subsets of murine CD4+ T cells. AB - Male BXSB mice develop lupus-like disease and die early in life (4 to 5 mo) whereas female mice do not. Others have demonstrated that CD4+ cells from male mice support B cell resistance to tolerance induction to human gamma-globulin (HGG). In this study, male and female mice tolerized at 2 mo of age with deaggregated HGG and subsequently immunized with HGG in comparison with mice immunized only were tested for anti-HGG Ab responses. CD4+ cells from draining lymph nodes of these mice were tested in culture for proliferation and production of cytokine mRNA and protein in response to HGG plus APC. Tolerized male but not female mice produced anti-HGG Abs of both the IgG1 and IgG2a isotypes. HGG stimulated CD4+ cells from immunized male and female mice that were not tolerized produced IL-2, IL-4, IL-5, IFN-gamma, and TNF-beta mRNA as well as IL-2 and IL-4 protein, whereas tolerized, immunized mice of both sexes failed to proliferate or produce either IL-2 or IL-4 or express any cytokine mRNA in response to HGG in vitro. A resistance in tolerance induction in male mice, as determined by anti HGG Abs, was also observed at 3 mo of age. Although a resistance to tolerance was also seen in terms of proliferation in the 3-mo-old males, production of IL-2 or IL-4 protein was still not observed. Thus, all T cell subsets identified by cytokine expression profiles were tolerized not only from females but also from males, of which the latter appeared to show some resistance to tolerance induction. PMID- 7537294 TI - Antigen-driven differentiation of naive Ig-transgenic B cells in vitro. AB - We have established a culture system in which naive B cells bearing a transgenic, chicken OVA (cOVA)-specific Ig differentiate to plasma cells in vitro after interaction with cOVA plus cOVA-specific helper T cells. B cell-enriched populations from Ig-transgenic mice, but not from nontransgenic mice, proliferated after presenting nanomolar concentrations of cross-linked cOVA to DO11.10 (cOVA plus IAd-specific) T cells. After 6 to 9 days of culture with Ag and specific T cells, the B cells acquired a plasma cell phenotype and secreted the transgene-derived Ig at high levels. Engagement of B cell surface Ig was not essential for primary B cell differentiation. Differentiating B cells enlarged, clustered, and acquired two plasma cell markers, Syndecan and CD43. B cell CD45 isoform expression changed: the B220 isoform was lost in a T cell-dependent manner, whereas the CD45RB isoform was gained in a T-independent manner. Although unstimulated B cells survived less than 72 h in vitro, those in Ag-stimulated cultures showed reduced early death, a surge of proliferation at 3 to 5 days, and increased death late in the culture. Using a large population of naive B cells of defined antigenic specificity permits us to study a primary immune response to an Ag, rather than to less physiologic polyclonal stimuli. Because all steps of differentiation occurred in vitro, they are easily accessible for study. This coculture system provides an opportunity to observe Ag-specific T cell-B cell collaboration. PMID- 7537289 TI - Good care of dying patients: the alternative to physician-assisted suicide and euthanasia. PMID- 7537291 TI - [In vitro fertilization neonates transferred to neonatal medicine. Mid-and long term outcome in 99 families]. AB - OBJECTIVE: To assess the physical and neurodevelopmental status of children born after in vitro fertilization (IVF) as well as the family condition. DESIGN: Follow-up study including physical and developmental examination in available families, and/or interviews of parents/pediatricians/teachers/social workers. SETTING: The Port-Royal neonatal intensive care unit and follow-up clinic. SUBJECTS: From 1.1.1987 to 31.12.1992, in 32 single, 71 twin, 28 triple, and one quadruple pregnancies resulting from IVF (total 132), one or more neonates were admitted to the Port-Royal NICU. The outcome is presented for the 99 families followed longer than two years (199 neonates, 166/199 born prematurely). MAIN OUTCOME MEASURES: Neurological sequelae (minor, moderate, major), developmental quotients, school level, behavioral disturbances, family conditions. RESULTS: From 21 singletons (2 deaths, 1 lost), 18 survivors were followed: 17 were normal above 2 years (10 above 3 years went to school), 1 had a severe cerebral palsy with low IQ. From 57 twin pregnancies (7 fetal and 9 neonatal deaths), the 98 survivors were followed: 78 were normal above 2 years (65 above 3 years went to school), 20 had some neuro-developmental deficits (of 14 above 3 years, 12 went to school); 25 families had had or still had problems (deaths and/or anomalies (9 cases) and/or sequelae); 3 mothers were single parents (2 divorces and one split). From 20 triple pregnancies (1 fetal and 3 neonatal deaths), 54 out of 56 survivors were normal above 2 years (33 above 3 years went to school); 3 siblings (1 deaf and 1 with cataract) were put in foster care after severe maternal beating; 2 mothers were single parents (one divorce and one split); 4 mothers had repeated breakdowns; one father tried suicide. From one quadruple pregnancy (one fetal and one neonatal deaths), the 2 survivors were normal above 3 years and went to school. Most sequelae involved very low birthweight babies, in association with a number of sleep and behavioural disturbances. From 98 mothers, 16 had 18 spontaneous pregnancies (11 full-term newborns, 5 miscarriages, 1 extra uterine pregnancy, 1 abortion) and 3 mothers had another IVF pregnancy (a few others had IVF failures). PMID- 7537297 TI - In CD8+ T cell-deficient lpr/lpr mice, CD4+B220+ and CD4+B220- T cells replace B220+ double-negative T cells as the predominant populations in enlarged lymph nodes. AB - Mice homozygous for lpr or gld develop autoimmunity and progressive lymphoproliferative disease characterized by the accumulation of two unusual populations of B220+ TCR-alpha beta+ T cells, a predominant CD4-CD8- double negative (DN) subset and a minor CD4dull+ subset. B220+ DN T cells appear to be derived from negatively selected thymocytes, but their immediate precursors have not been identified conclusively, and their relationship to CD4+B220+ T cells is unclear. Our previous studies of lpr and gld mice treated chronically with anti CD8 mAb provided evidence that the majority of B220+ DN T cells are unrelated to CD4+B220+ T cells and may be descended from peripheral thymus-derived CD8+ T cells. To investigate the contributions of MHC class I-selected thymus-derived T cells to the production of B220+ DN T cells and to the accumulation of CD4+ T cell subsets, we studied C3H-lpr and -gld mice rendered deficient in CD8+ T cells by the introduction of disrupted beta 2-microglobulin (beta 2-m) genes. These CD8+ T cell-deficient mice developed massively enlarged lymph nodes, in which CD4+B220+ T cells and CD4+ T cells replaced B220+ DN T cells as the dominant T cell subsets. As a population, the CD4+B220+ T cells were depleted of autoreactive populations specific for endogenous retroviral superantigens and were enriched for V beta 8.3+ T cells. The deficiency of CD8+ T cells in beta 2 m(-/-)-lpr mice had no effects on the accumulation of primed CD4+ T cells or autoreactive B cells. The selective reduction in B220+ DN T cells and corresponding accumulation of CD4+B220+ T cells in beta 2-m(-/-)-lpr mice provide strong evidence that 1) the majority of B220+ DN T cells are unrelated to CD4+ T cells and their development and/or accumulation is dependent on MHC class I expression; and 2) CD4+B220+ T cells are a remarkably similar, but separate, lineage of cells that develop independently of thymus-derived CD8+ T cells and class I MHC expression. PMID- 7537295 TI - IL-12 directly enhances in vitro murine erythropoiesis in combination with IL-4 and stem cell factor. AB - It has been demonstrated recently that in vivo administration of murine IL-12 to mice enhances the activity of cytotoxic NK cells and lymphocyte-activated killer cells, and that it has antitumor and antimetastatic activity. However, one side effect observed in response to systemic IL-12 treatment is anemia. In the present study, we examined for the first time the ability of IL-12 to affect directly the growth of murine erythroid progenitor cells in vitro. Whereas IL-12 alone or in combination with Erythropoietin (Epo) showed no stimulatory effect on erythroid progenitors, IL-12 potently enhanced the number of erythroid burst-forming unit (BFU-E) colonies formed in response to Epo+IL-4 by 63% and Epo+stem cell factor by 80%. The stimulatory effect of IL-12 occurred in a concentration-dependent fashion, with maximum enhancing effect observed at 50 ng/ml. Furthermore, single cell experiments suggested that the stimulatory effect of IL-12 on erythroid colony formation was directly mediated. Thus, IL-12 can directly enhance murine erythropoiesis in vitro, suggesting that IL-12-induced anemia is mediated through an indirect mechanism. PMID- 7537296 TI - T lymphocyte locomotion in a three-dimensional collagen matrix. Expression and function of cell adhesion molecules. AB - T cell locomotion within the extracellular matrix may be mediated by cell adhesion molecules. We investigated the expression and function of beta 1- and beta 2-integrins and CD44 on human peripheral CD4+ and CD8+ lymphocytes locomoting in a 3-D type I collagen matrix. Paths of randomly selected T cells were digitized from time-lapse videorecordings and were quantitatively analyzed. After the blocking of CD49b with mAb Gi9, the locomotion of a defined locomotor subset (50% of spontaneously locomoting cells) was inhibited. Anti-CD49d mAb HP2/1 and an activating anti-CD44 mAb (J173), respectively, induced transient recruitment (< 1 h) of previously nonmotile cells (10 to 35%). In contrast to the J173-induced short-term locomotion, hyaluronan incorporated within the matrix promoted locomotion for > 2 h. No significant effects were present for anti-CD49f (GoH3) and -CD11a (25.3) mAbs. After the addition of IL-8 to the matrix, rapid induction of locomotion in 20 to 30% of the cells (control) was evident, which was virtually abolished by anti-alpha 2- and alpha 6-integrin, and -CD11a mAbs. Thus, the locomotion of nonactivated and IL-8-activated T cells may involve different sets of integrins. Using flow cytometry, the development of a CD49b+CD29highCD44lowL-selectinlow T cell phenotype independent of activation markers including CD25, CD27, CD28, VLA-4, and CD45RA- to CD45RO-transition was observed after 4 days in the matrix. The initial development of spontaneous locomotion in the collagen matrix, however, was not accompanied by alterations in CAM surface staining and, therefore, may involve functional CAM activation rather than involving an increase in surface expression. PMID- 7537298 TI - The CD40 ligand expressed by human B cells costimulates B cell responses. AB - The possibility that activated B cells might express a ligand for CD40 that was of functional importance for B cell responses was examined by using highly purified human peripheral blood B cells, as well as a variety of B lymphoblastoid cell lines and hybridomas. Following stimulation with the combination of a calcium ionophore and a phorbol ester, human B cells bound a soluble fusion protein containing the extracellular portion of CD40 and the Fc region of IgG1 (CD40.Ig). A variety of B cell lines and hybridomas also bound CD40.Ig, either constitutively or after activation. In addition, CD40.Ig specifically immunoprecipitated a 33-kDa glycoprotein from surface 125I-labeled activated B cells. The nucleotide sequence of the coding region of the CD40 ligand mRNA amplified by RT-PCR from activated T cells and B cell lines was identical. The CD40 ligand expressed on human B cells was important functionally because homotypic aggregation of CD40 ligand-expressing B cells was inhibited by the CD40.Ig construct. Additionally, RNA and DNA synthesis as well as Ig production by polyclonally activated, highly purified peripheral B cells and a variety of B cell lines were inhibited significantly by the CD40.Ig construct. Finally, B cell lines expressing the CD40 ligand induced Ig production from resting normal B cells in a CD40-dependent manner. These results indicate that human B cells express a ligand for CD40 that is identical with that expressed by activated T cells and that the B cell-expressed CD40 ligand plays an important role in facilitating responses of activated B cells. PMID- 7537299 TI - Lytic reaction of in vivo primed peritoneal exudate CTL. Induction of high conductance single channels in the target cell membrane. AB - CTL, primary effectors in immune responses, deliver a "lethal hit" signal to target cells, causing their destruction. The precise membrane events associated with the lethal hit remain elusive. We investigated the signal(s) mediating destruction of tumor target cells (EL4) by perforin-deficient peritoneal exudate CTL (PEL). We utilized patch clamp techniques to record electrophysiological events associated with the cytolytic interaction of PEL and EL4 in isolated conjugates. PEL-EL4 interaction resulted in induction in EL4 cells, of single channels (followed by EL4 destruction), with a mean conductance of 437 pS and a reversal potential of -1.0 mV, suggestive of nonselective pathways. Similar channels were induced in EL4 cells conjugated with perforin-rich PEL blasts (PEB), by perforin, postnuclear extract from PEL (pnPEL) and from other cytotoxic lymphocytes, but not from noncytolytic lymphocytes. As similar channels were induced by pnPEL in EL4 membrane patches, we propose that these channels result from a direct effect of PEL-derived channel-forming substance(s) on the target cell's membrane. Importantly, postnuclear extracts from perforin-devoid cytotoxic PEL-hybridomas induced similar channels, suggesting the presence of a nonperforin, channel-forming activity in PEL and PEL-hybridomas. Based on the present study, we conclude that the delivery of the lethal hit by cytolytic PEL and PEL-hybridoma is associated with induction in the target cell of high conductance channels, which most likely mediate its destruction. We propose that these channels are related to the Fas pathway of lymphocytotoxicity. PMID- 7537300 TI - Up-regulation of recombination activating gene expression by signal transduction through the surface Ig receptor. AB - Recent evidence has demonstrated that a signal transduced through the lymphocyte Ag receptor may regulate the expression of the recombination activating genes (RAG). Although several groups have shown that such a signal may be required to down-regulate RAG-1 and RAG-2 after a functional Ag receptor has been generated in early mature T or B cells, recently it has been suggested that under some circumstances, cross-linking the B cell Ag receptor may result in up-regulation of RAG expression. To study this possibility, we used a unique set of cell variants isolated from a human mature B cell line, which differ in their expression of both the surface Ig receptor (sIg) and RAG-1 and RAG-2 genes. Two forms of stimulation were employed to generate a signal; either a soluble F(ab')2 anti-mu fragment or the combination of PMA and ionomycin. Northern blot analysis demonstrated that RAG-1 mRNA levels were increased in sIg+ variants after cross linking with anti-mu. Increases were also observed in all variants after stimulation with PMA and ionomycin. Further analysis of cross-linked sIg+ variants suggests that the observed up-regulation in RAG expression was a reversible event. Furthermore, we have determined that both increased transcription and transcript stabilization contributed to this inducible up regulation. We thus describe a mature B cell line in which RAG expression is up regulated after sIg cross-linking. This finding is discussed in the context of its potential role in situations where sIg+ B cells may undergo secondary rearrangements for the purpose of "editing" their sIg receptors. PMID- 7537301 TI - Dissociation of beta 2-microglobulin from HLA class I heavy chains correlates with acquisition of epitopes in the cytoplasmic tail. AB - A rabbit antiserum "ABR2" was raised against a peptide with sequence identity to 10 amino acids of the cytoplasmic tail of HLA class I heavy chains. Western blotting and immunoprecipitation analyses demonstrate that ABR2 reacts with HLA class I heavy chains. The antiserum reacts poorly with beta 2-microglobulin (beta 2-m)-associated heavy chains and reacts strongly with free heavy chains. ABR2 reacts with immature heavy chains from the endoplasmic reticulum that have yet to bind beta 2-m and mature heavy chains that have dissociated from beta 2-m at the plasma membrane. Comparison with HC10, a mAb that recognizes an epitope defined by polymorphism at residue 62 of the alpha 1 helix of free HLA class I heavy chains, shows that ABR2 reacts with overlapping populations of free heavy chains (for those allotypes that react with both Abs), but it also identifies populations that bind to one Ab and not the other. ABR2 induces dissociation of beta 2-m from HLA-B38 molecules expressed by the human B cell line "TEM," a phenomenon not detected with other allotypes or with the same allotype in a different cell line. This study shows that association of beta 2-m with the extracellular domains of HLA class I heavy chains can cause a change in the cytoplasmic tail that prevents binding of Abs present in the ABR2 antiserum. Similar findings have been made for mouse H-2 class I molecules, which suggests that this is a general property of class I MHC molecules. PMID- 7537302 TI - Myelin basic protein peptide complexes with the class II MHC molecules I-Au and I Ak form and dissociate rapidly at neutral pH. AB - The acetylated N-terminal peptide of myelin basic protein (MBP) is the immunodominant T cell epitope in the induction of experimental autoimmune encephalomyelitis in the I-Au- and I-Ak-expressing mouse strains. We used a direct binding assay to examine the kinetics of binding and dissociation of a series of MBP peptide analogues with the affinity-purified class II MHC molecules I-Au and I-Ak. We observe much faster in vitro rates of binding and dissociation than has been reported previously for other immunogenic peptides at neutral pH. The kinetics also reveal inactivation of the peptide-free class II MHC molecules. These results are consistent with previously proposed mechanisms for tolerance escape and autoimmune disease. PMID- 7537303 TI - Effect of amino acid substitutions within the region 62-76 of I-A beta b on binding with and antigen presentation of Torpedo acetylcholine receptor alpha chain peptide 146-162. AB - Previous study has shown that reduced T cell response to peptide alpha 146-162 of Torpedo californica acetylcholine receptor (tAChR) in B6.C-H-2bm12 (bm12) mice, a mutant of C57BL/6 (B6) mice, correlated with its nonsusceptiblity to experimental autoimmune myasthenia gravis. There are three amino acid differences between the I-A beta b of the two strains (positions 67, 70, and 71). We synthesized peptides I-A beta b62-76 (peptide b6), I-A beta bm1262-76 (peptide bm), and three additional peptides, b6(67F), b6(70Q), and b6(71K), and determined their ability to bind peptide alpha 146-162 and the dissociation constants (Kd) of the binding. Peptide alpha 146-162 bound with a significantly higher affinity to peptide b6 than to peptides bm or b6(71K), suggesting that the lower affinity of peptide alpha 146-162 to I-Abm12 is a factor in the reduced response to this peptide by bm12 T cells. This was confirmed by measurement of the Kd values of the binding of peptide alpha 146-162 to the I-A molecules of B6 and bm12. Furthermore, APC of bm12 presented the peptide, or tAChR, poorly to peptide-specific or to tAChR specific B6 T cells. The major effect is caused by the change of Thr-71 in I-A beta b to lysine in I-A beta bm12. However, APC of B6 also presented peptide alpha 146-162 much less efficiently to peptide-specific T cells of bm12. This demonstrated that these three amino acid changes also influence the T cell receptor recognition of peptide-MHC complex and that both B6 and bm12 T cells recognizing peptide alpha 146-162 or tAChR are under a high H-2 restriction. PMID- 7537304 TI - Alpha 4 integrin directs virus-activated CD8+ T cells to sites of infection. AB - This article examines the role of VLA-4 in directing lymphocytes to sites of viral infection using the murine lymphocytic choriomeningitis virus infection (LCMV) as the model system. This virus by itself induces little or no inflammation, but in most mouse/virus strain combinations a potent T cell response is induced, which is associated with marked CD8+ cell-mediated inflammation. Two expressions of LCMV-induced inflammation were studied: meningitis induced by intracerebral infection and adoptive transfer of virus specific delayed-type hypersensitivity. Our previous studies have shown that LCMV infection results in the appearance of activated CD8+ cells with an increased expression of VLA-4. In this study we have compared various T cell high and low responder situations, and these experiments revealed that acute inflammation correlates directly with VLA-4 expression on splenic CD8+ cells. This correlation could be extended to CD4+ and B cells in chronically infected low responder DBA/2 mice. The vascular ligand for VLA-4, VCAM-1, was found to be up-regulated on endothelial cells in sites of inflammation. Finally, preincubation of virus primed donor cells with mAb to VLA-4 completely blocked the ability to transfer virus-specific, delayed-type hypersensitivity when the donor cells were given i.v., but not when the cells were injected directly into the test site. Co transfer of CD8-depleted cells with anti-VLA-4-blocked cells did not reveal any cooperation. Taken together, these results indicate that VLA-4 play a critical role in lymphocyte homing during systemic virus infections and are involved in directing virus-specific CD8+ effector cells to sites of infection. PMID- 7537305 TI - Development of antimalaria immunity in mice lacking IFN-gamma receptor. AB - IFN-gamma receptor deficient (IFN-gamma R-/-) mice, immunized with different developmental stages of malaria parasites, were used to define the mechanisms of protection against the various stages of this infection. IFN-gamma R-/- mice failed to develop protective immunity against Plasmodium yoelii sporozoites or liver stages, upon immunization with a single dose of irradiated sporozoites, whereas in immunized wild-type mice, parasite development was strongly inhibited. Immunized wild-type mice expressed high levels of inducible nitric oxide synthase (iNOS) mRNA in their liver, upon challenge with viable sporozoites, whereas only background levels of iNOS were detected in immunized IFN-gamma R-/- mice. In contrast, after immunization with multiple doses of irradiated sporozoites, both IFN-gamma R-/- and wild-type mice mounted an immune response, which strongly inhibited the development of liver stage parasites. In both types of mice, protection occurred in the absence of appreciable expression of liver iNOS mRNA. As for the course of the erythrocytic phase of infection by nonlethal malaria species, P. yoelii yoelii and P. chabaudi adami, we observed only a moderately prolonged parasitemia in IFN-gamma R-/- mice compared with wild-type mice, indicating that IFN-gamma may only play a modest role in immunity against erythrocytic stages. These results indicate that IFN-gamma is the main mediator of the protective mechanism that develops first upon immunization with sporozoites. However, the nature of the anti-parasite mechanism(s) changes in the course of immunization, so that multiple immunizing doses elicit additional protective mechanisms, which are independent of IFN-gamma and its receptor. PMID- 7537306 TI - Lymph node (but not spleen) invasion by murine lymphoma is both CD44- and hyaluronate-dependent. AB - Similar to activated T cells, LB T cell lymphoma expresses the CD44 cell surface Ag. In addition, the vast majority of LB cells also express the beta 2 (CD18) and alpha L (CD11a) chains of LFA-1 integrin. In view of the finding that anti-CD18 mAb blocked spleen, but not lymph node invasion by LB cells inoculated s.c. into BALB/c mice, we tested the ability of anti-CD44 mAb (IM 7.8.1) to block the infiltration of LB cells into the lymph nodes. We found that, as opposed to anti CD18 mAb, anti-CD44 mAb, as well as its F(ab')2 or Fab fragment, prevented lymph node infiltration but had no effect on spleen invasion. This conclusion was based on histologic examination and [3H]thymidine incorporation into proliferating LB cells invading the lymphoid organs. Histologic analysis further demonstrated that LB cells invade the lymph node via the afferent lymphatics. The surface expression of CD44 molecules on LB cells was enhanced after PMA activation. PMA activation also enabled in vitro binding of the lymphoma to hyaluronic acid (HA), a known ligand of CD44. Because anti-CD44 mAb, its F(ab')2 or Fab fragment, and hyaluronidase blocked this binding, we also tested the ability of the enzyme to inhibit lymph node invasion by LB cells. We established through histologic examination and [3H]thymidine incorporation that hyaluronidase protected the lymph node, but not the spleen, from invasion by the lymphoma. PMID- 7537307 TI - Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions. AB - Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewisa (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca(2+)-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions. PMID- 7537308 TI - Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection. AB - Transplants performed between phylogenetically disparate species are subject to hyperacute rejection initiated by binding of xenoreactive natural Abs to endothelium in the donor organ. Binding of these Abs activates complement, leading to tissue injury and destruction of the graft. Human xenoreactive natural Abs recognize Gal alpha 1-3Gal beta 1-4GlcNAc (galactose alpha 1-3galactose beta 1-4-N-acetylglucosame); however, the relative importance of this Ag in graft rejection has not been proved. The present study was conducted to test the potential importance of alpha-galactosyl (alpha-Gal) determinants in the pathogenesis of hyperacute rejection. To this end, hearts (n = 3) from New World monkeys (Saimiri scureus, squirrel monkey), which can synthesize Gal alpha 1 3Gal, were transplanted heterotopically into Old World monkeys (Papio species, baboon), which do not synthesize Gal alpha 1-3Gal determinants and which have circulating anti-alpha Gal Abs. The xenografts were rejected in 51 to 56 min (mean +/- SD = 53.3 +/- 2.5), results similar to those observed in porcine grafts transplanted into baboons. Histologic analysis of the hearts revealed thrombosis and intraparenchymal hemorrhage and immune deposits consisting of IgM, Clq, C3, C4, C5b, and the membrane attack complex, but not properdin or factor B of the recipient deposited on graft endothelium. Sera obtained from baboons after perfusion of squirrel monkey kidneys revealed depletion of alpha-Gal-specific Abs and anti-pig endothelial cell Abs. These findings provide strong evidence that the Abs that accumulate in New World monkey organs during perfusion with baboon blood are the same Abs that would accumulate in a porcine organ transplanted into a primate and suggest that hyperacute rejection is not necessarily a reflection of phylogenetic distance but that the expression of terminal alpha-Gal residues provides an adequate target to initiate that process. PMID- 7537309 TI - Endogenous macrophage CSF production is associated with viral replication in HIV 1-infected human monocyte-derived macrophages. AB - Human monocyte-derived macrophages (MDM) cultured in medium containing macrophage (M) CSF are more susceptible to infection with HIV-1. M-CSF increases the frequency with which MDM become infected, the level of HIV mRNA expressed per infected cell, and the level of proviral DNA expressed per infected culture. Because of these effects of M-CSF on HIV-1 replication and the reported function of this factor as a survival and differentiation factor for human monocytes, we investigated whether HIV-1 could induce endogenous M-CSF production by MDM and the potential role of endogenous M-CSF on HIV-1 infection in these cells. MDM infected with HIV and maintained in the absence of exogenous M-CSF produced this cytokine endogenously at levels 5- to 24-fold higher than uninfected cells. In contrast, the proinflammatory cytokines IL-1, IL-6, and TNF-alpha and the growth factor granulocyte-macrophage CSF were not detected. The kinetics of M-CSF production following infection paralleled the kinetics of virus replication. Furthermore, enhanced production of M-CSF was dependent on viral entry and active replication of HIV-1. Thus, endogenous M-CSF production may contribute to the survival of HIV-infected MDM, enable them to function as a reservoir for HIV, and facilitate the spread of virus in vivo. PMID- 7537310 TI - Myelin oligodendrocyte glycoprotein induces a demyelinating encephalomyelitis resembling multiple sclerosis. AB - Chronic relapsing experimental autoimmune encephalomyelitis, a demyelinating disease induced by injection of central nervous system (CNS) tissue, is widely used as a model for multiple sclerosis. However, it is unclear which Ag or combination of Ags in the CNS induce the demyelinating immune response. We now show in Lewis rats that a single injection of myelin oligodendrocyte glycoprotein, a specific CNS myelin component, or an appropriately derived myelin oligodendrocyte glycoprotein peptide produces a relapsing remitting neurologic disease with extensive plaque-like demyelination. Igs from affected animals reacted specifically with myelin oligodendrocyte glycoprotein and stimulated a myelin protease activity, leading to myelin basic protein degradation. The demonstrated involvement of myelin oligodendrocyte glycoprotein as a new demyelinating neural Ag may provide a deeper insight into the pathogenesis of multiple sclerosis and its treatment. PMID- 7537311 TI - Denaturing and non-denaturing gel electrophoresis as methods for the detection of junctional diversity in rearranged T cell receptor sequences. AB - Two nucleic acid gel electrophoresis techniques were tested as a possible tool for analyzing junctional diversity in rearranged T cell receptor (TcR) sequences in order to define the extent of T cell heterogeneity. For this purpose denaturing gradient gel electrophoresis (DGGE) as well as non-denaturing gel electrophoresis (nDGE) techniques have been studied. Detection of junctional diversity is based on mobility shifts, caused by nucleotide sequence polymorphism, of polymerase chain reaction amplified rearranged TcR sequences. DGGE as well as nDGE procedures are suitable for the detection of junctional diversity in TcR V gene family sequences based on sequence dependent separation. Compared to DGGE, nDGE of DNA is a relatively simple and rapid procedure, with a high separation potential. nDGE permits separation of double stranded (homoduplexes) and/or single stranded DNA molecules of the majority of TcR chain encoding sequences. Formation and detection of unique heteroduplex molecules combined with single stranded DNA molecule analysis in nDGE permits the recognition of the remaining sequences, thus providing additional information on the degree of T cell heterogeneity. In conclusion, these nucleic acid gel electrophoresis techniques allow a direct assessment of the heterogeneity and clonality of T cell populations by the detection of junctional diversity in TcR chain encoding sequences. This analysis can be performed without the need of cell propagation and/or cellular cloning procedures, thereby eliminating the risk of introducing technical artefacts. PMID- 7537313 TI - An enzyme-linked immunosorbent assay to identify inhibitors of activation of platelet integrin alpha IIb beta 3. AB - The affinity of integrin alpha IIb beta 3 for adhesive ligands is tightly regulated by the platelet such that fibrinogen binding is observed only after platelet activation. Ligand binding is necessary for platelet aggregation, which contributes to vascular occlusion in pathological states. Therefore, we have developed an ELISA assay to screen for compounds that inhibit alpha IIb beta 3 activation. Washed platelets were incubated in microtitre wells with potential inhibitory compounds and stimulated with an agonist to activate alpha IIb beta 3. After the addition of biotin-PAC1, a fibrinogen-mimetic monoclonal antibody, the activation state of alpha IIb beta 3 was measured by sedimenting the platelets and quantitating the residual biotin-PAC1 in the cell-free supernatant in a streptavidin-based ELISA. This assay detected (1) specific PAC1 binding to activated platelets in response to a variety of agonists, and (2) dose-dependent inhibition of PAC1 binding by function-blocking anti-alpha IIb beta 3 monoclonal antibodies, by the tetrapeptide, RGDS, and by an alpha IIb beta 3-selective RGD peptidomimetic. Furthermore, the assay detected inhibition of PAC1 binding by intracellular inhibitors of platelet activation, including bisindolylmaleimide, a selective protein kinase C antagonist, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These studies demonstrate that this integrin activation ELISA can detect pharmacological blockade of platelet alpha IIb beta 3 by extracellular and intracellular inhibitors. Its use may facilitate the search for clinically useful anti-platelet drugs. PMID- 7537312 TI - Quantification of antigen specific CD8+ T cells using an ELISPOT assay. AB - An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h. PMID- 7537314 TI - Hydrocoating: a new method for coupling biomolecules to solid phases. AB - Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique--hydrocoating--for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays. PMID- 7537315 TI - [Transurethral incision of the prostate: an objective and subjective evaluation of long-term efficacy]. AB - The emergence of less invasive therapies has demanded the reassessment of surgical procedures for the treatment of benign prostatic hyperplasia. This study was designed to evaluate the long term efficacy of transurethral incision of the prostate (TUIP) using objective (urodynamic), and subjective (symptom score and assessment of satisfaction) parameters, and investigate sexual function. Forty one men after TUIP (mean age of 63.4 years) were reviewed, with a mean follow up of 53 months (range 12-96). Pre operative symptom score (based on Madsen-Iversen score) and urodynamic evaluation were compared to recent post TUIP symptom score, urodynamic evaluation, and satisfaction interview. Total symptom score, as well as obstructive and irritative components, significantly decreased after TUIP (p < 0.0001). Mean detrusor pressure at peak flow decreased from 85 to 44 cm H2O (p < 0.0001), and mean maximal detrusor pressure decreased from 114 to 55 cm H2O (p < 0.0001). Mean peak uroflow rates increased from 10.3 to 15.3 cc/sec (p = 0.019). Thirty two of the men (82%) reported long term improvement after TUIP, with an overall satisfaction rate of 67% (range 0-100). Regardless of objective urodynamic criteria (indicating obstruction or relief of obstruction), the number of men reporting subjective improvement, and the degree of improvement, were similar. Only 4 men (11%) reported new retrograde ejaculation. The proportion of men improved after TUIP compares favorably to long term data available on TURP. Assessing the degree of improvement (overall satisfaction) is unique and has not been previously reported. These results clearly demonstrate that, in selected patients, TUIP is an effective procedure for long term relief of outlet obstruction. PMID- 7537316 TI - Outcomes of epidural morphine treatment in cancer pain: nine years of clinical experience. AB - The outcome of epidural morphine therapy is described in 146 consecutive cancer patients who were treated by a community hospital-based pain service. The routine procedure used a standard epidural catheter that was tunneled subcutaneously. One hundred and twenty-one patients improved and stayed on lifelong or chronic epidural opioids. Mean treatment time was 92 days (median, 47; range, 2-2040); 49% of the time was spent as outpatients. Twenty-five patients failed to respond to the treatment. The oral daily morphine-equivalent dose prior to inclusion was 164 mg. The mean daily epidural start dose of morphine was 18 mg (range, 6-120), and the mean daily dose at termination was 69 mg (range, 2-540). The dose escalations, described as the ratio of the maximum dose to the minimum maintenance start dose, were moderate, with a mean of 4.1 (median, 2.5), which corresponded to a percent increase of 5.1 (median, 2.7) per patient per day. Lack of effect due to the character of the original symptoms or progression of pain was the main reason for withdrawal from epidural opioid therapy (N = 27), followed by catheter-related problems (N = 9) and drug-related complications (N = 5). Also due to drug-related complications, epidural morphine therapy was changed to buprenorphine or methadone in 19 patients. Adjuvant systemic opioids were given to ten patients and epidural local anesthetics were administered to 17 of the subjects. Neuropathic pain, certain visceral pain types, incident pain on movement, and pain from cutaneous ulcerations were characteristics of poor responders.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537317 TI - Unmet analgesic needs in cancer patients. AB - This study was designed to characterize unmet analgesic needs in a group of cancer patients. One hundred one inpatients were surveyed to document pain prevalence and intensity, satisfaction with pain control and factors associated with unmet analgesic needs during the week prior to admission. Forty-four percent had unmet analgesic needs, defined as pain of moderate or greater than moderate intensity. These patients had a significantly greater prevalence of bone and axillary node involvement, higher than pain intensity, and less satisfaction with pain control. Fifty-nine percent were satisfied with pain control. Dissatisfaction was associated with higher usual pain intensity, less satisfaction with physician information about pain etiology, less frequent attribution of pain to disease, sharp pain, constant and intermittent pain, patient perceptions of less physician concern about pain, patient and physician belief that the patient is receiving insufficient analgesic, and patient wanting more control over analgesic regimen. Patients may benefit from improved treatment of specific syndromes and by attention to communication. PMID- 7537318 TI - Psychosocial issues in palliative care: the patient, the family, and the process and outcome of care. AB - This article presents a synopsis of the psychosocial needs of patients and families in the terminal phase of malignant disease, as well as approaches to auditing the care of these clients. Recommendations for future research from the National Cancer Institute of Canada Workshop on Symptom Control and Supportive Care in Patients with Advanced Cancer are presented. These include recommendations on the need for more effective tools to measure the symptoms and burden of illness; an improved taxonomy to describe terminal illness; and studies to (1) measure the impact of interventions for improved symptom relief on the psychosocial distress of patients and families, (2) determine the impact of psychosocial interventions on the symptomatology associated with terminal illness, (3) operationalize "good palliative care" and the optimal delivery of palliative care, (4) assess the auditing of palliative care, (5) develop family typologies of adaptation to terminal illness, (6) describe high risk families, and (7) measure the impact of staff support programs on improving patient/family care. PMID- 7537320 TI - Assessing pain knowledge. PMID- 7537319 TI - Transdermal fentanyl in cancer pain: conversion from oral morphine. PMID- 7537322 TI - [Experimental and clinical study on chemotherapy for cervical adenocarcinoma]. AB - We examined the effectiveness of 5 anticancer agents in nude mice transplanted with 4 kinds of uterine cervical adenocarcinoma. MMC, CDDP, ACM, ACD and VP-16 were selected. The therapeutic effects were assessed in terms of growth inhibition rates and histological effects in the transplanted tumors of nude mice. From this study, the following results were obtained: In single agent administration, MMC was effective in all tumors, and CDDP was effective in 3 kinds of tumors as indicated by the results showing growth inhibition rates. Histological findings for the agents did not correlate with therapeutic findings. In combined administration, MMC + CDDP was the most effective as indicated by the results showing growth inhibition rates; this combination showed an additive effect and MMC and CDDP were considered to be key drugs for treating cervical adenocarcinoma. On the basis of these results, we used combination chemotherapy with MMC, CDDP and ACM (PAM regimen) in high risk and advanced patients. We found an improved prognosis in advanced patients. There were one complete response and three partial responses in 7 advanced cases including one suffering recurrence. The combination therapy including MMC, CDDP and ACM was found to be effective against cervical adenocarcinoma. PMID- 7537321 TI - Lumbar neurolytic sympathetic blockades provide immediate and long-lasting improvement of painless walking distance and muscle metabolism in patients with severe peripheral vascular disease. AB - Thirty patients with angiographically proven peripheral vascular disease (PVD) and intermittent claudication were treated with percutaneous lumbar neurolytic sympathetic blockade (NSB) using 1.5 mL ethanol 95%. Claudication had been progressive in all patients during conservative treatment. Median (range) painless walking distance increased from 95 (10-200) meters (m) before to 355 (25 1003) m immediately after NSB. Further improvement was seen during the 1-year follow-up, with two exceptions: one patient lost a leg after acute arterial embolism and another patient deteriorated after 6 months. In the latter case, a second NSB improved the walking distance again. One case of transient mild neuralgia of the L3 dermatome occurred. 31P-magnetic resonance investigations of the calf muscles before, during, and after a treadmill exercise were performed in seven patients: 1 week after NSB, the postexercise recovery of phosphocreatine was accelerated in all patients compared to the pre-NSB values. The accelerated recovery suggests an improved post-ischemic metabolic situation after chemical sympathectomy. PMID- 7537325 TI - Human papillomavirus type 16 capsids expose multiple type-restricted and type common antigenic epitopes. AB - The study of viral infectivity and detection of viral capsid antigens of the major cervical cancer-associated human papillomavirus (HPV) type, HPV-16, requires knowledge of which epitopes are exposed in clinical specimens of infected tissue or on intact capsids. To define the antigenic epitopes of HPV-16, antisera to 66 overlapping synthetic peptides corresponding to the HPV-16 capsid proteins L1 and L2 and to seven peptide analogues were tested in immunoperoxidase stainings of consecutive sections from formalin-fixed, paraffin-embedded HPV infected tissue. Antisera against eleven different peptides from L1 and against seven different peptides from L2 recognized the HPV capsid antigen. Most epitopes were only found on the capsid antigen of certain genital HPV types, but four antigenic epitopes in L1 were detectable also in cutaneous wart specimens. All antigenic epitopes in L2 were restricted to genital HPV types and four L2 epitopes were only detectable in HPV-16 or HPV-33 positive specimens. The surface exposure of the antigenic epitopes was investigated by comparing the reactivity of the antipeptide antisera with intact or disrupted virions or capsids of HPV 11, HPV-16 and bovine papillomavirus (BPV). Twenty antipeptide sera from L1 and seven antipeptide sera from L2 were reactive with intact HPV-16 capsids at titres up to 1:146,000. Sixteen of these antisera were also reactive with disrupted HPV 16 capsids. Cross-reactivity with disrupted HPV-11 and BPV was detected for eleven and six antisera, respectively, whereas intact HPV-11 or BPV virions showed only weak cross-reactivity. In conclusion, the HPV-16 L1 and L2 capsid proteins contained multiple antigenic epitopes, most of which were shared with one or several additional HPV types. PMID- 7537323 TI - Transfer of graded potentials at the photoreceptor-interneuron synapse. AB - To characterize the transfer of graded potentials and the properties of the associated noise in the photoreceptor-interneuron synapse of the blowfly (Calliphora vicina) compound eye, we recorded voltage responses of photoreceptors (R1-6) and large monopolar cells (LMC) evoked by: (a) steps of light presented in the dark; (b) contrast steps; and (c) pseudorandomly modulated contrast stimuli at backgrounds covering 6 log intensity units. Additionally, we made recordings from photoreceptor axon terminals. Increased light adaptation gradually changed the synaptic signal transfer from low-pass to band-pass filtering. This was accompanied by decreased synaptic delay and increased contrast gain, but the overall synaptic gain and the intrinsic noise (i.e., transmission noise) were reduced. Based on these results, we describe a descriptive synaptic model, in which the kinetics of the tonic transmitter (histamine) release from the photoreceptor axon terminals change with mean photoreceptor depolarization. During signal transmission, tonic transmitter release is augmented by voltage dependent contrast-enhancing mechanisms in the photoreceptor axons that produce fast transients from the rising phases of the photoreceptor responses and add these enhanced voltages to the original photoreceptor responses. The model can predict the experimental findings and it agrees with the recently proposed theory of maximizing sensory information. PMID- 7537326 TI - Hepatitis C virus core region: helper T cell epitopes recognized by BALB/c and C57BL/6 mice. AB - In this study, we characterized the B cell and T cell responses to the hydrophilic portion of hepatitis C virus (HCV) core protein in two strains of mice and identified the respective antigen determinants. BALB/c (H-2d) and C57BL/6 (B6:H-2b) mice were immunized by a subcutaneous injection of recombinant HCV core protein together with Freund's complete adjuvant. The level of antibody production, as determined by ELISA, was consistently higher in BALB/c than in B6 mice. However, antibodies in sera from each strain bound to the N-terminal region of the core protein within amino acids 1 to 28 (MSTNPKPQRKIKRNTNRRPQDVKFPGGG), according to an experiment using non-overlapping peptides that covered the hydrophilic portion of HCV core protein. The T cell responses were also higher in BALB/c than in B6 mice with respect to the proliferative responses of the draining lymph node cells in vitro. By limiting dilution cultures of the draining lymph node cells in vitro repetitively stimulated with recombinant core protein, T cell clones were established from both strains of mice and characterized. The surface markers of these clones were Thy-1.2+, CD3+, TCR alpha beta+, CD4+ and CD8+. The proliferative responses were inhibited in the presence of anti-CD4 or anti-MHC class II monoclonal antibodies. The T cell lines in BALB/c mice recognized an epitope in HCV core at amino acids 72 to 91 (EGRAWAQPGYPWPLYGNEGL). The T cell lines in B6 mice recognized an epitope at amino acids 55 to 74 (RPQPRGRRQPIPKARQPEGR). Thus, mice with different MHC haplotypes recognized different non-overlapping T cell antigenic determinants of HCV core proteins. PMID- 7537324 TI - Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes. AB - One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na-gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges. PMID- 7537327 TI - Evaluating a ten questions screen for childhood disability: reliability and internal structure in different cultures. AB - This paper uses five strategies to evaluate the reliability and other measurement qualities of the Ten Questions screen for childhood disability. The screen was administered for 22,125 children, aged 2-9 years, in Bangladesh, Jamaica and Pakistan. The test-retest approach involving small sub-samples was useful for assessing reliability of overall screening results, but not of individual items with low prevalence. Alternative strategies focus on the internal consistency and structure of the screen as well as item analyses. They provide evidence of similar and comparable qualities of measurement in the three culturally divergent populations, indicating that the screen is likely to produce comparable data across cultures. One of the questions, however, correlates with the other questions differently in Jamaica, where it appears to "over-identify" children as seriously disabled. The methods and findings reported here have general applications for the design and evaluation of questionnaires for epidemiologic research, particularly when the goal is to gather comparable data in geographically and culturally diverse settings. PMID- 7537328 TI - The apical border plaque in severe periodontitis. An ultrastructural study. AB - This study concerns the apical border (AB) plaque in relation to severe forms of periodontitis (SP), including juvenile, post-juvenile, and rapidly progressing periodontitis. Twenty-four (24) teeth from 16 patients with SP were examined by transmission electron microscopy (TEM). The AB was not discrete, with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB the established plaque consisted of a layer of Gram-positive cocci and ghost cells and a superficial layer mainly of Gram-negative morphotypes, including cocci, rods, filaments, fusiforms, and spirochetes. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with ghost cells in abundance. Ruthenium red, alcian blue-lanthanum nitrate, and safranin O were used to label matrix polyanionic macromolecules, and periodic acid (thiosemicarbazide) silver proteinate for intracellular polysaccharide (IPS). The matrix components were mainly fibrillar. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall, and cytoplasmic IPS granules. The latter varied in distribution and were evident even in the most apically advanced intact microorganisms. The results indicate that IPS and some matrix features of the apical border plaque in severe periodontitis in certain aspects resemble those of sub-contact area plaque on children's teeth, in health or associated with early chronic gingivitis, and with those in chronic adult periodontitis. They also suggest the establishment of acidic regions in the microniche at the bottom of the periodontal pocket in the various forms of periodontitis differing in rate of progression. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in severe periodontitis, similar to those in chronic adult periodontitis. PMID- 7537330 TI - Communicating through art: experience from the WHL Art Competition. AB - The World Hypertension League (WHL) International Art Competition started in 1991. The idea was to raise public awareness regarding hypertension prevention and treatment through unconventional means: Art. The purpose of this paper is to summarise the experience the WHL gained during preparation and follow-up activities of the Competition with emphasis on communication processes. PMID- 7537329 TI - The influence of hypoxia on transvascular leakage in the isolated rat heart: quantitative and ultrastructural studies. AB - 1. The multiple indicator dilution method was used to study the transvascular movement of gamma-globulin, bovine serum albumin, insulin and cyanocobalamin in the isolated rat heart. 2. Perfusion of the heart with well-oxygenated solution for 75 min (constant flow) did not produce a significant change either in the total area under the dilution curve or the 'leakage index' (an arbitrary measure of transvascular flux) for all the tracers. 3. Perfusion of the heart with hypoxic solution produced a significant increase in leakage of gamma-globulin of 38.6 +/- 18, 48.5 +/- 17.6, 60.5 +/- 24 and 58 +/- 20% after 15, 30, 45 and 60 min, respectively, compared with the well-oxygenated equilibration period. Permeability- surface area products (PS) for the smaller diffusible solutes, therefore, could not be estimated. 4. The flux of albumin, insulin and cyanocobalamin in response to hypoxia was similar to that of gamma-globulin. 5. Ultrastructural examination of well-oxygenated hearts revealed that Monastral Blue-labelled albumin remained within the lumen and that endothelial integrity remained intact. 6. Conversely biopsies from hypoxic hearts showed that the labelled albumin had passed to the interstitium through gaps (approximately 3 microns) in venular endothelium. 7. The results showed that, in intact hearts, hypoxia produced gaps in the endothelium of venules and that these gaps could be the possible route for transvascular leakage of macromolecules. PMID- 7537331 TI - The C-32 triacetyl-L-rhamnose derivative of ascomycin: a potent, orally active macrolactone immunosuppressant. PMID- 7537332 TI - Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides. AB - Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists. PMID- 7537333 TI - Conformationally constrained phosphotyrosyl mimetics designed as monomeric Src homology 2 domain inhibitors. AB - Inhibitors of specific src homology 2 (SH2) domain binding interactions could potentially afford new therapeutic approaches toward a variety of diseases, including several cancers. To date SH2 domain inhibitors have been confined to small phosphotyrosyl (pTyr)-containing peptides that appear to bind along the surface of SH2 domains with specific recognition features protruding into the protein. Among these protrusions is the pTyr residue itself, which is inserted into a well-formed binding pocket. In the present study monomeric pTyr mimetics were prepared having key aspects of their structure constrained to conformations of the bound pTyr residue observed in the previously reported X-ray structure of a pTyr-peptide bound to the Lck SH2 domain. The resulting constrained pTyr mimetics were examined for inhibitory potency in six SH2 domain constructs: Lck, Src, Grb2, and the C-terminal SH2 domains of PLC gamma (PLC gamma-C) and the p85 subunit of PI-3 kinase (p85-C), as well as the N-terminal SH2 domain of SH PTP2. Although inhibition constants were in the millimolar range, it was observed that capping pTyr as its N alpha-acetyl carboxamide [(L)-1] provided a roughly 2-3 fold increase in potency relative to free pTyr. Diastereomeric indanylglycine based analogues (+/-)-3a,b were essentially inactive. Of note was methanobenzazocine (+/-)-2. While being racemic and a partial pTyr structure, this analogue retained full binding potency of the enantiomerically pure N alpha acetyl pTyr amide (L)-1. Modification and elaboration of 2 could potentially result in small molecule inhibitors having greater potency. PMID- 7537334 TI - Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 3. Dipyrido[2,3 b:2',3'-e]diazepinones. AB - We have explored the potential of derivatives of the dipyrido[2,3-b:2',3' e][1,4]diazepinone ring system as inhibitors of HIV-1 reverse transcriptase (RT). These compounds are isomeric to the potent RT inhibitor nevirapine and are available via a novel Smiles rearrangement on intermediates used for the synthesis of nevirapine analogs. Derivatives of this isomeric series are weaker inhibitors of RT than corresponding nevirapine analogs, although with appropriate substitution of the A- and C-pyridine rings activity can be improved. PMID- 7537335 TI - Production and characterization of murine monoclonal antibodies specific for baboon IgG heavy and light chain epitopes. AB - We have generated a panel of murine monoclonal antibodies (MAbs) that recognize baboon IgG epitopes. The reactivity of the MAbs with IgG from other primate species was also examined. Specificity for IgG heavy (H) or light (L) chain epitopes was determined by Western blot analysis. The H chain-specific MAbs were analyzed for IgG subclass specificity and the L chain-specific MAbs for reactivity with baboon IgM and polymeric sIgA. Finally, an ELISA was developed to demonstrate the utility of the MAbs in analysis of humoral immune responses in baboons. PMID- 7537336 TI - Adenocarcinoma in the rectum of a capped langur (Presbytis pileata). AB - A rectal adenocarcinoma in a 22-year-old capped langur histologically resembling those in human cases is reported. An ill-defined diffuse tumor with fibrously firm rectal wall showed diffuse infiltrative growth of signet-ring cancer cells. Immunohistochemistry demonstrated positive staining for CEA, lysozyme, EMA, keratin and B72.3. PMID- 7537337 TI - Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes. AB - The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (Vm), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: KMI0.5, KNa0.5, and the Hill coefficient n. At 100 mM NaCl, KMI0.5 was about 50 microM and was independent of Vm. At 0.5 mM myo inositol, KNa0.5 ranged from 76 mM at Vm = -50 mV to 40 mM at Vm = -150 mV. n was voltage independent with a value of 1.9 +/- 0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent KI of 64 microM at Vm = -50 mV and 130 microM at Vm = 150 mV. To examine sugar specificity, sugar-induced steady-state currents (at Vm = -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scylloinositol > L-fucose > L-xylose > L-glucose, D-glucose, alpha-methyl-D glucopyranoside > D-galactose, D-fucose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose > D-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at Vm = -150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: D-glucose, alpha-methyl-D-glucopyranoside, D-galactose, D fucose, 3-O-methyl-D-glucose > D-xylose, L-xylose, 2-deoxy-D-glucose > myo inositol, L-glucose, L-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na(+) dependent uptake of 3H-D-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mM Na+ that was abolished by phlorizin (0.5 mM). This transient current decayed with a voltage sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in Vm. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. PMID- 7537339 TI - Identification of base-triples in RNA using comparative sequence analysis. AB - Comparative sequence analysis has proven to be a very efficient tool for the determination of RNA secondary structure and certain tertiary interactions. However, base-triples, an important RNA structural element, cannot be predicted accurately from sequence data. We show here that the poor base correlations observed at base-triple positions are the result of two factors. (1) Base covariation is not as strictly required in triples as it is in Watson-Crick pairs. (2) Base-triple structures are less conserved among homologous molecules. A particularity of known triple-helical regions is the presence of multiple base correlations that do not reflect direct pairing. We suggest that natural mutations in base-triples create structural changes that require compensatory mutations in adjacent base-pairs and triples to maintain the triple-helix conformation. On the basis of these observations, we devised two new measures of association that significantly enhance the base-triple signal in correlation studies. We evaluated correlations between base-pairs and single stranded bases, and correlations between adjacent base-pairs. Positions that score well in both analyses are the best triple candidates. This procedure correctly identifies triples, or interactions very close to the proposed triples, in type I and type II tRNAs and in the group I intron. PMID- 7537338 TI - Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells. AB - The conductance of pores induced by Staphylococcus aureus alpha-toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin induced conductance changes in lipid bilayers, namely rectification, voltage dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of alpha-toxin-induced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique. PMID- 7537341 TI - [Involvement of substance P in gastric mucosal lesions of patients with rheumatoid arthritis]. AB - We investigated the relation between gastric mucosal lesions and substance P (SP) in 64 patients with rheumatoid arthritis (RA) taking nonsteroidal antiinflammatory drugs (NSAIDs). In these patients, the incidence of gastric mucosal lesions was as high as 53.1%. Serum SP levels were significantly higher in patients with gastric mucosal lesions than in those without gastric lesions. Erythrocyte sedimentation rate, serum C-reactive protein and rheumatoid factor (RF) levels were also higher in patients with gastric mucosal lesions. A positive correlation between serum SP and RF levels was found in patients with RA. Experimental gastric mucosal lesions induced by an oral administration of indomethacin in rats were significantly enhanced by an additional intraperitoneal injection of SP. From these observations, it is suggested that, in addition to the effect of NSAIDs, SP elevation in blood has a role in the development of gastric mucosal lesion in patients with RA. PMID- 7537340 TI - Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibody-peptide interactions. AB - We previously described the use of a phage-displayed library of random hexapeptides to define and localise the epitope on the human tumor suppressor protein p53 recognised by the monoclonal antibody PAb240. Here we have extended these results to a further eight anti-p53 monoclonal antibodies and to two further libraries, which display 12-mer and 20-mer peptides, respectively. First, we showed that selection of PAb240 binding clones from the 12-mer and 20-mer libraries gives essentially identical results to those obtained by screening the 6-mer library. Second, we used the 6-mer and 12-mer libraries to define the detailed specificity profiles of six antibodies (DO-1, DO-2, DO-7, Bp53-11, Bp53 12 and Bp53-19), which recognise the same short, highly immunogenic N-terminal segment of p53. Finally, we employed all three libraries to reveal the distinct mechanisms by which PAb421 and PAb122, two monoclonal antibodies that allosterically activate sequence-specific DNA binding by p53, react specifically with the same positively-charged C-terminal segment. In each case the epitope locations inferred from the selected sequences were confirmed by probing an array of overlapping synthetic peptides representing the primary sequence of p53. The results emphasise the consequences for epitope mapping of screening random, as opposed to antigen-derived, peptide libraries; specifically (1) that comparison of selected sequences reveals the contribution of individual residues to binding energy and specificity; (2) that heteroclitic reactions can lead to definition of a consensus that is related to but distinct from the immunising epitope and (3) that isolation of non-immunogen-homologous "mimotope" sequences reveals discrete, alternative ligand structures. The results with PAb421 and PAb122 provide examples where, while selection from the 12-mer and 20-mer libraries leads to isolation of immunogen-homologous sequences, selection from the 6-mer library results in the isolation either of no binding clones (PAb122) or solely of "mimotope" sequences with no discernible homology to the original antigen (PAb421). In addition the results with PAb421 reveal that linear epitopes can be longer than previously thought and can be formally discontinuous, consisting of independent contact motifs, which show promiscuous relative positioning. PMID- 7537342 TI - [Interferon induced severe idiopathic thrombocytopenic purpura in a patient with chronic hepatitis C]. PMID- 7537343 TI - Transplantation of kidneys from donors with hepatitis C antibody into recipients with pre-transplantation anti-HCV. AB - Hepatitis C virus (HCV) is transmitted by organ transplantation. Consequently, several organ procurement organizations have imposed a moratorium on use of organs from anti-HCV positive donors. Because of the inadequate supply of cadaver kidneys for transplantation, we adopted a policy to transplant kidneys from anti HCV donors into anti-HCV positive recipients. During the period between March 1990 and December 1992, 24 anti-HCV positive dialysis patients received a kidney from anti-HCV positive donors (group I) and 40 anti-HCV positive patients received a kidney from anti-HCV negative donors (group II). We compared the prevalence of liver disease, anti-HCV, HCV RNA, graft and patient survival between groups. Pre-transplantation 17 of 24 (71%) patients in group I and 31 of 40 (79%) of patients in group II had serum HCV RNA. Post-transplantation follow up was 26 +/- 8 months and 30 +/- 10 months in groups I and II, respectively. During follow-up, elevated ALT levels were present in 7 of 24 (29%) and 16 of 40 (40%) of patients in groups I and II, respectively (P > 0.05). Post transplantation, all patients in both groups retained anti-HCV. The prevalence of HCV RNA post-transplantation was 22 of 23 (96%) patients in group I and 30 of 39 (77%) of patients in group II (P > 0.05). Graft and patient survival in group I (96% and 100%, respectively) were not significantly different from those in group II (93% and 98%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537344 TI - Neuraminidase promotes neutrophil, lymphocyte and macrophage infiltration in the normal rat kidney. AB - Neuraminidase (NA) is an enzyme produced by several microorganisms, which is capable of liberating sialic acid from glycoproteins and modifying cellular adhesion mechanisms. NA is considered a virulence factor in some bacterial species and has been implicated in the pathogenesis of acute poststreptococcal glomerulonephritis, a disease in which glomerular leukocyte infiltration is a prominent feature. We examined the effect of NA on kidney infiltration by neutrophils (PMN), T lymphocytes (TL) and monocyte-macrophages (MM). Intravenous injection of NA resulted in an early increase in the number of PMN (1 hr, 3.42 +/ 0.19 cells/cgs, mean +/- SEM; 3 hr, 3.63 +/- 0.13; 6 hr, 2.9 +/- 0.24; controls, 1.53 +/- 0.18; P < 0.001) and MM (1 hr, 3.49 +/- 0.16; 3 hr, 4.02 +/- 0.2; 6 hr, 3.88 +/- 0.27; controls 1.43 +/- 0.14; P < 0.001) in the glomeruli, while TL increased later (24 hr, 2.29 +/- 0.14; 48 hr, 2.4 +/- 0.2; 72 hr, 2.16 +/- 0.15; controls 0.7 +/- 0.07; P < 0.001). PMN and TL were also increased in the interstitium (up to ninefold for PMN and up to threefold for TL). Following i.v. injection of 51Cr-labeled NA-treated leukocytes, renal radioactive uptake was significantly increased at all times tested (percent radioactivity/gram of tissue after PMN injection, 3 hr, 5.57 +/- 0.46, mean +/- SEM; 12 hr, 5.38 +/- 0.77; 60 hr, 6.51 +/- 1.1; controls, 1.26 +/- 0.17, 1.75 +/- 0.25, and 2.46 +/- 0.08, respectively; P < 0.001 in each case.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537345 TI - Nonverbal communication and early language acquisition in children with Down syndrome and in normally developing children. AB - Many children with Down syndrome display asynchrony in development with the acquisition of language preceding at a slower pace than the acquisition of other cognitive skills. Recent research suggests that the expressive language delays that are displayed by these children may be associated with an earlier disturbance in the development of nonverbal requesting skills (Mundy, Sigman, Kasari, & Yirmiya, 1988; Smith & von Tetzchner, 1986). To test this hypothesis, a longitudinal study of 37 children with Down syndrome and 25 children with normal development was conducted. The results of the study indicated that this sample of children with Down syndrome exhibited a disturbance in nonverbal requesting. Furthermore, individual differences in nonverbal requesting were associated with the subsequent development of expressive language in these children. This association was observed even after taking into account initial variance in developmental level and language status. These data suggested that some of the processes involved in the expressive language delay of children with Down syndrome were not unique to linguistic development. Instead, some aspects of this delay appeared to be associated with problems in an earlier nonverbal phase of communication development. Additionally, the results suggested that measures of nonverbal communication skills also made a unique contribution to the prediction of language development among children with normal development. These data supported the hypothesis that the acquisition of nonverbal communication skills provides an important foundation for the emergence of language in atypical as well as typical development. PMID- 7537348 TI - Does an optimal strategy exist for patients with unresectable cholangiocarcinoma? PMID- 7537347 TI - Genotype-dependent serologic reactivities in patients infected with hepatitis C virus in the United States. AB - OBJECTIVE: To evaluate the serologic reactivities in patients infected with different hepatitis C virus (HCV) genotypes to four HCV proteins that are components of the second-generation recombinant immunoblot assay. MATERIAL AND METHODS: Serum samples from 36 patients with chronic HCV infection were obtained. RNA was extracted by using chaotropic lysis and isopropanol precipitation. Reverse-transcriptase polymerase chain reaction of the NS-5 region was performed, followed by automated single-pass dideoxy sequencing of desalted amplification products. Classification of isolated HCV subtypes was based on Simmonds' system. All samples were tested for antibodies to proteins 5-1-1, C100-3, C33c, and C22-3 with the second-generation recombinant immunoblot assay. RESULTS: Reactivity to protein 5-1-1 was significantly lower for patients with genotypes 2b and 3a than for those infected with HCV types 1a or 1b (P < 0.05). Antibody reactivity to the C100-3 protein was also reduced in patients infected with HCV types 2b and 3a. CONCLUSION: These data indicate that the genotype-dependent differences in serologic reactivities are substantial among patients with chronic HCV infection. PMID- 7537346 TI - "Natural history" of unresected cholangiocarcinoma: patient outcome after noncurative intervention. AB - OBJECTIVE: To clarify the "natural history" of unresected cholangiocarcinoma. DESIGN: We reviewed the outcome in 103 patients with biopsy-proven cholangiocarcinoma managed at our institution during a 5-year period (1980 through 1984) who did not undergo curative resection. MATERIAL AND METHODS: The study group of 56 men and 47 women had initial manifestations of jaundice (71%), abdominal pain (49%), and weight loss (44%). The histopathologic features were confirmed by operation in 73 patients, percutaneous biopsy in 16, endoscopy in 3, or autopsy in 11. The primary tumor site was the common hepatic duct in 27%, the hepatic bifurcation in 27%, the common bile duct in 26%, the liver in 13%, and the right or left hepatic duct in 6%. Biopsy-proven metastatic lesions were identified in 70 patients, including 18 with proven nodal involvement. Surgical intervention (N = 57) most commonly consisted of biliary decompression (26%), biliary bypass (16%), or cholecystectomy (11%). RESULTS: The operative mortality was 4% (N = 2). The hospital mortality of medically managed patients was 12%. The survival after the onset of symptoms was 53% at 1 year, 19% at 2 years, and 9% at 3 years. Only four patients (4%) lived more than 5 years. Univariate analysis of all hospitalized patients (N = 90) revealed a survival advantage for women, patients 62 years of age or younger, those with blood group A or O, surgical patients, patients who did not require biliary decompression, and those who received further palliative treatment after any type of biliary decompression. Multivariate analysis showed a survival advantage for patients who underwent surgical exploration and those who had further palliative treatment after either operative or percutaneous biliary decompression. CONCLUSION: Unresected cholangiocarcinoma is a rapidly fatal process, but early intervention affects the course of the disease and likely prolongs patient survival. PMID- 7537349 TI - From hospital to home: extra-mural hospital--hospital without walls. PMID- 7537351 TI - The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. AB - We have recently shown that during angiogenesis in situ, sprouting and newly formed capillaries appear to be composed of two cell types, endothelial cells and nonendothelial, pericyte-like cells. The effect of pericytes on the process of neovessel formation is largely unknown. To study the influence of nonendothelial cell types on endothelial tubule formation, we have performed coculture experiments in a fibrin-clot angiogenesis system. When seeded below a critical density on the surface of fibrin gels, endothelial cells (from macro- or microvascular origin) did not show spontaneous formation of sprouts. However, in superconfluent cell cultures or after stimulation of endothelial cells with basic fibroblast growth factor (bFGF), endothelial cells frequently acquired an elongated shape. By stimulation of endothelial cells with both bFGF and vascular endothelial growth factor (VEGF), development of short capillary-like structures was induced. When endothelial cells were cocultivated with a cell type of high fibrinolytic potential, i.e., fibroblasts, development of capillary-like formations could not be detected. Cocultivation of endothelial cells with vascular smooth muscle cells or with retinal pericytes also did not increase the number of capillary-like formations in fibrin gels. In contrast, vascular smooth muscle cells on their own could be demonstrated to give rise to branched capillary-like networks in fibrin, which easily could be mistaken for true capillaries. Our results indicate that periendothelial cells contribute to angiogenesis not only by fibrinolysis and proteolytic permeation of the extracellular matrix. Rather, the interactions of endothelial cells and pericyte like cells, as frequently observed during neovessel formation in situ, appear to be more specific and may require factors hitherto unknown. PMID- 7537350 TI - Sustained T-cell reactivity to Mycobacterium tuberculosis specific antigens in 'split-anergic' leprosy. AB - Split anergy represented by delayed-type hypersensitivity skin reaction to tuberculin, but not to leprosin, is known to occur in a distinct proportion of leprosy patients. The mechanism was originally attributed to Mycobacterium leprae specific suppression of T cells toward common mycobacterial antigens. This study ascertained an alternative explanation, attributing the phenomenon to selective responsiveness to M. tuberculosis-specific epitopes. Indeed, the results of blood T-cell proliferative responses in 11 split-anergic patients showed normal responsiveness to the M. tuberculosis-specific 38 kDa lipoprotein and peptide 71 91 of the 16 kDa antigen but diminished responsiveness to 2 common mycobacterial antigens, represented by the 65 kDa heat shock protein and the fibronectin binding Ag85 complex, as compared with leprosin responsive patients and healthy contacts. These findings support the hypothesis that split anergy is due to selective recognition of M. tuberculosis-specific epitopes and deletion of T cells reacting to shared mycobacterial antigens. PMID- 7537352 TI - [The treatment of hyperthyroidism with and indication for radioactive iodine]. PMID- 7537353 TI - Palliative chemotherapy with CMF after the same adjuvant regimen for breast cancer. The Breast Cancer Study Group. AB - BACKGROUND: The results of palliative chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (CMF) in patients with advanced breast cancer who received adjuvant therapy with the same regimen were investigated. RESULTS: Of 47 patients, 14 (30%) achieved an objective remission (median duration 9.5, range 5 21 months) and 8 (17%) stabilisation of disease (median duration 6, range 3-17 months). Objective remissions were observed in premenopausal as well as in postmenopausal women, in patients with all categories of dominant localisation of disease and regardless of the oestradiol receptor status of the primary tumour or eventual previous endocrine therapy. One of 4 and 13 of 43 patients who started palliative chemotherapy within or later than 12 months after the last adjuvant course obtained an objective remission. The median survival time from start of therapy of all treated patients was 12 (range 1-40) months. Patients with an objective remission or stable disease and patients with progressive disease had a median survival time of 20 (range 6-40) and 6 (range 1-35) months respectively (p < 0.0001). CONCLUSIONS: Palliative treatment with CMF should not be rejected for patients who have relapsed after adjuvant chemotherapy with the same modality. PMID- 7537354 TI - Cellular mechanism of melatonin action in neonatal rat pituitary. AB - Melatonin inhibits gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release from neonatal rat anterior pituitary. Melatonin has been shown to decrease the concentration of several second messengers in neonatal pituitary, but it is not known which of them transduces the melatonin effect on LH release. In order to determine the mechanism of melatonin action, we tested the effect of melatonin on GnRH-induced LH release in the presence of specific drugs affecting second messengers. The calcium channel antagonist nifedipine inhibited LH release from cultured pituitary to a similar degree as melatonin and prevented the inhibitory effect of melatonin on LH release. The calcium channel agonist Bay K potentiated the GnRH-stimulated LH release and reduced the inhibitory effect of melatonin on LH release. These data strongly suggest that melatonin inhibits LH release via inhibition of calcium influx through voltage sensitive channels. The cyclic AMP (cAMP) derivative 8-bromo-cAMP potentiated GnRH stimulation of LH release but did not prevent the melatonin-induced inhibition of LH release. However, when used in combination with Bay K, which reduced only partially the melatonin effect by itself, 8-bromo-cAMP completely blocked the melatonin effect. This observation suggests that decreased cAMP accumulation may also be involved in transduction of the melatonin effect on LH release. PMID- 7537355 TI - Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. AB - In a double-blind, placebo-controlled trial, the effects of recombinant human growth hormone were studied on cerebrospinal fluid concentrations of growth hormone, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), monoamine metabolites, neuropeptides and endogenous opioid peptides. Twenty patients, 10 patients in each of 2 groups, with adult-onset, growth hormone deficiency were treated for 1 month with recombinant human growth hormone (0.25 U/kg/week) or placebo. All the patients received the appropriate thyroid, adrenal and gonadal hormone replacement. In cerebrospinal fluid, the mean concentration of growth hormone increased from 13.3 +/- 4.4 to 149.3 +/- 22.2 muU/l (p = 0.002), during recombinant human growth hormone treatment. The cerebrospinal fluid IGF-I concentration increased from 0.67 +/- 0.04 to 0.99 +/- 0.10 micrograms/l (p = 0.005) and the IGFBP-3 concentration rose from 13.4 +/- 1.25 to 17.5 +/- 1.83 micrograms/l (p = 0.002). The dopamine metabolite homovanillic acid decreased from 282.1 +/- 36.0 to 234.3 +/- 26.5 nmol/l (p = 0.02) and the vasoactive intestinal peptide decreased from 4.1 +/- 0.6 to 3.7 +/- 0.4 pmol/l (p = 0.03). Cerebrospinal fluid immunoreactive beta-endorphin increased from 24.4 +/- 1.8 to 29.9 +/- 2.1 pmol/l (p = 0.002). There were no significant changes compared to baseline in the cerebrospinal fluid concentrations of enkephalins, dynorphin A, the norepinephrine metabolite 3 methoxy-4-hydroxyphenyl-ethyleneglycol, the serotonin metabolite 5 hydroxyindoleacetic acid, gamma-aminobutyric acid, somatostatin or corticotropin releasing factor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537356 TI - The patch clamp technique. AB - The introduction of the patch clamp technique less than two decades ago revolutionized the study of cellular physiology by providing a high-resolution method of observing the function of individual ionic channels in a variety of normal and pathological cell types. By the use of variations of the basic recording methodology, cellular function and regulation can be studied at a molecular level by observing currents through individual ionic channels. At a cellular level, processes such as signaling, secretion, and synaptic transmission can be examined. In addition, by combining the information from high-resolution electrophysiological recordings obtained by the patch clamp method with modern molecular biological techniques, further insight can be gained into the gene expression and protein structure of ionic channels. Given the ubiquity and importance of ionic channels, it is not surprising that their study has led to a new understanding of the mechanisms of certain disease processes and has given insight into treatments for these diseases. This review gives an historical perspective of the development of the patch clamp technique and an overview of the methodologies currently in use. Examples are shown to illustrate typical uses of the patch clamp technique with emphasis on the variety of recording configurations available and the advantages and drawbacks of each method. PMID- 7537357 TI - An inhibitor for calcineurin, FK506, blocks induction of long-term depression in rat visual cortex. AB - Long-term depression (LTD) of synaptic transmission, often used as an essential component in synaptic models for learning, memory and forgetting, can be produced in layer II/III of the visual cortex by a prolonged, low-frequency stimulation (LFS) of layer IV. The activation of Ca2+/calmodulin-dependent protein phosphatase, calcineurin, has been postulated to play a role in the induction of LTD. The recent introduction of a specific inhibitor for calcineurin, FK506, prompted the investigation of the involvement of this phosphatase in the induction of LTD in visual cortex. Thus, we administered FK506 at 1 microM to visual cortical slices of young rats, and found that it did not significantly affect field responses of layer II/III evoked by test stimulation of layer IV at 0.1 Hz, but prevented LTD of the responses from being induced by LFS (1 Hz for 15 min) in all the 10 slices tested. Without FK506, significant LTD was induced by the same parameters of LFS in 8 of the 12 slices. These results suggest the critical involvement of calcineurin in producing LTD in visual cortex. PMID- 7537358 TI - Neural somatostatin, vasoactive intestinal polypeptide and substance P in canine and human jejunum. AB - The distribution and co-localization of substance P immunoreactivity (SP-IR), somatostatin-IR (SS-IR) and vasoactive intestinal peptide-IR (VIP-IR) have been determined in canine and human jejunum by immunocytochemistry (ICC). Immunostaining with an antibody to protein gene product 9.5 revealed fewer neuronal cell bodies in the submucosal ganglion of human than in canine intestine. Double immunostaining demonstrated that SP-IR and SS-IR were always co localized in human but never in canine submucosal neurons. In both species, VIP IR was present in a separate population of neurons. In canine submucosal ganglia, each peptide represented approximately one-third of the neurons while the proportions of VIP and SP/SS-containing neurons in human were 40% and 42%, respectively. These results provide neuroanatomical evidence for different functions of neural SP and SS in canine and human jejunum. PMID- 7537359 TI - Expression of tetrodotoxin-resistant sodium channels in capsaicin-sensitive dorsal root ganglion neurons of adult rats. AB - We have used conventional whole-cell electrophysiological recording techniques to measure the level of expression of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in the capsaicin-sensitive and capsaicin-insensitive subpopulations of dorsal root ganglion neurons. We find that there are significantly higher levels of TTX-R expressed in the capsaicin sensitive group but no difference in the levels of TTX-S. Since capsaicin is a selective stimulant of polymodal nociceptors, these data raise the possibility that TTX-R sodium channels may be an important determinant of nociceptor phenotype. PMID- 7537361 TI - Association of phosphatidylinositol 3-kinase with SHC in chronic myelogeneous leukemia cells. AB - Expression of p210 BCR/abl oncoprotein transforms hematopoietic cells. P210 BCR/abl tyrosine kinase induces tyrosine phosphorylation of Shc, and activation of p21ras and PI 3-Kinase. Here we show that PI 3-Kinase associates with Shc in cells transformed by BCR/abl oncoprotein. Immunoprecipitation of Shc from cells expressing p210 BCR/abl had 7.5-fold increase in PI 3-Kinase activity compared to parental cells. Tyrosine phosphorylated Shc specifically bound to the C-SH2 domain of the p85 subunit of PI 3-Kinase. The p85 SH3 domain also interacted with Shc in cell lysates from parental and transformed cells. The binding of p85 SH3 domain to Shc was substantially higher in BCR/abl transformed than in parental cells. Phenylphosphate blocked p85 SH2 mediated association with Shc but enhanced the binding of the p85 SH3 domain to Shc. The N-terminal proline-rich region of Shc between A263 and N273 specifically blocked the interaction of p85 SH3 domain with Shc. Our results indicate that PI 3-Kinase interacts with Shc directly in hematopoietic cells which express p210 BCR/abl oncoprotein. PMID- 7537360 TI - The human spiral ganglion. AB - The adult normal human spiral ganglion (SG) was analyzed with regard to ultrastructure and immunohistochemistry. The cytoskeleton of the SG cells was found to comprise F-actin, intermediate filaments (IFs) and microtubules (MTs). The IF subgroups (cytokeratins, Cks; neurofilaments, NFs, vimentin, glial fibrillary acidic proteins, GFAP; desmin) displayed characteristic staining patterns. Ck No. 8 was found in all SG cells, whereas vimentin was lacking. GFAP stained only a small subpopulation of SG cells (type 2?). The light (68 kD) and medium-sized chains of NFs occurred in all SG cells and axons, whereas the 200-kD NF subunit was only found in the axonal hillock of (type 2?) SG cells, but in no other part of the cytoplasm, and regionally in nerve fibres. MAP-1 and MAP-2 occurred in all SG cells but only MAP-1 was found in the nerve fibres. The calcium-binding protein synaptophysin (SY) was expressed only in SG cells, in contrast to the S-100 which occurred more generally in the labyrinth. The neuropeptides VIP and substance P were identified in all SG cells, in contrast to NPY which was expressed in a small subpopulation of SG cell (type 2?). Staining for neuron-specific enolase (NSE) identified most (type 1?) but not all SG cells. The cell surface glycoprotein Thy-1 was expressed in SG cells in a way similar to that described for neurons in the CNS. The SG cells express a high degree of cytoskeletal complexity, allowing one to distinguish between type 1 and type 2 cells. The cell bodies and their adjacent nerve fibres show characteristic features of calcium-binding proteins, surface membrane glycoproteins, NSE and neuropeptides but the basic pattern is still similar to neurons in the CNS. PMID- 7537362 TI - Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. AB - The molecular interactions of the Src homology 2 (SH2) domain and the N-terminal proline-rich sequence motifs (pro-1 to pro-5) of the SH2 protein Shb with other components were presently characterised. Using a degenerate phosphopeptide library the preferred binding site for the Shb SH2 domain was determined to pTyr Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue. Experiments with competing peptides and platelet-derived growth factor (PDGF) beta-receptor mutants with Y to F substitutions in autophosphorylation sites revealed multiple binding sites for the Shb SH2 domain in the receptor. The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth factor receptor-1 (FGFR-1) mainly through position Y776. The receptor experiments suggest that other residues besides the +1 to +3 positions may also be of significance for Shb binding. The pro-4/pro-5 motif of Shb binds in vitro particularly well to the Src, p85 alpha PI3-kinase and Eps8 SH3 domains expressed as GST fusion proteins. However, the GST-SH3 domain fusion proteins tested bind in vitro to peptides corresponding to the pro-1 to pro-5 motifs of Shb with low affinity and selectivity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions. In vivo association between Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunoprecipitation. PDGF treatment did not affect the association between Eps8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins. PMID- 7537363 TI - Transcription elongation of the murine ornithine decarboxylase (ODC) gene is regulated in vitro at two downstream elements by different attenuation mechanisms. AB - Ornithine decarboxylase (ODC) plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels and is found to be deregulated in various malignancies. The rapid and dramatic induction of cellular ODC mRNA upon serum addition raised the possibility that a transcriptional attenuation mechanism may be involved in the regulation of ODC gene expression. Using transcription in HeLa nuclear extract and isolated transcription complexes, we have identified two sites of transcription arrest downstream to the transcription start site: Attenuator 1 (Att.1) located at +220, near two repeats of a USF/Myc-Max binding consensus sequence and attenuator 2 (Att.2) located at +1590 near a long stretch of T-residues. The two attenuators exhibit distinct properties as revealed by elongation of briefly initiated and partially purified transcription complexes: Att.1 serves as a transient pause site while arrest at Att.2 is more prolonged. The arrest at both attenuators is modulated by the general elongation factor TFIIS. In a promoter independent transcription system, using partially purified RNA polymerase II, only Att.2 was recognized efficiently. This suggests that the recognition of Att.2 is an intrinsic property of the polymerase while Att.1 recognition has to be facilitated by an auxiliary factor/s. PMID- 7537364 TI - Changes of cerebral biopterin and biogenic amine metabolism in leukemic children receiving 5 g/m2 intravenous methotrexate. AB - Acute or subacute neurologic disorders can be observed in patients receiving high dose methotrexate therapy for lymphoblastic leukemia or malignant tumor. Impairment of biopterin metabolism leading to decreased availability of monoamine neurotransmitters has been suggested to explain methotrexate neurotoxicity. To investigate such a mechanism, we have measured prospectively by HPLC the concentrations of total biopterin, homovanillic acid, and 5-hydroxyindolacetic acid in cerebrospinal fluid of 57 children with acute lymphoblastic leukemia. A sequential analysis of cerebrospinal fluid was performed for each patient: cerebrospinal fluid samples were obtained before therapy and after each of the four high-dose methotrexate infusions during the CNS prophylaxis phase. A significant increase of total biopterin concentrations in cerebrospinal fluid was observed after high-dose methotrexate therapy compared with the pretreatment values. No cumulative effect was noted. In contrast, no significant variation of the homovanillic acid and 5-hydroxyindolacetic acid levels was observed in cerebrospinal fluid. However, individual analysis revealed a transient decrease of homovanillic acid and 5-hydroxyindolacetic acid concentrations in cerebrospinal fluid of six children. The increase of total biopterin mimicking that observed in inherited dihydropteridine reductase deficiencies suggests that methotrexate inhibits the regenerating system of biopterin in the brain of patients undergoing high-dose methotrexate therapy. PMID- 7537365 TI - Transient elevation of granulocyte colony-stimulating factor levels in cerebrospinal fluid at the initial stage of aseptic meningitis in children. AB - At the early stage of aseptic meningitis, there is a transient increase in neutrophil counts in the cerebrospinal fluid. Some factors in the cerebrospinal fluid might induce migration of neutrophils into the cerebrospinal fluid. Granulocyte colony-stimulating factor (G-CSF) plays an important role, not only as a hemopoietic factor but also as a regulating factor for a biologic defense system by neutrophils in the foci of infection. To analyze the role of G-CSF on accumulating neutrophils in the cerebrospinal fluid, we have measured G-CSF levels in the cerebrospinal fluid of children with aseptic meningitis, paying particular attention to the phasal transition. Within the first 2 d from the onset, G-CSF levels in the cerebrospinal fluid were 223 +/- 97 ng/L, significantly higher than those of the patients without meningitis (p < 0.01). Beyond the second day after the onset, the G-CSF levels rapidly decreased to below the detectable level, even though the patients manifested meningeal signs and symptoms. There was a direct relationship between G-CSF levels and neutrophil counts in the cerebrospinal fluid (r = 0.763, p < 0.01). During the first 2 d after the onset, the G-CSF level in the cerebrospinal fluid in each case was remarkably higher than that in the serum. This finding suggests that the G-CSF in the cerebrospinal fluid was produced in the spinal cavity. From our results, the transient elevation of G-CSF levels might lead to the transient increase in neutrophil counts in the cerebrospinal fluid by recruiting them from the peripheral blood at the initial stage of aseptic meningitis. PMID- 7537366 TI - The relative roles of external taurine concentration and medium osmolality in the regulation of taurine transport in LLC-PK1 and MDCK cells. AB - Taurine is a beta-sulfonic amino acid that serves as a nutrient important for developing brain and retina and as an osmolyte in the medullary collecting duct. The activity of the taurine transport system is regulated by substrate supply and by the external osmolality; these two stimuli induce changes in taurine transport. Increased medium osmolality (500 mosmol) stimulates taurine uptake into MDCK cells but not LLC-PK1 cells. The enhanced taurine uptake that occurs in response to hyperosmolality is localized primarily to the basolateral surface of MDCK cells, whereas the adaptive response to medium taurine concentration is expressed on both the apical and the basolateral surfaces of both cell lines. The response of MDCK cells to medium osmolality requires protein synthesis and RNA transcription and is expressed in the presence of microtubular toxins. When cell monolayers were loaded with taurine by incubation in high-taurine medium before increasing medium osmolality, the expected increase in taurine uptake was blunted. Similarly, increased external beta-alanine (500 microM) also prevented the anticipated increase in taurine accumulation in response to hypertonicity; aminoisobutyric acid and betaine (500 microM) partially prevented the increase in taurine transport after hypertonicity, whereas L-alanine had no effect. The concentration of taurine or structurally similar analogs in the external medium might modify the response of taurine accumulation after exposure to hypertonic medium, in that taurine-replete cells behave differently than taurine-depleted cells. These studies indicate that there are at least tow distinct mechanisms involved in the regulation of taurine transport: external taurine concentration and medium osmolality, with taurine concentration seeming to be the predominant stimulus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537367 TI - Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase. AB - The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction. PMID- 7537368 TI - Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides. AB - Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA. PMID- 7537370 TI - Palliative care. Source of knowledge. PMID- 7537369 TI - RNA-binding activity of the matK protein encoded by the chloroplast trnK intron from mustard (Sinapis alba L.). AB - The chloroplast trnK gene for tRNALys(UUU) from mustard contains a 2574 bp group II intron with a long open reading frame for 524 amino acids. The encoded polypeptide appears to be structurally related to mitochondrial maturases which are involved in splicing. To study the properties of the intron encoded protein, we overexpressed the trnK ORF as a beta-galactosidase fusion protein in E. coli and carried out RNA-protein binding experiments with crude bacterial extracts and the purified fusion protein. Both gel-shift and UV-crosslinking experiments revealed preferential binding to the trnK precursor transcript. Of two other RNA probes containing chloroplast group II introns, the trnG precursor was recognized by the trnK ORF protein, but the rps16 precursor was not. Competition binding experiments indicate that G-residues seem to play a role in RNA-protein interaction. RNA-binding activity of the trnK intron encoded polypeptide is consistent with its suggested function as a plastid maturase, hence justifying the assignment matK for this gene. PMID- 7537371 TI - Palliative care. Dealing with constipation. PMID- 7537372 TI - The central nervous system of Grillotia erinaceus (Cestoda: Trypanorhyncha) as revealed by immunocytochemistry and neural tracing. AB - Immunofluorescent labeling and neural tracing techniques were employed in conjunction with confocal scanning laser microscopy to study the intact neuroanatomy of the central nervous system of the trypanorhynch tapeworm Grillotia erinaceus. Immunocytochemical labeling for the general nerve fibre marker PGP 9.5 showed a pattern of extensive labeling that paralleled findings obtained with the neural tracer DiI. In contrast, immunocytochemical labeling for 5-hydroxytryptamine (5-HT) was localised to cell bodies lying on the periphery of the ganglion, with fine immunoreactive fibres radiating out towards the bothridia. Following the retrograde transport of the fluorescent molecule DiI through axotomised nerve cords, it successfully labeled both the cerebral ganglion and associated nerve fibres within the scolex. The cerebral ganglion was shown to give rise to posterior nerve cords, an array of radial fibres that pass out to the bothridia, and to contain a centrally disposed group of cell bodies thought to be involved in efferent functions. PMID- 7537373 TI - Peripherally localized benign hyperplastic nodules of the prostate. AB - Approximately one-half of needle-core biopsy samples performed for palpable and/or ultrasonographically hypoechoic focal lesions of the prostate reveal carcinoma. A fraction of the negative biopsy samples are related to benign hyperplastic nodules, localized in the peripheral zone of the prostate. The present study examines the morphology of this particular lesion in surgical specimens obtained after cystoprostatectomy and radical prostatectomy performed for bladder and (small) prostate cancer, respectively. Peripheral hyperplastic nodules occur in 18.5% of this population and are unifocal in one-half of the cases. The mean diameter is 4 (+/- 1.3) mm. Peripheral hyperplastic nodules are characteristically localized posteriorly in the peripheral zone, between the midline and the lateral border of the prostate and often in the vicinity of the boundary of the transition zone. Histologically, they resemble glandulostromal hyperplastic nodules as observed in classic benign nodular hyperplasia of the transition zone. Cystic transformation may occur. The nodules are sometimes surrounded by condensed stroma and atrophic glands. Atypical adenomatous hyperplasia is rarely noticed in this lesion. No pure stromal (leiomyomatous) nodules without glands are seen. The peripheral hyperplastic nodule should be included in the differential diagnosis of focal lesions of the peripheral zone. PMID- 7537374 TI - Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. AB - Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predominated in late-passage cultures but were rare at early passages and apparently absent in milk. Surprisingly, a second cell type, present only in early-passage tissue-derived cultures, was fully immortalized by E7 alone. A third cell type, observed in tissue-derived cultures and in milk, showed a substantial extension of life span with E7 but eventually senesced. Finally, both E6 and E7 were required to fully immortalize milk-derived MECs and a large proportion of MECs in early-passage tissue-derived cultures, suggesting the presence of another discrete subpopulation. Identification of MECs with distinct susceptibilities to p53- and Rb-targeting human papillomavirus oncogenes raises the possibility that these cells may serve as precursors for different forms of breast cancer. PMID- 7537375 TI - The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice. AB - The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation. PMID- 7537377 TI - Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3. AB - Interferon alpha induction of transcription operates through interferon stimulated-gene factor 3 (ISGF), a transcription factor two components of which are members of the newly characterized Stat family of transcription factors. Interferon alpha induces tyrosine phosphorylation of Stat1 and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Stat1 and Stat2 is present in ISGF3 and that Stat1 and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3. PMID- 7537376 TI - Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3. AB - A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects. PMID- 7537378 TI - Evidence for a hypothalamothalamocortical circuit mediating pheromonal influences on eye and head movements. AB - A method for simultaneous iontophoretic injections of the anterograde tracer Phaseolus vulgaris leukoagglutinin and the retrograde tracer fluorogold was used to characterize in the rat a hypothalamothalamocortical pathway ending in a region thought to regulate attentional mechanisms by way of eye and head movements. The relevant medial hypothalamic nuclei receive pheromonal information from the amygdala and project to specific parts of the thalamic nucleus reuniens and anteromedial nucleus, which then project to a specific lateral part of the retrosplenial area (or medial visual cortex). This cortical area receives a convergent input from the lateral posterior thalamic nucleus and projects to the superior colliculus. Bidirectional connections with the hippocampal formation suggest that activity in this circuit is modified by previous experience. Striking parallels with basal ganglia circuitry are noted. PMID- 7537379 TI - Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. AB - Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels. PMID- 7537383 TI - Nursing management of the elderly surgical patient. AB - Older patients and their physicians are likely to choose surgery, when indicated, despite old age. To facilitate recovery, functional assessment provides data to plan immediate and long-term needs. Functional assessment includes physical, social, and environmental data. It also helps define "normal" functioning after surgery. Cognitive assessment is especially important and can be obtained during the initial interview of the patient. Cognitive assessment tools may be indicated if data are unclear. Postoperative complications occur in all body systems and include: MI, CHF dysrhythmias, pneumonia, atelectasis, hyponatremia, renal failure, confusion, deconditioning, skin tears, incontinence, and constipation. Referral may be needed before dismissal for significant changes in functional ability. PMID- 7537382 TI - A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription. AB - Members of the cytokine/growth hormone/prolactin (PRL) receptor superfamily are associated with cytoplasmic tyrosine kinases of the Jak family. For the PRL receptor (PRLR), after PRL stimulation, both the kinase Jak2 and the receptor undergo tyrosine phosphorylation. To assess the role of tyrosine phosphorylation of the PRLR in signal transduction, several mutant forms of the PRLR in which various tyrosine residues were changed to phenylalanine were constructed and their functional properties were investigated. We identified a single tyrosine residue located at the C terminus of the PRLR to be necessary for in vivo activation of PRL-responsive gene transcription. This clearly indicates that a phosphotyrosine residue in the cytoplasmic domain of a member of the cytokine/growth hormone/PRL receptor superfamily is directly involved in signal transduction. PMID- 7537380 TI - Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library. AB - Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries. PMID- 7537384 TI - [Malignant transformation of odontogenic cysts]. AB - Five out of 4,172 operated maxillary cysts (3 developed within the maxillary and 2 in the mandible) presented a malignant change of epithelium. In all these 5 cases histology demonstrated a transition from normal to cancerous epithelium. The overall percentage of malignant change was of 0.12%, with 0.077% for non keratinized epithelium lining odontogenic cysts (3 cases) and 0.65%, that is 8 times higher, for keratinized ones (2 cases). Keratinization of cystic epithelium and chronic inflammatory lesions were the main risk factors. PMID- 7537385 TI - Palliative laser treatment of rectal cancer. AB - BACKGROUND: Endoscopic neodymium-yttrium aluminum garnet laser treatment is a therapeutic modality for gastrointestinal neoplasms. METHODS: Patients given palliative therapy with laser for invasive rectal cancer during 1989-93 at the Dept. of Surgery, University of Turku, were analyzed. RESULTS: All 20 patients (median age, 81.5 years) were unsuitable for radical surgery owing to the poor clinical condition and/or advanced disease. In 14 (70%) patients laparotomy and diversion was avoided with laser treatment; 6 patients underwent laparotomy, 2 for pelvic infiltration and large-bowel obstruction, 2 for rectovaginal fistula formation, and 1 for severe pain. One patient underwent abdominoperineal resection for bleeding after laser treatment. Postoperative complications developed in three (15%) patients--two episodes of bleeding and one of sepsis; none of these complications was fatal. CONCLUSIONS: Local palliative laser treatment of rectal carcinoma is efficient in patients who are unsuitable for radical surgery. Operative diversion can be avoided without deaths and with minimal morbidity. PMID- 7537381 TI - Regulation of CD45 engagement by the B-cell receptor CD22. AB - The B-cell receptor CD22 binds sialic acid linked alpha-2-6 to terminal galactose residues on N-linked oligosaccharides associated with several cell-surface glycoproteins. The first of these sialoglycoproteins to be identified was the receptor-linked phosphotyrosine phosphatase CD45, which is required for antigen/CD3-induced T-cell activation. In the present work, we examine the effect of interaction between the extracellular domain of CD45 and CD22 on T-cell activation. Using soluble CD22-immunoglobulin fusion proteins and T cells expressing wild-type and chimeric CD45 forms, we show that engagement of CD45 by soluble CD22 can modulate early T-cell signals in antigen receptor/CD3-mediated stimulation. We also show that addition of sialic acid by beta-galactoside alpha 2,6-sialyltransferase to the CD22 molecule abrogates interactions between CD22 and its ligands. Together, these observations provide direct evidence for a functional role of the interaction between the extracellular domain of CD45 and a natural ligand and suggest another regulatory mechanism for CD22-mediated ligand engagement. PMID- 7537388 TI - Generation of memory B cells and plasma cells in vitro. AB - After germinal center B cells undergo somatic mutation and antigen selection, they become either memory B cells or plasma cells, but the signal requirements that control entry into either pathway have been unclear. When purified human germinal center cells were cultured with interleukin-2, interleukin-10, and cells expressing CD40 ligand, cells with characteristics of memory B cells were generated. Removal of CD40 ligand from the system resulted in terminal differentiation of germinal center B cells into cells with the characteristics of plasma cells. These results indicate that CD40 ligand directs the differentiation of germinal center B cells toward memory B cells rather than toward plasma cells. PMID- 7537386 TI - Exocrine pancreatic function in the early phase of human acute pancreatitis. AB - BACKGROUND: Little is known about exocrine pancreatic function during human acute pancreatitis. We aimed to evaluate interdigestive exocrine pancreatic secretion and pancreatic polypeptide (PP) release during the early phase of acute pancreatitis in humans. METHODS: Eight patients with acute pancreatitis (six biliary, one alcoholic, and one idiopathic acute pancreatitis) were studied within 72 h from the onset of symptoms. Four patients had necrotizing and four had edematous acute pancreatitis. Normal values were obtained from 26 normal subjects matched by sex and age. Interdigestive pancreatic secretion was studied by a duodenal intubation perfusion technique. Enzyme output was calculated for consecutive 15-min periods over 3-4 h and expressed as units per hour within a secretion cycle and units per hour around a secretion peak. Plasma PP concentrations were measured by radioimmunoassay in 15-min intervals. RESULTS: All variables studied were similar in patients with acute pancreatitis and in controls. PP release in acute pancreatitis was maintained in a normal cyclical pattern closely related to the secretory cycles. CONCLUSIONS: Interdigestive exocrine pancreatic secretion in the early phase of mild to moderate acute pancreatitis in humans remains within the normal range. This finding provides a rational basis for early therapeutic inhibition of pancreatic secretion in human acute pancreatitis. PMID- 7537389 TI - Utilization of superapubic catheters after visual laser ablation of the prostate. PMID- 7537387 TI - [Creutzfeldt-Jakob disease]. AB - Creutzfeldt-Jakob's disease is a transmissible encephalopathy manifesting with dementia and motor disturbances, which usually progresses rapidly and is lethal within months. It occurs mainly sporadically, but it can also be transmitted by proteinaceous infective particles called prions. The diagnosis has to rely on clinical symptoms, EEG and brain biopsy being the most suitable additional examinations. No therapy is yet known. "Naturally" occurring transmission has not been observed: all transmitted cases reported so far have been iatrogenic and followed administration of cadaveric hypophyseal hormones, transplantation of tissue from CNS or related organs, or brain surgery with contaminated instruments. Remarkable discoveries in the past decades with respect to the molecular and genetic characterization of the transmissible pathogen have led to a new understanding of the disease. The infective agent appears to be an abnormal isoform of a physiologically occurring protein: the cellular prion (PrPc). The crucial pathogenetic event is the conformational conversion of PrPc into its pathological isoform (PrPsc), an event thought to be triggered autocatalytically by the infectious agent itself. The disease can be elicited in experimental animals by inoculation of PrPsc. In the sporadic cases of Creutzfeldt-Jakob's disease, PrPsc is thought to arise through spontaneous conversion of PrPc. A growing body of evidence indicates that specific alleles of the prion gene confer a genetic predisposition to Creutzfeldt-Jakob's disease and to related pathologies. PMID- 7537390 TI - Basic fibroblast growth factor immunoreactivity in blood vessels and cells of disc herniations. AB - STUDY DESIGN: Basic fibroblast growth factor immunoreactivity was studied in disc herniation tissue. OBJECTIVES: The first objective was to analyze in which tissue components, if any, fibroblast growth factor is expressed in the disc herniation. The second objective was to compare such expression with that in fresh cadaver disc tissue. SUMMARY OF BACKGROUND DATA: Disc herniation tissue contains vascular ingrowth, which promotes the formation of granulation tissue. Fibroblast growth factor is a potent inducer of angiogenesis and also regulates extracellular proteolysis. METHODS: Twenty-seven disc herniation tissue and five macroscopically normal fresh cadaver discs were treated with an identical immunohistochemical protocol. Serial frozen sections were stained with a polyclonal basic fibroblast growth factor antibody and a polyclonal antibody to von Willebrand factor, which localizes endothelial cells. The immunostaining data were compared with relevant clinical data. RESULTS: Histologically, 74% of the samples contained anulus fibrosus and 59% nucleus pulposus. Basic fibroblast growth factor immunoreactivity was detected in 81% of the samples. There were immunopositive small blood vessels and scattered immunopositive disc cells (67%). Not all observed blood vessels were basic fibroblast growth factor immunopositive. In control discs, no immunoreactivity was observed. CONCLUSIONS: The observed presence of fibroblast growth factor in small blood vessels suggests an active angiogenesis as a result of disc injury. Cellular expression of fibroblast growth factor may be linked to proteolytic activity in disc extracellular matrix. PMID- 7537391 TI - Characterization of thermal hyperalgesia, c-fos expression, and alterations in neuropeptides after mechanical irritation of the dorsal root ganglion. AB - STUDY DESIGN: This study analyzed hypersensitization in sensory systems after mechanical irritation of the dorsal root ganglion. OBJECTIVES: To develop a reliable and reproducible animal model of hyperalgesia arising from the dorsal root ganglion and to understand the unique contributions of the dorsal root ganglion to clinical manifestations of sciatica. SUMMARY OF BACKGROUND DATA: The dorsal root ganglion likely plays an important role in disorders of sciatica. However, no previous study has analyzed sciatica after irritation of the dorsal root ganglion. Thermal hyperalgesia indicates a decrease in thermal nociceptive threshold and hypersensitization in sensory systems. METHODS: The left L4 and L5 dorsal root ganglia in rats (n = 22) were exposed circumferentially. Other rats (n = 22) also had the left L4 and L5 dorsal root ganglia ligated loosely with two 4-0 chromic gut sutures. Changes in thermal withdrawal latency were examined in the hindpaws across time. Substance P and vasoactive intestinal polypeptide contents were quantified in the dorsal root ganglion and spinal cord. Substance P, calcitonin gene-related peptide, and c-fos expression also were examined in the spinal cord by immunohistochemistry. In addition, histologic changes in myelinated nerve content were examined in the dorsal root ganglion. RESULTS: Thermal hyperalgesia occurred in rats with exposure of the dorsal root ganglion and in rats with loose ligation of the dorsal root ganglion, and was accompanied by an increase in c-fos expression and spontaneous pain-related behaviors. CONCLUSIONS: This experimental model reliably produced a disorder resembling an acute phase sciatica and should help further advance the understanding of pathomechanisms of spinal pain after irritation of the dorsal root ganglion in humans. PMID- 7537392 TI - The development of graphic symbols for medical symptoms to facilitate communication between health care providers and receivers. AB - Since there are a variety of communication barriers in health care settings in Japan, a study was designed to improve communication by the use of graphic symbols. At the beginning of this study, graphic symbols were developed to correspond to 26 basic symptoms. Seventy-six subjects voluntarily evaluated the comprehensibility of these symbols: nursing students (n = 29), manual sign language interpreters (n = 24), hearing impaired subjects with normal (n = 10), limited (n = 11), and minimal (n = 2) literacy abilities. The comprehension by each respondent of each symbol was compared with that of the authors. On the average, numbers of the matching meanings were 24.9 +/- 1.36 (mean +/- S.D.) for students, 24.5 +/- 1.77 for interpreters, 23.4 +/- 2.22, and 21.5 +/- 3.01 for the first two groups of the hearing impaired. Among the 26 symbols, 10 showed high levels of the matching rates (> 90%) for all groups. These symbols were considered to be effective alternatives to verbal expression. Further refinements of the graphic symbols were suggested to suppress the differences in interpretation of the remainder of the symbols. During this study, colleagues and subjects suggested cognitive strategies to clarify and enhance the meaning of the graphic symbols such as (a) the subtraction of excessive information, (b) the addition of further information, and (c) the simplification of the setting by minimizing social and cultural bias. PMID- 7537393 TI - [The treatment of localized rapidly progressing juvenile periodontitis by using endolymphatic infusions of drug preparations]. PMID- 7537394 TI - Anti-CD2 monoclonal antibody-induced receptor changes: down modulation of cell surface CD2. AB - Anti-CD2 mAbs suppress T cell immunity and prolong allograft survival in vivo while inducing the down-modulation of CD2 expression. Manipulation of cell surface molecules may be important in inducing tolerance, so down-modulation of CD2 expression on T cells by anti-CD2 mAbs was further defined with an in vitro model. The anti-CD2 mAb 12-15 caused CD2 expression on purified splenic T cells to decrease from 83.4 to 22.7% total positive cells while CD3, CD4, and CD8 expression remained unchanged. The expression of other adhesion molecules, LFA-1 alpha (CD11a), LFA-1 beta (CD18), Pgp-1 (CD44), CD45, MEL-14 (L-selectin), and VLA-4 alpha (CD49d), were all increased as a result of anti-CD2 treatment, whereas CD25 (IL-2R), CD48 (CD2 ligand), and ICAM-1 (CD54) remained unchanged. Kinetics showed that CD2 down-modulation was persistent and at the same magnitude from day 1 through day 7 of culture. Anti-CD2 mAb could down modulate CD2 on both CD4 and CD8 splenic lymphocyte subsets, thymocytes, and the T cell lymphoma EL-4; and, non-T cells did not seem to participate in the process of modulation. Mechanistic studies of mAb action showed that, in addition to 12-15, other anti CD2 mAbs could cause down-modulation of T cell CD2 expression in an epitope and isotype dependent fashion and that CD2 down-modulation correlated with inhibition of receptor-driven T cell stimulation. Intact antibody, including the Fc portion, was required to induce CD2 down-modulation, and additional experiments suggested an interaction with an Fc gamma R other than Fc gamma RII or Fc gamma RIII. CD2 down-modulation did not change with the addition of the cytokines IL-1, IL-2, IL 6, IL-10, TNF alpha, or TGF-beta 1. These results show that anti-CD2 mAbs significantly and persistently down-modulate CD2 on various T cell subpopulations. The mAbs must interact with both the CD2 receptor and an Fc gamma R. CD2 down-modulation is accompanied by changes in the array of other T cell surface receptors that may contribute to mechanisms of anti-CD2-induced immunosuppression. PMID- 7537395 TI - Transgenic expression of human complement regulatory proteins in mice results in diminished complement deposition during organ xenoperfusion. AB - Complement activation is an essential step in the hyperacute rejection of a vascularized xenograft. Endothelial cell-associated complement regulatory proteins limit complement activation in most settings, but are not able to limit the extensive complement activation that occurs in xenografts, at least in part due to their species specificity. To overcome this problem we and others have sought to express human complement regulatory proteins in the organs of potential donor animals. As an initial step toward evaluating this concept we tested organs from transgenic mice expressing human CD59 and/or decay-accelerating factor (DAF) in two in vitro perfusion systems for the ability to control activation of heterologous complement. In the first system, mouse hearts were perfused on a Langendorff circuit with 50% human plasma. Immunopathologic analysis of heart biopsies revealed deposition of human IgG, IgM, and C4 in both control and transgenic organs. The hearts from mice transgenic for human CD59 had substantially less and in some cases no membrane attack complex (MAC) and hearts from CD59/DAF transgenic mice had substantially less or no C5b and MAC. In the second system, mouse hearts were perfused with baboon blood through arterial lines inserted into baboons. Immunopathologic analysis of serial biopsies revealed the deposition of IgG, IgM, and C4 in control and transgenic hearts. Compared with controls, less MAC was deposited in many CD59-expressing hearts and less C5b and MAC in DAF-expressing hearts. These results demonstrate that human complement regulatory proteins expressed in a xenogeneic organ are able to contribute to the control of complement activation in that organ and support the concept that expression of these human molecules would help protect a xenogeneic organ transplanted into a human. PMID- 7537397 TI - The predictive value of epitope analysis in highly sensitized patients awaiting renal transplantation. AB - The accumulation of highly sensitized patients (HSP) on renal transplant waiting lists is a problem faced by all transplant registries. We have studied the HLA class I serological reactivity of 20 random HSP and have related antibody specificity to primary amino acid sequence. In six patients we identified significant correlations (chi 2 test, r > or = 0.93) between panel reactivity and specific amino acid substitutions characteristic of HLA-A, -B, and -C public epitopes. Antibody reactivity was associated with up to three public epitopes in each patient. The 12 separate antibody specificities identified were associated with 10 residues. Seven correlated with HLA-A locus substitutions (Glu-62/Gly-65, Lys-66, Arg-114, His-114/Tyr-116/Lys-127, Thr-142/His-145 [x2], and Thr-149), two with HLA-B locus substitutions (Thr-24, Ser-24) and three with interlocus antibodies associated with either HLA-A and B (Leu-82/Arg-83 [x2]) or with HLA-B and -C substitutions (Leu-163). This information allowed us to predict HLA class I allelic products of known primary sequence that would react negatively with each HSP serum. Windows of acceptable mismatches (WAMMs) can thus be delineated with a view to crossmatch negative transplantation without the need for exhaustive serological analysis. Surprisingly we found that WAMMs for these patients included up to 80% of the 10 commonest HLA class I haplotypes in the British population with four patients being crossmatch compatible with A1,3; B7,8. These observations lead us to propose a more intelligent approach to transplanting HSP based on epitope analysis and definition of WAMMs. PMID- 7537398 TI - Interactions between FK506 and rifampicin or erythromycin in pediatric liver recipients. PMID- 7537396 TI - Hamster to rat kidney xenotransplantation. Effects of FK 506, cyclophosphamide, organ perfusion, and complement inhibition. AB - Hamster to rat renal xenotransplantation was performed with recipient nephrectomies. Recipients were treated beginning on day 0 with continuous FK 506 monotherapy, a 7-day or open-ended monotherapeutic course of cyclophosphamide (CP), and the two drug regimens combined. CP alone (10 mg/kg/day) prevented a xenospecific antibody response and tripled median survival of the kidney (defined as recipient death) from 6 (control) to 18.5 days whereas FK 506 alone had no effect. The drugs in combination were no better than CP alone (15 days) unless the 5-day course of CP was given at a higher dose (15 mg/kg) and started 3 days preoperatively (79 days). In further experiments, adjuvant measures were added to the minimally effective FK 506/7-day CP regimen which gave a median survival of only 15 days. In the most successful modification, intraoperative antibody depletion by the temporary transplantation of third party hamster liver or en bloc kidneys increased median survival from 15 to 34 and 48 days, respectively. An intraoperative i.v. dose administration of the anticomplement drug K76 instead of antibody depletion increased survival to 26 days. Although the events of kidney rejection were similar to those of heart xenografts and partially forestalled by the antibody inhibiting CP treatment, or by antibody depletion, survival for > 100 days was accomplished in only 5 of 86 treated animals. The poorer survival previously reported with cardiac xenotransplantation is largely explained by the life support requirement of the kidneys. Renal failure was responsible for almost all deaths before 60 days, and subnormal renal failure was a pervasive adverse factor thereafter, frequently caused by pyelonephritis which is suspected to have had an immunologic etiology. PMID- 7537399 TI - Acute necrotico-hemorrhagic pancreatitis after famciclovir prescription. PMID- 7537400 TI - Anomalous pattern of IgG antibody response to primary cytomegalovirus infection after solid organ retransplantation. PMID- 7537401 TI - Current excitement from insect muscarinic receptors. AB - Recent electrophysiological, pharmacological and molecular studies suggest that muscarinic ACh receptors (mAChRs) in insects are related to, but distinct from, their mammalian counterparts. Insect mAChRs perform two primary roles that are distinguished by their locations. Presynaptic mAChRs, present on sensory terminals, inhibit transmitter release, thereby reducing the effectiveness of specific afferent inputs. In contrast, postsynaptic mAChRs depolarize and increase the excitability of motoneurons and interneurons, thereby acting as dynamic-gain controls. This postsynaptic modulation is achieved in different ways in specific neurons but generally results from the activation of persistent inward and outward currents. At the level of neural processing, these distinct roles enable insect mAChRs to regulate the transfer of sensory information, and modulate the contributions of central neurons to central pattern generators and reflexes. Because these phenomena can be studied in identified neurons, a combination of physiological and molecular studies of mAChRs in insects should help to elucidate some of their behavioral roles. Furthermore, such studies could lead to the identification of general mechanisms of functional plasticity in neuronal networks. PMID- 7537402 TI - Neuroscience-knowledge management: slow change so far. PMID- 7537403 TI - Neuropeptides: promise unfulfilled? PMID- 7537404 TI - Neural nitric oxide signalling. PMID- 7537405 TI - Reflections on Ca(2+)-channel diversity, 1988-1994. PMID- 7537407 TI - Excitotoxicity and the NMDA receptor--still lethal after eight years. PMID- 7537406 TI - Memories of NMDA receptors and LTP. PMID- 7537408 TI - Calcium: still center-stage in hypoxic-ischemic neuronal death. PMID- 7537409 TI - The basal ganglia. PMID- 7537410 TI - The functional anatomy of disorders of the basal ganglia. PMID- 7537411 TI - Phospholipase A2 and G proteins. PMID- 7537412 TI - Immediate-early genes: ten years on. PMID- 7537413 TI - 5-HT receptors: past, present and future. PMID- 7537414 TI - Opioid receptors: past, present and future. PMID- 7537415 TI - Neurotransmitter regulation of neuronal outgrowth, plasticity and survival in the year 2001. PMID- 7537416 TI - Neuronuggets: a selection and a prediction. PMID- 7537417 TI - The status of the neural segment. AB - A crucial phase of development in the vertebrate rhombencephalon involves transient organization into segments. Recent studies on the diencephalon and telencephalon pose the question of whether segmentation might also play a role in the development of more rostral brain regions. Criteria for segmentation formulated for the hindbrain might be met by the diencephalon, although there is disagreement as to the number and arrangement of segmental units. In contrast to the hindbrain, these segments appear when neurogenesis has begun, and might represent definitive functional units. Regarding the telencephalon, it is at present unclear whether domains of gene expression are associated with other features that are characteristic of segmental development, or whether other mechanisms control specification of this region. PMID- 7537418 TI - Molecular mechanisms of inherited startle syndromes. AB - Inherited neurological disorders involving an exaggerated startle response to unexpected sensory stimuli have been identified in mice, cows, dogs, horses and humans. Recent studies of the molecular pathology of a number of these startle syndromes have revealed that they are caused by defects in the inhibitory glycinergic pathways that mediate reciprocal and recurrent inhibition in the spinal cord. These defects arise from various mutations of the receptor for glycine, which either impair its sensitivity to agonists or reduce its expression in vivo. The emergent models of the molecular mechanisms that underlie startle disorders illustrate how diverse mutations can converge physiologically to produce a common phenotype. PMID- 7537419 TI - Cytokines and the nervous system. I: Expression and recognition. AB - Cytokines are a heterogeneous group of polypeptide mediators that have been associated classically with activation of the immune system and inflammatory responses. An increasing number of related mediators is now included in this category and most of them have been shown to act on a variety of tissues, including the PNS and CNS. Cytokines and their receptors are expressed in tissues of these nervous systems, and might derive from invading immune, or resident, cells. Trauma in peripheral tissues might also induce cytokine-mediated events in the CNS, via either the circulation or secondary induction within the brain. In this first of a two-part review, the general properties, expression and recognition of these cytokines with respect to the nervous system are discussed. PMID- 7537420 TI - Exocytotic Ca2+ channels in mammalian central neurons. AB - Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters. PMID- 7537421 TI - Do nerve impulses penetrate terminal arborizations? A pre-presynaptic control mechanism. AB - Is there clear evidence that action potentials travelling in a parent axon reach all of their terminals? In spinal afferents, many fibres extend their arborizations beyond the area in which cells can be shown to respond to the afferents. In addition, impulses fail to propagate in the long-range reach of myelinated fibres in dorsal columns when a GABA-operated Cl- shunt in the terminals is operating. When the mechanism is immobilized by antagonists of GABAA receptors, impulse blockade is relieved. This suggests an additional control mechanism, which is located in axons proximal to the synaptic area. Such a control could focus or defocus those parts of a terminal arborization that affect postsynaptic cells. PMID- 7537422 TI - TiPS on nomenclature. PMID- 7537423 TI - [Prostatic adenomectomy in patients with a history of myocardial infarct, acute cerebral circulatory disorder and pulmonary thromboembolism]. AB - Immediate and long-term outcomes of adenomectomy have been analyzed for patients with prostatic adenoma with a history of 1-3 myocardial infarctions (185 subjects), 1-2 episodes of cerebral circulation disorder (74 cases) on pulmonary artery thromboembolism (3 cases). The surgery was urgent because of acute urine retention for 88 patients, while it was scheduled in 174 patients. The postoperative developments were uneventful in 84.9%, 87.8%, 100% of postinfarction, postapoplectic and post-thromboembolism patients, respectively. Postoperative lethal outcomes occurred in 6.5% of patients with postinfarction cardiosclerosis, 5.4% of those with postapoplectic complications. As shown by long-term follow-up available for 225 patients, 19 of them died (8.4%), 296 survived 1-11 years after the surgery. One more myocardial infarction developed in 17, apoplexy in 3 patients. Long-term functional results of adenomectomy can be considered good in most of the patients in absence of a significant progression of cardiovascular disease. The authors think adenomectomy not contraindicated and promising in terms of short- and long-term outcomes if made in above kind of patients 4, 6 and 9-12 months after myocardial infarction, pulmonary artery thromboembolism and disturbance of cerebral circulation, respectively. PMID- 7537424 TI - Influence of codon usage and translational initiation codon context in the AcNPV based expression system: computer analysis using homologous and heterologous genes. AB - Codon usage by all the known gene sequences from Autographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (beta hCG) expressed to contrasting levels in the baculovirus system. The highly expressed luc gene showed a codon usage similar to AcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed beta hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base position. Alignment of the 20 nucleotides around the initiation codon of 23 AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system. PMID- 7537426 TI - MspI8, the repetitive sequence specifically interacting with nuclear matrix of rat testis cells. AB - The nuclear matrix bound DNA fraction of rat testis showed enrichment in repetitive sequences found in the 450 bp band after gel electrophoresis of the MspI digested rat DNA. DNA fragments isolated from this band were cloned. DNA of the clone pMspI8 showed homology to some representatives of rat LINE sequence family, and complexed in vitro more efficiently with testes nuclear matrix proteins than with yeast ARS1 sequence containing the matrix association region (MAR) or DNA from an other clone, MspI19. Western blot analysis showed that MspI8 sequence interacts with testes matrix protein of about 120 kDa. PMID- 7537425 TI - Prevention of amino acid racemization during guanidination--a prerequisite for measurement of protein digestibility by homoarginine labeling. AB - Homoarginine labeling (guanidination) is used to calculate true prececal protein digestibility. A particular worry is that guanidination of proteins at alkaline pH might cause formation of D-amino acids. If D-amino acids show decreased protein digestibility in vivo, as seen in vitro, then the homoarginine method would underestimate protein digestibility. Therefore, the degree of protein racemization was measured during guanidination of casein at pH values between pH 9 and 11 and temperatures between 4 degrees and 65 degrees C. Optimal conditions for the guanidination reaction were 4 degrees C and pH 10.5-11 or 22 degrees C and pH 10. A higher pH value at 22 degrees C or temperatures above 22 degrees C at each pH leads to the formation of appreciable amounts of D-amino acids. PMID- 7537427 TI - The dynamics of beta 1 integrin expression during peripheral nerve regeneration. AB - The aim of the present study was to examine the expression of beta 1 integrin subunit after peripheral nerve transection. After sciatic nerve transection two experimental procedures were used; changes in the freely regenerating rat sciatic nerve were compared to a situation in which spontaneous regeneration was prevented by suturing both ends of the nerve to the muscle next to the point of transection. Specimens for morphological analysis were collected 6 h, 1, 3, 5, 7 days and 2, 4, 6 and 8 weeks after the axotomy. Sections from the proximal (two zones) and distal (three zones) stumps next to the point of transection were stained with antibodies against beta 1 integrin subunit, macrophages, collagen types I and III, and S-100 protein. The control nerves showed beta 1 integrin stained cells in the perineurium and vasa nervorum but the endoneurium was negative. Positively stained endoneurial fibroblast-like cells could be seen in the proximal part of the nerve already at 24 h after transection. The number of these positively stained cells increased steadily; they were most numerous 4 weeks after transection in the distal zone 2. Subsequently, the number of positively stained endoneural cells declined sharply and 8 weeks after transection no positively stained cells could be found. The morphological appearance and the immunohistochemical properties of the cells suggest that the majority of beta 1 integrin-positive cells are endoneurial fibroblast-like cells. Thus, the process appeared to be dynamic, starting from the proximal part and continuing to the distal parts, and was similar in both experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537429 TI - Maternal serum alpha-fetoprotein screening for neural tube defects. Report of a program with more than 30000 screened pregnancies. AB - Maternal serum alphafetoprotein (MSAFP) screening has been set up in Asturias, in the north of Spain, in 1987 in order to make possible the prenatal diagnosis of neural tube defects (NTD) to overall pregnancy population. This large study shows the high sensitivity and specificity of MSAFP screening when it is done with absolute control of all variables such as gestational age, pregnant woman's weight, diabetes, etc. On the other hand, this study also shows a poor sensitivity second level ultrasound for the early diagnosis of NTD in the presence of spina bifidas with no bulge. We have also observed that the incidence of NTD in Asturias remained constant in the last six years, around 1.5-1.6 per 1000 pregnancies. However, due to MSAFP screening, there has been a decline in the prevalence of children born with these defects. We conclude that MSAFP screening is the best tool to identify and reduce NTD in our Region (Spain). PMID- 7537428 TI - Coexpression of stem cell factor and its receptor c-Kit in human malignant glioma cell lines. AB - Stem cell factor (SCF), a hematopoietic growth factor, is the ligand of the tyrosine kinase receptor encoded by the c-kit proto-oncogene. Beside the important role of this receptor-ligand complex in hematopoiesis, gametogenesis and melanogenesis, SCF and its receptor have been shown to be expressed in the brain. We have studied the expression of SCF and c-kit in 20 human malignant glioma cell lines at the mRNA as well as at the protein level. In addition, recombinant human (rh) SCF was tested in [3H]thymidine uptake assays for a mitogenic effect on these cells. SCF and c-Kit proteins were detected in the cytoplasm of glioma cells by alkaline phosphatase-monoclonal anti-alkaline phosphatase immunostaining and Western blot analysis. However, neither SCF nor c Kit were seen on the cell surface by flow cytometry. Furthermore, none of the proliferation assays showed a mitogenic effect for exogenously added rhSCF. Blocking studies using an anti-SCF antibody failed to demonstrate modulating effects on the growth of selected cell lines. These results suggest that SCF and c-Kit may mediate non-proliferative signals or may employ intracellular mechanisms for autocrine growth regulation of glioma cells. PMID- 7537430 TI - The diagnostic value of amniotic fluid Gram stain examination and limulus amebocyte lysate assay in patients with preterm birth. AB - The purpose of this study was to determine the value of Gram stain examination and Limulus amebocyte lysate (LAL) test in the detection of intraamniotic infection. Ninety women with preterm labor and intact membranes (n = 55) or preterm premature rupture of membranes (PROM) (n = 35) who delivered prematurely were included in the study. Amniotic fluid was cultured for aerobic and anaerobic bacteria as well as for mycoplasmas. Amniotic fluid analysis included Gram stain examination and limulus amebocyte lysate tests. The prevalence of positive amniotic culture was 32.2% (29/90) and the most common isolate was Ureaplasma urealyticum. Patients with preterm PROM group had a higher rate of infection than those with preterm labor and intact membranes 57.1% (20/35) vs. 16.4% (9/55), respectively (p = 0.0001). We found a lower gestational age at delivery and lower mean birth weight in neonates born to mothers with a positive amniotic fluid culture than those with negative amniotic fluid culture. The combined use of Gram stain examination and LAL test had a sensitivity and specificity of 51.7% (15/29) and 95.1% (58/61) respectively for the detection of positive amniotic fluid culture. We conclude that Gram stain examination and LAL test are rapid, simple and specific tests that can be used to detect microbial invasion of the amniotic cavity except in patients with mycoplasmas infections. PMID- 7537431 TI - Activation of xenobiotics into free radicals by prostaglandin-H-synthase and by rat liver microsomes. PMID- 7537432 TI - Antibodies to ICAM-1 ameliorate inflammation in acetic acid induced inflammatory bowel disease. PMID- 7537433 TI - A better understanding of anti-inflammatory drugs based on isoforms of cyclooxygenase (COX-1 and COX-2). PMID- 7537434 TI - Thromboxane A2 synthase inhibitor, DP-1904, decreases TNF alpha secretion from monocytes and inhibits E-selectin and ICAM-1 expression on the endothelial cell surfaces. PMID- 7537436 TI - Effects of 5-oxo-ETE on neutrophils, eosinophils, and intestinal epithelial cells. PMID- 7537435 TI - Augmented platelet-endothelial adhesion induced by PGI2 receptor desensitization. PMID- 7537437 TI - Modulation of EGF cell signaling tyrosine phosphorylation by linoleic acid metabolites. PMID- 7537438 TI - Streptokinase-prostacyclin--nitric oxide: in vivo interactions. PMID- 7537440 TI - Nitric oxide and the cyclooxygenase pathway. PMID- 7537439 TI - Prostaglandin-nitric oxide interactions in the microcirculation. PMID- 7537441 TI - Nitric oxide modulates angiogenesis elicited by prostaglandin E1 in rabbit cornea. PMID- 7537442 TI - On the mechanism of activation of PLA2 and PtdIns-PLC by fluoride in murine macrophages. PMID- 7537443 TI - Intrapericardial basic fibroblast growth factor induces myocardial angiogenesis in a rabbit model of chronic ischemia. AB - The objective of this study was to determine whether basic fibroblast growth factor (bFGF), a known angiogenic factor, can promote new vessel growth when infused within the pericardial space in a model of chronic myocardial ischemia. Intravenous angiotensin II (AII) was infused to induce left ventricular hypertrophy and concomitant ischemia in New Zealand white rabbits. Basic FGF was infused into the intrapericardial space with an osmotic pump. Animals were assigned to one of four groups: group 1 received intrapericardial bFGF and intravenous AII, group 2 received intrapericardial bFGF and intravenous saline solution, group 3 received intrapericardial albumin and intravenous AII, and group 4 received intravenous AII only. Epicardial angiogenesis was graded histologically on a scale of 0 to 2. Animals receiving intravenous administration of AII displayed left ventricular hypertrophy that disproportionately affected the interventricular septum with a wall thickness of 5.62 +/- 1.00 mm versus 3.98 +/- 0.61 mm in the AII group and the saline solution control group, respectively (p < 0.005). A highly localized angiogenic effect of bFGF was observed. The mean angiogenesis scores were 1.9, 1.4, 1.3, and 0.2 (p < 0.001) with an angiogenesis score of 2 (marked increase in vascularity) noted in 86%, 40%, 43%, and 0% of hearts in groups 1 through 4, respectively. We conclude that intrapericardial bFGF enhances new epicardial small-vessel growth in a rabbit model; furthermore this effect is enhanced in the presence of left ventricular hypertrophy. PMID- 7537446 TI - Fluctuations in serum amylase in patients with macroamylasemia. AB - OBJECTIVE: To report wide fluctuation of serum amylase in patients with macroamylasemia. It has generally been considered to remain constant. METHODS: Over the past 16 y, 18 patients have been diagnosed with macroamylasemia in our GI department. Of these, four patients were followed up with serial serum amylase determinations for a period of less than 1-4 y. Serum amylase was measured by the "Phadebas amylase test." Serum macroamylase was measured by "PEG precipitation technique." RESULTS: There was a wide fluctuation of serum amylase in three out of four patients. In the fourth patient, more persistent hyperamylasemia was noted during the shorter observation period. CONCLUSION: Marked fluctuation in serum amylase, ranging from 115 to 1160% in this study, may occur in patients with macroamylasemia. The reasons for these fluctuations are not clear but may be due to association-dissociation of amylase with serum proteins at variable time intervals. This fluctuation, especially when the amylase becomes normal (as in cases 1 and 3), may lead to confusion in differentiating macroamylasemia from other causes of hyperamylasemia. PMID- 7537447 TI - Cyclic nucleotides and vasoactive intestinal peptide production in a rabbit model of Escherichia coli septicemia. AB - Nitric oxide and vasoactive intestinal peptide (VIP) are potent vasodilators and postulated as inducers of hypotension. These mediators activate guanylate cyclase and adenylate cyclase, respectively, with subsequent biosynthesis of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) producing vascular smooth muscle relaxation and vasodilatation. Cyclic nucleotides and VIP were evaluated during Escherichia coli septicemia in two groups of rabbits; 1) sepsis alone and 2) sepsis and a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine. Arterial blood was obtained for determination of bacteremia, lactic acidemia, nucleotides, nitrites, and VIP levels. Significant bacteremia, endotoxemia, tachycardia, lactic acidosis, and hypotension occurred in all animals (P < 0.005). Circulating blood levels of cGMP, nitrites, cAMP, and VIP (P < 0.005) increased with development of shock. The NG-monomethyl-L-arginine treated animals had less cGMP, nitrites, cAMP, and VIP produced (P < 0.01). Plasma cGMP levels remained stable, suggesting that stimulated phagocytes in whole blood were responsible for increased cGMP levels. Infusion of VIP produced profound hypotension and lactic acidemia. Results of these experiments provide definitive evidence that nitric oxide and VIP are mediators during septic shock and their messengers are cGMP and cAMP, respectively. In addition, phagocytic stimulation with increased production of cGMP may initiate shock, with these mediators acting synergistically to prolong hypotension. PMID- 7537444 TI - Tacrolimus: a potential new treatment for autoimmune chronic active hepatitis: results of an open-label preliminary trial. AB - Autoimmune chronic active hepatitis (CAH-A) is a chronic liver disease of unknown etiology that is believed to have an autoimmune pathogenesis. The disease is slowly progressive until hepatic failure and portal hypertension develop and either death or liver transplantation occur. Currently, the only widely recognized therapy is the administration of glucocorticoids, which have both anti inflammatory and immunosuppressive actions. Many patients cannot tolerate such therapy because of the psychiatric, osteoporotic, and weight-enhancing actions of steroids. Tacrolimus (FK 506) is a new macrolide antibiotic that has an immunosuppressive activity that is estimated to be 10-200 times greater than that of cyclosporine. Because of its greater immunosuppressive activity, we have used it in the treatment of 21 patients with autoimmune chronic active hepatitis. Before each subject was treated, a liver biopsy and a panel of hematological, serological, and biochemical parameters were assessed. The Tacrolimus was administered orally at 12-h intervals, and the dose was controlled by monitoring plasma FK trough levels. After 3 months of therapy at an oral dose of 3 mg twice a day, having achieved a median blood level of 0.5 ng/ml, the serum ALT level was reduced by 80%, and the AST level was reduced by 70%. Modest change in the white blood cell count and platelet count were noted. The median BUN level increased from a level of 12 to 18 mg/dl, and the serum creatinine increased from 0.9 to 1.3 mg/dl. These preliminary data demonstrate that: 1) Tacrolimus can be used to successfully treat CAH-A; 2) the response of CAH-A to Tacrolimus treatment is rapid and sustained; and 3) a minor increase in the serum BUN and creatinine levels occurs as a consequence of Tacrolimus treatment. It is anticipated that with continued treatment for periods of 1-2 yr, the natural history of CAH-A will be changed such that hepatic failure and the requirement for liver transplantation may be averted. PMID- 7537445 TI - Transmission of hepatitis C virus by health care workers in a rural area of Japan. AB - OBJECTIVE: The prevailing route of hepatitis C virus (HCV) transmission in Japan is unclear, so we expected that a survey of routes of HCV infection in an endemic area might clarify the mode of transmission. METHODS: We screened 2049 inhabitants in an isolated rural village of Fukuoka, Japan using HCV markers. All serum samples were assayed for antibody to HCV (anti-HCV) (ELISA), and anti-HCV positive samples were assayed for HCV RNA and genotype (polymerase chain reaction). RESULTS: The prevalence of anti-HCV was 19.7% and increased with advancing age, from zero in the under 19 yr of age group to 30.7% in the 60-69 yr of age group. HCV RNA was detected in 82.9% of the anti-HCV-positive inhabitants, and 88.3% of them had genotype II. Anti-HCV was detected in 17.8% of the children of HCV-carrier mothers, similar to the rate (15.4%) for all inhabitants in the 40 49 yr of age group, the same age group for the mean age of the anti-HCV-positive children. Anti-HCV was detected in 34.8% of husbands of female HCV carriers and in 22.2% of wives of male HCV carriers, similar to the rates (36.2% in males and 26.6% in females) for all inhabitants in 60-69 yr of age group, the same age group for the mean ages of carriers' spouses. The prevalence of anti-HCV was the highest in inhabitants of one of three distinct areas of this village where patients had often been injected with insufficiently sterilized syringes and needles for treatment in the same clinic. CONCLUSION: These observations suggest that medical intervention probably played a more important role in the spread of the HCV infection in the village studied than did familial transmission. PMID- 7537449 TI - Diabetes insipidus as an early sign of pineal tumor. AB - A 13-year-old boy's acute onset of diabetes insipidus was the first manifestation of a pineal tumor. One year after the physical symptoms of diabetes insipidus occurred, neurological examinations, tumor markers, and computed tomography brain scan were all normal. Three years later, rapid development of neurological changes was observed with the prevailing symptoms of increased intracranial pressure. The changes were related to a pineal germ cell tumor identified by a high level of beta-human chorionic gonadotropin in plasma and neuroradiological investigations, including computed tomography and magnetic resonance imaging. The clinical course of this case is unusual and we advocate the following: (1) the follow-up of children with diabetes insipidus is important for determining whether or not it is an idiopathic type and (2) elevation of plasma beta-human chorionic gonadotropin and development of neurological signs in patients with so called idiopathic diabetes insipidus should arouse clinical suspicion of an intracranial germ cell tumor. It should be noted that the brain tumor is significant as the underlying pathology of childhood diabetes insipidus. PMID- 7537448 TI - Ovine bone marrow extracellular matrix and soluble protein extraction: fetuin amino terminus microheterogeneity. AB - In this article, the authors describe a new method using phosphate-buffered saline for the initial extraction of extracellular matrix (ECM) and soluble proteins from hematologic tissues. Direct comparisons between this method and previously reported methods demonstrate superior total protein yields with the novel technique in a fraction of the time for these ovine hematologic tissues: bone marrow, marrow aspirate, spleen, liver, and blood. Analysis by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining demonstrate comparable protein patterns with this method and a previously reported method. This method also successfully extracts the ECM glycoproteins fibronectin and laminin as well as the proteoglycan, chondroitin sulfate from marrow. These findings are demonstrated by Western and dot blotting. Bone marrow ECM and soluble proteins extracted by the novel method stimulate myeloid progenitor growth in a methylcellulose assay. Using an assay for elastase inhibitory capacity, the authors demonstrate that alpha 1-proteinase inhibitor, the principal inhibitor of neutrophil elastase, is present and that its activity is preserved in bone marrow samples extracted with this team's method. In contrast, very low total protein yields are obtained with the method used previously to recover hemonectin, a rabbit bone marrow ECM granulocytic cytoadhesion molecule. This team's novel procedure, which extracts ECM and soluble proteins from small samples of tissue in a rapid, efficient, and reproducible manner, greatly enhances the analysis of these proteins from tissue culture, animal, and human clinical samples. In addition, with purification by chromatofocusing chromatography and molecular sieving gel electrophoresis, N terminal amino acid sequencing could be performed on a developmentally regulated marrow protein with biochemical properties similar to those of hemonectin and the plasma protein fetuin. The authors propose that this novel technique be used for the initial extraction of ECM and soluble proteins from hematologic tissues and that subsequent, definitive recovery of insoluble proteins be accomplished using previously reported, though less efficient, methods. PMID- 7537450 TI - Complementary modalities/Part 2. Relaxation techniques for surgical patients. PMID- 7537451 TI - Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. AB - Activation of protein kinase C (PKC) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated fluid secretion in rat pancreatic ducts (N. Ashton, R. L. Evans, and B. E. Argent. J. Physiol. Lond. 452: 99P, 1992). Using the patch-clamp technique, we have investigated whether this inhibition of fluid secretion results from an effect of PKC on cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels. Exposure to 100 nM 4 beta-phorbol 12,13 dibutyrate (PDBu) had no effect on CFTR current density in unstimulated duct cells, but caused a 31% increase in the magnitude of CFTR currents recorded from cells stimulated with cAMP. Furthermore, prolonged (2-4 h) exposure of stimulated duct cells to 100 nM PDBu (a condition that should downregulate PKC) significantly slowed the rate at which CFTR currents run down after establishing a whole cell recording. A similar effect was observed with calphostin C (500 nM), a specific inhibitor of PKC. Thus, although inhibition of ductal fluid secretion by PDBu is unlikely to be explained by an effect on CFTR, modulation of PKC activity can affect both the magnitude and stability of CFTR currents in pancreatic duct cells. PMID- 7537452 TI - cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein. AB - Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway. PMID- 7537453 TI - Regulation of skeletal alpha-actin promoter in young chickens during hypertrophy caused by stretch overload. AB - Anterior latissimus dorsi (ALD) muscles of 3-wk-old male chickens were injected with plasmids containing various lengths of the chicken skeletal alpha-actin promoter (ranging from -2,090 to -77 relative to the transcription start site) driving luciferase. Hypertrophy of the left ALD muscle was induced by attaching a weight (11% of body wt) to the left wing of each chicken, with the unweighted contralateral wing serving the control. Six days of stretch overload significantly increased muscle mass 110%. Luciferase activity from the -2,090 actin-luciferase chimeric gene increased 127% compared with the contralateral control ALD muscle. Luciferase activities driven by the -424, -202, and -99 actin promoters were 179, 134, and 378% higher, respectively, in the stretched ALD muscle than in the contralateral control ALD muscle. Luciferase activity from the -77 deletion construct was not different between stretched and control muscles. These data indicate that the gene region responding to stretch is downstream of 99 and imply, but do not conclusively prove, that the region between -99 and -77, which contains serum response element 1, contributes to the stretch-induced increase in skeletal alpha-actin promoter activity in the ALD muscle. PMID- 7537454 TI - Moluccella laevis lectin, a marker for cellular differentiation programs in mouse gut epithelium. AB - We have assembled a system for testing the hypothesis that changes in glycoconjugate production represent markers for defining developmental, spatial, and environmental influences on the proliferation and differentiation programs of various mouse gut epithelial cell lineages. Multilabel immunohistochemical methods were used to survey the interactions of purified lectins with 1) normal fetal, neonatal, and adult FVB/N mouse gut, 2) gastric and intestinal isografts harvested at various developmental stages, and 3) transgenic mouse models of intestinal epithelial cell hyperplasia, dysplasia, and/or neoplasia. As a demonstration of the system's utility, we used the recently purified, alpha-N acetyl-D-galactosamine-specific, Moluccella laevis lectin (MLL). In the adult FVB/N mouse stomach, MLL only recognizes glycoconjugates produced by a population of nonproliferating neck and prezymogenic cells that occupy a pivotal point in the complex, migration-associated differentiation program of the zymogenic cell lineage. In the developing FVB/N stomach, MLL binds to members of the zymogenic and pit lineages even before morphogenesis of gastric units is completed. Expression of MLL epitopes in pit cells is restricted to the period before the gastric epithelium has completed its morphoregulatory program. Analysis of gastric isografts indicates that these lineage- and developmental stage-specific patterns of glycoconjugate accumulation are not influenced by normal luminal contents. In the adult FVB/N intestine, MLL binding can be used to operationally define variations in the differentiation programs of 1) members of the enteroendocrine and goblet cell lineages during their migration along the crypt to-villus axis and 2) cells comprising the follicle-associated epithelium overlying Peyer's patches. Accumulation of MLL epitopes in villus-associated enterocytes does not appear to be affected when these cells are induced to reenter the cell cycle by simian virus 40 large T antigen (SV40 TAg). MLL reactivity is not diminished when enterocytes begin to dedifferentiate as a result of production of SV40 TAg, human K-rasVal12, and a dominant negative human p53 mutant. The lack of change in MLL binding properties may reflect the brief residence time of enterocytes on the villus. These results indicate that glycoconjugate production represents a very useful tool for studying gut epithelial cell biology. Preliminary studies suggest that this is also true in the human gut. PMID- 7537455 TI - Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats. AB - Studies in acutely inflamed pancreatic tissue in humans and animals suggest that premature activation of proteases within the gland plays a key role in its pathophysiology. The present study aimed to detect such protease activation in relation to protease inhibition and to changes in the concentrations of the vital cellular compounds ATP and glutathione in pancreatic tissue during caerulein induced pancreatitis in rats. Within 1 h after supramaximal stimulation by intraperitoneal caerulein injection, pancreatic tissue activities of enzymatically active trypsin and elastase showed significant increases, accompanied by a twofold increase in trypsin inhibitory capacity. Over the same time course pancreatic ATP and glutathione concentrations dropped to 38% and 47%, respectively, after 1 h and reached a nadir of 22% and 28%, respectively, after 4 8 h. Intrapancreatic trypsin activation in this model, despite increasing trypsin inhibitory capacity, indicates concealed liberation of even more protease or enzyme-inhibitor complex instability. It is hypothesized that early acinar glutathione depletion, in part due to diminished ATP, could play a role in the premature activation of digestive enzymes by impairment of the integrity of the cytoskeleton and cell organelles or lowered defense capabilities against oxidant stress, finally leading to acute pancreatitis. PMID- 7537458 TI - Increased expression of sulfated gp300 and acinar tissue pathology in pancreas of CFTR(-/-) mice. AB - The CFTR (-/-) mouse model of cystic fibrosis (CF) has revealed that the mouse pancreatic duct has a Ca(2+)-regulated chloride conductance that allows ductal electrolyte transport to remain unaffected by loss of the cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, this model provides a unique opportunity to investigate effects of CF on the acinar tissue. It has been reported that exocrine secretions contain higher levels of sulfate in CF. We discovered in CFTR(-/-) acini that gp300, the major sulfated glycoprotein of the mouse acinar cell, has increased steady-state and biosynthetic levels. However, there are no apparent changes in sulfate or carbohydrate composition of gp300, indicating that posttranslational processing of this sulfated glycoprotein is not altered in CF. In addition to the increase in gp300, the morphology of CF acinar tissue is dramatically altered: acinar lumina of CFTR(-/-) mice are greatly dilated and filled with aggregated protein. gp300, which in the normal tissue is mainly localized to the zymogen granule membrane, was found to line the distended luminal membranes in the CF tissue. These results demonstrate that the acinar tissue is affected in the CF mouse and that expression of the major sulfated glycoprotein is also increased. It is suggested that increased expression of gp300 in CFTR(-/-) mice may cause poorly soluble exocrine protein secretion, contributing to the development of CF in the pancreas. PMID- 7537456 TI - Regulation and localization of the insulin-like growth factor system in small bowel during altered nutrient status. AB - Insulin-like growth factor-I (IGF-I) may regulate small bowel growth. Analyses here in ad libitum-fed, fasted, and refed rats demonstrate that during fasting and refeeding changes in jejunal mass correlate with changes in serum IGF-I and jejunal IGF-I mRNAs. These data indicate that circulating and locally expressed IGF-I contribute to nutrient regulation of jejunal mass. During refeeding, jejunal IGF binding protein 3 (IGFBP-3) mRNA abundance was reduced relative to that of IGF-I, possibly amplifying enterotrophic actions of IGF-I. Localization of IGFBP-3 to subepithelial cells in lamina propria of jejunum indicates that IGFBP-3 derived from lamina propria may modulate IGF-I action on adjacent epithelium. Ileum differed from jejunum in that refeeding did not increase bowel mass or IGF-I mRNA to ad libitum values. Differences in exposure to luminal nutrient may underlie distinct responses of the two segments. Rats fed elemental diet intravenously showed reduced jejunal mass but not reduced jejunal IGF-I mRNA compared with rats fed oral elemental diet. Elemental nutrient given intravenously or orally therefore does not differ in effects on jejunal IGF-I expression. Complex luminal nutrient may, however, regulate jejunal IGF-I expression. PMID- 7537457 TI - The selective beneficial effects of nitric oxide inhibition in experimental colitis. AB - We investigated the involvement of nitric oxide in trinitrobenzene-sulfonic acid (TNB) colitis. Every 24 h after TNB, rats were orally dosed with NG-nitro-L arginine methyl ester (L-NAME; 30 mg/kg), NG-nitro-D-arginine methyl ester (D NAME), or water, and food intake, body weight, and plasma nitrite levels were measured. On day 6, colonic nitric oxide synthase and myeloperoxidase (MPO) activity, histology, intestinal muscle growth, NADPH-diaphorase, and myenteric nerve function were assessed. Food intake and body weight were reduced during the first 72 h of colitis. On day 6 post-TNB, a fourfold increase in mucosal nitric oxide synthase, a 30-fold increase in MPO, and a fivefold elevation in plasma nitrite were measured. Smooth muscle hyperplasia and hypertrophy in both colonic muscle layers, numerous diaphorase-positive macrophages in the myenteric plexus, and a suppression of myenteric nerve function were also observed. Unlike D-NAME, oral L-NAME reduced MPO and intestinal muscle hyperplasia by > 90%. Likewise, plasma nitrite and colonic nitric oxide synthase were reduced by > 70%. L-NAME completely prevented macrophage infiltration into the muscle. Conversely, it had no effect on anorexia or intestinal smooth muscle hypertrophy, nor did it affect suppressed myenteric nerve neurotransmitter release. These results demonstrate the selective transmural protective effects of L-NAME in the inflamed colon, implicating nitric oxide as a mediator. PMID- 7537459 TI - NO and H2O2 mechanisms of guanylate cyclase activation in oxygen-dependent responses of rat pulmonary circulation. AB - Pulmonary hypoxic vasoconstriction appears to have both endothelium-dependent and -independent regulatory pathways. We have previously described a mechanism of guanylate cyclase activation in isolated pulmonary arteries that is smooth muscle contained and oxygen tension dependent. In this study we examine this mechanism, involving H2O2 metabolism by catalase, and its relationship to endothelial derived nitric oxide in the regulation of pulmonary artery pressure (PAP) by oxygen tension. Using probes selective for these two distinct mechanisms of guanylate cyclase activation, we found in the isolated buffer-perfused rat lung that 100 microM nitro-L-arginine (NLA), an inhibitor of NO formation, increased baseline PAP from 4.8 +/- 0.6 to 6.0 +/- 0.6 mmHg and hypoxic PAP from 6.8 +/- 0.8 to 8.56 +/- 0.6 mmHg. Aminotriazole (AT), an inhibitor of H2O2 metabolism by catalase, also increased PAP from 4.5 +/- 0.9 to 6.1 +/- 2.0 mmHg (P < or = 0.05) and hypoxic PAP from 6.0 +/- 1.7 to 8.7 +/- 2.7 mmHg (P < or = 0.05). Additionally, while NLA did not affect the vasodilation that occurs upon reoxygenation, AT inhibited the immediate response to reoxygenation. In the presence of both NLA and AT, baseline PAP increased from 4.25 +/- 0.8 to 9.9 +/- 0.92 mmHg (P < or = 0.05), but hypoxia did not significantly increase PAP and the reoxygenation response was inhibited. These data suggest that both NO and H2O2 catalase mechanisms contribute to a similar degree to maintain low PAP under normoxic conditions. The removal of either mediator may contribute to hypoxic vasoconstriction. PMID- 7537460 TI - Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung. AB - Peptidergic nerves in the respiratory tract release vasoactive intestinal peptide (VIP) and substance P (SP), which mediate physiological and immune functions. Antipeptide antibodies to type I VIP receptor (VIPR) and NK-1-type SP receptor (SPR) were used to identify these receptors in normal rat lungs. VIPRs and SPRs were detected on airway epithelium from the trachea to the respiratory bronchioles but not in alveoli, submucosal glands, or pulmonary smooth muscle, except for that of some pulmonary veins. VIPRs also were expressed on macrophages around capillaries, in tracheal and bronchial connective tissue, in alveolar walls, and in the subintima of pulmonary veins and some arterioles. The absence of receptors from airway smooth muscle and submucosal glands implies that mediation of some known effects of SP and VIP may be epithelial or macrophage dependent. Other types of VIPRs and SPRs on airway glands and smooth muscle may transduce direct effects. The similar localization of VIPRs and SPRs in rat lung suggests that VIP and SP may coordinately regulate some pulmonary functions. PMID- 7537461 TI - Constitutive endothelial nitric oxide synthase gene expression is regulated during lung development. AB - Nitric oxide (NO), a potent vasodilator, is a free-radical gas synthesized from L arginine by nitric oxide synthases (NOS). NO appears to have an important role in perinatal changes in pulmonary vascular resistance. We previously identified mRNA encoding the constitutive endothelial NOS (ceNOS) isoform in human pulmonary tissue. To begin investigating functions of this enzyme in perinatal pulmonary development, we measured ceNOS mRNA and immunoreactivity in the developing rat lung. With the use of RNA blot hybridization, abundant pulmonary ceNOS mRNA was detected during the late fetal and postnatal period. The highest levels were detected within 24 h after birth, and elevated mRNA levels persisted for 16 days. In contrast, much lower levels of ceNOS mRNA were found in adult rat lung. With the use of immunoblot techniques, ceNOS protein levels were found to be correlated with mRNA levels. To identify the pulmonary cell types expressing the ceNOS gene, in situ hybridization with a digoxigenin-labeled cRNA probe was performed on sections from lungs of 1-day-old and adult rats. In lungs from 1-day old rats, ceNOS mRNA was detected in alveolar and serosal epithelial cells as well as in endothelial cells lining small and medium-sized blood vessels. In contrast, in adult lungs, ceNOS gene transcripts were detected in rare endothelial cells. These observations suggest that ceNOS gene expression is regulated during lung development and that ceNOS is available to participate in the postnatal reduction of pulmonary vascular resistance. ceNOS gene expression in nonendothelial cells in the neonatal rat lung suggests that NO may also contribute to nonvascular functions in the developing lung. PMID- 7537462 TI - Ethanol stimulates apparent nitric oxide-dependent ciliary beat frequency in bovine airway epithelial cells. AB - The mucociliary apparatus of the lung provides an important host-defense function by clearing the upper airway of inhaled particles and infectious microorganisms. Because lung host defenses are impaired in alcoholics, we hypothesized that ethanol would decrease ciliary motility in airway epithelium. Ciliary beat frequency (CBF) was measured by videomicroscopy in primary cultures of ciliated bovine bronchial epithelial cells (BBECs). Ethanol rapidly stimulated ciliary motility in a time-dependent fashion with concentrations as low as 10 mM. No detectable decreases in ciliary motility were noted until ethanol concentrations exceeded 1,000 mM. Because many substances stimulate ciliary motility by releasing nitric oxide (NO) via upregulation of nitric oxide synthase (NOS), we preincubated ciliated BBECs with a stereospecific NOS inhibitor, NG-monomethyl-L arginine (L-NMMA). L-NMMA completely blocked ethanol-induced stimulation of CBF, which could be subsequently restored by adding either L-arginine or sodium nitroprusside, which is a direct NO donor. These results indicate that ethanol, at clinically relevant concentrations, stimulates the release of NO by airway epithelium that upregulates ciliary motility. The rapidity of this response suggests upregulation of the constitutive NOS, known to be present in airway epithelium, and may explain the increases in mucociliary clearance observed in previous studies of ethanol ingestion in animals and in humans. These data also suggest a novel signal transduction pathway, the NO/NOS system, by which ethanol may exert some of its diverse biologic effects. PMID- 7537463 TI - Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. AB - Hyperoxia causes a reproducible pattern of lung injury and repair in rodents, in which proliferation of alveolar epithelial cells (AEC) and fibroblasts is observed during recovery. We postulated that if quiescent cells are stimulated to reenter the cell cycle, then cyclin expression and cyclin-dependent protein kinase activity would be reactivated in AEC during the repair process after hyperoxic lung injury. To test this hypothesis, we exposed adult rats to short term hyperoxia, followed by recovery for various times in room air. Cellular proliferation in vivo was confirmed by 1) flow cytometric analysis of DNA content (FACS) of freshly isolated AEC and 2) immunohistochemistry of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA on lung sections. The percentage of freshly isolated AEC in S phase and G2/M phase on FACS analysis increased twofold to a maximum of 16.5%, after 48 h in 100% oxygen and 48 h recovery in air. Cyclins A and D and p34cdc2 protein expression were also increased during the recovery period; while p33cdk2 and p34cdk4 increased only slightly. p34cdc2 histone H1 kinase activity, both in whole lung and in AEC, decreased initially after 48 h in oxygen. However, a marked increase in p34cdc2 kinase activity was observed at 48 h recovery in whole lung and returned to baseline by 72 h. In isolated and cultured AEC, p34cdc2 kinase activity was maximal at 24 h of recovery in air. We conclude that cyclins A and D and p34cdc2 protein expression and p34cdc2 kinase activity are increased in vivo during recovery from hyperoxic lung injury in both adult rat lungs and in AEC isolated from these lungs. We speculate that the induction of cyclin-dependent protein kinase activity is a key event in mediating the proliferative cellular repair response to lung injury. PMID- 7537464 TI - Aberrant processing of surfactant protein C in hereditary SP-B deficiency. AB - Hereditary surfactant protein B (SP-B) deficiency causes lethal neonatal respiratory disease associated with abnormalities in pulmonary surfactant proteins and lipids. SP-C, a 4-kDa hydrophobic protein produced from a 21-kDa precursor, cooperates with SP-B to enhance the surface active properties of surfactant phospholipids. Anti-proSP-C polyclonal antisera were produced against fusion proteins containing 1) the amino terminus (amino acids 1-20), 2) the region carboxy-terminal to the mature SP-C peptide (amino acids 58-77), and 3) full-length 197-amino acid proSP-C and were characterized using immunoprecipitation, Western blot, and immunohistochemical techniques. Western blot analysis of bronchoalveolar lavage and amniotic fluid from hereditary SP-B deficient patients allowed identification of a 12-kDa form of SP-C that contained epitopes consistent with the amino-terminal and active peptide regions of SP-C (amino acids 1-57). The 12-kDa SP-C peptide was not detected in bronchoalveolar lavage from healthy adults or adults with alveolar proteinosis or pneumonia. We conclude that SP-B deficiency is associated with the aberrant processing and secretion of an immature SP-C peptide, which may contribute to the respiratory failure associated with hereditary SP-B deficiency. PMID- 7537465 TI - Glucocorticoid stimulation of fatty-acid synthase gene transcription in fetal lung: antagonism by retinoic acid. AB - Glucocorticoid hormones are known to stimulate the rate of fatty acid biosynthesis and to increase the activity and mRNA level of fatty-acid synthase (FAS) in late gestation fetal lung. We have now examined the effect of dexamethasone on FAS transcription in fetal rat lung. Explants of 19-day fetal rat lung cultured for 48 h in serum-free medium were exposed to dexamethasone (10(-7) M) for various time periods. Nuclei were isolated, and the rate of [32P]UTP incorporation into FAS and gamma-actin RNA transcripts was measured by transcription-elongation assay. Dexamethasone increased FAS transcription but had no effect on that of actin. The maximum effect of the hormone, approximately threefold increase, was observed 1-2 h after addition of the hormone but was still apparent up to 48 h. FAS transcription but not that of actin was inhibited by cycloheximide and puromycin in both control and dexamethasone-treated cultures. However, the stimulatory effect of the hormone was not significantly reduced by the inhibitors. Retinoic acid antagonized the stimulatory effects of dexamethasone on FAS activity, mRNA content as measured by Northern analysis, mass as measured by Western blotting, and rate of transcription. The effect of retinoic acid was dependent on concentration in the relatively narrow range of 5 x 10(-6) to 5 x 10(-4) M. These data show that glucocorticoids stimulate transcription of the FAS gene in late gestation fetal rat lung, that normal transcription of the FAS gene is dependent on ongoing protein synthesis, and that glucocorticoid stimulation of FAS gene expression is antagonized by retinoic acid. PMID- 7537466 TI - ANF and angiotensin II interact via kinases in the proximal straight tubule. AB - Atrial natriuretic factor (ANF) inhibits fluid absorption (Jv) in the proximal straight tubule (PST) only after stimulation with angiotensin II (ANG II). To investigate ANF's dependency on ANG II for transport inhibition, we blocked and mimicked angiotensin's second messenger cascades and then examined ANF's ability to inhibit Jv. ANG II (10(-10) M)-stimulated Jv was 0.47 +/- 0.10 nl.mm-1. min-1. After ANF (10(-10) M) was added to the bath, Jv fell by approximately 40% (P < 0.05). ANG II stimulates Jv via activation of protein kinase C (PKC) and decreasing protein kinase A (PKA) activity. We inhibited PKA with H-89. In the presence of only H-89, Jv was 0.75 +/- 0.11 nl.mm-1.min-1. After ANF was added to the bath Jv fell by 30% (P < 0.05). Intracellular adenosine 3',5'-cyclic monophosphate content was not affected by ANF in the presence of ANG II. ANF could not inhibit Jv in the presence of ANG II and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. KT-5823, a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase inhibitor, blocked the action of ANF on Jv (P > 0.30). PKC inhibition did not prevent the decrease in Jv induced by ANF. We conclude that ANF inhibits ANG II-induced stimulation of transport by a mechanism that requires phosphorylation mediated by cGMP-dependent protein kinase subsequent to a decrease of PKA activity. PMID- 7537467 TI - Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells. AB - The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis. PMID- 7537468 TI - Tumor necrosis factor induces E-selectin production in splanchnic artery occlusion shock. AB - Splanchnic arteries were clamped for 45 min to induce splanchnic artery occlusion (SAO) shock in anesthetized rats. Sham-operated animals were used as controls. Survival time, serum tumor necrosis factor-alpha (TNF-alpha), white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase (MPO) activity, and serum levels of soluble E-selectin (sE-selectin) were investigated. SAO-shocked rats exhibited decreased survival time (95 +/- 11 min, whereas sham-shocked rats survived for > 5 h), reduced mean arterial blood pressure, increased serum levels of TNF-alpha (185 +/- 8 U/ml) and MPO activity in the ileum (0.11 +/- 0.03 U x 10(-3)/g tissue) and lung (1.5 +/- 0.4 U x 10(-3)/g tissue), leukopenia, and enhanced serum levels of sE-selectin. Furthermore SAO-shocked rats showed histological alterations in the ileum and lung. Administration of cloricromene (2 mg/kg i.v.), an inhibitor of TNF-alpha, significantly increased survival time (225 +/- 10 min), decreased serum levels of TNF-alpha and sE-selectin, reduced leukopenia and MPO activity in the ileum (0.035 +/- 0.003 U x 10(-3)/g tissue) and lung (0.3 +/- 0.005 U x 10(-3)/g tissue), improved the cardiovascular changes, and reduced the histological changes in the ileum and lung. Finally, an anti-E-selectin antibody protected rats against SAO shock. Our findings are consistent with an involvement of E-selectin, "in vivo," in the pathogenesis of SAO shock. PMID- 7537469 TI - Modulation of reflex pressor response to contraction and effect on substance P release by spinal 5-HT1A receptors. AB - This study investigated whether activation of serotonin1A [5-hydroxytryptamine (5 HT)1A] receptors in the dorsal horn of the spinal cord attenuates the reflex pressor response to static contraction and passive muscle stretch. In addition, we determined if the attenuation of the response to contraction is mediated by inhibiting substance P (SP) release in the dorsal horn. Static contractions of the triceps surae muscle of chloralose-anesthetized cats were induced by stimulating the cut L7 and S1 ventral roots. Microdialysis (10 mM) of a selective 5-HT1A agonist [8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT)] into the L7 dorsal horn region produced a reversible attenuation of the reflex pressor response to a 1-min contraction (in mmHg: control = 36 +/- 3; 8-OH-DPAT = 17 +/- 3; recovery = 31 +/- 8; P = 0.013; n = 6) or passive stretch (in mmHg: control = 36 +/- 6; 8-OH-DPAT = 15 +/- 2; recovery = 32 +/- 6; P = 0.002; n = 6). However, a 5-HT1B agonist, 1-[3-(trifluoromethyl)-phenyl]piperazine, had no effect on the reflex pressor response. During 5-min contractions (n = 8), 8-OH-DPAT (10 mM) also blunted the pressor response but had no effect on the levels of SP-like immunoreactivity (in fmol/100 microliters: control = 0.492 +/- 0.026; 8-OH-DPAT = 0.501 +/- 0.034). These results suggest that activation of 5-HT1A receptors in the dorsal horn attenuates the reflex pressor response to contraction through a mechanism other than inhibition of SP release. PMID- 7537471 TI - Low-conductance anion channel activated by cAMP in teleost Cl- -secreting cells. AB - We studied characteristics and modulation of ion channels in primary cultures of opercular epithelium from the euryhaline marine killifish Fundulus heteroclitus. Primary cultures, 17-28 h old, retain mitochondria-rich Cl- cells identifiable by fluorescence microscopy. Cell-attached patches revealed frequent low-conductance 8.1 +/- 0.35 pS channels that usually became inactive on excision; high conductance anion channels were not apparent. Ion substitution experiments demonstrated selectivity for Cl- over gluconate of 1:0.07. With addition of 1 isobutyl-3-methylxanthine (0.1 mM) and dibutyryladenosine 3',5'-cyclic monophosphate (1.0 mM) to the bath, incidence of the channel increased from 35.3 to 61.9% of total patches (n = 156 and 21, respectively), and incidence of patches with multiple copies of the channel increased markedly from 2.2 to 38.5%. Epithelial Cl- transport was inhibited by mucosally added diphenylamine-2 carboxylic acid (1.0 mM) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1-1.0 mM). The anion channel was absent from cultured killifish corneal epithelium, a tissue that lacks Cl- cells. We conclude that a low-conductance anion channel of Cl- cells, likely in the apical membrane, may account for adenosine 3',5'-cyclic monophosphate-activated Cl- secretion by marine fish. PMID- 7537470 TI - Substance P increases microvascular permeability via nitric oxide-mediated convective pathways. AB - The goal of these studies was to examine the effects of substance P, a tachykinin neuropeptide, on pathways of microvascular permeability. Individual frog mesenteric venular capillaries were cannulated, and albumin apparent permeability coefficients (Ps) were determined by quantitative fluorescence microscopy. Ps of albumin (PsAlb) rose from 6.8 +/- 1.8 (SE) cm.s-1.10(7) at control to 22.3 +/- 2.3 cm.s-1.10(7) when substance P (10(-11) M) was perfused. The effect of increased microvessel permeability induced by substance P (10(-11) M) was blocked with the nonpeptide substance P receptor antagonist CP-96,345 and NG-nitro-L arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. PsAlb increased 0.99 cm.s-1.10(7) for every cmH2O increase in microvessel pressure after treatment of the vessel with substance P, demonstrating coupling of albumin flux to transvascular water flow. In conclusion, the mechanism of increased microvessel permeability in response to substance P appears to be the result of receptor-mediated increase in nitric oxide production and formation of water filled convective pathways presumably located between adjacent endothelial cells. PMID- 7537472 TI - Alterations in the insulin-like growth factor system in trauma patients. AB - The aim of the present study was to elucidate changes in the growth hormone (GH) insulin-like growth factor (IGF) axis in trauma patients throughout their stay in the surgical intensive care unit (SICU). The first venous blood sample was obtained within 24 h after admission to the SICU and before the start of nutritional support; the last sample was obtained within 24 h of each patient's discharge from the SICU. All patients were receiving nutritional support at this later time. Control subjects were healthy volunteers, matched for age and sex and fasted approximately 18 h before blood sampling. GH in trauma patients was increased 25-fold on the first day and was still elevated > or = 5-fold on the last day. Trauma decreased circulating levels of both IGF-I (50-60%) and IGF-II (33-45%) throughout the duration of the patients' stay in the SICU. A sustained reduction in plasma IGF-binding protein (BP)-3 (55-75%) was observed in trauma patients throughout the protocol. In contrast, IGFBP-1 levels increased more than threefold during this same period. Furthermore, IGFBP-1 in these patients had undergone posttranslational modification and existed primarily in a highly phosphorylated form. Blood, collected from a cohort (n = 3) of these patients within 24 h of their discharge from the hospital, indicated that IGF-I and IGF-II were still reduced (30%) and that the decrease in IGFBP-3 and the elevation in IGFBP-1 were still evident at this time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537473 TI - Method for the determination of cellular levels of guanosine-5'-diphosphate mannose based on a weak interaction with concanavalin A at low pH. AB - A rapid and simple two-step procedure for the quantitative analysis of GDP mannose (GDP-Man) recovered in ethanol extracts of cultured mammalian cells is described. GDP-Man is initially separated from water-soluble metabolites and other nucleotide sugars, including UDP-glucose (UDP-Glc) and GDP-fucose (GDP Fuc), due to a weak, alpha-mannoside-specific interaction with concanavalin A (Con A)-Sepharose at pH 3.5. The specificity and pH dependence of the GDP-Man-Con A interaction have been characterized. The partially purified fraction from Con A Sepharose can be further purified by high-performance anion-exchange chromatography on a Partisil-10 SAX silica gel column, and the concentration of GDP-Man was determined by monitoring the HPLC column eluate for absorbance at 254 nm. This procedure provides a simple means of calculating the specific activity of cellular GDP-[3H]Man pools, metabolically labeled with [2-3H]mannose. Using this new procedure, the relative rates of Glc3Man9Glc-NAc2-P-P-dolichol (Oligo-P P-Dol) biosynthesis and protein N-glycosylation were assayed in C 6 rat glial tumor cells, COS P6 cells, Chinese hamster ovary (CHO) cells, and mouse L929 cells by metabolic labeling with [2-3H]mannose. A comparison of the relative rates of incorporation of [2-3H]mannose into Oligo-P-P-Dol and N-linked oligosaccharides in four different cultured cell lines demonstrates that misleading results can be obtained if the calculation of the biosynthetic rates is not based on the specific activity of the nucleotide sugar pools. PMID- 7537474 TI - Characterization of post-translational modifications of brain tubulin by matrix assisted laser desorption/ionization mass spectrometry: direct one-step analysis of a limited subtilisin digest. AB - Matrix-assisted ultraviolet laser desorption/ionization (MALDI) mass spectrometry was used to investigate the molecular masses and heterogeneity patterns caused by post-translational modifications in tubulin from porcine brain. Direct analysis of the limited digest with subtilisn shows that the molecular masses of the majority of the carboxyterminal fragments are below 2 kDa, while the truncated tubulin subunits have lost approximately the same mass. The results confirm the cleavage sites previously postulated for this protease. The mass information on the peptides allows the degree of polyglutamylation to be measured directly and shows that molecules with two glutamyl residues in the side chain are the most abundant species. In addition it identifies the degree of tyrosination of alpha tubulin. This one-step monitoring of a complex digest provides information equivalent to that obtainable from the purified components, while the amount of material required is reduced by three orders of magnitude when compared to previous studies. MALDI spectra partially resolve the alpha and beta subunits of the highly homogeneous tubulin from turkey erythrocytes, which lacks polyglutamylation but does not separate alpha and beta subunits from the heterogeneous brain tubulin. Post-translational modifications of the brain tubulin result in shifting peaks to higher molecular masses, in broadening of the peaks, and in loss of resolution. PMID- 7537475 TI - A method to determine the minimal number of nucleotides required for the binding of a ribonucleoprotein to RNA. PMID- 7537476 TI - Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy-transfer fluorescent primers. AB - Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis. PMID- 7537478 TI - Polymerase chain reaction based genotyping for characterization of SLA-DQB and SLA-DRB alleles in domestic pigs. AB - Molecular genotyping of swine major histocompatibility complex SLA-DQB and SLA DRB genes using polymerase chain reaction (PCR)-based amplification is described. Locus-specific oligonucleotide primers were designed for the analysis of expressed SLA genes by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced, and the information gained was used to design primers for PCR genotyping of the exon 2 (beta 1) region from genomic DNA templates. A single segregating amplification product was detected for both DQB and DRB in all animals. PCR products were digested with restriction enzymes. Seven SLA-DQB PCR-restriction fragment length polymorphism (RFLP) pattern types were observed for both HaeIII and RsaI that defined 14 SLA-DQB alleles. A total of seven SLA-DRB PCR-RFLP pattern types were defined using MspI (3 RFLP pattern types) and RsaI (6 RFLP pattern types). In order to demonstrate their universal utility, the primers were tested on genomic DNA samples from 10 different swine breeds. No breed-specific alleles were observed. These results show that locus specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA-DQB and SLA-DRB from genomic DNA. PMID- 7537477 TI - A HindIII RFLP at the porcine inter-alpha-trypsin inhibitor locus (ITIH). PMID- 7537480 TI - [Roux loop in pancreatic surgery]. PMID- 7537479 TI - [Hydroxyethylstarch and osmotic nephrosis]. PMID- 7537481 TI - [Palliative endoscopic treatment of adenocarcinoma of Vater's ampulla: medium and long-term results]. AB - The palliative endoscopic treatment of tumors of the ampulla of Vater provides good short-term symptomatic results, while long-term results remain unknown. This study to assessed the course of 17 patients with carcinoma of the ampulla of Vater treated palliatively by endoscopy and monitored for a 5-year-period. From january 1985 to december 1989, 35 patients were diagnosed as having carcinoma of the ampulla of Vater. For 18 of them, curative surgery was performed, while for the 17 remaining 17 patients, palliative endoscopic treatment was proposed because of metastatic extension or surgical risk. Endoscopic treatment always included endoscopic sphincterotomy, and, in some cases, insertion of a biliary endoprosthesis. All patients were monitored until death or the end of follow-up on September 30, 1990. Endoscopic sphincterotomy was performed in 13 of the 17 patients, either alone in 10 cases, or with prosthesis in the other 3 cases. For the 4 other patients, endoscopic sphincterotomy could not be performed (large tumor in 2 cases, presence of duodenal diverticulum in 2 other cases). The 13 patients who underwent endoscopic treatment experienced rapid symptomatic improvement. Jaundice and cholangitis consistently recurred within a period of 1 to 44 weeks regardless of initial treatment. All recurrences except one, were successfully retreated by endoscopy (enlargement of initial sphincterotomy in 6 cases, insertion or replacement of prosthesis in 12 cases).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537482 TI - The effect of combined intravenous and oral clodronate treatment on bone pain in patients with metastatic prostate cancer. AB - Although osteosclerotic metastases are characteristic of prostate cancer, bone resorption is also accelerated. Clodronate is a specific inhibitor of osteoclastic bone resorption and relieves bone pain of osteolytic lesions in myelomatosis and breast cancer. The present open study included 16 prostate cancer patients who had painful bone metastases and who had failed hormonal therapy. Clodronate was given intravenously for six days (300 mg/day) followed by oral treatment for 21 days (3200 mg/day). A clear pain relief was found in nine of the 16 (56%) patients after intravenous administration. During the next three weeks with oral administration there was still pain reduction in five patients, while in three patients the pain increased. The treatment had no effect on conventional tumour markers but urinary hydroxyproline excretion decreased, indicating reduced bone resorption. Clodronate offers an alternative for treating patients with painful metastases from prostate cancer. PMID- 7537484 TI - Enhanced efficacy of Bleomycin adsorbed on silica particles against lymph node metastasis derived from a transplanted tumor. AB - A new drug delivery system using small silica (SI) particles which can adsorb Bleomycin (BLM) and are easily transferred to the lymphatics was established to deliver a large dose of anticancer agents to the regional lymph nodes. The effects of BLM-SI, BLM solution (BLM-SOL) and saline on transplanted tumor growth and lymph node metastasis were compared in animal experiments. The inhibitory effect on tumor growth and lymph node metastasis was more remarkable in the BLM SI group, the differences being statistically significant. The present study demonstrated that this new DDS using BLM adsorbed on SI particles was very useful for the control of transplanted tumor and lymph node metastasis. PMID- 7537483 TI - DT-5461a, an antitumor synthetic lipid a analog, causes selective blood flow reduction in tumor tissue. AB - We previously reported that a synthetic low-toxicity lipid A analog, DT-5461a, exhibited a significant antitumor effect was characteristically accompanied by extensive tumor necrosis, suggesting that DT-5461a causes a local circulatory disturbance in tumor tissues. In this study, we investigated the effect of DT 5461a on regional blood flow in various organs including tumor tissue with a radiolabeled tracer-distribution technique using 14C-iodoantipyrine. Intravenous administration of DT-5461a induced blood flow reduction in Meth A tumor subcutaneously implanted into BALB/c mice, but not in liver, spleen or lung of these mice. This tumor tissue-specific reduction in blood flow was significantly inhibited by pretreatment with antisera against tumor necrosis factor (TNF) alpha, interferon (IFN) alpha/beta, and IFN gamma. These results indicate that endogenously induced cytokines, namely TNF alpha and IFNs, are involved in the intratumor blood flow reduction caused by DT-5461a. PMID- 7537486 TI - Stem cell factor and interleukin-6, alone or in combination with granulocyte colony-stimulating factor, do not affect the growth of solid tumor cell lines. AB - Hematopoietic growth factors (HGFs) are glycoproteins that control hemopoiesis. They have potential usefulness in a range of clinical conditions including the treatment of patients with myelosuppression induced by chemotherapy. Among HGFs, Stem Cell Factor (SCF) and Interleukin 6 (IL-6) are attracting interest for their capacity to stimulate early hematopoietic progenitors. Furthermore, their use in combination with late-acting growth factors with a more lineage-restricted potential (such as granulocyte colony-stimulating factor, G-CSF) might be expected to offer optimal marrow stimulation and usefulness in clinical oncology. Since non-hematopoietic malignant cells may express receptors for HGFs and respond to these peptides in vitro, we investigated clonal growth 3H-thymidine incorporation and cell cycle analysis by flow cytometry of 5 human solid tumor cell lines under the influence of SCF and IL-6 with or without G-CSF. Our experiments show that these cytokines have no effects on the proliferative capacity of the cell lines tested. Based on our and previously reported data, the use of these HGFs can be considered safe in cancer patients. PMID- 7537485 TI - Phosphotyrosine, p62 c-myc and p21 c-Ha-ras proteins in colonic epithelium of normal and dimethylhydrazine-treated rats: an immunohistochemical analysis. AB - In the present study we used monoclonal antibodies to investigate the expression of phosphotyrosine, c-myc and c-Ha-ras proteins along the crypt continuum of normal and transformed rat colon tissue. Colon cancer was induced by administration of dimethylhydrazine. Particular attention was focused on the immunohistochemical pattern of murine colon mucosa during preneoplastic stages so as to permit the identification of putative changes in the expression/location of the oncoproteins prior to frank neoplasia. The immunohistochemical analysis of tyrosinephosphorylated proteins in the normal rat indicated that positive staining was mostly restricted to the lower colonic crypt zones. The carcinogenetic insult altered the magnitude and positional profile of phosphotyrosine along the colon crypt axis during the preneoplastic period. An intense positive reaction was observed in the upper crypt regions. Four weeks following the last DHM administration, viz. before tumor appearance, positive staining was evident in invasive adenocarcinoma tissue. In contrast to phosphotyrosine, the feeble c-myc immunohistochemical staining of normal rat colonic did not exhibit a focal topology. However, following DMH administration and prior to frank neoplasia, a substantial increase in the staining intensity for c-myc was noted, confined mostly to the supranuclear region of luminal cells. Invasive adenocarcinomas displayed intense cytoplasmic c-myc immunoreactivity. p21 c-Ha-ras expression and location along the colon crypt axis showed a different pattern when compared to p62 c-myc and phosphotyrosine. The p21 c-Ha ras protein was prominently expressed in surface epithelium of normal and DMH treated rats. Midcrypt colonocytes exhibited moderate p21 ras staining; in contrast, proliferating colonic cells resident in the lower crypt regions were consistently negative. These results suggest that c-Ha-ras gene product plays an important contributory role in determining the differentiated phenotype of the colonic cell. PMID- 7537487 TI - Mitochondrial calcium uptake stimulated by Cibacron blue F3GA in bovine sperm. AB - The triazine dye Cibacron Blue F3GA stimulated calcium uptake into an ionomycin sensitive compartment of washed bovine sperm. Cibacron blue-stimulated uptake was not sensitive to nifedipine, diltiazem, or verapamil, but was blocked by transition metals, 1 microM ruthenium red, rotenone, and the protonophore CCCP. Uptake was dependent on the maintenance of energized mitochondria but not on oxidative or substrate level ATP production and was not supported by glycolysis. The cibarcon blue-stimulated uptake was judged by these criteria to be mitochondrial. Uptake in the absence of Cibacron blue was 0.046 +/- 0.008 and 0.268 +/- 0.068 nmol/min/10(8) cells with 0.1 mM Cibacron blue at 15 microM Ca2+. Half-maximal stimulation occurred at 20-25 microM Cibacron blue. Cibacron blue also allowed efflux of mitochondrial calcium in the presence of external calcium. This efflux would be blocked by ruthenium red, Ni2+, and La3+, but not by Co2+, which effectively blocked Cibacron blue-stimulated influx. Permeabilizing the plasma membrane with filipin indicated that Cibacron blue stimulation of mitochondrial calcium uptake operates directly on the mitochondria. Cibacron blue does not stimulate mitochondrial respiration and thus, calcium uptake was not the result of increased mitochondrial flux. PMID- 7537488 TI - Comparative effects of alfuzosin versus Serenoa repens in the treatment of symptomatic benign prostatic hyperplasia. AB - OBJECTIVES: Sixty-three patients suffering from benign prostatic hyperplasia (BPH) entered a double-blind, comparative, parallel-groups study lasting 3 weeks, carried out to compare the efficacy and safety of alfuzosin 2.5 mg tid (n = 32) vs serenoa repens 160 mg bid (n = 31) in BPH. METHODS: Efficacy was assessed both on clinical symptoms (Boyarsky's scale, visual analogue scale, clinical global impression), urinary flow rates (uroflowmetry) and residual urinary volume (transabdominal ultrasound). Events and reported signs were recorded throughout the entire study. RESULTS: Statistically significant and clinically relevant differences were found between the two treatments in favour of alfuzosin for Boyarsky's total score (decrease from 9.6 +/- 3.0 to 5.9 +/- 3.0, 38.8% for alfuzosin and from 9.3 +/- 2.5 to 6.8 +/- 2.8, 26.9% for serenoa repens) and obstructive score (decrease from 4.9 +/- 2.1 to 3.0 +/- 1.9, 37.8% for alfuzosin; from 4.4 +/- 1.7 to 3.4 +/- 1.8, 23.1% for Serenoa repens; p = 0.01 for both). Clinically relevant differences were found between the two treatments for visual analogue scale and overall clinical impression at the end of the study. Furthermore, the increase in quality of micturition was better with alfuzosin. The proportion of responders (increase on day 21 in peak flow rate of at least 25% relative to the baseline values) was in favour of alfuzosin (71.8% and 48.4% for alfuzosin and Serenoa repens, respectively; p = 0.057). Both treatments were well tolerated. No patient treated with alfuzosin complained of any adverse event at any time during the study. One patient in the Serenoa group complained of mild pruritus which cleared spontaneously. Systolic, diastolic blood pressure and heart rate did not show any clinically relevant change during treatment with alfuzosin. CONCLUSIONS: The findings confirm the efficacy and safety of alfuzosin in symptomatic BPH and indicate the superiority of alfuzosin over Serenoa repens in the treatment of urinary signs and symptoms of BPH. PMID- 7537489 TI - Use of aprotinin in LVAD recipients reduces blood loss, blood use, and perioperative mortality. AB - Aprotinin, a bovine protease inhibitor, has been used extensively in patients undergoing cardiac surgical procedures in an effort to minimize blood loss and prevent the complications associated with blood replacement. We sought to evaluate the effect of aprotinin on postoperative blood loss, renal function, and the incidence of right ventricular failure in patients undergoing placement of a TCI Heartmate left ventricular assist device as a bridge to cardiac transplantation. Retrospective data analysis in 142 patients (42 receiving aprotinin and 100 untreated) demonstrated that the use of aprotinin was associated with a significant decrease in postoperative blood loss (p = 0.019) and in the intraoperative packed red blood cell transfusion (p = 0.019) and total blood product (p = 0.016) requirements. A transient, yet significant, increase in the postoperative creatinine level in the aprotinin group (p = 0.0006), but not in blood urea nitrogen level (p = 0.22), was noted. Interestingly, we noted an association between blood loss and the subsequent development of right ventricular failure; patients who required a right ventricular assist device bled significantly more than did those who did not suffer right ventricular failure (p = 0.02). Additionally, aprotinin recipients benefited by a reduction of nearly one half in the incidence of the need for a right ventricular assist device. The incidence of perioperative mortality was reduced in those receiving aprotinin compared with that in untreated patients, (p = 0.05). We conclude that aprotinin is safe and effective in decreasing postoperative blood loss and intraoperative blood product requirements, and in reducing perioperative mortality in patients undergoing left ventricular assist device placement as a bridge to cardiac transplantation. PMID- 7537490 TI - [Disorders of interferon status in bronchopulmonary diseases in children living under conditions of elevated background radiation]. AB - Impairments in the interferon status of children with chronic and recurring diseases of the respiratory organs were investigated and its correction was shown possible with the use of alpha 2-interferon (reaferon). Suppression of the interferon-producing capacity of the immunocytes was observed in all the cases of the disease aggravation. The level of the suppression correlated with the severity of the infection. In the patients affected by radiation the suppression of the interferonogenesis was more marked. However, in these cases no dependence of the detected impairments in the interferon status on the level of the radiation pollution of the territory of the children residence was shown. The inclusion of reaferon to the basal therapy resulted in a significant increase of the alpha-interferon production in the cases of bronchial asthma and asthmatic bronchitis as well as its recovery to the normal in the cases of recurring bronchitis and chronic pneumonia. In all the cases of bronchopulmonary diseases only a tendency towards normalization of the alpha-interferon indices was recorded. PMID- 7537491 TI - Sequence variation and biological activity of rubella virus isolates. AB - Haemagglutination (HA) by rubella virus is mediated by the E1 glycoprotein. Rubella isolates which haemagglutinate with different avidity have been characterised. A significant reduction of HA titre at pH 6.0 was observed in one isolate in which isoleucine is substituted for threonine at rubella E1 residue 280. This residue is located in an epitope (EP1) which we have previously identified and shown to bind HA inhibiting (HA1) monoclonal antibodies. The isolates studied are also distinguishable by plaque size but no sequence variations in the immunogenic region of E1 were identified which might account for this difference. No correlation was observed between infectivity and binding affinity of neutralising monoclonal antibodies for different rubella virus strains. PMID- 7537493 TI - HPLC demonstration that an all Trp-->Phe replacement in gramicidin A results in a conformational rearrangement from beta-helical monomer to double-stranded dimer in model membranes. AB - We have taken advantage of our previously reported high performance liquid chromatographic (HPLC) strategy to investigate the conformational behavior of the optically reversed gramicidin M (gM-), an analog of gramicidin A with all tryptophans replaced by phenylalanines, in different model membranes. It is quantitatively demonstrated for the first time that once inserted in the lipid environment, gM- (unlike the native peptide) undergoes a conformational transition from beta-helical monomers to thermodynamically stable double-stranded dimers. This transition is faster the higher the incubation temperature and can be neatly observed in both small unilamellar phospholipid vesicles and lysophospholipid micelles. The results of this study are discussed in the light of presently available data from other techniques, in the framework of the current efforts to understand structure-function relationships of linear gramicidins. PMID- 7537492 TI - Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro. AB - The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+. PMID- 7537495 TI - Autophosphorylation activity and association with Src family kinase of Sky receptor tyrosine kinase. AB - "Sky" is a putative receptor tyrosine kinase predominantly expressed in the brain. Sky, like Axl/Ufo/Ark and c-Eyk, has an extracellular domain composed of two immunoglobulin-like domains and two fibronectin type III domains. Immunoblot analysis using an antibody raised against a C-terminal peptide of Sky identified a 98-kDa Sky protein in COS cells transfected with sky cDNA (COS/sky cells). A 98 kDa protein in the immunoprecipitates with anti-Sky antibody was autophosphorylated on tyrosine, by in vitro kinase reaction. When the lysates of COS/sky cells were immunoprecipitated with anti-Sky antibody and immunoblotted with an anti-phosphotyrosine antibody, a 60-kDa phosphotyrosine-containing protein, in addition to the tyrosine-phosphorylated Sky, was detected. Using the anti-Src antibody, which is reactive to Src, Fyn and Yes, we obtained evidence for an association between the Src family tyrosine kinase and the tyrosine phosphorylated Sky receptor. These results suggest that the Src family kinase may play an important role in signal transduction of the Sky receptor. PMID- 7537494 TI - Alpha 1 B, but not alpha 1A, adrenoreceptor activates calcium influx through the stimulation of a tyrosine kinase/phosphotyrosine phosphatase pathway, following noradrenaline-induced emptying of IP3 sensitive calcium stores, in PC Cl3 rat thyroid cell line. AB - In PC Cl3 rat thyroid cell line noradrenaline-induced Ca2+ response, mainly due to the activation of alpha 1B receptors, is characterized by a rapid peak phase, due to the Ca2+ mobilization from inositol trisphosphate-sensitive internal stores, followed by a sustained plateau, representing the capacitative calcium entry. The plateau phase elicited by noradrenaline returns to the basal value within 100 sec from the removal of agonist. The tyrosine kinases inhibitor genistein completely abolishes the plateau upon noradrenaline withdrawal. On the contrary, the tyrosine phosphatases blocker, vanadate, potentiates the plateau phase of calcium response to noradrenaline and prevents the gradual decrease of [Ca2+]i after removal of noradrenaline. The noradrenaline-induced Ca2+ influx, due to the activation of alpha 1A receptor-operated Ca2+ entry is not affected by vanadate. The treatment with noradrenaline induced the tyrosine phosphorylation of specific substrates in lysates derived from PC Cl3 cells, an effect inhibited by genistein pretreatment. These results show that a balance between tyrosine phosphorylation and dephosphorylation is required for the regulation of capacitative calcium entry following noradrenaline stimulation of alpha 1B receptor, whilst the influx of Ca2+ directly operated by alpha 1A receptor activation seems to be independent of the tyrosine phosphorylating pathway. PMID- 7537496 TI - The amino acid sequence GPLY is not necessary for normal endocytosis of the human insulin receptor B isoform. AB - The human insulin receptor (hIR) cytoplasmic juxtamembrane domain contains two tyrosine (Y) residues which exist in GPLY and NPEY motifs that have been implicated in endocytic function. We have previously shown that the NPEY motif is not necessary for endocytosis of the B isoform (exon 11+) of hIR. To examine the role of the GPLY sequence in transmembrane insulin signaling and endocytic functions of hIR-B, we constructed a mutant receptor, hIR delta GPLY, that lacks the GPLY sequence (residues 962-965), and stably expressed it in CHO cells. When compared to wild type hIR-B (hIR-WT) similarly expressed in CHO cells, the hIR delta GPLY mutant exhibited higher insulin binding affinity (EC50 of 1.0 vs 3.5 nM) and normal insulin-stimulated receptor tyrosine autophosphorylation and kinase activity towards the endogenous 185 kDa insulin receptor substrate. The hIR delta GPLY receptor also exhibited normal endocytic functions as hIR-WT in that: a) the internalization of surface photoaffinity labeled hIR delta GPLY was similar to that of hIR-WT, and b) the rate and extent of 125I-insulin internalization and degradation at 37 degrees C were also unimpaired. Therefore, these results demonstrate that the GPLY sequence is not necessary for transmembrane insulin signaling and endocytic functions of the hIR-B isoform. PMID- 7537497 TI - Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus. AB - A cDNA library was constructed using poly(A+) RNA isolated from eyestalk neural ganglia of the blue crab, Callinectes sapidus. The library was screened using a probe generated by PCR based on the published amino acid sequence of molt inhibiting hormone from the shore crab, Carcinus maenas. DNA sequence analysis of one positive clone revealed a 339 bp open reading frame encoding a 78-residue putative molt-inhibiting hormone and a 35-residue signal peptide. The deduced amino acid sequence of C. sapidus molt-inhibiting hormone is 79% homologous with that of C. maenas. Northern blot analysis, using a fragment of the cloned molt inhibiting hormone cDNA as probe, revealed specific hybridization to a single band (approximately 1.4 kb) in RNA extracted from eyestalk but not control tissues. PMID- 7537498 TI - Mass spectrometric identification and quantification of endogenous 5 methoxyindole-3-acetic acid in quail tissues. AB - The endogenous levels of 5-methoxyindole-3-acetic acid (5-MIAA) in quail pineal gland, retina and serum were determined by capillary column gas chromatography/mass spectrometry/selected ion monitoring using a deuterated internal standard and the N-pentafluoropropionyl-O-pentafluorobenzyl ester derivative. Diurnal rhythms of pineal and serum 5-MIAA with high levels at mid light and low levels at mid-dark suggest a pineal origin of 5-MIAA in the blood. In contrast, retina displayed an opposite diurnal rhythm of 5-MIAA with high levels at night, suggesting that the biosyntheses of 5-MIAA in the quail pineal gland and retina were different. In vitro studies on incubation media indicated that both the pineal gland and retina are capable of synthesis/secretion of 5 MIAA. PMID- 7537499 TI - Myeloglycan, a series of E-selectin-binding polylactosaminolipids found in normal human leukocytes and myelocytic leukemia HL60 cells. AB - Sialosyl-Lex (SLex) is assumed to be the binding epitope of E- and P-selectin in normal human neutrophils and myelocytic leukemia HL60 cells. Glycosphingolipid (GSL) fractions from large quantities of normal human neutrophils and HL60 cell extract did not contain SLex GSLs having 6-10 sugar residues, as commonly found in solid tumor cells and tissues. Instead, the binding target of E-selectin was revealed to be a series of long-chain, unbranched polylactosamine GSLs with terminally sialylated, internally alpha 1-->3 polyfucosylated structure as the major component, or having SLex at the terminus and internally polyfucosylated structure as a minor component. These GSLs are hereby collectively termed "myeloglycan." Regardless of the site of fucosylation, all myeloglycans cross react strongly with "anti-SLex" monoclonal antibodies such as CSLEX, FH6, SNH3, and SNH4. PMID- 7537500 TI - Protein-tyrosine phosphatase inhibition by a peptide containing the phosphotyrosyl mimetic, L-O-malonyltyrosine. AB - Peptides containing phosphonate based non-hydrolyzable phosphotyrosyl (pTyr) mimetics previously have been shown to be competitive inhibitors of protein tyrosine phosphatases (PTPs). These agents suffer from low cellular penetration which is partially attributable to ionization of the phosphonate group at physiological pH. We have developed the non-phosphorus containing pTyr mimetic, L O-malonyltyrosine (L-OMT) and herein demonstrate using a PTP 1B enzyme assay that it is superior to phosphonomethyl phenylalanine (Pmp) as a pTyr mimetic when incorporated into the hexamer peptide Ac-D-A-D-E-X-L-amide (X = D,L-Pmp, IC50 = 200 microM; X = L-OMT, IC50 = 10 microM). Prodrug protection of L-OMT as its carboxylic acid diester could potentially increase cellular penetration, thereby making this a valuable reagent for cellular studies. PMID- 7537501 TI - Inhibition of anti-V3 domain antibody binding to human immunodeficiency virus type-1-infected cells by sulfated polysaccharides. AB - The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is an immunodominant region. Anti-V3 domain antibodies neutralize both HIV-1 infection and syncytium formation. The V3 domain has a high density of positive charge which is a potential binding site for anti HIV-1 sulfated polysaccharides. To investigate the inhibitory effect of sulfated polysaccharides on the binding of anti-V3 domain antibody, fluorescence activating cell sorting analysis was performed using two kinds of antibodies, NEA9284 (purified, 0.25 micrograms/ml) and 0.5 beta (ascite, 2.0 mg/ml), and HIV 1-infected CEM cells. When the binding assay with a 1:100 dilution of each antibody was performed in the presence of dextran sulfate, heparin, and inositol hexasulfate at concentrations which are antiviral, the compounds did not inhibit the binding of either antibody. As the antibody concentration was decreased with higher dilution, dextran sulfate was able to reduce antibody binding by 50-60%. Thus, antagonism of anti-V3 domain antibody binding by sulfated polysaccharides is not as extensive as reported previously by several groups. PMID- 7537502 TI - Further characterization of the 5'-flanking region of the rat lactase-phlorizin hydrolase gene. AB - The lactase-phlorizin hydrolase gene is widely used as a marker of intestinal differentiation. Recent evidence demonstrating that transcription plays a major role in the regulation of this gene suggests that study of the 5'-flanking region will allow an understanding of how the expression of this gene is controlled. However, sequence, restriction, and primer extension analysis of a rat genomic clone has revealed that previously published data are incomplete. In the present study, we used a directed sequencing strategy to carefully analyze this region. Our expanded analysis of the 5'-flanking region of the lactase-phlorizin hydrolase gene should facilitate future studies of its structure and function. PMID- 7537503 TI - Inclusion, observation, outcome. PMID- 7537504 TI - [In vitro simulation of rabbit cecal fermentation in a semi- continuous flow fermentor. III. Effect of the quantity of dry matter introduced daily in the fermentor and reproducibility of the method]. AB - In a preliminary experiment, 3 Rusitec-like fermentors of 1 L capacity were operated under identical conditions. The only difference was the quantity to treated substratum introduced daily into each fermentor: 15 g/d (M15); 40 g/d (M40); and 60 g/d (M60) on a dry matter basis. The fermentation substratum was a rabbit feed that had been digested with amylase and pepsin. The organic matter was lost over 48 h at a significantly higher rate in the M15 fermentor than in M40 and M60: 30.4%, 19.7% and 17.3%, respectively. The pH values observed in M40 and M60 (5.99 and 5.83) were similar to that observed in vivo under ad libitum feeding conditions. The pH was higher in M15 (6.7), as observed in vivo with restricted animals just before the daily meal. The volatile fatty acids (VFA) proportions for C2, C3 and C4 were similar to the in vivo proportion for the 3 fermentors. The daily total VFA production was the largest with M40 but was associated with a poor stability and an excess of minor VFAs. The introduction of 15 g/d was preferred by the authors because of organic matter disappearance rate, pH stability and VFA production. In a second experiment, 4 fermentors were used in the same way as the M15 one, in order to study the reproducibility of the method. Small but significant differences between fermentors were observed for all parameters in relation to a high fermentor stability from 1 day to the next.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537505 TI - Effect of age and exogenous amylase and protease on development of the digestive tract, pancreatic enzyme activities and digestibility of nutrients in young meat type chicks. AB - Day-old male meat-type chicks were fed a commercial starter diet supplemented with 2 levels of enzyme preparations containing amylase and proteases up to 14 d of age. Enzyme supplementation had no significant effect on feed intake or growth rate, and was accompanied by a significant decrease in gizzard content and small intestine weight. The intestine contents increased and this increase was accompanied by a significant decrease in its pH. Enzyme supplementation depressed the activity of chymotrypsin in the pancreas and the activity of amylase, trypsin and chymotrypsin in the intestinal contents. Some carry-over effects were observed on d 42, 4 weeks after the cessation of the enzyme supplements. These were mainly a significant depression in the activity of trypsin in the intestinal contents. In a balance study, diets supplemented with 0,250 and 1,000 micrograms/kg enzyme preparations were supplied. Exogenous enzyme supplements had no significant effect on the digestibility of all the nutrients studied except for the highest level of enzyme supplementation, which improved slightly but consistently the digestibility of amino acids. Some age effects were observed, mainly a decrease in the digestibility of fat and starch, and in the ME of the diet from weeks 1 to 2 followed by an increase during week 3. Protein digestibility and retention of nitrogen decreased with age. PMID- 7537506 TI - The expressed L1 proteins of HPV-1, HPV-6, and HPV-11 display type-specific epitopes with native conformation and reactivity with neutralizing and nonneutralizing antibodies. AB - Previous studies demonstrated that the human papillomavirus (HPV) type 1 L1 protein, expressed in cos cells by an SV40-based vector, displays conformational epitopes characteristic of native virions. In this study, we analyzed the expression of HPV-1, HPV-6, and HPV-11 L1 proteins in order to determine the forms of conformational epitopes expressed by recombinant L1 proteins. Using both immunofluorescence and immunoprecipitation techniques, polyclonal and monoclonal antibodies (MAbs) generated against native HPV-11 virions reacted with expressed L1 proteins of HPV-6 and/or HPV-11, but not HPV-1. Similarly, polyclonal antibodies and MAbs generated against HPV-1 virions reacted with the expressed L1 protein of HPV-1, but not HPV-6 or HPV-11. Of two MAbs that neutralized HPV-11 infection of murine fetal foreskin xenografts, one reacted with the expressed L1 protein of both HPV-6 and HPV-11, and the other reacted with HPV-11 only. A nonneutralizing conformationally dependent MAb reacted with the expressed L1 protein of both HPV-6 and HPV-11. These results demonstrate that expressed HPV L1 proteins retain type-specific, neutralizing, and nonneutralizing conformational epitopes and that cos cells may be utilized to evaluate host immune responses to such epitopes. PMID- 7537507 TI - Positive relationship between expression of CD44 and hepatic metastases in colorectal cancer. AB - This study was performed to determine the relationship between the expression of CD44, p53 and NM23 and the metastatic potential of colorectal cancer. Frozen sections of 56 cases of colorectal cancer were immunohistologically examined for the expression of CD44, p53 and NM23. Positive immunoreactivity was found in 30 of 56 cases (54%) in CD44, 26 of 56 (46%) in p53 and 20 of 56 in NM23 (36%), respectively. There was no significant relationship between the expression of these molecules and clinicopathological findings such as age, sex, size, location of tumor, histological type, serosal invasion, peritoneal dissemination, lymph node metastasis, extramural venous spread and tumor stage; however, when examining the relationship between the expression of these molecules and prognosis in terms of hepatic metastasis and recurrence after curative operation, a significant association was seen in the expression of CD44, but none in p53 and NM23. It was suggested that expression of CD44 could be used as a possible indicator to predict metastatic potential of colorectal cancer. PMID- 7537508 TI - Comparative studies on vascular endothelium in vitro. I. Cytokine effects on the expression of adhesion molecules by human umbilical vein, saphenous vein and femoral artery endothelial cells. AB - Endothelial cells (ECs) are very responsive to proinflammatory cytokines. ECs are stimulated by these substances to increase expression of cell surface adhesion molecules, leading to dramatically altered interactions with leukocytes. In these interactions, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are suggested to play the most important role. Recent evidence has suggested diversity in the responses of ECs from different regions of the vascular system. Human umbilical vein ECs (HUVECs) are the most often used EC culture model, although there are few studies comparing their response with other human EC types from the adult organism. In this study the expression of E-selectin, ICAM-1 and VCAM-1 on cultured human adult ECs from the saphenous vein (HSVECs) and from the femoral artery (HAFECs), as well as HUVECs was studied. Using a cell enzyme immunoassay as well as immunoelectron microscopical methods, we found that both HSVECs and HAFECs respond in a similar way to HUVECs to exogenous stimulation by IL-1 beta, TNF alpha or LPS. IL-1 beta and TNF alpha increased the expression of E-selectin on the cytoplasmic membranes of HUVECs, HSVECs and HAFECs and elicited even similar absolute quantities of this molecule, comparing the different cell types. ICAM-1 and VCAM-1 appeared to be regulated dose dependently by IL-1 beta, independent of the EC type. HUVECs as well as HSVECs and HAFECs gave a reproducible constitutive ICAM-1 expression, whereas E-selectin and VCAM-1 were absent on nonstimulated ECs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537509 TI - Corticostriatal and corticotectal projections from visual cortical areas 17, 18 and 18a in the pigmented rat. AB - Regions of the visual cortex and the deep layers of the superior colliculus (SC) have been suggested to be functionally linked via an 'indirect' pathway through the basal ganglia. The present report demonstrates projections from the striate (area 17) and extrastriate (areas 18 and 18a) visual cortex in Long-Evans hooded rats to the striatum (ST) and SC with anterograde (biocytin) and retrograde (WGA HRP and fluorescent dyes) tracers. Biocytin injections into each cortical area produced markedly different patterns of labeling in ST and SC. Injections into area 17 resulted in a dense labeling in the superficial layers of SC, with little labeling present in the deep layers of SC or ST. In contrast, injections into area 18a, which produced marked labeling in the deep layers of SC with moderate in the superficial layers, resulted in dense labeling in the caudal two-thirds of the dorsal region of ST; injections into area 18 produced numerous terminals mainly in the deep layers of SC and in the rostral two-thirds of the dorsal aspect of ST. WGA-HRP injections into ST resulted in numerous retrogradely labeled pyramidal neurons in areas 18 and 18a, but only a few labeled neurons were observed in area 17. Corticostriatal neurons in extrastriate visual cortex were distributed predominantly in layer V, with smaller numbers in layers II and III, whereas corticotectal neurons were located only in layer V of both the striate and extrastriate visual cortex. Although corticostriatal and corticotectal neurons were intermingled in layer V, no double-labeled neurons were observed following injections of different fluorescent dyes into ST and SC. It appears, therefore, that: 1) the major source of visual input to both ST and the deep layers of SC in the rat arises not from the primary visual cortex but from extrastriate visual areas; and that 2) the projections from the extrastriate visual area to ST and SC originate from different populations of corticofugal neurons. PMID- 7537510 TI - Identification of MIP-1 alpha/LD78 as a monocyte chemoattractant released by the HTLV-I-transformed cell line MT4. AB - It is known that the HTLV-I-transformed cell line MT4 releases chemotactic activity for monocytes spontaneously. The MT4 monocyte chemoattractant was purified to homogeneity and sequencing of 25 amino acids revealed identity with the C-C chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha/LD78). An anti-MIP-1 alpha/LD78 rabbit antiserum substantially inhibited chemotaxis of the MT4 chemoattractant. MT4 cells constitutively expressed MIP-1 alpha/LD78 but not the C-C chemokines MCP-1, RANTES, and MIP-1 beta/Act2 and the C-X-C chemokines IL 8, gro alpha, and gro beta. MT4-derived MIP-1 alpha/LD78 was active on monocytes but was a weak chemoattractant for polymorphonuclear leukocytes. Thus, MIP-1 alpha/LD78 is a major monocyte chemoattractant released by HTLV-I-transformed T cells. Expression of MIP-1 alpha/LD78, a leukocyte chemotactic and myelosuppressive molecule, may play an important role in the manifestations of HTLV-I-related diseases. PMID- 7537511 TI - Development of lymphoid hyperplasia in transgenic mice expressing the HIV tat gene. AB - During HIV infection, individuals experience multiorgan disorders such as adenopathy, splenomegaly, and lung and brain diseases. There is an increasing body of evidence that the HIV trans-activating tat gene product possesses multiple activities. First, it can activate several cellular genes; second, in its extracellular soluble form, it plays the role of growth factor in some cells such as Kaposi's sarcoma cells. Thus, we introduced the HIV tat gene, under the control of the cellular proteolipoprotein promoter, into the germline of mice and demonstrate that, when expressed, the tat gene product induces lymphoid hyperplasia in spleen, lymph nodes, and lung, as is observed in AIDS patients, but not in the brain or testes. Our findings indicate that HIV, through some of its genes, directly participates in the pathogenesis of AIDS. PMID- 7537512 TI - Recent advances in the genetics of metastasis. PMID- 7537514 TI - Thrombelastography reveals two causes of haemorrhage in HELLP syndrome. AB - We describe the use of thrombelastography in HELLP syndrome (Haemolysis, Elevated Liver Enzymes, Low Platelets). It differentiated between two possible causes of significant haemorrhage and revealed an accompanying underlying fibrinolysis. This allowed specific therapy to be directed at both abnormalities and, we believe, helped prevent this patient from undergoing radical surgery to curb blood loss. PMID- 7537513 TI - Toward the molecular details of the nuclear pore complex. PMID- 7537515 TI - Recommended composition of influenza virus vaccines for use in the 1995-1996 season. PMID- 7537516 TI - Expression of alpha-GalNAc glycoproteins by breast cancers. AB - The expression of complex carbohydrates recognised by Helix pomatia lectin (HPA, nominal monosaccharide binding specificity alpha-GalNAc) has been shown to predict unfavourable prognosis in breast and other cancers. It has been suggested that the prognostic significance of HPA binding may be through recognition of either Tn epitope (alpha-GalNAc-O-serine/threonine) or blood group A antigen (terminal alpha-1-->3GalNAc attached to the basic H-antigen, Fuc-alpha-1-->2-Gal beta-1-->4(or 3) GlcNAc-->R). In this study, the expression of glycoproteins terminating in alpha-GalNAc residues was investigated immunohistochemically using HPA and two monoclonal antibodies--BRIC 66 (anti-alpha-GalNAc) and BRIC 111 (anti Tn). In paraffin sections, 74/87 (85%) of breast cancers expressed HPA-binding ligands, while 28/87 (32%) were positive for BRIC 66 binding and 25/87 (29%) expressed Tn. Distribution of staining patterns were distinctive and different with the three markers. BRIC 66, BRIC 111 and HPA binding to glycoproteins derived from breast cancer homogenates and to blood group A and Tn positive glycoproteins in Western blots confirmed the immunohistochemistry data. The results suggest that the prognostic significance of HPA binding in breast cancer is unlikely to be simply through recognition of blood group A antigen or Tn epitope on cancer cells. Breast cancers may express a complex profile of related but distinct glycans sharing similar terminal immunodominant sugar GalNAc, which may be implicated in aggressive biological behaviour. PMID- 7537517 TI - Evidence for a weak angiogenic response to human colorectal cancers. AB - Many previous qualitative studies have shown that tumours are less vascular in the centre, and that host tissues become more vascular in close proximity to tumours. However, quantitative findings presented here for human colorectal cancer reveal some significant differences. Sections from 20 colorectal carcinomas (ten moderately and ten poorly differentiated) were immunostained with the QB/end/10 monoclonal to demonstrate blood vessels. These were measured by interactive morphometry and vascular volume density, surface density (Sv) and length density were recorded. In poorly differentiated carcinomas, the tumour centre was significantly less vascular than the periphery for all three parameters (P = 0.008 for Sv). However, no significant difference was seen for moderately differentiated tumours, which constitute the majority of colorectal cancers. Surrounding host tissues did not show a general increase in vascular density close to tumours. Furthermore, when total viable tissue was considered, the vascular density of carcinomas was not markedly different from normal mucosa. In the centre of moderately differentiated carcinomas for example, the mean value for Sv was only 1.4 times higher than the mean value for normal mucosa. These findings suggest that colorectal cancers may elicit a relatively weak angiogenic response, consistent with their exceptionally slow growth rate. PMID- 7537520 TI - A tumour-associated cell-surface glycoprotein accompanying p53 overexpression and higher growth potential for gastric cancer. AB - Tumour-associated cell-surface glycoprotein is associated with tumour progression in gastric cancer. We investigated the biological significance of tumour associated cell-surface glycoprotein, determined by the binding of Helix pomatia agglutinin (HPA), with regard to survival time and to the malignant potential of cancer cells in serosally invasive gastric cancer in 119 patients. HPA was positively stained in 75 of 119 patients (63.0%) with gastric cancer with serosal invasion. In patients with HPA-positive tissue, the tumour was larger than in HPA negative cases and was frequently located in the middle third of the stomach. The incidence of lymph node metastasis was higher than in patients with HPA-negative tissue. There were no differences between the cases staining negatively and positively with HPA with respect to the other factors examined. Gastric cancer tissues with HPA-positive staining revealed a higher positive rate of abnormal p53 staining and a higher concentration of proliferating cell nuclear antigen (PCNA) labelling. The survival time of the patients with HPA positive staining was shorter than for those whose tissues were HPA negative. Thus, tumour associated cell-surface glycoprotein is apparently closely related to the malignant potential of serosally invasive gastric cancer. PMID- 7537519 TI - Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694. AB - Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4 oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity. PMID- 7537521 TI - Changes in tissue distribution of rat alpha 1-macroglobulin and pregnancy associated alpha 1-glycoprotein after inflammatory injury. AB - Antiserum against rat alpha 1-macroglobulin (alpha 1MG) was produced in rabbits. Antiserum against rat pregnancy-associated alpha 1-glycoprotein (PAG) was obtained by immunization with a partly purified PAG preparation and absorption of the serum with male rat serum. Acute inflammation was produced in non-pregnant female rats by a single intramuscular injection of turpentine. The concentrations of both macroglobulins in the serum and in tissue extracts were measured by rocket immunoelectrophoresis at various times up to 7 days after injury. Inflammation produced in the rats resulted in moderately elevated serum levels of these proteins soon after injury. At first, alpha 1MG levels in a number of tissues (heart, lung, kidney, spleen, pancreas, uterus and ovary) were depressed markedly; they then stabilized. The elevated serum concentrations of alpha 1MG remained unchanged during inflammation. The store of PAG in the tissues was rapidly depleted and its serum level decreased to a normal value 7 days after injury. Our findings indicate that alpha 1MG plays a more important role in maintenance of the proteinase inhibitory potential in the rat than does PAG. PMID- 7537518 TI - Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines. AB - Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5 fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross resistance was found to those compounds known to be active through polyglutamation. PMID- 7537522 TI - Role of alpha-2-macroglobulin and bacterial elastase in guinea-pig pseudomonal septic shock. AB - An essential role of alpha-2-macroglobulin (alpha 2M) was revealed in the prevention of septic shock induced in guinea-pigs by an elastase producing strain (IFO-3455) of Pseudomonas aeruginosa. When bacterial peritonitis was induced by inoculating fibrin-thrombin clot containing viable bacteria at a dose of 10(9) c.f.u./kg body weight, the guinea-pigs (n = 6) died within 7-8 hours due to septic shock. Prior to the shock, consumption of two-thirds of the circulating alpha 2M was observed. When circulating alpha 2M was depleted 4 hours after the bacterial inoculation, the guinea-pigs immediately developed shock and died within one hour. This shock was prevented either with a specific elastase inhibitor, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, zincov (6 microM), or with human alpha 2M. Simultaneous depletion of circulating Hageman factor also prevented shock in the alpha 2M-depleted animals. These results indicate that septic shock was induced through activation of the Hageman factor dependent system by the bacteria-produced elastase which survived alpha 2M in the circulation. PMID- 7537523 TI - Induction and relief of nasal congestion in ferrets infected with influenza virus. AB - Susceptible ferrets intranasally infected with influenza virus consistently responded with maximal nasal secretion of virus, febrile reaction, and influx of inflammatory cells into nasal lumen on day 2 post infection (d.p.i.). Polymorphonuclear leucocytes were the earliest predominant cell, followed by monocytes/macrophages while lymphocytes were maintained as a minor population throughout the 7-day period. Nasal congestion level, continuously monitored by computer aided active anterior rhinomanometry, was reproducibly maximal at 2 d.p.i., diminished in intensity the next day and returned to the basal level within 7 d.p.i. Nasal congestion was effectively relieved by a single intranasal dose of 0.1% oxymetazoline or 0.2% phenylephrine, or a single intragastric administration of pseudoephedrine. Intranasal delivery of a single dose of 1% pyrilamine relieved nasal congestion while 0.8% ipratropium bromide and 30% cimetidine were ineffective. These results suggested that nasal congestion is regulated by alpha-adrenergic receptors in the mucosal vasculature or by H1 histamine receptor, but is unaffected by inhibitors of nasal secretion regulated by the cholinergic nervous system. The present study indicates that the infectious rhinitis ferret model provides a reproducible nasal congestion pattern that can be objectively measured by a refined active anterior rhinomanometric system. This labour intensive measurement, however, makes it difficult either to conduct a large population animal study or to use it for a rapid throughput screening of new drugs. The temporal relation between the influx of inflammatory cells into the nasal lumen and the onset of nasal congestion underlies the model's relevance to the exploration of the pathogenic mechanism(s) during viral rhinitis. PMID- 7537524 TI - Changes in erythroid progenitor cell and accessory cell compartments in patients with myelodysplastic syndromes during treatment with all-trans retinoic acid and haemopoietic growth factors. AB - Differentiation induction therapy is used in myelodysplastic syndromes (MDS) to improve maturation defects and to restore impaired function of malignant cells. To this end, 18 patients with MDS received either a combination therapy consisting in study 1 of all-trans retinoic acid (ATRA) and granulocyte-colony stimulating factor (G-CSF), or in study 2 of a combination with ATRA, G-CSF, erythropoietin (Epo) and tocopherol. The ANC increased in 19/20 patients in both studies, whereas an increase in haemoglobin concentration, platelet counts or reduction of transfusion requirement was seen in only 8/20 patients, correlating strongly with good BFU-E growth (P < 0.001). To assess the role of accessory cells in the modulation of the haemopoietic response to treatment, we analysed the capacity of peripheral blood monocytes to secrete cytokines (IL-1 beta, IL-6, IL-8, TNF alpha). Secretion of all cytokines was significantly reduced before therapy when compared with healthy controls, but increased during therapy, reaching normal levels for IL-8. These data indicate that a combination therapy with ATRA and cytokines improves impaired cytokine secretion from monocytes and induces a multilineage clinical response in a subgroup of MDS patients characterized by an almost intact erythroid compartment. In contrast, induction of TNF alpha might be responsible for treatment failure. PMID- 7537526 TI - Granulocyte colony-stimulating factor administration modulates the surface expression of effector cell molecules on human monocytes. AB - Granulocyte colony-stimulating factor (G-CSF) has been shown to stimulate human neutrophil functions, both in vitro and in vivo. We examined the effects of G-CSF administration on the surface expression of effector cell molecules on human neutrophils and monocytes. G-CSF (50 micrograms/m2/d) was administered subcutaneously to five healthy volunteers once a day for 7 d. Venous blood was obtained immediately before and after the completion of G-CSF administration and 1 week after the last G-CSF administration. The surface expression of complement receptors (CR), Fc receptors for IgG(FcR) and cellular adhesion molecules on human neutrophils and monocytes were determined by indirect immunofluorescence using flow cytometry and monoclonal antibodies. The expression of CR1, CR3, FcRI and FcRII on neutrophils increased significantly after G-CSF administration and then decreased after the last G-CSF administration. The expression of human leucocyte adhesion molecule-1 (LAM-1) on neutrophils reflected the above expression. On the other hand, the administration of G-CSF increased the expression of CR1, CR3, FcRI and FcRIII on monocytes. The expression of CR1, CR3 and FcRI on monocytes then decreased after the last G-CSF administration, whereas the expression of FcRIII remained at an increased level. These findings indicate that G-CSF administration modulates the expression of effector cell molecules on circulating monocytes as well as on neutrophils, resulting in enhanced defence against selected infections or in potentiation of the tumouricidal capacity of phagocytes in cancer patients. PMID- 7537525 TI - Diamond-Blackfan anaemia: three patterns of in vitro response to haemopoietic growth factors. AB - Culture of bone marrow from patients with Diamond-Blackfan anaemia (DBA) has previously shown a variable progenitor response to growth factor stimulation. An extensive standardized study has now been undertaken to investigate the presence of distinct sub-groups in this disorder. In vitro response of bone marrow progenitors to recombinant human growth factors, including stem cell factor, was examined in 18 DBA patients and five normal donors, assessing BFU-E, CFU-GM and CFU-GEMM development. In 16 of the DBA patients a synergistic response to combinations of growth factors was observed with optimal growth in cultures containing erythropoietin, interleukin-3 and stem cell factor. Growth factor induced erythroid response formed three distinct groups, based on BFU-E numbers: type I (mean age 4.87 years) showed > 70% normal erythroid response; type II (mean age 13.87 years) showed < 70% normal; and type III (mean age 15.29 years) < 5% normal. CFU-GM response also followed the trigrouping. The results suggest more than one pathogenic mechanism for the erythroid failure in DBA, indicating DBA may be composed of more than one distinct disorder, and further suggest the defect in DBA may not be confined to the erythroid series. PMID- 7537527 TI - Clinical and laboratory effects of long-term administration of hydroxyurea to patients with sickle-cell/beta-thalassaemia. AB - Hydroxyurea (HU), a widely used cytostatic, has been given over a long period of time to 14 adult Caucasian compound heterozygotes for beta s and various beta thalassaemia genes. All patients had severe pain crises and other complications prior to receiving the drug. After 4-8 weeks on high 'sub-toxic' doses of HU all patients responded with a multifold increase of fetal haemoglobin (HbF) and a marked increase of MCV and MCH; they also felt significantly better and ceased having pains or other complaints. Haematological toxicity was minimal and rapidly reversible. Follow-up of the patients has now exceeded 100 weeks and goes up to 180 weeks in two of them. Pain crises have never recurred. Maintenance of high levels of HBF requires continuous administration of high doses of HU; whenever the latter were decrease in various attempts to avoid potential long-term toxicity, the observed changes gradually faded. The effect of HU in HbS/beta thalassaemia may be better than that reported for homozygous HbS disease because the synthesized gamma-chains not only inhibit the sickling process but they also neutralize the noxious effects of the excess alpha-chains and cut down the ineffective erythropoiesis of the patients. PMID- 7537528 TI - Philadelphia-chromosome-negative peripheral blood stem cells can be mobilized in the early phase of recovery after a myelosuppressive chemotherapy in Philadelphia chromosome-positive acute lymphoblastic leukaemia. AB - Ten patients in first or second relapse with Philadelphia chromosome acute lymphoblastic leukaemia, ineligible for allogeneic sibling marrow transplantation, were treated with an intensive chemotherapy regimen including idarubicin, intermediate-dose arabinosylcytosine, etoposide and G-CSF. Peripheral blood stem cells were collected by leukapheresis during initial early WBC recovery from chemotherapy-induced aplasia. In 5/10 patients all metaphases in leukapheresis products were found to be Philadelphia-chromosome-negative and they have been used as autotransplants after conditioning with TBI/etoposide/cyclophosphamide (or idarubicin) and G-CSF. All five patients showed sustained engraftment and one of them is alive and well Philadelphia chromosome-negative 18 months after transplant. These preliminary results suggest that it is possible to recover Philadelphia-chromosome-negative blood stem cells after intensive chemotherapy, even in advanced patients, and to perform autografting with these cells. PMID- 7537529 TI - Flow cytometric determination of pre-transfusion red cell volume in fetuses and neonates requiring transfusion based on RhD+ dilution by transfused D- red cells. AB - We report a case of feto-maternal haemorrhage and describe a new flow-cytometric method of determining a fetus's or infant's pre-transfusion red cell volume (RCV). We validate this method against an established technique, employing fetal haemoglobin (HbF) dilution, for determining the RCV in fetuses and neonates requiring intravascular transfusion. We discuss advantages and other potential applications of this new method. PMID- 7537530 TI - Infection at the time of peripheral blood stem cell leukapheresis results in reduced granulocyte-macrophage colony forming unit yields. PMID- 7537531 TI - Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic staining methods with respect to colour segmentation. AB - The detection of human cytomegalovirus (HCMV) infected poly-morphonuclear leukocytes (PMNLs) for early finding of the pp65 antigen using automated image analysis has been improved. The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate. The number of automatically detected false positive objects due to incomplete inactivation of endogenous peroxidase and strong variations in counterstain intensity could be reduced by 81% using this AP staining method. The number of detected truly positive cells with both staining methods was not significantly different. Furthermore, a new image analysis system providing processing of colour images was evaluated. Since plain differences in absorption wavelength are required for colour segmentation, the red immuno-staining was combined with a green counterstain using methyl green. Screening of AP- instead of PO-stained slides in combination with colour segmentation resulted in a further reduction of the number of falsely detected alarms from 61% to 11%. Consequently, the sensitivity of the automated detection was improved. For AP staining detection of cells in frequencies of approximately one to one million was demonstrated. Screening for CMV-positive, alkaline phosphatase labelled cells using an image analysis system with colour segmentation resulted in a reduced false alarm rate, a better visual interpretation of the images and subsequently an increase in the sensitivity of the automated screening process. PMID- 7537532 TI - Induction of the transcription factors NF-kappa B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. AB - To address elements that might uniquely characterize CD40 mediated signaling, the nuclear expression of three transcription factors was evaluated following B cell stimulation by CD40L and by anti-Ig antibody. Cross-linked CD40L was found to induce nuclear expression of NF-kappa B, AP-1 and NF-AT with a time course and intensity similar to that produced by anti-Ig. Examination of NF-kappa B in more detail demonstrated that the CD40 mediated expression of DNA binding complexes correlated with induction of trans-activating activity which again attained similar levels following cross-linking of CD40 and slg. Despite the marked similarity in transcription factor induction triggered through CD40 and slg, differences in the intracellular signaling pathways utilized were apparent in that protein kinase C (PKC) depletion did not affect CD40 mediated induction of NF-kappa B even as induction by anti-Ig was abolished. These results suggest that a 'final common pathway' or convergence of transcription factor induction may exist for two distinct receptors, each of which is individually capable of triggering cell cycle progression, despite the use of separate intracellular signaling pathways that differ at the level of PKC. Although transcription factor induction was similar for CD40L and anti-Ig early on, subtle differences in expressed NF-kappa B and AP-1 nucleoprotein complexes were apparent at 24 h. Such differences may play a role in determining the variant effects on B cells of stimulation through these two receptors. PMID- 7537533 TI - Cloning the rat homolog of the CD28/CTLA-4-ligand B7-1: structural and functional analysis. AB - T cell activation involves the delivery of two independent signals to the naive T cell. The first signal occurs with engagement of the TCR. One of the best characterized second signals is ligation of CD28 on the surface of T cells by B7 molecules (B7-1, B7-2) present on the surface of activated antigen presenting cells (APCs). Recent studies have demonstrated that injection of a human fusion protein, CTLA-4-Ig, which in humans binds to both B7-1 and B7-2, prevents cardiac allograft rejection in a rat transplantation model when given 48 h after engraftment. In order to better characterize the role of B7-1 (which is maximally expressed 48 h after activation of APCs) in this model, as well as in models of tumor-induced immune responses, we have cloned the rat homolog of B7-1, and now report on its structure and function. A 1030 bp cDNA containing the entire coding sequence of the rat B7-1 was cloned with a polymerase chain reaction strategy utilizing degenerate primers derived from published murine and human B7-1 sequences. The rat B7-1 coding sequence is 67 and 81% homologous to human and murine B7-1 cDNAs, and the predicted peptide sequence is likewise 57 and 66% identical to the peptide sequences of human and murine B7-1 respectively. The greatest area of identity occurs in the extracellular portion of the molecule, particularly the Ig-C like domain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537534 TI - FcR cross-linking on monocytes results in impaired T cell stimulatory capacity. AB - Presentation of antigen to T lymphocytes without the appropriate co-stimulatory signals results in a state of antigen-specific unresponsiveness. Despite the presumed importance of the B7-CD28 interaction for the initiation and maintenance of T cell-mediated immune responses, relatively few studies have addressed the regulation of B7 expression. We have studied the expression of the CD80 (B7-1) and B7-2 molecules on peripheral blood monocytes following different activation signals, and it was demonstrated that not only IFN-gamma, but also granulocyte macrophage colony stimulating factor can induce CD80 expression on monocytes. In addition, we found that cross-linking of FcR on monocytes strongly inhibits the up-regulation of CD80 and B7-2, with as a functional consequence that the capacity to function as antigen presenting cells (APC) and to stimulate T cell activation is severely impaired. When cultures were prepared in 96-well plates coated with human IgG, stimulation of T cells with allogeneic monocytes resulted only in modest T cell proliferation and no detectable IL-2 secretion as compared with untreated culture plates or plates coated with Fab fragments of human IgG. Under these conditions cross-linking of CD28 on the T cells with specific mAb completely reverted the inhibitory effect observed after culture on IgG-coated plates. Furthermore, FcR cross-linking on monocytes strongly inhibited the capacity of monocytes to induce a specific memory T cell response to viral, bacterial and fungal antigens, whereas the treatment did not impair the capacity of the T cells to respond to pokeweed mitogen, phytohemagglutinin and concanavalin A. We conclude that after FcR cross-linking, the impaired APC function is most likely due to the inability of monocytes to provide the essential co-stimulatory signals to the T cells via the B7-CD28/CTLA-4 interaction. PMID- 7537535 TI - Induction of Th2 responses to soluble proteins is independent of B cell tolerance status. AB - Injection of high doses of monomeric human gamma globulins (dHGG) in naive, adult mice causes antigen-specific tolerance of B cell and Th1 lymphocytes, while inducing the selective expansion of antigen-specific Th2 cells. Several parameters of tolerance induction were analyzed in this work, in order to establish whether B cell tolerance and Th1 unresponsiveness were functionally related in this in vivo model. By varying the antigen form and site of injection, we demonstrate in this work that Th1 unresponsiveness to HGG is not a consequence of peripheral B cell tolerance. In particular, mice pretreated with heat aggregated antigen (HAHGG) or F(ab')2 HGG were found to develop a strong humoral response while displaying a defective Th1 response. In fact, these animals developed a strong Th2 response in vivo, demonstrating that selective expansion of antigen-specific Th2 cells in this model is not a consequence of B cell tolerance or antigen capture by Fc receptor-expressing cells. We conclude that while B cell tolerance in this model is only observed in response to deaggregated antigen, injection of all forms of adjuvant-free, protein antigens induces T helper precursor cells to differentiate into Th2-type helper cells in vivo irrespectively of the B cell tolerance status. PMID- 7537536 TI - Antigen mediated and polyclonal stimulation of human cytokine production elicit qualitatively different patterns of cytokine gene expression. AB - Polyclonal activators are widely used as surrogate antigens in analysis of human cytokine gene expression. An implicit assumption is that the T cell activation and cytokine production observed in response to polyclonal activation provides a more intense, but qualitatively identical, reflection of results that would be obtained with antigen. Here we demonstrate that stimulation using accessory cell independent (immobilized anti-CD3 mAb) or dependent [phytohemagglutinin (PHA) or soluble anti-CD3 mAb] polyclonal activators yields different conclusions from those that are obtained in response to antigen-specific T cell activation. Cytokine synthesis in 1-5 day bulk cultures of fresh peripheral blood mononuclear cells (PBMC) from 52 subjects evenly divided between grass pollen sensitive allergic rhinitis subjects and normal, non-atopic controls were examined. Antigen specific re-stimulation elicited elevated IL-4 and IL-10 production and lower IFN gamma synthesis among allergic subjects than normal non-atopic control subjects. This commitment of fresh PBMC towards a Th2-like response in atopics and the dominance of the IFN-gamma response seen in non-allergic subjects was reinforced when the ratio of IFN-gamma:IL-4 production in bulk culture was examined. Atopic individuals exhibited median IFN-gamma:IL-4 values of 0.07, whereas grass pollen stimulated cytokine production by normal subjects yielded a ratio of 4.8. In marked contrast, IL-2, IL-4, IL-10 and IFN-gamma production elicited using polyclonal activators, though much more intense, did not differ between allergic and non-allergic subjects (Wilcoxon rank sum test P > > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537537 TI - Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated. AB - It has recently been recognized that CD44 comprises a large family of alternatively spliced forms. In the thymus, CD44 has been postulated to play an important role in immature T cell migration and maturation. In this paper, we have studied the expression of CD44 molecules and two CD44 ligands, hyaluronan (HA) and fibronectin (FN), during human thymic fetal development. We found that mAbs against all CD44 isoforms (A3D8 or A1G3) reacted with both thymic epithelial (TE) cells and thymocytes beginning at the time of initial colonization of the human thymus by hematopoietic stem cells at 8.2 weeks of fetal gestation. However, mAbs specific for splice variants of CD44 containing membrane-proximal inserts (11.24, 11.10 and 11.9) reacted only with terminally differentiated TE cells in and around Hassall's bodies beginning at 16-19 weeks of fetal gestation. Studies of differentiated versus undifferentiated TE cells in vitro confirmed the selective expression of CD44 variant isoforms on terminally differentiated TE cells. Expression of HA and FN was determined by fluorescence microscopy using either biotinylated-HA binding protein or an anti-FN mAb. We found that whereas FN was present throughout the human fetal thymus beginning at 8.2 weeks, HA was not present until 16 weeks of gestational age. These data demonstrate the differential expression of standard versus variant CD44 isoforms during thymic ontogeny and implicate CD44 interactions with ligands other than HA as important in the earlier stages of human thymus development. PMID- 7537538 TI - Involvement of CD5 in Th1 and Th2 contact-mediated rescue of anti-mu and ionomycin induced growth inhibition in a B cell lymphoma. AB - Cross-linking of membrane Ig receptors by anti-mu antibodies (Ab) or treatment with ionomycin induced complete growth arrest and subsequent apoptotic cell death in an immature B cell lymphoma, BKS-2. The growth-inhibitory signals delivered by anti-mu and ionomycin were overcome by anti-CD3-activated Th2 clones D10.G4 and F1 and by Th1 cell clone S53. In this report the Th-mediated growth reversal in BKS-2 cells was shown to require contact-dependent interactions when the inhibition was caused by immobilized anti-mu or ionomycin. Th2 cells in transwells (lymphokines) failed to protect BKS-2 cells from the growth-inhibitory effect of immobilized anti-mu or ionomycin. Monoclonal antibodies to CD5 or CD40 ligands on activated Th cells partially inhibited the Th2 contact-dependent growth reversal of BKS-2 cells whereas simultaneous addition of both antibodies effectively prevented the delivery of contact-mediated growth signal. In contrast, anti-class I or class II Ab did not affect Th cell mediated growth reversal of BKS-2 cells. These data demonstrated that noncognate physical interaction with Th cells was essential for the recovery of BKS-2 cells when the latter were growth-arrested by strong inhibitory stimuli such as immobilized anti mu and ionomycin. Further CD5 as well as CD40 ligands on Th cells are important for signal transduction in this type of T-B interaction. PMID- 7537539 TI - In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice. AB - Hemophilia A is caused by blood coagulation factor VIII (FVIII) deficiency and is an attractive target for gene therapy. However, features of FVIII physiology, such as the instability of the mRNA and protein, have provided obstacles to the design of a feasible strategy for the transfer and expression of the human FVIII gene in vivo. We have constructed a recombinant adenoviral vector, Av1ALH81, that contains the human FVIII cDNA from which the B-domain has been deleted (BDD FVIII) and extensively characterized this vector in vitro and in vivo. In vitro, HepG2, human hepatoma cells, transduced with Av1ALH81 secreted high levels of biologically active human BDD FVIII measured by the Coatest bioassay (> 2,400 mU per 10(6) cells per 24 hr). Administration of Av1ALH81 to mice, via tail vein, resulted in expression of human BDD FVIII in the mouse plasma at levels averaging 307 +/- 93 ng/ml 1 week post-injection, measured by a sensitive human FVIII specific ELISA. Normal FVIII levels in humans are 100-200 ng/ml, and therapeutic levels are as low as 10 ng/ml. Purification of the human FVIII from the mouse plasma, and subsequent Coatest analysis, revealed that the human FVIII produced in the mice was biologically active. In addition, the duration of FVIII expression in vivo was followed, and high-level FVIII expression was sustained over a period of several weeks. The finding that an adenoviral vector can mediate high-level expression of human FVIII in an animal model provides the basis for the development of gene therapy for hemophilia A. PMID- 7537540 TI - Adenovirus-mediated gene transfer for cystic fibrosis: Part A. Safety of dose and repeat administration in the nasal epithelium. Part B. Clinical efficacy in the maxillary sinus. PMID- 7537541 TI - Complement in serum and dialysate in children on continuous ambulatory peritoneal dialysis. AB - OBJECTIVE: During continuous ambulatory peritoneal dialysis (CAPD), activation of complement in the peritoneal cavity may theoretically occur, with inappropriately high or low levels of certain complement factors in dialysate as a consequence. In a group of children on CAPD, it was tested whether levels of a number of complement factors in dialysate were in the range that was predicted on the basis of their molecular weight. DESIGN: Serum and dialysate levels of C1q, C3, C4, C3d, B, D, and P were measured after a night dwell in children on CAPD. Simultaneously, four non-complement proteins (beta 2-microglobulin, albumin, IgG, and alpha 2-macroglobulin) were also measured in dialysate and serum. Assuming a linear relationship between the log base 10 of the dialysate/serum ratio of these non-complement proteins and the log base 10 of their molecular weight, the expected ratios of all complement factors were determined. The differences between actual and predicted ratios were tested using a modified t-test, taking into account the inaccuracy of the estimate. SETTING: University hospital. PATIENTS: A group of 14 children on CAPD, with a median age of 7.8 years (range 2.1 - 13.2). These children had been on CAPD for a median period of 42.4 months (range 0.4 - 89.1). RESULTS: The ratios of factor D (p < 0.001) and C3d (p = 0.035) were elevated, whereas those of C3 (p < 0.001), C4 (p < 0.001), and factor P (p = 0.012) were decreased. CONCLUSIONS: Relatively low dialysate/serum ratios of C4, C3, and factor P could be caused by intraperitoneal consumption of complement. High levels of C3d are compatible with this. High dialysate/serum ratios of factor D indicate intraperitoneal production of factor D. These results provide evidence for activation of complement in the peritoneal cavity in children on CAPD. A further reduction of already low levels of complement factors in dialysate as a result of this may impair host defense. PMID- 7537543 TI - Male reproductive toxicity testing. PMID- 7537542 TI - Changes in salivary [K+], [Na+] and [K+]/[Na+] with varied test demands. AB - It was hypothesised that choice reaction-time (CRT) testing would cause salivary [K+]/[Na+] to increase. Relative contributions of [K+] and [Na+] to ratio changes were investigated in 23 hypertensives and ten hospital staff. Changes in post rest and post-test ionic concentrations and [K+]/[Na+], replicated earlier studies. Phasic [K+]/[Na+] changes were mainly due to [K+] changes. Significant increases in [K+] and decreases in [Na+] from a relaxed session, the day before CRT testing, to the testing session per se indicated test anticipation effects. In both groups, changes from pre-test "rest" to "on test" were significant only for [K+]. [K+] discriminated well between conditions in hypertensives. This was interpreted in terms of adaptive changes in sympathetic activation. Results show the robustness of salivary ion indices (especially of [K+]) as indicators of within-subject response to mental task demands. PMID- 7537545 TI - [Chronic hepatitis C. Predictive factors of response to treatment with interferon]. PMID- 7537544 TI - Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone. AB - The metabolism of arachidonic acid (AA) into vasoactive products by cyclooxygenase and lipoxygenase enzymes has been well described, as has their biological relevance. Recently, a number of studies have demonstrated the ability of cytochrome P-450 (P450) enzymes to metabolize AA into biologically important regulators of vascular tone. There are two categories of vasoactive P450 metabolites, namely those catalyzed by epoxygenase enzymes which generate epoxyeicosatrienoic acids (EETs) and those enzymes which generate hydroxyeicosatetraenoic acids (HETEs). Except for 20-HETE, P450 metabolites of AA occur as stereo- and regioisomers which determine, to some extent, their biological activity. 5,6-, 8,9-, 11,12- and 14,15-EETs are generally potent dilators in a number of vascular beds with a sensitivity which appears to increase as the vasculature decreases in size toward capillaries. HETEs, such as 12R- and 20-HETE, can be potent activators of vascular tissue with 20-HETE contracting cerebral and renal microvessels at concentrations of < 10(-10) M. Both EETs and HETEs can be made by vascular and extravascular tissue. Available data suggests that EETs are formed by endothelial and parenchymal tissue while HETEs can be endogenously formed in arterial muscle where they appear to act as second messengers. This review will discuss the molecular biology, stereochemistry, biological activity and importance of P450 metabolites of AA as para- and autocrine controllers of organ blood flow. We will also discuss the large diversity of P450 enzyme isoforms and how such diversity can provide for precise physiological control of vascular tone. PMID- 7537546 TI - Comparison of Giemsa-stained smear with Papanicolaou-stained smears of the same specimen. PMID- 7537547 TI - [Morbidity and mortality related to gastroenteroanastomosis in advanced gastric cancer]. AB - OBJECTIVE: Determination of the postoperative morbidity and mortality after gastroenterostomy in patients with unresectable gastric cancer. STUDY DESIGN: Retrospective review of clinical records of all patients with obstructive distal gastric cancer who underwent gastroenterostomy at the Instituto de Enfermedades Neoplasicas between 1980 and 1993. The following factors were analyzed: age, sex, hemoglobin, albumin, preoperative risk, ascites, extent of disease, operative time, hospital stay, morbidity and mortality. RESULTS: 198 gastroenterostomy were done with a morbidity and mortality rates of 20% and 10%, respectively. Pneumonia was the principal cause of postoperative morbidity and mortality. High operative risk, adjacent organ invasion by the tumor and peritoneal metastasis were factors associated with increased postoperative morbidity (p > 0.05). High operative risk was the only prognostic factor for postoperative mortality (p < 0.01). CONCLUSIONS: Because of high postoperative morbidity and mortality, gastroenterostomy should not be done in patients with unresectable gastric cancer and high preoperative risk. PMID- 7537548 TI - [Cellular and molecular mechanisms of the gastric mucosa: injury of the mucosa and the protective action of antacids]. AB - Gastrointestinal mucosa is exposed to many substances, some made by their own body as HCl, pepsin, etc, others from exogenous origin as NSAIDs, alcohol etc. that injury the mucosa. The body has build protective mechanism against the injury that we describe in the article. We know antacids acts neutralizing the acid a now we know it work as a powerful stimulant of the mucosal protection. This is called cytoprotection and is described in the article. PMID- 7537549 TI - Synergistic action of interleukin-10 (IL-10) with IL-3, IL-4 and stem cell factor on colony formation from murine mast cells in culture. AB - We examined the effects of interleukin-10 (IL-10) on colony formation from two different types of murine mast cells, bone marrow-derived mast cells (BMMC) and serosal mast cells (SMC), using a methylcellulose culture method. IL-10 alone did not induce colony formation from either phenotype. However, BMMC and SMC produced colonies in the presence of IL-10 in combinations with IL-3, IL-4 or stem cell factor (SCF), but they responded in different manners. IL-10 enhanced the IL-3 dependent colony formation from BMMC and induced the colony formation from BMMC synergistically with IL-4 or SCF dose-dependently, although IL-4 and SCF were not active alone. The most significant synergism was observed between IL-10 and IL-4. The addition of IL-10 to the cultures of BMMC in the presence of two or three factors enhanced the colony formation induced by IL-3 plus IL-4, and inhibited the colony formation induced by IL-3 plus SCF or IL-3, IL-4 plus SCF. In the colony formation from SMC, IL-10 synergized with IL-3 but not with IL-4 or SCF. IL-10 in combination with two or three factors enhanced the colony formation from SMC induced by IL-3 plus IL-4, but did not affect the colony formation induced by other combinations among IL-3, IL-4 and SCF. These findings indicate that IL-10 plays an important role in the proliferation of murine mast cells. PMID- 7537550 TI - Exogenously expressed granulocyte colony-stimulating factor (G-CSF) receptor on K562 cells can transduce G-CSF-triggered growth and differentiation signals. AB - Human granulocyte colony-stimulating factor (G-CSF) receptor cDNA was introduced into the erythroleukemic cell line K562, which normally does not express the receptor, using lipofection transfection of a G-CSF receptor expression plasmid vector. Transfected cells expressed the receptor with a dissociation constant of 130 pmol/L, and a maximum of 11,800 binding sites per cell. Culture of G-CSF receptor-expressing cells (GR-K562) in the presence of G-CSF enhanced DNA synthesis and a stimulation index of 4.61 was obtained in a 3H-thymidine uptake assay. Furthermore, flow cytometric studies revealed induced expression of CD11b (29% from 7%), and enhanced expression of CD13 (57% from 27%) on GR-K562 cultured in the presence of G-CSF. Our findings indicate that the exogenously expressed G CSF receptor can deliver signals and function efficiently on these immature cells on which the receptor is not normally expressed, which suggests the presence of intact intracellular signal transduction pathways that can be used by the ectopically expressed receptor. PMID- 7537551 TI - Induction of diaphorase-1 by dicoumarol in Drosophila virilis larvae. AB - Drosophila diaphorase-1 (DIA-1) is an enzyme similar to mammalian DT-diaphorase and is inhibited in vitro by dicoumarol. However, a ten-fold increase in DIA-1 activity was observed when third instar Drosophila virilis larvae were fed on a diet containing 0.1 M dicoumarol for 48 h. This induction was shown to be dose dependent and immunoprecipitation experiments with DIA-1 anti-serum demonstrated an increase in the DIA-1 protein level in dicoumarol-treated larvae. The induction of DIA1 by dicoumarol was found to be blocked by actinomycin D, which suggests a transcriptional mechanism of regulation. The opposite effect of dicoumarol on DIA-1 in vitro vs. in vivo suggests that a metabolic conversion takes place after the ingestion of this compound by D. virilis larvae. PMID- 7537552 TI - Expression of zfh-4, a new member of the zinc finger-homeodomain family, in developing brain and muscle. AB - We have identified zfh-4, a new member of a recently recognized zinc finger homeodomain (zfh) family of putative transcription factors. Zfh-4 expression is prominent in developing muscle and brain. In both tissues, zfh-4 RNA levels are highest embryonically, then decrease gradually to barely detectable levels in adults. In myogenic cell lines, far more zfh-4 is expressed in proliferating myoblasts than in myotubes, suggesting a cellular basis for the developmental regulation observed in vivo. In contrast, zfh-4 RNA in brain is more abundant in postmitotic cells of the marginal zone than in proliferating cells of the ventricular zone. Within the brain, zfh-4 RNA is regionally localized: expression is highest in midbrain, readily detectable in hindbrain, and very low in cerebral cortex. Its patterns of expression, and its homology to known DNA binding proteins, support the idea that zfh-4 may be a regulator of gene expression in developing brain and muscle. PMID- 7537553 TI - Angiogenic potential of the avian somite. AB - We have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH-1 antibody after 1-6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH-1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality. PMID- 7537554 TI - Interaction of lactoferrin with ascorbate and the relationship with bleomycin dependent DNA damage. AB - The interaction between bovine lactoferrin (bLf) and ascorbate (Asc) was investigated through malondialdehyde (MAD) formation in a solution containing DNA, bleomycin (BLM), and Fe2+ or Asc. The inhibition by bLf on MDA formation in the presence of Asc was not changed even by adding carbonate or oxalate ions to the solution. The percentage inhibition by the hydrolysates of bLf treated with pepsin, trypsin, and both enzymes on MDA formation was almost the same as that by the untreated bLf in the presence of Asc. The inhibition of MDA formation also occurred with the filtrate obtained from a solution containing bLf and Asc, but not with that from a solution of bovine serum albumin and Asc. The interaction of bLf and Asc was observed by gel filtration in a Sephadex G75 column. The binding amount of Asc was estimated to be 87 mol per mole of bLf. PMID- 7537556 TI - Structural requirements in heparin for binding and activation of FGF-1 and FGF-4 are different from that for FGF-2. AB - Size- and structure-defined oligosaccharides from heparin, 2-O-desulphated (2-O DS-) heparin, 6-O-desulphated (6-O-DS-) heparin, carboxy-reduced (CR-) heparin, and carboxyamidomethylsulphonated (AMS-) heparin were utilized in characterizing the structural properties of heparin to specifically bind to basic fibroblast growth factor (FGF-2) and to modulate the mitogenic activity of FGF-2 (Ishihara, M. et al., Glycobiology, 4, 451-458, 1994). The previous results showed that both 2-O-sulphate groups and the negative charge of the carboxy group in iduronate residues are required for specific interaction with FGF-2, but the 6-O-sulphate groups in N-sulphated glucosamine (GlcNS) residues do not influence the interaction with FGF-2. In the present study, the same oligosaccharides were fractionated on a FGF-1- or FGF-4-affinity column, and were assessed as promoters of FGF-1- or FGF-4-induced proliferation of adrenocortical endothelial (ACE) cells and chlorate-treated ACE cells. The present results suggest that the smallest heparin-derived oligosaccharide binding to these growth factors with the highest affinity and promoting their mitogenic activities is a fully N-sulphated decasaccharide enriched in 2-O- and 6-O-sulphated disaccharide units. In contrast to our results with FGF-2, a high content of 6-O-sulphate groups in GlcNS residues is required for specific interaction with FGF-1 and FGF-4. PMID- 7537559 TI - Determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in plasma and urine by HPLC with fluorimetric detection. AB - Using native fluorescence detection, 5-hydroxytryptamine (5-HT), 5 hydroxyindoleacetic acid (5-HIAA) and tryptophan were resolved from themselves and other naturally occurring compounds using reversed-phase HPLC within 5 min. Deproteinated platelet-poor plasma (PPP) and crude diluted urine were injected directly into the chromatograph. Careful selection of the HPLC column is important and various octadecyl silica (ODS) and base deactivated silic (BDS) columns were evaluated. Pre-treatment of an ODS column with tetrabutylammonium ions gave good selectivity. Between pH 5 and 6 the compounds were well resolved from each other. The limit of quantitative detection of 5-HT and 5-HIAA was 3.5 nmol/L. The overall chromatogram obtained using native fluorescence is cleaner than that obtained with the more commonly employed electrochemical (EC) systems although the chromatography is effectively the same. For analysis of 5-HT in plasma, collection in EDTA was more efficient than lithium heparin. Plasma 5-HT in healthy volunteers was mean 61 (SD = +/- 73) nmol/L, n = 20; urine 5-HIAA gave mean 28.95 (SD = +/- 0.98) mumol/L, (n = 12). Whole blood 5-HT analysis is unreliable in comparison with platelet-poor plasma. PMID- 7537557 TI - Differential regulation of CD43 glycoforms on CD4+ and CD8+ T lymphocytes in graft-versus-host disease. AB - Two distinct T-cell glycoforms of CD43 result from differential glycosylation of a single gene product in vivo. The 115 kDa glycoform carries mainly tetrasaccharides and is a pan T-cell marker, whereas the 130 kDa glycoform carries mainly hexasaccharides and is associated with T-cell activation. CD43 has been shown to play a role both in enhancing and inhibiting cell adhesion; however, the function of the individual glycoforms is unknown. We have examined the distribution and regulation of the CD43 glycoforms in a murine model of acute graft-versus-host disease (GVHD) using monoclonal antibodies (mAbs) S7 and 1B11 specific for the 115 and 130 kDa CD43 glycoforms, respectively. An increase in T lymphocyte CD43 130 kDa expression occurred during GVHD from day 4 onwards and coincided with splenomegaly and upregulation of the beta 1-6GlcNAc transferase (C2GnT), the key enzyme responsible for the addition of complex O-glycan branching to CD43. When T-lymphocyte subsets were examined for CD43 expression, we found that in GVHD, both CD43 glycoforms were upregulated on CD4+ T cells. However, in CD8+ T cells, CD43 115 kDa was downregulated while CD43 130 kDa was dramatically upregulated, such that two distinct CD8+1B11+ T-cell subsets were observed. These data demonstrate differential expression of the CD43 glycoforms in both resting and activated CD4+ and CD8+ T cells, and suggest that glycosylation differences between the CD43 glycoforms may reflect participation in the different functions of these T-cell subsets in immune disorders in vivo. PMID- 7537558 TI - Enzymatic synthesis of a 6'-sulphated sialyl-Lewisx which is an inhibitor of L selectin binding to peripheral addressin. AB - A sulphated form of sialyl-Lewisx, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1 3)GlcNAc6OSO3 beta 1-3Gal, was synthesized enzymatically from a precursor disaccharide, GlcNAc6OSO3 beta 1-3Gal, using sequential steps involving beta 1,4 galactosyltransferase, alpha 2,3-trans-sialidase and recombinant alpha 1,3 fucosyltransferase, respectively. Successful enzymatic fucosylation at the 3 position of the GlcNAc6OSO3 residue demonstrated that fucosyltransferase are capable of generating, in situ, sulphated sialyl Lewisx structures containing sulphate at the 6 position of GlcNAc. The sulphated sialyl-Lewisx pentasaccharide produced by this procedure inhibited binding of a soluble form of L-selectin to 35SO4-labelled peripheral addressin with an IC50 of 0.8 mM, whereas sialyl-Lewisx tetrasaccharide was a weaker inhibitor, displaying an IC50 of 3.2 mM. Hemmerich and Rosen (Biochemistry, 33, 4820-4829, 1994) recently reported the presence of Gal beta 1-4GlcNAcO6SO3 structures on murine peripheral addressin Sgp50, in addition to sialyl Lewisx structures sulphated at the 6-O-galactose position. Based on our data, we suggest that sialyl Lewisx sulphated at the 6-O-GlcNAc position may also exist on receptors and function as a ligand for L-selectin. PMID- 7537555 TI - Encapsulation of biologically active proteins in a multiple emulsion. AB - To improve the stability of IgY antibody in oral administration, encapsulation of IgY in a W/O/W emulsion was attempted. A stable W/O/W emulsion containing 1% IgY was prepared by using polyglyceryl condensed ricinolate (PGCR) and dextran-casein conjugate as the primary and secondary emulsifier, respectively. However, the activity of IgY antibody was reduced to less than 20% by encapsulation, suggesting that denaturation/inactivation of IgY had occurred at the oil/water interface. Adsorption of IgY to the inner water droplet surface was observed by electron microscopy. Rabbit IgG, alpha-amylase, and lysozyme also lost their activity after being encapsulated, although the rate of inactivation was lower than that of IgY. Molecular characterization of these proteins suggested that the rate of inactivation after encapsulation is likely to be dependent on the surface hydrophobicity and molecular stability of each protein. PMID- 7537560 TI - Further studies on the use of oligonucleotide facilitators to increase ribozyme turnover. AB - We showed previously that the turnover of a hammerhead ribozyme cleaving a substrate RNA could be increased by an oligonucleotide (facilitator) that bound to the substrate continguously with the ribozyme. This phenomenon has been investigated further by varying the temperature and the number of base pairs formed between the ribozyme and substrate. The results support the hypothesis that facilitators act by cooperative binding and are beneficial when the rate of cleavage is limited by the stability of the ribozyme/substrate complex. The facilitator also promotes cleavage at lower concentrations of magnesium. PMID- 7537561 TI - Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: a comparative study. AB - Phosphorodithioate (PS2) oligodeoxynucleotides (oligos) represent a relatively new class of backbone-modified oligo that have potential use as antisense agents. PS2 oligos are isoelectronic with phosphodiester (PO) and phosphoromonothioate (PS) oligos, and are nuclease resistant. However, unlike their PS congeners, PS2 oligos do not contain chiral centers. Little is known about the manner in which PS2 oligos interact with biological systems. In this study, we compare the cellular pharmacology of PS and PS2 oligos in HL60 cells. Cell surface binding, internalization, and compartmentalization are examined. Furthermore, the ability of PS and PS2 oligos to bind to rsCD4 and bFGF and to inhibit the activity of protein kinase C (PKC) is examined. Although the behavior of PS2 oligos closely parallels that of PS oligos, PS2 oligos appear to interact with some biological systems in a slightly different manner than PS oligos. These results indicate that PS2 oligos may have therapeutic potential other than as antisense agents. PMID- 7537563 TI - RNA-dependent DNA binding activity of the Pur factor, potentially involved in DNA replication and gene transcription. AB - The PUR element is a polypurine polypyrimidine motif that can stimulate transcription, encountered in the 5' regions of various genes and in the vicinity of several DNA replication initiation zones. We demonstrate that the PUR complex formation between the purine-rich strand of PUR and nuclear extracts can be prevented by pretreatment of nuclear extracts with RNA-damaging agents such as UV light or RNase A. A biochemical affinity method reveals that small RNA molecules copurify with the Pur factor. Moreover, the PUR binding activity of RNA-depleted nuclear extracts can be restored by addition of phenol-extracted RNAs. This work adds a new member in the emerging class of ribonucleoprotein particles as regulatory factors of the genetic expression. PMID- 7537562 TI - Identification of a stable RNA encoded by the H-strand of the mouse mitochondrial D-loop region and a conserved sequence motif immediately upstream of its polyadenylation site. AB - By using a combination of Northern blot hybridization with strand-specific DNA probes, S1 nuclease protection, and sequencing of oligo-dT-primed cDNA clones, we have identified a 0.8 kb poly(A)-containing RNA encoded by the H-strand of the mouse mitochondrial D-loop region. The 5' end of the RNA maps to nucleotide 15417, a region complementary to the start of tRNA(Pro) gene and the 3' polyadenylated end maps to nucleotide 16295 of the genome, immediately upstream of tRNA(Phe) gene. The H-strand D-loop region encoded transcripts of similar size are also detected in other vertebrate systems. In the mouse, rat, and human systems, the 3' ends of the D-loop encoded RNA are preceded by conserved sequences AAUAAA, AAUUAA, or AACUAA, that resemble the polyadenylation signal. The steady-state level of the RNA is generally low in dividing or in vitro cultured cells, and markedly higher in differentiated tissues like liver, kidney, heart, and brain. Furthermore, an over 10-fold increase in the level of this RNA is observed during the induced differentiation of C2C12 mouse myoblast cells into myotubes. These results suggest that the D-loop H-strand encoded RNA may have yet unknown biological functions. A 20 base pair DNA sequence from the 3' terminal region containing the conserved sequence motif binds to a protein from the mitochondrial extracts in a sequence-specific manner. The binding specificity of this protein is distinctly different from the previously characterized H-strand DNA termination sequence in the D-loop or the H-strand transcription terminator immediately downstream of the 16S rRNA gene. Thus, we have characterized a novel poly(A)-containing RNA encoded by the H-strand of the mitochondrial D-loop region and also identified the putative ultimate termination site for the H-strand transcription. PMID- 7537564 TI - Differentiation of B16 murine melanoma cells is associated with an increased level of c-SRC. AB - Elevated levels of c-SRC activity have been found in human melanocytes and some human melanoma cell lines. We examined c-SRC in B16 murine melanoma cells. B16-F0 non-metastatic melanoma cells contained threefold more c-SRC activity and protein than NIH 3T3 murine fibroblasts. Differentiation of B16-F1 metastatic melanoma cells with retinoic acid resulted in an elevation in c-SRC activity, protein and mRNA. The increase in c-SRC was detectable after about 48 h of retinoic acid treatment, as were changes in cellular morphology and growth rate. Thus, there is a correlation between differentiation and expression of c-SRC in B16 murine melanoma cells. These findings suggest a role for c-SRC in murine melanocyte differentiation or function. PMID- 7537566 TI - Proinflammatory cytokines in allergic rhinitis. AB - Allergic diseases such as allergen-induced rhinitis represent an inflammatory reaction that is characterized by the chemotaxis and activation of various cell populations. A high degree of cell-to-cell communication is needed to orchestrate this inflammatory immune response. A variety of cytokines and adhesion receptors seem to play an important role in the allergic late phase reaction. Here we demonstrate that proinflammatory cytokines such as interleukin(IL)-1, IL-8 and TNF-alpha (tumor necrosis factor-alpha) can be detected in nasal secretions and mucosa by enzyme-linked immunosorbent assay and immunohistochemistry. The increased expression of adhesion receptors in mucosa specimens of patients with seasonal allergic rhinitis points to their role in regulating the cellular migration and probably represents a key event in allergic inflammation. We established an in vitro model using freshly taken nasal mucosa to study the induction of adhesion receptors by proinflammatory cytokines. E-selectin, an endothelial receptor, was strongly upregulated by IL-1 beta, TNF-alpha and allergen. The induction due to allergen exposure of the mucosa was markedly inhibited by soluble cytokine receptors (sIL-1R, TNF-BP) or by a receptor antagonist (IL-1ra) and prednisolone, These findings indicate that proinflammatory cytokines may be key factors for the upregulation of adhesion processes in human nasal mucosa and the activation of various cell populations involved in the allergic inflammation. They therefore represent a main target for new therapeutic strategies. PMID- 7537567 TI - Cytokines in neutrophil-dominated airway inflammation in patients with cystic fibrosis. AB - Bronchopulmonary disease in patients with cystic fibrosis (CF) is a paradigm of neutrophil-dominated airway inflammation. We hypothesized that proinflammatory cytokines contribute to a localized neutrophil-dominated inflammatory state as present in CF airways. In a cross-sectional study, we analyzed 63 sputum samples from 33 CF patients for concentrations of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), and granulocyte colony stimulating factor (G-CSF) by means of enzyme-linked immunosorbent assay. Furthermore, the activity of neutrophil elastase (NE) in the sputum samples was determined using a specific chromogenic substrate. Compared to sputum samples from 10 healthy controls, there were significantly increased concentrations of IL 1 beta, IL-8 and TNF-alpha in the CF sputum samples. The concentration of IL-8 correlated significantly with NE activity in the CF sputum samples. In CF patients with airways chronically colonized with Pseudomonas aeruginosa, IL-8 concentrations in sputum were significantly enhanced. In glucocorticoid-treated patients, IL-1 alpha and G-CSF sputum concentrations were significantly lower when compared to levels in the other patients. These results show that there are high concentrations of proinflammatory cytokines in CF airways which may contribute to the localized neutrophil-dominated inflammatory state found clinically. PMID- 7537568 TI - [The structural characteristics of the polyribosomes in the rat liver studied by means of a tritium label]. AB - Polyribosomes isolated from the rat liver in a medium with low ionic strength were irradiated by "hot" tritium atoms under conditions providing for the replacement of the hydrogen atoms located at the surface of polyribosomes by tritium. After fractionation of such polyribosomes, the radioactivity of the obtained fractions was measured and their proportions were calculated for the total surface accessible for the tritium atoms (in %), as well as their specific radioactivity. The material loosely associated with the polyribosomes and containing amino acyl-tRNA-synthetases is more radioactive than rRNA and r proteins, especially concerning their specific radioactivity. This suggests that the material is organized as individual molecules located on the surface of ribosomes. The specific radioactivity of the RNA-component of this material (tRNA) is twice that of proteins, thus suggesting its surface localization in the composition of loosely associated material. Based on the pattern of labeling of rRNA and r-proteins of the native and preliminarily dissociated polyribosomes, we propose that the material, loosely associated with the polyribosomes, has affinity to both rRNA and r-proteins. PMID- 7537565 TI - Mast cells, eosinophils and IgE-positive cells in the nasal mucosa of patients with vasomotor rhinitis. An immunohistochemical study. AB - Vasomotor rhinitis (VMR) is a disorder of unknown pathogenesis. Forty patients with VMR were carefully selected on the basis of inclusion and exclusion criteria proposed by Mygind and Weeke. Nasal biopsy specimens were taken in the patient group as well as in a group of ten controls. Brush cytology was also taken in the VMR group. Inflammatory cells were identified and counted in the nasal mucosa, with the use of immunohistochemical techniques and a panel of monoclonal antibodies. Eosinophils were studied with the use of BMK13, EG2, and Giemsa. Mast cells were studied with anti-chymase (B7), anti-tryptase (G3) and toluidine blue. Sections were stained with IgE as well. There was no significant difference in the number of eosinophils, mast cells and IgE-positive cells between the two groups. Additionally, in contrast with other reports, in sections that were double-stained with anti-chymase and anti-tryptase, single chymase-positive cells were found. PMID- 7537570 TI - Pentamethyl-hydroxychromane, vitamin E derivative, inhibits induction of nitric oxide synthase by bacterial lipopolysaccharide. AB - Vitamin E, a lipophilic antioxidant, has effectively inhibited the activation of cytokine-induced nuclear factor kB (NFkB). Since NFkB plays a critical role in the induction of an isoform of nitric oxide synthase (iNOS) gene by lipopolysaccharide (LPS), we investigated the effect of a vitamin E derivative, pentamethyl-hydroxychromane (PMC), which is an extremely potent inhibitor of NFkB activation, on the induction of nitric oxide (NO) synthesis and iNOS mRNA by LPS. PMC inhibited the LPS-stimulated induction of NO production in a concentration dependent fashion in cultured J774 macrophages and rat vascular smooth muscle cells without evidence of cytotoxicity. However, the addition of PMC to J774 macrophages after the induction of iNOS did not inhibit NO production. Treatment of J774 macrophages with LPS resulted in a significant expression of iNOS mRNA, which was profoundly reduced by PMC. Data suggest that PMC inhibits the induction of iNOS by preventing iNOS gene expression through inhibition of NFkB activation. PMID- 7537569 TI - Acquisition of viable-like surface properties of sperm cells by adsorption of seminal plasma proteins revealed by centrifugal countercurrent distribution. AB - By using centrifugal countercurrent distribution in dextran/polyethylene glycol two-phase system, we show that the acquisition of adsorbed seminal plasma proteins by the sperm cell surface modifies the partition behaviour of bull spermatozoa reproducing that of alive cells. Thus, addition of increasing concentrations of proteins from the seminal plasma promoted a saturable enhanced affinity of the cells to the polyethylene glycol-rich phase in which alive cells preferentially partition. On the contrary, treatment with Tween provoked the opposite effect. PMID- 7537571 TI - Antigenic specificity of anti-ROS DNA antibodies: involvement of lysyl residues in antigen binding. AB - The antigenicity of native DNA modified with reactive oxygen species was examined. Goats were immunized with the modified polymer and the antibody response was estimated by direct binding and competition ELISA. The induced antibodies bound ROS-DNA and showed considerable binding to native DNA as well. Specificity analysis of the purified antibodies revealed the recognition of native B-, A- and allied conformations presented by various synthetic polynucleotides. The contribution of lysine residues to the immunochemical binding of purified IgG was investigated by modifying the free amino groups of lysine residues. The modification of lysine residues paralleled loss in IgG binding to ROS-DNA to the extent of 50%, suggesting that such residues might be involved in the antigen binding site of immunoglobulin molecule. PMID- 7537572 TI - Enhancement of cytokine production by macrophages stimulated with (1-->3)-beta-D glucan, grifolan (GRN), isolated from Grifola frondosa. AB - The ability of grifolan (GRN), a purified fungal (1-->3)-beta-D-glucan, to induce various cytokines from macrophages was examined in vitro. Interleukin-6 (IL-6) activity in supernatants from the culture of macrophage cell line, RAW264.7 was dependent on increasing doses of GRN. The level of IL-6 induced with 500 micrograms/ml of GRN was comparable to that induced with lipopolysaccharide (LPS) 10 micrograms/ml. Enhancement of the mRNA level of IL-6 by treatment with GRN was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of GRN on production of IL-6 was also observed using peritoneal macrophages from C3H/HeJ mice which did not respond to endotoxins. This data suggested that the ability of GRN to activate IL-6 production of macrophages is not due to contamination of endotoxins in the preparation. Enhanced production of cytokine by GRN was observed not only with IL-6, but also with interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). In the production of TNF alpha, GRN was more effective than LPS used in this study. Other soluble or gel-forming(1-->3) beta-D-glucans from various sources did not enhance the production of such cytokines although they are structurally similar to GRN. The above results indicate that GRN is a novel macrophage activator which augments cytokine production without dependence on endotoxins. PMID- 7537573 TI - Binding and aggregation of human gamma-globulin by cis diamminedichloroplatinum(II) through disulfide bond. AB - The incubation of gamma-globulin with cis-diamminedichloroplatinum (II) (cis-DDP) resulted in gradual formation of insoluble aggregates. Since the precipitates, composed of polymerized gamma-globulin and cis-DDP, were completely solubilized with urea, the reaction mixture containing precipitate was examined in terms of the binding of cis-DDP and the effect on disulfide (S-S) bonds in the gamma globulin. When gamma-globulin was incubated with 30 molar excess cis-DDP at pH 7.4 and 37 degrees C, cis-DDP gradually bound to as much as 12 mol per mol of gamma-globulin in 14 d. Concurrently, about four disulfide bonds were cleaved without reaching a certain plateau. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the aggregated gamma-globulin induced by cis-DDP was significantly different from that of the heat-denaturated aggregate form or the reduced form by sulfitolysis. The aquated complexes of cis-DDP also produced an insoluble precipitate and affected the S-S bond to a greater extent than the parent drug. PMID- 7537574 TI - Three isoforms of platelet-derived growth factors all have the capability to induce angiogenesis in vivo. AB - Three isoforms of platelet-derived growth factors (PDGFs) composing of AA, AB, and BB chains all exhibited angiogenic activity in a dose-dependent manner in an in vivo assay system involving the chorioallantoic membrane of chick embryo. The order of potency as BB > AB = AA. They, however, failed to stimulate proliferation of vascular endothelial cells, suggesting that their effects are indirect. These data suggest that possibility that three PDGF isoforms are indirect angiogenic factors. PMID- 7537575 TI - Analysis of antitumor properties of effector cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis. AB - Intestinal Bifidobacterium species are thought to be beneficial in animal and human intestines. We studied the mechanisms of Bifidobacteria in antitumor activity using a cell wall preparation (WPG) of B. infantis (Cancer Res., 45, 1300, (1985)). WPG enhanced the in vitro antitumor activities of mouse peritoneal exudate cells elicited with proteose-peptone (P-PEC) and thioglycollate broth (TG PEC), determined by cytostatic ([3H]thymidine uptake inhibition) and cytolytic ([3H]uridine release) assays. Tumor necrosis factor-alpha (TNF-alpha) and reactive nitrogen intermediates (RNI) play a role in such augmented cytotoxicity, because anti-TNF-alpha antibody almost completely blocked the increased cytolytic activity of P-PEC in the presence of WPG. Moreover, WPG induced RNI in the supernatant of TG-PEC in a dose-dependent manner. The mRNA expression of several cytokines (IL-1 beta, IL-6, IL-10, IFN-alpha and TNF-alpha) was induced in BALB/c mouse peritoneal cells 3 h after an intraperitoneal injection of WPG (3 h WPG PEC). However, this expression disappeared from 24 h WPG-PEC, except for that of IFN-alpha. IFN-gamma was not induced. Kinetic studies of the tumor neutralizing activities of the WPG-PECs by means of the in vivo Winn assay revealed that the activity emerged at 1.5 h, became maximal at 3 h and disappeared at 24h. These results indicated that Bifidobacterial WPG is a Biological Response Modifier (BRM) with characteristics similar to those of other bacterial BRMs. PMID- 7537576 TI - Effects of intrathecally injected glutamate and substance P antagonists on repeated cold stress-induced hyperalgesia in rats. AB - To determine the role of NK-1 substance P receptors and N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the spinal dorsal horn in the hyperalgesia induced by repeated cold stress (RCS), we examined the effects of intrathecal injections of antagonists to NK-1, NMDA and non-NMDA receptors on the nociceptive threshold of RCS rats for paw-pressure stimulation. Intrathecal injections of the NK-1 antagonist (2S,3S)-cis-2-(diphenylmethyl)-N-[(2 methoxyphenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine (CP-96,345, 0.3-3 nmol/rat), the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV, 1 10nmol/rat), and the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1-10 nmol/rat) suppressed RCS-induced hyperalgesia in a dose-dependent manner, without affecting the nociceptive threshold of normal rats. Combinations of any two of CP-96,345 (3 nmol/rat), APV (10 nmol/rat), and CNQX (10 nmol/rat) did not produce a larger inhibition than that produced by their single doses. The present results suggest that the enhancement of the substance P-NK-1 receptor system and glutamate-NMDA and non-NMDA receptor systems in the spinal dorsal horn is at least partly involved in the RCS-induced hyperalgesia. PMID- 7537579 TI - Invasive group A streptococcal infections: first report of enhanced surveillance. PMID- 7537577 TI - Substance P and serotonergic inputs to sympathetic preganglionic neurons. AB - Sympathetic preganglionic neurons are the final central links in the sympathetic pathways that control the heart and blood vessels. The neurotransmitters present in the supraspinal pathways that control the activity of sympathetic preganglionic neurons include amino acids, amines and peptides. In this paper we discuss evidence that suggests a role for serotonin and substance P in these pathways. Both of these neurotransmitters are present in bulbospinal neurons. Our results suggest that they have an important physiological role in the central regulation of blood pressure. PMID- 7537578 TI - Phosphorylation of microtubule-associated proteins by protein kinase CK2 in neuritogenesis. AB - Phosphorylation of microtubule-associated protein MAP1B and the neuronal-specific beta III-tubulin isoform takes place during neurite growth in neuroblastoma cells. Protein kinase CK2 (formerly referred to as casein kinase 2) is possibly involved in beta III-tubulin phosphorylation. As for MAP1B, there are at least two types of phosphorylation; one catalyzed by proline-directed protein kinases and another catalyzed by CK2. Protein kinase CK2 is primarily localized to the nuclei in proliferating neuroblastoma cells, whereas an increased amount of the enzyme is present in the cytoplasm of postmitotic cells bearing neurites. Treatment of neuroblastoma cells with an antisense oligonucleotide which specifically results in CK2 catalytic subunit depletion inhibits neuritogenesis. CK2 depletion is accompanied by dephosphorylation of MAP1B on the corresponding phosphorylatable sites. This dephosphorylation is paralleled by a release of MAP1B from microtubules. These results suggest that MAP1B phosphorylation by CK2 may be required for the assembly of microtubules within neurites. Other neuronal cytoskeletal proteins including MAP1A and tau are also substrates for CK2, indicating a role for the enzyme in the regulation of cytoskeletal functions also in mature neurons. PMID- 7537582 TI - Influenza activity in Great Britain. PMID- 7537580 TI - Legionnaires' disease: a cluster of cases in Reading. PMID- 7537581 TI - AIDS and HIV-1 infection in the United Kingdom: monthly report. PMID- 7537584 TI - Introduction to reverse transcription polymerase chain reaction. PMID- 7537583 TI - Two unrelated children with developmental delay, short stature and anterior chamber cleavage disorder, cerebellar hypoplasia, endocrine disturbances and tracheostenosis: a new entity? AB - Two unrelated children with developmental delay, anterior chamber-cleavage disorder, proportionate short stature and striking similarity in facial appearance appear to have an identical syndrome. Peters' plus syndrome has to be considered but additional abnormalities not described in this syndrome and their apparently different facies may be evidence for a hitherto undescribed condition. The further malformations both children have in common are cerebellar hypoplasia, hypothyroidism, tracheostenosis and dislocated hips. PMID- 7537586 TI - sVCAM-1 levels after segmental antigen challenge correlate with eosinophil influx, IL-4 and IL-5 production, and the late phase response. AB - Evidence from in vitro studies suggests a potential role for vascular cell adhesion molecule-1 (VCAM-1) in eosinophil trafficking. We hypothesized that induction of VCAM-1 occurs in the lung during IgE-mediated airway inflammation in humans. The technique of segmental antigen provocation followed by bronchoalveolar lavage (BAL) at 24 h was used to study 27 ragweed-allergic asthmatics (AA) and 18 atopic nonasthmatics (ANA). Total and differential cell counts were performed, and IL-4, IL-5, and soluble (VCAM) (sVCAM) levels in concentrated BAL fluid were measured by ELISA. A large increase in sVCAM levels after segmental challenge in both AA and ANA (1.79 +/- 0.31 to 139.39 +/- 68.58 ng/ml, p < 0.0005 and 2.85 +/- 0.80 to 98.25 +/- 77.35 ng/ml, p < 0.05, respectively) was observed. BAL IL-4 and IL-5 also increased after challenge (IL 4: 51.7 +/- 17.72 to 150.1 +/- 58.82 pg/ml, 0.05 < p < 0.10, n = 20 for AA, and 36.6 +/- 9.05 to 116.8 +/- 51.5 pg/ml, 0.05 < p < 0.10, n = 15 for ANA; IL-5: 0 to 2.67 +/- 1.62 ng/ml, p < 0.01, n = 16 for AA, and 0 to 2.87 +/- 2.16 ng/ml, 0.05 < p < 0.10, n = 10 for ANA). In both groups, the majority of the increase in sVCAM, IL-4, and IL-5 was accounted for by subjects who displayed a dual phase response after whole-lung antigen inhalation. This fact, plus the strong correlation observed between postchallenge sVCAM, IL-4, and IL-5 levels and eosinophil influx, suggests that VCAM, IL-4, and IL-5 play important roles in the recruitment of eosinophils to the lung of humans after antigen challenge. PMID- 7537585 TI - Organized type I collagen influences endothelial patterns during "spontaneous angiogenesis in vitro": planar cultures as models of vascular development. AB - Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo. PMID- 7537587 TI - Enhanced insulin-like growth factor molecules in idiopathic pulmonary fibrosis. AB - Idiopathic pulmonary fibrosis (IPF) is characterized by activated alveolar macrophages (AM), alveolar epithelial cell proliferation and interstitial matrix, and immune complex deposition. Spontaneous release of competence and progression type growth factors and their associated binding proteins may contribute to the pathologic features of IPF. To study the role of insulin-like growth factor (IGF) molecules in IPF we evaluated spontaneous release of IGF-I and IGFBP-3 in bronchoalveolar lavage cells from control subjects and from patients with IPF. IGF-I levels were similar compared with those in control subjects. In contrast, IGFBP-3 was significantly increased in IPF. In situ hybridization of open lung biopsies showed IGF-I to be abundant in IPF lung tissue in alveolar macrophages, interstitial mesenchymal cells, and epithelial cells. Northern, Western ligand blotting, reverse transcription PCR, and radioimmunoassay suggested that immune complexes stimulate expression of IGFBP-3 in mononuclear phagocytes in a time- and dose-dependent manner bearing strong similarities to stimulation by LPS. These data are compatible with the hypothesis that IGFBP-3 increases the bioactivity of IGF-I derived from a variety of lung tissues contributing to the fibrosis and remodeling seen in IPF. PMID- 7537588 TI - The effect of dextran 40 on patency following severe trauma in small arteries and veins. AB - Arteriotomy/intimectomy and venotomy/intimectomy were performed in the rabbit ear. Low molecular-weight dextran (dextran 40) was infused 2 h before reperfusion and on postoperative days 1, 3 and 5 using a standard clinical protocol. Bleeding times at reperfusion were recorded and patencies determined at intervals up to 2 weeks. Rabbits given single preoperative bolus doses of saline were used as controls. Dextran significantly prolonged bleeding-times in arteries and significantly improved early patency in both types of vessel, but the enhancements disappear by one week. Dextran 40 infusion thus has little effect on long-term patency. PMID- 7537589 TI - An investigation into the mechanism of capsaicin-induced oedema in rabbit skin. AB - 1. Oedema formation induced by intradermal capsaicin has been studied in rabbit skin. The effect of the anti-inflammatory steroid dexamethasone and also of a range of known inhibitors of oedema formation have been investigated in order to elucidate mechanisms involved in capsaicin-induced oedema formation. 2. Oedema formation, in response to intradermally-injected test agents, was measured by the local extravascular accumulation of intravenously injected 125I-labelled albumin. In separate experiments skin blood flow was assessed by the clearance of intradermally-injected 133xenon. 3. Oedema formation induced by intradermal histamine (3 nmol) and bradykinin (1 nmol), when in the presence of vasodilator doses of calcitonin gene-related peptide (CGRP) (3 pmol) or prostaglandin E1, (PGE1) (10 pmol), was significantly inhibited (P < 0.01) in rabbits pretreated with intravenous dexamethasone (3 mg kg-1, -4 h). In contrast dexamethasone had no effect on capsaicin (3 mumol)-induced oedema formation or, on capsaicin (30 100 nmol)-induced blood flow. 4. Oedema formation observed in response to intradermal capsaicin (3 mumol) was significantly inhibited (P < 0.01) when the selective capsaicin antagonist, ruthenium red (3 nmol) was co-injected. This suggests that the mechanism of capsaicin-induced oedema involves activation of sensory nerves. However, oedema was not inhibited when capsaicin was co-injected with the neurokinin NK1 receptor antagonist, RP67580 (10 nmol), the NK2 antagonist SR48960 (10 nmol) or the CGRP antagonist CGRP8-37 (300 pmol). 5. Oedema formation induced by capsaicin was not inhibited when co-injected with the histamine HI receptor antagonist, mepyramine (3 nmol), the PAF antagonist, WEB 2086 (100 nmol), the bradykinin B2 receptor antagonist, Hoel4O (1 nmol), or the cyclo-oxygenase inhibitor, indomethacin (10 nmol),suggesting that these mediators do not play a major role in the capsaicin-induced response.6. Histological analysis of capsaicin-treated skin sites revealed undamaged, intact microvessels and lack of haemorrhage. Further, co-injection of capsaicin with the hydrogen peroxide remover, catalase(2,200 u), had no effect on oedema formation. This suggests that capsaicin does not induce oedema formation secondary to free radical-induced damage.7. These results indicate that capsaicin-induced oedema in rabbit skin involves activation of sensory nerves. However, the oedema is not inhibited by pretreatment with the anti-inflammatory steroid,dexamethasone. Further the mechanisms which lead to the oedema formation observed after intradermal capsaicin remain unknown. PMID- 7537591 TI - Inhibition of muscarinic receptor-induced inositol phospholipid hydrolysis by caffeine, beta-adrenoceptors and protein kinase C in intestinal smooth muscle. AB - 1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1 oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H] Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX) sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2 diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H] inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7. Incubation with thapsigargin (1 microM) and ryanodine (10 microM) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8. The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate levels. Diacylglycerols may exert a negative feedback inhibition on inositol phosphate production. PMID- 7537590 TI - Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2. AB - 1. The effect of purified crotapotin, a non-toxic non-enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 micrograms/paw) and 5-hydroxytryptamine (5-HT) (3 micrograms/paw) in the rat hind-paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea-pig isolated lung were also investigated. 2. Subplantar co-injection of crotapotin (1 and 10 micrograms/paw) with carrageenin or injection of crotapotin (10 micrograms/paw) into the contralateral paw significantly inhibited the carrageenin-induced oedema. This inhibition was also observed when crotapotin (10 30 micrograms/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60 degrees C) failed to inhibit carrageenin-induced oedema. Subplantar injection of crotapotin (10 micrograms/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5-HT-induced oedema. 3. In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5-HT stores. 4. Crotapotin (30 micrograms/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 nM) and platelet activating factor (1 microM) in human platelet-rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml-1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 microg/paw) did not affect the release of 6-oxo-prostaglandin Fla, and TXB2 induced by ovalbumin in sensitized guinea-pig isolated lungs.5. Our results indicate that the anti-inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo-oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity. PMID- 7537592 TI - Influence of age on the signal transduction pathway of non-adrenergic non cholinergic neurotransmitters in the rat gastric fundus. AB - 1. The influence of aging on the relaxant response and the change in cyclic nucleotide content induced by vasoactive intestinal polypeptide (VIP), nitric oxide (NO), electrical field stimulation of the non-adrenergic non-cholinergic neurones and substances acting at different levels of the cyclic AMP and cyclic GMP transduction pathways was studied in longitudinal muscle strips of the rat gastric fundus. 2. The relaxant responses to VIP, sustained electrical stimulation, forskolin and 3-isobutyl-1-methylxanthine were reduced with age, while the responses to dibutyryl cyclic AMP were not. The increase in cyclic AMP content induced by sustained electrical stimulation and forskolin was lower in old rats. 3. The relaxant responses to NO and to short train electrical stimulation were similar in the three age groups. The inhibitory effect of NG nitro-L-arginine methyl ester on relaxations induced by short train electrical stimulation was more pronounced in old rats. The relaxant responses to sodium nitroprusside (SNP), 8-bromo-cyclic GMP and zaprinast were reduced with age. SNP induced a similar elevation of the cyclic GMP content in the three age groups. 4. These results suggest that aging differentially affects the cyclic AMP and cyclic GMP pathway for relaxation by VIP and NO in the rat gastric fundus, as the defect seems to occur at the level of the adenylate cyclase and cyclic GMP-dependent protein kinase respectively. PMID- 7537594 TI - Relaxation and decrease in [Ca2+]i by hydrochlorothiazide in guinea-pig isolated mesenteric arteries. AB - 1. We examined the effect of the thiazide diuretic, hydrochlorothiazide, on on intracellular calcium concentration ([Ca2+]i) and tone in guinea-pig mesentery arteries. Vessels were mounted on a microvascular myograph and loaded with the Ca(2+)-sensitive fluorescent dye, Fura-2. 2. Hydrochlorothiazide caused relaxation of noradrenaline-precontracted arteries associated with a fall in [Ca2+]i. Preincubation of arteries with hydrochlorothiazide inhibited both contraction and rise in [Ca2+]i in response to noradrenaline. Hydrochlorothiazide did not affect tone and [Ca2+]i when this was elevated by a combination of depolarizing potassium solution and noradrenaline. 3. Hydrochlorothiazide-induced vasorelaxation and decrease of [Ca2+]i was abolished by charybdotoxin, a blocker of large conductance Ca(2+)-activated K channels. 4. The rise in [Ca2+]i elicited by caffeine in Ca(2+)-free physiological salt solution, and presumably reflecting Ca2+ release from intracellular stores, was not altered by preincubation with hydrochlorothiazide. 5. Under depolarizing conditions hydrochlorothiazide did not alter the relationship between the extracellular concentration of Ca2+ and [Ca2+]i; however, hydrochlorothiazide caused a small reduction in the contraction produced for a given rise in [Ca2+]i suggesting hydrochlorothiazide may cause a slight desensitization of the contractile machinery. 6. These findings suggest that hydrochlorothiazide opens Ca(2+)-activated K channels leading to hyperpolarization and consequent closing of voltage-operated calcium channels. The result of this is an impaired influx of extracellular Ca2+, a decrease in [Ca2+]i and vasorelaxation. PMID- 7537593 TI - Sequential induction of nitric oxide synthase by Corynebacterium parvum in different organs of the mouse. AB - 1. The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2-/NO3- in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied. 2. C. parvum (100 mg kg-1, i.p.) induced a time-dependent expression of a Ca(2+)-independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta. 3. There was no significant induction of a Ca(2+)-independent NO synthase in the brain, nor was there any change in the Ca(2+)-dependent enzyme in this organ, during the study period. 4. C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 +/- 1 mmHg to 79 +/- 3 mmHg), which recovered gradually by day 28. 5. Plasma NO2-/NO3- was significantly elevated between days 8 and 24, with a maximum increase at day 12. 6. These results show that C. parvum induces a Ca(2+)-independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver. 7. Furthermore, the decrease in blood pressure induced by C. parvum is associated with the induction of NO synthase in the vasculature, whereas the increased concentration of plasma NO2-/NO3- seems to result from the generation of NO by a number of tissues. PMID- 7537596 TI - Lipid metabolism. PMID- 7537595 TI - Pharmacological analysis of the local and reflex responses to bradykinin on rat urinary bladder motility in vivo. AB - 1. The topical application of bradykinin (BK) (0.05-5000 pmol/rat) onto the serosal surface of the urinary bladder in urethane-anaesthetized rats, evoked low amplitude tonic contractions (not exceeding 25 mmHg) or high amplitude (about 50 mmHg), phasic reflex contractions (chemoceptive micturition reflex) which were abolished by bilateral ablation of the pelvic ganglia. In ganglionectomized rats, BK induced only a local, tonic-type contraction. 2. Systemic capsaicin pretreatment (164 mumol kg-1, 4 days before) reduced the incidence of chemoceptive reflex induced by BK (500 pmol/rat) but had no effect on the magnitude of the tonic-type contraction elicited by BK in ganglionectomized rats. Indomethacin (11 mumol kg-1, 20 min before) reduced the incidence but not the amplitude of the reflex contractions induced by topical application of BK (500 pmol/rat). In ganglionectomized rats, indomethacin (11 mumol kg-1, 20 min before) decreased the amplitude of the tonic contraction evoked by BK. Indomethacin did not affect the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat) onto the bladder. 3. Intrathecal administration of the tachykinin NK1 receptor antagonists, RP 67,580 (10 nmol/rat) or SR 140,333 (10 nmol/rat), abolished the chemoceptive reflex induced by BK without modifying the magnitude of the tonic contraction. SR 140,333 (10 nmol/rat) also abolished the occurrence of the chemoceptive reflex induced by capsaicin. 4. Intravenous administration of the B2 receptor antagonist, Hoe 140 (35 nmol kg-1, 10 min before) abolished the reflex and local effects induced by BK on bladder motility but failed to modify the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat). 5. Intrathecal administration of Hoe 140 (10 nmol/rat) reduced the incidence of the chemoceptive reflex induced by BK but had no effect on the amplitude of the local motor response. Likewise, Hoe 140(10 nmol/rat, i.t.) reduced the incidence of reflex bladder contractions induced by topical application of capsaicin (15 nmol/rat) without affecting the magnitude of the tonic-type contraction.6. These findings indicate that BK stimulates motility through B2 receptors in the rat urinary bladder.BK activates the reflex response by stimulating capsaicin-sensitive afferent nerves with a contribution from prostanoids. At the spinal cord level, tachykinin NK1 and BK B2 receptors could also be involved in the chemoceptive reflex induced by BK or capsaicin. PMID- 7537599 TI - [Alfuzosin in benign prostatic hyperplasia. A new principle is evaluated. Report of a study]. PMID- 7537598 TI - Short communication: the addition of carbogen and nicotinamide to a palliative fractionation schedule for locally advanced breast cancer. AB - Tumour cell hypoxia is a recognized cause of resistance to radiotherapy. Using clinically relevant dose-fractionation schedules in a mouse tumour model, the addition of carbogen and nicotinamide to overcome chronic and acute hypoxia results in a marked increase in radioresponsiveness with a lower degree of sensitization in normal tissue. Carbogen and nicotinamide were added to the palliative radiation treatment given to six patients with locally advanced breast cancer. The aim of the pilot study was to determine if patients tolerated the addition of carbogen and nicotinamide and to assess if there was any increase in radiosensitivity of the skin. Patients received 30 Gy prescribed to the intersection dose in six fractions over 17/18 days with full skin bolus to the tumour. All patients were given 6 g of nicotinamide orally 90 min before radiation treatment. Carbogen breathing was started 5 min prior to treatment and continued during it. Patients tolerated the treatment well, with vomiting in one patient being the only side effect that could be related to the nicotinamide, and this settled with an anti-emetic. No increase in skin reaction was noted with the addition of carbogen and nicotinamide, and good tumour regression was achieved. PMID- 7537597 TI - Computer planning of stereotactic iodine-125 seed brachytherapy for recurrent malignant gliomas. AB - At St Thomas' Hospital, we have developed a computer program on a Titan graphics supercomputer to plan the stereotactic implantation of iodine-125 seeds for the palliative treatment of recurrent malignant gliomas. Use of the Gill-Thomas Cosman relocatable frame allows planning and surgery to be carried out at different hospitals on different days. Stereotactic computed tomography (CT) and positron emission tomography (PET) scans are performed and the images transferred to the planning computer. The head, tumour and frame fiducials are outlined on the relevant images, and a three-dimensional model generated. Structures which could interfere with the surgery or radiotherapy, such as major vessels, shunt tubing etc., can also be outlined and included in the display. Catheter target and entry points are set using a three-dimensional cursor controlled by a set of dials attached to the computer. The program calculates and displays the radiation dose distribution within the target volume for various catheter and seed arrangements. The CT co-ordinates of the fiducial rods are used to convert catheter co-ordinates from CT space to frame space and to calculate the catheter insertion angles and depths. The surgically implanted catheters are after-loaded the next day and the seeds left in place for between 4 and 6 days, giving a nominal dose of 50 Gy to the edge of the target volume. 25 patients have been treated so far. PMID- 7537600 TI - Reflections on alpha blockade therapy for benign prostatic hyperplasia. PMID- 7537601 TI - Percentage area density of epithelial and mesenchymal components in benign prostatic hyperplasia: comparison of results between single biopsy, multiple biopsies and multiple tissue specimens. AB - OBJECTIVE: To determine whether histological analysis of six multiple random biopsies of the gland or analysis of only one biopsy provides a good estimate of the different components of the hyperplastic gland compared with the results obtained from tissue specimens (reference values). MATERIALS AND METHODS: The various components of prostate tissue obtained from 30 men undergoing suprapubic adenomectomy were investigated. The histological analysis was performed on multiple tissue specimens reflecting adenoma (reference values) and on one and six biopsies performed at random on the enucleated material of the hyperplastic gland. Immunohisto chemistry using anti-actin as a label of smooth muscle and specific histological staining coupled with computer-assisted quantitative morphometric analysis was used to ascertain the histological composition of the prostate. RESULTS: The mean ( +/- SD) area densities obtained from tissue specimens were 34.1 +/- 5%, 32.4 +/- 6.9%, 17.6 +/- 4.5% and 15.9 +/- 5.5% of smooth muscular and fibrous tissue, and epithelium and glandular lumen, respectively. The mean ratio of stromal to epithelial hyperplasia averaged 4.05 +/- 1.73. Both one and six biopsies gave a good estimate of fibrous tissue and glandular epithelium, but the percentage of smooth muscles was overestimated and the percentage of glandular lumen was underestimated. There was a significant relation between the prostate area densities of glandular epithelium (r = -0.41, P < 0.05), the percentage area density of prostate smooth muscle (r = 0.43, P < 0.05), and the weight of enucleated adenoma. No correlation was found with prostate-specific antigen (PSA). CONCLUSION: It seems feasible to propose medical treatment of benign prostatic hyperplasia (BPH) based on the histological composition of the prostate gland. One biopsy, reflecting in good proportions the nature of the adenoma, would be used to provide insight into the pathogenesis and therapy of BPH. PMID- 7537602 TI - Effects on the endocrine system of long-term treatment with the non-steroidal anti-androgen Casodex in patients with benign prostatic hyperplasia. AB - OBJECTIVE: To study the hormonal changes resulting from long-term use of the non steroidal anti-androgen Casodex. PATIENTS AND METHODS: A randomized, placebo controlled study was carried out on 27 patients with benign prostatic hyperplasia (BPH). Fourteen patients received Casodex 50 mg daily for 24 weeks and 13 received a placebo. The patients were followed up for a further 24-week period. RESULTS: Serum concentrations of luteinizing hormone (LH) increased by an average of 40% while follicle-stimulating hormone (FSH) remained unchanged. Testosterone increased by 35%, oestradiol by 29% and oestrone by 23%; all changes were statistically significant. Levels of androstenedione and dihydrotestosterone increased by 11% and 15%, respectively, but these increases did not reach statistical significance. A non-significant increase was also observed for sex hormone-binding globulin. The hormonal changes were reversible upon discontinuation of therapy. Prolactin and dehydroepiandrosterone-sulphate levels did not change. CONCLUSION: This study indicates that Casodex, due to competitive inhibition of central androgen receptors, increases LH secretion, thus causing increased production and increased metabolism of testosterone. PMID- 7537603 TI - What is the 'normal range' for prostate-specific antigen? Use of a receiver operating characteristic curve to evaluate a serum marker. AB - OBJECTIVE: To compare the relative sensitivity and specificity of prostate specific antigen (PSA) as a test for prostate cancer over a range of PSA values in a variety of patient groups, and to compare the sensitivity and specificity of PSA and prostatic acid phosphatase (PAP). SUBJECTS AND METHODS: Receiver operating characteristic (ROC) curves (sensitivity plotted against 1-specificity) were constructed to compare the ability of PSA to discriminate men with prostate cancer (n = 257) from those with benign prostatic hyperplasia (BPH) (n = 220) or control patients (n = 164). Receiver operating characteristic curves were also constructed to compare PSA and PAP in 173 men with either BPH or prostate cancer. RESULTS: When patients with symptomatic BPH and those with advanced prostate cancer are excluded, a PSA of 8 ng/mL has a sensitivity of 94% and a specificity of 98% for prostate cancer. In patients presenting with symptoms suggestive of bladder outflow obstruction, PSA remains a sensitive marker for prostate cancer (93% sensitivity at 10 ng/mL) but its specificity (65%) is poor. PSA is a sensitive test for skeletal metastases but levels of 60-80 ng/mL are required to achieve a specificity of 70% or more. The sensitivity of PSA is far superior to that of PAP. CONCLUSION: Serum PSA provides good discrimination between patients with and without prostate cancer. The sensitivity and specificity of PSA can be improved by excluding men with symptomatic BPH. The specificity of PSA as a diagnostic test for prostate cancer is reduced in men with symptoms of bladder outflow obstruction. For reasonable sensitivity and specificity, a PSA of 60-80 ng/mL is required for differentiating non-metastatic from metastatic prostate cancer. The ROC curve comparing PSA and PAP provides a graphical demonstration of the superiority of PSA as a tumour marker. The ability of PSA to identify prostate cancer can be improved by selecting out groups of patients and by adjusting the cut-off level of PSA to the population under study. PMID- 7537604 TI - Serum prostate-specific antigen in a community-based population of healthy Japanese men: lower values than for similarly aged white men. AB - OBJECTIVE: To determine whether the age-specific reference ranges for serum prostate-specific antigen (PSA) concentration generated for white men are applicable to other races. PATIENTS AND METHODS: Three-hundred and thirty-five healthy Japanese men, aged 40-79 years, residing in the small fishing village of Shimamaki-mura, Japan, agreed to enter this prospective, community-based study. All underwent a detailed clinical evaluation that included a serum PSA determination, a digital rectal examination and a transrectal ultrasound. Two hundred and eighty-six (85%) completed the prostatic evaluation and had no evidence of prostate cancer by any one of the three diagnostic tests; these men formed the study population on which all analyses were performed. RESULTS: The serum PSA concentration correlated directly with patient age (r = 0.33; P < 0.001) and prostatic volume (r = 0.57; P < 0.001). PSA density (PSAD) also was directly proportional to age (r = 0.30; P < 0.001). Adjusting for age, the serum PSA concentration was lower for Japanese men than for white men (P < 0.001). Thus, the recommended age-specific reference ranges (95th percentile) for serum PSA for Japanese were lower as well: 0.0-2.0 ng/mL for 40-49 years; 0.0-3.0 ng/mL for 50-59 years; 0.0-4.0 ng/mL for 60-69 years; and 0.0-5.0 ng/mL for 70-79 years. Based on transrectal ultrasound-volume determinations, the lower serum PSA concentrations in Japanese men are due in large part to their smaller prostate glands as compared with white men (P < 0.001). CONCLUSIONS: These findings confirm the earlier observations that serum PSA, prostatic volume and PSAD are age-dependent. However, because of physiological differences among the two races, partly due to the size of the prostate gland, the age-specific reference ranges for serum PSA are lower for Japanese men than for white men. Because of these racial differences, it is now crucial to conduct a similar investigation among black men. PMID- 7537605 TI - Failure of normalization of alphafetoprotein levels following the successful treatment of teratoma: a diagnostic trap for the unwary. PMID- 7537607 TI - Characterization and measurement of CD34-expressing hematopoietic cells. AB - Because of availability of anti-CD34 monoclonal antibodies, multiparameter flow cytometry has become the tool of choice for determination of hematopoietic stem and progenitor cells. This report describes general techniques for quantitation and characterization of CD34-expressing cells by flow cytometry. PMID- 7537606 TI - Comparison of the phenotype and clonogenicity of normal CD34+ cells from umbilical cord blood, granulocyte colony-stimulating factor-mobilized peripheral blood, and adult human bone marrow. AB - Bone marrow (BM) is most frequently used to transplant hematopoietic progenitor cells, but umbilical cord blood (UCB) and mobilized peripheral blood (PB) provide alternative sources of progenitor cells for transplantation. To study whether the clonogenicity and phenotype of progenitor cells vary between the compartments, CD34+ cells from UCB, mobilized PB, and BM were analyzed for in vitro colony formation and characterized by immunophenotyping for several lineage-associated and maturation-related cell surface molecules. We found that circulating CD34+ cells, either from PB after granulocyte colony-stimulating factor (G-CSF) mobilization or from UCB, contained a large proportion of cells (86-96%) with myeloid cell-associated molecules (CD33 and CD13) and clonogenic cells (colony forming unit-granulocyte-macrophage and burst-forming unit-erythrocyte) in excess of BM CD34+ cells. Further, UCB and PB CD34+ cells contained > or = 3% cells with a phenotype associated with immature (HLA-DR- and CD38-) progenitor cells, which was comparable to what is found among BM CD34+ cells. The proportion of CD34+ cells in UCB expressing the B cell-associated molecules (CD10 and CD19) was comparable to that found in mobilized PB (< or = 5%) but significantly lower than for BM CD34+ cells (19-24%). Further, we observed a higher proportion of CD34+ cells expressing the T cell-associated molecule CD7+ in UCB (7%) compared with both PB and BM (3-4%). In general, circulating CD34+ cells, either from UCB or G CSF-mobilized PB, display largely the same phenotypic profile and clonogenicity, being different from resident CD34+ cells in BM. PMID- 7537608 TI - Characterization of peripheral blood progenitor cells mobilized by cytotoxic chemotherapy and recombinant human granulocyte colony-stimulating factor. AB - The purpose of this study was to evaluate the antigenic profile of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). The mobilization regimens consisted of high-dose cytarabine/mitoxantrone for patients with NHL, DexaBEAM for patients with HD, and high-dose cyclophosphamide (4 or 7 g per m2) for patients with MM. Cytotoxic therapy was supported by recombinant human G-CSF (Filgrastim, 300 micrograms/day sc) to shorten the period of neutropenia and to increase the number of circulating hematopoietic progenitor cells. The mean numbers of circulating CD34+ cells/microliters during leukocyte recovery were different between patient groups, 80.5 +/- 9.8 (mean +/- SEM) in low-grade NHL and 51.2 +/- 9.7 in high grade NHL compared with 31.3 +/- 6.9 in HD and 24.4 +/- 4.1 in patients with MM. As a result, the greatest numbers of CD34+ cells/kg collected per leukapheresis were observed in patients with NHL, whereas the collection efficiency was substantially lower in patients with HD or MM. Patients with MM had also the smallest proportion of CD34+ cells in the mononuclear cell fraction (mean 0.79 +/ 0.10% versus 2.15 +/- 0.19% in low-grade NHL) but the greatest proportion of early CD34+ HLA-DR- progenitor cells (mean 2.38 +/- 0.51 versus 0.84 +/- 14% in low-grade NHL). Patients with MM had a mean proportion of CD34/c-kit+ cells that was twofold greater than that observed in patients with high- or low-grade NHL.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537609 TI - Peripheral blood progenitor cell transplantation: clinical, practical, and economic considerations. Symposium held at the London Regional Cancer Centre, London, Ontario, Canada, November 1993. PMID- 7537613 TI - Effects of the epileptogenic agent strychnine on membrane currents elicited by agonists of the NMDA and non-NMDA receptors in Xenopus oocytes. AB - The effects of strychnine (STRY) on ion channels activated by N-methyl-D aspartate (NMDA), kainate (KA), alpha-amino-3-hydroxy-5-methyl-4 isoxazolpropionate (AMPA) and quisqualate (QUIS) were studied using Xenopus oocytes, microinjected with mRNA from rats' brains. STRY reduced NMDA-, KA- and AMPA-induced membrane currents in a dose-dependent manner. The effect was more pronounced with NMDA than with KA and AMPA. QUIS-induced membrane currents were not affected by STRY. The depressive effect of STRY on NMDA responses was voltage dependent. The effect of STRY on the NMDA-induced membrane currents remains unchanged when the concentration of NMDA or glycine was increased. Intracellular injection of STRY did not alter the NMDA response. PMID- 7537610 TI - Transplantation of mobilized peripheral blood stem cells: role of filgrastim. AB - Autologous bone marrow transplantation, although successful, is limited in the rate of hematopoietic recovery achieved. Myeloablative chemotherapy results in prolonged pancytopenia with standard autologous bone marrow support. High-dose myelosuppressive chemotherapy results in severe but short-term pancytopenia, and hematopoietic recovery is routine without cellular support. Cellular support becomes necessary when multiple-cycle, severely myelosuppressive regimens are used because of the cumulative stem cell damage and unacceptably long duration of severe pancytopenia. Peripheral blood progenitor cells (PBPCs) may be used as cellular support, and colony-stimulating factors with or without chemotherapy are currently the most potent available PBPC-mobilizing tools. Filgrastim (rmethG CSF) has been shown to enhance the number of PBPCs for harvest in both cancer patients and normal donors. When Filgrastim is used in conjunction with chemotherapy, there appears to be greater PBPC mobilization, which seems to be dependent on dose and schedule of chemotherapy as well as the type of chemotherapeutic agent used. Platelet recovery has been shown to be more rapid when PBPCs are used compared with historical controls given autologous bone marrow infusions. It may be concluded that PBPC transplantation is useful supportive care following myeloablative chemotherapy. PMID- 7537611 TI - Clinical applications of filgrastim and stem cell factor in vivo and in vitro. PMID- 7537612 TI - Effects of intravenous infusions of cholecystokinin (CCK)-8 on exocrine and endocrine pancreatic secretion in conscious sheep. AB - The effects of cholecystokinin (CCK)-8 on both exocrine and endocrine pancreatic functions were examined simultaneously in five conscious sheep. Intravenous infusions of CCK-8 (0, 5, 10, 20, 30, 60, 120 and 240 pmol/kg/min for 40 min) induced dose-dependent increases in flow rate, and in protein and amylase outputs in pancreatic juice. The same CCK-8 infusions induced dose-dependent increases in plasma insulin, but no change in plasma glucagon concentrations. The threshold dose (10-30 pmol/kg/min) of CCK-8 infusion for stimulating insulin secretion was similar to that for stimulating amylase output. In conclusion, using amylase output as an indicator of physiological action, CCK is one of the potential candidates as a physiological regulator of insulin, but not glucagon secretion in sheep. PMID- 7537614 TI - Pre- and post-translational regulation of renal insulin-like growth factor binding protein-1 in insulin-deficient diabetes. AB - BACKGROUND: Renal size and production of insulin-like growth factor-I (IGF-I) increase rapidly after the onset of insulin-deficient diabetes, despite decreases in serum and hepatic levels of IGF-I and linear growth retardation in affected animals and humans. This increase in kidney IGF-I gene expression is mediated both by pre- and post-translational mechanisms, with the relative contributions of each locus of control varying with the severity and/or duration of diabetes. Since the actions of IGF-I are modified by specific circulating as well as locally produced IGF binding proteins (IGF BPs), and since kidney IGF BP1 content is increased in diabetes, we asked whether: 1) the time course of induction of increased BP1 expression paralleled that for induction of IGF-I; 2) severity and/or duration of diabetes affected pre- and post-translational renal expression of this protein as it does expression of IGF-I itself; and 3) insulin deficiency or hyperglycemia was responsible for this increase in kidney IGF BP1 content. METHODS: Adult rats were made diabetic by injection of streptozotocin (STZ), and kidney BP1 mRNA and protein were assessed by Northern and Western ligand blotting, respectively, in comparison with nondiabetic, insulin-treated diabetic, and phlorizin-treated diabetic animals. RESULTS: Rapid time- and STZ dose dependent increases in both pre- and post-translational renal IGF BP1 expression were noted in the untreated diabetic animals. Comparison of the relative changes in kidney BP1 mRNA and protein contents suggested that with increasing severity of diabetes, at least 20% of this effect was mediated pre-translationally and, therefore, did not merely reflect trapping of circulating BP1. Treatment with insulin completely inhibited the pre-translational and potently inhibited the post-translational component of the response, while correction of hyperglycemia with phlorizin did not. These observations were specific for BP1, with renal IGF BP3 mRNA and protein contents noted to be low basally and unaffected by diabetes. CONCLUSIONS: These data suggest that insulin strongly regulates pre- and post translational renal IGF BP1 gene expression and implicate BP1 as an important determinant of IGF-I activity in diabetic kidney. The similarity of the time course of BP1 induction to that of IGF-I in animals of the same age and severity of diabetes suggests that local IGF-I/BP1 interactions may potentiate kidney IGF I activity and promote initiation of the early stages of diabetic renal hypertrophy. PMID- 7537615 TI - Morphological aspects of the cytotoxic action of Helicobacter pylori. AB - OBJECTIVE: To morphologically investigate the cytotoxic action of Helicobacter pylori on gastric mucosal cells. METHODS: Twenty biopsy specimens were obtained from the antrum and greater curvature of the upper body of the stomach, and examined by electron microscopy to elucidate the process of mucus cell injury induced by H. pylori. RESULTS: Only H. pylori colonies in intimate contact with the mucosal cells caused various cell alterations, including loss of microvilli, mucus depletion, cell membrane injury and degeneration of the cytoplasm. In contrast, H. pylori that were not in contact with the mucosa had no effect on the surface mucus cells. CONCLUSIONS: The results of this study suggested the following mechanism of H. pylori-induced gastric mucosal injury. Intimate contact between H. pylori and the epithelial cell is necessary for cytotoxic action. During the process of cytotoxic action, degradation of the mucous gel layer and the glycocalyx are first caused by the proteolytic enzymes produced by H. pylori. Then the microorganism approaches the surface mucus cell, making intimate contact with the cell surface. Finally, H. pylori adheres directly to the cell membrane, effecting direct action of the toxins on the cell membrane. Subsequently, the degenerative changes induced destroy the cell membrane, resulting in degeneration of the cell itself. PMID- 7537616 TI - Comparative effects of 7% NaCl in 6% dextran 70 and 0.9% NaCl on oxygen transport in endotoxemic dogs. AB - We compared the effects of 7% NaCl in 6% dextran 70 (HSD) and 0.9% NaCl (IS) resuscitation of endotoxic dogs on hemodynamic and cardiorespiratory parameters and the oxygen consumption-delivery relationship. Escherichia coli endotoxin (3 mg.kg-1, intravenously) was infused over 5 min into 13 paralyzed, chloralose anesthetized, splenectomized dogs. Six additional dogs received a sham endotoxin infusion (saline) and served as controls. After 30 min, the endotoxic animals were resuscitated to 150% of their baseline cardiac output (CO) and maintained at this CO for 30 min using 7% NaCl in 6% dextran 70 (HSD at 1 ml.kg-1.min-1; n = 7) or 0.9% NaCl (IS at 4 ml.kg-1.min-1; n = 6). Oxygen consumption (VO2), measured by indirect calorimetry, hemodynamic parameters, and oxygen delivery (DO2), improved in similar temporal patterns in both groups during resuscitation and VO2 reached steady-state values. Oxygen delivery, VO2, mean arterial pressure, and cardiac output did not significantly differ between groups at the end of resuscitation, but VO2 increased significantly from baseline values only in the HSD group. The total volume of HSD administered averaged 10.0 +/- 0.2 ml.kg-1 which was significantly less than the volume of IS, which averaged 67.2 +/- 9.3 ml.kg-1. Incremental hemorrhages (2-5 ml.kg-1) were then performed in all dogs to determine the oxygen consumption-delivery relationship and the critical level of oxygen delivery (DO2Crit). The average DO2Crit values of the HSD, IS, and control groups were 9.42, 9.15, and 6.82 ml.min-1.kg-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537619 TI - Role of nitric oxide in the small intestinal microcirculation during bacteremia. AB - Nitric oxide (NO) is an important mediator of the hemodynamic effects of sepsis; however, its microcirculatory effects are unknown. To determine the role of NO in the small intestinal (SI) microcirculation, an intact SI loop was exteriorized from decerebrate rats into a controlled Krebs' bath. Bacteremic rats received 10(9) Escherichia coli intravenously. Videomicroscopy was used to measure arteriolar diameters (A1, A3) and optical Doppler velocimetry to quantitate flow. In controls, topical NO synthase (NO-S) substrate L-arginine (L-ARG; 10(-4) M) did not affect diameters or flow. Inhibition of NO-S by N omega-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) caused constriction (A1 = -18%; A3 = -24% from baseline diameter) and reduced A1 flow by 62%. These alterations were similar to bacteremic controls (A1 = -20%; A3 = -18%; A1 flow = -42%), despite the increased cardiac output (+21%). L-NAME treatment of bacteremic rats resulted in further constriction (A1 = -31%; A3 = -32%) and decreased A1 flow (-75%). Topical L-ARG (10(-4) M) ameliorated constriction (A1 = -6%; A3 = +7%) and improved blood flow (-5%) during bacteremia. We conclude that: 1) NO is important for basal SI microvascular tone; 2) bacteremia causes SI arteriolar constriction and hypoperfusion; 3) NO-S inhibition during sepsis may exacerbate SI vasoconstriction and hypoperfusion. PMID- 7537617 TI - Time sequence of histamine release and formation in rat endotoxic shock. AB - Increased histamine release and formation (induced histamine) are two hypotheses considered in the pathogenesis of endotoxic shock development. To prove both hypotheses a sequence of four randomized controlled studies in rats was performed. Histamine release was measured indirectly as a decrease in tissue histamine contents (lung, liver, spleen, stomach); histamine formation was estimated directly as an increase in histidine decarboxylase (HDC) activity in the same organs. Changes in contents and enzymatic activities were determined 4 and 8 h after shock induction; in addition, at the time of death, the activity of HDC was measured in heart, kidney, and small intestine. 4 h after shock induction, there was a significant decrease in the tissue-histamine content as measured only in the liver, with the same trend in lung and spleen. 8 h after endotoxin application, however, histamine concentration increased in the liver (significantly p < .05) and lung compared to the NaCl control group. The manifestation of changes in HDC activity in various organs was selective (i.e., not all organs showed alterations), not uniform (decreased as well as increased activities were measured), and time-dependent (no increase in HDC activity in animals dying at > 20 h). At 4 and 8 h, only the liver showed a strong increase in HDC activity which can explain the increase in histamine content. In lung, spleen, and stomach, a significant decrease occurred. The results on histamine release and formation let us conclude that histamine is involved in the pathogenesis of endotoxic shock development. PMID- 7537620 TI - [Whipple's disease]. PMID- 7537618 TI - Early endotoxic shock results in enhanced vasodilator responses to nitroglycerin but unaltered responses to neuropeptides calcitonin gene-related peptide and substance P. AB - To determine the role that vasoactive neuropeptides, calcitonin gene-related peptide, and substance P play in tissue-blood flow regulation during early septic shock, we examined the responsiveness of arteries removed from pigs 3 h after administration of Escherichia coli lipopolysaccharide or saline vehicle. The carotid, cranial mesenteric, and left anterior descending coronary arteries were excised, and rings were cut from each vessel. Constrictor responses were obtained to cumulative doses of norepinephrine or potassium chloride. Rings were reconstricted and challenged with acetylcholine, substance P, calcitonin gene related peptide, and nitroglycerin. Lipopolysaccharide significantly increased the cranial mesenteric artery's response to high concentrations of norepinephrine and the response to nitroglycerin in all vessels. This enhancement of responses to nitroglycerin suggests augmented smooth-muscle responsiveness to an exogenous source of nitric oxide, possibly associated with early depression of basal endothelial function. Depression of agonist-induced nitric oxide release may mask such enhancement with endothelial-dependent dilators and may enhance the response to adrenergic constrictors in some vascular beds. PMID- 7537621 TI - [AFP producing intestinal cancer]. PMID- 7537623 TI - Role of extracellular Ca2+ in the selective enhancement of contractile responses of arteries from diabetic rats to noradrenaline. AB - Maximum contractile responses of diabetic aortas incubated in the absence of extracellular Ca2+ to increasing Ca2+ (0.01-10 mM) in the presence of 1 microM noradrenaline, but not 40 mM KCl, were significantly increased compared with those of age-matched control rats. Maximum contractile responses of both aortas and mesenteric arteries from diabetic rats to noradrenaline, but not KCl, in the presence of extracellular Ca2+ (2.5 mM) were also significantly enhanced. The Ca2+ channel antagonists verapamil and nifedipine and the Ca2+ channel agonist BAY K8644 produced a similar percentage change in the magnitude of the noradrenaline response in arteries from both control and diabetic rats. These data confirm the selective nature of the enhancement of contractile responses of arteries from diabetic rats to noradrenaline and suggest that this may be mediated in part through enhanced noradrenaline-induced influx of extracellular Ca2+ through channels sensitive to the Ca2+ channel ligands. However, this does not appear to be the only explanation for the enhanced contractile responses of diabetic arteries to noradrenaline, since in the presence of maximum concentrations of nifedipine (3 microM) and verapamil (10 microM), responses of diabetic arteries to noradrenaline were still greater than those of control arteries. PMID- 7537622 TI - Nuclear phospholipids during the adaptation of human EUE cells to hypertonic stress. AB - The phospholipid component of interphase nuclei was analysed in EUE cells (an established cell line from embryonic human epithelium) grown in an isotonic culture medium and during the adaptation process to a hypertonic medium, using a highly specific ultracytochemical procedure, viz. labelling with the phospholipase A2 gold-complex. Within the nucleus, the phospholipids were localized in domains involved in different steps of the synthesis and processing of the RNA. These localizations did not vary at the two key steps of the adaptation process to hypertonic medium: short-term treatment (6 h) representing critical shock condition, and long-term growth (5 days) representing the adapted cells under survival conditions. On the contrary, deep changes of the labelling intensity of phospholipids at these sites occurred at the different times of hypertonic treatment and followed the same course as those observed in the ultramorphological patterns of transcription: the chromatin condensation, as evaluated by image analysis, the permanent nucleolar components, the interchromatin and the perichromatin granules. These data endorse the hypothesis that nuclear phospholipids could be involved in different steps of the transcriptional activity. They are indicative of the deep changes occurring in the EUE cells submitted to hypertonic stress. PMID- 7537624 TI - Topography of neovascularity in human prostate carcinoma. AB - BACKGROUND: All neoplasms require angiogenesis and resulting neovascularity for growth. The authors and others have confirmed the staging and prognostic significance of quantitative microvascularity density (MVD) in human prostate carcinoma (CAP). In the present investigation, the authors sought to identify the specific site of neovascularity within the neoplasm and adjacent benign tissue. METHODS: Histologically benign and malignant tissues from 14 random radical prostatectomy specimens were studied. The tumor edge was defined precisely by immunohistochemistry, suggesting a high molecular weight cytokeratin that stains only the basal cells of benign histology. Microvascularity density quantification was performed using von Willebrand factor antigen immunohistochemistry as previously defined. Five parallel arcs were defined along which vessel density was calculated including arcs within, on the edge, and removed from the neoplasm. RESULTS: In 13 of 14 cases, the highest vessel density was found within the tumor. Significant differences were observed between the edge of the tumor and 2.5 mm within the benign periphery, between the benign and malignant tissue at the border, and between CAP at the edge and CAP 2.0 mm within the neoplasm. These findings suggest a stepwise increase in MVD toward the center of the neoplasm. CONCLUSIONS: These data confirm the authors' previous observation that prostate cancer has approximately a two-fold increase in MVD compared with the benign tissue. Moreover, high vascularization of the center explains the rare finding of necrosis in CAP. These data suggest that angiogenic promoters may have their highest activity in the center of the neoplasm. PMID- 7537625 TI - Prediction of survival of patients terminally ill with cancer. Results of an Italian prospective multicentric study. AB - BACKGROUND: The individualization of prognostic factors in the various stages of cancer facilitates the planning of a therapeutic assistance program aimed at various subsets of patients. The prognostic factors for survival in patients terminally ill with cancer have been investigated in case studies that are often retrospective, monocentric and/or include a mixture of patients in advanced disease stages. The aim of this prospective multicentric study was to verify those clinical factors predictive of survival in a population of patients with terminal cancer. METHODS: This prospective and multicentric study was performed on 540 patients with solid tumors in the disseminated phase, no longer subject to specific therapy. Patients were evaluated at study entry and every 4 weeks thereafter. The analysis was performed for 23 clinical parameters. RESULTS: Of 530 assessable patients with a median survival of 32 days, 15 factors were found to be statistically significant prognostic factors. By univariate analysis, 13 factors were found to be indicators of a worse survival: age older than 65 years (P = 0.05); palliative corticosteroid treatment (P < 0.001), anorexia (P < 0.001); dry mouth (P < 0.001); dysphagia (P < 0.001); hospitalization (P < 0.001); transfusion (P < 0.001); weight loss greater than or equal to 10% (P = 0.001); dyspnea (P = 0.01); pain (P = 0.006); increasing amount of pain-killer treatment (P = 0.01); increasing number of symptoms (P < 0.001); and worse clinical prediction of survival (P < 0.001). Two factors that correlated with a better survival rate were palliative progestin treatment (P = 0.03) and a higher Karnofsky performance status (P < 0.001). Multiple regression analysis revealed that only clinical prediction of survival, anorexia, dyspnea, palliative steroidal treatment, Karnofsky performance status, and hospitalization were independent predictors of survival. CONCLUSIONS: The importance of certain clinical parameters as prognostic indicators for patients with terminal cancer (clinical experience, physical activity level, clinical symptoms relating to and unrelated to nutritional state) were confirmed; some others possible factors, such as treatment with corticosteroids and hospitalization, also were noted. These may be useful factors in the therapeutic, assistance decision-making process and may eliminate overtreatment and undertreatment resulting from philosophically preconceived attitudes, rather than from considering the patient's true pathologic condition. PMID- 7537626 TI - Serum CD44 splice variants in cervical cancer patients. AB - Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumours and has been shown to be associated with metastasis and poor prognosis in human malignancies. We evaluated serum levels of different soluble CD44 molecules (CD44 standard form and CD44 splice variants v5 and v6) in cervical cancer patients stage IB to IIIB. Two-hundred three serum samples were analysed. Serum levels of CD44st and CD44v5 showed no significant correlation with the presence or absence of cervical cancer. The splice variant CD44v6 showed a mean concentration of 227.3 +/- 90.9 (minimum 71.4, maximum 543.9) ng/ml when tumour was present and a mean concentration of 198.7 +/- 135.4 (minimum 67.2, maximum 696.3) ng/ml in cases of complete remission (P-value = 0.0001). However, in this preliminary study the sensitivity/specificity characteristic of CD44v6 was poor. PMID- 7537627 TI - Synthesis of specifically deoxygenated analogues of the methyl alpha-glycoside of the intracatenary monosaccharide repeating unit of the O-polysaccharide of Vibrio cholerae O:1. AB - Treatment of methyl alpha-D-perosaminide (1) with gamma-butyrolactone gave the 2' deoxy analogue of methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha-D mannopyranos ide (13), the methyl alpha-glycoside of the intracatenary monosaccharide repeating unit of the O-polysaccharide of Vibrio cholerae O:1. The analogous 4'-deoxy derivative was obtained by hydrogenolysis of a 4'-chlorodeoxy precursor, obtained by chlorination of methyl 2,3-di-O-benzyl-4-(2-O-benzyl-3 deoxy-L-glycero-tetronamido)-4,6-d ideoxy-alpha- D-mannopyranoside with methanesulfonyl chloride in DMF. To obtain the 3-deoxy analogue of 13, methyl 4 amino-2-O-benzyl-4,6-dideoxy-3-O-p-methoxy-benzyl-alpha-D-mannopyranos ide was converted into methyl 2-O-benzyl-4,6-dideoxy-4-(2,4-di-O-benzyl-3-deoxy-L-glycero tetronami do)- alpha-D-mannopyranoside, which was deoxygenated via the corresponding 3-O-(imidazol-1-ylthiocarbonyl) derivative. Subsequent catalytic debenzylation gave the deoxy compound (24). In an alternative synthesis, which is also generally useful for the preparation of 4-N-acyl-3-deoxy derivatives of 1, methyl 4-azido-4,6-dideoxy-alpha-D-mannopyranoside was converted through a series of transformations into methyl 4-amino-2-O-benzyl-3,4,6-tri-deoxy-alpha-D mannopyranoside. Subsequent reaction with 2-O-benzyl-3-deoxy-L-glycero tetronolactone, followed by hydrogenolysis of the formed tetronamido derivative, gave 24. PMID- 7537628 TI - Fructose-grafted amylose and amylopectin. AB - Amylose and amylopectin have been heated with sucrose in acidic methyl sulfoxide. The fructose oxocarbonium ion derived from sucrose was transferred as fructofuranosyl groups to 0-6 of the glucans, giving fructoglucans containing up to 7.9% (amylose) and 5.8% (amylopectin) of fructose. The fructosyl units on amylose prevented retrogradation from aqueous solution, resulted in a less-blue iodine complex, and decreased the extent of degradation by amyloglucosidase. The addition of fructosyl units to amylopectin reduced the extent of its degradation by beta-amylase. PMID- 7537629 TI - Structural studies of O-specific polysaccharide chains of the lipopolysaccharide from Yersinia enterocolitica serovar O:10. AB - Lipopolysaccharide (LPS) was isolated from Yersinia enterocolitica serovars O:10 and O:10 KL and the structural pattern of O-specific sugar chains elucidated. The rhamnan and L-xylulose (L-threo-pent-2-ulose) as constituents of the O-specific polysaccharide were obtained by autohydrolysis of the LPS. The rhamnan was shown to be a linear, alpha-(1-->3)-linked polysaccharide in the D configuration. L Xylulose was purified using paper chromatography on a preparative scale and its structure was confirmed by 13C NMR spectroscopy. Using sugar and methylation analysis and 13C NMR spectroscopy of the LPS and the rhamnan, the structural features of the disaccharide repeating unit of the Y. enterocolitica O:10 O specific polysaccharide were elucidated as: [formula: see text] PMID- 7537630 TI - Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using 90Y-labeled monoclonal antibodies in ovarian cancer animal models. AB - Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with 90Y-labeled monoclonal antifibrin antibody Fab fragment or with 90Y-labeled OC125-monoclonal antibody F(ab')2 fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning. PMID- 7537633 TI - NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion. AB - By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species. PMID- 7537631 TI - Glioblastoma therapy by direct intralesional administration of I-131 radioiodine labeled antitenascin antibodies. AB - Thirty patients with recurrent glioblastomas (29 brain, 1 spinal cord) received intralesional radioimmunotherapy aiming to control the progression of the tumor after surgery and radiotherapy. The BC-2 and/or BC-4 murine MAbs (Sorin Biomedica, Saluggia, Italy) were utilized. They strongly react against tenascin (TN), which is an extracellular antigen expressed in large amounts by the stroma of glioblastoma but not by normal brain. The MAbs were labeled with I-131 and were injected directly into the tumor mass to maximize the antibody concentration in the tumor and to irradiate the neoplastic cells. The dose consisted, on average, of 3 mg antibody and 1100 MBq I-131. In most cases the radioimmunotherapy (RIT) applications were repeated two, three, or four times. No systemic adverse reactions were recorded. The brain tolerance to direct antibodies injection was quite good. The antibody concentration in the tumor was high and the MAb residence time in neoplastic tumor was prolonged. Consequently the mean radiation dose to the tumor was high: > 25,000 cGy/cycle. Of 23 evaluable patients, we recorded 7 tumor stabilization (lasting, on mean, 9.1 mo), 4 partial remission (10 mo), and 4 complete remission (18 mo). The overall response rate was 34.7%. PMID- 7537632 TI - Ultrastructural localization of nitric oxide synthase and endothelin in coronary and pulmonary arteries of newborn rats. AB - This is the first report on the ultrastructural distribution of nitric oxide synthase and endothelin immunoreactivities in the coronary and pulmonary arteries of newborn Wistar rats. The distribution of nitric oxide synthase and endothelin was investigated using pre-embedding peroxidase-antiperoxidase immunocytochemistry. In both arteries examined, positive labelling for nitric oxide synthase was localized both in the endothelium and smooth muscle, whereas positive labelling for endothelin was localized in the endothelium exclusively. In the coronary artery, approximately 80% and 55% of the endothelial cells examined were positive for nitric oxide synthase and endothelin, respectively, whereas in the pulmonary artery, 77% and 60% of the endothelial cells were positive for nitric oxide synthase and endothelin, respectively. These findings indicate that nitric oxide synthase and endothelin are colocalized in some of the endothelial cells of the newborn rat. In the endothelium, nitric oxide synthase and endothelin immunoreactivities were distributed throughout the cell cytoplasm and in association with the membranes of intracellular organelles. In smooth muscle, a relationship of nitric oxide synthase immunoreactivity to endoplasmic reticulum was observed in the pulmonary artery. In summary, in the newborn rat, endothelial cells of the coronary and pulmonary artery are rich in nitric oxide synthase (neuronal isoform) and endothelin, and it is suggested therefore that they may be substantially involved in vasomotor control of the cardiac and pulmonary circulation during early stages of postnatal development. PMID- 7537634 TI - RNA export. PMID- 7537635 TI - Homology between reaper and the cell death domains of Fas and TNFR1. PMID- 7537636 TI - Suppression of intestinal neoplasia by DNA hypomethylation. AB - We have used a combination of genetics and pharmacology to assess the effects of reduced DNA methyltransferase activity on ApcMin-induced intestinal neoplasia in mice. A reduction in the DNA methyltransferase activity in Min mice due to heterozygosity of the DNA methyltransferase gene, in conjunction with a weekly dose of the DNA methyltransferase inhibitor 5-aza-deoxycytidine, reduced the average number of intestinal adenomas from 113 in the control mice to only 2 polyps in the treated heterozygotes. Hence, DNA methyltransferase activity contributes substantially to tumor development in this mouse model of intestinal neoplasia. Our results argue against an oncogenic effect of DNA hypomethylation. Moreover, they are consistent with a role for DNA methyltransferase in the generation of the C to T transitions seen at high frequency in human colorectal tumors. PMID- 7537638 TI - Distinct patterns of expression of the D-type cyclins during testicular development in the mouse. AB - The three D-type cyclins have been shown to be differentially expressed in a number of isolated cell types and cell lines, suggesting distinct roles in cell cycle regulation in particular cell lineages. The testis provides unique opportunities to study genes involved in cell cycle regulation, since it contains cells in both mitosis and meiosis as well as differentiated cells with little proliferation activity. Major transcripts of 4.2 kb, 6.8 kb, and 2.3 kb were detected in the adult mouse testis by Northern hybridization analyses for cyclin D1, cyclin D2, and cyclin D3, respectively. Additional transcripts of 1.8 and 2.7 kb were detected by Northern hybridization for cyclin D3 in the testis, but not in other tissues, and these transcripts were limited to germ cells. Northern and in situ hybridization analyses of normal and germ cell-deficient testes showed the surprising result that cyclin D1 was expressed in a pattern consistent with expression in the non-dividing Sertoli cells. Cyclin D2 levels appeared slightly enriched in germ cell-deficient testes as compared to intact testis, but in situ hybridization analysis did not reveal any distinct cellular localization. Also surprising was the observation that cyclin D3 expression was highest in the non dividing, haploid, round spermatids. The possible roles of these cyclins in the events of spermatogenesis are discussed. PMID- 7537637 TI - Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex. AB - A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump. PMID- 7537640 TI - Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part II: Pilot study for clinical application. AB - Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into 3 groups by fluorescence staining of the residual RNA. This identifies cells as 'high (HFR), medium (MFR) and low (LFR) fluorescence intensity' reticulocytes. In part II of the study we looked for the clinical applicability in paediatrics. Selected groups of patients with ineffective erythropoiesis, i.e. suffering from renal failure, oncologic patients with suppressed bone marrow activity caused by chemotherapy and anaemic new-born infants have been observed longitudinally for their reticulocyte maturity profiles. Data were compared to the commonly used parameters RBC, Hb and Hct. In all cases in which effective erythropoiesis returned documented by a normalization of standard blood parameters, HFR cells reacted significantly earlier than the traditional markers. These preliminary observations suggest the reticulocyte maturity pattern analysis can be used as an additional aid in diagnosis and as a helpful parameter for the monitoring of any anaemic situation. PMID- 7537639 TI - Role of protein kinase C in the anti-aggregatory effects of endothelin-1 on human platelets. AB - 1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10(-11) to 10(-6) mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 +/- 0.12). 3. Endothelin-1 at 10(-9) mol/l significantly decreased (P < 0.01) thrombin-stimulated aggregation from 81.4 +/- 1.5% (in the absence of endothelin-1) to 53.5 +/- 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 +/- 1.7 nmol/l to 140 +/- 1.8 nmol/l. 4. Preincubation of platelets with 10(-7) mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na(+)-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537641 TI - Visual hallucinations following treatment with vincristine. AB - Peripheral neuropathy is a common side effect of vincristine therapy. However, side effects due to central nervous system (CNS) toxicity following intravenous administration are rare. We report two patients who developed visual hallucinations during treatment with vincristine. PMID- 7537643 TI - Recording and control of ion channel currents in bilayer and patch-clamp experiments. AB - A program was developed for the control and recording of ion-channel activity in lipid bilayers and cell membranes. The software runs on a PC under DOS. Stimulation waveforms may be applied simultaneously with recording at a sample rate of 10 kHz and at 12-bit resolution. Comments may be stored with the recordings, also during timed repetitive pulse applications. The time critical parts and graphics are written in C and assembler whereas the I/O and control is written in Quick-Basic 4.5 (QB). A nested 16-level macro facility allows the use of complex protocols. Array-buffer arithmetic is supported. There is mouse support for cursor measurements and parabolic offset subtractions. DOS applications such as editing macro files may be executed without halting the program. The program runs in the QB interactive environment with the foreign language routines in a library and is open to customization. The library of drawing subroutines support up to 1024 x 768 resolution. PMID- 7537642 TI - Paraneoplastic cerebellar degeneration: a rare presentation of Hodgkin's disease. PMID- 7537644 TI - Resuscitation following trauma and hemorrhagic shock: is hydroxyethyl starch safe? PMID- 7537646 TI - Molecular cytogenetic analysis of the double-CMA3 chromosome of lake trout, Salvelinus namaycush. AB - The chromosome possessing two chromomycin A3 (CMA3) staining sites in lake trout (Salvelinus namaycush) was examined using FISH. All CMA3-bands in the karyotype contained ribosomal DNA (rDNA), including both sites on the double-CMA3 chromosome. One rDNA site on this chromosome was bordered by telomeric repeats, implicating a rearrangement in the origin of the second NOR. PMID- 7537645 TI - Effects of hydroxyethyl starch after trauma-hemorrhagic shock: restoration of macrophage integrity and prevention of increased circulating interleukin-6 levels. AB - OBJECTIVES: To determine the effects of resuscitation with the colloidal solution (hydroxyethyl starch) vs. crystalloid solution on cell-mediated immune functions after trauma-hemorrhage. DESIGN: Prospective, multiexperimental, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Thirty-six inbred male C3H/HEN (endotoxin-sensitive) mice, aged 6 to 7 wks, and weighing 18 to 23 g. INTERVENTIONS: Crystalloid (lactated Ringer's solution) with and without 6% hydroxyethyl starch after trauma-hemorrhage. MEASUREMENTS AND MAIN RESULTS: Mice underwent laparotomy, were bled to and maintained at a blood pressure of 40 mm Hg for 60 mins, then resuscitated with either 4x the shed blood volume as lactated Ringer's solution or 2x the shed blood volume as lactated Ringer's solution plus 1 x 6% hydroxyethyl starch. Sham mice were neither hemorrhaged nor resuscitated. At 2 or 24 hrs posthemorrhage, serum, splenocytes, peritoneal macrophages, and splenic macrophages were obtained. Bioassays were used to determine interleukin-2, interleukin-3, and interleukin-6 concentrations, while splenocyte proliferation was assessed by 3H-thymidine incorporation. Trauma hemorrhage markedly depressed splenocyte proliferation, interleukin-6 release by macrophages, and lymphokine release at 2 and 24 hrs postresuscitation. The combination of lactated Ringer's solution and hydroxyethyl starch neither restored, nor exacerbated lymphocyte functions. Interleukin-6 release by peritoneal macrophages was restored 24 hrs after hydroxyethyl starch infusion; serum interleukin-6 concentrations remained at sham levels. CONCLUSIONS: Since the use of lactated Ringer's solution and hydroxyethyl starch after hemorrhage did not adversely affect cell-mediated immune functions, but produced salutary effects on macrophage functions, hydroxyethyl starch is a safe and beneficial resuscitation adjunct. PMID- 7537648 TI - Flow cytometric detection of micronuclei by combined staining of DNA and membranes. AB - A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl 1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB and DPH were comparable to the results obtained with the combination of EB and HO. PMID- 7537647 TI - Construction of a pulsed-field map around D3S3 using partially demethylated DNA. AB - Heavy methylation of restriction sites in the relevant chromosomal region can be a major problem in the construction of a long-range restriction map. In the region around the locus D3S3 there appeared to be few accessible restriction sites. Therefore, we cultured cells in the presence of 5-azacytidine and used the partially demethylated DNA to construct a relatively detailed restriction map spanning approximately 1 Mb. Using partially demethylated DNA is a recommendable approach when a genome region appears inaccessible for pulsed-field analysis because excessively long restriction fragments are obtained. PMID- 7537649 TI - Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. AB - Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe. PMID- 7537652 TI - [The assessment of therapeutic success in chronic myeloid leukemia]. PMID- 7537650 TI - Computer software for testing drug susceptibility of malaria parasites. AB - A computer program is described for the automated analysis of data obtained by flow cytometry for in vitro antimalarial drug susceptibility testing. Samples of malaria-infected red blood cells (RBC), which were cultured in the presence of different concentrations of antimalarial drugs, were stained with Hoechst. The Hoechst fluorescence intensity of infected RBC corresponds to DNA content of the parasites and to their stage of development. After measurement of the samples by a FACStar flow cytometer equipped with a UV laser and an autosampler, FCS 1.0 data files were generated. The HP PAS-CAL program developed for these files identifies five different populations--uninfected RBC, infected RBC, free parasites, leukocytes, and debris--on the basis of their light scatter and fluorescence characteristics. The program calculates the percentage of infected cells, the total number of parasite nuclei, and the average number of nuclei per parasite. The results of each culture are presented as a drug dose-response curve. During data analysis, user interaction is limited to selecting the first file of the first culture. The algorithm then processes each culture automatically. Potential problems or difficulties in analysis are flagged. To date, a total of 862 drug tests have been evaluated and fall into two classes, an extended microtest and the World Health Organization standardized microtest. These tests gave satisfactory results in more than 99% of the cases. PMID- 7537651 TI - Sensitivity of monoclonal antibodies to carcinoembryonic antigen, tissue polypeptide antigen, alpha-fetoprotein, carbohydrate antigen 50, and carbohydrate antigen 19-9 in the diagnosis of colorectal adenocarcinoma. AB - PURPOSE: This study was designed to establish the sensitivity of monoclonal antibodies to carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tissue polypeptide antigen (TPA), carbohydrate antigen 50 (CA 50), and carbohydrate antigen 19-9 (CA 19-9) and the efficacy of the joint determination of several tumor markers, as well as the dynamics of postoperative normalization of each marker in the absence of recurrence. MATERIALS AND METHODS: A prospective study was carried out in 100 patients subjected to surgical resection of colon adenocarcinoma. Serum concentrations of these markers were determined the day before surgery and seven days, two months, and six months after surgery. RESULTS: The results demonstrate that sensitivity increased as the disease spread and that CA 19-9 was the most sensitive tumor marker. The rate of false negatives was 40 percent for Dukes Stage A lesions, 19 percent for Dukes Stage B, 7 percent for Dukes Stage C, and 0 percent for Dukes Stage D. Determination of two markers (CA 19-9 and CEA) provided the greatest sensitivity in Stages A and D tumors (60 percent and 100 percent, respectively); the incidence did not change when measurements of other antigens were associated. For Stages B and C, determination of at least three markers was necessary, the association of CEA, TPA, and CA 19-9 being that which showed the greatest sensitivity, 78 percent and 91 percent, respectively. CONCLUSIONS: It would be advisable to include monoclonal antibody determination of CEA, TPA, and CA 19-9 in the diagnosis of adenocarcinoma, despite the fact that ultimate sensitivity will depend on the degree of tumor extension or on the presence of metastasis. PMID- 7537653 TI - [Amantadine, rimantadine and influenza]. PMID- 7537654 TI - [Drug therapy for prostatic hyperplasia]. PMID- 7537655 TI - Rethinking thalidomide. PMID- 7537656 TI - Microheterogeneity of mouse antidextran monoclonal antibodies. AB - Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heterogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues. PMID- 7537657 TI - Electrophoretic, chromatographic and immunological studies of human urinary proteins. AB - Urinary proteins from both sexes were analyzed by high resolution two-dimensional gel electrophoresis (2-DE). For well reproducible 2-DE patterns, the samples were concentrated and desalted in one step by vacuum dialysis. A reference map for urine proteins was established by the analysis of urine from 10 healthy persons. Proteins in urine that share immunogenicity with serum proteins were identified by use of antibody to whole human-serum protein in an affinity-column fractionation of urine and differential analysis of the adsorbed (serum component) and unadsorbed (non-serum component) fractions. For identification of individual proteins, coelectrophoresis, immunoblotting and affinity chromatography with corresponding antibodies were used. Proteins identified in the map, besides known serum proteins, included: the subunit of Tamm-Horsefall protein, the secretory component of IgA, constant breakdown products of alpha 1 antitrypsin and retinol-binding protein, the five isoforms of the beta chain of human chorionic gonadotropin and the subunit of prostatic acid phosphatase. In addition, we could demonstrate three proteins which are markedly pronounced in female urine, especially pregnant women. To get more information about the native properties of various urinary proteins, they were separated into four main peaks according to their sizes using fast protein liquid chromatography equipment. Possible interpolypeptide disulfide bonds were studied using a nonreducing 2-DE system. 2-DE in combination with other methods seems to be a valuable tool for the characterization of urinary proteins in defined renal or extra-renal diseases. An example is given by analyzing the immune complexes from seven patients with a urinary tract infection. PMID- 7537658 TI - Peptides as standards for denaturing isoelectric focusing. AB - A set of commercially available peptides suitable for use as standards in denaturing isoelectric focusing (IEF) is described. The peptides N-procalcitonin fragment 1-57 (pI 3.98), Gln11-amyloid beta-protein fragment 1-28 (pI 5.76), gastric inhibitory polypeptide (pI 7.14), parathyroid hormone fragment 1-34 (pI 8.64) and human beta-endorphin (pI 9.49) can be focused to their isoelectric point in the presence of 8 M urea and 2% Nonidet P-40, and subsequently fixed and stained in polyacrylamide gels. The peptides give a linear standard curve in close agreement with a slope determined with a surface pH electrode. Under the same conditions some proteins focus to positions significantly at odds with their theoretical isoelectric point. The origins of these discrepancies and the implications for the determination of isoelectric points of unknown proteins by denaturing IEF are discussed. PMID- 7537659 TI - Detection of pectinesterase in polyacrylamide gels. AB - A rapid and sensitive method of detecting pectinesterase activity following electrophoresis or isoelectric focusing in polyacrylamide gels is described. The method uses ruthenium red and requires no addition of substrate when making the gels, thus obviating direct enzyme-substrate contact during electrophoresis. Because of its versatility, the method can be used in a wide variety of applications, such as plant and microbial taxonomy, enzyme purification and characterization, or as an analytical method in fresh and processed plant technology. PMID- 7537660 TI - A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes. AB - A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer. PMID- 7537663 TI - Application of cryopreserved golden hamster oocytes to in vitro genotoxicity assays for human sperm chromosomes. PMID- 7537664 TI - Tolerability and quality of life aspects with the anti-androgen Casodex (ICI 176,334) as monotherapy for prostate cancer. International Casodex Investigators. AB - Over 3,000 men, the majority of whom were patients with prostate cancer, were treated with Casodex (ICI 176,334), an oral anti-androgen, at doses ranging from 10 to 200 mg daily, corresponding to a total exposure to the drug of over 1,500 patient-years. Over this period, the tolerability of Casodex and its effect on quality of life were closely studied. Information on tolerability is presented from three large randomized trials of Casodex, 50 mg/day, in patients with prostate cancer, two large randomized trials of Casodex, 150 mg/day, in patients with prostate cancer and three double-blind, placebo-controlled trials of Casodex, 50 mg/day, in patients with benign prostatic hyperplasia (BPH). Information on quality of life and assessment of sexual functioning is presented from the trials using Casodex, 50 mg/day, for both prostate cancer and BPH. The most commonly reported adverse events were those that would be expected with an anti-androgen (i.e. breast tenderness, gynaecomastia and hot flushes). Overall, Casodex was well tolerated; there were no reports of light/dark adaptation problems or pulmonary fibrosis, and only one case of alcohol intolerance, which was not considered by the investigator to be treatment related. Only 0.3% of patients in the whole trial programme had to be withdrawn because of changes in liver function, and there were no clinically significant changes in mean liver function tests. Although there were no consistent differences between treatments for other aspects of quality of life, there was evidence of benefit from treatment with Casodex in maintaining both sexual interest and functioning. PMID- 7537661 TI - Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. AB - The crystal structure of a synthetic peptide representing the major antigenic loop of foot-and-mouth disease virus (FMDV), complexed with the Fab fragment of a neutralizing monoclonal antibody raised against the virus, has been determined at 2.8 A resolution. The peptide shows a high degree of internal structure with a nearly cyclic conformation. The conserved Arg-Gly-Asp motif, involved in the viral attachment of aphtoviruses to cells, participates directly in the interaction with several complementarity determining regions of the antibody molecule. The Arg-Gly-Asp triplet shows the same open turn conformation found in the reduced form of FMDV of another serotype and also in integrin binding proteins. The observed interactions provide a molecular interpretation of the amino acid replacements observed to occur in mutants resistant to neutralization by this antibody. The structure also suggests a number of restrictions to variation within the epitope which are imposed to keep the Arg-Gly-Asp motif in its functional conformation. PMID- 7537665 TI - Strontium-89 (Metastron) in the treatment of prostate cancer metastatic to bone. AB - Bone metastases that develop in patients with advanced prostate cancer often cause deep, unremitting pain. Palliative options for the control of this pain include analgesic support, cytotoxic chemotherapy and external-beam radiotherapy. In addition to external irradiation, interest in intravenously injected radioisotopes that are preferentially localized to bone has been mounting. Metastron (an isotope of strontium) imitates the biodistribution of calcium in vivo and is avidly taken up into bony metastases where it has a biological half life of just over 50 days. The biological half-life in undiseased bone is far shorter, approximately 14 days. Various studies have been conducted to evaluate the role of Metastron in metastatic prostate cancer. An optimum dose has yet to be finalized, but it is clear that the change of haematological toxicity becomes more significant at much larger doses. In the large, randomized Trans Canada study in which Metastron or placebo was given to patients as an adjunct to local field irradiation, those patients treated with Metastron had a significantly reduced intake of analgesics. Furthermore, progression of pain, as measured either by sites of new pain or by the requirement for further palliative radiotherapy, demonstrated statistically significant differences in favour of Metastron. There is thus increasing evidence of a useful role for Metastron in the treatment of prostate cancer metastatic to bone. PMID- 7537662 TI - Relationship between spatially restricted Krox-20 gene expression in branchial neural crest and segmentation in the chick embryo hindbrain. AB - Previous studies have suggested that the rostrocaudal patterning of branchial arches in the vertebrate embryo derives from a coordinate segmental specification of gene expression in rhombomeres (r) and neural crest. However, expression of the Krox-20 gene is restricted to neural crest cells migrating to the third branchial arch, apparently from r5, whereas this rhombomere contributes cells to both the second and third arches. We examined in the chick embryo how this spatially restricted expression is established. Expression occurs in precursors in both r5 and r6, and we show by cell labelling that both rhombomeres contribute to Krox-20-expressing neural crest, emigration occurring first from r6 and later caudally from r5. Krox-20 transcripts are not detected in some precursors in rostral r5, presaging the lack of expression in cells migrating rostrally from this rhombomere. After transposition of r6 to the position of r4 or r5, many Krox 20-expressing cells migrate rostral to the otic vesicle, whereas when r5 is transplanted to the position of r4, only a small number of migrating cells express Krox-20. These results indicate that, in the chick, Krox-20 expression in branchial neural crest does not correlate with rhombomeric segmentation, and that there may be intrinsic differences in regulation between the r5 and r6 Krox-20 expressing populations. PMID- 7537666 TI - A retrospective analysis of the cost effectiveness of treatment with Metastron in patients with prostate cancer metastatic to bone. AB - A retrospective study was performed on the cost-effectiveness of treatment for advanced prostate cancer metastatic to bone. Patients (n = 29) recruited into the trans Canada trial at the Cross Cancer Institute, Edmonton and randomized to treatment with Metastron (strontium-89 chloride) (n = 14) or placebo (n = 15) after local field irradiation therapy for pain palliation were studied over their entire survival time. Estimates were made of the direct costs of treatment, i.e. drugs (analgesics and hormonal agents) and external radiotherapy, and the indirect costs (investigations, outpatient visits and inpatient days, either total or for tertiary care) based on records from the referring hospital, the cancer clinic and any hospitals to which the patients may subsequently have been referred. Meaningful differences were apparent between the two groups in direct costs with the group receiving Metastron showing a reduction over the entire survival time of Can$ 1,720/person compared with placebo; it should be noted that in this analysis neither the costs of the Metastron, nor of the initial radiotherapy, have been included. The Metastron group also showed a reduction in costs of hospitalization for tertiary care of Can$ 5,696/person, though the total cost of hospitalization was similar in the two groups. These results suggest that treatment with Metastron can bring about reductions in management costs for patients with advanced prostate cancer and, coupled with the findings of the Trans Canada trial on the improvement in quality of life for patients given Metastron, they add financial support to the clinical rationale for the use of Metastron for the palliative treatment of patients with bone metastases resulting from prostate cancer. PMID- 7537667 TI - Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow. AB - We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on coimmobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV. PMID- 7537668 TI - Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide. AB - We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl 2, bcl-x, bax and IL-1 beta converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca++ flux, and ceramide production in the Jm and JMRE sublines. The tyrosine phosphorylation patterns and the Ca++ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD resistant and PCD-susceptible sublines. Ceramide is produced following cross linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD. PMID- 7537669 TI - MEM-59 monoclonal antibody detects a CD43 epitope involved in lymphocyte activation. AB - Previous studies on T cell activation via CD43 antigen stimulation were limited to the use of L10, a monoclonal antibody (mAb) recognizing a sialic acid independent epitope on the CD43 molecule. Here we study the CD43 mAb MEM-59, which recognizes a neuraminidase-sensitive epitope on the CD43 molecule, for its ability to activate T lymphocytes. The antibody by itself is able to stimulate proliferation of peripheral blood mononuclear cells (PBMC) in a monocyte dependent fashion, and to act synergistically with the mitogen phorbol 12 myristate 13-acetate. It is demonstrated that the monocyte dependence of MEM-59 induced proliferation of peripheral blood lymphocytes (PBL) cannot be attributed to cross-linking via Fc receptors on monocytes alone: F(ab')2 fragments of MEM-59 are at least as effective as intact IgG in the induction of PBMC proliferation. The effects of MEM-59 reported here are distinct in important ways from those reported for L10. Our proliferation data are extended by the observation that MEM 59 mAb induces mobilization of intracellular Ca2+ in PBMC and in the T cell line Jurkat, while the CD3/TcR-negative Jurkat derived-mutant J.TR3-T3.5 exhibits defective signaling compared to the parent cell line. Moreover, CD3 and CD43 are shown to be present jointly in a large complex in a mild detergent lysate of the T cell line HPB-ALL. These data indicate a physical and functional association between CD3/TcR and CD43 pathways, suggesting a role for CD43 as a co-stimulatory molecule in CD3/TcR signaling, especially in T cell-antigen-presenting cell interactions. PMID- 7537670 TI - Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T cell clones isolated from NOD mice. AB - T cells are known to play an important role in beta cell destruction in the nonobese diabetic (NOD) mouse model of Type I diabetes and islet-specific T cell clones have been demonstrated to be capable of adoptive transfer of diabetes. One important issue involves the identity of beta cell antigens that are recognized by nominally islet cell-specific T cell clones. We have previously reported that insulin-specific T cells are a predominant component of islet-specific T cells isolated from islet infiltrates of pre-diabetic NOD mice. In this report we examine six independently derived insulin-specific T cell clones established from islet infiltrates of pre-diabetic NOD mice in detail. All six clones were found to be specific to a region of the insulin molecule defined by a synthetic peptide encompassing residues 9-23 of the B chain. Despite this restricted specificity, each member of this panel exhibited a distinct receptor specificity defined either by V beta usage or antigen fine specificity. Five clones produced interferon (IFN)-gamma but not interleukin (IL)-4, placing them in the T helper type 1 (TH1)-like category whereas one clone produced both IL-4 and IFN-gamma, a characteristic of TH0 cells. All six clones were capable of either acceleration of diabetes in young NOD mice or adoptive transfer to NODscid mice. Taken together, these results suggest that spontaneously arising insulin-specific T cells participate in beta cell destruction during development of diabetes in NOD mice. PMID- 7537671 TI - Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. AB - We investigated the major histocompatibility complex (MHC) class I-restricted presentation of an epitope of the hepatitis B virus small surface (S) antigen particle to cloned murine cytotoxic T lymphocytes (CTL). Efficient Ld-restricted presentation of the S28-39 epitope to CTL is observed in cells of different tissue origin pulsed in vitro, either with the antigenic S28-39 12-mer S-peptide, or with particulate S-antigen. The kinetics of epitope presentation differ in S peptide-pulsed and in S-particle-pulsed cells: while a 15-min pulse with the antigenic peptide sensitizes targets for class I-restricted CTL lysis, presentation of S-particles requires 30-60 min to sensitize cells for CTL lysis. Uptake of antigenic material and active metabolism of the presenting cell are required for processing of S-particles, but not for sensitizing targets with S peptides. Intracellular processing and presentation of S-particles is blocked in cells treated with chloroquine, NH4Cl, primaquine, or leupeptin, but not by treatment with cycloheximide or brefeldin A. This processing pathway operates efficiently in peptide-transporter-deficient, Ld-transfected T2 cells, revealing a novel endosomal/lysosomal processing pathway for class I-restricted presentation of peptides derived from exogenous S-particles. PMID- 7537672 TI - Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. AB - Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively. PMID- 7537673 TI - Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. AB - Previous studies on the production of interleukin-12 (IL-12) have shown that it is released, together with other proinflammatory cytokines, shortly after exposure of phagocytic cells to a variety of pathogens. We here report that IL-12 is also released during the recall response to soluble antigen (Ag) devoid of intrinsic adjuvant activity. We show that activated T cells induce the production of IL-12 by monocytes via a mechanism involving the interaction of T cell associated CD40 ligand with CD40 on monocytes. The data suggest that Ag presentation on monocytes favors the persistence of type 1 responses. PMID- 7537674 TI - Induction in mice of serum IgE levels after treatment with anti-mouse IgD antibodies is preceded by differential modulation of tissue cytokine gene transcription. AB - Injection of mice with purified goat anti-mouse IgD (GAMD) leads to an interleukin (IL)-4-dependent increase of serum IgE levels. Challenge of GAMD primed mice with goat IgG (GIG) initiates a secondary immune response with elevated serum IgE. In this report, kinetic cytokine transcript profiles of murine lymphoid tissues in response to primary i.v. GAMD treatment, as well as GIG challenge are presented. For the first time, gene transcription patterns of the recently described cytokines IL-12 and IL-13 are shown and compared with the corresponding patterns for other cytokine genes involved in IgE regulation, i.e. IL-4, and interferon (IFN)-gamma. After GAMD injection, two groups of induction profiles were observed in spleen, mesenteric lymph nodes and Peyer's patches: while IL-4 and IL-12p35 gene transcription was strongly enhanced, IFN-gamma, IL 12p40 and IL-13 mRNA were only moderately induced. Generally, maximal mRNA induction was measured on days 3 to 4 after GAMD treatment. The data demonstrate a clear-cut difference between the IL-4 and IL-13 response on the transcriptional level although both gene products show similar biological activities. The cytokine mRNA profiles support the assumption of IL-4 playing the central role in generating an IgE response. However, they do not reflect a strict Th1 versus Th2 cytokine gene transcription pattern but rather point towards a concerted action of various, partially antagonizing cytokines with respect to the regulation of IgE synthesis. IL-12 may, possibly via stimulation of IFN-gamma synthesis, represent a counterbalancing factor in the IL-4-mediated IgE response. PMID- 7537675 TI - Clonal expansion of myelin basic protein-reactive T cells in patients with multiple sclerosis: restricted T cell receptor V gene rearrangements and CDR3 sequence. AB - Myelin basic protein (MBP)-reactive T cells are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In some patients with MS, these autoreactive T cells display a limited heterogeneity in their epitope recognition and T cell receptor (TCR) variable (V) gene usage. These individual-dependent properties of MBP-reactive T cells have led to the speculation that they may represent clonal expansion in vivo in some MS patients. In the present study, 51 MBP-reactive T cell clones derived from patients with MS and healthy individuals were examined for their epitope recognition and the TCR V alpha and V beta gene rearrangements. The V gene junctional region sequences of identified alpha and beta genes were further analyzed to probe their clonal origins, as the sequences are unique for individual clones. Our data showed that 26 clones derived from nine patients with MS shared a predominant reactivity to the immunodominant regions of MBP, 84-102, 110-129 and 143-168, and used various TCR V alpha and V beta rearrangements. The V gene usage of the clones was restricted to certain V alpha V beta combination(s) in a given MS patient, but varied among different patients. The sequence analysis revealed that the clones generated from a given patient shared a limited or a single junctional region sequence pattern(s), indicating their oligoclonal or monoclonal origin(s). In contrast, 25 MBP reactive T cell clones derived from normal individuals exhibited unfocused epitope recognition and V gene usage. Thus, the limited heterogeneity of MBP reactive T cells in their structural and functional characteristics reflects their clonal expansion in vivo in some patients with MS. PMID- 7537676 TI - Pharmacology of tachykinin receptors on neurones in the ventral tegmental area of rat brain slices. AB - The pharmacology of tachykinin receptors within the ventral tegmental area of rat brain slices was studied using in vitro electrophysiological techniques. The selective tachykinin NK3 receptor agonist senktide (100 nM) increased the action potential firing rate from 1.9 to 3.9 Hz in 70% of spontaneously active cells tested (n = 27). Senktide was the most potent agonist tested with an EC50 of 4 nM. In contrast the NK1 receptor agonists substance P-O-methyl ester (100-300 nM) or GR 73632 (1 microM) were inactive at the concentrations tested. Responses to neurokinin B (EC50 = 32 nM) were not blocked by the tachykinin NK1 receptor antagonist CP 99,994 (1 microM) nor by the tachykinin NK2 receptor antagonist SR 48968 (300 nM). Similarly responses to the tachykinin NK2 receptor agonist beta [Ala8]neurokinin A-(4-10) (EC50 = 427 nM) were not antagonised by the tachykinin NK2 receptor antagonist SR 48968 (300 nM) and thus were likely to be due to the activation of tachykinin NK3 receptors. These data demonstrate that NK3, and not NK1 or NK2 receptors, mediate the principal excitatory effects of exogenously applied tachykinin receptor agonists on dopamine neurones within the rat ventral tegmental area. PMID- 7537677 TI - Comparison of the induction of cyclooxygenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages. AB - Endotoxin causes the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2. We have compared the ability of endotoxin to increase the activities of these enzymes in bovine aortic endothelial cells and the macrophage cell line (J774.2). Endotoxin (1 microgram ml-1; for 24 h) caused a time dependent increase in the accumulation of cyclooxygenase metabolites from endogenous arachidonic acid, in both cell types. Cyclooxygenase activity towards exogenous arachidonic acid (30 microM; for 15 min) was also increased in both cell types. Endothelial cells and macrophages also contained comparable amounts of cyclooxygenase-2 protein after incubation with endotoxin for 24 h which was prevented by pretreatment with cycloheximide (10 micrograms ml-1; 30 min prior to endotoxin). Endotoxin for 24 h caused a time-dependent increase in nitrite accumulation in macrophages, but not in endothelial cells. Thus, endotoxin increased cyclooxygenase activity and induced cyclooxygenase-2 protein in endothelial cells and macrophages. Endotoxin also increased NO synthase activity in macrophages, but not in endothelial cells. PMID- 7537678 TI - Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. AB - Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10-12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl-L-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl-L-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl-L-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase. PMID- 7537679 TI - Role of nitric oxide in hyporeactivity to noradrenaline of isolated aortic rings in portal hypertensive rats. AB - To test the hypothesis that induction of nitric oxide synthase causes systemic vascular hyporesponsiveness to vasopressors in portal hypertension, we performed in vitro experiments on isolated thoracic aortic rings from partial portal vein ligated or sham operated rats at 3 weeks postoperatively. The concentration response curves to noradrenaline of intact and endothelium-denuded aortic rings from portal hypertensive rats were significantly shifted to the right as compared to those from sham operated animals. Maximal contractions did not significantly differ. Addition of NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, shifted the curves to the left in both sham operated and portal hypertensive rats, so that in intact rings, the concentrations of noradrenaline producing half-maximal response did not significantly differ any more between sham operated and portal vein ligated rats. In endothelium-denuded rings, a hyporeactivity to noradrenaline persisted in portal vein ligated rats. Furthermore, NG-nitro-L-arginine induced an additional significant increase in the maximal response to noradrenaline in sham operated as compared to portal hypertensive rats. The endothelium-dependent relaxations to acetylcholine were attenuated in portal hypertensive rats as compared to sham operated animals. From these results, it can be concluded that increased nitric oxide production in the vascular wall of thoracic aorta of portal hypertensive rats is involved in their hyporesponsiveness to noradrenaline. Our findings in endothelium-denuded rings indicate the involvement of the inducible nitric oxide synthase in the smooth muscle layer. Involvement of an inducible nitric oxide synthase in the endothelium cannot be excluded. The endothelial constitutive nitric oxide synthase, however, seems to be suppressed in portal vein ligated rats. PMID- 7537680 TI - Exogenous and endogenous cholecystokinin protects gastric mucosa against the damage caused by ethanol in rats. AB - Cholecystokinin (CCK) shows a potent influence on gastric secretion and motility but its role in gastric mucosal integrity has been little examined. In this study we found that exogenous CCK octapeptide protected gastric mucosa against ethanol induced gastric injury but was ineffective against aspirin-induced damage. The protective effects of CCK were dose-dependent and almost completely reversed by pretreatment with the specific CCKA receptor antagonist, loxiglumide, while the CCKB receptor antagonist, L-365,260, was not effective. The CCK-induced protection against ethanol injury was accompanied by a significant increase in gastric blood flow. The inhibition of nitric oxide (NO) synthase by NG-nitro-L arginine methyl ester attenuated the gastroprotection and gastric hyperemia induced by CCK while the concurrent treatment with L-arginine, but not D-arginine restored the protective activity of CCK and the accompanying increase in gastric blood flow. Endogenous CCK released by intraduodenal instillation of oleate prevented the formation of acute gastric lesions induced by both ethanol and aspirin and the protective effects were abolished by pretreatment with loxiglumide. We conclude that CCK exerts protective activity against ethanol induced damage and that this effect is mediated through specific CCKA receptors and hyperemia involving NO. PMID- 7537681 TI - Inhibition of plasma extravasation by abruquinone A, a natural isoflavanquinone isolated from Abrus precatorius. AB - Polymyxin B-induced hind-paw edema was suppressed by abruquinone A, an isoflavanquinone isolated from Abrus precatorius, in normal as well in adrenalectomized mice. Unlike dexamethasone, abruquinone A did not increase the liver glycogen content in fasting adrenalectomized mice. The volume of exuded plasma was significantly reduced by abruquinone A in neurogenic inflammation, passive cutaneous anaphylactic reaction and compound 48/80-induced ear edema. Histamine-, serotonin-, bradykinin- and substance P-induced plasma extravasation in ear edema was also suppressed by abruquinone A. Abruquinone A, like isoproterenol, significantly reduced the bradykinin- and substance P-induced plasma extravasation in normal as well as in compound 48/80-pretreated mice. In addition, abruquinone A suppressed the bradykinin- and substance P-induced ear edema to a significantly greater extent than diphenhydramine/methysergide did. In the in vitro experiments, abruquinone A suppressed the compound 48/80-induced histamine and beta-glucuronidase released from isolated rat peritoneal mast cell preparations. These results suggest that the anti-inflammatory effect of abruquinone A is mediated partly via the suppression of the release of chemical mediators from mast cells and partly via the prevention of vascular permeability changes caused by mediators. The glucocorticoid activity and the release of glucocorticoid hormones from the adrenal gland are probably not involved. PMID- 7537682 TI - Regulation of substance P release mediated via prejunctional histamine H3 receptors. AB - The involvement of the histamine H3 receptor in the regulation of substance P release in neurogenic inflammation was studied by using rat hindpaw skin. R-(-) alpha-Methylhistamine, a specific histamine H3 receptor agonist, significantly inhibited the increased vascular permeability induced by antidromic electrical stimulation of the sciatic nerve in a dose-dependent manner at doses of 0.5-3 mg/kg (i.v.), and thioperamide (2 mg/kg i.p.), a specific histamine H3 receptor antagonist, prevented the inhibitory effect of R-(-)-alpha-methylhistamine. The antidromic stimulation also caused a significant increase in immunoreactive substance P release in the subcutaneous (s.c.) perfusate in the rat hindpaw. R-( )-alpha-Methylhistamine (0.25-2 mg/kg) dose dependently inhibited the increase in release of immunoreactive substance P, and thioperamide (2 mg/mg i.p.) antagonized it. Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the perfusate, which was reduced by R-( )-alpha-methylhistamine and the antagonism of thioperamide was also observed. Histamine (in the presence of histamine H1 and H2 receptor antagonists) had an inhibitory effect on the electrically evoked release of immunoreactive substance P. These results strongly support the hypothesis that histamine regulates substance P release via prejunctional histamine H3 receptors that are located on peripheral endings of sensory nerves. PMID- 7537684 TI - Role of eicosanoids but not nitric oxide in the platelet-activating factor induced increase in vascular permeability in mouse skin. AB - We investigated the role of endogenous eicosanoids and nitric oxide (NO) in the platelet-activating factor (PAF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of PAF (45-180 pmol/site) induced a dose related increase in vascular permeability at the injection site. The vascular permeability induced by PAF (180 pmol/site) was significantly inhibited by pretreatment with an intraperitoneal injection of 1-O-hexadecyl-2-acetyl-sn glycero-3-phospho (N,N,N-trimethyl) hexanolamine (PAF receptor antagonist) (5 and 25 mg/kg) and indomethacin (cyclooxygenase inhibitor) (10 mg/kg), whereas it was not affected by concurrent intravenous administration of NO synthase inhibitors NG-nitro-L-arginine methyl ester (10 mg/kg) or methylene blue (100 micrograms/kg) nor by topical injection of NG-nitro-L-arginine methyl ester. The inhibitory effect of indomethacin was partially reversed by topical administration of prostaglandin E2. These results suggest that PAF increases venular permeability by activating PAF receptors and that plasma extravasation is potentiated by the release of prostanoids which cause arteriolar dilatation. However, NO is not involved in the effect of PAF in mouse skin. PMID- 7537683 TI - NG-nitro-L-arginine protects against hypoxia/hypoglycemia-induced decrease in CA1 presynaptic spikes in rat hippocampal slices. AB - The effects of nitric oxide (NO) synthase inhibitors on the hypoxia/hypoglycemia induced decrease in CA1 presynaptic fiber spikes elicited by stimulation of the Schaffer collaterals were investigated using rat hippocampal slices. Drugs were added to normal medium for 10 min before incubation under hypoxic/hypoglycemic conditions (15 min), and after a 3-h washout, the CA1 presynaptic potential was measured. Treatment with NG-nitro-L-arginine methyl ester but not with NG-nitro-D arginine methyl ester produced a concentration-dependent attenuation of the hypoxia/hypoglycemia-induced decrease in presynaptic fiber spikes. In contrast, treatment with precursors of NO in the arginine-to-NO pathway, such as sodium nitroprusside, S-nitro-N-acetylpenicillamine and N-morpholino sydnonimine exacerbated the 15-min hypoxia/hypoglycemia-induced decrease in the CA1 presynaptic potential. The neuroprotective effect of NG-nitro-L-arginine methyl aster was significantly attenuated by co-treatment with L-arginine. The present results suggest a facilitatory role of NO production in hypoxia/hypoglycemia induced presynaptic dysfunction in CA1 regions of hippocampal slices. PMID- 7537685 TI - NMDA receptor involvement in endothelin neurotoxicity in rat striatal slices. AB - The high K(+)-evoked dopamine release from rat striatal slices remained impaired by 50% up to 2 h after pulse exposure of the tissues to endothelin-3, under conditions of hypoglycemia/hypoxia. This striatal dysfunction was significantly improved by D-2-amino-5-phosphonopentanoic acid, a NMDA receptor antagonist, at a much lower concentration than that providing protection against NMDA-evoked dysfunction. In light of these findings, the important role of glutamatergic mechanisms, especially NMDA receptors, in mediating endothelin neurotoxicity warrants further attention. PMID- 7537686 TI - Effects of macromolecular crowding on nuclear size. AB - The concentration of macromolecules inside cells is high, and the resultant crowding of cytoplasm can be expected to affect many interactions involving macromolecular assemblies. Here, we have examined the effect of solute size and concentration on nuclear volume in saponin-permeabilized macrophages. Nuclei swelled in the presence of small solutes and shrank reversibly in the presence of larger permeant solutes. Remarkably, the smallest solutes capable of shrinking the nucleus were not excluded by the pores in the nuclear envelope. Indeed, nuclei shrank in the presence of such solutes even after the nuclear envelope had been sheared mechanically or permeabilized with detergent. Nuclei extracted with 1% Triton X-100 shrank in the presence of very high concentrations of small solute molecules (30% w/v) as well as in lower concentrations of larger solutes. Consistent with a macromolecular crowding effect, changes in nuclear volume were dependent on solute size and not simply dependent on the colligative properties of solutes or the exclusion of solutes by the nuclear envelope. Solute size dependent changes in nuclear volume were independent of the chemical nature of the solutes and of the activity of the ions in the buffer. Together, these observations indicate that high concentrations of macromolecules such as those found inside cells can influence the size of the nucleus by directly affecting nuclear structure. PMID- 7537687 TI - Growth regulation and cellular changes during differentiation of human prostatic cancer LNCaP cells as induced by T lymphocyte-conditioned medium. AB - Human prostatic epithelial cells from an androgen-dependent LNCaP cell line were examined in response to conditioned medium (CM) derived from phytohemagglutinin (PHA)-stimulated lymphocytes. Addition of CM caused a greater than 70% reduction of cell proliferation by cell counting and cell cycle. These cells showed G1 phase arrest and the clonogenicity was reduced. The growth-modulating effect was dose-dependent and not due to cell lysis or apoptosis. The binding of androgen to androgen receptor on these cells showed approximately 50% reduction, underlining a proliferation reduction mechanism. The prostate-specific antigen (PSA) was downregulated to approximately 75% during the process. Cell morphology showed dendritic processes extending from cytoplasm and other neuroendocrine cell characteristics. The expression of several cytoskeleton and intracellular proteins increased as determined by immunostaining on slides and by ELISA procedures. These included vimentin, correlating to cell shape changes, cytokeratins 8 and 18, associated with differentiated cell types of prostate epithelia, and neuron-specific enolase and serotonin, associated with neuroendocrine cells. From these cellular changes, we can infer that the cell growth was modulated along with induction of terminal differentiation. Activated T cells were demonstrated to be important in providing the modulating activity. This growth modulator was semipurified and had an estimated molecular weight 13,000 to 24,000 Da. The activity was determined to be distinct from TGF, TNF, and some commonly known lymphokines. The interaction between lymphoid and prostatic cells in growth and development is described. PMID- 7537688 TI - Cytoplasmic and nucleic calcium oscillations in immature mouse oocytes: evidence of wave polarization by confocal imaging. AB - Using confocal laser scanning microscopy and time-lapse reconstruction, we monitored [Ca2+]i in living mouse oocytes loaded with the calcium-sensitive fluorescent dyes fluo-3/AM or NuCa Green (specific for nuclear calcium) at the time of meiosis reinitiation. Our data confirm that spontaneous Ca2+ oscillations occurred in most of the immature fully grown mouse oocytes at least during the first half hour after release of the follicle and these oscillating oocytes underwent GVB in a large proportion after 1 h of culture. These spontaneous oscillations were disrupted when GVB was inhibited by dbcAMP and suppressed in the absence of external calcium. They were dependent on intracellular InsP3 sensitive Ca2+ stores since they were inhibited by heparin, an InsP3-receptor antagonist, or thapsigargin, which depletes InsP3-sensitive Ca2+ stores. A relation appeared between the germinal vesicle chromatin appearance and the oocyte's ability to exhibit calcium oscillations. NuCa Green-loaded oocytes exhibited clear fluorescence oscillations in the nuclear region which lead us to discuss the implication of the nuclear calcium in the meiotic process. Moreover, the observation of a clear polarization of the Ca2+ waves in 30% of the oocytes permits us to hypothesize the existence of a predetermined localization for the initial point of the calcium wave. PMID- 7537690 TI - Cisplatin resistance in a murine leukemia cell line is associated with a defective apoptotic process. AB - Apoptosis is characterized by typical morphological changes and most frequently fragmentation of DNA into oligonucleosome-size fragments. In order to investigate whether an alteration in the mechanisms involved in the process of apoptosis could contribute to cellular resistance, induction of apoptosis was studied in a cisplatin-resistant cell line (L1210/DDP) derived from a L1210 murine leukemia cell line (L1210/0). Treatments of the parental L1210/0 cell line with two DNA damaging agents (cisplatin and 5-azacytidine) or a protein kinase C inhibitor (staurosporine) led to biochemical events characteristic of apoptosis (as determined by the cell morphology and the oligonucleosomal DNA fragmentation). In contrast, the cisplatin-resistant L1210/DDP subline, which was cross-resistant to 5-azacytidine, did not exhibit any DNA fragmentation or morphological changes typical of apoptosis when exposed to toxic concentrations of either cisplatin or 5-azacytidine. The failure of these cells to undergo apoptosis upon cisplatin or 5-azacytidine exposure has been correlated with the lack of a nuclear endonuclease activity present in wild-type cell nuclei. However, staurosporine, which exerted the same toxicity on both cell lines, induced the internucleosomal DNA fragmentation and morphological features of apoptosis in both of them. This indicates that a functional pathway for apoptosis is preserved in the resistant cells. The induction of this pathway can be correlated with the presence of a cytoplasmic endonuclease activity whose specificity seems different from that operating in L1210/0 cells in terms of cation and pH dependence. Therefore, in these cell lines, different endonucleases are possibly involved in apoptosis. In response to treatment with drugs having different targets, the apoptotic cell death may operate through different signaling pathways, one of them being possibly defective in the L1210/DDP-resistant cells. PMID- 7537691 TI - FGF-1 is a heparin-independent mitogen for rat hepatocytes. AB - We have found that in the primary culture of rat hepatocytes, the potent mitogenic activity of native FGF-1 is independent of heparin. The well established characteristic of FGF-1 as a heparin-dependent mitogen is confirmed by human umbilical vein endothelial cells using the same preparation of FGF-1. Cross-linking experiments reveal that binding of FGF-1 to the hepatocyte cell surface receptors can be accomplished in the absence of exogenous heparin, in contrast to human endothelial cells for which it remains as a limiting factor. For both cell types, however, it is demonstrated that either endogenous or exogenous heparan sulfate/heparin moieties are essential for FGF-1 to establish receptor binding and mitogen action. Thus, the results suggest that hepatocytes harbor cell surface heparan sulfate moieties that are fully capable of utilizing FGF-1 in the environment. These results raise the possibility that FGF-1 is of differential potency for different cell types according to the nature and/or quantity of cell surface heparan sulfate moieties in vivo. PMID- 7537689 TI - Colchicine induces apoptosis in cerebellar granule cells. AB - Exposure to 1 microM colchicine, a microtubule disrupting agent, triggered apoptosis in rat cerebellar granule cells (CGC). Apoptotic nuclei began to appear after 12 h followed by oligonucleosomal DNA laddering, whereas inhibition of the mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide metabolism became significant between 18 and 24 h, when most cells already had apoptotic nuclei. These events were preceded by loss of tau protein and fragmentation of alpha and beta tubulins. Colchicine treatment also caused alterations in Ca2+ responses to chemical depolarization and a moderate, but progressive, increase in the resting intracellular Ca2+ concentration. Nearly all neurons expressed c-Fos after the treatment with colchicine. However, while in part of the cell population c-Fos levels subsequently declined, in the neurons undergoing apoptosis the protein was still expressed, but had an abnormal intracellular localization. An increased expression of the constitutive nitric oxide synthase (NOS-I) was also detected at 12 h and was followed by increased nitrite production. Treatment with 100 nM taxol to stabilize the microtubuli prevented DNA laddering and apoptotic body formation induced by colchicine. In contrast, pretreatment with the N-methyl-D-aspartate receptor-antagonist, MK-801, or L-type Ca2+ channel blockers did not prevent colchicine-induced CGC apoptosis. Inhibitors of NOS were also ineffective in preventing apoptotic body formation and DNA laddering, whereas they delayed the secondary cell lysis. These results support the idea that colchicine-induced cytoskeletal alterations directly initiate the genetic and structural modifications that result in CGC apoptosis. PMID- 7537692 TI - Alteration of DNA and RNA binding activity of human telomere binding proteins occurs during cellular senescence. AB - The loss of telomere sequences during in vitro and in vivo aging suggests that mechanisms affecting telomere length may have important consequences in cellular senescence. In this study, we have found that the activity of single-stranded telomere binding proteins is increased in nuclear extracts prepared from senescent human diploid fibroblasts compared to actively growing cells. Since single-stranded telomere binding proteins are closely related to RNA binding proteins, we examined the binding activity of several major RNA binding proteins to RNA by uv cross-linking. The level of activity was greatly diminished and the overall pattern of uv cross-linked products were altered in extracts prepared from senescent cells. Furthermore, Western analysis revealed a concurrent decrease in senescent extracts of the protein level for many RNA binding proteins, including those which bind to telomere sequence. Although the reduction in the level of these proteins parallels the reduced activity in RNA binding, the paradoxical increased telomere binding activity exhibited by extracts from older cells suggests a complex relationship between these proteins with RNA and DNA. Moreover, the reduced RNA binding activity of these proteins indicates that the biochemical function of several RNA binding proteins is compromised during cellular senescence, raising an intriguing possibility that a change in pre-mRNA metabolism may contribute to the multitude of changes in gene expression observed in cellular senescence. PMID- 7537693 TI - cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. AB - Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles. The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within 15 h of incubation at 37 degrees C in serum-free medium. The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death. In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%). Basic fibroblast growth factor (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death. Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone. In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related proteins. Cultures of three different cell lines established by SV40 transformation demonstrated resistance to 8-Br-cAMP- or forskolin plus IBMX-induced apoptosis, in contrast to the severe apoptotic response in primary cells. We suggest that stimulation of primary granulosa cells by high levels of cAMP catalyzes programmed cell death, while stimulation of the cells by gonadotropic hormones, which result in a moderate cAMP response, followed by desensitization to further stimulation, can prolong the lifespan of the luteinized granulosa cells. Moreover, one or more tumor suppressor proteins may mediate the cAMP generated signal leading to cell death. PMID- 7537694 TI - cAMP inhibits IGF-I-induced mitogenesis in fetal rat brown adipocytes: role of p21 ras. AB - Dibutyryl cAMP (DBcAMP) inhibited insulin-like growth factor I (IGF-I)-induced DNA synthesis of rat fetal brown adipocyte primary cultures. Its mitogenic inhibitory capacity, measured as [3H]thymidine incorporation into acid precipitable material, was dose-dependent, being maximal at 0.5 mM. The entry of cells into S + G2/M phases of the cell cycle and the increase in cell number induced by IGF-I were partially inhibited by DBcAMP at 24 h and totally prevented at 48 h. DBcAMP inhibited the mRNA expression of c-myc induced by IGF-I at 2 h. Moreover, DBcAMP inhibited in parallel H-ras mRNA expression, p21 ras, and proliferating cellular nuclear antigen protein content induced by IGF-I at 24 and 48 h of culture, respectively, suggesting the involvement of p21 ras in the progression of fetal brown adipocytes through the S phase of the cell cycle and DNA synthesis. PMID- 7537696 TI - Distinct patterns of cell cycle disturbance elicited by compounds interfering with DNA topoisomerase I and II activity. AB - DNA topoisomerases are enzymes governing the multitude of conformational changes DNA undergoes during the cell cycle. Several compounds are likely to interfere with specific steps of the catalytic cycle of these enzymes. Camptothecin arrests the activity of DNA topoisomerase I by provoking the formation of a single stranded DNA break with the enzyme molecule covalently attached to the DNA. Exposure to m-AMSA arrests DNA topoisomerase II by the formation of a ternary complex involving the drug, the enzyme, and DNA carrying a double-stranded break. Netropsin, distamycin A, and berenil inhibit DNA topoisomerase-mediated relaxation of supercoiled DNA by an as-yet unknown mechanism. Here, we analyze the cell cycle kinetic effects of exposure to camptothecin, m-AMSA, netropsin, distamycin A, and berenil by using continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Camptothecin elicits an accumulation of cells in all compartments of the cell cycle, while exposure to m-AMSA leads mainly to retention of cells in the G0/G1 compartment and to accumulation in the G2 phase. Neither camptothecin nor m-AMSA shows a synergism with bromodeoxyuridine incorporation into the DNA. These results point toward distinct functions of the two DNA topoisomerases in the process of cell cycle traverse. The compounds binding to the minor groove of DNA interfere with all phases of the cell cycle, but with a relative emphasis on the G2 phase. Neither camptothecin nor m-AMSA exhibits a synergistic effect in combination with berenil. Hence, at the level of perturbed cell cycle kinetics a distinction can be made between compounds provoking an abortive inhibition of the catalytic cycle of DNA topoisomerases (e.g., camptothecin, m-AMSA) and those interfering with the activity of the enzyme by a distinct mechanism. PMID- 7537697 TI - The E-cadherin complex contains the src substrate p120. AB - Using normal MDCK cells, and MDCK cells stably transfected with a temperature sensitive viral src allele (pp60 ts-v-src), we have examined the composition and tyrosine phosphorylation of the E-cadherin complex. E-cadherin is a transmembrane calcium-dependent cell-cell adhesion molecule that is complexed with cytoplasmic proteins including alpha-catenin, beta-catenin, plakoglobin (gamma-catenin), and actin. We have identified two heterodimeric complexes which demonstrate that alpha-catenin interacts directly with beta-catenin, or with plakoglobin, in the absence of E-cadherin. beta-Catenin has previously been shown to bind directly to E-cadherin. We propose that E-cadherin associates with alpha-catenin, and thereby the actin cytoskeleton, via either beta-catenin or plakoglobin. We have further identified three new but related protein components of the E-cadherin complex, which are each cross-reactive by Western blot analysis to antibodies directed against p120, a phosphotyrosine substrate of src, and a phosphotyrosine, phosphoserine, and phosphothreonine substrate of growth factor-stimulated signaling pathways. Greater quantities of the p120-related proteins were found present in the E-cadherin immunoprecipitates of ts-src MDCK cells compared to normal MDCK cells, while two of the p120 cross-reactive species were significantly tyrosine phosphorylated in both normal and ts-src MDCK cells. The association of p120-related species with the E-cadherin complex adds them to our consideration of possible modulators of cadherin function. Likewise, the newly identified alpha-catenin-beta-catenin and alpha-catenin-plakoglobin dimers may have interesting biological properties, conceivably including the titration of catenins between cadherin and APC complexes. PMID- 7537698 TI - Endogenous transcription occurs at the 1-cell stage in the mouse embryo. AB - In most animal species, the earliest stages of embryogenesis are regulated by maternally inherited components, at least until the activation of the zygotic genome. Although the first proteins derived from the activation of the embryonic genome have been detected long ago at the 2-cell stage in the mouse, the exact timing of transcriptional activity resumption after fertilization is still a matter of debate. Any new information about this critical event is relevant to the practice of transgenesis and cloning of embryos as well as to the general understanding of the regulation of nuclear processes following fertilization. Using a new fluorescent method allowing the detection of in vivo RNA synthesis, we show that endogenous transcription by RNA polymerase II takes place unambiguously as early as at the late 1-cell stage. Furthermore, we demonstrate that transcription is first initiated in the paternal pronucleus. PMID- 7537695 TI - IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. AB - IL-1 inhibits chondrocyte proliferation induced by TGF beta or serum. This study analyzed the role of nitric oxide (NO), which is induced at high levels by IL-1 in chondrocytes. NO, when administered through sodium nitroprusside (SNP), inhibited TGF beta or serum-induced chondrocyte proliferation. To determine whether IL-1-induced NO is responsible for growth inhibition by IL-1, chondrocytes were cultured in the presence of the nitric oxide synthase inhibitor N-monomethyl-L-arginine (NMA), which dose-dependently reduced the antiproliferative effects of IL-1. Analysis of interactions between PGE2, a known chondrocyte growth inhibitor, and NO showed that PGE2 does not induce NO and is not required for NO induction by IL-1. However, SNP induced high levels of PGE2, and NMA reduced IL-1-induced PGE2. This raised the possibility that PGE2 is a downstream mediator of the antiproliferative effects of NO. This was confirmed in experiments where the growth inhibitory effects of SNP were reduced by indomethacin. These results suggest that the chondrocyte growth inhibition by IL 1 in chondrocytes is due to the induction of NO, which stimulates the production of PGE2 as a mediator of its antiproliferative effects. PMID- 7537700 TI - The association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium. AB - The integrin alpha IIb beta 3-mediated redistribution of the tyrosine kinases pp125FAK and pp60Src and the small GTP-binding proteins CDC42Hs and Rap1B from the membrane skeleton to the cytoskeleton was found to be reversible: upon prolonged platelet aggregation (up to 15 min) induced by the thrombin-receptor activating peptide (TRAP) these signalling proteins dissociated from the cytoskeleton and reappeared in the membrane skeleton. Addition of the extracellular Ca2+ chelator EGTA and the intracellular Ca2+ chelator BAPTA/AM 30 s after TRAP allowed platelet aggregation and the association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton, but prevented their dissociation from the cytoskeleton. The results indicate that the prolonged elevation of cytosolic Ca2+ in stimulated platelets leads to the dissociation of signalling proteins from the cytoskeleton. PMID- 7537699 TI - Expression and function of beta 1 integrins on adherent and nonadherent Ehrlich ascites tumor cells. AB - Differences in integrin-mediated cell-matrix adhesion between two types of Ehrlich ascites tumor (EAT) cells, adherent and nonadherent EAT cells, have been studied. The adherent EAT (a-EAT) cells adhere to and spread on laminin- or fibronectin-coated plates, whereas the nonadherent EAT (na-EAT) cells do not. The adhesion of a-EAT cells to laminin or fibronectin requires the presence of both Ca2+ and Mg2+. Anti-human fibronectin receptor (alpha 5 beta 1 integrin) antiserum blocks a-EAT cells from adhering to both laminin- and fibronectin coated plates. An RGD-containing peptide (GRGDSP) inhibits the adhesion of a-EAT cells to fibronectin-coated but not to laminin-coated plates. Taken together, these data suggest that a-EAT cells interact with laminin and fibronectin via different beta 1 integrins. As determined by immunofluorescence flow cytometry assay, both a-EAT and na-EAT cells express similar amounts of surface antigen(s) recognized by the same anti-alpha 5 beta 1 integrin polyclonal antibody that blocks a-EAT cells from adhering to laminin and fibronectin. The alpha 5 and beta 1 subunits expressed on both types of EAT cells appear to have the same molecular weight when analyzed by immunoblotting. The same amount of 125I-labeled membrane protein was isolated from both types of EAT cells on immobilized laminin and fibronectin affinity columns. These data indicate that na-EAT cells express similar amounts of cell surface integrins, and these integrins, when isolated, react with laminin and fibronectin in the same manner as the integrins of a-EAT cells, even though na-EAT cells are unable to adhere to laminin and fibronectin. Previous work has shown that both types of EAT cells express Neu5Ac alpha 2,3 Gal beta 1, 4 GlcNAc residues on their cell surface and that they both react with Maackia amurensis lectin which specifically recognizes this carbohydrate structure. Here we show that both types of EAT cells attach to M. amurensis lectin-coated surfaces, but only a-EAT cells spread; this suggests that the differences between these two types of cells in responding to extra cellular matrix molecules may reside in some differences within the cell. PMID- 7537701 TI - Endotoxin triggers the expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in the rat aorta in vivo. AB - The free radicals nitric oxide (.NO) and superoxide (O2-) are known to react to form peroxynitrite (ONOO-), a highly reactive species. Peroxynitrite has been suggested to play an important role in the cellular damage associated with the overproduction of .NO, but there are very limited data regarding its in vivo formation. Here we demonstrate that injection of endotoxin into rats leads to the expression of an inducible isoform of .NO synthase (iNOS) in the thoracic aorta at 6 h and an increase in the circulating levels of nitrite/nitrate. Moreover, at the same time point, there is a marked increase in the immunoreactivity of nitrotyrosine, a marker of peroxynitrite in the aorta. The formation of nitrotyrosine was prevented by inhibiting the activity of NOS by NG-methyl-L arginine in vivo. Our data suggest that during endotoxin shock, part of .NO, produced following the induction of iNOS, is converted into peroxynitrite in the vicinity of large blood vessels. The demonstration of the in vivo formation of peroxynitrite at sites of .NO overproduction may necessitate the development of novel and additional approaches for limiting or preventing .NO-related cytotoxic or vasodilatory actions during circulatory shock. PMID- 7537702 TI - A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library. AB - From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885-890 of polyprotein or residues 111-116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide. PMID- 7537703 TI - The Salmonella ompC gene: structure and use as a carrier for heterologous sequences. AB - The Salmonella typhi (St) ompC gene codes for a major outer membrane protein (OMP) that is highly expressed in both low and high osmolarity. By hybridization studies with the entire gene or with segments thereof, ompC was found to be highly conserved within 11 different Salmonella serotypes, with the exception of S. arizonae. The study included several St isolates from Mexico and Indonesia. Variation was only detected in two (e and f) of the seven regions previously found to vary between St and E. coli ompC. Chimeric OmpC proteins, carrying a rotavirus VP4 capsid protein epitope, are well recognized by a specific monoclonal antibody (mAb) against this epitope, either in OMP preparations (by enzyme-linked immunosorbent assay; ELISA) or intact cells (by ELISA and immunogold-labelling), indicating that regions c and f are oriented towards the cell surface and are probably exposed. As has been shown before for other regulated OMP, this experimental approach could be useful for the presentation of heterologous epitopes in order to gain knowledge about porin topology, for testing the effect of altered porin surface epitopes on bacterial physiology, or else, in the development of multivalent vaccines. PMID- 7537704 TI - AUC is used as a start codon in Escherichia coli. AB - Overexpression of the polymerase II transcription factor E subunit alpha mRNA (TFIIE alpha) in Escherichia coli with the pET3a expression vector [Studier et al., Methods Enzymol. 185 (1990) 60-89] results in the production of a 50-kDa N truncated polypeptide in addition to the 57-kDa wild-type polypeptide. Microsequencing analysis of the 50-kDa truncated protein and verification of the TFIIE alpha mRNA, showed that a Shine-Dalgarno-like sequence present 200 nucleotides downstream from the normal start codon could direct the use of an internal AUC codon. This codon should thus be added to the list of the non-AUG start codons. PMID- 7537705 TI - Complete sequence of the gene encoding a chorionic gonadotropin-like protein from Xanthomonas maltophilia. AB - Our laboratory has previously reported that: (i) Xanthomonas maltophilia (Xm) produces a protein which has immunological resemblance to the beta-subunit of human chorionic gonadotropin (hCG) and (ii) possesses a high-affinity receptor which binds holo hCG, and the endogenous ligand, Xm chorionic gonadotropin (xCG), but does not bind human luteinizing hormone (hLH). We have also previously published a 492-bp partial nucleotide sequence of the gene (xcg) coding for xCG. We report herein the entire xcg sequence of 1362 bp, which codes for a 48-kDa protein. This sequence confirmed the 492-bp sequence, as well as two partial amino acid (aa) sequences which we have previously reported. The sequence has a region which is homologous to aa 56-139 of the beta-subunit of hCG, and a second region homologous to the C-terminal tail of hCG. This is the first report of a prokaryotic gene homologous to the hCG beta-subunit-encoding gene. PMID- 7537706 TI - Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila. AB - Some amino acid (aa) residues within the cytolytic enterotoxin (Act) of Aeromonas hydrophila essential for biological activity were identified. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and enterotoxic activities. By deletion analysis, generation of anti-peptide Ab, and site-directed mutagenesis we showed that two regions in Act (aa 245-274 and 361-405) were very important for biological functions. As shown by competitive inhibition assays, peptide 2 (aa 245-274) blocked cytotoxic activity of Act, and aa Tyr256, Trp270 and Gly274 were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp394 and Trp396 were important for biological activities. Mutations in other regions of the toxin (e.g., Gly169, Asp170, Gly171, Trp172, Asn177,178, Asp179 and His144,209,355) also decreased biological activity. The reactivity of these mutant toxins with Ab in immunoblots was not altered. Data reported in this study suggested the role of some aa residues in biological function(s) of Act. PMID- 7537708 TI - A three year follow up of self expanding metal stents in the endoscopic palliation of longterm survivors with malignant biliary obstruction. AB - Effective palliation of malignant biliary obstruction with conventional 10 or 12 French gauge straight polyethylene endoprostheses is limited by stent occlusion, which typically occurs four to five months after insertion. Short term follow up studies of self expanding metal stents (Wallstent, Schneider, UK) in the treatment of patients with malignant biliary obstruction have shown that their use is associated with fewer episodes of stent occlusion compared with plastic stents. There are few data, however, on the longterm patency and durability of metal stents in malignant disease. Between May 1989 and May 1992, metal stents were inserted in 28 patients with malignant bile duct strictures secondary to ampullary tumour (n = 10), pancreatic carcinoma (n = 10), cholangiocarcinoma (n = 7), and porta hepatis nodes from colorectal carcinoma (n = 1). The follow up of these patients until May 1993 is reported with a median follow up of 14.6 months. Twenty two of 28 (78.6%) patients remained free of jaundice or cholangitis. The median period of stent patency was 8.2 months (range 1.0-32.5). Thirteen patients represented with jaundice or cholangitis and endoscopic retrograde cholangiopancreatography showed evidence of stent occlusion due to tumour ingrowth. Successful clearance of metal stents was achieved by balloon trawling, or insertion of a polyethylene stent. In conclusion, metal stents provide improved longterm palliation for patients with malignant biliary strictures with fewer episodes of occlusion compared with conventional stents. PMID- 7537709 TI - Aetiology of late fetal death in Maputo. AB - BACKGROUND: To study the etiology of late fetal death in Maputo. PATIENTS: Stillbirths (n = 163) in Maputo were compared to 207 live births. RESULTS: A probable cause of fetal death could be determined in 112 cases (69%). Hypertension was the most common factor associated with stillbirth and occurred in 14% of the fetal deaths, followed by abruption of the placenta (13%), syphilis (8%), clinical intra-uterine infection (6%), malaria (4%), fetal malformation (4%), umbilical cord complications (4%) and anaemia (4%). Mothers of stillborns and referent mothers differed in the following parameters: fetal weight, gestational age, numbers of previous stillbirths, haemoglobin, packed cell volume (p < 0.001), age, number of pregnancies (p < 0.01), parity, number of live children and skinfold thickness (p < 0.05). In the stillborn cases where no probable cause of fetal death was established, newborn weight was significantly lower and estimated gestational age significantly shorter (p < 0.001), number of previous pregnancies and number of previous stillbirths were significantly larger (p < 0.05) than in the referent group, but otherwise no significant differences were found. In two randomly selected sub-groups, 28 mothers with stillbirth without probable diagnoses, and in 24 referent mothers, selected serum proteins and acute-phase reactants were determined. There were no statistically significant differences in the mean values of haptoglobin, pre-albumin, retinol binding protein and alpha 1-antitrypsin in the two subgroups. The stillbirth subgroup had significantly higher mean values of C-reactive protein and serum orosomucoid than the referent group (p < 0.01). CONCLUSION: Signs of maternal illness were present in one third of the mothers with stillbirth, and half of them required medical care. In one third there was no final diagnosis, but these mothers showed significantly higher mean values of acute phase reactants, suggesting low-grade infection. PMID- 7537707 TI - Adhesion molecule expression in primary sclerosing cholangitis and primary biliary cirrhosis. AB - There are conflicting reports regarding intercellular adhesion molecule-1 (ICAM 1) expression in primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Expression of adhesion molecules ICAM-1, lymphocyte adhesion molecule-1 (LFA-1), vascular cell adhesion molecule (VCAM), and E-selectin was examined together with HLA-DR in 16 liver biopsy specimens showing PSC and 12 specimens showing PBC. These were compared with biopsy specimens showing large duct obstruction (n = 7), chronic active hepatitis (n = 4), alcoholic liver disease (n = 4), and normal liver histological results (n = 5). ICAM-1 was detected on biliary epithelium in five of seven PSC specimens of histological stage 3 or 4, but not in nine early PSC specimens or in specimens from disease controls. In PBC, ICAM-1 was positive on three of 12 cases, two stage 2, and one stage 3. Nine of 16 PSC specimens (three of nine early, six of seven late disease) and six of 10 PBC specimens (three early, three late disease) were positive for HLA-DR. LFA-1 stained infiltrating inflammatory cells in PSC, PBC, and disease controls. In conclusion, ICAM-1 expression on biliary epithelium in PSC occurs mainly in late stage disease and therefore may be secondary to previous events inducing inflammation rather than of primary pathogenic importance. ICAM-1 expression in PBC is less common and not clearly associated with a particular disorder. Previous reports of ICAM-1 prevalence may have been biased towards end stage, pre-transplantation biopsy specimens. PMID- 7537710 TI - Cytogenetic toxicity and no-effect limit dose of pesticides. AB - The no-effect limit dose (NELD) of three commonly used pesticides with respect to their cytogenetic toxicity was determined in a number of test systems using a sufficient number of lower doses to characterize the dose-effect relationship. For lindane, malathion and metacid, this dose was 3.2, 7.0 and 3.0 mg/litre, respectively, for mitosis inhibition and 9.0, 55 and 60 mg/litre, respectively, for chromosome clastogeny in onion root-tip cells. For chromosome clastogeny in mice bone marrow cells, the NELDs of the three pesticides were 1.6, 1.5 and 2.0 mg/kg body weight/day, respectively. These values for dominant lethals and X chromosome-linked recessive lethals in Drosophila were 20 and 5 micrograms lindane/litre, 2 and 3.5 micrograms malathion/litre and 4 and 5.5 micrograms metacid/litre, respectively. Thus, the NELDs are not only pesticide specific but also organism specific, tissue specific and even damage specific. Furthermore, the NELD values determined are so small that the real human exposure to pesticides cannot be reduced below these levels without compromising the effectiveness of pesticides in use. PMID- 7537711 TI - Expression of CD44 in human hepatocellular carcinoma cell lines. AB - CD44 is a glycosylated cell surface adhesion molecule expressed on a diverse range of cells and has several variant forms, some of which are involved in metastasis of cancer cells. Because little is known about CD44 in human hepatocellular carcinoma (HCC), we investigated its expression in tissue specimens from primary lesions (12 cases), in smear specimens from peritoneal effusions (2 cases), and in cell lines (HCC cell lines, KIM-1, KYN-1, KYN-2, KYN 3, HAK-1A, and HAK-1B; combined hepatocholangiocarcinoma cell lines, KMCH-1 and KMCH-2; and bile duct carcinoma cell lines, KMC-1 and KMBC). Immunohistochemical studies using monoclonal antibody recognizing epitope Group 1 of human CD44 molecule showed that HCC cells in all tissue specimens, including the original tumors of one smear specimen and HAK-1A, were negative for CD44; whereas, HCC cells in two-smear specimens, KIM-1, KYN-2, KYN-3, HAK-1A, HAK-1B, KMCH-1, KMC-1, and KMBC, showed positive reactions on the cell membrane. Immunostain-positive cell lines showed a positive cell rate of 51.9% to 99.8% by flow cytometric analysis. Western blotting detected CD44 protein of hemopoietic type in KIM-1, KYN-3, HAK-1A, and HAK-B and epithelial type in KMC-1 and KMBC. Southern blotting of complementary DNA amplified after reverse transcriptase-polymerase chain reaction (RT-PCR) detected hemopoietic type and some variant forms with longer insertion in all cell lines but KMCH-2, whereas hemopoietic type and variants with minor insertion were only detectable in tissue specimens. These findings suggest that HCC cells in ascites and in culture often express CD44, but those in tissue do not at protein level. PMID- 7537712 TI - Thrombomodulin inhibits intrahepatic spread in human hepatocellular carcinoma. AB - Thrombomodulin (TM) converts thrombin from procoagulant into anticoagulant protein to activate protein C. Thrombin also plays an important role in the metastatic process of cancer cells. We performed an immunohistochemical and clinicopathological study of TM in 141 cases with resected hepatocellular carcinoma (HCC) measuring less than 6 cm in diameter. Twenty-five specimens (17.73%) stained positive for TM. TM was found in the cytoplasm and surface of cancer cells. The clinicopathological findings according to the positive of TM are examined in HCC. The preoperative plasma TM level of the patients with tissue that stained positive for TM was significantly higher than that of the patients with negative results; for the postoperative TM level, there were no differences between them. In addition, the frequencies of intrahepatic metastasis, tumor thrombus in the portal vein, and capsular infiltration were significantly lower in patients whose tissue stained positive for TM than in patients whose tissue stained negative for TM. The recurrence freedom rate was significantly higher in patients whose tissue stained positive for TM than patients whose tissue stained negative for TM. Thus, TM-producing HCC shows a slow intrahepatic spread. Therefore, these findings suggest that TM may inhibit the adhesion of tumor cells to the portal vein because of anticoagulant activity and thus prevent the spread of intrahepatic metastasis. PMID- 7537713 TI - Histidinemia in mice: a metabolic defect treated using a novel approach to hepatocellular transplantation. AB - Histidinemia in mice and in humans is an autosomal recessive disorder of histidine metabolism that leads to high-histidine levels in both plasma and urine and is caused by a lack of hepatic histidine-alpha-deaminase (histidase). We have used a novel approach to hepatocellular transplantation to effect a complete phenotypic cure of histidinemia in a mouse model. Mice lacking histidase were treated with isolated liver cells (approximately 18 x 10(6) hepatocytes and 9 x 10(6) nonparenchymal cells) from histidase-competent donors transplanted into the peritoneum (active transplant group). Recipient mice showed a dramatic decrease, by more than 75%, in urinary histidine levels from day one throughout the course of the experiment, resulting in levels within the normal range for wild-type mice. In comparison, there was no change in urinary histidine levels in the control group of histidase-deficient mice treated with isolated liver cells from mice lacking histidase (statistical comparison between the two groups, P < .003, two-way ANOVA). Histologically, ectopic liver tissue was seen in the peritoneum in association with abdominal wall, pancreas, and peritoneal connective tissue; immunohistochemical evidence showed expression of histidase in the ectopic liver tissue in the active transplant group. This report is the first to show complete correction of a defective biochemical phenotype achieved by hepatocellular transplantation. PMID- 7537714 TI - Role of cytoskeleton and acidification of endocytic compartment in asialoglycoprotein metabolism in isolated rat hepatocyte couplets. AB - The process of receptor-mediated endocytosis is common to a variety of species and cell types. One of the best characterized receptor-ligand systems is the hepatocyte receptor for asialoglycoproteins. We investigated the morphological features of the uptake and intracellular transport of gold-conjugated asialofetuin in isolated rat hepatocyte couplets. We assessed the effects of colchicine, lumicolchicine, cytochalasin B, and chloroquine on the uptake and intracellular transport of asialoglycoproteins. Isolated rat hepatocyte couplets were incubated with gold-conjugated asialofetuin, and transmission electron micrographs of these cells were analyzed to determine the density and distribution of gold particles in the peripheral and pericanalicular areas. Results were analyzed morphometrically. Colchicine significantly inhibited the uptake and intracellular transport of asialoglycoproteins, but did not affect membrane fusion of endocytic compartments in the peripheral area. Lumicolchicine and cytochalasin B had minimal effects on these processes. Chloroquine inhibited the uptake of asialoglycoproteins, but did not affect the intracellular transport of asialoglycoproteins. Results suggest that the microtubule is essential for intracellular movement of endocytosed asialoglycoproteins and receptor recycling, and that endocytic structures in the peripheral regions can fuse in the absence of intact microtubules. We also found that uptake and intracellular transport of asialoglycoproteins were independent of the microfilaments, and the pH gradient in endocytic compartments was important in receptor-mediated endocytosis of asialoglycoproteins. PMID- 7537716 TI - Keratin expression in cutaneous lichen planus. AB - The characteristic expression of keratins by keratinocytes is well documented. A typical 'hyperproliferative' profile of epidermal keratin expression occurs in psoriasis, wound healing and warts. This study analyses keratin expression in cutaneous lichen planus to determine abnormalities of differentiation occurring in this inflammatory disorder. Using a panel of monoclonal antibodies 28 samples (20 patients) were studied. The results showed that squamous differentiation was unaffected, with keratins K1 and K10 being expressed normally for the site sampled. The main abnormalities included extension of reactivity of the basal cell marker, LH8, into the suprabasal compartment. Keratin K17, usually restricted to adnexal structures, was variably expressed in the basal and suprabasal layers of the interfollicular epithelium of affected epidermis. Keratins K6 and K16, found suprabasally in hyperproliferative states, were detected both basally and suprabasally in all diseased samples. The keratin profile in lichen planus is analogous to the wound healing response. Suprabasal keratin K17 is found in psoriasis, wound healing and viral warts so the changes in keratin K17 may reflect hyperproliferative changes. It is likely that the changes in epidermal keratin expression are due to up-regulation of specific keratin genes by the production of cytokines and inflammatory mediators from the lymphocytic infiltrate typical of lichen planus. PMID- 7537715 TI - Distribution of extracellular matrix components and their receptors in human lymphoid tissue and B-cell non-Hodgkin lymphomas. AB - In this study the distribution patterns of various extracellular matrix components and their receptors (i.e. beta 1 integrins) in B-cell non-Hodgkin lymphomas were examined and compared to those in reactive lymphoid tissue. Neoplastic follicles within follicular lymphomas showed similar patterns to that observed in reactive follicles, which appeared to be strongly associated with the presence of follicular dendritic cells. Diffuse lymphomas of low and intermediate malignancy grade revealed features comparable to those of interfollicular areas of reactive lymphoid tissue, irrespective to which compartment the tumour cells were related. Highly malignant lymphomas, however, displayed unique extracellular matrix configurations, resulting from active matrix degradation by macrophages; this may support rapid tumour growth. Extranodal lymphomas showed virtually the same matrix patterns as their nodal counterparts, suggesting that (malignant) lymphoid cells generate (at least partly) their own specific microenvironment. In reactive lymphoid tissue beta 1 integrins were mainly found on resident cells and except for alpha 4, alpha 5 (and beta 1) the lymphoid cells expressed very little, if any, beta 1 integrins. In comparison, expression of these integrins on lymphoma cells was reduced (follicular lymphomas) or could not be detected at all (diffusely growing lymphomas); this might contribute to the growth pattern and metastatic properties of the tumours. PMID- 7537717 TI - Angiogenesis in invasive breast carcinoma: is it associated with parameters of prognostic significance? AB - Recent experimental and clinical studies suggest that tumour-induced angiogenesis may be an important step in the evolution of malignant tumours, and may be related to prognosis. In our study we examined 42 cases of breast carcinoma (mean age: 56.76 +/- 13.5), 21 with lymph node metastases and 21 without. Angiogenesis was evaluated after immunohistochemical staining of tumour vessels, using polyclonal antibody to factor VIII related antigen (VIIIR-Ag) and counting of the three most active areas of neovascularization. In the same manner we counted the microvessels in lymph node metastases. The mean vessel count of node-negative cases (51.16 +/- 19.32) did not differ significantly from node-positive cases (45.66 +/- 17.44). In contrast patients younger than 50 years had much higher mean vessel counts (54.04 +/- 16.47) than did patients older than 70 years (38.03 +/- 16.73) producing a P value of < or = 0.05. No association was found between tumour size and mean vessel count, nor was there any significant difference between grade I (45.94 +/- 16.54), grade II (53.13 +/- 23.22) and grade III tumours (51.71 +/- 20.64). When we compared the mean vessel count of primary tumours with those of node metastases, we found much lower counts in the latter (P < or = 0.01). The differences in our results from previous studies, probably reflect the heterogeneity which exists between different tumours in their ability to induce angiogenesis. Additionally, there is some evidence in our study that angiogenesis is possibly related to patient age and probably depends on differences in the tumour stroma. PMID- 7537718 TI - Alphafetoprotein-producing gastric adenocarcinoma. AB - We report a rare gastric tumour characterized morphologically by its hepatoid features and alpha-fetoprotein production and which presented clinically with gastric haemorrhage. Gastric fibroscopy showed a bleeding tumour of the antrum. The microscopic appearance of the tumor showed two different patterns. The most extensive presented hepatoid features. The second pattern showed undifferentiated features. The tumour cells showed immunohistochemical positivity for alphafetoprotein, EMA and p53 protein; 37% were aneuploid with a DNA index of 1.46. The serum level of alphafetoprotein was not measured before the gastrectomy but after ten days it was elevated at 1070 ng/ml. The patient died 6 months after the admission. This case provides, for the first time, information on the DNA content and the p53 expression of this unusual and aggressive variant of gastric adenocarcinoma. PMID- 7537719 TI - Allele-specific monoclonal antibodies against glutathione S-transferase Mu1-1. AB - IgG1 class mouse monoclonal antibodies (MAbs) were produced against human glutathione S-transferase Mu1-1 (GSTMu1-1). Eight MAbs of 16 are able to recognize only the native form of the enzyme; 4 MAbs bind to native and denaturated enzyme, and the remaining 4 can bind only to partially denatured antigen in direct ELISA or Western blot. The antibodies recognizing the native form of the enzyme bind to six different epitopes. Three overlapping epitopes are responsible for specific binding of MAbs to different allelic variants of GSTMu1 1. Three allele-specific antibodies, 2E1, 11F12, and 7D11, bind to GSTM1a monomer and the other two, 1H8 and 3H10, recognize GSTM1b monomer. PMID- 7537720 TI - Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies. AB - Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase dependent lipase activity. The dissociation constant of the inactive antibody antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid). PMID- 7537721 TI - Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens. AB - To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537722 TI - Evaluation of a cold staining method for acid-fast bacilli in sputum. AB - Comparison between the Ziehl-Neelsen staining method for acid-fast bacilli, applied with and without heating, was carried out in a controlled investigation using smears prepared from 306 sputum samples collected prior to treatment from suspected cases of pulmonary tuberculosis. Smear and culture positivity were graded and the colour intensity of bacilli recorded. Results showed that the chance corrected agreement (Kappa) between Z-N and cold methods was only 78%. The sensitivity of the Z-N and cold methods were 84% and 77% respectively when compared with culture results. Assuming 10% smear positivity among symptomatics reporting to Peripheral Health Institutions (PHIs), the positive predictive value of the cold method was very low (53%). When compared to culture, the positive predictive value is 71% for the Z-N method and 57% for the cold method for a symptomatic population with 15% culture positivity. In the absence of heating, penetration of the stain was significantly reduced and consequently the number of bacilli detected was less. The inability to take the stain without heating was seen in smears from all grades of culture positive samples; thus even heavy positives were missed by the cold method. The evaluation of the cold method against the standard Z-N method highlights its limitations and demonstrates that it is not as reliable as the standard Z-N method. PMID- 7537724 TI - Angiogenesis: models and modulators. AB - Angiogenesis in vivo is distinguished by four stages: subsequent to the transduction of signals to differentiate, stage 1 is defined as an altered proteolytic balance of the cell allowing it to digest through the surrounding matrix. These committed cells then proliferate (stage 2), and migrate (stage 3) to form aligned cords of cells. The final stage is the development of vessel patency (stage 4), generated by a coalescing of intracellular vacuoles. Subsequently, these structures anastamose and the initial flow of blood through the new vessel completes the process. We present and discuss how the available models most closely represent phases of in vivo angiogenesis. The enhancement of angiogenesis by hyaluronic acid fragments, transforming growth factor beta, tumor necrosis factor alpha, angiogenin, okadaic acid, fibroblast growth factor, interleukin 8, vascular endothelial growth factor, haptoglobin, and gangliosides, and the inhibition of the process by hyaluronic acid, estrogen metabolites, genestein, heparin, cyclosporin A, placental RNase inhibitor, steroids, collagen synthesis inhibitors, thrombospondin, fumagellin, and protamine are also discussed. PMID- 7537723 TI - Structure and function of nitric oxide synthases. AB - Nitric oxide (NO), which accounts for the biological activity of endothelium derived relaxing factor, is now thought to play a variety of roles in the nervous system and in immunologic reactions. NO is synthesized from L-arginine by nitric oxide synthase (NOS). There are three isoforms of NOS; type I (neuronal), type II (inducible), and type III (endothelial). The fundamental structure of the three isoforms, which contain calmodulin-, FMN-, FAD-, and NADPH-binding domains, is the same. Calmodulin is already bound to inducible NOS without requiring Ca2+, while the others are Ca2+/calmodulin-dependent. Endothelial NOS is bound to membranes by N-myristoylation, while the other isoforms are soluble. The human endothelial NOS gene has been cloned. It has several highly repetitive regions which could provide potential sites for DNA polymorphism. It might be of interest to examine the relationship between such polymorphism and cardiovascular disorders. PMID- 7537725 TI - Referred ocular pain relieved by suboccipital injection. AB - Pain originating from ophthalmic disease has been well documented. A series of patients presenting with eye or periorbital pain attributed to cervical region dysfunction were diagnosed and treated with injections of subcutaneous lidocaine followed by triamcinolone acetonide. Twelve patients, 11 women and 1 man, ranging in age from 20 to 82 years had an evaluation including a complete eye examination, and laboratory tests and neuroimaging as dictated by the history to exclude structural abnormalities or systemic disease. All patients had marked focal suboccipital tenderness ipsilateral to the side of their headache and eye pain. A subcutaneous injection with 2% lidocaine followed by triamcinolone acetonide 40 mg was administered directly to the site of focal tenderness. After injection, five patients described total relief of pain, five patients described some degree of pain relief, and two patients had no relief of headache. Duration of pain relief ranged from several hours to 3 months. Patients may present with periorbital or eye pain as a result of disease affecting the cervical sensory roots with subsequent stimulation of the trigeminal apparatus. Subcutaneous injection of lidocaine and triamcinolone acetonide may be of help in the diagnosis of these patients and provide temporary relief. PMID- 7537727 TI - Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection of the killer cells with the gamma-interferon gene. AB - Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human gamma-interferon (HuIFN-gamma) gene by means of liposomes having a positive charge on their surface. The cells secreted significant amounts of HuIFN-gamma (reaching more than 5 U/ml) into the culture medium. The HuIFN-gamma produced by the cells induced intercellular adhesion molecule-1 (ICAM-1) and enhanced the expression of Fas antigen on the surface of human glioma cells. Also, LAK cells transfected with HuIFN-gamma gene exhibited reinforcement of cytotoxicity toward human glioma cell lines (U251-MG and SK-MG-1). Furthermore, the reinforcement was significantly quenched by anti-ICAM-1 and/or anti-TNF-alpha monoclonal antibody. PMID- 7537726 TI - In vitro induction of cytotoxic T lymphocytes against HTLV-I-infected T-cells from adult T-cell leukemia patients, asymptomatic HTLV-I carriers and seronegative healthy donors. AB - We investigated an in vitro method to produce cytotoxic T lymphocytes (CTLs) against HTLV-I-infected T-cells using peripheral blood mononuclear cells (PBMC) of adult T-cell leukemia (ATL) patients, asymptomatic HTLV-I carriers (AC) and seronegative healthy donors. The PBMC were restimulated repeatedly for 4 weeks with HLA-matched HTLV-I-infected T-cells which had been pretreated at 56 degrees C for 30 min to inactivate infectious HTLV-I. The culture medium included 10-100 units/ml of recombinant lymphokines (rIL-1, rIL-2, rIL-4, rIL-6 and rIL-7) and 10% fetal calf serum in RPMI-1640 medium. The cytotoxic activity was measured against HLA-matched HTLV-I-infected T-cell lines after CD4+ or CD8+ cells were positively panned from the cultured PBMC. The PBMC of ATL, AC and healthy donors were able to produce either CD4+ or CD8+ CTLs against HTLV-I-related antigens (env, gag, p21x, p27rex and p40tax) as well as the antigen(s) of as-yet unknown specificity expressed on HTLV-I-infected T-cells. All the CTLs recognized the specific antigens in the context of either class I or class II HLA types. These results indicated that ATL patients, AC and healthy donors were immunocompetent to generate CTLs against HTLV-I-infected T-cells and probably against HTLV-I transformed T-cells. PMID- 7537731 TI - Lipopolysaccharide binding protein-mediated complexation of lipopolysaccharide with soluble CD14. AB - Endotoxin (lipopolysaccharide; LPS) activates a wide variety of host defense mechanisms. In mammals LPS binding protein (LBP) and CD14 interact with LPS to mediate cellular activation. Using sucrose density gradients and a fluorescent endotoxin derivative we have investigated the mechanism of LPS binding to LBP and the soluble form of CD14 (sCD14). LPS binds to LBP to form two types of complex; at low ratios of LPS to LBP complexes with one molecule of LBP and 1-2 molecules of LPS predominate, while at high ratios of LPS to LBP a large aggregate of LBP and LPS predominates. Complexes of LPS with sCD14 do not form large aggregates, consisting of only 1-2 LPS bound to a single sCD14 even at high multiples of LPS to sCD14. LBP catalyzes LPS binding to sCD14. Catalysis by LBP apparently occurs because LBP provides a pathway for LPS to bind to sCD14 which avoids the necessity for LPS monomers in aqueous solution. The dissociation constants for LPS.LBP and LPS.sCD14 complexes were determined to be 3.5 x 10(-9) and 29 x 10( 9) M, respectively. These numbers suggest that when LBP and sCD14 are present at roughly equal concentrations as they are in normal human plasma and compete for limited LPS, the LPS will predominantly associate with LBP. PMID- 7537732 TI - Syk is activated by phosphotyrosine-containing peptides representing the tyrosine based activation motifs of the high affinity receptor for IgE. AB - Engagement of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on the surface of mast cells induces tyrosine phosphorylation of numerous cellular proteins. Syk, one of several non-receptor protein tyrosine kinases implicated in Fc epsilon RI signaling, is activated following receptor cross-linking and associates with phosphorylated gamma subunits of Fc epsilon RI. We previously showed that the Src homology 2 (SH2) domains of Syk bind with high affinity to the conserved tyrosine-based activation motif (TAM) of the gamma subunit in vitro. In this report, we show that a tyrosine-phosphorylated gamma TAM peptide induced tyrosine phosphorylation of Syk in RBL-2H3 cell lysates and stimulated Syk kinase activity 10-fold in vitro, with half-maximal activation at 1-2 microM. A similar beta subunit TAM peptide showed much lower stimulation of Syk tyrosine phosphorylation and kinase activity. Phosphopeptide-induced activation was inhibited by an antiserum to the carboxyl-terminal tail of Syk, suggesting that those amino acids are also involved in Syk activation. These results indicate that the catalytic domain of Syk may be regulated by intramolecular interactions with adjacent domains and suggest that Syk binding to phosphorylated gamma subunits following Fc epsilon RI engagement in vivo stimulates Syk kinase activity. PMID- 7537729 TI - Localization of F-actin and fodrin along the organ of Corti in the chinchilla. AB - The distribution of the two cytoskeletal proteins, filamentous actin (F-actin) and fodrin, was investigated along the organ of Corti of the chinchilla using laser scanning confocal fluorescence microscopy. High intensity labeling of F actin was seen in outer and inner hair cells, including the stereocilia. High intensity staining was also seen for fodrin in outer and inner hair cells, but not in their stereocilia. Staining intensity of both proteins along the lateral cell wall of the outer hair cells appeared to be greater in the middle and basal cochlear turns than in the apical turn. Pillars and Deiters cells also exhibited high intensity labeling of F-actin. The lack of significant differences in the distribution of fodrin between outer and inner hair cells makes the role of this protein in the active processes still unclear. Comparison of the distribution of F-actin and fodrin in the chinchilla with those reported in the guinea pigs suggest possible species differences. PMID- 7537730 TI - cAMP-dependent phosphorylation stimulates water permeability of aquaporin collecting duct water channel protein expressed in Xenopus oocytes. AB - Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis. PMID- 7537728 TI - Acetylcholine activates a K+ conductance permeable to Cs+ in guinea pig outer hair cells. AB - Acetylcholine (ACh), the major neurotransmitter released by efferent nerve fibers in the cochlea, has been shown to activate a Ca(2+)-dependent K+ conductance in outer hair cells (OHCs). Previously we reported that this ACh operated conductance is permeable to Cs+. The purpose of the present study was to characterize further this Cs(+)-permeable channel and its dependency on Ca2+ using isolated OHCs and the patch clamp technique in the whole cell configuration. The changes in the ACh response were examined when Cs+, Ba2+, Cd2+, N-methyl-D-glucamine (NMG+) and tetraethylammonium (TEA+) were placed in the external or internal solutions. Cs+ substituted for K+ in carrying the ACh evoked Ca(2+)-dependent K+ current. When NMG+/TEA+ was substituted for internal K+ ACh-evoked an inward and an outward current, and Cs+ substituted for external K+ blocked the outward but not the inward current evoked by ACh suggesting it was carried by K+. In the NMG+/TEA+ condition, when the cell was held at different Vh values for an extended period of time, the ACh-induced K+ current rectified. In Ba2+ (3 mM) with zero Ca2+ ACh failed to induce any detectable current and the ACh response slowly recovered from the Ba2+ block, suggesting a block at an intracellular site. Cd2+ (1 mM) readily and reversibly blocked ACh-induced currents even when carried by Cs+. This data suggests that ACh opens a channel selective for K+, conductive to Cs+ and dependent on Ca2+. PMID- 7537733 TI - Receptors involved in carbohydrate binding modulate intestinal epithelial neutrophil interactions. AB - Neutrophil (polymorphonuclear neutrophil) migration across epithelial barriers is a common morphologic feature of many diseases. Previous studies show that PMN epithelial interactions are dependent on the PMN beta 2-integrin CD11b/18; however, nothing is known about surface carbohydrates and PMN-epithelial interactions. Here we investigate the role of carbohydrates on PMN-epithelial interactions using PMN and cultured monolayers of the intestinal epithelial cell line T84. Addition of the carbohydrates mannose 6-phosphate (Man-6-P) and glucose 6-phosphate (Glu-6-P), but not fructose 1-phosphate (Fru-1-P) inhibited transmigration by > or = 70%. Likewise, more complex carbohydrates, such as fucoidin and the Man-6-P-rich polysaccharide PPME selectively inhibited PMN transepithelial migration. These carbohydrates were found to be inhibitory in the apical-to-basolateral direction as well as the basolateral-to-apical direction, indicating a lack of polarity. This panel of related carbohydrates, however, was not effective in modulating short-term adhesion of PMN to epithelial monolayers, indicating that carbohydrate ligands may modulate different steps in the transmigration cascade. Finally, addition of functionally inhibitory monoclonal antibodies specific for the selectins (CD62E, CD62L, and CD62P) revealed no observable effect on PMN transmigration. These studies suggest that cell surface carbohydrates may play a role in inflammatory processes of the intestine. PMID- 7537734 TI - The receptor-like protein-tyrosine phosphatase, RPTP alpha, is phosphorylated by protein kinase C on two serines close to the inner face of the plasma membrane. AB - To determine whether the receptor-like protein-tyrosine phosphatase, RPTP alpha, which is widely expressed in both the developing and adult mouse, is regulated by phosphorylation, we raised antiserum against a C-terminal peptide. This antiserum precipitated a 140-kDa protein from metabolically 35S-labeled NIH3T3 cells. Using this antiserum, we showed that endogenous RPTP alpha is constitutively phosphorylated in NIH3T3 cells, predominantly on two serines, which we identified as Ser-180 and Ser-204, lying in the juxtamembrane domain. 12-O tetradecanoylphorbol-13-acetate (TPA) stimulation of quiescent NIH3T3 cells rapidly increased phosphorylation of Ser-180 and Ser-204. Purified protein kinase C (PKC) phosphorylated bacterially expressed RPTP alpha at Ser-180 and Ser-204. When wild type and S180A/S204A double mutant RPTP alpha S were transiently expressed in 293 human embryonic kidney cells, TPA stimulated phosphorylation of wild type but not of double mutant RPTP alpha. PKC down-regulation following prolonged exposure to TPA diminished TPA-stimulated RPTP alpha phosphorylation. Taken together, these results indicate that RPTP alpha is a direct substrate for (PKC). Examination of 293 cells expressing exogenous RPTP alpha using immunofluorescence confocal microscopy showed that RPTP alpha exists predominantly in two subcellular compartments: in dense intracellular granules or dispersed within the plasma membrane. TPA treatment caused redistribution of some intracellular RPTP alpha to the cell surface, but this did not require direct phosphorylation of RPTP alpha at Ser-180/Ser-204. Our results suggest that activation of PKC by cytokines modulates RPTP alpha function in several different ways. PMID- 7537735 TI - Structural diversity in the 5'-untranslated region of cytokine-stimulated human inducible nitric oxide synthase mRNA. AB - Inducible nitric oxide synthase, the critical enzyme responsible for the enhanced synthesis of nitric oxide in inflammatory states, is widely expressed in mammalian cells. To evaluate potential regulatory roles of the 5'-untranslated region (UTR) in the human inducible nitric oxide synthase gene, the transcription initiation sites and structure of the 5'-UTR of human inducible nitric oxide synthase were examined. Freshly isolated human alveolar macrophages, bronchial epithelial cells, and several types of cultured cells were evaluated following stimulation with cytokines (i.e. interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6). The mRNA was analyzed by reverse transcription-polymerase chain reaction. Northern analysis, and 5'-rapid amplification of cDNA ends. Despite the presence of a TATA box in the promoter region, multiple transcription initiation sites were observed, some extending several hundred base pairs upstream from the main TATA-directed initiation site. Alternative splicing in the 5'-UTR of human inducible nitric oxide synthase mRNA resulted in further diversity. The TATA-independent inducible nitric oxide synthase mRNA transcripts were up-regulated by cytokines. The long and complex 5' UTRs contain eight partially overlapping open reading frames upstream of the putative inducible nitric oxide synthase ATG, which may have an important role in translational regulation of human inducible nitric oxide synthase mRNA. PMID- 7537736 TI - Proline-rich sequence-mediated Jak2 association to the prolactin receptor is required but not sufficient for signal transduction. AB - The prolactin receptor (PRLR) belongs to the superfamily of cytokine/growth hormone/prolactin receptors. Members of this family do not contain a tyrosine kinase domain but are associated with cytoplasmic kinases of the Jak family. Here, we examine different mutants of the PRLR with respect to their ability to associate and activate the kinase Jak2 and the transcription factor Stat1. Moreover, using a biological assay system we are able to correlate these activities with activation of prolactin-responsive gene transcription. Our results indicate that interaction between Jak2 and PRLR requires a proline-rich sequence in the membrane proximal region of the receptor, which is conserved among the different members of the cytokine receptor superfamily. We also show that association of Jak2 with the receptor is sufficient for activation of the kinase as well as the transcription factor Stat1. Moreover, our findings indicate that association of PRLR with Jak2 is necessary but not sufficient for the transmission of a lactogenic signal. We have identified two other cytoplasmic regions of the PRLR that are required for activation of transcription. These two regions are located between boxes 1 and 2 and are in the carboxyl-terminal tail of the receptor. These sites probably involve specific interactions with other effector molecules. PMID- 7537737 TI - Skeletal growth factors regulate the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures. AB - Skeletal cells secrete insulin-like growth factors (IGFs) I and II and six known IGF binding proteins (IGFBPs). IGFBP-5 stimulates bone formation, and its synthesis correlates with changes in osteoblast cell growth. We tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF beta 1), and platelet-derived growth factor (PDGF) BB on IGFBP-5 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Treatment of Ob cells with bFGF, TGF beta 1, and PDGF BB caused a time- and dose-dependent decrease in IGFBP-5 mRNA levels and inhibited IGFBP-5 polypeptide levels in the extracellular matrix. The effects of bFGF, TGF beta 1, and PDGF BB on IGFBP-5 transcripts were independent of cell division and were observed in the presence and absence of hydroxyurea. bFGF, TGF beta 1, and PDGF BB did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells, and they inhibited IGFBP-5 heterogeneous nuclear RNA and the rate of IGFBP 5 transcription. In conclusion, bFGF, TGF beta 1, and PDGF BB inhibit IGFBP-5 expression in Ob cells independently of their mitogenic activity and through mechanisms that involve decreased transcription. PMID- 7537739 TI - Selective activation of the rat hepatic endosomal insulin receptor kinase. Role for the endosome in insulin signaling. AB - Insulin administration activates the insulin receptor kinase (IRK) in both plasma membrane (PM) and endosomes (ENs) raising the possibility of transmembrane signaling occurring in the endosomal compartment. Peroxovanadium compounds activate the IRK by inhibiting IR-associated phosphotyrosine phosphatase(s). Following the administration of the phosphotyrosine phosphatase inhibitor bisperoxo(1,10-phenanthroline)-oxovanadate (V) anion (bpV(phen)) activation of the hepatic IRK in ENs preceded that in PM by 5 min. When colchicine treatment preceded bpV(phen) administration IRK activation in ENs was unaffected but was totally abrogated in PM. Insulin receptor substrate-1 tyrosine phosphorylation followed the kinetics of IRK activation in ENs not PM and a hypoglycemic response similar to that achieved with a pharmacological dose of insulin ensued. These studies demonstrate that ENs constitute a site for IR-mediated signal transduction. PMID- 7537738 TI - Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand binding interactions, lipid cofactor requirements, and inhibitor sensitivity. AB - The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (Kd = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc 13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13. PMID- 7537740 TI - The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling pathway different from the Ras signaling pathway. PMID- 7537741 TI - A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by Fc epsilon R1 is not shared by a G protein coupled receptor. AB - Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric (alpha beta gamma 2) immunoglobulin E receptor (Fc epsilon R1) leads to the activation of cytosolic phospholipase A2 and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A2 pathway is linked to Fc epsilon R1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the Fc epsilon R1 gamma motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the Fc epsilon R1 beta motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A2 pathway and demonstrate the existence of the Fc epsilon R1-Syk-MAP kinase pathway. PMID- 7537743 TI - Bone-bonding behavior of titanium alloy evaluated mechanically with detaching failure load. AB - Although titanium (Ti) and Ti alloy are generally classified as bioinert materials in terms of their bonding to bone tissue, it is still unclear whether they bond chemically to bone. In this study, we examined the bone-bonding ability of Ti alloy (Ti-6Al-4V) using smooth-surfaced plates under non-load-bearing conditions. The bone-bonding behavior was evaluated mechanically by means of the detaching test reflecting tensile force. After implantation of the plates into the tibiae of rabbits for 4, 8, 16, and 25 weeks, detaching tests were performed. The failure load of the Ti alloy plates was close to 0 kg at 4 and 8 weeks, but gradually increased with time, reaching 0.334 kg at 16 weeks and 2.852 kg at 25 weeks on average. Histologic examination by Giemsa surface staining and SEM showed no differences between specimens at 8, 16, and 25 weeks, when Ti alloy plate made direct content with bone without any fibrous tissue. By SEM-EPMA, no clear calcium-phosphorus (Ca-P)-rich layer at the interface between the Ti alloy and bone tissue was evident, although a thin bone tissue was observed on the detached Ti alloy plate. The present results indicate that from both mechanical and histologic viewpoints, Ti alloy bonds directly to bone under static conditions after some time, probably more than 8 weeks. The possibility of chemical bone-bonding of Ti alloy was suggested. PMID- 7537742 TI - The phosphotyrosine phosphatase PTP1D, but not PTP1C, is an essential mediator of fibroblast proliferation induced by tyrosine kinase and G protein-coupled receptors. AB - PTP1C and PTP1D are non-transmembrane protein-tyrosine phosphatases (PTPs), which contain two src homology-2 domains. These enzymes are believed to play a role in regulating downstream signaling from receptors with intrinsic tyrosine kinase activity. The present study describes the tyrosine phosphorylation and the catalytic activity of both PTPs in CCL39 cells, a Chinese hamster lung fibroblast cell line, upon addition of a variety of growth factors. We demonstrate that PTP1C activity was significantly stimulated by insulin and the phorbol ester 12-O tetradecanoylphorbol-13-acetate but was not influenced by serum, platelet-derived growth factor (PDGF), or alpha-thrombin. However, tyrosine phosphorylation of PTP1C was increased in response to insulin, PDGF, and alpha-thrombin. PTP1D activity was slightly stimulated by insulin and 12-O-tetradecanoylphorbol-13 acetate but was significantly inhibited by serum, PDGF, and alpha-thrombin, although tyrosine phosphorylation is increased in response to these agonists. Mitogen-activated protein kinase phosphorylated PTP1C and PTP1D in in vitro kinase assays, suggesting that both PTPs are target proteins for mitogen activated protein kinase. We also show that overexpression of PTP1C or PTP1D had no effect on DNA synthesis stimulated by different growth factors. However, a mutated inactive form of PTP1D strongly inhibited the stimulatory effects of both PDGF and alpha-thrombin on early gene transcription and DNA synthesis. These results demonstrate for the first time that PTP1C and PTP1D may participate in signal transduction but in different manners and that only PTP1D is a positive mediator of mitogenic signals induced by both tyrosine kinase receptors and G protein-coupled receptors in fibroblasts. PMID- 7537744 TI - The A/B domain of truncated retinoic acid receptors can block differentiation and promote features of malignancy. AB - Recently, we discovered that stable introduction of a carboxyl-terminally truncated retinoic acid receptor gamma (tRAR gamma) into an epidermal keratinocyte line blocked the ability of these cells to differentiate, as judged by their failure to express late markers of squamous differentiation. We now demonstrate a correlation between the level of residual endogenous RAR activity of tRAR gamma-expressing keratinocyte lines and degree of terminal differentiation. Mutagenesis studies localize the effects to the A/B subdomain of the truncated receptor. Despite tRAR gamma's capacity to interfere with RAR mediated transactivation of retinoic acid response elements (RAREs) in keratinocytes, the effects of the truncated receptor are independent of its ability to bind DNA and directly interact with endogenous RARs. tRAR alpha also inhibits RARE-mediated gene expression in keratinocytes, even though its full length counterpart enhances RARE activity in these cells. Intriguingly, both tRAR gamma and RAR gamma suppress keratin promoter activity in epidermal cells, although for tRAR gamma, the effect is mediated through the A/B domain whereas for RAR gamma, the effects require DNA binding. Taken together, these findings suggest that the truncation allows for new and aberrant interactions with transcriptional proteins/cofactors that participate in governing RARE activity. This discovery may have relevance in tumorigenesis, where genetic lesions can result in mutant RARs or in loss of receptor expression. PMID- 7537745 TI - The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges. AB - A highly reproducible and technically straightforward technique for the isolation and long-term culture of normal human endometrial epithelial cells is described. The essential conditions for long-term culture are that the cells be seeded onto a gelatin matrix and that 'endothelial cell growth supplement' be present in the culture medium. Normal endometrial epithelial cells express cytokeratins and oestrogen receptors. They may be passaged five to six times without change in properties. Growth of normal endometrial epithelial cells was stimulated by 17 beta-oestradiol and epidermal growth factor. Expression of the mRNA coding for seven polypeptide angiogenic factors, by normal endometrial epithelial, stromal and three endometrial carcinoma lines, was examined. The endometrial epithelial and stromal cells express mRNA for the polypeptide angiogenic factors, basic fibroblast growth factor, vascular endothelial cell growth factor, transforming growth factor-beta 1 and pleiotrophin, as well as the cytokine midkine. Expression of the mRNA for both vascular endothelial growth factor and midkine by normal endometrial epithelial cells showed a 2-fold increase on treatment with a physiological dose of 17-beta-oestradiol (10(-10) M) while, in contrast, the mRNA of transforming growth factor-beta 1 decreased 4-fold on treatment with 17-beta oestradiol (10(-10) M) and was abolished by exposure to progesterone (5 x 10(-9) M). Expression of the mRNAs for angiogenic polypeptides by the endometrial carcinoma lines was more restricted. PMID- 7537746 TI - Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells. AB - Angiotensin II is a potent vasoconstrictor that has been also implicated in vascular hyperproliferative diseases, including atherosclerosis and restenosis following angioplasty. Treatment of cultured, serum-starved rat aortic smooth muscle cells with angiotensin II causes rapid protein tyrosine phosphorylation that precedes cell mitogenesis. We have identified two of the phosphoproteins as paxillin (75 kilodaltons) and the tyrosine kinase pp125Fak, both components of actin-associated focal adhesion sites. Angiotensin II stimulated a 5-fold increase in the tyrosine phosphorylation of paxillin and a smaller (1.5-fold) increase in pp125Fak tyrosine phosphorylation. Paxillin tyrosine phosphorylation was evident within 1 minute, and was maximal after 10 minutes. Similar elevated protein tyrosine phosphorylation levels of paxillin were obtained with exposure of the rat aortic smooth muscle cells to peptides endothelin-1 and alpha-thrombin that function, as angiotensin II, through binding to members of the seven transmembrane domain G protein coupled receptors. Angiotensin II treatment also stimulated the production of a well-ordered actin-containing stress fiber network and prominent paxillin-containing focal adhesions. The focal adhesions stained intensely with anti-phosphotyrosine antibody suggesting the tyrosine phosphorylation of paxillin and cytoskeletal reorganization were tightly coupled. Angiotensin II receptor occupancy has been shown previously to lead to protein kinase C activation. However, compared to angiotensin II stimulation, a smaller, delayed increase in paxillin tyrosine phosphorylation was observed following direct protein kinase C activation by the phorbol ester phorbol 12-myristate-13 acetate. Paxillin tyrosine phosphorylation was selective for certain agonists since no increase in tyrosine phosphorylation of this protein was observed following exposure to the potent mitogen PDGF. Thus, actin-based cytoskeletal changes involving sites of cell adhesion to the extracellular matrix may play an important role in normal and pathophysiologic smooth muscle cell growth regulation in response to certain angiotensin II-type vasoactive agonists. PMID- 7537749 TI - Arsenic speciation using capillary zone electrophoresis with indirect ultraviolet detection. AB - The toxicological or biological importance of many trace elements in biological and environmental samples depends to a great extent on their quantities as well as their oxidation states and chemical forms. In this work, arsenic species (arsenate, arsenite, dimethylarsinate [DMA], and monomethylarsonate [MMA]) are determined using capillary zone electrophoresis (CZE) with indirect ultraviolet absorption detection. Sodium chromate is used as an electrolyte solution and to provide the chromophore. The effect of the pH of the solution on the separation of species of interest is discussed. The absolute detection limits are 5.2, 3.5, 15.6, and 15.6 pg for arsenate, arsenite, monomethylarsonate, and dimethylarsinate, respectively. A solution extracted from coal fly ash is analyzed using this method. The arsenate concentration is determined to be 5 micrograms/g. Arsenite, monomethylarsonate, and dimethylarsinate are not found. The advantages of using CZE as an efficient and sensitive separation method are discussed. PMID- 7537748 TI - Leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro. AB - Using an in vitro model in which endothelial cells can be induced to invade a three-dimensional collagen gel to form capillary-like tubular structures, we demonstrate that leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro. The inhibitory effect was observed on both bovine aortic endothelial (BAE) and bovine microvascular endothelial (BME) cell, and occurred irrespective of the angiogenic stimulus, which included basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), the synergistic effect of the two in combination, or the tumor promoter phorbol myristate acetate. LIF inhibited bFGF- and VEGF-induced proliferation in BAE and BME cells. In addition, LIF inhibited BAE but not BME cell migration in a conventional two-dimensional assay. Finally, LIF decreased the proteolytic activity of BAE and BME cells and increased their expression of plasminogen activator inhibitor-1. These results demonstrate that LIF inhibits angiogenesis in vitro, an effect that can be correlated with a LIF mediated decrease in endothelial cell proliferation, migration and extracellular proteolysis. PMID- 7537747 TI - Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p dioxin. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity is particularly striking in adipose tissue, where it causes severe wasting. This phenomenon suggests that TCDD could have effects on adipocyte differentiation, now demonstrated using 3T3-L1 cells as a model system. When cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone (dex) and isobutylmethylxanthine (IBMX), a reduction occurred in the number of fat cell colonies measured 7-10 days later by Oil Red O staining. Northern blotting showed an accompanying reduction in amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on the maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by interaction of TCDD with the Ah receptor. The possibility that TCDD acts by inhibiting the signaling pathways activated by dex and IBMX was investigated. TCDD did not interfere with glucocorticoid-inducible transcription as judged by the unimpaired responsiveness of a transfected reporter construct. Treatment of cells with TCDD augmented the increase in protein kinase A (PKA) activity elicited by either IBMX or forskolin; therefore, if TCDD disrupts the cAMP signaling pathway, it must do so at a step after activation of PKA. PMID- 7537751 TI - Reactivity of five anti-HIV-1 subtype O specimens with six different anti-HIV screening ELISAs and three immunoblots. AB - Five anti-subtype O specimens were tested by anti-HIV-1/2 screening and confirmatory assays. They can be divided into three specimens, reactive with all ELISAs, independent of the nature of the antigen (recombinant proteins or peptides) and test configuration (indirect ELISA or double antigen/sandwich ELISA). One specimen was not detected by one peptide based ELISA. One specimen was only recognized by two ELISAs and should be considered as a marker sample for the weakness of currently used ELISAs with anti-subtype O. Three different immunoblot assays available commercially detected two of the specimens with a major binding of gp160 and other viral bands, especially the integrase and reverse transcriptase. Another two specimens lacked reactivity with glycoproteins almost completely, but showed some staining with the enzymes of HIV, and would most probably be interpreted as indeterminate. The fifth specimen, which was also missed by most of the ELISAs, had very faint staining of the gp160 and a very weak staining of p24, and would most probably be interpreted as negative. Adaption of currently available tests to anti-subtype O is needed for the future reliability of anti-HIV diagnostic reagents. PMID- 7537752 TI - P-selectin chases a butterfly. PMID- 7537750 TI - A simple procedure to generate chimeric Pr55gag virus-like particles expressing the principal neutralization domain of human immunodeficiency virus type 1. AB - The Pr55gag human immunodeficiency virus type 1 (HIV-1) precursor protein that is capable of auto-assembling was used as a carrier for a consensus sequence of the principal neutralization domain (PND) of the HIV-1 envelope. For this purpose, a modified HIV-1 gag gene with deletion of the sequence encoding a previously described p24 epitope (amino acids 196-228 of Pr55gag) was first obtained using PCR with degenerate primers, and then cloned. This deleted gag gene allowed in a second time the insertion of a synthetic oligonucleotide cassette encoding the North American/European consensus PND precisely in place of the p24 epitope. The chimeric gene was then inserted into a baculovirus transfer vector and expressed in insect cells. The construct formed 100-140 nm virus-like particles that were released into the extracellular medium. The use of a serum-free medium that supports growth of insect cells facilitated the downstream purification of the extracellular particles. The chimeric particles were recognized by monoclonal antibodies directed to V3 by Western blot but not by immune electron microscopy, suggesting that, although the inserted sequence was still antigenic it was not exposed at the surface of the particles. The results show the ability of Pr55gag to serve as a carrier for easy insertion, in a precisely defined region, of selected epitopes of gp120 surface envelope protein, and to still auto-assemble in virus-like particles. However, the data indicate that exposed epitopes of the mature p24 protein are not presented similarly in the Pr55 precursor, and therefore that different constructs with various insertions in different places must be generated. Such constructs offer an attractive approach for HIV vaccine development and will need evaluation for both antigenicity and immunogenicity. PMID- 7537754 TI - Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells. AB - An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na dependent solute transport. PMID- 7537753 TI - The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent. AB - Oxidized LDL has been previously reported to suppress the expression of genes induced in mononuclear phagocytes by inflammatory stimuli. In this study we extend these findings to demonstrate that the suppressive effects of oxidized LDL vary depending upon the gene being monitored and the stimulus being used to induce or enhance its expression. The expression of a selection of LPS-inducible genes exhibited differential sensitivity to pretreatment with oxidized LDL. Furthermore, the ability of oxidized LDL to suppress gene expression varied markedly with the inducing stimulus used. TNF alpha and IP-10 mRNA expression induced by IFN gamma and IL-2 was markedly more sensitive to suppression by oxidized LDL than that induced by LPS. The cooperative effects of IFN gamma and LPS on the expression of the inducible nitric oxide synthase gene were suppressed by oxidized LDL while the antagonistic effect of IFN gamma on LPS-induced expression of the TNF receptor type II mRNA was not altered. The suppressive activity of LDL was acquired only after extensive oxidation and was localized in the extractable lipid component. These results suggest a potent and direct connection between the oxidative modification of LDL and the chronic inflammation seen in atherogenic lesions. Furthermore, the appreciable selectivity of oxidized LDL in mediating secondary control of cytokine gene expression demonstrates that the active material(s) is targeted to disrupt specific intracellular signaling pathways. PMID- 7537755 TI - Coordinated expression of the vitronectin receptor and the urokinase-type plasminogen activator receptor in metastatic melanoma cells. AB - Integrin alpha v beta 3 is a marker of progression in malignant melanoma. Previously we reported that human melanoma cells derived from regional lymph node metastases had increased alpha v beta 3-mediated adhesion to lymph node vitronectin. In the present study, the expression and function of alpha v beta 3 were further investigated with emphasis on the functional relationship between alpha v beta 3 and the urokinase-type plasminogen activator system of proteolysis. We found that metastases-derived melanoma MeWo LNI 6I (6I) and MIM/8 LNI cells had a markedly increased expression of alpha v mRNA transcripts relative to the parent lines which was reflected in significantly elevated levels of the alpha v beta 3 heterodimers on the cell surface. These cells also expressed elevated levels of urokinase plasminogen activator receptor (uPAR) mRNA and had higher levels of surface bound urokinase plasminogen activator as detected by immunolabeling. To determine whether the expression of uPAR and alpha v were linked, alpha v synthesis in the metastatic melanoma cells was suppressed using alpha v antisense phosphorothioate oligonucleotides. This resulted in a marked decrease in detectable alpha v mRNA and protein and a corresponding substratum-specific reduction in cell adhesion to vitronectin. When uPAR expression in these cells was subsequently analyzed, we found a reduction of approximately 50% in uPAR mRNA levels. On the other hand, ligation of the alpha v beta 3 receptor on the melanoma cells by immobilized antibody resulted in a twofold increase in uPAR mRNA. The results suggest that the expression of uPAR in metastatic melanoma cells is linked to the expression and function of the vitronectin receptor. PMID- 7537756 TI - Locus for the inducible, but not a constitutive, nitric oxide synthase cosegregates with blood pressure in the Dahl salt-sensitive rat. AB - Alleles of the inducible nitric oxide synthase locus (Nos2) cosegregated highly significantly (P < 0.0001) with blood pressure in an F2 population [F2(S x MNS), n = 171] derived from a cross of inbred Dahl salt-sensitive (S) rats with Milan normotensive rats (MNS). In contrast, alleles at the constitutive brain nitric oxide synthase locus (Nos1) did not cosegregate with blood pressure in several F2 populations. Nos2 was mapped on rat chromosome 10. Nine genetic markers, including the angiotensin-converting enzyme (Ace) and Nos2 loci spanning roughly 46 cM on rat chromosome 10, all cosegregated strongly with blood pressure in the F2(S x MNS) population. Nos2 showed the highest LOD score of 6.3. Ace and Nos2 are 30 cM apart. In an F2 population [F2(S x WKY), n = 159] derived from a cross of S rats with Wistar-Kyoto (WKY) rats, Nos2 alleles did (P = 0.0070), but Ace alleles did not (P = 0.91), cosegregate with blood pressure. We conclude that the Nos2 locus rather than the Nos1 locus is a candidate for influencing blood pressure in the S rat. There are probably two separate but linked quantitative trait loci (QTL) for blood pressure on rat chromosome 10, one marked by Ace and the other marked by Nos2. In F2(S x MNS) functionally variant alleles at both QTL influence blood pressure, but in F2(S x WKY) only the QTL marked by Nos2 is segregating alleles influencing blood pressure. PMID- 7537758 TI - Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. AB - To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action. PMID- 7537759 TI - Luminal endocytosis and intracellular targeting by acinar cells during early biliary pancreatitis in the opossum. AB - Cell necrosis in acute experimental pancreatitis is preceded by a redistribution of digestive enzymes into a lysosomal subcellular compartment. We have investigated whether endocytosis from the acinar cell lumen might contribute to this disturbance of intracellular compartmentation. In an animal model of pancreatitis involving pancreatic bile duct ligation in opossums, we have studied in vivo endocytosis of dextran 40 and [14C]dextran 70, cationized ferritin, and horseradish peroxidase from the apical surface of acinar cells before the onset of necrosis. Marker solutions were instilled into the pancreatic duct of anesthetized animals at physiological pressure. Tissue samples obtained at intervals of up to 60 min after instillation of markers were studied by electron microscopy and electron microscope autoradiography. All markers were taken up by acinar cells in control animals and in animals with obstructed pancreatic bile ducts. Markers for membrane-mediated endocytosis (cationated ferritin and horseradish peroxidase) were transported to lysosomes in both groups. In contrast, the fluid-phase tracer dextran was transported to the secretory pathway in controls but to lysosomes after duct ligation. Since dextran and luminally present secretory proteins can be expected to follow the same route after endocytosis, our findings suggest that altered intracellular targeting of endocytosed proteases might be one mechanism by which digestive zymogens reach an intracellular compartment in which premature activation can occur. This phenomenon may be a critical and early event in the pathogenesis of biliary pancreatitis. PMID- 7537757 TI - VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis. AB - The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis. PMID- 7537760 TI - Adaptation of the growth hormone and insulin-like growth factor-I axis to chronic and severe calorie or protein malnutrition. AB - The hierarchy of diet components (e.g., protein, carbohydrate, vitamins, and minerals) influencing growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP) is not well defined. Young adult rats were fed diets for 1 mo that included low protein or 60% and 40% of carbohydrate calories. We hypothesized that levels of both hormones, their dominant BPs and liver IGF-I mRNA would fall, and that part of the mechanism for decreasing serum IGF-I would be enhanced IGFBP-3 protease activity. By day 30, caloric deprivation to 40% lowered serum GH, GHBP, IGF-I and IGFBP-3, and liver IGF-I mRNA. This was the only condition resulting in body weight loss (-15%) vs 39% gain in controls. Restriction to 60% calories had no impact on BP levels, slightly lowered IGF-I ( 12%) in the face of a 95% inhibition of GH levels, while allowing a modest 9% body weight gain. Protein deprivation lowered serum GH, IGF-I and IGFBP-3, and liver IGF-I mRNA, while GHBP levels were normal. The reduced total IGF-I under these dietary conditions could not be explained by an increase in IGFBP-3 protease activity, or a decrease in the association of IGF-I with IGFBP-3 and the acid labile subunit. PMID- 7537761 TI - Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing. AB - Congenital nephrogenic diabetes insipidus is a recessive hereditary disorder characterized by the inability of the kidney to concentrate urine in response to vasopressin. Recently, we reported mutations in the gene encoding the water channel of the collecting duct, aquaporin-2 (AQP-2) causing an autosomal recessive form of nephrogenic diabetes insipidus (NDI). Expression of these mutant AQP-2 proteins (Gly64Arg, Arg187Cys, Ser216Pro) in Xenopus oocytes revealed nonfunctional water channels. Here we report further studies into the inability of these missense AQP-2 proteins to facilitate water transport in Xenopus oocytes. cRNAs encoding the missense AQPs were translated with equal efficiency as cRNAs encoding wild-type AQP-2 and were equally stable. Arg187Cys AQP2 was more stable and Gly6-4Arg and Ser216Pro AQP2 were less stable when compared to wild-type AQP2 protein. On immunoblots, oocytes expressing missense AQP-2 showed, besides the wild-type 29 kDa band, an endoplasmic reticulum retarded form of AQP-2 of approximately 32 kD. Immunoblots and immunocytochemistry demonstrated only intense labeling of the plasma membranes of oocytes expressing wild-type AQP-2. Therefore, we conclude that in Xenopus oocytes the inability of Gly64-Arg, Arg187Cys or Ser216Pro substituted AQP-2 proteins to facilitate water transport is caused by an impaired routing to the plasma membrane. PMID- 7537762 TI - Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation. AB - Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. PMID- 7537766 TI - Inhibition of the proteinase activity in mastitic milk. AB - The antiproteolytic activity of selected proteinase inhibitors was studied in mastitic bovine milk and urokinase-activated normal milk using a caseolytic agar diffusion assay. The inhibition profiles of mastitic milk and urokinase-activated milk were compared with those of purified proteinases. The proteinase inhibition profile of mastitic milk did not resemble that of any of the pure proteinases, indicating a mixed type of proteinase system in mastitic milk. The trypanocidals diminazene (equivalent to Berenil) and pentamidine (equivalent to Lomidine), together with aprotinin (Trasylol), showed most promise when considering possible applications in mastitis to break up the proteolytic cascade within the inflamed udder. PMID- 7537764 TI - Hypertonicity, but not hypothermia, elicits substance P release from rat C-fiber neurons in primary culture. AB - Isocapnic dry gas hyperventilation provokes hyperpnea-induced bronchoconstriction in guinea pigs by releasing tachykinins from airway sensory C-fiber neurons. It is unknown whether dry gas hyperpnea directly stimulates C-fibers to release tachykinins, or whether this physical stimulus initiates a mediator cascade that indirectly stimulates C-fiber tachykinin release. We tested the hypotheses that mucosal hypothermia and/or hyperosmolarity--physical consequences of airway heat and water loss imposed by dry gas hyperpnea--can directly stimulate C-fiber tachykinin release. Neurons isolated from neonatal rat dorsal root ganglia were maintained in primary culture for 1 wk. Cells were then exposed for 30 min at 37 degrees C to graded concentrations of NaCl, mannitol, sucrose, or glycerol (0-600 mOsm) added to isotonic medium, or to isotonic medium at 25 degrees C without or with 462 mOsm mannitol added. Fractional release of substance P (SP) was calculated from supernatant and intracellular SP contents following exposure. Hyperosmolar solutions containing excess NaCl, mannitol, or sucrose all increased fractional SP release equivalently, in an osmolarity-dependent fashion. In marked contrast, hypothermia had no effect on fractional SP release under isotonic or hypertonic conditions. Thus, hyperosmolarity, but not hypothermia, can directly stimulate tachykinin release from cultured rat sensory C-fibers. The lack of effect of glycerol, a solute which quickly crosses cell membranes, suggests that neuronal volume change represents the physical stimulus transduced by C-fibers during hyperosmolar exposure. PMID- 7537765 TI - All-trans-retinoic acid stimulates synthesis of cyclic ADP-ribose in renal LLC PK1 cells. AB - Cyclic adenosine diphospho-ribose (cADPR) triggers Ca2+ release from intracellular stores and is therefore proposed to function as a second messenger in cellular signaling; however, an extracellular stimulus, i.e., first messenger (hormone or autacoid) that modulates cADPR metabolism has not been identified. We discovered that all-trans-retinoic acid (atRA) is a potent stimulus to increase cADPR synthesis by cultured LLC-PK1 cells. The stimulation of cADPR synthesis by atRA is dose dependent between 0.1 nM and 1 microM (maximum increase approximately delta + 600%), while atRA does not alter the rate of cADPR hydrolysis by LLC-PK1 cells. The activity of other intrinsic apical membrane enzymes was not significantly altered. The stimulation of cADPR synthesis by atRA occurs after a lag period of 6-8 h, and the stimulation is inhibited by actinomycin D and by cycloheximide. Our results therefore demonstrate that atRA in physiological concentrations is a potent extracellular stimulus, first messenger, that enhances cADPR synthesis, and the effect of atRA requires de novo protein synthesis. We suggest that some of the diverse biologic actions of atRA such as morphogenetic and cell differentiation may be mediated via cADPR. PMID- 7537768 TI - Further immunocytochemical characterization of cultured hair apparatus cells. AB - In immunocytochemical studies of cultured hair apparatus cells we employed anti hair keratin monoclonal antibodies (HKN-2, HKN-4, HKN-5, HKN-6 and HKN-7) and compared the results with electron microscopic studies. On day 1, the cultured cells positively stained with HKN-2 (52%), HKN-4 (50%), HKN-5 (43%), HKN-6 (33%), or HKN-7 (37%). On day 3, more than 88% of the cells showed no reaction to any antibody. On day 6, most of the cells stained with all of the antibodies: HKN-2 (96%), HKN-4 (92%), HKN-5 (80%), HKN-6 (72%) and HKN-7 (74%). These reactivities were maintained until day 13. Electron microscopic studies revealed that, on day 1, half of the observed cells were immature and 47% of them were already differentiated. Most (88%) of the cells showed immature features and the percentage of differentiated cells had decreased by day 3. The differentiated cells (87%) reappeared by day 6, and degenerated cells (63%) increased by day 13. These immunocytochemical results were consistent with those of simultaneous electron microscopic studies, demonstrating that immature cells proliferated and differentiated into subpopulations of each hair apparatus layer type, including the outer root sheath cells, in this culture system. PMID- 7537763 TI - Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A. AB - When AR42J cells, an amylase-secreting pancreatic exocrine cell line, were treated with activin A, cells extended neuritelike processes, and, concomitantly, amylase-containing vesicles disappeared. Immunofluorescence and immunoelectron microscopy revealed that these processes had neurite-specific cytoskeletal architectures: neurofilaments and microtubule bundles with cross-bridges of microtubule-associated protein 2. In addition to such morphological changes, activin-treated cells exhibited a marked increase in cytoplasmic free calcium concentration in response to depolarizing concentration of potassium. Moreover, activin-treated AR42J cells expressed mRNA for alpha 1 subunit of the neuroendocrine/beta cell-type voltage-dependent calcium channel. In naive AR42J cells, a sulfonylurea compound, tolbutamide, did not affect free calcium concentration, while it induced a marked elevation of free calcium in activin treated cells. Single channel recording of the membrane patch revealed the existence of ATP-sensitive potassium channel in activin-treated cells. These results indicate that activin A converts amylase-secreting AR42J cells to neuronlike cells. Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitive potassium channel as well as neuroendocrine/beta cell-type voltage-dependent calcium channel, activin treatment of AR42J cells may provide an in vitro model system to study the conversion of pancreatic exocrine cells to endocrine cells in islets. PMID- 7537769 TI - So-called mixed tumor of the skin on the wrist: an immunohistochemical study. AB - A case of so-called mixed tumor of the skin arising on the wrist was reported. Immunohistochemical staining of keratins of several molecular weights (AE1/AE3, RCK102, NCL-5D3 and 35 beta H11), carcinoembryonic antigen (CEA), S-100 protein, and desmin was performed. AE1/AE3 and RCK102 were positive in all the tumor cells; CEA, NCL-5D3 and 35 beta H11 were positive mainly in luminal cells of the tubuloalveolar structures. S-100 protein was positive in peripheral cells of the tubular lumina and in scattered cells in the mucous stroma. Desmin was negative in all the tumor cells. Immunohistochemical findings lent further support to the hypothesis that so-called mixed tumor of the skin differentiates into the secretory and ductal portions of the sweat gland. PMID- 7537767 TI - Junctional proteins of keratinocytes in Grover's disease, Hailey-Hailey's disease and Darier's disease. AB - Alterations of junctional structures in non-immune mediated acantholytic diseases (Grover's, Hailey-Hailey's and Darier's diseases) were examined using monoclonal antibodies against desmosomal attachment constituents (desmoplakin I & II and plakoglobin), desmosomal intercellular cement glycoprotein (desmoglein), protein of adherens junction (vinculin), and protein of gap junction (43Kd connexin). Universal cell surface (transmembrane) glycoprotein CD44 was also studied. In acantholytic foci of these diseases, attachment plaque proteins had dissolved and diffused into the acantholytic cells. The normal dotted linear pattern of immunostaining on the cell membrane was totally lost. In contrast, CD44 was well preserved on the cell membranes of acantholytic cells. Adherens junction and gap junction proteins were mostly preserved. Acantholytic cells of pemphigus vulgaris were similarly studied. In these cells, desmosomal attachment plaque proteins were very well preserved, while intercellular cement substance (desmoglein), adherens junctional proteins (vinculin), and gap junction protein (connexin) were totally absent, either on the cell membrane or in the cytoplasm. Electron microscopy confirmed an early dissolution of attachment plaque. Internalized desmosomal structures were seldom found in acantholytic cells of non-immune diseases. It was concluded that the primary event in acantholysis in these three diseases is the dissolution of desmosomal attachment plaque. PMID- 7537770 TI - Effects of substance P on cardiovascular regulation in the rabbit. AB - The effect of substance P on blood pressure and aortic reflex was investigated in rabbits. Microinjections of substance P and Sar9, Met(O2)11-SP (a selective NK1 receptor agonist) into the floor of the fourth ventricle led to a dose-dependent increase of blood pressure and a sharp enhancement of the baroreflex. These effects were abolished by pretreatment with SR 140333 (a selective NK1-receptor antagonist). Intraventricular injection of the antagonist alone significantly decreased the amplitude of the aortic reflex. After bivagotomy, the amplitude of the parasympathetic component of the baroreflex decreased dramatically and substance P injections were no longer effective. Our results demonstrate that substance P activation of NK1 receptors plays a major role in the modulation of the parasympathetic component of the baroceptor reflex. PMID- 7537771 TI - Substance P and serotonin independently affect intragastric pressure when microinjected into the nucleus raphe obscurus of the rat. AB - We have recently shown that microinjection of substance P (SP) into the nucleus raphe obscurus (NRO) of the rat decreases intragastric pressure, whereas microinjection of serotonin (5-HT) increases it. The purpose of the present study was to investigate whether there exists a functional interaction between SP and 5 HT in the NRO of the rat in their effects on gastric motor function. This was accomplished by microinjection of SP (135 pmol) and 5-HT (0.6 and 6 nmol) into the NRO in a rapid, sequential order in alpha-chloralose-anesthetized rats, while monitoring intragastric pressure and pyloric and greater curvature motilities. Substance P evoked significant decreases in intragastric pressure when microinjected into the NRO after vehicle and after 5-HT (at both 0.6 and 6 nmol). There was no difference in the magnitude of the SP effect after 5-HT when compared to the response after vehicle. Serotonin at a dose of 6 nmol, but not at a dose of 0.6 nmol, elicited significant increases in intragastric pressure when microinjected after vehicle or after SP, and there was no difference between the responses to 5-HT with respect to the initial treatment. We conclude that SP and 5-HT act independently in the NRO of the rat to affect intragastric pressure. PMID- 7537772 TI - Location of nitric oxide synthase in the developing avian ciliary ganglion. AB - A study has been made of the distribution of nitric oxide synthase (NOS) in the developing avian ciliary ganglion. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity first appeared in ciliary neurones at embryonic day 10 (E10). The number of NADPH-d positive neurones appeared maximal at this age and thereafter declined; at post hatched day 4 (P4) these neurones were found predominately in the periphery of the ganglion. At the light microscope level the NADPH-d stain appeared throughout the cell soma of the ciliary neurones. This was confirmed using tissue culture techniques. Ultrastructural delineation of horseradish peroxidase-labelled NOS antibodies was also found in the calyx where it was bound to the membranes of the endoplasmic reticulum as well as to the outer membranes of mitochondria. This distribution of NOS in the soma and calyx is consistent with the physiological role of NO as a co-transmitter and retrograde messenger that regulates the quantal secretion of the principal transmitter, acetylcholine, from the calyx. PMID- 7537773 TI - Homeobox genes Msx-1 and Msx-2 are associated with induction and growth of skin appendages. AB - The mechanism involved in the morphogenesis of skin appendages is a fundamental issue underlying the development and healing of skin. To identify molecules involved in the induction and growth of skin appendages, we studied the expression of two homeobox genes, Msx-1 and Msx-2, during embryonic chicken skin development. We found that i) both Msx-1 and Msx-2 are early markers of epithelial placodes for skin appendages; ii) both Msx-1 and Msx-2 are expressed in the growing feather bud epithelia but not in the interbud epithelia; iii) although mostly overlapping, there are differences between the expression of the two Msx genes, Msx-1 being expressed more toward the anterior whereas Msx-2 is expressed more toward the distal feather bud; iv) there is no body-position specific expression pattern as was observed for members of the Hox A-D clusters; v) in the feather follicle, Msx-1 and 2 are expressed in the collar and barb ridge epithelia, both regions of continuous cell proliferation; vi) when feather bud growth was inhibited by forskolin, an activator of adenylyl cyclase, the expression of both genes was reduced. These results showed that Msx genes are specifically expressed in epithelial domains destined to become skin appendages. Its function in skin-appendage morphogenesis may be twofold, first in making epithelial cells competent to become skin appendages and, second, in making epithelial cells maintain their potential for continuous growth. PMID- 7537774 TI - Human keratinocytes regulate their expression of B7/BB-1 antigen by a unique, calcium-dependent mechanism. AB - Previous studies of normal human keratinocytes have indicated that these cells express BB-1 antigen, an important adherence molecule usually associated with "professional" antigen-presenting cells. We studied freshly isolated epidermal cells and noted that the frequency of BB-1-positive cells in normal human skin varied from 2.6 to 7.4% of total epidermal cells. Two-color flow cytometry confirmed that keratinocytes were the major cell in the epidermis that expressed BB-1, because less than 10% of total epidermal Langerhans cells were positive for BB-1. Northern blot analysis of RNA extracted from normal human epidermis revealed low levels of 1.7-kb B7-1 transcripts, which were independent of the presence of epidermal Langerhans cells, again indicating that such transcripts were derived from keratinocytes. Keratinocytes cultured in medium containing low concentrations of extracellular calcium (0.07 mM) expressed low levels of cell surface BB-1. However, keratinocytes cultured in medium with higher levels of extracellular calcium (1.5 mM) lost cell-surface expression of BB-1. Similarly, low-calcium keratinocytes expressed the 1.7- and 2.9-kb B7-1 transcripts, whereas high-calcium keratinocytes expressed only the 1.7-kb transcript. Studies of the cell-surface expression of BB-1 by plastic adherent monocytes indicated that such cells do not respond to similar changes in extracellular calcium concentrations. Calcium-induced differentiation of keratinocytes regulates the expression of BB-1 antigen as well as transcripts, which is a novel mechanism for the regulation of this molecule. PMID- 7537775 TI - Changes in the concentrations of extracellular Mg++ and Ca++ down-regulate E cadherin and up-regulate alpha 2 beta 1 integrin function, activating keratinocyte migration on type I collagen. AB - We have demonstrated recently that shifts in the concentrations of extracellular Mg++ and Ca++ occur during cutaneous injury in vivo. These shifts correlate well with the timing of migration of various cell types involved in wound healing, including keratinocytes. In the present study, we examined the potential of such cation shifts to activate the keratinocyte migratory phenotype. In modified Boyden chamber migration assays, alpha 2 beta 1 integrin-mediated migration of human keratinocytes (HaCaT) on type I collagen was supported by Mg++ but not by Ca++ alone. Migration could be increased up to twofold, however, by using both cations in combination, as long as the Mg++ concentration was in the optimal range for migration in Mg++ only (1-3 mM) and Ca++ was present at concentrations of approximately 0.1-1 mM. Further examination of this divalent-cation-induced migratory keratinocyte phenotype demonstrated that, as Mg++ is elevated and Ca++ is reduced, mature E-cadherin and cell-cell contacts are reduced and the alpha 2 beta 1 integrin is redistributed from cell-cell contacts to the periphery. These in vitro observations corroborate what occurs in vivo at the keratinocyte migrating front during wound healing. Together these data suggest that changes in the concentrations of extracellular Mg++ and Ca++ can regulate the competitive interplay between Ca(++)-dependent E-cadherin-mediated and Mg(++)-dependent alpha 2 beta 1-integrin-mediated adhesion, promoting the development of an activated keratinocyte phenotype. PMID- 7537776 TI - Molecular analysis of elastic properties of the stratum corneum by solid-state 13C-nuclear magnetic resonance spectroscopy. AB - To elucidate the precise molecular mechanisms underlying stratum corneum (SC) elasticity, we investigated the molecular dynamics of chemical residues within keratin fibers of human plantar SC under various conditions by cross polarization/magic angle spinning 13C-nuclear magnetic resonance. The intensities of nuclear magnetic resonance spectra responsible for amide carbonyl, C alpha methine, and side-chain aliphatic carbons in the intact SC decreased markedly with increasing water content of up to 30% in dry SC, and then remained constant at greater than 30%. Lipid extraction of intact SC with acetone/ether (1:1) did not induce any significant change in the nuclear magnetic resonance spectrum, whereas additional treatment with water, which released natural moisturizing factors (mainly amino acids), caused the SC to lose elasticity. The observed decrease in elasticity of the SC recovered after treatment with basic and neutral amino acids, but not after treatment with acidic amino acid. With the latter treatment, movement of amino acid molecules was significantly disturbed, suggesting a strong interaction with keratin fibers. Parallel studies of the complex elastic modulus of a pig SC sheet with a rheovibron also demonstrated that removal of natural moisturizing factor reduced the elasticity of the SC; this effect was also reversed by the application of basic and neutral amino acids, but not by the application of acidic amino acid. These findings suggest that structural keratin proteins, mainly consisting of 10-nm filaments, acquire their elasticity with the help of hydrated natural moisturizing factor via the reduction of intermolecular forces, probably through nonhelical regions between keratin fibers. PMID- 7537777 TI - Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies. AB - Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation. PMID- 7537779 TI - Axon-reflex-mediated vasodilatation in the psoriatic plaque? AB - Blood flow in the psoriatic plaque is increased, but the underlying mechanisms are not known. The aim of the present study was to examine whether neurogenic factors are important for blood flow regulation in the plaque. Local neurogenic mechanisms were inhibited by surface anesthesia and central nervous control by conduction anesthesia of nerves to the psoriatic plaque. The differences in skin perfusion before and after anesthesia were measured with a laser Doppler perfusion imager. The skin perfusion in psoriatic plaques located in hairy skin was unaffected by conduction anesthesia, but surface anesthesia of the plaque evoked a marked blood flow reduction. The perfusion in ultraviolet-B-irradiated skin, used as a control for nonspecific phenomena, was reduced after local application of indomethacin but was unaffected or increased after surface anesthesia. The results are compatible with the idea that a local neurogenic mechanism (axon-reflex) contributes to the high blood flow in the psoriatic plaque. PMID- 7537780 TI - A common keratin 5 gene mutation in epidermolysis bullosa simplex--Weber Cockayne. AB - The Weber-Cockayne subtype of epidermolysis bullosa simplex is an inherited skin fragility disorder characterized by basal keratinocyte lysis and epidermal blistering confined primarily to the hands and feet. The disorder results from a mutation in either the keratin 5 or keratin 14 gene, which encode the peptide components of the obligate heterodimeric keratin intermediate filaments of the basal cell. We have determined that a T-->G substitution mutation in keratin 5, which results in a Ile-->Ser change at codon 161, is common among patients with the Weber-Cockayne disease variant, accounting for six of 13 cases tested. The observed high frequency of this mutation may result from either a mutational hot spot or a founder effect. The potential utility of this common mutation in confirming disease status in some at-risk individual is discussed. PMID- 7537781 TI - Autoimmune diseases: promising emerging therapies. PMID- 7537778 TI - Acidic fibroblast growth factor accelerates dermal wound healing in diabetic mice. AB - Acidic fibroblast growth factor (aFGF) is a potent mitogenic and chemotactic agent for vascular endothelial cells, dermal fibroblasts, and epidermal keratinocytes, the principal cellular constituents of skin. To explore its potential to heal chronic dermal wounds, we applied pure recombinant human aFGF topically to full-thickness excisional injuries in healing-impaired genetically diabetic mice. Transformation of the nonlinear percent initial wound areas as a function of time to linear rates of tissue ingrowth from the original wound edges showed that aFGF increased wound closure in a dose-dependent manner. Optimal 3 micrograms/cm2 doses of aFGF nearly tripled the linear rate of healing. The median time to complete closure decreased from 46 d in vehicle-treated wounds to only 16 d in those treated with aFGF. Histomorphometric analyses established that aFGF increased granulation tissue formation and reepithelialization throughout healing. Vehicle- and aFGF-treated wounds appeared to be histologically equivalent by the time of closure. Therefore, aFGF has potential therapeutic applications for promoting healing of dermal ulcers, especially in healing impaired individuals. PMID- 7537782 TI - Cytokine patterns in alopecia areata before and after topical immunotherapy. PMID- 7537783 TI - Analysis of gene expression in isolated single hair follicles: an approach using semiquantitative reverse-transcriptase-polymerase chain reaction. PMID- 7537784 TI - Structure and function of cutaneous nerves in alopecia areata. PMID- 7537786 TI - Hair follicle companion layer: reacquainting an old friend. PMID- 7537785 TI - HLA-linked skin disease: classical HLA genes or novel genes within HLA? PMID- 7537787 TI - Talin does not associate exclusively with alpha 2b beta 3 integrin in activated human platelets. AB - Talin is a high-molecular-weight protein that may stabilize connections between cytoplasmic actin and the submembrane portion of glycoprotein IIb-IIIa (GPIIb IIIa) (alpha 2b beta 3 integrin) in thrombin-stimulated human platelets. Using morphologic and electrophoretic techniques, we have examined the association of talin with the cytoskeleton of platelets activated by thrombin in the presence of fibrinogen-coated gold particles (Fgn/Au). Ultrastructural studies confirmed the presence of Fgn/Au firmly bound to the outside membranes of detergent-extracted platelets. Immunoblots of protein bands showed GPIIIa, but not talin, associated with cytoskeletons of activated platelets. Immunogold cytochemical techniques were performed on ultrathin cryosections of whole platelets to localize talin at the ultrastructural level. Studies were performed on normal platelets and platelets defective in GPIIb-IIIa (Glanzmann's thrombasthenia) and GPIb (Bernard Soulier syndrome). Talin was randomly distributed in the cytoplasm of resting platelets. Activation resulted in binding of Fgn/Au to the surface membrane and redistribution of talin to the submembrane region. However, no definitive colocalization between the two markers was noted. Activated thrombasthenic platelets failed to bind Fgn/Au, but talin was localized to the submembrane location. After activation, talin was confined to the submembrane zone of Bernard Soulier syndrome platelets. No definitive colocalization was observed between large clusters of Fgn/Au-occupied receptors and talin distributed in the submembrane region. GPIb and GPIIb-IIIa are not necessary for talin to localize in the submembrane region of activated cells. Talin does not redistribute exclusively with GPIIb-IIIa, and it may stabilize connections with other glycoproteins. PMID- 7537788 TI - Arachidonic acid metabolites are involved in mediating red blood cell adherence to endothelium. AB - As an initial investigation into the possible role of endothelial cell (EC) lipoxygenase and cyclooxygenase metabolites in the adherence of red blood cells (RBCs) to ECs, we evaluated the effect of nordihydroguaiaretic acid, (NDGA; 10 mumols/L, BW755c (30 mumols/L), aspirin (100 mumols/L), and indomethacin (10 mumols/L) on RBC-EC adherence using a static incubation system and 51Cr-labeled RBCs. NDGA and 3-amino-L-[3'-(trifluoromethyl)phenyl]-2-pyrazoline inhibitors of both the lipoxygenase and cyclooxygenase pathways, significantly decreased basal adhesion of RBCs to fetal bovine aortic ECs, whereas aspirin and indomethacin, selective inhibitors of the cyclooxygenase pathway, stimulated the adherence process. The inhibitor effect appeared to be mediated via an effect on EC functions, since preincubation of ECs with NDGA, in contrast to RBC-NDGA preincubation, inhibited the adherence process. Because bovine aortic ECs generate mainly prostacyclin and 15-HETE from arachidonic acid (AA) via the cyclooxygenase and the lipoxygenase pathways respectively, the role of these products (100 pmol/L to 1 mumol/L) on the adhesive process was further assessed. 15-HETE potentiated basal adhesion of RBCs to bovine aortic ECs in a concentration-dependent manner, with maximal responses of approximately 50% to 150% over baseline noted at concentrations between 1 and 100 nmol/L 12-HETE, a structural isomer of 15-HETE and the major platelet lipoxygenase product, also stimulated RBC adherence. In contrast, prostacyclin (assessed using carbacyclin, a stable synthetic analog of prostacyclin with similar biologic properties) had no significant effect on this process. In further studies, we demonstrated that the 12-HETE-induced adherence of sickle RBCs was mediated via an up-regulation of the vitronectin receptor on bovine aortic endothelium. Because microvascular capillary endothelium is the surface most likely to encounter erythrocytes in vivo, we extended our studies to human retinal capillary ECs to assess the involvement of eicosanoids in sickle RBC-microvessel adhesion. As with bovine aortic ECs, aspirin stimulated and NDGA decreased the adherence of sickle RBCs to human retinal capillary endothelium. These microvascular ECs generated prostacyclin, HHT, 15-HETE, and 15-HPETE from endogenous AA. Although carbacyclin and HHT had no effect on the adherence process, both 15-HETE and 15-HPETE (10 pmol/L to 100 nmol/L) stimulated RBC adhesion to capillary endothelium. Our studies document a role for the lipoxygenase metabolites in modulating basal adhesion of RBCs to both macrovascular and microvascular endothelium; the major cyclooxygenase metabolites appear to play no role in this process. PMID- 7537789 TI - Expression of E-selectin mRNA during ischemia/reperfusion injury. AB - To better understand molecular and cellular processes involved in tissue inflammation, we have examined expression of endothelial leukocyte adhesion molecule 1 (E-selectin) mRNA in adult male rats after ischemia/reperfusion (I/R) injury and after intravenous injection of lipopolysaccharide (LPS). The polymerase chain reaction was used to generate a rat E-selectin cDNA fragment by using heart total RNA from rats exposed to LPS. This partial cDNA fragment spanned sequences from complement repeat region-5 to the second cytoplasmic tail domain. Comparison of the predicted amino acid sequence from the rat cDNA fragment to mouse and human E-selectin protein sequences showed significant conservation. The rat E-selectin cDNA fragment was used as a probe to examine the regulation of E-selectin mRNA expression by Northern blot analysis. As previously described in other animal species, E-selectin mRNA expression was induced after intravenous injection of LPS. In contrast, ischemia did not induce E-selectin mRNA expression, except in the setting of I/R injury. I/R injury triggered expression of E-selectin mRNA in the kidney. These experiments represent a first in vivo examination of E-selectin mRNA expression after I/R injury and constitute an initial step in characterizing a model system for investigating inflammation in the setting of acute ischemic injury. PMID- 7537790 TI - Soluble CD14 promotes LPS activation of CD14-deficient PNH monocytes and endothelial cells. AB - Bacterial lipopolysaccharide (LPS) initiates the cascade of inflammatory events that, in infected patients, often result in a lethal systemic inflammatory response known as the sepsis syndrome. We studied LPS-stimulated expression of tissue factor (TF) in human peripheral blood mononuclear cells (PBMCs) and cultured endothelial cells or tumor necrosis factor-alpha (TNF-alpha) in PBMCs. CD14, a PBMC membrane protein, is involved in LPS signaling and is also present as a soluble molecule in serum. CD14 is absent from endothelial cells and, in varying degrees, from monocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH). LPS stimulation of TF in normal monocytes was enhanced > 30 fold by serum at low concentrations of LPS (< or = 10 ng/ml). The serum dependence of endothelial cells was even more pronounced; a full response to LPS was not observed in endothelium under serum-free conditions, even with LPS concentrations as high as 100 ng/ml. To better define the role of CD14, CD14 deficient PBMCs from two patients with PNH were compared with normal PBMCs. Although less than 3% of PNH monocytes expressed CD14, LPS-induced synthesis of TF and TNF-alpha by PBMCs from PNH patients was inhibited by anti-CD14 antibodies. Because patient serum samples were found to contain soluble CD14, we sought to determine whether PNH monocytes might respond to LPS through an activation pathway dependent on soluble CD14. Recombinant soluble CD14 substituted for serum to enable LPS stimulation of endothelium, PNH PBMCs, and surprisingly, CD14-replete normal PBMCs. In addition, a truncated sCD14 containing the N-terminal 152 amino acids similarly enabled LPS stimulation of normal PBMCs. These data underscore the importance of soluble CD14 and suggest that CD14 present in serum enables LPS responses in PNH monocytes and endothelial cells and may even influence the effects of LPS in normal human phagocytes. PMID- 7537792 TI - IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation. AB - It is well known that IGFs-I and -II stimulate both the proliferation and differentiation of myoblasts, but the role of the IGF binding proteins (IGFBPs) during these processes has not been established. In this study we show that IGF-I analogs with greatly reduced affinity for IGFBPs exhibited about a 10-fold increase in potency in stimulating proliferation (as in other cell types), but up to a 100-fold greater potency than native IGF-I in stimulating L6A1c differentiation. Analysis of conditioned media revealed that L6 cells secrete significant levels of IGFBPs that react with antisera to IGFBP-4, -5 and -6. Steady-state levels of IGFBP-4 mRNA were highest in proliferating myoblasts, while IGFBP-5 mRNA could not be detected in myoblasts although its levels were dramatically increased during IGF- or insulin-stimulated differentiation of myoblasts into myotubes. Elevated IGFBP-6 mRNA levels were found in quiescent cells in serum-free medium. IGF-I and IGF-II treatment elevated IGFBP-5 in conditioned media, but longR3IGF-I and insulin, which do not bind to IGFBPs, had smaller effects. This complex regulation of expression of different IGFBPs not only during different stages of muscle growth and differentiation, but also upon stimulation by IGFs or insulin, suggests that the IGFBPs play a specific and significant role in modulating the actions of the IGFs during myogenesis. PMID- 7537791 TI - Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus. AB - In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action. PMID- 7537793 TI - Risperidone in Parkinson's disease. PMID- 7537794 TI - B cell responses to acetylcholine receptor in rats orally tolerized against experimental autoimmune myasthenia gravis. AB - Oral administration of acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG), and decreased serum levels of anti-AChR antibodies. Using an ELISPOT assay, we have now determined numbers of cells in the popliteal, inguinal and mesenteric lymph nodes, spleen and thymus secreting anti-AChR IgG antibodies. Except for mesenteric lymph nodes, a marked diminution of such cells was detected in these lymphoid organs in rats orally tolerized with AChR compared to buffer-fed or vehicle-fed control rats with EAMG. Of note is that, after AChR feeding, the B cell response to AChR in thymus was diminished to the same low level as in CFA injected, buffer-fed control rats. The relative affinity of serum anti-AChR IgG antibodies measured by KSCN-ELISA was lower in the orally tolerized rats compared to buffer-fed or vehicle-fed rats. The observations showed that oral administration of AChR, besides preventing clinical EAMG, also counteracts the development of AChR-specific B cells, especially those with high affinity antibody production, in most lymphoid organs. PMID- 7537795 TI - Reactivity of normal T-cell lines to MBP isolated from normal and multiple sclerosis white matter. AB - T-cell reactivity to human myelin basic protein (MBP) has been extensively studied using T-cell lines and clones generated from both peripheral blood and cerebrospinal fluid, from normal controls and multiple sclerosis (MS) patients. These studies have largely utilized myelin basic protein isolated from control human adult white matter. In our study, we used MBP reactive T-cell lines as a probe to investigate antigenic differences in a series of MBP preparations isolated from either control human white matter or white matter from the central nervous system (CNS) of MS patients. Autologous peripheral blood derived mononuclear cells were used as antigen presenting cells (APC). Although the majority of T-cells were found to react equally well with all preparations of MBP isolated from both control and MS white matter, we were also able to identify T cell lines which reacted well with all preparations of MBP isolated from controls but failed to react with MBP isolated from MS white matter. These differences were unlikely to reflect differences in degradation products or excess peptides present in the MS brain since SDS-PAGE and HPLC did not show any difference in the MS samples compared to the controls, and the concentration response curves for a human T-cell clone specific for the 84-102 region of MBP were similar for all the MBP preparations. We did not detect differences in amino acid content amongst MBP preparations although single amino acid substitutions cannot be ruled out. These results raise the possibility that MBP isolated from MS brain may differ in charge microheterogeneity which would affect antigenic determinants.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537796 TI - Brief chemotherapy, Stanford V, and adjuvant radiotherapy for bulky or advanced stage Hodgkin's disease: a preliminary report. AB - PURPOSE: Although survival rates have improved for patients with bulky and advanced-stage Hodgkin's disease (HD), current treatments entail substantial acute morbidity and risks for late effects such as infertility, second malignancies, and cardiopulmonary toxicities. A novel, brief chemotherapy regimen (doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin, etoposide, and prednisone [Stanford V]) was designed to shorten the duration of treatment, significantly reduce cumulative doses of alkylating agents, doxorubicin, and bleomycin, and maintain dose-intensity (DI). This brief chemotherapy was combined with radiation therapy (RT) to bulky disease sites. METHODS: Since May 1989, 65 previously untreated patients were treated for stage II HD with bulky mediastinal involvement (n = 21) or for stage III or IV HD (n = 44). Patients received weekly chemotherapy for 12 weeks. Consolidative RT was given to the first 25 patients to sites of initial bulky disease or radiographic abnormalities that persisted after chemotherapy; in the remaining 40 patients, RT was limited to bulky disease (adenopathy > or = 5 cm and/or macroscopic splenic nodules defined by computed tomography [CT]). RESULTS: With a median follow-up period of 2 years, actuarial 3 year survival rate is 96% and failure-free survival (FFS) rate is 87%. The 3-year FFS rate is 100% for stage II patients with bulky mediastinal disease and 82% for patients with stage III to IV disease. There were no treatment-related deaths. In a preliminary analysis on a subset of patients, female and male fertility appears to be preserved. CONCLUSION: These preliminary results indicate that the Stanford V chemotherapy regimen with or without RT is well-tolerated and effective therapy for bulky, limited-stage, and advanced-stage HD. Less cumulative exposure to alkylating agents, doxorubicin, and bleomycin and limited use of radiation is expected to decrease risks for second neoplasms and late cardiopulmonary toxicity. Based on these results, the Stanford V chemotherapy with or without RT regimen deserves further study in the context of a randomized clinical trial. PMID- 7537799 TI - Dose escalation of paclitaxel with high-dose cyclophosphamide, with analysis of progenitor-cell mobilization and hematologic support of advanced ovarian cancer patients receiving rapidly sequenced high-dose carboplatin/cyclophosphamide courses. AB - PURPOSE: We commenced a phase I study of escalating dose Taxol (paclitaxel; Bristol-Myers Squibb Co, Wallingford, CT) in addition to cyclophosphamide, to assess its impact on both antitumor efficacy and mobilization of peripheral-blood progenitor cells (PBP). PATIENTS AND METHODS: Induction therapy consisted of two cycles of cyclophosphamide 3.0 g/m2 plus escalating-dose Taxol (dose levels I to IV, 150, 200, 250, and 300 mg/m2, respectively) in cohorts of three, plus filgrastim granulocyte colony-stimulating factor [G-CSF]) and leukaphereses to harvest PBP, followed by four courses of rapidly cycled carboplatin and cyclophosphamide (1,000 and 1,500 mg/m2 per course, respectively), for which hematopoietic rescue was achieved with PBP. RESULTS: Sixteen patients completed all planned cycles of Taxol/cyclophosphamide. Fifty-four cycles of carboplatin/cyclophosphamide were given and rescued with PBP. The median interval between treatments for Taxol/cyclophosphamide courses was 14 days (range, 13 to 21). Twelve patients completed all planned cycles of carboplatin/cyclophosphamide. The median inter-treatment interval for carboplatin/cyclophosphamide courses when rescue was achieved with Taxol/cyclophosphamide-primed PBP was 17 days (range, 14 to 25). The median number of days to recovery of an absolute neutrophil count (ANC) greater than 0.5 was 8 (range, 5 to 12), and of self-sustaining platelet count greater than 20 x 10(9)/L, 11 (range, 8 to 15). There was one episode of fatal sepsis. Of 13 patients assessable for response, there were five patients with pathologic complete responses (38.5%), six patients with microscopic residual disease (46%), and two patients with pathologic partial responses, for an overall response rate of 100%. CONCLUSION: The addition of escalating-dose Taxol to high-dose cyclophosphamide does not compromise PBP mobilization. The use of PBP mobilized in this fashion provides reliable engraftment after sequential administration of high-dose carboplatin/cyclophosphamide. Toxicities produced with this approach are manageable. The response rates demonstrated are promising and warrant further evaluation. PMID- 7537798 TI - Paclitaxel as second and subsequent therapy for metastatic breast cancer: activity independent of prior anthracycline response. AB - PURPOSE: Two phase II clinical trials were performed to determine efficacy and tolerability of paclitaxel (Taxol; Bristol-Myers Squibb Co, Wallingford, CT) and granulocyte colony-stimulating factor ([G-CSF] Neupogen; Amgen, Inc, Thousand Oaks, CA) as second or subsequent therapy for metastatic breast cancer. PATIENTS AND METHODS: Paclitaxel plus G-CSF was administered as a second stage IV regimen to 25 patients with metastatic breast cancer at a dose of 250 mg/m2 intravenously over 24 hours. Fifty-two patients received paclitoxel plus G-CSF at 200 mg/m2 as a third or subsequent regimen (no restriction on number of prior regimens or on prior high-dose chemotherapy). All patients had received prior anthracycline treatment, and ultimately had progressive bidimensionally measurable disease. RESULTS: Twenty-five of 76 patients (32.8%) had a major objective response (95% confidence interval [CI], 14% to 37%). The median duration of response was 7 months (range, 1 to 20+). Responses were as likely in patients with disease demonstrated to be unresponsive to anthracycline, ie, de novo resistance (11 of 37, or 30%) as in those with disease that once exhibited anthracycline sensitivity, ie, acquired resistance, (10 of 31, or 32%). G-CSF administration was associated with febrile neutropenic episodes in 36 of 402 cycles (9%) in 16 of 76 patients (21%). CONCLUSION: Paclitaxel's clinically significant activity against metastatic breast cancer extends to patients with many prior chemotherapy regimens. The lack of impact of prior doxorubicin therapy on the likelihood of subsequent response to paclitaxel suggests an important role for this agent in the treatment of refractory metastatic breast cancer. PMID- 7537800 TI - Late relapse of testicular cancer. AB - PURPOSE: This study analyzed a large group of patients with testicular germ cell cancer in complete remission, who relapsed more than 2 years after completion of treatment. PATIENTS AND METHODS: A review of all patients treated at Indiana University Medical Center from 1979 through 1992 for late relapse was conducted. Eighty-one patients were treated for late relapse of testicular cancer. Forty seven patients relapsed more than 5 years after successful management of their initial disease. RESULTS: At initial diagnosis, 35 patients had clinical stage I, 18 stage II, and 28 stage III disease. Twenty-three of 35 stage I, all 18 stage II, and all 28 stage III patients were treated by chemotherapy before their late relapse. The median follow-up duration of patients post-management of late relapse was 4.8 years. Twenty-one patients (25.9%) are continuously disease-free. Nineteen of these 21 patients had surgical resection of carcinoma or teratoma as a component of their therapy. Of sixty-five patients treated for late relapse by chemotherapy, 17 (26.2%) had a complete response, but only two have been continuously disease-free with chemotherapy alone. These two never received prior chemotherapy. CONCLUSION: Late relapse of testis cancer is more common than previously thought. Surgery is the preferred mode of therapy. Chemotherapy has only modest success in this entity, in contrast to the excellent results in de novo germ cell tumors. Patients treated for testicular germ cell cancer need annual follow-up evaluations throughout their life due to the possibility of late relapse. PMID- 7537797 TI - Worsening bone scan in the evaluation of antitumor response during hormonal therapy of breast cancer. AB - PURPOSE: Scintigraphic flare in association with response to therapy has been well described in the medical literature. During the course of a recent breast cancer trial, it became apparent that several patients with worsening bone scan but no other clinical evidence of disease progression might have potentially benefited from continued therapy, but had therapy discontinued. A retrospective analysis of this issue was performed to assess the magnitude and scope of this problem. MATERIALS AND METHODS: A total of 648 patients with hormone receptor positive or unknown advanced breast cancer were treated as part of a large-scale trial of first-line hormonal therapy. Patients were assessed for response to therapy, including response duration, progression-free interval (PFI), overall survival, and quality of life. The retrospective analysis presented here was performed to assess whether patients with a possible scintigraphic flare within the first 16 weeks of therapy might have had therapy discontinued prematurely due to a worsening bone scan attributable to tumor flare, rather than due to disease progression. RESULTS: Analysis of the hormonal trial showed that of 376 assessable patients 108 (29%) with bone disease had a possible scintigraphic flare by week 8 or 16 of the trial, based on data on the case report forms and radiology reports (bone scans and x-rays). Of these, 69 patients (64%) were continued on study therapy, which resulted in clinical benefit in 50 (72%) of those patients. In contrast, 39 patients (36%) with possible scintigraphic flare were removed from the trial. CONCLUSION: We conclude that changes in bone scintigraphy that mimic progressive disease early in the course of hormonal treatment of patients with breast cancer metastatic to bone may represent scintigraphic flare associated with response. Thus, clinicians must be cognizant of the phenomenon of scintigraphic flare to avoid premature discontinuation of a potentially beneficial treatment. PMID- 7537801 TI - Prediction of residual retroperitoneal mass histology after chemotherapy for metastatic nonseminomatous germ cell tumor: multivariate analysis of individual patient data from six study groups. AB - PURPOSE: To develop a statistical model that predicts the histology (necrosis, mature teratoma, or cancer) after chemotherapy for metastatic nonseminomatous germ cell tumor (NSGCT). PATIENTS AND METHODS: An international data set was collected comprising individual patient data from six study groups. Logistic regression analysis was used to estimate the probability of necrosis and the ratio of cancer and mature teratoma. RESULTS: Of 556 patients, 250 (45%) had necrosis at resection, 236 (42%) had mature teratoma, and 70 (13%) had cancer. Predictors of necrosis were the absence of teratoma elements in the primary tumor, prechemotherapy normal alfa-fetoprotein (AFP), normal human chorionic gonadotropin (HCG), and elevated lactate dehydrogenase (LDH) levels, a small prechemotherapy or postchemotherapy mass, and a large shrinkage of the mass during chemotherapy. Multivariate combination of predictors yielded reliable models (goodness-of-fit tests, P > .20), which discriminated necrosis well from other histologies (area under the receiver operating characteristic (ROC) curve, .84), but which discriminated cancer only reasonably from mature teratoma (area, .66). Internal and external validation confirmed these findings. CONCLUSION: The validated models estimate with high accuracy the histology at resection, especially necrosis, based on well-known and readily available predictors. The predicted probabilities may help to choose between immediate resection of a residual mass or follow-up, taking into account the expected benefits and risks of resection, feasibility of frequent follow-up, the financial costs, and the patient's individual preferences. PMID- 7537802 TI - Detection of recurrence in patients with clinical stage I nonseminomatous testicular germ cell tumors and consequences for further follow-up: a single center 10-year experience. AB - PURPOSE: A wait-and-see policy for patients with stage I nonseminomatous testicular germ cell tumors (NSTGCT) was evaluated in a prospective study. The frequency and time of recurrence, detection of recurrence, and presence of unfavorable prognostic factors were investigated. PATIENTS AND METHODS: During the period 1982 to 1992, 154 patients with stage I NSTGCT (median age, 29 years) underwent orchidectomy and were monitored at follow-up evaluation with physical examinations, alfafetoprotein (AFP) and beta-human choriogonadotropin (hCG) levels, chest x-rays (CXR), and computed tomographic (CT) scans of the abdomen and chest. Multivariate logistic regression analyses were performed to identify prognostic factors. RESULTS: During a median follow-up period of 7 years (range, 2 to 12), recurrence was found in 42 patients (27.3%). All cases of recurrence were detected within 2 years, 90% in the first year after orchidectomy. In 29 patients (69.0%), recurrence was detected in the abdominal lymph nodes. Nine patients (21.4%) had metastases in the retroperitoneum and mediastinum and/or lungs, and four patients (9.6%) had metastases only in the mediastinum or lungs. The majority of recurrences (97.6%) were detected by tumor markers and CT scans. Recurrence was related to the presence of vascular invasion, embryonal carcinoma (E), elevated preoperative hCG level, and absence of mature teratoma (M). Only vascular invasion was an independent risk factor. After polychemotherapy treatment for recurrence, the survival rate for the total group was 98.7%. CONCLUSION: The wait-and-see policy is a reliable method for follow-up monitoring of patients with stage I NSTGCT. Even in patients with unfavorable prognostic factors, it is justified to await the possible appearance of metastases. For the future, it is recommended that CXR be omitted from the schedule, and it might be feasible to discontinue follow-up evaluations after 5 years. PMID- 7537803 TI - Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications. AB - PURPOSE: To determine the frequency with which prostate-specific antigen (PSA) positive cells can be detected in the peripheral blood of patients with prostatic cancer in different stages and with different sensitivities to hormonal therapy. PATIENTS AND METHODS: Peripheral blood from 107 men with prostatic cancer and 27 non-prostate cancer controls was analyzed for PSA mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting. RESULTS: The lower limit of detection was one PSA-producing cell diluted into 1 x 10(6) blood mononuclear cells. The test detected PSA mRNA in four of 25 patients (16%) with clinically organ-confined (T1-2) disease, three of 10 (30%) with T3-4 or N+ tumors, and 25 of 72 (35%) with distant metastases. None of the control samples were positive. An increase in positivity was observed with increasing PSA levels. Within the subgroup of patients with distant metastases, positivity was observed in six of 16 patients (38%) with normal or undetectable PSA levels after hormonal therapy and, overall, in 37% of patients (21 of 57) with androgen-independent disease. CONCLUSION: An RT-PCR-based assay for PSA mRNA can detect circulating cells in the peripheral blood of patients with prostatic cancer. The frequency of positivity increases with tumor stage. A unique observation was the detection of cells in patients with no measurable PSA on hormonal therapy. This suggests that continued seeding of distant sites may still be occurring in these patients, despite seemingly successful therapy. The relationship between continued seeding, disease progression, and survival will require further study. PMID- 7537804 TI - Tin-117m(4+)-DTPA for palliation of pain from osseous metastases: a pilot study. AB - The physical and biological attributes of 117mSn(4+)-DTPA indicate that it should be an effective agent for palliative therapy of painful bony metastatic disease. The aim of this study was to evaluate whether or not this agent could effectively reduce pain while sparing the hemopoietic marrow from adverse effects. METHODS: Fifteen patients (10 males and 5 females) with painful bony metastases from various primary cancers were included in the study. Seven patients received 1.22 to 3.11 MBq/kg of 117mSn intravenously (Group 1) and eight patients received 4.85 to 5.77 MBq/kg (Group 2). All but one were treated as outpatients and followed for a minimum of 2 mo. RESULTS: In the first group, pain relief was non assessable in four patients because of death or additional treatment of soft tissue disease by another modality. One patient had no relief of pain, one had complete relief of pain and one had transient relief of pain. No myelotoxicity was observed. For Group 2, three patients achieved complete relief of pain, two good relief, two partial relief and one began to experience pain relief when he suffered a pathological fracture 2 mo post-treatment. None of these patients had myelotoxicity. CONCLUSION: Tin-117m(4+)-DTPA can reduce pain from metastatic disease to bone without inducing adverse reactions related to bone marrow. Further studies are needed to assess tolerance levels for the bone marrow and to evaluate response rates and duration of effect. PMID- 7537805 TI - Simultaneous measurement of one-carbon and polyglutamate derivatives of folic acid in rat liver using enzymatic interconversions of folates followed by ternary complex formation with thymidylate synthetase and 5-fluorodeoxyuridylic acid: standardization of the method. AB - A sensitive assay is needed for the measurement of individual folate derivatives in samples that contain low concentrations of folate. The ternary complex method for the determination of folylpolyglutamates has been combined with procedures for interconverting folate derivatives to provide a method capable of measuring 28 different folate derivatives in biological samples. The method takes advantage of the properties of the ternary complex formed with thymidylate synthetase, fluorodeoxyuridylic acid and 5,10-methylenetetrahydrofolic acid. In the presence of excess purified thymidylate synthetase and excess [3H]fluorodeoxyuridylic acid, limiting concentrations of folates were converted to 5,10 methylenetetrahydrofolate using purified folate interconverting enzymes. This process separated folate derivatives into four groups: Tetrahydrofolate + 5,10 methylenetetrahydrofolate; dihydrofolate; 5,10-methenyl-, 5-formyl-, and 10 formyltetrahydrofolates and 5-methyltetrahydrofolate. Within groups specificity was good, showing little overlap between folates. Sensitivities to 100 fmol of folate were achievable and to 1 pmol were standard. Recoveries were linear for each of the groups in this system to 50 pmol of folate. Ternary complexes containing different polyglutamates were separated by isoelectric focusing, visualized by fluorography and measured by densitometry. The densitometry was linear with folate concentration in the range 20-200 fmol for each of the polyglutamates. Primary and secondary coefficients of variation were determined. This method provides the sensitivity to measure individual folates in the femtomole range and the flexibility to determine the concentrations of 28 separate pools of folate derivatives, differentiating between derivatives of the pteridine moiety and glutamate chain length simultaneously. PMID- 7537806 TI - Protein synthesis is stimulated in nutritionally obese rats. AB - To investigate the tissue growth and the protein synthesis in vivo in nutritional obesity we used lipid-rich multichoice diet feeding. Young male rats of the Wistar strain were divided in two groups: control and obese. Control rats were fed pelleted nonpurified diet. Obese rats were fed a multichoice diet based on a variety of highly palatable energy-rich human foods for 30 d. Protein intake was kept equal in the groups to avoid its influence on protein turnover. The tissue growth pattern was evaluated by protein, DNA and RNA contents of liver, kidney, heart, skeletal muscles and small intestine. Protein synthesis in vivo was measured in these tissues by the phenylalanine flooding-dose technique. Rats fed the multichoice diet showed significantly greater body growth when compared with rats fed the nonpurified diet. Adipose and other tissue weights were significantly greater in the obese rats. The tissue growth pattern was characterized mainly by hyperplasia. In most tissues the net protein accretion found in obese rats resulted from an enhancement in the fractional rate of protein synthesis. The greater protein synthesis was due to an increase in the efficiency of ribosomes in kidney, heart and skeletal muscle and to an increase in the synthetic capacity in liver and small intestine. These data suggest that excess energy intake when protein intake is adequate stimulates tissue growth and protein synthesis in rats. PMID- 7537807 TI - Human growth hormone but not human insulin-like growth factor-I enhances recovery from neonatal malnutrition in rats. AB - The ability of hormonal therapy to enhance recovery from neonatal malnutrition was assessed in rats. Pups were malnourished via maternal food restriction (60% control intake). On d 16, restricted pups (n = 50) (62% control body wt) were refed until d 20 and were given growth hormone, insulin-like growth factor-I, destripeptide [1,3] insulin-like growth factor-I, or saline (placebo). Refed placebo pups attained 80% of control body weight by d 20. Growth hormone treatment caused a greater weight gain than refeeding alone (P < or = 0.05). Treatment with insulin-like growth factor-I and destripeptide [1,3] insulin-like growth factor-I did not affect body weight. All three hormones increased spleen and kidney weights (P < or = 0.05) compared with the refed-placebo group, whereas only growth hormone increased muscle weight (P < or = 0.05). Malnourished pups had lower serum insulin-like growth factor-I (P < or = 0.05) and insulin-like growth factor binding protein-3 (P < or = 0.05), and higher serum insulin-like growth factor binding protein-2 (P < or = 0.05) and hepatic insulin-like growth factor binding protein-2 mRNA (P < or = 0.05) than controls. Refeeding increased serum insulin-like growth factor-I compared with restricted pups (P < or = 0.05), and insulin-like growth factor-I treatment caused a further increase in serum insulin-like growth factor-I compared with the refed-placebo group (P < or = 0.05). These results show that growth hormone was moderately effective at increasing body weight gain and muscle growth during recovery from malnutrition. PMID- 7537808 TI - Use of L-arginine in the treatment of experimental necrotizing enterocolitis. AB - Although multifactorial in etiology, prematurity and feeding are two of the most common risk factors associated with necrotizing enterocolitis (NEC). To understand the pathogenesis of NEC, the complex interaction between intestinal contents and clearing mechanisms in the immature human gut must be elucidated. Nitric oxide (NO) is a proposed mediator of nonadrenergic noncholinergic neural inhibition, causing relaxation in the gut. In addition to its role as a neuroeffector substance, studies suggest that endogenous formation of NO maintains intestinal mucosal integrity, protecting the gut from blood-borne toxins and tissue-destructive mediators. Thus, NO has a dual role in both gut smooth muscle relaxation and mucosal protection. Because two of the primary risk factors in the development of NEC are prematurity (as it relates to gut dysmotility) and enteral feeding (as it relates to mucosal damage by intraluminal substrate), the authors chose to investigate the role of NO in the pathogenesis of NEC induced by intraluminal injection of acidified casein solution in neonatal piglets. METHODS: Having confirmed the consistent induction of NEC both macroscopically and histologically with this model (n = 32), the following were undertaken. Neonatal piglets (< 3 days old) were laparotomized, and intestinal loops were created from the terminal ileum to the proximal colon. The loops were injected with acidified casein solution and separated by saline-injected control loops. When the abdomen was closed, a continuous peripheral intravenous infusion of L-arginine, an NO synthase substrate (600 mg/kg/h [n = 6]), or N-omega-nitro-L arginine methyl ester (L-NAME), an NO synthase inhibitor (20 mg/kg/h [n = 6]), was begun. Gut segments were harvested 3 hours later and processed for evaluation of the extent of necrosis. RESULTS: Macroscopically, the L-NAME-treated group showed areas of hemorrhagic necrosis in the NEC-induced loops. The L-arginine treated group had greatly diminished or virtually absent lesions. H&E-stained sections were graded microscopically, using a scale from 0 to 4, ranging from intact villi (grade 0) to transmural necrosis (grade 4). In the untreated NEC group, intestinal damage in the acidified casein loops was exhibited by areas of necrosis (extending, in some cases, transmurally), submucosal edema, and inflammatory cell infiltrate (average grade, 3.5). In the L-NAME-treated group, the intestinal damage was similar to that of the NEC-induced group (average grade, 3.5), but also presented with marked hemorrhagic congestion. In the L arginine group, NEC-induced tissue damage was greatly attenuated, with necrosis limited primarily to the villus tips (average grade, 1). Nevertheless, inflammatory cell infiltrate and mild submucosal edema were still present. CONCLUSION: Continuous intravenous infusion with the NO synthase substrate L arginine markedly attenuates intestinal injury in this neonatal piglet model of NEC. Intravenous administration of the NO synthase inhibitor L-NAME causes hemorrhagic congestion of the gut wall. Based on these findings, the authors propose that treatment with the amino acid L-arginine should be considered as a potential therapeutic modality for NEC. PMID- 7537809 TI - Diagnosis, management, and outcome of cervicofacial teratomas in neonates: a Childrens Cancer Group study. AB - The management of cervicofacial teratomas in neonates is often complicated and may result in significant morbidity and death. A Childrens Cancer Group (CCG) retrospective study was conducted to evaluate a multiinstitutional experience with the treatment of these extremely rare neoplasms. Twenty neonates with cervicofacial teratomas, presenting from 1971 to 1994, were identified from nine CCG institutions. Fourteen neonates had cervical teratomas, and six had orofacial teratomas. There were 12 males and eight females. A diagnostic prenatal ultrasound examination was performed in six cases. Life-threatening airway obstruction occurred in seven infants (35%) in the early postnatal period. Two neonates died in the delivery room without ever having their airway secured. Two other infants with a prenatal diagnosis survived only because tracheostomies were performed by pediatric surgeons who were in the delivery room. Three other patients were orally intubated, one after sustaining hypoxic cardiac arrest. Eighteen infants had their primary tumor excised. Three patients required tracheostomy. After resection, two patients had evidence of unilateral recurrent laryngeal nerve injury, and two required prolonged thyroid hormone replacement. Histological examination showed eight mature and seven immature teratomas. Four infants (20%) clearly had malignant lesions. Pulmonary metastases occurred in two patients and contributed to one late death at 6 months of age. The overall survival rate was 85%, and the mean follow-up period was 5 years (range, 2 months to 16 years). Twelve of 17 surviving patients (70%) have had an excellent functional and cosmetic outcome. Four children have varying degrees of developmental delay and mental retardation. Hypoxia at birth was believed to have contributed to these problems in two cases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537810 TI - Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia. AB - The offspring of pregnant Sprague-Dawley rats exposed to nitrofen on gestational day 9.5 develop left-sided congenital diaphragmatic hernia (CDH). Twenty-four hours after treatment, on day 10.5, supravital staining with Nile blue sulfate and histological examination showed bilateral excessive cell death in cervical somites 2 through 4. After 48 hours, on day 11.5, cell death was absent in the cervical somites but was apparent in the mesoderm adjacent to the somites in the septum transversum and in the developing sympathetic ganglia adjacent to the dorsal aortae. Cell death was not apparent in the foregut or lung primordia on either day 10.5 or 11.5. The incidence of nitrofen-exposed embryos with such patterns of cell death closely paralleled that of left-sided CDH in similarly treated day 21.5 fetuses. Control animals treated with olive oil had normal programmed cell death patterns in the regions of interest and had no evidence of CDH on day 21.5. It is possible that these patterns of excessive cell death early in gestation may play a role in the genesis of diaphragmatic hernia. Mesoderm derived from cervical somites 3 through 5 contributes to the diaphragmatic anlage and forms the major portion of the muscle of the diaphragm. Because nitrofen damages mesodermal cell populations in cervical somites 2 through 4 and in the mesenchyme adjacent to the septum transversum 24 to 48 hours after administration, the authors propose that damage to these populations may reduce progenitor cells needed to populate the diaphragmatic anlage, thereby hindering pleuro-peritoneal canal closure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537811 TI - Neurocognitive outcome for neonates treated with extracorporeal membrane oxygenation: are infants with congenital diaphragmatic hernia different? AB - The neurocognitive outcome for infants with congenital diaphragmatic hernia treated with extracorporeal membrane oxygenation (ECMO) is compared with that of neonates treated with ECMO for other diagnoses. The mean age at the time of the latest assessment (for the 51 survivors with adequate follow-up) was 31 months. The neurological outcomes did not differ significantly. However, the cognitive outcome for infants with congenital diaphragmatic hernia was significantly poorer than for those without it. This was particularly true if the infant with congenital diaphragmatic hernia was a boy and his mother had limited formal education. PMID- 7537812 TI - Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by penasterol and penasterone. AB - Penasterol and penasterone, constituents of the Okinawan marine sponge Penares incrustans, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The concentrations of penasterol and penasterone required for 50% inhibition of anti-IgE-induced histamine release (IC50) were 0.5 and 1.5 microM, respectively. Both compounds dose-dependently inhibited phospholipase A2 (PLA2) activity. Moreover, they inhibited anti-IgE-induced [3H]arachidonic acid from rat mast cells. These results suggest that the mechanism of inhibition by these compounds of the histamine release induced by anti-IgE was through the inhibition of PLA2. PMID- 7537813 TI - Mechanosensitive ion channels in skeletal muscle from normal and dystrophic mice. AB - 1. We examined the activity of single mechanosensitive ion channels in recordings from cell-attached patches on myoblasts, differentiated myotubes and acutely isolated skeletal muscle fibres from wild-type and mdx and dy mutant mice. The experiments were concerned with the role of these channels in the pathophysiology of muscular dystrophy. 2. The predominant form of channel activity recorded with physiological saline in the patch electrode arose from an approximately 25 pS mechanosensitive ion channel. Channel activity was similar in undifferentiated myoblasts isolated from all three strains of mice. By contrast, channel activity in mdx myotubes was approximately 3-4 times greater than in either wild-type or dy myotubes and arose from a novel mode of mechanosensitive gating. 3. Single mechanosensitive channels in acutely isolated flexor digitorum brevis fibres had properties indistinguishable from those of muscle cells grown in tissue culture. The channel open probability in mdx fibres was approximately 2 times greater than the activity recorded from wild-type fibres. The overall level of activity in fibres, however, was roughly an order of magnitude smaller than in myoblasts or myotubes. 4. Histological examination of the flexor digitorum brevis fibres from mdx mice showed no evidence of myonecrosis or regenerating fibres, suggesting that the elevated channel activity in dystrophin-deficient muscle precedes the onset of fibre degeneration. 5. An early step in the dystrophic process of the mdx mouse, which leads to pathophysiological Ca2+ entry, may be an alteration in the mechanisms that regulate mechanosensitive ion channel activity. PMID- 7537814 TI - Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors. AB - 1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5 dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis. PMID- 7537817 TI - Selective regional vasoconstriction underlying pressor effects of galanin in anaesthetized possums compared with cats. AB - 1. Intravenous administration of porcine galanin (5 nmol kg-1) caused a rise in mean blood pressure in the brush-tailed possum, Trichosurus vulpecula, from 58 +/ 1.6 to 106 +/- 1.6 mmHg. This effect is in contrast to the cat, in which no significant change in blood pressure was recorded in response to galanin (88 +/- 2.3 vs. 86 +/- 2.4 mmHg). 2. Cardiac output and regional blood flow distribution were assessed by distribution of radioactive microspheres in four anaesthetized possums and four cats, before and after administration of galanin. 3. Cardiac output was 289.8 +/- 14.0 ml min-1 in the cat and 189.9 +/- 25.5 ml min-1 in the possum. Galanin administration did not significantly change cardiac output in either species. 4. In the possum, galanin administration caused large increases in resistance to flow in the spleen, gut, adrenal glands, kidney, skin and carcass. The largest increase was in the kidneys, where renal blood flow fell to 6% of control levels. 5. In the cat, changes in resistance were small. Small increases in resistance to flow in muscle and carcass were offset by small decreases in resistance in the lungs and kidneys. 6. The results suggest that the pressor effect of galanin in the possum is the result of direct vasoconstrictor action in several vascular beds, in contrast to the cat, in which such effects are few and weak. PMID- 7537815 TI - Voltage-clamp analysis of gap junctions between embryonic muscles in Drosophila. AB - 1. Intercellular communication between embryonic muscle fibres was examined in Drosophila melanogaster. 2. Injection of fluorescent dye revealed extensive coupling between muscle fibres which form a uniform communicating arrangement of cells without restriction at the segmental borders. 3. Dye transfer was blocked by octanol and membrane depolarization suggesting that it is mediated by gap junctions. 4. Double voltage-clamp experiments from cell pairs in situ showed that the ionic coupling is sensitive to the voltage difference between the cytoplasm and the extracellular space (transmembrane voltage, Vi-o) as well as between the cells (transjunctional voltage, Vj). 5. In steady-state conditions, the gap conductance (gj) was maximal for hyperpolarized Vi-o and decreased progressively to near zero as Vi-o became more positive than -50 mV. 6. Gap conductance decreased from a maximal value as Vj increased either in the positive or negative direction (by depolarizing or hyperpolarizing, respectively, one of the cells from a holding potential of -60 mV). In both cases, gj asymptotically approached a non-zero residual value which was different for negative and positive Vj (about 20% of the maximal conductance for negative transmembrane potentials and 10% for positive values). 7. Application of octanol (1 mM) resulted in an almost complete and reversible block of gj. PMID- 7537816 TI - Nitric oxide modulation of quantal secretion in chick ciliary ganglia. AB - 1. Long-term potentiation of quantal secretion was studied at ciliary ganglion synapses of post-hatched birds following tetanic stimulation of the oculomotor nerve and the effects of nitric oxide (NO) on quantal secretion were determined. 2. Tetanic stimulation of the oculomotor nerve at 30 Hz for 20 s at room temperature increased the amplitude of the excitatory postsynaptic potential (EPSP) by about 100%; 1-2 min after the tetanus the EPSP declined exponentially with a time constant of about 10 min (long-term potentiation; LTP). LTP was due to an increase in the quantal content of the EPSP not to a change in quantal size. 3. A component of LTP was shown to be due to the release of NO in the ganglion, as blocking the synthesis of NO with L-arginine methyl ester decreased the potentiation by 70%. 4. Exogenous application of NO using sodium nitroprusside increased the amplitude of the EPSP by more than 30% due to an increase in the quantal content of the EPSP. 5. Both 8-bromo-cGMP and 8-bromo cAMP increased the quantal content of the EPSP by more than 44% without changing the quantal size. 6. The results suggest that endogenous NO is involved in either the initiation or maintenance phase of LTP. This may occur through an increase in quantal secretion consequent on the action of an elevated cGMP increasing cAMP. PMID- 7537818 TI - Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels. AB - 1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta with alpha 1-subunits in oocytes and other expression systems. 4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha 1-subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties. PMID- 7537819 TI - Calcium-dependent inactivation of heteromeric NMDA receptor-channels expressed in human embryonic kidney cells. AB - 1. Whole-cell current through heteromeric NR1-NR2A and NR1-NR2B subunit combinations of NMDA channels transiently expressed in human embryonic kidney cells (HEK 293) were studied using the patch-clamp technique. 2. With 4 mM Mg-ATP in the internal pipette solution, the responses of cells expressing NR1-NR2A channels to glutamate application gradually decreased, reaching 50% of control during the first 20 min of recording. This process was accompanied by acceleration of desensitization. 3. Conditioning (5-15 s) applications of glutamate (100 microM) induced a transient inactivation of NR1-NR2A and NR1-NR2B channels (20-40%) with a slow time course of recovery (tau r = 10-60 s). Both the degree of inactivation and the time constant of recovery increased with the duration of conditioning applications of glutamate, and with an elevation of Ca2+ in the external solution. 4. These results show that both NR1-NR2A and NR1-NR2B recombinant NMDA receptor-channels expressed in HEK 293 cells can be transiently inhibited by Ca2+ ions in a similar way to that described for hippocampal neurones. PMID- 7537820 TI - The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat. AB - 1. The actions of dynorphin on N-methyl-D-aspartate (NMDA) responses were examined in acutely dissociated trigeminal neurons in rat. Whole-cell and single channel currents were recorded using the patch clamp technique. 2. Dynorphins reduced NMDA-activated currents (INMDA). The IC50 was 0.25 microM for dynorphin (1-32), 1.65 microM for dynorphin (1-17) and 1.8 microM for dynorphin (1-13). 3. The blocking action of dynorphin is voltage independent. 4. The inhibitory action of dynorphin cannot be blocked by high concentration of the non-selective opioid receptor antagonist naloxone, nor by the specific kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI). 5. Single-channel analyses indicate that dynorphin reduces the fraction of time the channel is open without altering the channel conductance. 6. We propose that dynorphin acts directly on NMDA receptors. PMID- 7537822 TI - Laparoscopic management of cancer patients. AB - All the reported experience indicates that laparoscopic staging of tumours provides additional information to the preoperative work-up. Its diagnostic yield is enhanced by contact ultrasonography. Staging laparoscopy may avoid needless laparotomy and permits palliative surgical treatment, bypass and resection. The major controversy concerns laparoscopic ablative procedure for potentially curative cancer in view of the potential but unproved risk of tumour dissemination and inadequate resection. Until further information becomes available from experimental investigations and centralized clinical studies, laparoscopic curative resections should not be performed routinely. Laparoscopic or retroperitoneoscopic lymphadenectomy appears to be useful by documenting node positive disease. Laparoscopic hepatic cryotherapy with insulated needle probes has the potential for control of secondary hepatic disease not amenable to surgical resection. PMID- 7537824 TI - Immunohistochemical analysis of beta 1-integrin receptors displayed by murine uterine natural killer cells over the course of successful pregnancy. AB - Granulated metrial gland (GMG) cells are a natural killer (NK) cell-like population present in large numbers in the pregnant rodent uterus. By day 8 of gestation GMG cells are large and granulated and localized to the mesometrial side of each implantation site. GMG cells appear to be highly migratory both in vivo and in vitro; however, little is known regarding their functions. Using indirect fluorescence immunohistochemistry, murine uteri and implantation sites were studied on successive days of gestation to characterize the extracellular matrix receptors of the VLA-integrin family displayed by GMG cells. On days 3 and 6 of gestation, double immunostaining using the monoclonal antibody LGL-1 was employed to recognize GMG cells because their morphology early in pregnancy resembles that of other lymphocytes. Between days 8-15 of gestation, GMG cells can be recognized by their unique morphology. The day 3 and day 6 LGL-1+ cells were positive for all antigens examined; that is, beta 1 plus alpha 1, alpha 3, alpha 4, alpha 5 and alpha 6. From days 8-15 of gestation, GMG cells were beta 1+, alpha 4+, alpha 5+ but alpha 1-, alpha 3-, alpha 6-. Thus, between days 6-8 of gestation, major changes occur in the uterine NK/GMG cell population which include the loss of the surface molecules VLA alpha 1, alpha 3 and alpha 6 or the rapid expansion of NK cells not expressing these proteins. It is postulated that major changes in the functions of uterine NK cells are likely to be associated with these alterations in cell surface phenotype and that functional studies of uterine NK cells should focus upon this relatively early gestational time point. PMID- 7537821 TI - Vasodilatation and smooth muscle membrane potential changes in arterioles from the guinea-pig small intestine. AB - 1. Dilatation of arterioles isolated from the guinea-pig small intestine was evoked by stimulation of a submucous ganglion and the application of acetylcholine, vasoactive intestinal peptide, galanin or dynorphin A. Changes in arteriole diameter and smooth muscle membrane potential were recorded simultaneously. 2. Ganglion stimulation caused vasodilatation and smooth muscle hyperpolarization that varied in both amplitude and time course from one arteriole to another. Vasodilatation could occur without hyperpolarization. 3. Vasodilatation caused by acetylcholine was accompanied by a rapidly developing hyperpolarization that began to decline before the maximum vasodilator effect had developed. 4. Vasoactive intestinal peptide caused dilatation without any change in smooth muscle membrane potential. 5. Galanin and dynorphin caused dilatation and a hyperpolarization of similar time course to the dilatation. 6. In 48% of arterioles tested the dilatation appeared to be mediated solely by acetylcholine. In 31% there was a cholinergic component, but no evidence for the involvement of acetylcholine in the remaining 21%. When the non-cholinergic dilatation occurred without a hyperpolarization we conclude that it was due to vasoactive intestinal peptide; otherwise it may have been due to either galanin or dynorphin. PMID- 7537828 TI - Assessment of quantitative models for plasmid ColE1 copy number control. AB - Two quantitative models of plasmid ColE1 copy number control are compared with respect to mathematical logic of derivation and application to experimental observations. Explanatory background material and clarifications are supplied for selected aspects of each model. Contrasting features are emphasized and experiments are suggested to distinguish between predictions of the models. PMID- 7537823 TI - A visual aid to assess diagnostic peritoneal lavage fluid. PMID- 7537825 TI - Human CD5+ B lymphocytes (B-1 cells) decrease in peripheral blood during pregnancy. AB - Pregnancy is a unique immunologic state where a natural homeostasis exists between antigenically different tissues. Several earlier studies have addressed the fluctuations in the number and/or function of lymphocytes, including B cells during pregnancy, but changes within the subsets of B lymphocytes, conventional (CD5-) and B-1 (CD5+), have not been addressed. Here we demonstrate that the frequency of B-1 cells decreases dramatically during pregnancy, whereas the frequency of conventional B cells remains relatively constant. The missing B-1 cells return to pre-pregnancy levels 8-10 weeks after parturition. The polyreactive autoantibodies secreted by B-1 cells have been implicated in autoimmunity and immune regulation. The possible role of B-1 cells during pregnancy will be discussed in that context. PMID- 7537827 TI - Immunologic assessment during treatment of rheumatoid arthritis with anti-CD5 immunoconjugate. AB - OBJECTIVE: To determine if sustained immunologic effects occurred after treatment of patients with rheumatoid arthritis (RA) with an immunoconjugate of murine anti CD5 monoclonal antibody with ricin A chain (anti-CD5). METHODS: We measured lymphocyte populations, mitogen induced peripheral blood mononuclear cell (PBMC) stimulation, cytokine levels, immunoglobulin levels, in vivo immune function, and clinical outcomes in 9 patients with RA treated with anti-CD5. RESULTS: The treatment of patients with RA with anti-CD5 was associated with marked acute depletion of peripheral blood lymphocytes (p < 0.01) during and immediately after treatment. A sustained decrease in the number of CD3, CD4, CD5, and CD8 bearing lymphocytes persisted for 2 months after treatment (p < 0.05). After 3 months a mild decrease in the number of these lymphocyte populations persisted, but when compared to baseline values, the differences were not found to be statistically significant. Phytohemagglutinin induced PBMC proliferation was decreased at the 3 month followup (p < 0.05). Evaluations of mitogen induced cytokine and immunoglobulin production, immunoglobulin level, autoantibody, and in vivo antibody response to tetanus toxoid did not show any consistent change from baseline. CONCLUSION: Anti-CD5 treatment of RA appears to be associated with a decrease in the population of cells bearing CD5, but does not appear to induce any persistent immunologic abnormalities. PMID- 7537826 TI - Complete response of a persistent ectopic pregnancy to dactinomycin after methotrexate failure. A case report. AB - Chemotherapy has become an accepted alternative for patients with persistent ectopic pregnancy, and methotrexate is the principal agent used. A patient with a persistent ectopic tubal pregnancy that failed to respond to methotrexate chemotherapy was treated with dactinomycin, with a complete response. PMID- 7537829 TI - The human proteasome subunit HsN3 is located in the inner rings of the complex dimer. AB - Subunit HsN3 of the human proteasome is a beta-type subunit homologous to PRE4 from yeast, X1 beta from Xenopus and RN3 from the rat. Using electron microscopy, the binding sites of a monoclonal antibody with specificity for subunit HsN3 have been located in the two juxtaposed inner rings of the human proteasome. Subunit HsN3 was present in two copies, one in each ring, in accordance with our concept of two identical halves making up the complete human proteasome. The subunit is involved in the trypsin-like as well as the peptidylglutamyl-peptide cleavage activities. PMID- 7537831 TI - [Current state of national or international standards of interferons and cytokines]. AB - Expert committee on Biological standardization (ECBS) of WHO had asked for an advisory committee for preparation of WHO international cytokine standards because of complexities by the rapid increase of number of cytokine molecules some of which are entered into clinical trials. The new meeting of the WHO informal Consultation on Cytokine standards was started from last spring and going to be held annually. The main mission of the committee was considered to be promoting the use of WHO international cytokine standards and reference materials etc., which must be available world wide. Various standard preparations of cytokines and growth factors are available at the National Institute of Biological Standards and Control (NIBSC) in the United Kingdom, some of which have been accepted by WHO as international standards (Table 1). Japanese national standards which had been prepared before establishment of WHO standards, and being labeled as Japanese reference Units (JRU) have to be calibrated with WHO standards to know International Units (IU). Japanese national references of IL-2 and TNF alpha were calibrated by collaboration with related companies and NIH Japan and the concluded results are obtained as follows. IL-2 (rec. DNA):1JRU = 1 IU TNF alpha (rec. DNA):1JRU = 35 IU PMID- 7537832 TI - [Quantitative flowcytometric analysis of B cell surface antigens in patients with autoimmune diseases]. AB - In 47 cases of autoimmune diseases (25 SLE, 9 MCTD, 6 PSS, 7 RA), two B cell surface antigens, CD19 and CD20 were quantitatively measured with a flowcytometer. When compared with the results of healthy controls, CD19 antigens were observed to be ca. 10% decreased in SLE and ca. 20% increased in RA. CD20 antigens were observed to be ca. 25% increased in SLE and no changes in RA. No significant increase nor decrease was observed with MCTD and PSS patients. In SLE patients, the amount of CD19 antigen expression was positively related to serum C3 levels, and that of CD20 antigen was negatively related to it. It is not obvious in what mechanism the expression of these antigens are regulated, however, from the point that the expression relates to the amount of serum C3 level, the quantitative measurement of CD19 and CD20 antigens is possibly be a good marker to detect the state of autoimmune disease such as SLE. PMID- 7537834 TI - [Phase II clinical trial of the radioactive strontium (89Sr) chloride agent, SMS. 2P for pain palliation in patients with prostate cancer with bone metastases]. AB - The phase II clinical trial of a strontium (89Sr) chloride agent (SMS. 2P) was performed in 9 patients with painful bone metastases secondary to prostate cancer. After an intravenous administration, 89Sr circulated in the plasma and was rapidly cleared. Urinary excretion varied widely among the patients. No serious acute side effects were observed. A mild transient increase in pain was reported by 4 patients 2-4 days after administration, two of whom complained of mild nausea or vomiting. All symptoms improved and never became a clinical problem. There were some abnormal hematological parameters. In particular, a decrease in the platelet level seemed to be a marrow suppression due to 89Sr irradiation. It is difficult to discriminate between the effects of 89Sr and the progress of the disease using tumor markers. The pain level improved within 2 weeks after administration and the effect continued for at least 8 weeks, which improved the quality of life for these patients. PMID- 7537833 TI - [The phenotypic expressions of AFP producing gastric carcinoma]. AB - The phenotypic expressions of AFP producing gastric carcinomas were investigated by histological, immunohistochemical, mucinhistochemical and biochemical studies. Intramucosal histology was well differentiated adenocarcinoma in fifteen out of seventeen cases. In the invasive area, papillary or papillary and solid structure of the cancer cells with clear cytoplasms were in fourteen cases, but there were only three cases that showed hepatoid structure. In the mucinhistochemistry, thirteen cases (76%) showed intestinal type mucin. And in the lectin binding property of serum AFP, five of seven cases showed yolk sac tumor like pattern and the other two cases showed hepatic pattern. These results suggests that most of the AFP producing gastric carcinomas derive from intramucosal intestinal type well differentiated adenocarcinoma, that retrodifferentiated to fetal intestine in the invasive area and produce fetal intestinal type AFP. PMID- 7537835 TI - Reliability of maximum number of simultaneously open channels as an estimator for the number of channels in single-channel recordings. AB - The single-channel recording technique has revolutionized the electrophysiological study of the ion channels. Interpretation of single-channel recordings often requires the exact number of channels present in a given patch- for example, derivation of the probability of a channel being open--and most theories handling gating kinetics assume that there is only one channel in the patch. Most investigators have estimated the number of channels as the number of maximal simultaneously open channels observed. Although this method is easy to understand and apply, examination of the validity of such estimation has not been much conducted before. As an attempt for such an examination, we have applied computer simulation method in order to obtain practically useful aids in judging the reliability of such estimates. Results show that (i) large estimated values (for example, larger than 10) are likely to be incorrect, and (ii) when the estimated value is small and the derived probability of a channel being open is within a certain range, the estimated number of channels is most likely to be correct. PMID- 7537836 TI - Aprotinin and vein graft occlusion after coronary artery bypass. PMID- 7537830 TI - Type I collagen packing, conformation of the triclinic unit cell. AB - The X-ray diffraction pattern of tendon collagen can contain a number of sharp Bragg peaks indicating three-dimensional crystallinity of the sample. Optimal diffraction images have been obtained with a high flux synchrotron X-ray source and a carefully maintained sample environment and staining techniques. The Bragg peaks are always superimposed on a diffuse background. This makes interpretation of data difficult and a number of conflicting models of collagen packing have been proposed. The removal of the diffuse scatter from the images allows the Bragg peaks to be seen on a relatively flat background. This was conducted by modelling the background points as a series of two-dimensional polynomial functions. The resultant set of observed Bragg reflections serves as an excellent basis to test the validity of two contradictory packing modes; (1) the triclinic model, Fraser et al., (2) the microfibril model, Kajava. From this it can easily be seen that the model proposed by Kajava is inappropriate, since there is limited agreement between predicted positions of reflections and the positions of observable reflections on film. The packing of collagen molecules on a triclinic lattice is favoured by this criterion. PMID- 7537837 TI - Peptide-based tachykinin NK2 receptor antagonists. PMID- 7537838 TI - In vitro systems for studying intestinal drug absorption. PMID- 7537839 TI - CD44v6 and CD44v3 variants in inflammatory bowel disease. PMID- 7537840 TI - Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn's disease. AB - Immune mechanisms, possibly involving cell-surface molecules such as CD44, have been invoked to explain the pathogenesis of inflammatory bowel disease. We used monoclonal antibodies against epitopes encoded within the variable region of CD44 to investigate CD44 isoform expression in colon, small intestine, and liver in patients with various intestinal disorders and in controls. Biopsy samples from patients with ulcerative colitis showed significantly increased epithelial expression of CD44 isoforms containing the v6 and v3 epitopes, detected with antibodies 2F10 and 3G5, respectively. CD44v6 was detected on colonic crypt epithelial cells in 23 of 25 ulcerative colitis samples compared with 3 of 18 colonic Crohn's disease samples (p = 3.0 x 10(-6); odds ratio 57.5 [95% CI 6.83 702]) and 3 of 52 controls (22 normal colon, 10 infective colitis, 2 radiation colitis, and 18 colonic Crohn's disease; p < 1 x 10(-8); odds ratio 199 [25.5 2294]). No significant expression of CD44v6, CD44v3, or CD44v8/9 was found in samples of normal proximal colon from 4 patients with distal ulcerative colitis, whereas samples from the affected area showed staining for CD44v6 and CD44v3. No expression of CD44 variants was found in 15 samples of normal small intestine, 11 small-bowel pouchitis, 8 coeliac disease, 3 small-bowel Crohn's disease, 6 normal liver, 6 primary biliary cirrhosis, or 9 primary sclerosing cholangitis. The high intensity of CD44v6 and v3 epitope expression on crypt epithelial cells in ulcerative colitis suggests that CD44 isoforms may have an important role in ulcerative colitis. Their detection could have diagnostic potential in differentiating ulcerative colitis from other forms of colonic inflammation including Crohn's disease. PMID- 7537841 TI - Transmission of hepatitis C virus to children and husbands by women infected with contaminated anti-D immunoglobulin. AB - In 1978-79 a single-source outbreak of hepatitis C occurred in 2533 women who had received virus-contaminated anti-D immunoglobulin. Children and husbands of 74 women with self-limited, and of 86 women with chronic, hepatitis C were followed up for over 10-15 years. In 3 of 231 investigated children (1.3%) serological evidence for HCV infection was found. However, none of the children developed an apparent or chronic hepatitis. Serum samples of the 94 husbands investigated showed no HCV antibodies or HCV RNA. We consider the risk of intrauterine or perinatal transmission of HCV, as well as that of transmission through close family contacts, to be low. No evidence was found for sexual transmission for women to men. PMID- 7537842 TI - CD44 isoforms in prognosis of breast cancer. PMID- 7537843 TI - Euthanasia. PMID- 7537844 TI - Felbamate selectively blocks in vitro hippocampal kainate-induced irreversible electrical changes. AB - The influence of the anticonvulsant felbamate has been tested on in vitro excitotoxicity induced by treatment of hippocampal slices with elevated concentrations of NMDA, AMPA and kainic acid. For comparison, the effects of the glutamate antagonist 7-chlorokynurenic acid and of the anticonvulsants pentobarbitone and lamotrigine, were also studied. Slice perfusion with 50 microM NMDA or 25 microM AMPA or 12 microM kainic acid produced within 30 min a disappearance or a pronounced irreversible amplitude reduction of the CA1 electrical synaptic responses. Slice perfusion with 1.2-1.6 mM felbamate or 100 microM lamotrigine significantly decreased the incidence of the irreversible disappearance of the CA1 electrical response induced by kainic acid. On the contrary, slice perfusion with the same concentrations of the drugs did not affect the irreversible disappearance of the CA1 electrical response induced by NMDA or AMPA. By contrast, slice perfusion with 100 microM of 7-chlorokynurenic acid significantly prevented the neurotoxic effects induced by both NMDA and kainic acid, while 100 microM of pentobarbitone failed to affect kainic acid induced neurotoxicity. The different profile of neuroprotection elicited by felbamate with respect to reference drugs indicates that a different mechanism of action than antagonism of NMDA response or potentiation of GABA response underlies the neuroprotectant effects of felbamate. PMID- 7537845 TI - Calcium efflux from cultured bovine adrenal chromaffin cells induced by pituitary adenylate cyclase-activating polypeptide (PACAP): possible involvement of an Na+/Ca2+ exchange mechanism. AB - The effect of pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. PACAP1 38 stimulated the efflux of 45Ca2+ from the cells in a concentration dependent manner (10(-9)-10(-7)M). This effect was inhibited by its potent receptor antagonist PACAP6-38. PACAP1-38 increased the formation of [3H]inositol phosphates and cyclic AMP in the cells. Forskolin, an activator of adenylate cyclase, also stimulated the efflux of 45Ca2+ from the cells. 3-Isobutyl-1 methylxanthine (IBMX), an inhibitor of phosphodiesterase, enhanced PACAP1-38 induced 45Ca2+ efflux from the cells. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, had no effect on the efflux of 45Ca2+ from the cells. The increases in 45Ca2+ efflux induced by PACAP1-38 and forskolin were reduced by deprivation of extracellular Na+ and the Na+/Ca2+ exchange inhibitor amiloride. In addition, PACAP1-38 stimulated 22Na+ influx into the cells, and this action was inhibited by amiloride. These results suggest that PACAP1-38 stimulates an Na+/Ca2+ exchange mechanism through activation of adenylate cyclase in cultured bovine adrenal chromaffin cells. PMID- 7537846 TI - [Granulocyte colony-stimulating factor: biologic effects and possibilities of therapeutic use]. AB - This review is a brief overview on recent advances in biology as well as in the potential clinical application of recombinant methionil human granulocyte colony stimulating factor (r-metHuG-CSF). Biologically active human granulocyte colony stimulating factor is a recombinant human protein expressed in Escherichia coli. It is localized on membrane of stroma cells in molecules of extracellular matrix. R-metHuG-CSF increases the number of circulating neutrophils, and to a much lesser extent macrophages. The clinically relevant uses of r-metHuG-CSF at present lie in two general areas: neutropenia and febrile netropenia present in patients with non-myeloid malignant diseases induced by anticancer agents. R metHuG-CSF produces dose-dependent and reversible side effects such as: musculoskeletal pain, urinary disorders, retention of fluid, pericarditis, dyspnea, hypoxia and hypotension. PMID- 7537847 TI - [The morphological and histochemical characteristics of the interprismatic structures and the human enamel. A light microscopy study (corrected)]. AB - Inter-rod structures of human enamel were investigated at the light microscopic level in order to define their morphology as well as the presence and distribution of both organic and inorganic molecules inside them. The histologic procedure employed in the present study allowed us to obtain 100 (microns) thick sections from permanent teeth without previous decalcification and paraffin or resin embedding. Three inter-rod structures were identified in the innermost area of the enamel on the basis of their morphologic aspect, namely the endings of dentinal tubules, the spindles and structures with a spherical or ovoidal shape never described before and by us denominated spheroids. Both spindles and spheroids were connected to one or more dentinal tubules which cross the dentin enamel junction. Measurements performed on all the specimens led to establish the real dimensions of the inter-rod structures. Histochemical staining carried out with selective dyes showed that spindles and spheroids were quite alike similar to each other as far as chemical composition and molecule distribution are concerned. Both structures contained calcium salts, glycosaminoglycans, glycoproteins, but not collagen. The endings of dentinal tubules among the rods showed histochemical characteristics similar to those of spindles and spheroids. On the basis of the above findings, spindles and spheroids could have a dentinal origin, and so likely derived from the metabolic activity of odontoblasts during the tooth-germ development. Whether they are embryonic vestigia without a functional role or are receptors which transmit signals to nerve endings in dentin and pulp remains to be clarified. PMID- 7537848 TI - An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line. AB - Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery. PMID- 7537849 TI - Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain. AB - The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR. PMID- 7537850 TI - Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens. AB - A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms. PMID- 7537851 TI - Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1. AB - Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene in endothelial cells is induced by lipopolysaccharide and the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3' of the TATA element. We have identified heterodimeric p65 and p50 as the NF kappa B species binding to the VCAM1 promoter in TNF-alpha-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and high mobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assay in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1. PMID- 7537852 TI - pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. AB - Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine. PMID- 7537853 TI - Mobile group II introns of yeast mitochondrial DNA are novel site-specific retroelements. AB - Group II introns aI1 and aI2 of the yeast mitochondrial COXI gene are mobile elements that encode an intron-specific reverse transcriptase (RT) activity. We show here that the introns of Saccharomyces cerevisiae ID41-6/161 insert site specifically into intronless alleles. The mobility is accompanied by efficient, but highly asymmetric, coconversion of nearby flanking exon sequences. Analysis of mutants shows that the aI2 protein is required for the mobility of both aI1 and aI2. Efficient mobility is dependent on both the RT activity of the aI2 encoded protein and a separate function, a putative DNA endonuclease, that is associated with the Zn2+ finger-like region of the intron reading frame. Surprisingly, there appear to be two mobility modes: the major one involves cDNAs reverse transcribed from unspliced precursor RNA; the minor one, observed in two mutants lacking detectable RT activity, appears to involve DNA level recombination. A cis-dominant splicing-defective mutant of aI2 continues to synthesize cDNAs containing the introns but is completely defective in both mobility modes, indicating that the splicing or the structure of the intron is required. Our results demonstrate that the yeast group II intron aI2 is a retroelement that uses novel mobility mechanisms. PMID- 7537854 TI - VH gene structure predicts a large potential anti-insulin repertoire. AB - The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5 + B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, also shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin. PMID- 7537857 TI - [Plasmids from certain strains of Streptomyces chrysomallus]. AB - Plasmid content has been studied in a number of Streptomyces chrysomallus strains producers of actinomycin C. The plasmids pSCH2 and pSCH3 have been isolated from nocardia-like mutants of Streptococcus chrysomallus BKM Ac-590 that are producing antibiotics macrotetrolides, bacteriocins and an inducer analogous to A-factor in addition to actinomycin. The size of the plasmids is 13.4 and 15.1 kb as found by restriction analysis. Plasmids differ in deletion and content in the cultures. The ability of the strains to produce antibiotics depends on plasmid content. PMID- 7537855 TI - The fate of human CD77+ germinal center B lymphocytes after rescue from apoptosis. AB - Germinal center (GC) B lymphocytes, defined by various criteria, have been shown to spontaneously undergo apoptosis in vitro unless they receive a positive signal. This rescue signal seems to be a multi-component process which involves not only the B cell receptor but also other cell surface receptors such as the CD40 antigen. In previous studies, we have shown that expression of the CD77 antigen is restricted to GC B lymphocytes and that CD77+ cells readily enter programmed cell death when cultured in vitro. In order to better characterize the CD77+ B lymphocytes, we have investigated the fate of these cells after rescue from apoptosis. Survival of CD77+ cells was achieved either with a combination of anti-CD40 mAb and IL4 (the CD40 system developed by Banchereau et al., (1991) Science 251, 70-72) or EBV infection. After 4 days of culture, similar phenotypic and functional changes of the CD77+ lymphocytes were observed in both systems: CD77 antigen was down-regulated, CD23 antigen which was originally negative became strongly expressed and the expression of CD38 and CD20 remained constant. Furthermore, large quantities of soluble CD23 were produced by the surviving cells. These results indicate that CD77 antigen is expressed by GC B cells which are highly susceptible to enter apoptosis but which are not doomed to die. PMID- 7537858 TI - Simple method for cDNA amplification starting from small amount of total RNA. AB - We describe a novel simple PCR-based technique for construction of cDNA libraries starting from the small samples of cells or tissues. This technique is based on the insertion of inverted terminal repeats (ITR) into amplified cDNA which causes a part of molecules to generate "pan"-type structures at each cycle of PCR amplification. This allows to avoid generation of the primer dimmer and makes possible the regulation of an average length of amplified sequences varying in concentration of primers. PMID- 7537856 TI - [Design of an expression vector for delivery of in vivo recombinant biologically active proteins. 1. Synthesis of the antigenic determinant of HIV-1 gp120 and gp41 proteins in enterobacteria]. AB - High-level expression vector pAZ was constructed for in vivo delivery of bioactive recombinant proteins, antigenic determinants, among other things. This vector meets the requirements to construction of recombinant bacteria as live oral carriers. It has a strong constitutive promoter, high stability in E.coli and vaccine strain Salmonella cells, and, moreover, encodes in addition for the marker protein (beta-galactosidase) which will later help follow up the fate of bacterial carriers and their interactions in the microorganism. Several recombinant plasmids encoding for beta-galactosidase variants with insertions of short fragments of HIV-1 gp41 and gp120 proteins, which were previously shown to be antigenic determinants, have been constructed on the basis of pAZ. E.coli and vaccine strain Salmonella cells were transformed by recombinant plasmids. To a considerable extent the level of hybrid protein synthesis depends on the structure of the antigenic determinant inserted. The maximal level of synthesis in E.coli is 16%. This hybrid protein could be isolated and purified (up to 90%) with the yield of 4 to 6 mg/g of wet cells. Almost all the hybrid proteins were immunologically reactive, as shown by ELISA with nonfractionated lysates and purified hybrids. In both strains in vitro stability of the vector and recombinant plasmids was at least 90% after 10 passages (about 140 generations) under random conditions. This paper sums up the first stage of construction of recombinant bacteria as live oral carriers. PMID- 7537859 TI - Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining. AB - The chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells. The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology. The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 'revertant' xrs-5 clones isolated following treatment with 5-azacytidine. nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity. Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity. Radiation sensitivity did correlate with DNA double-strand break rejoining ability. The radioresistant clones showing the over-condensed xrs-5 like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins. PMID- 7537860 TI - Series: current issues in mutagenesis and carcinogenesis, No. 54. Rodent germ cell mutagens and their activity in the rodent bone marrow micronucleus assay. PMID- 7537863 TI - Urinary excretion of aquaporin-2 in patients with diabetes insipidus. AB - BACKGROUND: Urine-concentrating ability is regulated by vasopressin. Recently, the specific water-channel protein of the renal collecting duct, known as aquaporin-2, was cloned. However, it is not certain whether this molecule is responsive to vasopressin. METHODS: We measured the urinary excretion of aquaporin-2 and its response to vasopressin in 11 normal subjects and 9 patients with central or nephrogenic diabetes insipidus. The urine samples were collected during periods of dehydration and hydration and after the administration of vasopressin. Urine samples were analyzed for aquaporin-2 by the Western blot assay and immunogold labeling, and the amount of aquaporin-2 was determined by radioimmunoassay. RESULTS: Aquaporin-2 was detectable in the urine in both soluble and membrane-bound forms. In the five normal subjects tested, the mean (+/- SE) urinary excretion of aquaporin-2 was 11.2 +/- 2.2 pmol per milligram of creatinine after a period of dehydration, and it decreased to 3.9 +/- 1.9 pmol per milligram of creatinine (P = 0.03) during the second hour after a period of hydration. In the six other normal subjects, an infusion of desmopressin (1 desamino-8-D-arginine vasopressin) increased the urinary excretion of aquaporin-2 from 0.8 +/- 0.3 to 11.2 +/- 1.6 pmol per milligram of creatinine (P < 0.001). The five patients with central diabetes insipidus also had increases in urinary excretion of aquaporin-2 in response to the administration of vasopressin, but the four patients with X-linked or non-X-linked nephrogenic diabetes insipidus did not. CONCLUSIONS: Aquaporin-2 is detectable in the urine, and changes in the urinary excretion of this protein can be used as an index of the action of vasopressin on the kidney. PMID- 7537861 TI - Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination. AB - The Chinese hamster lung V79-4 cell line was infected with a Moloney murine leukemia retrovirus and the infected cells were subsequently screened for mutants that were sensitive to X-rays using a toothpicking/96-well replica plating technique. Four independent mutants that were sensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 retrovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA double-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE experiments demonstrated that for any given X-ray dose all of the mutants incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensitivity to other DNA damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2, sxi-3 and sxi-4 appear to be specifically hypersensitive to genotoxic agents that cause DNA DSBs, whereas sxi-1 appeared to be hypersensitive to multiple types of DNA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antigen receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells. PMID- 7537865 TI - Extracellular amylase activities of Rhizomucor pusillus and Humicola lanuginosa at initial stages of growth. AB - Among thermophilic fungi, Rhizomucor Pusillus and Humicola lanuginosa have been reported to be among the most prolific producers of amylase, an apparently heat stable enzyme vital to the incorporation of carbon from macromolecular sources such as starch. Yet the highest levels of extracellular amylase in starch-yeast cultures of these fungi were measured after most of the growth had occurred; pre growth levels appeared to be very small. Since these low levels are the significant ones for growth, a procedure was devised to measure them: 1.162 x 10( 2) units (mg maltose/ml/min) were measured after two days of growth of R. pusillus and 6.230 x 10(-3) units measured after four days of the slower-growing H. lanuginosa. Re-assays of these after dialysis to remove most of the reducing sugars gave 1.689 x 10(-2) units and 1.234 x 10(-2) units, respectively, with all correlation coefficients 0.96 or better. PMID- 7537864 TI - The clinical importance of the urinary excretion of aquaporin-2. PMID- 7537862 TI - Screening of different keratin baits for isolation of keratinophilic fungi. AB - Twenty different keratin-rich substrates were screened as keratin baits for isolation of keratinophilic fungi from different soil sample of Chhindwara District, India. Fungi easily formed colonies on different baits. However, hair, feathers and horns were found to be more suitable than nails, fish fins and fish scales. PMID- 7537866 TI - The stories of the right hemisphere. PMID- 7537868 TI - Na,K-ATPase beta subunit isoform expression in the peripheral nervous system of the rat. AB - Using the RNase protection assay (RPA) to study the distribution of isoforms of the non-catalytic (beta) subunit of Na,K-ATPase in the peripheral nervous system, we found both beta 1 and beta 2 isoform mRNAs in dorsal root ganglion (DRG), but only beta 2 mRNA in sciatic nerve. Using Western blot to measure accumulation of the polypeptides at a ligature on the nerve we found that beta 1 but not beta 2 polypeptide is carried by rapid axonal transport in the sciatic nerve. These results imply that beta 1 is the prominent isoform of Na,K-ATPase in neurons and beta 2 the prominent isoform in Schwann cells. PMID- 7537867 TI - REM sleep deprivation induces galanin gene expression in the rat brain. AB - Rats were deprived of REM sleep for 24 h by keeping them on small platforms that were placed in a water bath (the platform method). Galanin coding mRNA was visualized using in situ hybridization, and cells expressing galanin mRNA were counted. In REM sleep-deprived animals the cell count was higher in the preoptic area and periventricular nucleus. Lesions of this area have been reported to induce wakefulness in cats and rats. Galanin administered into the lateral ventricle had no effect on sleep. We conclude that REM sleep deprivation can induce galanin gene expression in some brain areas, but galanin alone does not modify spontaneous sleep. PMID- 7537869 TI - A neuronal fibrillary inclusion shares the epitope of p24 of human immunodeficiency virus. AB - A monoclonal antibody to p24 of human immunodeficiency virus (HIV) was demonstrated to react with degenerated neurons immunohistochemically. In a case of Pick's disease, the neurons of the dentate gyrus were labeled with the antibody, while normal-looking ones and glial components were negative. The positive structures showed argyrophilia by the Bielschowsky and Bodian methods. Antibodies to ubiquitin, paired helical filament and tau did not react with the inclusions. Ultrastructurally, they consisted of skeins of fuzzy-surfaced fibrils with a diameter of 15 nm. In cases of Alzheimer type dementia, thread-like positive profiles were observed in some neurofibrillary tangle-bearing neurons. These studies revealed the cross-reactivity of the anti-HIV p24 antibody to unknown types of neuronal inclusions and provide a new aspect for research into neurodegenerative disorders. PMID- 7537870 TI - Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation. AB - Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution. PMID- 7537872 TI - Drosophila immunity. A sequence homologous to mammalian interferon consensus response element enhances the activity of the diptericin promoter. AB - Bacterial challenge of larvae or adults of Drosophila induces the rapid transcription of several genes encoding antibacterial peptides with a large spectrum of activity. One of these peptides, the 82-residue anti-gram negative diptericin, is encoded by a single intronless gene and we are investigating the control of expression of this gene. Previous studies using both transgenic experiments and footprint analysis have highlighted the role in the induction of this gene of a 30 nucleotide region which contains three partially overlapping motifs with sequence homology to mammalian NF-kappa B and NF-IL6 response elements and to the GAAANN sequence present in the interferon consensus response elements of some mammalian interferon-induced genes. We now show that the latter sequence binds in immune responsive tissues (fat body, blood cells) of Drosophila a approximately 45 kDa polypeptide which cross-reacts with a polyserum directed against mammalian interferon Regulatory Factor-I. Using a transfection assay of Drosophila tumorous blood cells, we show that the GAAANN sequence positively regulates the activity of the diptericin promoter. We propose that this motif cooperatively interacts with the other response elements in the regulation of the diptericin gene expression. PMID- 7537874 TI - An assessment of the antisense properties of RNase H-competent and steric blocking oligomers. AB - The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target. PMID- 7537875 TI - Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA. AB - Reverse transcription of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). A highly conserved polypurine tract (PPT) on the viral RNA serves as primer for plus-strand DNA synthesis and is a possible target for triple-helix formation. Triple-helix formation during reverse transcription involves either single-stranded RNA or an RNA.DNA hybrid. The effect of triple helix formation on reverse transcription has been analyzed here in vitro using a three-strand-system consisting of an RNA.DNA hybrid and triplex-forming oligonucleotides (TFOs) consisting either of DNA or RNA. Three strand triple helices inhibit RNase H cleavage of the PPT-RNA.DNA hybrid and initiation of plus strand DNA synthesis in vitro. Triple-helix formation on a single-stranded RNA target has also been tested in a two-strand-system with TFOs comprising Watson Crick and Hoogsteen base-pairing sequences, both targeted to the PPT-RNA, on a single strand connected by a linker (T)4. TFOs prevent RNase H cleavage of the PPT-RNA and initiation of plus-strand DNA synthesis in vitro. In cell culture experiments one TFO is an efficient inhibitor of retrovirus replication, leading to a block of p24 synthesis and inhibition of syncytia formation in newly infected cells. PMID- 7537873 TI - Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes. AB - Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding. PMID- 7537878 TI - Maternal urinary beta-core fragment of hCG/creatinine ratios and fetal chromosomal abnormalities in the second trimester of pregnancy. AB - Our aim was to evaluate the potential value of the ratio of the maternal urinary beta-core fragment of human chorionic gonadotropin (beta C-hCG) to creatinine (Cr) in discriminating between normal pregnancies and pregnancies associated with fetal chromosomal abnormalities. We hypothesized that pregnancies with fetal chromosomal abnormalities had abnormal quantities of beta C-hCG in the urine. The aims of the present study were to investigate retrospectively whether maternal urinary ratios of beta C-hCG/Cr are abnormal in women carrying fetuses with chromosome aberrations and to determine normative median values and a reference range for beta C-hCG/Cr between 14 and 19 weeks' gestation. Maternal urinary beta C-hCG and Cr concentrations were measured in 150 healthy women from 14 to 19 weeks and compared with ten cases of fetal chromosomal abnormalities matched for gestational age. The preliminary cut-off points corresponded to 0.29 multiple of the normal median (MOM) and 2.83 MOM, which were equivalent to the tenth and 90th centiles of the normal range. Of ten cases of fetal chromosomal abnormalities, one out of one (100 per cent) case with trisomy 18 and three of four (75 per cent) cases of variant 9 chromosomes had low beta C-hCG/Cr (< or = 0.29 MOM). One of five (20 per cent) cases with Down syndrome had elevated beta C-hCG/Cr (> or = 2.83 MOM). Urinary beta C-hCG/Cr ratios obtained in the second trimester may be useful for improved detection efficiency of Down syndrome, trisomy 18, and inversion of chromosome 9. Second-trimester maternal urinary beta C-hCG/Cr should be investigated further as a potential marker for fetal chromosome anomalies. PMID- 7537877 TI - [Changes in IGFBP-3 in relation to administration of rGH to deficient children]. AB - Serum IGFBP3 levels were tested in 7 GHD children before the rGH therapy, at the end of the same therapy and 1, 2, 4, and 8 weeks later. The IGFBP3 concentration returned to the pre-therapy levels only 6 weeks after the end of the therapy. PMID- 7537876 TI - Preparation of probe-modified RNA with 5-mercapto-UTP for analysis of protein-RNA interactions. AB - We report a modified synthesis for 5-mercapto-UTP (5-SH-UTP) and its use for analysis of protein-RNA interactions utilizing Escherichia coli and T7 RNA polymerases and yeast RNA polymerases I and III. 5-SH-UTP did not affect transcriptional pausing, Rho-independent termination or recognition of the E. coli transcription complex by NusA. RNA containing 5-SH-UMP did not crosslink to polymerase when irradiation was 302 or 337 nm. Transcription complexes containing RNA substituted with 5-SH-UMP were post-transcriptionally modified to attach a photocross-linking group to thiol-tagged nucleotides in the RNA on the surface of the polymerase of free in solution. The pKa for 5-SH-UTP was determined to be 5.6, so modification of the thiol groups in the RNA with p-azidophenacyl bromide could be carried out at pH 7. Addition of the transcription termination factor Rho, a RNA binding protein, to E. coli transcription complexes resulted in RNA crosslinking to Rho and to the beta and beta' subunits of polymerase. Using 5-SH UTP, one can distinguish RNA binding domains on the surface of RNA polymerases or other RNA binding proteins from those buried within the protein. PMID- 7537871 TI - Splicing of a circular yeast pre-mRNA in vitro. AB - We have tested the fate of a circularized synthetic pre-mRNA transcript in a whole cell splicing extract of Saccharomyces cerevisiae. Our results demonstrate that this circular precursor RNA is able to induce spliceosome formation in vitro and that the products of the following splicing reaction are the lariat-shaped intron, and a mature circular mRNA. Thus, it would appear that free 5' and/or 3' ends are not obligatory for a splicing reaction to occur, although we find its efficiency to be strongly influenced by the presence or lack of free ends. To our knowledge, this is the first demonstration that a circular pre-mRNA molecule is recognized as a suitable substrate by an eukaryotic mRNA splicing apparatus. PMID- 7537880 TI - Second-trimester maternal serum alpha-fetoprotein screening of pregnancies characterized by first-trimester isolated prominent fetal nuchal translucencies and normal karyotype. PMID- 7537879 TI - The influence of gravidity on Down's syndrome screening with free beta hCG. AB - Total human chorionic gonadotropin (hCG) levels have been shown in one previous small study to vary with gravidity. In order to establish if maternal serum free beta hCG was similarly affected, approximately 4500 pregnancies of varying gravidity were analysed. The results indicated that there was a 6 per cent higher median MOM in primigravid compared with multigravid women. The impact of such elevation in Down's syndrome screening programmes will be minimal. PMID- 7537881 TI - Down's syndrome screening: value of using free beta-hCG. PMID- 7537882 TI - The stability of blood samples for the measurement of the free beta subunit of chorionic gonadotrophin. PMID- 7537886 TI - Inhibition of nitric oxide synthesis attenuates alcohol consumption in two strains of alcohol-preferring rats. AB - The effect of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on voluntary alcohol consumption was examined in two different strains of alcohol-preferring rats, in a continuous-access, two-bottle-choice paradigm. Compared with the vehicle, intraperitoneal injections of L-NAME significantly and dose-dependently (10, 30, and 60 mg/kg) suppressed alcohol intake and preference in both alcohol-preferring (P) and Fawn-Hooded (FH) rats. The effect of the highest dose of L-NAME was nonspecific; it caused general decreases in consumption of alcohol, water, and food. Repeated injection of L NAME (30 mg/kg) for 4 consecutive days significantly attenuated alcohol intake, but tolerance developed after 3 days of treatment. A single administration of a high dose of L-NAME (60 mg/kg) did not influence the blood alcohol concentrations, which suggests a possible central effect. Furthermore, a moderate dose of 30 mg/kg L-NAME, which selectively inhibited alcohol intake, did not exert a significant effect on telemetrically measured heart rate, core body temperature, and gross motor activity of alcohol naive Fawn-Hooded rats. These results suggest an involvement of nitric oxide in alcohol drinking behavior. Although the true mechanism(s) of action is not yet clear, it can be speculated that L-NAME may exert its action indirectly by modulating neurotransmitters proposed to be involved in alcohol drinking and/or by influencing other neuronal factors, such as neuronal Ca2+ channels, which have been shown to be involved in alcohol drinking behavior. PMID- 7537883 TI - [Immunoanalysis of free alpha-subunits of glycoprotein hormones in human blood serum and its relationship with pituitary glycoprotein hormones]. AB - A radioimmunoassay test system has been designed for measurements of free alpha subunit (AS) of glycoprotein hormones in human blood serum with a sensitivity of 0.15 ng/ml. This test system revealed the absence of a direct correlation between the levels of free AS and levels of glycoprotein hormones in the blood sera of women with various endocrine profile, this being indicative of the specificity of this test system, on the one hand, and, on the other, confirming the possibility of independent secretion by the pituitary of free AS into the blood. Basal blood serum level of free AS in normal subjects is low: 0.9 ng/ml in women aged 17 to 30. The level of free AS secretion in the blood of many patients with nonfunctioning tumors of the pituitary was found increased, this demonstrating the diagnostic significance of measuring free AS as a marker of such tumors. PMID- 7537887 TI - Furocoumarin-photosensitized hydroxylation of guanosine in RNA and DNA. AB - Guanosine hydroxylation was used as a marker for assessing photooxidation of DNA and RNA sensitized by monofunctional and bifunctional furocoumarins. DNA or RNA, treated with sensitizer and UVA light, was enzymatically hydrolyzed, dephosphorylated and then analyzed by reversed-phase HPLC with electrochemical detection. Hydroxylated guanosine, i.e. 8-hydroxy-2'-deoxyguanosine (8-OHdG) or 8 hydroxyguanosine (8-OHG), was quantitated. 3-Carbethoxypsoralen (3-CP) was found to be an efficient photosensitizer for oxidation of guanosine in DNA, resulting in conversion of up to 0.4% of guanosine residues to 8-OHdG. In contrast, dramatically lower levels of guanosine hydroxylation were observed in 3-CP photosensitized RNA. Psoralen was found to be a more efficient photosensitizer than angelicin in both DNA and RNA. Additional studies of oxidation of 3-CP photosensitized DNA indicated that double-stranded DNA is 10 times more susceptible to photooxidation than single-stranded DNA, implicating 3-CP binding to DNA as an important mechanistic step in photooxidation of guanosine. The effects of D2O and degassing with argon on photooxidation of guanosine in DNA sensitized by 3-CP were inconsistent with a mechanism involving 1O2. In addition, chelation of adventitious metal ions present in preparations of DNA photosensitized by 3-CP had no effect on hydroxylation of guanosine. PMID- 7537884 TI - ATP activates cationic and anionic conductances in Schwann cells cultured from dorsal root ganglia of the mouse. AB - The whole-cell configuration of the patch-clamp technique was used to study membrane responses of mouse Schwann cells in organotypique culture to external application of adenosine 5'-triphosphate (ATP). ATP induced an inward current (IATP) which caused a membrane depolarization. IATP was dose dependent with a Kd of 8.4 mM. ATP analogues had the following relative agonist potency: ATP > ADP approximately alpha-beta methylene ATP. Neither AMP nor adenosine were effective. IATP was reversibly reduced by suramin, a P2 purinoceptor antagonist. Alteration of ionic gradients showed that ATP activated simultaneously cationic (potassium and calcium) and anionic (chloride) conductances. Values of the reversal potentials to ATP suggested the following permeability sequence through the anion channel: SCN- > I- > Br- approximately Cl- > aspartate > isethionate. IATP did not appear to be dependent on intracellular calcium, as inclusion or omission of calcium chelator (BAPTA, 10 mM) in the pipette solution had no effect on IATP. The observed ATP response resembles a P2-mediated response in its kinetics, its relative agonist potency and its blockade by suramin, but differs from known ones by its requirement for high concentrations of ATP and the activation of a cationic as well as an anionic conductance. PMID- 7537885 TI - Mediation of mydriasis in conscious rats by central postsynaptic alpha 2 adrenoceptors. AB - The alpha 2-adrenoceptor agonist, clonidine (0.001-1 mg/kg, IP), dose-dependently induced mydriasis in conscious rats (ED50 0.088 mg/kg). This response was maximal when measured 10 min after clonidine injection and was of about 30-min duration. The noradrenaline releasing agent, methamphetamine (0.75 mg/kg, IP), also increased pupil diameter. Clonidine (0.03 mg/kg, IP)-induced mydriasis was inhibited in a dose-related fashion by the alpha 2-adrenoceptor antagonists, idazoxan (0.03-3 mg/kg, IP) and yohimbine (0.03-3 mg/kg, IP), but was unaltered by the alpha 1- or beta-adrenergic antagonists, prazosin (1 and 3 mg/kg, IP) or pindolol (1 and 3 mg/kg, IP). Methamphetamine (0.75 mg/kg, IP)-induced mydriasis was similarly inhibited by idazoxan (1 mg/kg, IP) and yohimbine (1 mg/kg, IP). These data argued strongly that central alpha 2-adrenoceptors are involved in the mediation of mydriasis. The synaptic location of these receptors was determined using DSP-4 (50 mg/kg x 2, IP) to lesion noradrenergic neurones: this produced a 64% depletion of noradrenaline in the midbrain (containing the Edinger-Westphal nucleus responsible for mydriasis) and reduced the mydriatic effect of methamphetamine (0.75 mg/kg, IP) to a similar extent (72%), whereas clonidine mydriasis remained unaltered. Therefore, these results show that the mydriasis responses induced by either clonidine or methamphetamine are mediated by central postsynaptic alpha 2-adrenoceptors. PMID- 7537888 TI - Activin-A stimulates the expression of insulin-like growth factor binding protein 5 messenger RNA in human luteinizing granulosa cells. PMID- 7537889 TI - [Therapeutic vs. palliative logic]. PMID- 7537890 TI - [Quality assurance in palliative care. Comparison of the practice of 2 institutions in the framework of a retrospective pilot study]. PMID- 7537895 TI - Chemotherapeutic and Nonchemotherapeutic Palliative Approaches in the Treatment of Cancer. April 29-30, 1994. PMID- 7537893 TI - [Significance of hepatitis C virus antibodies in asymptomatic blood donors]. AB - The aim of the study was to determine the frequency and course of hepatitis C viremia in clinically healthy, anti-HCV positive test subjects, and to ascertain whether the HCV antibodies of the IgM type differed between viremia and immunity. In 21 anti-HCV positive blood donors (test subjects) with normal transaminase activity, two serum samples, taken at an interval of 25 +/- 10 months, have been investigated for HCV-RNA and HCV-IgM antibodies. In a total of 16 test subjects (76%) HCV-RNA was found during the first test and/or the follow-up: 14 of them were positive on both occasions, and one test subject each was HCV-RNA positive exclusively at the first test and the follow-up respectively. At the time of the follow-up the serum transaminase level was elevated in 4 test subjects. 3 of these 4 were HCV-RNA positive also. On the other hand, the results of the HCV-PCR were nonuniform in HCV-IgM antibody negative test subjects. The above results demonstrate that in the majority of clinically healthy, anti-HCV positive test subjects with normal transaminase activity, a viremia exists which persists and the course of which may include inflammatory phases. The proof of HCV-IgM antibodies correlates with a viremia. On the other hand, the lack of HCV-IgM antibodies does not exclude viremia. PMID- 7537892 TI - [Endoscopic palliation of malignant esophagus processes using a self-expanding metallic stent]. AB - We investigated the palliative effect of self-expanding metallic stents on malignant obstruction of the esophagus in 10 patients. All patients had high grade dysphagia and one had an esophago-bronchial fistula. Endoscopic insertions of the prosthesis was done under sedation. Dilatation of the stricture prior to insertion was rarely necessary. Coated stents were used as secondary treatment for patients with fistulae. The procedure related morbidity was low and no mortality was observed. The stents remained patent during the residual lifetime of the patients. Dislocation or perforation did not occur. The median survival of 8 deceased patients was 3.5 months (range 1.25-14.5 months). At present 2 patients are still alive 2.7 and 1.5 months after the procedure. Self-expanding stents in the esophagus provide good palliative therapy of dysphagia or fistulae caused by malignant tumors. Insertion is relatively simple and safer than in nonexpandable types. These improvements may justify the considerably higher price of these devices. PMID- 7537891 TI - [Transaminases and histological activity in chronic hepatitis C]. AB - The purpose of our study was to verify the correlation between biochemical and histological inflammatory activity in chronic hepatitis C. The histological activity index (HAI) of 42 patients with chronic hepatitis C was correlated with their transaminase activity. There was indeed a significant correlation between histological activity and the AST (r = 0.54, p = 0.0002) and ALT level (r = 0.36, p = 0.018) respectively. Due to considerable scatter of the results, transaminase activity did not allow reliable assessment of inflammatory activity in individual cases. In the majority, markedly elevated transaminase activity was associated with marked histological inflammatory activity, whereas normal or slightly elevated transaminase activity was found in all stages of inflammatory activity. To assess inflammatory activity in chronic hepatitis C, a liberal indication for liver biopsy should therefore be adopted. PMID- 7537894 TI - Enhancing cure and palliation: radiation therapy in the treatment of metastatic gestational trophoblastic neoplasia. PMID- 7537898 TI - Chemotherapy as a palliative treatment in carcinoma of the uterine cervix. AB - The vast majority of patients with cervix carcinoma present with either preinvasive or minimally invasive (stage IA) disease and are cured with surgery alone. The remaining patients can be classified into two groups: those with disseminated (extrapelvic) disease at the time of presentation or recurrence after initial treatment and those with advanced pelvic disease. In the first group, the goal of therapy is palliative and the principal modality is chemotherapy. A number of single agents have significant activity (> or = 15% response rate): the platinum compounds, certain alkylating agents, the anthracyclines, bleomycin, the vinca alkaloids, certain antifols, 5-fluorouracil, 5'-floxuridine, ICRF-159, hexamethylmelamine, and CPT-11. Although uncontrolled trials report high response rates with combination regimens, no such regimen has been shown to be superior to single-agent therapy. The current standard of care for initial treatment is single-agent cisplatin 50 to 100 mg/m2 every 3 weeks, with an expected 23% response rate. In the second group, palliation takes the form of prevention of recurrence. Patients who have stage IIIB or IVA disease benefit from the addition of concomitant chemotherapy to radiation. The current standard of care is hydroxyurea 80 mg/kg orally twice weekly during radiotherapy. For patients with stage IB or II disease, surgery and/or radiotherapy remains the standard of care. Although concomitant or neoadjuvant chemotherapy followed by either surgery or radiotherapy has been evaluated, no conclusive evidence proves the value of the addition of chemotherapy to the management of these patients. Ongoing phase III trials are evaluating both approaches. PMID- 7537896 TI - Palliative chemotherapy in advanced bladder cancer. AB - Transitional cell carcinoma of the urothelium is a chemosensitive tumor, and combination chemotherapy can provide not only palliation but a modest survival advantage in patients with advanced disease. While the four-drug regimen methotrexate/vinblastine/doxorubicin/cisplatin remains the standard combination therapy, its toxicity can be formidable. The overall response rate in phase III trials with this combination is in the 35% to 45% range, with a median survival duration in treated patients of only 12 months. Although attempts to decrease the toxicity of the regimen with the addition of hematopoietic growth factors have been successful, attempts to increase its efficacy by dose escalation have not. This has prompted a search for new active agents that can be incorporated into alternative combination regimens. Recently, activity has been noted for several drugs (including gallium nitrate, ifosfamide, and gemcitabine). In untreated patients, paclitaxel has demonstrated significant activity and is certainly among the most active single agents in the treatment of advanced bladder cancer. Studies incorporating these new agents into novel combination regimens are ongoing, with the goal of providing a regimen that has superior efficacy in advanced disease and, ultimately, in earlier stages of disease with curative intent. PMID- 7537897 TI - Pharmacologic management of cancer pain. AB - More than three quarters of cancer patients experience chronic pain during the course of their disease. With optimal pharmacotherapy alone, 70% to 90% could achieve adequate relief. Optimal pharmacotherapy begins with a comprehensive pain assessment, which defines the nature of the pain complaint and clarifies the degree to which pain and other factors contribute to impaired quality of life. Although the potential analgesic consequences of primary therapy are always considered, only radiotherapy is used commonly. Patients with persistent moderate to severe pain should be treated with an appropriate opioid regimen, which is based on careful selection of an opioid drug and route of administration, individualization of the dose through titration based on repeated assessment of the patient, and ongoing efforts to manage side effects. The use of adjuvant analgesics and the use of sequential opioid trials may improve the outcome of therapy for patients who fail to promptly attain a favorable balance between analgesia and side effects during an opioid trial. PMID- 7537899 TI - Chemotherapeutic palliative approaches in the treatment of breast cancer. AB - The palliative approach to the treatment of metastatic breast cancer deserves discussion because of the numerous patients diagnosed with this disease. Approximately 10% of the greater than 180,000 women diagnosed with breast cancer each year will present with metastatic disease, and an additional 50% to 70% will eventually relapse. The clinician must consider the treatment toxicity, efficacy, and impact on quality of life to arrive at the optimal therapeutic decision. The following discussion will provide an overview of palliative treatment options, including single-agent and combination chemotherapy, as well as new alternative drugs, such as paclitaxel and vinorelbine. Treatment toxicity and its impact on quality of life will be emphasized. PMID- 7537900 TI - The use of chemotherapy as palliative treatment for patients with advanced ovarian cancer. AB - Despite the high objective response rate of ovarian cancer to several reported platinum-based combination chemotherapy regimens, the majority of patients with advanced disease ultimately require consideration of a salvage regimen delivered for palliation of symptoms. A number of therapeutic strategies have been used in such patients, including re-treatment with cisplatin or carboplatin, or the use of paclitaxel, ifosfamide, hexamethylmelamine, oral etoposide, 5-fluorouracil plus leucovorin, or tamoxifen. Multiple factors and individual clinical features of the patient must be considered when selecting the most appropriate salvage therapy to be used in this clinical setting. PMID- 7537901 TI - Palliative chemotherapy of adult soft tissue sarcomas. AB - Sarcomas represent approximately 1% of adult malignancies. Surgery and local radiotherapy are the mainstays of therapy, as treatment of metastatic disease is palliative in intent in most cases. The value of adjuvant chemotherapy is controversial. Most adjuvant trials using single-agent doxorubicin have failed to show improved relapse-free and overall survival. Minor advantages in relapse-free survival have been reported in some adjuvant chemotherapy trials using combination chemotherapy. Further research is needed to develop effective adjuvant regimens. Surgery also may be important in the treatment of metastatic disease, as approximately 20% of patients with pulmonary metastases amenable to resection may be rendered disease free for prolonged periods. Chemotherapy is of modest value. Only three drugs (doxorubicin, dacarbazine, and ifosfamide) have clear activity in the treatment of adult soft tissue sarcomas. Combination chemotherapy may be associated with higher objective response rates than single agent chemotherapy, but toxicity also is greater with combination regimens and no survival advantage for the more aggressive alternatives has been reproducibly reported. In patients over the age of 50 years, single-agent chemotherapy may be preferable to combination chemotherapy. Combination chemotherapy, often with aggressive supportive measures, may be considered in younger patients with a good performance status. New active agents need to be identified if therapy of this group of diseases is to be improved. PMID- 7537902 TI - Palliative chemotherapy for non-small cell lung cancer. AB - The results of several randomized trials suggest a marginal survival benefit for patients with metastatic non-small cell lung cancer after treatment with chemotherapy. This intervention, however, has not become standard therapy. Toxicity, cost, and inconvenience have led many physicians to question whether chemotherapy has even a palliative role in this disease. Furthermore, it is well established that patients with a poor performance status, who are most in need of symptomatic palliation, are also those who are least likely to benefit and most likely to experience treatment-related toxicity. Nonetheless, evidence of a symptomatic benefit from combination chemotherapy has been presented. Indeed, symptomatic palliation can result even in the absence of a conventionally defined chemotherapy-induced response. Phase III trials supporting the palliative value of chemotherapy, however, have been limited and inconclusive. Fortunately, other palliative treatment modalities exist. These interventions should be carefully integrated with chemotherapy when an overall treatment plan appropriate for an individual patient is being developed. In addition to the symptomatic needs of each individual, this treatment plan must also address both physician and patient biases. PMID- 7537904 TI - Role of chemotherapy in the palliation of gastrointestinal malignancies. AB - In 1994, more than 200,000 patients in the United States were diagnosed with colon, gastric, or pancreatic cancer. Over half of these patients eventually will have recurrent, incurable disease and require palliative therapy. Although chemotherapy has no role in the primary treatment of most gastrointestinal cancers, and only a limited role as a form of adjuvant treatment in colon cancer, it is sometimes the only systemic treatment available for palliation. Effective chemotherapeutic agents for gastrointestinal cancers are few, and after more than three decades of research, 5-fluorouracil remains one of the few useful agents. This article reviews the role of chemotherapy in palliative therapy for the common gastrointestinal cancers. Discussion also includes recent advances in modulating 5-fluorouracil activity and the many attempts to study newer agents. PMID- 7537903 TI - Palliative therapy: extensive small cell lung cancer. AB - During the past two decades advances have been made in the systemic treatment of small cell lung cancer. Chemotherapy will produce objective responses in the majority of patients, yet few patients with extensive disease have prolonged survival. Combination regimens like cyclophosphamide/doxorubicin/vincristine, cisplatin/etoposide, and cisplatin/etoposide/ifosfamide have produced objective responses in 55% to 65% of patients. Recent clinical trials with single-agent chemotherapy suggest median lengths of survival that appear comparable to that obtained with combination therapy. Such approaches may be appropriate for elderly patients and will be evaluated in a prospective randomized trial. Ultimately, to provide the maximum palliative benefit for patients with extensive small cell lung cancer, the therapeutic benefit must be balanced against the costs of treatment (physical, psychological, and financial). PMID- 7537905 TI - Chemotherapeutic palliative approaches in the treatment of lymphoma. AB - A number of diverse therapies can be used to treat relapsed non-Hodgkin's lymphoma, with the most common treatment modality being conventional salvage chemotherapy using agents different from those used initially. Alternative therapies that may be palliative in certain clinical situations include localized radiation therapy, novel chemotherapeutic agents (like chlorodeoxyadenosine, fludarabine, or idarubicin), or conjugated monoclonal antibody therapy. The use of high-dose chemotherapy and bone marrow or hematopoietic stem cell transplantation can be a successful treatment option for patients with relapsed lymphoma; however, this should be considered as a therapy of curative intent and not only for palliative therapy. PMID- 7537906 TI - Cost aspects of palliative cancer care. AB - Increasing concern is evident about the cost of palliative cancer care, a concern stemming from both government interest in cost containment and the growing nongovernment use of payment rates that are fixed in advance. In general, data on the cost-effectiveness of palliative cancer care are mixed; some types of palliative care appear to increase costs, while other forms may reduce overall costs of treatment. Pain management, however, is an area that seems to present significant opportunity for cost savings in palliative care if oral medications are used instead of high-technology modalities. PMID- 7537907 TI - The symptoms of advanced cancer. AB - The incidence and mortality of cancer are increasing. In advanced cancer when cure is impossible, symptoms should be the focus of attention. We review our recent prospective studies on symptom prevalence and severity in 1,000 patients with advanced cancer. Advanced cancer patients are polysymptomatic. Symptom prevalence differs with age, gender, and cancer site. The pattern of gastrointestinal symptoms is related to gender and severe weight loss. Specific symptoms are associated with reduced survival. There is a gender difference in survival favoring females. Pain, easy fatigue, and anorexia are consistently among the 10 most prevalent symptoms at all 17 primary sites. When pain, anorexia, weakness, anxiety, lack of energy, easy fatigue, early satiety, constipation, and dyspnea were present, 60% to 80% of patients rated them as moderate or severe, ie, of clinical importance. The most common symptoms also are the most severe. In general, the same symptoms are clinically important at most primary sites. PMID- 7537908 TI - Measuring quality of life in palliative care. AB - In the palliative treatment of chronic illness, attention must extend beyond symptom control to include the overall quality of patients' lives. Symptom relief is highly valued by patients and usually is associated with improvement in general functioning and well-being (ie, overall quality of life). Side effects and costs of therapy, however, also must be included in the equation to fully balance the treatment decision. Patients tend not to think dichotomously in terms of toxicity versus efficacy; rather, they consider the aggregate of their capabilities, circumstances, and somatic sensations. In fact, they often confuse symptoms with side effects. We must therefore survey patients in terms of their disaggregated experience, using a valid self-report questionnaire, and help them make decisions about treatment by estimating which deficiencies or improvements are attributable to disease versus treatment. The dialogue that this demands is challenging, because it can threaten the adaptive suppression that many patients use to protect themselves from negative feelings. Unfortunately, the consequence of avoiding this dialogue often is unnecessary treatment given with misunderstood intentions. PMID- 7537909 TI - The palliative role of radiotherapy in the management of the cancer patient. AB - Radiotherapy (RT) plays a major role in the palliation of cancer symptoms. Cancer related bone pain, bronchial obstruction, superior vena cava syndrome, spinal cord compression, and central neurologic dysfunction due to brain metastases are all treated effectively and safely by RT. Although numerous techniques and radiation dose fractionation schemes are used in practice, substantial palliation often may be accomplished by just two to 10 appropriately targeted treatments. Thus, palliative RT regimens need not be highly protracted to be effective. Several new techniques, including strontium 89 radiopharmaceutical therapy, high dose-rate endobronchial brachytherapy, and stereotactic radiosurgery, are now being evaluated in clinical trials to determine their efficacy in the relief of various oncologic symptoms. Palliative RT continues to be a mainstay of treatment in the management of cancer patients with advanced disease. PMID- 7537910 TI - Surgery for support and palliation in patients with malignant disease. AB - Surgery is the major curative treatment modality for patients with malignant disease. However, surgery also plays an important role in supportive and palliative care of cancer patients. In settings in which maintaining quality of life rather than prolongation of survival is the major goal of treatment, the potential morbidity of more extensive surgical procedures must be carefully weighed against their possible benefits, and alternative options (if available and appropriate) should be carefully considered. PMID- 7537911 TI - Dose intensification--a phase I study of ifosfamide with vinorelbine (Navelbine): rationale and study design in advanced non-small cell lung cancer. AB - A phase I trial of a combination of vinorelbine (Navelbine; Burroughs Wellcome Co, Research Triangle Park, NC; Pierre Fabre Medicament, Paris, France) and ifosfamide given on a novel schedule on 3 consecutive days with granulocyte colony-stimulating factor was conducted to establish the maximum tolerated dose of vinorelbine and the dose-limiting toxicities of this regimen. Doses of vinorelbine were escalated in cohorts of patients. Of the 29 patients enrolled at the time of data analysis, 26 were evaluable for toxicity. At the first dose level (ifosfamide 2.0 g/m2 days 1, 2, and 3; vinorelbine 15 mg/m2 days 1, 2, and 3), two of three patients had dose-limiting toxicities. The dose of ifosfamide was decreased by 20%, and dose escalation of vinorelbine was restarted. At the vinorelbine dose level of 35 mg/m2/d for 3 days, three of four patients had dose limiting toxicities. The recommended phase II doses were established as 1.6 g/m2/d ifosfamide for 3 days and 30 mg/m2/d vinorelbine for 3 days. This study established that granulocyte colony-stimulating factor was needed for approximately 8 days. We are currently examining the feasibility of two dose levels of vinorelbine (25 mg/m2 and 30 mg/m2/d for 3 days) in combination with ifosfamide (1.6 g/m2/d) given every 2 weeks. PMID- 7537912 TI - A phase I/phase II trial of granulocyte colony-stimulating factor as bone marrow support in patients treated with vinorelbine (Navelbine): study design and goals. AB - Vinorelbine (Navelbine; Burroughs Wellcome Co, Research Triangle Park, NC; Pierre Fabre Medicament, Paris, France) has recently shown great promise in the treatment of advanced breast cancer. The dose-limiting toxicity of this agent is myelosuppression; nonhematologic toxicities are typically mild and easily managed. Because of this toxicity profile, support with growth factors such as granulocyte colony-stimulating factor may allow dose escalation of vinorelbine. To investigate this issue, a two-part phase I/II clinical trial has been designed. The first portion of this study addresses the need to develop a schedule of vinorelbine administration that can be more easily integrated with growth factor support than the currently used once-weekly schedule. In this part of the study, vinorelbine will be administered on a 21-day cycle once daily for 3 consecutive days at dosage levels beginning at 15 mg/m2/d and increasing at 5 mg/m2/d until dose-limiting toxicity is reached. In the next portion of the study, granulocyte colony-stimulating factor will be given in conjunction with vinorelbine. The schedule of vinorelbine administration will again be once daily for 3 days. Vinorelbine therapy will be initiated at one dose level below the previously determined maximum tolerated dose, and the dose will be escalated at 5 mg/m2/d until the maximum tolerated dose is reached. It is hoped that this phase I/II trial will provide valuable information about both an alternative schedule of vinorelbine administration and the potential of growth factor support allowing for dose escalation of vinorelbine. PMID- 7537913 TI - Treatment of metastatic breast cancer: present and future prospects. AB - Patients with recurrent breast cancer can be divided into three categories: those with locoregional recurrence, those with distant nonvisceral recurrence, and those with visceral recurrence. Survival from time of first relapse is clearly dependent on these categories. Selection of therapeutic modality should be based on considerations of site recurrence, symptomatology, anticipated response to therapy, and expected toxicities related to the therapy. Chemotherapy is appropriate for patients who are either unlikely to respond to hormone therapy, quite symptomatic, clearly hormone refractory, or have rapidly progressive visceral disease. Previously untreated patients are likely to respond to chemotherapy, with no clear-cut marker or clinical category associated with increased or decreased likelihood of benefit. Studies are ongoing to identify markers for response to chemotherapy, with recent investigations focusing on HER 2/neu expression. Standard combination chemotherapeutic regimens, consisting of either cyclophosphamide/methotrexate/5-fluorouracil (CMF) or cyclophosphamide/doxorubicin/5-fluorouracil (CAF), are associated with response rates in untreated patients of 35% to 80% and in previously treated patients of 10% to 40%. Although CAF probably has a slightly higher response rate than CMF, the toxicity of CAF is substantially higher. Newer agents are effective in both previously untreated and treated patients with breast cancer. These include paclitaxel, docetaxel, vinorelbine (Navelbine; Burroughs Wellcome Co, Research Triangle Park, NC; Pierre Fabre Medicament, Paris, France), and amonafide. Furthermore, modulation of previously existing agents, designed to overcome resistance, has been tested. Only leucovorin/5-fluorouracil has apparent clinical benefit. A number of novel approaches are being designed or are currently being used in clinical trials. These include differentiating agents, anti-angiogenesis factors, antitumor antigen-based therapy, growth factor receptor/ligand therapy, and gene therapy. PMID- 7537914 TI - [The palliative radiotherapy of Kaposi's sarcomas in AIDS patients]. AB - BACKGROUND: Follow-up of patients, who were irradiated because of AIDS-related Kaposi's sarcomas. PATIENTS AND METHODS: From 1983 to 1994 17 patients were irradiated because they suffered from AIDS-related neoplasms. Fifteen of these were irradiated because of Kaposi's sarcomas. The radiation fields were as small as possible, the total dose that was given was 30 Gy in the average, given 5 x 2 Gy or 4 x 2.5 Gy per week. RESULTS: The results concerning the cosmetic benefits were good, the pain could be reduced very well. In two patients the radiation therapy was cancelled: 1 patient suffered from a general tumor progression and 1 did not allow any further therapy. The remission rate was 3 to 4 months in the average, 3 patients did not show local progression for 4 respectively, 5 months now. One patient was in remission for 8 months. CONCLUSIONS: The radiation therapy is a local, but very effective method to treat patients with AIDS-related Kaposi's sarcomas. Whereas the side-effects are very little, we found quite a long remission rate. PMID- 7537915 TI - Partial inhibition of platelet aggregation and fibrinogen binding by a murine monoclonal antibody to GPIIIa: requirement for antibody bivalency. AB - We produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) IIIa. 7H2-IgG and 7H2-F(ab')2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, approximately 40,000 7H2-IgG antibody molecules bind per platelet. In contrast, approximately 80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s). PMID- 7537917 TI - A novel mechanism for exposure of fibrinogen binding sites on GPIIb-IIIa by a monoclonal antibody. AB - Conformational changes in platelet membrane glycoprotein (GP) IIb-IIIa, whose nature is not defined, lead to exposure of fibrinogen binding sites. We have reported previously that F(ab')2 fragments of a monoclonal antibody, PMA4, directed against the GPIIb-IIIa complex-specific domain, induced binding of fibrinogen to platelets without causing intracellular activation, whereas Fab did not. In this study, we examined the mechanism responsible for the difference in the ability of PMA4 F(ab')2 and Fab to expose fibrinogen binding sites. PMA4 Fab had affinity for GPIIb-IIIa similar to that of PMA4 F(ab')2. Addition of F(ab')2 goat anti-mouse Fab antibody to cross-link PMA4 Fab-bound GPIIb-IIIa molecules induced fibrinogen binding. There was a direct correlation between the number of molecules of PMA4 F(ab')2 and the amount of fibrinogen bound. PMA4 did not recognize ligand-induced binding sites (LIBS). These results suggest that the cross-linking of special sites on the GPIIb-IIIa complex-specific domain by bivalent antibody alters the conformation of GPIIb-IIIa to a state competent to bind soluble fibrinogen and that conformational changes in non-LIBS are involved in the mechanism for exposing fibrinogen binding sites on GPIIb-IIIa. PMID- 7537916 TI - Increased levels of soluble adhesion molecules in type 2 (non-insulin dependent) diabetes mellitus are independent of glycaemic control. AB - Patients with Type 2 (non-insulin dependent) diabetes mellitus are at increased risk of thrombosis and the premature development of atherosclerosis. This may be related to damage to the endothelium (which may be the primary target tissue for the disease process) resulting from a loss of normal glycaemic metabolic control. Thus changes in endothelial cell function, such as modified release of soluble leukocyte and platelet adhesion molecules, may be important. Accordingly, E selectin, von Willebrand factor (vWf), vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) were measured in serum from 60 patients and 76 controls. Raised levels of vWf (p = 0.0002), E-selectin (p < 0.0001) and VCAM (p = 0.003) in patient's samples failed to correlate with glycaemic control as assessed by levels of fructosamine and glycated haemoglobin, or with 24 h urine albumin. Levels of ICAM were not increased in our patients. Levels of the two endothelial cell products, vWf and E-selectin, failed to correlate although E selectin correlated with low density lipoprotein cholesterol (p = 0.016). vWf correlated with VCAM (p < 0.001) and hypertension (p = 0.032). We conclude that levels of soluble adhesion molecules vWf, E-selectin and VCAM are raised in Type 2 diabetes mellitus. The mechanisms for these changes appear to be independent of glycaemic control but may relate to concurrent hypertension and/or hypercholesterolaemia. PMID- 7537918 TI - Identification of novel peptide antagonists for von Willebrand factor binding to the platelet glycoprotein Ib receptor from a phage epitope library. AB - We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein Ib interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor. PMID- 7537919 TI - Autoimmune antiphospholipid antibodies are directed against a cryptic epitope expressed when beta 2-glycoprotein I is bound to a suitable surface. AB - The antiphospholipid antibodies (aPL) present in autoimmune disorders are associated with thromboembolic episodes, and their binding to phospholipids (PL) is mediated by a plasma cofactor, beta 2-glycoprotein I (beta 2GPI). Both PL and beta 2GPI seem necessary for binding, thus indicating that the two components comprise the epitope against which aPL are directed. Using an anti-beta 2GPI antibody ELISA with the antigen adsorbed onto polyvinylchloride (PVC) plates, we detected high antibody titres in 12 out of 12 plasma from patients with the antiphospholipid syndrome. No or very low positivity was obtained when the same ELISA was carried out in polystyrene (PST) plates, while an increasing positivity was found when processed (i.e. more hydrophilic) or COOH-surface PST plates were used. When beta 2GPI dependent IgG-aPL were purified using agarose-immobilized cardiolipin, 4 out of 4 preparations were highly positive in anti-beta 2GPI antibody ELISA using PVC plates, while beta 2GPI was not fully recognized by aPL IgG when adsorbed onto PST plates. These findings demonstrate that aPL are, in fact, anti-beta 2GPI antibodies directed against a cryptic epitope which is expressed when beta 2GPI is bound to anionic phospholipid, or another suitable surface. PMID- 7537922 TI - Study of different human and animal thromboplastins with human factor VIIa in the presence of aprotinin. PMID- 7537921 TI - Plasma levels of von Willebrand factor in non-insulin-dependent diabetes mellitus are influenced by dietary monounsaturated fatty acids. AB - We have previously demonstrated a lowering effect on von Willebrand Factor (vWF) of a diet rich in monounsaturated fatty acids (MUFA) as compared with a high carbohydrate diet. In the present study 16 non-insulin dependent diabetic (NIDDM) subjects participated in a cross-over experiment on an out-patient basis comparing the effects on vWF of three weeks treatments with two diets similar in carbohydrate and protein content, one rich in MUFA (30 energy %) and one rich in polyunsaturated fatty acids (PUFA) (30 energy %). Before and on the last day of the two diets, the levels of vWF, fibrinogen, fibronectin and alpha 2 macroglobulin were measured. After 3 weeks diet intervention vWF levels were lower after the MUFA regimen compared with the PUFA diet (mean +/- SD) (1.15 +/- 0.36 vs 1.32 +/- 0.42 U/ml, p = 0.003). Similar and unchanged levels of fibrinogen, fibronectin and alpha 2-macroglobulin were seen. In conclusion, three weeks on a diet rich in MUFA lowers vWF as compared with a PUFA rich diet, suggesting a beneficial effect of MUFA on the endothelium in NIDDM patients. PMID- 7537923 TI - Proteoglycans in cementum- and enamel-related predentin of young mouse incisors as visualized by cuprolinic blue. AB - In rodent incisors, dentin associated with cementum (root-analogue dentin) appears to differ considerably from that associated with enamel (crown-analogue dentin), both with respect to the composition of certain matrix components and the mineral. Since it is not known whether these dentin portions also differ with respect to their proteoglycans, the morphological appearance and spatial distribution of these components was studied in predentin by employing cuprolinic blue, a dye selective for proteoglycans. Lower incisors of five-day-old mice were stained with the dye and processed for electron microscopic examination. Cuprolinic blue-positive precipitates were found in both cementum- and enamel related predentin. In cementum-related predentin these structures were thick and short. In the enamel-related portion, however, they were long, slender and frequently stellate-shaped. The number of precipitates was similar between the two predentin portions and also no differences were found between the basal and the apical (adjacent to the mineralization front) aspects of the predentin layers. It is suggested that the differences in proteoglycan architecture among the predentin layers is somehow related to differences in the three-dimensional collagenous meshwork or to different patterns of mineralization. PMID- 7537920 TI - Membrane thrombomodulin levels are decreased during hypoxia and restored by cAMP and IBMX. PMID- 7537924 TI - Localization of alpha-fetoprotein in developing chick amniotic membrane. AB - The aim of this work was to investigate the localization of alpha-fetoprotein (AFP) in amniotic membrane (AM). By using the immunoperoxidase technique in several developmental stages, which reflected the changes of structure of the AM germinal layers, AFP was detected earliest in 7-day AM and localized selectively in the ectodermal cell layer. This was the only developmental stage at which AM occurred as a two-layer structure, ectoderm and somatic mesoderm, and was AFP positive. In the zone of fusion of the AM with the inner wall of the allantoic sac, cystlike cavities were observed which were markedly immunoreactive to AFP. In those membranes where fusion had consolidated and a four-layer structure could be distinguished: ectoderm, somatic mesoderm, splanchnic mesoderm and endoderm, AFP was localized in the ectodermal cells and in the splanchnic mesoderm resulting from the inner wall of the allantoic sac. Both mesodermal layers could be distinguished by means of the AFP immunoreaction since AFP labelled the splanchnic, but not the somatic mesoderm. At later developmental stages, e.g. 18 day, the AM had a three-layer structure and AFP was localized selectively throughout the splanchnic mesoderm. The disappearance of the somatic mesoderm coinciding temporarily with the disappearance of AFP from the ectodermal cells, suggests that the presence of AFP in such cells could depend on some factors related to the somatic mesoderm. PMID- 7537925 TI - Morphologic and morphometric evaluation of the effect of ICRF-187 on bleomycin induced pulmonary toxicity. AB - Morphologic and morphometric studies were made of the protective effects of ICRF 187 against the pulmonary damage induced by bleomycin in male and female C57/BL6 mice. Sixty minutes prior to the subcutaneous administration of 15 mg/kg of bleomycin, animals received either saline or ICRF-187 (300 or 150 mg/kg) intraperitoneally, twice a week for 4 weeks. The lungs of animals treated with bleomycin alone showed inflammation, hyperplasia of type II epithelial cells, squamous cell metaplasia and fibrosis. The extent of fibrosis was quantified by means of a color videometric system and histologic sections of lung stained according to a modified Masson trichrome method. The severity of these alterations, particularly of fibrosis, was reduced in all groups of animals pretreated with ICRF-187. The fibrosis was reduced to a similar extent in female mice treated with the 300 mg/kg and the 150 mg/kg doses of ICRF-187, from 39.3% to 17.6% and 13.3%, respectively. ICRF-187 induced significantly different degrees of reduction in fibrosis in the 2 groups of male mice treated with the 150 mg/kg and the 300 mg/kg doses, from 30% to 19.7% and 12.2%, respectively. In vitro studies indicated that both ICRF-187 and its open-ring hydrolysis product (ADR-925) remove iron slowly from the bleomycin-iron complex. This observation provides a basis for the concept that ICRF-187 protects by chelating iron involved in the formation of the bleomycin-Fe3+ complex that generates reactive oxygen radicals capable of causing pulmonary damage. PMID- 7537927 TI - Expression of mdm-2 and p53 protein in transitional cell carcinoma. AB - Amplification of the mdm-2 gene and overexpression of the mdm-2 protein might inactivate p53 function, and may have prognostic relevance. The present paper investigated the immunohistochemical overexpression of the mdm-2 and p53 proteins in 25 biopsy specimens of transitional cell bladder carcinomas (10 pT1 and 15 pT2 or higher stages). Five cases (20%) showed strong mdm-2 protein immunoreactivity in more than 5% of the tumor cells; 14 cases (56%) showed p53 immunoreactivity in more than 20% of the cells, and were considered as overexpressing p53 protein. Four of the five cases with strong mdm-2 immunoreactivity did not show p53 overexpression, and 13 of the 14 cases with p53 overexpression did not show mdm-2 immunoreactivity. Our data are consistent with the hypothesis that p53 overaccumulation (and hence possible p53 gene mutation) or mdm-2 overexpression (and hence possible mdm-2 gene amplification) may mirror two different and possibly complementary gene alterations, which might finally interfere with the control of cell proliferation and apoptosis. In this perspective, evaluation of the combined mdm-2/p53 protein phenotype in human bladder carcinomas could have prognostic relevance and give us better prognostic information than evaluation of the p53 protein alone. PMID- 7537929 TI - A specific oligonucleotide probe based on 5S rRNA sequences for identification of Vibrio anguillarum and Vibrio ordalii. AB - An oligonucleotide DNA probe based on 5S rRNA sequence data was constructed for the identification of the fish pathogens, Vibrio anguillarum and Vibrio ordalii. Specificity of the probe was tested in a colony blot hybridization assay. The respective probe was found to be specific for both V. anguillarum and V. ordalii. No cross hybridization was observed against other fish pathogens and the closely related Vibrionaceae genera. This specific probe may be useful for rapid identification of V. anguillarum and V. ordalii. PMID- 7537928 TI - [The late results of surgical interventions in radically inoperable stomach and colonic tumors]. AB - The long-term outcomes of palliative operations and diagnostic laparotomies in 98 patients with radically inoperable cancers of gastric-intestinal tract are analysed. Increased blood oncoantigene parameters are not to be considered as subclinical markers of oncological process. Though complex assessment of cancer patients' curability the parameters of oncoantigene content may occur to be useful in determination of the prognosis of life longitude of patients having undergone the primary tumour focus removal. PMID- 7537926 TI - Nitro-L-arginine analogues. Dose- and time-related nitric oxide synthase inhibition in brain. AB - BACKGROUND AND PURPOSE: The purpose of the present study was to measure cortical nitric oxide synthase (NOS) activity and determine the appropriate doses of N omega-nitro-L-arginine methyl ester (L-NAME) or N omega-nitro-L-arginine (L-NNA) for near-complete enzyme inhibition in dogs, cats, and pigs. We anticipated that NOS inhibition was dose- and time-dependent and questioned if the dose-response relationship was related to the specific drug or animal species. METHODS: Saline or L-NAME or L-NNA in escalating doses was administered to pentobarbital anesthetized pigs, dogs, and cats. Brain temperature and arterial blood gas, hemoglobin, and blood pressure levels were maintained within the physiological range. Cortical tissue was biopsied at baseline and 30, 120, and 360 minutes after agent administration for measurement of NOS activity by isotopic assay of the conversion of [14C]arginine to [14C]citrulline. RESULTS: L-NAME produced > 70% enzyme inhibition at a dose of 20 mg/kg across the species tested. Arterial blood pressure was elevated at 30 minutes after L-NAME treatment. However, consistent decreases in brain NOS activity required a longer period of time. Near complete inhibition was apparent in most animals by 120 minutes and persisted for 6 hours after administration. A smaller dose of L-NNA was required for > 70% enzyme inhibition in the cats and dogs (10 mg/kg). Near-complete NOS inhibition was evident in most animals at 30 minutes after L-NNA administration, which also persisted for 6 hours. In pigs, this same level of inhibition required 20 mg/kg. CONCLUSIONS: These results suggest that administration of L-NAME and L-NNA diminishes brain NOS activity in a dose- and time-dependent manner and that the duration of effect is at least 6 hours. PMID- 7537930 TI - Hospice and home health agency characteristics: United States, 1991. PMID- 7537931 TI - CD34 immunohistochemical staining of bone marrow biopsies in myelodysplastic syndromes. AB - Although it has been shown that the percentage of bone marrow blasts in myelodysplastic syndrome (MDS) constitute the only independent determinant of survival and progression to acute leukemia, the great variability in survival among patients with MDS of similar percentage of blasts has prompted us to investigate new objective, independent prognostic parameters for the selection of high-risk patients. It was suggested that CD34 antigen expression adversely affected the prognosis of acute myelogenous leukemia. However, no study has been published so far on clinical and prognostic significance of CD34 antigen expression in MDS. Bone marrow biopsies from 58 patients diagnosed as primary MDS were studied using QBEND/10, a monoclonal antibody which recognized the human progenitor CD34 antigen on routine aldehyde-fixed, paraffin-embedded samples. The high percentage of CD34-positive cells (above 3% of total bone marrow nucleated cells) was predominantly observed in cases with RAEB-T, CMML, and to a lesser degree in RAEB. But neither age, hemograms, bone marrow findings including percentage of blasts, ALIP, nor leukemic transformation correlated with the percentage of CD34-positive cells. The median actuarial survival time in the high positive group was significantly shorter (12.0 months) than that of the low group (30.0 months; p = 0.028). The high CD34 aggregate (> or = 3) was selectively found in cases with RAEB, RAEB-T, and CMML. The percentage of bone marrow blasts (p = 0.007) and ALIP (p = 0.030) significantly correlated with number of CD34 aggregates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537932 TI - Differential responses of CD34-positive acute myelogenous leukemic blasts to the costimulating effects of stem cell factor with GM-CSF and/or IL-3. AB - Stem cell factor (SCF), a c-kit ligand, has a preferential effect on the proliferation of several classes of immature hematopoietic progenitor cells in combination with GM-CSF or IL-3. To analyze the costimulatory role of SCF in leukemic growth, we investigated the effect of SCF in the presence of GM-CSF and/or IL-3 on isolated CD34-positive (CD34+) leukemic blasts from 15 patients with acute myelogenous leukemia (AML). Cultures of CD34+ cells from normal bone marrow were used as controls. When the proliferation of CD34+ AML blasts in the presence of GM-CSF and/or IL-3 were evaluated in vitro for the effects of SCF, two patterns emerged. In one pattern, CD34+ AML blasts responded with a significant increase in DNA synthesis and/or colony formation when SCF was used with GM-CSF and/or IL-3 relative to the growth with SCF alone; This result is consistent with those CD34+ bone marrow cells from normal donors. Six patients (40%) were included in this category. The addition of SCF as a single factor resulted in colony formation in all six of these cases. In the other pattern, nine of the patients (60%) had CD34+ leukemic cells whose growth with SCF plus either GM-CSF, IL-3, or GM-CSF+IL-3, was not significantly different from the growth noted in the presence of SCF alone. Among them seven cases that did not form colonies in response to SCF alone, and one case showing autocrine, background growth were included. In the six cases in which the costimulating effects of SCF were documented, CD34+ c-kit+ blasts comprised 50.5 +/- 18.7% of the CD34+ leukemic blasts-higher than 21.8 +/- 19.4% of cases in which the costimulating effect of SCF was not documented. In the cases showing high c-kit antigen expression (> or = 40%), SCF had a costimulatory effect in 71% (5/7) of the patients. In conclusion, our data indicate that CD34+ leukemic blasts from a good proportion of patients with AML did not respond to the costimulating effects of SCF in the presence of GM-CSF adn/or IL-3, in contrast to those CD34+ bone marrow cells from normal donors. The possible use of SCF for acute leukemia must await further cytogenetic and molecular studies, which should clarify the preferential costimulating role of SCF in normal hematopoiesis. PMID- 7537933 TI - [Evaluation of the value of triple diagnosis in prenatal medicine]. AB - The evaluation for every new method of early diagnostics is the criterion of quality of previous methods. The Triple diagnostics in the prenatal medicine however does not fulfill the criterion of standard for example for early diagnostics in gynecological oncology. Particularly, the precision in laboratories cannot be safely assumed so that false positive as well as false negative results are possible. The consequences of this method of early diagnostics are uncertainty for patients and physicians and of high expenditure for both. PMID- 7537934 TI - Immunohistochemical study of the distribution of endocrine cells in the gastrointestinal tract of the lesser mouse deer (Tragulus javanicus). AB - The occurrence and distribution of endocrine cells in the gastrointestinal tract of the lesser mouse deer, Tragulus javanicus, were studied immunohistochemically. Fourteen types of endocrine cells immunoreactive for serotonin, somatostatin, enteroglucagon, pancreatic glucagon, bovine pancreatic polypeptide (BPP), gastrin, substance P, motilin, gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), methionine-enkephalin-Arg6-Gly7-Leu8 (MENK-8), secretin, neurotensin, peptide tyrosine tyrosine (PYY) and chromogranin were revealed. Chromogranin-, serotonin-, somatostatin- and enteroglucagon-immunoreactive cells were detected in all regions examined, while pancreatic glucagon-immunoreactive cells, except in the proper gastric gland region, were not found in other regions of the gastrointestinal tract. Few BPP-immunoreactive cells in either the proper gastric gland or pyloric gland regions and abundant gastrin-immunoreactive cells in the pyloric gland region were observed. Restricted distributions of substance P-, GIP-, gastrin-, motilin-, CCK-, MENK-8-, secretin-, neurotensin- and BPP immunoreactive cells in the small intestine, and BPP-, substance P-, PYY- and motilin-immunoreactive cells in the large intestine were noted. The important findings include the presence of BPP-immunoreactive cells in the abomasum, pancreatic glucagon-immunoreactive cells in the proper gastric gland region, and substance P- and motilin-immunoreactive cells in the large intestine. It is suggested that the distribution pattern of gut endocrine cells in the lesser mouse deer is more similar to that in the pig than in the domestic ruminants so far reported. PMID- 7537935 TI - A staining method for bone canaliculi. AB - The modified Bodian method with protargol (silver protein) is ordinarily used to detect nerve fibers. With this technique, applied to decalcified rat bone sections, the bone canaliculi were clearly stained black with good contrast to the bone matrix in both lamellar and woven bone. In addition, the connections between the bone canaliculi and other canaliculi, osteoblasts, osteoclasts, and chondrocytes were easily detectable. We found that the bone canaliculi of woven bone were fewer in number and ran more irregularly than those of lamellar bone. We believe that this staining method for bone canaliculi in decalcified bone is superior to previously reported methods and may be useful in studies on bone pathology. PMID- 7537936 TI - Leukocyte migration and adhesion. PMID- 7537938 TI - [Occurrence of choroidal neovascularization following photocoagulation treatment for central serous retinopathy]. AB - Occurrence of choroidal neovascularization following laser photocoagulation treatment for central serous retinopathy (CSR) has been reported. We reviewed all the cases of photocoagulation treatment for CSR in our clinic during the past 25 years (1968-1993). Among 1,824 CSR-affected eyes which were treated, choroidal neovascularization occurred in 19 at the site of photocoagulation. In a careful reevaluation of pretreatment fluorescein angiograms, small choroidal neovascularizations were detected in 5 eyes, in which cases the diagnosis of CSR was incorrect. In 3 eyes, choroidal neovascularization was suspected and might have been masked. In the remaining 11 eyes, choroidal neovascularization was not seen. Our survey indicates that, in central serous retinopathy, when the age of the patient is over 50 years, and leakage is weak and parafoveal, choroidal neovascularization may be masked. Laser photocoagulation for these eyes should be conservative with careful, long-term observation. PMID- 7537939 TI - Use of the fluorochrome calcofluor white in the screening of stool specimens for spores of microsporidia. AB - Microsporidia are obligate intracellular protozoal pathogens associated with chronic diarrhea in individuals infected with HIV. Direct detection methods for microsporidial spores in stool include chromotrope-based, fluorochrome, and immunofluorescent stains. The authors compared the ability to detect microsporidial spores in 168 stool specimens using two stains: a chromotrope based modified trichrome stain and a fluorochrome stain, calcofluor white (Cellufluor, Polysciences, Warrington, PA). In addition to being faster and easier to perform, the calcofluor white stain was found to be more sensitive than the chromotrope-based stain, as 6 of 24 specimens positive by calcofluor white were negative by the chromotrope-based stain on initial smear evaluation. Repeat examination confirmed these six as being positive. To evaluate the specificity of the calcofluor white stain, 20 formalin-fixed stool specimens (5 positive and 15 negative for microsporidial spores) were evaluated in blinded fashion by two affiliated clinical laboratories using their own formulations of calcofluor white. A single discrepant result (falsely positive) was reported from one laboratory. The use of the calcofluor white stain is recommended as a simple and highly sensitive screening procedure for the detection of microsporidial spores in stool specimens. PMID- 7537940 TI - Auramine fluorescence for acid-fast bacilli in formalin-fixed, paraffin-embedded tissues. PMID- 7537937 TI - Effect of R59022, an inhibitor of diacylglycerol kinase, on IgE-mediated histamine release from human lung mast cells and basophils. AB - We have examined the effect of the diacylglycerol kinase inhibitor R59022 on histamine release from human lung mast cells and basophils. At 1 microM the drug increased the IgE-dependent release of histamine from human basophils from 19 +/- 5% to 60 +/- 13% (n = 5, p < 0.01). The increase in histamine release was dose dependent with maximum enhancement between 1 and 10 microM. 1 microM R59022 also increased f-met peptide-induced histamine release from 18 +/- 4% to 55 +/- 11% (n = 5, p < 0.05). However, the drug did not significantly increase the release of histamine when the non-physiologic stimulus PMA was used to initiate release. The effect of the drug on anti-IgE-induced release was most marked at lower concentrations of anti-IgE and declined when superoptimal concentrations of anti IgE were used. As anticipated there was a strong negative correlation (r = 0.764, p < 0.05) between anti-IgE-induced histamine release and the percentage enhancement in the presence of 10 microM R59022. In contrast, to these potent effects on the human basophil the drug failed to affect the anti-IgE-induced release of histamine from human lung mast cells. The data suggest that the R59022 increases the release of histamine induced by anti-IgE in human basophils but not in human lung mast cells. Furthermore, the ability of R59022 to potentiate basophil histamine release is restricted to receptor-mediated stimuli such as anti-IgE and does not extend to non-physiologic stimuli such as the phorbol ester PMA. PMID- 7537941 TI - Chemotaxis and phagocytosis of the neutrophils mobilized by granulocyte colony stimulating factor in healthy donors for granulocyte transfusions. PMID- 7537943 TI - Sequence-specific fragmentation generated by matrix-assisted laser desorption/ionization in a quadrupole ion trap/reflectron time-of-flight device. AB - Sequence-specific fragmentation for structural analysis has been generated by activation of ions via matrix-assisted laser desorption/ionization (MALDI) in an ion trap storage/reflectron time-of-flight device (IT/reTOF). The key to this work is that ion decay can be induced by MALDI activation but requires an extended period of time to occur in large peptides. This extended decay period, which may be in excess of 20 ms, is provided in these experiments using the long storage times of the ion trap device. The ions are stored until decay is complete and then rapidly pulsed into a reflectron TOF for analysis. Since the ions decay within the trap, they ultimately appear as stable ions in the reTOF rather than as metastable decay peaks. The ion fragmentation was found to depend strongly on laser power and the rf voltage placed on the ring electrode of the trap. The fragmentation obtained was shown to be similar to but different from that observed in FAB-low-energy CAD. In particular, enhanced fragmentation was obtained in the lower mass range and large species could be more easily fragmented than with FAB-low-energy CAD. The types of fragmentation for several target peptides are discussed. PMID- 7537942 TI - Hepatitis B, C, and D virus infections in patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma: a comparative study in Niger. AB - Eighty-nine Sahelian African patients with chronic active hepatitis (CAH) (14), cirrhosis (49), hepatocellular carcinoma (HCC) (26), and 47 controls were tested for hepatitis B virus (HBV, hepatitis B surface antigen [HBsAg]) and hepatitis D virus (HDV, anti-HDV antibody). Seventy-three percent of the patients were positive for HBsAg versus 29.8% of the controls (P < 0.0001). With anti-HDV test, 55.0% of the patients were positive versus 17.0% of the controls (P < 0.0001). To assess the prevalence of antibody to hepatitis C virus (HCV), we used an enzyme linked immunosorbent assay for screening (anti-HCV2): 19.1% of the patients were positive versus 6.4% of the controls (P < 0.05). An association between HBsAg and anti-HDV-positive test results was found in 46.1% of the patients versus 6.4% of the controls (P < 0.0001). A combination of HBsAg and anti-HCV2-positive test results was found in 13.5% of the patients versus 2.2% of the controls (P < 0.05). Anti-HDV and anti-HCV2 test results were positive in 13.5% of the patients versus 2.2% of the controls (P < 0.05). Triple-positive test results (HBsAg, anti HDV, and anti-HCV2) were found in 11.2% of the patients but in none of the controls (P < 0.025). Triple-negative test results were found in 14.6% of the patients versus 57.4% of the controls (P < 0.0001). The predominant association of the chronic HBV infection with CAH, cirrhosis, and HCC is confirmed in Sahelian Africa. The HDV superinfection (chronic HBV plus HDV infections) may be a major etiology.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537944 TI - Primary cultures of human hypertrophic prostate tissue in WAJC 404 medium: a study of cell morphology and kinetics. AB - 222 biopsy fragments of human hypertrophic prostate tissue were cultured in WAJC 404 serum-free medium for three weeks. Growth halos were examined after 7, 14 and 21 days of culture by optical and scanning electron microscopy. Colonies formed of two concentric areas showed in the inner halo elementary pseudo-lobular morphological units similar to the prostate structure. The cell morphological patterns of the halo turned out to be four in number. Every cell pattern was defined morphologically, morphometrically and phenotypically. Results indicate that all morphological differences must be attributed to the various phases of cell life, as all cell types were positive to cytokeratin. The nonconstant display of PSAP and PSA showed a moderate tendency to cell differentiation in WAJC 404 medium. Cell kinetics were also studied and revealed a decrease in proliferation after 14 days of culture. Primary cultures from biopsy fragments of human hypertrophic prostate tissue may be used as an experimental model up to the 14th day of culture. PMID- 7537945 TI - The cardiovascular effects and histamine-releasing properties of 51W89 in patients receiving nitrous oxide/opioid/barbiturate anesthesia. AB - BACKGROUND: Atracurium consists of a mixture of ten stereoisomers. One of these isomers, 51W89, is a potent intermediate-acting nondepolarizing neuromuscular blocking agent. Its ED95 is 0.05 mg.kg-1 in patients receiving nitrous oxide/opioid anesthesia. In preclinical trials, 51W89 did not show evidence of histamine release in cats at doses up to 80 times the human ED95. This study was undertaken to determine the cardiovascular effects and histamine-releasing properties of 51W89 in patients undergoing elective surgical procedures. METHODS: Sixty patients, ASA physical status 1 or 2, anesthetized with nitrous oxide/fentanyl/thiopental were studied. Patients received either 2 times the ED95 of atracurium or 51W89 or 4 or 8 times the ED95 of 51W89 as a rapid intravenous bolus under stable anesthesia, before surgical stimulation. Blood pressure and heart rate were measured by oscillometry and the electrocardiogram in patients receiving 2 times the ED95 of 51W89 or atracurium and by an intraarterial catheter and a tachograph triggered by the arterial pulse waveform in patients receiving 4 or 8 times the ED95 of 51W89. Maximal blood pressure and heart rate changes during the 5 min after administration of the muscle relaxant were recorded. Venous blood samples were obtained before the administration of relaxant and at 2 and 5 min after the administration of relaxant for determination of plasma histamine concentrations by radioenzymatic assay. RESULTS: Maximal blood pressure and heart rate changes in all groups of patients receiving 51W89 were small and similar to those observed in patients receiving 2 times the ED95 of atracurium. The mean maximum percent changes (+/- SE) in heart rate and mean arterial pressure were -0.6 +/- 1.5 and 0.4 +/- 2.5, respectively, in the group receiving 2 times the ED95 atracurium; -1.3 +/- 3.3 and 2.3 +/- 4.4, respectively, in the group receiving 2 times the ED95 51W89; -2.6 +/- 1.0 and 2.6 +/- 1.5, respectively, in the group receiving 4 times the ED95 51W89; and -2.4 +/ 1.5 and -1.0 +/- 1.3, respectively, in the group receiving 8 times the ED95 51W89. No patient developed a decrease in blood pressure > or = 20% or an increase in heart rate > or = 20% that was attributable to muscle relaxant administration. There was no dose-related change in plasma histamine concentration associated with the administration of 51W89. One patient in the study developed transient facial flushing after the administration of atracurium. CONCLUSIONS: 51W89 is a benzylisoquinolinium-type, nondepolarizing muscle relaxant that does not affect plasma histamine concentrations. No cutaneous flushing or clinically important cardiovascular effects were noted after rapid injection of doses up to and including 8 times its ED95 (0.4 mg.kg-1) in healthy patients undergoing elective surgical procedures. PMID- 7537946 TI - Effects of volume loading on pulmonary venous flow pattern in dogs with normal left ventricular function. AB - The effects of altered loading conditions on the pattern of pulmonary venous flow are poorly understood. The authors investigated such effects, therefore, by using volume loading in 6 open-chest dogs. The pulmonary venous flow volume rate curve was obtained with a transit-time ultrasonic flowmeter at a fixed heart rate. Measurements were performed in the control and several states during the intravenous infusion of dextran. The influences of volume loading on hemodynamic and pulmonary venous flow variables were compared between the control state and three interventional states in which mean left atrial pressure was approximately 1, 2, and 3 mm Hg above the control value. The systolic flow volume (SI), which corresponds to left atrial reservoir volume, significantly increased, but the early diastolic flow volume (DI), which corresponds to left atrial conduit volume, did not show significant change with volume loading. The flow volume during left atrial contraction significantly increased with volume loading. The flow volume during one cardiac cycle (PVF) significantly increased with volume loading. Approximately 73% of increased PVF was distributed to the systolic flow. The rest was distributed to the early diastolic flow (14%) and to the flow during left atrial contraction (12%). The change in the ratio of SI/DI significantly and positively correlated with the change in mean left atrial pressure (r = 0.87, P < 0.001). These findings indicate that increased pulmonary venous flow induced by volume loading in dogs with normal left ventricular function is mainly distributed to the left atrial reservoir volume. PMID- 7537947 TI - Toward pharmacologic modification of joint damage in osteoarthritis. PMID- 7537948 TI - [Cancer of the esophagus. Principles of treatment]. AB - Preoperative evaluation of esophageal cancer is fundamental to assess the degree of extension of the tumor and operability. Surgery remains the reference treatment for tumors with limited loco-regional extension. The prognosis depends on the degree of mural infiltration and lymph node involvement. Adjuvant chemotherapy or radiotherapy does not improve survival in randomized trials. In case of inoperability, good paliation can be achieved by electrocoagulation, laser or endoprosthesis, possibly combine with chemotherapy. PMID- 7537949 TI - Granulocyte colony stimulating factor significantly influences neutrophil recovery and duration of hospitalisation in bone marrow transplantation. AB - Eighty-eight consecutive patients undergoing bone marrow transplantation (BMT) from July 1985 to June 1993 were retrospectively studied for their bone marrow engraftment characteristics with and without granulocyte colony stimulating factor (R-metHUG-CSF, Filgrastim). Seventy-seven patients (87.5%) achieved engraftment, 55 out of 65 patients (84.6%) without R-metHUG-CSF and 22 out of 23 patients (95.7%) with R-metHUG-CSF (P > 0.1). The mean duration of administration of R-metHUG-CSF was 15.1 days. The mean time to engraftment was significantly reduced by 7.1 days, from 20.5 days to 13.4 days (P < 0.0001). The mean duration of hospitalisation was also significantly reduced by 11.1 days, from 52.6 days to 41.5 days (P < 0.0001). There were no side effects directly attributable to R metHUG-CSF encountered. We conclude that R-metHUG-CSF is very effective in shortening the duration of neutropenia in the immediate post-BMT period with lesser BMT morbidity, earlier discharge from hospital and lower cost of BMT. We recommend a routine 2-week course beginning on the day after marrow infusion. PMID- 7537951 TI - Peptide cholesystokinin receptor antagonist increases food intake in rats. AB - An analogue of the C-terminal heptapeptide of cholecystokinin (CCK) t-Boc-Tyr(SO3 )-Nle-Gly-D-Trp-Nle-Asp-a-2-phenylethylester is a potent, specific CCK receptor antagonist. Intraperitoneal injection of the antagonist abolished suppression of real feeding and sham feeding by exogenous CCK-8 (1.8 nmol/kg), and significantly increased real feeding. Assuming an antagonist distribution like that of exogenous CCK-8, our results suggest that exogenous CCK-8 and endogenous CCK reduce food intake by acting at a site(s) accessible to peripherally administered peptides. PMID- 7537952 TI - Rational therapy for cutaneous T-cell lymphomas in the 1990s. Fact or fancy? PMID- 7537950 TI - Antiviral effect of cyclopentenone prostaglandins on vesicular stomatitis virus replication. AB - Prostaglandins are potentially useful antiviral agents, however their mechanism of action is unclear. Recent evidence suggests that RNA transcription of vesicular stomatitis virus (VSV) is inhibited by prostaglandins (Bader and Ankel, J. Gen. Virol. 71, 2823-2832, 1990). Prostaglandins are known to have multiple effects on cells which may or may not be related to their antiviral action. We examined the effects of prostaglandins on cells and on VSV RNA polymerase in vitro to seek the mechanism of antiviral action. Actinomycin D inhibited cellular RNA synthesis but failed to block the antiviral activity of prostaglandins on VSV. Thus induction of host cell RNA transcription is not involved in the antiviral action. Neither modulation of the cellular glutathione level by prostaglandins nor formation of prostaglandin-glutathione conjugates was required for the antiviral action. The relative inhibition of VSV RNA polymerase in vitro by prostaglandins with different structures correlated to inhibition of VSV replication in infected cells. This result indicates that the same step in VSV replication is inhibited by prostaglandins both in the in vitro RNA polymerase assay and in the infected cell. PMID- 7537954 TI - A modified staining method to detect Wuchereria bancrofti microfilariae in thick smear preparations. PMID- 7537955 TI - Sperm nuclear maturity in spinal cord-injured men: evaluation by acidic aniline blue stain. AB - For many of the spinal cord-injured (SCI) men who are able to produce an ejaculate, infertility because of poor semen quality is a concern. Impaired spermatogenesis has been attributed as a possible reason for the poor semen quality. If so, events that occur during spermatogenesis may be used as a marker to evaluate the extent of spermatogenic alteration. During spermatogenesis, when the sperm nuclear condensation occurs, lysine-rich somatic histone are replaced by arginine-rich protamines in the DNA. Acidic aniline blue preferentially stains the immature sperm nucleus blue by binding to the lysine. Hence, each sperm can be individually evaluated for nuclear maturity. To test this concept, the nuclear maturity of sperm from 12 SCI men obtained by vibratory stimulation was compared with sperm samples obtained by self-masturbation from 104 non-SCI men. Sperm smears stained with acidic aniline blue were evaluated for nuclear maturity. The percent of unstained spermatozoa for non-SCI men (mean +/- SEM; 83.4 +/- 1.1%) was not statistically different from that of the SCI men (79.7 +/- 4.8%). However, the sperm motility (70.5 +/- 1.2%) and the percentage of normal sperm morphology (50.8 +/- 0.7%) of non-SCI men were significantly (P < .01) different from those of the SCI men (36.5 +/- 6.8% and 44.0 +/- 2.4%). It seems that the poor semen quality observed in SCI men is probably not caused by inadequate nuclear maturity of the spermatozoa. PMID- 7537953 TI - Immunostaining for CD34 to determine trichoepithelioma. PMID- 7537957 TI - The effect of finasteride on prostate volume, urinary flow rate and symptom score in men with benign prostatic hyperplasia. PMID- 7537956 TI - Urological tumours: recent changes. AB - The principles of urology and all surgical disciplines were, not too long ago, considered to be rigidly and permanently established. In this exciting era of change, the foundations of urology are now set on shifting sands rather than the bedrock of the past. The pertinent issue clinically is making discriminating evaluations of new developments so that, where appropriate, they are able to be incorporated into treatment practices to improve patient care. In this overview, a personal appreciation of the more important practical and conceptual changes relating to prostatic, urothelial and renal carcinoma during the past 5 to 10 years is presented. PMID- 7537958 TI - Enhanced complement susceptibility of avidin-biotin-treated human erythrocytes is a consequence of neutralization of the complement regulators CD59 and decay accelerating factor. AB - Biotinylation of erythrocytes (E) followed by avidin cross-linking at specific sites has been suggested as a novel means of drug delivery. Upon avidin cross linking, biotinylated E become complement-activating and highly susceptible to complement lysis, thus bringing about release of entrapped drug. We set out to examine the mechanisms of this biotin-avidin-induced lytic susceptibility, focusing on the effects of biotinylation and avidin cross-linking on the major E complement regulatory molecules, decay accelerating factor (DAF) and CD59. We demonstrate here that biotinylation of E, which does not render them complement activating, partially inhibits DAF but has little effect on CD59. Subsequent cross-linking with avidin causes complete inhibition of DAF and near complete loss of CD59 activity. Following cross-linking, DAF and CD59 become associated in high molecular mass avidin-containing complexes on the membrane. Incorporation of physiological amounts of CD59 into the membranes of biotinylated and avidin cross linked E is sufficient to render these cells resistant to complement lysis whereas incorporation of DAF has relatively little effect. An understanding of the molecular mechanisms underlying complement susceptibility of biotin-avidin treated E should allow a rational design of strategies for drug delivery using E or other large, potentially complement-activating carriers. PMID- 7537959 TI - Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro. AB - Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G 50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537961 TI - Structure-function relationships in Src family and related protein tyrosine kinases. AB - There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains. PMID- 7537962 TI - Regulation of P-selectin by tumor necrosis factor-alpha. AB - The levels of P-selectin mRNA and polypeptide were analyzed in bovine capillary cells treated with or without the cytokine tumor necrosis factor-alpha. The 3 kb P-selectin mRNA was upregulated three- to five-fold in cytokine-stimulated cells. The increase in mRNA correlated with a dramatic but short-lived increase in P selectin polypeptide as determined by metabolic-labeling and immunoadsorption. These data confirm earlier studies on mouse P-selectin expressed in a mouse endothelioma cell line and further indicate that P-selectin function can be regulated not only by rapid translocation to the cell surface but also by cytokine-stimulation of P-selectin biosynthesis. PMID- 7537960 TI - Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells. AB - The present paper described interactions of urinary-type plasminogen activator (u PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u PA-dependent adhesion of U937 cells to vitronectin is mediated. PMID- 7537963 TI - Hutchinson-Gilford progeria fibroblasts exhibit metabolically normal uridine uptake and RNA synthetic rates. AB - The accelerated aging disease Hutchinson-Gilford progeria syndrome displays altered messenger RNA levels in cultured fibroblasts, yet little is known of effects on transcription by RNA polymerases other than RNA polymerase II. Total RNA metabolism was examined by incubation of Hutchinson-Gilford progeria fibroblasts with [5-3H]uridine in asynchronous culture. Uptake of radiolabel was quantitative and was incorporated preferentially (99 +/- 0.3%) into newly synthesized RNA. Progeria and control cultures showed comparable rates of uptake of radiolabel, time courses of RNA synthesis, and relative intensity profiles of newly synthesized ribosomal RNA. These characteristics held over a greater than ten-fold range of cell densities (5 x 10(3) to 8 x 10(4) cells/cm2). Progeria and control fibroblasts thus have comparable metabolic capacities for uridine uptake and net RNA production, emphasizing the relative specificity of transcriptional changes previously identified in progeria cells. PMID- 7537964 TI - Hepatocyte growth factor specifically expressed in microglia activated Ras in the neurons, similar to the action of neurotrophic factors. AB - Hepatocyte growth factor (HGF) mRNA and its receptor (c-Met) mRNA were detected in the fetal and adult rat brain. Expression of c-Met mRNA was increased after birth. HGF mRNA was preferentially expressed in the microglia of the rat brain, while c-Met mRNA was expressed in neurons as well as astrocytes and microglia. Most of the neurons were c-Met positive, and HGF stimulated tyrosine phosphorylation of c-Met (140-kDa) in the neurons. HGF as well as bFGF also activated Ras in the neurons. These results suggest that HGF plays a biological role as one of the neurotrophic factors in the brain. PMID- 7537965 TI - Interferon gamma-inducible protein 10 (IP-10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis. AB - Angiogenesis is fundamental to a variety of physiological and pathological processes. While a number of factors have been identified that induce neovascularization, it is becoming increasingly apparent that endogenous angiostatic factors may play an important role in the regulation of angiogenesis during wound repair, chronic inflammation, and growth of solid tumors. In this study, we demonstrate the novel finding that IP-10, a member of the C-X-C chemokine family, is a potent inhibitor of both IL-8 and bFGF-induced angiogenic activity using in vitro and in vivo assays of angiogenesis. These findings support the contention that IP-10 may be a pivotal cytokine in the regulation of neovascularization. PMID- 7537966 TI - Slow accumulation of acetylcholinesterase in rat brain during enzyme inhibition by repeated dosing with chlorpyrifos. AB - When given to rats, O,O'-diethyl-O-[3,5,6-trichloro-2-pyridyl]- phosphorothionate (chlorpyrifos), a common insecticide, causes an unusually lengthy dose-dependent fall in the activity of brain acetylcholinesterase (AChE; EC 3.1.1.7). To determine whether the slow recovery involves impaired AChE synthesis, experiments were designed to measure AChE activity, immunoreactive AChE protein (AChE-IR) and AChE mRNA. Male, Long-Evans rats, maintained at 350 +/- 5 g, were dosed (s.c.) weekly for 4 weeks with 0, 15, 30, or 60 mg/kg chlorpyrifos in peanut oil. Brain tissue was harvested 1, 3, 5, 7 and 9 weeks after treatment began. AChE activity was measured by Ellman assay, and AChE-IR was estimated by two-site ELISA using monoclonal antibodies to rat brain AChE. While AChE activity fell significantly at all times and doses, AChE-IR increased at 3 and 5 weeks in the two higher dosage groups. Larger increases of AChE-IR were observed after chlorpyrifos was administered for 4 weeks by the oral route. Northern blots quantified with reference to cyclophilin were consistent with stable levels of AChE mRNA. Overall, it appears that chronically reduced brain AChE activity after chlorpyrifos reflects sustained enzyme inhibition, not loss of enzyme protein or suppression of AChE message. PMID- 7537969 TI - Adhesion receptor phenotypes of murine lung CD4+ T cells during the pulmonary immune response to sheep erythrocytes. AB - Understanding the molecular mechanisms of pulmonary lymphocyte recruitment is a crucial step toward selective control of immune lung diseases and infections in immunocompromised hosts. To dissect these mechanisms, we are studying the response induced in primed C57BL/6 mice by intratracheal challenge with the T cell-dependent antigen, sheep red blood cells (SRBC). This study used four parameter flow cytometry to examine expression by CD4+ murine T cells in peripheral blood and lungs of receptors known to be differentially expressed on primed human lymphocytes (CD2, CD11a, CD44, CD45RB, CD49d, and L-selectin). Compared with peripheral blood, more lung CD4+ T cells recovered by bronchoalveolar lavage (BAL) showed a primed phenotype. Judged by low expression of CD45RB or L-selectin, 76 to 90% of BAL CD4+ T cells were primed at all times. Adhesion receptor phenotype of CD4+ T cells in BAL and lung interstitium agreed closely, although BAL contained a greater percentage of primed cells. The percentage of CD4+ T cells with high expression of CD44+ and CD49d increased late in the response. However, when considering only upregulated adhesion receptors which might mediate recruitment, 22 to 52% of CD4+ T cells in BAL did not have increased adhesion receptor expression. Longer duration between priming and challenge did not increase adhesion receptor upregulation. High adhesion receptor expression was least evident during the periods of maximal lymphocyte influx, suggesting that factors other than increased surface density of organ-nonspecific adhesion receptors contribute to lymphocyte recruitment during pulmonary immune responses. PMID- 7537967 TI - A comparative review of the adverse effect profiles of heparins and heparinoids. AB - On the basis of the results of the 11 studies reviewed, thromboprophylaxis with unfractionated heparin, low molecular weight (LMW) heparin or a heparinoid (danaparoid sodium; Org 10172) in patients undergoing total hip replacement did not show any important clinical differences with respect to the tolerability profiles of the different compounds. However, as a result of the great variability in the presentation and evaluation of blood losses and bleeding complications in these studies, it is mandatory to perform a direct comparison of the different compounds in question in a double-blind, prospective clinical study. PMID- 7537968 TI - Glucocorticoid inhibition of RANTES expression in human lung epithelial cells. AB - An influx of eosinophils into the lungs occurs in several pulmonary disorders. However, the mechanisms involved remain unknown. Lung epithelial cell release of eosinophil chemotactic factors such as RANTES or macrophage inflammatory protein 1 alpha (MIP-1 alpha) could account for the influx of eosinophils into the lungs. In order to demonstrate the potential role for lung epithelial cells to release RANTES and/or MIP-1 alpha, we investigated the mRNA expression and protein release in cultured A549 cells. Tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) induced a time- and dose-dependent increase in RANTES mRNA expression and protein release. In contrast, MIP-alpha protein release was not detectable in these cells. As corticosteroids decrease the influx of eosinophils into the lungs in vivo, we also investigated the capacity of dexamethasone to decrease the TNF alpha-induced RANTES release and mRNA expression; both were decreased in a time- and concentration-dependent manner. Dexamethasone did not affect the TNF alpha-induced RANTES mRNA half-life and did not require protein synthesis to manifest an inhibitory effect. Supernatant from cells stimulated with TNF alpha and IL-1 beta increased eosinophil chemotaxis and this was also inhibited by dexamethasone. These findings suggest a role for RANTES release by lung epithelial cells in the recruitment of eosinophils into the lungs in pulmonary disorders such as interstitial lung diseases, idiopathic pulmonary fibrosis, or asthma and suggest that one beneficial effect of corticosteroids may be inhibition of lung epithelial cell RANTES mRNA expression and protein release. PMID- 7537970 TI - Distribution of integrins alpha v beta 6 and alpha 9 beta 1 and their known ligands, fibronectin and tenascin, in human airways. AB - We have previously identified two integrins, alpha 9 beta 1 and alpha v beta 6, from guinea pig airway epithelium. The extracellular matrix protein tenascin is a ligand for both of these receptors, and fibronectin is also a ligand for alpha v beta 6. In the present study, we used immunohistochemistry to examine the expression and spatial distribution of the alpha 9 subunit, alpha v beta 6, tenascin, and fibronectin in the proximal airways of 10 normal nonsmoking subjects and eight patients undergoing lung resection for cancer. We also performed the same analyses on sections of peripheral lung obtained from an additional seven subjects undergoing lung resection. alpha 9 was highly expressed throughout the airway epithelium (but not on alveolar epithelium) irrespective of clinical status. In contrast, alpha v beta 6 was expressed on proximal airway epithelial cells in four of eight smokers undergoing lung resection, but in none of the normal subjects and none of the distal airways examined. On bronchial epithelial cells cultured from resected airways, alpha v beta 6 was highly expressed on cells grown from patients who did not appear to express the receptor in vivo, as well as from subjects who did, suggesting that some component of the in vitro environment can induce expression. Although both tenascin and fibronectin were present below the proximal airway epithelium of both normal nonsmoking subjects and smokers, the spatial patterns of integrin and ligand expression were not congruent, because the integrins were present diffusely on the cell surface and on some cells that were not in contact with the basement membrane, whereas the ligands were present principally in the subepithelial layer. These findings are compatible with the existence of as-yet unidentified ligands for each of these integrins--for example, ligands involved in homotypic cell-cell interactions within the epithelium. PMID- 7537971 TI - [Pingyangmycin-induced chromosome damage in lymphocytes of laryngeal cancer patients]. AB - Lymphocytes from 28 untreated laryngeal cancer patients and 23 healthy controls were cultured in vitro and exposured to pingyangmycin (bleomycin A5), a clastogen. The lymphocytes were arrested in metaphase and analyzed. The total chromosome aberration rate, mean chromatid break rate per cell and cell aberration rate were 1.98% +/- 0.05%, 0.57% +/- 0.35%, and 42.8% +/- 12% respectively for laryngeal cancer patients. However, for healthy controls these values were 0.94% +/- 0.04%, 0.28% +/- 0.12%, and 27% +/- 12% respectively. Statistical analysis showed there are significant differences between the two groups. The data indicate that under our experimental conditions chromatid break rate 0.40 can be considered to be a borderline value, 0.80 hypersensitive value. For any individual, if the chromatid break rate is 0.40 or more, one should be ranked as having cancer risk. If 0.80 or more, then, highly cancer risk. PMID- 7537973 TI - Down-regulation of CFTR expression in an HT29-derived cell line by stable antisense RNA production. AB - In order to examine the interactions of the cAMP activated CFTR chloride ion channel with other chloride ion channels present in epithelial cells we have generated a model based on antisense mRNA down regulation of CFTR expression. The HT29-derived epithelial cell line CJC4-1 has integrated a plasmid that is constitutively expressing CFTR exons 1-6 in the antisense orientation, from the MoMULV LTR. This cell line shows a reduction in cAMP-activated chloride efflux, as measured by iodide efflux assay, in comparison to the parental HT29 cell line and a sense transfected control. PMID- 7537972 TI - Isolation from human brain of six previously unreported cDNAs related to the reverse transcriptase of human endogenous retroviruses. AB - cDNAs prepared from total RNA extracted from plaques of multiple sclerosis were amplified by the polymerase chain reaction. The 11-bp degenerate primers used were derived from conserved sequences of reverse transcriptase. Amplified cDNAs were fractionated according to size by electrophoresis in polyacrylamide gels under denaturing conditions. cDNAs of the proper size were cloned, grouped according to the sequence of their insert by differential hybridization, and sequenced. Six cDNAs were isolated and found to belong to new members of two groups of human endogenous retroviruses: the group related to ERV9 and that related to HERVK10 and HUMMTV. These sequences were expressed in all human organs tested, including normal white matter of brain. The approach described in this article is a powerful tool with which to isolate new members of the reverse transcriptase gene family. PMID- 7537975 TI - Differential effects of polyunsaturated fatty acids on cell growth and differentiation of premonocytic U937 cells. AB - The effect of long chain polyunsaturated fatty acids (PUFA) on cell growth and differentiation was assessed in human premonocytic U937 cells. Addition of either 10 microM arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) resulted in the rapid incorporation of these fatty acids into cellular phospholipids. Their uptake was greatest in the first 2 h. AA and EPA reached steady-state levels after 8 h, while levels of DHA increased steadily over 72 h. In parallel, fatty acid metabolites derived from AA and EPA, 22:4n-6, 22:5n-6 and 22:5n-3, 22:6n-3, respectively, increased continuously indicating an active fatty acid elongation and desaturation. The effects of PUFA on monocytic differentiation were examined in cells which had been enriched with AA, EPA or DHA for 8 h and subsequently treated with retinoic acid (RA), 1,25-(OH)2-vitamin D3 (1,25-D3), interferon-gamma (IFN-gamma) or their combinations for 72 h. Growth of differentiating or non-differentiating U937 cells was not affected by enrichment with PUFA. However, in cells differentiated with 1,25-D3 plus IFN-gamma, prior enrichment with all three PUFA slightly but significantly (P < 0.05) increased the expression of the monocytic surface antigens CD11b and CD14 and generation of superoxide anion. The data indicate that although n-6 and n-3 PUFA are rapidly incorporated into phospholipids, they do not affect cell growth. However, enrichment with PUFA increases monocytic differentiation of U937 cells when induced most effectively with 1,25-D3 plus IFN gamma. PMID- 7537977 TI - Xanthomonas maltophilia misidentified as Pseudomonas cepacia in cultures of sputum from patients with cystic fibrosis: a diagnostic pitfall with major clinical implications. AB - Pseudomonas cepacia infection in patients with cystic fibrosis (CF) has major significance in terms of infection control, psychosocial issues, and medical treatment. We describe three instances in which the diagnostic laboratory misidentified Xanthomonas maltophilia as P. cepacia in cultures of sputum from patients with CF. These errors were recognized when 3 (9%) of 32 isolates, which had all been identified as P. cepacia and had been submitted to the Canadian Pseudomonas Repository Laboratory (Vancouver, BC), were correctly identified there as X. maltophilia. Each of the three isolates grew well on P. cepacia media, turned a characteristic vivid pink color, were polymyxin-resistant, and were lysine-positive. All three were initially characterized incorrectly as oxidase-positive and DNase-negative. The diagnostic laboratory then reexamined 24 other isolates that had been identified as P. cepacia; complete biochemical testing confirmed that all were indeed P. cepacia. Because infection due to P. cepacia has major implications for patients with CF, when a possible strain of P. cepacia is isolated, careful and complete characterization should be performed. PMID- 7537976 TI - Inhibitors of protein and RNA synthesis block the cytotoxic effects of non steroidal antiestrogens. AB - Non-steroidal antiestrogens such as tamoxifen are known to exert cytotoxic effects against various cell lines in culture. When the antiestrogens are present at sufficiently high concentrations, their cytotoxicity cannot be reversed by estrogens and is demonstrable even with cell lines which lack the estrogen receptor. The mechanism of this cytotoxicity, which is clearly independent of estrogen antagonism, remains unknown. Using two murine cancer cell lines (the K36 leukemia and the EL4 lymphoma cell line), the human breast cancer cell line MCF7, and two non-steroidal antiestrogens (tamoxifen and clomiphene), our laboratory attempted to determine whether the cytotoxic action of non-steroidal antiestrogens was mediated by a mechanism requiring protein or RNA synthesis. In the case of K36 and EL4 cells, inclusion of tamoxifen or clomiphene in the culture medium regularly caused the viable call count to fall below 20-30% of control in 36-48 h. Under these conditions, the addition of inhibitors of protein or RNA synthesis consistently increased viable cell count in a dose-dependent manner. With cultures of K36 cells grown in the presence of 10 microM tamoxifen, for example, the addition of appropriate concentrations of emetine, cycloheximide, puromycin, or actinomycin D increased the percentage of viable cells to 5.0, 2.4, 4.0, and 4.0 times that of control, respectively. Additional experiments revealed that the macromolecular synthesis inhibitors, while effective in inhibiting protein or RNA synthesis to varying degrees, did not affect the cellular uptake of [3H]tamoxifen, suggesting that their ability to protect cells against antiestrogen-induced cell death was not due to an inhibition of cellular uptake of antiestrogens. In the case of MCF7 cells, however, inhibition of protein synthesis did not protect the cells against the cytotoxic effect of tamoxifen. These observations suggest that non-steroidal antiestrogens may exert their cytotoxic effect by at least two different mechanisms; only one of these require de novo protein synthesis. The effect of antiestrogens on K36 and EL4 cells may provide a useful system for the identification of proteins involved in cell death. PMID- 7537974 TI - The mouse homologue of the HTLV-I tax responsive element binding protein TAXREB107 is a highly conserved gene which may regulate some basal cellular functions. AB - We report the cDNA sequence of a mouse gene homologous to the HTLV-I tax responsive element binding protein TAXREB107 (M-TAXREB107). This gene is constitutively and ubiquitously expressed indicating a conserved biological function. We present evidence that its transcription is under strict control of a regulatory factor, which is rapidly metabolized. PMID- 7537978 TI - Immunogenicity of human recombinant acetylcholine receptor alpha subunit: cytoplasmic epitopes dominate the antibody response in four mouse strains. AB - In mysathenia gravis (MG) autoantibodies directed against acetylcholine receptors (AChR), at the neuromuscular junction lead to muscle weakness. These antibodies are directed against extracellular determinants, predominantly on the AChR alpha subunits. Similar antibodies can be induced in animals by immunisation with purified AChR, but immunisation of mice with recombinant human alpha subunit or its extracellular domain has produced conflicting results. To study further the immunogenicity of the human alpha subunit we immunised four inbred stains (C57B1/6, SJL, BALB/c, SWR) with almost full-length recombinant alpha subunit, r37-429, and looked at B cell epitopes by mapping with smaller recombinant fragments and synthetic peptides. The majority of anti-r37-429 antibodies bound to sequences within a region thought to be cytoplasmic, alpha 325-368, and reacted with human AChR. In two C57B1/6 sera, only, most antibodies were directed against an extracellular region, alpha 138-167, but the r37-429 used for immunisation of these two mice appeared to have lost the integrity of its cytoplasmic domain during preparation. Our results suggest that the antigenicity of the cytoplasmic region of the recombinant alpha subunit dominates the immune response in each of the four strains, and may even suppress the formation of antibodies to the extracellular domain. Moreover, although C57B1/6 and SJL mice were able to produce antibodies to alpha 138-167, these antibodies did not react with intact AChR, and none of the mice became weak. PMID- 7537979 TI - Intrathyroidal cell phenotype in murine lymphocytic and granulomatous experimental autoimmune thyroiditis. AB - In-vitro mouse thyroglobulin (MTg) activated spleen cells from immunized donor mice can induce experimental autoimmune thyroiditis (EAT) after transfer to recipient mice. The intrathyroidal cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Cells cultured with MTg together with anti IL2R antibody induce EAT with a granulomatous histopathology in which the thyroid infiltrate contains mononuclear cells (MNC) in addition to PMN's histiocytes, and multinucleated giant cells. Flow cytometric analysis of intrathyroidal MNC infiltrates demonstrated that both CD4+ and CD8+ T cells infiltrate the thyroid in both lymphocytic and granulomatous EAT and that CD8+ T cells outnumber CD4+ T cells. There were usually increased numbers of PMN's in the granulomatous thyroids, but low number of Ig+ and F4/80+ cells (macrophages) in the intrathyroidal infiltrate of both disease types. IL2R and Pgp-1 were expressed on both CD4+ and CD8+ intrathyroidal T cells. The majority of CD8+ cells were ICAM+, LFA-1+, and CD45RB+ whereas only a small percentage of CD4+ intrathyroidal T cells expressed these markers. There were no major differences in intrathyroidal MNC phenotype between lymphocytic and granulomatous EAT. Depletion of CD8+ T cells in recipient mice did not reduce EAT severity and resulted in an increased percentage of intrathyroidal CD4+ T cells expressing IL2R. These results suggest that CD8+ T cells are not functioning as effector cells in lymphocytic or granulomatous EAT. PMID- 7537980 TI - FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units. AB - The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell-depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages. PMID- 7537981 TI - Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow. AB - T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T progenitor cell potential within the CD34+ Lineage--(Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin- ABM cells with CD45RA, Thy 1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA- cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro. PMID- 7537982 TI - Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice. AB - The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice. PMID- 7537984 TI - C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo. AB - We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors. PMID- 7537983 TI - CD44 variant isoforms in non-Hodgkin's lymphoma: a new independent prognostic factor. AB - Isoforms of the transmembrane glycoprotein CD44, generated by alternative RNA splicing, have been correlated to tumor dissemination. For evaluation of the potential role of CD44 variant isoforms in non-Hodgkin's lymphoma (NHL), the presence of CD44 isoforms was analyzed in a large panel of reactive and neoplastic lymphoid tissues by immunohistochemical staining, as well as detection of CD44 variant RNAs by the reverse transcriptase-polymerase chain reaction. Whereas the CD44 standard or hematopoietic isoform (CD44s), devoid of the variant regions, was expressed in all leukocyte subpopulations, the variant isoforms (CD44v) showed a highly restricted pattern of expression, mainly observed in epithelial layers of lymphoid tissues and subpopulations of leukocytes after stimulation. In addition to a strong expression of CD44s, variant isoforms containing CD44-6v in combination with other variant exons were observed predominantly in aggressive lymphoma and were associated with a shorter overall survival of patients (n = 138; P < .0001). Moreover, multivariate analysis indicated CD44-6v as a new independent prognostic parameter in high grade NHL in comparison with the risk groups defined by the International NHL Lymphoma Prognostic Factors Project (N Engl J Med 329:987, 1993). PMID- 7537986 TI - Hemin-induced acceleration of hemoglobin production in immature cultured erythroid cells: preferential enhancement of fetal hemoglobin. AB - The effects of heme, when added as the ferric chloride salt, hemin, on human erythroid cells grown in a two-phase liquid culture system were studied. When added together with erythropoietin, on initiation of the second phase of the culture, hemin greatly accelerated hemoglobin (Hb) accumulation in these cells. The effect was greater during their early stages of maturation, suggesting that heme availability is then a rate-limiting step for Hb synthesis. Hemin increased preferentially the production of fetal Hb (HbF) compared with adult Hb; this was associated with a selective twofold elevation in gamma-mRNA levels. Using succinylacetone, a potent inhibitor of heme synthesis, we showed that exogenously supplied hemin could be incorporated into the de novo formed Hb. Therefore, the mechanism of hemin action may be several fold, including effects on globin gene transcription and posttranslational events, eg, supplying the prosthetic group for Hb assembly. Hemin increased HbF of cells derived from patients with sickle cell anemia and beta-thalassemia as well as that of cells from normal donors. Moreover, when added in combination with other HbF-augmenting agents such as the cytotoxic drug, hydroxyurea, a synergistic effect was obtained, with considerably less cytotoxicity than with hydroxyurea alone. These results have clinical potential in light of the ameliorating effect that increased HbF has in patients with genetic diseases of the beta-globin chain and raise the possibility of combined treatment with hemin and other drugs now being used to treat these diseases. PMID- 7537985 TI - Double-stranded RNA induces sickle erythrocyte adherence to endothelium: a potential role for viral infection in vaso-occlusive pain episodes in sickle cell anemia. AB - Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double-stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1--dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double-stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes. PMID- 7537990 TI - [Bleomycin is an antineoplastic agent capable of mimicking apoptosis]. AB - Bleomycin (BLM), a currently used anticancer drug, crosses the plasma membrane poorly. Electropermeabilization allows a defined number of BLM molecules to enter directly into the cell cytoplasm. Such a procedure has revealed that BLM is endowed with a very high intrinsic cytotoxicity. Here we show that when several million molecules of BLM are internalized, morphological changes identical to those usually associated with apoptosis are observed as well as very rapid DNA fragmentation into oligonucleosomal-sized fragments. We demonstrate that this fragmentation, which occurs within a few seconds after BLM internalization, is consistent with a direct internucleosomal cleavage of chromatin by BLM. This result reinforces the importance of DNA digestion as an early and essential step in the morphological changes associated with apoptosis. Our study also demonstrates that BLM cytotoxicity is actually due to the DNA double strand breaks generated by this drug. PMID- 7537987 TI - TCR-CD56+CD2+ nasal lymphomas with membrane-localized CD3 positivity: are the CD3+ cells neoplastic or reactive? PMID- 7537988 TI - FK 506 rescue therapy for hepatic allograft rejection: experience with an aggressive approach. AB - Although initial experiences with FK 506 rescue therapy for acute hepatic allograft rejection have provided promising results, analysis of available data indicates that inferior results are obtained when FK 506 rescue therapy is initiated in the latter stages of rejection. Since its initial availability, we have applied an aggressive approach towards FK 506 rescue therapy based on early conversion and assiduous dosing. We have reviewed our experience with this approach in patients with refractory hepatic allograft rejection to provide an assessment of this approach. Sixteen patients were treated for corticosteroid and OKT3-resistant acute hepatic allograft rejection. Fourteen patients were treated for cellular rejection and 2 for humorally-mediated rejection. Median follow-up was 7.3 months posttransplant and 6.0 months post-initiation of FK 506 therapy. Median time to first rejection was 8 days and median time to FK 506 therapy was 29 days. Laboratory values at the time of initiation of FK 506 therapy included: mean serum bilirubin, 4.0 +/- 3.1 mg/dl and SGPT 136 +/- 105 U/l. Prior to FK 506 therapy, patients received an average of 35.5 +/- 19.1 mg/kg of bolus/taper corticosteroids (prednisone equivalent) and 11.25 +/- 4.8 days of OKT3 therapy. FK 506 therapy was successful in reversing all episodes of rejection. Median time to rejection reversal with FK 506 rescue therapy was 23 days (mean +/- SD, 27.6 +/- 16.7 days) in patients with cellular rejection. Time to rejection reversal was 26 and 28 days in the 2 patients with humoral rejection. Patient and graft survival at 6 months were 100%/100%, and 94%/94% at 12 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537989 TI - [Pilot study of screening of prostate cancer by the assay of prostate specific antigen in occupational milieu]. AB - The goal of this study was to confirm the capacity of occupational medicine to become involved in cooperative screening programs with a dosage of the PSA (Prostate Specific Antigen) determined by immunoradiometric assay. Two thousands and five hundred seventy three salaried workers in the building sector, between 50 and 65 years old, participated in this investigation. Thirty seven individual ie 1.4% had a PSA level above or equal to 10 micrograms/l. Among them, 35 were checked within three months and 17 were found to have a persistently elevated PSA level. In this subgroup 15 pathologies including two cancers were found. We observed a great variability in the results of PSA determination in the groups of individuals whose initial assay level was above or equal to 10 micrograms/l. The linear correlation coefficient between the two assays (on the same individual), carried out at a six week interval on average, was low (r = 0.52 for N = 35). In our series, 3.5% of patients followed up had undergone a rectal examination less than a year previously. Occupational medicine seems to be an efficient setting for screening intervention. However, the people mainly concerned by our study, (salaried workers seen through the physicians interviewed) did not seem very aware of this type of action. PMID- 7537991 TI - [Comment apropos of the article: prognostic value of nutritional parameters in gastrectomy for cancer: a study of 203 cases]. PMID- 7537993 TI - Detection of prostate cancer. Recent evidence suggests screening may be justified in high risk younger men. PMID- 7537992 TI - Toxicological effects in rabbits induced by endosulfan, lindane, and methylparathion representing agricultural byproducts contamination. PMID- 7537994 TI - Immunoperoxidase detection of polycyclic aromatic hydrocarbon-DNA adducts in oral mucosa cells of smokers and nonsmokers. AB - An immunoperoxidase method using a polyclonal antiserum which recognizes benzo(a)pyrene and structurally related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts has been developed for the detection and quantitation of DNA damage in single cells. The method was used initially on 10T1/2 cells treated with [3H]anti-benzo(a)pyrene diol epoxide then applied to the detection of adducts in oral mucosa cells of smokers and nonsmokers. Levels of DNA damage were elevated in each of 16 smokers (mean relative staining, 503 +/- 104) compared to 16 age-, race- and sex-matched nonsmokers (251 +/- 82; P < 0.0001). There was an approximately 3-fold range in relative staining in both smokers (252 +/- 125 to 663 +/- 189) and nonsmokers (157 +/- 72 to 431 +/- 269) suggesting the importance of individual differences in capacity to metabolize the carcinogens and/or repair damaged DNA. This noninvasive method, requiring small numbers of cells, will be useful for routine monitoring of DNA damage in intervention studies as well as for biofeedback in smoking cessation programs. PMID- 7537997 TI - New method for stem cell quantification: applications to the management of peripheral blood stem cell transplantation. AB - A dramatic increase in peripheral blood stem cells (PBSC) is observed after high dose chemotherapy followed by haematopoietic growth factors. The degree of mobilisation of PBSC is quantified by the level of clonogenic cells detected by CFU assays (CFU-GM or CFU-GEMM) or CD34+ cell determination. Working under the hypothesis that, in peripheral blood, mononuclear cells in DNA synthesis (MCDS) are proliferating stem cells, we decided to detect these cells by flow cytometric measurement of their DNA content. The relations between the number of MCDS and well-known haematopoietic progenitor indicators such as CFU-GM or CD34+ cells were analysed. We studied the kinetics of recruitment of PBSC in cancer patients, treated with rmeHuG-CSF following VP-16 cytoxan chemotherapy, until the first day of leukapheresis. For the 31 patients studied the individual curves of peripheral MCDS and CFU-GM reconstitutions showed identical profiles and a good correlation was noted between the numbers of peripheral MCDS and CFU-GM (r = 0.73). In the leukapheresis product, the predictive value of MCDS was equivalent to CFU-GM for PBSC quantification (r = 0.70). In conclusion, MCDS analysis by flow cytometry provides reliable results and appears to be an alternative to CFU-GM assay or CD34+ cell determination for PBSC quantification. PMID- 7537996 TI - Fuzzy classification of nucleotide sequences and bacterial evolution. AB - A new method for reconstructing evolutionary relationship among bacteria by use of rRNA sequence data is proposed. The method is based on the concept of fuzzy classification of probabilities p(i), p(i/j) and p(i/j*) (i = A, G, C, U) of each sequence. The resulting partition tree shares common features of previous works but has some new peculiarities. PMID- 7537995 TI - Mutagen sensitivity as a biological marker of lung cancer risk in African Americans. AB - Cigarette smoking is the major determinant of lung cancer. However, only a fraction of smokers develops lung cancer; genetically determined susceptibility factors seem to play an important role also. Previous case-control studies have shown that in vitro bleomycin-induced mutagen sensitivity is an independent risk factor for head-and-neck cancers, and preliminary data suggest a similar association with lung cancer. However, these studies were almost exclusively performed on Caucasian populations. To test whether ethnic differences in cancer risk are due to differences in mutagen sensitivity, we are using the in vitro mutagen sensitivity assay to conduct a case-control study of mutagen sensitivity and lung cancer risk in low-risk (Mexican-American) and high-risk (African American) groups. Here we report the results of our ongoing study of 209 African Americans (90 cases and 119 controls) in the Houston-Galveston area. Mexican American data will be reported separately as case accrual increases. Predictably, all measures of cigarette smoking status (including intensity, duration, tar content, depth of inhalation, and type of cigarette) were significant predictors of risk. In addition, 55.3% of the cases were mutagen sensitive (defined as > or = 1 break/cell), compared with 24.6% of the controls, with an age-, sex-, and smoking-adjusted odds ratio (OR) of 3.7 (95% confidence limits = 1.4, 9.4). Of interest, higher risks were noted for former smokers (OR = 5.4) compared with current smokers (OR = 3.1) and especially for younger former smokers (< 55 years). By histologic-specific analysis, mutagen sensitivity was significantly associated with risk for adenocarcinoma (OR = 4.8) and squamous cell carcinoma (OR = 8.5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7537998 TI - Collection of progenitor cells for allogeneic transplantation from peripheral blood of normal donors. AB - Fourteen donors were given recombinant G-CSF to mobilize progenitor cells. Circulating CD34+ cells were monitored daily and leukapheresis was performed at 3 5 days when the level exceeded 20 x 10(6)/L. Monitoring of CD34+ cells collected at intervals during apheresis gave results within 20 min. Yields of 2.6-7.4 x 10(6) per kg recipient body weight were achieved in single aphereses of 2-4 h in all but two cases where the donor was substantially smaller than the recipient. These products were sufficient to establish engraftment, at least of granulocytes, in 11 five or six antigen matched recipients with high risk malignancy. Despite some complications donors tolerated the procedure well and the five individuals who had previously given marrow preferred these manoeuvres to bone marrow harvest. The ability to monitor CD34+ cells rapidly in the circulation and leukapheresis product facilitates an efficient collection technique for allogeneic BCT donors. Adequate yields could probably be achieved by a single harvest on days 2-4 in most donors. PMID- 7537999 TI - Allogeneic peripheral blood progenitor cells mobilized by G-CSF (filgrastim) for a second transplant in a patient with acute myeloid leukemia in relapse. AB - A 25-year-old man with AML, who relapsed 21 months after his first allogeneic bone marrow transplant (BMT), underwent a second transplant with peripheral blood progenitor cells (PBPC) obtained from his HLA-identical sibling. The donor cells were collected through four aphereses after G-CSF mobilization with 5 micrograms/kg/d for 5 days. The patient received BAVC conditioning regimen followed by non-T cell-depleted PBPC. Successful engraftment occurred with rapid hematopoietic recovery (time to reach 0.5 x 10(9)/L neutrophils and 50 x 10(9) platelets/L was 15 and 19 days, respectively). A bone marrow aspirate on day +19 showed trilineage engraftment. Erythrocyte phenotype showed that erythropoiesis was of donor origin. The patient developed grade II acute GVHD that responded to prednisone. Seven months after PBPC transplantation he remains in complete remission, alive and well, with just limited chronic GVHD. Allogeneic peripheral blood progenitor cell transplantation may be considered a suitable alternative to marrow transplant. PMID- 7538000 TI - The number of circulating CD34+ blood cells predicts the colony-forming capacity of leukapheresis products in children. AB - In children, only a few guidelines are available for optimizing peripheral blood progenitor cell (PBPC) harvesting. We analyzed by means of flow cytometry and clonogenic assays 60 harvest products obtained from 20 children by standardized leukapheresis after treatment with chemotherapy and CSF. In addition, 27 fresh blood samples obtained prospectively during the mobilization phase were studied. CFU-GM/kg significantly correlated with MNC/kg, CD34+ cells/kg and CD34+33- cells/kg in apheresis products (P < 0.001). In fresh blood samples, CFU-GM/ml significantly correlated with MNC/ml, CD34+ cells/ml and CD34+33- cells/ml (P < 0.001). The numbers of CD34+ cells/ml, CD34+33- cells/ml and MNC/ml in 19 blood samples taken prior to leukapheresis were compared with CFU-GM/kg harvested and thawed after cryopreservation applying multiple regression analysis with stepwise variable selection. The number of circulating CD34+ cells/ml prior to leukapheresis highly correlated with and was predictive for the number of collected CFU-GM/kg (P < 0.001). In addition, a significant correlation (P < 0.05) between the number of progenitor cells/kg reinfused and the time to myeloid and platelet recovery was found in children undergoing high-dose therapy. Our data indicate that a single leukapheresis will be sufficient to obtain a minimum number of 5 x 10(4) CFU-GM/kg if the pre-harvest number of circulating CD34+ cells is > or = 10(5)/ml. Thus, our results will help to optimize PBPC transplantation in children. PMID- 7538001 TI - Potential of autologous immunologic effector cells for bone marrow purging in patients with chronic myeloid leukemia. AB - Relapse is a major concern in autologous bone marrow transplantation (BMT). Therefore, purging of bone marrow to reduce the amount of tumor cells reinfused into the patient is widely used. Immunologic effector cells such as lymphokine activated killer (LAK) cells are attractive for purging of bone marrow since these cells might have an additional in vivo effect on tumor cells in contrast to other purging protocols. In patients with chronic myelogenous leukemia (CML), LAK cells can only be used in some patients for purging bone marrow since LAK cells possess no or only limited cytolytic activity against autologous CML tumor cells in most cases. In this study, we investigated the effect of autologous and allogeneic cytokine-induced killer (CIK) cells on tumor cells from patients with CML. CIK cells have been generated from peripheral blood lymphocytes by incubation with interferon-gamma on day 0, interleukin-1, interleukin-2 and a monoclonal antibody against CD3 on day 1. In contrast to LAK cells, CIK cells were able to lyse both autologous and allogeneic cells from patients with CML as determined by a 51Cr release and a tumor colony assay. The cytotoxicity of CIK cells against CML cells was confined to the CD56+ population. CIK cells showed no major toxic effect on hematopoietic progenitor cells when tested in CFU-GM assays. CIK cells eliminated three orders of magnitude of K562 cells and less than one order of magnitude of progenitor cells (25% reduction). This represents a differential effect of CIK cells on tumor and progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538002 TI - Low-dose granulocyte colony-stimulating factor enables the efficient collection of peripheral blood stem cells after disease-oriented, conventional-dose chemotherapy for breast cancer, malignant lymphoma and germ cell tumor. AB - Peripheral blood stem cells (PBSCs) were collected from 29 adult patients (median age 42 years, range 14-59 years) with breast cancer, germ cell tumor and malignant lymphoma after disease-oriented, conventional-dose chemotherapy combined with daily subcutaneous injections of low-dose (50 micrograms/m2 or 2 micrograms/kg) granulocyte colony-stimulating factor (G-CSF). The median number of colony-forming units-granulocyte macrophage (CFU-GM) collected in an apheresis was 2.37 (range 0-60.6) x 10(4)/kg body weight. Taking into consideration the minimum number of CFU-GM for hematopoietic reconstitution (at least 1 x 10(5) CFU GM/kg), it was suggested that sufficient PBSCs could be collected by a few leukaphereses, although the cell yields of PBSCs tended to differ among the chemotherapeutic regimens. Twelve patients subsequently received high-dose chemotherapy followed by peripheral blood stem cell transplantation (PBSCT), including four receiving PBSCT alone and eight both PBSCT and autologous bone marrow transplantation (BMT). When compared with the 20 patients who received high-dose chemotherapy followed by autologous BMT alone, the median day of recovery of a neutrophil count > 0.5 x 10(9)/l and a platelet count > 20 x 10(9)/l was significantly shortened in those who received PBSCT (9 vs 12 days; P < 0.01 and 14 vs 30.5 days; P < 0.001), resulting in a lower platelet transfusion requirement (4.5 vs 9; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538003 TI - Presence of hyaluronan binding protein in cardiac myoblasts and its altered level during myogenesis. PMID- 7538004 TI - Transport mechanisms of enoxacin in rat brush-border membrane of renal cortex: interaction with organic cation transport system and ionic diffusion potential dependent uptake. AB - The mechanism of the renal transport of enoxacin (ENX) has been investigated using brush-border membrane vesicles (BBMVs) isolated from the rat renal cortex. The initial rate and time-course of ENX uptake were quite dependent upon the medium pH (pH 5.5 > pH 7.5). The pH dependence was in accordance with the degree of cationic form. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) affected the transient uphill transport of ENX across the renal brush-border membrane in the presence of an outward-directed H(+)-gradient. The initial uptake was saturable, and transport kinetic parameters were given for a Km and Vmax of 0.59mM and 1.37nmol/(mg protein)/30s, respectively. On the other hand, an outward H(+)-gradient (pHin = 5.5, out = 7.5) dependent uptake of ENX was partially decreased by the voltage-clamped BBMVs. Furthermore, a valinomycin-induced K(+) diffusion potential (interior negative) was found to increase the uptake of ENX at pH 5.5, which is cationic form-rich. These results suggest that ENX uptake participates in not only the H+/organic cation antiport system for organic cation secretion but also the ionic diffusion potential (interior negative) dependent permeation through the membrane. PMID- 7538005 TI - The search for susceptibility genes of COPD. AB - Environmental factors, such as cigarette smoking, outdoor and indoor pollution, and childhood respiratory infections, are believed to play a major role as risk factors for developing chronic obstructive pulmonary disease (COPD). The only confirmed genetic risk factor for COPD is the inherited deficiency of alpha 1 proteinase inhibitor. However, the evidence of familial clustering of lung function and COPD occurrence and the development of COPD among susceptible smokers, at variance with the so-called resistant smokers, would suggest that the weight of genetic risk factors is greater than recognized. In this paper the role of candidate genes for increasing the risk of COPD (such as alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, cystic fibrosis transmembrane conductance regulator, and others) is reviewed. PMID- 7538006 TI - Molecular epidemiology: a key to better understanding of chronic obstructive lung disease. PMID- 7538007 TI - Psychotropic drugs in terminal care. AB - Respiratory patients require psychotropic drug administration to treat pain, cough and respiratory distress or to treat insomnia, anxiety, depression or psychosis. Terminal patients require thoughtful and compassionate use of these drugs, even when there is an expectation that such therapy may lead to an earlier death. Most psychotropic agents can be used safely in patients with respiratory disease, and careful use of selected drugs should always be employed if indicated for treating distressful conditions that may be benefitted. Guidelines to appropriate choices and doses are provided. PMID- 7538008 TI - Purification and characterization of an esterase isozyme from insecticide resistant and susceptible strains of German cockroach, Blattella germanica (L.). AB - The most active forms of esterases (E5, E6 and E7) from the German cockroach, Blattella germanica (L.) were purified from resistant and susceptible strains. About 45-155 fold purification with a 11-16% of total esterase recovery was achieved after different column chromatography and preparative gel electrophoresis. Elution profiles of resistant and susceptible strains were similar, but esterase E6 activity was higher in the resistant strains. Kinetic analyses indicate no differences in Km values between the resistant and susceptible strains. However Vmax was significantly higher in resistant strains. Inhibition of esterase activity by paraoxon, chlorpyrifos and propoxur did not suggest any structural differences in esterase E6 between strains. From these results we suggest that insecticide resistance in German cockroach is due to the increased production of E6 esterase. The role of E6 may be sequestration of toxic molecules rather than hydrolysis. PMID- 7538010 TI - Ubiquitinated cytokeratin inclusions in lichen amyloidosus: an immunohistochemical analysis. AB - Eosinophilic hyaline inclusions were consistently seen in the perinuclear cytoplasm of suprabasal keratinocytes in lichen amyloidosus. The inclusions, negative with amyloid staining, were immunoreactive for ubiquitin and cytokeratin, and ultrastructurally showed aggregations of fine filaments of two sizes (central thin and peripheral thick). The thin filaments were the main component in the upper epidermal layer. Four monoclonal antibodies (AE1, AE3, KL1 and CAM5.2) and one antiserum (WSS) were used for characterizing cytokeratin expression. The AE1 antibody normally stained the basal cells, but in lichen amyloidosus basal staining mostly disappeared. Instead, groups of suprabasal keratinocytes were labeled, with AE1-reactive inclusions distributed therein. In contrast, the KL1 antibody, showing suprabasal staining, failed to react with the inclusions. The inclusions were weakly reactive with the AE3 and WSS antibodies, which stained all keratinocytes. The CAM5.2 antibody was unreactive. The subepidermal amyloid deposits were negative with all the antibodies. The inclusions were ubiquitinated especially in the granular layer. Immunoelectron microscopy disclosed that ubiquitin was more densely localized in the thin filaments than in the thick ones. This indicated that cytokeratin expression and metabolism are altered in the affected epidermis, and that ubiquitin functions in the process of degradation of abnormal cytokeratin filaments. PMID- 7538009 TI - Nuclear pore complex ion channels (review). AB - It is currently thought that nuclear pore complexes (NPCs) primarily govern nucleocytoplasmic interactions via selective recognition and active transport of macromolecules. However, in various nuclear preparations, patch-clamp and fluorescence, luminiscence and ion microscopy support classical microelectrode measurements indicating that monoatomic ion flow across the nuclear envelope (NE) is strictly regulated. Gating of large conductance nuclear envelope ion channels (NICs) somewhat resembles that of gap junctional channels. In other respects, NICs are distinct in that they require cytosolic factors, are blocked by wheat germ agglutinin and are blocked and/or modified by antibodies to epitopes of NPC glycoproteins. Therefore, NIC activity, recorded as electrical current/conductance is likely to be intrinsic to NPCs. This observation suggests a potential use for the patch-clamp technique in establishing the mechanisms underlying nuclear pore gating in response to cytosolic and nucleosolic factors such as transcription and growth factors, oncogene and proto-oncogene products and receptors for retinoids, steroids and thyroid hormone. NIC activity may also be useful in evaluating the mechanisms of nuclear import of foreign nucleic acid material such as that contained in virons and viroids. Finally, in consideration to the electrophysiological data accumulated so far, the study of nuclear pore ion channel activity may help our understanding of other important issues such as cell suicide, programmed cell death or apoptosis. PMID- 7538012 TI - Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over expression of a chaperonin. AB - Based on 16S rDNA sequence comparison, intracellular mycetome-associated endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) form a distinct lineage within the gamma-3 subdivision of proteobacteria, related to the free-living bacterium Escherichia coli, midgut S-endosymbionts of various insects including tsetse flies, and to the P-endosymbiont lineage of aphids, Buchnera aphidicola. Gene organization and expression of several loci in intracellular microorganisms have revealed differences from free-living bacteria. This study analyses two of these characteristics in tsetse endosymbionts; the copy number and gene organization of rDNA operations and the nature of the abundant protein(s) synthesized by these microorganisms. Results indicate that Glossina morsitans morsitans S-endosymbionts have multiple (seven) rDNA operons coding for 16S (rrs) followed by 23S (rrl) gene sequences, whereas tsetse P-endosymbionts have a single, similarly organized rDNA operon. In tsetse mycetocytes in vitro, P endosymbionts synthesize a predominant protein of 60 kDa in size (p60) which by Western blot analysis shows immunological cross-reactivity with the abundant 63 kDa (p63) protein of B. aphidicola. p63 (also referred to as symbionin) has been characterized as a molecular chaperone, structurally and functionally similar to the groEL protein of E. coli. Under in vitro conditions, tsetse S-endosymbionts synthesize high levels of a similarly-sized protein that cross-reacts with p63 chaperonin. Antisera against the tsetse p60 protein also recognizes p63 protein of B. aphidicola, suggesting that the abundant tsetse endosymbiont protein is a chaperonin. PMID- 7538013 TI - The contribution of perivascular tissue to the neovascularization of full thickness skin grafts (prefabricated flaps): an experimental study. AB - The effect of varying amounts of perivascular tissue on the neovascularisation of full thickness skin grafts (prefabricated flaps) in rats was investigated. The femoral vasculature of adult Wistar rats (n = 48) was dissected to varying degrees: Group 1, perivascular areolar tissue and preadventitial tissue preserved; Group 2, perivascular areolar tissue removed, periadventitial tissue preserved; Group 3, femoral artery and vein skeletonised. Full thickness skin grafts harvested from the abdomen were laid on the femoral vessels. Silicone sheets were used to isolate the graft and its vascular supply from the surrounding tissue bed. At set times, from 24 h to 7 days, grafts from each group were harvested and the amount of neovascularisation was evaluated histologically. None of the grafts developed necrosis and all of them showed a progressive degree of neovascularisation. Using chi-square analysis, our study found no evidence that varying the amount of perivascular tissue significantly affected the degree of neovascularisation. In all 3 groups, the degree of neovascularisation was significantly improved over time (p = 0.002). PMID- 7538014 TI - Low-temperature solid-state 15N NMR characterization of polypeptide backbone librations. AB - The local molecular dynamics of gramicidin A in dimyristoylphosphatidylcholine bilayers were probed by a combination of low-temperature solid-state 15N NMR and rapid freezing of samples using liquid propane. It has previously been shown that this approach leads to sharp discontinuities in powder-pattern spectra of the gramicidin channel in lipid bilayers, and hence, an accurate determination of tensor element magnitudes is achieved. The static-tensor-element magnitudes determined here using hydrated bilayer samples at low temperatures are different from the values obtained from dry-powder samples at 308 K. However, for the one site studied, the orientation of the 15N chemical-shift tensor in the molecular frame is the same for both types of samples. Averaging of the chemical-shift tensor between 200 and 263 K is shown to be anisotropic and consistent with a motional axis parallel with the C alpha-C alpha axis for adjacent residues. The librational amplitudes determined here range from +/- 14 degrees to +/- 22 degrees at 263 K and are significantly larger than the nanosecond librations characterized by relaxation studies. The nanosecond motions are thought to represent correlated motions, while the librations described here are the sum of correlated (nanosecond) and uncorrelated (picosecond) motions. The picosecond librational amplitudes become considerably larger and more isotropic above 0 degree for the residues near the bilayer surface. PMID- 7538011 TI - Mesothelial cyst of the adrenal gland. AB - A case of mesothelium-lined cyst of the adrenal gland is reported. Although more than 300 adrenal cysts have been reported in the literature, epithelial cell lined cysts are rare and comprise only 9% of the cases. An adrenal cyst was found, the lining cells of which had features consistent with mesothelial cells. The inner surface of the cyst was lined by a single layer of cuboidal cells, which showed alcian blue positive cellular outline. Immunohistochemically, the lining cells were positively stained for keratin, epithelial membrane antigen and CA-125. Electron microscopic examination revealed many long complex microvilli and desmosomes in the lining cells. There was a basal lamina beneath the cell layer. These morphological and immunohistochemical findings indicated that the lining cell is derived from mesothelium. PMID- 7538015 TI - Esophageal cancer: diagnosis and treatment. AB - A review of diagnosis and treatment of esophageal cancer is presented. Symptomatic cancer of the esophagus is usually too far advanced for a cure to be offered. Alcohol and tobacco are the common risk factors of this disease. Endoscopy is the diagnostic method of choice. Management of cancer and survival of patients depend on the stage of the disease; therefore, accurate staging is vital in the diagnostic evaluation. Endoscopic ultrasound has become a powerful staging tool, and it must be complemented by conventional imaging. Surgery, chemotherapy, and radiotherapy are used in varying combinations for cure. Palliation of symptoms is important, and it is the only possible form of therapy for some patients. The therapeutic options need to be carefully considered to suit the individual needs of each patient. PMID- 7538016 TI - Hepatocellular carcinoma. AB - Hepatocellular carcinoma (HCC) is among the 10 most common tumors in the world. However, incidence is not evenly distributed across the world. In many instances, the proximate cause for the tumor can be identified. Chronic hepatitis B infection is probably the most common cause, followed by chronic hepatitis C. Other important causes are alcoholic liver disease, hemochromatosis, alpha 1 antitrypsin deficiency, and other chronic liver diseases. Although proximate causes may be identifiable, pathogenesis remains uncertain. Factors that may be important include the presence of Aflatoxin B1 in food, genetic changes induced by the hepatitis B virus, and repeated rounds of necrosis and regeneration, also induced by hepatitis viruses. The genes involved and the mutations necessary for hepatic carcinogenesis are unknown, with the sole exception of the p53 gene, which is probably a late phenomenon. Screening for HCC is widely practiced despite the lack of evidence of improved survival. The screening tests used include alphafetoprotein levels and ultrasonography. Screening can identify small tumors; however, survival may not be improved, because the presence of cirrhosis may limit the number of patients who can undergo resections; recurrences or second primary tumors are common; and the presence of chronic liver disease means that survival may be limited anyway. There are many different forms of therapy available; unfortunately, most have not been compared in randomized controlled trials. Surgery remains the therapy of choice if feasible. All other therapy is palliative, including chemotherapy, chemoembolization, hepatic artery embolization, various forms of radiotherapy, and various forms of ablative therapy. PMID- 7538017 TI - Expression of endothelial cell adhesion molecules in joints and heart during Borrelia burgdorferi infection of mice. AB - The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both peripheral- (PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart. PMID- 7538019 TI - Plasmodium falciparum: CD36 dependent cytoadherence or rosetting of infected erythrocytes is modulated by knobs. AB - A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K- line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K- lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K- line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells. PMID- 7538018 TI - Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition. AB - The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity. PMID- 7538023 TI - RNA structure. Describing the elephant. AB - The three-dimensional structures of RNAs are notoriously difficult to determine. Functional comparisons of variant molecules and cross-linking experiments are providing new information for structural modeling. PMID- 7538020 TI - Murine vascular cell adhesion molecule-1 (VCAM-1) proteins encoded by alternatively spliced mRNAs are differentially targeted in polarized cells. AB - VCAM-1 is an immunoglobulin (Ig) superfamily member expressed in endothelial cells that mediates adhesion to a variety of leukocytes in a VLA-4 dependent manner. In the mouse, two distinct forms of VCAM are produced. One form, VCAMTM, contains seven Ig domains followed by a single transmembrane region and a short cytoplasmic domain. A second form, VCAMGPI, which is preferentially induced by cytokines and LPS, contains only the first three Ig domains and is attached to the cell surface via a glycosylphosphafidylinositol (GPI) anchor. Both vascular and nonvascular expression of VCAM have been reported in a variety of normal and pathological settings. One possible role for the two VCAM isoforms is to allow for the targeted localization of VCAM to specific cell surface domains of polarized cells. This may be particularly relevant since VCAM is known to be expressed by two different polarized cell types, namely endothelial cells and kidney epithelial cells. In this study, MDCK cells permanently expressing either VCAMTM or VCAMGPI were established and used to examine the targeting of VCAM proteins to different polarized surface domains. VCAMTM was primarily located on the basolateral surface while VCAMGPI was located on the apical surface of polarized MDCK cells. Data is also presented that demonstrates that polarized expression is reversed in endothelial cells where VCAMTM was observed primarily on the apical surface. The differential localization of VCAM isoforms on the cell surface has direct implications for the ability of VCAM to mediate cell adhesion and transmigration. PMID- 7538022 TI - Nitric oxide synthases: biochemical and molecular regulation. AB - Nitric oxide synthases are a family of complex cytochrome P450-like hemeproteins that catalyze the five-electron oxidation of L-arginine to form nitric oxide. Nitric oxide synthase apoenzyme is dependent on molecular oxygen, nicotinamide adenine dinucleotide phosphate hydrogen, flavins and tetrahydrobiopterin, and it functions as a dimer. Three human nitric oxide synthase isoforms have been identified to date. The endothelial constitutive, neuronal, and inducible nitric oxide synthase isoforms are found on human chromosomes 7, 12 and 17, respectively. Characterization of the structural organization of the human nitric oxide synthase genes reveals that, although they are structurally related, the mechanisms by which they are regulated are distinct. Expression of the mRNA for endothelial constitutive nitric oxide synthase is regulated at the level of transcription and mRNA stability. The mRNA transcripts derived from the neuronal nitric oxide synthase gene are characterized by a remarkable degree of structural diversity. Levels of inducible nitric oxide synthase mRNA are controlled by interacting combinations of cytokines and biological mediators at the level of gene transcription and mRNA stability. PMID- 7538021 TI - The macrophage, TNF, and other cytokines. AB - A complex interplay of peptides known as the cytokines may have a tremendous influence over a number of inflammatory related conditions. Tumor necrosis factor occupies an early and central role in the initiation of cascades that ultimately influences a number of cell types involved in tissue inflammation, tissue rejection, cancer, and injuries from ischemia reperfusion. Only now are the cascades being defined and therapies being designed to interrupt the toxic effects of these cytokines and to treat malignancy. PMID- 7538027 TI - The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets. AB - The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [3H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na+,K(+)-ATPase activity was determined in a brain cell membrane preparation. [3H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5-50 nM [3H]MK-801 in the presence of 100 microM glutamate and glycine; and (2) binding assays at 10 nM [3H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 microM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na+,K(+)-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (Bmax), but the apparent affinity was significantly increased, as indicated by a decrease in the Kd (dissociation constant) from the control value of 8.1 +/- 1.6 nM to 5.5 +/- 2.1 nM (P < 0.05). Augmentation of [3H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538025 TI - Neurophysiology and neuropharmacology of projections from entorhinal cortex to striatum in the rat. AB - We studied projections from the entorhinal cortex (Ent) to the striatum in anesthetized rats using extra- and intracellular recording and multibarrel iontophoresis. The majority of recording were from the caudate-putamen (CPu) and core of the nucleus accumbens (AcbC). Electrical stimulation of the Ent evoked synaptic responses in 77% of tests with AcbC neurons and 48% of tests with CPu neurons. In the case of AcbC neurons, 61% of these tests proved to be excitatory and were often followed by inhibitory phases. In contrast to this, only 18% of tests from CPu neurons were excitatory. Intracellular HRP labeling showed that responsive cells were medium spiny neurons. During iontophoretic experiments, application of the glutamatergic AMPA antagonist DNQX could selectively decrease or block excitatory responses. The GABAA antagonist bicuculline methiodide increased cellular firing rates and could reveal excitatory responses, suggesting block of a short-latency, short-duration inhibitory component. Ejection of the GABAB antagonist CGP-35348 could attenuate a later, longer-duration component of inhibition. The results indicate that the Ent excites striatal neurons at least in part by glutamatergic receptors and suggest that this excitation is followed by secondary prolonged GABAergic inhibition. PMID- 7538024 TI - Primary, self-renewing erythroid progenitors develop through activation of both tyrosine kinase and steroid hormone receptors. AB - BACKGROUND: Self renewal in the hematopoietic system is thought to be restricted to a class of pluripotent stem cells. The capacity of cells with the properties of committed progenitors to self renew in many leukemias is thought to be an abnormal property resulting from the mutations responsible for leukemic transformation. It is not known how cells that can self-renew differ from cells that cannot. The notion that only pluripotent stem cells self renew has recently been challenged: normal committed erythroid progenitors capable of sustained self renewal have been described. These cells, called SCF/TGF alpha progenitors, co express the c-Kit receptor tyrosine kinase and c-ErbB, the avian receptor for epidermal growth factor and transforming growth factor (TGF) alpha, and they undergo continuous self renewal in response to TGF alpha and estradiol. In contrast, common erythroid progenitors (termed SCF progenitors) express only c Kit and undergo a limited number of cell divisions in response to the c-Kit ligand, stem cell factor (SCF). Both types of progenitor faithfully reproduce terminal erythroid differentiation in vitro when exposed to differentiation factors. Here, we have investigated the developmental origin of these two classes of self-renewing erythroid progenitors. RESULTS: We show that SCF progenitors can develop into SCF/TGF alpha progenitors. This developmental conversion requires 10 14 days and is accompanied by a gradual up-regulation of bioactive TGF alpha receptor. Using sera depleted of endogenous growth factors, we demonstrate that the development of SCF progenitors into SCF/TGF alpha progenitors absolutely requires the simultaneous presence of SCF, TGF alpha and estradiol, and is strongly enhanced by an unknown activity in chicken serum. CONCLUSIONS: SCF progenitors can be induced to develop into self-renewing SCF/TGF alpha progenitors. The development of self renewal is triggered by specific combinations of growth factors and hormones. This has important implications for understanding leukemogenesis, as the self renewal of leukemic cells may reflect the normal potential of certain committed progenitor cells and not, as has been thought, a unique abnormal property of leukemic cells. PMID- 7538026 TI - Nitric oxide induces an increased Na+ conductance in identified neurons of Aplysia. AB - The ionic mechanism of the effects of micropressure ejections of hydroxylamine (HOA) and sodium nitroprusside (SNP), nitric oxide (NO) generators, on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion-substitution techniques. Micropressure ejection of HOA and SNP onto the neurons caused a marked depolarization in the unclamped neurons. Clamping the same neurons at their resting potential level (-60 mV) and reejecting HOA and SNP with the same dose produced a slow inward current (Ii(HOA) and Ii(SNP), 3-7 nA in amplitude, 15 60 s in duration) associated with an increase in input membrane conductance. Bath applied hemoglobin (50 microM), a nitric oxide scavenger, almost completely blocked Ii(HOA) and Ii(SNP), and 3-isobutyl-1-methylxanthine (IBMX, 50 microM) prolonged and enhanced both Ii(HOA) and Ii(SNP). An intracellular injection of cyclic guanosine 3',5'-monophosphate (cGMP) into the same neurons produced a slow inward current (Ii(cGMP)) which resembled the responses to HOA and SNP, and this current was enhanced in IBMX. Bath-applied methylene blue (10 microM), an inhibitor of guanylate cyclase, significantly reduced Ii(HOA) and Ii(SNP). The inward currents induced by HOA, SNP and cGMP were sensitive to changes in the external Na+ concentration. These results suggest that extracellular NO can induce a slow inward current associated with an increase in Na+ conductance, mediated by an increase in intracellular cGMP. PMID- 7538028 TI - Changes of extracellular calcium concentration induced by application of excitatory amino acids in the human neocortex in vitro. AB - The influence of the glutamate subreceptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on cortical field potentials and on changes in extracellular free calcium concentration ([Ca2+]o) was tested on human neocortical slices (eleven from nine different patients). The tissue used was a small portion of that which is normally removed for the treatment of a brain tumor. [Ca2+]o and field potentials were measured by Ca(2+)-selective microelectrodes. Local pressure-microejection of NMDA (100 mumol/l)- and AMPA (1 mmol/l)-induced negative field potentials with maximal amplitudes of 0.9 +/- 0.1 mV (11 slices, mean +/- S.E.M.) and 1.0 +/- 0.1 mV (nine slices), respectively. The negative field potentials induced by NMDA were accompanied by monophasic decreases of [Ca2+]o (0.8 +/- 0.1 mmol/l, nine slices). AMPA elicited no (three slices) or only minor decreases of [Ca2+]o (0.2 +/- 0.1 mmol/l, five slices). The responses to the glutamate subreceptor agonists NMDA and AMPA were reversibly depressed by adding their specific antagonists DL-2 amino-5-phosphonovalerate (APV, 100 mumol/l, six slices) and 6-cyano-7 nitroquinoxalin-2,3-dion (CNQX, 5 mumol/l, four slices), respectively. The results correspond to findings in animal experiments and are consistent with the interpretation that in the human neocortex the Ca2+ permeability of channels gated by NMDA is higher than those gated by AMPA. PMID- 7538030 TI - To do no harm. PMID- 7538031 TI - Granulocyte colony stimulating factor treatment for neonatal neutropenia. AB - In a pilot study recombinant human granulocyte colony-stimulating factor (rhG CSF) was administered to 12 neutropenic preterm infants to determine if neonatal neutropenia is secondary to decreased endogenous G-CSF production. Respiratory variables were monitored because of the possible link between inflammatory cells and hyaline membrane disease. All infants showed increased neutrophil counts. The only possible side effect observed was an exacerbation of thrombocytopenia. PMID- 7538029 TI - The opioid peptide dynorphin modulates AMPA and kainate responses in acutely isolated neurons from the dorsal horn. AB - In freshly isolated spinal dorsal horn (DH) neurons (laminae I-IV) of the young rat, the effects of dynorphin A1-17, U-50,488H and U-69,593 on inward currents induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) were studied under whole-cell voltage-clamp conditions. When the cells were clamped to a holding potential of -60 mV, co-application of dynorphin A1-17 (10(-6) M) and AMPA (2 x 10(-5) M) reversibly decreased the peak amplitude of the initial transient component of the AMPA-induced current in 72% of the examined cells. In addition, dynorphin (10 microM) in perforated patch-recordings consistently produced a decrease in the steady-state component of the AMPA response. The depressant effect was concentration-dependent (IC50 = 86 nM) and reversible. The dynorphin A1-17-induced depression of the AMPA response was associated with slowing of the response kinetics, including both a 10-90% rise time and time constant of decay. The AMPA-induced currents were modulated by dynorphin not only during the co-administration but also after the removal of the peptide. Dynorphin increased the initial peak AMPA current in 42% of the examined cells. Similar as with dynorphin A1-17, the peak amplitude of the AMPA-induced current was reversibly suppressed in the presence of 1 microM U-50,488H and U 69,593 in 75% and 86% of the examined cells, respectively. Naloxone and the kappa 1-selective antagonist norbinaltorphimine (nor-BNI) blocked the initial depressant but not late excitatory effects of dynorphin A1-17 and U-50,488H. This antagonistic effect of naloxone and norbinaltorphimine suggests that the depressant effect of dynorphin A1-17 on the AMPA-activated conductance is a true opioid, probably kappa 1-opioid receptor-mediated event. In contrast, the dynorphin-induced late potentiation of AMPA/KA responses appears to be a non opioid effect since it was not inhibited by nor-BNI, CTAP and naltrindole, the selective kappa-, mu- and delta-opioid receptor blocking agents, respectively. Pretreatment of DH neurons with pertussis toxin blocked the depressant action of dynorphin A1-17, indicating that a Gi- or Go-type G protein was required for this effect on AMPA-activated currents. Intracellular dialysis with a highly specific peptide inhibitor (peptide 6-22) of the cAMP-activated protein kinase (PKA), and with Rp-cAMPS, prevented the depressant effect of dynorphin A1-17. In addition, staurosporine, a nonselective kinase inhibitor, blocked the dynorphin depression of the AMPA response.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538032 TI - Antibodies to hepatitis C virus: a cross-sectional study in patients attending a trauma unit or admitted to hospital for elective surgery. AB - OBJECTIVE: To assess the prevalence of hepatitis C virus (HCV) antibodies in patients attending a trauma unit or admitted to hospital for elective surgery, and to identify risk factors for acquiring HCV infection. DESIGN: A cross sectional study. SETTING: The emergency room of a Department of Orthopaedic Surgery and a hospital for elective (non-digestive) surgery. PATIENTS: A study population of 1824 subjects (mean age 40.1 +/- 18.1 years) and sera from a control group of 4703 blood donors giving their first donation. INTERVENTIONS: Serum samples were screened for HCV antibodies by second-generation enzyme-linked immunosorbent assays and recombinant immunoblot assays. MAIN OUTCOME MEASURES: HCV serostatus and the relationship between anti-HCV-positivity and age, intravenous drug use, tattoos, blood transfusion, a history of hepatitis and exposure to a contact who had had hepatitis. RESULTS: The overall prevalence of anti-HCV antibodies was 2.85% in the study population and 0.5% among blood donors (chi 2 = 64.5; P < 0.0001). Anti-HCV-positivity was more common in men (3.8 versus 1.9%; P < 0.05) and in subjects with history of hepatitis (13.5 versus 2.0%; P < 0.001), exposure to a contact who had had hepatitis (12.5 versus 1.9%; P < 0.001), blood transfusion (7.8 versus 2.3%; P < 0.001), tattoos (36 versus 2.4%; P < 0.001) and intravenous drug use (85.7 versus 1.9%; P < 0.001). However, one third of subjects had no risk factors for HCV infection. The logistic regression analysis confirmed a statistically significant association (P < 0.05) between anti-HCV-positivity and intravenous drug use, tattoos, blood transfusion, a history of hepatitis and exposure to a contact who had had hepatitis. CONCLUSIONS: These results show almost a five-fold higher prevalence of anti-HCV among patients attending a trauma unit or admitted to hospital for elective surgery than among blood donors. PMID- 7538033 TI - Cytokine expression in Peyer's patches following hemorrhage and resuscitation. AB - Intestinal dysfunction commonly occurs following hemorrhage and injury and appears to contribute to the development of multiple organ system failure in this setting. In order to examine possible mechanisms leading to intestinal dysfunction following blood loss, we investigated mRNA levels for cytokines with proinflammatory and immunoregulatory properties (interleukin 1 beta (IL-1 beta), IL-6, IL-10, TNF-alpha, TGF-beta, IFN-gamma) as well as mRNA expression for inducible nitric oxide synthase (NOS) over the 3 days following hemorrhage and resuscitation. Significantly increased levels of mRNA for IL-1 beta, IL-10, and IFN-gamma were found among cells isolated from Peyer's patches 3 days following hemorrhage. Amounts of mRNA for inducible NOS were not significantly altered 24 or 72 h after blood loss. In addition to being increased 72 h following hemorrhage, levels of mRNA for IL-10 also were increased 1 and 4 h posthemorrhage. No alterations in cytokine or NOS expression were found 24 h following blood loss. These results demonstrate that significant increases in proinflammatory and immunoregulatory cytokine mRNA levels among cellular populations in Peyer's patches are present at late posthemorrhage time points. These alterations in cytokine expression may contribute to the morphologic, immunologic, and functional changes in the intestines which are present following blood loss and injury. PMID- 7538034 TI - Endotoxin, cytokines, and nitric oxide in shock. PMID- 7538035 TI - L-arginine improves resting cardiac transmembrane potential after burn injury. AB - Previous studies from our laboratory have suggested that burn injury disrupts nitric oxide production and promotes a loss of cell membrane integrity. We hypothesized that administration of L-arginine, a precursor of nitric oxide (NO), would prevent postburn depolarization of the cardiac cell membrane and preserve cell membrane integrity. Third degree scald burn comprising 43% of the total body surface area was produced in rats (n = 22); sham burn controls (n = 11) were included. Burn rats were either untreated (N = 11) or given 300 mg/kg L-arginine immediately, 3, 6, and 23 h postburn. Untreated burn injury caused depolarization of the cardiac cell membrane (cardiac Em fell from 79.0 +/- 1.4 mV in shams to 67.0 +/- 1.5 mV 24 h after untreated burn, p < .05) and an increase in myocardial tissue lactate. Urine nitrate levels (assessed to provide a measure of NO production) fell after untreated burn (0.49 +/- 0.10 microM/24 h) compared with levels measured in sham burns (7.99 +/- 0.64 microM/24 h, p < .05), indicating that burn injury reduced NO production. Postburn administration of L-arginine improved cardiac Em (81.5 +/- 2.1 mV), reduced myocardial tissue lactate levels, and increased urinary nitrate levels above values measured for untreated burns. Our data indicate that L-arginine, in the absence of fluid resuscitation, provides postburn cardiac cell membrane protection, possibly due to enhanced nitric oxide production. PMID- 7538036 TI - High level of arginine vasopressin and 7.5% NaCl/6% dextran-70 solution: cardiovascular and renal effects. AB - A marked diuresis has been observed following resuscitation of hypotensive hemorrhaged animals with small volume hypertonic saline/dextran (HSD), 7.5% NaCl/6% dextran-70. We tested the hypothesis that high arginine vasopressin (AVP) levels associated with severe hemorrhage may exacerbate the diuretic effect of HSD infusion in euvolemic sheep. Following AVP infusion, a significant bradycardia (55% of baseline) and decreased cardiac output (62% of baseline) was observed (p < or = .05). Urine output increased during AVP infusion (25.4 +/- 2.3 ml/20 min) compared to control group (10.5 +/- 1.0 ml/20 min) (p < or = .0001). With HSD volume expansion, urine flow in the AVP group was initially 1.7 times greater than the control group (104.8 +/- 10 ml/20 min vs. 60.2 +/- 15 ml/20 min) (p < or = .05). High serum levels of AVP (600 +/- 33 pg/ml) may contribute to the diuresis seen with HSD resuscitation and possibly contribute to the bradycardia observed with severe hemorrhage. PMID- 7538037 TI - Treatment of hyperthermia and dehydration with hypertonic saline in dextran. AB - Hyperthermia may be accompanied by dehydration with or without electrolyte loss. To determine the efficacy of hypertonic saline in dextran solution (HSD, 7.5% NaCl in 6% dextran 70) for the treatment of heat stroke, rats were deprived of water for 24 h (DE) or not (ND), and then they were heat-stressed, and 4 mL/kg of saline (SAL) or HSD was administered via jugular cannula at the end of heat stress (a core temperature of 42.3 degrees C). The following four groups of 10 rats (Wistar-Furth) were used: NDSAL, DESAL, NDHSD, and DEHSD. Four control groups not subjected to heat stress were also studied. Percent change in plasma volume (PV) from baseline was significantly decreased by DE vs. ND (-6.8 +/- 1.2 vs. +4.6 +/- 1.6%) and increased by HSD vs. SAL (+10.1 +/- 4.1 vs. -3.5 +/- 1.6%) administration. Rats that were DE had significantly higher heating rates and less water loss during heat than ND rats, but hydration status was not correlated to 24 h survival. HSD groups had significantly higher PV and survival rates than their corresponding SAL groups regardless of hydrational status. PMID- 7538038 TI - Inhibition of endothelium-dependent vasodilation by Escherichia coli endotoxemia. AB - To test the hypothesis that release of endothelium-derived relaxing factor/nitric oxide is inhibited by Gram-negative lipopolysaccharide (LPS; endotoxin), we examined endothelium-independent and endothelium-dependent vasodilator agents in aortic vascular smooth muscle isolated from guinea pigs 4 h after injection of saline (controls) or induction of Escherichia coli endotoxemia. LPS significantly inhibited vasodilator responses to the endothelium-dependent agonists acetylcholine (ACh; 10(-10)-10(-5) M) and ADP (10(-8)-10(-5) M). However, LPS did not affect vasodilator responses to the endothelium-independent agonist nitroprusside (10(-10)-10(-4) M). The nitric oxide synthase (NOS) inhibitor N gamma-nitro-L-arginine methyl ester (L-NAME) inhibited the vasodilator response to ACh; whereas, the cyclooxygenase inhibitor indomethacin (INDO) did not reduce vasodilator effects of ACh. Neither L-NAME nor INDO affected the vasodilator effects of nitroprusside in LPS or control vessels. In contrast, L-NAME converted the vasodilator action of ADP to a vasoconstrictor response that was blocked individually by INDO and the thromboxane synthase inhibitor dazoxiben, suggesting that ADP releases NO and also the vasoconstrictor and platelet aggregating eicosanoid thromboxane A2. These findings suggest that acute (4 h) endotoxemia inhibits function of the constitutive isoform of NOS in vascular endothelial cells. Since L-NAME unmasked a vasoconstrictor action of the endogenous purinoceptor agonist ADP, pharmacologic agents that inhibit NOS may exacerbate LPS-induced inhibition of endothelial NOS; this series of events could lead to diminution of vasodilator reserves and perhaps to augmentation of platelet aggregation during Gram-negative sepsis. PMID- 7538040 TI - On the ethics of pain management. PMID- 7538041 TI - Prostate cancer: here and now. PMID- 7538039 TI - The interferon system: a review with emphasis on the role of PKR in growth control. PMID- 7538042 TI - Screening for prostate cancer: an analysis of the early experience. AB - Several common misconceptions have fueled the debate over the early detection and treatment of prostate cancer. While prostate cancer is often described as a common cancer that older men die with rather than of, the reality is that the incidence, mortality, and mean age and stage at diagnosis of prostate cancer are very similar to those of breast cancer, which is rarely the subject of similar concerns. Many studies have confirmed that given enough time, all clinically detected prostate cancers will inexorably progress locally and eventually metastasize to regional lymph nodes as well as to distant sites. The relatively slow doubling time compared to that of other cancers and the wide spectrum of biologic activity of prostate cancer have made retrospective studies reporting the long-term survival of conservatively treated patients highly suspect due to selection bias and inadequate follow-up. While it is accepted that a large number of men harbor clinically insignificant cancers in their prostate glands, these estimates have been based on careful pathologic step-sectioning studies of prostates obtained either at autopsy or after cystoprostatectomy for bladder cancer. Several studies have now demonstrated that currently available diagnostic modalities for detecting prostate cancer, DRE, PSA, and TRUS, are not able to detect a significant proportion of small, clinically unimportant cancers. Rather, studies have shown that while the traditional DRE has been largely unsuccessful in detecting prostate cancers at a sufficiently early stage for effective treatment with either radical prostatectomy or radiation therapy, a combination of the DRE and PSA followed by TRUS and ultrasound-guided biopsy in those with abnormal results can detect an increased proportion of clinically significant prostate cancers while they are still confined to the prostate gland and thus more likely to be eradicated by treatment. Several randomized trials are now under way in this country and in Europe that may settle many of these issues over the next decade. However, currently available data suggest that prostate cancer screening holds the promise of decreasing the considerable morbidity and mortality caused by this disease. PMID- 7538043 TI - How to use prostate-specific antigen in the early detection or screening for prostatic carcinoma. AB - At present, increased early detection of prostate cancer appears to be the most feasible way to reduce cancer-related mortality. As a result significant efforts have been made to identify more men with curable cancer. This article reviews the role of serum prostate-specific antigen in an early detection or screening strategy and describes efforts to enhance the specificity of prostate-specific antigen testing. PMID- 7538044 TI - The role of tumor rejection antigens in host antitumor defense mechanisms. AB - BACKGROUND: Normal cells undergo contact inhibition of growth when their surface molecules interact. Tumor cells, however, have undergone a mutation that prevents this arrest of growth upon contact inhibition and allows constant growth. Thus, growth inhibition fails to occur despite the interaction of surface molecules. In recent years a subgroup of these surface molecules has been of interest to cancer investigators. This subgroup has been termed the tumor rejection antigens (TRAs). As the name implies, these are specific to the tumor of origin and may direct the immune system of the host to target the tumor cells and kill them. METHODS: A literature search was carried out on TRAs to ascertain the current thinking on the subject. RESULTS: Initial studies of TRAs have revealed that some of them may be heat shock proteins (HSPs). In particular, grp96, a number of the HSP90 family, has been implicated. More recent studies, however, have shown that HSPs alone may not be immunogenic but may act as carrier proteins for tumor specific peptides. CONCLUSION: Such findings have led to speculation that HSPs or their associated peptides may have a role in the diagnosis and/or treatment of specific cancers. Immunotherapy and bispecific antibodies in particular are areas in which HSPs may prove to be useful. PMID- 7538045 TI - Spontaneous pneumothorax complicating chemotherapy for metastatic seminoma. A case report and a review of the literature. AB - BACKGROUND: Spontaneous pneumothorax complicating chemotherapy has been reported mainly in metastatic sarcoma, particularly of the osteogenic type. The main factor in the etiology of spontaneous pneumothorax could be related to tumor lysis and/or rapid rupture of chemosensitive peripheral or subpleural metastasis into the pleural cavity, thus leading to a bronchopleural fistula. METHODS AND RESULTS: A 49-year-old patient in whom spontaneous pneumothorax developed after successful chemotherapy for metastatic seminoma is described. On chest tube drainage, the lung reexpanded rapidly and the patient became clinically and radiographically symptom free. CONCLUSION: To the authors' knowledge, this is only the second case of spontaneous pneumothorax complicating chemotherapy induced rapid regression of lung and mediastinal metastases in patients with seminoma. Spontaneous pneumothorax should be included in the spectrum of chemotherapy-related side effects in chemosensitive solid tumors with lung metastases. PMID- 7538046 TI - Identification of a sister gene to P-glycoprotein. AB - The P-glycoproteins (Pgps) are a small family of transport proteins associated with the multidrug resistance phenotype of cell lines selected for growth in cytotoxic drugs. Utilizing low stringency screening, we have identified a novel gene closely related to the Pgps expressed in the pig and other mammalian liver which we have called Sister of P-glycoprotein (spgp). Sequence of this gene shows it to be a member of the ATP-binding cassette family of transporters and the gene most closely related to Pgp identified to date. The function of spgp is not known, but it can be recognized by at least one Pgp mAb, C219. This cross reactivity has implications for expression studies in tissues and tumors utilizing this and other Pgp antibodies. PMID- 7538047 TI - Prostate-specific antigen is a new favorable prognostic indicator for women with breast cancer. AB - Prostate-specific antigen (PSA) is thought to be produced exclusively by prostatic epithelial cells and is currently used as a tumor marker of prostatic adenocarcinoma. We recently found that 30% of breast cancers contain PSA immunoreactivity (IR-PSA). To examine the prognostic value of PSA in female breast cancer, we measured IR-PSA in tumor cytosols of 174 breast cancer patients and classified the breast cancers as either PSA positive or PSA negative based on an IR-PSA cutoff level of 0.03 ng/mg. IR-PSA was present in 27% of the patients. IR-PSA presence was associated with early disease stage, small tumors, and estrogen receptor-positive tumors. We used the Cox proportional hazards regression model to analyze survival of patients in association with PSA status and found that patients with IR-PSA-positive tumors had a reduced risk for relapse and death in univariate analysis (P = 0.02 and 0.06, respectively) and a reduced risk for relapse in multivariate analysis (P = 0.03). Further analysis indicated that the effect of IR-PSA on relapse-free survival was evident in node positive or estrogen receptor-negative patients. Our study suggests that IR-PSA is an independent favorable prognostic marker for breast cancer and may be used to identify a subgroup of estrogen receptor-negative and/or node-positive patients who have good prognoses. PMID- 7538048 TI - Nerve growth factor stimulates clonal growth of human lung cancer cell lines and a human glioblastoma cell line expressing high-affinity nerve growth factor binding sites involving tyrosine kinase signaling. AB - The growth of a panel of 22 different human tumor, leukemia, and lymphoma cell lines was examined in a human tumor cloning assay in agar or methylcellulose and a tritiated thymidine uptake assay. The cultures were performed in the absence or presence of increasing concentrations (0.5-500 ng/ml) of nerve growth factor (NGF). The growth of 17 of the 22 cell lines was not significantly and reproducibly affected by NGF. There was minor (1.2-fold) but reproducible stimulation of clonal growth in one glioblastoma cell line (86-HG-39) by NGF, but in this cell line NGF induced no growth modulation in a tritiated thymidine uptake assay. However, clonal growth of another glioblastoma cell line (87-HG-31) and all three lung cancer cell lines tested (HTB 119, HTB 120, CCL 185) could be stimulated up to 3-fold by NGF with a dose-response relationship for the growth factor. Growth stimulation by NGF could be completely reversed by neutralizing anti-NGF antibody and by the tyrosine kinase inhibitor genistein. Evaluation of secondary plating efficiency revealed the stimulation of colony formation as representing self-renewal and not terminal differentiation. Reverse transcriptase PCR experiments in the five responding cell lines showed expression of both low affinity NGF receptor (glycoprotein 75) and c-trk transcripts on the mRNA level. Of the five responding cell lines, only 86-HG-39, the cell line with the lowest responsiveness, revealed low-affinity NGF receptor on the protein level; the other four cell lines with high responsiveness, including the three lung cancer cell lines, expressed no low-affinity NGF receptor as shown by fluorescence activated cell sorter analysis and immunoprecipitation using the ME 20.4 antibody. Immunoprecipitation using anti-trk antibodies was negative in all five responding cell lines. However, binding studies with iodinated NGF showed only low-affinity binding on the 86-HG-39 cell line and only high-affinity binding on the high-responder cell lines CCL 185 and 87-HG-31. In summary, our data suggest that NGF can be operative in stimulation of clonal growth of malignant tumor cells. High-affinity but not low-affinity binding sites mediate signal transduction for clonal growth and signaling involves tyrosine kinase activity. PMID- 7538049 TI - Reinduction of experimental autoimmune encephalomyelitis in mice. AB - Experimental autoimmune encephalomyelitis (EAE) in Lewis rats and some strains of mice is typically a monophasic disease, and recovered animals are resistant to reinduction of disease. We demonstrate that SJL mice remain susceptible to disease after recovery, and suffer a second episode of disease when reinjected with spinal cord homogenate in complete Freund's adjuvant. Reinduced disease occurs earlier after injection than the initial disease (mean onset 7.3 days compared with 14.5 days), and has comparable severity and incidence. The susceptibility to reinduced disease is present for at least 20 weeks after the initial injection. If the initial episode of EAE is elicited using a synthetic peptide of proteolipid protein, then reinjection of the same peptide causes reinduced disease. PL/J mice and PL/J x SJL F1 mice are also susceptible to reinduced disease which occurs with an accelerated onset and higher incidence than the initial disease. We conclude that SJL and PL/J mice have a defect in immunoregulation which causes them to be susceptible to recurrent episodes of autoimmune disease. PMID- 7538051 TI - Enrichment for Th1 cells in the Mel-14+ CD4+ T cell fraction in aged mice. AB - CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-gamma upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN gamma and minute amounts of IL-4 and IL-10, suggesting that these "naive" cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-gamma as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells. PMID- 7538050 TI - Activation of murine T cells by bacterial superantigens requires B7-mediated costimulation. AB - Staphylococcus enterotoxins bind class II MHC molecules on antigen-presenting cells (APC) and stimulate T cells expressing appropriate V beta gene products. Although the role of non-TcR-associated costimulatory receptors during antigen specific T cell stimulation has been clearly established, the involvement of costimulatory activity in T cell activation by superantigens (SAgs) has been the matter of controversy. The aim of this study was to evaluate the role of the costimulatory-receptor ligand molecules CD28/B7 on bacterial SAg-mediated activation of naive murine T cells. We demonstrate in this report that a combination of monoclonal antibodies to murine B7.1 and B7.2 molecules inhibits the in vitro response of naive T cells to SAgs SEA, SEB, and TSST-1. The inhibition of T cell responses required simultaneous blocking of B7.1 and B7.2, suggesting that either B7.1 or B7.2 is sufficient to provide costimulatory signals to naive T cells in response to bacterial exotoxins. Inhibition of T cell activation by antibodies to B7-related molecules can be overcome by antibodies to CD28, a finding in agreement with the hypothesis that CD28-mediated signals participate in T cell activation by bacterial SAgs. These observations suggest that, as demonstrated for conventional antigen, T cell activation by SAgs requires the coordinated participation of TcR- and CD28-derived signals. PMID- 7538052 TI - Biology of Fas. PMID- 7538054 TI - The interaction of vascular endothelial cells and dorsal root ganglion neurites is mediated by vitronectin and heparan sulfate proteoglycans. AB - The interaction of peripheral nerve and blood vessels during development was studied by using DRG explant culture plated on confluent monolayer of vascular endothelial cells (VEC). The comparison of neurite length on various substrates showed a preference of DRG neurites in the following order; thrombospondin > laminin, vitronectin > fibronectin, VEC monolayer > collagen I, rat astrocyte monolayer. On layers of fibroblasts (3T3) or gliomas (C6), neurite extension was not observed. To identify the neurite outgrowth promoting adhesion molecules on VEC surface, several antibodies and synthetic peptides were added to the culture medium of DRG. With vitronectin antibody or with peptides containing the Arg-Gly Asp (RGD) sequence, 30-40% of neurite outgrowth was inhibited and these two effects were not additive. Therefore, a part of neurite outgrowth in this system is mediated by vitronectin in RGD dependent manner. Another molecule which promotes neurite outgrowth on VEC was identified by a new monoclonal antibody (MAb) EC1. In the Western blot analysis, the immunoreactive band which was over 400 kDa was intensified by guanidine HCl extraction. EC1 immunoreactive band disappeared after the treatment of heparitinase but not with other glycolyases, indicating that EC1 antigen is heparan sulfate proteoglycan(s). The DRG neurite outgrowth was inhibited by MAb EC1 by about 30-40%. By the combination of MAb EC1 and RGD peptide, the neurite outgrowth in explant culture was inhibited by about 50%, and in DRG dissociated culture nearly 100% inhibition was observed. Thus, for the DRG neurite elongation on VEC, vitronectin and heparan sulfate proteoglycan(s) are playing crucial roles. PMID- 7538053 TI - Role of nitric oxide in ischemia/reperfusion of the rat kidney. AB - This work studies the role that nitric oxide (NO) plays in ischemia/reperfusion (I/R) of the rat kidney. Sprague-Dawley rats, weighing 250-300 g, were subjected to 75 min of warm ischemia and contralateral nephrectomy. The animals were divided into six groups (n = 12 per group): ischemic control (IC) with normal saline, L-NG-mono-methyl-arginine (L-NMMA) 50 mg/kg, L-arginine (L-Arg) 300 mg/kg, Na-nitroprusside (Na-NP) 2.5 mg/kg, the combination of L-NMMA+Na-NP at the doses used before, and the sham group. All animals received the drug intravenously 60 min prior to ischemia. Survival was evaluated at seven days. Renal damage was assessed by kidney function tests (serum creatinine and blood urea nitrogen) and light histology. Lipid peroxidation was measured in renal tissue using the thiobarbituric acid assay. Significantly better survival was seen in the Na-NP group, as compared to the rest of the study. Serum creatinine at 24 and 48 hr showed a significant difference between the Na-NP group and the other groups. Histological damage was minimal in the Na-NP group. Clearly, the Na NP had the most beneficial effect in survival and histological structure. Lipid peroxidation was significantly different, with the lower levels seen in the L NMMA group and the higher levels in the Na-NP group. In base to these results, we conclude that exogenous NO has a beneficial and protective effect of the ischemically damaged rat kidney. This protection is independent of lipid peroxidation. Endogenous NO production does not play a role in I/R injury in our model. PMID- 7538055 TI - Postnatal development of the substance P-, neuropeptide Y- and serotonin containing fibers in the rat suprachiasmatic nucleus in relation to development of the retino-hypothalamic projection. AB - The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the development of substance P (SP)-, neuropeptide Y (NPY)- and serotonin (5 hydroxytryptamine, 5-HT)-immunoreactive fibers in the rat SCN and the development of the retino-hypothalamic tract using cholera toxin beta subunits (CTB), in order to understand which parts of the SCN participate in diurnal rhythm regulation and entrainment. In newborn rats, SP-, NPY- and 5-HT-immunoreactive fibers were scarcely detected in the SCN. The number of SP-immunoreactive fibers gradually increased between postnatal days (P) 15 and 30. At P30, the distribution pattern of SP-immunoreactive fibers in the SCN was similar to that in the adult rat. The number of NPY- and 5-HT-immunoreactive fibers increased greatly between P10 and P15, and the increase in NPY- and 5-HT-immunoreactive fibers continued until P20. CTB was injected into the unilateral eyeball of the rat at various postnatal stages. In neonates, several labeled retinal fibers already existed in the ventral part and ventro-lateral edge of the SCN. The number and density of labeled retinal fibers in the SCN gradually increased between P10 and P20. Between P20 and P30, a decrease in the labeling was observed in the dorsolateral part of the SCN. The adult pattern of labeled retinal fibers was achieved between P20 and P30. The development of SP-immunoreactive fibers was delayed about 10 days relative to that of NPY-, 5-HT-immunoreactive fibers and retinal fibers. PMID- 7538058 TI - Plastic-covered metallic endoprostheses in the management of oesophageal perforation in patients with oesophageal carcinoma. AB - OBJECTIVE: To evaluate the role of plastic-covered self-expanding metallic endoprostheses in patients with oesophageal perforation occurring during endoscopically guided dilatation prior to laser treatment for malignant obstruction. SUBJECTS AND METHODS: Six patients with oesophageal perforation following laser treatment for malignant obstruction were treated. Four patients received the polyurethane-covered Wallstent endoprosthesis (Schneider SA, Bulach, Switzerland) and two patients the barbed polyethylene-covered Gianturco stent (William Cook, Europe). RESULTS: All patients had successful stent placement under intravenous sedation and fluoroscopic guidance with immediate relief of dysphagia and sealing of the perforation. Following the procedure all patients could eat either a normal diet or soft food and five patients were discharged within 3-4 days. None of the serious sequelae usually associated with oesophageal perforation were observed. Two patients required second overlapping stents to be inserted within 1 week because of minor migration of the initial endoprostheses. In one patient two stents were necessary because the carcinoma extended over 17 cm. Five patients died after stent insertion (mean survival time = 49 days, range 16-80; median survival time = 37 days, range 16-80) due to a general deterioration in their condition, although all could swallow normally until death. The remaining patient was well and tolerating a light diet at 1 month. CONCLUSION: This technique is quick, safe and cost-effective and is now our preferred method of managing malignant oesophageal obstruction associated with perforation. PMID- 7538056 TI - Evidence for a CD14+ population of monocytes in inflammatory bowel disease mucosa -implications for pathogenesis. AB - Lipopolysaccharide (LPS) is abundant in the intestinal lumen. CD14 is the receptor for the LPS-LPS binding protein complex, and its presence on mononuclear phagocytes allows cell activation by pg/ml concentrations of LPS. We have shown that the recently recruited blood monocyte in inflammatory bowel disease mucosa is CD14+. This study examined the expression of CD14 on macrophages in inflamed (n = 13) and uninflamed (n = 7) intestine by immunohistochemistry, and on disaggregated lamina propria mononuclear cells (12 from inflamed, 17 from uninflamed intestine) and peripheral blood mononuclear cells (n = 26) by flow cytometry, using a panel of three MoAbs directed against CD14. Immunohistochemistry revealed that 3.7% of macrophages in uninflamed intestine were CD14+, while 25.1% of macrophages in active inflammatory bowel disease expressed CD14 (P < 0.02). Flow cytometry demonstrated that CD14 expression by macrophages from Crohn's disease and ulcerative colitis was augmented significantly (P = 0.02 and P = 0.01, respectively) compared with uninflamed intestine, with a discrete population of macrophages in inflammatory bowel disease, not present in normal intestine, which strongly expressed CD14. The characteristically high levels of CD14 on blood monocytes were unaffected by the presence of intestinal inflammation. Given the exposure of lamina propria cells to LPS present in the lumen of the terminal ileum and colon, the increased numbers of CD14+ macrophages in inflammatory bowel disease may result in greatly increased production of inflammatory mediators, thereby suggesting a mechanism for the perpetuation of mucosal inflammation. PMID- 7538059 TI - The use of the Wallstent endovascular prosthesis in the treatment of malignant inferior vena cava obstruction. AB - We report three cases of inferior vena cava (IVC) obstruction due to metastatic liver disease treated with a self-expanding Wallstent endoprosthesis (Schneider SA, Zurich, Switzerland). All three patients experienced symptomatic relief during their remaining few weeks of life. The Wallstent is effective and easy to deploy, and should be considered for use in the management of such patients. The literature to date on IVC stenting is also reviewed. PMID- 7538061 TI - Topical FK-506 prevents experimental corneal allograft rejection. AB - We evaluated the efficacy of topical cyclodextrin-encapsulated FK-506 in the prevention of experimental corneal allograft rejection. Two weeks after inducing corneal inflammation and neovascularization with 8-0 silk sutures, 23 albino rabbits received a unilateral 8-mm diameter central penetrating corneal allograft from pigmented donors. Rabbits were randomly assigned to no treatment (eight eyes), topical cyclodextrin four times daily for 28 days (seven eyes), or topical FK-506 0.3 mg/ml in a cyclodextrin suspension (eight eyes) four times daily for 28 days. Grafts were examined daily for degree of inflammation, neovascularization, edema, and signs of rejection for up to 100 days. Seven of eight (88%) untreated grafts and five of seven (71%) cyclodextrin-treated grafts rejected at a median of 3 weeks after transplantation, whereas only two (25%) of eight FK-506-treated grafts rejected and did so at a significantly longer interval (p < 0.005). Topical FK-506 prevents or delays corneal allograft rejection after experimental corneal transplantation. PMID- 7538057 TI - Blood monocytes of untreated asthmatics exhibit some features of tissue macrophages. AB - Airway macrophages are activated in asthmatic subjects. Peripheral blood monocytes from these subjects present some functional features of activation, but their membrane markers are not known. Recently a new subtype of blood monocytes, CD14+/CD16+, has been identified which possesses the characteristics of tissue macrophages. A study was carried out on nine normal subjects and 11 untreated asthmatics having variable severities of the disease to examine the phenotypic characteristics of monocytes. CD14, CD16, HLA-DR, CD11a, CD11b, CD44 and CD54 were studied using double fluorescence flow cytometry since these antigens have been defined in the CD14+/CD16+ monocytes. The functional activation of monocytes was examined using the release of superoxide anion. The co-expression of CD14 and CD16 by monocytes in terms of percentage and mean fluorescence intensity was significantly higher in asthmatics (P < 0.002 and P < 0.0001, respectively, Mann Whitney U-test). There was no difference for the other membrane markers between asthmatics and normal subjects. Superoxide anion release was significantly increased in asthmatic subjects (P < 0.01). This study shows that most blood monocytes of asthmatics are CD14+/CD16+ and are likely to present features of tissue macrophages. PMID- 7538060 TI - G-CSF in Felty's syndrome: correction of neutropenia and effects on cytokine release. PMID- 7538062 TI - Surgical options in the treatment of hepatic metastasis from colorectal cancer. AB - Current data indicate that liver resection is the only available treatment that regularly produces long-term survival with possible cure in patients with metastatic colorectal carcinoma to the liver. Although a number of clinical or pathologic factors predicts a poor outcome, the only absolute contraindications to liver resection are general health incompatible with recovery from major hepatic resection or clear evidence of wide dissemination of disease. Important areas for future study include the potential role of adjuvant regional chemotherapy after resection and cryoablation of "close" margins. For patients with unresectable disease, operative therapy also plays an important role. Multiple operative modalities hold promise in palliative treatment in the setting of clinically incurable disease. It is imperative that a large randomized trial of regional chemotherapy be performed allowing no crossover and with mortality as an endpoint. Additionally, the role of cryoablation begs systematic investigation to ensure proper use of this modality. PMID- 7538063 TI - [Treatment of maxillofacial cavernous hemangioma with pingyangmycin]. PMID- 7538064 TI - Can measurements of potential doubling time (Tpot) be compared between laboratories? A quality control study. AB - The purpose of this study was to investigate the reproducibility of potential doubling time measurements of human tumors in different laboratories and to distinguish which steps in the measurement procedure were subject to the greatest variation. This was achieved by comparing measurements on the same source material in two different laboratories in which three aspects of the technique were separately studied, namely, preparation and staining of the nuclear suspensions, running the samples on the flow cytometer (FCM), and analyzing the two-parameter FCM data. This involved exchange between the two centers of fixed tumor material, stained nuclear suspensions, and FCM data on floppy disks. The analysis step was found to be the least variable step. For DNA synthesis time, Ts, and the labeling index, LI, the coefficients of determination (R2) ranged from 92% to 95.4%. A small systematic bias was observed, with one center measuring approximately 15% higher values for both LI and Ts. Different criteria for window placements were found to be a contributing factor. Variations in the FCM step were approximately equal to those for analysis, with no systematic deviations. Variations for the preparation and staining step were the largest (R2 = 60.5% and 38.1% for LI and Ts, respectively). However, this step was the only one subject to intratumoral variability, which was the largest contributing factor to the variations observed. In addition, however, LI was on average 41% higher in one center compared to the other, resulting in a systematic bias. Differences in the level of green fluorescence of the labeled cells implicated antibody differences as a possible cause. The variations found here for the three procedural aspects were significantly smaller than variations observed between tumors, a requirement for a predictive assay. Suggestions for implementation of quality control procedures include objective (computer-assisted) data analysis on two-parameter histograms and optimization of antibody combinations. PMID- 7538065 TI - DNA staining changes associated with apoptosis and necrosis in blood lymphocytes of individuals with HIV infection. AB - We used flow cytometry to quantitate cells that die by apoptosis or necrosis. The method uses low concentrations of two DNA binding dyes that allow one to establish selective regions for live, apoptotic, and necrotic cells in a rat thymocytes model. Quantitative analysis of blood lymphocyte death in individuals with HIV infection by this technique shows the presence of nonviable cells that exhibit a spectrum of changes in staining by DNA binding dyes. These changes range from typical features of cells undergoing programmed cell death or apoptosis to changes observed in cells that die by accidental death or necrosis. The proportion of cells exhibiting these lethal changes increases significantly in patients who progress to AIDS, but, although cells with staining features associated with apoptosis and necrosis were both found to be increased in in vitro-activated cells from AIDS patients, spontaneous in vivo activation preferentially leads to apoptotic changes without a significant increase of cells exhibiting the staining changes associated with necrosis. PMID- 7538066 TI - Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture. AB - Embryonic mouse lung epithelium was separated from its mesenchyme and cultured under mesenchyme-free conditions. When covered with Matrigel, the cultured epithelium underwent branching morphogenesis in medium containing acidic fibroblast growth factor (aFGF), in which the epithelial cells constructed a simple columnar cell layer forming a lumen, as seen in normal development. The epithelial growth and branching morphogenesis induced by aFGF was completely inhibited by an antibody against aFGF. Heparin caused extra epithelial growth in cooperation with aFGF, but its use resulted in luminal expansion instead of enhanced branching. Basic FGF induced abnormal morphogenesis of the epithelium, though the lumen formed was lined by a simple columnar cell layer. Epidermal growth factor could not maintain epithelial cell growth, and the epithelium became a smaller and smoother ball than that at the start of cultivation. When covered with a collagen gel instead of Matrigel, the epithelium remained in its initial form, neither newly branching nor becoming a smooth ball, in the presence of aFGF. These results show that the epithelium of lung rudiments was able to branch under mesenchyme-free culture conditions in which a basement membrane matrix and aFGF were substitutes for the mesenchyme. PMID- 7538067 TI - Digit tip regeneration correlates with regions of Msx1 (Hox 7) expression in fetal and newborn mice. AB - We report that during mouse fetal development transcripts of Msx1 and Msx2 become progressively restricted to cells that will form more distal digit structures; the Msx2 expression domain is always more distal than Msx1. At birth both Msx1 and Msx2 are expressed in cells of the nail bed and hair follicle. We have found that the regenerative ability of mouse digit tips is restricted to levels in which the amputation plane is within the region of Msx1, but not Msx2, expression in early fetal digits and to levels where both Msx1 and Msx2 are expressed in late fetal and neonatal digits. Fetal digit tip regeneration is rapid and completed by birth, whereas neonatal digit tip regeneration requires 4 weeks and is sometimes imperfect. In both fetal and neonatal digits, we find that both Msx1 and Msx2 are expressed during regeneration, but not during wound healing associated with proximal amputations where no regenerative response is observed. These data support the hypothesis that the expression of Msx genes are important for digit cells to initiate and participate in a regenerative response. PMID- 7538068 TI - A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. AB - The murine homeobox-containing gene Hoxa-7 is expressed in restricted patterns during embryogenesis and plays an important role in the control of region specific differentiation. Previous studies have shown that separate elements specify lineage restriction and expression boundaries of Hoxa-7. In particular 3.6 kb of 5' flanking sequences were sufficient to establish an anterior boundary of Hoxa-7 gene expression. To identify the minimal regulatory element specifying the anterior boundary of expression, transgenic mice were generated carrying chimeric constructs with deletions of 5' flanking sequences fused to a thymidine kinase minimal promoter/E. coli lacZ reporter construct. By deletion analysis, a 470 bp long control element (AX 470) located 1.6 kb upstream of the transcription start site was identified that directed expression of the beta-galactosidase protein in a pattern reflecting the anterior boundary of expression of the endogenous Hoxa-7 gene. This element was active in either orientation and conferred region-specific expression to unrelated promoters, thereby behaving like an enhancer element. In contrast, transgenic mice carrying further 5' and 3' deletions of the 470 bp long element did not exhibit an anterior boundary of Hoxa 7 expression. Based on these results the minimal control element (AX 470) specifying the anterior boundary of Hox expression was designated as Hoxa-7 enhancer. Furthermore, 3 kb of the human HOXA7 upstream region were sequenced and compared to its mouse homologue in order to identify conserved regions. Sequence comparison revealed motifs that were strongly conserved between both species. The human homologue of the mouse Hoxa-7 enhancer was 70% identical at the nucleotide level and was also capable of directing an anterior boundary in transgenic mice. Using transgenic lines a detailed analysis of the Hoxa-7 enhancer-directed expression during embryogenesis was performed. lacZ expression was first detected in the allantois at day 7.5 p.c. and in mesoderm and ectoderm at day 8.5 of gestation. Between gestational ages E8.5 to E12.5 beta-gal expression was observed in the somites, spinal cord, spinal ganglia and paraxial mesoderm as well as in mesenchymal layers of the kidney. A distinct anterior limit of expression was noted in transgenic lines at level C4 (neural tube) and C5 (spinal ganglia). Our deletion experiments defined a minimal enhancer element specifying the anterior boundary of Hox gene expression in early and late phases of development. Further studies aim at characterizing the trans-acting factors that mediate the spatial and temporal expression of Hox genes in the developing embryo. PMID- 7538071 TI - In vivo and in vitro studies of the inhibition of the channel activity of colicins by gadolinium. AB - The primary effects of the ionophoric colicins A, E1 and B on Escherichia coli cells include triggering an efflux of cytoplasmic potassium, and a decrease of internal ATP as consequences of the opening of ionic channels in the cytoplasmic membrane. We report that micromolar concentrations of gadolinium and other members of the lanthanide family inhibited the efflux of potassium and the ATP decrease and that the cells recovered both ATP and potassium within a few minutes. Gadolinium, in the same concentration range also efficiently inhibited the channel activity of colicins A, E1, B and of the isolated channel-forming domain of colicin A in planar lipid bilayers. Colicin N was much less sensitive to the trivalent ion in planar lipid bilayers, consistent with the lack of effect of gadolinium on this colicin in vivo. Our data suggest that lanthanide ions act by direct binding to the colicin molecule and that this binding affects both its single-channel conductance and gating behaviour. PMID- 7538072 TI - Differential regulation of the cytochrome P450 3A1 gene transcription by dexamethasone in immature and adult rat liver. AB - We have previously shown that the in vivo induction of cytochrome P450 3A1 by dexamethasone occurs through a sharp and early transcriptional activation in the immature rat liver that is drastically impaired in adults [Telhada, M. B., Pereira, T. M. & Lechner, M. C. (1992) Arch. Biochem. Biophys. 298, 714-725]. In the present study we investigate the relative importance of cytochrome P450 3A1 gene transcription on the adaptive response to the synthetic glucocorticoid dexamethasone, by measuring the time-course run-on transcription rate and concomitant mRNA accumulation in the male rat liver at two different ontological developmental stages. The primary (direct) or secondary (dependent on protein neo synthesis) nature of the in vivo inductive response to dexamethasone and to pregnenolone 16 alpha-carbonitrile, is further investigated by inhibiting translation by cycloheximide pretreatment. The induction of cytochrome P450 3A1 gene transcription by the anti-glucocorticoid pregnenolone 16 alpha-carbonitrile is demonstrated to occur through a secondary mechanism, requiring ongoing protein biosynthesis, regardless of the developmental stage of the animals. Conversely, a significant developmentally controlled change is observed in the inductive response of the cytochrome P450 3A1 gene to dexamethasone, characterized by a markedly delayed transcriptional activation in the adult rat liver (90 day old) as compared to the immature rat liver (21 day old). This is consistent with the net primary response of the cytochrome P450 3A1 gene to dexamethasone demonstrated in this study to occur in the immature rat liver and almost lost at the adult stage, when protein neo-synthesis becomes essential for the inductive response. Our results demonstrate (a) a difference in the mechanisms underlying induction of the cytochrome P450 3A1 gene by the glucocorticoid agonist dexamethasone and by the antagonist pregnenolone 16 alpha-carbonitrile, and (b) an important change in the mechanisms of the inductive response to dexamethasone, associated with the immature/adult liver phenotype transition. This indicates the participation of specific labile transcription factors in the induction of cytochrome P450 3A1 gene by the synthetic glucocorticoids. PMID- 7538069 TI - Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. AB - In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways. PMID- 7538074 TI - A nuclear post-transcriptional event responsible for overproduction of argininosuccinate synthetase in a canavanine-resistant variant of a human epithelial cell line. AB - The Canr1 cell line, a canavanine-resistant variant of the cultured human epithelial cell line, RPMI 2650, overproduces argininosuccinate synthetase more than 200-fold. Run-on transcription assays showed no significant difference in transcription initiation of the argininosuccinate synthetase gene between Canr1 and RPMI 2650 cells. Furthermore, no difference in the relative transcription rate was seen along this 63-kb gene, suggesting that neither transcription initiation nor elongation is responsible for differential expression of argininosuccinate synthetase in these two cell lines. However, when isolated nuclei were labeled for a longer period of time in the transcription assay, precursor RNA of argininosuccinate synthetase in RPMI 2650 cells was found to be very labile. Apparently, a nuclear event affecting precursor RNA stability is responsible for the dramatic difference in argininosuccinate synthetase levels in these two cell lines. Using a microsatellite polymorphic marker, it was demonstrated that argininosuccinate synthetase from both alleles of the gene in Canr1 cells was overexpressed. This suggests that a trans-acting mechanism may be responsible for regulation of overproduction in this cell line. Furthermore, when protein synthesis was blocked by cycloheximide, less precursor RNA was observed in Canr1 cells. These data suggest that a labile protein factor(s) participates in the regulation. PMID- 7538075 TI - Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active. AB - We have expressed a cDNA for virus-induced chick interferon in Escherichia coli. The product, a 19-kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q-Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 10(8) IU/mg and is a powerful inducer of the interferon-responsive promoter of the chicken Mx gene. Culture medium of transfected COS cells expressing full length chick interferon cDNA contained up to 5 x 10(4) IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23-28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein. PMID- 7538073 TI - NMR mapping of the antigenic determinant recognized by an anti-gp120, human immunodeficiency virus neutralizing antibody. AB - The 24-amino-acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type-1, IIIB isolate (HIV-1IIIB, residues 308-331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5 beta, the complex of RP135 with an anti-gp120 HIV neutralizing antibody, 0.5 beta, which cross reacts with the peptide, was studied by using two dimensional NMR spectroscopy. A combination of homonuclear Hartmann Hahn two dimensional experiment and roating-frame Overhauser enhancement spectroscopy of the Fab/peptide complex measured in H2O was used to eliminate the resonances of the Fab and the tightly bound peptide residues and to obtain sequential assignments for those parts of the peptide which retain considerable mobility upon binding. In this manner, a total of 14 residues (Ser6-Thr19) were shown to be part of the antigenic determinant recognized by the antibody 0.5 beta. Lys5 and Ile20 were found to retain considerable mobility in the bound peptide while their amide protons undergo significant change in chemical shift upon binding. This observation suggests that these two residues are at the boundaries of the determinant recognized by the antibody. Competitive binding experiments using truncated peptides strongly support the NMR observations. PMID- 7538076 TI - Platelet-activating factor produced by endothelial cells. A molecule with autocrine and paracrine properties. AB - Endothelial cells (EC) participate in microenvironment homeostasis by regulating the trafficking of cells and molecules from the bloodstream to the tissues. The 'area codes' used to control the trafficking are adhesion molecules, receptors which pick up external signals, and mediators of cell-to-cell communication. Platelet-activating factor (PAF) is a mediator that belongs to this class of 'area-code' molecules. PAF is an acetylated derivative of phosphatidylcholine which is produced by EC and may act in an autocrine manner. Several stimuli may induce the synthesis of PAF by EC with different time courses. Thrombin, elastase and tumor necrosis factor (TNF) are prototypic molecules inducing very-early, early and delayed PAF synthesis, respectively. These stimuli activate the 'remodelling pathway' which requires the activation of phospholipase A2 and acetyl CoA:1-alkyl-2-lyso-sn-glycero-3-phosphate acetyltransferase. The effects of very-early and early stimuli are mediated by a direct stimulation of this pathway, whereas the action of TNF is mediated by the new synthesis of a serine protease. Stimulated EC produce PAF molecules with both an ether and an ester bond at the sn-1 position, but the former is predominant. The PAF produced is partially exposed on the external membranes and participates in adhesion or migration of neutrophils. PAF released can also stimulate EC themselves, by interacting with a specific receptor. PAF stimulates the migration and the shape change of EC by activating calcium influx and several serine/threonine and tyrosine kinases which regulate the cytoskeleton. PMID- 7538077 TI - Hepatocyte-derived interleukin-6 and tumor-necrosis factor alpha mediate the lipopolysaccharide-induced acute-phase response and nitric oxide release by cultured rat hepatocytes. AB - The regulation of acute-phase protein production and nitric oxide (NO) release in lipopolysaccharide-induced liver injury is thought to occur in response to monocytes/macrophages and Kupffer-cell-derived cytokines. In this study, we used primary cultured rat hepatocytes maintained as a differentiated phenotype to investigate the direct effects of endotoxin (lipopolysaccharide) on the production of the acute-phase proteins and on NO release. Lipopolysaccharide (10 micrograms/ml) increased the production of alpha 2-macroglobulin 2.5-fold compared to untreated cultures and decreased the production of albumin by 50%. The effect of lipopolysaccharide was mimicked by adding interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-alpha), cytokines being induced by treatment of hepatocytes with lipopolysaccharide. Maximal TNF-alpha (600 pg/ml) and IL-6 (1800 pg/ml) concentrations were observed 4 h and 6 h after lipopolysaccharide stimulation, respectively. The lipopolysaccharide-induced acute-phase protein response was blocked by anti-(IL-6) but not by anti-(TNF-alpha) IgG. The latter reduced the lipopolysaccharide-induced IL-6 production by 60%. Besides its effects on the acute-phase proteins, endotoxin caused a significant increase in NO production in cultured rat hepatocytes. Unlike anti-(IL-6) IgG, anti-(TNF alpha) IgG reduced the lipopolysaccharide-induced NO production by 50% indicating that endotoxin-induced NO production is partially mediated by TNF-alpha but not by IL-6. Preculture with gadolinium chloride (GdCl3), an inhibitor of Kupffer cells, did not change the response of hepatocytes to lipopolysaccharide indicating that the observed findings are direct endotoxin effects on hepatocytes. The data demonstrate that by their production of TNF-alpha and IL-6 rat hepatocytes respond to lipopolysaccharide treatment with an IL-6 mediated acute-phase protein and a TNF-alpha-mediated NO production. These features have previously been attributed to monocytes/macrophages and Kupffer cells. PMID- 7538078 TI - Immunochemistry of group A and Inaba C antigen factors constituting the O antigen of O1 Vibrio cholerae. AB - Serological cross-reactivity among intact lipopolysaccharides (LPS) from O1 Vibrio cholerae Inaba O-form (Inaba), Yersinia enterocolitica O9 (O9), non-O1 V. cholerae serogroup Hakata (Hakata) and Vibrio bio-serogroup 1875 Variant (1875 Variant) (all of which share Inaba antigen factor C), as well as a total of six kinds of chemically modified LPS (three from O9 and three from Inaba) was demonstrated by passive hemolysis and passive hemolysis inhibition by using these LPS as antigen for sensitizing sheep red blood cells and as inhibitor. These intact as well as chemically modified LPS contained, in their O polysaccharide chain, alpha(1-->2)-linked linear perosamine (4-amino-4,6-dideoxy-D-manno pyranose) homopolymers with different N-acyl groups: their acyl groups comprise 3 deoxy-L-glycero-tetronyl (Inaba LPS), formyl (O9 LPS), 3-hydroxypropionyl (1875 Variant LPS), acetyl (Hakata LPS and artificially introduced into Inaba and O9 LPS), propionyl and butyryl (both artificially introduced into Inaba and O9 LPS) groups. N-Deacylation of the alpha(1-->2)-linked N-(3-deoxy-L-glycero tetronyl)perosamine homopolymer of Inaba and the N-formyl one of O9 LPS resulted in virtual elimination of their serological reactivity with both homologous and heterologous antisera. Furthermore, when the resultant NH2 groups of the N deacylated perosamine homopolymers of both LPS were N-acylated with acetyl, propionyl or butyryl groups, they markedly recovered both of their serological reactivities. These results are compatible with the interpretation that the Inaba antigen factor C possessed by the four bacteria is substantially related to the common presence of N-acyl groups, regardless of their identity, residing in the perosamine residues constituting the O polysaccharide chain of their LPS. It was also indicated that the group antigen factor A of O1 V. cholerae is substantially related to the 3-deoxy-L-glycero-tetronyl groups residing in the perosamine homopolymer of Inaba LPS. PMID- 7538079 TI - Cardiac manifestations in mid-gut carcinoid disease. AB - The extent of heart disease and its relationship to the serotonin metabolite 5 hydroxyindoleacetic acid (5-HIAA), was studied with M-mode, 2D and Doppler echocardiography in 42 consecutive patients, 30 females and 12 males, median age 63 (range 23-75) years with histologically verified mid-gut tumour, liver metastases and 24-h urinary 5-HIAA excretion above 47 mumol.24 h-1. All patients had normal left ventricular ejection fractions, median 65% (interquartile range (IQR) 54-74%). Moderate to severe tricuspid regurgitation (TR) was diagnosed in 22 patients (59%); mitral or aortic regurgitation was found in nine (24%) and six (16%) patients, respectively. The mitral flow peak early (E) on late (A) velocity ratio was significantly decreased compared to age-matched normal subjects. The group of patients with 5-HIAA excretion exceeding 1000 mumol.24h-1 contained significantly more patients with severe TR than those with a lower excretion. The decrease in the E/A ratio may indicate reduced left ventricular compliance, possibly secondary to fibrous changes similar to those seen intra-abdominally and in the right side of the heart. As serotonin is degraded in the lung circulation, other mediators such as tachykinins and cytokines (PDGF) may be involved. PMID- 7538070 TI - Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. AB - The Bicaudal-C (Bic-C) gene of Drosophila melanogaster is required for correct targeting of the migrating anterior follicle cells and for specifying anterior position. Females lacking any wild type copies of Bic-C produce only eggshells open at the anterior end, because of the failure of the columnar follicle cells to migrate in the correct position at the nurse cell--oocyte boundary. Embryos which develop from eggs produced in females with only one wild type copy of Bic-C show defects in anterior patterning and an abnormal persistence of oskar RNA in anterior regions. We cloned Bic-C and found that, in ovaries, Bic-C RNA is expressed only in germline cells. Bic-C RNA is localized to the oocyte in early oogenesis, and later concentrates at its anterior cortex. The Bic-C protein includes five KH domains similar to those found in the human fragile-X protein FMR1. Alteration of a highly conserved KH domain codon by mutation abrogates in vivo Bic-C function. These results suggest roles for the Bic-C protein in localizing RNAs and in intercellular signaling. PMID- 7538080 TI - Primary chemotherapy with bleomycin, ifosfamide and cisplatinum (BIP) followed by radiotherapy in the treatment of advanced cervical cancer. A pilot study. AB - Bleomycin, Ifosfamide and Cisplatinum were combined in a 3 cycle regime of neoadjuvant chemotherapy given prior to radiotherapy in the treatment of 26 patients with late Stage (FIGO IIB-IIIB) cervical cancer. Seven patients were withdrawn for reasons of compliance and 1 patient due to toxicity. The response rate to chemotherapy in the remainder (18 patients) was 44.4%. Sixtyfour per cent of 17 patients who completed chemoradiotherapy responded completely. This regime was generally well tolerated and neurotoxicity was less problematic than in other reports, although fatal pneumotoxicity occurred in one patient. It is too early to comment on survival; this is an interim report of responses and toxicities. The results are less impressive than in other reported studies and cast doubt on the role of neoadjuvant chemotherapy in the management of advanced cervical cancer in developing countries. PMID- 7538081 TI - Precautions and complications in the use of the urolume wallstent. AB - The Urolume Wallstent was first used to treat urethral strictures in 1987. Its place in the treatment of urethral strictures is now well established and we have used it successfully in over 100 patients. Recent interest in alternative methods of treating benign prostatic hyperplasia (BPH) has led to the use of this and other stents in patients with symptomatic BPH who are a poor surgical risk. Over 270 patients have been treated by us with the Urolume Wallstent with satisfactory results. More recently we have also used this stent as an alternative to transurethral resection of the prostate in fat patients. Although the Urolume Wallstent is easy to use and clinically effective, complications can arise if proper care is not taken during its insertion. As the stent has become more widely used more problems and difficulties have been experienced. We discuss these and describe the precautions which should be taken in the use of this device in order to achieve the best results. PMID- 7538082 TI - Prostate-specific antigen detected prostate cancer: pathological characteristics of ultrasound visible versus ultrasound invisible tumors. AB - Most studies examining the issue of 'early detection of prostate cancer' advocate the combined use of serum prostate-specific antigen (PSA) and digital rectal examination (DRE). As a result, a significant number of new prostate cancers are diagnosed on the basis of an elevated serum PSA when the DRE is unremarkable. The purpose of this study is to determine if the PSA-detected tumors that are visible on transrectal ultrasound (TRUS) have the same pathological characteristics as PSA-detected tumors that are invisible on TRUS. One hundred and ninety-four patients with an elevated serum PSA concentration and nonpalpable prostate cancer who underwent radical retropubic prostatectomy (RRP) at our institution between March 1988 and December 1991 were reviewed. The patients were divided into two groups: 97 (50%) had no identifiable lesion on TRUS, and 97 (50%) had at least one hypoechoic area consistent with adenocarcinoma of the prostate. The pathological characteristics of the RRP specimens from the two groups were compared. There was no significant difference in the age (p = 0.14) or the preoperative serum PSA values (p = 0.18) between the groups. Also, there was no significant difference between the groups with regard to tumor volume (p = 0.89), focality of the cancer (p = 0.43), Gleason score (p = 0.81), DNA ploidy status (p = 0.96), pathological stage (p = 0.92), surgical margin involvement (p = 0.27), and tumor location (p = 0.64). These findings suggest that the clinical TNM staging system for prostate cancer may be simplified by eliminating the distinction between PSA-detected cancers visible on TRUS and PSA detected cancers not visible on TRUS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538085 TI - Production of granulocyte colony-stimulating factor by malignant mesothelioma. AB - We present the case of a male patient, aged 45 yrs, with malignant mesothelioma producing granulocyte colony-stimulating factor (G-CSF). The diagnosis was established by histopathological examination of the autopsied pleural tissues. Production of G-CSF was confirmed by immunoperoxidase staining, using a specific monoclonal antibody against recombinant G-CSF (rhG-CSF). PMID- 7538083 TI - Multicenter observational trial on symptomatic treatment of benign prostatic hyperplasia with alfuzosin: clinical evaluation of impact on patient's quality of life. The Italian Alfuzosin Cooperative Group. AB - The main aim of this multicenter observational study was to assess the impact on quality of life (QoL) of 3 months' treatment with an alpha 1-blocker, alfuzosin, 2.5 mg t.i.d., in patients suffering from symptomatic benign prostatic hyperplasia (BPH). Safety and efficacy evaluations were secondary objectives. Nine hundred and ninety patients were enrolled; 940 were evaluable for per protocol analysis. On day 84, all the three indices of the QoL self-questionnaire were significantly improved in comparison with baseline: mental health +0.4 (2.0%; p < 0.01); general health +0.6 (5.4%; p < 0.01, and, especially, activity +1.4 (13.4%; p < 0.01). Improvement was more marked in patients with severe symptoms at baseline (activity index: +25.6%). Alfuzosin also significantly relieved BPH symptoms according to the Maine Medical Assessment Program (total mean score: day 28 -3.07; day 84 -4.44) and Madsen-Iversen indexes (total mean score: day 28 -5.29; day 84 -7.98). Improvement was more marked in patients with severe symptoms at baseline. Subjective improvement was confirmed by objective measurements (uroflowmetry and residual volume). Fifty-two patients (5.2%) experienced one or more drug-related adverse medical events, mainly within the first 4 weeks of treatment (n = 46; 4.6%). Twenty-seven (2.7%) patients dropped out prematurely from the study for safety reasons. Forty vasodilatory events and 45 nonvasodilatory events were reported. Slight, not clinically significant decreases of blood pressure were observed but heart rate was not modified. In this study, 3 months' treatment with alfuzosin had a positive impact on patients' QoL by relieving their symptoms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538084 TI - The role of transrectal ultrasonography on the palpable and impalpable abnormal prostate. AB - Two hundred and three male patients underwent transrectal ultrasonography (TRUS) examination because of palpable nodule or hard consistency of the prostate. Of these, 56 of 65 (86.2%) digital rectal examination (DRE)-abnormal and 34 of 138 (24.6%) DRE-normal patients received transrectal sonoguided core needle biopsy. Among the DRE-abnormal patients, 18 (32.1%) had prostatic adenocarcinoma by biopsy with 2 additional patients who had initial negative biopsies eventually found to have cancer by transurethral resection of the prostate. In contrast, only 2 (5.9%) of 34 DRE-normal patients had cancers that were both hypoechoic and peripheral zone located. In 13 patients with transitional zone located lesions (hypoechoic 9, isoechoic 4), none had cancer. Of 27 patients who had normal DRE and unsuspected TRUS and received transurethral prostatectomy because of bladder outlet obstruction, 4 (14.8%) had cancer. In this study, hypoechoic lesions were found in 68 patients, among them 60 (88.2%) underwent biopsy but only 14 (23.3%) had cancer. From the results of this study, we concluded that TRUS can be a useful adjunct of DRE in detecting prostatic malignancy, especially when there has been an abnormal DRE, but its role on a digitally normal prostate requires further evaluation. Serum prostate-specific antigen can be an additional indicator to increase the prostate cancer detection rate. PMID- 7538086 TI - Nedocromil sodium reduces cigarette smoke-induced bronchoconstrictor hyperresponsiveness to substance P in the guinea-pig. AB - Acute exposure to cigarette smoke provokes airway hyperresponsiveness to substance P and inactivates neutral endopeptidase (NEP). To determine whether nedocromil sodium can prevent cigarette smoke-induced hyperresponsiveness to substance P, we studied two groups of anaesthetized guinea-pigs. One group of guinea-pigs was pretreated with aerosolized 0.9% NaCl solution (90 breaths), the other group was pretreated with aerosolized nedocromil sodium (10(-4) M, 90 breaths). In each animal, pretreatment was followed by either exposure to the smoke of one cigarette or exposure to air. After acute exposure to cigarette smoke or to air, we measured the change in total pulmonary resistance (RL) induced by increasing concentrations of aerosolized substance P. In the absence of nedocromil sodium, the bronchoconstrictor responses to substance P were greater in cigarette smoke-exposed guinea-pigs than in air-exposed animals. Aerosolized nedocromil sodium had no effect on the response to substance P in air exposed animals, but it reduced cigarette smoke-induced hyperresponsiveness to substance P. The preventive effect on cigarette smoke-induced hyperresponsiveness to substance P was observed at concentrations of aerosolized nedocromil sodium of 3 x 10(-5), 10(-4), and 3 x 10(-4) M. In vitro, cigarette smoke solution inhibited NEP activity from lung membrane preparations, but this inhibitory effect was not modified by nedocromil sodium (10(-4) M). We conclude that aerosolized nedocromil sodium reduces cigarette smoke-induced airway hyperresponsiveness to substance P in vivo. This action of nedocromil sodium is not due to a protective effect on cigarette smoke-induced inactivation of NEP in vitro. PMID- 7538087 TI - Anti-islet cell and anti-insulin antibody production by CD5+ and CD5- B lymphocytes in IDDM. AB - Although CD5 + B lymphocytes are mostly committed to the production of polyreactive natural autoantibodies, CD5 + B lymphocytes committed to the production of somatically mutated and monoreactive high-affinity IgM autoantibodies have been also shown. Increased proportions of CD5 + B lymphocytes in some autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM), have been noticed. The present study was undertaken to analyse the differences between CD5 + and CD5- B lymphocyte subsets for production of IDDM related autoantibodies, i.e. anti-human insulin antibodies (IA) and anti-human islet cell antibodies (ICA). For this purpose, Epstein-Barr Virus (EBV) transformation of FACS cell-sorted CD5 + and CD5- B lymphocytes and unfractionated enriched B lymphocytes from nine IDDM patients treated exclusively with recombinant human insulin, and from four healthy control subjects was performed; a mean of 102-216 microcultures with a mean of 1,000-2,333 cells/microculture for each B-lymphocyte fraction and individual was established. Data show that both CD5 + and CD5- B-lymphocyte subsets from either normal subjects or from IDDM patients receiving recombinant human insulin, contain B lymphocytes committed to the production of IA-IgM as a common element of their repertoire. In contrast, cells committed to the production of IA-IgG were only detected among the CD5- B lymphocyte subset from some IDDM patients. Only one microculture, out of a total of 6,211 screened (from control subjects and patients), in the CD5- B-cell subset from a recently-diagnosed IDDM patient, was found to produce ICA-IgM lambda. This might suggest that the frequency of circulating B lymphocytes committed to the production of ICA is very low even in IDDM patients bearing serum ICA. EBV-transformed B cells producing the ICA-IgM lambda were stabilized and cloned by somatic hybridization technique. This ICA IgM lambda human monoclonal antibody, designated HY1-MB91, is not polyreactive, but shows a restricted reactivity with human pancreatic islets, failing to react with other human tissues including cerebellar cortex, and lacking rheumatoid factor and anti-DNA antibody activities. It also lacks reactivity with pancreatic islets from other mammalian species (rat, mouse and monkey) as well as with other rat tissues, including cerebellar cortex. The antigen recognized by HY1-MB91 antibody in human islet cells is a cytoplasmic component mostly found in beta cells. PMID- 7538091 TI - Photosensitization with anticancer agents. 20--EPR studies on the photodynamic action of phleichrome: formation of semiquinone radical and activated oxygen species on illumination with visible light. AB - When phleichrome was illuminated with visible light, the semiquinone radical, singlet oxygen, and superoxide anion radical were detected. The formation of the semiquinone radical and activated oxygen species and the transformations and competitions between them depend on quinone and oxygen concentration, duration and intensity of illumination, and the nature of the substrate. In anaerobic solution, the semiquinone radical was predominantly photoproduced via the self electron transfer between the excited and ground species. In contrast, in aerobic solution, singlet oxygen is the principal product in the photosensitization of phleichrome. In addition to singlet oxygen, superoxide anion radical is also generated by the quinones upon illumination in aerobic solution, but to a lesser extent. The generation of the superoxide anion is significantly enhanced by the presence of electron donors. PMID- 7538093 TI - [Natural history of HCV seropositive cases found in health screening]. AB - In order to elucidate causative factors in HCV infection and natural history of HCV seropositive cases, examinees of adult health screening in two rural districts near Asahikawa (Groups A and B) were tested for HCV antibody and the clinical data of the HCV seropositive cases were retrospectively followed up. The examinees who showed cut-off index not less than 2.0 in C100-3 antibody test were taken as HCV seropositive cases. HCV seropositive rate was 5.1% for Group A, compared with 2.7% for Group B. HCV related factors were examined between HCV seropositive cases and age/sex-matched controls in the same group. As a result, blood transfusion and operation histories proved to be responsible for HCV seroreactivity in both Groups A and B. Although no significant difference was noted in blood transfusion and operation histories between Groups A and B, the rates with past history of liver disease, HBV exposure, and advanced age above 60 were significantly higher for Group A, suggesting HCV related factors other than blood transfusion and operation to be regionally different. Majority of HCV seropositive cases (79.2%) were ALT-normal, but 42.5% and 32.5% of this group showed ZTT- and ADA-abnormality, compared with 6.5 and 10.9% of the normal ALT group of HBV carriers respectively. HCV seropositive cases could be classified as ALT-abnormal (group I), ALT-transition from normal to abnormal (group II) and ALT persistently normal (group III), depending on the change in ALT level during follow-up period. Of these, group III represented 38% of HCV seropositive cases. The rate in which the HCV seropositive cases of this group III showed ZTT abnormality at least once during follow-up period was 81% and significantly higher than 43.5% for HBV carriers with persistently normal ALT level. Significant correlations between ZTT and IgG values were noted for both HCV seropositive cases and HBV carriers with persistently normal ALT level. For HCV seropositive cases, ZTT and IgG showed broad distributions from low to high values, compared with distribution in normal area for HBV carriers. ZTT abnormality in HCV seropositive cases may reflect some hepatic changes, because no other factors contribute to their ZTT abnormality. At least, some of group III HCV seropositive cases found in health screening may represent those who make no progress with chronic persistent hepatitis. PMID- 7538089 TI - [Atmospheric pollution and anthropometry in the newborn in Orenburg]. PMID- 7538090 TI - Localization of damage induced by reactive oxygen species in cultured cells. AB - N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process. PMID- 7538096 TI - [Normal synoviocytes and synoviocytes from osteoarthritis and rheumatoid arthritis bind extracellular matrix proteins differently]. AB - Extracellular matrix proteins are increased in inflammatory synovitis. We showed previously that the in situ expression of the corresponding extracellular matrix receptors (beta 1-integrins) is enhanced in synoviocytes (SC) of synovitis of different etiology (16). To investigate the adhesion of SC to extracellular matrix proteins, we examined the attachment of SC from normal and inflamed synovia to fibronectin, tenascin, laminin and collagen type IV. Compared to normal SC and SC of osteoarthritis, SC of rheumatoid arthritis showed an increased binding to tenascin, laminin, fibronectin and collagen type IV, suggesting a distinctive interaction of SC and extracellular matrix proteins in rheumatoid arthritis. Furthermore, the increased binding of SC of rheumatoid arthritis to extracellular matrix proteins may play a role in tissue remodelling associated with rheumatoid arthritis. PMID- 7538094 TI - [Transcript expression of the CPL 5x, BMD-3A, BMD-10 Interrogans leptospira]. AB - Total RNA of Leptospira interrogans SV Lai strain 017 was prepared by the method of Licl-Urea, and was used in dot hybridization with biotin-labelled DNA probes. The probes included BMD-3A, BMD-10, which were the leptospirial protective antigen genes, and CPL 5x, which was the genus specific gene of interrogans Leptospira. All of the three probes have shown various degrees of hybridization signs, proving that they all have transcript expression in leptospira. The transcript expression is the main way of regulating the gene expression in procaryotae, and it is significant in molecular genetics of Leptospira. The results indicate that the antigens encoded by the BMD-3A, BMD-10 may play an important role in immune reaction against leptospiral infection and provide a clue to the development of gene-engineering vaccine. The results also suggest that the antibody against the antigen encoded by CPL 5x is a useful tool in the classification of Leptospira interrogans. PMID- 7538095 TI - [Pharmacological modulation of the complement system: an opportunity for successful xenotransplantation]. AB - Hyperacute graft rejection triggered by the activation of the recipient's complement system represents the major obstacle to a successful xenotransplantation. Inhibition of complement activation is, therefore, considered as a prerequisite for xenograft survival. Support of the physiological regulation of the complement system appears to be the most promising strategy as indicated by first results from animal xenograft experiments. The transfer of human membrane-bound complement regulatory proteins offers new chances to protect the xenograft against the cytolytic complement attack. Another approach aims at interfering with receptor/ligand interaction of the adhesion molecules CR3 and CR4 (CD11b,c/CD18). All strategies of complement intervention have to consider the important function of complement within the immunological defense. PMID- 7538097 TI - [Experimental therapy of hypoplastic paroxysmal nocturnal hemoglobinuria]. AB - Paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia are associated either as a PNH-aplasia syndrome or the emergence of glycosylphosphatidylinositol (GPI)deficient blood cells in patients with severe aplastic anemia (SAA). It could be demonstrated that SAA patients with GPI-deficient cells in comparison to those without have a worse response to classical immunosuppressive therapy. Therefore, we treated a female PNH patient with severe thrombocytopenia, anemia and granulopenia with G-CSF and cyclosporine. Within 8 weeks, a trilineage response of hematopoiesis was observed. In addition, the proportion of normal to GPI-deficient granulocytes and monocytes increased significantly. PMID- 7538092 TI - [Corticosteroids in terminal cancer]. AB - Corticosteroids are frequently prescribed in terminal cancer. They were prescribed in more than 50% of 100 consecutive patients of our hospice for palliation of anorexia, weakness, symptoms of cerebral metastases and of hypercalcemia, and other problems. There was objective and/or subjective improvement in most, though usually of short duration. If there is no improvement after a few days, corticosteroids should be discontinued. PMID- 7538088 TI - Factor VIII, HIV and AIDS in haemophiliacs: an analysis of their relationship. AB - In this review, the association between the Acquired Immune Deficiency Syndrome (AIDS) and haemophilia has been carefully examined, especially the data that have been interpreted as indicating transmission of the human immunodeficiency virus (HIV) to the recipients of purportedly contaminated factor VIII preparations. In our view, the published data do not prove the hypothesis that such transmission occurs, and therefore HIV cannot account for AIDS in haemophiliacs. PMID- 7538098 TI - Hepatitis A, B and C seroprevalence in Pakistan. AB - A cross sectional study was conducted to determine the seroprevalence of Hepatitis A, B, and C virus in healthy Pakistani children. HAV IgG antibody was assayed in 258 subjects and it was found that 94% children by 5 years of age had HAV IgG-antibody. The overall seroprevalence of HAV IgG antibody was 55.8% and IgM 5.3%. HBVsAb levels assayed in 236 healthy children showed a seroprevalence of 2.97%. Similarly, HCV antibody seroprevalence was found to be a low 0.44% in healthy children. HAV is a major cause of Hepatitis, as compared to HBV and HCV which are of low endemicity. PMID- 7538099 TI - Anti-envelope antibodies in anti-hepatitis C virus (HCV) positive patients with and without liver disease. AB - Our aim was to verify whether the presence of antibodies to HCV envelope protein might mark the occurrence of liver damage, as recently suggested in the literature. Sera from 104 patients (62 male, 42 female) were tested: 84 were positive and 20 were negative to a second generation enzyme immunoassay for anti HCV antibodies; 51 patients had mild chronic liver disease (44 chronic hepatitis, seven steatosis), 43 had liver cirrhosis (superimposed by hepatocellular carcinoma in 18) and ten were asymptomatic anti-HCV positive subjects with normal liver function tests. Besides, all sera were tested by means of an enzyme immunoassay for the presence of serum antibodies to the synthetic peptide S24A (SIYPGHVSGH RMAWDMMMNW SPTA) derived from amino acids 307-330 of HCV polyprotein. Anti-S24A antibodies were detected in 40/84 sera positive and 1/20 negative at anti-HCV testing (Pearson chi 2 12.29; p = 0.005). Among anti-HCV positive sera, no significant difference existed in anti-S24A status with regard to clinical evidence of liver disease, ALT concentration or HCV RNA positivity. Thus, anti S24A antibodies are detectable in approximately half of HCV-positive sera, but they do not seem to add significant clinical information to existing tests or to be useful as putative markers of viraemia. PMID- 7538101 TI - Screening for prostate cancer. AB - In an attempt to detect prostate cancer when the tumor is still confined to the prostate, a screening program was established. We studied the efficacy of digital rectal examination (DRE) and serum prostate-specific antigen (PSA) in the early detection of prostate cancer. One thousand men aged 50-75 years underwent DRE and serum PSA determination. Transrectal ultrasound-guided biopsies were obtained in each case of a suspicious DRE. Six systematic biopsies were performed if the PSA level was > 10 ng/ml, even if DRE and transrectal ultrasonography revealed no areas suspicious of cancer. A suspicious DRE was noted in 11.5% of the subjects; 16% had elevated levels of serum PSA (> 4 ng/ml) and 3.9% had serum PSA > 10 ng/ml. Biopsies were obtained from 90 patients, of which 31 were positive for prostate cancer. The cancer detection rate was 2.2% for DRE, 2.0% for PSA > 10 ng/ml, and 3.1% for the two methods combined. Clinical staging revealed that in 29 of the 31 patients with prostate cancer, the tumor was confined to the prostate: Stage A in 9 cases and stage B in 20 cases. Only two patients had clinically advanced cancer, and 22 patients underwent radical prostatectomy. Pathological examination disclosed biologically significant tumors in 91% of the cases in terms of tumor volume and grade. Although there is little evidence that screening will result in the reduction of the disease-specific mortality rate, early detection of prostate cancer by DRE, serum PSA, and transrectal ultrasound should be encouraged. PMID- 7538100 TI - Hepatitis C virus RNA in serum and liver histology in asymptomatic anti-HCV positive subjects. AB - This study was designed to evaluate serum HCV-RNA, liver histology, and RIBA-II pattern in asymptomatic anti-HCV positive subjects with persistently normal or slightly (i.e. < or = 1.5 times the upper limit of the normal range) elevated serum ALT levels. To this purpose, 22 asymptomatic anti-HCV positive subjects (11 men and 11 women, median age 40, range 21-70 years) underwent liver biopsy and determination of serum HCV-RNA. Positivity for anti-HCV was determined by ELISA-2 and by RIBA-II. Serum HCV-RNA was determined by PCR. Our data show that: 1) 9/22 symptom-free, anti-HCV positive subjects had histological features of chronic liver disease associated with ongoing HCV infection; 2) four subjects had no histological signs of chronic hepatitis and normal serum ALT levels despite positivity for serum HCV-RNA; 3) serum ALT levels did not discriminate HCV-RNA positive subjects with from those without chronic hepatitis; 4) in anti-HCV positive subjects with normal serum ALT levels, a positive RIBA-II pattern was not always predictive of HCV viraemia or chronic hepatitis while an indeterminate RIBA-II pattern was frequently associated with nonspecific liver changes or normal histology. In conclusion, based on these findings, "true" healthy carriers of HCV (i.e. subjects with normal serum ALT levels and no histological features of chronic hepatitis despite HCV viraemia) may exist. PMID- 7538102 TI - Proportional amplification of individual HLA-A and -B antigens during upregulated expression of total class I HLA molecules. AB - Our recent studies demonstrated that each specific HLA-A or -B antigen is not expressed in equal quantity in cells of an individual and that the relative amounts of different HLA-A and -B antigens are genetically predetermined following Mendelian laws. These findings suggest the potential genetic importance of varied quantitative HLA expression on target cells in determining the sensitivity to cytotoxic T lymphocytes. It would be important to know whether the amounts of different HLA antigens are differentially or proportionally amplified after upregulated expression of total HLA antigens. We have therefore determined the effects of IFN treatment, EBV transformation, and influenza virus infection on the quantitative expression of total HLA antigens and the relative quantities of different specific HLA-A and -B antigens in human fibroblasts cell line and peripheral blood mononuclear leukocytes. In contrast to earlier studies using the transfected HLA genes, our results show that different individual HLA-A and -B antigens are proportionally and not differentially amplified during upregulated expression of total class I HLA molecules. This finding indicates that the genetic predetermination of varied quantitative expression of HLA antigens may play a role in influencing antiviral immunity and disease susceptibility. PMID- 7538104 TI - Augmentation of cancer chemotherapy by preinjection of human macrophage colony stimulating factor in L1210 leukemic cell-inoculated mice. AB - Human macrophage colony-stimulating factor (hM-CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF1 male mice inoculated with L1210 cells, a mouse B cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100 micrograms/kg/day) or hM-CSF (20 micrograms/kg/day) for 3 days from day 1. In mice preinoculated with 10(2) L1210 cells but not with 10(3) or more L1210 cells, a marked increment in survival rate was observed with hM-CSF treatment. We next examined the effect of hM-CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 10(5) L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumor-bearing mice, but all of the mice died within 20 days. When hM-CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM-CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO2-) in the serum. When L1210 cells were co-cultured with peritoneal macrophages from mice intraperitoneally injected with hM-CSF, the uptake of [3H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of NG monomethyl-L-arginine, an inhibitor of NO2- synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM-CSF-induced inhibition of L1210 growth. PMID- 7538105 TI - Specific antibody-mediated detection of Brochothrix thermosphacta in situ in British fresh sausage. AB - A rabbit polyclonal antibody-linked probe was developed which detected 76% of 800 food isolates of the spoilage bacterium Brochothrix thermosphacta when cells were bound to nitrocellulose. In slide cross-reaction tests all six environmental isolates tested were stained but the type strain was not. The antibody did not cross-react with Listeria grayi, L. monocytogenes, Lactobacillus plantarum, Lactococcus lactis, Streptococcus mutans, Bacillus cereus or B. subtilis. The antibody-linked probe detected Br. thermosphacta in thin sections of British fresh sausage when the viable count was greater than 10(6) g-1. Cells were detected mainly within 1 or 2 mm of the surface on the loose starchy material. They were not detected within muscle blocks or in the centre of the sausage. Such results suggest that growth of this organism occurs close to the surface of the sausage. PMID- 7538103 TI - Expression of CD44 variant exons 8-10 in colorectal cancer and its relationship to metastasis. AB - Splice variants of CD44 are overexpressed in human lung, breast, and colon carcinoma cell lines. This study was conducted to clarify the association between the expression of CD44 variant exons 8-10 and metastatic potential in human colorectal cancer. We found that the expression of a CD44 splice variant containing exons v8-10 was increased in all of 60 colorectal cancer specimens examined compared with matched normal colerectal mucosa, as determined by Northern blotting. Expression of CD44 variant exons 8-10 did not significantly correlate with histological type, depth of tumor invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, the level of CD44 variant exon 8-10 expression was significantly higher in carcinomas associated with liver metastasis than in those without liver metastasis. In addition, expression of CD44 variant exons 8-10 in the liver metastases was more intense than that in the primary colorectal cancers. These findings indicated that this domain of the CD44 glycoprotein encoded by exons v8-10 may play an important role in tumor hematogenous metastasis of human colorectal cancer. PMID- 7538106 TI - Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal DNA of human Capnocytophaga. AB - The confusion in the taxonomic status of the genus Capnocytophaga has made identification of strains and studies on the role of this genus in infectious diseases equivocal. In this study 33 strains of Capnocytophaga including reference strains and various clinical isolates, were studied using RFLP analysis of 16S ribosomal RNA genes. The 16S ribosomal RNA (rRNA) gene sequences from whole cell suspensions and isolated genomic DNA samples were amplified by the polymerase chain reaction (PCR) using eubacterial specific primers. PCR products were purified and characterized by single digestions with 12 restriction endonucleases. Five of these, BanI, CfoI, HaeIII, HphI and RsaII were found to discriminate reproducibly between strains, and restriction patterns (ribotypes) produced by these were analysed to clarify the classification of Capnocytophaga strains. Dendrograms inferring similarities were derived from these data by the UPGMA method. This analysis produced three major clusters of strains, each of which was associated with a previously proposed species type strain: C. gingivalis, C. sputigena and C. ochracea. The results support the division of Capnocytophaga into three species and demonstrate that, despite the heterogeneity of this genus, the modified ribotyping method provides a simple, rapid and reproducible way to identify Capnocytophaga strains. PMID- 7538107 TI - Characterization of monoclonal antibodies against Erwinia carotovora subsp. atroseptica serogroup I: specificity and epitope analysis. AB - The characteristics of two monoclonal antibodies (Mabs), A23/1221.59.44.d.3 (1221) and A23/1239.36.64.e.2 (1239), against Erwinia carotovora subsp. atroseptica serogroup I produced in this study were compared with those of two other independently obtained Mabs, 4G4 in Spain and 4F6 in Canada, using different strains as immunogen and different screening procedures. The reaction pattern of Mabs 1221 and 1239 determined by indirect ELISA on over 200 bacterial strains including five E.c. atroseptica and 36 E.c. carotovora serogroups, seven Erw. chrysanthemi biovars, 23 other plant bacterial pathogens and 33 saprophytic bacteria from potato was similar to that of 4G4. Specificity for E.c. atroseptica serogroup I was improved, especially when skimmed milk (Marvel) was used instead of bovine serum albumin as blocking agent. Mabs 1221, 1239 and 4G4 reacted positively with all 22 E.c. atroseptica serogroup I, the dominant E.c. atroseptica serogroup on potato, strains tested and only with two out of five E.c. atroseptica serogroup XXII strains, one E.c. carotovora serogroup XXI strain and one strain of a saprophytic bacterium, Comamonas sp. Essentially similar results were obtained when examined by immunofluorescence. Characterization of the four Mabs showed that they were IgG3 and SDS-PAGE/immunoblot results suggested that they were probably against the O-side chain of bacterial cell wall lipopolysaccharides. In competition ELISA between biotin-labelled and unlabelled Mabs, the competition pattern of the four Mabs was similar.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538108 TI - Leukocyte trafficking mediated by selectin-carbohydrate interactions. AB - The selectins have attracted intense interest because of their carbohydrate recognition properties and their pivotal roles in leukocyte trafficking. Future studies will center on the mechanisms for regulating the expression of the selectins and their ligands, the molecular details of selectin binding to glycoprotein ligands and small carbohydrates, and the biophysical principles that selectins employ to mediate attachment and rolling of leukocytes under flow. PMID- 7538109 TI - Activation of Fes tyrosine kinase by gp130, an interleukin-6 family cytokine signal transducer, and their association. AB - gp130 is a signal-transducing subunit of receptors for the interleukin-6 (IL-6) related cytokine subfamily including IL-6, leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor, indicating that gp130-mediated signals are involved in the immune response, hematopoiesis, inflammation, and endocrine and nervous system activity. We previously showed that gp130 stimulation rapidly activates Jak, Btk, and Tec tyrosine kinases, all of which constitutively associate with gp130. To further elucidate intracellular signal transduction through gp130, we examined the possible involvement of another nonreceptor tyrosine kinase, p92c-fes (Fes). We showed that gp130 stimulation rapidly induced tyrosine phosphorylation of Fes and actually activated its kinase activity in hematopoietic lineage cells. Furthermore, Fes associated with gp130 independently of ligand stimulation like Jak, Btk, and Tec tyrosine kinases. These results indicate that multiple nonreceptor tyrosine kinases are involved in the gp130 mediated signal transduction pathway. Because both gp130 and Fes are expressed not only in hematopoietic lineage cells but also in heart and nerve cells, Fes may play a role in signal transduction through gp130 in these tissues. PMID- 7538110 TI - Erythropoietin-induced recruitment of Shc via a receptor phosphotyrosine independent, Jak2-associated pathway. AB - Based on the recently implicated role of Shc as a signaling effector for type I cytokine receptors, factors which mediate the recruitment and phosphorylation of Shc in the erythropoietin receptor (EPOR) system have been studied. FDC-P1 cells stably expressing the wild type murine EPOR supported the EPO-induced association of Shc with Jak2 and its rapid tyrosine phosphorylation. However, this did not depend upon the presence of phosphotyrosine sites within the EPOR and was mediated by a mitogenically deficient receptor form (EPOR329) lacking cytoplasmic tyrosine residues. This was shown both by Western blotting of Shc and Jak2 co immunoprecipitates and through the development of an in vitro assay for cytokine induced Shc phosphorylation. The direct association of Shc with Jak2 also was observed and was shown to depend upon EPO-exposure and the SH2 subdomain of Shc. Together, these studies indicate that Jak2, in part, may mediate the EPO-induced phosphorylation of Shc. PMID- 7538111 TI - Characterization of carbohydrate binding proteins in Trypanosoma cruzi. AB - Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans and invades a great variety of mammalian cells. The nature of the ligand(s) and receptor components in both T. cruzi and target cells remains controversial, although it seems to involve an interaction with oligosaccharides. In an attempt to identify possible ligands on the parasite, we have searched for the presence of carbohydrate binding proteins (CBPs) in T. cruzi. By fluorescence-activated cell sorter analysis using a panel of fluoresceinated glyco- and neoglycopeptides with well characterized glycans, the presence of at least two different CBPs was identified on the surface of T. cruzi epimastigotes and trypomastigotes. The specificity of binding of the two CBPs seems to be mediated by galactose and mannose residues. The mannose- and galactose-mediated CBPs from epimastigotes and trypomastigotes were purified to homogeneity by affinity chromatography on immobilized thyroglobulin and identified as 60-70-kDa glycoproteins. Purified CBPs were able to specifically bind with high affinity to murine and human macrophages as well as other cell types susceptible to infection by T. cruzi but not to fat or neuronal cells. This binding was inhibited by the corresponding ligands. Moreover, the mannose mediated CBP binding was completely abolished by alpha-mannosidase treatment of the cells. These results suggest a possible role for the CBPs in the recognition events between the parasite and target cells and/or in the interaction of the epimastigotes with the insect gut cells. PMID- 7538112 TI - S-alkyl-L-thiocitrullines. Potent stereoselective inhibitors of nitric oxide synthase with strong pressor activity in vivo. AB - Nitric oxide synthase catalyzes the oxidation of a guanidino nitrogen of L arginine to nitric oxide with concomitant formation of citrulline. Enzyme activity is inhibited by a variety of N omega-monosubstituted L-arginine analogs including N omega-alkyl-, N omega-amino-, and N omega-nitro-L-arginine derivatives. We report here that both constitutive and inducible isoforms of nitric oxide synthase are strongly inhibited by S-alkyl-L-thiocitrullines (N delta-(S-alkyl)isothioureido-L-ornithines) with n-alkyl groups of one to three carbons. These compounds represent a novel class of inhibitors and are the most potent nitric oxide synthase-inhibiting amino acids described to date. Inhibition is reversible, stereoselective, and competitive with L-arginine. Spectral studies show no direct interaction of inhibitor sulfur with heme iron, a result in contrast to that seen previously with the parent compound, L-thiocitrulline. The S-alkyl-L-thiocitrullines have strong pressor activity in normotensive control rats; S-methyl-L-thiocitrulline reverses hypotension in a rat model of septic peritonitis and in dogs administered endotoxin. These latter findings suggest that the inhibitors may have therapeutic utility in treating hypotension due to the overproduction of nitric oxide. PMID- 7538113 TI - Control of the Escherichia coli rrnB P1 promoter strength by ppGpp. AB - Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2 combination, to a wild-type lacZ gene were constructed on plasmids and recombined into the mal region of the bacterial chromosome, close to the normal location and in the normal orientation of rrnB. The upstream activator region (Fis-binding sites) was always present with the P1 promoter, and all constructs contained the box A antitermination site of rRNA genes. Using these constructs, beta-galactosidase specific activities were measured in Escherichia coli strains carrying either both ppGpp synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+), or neither (delta relA delta spoT), using different media supporting growth rates between 0.6 and 2.8 doublings/h at 37 degrees C. The beta galactosidase activities were used to estimate the relative strength of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter. Promoter strength (transcripts initiated per min per promoter per free RNA polymerase concentration) was distinguished from promoter activity (transcripts initiated per min per promoter). In ppGpp-synthesizing (wild-type) bacteria, the relative strength of the rrnB P1 promoter increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus, at low or zero levels of ppGpp, the P1 promoter was 1.5 1.7 times stronger than the isolated P2 promoter. These results indicate that the normal growth rate control of the rrnB P1 promoter strength requires ppGpp, and that the strength is reduced at basal levels of ppGpp found during exponential growth. No additional ppGpp-independent control of the rrnB P1 promoter strength was evident. From the beta-galactosidase data and previously determined values of rRNA gene activities, the activities of the isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem combination, were estimated. With increasing growth rate, the activity of the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min, while the activity of the isolated P1 promoter increased 24 fold from 2 to 54 initiations/min. The increasing activity of the isolated P2 promoter is assumed to reflect the increasing RNA polymerase concentration at constant promoter strength, whereas the steeper increase in P1 promoter activity reflects increases in both polymerase concentration and promoter strength. When in tandem with P1, the P2 promoter activity is inferred to decrease as the P1 promoter activity increases. PMID- 7538115 TI - Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4. AB - Insulin-like growth factor binding protein 4 (IGFBP-4) is a 24-kDa protein that binds insulin-like growth factor 1 (IGF-1) and IGF-2 with high affinity and inhibits IGF action in vitro. We recently described a protease produced by the B104 neuronal cell line that cleaves IGFBP-4, yielding an approximate 16-kDa immunoreactive protein that binds IGFs with reduced affinity. We analyzed fragments produced by exposing pure IGFBP-4 to the protease to determine potential cleavage sites. Electrospray mass spectrometry and amino acid sequencing indicated the 16-kDa fragment spanned the NH2 terminus of native IGFBP 4 through Lys-120. There was evidence for an additional proteolytic fragment beginning at amino acid 132 and continuing to the COOH terminus. Proteolysis could be blocked by a synthetic peptide that spanned amino acids 117-126 but not by peptides that contained flanking sequences 111-120 or 125-135. Mutagenesis was used to alter the basic residue at position 120. The expressed mutant IGFBP-4 (K120A) was relatively resistant to cleavage, strongly suggesting that residues 120-121 represent the cleavage site. This region of IGFBP-4 is not homologous with other IGFBPs, explaining the apparent specificity of the protease for IGFBP 4. The 16-kDa IGFBP-4 fragment no longer inhibited IGF-1-stimulated thymidine uptake in vitro, suggesting that proteolytic processing of IGFBP-4 may have important functional consequences in vivo. PMID- 7538114 TI - Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts. AB - In rabbit aortic vascular smooth muscle cells (VSMC) platelet-derived growth factor BB (PDGF-BB) stimulated the tyrosine phosphorylation of phospholipase C gamma, p120 GTPase-activating protein, and the p85 alpha subunit of phosphatidylinositol 3'-kinase only at high concentrations (5-25 ng/ml). In contrast, PDGF-BB induced a rapid and concentration-dependent increase in p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, which was half-maximal and maximum at 1 and 2.5 ng/ml, respectively. Saliently, stimulation of p125FAK tyrosine phosphorylation was sustained at up to 100 ng/ml PDGF-BB and for prolonged times of treatment. With similar concentration dependence, PDGF-BB stimulated the tyrosine phosphorylation of the 68-kDa focal adhesion-associated protein, paxillin. PDGF-BB also induced p125FAK and paxillin tyrosine phosphorylation in human aortic VSMC. PDGF-BB caused no detectable disruption of the actin cytoskeleton in VSMC. PDGF-BB stimulated rabbit VSMC migration with a very similar concentration dependence to that for p125FAK and paxillin tyrosine phosphorylation. PDGF-BB was equally effective in stimulating p125FAK and paxillin tyrosine phosphorylation under conditions similar to those used for cell migration. In Swiss 3T3 fibroblasts, PDGF-BB and -AA stimulated p125FAK tyrosine phosphorylation and cell migration only at low concentrations, and stimulation was abolished at 10-25 ng/ml. PDGF-AA failed to stimulate tyrosine phosphorylation, mitogenesis, and chemotaxis in rabbit VSMC, and immunoblot analysis showed that rabbit VSMC expressed PDGF beta-receptors but no alpha receptors. These results implicate p125FAK in the chemotactic response to PDGF-BB and suggest that the ability of PDGF-BB to trigger the p125FAK pathway may be dependent both upon cell type and receptor isotype expression. PMID- 7538118 TI - Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig alpha/Ig beta immunoreceptor tyrosine activation motif binding and autophosphorylation. AB - Syk is a cytoplasmic protein-tyrosine kinase containing two amino-terminal Src homology 2 domains that is activated following ligation of the B cell antigen receptor. Syk activation in B cells correlates with Syk tyrosine phosphorylation as well as with Syk SH2-mediated association with the tyrosine-phosphorylated Ig alpha and Ig beta B cell antigen receptor subunits. Tyrosine-phosphorylated peptide 20-mers representing Ig alpha and Ig beta immunoreceptor tyrosine activation motifs were synthesized and found to stimulate the specific activity of Syk by as much as 10-fold in vitro. Maximal phosphopeptide-induced Syk activation required both Syk SH2 domains and phosphorylation of both tyrosine residues present in the immunoreceptor tyrosine activation motif. The biochemical mechanism responsible for the phosphopeptide-induced Syk enzyme activation appears to be a function of Syk autophosphorylation. Our observations suggest the association of Syk tandem SH2 domains with the tyrosine-phosphorylated Ig alpha and/or Ig beta immunoreceptor tyrosine activation motifs in B cells stimulates Syk autophosphorylation leading to Syk enzyme activation. PMID- 7538117 TI - Adhesive interactions between alternatively spliced CD44 isoforms. AB - Alternative splicing of a series of 10 contiguous exons present within the CD44 gene can generate a large number of differentially expressed CD44 isoforms that contain additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule proximal to the membrane spanning domain. Although distinct functions have been ascribed to certain of these isoforms, the effect of particular inserted domains on the ligand-binding specificity of the CD44 molecule remains unclear. In the present study, we demonstrate that while CD44H, the major CD44 isoform expressed on resting hemopoietic cells, and CD44R1, an alternatively spliced isoform present on transformed epithelial cells and certain activated and/or malignant hemopoietic cell types, can both bind avidly to hyaluronan, only CD44R1 can promote homotypic cellular aggregation when expressed in the CD44-negative murine lymphoma cell line TIL1. Experiments in which TIL1 cells transduced with different CD44 isoforms were tested for their ability to adhere to one another or to COS7 cells transfected with CD44R1, indicated that CD44R1 can recognize and bind a common determinant present on both CD44H and CD44R1. Monoclonal antibody blocking studies suggest further, that the determinant recognized by CD44R1 is located in a region of the CD44 molecule distinct from that involved in hyaluronan binding. PMID- 7538116 TI - Beta 1- and beta 3-class integrins mediate fibronectin binding activity at the surface of developing mouse peri-implantation blastocysts. Regulation by ligand induced mobilization of stored receptor. AB - Implanting mouse blastocysts adhere through their abembryonic surfaces to the endometrial extracellular matrix. Because blastocysts cultured on fibronectin in vitro dissociate to form trophoblast outgrowths, it is unclear whether this adhesion is initially mediated by fibronectin receptors on the apical or basolateral surface of the trophectoderm. Intact blastocysts were examined in a ligand binding assay utilizing the fibronectin cell binding domain attached to fluorescent microspheres. Fibronectin binding activity on the apical surface of the trophectoderm was confined to the abembryonic pole of the blastocyst, where trophoblast differentiation initiates, and was regulated temporally in accordance with blastocyst outgrowth. Soluble fibronectin (IC50 = 0.2 microM) or Gly-Arg-Gly Asp-Ser-Pro, but not laminin, competitively inhibited fibronectin binding activity. Addition of antibodies against the alpha v, alpha 5, beta 1, or beta 3 integrin subunits also inhibited binding activity. Blastocysts cultured in the absence of an adhesive substratum exhibited fibronectin binding activity only after exposure to immobilized or soluble ligand. Potentiation of binding activity by ligand was unaffected by cycloheximide but was sensitive to brefeldin A inhibition of protein trafficking. These findings suggest that the interaction of fibronectin with the trophectoderm induces a translocation event that up regulates fibronectin binding beta 1- and beta 3-class integrins on the apical surface. PMID- 7538120 TI - The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins. AB - P- and E-selectins belong to a family of Ca(2+)-dependent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca(2+)-dependent manner. This protein has a molecular mass of approximately 120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E selectins. PMID- 7538119 TI - Homology requirements for ligation and strand exchange by the FLP recombinase. AB - The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae belongs to the integrase family whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. Ligation takes place when the 5'-OH generated during the cleavage step attacks the phosphotyrosine bond and reforms a phosphodiester bond. When the incoming 5' OH is from the partner duplex, strand exchange occurs. The FLP recognition target (FRT) contains two inverted 13-base pair (bp) FLP binding sequences that surround an 8-bp core region. It has been shown that heterology in the core regions of the recombinase FLP recognition target sites can dramatically impair recombination. Therefore, it was of interest to study the homology requirements of the core sequence for FLP-mediated ligation. Using nicked duplex substrates containing mismatches in the core sequence, we have demonstrated that the FLP ligation reaction can tolerate mismatches at all positions in the 8-bp core except the position immediately adjacent to the cleavage site. Using half-FRT substrates that contain a single-stranded core sequence, we showed that 4 base pairs adjacent to the cleavage site in the core are required for FLP to execute ligation with a single-stranded oligonucleotide. FLP is also able to ligate the protruding single strand on a half-FRT site to the opposite strand to form a hairpin. We have studied the effect of the base composition of the protruding 8 nucleotide single strand upon the efficiency of hairpin ligation. These studies revealed the importance of intrastrand complementarity in the formation of hairpin by FLP. Hence we conclude that the homology in the position adjacent to the cleavage site is most important, and the degree of the homology required is dependent on the nature of the ligation assay. PMID- 7538121 TI - A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells. AB - Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity. PMID- 7538122 TI - Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library. AB - The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine activated alpha 2-macroglobulin interaction sites. PMID- 7538125 TI - The asparagine-linked oligosaccharides of the human chorionic gonadotropin beta subunit facilitate correct disulfide bond pairing. AB - The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)-beta subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-beta genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of p beta 1 into p beta 2, the rate-determining step in beta folding, was 7 min for wild-type beta but 33 min for beta lacking both N-linked glycans. The p beta 1-->p beta 2 half-time was 7.5 min in CHO cells expressing the beta subunit missing the Asn13-linked glycan and 10 min for the beta subunit missing the Asn30-linked glycan. The inefficient folding of hCG-beta lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG-beta-folding pathway. Unglycosylated hCG-beta was slowly secreted from CHO cells, and beta subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG-beta core fragment-like protein. However, coexpression of the hCG-alpha gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG-beta lacking N linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG-beta and may be involved in the folding of this beta form. These data indicate that N linked oligosaccharides assist hCG-beta subunit folding by facilitating disulfide bond formation. PMID- 7538123 TI - Lipopolysaccharide core structures in Rhizobium etli and mutants deficient in O antigen. AB - Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp. has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host. Many genes required for O-chain polysaccharide synthesis are in the lps alpha region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis. The LPSs from several strains mutated in the alpha region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(2-->6) alpha-Galp-(1-->6)-[alpha-GalpA-(1-->4)]-alpha-Ma np-(1-->5)- Kdop (3-deoxy-D manno-2-octulosomic acid) (Kdo)pentasaccharide and a alpha-GalpA-(1-->4)-[alpha GalpA-(1-->5)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue. These results indicate that the lps alpha region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region. Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis. PMID- 7538124 TI - Identification and mutational analysis of the glycosylation sites of human keratin 18. AB - Keratin polypeptides 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in glandular epithelia. We previously showed that K8/18 phosphorylation occurs on serine residues and that K8/18 glycosylation consists of O-linked single N-acetylglucosamines (O-GlcNAc) that are linked to Ser/Thr. Since the function of these modifications is unknown, we sought as a first step to identify the precise modification sites and asked if they play a role in keratin filament assembly. For this, we generated a panel of K18 Ser and Thr-->Ala mutants at potential glycosylation sites followed by expression in a baculovirus-insect cell system. We identified the major glycosylation sites of K18 by comparing the tryptic 3H-glycopeptide pattern of the panel of mutant and wild type K18 expressed in the insect cells with the glycopeptides of K18 in human colonic cells. The identified sites occur on three serines in the head domain of K18. The precise modified residues in human cells were verified using Edman degradation and confirmed further by the lack of glycosylation of a K18 construct that was mutated at the molecularly identified sites then transfected into NIH-3T3 cells. Partial or total K18 glycosylation mutants transfected into mammalian cells manifested nondistinguishable filament assembly to cells transfected with wild type K8/18. Our results show that K18 glycosylation sites share some features with other already identified O-GlcNAc sites and may together help predict glycosylation sites of other intermediate filament proteins. PMID- 7538126 TI - Differential regulation of protein-tyrosine phosphatases by integrin alpha IIb beta 3 through cytoskeletal reorganization and tyrosine phosphorylation in human platelets. AB - The major platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the alpha IIb beta 3 dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking alpha IIb beta 3 were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein tyrosine phosphatases in these fractions and whole cell lysates. First, alpha IIb beta 3-dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by alpha IIb beta 3-mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine phosphorylated in an alpha IIb beta 3-mediated aggregation-dependent manner. Thus, integrin alpha IIb beta 3 was involved differentially in the regulation of PTP1B and PTP1C. PMID- 7538127 TI - Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator. AB - To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR. PMID- 7538128 TI - Topography of ligand-induced binding sites, including a novel cation-sensitive epitope (AP5) at the amino terminus, of the human integrin beta 3 subunit. AB - Changes in ligand binding ability of the integrin alpha IIb beta 3 can be monitored by the concomitant expression of ligand-inducible binding sites (LIBS). A new LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of beta 3 recognized by the murine monoclonal antibody (mAb) AP5, is sensitive both to the binding of ligand and to micromolar differences in divalent cation levels. Calcium or magnesium can completely inhibit the binding of AP5 to alpha IIb beta 3 on platelets, with ID50 values of 80 and 1500 microM, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the presence of 1 mM calcium plus 1 mM magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The unique location of the AP5 LIBS was determined by comparing the binding of LIBS-specific mAb to recombinant human-Xenopus beta 3 chimeras produced in a baculovirus expression system. AP5 defines one region at the amino terminus beta 3 1-6. A second region, defined by mAb D3GP3, is probably located within beta 3 422-490, confirming the finding of Kouns et al. (Kouns, W. C., Newman, P.J., Puckett, K. J., Miller, A. A., Wall, C. D., Fox, C. F., Seyer, J. M., and Jennings, L. K. (1991) Blood 78, 3215-3223). The third region, encompassing at most residues 490-690, and perhaps more precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2, anti-LIBS3, anti-LIBS6, and P41. Since its exposure is uniquely regulated by both divalent cations and ligand, the amino terminus of beta 3 may be involved in control of ligand binding by divalent cation mobilization. PMID- 7538129 TI - Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer. AB - A 161-base pair fragment (AB1) approximately 10 kilobase pairs upstream of the transcription start site of the mouse heme oxygenase-1 gene functions as a basal level and inducer-dependent enhancer. AB1/chloramphenicol acetyltransferase fusion genes stably transfected into mouse hepatoma (Hepa) cells or L929 fibroblasts were activated 7-8- or 17-22-fold, respectively, after treatment of the cells with either CdCl2 or heme. The AB1 fragment is composed largely of three tandem repeats containing two conserved core elements, A and B. Part of core element A (TCCGGAGCTGTG) resembles the consensus-binding site for transcription factor AP-4, whereas core element B (GCTGAGTCANGG) includes the consensus-binding site (TGAGTCA) for the AP-1 family of transcription factors. Nuclear proteins from Hepa cells did not bind to any of the core A elements, but bound to all three copies of the core B element. AB1 derivatives with one or two mutant AP-1-binding elements exhibited reduced but measurable inducer-dependent enhancer activity, but mutation of all three AP-1-binding sites abolished activation by CdCl2 and heme and also by mercury chloride, zinc chloride, H2O2, sodium arsenate, and 12-O-tetradecanoylphorbol-13-acetate. Pretreatment of stably transfected L929 cells with protein kinase C inhibitors, but not with tyrosine kinase inhibitors or N-acetylcysteine, abrogated 12-O-tetradecanoylphorbol-13 acetate-dependent activation of the AB1/chloramphenicol acetyltransferase fusion gene. Induction by H2O2 was unaffected by the kinase inhibitors, but completely abolished by N-acetylcysteine. Heme-dependent induction was not significantly affected by any of these chemicals. PMID- 7538130 TI - Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. AB - We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed. PMID- 7538131 TI - Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L selectin. AB - L-selectin, the leukocyte selectin, mediates the carbohydrate-dependent attachment of circulating leukocytes to endothelium, preceding emigration into tissues. It functions in inflammatory leukocyte trafficking and in lymphocyte homing to lymph nodes. From previous work, the binding of L-selectin to endothelial-associated glycoprotein ligands, GlyCAM-1 and CD34, requires oligosaccharide sialylation, sulfation, and probably fucosylation. We have recently identified a major capping group in GlyCAM-1 as 6' sulfated sialyl Lewis x, a novel structure which potentially satisfies all of these requirements. In the present study, we define the complete structure of beta-eliminated chains of GlyCAM-1 using metabolic radiolabeling, plant lectin binding, and glycosidase digestions in conjunction with high pH anion-exchange chromatography. The majority of the O-glycans in GlyCAM-1 contain the T-antigen, i.e. Gal beta 1- >3GalNAc, which is incorporated into the core-2 structure, i.e. Gal beta 1- >3[GlcNAc beta 1-->6]GalNAc or larger core structures with additional GlcNAc residues. The structures of two O-glycans, based on core-2, were determined to be: [sequence: see text] The implications of these structures and more complex O glycans for binding by L-selectin are discussed. PMID- 7538132 TI - An incomplete program of cellular tyrosine phosphorylations induced by kinase defective epidermal growth factor receptors. AB - Although signaling by the epidermal growth factor (EGF) receptor is thought to be dependent on receptor tyrosine kinase activity, it is clear that mitogen activated protein (MAP) kinase can be activated by receptors lacking kinase activity. Since analysis of the signaling pathways used by kinase-defective receptors could reveal otherwise masked capabilities, we examined in detail the tyrosine phosphorylations and enzymes of the MAP kinase pathway induced by kinase defective EGF receptors. Following EGF stimulation of B82L cells expressing a kinase-defective EGF receptor mutant (K721M), we found that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all activated, and SHC became prominently tyrosine-phosphorylated. By contrast, kinase-defective receptors failed to induce detectable phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or p91STAT1, all of which were robustly phosphorylated by wild-type receptors. These data demonstrate that kinase-defective receptors induce several protein tyrosine phosphorylations, but that these represent only a subset of those seen with wild type receptors. This suggests that kinase-defective receptors activate a heterologous tyrosine kinase with a specificity different from the EGF receptor. We found that kinase-defective receptors induced ErbB2/c-Neu enzymatic activation and ErbB2/c-Neu binding to SHC at a level even greater than that induced by wild type receptors. Thus, heterodimerization with and activation of endogenous ErbB2/c-Neu is a possible mechanism by which kinase-defective receptors stimulate the MAP kinase pathway. PMID- 7538133 TI - Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies. AB - Antibodies against the four isoforms of the human plasma membrane Ca(2+)-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so. The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested. PMID- 7538136 TI - Tachykinin NK1 and NK2 receptors mediate the non-cholinergic bronchospastic response to capsaicin and vagal stimulation in guinea-pigs. AB - 1. The antibronchospastic activity against acetylcholine, capsaicin, electrical vagal stimulation and the selective tachykinin agonists ([beta Ala8]NKA-(4-10) and [Sar9]SP sulfone) of a novel NK2 receptor antagonist, MEN10,627 and/or the known NK1 receptor antagonist (+/-)-CP96,345 was studied in anaesthetized guinea pigs. 2. MEN10,627 (0.1 mumol kg-1 i.v.) and (+/-)-CP96,345 (3 mumol kg-1 i.v.) selectively reduced the bronchospasm induced by NK2 and NK1 tachykinin receptor agonists, respectively, without affecting the other tachykinin receptor agonist- or acetylcholine-induced bronchospastic response. 3. MEN10,627 (0.1 mumol kg-1 i.v.), in a dose-dependent manner, reduced the non-cholinergic response induced by bilateral stimulation of the vagi or by intravenous capsaicin. 4. The administration of (+/-)-CP96,345 (3 mumol kg-1 i.v.) alone did not affect these responses but, when administered in association with the NK2 antagonist, (+/-) CP96,345, was able to potentiate its inhibitory effect. 5. It is concluded that both NK1 and NK2 receptors are involved in the non-cholinergic bronchoconstriction induced by capsaicin or by stimulation of the vagi, although the NK2 receptor contribution is prominent. PMID- 7538135 TI - Evidence for serotonin involvement in the NANC excitatory neurotransmission in the catfish intestine. AB - 1. Four putative neurotransmitters (serotonin, substance P, ATP (alpha-beta methylene-ATP), and vasoactive intestinal peptide, VIP) of the non-adrenergic non cholinergic (NANC) innervation were examined for their role in the NANC excitatory neurotransmission in channel catfish intestine after adrenergic and cholinergic blockade. 2. VIP at concentrations ranging from 10(-12)M to 10(-4)M did not produce either a relaxant or a contractile response in these segments. 3. Serotonin, substance P and alpha-beta-methyl-ATP produced contractile responses in a dose-dependent manner. Their EC50 values were 5 x 10(-7)M, 5 x 10(-9)M and 5 x 10(-9)M and 5 x 10(-6)M, respectively. 4. Electrical field stimulation of the intestinal segments produced a predominant excitatory response after complete blockade of adrenergic and cholinergic divisions, suggesting a predominant NANC excitatory innervation. 5. Three types of serotonin receptor antagonists, namely methiothepin (predominantly a 5-HT1 antagonist), ketanserin (a selective 5-HT2 antagonist), methysergide and cyproheptadine (predominantly 5-HT2 blockers) and metoclopramide (a selective 5-HT3 blocker) were tested for their effectiveness against serotonin and EFS-induced contractions. Methiothepin, methysergide, cyproheptadine and metoclopramide produced significant blockade of the response to serotonin, whereas only methiothepin and cyproheptadine produced blockade of EFS-induced response. 6. Three agents tested for substance P blockade, namely spantide, 4-11 fragment of substance P, and methysergide (also a serotonin blocker), did not produce significant inhibition of the response to either substance P or EFS. 7. Suramin at a dose that blocked the ED50 concentration of ATP did not produce a significant blockade of the response to EFS suggesting that ATP-involvement in the NANC-e neurotransmission is unlikely. 8. This study confirmed the involvement of serotonin in the expression of non-adrenergic non cholinergic excitatory response of the channel catfish intestine. PMID- 7538134 TI - Mutants of Escherichia coli initiator tRNA defective in initiation. Effects of overproduction of methionyl-tRNA transformylase and the initiation factors IF2 and IF3. AB - We describe the effects of overproduction of methionyl-tRNA transformylase and initiation factors IF2 and IF3 on the activity, in vivo, of initiator tRNA mutants defective at specific steps of the initiation process in protein synthesis. The activity of the U35A36/G72 and U35A36/G72G73 mutants, which are defective in formylation, was increased by overproduction of methionyl-tRNA transformylase. In contrast, the activity of the C30:G40/U35A36 mutant, which is formylated normally but is defective in binding to the ribosomal P site, was not increased. Overproduction of IF2 had a strong stimulatory effect on the activity of virtually all the mutants carrying the U35A36 anticodon sequence change, including the U35A36, U35A36/G72, U35A36/G72G73, and the C30:G40/U35A36 mutants. In cells overproducing IF2, the amount of protein made by translation of a mutant mRNA, which uses the U35A36 mutant initiator tRNA, is severalfold higher than that made by translation of a wild type mRNA. We discuss the possible implications of this result on overproduction of proteins and on the order of assembly of the 30 S ribosome.mRNA.fMet-tRNA initiation complex in Escherichia coli. Over-production of IF3 did not affect the initiator activity of any of the tRNA mutants studied. PMID- 7538137 TI - A pharmacological analysis of calcium channels involved in phasic and tonic responses of the guinea-pig ureter to high potassium. AB - 1. Previous studies have established a marked difference in sensitivity to organic calcium channel blockers of the phasic compared with tonic component of the contraction to potassium chloride (KCl) in the guinea-pig ureter. The mechanisms responsible for this difference have remained unsettled. In particular, the possible involvement of non-L-type calcium channels in contractility of the ureter has not been determined. In this study we have re addressed this problem and, to eliminate any possible contribution of sensory neuropeptides released by KCl from peripheral endings of afferent nerves, all experiments were performed in ureters pre-exposed to the sensory neurone blocking agent, capsaicin (10 microM for 15 min). 2. Increasing concentrations of KCl (10 160 mM) produced phasic and tonic contractions of the guinea-pig isolated ureter: the L-type calcium channel agonist, Bay K 8644 (1 microM), enhanced both components of the contraction to KCl. 3. Nifedipine (1 microM) abolished all responses to increasing concentrations of KCl after 60 min contact time; after a shorter incubation period (15 min), the phasic contractions to low KCl concentrations were still observed, while the tonic responses were abolished. 4. The effects of nifedipine (0.1 nM-1 microM) on the phasic and tonic components of the response to 80 nM KCl were assessed after 15-120 min contact time. Nifedipine was equipotent in inhibiting the tonic response at all times tested, while a marked time-dependency of inhibition toward phasic responses was observed. After 15 min contact time, nifedipine was 181 times more potent in inhibiting tonic than phasic response to KCl, while after 120 min contact time the difference between EC50 values was only 5.4 times. 5. Cadmium chloride (3-30 microM) was equi-effective in inhibiting the phasic and tonic responses to KCl while nickel chloride was ineffective at 10-fold higher concentrations. omega-Conotoxin (0.1 microM) and tetrodotoxin (0.3 microM) were ineffective. 6. The present findings indicate that L-type voltage-dependent calcium channels mediate both phasic and tonic components of the response of the guinea-pig ureter to KCl while neither T type nor N-type voltage-dependent calcium channels are involved. The marked time dependency of inhibitory action of nifedipine suggests that L-type voltage dependent calcium channels which are responsible for the generation of phasic contraction of the ureter are in a low affinity state for interaction with nifedipine. PMID- 7538138 TI - The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin. AB - The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton. PMID- 7538142 TI - Prenatal screening for mutations in the insulin receptor gene. How reliably does genotype predict phenotype? PMID- 7538141 TI - Molecular size-fractionation during endocytosis in macrophages. AB - The sorting of macromolecules within and between membranous organelles is often directed by information contained in protein primary or secondary structure. We show here that absent such structural information, macromolecules internalized by endocytosis in macrophages can be sorted by size. After endocytosis, small solute probes of fluid-phase pinocytosis were recycled to the extracellular medium more efficiently than large solutes. Using macropinosomes pulse labeled with fluorescent dextrans, we examined the ability of organelles to exchange solute contents. Dextran exchange was optimal between organelles of similar age, and small dextrans exchanged more efficiently than large dextrans. Efferent solute movement, from lysosomes or phagolysosomes toward the plasma membrane, occurred through the same endocytic vesicles as afferent movement, toward lysosomes and this movement was solute size dependent. Remarkably, uniform mixtures of different-sized dextrans delivered into lysosomes separated into distinct organelles containing only one dextran or the other. Thus, the dynamics of endosomes and lysosomes were sufficient to segregate macromolecules by size. This intracellular size fractionation could explain how, during antigen presentation, peptides generated by lysosomal proteases recycle selectively from lysosomes to endosomes for association with class II MHC molecules. PMID- 7538139 TI - Endothelial receptor tyrosine kinases involved in angiogenesis. PMID- 7538144 TI - New biological functions of prostate-specific antigen? PMID- 7538145 TI - Diagnostic controversy: the diagnosis of childhood growth hormone deficiency revisited. PMID- 7538143 TI - Two mutations in the insulin receptor gene of a patient with leprechaunism: application to prenatal diagnosis. AB - Leprechaunism is an autosomal recessive disorder caused by mutations in the insulin receptor gene and characterized by intrauterine and postnatal growth restriction, abnormal glucose homeostasis, and severe insulin-resistance. Here we report the biochemical and molecular characterization of a male patient, NZ, who died at 2 yr of age with this syndrome. 125I-Insulin binding to fibroblasts from the proband, his mother, father, and unaffected sister was reduced to 8, 53, 38, and 35% of controls, respectively. Analysis of the insulin receptor gene by polymerase chain reaction amplification using primers flanking each of the 22 exons and direct DNA sequencing identified 2 different mutations in the proband. The paternal mutation was an in-frame deletion of base pairs 1159-1161 in exon 3, which resulted in the loss of the codon for Asn-281. The maternal mutation was a G-->A transition in the first nucleotide of the splice-donor junction in intron 13. The maternal mutation activated a cryptic splice site 27 base pairs upstream in exon 13 and caused an in-frame deletion of amino acids 859-867 of the extracellular domain of the insulin receptor beta subunit. Identification of both mutations enabled prenatal diagnosis in 2 subsequent pregnancies. In the first pregnancy, DNA from cells cultured from chorionic villus (CV) biopsies carried both mutations in the insulin receptor gene. In the second pregnancy, DNA from the CV biopsy cells was negative for both mutations, indicating that the fetus was unaffected by leprechaunism. Insulin binding could not be used in prenatal diagnosis because cells cultured from some control CV biopsies failed to bind insulin. These data indicate that patient NZ with leprechaunism was a compound heterozygote for 2 novel mutations in the insulin receptor gene and that direct DNA sequencing enables prenatal diagnosis for this lethal disorder. PMID- 7538140 TI - A conserved epitope on a subset of SR proteins defines a larger family of Pre mRNA splicing factors. AB - The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors. PMID- 7538147 TI - Identification of nitric oxide synthase in human uterus. AB - The aim of this study was to investigate the presence of nitric oxide synthase (NOS) in human uterus. Tissues were obtained at operation from 10 women undergoing hysterectomy for benign disease. In-situ hybridization was used to determine the distribution of mRNA for NOS with a 483 bp digoxigenin-labelled antisense riboprobe. Localization of NOS was detected by (i) immunocytochemistry using a monoclonal antibody raised against bovine constitutive endothelial NOS, and (ii) NADPH diaphorase, which has been suggested to co-localize with brain NOS. Messenger RNA for NOS was detected in endometrium and myometrium from nine of 10 women, predominantly in endometrial glandular epithelium and stroma and myometrial blood vessels. NOS-like immunoreactivity was seen in endometrial stroma and myometrial blood vessels, whereas NADPH diaphorase activity was localized mainly to endometrial glandular epithelium and myometrial blood vessels. These studies suggest that different forms of constitutive NOS are present in human endometrium and myometrium, and that nitric oxide may play a role in the paracrine control of the uterine vascular bed. PMID- 7538148 TI - Maternal plasma concentrations of insulin-like growth factor binding protein-1 and placental protein 14 in multifetal pregnancies before and after fetal reduction. AB - The aim of this study was to investigate the changes in maternal plasma insulin like growth factor binding protein-1 (IGFBP-1) and placental protein 14 (PP14) in multifetal pregnancies before and after embryo reduction. Maternal plasma IGFBP-1 and PP14 were measured serially in three groups of pregnant women at 8-21 weeks gestation. Groups 1 and 2 were 12 singleton and 12 twin pregnancies achieved after in-vitro fertilization (IVF). Group 3 comprised 26 women with multifetal pregnancies undergoing embryo reduction to twins. In the IVF pregnancies maternal plasma IGFBP-1 and PP14 increased with gestation to reach a peak at 20 and 10 weeks respectively; the mean concentrations in twin pregnancies were significantly higher than in singletons. In multifetal pregnancies the mean plasma concentration of both proteins was similar to that of IVF twin pregnancies before reduction; after reduction, the values fell to less than those of twins. These findings suggest that the maximum secretory capacity of the endometrium is achieved with twin pregnancies. In multifetal pregnancies undergoing iatrogenic reduction to twins, total residual endometrial function was less than in twin conceptions. PMID- 7538146 TI - Insulin-like growth factors and their binding proteins in the term and preterm human fetus and neonate with normal and extremes of intrauterine growth. AB - Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and insulin are believed to be important in the regulation of fetal and neonatal growth. We previously reported that the profiles of IGFBPs in fetal cord serum (FCS) were dependent on the growth/metabolic status of the fetus. The goals of the current study were to examine the IGF system in FCS from term fetuses with normal growth, those with intrauterine growth retardation (IUGR), and those who were large for gestational age (LGA) and in FCS from normal weight preterm (25-37 weeks) and term fetuses in the neonatal period from the day of birth (day 0) until 7 days of age (day 7). Western ligand blotting (WLB) of term FCS revealed IGFBPs with mol wt of 43 and 38 kilodaltons (kDa; IGFBP-3), 34 kDa (IGFBP-2), 28 kDa (IGFBP-1 and glycosylated IGFBP-4), and 24 kDa (IGFBP-4). In IUGR FCS, there was a 50% decrease in IGFBP-3 detected by WLB, which was shown not to be due to an IGFBP-3 protease in IUGR sera. In LGA FCS, IGFBP-3 levels were elevated 2-fold by densitometric analysis of ligand blots. In normal term FCS, the following levels (+/- SE) were present: IGF-I, 76 +/- 16; IGF-II, 401 +/- 38; IGFBP-3, 700 +/- 112; IGFBP-1, 77 +/- 10 ng/mL; and insulin, 3.8 +/- 1.6 microU/mL. In IUGR FCS, IGF-I, IGF-II, and IGFBP-3 were significantly reduced, and IGFBP-1 was 7-fold higher than in FCS from normal weight fetuses. In LGA FCS, IGF-I, insulin, and IGFBP-3 were significantly increased, whereas IGFBP-1 was significantly decreased. During the neonatal period, IGF-I levels on day 0 were 4-fold higher in FCS from term (38-40 weeks) compared to preterm (25-31 weeks) newborns. FCS IGF-II levels did not change significantly on day 0 between 25-40 weeks gestation. In the first 7 days of postnatal life, IGF-I levels were unchanged in preterm newborns, whereas in term neonates, IGF-I levels decreased precipitously on day 1, remained low during the first 3 days of life, and returned to birth levels by the end of the first week. In contrast, IGF-II and IGFBP-3 levels did not significantly change during the first week of life in preterm or term newborns.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538149 TI - Malignant myoepithelioma (myoepithelial carcinoma) of the breast: a detailed cytokeratin study. AB - AIMS: To study the expression of a range of cytokeratins by malignant myoepithelioma of the breast. METHODS: Immunophenotyping was carried out using a panel of antibodies on paraffin wax embedded and frozen material using immunohistochemistry and double-labelled immunofluorescence. Electron microscopy was also performed. RESULTS: The tumour cells were positive for CAM 5.2, actin, vimentin, and cytokeratin 14 and negative for cytokeratins 18 and 19. Electron microscopy showed well formed desmosomes and hemidesmosomes together with pinocytic vesicles, plentiful rough endoplasmic reticulum and 6 nM microfilaments with focal densities. CONCLUSIONS: The pattern of cytokeratin expression provides further evidence that tumours with a specific myoepithelial phenotype occur rarely in the breast. PMID- 7538151 TI - "Pseudoclusters" and typing by random amplification of polymorphic DNA of Aspergillus fumigatus. AB - This short report serves as a warning to the unwary of possible "pseudoclusters" of infection with Aspergillus fumigatus as shown by the typing system, random amplification of polymorphic DNA (RAPD). This was demonstrated by typing 10 epidemiologically distinct isolates of A fumigatus using two different preparations of Taq DNA polymerase. One of the enzymes did not discriminate between the isolates, giving the false impression that a cluster of infection had occurred. Enzyme source is thus a key variable when using RAPD to distinguish between isolates of A fumigatus. PMID- 7538152 TI - Neurochemical coding of enteric neurons in the guinea pig stomach. AB - The aim of this study was to investigate the neurochemical coding of myenteric neurons in the guinea pig gastric corpus by using immunohistochemical methods. Antibodies and antisera against calbindin (CALB), calretinin (CALRET), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), dopamine beta hydroxylase (DBH), beta-endorphin (ENK), neuropeptide Y (NPY), neuron-specific enolase (NSE), nitric oxide synthase (NOS), protein gene product 9.5 (PGP), parvalbumin (PARV), serotonin (5-HT), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. Double- and triple-labeling studies revealed colocalization of certain transmitters and enabled the identification of distinct subpopulations of gastric enteric neurons. NPY/VIP/NOS/ENK were present in 28% of all neurons, whereas 11% had NPY/VIP/DBH/ChAT; NOS-only neurons made up 2% of the population. The combination SP/ChAT/ENK occurred in 21% of the population, whereas SP/ChAT/ENK/CALRET and SP/CHAT/SOM/ +/- CALRET was identified in 5% and 6% of all cells, respectively. 5-HT-containing neurons comprised 2% of all cells and could be further classified by the presence of additional antigens as 5-HT/SP/(ChAT) or 5-HT/VIP/(ChAT). Approximately 21% of all neurons contained only ChAT with no additional antigen present and are referred to as ChAT/-. Gastric myenteric ganglion cells were not immunoreactive for CALB, PARV, CGRP, or TH. The results of this study indicate that gastric myenteric neurons can be characterized on the basis of different chemical coding. Neurochemical coding of corpus myenteric neurons revealed some similarities and significant differences in comparison with other regions of the gut. These differences might reflect adaptation of enteric nerves according to regional specialization and the distinct functions of the proximal stomach as a gastric reservoir. PMID- 7538150 TI - Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis. AB - A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a rapid diagnosis is most important. The specificity of the test was 100%. These results compared favourably with an enzyme immunoassay. This latex agglutination test is a simple, rapid and highly specific method of diagnosing melioidosis, and will be particularly useful in areas with limited laboratory facilities. PMID- 7538153 TI - MobyEd: a mobile educator unit. AB - A mobile educator unit is a cost-effective teaching tool that can be easily implemented in an acute hospital setting to assist in teaching patients, visitors, and personnel about health and wellness. The educator unit cannot replace the face-to-face interactions between professional and patient or visitor, but it can supplement and make information more readily available than a stationary display. PMID- 7538154 TI - The shadow knows: adventures with the overhead projector. PMID- 7538155 TI - An exploratory study of research utilization by nurses in general medical and surgical wards. AB - An exploratory study into research utilization is described. Firstly an agreed definition of research utilization was arrived at through consultation with a range of nurses in both academia and clinical practice. Potential factors that may influence research utilization were identified through a review of current research on research utilization and through a period of fieldwork carried out on two hospital wards in Scotland. The findings indicate that research utilization appears to be a complicated issue and cannot be decontextualized or fractionated in order to lead to an understanding but must address multiple factors simultaneously. This paper refers to research that may be utilized in clinical nursing practice rather than in education or nursing management. The impact of research in clinical practice on education and nursing management is not discounted but the focus of this study is on the influence that research may have on the actual delivery of patient care and, in this sense, education and management developments are encompassed. PMID- 7538156 TI - Preclinical safety studies with recombinant human interleukin 6 (rhIL-6) in the primate Callithrix jacchus (marmoset): comparison with studies in rats. AB - The haemopoietic and immunostimulatory properties of recombinant human interleukin 6 (rhIL-6) might be used clinically in cancer patients. For the preclinical assessment of the safety of such a therapy, we chose the primate marmoset (Callithrix jacchus) and Wistar rats. Recombinant hIL-6 given to marmosets at doses of up to 1000 micrograms kg-1 day-1 over 4 and 9 weeks did not induce fever and was very well tolerated. Haematological alterations included a sustained two- to threefold increase of thrombocyte counts, peaking at 4 weeks, as well as an increase in neutrophils and basophils. The number of bone marrow megakaryocytes at 4 and 9 weeks was not increased, but the ploidy grade was augmented. An acute-phase protein response was observed within 24 h after the first IL-6 administration, which reached a maximum after 1 week. The acute-phase protein response was not accompanied by functional or morphological signs of hepatocellular damage. Increased immunoglobulin and soluble IL-2 receptor in the serum levels reflected systemic immunostimulation. Recombinant hIL-6 was also given to rats at 500 micrograms kg-1 day-1 s.c. for 4 weeks, where it induced a stimulation of thrombopoiesis associated with increased platelet counts within 1 week. Furthermore, rhIL-6-treated rats had signs of immunostimulation and increased acute-phase reactants in serum, as in marmosets. There was no evidence of renal glomerular or hepatic pathology.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538159 TI - Fluorescent liposomes as quantitative markers of phagocytosis by alveolar macrophages. AB - A new phagocytic assay based on liposome ingestion by alveolar macrophages (AMs) is described. Fluorescent microspheres were encapsulated in liposomes, which allowed rapid enumeration by fluorometry. Liposomes made in the presence of vitronectin had the protein exposed on their outer surfaces, as determined by immunolabelling. Liposomes and rat AMs were incubated under conditions favorable for phagocytosis. Observation by light and electron microscopy showed AMs engulfing liposomes, with gradual transfer of fluorescent label from liposome to cell interior. This transfer was due to bona fide phagocytosis, as evidenced by (1) fluorescence of liposome-encapsulated dihydrofluorescein (DHF)-zymosan exclusively within AMs and (2) triggering of the respiratory burst by zymosan associated liposomes only under conditions that allowed phagocytosis. Phagocytic activity was expressed as liposomes/cell, the average number of liposomes phagocytosed per macrophage. We used this technique to follow phagocytosis over time and to measure the effects of lipopolysaccharide (LPS) and vitronectin on AM phagocytosis. PMID- 7538158 TI - Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes. AB - This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules. PMID- 7538157 TI - Expression of adhesion molecule ICAM-1 (CD54) in thyroid papillary adenocarcinoma. AB - The expression of adhesion molecules in thyroid specimens from 10 cases of papillary adenocarcinoma, 5 cases of follicular adenoma and 3 normal thyroid specimens was examined by an immunohistochemical method. Thyroid epithelial cells from all cases of papillary adenocarcinoma expressed the intercellular adhesion molecule 1 (ICAM-1, CD54). The ICAM-1-positive staining in these was detected predominantly on the apical site of malignant thyroid epithelial cells. However, no ICAM-1 expression was detected on thyrocytes of adenoma, and normal thyroid tissues. Furthermore, thyroid epithelial cells in patients with thyroid tumor and normal thyroid tissue did not react with anti-LFA-1, anti-VLA-4, anti-VCAM-1 and anti-ELAM-1 monoclonal antibodies. It is speculated that ICAM-1 expression in thyroid papillary adenocarcinoma may have a functional significance. PMID- 7538162 TI - Identification of tumour-specific translocations in archival material. AB - A reverse transcriptase nested polymerase reaction (RT-nPCR) method has been developed to detect the presence of a tumour-specific translocation in archival formalin-fixed, paraffin-embedded tissue. This technique can be applied to the large numbers of tumours in pathology departments' archives, allowing correlation of translocations with morphological diagnosis, response to therapy, and survival. PMID- 7538160 TI - [Evaluation of three kinds of assays for the presence of antibody to hepatitis C virus (anti-HCV) and the association of them with HCV RNA]. AB - In order to evaluate the feasibility for the detection of antibody to hepatitis C (anti-HCV), the first generation assay (c100-3 Ab) and two second generation assays (2nd EIA and 2nd PHA) were used to test 477 individuals who visited the medical hospital or clinics in Iki Island, Nagasaki Prefecture. HCV RNA, antibody titer by 2nd PHA and four kinds of antibody to epitope of HCV by RIBA II were also surveyed to determine their association with these three assays. Prevalence of anti-HCV was 26.6% by c100-3, 38.8% by 2nd PHA and 39.6% by 2nd EIA, indications that the 2nd generation assays are much more sensitive than c100-3. Prevalence of HCV RNA was 82.1% among 190, anti-HCV positive individuals; 100% among 52 individuals with liver disease, but only 75.4% in those without liver disease. HCV antibody titer, over 2(11) was higher among those who were positive for HCV RNA than those negative for HCV RNA. Four antibodies by RIBA II were all positive and reacted strongly when they were positive for HCV RNA, but only antibody to core antigen was observed among those negative for HCV RNA, suggesting that only antibody to core antigen remains in those with past HCV infection. PMID- 7538163 TI - Upregulation of integrin expression in benign vulvar warts. AB - Benign anogenital warts demonstrate features both of hyperproliferation and of aberrant differentiation associated with human papillomavirus (HPV) infection. The expression of integrins has been examined in normal vulvar epithelium and benign vulvar warts to determine whether HPV infection is associated with changes in integrin expression, either during terminal differentiation or among the proliferating cell population. The expression of integrins in normal vulvar epithelium was similar to that seen in the epidermis, with little expression of alpha 1 and expression of alpha 2, alpha 3, and beta 1 at the periphery of basal cells. In vulvar wart epithelium, alpha 1 was not significantly expressed, but upregulation of alpha 2, alpha 3, and beta 1 was seen at the periphery of cells throughout the basal and spinous layers. In addition, alpha 2 staining was greater at foci along the basal layer of normal vulva, whilst such heterogeneity was not detected in wart sections. Expression of beta 4 and alpha V was confined to the basal and lateral aspects of normal basal cells, but expressed at the periphery of basal cells and to a lesser extent in epibasal and spinous layers of warts. The observed differences in integrin expression are consistent with those reported in other hyperproliferative epithelia. PMID- 7538164 TI - Immunohistochemical analysis of adhesion molecules in the micro-environment of portal tracts in relation to aberrant expression of PDC-E2 and HLA-DR on the bile ducts in primary biliary cirrhosis. AB - We have examined immunohistochemically the expression of adhesion molecules in the micro-environment of portal tracts and their relationship to the expression of the pyruvate dehydrogenase E2 complex (PDC-E2) and HLA-DR in liver biopsy specimens. Ten cases of primary biliary cirrhosis (PBC) and 19 controls were examined, including four cases of extrahepatic biliary obstruction, six of chronic viral hepatitis, and nine normal livers. In PBC, the damaged small bile ducts demonstrated an increased expression of PDC-E2 and an aberrant expression of HLA-DR; about half of these damaged bile ducts also expressed intercellular adhesion molecules (ICAM)-1 and a few expressed vascular adhesion molecule (VCAM) 1. In addition, lymphocyte function-associated antigen (LFA)-1 and very late antigen (VLA)-4 were expressed on infiltrating lymphocytes around these bile ducts. In contrast, in control livers, these alterations in antigen expression on the bile ducts were either not observed or were only focal and weak, when present. These findings suggest that ICAM-1/LFA-1 and also VCAM-1/VLA-4 linkages between the damaged bile ducts and lymphocytes may facilitate antigen-specific reactions such as the presentation of antigens, possibly PDC-E2, to the periductal lymphocytes in PBC. ICAM-1, VCAM-1, and E-selectin were strongly expressed on the endothelial cells of some vessels in the portal tracts in PBC, suggesting the facilitation of the recruitment of lymphocytes around the bile ducts of PBC. VCAM-1, a member of the immunoglobulin superfamily, has not hitherto been reported on bile ducts. PMID- 7538165 TI - The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells. AB - Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mumol/liter) affects intracellular pH (pHi), Cl- (Cl-i) or cell volume of MDCK cells acutely exposed to normal (pHnorm = 7.4) and alkaline (pHalk = 7.7) conditions. At pHnorm, OTA increased Cl-i by 2.6 +/- 0.4 mmol/liter (n = 14, P < 0.05) but had no effect on pHi. At pHalk, application of OTA increased Cl i by 8.6 +/- 2.6 mmol/liter (n = 10, P < 0.05) and raised pHi by 0.11 +/- 0.03 (n = 8, P < 0.05). The Cl-/HCO3- exchange inhibitor DNDS (4,4'-dinitro-stilbene-2,2' disulfonate; 10 mumol/liter) eliminated the OTA-induced changes of pHi and Cl-i. OTA did not affect cell volume under both pHnorm and pHalk conditions. We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl-i without changing cell volume. The driving force of plasma membrane Cl-/HCO3- exchange dissipates, leading to a rise of pHi when cells are exposed to an acute alkaline load. Thus, OTA interferes with pHi and Cl-i homeostasis leading to morphological and functional alterations in MDCK cells. PMID- 7538167 TI - The biological reality of the interlacunar network in the embryonic, cartilaginous, skeleton: a thiazine dye/absolute ethanol/LR White resin protocol for visualizing the network with minimal tissue shrinkage. AB - Third toe phalanges of chicks aged 8-13 days in ovo and 7-day post-natal rat femoral growth plate were examined to determine whether the interlacunar network (IN), a structure with no lipoprotein membrane component or cytoplasmic organelles, is a genuine component of young growth cartilage. In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar network by toluidine blue and red staining by picro Sirius red indicate the presence of glycosaminoglycans and collagen. The network in phalanges dehydrated by 80% (v/v) ethanol appears little different; however, the network is much less widely detectable in phalanges dehydrated by 90% (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is permeated by a thiazine dye such as toluidine blue, using a solution of dye in the aldehyde fixative, the network is widely detectable, following dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to which whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by the extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrinkage areas are 33% or 35% of areas measured in buffer for 70% (v/v) ethanol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The network is thus present in radically shrunk tissue, but, significantly, is also fully represented in tissue shrunk by only a conventional margin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested. PMID- 7538166 TI - Inhibition of Mg2+ current by single-gene mutation in Paramecium. AB - "Eccentric" is a newly-isolated mutant of Paramecium tetraurelia that fails to swim backwards in response to Mg2+. In the wild type, this backward swimming results from Mg2+ influx via a Mg(2+)-specific ion conductance (IMg). Voltage clamp analysis confirmed that, as suspected, step changes in membrane potential over a physiological range fail to elicit IMg from eccentric. Further electrophysiological investigation revealed a number of additional ion-current defects in eccentric: (i) The Ca2+ current activated upon depolarization inactivates more slowly in eccentric than in the wild type, and it requires longer to recover from this inactivation. (ii) The Ca(2+)-dependent Na+ current deactivates significantly faster in the mutant. (iii) The two K+ currents observed upon hyperpolarization are reduced by > 60% in eccentric. It is difficult to envision how these varied pleiotropic effects could result from loss of a single ion current. Rather, they suggest that the eccentric mutation affects a global regulatory system. Two plausible hypotheses are discussed. PMID- 7538168 TI - AMPA-selective glutamate receptor subunits in the rat hippocampus during aging. AB - The levels of mRNAs for the subunits of alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, -2, -3, -4) in the rat hippocampus during aging were measured by Northern blotting. The distribution of these receptors was also examined at the protein level by immunoblotting using antibodies to GluR-1 and to an epitope common to GluR-2 and GluR-3 (denoted GluR-2/3). During aging a significant decrease of GluR-1 protein, but no change in the corresponding mRNA level, was observed. No differences in the level of GluR-2/3 protein and GluR-2, -3, and -4 mRNAs at the various ages examined (4, 12, and 24 months) were detected. Our results show that AMPA receptors are only slightly influenced by the aging process in the rat hippocampus. The slight decrease in GluR-1 protein content, not accompanied by a parallel decrease in the GluR-1 mRNA level, might be explained by a decreased translational efficiency or an increased protein degradation of the GluR-1 subunit. PMID- 7538161 TI - Prognostic significance of proteolytic enzymes in human brain tumors. AB - Proteases and their inhibitors have been shown to play roles in tumor invasion and metastasis in a number of experimental models. Recently, relative increases in the amounts of urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in tumor samples have been correlated with poorer, pathological grade, shorter disease-free interval, and shorter survival. To date, all studies investigating the prognostic significance of proteases and their inhibitors have been limited to extracranial cancer. In this article, we review the literature and present our data on the prognostic significance of proteases in human brain tumors. High levels of uPA were seen in malignant glioma and metastatic tumors (n = 82), whereas normal levels of uPA were found in low-grade gliomas. Analysis with magnetic resonance imaging (MRI) demonstrated a significant correlation between high levels of uPA and necrosis and edema (n = 50; P < 0.05). Similarly, patients with high levels of uPA had shorter survival than did patients with low levels of uPA. Tissue-type plasminogen activator (tPA), which was virtually absent in glioblastoma multiforme (GBM), colon lung, and breast metastasis, was found in normal quantities in anaplastic astrocytoma (AA), low-grade glioma (LGG), and meningioma. Melanoma had significantly more tPA activity than normal brain did. A reverse correlation was found between tPA and MRI findings of necrosis, enhancement, and edema. Similarly, patients with no detectable tPA activity had shorter survival than did patients with detectable tPA activity. We conclude that high levels of uPA and absent tPA activity correlate with histologically malignant brain tumors, aggressive characteristics, and shorter survival. PMID- 7538169 TI - Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P. AB - This study examined the sciatic nerve axonal transport of substance P-like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8- and 12-week alloxan-diabetic rats, respectively. One group of diabetic rats received acetyl-l-carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced boy growth. Axonal transport of SPLI was studied by measurement of 24-hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8-week diabetic rats. In the sciatic nerve of ALCAR-treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12-week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerves showed the typical sorbitol increase and myo-inositol loss that were significantly counteracted by ALCAR. This study suggests that ALCAR treatment prevents diabetes-induced sensory neuropathy by improving altered metabolic pathways such as polyol activity and myo-inositol synthesis, and by preventing the reduction of synthesis and axonal transport of substance P. PMID- 7538171 TI - Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120. AB - Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development. PMID- 7538172 TI - Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection. AB - Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection. PMID- 7538170 TI - Resistance of nitrogen metabolism to growth hormone treatment in the early phase after injury of patients with multiple injuries. AB - OBJECTIVES AND DESIGN: Several studies have shown an anticatabolic effect of recombinant human growth hormone (rhGH) in surgical patients. We investigated, in a prospective, randomized, double blind, and placebo-controlled study, the effect of r-hGH on hormone and nitrogen metabolism in 14 patients with multiple injuries in the early phase of injury. MATERIALS AND METHODS: All patients were treated in the intensive care unit, had mechanical ventilation, and were highly catabolic, with a mean daily nitrogen loss of 13.2 +/- 1.8 g. r-hGH was given subcutaneously (once a day, at 8 PM) in a dosage of 0.2 IU/kg.d for seven days, starting on the second day after injury. RESULTS: Administration of r-hGH evoked a significant increase in plasma concentrations of GH, insulin-like growth factor-I (IGF-I), and insulin-like growth factor binding-protein-3 (IGFBP-3). No significant differences were found for either daily or cumulative nitrogen balances (-103.1 +/- 14 g for patients receiving r-hGH and -92.1 +/- 18.1 for those with placebo). r-hGH therapy did not affect skeletal muscle extracellular water, nor did it affect plasma or muscle concentrations of total free amino acids or glutamine. Plasma albumin, prealbumin, and retinol-binding protein concentrations were also unchanged by r-hGH therapy, as were the urinary excretion of potassium and urea. CONCLUSIONS: We conclude that elevated plasma levels of GH, insulin, and IGF-I are unable to effect a protein anabolic drive in patients with multiple injuries during the early postinjury phase and assume that this r-hGH resistance to nitrogen metabolism takes place at the level distal to IGF-I. PMID- 7538174 TI - Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A. AB - Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex. PMID- 7538175 TI - Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A. AB - Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells. PMID- 7538173 TI - A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. AB - The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins. PMID- 7538176 TI - A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion. AB - We have previously reported that synthetic peptides representing the leucine zipper domain (DP107) and a second putative helical domain (DP178) of human immunodeficiency virus type 1 (HIV-1) gp41 exhibit potent anti-HIV activity. In this study we have used soluble recombinant forms of gp41 to provide evidence that the DP178 peptide and the DP178 region of gp41 associate with a distal site on the gp41 transmembrane protein whose interactive structure is influenced by the leucine zipper (DP107) motif. We also observed that a single coiled-coil disrupting mutation in the leucine zipper domain transformed the recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion and infectivity, which may be related to that finding. We speculate that this transformation results from liberation of the potent DP178-related sequence from a molecular clasp with a leucine zipper, DP107, determinant. The results are discussed in the context of two distinct conformations for the gp41 molecule and possible involvement of these two domains in structural transitions associated with HIV-1 mediated fusion. The results are also interpreted to suggest that the anti-HIV activity of the various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic processes. PMID- 7538177 TI - Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment. AB - Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication. PMID- 7538179 TI - [Effect of a combination treatment using imipenem/cilastatin sodium with G-CSF on infections in neutropenic patients with hematological malignancies]. AB - The clinical effectiveness of a combination treatment using imipenem/cilastatin sodium (IPM/CS) with G-CSF was studied in neutropenic patients (< 500/mm3) with hematological malignancies and secondary infections. Thirty seven patients were entered in the trial, and 30 patients were eligible. This combination was effective in 20 patients, thus the overall efficacy rate was 66.7 percent. The combination was effective in all 6 cases with septicemia, in 10 case out of 15 cases with fever after chemotherapy (efficacy rate; 66.7%), in 3 out of 8 cases with respiratory infections including 7 cases with pneumonia (efficacy rate; 37.5%), and a case with laryngopharyngitis. According to the order of the administration, the efficacy rates were 60.0% in 5 cases in whom G-CSF treatment was started before IPM/CS, 66.7% in 21 cases given both G-CSF and IPM/CS simultaneously, and 75.0% in 4 cases in whom IPM/CS was started before G-CSF. The difference was statistically not significant on the efficacy rates in the three groups. The efficacy in 18 cases treated with monotherapy on antibiotic was 72.2% and that in 12 cases treated with IPM/CS in combination with other antibiotics was 58.3%, and the difference in the efficacy rates in these two groups was not statistically significant. According to the neutrophil counts before and after the treatment, high response rate (60.0%) was obtained in cases of severe neutropenia (less than 100/mm3). Bacteriological examinations showed that all of bacteria detected as pathogens (10 strains of Gram-positive bacteria and 6 strains of Gram-negative bacteria) were eradicated, though 3 strains were replaced by other pathogens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538178 TI - Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies. AB - Reverse transcription of retroviral genomic RNA in a target cell is influenced by cellular factors, including the concentration of deoxyribonucleoside triphosphates (dNTPs). In addition, recent data have demonstrated that reverse transcription can be driven within human immunodeficiency virus type 1 virions, prior to infection of a cell, by increasing extracellular concentrations of dNTPs. In attempts to increase the transduction efficiency of recombinant murine leukemia virus vectors, endogenous reverse transcription was initiated within cell-free, recombinant murine leukemia virus virions in the presence of relatively high concentrations of dNTPs. As a result, the expression of transduced genes via these retroviral vectors was increased approximately 10-fold by treatment of virions with dNTPs. Combined with our previous data, these observations suggest that virion-associated DNA synthesis can occur in diverse groups of retroviruses and positively alter retroviral infectivity. As such, these manipulations may be useful for increasing the efficiency of retrovirus mediated gene delivery. PMID- 7538180 TI - [Clinicopathological studies of anti-HCV P1P4 core antibody]. AB - Anti-P1P4 core antibody, derived from a Japanese hepatitis C virus clone, was evaluated clinicopathologically in serum samples from 40 blood donors positive for anti-HCV antibody by 2nd generation assay and in 37 patients with HCV chronic hepatitis treated with interferon. The presence of anti-P1P4 antibody was highly correlated with the presence of HCV-RNA in the blood donors. In the patients with chronic hepatitis, more than a 50% reduction in P1P4 antibody titer after interferon therapy suggested the disappearance of HCV-RNA from the blood. Thus, anti-P1P4 antibody was useful in evaluating the virological effects of interferon therapy. However, clinically and pathologically, the titer of P1P4 antibody did not indicate the grade of liver inflammation. PMID- 7538181 TI - Enhanced expression of CD34 messenger RNA by developing endothelial cells of mice. AB - BACKGROUND: Immunohistochemical studies have demonstrated that CD34 antigen is present on the surface of vascular endothelial cells and hematopoietic cells. Roles of CD34 for angiogenesis and its function as a homophilic adhesion molecule between endothelial and hematopoietic cells have been speculated. EXPERIMENTAL DESIGN: Northern blotting was used to examine the expression levels of CD34 mRNA, and in situ hybridization was used to localize CD34 mRNA. First, we investigated changes in CD34 mRNA expression in developmental processes of mice. Second, we investigated the changes in skin tissues of adult mice in the process of wound healing and tumor growth. RESULTS: CD34 mRNA was strongly expressed by most of the vascular endothelial cells in developing organs. The magnitude of the expression decreased after birth but increased again in the process of wound healing and tumor growth. Although CD34 mRNA signals were observed in hematopoietic cells in the yolk sac and fetal liver, the endothelial cells of these tissues did not express CD34 mRNA signals. CD34 mRNA signals were detectable in neither hematopoietic nor endothelial cells of the bone marrow. In tissues other than hematopoietic ones, however, blood cells within the vessels did not express CD34 mRNA, although the endothelial cells expressed CD34 mRNA. CONCLUSIONS: The expression pattern of CD34 mRNA suggested its significant role in development of blood vessels not only in embryos but also in adults. Although CD34 mRNA was expressed both by endothelial cells and hematopoietic cells, the expression did not occur within the same organ, suggesting that the CD34 molecule may not be used as a homophilic adhesion molecule between endothelial and hematopoietic cells. PMID- 7538183 TI - Enhanced expression of decay accelerating factor and CD59/homologous restriction factor 20 on the colonic epithelium of ulcerative colitis. AB - BACKGROUND: The complement system is thought to be one of the factors involved in damaging the colonic mucosa in the pathogenesis of ulcerative colitis (UC). Several membrane-bound factors that regulate complement activation have been identified. EXPERIMENTAL DESIGN: To elucidate alteration(s) of the complement regulatory proteins and to add to the understanding of the role of the complement related immune responses in the pathogenesis of UC, we immunohistochemically examined the distribution of decay accelerating factor (DAF), CD59/homologous restriction factor 20 (HRF20), and membrane cofactor protein (MCP) in colonic mucosa with UC and compared it with that in normal and inflammatory control mucosae. RESULTS: In normal colonic epithelia, the cell surface distribution of DAF and CD59/HRF20 was confined to the apical domain, whereas MCP was present on the basolateral surface. Although MCP expression in active UC was not significantly different from normal mucosa, DAF and CD59/HRF20 expression on epithelial cells of UC was markedly enhanced in relation to the severity of mucosal inflammation. In the colonic epithelia of active UC, DAF and CD59/HRF20 were not only overexpressed on the apical surface but also were distributed to the basolateral membrane. The altered cell surface distribution of these molecules was also confirmed by immunoelectron microscopy. The enhanced expression of DAF and CD59/HRF20 was not specific to UC but was observed in colonic mucosa of inflammatory controls, such as ischemic colitis. CONCLUSIONS: Our results indicate that altered regulation of complement activation is present in UC mucosa, but whether it may play a causal role in the immunologic disorders leading to UC remains to be elucidated. PMID- 7538182 TI - Transmission of a chronic lymphoproliferative syndrome in ferrets. AB - BACKGROUND: Lymphomas and leukemias are caused by transmissible viruses in a wide variety of species, including humans, cattle, and cats. Features of lymphoma in ferrets suggest that it, too, may have an infectious etiology. No agent has been identified. EXPERIMENTAL DESIGN: Cells or cell-free inocula from a ferret with spontaneous malignant lymphoma were administered i.p. to six recipient ferrets. Two ferrets received fresh cells, two received frozen cells, and two received cell-free culture supernatant. The recipients were monitored routinely clinically and hematologically, and lymphoma was confirmed histologically. The lymphomas were characterized using cytology, cytochemistry, immunophenotyping, and histology. Cultivated cells from the donor and recipients were examined using reverse transcriptase assay, microscopy, and electron microscopy. RESULTS: All of the six recipient ferrets developed mild sustained lymphocytosis within 6 weeks of the inoculation. Two of six were euthanized 14 to 18 months after inoculation. Lymphoma was later diagnosed in three of the four remaining ferrets at 24 to 36 months after inoculation. All developed a chronic indolent syndrome featuring profound splenomegaly, lymphocytosis with atypia, and histologically polymorphous lymphoma. Two of the three who developed lymphoma had received fresh donor lymphoma cells, and the third had received supernatant from donor cell cultures with elevated reverse transcriptase activity. Cultivated cells from the affected ferrets demonstrated reverse transcriptase activity and retrovirus-like particles. CONCLUSIONS: This study demonstrates horizontal transmission of malignant lymphoma in ferrets using cell or cell-free inocula. Clinical and pathologic features of this syndrome in ferrets resembled virally induced lymphomas in other species. PMID- 7538186 TI - Postoperative selective bowel decontamination prevents gram-negative bacterial translocation in small-bowel graft recipients. AB - Gram-negative septic episodes are a potential risk of small-bowel transplantation; bacterial translocation through the graft is considered the mechanism. As a measure to prevent this complication, we evaluated postoperative selective bowel decontamination (SBD) in the rat model of orthotopic small-bowel transplantation [Lewis (LEW) and Brown-Norway (BN) rats as donors and recipients]. For 4 days after transplantation we gave FK 506, 2 mg/kg, which prevents rejection and results in indefinite recipient survival. For SBD, 24 mg/kg/day polymyxin E and 20 mg/kg/day tobramycin were administered via orogastric gavage to allograft recipients, both with and without FK 506 therapy. On Day 9, all rats were sacrificed, the peritoneal cavity was swabbed, and mesenteric lymph nodes (MLN), spleen, liver, and ileum were harvested for microbial qualitative and quantitative analysis. Animals with positive peritoneal swab cultures were excluded. SBD resulted in a significant reduction of the quantitative gram-negative bacterial flora in the ileum and cecum and of bacterial translocation to the MLN [0% versus 50% (no FK 506 therapy) and 8% versus 50% (FK 506 treated)]. In the allograft groups not treated with FK 506, SBD failed to significantly prolong survival, suggesting that acute rejection is not hastened by infection (bacterial translocation). We conclude that SBD in small-bowel-graft recipients prevents bacterial translocation by reducing intestinal gram-negative bacterial flora; this may reduce local and systemic infections by gut-derived organisms. PMID- 7538185 TI - Assessment of differential cytokine effects on angiogenesis using an in vivo model of cutaneous wound repair. AB - Angiogenesis, or new blood vessel formation, has been a subject of intense investigation in recent years. A major obstacle in this research has been the selection of an appropriate in vivo model with which comparisons to humans can be made as well as a reliable quantitative method. Using the porcine excisional wound healing model, we report a new and simple technique for obtaining objective assessments of the microvascular compartment. Factor VIII immunostaining of histological specimens was utilized for specific identification of endothelium devoid of background interference. This technique was coupled with morphometric analysis to quantitate the differential effects of tumor necrosis factor alpha (TNF alpha), transforming growth factor beta (TGF beta), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF) within healing porcine wounds. All cytokines stimulated angiogenesis, with low dose TNF alpha and bFGF treatments exhibiting the most profound effects at 7 days postwounding. With increasing levels of TNF alpha (1 ng, 10 ng, 100 ng, and 2.5 micrograms), a step-wise decrease in microvascular area was noted. Although no significant dose responsive differences in angiogenesis were noted following bFGF treatments, a profound increase in capillary area was shown. Significant yet less dramatic increases were noted in capillary area following treatment with EGF or IGF-1. Comparison of the angiogenic effects of TGF beta at 7 and 10 days postwounding showed a significant decrease in the microvasculature as wounds matured. Our data are consistent with previous qualitative in vitro and in vivo reports, thereby confirming the validity of this new model. The data furthermore provide the first quantitative evidence of differential angiogenic responses to cytokines within a clinically relevant model of cutaneous wound repair. PMID- 7538184 TI - Correlation of 133Xe clearance, blood flow and histology in the rat sponge model for angiogenesis. Further studies with angiogenic modifiers. AB - BACKGROUND: We have previously described a method of quantitating angiogenesis by using a simple 133Xe clearance technique for repeated measurement of relative blood flow changes through s.c. sponge implants over a period of 14 days. The quantitative requirement of this bioassay is that the measurements of 6-minute 133Xe clearance should provide a fast and reliable means to detect relative blood flow changes in the neovasculature, so a more vigorous validation of the use of the 133Xe clearance technique as an indicator of angiogenesis is needed. EXPERIMENTAL DESIGN: Four different techniques were used: (a) to measure absolute blood flow in the sponges using 113Sn microspheres; (b) to quantitate the levels of hemoglobin and total protein in the implants; (c) to determine the amount of neovasculature in the sponges by the carmine dye method; and (d) to carry out histologic and morphometric analysis of sponge implants. To confirm parallel changes in 133Xe clearance and in the other techniques, the effects of selected angiogenic promoters and inhibitors were also investigated. RESULTS: There was a good correlation between 133Xe clearance from the sponges and absolute blood flow (r = 0.952, p < 0.01); the levels of hemoglobin (r = 0.982, p < 0.01) and total protein (r = 0.962, p < 0.01); the amount of carmine dye (r = 0.974, p < 0.01); the fibrovascular growth areas (r = 0.992, p < 0.01); and the vascular density (r = 0.997, p < 0.01) in the implants. Daily administration of 3 pmol of IL-1 alpha or IL-8 caused intense neovascularization. When given alone, lower doses of IL-1 alpha (0.3 pmol) or bradykinin (10 pmol) produced no apparent effect. However, co administration of these doses to a single sponge together caused an increase in the rate of angiogenesis similar to that seen with a higher dose of IL-1 alpha (3 pmol) acting alone. In contrast, daily co-administration of a potent and selective protein kinase C inhibitor, calphostin C (4 micrograms), inhibited the neovascular response elicited by 3 pmol of IL-1 alpha. Furthermore, daily doses of 5 micrograms of dexamethasone for 14 days inhibited sponge-induced angiogenesis. CONCLUSIONS: The results clearly show that the 133Xe clearance technique not only gives an indication of the rate of perfusion of the sponges with blood but also gives a good estimate of its functional vascularity. Thus, the measurement of 133Xe clearance in the sponge implant provides a simple and objective method for routine studies of modifiers of angiogenesis. PMID- 7538187 TI - PSA-detected prostate cancer: contrasts with palpable disease. AB - The clinical and pathologic characteristics of 100 consecutive men with clinically localized prostate cancer and staged by radical prostatectomy were reviewed. Men with impalpable prostate-specific antigen (PSA) detected cancers (T1c) were contrasted against men with palpable disease (TB). Lesions were clinically staged as T1c in 53 men and TB in 47 men. Mean serum PSA for men with T1c cancers was 11.8 +/- 0.7 ng/dL (normal: 0 > 4) and 14.1 +/- 1.7 ng/dL for men with TB disease. Histologic evaluation revealed a mean Gleason's sum of 6.4 +/- 0.2 (scale: 2 > 10) for T1c lesions and 6.6 +/- 0.2 for men with TB cancers (P = NS). DNA content was diploid in 67% of T1c cancers and 62% of TB lesions (P = NS). An overall 43% clinical staging error was observed. Extraprostatic extension was demonstrated in 36% of T1c cancers and 51% TB lesions (P = NS). PSA-detected cancers were indistinguishable from palpable lesions by all measures evaluated. Prostate cancers detected in asymptomatic men by an elevated PSA should be considered biologically significant lesions. PMID- 7538188 TI - [Paradoxical myelosuppression with granulocyte-stimulating factor (G-CSF) and simultaneous administration chemotherapy]. PMID- 7538189 TI - Rat serum albumin modified by diflunisal acyl glucuronide is immunogenic in rats. AB - Acyl glucuronide metabolites of carboxylic acid drugs such as the salicylate derivative diflunisal (DF) have been shown to react with proteins in vitro and in vivo to produce covalent adducts. Such attachment of foreign compounds to endogenous molecules could be associated with toxic and/or immune consequences. In this study we have injected rats with rat serum albumin (RSA) modified (a) by DF using a carbodiimide reagent (-->DF-RSA-I, 4.9 micrograms DF/mg RSA) and (b) by incubation with DF acyl glucuronide (DAG) and its rearrangement isomers (iso DAG) (-->DF-RSA-II, 0.34 micrograms DF/mg RSA). All of the six rats injected with DF-RSA-I produced antibodies reactive with DF-modified keyhole limpet hemocyanin (KLH), the coating protein used in the ELISA. Three out of six animals injected with DF-RSA-II generated similar antibodies. Cross-reactivity with other non steroidal anti-inflammatory drugs (NSAIDs) such as naproxen and ketoprofen (as the free drugs) was not observed. This study shows that a self protein covalently modified by incubation with DAG and iso-DAG is immunogenic in rats. The data thus support the hypothesis that covalent modification of macromolecules by acyl glucuronide metabolites of acidic drugs in vivo can lead to the production of circulating antibodies which may be involved in aberrant immune responses such as drug hypersensitivity. PMID- 7538190 TI - The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. AB - The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation. Increased expression was associated with the appearance of a recA-specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (about 4.3 kb) transcript detected from non-competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA- lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae. PMID- 7538191 TI - Mediator-induced changes in macromolecular permeability in the rat mesenteric microcirculation. AB - An intravital fluorescence microscopic method for measurement of changes in macromolecular permeability has been established in the mesenterial microcirculation of the rat. After exteriorization of the fat-free distal part of the ileal mesentery, a 1-hr period of stabilization was followed by the injection of FITC-labeled macromolecules. Five minutes later, histamine, leukotriene B4, or leukotriene C4 was topically applied to the tissue by means of a micromanipulator. Areas of 1 mm2 were videotaped with a SIT camera. The fluorescence intensity of these areas was measured by an analogous video image processing system and displayed as gray value histograms. The shift of the frequency of gray levels from lower to upper regions could be attributed to an increase in light intensity in the mesentery, indicating an increase in vessel wall permeability. The sites of action of histamine and leukotriene C4 were very similar. Both mediators affected mainly the larger collecting venules. In contrast, leukotriene B4 exerted its effect at postcapillary venules. Moreover, leukotriene B4-induced extravasation was inhibited by superoxide dismutase, suggesting an involvement of oxygen radicals. The studies with histamine alone and with H1- and H2-antagonists demonstrated that histamine-induced extravasation in the rat mesentery was mediated by H1-histamine receptors. The present study introduces an experimental model for the measurement of changes in macromolecular permeability, which is useful for studying mediator effects and their pharmacological inhibition in the microcirculation of the rat mesentery. PMID- 7538192 TI - Ontogeny of microvascular permeability to macromolecules in the chick chorioallantoic membrane during normal angiogenesis. AB - Chick embryos were incubated using standard shell-less techniques for microscopic observations at Days 4.5, 5.0, and 5.5 of the normal 21-day gestation. The chorioallantoic membrane (CAM) was prepared for intravital fluorescence confocal microscopy. A graded series of FITC-dextrans (20, 40, 70, and 150 kDa) was injected via vitelline vein. The changes in interstitial optical intensity due to FITC-dextran extravasation were evaluated by computer-assisted image analysis. Apparent permeability coefficients (Ps) were calculated for first order postcapillary vessels from the changes in intensity as a function of time. On Day 4.5, Ps values (means +/- SE x 10(-7) cm/sec) in the CAM microvessels for FITC Dextran 20, 40, 70, and 150 were 11.8 +/- 1.0, 6.4 +/- 0.4, 3.1 +/- 0.5, and 1.5 +/- 0.5, respectively. The respective Ps values fell dramatically on Day 5.0 to 2.2 +/- 0.5, 0.7 +/- 0.2, 0.6 +/- 0.2, and 0.6 +/- 0.2. On Day 5.5, Ps values for all these FITC-dextrans were equal to 0.7 +/- 0.3. The evaluation of FITC-Dextran 10 on Day 5.5 yielded a Ps value of 1.9 +/- 0.3. Our data demonstrate a rapid reduction in microvascular permeability to macromolecules during normal angiogenesis in the early stages of CAM development. Our data also suggest that these changes in permeability may reflect functional adaptations of the CAM. A comparison of our data to those available in the literature for adult and tumoral tissues demonstrates that microvascular permeability properties in these tissues are different from those of the CAM. PMID- 7538193 TI - Evaluation of blood donors with equivocal hepatitis C serological results. AB - OBJECTIVE: To characterise blood donors with equivocal hepatitis C serological results and to develop an algorithm for their diagnosis and follow-up. DESIGN: Prospective case survey. SUBJECTS AND SETTING: 100 consecutive blood donors referred to the St Vincent's Hospital Liver Clinic, Victoria, with equivocal hepatitis C serological results (positive result for second generation Abbott Enzyme Immunoassay 2.0, but at least one negative result on supplemental testing by first generation Abbott neutralisation assay and Abbott Supplemental Assay for antibody to specific viral antigens). OUTCOME MEASURES: Percutaneous risk factors for hepatitis C exposure, peak serum alanine aminotransferase (ALT) levels, results of alternative immunoassay (Monolisa) and polymerase chain reaction (PCR) to detect hepatitis C viraemia. RESULTS: Thirty subjects had positive results for alternative immunoassay. A risk factor was identified for 32 subjects and was significantly associated (P < 0.01) with positive results for alternative immunoassay (23/32) and PCR (11/32), abnormal ALT levels (7/32), and strong reactivity on initial immunoassay (23/32). Presence of antibodies to both structural and non-structural antigens was also associated with risk factors and positive alternative immunoassay results. CONCLUSIONS: A definitive diagnosis was possible in 87% of subjects. A diagnosis of hepatitis C infection was based on positive alternative immunoassay results together with positive PCR results or presence of a risk factor. Hepatitis C was excluded for 60% of patients. The diagnosis for the remaining 13% remained indeterminate, indicating the need for a definitive diagnostic test for hepatitis C. PMID- 7538195 TI - Camptothecin induction of a time- and concentration-dependent decrease of topoisomerase I and its implication in camptothecin activity. AB - Camptothecin (CPT) has been shown to induce protein-linked DNA breaks (PLDB), which are stabilized intermediates of topoisomerase I (TOP1) activity. Due to the reversible nature of PLDB and the need for replication fork movement for CPT toxicity, both the time of CPT exposure and TOP1 levels are determinants of CPT toxicity. Therefore, the effects of CPT exposure on TOP1 over time were examined in an established human cell line, KB. Using an in vivo KCl-SDS co-precipitation assay, it was determined that 1 hr of CPT exposure resulted in a concentration dependent increase in PLDB that reached a maximum at 5 microM CPT. However, prolonged incubations with CPT revealed a concentration- and time-dependent decrease in CPT-induced PLDB formation. The most rapid loss of PLDB was within 6 hr. Neither aphidicolin nor cycloheximide cotreatments altered the PLDB decrease induced by CPT. Immunoblot analysis revealed a reduction in TOP1 protein upon CPT exposure, whereas RNA analysis revealed no changes, which suggested a post transciptional mechanism of TOP1 down-regulation. The CPT-induced reduction was specific for TOP1, because actin and tubulin levels were unaltered by CPT exposure. Finally, clonogenic assays revealed a small but statistically significant decrease in CPT toxicity throughout the CPT exposure period. Because PLDB formation based on TOP1 levels is an important step in the toxicity of CPT, the CPT-induced TOP1 reduction could be a transient mechanism of resistance for cells to avoid toxic levels of PLDB. PMID- 7538194 TI - Residue 78 in the second transmembrane domain of the neurokinin-1 receptor is important in coupling high affinity agonist binding to multiple second messenger responses. AB - The neurokinin-1 tachykinin receptor is a member of the G protein-coupled receptor superfamily. An unusual feature of the neurokinin-1 receptor is the presence of glutamic acid (residue 78) in the second putative transmembrane domain, at the location of a highly conserved aspartate residue in the G protein coupled receptor superfamily. The rat neurokinin-1 receptor cDNA was mutated to lysine, aspartate, and glutamine at this site and functionally expressed in Chinese hamster ovary cells, and clonal cell lines were isolated and characterized. Radioligand binding demonstrated that the Asp78 and Lys78 receptors have substance P binding affinities indistinguishable from those of the wild-type receptor and are expressed at roughly the same number of receptors per cell. The Gln78 receptor variant, on the other hand, exhibited no detectable agonist binding. Although wild-type and Asp78 receptors have essentially the same ability to stimulate inositol phospholipid turnover, cAMP production, and arachidonic acid release, the Lys78 variant is markedly attenuated in its ability to activate any of these pathways. These data indicate that residue 78 plays a role in the coupling of the rat neurokinin-1 receptor to cellular effectors. In addition, both Asp78 and Lys78 receptors show a greater percentage of high affinity binding that is resistant to guanosine-5'-O-(3-thio)triphosphate than does the wild-type receptor, indicating a potential difference in G protein coupling between wild-type and mutated receptors. PMID- 7538196 TI - Vasoactive intestinal peptide elevates pinealocyte intracellular calcium concentrations by enhancing influx: evidence for involvement of a cyclic GMP dependent mechanism. AB - Vasoactive intestinal peptide (VIP) receptor density is high in the pineal gland, which receives VIP innervation and responds to VIP with a relatively small increase in cAMP and cGMP levels. In the present study, we show that VIP (5-200 nM) treatment increased the intracellular calcium concentration ([Ca2+]i) in 64% of isolated individual pinealocytes; in comparison, norepinephrine (NE) elevated [Ca2+]i in 93% of the cells and produced more robust responses. Analysis of the role of second messengers indicated that [Ca2+]i was strongly elevated by cGMP analogs, but not by cAMP analogs. The nitric oxide-releasing agent S-nitro-N acetylpenicillamine and 2,2-diethyl-1-nitroxyhydraxine also elevated [Ca2+]i. Investigation of the mechanisms revealed that responses to VIP or 8-bromo-cGMP involved Ca2+ influx, as did the plateau component of the response to NE; the large rapid component of the response to NE, however, appeared to reflect release from intracellular stores. Pharmacological studies indicated that the VIP-induced Ca2+ influx was mediated by a retinal rod-type cyclic nucleotidegated cation channel, expression of which was confirmed by reverse transcription-polymerase chain reaction analysis. These observations indicate that fundamentally different mechanisms generate the responses to NE and VIP. The dominant effect of VIP causing transient elevation of [Ca2+]i appears to be through cGMP gating aI-cis diltiazem-sensitive rod-type cyclic nucleotide-gated cation channel. In contrast, the dominant effect of NE on [Ca2+]i is due to enhanced Ca2+ release from intracellular stores; the plateau component is due to influx through aI-cis diltiazem-insensitive channel. PMID- 7538198 TI - Molecular identification of microorganisms associated with parthenogenesis. AB - Cytoplasmically interited microorganisms are widespread in insects and have been implicated as causes of female parthenogenesis (females developing from unfertilized eggs) and cytoplasmic incompatibility. Normal sexual reproduction can be restored by treatment with antibiotics. Sequence analysis of the DNA encoding 16S ribosomal RNA has shown that cytoplasmic incompatibility bacteria from diverse insect taxa are closely related (they share >95% sequence sililarity) and belong to the alpha subdivision of Proteobacteria. Here we show that parthenogenesis-associated bacteria from parasitoid Hymenoptera also fall into this bacterial group, having up to 99% sequence similarity to some incompatibility microorganisms. Both incompatibility and parthenogenesis microorganisms alter host chromosome behaviour during early mitotic divisions of the egg. Incompatibility bacteria act by interfering with paternal chromosome incorporation in fertilized eggs, whereas parthenogenesis bacteria prevent segregation of chromosomes in unfertilized eggs. These traits are adaptive for the microorganisms. On the basis of their sequence similarities, we conclude that parthenogenesis bacteria and cytoplasmic incompatibility bacteria form a monophyletic group of microorganisms that 'specialize' in manipulating chromosome behaviour and reproduction of insects. PMID- 7538200 TI - Current bibliographies of neuropeptides prepared by the University of Sheffield Biomedical Information Services. PMID- 7538199 TI - Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons. AB - We have studied neurotrophic effects of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFGF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFGF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro. PMID- 7538197 TI - Suppression of the constitutive expression of cytochrome P-450 2C11 by cytokines and interferons in primary cultures of rat hepatocytes: comparison with induction of acute-phase genes and demonstration that CYP2C11 promoter sequences are involved in the suppressive response to interleukins 1 and 6. AB - Hepatic expression of various members of the cytochrome P-450 (CYP) superfamily is suppressed during inflammatory responses. We have shown that the specific expression of P-450 2C11 in male rat liver is suppressed transcriptionally by endotoxin treatment. To investigate the molecular mechanisms underlying this phenomenon, we studied the effects of the inflammatory cytokines interleukin (IL) 1, IL-6, tumor necrosis factor-alpha (TNF), interferon (IFN)-alpha, and IFN-gamma on the expression of P-450 2C11 and the mRNAs of two typical acute-phase protein genes, alpha 1-acid glycoprotein (AGP) and fibrinogen, in primary hepatocyte cultures. IL-1, IL-6, TNF, and IFN-alpha all suppressed P-450 2C11 mRNA, whereas IFN-gamma had no effect. IL-1 and TNF were more effective than IL-6 in the suppression of P-450 2C11 mRNA. Whereas IL-1 and IL-6 effects on P-450 2C11 were accompanied by induction of AGP and fibrinogen mRNAs, IFN-alpha and TNF treatments had no effects on AGP. The suppression of P-450 2C11 and the induction of AGP by IL-1 showed similar time courses. The combination of IL-1 and IL-6 showed additivity in suppression of P-450 2C11, at maximally effective concentrations of cytokines. The effects of IL-1 on P-450 2C11 and AGP expression were blocked by IL-1 receptor antagonist protein. We also studied the effects of IL-1 and IL-6 on the transient expression of chloramphenicol acetyl-transferase reporter gene constructs containing 200 or 1287 base pairs of the 5' flanking region of the CYP2C11 gene, transfected into primary hepatocytes. The chloramphenicol acetyltransferase activities in cells transfected with the 200 base pair construct were reduced to about 33% and 58% of control levels by treatment with IL-1 or IL-6, respectively, suggesting that sequences important for cytokine down-regulation lie within the proximal promoter region of the CYP2C11 gene. PMID- 7538202 TI - Pulmonary actions of the neurokinin1-specific agonist [Sar9,Met(O2)11]-substance P. AB - We examined the relationship between airway obstruction and plasma extravasation produced by the intravenous administration of the selective NK1 receptor agonist [Sar9, Met(O2)11]-substance P(SP). Conscious guinea-pigs were injected with Evans' blue dye followed by intravenous [Sar9,Met(O2)11]-SP. Animals were killed 3 min later and airway obstruction, determined via excised lung gas volumes, and plasma extravasation in the trachea, mainstem bronchi and intrapulmonary airways quantitated. Maximal plasma protein extravasation occurred at a dose about 30 times less than that required to elicit airway obstruction. Neither the neutral endopeptidase (NEP) inhibitor, thiorphan, or the angiotensin-converting enzyme (ACE) inhibitor, captopril, altered the extravasation response to [Sar9,Met(O2)11]-SP. However, thiorphan alone or combined with captopril produced a small but significant potentiation of the airway obstructive response. The marked difference between pulmonary gas trapping and Evans' blue extravasation responses suggest that [Sar9,Met(O2)11]-SP-induced airway obstruction is not secondary to increased pulmonary edema. PMID- 7538201 TI - Regulation of adenylate cyclase by galanin, neuropeptide Y, secretin and vasoactive intestinal polypeptide in rat frontal cortex, hippocampus and hypothalamus. AB - This study characterizes regional regulation of adenylate cyclase by galanin, neuropeptide Y (NPY), secretin and vasoactive intestinal peptide (VIP) in rat brain frontal cortex, hypothalamus and hippocampus. In our experimental system, galanin caused small detectable activation (10-20%) of basal adenylate cyclase activity in frontal cortex and hippocampus but had no effect on basal adenylate cyclase activity in hypothalamus. Galanin inhibited forskolin-stimulated adenylate cyclase in all three brain regions-hypothalamus, hippocampus and frontal cortex by 54.5%, 44.3% and 25.7%, respectively. NPY reduced basal and forskolin-stimulated enzyme activities by 35% only in frontal cortex, but not in the other two brain areas. Secretin had no effect in frontal cortex but caused similar adenylate cyclase activation in hypothalamus and hippocampus. VIP had a stimulatory effect of 32.8% and 32.4% in frontal cortex and hippocampus, respectively. The results indicate regional differences in adenylate cyclase modulation by the four peptides and reveal interesting relations in comparison with peptide and receptor densities in the three investigated brain regions. PMID- 7538204 TI - Current bibliographies of neuropeptides prepared by the University of Sheffield Biomedical Information Service. PMID- 7538203 TI - Galanin and somatostatin inhibition of substance P-induced airway mucus secretion in the rat. AB - Substance P is present in several neurons innervating the lung. Tachykinin receptors are expressed on submucosal gland cells. Substance P is known to be a potent stimulator of airway mucus secretion. In the present study we characterized the effects of galanin and somatostatin on basal and substance P induced mucus secretion. The stimulatory effect of substance P was concentration dependent (100 pmol/l: 112%, 1 nmol/l: 120%, 10 nmol/l: 153%, 100 nmol/l: 223%, 1 mumol/l: 275%, 10 mumol/l: 172%) and was inhibited by galanin and somatostatin (1 mumol/l substance P: 277%; 1 mumol/l substance P + 1 mumol/l somatostatin: 190%, p < 0.01; 1 mumol/l substance P + 1 mumol/l galanin: 206%, p < 0.05). In the presence of lower concentrations of substance P 1 mumol/l somatostatin and 1 mumol/l galanin did not modify mucus secretion. Lower concentrations of galanin and somatostatin did not significantly change mucus secretion stimulated by 1 mumol/l substance P. Both, galanin and somatostatin at 1 mumol/l left basal airway mucus secretion unaltered. These data suggest that mucus secretion into airways is regulated by a complex network of peptidergic stimulators and inhibitors including substance P, somatostatin and galanin. PMID- 7538205 TI - GR 73,632 and [Glu(OBzl)11]substance P are selective agonists for the septide sensitive tachykinin NK1 receptor in the rat urinary bladder. AB - The existence of a septide-sensitive subtype of the tachykinin NK1 receptor has been recently proposed. In the rat isolated urinary bladder, the non-peptide NK1 receptor antagonist RP 67,580 exhibits a higher affinity towards septide (pKB 7.57) than towards [Sar9]substance P sulfone (pKB 7.00). In this study we have investigated the pharmacological profile of the non-mammalian tachykinin physalaemin, of the synthetic NK1 receptor agonist GR 73,632 (delta aminovaleryl[LPro9,NMeLeu10]substance P(7-11)) and of [Glu(OBzl)11]substance P in relation to the putative existence of a septide-sensitive receptor. The activity of [Glu(OBzl)11]substance P at the NK1, NK2 and NK3 receptor was assayed in the guinea-pig ileum NK1 receptor assay (EC50 26 nM), in the rabbit pulmonary artery NK2 receptor assay (weak agonist activity) and in the rat portal vein NK3 receptor assays (no appreciable activity up to 1 microM). GR 73,632, [Glu(OBzl)11]substance P and physalaemin, all produced concentration-dependent contractions of the rat isolated urinary bladder, with EC50 values of 17, 79, and 9 nM, respectively. The responses to the three agonists were very slightly or not modified by the NK2 receptor antagonist SR 48,968 (1 microM). RP 67,580 (0.3-3 microM) produced a concentration-dependent rightward shift of the curve to GR 73,632, [Glu(OBzl)11]substance P and physalaemin without producing depression of their maximal response. Schild plot analysis indicated the competitive nature of the antagonism. The affinity (pKB) of RP 67,580 towards physalaemin, GR 73,632 and [Glu(OBzl)11]substance P was 7.12, 7.56 and 7.95, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538206 TI - A leucine-to-phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow-channel syndrome. AB - The slow-channel syndrome is one of several congenital myasthenic syndromes that result from inherited abnormalities of the ion channel of the skeletal muscle acetylcholine receptor (AChR). The ion channel is formed by the second transmembrane domains (M2) of the four AChR subunits. We screened the genomic DNA of one family with the slow-channel syndrome for mutations in the coding sequences for the M2 domains of the four AChR subunits and report the identification of a missense mutation that causes a leucine-to-phenylalanine substitution at position 269 of the epsilon subunit in three affected members of a family with the slow-channel syndrome. We propose that this mutation may be responsible for the disease. PMID- 7538207 TI - [Colorectal cancer in old age. Our experience]. AB - Two hundred and thirty-three patients treated for colorectal cancer during the period 1976-1991 were divided into three groups (A: < 65 yr; B: 65-74 yr; C: > 74 yr) in order to perceive possible statistically significant differences in patients older than 75 years. Epidemiological features are similar among the three groups, while a greater diagnostic delay (p = 0.013), a higher incidence of emergency procedures (p = 0.006) and a more advanced AP stage were found in group C. The high anesthesiological risk determined a conservative surgical approach only in 4.1% of patients, while a curative resection was performed on 51% of group C vs 72.7% of group A (p = 0.016). Postoperative complications and mortality for curative resections were 28% and 12% in group C vs 24.6% and 2.9% in group A (p = n.s.); the overall 5 years survival rate was 62.9%, 51.7% and 42.2% in groups A, B, and C. It is concluded that age alone should not be considered as a contraindication to curative surgery for colorectal cancer, for life expectancy and quality are considerably worse in the elderly undergoing derivative surgery. PMID- 7538208 TI - [The palliative surgery of periampullary neoplasms. Our experience]. AB - The increased incidence of peri-ampullar neoplasia and the low percentage of cases in which radical surgery is possible fuel interest in the use of palliative treatment for this pathology. The authors report their 10-year experience of palliative surgery for peri-ampullar cancer and evaluate immediate and long-term results. Between January 1980 and December 1990 the authors operated 91 patients with peri-ampullar carcinoma (head of the pancreas, Vater's ampulla, terminal choledochus). Eight (9%) underwent radical surgery and 83 (91%) underwent palliative surgery. The distribution of cases according to tumour origin in the latter group was as follows: head of the pancreas 60 cases (72%); intra pancreatic choledochus 13 (16%); Vater's ampulla 10 (12%). The most frequent symptoms were pain (70%) and jaundice (63%). The mean interval between the appearance of symptoms and diagnosis was 1 month (range: 5 days-12 months). The most frequently performed biliodigestive derivative surgery was cholecystojejunostomy (72% of cases). Complications were observed in 18% of cases. Mortality was 12%. Mean survival was 5 months (range: 2-43 months). The recent use of endoscopic techniques for the treatment of jaundice in patients with inoperable peri-ampullar carcinoma now requires prospective studies to compare these techniques with surgery. PMID- 7538209 TI - [Palliative treatments in inoperable pancreatic tumors. Technics compared]. AB - The authors experience of palliative treatment of advanced pancreatic cancer is reported. 32 patients underwent palliative surgical procedures of biliary decompression (20 choledochoduodenostomy and 12 cholecystoenteric bypass). In 26 patients a gastroenteric anastomosis was also performed. 8 patients underwent non surgical procedure because of their very poor conditions or short term survival. The authors point out the advantages offered by surgical treatment. Nevertheless a careful selection of patients submitted to this procedure is recommended. PMID- 7538210 TI - Comparative study of normotensive and hypertensive nitric oxide synthase inhibitors on morphine withdrawal syndrome in rats. AB - The effects of the normotensive, mainly centrally active nitric oxide synthase (NOS) inhibitor 7-nitro indazole and the hypertensive drug NG-nitro-L-arginine, which blocks both the endothelial and the central NOS, have been examined on naloxone-precipitated withdrawal in morphine-dependent rats. Both drugs attenuated the same withdrawal signs (teeth-chattering, penile licking, diarrhoea, chewing, wet-dog shakes, grooming), while other signs remained unaffected (rearing, jumping, ptosis, rhinorrhoea, irritability on touch). These findings indicate that mainly central (but not endothelial) nitric oxide is involved in the expression of some opioid withdrawal symptoms. PMID- 7538211 TI - Changes in expression of glutamate receptor subunits following photoreceptor degeneration in the rd mouse retina. AB - The effects of photoreceptor cell degeneration on the expression of nine alpha amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/kainate glutamate receptor subunit genes were investigated in the rd mouse retina by in situ hybridization. All known AMPA/kainate receptor subunits were found to be expressed in normal mouse retina. Following retinal degeneration, the expression of KA-1 was reduced, that of the GluR7 subunit was greatly increased, and that of the other seven subunits were not significantly affected. PMID- 7538213 TI - Patient care delivery: let's return to reality. AB - Practical solutions that promote humanistic patient care with cost-effective, reality-based foundations are represented in a sample plan for one surgical unit. A return to the basics provides the base to develop and implement principle centered leadership. PMID- 7538212 TI - A functionally active presynaptic high-affinity kainate receptor in the rat hippocampal CA3 subregion. AB - We studied the modulation of the intracellular free calcium concentrations ([Ca2+]i) by kainate/AMPA receptor activation in synaptosomes isolated from whole rat hippocampus, or from its CA1, CA3 or dentate gyrus subregions. The receptor was activated either by 100 microM S-alpha-amino-3-hydroxy-5-methyl-4 isoxazolopropionic acid (AMPA) (EC50 = 26.6 +/- 4.9 microM) or by 100 microM kainate (EC50 = 0.81 +/- 0.1 microM), but the effects of these agonists were not additive. The response to either AMPA or kainate was competitively inhibited by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine. Higher [Ca2+]i responses to 100 microM AMPA or to 100 microM kainate were observed in the CA3 subregion (43.2 +/- 2.5 nM or 42.8 +/- 2.3 nM, respectively) than in the whole hippocampus (22.4 +/- 1.1 nM or 22.4 +/- 1.6, respectively), in the CA1 subregion (26.4 +/- 1.1 nM or 26.6 +/- 2.6 nM, respectively) or in dentate gyrus (24.6 +/- 1.4 nM or 21.5 +/- 1.0 nM, respectively). These results indicate that the CA3 subregion of the hippocampus is enriched in a presynaptic high-affinity kainate receptor which modulates the [Ca2+]i in nerve terminals. PMID- 7538215 TI - Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR. AB - To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin embedded liver biopsies. PMID- 7538214 TI - Advanced proliferative retinopathy and iris neovascularization in an intravenous drug abuser. PMID- 7538217 TI - Effects of modified low density lipoprotein and hypoxia on the expression of endothelial leukocyte adhesion molecule-1. AB - OBJECTIVES: Expression of endothelial leukocyte adhesion molecule (ELAM-1 or CD62E) plays a role as an early event of atherogenesis. It is well known that interleukin-1 (IL-1) expresses ELAM-1 on vascular endothelial cells. We have examined pathological factors that induce ELAM-1 expression on cultured endothelial cells. METHODS: Examined factors were native low density lipoprotein (LDL), oxidized LDL, glycated LDL, hypoxia, and IL-1. Peroxidation of LDL was performed by ultraviolet radiation. Hypoxia was reproduced by adding a hypoxic cell-culture medium that was deoxygenated by use of a vacuum pump and nitrogen gas. Endothelial cells were harvested from a porcine aorta and were allowed to proliferate to be subconfluent in slide chambers. Expression of ELAM-1 was evaluated by counting the number of cells that were characterized by positive staining with the immunohistochemical technique. RESULTS: Without any stimulants, about 6.9% of the endothelial cells expressed ELAM-1. Weakly oxidized LDL (12 pmol/micrograms protein) significantly expressed ELAM-1 (14.8%) after an incubation period of 1 hour. Glycated LDL induced significant expressions (12.6%) in a fructosamine concentration of 65 pmol/micrograms protein. A one-hour incubation with a hypoxic culture medium expressed ELAM-1 in 16.3% of the cells. Native LDL did not cause any significant increases in the percentage. IL-1 expressed ELAM-1 in 30% of the cells even with as low a concentration as 3.1 U/ml. CONCLUSIONS: The present study shows that not only IL-1 but also weakly oxidized LDL, glycated LDL, and hypoxia may be possible factors that cause the expression of ELAM-1. PMID- 7538216 TI - Prostate disease: management options for the primary healthcare team. Report of a working party of the British Prostate Group. AB - The prostate gland has attracted a remarkable increase in interest in the past few years. The two most common diseases of this gland, benign prostatic hyperplasia and carcinoma of the prostate, have been brought into greater prominence by new diagnostic methods, public interest, and a wider choice of surgical and non-surgical treatments. Uncertainty about the significance of these changes has occurred because of the rapidity of change, the profusion of statements, opinions and promotions, and the relatively little guidance available from the profession. Ten urologists and two general practitioners have reviewed the relevant evidence about these two prostate diseases and the newer diagnostic methods; their conclusions are summarised here. Management options and guidance on clinical practice are also discussed. Because of a number of unresolved diagnostic and management issues, detailed requirements for practice guidelines have not been specified. PMID- 7538218 TI - Inhibition of leukotriene production by FK506 in rat basophilic leukemia-1 cells. AB - We investigated the inhibitory action of FK506 (0.0005-5 micrograms/ml) on the metabolism of arachidonate 5-lipoxygenase in rat basophilic leukemia-1 cells. Cells were stimulated with A23187 (10(-5) mol/l) for 15 min in the absence or presence of various concentrations of FK506. Arachidonate 5-lipoxygenase metabolites, peptide leukotrienes (LTs), leukotriene B4 (LTB4) and 5 hydroxyeicosatetraenoic acid (5-HETE) were measured by high-performance liquid chromatography. FK506 inhibited A23187-stimulated production of peptide LTs, LTB4 and 5-HETE in intact cells by up to 77, 73 and 60%, respectively. Phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), was not significantly inhibited by FK506. The synthesis of peptide LTs and LTB4 was not inhibited by FK506 when leukotriene A4-free acid was added to the culture medium. The synthesis of peptide LTs, LTB4 and 5-HETE was not affected by FK506 in a cell lysate study using AA as the substrate. These results indicate that FK506 inhibits the production of peptide LTs, LTB4 and 5-HETE by inhibiting 5 lipoxygenase activity in intact cells. The inhibition is not a direct action on 5 lipoxygenase but results from the activating processes of this enzyme. PMID- 7538220 TI - Combined spectroscopic and electrical recording techniques in membrane research: prospects for single channel studies. PMID- 7538219 TI - Nitric oxide synthase activity in brain regions and spinal cord of mice and rats: kinetic analysis. AB - Nitric oxide synthase (NOS) activity was determined by the rate of conversion of [3H]arginine to [3H]citrulline in brain regions (midbrain, hypothalamus, cerebellum, hippocampus, corpus striatum, cortex, pons-medulla and amygdala) and spinal cord of male Swiss Webster mice and male Sprague-Dawley rats. In mice, high activity of NOS was found in cerebellum, hypothalamus and midbrain; intermediate activity in pons-medulla, hippocampus, amygdala, corpus striatum and cortex; and low activity in the spinal cord. In rat, highest activity of NOS was observed in cerebellum followed in decreasing order by midbrain, hypothalamus, cortex, striatum, pons-medulla, hippocampus and spinal cord. In all tissues, NOS activity was higher in rat than in mouse. Analysis of the Eadie-Hofstee plot indicated that the Vmax and Km values of the enzyme in cortex and cerebellum of rats were higher than in mouse tissues. These studies show differential distribution of NOS activity in mouse and rat brain regions and spinal cord and higher activity of NOS in rat brain regions and spinal cord compared to mouse tissues. PMID- 7538223 TI - [How I treat... ventricular extrasystoles]. PMID- 7538222 TI - Necrotizing vasculitis and exacerbation of psoriasis after granulocyte colony stimulating factor for small cell lung carcinoma. PMID- 7538221 TI - Peptidylproline cis/trans isomerases. PMID- 7538224 TI - [Generalized pruritus]. PMID- 7538225 TI - [Aphasia caused by isolated lesion of the semi-ovale centre: contribution of the measurement of cerebral blood flow]. AB - Sudden onset stroke occurred in a right-handed vietnamese woman speaking, reading and writing french fluently. When first seen in our department, the patient had mild right facial paresis and non fluent atypical aphasia. CT scan and MRI showed a left subcortical infarct in the superficial territory of the middle cerebral artery; only white matter of the semiovale centre was involved. Neurological examination revealed linguistic impairment resembling transcortical motor aphasia, with unusual stuttering, hypophonia, occasional semantic paraphasias and phonological reading and writing abnormalities. Non verbal cognitive function, gestural and buccofacial praxes were normal. Cerebral blood flow study by SPECT was consistent with left sylvian functional deactivation. PMID- 7538226 TI - [Prostatic adenoma. Diagnosis, development, prognosis, treatment]. PMID- 7538227 TI - Articular cartilage: cause or victim in arthritis. PMID- 7538228 TI - Mapping of arthritogenic/autoimmune epitopes of cartilage aggrecans in proteoglycan-induced arthritis. AB - Immunization of BALB/c mice with chondroitin sulfate-depleted proteoglycan (aggrecan) of fetal human cartilage produces progressive polyarthritis and ankylosing spondylitis. The development of the disease in genetically susceptible BALB/c mice is dependent upon the expression of both cell-mediated and humoral immune responses against the host mouse cartilage proteoglycan (PG). Although cartilage PGs from various species have many biochemical and immunological similarities, only a select group of PGs from fetal and newborn human, fetal pig and canine articular cartilages, human osteophytes and human chondrosarcomas are able to induce arthritis in BALB/c mice. Arthritis develops only in mice that also develop autoantibodies to self-cartilage PGs, although autoantibodies occasionally are present in non-arthritic animals as well. The protease-sensitive auto/arthritogenic epitope(s) is located in, or close to, the chondroitin sulfate (CS) attachment region of the PG molecule. The primary structure of the core protein is responsible for the autoimmune/arthritogenic effect of this select group of PGs, whereas the core protein epitopes are masked by glycosaminoglycan (GAG)-side chains. The CS side chains seem to inhibit antigen recognition in all aggrecans with arthritogenic potential, whereas a similar effect with keratan sulfate (KS) appears only in PGs of aging cartilages. PMID- 7538229 TI - The treatment of lumbar spine pain syndromes diagnosed by discography: lumbar arthrodesis. PMID- 7538230 TI - Palliative operations for patients with unresectable endocrine neoplasia. AB - No good randomized studies exist for many types of endocrine tumors to prove that surgery increases the survival of patients with these tumors. However, many case reports and our own experience suggest that it does. Therefore, surgery may be performed to potentially increase the survival of patients with metastatic and localized malignant endocrine tumors. Subsequently, these tumors may still recur locally or distantly. Therefore, surgery is usually a palliative procedure but is occasionally curative. Because endocrine tumors secrete hormonal substances resulting in particular syndromes, debulking surgery may be necessary to control hormonal syndromes. The location of a tumor may cause mass symptoms that can also be relieved by surgery. Thus, many patients with malignant endocrine neoplasia benefit from aggressive surgery. The surgeon must determine the resectability of the tumor as well as the operative morbidity and benefit to the patient before proceeding. PMID- 7538231 TI - Dysmorphogenic effects of nitric oxide (NO) and NO-synthase inhibition: studies with intra-amniotic injections of sodium nitroprusside and NG-monomethyl-L arginine. AB - Sodium nitroprusside (SNP), a chemical that is readily converted to nitric oxide (NO) in biological systems, was microinjected into the amniotic fluids of cultured whole rat conceptuses on day 10.5 of gestation and dysmorphogenic/embryotoxic effects were evaluated after a 24 hr incubation period. Injections of 217 ng/embryo (approximately 800 microM) resulted in whitened zones of dead cells in a discretely circumscribed region within the mesencephalon closely associated with the neural tube. These zones were observed with a high incidence after SNP microinjections and were referred to as "white caps" because of their microscopic appearance. At higher concentrations, the whitened zone extended into the rhombencephalon and occasionally appeared to extend the full length of the dorsal midline. The whitened zones of tissue separated readily from the apparently normal underlying tissues upon removal or disturbance of the amniotic membrane. Coinjection of ferrous hemoglobin with SNP selectively prevented the appearance of "white caps" but not other embryotoxic manifestations. Microinjections of the breakdown products of light-exposed SNP elicited generalized embryotoxicity but "white caps" were not observed. In separate experiments, we found that embryonic enzymes catalyzed significant conversion of arginine to citrulline, indicating expression of NO-synthase during organogenesis. NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO synthase, was microinjected (50-150 ng/embryo; approximately 200-600 microM) on day 10.5 of gestation and produced malformations that differed markedly from those elicited by SNP. Failure of anterior and posterior neural tube closure and profound underdevelopment of the hyoid arch and optic cup were observed at concentrations that produced no apparent growth deficit. These studies with SNP and L-NMMA indicated that both an excess and a deficiency of NO can be embryotoxic/dysmorphogenic and suggest important roles for optimal levels of NO and NO synthases in normal embryonic development. PMID- 7538232 TI - Selective broths for the isolation of Pseudomonas pseudomallei from clinical samples. PMID- 7538233 TI - Isolation of Pseudomonas pseudomallei from soil in north-eastern Thailand. AB - In order to optimize the recovery from soil of Pseudomonas pseudomallei, the cause of melioidosis, 3 selective broths were compared. A basal salt solution containing L-threonine (TBSS) performed significantly better than trypticase soy broth containing crystal violet and colistin 50 mg/L (CVC50), both in isolation rate and suppression of overgrowth of other organisms, but the addition of colistin to TBSS gave the best results overall. In a survey in north-eastern Thailand, P. pseudomallei was recovered from 114 (68%) of the 167 sites tested. A detailed study of a single rice farm showed that the isolation rate increased with depth of soil sample, and P. pseudomallei could still be isolated during the dry season, although only from moist soil in areas where other crops were cultivated and around the water source. PMID- 7538234 TI - [Use of a turbidimetric method to study the kinetics of inhibition of fibrinolysis in diluted blood plasma]. AB - Dependence of fibrinolysis parameters on the concentration of inhibitors of fibrinolytic process has been studied when using turbidimetric method. It is shown that the process of destruction of fibrin clot formed in diluted blood plasma is subjected to the Michaelis-Menten ratio. The constant of inhibition of fibrinolytic process in diluted plasma activated by prothrombin and streptokinase is 10 and 0.5 microM for 6-aminohexanoic acid and contrykal, respectively. PMID- 7538236 TI - Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era. AB - Without question, much has been learned about the glycoprotein PSA in recent years. By increasing our understanding of this tumor marker's biochemical and physiologic properties, we will be able to improve its clinical utility. The discovery of the various molecular forms of PSA represents a significant advancement. Knowing the concentration and ratio of these PSA forms will be valuable in deciding which patients require further evaluation with transrectal ultrasound and prostate biopsy and which men can be monitored safely without undergoing further invasive testing. This information will be most valuable in treating the patient with a mildly elevated serum PSA level. Although assays are not yet available to detect specifically hK2, the striking similarities of hK2 to PSA, including selective expression in the prostate, suggest that this marker may also prove useful in prostate cancer management. Indeed, a new era of PSA testing has been entered, and the entire field of prostate cancer will benefit. PMID- 7538235 TI - Chemical compositions of 300 lower urinary tract calculi and associated disorders in the urinary tract. AB - The stones removed from 273 patients (220 males, 53 females) with bladder stones and 27 patients with urethral stones (males) were analyzed by infrared spectroscopy. Of these stones 50.0% contained magnesium ammonium phosphate (MAP), 39.9% calcium (oxalate and/or phosphate), 9.4% uric acid (urate), and 0.7% cystine. The disorders associated with bladder stones were prostatic hyperplasia and cancer (47.8%), neurogenic bladder (11.8%), urethral stricture (3.6%) and so on in males, and neurogenic bladder (48.1%), after operation of uterus (29.0%), bladder cancer (5.7%) and so on in females. Urinary infections were complications in 59.3% of patients with MAP stones, 41.2% with urate stones and 20.0% with calcium stones. The most often isolated bacteria from the infected urine were of the Proteus species. PMID- 7538238 TI - Transurethral resection versus incision of the prostate: a randomized, prospective study. AB - OBJECTIVES: To evaluate longer term effects of transurethral resection (TURP) and incision (TUIP) of the prostate in randomized patients. METHODS: In a randomized, prospective study, 120 patients with symptoms of bladder outlet obstruction caused by smaller benign prostates (estimated resectable weight less than 20 g) were assigned to TURP or TUIP. Patients were evaluated preoperatively and at intervals postoperatively as to urinary symptoms (Madsen's questionnaire), sexual function, and uroflowmetry. Overall evaluation of outcome of surgery was also assessed at follow-up visits. RESULTS: Fifty-six patients received a TURP and 61 a TUIP. Three patients refused to participate in the project after randomization, and 5 patients were lost to or excluded from follow-up. A group of 112 patients were obtainable for postoperative evaluation with a mean follow-up time of 34 months (1 to 82 months). Improvements in mean urinary peak flow rates were seen in both groups throughout the study period. The peak flow rates generally were higher (but not statistically so) in the TURP group. Postoperative irritative, obstructive, as well as total symptom scores decreased significantly at all follow-up visits after both TURP and TUIP (P < or = 0.034). Preoperatively and at all postoperative follow-up there was no statistically significant difference in irritative, obstructive, or total symptom scores between TURP and TUIP. The patients indicated an overall subjective improvement at all follow-ups in both groups, with no statistically significant difference between the treatment groups. Fifteen of 22 (68%) patients receiving TURP and 8 of 23 (35%) in the TUIP group who were sexually active before and after surgery developed postoperative retrograde ejaculation (P = 0.020). Postoperatively, 9 (16%) of the patients in the TURP and 14 (23%) in the TUIP group received further treatment for benign prostatic hyperplasia (BPH)-related infravesical obstruction. This difference was not statistically significant (P = 0.908). CONCLUSIONS: In small prostates TURP and TUIP were generally equally effective in relieving bladder outlet obstruction secondary to BPH. Most surgically treated BPH cases can be well managed by the incision technique, which is an underutilized procedure. PMID- 7538237 TI - A controlled trial of bicalutamide versus flutamide, each in combination with luteinizing hormone-releasing hormone analogue therapy, in patients with advanced prostate cancer. Casodex Combination Study Group. AB - OBJECTIVES: To compare the efficacy and safety of bicalutamide and flutamide, each used in combination with luteinizing hormone-releasing analogue (LHRH-A) therapy, in patients with untreated metastatic (Stage D2) prostate cancer. METHODS: Randomized, double-blind (for antiandrogen therapy), multicenter study with a 2 x 2 factorial design. Eight hundred thirteen patients were allocated 1:1 to bicalutamide (50 mg once daily) and flutamide (250 mg three times daily) and 2:1 to goserelin acetate (3.6 mg every 28 days) and leuprolide acetate (7.5 mg every 28 days). RESULTS: With a median duration of follow-up of 49 weeks, time to treatment failure, the primary endpoint, was significantly (P = 0.005) better for the bicalutamide plus LHRH-A group than for the flutamide plus LHRH-A group. Patients in the flutamide plus LHRH-A group were 34% more likely to fail treatment over the given time period, as indicated by the hazard ratio of 0.749 (95% confidence interval, 0.61 to 0.92) for bicalutamide plus LHRH-A to flutamide plus LHRH-A. Results for secondary endpoints (survival, quality of life, and subjective response) were similar between groups. Diarrhea occurred in 24% of patients in the flutamide plus LHRH-A group, compared with 10% of patients in the bicalutamide plus LHRH-A group (P < 0.001). CONCLUSIONS: In patients with metastatic prostate cancer, bicalutamide plus LHRH-A is well tolerated and provides superior efficacy to flutamide plus LHRH-A with respect to time to treatment failure. Assessment of the effects of these regimens on longer term survival requires additional time for follow-up. PMID- 7538240 TI - Randomized clinical trial comparing low power-slow heating versus high power rapid heating noncontact neodymium:yttrium-aluminum-garnet laser regimens for the treatment of benign prostatic hyperplasia. AB - OBJECTIVES: To evaluate and compare the safety and effectiveness of two noncontact laser regimens for the treatment of human benign prostatic hyperplasia (BPH), and to assess the impact of the spatial distribution of the laser-induced lesions on treatment outcome. METHODS: This was a prospective, double-blind, randomized study that included 29 patients with BPH (both the patients and the study physician assistant in charge of the follow-up evaluation were blinded to the treatment rendered). The patients were randomized to receive either a 15 W for 180 s (15 patients) or 50 W for 60 s (14 patients) laser regimen (powers measured at the fiber tip). There were two study phases for each treatment group: the irradiated sites were not overlapping during the first phase (lesions 1.5 cm apart), and were overlapping during the second phase (lesion less than 1.0 cm apart). RESULTS: Morbidity was minimal in both groups. At 1 year of follow-up, there was significant improvement of the American Urological Association-7 symptom score, the peak urinary flow rate, and the postvoid residual in both treatment groups. These improvements were not statistically significantly different regardless of time or the phase of the study for the two treatment groups. There were 3 treatment failures, 2 of whom were later successfully re treated with larger amounts of laser energy. Furthermore, the peak flows in the second phase of the study were statistically significantly higher than those in the first phase of the study, regardless of the treatment group. CONCLUSIONS: Our results suggest that both the 15 W for 180 s and the 50 W for 60 s are equally safe and effective treatments for BPH. Perhaps more importantly, they also suggest that the spatial distribution of lesions and overlapping of treated (irradiated) sites has significant impact on treatment outcome. PMID- 7538239 TI - Impact of prostate size on the outcome of transurethral laser evaporation of the prostate for benign prostatic hyperplasia. AB - OBJECTIVES: The aim of this study was to evaluate efficacy and safety of transurethral evaporation of the prostate (TUEP) using neodymium:yttrium-aluminum garnet (Nd:YAG) laser in prostate glands of various sizes. METHODS: One hundred consecutive patients with benign prostatic hyperplasia (BPH) and prostate volumes less than 40 cc (group I, n = 41), 41 to 80 cc (group II, n = 39), and more than 80 cc (group III, n = 20), who had preoperative prostate volume estimation by transrectal ultrasound and had completed a minimum of 3 months' follow-up, underwent TUEP. At baseline, and at 3 and 6 months, American Urological Association (AUA) score, peak flow rate (PFR), postvoid residual urine (PVR), and complications, if any, were documented. RESULTS: There were no significant differences in failure rates, complications, or ability to improve symptom score, PFR, and PVR between patients with prostate glands of various sizes. The mean improvement in PFR at 6 months was: group I, 9.9 cc/s (116%); group II, 7.4 cc/s (81%); and group III, 9.2 cc/s (107%). Reduction in AUA score was: group I, 14.6 (63%); group II, 17.7 (71%); and group III, 16.2 (70%). PVR was: group I, 62.5 cc (51%); group II, 31.4 cc (16%), and group III, 71 cc (83%) (differences not significant). The patients in urinary retention were separately analyzed (group I, 9, group II, 12, and group III, 5) and mean PFR at 6 months was: group I, 18.5 cc/s, group II, 15 cc/s, and group III, 17.1 cc/s. Mean AUA score at 6 months was: group I, 25.8; group II, 21; and group III, 23.6. Mean PVR score was: group I, 370 cc, group II, 439 cc; and group III, 400 cc (differences not significant). Mean postoperative catheterization time was higher in patients with glands larger than 80 cc (2.2 versus 2.9 versus 4.7 days in groups I, II, and III, respectively, P < 0.009 between groups II and III). Incidence of urinary tract infection (10 versus 0%) was greater in patients receiving only 48-hour as opposed to 10-day postoperative antibiotics. CONCLUSIONS: TUEP appears to be a safe and effective treatment for relief of symptoms of BPH and improvement of PFR in patients with all sizes of prostate glands. PMID- 7538241 TI - Relationship between changes in prostate-specific antigen and the percent of prostatic epithelium in men with benign prostatic hyperplasia. AB - OBJECTIVES: Pretreatment knowledge of prostate gland histology would allow a more scientifically based selection of medical therapy for men with benign prostatic hyperplasia (BPH) and may increase the effectiveness of the pharmacologic agents available. Changes in prostate-specific antigen (PSA), or PSA velocity, may reflect prostatic epithelial growth in BPH. Our objective was to determine if PSA velocity prior to diagnosis correlated with the relative amount of epithelium in BPH tissue. METHODS: We evaluated 39 men with BPH who had serial PSA determinations (mean, 5.4) on frozen sera from 2.3 to 25.1 years before diagnosis, and archival material from simple prostatectomy available for pathologic evaluation. We used an immunoenzymatic staining technique for PSA to bind prostatic epithelium selectively so that color differences in the stained tissue sections could be used to quantify stroma, epithelium, and glandular lumina. RESULTS: The average percentage of epithelium (%E) was 12.4 and the average stroma-epithelial ratio (SER) was 6.6. The correlation of PSA velocity for the three visits nearest to prostatectomy (n = 32) versus %E and SER was significant (P = 0.003 for both). The PSA value nearest to prostatectomy (n = 39) was directly correlated with %E and SER (P = 0.0001 and P = 0.001, respectively). CONCLUSIONS: These data suggest that PSA and PSA velocity are directly related to the histologic makeup of the prostate in men with BPH. Thus, pretreatment evaluation of PSA could be useful as part of an evaluation to direct BPH therapy. PMID- 7538242 TI - Structural comparison of prostate-specific antigen and human glandular kallikrein using molecular modeling. AB - OBJECTIVES: Prostate-specific antigen (PSA), the most useful tumor marker for prostate cancer, is one of three members of the human kallikrein family of serine proteases. PSA and human glandular kallikrein (hK2, previously called hGK-1) share extensive homology and are both produced in the prostate under androgen control. Our goals were to use molecular modeling techniques to generate models of the tertiary structure of PSA and hK2 and to compare their molecular features and areas of homology using these models. METHODS: Models of PSA and hK2 were generated by extrapolating from available crystallographic coordinates and amino acid sequences of homologous members of the serine protease family using standard comparative methods. RESULTS: Porcine kallikrein (57% homology) and rat tonin (53% homology) were used as templates for PSA. Porcine kallikrein (67% homology) was used as a template for hK2. The models were superimposed to define regions of nonhomology between PSA and hK2. CONCLUSIONS: Three-dimensional protein models of PSA and hK2 were generated. These models have potential uses in analyzing antigen antibody interactions, modeling of inhibitor complexes of both PSA and hK2, and furthering our understanding of the molecular interactions involved in the clinical detection of PSA and hK2. PMID- 7538243 TI - Use of prostate-specific antigen and tumor volume in predicting needle biopsy grading error. AB - OBJECTIVES: To study the discrepancy between histologic grades of needle biopsy and prostatectomy specimens and to define the potential role of prostate-specific antigen (PSA), PSA density, and tumor volume in predicting the tumor grade. METHODS: Histopathologic grades on needle biopsy and subsequent radical prostatectomy specimens were compared in 135 patients with clinically organ confined prostate cancer. The frequency of undergrading (difference of one or more grades) by needle biopsy was then compared to PSA, PSA density, and other preoperative parameters. RESULTS: The frequency of undergrading by one level was 45% using the M. D. Anderson Hospital (MDAH) system and 47% using the Gleason system. When the tumor grades were classified into two categories, undergrading rates of 26% (low to high grade by the MDAH system) and 21% (moderately well to poorly differentiated by the Gleason system) were noted. Serum PSA and PSA density correlated with the grading error rate: the higher the PSA and PSA density values, the higher the biopsy undergrading rate. PSA showed a significant correlation with undergrading (P = 0.0019). Undergrading occurred in 32% of patients with a PSA more than 4 and 10 ng/mL or less and in 47% of those with a serum PSA more than 10 ng/mL. In all cases when the PSA was 4 ng/mL or less, the needle biopsy results correctly predicted the final tumor grade category. Tumor volume was not associated with frequency of undergrading. CONCLUSIONS: The ability to predict tumor grade can be enhanced by considering the serum PSA value. PMID- 7538244 TI - Laparoscopic pelvic lymph node dissection, laparoscopically assisted seminal vesicle mobilization, and total perineal prostatectomy versus radical retropubic prostatectomy for prostate cancer. AB - OBJECTIVES: The outcomes of patients with prostate cancer who were candidates for radical prostatectomy were compared with patients who underwent either: (1) radical retropubic prostatectomy (RRP); or (2) laparoscopic pelvic lymph node dissection, laparoscopically assisted seminal vesicle mobilization, and total perineal prostatectomy (LN-SV-TPP). METHODS: The staging, surgical, and early postoperative characteristics of 10 consecutive patients treated by RRP were compared with 12 consecutive patients who underwent LN-SV-TPP. RESULTS: Patients who underwent LN-SV-TPP versus RRP had respective median blood loss of 450 versus 1250 cc (P = 0.001), median anesthesia time of 330 versus 287.5 minutes (P = 0.05), median surgical time of 237.5 versus 237.5 minutes (P = 0.6), median units transfused of 0 versus 1 (P = 0.05), median time to ambulation of 1 versus 2 days (P = 0.002), median time to oral intake of 1 versus 3.5 days (P < 0.001), median hospital stay of 3 versus 6 days (P < 0.001), and median morphine requirements of 44 versus 119 mg (P < 0.001). CONCLUSIONS: LN-SV-TPP is less morbid than RRP concerning blood loss, blood transfusions, pain, and postoperative recovery. Compared with LN-SV-TPP, RRP is faster and is particularly indicated for ease of performing a nerve-sparing radical prostatectomy. PMID- 7538246 TI - Intermittent androgen suppression in the treatment of prostate cancer: a preliminary report. AB - OBJECTIVES: To test the feasibility of using intermittent androgen suppression in the treatment of prostate cancer by taking advantage of the reversible action of medical castration. METHODS: Observations were made on a group of 47 patients (clinical Stage D2, 14; D1, 10; C, 19; B2, 2; and A2, 2) with a mean follow-up time of 125 weeks. Treatment was initiated with combined androgen blockade and continued for at least 6 months until a serum prostate-specific antigen (PSA) nadir was observed. Medication was then withheld until the serum PSA increased to a mean value between 10 and 20 ng/mL. This cycle of treatment and no treatment was repeated until the regulation of serum PSA became androgen independent. RESULTS: The first two treatment cycles lasted 73 and 75 weeks, with a mean time off therapy of 30 and 33 weeks and an overall mean percentage time off therapy of 41% and 45%, respectively. The mean time to achieve a nadir level of serum PSA was 20 weeks in cycle 1 and 18 weeks in cycle 2. Serum testosterone returned to the normal range within 8 weeks (range, 1 to 26) of stopping treatment. The off treatment period in both cycles was associated with an improvement in sense of well-being and the recovery of libido and potency in the men who reported normal or near-normal sexual function before the start of therapy. In 7 patients with Stage D2 disease, the cancer progressed to an androgen-independent state. The mean and median times to progression were 128 weeks and 108 weeks, respectively. Seven patients have died, 1 from a noncancer-related illness, with mean and median overall survival times of 210 weeks and 166 weeks, respectively. CONCLUSIONS: Prostate cancer is amenable to control by intermittent androgen suppression. This approach affords an improved quality of life when the patient is off therapy. It also results in reduced toxicity and cost of treatment and possibly delays tumor progression. Whether survival is affected in a beneficial or adverse way remains to be studied in a randomized, prospective study. PMID- 7538245 TI - Selection of men at high risk for disease recurrence for experimental adjuvant therapy following radical prostatectomy. AB - OBJECTIVES: Following surgery, men with recurrent prostate cancer have an isolated elevation in serum prostate-specific antigen (PSA) well in advance of measurable metastatic disease. Rational patient selection for new forms of adjuvant therapy, for example, gene therapy, is imperative. METHODS: In a retrospective study of two cohorts, we used proportional hazards regression analysis to develop and validate a multifactor model for identifying men who are at high risk of cancer recurrence. The model cohort consisted of 216 men with clinical Stage T2b and T2c treated by 1 urologist. The validation cohort consisted of 214 men with Stage T2b and T2c disease. RESULTS: A model for log relative risk, Rw, used serum PSA with a sigmoidal transformation (PSAST), radical prostatectomy Gleason score (GS), and pathologic stage (PS) as specimen confined or nonspecimen confined: Rw = (PSAST x 0.06) + (GS x 0.54) + (PS x 1.87). Recurrence risk categories were determined as low risk if Rw is less than 4.0, intermediate risk if it is 4.0 to less than 5.75, and high risk if Rw is more than 5.75. The observed Kaplan-Meier actuarial analysis of the three risk groups correlated well with the predictions determined for the model cohort. We then validated this model independently using a second cohort of 214 men with similar age, stage, and grade treated by 3 different urologists at two different institutions. CONCLUSIONS: The recurrence rates for men in the high-risk group are similar to those for men with positive lymph nodes and justifies exploration of experimental adjuvant therapy within this group using this model of patient selection. PMID- 7538247 TI - False-positive serum prostate-specific antigen values in a patient with non Hodgkin lymphoma of the kidney. AB - Prostate-specific antigen (PSA) is the clinically most useful tumor marker for prostate cancer. Although false-positive elevations have been reported due to disease processes outside the prostate gland with the use of the polyclonal assay, such false-positive test results have been exceedingly rare with the use of the monoclonal assay. We report the case of a patient diagnosed with a B-cell lymphoma of the kidney and a significant elevation of serum PSA levels by monoclonal assay in the absence of either inflammatory or malignant prostate disease. PSA returned to normal during lymphoma-specific chemotherapy with a cyclophosphamide, mechlorethamine, vincristine, procarbazine, prednisone regimen. Possible explanations and clinical implications are discussed. PMID- 7538248 TI - Expression, biological activity and kinetics of production of recombinant ovine TNF-alpha. AB - Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation. PMID- 7538249 TI - Characterisation of a membrane receptor on ruminants and equine platelets and peripheral blood leukocytes similar to the human integrin receptor glycoprotein IIb/IIIa (CD41/61). AB - This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band did not appear owing to the absence of labelling with sulpho-NHS-biotin. Co.35E4 and Co.20A1 recognised two different Ca2+ complex independent epitopes. The glycoprotein IIb/IIIa was present on ruminant and equine granulocytes, monocytes and platelets. However, binding on granulocytes and monocytes was due to the adsorption of membrane platelet fragments. PMID- 7538251 TI - Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. AB - It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1 specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1. PMID- 7538250 TI - Antigenic and sequence variation in the C-terminal unique domain of the Epstein Barr virus nuclear antigen EBNA-1. AB - The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 is essential for viral genome maintenance in vitro and may be the only EBV protein expressed by the majority of latently infected cells in vivo. EBNA-1 may therefore be critical to the evasion of host immunity which allows persistent infection. EBNA-1 includes a polymorphic internal repeat domain of unknown significance and unique regions which mediate all known functional activities and which have hitherto been assumed to be conserved between strains. Monoclonal antibodies were generated using a construct based on EBNA-1 of the prototype B95-8 strain, deleted for the repeat domain. These antibodies showed a limited profile of recognition of EBNA-1 in common laboratory EBV+ cell lines by immunoprecipitation and immunostaining. The observed antigenic heterogeneity also extended to spontaneously transformed B lymphoblastoid cell lines (LCLs) representing viral isolates circulating within US and UK populations. DNA fragments spanning the C-terminal unique domain of EBNA-1 from eleven spontaneous LCLs were amplified by polymerase chain reaction for sequencing, which directly demonstrated extensive and unexpected variability between diverse type 1 EBV isolates. The resulting polymorphism affects most of the putative MHC Class I binding epitopes which could be identified within this region using published sequence motifs, and influences MHC binding by variants of at least one such peptide in the processing mutant cell line T2. These findings could be related to the apparent lack of recognition of EBNA-1 by cytotoxic T lymphocytes. PMID- 7538253 TI - Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA. AB - A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2'-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form. PMID- 7538252 TI - HIV infection of choriocarcinoma cell lines derived from human placenta: the role of membrane CD4 and Fc-Rs into HIV entry. AB - We have shown previously that trophoblast cells from human placenta can be infected with HIV-1 and a productive infection established. Recently, (1991, J. Virol. 65, 2102-2107) Zachar et al. and D. M. Phillips and X. Tan (1992, AIDS Res. Hum. Retroviruses 8, 1697-1705) have described in vitro infection of choriocarcinoma cell lines. Using choriocarcinoma cell lines (JAR, BeWo, and FD25, a trophoblast-derived cell line) we have infected these cells with several laboratory strains of virus and have shown that this can be prevented either by sCD4 or by antibodies to CD4. This provides strong evidence that the infection may be through CD4. In addition, we have found that infection of JAR and FD25 cells by HIV-1/Lai was enhanced in the presence of human antisera to HIV-1. This supports an additional role for immunoglobulin receptors (Fc-R) in the entry of virus into the cell. We report here evidence that CD4 and Fc-R on the cell surface play crucial roles in the entry of HIV into such placenta-derived cell lines. PMID- 7538255 TI - Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17. AB - Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17 (BTV-17) were investigated with a panel of five neutralizing monoclonal antibodies (MAbs). One MAb (MAb 034) was originally raised to BTV serotype 10 (BTV-10) but also neutralizes BTV-17 (P. V. Rossitto, and N. J. MacLachlan, 1992, J. Gen. Virol. 73, 1947-1952). Competitive binding studies indicate that the MAbs recognize at least two epitopes on the neutralizing outer capsid protein VP2 of BTV-17. The MAbs were used to select neutralization resistant variant [escape mutant (EM)] viruses and to determine the phenotypic characteristics of these EM viruses by immunoprecipitation and neutralization assays. Sequencing of the L2 gene, which encodes VP2, identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. Four amino acids in three regions of VP2 are critical to the expression of the epitopes recognized by the panel of neutralizing MAbs. Amino acid 199 affects the binding of MAbs 17.82, 17.83, and 17.813; amino acid 213 affects the binding of MAb 17.85; and amino acids 327 and 582 synergistically affect the binding of MAb 034. Similarly, amino acids 327 and 402 synergistically affect the binding of MAb 034 to BTV-10 (C. D. DeMaula, H. W. Heidner, P. V. Rossitto, C. M. Pierce, and N. J. MacLachlan, 1993, Virology 195, 292-296), suggesting that the neutralizing epitope common to BTV-10 and BTV-17 has a similar location in VP2 of these two antigenically distinct viruses. PMID- 7538254 TI - Cyclosporin A and FK506 block induction of the Epstein-Barr virus lytic cycle by anti-immunoglobulin. AB - The Epstein-Barr virus (EBV) BZLF1 gene is expressed early upon induction of the viral lytic cycle and its protein product is unique in its ability to disrupt viral latency in some latently infected cell lines. Anti-immunoglobulin (anti-Ig) treatment of the Burkitt's lymphoma cell line Akata, which bears surface IgG, has previously been shown to synchronously induce transcription of the BZLF1 gene (K. Takada and Y. Ono, 1989, J. Virol. 63, 445-449). We have previously shown that anti-Ig induction of Akata cells activates expression of the tumor necrosis factor alpha (TNF-alpha) gene via a calcineurin-dependent mechanism (Goldfeld et al., 1992, Proc. Natl. Acad. Sci. USA 89, 12198-12201). Here, we report that anti Ig induction of the EBV lytic cycle in Akata cells can be blocked by the immunosuppressants cyclosporin A and FK506. Furthermore, we demonstrate that synergistic induction by phorbol ester and calcium ionophore of a BZLF1 promoter driven reporter construct in an EBV-negative BL cell line can be inhibited by addition of cyclosporin A. Thus, analogous to activation of TNF-alpha gene in Akata cells, anti-Ig induction of the BZLF1 promoter is most likely mediated by calcineurin and probably involves translocation to the nucleus of a transcription factor sequestered in the cytoplasm. As such, immunosuppressants may be useful probes for dissecting B cell activation pathways involved in regulating EBV gene transcription. PMID- 7538256 TI - Innervation of the skin of the forearm in diabetic patients: relation to nerve function. AB - Complications of diabetes include sensory and autonomic neuropathy. The aim of the present paper was to study the degree of sensory and autonomic neuropathy and correlate these findings with the distribution and density of neuropeptidergic nerve fibers in the skin of the forearm of diabetic patients and healthy controls. We investigated 30 diabetics (24 type 1 and 6 type 2) and compared them with 13 healthy controls. There were no differences between the groups with respect to density and distribution of nerve fibers displaying immunoreactivity to the pan-neuronal marker PGP 9.5 and sensory and parasympathetic neuropeptides (substance P, calcitonin gene-related peptide and vasoactive intestinal peptide). By contrast, nerve fibers containing neuropeptide Y, a marker of sympathetic neurons, were reduced in number in the diabetic patients. C-fiber function (measured as the axon-reflex-evoked flare response) became impaired with increasing age in all subjects. The diabetic patients, however, showed a reduced flare compared to age-matched healthy controls. The reduction was particularly prominent in the younger patients (20-50 years). There was a greater reduction of the flare in neuropathic patients than in non-neuropathic patients, but there was no correlation between the degree of functional impairment and the duration of the disease. PMID- 7538259 TI - The development of gene transfer for cystic fibrosis. PMID- 7538258 TI - Epidermal regeneration and occlusion. AB - Disruption of the stratum corneum, by tape stripping or chemical injury, results in epidermal recovery of the skin barrier. In human skin, 40-48 h after surface trauma, epidermal proliferation is maximal. In a previous study in mice, occlusion with plastic or the hydrocolloid Duoderm proved to inhibit the regenerative response. The aim of the present investigation was to find out whether occlusion modulates epidermal proliferation following removal of the stratum corneum in normal healthy volunteers by sellotape stripping. Epidermal proliferation was assessed, using a multiparameter approach, by measuring ornithine decarboxylase activity, keratin 16 expression and DNA synthesis. Following tape stripping without subsequent occlusion, ornithine decarboxylase activity, keratin 16 expression and DNA synthesis were induced to the same extent as observed in previous studies. However, in contrast to the experiments in mice, no indication of a modulation of these responses was observed by the application of the hydrocolloids Duoderm and Comfeel. In human skin, a direct effect of the artificial restoration of the skin barrier on epidermal regeneration remains unsubstantiated. PMID- 7538257 TI - Low dose versus medium dose UV-A1 treatment in severe atopic eczema. AB - Twenty-two patients with severe atopic eczema were included in a therapy study with UV-A1 (wavelengths > 340 nm) treatment. The patients were divided into two dose groups, each consisting of 11 patients. One group received 10 J/cm2 and the other 50 J/cm2 five times a week for 3 consecutive weeks. No topical or systemical steroids or antihistamines were allowed. Using the SCORAD index as a measure of disease activity before onset of therapy and after 10 and 15 treatments, we observed a significant improvement in both dose groups after 15 treatments (10 J/cm2: p < 0.05, 50 J/cm2: p < 0.005). After 10 treatments only the improvement in the 50 J/cm2 group was significant (p < 0.005); the difference between the two dose groups was significant (p < 0.05). The clinical efficacy of treatment was reflected neither by a decrease of serum IgE nor by a decrease of elevated serum levels of soluble adhesion molecules sICAM-1 and sELAM-1 in the two dose groups. In contrast, a marked but not significant decrease of serum ECP could be observed in the 50 J/cm2 group only. We conclude from these and other published data that although 10 J/cm2 UV-A1 has a limited effect on patients with severe atopic eczema, higher doses are of higher efficiency in the treatment of this condition. PMID- 7538262 TI - Familial Dandy-Walker malformation associated with macrocephaly, facial anomalies, developmental delay, and brain stem dysgenesis: prenatal diagnosis and postnatal outcome in brothers. A new syndrome? AB - Brothers are reported with an apparently new constellation of manifestations including Dandy-Walker complex (DWC), migrational brain disorder, macrocephaly, and facial anomalies. The first brother presented at birth, the second was detected prenatally with DWC and the pregnancy terminated. Fetal brain histopathology showed DWC associated with brainstem dysgenesis. Inheritance is likely autosomal or X-linked recessive. An extensive review of the differential diagnosis of DWC is provided. PMID- 7538260 TI - Epirubicin plus medroxyprogesterone as second-line treatment of advanced prostatic cancer. A study by the Italian Trials in Medical Oncology Group. AB - The evaluation of drug efficacy in patients with advanced prostatic cancer who have progressed to hormonal therapy is difficult, although palliation of the pain related to bone involvement still represents an important endpoint. In this study, epirubicin (EpiADM) plus medroxyprogesterone acetate (MPA) were given to advanced prostatic cancer patients with symptomatic bone involvement who had progressed to hormonal therapy. EpiADM was administered at a dose of 30 mg/m2 i.v. weekly and MPA at a daily dose of 1,000 mg p.o. for the first month and 500 mg thereafter. Fifty-four patients entered the trial, all of whom were evaluable. Amelioration of pain and a > or = 50% reduction in analgesic intake were observed in 52% of cases, with a mean duration of 4 months. Of the 28 responsive patients, 26 had already received two lines of hormonal therapy or were resistant to first line therapy. Of the 23 patients with measurable lesions, 6 obtained a > or = 50% tumor shrinkage at these sites. The treatment was well tolerated, and no cardiac toxicity was observed up to a total cumulative EpiADM dose of 660 mg/m2. In conclusion, this regimen seems to have a palliative effect in patients with advanced prostatic cancer who have progressed to hormonal therapy, and it is feasible in an outpatient setting. PMID- 7538261 TI - Distribution of extracellular matrix receptors in various forms of glomerulonephritis. AB - Integrins are heterodimeric transmembrane receptor glycoproteins consisting of alpha and beta subunits that mediate adhesion and interactions between cells and extracellular matrix. Such interactions may be perturbed in various pathologic states, resulting in the altered phenotypic expressions of the integrins in affected tissues. To ascertain the alterations in integrins in various renal diseases, their distribution was investigated in different forms of glomerulonephritis by indirect immunofluorescence and immunoelectron microscopy using specific antibodies directed against beta 1 integrins and integrin alpha v beta 3 (vitronectin receptor). In addition, the distribution of certain extracellular matrix components (ie, fibronectin, vitronectin, and type IV collagen) was examined. Integrin beta 1 and alpha v beta 3 were highly expressed in proliferating mesangial cells in immunoglobulin A nephropathy, membranoproliferative glomerulonephritis type I and diffuse proliferative lupus nephritis. Their putative ligands (ie, fibronectin, vitronectin, and type IV collagen) also were increased in the expanded mesangial regions. In immunoglobulin A nephropathy, integrin beta 1 and alpha v beta 3 were seen by immunoelectron microscopy to be localized to the mesangial cell membranes in close proximity to the immune complex deposits; however, fibronectin and vitronectin immunoreactivities were observed in the mesangial immune complex deposits. Similarly, vitronectin also was detected in the immune complex deposits of other forms of proliferative nephritis, ie, membranoproliferative glomerulonephritis type I and diffuse proliferative lupus nephritis. In diffuse proliferative lupus nephritis, the cellular crescents displayed immunoreactivity toward integrin alpha v beta 3 and vitronectin. In nonimmune complex glomerular disease associated with nephrotic syndrome (ie, minimal change nephrotic syndrome), integrin alpha 3 beta 1, which normally has a linear capillary distribution, was decreased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538263 TI - Joubert syndrome with congenital hepatic fibrosis: an entity in the spectrum of oculo-encephalo-hepato-renal disorders. AB - Joubert syndrome is an autosomal recessive inherited condition characterized by agenesis or hypoplasia of the cerebellar vermis, retinal dystrophy, chorioretinal colobomata, oculomotor abnormalities, episodic hyperpnea, ataxia, and mental retardation. Congenital hepatic fibrosis has not previously been described in Joubert syndrome. We report two unrelated children with Joubert syndrome and hepatosplenomegaly. On histopathological examination, both had congenital hepatic fibrosis. Both were also found to have congenital medullary cystic disease of the kidneys. Joubert syndrome appears to be one of a spectrum of congenital malformation syndromes involving the central nervous system, eye, liver and kidneys. PMID- 7538265 TI - The expression of variant CD44 in nasopharyngeal carcinoma is unrelated to expression of LMP-1. AB - CD44 expression in undifferentiated nasopharyngeal carcinoma has been analyzed by sequencing cDNA clones and by immunohistochemical staining using monoclonal antibodies specific for defined variant exons. Both sequencing and antibody staining revealed expression of CD44 variants containing exon v6, which has been linked to tumor metastasis. Although nasopharyngeal carcinoma typically contains a mixture of epithelial tumor cells and infiltrating lymphocytes, the CD44 v6 variant expression could be assigned to the tumor cells in biopsies and in a mouse xenograft system. Consistent with earlier studies, biopsies from about 60% of Chinese nasopharyngeal carcinoma cases expressed the Epstein-Barr virus LMP-1 protein, although all contained EBV DNA and expressed the EBER genes. The antibody specific for CD44 exon v6 generally gave a focal pattern of staining in epithelial tumor cells, but the expression of this variant did not appear to be related to the expression of the LMP-1 protein in the tumor. PMID- 7538267 TI - Living-donor liver transplantation at UCLA. AB - BACKGROUND: Living-donor (LD) liver transplantation has been developed as an alternative to overcome the shortage of cadaver donor organs for pediatric recipients. PATIENTS AND METHODS: We reviewed our experience with 9 LD transplants performed between August 25, 1993 and August 3, 1994. The median recipient age and weight were 13 months and 10 kilograms. Left lateral segments from parental donors, with aortic inflow via saphenous vein grafts, were used in all cases. RESULTS: At a median follow-up of 160 days, all donors were alive and well. Recipient and graft survival were both 89%. Rates of hepatic artery thrombosis, portal vein thrombosis, biliary complications, and acute rejection were 22%, 11%, 11%, and 67%, respectively. CONCLUSIONS: Excellent outcome can be achieved with LD liver transplantation in small children with minimal donor risk. This procedure has the potential to emerge as the preferred treatment for pediatric liver transplant candidates for whom it is an option. PMID- 7538264 TI - Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. AB - VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the ECs lining nearby microvessels to proliferate, to migrate, and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular ECs hyperpermeable so that they spill plasma proteins into the extravascular space, leading to the clotting of extravasated fibrinogen with deposition of a fibrin gel. Extravascular fibrin serves as a provisional matrix that favors and supports the ingrowth of new blood vessels and other mesenchymal cells that generate mature, vascularized stroma. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma derived matrix precede and accompany the onset of EC division and new blood vessel formation. It would seem, therefore, that tumors have "borrowed" fundamental mechanisms that developed in multicellular organisms for purposes of tissue defense, renewal, and repair. VPF/VEGF, therefore has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important, perhaps essential, elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in subtoxic concentrations that are more than sufficient to induce angiogenesis. Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating the expression of VPF/VEGF. In fact, there is already one clear example of this. TGF-alpha is a potent angiogenic factor but does not itself increase microvascular permeability. However, TGF-alpha strikingly upregulates VPF/VEGF expression in cultured keratinocytes and is thought to be responsible, at least in part, for the overexpression of VPF/VEGF in psoriasis. Moreover, overexpression of TGF-alpha, along with that of the EGF receptor with which it interacts, is characteristic of many malignant tumors, raising the possibility that TGF-alpha acts to stimulate VPF/VEGF expression in other types of epithelial cells and in this manner induces angiogenesis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538266 TI - Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. AB - We investigated the role of hepatocyte apoptosis in four different murine models of acute inflammatory liver failure. Liver damage induced in D-galactosamine sensitized mice by endotoxin infection was initiated by processes typical of apoptosis, ie, chromatin condensation, DNA fragmentation, and formation of intracellular apoptotic bodies. DNA was cleaved into oligonucleosomal fragments in the liver before a significant rise of alanine aminotransferase in plasma occurred. Passive immunization against tumor necrosis factor (TNF) completely inhibited the injury caused by endotoxin. Direct injection of recombinant TNF alpha also caused DNA fragmentation followed by alanine aminotransferase release into the plasma. Pretreatment of mice with interleukin-1 beta, which is known to suppress TNF-induced lethality, completely prevented apoptosis and liver failure in this model. These results demonstrate the causal role of TNF in endotoxin induced hepatic apoptosis. TNF-inducible hepatocyte apoptosis in vivo was not only observed in D-galactosamine-sensitized mice, but also when the alternative transcriptional inhibitor actinomycin D was used. In mice injected with the TNF inducing T cell mitogen concanavalin A, hepatic apoptosis was even noticed without requirement of additional sensitizers. We conclude that TNF-induced hepatocyte apoptosis is an early, general, and possibly causal event during experimental liver failure triggered by inflammatory stimuli. PMID- 7538268 TI - Laparoscopic gastroenterostomy for duodenal obstruction. AB - BACKGROUND: It is not yet clear where laparoscopic procedures will fit into the armamentarium of the surgeon. Over the past decade, there has been a clear trend toward minimally invasive procedures for palliation of inoperable cancer. Traditionally, when duodenal obstruction occurs secondary to a disease process, gastric bypass through laparotomy is required. PATIENTS AND METHODS: Between November 13, 1992 and September 13, 1994, 10 patients underwent laparoscopic gastroenterostomy for duodenal obstruction. In 9 patients, the procedure was carried out for malignant obstruction; in 1 patient, duodenal obstruction was secondary to chronic scarring from benign peptic ulcer disease. Eight of these patients already had biliary decompression through radiologic or endoscopic means. One patient underwent laparoscopic cholecystenterostomy for biliary obstruction in addition to the laparoscopic gastroenterostomy. RESULTS: Laparoscopic gastroenterostomy was successfully completed in 8 of the 10 patients. In 2, conversion to open surgery was necessary. There was no mortality related to this operative approach. CONCLUSIONS: Laparoscopic gastroenterostomy is a safe procedure for treatment of duodenal obstruction. Good palliation can be expected in patients with obstruction of the duodenum secondary to advanced malignancies. PMID- 7538269 TI - Aprotinin reduces the need for blood products during liver transplantation. AB - BACKGROUND: Bleeding complications and blood product consumption have been a major concern during liver transplantation. Prevention of plasminogen activation and fibrinolysis by aprotinin administration has been shown to reduce perioperative bleeding during operations associated with high blood-product consumption. PATIENTS AND METHODS: Use of blood-products (packed red cells, frozen plasma, platelets, and cryoprecipitate) was analyzed both during the three stages of orthotopic liver transplantation and during total hospitalization of the 26 patients transplanted without aprotinin and the subsequent 40 patients with aprotinin. A similar analysis was performed for 15 patients immediately before and after the introduction of aprotinin to eliminate the "learning curve" effect for liver transplantation. The effect of epsilon-amino-caproic acid was analyzed as 13 patients received neither epsilon-aminocaproic acid nor aprotinin and 13 patients received epsilon-aminocaproic acid but not aprotinin. RESULTS: There was a significant reduction in total hospital use of cryoprecipitate, frozen plasma, platelets, and red cells in the aprotinin-treated patients. This reduction was seen during the anhepatic and reperfusion stages of liver transplantation. There was no difference in blood product consumption between the groups who were or were not treated with epsilon-aminocaproic acid. CONCLUSION: Aprotinin significantly reduces the need for red cell, frozen plasma, platelet, and cryoprecipitate transfusion use during orthotopic liver transplantation, and appears to be more efficacious than epsilon-aminocaproic acid. PMID- 7538270 TI - Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution. AB - Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538271 TI - Survival and activity of Pseudomonas sp. strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton. AB - Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate. PMID- 7538272 TI - Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences. AB - Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed. PMID- 7538273 TI - Sequence analysis of a gene cluster involved in metabolism of 2,4,5 trichlorophenoxyacetic acid by Burkholderia cepacia AC1100. AB - Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases. PMID- 7538274 TI - Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. AB - A total of six 16S rRNA targeted oligonucleotide probes were used to quantify Fibrobacter abundance and diversity in the gastrointestinal contents of a pony. Approximately 12% of the total 16S rRNA extracted from cecal contents hybridized with a Fibrobacter genus-specific probe and a Fibrobacter succinogenes species specific probe. However, no significant hybridization was observed with a probe for the species. Fibrobacter intestinalis or with three probes for F. succinogenes subspecies. This suggested the presence of a previously undescribed population of F. succinogenes-like organisms. Novel lineages of F. succinogenes were subsequently identified by using PCR primers specific for the genus to amplify sequences coding for 16S rRNA from DNA extracted from cecal contents. Sequences of the cloned amplification products were shown to be affiliated with F. succinogenes but represented two distinct, and novel, lines of descent within the species. PMID- 7538276 TI - Application of a strain-specific rRNA oligonucleotide probe targeting Pseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment. AB - Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain. The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P. fluorescens Ag1. The correlation between the ribosomal content of P. fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h. Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content. The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy. After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell. After 1 day of carbon starvation, the rRNA content had decreased to 20%. When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30 degrees C. P. fluorescens Ag1 was used in a mesocosm release experiment. The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces. The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P. fluorescens Ag1 in the mesocosm experiment. PMID- 7538275 TI - TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4. AB - Burkholderia (Pseudomonas) cepacia PR1(23) has been shown to constitutively express to toluene catabolic pathway distinguished by a unique toluene ortho monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of < 70 and > 100 kb. A derivative strain cured of the largest plasmid, PR1(23) Cure, was unable to grow on phenol or toluene as the sole source of carbon and energy, which requires expression of the Tom pathway. Transfer of the larger plasmid from strain G4 (the parent strain inducible for Tom) enabled PR1(23) Cure to grow on toluene or phenol via inducible Tom pathway expression. Conjugal transfer of TOM23c from PR1(23) to an antibiotic-resistant derivative of PR1(23) Cure enabled the transconjugant to grow with either phenol or toluene as the sole source of carbon and energy through constitutive expression of the Tom pathway. A cloned 11.2-kb EcoRI restriction fragment of TOM23c resulted in the expression of both Tom and catechol 2,3-dioxygenase in Escherichia coli, as evidenced by its ability to oxidize trichloroethylene, toluene, m-cresol, o-cresol, phenol, and catechol. The largest resident plasmid of PR1 was identified as the source of these genes by DNA hybridization. These results indicate that the genes which encode Tom and catechol 2,3-dioxygenase are located on TOM, an approximately 108-kb degradative plasmid of B. cepacia G4. PMID- 7538277 TI - Detection of ammonium-oxidizing bacteria of the beta-subclass of the class Proteobacteria in aquatic samples with the PCR. AB - The PCR was used as the basis for the development of a sensitive and specific assay for the detection of ammonium-oxidizing bacteria belonging to the beta subclass of the class Proteobacteria. PCR primers were selected on the basis of nucleic acid sequence data available for seven species of nitrifiers in this subclass. The specificity of the ammonium oxidizer primers was evaluated by testing known strains of nitrifiers, several serotyped environmental nitrifier isolates, and other members of the Proteobacteria, including four very closely related, nonnitrifying species (as determined by rRNA sequence analysis). DNA extracts from 19 bacterio-plankton samples collected from Lake Bonney, Antarctica, and the Southern California Bight were assayed for the presence of ammonium oxidizers. By using a two-stage amplification procedure, ammonium oxidizers were detected in samples collected from both sites. Chemical data collected simultaneously support the occurrence of nitrification and the presence of nitrifiers. This is the first report describing PCR primers specific for ammonium-oxidizing bacteria and the successful amplification of nitrifier genes coding for rRNA from DNA extracts from natural samples. This application of PCR is of particular importance for the detection and study of microbes, such as autotrophic nitrifiers, which are difficult or impossible to isolate from indigenous microbial communities. PMID- 7538278 TI - Biochemical analysis of starch degradation by Ruminobacter amylophilus 70. AB - Ruminobacter amylophilus is an obligate anaerobe that uses only alpha-linked glucose molecules (i.e., maltose, maltodextrins, and starch) as a source of energy, making it an excellent model for the study of bacterial starch degradation. Constitutive amylase, amylopectinase, and pullulanase activities were found in intracellular and extracellular fractions of R. amylophilus. However, extracellular activities apparently resulted from cell lysis. Both soluble and membrane-bound polysaccharidase activities were detected. Most of the soluble polysaccharidase activity partitioned with the periplasmic cell fraction. No alpha-glucosidase or maltase activity was detected in either the cellular or extracellular fraction. In addition, intact cells of R. amylophilus bound U-14C starch. This binding could be saturated and was constitutive and sensitive to proteinase K, indicating protein or protein complex mediation. Competition experiments showed that these starch-binding sites had equally high affinities for starch and maltodextrins larger than maltotriose. The sites had a reduced affinity for maltose and virtually no affinities for glucose and nonstarch polysaccharides. These findings suggest that R. amylophilus binds starch molecules to the cell surface as an initial step in transporting the molecule through the outer membrane and into the periplasmic space. Extracellular polysaccharides do not appear to be involved in starch degradation. PMID- 7538282 TI - Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes. AB - Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia. PMID- 7538279 TI - Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. AB - The phylogenetic diversity of small-subunit rRNA genes associated with the domain Bacteria was examined (by using previously defined operational taxonomic units [C. L. Moyer, F.C. Dobbs, and D. M. Karl, Appl. Environ. Microbiol. 60:871-879, 1994]; those for Pele's Vents Bacteria are hereafter abbreviated PVB OTUs) with samples from a microbial mat at an active, deep-sea hydrothermal vent system. A cluster of phylogenetically related PVB OTUs (OTUs 2, 3, 6, and 8) was closely affiliated with Thiovulum sp. contained within the epsilon subclass of the class Proteobacteria and accounted for 60.5% of the small-subunit rRNA bacterial clone library from Pele's Vents. A second, smaller cluster of PVB OTUs (OTUs 1 and 11) was closely affiliated with Xanthomonas sp., contained within the gamma subclass of the Proteobacteria and accounted for a total of 27.1% of the bacterial clone library. The remaining five PVB OTUs each accounted for 2.1% of the clones recovered and were affiliated with the following phylogenetic groups: PVB OTU 5 was a member of the Alteromonas group; PVB OTU 12 was a member of the Colwellia assemblage; PVB OTU 4 was loosely determined to be a member of the Thiothrix group, with the endosymbiotic bacteria from Bathymodiolus thermophilus and Calyptogena magnifica as the nearest relatives; PVB OTU 10B was a member of the Myxobacterium group; and PVB OTU 9A was a member of the Paraphyletic assemblage, with the Octopus Spring microbial mat type K clone as the closest known relative.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538281 TI - Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species. AB - Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe. PMID- 7538280 TI - Genetic and phenetic analyses of Bradyrhizobium strains nodulating peanut (Arachis hypogaea L.) roots. AB - Seventeen Bradyrhizobium sp. strains and one Azorhizobium strain were compared on the basis of five genetic and phenetic features: (i) partial sequence analyses of the 16S rRNA gene (rDNA), (ii) randomly amplified DNA polymorphisms (RAPD) using three oligonucleotide primers, (iii) total cellular protein profiles, (iv) utilization of 21 aliphatic and 22 aromatic substrates, and (v) intrinsic resistances to seven antibiotics. Partial 16S rDNA analysis revealed the presence of only two rDNA homology (i.e., identity) groups among the 17 Bradyrhizobium strains. The partial 16S rDNA sequences of Bradyrhizobium sp. strains form a tight similarity (> 95%) cluster with Rhodopseudomonas palustris, Nitrobacter species, Afipia species, and Blastobacter denitrificans but were less similar to other members of the alpha-Proteobacteria, including other members of the Rhizobiaceae family. Clustering the Bradyrhizobium sp. strains for their RAPD profiles, protein profiles, and substrate utilization data revealed more diversity than rDNA analysis. Intrinsic antibiotic resistance yielded strain specific patterns that could not be clustered. High rDNA similarity appeared to be a prerequisite, but it did not necessarily lead to high similarity values between RAPD profiles, protein profiles, and substrate utilization. The various relationship structures, coming forth from each of the studied features, had low compatibilities, casting doubt on the usefulness of a polyphasic approach in rhizobial taxonomy. PMID- 7538283 TI - Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep sea deposit feeder. AB - In 1992, two independent reports based on small-subunit rRNA gene (SSU rDNA) cloning revealed the presence of novel Archaea among marine bacterioplankton. Here, we report the presence of further novel Archaea SSU rDNA sequences recovered from the midgut contents of a deep-sea marine holothurian. Phylogenetic analyses show that these abyssal Archaea are a paraphyletic component of a highly divergent clade that also includes some planktonic sequences. Our data confirm that this clade is a deep-branching lineage in the tree of life. PMID- 7538285 TI - Oral iloprost as a treatment for Raynaud's syndrome: a double blind multicentre placebo controlled study. AB - OBJECTIVE: To compare the efficacy, tolerance and safety of 50-150 micrograms orally administered iloprost given twice a day versus placebo in patients with Raynaud's syndrome. METHODS: The study was multicentre (n = 3), double blind and placebo controlled. Sixty three patients who had eight or more vasospastic attacks per week were enrolled. After a one week run-in period, all patients received either iloprost or placebo treatment to a maximum tolerated dose of 150 micrograms twice a day for 10 days. Diary cards assessed the duration and severity of the vasospastic attacks. Side effects were monitored by direct questioning. A global assessment of treatment efficacy was made by the patient at the end of treatment and two weeks later. RESULTS: Patient opinion tended to favour iloprost at the end of the 10 day treatment phase (p = 0.09) and this was significant at day 24 (the follow up visit) (p = 0.011). Although the duration and severity of attacks tended to decrease in the iloprost treated group, these results tended not to reach statistical significance (for severity p = 0.06 at end of treatment, p = 0.09 on day 24). CONCLUSION: Iloprost administered intravenously has been shown to be of benefit in the treatment of the Raynaud's syndrome associated with systemic sclerosis, but this route of administration is inconvenient. This study evaluated the use of iloprost administered orally to patients with Raynaud's syndrome. Patient documented improvement was significantly improved by iloprost. Diary card analysis showed a trend in favour of iloprost, but these results did not reach statistical significance. PMID- 7538284 TI - [Significance of tumor angiogenesis as an independent prognostic factor in axillary node-negative breast cancer]. AB - The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies. PMID- 7538286 TI - Neovascularisation and the induction of cell adhesion molecules in response to degradation products from orthopaedic implants. AB - OBJECTIVES: To characterise the cellular interactions and the mechanisms involved in the recruitment of inflammatory macrophages and T cells to the bone implant interface in 30 patients with aseptically loosened orthopaedic prostheses. METHODS: Cell adhesion molecules E-selectin, VCAM-1, ICAM-1 and the receptors LFA 1 and CR3 were immunolocalised on cryostat sections of the bone-implant interface obtained during revision arthroplasty. The percentage of expression on vascular endothelium was determined on serial sections. RESULTS: E-selectin was upregulated on different vessels in 21 patients. Its expression correlated strongly with the presence of metal wear debris. VCAM-1 was detected on vessels in six patients only, and was coexpressed with E-selectin in three patients with metal debris. VCAM-1 was more frequently observed in the lining cells on the implant side. ICAM-1 was upregulated on vessels on the bone side only in 13 patients, and was more strongly expressed on aggregates of macrophages and multinucleated giant cells on the implant side. These macrophage aggregates coexpressed both ICAM-1 and CR3. CONCLUSION: Our findings implicate the contribution of three different pathways in the recruitment of inflammatory cells to the joint in response to orthopaedic implant wear particles. The association of E-selectin expression and metal debris may suggest hypersensitivity reactions. Finally, the simultaneous expression of ICAM-1 and CR3 on the same macrophages on the implant side may predict an additional function of these molecules in homotypic adhesion/cell aggregation that precede differentiation of phagocytes to multinucleated giant cells. PMID- 7538288 TI - Characteristics of single muscle fibers in m. extensor caudae longus in the cat. PMID- 7538289 TI - Hodgkin's disease, mixed cellularity type, with a B-cell immunophenotype. Report of a case and literature review. AB - The origin of the Reed-Sternberg cell, the neoplastic cell of Hodgkin's disease, has not been defined. We evaluated a case of Hodgkin's disease, mixed cellularity type, which presented in the retroperitoneum of a 45-year-old woman. Reed Sternberg cells and Hodgkin's cells expressed the characteristic markers CD15 and CD30. In addition, they expressed the B-cell antigens CD19 and CD20, as well as CD45/leukocyte common antigen. Clonal rearrangement of the immunoglobulin heavy chain gene was detected by Southern blot analysis. These results suggest that some cases of Hodgkin's disease are derived from an activated cell of lymphoid origin. This case documents a close relationship between Hodgkin's disease and non-Hodgkin's lymphoma, and it demonstrates that even when newer ancillary techniques are employed these two entities can have overlapping features. PMID- 7538291 TI - Laser photocoagulation of subfoveal choroidal neovascularization secondary to age related macular degeneration. PMID- 7538287 TI - TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C. AB - OBJECTIVE: The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. SUMMARY BACKGROUND DATA: The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. METHODS: Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. RESULTS: Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated. PMID- 7538290 TI - [Tumor of the pancreas. Support treatment]. AB - Carcinoma of the pancreas has a poor prognosis with a short survival time. Despite diagnostic advances, diagnosis is often delayed because early symptoms are frequently vague and non-specific. Symptomatic treatment is the only possibility in the large majority of patients with pancreatic cancer since curative surgical excision of the tumor is only possible in few cases. Symptoms managed, in the aim to improve the quality of life, include weight loss and anorexia, abdominal pain, jaundice secondary to biliary compression and digestives consequences of pancreatic surgery. Anorexia is a frequent complaint in patients with pancreatic cancer, and contributes to decreased caloric intake, weight loss and malnutrition. Patients who are unable to eat, due to obstruction or dysfunctional gastrointestinal tract, may benefit from enteral or parenteral nutrition. Patients whose main reason to not obtain adequate food intake is anorexia may benefit from recently developed pharmacologic strategies. Megestrol acetate often produce an improvement in appetite and weight gain in patients with advanced cancer. Jaundice can be treated by surgical biliary drainage or better still by palliative biliary endoprosthesis. Percutaneous transhepatic or endocopic biliary drainage are now well established methods for decompressing malignant biliary obstruction. Pain occurs in 80-85% of patients with advanced disease. Abdominal pain should be treated by oral non-narcotic analgesics, or, if necessary, potent narcotic analgesics, and sometimes by percutaneous block of the celiac ganglion, splancnicectomy or abdominal radiotherapy. Celiac plexus block with alcohol is the most common and well described therapy for the specific pain from carcinoma of the pancreas. Digestive consequences of pancreatic surgery also require symptomatic treatment. PMID- 7538294 TI - [Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties]. AB - We have prepared two lipophilic derivatives of caffeic acid at the carboxylic function--caffeic acid phenethyl ester, an active component of propolis, and N,N' dicyclohexyl-O-(3,4-dihydroxycinnamoyl)-isourea. Both substances inhibit barley 5 lipoxygenase and soybean 15-lipoxygenase at micromolar concentrations. The inhibition is uncompetitive, dose-dependent and reversible. The caffeic acid derivatives also exhibit antioxidant properties and at a concentration 5-10 microM completely block the production of the reactive oxygen species in human neutrophils and in the cell-free xanthine/xanthine oxidase system. PMID- 7538293 TI - Ductin--a proton pump component, a gap junction channel and a neurotransmitter release channel. AB - Ductin is the highest conserved membrane protein yet found in eukaryotes. It is multifunctional, being the subunit c or proteolipid component of the vacuolar H(+)-ATPase and at the same time the protein component of a form of gap junction in metazoan animals. Analysis of its structure shows it to be a tandem repeat of two 8-kDa domains derived from the subunit c of the F0 proton pore from the F1F0 ATPase. Each domain contains two transmembrane alpha-helices, which together may form a four-helix bundle. In both the V-ATPase and gap junction channel, ductin is probably arranged as a hexamer of subunits forming a central channel of gap junction-like proportions. The two functions appear to be seggregated by ductin having two orientations in the bilayer. Ductin is also the major component of the mediatophore, a protein complex which may aid in the release of neurotransmitters across the pre-synaptic membrane. It is also a target for a class of poorly understood viral polypeptides. These polypeptides are small and highly hydrophobic and some have oncogenic activity. Ductin thus appears to be at the crossroads of a number of biological processes. PMID- 7538292 TI - Cell-cell signalling, microtubule organization and RNA localization: is PKA a link? AB - Specification of the anterior-posterior axis of the Drosophila embryo is brought about by the asymmetric localization of specific maternally expressed RNAs and proteins within the oocyte. While many of these localized molecules have been identified and progress has been made towards understanding their functions, how the localization process is instigated remains unclear. A recent paper reports that protein kinase A (PKA) activity is essential for many of these RNA localizations and for the correct polarization of the microtubule cytoskeleton. These and other results support a model for anterior-posterior axis establishment which involves intercellular signalling between the oocyte and certain neighbouring somatic cells. PMID- 7538295 TI - CD28 expression on T cell subsets in vivo and CD28-mediated T cell response in vitro in patients with rheumatoid arthritis. AB - OBJECTIVE: In view of the critical importance of the CD28-CD80 (B7/BB1) costimulatory pathway in antigen-specific T cell activation and clonal expansion, we examined CD28 surface molecule expression in vivo, and T cell receptor/CD3 mediated and B7/BB1-costimulated T cell proliferation in vitro, in rheumatoid arthritis (RA). METHODS: Two-color immunofluorescence analyses of peripheral blood and synovial fluid-derived T cells, as well as 3H-thymidine incorporation assays, were performed. RESULTS: In the peripheral blood of 31 patients with active, untreated RA, a mean of 91% (range 48-100%) of CD4+ and 46% (range 13 82%) of CD8+ T cell subsets were CD28+, which was not significantly lower than normal. Although an overall decrease in the number of T cells was not observed, the numbers of CD28+CD8+ T cells were significantly lower in RA patients (mean 233/microliters, versus 292/microliters in controls), and this decrease was more pronounced in patients with severe disease (mean 172/microliters). CD28 expression on peripheral CD8+ T cells in RA patients, but not in healthy individuals, correlated inversely with T cell activation as assessed by HLA-DR antigen expression. In contrast to the peripheral blood, RA synovial fluid T cells were almost exclusively CD28+, suggesting that migration of CD28+CD8+ T cells to active sites of inflammation may occur. In vitro proliferative responses of peripheral blood T cells to B7/BB1 costimulation in the presence of mitogenic doses of anti-CD3 monoclonal antibody were identical in patients with RA and healthy individuals. CONCLUSION: Functionally intact CD28+ T cells may contribute to the abnormal immunoregulation and joint inflammation in RA. PMID- 7538297 TI - Large and small proteoglycans of osteoarthritic and rheumatoid articular cartilage. AB - OBJECTIVE: To identify characteristic changes in large aggregating (aggrecan) and small proteoglycan (PG) populations in articular cartilages during osteoarthritis (OA) and rheumatoid arthritis (RA). METHODS: Aggrecan populations in guanidine extracts of femoral condylar cartilages of 46 OA and 8 RA patients who underwent total knee arthroplasty, as well as of 2 fetuses and 6 normal adults, were separated in agarose-polyacrylamide composite gels. Small PGs (biglycan, decorin, and fibromodulin) in the same extracts were analyzed in 12% polyacrylamide gels. Gels were stained or electrophoretically transferred and probed with antibodies to aggrecan epitopes and to small PGs. Epitope contents of the samples were also compared by inhibition radioimmunoassay. RESULTS: There were significant differences found among normal and diseased samples in their electrophoretic mobilities, band distributions, and antibody staining. OA and especially RA samples were heavily degraded, lacked certain aggrecan populations, and contained fewer keratan sulfate and chondroitin-6-sulfate epitopes compared with normal samples. Levels of chondroitin-4-sulfate and "fetal-type" epitopes were elevated in the OA samples compared with the normal ones. More core proteins of small PGs were found in diseased than in normal cartilages, but they were more heterogeneous in size and glycosaminoglycan substitution. CONCLUSION: There is extensive degradation of both large and small PGs in diseased cartilages, but a repair process does exist, especially in OA cartilages. Chondrocytes of diseased cartilages are able to synthesize fetal-type aggrecans. Small PGs are glycosylated differently in diseased cartilages than in normal ones. PMID- 7538298 TI - Increased levels of substance P and calcitonin gene-related peptide in rat adjuvant arthritis. A combined immunohistochemical and radioimmunoassay analysis. AB - OBJECTIVE: To analyze the occurrence of substance P (SP) and calcitonin gene related peptide (CGRP) in ankle joints and corresponding dorsal root ganglia (L2 L6) of rats with adjuvant arthritis. METHODS: Arthritis was induced by inoculation with heat-killed mycobacteria. The morphologic distribution of SP and CGRP was assessed by immunohistochemical analysis. Tissue concentrations of the neuropeptides were determined by radioimmunoassay. RESULTS: Neuronal CGRP-like immunoreactivity was clearly increased in the synovium and the dorsal root ganglia, whereas the increase in SP-positive structures was less pronounced. The tissue concentrations of SP and CGRP were significantly increased both in ankle joints and in dorsal root ganglia. CONCLUSION: Levels of sensory neuropeptides are increased under conditions of joint inflammation. PMID- 7538296 TI - Monoclonal antibodies that detect biochemical markers of arthritis in humans. AB - OBJECTIVE: To evaluate the potential of using monoclonal antibodies (MAb) 3-B-3( ) and 7-D-4 to detect biochemical markers of altered cartilage metabolism in human arthritides. METHODS: Fifty-five samples of normal articular cartilage (subjects' age range 18 weeks of gestation to 83 years of age) and 89 samples of arthritic cartilage (patients' age range 20-81 years) were collected, and their proteoglycans were extracted and analyzed for the presence of the epitopes recognized by MAb 3-B-3 and 7-D-4. RESULTS: Native 3-B-3(-) mimotope was expressed at a high incidence in proteoglycans extracted from the cartilage of patients with most of the arthritic diseases examined (osteoarthritis, juvenile rheumatoid arthritis, rheumatoid arthritis, avascular necrosis, and degenerative meniscal tears). Its expression in normal cartilage specimens was very low or absent, occurring mainly in the young, skeletally immature individuals. In contrast, expression of the 7-D-4 epitope was more variable in patients with different arthritides and was also frequently found in normal cartilage specimens. Immunohistochemical analyses with both 3-B-3(-) and 7-D-4 showed strong focal positive staining in superficial areas, where cartilage degeneration, remodeling, and repair were greatest. CONCLUSION: The biochemical markers recognized by MAb 3-B-3(-) and 7-D-4 are indicative of altered proteoglycan synthesis and metabolism in human articular cartilage. The data suggest that in human cartilage, the 3-B-3(-) epitope might be a better marker of biochemical changes than the 7-D-4 epitope. PMID- 7538299 TI - Clinical impact of drug addiction in alcoholics. AB - Careful interviewing of alcoholics who wish to undergo alcohol withdrawal programmes reveals that some are past intravenous drug abusers. As these two potentially hepatotoxic types of substance abuse could cause liver disease or influence its clinical course, we studied biological, histological and virological features in 26 alcoholics with a past history of intravenous (i.v.) drug abuse, compared with paired controls (alcoholics without i.v. drug abuse). There were no differences with regard to routine liver test results. In contrast, the former drug abusers had a significantly higher prevalence of serum markers of hepatitis C (76.9%) and hepatitis B viruses (76.9%) than the other patients (16.7 and 12.5%, respectively). Eight patients, all of whom were HBs Ag negative, were positive for serum HBV-DNA; three were former drug abusers and five were not, giving an overall prevalence of HBV markers in the two groups of 80.8 and 25%, respectively. Two former drug abusers had anti-HIV antibodies and one had anti hepatitis delta virus antibodies. Ten of the 17 former drug abusers who underwent liver biopsy had histological signs of viral infection. These data underline the need for careful interviews of alcoholic patients, together with serological tests for viral infections and histological analysis of the liver, as some will have liver-damaging viral diseases and may be candidates for anti-viral (i.e. interferon) treatment. PMID- 7538300 TI - Glutamatergic neurotransmission in hepatic encephalopathy. AB - There is increasing evidence that glutamatergic neurotransmission is perturbed in hepatic encephalopathy (HE). Studies of the glutamate receptor system suggest that glutamate receptor density is reduced in different animal models of HE. Glutamate release and/or uptake is also believed to be altered in the disorder resulting in increased glutamate concentrations in the synaptic cleft. The relevance of these findings to the pathogenesis of HE remains to be clarified. PMID- 7538301 TI - The neuroanatomical specificity of ethanol action on ligand-gated ion channels: a hypothesis. AB - The studies described will demonstrate that the subunit composition of a GABAA receptor allows ethanol to enhance responses to GABA. Since we have determined that ethanol will influence responses to glycine, nicotine and NMDA in some, but not all, neurons with receptors to these agonists, we hypothesize that specific receptor subtypes of these ligand-gated ion channels will be affected by ethanol. PMID- 7538302 TI - Ethanol action on excitatory amino acid activated ion channels. AB - The effects of ethanol on excitatory amino acid activated ion channels were investigated using patch-clamp recording methods. Intoxicating concentrations of ethanol (5-50 mM) inhibited ion current activated by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in a concentration-dependent manner (IC50 = 30 mM). The intoxicating potency of different alcohols was correlated with their potency for inhibiting NMDA-activated current, suggesting that alcohol-induced inhibition of NMDA channel function may contribute to the neural and cognitive impairments associated with intoxication. Analysis of mechanism suggests that ethanol inhibits NMDA-activated current by altering gating of the channel, rather than by affecting channel conductance, ion permeance or regulatory sites on the channel. Anesthetic concentrations of ethanol (> 50 mM) produced a concentration dependent inhibition of kainate- and quisqualate-activated currents (IC50 = 220 mM), suggesting that this inhibition may contribute to the general anesthetic effects of ethanol. This hypothesis is supported by the observations that the general anesthetic agents, trichloroethanol (the active metabolite of chloral hydrate), pentobarbital and halothane, all inhibit kainate- and quisqualate activated currents. PMID- 7538303 TI - Biochemical markers for the effects of ethanol. PMID- 7538304 TI - Behr syndrome. PMID- 7538306 TI - Pathology from hydroxyapatite deposits in periarticular tissues. First conclusions on etiopathogenesis. AB - Biopsies of periarticular tissues from patients with episodes of chronic periarthritis were examinated by histological, ultrastructural and chemico crystallographic methods. In all samples the mineralogical observations at the optical microscope, showed aggregates of microcrystalline incrustations, whose ultrastructural morphology has been characterized by SEM. The histological observations showed necrosis of collagen fibres and microcrystallization process in cavities and in metaplastic fibrocartilage. The HA deposits in the periarticular tissues, in the next stage of the process, are surrounded and demolished by macrophages and multinucleated giant cells. At the same time granulation tissue carries out the repair process, that begins with the formation of thin parallel collagen fibers interposed with many fibroblasts and numerous small vessels. PMID- 7538305 TI - Influence of gonadal hormones on sexual differences in sensitivity to methamphetamine-induced neurotoxicity. AB - The administration of high doses of methamphetamine to mice causes long-lasting depletions of striatal dopamine to a greater extent in males than in females. Likewise, the incidence of Parkinson's disease is higher in males than in females. The present study investigated the roles of estrogen and testosterone in mediating the dopamine depletion induced by methamphetamine. Male and female mice received four cumulative SC doses of methamphetamine (10 mg/kg) at two hour intervals and were sacrificed two weeks later for HPLC analysis of striatal monoamines. Intact male mice were found to have a 76% dopamine depletion, which was significantly greater than the 37% depletion exhibited by the intact female mice. Neither removal of the ovaries nor removal of the testes one month prior to the methamphetamine treatment significantly changed the magnitude of the methamphetamine-induced dopamine depletion. Thus, the reduced sensitivity of female mice to methamphetamine may be independent of physiological gonadal hormones. PMID- 7538307 TI - Cloning and nucleotide sequence comparison of the groE operon of Pseudomonas aeruginosa and Burkholderia cepacia. AB - By alignment of GroEL amino acid sequences from four distantly related bacteria two highly conserved domains were identified. Two oligonucleotides complementary to the conserved domains were designed based on the preferred Pseudomonas aeruginosa codon usage. The primers were used in the PCR to amplify a 900-base fragment of the P. aeruginosa groEL gene. The fragment was sequenced and the partial GroEL sequence was expanded by vectorette PCR upstream and downstream to cover the complete P. aeruginosa groE operon. The same technique was used to sequence the Burkholderia cepacia (formerly Pseudomonas cepacia) groE operon and the region immediately upstream of groES. The B. cepacia groE operon is preceded by typical -10 and -35 heat shock expression signals. A total of 2041 and 2139 bp was sequenced from P. aeruginosa and B. cepacia respectively. Each revealed two open reading frames encoding two proteins with a predicted molecular mass of 10 and 57 kDa, corresponding to GroES and GroEL respectively. The GroEL proteins show an interspecies amino acid homology of 71%, and 73% with E. coli GroEL. Both GroEL proteins are 52% homologous to the corresponding human mitochondrial GroEL protein. The sequence data confirm the existence of highly conserved structures, which could be functionally important for the concerted action of GroEL and GroES in the folding and assembly of other proteins, and possibly in the initiation of autoimmune diseases. PMID- 7538309 TI - Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: subtype-specific coupling to ion channels. AB - A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways. PMID- 7538308 TI - Why associations among traits do not necessarily indicate their common etiology: a comment on the Geschwind-Behan-Galaburda model. PMID- 7538310 TI - Pore loops: an emerging theme in ion channel structure. PMID- 7538312 TI - The effect of "high dose" aprotinin and other factors on bleeding and revisions for bleeding in adult coronary and valve operations: an analysis of 2190 patients during a five-year period (1987-1991). AB - We retrospectively evaluated risk factors for postoperative bleeding and for revisions due to bleeding in 2190 adult coronary and valve patients who underwent surgery at our hospital during the 5-year period from 1987 to 1991. During this period 889 (40.6%) patients were given "high dose" aprotinin. Their mean age was 59.3 +/- 8.8 years, 1636 (74.7%) were males, 200 (9.1%) underwent surgery on an emergency basis and 72 patients (3.3%) underwent redo-operations. The patients were divided into four groups according to the type of surgery: all patients pooled together (2190), coronary artery surgery patients (1384, 63.2%, group I), valve surgery patients (706, 32.2%, group II) and combined (coronary plus valve) surgery patients (100, 4.6%, group III). Stepwise logistic regression analysis, performed to assess the risk factors for revisions due to bleeding showed aprotinin treatment to be the sole protective factor in all patients, group I and group II. In group III only the use of a hollow fiber membrane oxygenator proved a protective factor. Risk factors for revisions for bleeding were found to be aortic cross-clamp time in all patients, group I and group II. Use of the internal thoracic artery (ITA) was significant in group I patients and age at operation in group II. Multiple stepwise linear regression analysis, performed to evaluate the effect of various risk factors on cumulative postoperative blood loss in all patients, confirmed aprotinin as the only factor capable of reducing blood loss, while aortic cross-clamp time, coronary surgery and male gender showed a positive linear relation with postoperative bleeding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538311 TI - Protein kinase C-mediated inhibition of an inward rectifier potassium channel by substance P in nucleus basalis neurons. AB - In nucleus basalis neurons, substance P (SP) causes a slow excitation, mediated through a pertussis toxin-insensitive G protein, by suppressing an inward rectifier K+ channel. Here we report that SP applied outside the patch pipette inhibited the single-channel activity, recorded on-cell, of the inward rectifier. The PKC inhibitors staurosporine and PKC(19-36) suppressed this effect in whole cell mode and in on-cell single-channel mode. A diacylglycerol analog mimicked the SP effect, and PKC(19-36) suppressed this analog effect. SP irreversibly suppressed the inward rectifier in neurons treated with okadaic acid. These results indicate that a diffusible messenger mediates the SP effect, that its signal transduction involves phosphorylation by PKC, and that dephosphorylation by a serine/threonine protein phosphatase mediates its recovery. PMID- 7538313 TI - The use of non-human primates in the study of PCB immunomodulation. PMID- 7538314 TI - The immunotoxicity of pesticides in rodents. AB - Many pesticides that are widely used and have great potential for occupational and public exposure have received only a cursory examination with regards to their immunotoxic potential. Many of the studies that are available were done during the early period of immunotoxicology and many reports do not state whether or not any other toxic signs were observed and the dose-response relationships were not generally examined. Most studies were done in multiple species, through various routes of administration and using a variety of assays of immune function and do not allow a comparison. For the compounds extensively studied, the site of action has been determined at the cellular and perhaps the biochemical level, but the molecular site of action has not been determined. The approach to evaluate the mechanism of action will vary from compound to compound. PMID- 7538315 TI - Palliative care in the community: setting practice guidelines for primary care teams. AB - BACKGROUND: Previous studies have demonstrated deficiencies in palliative care in the community. One method of translating the results of research into clinical practice, in order to produce more effective health care, is the development of clinical guidelines. Setting standards for such care has been performed by care teams in both hospital and hospice settings but not in primary care. AIM: This study set out to develop guidelines for primary care teams to follow in the provision of palliative care in the community using facilitated case discussions with the members of such teams, as a form of internal audit. METHOD: Five practices were randomly chosen from the family health services authority medical list. Meetings between the facilitators and primary care teams were held over a period of one year. The teams were asked to describe good aspects of care, areas of concern and suggestions to improve these, in recent cases of patient deaths. RESULTS: In total 56 cases were discussed. All practices felt that cohesive teamwork, coordinated management, early involvement of nursing staff and the identification of a key worker were essential for good terminal care. Concerns arose in clinical and administrative areas but the majority were linked to poor communication, either between patient and professionals within the primary care team or between primary and secondary care. All the positive aspects of care, concerns and suggestions were collated by the facilitators into guidelines for teams to refer to from the initial diagnosis of a terminal illness through to the patient's death and care of the relatives afterwards. CONCLUSION: Developing multidisciplinary as opposed to medical guidelines for palliative care allows primary health care teams to create standards that are acceptable to them and stimulates individuals within the teams to accept responsibility for initiating the change necessary for more effective care. The process of facilitating teams to discuss their work allows for recognition and respect of individuals' roles and more importantly provides shared ownership, an important contributory factor in the implementation of guidelines. PMID- 7538316 TI - Consultation patterns in a community survey of men with benign prostatic hyperplasia. AB - BACKGROUND: The Stirling benign prostatic hyperplasia natural history group have previously reported a prevalence of this condition of 255 per 1000 in a community study of 1610 men aged 40-79 years. AIM: It was decided to examine the consultation patterns of men with benign prostatic hyperplasia in greater detail. METHOD: All participating men were invited to complete a previously validated lifestyle questionnaire including questions on consultations with their general practitioner during the previous year and previous history of prostatic problems. The men who had a urinary symptom score greater than 11, or who had a urinary flow rate of less than 15 ml per second were examined by transurethral ultrasonography for prostate size. RESULTS: Of 364 men with benign prostatic hyperplasia, 89% had not consulted their doctor about urinary symptoms in the year prior to the study. Men with moderate to severe urinary symptoms were six times more likely to have consulted their doctor than those with mild symptoms. Moderate to severe symptoms and greater interference with daily living activities were both associated with a greater likelihood of consultation, independent of age. Of all the men in the study referred to the specialist clinic for assessment of prostate size by transurethral ultrasonography, two thirds were referred because of low urinary flow rate and one third because of high urinary symptom scores. The reported consultation data showed a reverse ratio of one third of those consulting having a low urinary flow rate and approximately two thirds having urinary symptoms. CONCLUSION: While mass screening is unjustified, there is a need for patient education about benign prostatic hyperplasia in general and the recognition of declining strength of urinary flow as a symptom of benign prostatic hyperplasia and not of ageing alone. Furthermore, evaluation of primary care use of urinary flowmeters and the development of local protocols are suggested as elements of a case finding strategy for benign prostatic hyperplasia based on patient led consultation. PMID- 7538318 TI - The endothelium in psoriasis. PMID- 7538320 TI - Recent advances in the treatment of human immunodeficiency virus infections with interferons and other biological response modifiers. PMID- 7538319 TI - Visceral endoderm-1 (VE-1): an antigen marker that distinguishes anterior from posterior embryonic visceral endoderm in the early post-implantation mouse embryo. AB - We describe here an antigen marker, designated VE-1, that is detected early in gastrulation (approximately E6.5 through approximately E7.25) in the anterior visceral endoderm overlying the embryonic ectoderm opposite the primitive streak. The antibody-positive domain extends from the embryonic-extraembryonic junction to the distal tip of the embryo, and laterally around approximately one-third of the circumference of the egg cylinder. Analysis of embryos at earlier stages indicates that VE-1 is first expressed shortly after implantation, at approximately E5.0, in the visceral endoderm on one side of the embryo and thus is the earliest molecular marker of A-P asymmetry in the post-implantation mouse embryo described to date. Although VE-1 was detected with a polyclonal antiserum raised against a 24 amino acid polypeptide sequence of FGF2, we provide evidence that the VE-1 antigen is not FGF2. The data reported here are the first to provide molecular evidence that A-P polarity in the mouse embryo is established by E5.0 and that the visceral endoderm has A-P polarity. PMID- 7538317 TI - [HCV-positive blood donors. Risk factors]. PMID- 7538321 TI - Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. AB - We have shown previously that the ras and myc oncogenes can induce poorly differentiated mouse prostate carcinomas in vivo with high frequency (greater than 90%) using inbred C57BL/6 mice in the mouse prostate reconstitution model system. To study the androgen sensitivity of these carcinomas, we have developed an in vitro model system which includes a cell line from normal urogenital sinus epithelium (CUGE) and cell lines from three ras + myc transformed mouse prostate carcinomas (RM-9, RM-1, and RM-2). CUGE cells, as well as all prostate carcinoma cell lines, were positive for cytokeratin 18 mRNA and immunoreactive to cytokeratin-specific antiserum. Two out of three of the early passage carcinoma cell lines were clonal with respect to Zipras/myc 9 retrovirus integration as determined by Southern blot analysis. Whereas significant mitogenic effects of testosterone (10 nM) were not seen in CUGE cells grown in serum-free medium, under similar conditions approx. 2-fold increases in cell number were seen in all low passage prostate carcinoma cell lines. Also, in the presence of growth inhibitory levels of suramin (50 micrograms/ml), testosterone was capable of significant growth stimulation in the carcinoma cell lines. With further propagation from low passage [20-25 population doublings (PD)] to high passage (75-100 PD), all carcinoma cell lines demonstrated increased and similar growth rate in the presence and absence of testosterone. These cell lines maintained stable androgen receptor numbers and binding kinetics during the transition from testosterone-responsive growth to reduced responsivity over multiple passages in culture (> 150 PD). Overall, our studies indicate that the capacity to bind testosterone is stably maintained through the transition of the androgen sensitive to insensitive phenotype and raise the possibility that androgen sensitivity can persist throughout progression but is masked by the acquisition of autocrine pathways. PMID- 7538322 TI - Peptidergic nerves in Hirschsprung's disease and its allied disorders. AB - To clarify the significance of peptidergic nerves in Hirschsprung's disease (aganglionosis), hypoganglionosis, and neuronal intestinal dysplasia (NID), we investigated enteric nerve responses in colonic tissues obtained from patients with these diseases. Colonic tissue specimens were obtained from 12 patients with aganglionosis, 8 patients with hypoganglionosis, and 6 patients with NID. Colon specimens from 20 patients without constipation were used as controls. A mechanograph was used to evaluate in vitro colonic responses to electrical field stimulation (EFS) of the adrenergic and cholinergic nerve blockers and gastrointestinal hormones. The following results were obtained: (1) Non adrenergic inhibitory nerves were found to act on the normal human colon and to a lesser extent in colons with hypoganglionosis or NID, but had no effect on the enteric nerves in colons with aganglionosis. (2) Peptidergic neurotransmitters such as VIP, substance P, and neurotensin apparently act in the normal human colon, and to a lesser extent in the colons with hypoganglionosis or NID, but their effect was almost absent in aganglionosis. (3) VIP acts via neural mechanisms, while substance P and neurotensin may act both via nerves and also directly on the bowel smooth muscle. The diminution of action of non-adrenergic inhibitory nerves and peptidergic nerves may be largely related to the impaired motility observed in hypoganglionosis, NID and aganglionosis. PMID- 7538323 TI - Nucleotide modification and base conversion of RNA. Part I. Savoie, France, May 1994. Workshop proceedings. PMID- 7538324 TI - Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin. AB - An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition. PMID- 7538326 TI - Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis. AB - Synthetic oligoribonucleotides have become increasingly valuable in studies of RNA structure and function. A range of nucleotide analogues is available which carry modifications in the base, sugar or phosphate moieties. Such analogues have been incorporated into synthetic RNA structures to eliminate or alter individual functional groups in the RNA which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparisons of the properties of the modified RNAs with unmodified RNA models allow conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA. These methods have been applied to studies of RNA interactions with proteins, RNA catalysis and RNA structure. PMID- 7538327 TI - Recombinant photoreactive tRNA molecules as probes for cross-linking studies. AB - Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented. PMID- 7538325 TI - Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA. AB - Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one dimensional difference nuclear Overhauser effect (NOE) methods. In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications. PMID- 7538328 TI - The effect of interleukin-4 with or without mast cell growth factor on peripheral blood granulocyte-macrophage colony-forming cells in healthy controls and in myeloproliferative disorders. AB - The effect of interleukin-4 (IL-4) on peripheral blood (PB) granulocyte macrophage (GM) progenitors was investigated in the presence and absence of other hematopoietic growth factors, especially the mast cell growth factor (MGF), in eight healthy controls and in 26 patients with myeloproliferative disorders (MPDs) using a clonogenic cell culture assay. In the controls IL-4 was effective alone, stimulating myeloid colony growth in 50%, while MGF had no effect as a single factor. When either IL-4 or MGF was added to the combination of IL-3, GM CSF, G-CSF, and IL-6, a statistically significant increase in the colony number was observed. The most potent colony formation took place when all these GFs were combined. In the combinations, the effect of IL-4 was additive, while MGF worked synergistically. In the MPDs, IL-4 had no effect at all on the GM progenitors in the whole group of MPDs or on the different subgroups. PMID- 7538329 TI - Effect of pentoxifylline on nitric oxide released by murine macrophages. AB - The growing knowledge on the pathological role of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in septic shock stimulated efforts to control their generation pharmacologically in clinical situations. Pentoxifylline (PTXF) is well known as an inhibitor of TNF synthesis, whereas information about its role in suppression of NO generation is much less available. In our study, we have shown that PTXF suppresses the synthesis of both mediators, TNF and NO, released by macrophages activated with different stimuli. However, in contrast to N monomethyl-L-arginine (an inhibitor of NO synthase), PTXF influenced NO generation only during the induction phase. In conclusion, we suggest that a possible new therapeutic approach in septic shock may result from the inhibition of these two major mediators by simultaneous application of PTXF and a specific inhibitor of NO generation. Further experimental investigations and clinical trials are necessary to evaluate the safety and effectiveness of application of these inhibitors. PMID- 7538330 TI - [Filgrastim in the treatment of severe neutropenia in a patient with bilobar pneumonia]. PMID- 7538331 TI - IgG+, CD5+ human chronic lymphocytic leukemia B cells. Production of IgG antibodies that exhibit diminished autoreactivity and IgG subclass skewing. AB - Several questions exist regarding CD5+ B cells. These include the ability of these cells, as compared to CD5- B cells, to undergo an Ig isotype class switch, the subclasses utilized, and the effects that switching may have on antigen binding. To address these issues, ten patients with chronic lymphocytic leukemia (CLL) whose CD5+ leukemic B cell clones produced IgG were studied. Monoclonal IgG was collected from PMA-stimulated CLL cells and from heterohybridomas constructed with these cells, and then analyzed for IgG subclass utilization, autoreactivity, and DNA idiotype expression. The monoclonal B cells from 80% of the CLL patients produced IgG1 and those from 20% produced IgG3. None produced IgG2. In contrast to the known autoreactivity of IgM-producing CD5+ CLL cells (> 50% autoreactive), none of these IgG antibodies reacted significantly with the autoantigens tested. However, three did react significantly with autoantigen after artificially increasing antibody valency by crosslinking. Whereas five of the IgG molecules expressed a cross reactive idiotypic (CRI) marker characteristic of non-mutated kappa anti-DNA antibodies, three expressed a CRI displayed primarily on mutated IgG anti-DNA antibodies. Thus, some CD5+ human B cells can undergo an isotype class switch that for these CLL cells is biased against IgG2 and in favor of the IgG1 and IgG3. In their native state the IgG molecules secreted by these isotype switched CD5+ cells have diminished autoreactivity, as compared to IgM-producing CLL cells. Since some of the IgG antibodies could be made auto- and poly-reactive by increasing antigen-binding valency, while others expressed idiotypic markers of mutated antibodies, certain of these CD5+ B cells probably utilize non-mutated Ig V genes coding for polyreactive antibodies, whereas others may use genes that have undergone somatic mutation and that code for more restricted specificities. Therefore, both valency and VH gene mutation may account for the diminished autoreactivity of these CD5+ B cell-derived IgG antibodies. PMID- 7538332 TI - Autoantibodies-like antigen binding characteristics of induced antibodies against polylysine-polyglutamate complex. AB - Rabbits immunized with polylysine-polyglutamate complex induced high titer antibodies. The immune IgG exhibited high degree of specificity towards the immunogen. In a competition assay, nDNA, heat denatured DNA and total buffalo thymus RNA were inhibitory. An appreciable binding with polyglutamate, poly(rG).poly(dC) poly(dG).poly(dC), cardiolipin, poly(dA-dU). Poly(dA-dU) and Z- or Z-like conformations demonstrates the polyspecificity of induced antibodies. The immunological specificity of induced antibodies was comparable to autoantibodies derived from SLE. The results might be of some help in understanding the etiology of autoimmune process. PMID- 7538333 TI - The mouse/human chimeric monoclonal antibody cA2 neutralizes TNF in vitro and protects transgenic mice from cachexia and TNF lethality in vivo. AB - The pleiotropic cytokine tumour necrosis factor-alpha (TNF) is thought to play a central role in infectious, inflammatory and autoimmune diseases. Critical to the understanding and management of TNF-associated pathology is the development of highly specific agents capable of modifying TNF activity. We evaluated the ability of a high affinity mouse/human chimeric anti-TNF monoclonal antibody (cA2) to neutralize the in vitro and in vivo biological effects of TNF. cA2 inhibited TNF-induced mitogenesis and IL-6 secretion by human fibroblasts, TNF priming of human neutrophils, and the stimulation of human umbilical vein endothelial cells by TNF as measured by the expression of E-selectin, ICAM-1 and procoagulant activity. cA2 also specifically blocked TNF-induced adherence of human neutrophils to an endothelial cell monolayer. Receptor binding studies suggested that neutralization resulted from cA2 blocking of TNF binding to both p55 and p75 TNF receptors on the cells. In vivo, repeated administration of cA2 to transgenic mice that constitutively express human TNF reversed the cachectic phenotype and prevented subsequent mortality. These results demonstrated that cA2 effectively neutralized a broad range of TNF biological activities both in vitro and in vivo. PMID- 7538335 TI - Symposium. Growth factors and benign prostatic hyperplasia. 6th November 1993, Paris, France. PMID- 7538334 TI - Role of cyclic nucleotides and nitric oxide in blood mononuclear cell IgE production stimulated by IL-4. AB - The involvement of cyclic nucleotides and of phosphodiesterase activities in IL-4 induced IgE production and release of the soluble form of the low affinity receptor for IgE (sCD23) by normal human peripheral blood mononuclear cells (PBMC) was evaluated. PBMC were stimulated by a suboptimal dose of IL-4 (10 ng/ml) cAMP inducers, adrenaline (ADR) and cholera toxin (CTx), which were found to potentiate IL-4-induced IgE production and sCD23 release after 12 days of culture. In the presence of an optimal dose of IL-4 (30 ng/ml), both ADR and CTx inhibited the production of both IgE and sCD23. In the presence of a chemical cGMP inducer, Sin-1, the production of IgE induced by 10 ng/ml IL-4 appeared to be potentiated whereas in the same experimental situation the sCD23 production was decreased. Sin-1 was found to inhibit the production of both IgE and sCD23 as effectively as cAMP inducers when an optimal dose of IL-4 was used. Since Sin-1 is a nitric oxide (NO) generating compound, we evaluated the possible involvement of the L-arginine metabolic pathway using a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine (LNMMA). In the presence of 1 mM LNMMA both IgE and sCD23 production induced by either a sub-optimal or an optimal dose were partially inhibited (from 50 to 80% inhibition depending on the donor). The generation of cAMP and cGMP in the cells is controlled by cyclic nucleotide phosphodiesterases (CN-PDE), so we evaluated the effect of a CN-PDE inhibitor, isobutyl-methyl xanthine (IBMX), on the IL-4-induced IgE and sCD23 production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538336 TI - HLA-mismatched cord blood transplantation: immunological studies. AB - Two cases of HLA partially matched related cord blood transplantation were reported. The first patient remains healthy more than 4 years after graft. The second patient died from acute GVHD. In the first case, the immune reconstitution was studied over a 2 year period after graft: the alloreactive cytotoxic T-cell precursor (CTLp) frequencies were comparable between the donor and the recipient. Indirect evidence suggested that the resolution of GVHD was not due to a specific deletion of CTLp against the HLA antigens unshared between donor and recipient. PMID- 7538338 TI - Cytokine regulation of early B-lymphopoiesis assessed in culture. AB - Lymphohemopoietic progenitors that are capable of expressing B-cell and myeloid lineages may be cultured from bone marrow cells of adult mice by using a two-step methylcellulose culture system. In this system, the primary colonies expressing myeloid lineages are plated in secondary culture for B-lymphoid colony formation. We have observed that combinations of two factors based on steel factor (SLF), such as SLF plus interleukin (IL)-6, SLF plus granulocyte colony-stimulating factor (G-CSF), and SLF plus IL-11 support the differentiation and proliferation of B-cell progenitors from the lymphohemopoietic progenitors. Surprisingly, IL-3 failed to support B lymphopoiesis either alone or in combination with other factors. In addition, when added to permissive culture conditions, IL-3 and IL-1 independently inhibited the B-cell potential of the primary colonies. The inhibitory effects of IL-3 and IL-1 observed in this in vitro system may be significant in the selection of cytokine combinations for in vitro expansion of hemopoietic stem cells. PMID- 7538337 TI - SCID mice as an in vivo model of human cord blood hematopoiesis. AB - Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation. PMID- 7538339 TI - Primitive hematopoietic progenitor cells are present in peripheral blood autografts. AB - In this report we have used an in vitro assay for long-term culture-initiating cells (LTC-IC) to detect primitive hematopoietic progenitor cells (HPC) in the peripheral blood (PB) of cancer patients who received high-dose cyclophosphamide (HD-CTX) followed by a combination of recombinant hematopoietic growth factors (C HGF) including either interleukin-3 (IL-3) + granulocyte colony-stimulating factor (G-CSF), IL-3 + granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3/GM-CSF fusion protein (PIXY-321). In addition, we have developed a quantitative assay for cells capable of generating additional colony-forming cells (pre-CFC) as a means of determining primitive HPC present in mobilized PB cells. CD34+ human leukocyte A (HLA)-DR- cells isolated from the mobilized PB were capable of initiating long-term hematopoiesis in vitro that persisted for 10 weeks, while CD34+ HLA-DR- cells obtained from the nonmobilized PB or BM were capable of sustaining long-term hematopoiesis in vitro for only 4 weeks and 8 weeks, respectively. As determined by a limiting dilution analysis of mobilized PB CD34+ HLA-DR- cells, the frequency of pre-CFC was 4.3% (range, 1.0-8.3%). Pre CFC comprised 0.01% (range, 0.001-0.02%) of mobilized PB mononuclear cells, and 151 pre-CFC were calculated to be present in one milliliter of mobilized PB (range, 20-310/ml). These results suggest that PB mononuclear cells collected by leukapheresis following mobilization with HD-CTX + C-HGFs contain not only differentiated HPCs but also more primitive HPC. PMID- 7538341 TI - Evaluation of the Ceprate LC34 affinity system for the enrichment of CD34+ hematopoietic stem cells from cord blood. AB - Umbilical cord blood (CB) has been shown to contain sufficient hematopoietic stem cells for allogeneic bone marrow transplant (BMT) and to contain progenitor cells susceptible to retrovirally mediated gene transduction. Enrichment of CD34+ cells from fresh unseparated or thawed unseparated CB could offer several advantages for (1) the storage of CB samples in an unrelated stem cell bank, which includes a decrease in volume and thus less storage space and (2) gene transfer into these cells. Cord blood was collected from the umbilical cord vein immediately after vaginal full-term delivery and samples of 5-12 ml (total leucocytes: 100 +/- 50 x 10(6)) of fresh unseparated (n = 6) and thawed unseparated (n = 8) CB were processed to enrich CD34+ cells using the Ceprate LC Biotin-Avidin affinity system. CD34+ cells, present at a frequency of 1.2 +/- 0.8% among leukocytes from CB (calculated by immunophenotyping and fluorescence-activated cell sorter (FACS) analysis), can be rapidly enriched to 54.4 +/- 12.3% (range, 38.6-87.1%) from fresh unseparated CB and 44.6 +/- 28% (range, 13.6-76%) from thawed unseparated CB. Hematopoietic progenitor assays for unseparated (cell concentration 3 x 10(4), 1 x 10(5), 3 x 10(5)) and CD34-enriched cells (cell concentration 1 x 10(3), 3 x 10(3), 1 x 10(4)) were performed in the presence of 5 U human interleukin-3 (hu IL-3), 30 U hu-IL-6, 10 U human granulocyte colony-stimulating factor, 6 U erythropoietin, and 15 ng human stem cell factor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538340 TI - Analysis of human hematopoietic stem cell populations. AB - Purification of human hematopoietic stem cells (HSC) may be useful clinically for preparation of tumor-free grafts to be used for autologous transplantation and as targets for gene therapy. To analyze the phenotype of the human HSC, assays were used that measure the unique properties of stem cells, i.e., their long-term repopulating ability and their multilineage potential. These assays include: (1) an in vitro long-term hematopoietic culture system, using the murine bone marrow stromal cell line SyS1, which supports both B lymphopoiesis and myelopoiesis; (2) fetal human bone grafts implanted in SCID-hu mice, in which maintenance of CD34+ cells and B and myeloid differentiative capacity of candidate stem cell populations may be measured; (3) fetal human thymus grafts in SCID-hu mice, which allow the analysis of in vivo T-cell potential of a candidate stem cell population. Stem cells in adult bone marrow (ABM) or cytokine-mobilized peripheral blood (MPB) are thought to express CD34 but lack expression of markers indicating lineage commitment. This CD34+ Lineage- (Lin-) subpopulation has been isolated by fluorescence-activated cell sorting and tested for activity in the assays described here. CD34+ Lin- cells from both ABM and MPB demonstrated long term engraftment in the SCID-hu bone model. This CD34+ Lin- population can be subfractionated further using an antibody to Thy-1. The Thy-1+ subset of CD34+Lin cells is enriched for both long-term culture-initiating cells (LTC-1C) and has the ability to engraft in vivo. PMID- 7538342 TI - Age-related decline in proliferative potential of purified stem cell candidates. AB - Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here. Extensive qualitative changes in hematopoietic cells from various stages of development should be taken into account in the design of novel therapeutic strategies. PMID- 7538344 TI - Commentary: hematopoietic progenitors in fetal and adult tissue. PMID- 7538345 TI - Isolation and ex vivo expansion of CD34+ cells from cord blood using dextran sedimentation and avidin column selection. AB - Human umbilical cord bloods were fractionated by unit gravity sedimentation in 1% (v/v) dextran, followed by immunoaffinity selection for CD34+ stem and progenitor cells. Dextran sedimentation alone enabled recovery of more than 80% of the nucleated cells present and 90% of the CD34+ cells, as determined by flow cytometry. The addition of an immunoaffinity selection step for CD34+ cells resulted in a 134-fold enrichment for CD34+ cells, with a mean yield of 64 +/- 15%. The resultant CD34+ population contained almost half the CFU-GM activity initially present in the cord bloods and could be expanded ex vivo in liquid culture. PMID- 7538343 TI - Human umbilical cord blood hematopoietic progenitor cells: are they the same as their adult bone marrow counterparts? AB - In an attempt to expand the hematopoietic progenitor cell (HPC) content of a single collection of umbilical cord blood (CB), we investigated the ex vivo proliferative potential of CB CD34+ cells and the rate of exit of these cells from G0/G1 phases of cell cycle in response to different cytokine combinations. Initial experiments in which phenotypically defined populations of CB and adult bone marrow (BM) CD34+ cells were examined for their HPC content revealed that, contrary to BM, CB CD34+ human leukocyte A (HLA)-DR+ cells appeared to contain the majority of primitive HPC. In cultures of BM CD34+ HLA-DR+ cells incubated with stem cell factor (SCF)+interleukin-3 (IL-3), CD34+ cells increased five-fold over 5 days, while CD34+ cells from CB CD34+ HLA-DR+ cultures increased 11-fold under these same conditions, illustrating an enhanced proliferative potential of CB CD34+ HLA-DR+ cells vs. similar cells from adult BM. Furthermore, a 6.2-fold increase in the number of CB CD34+ still residing in G0/G1 was observed on day 5 in cultures supplemented with SCF and IL-3, suggesting the generation of large numbers of primitive HPC in vitro. The effect of SCF on the exit of CB and BM CD34+ HLA-DR+ cells from G0/G1 was then examined. Following 36- to 48-hour exposure to SCF, 45% of quiescent CB cells exited G0/G1 in contrast to only 13% of quiescent BM cells. In serum-free media supplemented with either SCF or IL-3 alone, CB CD34+ HLA-DR+ cells did not exit G0/G1 phases of cell cycle as rapidly as when CB plasma was present, unless SCF and IL-3 were added simultaneously. Collectively, these results suggest that CB CD34+ cells are more responsive to cytokine stimulation, especially SCF, and may represent more suitable candidates for ex vivo expansion of HPC than BM cells. Furthermore, these data illustrate potentially important biologic differences between the HPC content of subpopulations of BM and CB cells, and the response of these subpopulations to cytokine stimulation. PMID- 7538346 TI - Rapid isolation and serum-free expansion of human CD34+ cells. AB - Human CD34+ cells were isolated from bone marrow from normal volunteers and expanded under serum-free culture conditions. CD34+ cells were cultured with interleukin-3 (IL-3), IL-1, and stem cell factor and expanded in granulocyte macrophage colony-forming units, erythroid blast-forming units, and CD34+ cell number during the first 7-14 days of incubation. By contrast, cultures maintained in fetal calf serum under identical conditions showed much reduced expansion, as measured by all of the above parameters. The level of expansion of the CD34+ cells was dependent on the combination of growth factors used during culture. The data establish the feasibility of serum-free expansion of progenitors and suggest the clinical use of this procedure for the generation of expanded progenitor cell products for transfusion after chemotherapy to minimize treatment-related cytopenias. PMID- 7538347 TI - Harvesting, characterization, and culture of CD34+ cells from human bone marrow, peripheral blood, and cord blood. AB - Stem and progenitor cells from a variety of sources including bone marrow, cord blood, and peripheral blood have been used for transplantation. This study compares CD34 cells from all three sources. Flow cytometry analysis of CD34 cells in multiple samples of normal peripheral blood and patient peripheral blood mobilized with chemotherapy (cyclophosphamide/VP16), chemotherapy plus granulocyte colony stimulating factor (G-CSF), and G-CSF alone were compared to bone marrow and cord blood. Although the relative distribution of CD34 percentages in each preparation of cells varied widely, on average the percentage of CD34 cells in these different preparations was 0.15%, 0.6%, 2%, 0.45%, 1.68%, and 0.83% respectively. CD34 subset analysis was performed on these cell preparations using multicolor flow cytometry and antibodies to CD33, CD13, CD45RA, CD19, CD71, and CD38. The major differences observed were that bone marrow CD34 cells contain high percentages of CD19+ cells not found in significant quantity in the other cell preparations and cord blood CD34 cells contained a higher percentage of CD38-cells than the other cell preparations. A magnetic bead system was used with anti-CD34 antibody to purify CD34 cells from mobilized peripheral blood apheresis products, cord blood, and bone marrow. Efficient selection with high purities of CD34 cells was achieved with each of the cell preparations. Comparison of colony-forming activity of each of the cell preparations showed cord blood and mobilized peripheral blood to have slightly higher cloning efficiencies than bone marrow with higher numbers of erythroid blast-forming units (BFU-E) also observed in cord blood CD34 cells. Culture of isolated CD34 cells in liquid culture with interleukin-3, stem cell factor, G CSF, and granulocyte-macrophage GM-CSF showed over a 100-fold expansion in cell numbers after 25 days, with the peak expansion of colony-forming cells occurring between days 11 and 16. Analysis of day-10 cells from these cultures showed them to be predominantly promyelocytes, myelocytes, and metamyelocytes, with cord blood CD34 cultures showing more promyelocytes than peripheral blood or bone marrow and bone marrow showing more metamyelocytes. Comparison of the proliferation of CD34 cells from these different cell preparations showed that cord blood CD34 cells cultured for 10 days averaged an 85-fold increase in cell numbers followed by mobilized peripheral blood CD34 cells, with an average 56 fold increase, and bone marrow CD34 cells, with an average 49-fold increase. PMID- 7538348 TI - Expansion of human neonatal progenitor cells in vitro under serum-deprived conditions. AB - Over time CD34+ cells purified from human cord blood generate large numbers of progenitor and precursor cells in liquid culture under serum-deprived conditions if stimulated with a cocktail of growth factors which include stem cell factor (SCF). The ex vivo expansion observed in liquid cultures is not homogeneous over time but involves the recruitment of different cell compartments and can be triggered by different growth factor combinations. We have recognized at least three phases in these liquid cultures. Phase I spans the first 20 days of culture. In this phase, progenitor and precursor cells are generated from the progenitor cell compartment itself in response to SCF in combination with either IL-3, erythropoietin, or G-CSF. Phase II spans the second month of culture and involves the recruitment of less and less differentiated cells by IL-3 and SCF. Phase III spans from the third month on and results in the indefinite proliferation of human mast cells. These results raise caution on the biological equivalence of liquid culture en vivo expanded hematopoietic cells at different time points. PMID- 7538349 TI - Purification and release from quiescence of umbilical cord blood early progenitors reveal their potential to engraft adults. AB - Steel factor (SF) increases the frequency of colony formation by CD34+ CD38- cycling cells, but it does not reverse the effect of an autocrine production of transforming growth factor (TGF)-beta 1 by early progenitors of the stem cell compartment. We have used optimal culture conditions supplemented with SF and anti-TGF-beta serum to estimate the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU), twice as many granulocyte macrophage (GM) CFU, and three times as many erythroid burst-forming units as the same population from an average bone marrow sample used in adult transplantation. These results suggest that umbilical cord blood is a suitable source of cells for adult transplantation. PMID- 7538350 TI - Cytokine-dependent ex vivo expansion of early subsets of CD34+ cord blood myeloid progenitors is enhanced by cord blood plasma, but expansion of the more mature subsets of progenitors is favored. AB - Expansion of stem/progenitor cells has important implications for transplantation. We recently reported that a factor or factors in cord blood (CB), but not adult peripheral blood (PB), plasma enhanced replating of granulocyte erythroid macrophage megakaryocyte colony-forming units (CFU-GEMM) progenitors, a measure of self-renewal capacity. In this context, we evaluated effects of CB plasma, in comparison with PB plasma and fetal bovine serum (FBS), on ex vivo expansion of CD34+ column-separated (72-98% CD34+) CB cells using stroma-free cultures in the absence and presence of either PIXY321 (a granulocyte macrophage colony-stimulating factor/interleukin-3 [GM-CSF/IL-3] fusion protein), IL-3+IL-6+IL-1, or steel factor (SLF) -/+ PIXY. CB plasma, PB plasma, or FBS alone did not sustain cell numbers. Combinations of CB plasma +SLF+PIXY induced maximal cumulative nucleated cell expansion (1044-fold), which was greater than that of PB plasma plus cytokines (633-fold) and FBS plus cytokines (142-fold). Total CD34+ cells peaked by day 7 with 7-fold expansion in the presence of CB plasma+SLF+PIXY compared with PB plasma or FBS with these same cytokines (threefold each). By day 7, total CFU-GEMM production in the presence of either PIXY, SLF+PIXY, or IL-3+IL-6+IL-1 was greater with CB plasma (maximum 11.4-fold average increases) than with PB plasma (6.8-fold increase). These increases were greater than with FBS. However, PB plasma was at least as good as CB plasma for expansion of immature and mature subsets of CFU-GM. The frequency of progenitors decreased with time, and expansion was coupled with differentiation. Although the proliferative capacity of CFU-GEMM was maintained, the capacity of CFU-GEMM to be replated decreased after time in suspension culture, suggesting age-related commitment of cells. Moreover, with plasma +SLF+PIXY for 7 days, expansion of more mature CFU-GM (responsive to GM-CSF) was greater (16-146-fold with CB plasma and 31-208-fold with PB plasma) than immature CFU-GM (responsive to GM-CSF+SFL) (4- to 14-fold with CB plasma and 6- to 17-fold with PB plasma). The results suggest that CB plasma enhances expansion of CFU-GEMM to a greater extent than PB plasma or FBS, but expansion in these cultures favors more mature subsets of cells. PMID- 7538352 TI - Ex vivo expansion of cord blood-derived stem cells and progenitors. AB - Cord blood-derived CD34+ cells have proved superior to adult marrow or elicited peripheral blood in ex vivo cell and progenitor expansion. In addition, cord blood-derived cells with a phenotype and function of early stem cells (e.g., long term culture-initiating cells) can be expanded in vitro in the presence of a combination of cytokines by 15- to 20-fold over 7-14 days. These observations suggest that ex vivo expansion of cord blood CD34+ cells will permit improved engraftment of adults and will prove effective in retroviral-mediated gene transfection of early stem cell populations. PMID- 7538351 TI - Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture. AB - Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL 1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells. PMID- 7538354 TI - Comparative analysis of retroviral-mediated gene transduction into CD34+ cord blood hematopoietic progenitors in the presence and absence of growth factors. AB - Hematopoietic stem and progenitor cells present in umbilical cord blood at the birth of a child are efficiently transduced ex vivo by genes using retroviral vectors in combination with exposure of these cells to combinations of growth factors. Because retroviral-mediated gene transduction of adult bone marrow and blood hematopoietic stem and progenitor cells is greatly enhanced by growth factors, we evaluated the possibility that cord blood progenitors, which have extensive proliferative and replating capacity, could be efficiently transduced with a TK neo gene in a retroviral vector in the absence of growth factors, and also determined the influence of exogenously added growth factors on this transduction. Highly purified CD34+ (62% pure) cord blood cells isolated by magnetic bead separation were cultured in suspension for 72 hours with viral supernatant in the absence and presence of interleukin-3 (IL-3), IL-6, and steel factor. Evaluation of progenitor cell-derived colonies and polymerase chain reaction (PCR)/Southern analysis of the TK neo gene in resultant colony cells demonstrated that some gene transduction was apparent in the absence of growth factors (12.8-14.3% by PCR), but that this was greatly enhanced (40.0-44.4%) by addition of growth factors. Reverse transcription PCR analysis of the expression of IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor genes in this population of cells suggested that the transduction, although at a lower efficiency, in the absence of added growth factors might in part be due to "constitutive" and viral supernatant-induced expression of these cytokine genes in the CD34(+)-enriched cell population.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538353 TI - Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood. AB - Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20 fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma. Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor. Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum. In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU GM were expanded only 10- to 14-fold by day 14. As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538355 TI - Parvovirus-based vectors for human gene therapy. AB - It is becoming increasingly clear that the parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors in human gene therapy. Specifically, the adeno-associated virus 2 (AAV), a human parvovirus, has gained particular attention in view of its nonpathogenic nature as well as its remarkable site-specificity of integration into the human chromosome. Using the recombinant AAV vector system, it is feasible to obtain high-efficiency transduction of slow- or non-cycling primary hematopoietic stem and progenitor cells, without the need for prestimulation with cytokines, which could potentially lead to differentiation of these cells before transplantation. PMID- 7538356 TI - Umbilical cord blood cell transduction by retroviral vectors: preclinical studies to optimize gene transfer. AB - Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogeneic bone marrow transplantation, for the treatment of a number of genetic diseases. To determine conditions that yield maximal gene transfer into UCB progenitor cells, we examined a number of variables. We used cell-free retroviral vector supernatants that convey neomycin (G418) resistance and measured the percentage of G418-resistant progenitor derived colonies. Adding retroviral supernatant to the UCB cells in basal medium once a day for 3 days produced a threefold increase of G418-resistant colonies (9.8%) compared to a single exposure to supernatant (3.1%). To establish whether recombinant human growth factors are beneficial during transduction, the presence of interleukin-3 (IL-3), IL-6, and mast cell growth factor (MGF, a c-kit ligand) were compared in different combinations. Inclusion of the three factors together caused a threefold increase of gene transfer (30.4%) compared to transduction in basal medium. When the UCB cells were precultured in medium containing IL-3, IL 6, and MGF for 3 days before addition of the retroviral supernatant on days 4, 5, and 6, the average extent of gene transfer was 21.8%, compared with an average of 34.4% when UCB cells were transduced on days 1, 2, and 3. The presence of marrow stroma during the transduction of the UCB cells did not further increase gene transfer. We conclude that UCB progenitor cells can be efficiently transduced with the use of recombinant human growth factors IL-3, IL-6, and MGF and may be a suitable source for gene therapy. PMID- 7538358 TI - Relationship between staining by digital subtraction angiography and vascularity in breast cancer: analysis of the time-density curve and the results of staining for factor VIII-related antigen/von Willebrand factor. AB - We performed intravenous digital subtraction angiography (IV-DSA) in 31 patients with primary invasive breast cancer, and calculated the maximum density of tumor staining from the time-density curve obtained. Specimens resected from the same patients were stained for factor VIII-related antigen/von Willebrand factor (vWF), and the amount of microvessels was calculated by image processing. A significant correlation (correlation coefficient: 0.85) was found between the maximum density of tumor staining and the vascularity determined by staining for vWF, indicating that the maximum density indirectly reflects the vascularity in the tumor. PMID- 7538357 TI - The relationship of cytoplasmic intermediate filaments and membrane antigens with hormone receptors, nuclear staining density, and mode of stromal invasion in human breast cancer. AB - Serial sections on biopsies of 26 women with invasive breast carcinoma were stained for low molecular weight keratins (LKER), high molecular weight keratins (HKER), epithelial membrane antigen (EMA), vimentin (VIM), carcinoembryonic antigen (mCEA and pCEA), and nuclear estrogen (mER) and progesterone receptors (mPGR), using monoclonal and polyclonal sera. Specified areas were identified on serially sectioned slides and staining reactions were compared among areas as well as among patients. The study concludes: (1) LKER staining was positively related to (a) the percentage of tumor cells with densely stained nuclei, (b) a trabecular mode of stromal invasion, and (c) HKER and EMA staining, and inversely related to (d) VIM staining and gross tumor size. (2) HKER was also inversely related to gross tumor size. (3) VIM staining was positively related to pCEA staining. (4) VIM staining was inversely related to staining ER and PGR. (5) LKER, HKER, and EMA staining was stronger in areas of trabecular rather than confluent areas of stromal invasion within the same biopsy. (6) Staining for ER and PGR was not related to mode of stromal invasion, but showed a strong inverse relationship with mitotic index. Positive staining for LKER may be an indicator of better differentiation together with densely staining nuclei and trabecular mode of stromal invasion, whilst VIM and pCEA appear to be related to features indicative of lack of differentiation. Hormone receptor positivity seems to be strongly related to mitotic activity rather than differentiation. PMID- 7538362 TI - Localization of the tenascin-C gene to pig chromosome 1. PMID- 7538360 TI - [Characterization of chromosomal rearrangements by in situ hybridization in glioblastoma]. AB - In a case of glioblastoma, the following karyotype was determined: 47, X, - Y, + der(1) t(1;9)(p21;p23), t(1;9)(p21;p23), + 3, + 7, der(9) t(Y;9)(q11;p21), - 13, t(13;16)(p13,p11), del(14)(q11q22). Classical satellite DNAs are mainly located in chromosomes 1, 9, 15, 16 and Y. Because, most of these chromosomes were implicated in the rearrangements, a detailed cytogenetic study was undertaken. This study included in situ hybridization of the satellite and alphoid DNAs of chromosomes 1, 9, 16 and Y combined with various chromosome banding methods (DA DAPI, quinacrine mustard and R-banding). The data obtained, demonstrated that the breakpoints were always located outside the areas containing the satellite and alphoid DNAs. The situation observed here differs from that reported in breast cancers for which a high proportion of the breakpoints occur within these areas. These findings suggest that in glioblastoma, chromosome rearrangements result from different mechanisms than those implicated in breast cancers. Thus, in cancers, chromosomal instabilities may result from several mechanisms. PMID- 7538361 TI - [Contribution of chemotherapy in the treatment of cancer of the cavum (UCNT): apropos of 47 cases]. AB - The authors present, from a 47 patients retrospective study, the place of BAC chemotherapy in the treatment of undifferenciated nasopharyngeal carcinoma. Twenty three patients treated with exclusive radiation therapy are compared with 24 patients treated with three courses of BAC chemotherapy prior to and after radiation therapy. The procedure using associated chemotherapy permits to increase the 5 years survival rate up to 20% more and to obtain a 100% primary response of the tumor. This encouraging results should be confirmed by a prospective randomized study about a great number of patients. PMID- 7538359 TI - Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms. AB - The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport. PMID- 7538363 TI - Organochlorine pesticide residues in cereals in Nigerian markets. PMID- 7538364 TI - Surgical treatment of tumor metastases: general considerations and results. AB - The prerequisite for a curative resection of metastases is their restriction to the key organs, the liver and lungs, in the sense of a limited dissemination. For long-term prognosis, the type of primary tumor as well as the radical resection of lung and liver metastases is essential. To improve the process of surgical indication and therapy of tumors, clear definitions for the terms "tumor recurrence" and "metastases" have been agreed upon. Research and clinical investigation have led to a better understanding of tumor-regulating factors, some of which are briefly described: Metastasis promoting factors include the lack of E-cadherin, which leads to a local penetration of basal membranes by tumor cells; CD44 seems to play an important role in cell-cell and cell-matrix interactions, apparently increasing the metastatic potential of tumors and reducing the long-term survival of patients. High levels of urokinase in primary tumors are also associated with a poorer prognosis, as well as plasminogen inactivator inhibitor PAI II, which plays a crucial role in tumor growth. Positive findings in bone marrow aspirates of patients with different malignancies, stained for cytokeratin 18, either are associated with higher recurrence rates in colon and breast cancer or can be correlated to the prognosis of patients with gastric cancer. Technical aspects of surgery for hepatic, pulmonary and skeletal metastases are presented and discussed with respect to curative and palliative indications. PMID- 7538366 TI - Perforated acute appendicitis in a patient with AIDS/HIV infection: report of a case. AB - We report herein the case of a 40-year-old man with AIDS who was admitted to hospital with severe abdominal pain, fever, and chills. He underwent an emergency laparotomy which revealed a perforated appendix with suppurative peritonitis. An appendectomy with peritoneal drainage was carried out, but the postoperative course was complicated by fever without leukocytosis; however, he gradually improved following treatment with intravenous antibiotics, granulocyte colony stimulating factor (G-CSF) and immunoglobulins, and made a complete recovery. His postoperative course demonstrates the effectiveness of this treatment regimen for patients with AIDS complicated by infection without an increase in the white blood cell count (WBC). PMID- 7538365 TI - Serum-soluble interleukin-2 receptor levels before and after surgical treatment for Graves' disease. AB - Serum-soluble interleukin-2 receptor (ssIL-2R) is released from its specific affinity membrane receptor on activated T lymphocytes and is detected as a form of the alpha-chain of IL-2R in the peripheral blood. We measured the ssIL-2R levels by enzyme-linked immunosorbent assay in 29 healthy subjects and 39 patients with Graves' disease who were given antithyroid drugs to prepare them for surgical treatment. The preoperative ssIL-2R levels in Graves' patients were significantly higher than those in the healthy controls (681 +/- 387 vs 369 +/- 149 U/ml); in particular, the preoperative levels in hyperthyroid patients were significantly higher than those in euthyroid patients. On the 5th day after thyroid resection, their ssIL-2R levels (560 +/- 296 U/ml) were significantly lower than the preoperative levels. There were no correlations between the preoperative ssIL-2R levels and the immunological parameters such as thyroglobulin antibody, microsomal antibody, and thyroid-stimulating hormone receptor antibody. Thus, we conclude that ssIL-2R levels can be decreased by surgical resection of goiter in Graves' patients whose hyperthyroid status does not respond to the administration of antithyroid drugs, and this may be a useful indicator of immunological status after surgical treatment of Graves' disease. PMID- 7538367 TI - IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain. AB - IGFBP-3 and IGFBP-5, but not the other 4 IGF binding proteins, specifically bound to endothelial cell (EC) monolayers. Charged compounds, such as heparin and heparan sulfate, competed for this binding. Of the 6 IGFBPs, IGFBP-3 and IGFBP-5 had the greatest heparin affinity. Peptides of 18 amino acids were synthesized, corresponding to a common basic region of IGFBP-3 (P3), IGFBP-5 and IGFBP-6 (P6) which contained a heparin binding sequence. P3 and P6 inhibited IGFBP-3 and -5 binding to endothelial cell monolayers and the peptides bound directly to EC extracellular matrix. This suggested that the C-terminal basic segment of IGFBP 3/-5 is important for the association of the binding protein with the EC monolayer. PMID- 7538368 TI - Regulation of chick muscle satellite cells by fibroblast growth factors: interaction with insulin-like growth factor-I and heparin. AB - This study describes the effect of acidic and basic fibroblast growth factor (FGF) on DNA synthesis in chick satellite cells in vitro and interactions with insulin-like growth factor-I (IGF-I) and exogenous heparin. Basic bFGF stimulated incorporation of [3H]thymidine into DNA with a half-maximum concentration (ED50) of 3.23 +/- 0.33 pmol/l, more than 500-fold more potent than acidic FGF (ED50 = 2.13 +/- 0.5 nmol/l). Both bFGF and IGF-I allowed the cells to traverse the cell cycle with an approximate length of the G1 phase of 12 h. When cells were incubated with bFGF and IGF-I together their effects on DNA synthesis were additive rather than synergistic throughout the full concentration range. Incubation of satellite cells with low concentrations of heparin (ng/ml) to mimic the effect of endogenous heparan sulphate proteoglycan caused a small increase in DNA synthesis, whereas higher concentrations (microgram/ml) inhibited DNA synthesis in a dose-related manner. A low concentration of heparin increased DNA synthesis at the highest concentration of bFGF, but high doses of heparin inhibited the response to bFGF throughout the dose-response curve but without altering the ED50. RNAse protection assay showed the expression of bFGF mRNA in proliferating cells which appeared to decrease on differentiation. The results suggest that aspects of neonatal muscle development are regulated by interactions between autocrine/paracrine growth factors such as IGF-I and bFGF, perhaps IGF-I derived from the circulation, and components of the extracellular matrix. Concentrations of the matrix components may change throughout the neonatal period and into adulthood and have an important effect on the regulatory role played by the growth factors. PMID- 7538370 TI - Development of smooth muscle hypertrophy is closely associated with increased gene expression of insulin-like growth factor binding protein-2 and -4. AB - The aim of the present investigation was to study the role of the IGF system in the development of smooth muscle hypertrophy. Hypertrophy was initiated by partial ligation of the urethra in female Sprague-Dawley rats. The solution hybridization assay was used to analyse the mRNA levels. Ligation of the urethra induced a sustained increase in bladder wet weight during the following 6-week period studied. IGF-I mRNA increased from 1 to 3 amol/micrograms DNA 1-day after ligation, peaked at 3-days (4-fold), and normalized 6-weeks after ligation. In hypertrophying bladder, IGFBP-2 and -4 mRNA increased sharply during the first 10 days (5-fold) and remained elevated during the 6-week period. The IGF-I receptor mRNA did not change significantly. Removal of the obstruction 10 days after ligation caused a regression of bladder wet weight, and resulted in normalization of IGF-I, IGFBP-2 and -4 mRNA. The results show a close correlation between progression or regression of smooth muscle hypertrophy and differential regulation of IGF-I, IGFBP-2 and -4 mRNA, indicating that the IGF system may play a role in smooth muscle hypertrophy. PMID- 7538369 TI - The development of an eluted stain bioassay (ESTA) for human growth hormone. AB - The basic characteristics of MTT-formazan production by both quiescent Nb2 cells and those activated by fetal calf serum or human growth hormone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lactogens such as growth hormone. The resulting in vitro bioassay is sensitive and precise, with a detection limit of about 0.05 mU hGH/l (19 ng/l) and a within-assay imprecision of 2.5% in the presence of 0.3 mU hGH/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, large magnitudes of response are observed. The major component of the response is clearly derived from metabolic activation of the cells, rather than increased cell proliferation. The response was abolished by anti-human growth hormone. Delayed addition of the latter demonstrated that the presence of the hormone is required for the entire 96 h of the recommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plateau which covers 4 orders of magnitude of incremental hormone concentrations. A decline in response is observed at the highest dose tested, 10(6) mU hGH/l (385 mg/l). This auto-inhibition is consistent with recent reports of a reduction in response due to stoichiometric blockade of sequential receptor dimerisation which is crucial for activation of both somatogenic and lactogenic receptors by hGH. PMID- 7538371 TI - Valid measurements of total IGF concentrations in biological fluids. Recommendations from the 3rd International Symposium on Insulin-like Growth Factors. PMID- 7538372 TI - Bradykinin-B2 receptors in humans and rats: cDNA structures, gene structures, possible alternative splicing, and homology searching for subtypes. AB - 1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolated a human bradykinin receptor cDNA homologous to a rat B2 receptor cDNA. 2. The cDNA was expressed in the bradykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the known rat B2 receptor. In addition, the expressed receptor has a low affinity for des-Arg9-bradykinin. Thus, the cDNA encodes a human B2-bradykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, sequencing and characterization of parts of the human and rat B2-bradykinin receptor genes demonstrated the postulated three-exon structure. This structure includes two 5' exons upstream of the most favorable translation initiation methionine in exon-3. 4. The two 5' exons each contain methionines, which if independently spliced to the third exon, would yield an open reading frame that includes all of exon-3. This arrangement could thus vary the amino-terminal region of the protein. Do these potential arrangements occur in human RNAs, and will they lead to proteins with differing amino-termini? 5. Reverse transcriptase polymerase chain reactions (RT-PCR) using human mRNA, nested primers from exon-1 and exon-3, and detection of the products by hybridization using an independent exon-1 oligonucleotide showed that the arrangement of exon-1 with exon-2 and exon 3 could not be detected in eight human RNAs. Furthermore, exon-1 spliced with exon-3 was a common arrangement. 6. Low stringency examination of human and rat Southern blots revealed only bands attributable to the known human or rat B2 bradykinin receptor. 7. Reduced stringency hybridization searches of seven different genomic and cDNA libraries--including two different human genomic libraries, a rat genomic library, two different rat uterus cDNA libraries, a rat brain library and a human lung library--yielded only rat or human B2-bradykinin receptors. The results of our low stringency hybridization experiments suggest that other bradykinin receptors are less than 60% identical, on the nucleotide level, to the known B2 receptor. 8. Degenerate polymerase chain reactions using rat genomic DNA as a template and degenerate primers, designed based on the homology of a B2-bradykinin receptor with angiotensin-II type-1 receptor, identified B2-bradykinin receptors, angiotensin-II-type-1 receptors and three novel orphan receptors. PMID- 7538375 TI - Plasma kallikrein clearance by the liver: a review. AB - 1. The liver is the main organ clearing both plasma and tissue kallikreins from the circulation. Hepatocytes are responsible for the internalization of rat plasma kallikrein (RPK) and the clearance of plasma kallikrein by the liver is Ca(2+)-independent. The binding site of RPK to the liver cell is located on its heavy chain which is not exposed on prokallikrein. An S-type lectin accounts for the receptor-mediated endocytosis of RPK. 2. These properties of the liver are affected by pathological situations, particularly the acute-phase response to inflammation, in which the kallikrein-kinin system plays a major role. The hepatic clearance of the alpha 2-macroglobulin-plasma kallikrein complex is less efficient than the clearance of the free enzyme. PMID- 7538374 TI - Involvement of the kallikrein-kinin system in the salivary secretion elicited in rats by heat stress. AB - 1. During heat exposure, rats secrete large amounts of saliva. Salivation started when body temperature exceeded 39 degrees C and was reduced by kininogen deficiency or by HOE 140, a bradykinin antagonist. This secretory response was associated with a partial depletion of glandular kallikrein from the submaxillary glands. The depletion was abolished by simultaneous treatment of the animals with an alpha- and a beta-adrenergic antagonist. During heat exposure, plasma levels of kininogens were reduced. 2. Pilocarpine and substance P induced a similar flow of saliva in normal and kininogen-deficient rats and released low amounts of kallikrein from salivary glands. Phenylephrine and isoproterenol induced a larger flow of saliva in normal rats than in kininogen-deficient rats. Both agents released large amounts of kallikrein in saliva but isoproterenol was only active at large doses. 3. During heat exposure, the blood content of submaxillary glands in normal as well as in kininogen-deficient rats increased as a function of the ambient temperature. This increase was suppressed by atropine and NG-nitro-L arginine, a NO-synthase inhibitor, but was not modified by HOE 140. Simultaneously, a swelling of the glands and of the surrounding soft tissues occurred in normal but not in kininogen-deficient rats. Kallikrein was present in the edema fluid. 4. The kallikrein-kinin system would thus participate in heat induced salivary secretion and kinins may be a factor responsible for electrolyte and water exchanges in the glands. PMID- 7538376 TI - Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations. AB - Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of > 400 mutations associated with disease. Except for the delta F508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers. PMID- 7538373 TI - Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9. AB - 1. The kinetics of the degradation of the kinins bradykinin and Met-Lys bradykinin, angiotensins I and II and the tachykinin substance P by PMNL collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min 1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate. PMID- 7538379 TI - [Retropubic adenectomy (Millin's technique). Our experience]. AB - From January 1983 to December 1993, on 1917 ptz who underwent to operations for BPH, 1532 pts (80%) had transurethral adenomectomy, and 385 pts (20%) had retropubic adenomectomy. The prostatic weight, obtained by ultrasound, is the factor which determines the kind of operations: transurethral adenomectomy if the prostate weight is lower than 50 gr, open surgery if it is more than 50 gr. In our experience about retropubic adenomectomy (Millin) we never had patient's death during operation or in the immediate post-operatively period. The early complications were 13.7%, and the late complications were 3.8%. The retropubic adenomectomy, when performed with right indications, is still valid. PMID- 7538378 TI - [Technical considerations on optimization of TURP]. PMID- 7538380 TI - [Transurethral needle ablation (TUNA) of the prostate: preliminary results]. AB - A multicentric prospective study was designed in Italy to verify the efficacy and the safety of TUNA-BPH patients treatment. The study is co-ordinate by Progress in Urology Association was started in January 1994. Thirty five patients were treated (Mean age 68.4 +/- 4.1 aa). All patients were selected and evaluated to respect the recommendations of the International Consensus Committee (Paris 1993). The procedure was performed with lidocaine 2.5% urethral gel, oral intake of 10 mg of diazepam and steroidal antiphlogistic drugs 1 hour before treatment. Maximal number of prostatic lesions were 6; in 2 BPH initial cases were done 2 lesions alone. The treatment was interrupted at the third lesion in 2 patients because uncomfortable. Hematuria was observed in all patients, but it was resolved within 12 hours. Irritative symptoms were referred by 10 patients (28.6%) and they spontaneously were insignificant within 24 hours. One patient had an acute prostatitis. In 15 patients (42.6%) was necessary to put a suprapubic cystostomy, but all except one voided within 36 hours. We noted narrow connection between prostatic lesions number and urinary acute retention after TUNA. The average follow-up is 3.2 months (range 0.5-6). At the follow-up urine culture was always negative and PSA values increased in the first month. The value of PSA was always higher than pre-treatment, but from 1 month to 3 months progressively reduced.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538382 TI - [Symptoms of prostatic hypertrophy: evaluation parameters]. AB - To evaluate BPH symptoms many questionnaires have been proposed. The World Health Organisation Consultation on BPH recommended the "AUA-7 questionnaire" to which and additional on the quality of life was added, creating the "International Prostate Symptom score" (I-PSS). In any case, before proposing a surgical therapy, you have to correlate between symptoms, gland size and urodynamics. The only method to accurately evaluate outflow obstruction is pressure/flow study. PMID- 7538381 TI - [Physiopathology of BPH obstruction]. AB - The underlying BPH related mechanisms of outlet obstruction are outlined (modification of the shape of the bladder neck, failure of funnel formation, geometric variations of the urethra, alpha 1 adrenoceptors mediated sympathetic hyperactivity at the level of the lower genito-urinary tract). Bladder voiding dysfunction in response to prostate obstruction is also discussed and correlated to ultrastructural patterns and clinical symptoms. Finally, a pathogenetic mechanism for detrusor overactivity in obstructed bladder is suggested. PMID- 7538377 TI - Novel frameshift mutation in exon 4 of CFTR gene. PMID- 7538383 TI - [Benign prostatic hypertrophy. Optimized diagnosis]. AB - The optimal diagnostics of B.P.H. must be efficient, cheap and of spare invasiveness either physical than psychological. The optimization of diagnosis is the obtainment of high quality in the services provided, matched to a correct and shrewd utilisation of resources. To optimise a diagnosis efficiency is necessary, which means the largest degree of standardization of diagnostical procedures, with responsible management of economical factors. A course of standardization, cause his excessive simplification, cannot shared to a lot of Urologists, because in their opinion there is a risk to lose informations in every single patient, but is essential to use a universal language that make easy the comparison with patients and with results whether in order to clinics or trails. The standardisation of diagnostics consists to obtain the maximum of informations from present methodology and that is possible to realize in two way: improving the technique of execution and including the diagnostic test in the appropriate point of algorithm. PMID- 7538384 TI - [Complex urodynamic diagnosis of prostatic obstruction]. AB - Pressure/flow study is nowadays essential in the routine diagnostic workup of prostatic obstruction. It is the single most useful investigation showing evidence of bladder outlet obstruction. Therefore it should always be performed before any surgical treatment aimed to relieve the obstruction and it should always be referred to whenever the results of a medical treatment are judged. Lack of indication to pressure/flow study is limited to few clinical conditions such as complete urinary retention, large bladder diverticulum, high grade vesicoureteral reflux, bladder stone and/or urinary tract infection. The investigation needs much experience while is carrying out in order to avoid artifacts and then to read the pressure/flow data obtained. Results evaluation is still debatable. Nevertheless recent computerized programs allow to reduce artifacts and to obtain more reproducible and comparable data in order to make more reliable the indication to treatment or to judge its results. PMID- 7538385 TI - [PSA values before and after finasteride treatment in patients with benign prostatic hypertrophy]. AB - A total of 21 patients with benign prostatic hyperplasia was treated with finasteride to evaluate the variation of prostatic volumes and PSA values. After 6 months prostatic volumes showed a decrease of 16% while PSA values of 27%. No variations of PSA density were observed. PMID- 7538386 TI - [Terazosin in the treatment of benign prostatic hypertrophy]. AB - Benign prostatic hypertrophy provokes clinical manifestations correlated on one hand to a static obstructive component, due to the increase in glandular size, and on the other hand to a dynamic component, controlled by the alpha adrenergic autonomic system which gives the smooth muscular tonus of the prostatic adenoma. The alpha adrenergic receptor block reduces the dynamic component, improves the clinical and urodynamic parameters determined by the infra-bladder obstruction in patients with BPH. The selective alpha 1, long acting antagonists especially, such as terazosin, offer a safe and efficient therapy for selected patients suffering from BPH. They also have the indisputable benefit of mono administration. In this study the basic concepts of BPH treatment with terazosin are discussed. PMID- 7538387 TI - [Alpha blockers, TUS-P and TUR-P in the treatment of benign prostatic hypertrophy. A comparison using multivariate statistical analysis]. AB - To evaluate the effectiveness of Alpha-blockers, TUI-P and TUR-P in the treatment of obstruction due to BPH, 50 patients, never before treated, were considered. Fifteen were treated with alfuzosin chlorhydrate 7.5 mg/day for four months, 15 were submitted to TUI-P and 20 to TUR-P. In all patients linear purr was carried out before treatment and was repeated from 60 to 90 days after intervention in surgical patients and during the fourth month of treatment in patients treated with alfuzosin. The data obtained were analyzed with the T-test both for dependent and independent samples. The results show that Alpha-blocker contain an increase in maximal flow, without decreasing bladder voiding pressures. On the contrary TUI-P and TUR-P, besides the increase in maximal flow obtain a significant reduction of bladder pressures. The conclusions are the following: maximal flow alone is not a sufficient parameter to evaluate the work of the bladder, the entity of the obstruction and the effectiveness of the therapy. The treatment with Alpha-blockers is unable to reduce the obstruction due to BPH. TUI P and TUR-P realize an effective deobstruction. Under the same deobstructing effect TUR-P assures a better voiding performance by obtaining higher flow values. PMID- 7538389 TI - BPH: how it is managed in Lombardia today. PMID- 7538390 TI - [Transurethral resection of the prostate: when?]. AB - Over a period of 5 years (from 1989 to 1993) 410 patients with benign prostatic hypertrophy (BPH) were treated by transurethral resection of the prostate (TURP). A retrospective study was conducted by the Authors to evaluate indications, surgical approaches and results. The Authors' experience is compared with literature reports. PMID- 7538388 TI - [Radical versus conservative therapy in prostatic adenoma. Open adenomectomy]. AB - Our technical procedure for surgical adenomectomy of prostate is the Hey Delinotte one. Indications for this type of procedure are large adenomas with a volume, estimated by ultrasonography, more than 50 cc. and with significant obstruction and urinary retention. In our experience during the past 3 years, in patients without severe operating risk, it was utilized a little more frequently than transurethral resection. We hope that in the future the new procedures will be as useful and resolutive as traditional ones. PMID- 7538392 TI - [Turbo TURP]. AB - Transurethral resection of the prostate (TURP) is just one of the numerous options available in the modern treatment of benign prostatic hyperplasia (BPH), but it's still now the "gold standard". Absorption of irrigating fluid is the greatest complication and results in clinical manifestations in 2% of the TURPs performed. There is a statistically significant relationship between gland size and the total volume of irrigant absorbed. To reduce this absorption Reuter introduced suprapubic trocar drainage to obtain the same results from both the small and the large prostates. Operating time, in fact, is reduced because, also at the same time, this large tube drains both irrigation fluid and prostatic chips. PMID- 7538391 TI - [TURP: what and when. Indications, results, complications: our experience]. AB - Transurethral resection of the prostate (T.U.R.P.) has indeed replaced open surgery in the great majority of cases, particularly when the gland is estimated to weight 50-60 g or less. After prostatectomy the patient resumes a normal voiding pattern and obstructive symptoms quickly disappear, although irritative symptoms tend to persist for some time. PMID- 7538393 TI - [Etiopathogenesis of benign prostatic hypertrophy]. AB - The pathogenesis of benign prostatic hyperplasia (BPH) remains largely unresolved. The natural history of the disease involves two distinct phases, a pathological and a clinical one, whose pathogenesis is different. The pathologic phase is composed of two stages microscopic and macroscopic neither of which produces clinical dysuria. Nearly all men develop microscopic BPH if they live long enough but only in 50% of men microscopic BPH grows to produce a macroscopic enlargement of the gland and the evolvement happens in a period between 5 and 7 years. So different etiologies for the microscopic and macroscopic evolution of prostate can be argued. The clinical phase of BPH involves the progression of pathologic BPH to the clinical form in which the patients develop symptomatic dysuria. Again only about 50% of the men with macroscopic BPH progress to the clinical form: although macroscopic enlargement of the prostate is necessary for the development of clinical BPH it is not sufficient by itself for the progression to clinical phase and additional factors are required such as phlogosis, vascular infarct, enanchement of alpha adrenergic tone. These remarks on natural history suggest a multiple approach to BPH not addressed to treat the pathologic phase but the clinical one.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538394 TI - [Surgical treatment of benign prostatic hypertrophy: what and when TURP vs adenomectomy]. AB - Surgical treatment is the gold standard for Benign Prostatic Hyperplasia (BPH) therapy. At the present diagnostic approach allows better patient selection and treatment assignment. In this work we have studied retrospectively the outcome of 120 BHP patients who underwent trans urethral resection (TURP) and of 145 BPH patients underwent "open" prostatectomy. In all the patients surgical time, prostate weight, indwelling catheter standing, rest in bed, early and late complications were evaluated. Irritative symptoms occurred after TURP in 10% of the cases. The two therapeutical options are not comparable for they technically different. The choice between the two depends on the accurate patient characterization and selection. PMID- 7538396 TI - [Incision of the bladder neck]. AB - Transurethral incision of the bladder neck (TUIBN) is a simple method of relieving bladder outflow obstruction producing similar results to transurethral resection of the prostate (TURP) when obstruction is caused by small prostate (< 40 gr). The complication rate is low and retrograde ejaculation has a far less degree than after TURP, so less bleeding. The major advantage of TUIBN are: short hospitalization, less bladder neck contractures. The drawback of the absence of prostate tissue for an earlier diagnosis of cancer can be balanced performing a biopsy before treatment. PMID- 7538397 TI - [Role of urodynamics in implantation of prostatic stents]. AB - Recent anxieties about the risk of prostate surgery in elderly and unfit patients have generated considerable interest in a number of new permanent prostatic stents. The prostatic Urolume Wallstent was positioned in 34 patients affected by BPH, all with acute or chronic retention of urine, most of whom were unfit for conventional prostatic surgery; the average follow-up is 14 months (range 9-24 months). The stents were inserted with the patients under local or regional anesthesia. 14 patients had prevalent irritative pre-retention symptoms, while in 18 patients obstructive symptoms were dominant; in the remaining 2 patients symptoms were minimal. Cystometry was performed in 29 patients before treatment for evaluation of detrusor activity. Following stent insertion, 31 patients were able to empty satisfactorily, while the remaining 3 patients presented chronic retention because of detrusor failure, evidenced before treatment. Most patients experienced frequency and urgency for several months (90% of 14 patients with pre treatment irritative symptoms; 25% of the other 20 patients). No incrustation occurred, no serious urosepsis was registered. In 3 patients we need to put in a second stent. In the first weeks, 4 stents were removed for dislocation and then replaced endoscopically without difficulty or damage to the urethra. After 6 months, other 5 stents were definitively removed because of persistent unbearable irritative symptoms with poor quality of life (all these patients had severe pre operative detrusor overactivity).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538398 TI - [Role of intraprostatic stents in benign prostatic hypertrophy]. AB - We report our indications and experience in the use of intraprostatic endoprosthesis. We report also our preliminary experience in the use of a new intraprostatic endoprosthesis which has been developed at our institute. This prosthesis can be easily positioned, shows little invasiveness and does not require ultrasonic, radiological or endoscopical guidance. PMID- 7538395 TI - [Prostatic prosthesis in the treatment of prostatic hypertrophy: our experience]. AB - Prostatic stenting is a therapeutic option in the management of obstruction in patients unfit for surgical treatment. At present different devices are available, permanent or temporary, the former being covered by epithelium. The Authors report their experience on 40 patients, 27 of which treated with Fabian spiral. They conclude considering that permanent stenting is likely to be a better solution; temporary spirals, however, give good results and can be the first choice in some situations. PMID- 7538400 TI - [Initial experiences in the treatment of benign prostatic hypertrophy with laser (side firing)]. AB - After a short literature review about the topic, the AA. present a 6 months follow up of 15 patients treated with prolase II fiber (cytocare) for BPH. In 80% we got good results about the symptom score and urinary flow, but at the endoscopic control in some cases we found an irregularity in the prostatic urethral as there was some prostatic residual tissue. 20% of the patients were submitted to TURP to relieve the symptoms. PMID- 7538401 TI - Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. AB - Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification. PMID- 7538399 TI - [Intraurethral devices in the treatment of prostatic obstruction]. AB - We describe our experience (started in 1988) about the use of urethral stents implanted into patients with prostatic outflow obstruction, unit for prostatic surgery. This technique has been applied successfully with few complications. The use of different stents (Fabian's spiral, mesh stent) allowed us to get the right choices in the different shapes of prostatic urethra and in the different expectancies of the patients. Lastly we can offer an useful recommendation about the patients evaluation and the stents selection. PMID- 7538402 TI - Purification of antigenized immunoglobulins derivatized with monomethoxypolyethylene glycol. AB - Genetically engineered immunoglobulins (Igs) carrying viral B or T cell peptides in the CDR3 loop, function as efficient delivery system of the defined viral epitopes. Two of these antigenized Igs (AIgs) were derivatized with 2-O monomethoxypolyethylene glycol-4,6-dichloro-s-triazine (mPEG). Herein, we describe a two-step strategy to purify mPEG-derivatized AIgs (AIgs-mPEG). Unreacted mPEG polymers were removed by size-exclusion chromatography using ammonium hydrogencarbonate as a buffer system. Mildly PEGylated AIgs were isolated from free and highly derivatized AIgs by anion-exchange chromatography. Electrophoretic analysis indicated that the AIgs-mPEG preparation contained less than 4 x 10(-4) M unreacted mPEG. This strategy may be applied to other mPEG derivatized monoclonal antibodies. PMID- 7538403 TI - Growth hormone administration in addition to a very low calorie diet and an exercise program in obese subjects. AB - A major problem of weight reduction in obesity is the undesirable loss of lean body mass that accompanies fat loss, particularly in severe calorie restriction. In order to achieve maximal fat loss, but without great loss of lean tissue, growth hormone (GH) in a dose of 6 U/day subcutaneously was added to a very low calorie diet and an exercise program for moderately obese subjects. Body weight, body composition and hormonal status were studied during an eight-week period. The results of seven patients using GH (seven females; mean age 39.1 +/- 7.9 years; mean body weight 94.2 +/- 10.7 kg; mean body mass index 35.1 +/- 2.3 kg/m2) were compared to the results of eight patients using placebo (two males, six females; mean age 38.9 +/- 10.4 years; 100.0 +/- 11.0 kg; mean body mass index 32.9 +/- 1.9 kg/m2). The groups were comparable for demographic data. Both serum insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) levels became significantly higher in the GH group (p = 0.001 and p = 0.014, respectively). Mean serum IGF-I levels increased from 29.0 +/- 8.19 nmol/l at randomization to 50.14 +/- 14.66 nmol/l after 2 weeks in the GH group, whereas the levels decreased from 34.25 +/- 10.26 nmol/l to 27.63 +/- 8.14 nmol/l in the placebo group. After two weeks, IGF-I and IGFBP-3 levels stabilized. In the first half of the study serum free triiodothyronine (T3) levels remained stable in the GH group, whereas a decrease was found in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538404 TI - Interactions between liver nuclear proteins and the human insulin-like growth factor binding protein 1 promoter in the course of development. AB - Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of the IGFs. Among the six IGFBPs known to date, IGFBP-1 is the most tissue specific, its expression being limited to the liver and the endometrium. In the liver, IGFBP-1 gene expression is maximal during the perinatal period, with its peak corresponding to a transient rise in gene transcription activity. In this study, interactions between rat liver nuclear proteins and the human IGFBP-1 promoter have been analysed in the course of development, using in vitro DNase I protection and mobility shift assays. Only the interactions between DNA and proteins localized between nt -305 and -268 varied through the period studied (16 days in utero to 70 days postnatally). Three proteins, named Pa, PC1 and PC2, interacted with sequences between nt -295 and -285, nt -305 and -295 and nt -285 and -268, respectively. There was a marked perinatal increase in phenotype expression of Pa, which was parallel to that in IGFBP-1 gene transcription activity. In addition, DNA-Pa interactions and DNA-PC2 interactions were mutually exclusive. These results suggest that the interaction of Pa with its target sequence(s) prevent PC2 binding and thereby contribute towards increased IGFBP-1 gene transcription. PMID- 7538406 TI - Chromosome identification and mapping in the grass Zingeria biebersteiniana (2n = 4) using fluorochromes. AB - The grass Zingeria biebersteiniana is one of five angiosperms known with 2n = 2x = 4. Its chromosomes were studied using fluorochrome banding and fluorescence in situ hybridization (FISH). The large pericentromeric region fluoresced much more brightly on chromosome 2 than on chromosome 1, using two different fluorochrome banding methods. These offer rapid and reliable means for identifying chromosomes and work throughout mitosis. FISH located the major site of 18S-26S rDNA sequences at the secondary constriction, which is proximal to two minor sites, all on the short arm of chromosome 1. Two 5S sites were also detected, the most distinct on the short arm of chromosome 2 and the other apparently co-localized with part of the major 18S-26S rDNA cluster on chromosome 1. These results constitute the first steps in constructing a physical gene map for Z. biebersteiniana. Such information may facilitate future studies of the organization and reorganization of grass genomes, including research into the spatial arrangement of the genome in Zingeria nuclei and much wider comparisons of synteny and genome evolution in grasses. PMID- 7538405 TI - Patterns of care for carcinoma of the prostate gland: results of a national survey of 1984 and 1990. AB - BACKGROUND: The annual incidence of carcinoma of the prostate gland increased from an estimated 76,000 cases in 1984 to 200,000 in 1994. Part of this increase may be the result of increased detection. Management of the disease has also changed. To measure such changes, the American College of Surgeons conducted a patient care evaluation study of carcinoma of the prostate gland. STUDY DESIGN: Information was voluntarily submitted by cancer registrars on forms designed by a team of specialists. Data were received from 730 hospitals (of 2,000 hospitals invited for the study) on 14,716 patients with newly diagnosed adenocarcinomas of the prostate gland in 1984 and from 1,035 hospitals for 23,214 patients with carcinoma of the prostate gland in 1990. RESULTS: From 1984 to 1990, there was increased diagnostic use of the prostate specific antigen (PSA) test (from 5.1 to 66.4 percent of incident carcinomas) and transrectal ultrasound (TRUS) (0.9 to 19.7 percent). Use of the prostatic acid phosphatase assay declined from 62.4 to 47 percent. Although the proportion of early stage (0, I, II) disease increased for all racial or ethnic groups combined, the greatest increase was for whites (from 57.3 to 60.6 percent), while the increase for African-Americans was less (from 46.9 to 48.3 percent). The use of radical prostatectomy without radiation therapy or chemotherapy increased from 7.3 to 20.3 percent and the proportion of patients receiving no carcinoma-directed treatment decreased from 37.8 to 30 percent. Radiation therapy remained the same. Hormone therapy without radical prostatectomy declined from 24.4 to 19.7 percent. African-Americans had a lower five-year survival rate than whites, even when stratified for stage. CONCLUSIONS: The diagnostic use of the PSA test and TRUS increased markedly by 1990 and may have contributed to the increased diagnosis of carcinomas of the prostate gland and the earlier stage at diagnosis. The overall use of radical prostatectomy has increased and the proportion of patients receiving no treatment has decreased. African-Americans had a lower five-year survival rate than other groups, even when stage was controlled. PMID- 7538408 TI - Prevention of anergy induction in cloned T cells by interleukin 12. AB - A variety of tumors are potentially immunogenic but do not stimulate an effective antitumor immune response in vivo. Tumors may be capable of delivering antigen specific signals to T cells, but may not deliver the costimulatory signals necessary for full activation of T cells. In this regard, we recently reported that a human melanoma cell line (sMC) expressing MHC class II, was able to induce clonal anergy in a specific, MHC-restricted CD-4+ T cell clone (sTC3). We used this system to investigate the influence of interleukin (IL)-12 on induction of this T cell unresponsiveness. The presence of 10 to 100 U IL-12 during the induction phase of anergy leads to a primary proliferative response of sTC3, which was significantly higher than that induced by IL-12 alone; however, in the absence of IL-12 no proliferation was seen during the induction of anergy. Subsequent optimal stimulation of IL-12 treated cells, but not of those cultured without IL-12, led to substantial IL-2 production and cell proliferation. This indicates that induction of the unresponsive state could be inhibited by IL-12. In addition, we have recently demonstrated that anergic T cell clones can produce high amounts of IL-10 and that this event was correlated with their impaired ability to produce IL-2. This marked induction of IL-10 can be suppressed if IL 12 is present during initiation of unresponsiveness. However, IL-12 was not able to prime the T cell clone, sTC3, to become resistant against the anergizing stimulus, as this cytokine was only effective when present at the time of anergy induction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538407 TI - Expression of murine VCAM-1 in vitro and in different models of inflammation in vivo: correlation with immigration of monocytes. AB - VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vivo, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism and the subsequent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8 positive lymphocytes was only observed in ACD and Leishmaniasis. PMID- 7538409 TI - Analysis of mast cell subpopulations (MCT, MCTC) in cutaneous inflammation using novel enzyme-histochemical staining techniques. AB - In order to gain insights into the dynamics of mast cell subpopulations in normal and diseased skin, a novel enzyme-histochemical double and triple staining method was employed that allowed the detection of metachromasia (toluidine blue) and the mast cell proteases tryptase and chymase within the same cell. Cryostat sections were used of skin biopsies from the following specimens: normal skin (N = 4), psoriasis (N = 13), atopic eczema (N = 7), lichen planus (N = 6), interferon alpha 2a injection sites (N = 1) of a leukemic infiltrate and corresponding normal skin of the same patient before and after treatment. (i) Equal numbers of tryptase- and chymase-positive mast cells (MCTC) were obtained in all normal and diseased specimens in papillary and reticular dermis, with threefold increases around appendages. (ii) Tryptase-positive mast cells (MCT) were absent in normal skin, but were markedly increased in a disease-specific pattern within the papillary dermis, the inflammatory infiltrate and around appendages. (iii) Marked increases of MCT were also noted at interferon injection sites within the leukemic infiltrate, but not in the normal skin of the same patient. These data suggest that disease-dependent mast cell dynamics involve only MCT in cutaneous inflammation and that MCT numbers are controlled by distinct, disease-specific local tissue factors. PMID- 7538410 TI - Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans. AB - In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd2+ is strongly inhibited by Ca2+ ions; furthermore, Ca2+ reduces the net accumulation of Cd2+ by worms. Both Ca2+ and a variety of calcium uptake inhibitors (nifedipine, La3+, verapamil) depress the dose response of the transgene to Cd2+. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd2+ treated worms, but has a much larger effect in the case of Mn2+, reflecting its much greater affinity for this ion. PMID- 7538412 TI - Effect of ibuprofen on the acute-phase response and protein metabolism in patients with cancer and weight loss. AB - The aim of this study was to determine whether administration of the non steroidal anti-inflammatory agent ibuprofen might attenuate the acute-phase response in patients with colonic cancer. Cytokines and acute-phase proteins were measured before administration of ibuprofen and again 3 days later, when protein synthesis was measured using 15N-glycine. In patients with cancer, ibuprofen caused a significant reduction in the plasma concentration of all five acute phase proteins studied. Although interleukin 6 levels were raised, they did not change following administration of ibuprofen. Unlike the situation in patients with cancer who did not receive ibuprofen, whole-body protein kinetics were similar to those of control subjects in patients with cancer who received ibuprofen. Whether or not ibuprofen had been administered, non-export hepatic protein synthesis rates were significantly lower in patients with than in those without cancer. These results suggest that short-term administration of ibuprofen can attenuate accelerated whole-body protein kinetics and the acute-phase response in patients with advanced cancer. PMID- 7538411 TI - Effect of EGF on rat parotid gland secretory function. AB - The present study reports changes in saliva composition from the rat parotid gland in response to single and repeated administration of epidermal growth factor (EGF). Treatment of rats with EGF (10 micrograms/kg, i.p., twice daily for 3 days) caused an increase in amylase activity in saliva collected from cannulated parotid duct, following stimulation of secretion with pilocarpine, with a corresponding decrease in enzyme activity in the gland. Analysis of parotid gland RNA by reverse transcriptase-PCR generated a single predicted amylase-derived cDNA product of 576 bp. The steady-state levels of mRNA for amylase from EGF-treated parotid total RNA showed a 1.8-fold increase compared to untreated controls. A single dose of EGF (15 min following i.p. injection) elicited an activation of both protein kinase A and protein kinase C activities. While the activation of protein kinase A was still maintained under the chronic EGF regimen, the activity levels of protein kinase C showed down-regulation to untreated control values. PMID- 7538413 TI - Treatment of lymphocele in renal transplant recipients by laparoscopic fenestration after transcutaneous staining. AB - Lymphocele is a rare cause of impairment of graft function after renal transplantation. Treatment ranges from external drainage to laparoscopic internal marsupialization. A therapeutic approach to the treatment of symptomatic lymphocele is described, which employs external drainage, if necessary followed by methylene blue staining and laparoscopic internal drainage, should conservative treatment fail. Five patients developed post-transplant lymphocele of varying size and were treated according to this protocol. Identification of the blue lymphocele at laparoscopy was straightforward and complete marsupialization was performed. After a mean follow-up of 8.4 months all patients are free from symptoms and have well functioning grafts. PMID- 7538414 TI - Function and receptor specificity of a minimal 20 kilodalton cell adhesive fragment of fibronectin. AB - Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin. PMID- 7538416 TI - Expression of the alternatively spliced EIIIB segment of fibronectin. AB - Previous descriptions of the expression and distribution of the alternatively spliced EIIIB segment of fibronectin (FN) relied upon an antibody which, on subsequent testing, was shown not to recognize this segment directly. This raises concerns regarding the reliability of all such previous descriptions. In order to prepare reagents directly reactive with this segment, we raised polyclonal antibodies to two different bacterial fusion proteins containing intact EIIIB segments, and to a synthetic 36 amino acid peptide from the center of the EIIIB segment. Antibodies raised to each of these three immunogens recognized fusion proteins containing the EIIIB segment, but failed to recognize full length EIIIB+ FNs produced by mammalian cells, suggesting that oligosaccharide linked to Asn1359 within the EIIIB segment, or potentially to other residues in FN, might interfere with antibody recognition of this segment. Consistent with this hypothesis, N-deglycosylation of recombinant full and partial length EIIIB+ FNs permitted their specific recognition by the anti-fusion protein (but not anti peptide) antibodies. Using anti-fusion protein antibodies coupled with deglycosylation procedures, we provide a series of new results relevant to the functions of the EIIIB segment: 1) Endogenously synthesized EIIIB+ FN is incorporated into the extracellular matrix of cultured fibroblasts, where it appears by immunofluorescence microscopy and radioimmunoprecipitation analyses to have a distribution very similar to both EIIIA+ forms and the total pool of FNs. 2) No reproducible changes can be shown to occur in the extent of synthesis or matrix incorporation of EIIIB+ FNs upon cellular transformation. 3) Low levels of EIIIB+ FN are normally present in blood plasma and consequently also in purified preparations of plasma FN. 4) EIIIB+ FN is present in blood platelets, where it constitutes a minor fraction of total platelet FN, yet is greater than 4-fold enriched relative to plasma FN. 5) EIIIB+ FN is synthesized by first passage cultured endothelial cells, suggesting that the endothelium could constitute a source for this FN isoform in the circulating blood. The antibodies and methods used in this study constitute the first direct assays of EIIIB+ FN protein expression and are applicable to a variety of species. PMID- 7538417 TI - Activity-dependent regulation of a ribosomal RNA epitope in the chick cochlear nucleus. AB - Elimination of auditory nerve activity results in rapid metabolic changes, cell atrophy, and cell death in nucleus magnocellularis (NM), the cochlear nucleus of the chick. The transneuronal signals involved in the activity-dependent regulation of NM neurons are not well understood. One of the most rapid transneuronal effects is alteration in protein synthesis by NM neurons. Previous studies using an in vitro preparation of the brain stem auditory system suggested that up-regulation of protein synthesis in NM neurons requires the action of some trophic substance released by active auditory nerve fibers. Here, similar results were obtained when measuring changes in immunoreactivity using a monoclonal antibody (Y10B) that recognizes ribosomal RNA. This immunolabeling assay has advantages over the global protein synthesis assay in that it is not sensitive to possible changes in specific activity of the precursor pool or possible differences in the uptake of the labeled amino acids. Unilateral stimulation of the auditory nerve for 1 h resulted in greater immunolabeling of NM neurons on the stimulated side of the slice. This is consistent with previous in vivo results after unilateral deafferentation. Blockade of synaptic transmission by maintaining the slice in a low-Ca2+/high Mg2+ medium prevented the stimulation induced difference in immunolabeling. Electrical stimulation of the postsynaptic NM neurons alone (antidromic stimulation, via electrical stimulation of NM neuron axons) did not result in greater immunolabeling. Rather, antidromically stimulated neurons tended to show lighter labeling. Thus, the transneuronal regulation of ribosomes in NM neurons appears to require some substance released from the active auditory nerve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538415 TI - Localization of multiple functional domains on human PECAM-1 (CD31) by monoclonal antibody epitope mapping. AB - PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily, has been implicated in white cell transmigration, integrin activation on lymphocytes, and cell-cell adhesion. The purpose of this investigation was to identify specific regions of the PECAM-1 extracellular domain mediating these functions by identifying the location of epitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding regions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epitopes of monoclonal antibodies that activated integrin function on lymphocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N terminus of the molecule. The binding regions of mAbs that blocked PECAM-1 mediated heterophilic L-cell aggregation were located either in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpointed the epitope of the 4G6 mAb to a hexapeptide, CAVNEG, within Ig-like domain 6. These results demonstrate that PECAM-1 contains multiple functional domains. Regions within N-terminal Ig-like domains appear to be required for transmigration. In contrast, two distinct regions were implicated in L-cell mediated heterophilic aggregation. PMID- 7538419 TI - Ultrastructural relationships between terminals immunoreactive for enkephalin, GABA, or both transmitters in the rat ventral tegmental area. AB - The ventral tegmental area (VTA) receives extensive afferent input from neurons containing the opioid peptide enkephalin (Enk) and/or GABA. We examined the ultrastructural basis for known functional interactions between these inhibitory neuromodulators using a combined immunoperoxidase and immunogold-silver technique. As visualized with either marker in single sections, Enk-immunolabeled terminals contained numerous small clear vesicles and one or more intensely immunoreactive dense-cored vesicles. Enk-labeled terminals formed either symmetric or asymmetric synapses on small or large unlabeled dendrites. The immunoreactive dense-cored vesicles were usually detected away from these sites of synaptic contact. Terminals singly immunoreactive for GABA, or dually labeled for Enk and GABA, showed similar morphological features but formed primarily symmetric axo-dendritic synapses. In many instances, GABA- and/or Enk immunolabeled terminals were in direct apposition to each other and formed synapses on immediately adjacent parts of a common dendrite. Close appositions were also noted between GABA- and Enk-immunoreactive axons and varicosities that did not form synapses with either common or divergent dendrites in single sections. Immunoreactive dense-cored vesicles were frequently detected at the apposed plasmalemmal surfaces between these axon terminals. The findings suggest that Enk and GABA are released from the same or convergent terminals and co regulate the activity of common target neurons within the rat VTA. The results are also consistent with potential presynaptic interactions between these transmitters. PMID- 7538420 TI - Nerve growth factor immunoreactivity and sympathetic sprouting in the rat hippocampal formation. AB - Several lines of evidence support a role for nerve growth factor (NGF) in the sympathetic sprouting response that occurs following septal cholinergic denervation of the rat hippocampal formation. The present study was undertaken to compare the distribution of NGF-like immunoreactivity and the topography of sympathetic sprouting in rats receiving medial septal lesions. Comparisons were made using adjacent sections of the hippocampal formation stained either for NGF like immunoreactivity or for NGF receptor-immunoreactivity (p75, to visualize sympathetic fibers). p75-immunoreactive sympathetic axons were localized within the same regions exhibiting NGF-like staining, i.e., the hilus of the dentate gyrus and stratum lucidum in the CA3 area. Furthermore, the sympathetic fibers that invaded the hippocampal formation exhibited NGF-like immunostaining. These results provide additional evidence in support of NGF's role in this collateral sprouting response in the mature rat CNS. PMID- 7538418 TI - Organization of the basal forebrain in the cat: localization of L-enkephalin, substance P, and choline acetyltransferase immunoreactivity. AB - The present study uses immunocytochemical techniques to determine whether cholinergic basal forebrain neurons in the cat are in a position to receive a homogeneous pattern of inputs, or if specific immunocytochemically defined afferent systems are localized to only selected regions of the basal forebrain. Monoclonal antibodies against choline acetyltransferase (ChAT) were used to identify the location of putative cholinergic neurons which are known to project to the cerebral cortex. In addition, polyclonal antibodies against substance P (SP) or enkephalin (Enk) were used on either adjacent or on the same histological sections reacted for ChAT to identify the neuropeptide plexuses that provide input to the basal forebrain. ChAT-immunoreactive (ChAT-IR) perikarya were located throughout the vertical limb, genu and horizontal limb of the diagonal band of Broca. ChAT-IR neurons also were located within the substantia innominata (SI), within the peripallidal zone around the globus pallidus, and were intercalated within the internal capsule. Enk-IR and SP-IR were used to determine the distribution of putative peptidergic terminals within the basal forebrain. Extensive Enk-IR and SP-IR terminal label was localized within the globus pallidus and the surrounding peripallidal zones, as well as within the SI, whereas the components of the diagonal band of Broca demonstrated negligible Enk IR and SP-IR label. These data predict that the subdivisions of the cholinergic basal forebrain in the cat do not share a uniform afferent system, and only selective portions of this cholinergic system are in an anatomical position to receive a major direct input from the identified subcortical peptidergic afferents. The segregation of afferents has important consequences in the selective control of cortical function by the cholinergic basalocortical pathway. PMID- 7538421 TI - A longitudinal study of granulocyte colony-stimulating factor levels and neutrophil counts in newborn infants. AB - PURPOSE: The goal of this study was to longitudinally measure endogenous granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) levels in newborn infants and to attempt to correlate these levels with neutrophil counts. PATIENTS AND METHODS: Samples for complete blood count, G-CSF, and GM-CSF were obtained from groups of healthy full-term infants at 0 (cord blood or nursery admission), 12, 24, 48, and 72 h. Samples were also obtained from premature infants at the above times and at 1 week. G-CSF and GM-CSF levels were measured using bioassays. RESULTS: Levels of G-CSF ranged from < 5 to 53,800 pg/ml. Levels were significantly higher (p < 0.001) in premature infants and decreased over time in all infants. White blood cell counts also decreased over the first week of life. All GM-CSF levels were below the detectable range. CONCLUSIONS: Levels of G-CSF at birth are higher than those seen in adults. These increased levels may partially explain the leukocytosis seen in the first week of life. This, as has been shown in preliminary studies, suggests that infants are capable of an increase in neutrophil count after administration of exogenous G-CSF. PMID- 7538422 TI - Characterization of LDL and VLDL binding sites on human basophils and mast cells. AB - Recent data suggest that basophils and mast cells play a potential role in the processing and accumulation of plasma lipoproteins. This study investigated the interactions of 111In-low-density lipoprotein (LDL), 111In-acetyl-LDL, and 111In very-low-density lipoprotein (VLDL) with purified primary human blood basophils, immortalized human basophils (KU812 cell line), and a human mast cell line, HMC 1. Binding sites for 111In-LDL resolved into curvilinear Scatchard plots indicating two classes of specific binding sites on primary basophils (Bmax1, 7404 sites/cell; Kd1, 1.9 nmol/L; Bmax2, 39,611 sites/cell; Kd2, 29 nmol/L), on KU812 cells (Bmax1, 8290 +/- 2690 sites/cell; Kd1, 2.4 +/- 0.6 nmol/L; Bmax2, 46,470 sites/cell; Kd2, 33.4 +/- 7.8 nmol/L), and on HMC-1 cells (Bmax1, 7840 +/- 360 sites/cell; Kd1, 1.8 +/- 0.8 nmol/L; Bmax2, 61,450 +/- 9900 sites/cell; Kd2, 28.4 +/- 9.4 nmol/L). On KU812 cells, binding of 111In-LDL was displaced by apolipoprotein (apo)-E-rich high-density lipoprotein (HDL) (IC50, 14 +/- 6 nmol/L), LDL (IC50, 29 +/- 11 nmol/L), VLDL (IC50, 55 +/- 21 nmol/L), HDL2 (IC50, 420 +/- 140 nmol/L), and heparin (IC50, 67 +/- 28 nmol/L), whereas no competition was produced by HDL, HDL3, or acetyl-LDL (IC50, > 1 mumol/L). Western blot analysis using the monoclonal antibody C7 confirmed the presence of the LDL receptor on human basophils and HMC-1 cells. 111In-acetyl-LDL binding sites (scavenger receptor) could be detected neither on human basophils nor on HMC-1 cells. 111In-VLDL bound to a single class of high-affinity binding sites on primary basophils (Bmax, 4320 sites/cell; Kd, 10 nmol/L), KU812 cells (Bmax, 4020 +/- 840 sites/cell; Kd, 8 +/- 3 nmol/L), and HMC-1 cells (Bmax, 6143 +/- 1866 sites/cell; Kd, 4 +/- 2 nmol/L). 111In-VLDL binding was displaced by VLDL > LDL > apoE-rich HDL but not by heparin (IC50 > 1 mmol/L). In the presence of prostaglandin E1, the number of 111In-LDL receptors increased by 150% (P < .05) in the high-affinity range and by 170% (P < .01) in the low-affinity range, whereas the number of 111In-VLDL binding sites remained unchanged. VLDL, LDL, HDL, and the subclasses HDL2 and HDL3 inhibited immunological histamine release by primary normal basophils (n = 3) and mast cells (n = 3). Our results provide evidence for the existence of LDL and VLDL binding sites on human basophils and HMC-1 mast cells. The exact biological and pathophysiological roles of these sites remain to be elucidated. PMID- 7538423 TI - Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. AB - Local shear stresses generated by blood flow exert direct mechanical effects on adhesion of circulating leukocytes to vascular endothelium, but their effects on expression of endothelial-leukocyte adhesion molecules have not been determined. Shear stress in rabbit carotid arteries was increased by 170% or decreased by 73% in 5 days by surgical manipulations. En face immunofluorescence staining with the monoclonal antibody Rb1/9 revealed that vascular cell adhesion molecule-1 (VCAM 1) expression was greatly increased under low shear stress, but the distribution of staining was patchy. Thus, 71.4 +/- 7.8% of fields were VCAM-1 positive versus 2.4 +/- 0.47% of fields in control arteries. Frequently, large regions showed consistent but heterogeneous staining. Occasionally, small islands of cells were labeled intensely. Monocytes, detected by use of the monocyte-specific antibody HAM 56, adhered to endothelium under low shear stress; 64.5 +/- 8.2% of the monocytes colocalized with detectable VCAM-1, although many (83.2 +/- 2.8%) VCAM 1-positive regions were devoid of monocytes. VCAM-1 expression also increased significantly but to a lesser extent when shear stress was approximately doubled. Thus, 8.7 +/- 1.5% of fields were VCAM-1 positive under high shear versus 2.5 +/- 0.87% under normal shear stress. No monocytes were detected at high shear stress. At normal shear stresses, intercellular adhesion molecule-1 (ICAM-1), detected by use of the monoclonal antibody Rb2/3, was extensively distributed; thus, 53.5 +/- 5.5% of fields contained ICAM-1-positive cells. The junctional regions of the cells were heavily stained.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538424 TI - Lack of role for nitric oxide (NO) in the selective destabilization of endothelial NO synthase mRNA by tumor necrosis factor-alpha. AB - The constitutive expression of endothelial nitric oxide (NO) synthase (cNOS) is essential for the physiological regulation of vascular tone and structure. The mechanism of downregulation of steady state cNOS mRNA in human umbilical vein endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha) was investigated by using Northern blot analysis of total cellular RNA. TNF-alpha produced a dose- and time-dependent decrease in cNOS mRNA expression that was near maximal at 10 U/mL and 6 hours of exposure, respectively. In contrast, steady state expression of endothelin-1 and plasminogen activator inhibitor-1 (PAI-1) mRNA was upregulated by TNF-alpha. The pharmacological generation of NO using sodium nitroprusside (10 mumol/L) and S-nitroso-acetylpenicillamine (100 to 400 mumol/L) had no effect on cNOS mRNA levels, and TNF-alpha-induced downregulation of cNOS was not prevented by coincubation with the inhibitors of NO synthesis N omega-nitro-L-arginine methyl ester (1 mmol/L) and NG-monomethyl L arginine (10 mmol/L). Under control conditions, cNOS and PAI-1 mRNA were stable after treatment with actinomycin D for periods greater than 24 hours, whereas endothelin-1 message was rapidly degraded (half-life, < 1 hour). Pretreatment with TNF-alpha (30 U/mL) selectively reduced that half-life of cNOS mRNA to less than 12 hours without altering the stability of PAI-1 message. TNF-alpha-induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 mumol/L) but was not reproduced by addition of sodium nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538425 TI - Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site. AB - Accumulating evidence strongly implicates oxidized LDL (Ox-LDL) in the pathogenesis of atherosclerosis. Several receptors have been identified that bind and internalize Ox-LDL, but their relative importance in vivo is unclear. CD36 is an 88-kD transmembrane glycoprotein expressed on monocytes/macrophages, platelets, and microvascular endothelium that has been implicated as a putative receptor for Ox-LDL. We demonstrate that an anti-CD36 monoclonal antibody inhibited 50% of the specific binding and 26% of the specific degradation of Ox LDL by human monocyte-derived macrophages. To characterize more completely this binding we evaluated interactions between CD36 and Ox-LDL in murine NIH-3T3 cells stably transfected with human CD36 cDNA. Ox-LDL bound to CD36-transfected 3T3 cells in a saturable manner. Specific binding, internalization, and degradation of Ox-LDL were increased fourfold in CD36-transfected cell lines compared with 3T3 cells transfected with vector alone. Binding of Ox-LDL to CD36-transfected 3T3 cells was inhibited by a panel of anti-CD36 antibodies and by soluble CD36 but not by thrombospondin. Specificity of binding was demonstrated by the equivalent binding of LDL and acetylated LDL to control and CD36-transfected 3T3 cells. The epitope or epitopes on Ox-LDL recognized by CD36 are undefined. Two observations suggest that CD36 recognizes a lipid moiety or that the lipid portion of the lipoprotein is essential for apoprotein recognition. The first is that the increased binding of Ox-LDL to CD36-transfected 3T3 cells is abrogated by delipidation of the lipoprotein, and the second is that oleic acid competes for the binding of Ox-LDL to CD36-transfected 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538426 TI - Lipoprotein lipase synergizes with interferon gamma to induce macrophage nitric oxide synthetase mRNA expression and nitric oxide production. AB - Lipoprotein lipase (LPL) induces macrophage tumor necrosis factor-alpha (TNF alpha) gene expression and protein secretion. Since TNF-alpha can increase interferon gamma (IFN-gamma)-dependent nitric oxide (NO) production, we studied whether LPL may synergize with IFN-gamma for the induction of macrophage NO production. Although ineffective by itself, LPL in combination with IFN-gamma increased L-arginine-dependent NO production in a dose-dependent manner. Preincubation of LPL with an anti-LPL neutralizing antibody totally suppressed this effect. Increased NO synthetase (NOS) mRNA expression was also observed after macrophage treatment with IFN-gamma and LPL. Protein synthesis was required for the induction of NOS mRNA, and a TNF-alpha-mediated effect of LPL on NOS gene expression and NO production was observed. The ability of LPL to augment IFN gamma-dependent NOS mRNA expression was associated with an increase in the NOS gene transcriptional activity but not in the NOS mRNA stability. Finally, binding of nuclear proteins to the nuclear factor-kappa B- and TNF-alpha-responsive sequences of the macrophage NOS promotor was decreased by treatment of the cells by IFN-gamma alone or in combination with LPL. These data provide evidence for a link between LPL and arginine metabolism in macrophages and further stress the role of LPL in the regulation of macrophage activation. PMID- 7538427 TI - Docosahexaenoic acid selectively attenuates induction of vascular cell adhesion molecule-1 and subsequent monocytic cell adhesion to human endothelial cells stimulated by tumor necrosis factor-alpha. AB - Incorporation of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) but not eicosapentaenoic acid or n-6 arachidonic acid into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF-alpha)-induced surface expression of vascular cell adhesion molecule-1 (VCAM-1). In parallel, DHA inhibited TNF-alpha-stimulated monocytic U937 cell adhesion to HUVECs but did not affect TNF-alpha- or interferon gamma induced expression of intercellular adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 or VCAM-1 induction by interleukin-1 beta. DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-1 mRNA by TNF alpha. VCAM-1 induction is regulated by activation of nuclear factor-kappa B, which can be mediated by a TNF-alpha-responsive phosphatidylcholine-specific phospholipase C (PC-PLC). Gel-shift analysis showed inhibition of TNF-alpha induced nuclear factor-kappa B mobilization by DHA. While the PC-PLC inhibitor D609 dose-dependently prevented VCAM-1 induction by TNF-alpha, 1,2-diacyl glycerol (DAG) stimulated VCAM-1 expression, suggesting that VCAM-1 induction by TNF-alpha may be mediated by activation of PC-PLC. Treatment with DHA resulted in a fourfold enrichment in PC. In addition, DHA or D609 but not eicosapentaenoic acid or arachidonic acid suppressed activation of PC-PLC by TNF-alpha, estimated as [14C]DAG synthesis in prelabeled HUVECs. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF-alpha and subsequent monocytic cell adhesion by inhibition of TNF-alpha-stimulated PC-PLC activation in HUVECs. PMID- 7538428 TI - Megakaryocytes from patients with coronary atherosclerosis express the inducible nitric oxide synthase. AB - Endothelial and platelet generation of nitric oxide (NO) plays an important role in the regulation of hemostasis. Alterations in NO biosynthesis are described in atherosclerosis. We have investigated the NO pathway in megakaryocytes and platelets from patients with atherosclerosis and age-matched control subjects. Megakaryocytes and platelets were isolated from patients with severe coronary atherosclerosis (n = 19) and normal coronary arteries (n = 9) as demonstrated by selective angiography. Constitutive (Ca(2+)-dependent) and inducible (Ca(2+) independent) NO synthase (cNOS and iNOS, respectively) activities were measured by using the citrulline assay and by immunostaining techniques using an anti peptide antibody to iNOS. Megakaryocytes from patients with atherosclerosis expressed significantly greater amounts of iNOS (1.28 +/- 0.46 pmol citrulline.mg 1.min-1) than cNOS (0.29 +/- 0.40 pmol.mg-1.min-1). In contrast, megakaryocytes from patients with normal coronary arteries expressed significantly more cNOS (1.48 +/- 0.23 pmol.mg-1.min-1) than iNOS (0.49 +/- 0.40 pmol.mg-1.min-1). Platelets isolated from both groups showed no significant difference in cNOS expression, and no iNOS was seen in either group. Immunostaining confirmed the presence of the iNOS in megakaryocytes. These results suggest there is a link between the expression of iNOS in the megakaryocyte and atherosclerosis. PMID- 7538430 TI - Effects of stroma-free hemoglobin solutions on pulmonary vascular resistance and mediator release in the isolated perfused rabbit lung. AB - The aim of this study was to evaluate the effect of an ultrapure bovine stroma free hemoglobin (SFH) on pulmonary vascular resistance and mediator release and to analyze potential mechanisms of action in the isolated perfused rabbit lung model. Repetitive bolus applications of small amounts of bovine SFH were examined which resulted in a reproducible acute increase of pulmonary vascular resistance of approx. 9 mmHg (controls, n = 6). It was tested whether the platelet activating factor (PAF) antagonist WEB 2086 (50 microM; n = 6), the cyclooxygenase blocker diclofenac (10 micrograms/ml; n = 6), the iron-chelating agent deferoxamine (500 micrograms/ml, n = 6) and the radical scavenger catalase (5000 U/ml; n = 6) exert a protective effect on vasoconstrictor response to SFH. The pressure increase was completely suppressed in the lungs pretreated with WEB 2086, whereas diclofenac, deferoxamine and catalase failed to inhibit the vasoconstriction due to SFH. No significant differences in either TXB2 generation or in histamine release were found in the WEB 2086 group compared with untreated lungs. The results point towards the crucial role of PAF in mediation of vasoconstrictor side effects due to SFH. PMID- 7538429 TI - A self consistent mean field approach to simultaneous gap closure and side-chain positioning in homology modelling. AB - A new computational procedure which simultaneously provides gap closure and side chain positioning in homology modelling is described. It uses a database search scheme to generate fragments to model gaps, a rotamer library to define side chain conformations, and iteratively refines a conformational matrix CM, such that its elements CM(i,j,o) and CM(i,j,k) give the probabilities that the backbone of residue i adopts the conformation described by fragment j and that its side-chain adopts the conformation of its possible rotamer k. Each residue experiences the average of all possible environments, weighted by their respective probabilities. The method converges, thereby deserving the name of 'self consistent mean field' approach. PMID- 7538431 TI - A recombinant amino-terminal fragment of bactericidal/permeability increasing protein (rBPI23) inhibits soluble CD14-mediated lipopolysaccharide-induced endothelial adherence for human neutrophils. AB - Exposure of cultured human umbilical vein endothelial cells (HUVEC) to lipopolysaccharide (LPS) or interleukin 1 (IL-1) causes increased expression of adhesion molecules such as E-selectin and CD54 by HUVEC and consequently increased adherence of peripheral blood neutrophils. A recombinant aminoterminal fragment of bactericidal/permeability increasing protein (rBPI23) was shown to specifically block the LPS-induced adhesiveness of HUVEC for neutrophils. rBPI23 also prevented the LPS- but not IL-1 beta-induced upregulation on HUVEC of E selectin and CD54. Furthermore, this inhibition was evident even when the endothelial cells were exposed to LPS for up to 1-2 h prior to rBPI23 addition. The inhibitory effects of an anti-CD14 monoclonal antibodies (mAb) were similar to those of rBPI23. Combination of the anti-CD14 mAb and rBPI23 resulted inhibition greater than either one used alone. These studies demonstrate that rBPI23 acts as a specific and potent inhibitor of soluble CD14-mediated LPS induction. PMID- 7538432 TI - Diltiazem and superoxide dismutase modulate hepatic acute phase response in gram negative sepsis. AB - This study assessed the hepatic acute phase response and cellular Ca2+ regulation in septic animals and in hepatoma cell lines in vitro. Sepsis was induced in male Sprague-Dawley rats by implanting in their abdominal cavities fecal pellets impregnated with live Escherichia coli and Bacteroides fragilis. 8 h after implantations, rats were treated with diltiazem (1.2 mg/kg) or superoxide dismutase (SOD) (5 x 10(3) units/kg). After 24 h, plasma acute phase proteins (APP) were determined by immunoelectrophoresis, and hepatic APP-mRNAs by Northern blot hybridization. Effects of diltiazem, verapamil, or SOD on hepatic cells were determined in rat Reuber H-35 and human HepG2 hepatoma cells. Sepsis induced a significant increase in plasma APP and their hepatic mRNAs. Diltiazem and SOD reduced the sepsis-induced elevations in plasma lactate, the febrile response and mortality. APP expression in H-35 and HepG2 cells, stimulated by interleukin 1 (IL-1), IL-6, and dexamethasone, was inhibited by diltiazem or verapamil but not SOD. The results suggest that a heightened hepatic APP response in septic animals accompanies systemic/metabolic derangements and a significant animal mortality. Because diltiazem was previously shown to prevent sepsis-related disturbances in hepatic cellular Ca2+ regulation, its mediation of decrease in APP, systemic/metabolic response, and mortality may be effected through modifications in cellular Ca2+ regulation. The data from hepatoma cells show an attenuation of the AAP can result from direct effects of a calcium blocker. However, whether the blocker primarily modifies cellular Ca2+ regulation and secondarily effects APP gene expression, or directly effects gene expression remains unknown. PMID- 7538433 TI - Dose response effects of hypertonic saline and dextran on cardiovascular responses and plasma volume expansion in sheep. AB - Despite the established efficacy of 7.5% NaCl/6% dextran-70 in the treatment of hypovolemia, the optimal formulation of a hyperosmotic/hyperoncotic small volume resuscitation solution has yet to be defined. The present study investigates the cardiovascular effects of hypertonic saline ranging from 3.75%-25% NaCl (HS) and dextran-70 (D-70) ranging from 3 to 24%. HS and D-70 were studied alone or in specific combinations at a dose of 4 mL/kg, in euvolemic sheep. Blood samples were collected before, during and up to 60 min after infusion of the test solutions. Dose-dependent effects of HS were immediate increases in cardiac output (CO) of 30-85%, falling to 10-35% over baseline after 60 min. HS concentrations over 3.75% significantly reduced systemic vascular resistance, but HS had no significant effect on mean arterial pressure (MAP). Plasma volume (PV) expansion with HS was an immediate, but transient increase of 12-35%. Infusion of D-70 induced sustained 10-20% increases in CO and 10-30% increases in PV, peaking 10 min post-infusion. D-70 also resulted in small (5-12 mmHg) increases in MAP. Cardiovascular effects of D-70 correlated with a dose-dependent increase in plasma dextran concentrations. All HS solutions significantly increased plasma Na, which peaked at > or = 200 mEq/L in the 25% group. The effects of D-70 and HS combined were additive on PV expansion and CO. These data indicate that concentrations of HS and D-70 which are higher than those currently used have a greater capability for expanding PV, but use of HS > 7.5% may be limited by resulting hypernatremia. PMID- 7538435 TI - Brief comparison of the pharmacokinetics and pharmacodynamics of the traditional and newer antipsychotic drugs. AB - The pharmacokinetics and pharmacodynamics of chlorpromazine, haloperidol, clozapine, and risperidone are described. Most traditional antipsychotic drugs have similar pharmacokinetic profiles that differ from the newer agents in several key respects. Traditional agents tend to be completely absorbed and undergo extensive presystemic elimination, but clearance tends to be almost exclusively by hepatic-enzyme metabolism. The volume of distribution is large, steady-state plasma concentrations are highly variable, and many traditional antipsychotic drugs have pharmacologically active metabolites. The newer antipsychotic agents clozapine and risperidone are also well absorbed and undergo extensive presystemic elimination. They are cleared mostly through hepatic-enzyme metabolism, but a small portion of risperidone is cleared renally. Steady-state plasma concentrations of clozapine and risperidone exhibit large interpatient differences at therapeutic dosages. The primary metabolite of risperidone, 9 hydroxyrisperidone, has activity equipotent to that of the parent drug. It is not clear whether clozapine produces active metabolites. Basic information on the pharmacokinetics and pharmacodynamics of clozapine and risperidone is known; however, more information is needed. PMID- 7538434 TI - The role of nitric oxide in Kupffer cell-hepatocyte interactions. AB - A certain level of normal or near-normal hepatic function is essential for survival. Over the last few years, the importance of the regulation of hepatic function by the interaction of specific cell populations in the liver has become increasingly evident. Kupffer cells, the fixed hepatic macrophages, have the ability to profoundly affect hepatocyte function, while hepatocytes, in turn, possess the capacity to modify Kupffer cell function. Nitric oxide, produced from the amino acid L-arginine, is a short-lived radical that is a potent mediator of cellular function and cell-cell interaction, and is synthesized by both Kupffer cells and hepatocytes. The purpose of this review is to examine the numerous areas in which nitric oxide may mediate the interactions between Kupffer cells and hepatocytes, and therefore regulate both normal and abnormal hepatic physiology and function. PMID- 7538437 TI - Clinical use of the newer antipsychotic drugs. AB - The clinical use of clozapine and risperidone is reviewed. Traditional antipsychotic drugs are effective for treating the positive symptoms of schizophrenia but have little or no effect on the negative symptoms of this disease. Newer antipsychotic agents, such as clozapine and risperidone, are effective for treating both positive and negative symptoms in acutely ill and treatment-resistant patients. Clozapine and risperidone also have a lower incidence of extrapyramidal symptoms (EPS) than traditional antipsychotic drugs. Three hypotheses have been proposed to account for the newer agents' ability to relieve negative symptoms: (1) Improvement in negative symptoms may be linked to improvement in positive symptoms. (2) Negative symptoms may improve in the absence of the EPS often caused by traditional agents. (3) The newer antipsychotic agents may directly affect the neural circuits that trigger negative symptoms. PMID- 7538436 TI - Recent developments in antipsychotic therapy. Introduction. PMID- 7538439 TI - Activation of Bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain. AB - Bruton's tyrosine kinase (BTK) is a nonreceptor tyrosine kinase critical for B cell development and function. Mutations in BTK result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Using a random mutagenesis scheme, we isolated a gain-of-function mutant called BTK* whose expression drives growth of NIH 3T3 cells in soft agar. BTK* results from a single point mutation in the pleckstrin homology (PH) domain, where a Glu is replaced by Lys at residue 41. BTK* shows an increase in phosphorylation on tyrosine residues and an increase in membrane targeting. Transforming activity requires kinase activity, a putative autophosphorylation site, and a functional PH domain. Mutation of the SH2 or SH3 domains did not affect the activity of BTK*. Expression of BTK* could also relieve IL-5 dependence of a B lineage cell line. These results show that transformation activation and regulation of BTK are critically dependent on the PH domain. PMID- 7538438 TI - Food anaphylaxis following ingestion of carmine. AB - BACKGROUND: The risk of sensitization to reactive dyes is well established. The clinical situation is caused most often by synthetic azo dyes and triphenylmethane derivatives but natural dyes such as carmine extracted from dried female insects, Coccus cacti (cochineal), have been incriminated. OBJECTIVE: Study of a case of anaphylaxis after ingestion of yogurt to establish the responsibility of carmine. METHOD: Case report of a patient who received skin prick test and leukocyte histamine release test with carmine and yogurt. CONCLUSIONS: This case provided evidence of an IgE-dependent mechanism and draws attention to the triggering dose of carmine (1 mg) although the acceptable daily intake is up to 5.0 mg per kg of body weight. PMID- 7538440 TI - Reconstitution of Syk function by the ZAP-70 protein tyrosine kinase. AB - ZAP-70 and Syk are PTKs required for TCR and BCR function, respectively. Loss of the Syk PTK results in a nonfunctional BCR. We provide evidence here that ZAP-70 and Syk are functionally homologous in antigen receptor signaling by demonstrating that expression of ZAP-70 in Syk- B cells reconstitutes BCR function. Reconstitution required the presence of functional Src homology 2 (SH2) and catalytic domains of ZAP-70. Thus, drug targeting of a single SH2 domain within ZAP-70 should be sufficient to inhibit hematopoietic antigen receptor function. In addition, we demonstrate that both ZAP-70 and Syk can bind directly to the phosphorylated Ig alpha and Ig beta subunits with affinities comparable to their binding to the TCR CD3 epsilon subunit. These data suggest that ZAP-70 and Syk are comparable in their abilities to mediate hematopoietic antigen receptor signaling. PMID- 7538441 TI - The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. AB - Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF kappa B, block TNF alpha induction of the leukocyte adhesion molecules E selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented. PMID- 7538443 TI - Substance P induces histamine release from human pulmonary mast cells. AB - Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 microM) and A23187 caused histamine release (median 26.7% range 6.2-62.8% and 32.1%, 7.7-56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1-33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen. PMID- 7538444 TI - [ATP as a neurotransmitter in the brain: its possibility based upon recent findings]. AB - We discuss here possible neurotransmitter roles of extracellular ATP in the central nervous system. Important findings concerning the effects of ATP on central neurons have been made in the last few years. A fast neurotransmitter role of ATP in rat medial habenula reported by Edwards et al (1992) may be the best example. In rat cultured hippocampal neurons, we have found that ATP evokes postsynaptic currents, and increases the intracellular Ca2+ concentration. In addition, ATP also produces a "long-term potentiation like effect" in these cells. Receptors for ATP are generally divided into two classes: one class is coupled with ion channels and the other class is coupled with GTP-binding proteins. In cultured hippocampal neurons, the GTP-binding protein-coupled receptors are present and promote the inhibition of K+ channels. Although the channel-coupled receptors have not been identified in the hippocampal neurons, these channels have been found in nucleus solitarii and locus coeruleus. Several types of ATP receptors have been cloned very recently. These cloned receptors, combined with molecular biological techniques, will allow great progress in this field. PMID- 7538442 TI - B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. AB - The functional necessity for two CD28 counterreceptors (B7-1 and B7-2) is presently unknown. B7-1 and B7-2 equivalently costimulate IL-2 and interferon gamma (IFN gamma) production and IL-2 receptor alpha and gamma chain expression. B7-2 induces significantly more IL-4 production than B7-1, with the greatest difference seen in naive T cells. Repetitive costimulation of CD4+ CD45RA+ T cells with B7-2 results in moderate levels of both IL-4 and IL-2, whereas repetitive costimulation with B7-1 results in high levels of IL-2 and low levels of IL-4. Therefore, B7-1 and B7-2 costimulation mediate distinct outcomes, since B7-2 provides an initial signal to induce naive T cells to become IL-4 producers, thereby directing the immune response more towards Th0/Th2, whereas B7-1 is a more neutral differentiative signal. PMID- 7538447 TI - Alteration of expression in integrin beta 1-subunit correlates with invasion and metastasis in colorectal cancer. AB - We have investigated the expression of the integrin beta 1 subunit in 51 colorectal cancer tissues using immunoblotting and have determined the relationship between expression and clinical stage. In comparison with normal mucosa the pre-beta-subunit (the precursor of beta 1-subunit) was increased in 15 cases and decreased in two cases, and expression of the beta 1-subunit was decreased in two cases. These alterations of expression of the beta 1-subunit were significantly correlated with lymph node metastasis and depth of invasion (P < 0.01). These results suggest that integrin plays an important role in the invasion and metastasis of colorectal cancer. PMID- 7538445 TI - The number of tartrate-resistant acid phosphatase-positive osteoclasts on neonatal mouse parietal bones is decreased when prostaglandin synthesis is inhibited and increased in response to prostaglandin E2, parathyroid hormone, and 1,25 dihydroxyvitamin D3. AB - The culture of parietal bones from 4-day old mice in indomethacin (Ind) for 1 day caused a large reduction in the number of tartrate-resistant acid phosphatase positive osteoclasts (TRAP + OC) relative to both control bones and to freshly isolated bones. This reduction did not occur if prostaglandin E2 (PGE2) was present. When 5-bromo-2'-deoxyuridine (BDU) was injected into 4-day old mice, newly formed TRAP + OC nuclei became labeled 1 day later; these bones were then cultured with Ind for 1 day. TRAP + OC and newly labeled TRAP+OC nuclei were commensurately decreased in number. This suggests an active down-regulation rather than merely the inhibition of new TRAP+OC formation. Incubation of bones with Ind and either PGE2, parathyroid hormone, or 1,25 dihydroxyvitamin D3 for 6 hours following a 1-day preincubation in Ind, resulted in an increase in TRAP + OC compared with Ind alone. Using BDU labeling in vitro and in vivo, we show that this increase in number of TRAP+OC is not the result of cell proliferation, but rather differentiation of postmitotic precursors. PMID- 7538446 TI - A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures. AB - Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To overcome these obstacles, we developed a new method of isolating nucleic acids from chondrocytes which facilitates measurement of cell number by DNA analysis. Chondrocytes were isolated from chick embryo sterna and grown continuously without subculturing for 2-3 weeks in monolayer. The cells were treated with triton X-100 and the nucleic acid content of the extract was determined by measuring DNA fluorescence in the presence of Hoechst dye 33258. To minimize background fluorescence due to the triton, we precipitated the DNA with alcohol and then solubilized the nucleic acids in EDTA. This simple procedure removed the detergent and substantially increased the sensitivity of the method. Thus, we could measure with high precision and high recovery, the DNA content of cultures of 10,000-50,000 cells. In a single well containing 0.5-1.0 million cells, sufficient material remained for subsequent measurements of alkaline phosphatase activity and protein and calcium content. As the mineral present in the triton-treated samples was soluble in EDTA, we experienced no problems in measuring the calcium content of the culture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of the triton maintained the catalytic state of alkaline phosphatase. We conclude that this procedure provides a simple and rapid approach to measuring major indicators of chondrocyte maturation and function. PMID- 7538448 TI - Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells. AB - We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC50 of 41-53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a comparable efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the short term assay, is due to inefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution. PMID- 7538449 TI - Biological response modifiers (BRM) as antigens. III. T cell lines specific for BRM kill tumor cells in a BRM-specific manner. AB - In order to investigate tumoricidal effector cells in therapy by biological response modifiers (BRM) such as Propionibacterium acnes, bacillus Calmette Guerin (BCG), Streptococcus pyogenes and a protein-bound polysaccharide (PSK), we established T cell lines specific for each BRM from BALB/c mice immunized with the corresponding BRM. These T cell lines proliferated and produced interleukin-2 (IL-2) and/or IL-4, but only in the presence of the relevant BRM and BALB/c spleen cells as the antigen and antigen-presenting cells respectively. Cross functional experiments indicated that each BRM acts as a nominal antigen, but not as a non-specific immunostimulator. In addition, the T cell lines killed Ia positive syngeneic B lymphoma cells, but only in the presence of the relevant BRM. These experiments excluded the possibility of cytotoxic effects by each BRM. The T cell lines and clones also killed Ia-negative bystander target cells, but only in the presence of both a relevant antigen and antigen-presenting cells. The T cell clones specific for S. pyogenes or P. acnes tested were Thy1+, L3T4+ and Lyt2-. These results indicate that some BRM exert tumoricidal activity by inducing T cells that recognize them as an antigen and kill tumor cells in an antigen-specific manner. The T cells killed tumor targets in either a tumor necrosis-factor(TNF)-dependent or a TNF-independent manner. The mediator of the latter pathway remains to be elucidated. PMID- 7538450 TI - Location of neurons projecting to the hypophysial stalk--median eminence in ring doves (Streptopelia roseogrisea). AB - The median eminence/pituitary stalk represents the final common pathway for fibers from neurons that project to the pituitary gland. We have used the lipophilic fluorescent tracer 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate (DiI) to determine the location of neurons projecting to the median eminence/pituitary stalk in ring doves. The tracer can be precisely applied to fixed tissue, in areas to which it is otherwise difficult to gain access. Following application of DiI to the median eminence/pituitary stalk, labeled neurons were detected in six distinct regions: the ventro-medial hypothalamic nucleus, paraventricular nucleus, supraoptic nucleus, in and ventral to the lateral forebrain bundle, preoptic area, and lateral septum. Labeled fibers branched extensively in the diencephalon, particularly along the third ventricle and in the septal-preoptic area. Sparse fiber labeling occurred caudal to the tuberal hypothalamus, even though these regions were close to the application site of the tracer. Labeled cerebrospinal fluid-contracting cells were seen in the paraventricular region of the third ventricle. The results indicate that the avian neuronal system that projects to the median eminence and neural lobe occurs in diffuse clusters largely along the midline region of the hypothalamic septal-preoptic area. The paucity of fiber staining caudal to the tuberal hypothalamic region indicates that cells of these regions do not project to the median eminence/pituitary. PMID- 7538451 TI - c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. AB - In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells. PMID- 7538452 TI - Alterations in the heme biosynthetic pathway from the III-V semiconductor metal, indium arsenide (InAs). AB - The effects of indium and arsenic on the heme biosynthetic pathway have been well documented but the effects of indium arsenide (InAs), the next possible generation of the III-V semiconductors, are unknown. Male Syrian golden hamsters were given s.c. injections of sodium arsenite (As3+), indium chloride (In3+) or indium arsenide (InAs). Erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity was inhibited in all exposure groups, while hepatic ALAD activity was not significantly changed. In contrast, the activity of renal ALAD was found to be statistically decreased by As3+ at 10 days, but increased at 30 days, while In3+ and InAs inhibited this enzyme activity at all time points. In vitro studies showed that hepatic ALAD activity was more sensitive to In3+ than As3+, suggesting that the effects of InAs in vivo on this enzyme are due primarily to the In rather than the As moiety. Studies of urinary porphyrin excretion patterns in animals treated with InAs showed marked, early 2-4-fold increase in the excretion of the penta-, hexa- and heptacarboxyl porphyrin at 1-5 days which continued through day 30 of the study. In contrast, there was a slow and steady rise in the excretion of coproporphyrin I and III which reached a maximum at day 30. The results of these studies indicate that both the In and As moieties of InAs are biologically active following InAs exposure and that the enzymes in the heme pathway, such as ALAD, may have great utility as markers of exposure/toxicity for these agents. PMID- 7538456 TI - Measurements of plasma elastase alpha 1-proteinase inhibitor complexes in patients receiving cancer chemotherapy with granulocyte colony-stimulating factor. AB - We monitored the plasma elastase alpha 1-proteinase inhibitor complex levels in 21 patients with primary lung cancer who received combination chemotherapy with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF), and 15 normal nonsmokers as controls. Of the 21 patients, 14 received combination chemotherapy without rhG-CSF (among them, 6 developed pneumonia) and 7 received combination chemotherapy with rhG-CSF (among them, 1 developed pneumonia). We measured peripheral WBC counts, C-reactive protein (CRP) levels, plasma elastase alpha 1-proteinase inhibitor complex (complex) levels, and complex/WBC values during cancer chemotherapy. In patients who received cancer chemotherapy without rhG-CSF and had no complications (n = 8), WBC values decreased after chemotherapy, and then gradually increased. Complex levels also decreased slightly after chemotherapy and gradually recovered. The value obtained from dividing the complex concentration by WBC count (complex/WBC value) remained stable during cancer chemotherapy. In patients who received cancer chemotherapy with rhG-CSF and had no complications (n = 6), WBC values decreased after chemotherapy, and then rapidly increased to abnormally high values. Complex levels also decreased slightly after chemotherapy and rapidly increased to abnormally high values together with the WBC counts. The complex/WBC values remained stable during cancer chemotherapy. In patients who developed pneumonia during cancer chemotherapy with or without rhG-CSF (n = 7), their complex levels, complex/WBC values, and CRP levels were elevated at the onset of pneumonia. The maximum complex levels (the highest levels during chemotherapy) were significantly higher in patients who received cancer chemotherapy with rhG-CSF and did not develop pneumonia (583.1 +/- 114.5 ng/mL) and in patients who developed pneumonia during cancer chemotherapy (516.7 +/- 113.2 ng/mL), compared with normal nonsmokers (130.2 +/- 5.5, p < 0.01) and patients who received cancer chemotherapy without rhG-CSF and did not develop complications (211.5 +/- 23.3, p < 0.01). The maximum complex/WBC values were not increased in patients who received cancer chemotherapy with rhG-CSF (0.08 +/- 0.01) and patients who received cancer chemotherapy without rhG-CSF (0.092 +/- 0.01, p < 0.01). The maximum complex/WBC values were significantly higher in patients with pneumonia (0.56 +/- 0.12) compared with normal nonsmokers (0.026 +/- 0.002, p < 0.01) and patients without complications. These findings suggest that although rhG-CSF increases total plasma elastase burden, increased release of neutrophil elastase from individual neutrophils does not take place in vivo in the absence of pneumonia. PMID- 7538455 TI - Enhancement of hematopoietic uptake by granulocyte colony-stimulating factor in Ga-67 scintigraphy. AB - The authors report on two non-Hodgkin's lymphoma cases which showed markedly enhanced Ga-67 uptake of granulopoietic area induced by recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration. The rhG-CSF is a newly developed agent for reduction of infections, which stimulates the proliferation and differentiation of granulopoiesis. Use of rhG-CSF is becoming increasingly frequent because the indications are so broad that most patients with intensive chemotherapy for malignancies benefit from undergoing this treatment. In this study, the results of the two cases were: 1) G-CSF enhanced the accumulation of Ga-67 citrate in the granulopoietic area; 2) the marked uptake of red marrow looked like involvement; and 3) in contrast, the involved area became relatively "cold." These findings should be considered in the interpretation of Ga-67 scintigraphy. PMID- 7538457 TI - Calcium entry attenuates adenylyl cyclase activity. A possible mechanism for calcium-induced catecholamine resistance. AB - In experimental animals, coadministration of calcium (Ca) salts with beta adrenergic receptor agonists reduces the increased blood pressure and cyclic AMP (cAMP) produced by beta-adrenergic receptor agonists alone. In patients, coadministration of these drugs reduces the increased cardiac output and blood glucose produced by selective administration of beta-adrenergic agonists. The mechanism by which Ca might produce catecholamine resistance remains unclear. Healthy volunteers donated venous blood from which lymphocytes were isolated. The cAMP production was measured by radioimmunoassay under control conditions and after incubation with epinephrine or colforsin (forskolin) in the presence and absence of inhibitors. Epinephrine and colforsin produced concentration-dependent increases in cAMP production. Extracellular Ca concentration over the range from 0 to 8 mM did not inhibit basal cAMP production or that stimulated by either colforsin or epinephrine. The calcium channel agonist Bay K 8644 (50 microM) combined with normal extracellular Ca concentration significantly attenuated colforsin-induced increases in cAMP production. When barium was substituted for Ca in the extracellular fluid, the cAMP response to colforsin was restored, despite Bay K 8644. Inhibition of Ca channel permeability with cadmium or cobalt ions partially restored colforsin-stimulated cAMP production, despite the presence of extracellular Ca and Bay K 8644. These results suggest that entry of Ca ions through Ca channels attenuates adenylyl cyclase. The inhibition appears specific for Ca ions over other permeant divalent cations, and favors a possible physiologic role for the recently cloned Ca-inhibited adenylyl cyclase. PMID- 7538459 TI - [Chemoembolization in primary liver cell carcinoma. Results of a prospective study]. AB - A remarkable progress was made in the palliative treatment of unresectable hepatocellular carcinoma using transcatheter arterial chemoembolization (TAC). The combination of doxorubicin as cytotoxic drug and of Lipiodol as carrier, which is selectively accumulated within the tumors is an effective treatment. Prolonged survival has been demonstrated in several clinical studies. In a prospective trial the effect of regional TAC with temporary ischemia of the liver was evaluated concerning liver function, systemic reactions and tumor response. Temporary ischemia of the liver was well tolerated even in patients with liver cirrhosis. Revascularization of the liver after TAC was observed in all cases. Systemic side effects of the cytotoxic drug were mild. In patients with Child C cirrhosis or tumor volume of more than 60% of the liver no impact of the treatment on the survival time was observed. In the entire patient group a survival rate of 70% after 15 months was achieved. PMID- 7538461 TI - Bone morphogenetic protein-2 inhibits the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures. AB - Previous work from our laboratory indicated that bone morphogenetic protein-2 (BMP-2) enhances the synthesis of insulin-like growth factor-I (IGF-I) and IGF-II by skeletal cells. The activity of IGF-I and -II is regulated by six known IGF binding proteins (IGFBPs). Although most IGFBP's inhibit the actions of IGF on bone growth, IGFBP-5 is stimulatory, and its synthesis correlates with changes in osteoblast cell growth. We tested the effects of BMP-2 on IGFBP-5 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Treatment of Ob cells with BMP-2 caused a time- and dose-dependent decrease in IGFBP-5 messenger RNA (mRNA) levels, as determined by Northern blot analysis. The effect was maximal after 24 h of treatment and occurred at BMP-2 concentrations of 0.03-3.3 nM. Treatment with BMP-2 for 24 h also decreased IGFBP 5 polypeptide levels in the extracellular matrix, as determined by Western blot analysis. The effects of BMP-2 on IGFBP-5 transcripts were independent of cell division, as they were observed in the presence and absence of hydroxyurea (1 mM). IGFBP-5 transcripts were barely detectable in the presence of the protein synthesis inhibitor cycloheximide at 3.6 microM, and further suppressive effects of BMP-2 on IGFBP-5 mRNA could not be determined. BMP-2 did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells. In addition, BMP-2 inhibited IGFBP-5 heterogeneous nuclear RNA, determined by reverse transcription polymerase chain reaction, after 2-6 h of treatment, suggesting an inhibition of IGFBP-5 transcription or processing. In conclusion, BMP-2 inhibits IGFBP-5 expression in Ob cells through pathways that are independent of its mitogenic activity and through mechanisms that may involve decreased transcription or altered RNA processing. PMID- 7538458 TI - [Pathology of wound healing]. AB - In this review the morphologically defined steps of wound healing are described. Then the mechanisms of molecular regulation are discussed, which are the basis of the phases of wound healing. A main topic is the role of signal peptides in the regulation of cellular and stromal alterations during wound healing. An overview is given about the respective growth factors, their main function during wound healing and their receptors. Finally, analogies with fetal wound healing and the therapeutic modulation of wound-healing disturbances are discussed. PMID- 7538460 TI - [Histopathological and immunohistochemical study of ovarian polyembryoma]. AB - A case of polyembryoma which is rare in ovarian malignant germ cell tumors was reported. Microscopically, numerous and variable embryoid bodies, surrounded by embryonic hepatoid tissues, trophoblastic cells and multiple elements, were observed. The results of immunohistochemical analysis were: (1) alpha fetoprotein (AFP) was not only shown positive in yolk sac cells among the embryoid bodies, in the endoderm and in the margins of the ectodermal cells, but also in multiple elements in the mucinous matrix. (2) Hepatoid tissues reacted positively to AFP and alpha antitrypsin (AAT) as well as to hCG. (3) By using hCG and human placental lactogen (hPL) as markers it is proven that among trophoblastic cells of the tumor, not only syncytiotrophoblastic cells but also the intermediate cells and the cytotrophoblastic cells could be identified. PMID- 7538453 TI - Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency. AB - OBJECTIVE: The aim of this investigation was to study the effect of relatively high dose IGF-I therapy given for several months, on serum levels of IGF-I, IGF II and IGFBP-3, and on IGF-I pharmacokinetics in patients with growth hormone insensitivity due to GH receptor dysfunction. DESIGN AND PATIENTS: Two adolescent subjects from Ecuador were treated with recombinant IGF-I at a dosage of 120 micrograms/kg s.c. twice daily, in combination with a GnRH analogue for 8 months. MEASUREMENTS: Serum was sampled at baseline and at 3-8 months, for determination of IGF-I, IGF-II and IGFBP-3 by radioimmunoassay, and for evaluation of IGFBPs and IGFBP-3 protease activity by Western ligand blot and protease assay, respectively. RESULTS: Peak serum IGF-I levels ranged from 272 to 492 micrograms/l. Mean serum IGF-II levels were decreased concurrently with the increase in IGF-I. Serum IGFBP-3 levels failed to rise with prolonged IGF-I treatment. There was no apparent change in the half-life of IGF-I during the treatment period. CONCLUSIONS: IGF-I administration does not increase serum levels of IGFBP-3 or significantly alter IGF-I pharmacokinetics. PMID- 7538454 TI - The role of beta 1 integrins in adhesion of two breast carcinoma cell lines to a model endothelium. AB - Interactions between tumour cells and the endothelium are vital to the formation of haematogenous metastases. Binding to model endothelium of one oestrogen receptor positive breast carcinoma cell line (MCF-7) and one receptor negative line (HS578T) was examined in vitro together with endothelial retraction induced by these tumour cells. Adhesion was inhibited by monoclonal antibodies specific for the VLA integrins and by peptides containing the RGD motif which is commonly recognised as a ligand by the VLA adhesion molecules. However, binding of the two tumour cell lines was inhibited by monoclonal antibodies specific for different VLA molecules; anti-alpha 6 beta 1 inhibited MCF-7 adhesion but anti-alpha 5 beta 1 inhibited Hs578T. These results were consistent with flow cytometric quantification of the expression of these VLA integrins on the surfaces of the two tumour cell lines. Enzyme-linked immunosorbent assays (ELISA) demonstrated that laminin was present on the endothelial cell surface but collagen IV was absent. ELISA failed to detect increased exposure of the subendothelial matrix during the first hour after addition of either cancer cell type. This was supported by assays which demonstrated maintenance of the endothelial permeability barrier during this period. Slight endothelial retraction was detected within 2 hours of the addition of tumour cells. It is concluded that binding between tumour cells and confluent endothelium is inhibited by the blockade of adhesion molecules which are normally associated with interactions between the cell and the subendothelial matrix. Tumour cell to matrix interactions rather than direct tumour to endothelial cell adhesion may be the limiting step in tumour cell binding to the endothelium. PMID- 7538462 TI - Isolation, culture, and characterization of the two cell subpopulations forming the rat decidua: differential gene expression for activin, follistatin, and decidual prolactin-related protein. AB - The decidual tissue of the pregnant rat is formed primarily by two markedly different decidual cell populations located in the mesometrial and antimesometrial sites of the uterus. The antimesometrial decidua functions as an endocrine organ, secreting PRL-like hormones and expressing activin and follistatin. In contrast, the mesometrial decidua has no apparent endocrine activity, but secretes abundant alpha 2-macroglobulin. To determine whether the profound difference in morphology and gene expression is inherent to each cell population or whether it results from the position of these cells in the uterus and their subjection to regionally restricted information, we isolated the giant antimesometrial decidual cells from the small mesometrial decidual cells by elutriation. We examined, using Northern and reverse transcription-polymerase chain reaction analysis, the expression of PRL-related protein (DPRP), follistatin, activin, and alpha 2-macroglobulin in the two cell populations just after cell separation and after primary culture. We also examined, using coculture, the possible cross-talk between the two cell populations. In culture, the cells maintained the morphological appearance seen in the decidua; the mesometrial cells remained less differentiated, whereas the antimesometrial cells formed giant cells. In addition, the antimesometrial cells, which have been shown previously to be the only site of DPRP messenger RNA (mRNA) expression, remained, after several days of culture, the only cells able to express this gene. The coculture experiment suggested that the absence of DPRP mRNA in the mesometrial cells is not due to inhibitory signals from the antimesometrial cells, but, rather, to the inherent inability of the mesometrial decidual cells to express the DPRP gene. The cellular expression pattern of follistatin was also not changed by cell separation. However, in sharp contrast to that of DPRP and follistatin, the cell-specific expression of activin-beta A and alpha 2 macroglobulin was markedly affected by cell separation. A remarkable switch in activin-beta A cell expression was observed when mesometrial cells were separated from antimesometrial cells and maintained in culture. Activin-beta A mRNA, which was barely detectable in these cells in situ, became expressed in higher levels than in antimesometrial cells. The isolated antimesometrial cells also acquired the ability to express the alpha 2-macroglobulin gene, whose expression is highly restricted to the mesometrial cells in vivo. No down-regulation of antimesometrial alpha 2-macroglobulin mRNA could be induced by mesometrial cells in a coculture experiment. However, alpha 2-macroglobulin mRNA levels in mesometrial cells were clearly up-regulated by antimesometrial cell signal(s).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538463 TI - Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells. AB - Smooth muscle cells (SMC) secrete insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) and an IGFBP-4 protease. The purpose of this study was to determine the characteristics of this IGFBP-4 protease and to compare its inhibitor profile to those of IGFBP-5 and IGFBP-2 proteases, which are also present in SMC-conditioned medium. Cultured SMC were exposed to serum-free medium for periods of 24-72 h, and the amount of proteolytic activity in the conditioned medium was assessed by its capacity to degrade pure IGFBP-4. Minimal activity (e.g. < 20% of IGFBP-4 degraded in 24 h at 37 C) was present in conditioned medium unless IGF-I or IGF-II was added. This resulted in more than 60% of the intact IGFBP-4 being degraded in 14 h. The activity was a calcium-dependent serine protease and was inhibited by EDTA or 3,4-dicloroisocoumarin. Calcium, but not zinc, could restore proteolytic activity. Heparin alone inhibited IGFBP-4 proteolysis by more than 60%. When heparin cofactor-II and antithrombin-III (AT III) were added alone, they each had an effect. The combination of heparin plus AT-III was no more active than heparin alone, but the combination of heparin cofactor-II and heparin resulted in near complete inhibition. Peptides that contained the active sites of AT-III or alpha 1-antichymotrypsin were potent inhibitors of the IGFBP-4 protease. The medium also contained proteolytic activities for IGFBP-2 and IGFBP-5. Comparison of the inhibitor profiles for the IGFBP-4 and IGFBP-5 proteolytic activities revealed major differences, but the IGFBP-2 proteolytic activity was very similar to that of the IGFBP-4 protease. IGFBP-4 zymography showed a band with a molecular mass estimate of 48 kilodaltons. In contrast, when IGFBP-2 was used as the substrate, a single band at 36 kilodaltons was visualized. These data taken together with the protease inhibitor results suggest that the IGFBP-2, IGFBP-4, and IGFBP-5 proteases are members of a similar family of calcium-dependent serine proteases, but they are distinct proteases. As IGFBP-4 is a potent inhibitor of IGF action, and the activity of this protease is regulated by IGF exposure, the protease represents a novel system for regulating the actions of IGF-I in this cell type. PMID- 7538464 TI - Preprotachykinin-A gene expression occurs transiently in the developing rat endocrine pancreas and can be regulated in RINm5F cells. AB - We have reported previously that the rat insulinoma cell lines, RINm5F and RINr1046-38, express the preprotachykinin(PPT)-A gene, which encodes the tachykinin peptides, substance P and neurokinin A. Because endocrine cells of the adult rat pancreas do not appear to express PPT-A, we investigated whether the gene is expressed by rat pancreatic endocrine cells during development. We used immunohistochemistry, employing different substance P and neurokinin A antibodies, to show that many endocrine cells of the fetal and neonatal rat pancreas synthesise these products of PPT-A gene expression. Colabeling experiments revealed that a significant number of both insulin-containing and non insulin-containing cells express tachykinins. After postnatal day 20, the number of tachykinin-immunoreactive pancreatic islet cells declines and, as already reported, none were detected in the adult rat pancreas. The transient expression of PPT-A by the developing endocrine pancreas is a novel finding. Substance P and neurokinin A are known to have trophic actions and may serve as growth factors during pancreatic islet development. PPT-A gene expression by RINm5F and RINr1046 38 cells is further evidence that these cells resemble developing pancreatic endocrine cells. They are potentially valuable as unique models for studying the regulation of tachykinin biosynthesis. We provide evidence in this study, using quantitative PPT-A messenger RNA analysis, that PPT-A expression in RINm5F cells may be up-regulated by activation of protein kinase C, down-regulated by activation of glucocorticoid receptors, and is not significantly affected by changes in intracellular cAMP levels. PMID- 7538465 TI - Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)-phthalate treatment. III. Fusion of early autophagosomes with lysosomal compartments. AB - Fusion of early autophagosomes containing peroxisomes with endosomal and lysosomal structures in rat liver cells was investigated. Male Wistar rats were administered di-(2-ethylhexyl)phthalate (DEHP) for 14 days to induce proliferation of peroxisomes, and then the animals were injected intravenously with horseradish peroxidase (HRP)-conjugated asialofetuin to label the lysosomal compartment. Either 30 min or 60 min after the injection, the animals were treated with leupeptin for 20, 40 and 60 min, respectively. Most of autophagic vacuoles containing peroxisomes were stained with diaminobenzidine (DAB) endocytosed HRP-asialofetuin 60 min after leupeptin injection, whereas many of them were negative for DAB reaction 20 min after leupeptin injection. Between 20 to 40 min after leupeptin treatment many autophagic vacuoles fused with DAB positive lysosomal compartments, including late endosomes. Percoll gradient centrifugation showed that particles containing HRP activity migrated from a density of 1.09 g/ml to that of 1.14 g/ml as the time after leupeptin injection passed. Acid phosphatase activity migrated in the same manner. These results clearly show that early autophagosomes obtain the lysosomal proteinases by fusion with lysosomal compartments, including the late endosomes and that peroxisomes trapped in autophagosomes are degraded by these proteinases. PMID- 7538466 TI - Production of angiogenesis inhibitors and stimulators is modulated by cultured growth plate chondrocytes during in vitro differentiation: dependence on extracellular matrix assembly. AB - Secretion of angiogenesis inhibitors and stimulators is modulated during in vitro differentiation of embryonic chick growth plate chondrocytes. Supernatants from dedifferentiated cells undergoing maturation to hypertrophic chondrocytes in suspension progressively inhibited vascular cell random migration and invasion of basement membrane matrix by endothelial cells. Maximal inhibition was exhibited by conditioned medium from hypertrophic chondrocytes. The same medium also repressed vascular cell migration induced by highly angiogenic Kaposi's sarcoma cell supernatants and prevented formation of an anastomosed network of tube-like structures by endothelial cells plated on matrigel. On the contrary, when the suspension culture of hypertrophic chondrocytes was supplemented with ascorbic acid, a condition leading to the formation of a mineralized tissue similar to calcified cartilage, a dramatic switch to production of angiogenic activity was observed. Medium conditioned by osteoblast-like cells derived from hypertrophic chondrocytes also induced vascular cell migration and invasion of basement membrane matrix. The presence of angiogenic activity in the conditioned medium was assessed also by an in vivo assay in mice using reconstituted basement membrane associated with heparin. Therefore, interactions of chondrocytes with their extracellular matrix are an absolute requirement for the expression of angiogenic activities by hypertrophic chondrocytes at late developmental stages. PMID- 7538467 TI - Fas antigen signals proliferation of normal human diploid fibroblast and its mechanism is different from tumor necrosis factor receptor. AB - Recent cloning of the cDNA for Fas/Apo-1 and its ligand has revealed that they belong to the tumor necrosis factor (TNF) receptor and TNF family, respectively, and play an important role in apoptosis (programmed cell death). Like TNF, antibodies against the Fas antigen (anti-Fas) have been shown to be cytotoxic to Fas-expressing cells. Whether Fas, like TNF receptor, also mediates proliferation of normal human diploid fibroblasts (HDF), is not known. In this study, we show that HDF expresses Fas antigen and the engagement of this antigen signals proliferation of these cells in a dose-dependent manner. Unlike TNF receptor, however, Fas-mediated proliferation of HDF could not be blocked by orthovanadate, a tyrosine phosphatase inhibitor. The difference in the signaling was further evident from our observation that TNF induced the expression of interleukin-6 but anti-Fas did not. Overall, our results demonstrate for the first time that besides cell killing, Fas also mediates proliferation of HDF and that its mechanism is different from that of TNF receptor. PMID- 7538468 TI - High-pressure stabilization of alpha-chymotrypsin entrapped in reversed micelles of aerosol OT in octane against thermal inactivation. AB - alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2 ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity. PMID- 7538471 TI - Structural properties of alpha-fetoprotein from human cord serum: the protein molecule at low pH possesses all the properties of the molten globule. AB - Structural studies of alpha-fetoprotein (AFP) from human cord serum have shown that a decrease in pH to 3.1 leads to a considerable conformational rearrangement of the protein molecule. The acid form of AFP belongs to the class of denatured conformations and fulfills all the requirements of the molten globule state. The possible functional role of such a transformation is discussed. PMID- 7538472 TI - Evidence for a cGMP gated cation channel in photoreceptor cell membranes of Sepia officinalis. AB - It is assumed that cyclic nucleotides are involved in signal transduction of invertebrate photoreceptors. In this study, membranes of photoreceptor cells from freshly caught cuttlefish were isolated, and the membrane proteins were reconstituted into proteoliposomes. With the dye Neutral red it was possible to measure cyclic nucleotide induced Na+ fluxes into the liposomes. cGMP and cAMP concentrations for half maximal activation of Na+ fluxes are 77 microM and 224 microM, respectively, with Hill coefficients of 2.0 for cGMP and 2.4 for cAMP. These fluxes may demonstrate the presence of at least one cyclic nucleotide gated cation channel in the membranes of the photoreceptor cells of the invertebrate Sepia officinalis. PMID- 7538469 TI - Involvement of multiple proteases during Fas-mediated apoptosis in T lymphocytes. AB - The mechanism of Fas antigen-mediated apoptosis is at present unclear. We show here that the 100,000 x g supernatant from cell lysates prepared from anti-Fas stimulated JUR-KAT T cells, induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors, TPCK and DCI, and by the interleukin 1-beta-converting enzyme inhibitor, VAD-FMK. In addition, chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK, but not by DCI or VAD-FMK. These results suggest that Fas-mediated apoptosis in T cells involves the activation of a cascade of proteases. PMID- 7538470 TI - Isolation and partial characterization of a protease involved in Fas-induced apoptosis. AB - Protease involvement has been implicated in the signalling process of activation induced apoptosis. Here we report the isolation of a protease from Jurkat T cells undergoing Fas-induced apoptosis. Although the protease probably is a serine protease, it seems to be distantly related to members of the ICE/ced-3/Ich-1(nedd 2) family. In a cell-free system using isolated thymocyte nuclei, the protease rapidly induces DNA fragmentation and morphological changes typically seen in apoptosis. Our results clearly show protease activation downstream to Fas ligation and implicate an important role for the isolated protease in signalling of Fas-induced apoptosis. PMID- 7538473 TI - Serum levels of insulin-like growth factor I and insulin-like growth factor binding protein-1 and -3 in women with regular menstrual cycles. AB - OBJECTIVE: To investigate the cyclic changes of serum insulin-like growth factor (IGF-I), IGF-binding protein-1 (IGFBP-1) and IGFBP-3 levels during ovulatory menstrual cycle. DESIGN: A prospective study following a preset protocol. SETTING: A tertiary-care academic medical center. PARTICIPANTS: Thirty young female adults with regular menstrual cycles were recruited (18 with normal luteal phase and 12 with inadequate luteal function confirmed by serum P levels). MAIN OUTCOME MEASURES: Serum concentrations of IGF-I, IGFBP-1, and IGFBP-3 from women with regular menstrual cycles were assayed. Circulating E2 and P levels also were determined to certify the ovulatory cycles. RESULTS: In women with normal luteal function, there was a peak of serum IGFBP-1 levels before ovulation concomitant with the preovulatory E2 peak. The nadir of serum IGFBP-1 levels was in the midluteal phase. Circulating IGFBP-1 elevated rapidly during late luteal phase and reached a peak on the 1st day of menstruation then declined slightly until a preovulatory IGFBP-1 peak occurred. In women with inadequate luteal function (midluteal serum levels of P < 10 ng/mL [conversion factor to SI unit, 3.180]), the preovulatory increase in serum IGFBP-1 was not significant and the circulating IGFBP-1 levels fluctuated throughout the menstrual cycle except for a unique peak of serum IGFBP-1 on the 1st day of menstruation. By contrast, there were no cyclic changes of serum IGF-I and IGFBP-3 levels in women with regular menstrual cycles, including both normal nad inadequate luteal functions. CONCLUSIONS: The preovulatory increase in serum IGFBP-1 levels may be of follicular origin and associated with the subsequent luteal function in females with ovulatory cycles. However, the involvement of IGFBP-1 in the process of follicular maturation and luteogenesis, as well as the regulation of luteal function, needs to be explored further. PMID- 7538475 TI - Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination. AB - OBJECTIVE: To determine the relationships between sperm function tests and fertilization of human oocytes in vitro. DESIGN: Analysis of infertile patients undergoing IVF therapy. SETTING: Diagnostic Andrology Laboratory and Assisted Conception Service. PATIENTS: Forty-one couples who underwent IVF-ET therapy were studied. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to numerous criteria of semen quality, including the conventional semen profile, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, sperm morphology, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS: Significant correlations were observed between fertilization rates and several attributes of the sperm preparations, including elements of sperm function (acrosome reaction), movement (percentage motile, hyperactivation, the amplitude of lateral sperm head displacement), morphology (normal morphology, midpiece defects, multiple anomalies index), nuclear normality (acridine orange staining), and reactive oxygen species generation (chemiluminescence induced by phorbol ester and FMLP). In a stepwise multiple regression analysis, an accurate prediction of fertilization rates was obtained using a multiple regression equation incorporating six variables of which sperm morphology and FMLP-induced chemiluminescence were the most informative. CONCLUSIONS: A set of criteria have been identified that accurately predict the fertilizing potential of human sperm suspensions in vitro and that place particular emphasis on sperm morphology and the degree of leukocyte contamination. PMID- 7538476 TI - Nematocyte differentiation in hydra: commitment to nematocyte type occurs at the beginning of the pathway. AB - In hydra the four types of nematocytes arise by differentiation from the multipotent stem cells among the interstitial cells. It has been unclear where along the nematocyte pathway commitment to type occurs. Some evidence suggests that this commitment occurs at the beginning of the pathway, while other data suggest that it occurs at the terminal cell cycle midway through the pathway. Upon reduction of cell population sizes of the interstitial cell lineage by treatment with hydroxyurea, interstitial cells entering nematocyte pathways frequently undergo an amplification division. A nearest-neighbor analysis of pairs of nematoblast nests in such depleted animals has shown that the fraction of the nearest-neighbor pairs that are matched pairs, in which both nests are of the same type, is higher than predicted. A very high fraction of the matched pairs were identical pairs in which the number of cells in each nest was the same. Also, in a large majority of the identical pairs the nests were shown to be in the same stage of development. The simplest interpretation of these results is that the two daughters of the amplification division giving rise to the matched pair were committed to nematocyte type before the division occurred. In another experiment we show that the length of the G2 phase of the next-to-terminal cell cycle differs between desmonemes and stenoteles. This indicates that differences in the differentiation pathways of these two types exist before the terminal cell cycle. This result also supports the idea that commitment to type occurs at the beginning of the pathway. A means of reconciling the view that commitment occurs early with the view that it occurs late in teh differentiation pathway is discussed. PMID- 7538474 TI - Expression of cadherins and integrins in human endometrium throughout the menstrual cycle. AB - OBJECTIVE: To detect cadherin and integrin expression in biopsies of endometrium in the phases of the cycle. Cell adhesion molecules may be involved in endometrial shedding during menstruation and attachment of shed endometrial tissue to the peritoneal lining in endometriosis patients. DESIGN: An immunohistochemical study on fresh frozen sections. SETTING: Tertiary-care university medical center. PATIENTS: Sixteen patients undergoing monitoring of their cycle as part of a subfertility workup. All patients had regular and ovulatory cycles. INTERVENTIONS: Endometrium samples were obtained at well defined phases of the cycle. Simultaneously, blood samples were collected for E2 and P assay. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including E- and P-cadherin and the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and the expression of estrogen receptor (ER) and P receptor (PR). RESULTS: E- and P-cadherin expression was demonstrated in all endometrium samples. Integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were detected in samples from all cycle phases, whereas integrin alpha 2 beta 1 was not detected in midluteal samples. The serum levels of E2 were 24.7 pg/mL (range: 10.9 to 35.4 pg/mL) in the early follicular phase and 190.7 pg/mL (range: 152.5 to 256.1 pg/mL) in the preovulatory phase (conversion factor to SI unit, 3.671). Serum P was 13.7 ng/mL (range: 10.3 to 16.7 ng/mL) in the midluteal phase and 6.4 ng/mL (range: 1.2 to 13.7 ng/mL) in the premenstrual phase (conversion factor to SI unit, 3.180). The portion of cells staining for ER and PR was at a maximum during the preovulatory phase, both for epithelial and stromal cells. CONCLUSIONS: E- and P-cadherin expression was detected in all samples and did not vary throughout the menstrual cycle. If their expression is involved functionally in the cyclic menstrual shedding, the loss of expression is limited to a short period of time. Of the beta 1 integrins, only alpha 2 beta 1 expression was modulated during the menstrual cycle and found to be absent in the midluteal phase. No relation was found between the expression of cell adhesion molecules and the expression of ER and PR. Because the cadherins and beta 1 integrins could be detected in late luteal phase endometrium, these cell adhesion molecules could be involved in the attachment of endometrial fragments to the peritoneal lining as a result of retrograde menstruation. The potential function in the pathogenesis of endometriosis remains to be elucidated. PMID- 7538478 TI - [Liver metastases from colorectal adenocarcinoma: does systemic chemotherapy have any value?]. PMID- 7538477 TI - Regulation of integrin alpha 5 beta 1 affinity during myogenic differentiation. AB - The antigen recognized by U1 alpha, a monoclonal antibody to the alpha chain of a chicken integrin fibronectin receptor, was identified as alpha 5. It identifies the same polypeptide as antisera raised to a sequence from the alpha 5 cytoplasmic domain. The U1 alpha antibody has the unusual functional property for alpha chain antibodies of enhancing the binding of alpha 5 beta 1 for its ligand fibronectin. U1 alpha was used to examine the function of alpha 5 beta 1 during myogenic differentiation. As myogenic cells differentiated from replicating myoblasts to bipolar myocytes there was a decrease in their adhesion to the substrate caused by inactivation of alpha 5 beta 1, which could be reversed by treatment of the cells with U1 alpha. The U1 alpha induced increased adhesion to fibronectin but did not inhibit the differentiation process as measured by formation of myotubes. However, U1 alpha did interfere with both cell migration and morphogenesis of myotubes. The resulting myotubes were smaller, more branched, and showed less regular alignment of nuclei. The results suggest that the ability of the cell to regulate alpha 5 beta 1 affinity is critical to myogenic morphogenesis. PMID- 7538479 TI - A rapid permeabilization procedure for accurate quantitative determination of beta-galactosidase activity in yeast cells. AB - A procedure is described which allows the rapid permeabilization of yeast cells, Schizosaccharomyces pombe and Saccharomyces cerevisiae, for quantitative in situ assays of beta-galactosidase activity. Yeast cells are permeabilized by incubation in buffer containing 0.2% of the detergent sodium lauroyl sarcosinate without any need for washing or vortexing. This procedure is equally applicable to fresh and frozen samples. It is compared to earlier reported methods and found to be superior by being more accurate and less time-consuming. PMID- 7538480 TI - DNA base modifications and antioxidant enzyme activities in human benign prostatic hyperplasia. AB - The authors have studied DNA base damage and activities of antioxidant enzymes in human benign prostatic hyperplasia (BPH) tissues and surrounding disease-free tissues removed from prostate glands of 15 patients. In these tissues, endogenous levels of various typical hydroxyl radical-induced products of DNA bases and activities of catalase and superoxide dismutase were measured. The majority of patients had higher levels of DNA base lesions and lower activities of enzymes in BPH tissues than in normal prostate tissues. When activities of both enzymes were lower in BPH tissues than in normal tissues, the increases in the amounts of DNA base lesions over control levels were most prominent. In the case of similar enzyme activities in both BPH and normal tissues, no changes in levels of DNA base lesions were observed. These results suggest a possible association between antioxidant enzyme activities and levels of DNA base lesions in BPH tissues. Some of the identified DNA lesions are known to be premutagenic and may play a role in carcinogenesis. Although a possible link between BPH and prostate cancer is controversial, BPH patients with both decreased antioxidant enzyme activities and increased levels of DNA lesions may be at risk of developing prostate cancer. PMID- 7538481 TI - Simple detection of the inter-alpha-trypsin-inhibitor (ITI) polymorphism by isoelectric focusing with direct immunofixation. AB - The inter-alpha-trypsin inhibitor (ITI) polymorphism was analysed in a German population, using polyacrylamide gel isoelectric focusing with subsequent direct immunofixation with monospecific ITI antisera. Gene frequencies of ITI*1, ITI*2, ITI*3, and ITI*4 were calculated to be 0.6150, 0.3753, 0.0078 and 0.0019, respectively. In our study the allele ITI*4 is described for the first time in a German population. PMID- 7538482 TI - Novel leukemic lymphoma with probable derivation from immature stage of natural killer (NK) lineage in an aged patient. AB - A 66-year-old male patient was admitted with dyspnea; physical examination revealed petechiae and systemic lymphadenopathy. Laboratory findings showed leukemia. The blasts in the peripheral blood were negative for cytochemical myeloperoxidase, and had condensed nuclear chromatin with a nucleolus. The histological diagnosis of the biopsied neck lymph node was lymphoblastic lymphoma. The leukemia cells expressed CD2, CD6, CD7, CD13low, CD56, beta chain of IL-2 receptorlow (IL-2R beta), and HLA-DR antigens, but not other pan-T (CD5, CD3, CD4, and CD8); pan-B (CD10, CD19, CD20, and CD24); natural killer (NK) (CD16, CD57); or myeloid (CD33) antigens. Electronmicroscopy revealed convoluted nuclei with conspicuous nucleoli and peripherally condensed heterochromatin. Membrane-bound granules containing an electron dense matrix were observed in the cytoplasm, indicating the NK cell nature of the neoplastic cells. While terminal deoxynucleotidyl transferase (TdT) and cytoplasmic CD3 were not detected by immunofluorescence on fixed smears, Northern blot analysis revealed the gene expression of CD3 epsilon, CD3 zeta, and TdT. Gene rearrangement analysis revealed that the beta, gamma, and delta chains of T-cell receptor (TCR) and immunoglobulin heavy chain (IgH) were of germline genotype. While the overall interpretation of the phenotype and genotype was difficult, the derivation of an immature stage of NK lineage was strongly suggested, based predominantly on the electronmicroscopic features. Despite initially successful chemotherapy, the patient died 14 months after initial presentation. PMID- 7538483 TI - Immunolocalization of interleukin-6 in salivary gland tumors. AB - Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses and acute phase reactions. It has demonstrated a growth factor function in several tumors, including those of salivary, plasma cell, and renal origin. We performed immunohistochemical staining for IL-6 localization on 57 salivary tumors. Reactivity was scored by intensity (0 to 4+) and percentage of cells staining, and the tumors were classified into three groups representing low (0 to 1+, 0% to 30%), moderate (2 to 3+, 31% to 75%), or high (> 3 to 4+, 76% to 100%) reactors. High reactivity was found in all primary pleomorphic adenomas (N = 10), five of eight recurrent pleomorphic adenomas, and all polymorphous low grade adenocarcinomas (N = 4). Moderate reactivity was observed in four of seven basal cell adenomas and three of five myoepitheliomas. Low reactivity characterized all acinic cell carcinomas (N = 3) and mucoepidermoid carcinomas (N = 3) as well as six of nine primary adenoid cystic carcinomas and all metastatic adenoid cystic carcinomas (N = 3). Carcinoma ex pleomorphic adenoma (N = 5) had three low and two moderate reactors. A pattern emerged in which the benign and low grade malignant tumors showed stronger reactivity than the metastatic or high grade malignant tumors. This suggests an inverse relationship between the presence of IL-6 and the biological aggressiveness of salivary gland tumors. The function of IL-6 in salivary gland neoplasia awaits further study and elucidation. PMID- 7538485 TI - Expression of muscle actins in diffuse mesotheliomas. AB - Ultrastructural examinations have shown myofibroblastoid differentiation in sarcomatoid/desmoplastic mesotheliomas, but immunohistochemical expression of muscle actins seldom has been documented. We examined 10 sarcomatoid, 12 epithelial, and five biphasic mesotheliomas immunohistochemically for the expression of muscle-specific actin (MSA) and smooth muscle actin (SMA) and compared it with that in 12 specimens of lung cancer. All of the sarcomatoid mesotheliomas were found to be positive for both MSA and SMA. The epithelial cells in nine epithelial and two biphasic mesotheliomas were positive for MSA, but SMA was only positive in one epithelial mesothelioma. Conversely, the lung cancers were negative for both MSA and SMA in the epithelial cells, except for one specimen that was weakly positive for MSA. The stromal cells in both the epithelial mesotheliomas and lung cancers were negative for cytokeratin but were positive for MSA and SMA, whereas the sarcomatoid and biphasic mesothelioma spindle cells were positive for all three antibodies. We concluded that sarcomatoid mesothelioma was positive for MSA and SMA, which is in support of its myofibroblastic differentiation, and that positivity for MSA in some epithelial mesotheliomas might be of diagnostic value in differentiation from lung cancers. PMID- 7538484 TI - Site-specific comparison of p53 immunostaining in squamous cell carcinomas. AB - The p53 tumor suppressor gene is believed to be the most commonly mutated gene in human cancer. p53 is thought to function as a negative regulator of the cell cycle, arresting cells in the G1 phase. This study examined the effects of different mutagenic environments on the incidence of p53 overexpression in squamous cell carcinomas (SCCs) from sun exposed and non-sun exposed squamous epithelium. An immunohistochemical analysis was undertaken in an attempt to assay SSC for p53 overexpression, an indirect measure of missense mutant p53. Positive nuclear staining for p53 was observed in 14 of 21 sun exposed SCCs, two of 19 vulvar/perianal SCCs, and 15 of 20 oral cavity SCCs. The number of positive anogenital tumors was low compared with that of both sun exposed (chi-squared, 1 df, P = .0004) and oral (chi-squared, 1 df, P < .0001) sites. It was concluded that p53 protein accumulation is common in sun-exposed cutaneous SCC and oral SCC compared with anogenital SCC, and thus it is hypothesized that the nature of the mutagenic environment in which SCC develops directly affects the incidence of immunohistochemically detectable p53-positive cells. PMID- 7538487 TI - Silicone-induced arthritis in rats and possible role for T cells. AB - Intra-articular injection of silicone into the ankle joint of the rat induced persistent arthritis. Histologically, at 24 h after silicone injection, there was marked edema of the synovial tissue containing many inflammatory cells including polymorphs, monocytes and macrophages. This was followed by marked proliferation of synovium with dense infiltration of mononuclear cells and destruction of cartilage. Immunohistological studies showed that a large number of CD5+ and alpha beta+ T cells infiltrated in synovial tissues in the chronic phase of joint inflammation. Treatment of rats with a monoclonal antibody against TCR alpha beta significantly suppressed the development of chronic, but not acute arthritis. Thus, T cells may play a role in silicone-induced chronic arthritis. PMID- 7538488 TI - Antibodies to Escherichia coli 06 porins cross-react with urinary pathogens. AB - Antibodies to partially purified E. coli 06 35-40 KDa porin trimers recognized the reactive epitopes in the intact porin surface molecule present in various wild-type, heterologous, urinary pathogens. The presence of lipopolysaccharide in the membrane did not shield the antibody binding sites. The reactivity was shown to be specific for porins since LPS-absorbed porin antisera reacted with porins on immunoblots and showed no reactivity with LPS. Additionally, the cross reactions were abolished by absorption of the porin antisera with E. coli 06 containing porin trimers. These data strengthen the rationale for exploring the enhancement of immunoprotection by monoclonal antibodies to specific immunoreactive antigens in the porin molecule. PMID- 7538486 TI - Variation outside variable segments of the major outer membrane protein distinguishes trachoma from urogenital isolates of the same serovar of Chlamydia trachomatis. AB - OBJECTIVES--Whereas serovars A, B, Ba and C of Chlamydia trachomatis are usually associated with trachoma, two of these serovars (Ba and C) are occasionally observed in urogenital infections. Variation in the gene encoding the major outer membrane protein (MOMP) was explored to distinguish urogenital from trachoma specimens of the same serovar. METHODS--A large portion of the MOMP gene was amplified by nested PCR directly from clinical samples from trachoma or urogenital infection and the serovar of the infecting C trachomatis was determined by restriction fragment length polymorphism (RFLP). Amplified DNA from trachoma serovars B, Ba and C and from urogenital serovars Ba, C, D and E was sequenced by the dideoxy chain termination method. RESULTS--While almost identical in variable segment (VS)I, three urogenital Ba samples differed from all trachoma B and Ba samples at eight nucleotides including two sites which changed amino acids in the constant region upstream of VSI. An identical sequence in this region was observed for the reference urogenital D serovar. Variation in this same region upstream of VSI also distinguished 40% of serovar D samples from prototype D including three that were sequenced. Two urogenital C differed from trachoma C samples at four sites that changed the MOMP amino acid sequence including two changes in the constant region between VSII and III and single changes in VSII and III. On the basis of these sequence determinations, RFLP was predicted which allowed extension of these observations to 20 other urogenital Ba, 12 trachoma B or Ba, seven variant D, 12 D, four urogenital C and three trachoma C samples without further sequencing. CONCLUSION--Urogenital Ba and C samples have VSI or II and III sequences identical or very similar to trachoma strains of the same serovar, but resemble more closely other serovars in the constant regions. Urogenital serovar D samples can also be divided into two genotypes on the basis of sequence differences in the constant region preceding VSI. PMID- 7538490 TI - Isotype-associated recognition of allergen epitopes and its modulation by antigen dose. AB - The existence of competing and blocking IgG antibodies and their interrelationship with IgE antibodies was investigated in the IgE immune response of CBA/J mice to bee venom phospholipase A2 (PLA2) and keyhole limpet haemocyanin (KLH). Minute doses of antigen induced high titres of both IgE and IgG antibodies, whereas large doses elicited high IgG responses but only weak IgE antibody titres. Immunization with minute doses of antigen led to the production of antibodies with class-associated epitope specificities (designated epitopes G for IgG and E for IgE antibodies), and therefore no competition was observed. For IgG antibody production, G epitopes were immunodominant and IgG antibodies directed to E epitopes were produced only after immunization with an increased immunogen dose. Under this condition IgG antibodies recognizing the E epitopes acted as blocking antibodies competing with IgE antibodies for binding to E epitopes, the immunodominant epitopes for the IgE response. The data demonstrate the existance of distinct immunodominant antigenic determinants for IgE and IgG antibodies on PLA2. PMID- 7538489 TI - Priming of human neutrophils by tumour necrosis factor-alpha and substance P is associated with tyrosine phosphorylation. AB - The mechanisms involved in neutrophil 'priming' are unknown. 'Priming' by substance P and tumour necrosis factor-alpha (TNF-alpha) occurred without effecting cytosolic-free Ca2+ signalling and was independent of actin polymerization. We demonstrate here that these two primers, which act on different receptor classes, both cause tyrosine phosphorylation of a number of protein substrates including a prominent 74,000 MW protein. This protein was not recognized by anti-c-raf antibodies. The concentration relationship and time course of tyrosine phosphorylation were consistent with a role in mediating priming. Pretreatment with genistein both inhibited tyrosine phosphorylation and abolished the priming by either substance P or TNF-alpha. PMID- 7538491 TI - Recognition of Mycobacterium leprae recombinant 18,000 MW epitopes by IgG subclasses in leprosy. AB - IgG subclasses are known to be differentially regulated by cytokines (elaborated by activated T cells), which act as growth factors and immunoglobulin switch factors on B cells. In leprosy, we have previously shown that IgG subclass antibodies to a purified recombinant antigen of Mycobacterium leprae (18,000 MW) are restricted to IgG1 and IgG3 across the disease spectrum. The only significant difference observed was that lepromatous patients with low to undetectable T-cell responses showed a strong correlation between IgG1 and IgG3 (P < 0.001) antibodies while tuberculoid patients who showed strong T-cell responses did not show such a correlation. To examine if these differences were related to T-cell mediated class switching in tuberculoid leprosy patients, we have studied epitope recognition by IgG1 and IgG3 using a panel of synthetic peptides spanning the 18,000 MW molecule in an enzyme-linked immunosorbent assay (ELISA). In lepromatous patients there was little similarity in peptide recognition by IgG1 and IgG3, with IgG1 recognition being restricted to a single dominant carboxy terminal peptide while the IgG3 antibodies recognized a diverse set of peptides in the N-terminal half of the 18,000 MW molecule. In tuberculoid patients both IgG1 and IgG3 antibody showed recognition of similar peptides in the N-terminal half of the 18,000 MW molecule. Our results therefore support the hypothesis that immunoglobulin class switching is occurring in tuberculoid but not in lepromatous patients. PMID- 7538493 TI - Biological therapies in bone marrow transplantation. AB - Biological materials are increasingly being utilized in bone marrow transplants. Alpha interferon is given post autotransplants, in an attempt to eradicate residual cancer cells. Colony stimulating factors (granulocyte macrophage colony stimulating factor: GM-CSF and granulocyte colony stimulating factor: G-CSF) are widely used after bone marrow transplants to accelerate bone marrow recovery. Their use has been associated with fewer infections, shorter hospital stay and lower procedural costs. Both these factors benefit some patients with graft failure as well. The recent demonstration that both GM-CSF and G-CSF can mobilize large numbers of haematopoietic stem cells into the peripheral blood has resulted in the widespread use of peripheral blood stem cell transplantation as an alternative to autologous bone marrow transplantation. Identification of tumour cells in the mobilized peripheral blood stem cells, however, has raised some concern. PMID- 7538492 TI - Inhibitory effect of cyclosporin A and FK506 on nitric oxide production by cultured macrophages. Evidence of a direct effect on nitric oxide synthase activity. AB - Casein-elicited peritoneal macrophages from mice were cultured either alone or with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), and the effect of cyclosporin A (CsA) and FK506 on NO2- production (due technical difficulties NO2- was taken as the index for NO) was analysed. We observed an inhibitory effect of CsA and FK506 on NO2- production. The IC50 for NO2- production by casein-elicited macrophages was 0.1 microgram/ml for CsA and 0.3 microgram/ml FK506. The effect of both drugs was dose-dependent and was more clear in non-stimulated macrophages. The presence of IFN-gamma and LPS in the culture increased NO2- production by casein-elicited macrophages and partially eliminated the inhibition exerted by CsA and FK506. Both drugs acted directly on the nitric oxide synthase (NOS), since CsA and FK506 reduced by 35% and by 17%, respectively, NOS activity in the crude cytosolic fraction. However, CsA and FK506 did not alter 14CO2 production from [1-14C]glucose, suggesting that the pentose monophosphate pathway activity was not modified. These data add new insight into the interpretation of the immunosuppressive properties of both drugs. PMID- 7538494 TI - Prevalence of anti-HCV antibodies in western India. AB - Nearly 2000 serum samples collected from different risk groups from Pune and Bombay metropolitan areas were tested for antibodies to hepatitis C virus (anti HCV) by Recombinant Immunoblot Assay-3 (RIBA-3). Patients undergoing haemodialysis showed 24.5 per cent seropositivity whereas 5.7 and 5.3 per cent of multiply transfused patients (>2 units) and chronic liver disease patients respectively were anti-HCV positive. Leprosy patients had almost 0.7 per cent seropositivity. In other risk groups the positivity rate was nil. In normal population only one out of 830 persons had anti-HCV antibodies. It is therefore apparent that the prevalence of hepatitis C virus (HCV) in western India is not high. However, special care needs to be taken for dialysis patients. As none of the 430 pregnant women and 86 children below the age of 5 yr were anti-HCV positive, vertical mode of HCV transmission seems to be negligible. PMID- 7538495 TI - The role of mast cells in treatment of scabies. AB - OBJECTIVE: The purpose of this study was to recognize the role of mast cells in the pathogenesis of scabies. METHODS: One hundred and fifty patients and 10 controls were included in the study. Group 1 included 20 patients without previous treatment. In group 2, 80 patients were treated with antiscabietic drugs. Group 3 had 50 patients who received an antiscabietic drug followed by 3 days of crotamiton. Diurnal and nocturnal skin biopsies were taken from group 1. In groups 2 and 3, the biopsies were taken after 2 weeks of treatment. Sections were cut and stained by hematoxylin and eosin and Giemsa stains. RESULTS: Mast cells were increased in diurnal and nocturnal biopsies. Evident degranulation of mast cells was detected in nocturnal biopsies. The mast cell number decreased to half its pretreatment number in patients treated with antiscabietic drugs and to its normal number in patients treated with antiscabietic drugs followed by 3 days with crotamiton. CONCLUSION: The number of mast cells are increased in scabietic lesions. This plays a role in the pathogenesis of the clinical and histologic picture of scabies. We recommend that an antiscabietic drug should be followed by 3 days of crotamiton in the treatment of scabies. PMID- 7538496 TI - Reticulate pigmentation due to 5-fluorouracil. PMID- 7538498 TI - Prostate-specific antigen and radiation therapy for clinically localized prostate cancer. AB - PURPOSE: This study was undertaken to: (a) define the prognostic significance of pretreatment serum prostate-specific antigen (PSA) levels in localized prostate cancer treated with radiation; (b) define the prognostic usefulness of postradiation PSA levels; (c) evaluate the outcome of radiation using PSA as an endpoint. METHODS AND MATERIALS: Disease outcome in 707 patients with Stages T1 (205 men), T2 (256 men), T3 (239 men), and T4 (7 men), receiving definitive external radiation as sole therapy, was evaluated using univariate and multivariate techniques. RESULTS: At a mean follow-up of 31 months, 157 patients (22%) developed relapse or a rising PSA. Multivariate analysis revealed pretreatment PSA level to be the most significant prognostic factor, with lesser though significant contributions due to Gleason grade (2-6 vs. 7-10) and transurethral resection in T3/T4 disease. The following four prognostic groupings were defined: group I, PSA < or = 4 ng/ml, any grade; group II, 4 < PSA < or = 20, grades 2-6; group III, 4 < PSA < or = 20, grades 7-10; group IV, PSA > 20, any grade. Five-year actuarial relapse rates in these groups were: I, 12%; II, 34%; III, 40%; and IV, 81%. Posttreatment nadir PSA was an independent determinant of outcome and only patients with nadir values < 1 ng/ml fared well (5-year relapse rate 20%). Using rising PSA as an endpoint the 461 patients with T1/T2 disease had an actuarial freedom from disease rate of 70% at 5 years, which appeared to plateau, suggesting that many were cured. No plateau was evident for T3/T4 disease. CONCLUSION: Pretreatment serum PSA is the single most important predictor of disease outcome after radiation for local prostate cancer. Tumor grade has a lesser though significant prognostic role. Postirradiation nadir PSA value during the first year is a sensitive indicator of response to treatment. Only nadir values < 1 ng/ml are associated with a favorable outlook. A significant fraction of men with T1/T2 disease may be cured with radiation. There was no evidence for a cured fraction among patients with T3/T4 disease. PMID- 7538497 TI - A human hybridoma monoclonal antibody (TrJ11) recognizing a new HLA-DR epitope shared by DR4, DR8, DR11, and DRB1*1303. AB - The cytotoxic IgM lambda human hybridoma mAb TrJ11 reacts with lymphoblastoid B cell lines expressing DR4, DR8, DR11, and DRB1*1303. However, TrJ11 was monospecific when normal B cells freshly isolated from blood served as targets in that it only killed HLA-DR4-positive cells. Thus, of 235 HLA-typed persons TrJ11 was strongly cytotoxic for normal B cells of all 90 DR4-positive individuals, but it did not react with B cells from any of the 145 DR4-negative donors. Hence, mAb TrJ11 proved to be suitable for routine DR4 typing. The specific binding of TrJ11 to a DR4-positive cell line was profoundly blocked by the mouse HLA-DR beta chain specific monomorphic mAb TAL 14.1, indicating that the epitope recognized by TrJ11 is located in the DR beta chain. The possibility that amino acids located in the floor of the peptide-binding site are critical for the TrJ11 epitope is discussed. PMID- 7538499 TI - Prostate-specific antigen for pretreatment prediction and posttreatment evaluation of outcome after definitive irradiation for prostate cancer. AB - PURPOSE: This study was undertaken to assess the predictive value of pretreatment prostate-specific antigen (PSA) and the difference between clinical and PSA disease-free status in patients with long-term follow-up after irradiation for prostatic carcinoma. Comparison of the distribution of prognostic factors between surgical and radiation series was also made. METHODS AND MATERIALS: From 1975 1989, 652 patients with clinical Stage A2-C prostatic adenocarcinoma were definitively irradiated using external beam therapy. One hundred and fifty patients with banked serum and up to 14 years follow-up have pretreatment PSA levels and 355 patients with up to 17 years follow-up have posttreatment values. Treatment failure was analyzed by tumor stage, grade, and four pretreatment PSA categories. Disease-progression was evaluated by clinical and biochemical (PSA) endpoints. Prognostic factors were compared to two surgical series. RESULTS: A significant difference was seen in clinical and PSA disease-free (PSA < or = 4.0 ng/ml) status based on tumor grade, stage, and pretreatment PSA category. Although the expected clinical outcome has been well-documented previously, results based on posttreatment PSA levels show 5-year disease-free survivals reduced by 10-16% and 10-year survivals lessened by 15-39% depending upon the particular tumor grade and stage. The earlier stage, lower grade tumors showed the largest difference between clinical and biochemical recurrence rates at the longest interval from treatment. Even more notable were the differences in the clinical and PSA disease-free rates based on the pretreatment PSA level. Comparing the irradiated patients to two surgical series showed that the former had a larger percentage of more advanced stage tumors with more unfavorable PSA levels as compared to prostatectomy patients. CONCLUSION: With long-term follow up, the pretreatment PSA level continues to be a powerful predictor of clinical and biochemical outcome in patients irradiated for apparently localized prostate cancer. Differences between clinical and PSA outcome can be considerable, but oftentimes clinically insignificant. The distribution of prognostic factors between radiation and prostatectomy series seems to favor the latter. PMID- 7538501 TI - Intraluminal brachytherapy in the treatment of pancreas and bile duct carcinoma. AB - PURPOSE: A new method of palliation of malignant obstructive jaundice is presented. METHODS AND MATERIALS: Twelve patients with carcinoma of the extrahepatic bile ducts (EHBD-five patients) or pancreatic head (PH-seven patients) received radiation therapy between 1988 and 1991. Percutaneous transhepatic biliary drainage was performed in four EHBD patients and an endoprosthesis was placed during endoscopic retrograde cholangiopancreatography (ERCP) in the other eight patients. All 12 received intraluminal brachytherapy (ILBT): 20-50 Gy calculated at 1 cm from the Iridium-192 (192Ir) wire. In four PH patients the source was placed in the duct of Wirsung; in the other eight patients ILBT was performed via the common bile duct. Five of the seven PH patients and one of the five EHBD patients received External Beam Radiation Therapy (EBRT): 26-50 Gy, alone or with concomitant 5-Fluorouracil (5-FU). RESULTS: Cholangitis occurred in six patients. Three PH patients treated with EBRT+ILBT developed gastrointestinal toxicities. With a minimum follow-up of 18 months, median survival times were 14 months (EHBD) and 11.5 months (PH); one of the seven PH patients is alive (29 months) and two of the EHBD patients are alive (18 and 43 months). All patients had satisfactory control of jaundice. CONCLUSIONS: The results in the EHBD patients suggest that the addition of ILBT after biliary drainage prolongs survival. Further experience is necessary to determine whether ILBT in the common bile duct and/or in the duct of Wirsung may be, in PH patients, an alternative boost technique to Interstitial Brachy-therapy (IBT) or Intraoperative Electron Beam Radiation Therapy (IOEBRT). PMID- 7538500 TI - The efficacy of postprostatectomy radiotherapy in patients with an isolated elevation of serum prostate-specific antigen. AB - PURPOSE: To determine the efficacy of radiotherapy (RT) in patients with an isolated elevation of prostate specific antigen (PSA) after radical prostatectomy (RP). METHODS AND MATERIALS: Between November 1987 and May 1993, 53 patients with adenocarcinoma of the prostate were referred for pelvic RT for an elevated PSA after RP. No patient had clinically or radiographically apparent local or distant disease, nor had an undergone prior androgen ablation. Patients received a median dose of 61.2 Gy to the prostatic bed. An undetectable PSA was required to be considered disease free (NED). Univariate and multivariate analyses were performed to identify factors predictive of becoming disease free after RT. RESULTS: The median follow-up was 15 months. Of the 53 patients, 16 (30%) became NED after RT and 15 (28%) had a declining (n = 11) or stable (n = 4) PSA at last evaluation. The median time after RT to achieve an undetectable PSA was 9.3 months. At 12 and 24 months, the actuarial disease-free survival was 30 and 23%, respectively; actuarial progression-free survival was 71 and 26%, respectively. By univariate analysis, the last PSA level before RT (i.e., the pre-RT PSA) and an undetectable PSA after RP were significant predictors of becoming NED (p = 0.0001 and 0.04, respectively). However, on multivariate analysis, only the pre RT PSA remained significant (p = 0.01). The mean pre-RT PSA differed significantly between patients who became NED after RT and those who did not (1.5 +/- 0.2 ng/ml vs. 7.6 +/- 1.6 ng/ml, respectively; p = 0.018). Fourteen of 27 (52%) patients with pre-RT PSA levels < or = 2.5 ng/ml became NED, vs. only 2 of 26 (8%) patients with higher levels. There were no severe acute or late sequelae of RT. CONCLUSION: Prostatic-bed RT for an elevated serum PSA after RP is most effective in patients with a pre-RT PSA < or = 2.5 ng/ml. Patients with significantly higher PSA values are unlikely to benefit from RT, possibly due to the presence of occult distant metastases. The optimal therapy for this latter group remains to be determined. PMID- 7538502 TI - A method for determining a prostate-specific antigen cure after radiation therapy for clinically localized prostate cancer. AB - PURPOSE: A method that allows the determination of a prostate-specific antigen (PSA) cure after definitive management for prostate carcinoma with radiation therapy is presented and tested. METHODS AND MATERIALS: The method involves a calculation of the patient's theoretical baseline PSA prior to the development of prostate cancer by using three serial rising PSA determinations obtained prior to the institution of therapy. The rate of rise of the PSA prior to therapy and the rate of decline of the PSA posttherapy are calculated, using an exponential model. Two criteria must be satisfied to define a PSA cure. First, the PSA nadir after treatment should be less than the calculated theoretical baseline PSA. Second, the rate of decline of PSA posttreatment should be greater than the rate of rise of the PSA prior to treatment. RESULTS: Applying these two criteria to the patient data base (n = 16) at a median follow-up of 19 months enabled the accurate prediction of 6 out of 6 (100%) of patients with documented PSA failure and 7 out of 10 (70%) of patients without PSA failure. Therefore, despite short follow-up, all six patients with PSA failure were predicted. Further follow-up is needed to ascertain if the seven patients predicted to be cured will remain PSA failure free and if the three patients currently without PSA failure in whom the model predicts failure, will subsequently fail. CONCLUSION: Therefore, using each patient's PSA history as the natural control may eliminate the error that is introduced with defining a PSA cure by using a single value for the PSA nadir at a specified time after radiation therapy. PMID- 7538503 TI - Comments on the treatment of prostate cancer by conventional radiation therapy: an analysis of long-term outcome--regarding Zietman et al., IJROBP 32:287-292; 1995. PMID- 7538504 TI - Are only low pretreatment PSA patients curable? Regarding Kuban et al., IJROBP 32:307-316; 1995. PMID- 7538505 TI - Therapeutic effect of argon plasma coagulation on small malignant gastrointestinal tumors. AB - In endoscopy, argon plasma coagulation (APC) is a new principle of non-contact electrocoagulation and has proved to be a sufficient tool for palliative endoscopic treatment of gastrointestinal neoplasms, predominantly of the oesophagus and colorectum. In a study of 67 patients suffering from histologically confirmed and endosonographic T1-staged tumours of the gastrointestinum, 10 patients were selected for endoscopic APC treatment because of the impossibility of surgical therapy. Although the application was primarily of a palliative nature, in 9 of 10 cases of minor neoplasms, no further tumour could be detected in biopsies during the observation period (9.45 +/- 2.8 months). One patient was not cured locally. In none of the patients was any serious complication noticed during the outpatient follow-up. The effective results and lack of severe complications suggest this technique as an alternative therapy in selected patients with smaller gastrointestinal tumours. PMID- 7538506 TI - CM101, a polysaccharide antitumor agent, does not inhibit wound healing in murine models. AB - CM101 (previously called GBS toxin), a new anticancer polysaccharide that induces inflammatory reactions in neovasculature of tumors, does not cause similar reactions in neovasculature of healing wounds. It appears that treatment with CM101 will not interfere with normal wound healing in cancer patients. PMID- 7538507 TI - Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. AB - Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis. PMID- 7538508 TI - Specific detection of Brucella DNA by PCR. AB - A PCR assay with primers derived from the 16S rRNA sequence of Brucella abortus was developed. Nine different combinations between six primers were tested. One pair of primers, which amplified a 905-bp fragment, was selected. As little as 80 fg of Brucella DNA was detected by this method. DNAs from all of the representative strains of the species and biovars of Brucella and from 23 different Brucella isolates were analyzed and yielded exclusively the 905-bp fragment. No amplification was detected with DNAs from 10 strains phylogenetically related to Brucella spp., 5 gram-negative bacteria showing serological cross-reactions with Brucella spp., and 36 different clinical isolates of non-Brucella species. Only Ochrobactrum anthropi biotype D yielded a PCR product of 905 bp, suggesting a closer relationship between Brucella spp. and O. anthropi biotype D. The specificity and high sensitivity of the PCR assay may provide a valuable tool for the diagnosis of brucellosis. PMID- 7538509 TI - Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. AB - Shiga-like toxin (verotoxin)-producing strains of Escherichia coli (SLTEC) originating from healthy cattle, sheep, goats, pigs, cats, and dogs were investigated for properties which are related to virulence of E. coli for humans. The slt-II (Shiga-like toxin II) and slt-IIc genes were frequent in SLTEC from healthy cattle and dogs but were rarely found in SLTEC from other animals. The slt-IIe gene was detected only in porcine SLTEC. SLTEC from goats and SLTEC from sheep were found to carry different SLT-II determinants which were not further characterized genetically. Sixty (28.8%) of 208 SLTEC from healthy animals showed diffuse adherence to HEp-2 cells. However, none of the strains was positive for genes specific for the local adherence (eaf), diffuse adherence (daa), or enteroaggregative (EAggEC) E. coli type. Only 3 (1.4%) of the 208 SLTEC were positive for attaching and effacing E. coli (eae) sequences. The enterohemolytic phenotype was present in 128 of the 208 SLTEC. Almost all enterohemolytic animal SLTEC were found to carry DNA sequences specific for the plasmid-encoded enterohemorrhagic E. coli hemolysin of E. coli O157. Bacteriophage-associated enterohemolysin (Ehly1 and Ehly2)-specific sequences were detected only in 14.4% of the 208 SLTEC and were linked with certain serotypes. The SLTEC from healthy animals constitute a very heterogeneous group of E. coli, and many of these strains appeared to be specific for their hosts. The absence of eae sequences in most animal SLTEC could indicate that these strains are less virulent for humans than the classical eae-positive enterohemorrhagic E. coli types. PMID- 7538510 TI - Detection of Anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay. AB - A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on a major surface protein 5 (MSP5) B-cell epitope conserved among Anaplasma species was used to detect goats infected with Anaplasma ovis. We examined strains of A. ovis isolated from goats in Kenya and demonstrated that MSP5 and the target B cell epitope, bound by monoclonal antibody ANAF16C1, were conserved. Sera from 149 goats in four regions of Kenya and from 302 goats in six U.S. states were tested for the presence of epitope-specific antibodies with the MSP5 competitive inhibition ELISA. Evidence that the assay can be used to detect A. ovis-infected goats includes the following: (i) 53 goats raised in confinement with arthropod control were all seronegative; (ii) six goats experimentally infected with A. ovis seroconverted at the same time that they developed detectable rickettsemia; (iii) seroconverted goats remained seropositive, consistent with the persistence of A. ovis in goats and the presence of anti-MSP5 antibody in cattle persistently infected with Anaplasma marginale; and (iv) 119 of 127 known A. ovis-infected goats in Kenya were seropositive. A. ovis infection, as determined serologically and by demonstration of infected erythrocytes, in goats from the four regions in Kenya was highly prevalent. In contrast, despite the presence of A. ovis and competent arthropod vectors in the United States, the prevalence of infection appeared to be very low. The high prevalence in Kenya and the occurrence of anemia in persistently infected goats may be impediments to current efforts to increase milk yields on small farms. PMID- 7538511 TI - A chemically defined liquid medium that supports primary isolation of Rochalimaea (Bartonella) henselae from blood and tissue specimens. AB - Rochalimaea (Bartonella) henselae is a fastidious, slowly growing, gram-negative bacillus that is an etiologic agent of bacillary angiomatosis, cat scratch disease, and related syndromes. Accumulation of direct microbiologic evidence of the relationship between the organism and the syndromes compatible with cat scratch disease has been hindered by the difficulties in the primary isolation of the organism from infected tissue specimens. A chemically defined liquid medium was developed to support the growth of Rochalimaea species to facilitate study of the organism. This medium was also used successfully to isolate R. henselae from clinical specimens from infected patients and a domestic cat. Recovery of R. henselae in this was more successful than when recovery was attempted on solid agar. This cell-free, extract-free, defined medium additionally supported the growth of Rochalimaea quintana and Afipia felis. PMID- 7538512 TI - Techniques for controlling variability in gram staining of obligate anaerobes. AB - Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes. PMID- 7538513 TI - Levels of hepatitis C virus (HCV) RNA in serum and their relationship to levels of immunoglobulin M and G antibodies against HCV core protein. AB - The presence and levels of hepatitis C virus (HCV) RNA and immunoglobulin M (IgM) and IgG antibodies against the virus core protein were determined in 449 serum specimens. Despite the fact that a relationship between the presence, but not the levels, of HCV RNA and HCV IgM was observed, the significance of HCV core IgM assays seems limited. PMID- 7538515 TI - Preservation of topography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats. AB - In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV-VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex. PMID- 7538514 TI - Steady-state pharmacokinetics of hydromorphone and hydromorphone-3-glucuronide in cancer patients after immediate and controlled-release hydromorphone. AB - Although the pharmacokinetics of oral hydromorphone has been evaluated in healthy volunteers after small single oral doses, data are not available regarding the disposition of hydromorphone and its principal metabolite, hydromorphone-3 glucuronide (H3G), at steady-state and after large oral doses. The authors studied the pharmacokinetics of hydromorphone and H3G after oral administration of an immediate-release (IR) and controlled-release (CR) formulation of hydromorphone at a daily dose of 48 +/- 11 mg (range 6-216 mg) in a randomized, double-blind, steady-state, two-way crossover evaluation in 18 patients with chronic cancer pain. Controlled-release hydromorphone demonstrated equivalent bioavailability and acceptable CR characteristics, when compared with IR hydromorphone (CR vs. IR: AUC0-12 123.10 +/- 20.38 vs. 118.98 +/- 20.92 ng.hr.mL 1, P = NS, Cmax 17.76 +/- 3.07 vs. 19.70 +/- 4.04 ng.mL-1, P = NS, Cmin 6.04 +/- 1.01 vs. 5.28 +/- 1.000 ng.mL-1, P = NS, and Tmax 4.78 +/- 0.78 vs. 1.47 +/- 0.22 hr, P = 0.0008). A significant linear relationship existed between hydromorphone dose and hydromorphone AUC (r = 0.8315, P = 0.0001) and between hydromorphone AUC and H3G AUC (r = 0.8048, P = 0.0001) over a wide dose range. The steady-state molar ratio of H3G to hydromorphone was 27:1. The authors conclude that CR hydromorphone provides a pharmacokinetic profile consistent with 12 hourly dosing and that at steady state, oral hydromorphone is extensively metabolized to H3G, although the pharmacologic activity of this metabolite remains unknown. PMID- 7538516 TI - Thalamic distribution of projection neurons to the primary motor cortex relative to afferent terminal fields from the globus pallidus in the macaque monkey. AB - To examine quantitatively the pathway from the internal segment of the globus pallidus to the primary motor cortex through the thalamus, we compared the distribution of thalamocortical neurons projecting to the motor cortex with the distribution of afferent terminal fields from the pallidum in the ventrolateral nuclear group of the thalamus in four Japanese monkeys by using the anterograde and retrograde double-labeling method. In each monkey, different fluorescent retrograde tracers (Fast Blue and Diamidino Yellow) were injected separately into the distal and proximal forelimb areas of the primary motor cortex after physiological mapping with intracortical microstimulation. In the same individual monkeys, an anterograde tracer, wheatgerm agglutinin conjugated to horseradish peroxidase, was injected into the internal segment of the globus pallidus after the forelimb part was identified physiologically. A small group of projection neurons to the distal and proximal representations of the motor cortex were found in the terminal fields from the pallidum, but a majority of the projection neurons were distributed outside the terminal area in the thalamus. These results confirm the existence of the pathway from the pallidum through the thalamus to the primary motor cortex, but also indicate that the primary motor cortex receives its major thalamic inputs from outside of the pallidal projection area, and that the pallidum sends its major outputs to nonprimary motor areas through the thalamus. PMID- 7538517 TI - Role of radiotherapy in the treatment of epidemic Kaposi's sarcoma: experience with sixty-five cases. AB - BACKGROUND: Treatment of Kaposi's sarcoma (KS) associated with HIV infection should improve often disfiguring lesions, with an acceptable cosmetic outcome; relieve associated signs and symptoms (pain and edema); and have no adverse effects on the patient's already impaired immune status. OBJECTIVE: Our purpose was to determine the role of x-ray therapy in the treatment of KS. METHODS: Contact x-ray therapy and half-deep x-ray therapy were used to treat 594 lesions in 65 patients with KS, who were observed for 1 to 43 months (mean, 9 months). RESULTS: Complete remission was achieved with pigmentation in 405 lesions (68.3%), with good cosmetic results in 105 (17.7%), and with hypopigmentation in three (0.5%). In 80 lesions (13.5%) only size reduction or pain palliation were achieved. Fourteen lesions (2.4%) relapsed 2 to 9 months after treatment. CONCLUSION: X-ray therapy is well tolerated and meets the specified requirements for the treatment of KS. PMID- 7538518 TI - Major differences in specificity among naturally occurring human IgG-subclass anti-IgE autoantibodies. AB - BACKGROUND: The specificity of naturally occurring IgG-subclass anti-IgE autoantibodies (a-IgE Ab) was studied by ELISA inhibition assay with a highly purified IgE-DES myeloma protein as a solid-phase antigen. Because the IgG isotypes differ in effector functions, detailed specificity studies of IgG subclass anti-IgE antibodies might clarify their putative role. METHODS: Selected sera containing a high concentration of a single IgG a-IgE Ab subclass were allowed to react with purified immunoglobulins of all five classes including five different IgE myeloma proteins and papain-derived Fab epsilon and Fc epsilon fragments of IgE-DES. RESULTS: The inhibition results indicated that IgG1, IgG2, and IgG3 a-IgE Ab reacted in a low-affinity reaction with IgE myeloma protein restricted determinants because only IgE-DES was inhibitory. Moreover, the reacting epitopes were heat resistant (2 hours, 56 degrees C) and localized in the Fab epsilon-DES fragment. In sharp contrast, IgG4 a-IgE Ab reacted with high affinity to all five IgE myeloma proteins involving heat-susceptible epitopes confined to the Fc epsilon fragment. CONCLUSIONS: It seems that a majority of IgG a-IgE Ab have a specificity for nonisotypic myeloma-restricted determinants whereas IgG4 a-IgE Ab are mainly isotype specific. These findings ought to be taken into account in the consideration of physiologic implications of IgG a-IgE Ab and in the interpretation of previous investigations in which intact IgE myeloma proteins have been used to detect IgG a-IgE Ab. PMID- 7538519 TI - An overview of current pharmacotherapy in perennial rhinitis. AB - BACKGROUND: Individuals with rhinitis experience significant morbidity due to their disease. Research and clinical care to reduce this suffering are important. OBJECTIVE: To review the pharmacological agents that have been developed for the treatment of perennial rhinitis. METHOD: Literature review of human studies. RESULTS: Medication classes include antihistamines, decongestants, anticholinergics, cromolyn sodium, and corticosteroids. These vary in regard to their pharmacology, efficacy, and adverse effects. Compliance and cost issues are also critical components of the therapeutic regimen. CONCLUSION: Carefully chosen pharmacotherapy based on an understanding of the pathophysiology of the disease, knowledge of the potential of medications, and commitment to an ongoing patient/physician education and monitoring program can lead to improved well being for individuals with perennial rhinitis. PMID- 7538520 TI - Use of ipratropium bromide nasal spray in chronic treatment of nonallergic perennial rhinitis, alone and in combination with other perennial rhinitis medications. AB - To study the long-term safety and effectiveness of ipratropium bromide nasal spray 0.03% in the treatment of nonallergic perennial rhinitis, we administered this medication for 1 year in an open-label trial involving 285 patients. Our intention was to maintain the highest protocol dose possible to gain a clearer picture of the long-term side effect profile of the compound. Ipratropium bromide was well tolerated with no serious side effects in this patient population. It provided a significant improvement in rhinorrhea throughout the year-long trial; only 17 of 285 patients (6%) were considered treatment failures. There was an improvement in patient quality of life, as well as a substantial reduction in the need for other medications (antihistamines, decongestants, and nasal steroids) used to treat perennial rhinitis symptoms. PMID- 7538522 TI - Etiologic conditions as predictors of children's problems and competencies in elementary school. PMID- 7538521 TI - A pharmacologic continuum in the treatment of rhinorrhea: the clinician as economist. AB - The economics of medications are now of great concern to health-care providers. Pharmacoeconomic issues are by no means simple, and yet, ironically, they assume greater importance in prescribing for modest disorders like rhinorrhea than for life-threatening conditions. The therapeutic continuum of quality and cost becomes foreshortened, and safety is an additional concern. Choosing the appropriate medication for rhinorrhea, then, can pose a challenge to the clinician, just as choosing a vital medication. This paper reviews the usage, quality, and cost of major therapies for the rhinorrhea that occurs secondary to various conditions, including nasal steroids, antihistamines and anticholinergics, and discusses the role of the clinician in factoring costs into therapy. PMID- 7538523 TI - Pray that it is not a neurologic problem. PMID- 7538524 TI - Physicians' testing of children with developmental disabilities. PMID- 7538525 TI - Developmental dysgraphia and motor skills disorders. PMID- 7538526 TI - Neuroanatomic substrate of developmental specific learning disabilities and select behavioral syndromes. PMID- 7538527 TI - Regulation of differentiation of peritoneal B-1a (CD5+) B cells. Activated peritoneal macrophages release prostaglandin E2, which inhibits IgM secretion by peritoneal B-1a cells. AB - The B-1a (CD5+) subset of B cells comprises the majority of B cells in the peritoneal cavity and is implicated in the pathogenesis of certain autoimmune diseases and lymphoproliferative disorders. When we stimulated purified B-1a cells with LPS, they produced more than four times as much IgM as similarly stimulated whole peritoneal cells (containing the same number of B-1a cells). Reconstitution experiments using FACS-purified peritoneal cell populations revealed that resident peritoneal macrophages (Mac1+, B220-) profoundly inhibited the LPS response of peritoneal B-1a cells. Culture of B-1a cells with peritoneal macrophages at a ratio of 3:1 (reflecting the in vivo ratio) resulted in a fivefold or greater reduction in the IgM response to LPS. LPS activation of macrophages resulted in production of a soluble factor that inhibited LPS-induced B cell differentiation by 86% when used at a concentration of 5%. When [3H]arachidonic acid-pulsed macrophages were stimulated with LPS, the major arachidonic acid metabolite secreted was PGE2 (a potent inhibitor of B cell differentiation). The inhibitory capacity of the macrophage-derived supernatant was reversed by the addition of anti-PGE2. These findings indicate that macrophage-derived PGE2 functions as an important regulator of polyclonal response of B-1a cells to LPS. PMID- 7538528 TI - Costimulation with B7-1, IL-6, and IL-12 is sufficient for primary generation of murine antitumor cytolytic T lymphocytes in vitro. AB - The requirements for generating CD8+ CTLs against the mastocytoma P815 were first defined by using an allogeneic mixed lymphocyte tumor culture (MLTC). Both the expansion of effector lymphocytes and the acquisition of lytic activity were dependent on the presence of accessory cells, but not CD4+ lymphocytes. Several factors were examined for their ability to replace accessory cell function. Expression of B7-1 by P815 was sufficient to induce IL-2 production by CD8+ cells, but substantial proliferation was achieved only if IL-6 was provided as well. Although IL-12 had little effect on the net proliferation of developing effector cells, it increased specific lytic activity 10-fold, acted synergistically with B7-1 in induction of IFN-gamma production during primary stimulation, and resulted in a shift toward a Th1 cytokine profile following secondary stimulation. These costimulatory factors were then studied in a primary syngeneic MLTC by using splenocytes from nonimmunized DBA/2 mice, an approach that had never before succeeded in generating specific CTLs. The combination of B7-1, IL-6, and IL-12 was sufficient to induce P815-specific CTL activity after a 5-day MLTC, which was expanded optimally following secondary stimulation in the presence of B7-1, IL-2, and IL-7. The irrelevant syngeneic tumor L1210, constructed to express B7-1 and the P815-derived tumor Ag gene P1A, also generated CTLs that lysed the parental P815. The combination of B7-1, IL-6, and IL-12 should be useful in the derivation of other tumor-specific CTLs in vitro, and may constitute the optimal stimuli for rapid proliferation and differentiation of helper-independent, tumor-specific CTLs in vivo. PMID- 7538529 TI - Regulation of germinal center B cell differentiation. Role of the human APO-1/Fas (CD95) molecule. AB - We previously described the existence of a tonsillar IgD- B cell subset with memory B cell features. To test the possibility that these cells could derive from germinal center (GC) B cell precursors, we examined the proliferation, differentiation, and phenotype of GC B cells after culturing with either anti CD40 Abs or activated T cells, presumably mimicking the signals received by centrocytes in the light zone of GC. We show in this work that GC B cells proliferate and secrete Igs in both activation systems, thus indicating that CD40 ligation is also required for differentiation of GC B cells along the plasmacytoid pathway. T cell-dependent activation of GC B cells induced down regulation of most GC-related markers (CD10, CD38, and CD77) and up-regulation of CD44 and CD62-L which are both expressed on the putative memory B cells subset. Moreover, T cell-mediated stimulation of GC B cells resulted in the strong induction of CD5 and up-regulation of APO-1/Fas (CD95). In contrast, stimulation performed with immobilized anti-CD40 Abs did not affect expression of CD10 and CD38 and failed to induce CD62-L and CD5, suggesting that the CD40 signaling pathway is necessary but not sufficient for the development of memory B cells. CD95 ligation on GC B cells was found to antagonize the stimulatory effect of immobilized anti-CD40 Abs on their proliferation, survival, and Bcl-2 expression. The possible role of CD95 in the expansion and selection of the Ag-activated B cell clones in GC is discussed. PMID- 7538530 TI - Influence of TGF-beta on murine thymocyte development in fetal thymus organ culture. AB - TGF-beta is a multifunctional growth regulator that can either inhibit or stimulate the growth and differentiation of lymphocytes. For several cell types the effect of TGF-beta was found to correlate with the differentiation stage of the cells. We have studied the influence of TGF-beta on the differentiation of murine thymocytes by evaluating the effect of TGF-beta on the generation of thymocyte subpopulations in fetal thymus organ culture. TGF-beta inhibited the growth and differentiation of CD4-CD8- double-negative thymocytes. In the CD4-CD8 double-negative cell population, most cells remained CD44+CD25-, with CD44+CD25+ and CD44-CD25- subpopulations dramatically decreased in cell numbers. The accumulation of cells with a phenotype characteristic of cells in early stage of differentiation suggests a block at very early transition steps. These observations were confirmed in experiments with precursor cells from fetal liver transferred to 2-deoxyguanosine-treated alymphoid thymic lobes, inasmuch as addition of TGF-beta caused a complete inhibition of T cell development. Differentiation into CD4+CD8+ double-positive thymocytes and CD4+ single-positive thymocytes was impaired because these cell numbers were greatly reduced. In contrast, the CD8+ single-positive subpopulation retained normal cell numbers. This CD8+ population had characteristics of a mature subset as the cells expressed CD8 beta and high levels of TCR-alpha beta and CD3. This TCR-alpha beta + cell population was not actively dividing, suggesting that these cells arise de novo by differentiation. PMID- 7538531 TI - Identification of the TS2/18-recognized epitope on the CD2 molecule as a target for suppression of T cell cytokine synthesis. AB - CD2 mAb can inhibit T cell activation, but the mechanisms involved are still unclear. In this study, we identify the mAb TS2/18, previously reported to bind to an epitope on the distal domain of the CD2 molecule at amino acids 87-99 (1), as a particularly potent inhibitor of T cell cytokine synthesis. Although TS2/18 is comitogenic with the CD2R mAb VIT13, this mAb combination does not induce the secretion of substantial amounts of cytokines. When added to T cells stimulated with the CD2 mAb pair OKT11-VIT13, TS2/18 efficiently blocks the induction of cytokine synthesis induced by that CD2 mAb pair, although it does not interfere with the binding of OKT11. In addition, TS2/18 inhibits the increase in protein tyrosine phosphorylation and the accumulation of phosphatidic acid induced by either OKT11-VIT13 or a cross-linked CD3 mAb. Finally, TS2/18 disrupts CD2 clusters induced by the CD2 mAb pair OKT11-VIT13. We conclude that TS2/18 blocks T cell cytokine synthesis by interfering with early signal transduction, possibly by impairing the formation of signal-transducing molecule complexes on the T cell surface. Together, these data identify the CD2 epitope recognized by the mAb TS2/18 as a candidate epitope for T cell-specific immunosuppressive ligands. PMID- 7538532 TI - Activation of gamma delta T cells for production of IFN-gamma is mediated by bacteria via macrophage-derived cytokines IL-1 and IL-12. AB - Gamma delta T cells are found at sites of microbial infection and have been reported to proliferate in response to bacterial Ags. We show here that although the response by Listeria-elicited peritoneal gamma delta T cells to heat-killed bacteria in the presence of macrophage accessory cells may be partially mediated via the TCR, it is predominantly mediated via cytokines produced by the macrophages. Macrophage cytokines IL-12 and IL-1 synergize to induce some proliferation and considerable IFN-gamma production by peritoneal gamma delta T cells. This cytokine synergy pattern differs from that reported for NK cells, in which IL-12 in combination with either IL-2 or TNF-alpha induces NK cells to produce IFN-gamma. The combination of IL-12 and IL-1 provides a strong stimulus for IFN-gamma production by gamma delta T cells, but a relatively weak signal for proliferation. This is in contrast to the strong proliferative signal from the combination of IL-7 and IL-1 and the relatively weak stimulation of IFN-gamma production by the IL-7/IL-1 combination. Thus, there is differential regulation of NK and gamma delta T cells by cytokines and differential regulation of activation functions within the gamma delta T cell population by combinations of cytokines. These data provide evidence for a potentially important pathway for augmentation of IFN-gamma secretion at sites of infection where gamma delta T cells are found in abundance and where IFN-gamma may play a major role in the control of the infection. PMID- 7538533 TI - IL-3 and granulocyte-macrophage colony-stimulating factor strongly induce Ig secretion by sort-purified murine B cell activated through the membrane Ig, but not the CD40, signaling pathway. AB - Sort-purified resting murine B cells proliferate in response to dextran conjugated anti-IgD Abs (alpha delta-dex) but fail to secrete significant amounts of Ig even after the addition of IL-1 + IL-2. We show that either IL-3 or granulocyte-macrophage CSF (GM-CSF) stimulates 10- to 50-fold enhancements in IgM secretion by sort-purified B cells treated with alpha delta-dex + IL-1 + IL-2, and that the combined actions of IL-3 and GM-CSF are typically greater than additive. Both IL-3 and GM-CSF act primarily as B cell differentiation factors, although IL-3 induces a modest enhancement in cellular outgrowth. The enhancing effects of IL-3 and GM-CSF require multivalent Ag receptor cross-linkage, mediated by alpha delta-dex, as neither cytokine induces IgM secretion in the presence of unconjugated anti-IgD Abs. Although both alpha delta-dex and IL-1 + IL-2 are required for optimal IL-3- and GM-CSF-mediated IgM secretion, both IL-3 and GM-CSF stimulate a modest IgM secretory response by cells activated with alpha delta-dex alone. In this regard, supernatant from either an activated CD4+ Th1 or Th2 clone potently induces IgM secretion by alpha delta-dex + IL-1 + IL-2 activated B cells and this is due, in large part, to the presence in these supernatants of either IL-3 and/or GM-CSF. Neither IL-3 nor GM-CSF stimulates significant IgM secretion by B cells activated through the CD40 signaling pathway alone, although the combination of CD40 and membrane Ig signaling leads to a strong enhancement of the IL-3 + GM-CSF-mediated IgM synthesis above that obtained with membrane Ig signaling alone. The demonstration that IL-3 and GM-CSF act directly as differentiation factors for B cells activated through their Ag receptor establishes a novel cytokine pathway for induction of humoral immunity. PMID- 7538535 TI - Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8+ memory T cells. AB - Whether the L-selectin-low (L-sel-lo) phenotype of acutely stimulated CD8+ T cells is a permanent characteristic of long-term memory CTL precursors (p) is addressed for mice primed with an influenza A virus or the murine parainfluenza type 1 virus, Sendai virus. In both cases, many of the splenic CD8+ CTLp gradually lose the predominantly L-sel-lo profile associated with recently generated CTLp populations. The influenza-specific CTLp also tend to revert from the activated alpha 4-integrin-high to the resting alpha 4-integrin-low form. The kinetics of the switch back to the "naive" L-sel-hi phenotype differs for the influenza and Sendai virus models, perhaps reflecting events occurring during the acute phases of these responses. The return to being L-sel-hi is not due to irreversible lymphocyte senescence, because restimulation of this set with the inducing virus in vitro causes most of the cells to become L-sel-lo. Also, despite the time-related drift of these particular memory CTLp to the L-sel-hi state, the size of the total pool of L-sel-lo CD8+ T cells increases with age. PMID- 7538536 TI - T lymphocyte adhesion to human brain pericytes is mediated via very late antigen 4/vascular cell adhesion molecule-1 interactions. AB - T cell adhesion to the brain microvascular endothelium and subsequent migration into the brain parenchyma is one of the major events in the development of multiple sclerosis (MS). Interactions of the T cell integrin very late antigen-4 (VLA-4) with its receptor, vascular cell adhesion molecule-1 (VCAM-1) have been described to be of crucial importance for the development of MS. We investigated the expression of these adhesion molecules in MS brain tissue by immunohistochemical analysis, and studied their functional involvement in an in vitro T cell adhesion assay. A number of other adhesion molecules were studied for comparison. In cryosections of several MS brains, expression of VCAM-1 was demonstrated not only on the endothelium of vessels surrounding MS plaques, but also in perivascular positions, suggesting expression by pericytes. T cells adhered to both cell types in vitro. Both LFA-1/intercellular adhesion molecule-1 and VLA-4/VCAM-1 interactions were equally involved in the adhesion of T cells to TNF-alpha-stimulated endothelial cells. However, adhesion of T cells to TNF-alpha stimulated pericytes was clearly dominated by VLA-4/VCAM-1 interactions. These results indicate that pericytes, next to endothelial cells, may play an important role in regulating T cell infiltration into the central nervous system. PMID- 7538537 TI - Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene. AB - The intestinal epithelium of neonatal mice and rats expresses an Fc receptor that mediates selective uptake of IgG in mothers' milk. This receptor (FcRn), which helps newborn animals to acquire passive immunity, is an MHC class I-like heterodimer made up of a heavy chain and beta 2-microglobulin. In the present study, we determined the genomic structure of a mouse gene (Fcrn) encoding the heavy chain of FcRn. The overall exon-intron organization of the Fcrn gene was similar to that of the MHC class I gene, thus providing structural evidence that Fcrn is a bona fide class I gene. The 5'-flanking region of the Fcrn gene contained the binding motifs for two cytokine-inducible transcription factors, NF IL6 and NF1. However, regulatory elements found in MHC class I genes (enhancer A, enhancer B, and the IFN response element) were absent. Phylogenetic tree analysis suggested that, like the MICA, AZGP1, and CD1 genes, the Fcrn gene diverged from MHC class I genes after the emergence of amphibians but before the split of placental and marsupial mammals. Consistent with this result, Southern blot analysis with a mouse Fcrn cDNA probe detected cross-hybridizing bands in various mammalian species and chickens. Sequence analysis of the Fcrn gene isolated from eight mouse strains showed that the membrane-distal domain of FcRn has at least three amino acid variants. The fact that Fcrn is a single copy gene indicates that it is expressed in both the neonatal intestine and the fetal yolk sac. PMID- 7538534 TI - Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-alpha. AB - Human CD34+ bone marrow progenitors cultured in the presence of granulocyte macrophage CSF (GM-CSF) develop along a myeloid pathway, and the addition of exogenous TNF-alpha leads to the differentiation of dendritic cells among the myeloid progeny. These bone marrow CD34+ -derived dendritic cell that develop during 2-wk culture have the same morphologic, phenotypic, and functional properties that distinguish mature dendritic cells in blood. c-kit ligand does not directly influence dendritic cell differentiation per se, but rather increases the total cell number in synergistic combination with GM-CSF and TNF alpha. This degree of expansion translates into an effective yield of approximately 1.7 x 10(6) mature dendritic cells per single ml of normal adult human bone marrow, compared with approximately 10(6) dendritic cells usually obtained from 450 to 500 ml of peripheral blood. In addition to dendritic cells that constitute approximately 10 to 15% of the total myeloid progeny, the cultures contain monocytes/macrophages and intermediate granulocytic precursors. Monocytes/macrophages and dendritic cells together comprise all of the class II MHC-positive progeny. Sorted cells bearing the CD14+ HLA-DR+ phenotype of mature monocytes are at least 1.5 to 2 logs less active than CD14- HLA-DR+ dendritic cells as stimulators in the allogeneic MLR, even though both CD14+ and CD14- subpopulations share expression of several costimulatory ligands. The synergistic combination of c-kit ligand, GM-CSF, and TNF-alpha therefore expands substantial numbers of immunostimulatory CD14- HLA-DR+ dendritic cells from defined CD34+ progenitors in human bone marrow. This should facilitate the use of dendritic cells in the manipulation of T cell-mediated immune responses. PMID- 7538538 TI - Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides. AB - Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16 induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201 binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7 containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma. PMID- 7538539 TI - Self and foreign 60-kilodalton heat shock protein T cell epitope peptides serve as immunogenic carriers for a T cell-independent sugar antigen. AB - Healthy individuals manifest natural T cell reactivity to epitopes of the 60-kDa heat shock protein (hsp60) of both self and bacterial origin. The present studies were done to learn whether defined peptides of hsp60 could function as T cell carrier epitopes for a poorly immunogenic T-independent capsular polysaccharide, the Vi Ag of Salmonella typhi. Homologous peptides were synthesized from the mouse self-hsp60 molecule (CP1m), from the closely related human hsp60 molecule (CP1h), and from the more distant Escherichia coli (CP1ec) and mycobacterial (CP1mt) hsp60 molecules. The peptides were conjugated to Vi and tested for their immunogenicity in BALB/c (H-2d) and H-2 congenic mice (H-2k and H-2b). We now report that the self-CP1m and cross-reactive CP1h peptides were as immunogenic as was the non-cross-reactive foreign CP1ec peptide. Small amounts of the CP1 peptide, even in PBS, sufficed to induce anti-Vi Abs of the IgG1 (T-dependent) isotype in naive mice. The carrier effect was associated with the ability of the peptides to bind to APC and to induce T cell proliferation. H-2d and H-2k mice, but not H-2b mice responded to CP1m/h and CP1ec. None of the mice responded to CP1mt. No signs of inflammation or autoimmune disease were detected. Thus, natural T cell autoimmunity exists and can be harnessed to provide T cell help for Ab production to a foreign bacterial molecule in a synthetic vaccine. PMID- 7538540 TI - Identification of T and B cell epitopes recognized by humans in the C-terminal 42 kDa domain of the Plasmodium falciparum merozoite surface protein (MSP)-1. AB - The 42-kDa, C-terminal region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a putative malaria vaccine candidate Ag. Nine synthetic peptides corresponding to predicted T cell sites of MSP-1 in blocks 15 and 16 and eight overlapping peptides representing the conserved block 17 were used to identify naturally immunogenic epitopes. These peptides were tested for their ability to induce proliferation of PBMC from residents in western Kenya, where malaria transmission is holoendemic. Six peptides (PL145, PL146, PL147, PL148, PL149, and PL150) from blocks 15 and 16 induced a positive proliferative response in > 30% of the individuals tested, and three peptides (PL151, PL152, and PL153) induced a proliferative response in < 25% of the donors. Among these peptides, PL146 was from the highly conserved region, PL150 was from a polymorphic region, and all other peptides were from a dimorphic region of blocks 15 and 16. In block 17, only three peptides, PL99, PL100, and PL103, induced proliferation in 30 to 37% of the volunteers. The rest of the peptides induced a proliferative response in approximately 13 to 25% of the donors. The plasma from these donors widely reacted with different allelic forms of 19-kDa recombinant proteins representing block 17 and recognized at least two linear B epitopes, PL104 and PL97. In summary, this study revealed that a majority of immunodominant T and B epitopes are localized in the conserved or dimorphic regions that are nonpolymorphic in the 42-kDa protein of MSP-1. This study suggests that incorporation of T epitopes from the dimorphic blocks 15 and 16 in a vaccine construct may be useful to ensure Ag-specific memory responses. PMID- 7538541 TI - Human skin mast cells express functional beta 1 integrins that mediate adhesion to extracellular matrix proteins. AB - We have evaluated the adhesion of human cutaneous mast cells to several components of the extracellular matrix (plasma fibronectin, laminin, collagen type I and IV) and studied whether these cells express the beta 1 integrins potentially involved in the adhesion to these proteins. Human skin mast cells (5.1 +/- 1.5% pure) spontaneously adhered to fibronectin and laminin (0.1 to 10 micrograms/ml) immobilized on plastic surfaces (e.g., 14 +/- 7.2% and 14 +/- 4.4% adhesion at 10 micrograms/ml, respectively). Similar results were obtained with a 90% pure mast cell preparation. In contrast, cutaneous mast cells did not adhere to collagen type I (1.6 +/- 0.5% adhesion) or type IV (1.2 +/- 0.8% adhesion). Control adhesion in BSA-coated wells was < 5%. Mast cell adhesion to fibronectin was optimal after an incubation period of 60 to 90 min (t1/2 = 28.2 +/- 6.2 min), whereas adhesion to laminin was faster (t1/2 = 10.1 +/- 1.2 min), being nearly optimal after a 15-min incubation period. Human skin mast cell adhesion to fibronectin and laminin was found to be dependent on the presence of divalent cations in the extracellular medium. Dual-color immunofluorescence and flow cytometry were used to evaluate whether human skin mast cells (51.3 +/- 3.9% pure) express beta 1 integrins that may mediate cell adhesion to extracellular matrix proteins. These mast cells were found to express VLA (very late Ag)-3 (75.3 +/- 35.6 specific fluorescence intensity) and, to lesser degree, VLA-4 and VLA-5 receptors (8.0 +/- 2.5 and 6.9 +/- 3.2 specific fluorescence intensity, respectively). In contrast, VLA-1, VLA-2, and VLA-6 integrins were not expressed significantly. mAb to VLA-3, VLA-4, and VLA-5 each inhibited by 70% skin mast cell adhesion to fibronectin. mAb to VLA-3 nearly abolished mast cells adhesion to laminin, whereas anti-VLA-4 and anti-VLA-5 were ineffective. Finally, immunosuppressant cyclosporin A (100 nM) and FK-506 (10 nM) significantly inhibited mast cell adhesion to both fibronectin and laminin (p < 0.05). Our data demonstrate that human skin mast cells spontaneously adhere to fibronectin and laminin, and that this adhesion is mediated by VLA-3, VLA-4, and/or VLA-5 integrins on these cells. Interactions between these beta 1 integrins and extracellular matrix proteins may be involved in perivascular tissue localization of human mast cells in vivo. PMID- 7538542 TI - A monoclonal antibody that blocks the complement regulatory activity of guinea pig erythrocytes and characterization of the antigen involved as guinea pig decay accelerating factor. AB - MCA44 is a mAb with the capacity to sensitize neuraminidase-treated guinea pig E for hemolysis by homologous guinea pig C, and the Fab fragments of this mAb could also sensitize guinea pig E interfering with the function of a membrane inhibitor of C on guinea pig E. Using an immunosorbent column to which MCA44 was coupled, the antigenic molecule termed 44Ag was purified from the glycoprotein fraction extracted from E membranes. C intermediate sheep E treated with guinea pig C1 and C4 after sensitization with Ab (EAC14b cells) lost the ability to generate C3 convertase with C2 after incubation with 44Ag. Treatment of guinea pig E and PBL with phosphatidyl-inositol specific phospholipase C (PIPLC) partially removed 44Ag, as determined by flow cytometric analysis after immunofluorescence staining with MCA44. However, 125I-labeled 44Ag adsorbed to human E was efficiently removed by PIPLC treatment with a slight reduction in M(r). The 44Ag purified on an immunosorbent column showed three bands on SDS-PAGE. However, partial N terminal amino acid sequences of the 55-kDa, 70-kDa, and 88-kDa bands under nonreducing conditions were identical and the sequence was 55% homologous to the N-terminal sequence of human decay accelerating factor (CD55). Intracutaneous administration of MCA44 or its F(ab')2 fragment resulted in increased capillary permeability, even after 3 days, as determined by the appearance of Evans blue spots after i.v. administration of the dye. Because control Abs including anti class I-MHC did not cause such increased capillary permeability, the increase in permeability caused by MCA44 was likely induced by blocking the function of 44Ag in vivo, indicating a crucial role for these molecules in preventing over activation of C at the site. PMID- 7538544 TI - Oligoclonal CD8+ T cells are preferentially expanded in the CD57+ subset. AB - A number of recent reports have established that oligoclonality and/or clonal expansion is a common feature of the CD8+ T cell population. Oligoclonal expansion has also been observed in bone marrow transplant recipients and rheumatoid arthritis patients, disease states in which CD57+CD8+ T cells are occasionally elevated. In this study we have compared the TCR repertoire of the CD57+ and CD57- subsets of CD8+ T cells in normal persons by using three-color FACS analysis with a panel of 16 mAbs specific for TCR V segments. The CD57 surface marker was highly variable in frequency but generally present on a minority of CD8+CD3+ T cells (mean 16.3%, SD 12.7) in a group of 41 normal volunteers. Dramatic oligoclonal expansion was present in the CD57+CD8+ T cell population in 15 of 41 (37%) of our study population and thus is a characteristic feature of the normal immune system. No such prominent oligoclonal expansions were observed in the CD57-CD8+ subset, although preliminary experiments suggest that oligoclonality per se is occasionally present at a lower frequency in CD57- cells. The reasons for this persistent accumulation of oligoclonal CD8+CD57+ T cells and their function in immune homeostasis are unclear. PMID- 7538545 TI - Two mechanisms of antigen-specific apoptosis of myelin basic protein (MBP) specific T lymphocytes derived from multiple sclerosis patients and normal individuals. AB - Several stimuli induce mature T cells to undergo apoptosis or programmed cell death (PCD) including specific Ag. We have demonstrated previously that Ag induces the death of encephalitogenic T cells in vitro and deletion in vivo, leading to amelioration of autoimmune encephalomyelitis. We have now examined whether activated, myelin basic protein (MBP)-specific human T cells may be eliminated by Ag-induced PCD. We demonstrate that activated MBP-specific T cell lines (TCL) undergo the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when given a TCR challenge. We found evidence that two mechanisms led to apoptosis: a propriocidal mechanism that was highly Ag specific and dependent on the dose of exogenously added rIL-2, and a cytolytic mechanism in which MBP-specific TCL lysed B cell targets and engaged in considerable "fratricidal" cytolysis of other MBP-specific T cells. These two pathways leading to MBP-specific apoptotic death could be distinguished by their glucocorticoid sensitivity. Glucocorticoid treatment significantly blocked MBP induced propriocidal apoptosis but had no effect on T cell cytolysis of B cell targets. Although it has been proposed that autoimmune disease could result from the failure of normal deletional mechanisms, this preliminary survey of MBP reactive mature TCL from multiple sclerosis patients revealed that such cells are highly susceptible to TCR-induced PCD and comparable with TCL from normal subjects. Thus, therapeutic strategies based on Ag-induced PCD of T lymphocytes may be feasible in man. PMID- 7538546 TI - A randomized, double-blind, placebo-controlled trial of systemically administered interferon-alpha, -beta, or -gamma in combination with cryotherapy for the treatment of condyloma acuminatum. AB - One hundred fifty-two patients were enrolled in a study to evaluate 3 interferon (IFN) preparations used in combination with cryotherapy for treatment of anogenital warts. Subjects received subcutaneous injections (2 x 10(6) units/m2) of IFN-alpha n1, -beta, -gamma or placebo 3 times a week for 6 weeks and cryotherapy with liquid nitrogen. Subjects were followed < or = 1 year. Among patients followed > or = 12 weeks, two-thirds had a complete response. No significant differences in rates of complete response (P = .37) or reappearance of a wart at the initial site (P = .20) were noted among the treatment groups. However, patients who received IFN-beta or -gamma developed new warts at a significantly lower frequency (P = .02). IFN administration was associated with side effects but was well tolerated. IFN-beta was the least toxic of the 3 preparations and had the best therapeutic ratio. PMID- 7538543 TI - An epitope-selective, transporter associated with antigen presentation (TAP)-1/2 independent pathway and a more general TAP-1/2-dependent antigen-processing pathway allow recognition of the HIV-1 envelope glycoprotein by CD8+ CTL. AB - The lysis of virally infected cells by CTLs requires the recognition of processed fragments of viral proteins presented in association with class I MHC molecules on the surfaces of infected cells. Processing begins in the cytosol with the degradation of viral proteins into peptides that are then transported into the endoplasmic reticulum (ER) for association with newly synthesized class I molecules. Transport is mediated by a heterodimer of the MHC-encoded proteins, transporter associated with Ag presentation (TAP)-1 and TAP-2. Uncertainty exists over the site of processing of viral envelope (env) proteins. The extracellular domains of env proteins are not present in the cytosol, the site in which the class I-restricted Ag-processing pathway begins. Rather, the ecto-domains of env proteins are cotranslationally translocated into the ER during biosynthesis. We have analyzed the processing of the HIV-1 env protein by using a large series of env-specific human CD8+ CTL clones. These studies have led to the delineation of two distinct processing pathways. The first pathway permits a subset of class I restricted epitopes in the ecto-domain of the env protein to be generated efficiently by a TAP-1/2-independent mechanism localized to the ER or a premedial Golgi compartment. A second, more general pathway that is capable of generating all env epitopes uses as a substrate env protein mislocalized to the cytosol and produces peptides that are transported from the cytoplasm to the ER in a TAP-1/2 dependent fashion. PMID- 7538547 TI - Combination therapy with zidovudine prevents selection of human immunodeficiency virus type 1 variants expressing high-level resistance to L-697,661, a nonnucleoside reverse transcriptase inhibitor. AB - L-697,661 is a human immunodeficiency virus type 1 (HIV-1)-specific nonnucleoside reverse transcriptase (RT) inhibitor. Its tolerability and activity in combination with zidovudine were evaluated in a 48-week double-blind study. One hundred nineteen zidovudine-naive HIV-1-infected patients with CD4 cell counts of 200-500/mm3 received either combination therapy, L-697,661 alone, or zidovudine alone. Activity was assessed by CD4 cell count changes. Selection for L-697,661 resistant virus was monitored by susceptibility testing of RT expressed by circulating viral RNA. Therapy was generally well tolerated. All groups receiving zidovudine exhibited transient increases in CD4 cell counts, while the L-697,661 monotherapy group showed a significant decline and yielded RT > 100-fold resistant to L-697,661 and associated with substitutions at RT residue 181. The RT from patients receiving combination therapy was maximally 15-fold less susceptible to L-697,661. Hence, cotreatment with zidovudine prevents selection of HIV-1 variants that are highly resistant to L-697,661 in patients naive to both compounds. PMID- 7538548 TI - Prevalence and clinical significance of zidovudine resistance mutations in human immunodeficiency virus isolated from patients after long-term zidovudine treatment. AIDS Clinical Trials Group 116B/117 Study Team and the Virology Committee Resistance Working Group. AB - Zidovudine resistance mutations at reverse transcriptase codons 215 or 41 were found in two-thirds of human immunodeficiency virus type 1 (HIV-1) isolates obtained at baseline from patients enrolled in an AIDS Clinical Trials Group (ACTG) protocol that compared didanosine with continued zidovudine in patients with > or = 16 weeks of previous zidovudine therapy (ACTG 116B/117). The combined presence of mutations at both codons 215 and 41 conferred an increased risk for progression (relative hazard, 1.82; 95% confidence interval [CI], 1.02-3.26) and an increased risk for death (RH, 5.42; 95% CI, 1.92-15.30) in analyses that controlled for other factors predictive of progression. However, the benefit of switching to didanosine compared with continued zidovudine therapy was independent of the presence of these mutations. Although this information is not helpful in determining when to alter therapy, detection of zidovudine resistance mutations provides prognostic information in patients with advanced HIV disease. PMID- 7538549 TI - Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type 1. AB - In an ultrasensitive assay for reverse transcriptase (RT), an in vitro transcribed heteropolymeric RNA sequence was used as a template and polymerase chain reaction (PCR) amplification with Southern blot hybridization served as a detection system for the cDNA reaction product. The assay, called Amp-RT, detected 9 tested retroviruses in unconcentrated culture supernatants diluted 10(2)- to 10(5)-fold. A comparative analysis using human immunodeficiency virus type 1 (HIV-1) revealed that Amp-RT was 100,000 times more sensitive than the standard RT assay, 10,000 times more sensitive than p24 antigen capture and branched DNA assays, and 100 times more sensitive than RT-PCR or TCID50 assays. Analysis of serum specimens from 42 HIV-1-infected persons by Amp-RT showed that 36 samples (85.7%) were RT-positive. In contrast, 41 serum specimens from persons seronegative for HIV-1 and human T lymphotropic virus types I and II were all Amp RT-negative. PMID- 7538550 TI - Lipopolysaccharide toxicity-regulating proteins in bacteremia. AB - The toxicity of lipopolysaccharide (LPS) is modified by several proteins, such as bactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP). BPI and LBP plasma levels were measured in patients with gram-negative (n = 36) or gram-positive (n = 28) bacteremia. Levels of BPI and LBP, which are proteins that neutralize and enhance LPS effects, respectively, were increased before bacteremia was first detected. The BPI/neutrophil ratio, reflecting neutrophil activation, was significantly associated with the presence of sepsis syndrome and death in bacteremic patients: 1.06 (0.11-6.49) versus 0.57 (0.06-3.82) in patients with and without sepsis syndrome (P < .01), respectively, and 0.64 (0.06 3.82) versus 1.02 (0.12-6.49) in survivors and nonsurvivors (P < .05), respectively (ratio in nanograms of BPI per 10(6) neutrophils). High LBP peak levels were significantly associated with the presence of sepsis syndrome (P < .01). No differences in BPI and LBP levels were observed in patients with gram negative versus gram-positive bacteremia. BPI/neutrophil ratio, as a parameter of neutrophil activation, may be useful in monitoring infectious disease. PMID- 7538551 TI - Efficacy of 9-(2-phosphonylmethoxyethyl)adenine treatment against chronic simian immunodeficiency virus infection in macaques. AB - Long-tailed macaques chronically infected with simian immunodeficiency virus (SIV) were treated for 4 or 8 weeks with daily subcutaneous doses of the antiretroviral compound 9-(2-phosphonylmethoxyethyl)adenine (PMEA). The efficacy of PMEA was evaluated by monitoring cell-free virus in plasma, virus titer and viral DNA in peripheral blood mononuclear cells, and absolute numbers of lymphocyte subsets. In mock-treated control macaques, virus titers changed minimally. However, in treated macaques, PMEA exhibited impressive effects, leading to the disappearance of virus in the blood within the first week of treatment and lasting through the fourth week of treatment. The results indicate that PMEA can effectively reduce SIV in chronically infected macaques and offer an optimistic perspective for therapeutic intervention against human immunodeficiency virus infection. PMID- 7538552 TI - Distinguishing acute from symptomatic chronic hepatitis C virus (HCV) infection by site-directed serology of the HCV structural proteins. AB - To distinguish acute from symptomatic chronic hepatitis C virus (HCV) infection, site-directed serology was done on the complete core, envelope 1, and envelope 2 proteins using multiple peptide assays. Serum samples were obtained from 7 patients with clinically confirmed acute HCV infections and from 36 patients with symptomatic chronic HCV infections. At onset of symptoms, all patients with acute infection had HCV RNA in serum, 1 had > 2 strong core peptide reactivities (A405 > 1.0), and the number of core reactivities increased with time in 6 patients. In contrast, 35 of 36 chronic-phase patients with HCV RNA in serum had 3-12 strong core reactivities (P < .001). Envelope 2 antibodies were found in 2 acute-phase and in 32 chronic-phase HCV patients (P < .01). Thus, a serum sample with HCV RNA from a patient with hepatitis can be classified as acute or chronic using site directed serology of the HCV structural proteins. PMID- 7538553 TI - [Observation of Pseudomonas aeruginosa biofilm with confocal laser scanning microscope]. AB - Confocal laser scanning microscope (CLSM) is a new type of microscope, which permits non-invasive optical sectioning of biomaterials by reducing out-of-focus haze. A biofilm of Pseudomonas aeruginosa was developed on the silicon disks using a modified Robbins device and used to visualize hydrated living biofilm with CLSM. In order to examine optimum staining agents and conditions, acridine orange, fluorescein isothiocyanate (FITC), FITC-conjugated concanavalin A (FITC ConA), Evans blue, safranine and rhodamine-conjugated concanavalin A were used in three different medium (pH 4.5, pH 7.5, pH 9.5). Only extracellular matrix was stained as a net-like structure for FITC-ConA and only bacteria for acridine orange and safranine. Although the staining patterns with FITC and Evans blue were affected markedly by the medium pH, those with other staining agents were not affected significantly. It was considered that the staining characteristics specific for each agent and changes of staining pattern by pH were probably due to relative differences among matrix, bacteria and staining agents in their electrostatic charges. In addition to sagittal sectioning images of P. aeruginosa biofilm, clear doubles staining images, which differentiates bacteria from matrix in the identical material, could be obtained with the combination of safranine and FITC-ConA. PMID- 7538554 TI - [Diagnosis and therapy of glomerulonephritis and nephrotic syndrome]. PMID- 7538555 TI - Granulocyte colony-stimulating factor accelerates neutrophil engraftment following peripheral-blood stem-cell transplantation: a prospective, randomized trial. AB - PURPOSE: It is well-established that the infusion of hematopoietic growth factors (HGF) accelerates neutrophil recovery in patients undergoing high-dose therapy followed by autologous bone marrow infusion. In addition, there is evidence that the infusion of autologous peripheral-blood stem cells (PBSC) accelerates engraftment in comparison to patients who receive bone marrow alone. However, few data are available regarding the ability of HGF to accelerate engraftment further in patients who receive PBSC following high-dose therapy. PATIENTS AND METHODS: Forty-one patients undergoing high-dose therapy followed by infusion of autologous PBSC with or without bone marrow were randomized to receive granulocyte colony-stimulating factor (G-CSF) 5 micrograms/kg/d beginning on day + 1 following transplant or standard posttransplant supportive care without HGF. RESULTS: The median time to a neutrophil count > or = 500/microL was 10.5 days in the G-CSF group versus 16 days in the control group (P = .0001). G-CSF was associated with statistically significant reductions in the time to neutrophil engraftment among patients who received PBSC alone (11 v 17 days, P = .0003) and in patients who received PBSC in conjunction with bone marrow (10 v 14 days, P = .02). The median duration of posttransplant hospitalization (18 v 24 days, P = .002) and the median number of days on nonprophylactic antibiotics (11 v 15, P = .03) were also significantly reduced. CONCLUSION: Administration of G-CSF in the posttransplant period accelerates the rate of neutrophil engraftment, shortens the duration of hospitalization, and reduces the number of days on nonprophylactic antibiotics in patients who receive autologous PBSC with or without autologous bone marrow following high-dose therapy. PMID- 7538556 TI - Hematopoietic rescue after high-dose chemotherapy using autologous peripheral blood progenitor cells or bone marrow: a randomized comparison. AB - PURPOSE: To compare autologous bone marrow (BM) with peripheral-blood progenitor cells (PBPC) as hematopoietic rescue after high-dose chemotherapy (HDCT). PATIENTS AND METHODS: From January 1991 until April 1993, 47 consecutive patients with relapsed or refractory germ cell tumors were randomized to either BM harvest or collection of PBPC mobilized by chemotherapy plus granulocyte colony stimulating factor (G-CSF). After additional conventional-dose salvage treatment, all patients received HDCT with carboplatin 1,500 mg/m2, etoposide 2,400 mg/m2, and ifosfamide 10 g/m2 with either BM or PBPC rescue. RESULTS: Forty-six patients were assessable for hematologic reconstitution, and one patient died on day +4 before engraftment. Rescue using PBPC resulted in a significantly shorter recovery time to neutrophil counts more than 500/microL (10.0 v 11.0 days, P < .01), neutrophil counts more than 1,000/microL (10.0 v 12.0 days, P = .001), and platelet counts more than 20,000/microL (10.0 v 17.0 days, P < .01), as well as in fewer days to transfusion independence from RBCs (8.0 v 12.0, P < .05) and platelets (9.0 v 12.0, P < .01) and fewer days of intravenous (IV) antibiotics (9.0 v 11.0, P < .05). However, no statistical differences in transfusion requirements or in other clinical outcome variables were observed. Overall survival and event-free survival also were not different in the two study arms. CONCLUSION: We conclude that the use of PBPC mobilized by chemotherapy plus G-CSF results in sustained trilineage reconstitution after HDCT, which occurs more rapidly as compared with BM. The earlier hematologic reconstitution in patients with PBPC rescue significantly reduces the time to transfusion independence. PMID- 7538557 TI - Malignant germ cell tumors in men infected with the human immunodeficiency virus: natural history and results of therapy. AB - PURPOSE: To determine how men infected with the human immunodeficiency virus (HIV) tolerate and respond to treatment for malignant germ cell tumors (GCTs), and how GCT histology and stage compare among HIV-infected versus non-HIV infected men. PATIENTS AND METHODS: Two hundred ninety-four cases of GCT diagnosed or treated from 1980 to 1993 were reviewed. Nine new cases among HIV infected men were identified; these were analyzed together with six cases previously reported from our institution. RESULTS: Low-stage tumors (stages I and IIA) comprised 67% of HIV-infected and 63% of non-HIV-infected cases. Sixty-seven percent of HIV-infected cases were seminomas versus 51% of non-HIV-infected cases. Ten patients had AIDS at the time of GCT diagnosis. Five patients underwent radiation therapy and one patient underwent retroperitoneal lymphadenectomy without complications. Seven patients received chemotherapy with four cycles of cisplatin, etoposide, and bleomycin (PEB) or cisplatin, vinblastine, and bleomycin (PVB) without excess cytopenias or new opportunistic infections. Of seven patients treated for advanced disease, there were five complete and two partial responses. Six patients have died of AIDS at a median of 20 months after diagnosis of GCT. The median follow-up time for surviving patients has been 42 months (range, 8 to 87) and all but one remain without evidence of active disease. In no case was a patient's HIV disease classification altered by antitumor therapy. CONCLUSION: The natural history of GCTs is comparable in HIV-infected and non-HIV-infected men and standard therapy including orchiectomy, retroperitoneal lymph node dissection, radiation therapy, and chemotherapy is well tolerated. PMID- 7538558 TI - Topographic organization of corticospinal projections from the frontal lobe: motor areas on the medial surface of the hemisphere. AB - We examined the topographic organization of corticospinal neurons in the four premotor areas on the medial wall of the hemisphere of macaques. These motor areas include the supplementary motor area (SMA) and three areas buried within the cingulate sulcus: the caudal cingulate motor area on the dorsal bank (CMAd), the caudal cingulate motor area on the ventral bank (CMAv), and the rostral cingulate motor area (CMAr). In one set of animals, we injected one fluorescent tracer into lower cervical segments of the spinal cord and another fluorescent tracer into lower lumbosacral segments to define the topographic organization of arm and leg representation within each premotor area. Similarly, in another set of animals, we injected different tracers into upper cervical and lower cervical segments to provide an indication of the topographic organization of proximal and distal arm representation within the arm representation of each premotor area. We found that all four of the premotor areas on the medial wall project to cervical and lumbosacral segments of the spinal cord. Three of these areas (SMA, CMAd, and CMAv) are like the primary motor cortex in having distinct arm and leg representations. The arm representation in each of the four motor areas on the medial wall contains separate regions that project densely to upper or to lower cervical segments. This observation suggests that each motor area contains distinct proximal and distal representations of the arm. Surprisingly, the size of the distal representation is comparable to or larger than the size of the proximal representation in each motor area. Thus, contrary to some previous hypotheses, the anatomical substrate exists for the premotor areas on the medial wall to be involved in the control of distal, as well as proximal arm movements. Our results provide a new map for guiding the exploration of the motor functions of the medial wall of the hemisphere. Furthermore, the observations of the present study support our suggestion that each of the premotor areas may be an important source of descending commands for the generation and control of movement. PMID- 7538559 TI - Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons. AB - Axon regeneration after injury and long-term alterations associated with learning both require protein synthesis in the neuronal cell body, but the signals that initiate these changes are largely unknown. Direct evidence that axonal injury activates molecular signals in the axon was obtained by injecting axoplasm from crushed or uncrushed nerves into somata of sensory neurons with uncrushed axons. Those injected with crush axoplasm behaved as if their axons had been crushed, exhibiting increases in both repetitive firing and spike duration, and a decrease in spike afterhyperpolarization 1 d after injection. Because similar changes occur in the same cells after learning, these data suggest that some of the long lasting adaptive changes that occur after injury and learning may be induced by common axoplasmic signals. Since the signals in axoplasm must be conveyed to the cell soma, we have begun to test the hypothesis that at least some of these signals are proteins containing a nuclear localization signal (NLS). Axoplasmic proteins at the crush site and those that accumulated at a ligation proximal to the crush were probed with an antibody to an amino acid sequence (sp) containing a NLS that provides access to the retrograde transport/nuclear import pathway. One protein, sp97, displayed properties expected of an axonal injury signal: it responded to injury by undergoing an anterograde-to-retrograde change in movement and, when the ligation was omitted, it was transported to the cell bodies of the injured neurons. PMID- 7538562 TI - Modular organization of the pontine nuclei: dendritic fields of identified pontine projection neurons in the rat respect the borders of cortical afferent fields. AB - Cortical afferents transferring information destined for the cerebellum terminate in the pontine nuclei (PN) in a divergent and patchy fashion. We investigated whether the form of dendritic fields of pontine projection neurons which are postsynaptic to the cortical afferents are related to this patchy pattern. To this end we used a triple combination of (1) retrograde labeling (injection of Fluorogold into the brachium pontis), (2) anterograde labeling [injection of Dil into cortical areas A17 and Sml(forelimb)], and (3) subsequent intracellular fills of identified projection neurons (Lucifer yellow) in slightly fixed slices of pontine brainstem. In 64 projection neurons whose somata were located within 160 microns of the border defined by cortical afferent fields, most of the dendritic trees were found to respect the border. Strikingly, proximal dendrites which were oriented toward the border often bent in order to avoid the boundary. This observation was supported by a quantitative analysis. It revealed that overlap areas of dendritic fields with the neighboring compartment were significantly smaller than those of hypothetical, radially organized dendritic fields of the same size, indicating that the dendritic fields are indeed confined to single compartments. In a second series of experiments, double injections of the anterograde tracers Dil and DiAsp into adjacent sites within one cortical area (A17 or Sml) were made in order to test if the topology of the cortical map is preserved within individual pontine compartments. This, however, does not seem to be the case, since the terminal fields displayed a complex pattern of overlap and nonoverlap rather than a consistent shift of terminal fields expected in the case of preserved topology. The results of the present study are consistent with the view that pontine modules independently process information from different parts of individual cortical areas. We suggest that this characteristic property of the corticopontine projection system might be the morphological basis of the well established fact that somatotopically continuous sensory maps in the cortex are transformed into maps at the level of the cerebellar cortex, showing a fractured somatotopy. PMID- 7538561 TI - Interleukin-1 beta attenuates excitatory amino acid-induced neurodegeneration in vitro: involvement of nerve growth factor. AB - Certain cytokines have been reported to exert neurotrophic actions in vivo and in vitro. In the present study, we investigated the possible neuroprotective actions of the cytokine human recombinant interleukin-1 beta (hrIL-1 beta) against excitatory amino acid (EAA)-induced neurodegeneration in cultured primary cortical neurons. Brief (15 min) exposure of cultures to submaximal concentrations of glutamate, NMDA, AMPA, or kainate caused extensive neuronal death (approximately 70% of all neurons). Neuronal damage induced by the EAAs was significantly reduced (up to 70%) by pretreatment with 500 ng/ml (6.5 x 10(3) U/ml) hrIL-1 beta for 24 hr. The neuroprotective effect of hrIL-1 beta was reversed by coapplication of an IL-1 receptor antagonist (IL-1ra, 50 micrograms/ml). Neuroprotective actions of hrIL-1 beta were also reduced by administration of a neutralizing monoclonal antibody to NGF (65% inhibition). In concordance, the neurotoxic actions of EAAs were significantly reduced (by 40%) after pretreatment with NGF (100 ng/ml for 48 hr). Furthermore, an additive neuroprotective effect of approximately 75% was observed when cultures were exposed to a combination of hrIL-1 beta and NGF. In contrast, exposure of cultures to high concentrations hrIL-1 beta alone (100 micrograms/ml, 1.3 x 10(6) U/ml) for periods up to 72 hr resulted in neurotoxicity, which was reversed by IL 1ra (1 mg/ml). These findings suggest that hrIL-1 beta can limit EAA-induced neuronal damage. These effects appear to be may be mediated, at least in part, via NGF. These findings may be relevant to the understanding of neurodegenerative diseases. PMID- 7538560 TI - Modulation of inspiratory drive to phrenic motoneurons by presynaptic adenosine A1 receptors. AB - The involvement and mechanisms of adenosine A1 receptors in regulating bulbospinal synaptic transmission of inspiratory drive to phrenic motoneurons were investigated. The adenosine analog N6-cyclopentyladenosine (CPA) induced a dose-dependent decrease of both inspiratory-modulated activity of C4 ventral roots and synaptic currents of phrenic motoneurons in an in vitro brainstem/spinal cord preparation from neonatal rats. No significant changes were observed in steady-state membrane current (during the expiratory phase). The depressant action of CPA on inspiratory drive was blocked by the selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT). The adenosine receptor antagonist 3-isobutyl-1-methylxanthine (IBMX) induced varying degrees of enhancement of inspiratory-modulated synaptic current, as did CPT. This suggests a role of endogenous adenosine in synaptic transmission of respiratory drive to phrenic motoneurons. The relative contribution of pre- and postsynaptic adenosine receptors was examined by looking at the effects of CPA on postsynaptic membrane properties and on spontaneous or miniature excitatory postsynaptic currents (EPSCs). CPA had no detectable effect on the input resistance of phrenic moto neurons. Moreover, the inward currents of phrenic moto-neurons in response to exogenously applied glutamate were not affected by adenosine-related compounds. On the other hand, CPA produced a significant decrease in the frequency of spontaneous and of miniature EPSCs. We conclude that adenosine can modulate transmission of inspiratory drive from bulbospinal neurons to phrenic motoneurons via presynaptic A1 receptors. PMID- 7538563 TI - Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway. AB - Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6 hydroxydopamine (6-OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long distance axon growth in the brain. PMID- 7538564 TI - L-glutamate-induced responses and cGMP-activated channels in three subtypes of retinal bipolar cells dissociated from the cat. AB - Effects of L-glutamate (Glu), the neurotransmitter released by photoreceptors, on isolated cat bipolar cells were examined. Membrane currents of bipolar cells were recorded by the patch-clamp technique in a conventional whole-cell recording configuration using pipettes containing 1 mM cGMP, which has been known to activate a cationic current sensitive to Glu in ON-type bipolar cells. ON-type bipolar cells (depolarized by light in in situ) and OFF-type bipolar cells (hyperpolarized by light) were identified by their response polarity to Glu. When the whole-cell configuration was established, ON-type bipolar cells showed a steady inward current which was suppressed by Glu, consistent with the response polarity observed in in situ recordings. In contrast, OFF-type cells did not show a steady current during the recordings. However, they responded to Glu with an increase in cationic conductance. Among recorded cells, rod-driven bipolar cells were identified by their immunoreactivity to anti-protein kinase C (PKC-IR) antibody. Examination of PKC-IR revealed that ON-type bipolar cells included both rod- and cone-driven bipolar cells, while OFF-type cells were all cone-driven bipolar cells. The cGMP-activated current observed in ON-type cells was accompanied by a change in the current fluctuation due to the opening and closing of underlying channels. Fluctuation analysis gave a unitary conductance value of 13 pS. In half of the cells examined, maximum open probability reached almost 100%. The cGMP-activated channel in bipolar cells seems novel, fundamentally different from those found in photoreceptor cells or olfactory receptor cells. PMID- 7538565 TI - Selective regulation of current densities underlies spontaneous changes in the activity of cultured neurons. AB - We study the electrical activity patterns and the expression of conductances in adult stomatogastric ganglion (STG) neurons as a function of time in primary cell culture. When first plated in culture, these neurons had few active properties. After 1 d in culture they produced small action potentials that rapidly inactivated during maintained depolarization. After 2 d in culture they fired large action potentials tonically when depolarized, and their properties resembled very closely the properties of STG neurons pharmacologically isolated in the ganglion. After 3-4 d in culture, however, their electrical properties changed and they fired in bursts when depolarized. We characterized the currents expressed by these neurons in culture. They included two TTX-sensitive sodium currents, a calcium current, a delayed-rectifier-like current, a calcium dependent potassium current, and two A-type currents. The changes in firing properties with time in culture were accompanied by an increase in inward and decrease in outward current densities. A single-compartment conductance-based model of an STG neuron was constructed by fitting the currents measured in the biological neurons. When the current densities in the model neuron were matched to those measured for the biological neurons in each activity state, the model neuron closely reproduced each state, indicating that the changes in current densities are sufficient to account for the changes in intrinsic properties. These data indicate that STG neurons isolated in culture change their intrinsic electrical properties by selectively adjusting the magnitudes of their ionic conductances. PMID- 7538566 TI - A glutamate-activated chloride current in cone-driven ON bipolar cells of the white perch retina. AB - Cone-driven ON-type bipolar cells were patch clamped in white perch retinal slices. Application of glutamate activated a current (IGlu) that was mediated by a conductance increase. The reversal potential for IGlu followed ECl closely when the intracellular chloride concentration was varied. IGlu was not blocked by 100 microM picrotoxin or 1 microM strychnine, indicating that it was not caused by inhibitory input. IGlu is not mediated by a typical ionotropic glutamate receptor since it was not activated by kainate, AMPA, or NMDA, or blocked by kynurenic acid, CNQX, DNQX, or AP-V. Further, IGlu is not mediated by a known metabotropic glutamate receptor since it was not activated by quisqualic acid, AP-4, ACPD, or ibotenate. IGlu required the presence of extracellular sodium and could be partially inhibited by the glutamate uptake inhibitors THA and tPDC. This is suggestive of sodium-dependent glutamate transport. However, when intracellular sodium was greatly increased, neither the magnitude nor reversal potential of IGlu was substantively affected. Thus, IGlu appears to involve a chloride channel activated by a glutamate receptor with transporter-like pharmacology. IGlu is localized to the dendrites of the bipolar cell, where bipolar cells receive an endogenous glutamatergic input from photoreceptors. Further, the reversal potential of the light response in these cells is the same as that of IGlu. Thus, it seems likely that IGlu is the current responsible for the cone component of the ON bipolar cell light response in the teleost retina. PMID- 7538567 TI - Dopamine enhances glutamate-activated currents in spinal motoneurons. AB - In cultured embryonic chick motoneurons, glutamate-activated currents rise quickly and then decay rapidly to relatively small steady-state current levels. Dopaminergic modulation of these receptors was studied using patch-clamp recording techniques. At concentrations > or = 10 nM, dopamine enhanced glutamate activated currents by about 200%. This enhancement was diminished by the nonspecific dopamine receptor antagonist S(+)-apomorphine and the more specific D1 receptor antagonist SCH23390, and it was mimicked by the D1 partial agonist SKF38393. Glutamate receptor desensitization rate was not altered in the presence of dopamine. Enhancement was specific to the kainate component. Current-variance analysis indicated that in the presence of dopamine the conductances of glutamate activated channels were not altered but that the relative fraction of kainate type channels activated by glutamate increased. Intracellular cAMP levels increased by 33% following exposure to 100 microM dopamine. The effects of elevated cAMP or protein kinase A (PKA) were tested by including 100 microM cAMP or PKA, respectively, in the recording pipette solution. This increased the kainate-activated currents specifically. Dopaminergic enhancement was not observed when a PKA inhibitor was in the pipette. mRNA encoding D1 was detected in the spinal cord by a reverse transcription, polymerase chain-reaction amplification procedure. Thus, dopamine is acting most probably on an avian homolog of the D1 receptor family. We conclude that dopamine causes cAMP to increase, which results in increased activation of kainate-gated channels during glutamate-mediated transmission. PMID- 7538568 TI - Relationships between dendritic fields and functional architecture in striate cortex of normal and visually deprived cats. AB - We examined relationships between the pattern of geniculocortical innervation and the dendritic fields of cells in layer 4 of in cat primary visual cortex. Experiments were performed on normal animals and on cats in which the geniculocortical projection was altered by monocular deprivation or by the induction of divergent squint during the critical period. Thalamic afferents providing the input from the contralateral eye were anterogradely labeled by injecting the fluorescent tracer Dil into lamina A of the lateral geniculate nucleus. Intracellular staining with Lucifer yellow in slice preparations allowed simultaneous visualization of the morphology of individual cells and the thalamic afferents. Our results demonstrate that spiny stellate cells close to the upper and lower margin of the geniculocortical input have highly asymmetric dendritic fields, and thereby confine their dendrites to the termination zone of these afferents. This effect was specific for the cell class; it was not observed in pyramidal neurons. These dendritic asymmetries perpendicular to the laminar borders of spiny stellate cells were not altered by monocular deprivation or strabismus. In contrast, visual deprivation strongly influenced the dendritic arbors of spiny stellate cells near the borders between adjacent ocular dominance columns. In normal animals, the dendrites of cells near columnar borders remained preferentially within one column. These dendritic asymmetries became much more pronounced in strabismic animals. Monocular deprivation weakened the influence of the columnar borders on dendritic fields. Spiny stellate cells within the columns of the open eye exhibited a slight tendency to confine their dendrites to these columns. Cells in the columns of the deprived eye showed the opposite effect; they extended their dendrites preferentially into the adjacent columns of the open eye. These results demonstrate that the segregation of geniculocortical afferents into ocular dominance columns and its perturbation by manipulation of the visual input plays an important role in defining the morphology of cortical target cells. Thus, activity-dependent structural changes not only occur at the level of the presynaptic terminals, but also at the level of the postsynaptic target cells, and thereby contribute to build up the functional architecture of the cortex. PMID- 7538569 TI - Increased expression of neuronal nitric oxide synthase (NOS) in visceral neurons after nerve injury. AB - The distribution of nitric oxide synthase immunoreactivity (NOS-IR) and the changes in this distribution after peripheral axotomy were examined in lumbosacral afferent and preganglionic neurons (PGNs) innervating the pelvic viscera of the male rat. The visceral neurons in L6-S1 and L1-L2 dorsal root ganglia (DRG) and in the spinal cord were identified by retrograde axonal transport following injection of Fluorogold (FG) into the major pelvic ganglion (MPG). Axotomy was performed by removing the MPG on one side 2-4 weeks prior to sacrificing the animals. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of control animals. Significantly greater numbers of NOS-IR cells were present in thoracic (T8, T10, T12; 30-44 cell profiles/section) and rostral lumbar DRGs (L1-L2; 3-15 NOS-IR cell profiles/section) compared to caudal lumbosacral (L5-S1) DRGs (0.2-0.7 cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 11 NOS-IR cell profiles/section) but not in the L2 or L5 DRG ipsilateral to axotomy. In these ganglia, an average of 37.0 +/- 4.0% (L6) and 20.6 +/- 2.2% (S1), respectively, of FG-labeled pelvic afferent neurons were NOS-IR compared to 1.1 +/- 0.5% (L6) and 2.5 +/- 1.4% (S1) contralateral to the axotomy. Following axotomy, a significantly greater percentage of dye-labeled pelvic visceral afferents in the L1 and L2 DRG also exhibited NOS-IR in comparison to the contralateral side. Following axotomy, NOS IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the sacral parasympathetic nucleus (SPN) on the ipsilateral side of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L4, L5, or S2). Axotomy also changed the numbers of NADPH-d-positive and NOS-IR cells in the region of the SPN in the L6 spinal segment. Contralateral to the axotomy 38.3 +/- 4.0% of PGNs in the L6 spinal segment were colabeled with NOS-IR; however, ipsilateral to axotomy, a significantly greater percentage (61.0 +/- 3.0%; p < or = 0.01) of PGNs exhibited NOS-IR. Axotomy did not alter the distribution of PGNs in the S1 segment exhibiting NOS-IR. These results indicate that NOS-IR in visceral afferent and PGNs is plastic and can be upregulated by peripheral nerve injury. PMID- 7538570 TI - A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death. AB - Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4 piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult. PMID- 7538572 TI - The role of radiation therapy in palliative care. AB - Radiation therapy plays a significant role in the palliation of symptoms of patients with malignant disease. This article looks at the scientific and clinical rationales for its use and discusses some of the current controversies in palliative radiotherapy. The utility of radiation therapy is discussed and potential side effects are described. Radiation oncologists are important members of the multidisciplinary team providing palliative care. Their involvement in the care of patients with advanced or metastatic malignant disease allows patients to benefit from a modality of treatment which can be both effective and minimally inconvenient when used appropriately. PMID- 7538571 TI - Fast, local signal transduction between the mu opioid receptor and Ca2+ channels. AB - We used patch-clamp methods to describe signal transduction between the mu opioid receptor, the binding site for morphine, and high-threshold Ca2+ channels in dorsal root ganglion (DRG) sensory neurons from adult rats. Opioid signaling persists in excised membrane patches, and an activated opioid receptor can only inhibit nearby Ca2+ channels; thus, no readily diffusible second-messenger molecule mediates between the mu receptor and Ca2+ channels. Inhibition of Ca2+ channels begins several hundred msec after application of opioid and it is maximal by 5 sec; this is faster than typical phosphorylation cascades. Blockade of the known serine-threonine kinases and phosphatases does not affect this opioid signaling and, as shown previously by Seward et al. (1991) and Moises et al. (1994a), pertussis toxin eliminates virtually all of the effect. Inhibited channels can open, but their half-activation voltage is unphysiologically positive. The link between the mu receptor and Ca2+ channels is clearly unlike the protein kinase C-dependent paths that couple mu receptors to NMDA channels in dorsal horn neurons (Chen and Huang, 1991) and alpha-adrenergic receptors to Ca2+ channels in DRG neurons (Diverse-Pierluissi and Dunlap, 1993). The rapid kinetics and tight localization of the signaling path are properties expected if receptor and channel are linked directly by a G-protein, but these properties do not constitute proof of such a pathway. PMID- 7538575 TI - Pancreatitis and organic acidemias. PMID- 7538573 TI - When does palliative care begin? A needs assessment of cancer patients with recurrent disease. AB - This cross-sectional study compared the prevalence and intensity of needs expressed by cancer patients at the time of first recurrence (n = 75) with those of patients at the time of disease progression (n = 75). On a 72-item Likert-type scale participants rated from "none" to "very much" the severity of physical symptoms; emotional, psychological, and social problems; and difficulties with activities of daily living. The progressive disease group reported more problems than the first recurrence group in 11 of 24 (47%, p < 0.05) symptom-related questions. The progressive disease group also expressed greater needs in 19% of the function-related questions. There were no differences in reported psychological problems, responses to recurrence, or greatest concern between the two groups. These results support the need for palliative care throughout the entire course of illness. PMID- 7538576 TI - Assessment of human immunodeficiency virus/acquired immunodeficiency syndrome audiovisual materials designed for grades 7 through 12. AB - Nurses are often involved in formulating and implementing HIV/AIDS education programs in the public schools. A tool was developed to assess the inclusion of components that deal with factual information, social implications of HIV/AIDS, abstinence, condom usage, and attitudes in audiovisual materials. This information, in checklist format, can assist nurses in evaluating audiovisual materials and in selecting the most appropriate materials dealing with HIV/AIDS for groups of adolescents. Twenty-four audiovisual materials from a rural education agency were evaluated using this checklist; the tool provided a method to analyze and convey information regarding the components of the HIV/AIDS audiovisual materials, which can assist nurses in choosing or recommending educational resources appropriate in programs for teaching adolescents about sexuality and HIV/AIDS prevention. PMID- 7538574 TI - Disseminated intravascular coagulation associated with granulocyte colony stimulating factor therapy in a child with human immunodeficiency virus infection. AB - We report a case of disseminated intravascular coagulopathy, apparently caused by exposure to granulocyte colony-stimulating factor (G-CSF). The medical records of patients treated for more than 30 consecutive days with subcutaneously administered G-CSF were reviewed for the occurrence of thrombocytopenia or coagulation abnormalities. New-onset thrombocytopenia with a platelet count less than 100 x 10(9) cells/L (< 100,000 cells/mm3) developed in 9 of 23 patients (39%) after a median of 11 weeks of treatment with G-CSF at dosages between 1 and 10 micrograms/kg per day. PMID- 7538578 TI - Neurokinin1 receptor mediation of the vasodepressor effects of substance P in the nucleus of the tractus solitarius. AB - The involvement of substance P (SP) in cardiovascular regulatory mechanisms was investigated in the nucleus of the tractus solitarius (nTS) of urethane anesthetized Wistar rats. Unilateral microinjections of SP (0.25 pmol in 50 nl) or the neurokinin (NK) 1 agonist [Sar9,Met(O2)11]-SP (0.25 pmol) resulted in decreases of blood pressure and heart rate when placed in the nTS but not in neighboring structures. Bilateral vagotomy abolished SP-induced bradycardia but did not prevent the vasodepression. Microinjection of the NK2 agonist alpha-NKA[4 10] (0.25 and 1.25 pmol) or the NK3 agonist senktide (0.25 and 1.25 pmol) had no effect on arterial pressure or heart rate. The relatively non-selective tachykininergic antagonist [D-Pro2,D-Trp7.9]-SP (10 pmol in 100 nl) and the NK1 antagonist cyclo(Gln-D-Trp-(N-Me)-Phe(R)Gly[ANC-2]Leu-Met)2 (1.25 pmol) prevented the cardiovascular effects of both SP and the NK1 agonist, whereas the NK2 antagonist cyclo(GlnTrpPheGlyLeuMet) was ineffective. Neither of the effective antagonists affected resting blood pressure, heart rate or the gain or threshold of the baroreflex. These data indicate that SP acts in the nTS via NK1 receptors to evoke a vagally-mediated bradycardia and a hypotension that is independent of changes of heart rate and suggest further that SP is not integral to the circuitry subserving the baroreflex. PMID- 7538577 TI - Inhalational anesthetics do not alter nitric oxide synthase activity. AB - Inhalational anesthetics inhibit the nitric oxide (NO)-soluble guanylate cyclase signaling pathway in vascular and neuronal tissues and it has been proposed that this inhibition is due to several mechanisms, which include a direct inhibition of NO synthase. To determine the direct interaction of anesthetics with NO synthase, the effects of halothane, isoflurane and enflurane on NO synthase activity of bovine and rat brains and cultured bovine aortic endothelial cells were investigated. Halothane and enflurane at 1% to 3% concentrations produced no significant effect on crude bovine brain NO synthase activity, as measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Similarly, crude rat brain NO synthase activity was not affected by exposure to 1% to 4% halothane or isoflurane. The effects of inhalational anesthetics on the crude bovine brain NO synthase activity were not altered when assayed at two different temperatures (22 degrees C and 37 degrees C). Halothane and isoflurane produced no significant effects on the activity of partially purified rat brain NO synthase at different concentrations of L-[3H]arginine in the reaction mixture. Partially purified endothelial NO synthase, when equilibrated with halothane or isoflurane (0.5-2%), exhibited no significant alteration in enzyme activity. This study suggests that the effects of inhalational anesthetics on NO synthesis in rat and bovine brains and in vascular endothelial cells are not due to their direct interaction with NO synthase. PMID- 7538579 TI - The monoamine oxidase-B inhibitor L-deprenyl protects against 3,4 methylenedioxymethamphetamine-induced lipid peroxidation and long-term serotonergic deficits. AB - 3,4-Methylenedioxymethamphetamine (MDMA)-induced serotonergic neurotoxicity was assessed in the striatum, hippocampus and frontal cortex of rats by using [3H]paroxetine binding to label serotonin (5-HT) uptake sites and 5-HT and 5 hydroxyindoleacetic acid (5-HIAA) levels as markers of serotonergic function. NMDA (40 mg/kg) induced a significant decrease in both [3H]paroxetine binding Bmax and 5-HT and 5-HIAA levels 7 days after treatment. The monoamine oxidase-B inhibitor L-deprenyl (2 mg/kg) administered 30 min before MDMA blocked these decreases. MDMA (40 mg/kg) also maximally increased the formation of thiobarbituric acid reactive substances (an indicator of lipid peroxidation) 12 hr after treatment in all three brain regions studied. This increase in malondialdehyde formation was also blocked by pretreatment with L-deprenyl. Tryptophan hydroxylase (TPH) activity was also significantly reduced 18 hr after MDMA. L-Deprenyl reversed this decrease in TPH activity. Another experiment confirmed that a significant fraction of [3H]dopamine uptake into hippocampal synaptosomes was blocked by 500 nM fluoxetine, a selective 5-HT uptake inhibitor. These data suggest that the deamination by monoamine oxidase-B of excessive dopamine within the 5-HT terminal generates hydrogen peroxide that may lead to membrane lipid peroxidation, and perhaps other oxidative insults, resulting in selective 5-HT terminal degeneration subsequent to MDMA treatment. PMID- 7538580 TI - Metabolism of exogenous cyclic AMP to adenosine in the rat kidney. AB - Although adenosine contributes importantly to the regulation of renin release, renal vascular resistance and renal tubular reabsorption, the metabolic pathways that control the intrarenal production rate of adenosine remain ill defined. The objective of this study was to determine whether extracellular metabolism of cyclic AMP to AMP by extracellular phosphodiesterase and hence to adenosine by ecto-5'-nucleotidase can occur in the intact kidney. To test this hypothesis, five experimental series were conducted in kidneys from male Sprague-Dawley rats perfused in a nonrecirculating system (5 ml/min) in vitro with oxygenated Tyrode's solution at 37 degrees C. In each experimental series, cyclic AMP was added to the Tyrode's solution and the renal secretion rates (i.e., the renal venous concentration of purine x the perfusion flow rate) of AMP, adenosine and inosine were determined using high-performance liquid chromatography. In the first experimental series, only cyclic AMP was added to the perfusate. In the second, third, fourth and fifth experimental series, kidneys were perfused with Tyrode's solution containing both cyclic AMP and either 3-isobutyl-1 methylxanthine (a phosphodiesterase inhibitor), alpha,beta-methyleneadenosine-5' diphosphate (an ecto-5'-nucleotidase inhibitor), dilazep (an adenosine transport inhibitor) or 1,3-dipropyl-8-p-sulfophenylxanthine (a xanthine that is restricted to the extracellular compartment). In the first experimental series (n = 8), addition of cyclic AMP to the perfusate resulted in significant concentration related increases in the renal secretion rates of AMP, adenosine and inosine, with the increase in AMP secretion being significantly greater than the increases in adenosine or inosine secretions (delta adenosine secretion/delta AMP secretion = 0.38 +/- 0.10).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538581 TI - Cannabinoids modulate voltage sensitive potassium A-current in hippocampal neurons via a cAMP-dependent process. AB - Previous studies have shown that cannabinoid receptor analogs increase voltage dependent potassium A-current (IA) in cultured hippocampal cells. Because cannabinoid receptors inhibit adenylate cyclase, the present study explored whether cAMP played a role in mediating this effect on IA. The specific issue of whether cannabinoid receptor modulation of voltage-dependent IA acts via a cAMP dependent process was investigated. The cAMP analog, 8-bromo-cAMP, as well as the adenylate cyclase stimulant forskolin, produced concentration-dependent shifts in IA that were opposite those produced by cannabinoid receptor ligands. Moreover, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also produced a marked negative shift in the steady-state voltage dependence of IA and increased the effect of forskolin on IA. As shown in previous studies, the cannabinoid agonist WIN 55,212-2 increased IA via a decrease in steady-state voltage dependent inactivation of IA. WIN 55,212-2 also reversed the effects of forskolin on IA. The electrophysiological studies were paralleled by direct assays of cAMP in these cells, where cannabinoids inhibited forskolin-stimulated cAMP by 50% in a pertussis toxin-sensitive manner. The results confirmed that pertussis toxin sensitive cannabinoid receptor-mediated changes in IA were probably the result of inhibition of adenylate cyclase. The findings are discussed in terms of modulation of IA conductance properties via cannabinoid receptor-mediated inhibition of cAMP levels within the cell. PMID- 7538582 TI - Effects of recombinant human hemoglobin on motor functions of the opossum esophagus. AB - Chemically altered hemoglobins are being investigated as blood substitutes. They may affect numerous biological processes since free hemoglobin binds nitric oxide (NO). Nitric oxide is a neural mediator of relaxation of the lower esophageal sphincter (LES) and esophageal peristalsis. We hypothesize that recombinant human hemoglobin (rHb1.1) alters esophageal motor function by scavenging NO. Contraction of transverse muscle strips from the opossum esophagus and LES was monitored. Transmembrane potential differences of circular smooth muscle from the esophagus were recorded using glass microelectrodes. Intrinsic esophageal nerves were stimulated electrically. Esophageal manometries were performed with a low compliance perfused-catheter system. The activity of the enzyme NO synthase was determined with the citrulline assay. Recombinant hemoglobin diminished nerve induced relaxation of LES muscle but did not alter LES tone. Circular esophageal muscle responded to nerve stimulation with an inhibitory junction potential and a mechanical off response. Recombinant hemoglobin diminished the inhibitory junction potential and shortened the latency of the off response. It increased the velocity of esophageal peristalsis, decreased the amplitudes of these contractions and diminished LES relaxation. Cyanomethemoglobin had little effect on nerve- or swallow-induced responses. Hemoglobin did not inhibit the activity of NO synthase. Recombinant human hemoglobin appears to alter esophageal motor function by scavenging NO. PMID- 7538583 TI - Induction of calpain-mediated spectrin fragments by pathogenic treatments in long term hippocampal slices. AB - The use of cultured hippocampal slices for studies of calpain-mediated pathogenesis was investigated. Breakdown products (BDPs), which result from proteolysis of spectrin by calpain I, were assayed with BDP-specific antibodies developed against peptide sequences on either side of the calpain cleavage site. The antibodies recognized either amino- or carboxy-terminal BDPs (147-kD BDPN and 152-kD BDPC, respectively). Various pathogenic manipulations, including trimethyltin, certain snake venoms and agonists for excitatory amino acid receptors, were found to cause rapid and pronounced increases in the proteolytic fragments. These effects were selective, i.e., chemicals or toxins directed at nonglutamatergic neurons had little effect on BDP concentrations. Transient accumulations of spectrin fragments were obtained with brief applications of N methyl-D-aspartate; longer infusions resulted in lasting increases. Results similar to these have been observed in vivo with ischemic episodes of varying duration. Agonists of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subclass of glutamate receptors produced significant increases in spectrin proteolysis; however, prolonged exposure of the slices to centrally active drugs that enhance the currents passed by the receptors did not. The sensitivity, selectivity and temporal properties of the proteolytic response support the idea that cultured slices can be used to analyze the events leading to and following from calpain activation in the adult brain. PMID- 7538584 TI - Modulation of sympathetic vasoconstriction by sensory nerves and nitric oxide in rat irideal arterioles. AB - Transmural nerve stimulation of arterioles in the rat iris produces a vasoconstriction mediated via alpha-1B adrenoceptors and the mobilization of intracellular calcium. This study has investigated the role of sensory nerves in modulating this vasoconstriction using isolated preparations of iris perfused with Krebs' solution. Repeated stimulation of the transmural nerves for 1 sec (10 Hz), at intervals less than 2 min, produced a rapid and long-lasting loss of the vasoconstriction. This inhibition was not seen in arterioles taken from rats treated neonatally with capsaicin (50 mg/kg) nor after application of capsaicin (10(-5) M) to control preparations. In arterioles from control rats, both the substance P analog Sar9 and calcitonin gene-related peptide (CGRP) were effective in inhibiting nerve-mediated vasoconstriction. L-NG-nitroarginine methyl ester (10(-5) M), but not D-NG-nitroarginine methyl ester (10(-5) M), prevented the loss of vasoconstriction during repetitive stimulation, which suggested the involvement of nitric oxide. Antagonists to the NK1 and NK2 neurokinin receptor subtypes, RP 67580 and L-659877, respectively, were without effect, whereas the antagonist to the CGRP1 receptor, CGRP8-37, prevented the loss of vasoconstriction during repetitive nerve stimulation. The effects of both Sar9 and CGRP (10(-8) M) in inhibiting the nerve-mediated vasoconstriction were prevented by preincubation in L-NG-nitroarginine methyl ester (10(-5) M), which suggested that the mechanism of action of both peptides involved the release of nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538585 TI - Effect of ruthenium red on voltage-sensitive Ca++ channels. AB - The organometallic dye, ruthenium red (RR) inhibited Ca++ influx, omega-conotoxin GVIA-sensitive Ca++ binding and Ca(++)-dependent neurotransmitter release in a qualitatively similar manner in rat and chicken synaptosomes and in mammalian neuromuscular preparations, but had no effect on Ca(++)-dependent processes mediated by the dihydropyridine-sensitive Ca++ channel. These effects are specific for the RR complex, as RuCl3 affected neither Ca++ influx, omega conotoxin GVIA binding nor neurotransmitter release, but did, however, in contrast to RR, displace [3H]nitrendipine from synaptosomes. RR, in a manner similar to omega-conotoxin MVIIC and omega-agatoxin IVA (AgaIVA), also effectively inhibited the response of the rat diaphragm to nerve stimulation and blocked AgaIVA-sensitive Ca++ influx in the rat brain, suggesting a significant interaction at the P-type voltage-sensitive Ca++ channel. These effects of RR suggest that this amino complex affects both the N and the P domain of the Ca++ channel in the chicken brain and both the N- and P-type Ca++ channel which is intimately coupled to the Ca++ influx and neurotransmitter release in rat synaptosomes. Its ability to block all of the Ca++ influx in mammalian brain preparations and to inhibit completely the nerve-stimulated rat neuromuscular junction certainly indicates a significant action at the P-type voltage-sensitive Ca++ channel, similar to omega-conotoxin MVIIC or AgaIVA. RR should prove to be a valuable synthetic, inexpensive tool with which to probe the neuropharmacology of the mammalian neurotransmitter-linked voltage-sensitive Ca++ channels. PMID- 7538586 TI - Cartilage metalloproteases in disuse atrophy. AB - A canine knee model of disuse atrophy produced by nonrigid fixation (sling) was characterized in respect to variables of proteoglycan size distribution, as well as biomechanical properties versus controls. Using this model, we found, in addition to the accepted dogma attributing changes to reduced protein synthesis by chondrocytes, that there is elevation of proteases and depression of tissue inhibitor of metalloproteases (TIMP) in atrophic knee cartilage. The findings are suggestive of cartilage remodelling reminiscent of bone remodelling in disuse atrophy reported by others. Whether the abnormal changes of protease-TIMP balance in knee cartilage can be retarded prophylactically by concurrent treatment with pentosan polysulfate and insulin like growth factor 1 remains uncertain. PMID- 7538587 TI - Chondrocytes and antirheumatic drugs. AB - Effects of antirheumatic drugs upon cartilage matrix metabolism have been studied in a variety of chondrocyte in vitro systems. When compared longterm in 60 experiments under standardized conditions, articular chondrocytes cultured in agarose exhibit variability in proteoglycan synthesis, and its suppression by interleukin 1 (IL-1), but a high reproducibility in the modulation of these effects by antirheumatic drugs. Pentosan polysulfate, tenidap, tiaprofenic acid, and RO 31-9790 all compensated to a certain extent the IL-1 induced suppression of matrix synthesis in bovine chondrocytes, but only for tiaprofenic acid could this be confirmed using chondrocytes of human origin. Culture conditions as well as species differences should therefore be considered carefully when chondrocyte cultures are used as pharmacological models. PMID- 7538588 TI - Screening tests for growth hormone deficiency. PMID- 7538590 TI - Benzophenone derivatives: a novel series of potent and selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase. AB - A series of benzophenone derivatives has been synthesized and evaluated as inhibitors of HIV-1 reverse transcriptase (RT) and the growth of HIV-1 in MT-4 cells. Through the use of the structure-activity relationships within this series of compounds and computational chemistry techniques, a binding conformation is proposed. The SAR also indicated that the major interactions of 1h with the RT enzyme are through hydrogen bonding of the amide and benzophenone carbonyls and pi-orbital interactions with the benzophenone nucleus and an aromatic function separated from the benzophenone by a suitable spacer group. The crystal structure of compound 1h has been determined. A number of compounds with potent inhibitory activity against HIV-1 RT and HIV in cellular assays at levels comparable with AZT and our efforts to identify a metabolically stable analogue are described. PMID- 7538589 TI - Synthesis and anti-HIV activity of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential multifunctional inhibitors of HIV-1 reverse transcriptase. AB - In an attempt to combine the HIV-inhibitory capacity of 2',3'-dideoxynucleoside (ddN) analogues and non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), we have designed, synthesized, and evaluated for their anti-HIV activity several dimers of the general formula [ddN]-(CH2)n-[NNRTI]. These dimers combine in their structure a ddN such as AZT and a NNRTI such as TSAO-T and HEPT linked through an appropriate spacer between the N-3 of the thymine base of both compounds. The [TSAO-T]-(CH2)n-[AZT] dimers proved markedly inhibitory to HIV-1. Also, if AZT was replaced by thymidine in the dimer molecules, potent anti-HIV-1 activity was observed. However, although the compounds proved inhibitory to HIV-1, they were less potent inhibitors than the parent compounds from which they were derived. None of the dimers were endowed with anti-HIV-2 activity. In contrast with the TSAO-T monomers, none of the TSAO-T-containing dimers proved markedly cytotoxic to the cells. There was a clear trend toward decreased antiviral potency with lengthening the methylene spacer in the [TSAO-T]-(CH2)n-[AZT] dimers. PMID- 7538591 TI - Determination of the differential effects of hydrogen bonding and water release on the binding of FK506 to native and Tyr82-->Phe82 FKBP-12 proteins using free energy simulations. AB - We use the thermodynamic integration technique to calculate the free energy associated with the Tyr82-->Phe82 mutation (Y82F) in the protein FKBP-12, both free and bound to known inhibitor FK506 (tacrolimis). We find that the net difference in free energy for the two changes is 0.85 kcal/mol, with the binding of FK506 relatively more favorable for the native protein than the mutant. This net energy compares very favorably with the experimentally measured value of 0.60 kcal/mol. The results indicate that the relatively better binding of FK506 to the native protein is driven by the favorable entropy associated with the release of water molecules from the protein when the ligand binds. For a variety of reasons, modest size of the system, smallness of the change being examined, rapid convergence of the ensemble that needs to be determined and use of statistical estimates to control sampling, we have been able to carry out atypically reliable and reproducible free energy calculations for this protein system. Free energy changes for the two simulations (Y82F FKBP-12/FK506 and Y82F FKBP-12) have been calculated a total of eight times each, to compare a variety of different methodological choices and to ensure that the results are statistically significant. Detailed analysis of the free energy results has been carried out, and indicates that even when applicable, deconvolution of the total free energy into components can be very difficult, that the statistical error estimates can give a reasonable bound on the error in a simulation, and that one must be careful to use the same simulation protocol in all simulations being compared. PMID- 7538592 TI - Interleukin 12: newest member of the antiangiogenesis club. PMID- 7538593 TI - Inhibition of angiogenesis in vivo by interleukin 12. AB - BACKGROUND: In previous animal studies, interleukin 12 (IL 12) was shown to inhibit the growth of a wide spectrum of tumors in vivo but to have no direct effect on tumor cells in vitro. Also, contrary to the expectation of a T-cell mediated effect, the antitumor activity of IL 12 was not completely abrogated in tests of T-cell-deficient mice. These observations suggest that IL 12 may possess antiangiogenic properties that account for its tumor-inhibitory effects in vivo. PURPOSE: Our goal was to investigate the hypothesis that IL 12 has antiangiogenic properties. METHODS: A model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL 12 and interferon gamma (IFN gamma) on angiogenesis in vivo. Different strains of male mice, e.g., immunocompetent C57BL/6 mice, severe combined immune-deficient (SCID) mice, natural killer cell-deficient beige mice, and T-cell-deficient nude mice, were treated with IL 12 (1 microgram/day) intraperitoneally for 5 consecutive days. The extent of neovascularization in response to a basic fibroblast growth factor pellet and the inhibition of neovascularization by IL 12 or IFN gamma were assessed by measuring the maximal vessel length and the corneal circumference involved in new blood vessel formation. The antitumor activities of IL 12 and of the angiogenesis inhibitor AGM-1470 were evaluated in Lewis lung carcinoma bearing mice. In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreatic islet endothelial cells, and mouse hemangioendothelioma cells. RESULTS: IL 12 treatment almost completely inhibited corneal neovascularization in C57BL/6, SCID, and beige mice. This potent suppression of angiogenesis was prevented by the administration of IFN gamma neutralizing antibodies, suggesting that the suppression was mediated through IFN gamma. In addition, the administration of IFN gamma reproduced the antiangiogenic effects observed during treatment with IL 12. Treatment with IL 12 and AGM-1470 combined did not increase toxicity and showed a trend toward enhanced antitumor efficacy in Lewis lung carcinoma-bearing mice. CONCLUSIONS: IL 12 strongly inhibits neovascularization. This effect is not mediated by a specific cell type of the immune system. Instead, IL 12 has been shown to induce IFN gamma, which, in turn, appears to play a critical role as a mediator of the antiangiogenic effects of IL 12. IMPLICATIONS: Recognition of the mechanisms of the antiangiogenic properties of IL 12 may be crucial in planning its clinical applications, including a possibility of coadministration with other inhibitors of neovascularization. PMID- 7538595 TI - Enhanced chemoresistance by elevation of p185neu levels in HER-2/neu-transfected human lung cancer cells. PMID- 7538594 TI - Vasectomy and prostate cancer: results from a multiethnic case-control study. AB - BACKGROUND: Vasectomy, a widely used form of contraception, has been associated in some studies with increased prostate cancer risk. PURPOSE: We assessed this association on the basis of data collected in a large multiethnic case-control study of prostate cancer that was conducted in the United States (Los Angeles, San Francisco, and Hawaii) and Canada (Toronto and Vancouver). METHODS: In home interviews conducted with newly diagnosed prostate cancer case patients and population control subjects, we obtained information on the participants' medical history, including a history of vasectomy and the age at which the procedure was performed, as well as other potential risk factors. Blood samples were collected from control subjects only and were assayed for concentration of sex hormones and sex hormone-binding globulin. RESULTS: The present analysis was based on 1642 prostate cancer patients and 1636 control subjects. A history of vasectomy was not significantly associated with prostate cancer risk among all racial/ethnic groups combined (odds ratio [OR] = 1.1; 95% confidence interval [CI] = 0.83-1.3), whites (OR = 0.94; 95% CI = 0.69-1.3), blacks (OR = 1.0; 95% CI = 0.59-1.8), or Chinese-Americans (OR = 0.96; 95% CI = 0.42-2.2). Among Japanese-Americans, the OR was 1.8 (95% CI = 0.97-3.4), but the statistically nonsignificant elevation in risk was limited to more educated men and those with localized cancers. ORs did not vary significantly by age at vasectomy or years since vasectomy. We found a lower serum concentration of sex hormone-binding globulin and a higher ratio of dihydrotestosterone to testosterone among vasectomized control subjects than among nonvasectomized control subjects. CONCLUSIONS: The findings of this study do not support previous reports of increased prostate cancer risk associated with vasectomy. However, the altered endocrine profiles of vasectomized control subjects seen in this cross-sectional comparison warrant further evaluation in longitudinal studies. PMID- 7538596 TI - Evaluation of the use of uncertainty factors in deriving RfDs for some chlorinated compounds. AB - Risk assessment of exposure to chemicals having a toxic endpoint routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. The purpose of this investigation was to evaluate whether the magnitude of the U.S. Environmental Protection Agency (EPA) 10x uncertainty factors has scientific merit when compared with data from recent human and animal experimental studies. A compilation and comparison of ratios between LOAEL/NOAEL (lowest observed adverse effect level/no observed adverse effect level) and subchronic/chronic values was made for six chlorinated compounds, namely, carbon tetrachloride, methylene chloride, pentachlorophenol, monochlorobenzene, chlorpyrifos, and 1,1 dichloroethane. Data sets demonstrated that 91.3% of the LOAEL/NOAEL ratios were < or = 6 while 87% of the ratios for the same parameter were < or = 5. Furthermore, subchronic/chronic ratios were < or = 3.5. From our investigation we concluded that automatic safety factors of 10x are not scientifically supportable and are overly conservative for the chlorinated compounds studied here. PMID- 7538597 TI - Pathogenesis and prophylaxis of postoperative thromboembolic disease in urological pelvic surgery. PMID- 7538600 TI - Prostate cancer. PMID- 7538598 TI - Prostate specific antigen density is not an independent predictor of response for prostate cancer treated by conformal radiotherapy. AB - Although prostate specific antigen (PSA) density appears to be an important discriminator between benign and malignant prostatic disease, conflicting data exist concerning its prognostic value. The present study was undertaken to confirm whether PSA density represents a new prognostic indicator of disease-free survival for prostate cancer treated with conformal radio-therapy. Between April 1989 and December 1992, 186 patients with organ confined prostate cancer were treated with definitive irradiation according to previously published conformal guidelines. The PSA density was defined as the ratio of the pretreatment serum PSA (ng./ml.) to the prostate volume (ml.) as determined from treatment planning computerized tomography. The median PSA density was 0.15 with a range of 0.02 to 2.12. A statistically significant advantage in actuarial freedom from biochemical relapse was noted for patients with pretreatment PSA levels less than 15 ng./ml. when compared to those with higher pretreatment PSA levels (3-year freedom from biochemical relapse 85% versus 28%, p < 0.001). Also, patients with PSA density of 0.15 or less had statistically superior freedom from biochemical relapse compared to their counterparts with higher PSA density (3-year freedom from biochemical relapse 88% versus 28%, p < 0.001). In a multivariate analysis only the baseline PSA (p < 0.002) and the Gleason score (p < 0.002) emerged as significant predictors of prolonged freedom from biochemical relapse. The PSA density had no impact on freedom from biochemical relapse whether it was entered into this multivariate model as a continuous or a dichotomous variable. In our data base baseline PSA levels remain the most powerful independent discriminant of response to conformal irradiation. PSA density is only a surrogate for baseline PSA levels and does not refine the ability to predict prolongation of freedom from biochemical relapse following conformal radiotherapy. PMID- 7538599 TI - Prostate specific antigen regression and progression after androgen deprivation for localized and metastatic prostate cancer. AB - To identify prostate specific antigen (PSA) functions of prognostic significance in regard to treatment with androgen deprivation for prostate cancer we analyzed the pretreatment PSA, PSA half-life, PSA nadirs, times to PSA elevation and PSA doubling times in 245 patients with localized and 78 with metastatic disease who were treated with this modality. There was a direct correlation between the pretreatment PSA and the time to PSA elevation in patients with localized cancer (p = 0.000003) but no significant correlation in those with metastatic cancer. The PSA half-life was highly variable and did not correlate with other PSA functions of prognostic significance. Incremental increases in the PSA nadir correlated with the time to PSA elevation in patients with localized and metastatic cancer (p < 0.000001 and p = 0.00009, respectively), and with other parameters of prognostic significance. The median PSA doubling time in 26 patients with localized cancer in whom distant metastases did not develop (7.5 months) was significantly longer than that in 7 in whom new metastases developed (2.5 months) and in 43 with preexisting metastatic cancer (2.5 months) (p < 0.05 and p < 0.0001, respectively). In the 7 patients with localized cancer in whom metastases developed the median of the ratios of the PSA when the metastases were manifest and the pretreatment PSA was 0.14, and in 24 patients with preexisting metastatic cancer the median of the ratios of the antemortem PSA and the pretreatment PSA was 1.2. These data show that PSA synthesis by prostate cancer is reduced after androgen deprivation but that the PSA nadir and PSA doubling time following treatment provide important prognostic information. PMID- 7538602 TI - Re: The value of serial prostate specific antigen determinations 5 years after radiotherapy: steeply increasing values characterize 80% of patients. PMID- 7538601 TI - Dramatic prostate specific antigen decrease in response to discontinuation of megestrol acetate in advanced prostate cancer: expansion of the antiandrogen withdrawal syndrome. AB - We report a dramatic decrease in prostate specific antigen in response to the discontinuation of megestrol acetate in a patient with progressive metastatic prostate cancer. Our case demonstrates that withdrawal responses may occur with steroidal and nonsteroidal antiandrogens. PMID- 7538603 TI - Expressions of E-cadherin and exon v6-containing isoforms of CD44 and their prognostic values in human transitional cell carcinoma. AB - Cell surface adhesion molecules, E-cadherin and exon v6 containing CD44 isoforms (CD44v6), were readily found in well-to-moderately differentiated urothelial cell lines but were down-regulated in poorly differentiated cell lines. One hundred and fifteen tumors of transitional cell carcinoma (TCC) were examined with E cadherin and CD44v6 specific antibodies. Sixty-five (56.5%) tumors exhibited a preserved type while 50 (43.5%) showed a reduced type for CD44v6. Sixty-seven (58.3%) tumors were classified as the preserved type, and 48 (41.7%) were classified as the reduced type for E-cadherin. The staining pattern of E-cadherin was the same as that of CD44v6 in 87.0% (100 of 115) of tumors. The frequency of the reduced type was higher in poorly differentiated carcinomas (32 of 52 for CD44v6, p = 0.001; 27 of 52 for E-cadherin, p = 0.112) and tumors with an invasive growth pattern (22 of 27 for CD44v6, p < 0.001; 20 of 27 for E-cadherin, p < 0.001) than it was in well-differentiated carcinomas and tumors with expansile growth. However, the association with lymph node involvement or distant metastasis did not reach statistical significance. There was no difference in survival in reference to the expression patterns of CD44v6 and E-cadherin. Furthermore, neither marker was a significant prognostic factor for tumor recurrence and survival according to Cox's multiple variant regression analysis. PMID- 7538604 TI - Glycine responses in rat CNS neurons studied with gramicidin perforated patch recording. AB - The conventional whole-cell recording of the patch-clamp technique has a great advantage over other electrical recording methods in that it could perfectly control the extra- and intracellular concentrations of ions (such as Na+, K+, Ca2+, and Cl-). However, this advantage is a two-edged sword: it disrupts the physiological ionic environment inside cells. Thus, various Cl(-)-mediated responses in neurons, such as those elicited by inhibitory amino acids, gamma amino butyric acid (GABA) and glycine (Gly), have been recorded only under an artificial condition. Subsequently, measurement of the normal concentration of intracellular Cl- ([Cl-]i) has been much hampered. [Cl-]i is suggested to play a role in membrane excitability (Deisz and Lux: J Physiol 326: 123-138, 1982) and intracellular signal transduction (Higashijima, Ferguson, and Sternweis: J Biol Chem 262: 3597-3602, 1987), yet the detailed analysis of these fundamental roles has been left undone due to technical difficulties. We have recently developed a "gramicidin perforated patch recording", a novel method of patch-clamp technique that enables analysis of membrane currents with intact [Cl-]i. We here report that by applying this method to acutely dissociated CNS neurons of the rat, we could analyze the Gly-induced membrane currents in the normal ionic environment. And using the reversal potentials, we could deduce the actual [Cl-]i of central neurons. PMID- 7538605 TI - Modulation of ionic currents through GABAA receptor subtypes by endogenous steroids. AB - GABAA receptors were expressed in Xenopus oocytes by injecting messenger RNAs from chick retina and chick cortex, and the potency of 3 alpha-hydroxy-5 alpha pregnan-20-one (3 alpha-OH-DHP) and 5 alpha-pregnane-3 alpha, 21-diol-20-one (THDOC) was investigated by electrophysiology under voltage clamp conditions. For the receptors of chick retina, 3 alpha-OH-DHP (100 nM) and THDOC (100 nM) augmented GABA actions (peak current) 1.6 times (n = 6) and 1.5 times (n = 7), respectively. The currents induced by 0.3-10 microM GABA for the receptors of the chick retina were sensitive to bicuculline, pentobarbital, and diazepam. GABAA receptors in the chick retina in vivo as well as the receptors in the brain may be functionally modulated by endogenous steroids. PMID- 7538606 TI - Three classes of GABAergic interneurons in neocortex and neostriatum. AB - We have compared the cellular organization of GABAergic interneurons between frontal cortex and neostriatum of rats by immunohistochemistry for calcium binding proteins, somatostatin and nitric oxide synthase (NOS). GABAergic interneurons in both neocortex and neostriatum could be divided into three separate populations containing parvalbumin, somatostatin, or calretinin. NOS cells were considered to be GABAergic and belong to somatostatin cells in both structures. Distributions of calbindin D28k in three classes of interneurons were different among neocortical layers and neostriatum. PMID- 7538607 TI - Autonomic regulation of cardiac chloride current. AB - The cellular mechanism of autonomic regulation of the catecholamine-induced chloride current (ICl) was investigated by recording the whole-cell current in isolated guinea-pig ventricular cells. The beta-adrenergic and related stimulations such as adrenaline, isoprenaline, forskolin, or internal application of cyclic AMP (cAMP), induced the ICl. Acetylcholine (ACh) depressed the responses of ICl induced by the beta-adrenergic stimulation or forskolin, which was followed by a transient overshoot of the ICl response upon washing out ACh. ACh failed to interfere with ICl induced by cAMP, and the pretreatment of myocytes with pertussis toxin (PTX) abolished the inhibitory effect of acetylcholine. Intracellular application of cGMP, which levels are known to be elevated by the muscarinic stimulation, enhanced ICl activated by submaximal doses of isoprenaline. In the absence of agonists, cGMP failed to induce ICl. Thus, the antagonistic interaction between the beta-adrenergic stimulation and the muscarinic stimulation is at the membrane level, most probably via PTX sensitive GTP-binding proteins. cGMP might contribute to the post-ACh rebound phenomenon by facilitating the beta-stimulation. PMID- 7538608 TI - Regulation of absorption by phosphorylation of CFTR. AB - In contrast to fluid secretion which in many tissues is well known to be regulated by phosphorylation, little is known of phosphorylation regulation of electrolyte absorption. It is now well established that CFTR (cystic fibrosis transmembrane regulator) is a Cl- channel which is activated by phosphorylation. The human eccrine sweat duct is an absorptive epithelium which expresses relatively large amounts of CFTR. We find that the Cl- conductance (GCl) expressed in the isolated, microperfused sweat duct is also dependent upon phosphorylation activation which can be mediated via cAMP. The activation is Mg2+ dependent, Ca2+ independent, and inhibited by the kinase inhibitor staurosporine. Deactivation appears to occur via endogenous phosphatases that can be inhibited by okadaic acid. We surmise that phosphorylation regulation of CFTR GCl is an important mechanism for shifting Cl- absorption efficiently between electroconductive and carrier mediated transport. PMID- 7538609 TI - Human P-glycoprotein as a multi-drug transporter analyzed by using transepithelial transport system. AB - To analyze the mechanism of drug transport, mechanism of inhibitors, and physiological substrates of human P-glycoprotein, we established a transepithelial transport system by introducing MDR1 cDNA into LLC-PK1, a pig kidney epithelial cell line. P-glycoprotein functions as a steroid transporter as well as a drug transporter as physiological functions. P-glycoprotein also transports MDR modulators such as cyclosporin A, FK506, and calcium channel blockers. PMID- 7538611 TI - Tubular hypertrophy due to work load induced by furosemide is associated with increases of IGF-1 and IGFBP-1. AB - We have examined the expression of insulin-like growth factor 1 (IGF-1), IGF binding protein-1 (IGFBP-1), and IGF binding protein-3 (IGFBP-3) in the rat distal nephron during increased cell work load and hypertrophy, induced by the diuretic, furosemide. Furosemide was given for six days to increase distal sodium delivery and uptake. To mitigate salt loss, the animals drank 0.8% NaCl and 0.1% KCl. Control rats were infused with vehicle (0.9% saline) and drank tap water. Furosemide increased urinary volume (13-fold) and sodium excretion (eightfold), and decreased urine osmolarity (fourfold). By immunocytochemistry, staining for IGF-1 and IGFBP-1 was markedly increased in distal convoluted tubules and cortical collecting ducts; both segments also underwent hypertrophy. Increased staining for the peptides was evident early (1 hr, 18 hr) after furosemide, prior to hypertrophy of cells. Whereas transcripts of IGF-1 and IGFBP-3 mRNA showed little or no increase in extracts from furosemide-treated kidney cortices, IGFBP 1 mRNA was increased threefold 18 hours after furosemide. Alterations of IGF-1 and IGFBP-1 were independent of changes in plasma aldosterone, glucocorticoids or arginine vasopressin. That IGFBP-1 mRNA increased threefold without significant changes in IGF-1 mRNA suggests that hypertrophic stimuli might initially induce the synthesis of IGF binding protein followed by the trapping of extracellular IGF-1. The present study raises the possibility of IGF-1 and IGFBP-1 being involved in processes that lead to tubular hypertrophy. IGFBP-1 may regulate these effects by binding to and interaction with IGF-1. PMID- 7538610 TI - Activation of Na:2Cl:K cotransport by luminal chloride in macula densa cells. AB - Changes in macula densa intracellular pH (pHi) were used to monitor the direction of flux mediated by the apical Na:2Cl:K cotransporter. At the macula densa, a decrease in luminal [Cl] ([Cl]1) from 60 to 1 mM produced cellular alkalinization secondary to a cascade of events involving a decrease in apical Na:2Cl:K cotransport, a fall in intracellular [Na] ([Na]i) and a stimulation of Na:H exchange. This is supported by the fact that 97% of the change in macula densa pHi with reduction in [Cl]1 was bumetanide-sensitive whereas 92% of this pH change was amiloride-sensitive. We found that, in the presence of 20 mM Na and 5 mM K, a [Cl]1 of 14.3 +/- 2.4 mM (N = 7) produced equilibrium of the apical cotransporter since the pHi obtained under this condition was identical to the pHi found after reducing the net ionic flux to zero with bumetanide. Using this value together with the expected stoichiometry for the bumetanide-sensitive cotransporter, it was estimated that the intracellular [Cl] ([Cl]i) at equilibrium (or in the presence of bumetanide) could be as low as 5 mM. Also, using a Hill number of 2 which is consistent with the present data, the affinity for [Cl]1 was found to be 32.5 mM. Under physiological luminal conditions prevailing at the end of the thick ascending limb (approximately 3.5 mM K, and approximately 25 to 30 mM NaCl), macula densa cells are probably operating close to equilibrium while maintaining a small net reabsorption of Na/K and Cl. Since macula densa cells appear capable of reducing [Cl]i to very low levels, a reabsorptive flux should continue to occur until [NaCl]1 is reduced to 18 mM. PMID- 7538612 TI - Biosynthesis of C3 by human mesangial cells. Modulation by proinflammatory cytokines. AB - Deposits of complement (C) components are found in the glomeruli of patients with various glomerulonephritides without detectable immunoglobulins, thus suggesting a pathogenetic role of the locally produced proteins of this system. In the present study, we have examined human mesangial cells (HMC) for their ability to secrete C3. Three different cell lines were examined and all showed a basal production of C3, which was up-regulated following stimulation with IL-1 beta. IL 6 had no direct stimulatory effect on its own, but synergized with IL-1 to induce an increased production of C3 in the culture supernatant and its relative amount was confirmed by SDS-PAGE and immunoblot. Another agonist such as lipopolysaccharide was not able to induce any significant C3 synthesis. Analysis of C3 HMC gene expression, performed by both reverse transcription-polymerase chain reaction of isolated RNA and Northern blot, confirmed the parallel increase of the specific transcript under IL-1 beta and IL-1 beta + IL-6 stimulation. From these data we conclude that production of C3 in the mesangium could have a pathophysiologic relevance. PMID- 7538613 TI - [Cognitive development of children and adolescents after correction of transposition of great vessels]. AB - BACKGROUND: To study the effect of transposition of the great arteries on later cognitive functioning. PATIENTS: Twentyeight children and adolescents underwent psychometric testing 3.5 to 13.7 years following operation. METHODS: A battery of intelligence, attention and visual memory tasks as well as the Draw-a-Man-Test were administered. Parents completed standardized questionnaires on developmental milestones. RESULTS: Both, mean verbal (93 +/- 15) and performance IQs (97 +/- 21) were normal. The length of interval between operation and psychologic test but not age at repair was a significant predictor of intelligence quotient. Half of the children (9/18) showed attention deficits, 7/11 (64%) were identified as suspect of brain dysfunction. Performance on the Draw-a-Man-Test disclosed 12 (43%) as slightly mentally retarded. CONCLUSIONS: (1) Cognitive functions in children with TGA are more impaired than suggested by the results of intelligence tests. (2) Psychological and neurologic follow-up should be mandatory from early on. (3) Previous studies have overestimated children's intelligence due to unrecognized changes of test norms. PMID- 7538614 TI - Characterization of cilia-associated respiratory bacillus in rabbits and analysis of the 16S rRNA gene sequence. AB - The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative bacterium that has been implicated as an etiologic agent of respiratory tract disease in laboratory rodents. A morphologically and antigenically similar organism has been identified in rabbits and is thought to be a related bacterium, although clinical signs of disease and histologic lesions are absent in infected rabbits. To compare the pathogenicity of rat- and rabbit-origin CAR bacillus isolates in rabbits, neonatal rabbits were experimentally infected with CAR bacillus isolates obtained from an infected rat and rabbit. Rabbits experimentally inoculated with rabbit-origin CAR bacillus had a nasal discharge, seroconverted and developed histologic lesions, whereas rabbits inoculated with rat-origin CAR bacillus seroconverted but did not have evidence of colonization of the respiratory tract. The CAR bacillus isolates were further examined at the genetic level by sequencing 1,261 base pairs of the 16S rRNA gene from six CAR bacillus isolates obtained from infected rabbits. A consensus sequence was obtained and compared with the analogous gene sequence data from rat-origin CAR bacillus isolates. Results indicated that these two organisms are distinctly different, with only 48.8% sequence homology. Comparison of the rabbit-origin 16S rRNA gene sequence with the database Genbank indicated that the organism is most closely related to members of the genus Helicobacter. Bacteria with the highest percentage of similarity with the rabbit-origin CAR bacillus were Helicobacter sp. strain Seymour and H. felis, with 91.1 and 90.8%, respectively. Findings of this study indicate that CAR bacillus isolates from rats and rabbits are host specific and are different bacteria that belong to distinct genera. PMID- 7538615 TI - Developmental changes in modulation of contractility of rabbit hearts. AB - We recently described postnatal developmental differences of beta-adrenergic- and G-protein-mediated modulation of L-type calcium currents (ICa) in newborn and adult rabbit heart. To extend the results obtained by ICa experiments, we studied developmental changes in modulation of contractility induced by isoproterenol (ISO), forskolin (FOR) and isobutyl-methylxanthine (IBMX), all of which work through the cyclic AMP-dependent pathway. Left ventricular developed pressure (LVDP) and its first derivative (dP/dt) were measured with an intraventricular fluid-filled balloon in isolated adult and newborn (ages 1-3 days) rabbit hearts under Langendorff perfusion. ISO increased LVDP and +/- dP/dtmax dose dependently, with much greater positive inotropic effect on adult than on newborn heart. Concomitant use of a subthreshold concentration of IBMX (0.1 microM), a nonselective phosphodiesterase inhibitor (PDEI), did not significantly potentiate the dose-dependent inotropic effect of ISO in adult heart, but did markedly potentiate such effect in newborn heart. ISO 10 microM and FOR 10 microM had comparable inotropic effects on adult heart, whereas 10 microM ISO had a much weaker inotropic effect than 10 microM FOR on newborn heart. However, the increase in LVDP and +dP/dtmax and the percentage increase in these values induced by 10 microM FOR in newborn heart was still significantly lower than that in adult heart. Newborn heart has less effective signal transduction from beta adrenergic stimulation (ISO) to increased contractility than does adult heart. Although newborn heart responds more to direct stimulation of adenylyl cyclase activity (FOR) than to beta-adrenergic stimulation, it still has less overall contractile reserve than adult heart. PDE isozymes of newborn heart are much more sensitive to IBMX than are those of adult heart, producing a significant potentiation of ISO effect by low doses of IBMX in newborn, but not adult, heart. PMID- 7538617 TI - Exogenous expression of human granulocyte colony-stimulating factor receptor in a B-lineage acute lymphoblastic leukemia cell line: a possible model for mixed lineage leukemia. AB - We report evidence that the granulocyte colony-stimulating factor (G-CSF) receptor is present and may be functioning on blast cells from some patients with myeloid surface antigen positive (My+) acute lymphoblastic leukemia (ALL). In the present study, a human G-CSF receptor expression plasmid was transfected into a newly established B-lineage ALL cell line 'Tanoue' and its subclone 'ST' by lipofection to investigate whether expression of the G-CSF receptor and G-CSF stimulation would induce myeloid characteristics on myeloid surface antigen negative (My-) ALL cells. The G-CSF receptor became detectable on the transfected cells (GR-Tanoue and GR-ST), with dissociation constant values of 50-130 pmol/l, and maximal binding sites (Bmax of 77-6100 sites/cell on receptor binding assays. Short term culture with recombinant human G-CSF induced myeloid differentiation (a two to three-fold increase in CD33 and CD15 expression), and a moderate 3H thymidine uptake (stimulation index, 1.75) only in the GR-ST clone no. 15 which expressed a high number of G-CSF receptors (Bmax, 6100 sites/cell). Our data show that (a) exogenous expression of the G-CSF receptor and G-CSF stimulation can induce myeloid characteristics on ALL cells; and (b) in the G-CSF receptor expressing cells, there is a correlation between the number of G-CSF receptors and cell responsiveness to G-CSF in either proliferation or differentiation. PMID- 7538616 TI - The insulin-like growth factor system in regulation of normal and malignant hematopoiesis. AB - The published data available on the insulin-like growth factor (IGF) family of peptides, its receptors and binding proteins in connection with normal and neoplastic hematopoietic processes are reviewed. It is found that nearly all the different hematopoietic cells, either normal or neoplastic, express IGF receptors. The IGFs are involved in normal erythropoiesis, granulopoiesis and lymphopoiesis. IGFs, especially IGF-1, are mitogenic for cell lines of myeloid and lymphoid leukemias and Burkitt's lymphoma. Differentiation of certain immature malignant cell lines is associated with a decrease in the number and affinity of IGF receptors. It might be concluded that the IGFs have an important role in the physiologic and neoplastic processes of the hematopoietic system, and the expanding knowledge of this issue will contribute to the understanding of proliferation and differentiation processes in specific hematologic disorders. PMID- 7538618 TI - Biochemical effect of three different inhibitors of purine/pyrimidine metabolism on differentiation in HL60 cells. AB - The effects of three different nucleotide biosynthesis inhibitors were tested on differentiation and purine/pyrimidine metabolism in HL60 cells. On the three nucleotide biosynthesis inhibitors, acivicin and mycophenolic acid were able to differentiate HL60 cells, while alanosine failed to do so. Differentiation of HL60 cells by acivicin and mycophenolic acid was associated with substantial decreases in both the guanylate and adenylate pools and appeared to be dependent on the state of depletion of intracellular GTP. Simultaneous addition of guanosine or guanine to mycophenolic acid-treated cells restored the GTP pool and prevented differentiation from occurring. Adenine or adenosine had no such effect, while hypoxanthine and inosine partially reversed the differentiation. In acivicin-treated cells, simultaneous addition of guanine caused partial prevention of differentiation. Even though treatment of HL60 cells with alanosine resulted in the depletion of guanylates, this effect was secondary to the depletion of adenylates and developed only upon prolonged exposure. In all the inhibitor-treated cells the activities of the key regulatory enzymes of de novo purine biosynthesis were affected. Even though the measurable activity of hypoxanthine/guanine phosphoribosyl transferase was enhanced in inhibitor-treated cells, the activity of the salvage pathway was inhibited in mycophenolic acid and alanosine-treated cells. Besides de novo purine nucleotide biosynthesis, de novo pyrimidine nucleotide biosynthesis was also inhibited in inhibitor-treated cells. The inhibition of purine and pyrimidine nucleotide biosynthesis in mycophenolic acid, acivicin and alanosine-treated cells resulted in an increase in the steady state concentration of PRPP. Since purine and pyrimidine nucleotides play an important role in the synthesis of important macromolecules, it can be suggested that depletion of guanine ribonucleotide as a result of inhibition of early de novo purine biosynthesis, or due to specific inhibition of de novo guanine nucleotide biosynthesis, may be an obligatory step in the initiation of differentiation in mycophenolic acid and acivicin-treated HL60 cells. PMID- 7538620 TI - Design of the EPC-9, a computer-controlled patch-clamp amplifier. 1. Hardware. AB - The EPC-9 patch-clamp amplifier is a digitally controlled analog device for recording currents in membrane patches and in small cells. It has neither front panel controls nor internal trim adjustments; instead all gain, range-changing, transient cancellation, calibration and other functions are computer controlled. Novel aspects of the circuit design of this instrument are discussed, with special reference to the issues of allowing computer control of all functions. PMID- 7538621 TI - Design of the EPC-9, a computer-controlled patch-clamp amplifier. 2. Software. AB - The EPC-9 computer-controlled amplifier has no front-panel controls; therefore the user interface to the EPC-9 patch-clamp amplifier is defined entirely by software. This paper describes various user interfaces that have been implemented, including a high-level programming interface, a user interface based on the PostScript language, and graphical user interfaces that control the EPC-9 from data-acquisition programs. Also described are the algorithms used for automatic adjustment of the C-Fast and C-Slow transient cancellation circuitry. An overview of the procedures that perform automatic testing and calibration is given. PMID- 7538619 TI - Analysis of c-kit expression of human erythroleukemia cell line, HEL: clonal variation and relationship with erythroid and megakaryocytic phenotype. AB - The proto-oncogene c-kit encodes the receptor for a stem cell factor. We examined HEL, a human erythroleukemia cell line, in order to clarify the correlation between the c-kit receptor (KR) expression and lineage-specific phenotype. Although HEL cells are known to express KR, we found two relatively distinct HEL cell populations in terms of KR expression. We then subcloned HEL cell lines with clone sorting on the basis of KR expression and compared their various characteristics. The highly KR-expressing subline, HEL-P1, expressed a high level of glycophorin A (GPA), a known erythroid lineage marker. HEL-N1, in which most of the cells were KR-negative, showed a higher megakaryocytic lineage marker CD41b expression than HEL-P1. However, the expression of granulomonocytic lineage markers were not significantly different between the two subclones. Cell growth rate and cell cycle analysis also did not detect significant differences between the sublines. HEL-P1 cells gradually lost their KR expression in serum-containing culture, while the percentage of KR-positive HEL-N1 cells increased in serum-free culture. These observations indicate that KR expression was associated with the synchronous expression of GPA and inversely correlated with CD41b, and reversible transitions between KR-positive cells and KR-negative cells exist. We suggest that KR plays an important part in commitment of erythroid and megakaryocytic precursor cells. PMID- 7538622 TI - Tuberculosis and cancer: parallels in host responses and therapeutic approaches? AB - Mycobacteria elicit two quite different immune responses. One is protective and is partly based on recognition and lysis of stressed, bacilli-laden cells expressing heat-shock proteins. The other suppresses this recognition and instead leads to indiscriminate necrosis of tissues containing mycobacteria (the Koch phenomenon). The type of response depends on the predominant T-cell maturation pathway, Th1 or Th2, which in turn is determined by priming by prior contact with environmental mycobacteria. Vaccination by BCG induces whichever response the recipient is primed to make, and this is a likely explanation of the variable efficacy of this vaccine in prevention of tuberculosis and therapy of cancer. Thus; BCG is a two-edged sword. We postulate that by using other mycobacterial preparations, such as killed Mycobacterium vaccae, it might be possible to suppress the indiscriminate necrosis and enhance Th1-regulated selective destruction of tumour cells. PMID- 7538623 TI - The relative essentiality of the antioxidative function of coenzyme Q--the interactive role of DT-diaphorase. AB - This paper will address two aspects regarding the antioxidative role of coenzyme Q (CoQ): (1) Is the antioxidant function of CoQ primary or secondary (coincidental), i.e. was this molecule selected during evolution to function primarily as an essential functional component of the mitochondrial electron transfer chain and oxidative phosphorylation processes, is its antioxidative capability merely a coincidence of its hydroquinone structure, or was its synthetic enzyme sequence selected on the basis of the advantage to the evolving organism of both functions of CoQ? (2) What is the mechanism whereby the hydroquinone (antioxidant) form of CoQ (CoQH2) is maintained in high proportion in the various and many membranes in which it resides, and in which an obvious electron transfer mechanism to reduce it is not present? The essentiality of the antioxidative role of CoQH2 will be explored and compared to other primary and secondary antioxidants. Recent evidence implicating the two-electron quinone reductase, DT-diaphorase, in the maintenance of the reduced, antioxidant state of CoQ during the oxidative stress of exhaustive exercise will be presented, and a hypothesis concerning the evolutionary significance of DT-diaphorase will be offered. PMID- 7538626 TI - Raman spectroscopy of DNA and proteins. PMID- 7538627 TI - Oculofacial-skeletal myorhythmia in central nervous system Whipple's disease: additional case and review of the literature. AB - A case of oculofacial-skeletal myorhythmia associated with cerebral Whipple's disease is presented. This peculiar abnormal movement disorder consists of a convergent-divergent pendular nystagmus associated with a synchronous, rhythmic movement of the mouth, jaw, and extremities. The movements in previous cases have responded inconsistently to a variety of broad-spectrum antibiotic drugs, and antiepileptic, muscle relaxant, antispastic, and psychotropic drugs. The current patient's mental status improved after starting intravenous ceftriaxone but improvement of the abnormal movements occurred only after the addition of oral valproate. Current treatment consists of intravenous trimethoprim sulfamethoxazole for 2 weeks followed by oral trimethoprim-sulfamethoxazole twice daily for 1 year. Presented here is an alternative treatment of intravenous ceftriazone followed by oral trimethoprim-sulfamethoxazole in combination with valproate for abnormal movements not responsive to antibiotic drugs alone. PMID- 7538628 TI - A method for chromosome preparation of guinea-pig oocytes. PMID- 7538624 TI - Absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes. AB - Absorption and CD measurements of complementary oligomers and mixtures are described. The concentrations of oligomers may be estimated from absorption measurements and nearest-neighbor calculations of molar extinction coefficients. Interactions between complementary strands in mixtures can lead to obvious differences between measured CD spectra and the average of the spectra of the individual strands. CD spectra also allow an assessment of whether the individual strands are in self-complexes, which could compete with duplex or triplex formation. Isodichroic and isoabsorptive points provide important indicators of the stoichiometry of the strands in base-paired complexes. CD spectra provide an important means of characterizing differences in the conformations of DNA, RNA, and hybrid duplexes or triplexes having analogous sequences. PMID- 7538629 TI - Immediate surgery is the treatment of choice for carpal tunnel syndrome. PMID- 7538625 TI - Circular dichroism. PMID- 7538630 TI - Axonal neuropathy and late detection of Refsum's disease. PMID- 7538631 TI - Renal transplantation in asymptomatic carriers of hepatitis B surface antigen. AB - The aim of the present study was: (a) to assess the effect of HBsAg on the survival of both renal grafts and patients, and (b) to determine the outcome of HBV chronic infection after renal transplantation. Fourteen patients seropositive for HBsAg but asymptomatic before renal transplantation (group A) were included in the study. The results were compared to those of 14 transplanted patients (group B) seronegative for HBsAg with similar age and immunosuppressive treatment. Four patients received a graft from a living-related donor and 10 patients from a cadaver donor in each group. Eight of 14 patients of group A showed, after renal transplantation, chronic hepatitis, which was not observed in any of the group B patients (p < 0.01). The rate of acute rejection episodes was significantly greater (p < 0.05) in group B than in group A. The graft survival was found to be similar in both groups at the 1st year, but significantly less (p < 0.01) in group B than in group A at the 5th year after transplantation. The survival of patients was found to be significantly less in group A than in group B at the 1st (p < 0.05) and 5th years (p < 0.01) after transplantation. In 2 patients of group A and 1 of group B anti-HCV was found, while HDAg plus anti-HD was found in 1 patient of group B. The HBV-DNA was found in 4 of 8 alive patients of group A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538632 TI - MDR1 gene expression in lymphocytes of patients with renal transplants. AB - The MDR1 gene, a multidrug resistance gene, codes for P-glycoprotein which pumps hydrophobic drugs out of the cells. Since cyclosporins also bind to P glycoprotein and might be pumped by this transmembrane protein, we determined the expression of the MDR1 gene in the lymphocytes of 32 patients with renal transplants. MDR1 RNA expression of lymphocytes was measured by slot blot analysis and compared to the expression of drug-sensitive KB-3-1 cells and multidrug-resistant KB-8-5 cells. MDR1 RNA expression was detected in the lymphocytes of 9 (28%) patients, whereas no expression was seen in the remaining 23 patients. No association between MDR1 RNA expression and transplant function or hematological parameters was observed. However, none of the 6 patients who had transplants for more than 40 months expressed the MDR1 gene in their lymphocytes. In conclusion, expression of the MDR1 gene does occur in lymphocytes of patients with renal transplants and might reduce the immunosuppressive efficacy of cyclosporins through enhanced efflux of cyclosporins. PMID- 7538633 TI - Immunohistochemical and biochemical detection of low density lipoprotein receptors in cultured rat mesangial cells. AB - The expression of low density lipoprotein (LDL) receptors by cultured rat mesangial cells was demonstrated using immunohistochemical and biochemical methods. Using a mouse monoclonal anti-human LDL receptor antibody, the immunofluorescence technique was applied to cultured mesangial cells and showed granular staining. Immunoblotting analysis of the membrane fraction of cultured mesangial cells showed a single band with a protein of approximately 130,000 molecular weight. In addition, [125I]LDL binding and the uptake of [125I]LDL by cultured mesangial cells were studied. Binding and uptake both reached an equilibrium after about 30 min of incubation. A 50% reduction in [125I]LDL (20 micrograms protein/ml) binding and uptake occurred when unlabelled LDL was added to cultures at 20-40 micrograms protein/ml. Addition of high density lipoprotein without apoE to the culture medium did not induce competitive inhibition of [125I]LDL binding and uptake by mesangial cells. These findings suggest that cultured mesangial cells have the capacity to express an LDL receptor that regulates the cellular uptake of LDL. PMID- 7538635 TI - Treatment of intracranial nongerminomatous malignant germ cell tumors producing alpha-fetoprotein. AB - We treated 10 patients with intracranial nongerminomatous malignant germ cell tumors producing alpha-fetoprotein between 1969 and 1992. Two patients were treated with radiotherapy (RT) only (RT group), and three were treated with RT and cisplatin plus vinblastine plus bleomycin therapy with or without surgery (cisplatin plus vinblastine plus bleomycin group). The most recently treated five patients received cisplatin plus etoposide (PE) therapy with or without RT and/or surgery (PE group). The level of alpha-fetoprotein in serum was elevated in all 10 patients. In the PE group, PE therapy consisted of cisplatin (20 mg/m2) and etoposide (60 mg/m2) daily for 5 days (one course) given two or three times at 4 week intervals and then once every 4 months; the patients received three to six courses (mean, 4.2 courses). In the RT group (n = 2), one patient died 3 months after diagnosis and the other died at 12 months. In the cisplatin plus vinblastine plus bleomycin group (n = 3), complete remission was obtained in one patient, but the other two patients died 12 and 24 months after diagnosis. In contrast, in the PE group (n = 5), complete remission was obtained in all patients who are all currently alive without recurrence, at 35 to 71 months (average, 53.6 mo) after diagnosis. The results indicate that multidisciplinary treatment including combination chemotherapy with cisplatin and etoposide with or without surgery and/or RT is highly effective in the treatment of patients with alpha-fetoprotein-producing intracranial nongerminomatous malignant germ cell tumor. PMID- 7538634 TI - Differential cellular regulation of pro-opiomelanocortin by interleukin-1-beta and corticotropin-releasing hormone. AB - Considerable evidence supports the existence of a bidirectional communication between the immune system and the hypothalamo-pituitary-adrenal (HPA) axis. In the present study, we examined the interleukin-1 beta (IL1 beta)-mediated regulation of pro-opiomelanocortin (POMC) at a cellular level, from secretion to gene expression, using murine anterior pituitary corticotroph tumor (AtT20) cells as a model system. The regulatory effects of IL1 beta were compared to those of the classical POMC regulator, corticotropin-releasing hormone (CRH). IL1 beta was found to evoke an early, preferential release of beta-lipotropin (beta LPH) which was accompanied by elevations in POMC heteronuclear (hn)RNA and c-fos and c-jun mRNAs. IL1 beta also elicited a late, preferential release of beta LPH which was associated with only an enhanced expression of POMC hnRNA. Additionally, IL1 beta stimulated an intermediate, preferential release of beta-endorphin (beta E) which was not accompanied by any changes in gene expression. In marked contrast to IL1 beta, CRH evoked an early, preferential beta E secretory response which was associated with elevations in POMC hnRNA and c-fos mRNA. CRH also elicited a late, preferential beta E release which was associated with only an enhanced POMC hnRNA expression. These findings show that although both IL1 beta and CRH activate the corticotrophs, they elicit dramatically different patterns in the regulation of the biochemical dynamics of POMC. Such distinct patterns of corticotroph activation in response to IL1 beta or CRH exposure in vivo would allow the pituitary not only to indicate that it has been activated, but also how it has been activated. This characteristic may be critically important in the function of the HPA axis and in the interaction of the HPA axis with the immune system. PMID- 7538636 TI - Effects of protein kinase C modulators on multidrug resistance in human glioma cells. AB - To identify the role of protein kinase C (PKC) in multidrug resistance, the effects of phorbol-12-myristate-13-acetate (PMA), a PKC activator, or calphostin C, a PKC inhibitor, on intracellular vincristine accumulation and expression of P glycoprotein phosphorylation were studied in one multidrug-resistant and three multidrug-sensitive human glioma cell lines. Basal PKC activities and immunoreactivities of PKC-alpha and -zeta were higher in multidrug-resistant cells than in multidrug-sensitive cells. There was no significant difference in the immunoreactivity of PKC-delta between multidrug-resistant and -sensitive cells, and immunoreactive PKC-beta, -gamma, and -epsilon were not detected in either multidrug-resistant or -sensitive cells. The treatment of multidrug resistant cells with 100 nM PMA for 2 hours resulted in the activation not of PKC zeta but of PKC-alpha, with concomitant decrease in vincristine accumulation and increase in P-glycoprotein phosphorylation. The exposure of multidrug-resistant cells to 100 nM PMA for 24 hours induced down-regulation not of PKC-zeta but of PKC-alpha, with concurrent decrease in vincristine accumulation, and reduced but still increased P-glycoprotein phosphorylation. The treatment of multidrug resistant cells with 100 nM calphostin C for 2 hours decreased immunoreactive PKC zeta and not immunoreactive PKC-alpha, inducing increase in vincristine accumulation, with concomitant decrease in P-glycoprotein phosphorylation. There was no evidence of significant change in vincristine accumulation in multidrug sensitive cells treated with PMA or calphostin C. This may suggest that at least two isozymes of PKC, PKC-alpha and -zeta, are involved in P-glycoprotein phosphorylation and that vincristine efflux function in multidrug-resistant human glioma cells is closely associated with P-glycoprotein phosphorylation and is decreased by PKC inhibitor. PMID- 7538637 TI - Relationship between dopamine release in nucleus accumbens and place preference induced by substance P injected into the nucleus basalis magnocellularis region. AB - The activity of the neurotransmitter dopamine in the nucleus accumbens is considered to be an important element in the central processing of reinforcement. Unilateral administration of the neurokinin substance P into the area of the nucleus basalis magnocellularis of rats was found to be reinforcing, as assessed by the conditioned place preference paradigm. Simultaneous in vivo microdialysis showed that administration of substance P into the area of the nucleus basalis magnocellularis could increase extracellular concentrations of dopamine in the contralateral nucleus accumbens. Only those animals in which the administration of substance P induced this increase in dopamine levels acquired place preference. Furthermore, the changes in extracellular dopamine levels after substance P administration had a bimodal time course with an acute increase (to about 160% of baseline) during the first hour after injection, with a low (to 120 130%) and enduring increase occurring thereafter. Interestingly, during this second increase there were indications for positive correlations with the degree of place preference induced by substance P. Further positive correlations with place preference were found in the levels of the serotonergic metabolite 5 hydroxyindoleacetic acid. In contrast to dopamine, these were observed ipsi- and contralateral to the side of substance P administration. By combining the methods of in vivo microdialysis and conditioned place preference it was shown that the reinforcing effect induced by unilateral substance P injection in the nucleus basalis magnocellularis is related to dopaminergic (and possibly serotonergic) mechanisms in the nucleus accumbens. PMID- 7538638 TI - Galanin induces a hyperpolarization of norepinephrine-containing locus coeruleus neurons in the brainstem slice. AB - Galanin applied in the bath or by micropipette directly on to locus coeruleus neurons in an in vitro slice preparation caused a hyperpolarization accompanied by a small decrease in membrane resistance. Immunohistochemical staining of intracellularly filled neurons indicated that the effect of galanin was exerted on norepinephrine neurons of the locus coeruleus. The galanin effect was variable in amplitude and duration and often showed desensitization, with subsequent applications producing a smaller response. When cells were exposed to tetrodotoxin or tetrodotoxin/low calcium media, the galanin response was still present. Under voltage clamp galanin application caused a net outward current that did not reverse in normal potassium concentrations; however, by increasing extracellular potassium concentrations the net outward current was reversed and the reversal potential shifted to a less negative potential. The response to galanin was identical when either KCl or KAc was used as the intracellular electrode solution. Tetraethylammonium chloride significantly reduced or abolished the response to galanin in most cells, although in a few cells the galanin response was not affected. Glibenclamide, a blocker of ATP-sensitive potassium channels, did not affect the galanin hyperpolarization. In addition, diazoxide had no effect on the membrane properties of locus coeruleus neurons. These results demonstrate that galanin exerts its inhibitory effect in the locus coeruleus via an increase in K+ conductance; however, not via the pancreatic type of ATP-sensitive K+ channels. Cryostat sections of the locus coeruleus incubated in 125I-labeled galanin revealed binding sites in the locus coeruleus at all levels. Sections of the locus coeruleus processed for ultrastructural immunocytochemistry showed galanin immunoreactivity in many neuronal somata and dendritic processes within the nucleus, confirming earlier evidence for the coexistence of galanin and noradrenaline in locus coeruleus neurons. Galanin immunoreactive soma and dendrites in the locus coeruleus less frequently received galanin-immunoreactive synapses of axonal origin. These findings suggest that endogenous galanin in the locus coeruleus is mainly released from noradrenaline galanin somata and/or dendrites to act on autoreceptors or on receptors on adjacent neurons. PMID- 7538639 TI - Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord- II. Co-existence of galanin with other peptides in local neurons. AB - Using light microscopic immunoperoxidase and immunofluorescence histochemistry, double-staining methodology, and electron microscopic pre-embedding and post embedding immunocytochemistry, we studied galanin-immunoreactive neurons in the superficial dorsal horn of the rat spinal cord. Co-existence of galanin with other neuropeptides was also analysed. The lumbar 4 and 5 segments of normal rats and after rhizotomy or spinal cord transection were studied. Galanin-positive local neurons in lamina II were often islet cells and could be classified as type A, which had abundant electron-dense cytoplasm containing many large dense-core vesicles, and type B, which had electron-lucent cytoplasm with only a few large dense-core vesicles. Galanin-positive and -negative peripheral afferent terminals made synaptic contact mostly with galanin-negative dendrites and cell bodies, but also with type B galanin cell bodies and with galanin-positive dendrites of unidentified type. Galanin-immunoreactive terminals from local neurons could also be classified into two types. Type alpha terminals were most common; they contained densely packed synaptic vesicles and many large dense-core vesicles, were strongly immunostained and most frequently made synaptic contact with galanin-negative dendrites. Type beta terminals contained loosely packed synaptic vesicles and a few large dense-core vesicles, and were weakly immunostained. Axosomatic synaptic contact were sometimes found between type beta terminals and type B galanin-positive cell bodies, but were most often associated with galanin negative dendrites. Double immunostaining showed that galanin-like immunoreactivity co-localized mainly with enkephalin-like, but sometimes also with neuropeptide Y-like immunoreactivity in some local neurons in lamina II. Galanin-like and substance P-like immunoreactivities were identified in the same neurons in deeper layers of the dorsal horn. Coexistence of these neuropeptides and neurotensin with galanin was demonstrated not only in terminals in lamina II but also in large dense-core vesicles, as revealed by post-embedding immunocytochemistry. These results show that galanin-immunoreactive neurons in lamina II receive inputs directly from primary afferents and frequently make synaptic contacts with other intrinsic neurons. Galanin in the superficial dorsal horn may be released both from primary afferents and local neurons to modulate sensory processing in many different ways, including interacting with enkephalin, neuropeptide Y, neurotensin and substance P released from the same and/or other local neurons. PMID- 7538641 TI - Antagonism of nociceptive responses of cat spinal dorsal horn neurons in vivo by the NK-1 receptor antagonists CP-96,345 and CP-99,994, but not by CP-96,344. AB - Extracellular and intracellular studies were undertaken to test the effects of the non-peptide, substance P (NK-1) receptor antagonists CP-96,345 and CP-99,994, and of CP-96,344, the inactive enantiomer of CP-96,345, on the responses of spinal dorsal horn neurons to peripheral noxious and non-noxious cutaneous stimuli in spinalized cats anesthetized with alpha-chloralose. The effect of these agents on the response of dorsal horn neurons to iontophoretic application of substance P was also tested in extracellular studies. The substance P-induced slow, prolonged discharge of dorsal horn neurons was blocked by administration (0.5 mg/kg, i.v.) of CP-96,345 (n = 10) or CP-99,994 (n = 9), but was unaffected by CP-96,344 (n = 9). The response of substance P-sensitive neurons to noxious thermal stimulation of the cutaneous receptive field, especially the late afterdischarge phase, was also significantly inhibited by CP-96,345 (n = 10) and by CP-99,994 (n = 7). The response of such neurons to noxious pinch stimulation of the receptive field was also significantly inhibited by CP-96,345 (n = 7) and CP-99,994 (n = 8), but the response of three other substance P-sensitive neurons to pinch was unaffected by CP-96,345. CP-96,344 did not affect the response of any neuron tested to either of these noxious stimuli (noxious thermal, n = 7; pinch, n = 6). The response to hair afferent stimulation was unaffected by any of these compounds (CP-96,345, n = 16; CP-96,344, n = 5; CP-99,994, n = 6). In intracellular studies, the effect of these antagonists was tested on responses of dorsal horn neurons to noxious pinch stimulation or to a train of high intensity electrical stimulation of the superficial peroneal nerve. Both stimuli produced an initial fast depolarization followed by a slow and prolonged depolarization with corresponding discharge patterns. CP-96,345 (n = 3) and CP-99,994 (n = 6) selectively blocked the late, slow components of the stimulus-evoked response without affecting the early components. Responses to single electrical pulses of the same intensity and duration were not affected. CP-96,344 did not affect any of the responses tested (n = 5). The data indicate that nociceptive responses of a subset of spinal dorsal horn cells are selectively blocked by the NK-1 receptor antagonists, CP-96,345 and CP-99,994, thus confirming the involvement of NK-1 receptors in these responses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538640 TI - Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord- III. Effects of peripheral axotomy with special reference to galanin. AB - In this study co-localization of galanin- with calcitonin gene-related peptide (CGRP)-like immunoreactivity was examined in dorsal root ganglion neurons 14 days after sciatic nerve cut using a laser scanning confocal microscope. CGRP- and galanin-like immunoreactivities were also analysed in the dorsal horn of the spinal cord of these animals with immunofluorescence microscopy. The ultrastructural changes in galanin-immunoreactive, presumably primary afferent terminals in the superficial dorsal horn, were studied as well as the relationship between galanin-, substance P- and CGRP-like immunoreactivities in primary afferent terminals. Local galanin-positive neurons in lamina II were also analysed after peripheral axotomy. Under the confocal microscope, CGRP-like immunoreactivity was located in the perinuclear region, probably the Golgi complex, and in dot-like structures, probably representing large dense-core vesicles, in normal dorsal root ganglion neurons. However, after peripheral axotomy CGRP was mainly detected in dot-like structures. Only a slight decrease in percentage of CGRP neurons in dorsal root ganglion was seen after axotomy, and about 84% of the galanin-positive neurons contained CGRP. The field of galanin positive nerve fibres in the superficial lumbar (L)4 and L5 dorsal horn expanded and the intensity of staining for CGRP was reduced in these regions 14 days after sciatic nerve cut. Using pre-embedding immunoelectron microscopy, several morphological changes were observed in galanin-positive terminals in laminae I and II ipsilateral to the lesion. Most importantly, the most frequently occurring type of galanin-positive terminals (type 1) showed distinct changes with a granular matrix, many immunoreactive, peripherally located large dense-core vesicles, empty large vesicles and synaptic vesicles which were displaced from the presynaptic zone. Other galanin-positive terminals underwent even more pronounced morphological changes, including extensive vesiculolysis, also of large dense-core vesicles, filamentous degeneration or formation of axonal labyrinths. An increased number of galanin-positive nerve terminals was observed in lamina III of the ipsilateral dorsal horn after axotomy. They did not form glomeruli and contained few large dense-core vesicles. Post-embedding immunocytochemistry combined with quantitative analysis revealed that significant changes occurred in a proportion of terminals also with regard to peptide content in large dense-core vesicles after axotomy. Thus, the percentage of galanin positive large dense-core vesicles increased in several cases and that of substance P- and CGRP-immunoreactive ones decreased.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538642 TI - Differential brain area vulnerability to long-term subcortical excitotoxic lesions. AB - To investigate the long-term effects of excitatory amino acid microinjections into the basal forebrain and its correlation with a possible Ca2+ imbalance associated with the excitotoxic process, ibotenic acid, mainly an N-methyl-D aspartate receptor agonist, and quisqualic acid, an agonist of non-N-methyl-D aspartate receptors, were injected into two regions rich in cholinergic neurons, namely the medial septal nucleus and the ventral globus pallidus. Within the globus pallidus but not within the medial septal nucleus, 13 days and one year postlesion, nerve cell death was associated with the appearance of calcium deposits within the large putative GABAergic pallidal neurons, being more pronounced in ibotenic acid than quisqualic acid-lesioned rats. An intermediate two month post-lesion study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid microinjections in globus pallidus demonstrated that the AMPA subtype of glutamate receptor may also be involved in this Ca2+ imbalance, together with the N-methyl-D-aspartate and metabotropic subtype receptors. Quisqualic acid lesions in globus pallidus and medial septum were associated with a substantial disappearance of cholinergic cell bodies and their nerve terminal networks within the cerebral cortex and hippocampal formation respectively, as assessed by choline acetyltransferase and acetylcholine esterase immunocytochemistry. Ibotenic acid lesions resulted in a lower reduction of cholinergic markers. One year after septal lesions induced either by ibotenic or quisqualic acid, a marked atrophy of the entire dorsolateral septal nucleus was observed. Our results support the hypothesis that brief and intense glutamate exposure can induce long-term neurodegenerative processes and give evidence that long-term excitotoxic lesions of the two areas studied result in marked differences in neuronal damage, including intracellular calcium deposits which do not correlate with the cholinergic deficits produced by multiple glutamate receptor subtypes. PMID- 7538643 TI - Inhibitory synaptic inputs to the oculomotor nucleus from vestibulo-ocular-reflex related nuclei in the rabbit. AB - Studies of the pathways involved in the vestibulo-ocular reflex have suggested that the projection from the superior vestibular nucleus to the ipsilateral oculomotor nucleus is inhibitory, whereas the medial vestibular nucleus, the abducens nucleus and the contralateral superior vestibular nucleus most likely exert excitatory effects on oculomotor neurons. In order to determine directly the termination pattern and the neurotransmitter of these afferents, we studied their input to the oculomotor nucleus in the rabbit at the light microscopic level with the use of anterograde tracing of Phaseolus vulgaris-leucoagglutinin combined with retrograde tracing of horseradish peroxidase from the extraocular muscles, and at the ultrastructural level with the use of anterograde tracing of wheatgerm-agglutinated horseradish peroxidase combined with GABA and glycine postembedding immunocytochemistry. The general ultrastructural characteristics of the neuropil and the types of boutons observed in the rabbit oculomotor nuclei are in general agreement with the descriptions for the oculomotor complex of other mammals. The superior vestibular nucleus projected bilaterally to the superior rectus and inferior oblique subdivisions, and ipsilaterally to the inferior rectus and medial rectus subdivision; the medial vestibular nucleus projected bilaterally to the medial rectus, inferior oblique, inferior rectus and superior rectus subdivisions with a strong contralateral predominance. The abducens nucleus projected contralaterally to the medial rectus subdivision. More than 90% of all the anterogradely labeled terminals from the ipsilateral superior vestibular nucleus were GABAergic. These terminals were characterized by flattened vesicles and symmetric synapses, and they contacted somata, as well as proximal and distal dendrites of motoneurons. All terminals derived from the medial vestibular nucleus the abducens nucleus and the contralateral superior vestibular nucleus were non-GABAergic. These non-GABAergic terminals showed spherical vesicles and asymmetric synapses, and they contacted predominantly distal dendrites. None of the anterogradely labeled terminals from the studied vestibular nuclei or abducens nucleus were glycinergic. The present study provides the first direct anatomical evidence that most, if not all, of the synaptic input from the superior vestibular nucleus to the ipsilateral oculomotor nucleus is GABAergic, and that the medial rectus subdivision is included in the termination area. Furthermore, it confirms that the projections from the medial vestibular nucleus, the abducens nucleus and the contralateral superior vestibular nucleus are exclusively non-GABAergic. PMID- 7538644 TI - Acidic fibroblast growth factor stimulates motor and sensory axon regeneration after sciatic nerve crush in the rat. AB - A time course study in untreated male Sprague-Dawley rats showed that there was no significant difference in the rate of regeneration of motor and sensory axons after a crush injury. Acidic fibroblast growth factor, given topically directly to the site of the crush injury (osmotic minipump for three days) or systemically (i.v. once daily for three days), stimulated the regeneration of motor axons and myelinated sensory axons in the sciatic nerve of the rat. Dose-dependent increases in regeneration distance were seen after 3.6, 36 or 360 ng/day were applied locally and 3 or 10 micrograms/kg per day were given systematically. The greatest effects were achieved with 36 ng/day locally or 10 micrograms/kg per day systematically, when the increase in regeneration distance over three days compared to untreated rats was 47% and 48%, respectively. Administration of heparin vehicle had no significant effect on regeneration. We conclude that since a crush injury has little effect on the endoneurial tubes and supporting cells, the stimulatory effects of acidic fibroblast growth factor on peripheral nerve regeneration seen in this study are likely to be due to a direct acceleration of axonal extension. This is in contrast with the axonal regeneration that occurs across a gap after nerve transection, where axonal extension may be secondary to stimulatory effects on non-neuronal cells providing a supporting "bridge" across the gap. These results suggest that acidic fibroblast growth factor may be clinically useful in the treatment of peripheral neuropathy in man, particularly since systemic treatment for short periods may be effective. PMID- 7538646 TI - Calbindin D28k-containing neurons are restricted to the medial substantia nigra in humans. AB - A controversy exists in the literature as to whether neurons containing the calcium binding protein calbindin-D28k are located within the human substantia nigra. The point of variance between reports, however, is not the anatomical distribution of these neurons, but rather the delineation of the dorsal border of the substantia nigra. It has been suggested that the dense substance P striatonigral innervation delimits the substantia nigra in the human. The aim of the present study is to re-examine the distribution of calbindin-D28k-positive neurons throughout the substantia nigra using substance P to delimit its borders. Although a few calbindin-D28k-positive neurons were found in the medial cell group of the substantia nigra, the vast majority of positive neurons were located in the adjacent A8 and A10 dopaminergic cell groups. This anatomical location of calbindin-D28k-positive neurons is consistent with previous reports, though our results indicate that when the striatonigral projection is used to define the substantia nigra, calbindin-D28k is not a notable feature of these neurons. This questions the neuroprotective role of this protein in Parkinson's disease. PMID- 7538645 TI - Nitric oxide synthase-immunoreactive vagal afferent fibers in rat superior cervical ganglia. AB - Chronic (5-14 days) preganglionic denervation of the rat superior cervical ganglia by sectioning the cervical sympathetic trunk resulted in a time-related partial or complete loss of nitric oxide synthase (isoform I)-immunoreactive fibers and terminals surrounding many sympathetic ganglionic neurons. Unexpectedly, denervation unmasked many varicose nitric oxide synthase immunoreactive fibers, some of which could be traced the entire length of the superior cervical ganglia. Injection of the retrograde tracer Fluorogold into the superior cervical ganglia labeled a population of nodose ganglion cells and of dorsal root ganglion cells from C8 to T3 segments. When the same sections were processed for nitric oxide synthase-immunoreactivity, 40% of the Fluorogold containing nodose ganglion cells also expressed nitric oxide synthase immunoreactivity, whereas colocalization was observed in only a few dorsal root ganglion cells. Similarly, injection of Fluorogold into denervated superior cervical ganglia labeled a population of nodose ganglion cells. Sectioning of all nerve trunks associated with the superior cervical ganglion prior to injection of Fluorogold, except the cervical sympathetic trunk, resulted in no detectable labeling of Fluorogold in the ipsilateral nodose ganglion cells. These results indicate that a population of rat nodose ganglion cells contain nitric oxide synthase and that some of these neurons project their axons through the superior cervical ganglion and terminate in the peripheral target tissues. The possibility that nitric oxide synthase-immunoreactive vagal afferent fibers may participate in nociception is considered. PMID- 7538648 TI - Characterization of the mononuclear cell infiltrate in the glomerulus of rats with glomerulonephritis. AB - Bone marrow-derived inflammatory cells contribute to glomerular damage in experimental glomerulonephritis. The time sequential appearance and the pattern of inflammatory cells in the diseased glomerulus is, however, unclear. We therefore characterized the inflammatory cell infiltrate in a model of unilateral in situ immune complex glomerulonephritis. The cellular infiltrate was specific for the diseased kidney and independent of changes in peripheral blood or spleen. We found an influx of leukocyte common antigen (LCa)-positive and ED1-positive (monocytes/macrophages) cells in isolated glomeruli as early as 24 h after induction of the disease. Lymphocytes of the CD4-, CD8- and CD20-positive phenotypes were present in diseased glomeruli at Day 10. Complement depletion prevented the influx of monocytes/macrophages at 24 h in the glomerulus, which indicates that complement activation is important for the glomerular cell infiltrate at this early time point. The results demonstrate that the inflammatory cell infiltrate is regulated in situ at the glomerular level and not accompanied by similar changes in peripheral blood and spleen cells. PMID- 7538649 TI - Interleukin-1 beta induces the formation of nitric oxide in isolated juxtaglomerular cells: influence on renin secretion. AB - Renin secretion may be modulated by nitric oxide (NO). We studied whether interleukin-1 beta (IL-1 beta) induces endogenous NO synthesis in mouse juxtaglomerular cells (JGC) in primary culture, and whether endogenous NO or NO applied exogenously via sodium nitroprusside (SNP) influences renin secretion. JGC seeded on culture plates were stimulated by IL-1 beta or by SNP. Cyclic guanosine 3,5'-monophosphate (cGMP) in the cell supernatant was determined as indicator for NO effects. Stimulation of JGC with IL-1 beta or SNP increased cGMP in the supernatant significantly. The NO synthase inhibitors NG-nitro-L-arginine or NG-monomethyl-L-arginine, or the NO scavenger oxyhaemoglobin prevented the IL 1 beta-induced increase of cGMP. The biological activity of NO was shown in a bioassay by the vasodilatory effect of the effluent from an IL-1 beta-stimulated JGC column on a precontracted rat aortic ring and was prevented by oxyhaemoglobin and methylene blue. Renin activity of JGC was detected in the culture supernatants and the cells. Spontaneous renin secretion into the cell supernatant was 26 +/- 1% of total activity. Melittin or forskolin concentration dependently increased renin secretion up to 90 +/- 2%. Incubation of JGC with IL-1 beta in the absence or presence of NO inhibitors did not alter spontaneous or stimulated renin secretion. SNP (30 microM) had a dual effect on renin secretion. After 1 h of incubation, it inhibited basal renin secretion, whilst it had a stimulatory effect after 20 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538650 TI - Hepatitis C virus (HCV) infection in haemodialysed patients: HCV-RNA and anti-HCV antibodies (third-generation assays). AB - Samples from 128 haemodialysed patients were tested by anti-HCV 2nd- and 3rd generation assays from Ortho: 53 were positive by ELISA 2.0 and 54 (42%) by ELISA 3.0. The 54 anti-HCV-positive patients were tested by RIBA-2 and RIBA-3 and by PCR for the detection of HCV-RNA: 46 of the 47 patients (98%) reactive by RIBA-2 and 48 of the 51 patients (94%) reactive by RIBA-3 were HCV-RNA positive. Three patients with RIBA-3 indeterminate results were HCV-RNA negative. Among the 74 anti-HCV negative patients, 29 were tested by PCR with negative results. Two distinct episodes of hepatitis C have been observed in two patients during the follow-up and 44 of the 50 patients (88%) known positive for anti-HCV since at least 1989 were still viraemic in 1993. A very high correlation was found between anti-HCV antibodies reactive by RIBA and the presence of HCV-RNA. A lack of protection after a resolved infection and a high frequency of chronic disease have been observed as well as a reinfection or a reactivation of the infection in two patients. PMID- 7538651 TI - Incidence of and risk factors for hepatitis B virus and hepatitis C virus infection among haemodialysis and CAPD patients: evidence for environmental transmission. AB - Hepatitis B virus (HBV) serum markers (HBsAg, anti-HBs, anti-HBc) and antihepatitis C antibody (anti-HCV) were prospectively followed in haemodialysis and CAPD patients. From January 1987 to January 1990, 185 patients on haemodialysis and 124 on CAPD were analysed. Among patients susceptible to HBV (69 on haemodialysis and 70 on CAPD), there were 17 HBsAg seroconversions on haemodialysis (0.19/patient-year) and 1 on CAPD (0.01/patient-year). A Cox proportional hazards model showed that haemodialysis treatment was the only risk factor significantly associated with HBV infection, thus suggesting transmission through the environment. Regarding hepatitis C, 83 anti-HCV-negative patients on haemodialysis and 46 on CAPD were followed. There were 18 seroconversions on haemodialysis (0.15/patient-year) and two seroconversions on CAPD (0.03/patient year). Haemodialysis treatment was also the only risk factor significantly associated with a higher risk of HCV infection. The hazard ratio for HCV infection in haemodialysis patients was 5.7 compared to CAPD patients. Nevertheless, for one patient on CAPD treatment transfusions were the only possible source of HCV infection. In conclusion, both viruses were transmitted mainly through the haemodialysis environment, but the role of transfusions could not be excluded. PMID- 7538652 TI - Proportion of NADPH-diaphorase reactive sympathetic efferents innervating the normal and inflamed knee joint of the cat. AB - To determine whether postganglionic sympathetic efferents may be a source of nitric oxide in normal and inflamed knee joints, the distribution of NADPH diaphorase was studied in retrogradely labelled neurons of the paravertebral sympathetic ganglia in the cat. In these ganglia, a strong NADPH-diaphorase staining reaction was found in 3-7% (segments L2-L5) and 14-17% (segments L6 and L7) of the neurons. Only 2.5 +/- 1.2% (mean +/- SD, 4 normal joints), of 2207 labelled neurons showed a positive staining reaction. During a unilateral inflammation (32 h), this proportion slightly increased to 5.0 +/- 1.2% (unaffected joints of 4 animals, 2510 labelled perikarya) and 5.4 +/- 2.8% (inflamed joints of 4 animals, 2578 labelled perikarya). However, these increases only attained statistical significance between the values obtained from control animals and from the normal sides of animals with a monoarthritis. Thus, the data indicate that in the sympathetic innervation of the normal and inflamed knee in the cat, the release of nitric oxide has little or no importance. PMID- 7538654 TI - Expensive medicines. PMID- 7538653 TI - Substance P is a possible neurotransmitter in the rat spinothalamic tract. AB - In order to shed some light on the neurotransmitters in the spinothalamic tract (STT), we examined, biochemically and immunohistochemically, the contents of various neurotransmitter candidates in the terminal field of the STT after cervical hemi-chordotomy (HC) and dorsal quadrant-chordotomy (dQC) in the rat. Substance P (SP), calcitonin gene-related peptide (CGRP), enkephalin, neuropeptide Y, neurotensin, oxytocin and dynorphin A were analyzed immunohistochemically. The contents of neuropeptides (SP, CGRP and cholecystokinin octapeptide) were measured by radioimmunoassay and those of amino acids (aspartic acid, glutamic acid, gamma-aminobutyric acid (GABA) and glycine) and noradrenaline were determined using high-performance liquid chromatography. Cervical hemi-chordotomy, but not dQC, caused significant decreases of the SP like immunoreactivity in and SP content of the ventral thalamus on the ipsilateral side, compared with that on the contralateral side and of rats subjected to sham-operation. However, neither HC nor dQC resulted in any changes in the ventral thalamic contents of other putative neurotransmitters examined. These results suggest that, in rats, the STT contains SP and that SP-positive fibers run in the ventral half of the ascending spinal tract at the cervical level. PMID- 7538655 TI - JAK3 associates with the human interleukin 4 receptor and is tyrosine phosphorylated following receptor triggering. AB - In human B cells, interleukin 4 (IL4) acts in regulating proliferation, antigen expression, isotype switching and differentiation. These different effects are mediated through the IL4R complex including the IL2R gamma chain (gamma c) and a specific p130/140 binding unit referred below as human Interleukin 4 Receptor (IL4-R). Here, we studied the signal transduction events following IL4R activation and leading to CD23 expression on resting B cells. We demonstrate that IL4R triggering induced the tyrosine phosphorylation of JAK3 and of a p170 protein. Coimmunoprecipitation of JAK3 with the IL4R suggests a physical association which exists prior to IL4R complex stimulation. Orthovanadate treatment, while having no effect on IL4-induced p130 phosphorylation, leads to the hyperphosphorylation of the p170 and inhibits IL4-induced CD23 expression. These suggest that two mandatory steps exist in early IL4 signaling: one controlled by JAK3 activation and the other by the p170 phosphoprotein. PMID- 7538647 TI - Crossed aphasia. An update. AB - The aim of this article is to present an update of a rare but interesting problem: "crossed aphasia". This term indicates the presence of aphasia after unilateral cerebral lesion of the hemisphere ipsilateral to the patient's dominant hand. We report two cases, review the most relevant literature, and analyze clinical, neuroanatomical, and neurophysiological aspects, taking in consideration the various interpretations proposed to explain this unusual language disorder. PMID- 7538657 TI - Oxantel-activated single channel currents in the muscle membrane of Ascaris suum. AB - The patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10-100 microM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of -75 mV and a concentration of 10 microM was 1.34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2.41 x 10(7) M-1 s-1 at -50 mV and 2.64 x 10(7) M-1 s-1 at -100 mV. The unblocking rate constant, K-B, did exhibit voltage sensitivity being 443.6 s-1 at -50 mV and 86.8 s-1 at -100 mV. Thus the blocking dissociation constant KB (= K-B/K + B) was 18.5 microM at -50 mV and 3.3 microM at -100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed. PMID- 7538656 TI - Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. AB - In the present study we demonstrate that erbB-3 and erbB-2 cooperate in neoplastic transformation. Under conditions in which neither gene alone induced transformation, they readily transformed NIH3T3 cells if co-expressed. Furthermore, at high expression levels of ErbB2 which cause transformation, ErbB3 enhanced focus formation by one order of magnitude. Synergy required an intact ErbB2 extracellular domain and tyrosine kinase activity. Cooperation between ErbB3 and ErbB2 involved heterodimerization and increased tyrosine phosphorylation of ErbB3. Signaling by the heterodimer resulted in increased PI 3 kinase recruitment as well as quantitative and qualitative differences in substrate phosphorylation. Evidence for signaling by an active ErbB3-ErbB2 heterodimer in four mammary tumor cell lines indicated relevance of this mechanism for human neoplasia. Our detection of the NDF/heregulin transcript in NIH3T3 cells implicates an autocrine loop involving this ligand in signaling by the ErbB3-ErbB2 heterodimer in the model system, whereas heregulin-independent mechanisms likely exist for cooperative signaling by ErbB3 and ErbB2 chronically activated in some human mammary carcinomas. PMID- 7538658 TI - A phase II study of recombinant human granulocyte-colony stimulating factor (rHuG CSF, lenograstim) in the treatment of agranulocytosis in children. AB - The present study evaluated the clinical efficacity and tolerability of the subcutaneous (SC) administration of lenograstim, a glycosylated form of rHuG-CSF identical to human G-CSF, in the treatment of congenital agranulocytosis. Assessment criteria included neutrophil response and response stability, incidence and severity of infection and gingivostomatitis and quality of life. Lenograstim, at induction dosages of 5 (n = 9), 10 (n = 2) or 20 (n = 1) microgram/kg/day SC, produced neutrophil recovery in all of 12 children with congenital agranulocytosis. There was a median delay of 7 days to recovery after establishment of the effective induction dose. Whereas this dosage maintained a stable neutrophil response in 7 patients, the remaining 5 required dosage increases and dose reduction during maintenance therapy was not possible in these 5 cases. Among 4 patients stabilised at a dosage of 5 micrograms/kg/day, in 2 cases a lower minimum effective dose of 2 micrograms/kg/day was attained over the maintenance phase. Administration of twice the daily dose of lenograstim on alternate days was feasible in 3 of 8 patients. Lenograstim therapy reduced the incidence of infection and hospitalisation for infection relative to the prestudy period, while in 6 of 9 cases there was complete recovery from gingivostomatitis. Only one patient discontinued treatment on account of adverse events. Finally, perceived health and disease related symptoms showed a significant (p < 0.001) amelioration in the course of the study. Thus, lenogastrim produced sustained neurotrophil recovery in patients with congenital agranulocytosis, decreased the incidence and severity of infection and improved the quality of life. PMID- 7538661 TI - RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain. AB - The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein. PMID- 7538660 TI - Efficient extension of a misaligned tRNA-primer during replication of the HIV-1 retrovirus. AB - The human immunodeficiency virus (HIV) and other retroviruses show extensive genomic variation, which is primarily due to error-prone replication by the viral reverse transcriptase (RT) enzymes. RT errors include misincorporation with subsequent extension of the mismatched terminal base, and extension of realigned primer-template duplexes. Whereas both RT-mediated mechanisms have been extensively studied in vitro, almost no in vivo experiments have been performed. In this work, we analyzed the ability of HIV-1 RT to extend a misaligned tRNA(Lys3) primer in vivo. This tRNA binds with its 3'-terminal 18 nt to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). We constructed a series of mutant viral genomes with small insertions or deletions in the PBS sequence, resulting in misalignment of the tRNA primer. Extension of the misaligned primer did occur with reasonable efficiency for some of the mutants, resulting in reversion to the wild-type viral sequence. The infectivity and reversion frequency of the PBS mutants is therefore a measure of the efficiency of extending a misaligned primer in vivo. Using virion-derived primer-template complexes, we also measured the tRNA-priming efficiency in vitro. The combined results show that HIV-1 RT can elongate a misaligned primer and that the efficiency of primer extension is determined by the extent of the mismatch. PMID- 7538663 TI - Inhibition of tumor angiogenesis and the therapeutic ability of linomide against rat prostatic cancers. AB - Linomide, a quinoline-3-carboxamide, has growth-inhibitory effects against a series of Dunning R-3327 rat prostatic cancers in vivo [Ichikawa et al.: Cancer Res 52:3022-3028, 1992]. In addition, we have demonstrated that daily linomide treatment can inhibit angiogenic responses in nontumor-bearing rats and reduce tumor blood flow in tumor-bearing rats [Vukanovic et al.: Cancer Res 53:1833, 1993]. In the present study we have demonstrated that the reduced tumor blood flow is due to linomide's ability to inhibit tumor angiogenesis, as documented by decreased number of blood vessels in prostatic carcinomas growing in rats treated daily with linomide. Due to linomide's ability to inhibit tumor angiogenesis, and since tumor angiogenesis is required not only for the growth of the primary cancer but also for its ability to metastasize, the effect of linomide on metastasis was directly tested using a quantitation metastasis assay. These in vivo experiments demonstrated that daily linomide treatment decreased by 3-fold the extent of dissemination of cancer cells to the lungs. To test if this antimetastatic response is due to direct effects of linomide on the metastatic cells themselves as well as an induced effect upon inhibition of tumor angiogenesis, additional studies were performed. These studies demonstrated that linomide is not converted in vivo to metabolite(s) which are directly cytotoxic or cytostatic to the prostatic cancer cells themselves. These studies also demonstrated that linomide does not decrease the attachment, migration, or invasive abilities of metastatic cancer cells. These results suggest that the major mechanism for the antitumor and antimetastatic effects of linomide is via its inhibition of tumor angiogenesis. Additional studies have demonstrated that in vivo linomide treatment results in the apoptotic death of thymocytes. This cytotoxic effect is not required for linomide's antitumor effect, nor is it due to elevated plasma levels of glucocorticoid. PMID- 7538662 TI - DNA and RNA-DNA annealing activity associated with the tau subunit of the Escherichia coli DNA polymerase III holoenzyme. AB - The DNA polymerase III (pol III)holoenzyme is the 10 subunit replicase of Escherichia coli. The 71 kDa tau subunit, encoded by dnaX, dimerizes the core polymerase (alpha epsilon theta) to form pol III'[(alpha epsilon theta)2 tau 2]. tau is also a single-stranded DNA-dependent ATPase and can substitute for the gamma subunit during initiation complex formation. We show here that tau also possesses a DNA-DNA and RNA-DNA annealing activity that is stimulated by Mg2+, but neither requires ATP nor is inhibited by non-hydrolyzable ATP analogs. This suggests the tau may act to stabilize the primer-template interaction during DNA replication. PMID- 7538659 TI - Administration of granulocyte colony-stimulating factor from day 7 after autologous bone marrow transplantation: effects on neutropenia and duration of hospitalization. AB - G-CSF (5 mg/kg/day Filgrastim) was administered from day 7 after autologous bone marrow transplantation (ABMT) in a series of 17 patients treated for multiple myeloma or non-Hodgkin's lymphoma. In comparison with retrospective controls receiving ABMT without G-CSF and matched for age, underlying disease, disease status at ABMT, number of CFU-GM/kg reinfused, conditioning regimen and number and type of chemotherapy courses prior to ABMT, the duration of neutropenia, intravenous antibiotics and hospitalization was significantly reduced in the G CSF group (p < 0.001). Delaying the administration of G-CSF after ABMT is an interesting possibility which merits further exploration in prospective randomized studies. PMID- 7538664 TI - Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver. AB - Intraperitoneal injection of epidermal growth factor into mice results in the appearance of multiple tyrosine-phosphorylated proteins in liver nuclei within minutes after administration. We have previously identified three of these proteins as Stat 1 alpha, Stat 1 beta (p91, p84), and Stat 3 (p89). In the present report we demonstrate that Stat 5 (p92), the recently described prolactin inducible transcription factor detected in mammary glands, is the major tyrosine phosphorylated protein translocated to the nucleus in mouse liver in response to epidermal growth factor. Furthermore, gel-shift analysis and affinity purification revealed that Stat 5, Stat 1 alpha, and Stat 1 beta specifically bind to the prolactin inducible element upstream of the beta-casein promoter. PMID- 7538665 TI - Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes. AB - Two water channel homologs were cloned recently from rat kidney, mercurial insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expression of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels. PMID- 7538666 TI - CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. AB - Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3 fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions. PMID- 7538667 TI - Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations. AB - Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions. PMID- 7538669 TI - Catalytically critical nucleotide in domain 5 of a group II intron. AB - Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems. PMID- 7538668 TI - Roles of nitric oxide in tumor growth. AB - A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type parental cells. This growth retardation was reversed by the addition of N-iminoethyl-L-ornithine. In nude mice, however, the tumors from these cells grew faster than those derived from wild-type cells and were markedly more vascularized, suggesting that NO may act as part of a signaling cascade for neovascularization. Recent observations that the generation of NO in human breast and gynecological cancers correlates positively with tumor grade are consistent with this hypothesis. We suggest that NO may have a dual pro- and antitumor action, depending on the local concentration of the molecule. PMID- 7538670 TI - Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein. AB - Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death. PMID- 7538671 TI - Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide. AB - In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1 1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity. PMID- 7538672 TI - The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein. AB - The retinoblastoma protein (Rb) is a target of viral oncoproteins. To explore the hypothesis that viral proteins may be structural mimics of cellular proteins, we have searched cDNA libraries for Rb-binding proteins. We report here the cloning of a cDNA for the protein RIZ from rat and human cells. RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs. It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A. A domain is conserved between RIZ and the PRDI-BF1/Blimp-1 differentiation factor. Other motifs of RIZ include putative GTPase and SH3 (src homology domain 3) domains. RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues. It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined. While the function of RIZ is not yet clear, its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma. PMID- 7538674 TI - Activation of the Lck tyrosine protein kinase by hydrogen peroxide requires the phosphorylation of Tyr-394. AB - Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells. PMID- 7538673 TI - Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid. AB - mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor suppressive role may involve a senescence pathway. PMID- 7538676 TI - Intrinsic transcript cleavage activity of RNA polymerase. AB - The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage constitutes the second enzymological activity of RNA polymerase along with polymerization/pyrophosphorolysis of RNA, whereas the Gre proteins merely enhance this intrinsic property. PMID- 7538679 TI - Twin analysis of odor identification and perception. AB - Findings from the first twin analysis using the University of Pennsylvania Smell Identification Test (UPSIT) and a phenyl ethyl alcohol (PEA) threshold detection test are presented. A genetic influence on odor identification was suggested for males, but not for females, consistent with previous twin research on physical measures. In addition, females scored significantly higher on the UPSIT than males. A curvilinear age trend for odor identification was detected for males, but not for females. In contrast, neither genetic, age, nor gender effects were suggested for PEA sensitivity. Performance on the olfactory tests was unrelated to scores on standard measures of intelligence. PMID- 7538678 TI - Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. AB - Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments. PMID- 7538681 TI - Self-expandable metallic stents in high-risk patients with benign prostatic hyperplasia: long-term follow-up. AB - PURPOSE: To evaluate the long-term clinical utility of self-expandable metallic Z stents in benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Under fluoroscopic guidance, 14 multiple-connected Z stents (10 mm in diameter fully expanded, 30-60 mm in length) were placed in 13 patients with bladder obstruction from BPH and high operative risks. The stents were placed in the prostatic urethra with 2-20-mm protrusion into the urinary bladder in six patients (group A) and entirely within the prostatic urethra in seven patients (group B). RESULTS: Eleven of 13 patients could void immediately, and the other two patients with atonic bladder voided within 8 weeks. Maximum urine flow rates just after stent placement were 8-27 mL/sec. Two patients died of unrelated causes within 2 months. During the follow-up period (mean, 37 months) in the other 11 patients, all patients in group A but none in group B underwent surgery owing to stone formation where the stent protruded into the bladder. CONCLUSION: Expandable Z stents are effective in patients with BPH but should not protrude into the urinary bladder because of stone formation. PMID- 7538677 TI - Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA 2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7. AB - Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c kitBR). We used reverse transcriptase polymerase chain reaction to assay the c kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation. PMID- 7538675 TI - Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo. AB - The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo. PMID- 7538680 TI - Toward an understanding of unmentalized experience. AB - The term unmentalized experience is defined, elaborated, explicated, and illustrated with clinical examples from the analysis of adult patients. The origins of the author's conceptualization of the term are traced from Freud's early notion of the "anxiety equivalent" through the work of present-day object relations theorists. A model for the somatic recording of early pre- and postnatal experience is derived from Stern's research in the field of infant development, and some implications for treatment are noted. PMID- 7538683 TI - Genetic dissection of synthesis and function of modified nucleosides in bacterial transfer RNA. PMID- 7538682 TI - Esophageal carcinoma: initial results of palliative treatment with covered self expanding endoprostheses. AB - PURPOSE: To assess the effectiveness of a polyurethane-covered self-expanding metallic endoprosthesis in the relief of dysphagia due to irresectable esophageal carcinoma. MATERIALS AND METHODS: Thirty-two patients (20 men, 12 women) aged 41 89 years (median, 70 years) with inoperable esophageal carcinoma underwent stent placement (44 stents). All patients underwent both clinical and radiologic examination before and after treatment. Clinical follow-up was performed at 4 week intervals. RESULTS: Stent placement was successful in all patients, with good symptomatic relief and no serious complications. Eleven patients needed more than one stent because of early partial migration, late complete migration, tumor overgrowth, or long stricture. Seven patients had associated esophageal fistulization or perforation; leaks were successfully sealed after stent insertion. The mean dysphagia score was 3.38 +/- 0.49 (standard deviation) before treatment and 0.81 +/- 0.88 at 3-4 days after insertion. Nineteen patients died, with a median survival time of 78 days (range, 12-245 days), and 13 were alive 14 67 days (median, 30 days) after treatment and were swallowing a near normal diet. CONCLUSION: The insertion of plastic-covered endoprostheses provides rapid, safe, and effective palliation of malignant esophageal obstruction. PMID- 7538684 TI - Calcitonin gene-related peptide (CGRP) and its effects on protein release in vitro in the developing submandibular gland of the rat. AB - Indirect immunohistochemical methods were used to study presence and number of CGRP-immunoreactive (CGRP-IR) nerve fibers in the submandibular gland and ganglion cells of the superior cervical, submandibular and trigeminal ganglia of the developing rat. The effect of CGRP on peroxidase and total protein release was also studied in the developing postnatal submandibular glands of 1, 5, 12 and 30-day-old, as well as adult rats by in vitro methods. The possible costimulation of CGRP with SP, NKA or carbachol on 5-day-old and adult rats was also tested. The stimulatory effects of these compounds were compared to the basic release of peroxidase and total amount of proteins from submandibular gland fragments in incubation solution. CGRP-IR nerve fibers were found in relatively high number during post-natal development, mainly around blood vessels and ducts. Some CGRP IR nerve fibers were also detected around acini. The number of these fibers was quite low and remained constant during the post-natal development. The number of CGRP-IR trigeminal ganglion cells was higher on the 5th and 12th post-natal day than later in development and in adult animals. At the same time, superior cervical- and submandibular ganglion cells were non-reactive for CGRP, suggesting trigeminal origin of CGRP-IR nerve fibers during the development in the submandibular gland. In the secretory studies, CGRP per se stimulated both peroxidase and total protein release in the submandibular gland most effectively on 5th and 12th post-natal days, while there was no clear secretory response in the adult glands. In the 5-day-old submandibular gland CGRP in combination with SP, NKA or carbachol clearly enhanced the total protein secretory response when compared with the release caused by these substances alone. However, in the adult submandibular gland, the combinations did not enhance total protein release more than any of the substances alone. Furthermore, it can be concluded that the presence of a more dense CGRP-IR innervation during the early postnatal period in the developing submandibular gland is accompanied with an increased responsiveness of the secretory elements to CGRP. PMID- 7538686 TI - Role of interferons in the restriction of HIV replication in human monocytes/macrophages. PMID- 7538687 TI - Interferon interactions with HIV1 in macrophages. PMID- 7538689 TI - Competition of viral and satellite RNAs of cucumber mosaic virus for replication in vitro by viral RNA-dependent RNA polymerase. AB - Cucumber mosaic virus (CMV) satellite RNA-induced viral symptom modulation is usually accompanied by a significant reduction of virus accumulation in plant tissue, which has led to the hypothesis that satellite RNA competes with the viral RNAs for replication by the viral replicase and thereby reduces viral RNA synthesis and viral symptoms. In this report, the RNA synthesis of the viral and satellite RNAs of CMV was studied in vitro using an RNA-dependent RNA polymerase (RdRp) purified from CMV-infected plants. Comparison of the kinetics of the CMV RdRp-catalysed RNA synthesis using as templates viral RNAs and satellite RNA, alone or in an appropriate mixture, showed that these RNAs competed with each other for RNA synthesis by the CMV RdRp. Determination of the rates of 32P incorporation into the viral and satellite double-stranded RNA products revealed an apparent replication advantage of the satellite RNA over viral RNAs. The results provide strong support for a previously proposed biochemical mechanism that attributes CMV satellite-induced viral symptom modulation to the replication competition between the satellite and viral RNAs. PMID- 7538685 TI - Ca2+ signaling through secretagogue and growth factor receptors on pancreatic AR42J cells. AB - Intracellular signaling by an increase in [Ca2+]i was observed in pancreatic AR42J cells in response to agonists whose receptors are G-protein coupled including cholecystokinin (CCK), bombesin, carbachol, substance P, pituitary adenylate cyclase activating peptide (PACAP), bradykinin, ATP, calcitonin gene related peptide (CGRP), and in response to growth factors EGF and FGF whose receptors are tyrosine kinases. The response to growth factors was smaller both in magnitude and in the percentage of cells responding but was independent of extracellular Ca2+. CCK and carbachol induced sizeable increases in inositol phosphates while growth factors did not. The responses to both carbachol and EGF, however, were blocked by the phospholipase C inhibitor U73122. The tyrosine kinase inhibitor, genestein, blocked the response to EGF but not that to CCK. These data are consistent with two types of signaling mechanisms in AR42J cells. Secretagogues act on receptors which couple through G proteins to induce a large amount of inositol phosphate production and subsequent intracellular Ca2+ mobilization. Growth factors act on receptors which signal through tyrosine kinase activity and in this cell type produced limited amounts of inositol phosphate and a smaller increase in intracellular Ca2+. PMID- 7538688 TI - Antimicrobial activity of 9-oxo and 9-thio acridines: correlation with interacalation into DNA and effects on macromolecular biosynthesis. AB - The antimicrobial activity of several new 9-acridinones and 9-thioalkylacridines towards Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis and Candida albicans was investigated. Minimal inhibitory, bactericidal and fungicidal concentrations were determined using a microplate assay which enabled inhibitory, bactericidal and fungicidal indices to be calculated. These indices facilitated structure/activity relationship studies. DNA-intercalating capability and DNA supercoiling inhibitory effects as well as inhibitory effects on macromolecular synthesis were determined. Results showed that intercalation into DNA, which is the mechanism of action usually postulated for acridines, cannot be correlated with the properties examined. However, inhibition of RNA synthesis may be involved in the antimicrobial activity of the drugs. PMID- 7538690 TI - Clinicopathologic studies of an eye after submacular membranectomy for choroidal neovascularization. AB - BACKGROUND: Submacular membranectomy has been suggested as an alternative treatment for subfoveal choroidal neovascularization (CNV). Clinicopathologic features of the right eye of a 59-year-old man with recurrent subfoveal CNV who underwent submacular membranectomy after two unsuccessful laser photocoagulation treatments are reported. METHODS: The surgically excised subfoveal membrane was sectioned serially and evaluated by light microscopy. The globes were obtained postmortem and serial sectioned through the macula and optic nerve head for light microscopy. Ultrastructural study of a tissue section in the center of the lesion was performed. RESULTS: Histopathologic study of the surgically excised membrane disclosed a thin two-component fibrovascular membrane with the larger component internal to residual retinal pigment epithelium and basal laminar deposit. Photoreceptor outer segments were present on the internal surface of the membrane near one margin. Light and electron microscopic study of the postmortem globe revealed a very thin subfoveal subretinal pigment epithelial fibrovascular membrane with loss of photoreceptor cell layer in a central 0.5 mm area, loss of outer segments, reduction of inner segments, and thinning of the outer nuclear layer in the remainder of the lesion. There was moderate retinal pigment epithelial attenuation and mild basal laminar and basal linear deposits. CONCLUSION: Submacular membranectomy for recurrent subfoveal CNV secondary to age related macular degeneration after two unsuccessful laser photocoagulation treatments appeared to be effective with repopulation of two thirds of the area of membranectomy by extension of attenuated retinal pigment epithelium from adjacent areas. There was, however, persistence or recurrence of CNV, moderate atrophy of the overlying retina with total loss of the photoreceptor cells over the central 0.5 mm of the membrane, and moderate loss of the photoreceptor cells over the remaining area. PMID- 7538691 TI - The potential of gene therapy for treatment of kidney diseases. AB - As knowledge of the genes and molecular events involved in specific kidney disorders expands, so do the potential applications for treating the disorders on fundamental levels. Progress in gene therapy for genetic diseases for which molecular events are simpler or better characterized has already been substantial. In addition, exciting first steps in targeting genes specifically to the kidney have emerged, although much work in defining kidney-specific genes and gene regulatory elements still needs to be done. To provide a framework for understanding the concepts and problems involved in gene therapy, we will discuss basic aspects of gene structure and regulation, gene delivery vectors, steps necessary to achieve tissue- and cell-specific expression of delivered genes, and some present and future applications of gene therapy in kidney diseases. PMID- 7538693 TI - Incidental carcinoma of the prostate. AB - Transrectal ultrasonography (TRUS), digital rectal examination (DRE), and quantification of serum prostate-specific antigen (PSA) are accepted and evaluated methods for detecting prostate cancer. Positive predictive values (PPV) of DRE and TRUS are low, and only slightly enhanced when used in combination with PSA. PSA lacks sufficient sensitivity and specificity to be used alone as a screening test for prostate cancer. The parameters PSA-density and PSA-velocity make PSA a better tumor marker, but they are not reliable on an individual basis. Age-specific reference ranges have the potential to make PSA a more sensitive tumor marker for men less than 60 years of age and a more specific one for men beyond 60 years. With currently available diagnostic methods approximately 10% of patients undergoing transurethral or open resection of the prostate for presumed benign prostatic hyperplasia will have carcinoma detected in the histologic material. In 392 patients successively treated in our clinic for presumed BPH and thoroughly investigated to exclude prostatic carcinoma (DRE, TRUS, biopsy when PSA > 4 ng/ml or PSA-D > 0.15), the tumor was found incidentally in 4%. Another finding in this study was the detection of prostatic carcinoma by random biopsy in patients without a palpable or visible tumor by imaging and without PSA increase (> 4 ng/ml). Biopsies were performed because of a hypoechoic zone in the opposite lobe which turned out to be negative. Such tumors cannot be properly classified in the current TNM system. Treatment options for patients with incidental prostatic carcinoma are age- and stage-dependent. Patients less than 60 years old may be treated with a curative approach, irrespective of the T category (T1a or T1b); patients with a life expectancy longer than 10 years and a pT1b incidental carcinoma likewise should be offered a curative therapy. PMID- 7538694 TI - Radical prostatectomy or deferred treatment? AB - Screening for prostate cancer has intensified, due both to increased patient and physician awareness and to the availability of new, more sensitive diagnostic tools (prostate-specific antigen [PSA], rectal ultrasound, etc.). Consequently, the number of newly diagnosed cases of prostatic cancer is rising rapidly, whereas the frequency of death due to prostate cancer remains almost stable. It must therefore be assumed that the number of patients in whom a diagnosed prostate cancer will not be fatal is also increasing. Consequently, not every prostatic carcinoma requires radical treatment when diagnosed. Also, it must be concluded that not every man who is a long-term survivor after radical prostatectomy owes his survival to the treatment. Long-term survival may reflect the relatively benign biological potential of this disease in an individual patient. Therefore there is an inherent risk of overtreating patients and this must be weighed against the costs, the postoperative morbidity, and the mortality, albeit low, of a radical prostatectomy. Nevertheless, as long as we do not have diagnostic tools which, at an early stage of prostatic cancer, enable us to determine whether a carcinoma will ultimately have a fatal outcome, we are obliged to offer a radical prostatectomy to younger patients (who have a life expectancy of more than 10 years) as long as they have organ-confined disease. PMID- 7538692 TI - Diagnostic markers of prostate cancer: utility of prostate-specific antigen in diagnosis and staging. AB - The optimal tumor marker for prostate cancer would be effective for early detection, staging, and monitoring patients after definitive treatment. This marker would have a high sensitivity, specificity, and positive predictive value for distinguishing men with benign prostatic hyperplasia (BPH) from men with early prostate cancer. Such a marker would consistently detect biologically significant disease, correlate with clinical and pathologic staging, and predict prognosis. In addition, this marker would be accurate at indicating cure or progression of disease after treatment. Certainly, the ideal marker also would be reproducible, inexpensive, generate results rapidly, be easy to perform, be accessible to clinicians, and tolerable to patients. Unfortunately, such a "super" marker does not exist at this time. However, prostate-specific antigen (PSA) has many of the aforementioned capabilities. This article will describe the current utility of PSA in the diagnosis and staging of prostate cancer. PMID- 7538696 TI - Umbilical cords: turning garbage into clinical gold. PMID- 7538695 TI - Hormone refractory disease. AB - Hormone refractory disease is observed in less than 20% of newly diagnosed cases of advanced prostatic cancer. In the majority of cases, hormone refractory disease appears after a median time of 18 months of endocrine manipulation and is attributed to the selection and/or cloning of pre-existing or de novo appearing hormone-independent or resistant cell lines. There are no generally accepted rules for second-line management. The varying sets of criteria used by different study groups make comparisons of widely different regimens very difficult. Actually, it seems reasonable to consider length of survival as the only objective response criterion. This implies, however, that there should be an unanimous definition about the moment of primary treatment failure. Indeed, the detection of hormonal escape is a gradual event and the relative length of survival time depends on the chosen moment of therapy administration. To date, monitoring of prostate specific antigen (PSA) has become the best and primary tool to document progression of disease. Earlier diagnosis based on a rise in PSA levels in patients that are still asymptomatic with a good performance status, might provide the opportunity to treat patients that could profit from therapy and give a fair chance to the investigated drug to show efficacity and tolerability. In this paper we will discuss the rationale of the current available second-line therapeutic options in relapsed prostatic cancer. PMID- 7538697 TI - CD1 recognition by mouse NK1+ T lymphocytes. AB - Rare major histocompatibility complex (MHC) class I-like CD1-specific T cells have been isolated from human blood, but it has not been determined whether these clones are part of a defined subset of CD1-specific T cells selected during T cell development, or whether their recognition of CD1 is a fortuitous cross reaction. In mice, an entire subset of alpha beta thymocytes with a unique phenotype was found to be CD1-specific. This particular subset, and its human counterpart, provide evidence that CD1 has a general role in selecting and interacting with specialized alpha beta T cells. PMID- 7538698 TI - Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon. AB - An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes. PMID- 7538699 TI - Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. AB - A bacterial spore was revived, cultured, and identified from the abdominal contents of extinct bees preserved for 25 to 40 million years in buried Dominican amber. Rigorous surface decontamination of the amber and aseptic procedures were used during the recovery of the bacterium. Several lines of evidence indicated that the isolated bacterium was of ancient origin and not an extant contaminant. The characteristic enzymatic, biochemical, and 16S ribosomal DNA profiles indicated that the ancient bacterium is most closely related to extant Bacillus sphaericus. PMID- 7538701 TI - Molecular aspect of HIV-1 resistance to zidovudine and its reversal. PMID- 7538700 TI - Ion channels and the control of myometrial electrical activity. AB - Understanding the role of ion channels in the generation of slow waves and action potentials in the myometrium is critical in designing strategies to regulate uterine contractile activity. The development of the patch clamp technique has allowed the identification of specific types of channels in the myometrium and provided insights into their regulation by hormones and drugs. Specifically, new studies suggest that KATP and KCa channel openers could be important tools in the management of inappropriate uterine contractions, but peripheral effects will have to be controlled. Conversely, blockers of these same channels may have some effects on dystocia. The study of contractant-operated channels in the myometrium is still in its infancy, but promises new insights into possible modes of regulation as well. Myometrial activity is controlled at a number of levels. The regulation of ion channels is an important aspect, but receptor-mediated actions that do not appear to be voltage- or ion-dependent presumably are also important contributors and hence are sites of potential modulation as well. Clearly, future multifaceted approaches to tocolysis, and perhaps also dystocia, may well include agents targeting the activity of ion channels. PMID- 7538704 TI - Intensive weekly chemotherapy for elderly gastric cancer patients, using 5 fluorouracil, cisplatin, epi-doxorubicin, 6S-leucovorin and glutathione with the support of G-CSF. AB - AIMS AND BACKGROUND: Elderly patients constitute the largest group of advanced gastric cancer patients. Since there is a widespread misconception that the elderly are poorly tolerant of chemotherapy, most of them are untreated or receive a less aggressive but also less efficacious chemotherapy. However, because tolerance and response to cancer seem to vary more with physiologic age than chronologic age, we evaluated the feasibility and activity of an intensive weekly chemotherapy in elderly gastric cancer patients. METHODS: 23 advanced gastric cancer patients (13 males and 10 females), older than 70 years, received weekly: cisplatin, 40 mg/m2 i.v.; epi-doxorubicin, 35 mg/m2 i.v.; 6S-leucovorin, 250 mg/m2 i.v.; 5-fluorouracil, 500 mg/m2 i.v.; and glutathione, 1.5 g/m2 G-CSF, at the dose of 5 micrograms/kg, was administered daily from the day after to the day before each chemotherapy administration. RESULTS: Toxicity was mild. The most common adverse effects were leukopenia (grade 3 in 2 patients); thrombocytopenia (grade 3 in 1 patient) and anemia (grade 3 in 3 patients). Five patients (20%) achieved a complete response and 9 (39%) a partial response, resulting in an overall response rate of 59% (95% CI, 40% to 78%). The median survival time was 13 months for all the patients and 15 months for patients with objective responses. CONCLUSIONS: This regimen appears feasible also in elderly gastric cancer patients and, because of its activity, suitable for further studies. PMID- 7538703 TI - Preoperative hyperthermia combined with chemotherapy and irradiation for the treatment of patients with esophageal carcinoma. AB - AIMS AND BACKGROUND: The purpose of this study was to investigate the combined effects of hyperthermia, chemotherapy and irradiation on esophageal cancer. METHODS AND MATERIAL: Since 1978, we have clinically applied hyperthermia combined with chemotherapy and irradiation (HCR therapy), to patients with carcinoma of the esophagus. The clinical results of 136 patients receiving preoperative HCR therapy were then compared with those of 107 cases undergoing preoperative chemo-radiotherapy (CR). RESULTS: A histological examination of the resected esophagus after preoperative treatment revealed that 65.4% and 50.5% of the patients responded markedly (no viable cancer cells) and moderately (more than two thirds of all cancer cells destroyed) to HCR and CR therapies, respectively (p < 0.05). The five-year survival rates were 22.3% and 13.7% in the HCR and CR groups, respectively, and the difference was statistically significant (p < 0.01). In particular, for the patients classified as TNM Stages III and IV, a significantly longer survival period was obtained with HCR therapy (p < 0.05). In addition, no severe side effects were encountered in the patients given just hyperthermia. CONCLUSION: Our clinical results suggest that preoperative hyperthermo-chemo-radiotherapy shows great promise for treatment of patients with advanced carcinoma of the esophagus. PMID- 7538702 TI - Nitric oxide is a key molecule in migraine and other vascular headaches. AB - Nitric oxide (NO) may play a key role in migraine and other vascular headaches since glyceryl trinitrate (a donor of NO) and histamine (which probably activates endothelial NO formation) both cause a pulsating dose-dependent headache with several migrainous characteristics. At relatively high doses of glyceryl trinitrate, migraine sufferers develop stronger and more migraine-like headaches and more pronounced cerebral arterial dilatation than controls. After the infusion of glyceryl trinitrate, non-migraineurs remain headache-free while migraineurs develop a migraine-like attack. In this review, Jes Olesen, Lars Thomsen and Helle Iversen suggest that migraine may be caused by increased amounts and/or affinity of an enzyme in the NO-triggered cascade of reactions. NO may also be involved in the pathogenesis of other vascular headaches. PMID- 7538707 TI - Magnetic resonance imaging following microwave thermotherapy, laser ablation and transurethral resection in patients with BPH. AB - Morphologic changes in hyperplastic prostate tissue following three different treatments were analysed by MRI using an endorectal surface coil. In cases treated by TUMT, the MRI findings of the prostate showed hemorrhagic necrosis, which was absorbed in 12 weeks, and a widened prostatic urethra. In cases treated by laser ablation, the thermal effect went deep, causing necrotic changes, and it took more than 8 weeks for the tissue to be shed and re-epithelization completed. In TURP, degenerative changes were seen only near the surface following mass reduction of the hyperplastic tissue, and the capsule shrinkage resulted in normalization of the prostatic urethra. It is suggested that improvement of the other parameters, such as IPSS and peak flow, in patients with obstructive BPH basically depends on these morphologic changes shown by MRI. PMID- 7538708 TI - [Initial experiences with the Memotherm Stent in treatment of benign prostatic hyperplasia]. AB - We report our experience with a new thermosensitive stent (Memotherm), which has been invented recently as an alternative method for treatment of benign prostatic hyperplasia. The wall of this stent is a thermoreactive mesh made of nitinol, which reaches its maximum force of expansion at body temperature. Its high degree of flexibility allows the Memotherm stent to fit the natural course of the prostatic urethra. This stent is available in different sizes between 2 and 8 cm to meet the demand for different lengths of prostatic urethra. Furthermore, the meshed structure of this stent allows its atraumatic removal. From April 1992 to September 1993, 54 consecutive patients with benign prostatic hyperplasia and Memotherm stents inserted. The mean patient age was 76.1 years (range 61-98 years). Patients were considered suitable for treatment with the Memotherm stent when they presented with a poor operative risk. Mean prostatic volume was 51.9 +/ 25 ml (20-150 ml), and on average a stent measured 32.3 +/- 9.5 mm (20-70 mm). Preoperatively, only 14 patients (26%) were able to void, and 40 patients (74%) had had a transurethral or suprapubic catheter placed. After placement of the Memotherm stent, 53 of 54 patients were able to void. In the patients who were able to void preoperatively, the mean maximum flow rate increased from 4.5 ml/s to 15.8 ml/s within the first 6 months after treatment. Within the same period of time, the mean residual urine volume decreased from 194.4 ml to 11.8 ml, and the AUA 6-Symptom Score improved from 24 to 3.5 points. All differences are statistically significant (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538705 TI - [Methodologic aspects of research on amidation of protein molecules]. AB - The paper deals with methodical aspects of protein amidation study. Critical review of some methodical procedures described in literature has been made. Presence of catalytic activity in medical drugs of serum albumin, human gamma globulin--as well as in commercial drugs of bovine serum albumin have been detected by means of amino acid analysis (automatic analyzer of amino acids AAA 339T). PMID- 7538706 TI - Changes in ultrasonic Doppler backscattered power downstream of concentric and eccentric stenoses under pulsatile flow. AB - The main objective of the present work was to investigate, under pulsatile flow, the patterns of variation of the Doppler power backscattered by blood and Sephadex particles upstream and downstream of concentric and eccentric stenoses ranging from 47% to 91% area reduction. Doppler measurements were performed at 5 diameters upstream and 5, 10, 15 and 20 diameters downstream of the constriction. For the concentric 75% and 85%, and the eccentric 79% and 91% area reduction stenoses, a progressive increase of the power backscattered by red cell suspensions at 40% hematocrit was measured downstream of the narrowing. The maximal power usually occurred around 10 diameters after the stenosis and dropped further downstream. In addition to the increase in the power, a cyclic variation of the backscattered intensity was observed within the flow cycle. For the concentric 52% and eccentric 47% area reduction stenoses, no variation of the Doppler power was measured during flow acceleration and deceleration for all recording sites. A coefficient of correlation of 0.82 was measured between the percentage of area reduction and the ratio of the Doppler mean power at 10 diameters downstream to that at 5 diameters upstream of the stenoses. Using Sephadex particles at low concentration, no increase of the Doppler power was found downstream of the 85% and 91% area reduction stenoses. The possible link between the intensity of turbulence and the power backscattered by blood is discussed along with the influence of the correlation between the scattering particles, under turbulent flow. PMID- 7538709 TI - [Status of phytotherapeutic drugs in treatment of benign prostatic hyperplasia]. AB - Phytotherapeutic preparations are still commonly used for the treatment of symptomatic benign prostate hyperplasia (BPH) in Germany; in recent years there has even been an increase in their use, so that sales now amount to more than DM 220 millions per year. The preparations most frequently used are extracts of Hypoxis rooperi, the roots of the stinging nettle, the fruits of the saw palmetto, pumpkin seeds and rye pollen. The suggested mechanisms of action have not been documented by scientific observation. This applies especially to the blocking effect on 5 alpha-reductase postulated with the doses used. Moreover, a critical analysis of the data available suggests that the effects of phytotherapy are no better than those of placebo treatment. Further studies are urgently needed, to compare the effects of phytotherapy with those of chemically defined drugs (alpha 1-receptor antagonists, 5 alpha-reductase blocker) that seem to have a beneficial influence on the pathomechanism underlying symptomatic BPH. PMID- 7538711 TI - [Transurethral laser ablation of the prostate (TULAP). Initial results with 188 patients]. AB - Laser therapy for benign prostatic hyperplasia (BPH) is now the most impressive alternative to transurethral resection of the prostate (TUR-P). Transurethral laser ablation of the prostate (TULAP) is a new technique that combines non contact and contact laser treatment of BPH. From November 1992 to August 1994, 188 patients were treated by the TULAP technique. Their median age was 70.3 (58 87) years. The mean prostatic volume determined by transrectal ultrasound, was 49.2 ml. A 6-month follow up was possible for 102 patients. The AUA 7-Symptom Score decreased from 29 points preoperatively to 6 points after 6 months. Residual urine volume decreased from 205 ml to 27 ml after 6 months. Maximum flow rate increased from 7.2 ml/s to 19.3 ml/s after 6 months. No severe complications were observed, and in particular no major bleeding or TUR syndrome. We regard the TULAP procedure as an effective treatment alternative for obstructive symptoms caused by BPH. PMID- 7538713 TI - [Benign prostatic hyperplasia--an old disease picture--from a new viewpoint]. PMID- 7538714 TI - [TULIP (transurethral ultrasound-controlled laser-induced prostatectomy)- experiences with over 80 patients]. AB - Of the many methods currently under investigation for the treatment of symptomatic and obstructive benign prostatic hyperplasia (BPH), laser treatment seems the most likely to yield results comparable to those achieved with transurethral resection of the prostate (TURP). Between May 1991 and December 1993 a total of 83 men were treated in our department with the TULIP (transurethral ultrasound-guided laser-induced prostatectomy) device. Within the first year of follow-up there was a marked improvement in both signs and symptoms. The average urinary peak flow increased from 6 to 16 ml/s, the residual urine volume decreased from 218 ml to 36 ml, and the symptom score (assessed against a modified Boyarsky score) decreased from 17 to 4 points. Our experience so far indicates that the advantages of TULIP are minimal blood loss, no post transurethral resection syndrome, and a low rate of retrograde ejaculation postoperatively. In addition, the procedure can be performed with analgosedation. Disadvantages are the delayed onset of improvement, combined with irritative symptoms in the early weeks after the procedure. In addition, no tissue can be obtained for histological examination, which may be important in the long-term. Randomized prospective studies, including cost-benefit analyses, are needed to check for advantages of TULIP over TURP. PMID- 7538710 TI - [The effectiveness of beta-sitosterol in treatment of benign prostatic hyperplasia]. PMID- 7538712 TI - [Long-term results of transurethral prostate incision (TUIP) and transurethral prostate resection (TURP). A prospective randomized study]. AB - TUIP (transurethral incision of the prostate) or TURP (transurethral resection of the prostate) was performed in 120 patients to treat small-volume (< 20 g) benign prostatic hyperplasia in a prospective randomized study. The mean follow up was 43 months. Preoperatively and at regular follow-up visits obstructive and irritative symptom scores were obtained, sexual function was evaluated, and a uroflow study was performed. Both treatments achieved a marked improvement of symptoms. The obstructive, irritative and the total symptom scores did not differ significantly between TURP and TUIP. After 2-3 years the scores slowly increased in both groups. Subjective satisfaction rates averaged about 60% after more than 3 years and remained at this level. Initial significant differences in the peak flow rate in favour of TURP were no longer seen after more than 2 years. In smaller prostates TURP and TUIP are equally effective and since TUIP has fewer side effects and entails lower costs it appears to be an acceptable alternative to TURP. PMID- 7538715 TI - [Interstitial laser-induced coagulation of the prostate for therapy of benign hyperplasia]. AB - Interstitial laser-induced coagulation (ILC) is a new, minimally invasive, procedure for the treatment of benign prostatic hyperplasia (BPH). It is based on the placement of special light guides in the prostatic tissue, either transurethrally or perineally. The radiation source is a Nd-YAG laser, which is active for 3 or 5 min per fiber placement, respectively, with the power reduced stepwise. The coagulation necroses caused shrink during follow-up, leading to a decrease in the obstruction. In 239 patients treated in this way the mean I-PSS Score fell from 25.4 to 8.1 points and the quality of life index from 4.1 to 1.6, the urinary peak flow rate increased from 7.7 to 16.3 ml/s, the residual urine volume fell from 151 to 32 ml, and the mean prostate volume declined from 47.4 to 32.2 ml in the first 3 months of follow up; 9.2% of the patients required further treatment within 1 year because of persistent obstruction. The complication rate was low. Statistical analysis showed no factors allowing prediction of the outcome. ILC is an effective method with few side effects and complications, and it has a wide range of indications for all BPH patients. PMID- 7538716 TI - [Minimally invasive therapy of benign prostatic hyperplasia with focussed ultrasound]. AB - To determine the morphologic impact and safety of high-intensity focused ultrasound (HIFU) administered transrectally for tissue ablation, a total of 22 prostates were treated in vivo with transrectal HIFU prior to surgical removal. Location and size of the tissue lesions correlated well with the predefined target area and revealed sharply delineated coagulative necrosis in all cases. In a subsequent phase II clinical trial the clinical effectiveness of transrectal HIFU as a minimally invasive treatment modality for patients with symptomatic benign prostatic hyperplasia (BPH; n = 50) was studied. The maximum urinary flow rate (Qmax, ml/s) increased from 8.9 +/- 0.6 (n = 50; mean +/- SEM) to 12.8 +/- 0.9 (3 months, n = 46), 11.9 +/- 0.9 (6 months, n = 44) and 12.2 +/- 1.0 (12 months, n = 32). Within the same time period, the post-voiding residual volume (ml) decreased from 113 +/- 17 (mean +/- SEM) to 47 +/- 6, 57 +/- 7, and 49 +/- 6. These data demonstrate that transrectal HIFU can induce intraprostatic coagulative necrosis. The phase-II clinical data presented in this paper reveal that transrectal HIFU is an effective and safe minimally invasive treatment option for BPH. Nevertheless, a randomised phase-III trial comparing HIFU and TURP is mandatory to define the definitive role of transrectal HIFU in BPH, and is presently planned. PMID- 7538717 TI - Pretransplant intrathymic inoculation of donor antigen combined with FK506 treatment: prolongation of survival of cardiac, but not renal, allografts in rats. AB - It has been shown that pretransplant intrathymic injection of donor antigen prolongs rat cardiac allograft survival. The purpose of the present study was to evaluate whether co-administration of FK506 could enhance the beneficial effect of intrathymic preimmunization; we also wished to clarify whether this combined treatment could be effective for renal allografts. Seven days prior to transplantation 5 x 10(7) donor splenocytes were injected into the thymus of LEW (RT1l) recipients of WKA (RT1u) cardiac or renal allografts. Using a miniosmotic pump, FK506 was administered for 7 days before transplantation at a dose of 3 mg/kg/day. Whereas intrathymic injection of donor splenocytes or subcutaneous FK506 alone showed limited (15.2 days) or no (7.3 days) prolongation of cardiac allograft survival, the combined treatment with these two modalities synergistically prolonged the survival of the cardiac allograft (> 82.0 days). This combined treatment, however, failed to prolong renal allograft survival (8.4 days). Although donor-specific prolongation of a second cardiac allograft was obtained in LEW rats with a well beating WKA heart, two of the three recipients ultimately rejected both first and second WKA cardiac allografts. Additionally, LEW rats with a long-surviving WKA heart rejected a WKA renal allograft in a normal fashion. These data demonstrate a synergistic effect of intrathymic injection of donor splenocytes and subcutaneous FK506 on cardiac, but not renal, allograft survival and suggest that quantitative or qualitative differences of immunogenicity of kidney may contribute to the rejection of renal allografts. PMID- 7538719 TI - Partial sacral resection and reconstruction with bone cement. Discussion of an operative experience. AB - Sacral resection is often the only way for effective palliation of the severe pain in tumors involving sacrum. However the operation hardly seems to be acceptable with the reported operative times, blood losses and complication rates. A case of retrorectal leiomyosarcoma invading sacrum, with severe pain resistant to combined irradiation and chemotherapy is reported. The tumor was removed with resection of the right two thirds of S2, S3 and S4, and the sacrum was reconstructed with bone cement. Complete relief of the pain was obtained with no postoperative complications. It is concluded that incomplete resection of the sacral vertebrae should be performed for sacral root pain palliation if other methods fail. Bone cement reconstruction seems to be a valuable alternative to muscle flaps in covering sacral defects. PMID- 7538718 TI - Methylation of cottontail rabbit papillomavirus DNA and tissue-specific expression in transgenic rabbits. AB - Transgenic rabbits carrying multiple copies of CRPV genomic DNA were described previously (Peng et al. (1993) J. Virol. 67, 1698-1701). CRPV DNA was detectable in all tissues of the transgenic rabbits, however, the transcripts of CRPV genes only were found in skin and skin tumors. Tumor development was also restricted to skin. To study the mechanism involving tissue-specific expression of CRPV genes in rabbits, cellular DNAs, isolated from different normal tissues and skin tumors, were digested with the two isoschizomeric restriction endonucleases MspI (methylation resistant) and HpaII (methylation sensitive), respectively, and analyzed by Southern blot. CRPV DNA, especially its upstream regulatory region (URR), was extensively methylated in all normal tissues, but methylation was remarkably reduced in skin tumors. These data suggest that extensive methylation of CRPV genome, especially in the URR, might be a factor in controlling its tissue-specific expression. PMID- 7538720 TI - Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells. AB - Gelatinase A is an enzyme capable of cleaving soluble beta-amyloid protein (beta AP), and may function as an alpha-secretase to produce secretory forms of amyloid precursor protein. We examined gelatinase A immunoreactivity in the brains and posterior roots of neurologically normal, lacunar stroke, Alzheimer disease (AD), amyotrophic lateral sclerosis, progressive supranuclear palsy and myasthenia gravis cases. The gelatinase A antibody stained only microglial cells in the white matter in all the brain tissues. In AD brain, the reactive microglia located in the center of classical senile plaques, as well as in other microglial cells in the gray matter, showed no immunoreactivity. Gelatinase A in white matter microglial cells may play a role in preventing local deposition of beta AP. In the posterior root, Schwann cells had positive immunoreactivity. As with other metalloproteases, gelatinase A in Schwann cells may play an antiproliferative role. PMID- 7538721 TI - Axonal regeneration into chronically denervated distal stump. 2. Active expression of type I collagen mRNA in epineurium. AB - During the first 2 weeks after an injury to peripheral nerve, endoneurial cells proliferate and express integrin beta 1 and mRNA for collagen types I and III. Clinical results for surgical repair within this time are clearly better than those obtained after delayed (months after original injury) surgery. The question of whether this is due to changes in the proliferative capacity of endoneurial cells or to changes in expression of mRNA for collagen types I and III or integrin beta 1 was studied using rats. The left common peroneal nerve was transected and allowed to degenerate for 3 and 6 months. After these times, the tibial nerve of the same animals were transected, and the fresh proximal stump of the transected tibial nerve was sutured into the chronically denervated distal stump of the common peroneal nerve. At 3 and 6 weeks after the reoperation, samples were collected from the distal stump for morphometry, immunohistochemistry and in situ hybridization. Proliferating cells and Schwann cells were identified by immunohistochemistry. These cells increased markedly in number during the axonal reinnervation. In situ hybridization revealed that in the epineurium and perineurium, which were fibrotic, especially type I but also type III collagen mRNA were highly expressed. The amount of type I collagen mRNA in the endoneurium seemed to increase with progressing axonal reinnervation. Immunostaining for integrin beta 1 was negative in these distal stumps. In the present study the proliferation of endoneurial cells and expression of type I collagen mRNA in the endoneurium were similar to those found after immediate regeneration of transected peripheral nerve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538723 TI - Effects of cytokines on hematopoietic progenitor cells in cord blood, in bone marrow, and in peripheral blood mobilized by chemotherapy and G-CSF. AB - We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1-3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential. PMID- 7538724 TI - Marked increase of peripheral blood myeloblasts following G-CSF therapy in a patient with acute lymphoblastic leukemia. AB - A 9 year old boy with acute lymphoblastic leukemia (ALL) received recombinant human granulocyte colony-stimulating factor (rhG-CSF) and showed a marked increment of myeloblasts in the peripheral blood. He was administered repeated courses of intermediate-dose cytosine arabinoside (Ara-C) therapy (1500 mg/m2, days 1-5) for frequent central nervous system (CNS) relapse of ALL. The peripheral white blood cell nadir was less than 1000/microL, so he was treated with rhG-CSF. A marked increment of peripheral blood blasts was noted 3-5 days after rhG-CSF treatment. These cells decreased with the appearance of mature myeloid cells and disappeared about 2 weeks after the start of treatment. These findings suggested that the blasts might have the ability to differentiate into mature myeloid cells. A control patient with repeated CNS relapse of ALL showed no increment of peripheral blood blasts after similar repeated courses of Ara-C without rhG-CSF treatment. Cultured peripheral blood blasts obtained from the present patient showed differentiation into mature myeloid cells by morphological studies and surface marker analysis. These findings indicate that the peripheral blood blasts drawn by G-CSF were not leukemic blasts but normal myeloblasts. PMID- 7538725 TI - Basic fibroblast growth factor regulates IGF-I binding proteins in the clonal osteoblastic cell line MC3T3-E1. AB - In previous studies, we reported that basic fibroblast growth factor (bFGF) regulates insulin-like growth factor messenger RNAs and protein levels in the osteoblastic MC3T3-E1 cells. In the present study, we examined the expression of insulin-like growth factor binding proteins (IGFBPs) in MC3T3-E1 cells and determined whether bFGF altered IGFBP mRNAs and protein levels. Since previous studies suggested that IGFBPs can inhibit DNA synthesis stimulated by IGF-I, we wondered whether the mitogenic effect of bFGF was altered by exogenous IGFBP-3. Confluent MC3T3-E1 cells were serum-deprived for 24 h and then treated with bFGF for 6-24 h. In control cultures, MC3T3-E1 cells expressed the mRNAs for IGF-I, IGF-II, and IGFBP-2, 4, 5, and 6 but not IGFBP-1 or 3. A 24 h treatment with bFGF at 10(-8) M decreased IGF-I mRNA by 97%, IGF-II mRNA by 73%, IGFBP-2 by 64%, IGFBP-4 by 73%, IGFBP-5 by 95%, and IGFBP-6 by 65%. The inhibitory effect of bFGF on IGF-I and IGFBP mRNA levels was not altered by aphidicolin, an inhibitor of cell replication. bFGF 10 nM decreased IGF-I levels determined by radioimmunoassay after acidification by 45% and 72% at 24 and 48 h, respectively. Western ligand blot for IGF binding proteins revealed that MC3T3-E1 cells expressed IGFBPs of 24, 30, and 34 kD. Treatment with bFGF 10(-8) M decreased the levels of the 24 and 30 kD band at 24 h but increased the 34 kD band.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538722 TI - Recurrent anaplastic ependymoma with an abnormal karyotype and c-myc proto oncogene overexpression. AB - Cytogenetic analysis on a supratentorial, recurrent, anaplastic ependymoma from a 29-year-old female disclosed the presence of an abnormal clone with the karyotype 46,XX,der(8)t(8;11)(q24;p11),-11,add(?)t(?;11)(?;q13). By the Northern hybridization assay and immunohistochemical staining, tumor cells revealed overexpression of c-myc proto-oncogene, although no evidence of amplification or structural rearrangement of this gene was found. PMID- 7538726 TI - Alterations in perivascular dilatory neuropeptides (CGRP, SP, VIP) in the external jugular vein and in the cerebrospinal fluid following subarachnoid haemorrhage in man. AB - A possible involvement of perivascular vasodilatory neuropeptides in subarachnoid haemorrhage (SAH) has been evaluated in man by measuring the levels of calcitonin gene related peptide (CGRP)-, substance P (SP)- and vasoactive intestinal peptide (VIP)-like immunoreactivity (LI) in the cranial venous outflow and in CSF in 34 patients admitted to the hospital after an acute SAH. After operation with aneurysm clipping and nimodipine treatment, blood samples were taken from the external jugular vein (EJV) or cerebrospinal fluid (CSF) and analysed for neuropeptide levels with specific radioimmuno assays (RIA) during the postoperative course. The degree of vasoconstriction in the patients was monitored with Doppler ultrasound recordings bilaterally from the middle cerebral (MCA) and internal carotid arteries (ICA) following the EJV blood sampling every second day. The mean value of all CGRP-LI measurements in EJV during the entire course of SAH (n = 20) revealed a significantly higher level as compared to controls. The highest CGRP-LI levels were found in patients with the highest velocity index values (vasospasm). The relationship Vmean MCA/Vmean ICA was used as an index of vasoconstriction. In patients with MCA aneurysms (n = 10), a significant correlation (r = 0.65, p < 0.05) was found between the vasospasm index and CGRP-LI levels. There were no changes observed in the SP- and VIP-LI levels. Alterations in cerebrovascular tone induced by changing arterial CO2 tension or lowering of blood pressure (ketanserin infusion test) did not alter the levels of the perivascular peptides in the EJV. In addition, CGRP-, SP-, VIP- and neuropeptide Y (NPY)-LI were analysed in CSF in the post-operative course after subarachnoid haemorrhage (SAH) in 14 patients. The CSF VIP-LI was lower in SAH than in control (p < 0.05). The CGRP-LI level was measurable in SAH CSF but not in CSF of controls. In individual patients with marked vasoconstriction increased levels of CGRP-LI (up to 14 pmol/L) and NPY-LI (up to 232 pmol/L) were observed. The results of this study are in support of our hypothesis that there is an involvement of the sensory peptide CGRP in a dynamic reflex aimed at counterbalancing vasoconstriction in SAH. PMID- 7538727 TI - Endoscopic laser ablation of the prostate: MR appearances during and after treatment and their relation to clinical outcome. AB - OBJECTIVE: Endoscopic laser ablation is a new treatment for benign prostatic hypertrophy. The objectives of this study were to determine the appearances of the prostate on MR images obtained during, 1 week after, and 3 months after this procedure and to determine if a correlation exists between the MR findings and the clinical outcome. Such appearances could then be used to guide the application of laser energy during the procedure in order to optimize the clinical result. SUBJECTS AND METHODS: Eight consecutive men 58-74 years old with symptoms of bladder outflow obstruction caused by benign prostatic hypertrophy underwent endoscopic laser ablation of the prostate under spinal or epidural anaesthesia. Imaging was done on a 0.5-T Picker Asset system with an endorectal receiver coil and conventional T1-weighted spin-echo, T2-weighted spin-echo, and gradient-recalled-echo sequences. T1-weighted magnetization transfer images were obtained in three patients. Images were obtained preoperatively, after ablation of the left-sided quadrants, immediately after completion of the procedure, and 1 week and 3 months later. Preoperative and 3-month postoperative symptom scores, peak urine flow rates, and bladder residual volumes were studied. Images were visually assessed for signal-intensity changes and the presence of cavitation by three radiologists in conference. The results were quantitatively analyzed by measuring prostatic volumes on the gradient-recalled-echo images and by measuring the width and area of regions of signal-intensity change on the T2-weighted images. RESULTS: MR images made immediately after treatment showed an increase in the volume of the prostate (mean, 34%) and a poorly defined, low-signal-intensity region around the urethra on the T2-weighted images in six patients. This probably represented coagulative necrosis. The prostate was smaller on MR images made 1 week after treatment, and after 3 months the prostate returned to its preoperative size. After 1 week, the low-signal-intensity periurethral region on the T2-weighted images was less obvious, and at 3 months it was replaced in four patients by a well-demarcated low-signal-intensity ring on the T2-weighted and gradient-recalled-echo images. No evidence of cavity formation in the prostate was seen on MR images in any patient. Symptom scores and peak urinary flow rates improved after 3 months, with a significant difference between the mean increase in symptom scores in the patients with and without the periurethral changes seen immediately after treatment. However, we found no significant difference between the mean increase in peak urinary flow rates in the patients with and without periurethral changes seen either immediately or at 3 months after treatment. No statistically significant correlation was found between the amount of prostatic swelling and the improvement in symptom scores or peak urine flow rates. CONCLUSION: In patients who have had laser prostatectomy, MR imaging shows significant immediate glandular swelling, which may account for the delayed improvement in symptoms reported with this technique. The presence of the periurethral changes immediately after treatment was correlated with a subsequent improvement in symptom scores. After 3 months, no cavity could be seen in the prostate. This may account for the poorer long-term clinical outcome reported with endoscopic laser ablation of the prostate compared with transurethral prostatectomy. PMID- 7538728 TI - Monoclonal antibody X18A4 identifies an oncofetal fibronectin epitope distinct from the FDC-6 binding site. AB - OBJECTIVE: Oncofetal fibronectin reactive with antibody FDC-6 has been associated with trophoblastic implantation and chorion structural stability. Abnormal release of this fibronectin into cervical and vaginal secretions has identified patients at risk for preterm labor and delivery. The aim of this study was to determine whether trophoblast-derived oncofetal fibronectin contains other novel epitopes distinct from the FDC-6 binding site. STUDY DESIGN: Antitrophoblast fibronectin hybridomas were generated and screened by comparative immunoassays. One specific monoclonal antibody, X18A4, was identified and compared with antibody FDC-6 by immunocytochemical and immunoblot analyses. Both antibodies were also evaluated in "sandwich"-type double monoclonal immunosorbent assays. RESULTS: X18A4 and FDC-6 bind avidly and noncompetitively to distinct epitopes within oncofetal fibronectin. They exhibit similar immunohistochemical staining of the extracellular matrix within placental tissue, ovarian epithelial tumors, and cultured trophoblasts. However, in contrast to FDC-6, X18A4 has no detectable binding activity to human plasma fibronectin, and its binding to oncofetal fibronectin was unaffected by enzymatic deglycosylation. Immunoblot analyses of oncofetal fibronectin proteolytic digests suggest that X18A4 binds near or within the alternatively spliced type III connecting segment domain. CONCLUSIONS: X18A4 identifies and binds with high affinity to a new epitope within oncofetal fibronectin, distinct from the FDC-6 binding site. Because X18A4 displays no detectable binding to plasma fibronectin, it could be used as an important adjunctive antibody for enhancing the specificity of clinically based oncofetal fibronectin diagnostic assays. PMID- 7538730 TI - Entorhinal cortex in temporal lobe epilepsy. PMID- 7538732 TI - Florid vascular proliferation associated with neural and neuroendocrine neoplasms. A diagnostic clue and potential pitfall. AB - We report the light microscopic and immunohistochemical features of vascular proliferations associated with 26 extracranial neural and neuroendocrine neoplasms including esthesioneuroblastoma, neuroblastoma/ganglioneuroblastoma, the primitive neural component of immature teratoma, mediastinal teratoma, primitive neuroectodermal tumor, intra-abdominal desmoplastic small cell tumor, Merkel cell carcinoma of the skin, and thyroid medullary carcinoma. These vascular proliferations were similar to those associated with high-grade glial neoplasms and were characterized by tufts of vessels with a glomeruloid configuration or by long cords of vessels. Immunohistochemical evaluation documented the presence of endothelial cells, perithelial cells, and basement membrane components within the foci of proliferating vessels. We propose that these vascular proliferations represent a characteristic feature of the neuroendocrine/neural neoplastic phenotype and that they possibly arise as the result of angiogenic factors produced by the neoplastic cells. The presence of these distinctive vascular lesions in the stroma of a poorly differentiated neoplasm should alert the pathologist to the possibility of the neoplasm being of a neural or neuroendocrine nature. PMID- 7538731 TI - Correlation of CSF 5-HIAA concentration with sociality and the timing of emigration in free-ranging primates. AB - OBJECTIVE: The purpose of this study was to examine the relationship between behavior and serotonin in nonhuman primates. METHOD: During a routine capture and medical examination, 26 adolescent male rhesus macaques (Macaca mulatta) were selected as subjects from a free-ranging population of 4,500 rhesus monkeys inhabiting a 475-acre sea island. Blood samples (N = 23) and CSF samples (N = 22) were obtained, and each subject was fitted with a radio transmitter collar for rapid location. The subjects were released into their social groups, and quantitative behavioral observations were made over a 3-month period. RESULTS: CSF 5-hydroxyindoleacetic acid (5-HIAA) concentration was positively correlated with three measures of sociality: 1) total time spent grooming others, 2) total time spent in close proximity to other group members, and 3) mean number of neighbors within a 5-m radius. In addition, CSF 5-HIAA concentration was positively correlated with age at emigration from the natal group (in months). CONCLUSIONS: In adolescent male rhesus macaques living in naturalistic settings, CSF 5-HIAA concentration is positively correlated with affiliative sociality. Rhesus males with low CSF 5-HIAA concentrations exhibit less social competence and emigrate from their social groups at a younger age than do males with higher concentrations of CSF 5-HIAA. PMID- 7538733 TI - Visualization of injected embolic material (polyvinyl alcohol) in paraffin sections with Verhoeff-Van Gieson elastica stain. AB - Therapeutic embolization of blood vessels is widely used to treat vascular malformations and to preoperatively decrease the size of bulky tumors by creating iatrogenic infarction. Surgical pathologists are often called upon to identify the presence of embolized material in microscopic slides. Polyvinyl alcohol particles are often used as the material for embolization. On hematoxylin-eosin and other routine stains this substance is almost invisible. These particles stain black and can be sharply outlined by using the Verhoeff-Van Gieson stain. PMID- 7538729 TI - A model of intrauterine infection and preterm delivery in mice. AB - OBJECTIVE: Our purpose was to determine whether intrauterine bacterial inoculation leads to preterm delivery in mice. STUDY DESIGN: Fifty-four female CD 1 mice at 75% of the length of the gestational period (14.5 days) received either an intrauterine bacterial inoculum of 2 to 10 x 10(3) Escherichia coli (n = 33), an intraperitoneal bacterial inoculum (n = 7), or an intrauterine injection of a sterile solution (n = 14). RESULTS: Delivery within 48 hours of surgery occurred in 91% of mice after intrauterine bacteria, in 0% after intraperitoneal bacteria, and in 7% after sterile intrauterine injection (p < 0.001). Intrauterine bacterial inoculation produced systemic infection (i.e., recovery of organisms from culture of the heart) in 50% of animals post partum. Intraperitoneal bacteria and intrauterine saline solution injections resulted in systemic infection rates of 20% and 0%, respectively, 48 hours after surgery. Five of seven animals injected with bacteria into the uterus had histologic evidence of metritis, mild in all cases. Intrauterine bacterial inoculation resulted in induction of ribonucleic acid transcripts for tumor necrosis factor-alpha, interleukin-1 alpha, interleukin-1 beta, and cyclooxygenase-2. CONCLUSIONS: Intrauterine inoculation with Escherichia coli in mice leads to preterm delivery and the local induction of factors known to be involved in human preterm labor with infection. The observation that intraperitoneal bacterial inoculation does not result in preterm delivery suggests that in this model labor is the product of a local (uterine) stimulus. PMID- 7538735 TI - Comparative studies on various modes of classification of morphology of sperm heads and results in in vitro fertilization--a preliminary report. AB - In a retrospective blind study, Papanicolaou's stained semen smears of the husbands of 105 patients who took part in the in vitro fertilization/embryo transfer (IVF/ET) program of the Department of Gynecology and Obstetrics at the Heinrich Heine University Dusseldorf in 1991, were evaluated for the rates of normal sperm heads. The patients were divided into three groups according to the results of the IVF/ET: A = no fertilized egg; B = only fertilization, no pregnancy; C = pregnancy/birth. Four different categories of the sperm heads' morphology were defined: 1) 'Ideally' normal heads (normal size, relations width to length 2/3 to 3/5, acrosomal region > or = 40 < or = 70%); 2) Normal heads with only minor deviations from the 'ideally' normal form, strictly excluding spermatozoa with inhomogeneously stained acrosomal or pointed post-acrosomal regions; 3) Sperm heads with major alterations which are nevertheless widely considered as still normal in the literature; 4) Heads which are generally accepted as pathologically formed. It could be shown that the application of the strict criteria in the categories 1 plus 2 lead to significant differences not only between the IVF/ET groups A and B or C, but also between the groups B and C. PMID- 7538734 TI - Cathepsin B in angiogenesis of human prostate: an immunohistochemical and immunoelectron microscopic analysis. AB - BACKGROUND: Angiogenesis (or neovascularization) is required for the growth of solid organ tumors and precedes invasion of the adjacent stroma by neoplastic cells. We investigated the relative density and distribution of cathepsin B (CB) immunostained microvessels (i.e., small blood vessels and capillaries) in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma (CAP) by immunocytochemical localization of an antibody directed against a cathepsin B-derived synthetic peptide (Syn-CB). METHODS: We studied 16 formalin-fixed, prostatectomy specimens that were embedded in paraffin/paraplast for histological examination by hematoxylin and eosin and immuno-localization of the Syn-CB antibody. Selected paraformaldehyde-fixed specimens were embedded in K4M Lowicryl or LRWhite resins. We localized the antibody in thin sections using immunoelectron microscopy techniques. RESULTS: Eight patients had BPH [4 patients with BPH alone, 2 with BPH and PIN, and 2 with BPH and CAP]. Ten cancer cases included one with Gleason histologic score 4, two with score 6, four with score 7, and three with score 8. In CAP cases, Gleason score 6 and 7 tumors had more microvessels than the score 4 or 8 tumors. In both BPH and CAP cases, the antibody was localized chiefly in the endothelial cells of microvessels, but occasionally in ductal and glandular epithelial cells. Ultrastructurally, CB-immunoreactive gold particles were markedly increased at the luminal and basal plasma membrane surfaces and folds of endothelial cells in neoplastic prostate, but not in the endothelial cells of BPH. Furthermore, the presence of CB localizing gold particles in collagen and smooth muscle fibers near the microvessels indicated leakage of the enzyme in prostatic stroma of neoplastic prostate. Similar leakage was not observed in BPH. Morphometric analysis showed that the relative density of microvessels increased two to three times in cancer patients when compared to patients with BPH alone. Our study also indicated that BPH associated with PIN or CAP had an increased density of microvessels when compared to BPH alone. CONCLUSIONS: Our study showed that the relative density and distribution of microvessels are the most important features of neovascularization in prostatic tumors. The relative density of microvessels increased in PIN and CAP when compared to BPH alone. Although the localization of CB is associated with lysosomes of endothelial cells in both BPH and CAP, there is a greater association of CB with the plasma membranes of endothelial cells in CAP than BPH. Immunoelectron microscopy provided evidence that CB might be involved in dissolution of basement membranes in neoplastic tumors during angiogenesis. CB localization has the potential of defining a role for this protease in degradation of extracellular matrix constituents during early steps of angiogenesis. PMID- 7538738 TI - Coronary angiogenesis. From morphometry to molecular biology and back. AB - We have reviewed briefly the current state of knowledge relating to the regulation of angiogenesis, including the role of angiogenic growth factors, and we have described the major structural components of the vascular wall and their changes during formation of new channels. Finally, we have described quantitative evaluations of vascular growth in cardiac muscle. Examples of substantial angiogenesis during the early postnatal stages of normal development were used together with examples depicting a more moderate stimulation of capillary growth in adult rat hearts treated with nifedipine or thyroxine. Adequate capillary supply is a precondition for tissue survival and proper function. This is probably why vascular growth is so tightly regulated by several systems which may alternately stimulate or inhibit the formation of new vessels. Recent advances in molecular biology have enabled us to study the mechanisms of angiogenesis under both in vitro and in vivo conditions, but the final assessment of vascular growth should be accomplished through morphometric analysis. PMID- 7538739 TI - Development of cardiomyocytes expressing cardiac-specific genes, action potentials, and ionic channels during embryonic stem cell-derived cardiogenesis. PMID- 7538740 TI - [Lichen and liver]. PMID- 7538737 TI - The regulation of blood vessel growth by vascular endothelial growth factor. PMID- 7538741 TI - [Lichen planus and hepatitis C]. AB - Cases associating lichen planus and hepatitis C are increasingly being reported. We observed three new cases, two males and one female aged 46, 63 and 56 years respectively. In all three cases, the lichen planus was limited to the oral cavity with erosion in two cases. Hepatitis C was confirmed by 2nd generation ELISA. Two of the patients consulted for lichen planus during the recovery phase after hepatitis C. In the third patient, lichen planus preceded the hepatitis. These three cases are further evidence favouring the hypothesis of a non fortuitous association between lichen planus and hepatitis C. The hepatitis C virus might induce an immune reaction leading both to lichen planus and chronic hepatitis. PMID- 7538736 TI - Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation. AB - This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TuS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TuS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotin streptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TuS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit. PMID- 7538743 TI - [Cutaneous cysts and pseudocysts. I]. PMID- 7538742 TI - [When should hepatitis C serology be required in dermatology?]. PMID- 7538744 TI - [Improvement of survival of testicular cancer patients through chemotherapy]. AB - Because treatment of testicular cancer has improved dramatically during the past 15 years, about 70% of the patients can be relieved of this disease today. Although this can be attributed to the availability of various tumor markers, better imaging diagnosis and improvement of surgical techniques, the improvement of chemotherapy, mainly based on cisplatin, has made the largest contribution. Hopefully, more powerful chemotherapy regimens will be available for patients who fail to respond to primary chemotherapy or relapse within a short period after primary chemotherapy. Supportive therapy, such as bone marrow transplantation, will serve to enhance the treatment for testicular cancer. On the other hand, milder therapy will given, if it is determined sufficient to treat the patient with an excellent response to the present therapeutic regimen. In the future, more therapeutic regimens will be prescribed to meet the exact needs of individual patients according to their prognoses. PMID- 7538745 TI - [Role of radiotherapy in the treatment of localized prostatic carcinoma]. AB - OBJECTIVES: Radical treatment for prostate cancer aims at complete eradication of tumor. Over the last 30 years, a vast literature has been published on the available therapeutic approaches for loco-regionally confined prostate cancer. There is no consensus on the appropriate management of either early stage or locally advanced disease and treatment is still given on the basis of physician preconception, training and instinct. METHODS: The indications and results of postradical prostatectomy adjuvant pelvic radiation therapy and radical radiation therapy are reviewed. RESULTS: Following radical prostatectomy, extracapsular disease, carrying a significant risk of local recurrence, is found from 12-68% of the time depending on the clinical tumor stage. External beam radiotherapy is a reference for treatment standards for prostate cancer. CONCLUSIONS: External irradiation remains the unquestioned standard for the treatment of locally advanced disease. For early stage disease, patients have the option of selecting radical prostatectomy or radiotherapy. PMID- 7538746 TI - [Clinical, urodynamic, and radiologic results in the study of prostatism with the PURR measure (passive urethral resistance ratio)]. AB - OBJECTIVE: The present study was conducted to determine the correlation between the urinary symptoms and lower urinary tract dynamics in patients with prostatism. METHODS: 105 males with prostatism (mean 66.19 years) were evaluated clinically, urodynamically and radiologically by PURR (passive urethral resistance ratio) determination. RESULTS: The intensity of the irritative symptoms was slightly higher (21%) when bladder instability was associated. The obstructive symptoms were associated with diminished urinary flow rate and could be observed in bladder outlet obstruction (81%) or diminished bladder contractility (79%). Both conditions were found in 49% of the cases. The postvoiding urine depended on diminished bladder contractility (37%), but not on bladder outlet obstruction. Bladder instability did not depend on obstruction, although 88% of the patients with unstable bladder were obstructed. Compressive obstruction was associated with a lesser degree of diminished contractility (55%) than the other forms of obstruction. Prostatism was associated with diminished contractility without bladder outlet obstruction in 15% of the cases. The irregular bladder was more frequent in bladder outlet obstruction (92%) and bladder diverticula were more frequent in bladder instability (36%). In all the cases with compression and lengthening of the prostatic urethra, the diagnosis was compressive obstruction. In all the cases with segmental, diminished urethral diameter, the diagnosis was obstruction, although only 30% were diagnosed as constrictive compression. No pathological urethral changes were demonstrable on cystourethrography in 33% of the cases with obstruction. CONCLUSIONS: Urinary symptoms correlate poorly with lower urinary tract dynamics. PURR determination is a very reliable method of diagnosis in urinary obstruction, for both segmentally diminished and more extensive involvement (elongation and compression of the prostatic urethra) of the urethral lumen. PMID- 7538748 TI - [Neuroendocrine cells in the morphogenesis of prostatic pathology]. PMID- 7538747 TI - Nd: YAG laser thermocoagulation of the prostate: the barbecue effect. AB - OBJECTIVE: To determine systematically the depth of coagulation necrosis induced during Nd: YAG laser irradiation in the canine prostate model, and to define the correlation of laser dose with depth of the lesions. METHODS: We investigated the effect of various dosimetries on prostatic tissue of 17 mongrel canines using laser power in the range of 8 to 60 watts (W) at varying exposure times, from 20 to 300 seconds (S). Applied energy ranged from 1,000 to 5,500 Joules (J). RESULTS: Three distinct features were observed. First, deep coagulation necrosis was induced only with laser regimens of > or = 2,100J, suggesting that there is a threshold of energy that must be overcome in order to achieve significant coagulation depth. Second, the deepest coagulation necrosis was achieved with a low power slow-heating regimen (15W X 180S). Tissue surface changes appeared to be responsible for the reduced depth of coagulation necrosis seen with higher power regimens (> 20 W). Third, increasing the energy above 3,600J did not augment the depth of coagulation, suggesting that a steady state for temperature distribution is reached at that rate of heating. CONCLUSIONS: Our study suggests that in the canine prostate, a low power slow-heating regimen of 15W X 180S may yield larger volumes of coagulation necrosis than other regimens. PMID- 7538749 TI - [The digital rectal exam, prostate-specific antigen and transrectal echography in the diagnosis of prostatic cancer]. AB - OBJECTIVES: This study analyzes the three available clinical methods in the diagnosis of prostate cancer (PC): digital rectal examination (DRE), serum prostate specific antigen (PSA) and transrectal ultrasound (TRUS), and attempts to establish the best system in the early detection of PC. METHODS: The findings of DRE, serum PSA and TRUS of the prostate in patients in which a TRUS-guided prostate biopsy was performed initially, and prostate surgery afterwards (TURP in 72%, radical retropubic prostatectomy in 20% and simple prostatectomy in 8%), are retrospectively analyzed. RESULTS: PC was eventually found in 50 patients (56%). Seventy-eight patients had an abnormal DRE, 44 of which had a final diagnosis of PC (sensitivity = 94%, specificity = 20.5%). Serum PSA levels were available in 60 cases before diagnosis. Levels > 4 ng/ml were found in 32 of 38 cases with PC (sensitivity = 84%, specificity = 31%). The TRUS finding with the highest positive predictive value (PPV) for PC was diffuse prostate heterogeneity (77.8%). When a hypoechoic nodule was seen in the prostate peripheral zone, PC was found in 53.7%. Different combinations of the three methods are analyzed, trying to increase the diagnostic capabilities of each isolated test. Transrectal ultrasound-guided prostate biopsy detected 44 (88%) of the 50 cases with PC, its negative predictive value being 86.7%. Prostate tissue valid for histologic diagnosis was obtained in 97% of the cases, and the predictive value of the true tumor grade was 84.1%. The general complication rate attained 3.4%, none being of the septic type. CONCLUSIONS: When parallel testing was examined (one or more tests being abnormal), the most efficient combination in the early detection of PC was when DRE was suspicious or serum PSA > 4 ng/ml (sensitivity = 95%, PPV = 62%). When at least two diagnostic tests were abnormal (serial testing), the most efficient combination to confirm the presence of this tumor was an abnormal TRUS and a serum PSA > 10 ng/ml (specificity = 86%, PPV = 88%). PMID- 7538750 TI - [Role of endothelial nitric oxide synthases in the contractile response to angiotensin II of the aorta in rats. Wistar Kyoto and hypertensive rats]. AB - Dysfunctions of EDRF/L-arginine-NO pathway have been demonstrated in genetic and experimental hypertension. NO is produced through the conversion of L-arginine to L-citruline by NO synthases (NOS) which exist at least in two isoforms. The first termed constitutive (NOSc) and located in the endothelium of the vascular wall results in the basal and stimulated NO production. The second termed inducible (NOSi), which produces large amounts of NO, can be expressed in both smooth muscle and endothelial cells. The aim of the study was to examine and compare in isolated aortic rings of WKY and SHR rats, the activity of the two isoforms of endothelial NO synthases and their influence on the constrictor response induced by angiotensin II. On phenylephrine preconstricted endothelium intact aortic rings (10(-6) M, WKY = 1.2 +/- 0.04 g; SHR = 1.2 +/- 0.07 g; n = 7), carbachol (10(-5) M) induced a greater relaxation in WKY (84 +/- 2.5%) than in SHR (63 +/- 8.5%) rat. This suggests the presence of a low NOSc stimulated activity in the hypertensive strain. When the incubation period was limited to 30 min after equilibration period, L-arginine (3.10(-4) M) did not induce relaxation. When the incubation period was prolonged (180 min), L-arginine induced a relaxation (WKY = 75 +/- 8%; SHR = 58 +/- 10%; n = 7). This relaxation was not observed in a medium containing actinomycin D (10(-6) M) or after endothelium removal, indicating the induction of an endothelial NOSi in the two strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538751 TI - Identification of T-cell epitopes using allele-specific ligand motifs. AB - In order to facilitate the identification of T-cell epitopes as useful components of synthetic vaccines, we investigated the role of MHC molecules as the restriction element for the recognition of epitopes by the alpha beta receptor of T cells. MHC molecules are able to present thousands of different peptides to T cells, with all the peptides presented by one distinct type of MHC sharing common structural features. Our group analyzed these common characteristics concerning peptide length (only MHC I ligands) and anchor positions (MHC I and II ligands) occupied by a small set of closely related amino acids. Until now, for more than fifty MHC proteins allele-specific "peptide motifs" have been defined. The exact knowledge of MHC I peptide motifs allows for a prediction of CTL epitopes, and this kind of prediction has been successful in many cases over the last three years. PMID- 7538753 TI - Chronic pain and distraction: an experimental investigation into the role of sustained and shifting attention in the processing of chronic persistent pain. AB - Although there is anecdotal evidence for the psychoanalgesic properties of distraction, research evidence is equivocal. Drawing on the clinical and experimental studies of attention-based coping strategies for pain control, and the theoretically driven 'cognitive' models of the human attention system, two experiments are reported. Experiment One demonstrates that chronic pain patients suffering high intensity pain show significantly impaired performance on an attentionally demanding task when compared to low pain patients and normal controls. Experiment Two tests the hypothesis that the low intensity pain patients in Experiment One are coping with the dual demand of processing the pain and processing the task by switching quickly between these attentional demands. The results of both experiments are discussed in terms of the evidence for the analgesic properties attention based coping strategies with clinical pain populations and re-addresses the literature on coping with pain in terms of cognitive theories of attention. PMID- 7538754 TI - Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes. AB - The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites. PMID- 7538752 TI - Outer membrane proteins of Pseudomonas aeruginosa as vaccine candidates. AB - We tested the ability of recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this gram negative pathogen under two main pathophysiological events leading to P. aeruginosa sepsis. i) systemic infection during immunosuppression, and ii) bacterial translocation. A hybrid vaccine was cloned combining protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice, with recombinant Salmonella dublin expressing OprI induced s-IgA antibodies in the gut mucosa against OprI and provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is safe for use in humans, recombinant OprI was purified and used for immunization of volunteers. Vaccination was well tolerated and no major side effects were observed. The induction of serum antibodies against OprI was found to be dose-dependent and was observed in total in 65% of the volunteers. PMID- 7538756 TI - Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and beta 2-adrenoceptor-expressing NG108-15 cells. AB - Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2 adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor. PMID- 7538755 TI - Ethanol potentiates interleukin-1 beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells. AB - Experiments were performed to examine the effect of ethanol on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in the release of nitrite and in the intracellular accumulation of L citrulline. In parallel with this, IL-1 beta increased inducible nitric oxide synthase (iNOS) mRNA and protein. Ethanol (6.5-650 mM) potentiated the IL-1 beta mediated stimulation of iNOS mRNA production, the appearance of iNOS protein and the generation of nitrite and L-citrulline from smooth muscle cells in a concentration-dependent manner. In the absence of IL-1 beta, ethanol failed to induce iNOS expression. These results demonstrate that pharmacologically relevant concentrations of ethanol enhance the IL-1 beta-induced expression of the iNOS gene in vascular smooth muscle. The ability of ethanol to augment the release of the platelet inhibitor and vasodilator nitric oxide may, in part, contribute to the beneficial cardiovascular effects associated with moderate alcohol consumption. PMID- 7538757 TI - Activation and tyrosine phosphorylation of p72syk as well as calcium mobilization after hydrogen peroxide stimulation in peripheral blood lymphocytes. AB - To determine the regulatory role of p72syk in lymphocyte activation, peripheral blood lymphocytes were treated with 10 mM hydrogen peroxide. Hydrogen peroxide induced a rapid elevation of p72syk activity (4-6-fold) and a dramatic increase in tyrosine phosphorylation of a number of cellular proteins, including phospholipase C gamma 1 (PLC gamma 1) and p72syk. Monoclonal antibodies to PLC gamma 1 co-precipitated p72syk from hydrogen peroxide-stimulated cell lysates, but not from unstimulated cell lysates. Furthermore, we observed a rise in intracellular Ca2+, corresponding to the combination of extracellular Ca2+ influx and the release from intracellular Ca2+ stores. Extracellular Ca2+ influx was necessary for the sustenance of p72syk activity, but not for the initiation of p72syk activation induced by hydrogen peroxide. Taken together, these data suggested that one possible role of p72syk was to activate PLC gamma 1, at least in part through tyrosine phosphorylation, and then to trigger calcium mobilization in pig peripheral blood lymphocytes in response to hydrogen peroxide stimulation. PMID- 7538758 TI - Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin. AB - A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577. PMID- 7538760 TI - Interaction of thyroid hormone nuclear receptor with antibody: characterization of the thyroid hormone binding site. AB - To understand the structural basis in the hormone-dependent transcriptional regulation of human beta 1 thyroid hormone receptor (h-TR beta 1), we characterized the region which interacted with the thyroid hormone, 3,3',5 triiodo-L-thyronine (T3). Using the hormone binding domain of h-TR beta 1 (K206 D461) as an immunogen, we screened for monoclonal antibodies which inhibited the binding of T3 to h-TR beta 1. mAb C3, which recognized native h-TR beta 1, was obtained. Analyses of the binding data indicate that binding of T3 to h-TR beta 1 was competitively inhibited by mAb C3. Using a series of truncated mutants of h TR beta 1 and synthetic peptides, we mapped the binding site of mAb C3 to the region of E248-V256. Thus, part of T3 binding site in h-TR beta 1 is in this nine amino acid segment, which was shown by circular dichroism spectroscopy to be a random coil. Based on the proposed model of the hormone binding domain as an alpha/beta barrel, E248-V256 contains part of Loop 1 which is on the same side of the DNA binding domain. These results raise the possibility that Loop 1 could be in direct contact with the nearby DNA binding domain to affect the interaction of DNA binding domain with the T3 target genes. PMID- 7538761 TI - DnaK/DnaJ supplementation improves the periplasmic production of human granulocyte-colony stimulating factor in Escherichia coli. AB - Human granulocyte-colony stimulating factor (hG-CSF) can be efficiently synthesized in E. coli as a fusion to a quimeric signal peptide, but this fusion is only partially exported to the periplasm. We have studied the effects of supplementing the host content in chaperonins on the secretion of prehG-CSF in E. coli. We have found that the DnaK/DnaJ chaperonin system improves the secretion of this recombinant protein. This improvement correlates with an increase in the solubility of the precursor. Only a small fraction of the mature protein is released from the periplasm by an osmotic shock. PMID- 7538759 TI - Depletion of small cytoplasmic RNA confers fusidic-acid resistance on Bacillus subtilis. AB - Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of a signal recognition particle (SRP)-like RNA family. To analyze the function of scRNA in protein synthesis, a B. subtilis strain SC201NA was constructed in which the expression of intact scRNA is regulated by an IPTG-inducible promoter. In this strain, depletion of scRNA leads to deficient translation and sporulation as well as morphological changes. In addition, the growth of SC201NA in the absence of IPTG became fusidic-acid resistant. The acquisition of fusidic-acid resistant phenotype by depletion of scRNA suggested that scRNA is associated with elongation factor G (EF-G) in the translation process. PMID- 7538762 TI - Adhesion properties of Mono Mac 6, a monocytic cell line with characteristics of mature human monocytes. AB - Progress in the understanding of blood cell--endothelial cell interactions has been achieved by the development of in-vitro model systems. We describe adhesion properties of the recently established human monocytic cell line Mono Mac 6. These cells showed increased adherence to unstimulated and tumour necrosis factor (TNF)-alpha (50 U/ml) stimulated human umbilical vein endothelial cells (HUVEC) (9.4% +/- 0.4% and 56.5% +/- 3.3%), as compared to U937 cells (2.6% +/- 0.8% and 40.0% +/- 8.4%). The values were similar to freshly isolated human blood monocytes (18.8% +/- 7.5% and 55.7% +/- 9.3%, respectively). Maximal binding was 6.2 +/- 0.6 Mono Mac 6 cells per HUVEC, which was 34% less than U937 cells (8.9 +/- 0.3). The lower number of adherent Mono Mac 6 cells per HUVEC could be due to their larger size, as assessed by flow cytometry. Blocking experiments with monoclonal antibody (mAb) directed against E-selectin, VCAM-1 and ICAM-1 on HUVEC and CD11b or CD14 on Mono Mac 6 cells demonstrated the contribution of these molecules to Mono Mac 6 adherence. Reduced binding after 24 h parallels the decline of E-selectin expression in HUVEC. Linearity of cell binding was confirmed from 0.2 x 10(6) to 1.0 x 10(6) Mono Mac 6 cells. Expression of CD11b and CD14 in Mono Mac 6 cells and in isolated human monocytes but not in U937 cells leading to interaction with ICAM-1 on HUVEC appears to be responsible for the increased adhesion of Mono Mac 6, as compared to U937 cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538763 TI - [Importance of Perls stain in histologic diagnosis of Kaposi sarcoma]. AB - The authors studied 27 cases of Kaposi's sarcoma, 23 cases of pyogenic granuloma, 18 cases of hemangioma, 17 cases of stasis dermatitis and 9 cases of dermatofibroma. In all these cases, they used the hematoxylin-eosin stain and Perls' stain', and they showed the presence of hemosiderin in all the cases of Kaposi's sarcoma and stasis dermatitis. The hemangioma and the dermatofibroma presented 50% of positivity, and the pyogenic granuloma, only 26.1%. PMID- 7538764 TI - [Immunohistochemical characterization of acute leukemia. Study of 31 bone marrow biopsies]. AB - An immunohistochemical study by avidin-biotin-peroxidase was performed on paraffin-embedded and decalcified bone marrow biopsies in 31 acute leukemias (19 myeloid and 12 lymphoblastic). The Ulex Europaeus lectin and 14 antibodies (anti CD45, -CD34, -myeloperoxidase, -lysozyme, -CD15, -CD68, -carcinoembryonic antigen, -factor VIII-related antigen, BNH9, anti-CD45RO, -CD3, -CD20, DBB42 and DBA44) were tested. All acute myeloid leukemias from M0 to M5 type were stained by either the anti-myeloperoxidase or anti-lysozyme antibodies. CD68, CD15 and the carcinoembryonic antigen were respectively expressed in 80%, 40% and 20% of myeloid leukemias from M1 to M5 type. The Ulex Europaeus lectin and the anti factor VIII-related antigen antibody stained only the M7 leukemia and the anti CD3 antibody stained only the T acute lymphoblastic leukemia. DBB42 was expressed by 63% of B-lineage lymphoblastic leukemias and CD20 by 36%. No leukemia was stained by DBA44. Immunohistochemistry on bone marrow biopsy can assess the lineage of most acute leukemias with the use of a panel of antibodies such as the anti-myeloperoxidase, -lysozyme, -CD68, -CD20, DBB42, -CD3, BNH9, anti-factor VIII-related antigen antibodies and the Ulex Europaeus lectin. PMID- 7538767 TI - B-cell activation by helper T-cell membranes. AB - Resting B cells can be stimulated to proliferate and differentiate to antibody producing cells by the combination of cell contact and soluble signals provided by activated primed helper T (Th) cells. The ability of purified plasma membranes from activated Th cell clones and recombinant lymphokines to reconstitute B cell proliferation and differentiation has allowed an increased understanding of B cell activation and characterization of the molecules involved. B cell-Th cell contact appears sufficient for delivering the proliferative signal to B cells in the absence of lymphokines. A receptor ligand pair that plays a critical role in delivery of the contact signal is CD40 on the B cell surface and the ligand for CD40 on activated Th cells. Lymphokines alone do not drive resting B cell differentiation, however, when these soluble signals are delivered during the time of B cell DNA replication, they effect B cell differentiation and isotype switching. Delivery of the CD40-dependent contact signal to resting B cells appears to require a high degree of CD40 crosslinking on the B cell surface. Providing contact signals to naive B cells with recombinant molecules in membrane fractions may allow the generation of methodology to support the production of novel antibodies in vitro. PMID- 7538768 TI - Cellular mucins: targets for immunotherapy. AB - Mucins are attracting great interest as potential targets for immunotherapy in the development of vaccines for cancers expressing Mucin 1 (MUC1) (e.g., breast, pancreas, ovary, and others) as there is (1) a 10-fold increase in the amount in adenocarcinomas; (2) an alteration in expression where they become ubiquitous, and (3) due to altered glycosylation, new epitopes appear on the cell surface that are absent in normal tissues. These new epitopes can be carbohydrate; others are peptide in nature. The cloning of the cDNAs from mucins, particularly MUC1, has led to rapid advances being made, and it is clear that a highly immunogenic peptide exists within the variable number of tandem repeats (VNTR) found in all mucins. This peptide is immunogenic in mice, giving rise to strong antibody production, and most monoclonal antibodies made to breast cancer, which react with the protein core, react with the peptide APDTR. It is now also clear that humans with breast cancer have, in their draining lymph nodes, precursors of cytotoxic T cells that can be stimulated in vitro to react against breast cancer and indeed against the APDTR or a closely related peptide--shown from antibody blocking studies. These CTLs are unique in that they are non-MHC restricted. The identification of suitable targets, coupled with the known immunogenicity of both the peptide and neo-carbohydrate epitopes, has led to the development of several different programs to immunize humans against breast cancer using either synthetic carbohydrates or peptides conjugated with adjuvants, and clinical trials are now in progress to evaluate their immunogenicity and anti-cancer effects. PMID- 7538765 TI - [Clinical and electron microscopy study of sudden deafness treatment with the 10% HES 200/0.5 and pentoxifylline combination]. AB - The incidence of sudden hearing loss has increased. The pathogenetic mechanisms are still unknown, but viral infections and vascular phenomena with acute impairment of microvascular perfusion are thought to play a major role. Infusion of hydroxyethyl starch (HES) is used as a regimen to treat sudden hearing loss. In our clinic, anaphylactic reactions due to HES have not been observed so far. However, the use of HES is still discussed controversially due to long-term storage of HES molecules in tissue and due to high incidence of long-lasting pruritus. In a retrospective analysis of 118 patients treated with HES for sudden hearing loss, we observed pruritus starting in 64% of patients one to three weeks after therapy. This symptom with a duration between two weeks and four months was refractory to medical interventions. During therapy with HES improvement of hearing was observed in 75% of patients, in 62% improvement of hearing persisted still at the end of the observation period (7 months post infusionem). Light and electron microscopic assessment of human skin biopsies of one patient after treatment with HES showed storage of HES especially within dermal macrophages. Pathogenetically a pathway independent of histamin seems responsible for the induction of pruritus. Accordingly, classic antihistaminic drugs had no therapeutic effect in our patients. Dextran is used as an alternative to hydroxyethyl starch. In contrast to HES, the often mentioned higher incidence of severe anaphylactic reactions due to dextran has dramatically decreased with hapten inhibition (after preinjection of monovalent haptendextran Promit).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538766 TI - Preparation and pharmacological activities of homolupinanoyl anilides. AB - On the pattern of well known dialkylaminoacyl anilides, a set of N-homolupinanoyl anilides was prepared and subjected to a broad pharmacological screening with in vivo and in vitro assays. As expected most compounds exhibited a strong antiarrhythmic activity, often comparable or superior to that of lidocaine and quinidine. Compound 1 exhibited an unusual profile as antiarrhythmic, being devoid of local anesthetic activity, calcium channel and beta adrenoceptor antagonism. Calcium channel blocking activity was seen in all aminobenzophenone derivatives, but not in the simpler anilides. Noteworthy are also the capacity of compound 7 to protect mice from a lethal dose of KCN, the moderate antihypertensive activity of 10 and, above all, the antagonism to guinea pig ileum contractile responses induced by several agents exhibited by compound 11, which deserves further investigation for a potential use in irritable bowel syndrome. Compound 11 showed also good relaxant activity on tracheal strips and inhibitory activity against arachidonate induced platelet aggregation. Finally, compound 11 displaced several radioligands from their respective binding sites. Most potent was displacement of [3H]pirenzepine (IC50 < or = 0.01 microM) from M1 binding sites of rat brain, while displacement of [3H] methylscopolamine from rat heart (M2) and submaxillary salivary glands (M3) preparations was much weaker (IC50 approximately equal to 2.4 and 1.3 microM, respectively). PMID- 7538769 TI - The role of neuropeptides in learning: focus on the neurokinin substance P. AB - The neurokinin substance P (SP) can have neurotrophic as well as memory-promoting effects. The study of its mechanisms may provide new insights into processes underlying learning and neurodegenerative disorders. Our work shows that SP, when applied peripherally (i.p.), promotes memory and is reinforcing at the same dose of 37 nmol/kg. Most important, however, is the finding that these effects seemed to be encoded by different SP-sequences, since the N-terminal SP1-7 (185 nmol/kg) enhanced memory, whereas C-terminal hepta- and hexapeptide sequences of SP proved to be reinforcing in a dose equimolar to SP. These differential behavioral effects were paralleled by selective and site-specific changes in dopamine (DA) activity, as both SP and its C-, but not N-terminus, increased extracellular DA in the nucleus accumbens (NAc), but not in the neostriatum. The neurochemical changes lasted at least 2 h after injection. Direct application of SP (0.74 pmol) into the region of the nucleus basalis magnocellularis (NBM) was also memory promoting and reinforcing, and again, these effects were differentially produced by the N-terminus and C-terminus, supporting the proposed structure-activity relationship for SP's effects on memory and reinforcement. In addition, it was found that a single injection of SP into the NBM led to an increase of extracellular DA in the contralateral NAc. This effect of SP was observed only in those animals where SP was reinforcing, providing evidence for a lateralized relationship between reinforcement induced by injection of SP into the NBM and DA activity in the NAc. Furthermore, the outcome of a series of experiments suggests, that SP may not only be considered to have memory-promoting effects in normal animals, but can also improve functional recovery after unilateral 6-OHDA lesion of the substantia nigra and after lesions of the hippocampus, and can counteract age-related performance deficits. PMID- 7538772 TI - Intermediate filaments in disease. AB - Intermediate filaments are major structural proteins encoded by a large multigene family. Their tissue-specific expression makes them important in studies of development, differentiation and pathology. Most intermediate filaments are keratins; recent discoveries of keratin mutations in a range of genetic skin disorders have clarified their role as providing essential structural support for cells in different physical settings. PMID- 7538771 TI - Role of interferons and other cytokines in the regulation of the immune response. AB - Cytokines represent the major factors involved in the communication between T cells, macrophages and other immune cells in the course of an immune response to antigens and infectious agents. A number of studies on mouse and human T helper (Th) clones have recently provided extensive evidence for the existence of different activities exhibited by Th cells (called Th1 and Th2), which was apparently inferred from the profile of cytokine secretion. The Th1-type immune response is generally associated with IgG2a production and the development of cellular immunity, the Th2-type response with IgE production, eosinophils and mast cell production. This review focuses on the role of different cytokines produced by macrophages (especially interferons (IFNs), TNF-alpha, IL-10 and IL 12) or T cells (IFNs, IL-2, IL-4, IL-10, IL-13 and TGF-beta) in macrophage-T cell interactions and the cytokine relevance in the differentiation of Th cells towards the Th1 or Th2 type of immune response. Th1-derived cytokines (IFN-gamma, IL-2, TNF-alpha) favor macrophage activation, whereas the Th2 cytokines (IL-4, IL 10, IL-13) exhibit suppressive activities on macrophage functions. A key role in the differentiation towards the Th1-type response is now attributed to IL-12, a recently described cytokine produced mainly by macrophages. Its production can be upregulated by IFN-gamma and is inhibited by IL-10 and IL-4. All this emphasizes the importance of macrophage-cytokine interactions in determining the type of immune response. This article also aims to review recent data concerning the roles of IFNs alpha/beta (type I) and IFN-gamma (type II) in the regulation of the immune response. While there is much information on the regulatory effects of IFN-gamma (also called "immune IFN") on the immune response, little is so far known of the role of type I IFNs. These cytokines, originally described as simple antiviral substances, are now taken to be important regulators of the immune response. Recent data indicate that these molecules (especially IFNs-alpha) specifically promote the differentiation towards the Th1-type response. The stimulatory effects of IFN-alpha on the generation of the Th1-type response may be involved in its therapeutic effects in some human diseases, including early AIDS, hypereosinophilia and certain tumors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538770 TI - Molecular mechanisms of associative memory and their clinical implications. AB - In order to study how the human brain acquires, records, and recalls the relationships that comprise the images of human memory, our laboratory initiated a research strategy more than two decades ago. The strategy began with the hypothesis that the complex patterns of human memory are constructed from numerous simple relationships that are distributed over sensory space in our experience. This hypothesis further proposed that repeatable fundamental network architectures are distributed over brain structures to create internal images of our external and internal sensory experience. Based on this hypothesis, the first element of our research strategy was to (1) identify fundamental network architectures that learn and remember simple associative relationships such as those of Pavlovian conditioned responses; (2) demonstrate that the network biophysical and biochemical mechanisms of associative learning and memory in fundamental network architectures are conserved across species as diverse as those of snails, rabbits, and other mammals; (3) demonstrate that conserved memory mechanisms are targets of pathologic involvement in a human disease characterized by memory loss such as early Alzheimer's disease; (4) and derive mathematical and logical descriptions of the functions of biological associative network architectures during learning and memory. These descriptions would then be used to design artificial neural networks that would be implemented within computer programs. Observations demonstrating the plausibility of this research strategy are presented and discussed. PMID- 7538773 TI - Intermediate filaments: new proteins, some answers, more questions. AB - The past year has seen significant progress in the characterization of intermediate filament proteins. New proteins have been identified and physiologically significant differences between known proteins have been revealed. Changes in intermediate filament organization have been linked to changes in cell behavior, and mutational analyses are beginning to reveal the connection between intermediate filament expression, network formation, cellular behavior and disease. PMID- 7538774 TI - Somatostatin receptor activation of cellular effector systems. AB - Somatostatin (SRIF) induces its multiple biological actions by interacting with a family of receptors, referred to as SSTR1-SSTR5. These receptors are capable of associating with particular guanine nucleotide binding proteins to couple the receptors to distinct cellular effector systems. Therefore, G proteins have an important role in directing SRIF signalling and may provide the molecular basis for the diverse cellular actions of SRIF. PMID- 7538775 TI - Histamine and the cAMP pathway in the guinea-pig pancreas. AB - This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase from the guinea-pig pancreatic lobules. Histamine increased amylase release, reaching a maximum response at 10(-5) M. Similarly, vasoactive intestinal peptide (VIP) evoked significant increase in amylase release, though not in a dose-dependent fashion. When the pancreatic lobules were incubated with histamine in combination with VIP, forskolin or 3-isobutyl-1-methylxanthine (IBMX), amylase secretion was increased as compared to histamine alone. The stimulatory effect of VIP was also increased by the presence of forskolin or IBMX. These findings suggest that in guinea-pig pancreatic lobules, VIP, forskolin and IBMX, agents involved in the cyclic adenosine monophosphate (cAMP) pathway, potentiate histamine stimulated amylase release. PMID- 7538776 TI - Immunolocalization of adhesion molecules in psoriatic arthritis, psoriatic and normal skin. AB - Adhesion molecule expression in synovial membrane obtained from patients with psoriatic arthritis (PA) has previously been compared with rheumatoid arthritis (RA). Although expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was similar in both psoriatic and rheumatoid synovium, in contrast, little or no endothelial leucocyte adhesion molecule-1 (ELAM-1) was observed in psoriatic synovium. In the present study, the expression of ICAM-1, ELAM-1 and VCAM-1 was examined in the involved and uninvolved skin from patients with PA (n = 15), patients with psoriasis (Ps) but no arthritis (n = 5) and in normal skin (n = 4). ICAM-1 was intensely expressed on endothelium and keratinocytes of involved skin from patients with Ps with or without arthritis. There was constitutive expression of ICAM-1 on endothelium only in uninvolved and normal skin. In contrast, ELAM-1 expression was restricted to endothelial cells; it was widespread and intense in involved skin, but was minimal in uninvolved and normal skin. VCAM-1 was expressed on endothelium, and also on some dendritic cells in involved psoriatic skin. There was minimal VCAM-1 staining on endothelial cells in uninvolved and normal skin. In conclusion, in involved psoriatic skin from patients with and without arthritis ICAM-1, ELAM-1 and VCAM-1 expression is up-regulated on vascular endothelium, and ICAM-1 is expressed on keratinocytes. However, ELAM-1 and VCAM-1 expression seen in dermal vessels is not found in psoriatic synovial vessels. These differences suggest a mechanism for controlling cellular traffic in Ps and in PA. PMID- 7538777 TI - Substance P binding in normal neonatal foreskin. AB - The distribution of binding sites for NTE-biotinyl-[Arg3]-substance P (SPB) was demonstrated in neonatal foreskin using a conjugate of streptavidin with horseradish peroxidase. The observed binding is reversible, and may be abrogated by either the non-peptide substance P receptor antagonist, CP-96,345, or by unlabelled substance P. The generalized epidermal distribution and focal dermal localization of SPB binding suggest that although NK-1 receptors are abundant in human neonatal foreskin, neuromodulatory mechanisms may play a significant role in epidermal physiology. PMID- 7538779 TI - Extravasation of macromolecules and possible trapping of transforming growth factor-beta in venous ulceration. AB - The pathogenesis of venous ulceration is thought to involve formation of pericapillary fibrin cuffs as a result of venous hypertension, and a recent hypothesis suggests that extravasated plasma proteins may bind or trap growth factors. We have compared the tissue distribution of fibrin cuffs, plasma proteins, procollagen, and transforming growth factors (TGF-beta 1 and TGF-beta 2) within venous ulcers and normally healing graft donor sites. In venous ulcers, the papillary dermis and the ulcer bed contained convoluted capillaries with phosphotungstic acid haematoxylin-positive pericapillary fibrin cuffs. By immunohistochemical staining, the cuffs were positive for actin, and contained massively redundant lamellae of basement membrane material which stained positive for type IV collagen. Extravasated factor XIIIa and alpha 2-macroglobulin were present within the fibrin cuffs. Increased numbers of type I procollagen positive fibroblasts, and increased TGF-beta 1 immunoreactivity were present within the fibrin cuffs, but not in the provisional matrix in the ulcer bed around the cuffs. In contrast, in normally healing graft donor sites, tortuous capillaries and fibrin cuffs were absent, factor XIIIa and alpha 1-macroglobulin were restricted to the lumina of vessels, and procollagen and TGF-beta immunoreactivity were present within the granulation tissue and adjacent dermal matrix at the wound margin. These observations suggest that growth factors critical in wound healing, such as TGF-beta, are present within venous ulcers, but are abnormally distributed. Their distribution within fibrin cuffs and co localization with extravasated plasma proteins, particularly alpha 2 macroglobulin, which is a recognized scavenger molecule for TGF-beta and other growth factors, provides evidence for a possible 'trapping' of growth factors in venous ulcers. PMID- 7538778 TI - Mammary and extramammary Paget's disease: expression of Ca 15-3, Ka-93, Ca 19-9 and CD44 in Paget cells and adjacent normal skin. AB - The histogenesis of mammary Paget's disease (MPD) and extramammary Paget's disease (EMPD) cells remains controversial. The purpose of this study was to investigate MPD and EMPD immunohistochemically with antibodies to some tumour markers (Ca 15-3, KA-93 and Ca 19-9), and a cell surface receptor for hyaluronate (CD44), as these have been shown to be expressed in normal eccrine or apocrine glands and/or the epidermis, as well as some tumours. Surgically excised, formalin-fixed, paraffin-embedded tissues, or frozen tissues, from seven mammary, five vulvar, two scrotal and two axillary lesions were studied. Paget cells stained strongly with antibodies to Ca 15-3 and KA-93, but did not stain with those to Ca 19-9 and CD44. Staining with the antibody to Ca 15-3 was also observed in the ductal and secretory portions of the eccrine and apocrine glands, and in the sebaceous gland cells. Staining with the antibody to KA-93 was also seen in the apocrine secretory coils, lactiferous duct, epidermal dendritic cells, and cells in the dermal inflammatory infiltrate. Staining with the antibody to Ca 19-9 was observed only in the eccrine duct, and that to CD44 was seen in eccrine secretory cells and epidermal keratinocytes. These findings suggest that the origin of Paget cells may be the secretory cells of apocrine sweat glands (in EMPD) or the luminal lactiferous ducts (in MPD). We found that the antibodies to Ca 15-3 and CD44 were useful in differentiating Paget cells from surrounding keratinocytes, by showing positive and negative immunoreactivity, respectively. PMID- 7538780 TI - [The presence of hepatitis C virus antibodies in various risk groups]. PMID- 7538783 TI - The cell adhesion molecule, VCAM-1, is selectively elevated in serum in pre eclampsia: does this indicate the mechanism of leucocyte activation? PMID- 7538781 TI - Mechanism of c-SRC activation in human melanocytes: elevated level of protein tyrosine phosphatase activity directed against the carboxy-terminal regulatory tyrosine. AB - Normal human melanocytes, and some human melanoma cell lines, contain c-SRC which is constitutively activated by hypophosphorylation of tyrosine 530. We investigated the possibility that the activation of c-SRC in melanocytes might be attributable to elevated levels of tyrosine 530-directed protein tyrosine phosphatase activity in these cells. We found three times more of this phosphatase activity in cell extracts from melanocytes compared to fibroblasts. The majority of the tyrosine 530-dephosphorylating activity was present in the particulate fraction of cell homogenates, where c-SRC is also located. Treatment of melanocytes with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, caused inactivation of c-SRC. From these results, we conclude that activation of c-SRC in human melanocytes may be attributed to an elevated level of protein tyrosine phosphatase activity directed against tyrosine 530. PMID- 7538782 TI - Maternal serum hCG and fetal nuchal translucency thickness for the prediction of fetal trisomies in the first trimester of pregnancy. AB - OBJECTIVE: To compare the potential value of maternal serum total hCG and free beta-hCG in predicting the risk for fetal trisomies during the first trimester of pregnancy and to examine whether data on maternal hCG and fetal nuchal translucency thickness can be combined to derive risks. METHODS: Maternal serum total hCG and free beta-hCG were measured in samples from 83 singleton pregnancies with fetal chromosomal abnormalities (trisomy 21 (n = 41), trisomy 18 (n = 19), trisomy 13 (n = 8) sex chromosome aneuploidies (n = 11), triploidy (n = 4) and 394 chromosomally normal controls at 10 to 13 weeks gestation. In all cases, the fetal nuchal translucency thickness was measured at the time of fetal karyotyping. RESULTS: In the 249 chromosomally normal controls with fetal nuchal translucency less than 3 mm, total hCG and free beta-hCG decreased significantly with increased fetal crown-rump length. In 145 chromosomally normal fetuses with nuchal translucency 3 to 9 mm total hCG and free beta-hCG were not significantly different from the 249 with nuchal translucency less than 3 mm. In fetuses with trisomy 21, total hCG and free beta-hCG were significantly higher, whereas in trisomies 18 and 13 levels were lower than in chromosomally normal controls. When the cutoff levels for total hCG and free beta-hCG were selected to include 4% of chromosomally normal fetuses, the detection rates for trisomy 21 were 24% and 32%, respectively. There was no significant association between hCG and nuchal translucency thickness in either the chromosomally normal (r = -0.01) or abnormal group (r = -0.15). CONCLUSION: An improved estimate of risk for fetal trisomies at 10 to 13 weeks gestation can be derived by combining data on maternal age, maternal serum total or free beta-hCG and fetal nuchal translucency thickness. PMID- 7538784 TI - Shadow cell differentiation in tumours of the colon and uterus. AB - We report on six patients with tumours of visceral organs (three patients with endometrial adenocarcinoma with squamous cell differentiation, one patient with atypical hyperplasia of endometrium, and two patients with adenocarcinoma of the colon with squamous cell differentiation), where unquestionable differentiation into shadow cells was observed. In all six cases the shadow cells were found mostly in the morules of immature squamous cells. The shadow cells were morphologically identical, on the light microscopical and ultrastructural level, to similar cells found in pilomatricomas. They were often accompanied by granulomatous giant cell reaction and calcification. PMID- 7538785 TI - [Polyethylene glycol-thiosulfate (PEG-Th) staining--a modification of the AgNOR method]. AB - Visualization and analysis of nucleolus organizer regions (NOR) by a silverstaining is a well accepted method in cytological and histological tumor diagnostic. For specific staining of the NOR the AgNOR-method developed by HOWELL and BLACK (1980) and PLOTON et al. (1986) is performed. One major disadvantage of the AgNOR-method is the instability of the reaction products and therefore the short durability of the slide preparations. A modification of the AgNOR-method, the PEG-Th-staining, leads not only to a longer durability of the reaction products but also to a better contrast within the sections. Main characteristic of the PEG-Th-staining is a postfixation of the specific silver precipitations for 5 min in a 5% sodium thiosulfate solution. It could be proven by image analysis that there is no significant difference between the morphometric parameters evaluated on AgNOR- and PEG-Th-stained sections. Therefore results evaluated on PEG-Th-stained sections are absolutely comparable to those obtained on sections stained with the AgNOR-method thus providing the advantage of a longer durability of the reaction products and easier evaluation. PMID- 7538786 TI - Translocation of a channel-forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore. AB - A channel-forming antimicrobial peptide, magainin 2, has been shown to translocate across phospholipid bilayers by forming a pore comprising multimeric peptides. The translocation was demonstrated by four sets of experiments by use of resonance energy transfer from tryptophan introduced into the peptide to a dansyl chromophore incorporated into the lipid membrane. The translocation was coupled to pore formation, as detected by the dye efflux from the lipid vesicles; about 30% of the total peptide molecules translocated into the inner leaflets over 10 min, while 80% of the dye molecules leaked out at a lipid to peptide ratio of 57. This novel model can explain the problems debated so far, i.e., the peptide forms an ion channel whereas the magainin helix essentially lies parallel to the membrane surface. Channel (pore) formation in the vesicles is a transient process observable mainly during the early stage of the peptide membrane interactions. PMID- 7538787 TI - Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid. AB - The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues. PMID- 7538788 TI - Stabilizing effect of D-alanine2 in gramicidin channels. AB - We have investigated the effects of replacing Gly2 by D-Ala2 in gramicidin A (gA) analogues that have either L-Val, L-Ala, or Gly as the formyl-N-terminal residue. Circular dichroism, two-dimensional nuclear magnetic resonance, and hybrid channel experiments all show that [Ala1,D-Ala2]gA channels are structurally equivalent to the native [Val1,Gly2]gA channels, being formyl-NH-to-formyl-NH dimers of single-stranded, right-handed beta 6.3 helices. Replacing the Val1 of gA by Ala or Gly decreases the average channel duration. Replacing Gly2 by D-Ala in [Val1,Gly2]gA increases the average channel duration 4-fold and the single channel conductance by approximately 15%; replacing Gly2 with D-Ala in [Ala1,Gly2]gA or [Gly1,Gly2]-gA leads in each case to a 10-fold increase in the average channel duration with only modest changes in the single-channel conductance, which depends on the identity of the position-one residue and the permeant ion. These results illustrate the importance of neighboring-residue side chain and backbone interactions for the modulation of channel properties. PMID- 7538790 TI - Ethanol- and acetonitrile-induced inhibition of water diffusional permeability across bovine red blood cell membrane. AB - The effect of 0-3% (v/v) ethanol and acetonitrile on water diffusional permeability of bovine and chicken red blood cells (RBCs) was studied using a pulse 1H-T2 NMR technique. Transmembrane water diffusional exchange times, tau exch, of 9.2 +/- 0.46 ms and 18.3 +/- 1.0 ms were determined for bovine and chicken erythrocytes at 27.5 degrees C, respectively. Arrhenius activation energies Ea of water diffusion were 20.4 and 35.8 kJ mol-1. Ethanol, and acetonitrile being 2-fold more effective, markedly increased both tau exch and Ea in bovine RBC as compared to the well-known mercurial inhibitor of water channels, p-chloromercuribenzene sulfonate. Chicken RBCs that have no protein water channels, were found to be completely insensitive for either agent. It was suggested that ethanol and acetonitrile partitioning into the lipid phase of bovine RBC membrane affects the permeability of CHIP28 water channel but not the lipid confined water diffusion. The results suggest that the inhibition of transmembrane movement of water via CHIP28 channels might be involved in the anti hemolytic action of anaesthetics such as ethanol. PMID- 7538789 TI - Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix. AB - Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne (Von Heijne, G. (1992) J. Mol. Biol. 225, 487-494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN NH2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic alpha helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% alpha helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (> 250 microM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope. PMID- 7538792 TI - Therapeutic options in malignant mesothelioma. AB - No significant advances have been made in the treatment of diffuse malignant mesothelioma in the past 12 months. For fit patients with early disease (stage I), radical surgery remains the best option, but doubt remains concerning optimum adjuvant therapy. A collaborative approach with multicentre trials of standardized treatment protocols is essential if any progress is to be made in the future. PMID- 7538794 TI - Short-term oral treatment of canine benign prostatic hypertrophy with chlormadinone acetate. AB - Ten beagle dogs (4-8 years of age) with benign prostatic hypertrophy were treated daily for 7 or 14 days by a daily oral administration of chlormadinone acetate (CMA), an anti-androgenic preparation, at a dose of 0.5 or 2.0 mg/kg/day. The groups of dogs treated with CMA at a dose of 2.0 mg/kg showed a greater reduction of the prostatic volume during 7 or 14 days of treatment than the group treated with 0.5 mg/kg of CMA. At 56 and 84 days after the start of CMA treatment, prostate volume was reduced to 63 and 79% of the pre-administration volume, respectively. No marked decrease in plasma LH or testosterone was found after the CMA treatment. These results indicate that the therapeutic effects of 7-day oral treatment with CMA at 2.0 mg/kg/day in dogs with BPH last for about three months or longer without side effects. PMID- 7538791 TI - Gene therapy and bone marrow transplantation. AB - Retrovirus-mediated gene transfer into hematopoietic stem cells has been shown in mice, large animals, and humans. Transduction efficiency has been high in mice but has remained low in large animals and humans. Improved transduction efficiency into hematopoietic progenitor cells of large animals and humans has been achieved in vitro by enriching for CD34+ cells, adding growth factors to the transduction culture, extending the exposure time of hematopoietic cells to retrovirus particles, and by using retrovirus vectors pseudotyped with the gibbon ape leukemia virus envelope. Whether these modifications will also result in increased transduction of pluripotent hematopoietic stem cells has yet to be demonstrated by in vivo transplantation studies. Current transduction efficiency of hematopoietic stem cells in large animals and humans appears to be sufficiently high (0.1% to 1%) for gene marking studies. Efficiency needs to be further increased before gene transfer can be used for therapeutic applications. PMID- 7538793 TI - Genes involved in melanoma susceptibility and progression. AB - Despite the ever-increasing levels of biologic complexity, major strides have been made in the past few years in three fundamental areas of research critical to understanding the biology of melanoma development and progression: 1) identifying a potential susceptibility gene on 9p21 and defining its role in control of the cell cycle; 2) the cataloguing of cytoplasmic, cell surface, and secreted proteins that play major roles in the ability of melanoma cells to migrate from the primary tumor to distant sites; and 3) the elucidation of the interconnected signal transduction pathways that couple the functions of these proteins to basic cellular processes such as architecture, locomotion, and gene transcription. This review briefly discusses each of these areas. PMID- 7538795 TI - Changes in the nucleosomal structure of the Marek's disease virus genome in lymphoblastoid cell line MDCC-MSB1 induced by 5-azacytidine. AB - Marek's disease virus (MDV) DNA in latently infected lymphoblastoid cell lines is considerably methylated. Treatment of the MDV-derived lymphoblastoid cell lines MDCC-MSB1 (MSB1) and MDCC-RP1 (RP1) with 5-azacytidine (5-AzC) results in hypomethylation of MDV DNA. An increase in mRNA from certain portions of MDV DNA, including the BamHI-H region, was observed in 5-AzC-treated MSB1 cells, but not in the agent-treated RP1 cells. After the treatment of cells with 5-AzC, a site hypersensitive to digestion with DNaseI appeared in the BamHI-H region of MDV DNA in MSB1 but not in RP1. These results suggested that the enhancement of mRNA synthesis by 5-AzC is associated with changes in the nucleosomal structure of MDV DNA in lymphoblastoid cell line MSB1. PMID- 7538796 TI - Horse trophoblasts produce tumor necrosis factor alpha but not interleukin 2, interleukin 4, or interferon gamma. AB - The distribution of four cytokines was analyzed in the endometrium and trophoblast of the horse between Days 30 and 55 of gestation. Endometrial tissues, invasive trophoblast (chorionic girdle), and noninvasive trophoblast (chorion and allantochorion) were examined separately. Cytokine expression was determined by amplification of specific mRNA via the reverse transcriptase polymerase chain reaction (RT-PCR). Messenger RNA for interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN gamma) was detected in endometrial tissues, unstimulated peripheral blood lymphocytes, and control kidney tissue, but not in trophoblasts. leukocytes resident in the endometrium or traversing the uterus via blood vessels might be the source of these cytokines. Endometrial tissues and invasive and noninvasive trophoblasts expressed mRNA for tumor necrosis factor alpha TNF alpha). Immuonoreactive TNF alpha protein was detected in different cell types of the endometrium and in the invasive and noninvasive trophoblast. The ubiquitous expression of TNF alpha by the endometrium and trophoblasts suggests that this cytokine might have an important role in regulating placental growth and differentiation or maternal leukocyte responses to trophoblasts. IL-2, IL-4, and IFN gamma might have important immunoregulatory roles within the endometrium. PMID- 7538797 TI - Purification of heparin-binding epidermal growth factor-like growth factor from pig uterine luminal flushings, and its production by endometrial tissues. AB - Pig uterine luminal flushings contain at least four heparin-binding growth factors (HBGF) that stimulate fibroblast [3H]thymidine incorporation. One of these factors, which appeared to be a relatively minor HBGF, was eluted from heparin affinity columns by 1.0 M NaCl and was found to compete with 125I epidermal growth factor (EGF) for binding to an endometrial carcinoma cell line. This EGF receptor (EGF-R)-binding property was abolished by an antiserum to heparin-binding EGF-like growth factor (HB-EGF) that specifically blocks binding of HB-EGF to the EGF-R. Reverse-phase HPLC resulted in the purification of two EGF-R-binding activities correlated with 13,500 and 17,000 M(r) proteins that reacted with an antiserum raised against residues 9-26 of human HB-EGF. Uterine extracts also contained an EGF-R-binding factor that was eluted from heparin by 1.0 M NaCl and was antagonized by HB-EGF antiserum. Endometrial mRNA subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR through the use of HB-EGF-specific primers yielded fragments of the predicted size. Cloning of the nested PCR product revealed a 380-bp porcine HB-EGF cDNA sequence that was 78-85% homologous to primate or rodent HB-EGF. HB-EGF was immunohistochemically localized primarily to the luminal epithelium in both pregnant and nonpregnant animals. PMID- 7538798 TI - Bovine SRY gene locus: cloning and testicular expression. AB - The bovine SRY gene was cloned by a combination of anchored polymerase chain reaction (PCR) amplification of genomic restriction fragments and reverse transcription-PCR (RT-PCR) of testicular RNA. We report 1800 bp of combined genomic and cDNA sequences including 911 bp of 5' upstream sequences, an open reading frame of 687 bp, and 202 bp of sequences corresponding to the 3' end of the mRNA. The bovine SRY gene encodes a deduced (predicted on the basis of a cDNA sequence) protein product of 229 amino acids, with sequence conservation between species, notably in the region of the high-mobility group (HMG) domain or HMG box. Outside of the HMG box, the bovine SRY structure shows greater resemblance to the human SRY than to the mouse Sry. As with human SRY promoter sequences, putative binding sites for Sp1 and for SRY itself are seen in the bovine SRY promoter region. Unlike the human SRY promoter, CAAT and TATA box motifs are present in the bovine sequences. Southern analysis and PCR amplification of male and female bovine genomic DNA show that the described sequences are specific to the Y chromosome. Northern analysis of bull testicular RNA demonstrated low levels of expression of the bovine SRY gene in adult testes with a major poly(A) species at 1.9 kb. RT-PCR amplification of bull testicular RNA revealed multiple sites of polyadenylation, but sequencing showed no consensus polyadenylation signal. PMID- 7538799 TI - Distribution of neuronal and non-neuronal NADPH diaphorases and nitric oxide synthases in rat uterine horns under different hormonal conditions. AB - Since pharmacological evidence indicates that nitric oxide (NO) operates in the control of uterine motility, we have studied the distribution of NADPH diaphorase and NO synthases in the rat uterus using histochemical and immunohistochemical methods. Numerous nerve fibers displayed NADPH diaphorase activity and immunoreactivity to antisera raised against neuronal NO synthase. Nerve fibers appeared in all stages of the estrous cycle and also after ovariectomy. NADPH diaphorase activity was also present in endothelia and cells dispersed in the different uterine layers. Most NADPH diaphorase-positive (ND) cells had eosinophilic granules with occasional cells expressing the ED1 macrophage monocyte marker. Immunoreactivity for an inducible NO synthase was found in a small number of macrophage-like cells without NADPH diaphorase activity. Thus, ND cells may express another NO synthase isoform not detected by the available antisera. In normal cycling rats, ND cells were most abundant during proestrus, and their number further increased after estrogen treatment. ND cells were not observed after ovariectomy but were present after estrogen replacement therapy. ND cells could be involved in the estrogenic control of in vivo and in vitro uterine. PMID- 7538800 TI - An ATP-activated cation conductance in human epididymal cells. AB - An outwardly rectifying conductance was observed in primary cultured human epididymal cells under the patch-clamp whole-cell configuration in KCl pipette and bath solutions. Removal of Cl- from intracellular and extracellular solutions did not affect this conductance, but substitution of K+ with N-methyl-D-glucamine in both solutions completely blocked the current. The current magnitude was also found to be affected by intracellular but not extracellular K+ concentrations. The conductance could be inhibited by the cation channel blockers, barium and tetraethylammonium chloride. Single-channel recordings from the same cell population also revealed the presence of a conductance of about 150 pS with voltage dependence and blocker sensitivity similar to those observed for the whole-cell current. This cation conductance was not affected by changes in solution osmolarity but could be activated by extracellular ATP. ATP activation of the conductance could be mimicked by ionomycin and inhibited by BAPTA-AM, an agent that suppresses intracellular free Ca2+. This ATP-regulated cation conductance may be responsible for secreting K+ across the epididymal epithelium, resulting in an observed K+ concentration much higher in the lumen of the epididymis than in the blood. PMID- 7538803 TI - Dimensions of the narrow portion of a recombinant NMDA receptor channel. AB - Glutamate-activated single-channel and ensemble currents were recorded from Xenopus laevis oocytes and HEK 293 cells expressing a recombinant NMDA receptor, assembled from NR1 and NR2A subunits. Cesium was the main charge carrier, and organic cations were used to determine the presence of vestibules of this channel and to estimate its pore diameter. The large organic cations tris-(hydroxymethyl) aminomethane (Tris), N-methyl-glucamine (NMG), arginine (NMG), arginine (Arg), choline, and tetramethylammonium (TMA), when added in millimolar concentrations to the extracellular or cytoplasmic side, produced a voltage-dependent blockade of single-channel Cs+ currents. These molecules behaved as impermeant ions that only partially traverse the channel from either side. The smaller cations trimethylammonium (TriMA) and dimethylammonium (DMA) produced a small and nearly voltage-independent reduction in current amplitude, suggesting that they are permeant. In biionic experiments with Cs+ as the reference ion, the large blocking cations NMG, Arg, Tris, TMA, choline, hexamethonium (Hme), triethylammonium (TriEA), and tetraethylammonium (TEA) showed no measurable permeability. TriMA and smaller ammonium derivatives were permeant. Both the permeability and single-channel conductance of organic cations, relative to Cs+, decreased as the ion size increased. The results suggest that the NMDA receptor has extracellular and cytoplasmic mouths that can accommodate large cations up to 7.3 A in mean diameter. The narrow portion of the pore is estimated to have a mean diameter of 5.5 A. PMID- 7538802 TI - Cross-linking of CD4 in a TCR/CD3-juxtaposed inhibitory state: a pFRET study. AB - Instances when T cell activation via the T cell receptor/CD3 complex is suppressed by anti-CD4 Abs are generally attributed either to the topological separation of CD4-p56lck from CD3, or their improper apposition. Photobleaching fluorescence resonance energy transfer measurements permitted direct analysis of these alternatives on human peripheral blood lymphocytes. Distinction between changes of relative antigen densities or positioning was made possible by simultaneously recording donor and acceptor fluorescence in the energy transfer experiment performed on homogeneous populations of flow-sorted cells. We show here that CD4 stays in the molecular vicinity of CD3, while anti-CD3 stimulation is suppressed by anti-CD4 or cross-linked HIV gp120. Our data suggest that cross linking of CD4 through particular epitopes is capable of inhibiting activation driven by Abs binding to specific sites on CD3 without major topological sequestration of the Ags, in such a way that additional positive signals will also be affected. Thus, these and other related cases of negative signaling via CD4 may be interpreted in terms of functional uncoupling rather than a wide physical separation of CD4 from the T cell receptor-CD3 complex. PMID- 7538804 TI - Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy. AB - The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions. PMID- 7538805 TI - Sodium in gramicidin: an example of a permion. AB - The reaction path and free energy profile of Na+ were computed in the interior of the channel protein gramicidin, with the program MOIL. Gramicidin was represented in atomic detail, but surrounding water and lipid molecules were not included. Thus, only short range interactions were investigated. The permeation path of the ion was an irregular spiral, far from a straight line. Permeation cannot be described by motions of a single Na+ ion. The minimal energy path includes significant motion of water and channel atoms as well as motion of the permeating ion. We think of permeation as motion of a permion, a quasi-particle that includes the many body character of the permeation process, comparable with quasi particles like holes, phonons, and electrons of solid-state physics. Na+ is accompanied by a plug of water molecules, and motions of water, Na+, and the atoms of gramicidin are highly correlated. The permion moves like a linear polymer made of waters and ion linked and moving coherently along a zigzag line, following the reptation mechanism of polymer transport. The effective mass, free energy, and memory kernel (of the integral describing time-dependent friction) of short range interactions were calculated. The effective mass of the permion (properly normalized) is much less than Na+. Friction varies substantially along the path. The free energy profile has two deep minima and several maxima. In certain regions, the dominant motions along the reaction path are those of the channel protein, not the permeating ion: there, ion waits while the other atoms move. At these waiting sites, the permion's motion along the reaction path is a displacement of the atoms of gramicidin that prepare the way for the Na+ ion. PMID- 7538808 TI - The ex vivo production of human monoclonal antibodies to glycoprotein IIb-IIIa complexes of blood platelets. AB - The integrin alpha IIb beta 3 (GPIIb-IIIa complex) of blood platelets mediates platelet aggregation by binding adhesive proteins which form bridges between activated cells. This same process is implicated in arterial thrombosis. The goal of our research is to take B-lymphocytes from patients possessing inhibitory antibodies to GPIIb-IIIa and develop technology permitting their production ex vivo. Starting point is the peripheral blood from two patients with Glanzmann's thrombasthenia, an inherited disorder in which platelets lack these complexes, and where high titre antibodies to GPIIb-IIIa have formed following contact with normal platelets after transfusion and/or pregnancy. We describe a strategy of in vitro stimulation to overcome the following constraints: (i) peripheral blood contains a low concentration of antigen-reactive specific B-cells, and (ii) the circulating B-cells are arrested in a phase in which additional stimuli are required to induce antigen-specific clonal activation. Optimal conditions involve the use of a combination of growth factors, polyclonal activators and soluble GPIIb-IIIa prior to the fusion of activated B-cells with either (a) the murine myeloma cell line X63 Ag 8,653 or (b) the heteromyeloma cell line SPM4-0. In this way, we have obtained several cell lines secreting antibodies specific for the GPIIb-IIIa complex. Our next aim is to rescue the relevant human immunoglobulin genes from these hybridoma cells. PMID- 7538801 TI - [Simulation of myocardial action potentials of various cell types in relation to neural parameters]. AB - Action potentials of various myocardial cell types were simulated in a computer model based on current knowledge of the electrical properties of ionic channels and pumps in the ventricular cell membrane and the sarcoplasmic reticulum. The transport mechanisms of sodium, potassium, calcium and chlorine ions through the cell membrane are described mathematically, as is the exchange of calcium between the myoplasm and sarcoplasmic reticulum. Ten ionic channels and three pumps of the cell membrane are taken into account, while three channels and one pump of the sarcoplasmic reticulum are considered in the computations. For the first time, the transient outward potassium current IK,to was simulated, the effect of which on the early repolarisation phase of the action potential was reproducible in the model. Calcium buffers in the myoplasm and the sarcoplasmic reticulum are also considered. From the resulting ionic currents through the channels and pumps, the membrane potential is computed using an equivalent circuit diagram of the cell membrane. In particular the influence of neural activity on channel conductance and the probability of channel patency were taken into account. By means of this model, different shapes of ventricular action potentials were simulated. The action potentials of epicardial cells, M-cells, endocardial cells and Purkinje fibres were accurately simulated. In addition, the effects of sympathetic drive and various drugs were demonstrated in the model as well as the shortening of the action potential duration with increasing stimulation frequency. PMID- 7538807 TI - Human monoclonal anti-Rh antibodies produced by human-mouse heterohybridomas express the Gal alpha 1-3 Gal epitope. AB - The presence of the Gal alpha 1-3 Gal structure (Gal epitope) in the carbohydrate component of fifteen human monoclonal antibodies with specificity for the Rh blood group factor and produced by human-mouse heterohybridomas was evaluated. To do that, an antiglobulin-like agglutination test and an enzyme linked immunosorbent assay were performed using an affinity-purified anti-Gal antibody obtained from the serum of an AB blood group donor. Using the antiglobulin reaction, results were obtained showing that only five of the fifteen human monoclonal antibodies tested contained the structure at levels sufficient to allow agglutination. However, all fifteen monoclonals were positive using the more sensitive enzyme linked immunosorbent assay. By means of an indirect immunofluorescence assay the same anti-Gal antibody was used to test the presence of Gal epitopes on the surface of the producer heterohybridomas. Results were obtained indicating that twelve out of the fifteen hybridomas studied do express the Gal structure on its surface. The relevance of these findings is discussed in the context of therapeutic applications of human monoclonal antibodies. PMID- 7538809 TI - Overexpression of transforming growth factor-beta 1 mRNA is associated with up regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice. AB - Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes. PMID- 7538806 TI - Early afterdepolarizations in cardiac myocytes: mechanism and rate dependence. AB - A model of the cardiac ventricular action potential that accounts for dynamic changes in ionic concentrations was used to study the mechanism, characteristics, and rate dependence of early after depolarizations (EADs). A simulation approach to the study of the effects of pharmacological agents on cellular processes was introduced. The simulation results are qualitatively consistent with experimental observations and help resolve contradictory conclusions in the literature regarding the mechanism of EADs. Our results demonstrate that: 1) the L-type calcium current, ICa, is necessary as a depolarizing charge carrier during an EAD; 2) recovery and reactivation of ICa is the mechanism of EAD formation, independent of the intervention used to induce the EADs (cesium, Bay K 8644, or isoproterenol were used in our simulations, following similar published experimental protocols); 3) high [Ca2+]i is not required for EADs to develop and calcium release by the sarcoplasmic reticulum does not occur during the EAD; 4) although the primary mechanism of EAD formation is recovery of ICa, other plateau currents can modulate EAD formation by affecting the balance of currents during a conditional phase before the EAD take-off; and 5) EADs are present at drive cycle lengths longer than 1000 ms. Because of the very long activation time constant of the delayed rectifier potassium current, IK, the activation gate of IK does not deactivate completely between consecutive stimuli at fast rates (drive cycle length < 1000 ms). As a result, IK plays a key role in determining the rate dependence of EADs. PMID- 7538811 TI - Gangliosides of anencephalic and fetal brain--immunostaining on thin-layer chromatograms. AB - The expression of gangliosides of ganglio-series as well as neolacto-series gangliosides in anencephalic and in normal human fetal brain was compared with that in adult brain by immunostaining on thin-layer chromatograms. A difference in the expression of ganglio-series gangliosides with GM1a core was found between anencephalic and normal fetal brain, with less expression of GM1a and GD1a in anencephaly compared with normal fetal brain, in which these gangliosides dominate. Small amounts of GM1b were detected in fetal brain whereas only traces were found in anencephalic brain. Lactosamine-containing gangliosides were present in fetal and in anencephalic brain as alpha 2-3 as well as alpha 2-6 sialylated nLcOse4Cer structures. A heterogeneous group of neolacto-series gangliosides was expressed in anencephalic brain in both the monosialo- and presumed disialoganglioside range. These findings demonstrate a significant change in ganglioside pattern in anencephaly where the process of cell differentiation and maturation has been severely disturbed. PMID- 7538810 TI - NADPH-diaphorase expression in neurones and glial cells of the locust brain. AB - Using NADPH-diaphorase (NADPH-d) staining as a marker for the enzyme nitric oxide synthase (NOS) we investigated the possible sites of nitric oxide (NO) synthesis in the olfactory pathways of an insect brain. Staining of frozen sections revealed NADPH-d activity in neurones and in glial cells. A cluster of intensely stained interneurones innervates the neuropile of the antennal lobe. NADPH-d expression in the mushroom bodies showed a compartmentalized pattern. The mushroom body intrinsic Kenyon cells did not express NADPH-d. The pedunculus and lobes showed fine granular staining and were invaded by NADPH-d-positive mushroom body extrinsic fibres. The expression of NADPH-d in glial cells enclosing the mushroom bodies suggest that insect glial cells may release NO as a messenger molecule. PMID- 7538812 TI - Chronic (-)baclofen or CGP 36742 alters GABAB receptor sensitivity in rat brain and spinal cord. AB - Administration of the GABAB receptor agonist, (-)-baclofen 10 mg kg-1, i.p. daily for 21 days to rats prevented (-)-baclofen-induced hyperpolarizing responses and synaptically-evoked late inhibitory potentials (IPSPs) in olfactory cortical neurones recorded intracellularly from 450 microns brain slices. In contrast, pre treatment with CGP 36742 induced a significant increase in (-)-baclofen-mediated post-synaptic responses and late IPSP amplitude. In the spinal cord, the potency of (-)-baclofen in inhibiting electrically-evoked substance P-like immunoreactivity or amino acid release was significantly reduced or increased in slices from rats pre-treated with the GABAB agonist or antagonist, respectively. These data suggest that functional responses to GABAB receptor activation in the mammalian central nervous system can be up- or down-regulated. PMID- 7538813 TI - Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells. AB - CD34 is expressed on human and murine hematopoietic stem and progenitor cells and its clinical usefulness for isolation of stem/progenitor cells has been well established. Although expression of CD34 is regulated in a developmental stage specific manner, the function of CD34 is not known. Recently we have shown that both a full-length and truncated form of CD34 protein is expressed by hematopoietic cells (Blood 84:691, 1994). To test whether failure to suppress either form of CD34 could affect terminal myeloid differentiation, we constitutively expressed these CD34 proteins in murine M1 myeloid leukemia cells, which can be terminally differentiated to macrophages by treatment with interleukin-6 of leukemia inhibitory factor. Surprisingly our results show that forced expression of the full-length but not the truncated form of CD34 impedes terminal differentiation by these agents. Because the difference between the two forms of CD34 protein resides in the length of their respective cytoplasmic tail domains, our findings strongly suggest that the cytoplasmic domain region of full length CD34 is responsible for the observed maturation arrest phenotype. These findings suggest a potential negative regulatory role for full-length CD34 in hematopoietic cell differentiation and may explain, at least in part, the block in maturation observed in CD34+ acute myeloid leukemia. PMID- 7538814 TI - Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. AB - We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages. PMID- 7538815 TI - A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia. AB - To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway. PMID- 7538816 TI - Hepatocyte growth factor as a hematopoietic regulator. AB - Hepatocyte growth factor (HGF) was originally isolated as a mitogen for adult hepatocytes, but this cytokine is now regarded as a multi-functional factor. In the present study, we show that the mouse liver in the middle and/or late stage of the fetal life expresses both HGF and c-met (its receptor) messages. HGF and c met mRNA are coexpressed not only in the adherent layers of fetal liver long-term cultures (FL-LTCs) and adult bone marrow long-term cultures (BM-LTCs), but also in the stromal cell lines MS-5 and PA-6. Addition of human HGF (2 and 20 ng/mL) to the LTCs enhances (1) nonadherent cell counts (ninefold in FL-LTCs and sixfold in BM-LTCs), (2) nonadherent colony-forming unit-in culture (CFU-C) counts (eightfold in FL-LTCs and fivefold in BM-LTC), and (3) cobblestone colony counts. However, HGF slightly inhibits the proliferation of stromal cells. No direct effect of HGF on freshly isolated BM and/or FL cells is found in the CFU-C assay. However, an approximately 1.5-fold synergistic increase in CFU-C counts is noted when the BM or FL cells are cocultured with HGF in the presence of interleukin-3. These findings strongly suggest that HGF plays a crucial role as a hematopoietic regulator in the proliferation and differentiation of hematopoietic progenitors. PMID- 7538817 TI - Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro. AB - The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ. PMID- 7538818 TI - Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel. AB - The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR. PMID- 7538819 TI - Role of extracellular adenosine triphosphate in the cytotoxic T-lymphocyte mediated lysis of antigen presenting cells. AB - The lysis of antigen presenting cells (APCs) by cytotoxic T lymphocytes (CTLs) may be one mechanism whereby an immune response is downregulated by Staphylococcus superantigens. Disappearance of monocytes/macrophages from staphylococcal enterotoxin A (SEA)-activated peripheral blood mononuclear cell (PBMC) cultures, but not from control PBMC cultures was seen by flow cytometry. Recently, adenosine triphosphate (ATP) has been described as an effector molecule in CTL-mediated lysis of some murine tumor target cells. We have also shown that ATP caused the lysis of human macrophages, and that treatment of cells with interferon gamma (IFN gamma) rendered macrophages significantly more sensitive to ATP than untreated cells. To show that this purine nucleotide may play a role in modulating the immune system, we generated human CTLs that were stimulated with SEA, and used them as effector cells against SEA-pulsed autologous macrophages. CTLs were found to specifically lyse SEA-pulsed macrophages, while control, unpulsed, macrophages were unaffected. The addition of hexokinase, an enzyme that hydrolyzes ATP, significantly abrogated the killing of SEA-pulsed cells during the assay. In examining the mechanism of cytotoxicity, electron microscopy showed that macrophages incubated with both ATP and CTLs underwent necrosis, rather than apoptosis. From these results, it is suggested that ATP is released from CTLs during antigen presentation, and that IFN gamma-activated macrophages, which are inherently more sensitive to this mediator, are readily lysed and therefore removed from circulation, thus downregulating an immune response. PMID- 7538820 TI - Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro. AB - Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538821 TI - The efficacy of CD3 x CD19 bispecific monoclonal antibody (BsAb) in a clonogenic assay: the effect of repeated addition of BsAb and interleukin-2. AB - To evaluate the potency by which human T cells are targeted and activated by bispecific monoclonal antibodies (BsAbs) to lyse tumor cells, a clonogenic assay was developed. The efficacy of a CD3 x CD19 BsAb binding to both the CD3 T-cell antigen and the CD19 B-cell antigen was already proven in 51Cr-release assays and in 3-day activation cultures. To achieve more quantitative results, a 14-day clonogenic assay, based on limiting-dilution, was performed for the determination of the initial and residual number of clonogenic units obtained with a CD19+ pre pre-B acute lymphoblastic leukemia (ALL-B) cell line. Elimination of up to 5 logs of ALL-B cells by freshly isolated peripheral blood mononuclear cells (PBMCs) cultured with BsAb plus interleukin-2 (IL-2) could be detected. The presence of human IgG did not abolish the effect. Repeated addition of each of the two agents was necessary, because a single treatment produced only a 1- to 2-log kill. CD3 monoclonal antibody and IL-2 stimulation ("lymphokine-activated killer cell" conditions) resulted in only a 2-log kill. The number of T cells proved critical in lysis of ALL-B cells, with a 5-log kill using a T-cell:B-cell ratio of 3:1 but with only a 1-log kill using a ratio of 1:1. PBMCs isolated from patients with non-Hodgkin's lymphoma, both in relapse or remission, proved to be as competent as those from healthy donors in removing ALL-B cells. This clonogenic assay shows the importance of repeated administration of CD3 x CD19 BsAb and IL-2 and offers the possibility to compare it with other therapies in B-cell malignancy. PMID- 7538822 TI - Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis. AB - We have investigated the adhesive capacity of neutrophils after spontaneous apoptosis, which occurs during in vitro culture. Apoptotic neutrophils show reduced adhesion to E selectin and the CD18 integrin ligand fibrinogen. Neutrophil apoptosis is associated with changes in the levels of surface expression of key receptors that mediate neutrophil adhesion events. Notably, apoptotic neutrophils show reduced expression of L-selectin/selectin ligand. In contrast, CD11b/CD18 and CD11c/CD18 integrins are expressed at increased levels. The reduced capacity for adhesion of apoptotic neutrophils may be achieved by very different mechanisms. Regulation of the levels of surface expression of receptors/ligand may control selectin-mediated adhesion, possibly as a result of protease/sialidase activity. In contrast, modulation of integrin-mediated adhesion may involve functional uncoupling of receptors present on the surface of the apoptotic cell without alteration in levels of surface expression. The altered adhesive potential of the apoptotic neutrophil may serve to limit release of its histotoxic contents and reduce inappropriate tissue injury. PMID- 7538823 TI - Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. AB - Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA 4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony-stimulating factor (G CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady-state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = -.86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = -.70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation. PMID- 7538825 TI - Response to granulocyte-macrophage colony-stimulating factor (GM-CSF) but not to G-CSF in a case of agranulocytosis associated with large granular lymphocyte (LGL) leukemia. PMID- 7538824 TI - Transplantation of enriched and purged peripheral blood progenitor cells from a single apheresis product in patients with non-Hodgkin's lymphoma. AB - High-dose chemotherapy with or without radiotherapy followed by autologous transplantation of hematopoietic progenitor cells is an effective treatment for patients with high-risk or relapsed non-Hodgkin's lymphoma. Chemotherapy and/or hematopoietic growth factors have been used to mobilize progenitor cells in the peripheral blood for transplantation. However, the mobilized blood cell products have been found to be frequently contaminated with tumor cells, and techniques have not been developed to purge tumor cells from these products. In addition, the minimum number of hematopoietic progenitor cells required for engraftment has not yet been fully elucidated. We treated 21 patients with a single infusion of cyclophosphamide (4 g/m2) followed by daily administration of granulocyte colony stimulating factor (G-CSF). After recovery of the white blood cell count, a single 3-hour apheresis collection was performed. The apheresis product was then applied to a discontinuous Percoll gradient. The low-density fractions resulting from this separation procedure were enriched for CD34+ progenitor cells (total cell yield, 19.5%; CD34+ cell recovery, 81.2%). These enriched cellular products were treated with a panel of anti-B cell or anti-T cell monoclonal antibodies and complement in an effort to remove residual tumor cells. After treatment of the patient with myeloablative therapies, the enriched and purged cells were reinfused. Hematologic recovery was rapid, with median neutrophil engraftment in 10 days [absolute neutrophil count (ANC), greater than 0.5 x 10(9)/L] and 11 days (ANC, greater than 1.0 x 10(9)/L). Median platelet transfusion independence required 13 days. The rapidity of multilineage engraftment correlated with the number of CD34+ cells per kilogram that were infused. Patients who received more than 2 x 10(6) CD34+ cells per kilogram had rapid hematologic engraftment, whereas those patients transplanted with less than 2 x 10(6) CD34+ cells per kilogram had slower platelet recovery. Modeling studies using a lymphoma cell line with a t(14; 18) chromosomal translocation demonstrated the successful removal of tumor cells assayed using the polymerase chain reaction (PCR) after the processing and purging. Four of the 21 patients had PCR-detectable lymphoma cells in the bone marrow and peripheral blood; however, the enriched and purged blood products reinfused in all four did not contain detectable tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7538827 TI - Comparison of sulforhodamine B, tetrazolium and clonogenic assays for in vitro radiosensitivity testing in human ovarian cell lines. AB - The radiation sensitivity of six human ovarian tumor cell lines was evaluated using sulforhodamine B (SRB), tetrazolium (MTT) and clonogenic assays. Radiobiological parameters calculated from a linear quadratic model (SF2, alpha, beta) as well as from a single-hit multitarget model (D(o), Dq, n) and from the area under the dose-response curve (mean inactivation dose; MID) were compared. If the values deduced from MTT experiments were statistically comparable to those obtained from clonogenic assays, significant differences were observed between SRB and the two other assays that concerned the results achieved with the highest radiation doses tested (6-8 Gy), yielding a surviving fraction of approximately 20%. In addition, the intra- and inter-experimental variation of SRB dramatically increased within this range of radiation doses. However, up to 6 Gy, the SRB assay proved to be statistically comparable to MTT and clonogenic assays, and allowed the calculation of SF2, alpha and MID radiobiological parameters. PMID- 7538826 TI - CD7 expression does not predict for poor outcome in acute myeloblastic leukemia. PMID- 7538828 TI - Bone marrow aplasia and severe skin rash after a single low dose of methotrexate. AB - A 64 year old man with recurrent metastatic squamous cell carcinoma of the head and neck developed severe skin rash and bone marrow aplasia 4 and 7 days, respectively, following a single dose of 40 mg/m2 methotrexate (MTX). Skin rash involved regions of the face, lower abdomen, back, buttocks and both upper thighs. Biopsy of the skin rash demonstrated superficial perivascular lymphocytic infiltrate and was consistent with a drug reaction. Peripheral blood count revealed pancytopenia and a bone marrow biopsy was consistent with aplasia. Blood counts returned to normal 6 days after institution of granulocyte colony stimulating factor therapy. In the absence of mucositis or diarrhea, severe dermatologic toxicity following a single low dose of the drug suggests an 'allergic' or acute hypersensitivity reaction to MTX in this patient. Development of an extensive skin rash following a single dose of MTX may be an early warning sign for life-threatening bone marrow aplasia. PMID- 7538829 TI - Consequences of angiogenesis for tumor progression, metastasis and cancer therapy. AB - The growth of solid tumors to a clinically relevant size is dependent upon an adequate blood supply. This is achieved by the process of tumor stroma generation where the formation of new capillaries is a central event. Progressive recruitment of blood vessels to the tumor site and reciprocal support of tumor expansion by the resulting neovasculature are thought to result in a self perpetuating loop helping to drive the growth of solid tumors. The development of new vasculature also allows an 'evacuation route' for metastatically-competent tumor cells, enabling them to depart from the primary site and colonize initially unaffected organs. Several molecular and cellular mechanisms have been identified by which tumor parenchyma may exert its angiogenic effect on host endothelial cells. As a result of this paracrine influence, tumor-associated endothelial cells acquire an 'immature' phenotype manifested by rapid proliferation, migration, release of proteases and expression of cytokines, endothelial-specific tyrosine kinases (e.g. flk-1, tek and others) as well as numerous other molecular alterations. Consequently a network of structurally and functionally aberrant blood vessels is formed within the tumor mass. There is also evidence that endothelial cells themselves, and likewise other stromal cells, may act reciprocally to alter the behavior of adjacent tumor cells in a paracrine or cell contact mediated fashion. For example, production of interleukin 6(IL-6) by endothelial cells may have a differential effect on human melanoma cells expressing different degrees of aggressiveness. In this manner endothelial derived cytokines could conceivably contribute to tumor progression by suppressing the growth of the less aggressive tumor cells and promoting dominance of their malignant counterparts in 'strategic' perivascular zones. Distinct biological features expressed by tumor-associated vasculature may serve as potential prognostic markers of disease progression as well as novel targets for therapeutic intervention. PMID- 7538830 TI - Angina pectoris following cisplatin, etoposide, and bleomycin in a patient with advanced testicular cancer. AB - OBJECTIVE: To report a case of angina pectoris associated with chemotherapy for testicular cancer. CASE SUMMARY: An HIV-infected patient with massive retroperitoneal metastases of a mixed embryonal and undifferentiated teratocarcinoma was treated with cisplatin, etoposide, and bleomycin. While the patient was receiving the second course of chemotherapy, he developed several episodes of angina pectoris that responded to nitroglycerin. Coronary angiography excluded structural abnormalities in the coronary arteries. The patient was treated prophylactically with nifedipine during the 2 following courses of chemotherapy with no new ischemic events. DISCUSSION: Coronary vasospasm seemed to be responsible for the angina in this patient. Several pathogenetic mechanisms that could explain these vascular events are discussed, including the possible role of bulky metastatic disease. CONCLUSIONS: The combination of cisplatin, etoposide, and bleomycin for testicular cancer, perhaps associated with bulky metastatic disease, can induce vasospastic phenomena that might be life threatening. PMID- 7538831 TI - Epithelial lung cell marker: current tools for cell typing. AB - This review discusses current immunohistochemical and lectin histochemical approaches to identify and to distinguish the different epithelial cell populations of the pulmonary tissue. Special emphasis is given to the characterization of pulmonary alveolar and bronchial epithelial cells and mesothelial cells. Structural proteins, membrane molecules and secretory products of the alveolar epithelium, which have already been characterized and which may be useful for monitoring the developmental or pathological processes, are listed and briefly described. PMID- 7538834 TI - The use of microwave irradiation for immunohistochemistry: a new methodological proposal. AB - We developed a rapid immunohistochemical method using a microwave oven in formalin-fixed, paraffin-embedded sections from normal and pathological tissues. The strongest immunoreactivity was obtained for actin, Ca 125, CEA, pan cytokeratin, chromogranin A, EMA, GFAP, thyroglobulin, kappa and lambda chains. In control tissues, processed with conventional immunocytochemical procedure, the reactivity was found to be qualitatively and quantitatively similar. Dako EPOS kits were also assayed with good staining intensity, shortening the original technique to 16 min. Our microwave immunohistochemical method is simple, rapid and it may be recommended for use in routine laboratories. PMID- 7538832 TI - Structural organization of enteric nervous system in human colon. AB - The organization of the Enteric Nervous System (ENS) was studied in the human colon. Fragments of the whole colonic wall were either routinely processed or Zinc-Iodide Osmium impregnated. Single-layer preparations were also obtained from some of the Zinc-Iodide Osmium-impregnated specimens. The results showed some differences in the organization of human colonic ENS from that of other mammals. In fact, the human submucous plexus was made up of three interconnected ganglionated networks arranged along three different planes. With respect to the myenteric plexus, its ganglia were large sized and irregularly shaped. Moreover, during the microdissection of the colonic wall, we found the absence of a cleavage plane between the circular and longitudinal muscle layers; on the other hand the cleavage plane between mucosa and submucosa was not immediately below the muscularis mucosae, but slightly deeper, since the innermost part of the submucosa remained adhering to overlying layers. PMID- 7538833 TI - Vessel wall-dependent metabolic pathways of the adhesive proteins, von-Willebrand factor and vitronectin. AB - The integrity of the vessel wall under quiescent conditions as well as its appropriate responsiveness under conditions of stimulation, inflammation or vascular injury is controlled by a number of adhesive interactions involving distinct cellular receptor systems and various multifunctional adhesive ligands. While a number of these extracellular matrix components of the vessel wall are endogenously produced, secreted and deposited, exogenous adhesion proteins may become translocated from the intra- to the extravascular space by virtue of endothelial cell-specific transport systems. Two prominent examples for each metabolic pathway are discussed. Endothelial cell-specific biosynthesis and secretion as well as deposition of multimeric von-Willebrand-factor within intracellular granules (Weibel-Palade bodies) relates to the first possibility of processing, whereas binding of reactive forms of circulating vitronectin to diverse cellular receptors with subsequent extravasation and deposition into the extracellular matrix appears to be characteristic for the second case. In this review the known features of the metabolic routes of both adhesion proteins are discussed and set in perspective to their functional properties. Their localized mode of action in the vessel wall appears to be crucial for balanced haemostasis and immune systems, two major defence mechanisms of the organism. PMID- 7538835 TI - Lindane and methylparathion residue degradation in carnation byproduct (Dianthus caryophyllus, L.) under different environmental conditions. PMID- 7538836 TI - Acute toxicities of selected insecticides to the aquatic arthropod Artemia salina. PMID- 7538837 TI - High-calorie, rice-derived, short-chain, glucose polymer-based oral rehydration solution in acute watery diarrhea. AB - In this study, we have compared the effects of the World Health Organization oral rehydration solution (WHO ORS) and an ORS containing short polymers of glucose (Amylyte ORS) at a high caloric density (five times) and comparable osmolality, on stool output, duration of diarrhea, weight gain and fluid and electrolyte balance, in randomized, open-labeled, controlled clinical trials in five centers. A total of 198 male children (4 months to 10 years) with acute diarrhea ( <72 h after onset) were assigned by random allocation to either WHO ORS or Amylyte ORS at five centers in Asia. Children were stratified according to grade of dehydration (mild, moderate or severe) and the initial purging rates during the first 6 h (low ( < 2 ml/kg/h), moderate (2-5 ml/kg/h) and high ( > 5 ml/kg/h) purgers). The clinical characteristics of the children in the two treatment groups were comparable. Amylyte ORS reduced stool volumes significantly in children with severe dehydration (285.4 +/- 74.2 versus 75.5 +/- 20.0 ml/kg; p < 0.05) and in children with a high initial purging rate (200.3 +/- 42.8 versus 130.5 +/- 9.1 ml/kg; p < 0.05). This was accompanied by a significant (276.4 +/- 14.6 versus 227.6 +/- 11.8 ml/kg; p < 0.01) reduction in ORS requirements in the Amylyte ORS treated group, the effect being greatest in children with severe dehydration (491.5 +/- 108.5 versus 155.7 +/- 27.3 ml/kg; p < 0.01) or high initial purging rates (394.2 +/- 66.2 versus 316.8 +/- 34.8 ml/kg; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538838 TI - [Possibilities and limits of a new color technique: ultrasound angiography- results of the "Heidelberg Round Table Discussion"]. AB - Sonographic diagnostics has been essentially enlarged by blood flow measurements with the color Doppler technique. Nevertheless, the method has certain limitations, especially when employed to visualize extremely slow blood flow velocities, which occur e.g. in malignant tumors due to neovascularization. Recently, a new technique, the so-called angio-color procedure, has been presented, which is supposed to overcome the above-mentioned limitations. The way of coding the signal differs considerably from that in conventional color Doppler: The amplitude is color coded, not the frequency deviations. Results and first experiences with the new procedure in various domains were discussed at a 'Heidelberg Round Table' and are summarized in the present article. In the obstetrics/gynecology there are clear advantages in the representation of blood flow in the placenta. In eutrophic fetuses a blood flow over the entire placental breadth was detected, in dystrophic fetuses, however, only on the margins of the placenta. Conventional color Doppler could not visualize blood flow in the placenta at all. Blood flow assessment in the fetal organs also provided different results with the two procedures: the angio-color method showed color pixels even in the peripheral areas. In 8 malignant breast tumors the same observation was made, the angio-color procedure seemed to visualize more color pixels. In 4 of 6 benign lesions blood flow could be measured with the traditional color Doppler, but in 5 of 6 patients with the angio-color Doppler. This was also observed in ovarian tumors. Angio-color representation of renal transplants with normal function resulted in blood flow information reaching deep into the renal capsule, whereas in cases of renal transplants with impaired function a clearly reduced perfusion was visualized. These differences were not so evident with the conventional Doppler method. In lymph node diagnosis, too, marked differences between malignant and reactive lymph nodes could be visualized. In the diagnosis of arteriosclerotic plaques, blood flow in the residual lumen could also be detected more exactly with the angio-color technique. Color representation of liver tumors also showed a higher color signal density with ultrasound angiography. In summary, ultrasound angiography can provide additional information in the color representation of vessels with slow flow velocities. However, an obvious problem is the unability to quantify detected low blood flow. The new procedure will not replace but usefully complete the established sonographic techniques. PMID- 7538839 TI - Nucleic acid affinity chromatography: preparation and characterization of double stranded RNA agarose. AB - An improved method for the preparation of a double-stranded RNA-coupled resin is described. Periodate-oxidized double-stranded RNA, in the form of either purified reovirus double-stranded RNA or poly(rl). poly(rC), was covalently attached to agarose-adipic acid hydrazide. The resulting affinity resin had increased specificity for known double-stranded RNA binding proteins, as compared to a similar commercially available resin. The application of this resin in the purification and characterization of double-stranded RNA binding proteins is described. PMID- 7538840 TI - Modified immunoassays for polyether toxins: implications of biological matrixes, metabolic states, and epitope recognition. AB - Polyether marine toxins are responsible for the seafood intoxication phenomena known as neurotoxic shellfish poisoning (due to brevetoxins), ciguatera (due to ciguatoxin), and diarrheic shellfish poisoning (due to okadaic acid). Using traditional techniques of hapten (pure toxin) conjugation to protein to create complete antigen, animal immunization and antibody isolation, and specific antibody subpopulation purification, discriminating antibodies have been isolated that detect brevetoxins and ciguatoxin, but not okadaic acid, in a dose-dependent fashion. Using microorganic chemistry and purified toxins, a unique set of tools has been created for the study of polyether ladder toxin accumulation; depuration; and specific site localization in tissues, food sources, and clinical samples. Developed test protocols can detect toxin in dinoflagellate cells, in extracts from food sources, in seawater and culture media, and in human serum samples. Enzyme-linked immunosorbent assay protocols developed for eventual collaborative testing have been successful in limited applications within the laboratory (correlation coefficient of 0.92 excluding 2 outliers), and alternative formats are being developed to optimize the basic test for use in research laboratories, regulatory laboratories, and field inspections. PMID- 7538841 TI - Radioimmunoassay for PbTx-2-type brevetoxins: epitope specificity of two anti PbTx sera. AB - Antiserum against PbTx-2-type brevetoxins was produced by immunizing rabbits with a PbTx-2-bovine serum albumin (BSA) conjugate. This serum had a higher affinity, but lower titer, than our current goat serum. Using 4 natural brevetoxins and 6 synthetic derivatives as competitors in our brevetoxin radioimmunoassay, we determined the epitope specificity of both sera. Modification of the backbone structure at C-42 on the K-ring had little or no effect on the antigen-binding capability of either serum. Reduction of the double bond between C-2 and C-3 on the A-ring by reduction of the lactone decreased binding 500 to 750-fold. Epoxidation of the double bond between C-27 and C-28 on the H-ring did not affect binding, which suggested that the goat serum is specific for the A-ring region of the brevetoxin backbone. In contrast, modifying the A-ring had no effect on rabbit serum binding. However, epoxidation of the H-ring decreased binding 5 to 20-fold, which suggested that the rabbit antiserum is specific for the H-ring region of the molecule. These results suggest that assays utilizing only one antibody may not adequately detect toxin metabolites if molecules are altered in the critical region of antibody recognition. PMID- 7538842 TI - [Surface of cytochrome P-450 2B4: structure and function]. AB - The review is devoted to the identification and structure of one of the cytochromes P450s-cytochrome P-450 2B4 derived from the rabbit liver endoplasmic reticulum. A critical review is made of the data on this enzyme membrane topology, its active site's structure and localization of its membrane and water exposed regions. The paper is based on the data available in the literature and the authors' own findings. Various experimental and calculating methods used to identify the topography of cytochrome P450 are covered in the paper. PMID- 7538843 TI - Preferential binding of insulin-like growth factors to a binding protein rather than to receptors on chicken hepatoma cell (LMH) membranes. AB - [125I]IGF-I binding to chicken hepatoma cell (LMH) membranes was displaced by unlabelled IGF-I or IGF-II, but not by insulin. Cross-linking revealed specific binding sites of 128 and 28-31 kDa, which following solubilization could be separated by wheat germ agglutinin (WGA) chromatography. [125I]IGF-I binding to the WGA eluate (128 kDa) could be displaced by insulin although with a 30-fold lower potency than IGF-I. Binding to the WGA flow-through (28-31 kDa) was not inhibited by insulin. This suggested that IGF binding to LMH was due mainly to membrane bound IGFBP rather than to type 1 IGF receptors. A reverse proportion was observed in normal chicken liver. A predominant 28 kDa IGFBP was synthesized and secreted by LMH cells, together with an unusual 60 kDa IGF binding entity which only bound [125I]IGF-II (with weak affinity). This process was not affected by the presence or absence of glucose, dexamethasone, glucagon, insulin or IGF-I. PMID- 7538844 TI - Biochemical analysis of prostate specific antigen-proteolyzed insulin-like growth factor binding protein-3. AB - Prostate specific antigen (PSA) has been shown to proteolyze IGFBP-3. However, the cleavage sites and mechanism of proteolysis are unknown. In this study, we proteolyzed recombinant human IGFBP-3 with PSA bound to a solid phase support. The reaction mixture was separated by centrifugation, with PSA remaining in the solid phase and the proteolyzed IGFBP-3 in the aqueous phase. The IGFBP-3 fragments were functionally analyzed by affinity labeling and Western ligand blotting (WLB). Further biochemical analyses were provided by silver staining of total protein and Western immunoblotting (WIB) of immunoreactive fragments with an IGFBP-3 specific antiserum (alpha-BP-3 g1). N-terminal sequence analysis was performed on filter-immobilized IGFBP-3 fragments, following size separation by SDS-polyacrylamide electrophoresis. PSA proteolyzed IGFBP-3 into at least 7 fragments (M(r) of 26 kDa to 13 kDa) as identified by silver staining and WIB. At least 3 fragments were visible by affinity labeling with radiolabeled IGF-I or IGF-II and 4 were weakly visible by WLB. These data indicate that some IGFBP-3 fragments retain their ability to bind IGF. N-terminal sequence analysis revealed at least 5 different proteolytic recognition sites for PSA in IGFBP-3. Three of the 5 sites were consistent with a 'kallikrein-like' enzymatic activity and 2 sites were consistent with a 'chymotryptic-like' enzymatic activity. The chymotryptic activity of PSA was further confirmed by the ability of alpha-1 antichymotrypsin and chymostatin to block PSA cleavage of radiolabeled IGFBP 3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538845 TI - Hydrogen exchange in BPTI variants that do not share a common disulfide bond. AB - Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14 38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond. PMID- 7538846 TI - Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. AB - The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed. PMID- 7538848 TI - Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting. AB - Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851 865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7-16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10-fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6- to > 500-fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10 fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538847 TI - Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. AB - Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472 1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL 6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented. PMID- 7538850 TI - Molecular studies on phospholipid-binding sites and cryptic epitopes appearing on beta 2-glycoprotein I structure recognized by anticardiolipin antibodies. PMID- 7538849 TI - NMR structure determination of tick anticoagulant peptide (TAP). AB - Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa. PMID- 7538852 TI - Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase. AB - HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme. PMID- 7538851 TI - Enhancement of neutrophil function by in vivo filgrastim treatment for prophylaxis of sepsis in surgical intensive care patients. AB - PURPOSE: To determine the kinetics of leukocyte counts and of oxygen radical production of neutrophils from postoperative/posttraumatic patients with or without infusion of filgrastim (recombinant human granulocyte colony-stimulating factor, rhG-CSF) as prophylaxis against sepsis. METHODS: Twenty postoperative/posttraumatic patients with a Therapeutic Intervention Scoring System (TISS) score greater than 30 were included in this study. In the 10 patients of the study group, filgrastim (1 microgram/kg/d) was infused continuously within the first 3 days and tapered to 0.5 microgram/kg/d on the following 4 days or until discharge from the surgical intensive care unit. Ten patients without administration of filgrastim served as controls. Oxygen radical production of isolated neutrophils of these patients was tested by N-formyl methionyl-leucyl-phenylalanine (FMLP)- and zymosan-induced chemiluminescence from serial blood samples, taken until the 16th postoperative day. RESULTS: Compared with the first postoperative day, in vitro FMLP-induced neutrophil chemiluminescence was significantly increased during the following 4 postoperative days in the patients with filgrastim infusion; however, only during the first 2 postoperative days in the control group. The increase in the FMLP induced neutrophil chemiluminescence was significantly greater (P < .05) in the study group than in the control group on the third and on the fourth postoperative day. Tapering of filgrastim by 0.5 microgram/kg/d in the study group resulted in a reduction of FMLP-induced neutrophil oxygen radical production within 48 hours. In contrast, zymosan-induced neutrophil chemiluminescence was not measurably affected in both groups. Leukocyte count of the study group significantly (P < .05) exceeded the leukocyte count of the control group from the third up to the 10th postoperative day. None of the patients treated with filgrastim developed sepsis; however, three patients within the control group did. CONCLUSIONS: Prolonged enhancement of neutrophil count and function induced by rhG-CSF may be useful in the prophylaxis of sepsis in posttraumatic/postoperative patients at high risk of sepsis. PMID- 7538853 TI - Effects of changes in rat brain glucose on serotonergic and noradrenergic neurons. AB - Microdialysis was used in the freely moving rat to measure the effects of graded changes in brain glucose on the serotonergic and noradrenergic projections to the hippocampus. The concentration of glucose in the dialysate was monitored using an enzyme-based assay. A systemic injection of insulin caused a steep decline in glucose level which was restored to the control level by oral administration of glucose solution. The changes in 5-hydroxytryptamine (5-HT) and noradrenaline were a mirror image of the glucose changes: they rose after insulin injection and returned to control during glucose administration. A delayed increase was shown by 5-hydroxyindoleacetic acid (5-HIAA) which did not return to baseline on glucose administration. The metabolite dihydroxyphenylacetic acid (DOPAC) decreased after insulin administration and increased above control during glucose administration. While the responses of 5-HT, noradrenaline and 5-HIAA to hypoglycaemia resemble those to mild stress, the changes in DOPAC are the reverse of those produced by stress. PMID- 7538854 TI - Ischaemia-induced long-term hyperexcitability in rat neocortex. AB - The long-term structural and functional consequences of transient forebrain ischaemia were studied with morphological, immunohistochemical and in vitro electrophysiological techniques in the primary somatosensory cortex of Wistar rats. After survival times of 10-17 months postischaemia, neocortical slices obtained from ischaemic animals were characterized by a pronounced neuronal hyperexcitability in comparison with untreated age-matched controls. Extra- and intracellular recordings in supragranular layers revealed all-or-none long latency recurrent responses to orthodromic synaptic stimulation of the afferent pathway. These responses were characterized by durations up to 1.7 s, by multiple components and by repetitive synaptic burst discharges. The reversible blockade of this late activity by DL-amino-phosphonovaleric acid (APV) suggested that this activity was mediated by N-methyl-D-aspartate (NMDA) receptors. The peak conductance of inhibitory postsynaptic potentials was significantly smaller in neurons recorded in neocortical slices obtained from ischaemic animals than those from the controls. However, the average number of parvalbumin (PV)-labelled neurons per mm3, indicative of a subpopulation of GABAergic interneurons, and the average number and length of dendritic processes arising from PV-containing cells was not significantly different between ischaemic and control cortex. The prominent dysfunction of the inhibitory system in ischaemic animals occurred without obvious structural alterations in PV-labelled cells, indicating that this subpopulation of GABAergic interneurons is not principally affected by ischaemia. Our data suggest a long-term down-regulation of inhibitory function and a concurrent NMDA receptor-mediated hyperexcitability in ischaemic neocortex. These alterations may result from structural and/or functional properties of inhibitory non-PV-positive neurons or permanent functional modifications on the subcellular molecular level, i.e. alterations in the phosphorylation status of GABA and/or NMDA receptors. The net result of these long-term changes is an imbalance between the excitatory and inhibitory systems in the ischaemic cortex with the subsequent expression and manifestation of intracortical hyperexcitability. PMID- 7538855 TI - Radiation-induced reductions in macrophage recruitment have only slight effects on myelin degeneration in sectioned peripheral nerves of mice. AB - Macrophage recruitment into the distal nerve stump of the cut or crushed sciatic or saphenous nerves of C57BL/6J mice was reduced by prior whole body irradiation. This procedure was successful in keeping the numbers of cells stained with the mouse macrophage-specific antibody F4/80 to the levels found in unsectioned nerves. Quantitative image analysis of immunostained sections showed that the rate of loss of myelin basic protein was identical in nerves from irradiated and unirradiated mice up to 5 days but thereafter was slower in macrophage-deprived nerves. Similar analysis of semithin sections stained with toluidine blue detected more undegenerated myelin in the nerves from irradiated mice 10 days after operation. Quantitative counts made from electron micrographs of the sectioned nerves at 7 days also showed slightly less extensive myelin breakdown in the nerves from irradiated mice. Complete removal of myelin from some Schwann cells can occur without macrophages, but macrophages accelerate the removal of myelin in the later stages of Wallerian degeneration. It is concluded that there are two phases to the breakdown of myelin in peripheral nerves undergoing Wallerian degeneration: an initial stage entirely dependent on the activity of Schwann cells and a later stage dependent on both Schwann cells and the presence of macrophages. PMID- 7538856 TI - The effects of AMPA-induced lesions of the septo-hippocampal cholinergic projection on aversive conditioning to explicit and contextual cues and spatial learning in the water maze. AB - The environmental context of an animal both subsumes and is associated with the explicit cues that guide its behavioural responses. Recent work in this laboratory suggests that learning about the relationship between the cues which comprise a context depends on the hippocampus. In the present study the role of the cholinergic input to the hippocampus in contextual learning was assessed in rats using a conditioned stimulus/context conditioning paradigm and spatial learning in the Morris water maze. In the former, a place preference apparatus provided the context. The subject was confined in the black chamber and a 'clicker' conditioned stimulus was presented five times in a 20 min period. A trace interval of 5 or 30 s, depending on the group, was interposed between the end of the clicker and a footshock. Theory predicts that animals in the 5 s condition will learn more about the clicker as a predictor of shock and become strongly conditioned, while those in the 30 s condition learn relatively more about the context. Conditioning to the clicker (conditioned stimulus) was measured in a separate lick suppression chamber--presentation of the clicker suppresses drinking, and contextual learning was determined by recording the time spent on the black side of the place preference apparatus when both the black and a familiar white chamber were accessible. Lesions of the medial septum/diagonal band induced by RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) enhanced contextual learning in this paradigm but disrupted conditioned stimulus conditioning in the 30 s condition. Acquisition of the Morris water maze was largely unimpaired. The results are suggested to reflect a shift towards the use of hippocampal-dependent contextual learning strategies in lesioned animals. PMID- 7538859 TI - [Rapid diagnosis of bacteremia]. PMID- 7538857 TI - Gelatin sponge-supported histoculture of human nasal mucosa. AB - Considerable progress has recently been made in the understanding of airway inflammation by cell culture assays and in vivo provocation studies. Inasmuch as ethical considerations limit experimental work in humans, physiologically relevant in vitro models are required to better understand cellular and molecular tissue interactions in human nasal mucosa. Here we describe a human nasal mucosa culture model utilizing a simple gelatin sponge-supported histoculture system at the air-liquid interface. Viable mucosa was preserved for at least 48 h, as shown by morphology and immunohistochemical staining with Ki-67 as marker for proliferation. Pro-inflammatory mediators (kinins, histamine, thromboxane B2, prostaglandin F2 alpha, and substance P) are detectable in serum-containing as well as serum-free culture medium. Incubation with 10(-8) M substance P increases the number of degranulated mast cells after 48 h by 26% (P < 0.01). In this model, biochemical responses can be correlated with histologic alterations of the target tissue. Inflammatory parameters can be examined and compared in various patient groups and different stimulators/inhibitors. This culture method provides a valuable research tool for analyzing all compartments present in nasal mucosa under physiologically relevant conditions, and for studying complex interactions and responses of mucosal cell populations in their natural tissue environment. PMID- 7538858 TI - [Transport medium for pathogenic pseudomonads]. AB - A transporting medium was designed for the transportation and preservation for 5 to 7 days of material infected with agents of malleus and melioidosis. Foreign contaminating microorganisms gradually die in this medium, whereas solitary cells of malleus and melioidosis agents (100 bacterial cells per 0.1 ml) survive. PMID- 7538860 TI - [Differentiation between glanders and melioidosis]. PMID- 7538861 TI - [Micromethod of measurement of blood alpha 1-proteinase inhibitor and alpha 2 macroglobulin]. AB - A micromethod for simultaneous measurements of alpha 1-proteinase inhibitor and alpha 2-macroglobulin in human blood serum and plasma is suggested. The method is time-and reagent saving, accurate, and easily reproducible. It can be used to investigate other biological material and fluids, such as bronchial and vaginal lavage fluid, tissue homogenates, etc. PMID- 7538864 TI - Critical periods of early development created by the coordinate modulation of ion channel properties. AB - The development of ion channel properties in excitable cells begins in the very early embryo and continues throughout differentiation. The pattern of ion channel development in a given cell type is not a simple linear progression to the mature state, but rather is a complex sequence of modulatory events that create windows of time during which excitability is qualitatively different from that in the mature cell. These windows are likely candidates for critical periods when electrical activity influences later development. PMID- 7538862 TI - Report from a Nordic workshop on CD34+ cell analysis: technical recommendations for progenitor cell enumeration in leukapheresis from multiple myeloma patients. Nordic Myeloma Study Group Laboratories. AB - Recently, high-dose chemo- and radiotherapy for newly diagnosed young patients with multiple myeloma has been established in member centers of the Nordic Myeloma Study Group (NMSG). The treatment includes supportive use of autologous hematopoietic blood progenitors harvested by leukapheresis. Safe and adequate hematopoietic support with minimal toxicity requires optimization of the number and quality of harvested progenitors. We have therefore established a workshop consisting of the 11 laboratories within the NMSG with the aim of developing recommendations for enumeration of CD34-positive cells. In this first workshop report, we discuss technical variables affecting the enumeration of progenitors harvested by leukapheresis and make specific recommendations. The products are analyzed within 4 h following erythrocyte lysis using the Ortho lysing solution for 8-10 minutes. A sample containing 0.5-1.0 x 10(6) nucleated cells is incubated with the test or control antibody (anti-CD34 = HPCA-2PE and Ms IgG1PE from Becton Dickinson) at dilutions of 1:10 to 1:40 in a final volume of 1 ml. The samples are incubated 15 minutes at ambient temperature, washed two to three times, and fixed with 1% paraformaldehyde (150 microliters/pellet) for a minimum of 10 minutes. Flow cytometric analysis is performed on 50,000 cellular events with debris eliminated. The estimation of CD34-positive cells can be performed on an FL2-side-scatter plot after marking of a positive population containing > 50 events. Using quadrant statistics, this population can be identified in the upper left quadrant, among cells with the side-scatter profile of small lymphocytic cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538863 TI - Voltage-gated ion currents in embryogenesis. AB - Excitability is not an exclusive property of differentiated cells, as egg cells already express a surprising variety of species-specific combinations of voltage gated ion channels. After fertilization, ion currents are generally down regulated and reappear at about the time of the last mitosis of neuronal precursor cells. When ganglia have formed, Na+ and Ca2+ currents are generally present. The first voltage-gated ion currents in differentiating neurons are unambiguously identified by kinetic and pharmacological criteria according to the channel classifications defined for ion currents in more mature cells. Specialization of different neuronal subtypes with respect to the expression of characteristic patterns of ion channels occurs before neurites grow out and contact their targets. During maturation of committed neurons, the densities of ion currents change. Whereas low voltage-activated Ca2+ current are only transiently expressed in several types of cells Na+, high voltage activated Ca2+, and K+ currents are usually up-regulated until stable final values are reached in adult cells. The most challenging questions to be answered by future research concern the molecular mechanisms regulating the expression of the specific patterns of ion channels characteristic of different subtypes of neurons. PMID- 7538865 TI - Neuronal excitability is induced by cell-cell interactions during early embryogenesis. AB - Neural induction in vertebrate embryos has been mainly studied in multicellular systems composed of pieces excised from ectoderm and mesoderm of amphibian embryos. A simple model system for neural induction has been established from the cleavage-arrested 8-cell ascidian embryo by pairing a single ectodermal with a single vegetal blastomere, as a competent and a inducer cell, respectively. This induction, mediated by specific cell contact, can be mimicked by treating with serine protease. In this simple model system, the turnover of subtypes of Na+ channels occurs, dependent on new transcriptional activities after neural induction. A cloned ascidian Na+ channel gene, TuNa I, is especially useful for analysis of the neural induction on the gene regulation level, and can serve as a specific marker for neuronal differentiation in the ascidian tadpole larva. In contrast to Na+ channels, transcription of inward rectifier K+ channels is suppressed immediately after neural induction. Since the inward rectifier is one of several genes expressed early in epidermal cells, we conclude that the inductive signal not only enhances the transcription of neural characters, but also suppresses that of non-neural characters. PMID- 7538866 TI - Gap junctions in the developing nervous system. AB - Cell-cell interactions are important in the development of the nervous system. Gap junctions (GJs) form direct intercellular channels that permit diffusion of ions and small molecules and thus cells linked by GJs can influence each other's properties or behavior either through transmission of electrical signals or through transfer of signaling molecules. In the developing nervous system, widespread GJ communication occurs at the time of neural induction, but in the adult nervous system it is much more restricted. In addition, certain events in neural development appear to involve the formation of transient junctional connections. This review examines briefly four aspects of neural development in which GJs may be involved, namely, neurulation, regional differentiation, migration, and axon guidance. PMID- 7538867 TI - Developmental regulation of ion channels and receptors on glial cells. AB - The electrophysiological properties of glial precursor cells and oligodendrocytes were studied in an intact tissue preparation, the corpus callosum slice, with the patch-clamp technique. The pattern of voltage-gated currents exhibited by different cell types in this white matter tract was compared with that from cultured cells of the O-2A lineage. Precursor cells from the in vitro and the in situ preparation were strikingly similar in most aspects. In contrast, oligodendrocytes in the intact tissue differed from their culture correlates and displayed the ability to redistribute excess K+. To study glial transmitter receptors in neuronglial interactions during development, Bergmann glial cells were investigated in a slice preparation of the cerebellum. PMID- 7538869 TI - Modulation of antibody affinity by synthetic modifications of the most exposed pyranose residue of a trisaccharide epitope. AB - When the Salmonella trisaccharide epitope, methyl 3-O-(3,6-dideoxy-alpha-D-xylo hexopyranosyl)-2-O-(alpha-D-galactopyra nos yl)- alpha-D-mannopyranoside 12 is bound by a monoclonal antibody Se155.4, the 3,6-dideoxy-alpha-D-hexose is completely buried, while the galactopyranosyl mannopyranosyl units lay across the protein surface. Crystallography of an antibody complexed with 12 also shows that the galactose residue is the most exposed saccharide. A simplified strategy to synthesize 12 and analogues modified at the galactose residue is described. Monosaccharide building blocks containing benzyl ether and ester protecting groups were used for efficient assembly of trisaccharides that can be deprotected by a single hydrogenolysis step, or occasionally preceded by a transesterification stage. Glycosylation of methyl 2-O-benzoyl-4,6-di-O-benzyl alpha-D- mannopyranoside 4 by 2,4-di-O-benzyl-3,6-dideoxy-D-xylo-hexopyranosyl chloride 8 affords after transesterification the disaccharide acceptor 10. This disaccharide serves as a universal acceptor for glycosylation by glycosyl donors that lead, following facile deprotection, to the alpha- and beta-D-galacto, alpha and beta-D-gluco, and 2-amino-2-deoxy-alpha-D-galacto trisaccharides 12, 14, 17, 18 and 21. Only a small change in binding energy delta (delta G) occurs when the alpha-D-galactopyranosyl residue of 12 is replaced by either an alpha-D glucopyranosyl 17 or a 2-amino-2-deoxy alpha-D-galactopyranosyl unit 21. Whereas binding of the beta-D-glucopyranose congener 18 was tolerated by the antibody, the beta-D-galactopyranose analogue 14, showed a 250 fold loss of affinity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538868 TI - First synthesis of the 3'-sulfated Lewis(a) pentasaccharide, the most potent human E-selectin ligand so far. AB - Tri- and pentasaccharides of Lewis(a)-type, sulfated at position 3 of the outer galactose, have been prepared using the new 4-methoxybenzyl glycoside of N acetylglucosamine 5 as starting material. The synthesis of the pentasaccharide 2 was achieved through a beta-stereoselective coupling of an alpha trichloroacetimidate activated form of the N-acetamido protected trisaccharide 18 on to a 3',4'-unprotected lactose derivative. PMID- 7538870 TI - Systematic synthesis of N-methyl-1-deoxynojirimycin-containing, Le(x), Le(a), sialyl-Le(x) and sialyl-Le(a) epitopes recognized by selectins. AB - A systematic synthesis of the N-methyl-1-deoxynojirimycin-containing oligosaccharides related to the Lewis x, Lewis a, sialyl-Lewis x and sialyl-Lewis a antigens has been achieved. The couplings of the suitably protected 1 deoxynojirimycin derivative 10 with methyl-1-thioglycosides (glycosyl donors) of L-fucose (11), D-galactose (15) and alpha-sialyl-(2-->3)-D-galactose (27) were carried out by using dimethyl(methylthio)sulfonium triflate (DMTST) or N iodosuccinimide/trifluoromethanesulfonic acid (NIS/TfOH) as the glycosyl promoter. The resulting di- and tri-saccharides were each converted, by further cross glycosylations with 11, 15 or 27, to the desired tri- and tetra-saccharides 3-6 that inhibit the recognition between sialyl-Lewis x and selectins, a family of leukocyte cell adhesion molecules. PMID- 7538871 TI - Neurokinin actions on substantia gelatinosa neurones in an adult longitudinal spinal cord preparation. AB - We have used an adult longitudinal spinal cord preparation to study the effects of a range of selective neurokinin analogues on single neurones located exclusively within the substantia gelatinosa. Since the preparation retained attached dorsal roots it was possible synaptically to activate the substantia gelatinosa neurones by electrical stimulation of their afferent fibres, thus providing a means of studying directly the role of neurokinins in mechanisms of primary afferent transmission. The actions of three agonists selective for the three NK receptor subtypes (NK1, GR73632; NK2, GR64349; NK3, senktide), and a highly selective antagonist at NK1 receptors (GR82334) were investigated. Experiments were performed on a total of 274 substantia gelatinosa neurones, estimates of conduction velocity for evoked responses suggested that the majority of these neurones were innervated by unmyelinated afferents. A large proportion responded to iontophoretically applied neurokinin agonists. The majority responded to NK1, fewer responded to NK2; some, although not all, of the neurones tested responded to both NK1 and NK2 agonists. In most cases the responses were excitatory, although inhibitory effects were observed in some neurones. None of the neurones tested responded to NK3 agonist. Excitatory and inhibitory actions could be demonstrated following abolition of synaptic transmission by removal of calcium, suggesting direct mechanisms for both effects. The antagonist alone failed to modify either spontaneous firing or firing in response to afferent stimulation in any of the neurones studied, even though the doses used were shown to be effective in selectively antagonising responses to the NK1 agonist, suggesting that neither relied on the endogenous release of neurokinins. PMID- 7538874 TI - Magnetic resonance imaging of acute trauma of the cervical spine: spectrum of findings. AB - This pictorial essay illustrates the magnetic resonance imaging (MRI) spectrum of acute injury to the cervical spine. The authors discuss the appearance of the traumatized cord, including intramedullary hemorrhage, and the causes of spinal cord compression, such as disk herniation, epidural hematoma, fracture, dislocation and underlying spinal stenosis. The ability of MRI to directly reveal the severity of cord injury and, at the same time, to indicate the cause of cord compression is particularly useful in the management of incomplete injury, for which surgical intervention may prevent further deterioration. PMID- 7538872 TI - Brain RNA synthesis, long-term potentiation and depression at the perforant path granule cell synapse in the guinea pig. AB - The effects of long-term changes in synaptic efficacy at the perforant path granule cell synapse on the de-novo synthesis of ribonucleic acid (RNA) were investigated in hippocampal and cortical areas in anaesthetized Guinea pig preparations. Two experiments were run with stimulating and recording microelectrodes aimed at the perforant bundle and dentate gyrus hilus on both sides. In Experiment 1, a low-frequency (LFS; 0.02 Hz, 3 h) or high-frequency stimulation (HFS; 400 Hz, 250 ms) was delivered to the left perforant bundle with the contralateral side as control. In Experiment 2, animals received LFS or HFS trains with implanted nonstimulated animals used as controls. The latency and amplitude of the field postsynaptic potentials (FPSP) and population spike (POPS) were monitored under baseline conditions and following stimulation over a 3 h period. In addition, two HFS groups were tested with few (HFS-F: every 15 min) or several test stimuli (HFS-S: every 3 min). In both experiments RNA synthesis was determined by measuring the amount of 3H-5,6-uridine incorporated into the RNA 3 h after bilateral intraventricular injection. In Exp. 1 the LFS group showed a higher synthesis of RNA than both HFS groups. The rate of RNA synthesis did not differ between the stimulated and nonstimulated side. In Exp. 2 the HFS groups showed a decreased RNA synthesis. In the HFS-F group, it pertained to the dorsal dentate area, CA1, subiculum, cingulate and dorsal cortices bilaterally, and to the ventral dentate area and CA3 on the nonstimulated side. In contrast, the HFS S group showed decreased RNA synthesis at the dorsal dentate area and dorsal cortex on the stimulated side, and at CA1, subiculum, and cingulate cortex bilaterally. The decrease was stronger in the HFS-F than in the HFS-S group. Moreover, the subgroup with a low (0-60%) and that with a high (61-240%) level of long-term potentiation of FPSP revealed lower and higher RNA synthesis, respectively, both in homosynaptic target areas, and in heterosynaptic sites. Further, correlative analyses between FPSP, POPS and RNA synthesis revealed a complex pattern, depending upon the type of stimulation and on the brain side. Finally, cross-correlation analyses revealed a high degree of coupling among brain sites in the stimulated groups, indicating distributed covariant changes in RNA synthesis across different brain sites. Thus, changes in synaptic efficacy covary with changes in RNA synthesis, and presumably exert a modulatory role on gene expression. PMID- 7538873 TI - Systemic 3-nitropropionic acid: behavioral deficits and striatal damage in adult rats. AB - Previous animal studies have demonstrated that systemic administration of 3 nitropropionic acid (3-NP) leads to neuropathological changes similar to those seen in Huntington's disease (HD). Recently, we reported hypoactivity in 6- and 10-week old rats treated with systemic 3-NP (IP, 10 mg/kg/day) once every 4 days for 28 days. Although these behavioral results seem to differ from the observed hyperactivity in most excitotoxic models of HD, 3-NP may provide a better model of juvenile onset and advanced HD. In the present study, older rats were similarly treated with 3-NP to further characterize the reported age dependency of striatal neuronal death caused by 3-NP. Hypoactivity was observed in 14- and 28-week old rats with the latter demonstrating more profound features. The present study also provided the first direct evidence of a 3-NP effect on passive avoidance behavior. Experimental and control animals showed no significant difference in daytime acquisition and retention of a passive avoidance task. However, when the retention tests were conducted during the night time (in contrast to previous daytime tests), 3-NP-treated animals exhibited significant retention deficits. In addition, the neuropathological effects of 3-NP were determined by Nissl, AChE and NADPH-diaphorase histochemistry. Metabolic activity was studied using cytochrome oxidase activity as an index. Results revealed striatal glial infiltration, loss of intrinsic striatal cholinergic neurons, but some sparing of large AChE positive neurons, minimal damage of NADPH-diaphorase containing neurons, and very slight, if any, alterations in cytochrome oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538877 TI - Non-neoplastic chorioretinal enhancement patterns in magnetic resonance imaging of the eye. AB - OBJECTIVE: To define the role of contrast-enhanced magnetic resonance imaging (MRI) as a possible adjunct to funduscopy and ultrasonography in a selected sample of non-neoplastic disorders of the chorioretina. PATIENTS AND METHODS: The study group consisted of five patients (ranging in age from 3 to 78 years) with one of the following diagnoses: ocular toxocariasis, staphyloma, glaucoma, or ocular involvement of cytomegalovirus (CMV) infection or AIDS. All of the patients underwent MRI, and the findings were correlated with those of funduscopy and ultrasonography when possible. RESULTS: There were two abnormal MRI enhancement patterns, one with and the other without major distortion of the chorioretina. Areas of abnormal enhancement correlated well with the funduscopic findings. For the patient with CMV infection and the one with AIDS, who were not examined with ultrasonography, MRI showed subtle chorioretinal abnormalities. In the other three cases, for which both ultrasonography and MRI were performed, the findings of the two methods correlated well. CONCLUSIONS: Ultrasonography remains the imaging modality of choice in the work-up of most ocular abnormalities. Ultrasonography, MRI and computed tomography are recommended when funduscopy is technically not possible. Because contrast-enhanced MRI is often performed to define the remainder of the orbit, as well as extra-orbital structures, and because of its capability to demonstrate abnormalities of the chorioretina, this modality may serve as a useful adjunct to ultrasonography. Further studies are needed to compare the efficacy of contrast-enhanced MRI and ultrasonography in the evaluation of small, nonneoplastic lesions of the chorioretina. PMID- 7538875 TI - Magnetic resonance imaging of brain death. AB - OBJECTIVE: To demonstrate the magnetic resonance imaging (MRI) appearance of the brain in patients with clinical brain death. PATIENTS AND METHODS: High-field (1.5-T) MRI was performed on five patients who were subsequently proven clinically brain dead. Conventional T1-weighted and T2-weighted imaging was performed. RESULTS: MRI exhibited similar features for all of the patients: loss of the subarachnoid spaces of the brain; slow flow in the intracavernous and cervical internal carotid arteries; and loss of flow void in the small and large intracranial arteries, as well as in the major intracranial venous sinuses. The differentiation between grey and white matter in the brain was preserved, although the brain had a "supernormal" appearance due to the absence of cerebrospinal fluid and arterial pulsations. These findings have not been observed in MRI of comatose patients who were not clinically brain dead. CONCLUSION: With the advent of MRI-compatible ventilators and noninvasive monitoring, which facilitate imaging of patients under intensive care, MRI may offer another method of confirming the clinical diagnosis of brain death. PMID- 7538876 TI - Intramedullary schwannoma. AB - OBJECTIVE: To present the imaging findings for two cases of intramedullary schwannoma involving the thoracic cord and the conus medullaris. PATIENTS AND METHODS: a 34-year-old man and a 53-year-old woman presented with symptoms of weakness and pain in the lower limbs. Myelography and magnetic resonance imaging as well as other examinations, were performed. RESULTS: The imaging failed to indicate the correct diagnosis in either patient. However, intramedullary schwannoma was confirmed pathologically in both patients. Subtotal excision resulted in local tumour recurrence at 5 and 3 years, respectively. CONCLUSIONS: Intramedullary schwannoma should be considered in the differential diagnosis of spinal cord tumours. The importance of accurate diagnosis from frozen sections at surgical biopsy is emphasized . PMID- 7538878 TI - Periurethral injection of collagen in the treatment of urinary stress incontinence: ultrasonographic appearance. AB - OBJECTIVE: To describe the ultrasonographic appearance of collagen injected periurethrally to treat urinary stress incontinence. PATIENTS AND METHODS: Transvesical and transvaginal ultrasonography (US) was performed 26 times in 23 patients 3 to 36 months after periurethral injection of collagen to treat symptomatic urinary stress incontinence. The patients ranged in age from 23 to 86 (median 54) years. The appearance, location and volume of the collagen were recorded. Clinical data were also obtained. RESULTS: Transvesical US demonstrated the collagen in 17 of the patients, whereas transvaginal US demonstrated the collagen in all of them. The collagen collections appeared as circumscribed masses at the bladder base and showed various levels of echogenicity with both techniques. However, in patients with more than one deposit of collagen, the collections had similar echogenicity, and echogenicity increased over time in the two patients who underwent serial imaging. In 21 of the patients, the collagen collections were located posterior or lateral to the urethra, and these patients recorded complete or moderate resolution of incontinence. In two of the patients the collections were exophytic, projecting into the bladder lumen; these patients experienced little improvement in their continence. CONCLUSIONS: US provides a rapid, noninvasive method of assessing collagen after periurethral injection. Transvaginal US is the best method of visualizing such collections. PMID- 7538879 TI - Percutaneous gastrostomy without gastropexy. AB - OBJECTIVES: To describe in detail the technique for percutaneous gastrostomy without gastropexy, to determine the overall success rate for this technique in providing access to the gastrointestinal tract for feeding and to determine if routine gastropexy is necessary. PATIENTS AND METHODS: The authors reviewed all cases of percutaneous gastrostomy performed over a 14-month period. In total, 24 patients (12 males and 12 females) underwent insertion of gastrostomy tubes in the radiology department during this period. Eighteen of the patients had ear, nose and throat tumours and the other 6 had feeding difficulties as a result of neurologic disorders. All gastrostomy tubes were inserted under fluoroscopic guidance without sedation or administration of glucagon. Gastric fixation was not employed. RESULTS: All of the procedures were successful. No major complications occurred. One minor episode of chemical peritonitis did not require treatment. CONCLUSIONS: Percutaneous gastrostomy, performed in the radiology department, is safe and effective. On the basis of these findings and a literature review, the authors suggest that routine gastric fixation is not required. However, gastropexy may be appropriate in some circumstances, and its role in these situations has still to be confirmed. PMID- 7538881 TI - Needle size and sample adequacy in ultrasound-guided biopsy of thyroid nodules. AB - OBJECTIVES: To determine the optimal needle size (23-gauge or 27-gauge) for ultrasound-guided fine-needle aspiration biopsy of thyroid nodules and to compare the interoperator yield for this procedure. PATIENTS AND METHOD: Over an 11-month period, 123 patients underwent biopsy of a thyroid nodule. Three experienced radiologists were assigned at random to sample the nodules. For each nodule, four passes were performed in random order, two with 23-gauge needles and two with 27 gauge needles. If a specific pass yielded no tissue or blood, as determined by visual inspection (i.e., the sample was dry), the procedure was repeated until a satisfactory sample was obtained. After each patient had left the department, the aspirates were reviewed by a cytopathologist (who was not aware of needle size or operator identity) to determine diagnostic adequacy. RESULTS: Among the 123 nodules, 88 were solid, and 35 were complex cysts. There was no significant difference between the two sizes of needle in the adequacy of the samples obtained (102 nodules were adequately sampled with the 23-gauge needle and 95 with the 27-gauge needle; McNemar chi 2 test, p = 0.1456). However, there were significantly fewer dry passes with the larger needle (2 with the 23-gauge needle and 16 with the 27-gauge needle; chi 2 test, p = 0.0022). Sixteen nodules were inadequately sampled with both needles. Eight of these were less than 1 cm in greatest dimension. Only one solid nodule greater than 1 cm in greatest dimension was inadequately sampled. There was no difference in yield among the three radiologists (chi 2 test, p = 0.5192). No significant complications were encountered. CONCLUSIONS: Needles of both 23 and 27 gauge can be used to obtain fine-needle aspiration biopsy samples from thyroid nodules. Using both sizes is recommended, because the number of dry passes is lower with the larger needle, but the diagnostic quality of the aspirate may be better with the smaller one. Experienced physicians can perform fine-needle aspiration biopsy with equal proficiency. PMID- 7538880 TI - Ultrasound guidance in percutaneous gastrostomy and gastrojejunostomy. AB - OBJECTIVE: To evaluate the utility of ultrasound (US) guidance in the percutaneous placement of gastric feeding tubes in patients in whom placement of a nasogastric tube is not possible. PATIENTS AND METHODS: Records from feeding tube placements performed between January 1991 and August 1994 were reviewed. Of the 238 procedures performed, 27 cases (11%) involved initial US guidance, rather than nasogastric tube assistance, because of upper gastrointestinal obstruction. RESULTS: US allowed rapid puncture and subsequent insufflation of the stomach in 26 of the 27 patients, and there were no complications related to its use. In the other patient the position of the transverse colon prevented suitable visualization for puncture, and surgical placement of the feeding tube was necessary. CONCLUSION: US guidance is a safe and effective means by which the stomach can be punctured and distended before placement of a percutaneous feeding tube for patients in whom nasogastric intubation is not possible. PMID- 7538882 TI - Metachondromatosis. AB - OBJECTIVES: To draw attention to metachondromatosis, which may be misdiagnosed as multiple osteochondromatosis, and to point out several findings that have not been emphasized in previous reports. PATIENTS AND METHOD: The authors reviewed the relevant clinical and detailed radiographic findings for five patients from different countries, who underwent imaging at various ages during childhood. RESULTS: Deformities resembling exostoses and punctate calcification were distinctive and represent previously unemphasized features of vertebral involvement in metachondromatosis. Widespread metaphyseal changes were best seen in the femoral necks, which were broad and showed prominent cartilage columns in all of the patients. Flattening of the femoral heads was observed in three patients; in one of these the flattening progressed to epiphyseal necrosis and coxa magna. In one patient the hands and feet showed no enchondromatous involvement. CONCLUSION: Metachondromatosis is a generalized bone dysplasia predominantly affecting the tubular bones and, to a lesser degree, flat bones and the vertebral column. Significant complications may include avascular necrosis of the femoral head and progressive deformity of the small joints due to expanding local exostoses. The typical involvement of the hands and the feet may not always be seen. PMID- 7538883 TI - Perforated atherosclerotic ulcer of the aorta presenting with upper airway obstruction. AB - The authors describe an unusual presentation of penetrating atherosclerotic ulcer of the aorta in an 84-year-old man. The ulcer perforated the aorta, and hematoma extended into the soft tissues of the neck, causing upper airway and esophageal obstruction. The results of autopsy proved the findings of prior chest radiography and computed tomography. PMID- 7538884 TI - Dynamic computed tomography evaluation of tracheobronchomegaly. AB - The authors report a case of tracheobronchomegaly in a 62-year-old man with chronic expiratory cough. Computed tomography showed dilatation of the trachea on inspiration and complete collapse of the trachea and the proximal bronchi on full expiration. The authors discuss the importance of the collapse on expiration as the cause of the patient's symptoms. PMID- 7538885 TI - Expandable metallic bronchial endoprosthesis. AB - The authors report the successful insertion, under fluoroscopic guidance, of an expandable metallic endoprosthesis in a 60-year-old woman with recurrent obstructive pneumonia secondary to metastasis. This procedure allowed 93 days of palliation for the patient, who had previously required multiple endoscopic treatments for the recurrent bronchial obstruction. PMID- 7538887 TI - Herniation of the transverse colon: an unusual complication of pedicled omentoplasty. AB - Pedicled omentoplasty is frequently used in chest reconstruction and treatment of sternal infection. The procedure is effective, but it is associated with several postoperative complications. The authors describe a 49-year-old man in whom sternal infection after heart surgery was successfully treated by sternectomy and omentoplasty. Several months later, the transverse colon herniated into the transposed omentum. To the authors' knowledge, this complication has not been previously reported. PMID- 7538886 TI - Percutaneous stent placement for treatment of stenosis of a portacaval H-graft shunt. AB - Surgical treatment of an occluded or stenotic portacaval shunt carries a high risk of mortality, but the rate of restenosis after transluminal angioplasty is also high. The authors report high-grade stricture of a portacaval H-graft shunt in a 51-year-old man, who presented with hematemesis and melena. The patient was treated with concomitant balloon angioplasty and placement of a metallic stent through a percutaneous venous approach. The procedure was tolerated well by the patient, and stenosis had not recurred at follow-up 1 year later. PMID- 7538891 TI - Residents' corner. Answer to case of the month #31. Aortoduodenal fistula associated with prosthetic graft for abdominal aortic aneurysm. PMID- 7538892 TI - Malignant hyperthermia and injection of iodine-containing contrast media. PMID- 7538889 TI - Surgically transposed ovary presenting as an intraperitoneal mass on computed tomography. AB - The authors present a case of surgical transposition of the left ovary, which presented on computed tomography (CT) as an intraperitoneal cystic mass. To their knowledge, there is only one other report in the radiologic literature of the CT appearance of a transposed ovary, but in that case the ovary appeared as a retroperitoneal mass. PMID- 7538888 TI - Ruptured spleen as a complication of insertion of an automatic implantable cardioverter-defibrillator. AB - The authors describe a previously unreported complication of insertion of an automatic implantable cardioverter-defibrillator via the left subcostal surgical approach. Splenic hematoma, intraperitoneal hemorrhage and hypotension developed in a 66-year-old man within 5 days of implantation, and the patient underwent splenectomy. PMID- 7538890 TI - Blastic osseous metastases in ovarian carcinoma. AB - A 75-year-old woman with histologically proven ovarian adenocarcinoma and no evidence of other malignant lesions was found to have disseminated blastic (sclerotic) metastases to bone without clinical evidence of gross peritoneal spread or lung involvement. The patient's survival for 28 months after the earliest evidence of bone involvement is noteworthy, because osseous metastasis in ovarian malignancy is usually a late manifestation of the disease. PMID- 7538893 TI - Synaptic regeneration and functional recovery after excitotoxic injury in the guinea pig cochlea. AB - Cochlear ischemia and acoustic trauma result in an immediate hearing loss accompanied by the complete disruption of the terminal dendrites of primary auditory neurons postsynaptic to the sensory inner hair cells (IHCs). This synaptic uncoupling, due to an acute glutamate (IHC neurotransmitter) excitotoxicity process, can be mimicked by glutamate agonists. Thus, we have followed over a 5-day period the responses of guinea-pig cochleas to a local application of 200 microM AMPA. This application immediately results in a destruction of all postsynaptic endings of the auditory nerve, resulting in a total loss of cochlear potentials. Twenty-four hours after this excitotoxic injury, the inner hair cells were contacted by repaired postsynaptic dendrites and the cochlear potential had partially recovered. This process of neo synaptogenesis was completed and the potentials were fully restored at 5 days post exposure. As shown by in situ hybridization, an up-regulation of NMDA and metabotropic glutamate receptors in the primary auditory neurons occurred during this process of recovery. This process of neo-synaptogenesis and functional recovery probably accounts for restoring hearing after temporary losses due to excitotoxic-related pathologies. PMID- 7538894 TI - Hepatocyte growth factor and retinoic acid exert opposite effects on synthesis of type 1 and type 2 acute phase proteins in rat hepatoma cells. AB - Rat hepatoma cells H-35 cultured in serum-free medium were exposed to interleukin 6 (IL-6), interleukin-1 (IL-1), hepatocyte growth factor (HGF), retinoic acid (RA), or a mixture of these factors. Production of acute phase proteins, responding to IL-6 alone (type 2) or to the mixture of IL-6 and IL-1, was assessed by electroimmunoassay and the corresponding mRNAs were compared by Northern blot analysis. HGF enhanced IL-6-induced synthesis of alpha-2 macroglobulin but reduced synthesis of C3 complement and alpha-1-acid glycoprotein. Retinoic acid reduced the response to IL-6 of alpha-2-macroglobulin but enhanced that of alpha-1-acid glycoprotein and especially of C3 complement. In general, changes in protein secretion were correlated with the contents of their corresponding cellular mRNAs. These results indicate that hepatocyte growth factor can enhance basal or IL-6-induced gene expression of type 2 and reduce the expression of type 1 acute phase proteins, whereas the action of retinoic acid is opposite. The modulation of acute phase response by HGF and RA likely involves transcriptional factors and regulatory sequences in the genes coding for these two types of acute phase proteins. PMID- 7538895 TI - The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas. AB - Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in several types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and that dThdPase has angiogenic activity. We measured dThdPase activity and the level of thrombomodulin (TM) as a marker for endothelial cells in colorectal carcinomas and adjacent normal tissues from 21 patients, and in adenomas from 13 patients. The average dThdPase activity of colorectal carcinomas (11.58 +/- 6.30 nmol/100 micrograms protein/h) was significantly higher than that of adenomas (8.57 +/- 4.14 nmol/100 micrograms protein/h) or normal tissues (4.89 +/- 3.16 nmol/100 micrograms protein/h). In immunohistochemical study, the expression of dThdPase was observed more frequently in colorectal carcinomas than in adenomas or normal mucosas. The amount of TM in colorectal carcinomas (8.32 +/- 5.07 ng/100 micrograms protein) was significantly higher than that of adenomas (4.51 +/- 4.49 ng/100 micrograms protein) or normal tissues (3.51 +/- 2.78 ng/100 micrograms protein). dThdPase activity in human colorectal carcinomas, adenomas and normal tissues was significantly correlated with the expression of TM in these tissues. These results indicate that the expression levels of both dThdPase and TM in colorectal carcinomas are higher than those in colorectal adenomas and normal tissues and suggest that dThdPase may be involved in angiogenesis in human colorectal carcinomas, adenomas and normal tissues. PMID- 7538896 TI - Immunohistochemical analysis of dichloroacetic acid (DCA)-induced hepatocarcinogenesis in male Fischer (F344) rats. AB - We examined the incidence of proliferative lesions, hyperplastic nodules and altered hepatic foci, in male F344 rat liver, to determine their preneoplastic potential during dichloroacetic acid (DCA)-induced hepatocarcinogenesis. Immunohistochemical and image analysis methods were used to detect the expression of 6 histochemical markers of neoplastic cells; p21 ras, p39 c-jun, p55 c-fos, aldehyde dehydrogenase (ALDH), glutathione s-transferase (GST-p), and alpha fetoprotein (AFP) during DCA-induced hepatocarcinogenesis. Our results were consistent with our previous data and suggested that the hyperplastic nodules, rather than altered hepatic foci, is a putative preneoplastic lesion during DCA induced hepatocarcinogenesis in the male F344 rat. PMID- 7538898 TI - B7-1 expression by a non-antigen presenting cell-derived tumor. AB - The existence of a naturally occurring immunosurveillance against neoplastic cells is controversial. A difficulty with this concept is that tumor-specific antigen-reactive T cells would not be expected to become activated after encountering tumor cells, since T cells that bind to antigen in the absence of the costimulation provided by antigen-presenting cells may be inactivated. We studied a transgenic model of tumorigenesis where T cells reactive to a particular tumor-specific antigen are lost prior to the development of non antigen-presenting cell-derived tumors; therefore, the tumors that develop are not subjected to immunosurveillance. We found that a tumor cell line derived from one such tumor expresses the T-cell costimulatory molecule B7-1, the expression of which is normally restricted to antigen-presenting cells. In addition, we found that several immortalized cell lines, which are nontumorigenic and thus have suffered only early genetic events in the tumorigenesis process, express B7. This suggests that a host cell can be induced to express surface B7-1 molecules after suffering an oncogenic insult, which might possibly be a primary mechanism of immunosurveillance against tumors. PMID- 7538897 TI - Angiogenesis-directed implantation of genetically modified endothelial cells in mice. AB - By virtue of their location within blood vessels and their ability to express foreign genes, endothelial cells are attractive vehicles for the delivery of therapeutic molecules in vivo. We wished to determine whether i.v.-injected, genetically modified endothelial cells can become incorporated into sites of active angiogenesis in vivo. To do so, we studied the fate of i.v.-injected, lacZ expressing human umbilical vein endothelial cells in athymic nude mice bearing lethally irradiated NIH 3T3 murine fibroblast cells transfected with a sp hst/KS3:fibroblast growth factor-1 chimera that forces the secretion of the angiogenic protein, fibroblast growth factor-1. Following i.v. injection, lacZ labeled human umbilical vein endothelial cells accumulated at sites of fibroblast growth factor-1-induced angiogenesis, persisting for at least 4 weeks. These results suggest that i.v.-administered, genetically modified endothelial cells can migrate into and survive within an angiogenic site. This strategy may be useful for delivery of therapeutic molecules to sites of pathological angiogenesis during tumor metastasis. PMID- 7538899 TI - Does pregnancy immunize against breast cancer? AB - Multiparity has been linked with protection against breast cancer. T cells from biparous women, but not T cells from nulliparous women or men, specifically proliferated in response to core peptide sequences of a human breast cancer associated mucin (MUC-1). Two of the nulliparous women were retested during the first trimester of their first pregnancy, and their T cells proliferated specifically in response to MUC-1 mucin. These observations support the hypothesis that there is a natural immunization against MUC-1 peptide epitopes during pregnancy which provides some protection against the development of breast cancer. These data also suggest that certain MUC-1 synthetic peptides might be effective components of "vaccines" for therapy or prevention of breast cancer. PMID- 7538900 TI - Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. AB - Approximately one third of breast cancers grow independently of estrogen, lack detectable estrogen receptor (ER) protein, and rarely respond to hormonal treatment. Previous studies correlated the lack of ER gene expression in ER negative breast tumor cells with hypermethylation of a CpG island in the 5' region of the ER gene. In order to determine whether demethylation of the ER gene in the ER-negative human breast cancer cell line MDA-MB-231 could affect ER transcription, cells were treated with two inhibitors of DNA methylation, 5 azacytidine or 5-aza-2'-deoxycytidine. DNA from cells treated with either drug became partially demethylated at several methylation-sensitive restriction enzyme sites, including HhaI, NotI, and SacII, within the ER CpG island. This demethylation correlated with reexpression of the ER gene as detected by reverse transcriptase-PCR and production of ER protein as detected by Western blot analysis. ER produced in drug-treated cells was functionally active as demonstrated by its ability to activate transcription of estrogen-responsive genes. These results suggest that DNA methylation of the ER CpG island may play a role in suppression of ER gene expression in ER-negative breast cancer cells. PMID- 7538901 TI - Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen. AB - Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules. PMID- 7538902 TI - Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1. AB - Although the cyclin-dependent kinase inhibitor p21SDI1 (WAF1/CIP1) has been proposed as the mediator of p53-induced cell cycle arrest following DNA damage, several stimuli now appear to induce SDI1 independent of p53 function. We have examined the behavior of p53 and SDI1 in an isogeneic model by manipulating p53 status in normal diploid human fibroblasts using an amphotropic retroviral vector. Following DNA strand break damage induced by bleomycin, both SDI1 induction and G1-S cell cycle arrest are p53 dependent, consistent with SDI1 being the key mediator. In contrast, in cellular senescence (and following UV irradiation), induction of SDI1 occurs independent of p53 function yet growth arrest is still p53 dependent. We conclude (a) that redundant pathways exist for induction of SDI1, but that (b) SDI1, while perhaps necessary, is not sufficient for inhibition of cell cycle progression, requiring the cooperation of an additional factor (possibly another cyclin-dependent kinase inhibitor) whose expression, at least in the case of senescence, is strictly p53 dependent. PMID- 7538903 TI - The presence of prostate-specific antigen-related genes in primates and the expression of recombinant human prostate-specific antigen in a transfected murine cell line. AB - Human prostate-specific antigen (PSA) has been shown as an aid in the early detection of prostate cancer (W. J. Catalona et al., J. Am. Med. Assoc., 270: 948 954, 1993) and was approved in 1994 by the Food and Drug Administration for early detection of prostate cancer. Immunotherapies directed against PSA have been suggested in patients with metastatic prostate cancer. One of the essential questions is to define which nonhuman species express PSA for experimental studies. Using Southern blot analyses, genes related to human PSA have been detected in several nonhuman primate species, including chimpanzee, orangutan, gorilla, macaque, and rhesus monkey, but not in other mammalian species, including rabbit, cow, pig, dog, rat, or mouse. Immunohistochemical staining with anti-human PSA antisera detected strong staining in both human and monkey prostatic epithelial cells with no reactivity to rat prostate cells. Because the PSA gene is not present in the murine genome, a matched set of murine cell lines has been developed that may be useful to study the biochemical functions of PSA and as an experimental target for PSA-directed immunotherapy. To establish such cell lines, a C57BL/6 murine colon adenocarcinoma cell line, MC-38, was transfected with a retroviral vector containing cDNA encoding the human PSA gene. Genetic analysis of a PSA-secreting clone, PSA/MC-38, demonstrated that the PSA gene had been stably integrated into the MC-38 genome. The PSA/MC-38 cell line was found to secrete PSA into tissue culture medium, producing a protein of approximately M(r) 30,000. In vivo, PSA/MC-38 grew as a s.c. tumor in male and female mice. PSA/MC-38 tumors grew more rapidly in athymic mice than in syngeneic C57BL/6 mice, and in both mouse strains, the PSA/MC-38 tumors grew more slowly than control vector-transduced tumors. PSA was detected in the serum and tumors of PSA/MC-38 tumor-bearing mice. It is proposed that PSA/MC-38 cells may be used as a murine tumor model to test potential therapeutic vaccines and other experimental therapies directed against PSA. PMID- 7538904 TI - Clinical evaluation of long-term treatment using cernitin pollen extract in patients with benign prostatic hyperplasia. AB - Seventy-nine patients with benign prostatic hyperplasia (BPH) were treated with cernitin pollen extract. Patient ages ranged from 62 to 89 years (mean, 68 years). Mean baseline prostatic volume was 33.2 cm3. Cernitin pollen extract was administered in a dosage of 126 mg (2 tablets, 63 mg each), three times a day, for more than 12 weeks. Symptom scores, based on a modified Boyarsky scoring scale, uroflowmetry, prostatic volume, residual urine volume, and urinalysis results were examined before and after administration of cernitin pollen extract. Symptom scores significantly decreased from baseline, and the favorable results continued during the treatment period. Urine maximum flow rate and average flow rate increased significantly from 9.3 mL/s to 11 mL/s and from 5.1 mL/s to 6 mL/s, respectively. Residual urine volume decreased significantly from 54.2 mL to less than 30 mL. There was no change in prostatic volume. However, 28 patients treated for more than 1 year showed a mean decrease of prostatic volume to 26.5 cm3. No adverse reactions were observed. Clinical efficacy at 12 weeks was rated excellent, good, satisfactory, and poor in 11%, 39%, 35%, and 15% of patients, respectively. Overall clinical efficacy was 85%. In conclusion, cernitin pollen extract showed a mild beneficial effect on prostatic volume and urination variables in patients with symptomatic BPH. PMID- 7538905 TI - [Detection of the G551D mutation in a patient with nasal polyps]. AB - Atypical forms of cystic fibrosis can be manifested by a mild affection of the airways in adult age. They are the phenotypic manifestation of less frequent mutations for CF. The authors present the case-history of a man who developed the first symptoms of respiratory disease at the age of 16 years. He had relapsing nasal polyps, sinusitis, bronchial asthma, intolerance of acetylsalicylic acid. Bacteriological examination revealed repeatedly Pseudomonas aeruginosa on the respiratory mucosal membranes. The levels of the chloride ion in sweat were normal, genetic examination revealed a mutation of the gene for cystic fibrosis, G 551 D. PMID- 7538906 TI - Detachment of transformed cells. Role of CD44 variants. AB - A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts, ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20-24 dyn/cm2 were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells' ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell's ability to metastasize. PMID- 7538907 TI - FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. AB - Using the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8. FADD contains a death domain homologous to the death domains of Fas and TNFR-1. A point mutation in FADD, analogous to the lpr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction. Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interleukin-1 beta-converting enzyme. These findings suggest that FADD may play an important role in the proximal signal transduction of Fas. PMID- 7538909 TI - Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. AB - Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death. PMID- 7538908 TI - RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. AB - Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, we have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein we have named RIP, for receptor interacting protein. RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein. PMID- 7538910 TI - Antigen-presenting cell lines internalize peptide antigens via fluid-phase endocytosis. AB - In this report, we present experimental evidence that antigen-presenting cell lines take up peptide antigens in a manner consistent with fluid-phase endocytosis. Using the fluid phase endocytic marker inulin and a mathematical model for fluid phase uptake, we have found a basal uptake rate constant of approximately 0.9-2 microns 3/cell minutes in A20, TA3, and J774 cells. An influenza virus peptide, PB2(303-313), the octapeptide, angiotensin II, an ovalbumin peptide, OVA(323-339), and a guinea pig myelin basic protein peptide, MBP(72-86), have uptake rate constants comparable to inulin, i.e., between 1 and 4 microns3/cell minutes in A20 cells. However, another influenza virus peptide, PB2(146-159), has an uptake rate constant approximately sixfold higher than that found for inulin in A20 cells. We have also determined that the peptide antigens we tested are retained in A20 cells similarly to inulin, with half-times calculated to be from 2 to 13 min as compared to 2 min for inulin. Notably, these results were obtained over short incubation times (up to 20 min) and under conditions that restrict peptide proteolysis and also protein synthesis. We conclude from these studies that peptide antigens enter antigen-presenting cells via fluid-phase endocytosis. PMID- 7538911 TI - Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma. AB - Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL 4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented. PMID- 7538912 TI - Monoclonal anti-dipeptide antibodies cross-react with detyrosinated and glutamylated forms of tubulins. AB - Two monoclonal antibodies, GLU-1 and A1.6, raised against gamma-L-glutamyl-L glutamic acid dipeptide (Glu-Glu) and Ca(2+)-dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu-Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross-reaction of the antibodies to the alpha tubulin subunit. alpha-Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and A1.6 specifically recognize the detyrosinated form of alpha-tubulin. When microtubule protein purified from brain was probed, not only alpha-but also, to a lesser extent, beta tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the gamma position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class III beta isotype at amino acid position 437 to 439, it is suggested that GLU-1 and A1.6 are able to recognize the glutamylated form of beta-tubulin. These results indicate that the C-terminal Glu-Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of alpha-tubulin and the glutamyl side chain of beta-tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post-translationally modified tubulin subunits. PMID- 7538915 TI - Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes. AB - Whole-cell patch clamp was used to look for cystic fibrosis transmembrane conductance regulator (CFTR)-like chloride currents in calcium-tolerant human cardiac myocytes. Potassium-containing solutions were used initially. Steady state currents were measured with hyperpolarizing ramps (-16.25 mV/s). Peak net inward currents during voltage steps from -50 to +5 mV were used as an index of L type calcium current. Isoproterenol (1 mumol/L) or forskolin (10 mumol/L) were used in attempts to evoke CFTR-like chloride current. No forskolin- or isoproterenol-induced steady state current was found in any of 17 atrial cells from seven patients in the absence of cell swelling. Every cell exhibited a large increase in net inward current in response to forskolin, suggesting that cAMP dependent stimulation of L-type calcium current was present. Swelling with osmotic stress induced an outwardly rectifying steady state current with a reversal potential close to the chloride equilibrium potential. Once this current was activated, exposure to forskolin caused a further increase that subsided on washout (four of four cells, two patients). The atrial swelling-induced current was studied in more detail by using cesium-containing solutions. The current was determined to be a chloride current because the reversal potential was sensitive to changes in intracellular chloride and outward currents were blocked by 150 mumol/L DIDS. Ventricular cells were isolated from five failing human hearts. No CFTR-like current was found in any of 17 cells. In eight of eight ventricular cells, a swelling-induced current was found. The amplitude of the swelling induced current was enhanced by forskolin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538913 TI - Protein synthesis in the hypertrophied heart of spontaneously hypertensive rats and a comparison of the effects of an ACE-inhibitor and a calcium channel antagonist. AB - The aim of the investigation was to assess and compare the effects of a calcium channel antagonist, (i.e. amlodipine) and an ACE-inhibitor (i.e. lisinopril) in reducing chronic left ventricular hypertrophy in 15-week old spontaneously hypertensive rats (SHR). Changes in cardiac hypertrophy were assessed after 8 weeks by measuring the fractional rates of protein synthesis using a 'flooding dose' of [3H]-phenylalanine for 10 min. Blood pressure was monitored throughout the treatment period in both SHR and Wistar-Kyoto control rats (WKY). The results showed a decrease in blood pressure by amlodipine after 1 week of treatment which was further reduced at 4 to 8 weeks. Lisinopril caused immediate and sustained reductions in blood pressure (190 mmHg to 130 mmHg, P < 0.001). After 8 weeks of treatment in SHR rats, amlodipine had no significant effect on left ventricular weight (P > 0.05), whereas lisinopril caused a marked reduction. The protein content and RNA were also not changed by amlodipine. In contrast, lisinopril significantly lowered the tissue protein, RNA and DNA content (P < 0.001). The changes in the left ventricles of lisinopril-treated SHR rats were accompanied by an increase in the fractional synthesis rate of left ventricular myofibrillar proteins (+12 per cent, P < 0.025). The synthesis rate per unit RNA was also increased in right ventricular tissue of lisinopril-treated SHR rats. However, amlodipine had no effect on the fractional synthesis rates of any of the left ventricular fractions of SHR rats (P > 0.05). The cellular efficiency in the right ventricle was also increased in amlodipine-treated SHR rats, indicating a moderate effect on protein metabolism. In conclusion, amlodipine had minimal effects in the reduction of established left ventricular hypertrophy (LVH), despite reducing the blood pressure, whereas lisinopril caused regression of LVH. These events were associated with small changes in protein synthesis rates, with the contractile protein showing an increase. PMID- 7538914 TI - Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro. AB - Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate. PMID- 7538916 TI - Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells. AB - Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID- 7538917 TI - Contraction-induced cell wounding and release of fibroblast growth factor in heart. AB - The heart hypertrophies in response to certain forms of increased mechanical load, but it is not understood how, at the molecular level, the mechanical stimulus of increased load is transduced into a cell growth response. One possibility is that mechanical stress provokes the release of myocyte-derived autocrine growth factors. Two such candidate growth factors, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively), are released via mechanically induced disruptions of the cell plasma membrane. In the present study, we demonstrate that transient, survivable disruption (wounding) of the cardiac myocyte plasma membrane is a constitutive event in vivo. Frozen sections of normal rat heart were immunostained to reveal the distribution of the wound event marker, serum albumin. Quantitative image analysis of these sections indicated that an average of 25% of the myocytes contained cytosolic serum albumin; ie, this proportion had suffered a plasma membrane wound. Wounding frequency increased approximately threefold after beta-adrenergic stimulation of heart rate and force of contraction. Heparin-Sepharose chromatography, enzyme linked immunosorbent assay, growth assay coupled with antibody neutralization, and two-dimensional SDS-PAGE followed by immunoblotting were used to demonstrate that both aFGF and bFGF were released from an ex vivo beating rat heart. Importantly, beta-adrenergic stimulation of heart rate and force of contraction increased FGF release. Cell wounding is a fundamental but previously unrecognized aspect of the biology of the cardiac myocyte. We propose that contraction-induced cardiac myocyte wounding releases aFGF and bFGF, which then may act as autocrine growth-promoting stimuli. PMID- 7538918 TI - Low concentration of oxidized low-density lipoprotein and lysophosphatidylcholine upregulate constitutive nitric oxide synthase mRNA expression in bovine aortic endothelial cells. AB - Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries. Recently, the endothelium-dependent relaxing factor has been identified as nitric oxide (NO). We used RNase protection assay and immunoblotting to elucidate the effect of atherogenic lipoprotein on the expression of constitutive NO synthase (cNOS) mRNA and protein levels in bovine aortic endothelial cells. Twenty-four hour exposure to a low concentration of oxidized low-density lipoprotein (10 micrograms protein/mL) upregulated cNOS mRNA levels (2.4 +/- 0.4-fold, P < .01). However, native low-density lipoprotein and high-density lipoprotein did not have any effect on cNOS mRNA levels. Furthermore, 5 micrograms/mL of lysophosphatidylcholine (LPC) also upregulated cNOS mRNA levels (2.6 +/- 0.5 fold, P < .01) at 8 hours. This action of LPC was abolished with cycloheximide but not with staurosporine. We concluded that atherogenic lipoproteins upregulate cNOS mRNA and protein levels in bovine aortic endothelial cells. This observation supports the hypothesis that an impairment of endothelium-dependent vasodilatation in atherosclerotic vessels may not be due to a decrease in cNOS expression. Moreover, the LPC action on cNOS mRNA levels requires new protein synthesis. PMID- 7538919 TI - Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. PMID- 7538921 TI - Sialyl salivary-type amylasemia associated with immunoglobulin D-type multiple myeloma. PMID- 7538920 TI - Growth factor therapies for vascular injury and ischemia. PMID- 7538922 TI - Multi-enzyme reference material from established human cell lines and human sources. AB - A multi-enzyme reference material was prepared from seven enzymes of asparatate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LD, EC 1.1.1.27), creatine kinase (CK, EC 2.7.2.2), gamma-glutamyltranspeptidase (gamma-GT, EC 2.3.2.2) and amylase (AMY, EC 3.2.1.1) which were purified from human sources including established human cell lines. The enzymatic properties of the material closely resembled those of human serum. In lyophilized form the preparation was stable for at least 200 days when stored at 40 degrees C. Intermethod comparisons of the enzyme activities in 80 clinical specimens were done by correcting the mean values with calibration constants for different assay methods resulting from use of a human serum, the multi-enzyme reference and a commercial control serum. The results from the comparison for the six enzymes of AST, ALT, LD, CK, gamma-GT and AMY in use of the multi-enzyme reference were almost the same as those with use of a human serum as a calibrator, but were not satisfactory for ALP. Even though further search for more reliable material for ALP is required the multi enzyme reference material can be used for standardization in clinical chemistry. PMID- 7538923 TI - Human chorionic gonadotrophin immunoactivity in cystic intracranial tumours. AB - BACKGROUND AND OBJECTIVE: With regard to intracranial tumours, elevated hCG in CSF or serum has been considered to be specific for germ-cell tumours. Recently however, elevated hCG has also been shown to be present in cyst fluid and CSF of patients with craniopharyngiomas. While germ-cell tumours are generally non cystic, the aim of our study was to determine the significance of hCG in cystic intracranial lesions. DESIGN: In a prospective study, hCG immunoactivity and subunits of hCG were measured in cyst fluid, CSF, and serum of patients harbouring intracranial cyst lesions. PATIENTS AND MEASUREMENTS: hCG immunoactivity was measured in cyst fluid and serum samples of 42 patients. CSF samples were available from 12 patients with craniopharyngiomas. In order to fully characterize the hCG immunoactivity, we used immunoradiometric assays for total hCG activity (measuring both intact hCG and the free beta-subunit of hCG), and those specific for intact, dimeric hCG (hCG), free beta-subunit of hCG (hCG beta) and free alpha-subunit. Furthermore, immunostaining of tumour tissue was performed using monoclonal antibodies directed against the free beta-subunit of hCG. RESULTS: Total hCG immunoactivity was markedly elevated in cyst fluid of all 17 craniopharyngiomas (range 36.7-4558 IU/I; normal < 5 IU/I). Moderately elevated levels of hCG in cyst fluid were detected in three of four pituitary adenomas, in two metastases from lung cancer and in two arachnoid cysts. hCG beta was detected in cyst fluid from all hCG positive cysts, while specific determination of intact (dimer) hCG and alpha-subunit mostly yielded negative results. No hCG immunoactivity was found in cystic gliomas, meningiomas or haemangioblastomas. hCG was elevated in CSF of two patients with craniopharyngiomas, but no hCG immunoactivity was detected in any serum sample. Subtle immunostaining of epithelial cell groups was shown in five of ten craniopharyngiomas. Clear immunostaining for hCG beta was also found in scattered epithelial cells of one pituitary adenoma. CONCLUSIONS: hCG immunoactivity in cystic intracranial lesions is due mainly to hCG beta. Measurement of hCG immunoactivity in cyst fluid can be helpful in the differential diagnosis of intracranial cystic lesions, if surgery is restricted to cyst decompression and no histology is available. High levels suggest a craniopharyngioma. PMID- 7538925 TI - High prevalence of hepatitis C infection in Afro-Caribbean patients with type 2 diabetes and abnormal liver function tests. AB - Moderate elevations of serum transaminases are frequently found in patients with diabetes mellitus and are often attributed to fatty infiltration of the liver without further investigation. Recent studies of patients with end-stage liver disease have suggested a possible association between Hepatitis C virus (HCV) antibody positivity and the development of diabetes (mostly Type 2). As a first step in the examination of any potential association between HCV and Type 2 diabetes in subjects without overt liver disease, we examined 200 British patients with Type 2 diabetes (100 White Caucasians, 50 Asians, and 50 Afro Caribbeans), recruited from the United Kingdom Prospective Study of Diabetes, half of whom had a significant elevation of alanine aminotransferase (ALT) on at least two occasions and half of whom had consistently normal ALT levels. In Afro Caribbean Type 2 diabetic subjects 7/25 (28%) patients with abnormal ALT and 1/25 (4%) with normal ALT were HCV antibody positive. Among White Caucasian subjects 6/50 (12%) patients with abnormal LFTs and 0/50 with normal LFTs were HCV antibody positive and in Asians the prevalence was 2/25 (8%) and 0/25, respectively. This study suggests that persistent mild to moderate elevation of serum transaminases in a patient with Type 2 diabetes should not automatically be attributed to the metabolic disturbances of diabetes. Particularly in Afro Caribbean subjects, HCV infection is a major diagnostic consideration. The question of whether HCV infection itself may have a diabetogenic action is worthy of further investigation. PMID- 7538926 TI - Allergic contact dermatitis from purified eosin. AB - We report on a patient, seen in 1993, who developed sensitization to eosin from topical preparations. The patient had a positive patch test reaction to pure tetrabromofluorescein but not to its impurities separated by thin-layer chromatography. Our findings suggest that the allergen was eosin itself. PMID- 7538927 TI - Dysfunctions of Fc alpha receptors by blood phagocytic cells in IgA nephropathy. PMID- 7538924 TI - Polarization of mitochondria in the unfertilized mouse oocyte. AB - Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. The localization of mitochondria during maturation was studied in live mouse oocytes by confocal laser scanning microscopy (CLSM). Mitochondria were labeled with rhodamine 123 or Mitotracker (Molecular Probes, Eugene, OR) both of which are cell permeant and accumulate in mitochondria; acridine orange was used to mark chromatin. Prior to maturation, oocytes appeared to be radially symmetrical with no evident polarity; fully mature oocytes exhibited obvious polarity marked by the position of the metaphase II spindle in the cortex. CLSM revealed several interesting features of mitochondrial distribution: 1) A cortical clump of mitochondria was seen approximately 30-45 degrees to one side of the metaphase II spindle and marked the region of polar body I extrusion. 2) Large foci of mitochondria (7-14 microM) were frequently found around the central region of the mature oocyte, while the central region often exhibited markedly fewer mitochondria. 3) Small mitochondrial foci (3 microM) in the cortex and near the GV characterized several oocytes which failed to mature. 4) Non-spindle-associated mitochondria were not uniformly distributed in the mature oocyte but were concentrated in the hemisphere containing the metaphase II spindle. 5) The distal margins of this mitochondrial hemisphere were sharply demarcated at the cortex. These findings should help us understand organelle localization during mammalian oocyte maturation, and may give insights into possible causes of infertility and into early events of preimplantation development. PMID- 7538929 TI - Experimental IgA mesangial nephropathy: the role of antigen and antibody. PMID- 7538928 TI - Altered mucosal immunity in IgA nephropathy investigated using the ELISA spot method in peripheral blood. PMID- 7538930 TI - Molecules controlling B-lymphocyte growth and differentiation. PMID- 7538931 TI - [Prostate-specific antigen]. PMID- 7538932 TI - [Palliative nursing is looking for a new vision of patient care]. PMID- 7538933 TI - Diagnostic and prognostic value of the new tumour marker CYFRA 21-1 in patients with squamous cell lung cancer. AB - We wanted to investigate the diagnostic and prognostic significance of serum CYFRA 21-1, especially in predicting the risk of recurrence in patients with operable squamous cell lung cancer. Serum levels of CYFRA 21-1 were measured using an immunoradiometric assay (CIS bio) in 76 patients with squamous cell lung cancer (64 operable and 12 with unresectable tumours), 22 with other non-small cell type (12 with adenocarcinoma and 10 with large-cell type) and 45 with nonmalignant lung diseases. Elevated preoperative CYFRA 21-1 levels were identified in 63% of patients with squamous cell type (SqCC), 33% with adenocarcinoma, and 30% with large-cell carcinoma type. The diagnostic specificity of the assay was 96%. Positive CYFRA 21-1 levels were observed in 33% of stage I, 52% of stage II, 76% of stage IIIa and 83% of stage IIIb patients with SqCC type. Statistically significant differences were obtained between stages I and II and between II and IIIa, but not between stages IIIa and IIIb. Recurrence-free survival probability for patients with elevated serum CYFRA 21-1 levels before surgery was 63% (24/38) versus 92% (24/26) for patients with normal serum CYFRA 21-1 levels. However, the difference was not statistically significant when adjusted for the TNM stage (primary tumour, regional lymph node involvement, occurrence of distant metastasis). In 9 of the 10 patients with increased trend for CYFRA 21-1 during follow-up, elevated serum CYFRA 21-1 levels preceded (7) or coincided (2) with the clinical detection of tumour recurrence, providing a predictive value of an increased trend of 90%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7538934 TI - Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. AB - Viral infection may induce the expression of nitric oxide (NO) synthase, resulting in increased NO formation that has an antiviral effect. NO may be produced by various cells of the upper and lower respiratory tract, and may be detected in the exhaled air. We have studied the levels of exhaled NO in 18 normal subjects during symptomatic upper respiratory tract infections and during recovery 3 weeks later. Exhaled NO was measured using a modified chemiluminescence analyser. At the time of symptoms of upper respiratory tract infection, the peak exhaled NO values were 315 +/- 57 ppb (mean +/- SEM) and decreased to 87 +/- 9 ppb during recovery. Recovery values of exhaled NO were similar to those reported in age-matched normal control subjects (88 +/- 3 ppb, n = 72). These findings suggest that symptomatic upper respiratory tract infections markedly increase the concentration of NO in exhaled air. This may reflect the induction of nitric oxide synthase (NOS) in upper and lower respiratory tract, and may be relevant to viral exacerbations of asthma. PMID- 7538935 TI - A rapid, simple method for the isolation and characterization of the photoreceptor of Dictyostelium discoideum amoebae. AB - A membrane-bound 45.5 kDa protein has been isolated from Dictyostelium discoideum amoebae. It shows an absorption spectrum, which closely resembles the action spectrum for amoebal phototaxis, leading to the conclusion that this protein might play an important role in the photoreception of Dictyostelium amoebae. For further characterization we employed phase partition in an aqueous polymer two phase system, which was developed by Widell and Larsson for the separation of plasma membrane proteins of higher plants. This method clearly shows that the 45.5 kDa protein is a plasma membrane protein and not an intracellular protein. Furthermore, by using phase systems with increasing polymer concentrations, this simple and rapid purification of plasma membrane proteins allowed us to isolate the putative photoreceptor in one single step. Compared to standard biochemical methods phase partition provides an enormous facilitation of the isolation of D. discoideum membrane proteins. PMID- 7538937 TI - Interleukin 6 mediated differentiation and rescue of cell redox in PC12 cells exposed to ionizing radiation. AB - The differentiation of PC12 cells to a neuron-like morphology was induced by ionizing radiation in the presence of serum. This effect was detectable at 5 grays (Gy) and reached a maximum at 10-20 Gy. Increases in the DNA binding activity of nuclear factor kappa B (NF-kappa B) and increased Interleukin 6 (IL 6) mRNA levels were observed at a dose of 15 Gy. Neutralization of supernatant IL 6 by the addition of anti-IL-6 antibody inhibited the neuronal differentiation and decreased cellular redox. Ionizing radiation and serum may act synergistically as neurotropic factors. PMID- 7538938 TI - Down-regulation but not phosphorylation of stathmin is associated with induction of HL60 cell growth arrest and differentiation by physiological agents. AB - Stathmin is a cytosolic phosphoprotein that has an important but, as yet, undefined role in cell proliferation and differentiation. Induction of growth arrest and differentiation of HL60 cells to monocytes by phorbol 12-myristate 13 acetate is associated with rapid phosphorylation of the protein. Stathmin phosphorylation was not seen when HL60 cells were induced to differentiate to monocytes, by 1 alpha, 25-dihydroxyvitamin D3, and to neutrophils, by all-trans retinoic acid and granulocyte colony stimulating factor. In all the above instances, stathmin expression was down-regulated. Thus, increased stathmin phosphorylation is not required for cell growth arrest or differentiation or down regulation of stathmin expression. PMID- 7538936 TI - Demonstration of human Borna disease virus RNA in human peripheral blood mononuclear cells. AB - BDV naturally infects horses and sheep, and causes sporadic neurological disease. Serological evidence suggests an association of BDV, or a related virus, with specific psychiatric diseases in humans. Here, by using a nested RT-PCR technique, we demonstrate that human BDV RNA is present in the PBMC of psychiatric patients. In an examination of a total of 60 patients from 5 wards of a hospital in Japan, the detection rate differed within each ward, ranging from 8% to > 50% (37% on the average). Of particular note was the finding that the human derived BDV sequences, which included deleted forms in about 23% of the positive samples, were slightly different from those derived from horse BDV. These results suggest urgent consideration of the measures to be taken to cope with the effects of blood transfusion. In addition, the detection of a high level of BDV in the PBMC of patients will help our understanding of the pathogenesis in the disease. PMID- 7538939 TI - Diagnosis of preterm premature rupture of the membranes using a newly developed AFP monoclonal antibody test kit. AB - OBJECTIVE: We have developed a new anti-AFP monoclonal antibody kit which is easier to use than other examination methods for detecting AFP in the leaked amniotic fluid. In this study, we investigate the clinical value of this test in the diagnosis of preterm premature rupture of the membranes (PROM). METHODS: We employed 103 patients of less than 37 weeks of gestational age for this preliminary study. We compared the clinical usefulness of this new AFP test with that of the nitrazine test and measured the concentration of AFP in vaginal fluid or cervical secretion by EIA. RESULTS: The nitrazine test showed a correct diagnostic rate of 62.1%, in contrast the AFP test kit had a 98.0% rate (P < 0.001). The reaction time using the kit is 3 min. CONCLUSION: The AFP test is a simple and non-invasive test which can be easily carried out repeatedly as a bedside examination. This study has confirmed the high clinical efficacy of the newly developed AFP test kit as a method of PROM diagnosis. PMID- 7538940 TI - Evidence for direct modification of NF kappa B by the tyrosine kinase inhibitor, herbimycin A. PMID- 7538941 TI - Mercuric chloride modulates [3H]AMPA binding to Xenopus brain membranes. PMID- 7538943 TI - Nitric oxide synthase activation is a unique mechanism of garlic action. PMID- 7538942 TI - Exercise induced modulation of immune parameters. PMID- 7538945 TI - Mutational analysis of the epitopes recognized by anti-(rat CD2) and anti-(rat CD48) monoclonal antibodies. PMID- 7538944 TI - LHRH-induced tyrosine phosphorylation and MAP kinase activation in alpha T3-1 cells. PMID- 7538946 TI - Salt sensitivity and arginine toxicity in Aspergillus nidulans. PMID- 7538947 TI - The effect of colchicine on cyclic AMP accumulation in NG108-15 cells. PMID- 7538948 TI - Effect of cAMP elevation on the NPY gene transcription. PMID- 7538949 TI - Beta-amyloid inhibition of MTT reduction is not mimicked by inhibitors of mitochondrial respiration. PMID- 7538950 TI - Aminopeptidase P: cation activation and inhibitor sensitivity are substrate dependent. PMID- 7538952 TI - Agonist regulation of high affinity [3H] forskolin binding as a measure of GS alpha-adenylyl cyclase interactions. PMID- 7538951 TI - Interaction of the beta 2-adrenoceptor with epitope-tagged Gs alpha in NG108-15 cells. PMID- 7538953 TI - Expression of epitope-tagged D2 dopamine receptors in Sf21 cells. PMID- 7538955 TI - Pharmacological characterisation of high affinity [35S]GTP gamma S binding in membranes from CHO-K1 cells stably expressing rat D2(short) dopamine receptors. PMID- 7538954 TI - Comparison of [3H]nemonapride and [3H]spiperone binding to D2(long) dopamine receptors. PMID- 7538956 TI - Tachykinin non-peptide antagonists: binding domain and molecular mode of action. PMID- 7538958 TI - [Hematoma of the abdominal wall as differential diagnosis of cystic pelvic tumor]. AB - In a woman patient aged 80 years under anticoagulation with dicumarol (Marcumar), abdominal pain suddenly occurred which was located on the right side as well as signs of acute bleeding. Preoperative sonography and computer scan showed a large, cystic tumour, most likely originating from the right ovary with infiltration of the abdominal wall. Intraoperative diagnosis was a large haematoma of the abdominal wall and the retroperitoneum penetrating into the free abdomen. PMID- 7538959 TI - [Hemodilution in treatment of intrauterine dystrophy]. AB - It was the aim of the study to show that HES (Hydroxy-Ethyl-Starch) in intrauterine dystrophy identified by ultrasonography can increase fetal growth. Twenty pregnant women were treated in two groups (outpatients versus inpatients). Clinically, the result was satisfying. PMID- 7538957 TI - CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines. AB - In the present report, we have investigated the role of DNA methylation on the binding and trans-acting properties of transcription factors involved in the regulation of the rat prolactin (rPRL) gene, specifically Pit-1. To this aim we took advantage of a model system composed of three GH3 rat pituitary tumor cell lines that greatly differed in the extent of rPRL gene methylation and in the level of rPRL gene expression. Northern blot analyses indicated that identical species of Pit-1 mRNA were present to similar extent in the three GH3 cell lines. Electrophoretic mobility shift assays further demonstrated that Pit-1 was present in nuclear extracts and displayed equal affinities to bind the 1P responsive element encompassing the -65 to -38 region of the rPRL promoter, whatever the GH3 cell line tested. These data suggested that differential expression of the rPRL gene among cell lines did not result from variable amounts of Pit-1. By combining in vitro methylation and transient transfection experiments with a rPRL promoter driven CAT construct, we showed that extensive methylation at CpG sites abolished the expression of the reporter gene. Furthermore, in vivo competition assays demonstrated that CpG methylation inhibited gene expression by preventing the binding of transcription factors We propose that related mechanisms linked to DNA methylation might alter the activity of the endogenous PRL gene in the low expressing cell line. PMID- 7538960 TI - [Doppler ultrasound findings in hemodilution with hydroxyethyl starch in intrauterine fetal retardation]. AB - The surveillance of IUGR is one of the main indications of Doppler ultrasound for use in obstetrics. The volume replacement with hydroxyethyl starch is an effective therapy against plasma volume contraction diagnosed by an increase of hematocrit above 38%, which is often combined with IUGR. 48 patients with those problems were treated with middle molecular hydroxyethyl starch for at least 10 days. Doppler ultrasound was used before, within and after therapy. 39 of 48 patients (81%) showed pathological Doppler results in at least one of the examined vessels (uterine artery, umbilical artery, fetal aorta and middle cerebral artery). There is a correlation between normalisation of Doppler results, decrease of hematocrit and increase of the fetal growth indicated by ultrasound or birth weight, respectively. This therapy seems to be useful if it starts between 28th and 34th week of pregnancy; if we find at most two vessels with pathological Doppler results; and if there is an improvement of the Doppler results within therapy. A poor effect is to be expected if therapy begins after 36 weeks of pregnancy, if no change in pathological Doppler results can be found, if fetal vessels show pathological Doppler results and if there is a "notching" in the uterine artery. The group of patients with intrauterine growth retardation can be specified by Doppler ultrasound, which may profit from volume replacement. Doppler examinations are helpful to fetal surveillance within this therapy. PMID- 7538961 TI - Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3. AB - The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl polyoxyethylene media. Both proteins, characterized by the same size, heat modifiability and N-terminal sequence were re-incorporated in virtually solvent free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3). PMID- 7538962 TI - FKBP39, a Drosophila member of a family of proteins that bind the immunosuppressive drug FK506. AB - A cDNA, coding for the first Drosophila melanogaster homolog of a family of proteins (FK506-binding proteins, FKBPs) which bind to the immunosuppressive drug FK506, was isolated. The deduced aa sequence corresponds to a 39-kDa product (FKBP39) which, besides a domain with similarity to FKBPs, has a highly charged domain with two strongly acidic stretches. The transcript could be detected in all developmental stages, with the highest expression in the embryo. In adult flies, the strongest signal was detected in the ovaries. Although the FKBP39 gene is expressed in the immunocompetent D. melanogaster blood cell line, mbn-2, the antibacterial defense reaction of these cells is unaffected by FK506. PMID- 7538963 TI - A novel alternative splice domain in zebrafish tenascin-C. AB - We have identified and sequenced clones from a zebrafish library that correspond to tenascin-C (TN-C). The 2036-bp sequence covers the C-terminal segment of the protein. Comparison of this sequence to TN-C from other vertebrates indicates that our sequence starts in FN-III domain 5, continues with one alternative splice domain, then with FN-III domains 6-8 and, finally, the complete fibrinogen like domain. The alternative splice domain, designated TNfnQ, is not closely identified to any of the domains in other species, and may represent a novel descendent of the alternative splice-domain precursor. PMID- 7538965 TI - Effect of histamine and substance P on the rabbit and human iris sphincter muscle. AB - BACKGROUND AND METHODS: In an attempt to clarify the functional action of histamine and substance P on atropine-resistant miosis, we isolated rabbit and human iris sphincter muscles and investigated their mechanical properties using the isometric tension recording method. RESULTS: Substance P dose-dependently contracted the rabbit iris sphincter, but had no effect on the human iris sphincter. In the rabbit iris sphincter, histamine reduced the amplitude of twitch contraction evoked by field stimulation but had no effect on carbachol induced contraction. Thioperamide, but not mepyramine or cimetidine, partially antagonized the histamine-induced reduction in the amplitude of twitch contractions. In the human iris sphincter, on the other hand, histamine dose dependently provoked contraction and the amplitude of histamine-induced contraction was affected neither by atropine nor by indomethacin. CONCLUSIONS: These results provide evidence that histamine has strong contractile effect on the human iris sphincter muscle; the rabbit iris sphincter muscle, however, apparently lacks functional histamine receptors. In rabbits, exogenously applied histamine only activates H3 receptors located on the cholinergic nerve terminal, hence the excitatory neuro-effector transmission is suppressed. Thus, histamine may have an important roles in atropine-resistant miosis in humans, but not in rabbits. PMID- 7538964 TI - Cellular fibronectin and tenascin in experimental perforating scleral wounds with incarceration of the vitreous. AB - BACKGROUND: Posterior perforating eye injury carries a high risk of visual loss due to the formation of intravireal and epiretinal scar tissue. Intraocular scar formation in patients with retinal detachment has been shown to be associated with elevated intravitreal FN levels. The extracellular matrix glycoproteins fibronectin (FN) and tenascin (TN) have been located in epiretinal scar membranes. As both FN and TN are also involved in healing of cutaneous and corneal wounds, we undertook to study their expression in rabbit perforating scleral wounds with vitreous incarceration. METHODS: A perforating scleral wound was produced and sutured without removal of vitreous from the wound in 18 pigmented rabbits. The rabbits were killed at various times (1 h to 21 days) after the operation, and the indirect immunohistochemical method was used for demonstration of FN and TN. Monoclonal mouse hybridoma antibodies 52 DH1 and 100 EB2, recognizing the cellular form of FN (cFN) and TN, respectively, were used. RESULTS: During the first post-operative week immunoreaction for glycoproteins, both the locally produced cFN and TN, were observed at the scar tissue containing the prolabed vitreous and the adjacent sclera. Subsequently, the reaction gradually shifted to the vitreal side of the wound, and 3 weeks after the operation it was almost completely restricted to a separated mass of vitreous beneath the scar. CONCLUSION: The expression of cFN and TN in the scleral scar and vitreous is indicative of their local synthesis. The shift of the expression of those proteins to the vitreal side of the wound with time suggests that the scarring process in the vitreous is delayed compared to the sclera. PMID- 7538967 TI - In vivo administration of stem cell factor enhances both proliferation and maturation of murine megakaryocytes. AB - BACKGROUND: Stem cell factor (SCF) has already been shown to participate in the regulation of erythro- and granulopoiesis. The aim of this study was to define the possible role of SCF in the regulation of megakaryocytopoiesis. METHODS: Stem cell factor activity has been assessed in an in vivo murine model, in which different doses of the factor were either given alone or in association with recombinant human erythropoietin (rhEpo). Mice were sacrificed after a six-day treatment to evaluate the effect of SCF on the number of bone marrow and spleen colony-forming units-megakaryocyte (CFU-Mk), and after a two-day treatment for evaluation of thrombopoietin-like activity. RESULTS: We found that SCF induces a dose-related increase in the number of CFU-Mk in both the bone marrow and spleen of the treated mice, and that in the range of the doses used (from 25 to 200 mg/kg/day) the greatest activity was observed when a dose of 200 mg/kg/day was injected. The effect was enhanced by adding rhEpo to optimal SCF concentrations. SCF also stimulated megakaryocyte maturation as assessed by the megakaryocyte number, the size of acetylcholinesterase-positive cells, 35Sulphur (35S) incorporation into the newly formed platelets. All these parameters were only minimally affected by the addition of rhEpo. CONCLUSIONS: These data suggest that SCF participates in the regulation of megakaryocytopoiesis and that its administration might have a role in the treatment of disorders of platelet production. PMID- 7538966 TI - Superoxide production by neutrophils in children with malignant tumors treated with recombinant human granulocyte colony-stimulating factor. AB - BACKGROUND: Human recombinant granulocyte colony-stimulating factor (rhG-CSF), widely used to combat chemotherapy-induced neutropenia, stimulates both in vivo and in vitro intra- and extra-cellular O2- production in human polymorphonuclear cells (PMNs). PATIENTS AND METHODS: Twelve patients with solid tumors or acute lymphoblastic leukemia were treated during induced aplasia with rhG-CSF (5 micrograms/kg/day). Intra- and extracellular O2- production by PMNs isolated from these patients after 5 days of rhG-CSF therapy was assessed following both fMLP and PMA stimulation. RESULTS: All patients showed a rise in PMN count; administration of rhG-CSF enhanced intra- and extracellular O2- release after fMLP but not after PMA stimulation. CONCLUSIONS: rhG-CSF potentiates in vivo O2- production by PMNs stimulated with receptor-mediated agonists via G-protein (e.g. fMLP), but not by those stimulated with agonists that bypass receptors via protein kinase C (e.g. PMA). PMID- 7538969 TI - [Idiopathic external hydrocephalus]. AB - 5 male children who had idiopathic external hydrocephalus are described; all were all in the first year of life. Macrocephaly and hypotonia were present in all, as well as mild to moderate delay in language and gross motor development; cognition was normal. CT-scan showed in all enlarged subarachnoid spaces but normal or mildly dilated lateral ventricles. Macrocephaly was present in the fathers of 4 of the 5 families. In all cases the course of this condition was benign. There was clinical and roentgenological improvement between the ages of 2 and 2.5 years; the developmental delay resolved at about age 4, at which time the CT pattern had become normal. PMID- 7538968 TI - Allogeneic transplants of rhG-CSF-mobilized peripheral blood stem cells (PBSC) from normal donors. GITMO. Gruppo Italiano Trapianto di Midollo Osseo. AB - There is a growing interest in the use of peripheral blood stem cells (PBSC) for allogeneic transplantation. This is due in part to the idea that, as with autologous transplantation, increasing the number of allogeneic hemopoietic progenitors infused may lead to reduced complications. However, introducing the PBSC technique into allogeneic transplants implies theoretical as well as ethical problems involving both patient and donor. We are still uncertain whether the PBSC technique will result in an increase of GVHD or (better) of GVL. G-CSF, necessary for effective PBSC mobilization, is safe but its use in normal subjects should be regarded with caution. For this reason, a Study Committee promoted by the GITMO (Gruppo Italiano Trapianto di Midollo Osseo) evaluated the key aspects of allogeneic PBSC collection and transplantation. The present paper summarizes the scientific data and suggests some guidelines for the introduction of allogeneic PBSC transplantation into clinical practice. The procedure should be considered experimental and the Committee strongly recommends the use of allogeneic PBSC in experienced centers, initially in patients with advanced disease. The donor should be given a complete explanation of the advantages and risks of G-CSF therapy, leukapheresis and general anesthesia. A careful monitoring of both patient and donor should also be included to watch for short term and long-term side effects. PMID- 7538970 TI - Autoantibodies to insulin and to proinsulin in type 1 diabetic patients and in at risk probands differentiate only little between both antigens. AB - To answer the question whether insulin or proinsulin would be the true antigen for both insulin and proinsulin autoantibodies, displacement experiments of 125I insulin and -proinsulin binding with both unlabeled antigens were performed in sera of four groups of antibody-positive probands: first-degree relatives of Type 1 diabetic patients, pre-Type 1 diabetic persons, recent-onset Type 1 diabetic patients, insulin-treated Type 1 diabetic patients. In subjects who were primarily screened to constitute these groups, prevalences of insulin and proinsulin autoantibodies were nearly identical. In antibody-positive sera, 125I insulin and -proinsulin binding values in general were closely correlated to each other with regression coefficients near 1.0. In all groups of probands, mean values of 125I-insulin and -proinsulin binding did not significantly differ. With the exception of a few sera, insulin and proinsulin antibodies differentiated only little between both antigens. Epitopes of the insulin molecule are therefore preferred. Nevertheless, insulin and proinsulin autoantibodies are not completely identical nor are insulin autoantibodies merely a subgroup of proinsulin autoantibodies: In each group, in the mean, insulin antibodies as well as proinsulin antibodies reacted somewhat (but significantly) stronger with their respective antigen. In some cases a distinct (relative) specificity for either antigen of insulin and proinsulin autoantibodies were observed, the latter being still present after some months of insulin treatment. In conclusion, despite detectable differences in antigen specificity, insulin and proinsulin autoantibodies seem to be equally potent markers of Type 1 diabetes mellitus. PMID- 7538971 TI - Number and sex of offspring of delta F508 carriers outside cystic fibrosis families. AB - Number and sex of offspring were determined in a group of 7,841 randomly selected blood donors who were screened for the delta F508 mutation. We did not find any evidence for differences in number or sex ratio of offspring between delta F508 carriers and non-carriers. PMID- 7538973 TI - An improved method for chemical devitellinization of X-gal stained Drosophila embryos. AB - In Drosophila developmental biological studies, X-gal staining is commonly employed to study the spatio-temporal expression of the lacZ reporter gene in the transformed flies or their embryos. Study of the lacZ pattern in embryos often suffers from the lack of an efficient and high yielding technique for devitellinization of X-gal stained embryos. Devitellinization techniques employed during antibody staining, in situ hybridization or embryonic cuticular preparations generally do not give satisfactory results when used for similar purpose in X-gal stained embryos. This results in the flaky appearance of the blue stain. We present here an improved chemical devitellinization technique which gives a high yield of devitellinized embryos and a better resolution of the X-gal staining pattern. PMID- 7538972 TI - Hexachlorocyclohexane-induced changes in lipid peroxidation, superoxide dismutase and catalase activities and glutathione content in chick liver. AB - An elevation in the level of lipid peroxides in nuclear, mitochondrial and microsomal fractions of chick liver was recorded 6 hr after hexachlorocyclohexane (HCH; 50 mg/kg body wt., ip) treatment. The magnitude of increase remained more or less same even 24 hr after the pesticide treatment. Total glutathione content also increased by 6 hr of HCH treatment and did not change even 24 hr after the pesticide treatment. Protein content of crude homogenate and 10000 g supernatant decreased significantly 6 hr after the pesticide treatment. The magnitude of decrease was more or less same even 24 hr after the pesticide treatment. Although cytoplasmic superoxide dismutase and catalase activities (expressed as units/mg protein) did not change 24 hr after HCH treatment, a small but significant increase in superoxide dismutase activity was recorded 6 hr after HCH treatment. On the other hand when activities were expressed as units/mg tissue wt., a significant decrease in the activities of both the enzymes was recorded 6 and 24 hr after HCH treatment. Therefore, the decrease in the activities of both the enzymes in response to HCH in chick liver may be due to decrease in tissue protein content in general rather than specific decrease in the activities of the enzymes. PMID- 7538974 TI - Tenascin-C in serum: a questionable tumor marker. AB - In normal adult tissue tenascin-C (TN-C) is usually expressed at low levels. However, it is strongly induced in many tumors as well as in other pathological conditions often associated with inflammation. To evaluate the diagnostic significance of TN-C, we established a sensitive sandwich ELISA to determine TN-C levels in serum. Furthermore, we investigated the distribution of TN-C variants in serum and found the large TN-C isoforms to be predominant. We measured TN-C in sera from 15 healthy persons, 75 tumor patients and 84 patients selected due to their elevated levels of the acute-phase protein C-reactive protein (CRP), which is a very specific marker for infection and inflammation. It was found that sera from cancer patients can have elevated TN-C levels; however, the increase was more pronounced in persons with high levels of CRP. There appeared to be a correlation of TN-C levels with the levels of CRP. In view of these facts, the diagnostic value of TN-C levels in serum as a potential tumor marker seems to be questionable, since our data show that TN-C levels can be elevated as a consequence of infection and inflammation. PMID- 7538976 TI - CD69+ and HLA-DR+ activation antigens on peripheral blood lymphocyte populations in metastatic breast and ovarian cancer patients: correlations with survival following active specific immunotherapy. AB - Lymphocyte activation markers CD69 and HLA-DR were studied in metastatic breast and ovarian cancer patients who received active specific immunotherapy (ASI) using cancer vaccines containing the synthetic tumor-associated antigen sialyl-Tn or the Thomsen-Friedenreich antigen conjugated to KLH plus DETOX adjuvant. Breast cancer patients who showed prolonged survival following ASI had lower numbers of total CD69+ and CD4+CD69+ cells prior to ASI compared to patients who died. However, following ASI, the surviving patients showed an increase in CD69+ and CD4+CD69+ cells and the deceased patients showed a decrease. A greater than 50% increase in the percentage of cells bearing the activation marker CD69 is associated with an increase in survival in both ovarian and breast cancer patients. In the surviving breast cancer patients there was a significant decrease in the percentage of non-B lymphocyte HLA-DR+ (CD20-HLA-DR+) cells following cyclophosphamide treatment. A strong positive correlation was found between lymphocyte populations CD20- HLA-DR+ and CD8+CD57+, a putative suppressor cell population. Breast cancer patients who showed a greater than median decrease in CD20-HLA-DR+ lymphocytes following cyclophosphamide treatment had a survival advantage over patients who had less than the median decrease in the percent CD20 HLA-DR+ lymphocytes. PMID- 7538975 TI - Expression of E-selectin on endothelial cells of small veins in human colorectal cancer. AB - E-selectin is an adhesion molecule of endothelial cells that binds to cancer cells mediated by sialyl Lewis A (sLea) or sialyl Lewis X (sLe(x)). It is suspected to be involved in hematogenous metastasis of tumors. Therefore, it is worth examining E-selectin expression in human colorectal cancer and its hepatic metastasis. In the present study, E-selectin was clearly revealed on the endothelial cells of small vessels adjacent to cancer nests both in primary and in metastatic nests in immunohistochemistry. In these tissues, E-selectin was observed on the endothelial cells lining the lumen of small vessels. Its expression adjacent to cancer nests appears to be induced through some stimuli by cancer cells, since its degree of expression is inversely correlated to the distance of the blood vessels from the cancer nests (p < 0.001). Endothelial cells adjacent to the metastatic lesion expressed E-selectin more extensively than those adjacent to the primary foci. This is also in line with the finding on serum E-selectin levels which were significantly elevated in the metastatic group as compared with the non-metastatic group. The serum E-selectin level may provide useful information in the diagnosis for hepatic metastasis of colorectal cancer, although the results are still tentative. PMID- 7538977 TI - Alpha v-integrins in human melanoma: gain of alpha v beta 3 and loss of alpha v beta 5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice. AB - We investigated the expression of alpha v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that alpha v beta 5 was the alpha v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of alpha v beta 5 expression was related to melanocytic tumor progression. In line with earlier reports, alpha v beta 3 was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-alpha v monoclonal antibodies (MAbs) in lesions where both alpha v beta 3 and alpha v beta 5 were absent showed that alternative alpha v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of alpha v beta 5 and alpha v beta 3 in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed alpha v beta 5 at their surface. Surprisingly, alpha v beta 3 was detected exclusively in the non-metastatic cell lines. Absence of alpha v beta 3 in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from 35S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that alpha v beta 5 expression is often lost in advanced stages of melanocytic tumor progression in situ, while alpha v beta 3 is acquired, but that a decrease in alpha v beta 5 and an increase in alpha v beta 3 expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice. PMID- 7538979 TI - Escape mechanisms of human leukemic cells to long-term immunotoxin treatment in an in vitro experimental model. AB - In kinetic assays, an anti-CD5-ricin A chain (ST.I-RTA) immunoconjugate (immunotoxins, IT) specifically inhibited up to 40% the protein synthesis of Jurkat target cells within the first 40 hr. Longer exposures of leukemia cells to ST.I-RTA resulted in a progressively higher number of target cells escaping IT treatment and becoming resistant to further treatment with ST.I-RTA even in the presence of the RTA-IT enhancer monensin. Resistant Jurkat cells proliferated at the same rate as control untreated cells, and were as sensitive as control cells to a transferrin-RTA IT, indicating that the ST.I-RTA-resistant tumor-cell population did not become insensitive to the enzymatic activity of RTA. Binding studies revealed that the anti-CD5 IT treatment induced a transient modulation of CD5 antigens but not of the functionally related CD3 antigens. The CD5 antigens were re-expressed at the cell surface following removal of the IT molecules from the culture medium with 1.1% of the total CD5 Ag being re-expressed per hr. When our experimental data on the kinetics of cell intoxication by the IT were corrected for the proliferative potential of the resistant and of the sensitive tumor-cell populations, it appeared that the effect of ST.I-RTA treatment on Jurkat cells was only to delay cell growth for a limited time period (20 hr) without reducing effectively the tumor-cell burden. Our results may have implications for the long-term treatment of target tumor cells with IT. PMID- 7538980 TI - Expression of protectin (CD59) in human melanoma and its functional role in cell- and complement-mediated cytotoxicity. AB - Immunohistochemical and/or indirect immunofluorescence analysis with monoclonal antibody (MAb) H19 demonstrated the expression of protectin (CD59) in 54 surgically removed metastatic melanoma lesions and on 8 out of 12 melanoma cell lines. CD59 expression had a low degree of intra- and intertumor heterogeneity. SDS-PAGE analysis showed that the molecular weight of CD59 expressed on melanoma cells is about 20 kDa. Treatment of melanoma cells with 5U/ml of phosphatidylinositol-specific phospholipase C completely abolished cell-surface expression of CD59. Interferon-gamma and/or tumor necrosis factor-alpha or phorbol 12-myristate 13-acetate neither modulated the expression of CD59 by melanoma cells nor influenced the amounts of CD59-specific mRNA. F(ab')2 fragments of anti-CD59 MAb YTH53. I did not inhibit the lysis of melanoma cells by allogeneic natural killer (NK) cells or lymphokine-activated killer (LAK) cells. In contrast, the whole Ig molecule of MAb HI9 or YTH53.I significantly (p < 0.05) enhanced NK-cell-mediated lysis of melanoma cells, suggesting the induction of antibody-dependent cell-mediated cytotoxicity. Lastly, masking of CD59 by MAb YTH53.I or its F(ab')2 fragments significantly (p < 0.05) enhanced, in a dose-dependent fashion, the lysis of anti-GD3-sensitized melanoma cells by homologous complement. These data demonstrate that CD59 expressed by human melanoma cells might regulate host-tumor interaction by protecting neoplastic cells from complement-mediated lysis. PMID- 7538978 TI - Bispecific monoclonal antibody anti-CD3 x anti-tenascin: an immunotherapeutic agent for human glioma. AB - Besides surgery, the therapeutic possibilities for the treatment of human gliomas include adoptive cellular immunotherapy, radioimmunotherapy, immunotherapy mediated by chemoimmunoconjugates and, more recently, bispecific monoclonal antibodies (biMAbs). Anti-CD3 x anti-tenascin (TN) is the first reagent of a number of biMAbs under investigation for prospective use in vivo to maximize the cell-mediated cytolytic potential of glioma patients. This biMAb originated from the fusion of 2 parental hybridomas, made resistant by retrovirus-mediated infection to the different metabolic drugs, geneticin and methotrexate, respectively. The resulting hybrid hybridomas were selected on the basis of the double specificity for CD3 and TN, cloned several times and grown under continuous metabolic pressure. The different families of recombinant antibodies were then purified by high-pressure liquid chromatography on hydroxylapatite columns. Immunohistochemical studies on tumor specimens of different origin and histotype have shown that the selected biMAb presented a distribution pattern similar to that of the parental anti-TN MAb, maintaining the same staining homogeneity and intensity. Moreover, the mitogenic activity of anti-CD3 x anti-TN biMAb on peripheral blood mononuclear cells was similar to that featured by the parental anti-CD3 MAb. Furthermore, the hybrid molecule induced TNF-alpha gene expression in activated PBMC. Finally, the anti-CD3 x anti-TN featured the desired killer targeting ability, being able to induce a significantly increased cytotoxic activity against TN+ tumor cells. PMID- 7538981 TI - Relationship between histologic grading and serum prostate specific antigen in prostatic carcinoma. AB - Prostate specific antigen (PSA) is the most important tumour marker which, used routinely, proved to have a positive correlation with tumour volume and pathological stage. To evaluate the relationship between serum PSA and histologic grading of prostatic carcinoma, preoperative PSA determinations were made in 25 patients with prostatic cancer. Biopsy materials were evaluated and the Gleason scores were adjusted for histologic grading. The higher values of PSA were proportional to the Gleason scores of the primary prostatic biopsy. PMID- 7538982 TI - The new syndrome of congenital hypoparathyroidism associated with dysmorphism, growth retardation, and developmental delay--a report of six patients. AB - We describe six children of Arab origin with a syndrome of congenital hypoparathyroidism, severe growth retardation, developmental delay, and dysmorphism. The most prominent dysmorphic features were microcephaly, facial and dental anomalies, and small hands and feet. Bone age was markedly retarded. Neither immunological nor chromosomal abnormalities were found. Insulin-like growth factor-1 serum levels, measured in two patients, were abnormally low. PMID- 7538983 TI - Six year neurodevelopmental follow-up of very low birthweight children. AB - Twenty-four children born preterm with very low birthweight (VLBW) in 1985 at Bikur Holim Hospital were followed until age 6 years. Their neurological status and developmental and cognitive abilities were examined at 1, 2 and 6 years of age respectively and were compared with a control group at age 6 years. Of the 24 VLBW children, 4 had major disabilities. Of those without major disabilities, mean total IQ (WPPSI) at six years was 101.5 +/- 11.3, not significantly different from their mean Mental Development Index (Bayley) at age 2 years which was 96.1 +/- 19.6, or from the mean total IQ of a control group of 6-year-old children which was 109.8 +/- 14.7. However, the mean verbal IQ of the VLBW children (95.3 +/- 11.7) was significantly lower than that of the control group (106.2 +/- 14.3) (P = 0.02). Minor neurological deficit was found in seven of the VLBW children and in only one of the controls (P = 0.05). These findings point to possible future learning difficulties and should alert both pediatricians and educationalists to the importance of long-term follow-up of VLBW children in order to identify and address their specific educational needs. PMID- 7538984 TI - Survival rate and 2 year outcome in very low birthweight infants. AB - The purpose of this study was to determine the mortality rate of 282 infants with a birthweight (BW) of < 1,500 g during a 5 year period (1987-92), and to evaluate 2 year neurodevelopmental outcome in 114 infants (born 1987-90) and its correlation with intracranial pathologies diagnosed by ultrasonography. Overall survival rate was 76.6% (216/282). With the introduction of exogenous surfactant therapy in 1991 a significant increase in survival was noted in the 751-1,000 g BW infants (from 20/36 to 20/22, P < 0.002). Two year evaluation was performed in 114 of 119 infants (95.7%) who survived this age. Twenty-two children (22/114, 19.3%) were functionally disabled, of whom 15 (13%) had a major disability. Of these 15 infants, 5/28 weighed < 1,000 g and 10/86 weighed 1,001-1,500 g (P = 0.6). Severe intraventricular hemorrhage (IVH) and cystic periventricular leukomalacia (PVL) were significantly associated with adverse outcome at 2 year (5/11 and 10/10 respectively were handicapped, P < 0.001). We therefore conclude that every effort should be made to save the extremely premature infants (< 1,000 g BW). Since adverse outcome was significantly associated with severe brain pathologies (large IVH and cystic PVL) rather than with lower BW, brain ultrasonography as a diagnostic mode should be taken into clinical consideration. PMID- 7538985 TI - Outcome of very low birthweight infants. PMID- 7538986 TI - [Multiple palmoplantar keratoses, basaliomas and porocarcinomas after arsenic therapy]. PMID- 7538987 TI - The social context of critical care clinical judgment. AB - BACKGROUND: Clinical judgment in critical care is supported by a rich social network of care providers. The purpose of this study was to describe the social context in which the process of critical care clinical judgment occurs from the nurse's perspective. METHODS: An ethnographic study was conducted that included interviews with 10 nurses and participant observation in an open heart surgery unit with 59 nurses and two surgical teams during a 2-year period. RESULTS: Nurses and physicians were organized in hierarchies of nurse manager, resource nurse, charge nurse, and staff nurse or attending surgeon, fellow, chief resident, and resident. These parallel hierarchies allowed for checks on judgment both within and across professional lines. Rituals, such as nursing report, physician rounds, and flow sheet use, provided a context for a critique on judgment processes. Communication of judgment was frequently a casual, open conversation. At other times, differences in perspective could result in conflict. Communication between nurses and physicians has been associated with better patient outcomes. Critical care unit directors and managers can use an analysis of communication patterns to develop supports to clinical judgment. PMID- 7538988 TI - The same esterase B1 haplotype is amplified in insecticide-resistant mosquitoes of the Culex pipiens complex from the Americas and China. AB - In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B, and overproduction of all esterases B is due to gene amplification. In order to determine if the esterase B1 identified by electrophoretic studies in Culex pipiens mosquitoes from different countries is overproduced due to the amplification of the same DNA haplotype, the amplified region encompassing the structural esterase B1 gene was characterized by restriction mapping and RFLP. The same amplified haplotype was found in mosquitoes with an esterase B1 protein, independently of their geographical origin: French Guiana, Venezuela, Puerto Rico, California and China. Large variations in amplification levels were observed. It is concluded that B1 amplification has a unique origin, either in America or in Asia, and has subsequently spread by migration. This migration is more limited than that of A2 B2 esterases, since B1 is confined to the Americas, the Caribbean and part of China, whereas the A2-B2 distribution now includes the Americas, the Caribbean, Asia, Africa, the Pacific Islands and Europe. PMID- 7538989 TI - Developmental considerations of adolescents with developmental disabilities. AB - Adolescents with developmental disabilities have pertinent developmental and psychosocial needs that require consideration for optimal long-term planing and intervention. Unless the special needs of these adolescents are addressed, obstacles to living meaningful and satisfying lives will occur. This article examines the major issues facing adolescents with developmental disabilities. Nursing implications are discussed. PMID- 7538990 TI - Cellular and antibody responses to equine herpesviruses 1 and 4 following vaccination of horses with modified-live and inactivated viruses. AB - The ability of monovalent and bivalent equine herpesvirus (EHV) vaccines to stimulate cellular and antibody responses to EHV-1 and EHV-4 was compared in healthy horses. Comparison of data from lymphocyte blastogenesis tests in which live viruses were used as antigens and that were conducted prior to vaccination and after 2 vaccinations revealed that horses given modified-live EHV-1 had significant increases in proliferative responses to EHV-1 (P = 0.03) and EHV-4 (P = 0.04). Responses to EHV-1 and EHV-4 in horses given the inactivated-virus bivalent vaccine were less; however, significant differences were not noticed when postvaccinal lymphocyte blastogenesis tests were compared between the groups of vaccinees. Interleukin-2 activity was not detected in leukocyte cultures from either group of vaccines following stimulation with live EHV-1 or EHV-4; however, interferon activity was found in similar cultures from both groups of vaccinees. For EHV-4, interferon activity in cultures from both groups of vaccinees was significantly (P < 0.05) greater than that in leukocyte cultures from unvaccinated controls. Both vaccines induced significant (P < 0.05) increases in serum antibodies that neutralized EHV-1 infectivity. The ELISA for EHV-1 and EHV 4 antibodies revealed that both vaccines induced significant (P < 0.05) increases (compared with preinoculation values) in antibodies reactive with these 2 types of EHV. Total serum antibody responses, as measured by ELISA, to EHV-1 and EHV-4 were significantly (P < 0.05) higher in horses that received the bivalent inactivated-virus vaccine, compared with that in horses that received monovalent vaccine. Evaluation of these data revealed that vaccination with modified-live EHV-1 can stimulate cellular and antibody responses that cross-react with EHV-4. PMID- 7538991 TI - A PstI restriction fragment length polymorphism in sheep detected with a human myelin basic protein (MBP) probe. PMID- 7538992 TI - Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods. AB - DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella. PMID- 7538993 TI - Expression of novobiocin resistance genes in the novobiocin-producing organism Streptomyces niveus. AB - RNA isolated at intervals during fermentation from the novobiocin-producing wild type strain of Streptomyces niveus and from a series of novobiocin-non-producing (Nov-) mutants was hybridized to DNA probes containing sequences which specify novobiocin resistance. The probes were made from inserts contained in the clones pGL101 and pGL103 which increase the level of novobiocin resistance of S. lividans transformants from 10 micrograms ml-1 to 50 micrograms ml-1 and 150 micrograms ml-1, respectively. No hybridization was detected with the pGL101 probe. The pGL103 probe hybridized to RNA extracted during the later stages of growth--a pattern corresponding to the transition from low to high level novobiocin resistance during growth of S. niveus wild-type cultures. Neither probe hybridized to RNA extracted from four Nov- mutants. These mutants showed variable levels of novobiocin resistance but none expressed the high wild-type levels. The authors conclude that expression of the DNA sequence in pGL103 is associated with high level novobiocin resistance. PMID- 7538994 TI - Responses of brown adipose tissue activity to unloading in rats. AB - To study the responses of thermogenic activity in brown adipose tissue (BAT) to unloading, male Wistar rats were hindlimb suspended for 10 days. Compared with control rats, a significant increase in the BAT-to-body mass ratio and considerable differences in chemical components in BAT were observed in the hindlimb-suspended rats. These findings indicate a marked increase in the thermogenic capacity in BAT of the experimental group. Likewise, the thermogenic activity (which was assessed by guanosine 5'-diphosphate binding to BAT mitochondria) was markedly greater in the mitochondria recovered from BAT of the hindlimb-suspended rats than in those from the control rats (1,610 +/- 450 vs. 202 +/- 132 pmol recovered). Moreover, the uncoupling protein content in the BAT mitochondrial fraction of the hindlimb-suspended rats was significantly higher (1.6-fold) than that in the control rats. As was expected, the uncoupling protein mRNA expression was greater in hindlimb-suspended rats than in control animals. These results suggest that chronic hindlimb suspension leads to an increase in both the thermogenic capacity and the activity in BAT of rats. PMID- 7538995 TI - Inhibition of neutral endopeptidase increases airway responsiveness to ACh in nonsensitized normal rats. AB - The effects of sensory neuropeptides on the airway responsiveness to acetylcholine (ACh) were investigated in normal nonsensitized rats. The airway responsiveness to inhaled ACh was significantly increased after treatment with neurokinin A (NKA; 0.001%) or substance P (SP; 0.01%) aerosol in the presence of the neutral endopeptidase (NEP) inhibitor. NKA had a more potent effect than SP. Interestingly, the intravenous treatment with NEP inhibitor alone also induced airway hyperresponsiveness (AHR) to inhaled ACh. This AHR was significantly attenuated by pretreatment with a nonselective NK-receptor antagonist, [D-Pro2,D Trp7,9]SP, systemic capsaicin, or bilateral cervical vagotomy, indicating that decreased NEP activity results in accumulation of endogenous sensory neuropeptide(s) and enhancement of vagal reflex to cause AHR. The airway responsiveness to ACh of isolated left main bronchus was also increased after treatment with 10(-6) M NKA, but not SP, together with 10(-6) M phosphoramidon. This in vitro AHR to ACh induced by phosphoramidon plus NKA was significantly attenuated by pretreatment with 10(-6) M tetrodotoxin. These findings suggest that overaccumulated sensory neuropeptides, especially NKA, may enhance the probability of transmitter release, probably via NK2 receptors, and that the enhanced transmitter release might be involved in AHR in rats. PMID- 7538996 TI - SP-induced contraction of airway smooth muscle in normal and allergen-sensitized rabbits: mechanism of action. AB - We studied the mechanisms involved in the airway smooth muscle (ASM) contraction to substance P (SP) in normal (control) and allergen-sensitized (immune) rabbits as well as immune rabbits exposed to allergen via the airways (immune challenged). Cumulative concentration-response curves to SP (1 x 10(-9) to 1 x 10(-4) M) were performed in ASM segments in the absence and presence of atropine (10(-5) M) in vitro. The maximal contractile response (g tension/g tissue) at 10( 4) M SP and ASM contractions at various concentrations of SP were expressed as means +/- SE. We found no difference in the contractile response to SP between control and immune animals. ASM segments obtained from immune-challenged rabbits were more responsive to SP. Atropine shifted to the right the concentration response curves and decreased the maximal ASM contraction at 10(-4) M SP in all three groups; this effect, however, was greater in immune-challenged tissues. These findings demonstrate an increased contractile response to SP in immune challenged animals mediated by a more pronounced facilitation of cholinergic neurotransmission. We conclude that the final ASM response to SP is the result of a complex interaction between direct effects on ASM and indirect effects through modulation of cholinergic neurotransmission. PMID- 7538997 TI - Neutral endopeptidase modulates substance P-induced vasodilation in vivo. AB - The purpose of this study was to investigate whether neutral endopeptidase (NEP; EC 3.4.24.11) modulates substance P-induced vasodilation in the oral mucosa in vivo. Using intravital microscopy, we measured the diameter of second-order arterioles (44-70 microns) in the hamster cheek pouch during suffusion of capsaicin and substance P. We found that capsaicin (0.1 and 10.0 nM) induced significant concentration-dependent vasodilations (13 +/- 4 and 39 +/- 7% increase from baseline, respectively; P < 0.05) that were significantly potentiated by phosphoramidon (10.0 nM), a selective NEP inhibitor (35 +/- 15 and 61 +/- 12% increase from baseline, respectively; P < 0.05). Substance P (0.1 and 10.0 nM) also induced significant concentration-dependent vasodilations (7 +/- 3 and 25 +/- 8% increase from baseline, respectively; P < 0.05) that were mediated by the COOH-terminal of the molecule. Substance P-induced responses were significantly potentiated by phosphoramidon (34 +/- 9 and 53 +/- 10% increase from baseline, respectively; P < 0.05) and thiorphan (10.0 microM), a selective NEP inhibitor (44 +/- 11 and 53 +/- 10% increase from baseline, respectively; P < 0.05). Substance P-(1-9) had no significant effects on arteriolar diameter. Suffusion of captopril, leupeptin, Bestatin, and DL-2-mercaptomethyl-3 guanidinoethylthiopropanoic acid together had no significant effects on substance P-induced vasodilation. Phosphoramidon did not potentiate nitroglycerin-induced vasodilation. These data indicate that NEP modulates substance P-induced vasodilation in the hamster cheek pouch in vivo. We suggest that any decrease in tissue NEP activity may amplify neurogenic vasodilation in the oral mucosa. PMID- 7538999 TI - Plasmapheresis combined with interferon: an effective therapy for multiple sclerosis. AB - The rationale for the use of interferon (IFN) in the treatment of multiple sclerosis (MS) is based on its recognized antiviral and immunomodulating actions. The pathogenesis of MS is believed to be due to an immunologic response in a genetically predisposed individual, localized within the central nervous system white matter, and triggered by exposure to an environmental agent such as a virus. Based on our personal experience we find that the efficacy of IFN therapy is hampered in MS patients by the presence of an interferon inhibitor factor (IIF) in the patients' sera which we have isolated and characterized. When plasmapheresis (PP) was done on 24 MS patients with intermittent 3-day administration of IFN-alpha and human leukocyte IFN, marked increase of IFN in 18 patients and modest increase in three patients correlated with clinical improvement. Three clinical nonresponders showed no increase in IFN levels following therapy. The ability to remove IIF and lymphokine inhibitor factor (LIF) by PP may explain the successful treatment of our patients. We describe the evaluation of helper T cells, suppressor T cells, HLADR antigen, natural killer cells, and monocyte/macrophage cell populations by flow cytometry before and after PP. A significant increase in these immune-competent cells correlated with marked improvement in Kurtzke disability status scale in 13 patients, while eight stabilized. Patients showing progression of the disease either showed decrease or no change in these parameters after therapy. Encouraging results from this pilot study suggest that PP combined with immunomodulatory regimens of IFN may be an effective therapy for MS. PMID- 7538998 TI - The source of nitric oxide in skeletal muscle. PMID- 7539000 TI - Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. AB - CD40 activation is critical for B-cell function, leading to activation and expression of cell surface markers, proliferation, immunoglobulin class switching and inhibition of programmed cell death (PCD). Germinal center B-cells, for example, can be prevented from undergoing PCD by CD40 activation. The mechanism by which PCD is inhibited has been an enigma. A potential role for A20, a novel zinc finger protein, in inhibiting B-cell apoptosis was suggested by our previous finding that it is induced by the Epstein-Barr virus LMP-1 gene product, a potent cell death inhibitor. We now show that CD40 activation induces A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, we show that CD40 activation of A20 expression is mediated by inducible binding of NF kappa B complexes to the A20 promoter and provide evidence for a critical role for Thr234 (in the CD40 cytoplasmic domain) in activating NF-kappa B. PMID- 7539001 TI - Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. AB - Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (delta F508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both delta F508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis. PMID- 7539002 TI - A human keratinocyte cell line produces two autocrine growth inhibitors, transforming growth factor-beta and insulin-like growth factor binding protein-6, in a calcium- and cell density-dependent manner. AB - Two growth inhibitors were identified in culture medium conditioned by a human keratinocyte cell line, HaCat. TGF-beta was detected in media conditioned by growing or confluent HaCat cells, as well as in media conditioned at physiological (1 mM) or low (0.03 mM) Ca2+ concentrations. However, a considerable part of transforming growth factor beta (TGF-beta) in media conditioned at a physiological Ca2+ concentration was in active form, whereas most TGF-beta in media conditioned at a low Ca2+ concentration was latent. The other growth-inhibitory activity, which was detected only in media conditioned by confluent cells at a physiological Ca2+ concentration, was purified to homogeneity by a four-step procedure. The N-terminal amino acid sequence of the 33-kDa protein was identical with that of insulin-like growth factor binding protein-6 (IGFBP-6). Purified IGFBP-6 inhibited the growth of HaCat and Balb/MK keratinocyte cell lines, as well as Mv1Lu cells. The growth activity was also demonstrated by human recombinant IGFBP-6. In summary, HaCat cells secrete at least two possible autocrine growth inhibitors: TGF-beta which is secreted constitutively, but activated in a Ca(2+)-dependent manner, and IGFBP-6 which is secreted in a cell density- and Ca(2+)-dependent manner. PMID- 7539003 TI - Identification of a sequence-specific single-stranded DNA binding protein that suppresses transcription of the mouse myelin basic protein gene. AB - The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed. PMID- 7539004 TI - Critical threonine and aspartic acid residues within the I domains of beta 2 integrins for interactions with intercellular adhesion molecule 1 (ICAM-1) and C3bi. AB - Integrins mediate signal transduction through interactions with multiple cellular or extracellular matrix ligands. Evidence is accumulating that the I (or A) domain, a approximately 200-residue inserted sequence in some integrin alpha subunits, mediates ligand binding. We have previously shown that Thr-221 of the putative ligand binding sites within alpha 2 I domain of alpha 2 beta 1 is critical for binding to collagen (Kamata, T., and Takada, Y. (1994) J. Biol. Chem. 269, 26006-26010). Here we report that the mutation of Thr-206 of alpha L blocks intercellular adhesion molecule 1 (ICAM-1) binding to alpha L beta 2 and mutation of Thr-209 of alpha M blocks ICAM-1 and C3bi binding to alpha M beta 2. The data indicate the Thr residues of alpha M and alpha L corresponding to Thr 221 of alpha 2 are critically involved in the ligand interaction with beta 2 integrins. The mutations of the Asp-137 and Asp-239 of alpha L also block ICAM-1 binding to alpha L beta 2, as do the corresponding Asp residues of alpha 2 or alpha M in collagen/alpha 2 beta 1 or C3bi/alpha M beta 2 interactions, respectively. These data suggest that these Thr and Asp residues, conserved among I domains, are critical for interaction with structurally distinct ligands (e.g. ICAMs, C3bi, and collagen). PMID- 7539005 TI - Identification of amino acids in the CD11a I-domain important for binding of the leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) AB - Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM 1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1. PMID- 7539006 TI - pp60src-mediated phosphorylation of connexin 43, a gap junction protein. AB - Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60c-src is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43. PMID- 7539007 TI - Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL. AB - The hematopoietic neutral serine proteases leukocyte elastase and cathepsin G are synthesized as inactive precursors, but become activated by removal of an amino terminal dipeptide and are stored in granules. Moreover, the pro forms of elastase and cathepsin G show carboxyl-terminal prodomains of 20 and 11 amino acids, respectively, which are not present in the mature enzymes. To investigate mechanisms of processing, activation, and granular targeting, we have utilized transgenic expression of myeloid serine proteases in the rat basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I. (1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastase was stably expressed in RBL-1 cells, and the translation products were characterized by biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Processing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing fractions was shown by subcellular fractionation experiments. The processed forms were enzymatically active, judging by the occurrence of amino terminal processing demonstrated by radiosequence analysis, the acquisition of affinity for the protease inhibitor aprotinin, and the appearance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl terminal prodomains, deletion mutants of leukocyte elastase and cathepsin G lacking the carboxyl-terminal extension were constructed and transfected into RBL cells. Our results show that as full-length proteins, the deletion mutants were converted to active enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. We conclude that human leukocyte elastase and cathepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-terminal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells. PMID- 7539008 TI - SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca(2+)-binding EF-hand. AB - SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy-Ullrich et al. (1991): J Cell Biol 115:1127-1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose dependent loss of focal adhesion-positive cells, that was maximal at approximately 1 microgram/ml (0.03 microM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F-actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin alpha v beta 3 remained clustered in a plaque-like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin-extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine-rich peptide 2.1 (amino acids 54-73) and the Ca(2+)-binding EF-hand-containing peptide 4.2 (amino acids 254 273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion-labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor-mediated event. PMID- 7539009 TI - Tyrosine phosphorylations specific to mitosis in human and hamster cells. AB - Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and approximately 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments. PMID- 7539011 TI - Elimination of B-lineage leukemia and lymphoma cells from bone marrow grafts using anti-B4-blocked-ricin immunotoxin. AB - Bone marrow is the primary site of disease in patients with acute lymphoblastic leukemia (ALL) and is frequently involved in patients with non-Hodgkin's lymphoma (NHL). At the time of autologous bone marrow transplantation, marrow grafts from patients with leukemia and lymphoma are often still contaminated by malignant cells, even when such patients achieve complete clinical remission. In this study, we evaluated the potential of anti-B4-blocked-ricin (anti-B4-bR) immunotoxin to eliminate residual ALL and NHL cells from bone marrow. Anti-B4-bR binds to the CD19 antigen, which is B-lineage specific, and, at concentrations of 5 x 10(-9) M or greater, could eliminate more than 3 logs of CD19+ Nalm-6 or Namalwa cells in a 20-fold excess of normal irradiated bone marrow after only 5 hr of incubation. This activity was abrogated by the addition of anti-B4 but not by the presence of galactose, which is the natural ligand for native ricin. Also, when used at these high concentrations, anti-B4-bR showed little nonspecific toxicity against normal hematopoietic progenitors. In conclusion, a single short exposure to anti-B4-bR is capable of inducing high levels of depletion of CD19+ leukemia and lymphoma cells without significant nonspecific toxicity against normal marrow progenitors. Therefore, anti-B4-bR offers an interesting approach to the elimination of B-lineage malignant cells prior to autologous bone marrow transplantation. PMID- 7539012 TI - Chemokine expression in murine experimental allergic encephalomyelitis. AB - Chemokines are a family of low molecular mass proteins with chemotactic and cell activating activities. Reverse transcription-polymerase chain reaction and Northern hybridization were used to examine their expression during murine experimental allergic encephalomyelitis (EAE), an autoimmune disease used as a model of multiple sclerosis. The mRNAs encoding RANTES, MIP-1 alpha, MIP-1 beta, TCA3 (I-309), IP-10, JE (MCP-1), KC (MGSA/gro), and MARC (MCP-3) were induced in the spinal cord 1-2 days before clinical signs were apparent. SDF, a cDNA predicted to encode a chemokine-like product, was expressed in normal as well as diseased spinal cords. No expression of C10 or MIP-2 was detected. Activated encephalitogenic T cells expressed message for RANTES, MIP-1 alpha, MIP-1 beta, and TCA3. These results define a subset of chemokines that may play an important role in the inflammatory process during murine EAE. PMID- 7539010 TI - Regulation of apoptosis in immune cells. PMID- 7539013 TI - Cervical primary afferent input to vestibulospinal neurons projecting to the cervical dorsal horn: an anterograde and retrograde tracing study in the cat. AB - Vestibulospinal neurons in the caudal half of the medial and descending vestibular nuclei terminate in the cervical spinal cord, not only in the ventral horn and intermediate zone but also in the dorsal horn. The purpose of the present study was to examine whether the areas containing these vestibulospinal neurons are reached by cervical primary afferents. In one group of experiments, wheat germ agglutinin-horseradish peroxidase conjugate and horseradish peroxidase were pressure injected into spinal ganglia C2-C8 and revealed anterogradely labeled fibers and boutons in the caudal part (caudal to the dorsal cochlear nucleus) of the ipsilateral medial and descending vestibular nuclei. This projection was verified in experiments in which wheat germ agglutinin-horseradish peroxidase conjugate was microiontophoretically injected into the caudal half of either the medial or the descending vestibular nuclei and revealed retrogradely labeled cells only in ipsilateral spinal ganglia C2-C7, with a maximum of cells in C3. In another group of experiments, after microiontophoretic injections of Phaseolus vulgaris leucoagglutinin or Biocytin into either the medial or the descending vestibular nuclei, anterogradely labeled fibers and boutons were present in the cervical spinal cord, mainly bilaterally in the dorsal horn (laminae I-VI) but also, to a lesser extent, in the ventral horn and intermediate zone. The existence of a loop that relays cervical primary afferent information to vestibulospinal neurons projecting to the cervical spinal cord, in particular the dorsal horn, may have implications for vestibular control over local information processing in the cervical dorsal horn. PMID- 7539014 TI - Spontaneous sequences of onset of torsade de pointes in patients with acquired prolonged repolarization: quantitative analysis of Holter recordings. AB - OBJECTIVES: This study investigated the cycle length changes preceding the spontaneous onset of torsade de pointes in patients with acquired prolonged ventricular repolarization. BACKGROUND: Torsade de pointes is a polymorphic ventricular tachycardia generally associated with prolonged ventricular repolarization. Because torsade de pointes is not inducible by programmed electrical stimulation, quantitative analysis of Holter recordings of spontaneous episodes may clarify the mechanisms favoring the onset of torsade de pointes in actual clinical conditions. METHODS: The digitized Holter recordings of 12 patients were analyzed by a computerized Holter system (ATREC). All arrhythmias were grouped according to three classes: 1) isolated premature ventricular beats (n = 47,147, mean/patient [+/- SD] 3,929 +/- 11,571); 2) salvos of 2 to 4 consecutive beats (n = 2,003, mean/patient 167 +/- 359); 3) torsade de pointes > or = 5 beats (n = 105, mean/patient 9 +/- 11). For each patient and class of arrhythmias, six variables were computed from the 10 min and the 10 cycles preceding the event onset. RESULTS: A significant heart rate increase in the last minute (p < 0.01) and typical oscillatory short-long-short cycle length sequences preceded the onset of arrhythmias, with greater oscillation preceding torsade de pointes than salvos and premature ventricular beats. The cycle lengths preceding the onset were highly correlated with the class of arrhythmias (r = 0.65, p < 0.005) and allowed the correct classification of 69% of events by discriminant analysis (p < 0.0001). A significant negative correlation was observed between the duration of torsade de pointes and the mean length of the initial cycles (r = -0.62, p < 0.001), indicating that longer torsade de pointes had a faster rate than that at onset. CONCLUSIONS: In patients with acquired prolonged repolarization, the spontaneous onset of ventricular arrhythmias was preceded by an increasing heart rate in the last minute and escalating oscillatory "short long-short" cycle length patterns, with greater oscillations preceding torsade de pointes than salvos and isolated ventricular beats. These findings suggest that adrenergic- and pause-dependent mechanisms (possibly inducing afterdepolarizations and triggered activity) may have a synergetic role in the genesis of complex ventricular arrhythmias associated with delayed ventricular repolarization. PMID- 7539015 TI - The coagulation system is activated in idiopathic cardiomyopathy. AB - OBJECTIVES: We investigated the plasma levels of molecular markers for platelet activity and the thrombotic and fibrinolytic status in patients with hypertrophic cardiomyopathy and dilated cardiomyopathy to determine the activating site of coagulation in these disorders. BACKGROUND: A thromboembolic event is a serious complication in patients with idiopathic cardiomyopathy. However, the activating site of the coagulation system in idiopathic cardiomyopathy has not been fully investigated. METHODS: We determined the plasma levels of molecular markers for platelet activity (platelet factor 4 and beta-thromboglobulin), thrombotic status (fibrinopeptide A and thrombin-antithrombin III complex) and fibrinolytic status (D-dimer and plasmin-alpha 2-plasmin inhibitor complex) in 13 patients with hypertrophic cardiomyopathy, 17 patients with dilated cardiomyopathy and 20 normal subjects. RESULTS: Plasma levels of platelet factor 4, beta thromboglobulin and plasmin-alpha 2-plasmin inhibitor complex did not differ significantly among the three groups, whereas plasma levels of fibrinopeptide A and thrombin-antithrombin III complex in both patient groups were significantly higher than those in normal subjects. Plasma levels of D-dimer in patients with dilated cardiomyopathy were significantly higher than those in patients with hypertrophic cardiomyopathy and normal groups. In patients with hypertrophic cardiomyopathy, both fibrinopeptide A and thrombin-antithrombin III complex levels were significantly correlated with left atrial diameter. In patients with dilated cardiomyopathy, fibrinopeptide A and thrombin-antithrombin III complex levels showed a positive correlation with left ventricular end-diastolic volume and a negative correlation with fractional shortening of the left ventricle. CONCLUSIONS: The activated coagulation system in patients with hypertrophic and dilated cardiomyopathy may be triggered by left atrial dilation in hypertrophic cardiomyopathy and left ventricular enlargement and dysfunction in dilated cardiomyopathy. PMID- 7539016 TI - Computer-simulated and clinical models agree on optimal postoperative management for hypoplastic left heart syndrome. PMID- 7539017 TI - Influence of ion channel modulation on in vitro interferon-gamma induced MHC class I and II expression on macrophages. AB - The in vitro effect of K+ channel blockers quinidine and verapamil, anion channel blocker SITS and K+ channel openers diazoxide, pinacidil, and BRL 38227 on interferon-gamma (IFN-gamma) induced MHC class I and II expression of Lewis rat peritoneal macrophages was investigated by cell ELISA assay. MHC class I expression was significantly enhanced by diazoxide at concentrations of 10(-5)M to 10(-6)M and by pinacidil and BRL 38227 at the concentration of 10(-6)M. MHC class II expression was enhanced by pinacidil and BRL 38227 at concentrations of 10(-5)M to 10(-6)M. The enhancing effect of pinacidil could be blocked by inhibitors of the protein kinases PKA and PKC suggesting that activation of both is required for optimum induction of MHC molecule expression. K+ and anion channel blockers were less active in modulation of MHC molecule expression. Verapamil had no influence, quinidine suppressed MHC class I expression at concentrations of 10(-4)M to 10(-5)M, and SITS suppressed MHC class I expression at the concentration of 10(-3)M. Since MHC class II expression is essential for efficient antigen presentation to T helper cells and MHC class I expression is required for target cell lysis by cytotoxic T cells, ion channel modulating drugs may be potential candidates for immunopharmacological intervention in inflammatory diseases. PMID- 7539018 TI - Calcineurin activation protects T cells from glucocorticoid-induced apoptosis. AB - In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway. PMID- 7539019 TI - Sequence analysis of anti-AChR antibodies in experimental autoimmune myasthenia gravis. AB - Autoantibodies directed against the acetylcholine receptor (AChR) lead to AChR loss and muscular weakness in myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis (EAMG). The role of different anti-AChR sequences and specificities in the pathogenesis of EAMG was investigated by sequencing a panel of 19 mouse mAbs, previously elicited against Torpedo and human AChR, that bound to at least four different epitope regions. The pathogenicity of eight mAbs that cross-reacted with mouse or rat AChR was tested. EAMG was induced by four mAbs against the main immunogenic region (MIR). Sequence analysis of different anti-AChR specificities showed a large diversity of H and L chain sequences. Highly homologous H chain sequences (> 90%) were found among some mAbs with similar specificities, whereas highly homologous L chain sequences were not restricted to Abs of a particular fine specificity. Sharing of a highly homologous VH gene or an identical DJH region was observed among three of four pathogenic anti-MIR mAbs, obtained by immunization with AChRs from different species. The VH genes of these three pathogenic mAbs were closely related to PC7183 germline genes indicating that some pathogenic Abs may already be present in the germline repertoire. PMID- 7539020 TI - The natural human IgG anti-F(ab')2 antibody recognizes a conformational IgG1 hinge epitope. AB - Natural IgG anti-F(ab')2 Abs are part of the physiologic immune repertoire and have important immunoregulatory functions. Although previous work suggested that some of these Abs recognize epitopes located in the constant region of the F(ab')2 molecule, an exact epitope mapping has not been performed. We found that the anti-F(ab')2 Ab binds strongly to F(ab')2 but only weakly to Fab fragments. Fab fragments are lacking the core and lower hinge region. In our experiments, we show that the IgG anti-F(ab')2 Ab binds strongly to a synthetic double chain peptide (225-237/225'-237') comprising the core and lower hinge region of the human IgG1 molecule. In contrast, it binds only weakly to the same peptide in monomeric form (225-237) or to a short double chain hinge peptide (225-232/225' 232'). The double chain peptides comprise a cyclic region between the two cystine bridges and an exocyclic region. Previous nuclear magnetic resonance analyses showed that the cyclic portion of the short double chain hinge peptide adopts the same conformation as that found in the intact IgG1 molecule. The dichroic properties of the short and long double chain hinge peptides indicate that they have identical conformations in their cyclic regions, but have different conformations in their exocyclic regions. The conformational differences in the exocyclic regions explain the binding of the Ab to the long double chain hinge peptide and the lack of binding to the short one. The circular dichroism spectrum of the monomeric hinge peptide, which is not recognized by the Ab, is consistent with the absence of an ordered peptide structure. These findings lead us to conclude that the IgG anti-F(ab')2 Ab recognizes a conformational IgG1 hinge epitope. PMID- 7539022 TI - Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism. AB - The aim of the present study was to investigate the angiogenic properties of platelet-activating factor (PAF). In vitro PAF was shown to induce a dose dependent migration of human endothelial cells (EC) across the polycarbonate filters in Boyden's chambers. In contrast, D-PAF, the biologically inactive enantiomer, and Lyso-PAF did not stimulate a significant migration of EC. This effect of PAF was not associated with a proliferative response of EC to this mediator. Moreover, the ability of PAF to stimulate the migration of EC was independent of the presence of heparin in the medium. WEB 2170, a specific PAF receptor antagonist, prevented the migration of EC induced by PAF, thus suggesting a receptor-dependent stimulation. The expression of PAF receptor gene by EC was confirmed by reverse transcriptase-PCR and Southern blot analysis. The in vivo angiogenic effect of PAF was studied in mice using a model in which Matrigel was used for the delivery of mediators. PAF induced a dose-dependent angiogenic response, which at pharmacologic concentrations (1-5 microM) did not require heparin, but at physiologic concentrations (5-50 nM) required the presence of heparin at doses that were not angiogenic per se. The angiogenesis induced by 50 nM PAF was, indeed, inhibited both by protamine and by the PAF receptor antagonist WEB 2170. The angiogenic effect of D-PAF and Lyso-PAF was not significant. Neutralizing Abs to basic fibroblast growth factor induced a slight but not statistically significant reduction of the angiogenesis induced by PAF. PMID- 7539021 TI - Nitric oxide synthesis in rat peritoneal macrophages is induced by IgE/DNP complexes and cyclic AMP analogues. Evidence in favor of a common signaling mechanism. AB - The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes. PMID- 7539023 TI - Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b. AB - Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes. PMID- 7539024 TI - Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. AB - Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory diseases. In the present study, we investigated the potential role of NO in an ocular model of inflammation, endotoxin-induced uveitis (EIU), in Lewis rats. Injection of LPS in one footpad induces severe uveitis after 16 h, which is accompanied by an increase of NO in the aqueous and vitreous humors, as evaluated by nitrite assay. Reverse transcriptase-PCR experiments reveal a large increase of inducible NO synthase (iNOS) mRNA in the iris/ciliary body, from 2 to 24 h after LPS treatment. In the retina, maximal increase of iNOS mRNA was detected 16 h after LPS treatment. Two i.p. injections of the NOS inhibitor, NG-nitro-L arginine methyl ester (L-NAME), which inhibits nitrite release in the aqueous and vitreous humors, profoundly reduce clinical and histologic inflammation in EIU rats. These results implicate the NO pathway in the pathogenesis of EIU and demonstrate the possibility of modulating this inflammatory disease by injection of a NOS inhibitor. PMID- 7539025 TI - Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide). AB - Endotoxic shock is a life-threatening condition mediated by cytokines released after exposure to bacterial LPS/endotoxin. Activation of monocytes and neutrophils by the binding of LPS to the membrane receptor, CD14, plays a key role in this response. Furthermore, a soluble form of the CD14 receptor enhances the endothelial cell response to LPS. We show here that despite the agonist effects of soluble CD14 on the endothelial cell response to LPS, recombinant soluble CD14 is able to protect mice from LPS-induced lethality. This protection appears to be associated with the inhibition of TNF-alpha release. These results suggest that the soluble CD14 receptor may represent a new form of therapy for endotoxic shock in humans. PMID- 7539026 TI - Immunohistologic analysis of ineffective CD40-CD40 ligand interaction in lymphoid tissues from patients with X-linked immunodeficiency with hyper-IgM. Abortive germinal center cell reaction and severe depletion of follicular dendritic cells. AB - The CD40 ligand (CD40L) is an activation-induced surface membrane protein expressed by CD4+ T helper cells in lymphoid follicles, and is involved in the contact-dependent signaling-mediated activation, proliferation, and differentiation of CD40+ B cells. Using immunohistochemistry, the present study analyzes the cell microenvironment of lymphoid tissues in two cases of X-linked hyper-IgM syndrome, a congenital immunodeficiency caused by mutations of the CD40L gene, and which represents a unique model to dissect the functional and morphologic consequences of disrupted CD40/CD40L interactions. Prominent primary B follicles are identified in the lymph nodes and in the extranodal lymphoid tissues from both cases, but tiny collections of Bcl-2-, MIB1/Ki67+ centroblasts are also found in one case. Despite the CD40L defect, intrafollicular CD4+CD57+ T helper cells, identified by anti-parvalbumin mAb, are normally present. However, a severe depletion of follicular dendritic cells, recognized by Abs against NGFR, CD21 and CD23, and lack of expression of the Ag recognized by KiM4p on these cells, are noticed. Finally, no major alterations of the architecture and cellular composition of the paracortical T cell area are found. A large number of plasma cells exclusively expressing IgM were detected in the colon lamina propria in one of the patients, who also had extremely elevated IgM serum levels. Taken together, these data support the idea that ineffective CD40/CD40L interactions determine both abortive germinal center cell reaction as well as severe depletion and phenotypical abnormalities of follicular dendritic cells, thus impairing the functional development of B follicles. Recurrent or persisting antigenic stimulation in mucosal tissues is likely to play a major role in determining and maintaining elevated IgM serum levels. PMID- 7539027 TI - Differences in the degree of depletion, rate of recovery, and the preferential elimination of naive CD4+ T cells by anti-CD4 monoclonal antibody (GK1.5) in young and aged mice. AB - We compared the degrees of depletion and the rates of recovery of CD4+ T cells in young and old mice given a single injection of an anti-CD4 (GK1.5) mAb. We found that in young mice the CD4+ population of T cells was completely depleted from the peripheral blood and, subsequently, the lymph nodes and spleen. Young mice recovered 80% of CD4+ T cells within 100 days after the injection. Although aged mice have less CD4+ T cells, CD4+ T cells were depleted by only 60% after mAb injection. This finding was associated with prolonged circulation of Ab-coated T cells. The rate of repopulation of peripheral CD4+ T cells was also affected by aging, because 5 times less CD4+ T cells appear in aged than in young mice between days 14 and 100 post injection. Further, we found that the CD4+ T cells that are depleted in both young and old mice by injection of anti-CD4 mAb are CD45RBhigh, suggesting a preferential depletion of immature cells. Finally, the thymus appears to be required to repopulate the T cell population post injection, because thymectomized young mice failed to recover CD3+CD4+ cells. PMID- 7539028 TI - Cytokine regulation of angiogenesis in breast cancer: the role of tumor associated macrophages. AB - Studies over the past 20 years have established that the development of new capillaries from an existing vascular network (a process called angiogenesis) is an essential component of tumor growth. Malignant tumors do not grow beyond 2-3 mm3 in size unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products. Tumor angiogenesis also provides an essential exit route for metastasizing tumor cells from the tumor to the bloodstream. Indeed, extensive neovascularization is a poor prognostic factor in several forms of human cancer. Angiogenesis is a complex, multistep process driven by many local signals within the tumor. This involves the degradation of the extracellular matrix around a local venule after the release of collagenases and proteases, the proliferation and migration of capillary endothelial cells, and their differentiation into functioning capillaries. Cytokines produced by various cell types present within the microenvironment of solid tumors form a complex, dynamic network in which they have multiple effects on tumor progression. Herein we review our work on the presence, and possible regulatory influence on tumor angiogenesis, of a number of these cytokines within invasive breast carcinomas. We have combined immunocytochemistry with a single cell cytokine release assay called the reverse hemolytic plaque assay to investigate the cellular sources of the key angiogenic cytokines, vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor-alpha. Tumor-associated macrophages in the stromal compartment of these tumors and/or malignant epithelial cells were seen to be a major producer cell for these cytokines, whereas tumor necrosis factor-alpha receptors were expressed by leukocytes, malignant cells, and endothelial cells in tumor blood vessels. PMID- 7539029 TI - Role of C-X-C chemokines as regulators of angiogenesis in lung cancer. AB - Lung cancer is the leading cause of malignancy-related mortality in the U.S. and is predicted to increase over the remainder of this decade. Despite attempts to advance early diagnosis and use combination therapies, the clinical response of this cancer yields an overall 5-year survival rate of less than 15%. Clearly, new strategies for therapy are indicated. Although carcinogenesis is complex, tumor growth beyond 1-2 mm3 is dependent on angiogenesis. One of the potential mechanisms that allows for tumorigenesis is dysregulation of the balance of angiogenic and angiostatic factors that favors net neovascularization within the primary tumor. Numerous studies have investigated the role of a variety of molecules in the regulation of angiogenesis. Recently, interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be an angiogenic factor. In contrast, platelet factor 4 (PF4), another C-X-C chemokine, has been shown to have angiostatic properties. It is interesting that the major structural difference between IL-8 and PF4 is the presence of the NH2-terminal ELR (Glu-Leu Arg) motif that precedes the first cysteine amino acid residue of IL-8 and is important in ligand/receptor interactions. We hypothesize that angiogenesis associated with tumorigenesis is dependent on members of the C-X-C chemokine family acting as either angiogenic or angiostatic factors. This paradigm predicts that the biological balance in the expression of these C-X-C chemokines dictates whether the neoplasm grows and develops metastatic potential or regresses. In this review we discuss our recent laboratory findings that support this contention and suggest that further elucidation of the biology of C-X-C chemokines in the context of neovascularization of nonsmall cell lung cancer will permit novel targeted therapy aimed specifically at attenuating tumor growth and metastasis. PMID- 7539030 TI - A role for C-C chemokines in fibrotic lung disease. AB - Pulmonary fibrosis is the end point of a chronic inflammatory process characterized by leukocyte recruitment and activation, fibroblast proliferation, and increased extracellular matrix production. Previous studies of models of pulmonary fibrosis have investigated the role of cytokines in the evolution of the fibrotic response. The involvement of tumor necrosis factor and interleukin-1 in bleomycin-induced lung injury, a model of idiopathic pulmonary fibrosis, has been well established, suggesting that cytokines mediate the initiation and maintenance of chronic inflammatory lesions. However, the aforementioned cytokines alone cannot account for the recruitment and activation of specific leukocyte populations found in the bleomycin model. Recently, a family of novel proinflammatory cytokines (chemokines) was cloned and characterized, yielding many putative mediators of leukocyte functions. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemoattractant protein-1 (MCP-1) belong to the C-C chemotactic cytokine family, a group of low-molecular-weight peptides. These molecules modulate chemotaxis, proliferation, and cytokine expression in leukocyte subsets. Our group has investigated the roles of MCP-1 and MIP-1 alpha in the bleomycin model. Both MCP-1 and MIP-1 alpha are expressed in a time dependent manner after bleomycin challenge, and passive immunization of these animals with either anti-MIP-1 alpha or anti-MCP-1 antibodies attenuated leukocyte accumulation. In addition, we have identified specific cell types expressing MCP-1 or MIP-1 alpha by in situ hybridization and immunohistochemical localization, respectively. Furthermore, our results indicate that MIP-1 alpha expression is mediated by alveolar macrophage-derived tumor necrosis factor, identifying an important cytokine pathway in the initiation of pulmonary fibrosis. Finally, anti-MIP-1 alpha therapy attenuated fibrosis, providing direct evidence for its involvement in fibrotic pathology. Our work has clearly established that the C-C chemokines MCP-1 and MIP-1 alpha are expressed and contribute to the initiation and maintenance of the bleomycin-induced pulmonary lesion. PMID- 7539031 TI - Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin 5 transduce signals through two forms of STAT5. AB - Recently, JAK2 kinase was found to be one of the tyrosine kinases activated by interleukin-3 (IL-3) in target cells. JAK2 belongs to a family of kinases that act upstream of transcription factors called STATs. STATs exist in the cytoplasm as latent, transcriptionally inactive forms until, in response to extracellular signals, they become phosphorylated on tyrosine residues, translocate to the nucleus, and bind to specific DNA elements. Because IL-3 activates JAK2, we searched for the STAT(s) that might transduce IL-3 signals. Several lines of evidence suggest that IL-3 uses the murine homologue of STAT5, a factor originally purified from sheep. Unexpectedly, during isolation of the murine homologue, we found two highly related molecules that we have designated STAT5A and STAT5B. PMID- 7539032 TI - How can cryptic epitopes trigger autoimmunity? PMID- 7539033 TI - Of ITAMs and ITIMs: turning on and off the B cell antigen receptor. PMID- 7539034 TI - Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell determinants. AB - Bound antibodies can modulate antigen processing but it is not clear to what extent this affects antigen presentation. Here we show that presentation of T cell determinants in tetanus toxin can be either enhanced or suppressed as a direct consequence of antibody modulation of antigen processing in human B lymphoblastoid cells. Remarkably, a single bound antibody or its Fab fragment can simultaneously enhance the presentation of one T cell determinant by more than 10 fold while strongly suppressing the presentation of a different T cell determinant. Biochemical analysis demonstrates that both the suppressed and boosted determinants fall within an extended domain of antigen stabilized or "footprinted" by this antibody during proteolysis. These results demonstrate that bound antibodies can modulate the capture of peptides by class II major histocompatibility complex (MHC), thus manipulating the T cell response towards or away from particular determinants. Altered processing of protein-protein complexes leading to enhanced loading of class II MHC and substantially lowered threshold for T cell activation suggests a novel mechanism that might reveal "cryptic" self determinants. PMID- 7539035 TI - Syk and ZAP-70 mediate recruitment of p56lck/CD4 to the activated T cell receptor/CD3/zeta complex. AB - During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP 70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2-mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes. PMID- 7539036 TI - T cell determinants from autoantibodies to DNA can upregulate autoimmunity in murine systemic lupus erythematosus. AB - (NZB x NZW) F1 (BWF1) mice develop spontaneous T cell autoimmunity to VH region determinants of syngeneic anti-DNA before the onset of clinical disease. In this study, we characterized the immunogenicity, MHC binding, and lymphokine secretion patterns induced by T cell determinants from the VH region of one such anti-DNA mAb (A6.1) and examined their role in the regulation of autoimmunity. Determinants were identified by proliferation of syngeneic splenic T cells from young, unprimed BWF1 mice in response to overlapping 12-mer peptides representing the entire VH region sequence. Immunization of young BWF1 mice with any of three determinants (A6H 34-45 [p34], A6H 58-69 [p58], and A6H 84-95 [p84]) elicited proliferative responses upon in vitro recall. Upon immunization with the whole A6.1 molecule, however, proliferative responses could be recalled only to the p58 peptide, defining this as immunodominant. The other two peptides (p34 and p84) elicited minimal or no proliferation and could be termed cryptic. Proliferative responses elicited by the cryptic determinants were restricted by a single class II (I-Ed for p34 and I-Au for p84), whereas the immunodominant p58 determinant was restricted by both I-Ed and I-Eu. The cryptic p34 and p84 bound strongly to I Ed and I-Au, respectively, whereas the immunodominant p58 peptide bound poorly to I-Ed. A6H p84 elicited T cells that secreted lymphokines in a pattern consistent with a Th1-like phenotype, whereas p58 induced a Th2-like cytokine pattern. Immunization with p34 or p84, or adoptive transfer of a p84-reactive T cell line to young BWF1 mice significantly increased IgG anti-DNA levels, accelerated nephritis, and decreased survival. In conclusion, in BWF1 mice, autoreactive T cells recognizing both cryptic and dominant self-determinants on anti-DNA autoantibodies escape deletion or anergy induction. Furthermore, since these cells are spontaneously activated before the onset of clinical disease, they may be involved in the development of the autoimmune process. PMID- 7539037 TI - Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals. AB - Apoptosis (programmed cell death) of T lymphocytes has been proposed as a mechanism which plays an important role in the pathogenesis of human immunodeficiency virus (HIV) disease. Activation of Fas (CD95) can either result in costimulation of proliferation and cytokine production or in the induction of apoptosis of T lymphocytes. This raises the possibility that Fas is involved in the observed T cell apoptosis during HIV disease. In this report we show that peripheral blood CD4+ and CD8+ T lymphocytes from HIV-infected individuals undergo apoptosis in vitro in response to antibody stimulation (cross-linking) of Fas at a much higher frequency than from uninfected controls. This anti-Fas induced T cell apoptosis is markedly higher than spontaneous T cell apoptosis in HIV-infected individuals. Antibodies against other members of the tumor necrosis factor (TNF)/nerve growth factor receptor family such as CD27, CD30, CD40, 4-1BB, p55 TNF receptor, p75 TNF receptor, and TNF receptor-related protein did not result in any increase of T cell apoptosis above that spontaneously observed in HIV+ individuals. Anti-Fas-induced apoptosis was much higher in symptomatic HIV infected individuals; and the magnitude of anti-Fas-induced CD4+ T cell apoptosis correlated inversely with peripheral blood CD4+ T cell absolute counts. Surface expression of Fas on T cells was also found to be higher in HIV-infected individuals. Resting and activated CD4+ and CD8+ T cells both underwent apoptosis in response to anti-Fas antibody. L-Selectin positive memory CD4+ T cells were especially susceptible to anti-Fas-induced apoptosis. These findings show that CD4+ and CD8+ T lymphocytes in HIV-infected individuals are primed in vivo to undergo apoptosis in response to Fas stimulation, suggesting that Fas signaling may be responsible for the T lymphocyte functional defects and depletion observed in HIV disease. PMID- 7539038 TI - Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor associated protein involved in the regulation of B cell signaling. AB - Recent data implicating loss of PTP1C tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (mev) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCR) ligation in me and mev splenic B cells and in CD5+ CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional PTP1C, me and mev B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc gamma IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTP1C with the resting BCR complex, as evidenced by coprecipitation of PTP1C protein and phosphatase activity with BCR components and the depletion of BCR-associated tyrosine phosphatase activity by anti-PTP1C antibodies. These results suggest a role for PTP1C in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-alpha. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an approximately 140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells. PMID- 7539039 TI - Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries. AB - Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general. PMID- 7539040 TI - Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. AB - Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens. PMID- 7539041 TI - The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 and RANTES in the skin of human atopic subjects: relationship to eosinophil, T cell, and macrophage recruitment. AB - The C-C chemokines RANTES and monocyte chemotactic protein-3 (MCP-3) are potent chemoattractants in vitro for eosinophils and other cell types associated with allergic reactions. We tested the hypothesis that the allergen-induced infiltration of eosinophils, T cells, and macrophages in the skin of atopic subjects is accompanied by the appearance of mRNA+ cells for RANTES and MCP-3. Cryostat sections were obtained from skin biopsies from six subjects 6, 24, and 48 h after allergen challenge. Tissue was processed for immunocytochemistry (ICC) and for in situ hybridization using 35S-labeled riboprobes for RANTES and MCP-3. In contrast to diluent controls, allergen provoked a significant increase in mRNA+ cells for MCP-3, which peaked at 6 h and progressively declined at 24 and 48 h. This paralleled the kinetics of total (major basic protein positive [MBP]+) and activated (cleaved form of eosinophil cationic protein [EG2]+) eosinophil infiltration. The allergen-induced expression of cells mRNA+ for RANTES was also clearly demonstrable at 6 h. However, the numbers were maximal at 24 h and declined slightly at the 48-h time point. The number of mRNA+ cells for RANTES paralleled the kinetics of infiltration of CD3+, CD4+, and CD8+ T cells whereas the number of CD68+ macrophages was still increasing at 48 h. These data support the view that MCP-3 is involved in the regulation of the early eosinophil response to specific allergen, whereas RANTES may have more relevance to the later accumulation of T cells and macrophages. PMID- 7539042 TI - Inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites. AB - IFN-gamma-induced nitric oxide (NO) in the murine macrophage-derived cell line RAW 264.7 was previously shown to inhibit replication of the poxviruses ectromelia and vaccinia (VV) and HSV-1. In the current study we demonstrate that murine macrophages activated as a consequence of VV infection express inducible nitric oxide synthase. These activated macrophages were resistant to infection with VV and efficiently blocked the replication of VV and HSV-1 in infected bystander cells of epithelial and fibroblast origin. This inhibition was arginine dependent, correlated with nitrite production in cultures, and reversible by the NOS inhibitor N omega-monomethyl-L-arginine. NO-mediated inhibition of VV replication was studied by treatment of virus-infected human 293 cells with the NO donor S-nitroso-N-acetyl-penicillamine. Using a VV-specific DNA probe, antibodies specific for temporally expressed viral proteins, and transmission electron microscopy, we have shown that NO inhibited viral late gene protein synthesis, DNA replication, and virus particle formation, but not expression of the early proteins that were analyzed. Putative enzymatic targets of NO were identified by reversing the NO-mediated inhibition of VV replication in the 293 cells with exogenous ferrous sulfate and L-cysteine. Reversal of inhibition may derive from the capacity of these reagents to protect or regenerate nonheme iron or thiol groups, respectively, which are essential for the catalytic activities of enzymes susceptible to inactivation by NO. PMID- 7539043 TI - Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. AB - Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth. PMID- 7539045 TI - L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. AB - L-selectin, a cell surface adhesion molecule that is expressed by most leukocytes, mediates leukocyte rolling along vascular endothelium at sites of inflammation. The contribution of L-selectin to leukocyte migration in models of chronic inflammation was assessed by using mice that lack cell surface L-selectin expression. Significant inhibition of neutrophil (56-62%), lymphocyte (70-75%), and monocyte (72-78%) migration into an inflamed peritoneum was observed 24 and 48 h after administration of thioglycollate, an inflammatory stimulus. L-selectin deficient mice were also significantly impaired in delayed-type hypersensitivity reactions. Footpad swelling in response to sheep red blood cell challenge was reduced 75% in L-selectin-deficient mice compared with wild-type mice. Ear swelling in a model of contact hypersensitivity induced by oxazolone challenge was also reduced by 69% compared to wild-type mice. Consistent with L-selectin mediating leukocyte migration into diverse vascular beds during inflammation, L selectin-deficient mice were significantly resistant to death resulting from lipopolysaccharide (LPS)-induced toxic shock. LPS administration resulted in a 90% mortality rate in control mice after 24 h, while there was a 90% survival rate in L-selectin-deficient mice. These results demonstrate that L-selectin plays a prominent role in leukocyte homing to nonlymphoid tissues during inflammation and that blocking this process can be beneficial during pathological inflammatory responses. PMID- 7539044 TI - Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised. AB - We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release. PMID- 7539046 TI - Reduced recruitment of inflammatory cells in a contact hypersensitivity response in P-selectin-deficient mice. AB - The inflammatory response at sites of contact hypersensitivity induced by oxazolone was examined in the ears of P-selectin-deficient and wild-type mice. Accumulation of CD4+ T lymphocytes, monocytes, and neutrophils was reduced significantly in the mutant mice, as well as mast cell degranulation. In contrast, there was no significant difference in vascular permeability or edema between the two genotypes. The results demonstrate a role for P-selectin in recruitment of CD4+ T lymphocytes and show that P-selectin plays a role in long term inflammation as well as in acute responses. PMID- 7539047 TI - Rescue from granzyme B-induced apoptosis by Wee1 kinase. AB - Granzymes are a family of granule-associated serine esterases that mediate apoptosis by cytotoxic T lymphocytes and natural killer cells. We have previously shown that cdc2, the mitosis-regulating cyclin-dependent kinase, is required for granzyme B-induced apoptosis in target cells. In addition, granzyme B induces premature activation and tyrosine dephosphorylation of cdc2 during apoptosis. Throughout most of the cell cycle and until the cell is prepared to enter mitosis, cdc2 kinase activity is negatively regulated by phosphorylation of a residue within its adenosine triphosphate-binding domain by Wee1, a nuclear kinase that maintains mitotic timing in eukaryotic cells. We have transiently expressed c-myc epitope-tagged Wee1 cDNA in BHK cells. Cells that expressed Wee1 in the nucleus became resistant to apoptosis induced by granzyme B and perforin. Wee1-transfected cells also exhibited markedly increased cdc2 tyrosine phosphorylation. Thus, Wee1 can rescue cells from granzyme-induced apoptosis by preventing cdc2 dephosphorylation. PMID- 7539048 TI - Single calcium channel behavior in native skeletal muscle. AB - The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14 pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single channel correlates of T current and L current, respectively. PMID- 7539050 TI - Expression of proteolipid protein gene is directly associated with secretion of a factor influencing oligodendrocyte development. AB - Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development. PMID- 7539049 TI - Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of alpha 1 and beta subunits. AB - The cardiac dihydropyridine-sensitive calcium channel was transiently expressed in HEK293 cells by transfecting the rabbit cardiac calcium channel alpha 1 subunit (alpha 1C) alone or in combination with the rabbit calcium channel beta subunit cloned from skeletal muscle. Transfection with alpha 1C alone leads to the expression of inward, voltage-activated, calcium or barium currents that exhibit dihydropyridine sensitivity and voltage- as well as calcium-dependent inactivation. Coexpression of the skeletal muscle beta subunit increases current density and the number of high-affinity dihydropyridine binding sites and also affects the macroscopic kinetics of the current. Recombinant alpha 1C beta channels exhibit a slowing of activation and a faster inactivation rate when either calcium or barium carries the charge. Our data suggest that both an increase in the number of channels as well as modulatory effects on gating underlie the modifications observed upon beta subunit coexpression. PMID- 7539051 TI - Molecular cloning and tissue expression of a cDNA encoding IP1--a P0-like glycoprotein of trout CNS myelin. AB - A full-length cDNA encoding a major structural glycoprotein of trout CNS myelin (IP1) was cloned and sequenced. The deduced amino acid sequence exhibited a significant structural homology with the P0 proteins of rat PNS and shark CNS. Sequence conservation was strongest in the extracellular domain, and it included the position of the two cysteine residues required for stabilization of an immunoglobulin-like secondary structure as well as those of the single N glycosylation acceptor site. The cytoplasmic domain was shorter by 38 amino acids than those of rat and shark P0 and except for a high proportion of basic amino acids did not show any appreciable sequence homology. A single mRNA species of 2 kb was identified by northern blotting, which was expressed in brain tissue but not in liver. By in situ hybridization a selective labeling of myelinating glial cells in the trout CNS and PNS was revealed. The developmental appearance of the IP1 transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. PMID- 7539052 TI - Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage. AB - We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA 1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy 5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs. PMID- 7539053 TI - Galectin-3, a beta-galactoside-binding animal lectin, binds to neural recognition molecules. AB - In this study, we have investigated the ability of galectin-3, a beta-galactoside binding animal lectin, to interact in vitro with different neural tissue-derived glycoproteins involved in cell-cell and cell-substrate adhesion. Galectin-3 interacted to varying degrees with the cell recognition molecules L1, the myelin associated glycoprotein, and the neural cell adhesion molecule and the extracellular matrix molecules tenascin-C and tenascin-R but not with collagen type I. Binding of galectin-3 to the different glycoproteins tested was carbohydrate dependent and could be specifically inhibited by the addition of lactose and, to a lesser extent, galactose. PMID- 7539054 TI - Involvement of different types of voltage-sensitive calcium channels in the presynaptic regulation of noradrenaline release in rat brain cortex and hippocampus. AB - Transmitter release at the nerve terminal is mediated by the influx of Ca2+ through voltage-sensitive calcium channels (VSCCs). Many types of VSCCs have been found in neurons (T, N, L, and P), but uncertainty remains about which ones are involved in neuronal excitation-secretion coupling. Specific ligands for the L- and N-type VSCCs were used to determine which of these subtypes might be involved in the K(+)-evoked [3H]noradrenaline release from superfused rat brain cortical and hippocampal synaptosomes. In cortical presynaptic terminals the 1,4 dihydropyridine agonist Bay K8644 enhanced the K+ (15 mM)-evoked [3H]noradrenaline release. This effect was reversed by the 1,4-dihydropyridine antagonists nimodipine and nitrendipine. The L-type VSCC ligands had no effect on hippocampal synaptosomes. In contrast, the N-type VSCC blocker omega-conotoxin markedly reduced the K(+)-evoked [3H]noradrenaline release in nerve terminals from both regions. Inhibition was greater in hippocampal synaptosomes. When applied together the inhibitory actions of nimodipine and omega-conotoxin were approximately additive. These findings indicate that both L- and N-type VSCCs participate in noradrenaline release in rat brain cortex and suggest that noradrenergic terminals in the two regions examined may have distinct populations of VSCCs: L type in cortex and N type in hippocampus. PMID- 7539055 TI - Characterisation of antibodies specific for chick brain neural cell adhesion molecules which cause amnesia for a passive avoidance task. AB - Antisera were prepared against six postsynaptic density glycoprotein fractions (150-180, 62-80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day-old chicks in a one-trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R-1 and R-6, or their IgGs (IgG-1 and IgG-6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later, IgG-1 and IgG-6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti-N-CAM antibodies. IgG-1 and IgG-6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N linked mannose and fucose, and a very low amount of polysialic acid and O-linked galactose. Absorption of IgG-6 antibodies with neural cell adhesion molecule (N CAM) isolated from synaptic plasma membranes derived from day-old chick brain resulted in loss of amnestic effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539056 TI - Participation of noradrenergic neurotransmission in the enhancement of baroreceptor reflex response by substance P at the nucleus tractus solitarii of the rat: a reverse microdialysis study. AB - We applied reverse microdialysis and HPLC analysis to evaluate the participation of noradrenergic neurotransmission in modulation of the baroreceptor reflex response by substance P at the nucleus tractus solitarii in Sprague-Dawley rats anesthetized with pentobarbital sodium (50 mg/kg, i.p., with 20 mg/kg/h.i.v. supplement). Continuous infusion of substance P (600 microM) at 1 microliter/min into the nucleus tractus solitarii through a stereotaxically positioned microdialysis probe (active exchange length, 180-200 microns; diameter, 220 microns) for 1 h elicited an enhancement of the baroreceptor reflex response. This facilitatory effect correlated positively, during the 60-min infusion period, with the time course of increase in the extracellular concentration of substance P and noradrenaline in the nucleus tractus solitarii. Experimentally elevating the concentration of noradrenaline at this medullary nucleus also augmented the baroreceptor reflex sensitivity. On the other hand, depletion of the noradrenergic fibers and nerve terminals at the nucleus tractus solitarii with DSP4 diminished the enhancement of baroreceptor reflex response and the corresponding elevation in extracellular concentration of noradrenaline by substance P. Microinfusion of noradrenaline into the nucleus tractus solitarii in DSP4-treated animals, however, potentiated the baroreceptor reflex response. These results suggested that the enhancement of baroreceptor reflex response by substance P may involve an increase in the concentration of noradrenaline at the nucleus tractus solitarii via a presynaptic mechanism. PMID- 7539057 TI - Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease. AB - We present the first evidence for carbonyl-related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimer's disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress-type mechanisms, through the formation of cross-links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo. PMID- 7539058 TI - Properties of networks controlling locomotion and significance of voltage dependency of NMDA channels: stimulation study of rhythm generation sustained by positive feedback. AB - 1. We have built a realistic 24-neuron model based on data from the spinal pattern generator for swimming in Xenopus embryos with the use of the SWIM programs. The neurons have dendrite, soma, and axon compartments with voltage gated Na+ and K+ channels. Dendritic synapses were modeled as modulated ionic conductances with currents that have different reversal levels. One of these conductances was voltage dependent to model N-methyl-D-aspartate ("NMDA") synapses in the presence of Mg2+. 2. In this model, rhythm generation is initiated by a brief excitation, depends on rebound from reciprocal inhibition, and is sustained by long-duration "NMDA-dependent" feedback excitation. 3. Without NMDA voltage dependency, rhythmic activity is stable over a wide range of synaptic conductances. Its frequency decreases with more inhibition and increases with more excitation. The introduction of normally distributed variation in soma size or excitatory synaptic conductance extends the lower stable frequency range. Without such variation the frequency of the 24-neuron model is the same as a 4 neuron model provided that the synaptic conductances for each neuron are the same. 4. The effect of introducing NMDA voltage dependency on rebound after negative current injections or synaptic inhibition was investigated in single depolarized model neurons. With NMDA voltage dependency, hyperpolarizations and rebound spike responses were increased. 5. Network activity with NMDA voltage dependency was similar to that without it, but inhibitory postsynaptic potentials (IPSPs) and spikes were larger, and frequencies were lower and more sensitive to changes in excitatory and inhibitory conductance. 6. We conclude that in the model, mutual reexcitation among excitatory spinal interneurons can sustain rhythm generation by positive feedback and that NMDA voltage dependency can enhance postinhibitory rebound, stabilize swimming activity and extend its lower frequency range, and steepen the dependency of frequency on synaptic drive. PMID- 7539059 TI - Contraction characteristics and myosin heavy chain composition of rabbit masseter motor units. AB - 1. We studied isometric twitch peak force (TPF) and twitch contraction time (TCT) of 249 motor units of the masseter muscle in 41 rabbits after extracellular electrical stimulation of single trigeminal motoneurons in the brain stem. In 41 of these units we determined the amount of tension decrease during a partially fused tetanus (sag) and the ratio between peak tetanic force after 2 min of intermittent tetanic stimulation and initial tension (fatigue index). Muscle fibers of 24 motor units were identified by the glycogen depletion method and characterized in serial sections with monoclonal antibodies against type IID, IIA, "cardiac" alpha, and I isoforms of myosin heavy chain (MHC). 2. The motor units had TCTs ranging from 13 to 32 ms. The majority of the units showed forces < 35 mN. The TPFs were larger and varied more for motor units with short and intermediate TCTs than for units with long TCTs. There is a small but statistically significant negative correlation between the motor unit TPF and the TCT. 3. All units exhibited "sag" and, with the exeption of one, had fatigue indexes > 0.75. The studied rabbit masseter motor units can therefore be classified as fast, fatigue-resistant, except for one that belonged to the FF (fast, fatigable) category. No slow units were represented in the sample pool. Significant correlations were not found either between TCT and the amount of sag or between TCT and the fatigue index. 4. Immunohistochemical analysis showed that the FF unit had fibers containing only IID-MHC. Five other units were found with a single MHC--three with IIA-MHC and two with alpha-MHC. In three other units all fibers showed one combination of two MHCs (1 IIA/IID, 1 IIA/alpha, and 1 alpha/I). The remaining 15 units contained two MHCs spread unevenly over the constituting fibers. Large variations in myosin composition of fibers within one motor unit cast doubts on the presumed dominant neuronal influence on myosin expression in the adult animal. 5. We found a close, statistically significant correlation between the TCT and the estimated MHC content of the units: the TCT was 13 ms for the IID unit, 18 ms for the pure IIA units, and 28 ms for the pure alpha units. Units with two MHCs had intermediate TCTs; units with alpha/I-MHC mixtures had TCTs of 29-30 ms. No pure MHC-I units were identified.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539060 TI - Nonselective cation channels coupled with tachykinin receptors in rat sensory neurons. AB - 1. Effects of substance P (SP) and other tachykinins on membrane currents were investigated using whole cell voltage clamp in cultured sensory neurons isolated from rat dorsal root ganglia. 2. SP (100 nM) evoked an inward current in two thirds of the cells at negative potentials. In most of the cells that generated the inward current in response to SP, capsaicin also activated an inward current. The SP-evoked inward current was not observed in cells loaded with 2 mM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). 3. Neurokinin A (NKA) or neurokinin B (NKB) also activated an inward current. At 100 nM of each agonist, the order was NKB > NKA > SP with respect to activated current amplitude. 4. The tachykinin activated current was reversed around +10 mV with a standard extracellular solution containing 140 mM NaCl. The reversal potential became more negative when extracellular NaCl was reduced by substituting with sucrose. The inward current was also activated in cells bathed in an extracellular solution containing Cs+, tetraethylammonium (TEA) or N-methyl-D-glucamine (NMDG) as a major cation instead of Na+. The order of permeability, determined from the reversal potential of the current, was Cs+ not equal to Na+ > TEA > NMDG. The amplitude of the inward current activated by NKB was increased when extracellular Na+ was replaced with Cs+, TEA or NMDG. 5. Permeability of Ca2+ was tested using an extracellular solution containing Ca2+ as the only cation (111.8 mM Ca2+ outside). Under this condition, NKB evoked an inward current that reversed around +30 mV. 6. The results suggest that SP and other tachykinins activate nonselective cation channels, which are also permeable to Ca2+, through receptors which are more responsive to NKB than to SP or NKA. The channel activation may serve as a mechanism underlying tachykinin-mediated excitatory neurotransmission in sensory neurons. PMID- 7539061 TI - Whole cell and single channel properties of a new GABA receptor transiently expressed in the Hippocampus. AB - 1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis. PMID- 7539062 TI - Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion. AB - Many novel experimental therapeutic agents, such as neurotrophic factors, enzymes, biological modifiers, and genetic vectors, do not readily cross the blood-brain barrier. An effective strategy to deliver these compounds to the central nervous system is required for their application in vivo. Under normal physiological conditions, brain interstitial fluid moves by both bulk flow (convection) and diffusion. It has recently been shown that interstitial infusion into the white matter can be used to increase bulk flow, produce interstitial convection, and efficiently and homogeneously deliver drugs to large regions of brain without significant functional or structural damage. In theory, even more uniform distribution is likely in gray matter. In the current study, four experiments were performed to examine if convection-enhanced delivery could be used to achieve regional distribution of large molecules in gray matter. First, the volume and consistency of anatomical distribution of 20 microliters of phaseolus vulgaris-leukoagglutinin (PHA-L; molecular weight (MW) 126 kD) after continuous high-flow microinfusion into the striatum of five rats over 200 minutes were determined using immunocytochemistry and quantified with image analysis. Second, the concentration profile of 14C-albumin (MW 69 kD) infused under identical conditions was determined in four hemispheres using quantitative autoradiography. Third, the volume of distribution after convection-enhanced infusion of 250 or 500 microliters biotinylated dextran (b-dextran, MW 10 kD), delivered over 310 minutes into the caudate and putamen of a rhesus monkey from one (250 microliters) or two (500 microliters) cannulas, was determined using immunocytochemistry and quantified with image analysis. Finally, the ability to target all dopaminergic neurons of the nigrostriatal tract via perfusion of the striatum with subsequent retrograde transport was assessed in three experiments by immunohistochemical analysis of the mesencephalon following a 300-minute infusion of 27 microliters horseradish peroxidase-labeled wheat germ agglutinin (WGA-HRP) into the striatum. Convection-enhanced delivery reproducibly distributed the large-compound PHA-L throughout the rat striatum (the percent volume of the striatum perfused, Vs, was 86% +/- 5%; mean +/- standard deviation) and produced a homogeneous tissue concentration in the perfused region (concentration of 14C-albumin relative to infusate concentration 30% +/- 5%). In the monkey, the infusion widely distributed b-dextran within the striatum using one cannula (caudate and putamen Vs = 76% and 76%) or two cannulas (Vs = 90% and 71%).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539063 TI - Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines. AB - The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cytostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against alpha-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR. PMID- 7539064 TI - In vivo inhibition of angiogenesis and growth of the human U-87 malignant glial tumor by treatment with an antibody against basic fibroblast growth factor. AB - The effectiveness of in vivo suppression of neovascularization and growth of malignant glial tumors by in situ administration of an antibody directed against basic fibroblast growth factor (bFGF), a strong mitogen for cells of mesodermal origin, was tested. One hundred fifty congenitally athymic nude rats (han rnu/rnu) were implanted intracerebrally with U-87MG tumor cells, known constitutive producers of bFGF. The animals were randomly assigned to six groups of 25 animals each. Animals were treated by in situ application of saline (Group F), control antibody (Group D), or polyclonal anti-bFGF antibody (Group B). In additional groups a putative effect on tumor growth caused by the treatment application device itself (between growth control Groups A and E), and the effect of heat-inactivated tumor cells (negative control Group C) were tested. After 3 weeks of treatment, tumor progression and degree of neovascularization were morphometrically recorded. In the untreated Groups A and E massive tumor growth was recorded, consisting of 19.9% +/- 0.4% and 27.1% +/- 0.5%, respectively, of the total brain cross-sectional area. In Group C, no tumor growth occurred. In control Groups D and F tumor progression consisted of 18.6% +/- 0.4% and 18.5% +/ 0.4%, respectively, of the total brain cross-sectional area; whereas in the anti bFGF treated Group B, significantly smaller tumor masses measuring 7.2% +/- 0.1% were recorded. New blood vessels were located both peritumorally and intratumorally and defined as numerical density and area fraction (number/area and area/area). Significantly more new blood vessels were found in Groups A, D, E, and F, ranging from 41,380/mm2 +/- 464/mm2 to 53,442/mm2 +/- 150/mm2 peritumorally and 51,846/mm2 +/- 495/mm2 to 64,660/mm2 +/- 183/mm2 intratumorally than in the anti-bFGF treated Group B, which numbered 8220/mm2 +/- 225/mm2 peritumorally and 16,554/mm2 +/- 236/mm2 intratumorally. The authors conclude that treatment with anti-bFGF antibody is effective in inhibiting tumor-induced angiogenesis and correlated tumor progression. However, owing to the character of the experimental system used, one cannot exclude the possibility that application of the specific anti-bFGF antibody also counteracts device-induced neovascularization. The authors suggest that combined surgical excision and adjuvant immunotherapy of tumors such as glioblastoma and other malignant brain tumors that express bFGF might prevent tumor recurrence. PMID- 7539065 TI - Structural requirements, synthesis and interaction of secondary nitrosamine inhibitors with acetylcholinesterase from Torpedo fuscomaculata. AB - A series of secondary diaryl and dialkyl nitrosamines have been synthesised and tested as substrates and/or inhibitors of highly purified acetyl- cholinesterase from Torpedo fuscomaculata. None were found to act as substrate, but many could selectively inhibit the enzyme. Kinetic analysis has shown that all the nitrosamines act as reversible competitive inhibitors with respect to the substrate, acetylthiocholine chloride; with time they act as irreversible covalent inhibitors. Scatchard analysis indicates that aliphatic nitrosamines have a weaker affinity for the enzyme compared to the aromatic and heterocyclic nitrosamines. In all cases the number of binding sites was four. Pseudo first order kinetics are observed with the rate constant being proportional to the concentration of the nitrosamine and the order of reaction being equal to one. PMID- 7539066 TI - Evaluation of some 4-fluoro- and 4-cyano derivatives of delta 4,3-ketosteroids as inhibitors of testosterone 5 alpha-reductase. AB - Some 4-fluorinated analogues of 3-oxo-delta 4 steroids and 4-cyano derivatives of progesterone and androstenedione were evaluated as inhibitors of steroid 5 alpha reductase activity. Inhibitors of this enzyme may be useful in treating prostatic cancer. 4-Fluoroandrostenedione was a modest inhibitor of the rat enzyme (IC50 = 4.08 microM), while 4-cyanoprogesterone was a potent inhibitor of both the rat and human enzymes (IC50 values = 0.045 microM and 0.050 microM respectively). These two steroids were tested in vivo for activity against androgen sensitive organs in WHT mice. 4-Fluoroandrostenedione caused increases in organ weights, suggesting it is an androgen agonist, while the 4-cyano compound displayed modest androgen ablation. Therefore substitutions at the 4-position may produce compounds of therapeutic use in treating prostate cancer. PMID- 7539067 TI - Antibacterial effects of esters of guanidino- and amidino-acids trypsin inhibitors. AB - Inhibitory effects of various esters of trans-4 guanidinomethylcyclohexanecarboxylic acid and the 4-tert- butylphenyl esters of amidinopiperidine-4-alkanoic, trans-4- amidinocyclohexanealkanoic, trans-4 guanidinoethylcyclohexanecarboxylic and trans-4-guanidinocyclohexanealkanoic acids, all trypsin inhibitors, on the growth of E. coli, B. subtilis, S. aureus and S. epidermidis were examined. 4-tert-Butylphenyl esters strongly inhibited the growth of E. coli and the order of the inhibitory effects correlated with that for the inhibitory effects on proteinase In, which appears immediately before initiation of DNA synthesis in E. coli and closely correlates with the onset of DNA synthesis. No correlation was observed between the inhibitory effects and Ki values for trypsin. The 4-tert-butylphenyl esters also strongly inhibited B. subtilis, S. aureus and S. epidermidis, and the order, of the inhibitory effects on these bacteria roughly coincided with that on E. coli. The order of the inhibitory effects of each ester, on these bacteria was S. epidermidis > S. aureus > B. subtilis > E. coli. Among the esters examined, the biphenyl ester of trans-4-guanidinoethylcyclohexanecarboxylic acid was the most inhibitory on these four bacteria and proteinase In. Hydrolysis of tert butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide, which is a substrate for proteinase In, in crude extracts of E. coli, B. subtilis and S. epidermidis was examined. The order of this activity in these bacteria was E. coli > B. subtilis > S. epidermidis. PMID- 7539068 TI - Investigation of the nature of o-phthalaldehyde reaction with octopine dehydrogenase. AB - The effect of o-phthalaldehyde on octopine dehydrogenase inactivation has been studied. o-Phthalaldehyde binds to the proximal cysteine and lysine residues of the enzyme leading to the formation of isoindole derivative. Double inhibition studies with o-phthalaldehyde and p-chloromercuricphenyl sulfonic acid have indicated that o-phthalaldehyde does not bind to the functional cysteine present at the active site. Protection experiments have shown that L-arginine prevented o phthalaldehyde inactivation. This could be only due to the reaction of the amino group of L-arginine with o-phthalaldehyde as per the mechanism proposed elsewhere since L-arginine cannot bind to the enzyme prior to NADH. Other substrates such as pyruvate oR NADH could not prevent the o-phthalaldehyde reaction with the enzyme. Fluorescence spectral studies demonstrated that in the presence of externally added amino acid no isoindole derivative formation occurs. However, a characteristic isoindole derivative is formed in the presence of beta mercaptoethanol although the enzyme does not lose its activity. This indicated that o-phthalaldehyde can bind with lysine of the enzyme and thiol of externally added beta-mercaptoethanol. Pyridoxal 5'-phosphate, a lysine specific reagent also binds to the enzyme giving the characteristic absorption and fluroescence peak at 325 nm and 395 nm respectively. However, no loss of enzyme activity was observed. On the basis of these experiments we would suggest that o phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation. PMID- 7539069 TI - Retinoic acid inhibition of a tumour protease immobilised on cell surfaces and in free solution. AB - Retinoids are inhibitors of tumour cell proliferation in culture and have been shown to suppress carcinogenesis and decrease the levels of proteases. The present study has demonstrated that retinoic acid is a potential non-competitive inhibitor of a protease (GB) immobilised on the surfaces of tumour cells in thin sections and free GB in solution. The enzymic status of GB on the cell surfaces in sections has been determined by challenging the retinoic acid-treated cells with a second fluorescent inhibitor (9-AA), followed by fluorescence microscopic analysis. The inhibition of cell surface GB by retinoic acid was demonstrated to be reversible. The activity of soluble GB has been measured by the MUGB assay in the presence and absence of retinoic acid. It is suggested that retinoic acid acts on GB by interacting with a binding site, different from the active site, and causes major conformational changes, resulting in enzyme inhibition. It is possible that the modulation of GB activity by retinoic acid may play a role in the control of cell migration and metastasis. PMID- 7539070 TI - Xanthine oxidase inhibitors from the leaves of Alsophila spinulosa (Hook) Tryon. AB - Diploptene(1), beta-sitosterol(2), a mixture of 6'-O-(E-P-coumaroyl)-alpha glucopyranose and 6'-O-(E-P-coumaroyl)-beta-glucopyranose(3), a mixture of 6'-O (E-P-caffeoyl)-alpha-glucopyranose and 6'-O-(E-P-caffeoyl)-beta-glucopyranose(4), caffeic acid(5) and astragalin(6) were isolated from an ethanolic extract of the leaves of Alsophila spinulosa Hook Tryon (Cyatheaceae). The plant has been used in folk medicine for hepatitis, gout, rheumatism, and tumor and these compounds were tested for their inhibitory effect on xanthine oxidase. Caffeic acid was the most potent constituent (IC50 = 39.21 microM; Ki = 28.2 microM) and was an uncompetitive inhibitor of the enzyme with respect to the substrate xanthine. PMID- 7539072 TI - Kinetics of the reversible tight-binding inhibition of pig liver catechol-O methyltransferase by [2-(3,4-dihydroxy-2- nitrophenyl)vinyl]phenyl ketone. AB - The inactivation of partially purified pig liver catechol-O-methyltransferase (COMT) by [2-(3,4-dihydroxy-2-nitrophenyl)vinyl]phenylketone has been studied. The results demonstrated that COMT is inhibited in a reversible tight-binding fashion with an apparent Ki of 0.2 microM. IC50 values were determined at different concentrations of both substrates of the enzymatic reaction, pyrocatechol and S-adenosyl-L-methionine (AdoMet). The plot of IC50 versus pyrocatechol concentration gave a straight line, suggesting competitive inhibition. However the nitrocatechol derivative showed an uncompetitive inhibition pattern when measured as a function of AdoMet concentration. PMID- 7539071 TI - Pyridyl substituted benzocycloalkenes: new inhibitors of 17 alpha hydroxylase/17,20-lyase (P450 17 alpha). AB - Compounds capable of inhibiting 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha) are of great interest for the therapy of prostatic cancer since they block androgen biosynthesis. In order to evaluate the inhibitory activity of a series of benzocycloalkenes developed in our group, an in vitro assay was established using rat testicular microsomes as source of the enzyme, non labelled progesterone as substrate and a HPLC procedure for separation of the steroids. The inhibitory activities of 33 test compounds were compared to ketoconazole (IC50 67 microM), a known inhibitor of P450 17 alpha, which recently has been successfully used in prostate cancer patients. Several compounds of the present study were stronger inhibitors of P450 17 alpha than ketoconazole. The most active compounds were compound 12(5-methoxy-2-(4-pyridylmethyl)-1-tetralone: IC50 13 microM) and compound 13(5-methoxy-2-(4-pyridyl)-1-tetralone: IC50 13 microM). PMID- 7539073 TI - Inactivation of yeast alcohol dehydrogenase by nitrilopropionamides. AB - A series of halonitrilopropionamides have been examined as potential inhibitors of yeast alcohol dehydrogenase. Analogues with a good leaving group on the alpha carbon, and a geminal electronegative atom, were found to be initial competitive inhibitors against NAD with inhibition constants as low as 0.6 microM. Incubation of the enzyme with these inhibitors leads to a slow, irreversible inactivation, with an inactivation constant for 2,2-dibromo-3-nitropropionamide of about 100 microM. No protection against inactivation was observed with the substrate ethanol, while the presence of saturating levels of NAD slowed the rate, but not the final extent, of enzyme inactivation. The resulting enzyme-inactivator complex is stable to a range of conditions that are known to denature the enzyme, indicating that a covalent modification of yeast alcohol dehydrogenase has led to the inactivation. A bimodal inactivation model is proposed to account for the observed interactions of these halonitrilopropionamides with yeast alcohol dehydrogenase. PMID- 7539074 TI - A tridimensional representation of enzyme inhibition useful for diagnostic purposes. AB - A new three dimensional representation of enzyme inhibition, applied to Lineweaver-Burk, Hanes and Eadie-Hofstee plots is presented. This type of representation has advantages for enzyme inhibition diagnosis, showing graphic characteristics that pass unnoticed in linear plots. PMID- 7539075 TI - Effect of the antiallergic drug disodium cromoglycate and various derivatives on alkaline phosphatase. AB - Disodium cromoglycate (DSCG) inhibits alkaline phosphatase in a non-competitive manner, the enzyme undergoing a conformational change which is attenuated by the presence of calcium ions. The structurally related pyranone and benzoic acid are weak inhibitors of the enzyme and produce a similar conformational change. Coumarin does not induce any conformational change in the enzyme suggesting that the 4-oxo group in DSCG may be essential for its effect. PMID- 7539076 TI - Inhibition of HIV-1 reverse transcriptase by defined template/primer DNA oligonucleotides: effect of template length and binding characteristics. AB - The interaction of partially double stranded DNA oligonucleotides with HIV-1 RT was studied by investigating their ability to inhibit the homopolymeric poly(rC) directed (dG) synthesis reaction. A 20/18mer oligonucleotide, with a sequence based on the Lys3-tRNA primer region, showed stronger inhibition of the homopolymeric RT reaction than a G/C rich oligonucleotide series lacking or possessing a hairpin moiety. Interaction of the enzyme with the G/C rich oligonucleotides, as determined by IC50 measurements, was insensitive to the extent of the unpaired template region at the 3' or 5' position. Addition of a hairpin moiety, composed of four thymidine bases, onto G/C rich oligonucleotides increase their inhibitory potency (at least six times) and shifted the mode of inhibition of RT to competitive with respect to poly (rC).(dG), which was otherwise mixed (competitive/noncompetitive) for the linear G/C rich and 20/18mer oligonucleotides. The results indicate that interaction of the enzyme with the primer/template stem, but not with the unpaired template region, is an important step in complex formation. PMID- 7539077 TI - Liver transplantation for HBV-related disease under immunosuppression with tacrolimus: an experience with 78 consecutive cases. AB - Hepatitis B viral liver disease (HBVLD) is a major worldwide health problem. It is estimated over 300 million people have had hepatitis B virus infection and that one-third of these have chronic HBVLD. Little effective therapy exists for HBVLD even though high dose interferon (IFN) has been advocated. For those who either are untreated or do not respond to IFN, HBVLD is steadily progressive and orthotopic liver transplantation (OLTx) is the only available therapy. Until quite recently, all OLTx recipients received cyclosporine (CyA) and prednisone. The consequence of OLTx for HBV disease in individuals immunosuppressed with tacrolimus has not previously been reported. A total of 78 consecutive patients with HBV-related liver diseases who were transplanted between January 1, 1990, and December 31, 1991, and treated with tacrolimus were studied. The clinical records of these patients were reviewed retrospectively. HBV disease recurrence was documented with serologic and histologic methods. As of April 1, 1993, 57 of 78 (73%) of the patients were still alive. Thirty-one of the alive patients have documented HBV recurrence. Eighteen of these 31 patients, however, have normal liver function. With a median follow-up of 24 months, 8 patients (10.9%) have died of recurrent HBVLD. Seven of 8 patients, who preoperatively were HBeAg+, developed recurrence and 4 of these patients have already died of recurrence. Patients who were HBsAg+ rarely recurred (1 of 16 patients). The use of HBIG did not prevent recurrence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539078 TI - A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung's disease. AB - Recent experimental studies in mice have shown that the proto-oncogene c-kit plays a key role in the development of a component of the pacemaker system that is required for generation of autonomic gut motility. These studies further suggest that interaction of the c-kit receptor and its ligand (stem cell factor, SCF) is critical for the development of the enteric nervous system. The authors investigated the presence of c-kit-positive (c-kit+) cells as well as the expression of SCF in bowel from 12 patients with Hirschsprung's disease (HD), 4 patients with total colonic aganglionosis (TCA), 2 patients with extensive aganglionosis (EA) and 14 controls. Our methods involved the use of immunohistochemistry with antihuman c-kit sera and antihuman SCF sera. A few c kit+ cells were found in the muscle layers of aganglionic bowels from HD, TCA and EA, in contrast to many c-kit+ cells in ganglionic bowel segments from control, HD, and TCA patients. Expression of SCF was identified in the muscle layers as well as in myenteric plexus of ganglionic bowel, in contrast to its absence in the muscle layers of aganglionic bowel specimens. A lack of c-kit and SCF might be of significance for autonomic gut dysmotility in aganglionic bowel segments of patients with HD and allied disorders such as chronic idiopathic intestinal pseudo-obstruction. PMID- 7539079 TI - Biliary amylase and congenital choledochal dilatation. AB - The relationship between levels of biliary amylase measured at operation and clinical features was studied in a series of 55 children with congenital biliary dilatation (choledochal cyst) who presented between 1976 and 1993. There were 36 cystic and 19 fusiforms dilatations in the series. The most common modes of presentation were painless jaundice (n = 23) and pancreatitis (n = 22). Five infants presented with abnormal antenatal ultrasound examinations. Children with pancreatitis were older than those with painless jaundice (4.2 versus 1.5 years; P = .005), and a higher proportion had raised levels of biliary amylase (100% versus 44%; P < .0001). There was no difference in the age at presentation (P = .32), clinical mode of presentation (P = .3), or the level of biliary amylase (P = .25) between cystic and fusiform dilatations. A correlation was found between age at surgery and biliary amylase in the cystic (rs = 0.55; P = .001) but not in the fusiform group (P = .22). All infants with antenatal diagnoses were cystic dilatations. Choledochal cystic dilatations that were diagnosed antenatally did not have significant amylase reflux, suggesting that the aetiology of this subgroup is truly congenital. Children who present at a later age with pancreatitis invariably have high levels of biliary amylase, which is presumed to occur because of a common channel and reflux of biliary and pancreatic secretions. PMID- 7539080 TI - Two CF patients, one homozygous for the 621 + 1G > T splice mutation, the other homozygous for the 1898 + 1G > A splice mutation. PMID- 7539081 TI - Determinants of RNA hairpin loop-loop complex stability. AB - Complexes formed by RNA hairpin loops with complementary loop sequences derived from Escherichia coli RNA I and RNA II, which are involved in the control of DNA replication of plasmid ColE1, have been analyzed to determine the sequence and structural elements required to achieve full affinity. Of particular interest is the origin of the enhanced stability of the complex formed by hairpin loops whose loop sequences have been inverted 5' to 3' with respect to wild-type sequences. Full complementarity of the two interacting loops is required to achieve full or enhanced affinity, while the stems of the two hairpins can differ. The major determinant of enhanced affinity lies in the base-pairs formed at positions 1 and 7 of the loops, together with the two base-pairs of each stem which are closest to the loop. Sequence variation in the middle of the loops, or further down the stem away from the loops, exerts only a modest influence on complex stability. We incorporate these results into a model for the loop-loop interaction which accounts for the importance of positions one and seven and the first two nucleotides of the stem, while providing potentially unique structures for recognition by the RNA one modulator protein. PMID- 7539082 TI - Myocardial contractile dysfunction in the systemic inflammatory response syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes. AB - A major determinant of survival in patients with advanced viral or bacterial infection, or following severe trauma or burns complicated by multiple organ failure, is the combination of clinical signs termed the systemic inflammatory response syndrome (SIRS). SIRS is characterized by hypotension, tachypnea, hypo- or hyperthermia and leukocytosis as well as other clinical signs and symptoms, including a depression in myocardial contractile function. Heart failure complicating systemic sepsis or other causes of SIRS is usually not accompanied by coronary artery ischemia due to hypotension, myocardial necrosis, or marked cardiac interstitial inflammatory infiltrates, and thus the cause of cardiac contractile dysfunction in this syndrome has remained unclear. However, recent evidence has implicated an endogenous nitric oxide (NO) signalling pathway within cardiac myocytes and other cellular constituents of cardiac muscle, including the microvascular endothelium, as a possible contributor to the pathogenesis of heart failure in this syndrome. Cardiac myocytes are now known to express both constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) activities. Activation of cNOS appears to modulate cardiac myocyte responsiveness to muscarinic cholinergic and beta-adrenergic receptor stimulation. Induction of iNOS by soluble inflammatory mediators, including cytokines, causes a marked depression in myocyte contractile responsiveness to beta-adrenergic agonists. Thus, inappropriate activation of cNOS or excessive or prolonged induction of iNOS in the myocardium may contribute to cardiac dysfunction complicating SIRS. PMID- 7539083 TI - Immunofluorescence techniques for the identification of immune effector cells in rat heart: applications to the study of the myocarditis induced by interleukin-2. AB - A detailed description is presented of immunohistochemical methods for identification of various types of immune effector cells in rat heart, involving the use of antibodies conjugated with different fluorochromes for the simultaneous demonstration of 2 or 3 different antigens by means of fluorescence microscopy. The initial results of the application of these techniques to the study of the myocarditis induced by interleukin-2 (IL-2) are also presented. Antibodies used included: OX6 antibody (for MHC class II molecules, mainly expressed by dendritic cells): W3/25 and OX8 antibody, for the demonstration of the rat equivalents of CD5 and CD8, respectively: asialo-GM1 ganglioside antibody for the identification of natural killer (NK) cells and lymphokine activated killer (LAK) cells, and ED2 antibody for labeling of macrophages. Fluorochromes used were: fluorescein isothiocyanate (green), tetramethylrhodamine isothiocyanate (red), Texas red sulfonyl chloride (red), and 7-amino-4 methylcoumarin-3-acetic acid (blue). IL-2-induced myocarditis was characterized histologically by infiltration of the myocardium by mononuclear inflammatory cells, microvascular alteration, interstitial edema, and myocyte damage and necrosis. In the initial stages, NK/LAK cells were the predominant type of infiltrating lymphocytes; however, the numbers of these cells decreased sharply in subsequent stages. Macrophages also were initially abundant, and continued to be prevalent throughout the late stages. CD8+ lymphocytes were more numerous than CD4+ lymphocytes. Dendritic-cells showed a diffuse increase in number and also accumulated around foci of myocyte necrosis. Three phenotypes of dendritic cells were recognized, and the possible implications of these findings are discussed. It is hoped that these techniques will prove useful for the immunohistochemical evaluation of various inflammatory diseases of the heart. PMID- 7539084 TI - Anti-beta 1-adrenoceptor autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: mapping of epitopes in the first and second extracellular loops. AB - In a preceding communication (Wallukat et al., 1992, Z Kardiol 81 [Suppl. 4]: 79 83), it was reported that synthetic peptides, corresponding in amino acid sequence to either the first or the second extracellular loop of the human beta 1 adrenoceptor, selectively suppressed the metoprolol- and bisoprolol-sensitive positive chronotropic action exerted in cultures of beating neonatal rat cardiomyocytes by the serum immunoglobulin fraction of patients with myocarditis and idiopathic dilated cardiomyopathy (DCM) and by affinity-purified autoantibodies from that fraction. These observations added to existing evidence that these antibodies were directed against the beta 1-adrenoceptor and might thus contribute to the harmful chronic cardiac adrenergic drive to which patients with DCM are believed to be exposed. Specifically, they pointed to the putative first and second extracellular loops of this receptor (these loops are each identical in man and the rat) as the sites of epitopes recognized by the chronotropically active, beta 1-agonistic autoantibodies. Now we report on the mapping of these epitopes with the help of two series of short synthetic overlap peptides, one series forming part of the first and the other of the second extracellular loop of the beta 1-adrenoceptor. Inhibition of the positive chronotropic response of cultured rat cardiomyocytes to the anti-beta 1-receptor autoantibodies (EC50 = 0.14 +/- 0.01 nM) from the serum immunoglobulin fraction of patients with DCM was taken as reflecting the neutralization of these antibodies by a particular overlap peptide. In this way the sequences S-F-F-C-E-L (residues 129-134) and A-R-R-C-Y-N-D (residues 206-212) emerged as the dominant epitopes in the first and second extracellular loops, respectively, followed with respect to neutralizing ability by the first loop sequence E-Y-G-S-F-F (residues 126-131) and the second loop sequences H-W-W-R-A-E (residues 197-202) and P-K-C-C D-F (residues 213-218). Synthetic peptides corresponding to the sequences of the third extracellular loop of the beta 1-receptor (residues 346-356) and of the second extracellular loop of the human beta 2-receptor (residues 172-197) failed to neutralize the beta 1-agonistic autoantibodies. Using dithiothreitol as a reducing agent a disulfide bridge between cysteine 132 in the first and cysteine 209 in the second extracellular loop was considered to be essential for the chronotropic action of these autoantibodies.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539085 TI - Functional epitope analysis of the second extracellular loop of the human heart muscarinic acetylcholine receptor. AB - Two synthetic peptides corresponding to amino acids 172-181 and 169-193 of the second extracellular loop of the human M2 muscarinic receptor respectively were used to raise antibodies in rabbits. Affinity-purified antibodies were able not only to recognize a major band with a molecular weight of about 80 kDa on the electrotransferred membrane proteins of rat ventricular membranes but also to localize the muscarinic receptors on the sarcolemma and t-tubules of rat cardiomyocytes. Antibodies were also able to mimic muscarinic agonist stimulation as demonstrated by a negative chronotropic effect on cultured neonatal cardiomyocytes. In contrast with the antibodies raised against the peptide 169 193, the antibodies against the peptide 172-181 were unable to inhibit muscarinic ligand binding. These results suggest that the decapeptide 172-181 contains the B cell epitope responsible for the functional effect of antibodies directed against the second extracellular loop of the receptor. Coupling this peptide by cystein 177 blocks the induction of antibodies with pharmacological effects but induces antibodies which are able to recognize the denatured receptor protein and to exert a negative chronotropic effect. PMID- 7539086 TI - Stretch-activated channel blockers modulate cell volume in cardiac ventricular myocytes. AB - Stretch-activated channels (SAC) are postulated to regulate cell volume. While this hypothesis is appealing, direct evidence is lacking. Using digital video microscopy, we found that pharmacological blockade of SACs alters the cell volume of isolated rabbit ventricular myocytes during hypoosmotic stress. Under control conditions, relative cell volume increased from 1.0 to 1.311 +/- 0.019 after 10 min in 195 mosmol/l solution. The cation SAC blocker gadolinium (Gd3+; 10 microM) reduced the amount of swelling in hypoosmotic solution by 24% and induced a regulatory volume decrease otherwise not observed. In contrast, the anion SAC blocker 9-anthracene carboxylic acid (9-AC; 1 mM) increased swelling by 44% under the same conditions. Based on the direction of SAC currents, Gd3+ and 9-AC are expected to have opposite effects on cell volume. Furthermore, Gd3+ and 9-AC changed cell volume by only approximately 2% in isosmotic solutions when SACs are expected to be closed. This supports the idea that Gd3+ and 9-AC affect stretch activated transport processes. In contrast, omitting bath Ca2+ did not alter cell volume under iso- or hypoosmotic conditions suggesting stretch-activated Ca2+ influx is not important in setting cell volume. Not all channels can affect cell volume. Opening ATP-sensitive K+ channels with aprikalim (100 microM) or blocking them with glibenclamide (1 microM) did not alter cell volume under isosmotic or hypoosmotic conditions. These data support the idea that SACs are involved in cardiac cell volume regulation. PMID- 7539087 TI - Immediate relief of spontaneous coronary artery spasm by intracoronary infusion of an endothelium-dependent vasodilator, substance P. AB - This report describes a 45-year-old Japanese man who had episodes of anginal chest pain on effort. Coronary arteriography in the baseline state revealed subtotal occlusion in the mid-portion of the left anterior descending coronary artery. After intracoronary infusion of an endothelium-dependent vasodilator, substance P, the subtotal occlusion was immediately abolished. We concluded that endothelium-dependent vasodilation evoked with substance P was present at the site where coronary vasospasm occurred spontaneously in our case. PMID- 7539088 TI - Antibody-induced alterations of protein and nucleic acid metabolism during experimental thyroglobulin immunization. AB - The protein and nucleic acid contents of liver, spleen, muscle, kidney, and brain were studied in experimental autoimmune thyroiditis. Protein and ribonucleic acid levels showed a positive increase in the case of liver, spleen, and muscle on days 56 and 98 after immunization and on day 147 they reached control levels. Spleen deoxyribonucleic acid showed a significant increase on day 56, whereas the other days showed no change. Kidney and brain tissue revealed no change. PMID- 7539090 TI - Sputum substance P in aspiration pneumonia. PMID- 7539089 TI - Fulminant reactivation of hepatitis B due to envelope protein mutant that escaped detection by monoclonal HBsAg ELISA. AB - The detection of a fatal case of reactivation of hepatitis B, in a previously vaccinated Indonesian patient after withdrawal of chemotherapy for lymphoma, was delayed because HBsAg was negative in a widely used monoclonal-antibody-based ELISA. The serum was later found to be strongly reactive for HBsAg by the polyclonal radioimmunoassay and for HBV DNA. PCR sequencing revealed a substitution of arginine for glycine at position 145 of HBsAg in the major neutralising epitope cluster, the a determinant, as well as a 2-aminoacid insertion of asparagine and threonine between positions 122 and 123, immediately upstream of this determinant. PMID- 7539091 TI - Aggrecan-versican-neurocan family proteoglycans. PMID- 7539092 TI - Hyaluronic acid receptors. PMID- 7539093 TI - Identification of recognition sequences of adhesion molecules using phage display technology. PMID- 7539094 TI - Tenascins. PMID- 7539095 TI - Thrombospondins. PMID- 7539096 TI - Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32 and detection of specific antibodies by ELISA. AB - We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides can be valuable diagnostic material in the serology of HCMV infection (5, 6, 13). In this work we fused sequences expressing two different epitopes (aa 1005-1048 and aa 595-614) contained in the basic phosphoprotein of 150 KD coded by UL32 (1, 2), (ppUL32), which has repeatedly been shown to be the strongest immunogen present in the viral particle. The fusion protein was tested by ELISA with HCMV-positive human sera in comparison with other fusion proteins of ppUL32. We found that the double epitope fusion protein was recognised by IgM present in a larger number of sera and with more intense reactions than all the other ppUL32 fusion proteins. The double epitope reacted positively with 81.3% and, when denatured, with 94.7% of IgM-positive sera respectively. IgG reactivity was also high, reaching a percentage of 90.7. PMID- 7539097 TI - Repeated exposure to lindane leads to behavioral sensitization and facilitates electrical kindling. AB - Repeated intermittent exposure to some chemicals produces behavioral sensitization and seizure induction through a kindling mechanism. Although many pesticides are convulsant at high dosages, the persistent neurological effects of chronic low level exposure are unclear. The impact of intermittent exposure to lindane on behavioral seizure development and subsequent electrical kindling was assessed in the present study. Rats were administered lindane (0 or 10 mg/kg, po) for 30 days, or 3 times/week for 10 weeks. Enhanced behavioral responsiveness to lindane (myoclonic jerks, clonic seizures) emerged over the course of dosing and persisted 2 to 4 weeks after the last dose. The incidence of generalized convulsions was increased from 0% to 15% between the first and final day of dosing. In addition, electrographic recordings from the amygdala revealed brief rhythmic bursts and isolated interictal spike and wave discharge in the absence of overt behavioral seizures. Electrical kindling of the amygdala, beginning 4 to 6 weeks after the final dose, was facilitated. In contrast, prior administration of a single convulsive dose of lindane (20 mg/kg) was without effect on kindling development. These data indicate that repeated exposure to subconvulsant doses of lindane produces a persistent alteration in the central nervous system as evidenced by an enhanced susceptibility to kindled seizures. The pattern of behavioral development whereby the sensitivity is built up gradually over time is suggestive of a chemical kindling mechanism. Savings in the number of stimulation sessions required to induce electrical kindling following a history of lindane treatment provides further evidence that prior lindane exposure may lead to a state of partial kindling. Thus, intermittent subconvulsive lindane treatment induces alterations in limbic excitability that persist for at least 1 month. PMID- 7539098 TI - Seizure thresholds in kindled animals are reduced by the pesticides lindane and endosulfan. AB - Lindane and endosulfan are chlorinated hydrocarbon pesticides that bind to the gamma-aminobutyric-acidA (GABAA) receptor ionophore complex. We have previously demonstrated development of a persistent predisposition to the seizure-inducing properties of these pesticides following repeated administration of initially subconvulsive doses. A subsequent facilitation of electrical kindling development indicated that a chemical kindling mechanism may contribute to this enhanced sensitivity. We tested the bidirectionality of kindling transfer induced by chemical and electrical means by comparing the convulsive thresholds of lindane and endosulfan in electrically kindled and nonkindled animals. Amygdala kindled, implanted/nonstimulated, and nonimplanted controls were administered 0, 5, 10, or 20 mg/kg of lindane or endosulfan in corn oil by gavage, and each animal's behavior was scored for the presence of myoclonic jerks (MCJ) and clonic seizures. Dose-related increases in the number of animals displaying convulsions in response to both of these pesticides were observed. The dose of lindane (4.5 mg/kg) and endosulfan (4.3 mg/kg) producing MCJs in 50% of the animals (ED50) was reduced by more than 60% in kindled animals relative to controls. A second experiment evaluated the generalized seizure threshold in previously kindled animals treated with low doses of endosulfan (0, 2.5, and 5 mg/kg, po by gavage). Endosulfan reduced the intensity of electrical stimulation required to evoke seizures in amygdala kindled animals. These data indicate that these chlorinated hydrocarbon insecticides reduce seizure thresholds in amygdala-kindled animals with acute administration, and together with previous findings (see ref. 20) demonstrate bidirectional transfer between chemical and electrical kindling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539099 TI - Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure. AB - The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications. PMID- 7539100 TI - Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase. AB - RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation. PMID- 7539101 TI - Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. AB - To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation. PMID- 7539102 TI - Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. AB - Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO 1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His 175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53. PMID- 7539103 TI - Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation. AB - Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD 1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1 minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation. PMID- 7539104 TI - Regulation of interleukin 2 gene expression by CD28 costimulation in mouse T-cell clones: both nuclear and cytoplasmic RNAs are regulated with complex kinetics. AB - T-cell receptor (TCR) signalling is required to induce expression of the interleukin 2 (IL-2) gene in mouse T cells. Additional costimulation through CD28 augments IL-2 production by 30- to 100-fold. Using IL-2 RNA accumulation and transcription reporter assays, we have addressed potential mechanisms of CD28 regulation at various time points of stimulation. The kinetic regulation of IL-2 mRNA by TCR and CD28 signals is complex: (i) at the earliest detectable time point, CD28 signalling causes a 20-fold increase compared with TCR signalling alone; (ii) both groups rapidly accumulate mRNA for the first 4 h; (iii) IL-2 mRNA then disappears from cells stimulated through the TCR alone but plateaus or increases slightly in cells costimulated through CD28; and (iv) after 8 h, the mRNA disappears in cultures with the anti-CD28 antibody. Transcription reporter assays did not show a specific effect of CD28 signalling on IL-2 enhancer driven transcription. This was true for either a 353- or a 1.9-kb enhancer, over a broad range of kinetics and TCR occupancy, and with several TCR signal mimics. The early component of CD28 costimulation is nuclear, however, since the initial enhancement of mRNA is also found in unspliced IL-2 RNA. Between 2 and 6 h, there is a marked difference in the rates of decay of IL-2 mRNA in the presence and absence of the CD28 signalling. Rapid decay of IL-2 mRNA commences after 8 h even in the presence of CD28 signals, although the decay occurs at a rate slower than that seen after 4 h of anti-TCR stimulation alone. This complexity suggests the existence of two interesting molecular mechanisms by which CD28 costimulates lymphokine gene expression. PMID- 7539106 TI - Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro. AB - Its reactivity to the antiphosphotyrosine 4G10 monoclonal antibody by Western blot analysis demonstrated that the human estrogen receptor (hER) from human MCF 7 cells and the recombinant hER expressed in Sf9 insect cells were phosphorylated on tyrosine(s). Reverse phase-HPLC separation of a tryptic digest of the 32P labeled purified hER from Sf9 and MCF-7 cells followed by amino acid and radiolabel sequencing revealed that tyrosine-537 was phosphorylated. The phosphorylation on tyrosine-537 was independent of estradiol treatment of MCF-7 cells, indicating that tyrosine-537 is a basal phosphorylation site. Two src family tyrosine kinases, p60c-src and p56lck, phosphorylated the purified recombinant hER on tyrosine-537 in vitro. In addition, two tyrosine phosphatases, protein tyrosine phosphatase-1B and src homology-2 protein tyrosine phosphatase 1, dephosphorylated phosphotyrosine-537 of the hER in vitro. These data suggest that tyrosine phosphorylation of the hER is regulated by potentially oncogenic tyrosine kinases and phosphatases that may modulate the function of ER in normal and/or abnormal cell growth. PMID- 7539105 TI - Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing. AB - We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA. PMID- 7539107 TI - Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta subunit to the common alpha-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG. AB - The hCG beta-subunit contains a carboxy-terminal extension bearing four serine linked oligosaccharides [carboxy-terminal peptide (CTP)], which is important for maintaining its longer half-life compared with the other glycoprotein hormones. Previously, we enhanced the in vivo half-life of FSH by fusing the CTP to the carboxy end of FSH beta coding sequence. The alpha-subunit is common to the glycoprotein family. We constructed alpha-subunit CTP chimeras, since such analogs with the appropriate O-linked glycosylation and conformation would increase the in vivo stability of the entire glycoprotein hormone family. Two chimeras were constructed using overlapping polymerase chain reaction mutagenesis: a variant with CTP at the carboxy end and another analog with the CTP at the N-terminal region of the subunit, between amino acids 3 and 4. The latter design was based on models showing that the amino-terminal region of alpha is not involved in assembly with the beta-subunit, nor is it essential for receptor binding and signal transduction. These chimeras were cotransfected with the hCG beta gene into Chinese hamster ovary cells. The chimeras were secreted and combined efficiently with the CG beta-subunit, comparable to the wild type alpha-subunit. CG dimers containing the alpha-subunit chimera with CTP at the carboxy end of the subunit had a much lower binding affinity for the hLH-hCG receptor in vitro, whereas the binding of the dimer containing the CTP at the amino-terminal end of the subunit was similar to wild type hCG. Furthermore, the in vivo activity of this analog was enhanced significantly. Moreover, regardless of the two insertion points in the alpha-subunit, the CTP sequence was O glycosylated. These data suggest that the entire signal for O-glycosylation is primarily contained within the CTP sequence and is not dependent on the flanking regions of the recipient protein. The transfer of CTP to the alpha-subunit of hCG results in an agonist with prolonged biological action in vivo. These data further support the rationale for using the CTP as a general target to increase the potency of bioactive glycoproteins. PMID- 7539108 TI - Detection of rifampin-resistant bacteria using DNA probes for precursor rRNA. AB - Ribosomal RNA precursor (pre-rRNA) molecules have terminal domains (tails) which are removed during late steps in rRNA processing, to yield the mature rRNA subunits. Transcriptional inhibitors such as rifampin can deplete pre-rRNA in sensitive cells by inhibiting de novo pre-rRNA synthesis while allowing maturation to proceed. We developed direct DNA probe assays for pre-rRNA tail sequences of Escherichia coli, and evaluated their ability to rapidly distinguish rifampin-resistant from rifampin-sensitive strains in cultures treated with the drug. Pre-rRNA became undetectable in sensitive cells less than a generation time after rifampin exposure, but remained abundant in resistant cells. Resistant cells were detectable by this method against a 100-fold excess of sensitive cells, showing that this method can detect resistant mutants even when present as a small percentage of a pathogen population. Our data indicate that the response of pre-rRNA to antibiotic treatment is sufficient in rate and magnitude to make it a useful metabolic marker for antibiotic sensitivity. PMID- 7539109 TI - A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. AML Cooperative Study Group. AB - BACKGROUND: Intensive chemotherapy for acute myelogenous leukemia (AML) continues to yield low rates of complete remission and survival among patients over the age of 65 years. Infection-related mortality is particularly high among these patients during the period of neutropenia that follows chemotherapy. We determined the effect of lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) on mortality at eight weeks (the main end point) and the rate of complete remission among patients with AML who were 65 years old or older. METHODS: After induction chemotherapy with daunorubicin (45 mg per square meter of body-surface area per day for 4 days) and cytarabine arabinoside (200 mg per square meter per day for 7 days), 173 patients with newly diagnosed AML were randomly assigned on day 8 to receive either lenograstim (5 micrograms per kilogram of body weight per day) or placebo, starting on day 9, until there was neutrophil recovery or a treatment failure, or for a maximum of 28 days. Salvage chemotherapy was also followed by lenograstim or placebo. Patients with a complete remission received two consolidation courses of chemotherapy without lenograstim or placebo. RESULTS: The mortality rate at eight weeks was similar in the lenograstim and placebo groups (23 and 27 percent, respectively; P = 0.60), as was the incidence of severe infections. The median duration of neutropenia (absolute neutrophil count < or = 1000 per cubic millimeter) was shorter in the lenograstim group (21 days, as compared with 27 days in the placebo group; P < 0.001). Eight percent of the patients in both groups had regrowth of AML cells. The rate of complete remission was significantly higher in the lenograstim group (70 percent, as compared with 47 percent in the placebo group; P = 0.002). Overall survival, however, was similar in the two groups (P = 0.76). CONCLUSIONS: The administration of lenograstim after chemotherapy for AML did not decrease the mortality rate at eight weeks among patients over the age of 65 years. The patients who received lenograstim had a significantly higher rate of complete remission than those who received placebo. Nevertheless, the overall survival in the two groups did not differ significantly. PMID- 7539110 TI - Disappointments in treating acute leukemia in the elderly. PMID- 7539111 TI - Treatment of benign prostatic hyperplasia. PMID- 7539112 TI - Male infertility. PMID- 7539113 TI - Altered immune responses in mice lacking inducible nitric oxide synthase. AB - Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality. PMID- 7539114 TI - Procaine impairs the function of 5-HT3 receptor-ion channel complex in rat sensory ganglion neurons. AB - Previous studies have shown that local anesthetics block voltage-dependent Na+ channels and nicotinic acetylcholine receptors. The present study investigated the effect of the local anesthetic, procaine on another ligand-gated ion channel, the 5-HT3 receptor, in rat nodose ganglion neurons. Procaine (0.01-100 microM) inhibited the 5-HT3 receptor-mediated inward current in the whole-cell patch clamp recording. The inhibition was fully reversible, concentration-dependent but not sensitive to changes in membrane potential. Concentration-response curves indicated that procaine appears to produce a competitive inhibition on 5-HT3 receptors with a KD of 1.7 microM. These observations suggest that one of the actions of procaine in nervous system is on 5-HT3 receptors. PMID- 7539115 TI - Chlormethiazole, dizocilpine and haloperidol prevent the degeneration of serotonergic nerve terminals induced by administration of MDMA ('Ecstasy') to rats. AB - An investigation has been made into the effect of 3,4 methylenedioxymethamphetamine (MDMA or 'Ecstasy') administration on the concentration of 5-hydroxytryptamine (5-HT), uptake of [3H]5-HT and [3H]paroxetine binding in rat cerebral cortex tissue. Four days after 2 injections of MDMA (20 mg/kg i.p., 6 hr apart) the concentrations of 5-HT and its metabolite 5-HIAA were reduced by 60%. The binding of [3H]paroxetine to the presynaptic 5-HT transporter was decreased and high affinity uptake of [3H]5-HT was reduced by a similar amount, indicating neurodegeneration of 5-HT terminals. Pretreatment with chlormethiazole (100 mg/kg i.p.), 10 min before each MDMA injection prevented the decrease in both [3H]parotextine binding and uptake of [3H]5-HT. The loss in 5-HT and 5-HIAA content was also attenuated. Pretreatment with dizocilpine (1 mg/kg i.p.) or haloperidol (2 mg/kg i.p.) also prevented the MDMA-induced loss of [3H]paroxetine binding and attenuated the loss of 5-HT and 5 HIAA content. All three compounds also decreased the degree of hyperthermia that follows MDMA administration, although previous studies suggest that the long term neurodegeneration is not associated with the acute hyperthermic response. These data support the findings of others that MDMA injection produces degeneration of 5-HT nerve terminals in the cortex, confirm that chlormethiazole, dizocilpine and haloperidol attenuate MDMA-induced neurotoxic loss of 5-HT and demonstrate for the first time that these compounds prevent the neurodegeneration of 5-HT nerve terminals that follows MDMA administration. PMID- 7539116 TI - Glycosaminoglycan polysulfate in primary degenerative dementia. Pilot study of biologic and clinical effects. AB - To test the hypothesis that the therapeutic effects of glycosaminoglycan polysulfate (GAP) in primary degenerative dementia of the Alzheimer type (PDD) is associated with a reversal of biochemical changes seen in PDD, a two-phase, clinical-biochemical study was conducted. In the first phase of this study a number of biochemical parameters were compared in 12 patients with PDD and their sex- and age-matched controls, and it was found that platelet monamine oxidase B activity was significantly higher and cerebrospinal fluid (CSF) homovanillic acid levels significantly lower in the PDD than in the normal control group. In the second phase of this study the same 12 PDD patients were treated with GAP at a daily dosage of 250 lipasemic-releasing units for a period of 1 month and it was found that all four biochemical parameters shifted towards normal values during therapy with the changes in CSF 5-hydroxy-indole acetic acid levels attaining statistical significance. Although clinical changes were minimal, in light of prior clinical findings in studies conducted with GAP in similar populations, the possibility was entertained that clinical improvement with GAP in PDD patients is preceded by biochemical changes. PMID- 7539117 TI - [An undying civilization damage: lead poisoning]. AB - The authors summarise the history, the sources, the pathophysiology, the symptoms and signs, the diagnostic methods, the differential-diagnostic possibilities, the therapy and prevention of the lead poisoning. They suggest that besides the classical professional and alimentary sources the environmental pollution caused first of all by leaded petrol plays a more and more important role in the lead exposure of the people. They draw the attention of the physicians to the lead intoxication as a possible cause of abdominal pain and/or anemia. PMID- 7539118 TI - Regulation of Ras-related RhoB protein expression during the cell cycle. AB - The immediate-early gene rhoB codes for a small GTP-binding protein highly homologous to the RhoA protein. While RhoA is known to regulate the assembly of focal adhesions and stress fibers in response to growth factors, the function of RhoB remains unknown. In a first attempt to elucidate its function, we examined the variation of the RhoB protein expression in response to induction of its mRNA. We report here that RhoB is an unstable protein rapidly and transiently induced by growth factors in PC12 and HeLa cells. Moreover, RhoB protein accumulation is periodic through the cell cycle. First detected at the G1/S phase transition, the level of the RhoB protein is maximal during the S phase and declines at the S/G2-M transition. This timing suggests that RhoB plays a role in the G1/S phase transition and/or in the S phase of the cell cycle. We also confirm here a vesicular and perinuclear localization of the endogenous RhoB protein induced by growth factors. PMID- 7539119 TI - Introduction of a loss-of-function point mutation from the SH3 region of the Caenorhabditis elegans sem-5 gene activates the transforming ability of c-abl in vivo and abolishes binding of proline-rich ligands in vitro. AB - We have introduced two loss-of-function point mutations from highly conserved regions of the src homology 3 (SH3) domains of the Caenorhabditis elegans sem-5 gene into the SH3 domain of the murine type IV c-abl tyrosine kinase proto oncogene. One of the mutations, P131L, activated abl to transform fibroblasts while the other, G128R, did not. When combined with independent activating mutations in the c-abl kinase domain or NH2-terminus, the G128R mutation blocked transformation by the double mutant, suggesting that the G128R mutant was unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c Abl protein, was localized to the nucleus and actin cytoskeleton and had normal tyrosine kinase activity in vitro, while the transforming c-Abl P131L protein was localized exclusively to the cytoplasm and exhibited decreased in vitro kinase activity. By real-time biospecific interaction analysis, the wild type Abl SH3 domain bound to two proteins containing proline-rich motifs with dissociation constants of 0.2 and 17 microM; the G128R mutant bound with 50-fold lower affinity, and no binding was detected by the P131L mutant. Both mutations completely abolished binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay. These results suggest that the transforming activity of Abl is regulated in vivo by an inhibitor protein which associates with the SH3 domain via a proline-rich sequence. PMID- 7539120 TI - Evaluation of the cytokine response in Kawasaki disease. AB - Intracellular cytokines interleukin 6, tumor necrosis factor (TNF) alpha and TNF beta were studied in peripheral blood mononuclear cells of patients with Kawasaki disease during the acute, subacute and convalescent stages of the disease utilizing cytokine-specific monoclonal antibodies and indirect immunofluorescence. Intracellular cytokines TNF-alpha and -beta were present only during the acute stage before initiation of therapy and not in the subacute or convalescent phase. Intracellular interleukin 6 was seen in both the acute phase and, in small numbers of patients, in the subacute stage of the illness. Overall 15 of 25 (60%) patients produced at least one intracellular cytokine. In the acute stage both monocyte and lymphocyte cytokines were detected intracellularly, TNF-alpha and TNF-beta in 58% of patients whereas interleukin 6 was seen in only 16%. This study provides evidence to support the involvement of activated mononuclear cells, both T cells and monocytes and their secreted soluble products, cytokines, in the pathogenesis of Kawasaki disease. PMID- 7539121 TI - A randomized clinical trial of home intervention for children with failure to thrive. AB - OBJECTIVE: To evaluate the efficacy of a home-based intervention on the growth and development of children with nonorganic failure to thrive (NOFTT). DESIGN: Randomized clinical trial. PARTICIPANTS: The NOFTT sample included 130 children (mean age, 12.7 months; SD, 6.4) recruited from urban pediatric primary care clinics serving low income families. All children were younger than 25 months with weight for age below the fifth percentile. Eligibility criteria included gestational age of at least 36 weeks, birth weight appropriate for gestational age, and no significant history of perinatal complications, congenital disorders, chronic illnesses, or developmental disabilities. Children were randomized into two groups: clinic plus home intervention (HI) (n = 64) or clinic only (n = 66). There were no group differences in children's age, gender, race, or growth parameters, or on any of the family background variables. Most children were raised by single, African-American mothers who received public assistance. Eighty nine percent of the families (116 of 130) completed the 1-year evaluation. INTERVENTIONS: All children received services in a multidisciplinary growth and nutrition clinic. A community-based agency provided the home intervention. Families in the HI group were scheduled to receive weekly home visits for 1 year by lay home visitors, supervised by a community health nurse. The intervention provided maternal support and promoted parenting, child development, use of informal and formal resources, and parent advocacy. MEASUREMENTS: Growth was measured by standard procedures and converted to z scores for weight for height and height for age to assess wasting and stunting. Cognitive and motor development were measured with the Bayley Scales of Infant Development, and language development was measured by the Receptive/Expressive Emergent Language Scale. Both scales were administered at recruitment and at the 12-month follow up. Parent-child interaction was measured by observing mothers and children during feeding at recruitment and at the 12-month follow-up, and the quality of the home was measured by the Home Observation Measure of the Environment 18 months after recruitment. ANALYSES: Repeated-measures multivariate analyses of covariance were used to examine changes in children's growth and development and parent-child interaction. Analyses of covariance were used to examine the quality of the home. Independent variables were intervention status and age at recruitment (1.0 to 12.0 vs 12.1 to 24.9 months). Maternal education was a covariate in all analyses. When changes in developmental status and parent-child interaction were examined, weight for height and height for age at recruitment were included as covariates. RESULTS: Children's weight for age, weight for height, and height for age improved significantly during the 12-month study period, regardless of intervention status. Children in the HI group had better receptive language over time and more child-oriented home environments than children in the clinic-only group. The impact of intervention status on cognitive development varied as a function of children's ages at recruitment, with younger children showing beneficial effects of home intervention. There were no changes in motor development associated with intervention status. During the study period, children gained skills in interactive competence during feeding, and their parents became more controlling during feeding, but differences were not associated with intervention status. CONCLUSIONS: Findings support a cautious optimism regarding home intervention during the first year of life provided by trained lay home visitors. Early home intervention can promote a nurturant home environment effectively and can reduce the developmental delays often experienced by low income, urban infants with NOFTT: Subsequent investigations of home intervention should consider alternative options for toddlers with NOFTT: PMID- 7539122 TI - The role of parents in the detection of developmental and behavioral problems. AB - OBJECTIVE: The success of early identification of children with developmental and behavioral problems is influenced by the manner in which pediatricians elicit, recognize, and select clinical information and derive appropriate impressions. Parents are ready sources of clinical information, and they can be asked to provide two broad types of data: appraisals, including concerns, estimations, and predictions; and descriptions, including recall and report. The purpose of this article is to help pediatricians make optimal use of clinical information from parents to increase the accuracy of clinical judgment in detecting children with developmental and behavioral problems. DESIGN: Review of 78 research articles and tests relying on parent information from pediatric, psychological, and education literature. RESULTS AND CONCLUSION: There are several formats for eliciting parental information that are superior in terms of accuracy and ease of evocation. Specifically, parents' concerns and good-quality standardized parent report measures such as the Child Development Inventories capitalize best on parents' observations and insights into their children. In combination, these two types of parental information offer an effective method for the early detection of behavioral and developmental problems in primary-care settings. PMID- 7539123 TI - Risperidone: the search for a better antipsychotic. AB - Risperidone, the latest agent in the continuing search for a better antipsychotic drug, seems to be an important addition to our treatment armamentarium for several reasons. First, risperidone is as effective as traditional antipsychotics for treating schizophrenia and perhaps more effective for treating refractory schizophrenia. Second, and related to the first point, risperidone is effective in the treatment of the negative symptoms of schizophrenia. Third, risperidone has a superior side-effect profile causing relatively few, if any, EPSEs. Fourth, risperidone may have an antidyskinetic property that, rather than simply not causing tardive dyskinesia, may actually improve those symptoms. PMID- 7539124 TI - On speaking one's truth. PMID- 7539125 TI - ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells. AB - The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539126 TI - Effects of cell differentiation on ion conductances and membrane voltage in LLC PK1 cells. AB - LLC-PK1 cells serve as a widely used model for the renal proximal tubule. Until now, little has been found out about their membrane voltage (Vm) and ionic conductances (g). Several studies have shown changes in cell properties during differentiation and ageing. The aim of this study was to examine the relationship between Vm or g and the age of these cells. Therefore, we investigated single cells, subconfluent and confluent monolayers of LLC-PK1 cells aged 1-8 days with the slow-whole-cell patch-clamp technique. The Vm of all cells was -34 +/- 2 mV (n = 75) and the membrane conductance (gm) was 2.3 +/- 0.3 nS (n = 30). Vm in cells aged up to 2 days was -24 +/- 3 mV (n = 22) whereas Vm in cells aged 5-8 days was -50 +/- 3 mV (n = 15). An increase of extracellular K+ from 3.6 to 18.6 mmol/l led to a depolarization in all cells of 4 +/- 1 mV (n = 31) and an increase of gm by 17 +/- 13% (n = 15). Complete replacement of extracellular Na+ by N-methyl-D-glucamine (NMDG) led to a hyperpolarization of 19 +/- 2 mV (n = 38) and gm was lowered by 27 +/- 14% (n = 17). A reduction in extracellular Cl- from 147 to 32 mmol/l showed no significant effect on Vm (n = 16) or gm (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539127 TI - Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization. AB - Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13 acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539128 TI - Improved micro-perfusion chamber for multiple and rapid solution exchange in adherent single cells. AB - A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 microliters) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 microliters) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of Silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 microliters) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the chamber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M omega). Additionally, a radioactive calcium channel ligand [3H]isradipine was added to the MPC and the appearance of radioactivity in the Petri dish (outside the MPC) subsequently measured.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539129 TI - Effect of lyophilization in sucrose plus dextran and rehydration in thioglycollate broth on performance of competitive exclusion cultures in broiler chicks. AB - Cecal bacteria grown under continuous-flow anaerobic conditions were lyophilized in skim milk (SM), Reagent 20 (R-20; sucrose and bovine serum albumin fraction V), or sucrose plus dextran (SDx), rehydrated in drinking water or thioglycollate beef broth (TGB), and compared with fresh broth culture for control of Salmonella typhimurium enteric colonization in broiler chicks. All groups were challenged on Day 3 with 10(4) cfu of S. typhimurium per chick. Mean Salmonella colony-forming units in cecal contents of groups provided lyophilized cultures rehydrated in TGB were not different from those in groups provided fresh broth culture and were reduced (P < .05) compared with controls at 10 d of age. Likewise, groups treated with the cultured lyophilized in R-20 and SDx and rehydrated in TGB had fewer Salmonella than similar groups treated with culture rehydrated in water. The results of this study indicated that the culture was as effective when lyophilized in SDx as it was when lyophilized in SM or R-20; culture lyophilized in SDx, SM, or R-20 and rehydrated in TGB was more effective than when it was rehydrated in water for the reduction of Salmonella colonization in broiler chicks. PMID- 7539130 TI - O-methylation of L-dopa in melanin metabolism and the presence of catechol-O methyltransferase in melanocytes. AB - O-Methylation of L-dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L-dopa, 3-O-methoxytyrosine was detected in extracts of cultured human melanocytes. The enzyme catechol-O methyltransferase is responsible for this O-methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane-bound isoforms. Immuno-electron microscopy suggests the presence of the membrane-bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol-O-methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O methylation of L-dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum. PMID- 7539131 TI - [Hodgkin cells are clonal B-cells in various stages of differentiation]. AB - Hodgkin's disease, especially its biology and pathogenesis, has been under discussion for more than 160 years. Numerous investigations have focused on the nature and clonality of Hodgkin cells, but so far no definitive answer have been yielded by immunohistochemistry, Southern blotting and PCR. However, the use of single-cell PCR has now made it possible to answer the question of the derivation of Hodgkin cells. Using a micromanipulator, Hodgkin cells can be picked out of histological sections and B-cell specific gene rearrangements (VDJ) can be amplified. With this technique it has proved possible to demonstrate the B-cell derivation of Hodgkin cells and their clonality. Mutated immunoglobulin genes in lymphocyte-predominant Hodgkin's disease indicate a germinal center cell origin of Hodgkin cells of this type. These findings, however, do not exclude cases of Hodgkin's disease with Hodgkin cells with a T-cell genotype. PMID- 7539132 TI - [Mucin-associated antigens as markers of gastrointestinal differentiation and carcinogenesis]. AB - Mucins are heavily glycosylated glycoproteins which exhibit a variety of antigenic determinants consisting of carbohydrates and/or peptide sequences. The application of monoclonal antibodies and lectins in immunohistochemistry resulted in a considerable extension of knowledge regarding their topography during histogenesis. Additionally, typical alterations of antigenic profiles during neoplastic transformation of cells and tissues were described. A number of new results are in keeping with the assumption that mucin-associated antigens play an important role in tumor biology, for example metastasis, and as markers of prognosis. The purpose of the present paper is to give a review, including the authors' own results, of knowledge on the gastro-intestinal mucin antigens in experimental and clinical pathology. PMID- 7539133 TI - Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients. AB - Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733 2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo. PMID- 7539134 TI - A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice. AB - Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue- and development-specific manner, although little is known about the molecular mechanisms underlying this regulation. The expression pattern of keratin 6 is particularly complex, since besides being constitutively expressed in hair follicles and in suprabasal cells of a variety of internal stratified epithelia, it is induced in epidermis in both natural and artificially caused hyperproliferative situations. Therefore, the regulatory sequences controlling keratin 6 gene activity are particularly suitable for target gene expression in a tissue-specific manner. More interestingly, they can be skin induced in transgenic animals or in gene therapy protocols, particularly those addressing epidermal hyperproliferative disorders. To delimit the regions containing these regulatory elements, different parts of the bovine keratin 6 gene linked to a beta-galactosidase reporter gene have been assayed in transgenic mice. A 9-kbp fragment from the 5' upstream region was able to provide both suprabasal tissue-specific and inducible reporter expression. PMID- 7539135 TI - Angiogenic properties of human immunodeficiency virus type 1 Tat protein. AB - Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1 infected individuals and in KS lesions. Here we show that Tat also promotes AIDS KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS. PMID- 7539136 TI - Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. AB - To investigate the physiological roles of gp130 in detail and to determine the pathological consequence of abnormal activation of gp130, transgenic mice having continuously activated gp130 were created. This was carried out by mating mice from interleukin 6 (IL-6) and IL-6 receptor (IL-6R) transgenic lines. Offspring overexpressing both IL-6 and IL-6R showed constitutive tyrosine phosphorylation of gp130 and a downstream signaling molecule, acute phase response factor/signal transducer and activator of transcription 3. Surprisingly, the distinguishing feature of such offspring was hypertrophy of ventricular myocardium and consequent thickened ventricular walls of the heart, where gp130 is also expressed, in adulthood. Transgenic mice overexpressing either IL-6 or IL-6R alone did not show detectable myocardial abnormalities. Neonatal heart muscle cells from normal mice, when cultured in vitro, enlarged in response to a combination of IL-6 and a soluble form of IL-6R. The results suggest that activation of the gp130 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in physiological regulation of myocardium. PMID- 7539137 TI - Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. AB - Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. & Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes. PMID- 7539138 TI - Cyclosporin A potentiates the dexamethasone-induced mouse mammary tumor virus chloramphenicol acetyltransferase activity in LMCAT cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression. AB - As previously observed for FK506, we report here that cyclosporin A (CsA) treatment of mouse fibroblast cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid (LMCAT cells) results in potentiation of dexamethasone (Dex)-induced CAT gene expression. Potentiation by CsA is observed in cells treated with 10-100 nM Dex but not in cells treated with 1 microM Dex, a concentration of hormone which results in maximum CAT activity. At 10 nM Dex, 1-5 microM CsA provokes an approximately 50-fold increase in CAT gene transcription, compared with transcription induced by Dex alone. No induction of CAT gene expression is observed in cells treated with CsA or FK506 in the absence of Dex. The antisteroid RU 486 abolishes effects obtained in the presence of Dex. Using a series of CsA, as well as FK506, analogs, including some devoid of calcineurin phosphatase inhibition activity, we conclude that the potentiation effects of these drugs on Dex-induced CAT gene expression in LMCAT cells do not occur through a calcineurin-mediated pathway. Western-blotting experiments following immunoprecipitation of glucocorticosteroid receptor (GR) complexes resulted in coprecipitation of GR, heat shock protein hsp90 and two immunophilins: the FK506 binding protein FKBP59 and the CsA-binding protein cyclophilin 40 (CYP40). Two separate immunophilin-hsp90 complexes are present in LMCAT cells: one containing CYP40-hsp90, the other FKBP59-hsp90. Thus, both FKBP59 and CYP40 can be classified as hsp-binding immunophilins, and their possible involvement as targets of immunosuppressants potentiating the GR-mediated transcriptional activity is discussed. PMID- 7539139 TI - Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries. AB - A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv. PMID- 7539140 TI - NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes. AB - The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4 azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha. PMID- 7539142 TI - Ganglioside GM1 binds to the Trk protein and regulates receptor function. AB - Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk. PMID- 7539141 TI - The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids. AB - Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin. PMID- 7539143 TI - Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs. AB - Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family. PMID- 7539144 TI - Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages. AB - We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation. PMID- 7539145 TI - Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes. AB - To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species. PMID- 7539146 TI - Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences. AB - By using taxonomic characters derived from EcoRI restriction endonuclease digestion of genomic DNA and hybridization with a labeled rRNA operon from Escherichia coli, a polymorphic structure of Listeria monocytogenes, characterized by fragments with different frequencies of occurrence, was observed. This structure was expanded by creating predicted patterns through a recursive process of observation, expectation, prediction, and assessment of completeness. This process was applied, in turn, to normalized strain patterns, fragment bands, and positions of EcoRI recognition sites relative to rRNA regions. Analysis of 1346 strains provided observed patterns, fragment sizes, and their frequencies of occurrence in the patterns. Fragment size statistics led to the creation of unobserved combinations of bands, predicted pattern types. The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences. Each EcoRI site had a frequency of occurrence, and unobserved fragment sizes were postulated on the basis of knowing the restriction site locations. The result of the recursion process applied to the components of the strain data was an extended classification with observed and predicted members. PMID- 7539148 TI - Different calcium channels are coupled to potassium channels with distinct physiological roles in vagal neurons. AB - Whole-cell and sharp microelectrode recordings were obtained from neurons of rat dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. Calcium currents were studied with sodium currents blocked with tetrodotoxin, potassium currents blocked by perfusing the cell with caesium as the main cation and using barium as the charge carrier. From a holding potential of -60 mV, inward currents activated at potentials positive of -50 mV and peaked around 0 mV. Voltage clamping the neuron at more hyperpolarised potentials did not reveal any low-threshold inward current. The inward current was effectively blocked by cadmium (100 microM) and nicked (1 mM), suggesting that it is carried by voltage-dependent calcium channels. The inward current could be separated into three pharmacologically distinct components: 40% of the whole cell current was omega-conotoxin sensitive; 20% of the current was nifedipine sensitive; and the rest was blocked by high concentrations of cadmium and nickel. This remaining current cannot be due to P-type calcium channels as omega-agatoxin had no effect on the inward current. Nifedipine had no significant effect on the action potential. Application of omega-conotoxin reduced the calcium component of the action potential and significantly reduced the potassium current underlying the afterhyperpolarization. Application of charybdotoxin slowed action potential repolarization. When N-type calcium channels were blocked with omega-conotoxin, charybdotoxin was still effective in slowing repolarization. In contrast, charybdotoxin was ineffective ineffective when calcium influx was blocked with the non-specific calcium channel blocker cadmium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539147 TI - Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance. AB - We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism. PMID- 7539149 TI - Replantation of the avulsed pinna: 100 percent survival with a single arterial anastomosis and substitution of leeches for a venous anastomosis. AB - We have presented an alternative form of management for the difficult ear replant in which conventional venous outflow cannot be reestablished. Early and aggressive medicinal leeching provided an excellent means of venous decompression until neovascularization had taken place. The primary use of leeches in such small composite replants may be superior to the time-consuming and technically difficult exercise of trying to find and anastomose suitable veins. In selected patients this technique may allow successful replantation of the amputated ear, thereby providing a superior aesthetic result to most forms of secondary reconstruction. PMID- 7539150 TI - [Amylasemia in uremics: a complex interpretation]. AB - Plasmatic amylase increases quite often in uremia. Hyperparathyroidism is also present in uremic patients and it is thought to be the main factor of an increased of amylase; its effect is mediated by an increase of cytosolic calcium. The authors measured the plasmatic level of amylase, lipase, calcium phosphates and parathormone before and after administration of Ca-antagonists (Verapamil and Diltiazem). The results did not shown any correlation. Hyperparathyroidism increased the pancreatic secretion but don't influences the amount of enzymes. PMID- 7539151 TI - Identification of the causative agent of granulocytic ehrlichiosis in Swedish dogs and horses by direct solid phase sequencing of PCR products from the 16S rRNA gene. AB - Seven Swedish isolates of Ehrlichia species from the blood of four dogs and three horses with clinical granulocytic ehrlichiosis, were identified by direct solid phase DNA sequencing of polymerase chain reaction (PCR) products from the 16S rRNA gene. The amplified DNA fragments were produced with primers complementary to the universal regions, U1, U2, U5 and U8 of the 16S rRNA molecule. Identical sequences were obtained from all seven isolates. This nucleotide sequence was similar to the sequences deposited in GenBank for Ehrlichia phagocytophila and E equi. The sequence of the Swedish ehrlichiae differed in two nucleotide positions from the E phagocytophila sequence and in three positions from the E equi sequence, and it is tentatively proposed that it is a subspecies of one of these two. The alignment of the sequence of the Swedish isolates with a recently deposited sequence from human cases of ehrlichiosis in the USA revealed 100 per cent identity in a segment of about 1400 bp. PMID- 7539152 TI - [Evaluation of prostatic specific antigen after a digital rectal examination: experience with 36 patients]. AB - Levels of prostatic specific antigen may be modified with procedures over the prostatic gland. We measured this antigen in 36 hospitalized patients before and immediately after a digital rectal examination. Antigen levels were 8.3 +/- 21.3 ng/ml before and 13.8 +/- 27.5 ng/ml after the examination (NS). It is concluded that digital rectal examination did not modify antigen levels in our sample. PMID- 7539153 TI - [Palliative treatments of cancers of upper respiratory and digestive tracts]. AB - Palliative treatment of head and neck cancers needs an initial evaluation based upon clinical symptoms and tumoral behaviour. Local recurrences are the most frequent presentation of tumoral failures. The therapeutic management of these local recurrences requires a mixture of unspecific and specific oncologic procedures. Radiotherapy and chemotherapy are frequently used in those circumstances in order to reduce the tumoral volume and to facilitate the effectiveness of unspecific treatments. Management of metastatic disease is often impaired by the moderate efficiency of chemotherapy while this treatment leads to marked side effects. Basically, the best therapeutic choice depends on the expected positive balance between the improvement of symptoms and the level of treatment-related side effects. However, daily clinical approach is a better way to care these patients than predefined rigid protocols. PMID- 7539154 TI - The biophysics of sickle cell hydroxyurea therapy. PMID- 7539155 TI - PTB domain binding to signaling proteins through a sequence motif containing phosphotyrosine. AB - Src homology 2 (SH2) domains mediate assembly of signaling complexes by binding specifically to tyrosine-phosphorylated proteins. A phosphotyrosine binding (PTB) domain has been identified which also binds specifically to tyrosine phosphorylated targets, but is structurally different from SH2 domains. Expression cloning was used to identify targets of PTB domains. PTB domains bound to phosphotyrosine within a sequence motif, asparagine-X1-X2-phosphotyrosine (where X represents any amino acid), that is found in many signaling proteins and is not recognized by SH2 domains. Mutational studies indicated that high affinity binding of PTB domains may require a specific conformation of the motif. PMID- 7539156 TI - Anharmonic wave functions of proteins: quantum self-consistent field calculations of BPTI. AB - The harmonic approximation for the potential energy of proteins is known to be inadequate for the calculation of many protein properties. To study the effect of anharmonic terms on protein vibrations, the anharmonic wave functions for the ground state and low-lying excited states of the bovine pancreatic trypsin inhibitor (BPTI) were calculated. The results suggest that anharmonic treatments are essential for protein vibrational spectroscopy. The calculation uses the vibrational self-consistent field approximation, which includes anharmonicity and interaction among modes in a mean-field sense. Properties obtained include the quantum coordinate fluctuations, zero-point energies, and the vibrational absorption spectrum. PMID- 7539157 TI - Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. AB - Fas (also known as Apo1 and CD95) is a cell surface receptor involved in apoptotic cell death. Fas expression and function were analyzed in three children (including two siblings) with a lymphoproliferative syndrome, two of whom also had autoimmune disorders. A large deletion in the gene encoding Fas and no detectable cell surface expression characterized the most affected patient. Clinical manifestations in the two related patients were less severe: Fas mediated apoptosis was impaired and a deletion within the intracytoplasmic domain was detected. These findings illustrate the crucial regulatory role of Fas and may provide a molecular basis for some autoimmune diseases in humans. PMID- 7539158 TI - [Combined methods for economic blood utilization in cardiac reoperations in children]. AB - During 47 re-operations on the open heart the authors used the method of autotransfusion, predeposition of the patient's blood and intravenous administration of aprotinin to reduce postoperative haemorrhage and blood consumption. The patients were classified according to the methods or their combinations used into three groups and the authors compared, using statistical methods the blood losses, blood consumption, haemolysis, renal function and the effect of the methods used on the morbidity. The results were compared with a control group where the mentioned methods of economizing on blood were not used. The authors recorded significantly higher haematocrit values in all three groups where economic methods were used at the end of the extracorporeal circulation (EC), as compared with the control group. The volume of the administered blood transfusion was significantly lower in group 1 where an autotransfusion apparatus Cell saver was used. The volume of the administered blood transfusion in the other groups did not differ when evaluated by statistical methods. The filling of the apparatus for extracorporeal circulation was blood free in 90% in group 1, in 75% in group 2, in 92.2% in group 3 and in 75% in the control group. Blood losses via thoracic drains did not differ significantly in different groups though there was a wide range of recorded values. In both groups 2 and 3 where patients were given aprotinin haemoglobinuria was more frequent, as confirmed by laboratory tests. The authors observed also a greater diuresis, without laboratory evidence of impairment of renal function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539159 TI - Differential antigen presentation of hepatitis B surface antigen on cell membranes of responder and nonresponder mice. AB - In several systems it has been shown, that non-responsiveness to an antigen in mice of a particular haplotype is due to the lack of binding of an immunogenic peptide to class II molecules. Such studies have been done using detergent solubilized, affinity purified class II molecules. It has been reported, that the presence of certain phospholipids around class II molecules dramatically alters the extent of peptide binding to these molecules. It thus appears that the milieu in which the class II molecules are inserted influences to a considerable extent the level of peptide binding. Hence it is likely that the kinetics of binding of immunogenic peptides to class II on the cell surface, may be different from that of molecules inserted in detergent micelles. We therefore decided to test this by studying the binding of radiolabeled peptides to class II molecules on cell membranes. We report here a rapid and sensitive assay for peptide binding to murine class II molecules on cell membranes. Further, we have used this assay to study the nature of the interaction of immunogenic peptides and class II molecules on cell membranes of mice who are responders and non-responders to Hepatitis B surface Antigen (HBsAg). Interestingly, we find that immunogenic peptides bind in good correlation with their MHC restriction. We also observed that the HBsAg derived peptide which is unable to elicit a T cell response in the non-responder but fails to make a stable class II-peptide complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539160 TI - RG1, a new murine monoclonal antibody recognizing a "supertypic" determinant on HLA-A molecules. AB - Monomorphic and polymorphic anti-HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA-A2 transfected cell line but not on cells transfected with HLA-E, -F or -G molecules. MAb reactivity with reference B-lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA-A molecules. Cells expressing HLA-A2, A24(9) and -A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA-A1, -A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA-A23(9), -A25(10), -A26(10), A29(19), -A30(19), -A31(19), -A32(19), -A33(19) and some HLA-A3 molecules. Based on class I alpha sequence data, these results suggest that the RG1 epitope is localized to a region of the alpha 2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding. PMID- 7539161 TI - Acidic FGF expression in the surroundings of senile plaques. AB - Immunohistochemical examination of postmortem brain tissue of Alzheimer's disease revealed that acidic fibroblast growth factor (aFGF) was specifically expressed in a subpopulation of reactive astrocytes which were congregated at the margin of the senile plaque. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than beta-amyloid protein deposits. Although, on the other hand, immunohistochemical staining for fibroblast growth factor receptor-1 occurred in some cortical neurons of Alzheimer's disease, the staining pattern did not differ from that in age-matched controls. Possible significance of aFGF-positive astrocytes in the surroundings of the senile plaque will be discussed in relation to receptor mediated or non-mediated mechanisms. PMID- 7539162 TI - Inflammatory response in Alzheimer's disease. AB - Microglia belong to the mononuclear phagocyte system. They represent the brain resident tissue macrophages and function as the scavenger cells in brain. In Alzheimer's disease (AD), microglia become activated. Reactive microglia aggregate around senile plaque beta-amyloid and neurofibrillary tangles. Heavy accumulation of these pathological debris in postmortem, however, indicates the failure or, at best, partial success of the removal. It is supposed that continued activation of microglia in these lesions elicits a persistent inflammatory response. In fact, activation fragments of the complement system have been detected in association with beta-amyloid deposits and extracellular ghost tangles. Thrombin, a central serine protease of the coagulation pathway, is also deposited in these pathological debris. Both complements and thrombin could augment the biochemical, synthetic and phagocytic capacities of microglia. Microglia, in turn, might play a major role for the activation of complement and coagulation systems in brain. The available evidence strongly suggests a significant similarity between the chronic inflammation and the tissue response in AD lesions, supporting a notion that the inflammatory process is a part of Alzheimer pathology. PMID- 7539163 TI - On the effects of paraquat on isolated mitochondria. Evidence that paraquat causes opening of the cyclosporin A-sensitive permeability transition pore synergistically with nitric oxide. AB - This paper reports an investigation on the effects of the bipyridylium herbicide, paraquat, on rat liver mitochondria in vitro. We show that paraquat induces a Ca(2+)-dependent permeability increase of the inner mitochondrial membrane leading to membrane depolarization, uncoupling and matrix swelling. The permeability increase is not observed in the absence of Ca2+ accumulation, and is not due to a direct effect of paraquat on the membrane energy level, as assessed by measurements of membrane potential, respiration and mitochondrial permeability to solutes at high concentrations of paraquat in the presence of excess ethylene bis(oxoethylenenitrilo)tetraacetic acid (EGTA), a Ca2+ chelator. The Ca(2+) dependent permeability increase is due to inappropriate opening of the endogenous permeability transition pore (MTP), a regulated, voltage-dependent channel of the inner mitochondrial membrane. The pore is primarily affected by paraquat through a shift of the gating potential to more negative values, allowing pore opening at physiological membrane potential. This effect apparently involves oxidation of a critical dithiol in the pore voltage sensor, while other regulatory aspects of the MTP (matrix pH and Ca2+) are unaffected by paraquat, which is not transported inside the mitochondrial matrix. The effects of paraquat on MTP opening depend on inhibition of electron transfer at Site I by rotenone, or by respiratory chain inhibition by nitric oxide, one of the proposed endogenous mediators of paraquat toxicity to the lung (Berisha, H.I., Hedayatollah, P., Absood, A., and Said, S.I. (1994) Proc. Natl. Acad. Sci. USA 91, 7445-7449). Taken together, these data provide an additional biochemical mechanism by which paraquat may affect cell function, and support the idea that mitochondrial damage is an important determinant in paraquat toxicity (Hirai, K.-I., Ikeda, K., and Wang, G.-Y. (1992) Toxicology 72, 1-16). PMID- 7539164 TI - Reversion by polyclonal antibodies of alpha effects of Tityus serrulatus venom on frog sciatic nerve. AB - Fraction T2 from Tityus serrulatus venom produced a marked lengthening of action potentials recorded with the single sucrose-gap technique, a characteristic effect of alpha scorpion toxins. This effect was not reversed by thorough washing of the nerve. On the other hand, T2 fraction deactivated by complete iodination did not cause any alteration of the compound action potential, even if applied in concentration as high as 4000 times the half saturation dose of the unmodified T2 fraction. This high dose of deactivated T2 did not hinder the onset of the full effect of a single dose of T2 fraction applied subsequently. Polyclonal antibodies against native or against iodinated venom reverted the action of T2 fraction, restoring normal electrical response. We conclude that both types of antibodies may remove the effect of alpha toxins on sodium channel. PMID- 7539166 TI - Paraoxonase protects against chlorpyrifos toxicity in mice. AB - Paraoxonase can hydrolyze paraoxon (PO), chlorpyrifos-oxon (CPO) and other organophosphates. Previous studies have indicated that the levels of serum paraoxonase can influence the toxicity of PO and CPO. In the present study we have investigated whether exogenous paraoxonase administered to mice would offer protection toward the acute toxicity of a phosphorothioate, chlorpyrifos (CPS). Paraoxonase was purified from rabbit serum and injected i.v., or i.v. plus i.p., in mice. Inhibition of acetylcholinesterase (AChE) in brain, diaphragm, plasma and red blood cells was measured as an index of CPS (100 mg/kg) toxicity. Administration of paraoxonase 30 min before CPS increased plasma enzyme activity toward CPO by 35-fold, and protected against its toxicity; protection was still present at 24 h, when enzyme activity was still 20-fold over basal. When paraoxonase was given 30 min after CPS, a significant protection against CPS toxicity was still observed, while after 3 h the protective effect was decreased. To mimic conditions of severe acute poisoning, a higher dose of CPS (150 mg/kg) was also administered. Administration of paraoxonase 30 min after this exposure abolished cholinergic signs and significantly protected against AChE inhibition. These results indicate that exogenous paraoxonase offers significant protection against CPS toxicity when administered both before and after the organophosphate, suggesting that it may be considered as a potential additional treatment of organophosphate poisoning. PMID- 7539165 TI - Effects of the herbicide chlorthiamid on the olfactory mucosa. AB - Chlorthiamid (2,6-dichlorothiobenzamide) and its major metabolite 2,6 dichlorobenzonitrile are olfactory toxicants with a high in vivo covalent binding in the olfactory mucosa of mice. This study showed that the cytochrome P450 (P450) inhibitors, metyrapone and sodium-diethyldithiocarbamate, abolished the chlorthiamid-induced toxicity (12 mg/kg; 0.06 mmol/kg) in C57B1/6 mice suggesting a P450-dependent toxicity. Incubation of [14C]-labelled chlorthiamid with rat olfactory microsomes showed a low NADPH-dependent oxidative covalent binding which was only 3-fold higher than that in liver microsomes. Thus the results do not support a major in situ metabolic activation of chlorthiamid and it is suggested that metabolic activation of the major chlorthiamid metabolite (2,6 dichlorobenzonitrile) is responsible for most of the covalent binding and toxicity of chlorthiamid at this site in vivo. Thiobenzamide (16 mg/kg; 0.12 mmol/kg), a dechlorinated chlorthiamid-analog, induced no marked morphological changes in the olfactory mucosa demonstrating that chlorines in the 2,6-position are important for the chlorthiamid-induced toxicity at this site. PMID- 7539167 TI - Cerebral blood flow during inhibition of brain nitric oxide synthase activity in normal, hypertensive, and stroke-prone rats. AB - BACKGROUND AND PURPOSE: Because tonic production of nitric oxide (NO) is important in regulating cerebrovascular tone and NO may be important in the mechanism of brain injury from focal ischemia, we speculated that stroke predisposition in spontaneously hypertensive stroke-prone rats (SHR-SP) may be related to impaired tonic production of NO. This study was designed to test the hypothesis that the cerebral blood flow (CBF) response to inhibition of NO synthase in SHR-SP would be different than that observed in normal Wistar-Kyoto (WKY) rats and non-stroke-prone spontaneously hypertensive rats (SHR). METHODS: Pentobarbital-anesthetized, mechanically ventilated rats were tested for CBF response to saline, 5 or 20 mg/kg IV of NG-monomethyl-L-arginine (L-NMMA), or 20 mg/kg IV of N omega-nitro-L-arginine (L-NA). In addition, specificity for an NO dependent mechanism was assessed by determining the ability to reverse any alteration in CBF with L-arginine. Hemorrhage was used to minimize any increase in mean arterial blood pressure (MABP) from NO synthase inhibition. In a separate cohort of rats, differential sensitivity of NO synthase for inhibition by nitro arginine analogues was determined. RESULTS: Baseline MABP was greater in SHR-SP (185 +/- 3, n = 38) and SHR (169 +/- 3, n = 38) compared with WKY rats (101 +/- 2 mm Hg, n = 38, P < .05). Baseline CBF was similar between strains; however, cerebrovascular resistance was higher in SHR-SP (2.16 +/- 0.09, n = 27) and SHR (1.94 +/- 0.07, n = 27) compared with WKY rats (1.23 +/- 0.06 mm Hg/mL per minute per 100 g, n = 27, P < .05). CBF was unchanged with 5 mg/kg L-NMMA or with L arginine in the absence of L-NMMA in each strain. CBF decreased similarly in SHR and SHR-SP (n = 9 each) in response to 20 mg/kg L-NMMA (SHR, 85 +/- 6 to 67 +/- 6; SHR-SP, 87 +/- 7 to 69 +/- 5 mL/min per 100 g) and was completely reversed by L-arginine. CBF did not decrease with 20 mg/kg L-NMMA in WKY rats. Administration of L-NA (n = 5 each) produced similar reduction of CBF (WKY rats, 67 +/- 6%; SHR, 49 +/- 9%; SHR-SP, 61 +/- 6% of baseline) and inhibition of NO synthase in each strain (approximately 80% inhibition). CONCLUSIONS: There was no difference in the cerebrovascular response to NO synthase inhibition in SHR-SP and non-stroke prone SHR. Therefore, it is unlikely that an altered sensitivity of NO synthase to inhibition can explain predisposition to stroke in SHR-SP. PMID- 7539169 TI - Rapid hair regrowth in refractory alopecia universalis associated with autoimmune disease following liver transplantation and tacrolimus (FK506) therapy. PMID- 7539168 TI - Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection. AB - Since complement-mediated hyperacute rejection of xenografts prevents the use of pigs as organ donors to man, the development of transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in pig tissues, specifically expression in endothelial cells of pigs transgenic for hDAF, is described. Twenty seven transgenic pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 transgenic pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur. PMID- 7539170 TI - Soluble vascular cell adhesion molecule-1 following cardiac transplantation. PMID- 7539171 TI - Controlling the vasculature: angiogenesis, anti-angiogenesis and vascular targeting of gene therapy. AB - Angiogenesis is the development of new blood vessels from an existing vascular bed. Normal vascular proliferation occurs only during embryonic development, the female reproductive cycle and wound repair. By contrast, many pathological conditions (for example, cancer, atherosclerosis and diabetic retinopathy), are characterized by persistent, unregulated angiogenesis. Conversely, inadequate angiogenesis can lead to failure of ulcers to heal and myocardial infarction. Control of vascular development could permit new therapeutic approaches to these disorders. For example, several anti-angiogenic drugs are currently undergoing clinical trials for the treatment of cancer, whereas enhancement of angiogenesis by exogenous growth factors can prevent or limit the damage in chronic wounds and duodenal ulcers. Here Tai-Ping Fan, Rhys Jaggar and Roy Bicknell highlight recent achievements and discuss the prospects of receptor antagonists, enzyme inhibitors, tumour suppressor genes and vascular targeted approaches, especially that of gene therapy, in the future development of angiotherapy. PMID- 7539172 TI - Etiology of benign prostatic hyperplasia. AB - Benign Prostatic Hyperplasia (BPH) is the most common neoplastic condition that afflicts men, and it constitutes a major factor impacting the health of the American male. This article reviews voiding dysfunction and the role of aging, the testis, and androgen in the development of BPH. Emphasis is placed on new concepts in the basic aspects of BPH etiology as a result of recent investigations. PMID- 7539173 TI - Epidemiology and natural history of benign prostatic hyperplasia. AB - Clinical manifestations of BPH include the symptoms, signs, and sequelae of urinary obstruction caused by the abnormal growth of non-malignant nodules that arise in a small region around the proximal segment of the prostatic urethra. Although the pathologic diagnostic criteria are relatively specific, clinical diagnostic criteria are much less specific, and no single BPH case definition has gained wide acceptance for use in epidemiologic studies. PMID- 7539174 TI - Pathophysiology of clinical benign prostatic hyperplasia. AB - The pathophysiology of clinical BPH has been attributed to bladder outlet obstruction resulting from the enlarged prostate. A direct relationship does not exist between prostate size, symptom severity, or bladder outlet obstruction. The pathophysiology of prostatism is most likely multifactorial. Elucidating the factors contributing to symptoms will likely provide the foundation for the development of new pharmacological alternatives for the medical treatment of BPH. PMID- 7539175 TI - Clinical manifestations of benign prostatic hyperplasia. AB - The clinical diagnosis of BPH is usually established after completing the initial evaluation of patients who present with symptoms of prostatism. The steps recommended in the initial evaluation include a medical history, symptom assessment using a Symptom Score Index, a digital rectal examination, a focused neurological examination, urinalysis, measurement of serum creatinine, and an optional PSA measurement. Further examinations such as uroflowmetry and cystoscopy are indicated only if the diagnosis is uncertain, or to determine the feasibility of specific invasive therapies. PMID- 7539176 TI - Advances in benign prostatic hyperplasia. The developmental and clinical utility of symptom scores. AB - Urinary symptom severity can be documented objectively among men with BPH using validated instruments such as the AUA Symptom Index. The AUA Index was developed to discriminate among BPH patients who are more or less bothered by their condition, and to measure change in symptom levels over time or with treatment. The AUA Index is reliable and valid for these purposes and can be useful in both clinical practice and research. The role of symptom measurement in general and the AUA Index in particular in the diagnosis of BPH is unclear and may be limited. Additional research is needed to maximize the utility of symptom measurement in urology. PMID- 7539177 TI - Uroflowmetry and urodynamics. AB - The ability to define and understand the relationship and significance of bladder outlet obstruction to the diagnosis and treatment of BPH continues to be a clinical problem that needs examination. Urodynamics remain the best tool to diagnose bladder outlet obstruction. However, controversies exist in its role as a diagnostic tool for therapeutic intervention. This article reviews the various issues and state of the art urodynamic modalities that pertain to the application of urodynamics to BPH. Specifically, a critical review is provided for the various urodynamic modalities such as uroflowmetry and multichannel urodynamics as it pertains to its clinical application and study of BPH. PMID- 7539178 TI - Imaging of the prostate. Benign prostatic hyperplasia. AB - Imaging studies of the prostate and urinary tract in patients with BPH include ultrasonography, intravenous urography, urethrography, CT scans, and MR imaging. Ultrasound can assess for upper tract changes, determine post void residuals, estimate prostatic volume, and demonstrate zonal anatomy and internal changes within the prostate. Intravenous urography is indicated for patients with hematuria, urinary tract stones, and other atypical histories. Urethrography provides information in the post prostatectomy patients with residual symptoms. CT scans and MR imaging are expensive and have no routine use in evaluating patients with BPH. PMID- 7539179 TI - The role of prostate-specific antigen in the evaluation of benign prostatic hyperplasia. AB - This article reviews the role of prostate-specific antigen (PSA) in the evaluation of BPH. Efficacy of the PSA test in differentiating patients with BPH from patients with early, potentially curable prostate cancer is discussed. In addition, this complete overview provides a critical analysis of various concepts (PSA density, PSA velocity, age-specific reference ranges, and free versus complexed serum PSA), that are devised to optimize the diagnostic value of the serum PSA concentration in a population in which prostate cancer and BPH are almost equally prevalent. A clinical algorithm, useful to the practicing clinician for the early detection of prostate cancer in the BPH patient, is also proposed. PMID- 7539180 TI - Assessing treatment results in benign prostatic hyperplasia. AB - This article discusses general and specific principles for the evaluation of BPH. General principles include bias elimination; sample size; appropriate and sensitive parameters of evaluation; blind, double-blind, controlled, and placebo controlled studies; and statistical versus clinical significance. Specific considerations include prostate volume and size; flowmetry; residual urine volume; cystometry and pressure flow studies; and symptomatic versus urodynamic improvement. PMID- 7539181 TI - Transurethral incision of the prostate. AB - Transurethral incision of the prostate (TUIP) is a safe and effective therapeutic modality for treating symptoms of bladder outlet obstruction in patients whose prostates have an estimated weight loss of less than 30 g. New minimal invasive techniques should be compared with TUIP instead of with transurethral resection of the prostate (TURP). PMID- 7539182 TI - Alpha blockade for the treatment of benign prostatic hyperplasia. AB - Randomized double-blind placebo controlled studies have consistently demonstrated the safety and effectiveness of selective alpha 1 blockers for the treatment of clinical BPH. Selective alpha 1 blockers relieve the symptoms of prostatism and decrease bladder outlet obstruction. The advantages of this class of drugs for the medical treatment of BPH include the extremely rapid onset of action, statistically and clinically significant effects on relevant outcome measures, adverse experiences that are generally infrequent, mild, and reversible, serum PSA levels are not affected, and hypertension is also effectively treated. The long-term effectiveness of selective alpha 1 blockers has recently been reported. Prostate smooth muscle tension is mediated by the alpha 1a (previously defined as alpha 1c) AR. The pharmaceutical industry is aggressively pursuing development of alpha 1a "prostate" selective AR antagonists. PMID- 7539183 TI - Benign prostatic hyperplasia. Hormonal treatment. AB - Androgen withdrawal therapy appears to produce a 20% to 30% decrease in the volume of the hyperplastic prostate after 3 to 6 months of therapy. Longer periods of treatment do not result in further prostatic regression, but further growth of the gland is not apparent in men treated with finasteride for 4 years. Although biopsy studies indicate that epithelial regression occurs to a much more significant degree than stromal regression, this may simply reflect the relatively longer turnover of the stromal cell population. The significant placebo effect of oral medication in patients with BPH makes interpretation of clinical symptomatology and uroflow data difficult. Analysis of symptom improvement is further complicated by the relatively slow improvement of patients on hormonal therapy, as opposed to surgery, where relief is immediate. In addition to limited stromal involution and inadequate treatment duration, other biologic factors may limit the clinical efficacy of androgen withdrawal therapy. Most importantly, partial involution may not necessarily decrease urethral resistance. In addition, obstruction-induced detrusor dysfunction may persist after relief of outflow obstruction in some patients, as it does following surgery. Incomplete blockade of androgen action, as well as compliance issues, may also limit efficacy. Long-term studies validate a modest but significant clinical response rate of finasteride therapy with preservation of sexual function in most men. Of the available hormonal therapeutic agents, only finasteride appears to have an acceptable risk-benefit ratio. Other 5 alpha reductase inhibitors in the "pipeline," such as episteride, may have similar benefits. In the long term, this class of drugs may have a much larger role in patients who present with the early signs of BPH, to prevent any further progression of the disease. Presently, no form of antiandrogen therapy is as effective as transurethral resection of the prostate in relieving symptomatology. Because of patient preferences, however, medical therapy will play an increasingly important role in the management of patients with BPH. PMID- 7539184 TI - Combination medical therapy for benign prostatic hyperplasia. AB - Preliminary clinical observations suggest that the efficacy of combination therapy (alpha 1 blockade + androgen suppression) is superior to the individual monotherapies. Several randomized double-blind multicenter placebo controlled studies are being done to define the efficacy and safety of combination therapy relative to the monotherapies. The ultimate role of combination therapy in clinical practice depends on a critical assessment of relative efficacy, safety, and cost. PMID- 7539185 TI - Phytotherapeutic agents in the management of symptomatic benign prostatic hyperplasia. AB - Phytotherapeutic agents constitute a large part of the market for the pharmacological treatment of symptomatic BPH. At present, these agents are being used on an empirical basis with some success in relieving symptoms. They have not been shown to have any effect on objective indices. Several agents are presently undergoing randomized trials. PMID- 7539186 TI - Lasers for the treatment of benign prostatic hyperplasia. AB - In our experience, efficacy favors the TURP and safety the laser. Unlike balloon dilatation of the prostate, there are likely to be technologic advancements that will allow more efficient tissue ablation, which will presumably result in more TURP-like efficacy. In addition, each of the many side-firing fibers has different physical properties that require fiber specific dosimetry studies. It is not clear how this will impact clinical efficacy; however, it is presumed that this will optimize efficacy while maintaining safety and minimizing catheterization time and postoperative irritative symptoms. VLAP is the surgical procedure of choice in high-risk patients and is an effective alternative for the treatment of symptomatic BPH. In high-risk patients, other devices such as stents may play an important role. From our assessment, TURP seems to provide greater symptomatic relief and probably is more durable than the VLAP procedure. Patients will often choose VLAP over TURP because of the lower risks of major complications and the perceived advantages of laser therapy over more conventional surgery. Other ablation techniques that are less invasive, more efficient, or do not require anesthesia but have equivalent efficacy could challenge the role of laser devices. Similarly, the continued development of more effective medical therapy will continue to challenge the role of all surgical interventions for BPH. Future investigations will focus on issues of dosimetry, tissue coagulation versus vaporization as well as fiber design and durability. The continued efforts to define the optimal technique reflect the general perception of the urologic community to improve the clinical outcome so that efficacy is more TURP-like. New delivery systems are being investigated as part of the effort to improve outcome. This includes the use of the holmium and diode lasers. The efficacy of these future delivery systems awaits proper study. PMID- 7539187 TI - Prostatic stents. Current perspectives for the management of benign prostatic hyperplasia. AB - The urethral stent is a relatively new treatment modality for symptomatic BPH. Our knowledge and experience using various stenting devices for this indication have been enhanced considerably within the last 12 to 18 months as the results from more investigations have become available. These preliminary data indicate that the intraurethral stent is an effective and safe treatment for symptomatic BPH in properly selected patients. Although the majority of clinical data currently available involve the UroLume Endourethral Prosthesis (American Medical Systems), other product designs such as thermosensitive and biodegradable prostheses are being investigated in animal models and human clinical trials. The ultimate urethral endoprosthesis for the treatment of an enlarged prostate gland resulting in symptomatic bladder outlet obstruction has yet to be developed. PMID- 7539188 TI - Microwave therapy of benign prostatic hyperplasia. AB - Thermotherapy of the prostate proves to be a safe and effective treatment for patients with symptomatic prostatism secondary to BPH. Present treatment regimens yield results and side effects intermediate between drug therapy and prostatectomy. Future enhancements of the thermotherapy technique are likely to improve its results to the level of surgery. PMID- 7539189 TI - Transurethral needle ablation for the treatment of benign prostatic hyperplasia. AB - The TUNA device is a unique system that uses radiofrequency energy to ablate the obstructing prostate adenoma. Although the clinical data are preliminary, they appear to provide improvement in symptom scores and peak flow rates, although somewhat less than TURP. A prospective, randomized trial comparing TUNA to TURP is under way, which should answer many of the questions regarding the safety and efficacy of this device for BPH. It is a potentially attractive device because of the limited anesthesia requirements, ease of the procedure, and the possibility of making this an office-based technique. The definitive role of this unique device awaits the results of ongoing clinical trials. PMID- 7539190 TI - The Agency for Health Care Policy and Research. Clinical guidelines for the diagnosis and treatment of benign prostatic hyperplasia. AB - The BPH guidelines provide a rational diagnostic and treatment algorithm for practitioners consulting with patients presenting with symptoms of prostatism. The guidelines also serve several very important purposes: They represent the first attempt at a comprehensive review of the English-language literature regarding the evaluation and treatment of men presenting with symptoms of BPH. They are presented in a comprehensible and transparent fashion to the reader, inasmuch as each piece of supporting evidence is explicitly listed. A decision diagram is presented, allowing health care providers to evaluate and treat patients with BPH in a rational fashion. A balance sheet of treatment outcomes should help patients and health care providers in their shared decision-making regarding treatment alternatives. They uncovered significant knowledge gaps and areas for future basic and clinical research. As more and more treatment alternatives become available, the guidelines will have to be updated to reflect both the changing health care environment and the ever-expanding armamentarium of treatment choices. PMID- 7539191 TI - The treatment of benign prostatic hyperplasia: a glimpse into the future. AB - The treatment of BPH has changed dramatically over the last decade. Industry has recognized the potential market for BPH therapies. Urologists have accepted and embrace the development of alternative treatment strategies. The combined resources and talents of industry and academic urology will lead to exciting future advances in this field. The optimal treatment of BPH requires not only new technology but a better understanding of the pathophysiology and natural history of this disease. Urologists are presently engaged in studies to better define these fundamental aspects of the disease process. The end result of these investigative efforts will be to the advantage of patients afflicted with BPH. PMID- 7539192 TI - [Euthanasia: refusal requires alternatives. The home hospital model could be a solution for some cancer patients]. AB - Maybe more important than an emotional debate on "pro and con" of euthanasia is search of alternatives for all who would request for physician-assisted suicide. Obviously it is not easy to find such alternative approaches. However only these justify a position "contra euthanasia". As one alternative Franco Pannuti introduced 1985 the concept of Eubiosia. Eubiosia, what means, the set of qualities that give life dignity, was proposed as a fundamental right of all patients. And dying in dignity as part of life in dignity excludes euthanasia. In the same way as respecting beginning life we have to respect ending life. A possible approach to guarantee Eubiosia for cancer patients is the hospital at home. A hospital at home is a part of the health care system having his own structural and organisational characteristics. It guarantees for a certain group of patients clinical level of care at the comfort of their own homes. The evaluation of 10,236 patients admitted in the Bologna home hospital, show that a majority of patients favour this care model which additional can have economical advantages. PMID- 7539193 TI - [Comment on Grond, A., M. Ostermann-Myrau: Benign and malignant diseases of the prostate]. PMID- 7539194 TI - [Anti-HCV positivity in blood donors]. AB - At the blood transfusion department of the Faculty Hospital in Olomouc in 1992 and 1993 18,293 blood donors were examined and in 70, i.e. 0.38% anti-HCV positivity was found. All positive subjects were subjected to clinical, laboratory and sonographic examination and are checked regularly at the Second Medical Clinic. In none of the patients liver biopsy was performed so far. The clinical, laboratory and sonographic findings provide evidence of liver damage in 15, i.e. 21.4%. In two of them, i.e. in 2.8%, the findings support the diagnosis of chronic active hepatitis; this will be verified by biopsy. The authors consider dispensarization of anti-HCV positive subjects useful. In all examination of RNA HCV however must be made. PMID- 7539195 TI - [Epitope specificity and protective activity of monoclonal antibodies to Venezuelan equine encephalomyelitis virus]. AB - Epitope specificity and protective activity of 5 types of monoclonal antibodies (MAB) to strain TM-14 of Venezuelan equine encephalomyelitis (VEE) virus were studied. The competing VEE A4 and VEE B5 MAB were shown to recognize the conformation-dependent epitope of glycoprotein E2 third site and to possess an extremely high protective activity. VEE C6 MAB were active in all the studied biological tests (hemagglutination inhibition, neutralization tests, passive defence) and were directed to glycoprotein E2 site six. Competitive radioimmunoassay demonstrated that VEE A5 MAB also interacted with E2-6 site and by the sum of characteristics detected a new epitope of this site. VEE G1 MAB specific to glycoprotein E1 were characterized by protective, but not neutralizing activity, and competed with MAB to sites E2-2 and E2-6. This means that VEE G1 MAB detect a heretofore not described epitope E1 overlapping with two E2 sites. PMID- 7539196 TI - [Phenotypic characterization of human hematopoietic stem cells]. AB - Mature hematopoietic cells of all lineages are derived from committed progenitor cells, which in turn are thought to originate from pluripotential stem cells. On the basis of in vitro clonogenic assay systems, different stages within this progenitor or stem cell compartment have been identified. Using cell separation techniques, it has been shown that all in vitro clonogenic cells are included in the minor population of bone marrow or peripheral blood cells expressing the CD34 molecule. CD34 positive cells have further been subdivided into subsets of clonogenic cells by their coexpression of certain characteristic molecules. Based on these immunophenotypic features, it has been shown that most immature multipotential clonogenic cells may be separated from the majority of already lineage committed colony forming cells. Additionally, within the committed progenitor compartment, molecules have been described allowing the specific identification of the earliest stages of granulomonocytic, erythroid or lymphoid cell differentiation. Apart from these lineage or differentiation stage specific molecules, CD34+ cells express a number of molecules that are supposed to function as cytoadhesion structures or cytokine receptors enabling these cells to interact with their stroma microenvironment. PMID- 7539197 TI - [Autologous stem cell harvesting after high dosage chemo- and/or growth factor therapy]. AB - 5 patients with metastatic tumors and 4 patients with hematological malignancies were treated either with G- or GM-CSF alone (5 micrograms/kg s.c.) or in combination with cyclophosphamide (7 g/m2) or epirubicin (120 mg/m2) to reduce either tumor mass or to maintain remission and to prime for peripheral stem cell collection. PMID- 7539198 TI - Does envelope V3 peptide comprise the principal neutralizing determinant for HIV? PMID- 7539199 TI - Safety and immunogenicity of a prototype oral whole-cell killed Campylobacter vaccine administered with a mucosal adjuvant in non-human primates. AB - The safety and immunogenicity of two prototype oral Campylobacter killed whole cell (CWC) vaccines were tested in rhesus monkeys. Animals were immunized with a primary two-dose series (days 0 and 14) of vaccine consisting of CWC (10(10) particles/dose) given alone or in combination with 0.5-1000 micrograms of the heat-labile enterotoxin of Escherichia coli as an oral adjuvant (OA). A booster vaccination, 4 weeks after primary immunization, was given to animals receiving CWC alone or supplemented with 0.5, 5 or 50 micrograms of OA. Both CWC and CWC-OA were well tolerated, with no adverse side-effects noted. Campylobacter-specific as well as adjuvant-specific antibody-secreting cells (ASCs) were determined in peripheral blood collected 7 days after each vaccine dose. Campylobacter-specific IgA ASC responses were enhanced by OA in a dose-dependent manner (p = 0.025), while IgG ASC responses were not. Seroconversions (both IgA and IgG) to Campylobacter antigens were also enhanced in monkeys receiving adjuvanted vaccine. No significant booster vaccination effect was observed in circulating ASCs in any of the immunization groups. In vitro T-cell proliferative responses to Campylobacter jejuni antigens were somewhat enhanced in both the CWC and CWC OA immunization groups. These results demonstrate that CWC-OA is safe and superior to CWC alone in its ability to stimulate both local and systemic Campylobacter-specific IgA and IgG responses in primates and they support its further evaluation in human clinical studies. PMID- 7539200 TI - Immunocytochemical staining of unstained versus previously stained cytologic preparations. AB - Immunoperoxidase (IP) staining of fine needle aspiration specimens may provide useful adjunct information in cytodiagnosis. IP may be used on unstained direct smears or cell block material if available. In the absence of such material, IP is sometimes used on previously stained (Papanicolaou) smears. Few studies have examined the adequacy of such preparations, however. We compared IP staining results on unstained, alcohol-fixed or air-dried smears versus alcohol-fixed, previously Papanicolaou stained smears and versus alcohol-fixed, decolorized, Papanicolaou-stained smears, using antibodies to keratin (CAM 5.2, AE1/AE3 and K903), epithelial membrane antigen, monoclonal carcinoembryonic antigen, desmin, muscle-specific actin (HHF-35), vimentin, leukocyte common antigen, B and T cell markers L26 and UCHL-1, and prostate-specific antigen. Seventy-five sets of smears were made from various neoplastic or nonneoplastic tissues received as surgical specimens, and antibodies were selected for anticipated positive staining. The results were graded in a semiquantitative fashion. All the methods gave comparable results regarding expected positive staining, background staining and preservation of cellularity. We conclude that these methods of smear preparation are of equal utility in adjunctive immunocytochemical studies. PMID- 7539201 TI - Value of immunoperoxidase staining of thyroglobulin in fine needle aspiration cytology of thyroid diseases. AB - To elucidate the value of thyroglobulin staining by the immunoperoxidase method in fine needle aspiration cytology of thyroid diseases, it was performed on fine needle aspiration smears of 57 cases of various thyroid diseases. Thirteen of 22 cases (59%) with benign nodular goiter were positive. Eight of 14 cases (57%) with papillary thyroid carcinoma were positive. Among these eight cases with positive staining, seven were at clinical stage II or less. Among the other six cases with negative staining, five cases were in clinical stage III or more. There was a significant relationship between clinical stage and thyroglobulin staining (P < .05). One of 10 cases with thyroid cysts was positive. One of four cases with anaplastic carcinoma was positive. One of two cases with follicular thyroid carcinoma was positive. Two cases of subacute thyroiditis were positive. One case of Hashimoto's thyroiditis was positive. Two cases of metastatic thyroid cancer from the ovary were negative. These results reveal that positive thyroglobulin staining was helpful in defining the source of tissue from the thyroid. However, negative staining could not definitively exclude a thyroid origin. Positive thyroglobulin staining in papillary thyroid carcinoma correlated with less advanced clinical stages. PMID- 7539202 TI - Role of nuclear grooves in the diagnosis of papillary thyroid carcinoma. A quantitative assessment on fine needle aspiration smears. AB - Thirty-four cases of papillary thyroid carcinoma and 69 control cases consisting of 17 follicular neoplasms, 5 Hurthle cell neoplasms, 7 medullary carcinomas, 6 thyrotoxic goiters, 19 chronic lymphocytic thyroiditis cases, 3 subacute thyroiditis cases and 12 colloid goiter cases were studied for nuclear grooves. Such grooves were seen in 100% of papillary carcinoma and in 75-100% of other thyroid disorders, but their number was strikingly higher in papillary carcinoma. A quantitative assessment for nuclear grooves was made based on counting 500 follicular cells in hematoxylin and eosin (H&E)-stained and May-Grunwald-Giemsa (MGG)-stained smears. In H&E-stained smears of papillary carcinoma the number of cells with nuclear grooves (227.3 +/- 99.96 SD) was significantly higher as compared to other thyroid disorders (P < .01 to < .001). Eighty-eight percent of papillary carcinoma had nuclear grooves in > or = 20% tumor cells, whereas none of the other thyroid diseases exceeded this level. In MGG-stained smears the number of cells with nuclear grooves (40.7 +/- 32.83 SD) was also significantly higher as compared to other thyroid disorders (P < .05 to < .001). Of papillary carcinomas, 67.6% had > or = 4% cells with nuclear grooves, whereas 0-40% of other thyroid diseases exceeded this level. Nuclear grooves were significantly higher in H&E-stained smears of papillary carcinoma as compared to MGG-stained smears (P < .001). Based on this objective assessment, nuclear grooves were a useful criterion in the diagnosis of papillary thyroid carcinoma in H&E-stained smears but were not as reliable in MGG-stained smears. PMID- 7539203 TI - Cytologic features of intraabdominal desmoplastic small round cell tumor. A case report. AB - Intraabdominal desmoplastic small round cell tumor (IAD-SRCT) is a highly aggressive, malignant neoplasm that affects predominantly young adult males. This report describes the fine needle aspiration and ascitic fluid cytology findings in a 15-year-old male with IAD-SRCT. The tumor cells consisted of small, round to oval cells with a scant amount of basophilic cytoplasm. No cytologic evidence of cellular differentiation was identified. The similarities of cytologic features with other small round cell tumors in the pediatric age group may lead to differential diagnostic difficulties that require the application of ancillary diagnostic methods, such as immunohistochemistry, electron microscopy and cytogenetic techniques. In this case, immunohistochemical stains were positive for cytokeratin, desmin and neuron-specific enolase. Flow cytometry revealed a tetraploid DNA stemline and proliferation index of 21% (S + G2M). Ultrastructural examination revealed undifferentiated small cells with large, perinuclear whorls of intermediate filaments. Cytogenetic studies showed a grossly abnormal karyotype with a t(11;17)(p13;q11.2) translocation and a near-tetraploid modal chromosome number. This karyotype differed from those in previous cases in that it lacked a 22q12-13 fusion partner but provided further evidence for involvement of the 11p13 region in the pathogenesis of this unusual tumor. PMID- 7539204 TI - Reticulin stain in the fine needle aspiration differential diagnosis of liver nodules. PMID- 7539205 TI - Proposed mechanisms for the involvement of lactoferrin in the hydrolysis of nucleic acids. AB - Lactoferrin has recently been proposed to have ribonuclease activity in the absence of bound iron. We and others have demonstrated previously that lactoferrin interacts with DNA and will bind a number of transition metal ions via surface-exposed histidyl residues. In the present study, we investigated the possibility that surface-bound copper ions on lactoferrin may catalyze the production of active oxygen species responsible for the hydrolysis of nucleic acids. Purified lactoferrin (apo- and holo-forms) was incubated with CuCl2 in solution to obtain lactoferrin with surface binding sites saturated by Cu(II)ions. the lactoferrin-Cu(II) complex was purified by Bio-Gel P-6 chromatography columns and tested for hydrolytic activity against DNA and RNA as analyzed by agarose gel electrophoresis. Incubation of lactoferrin-Cu(II) complexes with supercoiled plasmid Bluescript II SK DNA led to the rapid formation of relaxed open circular or linear forms of DNA characterized by changed electrophoretic mobility. Lactoferrin with bound Cu(II) also caused extensive degradation of yeast tRNA molecules in the presence of hydrogen peroxide. Covalent modification of surface-exposed histidyl residues by carboxyethylation with diethylpyrocarbonate abolished the lactoferrin-associated hydrolytic activity. These results indicate that lactoferrin-bound Cu(II) can indeed facilitate the hydrolysis of DNA and RNA molecules. Copper-binding sites on lactoferrin appear to serve as centers for repeated production of hydroxyl radicals via a Fenton-type Haber-Weiss reaction. Enhanced nuclease activity associated with elevated local concentrations of lactoferrin would promote microbial degradation. PMID- 7539206 TI - Genetic disorders of pigmentation. PMID- 7539207 TI - Implication of cytokines in the aggravation of malnutrition and hypercatabolism in elderly patients with severe pressure sores. AB - The objectives of this work were to study the production of the cytokines Interleukin 1 (IL-1), Interleukin 6 (IL-6), and tumour necrosis factor (TNF) in elderly patients with severe pressure sores and to assess their potential contribution to the aggravation of malnutrition. Nineteen bedridden patients with stage III or IV pressure sores, 12 bedridden patients free from pressure sores, but at risk of them, and 12 control patients without risk of pressure sores were studied. Nutritional status was evaluated using anthropometry, serum albumin, prealbumin, retinol-binding protein analyses, and delayed hypersensitivity skin tests. Acute-phase proteins (APP), cortisol, and cytokines blood levels together with cytokine production were measured. Nutritional status was poor in patients with sores and their APP, and IL-6 blood levels were significantly increased; IL 1 and TNF serum concentrations were not elevated in these patients. A significant difference in cortisol levels was observed between patients with stage III and IV sores. A local cytokine origin (especially IL-6, and also IL-1) was demonstrated. Thus cytokines, mainly IL-6, produced by tissue cells in damaged areas together with cortisol may aggravate malnutrition and hypercatabolism in patients with sores. PMID- 7539208 TI - UVs syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group I. AB - Previously, we reported two DNA repair-defective siblings who did not belong to any complementation group of xeroderma pigmentosum (XP) or Cockayne syndrome (CS). By surveying other photosensitive patients whose fibroblasts showed similar biochemical phenotypes, we found another nonconsanguineous Japanese patient belonging to the same complementation group as our previous cases. Postreplication repair of the cells derived from these patients was normal, indicating that they cannot be classified as XP variant. Neither transfection nor microinjection of the cells with the human DNA repair gene ERCC1, which is known not to correct any complementation groups of XP or CS, failed to correct the defect of these cells, indicating that they do not belong to the rodent complementation group 1. However, the defect in recovery of RNA synthesis (RRS) after UV irradiation was restored by microinjection of HeLa cell extract. Although clinical manifestations of these patients--such as acute sunburn, dryness, freckling, pigmentation anomalies on sun-exposed skin, and teleangiectasia without neurological abnormalities or tumors--are similar to a mild XP phenotype, cellular characteristics such as UV sensitivity and defective RRS after UV irradiation with normal unscheduled DNA synthesis (UDS) are reminiscent of CS. On the basis of these results, we propose that these patients be included under a general category designated "UV-sensitive" (UVs) syndrome. PMID- 7539209 TI - Mutations in the SLC3A1 transporter gene in cystinuria. AB - Cystinuria is an autosomal recessive disease characterized by the development of kidney stones. Guided by the identification of the SLC3A1 amino acid-transport gene on chromosome 2, we recently established genetic linkage of cystinuria to chromosome 2p in 17 families, without evidence for locus heterogeneity. Other authors have independently identified missense mutations in SLC3A1 in cystinuria patients. In this report we describe four additional cystinuria-associated mutations in this gene: a frameshift, a deletion, a transversion inducing a critical amino acid change, and a nonsense mutation. The latter stop codon was found in all of eight Ashkenazi Jewish carrier chromosomes examined. This report brings the number of disease-associated mutations in this gene to 10. We also assess the frequency of these mutations in our 17 cystinuria families. PMID- 7539210 TI - CFTR haplotype analysis reveals genetic heterogeneity in the etiology of congenital bilateral aplasia of the vas deferens. AB - Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations. PMID- 7539211 TI - Girl with signs of Pelizaeus-Merzbacher disease heterozygous for a mutation in exon 2 of the proteolipid protein gene. AB - We studied a female infant with clinical signs of Pelizaeus-Merzbacher disease (PMD), who has a familial mutation (C41-->T) in exon 2 of the proteolipid protein gene (PLP), and selected relatives. While the carrier mother and grandmother of the proposita currently are neurologically normal and show normal T2 magnetic resonance imaging (MRI) of the brain, the infant has a neurological picture, MRIs, and brain auditory evoked response (BAER) consistent with that diagnosis. The data here presented show that PMD can occur in females carrying a mutation in the PLP gene. Our experience with the MRIs of this patient, her mother and grandmother, and those of a previously reported family [Pratt et al.: Am J Med Genet 38:136-139, 1991] show that molecular genetic analysis and not MRI is the appropriate means for carrier detection. PMID- 7539212 TI - Pelizaeus-Merzbacher disease in a family of Portuguese origin caused by a point mutation in exon 5 of the proteolipid protein gene. AB - Single-strand conformational polymorphism analysis of an affected male with Pelizaeus-Merzbacher disease (PMD) showed a slight change in mobility of amplified exon 5 of the proteolipid protein (PLP) gene. The exon was sequenced and a G-->A transition at codon 216 was found. This mutation eliminates a BstNI restriction site and creates a MaeI restriction site. In 1989, Gencic et al. reported a mutation that destroyed the same BstNI site, but resulted in a substitution at codon 215 [Am J Hum Genet 45:435-442]. The mutation we report here is also present in the patient's mother and her male fetus as determined by polymerase chain reaction analysis of amniocytes. PMID- 7539213 TI - In-frame deletion in the proteolipid protein gene of a family with Pelizaeus Merzbacher disease. AB - We describe an in-frame deletion of parts of exons 3 and 4 of the proteolipid protein gene (PLP), with all of the intervening sequence, in a 3-generation family with Pelizaeus-Merzbacher disease. The mutation removes 49 amino acids of the PLP. PMID- 7539214 TI - Multifunctional transporter models: lessons from the transport of water, sugars, and ring compounds by GLUTs. AB - Facilitative glucose transporters (GLUTs) have recently been shown to be multifunctional, transporting substrates other than sugars, such as water and ring compounds as large as nitrobenzene-diazol-aminoglucose. Other membrane proteins, including transporters and cystic fibrosis transmembrane conductance regulator, have also revealed a finite permeability to water. We compare the alpha-helical and beta-barrel models for the structure of GLUTs, discuss recent evidence, and argue that a beta-barrel fold explains it better. We show a model for GLUTs consisting of a relatively rigid beta-barrel translocation unit ("channel") of diameter ample enough to allow permeation of the above substrates (approximately 20 A) but gated shut by mobile loops at both ends. Such gates would open only after aromatic interactions would lead to binding of the ring substrates for GLUTs; water would, however, traverse crevices in the closed gates. Using the insights gained from GLUTs, we propose that other transporters may share with GLUTs the motif of a beta-barrel channel and would be permeable to water due to the presence of such channels together with similarly behaving gates. PMID- 7539215 TI - Nonselective cation channel activation during wound healing in the corneal endothelium. AB - Rabbit corneas were injured by mechanical or thermal trauma. At several time points after wounding, corneal endothelial cells were isolated and their ion channels examined using standard and amphotericin perforated-patch whole cell patch-clamp configurations. Within 15-24 h after mechanical or thermal trauma, a nonselective cation current was observed in 79% of the cells examined that was not present in unwounded or sham-wounded corneas. By 73 h postwounding, the current was present in only 10% of the cells examined. The wound healing-induced current is outwardly rectifying, activates at depolarized voltages, shows no sign of inactivation, and is inhibited by flufenamic acid, quinidine, and acetate. In addition to this new current, it was observed that endothelial cells from freeze wounded corneas no longer expressed the transient K+ current seen in control, sham, and mechanically wounded corneas. Corneal endothelial superfusion experiments found no significant difference in swelling rates between control and flufenamic acid-superfused wounded corneas, indicating that the wound healing induced channel is not involved in the stromal hydration maintenance function of the corneal endothelium. PMID- 7539216 TI - Boar sperm plasma membrane Ca(2+)-selective channels in planar lipid bilayers. AB - Entry of Ca2+ through Ca2+ channels is thought to trigger the acrosome reaction of spermatozoa during fertilization. Antagonists of the L-type Ca2+ channel are known to prevent the intracellular Ca2+ (Ca2+) increase and inhibit acrosomal exocytosis in mammalian sperm. Planar bilayer recordings were used to study Ca2- channels incorporated from partially purified boar sperm plasma membranes. With symmetrical 50 mM NaCl and 100 mM BaCl2 on the cis side, single-channel events consistent with Ba2+ flux from cis to trans were observed. These channels were activated by the dihydropyridine agonist (+/-)BAY K 8644 and blocked by the antagonist nitrendipine. Sperm Ca2- channels did not require depolarization for activation and did not inactivate. The (+/-)BAY K 8644 and (S-)BAY K 8644 enantiomers increased apparent open time in a dose-dependent [half-maximal activity constant (K0.5) = 0.9 and 0.3 microM, respectively] manner. Dihydropyridine antagonists nitrendipine (K0.5 = 0.5 microM) and (R+)BAY K 8644 (K0.5 = 2.8 microM) decreased apparent open times. The channels described in this report share some properties with brain, cardiac, and skeletal muscle t tubule Ca2+ channels and may be involved in increasing Cai2+ before the acrosome reaction. PMID- 7539217 TI - Processing of pro-PTHRP by the prohormone convertase, furin: effect on biological activity. AB - We have examined the conversion of parathyroid hormone-related peptide (PTHRP) from its NH2-terminally extended precursor pro-PTHRP. Within pro-PTHRP, an amino acid sequence exists that can serve as a substrate for the prohormone convertase, furin. To evaluate the potential role of furin in processing of this entity, we expressed pro-PTHRP in COS-7 cells, which normally produce this enzyme. Transiently transfected COS-7 cells secreted high levels of PTHRP into conditioned culture medium. Cotransfection of these cells with antisense furin cDNA resulted in marked inhibition of furin mRNA expression and secretion of an NH2-terminal fragment of pro-PTHRP, which comigrated with synthetic pro-PTHRP-( 12-->+36) on gel-permeation high-pressure liquid chromatography. In an adenylate cyclase bioassay, condition medium containing this fragment and synthetic pro PTHRP-(-12-->+36) both exhibited lower potency than synthetic PTHRP-(1-36) and conditioned medium containing PTHRP produced by COS-7 cells in the absence of antisense furin. These results demonstrate the capacity of furin to convert pro PTHRP to a more active product and suggest a role for this enzyme in the normal intracellular processing of this hormone. PMID- 7539218 TI - Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. AB - Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome. PMID- 7539219 TI - Distension-induced electrogenic Cl- secretion is mediated via VIP-ergic neurons in rat rectal colon. AB - Distension of rat rectal colon causes electrogenic Cl- secretion via the plexus submucosus Meissner. This study aimed to identify the neurotransmitter(s) of this reflex pathway. Distension was applied to partially stripped rat rectal colon in Ussing chambers. Baseline short-circuit current (Isc) increased and then slowly declined again within 30 min. The increase in Isc 10 min after distension (delta Isc10) was 1.8 +/- 0.3 mumol.h-1.cm-2. Atropine (1 microM) did not alter delta Isc10. Thus cholinergic neurons with muscarinic synapses were not involved. Tissues were then desensitized to vasoactive intestinal peptide (VIP) or substance P. This required continuous infusion of VIP or substance P into the chamber; otherwise, desensitization was only temporary due to rapid degradation of VIP or substance P. During substance P desensitization, distension still induced a secretory response (delta Isc10 not significant vs. control), whereas during VIP desensitization distension no longer had an effect. Furthermore, a polyclonal anti-VIP antiserum blocked 81% and the VIP antagonist [p-Cl-D Phe6,Leu17]VIP blocked 89% of the distension-induced delta Isc10, supporting the results of the desensitization experiments. To localize the site of VIP action, tetrodotoxin (TTX) was used. The TTX effect on Isc during VIP stimulation was not different from its effect on baseline Isc. This is in accord with the concept that the VIP receptors are mainly located on the enterocytes. We conclude that VIP, but not substance P or acetylcholine (via muscarinic receptors), acts as a neurotransmitter in the distension-induced reflex pathway, causing Cl- secretion in rat rectal colon. PMID- 7539220 TI - A forskolin-activated Cl- current in mouse mandibular duct cells. AB - We have previously shown that unstimulated granular duct cells of mouse mandibular gland contain a hyperpolarization-activated Cl- conductance with characteristics resembling the hyperpolarization-activated volume-sensitive Cl- channel (ClC-2). We now show that stimulation of these cells with forskolin, but not 1,9-dideoxyforskolin, activates a second whole cell Cl- conductance with properties resembling the cystic fibrosis transmembrane conductance regulator (CFTR). This conductance has a linear current-voltage relation and is not voltage activated. Its anion permeability sequence is Br- (1.96) > NO3- (1.36) > Cl- (1) > I- (0.44), and its conductance sequence is Cl- (1) > NO3- (0.66) > Br- (0.34) > I- (0.21). The current carried by this conductance is attenuated 65% by 1 mmol/l diphenylamine-2-carboxylate but is not affected by 0.1 mmol/l4,4' diisothiocyanatostilbene-2,2'-disulfonic acid or 0.1 mmol/l glibenclamide. The current can be activated by norepinephrine (1 mumol/l), evidently acting via beta adrenergic receptors, since the effect of norepinephrine is inhibited by propranolol (1 mumol/l). We conclude that this adrenergically evoked conductance is due to CFTR, which has previously been shown to be expressed in salivary duct cells, and suggest that it may form part of the mechanism by which beta adrenergic agonists modulate NaCl absorption by salivary ducts. PMID- 7539221 TI - Extracellular heterotrimeric laminin promotes differentiation in human enterocytes. AB - To investigate the role of laminin (Ln) and merosin (a Ln variant) in the modulation of intestinal epithelial cell differentiation, we have analyzed their functional expression as well as their potential influence during the differentiation process of Caco-2 cells, a human cell line unique in its property to differentiate into a mature enterocyte-like cell type in vitro. By indirect immunofluorescence and Western blotting, Caco-2 cells have been found to express the B1 and B2 chains of Ln, as well as a heavy approximately 350- to 370-kDa chain related to the human A chain, but not the M chain, of merosin. A gradual deposition of this A-like chain-containing Ln molecule has been observed as Caco 2 cells undergo differentiation. At the cellular level, a clear relationship between basal staining for the A chain and apical staining for the brush-border membrane enzyme sucrase-isomaltase (SI) has been observed. Human Ln, used as substratum, has been found to increase significantly the expression of SI and lactase in Caco-2 cells, whereas both Ln and human merosin have been shown to stimulate aminopeptidase N and alkaline phosphatase expression. Taken together, these data indicate that enterocytic differentiation-related gene expression is promoted by an extracellular deposition of Ln and may be susceptible to a differential modulation by variant forms of the molecule. PMID- 7539222 TI - Neurokinin depletion attenuates pulmonary changes induced by antigen challenge in sensitized guinea pigs. AB - The role of neurokinins in the acute pulmonary response to antigen was studied in guinea pigs that received ovalbumin (50 mg/kg ip) on days 1 and 3 and capsaicin (50 mg/kg sc) on day 21 (OAC); ovalbumin on days 1 and 3 (OA1); capsaicin on day 1 and OA on days 8 and 10 (COA); and ovalbumin on days 8 and 10 (OA2). On day 28, guinea pigs were submitted to ovalbumin aerosol challenge. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) were significantly lower in OAC and COA groups compared with OA1 and OA2 groups (P < 0.001). There was no difference between maximal Edyn and RL values obtained in OAC and COA groups. Morphometric analysis of lungs showed significantly (P < 0.05) lower values of contraction index of airways, peribronchial edema, and alveoli over inflation in guinea pigs that received capsaicin compared with intact guinea pigs. Capsaicin treatment did not influence the formation of specific IgG1 anaphylactic antibodies. We conclude that neurokinin depletion results in a decrease in the pulmonary mechanical and inflammatory responses to antigen challenge in sensitized guinea pigs. These effects are observed when capsaicin is given either before or after sensitization. PMID- 7539223 TI - Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel endothelial cells. AB - The development of new vessels (angiogenesis) is essential to wound healing. The center of a wound space is hypoxic, a condition that has been shown to stimulate angiogenesis in animal models of coronary artery occlusion. Because the mechanisms involved in this complex process are difficult to study in situ, an in vitro model would provide a useful complement to in vivo studies. This laboratory has developed and characterized calf pulmonary microvessel endothelial cell (PMVEC) cultures and an in vitro model system of angiogenesis using collagen three-dimensional gels that permit migration of cells into vessel networks. This system was used to study the direct effect of normoxia (20% O2) or hypoxia (5% O2) on PMVEC ability to undergo angiogenesis in vitro. Major changes leading to formation of capillary-like networks occurred during the first 3 days of hypoxic exposure only and included restructuring of actin filament networks, focal changes in distribution of basic fibroblast growth factor, and orientation and migration of cell tracts into a collagen gel matrix to form vessel networks. PMID- 7539224 TI - TGF-beta regulates production of NO in pulmonary artery smooth muscle cells by inhibiting expression of NOS. AB - We have previously reported that a mixture of lipopolysaccharide and cytokines stimulates cultured rat pulmonary artery smooth muscle cells (RPASM) to express elevated levels of mRNA for inducible nitric oxide synthase (iNOS), and to produce large amounts of nitric oxide (NO). The current study tests the hypothesis that transforming growth factor-beta (TGF-beta) modulates this process. Accordingly, RPASM were treated with a mixture of LPS (10 micrograms/ml) and the cytokines interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (500 U/ml), and interferon-gamma (100 U/ml). In the absence of TGF-beta 1, NO production (indicated by colorimetric assay of cumulative nitrite levels at 24 h) was greatly increased, as previously observed. Under identical conditions, TGF beta 1 caused a concentration-dependent decrease in NO production. The addition of neither excess L-arginine nor sepiapterin reversed the inhibition, indicating that the effect of TGF-beta 1 was not due to limitation of enzyme substrate or cofactor tetrahydrobiopterin, respectively. Northern and Western analyses showed that TGF-beta 1 reduced levels of iNOS mRNA and protein to baseline at all time points examined up to 24 h. Complete suppression of iNOS protein expression was evident even when TGF-beta 1 was added at postinduction time points. One mechanism of action of TGF-beta 1 was demonstrated in experiments in which degradation of iNOS protein was greatly increased by the addition of TGF-beta 1. These results demonstrate that TGF-beta 1 regulates production of NO in RPASM by inhibiting iNOS expression in part by increasing degradation of existing iNOS protein. PMID- 7539225 TI - Clockwork and the symbolization of time. PMID- 7539226 TI - Feedback in dynamic psychotherapy: notes on selected clinical issues and practical applications. PMID- 7539227 TI - [Proliferative activity of epithelium of cervix uteri in sexually-transmitted diseases]. AB - Comprehensive examination of patients with pseudoerosions of the cervix uteri showed that 71.1% of young nulliparous women have sexually-transmitted infections. Comparative study of proliferative and metabolic activity of integumental epithelial cells using historadioautography showed the highest level of DNA and RNA synthesis in the epithelial cells of the cervix uteri in the patients suffering from chlamydiasis, viral infections, and their combinations. Such patients should be referred to a group at risk of developing dysplasia of the cervix uteri and are in need of active specific treatment. PMID- 7539228 TI - A study and review of developmental dysgraphia in relation to acquired dysgraphia. AB - A survey of developmental dysgraphia in a population of 259 normal 13- and 14 year-old schoolchildren has been carried out. The neurological characteristics of handwriting impairments in the 25 children with the poorest handwriting skills are described. These data form the basis of a clinical classification of developmental dysgraphia. A group of simple clinical tests of handwriting and associated functions are offered for routine use. Because they are based on neurological principles relating to acquired dysgraphia, the classification and testing format may be used for correlation with neuro-imaging in research studies. More importantly, they may also facilitate both counselling and remediation for a particular child. The test battery could be modified for use in epidemiological surveys of neurodevelopmental disorders. PMID- 7539229 TI - Ribonuclease protection assay on in situ hybridization tissue: an alternative to polymerase chain reaction analysis. PMID- 7539230 TI - Estimation of extremely low amounts of single mRNAs by quantitative noncompetitive reverse transcription--polymerase chain reaction assay in biological specimens from normal and neoplastic cells. PMID- 7539231 TI - Hypertonic saline dextran does not increase cardiac contractile function during small volume resuscitation from hemorrhagic shock in anesthetized pigs. AB - Small volumes of hypertonic saline dextran (10% of shed blood volume [SBV] restore cardiac output (CO) and increase arterial pressure in hemorrhagic shock. Besides rapid expansion of plasma volume, a positive inotropic effect has been proposed as an additional mechanism for the immediate onset of the cardiovascular response. This study compares the effects of 7.2% saline/10% dextran 60 (HSDex, n = 8) and normal saline (NS; n = 6) on central hemodynamics and cardiac contractility assessed by end-systolic elastance (Ees; conductance technique) and segmental preload recruitable stroke work (sPRSW; sonomicrometry). In anesthetized open chest pigs (28 +/- 1 kg, mean +/- SEM) shock was induced by blood withdrawal (40% of blood volume) to maintain mean arterial pressure (MAP) at 45 mm Hg for 75 min. Resuscitation was started by bolus infusion (2 min) of either HSDex (10% of SBV) or the identical sodium load of NS (80% of SBV); 30 min later both groups received 6% dextran (10% of SBV). Hemorrhagic shock reduced CO (-45%) and left ventricular end-diastolic volume (Ved; -70%) while Ees increased (NS:2.2 +/- 0.4 to 7.5 +/- 1.8 mm Hg/mL, P < 0.05; HSDex: 1.9 +/- 0.2 to 9.1 +/- 2.6 mm Hg/mL, P = 0.085). Within 5 min after infusion of either solution CO returned to baseline values and MAP (NS +55%, HSDex +64%) and Ved (+100%) increased. Neither HSDex nor NS increased Ees above shock levels (NS, 8.7 +/- 4.9 mm Hg/mL; HSDex, 7.3 +/- 2.6 mm Hg/mL) and no group differences occurred in other measurements of contractility (dP/dt40,sPRSW). Plasma osmolality increased to 328 +/- 3 mOsmol/kg with HSDex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539232 TI - Balloon dilatation through the subcutaneously placed afferent limb of a hepaticojejunostomy in patients with resected Klatskin tumors. AB - It is generally conceded that palliation for proximal bile duct tumors (Klatskin) is exceptional if obstruction and the resultant infections can be prevented. Our experience with balloon dilatations thru the subcutaneously placed afferent limb of a choledocho or hepatico jejunostomy in patients with benign strictures suggests that this approach will be effective in patients with malignancies and thus provide long-term control of the obstruction without the need for external tubes. This is a report on one patient who, following a resected Klatskin tumor with positive margins, was treated with transhepatic internal external stents and was converted to a subcutaneous limb following numerous bouts of cholangitis. A schedule for repeat dilatations thru the jejunal limb was established. The patient has remained afebrile with a normal bilirubin and a moderately elevated alkaline phosphatase. Recurrent tumors or postirradiation strictures in patients with resected Klatskin tumors can be effectively controlled by repeated balloon dilatation without the need for external stents. PMID- 7539234 TI - Measurement of 5-HIAA in urine. PMID- 7539233 TI - Comparing subcutaneous danaparoid with intravenous unfractionated heparin for the treatment of venous thromboembolism. A randomized controlled trial. AB - OBJECTIVE: To compare the efficacy and safety of two subcutaneous doses of danaparoid with that of continuous intravenous administration of unfractionated heparin in the treatment of venous thromboembolism. DESIGN: An open-label, randomized, multicenter clinical trial. SETTING: One university hospital and two university-affiliated hospitals. PATIENTS: 209 patients suspected to have venous thromboembolism. Of these, 188 had a confirmed diagnosis (by ventilation perfusion lung scan and ultrasonography or contrast venography of the leg) and received study medication. INTERVENTIONS: Patients were randomly assigned to either low-dose danaparoid (intravenous loading dose of 1250 U followed by 1250 U administered subcutaneously twice daily [n = 65]); high-dose danaparoid (intravenous loading dose of 2000 U followed by 2000 U administered subcutaneously twice daily [n = 63]); or unfractionated heparin (intravenous loading dose of 2500 U followed by dose-adjusted continuous infusion [n = 60]). Treatment lasted at least 5 days and was continued until anticoagulation (achieved with acenocoumarol) was adequate. MEASUREMENTS: Efficacy determined clinically and by repeated imaging tests on treatment days 5 to 8; safety determined by daily assessment for bleeding. RESULTS: Two lung scans were done in each of 179 patients; ultrasonography or venography of the leg was done twice in each of 173 patients; and both repeated leg and lung tests were done in 166 patients. A significant reduction in recurrence or extension of venous thromboembolism was seen in patients receiving high-dose danaparoid (8 of 63 [13%]) compared with patients receiving intravenous unfractionated heparin (17 of 60 [28%]; relative risk, 0.45 [95% CI, 0.21 to 0.96]). Four of 61 patients receiving high-dose danaparoid (7%) and 14 of 58 patients receiving unfractionated heparin (24%) had recurrence of pulmonary embolism (relative risk, 0.27 [CI, 0.09 to 0.78]); 3 of 58 patients receiving high-dose danaparoid (5%) and 6 of 54 patients receiving unfractionated heparin (11%) had recurrence of deep venous thrombosis (relative risk, 0.47 [CI, 0.12 to 1.77]). Occurrence of major and minor bleeding was similar in the three groups; major bleeding occurred in 1 patient receiving low-dose danaparoid, 1 patient receiving high-dose danaparoid, and 2 patients receiving heparin. CONCLUSIONS: Our results suggest that high-dose danaparoid is safer and more effective than unfractionated heparin for the treatment of venous thromboembolism. PMID- 7539235 TI - Expression of cytokeratin 8 and low molecular weight cytokeratins in human basal cell carcinoma. AB - The expression of low molecular weight cytokeratins (Cks) 7,8,18, 18 and high molecular weight cytokeratin 10 in 23 basal cell carcinomas (BCCs) was investigated using a panel of 14 different commercially available monoclonal antibodies (MoAbs) with specific anti-cytokeratin activity. Four of these MoAbs were directed against Ck 8. The results showed that Ck 8 was detected in all 23 BCCs using MoAb 4.1.18. Two of the MoAbs showed inconsistent staining for Ck 8 and one of them did not show any staining at all. Cytokeratins 7 and 19 were detected incosistently. Cytokeratins 18 and 10 were not detected in any of the 23 BCCs that were examined. The incosistent observations on the expression of Ck 8 in BCCs in this study could have been due to different epitopes of the different cytokeratins that were detected by the different MoAbs. The results of this study lead to recommendation that whenever possible a panel of different MoAbs directed against the same Ck(s) should be used in order to minimize the risk of obtaining incorrect experimental results. PMID- 7539236 TI - Radiotherapy plus carboplatin and teniposide in patients with brain metastases from non small cell lung cancer. AB - BACKGROUND: The role of chemotherapy alone or added to radiation treatment for the palliation of multiple, unresectable brain metastases from non small cell lung cancer (NSCLC), is not yet well defined. Carboplatin and teniposide, however, are an interesting combination in this setting since they are active in NSCLC and because of encouraging results against brain metastases in other tumor types. METHODS: Twenty patients with brain metastases from NSCLC were treated with whole brain irradiation (total dose of 45 Gy) and chemotherapy (carboplatin, 300 mg/sm on day 1 and teniposide, 60 mg/sm on days 1, 2 and 3). RESULTS: Nine patients (45%) showed a complete remission of neurologic symptoms, and 7 (35%) an improvement. Neurologic signs disappeared in 8 patients (40%) and improved in 7 (35%). Three patients (15%) had partial (50%) regression of brain metastases at CT scan, and also showed response in other tumor sites. One other patient had a response of chest and liver lesions, while the cerebral metastases remained stable. Median survival was 7 months with a range of 1-9 months. Toxicity was mild, with no toxic deaths. CONCLUSIONS: Aggressive treatment can be taken into consideration also in the case of NSCLC patients with brain metastases and negative prognostic features. Their participation in clinical trials should be encouraged, since this will allow definition of the contribution of combined radiotherapy, chemotherapy and supportive care to the quality and duration of the patient's life. PMID- 7539237 TI - Hepatocellular carcinoma: surgical resection versus surgical resection combined with pre- and post-operative locoregional immunotherapy-chemotherapy. A prospective randomized study. AB - Hepatocellular carcinoma remains a disease with poor prognosis. Liver resection, although the optimal method of management, is associated with a high incidence of intrahepatic tumour recurrence ranging between 50-70%, 12 to 18 months following surgery. This study assesses prospectively the results of liver resection as compared to liver resection combined with pre- and post-operative locoregional chemotherapy-immunotherapy in 40 patients suffering from hepatocellular carcinoma. Patients were randomly assigned to two groups. Group A (20 patients) had liver resection only, while Group B (20 patients) had liver resection combined with pre- and past-operative targeting locoregional chemotherapy immunotherapy. Five (5) patients died in total: two from Group A and two from Group B, during the first 30 days following surgery due to reasons related to the procedure (post-operative liver failure in three, pulmonary embolism in one). The remaining patient from Group A died 10 months following liver resection due to intrahepatic tumour recurrence. From Group A, 17 patients are alive from 3 to 26 months after surgery. Of the 17 alive patients, 10 are free of disease and 7 show intrahepatic recurrence. Thus, tumour intrahepatic recurrence occurred in 8 patients of group A. From Group B, all 18 patients are alive and free of disease from 4 to 27 months after surgery. No patient died because of the disease nor has any patient shown intrahepatic tumour recurrence. As a conclusion of the present results, liver resection combined with pre- and post-operative targeting locoregional immunotherapy-chemotherapy appears to offer substantial advantages for patients undergoing surgery because of hepatocellular carcinoma. PMID- 7539238 TI - Expression of adhesion receptors in epidermal tumors: correlation with TNF alpha expressing cells. AB - The expression of intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (E-selectin) and vascular cell adhesion molecule-1 (VCAM-1) was investigated in a series of basal cell carcinomas and squamous cell carcinomas in comparison to benign viral-induced lesions (cutaneous warts, condyloma) and was correlated to the infiltration density and the presence of TNF alpha expressing cells. Tumor cells expressing ICAM-1 were frequently detected in squamous cell carcinoma and condyloma and were correlated with a dense infiltration of HLA-DR and LFA-1 positive cells. Tumor cells were devoid of ICAM 1 in other lesions. Tumor microvessels detected by Von Willebrand factor were consistently present but vessels expressing adhesion receptors were less frequent. E-selectin was usually intense and frequent, whereas VCAM-1 was rare except in dense infiltrated zones and in viral-induced lesions. In all samples, tumor cells strongly expressed TNF alpha. Though the cytotoxin has been reported to be an inducer of adhesion receptors, its expression in epidermal tumors did not correlate with a concomitant up-regulation of epithelial ICAM-1 or endothelial VCAM-1. This may contribute to a reduced influx of T cells into the tumors. PMID- 7539239 TI - Differential expression of transforming growth factor alpha, adhesions molecules and integrins in primary, metastatic liver tumors and in liver cirrhosis. AB - Changes in cytokines, intercellular cell-matrix adhesion molecules and integrins may influenced tumor cell invasion and metastases. This study described the distribution, pattern and intensity of cytokine TGFa, adhesion molecules CD 34 and CD 44 and integrins a2, a3, CD 29 (beta 1 chain) and CD 61 (beta 3 chain) in hepatocellular carcinoma (HCC), metastatic liver tumors and hepatic cirrhosis. Fresh snap-frozen tissue from 20 cases of HCC, 5 metastatic adenocarcinomas and 10 cirrhotic livers was studied immunohistochemically using available antibodies. The most intense staining of TGFa was found in metastatic adenocarcinoma, following by regenerating hepatocytes in cirrhotic liver and well differentiated HCC. Insignificant differences in activity of CD 34 in various pathologies, up regulation of CD 44 in poorly differentiated HCC and down-regulation in metastatic tumors were found. All integrins studied showed down-regulation in poorly differentiated HCC, relatively high activity of a2, a3 and beta 1 in metastatic tumors and the presence of all integrins in cirrhotic liver. PMID- 7539240 TI - Combination chemotherapy with carboplatin, cyclophosphamide and fluorouracil in advanced breast cancer. AB - Single agent carboplatin has demonstrated antitumoral activity in patients with advanced breast cancer. Thirty patients with inoperable locally advanced and/or metastatic breast cancer were treated with carboplatin (300 mg/m2) in combination with cyclophosphamide (600 mg/m2) and 5-fluorouracil (500 mg/m2) given on day 1 in a 4-weekly schedule. Of 29 patients evaluable for response, 4 presented CR and 4 PR (28%). Seven out of 19 chemotherapy-naive patients achieved CR (4) or PR (3) (37%). In contrast, only one patient out of 10 achieved PR in the group with previous adjuvant chemotherapy (10%). Responses were observed in primary tumours as well as in metastatic sites, including lymph nodes, lung, liver and skin. Median duration of response was 7.5+ and 3.8 months in CR and PR patients respectively. Toxicity was generally mild. Only 2 patients presented with clinically relevant hematologic toxicity. No significant non-hematologic toxicity was observed. It appears that this regimen, at the dosage and schedule studied, possesses only modest activity in patients with breast cancer, while being relatively atoxic. Carboplatin merits further investigation in this disease, but dosing should be individualised using e.g. a pharmacokinetic formula. PMID- 7539241 TI - Dose-intensive chemotherapy with cisplatin, vincristine, doxorubicin and etoposide against lung cancer: a pilot study. AB - We carried out the present study for the purpose of investigating the toxicity and efficacy of dose-intensive chemotherapy (Tokushima-CODE; T-CODE) against advanced lung cancer. Ten patients (8 with small-cell lung cancer and 2 with non small-cell lung cancer) received chemotherapy consisting of cisplatin (27 mg/m2 on days 1-3), vincristine (0.7 mg/m2 on days 1 and 8), doxorubicin (40 mg/m2 on day 1) and etoposide (133 mg/m2 on days 1-3). Recombinant granulocyte-colony stimulating factor (75 micrograms/body on days 4-15) was injected subcutaneously. Nine patients had measurable lesions, and they all experienced partial responses. WHO grades 3 and 4 leukopenia was found in 7 out of 10 patients; thrombocytopenia (grades 3 and 4) was seen in 5 out of 10 patients during the first cycle. These results suggest that T-CODE chemotherapy is a promising induction chemotherapy. PMID- 7539242 TI - Prolonged 5-fluorouracil infusion in patients with metastatic colon cancer pretreated with bolus schedule of the same agent. AB - 5-Fluorouracil (5FU) is the most important drug in the treatment or gastrointestinal cancer. 5FU can be administered by bolus or continuous infusion. It seems that continuous infusion is capable of producing responses in patients pretreated with bolus of the same drug. To overcome drug resistance in metastatic colon cancer patients, we have administered (via programmable pump) 5FU by prolonged infusion with doses of 250 mg/m2/die for six weeks with a one week rest period. Twenty-one patients with disease progression following bolus 5FU leucovorin were enrolled. The treatment was well tolerated with mucositis (grade I-II) in five patients and hand-foot syndrome in four; these side effects were managed with brief interruption of the infusion. Four partial responses and six stable disease were obtained. Two patients are alive after 14 months. The data of this study suggest that it is possible to overcome acquired 5FU bolus resistance by use of different schedules of the same drug. PMID- 7539243 TI - Assessment of apoptosis in oesophageal carcinoma preoperatively treated by chemotherapy and radiotherapy. AB - Apoptosis, programmed cell death, was immunohistochemically determined in 55 samples of oesophageal squamous cell carcinoma using the BM1 Mab. Sections from patients not treated (group 1, n = 12) or preoperatively treated by chemotherapy (group 2, n = 11), radiation (group 3, n = 13) or both (group 4, n = 8), and 11 additional cases of high-grade dysplasia or early cancer were examined. Most of the apoptotic cells were BM1-positive and checked by TUNEL proved to be nick end positive. They accounted for 7 (11%), 19 (29%), 21 (32%) and 26 (38%) cells per field in those 4 groups respectively. Chemotherapy and/or radiation significantly increased the number of apoptotic cells as compared to controls (p = 0.029 and p = 0.029, respectively). To assess the implications of the oncogene expression in the apoptotic pathway, additional section stained with bcl2 and p53 were negative for bcl2 and were positive for p53 in 16 samples (37%). Overall, positive cases for p53 mutation showed a significantly decreased incidence of apoptotic cells (p = 0.03). These results suggest that in situ assessment of apoptotic response better correlates to the apoptosis induced by radiation than that by chemotherapy, that abnormalities of the p53 protein decrease the apoptotic response in oesophageal carcinoma, and that immunohistochemical analysis of p53 protein helps to determine the sensitivity to these anticancer agents. PMID- 7539244 TI - Liver disease in cystic fibrosis. PMID- 7539245 TI - An assessment of growth hormone provocation tests. PMID- 7539246 TI - Keratin 14 gene point mutation in the Kobner and Dowling-Meara types of epidermolysis bullosa simplex as detected by the PASA method. AB - Recent advances in molecular biology have enabled the association of epidermolysis bullosa simplex (EBS) with point mutations of keratin 14 and/or keratin 5 genes to be established. We describe here the detection of point mutations in genomic DNA from formalin-fixed and paraffin-embedded sections from five cases of epidermolysis bullosa using the PCR amplification of specific alleles (PASA) method. In two of four cases of Kobner-type EBS a point mutation of helix 2b (384 Leu-Pro) was detected and in one case of Dowling-Meara-type EBS a mutation in helix 1a (125 Arg-Cys) was detected. The results of this study are consistent with previous reports and they demonstrate that the PASA method is a rapid and reproducible method for the detection of single-base changes and small deletions. PMID- 7539247 TI - Comparative immunoreactivity of the eosinophil constituents MBP and ECP in different types of urticaria. AB - In order to elucidate the role of eosinophil constituents in urticaria, we investigated major basic protein expression immunohistologically in comparison with that of eosinophilic cationic protein and the low-affinity IgE receptor in lesional and uninvolved skin of different types of urticaria. Eosinophil activation was studied with the markers EG1 and EG2. Different eosinophil constituents were found in all urticarial lesions except those of urticaria pigmentosa. MBP staining tended to be distributed diffusely throughout the tissue, whereas EG1 and EG2 antibodies were located at or close to individual cells. Staining with the low affinity IgE receptor antibody was rare. In uninvolved skin, major basic protein and particularly eosinophilic cationic protein reactivity was found in chronic recurrent urticaria, delayed pressure urticaria and, to a minor degree, in cholinergic urticaria. No correlation was found between antibody reactivity and eosinophil counts. Reactivity with either of the eosinophil constituents is thus a better marker for eosinophil involvement than routine H&E staining of the cells. The demonstration of eosinophil constituents in non-lesional skin of some urticaria patients suggests generalized eosinophil activation in certain subtypes of the disease. PMID- 7539248 TI - Investigation of epidermotropism in canine mycosis fungoides: expression of intercellular adhesion molecule-1 (ICAM-1) and beta-2 integrins. AB - In human mycosis fungoides (MF), interactions between LFA-1 (CD11a/CD18) and ICAM 1 (CD54) are involved in lymphocyte adhesion to keratinocytes. The purpose of this study was to evaluate the expression of ICAM-1, beta-2 integrins and class II major histocompatibility complex molecules (MHC II) on keratinocytes and infiltrating lymphocytes in canine MF. Sections of frozen skin biopsy specimens from normal dogs (n = 3) and dogs with MF (n = 17) were evaluated by immunohistochemistry for expression of ICAM-1, beta-2 integrins, and class II MHC molecules. Our results demonstrated that in canine MF, ICAM-1 was expressed variably on epidermal and follicular keratinocytes. The extent of keratinocyte ICAM-1 expression did not correlate with the degree of lymphocyte epithelial infiltration, nor with lymphocyte LFA-1 expression. This was especially evident in cases of Pagetoid reticulosis-like disease in which prominent lymphocyte epidermotropism was not accompanied by keratinocyte ICAM-1 expression. Keratinocyte class II MHC molecule expression did not correlate with keratinocyte ICAM-1 expression. In conclusion, in canine MF, the lack of statistically significant correlations between epithelial lymphocyte infiltration and keratinocyte ICAM-1 expression, and between keratinocyte ICAM-1 and lymphocyte LFA-1 staining, suggests that the LFA-1/ICAM-1 pathway is not the major adhesion mechanism between lymphocytes and keratinocytes. It is suspected that different ligands of the LFA-1 integrin (e.g. ICAM-2) or other adhesion molecules (e.g. CD2/LFA-3, VLA-1) might be involved in the epitheliotropism phenomenon in canine MF. These hypothesis cannot be evaluated in the dog at this time owing to the lack of specific monoclonal antibodies. PMID- 7539249 TI - Transforming growth factor beta 1 inhibits IL-3- and IL-4-dependent mouse connective tissue-type mast cell proliferation. AB - Transforming growth factor beta 1 (TGF beta 1) is a regulator of cell proliferation and differentiation. Using a mouse peritoneal cell-derived mast cell culture system, we investigated the effects of TGF beta 1 on mast cell proliferation. TGF beta 1 inhibited IL-3- and IL-4-dependent connective tissue type mast cell proliferation. The effect was concentration dependent: 50% inhibition was observed with 1.0 ng/ml TGF beta 1 and the maximal inhibitory effect (no proliferation), was observed with 10 ng/ml. Flow cytometric analysis suggested that the inhibitory effect of TGF beta 1 was due to blocking of both G1 and G2 phases. Both control and TGF beta 1-treated mast cells showed similar histamine release induced by the calcium ionophore, A23187. TGF beta 1 seems to be an important negative regulator of connective tissue-type mast cell proliferation with apparently no appreciable effect on mast cell function. PMID- 7539250 TI - Synthesis of pyrryl aryl sulfones targeted at the HIV-1 reverse transcriptase. AB - Various aryl 1-pyrryl sulfones were synthesized and tested as inhibitors of HIV 1. 2-Nitrophenyl-2-ethoxycarbonyl-1-pyrryl sulfone, the most active among test derivatives, was selected as lead compound of the aryl pyrryl sulfone series. The in vitro anti-HIV-1 activity and cytotoxicity of 41 compounds is reported. Some structure-activity relationships are discussed also in comparison with the known NPPS (2-nitrophenyl phenyl sulfone). PMID- 7539251 TI - Synthesis and biochemical evaluation of (carbamoylalkenyl)phenyloxy carboxylic acid derivatives as non-steroidal 5 alpha-reductase inhibitors. AB - Several (carbamoylalkenyl)- and (carbamoylalkenyl)phenyloxy carboxylic acids (Table 1) and some of their ethyl esters (Table 2) were synthesized and evaluated in vitro as inhibitors of steroid 5 alpha-reductase. Inhibitors of this enzyme may be useful in treating dihydrotestosterone-related diseases such as prostate cancer and benign prostatic hyperplasia. Using an enzyme preparation obtained from human prostate carcinoma tissue, the inhibition values ranged from 0 to 57% at the given dose of 100 microM. In the series of free acids, surprisingly, the compounds showed only modest inhibitory potency (0-26%). By contrast, the ethyl esters displayed inhibition values up to 57%. Structure-activity relationships are discussed. PMID- 7539252 TI - Minimal-access surgery for staging of malignant melanoma. AB - OBJECTIVE: To develop a simple, minimally invasive technique of determining whether regional node metastasis has occurred in patients with melanoma. SETTING: Teaching hospital tertiary care and private practice settings. PATIENTS: Between February 1993 and October 1994, 121 patients with invasive malignant melanoma and clinically negative lymph nodes were enrolled in this clinical trial. DESIGN: Consecutive sample clinical trial. Within 24 hours prior to lymph node resection, a radioactive tracer was injected into the dermis around the site of the primary melanoma. Forty-four patients also had blue dye injected immediately prior to surgical resection. Measurement of radioactivity in the lymph nodes and surgical localization were made using a handheld gamma detector. Radiolabeled nodes were selectively removed with the least dissection possible. In patients with pathologically positive radiolabeled nodes, regional lymphadenectomy was performed. OUTCOME MEASURES: Successful identification of radiolabeled sentinel lymph nodes, correlation of radiolabeling with injection of blue dye, and regional node recurrence rate. RESULTS: Surgeons successfully resected the radiolabeled sentinel lymph nodes in 118 (98%) of 121 patients. One hundred percent of blue-stained lymph nodes were successfully radiolabeled. Fifteen patients had pathologically positive sentinel lymph nodes. In 10 patients, the sentinel node was the only node with metastasis. Two systemic and one regional node recurrences occurred during a mean follow-up of 220 days. CONCLUSIONS: Selective gamma probe-guided resection of the radiolabeled sentinel lymph node is possible in over 95% of patients with melanoma. This technique offers a simple and reliable method of staging of regional lymph nodes in these patients without performing a regional lymphadenectomy. PMID- 7539253 TI - Co-induction of arginase and nitric oxide synthase in murine macrophages activated by lipopolysaccharide. AB - In view of studies showing that not only nitric oxide synthase (NOS) activity but arginase activity is induced in rodent macrophages by lipopolysaccharide (LPS), the objective of this study was to investigate the co-induction of these two enzymes and to ascertain whether common mechanisms are involved. RAW 264.7 cells were activated by 2 micrograms LPS/ml and incubated for up to 48 hr. Inducible NOS (iNOS) and inducible arginase II (AII) activities were monitored, respectively, by measuring NO2-/NO3- accumulation in cell culture media and formation of urea (as CO2) from L-arginine by cell lysates. AII activity increased linearly up to at least 48 hr, whereas NO2-/NO3- formation reached a plateau well before 48 hr. Immunoprecipitation experiments revealed that AII accounted for 90-100% of arginase activity in LPS-activated macrophages. The inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, inhibited the induction of iNOS but not AII. Moreover, whereas IFN-gamma caused iNOS induction, AII induction was nearly abolished by IFN-gamma, perhaps by inhibiting transcription of the AII gene. These observations indicate that co-induction of iNOS and AII occurs by distinct transcriptional mechanisms, AII induction could diminish NO production by decreasing L-arginine availability, and IFN-gamma can prevent AII induction. PMID- 7539254 TI - Lack of critical domains in the beta-chain of hepatocyte growth factor. AB - Hepatocyte growth factor (HGF) is a cytokine with pleiotropic effects on many different cell types. Its biological activities depend on the disulfide-linked alpha beta heterodimeric molecule. To study the functions of the beta-chain of HGF, insect cell-expressed HGFs prepared from nested deletions from C-terminus of beta-chain were studied for their biological activities and ligand-binding functions. The results demonstrated that the functions of HGF are dependent on the intactness of its beta-chain. The loss of HGF activities is correlated with the progressive deletion of beta-chain. There is not a single critical domain in the beta-chain that is essential for the functions of HGF. We propose that beta chain may act to stabilize the ligand-receptor binding or contribute to the proper conformation of HGF to interact with its receptor. PMID- 7539255 TI - The protein kinase C isoforms leading to MAP-kinase activation in CHO cells. AB - Stimulation of CHO cells with phorbol ester caused transient activation of mitogen-activated protein kinase (MAP-kinases with 42 and 44 kDa). A similar treatment of CHO cells which overexpress a massive amount of the alpha- or delta isoform of protein kinase C (PKC) resulted in the prolonged activation of MAP kinase and eventually in the appearance of mostly dikaryotic, sometimes tetrakaryotic cells. The results suggest that the delta-isoform, as well as the alpha-isoform, has a potential to cause MAP kinase activation. Unusual activation of the PKC/MAP-kinase pathway, however, may lead to abnormal cytokinesis. PMID- 7539256 TI - Particulate and soluble forms of the inducible nitric oxide synthase are distinguishable at the amino terminus in RAW 264.7 macrophage cells. AB - We report here on the characterization of soluble and particulate forms of the inducible nitric oxide synthase in RAW 264.7 macrophage cells. Stimulation of these cells with E. coli lipopolysaccharide and interferon-gamma resulted in a significant induction of nitric oxide synthase activity with approximately 20% of the total activity localized to the cell membrane. Like the soluble enzyme form, the membrane-associated nitric oxide synthase activity was inhibited by NG monomethyl-L-arginine and did not require the addition of calcium. Both protein forms were immunoreactive on Western blots with antibodies specifically recognizing the carboxyl terminus of the protein. In contrast, antibodies specific for the amino terminus recognized inducible nitric oxide synthase from the cytosol, but failed to recognize the membrane-associated protein. Thus, macrophage cells are capable of expressing two forms of the inducible nitric oxide synthase that are definable by an intracellular distribution that correlates to antigenic differences at the amino terminus. PMID- 7539257 TI - Caffeine inhibits the binding of thyrotropin-releasing hormone in GH4C1 pituitary cells. AB - Caffeine can modulate intracellular Ca2+ concentration ([Ca2+]i) by triggering the mobilization of Ca2+ from intracellular Ca2+ stores. In the present study we show that in Fura 2 loaded GH4C1 cells, caffeine inhibited, in a dose-dependent manner, the Ca2+ response induced by a submaximally effective dose (3 nM) of thyrotropin-releasing hormone (TRH). We also show that caffeine decreased the specific binding of [3H]TRH. Equilibrium binding studies with [3H]TRH and Scatchard analysis of the binding data showed that caffeine increased the dissociation constant (Kd) from 8 +/- 1 nM to 26 +/- 3 nM, while the maximum amount of [3H]TRH bound to the cells was increased by 32%. Thus, caffeine inhibited the TRH-evoked increase in [Ca2+]i by inhibiting the binding of TRH to its receptor. PMID- 7539258 TI - Bradykinin induces tyrosine phosphorylation of epidermal growth factor-receptor and focal adhesion proteins in human keratinocytes. AB - Bradykinin is an inflammatory mediator which activates signalling pathways in human keratinocytes via a receptor linked to a GTP-binding protein. In the HaCaT human keratinocyte cell line, we observed bradykinin-stimulated tyrosine phosphorylation of several cellular proteins with peak response at 15 min. The focal adhesion proteins paxillin and p125FAK were tyrosine phosphorylated in response to bradykinin but not in response to epidermal growth factor. Interestingly, we identified the epidermal growth factor receptor as a novel target for bradykinin-induced tyrosine phosphorylation. The tyrosine kinase inhibitor genistein and the protein kinase C inhibitors staurosporine and Ro31 7549, all blocked bradykinin-induced tyrosine phosphorylation. Our data suggest that stimulation of the bradykinin receptor leads to activation of a tyrosine kinase activity via a protein-kinase-C-dependent pathway in human keratinocytes. PMID- 7539259 TI - An animal model of congenital defect of gene expression of cholecystokinin (CCK) A receptor. AB - Expressions of the CCK-A and B receptor genes in fetal and adult pancreas of OLETF rats were examined by the reverse transcriptase polymerase chain reaction followed by Southern blot hybridization. The pancreatic responses to various stimulants were examined in vitro and results were compared with those of control (LETO) rats. CCK-A receptor mRNA was not expressed in the fetal pancreas of either strain or in the adult pancreas of OLETF rats, but was expressed in the adult pancreas of LETO rats. CCK-B receptor mRNA was expressed in fetal and adult pancreas in both strains. Southern blot hybridization indicated a difference in gene structure in the two strains. The maximal effective concentrations of neuromedin C, carbachol, and secretin for amylase secretion and intracellular Ca2+ movement stimulated by carbachol and neuromedin C were similar in the two strains. CCK-8 and the non-sulfated form stimulated amylase secretion only in LETO rats. These results suggest that OLETF rats are a new model of a congenital defect of the CCK-A receptor gene and should be useful for determining CCK receptor function. PMID- 7539260 TI - Pyrrolidine dithiocarbamate prevents IL-1-induced nitric oxide synthase mRNA, but not superoxide dismutase mRNA, in insulin producing cells. AB - We presently investigated the effects of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF-kappa B), on the induction of nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) mRNAs by IL-1 beta in insulin-producing RIN cells. PDTC decreased by 90% both IL-1 beta induced increase in medium nitrite accumulation (an indicator of NO production) and induction of iNOS mRNA expression. However, PDTC did not prevent induction of MnSOD mRNA by IL-1 beta. PDTC induced an early (45 min) expression of c-fos mRNA and potentiated IL-1 beta-induced c-fos expression. Our data suggest that NF kappa B activation is necessary for iNOS, but not for MnSOD, mRNA expression in insulin producing cells. PMID- 7539261 TI - An inward rectifier K+ current modulates in neuroblastoma cells the tyrosine phosphorylation of the pp125FAK and associated proteins: role in neuritogenesis. AB - The relationships between the integrin-mediated activation of inward rectifyier K+ channels (KIR), the phosphorylation of pp125FAK and the rescue of neuritogenesis were studied in 41A3 mouse neuroblastoma cells. Neuritogenesis, elicited by adhesion to FN-enriched substrata, was reversibly impaired by pretreating these cells with the tyrosine kinase inhibitor Herbimycin A. This impairment mimicked that operated by Cs+ ions, which selectively inhibited the integrin-mediated activation of KIR channels. Various phosphotyrosine containing cellular proteins underwent a marked increase upon cell adhesion to FN-coated dishes. This increase was significantly reduced by Cs+ addition. Immunoprecipitation of pp125FAK revealed that the phosphorylation of this kinase and several associated proteins was significantly and reversibly inhibited by Cs+, indicating that integrin-mediated activation of KIR channels is a limiting step upstream to the phosphorylation of pp125FAK in the commitment to neuritogenesis. PMID- 7539262 TI - Site-specific association of c-Src with epidermal growth factor receptor in A431 cells. AB - We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100 solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor. PMID- 7539263 TI - Access to peptide regions of a surface mucin (MUC1) is reduced by sialic acids. AB - Mucinous glycoproteins are present on the surfaces of tumor cells. Knowledge of which parts of the mucin molecule are accessible targets for cells of the immune system is important in the development of successful therapeutic approaches. One breast (ZR-75-1), two colon (Colo 205 and SW1116), and three pancreas (Capan-2, HPAF and SW1990) cancer cell lines were examined. The reactivities of antibodies HMFG-2, specific for the tripeptide (DTR) in the 20 amino acid tandem repeat of MUC1, and SM-3 (PDTRP) were greatly enhanced by pre-treating cells with an inhibitor of O-glycosylation, benzyl-alpha-N-acetylgalactosamide. However, desialylation of cell surfaces with neuraminidase or pre-treatment with an inhibitor of carbohydrate processing, monensin, also greatly enhanced the reactivities of HMFG-2, SM-3 and HMFG-1 (PDTR). Thus, sialic acids on termini of neighboring oligosaccharides significantly limit access to the peptide region recognized by antibodies HMFG-1/2 and SM-3. PMID- 7539264 TI - NO synthase activity does not influence electrical activity of mouse pancreatic B cells. AB - The membrane potential of mouse pancreatic B-cells was recorded with intracellular microelectrodes. At a low concentration the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 mumol/l) did not alter electrical activity induced by 15 mmol/l glucose. At 5 mmol/l L-NAME depolarized B-cells similarly to L-arginine. The depolarization of the B-cell membrane induced by 5 mmol/l L-arginine in the presence of 15 mmol/l glucose was not inhibited but further increased by the addition of 15 mmol/l L-NAME. D-arginine which is not used as a substrate by the NO synthase also depolarized the B-cells. However, higher concentrations were necessary than for L-arginine pointing to a preferential transport of the L-enantiomer. It is concluded that the electrical activity of pancreatic B-cells is not influenced by the NO synthase and that the enzyme is not involved in the arginine-induced depolarization of B-cells. PMID- 7539265 TI - The effects of a novel and selective inhibitor of tryptophan 2,3-dioxygenase on tryptophan and serotonin metabolism in the rat. AB - The effects of a novel inhibitor 680C91 ((E)-6-fluoro-3-[2-(3- pyridyl)vinyl]-1H indole) of the key enzyme of tryptophan catabolism tryptophan 2,3-dioxygenase (TDO) (EC 1.13.11.11), were examined on tryptophan catabolism in vitro and in vivo and on brain levels of tryptophan, serotonin (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA). 680C91 was a potent (Ki = 51 nM) and selective TDO inhibitor with no inhibitory activity against indoleamine 2,3-dioxygenase (EC 1.13.11.17), monoamine oxidase A and B, 5-HT uptake and 5-HT1A,1D,2A and 2C receptors at a concentration of 10 microM. 680C91 had no effect on the binding of tryptophan to serum albumin in plasma and inhibited TDO competitively with respect to its substrate tryptophan. 680C91 inhibited the catabolism of tryptophan by rat liver cells and rat liver perfused in situ. The catabolism of L [ring-2-14C]-tryptophan and a load dose of tryptophan (100 mg/kg) in vivo were inhibited by prior administration of 680C91. Administration of 680C91 alone produced marked increases in brain tryptophan, 5-HT and 5-HIAA. A load dose of tryptophan (100 mg/kg), producing increases in brain tryptophan 4-fold greater than that seen with 680C91, did not increase brain 5-HT and 5-HIAA to levels greater than those seen with 680C91 and produced a shorter-lasting increase in these parameters. These data therefore demonstrate the importance of TDO as a regulator of whole-body tryptophan catabolism and brain levels of tryptophan and 5-HT and suggest that a greater antidepressant efficacy might be achieved with inhibitors of TDO than tryptophan administration alone. PMID- 7539266 TI - Tubulin alterations in taxol-induced apoptosis parallel those observed with other drugs. AB - We have described previously that during apoptosis cellular tubulin is reorganized into visible tubulin structures that correlate with apoptotic morphology. Such changes have been observed in human leukaemic cells treated with a variety of cytotoxic agents. These structures are unlike those seen in untreated non-mitotic or mitotic cells. As taxol is known to act by enhancing the polymerization of tubulin in the initiation and extension of microtubules (MTs), and has been shown to induce and stabilize the formation of tubulin structures in a variety of cells, we examined the involvement of tubulin in apoptosis induced by taxol. Apoptosis was induced in a human T-cell leukaemic line, CCRF-CEM, following treatment with 10 nM taxol. The morphological features typical of apoptosis were apparent in taxol-treated cells after drug addition. Immunocytochemical analysis using a monoclonal antibody to beta-tubulin indicated that taxol induced visible tubulin polymerization. DNA fragmentation was detected at 10 hr post-treatment. Flow cytometric analysis of taxol-treated cells showed a time-dependent accumulation of cells in G2/M phase with the appearance of a hypodiploid peak coincident with the detection of DNA fragmentation. Microtubule structures observed following taxol treatment were of three types. At the time of DNA fragmentation, 50% of the cells displayed tubulin structures associated with apoptotic morphology similar to those seen in apoptosis induced by treatment with methotrexate (10(-8) M) or etoposide (17 microM). Twenty percent of the cells were arrested in mitosis at this time. These cells contained either multiple asters or disordered mitotic spindles, and did not display apoptotic morphology. The remaining cells, while normal in morphology, had extensive tubulin polymerization in the cytoplasm and around the nucleus. We examined the time course of tubulin mRNA expression in apoptosis induced by taxol, methotrexate and etoposide. The level of tubulin mRNA displayed a transient increase after treatment, and prior to the onset of DNA fragmentation with each of the three drugs. These results suggest that during apoptosis taxol induces tubulin changes that display characteristics similar to those observed during apoptosis following treatment with drugs that do not interact directly with tubulin. PMID- 7539267 TI - Gliotoxin inactivates alcohol dehydrogenase by either covalent modification or free radical damage mediated by redox cycling. AB - The fungal metabolite gliotoxin shows selective toxicity to cells of the immune system and has been implicated in the aetiology of invasive aspergillosis. The related toxin sporidesmin is the causative agent of facial eczema in sheep. The toxicity of these compounds has been related to their ability to redox cycle intracellularly and thus produce damaging free radicals. These toxins are also potentially capable of forming mixed disulphides with thiol groups on proteins by virtue of their bridged disulphide structure. We show here that gliotoxin can inactivate horse liver alcohol dehydrogenase by either oxidative damage or covalent modification of thiol groups on the enzyme. Either Cys-281 or Cys-282 is selectively modified. Neither of these residues are at the active site. Covalent modification occurs in the absence of reducing agents such as dithiothreitol. In the presence of dithiothreitol no protection is observed and the rate of inactivation is enhanced although as expected no covalent modification occurs. Gliotoxin can therefore inhibit alcohol dehydrogenase by either pathway and this will depend on the availability of reducing agents such as glutathione and/or how readily the reactive oxygen species generated are removed. PMID- 7539268 TI - [Cleavage of a double-stranded DNA target by bleomycin derivatives of oligonucleotides, forming a ternary complex]. AB - Hexadecathymidylate derivatives, containing covalently-bound antitumor antibiotic bleomycin A5, were shown to form a triple-helix complex with double-strand 30-bp DNA-target and to carry out within this complex complementary-addressed DNA modification. Fivefold excess of reagent in relation to target leads to non specific cleavage mainly of pyrimidine-rich DNA strand. Total degrees of the target-strand cleavage by 5'- and 3'-bleomycin derivatives of hexadecathymidylate were 25 and 35% for purine-rich strand and 47 and 36% for pyrimidine-rich strand. Degrees of non-specific cleavage by 5'-bleomycin derivative of hexadecanucleotide that does not form triple-helix were 6 and 16% for purine- and pyrimidine-rich strands, respectively. Comparison of these data has shown that site-specific cleavage prevailed nonspecific one. Triplex of 5'-bleomycin derivative with DNA melted by 5 degrees C lower (m.p. 40 degrees C) than the similar triplex of hexadecathymidylate. Temperature lowering from 50 to 20 degrees C increases the DNA-cleavage degree according to the increase in the part of target molecules involved in triple-helix formation. PMID- 7539269 TI - Influence of temperature on long-term keratin immunoreactivity for oral exfoliative cytology. AB - We previously showed that keratin profiles can be of value in the diagnosis of oral cancer when using exfoliative cytology. In the future, they may form part of a screening program for oral cancer. This study evaluated the influence of long term storage on keratin expression. Smears were collected from the clinically normal buccal mucosa and dorsal tongue of 22 patients. Half were stored in a refrigerator (5 degrees C) and half in a freezer (-70 degrees C). A total of 528 smears were collected. A panel of three antikeratin antibodies (LP34, AE8 and 1C7) was used to identify the preservation of keratin expression (graded as absent, few cells positive or many cells positive). The results for smears from dorsal tongue indicated that many cells were impermeable by the antikeratin antibodies. However, a satisfactory level of keratin immunoreactivity was observed in smears from buccal mucosa stored at -70 degrees C for over one year. Results for storage at 5 degrees C for both sites were inadequate after one month. Thus, smears from nonkeratinized oral sites may be stored at -70 degrees C for at least one year without a profound loss of keratin immunoreactivity, thus allowing examination of archival material. PMID- 7539270 TI - FK 506 versus cyclosporin in the prevention of renal allograft rejection- European pilot study: six-week results. AB - FK 506 was compared with cyclosporin in a randomised trial in good-risk cadaveric renal transplant recipients. The objective was to evaluate whether oral FK 506 dosing was viable and whether blood concentrations in the range 10-20 ng/ml would prove to be practical. Thirty-one adult patients were randomised to FK 506 and 16 to cyclosporin. Both groups received an identical regimen of azathioprine and corticosteroids. Serum creatinine concentrations decreased rapidly in both groups with mean values below 200 mumol/l within 2 weeks. One graft in the cyclosporin group was lost due to renal vein thrombosis. During the 6-week study period, 19.4% of patients on FK 506 and 31.3% on cyclosporin experienced acute rejection. One patient in each group experienced corticosteroid-resistant rejection that responded to anti-lymphocyte therapy. Infections were reported in 51.6% of the FK 506 group compared with 37.5% of the cyclosporin group. The spectrum of adverse events was similar in both groups. However, minor neurological disorders were more common in the FK 506 group (54.8% versus 6.3%) whereas hypertension was less common (48.8% versus 75.0%). The results indicate that oral FK 506 rapidly achieves therapeutic blood concentrations and is an effective immunosuppressant for the initial treatment of renal allograft recipients. PMID- 7539271 TI - Combined effects of ionizing radiation and cycloheximide on gene expression. AB - We performed experiments to determine the effects of ionizing radiation exposure on expression of genes such as beta-actin, c-fos, histone H4, c-myc, c-jun, Rb, and p53 after exposure of Syrian hamster embryo (SHE) cells to the protein synthesis inhibitor cycloheximide. The purpose of these experiments was to determine the role of a labile protein in the radiation-induced response. The results revealed that when ionizing radiation (either fission-spectrum neutrons or gamma rays) was administered 15 min after cycloheximide treatment of SHE cells, the radiation exposure reduced cycloheximide-mediated gene induction of c fos, histone H4, and c-jun. In addition, dose-rate differences were found when radiation exposure most significantly inhibited the cycloheximide response. Our results suggest that ionizing radiation does not act as a general protein synthesis inhibitor and that the presence of a labile protein is required for the maintenance of specific gene transcription and mRNA accumulation after radiation exposure, especially at high dose-rates. PMID- 7539272 TI - A risk-benefit assessment of risperidone in schizophrenia. AB - The atypical antipsychotic risperidone combines dopaminergic and serotonergic antagonism. This results in a drug that is both clinically effective, reducing positive and negative symptoms of schizophrenia, and has a low incidence of adverse effects. At a dosage of 4 to 8 mg/day, risperidone is comparable to 10 mg/day of haloperidol. This dosage has a low incidence of extrapyramidal adverse effects and is nonsedative, although it may cause orthostatic hypotension. There is no current evidence for specific biochemical and haematological abnormalities associated with risperidone. Although the clinical benefits appear to outweigh the risks, this drug continues to be a relatively expensive treatment option in the UK. There is therefore a need for a formal cost-utility assessment of risperidone and for comparisons between this drug and other atypical neuroleptics. PMID- 7539273 TI - Eosinophil infiltration is related to increased expression of vascular cell adhesion molecule-1 in nasal polyps. AB - Endothelial adhesion molecules are important in the recruitment of leukocytes to inflammatory sites. Nasal polyps characteristically contain a leukocyte infiltrate in which eosinophils often are remarkably prominent. We have studied whether this feature is related to a particular profile of adhesion molecules on the local microvascular endothelium. Nasal polyps were obtained from 15 patients. Mucosal biopsy specimens of the lower and the middle turbinate from the same patients as well as from three control subjects served as reference tissue. Expression of endothelial adhesion molecules and the relative numbers of eosinophils and neutrophils were examined by two- and three-color immunofluorescence staining. Both the number of eosinophils and the proportion of vessels positive for vascular cell adhesion molecule-1 (VCAM-1) were significantly increased in nasal polyps compared with the turbinate mucosa of the same patients (P = 0.008 and P = 0.001, respectively). By contrast, the number of neutrophils and the relative expression of E-selectin and intercellular adhesion molecule-1 were similar at both tissue sites. Furthermore, the relative number of eosinophils in nasal polyps was well correlated (rs = 0.73, P = 0.006) with the percentage of vessels positive for VCAM-1, but this was not true for neutrophils. Taken together, this direct in situ observation strongly supports the crucial role suggested for VCAM-1 in human eosinophil extravasation at inflammatory sites. PMID- 7539274 TI - Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase. AB - Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon gamma (IFN-gamma), tumor necrosis factor alpha, interleukin 1 alpha, (IL-1 alpha), lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate- (NO2-/NO3-) in a dose- and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-10 reduced NO2-/NO3- generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-gamma, also immunostained for iNOS and generated NO2-/NO3-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of NO2-/NO3- and macrophages that spontaneously released NO2 /NO3- and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced NO2-/NO3- and both cell types immunostained for iNOS (approximately 20% of all type II cells and 35% of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of NO2-/NO3- was dependent on the in vivo availability of tumor necrosis factor alpha, IL-1, and IFN-gamma. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines. PMID- 7539275 TI - Iron and reactive oxygen species in the asbestos-induced tumor necrosis factor alpha response from alveolar macrophages. AB - Free radicals and other reactive oxygen species (ROS) are important mediators in asbestos-induced lung toxicity. Asbestos fibers are thought to stimulate cells to generate ROS via iron that is present on fibrous silicates. The pathophysiologic responses in the lung after asbestos exposure are characterized by the accumulation of macrophages at the site of fiber deposition and the release of growth factors and proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). We have examined the role of iron-catalyzed ROS in asbestos induction of TNF-alpha from rat alveolar macrophages. Treatment of alveolar macrophage cultures with asbestos stimulated dose-dependently TNF-alpha secretion, which was inhibited by the addition of deferoxamine, an iron chelator. Asbestos fibers, pretreated with deferoxamine to remove iron from the fibers before addition to alveolar macrophages, also significantly reduced the TNF-alpha response. Consistent with the role of iron on asbestos fibers in catalyzing hydroxyl radical generation, membrane-permeable hydroxyl radical scavengers (tetramethylthiourea, dimethyl sulfoxide) inhibited the asbestos-induced TNF alpha response. The asbestos-induced increase in TNF-alpha, as well as in interleukin-1 alpha, and their inhibition by tetramethylthiourea occurred at the transcriptional level. The role of ROS in signaling TNF-alpha stimulation was confirmed by use of free radical-generating systems (hypoxanthine-xanthine oxidase, hydrogen peroxide, glucose-glucose oxidase, or ferrous plus hydrogen peroxide). These results suggest that intracellularly generated ROS can stimulate TNF-alpha in alveolar macrophages and that asbestos-induced TNF-alpha gene expression and secretion are mediated by iron-catalyzed product of ROS. PMID- 7539276 TI - Operative treatment of elbow arthritis. AB - There have been a number of recent developments in elbow surgery for arthritis and stiffness. Advances have occurred in our understanding of the basic biomechanics of elbow arthroplasty as well as in the long-term clinical follow-up of patients treated with these implants. In addition, treatment of elbow arthritis by nonprosthetic means, including arthroscopy and a variety of open procedures, has been further advanced by reports in the literature. An escalating interest in the role of and techniques for elbow arthroscopy has broadened the indications for this technique to expand into the area of treatment of the stiff elbow as well. This paper will discuss these three areas of elbow surgery as they relate to the treatment of elbow arthritis. PMID- 7539277 TI - Interphase cytogenetics of prostatic adenocarcinoma and precursor lesions: analysis of 25 radical prostatectomies and 17 adjacent prostatic intraepithelial neoplasias. AB - Twenty-five radical prostatectomy specimens were screened for the presence of numerical chromosome changes within the adenocarcinoma as well as 17 adjacent prostatic intraepithelial neoplasias (PIN) by means of interphase in situ hybridization (ISH) to routinely processed tissue sections. To this end a defined alfoid repetitive DNA probe set was used, specific for the centromeres of chromosomes 1, 7, 8, 10, 15, and Y. The cytogenetic information was correlated with histopathological and clinical features as well as with DNA ploidy. Numerical aberrations of at least one chromosome were shown in 13 of 25 cases (52%). Alterations of chromosome 8 and loss of the Y chromosome were the most frequent findings (both 20%), followed by loss of chromosomes 15 (16%) and 10 (12%). Gain of chromosome 7 was seen in 8% of cases. No aberrations of chromosomes 7, 8, 10, and 15 were found in the adjacent PIN lesions, whereas loss of the Y chromosome in both PIN and tumor occurred in two cases. Also, (low level) aneuploidy was observed in 76% of these PIN lesions. Ploidy of the carcinomas as assessed by ISH correlated well with ploidy measured by DNA flow cytometry (FCM; P < 0.02). Due to the more specific correspondence between ISH and tumor pathology, pathologic grade correlated with ISH aneuploidy (P < 0.05), whereas FCM ploidy did not. Furthermore, genetic heterogeneity within a tumor was seen, as judged by the focal appearance of chromosomal aberrations. Chromosomal alterations occurred in all grades and stages, although loss of chromosome 10, gain of chromosome 7, and aberrations of chromosome 8 tended to predominate in more advanced cancers. PMID- 7539278 TI - An Alu VpA marker on chromosome I demonstrates that replication errors manifest at the adenoma-carcinoma transition in sporadic colorectal tumors. AB - Widespread mutations in simple tandemly repeated (STR) DNA sequences are frequently found in colorectal tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) and less frequently in sporadic colorectal cancers. This aims of this study were to determine the type of DNA sequence most commonly affected by such mutations and to examine the point in the natural history of the tumor where replication errors (RERs) appear. An unselected series of colorectal tumors (49 adenomas and 108 carcinomas) was examined with 4 different STR markers: one Alu VpA polymorphism (MYCLI), one tetranucleotide repeat (D17S846), one dinucleotide repeat (D3S1029), and one polyA repeat (AP delta 3 delta). All 3 positive adenomas and 18 of 20 positive carcinomas showed replication errors in the Alu VpA sequence at the MYCLI locus, making this marker more than twice as sensitive as the best of the other 3 markers. Importantly, all positive adenomas showed small foci of carcinoma in situ. This suggests that replication errors manifest at the adenoma/carcinoma transition in sporadic colorectal tumors. PMID- 7539279 TI - Two tumor suppressive loci on chromosome 10 involved in human glioblastomas. AB - A number of cytogenetic and molecular analyses have revealed very frequent and extensive losses of regions of chromosome 10 in human glioblastomas. Our recent studies have demonstrated that the transfer of a chromosome 10 into human glioblastoma cells resulted in suppression of their transformed and tumorigenic phenotype. To localize the suppressive region further, we isolated and characterized certain hybrid cells that had undergone chromosomal rearrangements to yield hybrid cells retaining only various regions of the inserted chromosome 10. One series of subclones showed the loss of the majority of the long arm of chromosome 10 (10q21-10qter) and regained the ability to grow under anchorage independent conditions, but the cells still failed to exhibit significant tumorigenicity in nude mice. Another set of subclones exhibited major deletions of large segments of the long arm of chromosome 10 (10q21-q23; 10q26-qter), yet retained certain distal alleles associated with 10q24 to 10q26. These subclones were identical in their biological characteristics to the hybrids containing an intact chromosome 10, exhibiting no growth in soft agarose or in nude mice. These results implicate the presence of two independent phenotypically suppressive regions on chromosome 10 (10pter-q11 and 10q24-q26) that are involved in glioma progression. PMID- 7539280 TI - Electrophoretic isolation of extrachromosomal DNA from tumor cells. AB - Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression. PMID- 7539281 TI - DNA-repeat instability is associated with colorectal cancers presenting minimal chromosome rearrangements. AB - The DNA-repeat [(CA)n] instability of colorectal cancer cells was studied relative to our previously defined classification based on chromosome alterations. Of the 23 tumors analyzed, 13 belonged to the "monosomic" type (MT) characterized by simultaneous loss of chromosome 18 and chromosome arm 17p, and many structural rearrangements, 7 to the "trisomic" type (TT) with many chromosome gains but few rearrangements, and 3 had a normal karyotype (NT). (CA)n repeat sequences were examined on chromosomes 2, 5, 11, 13, 18, and 20. We found sequence alterations in 12 tumors at 1 or several loci, 9 of which (1/13 MT, 5/7 TT, and 3/3 NT) exhibited a typical shift in allele size defined as microsatellite instability. Furthermore, a single alteration was observed for the MT tumor, whereas one NT tumor displayed instability on two and all the other tumors on three or more loci. These results suggest an inverse relationship between the occurrence of chromosome structural rearrangements and microsatellite instability, providing another argument for the subdivision of colorectal cancers into groups of distinct oncogenic pathways. PMID- 7539282 TI - Region-specific loss of heterozygosity on chromosome 19 is related to the morphologic type of human glioma. AB - Allelic mutation on chromosome 19 has previously been reported as a frequent genetic event in human glial tumors. In an effort to localize specific regions of importance on this chromosome better, 13 highly polymorphic genetic markers distributed along the length of chromosome 19 were used for evaluation of loss of heterozygosity (LOH) and microsatellite instability in a total of 100 brain tumors, including 75 astrocytomas (55 grade 4; 7 grade 3; 5 grade 2; 6 grade 1; and 2 other), 17 oligodendrogliomas (1 grade 4; 5 grade 3; 10 grade 2; and 1 grade 1), and 8 mixed oligoastrocytomas (MOA) (3 grade 4; 2 grade 3; and 3 grade 2). No microsatellite expansion was observed in these glial tumors for any of the chromosome 19 loci examined. LOH for loci on chromosome 19 was detected in 23/74 informative astrocytomas (31%), 11/17 oligodendrogliomas (65%), and 3/8 MOA (38%). Partial deletion of chromosome 19 occurred more frequently (31/37 cases) than did loss of one whole copy of the chromosome, and a morphology-specific pattern of LOH was observed. In 12/14 (86%) instances of chromosome 19 deletion in oligodendrogliomas and MOA, the 19q arm showed LOH, whereas the 19p arm showed no loss for all informative loci. Conversely, in 17/23 (74%) instances of chromosome 19 deletion in astrocytomas, the 19p arm showed LOH, whereas the 19q arm showed no loss for one or more loci. Thus, loss of 19q and retention of 19p are strongly associated with oligodendroglioma and MOA, whereas loss of 19p and retention of distal 19q is associated with astrocytoma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539283 TI - Localization of the 8;13 translocation breakpoint associated with myeloproliferative disease to a 1.5 Mbp region of chromosome 13. AB - There are five reported cases of an atypical myeloproliferative disorder in which the leukemia cells have a consistent t(8;13)(p11;q12) translocation. We analyzed the breakpoint in metaphases from two of these patients by fluorescence in situ hybridization using a series of yeast artificial chromosomes (YACs) derived from the 13q12 region. We found that a YAC containing the FLT1 and FLT3 oncogenes was localized distal to the 13q12 breakpoint and was not rearranged. YAC66, a YAC that lies immediately adjacent to the chromosome 13 centromere, was localized proximal to the 13q12 breakpoint and was not rearranged. A third YAC, which is located between FLT1 and YAC66, was unrearranged in normal metaphase chromosomes, but showed hybridization signals on both derivative chromosomes in both cases. Thus, the breakpoints in these two cases are localized to the same 1.5 Mbp region of 13q12. This may be the site of an unidentified gene involved in the pathogenesis of some types of leukemia. PMID- 7539284 TI - Visualization of INT2 and HST1 amplification in oral squamous cell carcinomas. AB - Oral squamous cell carcinoma (OSCC) develops along a multistep genetic pathway including loss of tumor suppressor genes and alteration of oncogenes. We characterized seven OSCC cell lines by classical and molecular cytogenetic analysis and fresh tumor and adjacent oral mucosa corresponding to three of the cell lines by molecular cytogenetics. We observed homogeneously staining regions (hsrs) in four of the seven cell lines, at 11q13 in three and at 11q23 and in an unidentified marker chromosome in the fourth. Amplification of band 11q13 occurs in 30-60% of head and neck squamous cell carcinomas. To determine whether INT2 and HST1, both located in band 11q13, are amplified in the tissues and cell lines and to confirm the chromosomal location(s) of the amplification, we used dual color fluorescence in situ hybridization (FISH) with DNA probes for these genes and the chromosome 11 centromere. We report chromosomal localization of INT2/HST1 amplification in OSCC. Coamplification of INT2 and HST1 was detected in the hsrs in cultured tumor cells from the four hsr-containing tumors and in directly harvested tumor cells, which were available from only two of these tumors. Amplification was not present in tumors lacking hsrs or adjacent oral mucosa corresponding to any of the seven tumors. The observation of amplification in fresh tumor cells suggests that the amplification was present in the patients, may play a key role in the development and/or progression of OSCC, and is not due to karyotypic evolution in vitro. The absence of amplification in the adjacent mucosa suggests that 11q13 amplification is a relatively late event in OSCC tumorigenesis. PMID- 7539285 TI - Molecular characterization of MAR, a multiple aberration region on human chromosome segment 12q13-q15 implicated in various solid tumors. AB - Chromosome arm 12q breakpoints in seven cell lines derived from primary pleomorphic salivary gland adenomas were mapped by FISH analysis relative to nine DNA probes. These probes all reside in a 2.8 Mb genomic DNA region of chromosome segment 12q13-q15 and correspond to previously published sequence-tagged sites (STS). Their relative positions were established on the basis of YAC cloning and long range physical and STS content mapping. The 12q breakpoints of five of the cell lines were found to be mapping within three different subregions of the 445 kb DNA interval that was recently defined as the uterine leiomyoma cluster region of chromosome 12 breakpoints (ULCR12) between STS RM33 and RM98. All seven breakpoints appeared to map within the 1.7 Mb DNA region between STS RM36 and RM103. Furthermore, the chromosome 12 breakpoints of three primary pleomorphic salivary gland adenomas were also found to be mapping between RM36 and RM103. Finally, FISH analysis of two lipoma cell lines with 12q13-q15 aberrations pinpointed the breakpoints of these to relatively small and adjacent DNA segments which, as well as those of two primary lipomas, appeared to be located also between RM36 and RM103. We conclude from the observed clustering of the 12q breakpoints of the three distinct solid tumor types that the 1.7 Mb DNA region of the long arm of chromosome 12 between RM36 and RM103 is a multiple aberration region which we designate MAR. PMID- 7539286 TI - Loss of heterozygosity on 7q31 occurs early during breast tumorigenesis. AB - Although human breast tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumor suppressor genes, the identity of the genetic alterations that are critical in the early stages of the breast tumorigenic process remains obscure. A high frequency (27-41%) of loss of heterozygosity (LOH) occurrence has been shown at the MET locus on chromosome band 7q31 and this specific alteration is associated with poorer survival. Here, we report that restriction fragment length polymorphism (RFLP) analysis on 221 informative (heterozygous) primary breast tumors and 57 informative relapses (13 local recurrences and 44 distant metastases) revealed a similar frequency of 7q31 LOH as tumors progress from primary cancer to relapse, in marked contrast to other changes such as 11p15.5 LOH. This finding suggests that inactivation of a putative tumor suppressor gene located in 7q31 is a very early event in breast tumorigenesis. Our results also show that metastatic potential is an induced phenomenon that occurs at a relatively early stage, rather than a marker of tumor progression. PMID- 7539287 TI - Fusion of the EWS gene to a DNA segment from 9q22-31 in a human myxoid chondrosarcoma. AB - Southern blot analyses revealed a rearrangement of the EWS gene in a skeletal human myxoid chondrosarcoma. Interphase fluorescence in situ hybridization (FISH) studies, using cosmid clones F7 and G9 that flank the EWS locus on 22q12, confirmed the presence of this EWS gene abnormality. Cloning the rearranged EWS DNA fragment and mapping by FISH demonstrated that the EWS gene is joined to DNA sequences localised in 9q22-31. These findings are consistent with previous cytogenetic reports of a recurrent t(9;22)(q22-31;q11-12) in the myxoid variant of chondrosarcoma and reveal involvement of the EWS gene in a fourth type of human sarcoma. PMID- 7539288 TI - Serotonin, schizophrenia and antipsychotic drug action. AB - A rapidly growing body of data suggests that dysfunction in serotonergic (5-HT) function may be involved in the pathophysiology of schizophrenia, and that pharmacologic agents for this illness have their therapeutic effects mediated through serotonergic mechanisms. The purpose of this paper is to critically review data relevant to 5-HT's role in the pathophysiology and drug treatment of schizophrenia. Pathophysiologic evidence includes the psychotomimetic effects of lysergic acid (LSD), postmortem studies, single-dose 'challenge' studies and investigations of CSF and peripheral levels of 5-HT and its metabolites. The current nomenclature, potential therapeutic effects and importance of 5-HT receptor subtype antagonism will be examined. In addition, relatively novel strategies of 5-HT uptake blockade and direct acting 5-HT agonists will be assessed. A hypothesis of cortical-subcortical imbalance with an increase in subcortical 5-HT function responsible for positive symptoms and a decrease in prefrontal 5-HT function responsible for negative symptoms is proposed. Future implications of these data are discussed. PMID- 7539289 TI - Distribution of activity of hydrolytic enzymes in the digestive tract of rabbits. AB - Activities of twelve hydrolytic enzymes in the digestive tract of young rabbits before weaning (4 weeks old) and adult rabbits (3 months old) were measured. The principal digestive enzymes in both groups of rabbits appeared to be amylase (EC 3.2.1.1), maltase (EC 3.2.1.20), pectinase (EC 3.2.1.15) and proteinases. The stomach of young rabbits contained most of the lipolytic activity and 45.7% of the total proteolytic activity of the digestive tract. The highest specific activities (per g digesta) of amylase, maltase and proteinase in young rabbits were found in the small intestine. Total activities (per segment) of amylase and maltase in the small intestine and the caecum were similar. Activities of cellulase (EC 3.2.1.4), inulinase (EC 3.2.1.7) and beta-glucosidase (EC 3.2.1.21) were low and activity of pectinase was fairly high in all segments of the digestive tract. The highest activity of urease (EC 3.5.1.5) was found in the caecum. Enzymic profiles of the colonic chymus resembled those of the caecum. Total hydrolytic activity was lower in the colon than in the caecum. Specific activities of amylase and invertase (EC 3.2.1.26) were lower and those of inulinase and lactase (EC 3.2.1.23) higher in 4-week-old rabbits than in 3-month old rabbits. Gastric proteinase represented almost half of the total proteolytic activity of the digestive tract, whereas lipolytic activity of gastric contents was not found in measurable quantities in adult rabbits. The caecal contents of adult rabbits contained most of the total activity of lipase (EC 3.1.1.3), cellulase, xylanase (EC 3.2.1.32), pectinase, lactase, invertase, beta glucosidase and urease present in the digestive tract.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539290 TI - [Ocular manifestations in progeria]. AB - The observation of a 17-year-old teenager with progeria, ventricular septal shortcoming with wide anterolateral [correction of arteriolateral] myocardial ischemia. From an ocular point of view, he shows senescence with senile ectropion, evolutive cataract, retinal angiosclerosis. The role of the manifestation of ocular senescence in Hutchinson-Gilford Syndrome is emphasized. PMID- 7539292 TI - Non-sialate inhibitor of influenza A/WSN/33 neuraminidase. AB - An N1 strain of influenza A virus neuraminidase (A/WSN/33 NA) was purified and used to screen for inhibitors. As a result, a well-known tuberculostatic, 4' formylacetanilide thiosemicarbazone (or thiacetazone), was identified. Thiacetazone is a non-sialate compound and inhibits the enzyme in a noncompetitive manner with respect to the substrate sialic acid. Mechanistic studies indicate that the inhibition was due to the competition of thiacetazone with Ca2+, which maintains N1 neuraminidase in an active conformation. The Ki for the inhibition was estimated to be about 4 microM. Equilibrium exchange experiments revealed that when purified A/WSN/33 NA was incubated with 5 microM 45CaCl2, 2 mol of 45Ca2+ ion was exchanged into each mole of NA tetramer and subsequently displaced from the enzyme upon the introduction of the inhibitor. Inhibition of plaque formation by thiacetazone in an MDCK cell culture that had been infected with the influenza A/WSN/33 virus was demonstrated. Thiacetazone was highly specific for A/WSN/33 neuraminidase, since little effect was noted when it was tested against NAs from the other strains of influenza virus or from bacteria. This compound might represent a group of non-sialate inhibitors of influenza NA that bind to a noncatalytic or an allosteric site on the enzyme. PMID- 7539291 TI - Tetrahydrobiopterin-deficient nitric oxide synthase has a modified heme environment and forms a cytochrome P-420 analogue. AB - Optical absorption and resonance Raman spectra of neuronal nitric oxide synthase (b-NOS) isolated in the absence of tetrahydrobiopterin demonstrate that the enzyme preparation is very unstable. This unstable form of the enzyme has properties analogous to those of cytochrome P-420cam, an inactive form of cytochrome P-450cam. Although cysteine is preserved as the proximal ligand in both the ferric and ferrous forms of unstable b-NOS, the lack of tetrahydrobiopterin significantly increases the hexacoordinate low-spin fraction of the heme content, resulting in a loss of the enzymatic activity. Upon the addition of CO, the unstable b-NOS converts from a species exhibiting a Soret absorption maximum at 443 nm, as reported for the CO adducts of stable b-NOS and cytochrome P-450cam, to a species with a Soret maximum at 421 nm. The resonance Raman spectrum of the 421-nm form is the same as those of CO-bound myoglobin at low pH and CO-bound cytochrome P-420cam. The heme in this form of the enzyme is coordinated by a weaker ligand than thiolate; histidine coordination in the CO bound form of the P-420-like species of NOS is consistent with all of the available data. A similar unstable form of the macrophage (i-NOS) enzyme was also detected. Not only does the heme pocket of NOS have the same coordination as cytochrome P-450 in its stable form, but the partially denatured form has the same properties as cytochrome P-420, the inactive form of cytochrome P-450. Among other possible roles, tetrahydrobiopterin may play a significant role in the stabilization of the active enzyme. PMID- 7539293 TI - Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase. AB - In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT. PMID- 7539294 TI - A new approach to examine conformational changes occurring upon binding of ligand by biomolecules. AB - Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques. PMID- 7539295 TI - Expression of the human complex-forming glycoprotein HC (alpha 1-microglobulin) in Escherichia coli. AB - The mature form of human protein HC, or alpha 1-microglobulin, has been expressed in Escherichia coli. Protein HC is a member of the lipocalin superfamily of hydrophobic ligand-binding proteins, and carries a heterogeneous chromophore linked covalently by a reduction-resistant bond. Protein HC was first overexpressed as a C-LytA/HC fusion protein containing the C-terminal moiety of the pneumococcal lytic amidase (LytA). Recombinant C-LytA/HC was found to be an insoluble aggregate that was solubilized with 6 M guanidinium chloride and renatured by the addition of thiol reagents in the presence of L-arginine. Recombinant protein HC (rHC) was released from C-LytA/HC by trypsin digestion and purified by size-exclusion chromatography. rHC protein possesses an N-terminal amino-acid sequence identical to that of human protein HC, and a slightly lower molecular mass as determined by SDS-PAGE. Both C-LytA/HC and rHC reacted with polyclonal antibodies raised against native protein HC. A photodiode array detection system on-line with a HPLC system has allowed the identification of a chromophore associated to rHC protein displaying significant absorption in the visible region of the spectrum in resemblance to that found in the natural form of human protein HC. PMID- 7539297 TI - A comparative study of the interaction of Myocrisin with albumin and gamma globulin. AB - The reactions of Myocrisin with albumin and gamma-globulin have been investigated using atomic absorption spectrometry and gel filtration. Albumin rapidly binds gold up to the levels predicted from the concentration of free sulphydryl groups present in the protein. However, in the presence of glutathione, gold incorporation is increased, suggesting that in vivo, free thiols (glutathione, thiomalate) may facilitate gold uptake by the protein. In comparison, gamma globulin is found to be capable of binding up to one atom of gold per molecule of protein in a slow reaction which requires high Myocrisin concentrations. PMID- 7539298 TI - Maternal dietary carbohydrate restriction influences the developmental profile of postnatal rat brain indoleamine metabolism. AB - Dietary glucose restriction during pregnancy can retard fetal brain development, lower term brain glycogen levels and adversely affect the serotonergic neurotransmitter system in the fetus. To study if the postnatal profile of brain indoles continues to respond to these diet-induced changes, pregnant rats were fed graded levels (0, 12, 24, 60%) of glucose from impregnation to day 15 postpartum, and neonatal brain measurements were made. A steady decrease in tryptophan levels, a steady increase in 5-hydroxytryptamine (5-HT) levels and a U shaped change in 5-hydroxyindoleacetic acid (5-HIAA) were observed during the first 15 postpartum days. Superimposed on these development profiles was a temporary surge in the concentrations of all three indoles 24 h after birth, which was dramatic for tryptophan and more modest for 5-HT and 5-HIAA. The level of carbohydrate in the maternal diet significantly influenced the magnitude of this increase in tryptophan, 5-HT and 5-HIAA at 24 h: the values were significantly higher in the carbohydrate-restricted (12 or 24%) rat pups when compared with control or carbohydrate-free (0% glucose) offspring. No effects of dietary treatment were apparent by day 6. However, the reemergence of a significant difference in brain 5-HT content at day 15 postpartum indicates that even when energy intake is adequate the level of carbohydrate in the maternal diet may continue to play a role in modulating serotonergic neurotransmitter levels later in development. PMID- 7539296 TI - Protein tyrosine phosphorylation as a mechanism which regulates cytokine activation of early response genes. AB - Two well-defined rapid responses which occur as a consequence of growth factors binding to their cell surface receptors involve tyrosine phosphorylation of cellular proteins and the induction of the transcription of cellular genes. Recent advances have been made in purification and cloning of Src homology 2 and 3 (SH2/SH3) domain-containing transcription factors which are required for the activation of early response genes by interferons. These transcription factors are covalently modified by tyrosine phosphorylation such that they interact with enhancers needed for interferon-stimulated gene expression. The Jak family of tyrosine kinases are also an integral component in these signalling cascades. The information gained concerning interferon signalling has now been extended to include a broad network of cytokine-regulated signalling systems which use tyrosine phosphorylation of a family of structurally related proteins to activate transcription of early response genes. PMID- 7539299 TI - [Approach to benign hypertrophy of the prostate in primary health care]. PMID- 7539300 TI - Evaluating multiple diagnostic tests with partial verification. AB - To evaluate diagnostic tests, one would ideally like to verify, for example, with a biopsy, the disease state of all subjects in a study. Often, however, no all subjects are verified. Previous methods for evaluation assume that the decision to verify depends only on recorded variables. Sometimes, particularly if the disease process is not well understood, the decision to verify may also depend on unrecorded variables related to disease. We propose a method to estimate the true and false-positive rates of multiple tests while adjusting for the effect, on the decision to verify, of unrecorded variables related to disease. To put the estimates into a more usable form, we develop a simple algorithm for creating a receiver-operating curve which maximizes the true-positive rate, given the false positive rate. We apply the methodology to data on the early detection of prostate cancer using ultrasonography, digital rectal exam, and prostate specific antigen. PMID- 7539301 TI - Exogenous testosterone alters expression pattern of cell recognition molecules in brains of juvenile zebra finches. AB - We assessed spatio-temporal expression of tenascin and janusin by immunocytochemistry in testosterone-treated zebra finches and in untreated controls. These cell recognition molecules and components of the extracellular matrix mediate neurone-glia interactions, which are crucial for differentiation of ordered neural structures. We describe boundaries for the spatio-temporal expression of cell recognition molecules that highly correlate with the song motor centres as defined by combined neuroanatomical and ethological studies on song vocalization. The expression of janusin is accelerated by exogenous testosterone. PMID- 7539302 TI - Kindling induces a long-lasting increase in brain nitric oxide synthase activity. AB - Generalized seizures induced by kindling are associated with a long-term increase in vasopressin mRNA expression in vasopressin neuroendocrine cells. Since nitric oxide synthase activity is strongly expressed in these neurons and may play a role in mechanisms of plasticity, we used NADPH-diaphorase histochemistry to examine nitric oxide synthase activity 1 month after amygdala kindling. Both the number of stained neurons and average intensity of cellular labeling in the supraoptic nucleus were increased in the kindled rats. In adjacent limbic regions, terminal-like staining was also increased, suggesting a general elevation of limbic nitric oxide synthase activity. Thus, increased nitric oxide production may play a role in sustaining the increase in vasopressin mRNA and plastic changes in the amygdala associated with kindling. PMID- 7539303 TI - Callosal neurones give rise to zinc-rich boutons in the rat visual cortex. AB - Sodium selenide was used as a retrograde tracer to assess the callosal origin of zinc-rich boutons in the neocortex of the rat. Selenide injections were placed in the lateral end of area Oc1 (area 17). Zinc present in synaptic boutons precipitated as zinc selenide and was transported retrogradely to the parent cell bodies. In addition to the ipsilateral labelling, contralateral retrogradely labelled somata were mainly observed along the border between areas Oc1 and Oc2L (area 18a). Labelled neurones were present in layer 2-3 and layer 6. In contrast to tracing studies with peroxidase, no labelled neurone was observed in layers 4 and 5 except at the inner border of layer 5. This study reveals the chemical heterogeneity of callosal projections, which may be divided into zinc-rich and zinc-poor systems. PMID- 7539305 TI - Inhibitory effect of arcaine on nitric oxide synthase in the rat brain. AB - Nitric oxide is synthesized by nitric oxide synthase (NOS) from L-Arg, which contains a guanidino group. Arcaine is the diguanidino compound and a derivative of Arg. This study was conducted to investigate the effects of arcaine on rat brain NOS activity, using nitrite and nitrate as indicators. Arcaine inhibited NOS activity in a linear mixed manner (K1 = 18.68 microM). Almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, but the guanidino nitrogens of arcaine were not substituted. Arcaine was also reported to be a competitive antagonist of the polyamine site on the N methyl-D-aspartic acid (NMDA) receptor. Arcaine appears to be an excellent drug to investigate not only the chemical nature of NOS but also the functional and structural relationship between NOS and NMDA receptors. PMID- 7539304 TI - Single glutamate channels in CA1 pyramidal neurones after transient ischaemia. AB - Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones. PMID- 7539306 TI - Involvement of inositol trisphosphate in cerebellar long-term depression. AB - We examined the role of increases in Ca2+ from different sources in the induction of long-term depression (LTD) of glutamate or AMPA responsiveness in cultured Purkinje neurones. Photolysis of caged Ca2+ or caged inositol 1,4,5-trisphosphate (InsP3) as well as depolarization was used to increase Ca2+ concentration. Heparin, contained in a patch pipette to block InsP3 binding to its receptor, prevented LTD induction by coupling of glutamate application and depolarization. Although pairing of depolarization and AMPA application did not induce LTD, photolysis of caged InsP3 in conjunction with depolarization and AMPA application induced LTD. The results suggest that not only Ca2+ influx through voltage-gated Ca channels but also InsP3-induced Ca2+ mobilization are involved in LTD induction. PMID- 7539308 TI - Methods for studying neurotransmitter transduction mechanisms. AB - This review describes the methodologies used to study the transduction mechanisms that are activated in excitable cells by G-protein-coupled agonists. In view of the complexity of second-messenger systems, it is no longer relevant to ask, "What is the transduction mechanism involved in the action of a given neuromodulator?" because, in many cases, a variety of transduction mechanisms and physiological responses are invoked following receptor activation. This means that a single aspect of the physiological response must be selected for study in order to address the question of transduction mechanism. This review is therefore concerned with a description the use of patch- and voltage-clamp procedures to study transduction mechanism because they are designed to isolate one aspect of the physiological response: the change in activity of a single type of membrane ion channel. PMID- 7539309 TI - The phenylalanine response curve in relation to growth and mental development in the first year of life. AB - Thirty-three children with classical phenylketonuria were treated from 9 to 21 days of age onward. The standard for phenylalanine concentration was 0.2-0.5 mmol/l. During the first years of life phenylalanine concentrations were measured once every two to six weeks. For each child these values were graphically plotted in a so-called phenylalanine response curve. Growth and mental development indices were assessed at about one and two years of age. Measurements for weight, height and head circumference were normal at one month of age, showed a significant decline at the age of one year but were restored for normal values at the age of two. No associations were found with phenylalanine parameters. Mental development indices at one and two years were not different from normal. Significant negative correlations were found between mental development indices at one year and phenylalanine values above 0.5 mmol/l. Changes in mental development indices between the first and second years of life are significantly related to the phenylalanine levels during the second year of life. PMID- 7539307 TI - Impaired splicing of phytochrome B pre-mRNA in a novel phyB mutant of Arabidopsis. AB - Phytochrome is the red/far-red absorbing photoreceptor active in photomorphogenesis, the apoprotein of which is encoded by a small gene family (PHYA, PHYB, PHYC, PHYD and PHYE). A novel phytochrome B-deficient mutant, phyB 103, was isolated from a screen of EMS-mutagenised Arabidopsis M2 seed. phyB-103 carries a G-to-A base substitution at the 5' splice site +1 G nucleotide of intron 1 of PHYB. The phyB-103 PHYB transcript is larger than the wild-type PHYB transcript and DNA sequence analysis showed that the entire intron is retained in the mature PHYB transcript of phyB-103. Thus the phyB-103 G-to-A substitution prevents intron splicing. The retained intron contains within it an in-frame stop codon, and the predicted PHYB-003 apoprotein thus terminates prematurely. phyB 103 is therefore likely to be a null allele of PHYB, consistent with the observation that the phenotype conferred by phyB-103 is as severe as that conferred by previously described phyB null alleles. PMID- 7539310 TI - The rat athymic nude (rnu) locus is closely linked to the inducible nitric oxide synthase gene (Nos2). PMID- 7539311 TI - Endothelial nitric oxide synthase (Nos3) maps to the proximal region of mouse chromosome 5. PMID- 7539312 TI - Organochlorine pesticide contaminants in human adipose tissue collected in Tebriz (Iran). PMID- 7539313 TI - Organochlorine pesticide residue concentrations in biota and sediments from Rio Palizada, Mexico. PMID- 7539314 TI - Changes induced in the gills of milkfish (Chanos chanos Forsskal) fingerlings after acute exposure to nifurpirinol (Furanace; P-7138). PMID- 7539315 TI - Bioethics in the news ... (with commentary). PMID- 7539316 TI - Sarcolemmal indentation in cardiomyopathy with mental retardation and vacuolar myopathy. AB - Muscle biopsies from three patients with cardiomyopathy, mental retardation and increased serum creatine kinase levels revealed scattered fibers with tiny intracytoplasmic vacuoles containing basophilic and acid phosphatase-positive material and slightly increased amounts of PAS-positive granules. These findings are consistent with those seen in the so-called lysosomal glycogen storage disease with normal acid maltase. In addition to the vacuoles, there were occasional folds or indentations in the sarcolemma which were connected to the membrane enclosing the vacuoles. These membranes were well demonstrated histochemically by the nonspecific esterase and acetylcholinesterase stains. On electron microscopy, most of the vacuoles were bounded by membranes with basal lamina. The vacuolar membrane stained positively with antibodies raised to dystrophin, dystrophin-associated glycoproteins, laminin and type 4 collagen, and it was identical to the sarcolemma and its basal lamina. Therefore, the membrane abnormality which causes sarcolemmal folding is probably critical to understanding the pathomechanism of this disease. PMID- 7539317 TI - Clinical tumour markers in lung cancer. AB - Within the past few years, the measurement of serum and tissue markers has had an increasing influence on clinical decisions about initial treatment and follow-up. Lung cancer illustrates the types and importance of these various markers. This review presents data concerning the most studied and interesting markers in non small cell (NSCLC) and small cell lung cancer (SCLC). CEA, TPA, SCC-Ag, CYFRA 21 1, ferritin, CA19-9, CA50, CA242, H-K-N-ras mutations and p53 mutation seem to be the most prolific in NSCLC, while NSE, BN/GRP, CK-BB, NCAM, IL-2R, IGF-I, transferrin, ANP, mAb (cluster 5), Le-y and c-N-L-myc mutation are markers in SCLC patients. Some of these serum markers might be useful adjuncts for monitoring response to therapy, including early detection of tumour reactivation to allow curative therapy and rapid detection of treatment failure to allow change of the regimen. The study of these markers also may lead to a better understanding of the biological characteristics of lung cancer. The information derived from these biological studies represents the most promising avenue towards new treatment strategies, as well as attempts at secondary prevention. PMID- 7539319 TI - Using a matrix to communicate quality progress. PMID- 7539318 TI - Autologous stem cell transplantation as adjuvant treatment vs no further therapy for poor-risk Hodgkin's disease in first complete remission after MOPP/ABVD. PMID- 7539320 TI - Getting the most out of poster presentation. PMID- 7539321 TI - Labelling of polysaccharides using [11C]cyanogen bromide. In vivo and in vitro evaluation of 11C-hyaluronan uptake kinetics. AB - A method for the 11C-labelling of polysaccharides in high specific radioactivity is described. Dextran and hyaluronan were treated with [11C]cyanogen bromide in aqueous solution at pH 11.5 to give 30-47% radiochemical yields with higher than 98% radiochemical purity in synthesis times of 24-26 min counted from the end of bombardment. Specific radioactivities at the end of synthesis ranged from 0.12 to 3.1 Ci/mumol. The biodistribution kinetics of [11C]hyaluronan injected intravenously was studied in rats by means of positron emission tomography, showing a rapid and displaceable uptake in liver. Uptake and displacement of [11C]hyaluronan was also demonstrated in cultured rat liver endothelial cells. PMID- 7539322 TI - Hypergammaglobulinemic purpura of Waldenstrom associated with systemic lupus erythematosus: report of a case and review of the literature. AB - A patient with hyperglobulinemic purpura of Waldenstrom and systemic lupus erythematosus is reported. The coexistence of these two conditions which share a number of common clinical and laboratory features is rare. Treatment of the patient with prednisone, colchicine and hydroxychloroquine led to the improvement of the cutaneous vasculitis and a drop in ESR, serum gamma globulins and IgM and IgG rheumatoid factors. The features of nine other cases and the immunopathogenesis of the disease are reviewed. PMID- 7539323 TI - Heparan sulphate-ELISA gives false positive results for anti-DNA-DNA/histone immune complexes in sera of patients with SLE. AB - Heparan sulphate-reactive antibodies in lupus sera have been suggested to be anti DNA-DNA/histone immune complexes and to be associated with lupus nephritis. In this study, 23 anti-DNA-positive lupus sera including 13 active nephritis sera were tested for the presence of circulating anti-DNA-DNA/histone immune complexes by solid phase heparan sulphate-ELISA. Because of high background binding to protamine chloride-linked heparan sulphate plates, poly-L-lysine (PLL) was used as a linker and the remaining active sites of PLL were blocked with poly-L glutamic acid. The ELISA was capable of detecting small amounts of anti-DNA IgG DNA/histone immune complexes formed in vitro. However, only three active nephritis sera of the 23 sera tested showed significant binding to heparan sulphate plates. This binding was found to be non-specific, the result of high background binding of IgG to PLL. Anti-heparan sulphate ELISA using positively charged linkers detects non-specific binding when lupus sera are tested. Specific assays need to be developed for DNA/histone-related immune complexes present in lupus sera. PMID- 7539324 TI - Separation of transfer RNA and 5S ribosomal RNA using capillary electrophoresis. AB - Capillary gel electrophoresis and capillary electrophoresis using entangled polymer solutions was investigated for their applicability for the separation of low-molecular-mass RNAs (transfer RNA and 5S ribosomal RNA), with a size range of 70-135 nucleotides, from bacteria. Cross-linked polyacrylamide gel-filled capillaries (3 and 5%) were used for capillary gel electrophoresis. Good resolution was obtained using gel-filled capillaries only for small tRNAs with lengths to 79 nucleotides, larger tRNAs and 5S rRNA could not be resolved using this method. Buffers containing sieving additives were employed to improve separations of RNA by capillary electrophoresis using entangled polymer solutions. The use of linear sieving polymers in buffers resolved 5S rRNA and tRNAs, even when they possessed only different secondary structure or small differences in length (1-5 nucleotides). PMID- 7539325 TI - Astrocytes upregulate glial fibrillary acidic protein (GFAP), but not insulin like growth factor-I (IGF-I) during experimental autoimmune neuritis (EAN). AB - T cell-mediated autoimmune neuritis produces rapid activation of spinal cord microglia. To determine whether this microglial response upregulates astrocytic expression of IGF-related proteins, we induced EAN and used in situ hybridization and immunocytochemistry to examine the mRNAs and peptides for glial fibrillary acidic protein (GFAP), insulin-like growth factor-I (IGF-I), IGF-I receptor (IGFR I) and IGF binding protein-2 (IGFBP-2). Relative levels of GFAP mRNA and peptide were highest in the lumbar spinal cord 4-10 d following T cell transfer and significant GFAP elevations were still present after three weeks. The astrocytes expressing GFAP mRNA and peptide were localized around motoneurons which were related topographically to axons in peripheral nerve inflammatory lesions. In the nucleus gracilis, where terminals of dorsal root ganglion neurons are located, astrocytic levels of GFAP mRNA and peptide rose later and did not reach their highest levels until 21 d after T cell transfer. Even though microglia were activated in both locations 2-4 d after transfer, astrocytic levels of IGF-I, IGFR-I and IGFBP-2 mRNA and peptide did not differ significantly from those observed in controls. The dissociation of GFAP and IGF-I expression in EAN suggests that these astrocytic responses may be independently regulated. We also suggest that the type and severity of remote neuronal injury are probably more important inducers and regulators of these astrocytic responses than microglial cell activation. PMID- 7539326 TI - Modulation of cytokine-induced eosinophil infiltration by phosphodiesterase inhibitors. AB - The effects of selective phosphodiesterase (PDE) isoenzyme inhibitors on eosinophil airway infiltration induced by intratracheal administration of recombinant human cytokines were investigated in the guinea pig. Recombinant human IL-5 and IL-8 elicited a concentration-dependent increase in the number of eosinophils in the bronchoalveolar lavage (BAL) fluid. In contrast, no effect was observed after intratracheal injection of recombinant human IL-3 or recombinant human RANTES. Pretreatment with the PDE IV inhibitors rolipram or Ro 20-1724 or the nonselective PDE inhibitor theophylline 1 h before intratracheal injection of IL-5 significantly reduced the number of eosinophils in the BAL fluid at 48 h. In contrast, the selective PDE III inhibitors milrinone and SK&F 94-836 and the PDE I/V inhibitor zaprinast did not inhibit the airway eosinophil infiltration induced by IL-5. Betamethasone also significantly inhibited the IL-5-induced eosinophil infiltration in BAL fluid. Administration of rolipram or betamethasone 1 h before IL-8 significantly reduced airway eosinophil infiltration. Because the selective PDE IV inhibitors markedly inhibited eosinophil infiltration in guinea pig airways induced by cytokines, it is suggested that PDE IV inhibitors have antiinflammatory effects in the airways and may be useful in the treatment of asthma. PMID- 7539327 TI - Soluble form of P-selectin in plasma is elevated in acute lung injury. AB - A number of adhesion molecules on neutrophils and the pulmonary capillary endothelium mediate the neutrophil accumulation in the lungs at the onset of adult respiratory distress syndrome or acute lung injury (ALI). P-selectin, located on both vascular endothelial cells and platelets, has been shown to be one of these neutrophil-endothelial cell adhesion molecules. In this study, we measured the soluble form of P-selectin in plasma (PPS) from 19 patients (surviving, 11; deceased, 8) with ALI due to various causes and assessed the clinical significance of this measurement. Twelve healthy subjects and 29 patients with other pulmonary diseases, including idiopathic pulmonary fibrosis (IPF) (n = 8), sarcoidosis (n = 5), pneumonia (n = 8), and sepsis without ALI (n = 8) were also studied for comparison. PPS in patients with ALI (474.5 +/- 366.8 ng/ml, mean +/- SD) were significantly higher than those in control subjects (98.8 +/- 39.7, p < 0.01) and in patients with IPF (210.4 +/- 76.6, p < 0.05), sarcoidosis (135.2 +/- 71.5, p < 0.05), pneumonia (225.3 +/- 81.0, p < 0.05), and sepsis without ALI (271.8 +/- 46.5, p < 0.05). There was no significant difference in PPS levels between seven patients with and 12 patients without multiple organ failure. Lung injury scores correlated significantly with the PPS level (r = 0.605, p < 0.05). PPS levels of deceased patients with ALI (841.0 +/- 252.4) were significantly higher than those of surviving patients with ALI (208.0 +/- 109.2, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539328 TI - [Prenatal diagnosis of spina bifida and abdominal wall defects]. PMID- 7539329 TI - Non-viral approaches to gene therapy. AB - There have been rapid advances in the development of non-viral techniques for gene transfer. Although viruses are highly evolved to infect mammalian cells, they have several limitations. The general theme is to mimic the advantageous components of viral systems whilst separating them from their limiting functions. The systems described here are broadly divided into true non-viral techniques and viral-assisted technologies. The merits and limitations of each method are presented, and examples of their application to current developments in gene therapy are discussed. At present no preferred technique has emerged as a clear favourite for non-viral delivery. Further refinement of the technology with particular emphasis on achieving long-term gene expression is required before initiating clinical trials of non-viral gene delivery. PMID- 7539330 TI - Deletion and insertion mutations in short tandem repeats in the coding regions of human genes. AB - In vitro studies in bacterial, yeast and eukaryotic systems have demonstrated the existence of deletion and insertion 'hot-spots' involving repetitive sequences. Slipped-strand mispairing (SSM) has been suggested to be the mechanism involved. Progress in human molecular genetics has allowed the identification of many mutations causing diseases. Analysis of sequences involved in these mutations provides an opportunity to investigate the contribution of short tandem repeats to the naturally occurring mutations in coding regions of human genes. We have analyzed the sequences surrounding 625 disease-causing mutations in the coding regions of three genes: the cystic fibrosis transmembrane conductance regulator, beta globin and factor IX. Altogether, 134 (21%) insertion and deletion mutations of 4 base pairs or less were identified. In 47% of these mutations, the deletions and insertions occurred within a unit repeated tandemly 2- to 7-fold. These were classified as SSM mutations. The proportion of SSM mutations was significantly higher than expected by chance. The estimated net proportion of deletion and insertion mutations attributed to SSM was 27%. These results indicate that very short repetitive sequences contribute significantly to the generation of deletion and insertion mutations in human genes, and to the evolution of diversity of their coding regions. PMID- 7539331 TI - The B lymphocyte as an immunotherapeutic target. PMID- 7539333 TI - News of the paper pelvis. PMID- 7539332 TI - Anatomic relationship between substance P- and CGRP-immunoreactive nerve fibers and mast cells in the palatal mucosa of the rat. AB - The study describes the distribution of mast cells and of substance P (SP) and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the rat palatal mucosa, focusing on the anatomic relationship between these tissue elements. The maxilla of 10-14-wk-old rats was dissected free, fixed, demineralized and frozen. Consecutive sections were stained with avidin peroxidase or processed for immunohistochemistry. In order to define the correlation between nerve fibers and mast cells, double staining techniques were used. The distance between each avidin-positive mast cell and the nearest detectable nerve fiber was determined. 5-Hydroxytryptamine- (5-HT) and avidin peroxidase-positive mast cells were frequently seen in the palatal mucosa but were rarely found in the gingival area. A large number of nerve fibers showing SP and CGRP-like immunoreactivity were seen, particularly in association with blood vessels. Some nerve fibers were located in contact with or very close to the mast cells but the vast majority of mast cells showed no close anatomic association to nerve fibers. The nerve fibers and mast cells were mainly concentrated to the same regions in the palatal mucosa where blood vessels occurred. The observations suggest that in the rat palatal mucosa the main functional relationship relates to SP/CGRP and the blood vessels, and only to a minor degree to SP/CGRP and mast cells. PMID- 7539334 TI - Relationship of the expression of the S20 and L34 ribosomal proteins to polyamine biosynthesis in Escherichia coli. AB - Polyamine biosynthesis in Escherichia coli is regulated transcriptionally and post-translationally. Antizyme and ribosomal proteins S20 and L34 participate in post-translational inhibition of the polyamine biosynthetic enzymes ornithine and arginine decarboxylase. The aim of the present study was to investigate the significance of S20 and L34 in polyamine regulation in vivo. In vivo overexpression of S20 and L34 lowered the activities of ornithine and arginine decarboxylases and decreased total polyamine production. The levels of cadaverine, a related diamine whose synthesis is not regulated by S20 and L34, did not decrease but increased. The diminished ornithine and arginine decarboxylase activities are shown to result from reversible post-translational inhibition since the enzymes could be reactivated to normal levels upon titration of the inhibitors. The effects were specific as overexpression of eight other ribosomal proteins had no influence. Overexpression of ornithine decarboxylase results in elevated polyamine production and it increases S20 and L34 levels but not those of other ribosomal proteins. Ornithine depletion decreases S20 and L34 to normal levels in the ornithine decarboxylase overproducing cells. Immunoprecipitation experiments coupled with immunoblots indicated that ornithine and arginine decarboxylases physically interact with S20 and L34. This study shows that ribosomal proteins S20 and L34 can inhibit ornithine and arginine decarboxylases and polyamine biosynthesis in vivo. It is concluded that, unlike other basic ribosomal proteins and polycationic compounds which inhibit the activities of these enzymes only in vitro, S20 and L34 are biologically relevant in the regulation of the polyamine biosynthetic pathway. PMID- 7539336 TI - Nitric oxide synthase induction and cytoprotection of rat gastric mucosa from injury by ethanol. AB - The present study determined the effects of nitric oxide (NO) synthase induction on ethanol-mediated damage to rat gastric mucosa. NO synthase activity was determined by [14C]arginine conversion to radiolabeled citrulline. Ca(2+) independent NO synthase activity was determined by citrulline formation in the presence of EGTA (1 mM) in the incubation mixture. Intraluminal ethanol administration (2 mL; 40% w/v) to control rats resulted in an increase in mucosal damage characterized as vasocongestion and hemorrhagic necrosis and a reduction in Ca(2+)-dependent NO synthase activity. Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg i.v.) augmented Ca(2+)-independent NO synthase activity (determined 4 h later) and reduced damage in response to intraluminal ethanol instillation. Ethanol treatment did not significantly affect induction of NO synthase activity. Dexamethasone pretreatment (1 mg/kg i.v. 2 h before LPS administration) reduced both Ca(2+)-independent NO synthase activity and the gastroprotective effect of LPS against ethanol-mediated mucosal injury. Likewise, concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg s.c.) inhibited the gastroprotection associated with LPS treatment, an effect abolished by pretreatment with the NO substrate L-arginine (300 mg/kg s.c.). Indomethacin (5 mg/kg i.v.) was ineffective in suppressing LPS mediated gastroprotection. These results suggest that while Ca(2+)-dependent NO formation is inhibited by ethanol treatment, the inducible Ca(2+)-independent NO synthase plays a role in LPS-mediated gastroprotection against ethanol-mediated damage to the gastric mucosa. PMID- 7539335 TI - Acute phase-dependent changes in the binding of rat liver nucleoproteins to the cytokine response element of the rat haptoglobin gene. AB - Hormones released during the acute phase reaction promote the transcriptional activation of the haptoglobin (Hp) gene and a consequent increase of Hp protein synthesis in the liver. The mechanisms underlying the alterations of basal transcription rates of eukaryotic genes are assumed to result from modulations of the binding affinities between nucleoproteins and specific DNA sequences in the enhancer and promoter elements. In order to characterize the changes in the interaction of nucleoproteins with the promoter that accompany the induction of the Hp gene, nuclear extracts from normal and inflamed livers were probed with hormone responsive element (HRE) of the rat Hp gene by gel mobility shift and Southwestern assays. Each of the three cis-acting sequences of the HRE, elements A, B, and C, recognized a distinct set of proteins. Together they conferred an additional level of specificity to the protein binding sites of the entire ABC element. These sites were recognized by proteins in liver nuclear extracts isolated from both control and treated rats. The differences in the gel shift and Southwestern patterns of the corresponding DNA-protein complexes suggested that transcriptional activation of the Hp gene relied on changes in the concentrations and/or functional modifications of preexisting proteins rather than on the induction of new trans-acting factors. PMID- 7539337 TI - Expression of cell adhesion molecules on ovarian tumour cell lines and mesothelial cells, in relation to ovarian cancer metastasis. AB - A major route for the spread of ovarian cancer is by the attachment of tumour cells to the mesothelium lining in the peritoneal cavity. The expression of various adhesion molecules has been measured on freshly-prepared mesothelial cells, two mesothelial cells lines and 13 established ovarian tumour cell lines. The integrins beta 1 and beta 3, ICAM-1, and CD44 were detected on all mesothelial preparations and on many or all of the tumour lines. VCAM-I was expressed exclusively on the mesothelial cells and Lewis x was expressed on half of the tumour lines. There was low or no expression of sialyl Le(x), sialyl Le(a), integrins alpha 4, beta 1, beta 4, or E and P selectins. Only CD44 expression was significantly affected by trypsin treatment. From the known interactions of adhesion molecules, the results suggest that CD44, and beta 1 and beta 3 integrins may be important in tumour/mesothelial interactions. PMID- 7539338 TI - Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis. AB - Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-alpha (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6 x DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was covered rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrate, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71 +/- 0.04 l/kg and the clearance was 0.32 +/- 0.02 l/h-1/kg-1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo. PMID- 7539339 TI - Treatment of advanced, high-grade soft-tissue sarcoma with ifosfamide and continuous-infusion etoposide. AB - A total of 33 patients (median age, 44 years) with high-grade, adult soft-tissue sarcoma were treated with etoposide given at 600 mg/m2 in a 72-h continuous infusion and ifosfamide given at 1500 mg/m2 per day for 3 days every 3 weeks. Dose escalation/reduction was protocolled depending on the level of hematological toxicity observed in the preceding course. Overall, 90% of patients had metastatic disease, and the most common histologies were malignant fibrous histiocytoma and leiomyosarcoma. A median of 5 (range, 1-9) courses were given. Of 30 patients who were evaluable for response, 12 (40%) obtained a partial remission, and the median time to progression was 8 (range, 4-13) months. Grade 3 4 leukopenia and thrombocytopenia were seen after 89% and 8% of the courses, respectively; neutropenic fever was seen in half of the patients (15% of courses); and 32% of courses had to be postponed by 7 days or more due to myelosuppression. Dose reduction to below the standard had to be performed in 46% of courses, and dose escalation was achieved in only 13%. The reduced toxicity seen after the addition of granulocyte colony-stimulating factor (G-CSF) in five patients indicates that growth-factor support may enhance the dose intensity of the regimen. The results indicate significant activity for this regimen in adult soft-tissue sarcoma, which may in part be a result of the escalated dose and prolonged mode of administration of the phase-specific agent etoposide. As a result of this pilot series, a phase II study with ifosfamide, etoposide, and G CSF in advanced adult soft-tissue sarcoma has been initiated by the Scandinavian Sarcoma Group. PMID- 7539341 TI - Nonisotopic competitive RT-PCR assay to measure MDR1 gene expression. AB - We report an original application of competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of MDR1 mRNA in clinical specimens by simultaneous reverse transcription and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The competitor RNA shares the same MDR1 primer sequences as the cellular mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments after agarose gel electrophoresis and ethidium bromide staining. The concentration of MDR1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. We have used this assay to measure MDR1 expression in breast carcinomas and assessed the precision, sensitivity, and accuracy of the method. Competitive RT-PCR is a simple, highly specific, nonradioactive procedure for the quantification of MDR1 mRNA and is particularly suitable for use in the clinical laboratory. PMID- 7539340 TI - Low frequency of alterations in p53, K-ras, and mdm2 in rat lung neoplasms induced by diesel exhaust or carbon black. AB - Inhalation of diesel exhaust (DE), which contains soot particles with adsorbed mutagenic organic compounds, and its virtually mutagen-free soot particle analog, carbon black (CB), produce similar types and prevalences of pulmonary neoplasms in chronically exposed F344 rats. This result suggests that DE-induced neoplasia develops from the effects of a high lung burden of carbonaceous particles rather than from the genotoxicity of organic constituents. In this investigation, pulmonary carcinomas from rats exposed to DE or CB were analyzed for alterations in K-ras and p53 to determine if mutations caused by these agents are also similar. K-ras and p53 were chosen for this study because mutation patterns of these genes in lung neoplasms have been associated with specific exposures. A low frequency (3/50) and variable pattern of activating mutations were identified in codons 12 and 61 of the K-ras gene. Immunoreactive levels of p53 protein, suggesting gene dysfunction, were present in 7/13 squamous cell or adenosquamous carcinomas, regardless of the associated exposure. However, single-strand conformational polymorphism analysis and direct sequencing of p53 did not detect any mutations in these neoplasms. No immunoreactivity or mutations in p53 were observed in adenocarcinomas. The increased level of p53 protein in the squamous carcinomas is not explained by stabilization by the mdm2 gene product, because this protein was not overexpressed based on immunohistochemical analysis. No pattern of mutation was detected that would suggest a differential mechanism of carcinogenicity between DE and CB; however, inactivation of the p53 pathway may have a role in the development of rat lung neoplasms with a squamous cell carcinoma component. PMID- 7539342 TI - Structural analysis of CFTR gene in congenital bilateral absence of vas deferens. AB - Congenital bilateral absence of the vas deferens (CBAVD) is found in most males with cystic fibrosis (CF), but this malformation can be observed without any pulmonary or digestive features. We have analyzed 13 exons of the CF gene in a cohort of 25 CBAVD patients. Among the 50 chromosomes studied, 24 mutations were identified: delta F508 (14 cases), R117H (7 cases), R1070W (2 cases), 621 + 1 G - > T (1 case), and A1067V (1 case). Except for delta F508, the most frequent mutations (R117H, R1070W) were not observed in the CF group (109 patients) studied in our laboratory. We discuss the significance of these results. PMID- 7539343 TI - Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 1: Preparation and properties. AB - We have developed a new multienzyme control serum, Seraclear-HE, which was designed to function not only as an accuracy and precision control serum but also as an intermethod calibrator for unifying interlaboratory clinical enzyme data in terms of reference method values. Seraclear-HE contains as analytes the following enzymes of human origin only: aspartate aminotransferase (AST, EC 2.6.1.1) and lactate dehydrogenase (LD, EC 1.1.1.27) from erythrocytes; alanine aminotransferase (ALT, EC 2.6.1.2) from a hepatoma cell line; alkaline phosphatase (ALP, EC 3.1.3.1) from an amnion cell line; creatine kinase (CK, EC 2.7.3.2) from an embryo kidney cell line; gamma-glutamyltransferase (GGT, EC 2.3.2.2) from a macrophage cell line; and amylase (AMY, EC 3.2.1.1) from urine and saliva. The seven partly purified enzymes were lyophilized in partially delipidated human serum containing sucrose (50 g/L), pyridoxal 5'-phosphate (30 mmol/L), and other stabilizers. The material is stable for at least 2 years at temperatures < or = 10 degrees C. For two concentrations of this preparation, reference method values (mainly International Federation of Clinical Chemistry and Japan Society of Clinical Chemistry) obtained at both 30 degrees C and 37 degrees C are assigned. PMID- 7539344 TI - Accuracy of luteinizing hormone immunoassay is improved by changing epitope specificity of the labeled monoclonal antibody. PMID- 7539345 TI - Demonstration of macroamylasemia by polyethylene glycol (PEG) precipitation requires correct PEG concentration. PMID- 7539346 TI - Zatebradine attenuates cyclic AMP-related positive chronotropic but not inotropic responses in isolated, perfused right atria of the dog. AB - 1. Inhibition of I(f) or ICa by zatebradine has been reported in mammalian SA nodal cells. We thus investigated whether zatebradine differentially attenuates the positive chronotropic and inotropic responses to norepinephrine, isoproterenol, NKH 477 (an adenylyl cyclase activator), 3-isobutyl-1 methylxanthine (IBMX) and Bay k 8644 (a calcium channel agonist) in the isolated, blood-perfused dog atrium. 2. When zatebradine (0.03-1 mumol) decreased sinus rate from 104 +/- 4.5 to 73 +/- 4.9 beats/min dose-dependently, it selectively attenuated the positive chronotropic but not inotropic responses to norepinephrine in a dose-related manner. Zatebradine decreased the norepinephrine induced tachycardia (by approximately 80% from the control) more effectively than the spontaneous sinus rate (by approximately 30% from the control). 3. Zatebradine similarly attenuated the positive chronotropic but not inotropic responses to isoproterenol, NKH 477 and IBMX. Fifty per cent inhibition doses of zatebradine (0.10-0.18 mumol) for the chronotropic responses to each substance were not significantly different. 4. On the other hand, zatebradine attenuated neither positive chronotropic nor inotropic responses to Bay k 8644. 5. We therefore suggest that zatebradine selectively attenuates the positive chronotropic but not inotropic responses to cyclic AMP-related substances due to inhibition of I(f) but not ICa in the dog heart. PMID- 7539347 TI - Role of the low zinc bioavailability on cellular immune effectiveness in cystic fibrosis. AB - An altered cellular immune response as a secondary phenomenon has been suggested to be probably involved in the bronchopulmonary infections by Pseudomonas aeruginosa in cystic fibrosis (CF). The difficulty to eradicate with modern anti pseudomonal antibiotics the bronchopulmonary infections has led us to further investigate the possible existence of other cellular immune defects and their cause. Alterations in zinc turnover are present in CF. Zinc is relevant for good immune functioning. In particular, zinc is required to confer biological activity to thymulin (ZnFTS), a biochemically defined thymic hormone with a modulating action on cell-mediated immunity. The zinc-unbound form (FTS) is inactive and it can be unmasked by in vitro zinc addition to the plasma samples revealing the total amount of circulating thymulin (active + inactive). Marginal zinc deficiencies may prevent peripheral biological activation of active thymulin. Total zinc-saturable thymulin fractions in CF are similar to those observed in normal subjects, whereas the active quota is strongly reduced associated with concomitant high plasma levels of inactive thymulin compared to the values of healthy children (P < 0.01). A strict correlation exists between zinc and thymic hormone-saturable fraction (r = 0.87, P < 0.01) in CF. These findings suggest that the defect is not due to a thymic failure but to a reduced peripheral saturation of thymulin by zinc ions. This defect might depend on augmented plasma concentration of alpha 2-macroglobulin, which has a higher binding affinity for zinc than thymulin. T cell subsets are normal in CF. Reduced NK cell number and activity are present. Also, plasma IL-2 levels are reduced. The existence of positive correlations between zinc and IL-2 (r = 0.79, P < 0.01) and between zinc or active thymulin and NK activity (r = 0.70, P < 0.01 and r = 0.88, P < 0.01, respectively) suggest a close link among zinc failure, impaired IL-2 activity, low thymulin level, and reduced NK activity in CF patients with both normal and growth retardation. Although the role of NK cells is unknown in CF, a zinc supplementation, in order to induce a complete saturation of thymulin molecules, to correct some cellular immune defects and to improve the growth, may be suggested. PMID- 7539348 TI - The SS-A/SS-B autoantigenic complex: localization and assembly. AB - The subcellular distribution of Ro ribonucleoprotein particles (Ro RNPs) and their constituents was determined using mammalian tissue culture cells and Xenopus laevis oocytes. Cell fractionation revealed that Ro RNPs are restricted to the cytoplasm, while the Ro RNP-specific proteins were also present in the nucleus. Transport studies indicated that the protein components of Ro RNPs are actively imported into the nucleus while the RNA component accumulates in the cytoplasm. These results suggest a model for the assembly of Ro RNPs. PMID- 7539349 TI - Autoantibody-defined epitopes on nuclear antigens are conserved, conformation dependent and active site regions. AB - Antinuclear antibodies in the systemic rheumatic diseases have been powerful reagents for identifying and characterizing nuclear antigens and for elucidating immune mechanisms which drive the autoimmune response. Concepts which have emerged from these studies include the following: the autoimmune response is antigen driven, autoantigens are components of subcellular particles, autoantigens are involved in important biosynthetic functions and epitopes recognized by autoantibodies are active sites or functional domain regions. PCNA (proliferating cell nuclear antigen) is a nuclear protein of 36 kDa and autoantibodies are present in patients with systemic lupus erythematosus. PCNA is a component of the DNA replication complex and is associated with DNA polymerase delta in continuous strand DNA synthesis at the replication fork. Studies have shown that epitopes on PCNA recognized by lupus antibodies are conformation dependent. Composite peptides synthesized by joining discontinuous linear sequences were used to immunize rabbits and one such composite peptide induced antibodies with properties strikingly similar to human autoantibodies. Immunogens in the systemic autoimmune diseases are likely to be intranuclear or other intracellular particles composed of protein-protein or protein-nucleic acid complexes which are involved in cellular biosynthetic functions. PMID- 7539350 TI - Idiotypes as markers for immunoglobulin genes contributing to the production of rheumatoid factors: theoretical and practical implications. AB - Monoclonal antibodies have been produced that recognise conventionally defined cross-reactive idiotopes (CRI) expressed on rheumatoid factors. However, it is now evident that these "CRI" are markers for variable region sequences encoded by germline V gene segments and, therefore, that most of these reagents recognise isotypic epitopes rather than CRI. The implications of these findings for the identification and manipulation of auto-reactive B cell clones and the definition of regulatory idiotypic networks are discussed. PMID- 7539351 TI - Anti-RA33 autoantibodies may recognize epitopes in the N-terminal region of hnRNP A2 (RA33). AB - The nuclear autoantigen RA33, which is identical to the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP-A2), is a nucleic acid binding protein of 341 amino acids. The N-terminal part contains two RNA binding domains whereas the C-terminal part consists of a long glycine-rich region starting around amino acid 192. Autoantibodies to hnRNP-A2/RA33 can be detected in 20-40% of sera from RA, SLE and MCTD patients. So far, it has not been known which regions of A2/RA33 are recognized by these autoantibodies. To address this issue, tryptic fragments of natural A2/RA33 were investigated by immunoblotting using 14 sera from anti-RA33 positive patients with RA (n = 5), SLE (n = 5) and MCTD (n = 4). Most sera reacted with a 22 kD fragment comprising the N-terminal part of the protein. However, a smaller 18 kD fragment was recognized only by 3 RA and 3 MCTD sera whereas two of five SLE sera were reactive with two larger fragments of 26 and 29 kD. In order to further characterize the epitope(s) C-terminal deletion mutants of recombinant A2/RA33 were investigated by immunoblotting employing 27 sera from anti-RA33 positive patients with RA (n = 10), SLE (n = 8), and MCTD (n = 9). All sera recognized a fragment terminating at amino acid 212 which contained the complete N-terminal region as well as 20 amino acids of the glycine rich section. Thus, these data indicate that the N-terminal part of A2/RA33 contains epitopes for antiA2/RA33 autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539352 TI - Immuno-localization of the calcitriol receptor, calbindin-D28k and the plasma membrane calcium pump in the human eye. AB - Using immunohistochemical methods, we detected epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump and the 28kD vitamin D dependent calcium-binding protein in sections of the human eye. In retinal photoreceptors, vitamin D receptor, plasma membrane calcium pump and calcium binding protein epitopes were detected in the outer nuclear layer. Epitopes for the vitamin D receptor and the calcium-binding protein were present in the inner and outer segments of the photoreceptors, where visual transduction occurs. All three proteins were detected in some cells of the ganglion cell layer, the inner nuclear layer, and the retinal pigment epithelium. Epitopes for these proteins also were noted in the ciliary body epithelium. VDR epitopes were seen in lens epithelium. Some immunostaining for VDR, PMCA and calbindin-D28k also was present in the endothelium and in the basal epithelium of the cornea. The presence of these proteins in several tissues of the human eye suggests that the proteins may play a role in the cellular physiology of the eye. Their exact functions in the eye remain undetermined. PMID- 7539354 TI - Establishment of a leukemic lymphocyte culture from human aqueous humor. AB - We have recently developed new techniques for culturing chronic lymphocytic leukemia (CLL) lymphocytes from human aqueous humor. We used leukemic lymphocytes collected from the aqueous humor of a patient with CLL and leukemic glaucoma. We grew these leukemic cells in combination with a feeder cell layer and other technical refinements. Microscopy and immunoassay indicate success in obtaining a homogenous population of B-type CLL cells through the 12th passage of the culture. No significant effect on cell growth was found with either of two mitogens (PWM and PHA), or between culture with and without autologous serum. Our new techniques for culturing leukemic cells derived from the aqueous humor provide a reliable resource for the study of chronic lymphocytic leukemia lymphocytes and leukemic glaucoma. PMID- 7539355 TI - [Adjuvant and palliative regional therapy of liver metastases in colorectal tumors]. PMID- 7539353 TI - Identification and partial characterization of a proteinase specific for insulin like growth factor binding protein-3 in aqueous and vitreous humors. AB - The IGFs (-I and -II) are normally found in serum and other extracellular fluids complexed to specific binding proteins (IGFBPs). While several IGFBPs have been identified in vitreous and aqueous humors, the major serum carrier of IGF, IGFBP 3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (IGFBP-3ase), samples of bovine vitreous or aqueous humor were mixed with serum and incubated at 37 degrees C for 4 h followed by western ligand blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sensitive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid treatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chromatography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially-purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable by western ligand blotting but are readily visualized using an IGFBP 3 specific antibody. Comparison of normal and diabetic vitreous humor reveals the presence of an increased amount of IGFBP-3 proteolytic fragments in the diabetic as compared to control. These findings indicate the presence of a IGFBP-3 proteinase in aqueous and vitreous humors that may be important in regulating ocular homeostasis. PMID- 7539356 TI - Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. AB - We found that there are two major pathways by which RNAs are localized at the vegetal cortex during oogenesis of Xenopus laevis. One of these, through which Xlsirts, Xcat2 and Xwnt11 are localized, involves transport during stages 1 and 2 of oogenesis via a region of the mitochondrial cloud that we call the message transport organizer or METRO. This pathway involved three steps, transport of RNA from the GV to the mitochondrial cloud, sorting of the RNAs to specific regions of the METRO, and translocation to and anchoring at the vegetal cortex. These three RNAs exhibit a distinct pattern of spatial localization within the METRO when they approach the vegetal cortex. The other pathway is used by Vg1. We detected Vg1 throughout the oocyte cytoplasm during stages 1 and 2. During stage 3 it was translocated to the vegetal cortex and associated with the cortex overlapping the region at which the Xlsirt, Xcat2, and Xwnt11 RNAs are anchored. Our results also showed that anchoring of these RNAs was dependent in part on actin microfilaments, but was independent of microtubules. These results demonstrate a novel mechanism of translocation and RNA sorting used by RNAs several of which may be involved in the establishment of the embryonic body axis. PMID- 7539357 TI - Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. AB - VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing alpha 4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. alpha 4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent alpha 4 expressing cells. These data suggest important roles for VCAM-1 and alpha 4 in the development of the placenta and the heart. PMID- 7539358 TI - Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice. AB - The vertebrate ocular lens undergoes a spatially defined pattern of differentiation which may be regulated by the ocular distribution of proteins from the fibroblast growth factor (FGF) family. The ability of altered FGF-1 (acidic FGF) distribution to disrupt the normal pattern of lens differentiation was evaluated by the production of transgenic mice which express FGF-1 under the control of the lens-specific alpha A-crystallin promoter. Since FGF-1 lacks a classical signal peptide consensus sequence, transgenic mice were also produced with a chimeric construct containing the signal peptide sequence of the FGF-4 gene fused in frame to the coding sequences of the FGF-1 cDNA in order to obtain extracellular expression of the transgene. The presence of transgenic mRNA and protein was confirmed by in situ hybridization, Western analysis and immunohistochemistry. The ocular histology of newborn and young adult transgenic mice expressing FGF-1 without a signal peptide appeared normal. In contrast, mice expressing secreted FGF-1 exhibited lens abnormalities including the elongation of anterior epithelial cells. Epithelial cell elongation was accompanied by expression of the fiber cell differentiation marker, beta-crystallin. These observations provide an in vivo demonstration that FGF-1 can induce anterior lens epithelial cells to express characteristics consistent with the onset of fiber cell differentiation. The transgenic induction of differentiation confirms that normal lens morphology reflects an asymmetric distribution of inductive factors within the eye. PMID- 7539359 TI - Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. AB - alpha 4 integrins are cell surface receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesions by interacting with fibronectin (FN) and vascular cell adhesion molecule 1 (VCAM-1), respectively. We have generated a null mutation in the gene for the alpha 4 integrin subunit. Homozygous null embryos express no alpha 4 integrins and show two unexpected defects, both of which lead to embryonic lethality. The first defect is failure of fusion of the allantois with the chorion during placentation. The second is in the development of the epicardium and coronary vessels leading to cardiac hemorrhage. Both processes clearly involve alpha 4 integrin interactions that were previously unsuspected. alpha 4 integrin and VCAM-1 are expressed at the sites of these interactions. These results raise the possibility of abortifacients targeting alpha 4 integrins, and raise serious questions about potential side effects of drugs currently being designed to block alpha 4 integrin functions in inflammation. PMID- 7539360 TI - Development of cortical fibres into the gracile nucleus of the rat: an ultrastructural study. AB - A study was made on the development of cortical synapses in the gracile nucleus of rats using degeneration methods. A total of 46 animals, 1 adult and 45 neonates whose ages varied from 1 to 7 days, had the right somatosensory motor cortex destroyed. The survival period varied from 1 to 30 days. Identification of axonal terminals in the gracile nucleus was also achieved by tracing the cortical fibres of 1 adult rat using horseradish peroxidase-wheat germ agglutinin (HRP WGA). Degenerating axodendritic and axosomatic terminals that originated from cortical fibres were seen in the adult animal which survived 2 days. Their origin was confirmed by the presence of HRP-WGA inside the terminals. Light or electron microscopic changes were not seen, and in particular, the gracile nucleus was not smaller than in the control adult animals which survived 30 days or in neonates which survived 8-30 days, consistent with the small component of cortifugal fibres believed to terminate in secondary sensory nuclei. In neonates that survived a shorter period, terminal degeneration was only seen in cases operated at 4 days and later, indicating that cortical axons do not synapse in the gracile nucleus until postnatal day 4. Our results provide further support for the existence of a period in which the fibres approach their target but do not penetrate it to form synaptic junctions during the growth of cortical axons toward their targets, since previous studies have demonstrated that cortical fibres persisting to adulthood decussate completely at the level of the medulla at 12-36 h after birth.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539361 TI - The neurotoxic effects of lindane at acute and subchronic dosages. AB - Lindane (gamma-HCH), a multipurpose organochlorine pesticide, was administered to Rattus norvegicus at acute and subchronic dosages. The dosage given was half that corresponding to LD50 (half the lethal dose). For rats, this is 60 mg/kg of body weight. The acute dose was administered on a single occasion, while the subchronic dosage was administered on 10 occasions over a 30-day period. Six types of behavior pattern based on the neurotoxic register were established for the acute dosage. Significant decreases in the levels of the neurotransmitter dopamine were also found, in similar proportions for both types of dose. The difference between single dose intoxication and poisoning by overlapping doses must be underlined, as the second type may produce the same effects without giving rise to clear symptoms. PMID- 7539362 TI - Hexavalent chromium induces gametogenesis in the freshwater alga Scenedesmus acutus. AB - The aggregates formed by Scenedesmus acutus cultivated in the presence of 1 mg/liter Cr(VI) persist even after 7 days of treatment. They were examined by fluorescence and electron microscopy and were found to be an early stage of sexual reproduction. They consist of four small cells deprived of a true cell wall and enclosed in a common envelope. When the envelope breaks up they are released in the culture medium as biflagellated mobile cells. These cells have been identified as gametes. PMID- 7539363 TI - Use of a Gammarus pulex bioassay to measure the effects of transient carbofuran runoff from farmland. AB - There is now much information on the presence of pesticides in surface waters, but very little about their actual effects on aquatic life. This paper reports on the transient concentrations of a carbamate insecticide carbofuran, which were observed in a head-water stream draining treated farmland, and describes the resulting effects on a bioassay organism. One month after an application of 3 kg carbofuran/ha as broadcast granules to an oilseed rape crop, carbofuran concentrations of up to 26 micrograms/liter were measured in a nearby headwater stream after heavy rainfall. The majority of the carbofuran was translocated via field drains (where concentrations up to 264 micrograms/liter were detected), although the possibility of some surface runoff cannot be ruled out. Peak carbofuran concentrations only persisted for about 24 hr after the rainfall event, although measurable levels could be detected for at least 4 days. An in situ bioassay of streamwater which monitored the feeding rate of the gammarid amphipod crustacean Gammarus pulex showed that feeding stopped completely during the rainfall event, and was rapidly followed by death of all the caged organisms. Subsequent laboratory studies of toxicity showed that the peak concentrations of carbofuran had exceeded the G. pulex 24-hr LC50 of 21 micrograms/liter, and that concentrations as low as 4 micrograms/liter could reduce feeding rate. These findings are discussed with reference to regulatory risk evaluation procedures. PMID- 7539364 TI - Ketone EC50 values in the Microtox test. AB - The Microtox EC50 values for the following ketones are reported in the following homologous series: straight chain methyl ketones (acetone, 2-butanone, 2 pentanone, 2-hepatonone, 2-octanone, 2-decanone, and 2-tridecanone); methyl ketones substituted at one alpha carbon (3-methyl-2-butanone; 3,3-dimethyl-2 butanone); methyl substituted at two alpha carbons (2,4-dimethyl-3-pentanone; 2,2,4,4-tetramethyl-3-pentanone); phenyl groups replacing methyl in acetone (acetophenone; benzophenone); methyl groups substituted at the alpha carbons of cyclohexanone; and 2,3- 2,4-, and 2,5-hexanediones, most for the first time. While there were linear relationships between log EC50 and MW for the straight chain methyl ketones, and for methyl substitution at the alpha carbon for methyl ketones, there were no other linear relationships. As molecular weight increased, the EC50 values of soluble ketones decreased; as distance between two carbonyl groups decreased so too did EC50 values. Thus, for the ketones the geometry around the carbonyl group is an important determinant of toxicity as well as MW, water solubility, and octanol/water coefficient. PMID- 7539365 TI - Atrazine toxicity on transport properties of hemocyanin in the crab Oziotelphusa senex senex. AB - The current study reports the possible changes in the copper metabolism of the crab Oziotelphusa senex senex exposed to a sublethal concentration of atrazine. The alterations observed in the synthesis of pigment, hemocyanin, and its affinity to oxygen suggest the existence of respiratory distress in the crab as a consequence of atrazine toxicity. PMID- 7539366 TI - Comparison of forest soil microcosm and acute toxicity studies for determining effects of fenitrothion on earthworms. AB - The effects of the organophosphate pesticide fenitrothion on Eisenia fetida Savigny and Dendrobaena octaedra Savigny were studied using the OECD acute toxicity test and microcosms containing forest organic matter. In the acute toxicity study, both species were relatively susceptible to fenitrothion, although D. octaedra (LC50 = 393.9 ml/cm2) was approximately eight times more sensitive than E. fetida (LC50 = 54.1 ml/cm2). E. fetida was unable to utilize oak/maple litter for growth, and attempts to use this species in microcosms were abandoned. In microcosm studies, reproduction of D. octaedra was affected at the expected environmental concentration (EEC), whereas growth and maturation rates did not decrease until 100x the EEC was used. Even at 100x EEC, a significant effect on survival of D. octaedra in microcosms was not demonstrated. The use of microcosm studies to provide insight into possible ecological effects of pesticides is discussed. PMID- 7539368 TI - Cadmium disposition in the earthworm Eisenia fetida. AB - The disposition of cadmium was examined in the earthworm Eisenia fetida using two exposure media, filter paper, and artificial soil. Uptake and elimination rates were estimated to be 0.03039 and 0.00895 hr-1, respectively, for 1.25 micrograms Cd/cm2 filter paper exposure and 0.00512 and 0.00029 hr-1, respectively, for 10 micrograms Cd/g artificial soil exposure. The distribution of cadmium was examined using whole body autoradiography, tissue distribution, and subcellular localization. Cadmium tissue distribution was exposure-dependent, while cadmium subcellular localization apparently was not exposure-dependent. Acid-soluble thiol levels, examined as an index of cadmium exposure, were too variable in nonexposed earthworms to serve as an indicator of cadmium exposure. PMID- 7539367 TI - Hazard assessment of inorganics to three endangered fish in the Green River, Utah. AB - Acute toxicity tests were conducted with three life stages of Colorado squawfish (Ptychocheilus lucius), razorback sucker (Xyrauchen texanus), and bonytail (Gila elegans) in a reconstituted water quality simulating the middle part of the Green River of Utah. Tests were conducted with boron, lithium, selenate, selenite, uranium, vanadium, and zinc. The overall rank order of toxicity to all species and life stages combined from most to least toxic was vanadium = zinc > selenite > lithium = uranium > selenate > boron. There was no difference between the three species in their sensitivity to the seven inorganics based on a rank-order evaluation at the species level. Colorado squawfish were 2-5 times more sensitive to selenate and selenite at the swimup life stage than older stages, whereas razorback suckers displayed equal sensitivity among life stages. Bonytail exhibited equal sensitivity to selenite, but were five times more sensitive to selenate at the swimup life stage than the older stages. Comparison of 96-hr LC50 values with a limited number of environmental water concentrations in Ashley Creek, Utah, which receives irrigation drainwater, revealed moderate hazard ratios for boron, selenate, selenite, and zinc, low hazard ratios for uranium and vanadium, but unknown ratios for lithium. These inorganic contaminants in drainwaters may adversely affect endangered fish in the Green River. PMID- 7539369 TI - Toxicity of complex chemical mixtures: acute and long-term effects on different life stages of zebrafish (Brachydanio rerio). AB - Acute and long-term toxicity of 3,4-dichloroaniline and lindane to zebrafish were examined in tap water and water from the Rhine River and the toxicity of the binary mixture 3,4-dichloroaniline/lindane in tap water was investigated. The acute toxicity of 3,4-dichloroaniline and lindane was not influenced by the complex matrix of river water compared with tap water. The binary mixture of 3,4 dichloroaniline and lindane demonstrated an additive effect in the acute test. The survival rate of early life stages in river water was reduced by lindane (80 micrograms/liter), whereas the effects of 3,4-dichloroaniline on survival and growth were visible but not significant. The binary mixture of 2 micrograms/liter, 3,4-dichloroaniline and 40 micrograms/liter lindane in tap water had an influence on growth on the early life stages of zebrafish. PMID- 7539370 TI - Does a heterogeneous distribution of food or pesticide affect the outcome of toxicity tests with Collembola? AB - The reproduction of two closely related soil microarthropods, Folsomia candida Willem and Folsomia fimetaria L. (Insecta: Collembola), was tested under the influence of the insecticide dimethoate. Dimethoate had an adverse effect on the survival of adults and their reproduction in concentrations of about the recommended field dose, with F. fimetaria being more sensitive than F. candida. The experimental conditions were altered to evaluate the realism in the basic single species/single chemical reproductive test system. The importance of the spatial distribution of dimethoate was studied with food applied to the surface (original procedure), mixed homogeneously in the whole soil profile or only in the top layer, or mixed heterogeneously into the soil preserving the small granula of the yeast originally in the commercial formulation. Toxicity decreased significantly when exposure could be avoided in an uncontaminated bottom layer and even more if food was available in this soil horizon. But the results indicate that Collembola were not able to completely avoid dimethoate when they had the choice. For extrapolation purposes a simple test system may be sufficient as EC50 was changed less than one order of magnitude with the different test designs. In terms of EC50 the outcome of a toxicity test with a heterogeneous distribution of food and dimethoate was changed only slightly but the effects to suboptimally fed populations should be considered because they may be more vulnerable. PMID- 7539371 TI - Biochemical and physiological effects in fish exposed to bleached kraft mill effluents. AB - The studies were performed on perch (Perca fluviatilis) caught outside Husum and Iggesund kraft pulp mills in 1985 and 1987, respectively, and the results were compared to previous observations from 1984 to 1985 in the receiving waters of the Norrsundet kraft pulp mill. The studies revealed that the biochemical and physiological variables, i.e., EROD activity, gonad size, number of lymphocytes, carbohydrate metabolites, and plasma ion composition, can be used as sensitive and reliable indicators of toxic effects on natural populations of fish exposed to conventional chlorine-bleached kraft mill effluents. However; the effects were generally less pronounced and indicated less geographic extension than those seen in perch previously investigated in the receiving waters of the Norrsundet kraft pulp mill. Based on these studies on perch at Husum and Iggesund and the previous studies at Norrsundet it can be concluded that the chlorine-bleached kraft mill effluents cause serious disturbances to vital biochemical and physiological functions, which can affect the health status and presumably also the survival of the fish. Finally, the responses do not reflect the present situation in the receiving waters since all three mills have taken extensive measures for process alterations and discharge reductions after the time of the investigations. PMID- 7539372 TI - DNA dosimetry in biological indicator species living on PAH-contaminated soils and sediments. AB - A large variety of environmental carcinogens are metabolically activated to electrophilic metabolites that can bind to nucleic acids, forming covalent adducts. In organisms possessing active metabolic systems for a particular carcinogen, DNA adducts generally have longer biological half-lives than the substrate carcinogens. Thus, measurement of specific DNA adduct concentrations in terrestrial and water organisms may provide a relevant biological indicator of prior exposure to environmental carcinogens. Analysis of carcinogen load in indicator species with specific behavioral patterns may indicate human exposure risk to environmental carcinogens. Recently, sensitive assays have been developed to measure carcinogen-DNA adducts in organisms exposed to complex mixtures such as polycyclic aromatic hydrocarbons (PAH). At first instance, the nuclease P1 version of the 32P-postlabeling assay was used to examine the liver of eel (Anguilla anguilla) for the presence of aromatic DNA adducts. The fish were collected from six freshwater sites in the Amsterdam area with different levels of PAH contamination in their sediments. Chromatograms derived from DNA of fish from polluted sites revealed a broad diagonal zone indicating the presence of DNA adducts containing aromatic or bulky hydrophobic moieties not present in DNA of fish from an unpolluted reference site. Significant correlations were found between the aromatic DNA adducts levels and the levels of PAH in sediments (P < 0.001). To examine the validity of DNA adduct dosimetry in terrestrial organisms earthworms (Lumbricus terrestris) were kept on industrially contaminated PAH soils for several weeks. Several aromatic DNA adducts could be detected in DNA from the exposed earthworms; adduct levels were significantly increased with increasing exposure time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539373 TI - Changes in periwinkle (Littorina littorea) populations following the ban on TBT based antifoulings on small boats in the United Kingdom. AB - In 1987 the UK Government banned the use of tributyl tin (TBT)-based antifouling paints on small boats of less than 25 m. Following initial control measures taken in 1986, a program of research was started to monitor concentrations of TBT residues in estuarine waters and sediments and to observe faunistic changes in highly contaminated estuaries. As part of this program, the size-frequency and abundance of Littorina littorea populations have been recorded in the estuaries of the rivers Crouch (Essex) and Hamble (Hampshire). Since the TBT ban, the concentration of TBT residues in water and sediments has been steadily declining. In both L. littorea populations, the frequency of O-group individuals has increased markedly, and there has been a simultaneous decrease in TBT residues in L. littorea tissues. Furthermore, plankton surveys of the River Crouch show that the numbers of L. littorea eggs and veliger larvae have progressively increased, suggesting that TBT may have impaired periwinkle reproduction and/or survival of the eggs and larvae. Subsequent laboratory experiments have indeed shown that reduced egg production was the probable mechanism of action, but the imposex associated with exposure to TBT in dogwhelks (Nucella lapillus) has not been seen. PMID- 7539374 TI - Tolerance induction and life cycle changes in cadmium-exposed Chironomus riparius (Diptera) during consecutive generations. AB - Cultures of Chironomus riparius were exposed to cadmium during nine consecutive generations to determine whether cadmium tolerance could be induced. Selection for cadmium tolerance was assumed to influence the population dynamics of this species. Therefore, the responses and interactions of different population parameters (such as mortality, growth, and reproduction) were studied during the selection process. Exposure to cadmium during consecutive generations caused increasing effects on some life cycle parameters compared to a one-generation experiment. Tolerance to cadmium increased during exposure to 54.2 nM Cd and the tolerant population seemed to be stimulated by low cadmium concentrations (based on an acute growth experiment). Despite this tolerance development, mortality among cadmium-exposed tolerant chironomids remained high. These experiments illustrated that changes of the life cycle and tolerance can be expected as soon as single-generation NOEC values are exceeded, and in addition that "safe concentrations" based on a one-generation toxicity experiment could well underestimate the potential effects of a toxicant on midge populations. PMID- 7539376 TI - Evidence against direct activation of chloride secretion by carbachol in the rat distal colon. AB - Carbachol (5 x 10(-5) mol.1-1) induced a biphasic increase in short-circuit current (Isc) consisting of an initial peak phase followed by a long-lasting plateau. Complete dependence on the presence of Cl- ions and sensitivity to bumetanide confirmed that carbachol induces Cl- secretion. The plateau phase was blocked by indomethacin, and both the plateau and the peak phase were suppressed in the combined presence of indomethacin and tetrodotoxin. Inhibition of the carbachol response could be overcome by agonists of the cAMP pathway like prostaglandin E2, forskolin or 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate. The increase in Isc was inhibited by a blocker of cAMP-activated Cl- channels, glibenclamide, but was resistant to an inhibitor of Ca(2+) activated Cl- channels, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). The K+ channel blockers Ba2+ and charybdotoxin inhibited the first and suppressed the second phase of the carbachol response, whereas a less specific K+ channel blocker, quinine, suppressed both phases. These results suggest that the dominant effect of carbachol in the intact colonic mucosa is an opening of Ca(2+) dependent, charybdotoxin- and Ba(2+)-sensitive K+ channels, which leads to hyperpolarization of the epithelial cells. This stimulates Cl- secretion only if there are spontaneously open apical Cl- channels which are basically stimulated by a continuous release of neurotransmitters and prostaglandins. Only during the first phase of the carbachol effect is there indirect evidence for activation of a Cl- conductance synergistically with the cAMP pathway as shown by the increase in tissue conductance resistant to K+ channel blockers. PMID- 7539375 TI - Influence of the energy relationships of trophic levels and of elements on bioaccumulation. AB - A concern of ecotoxicology is to predict to which trophic levels in biocenoses bioaccumulation of compounds or of elements occurs. Transformity, a measure of the energy required to produce and maintain a component or a flow resulting from an energy transformation process, may help predict bioaccumulation potential. This notion derives from two concepts. First, common substances are more likely to be processed by the biosphere. Moreover, the uptake of rare ones from the physical environment by organisms of low trophic levels makes them less unusual to organisms of high trophic levels, which may evolve a capability of processing them. Second, transformity expresses energy relationships between parts of a system. Substances that require more energy to form or concentrate are also the more unusual. The hypothesis was formulated that there is a correlation between the rarity, complexity, and energy required for concentrating a substance, and thus its transformity, and the transformity of the trophic level to which it bioaccumulates. This hypothesis was tested for a set of elements with published data on their biogeochemistry and bioaccumulation and on energy transfers between trophic levels in ecosystems. The transformities of the elements were calculated from the energy required by the biosphere for maintaining a difference in concentration compared to its physical environment. Transformities of corresponding trophic levels were calculated from the energy driving the energy flows. There was a significant rank correlation between the transformity of elements and that of trophic levels. This may be an important generalization in ecotoxicology because it may lead to the possibility of predicting bioaccumulation tendency. PMID- 7539378 TI - Dose-dependent anticonvulsant and proconvulsant effects of nitric oxide synthase inhibitors on seizure threshold in a cortical stimulation model in rats. AB - In the central nervous system, nitric oxide (NO) is increasingly being considered as a trans-synaptic retrograde messenger, being involved for instance in cellular responses to stimulation of glutamate receptors of the NMDA subtype. Thus, compounds that modify NO production, such as NO synthase inhibitors, may provide a means of altering NMDA receptor function. The functional consequences of NO synthase inhibition are, however, complicated by the fact that NO not only serves as a messenger to activate guanylyl cyclase and so to raise cGMP in target cells in response to NMDA receptor stimulation but also to induce feedback inhibition of the NMDA receptor via a redox modulatory site on the receptor complex. This may explain the contrasting results obtained previously with NO synthase inhibitors in animal models of ischaemia and seizures. In the present study, we tried to resolve the reported discrepancies about the effects of NO synthase inhibitors in seizure models by studying such drugs at various doses in a novel model of cortical seizure threshold. In this model, the threshold for seizures in rats is determined at short time intervals by applying ramp-shaped electrical pulse-trains directly to the cerebral cortex, allowing one to determine the time course of anti- or proconvulsant drug effects in individual rats. Two NO synthase inhibitors, NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester, were compared with a clinically effective antiepileptic drug, i.e. valproate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539379 TI - Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. AB - We compared the behavioral effects of treatment with pruritogenic and algesiogenic agents in mice. The animals were given subcutaneous injections of pruritogenic agents, compound 48/80 (3-100 micrograms), substance P (10-300 micrograms) and histamine (3-300 micrograms), and algesiogenic agents, capsaicin (30 and 100 micrograms) and dilute formalin (5 mg of formaldehyde), into the rostral back, and scratching of the injected site by the hind paws was counted. Compound 48/80 and substance P dose dependently elicited the scratching behavior, but histamine, capsaicin and dilute formalin were without significant effects at the doses examined. These results suggest that compound 48/80- and substance P induced scratching of the injected site is due to itch, but not to pain. The data did not provide support for the idea that histamine produces itch in the mouse. PMID- 7539381 TI - Separation and characterization of mobilized and unmobilized peripheral blood progenitor cells by counterflow centrifugal elutriation. AB - Peripheral blood stem cell (PBSC) harvests are highly contaminated with lymphocytes. In allogeneic transplantations with PBSC, this might lead to an increased risk of graft-vs.-host disease (GVHD). Marrow graft T cells depletion (TCD) by counterflow centrifugal elutriation (CCE) has been performed to decrease the incidence of GVHD, but in some cases, it has also led to a reduction in the PBSC content of the graft due to the distinct elutriation properties of these cells. We studied the ability of CCE to deplete lymphocytes from recombinant human granulocyte colony-stimulating factor (rhG-CSF)-mobilized PB mononuclear cells (PBMNC) and from unmobilized PBMNC from healthy volunteers. We characterized the elutriation properties of circulating mobilized and unmobilized hematopoietic progenitors. With an experimental three-flow rate CCE procedure, originally described for separation of bone marrow, fractions 24, 28, and Rotor Off (RO) were obtained without impairing yield or viability. This CCE procedure can also be used for the separation of PBMNC harvests by reducing the T cell contamination to or below the range seen in bone marrow grafts; CD3+ cell contamination in the RO fractions of mobilized and unmobilized PBMNC was reduced to < 10% of the input. This could bring allogeneic PBSCT within the range of feasibility. In unmobilized PBMNC, 25% of the recovered CD34+ cells were found in fraction 24, where no colonies were detected, and 50% of the recovered CD34+ cells were found in fraction RO, where most of the colonies were detected. In mobilized PBMNC, 80% of the recovered CD34+ cells were found in fraction RO, together with 90% of the colonies, but almost no colonies or CD34+ cells were detected in fraction 24. CD34+ cells in fraction 24 expressed less CD13, and antigen found on more mature myeloid cells. Mobilization with rhG-CSF causes a shift of CD34+ cells from smaller cells (fraction 24) to larger cells (fractions 28 and RO). PMID- 7539380 TI - Growth-inhibitory effect of 2-CdA on myeloid CFU-GM progenitors in normal human long-term bone marrow cultures and its compensation by G-CSF or IL-3. AB - As neutropenia is a common side effect of treatment with 2-chlorodeoxyadenosine (2-CdA), we investigated the myelosuppressive action of 2-CdA in Dexter-type human long-term bone marrow cultures (LTBMCs). LTBMCs were incubated with varying doses of 2-CdA (5 to 20 nM/L) during the first week. At 20 and 10 nM/L 2-CdA, we found a marked reduction in colony-forming unit-granulocyte/macrophage (CFU-GM) production throughout the culture period of 7 weeks (maximum reduction to 3.5% of untreated control cultures with 20 nM/L and to 27.2% with 10 nM/L, respectively). Even the lowest 2-CdA dose tested (5 nM/L) strongly reduced the number of CFU-GM progenitors during the first 3 weeks (maximum reduction to 52.4% of untreated controls), but this effect was transient, and values had recovered to normal within in 5 weeks. 2-CdA was also shown to cause a dose-dependent decrease in long-term culture-initiating cell (LTCIC) detections after 5 weeks in culture (49.6% of control cultures with 10 nM/L 2-CdA and 14% with 20 nM/L 2-CdA, respectively). When 2-CdA was added to LTBMCs initiated on preformed irradiated stromal feeder layers, similar results on CFU-GM production were obtained, indicating that the effects observed were not secondary to effects on the formation of a supportive layer. In addition, IL-6-concentrations in the supernatant of LTBMCs measured at various intervals after the addition of fresh medium with or without 2-CdA showed no significant decrease in cultures treated with 2-CdA. As neutropenia has been shown to be associated with a small but significant risk of fatal infection, we subsequently investigated the reversal potential of the 2-CdA effect by addition of recombinant human granulocyte colony stimulating factor (rhG-CSF) or rh interleukin-3 (rhIL-3). The weekly addition of 100 ng/mL rhG-CSF counteracted the 2-CdA-mediated decrease in CFU-GM numbers during the entire period of 7 weeks, reaching statistical significance from weeks 3 to 7 (p < 0.05). Addition of rhIL-3 (100 ng/mL) showed an enhancement of CFU-GM output in 2-CdA-treated cultures that resulted in their numbers exceeding those in control cultures (without 2-CdA) from weeks 1 to 5 (p < 0.05) with a maximum increase of 5.1-fold over the parallel control value at week 3.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539377 TI - Dissimilarities between cholinergic and dopaminergic turning elicited by nucleus accumbens stimulation in freely moving rats. AB - Contralateral turning was produced by unilateral injection of carbachol (0.5, 2.5, 5 micrograms) into the nucleus accumbens, but not into the dorsal or ventral striatum. This behaviour was inhibited by muscarinic M1 acetylcholine receptor blockade in the nucleus accumbens, and less effectively by blockade of muscarinic M2 and nicotinic acetylcholine receptors. Unilateral injection of a mixture of the dopamine D1 receptor agonist 1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8 diol (SKF 38393, 5 micrograms) and the dopamine D2 receptor agonist quinpirole (10 micrograms) also produced contralateral turning. The stepping pattern, however, completely differed from that induced by carbachol. The number of carbachol-induced turnings was reduced by dopamine D1 or D2 receptor blockade (8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol (SCH 23390) and l-sulpiride, respectively) in the nucleus accumbens. However, the reduction was due to a change in the turning pattern. Blockade of muscarinic acetylcholine receptors in the nucleus accumbens did not change the contralateral turning induced by unilateral injection of dopamine receptor agonists into the nucleus accumbens. The results demonstrate that there is no functional interaction between the cholinergic and dopaminergic substrates involved, although blockade of the dopamine receptors elicited behavioural deficits that competed with the turning elicited by carbachol. The contralateral turning elicited by carbachol injection into the nucleus accumbens requires an intact dopamine activity at the level of dopamine D1 and D2 receptors in the ipsilateral, but not contralateral, ventrolateral striatum. PMID- 7539382 TI - IL-11 directly stimulates murine and human erythroid burst formation in semisolid cultures. AB - The effect of interleukin-11 (IL-11) on cultures of bone marrow cells was investigated. We found that IL-11 increased, in a dose-dependent manner, the number of bursts in the presence of Epo in murine or human cells cultures. This effect was also observed in cultures of murine cells established in serum-free conditions as well as in cultures of CD34+ enriched human cells in serum containing (but not serum-free) cultures. A linear relationship between the number of bursts and the plated cell number was observed with murine bone marrow cells or non-adherent mononuclear cells (NA-MNC) human bone marrow cells. Moreover, the effect of IL-11 was not abrogated when either anti-stem cell factor receptor (anti-SCF-R), anti-IL-3, or anti-granulocyte/macrophage colony stimulating factor (GM-CSF), three cytokines known to greatly synergize with IL 11, was added to cultures. These results lead us to conclude that IL-11 directly stimulates the proliferation of murine and human burst-forming units-erythroid (BFU-E). PMID- 7539383 TI - Comparison of the inhibitory effect of AcSDKP, TNF-alpha, TGF-beta, and MIP-1 alpha on marrow-purified CD34+ progenitors. AB - The aim of this study was to compare the inhibitory effect of the tetrapeptide AcSDKP, tumor necrosis factor-alpha (TNF-alpha), which contains the sequence of the peptide, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on sorted CD34+ cells using both proliferation and clonogenic assays. Although a short treatment with any of the molecules decreased the growth of colony-forming unit granulocyte/macrophage (CFU GM) and burst-forming unit-erythroid (BFU-E) progenitors (except for TNF-alpha as it is a greater inhibitor for CFU-GM), further experiments using a 6-day liquid culture in the presence of a combination of growth factors (recombinant human interleukin-3 [rhIL-3], IL-6, IL-1 beta, GM colony-stimulating factor [GM-CSF], G CSF, erythropoeitin [Epo], and stem cell factor [SCF]) allowed us to determine a number of differences between their effects: 1) TGF-beta and TNF-alpha induced a stronger decrease in the proliferation and clonogenicity of CD34+ subsets than MIP-1 alpha and AcSDKP, 2) the dose-response curves appeared different, and 3) contrary to TGF-beta and TNF-alpha, AcSDKP and MIP-1 alpha required repeated addition to induce inhibition. Therefore, our data clearly show that while the inhibitory effect of TNF-alpha and AcSDKP appeared to be different, there is a close similarity in the effect of AcSDKP and MIP-1 alpha on normal human progenitor response to the combination of growth factors used. PMID- 7539384 TI - MEG-01s cells have receptors for and respond to IL-3, IL-6, and SCF. AB - An established megakaryoblastic cell line, MEG-01s, was used to study receptor expression and receptor-mediated responses to factors known to affect megakaryocytopoiesis. In addition, the antigenic characteristics of this cell line were further defined. MEG-01s cells were CD34+CD33+CD38 +/- HLA-DR- and expressed erythroid and granulocytic differentiation antigens as well as many megakaryocytic lineage-restricted antigens. These cells also expressed receptors for interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), as measured by flow cytometry and/or RNA expression. MEG-01s cell proliferation or survival was only marginally influenced by these factors and their combinations. c-kit, the receptor for SCF, was downmodulated by its ligand. This modulation was time dependent, appeared to involve receptor conformational changes, and became concentration-dependent by day 3. Northern blot analysis indicated that amounts of c-kit RNA increased as downmodulation proceeded. IL-3 induced IL-6 secretion in these cells, which was augmented by a protein kinase-C (PKC) inhibitor, H7, and reduced by a tyrosine kinase inhibitor, genistein. Evidence for autocrine regulation of this cell line by IL-6 was demonstrated by the inhibitory effects of an antisense oligonucleotide on 3H-thymidine (3H-TdR) incorporation. These cells should prove useful for studies of the early signal transduction mechanisms involved in cytokine function. PMID- 7539385 TI - Noradrenergic and peptidergic innervation of the amphibian spleen: comparative studies. AB - Spleens from representatives of the three amphibian orders were examined using sucrose-potassium phosphate-glyoxylic acid (SPG) histofluorescence to detect catecholamines and immunocytochemistry to detect several neural antigens. Nerve fibers are scattered throughout the spleens of adult salamanders (Taricha torosa, Notophthalmus viridescens, and Ambystoma mexicanum). A less abundant but similarly diffuse pattern of innervation characterizes the spleen of the caecilian, Typhlonectes sp. The spleen of the adult frog, Xenopus laevis, is separated into clearly defined compartments of red pulp and white pulp, much as is seen in the mammalian spleen. As in mammals, sympathetic innervation of the Xenopus spleen is noradrenergic (NA) and confined to the white pulp. The white pulp of Xenopus spleen also contains fibers which stain for neuropeptide Y and substance P. The spleen of the anuran, Rana pipiens, is also highly compartmentalized, with tyrosine hydroxylase positive fibers in proximity to blood vessels. These findings provide an anatomical substrate for neural-immune interactions in the Amphibia. PMID- 7539386 TI - Metabolism of exogenous cholesterol by rat adrenal mitochondria is stimulated equally by physiological levels of free Ca2+ and by GTP. AB - Adrenal mitochondria metabolize cholesterol at inner membrane (IM) cytochrome P450scc. Exogenous and outer membrane (OM) cholesterol are metabolized more slowly due to a limiting transfer of cholesterol from OM to IM. This process is stimulated by in vivo ACTH treatment and inhibited by cycloheximide (CX)-induced depletion of labile regulatory proteins. In isolated rat adrenal mitochondria, GTP enhances the metabolism of exogenous cholesterol, consistent with enhanced intermembrane cholesterol transfer (Xu et al. (1989) J. Biol Chem. 264, 17674), but metabolism of 20 alpha-hydroxycholesterol, which readily traverses mitochondrial membranes, is not affected. The non-hydrolyzable analog, GTP gamma S, completely inhibits the activation of cholesterol metabolism by GTP, suggesting a requirement for GTP hydrolysis. Low concentrations of Ca2+ (0.4-4 microM) stimulate two independent cholesterol transport processes. For exogenous cholesterol, a Ca(2+)-mediated process can replace GTP since each produces comparable stimulation and the combination produces little additional activity. This Ca2+ stimulation is insensitive to GTP gamma S and also to Ruthenium Red (RR), which prevents Ca2+ entry into the matrix. Ca2+ also enhances availability to P450 scc of endogenous OM cholesterol, which accumulates during in vivo CX inhibition. This stimulation is, however, distinguished by insensitivity to GTP and complete inhibition by RR. Ca2+, therefore, enhances intermembrane transfer of exogenous cholesterol from OM without entry into the matrix through a process which is independently stimulated by GTP. Ca2+ induces transfer of endogenous OM cholesterol through a completely different mechanism involving RR-inhibited matrix changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539387 TI - Nitric oxide modulates pancreatic basal secretion and response to cerulein in the rat: effects in acute pancreatitis. AB - BACKGROUND/AIMS: Nitric oxide synthase activity is detected in the pancreas, but the role of NO on pancreatic function has not been fully characterized. The aim of this study was to evaluate the role of NO in normal and diseased pancreatic function. METHODS: Amylase and NO secretion were measured in vivo in rats and in vitro in dispersed acini, with and without NO synthesis blockade, by NG-nitro-L arginine methyl ester (L-NAME). Rats were subjected to cerulein-induced pancreatitis, and the effects of L-NAME or NO donors were assessed. RESULTS: L NAME reduced amylase output to 60% of basal. This effect was reversed by L arginine. The secretory response to optimal doses of cerulein induced a poor amylase secretion and a marked release of NO. High doses of cerulein in combination with L-NAME inhibited NO formation and amylase secretion. In dispersed acini, supramaximal cerulein concentrations induced NO release, but the amylase dose-response curve was not modified by NO inhibition. In acute pancreatitis, L-NAME increased amylasemia and tissue myeloperoxidase activities, whereas NO donors reduced amylasemia, lipasemia, and the histological damage score. CONCLUSIONS: The L-arginine/NO pathway facilitates basal and stimulated pancreatic secretion in vivo. NO donor drugs may improve the course of acute pancreatitis. PMID- 7539388 TI - Effect of cerulein hyperstimulation on the paracellular barrier of rat exocrine pancreas. AB - BACKGROUND/AIMS: Cerulein-induced pancreatitis causes a rapid increase in pancreatic enzyme levels in serum and decreases in pancreatic duct secretion and interstitial edema. One mechanism to explain these early events is disruption of the actin tight junction paracellular seal of acinar and intralobular pancreatic duct cells. METHODS: To examine the paracellular barrier of the proximal exocrine pancreas, rats were hyperstimulated with 5.0 micrograms.kg-1.h-1 of cerulein. Actin was visualized with rhodamine phalloidin and by electron microscopy and tight junctions were visualized with antibodies to the tight-junction protein ZO 1. Paracellular permeability was measured by movement of horseradish peroxidase from interstitium into duct or acinar lumens. RESULTS: In controls, linear actin and ZO-1 staining occurred along the apical membrane of intralobular duct cells and extended to the apical pole of acinar cells. Hyperstimulation caused progressive disruption of the linear staining of f-actin and ZO-1. Actin disruption in duct cells was confirmed by electron microscopy. Horseradish peroxidase entered intralobular ducts and acinar lumens of hyperstimulated animals more frequently than those of controls. CONCLUSIONS: The structure and function of the paracellular barrier of acinar and intralobular pancreatic duct cells are disrupted early during cerulein pancreatitis and may contribute to early clinical features. PMID- 7539389 TI - Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. AB - BACKGROUND/AIMS: Inflammatory events are believed to play an important role in the pathogenesis of acute pancreatitis. Interleukin 10 (IL-10) recently emerged as a major anti-inflammatory cytokine, inhibiting the secretion of proinflammatory cytokines by monocytes and/or macrophages. The potential protective role of IL-10 in a model of acute necrotizing pancreatitis in mice was tested. METHODS: Animals received two intraperitoneal injections of either 1000 U recombinant IL-10 or control supernatant before and during induction of acute pancreatitis with repeated cerulein injections (seven intraperitoneal injections of 50 micrograms/kg at hourly intervals). RESULTS: Systemic amylase and lipase release peaked 9 hours after the first cerulein injection. This peak was significantly reduced by IL-10 treatment. Histologically, edema and inflammation of the pancreas were observed in both groups, whereas necrosis was dramatically reduced in IL-10-treated animals. Serum tumor necrosis factor levels were undetectable in this model; reverse-transcriptase polymerase chain reaction analysis of resected pancreatic tissues performed at the time of maximal morphological alterations showed a dramatically decreased expression of tumor necrosis factor alpha messenger RNA after IL-10 treatment compared with control pancreatitis. CONCLUSIONS: IL-10 is able to decrease the severity of experimental acute pancreatitis, mainly by inhibiting the development of acinar necrosis. Inhibition of local tumor necrosis factor alpha might explain, at least in part, the protective effect of IL-10. PMID- 7539391 TI - Angiostatin: candidate for molecule of the year! PMID- 7539390 TI - Autoimmune hepatitis and chronic hepatitis C: latent or initiated by interferon therapy? PMID- 7539392 TI - The unc-8 and sup-40 genes regulate ion channel function in Caenorhabditis elegans motorneurons. AB - Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels. PMID- 7539393 TI - Low prevalence of hepatitis C virus infection in porphyria cutanea tarda in Germany. AB - Previous studies from Spain, Italy, and France have demonstrated a high prevalence (71% to 91%) of antibodies against hepatitis C virus in patients with porphyria cutanea tarda (PCT). To determine the role of hepatitis C virus (HCV) in PCT in Germany, we have assessed the prevalence of antibodies against HCV and hepatitis B virus (HBV) in 106 patients (mean age, 60 +/- 14 years) with the disease. Eight of 106 patients (8%) were positive for HCV antibodies and HCV RNA using second-generation enzyme-linked immunosorbent assay (ELISA), recombinant immunoblot assay, and polymerase chain reaction. Antibodies against HBV core antigen were found in 14 patients (13%). Of the patients with antibodies against HCV alanine transaminase (ALT) (aspartate transaminase [AST]) levels above normal occurred in 71% (86%). Because elevated ALT (AST) levels were also found in 51% (64%) of 88 patients without markers of HCV or HBV, we suggest that liver damage in PCT may exist in absence of these viruses. This is supported by the finding that in patients without HCV or HBV markers, higher serum ALT and AST activities were found in patients with overt disease or relapse (ALT, 59 +/- 44 U/L; AST, 37 +/- 21 U/L), whereas patients in remission displayed significantly lower serum enzyme activities (ALT, 16 +/- 8 U/L; AST, 16 +/- 7 U/L), (P < .001). These results indicate that HCV infection does not play a major role in the pathogenesis of PCT in Germany. PMID- 7539394 TI - Hematopoietic stem cell markers are expressed by ductal plate and bile duct cells in developing human liver. AB - The identification of ductal plate cells as likely progenitors for bile duct epithelium and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities. We have examined the distribution of three hematopoietic stem cell markers, c-kit, CD34, and CD33 in addition to laminin, the standard cytokeratin markers CAM 5.2, CK 18, and CK 7 and the oval cell marker OV-6 in fetal liver during various stages of development. Hematopoietic stem cell markers were expressed in ductal plate cells in a pattern similar to the early cytokeratin markers CAM 5.2 and CK 18. Cells stained strongly for these early cytokeratin markers until 22 weeks. Thereafter, the expression of these markers decreased while positivity for CK 7 increased. Bile duct cells showed a distribution of hematopoietic and cytokeratin markers resembling that of ductal plate cells. Both ductal plate cells and bile duct cells expressed OV-6 strongly throughout development. This study showed similarity between hepatic and bile duct precursors and bone marrow stem cells. The comparable distribution of markers in bile duct epithelium and ductal plate cells may imply fewer transitional stages between ductal plate cells and bile duct epithelium than between the putative stem cells and hepatocytes. PMID- 7539396 TI - Roles of prostaglandin production and mitogen-activated protein kinase activation in hepatocyte growth factor-mediated rat hepatocyte proliferation. AB - Hepatocyte growth factor (HGF) and epidermal growth factor (EGF)-stimulated DNA synthesis in primary cultured rat hepatocytes. HGF-induced DNA synthesis was concentration-dependently inhibited by a cyclooxygenase inhibitor, indomethacin. BW755C, a dual inhibitor for cyclooxygenase and lipoxygenase activities, also inhibited hepatocyte growth. Prostaglandin E1 (PGE1), PGE2, and PGF2 alpha induced DNA synthesis even at such a low concentration as 5 nmol/L and potentiated [3H]thymidine incorporation induced by HGF in an additive manner. HGF caused arachidonic acid (AA) release and eicosanoid production. These events were detectable within 10 minutes after stimulation and lasted for at least 60 minutes. Furthermore, two proteins with approximately 40 kd were tyrosine phosphorylated by HGF. These proteins were identified as p42/p44 mitogen activated protein (MAP) kinases by anti-MAP kinase immunoblots, which were known to activate cytosolic phospholipase A2 (cPLA2), a key enzyme in AA release. Activation of MAP kinases was detectable within 5 minutes after stimulation with HGF and lasted for at least 60 minutes. EGF-mediated DNA synthesis was also inhibited by the above cyclooxygenase inhibitors. EGF caused AA release and tyrosine phosphorylation of MAP kinases. These results suggest that HGF as well as EGF causes AA release, probably through activation of cPLA2 mediated by MAP kinases, and that PGs, metabolites of AA, might play a pivotal role in hepatocyte proliferation in an autocrine mechanism. PMID- 7539397 TI - Regulation of human Gc (vitamin D--binding) protein levels: hormonal and cytokine control of gene expression in vitro. AB - Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response. PMID- 7539395 TI - Further characterization and comparison of inducible nitric oxide synthase in mouse, rat, and human hepatocytes. AB - Marked differences in induced nitric oxide (NO) synthesis occur between species. We have previously shown that both human and rat hepatocytes express an inducible NO synthase in response to cytokines and lipopolysaccharide. In this study, we compare the expression and regulation of cytokine-induced NO synthase in hepatocytes isolated from three species, human, rat, and mouse. On stimulation with tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS), it was found that hepatocytes from all three species produce high levels of NO with levels of production exhibiting the following hierarchy: rat hepatocytes > mouse hepatocytes > human hepatocytes. Whereas rat and mouse hepatocytes express inducible NO synthase messenger RNA (mRNA) in response to TNF alpha, IL-1 beta, or IFN gamma as a single stimulus, human hepatocytes respond to LPS alone. Inhibition of NO generation through transforming growth factor (TGF-beta 1) was seen in mouse (77% +/- 5.9) and rat hepatocytes (17% +/- 2.6) whereas only about 10% was seen in human hepatocytes. Epidermal growth factor (EGF) was shown to inhibit NO synthesis in human and mouse hepatocytes but not rat. A marked NO dependent inhibition of total protein synthesis was seen in rat and human hepatocytes, whereas mouse hepatocytes showed almost no inhibition in protein synthesis when stimulated. NO-dependent cyclic guanosine monophosphate (cGMP) release was found in all three species.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539399 TI - Detection of hepatitis C virus by polymerase chain reaction and recombinant immunoblot assay 3.0 in porphyria cutanea tarda. PMID- 7539398 TI - Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol. AB - To determine the involvement of different hepatocyte populations in response to periportal injury, the restitutive response to allyl alcohol (AA) injury was examined. Adult female Sprague-Dawley rats were injected intraperitoneally (IP) with 0.62 mmol/kg AA, killed at 6, 9, 12, 33, 57, 81, and 153 hours after injection, and the livers were examined for injury and for restitutive proliferation by histology, autoradiography, and immunohistochemistry to detect alpha-fetoprotein (AFP), glutathione-s-transferase-p (GST-p), desmin, leukocyte common antigen, albumin, and monoclonal antibodies to liver cells: OV-6, H-4, and T-6. AA produces variable periportal liver necrosis predominantly at 6 to 12 hours. Proliferation of hepatocytes throughout the hepatic cord is seen early after injury in nonnecrotic areas: predominantly in zone II, but also in zones I and III, including some cells adjacent to the central vein. Within 2 to 3 days the necrotic zones are filled with small cells and by 1 week the liver architecture is essentially restored. During the active restitutive reaction from the immediate periportal rim the following cell phenotypes are seen: null cells: ->(AFP+, OV-6-, GST-p-) cells-->(AFP-, OV-6+, GST-p+) cells-->large (AFP-, OV-6-, GST-p-, H-4+) liver cells. Albumin staining was negative. We conclude that restitutive proliferation of periportal necrosis induced by AA appears to be accomplished by proliferation of intraportal (?stem) cells whose progeny differentiate and eventually repopulate the necrotic zone. PMID- 7539400 TI - A new monoclonal antibody (A78-G/A7) to the Thomsen-Friedenreich pan-tumor antigen. AB - A new monoclonal antibody to the Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope; Gal beta 1-3GalNAc-) has been developed that is specific for both anomeric forms of this disaccharide (TF alpha and TF beta, including related structures on glycolipids), and not assay restricted. We demonstrate that this avid antibody (A78-G/A7) is well suited for immunohistochemistry on paraffin embedded and cryosectioned tissues, immunoblotting, ELISA techniques, and hemagglutination. Immunohistochemistry on paraffin sections does not require proteolytic or microwave pretreatment. The binding characteristics of this antibody are largely independent of variations in pH (6.0-8.2) and temperature (4 37 degrees C). Immunoblotting with KG-1 (human acute myelogenous leukemia) cells revealed a series of TF-active glycoproteins with a main band at about 155 kDa. Immunoprecipitation was performed using a new technique applicable to IgM-type antibodies. PMID- 7539402 TI - Phorbol ester activates CD5+ leukaemic B cells via a T cell-independent mechanism. AB - B cell chronic lymphocytic leukaemia (B-CLL) is characterized by the proliferation and accumulation of sIgM+ CD5+ lymphocytes that fail to progress to the final stages of B cell development. Stimulation of unfractionated PBL from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and IgM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of IgM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis was also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on CD5+ CLL B cells was independent of T cells. In contrast, activation and differentiation of the malignant CD5+ B cells into IgM-secreting cells following culture with mitogens did not occur in the absence of T cells. PMID- 7539401 TI - Peptides with carboxyl-terminal sequence of alanine-proline: detection by a human monoclonal antibody. AB - A human B lymphoblastoid cell line JWCI-L94 secretes an IgM human monoclonal antibody (HuMAb) that reacts with human melanoma cell lines, M14 and M12. To identify the antigenic epitope of this antibody, we screened lambda gt11 expression libraries constructed from M14 and M12. A total of 12 immunoreactive clones were isolated, and their DNA sequences were determined. The only sequence shared by all these clones was alanine-proline (A-P) at the carboxyl (C) terminal. HuMAb L94 reacted not only with C-terminal A-P-containing fusion proteins, but also with the synthetic dipeptide A-P. None of the peptides containing A-P internally or amino terminally reacted to HuMAb L94. Proline or alanine alone had no ability to bind to HuMAb L94. When alanine was replaced by glycine (G-P) or proline (P-P), the binding activity of these peptides was similar to that of A-P. On the other hand, when alanine was replaced by serine, valine, leucine, glutamine, lysine, methionine, phenylalanine, or hydroxyl proline, the resulting peptide completely lost the antigenic activity of HuMAb L94. These results demonstrate that HuMAb L94 recognizes C-terminal A-P, G-P, or P-P, and that a human antibody can recognize peptides as small as a two-amino acid residue. PMID- 7539403 TI - Protease-modified erythrocytes: CD55 and CD59 deficient PNH-like cells. AB - The increased susceptibility to homologous complement in paroxysmal nocturnal haemoglobinuria (PNH) is known to be associated with the deficiency of the membrane complement inhibitors CD59 and CD55. Proteases have been used in this study to modify normal human RBC to complement sensitive PNH-like cells. To investigate the protective role of CD59 and CD55, the relationship between the content of CD59 and CD55 and the complement susceptibility of the PNH-like cells has been determined. The differential resistance of the enzyme-treated RBC to complement-mediated injury was measured by acidified serum lysis. Pronase-treated erythrocytes lacked both CD59 and CD55 and were very susceptible to complement mediated lysis. Papain treatment of RBC reduced the CD55 content but did not affect CD59 and induced slight susceptibility to complement-mediated lysis. Trypsin treatment of RBC destroyed 80% of CD59, had little effect on CD55 (unless incubation was extended) and slightly increased susceptibility to lysis. Thus, partial CD55 and CD59 activity was sufficient to protect cells from complement mediated lysis. In the reactive lysis assay, anti-CD55 and anti-CD59 induced haemolysis, anti-CD59 having the more pronounced effect. Lysis was enhanced when RBC were treated by both antibodies simultaneously. PMID- 7539404 TI - Influence of compounds affecting synthesis, modification and transport of proteins on the expression and release of interleukin-2 receptor. AB - One of the most central events during lymphocyte activation is the synthesis and release of IL-2. IL-2 induces the synthesis and expression of the IL-2 receptor alpha-chain (IL-2R) on the lymphocyte as well as the release of a truncated form of IL-2R (sIL-2R). The two proteins are identical except for the absence of the transmembrane and intracellular part on sIL-2R. We have in an in vitro model investigated the influence of certain compounds, affecting different parts of the proliferative response, on the release and expression of IL-2R. We found the generation of the two molecules to have different sensitivity to blocking of protein synthesis, glycosylation, microtubular assembly and proteolytic activity. However, blocking of intracellular transport from Golgi to the endoplasmic reticulum, disassembly of actin filaments and disturbances in the intracellular sodium/calcium balance had identical effects on expression and release of the respective IL-2R. These findings indicate a more complex and specific mechanism behind the generation of sIL-2R than simply by shedding through the action of proteases on the membrane-bound IL-2R. PMID- 7539405 TI - Nitric oxide and its putative role in hypertension. PMID- 7539408 TI - Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi. AB - The neutralizing epitopes of the major outer surface proteins A and B (OspA and OspB) of Borrelia burgdorferi B31 were investigated by epitope mapping using overlapping synthetic peptides, encompassing full-length OspA and OspB, and antiborrelial monoclonal antibodies (MAbs). OspA MAb N4B12 and OspB MAbs N5G5, W7C2, and P4D1 displayed a complement-independent antiborrelial activity, and complement failed to enhance the antiborrelial activity, as measured by a sensitive colorimetric assay. A combination of N4B12 with N5G5 displayed a higher antiborrelial activity than did the MAbs individually. OspA MAbs B3G11 and L3B5, however, exhibited a significant antiborrelial activity only in the presence of complement. Epitope mapping showed that B3G11 bound to one OspA synthetic peptide with the sequence of amino acids 247 to 256 (QYDSNGTKLE) and produced more than sixfold-higher reactivity than with other sequences, as measured by an enzyme linked immunosorbent assay. OspB MAb N5G5 bound to an OspB peptide with the sequence of amino acids 211 to 220 (TLKREIEKDG), yielding at least threefold higher reactivity than with other sequences. These two peptide sequences were found to contain neutralizing epitopes. Other MAbs had weak binding activities with the synthetic peptides, and their specific epitopes remain to be further analyzed. Thus, this study demonstrated both complement-independent and complement-dependent antiborrelial MAbs and identified the linear epitopes on OspA and OspB capable of inducing neutralizing antibody responses. PMID- 7539409 TI - Involvement of cysteine residues in the biological activity of the active fragments of guinea pig neutrophil cationic peptides. AB - Guinea pig neutrophil cationic peptides (GNCPs) are single-chain polypeptides with 31 amino acid residues containing six cysteine residues, which exhibit both antibacterial and histamine-releasing activities in vitro. In this study, the role of the sulfhydryl groups in defining the antibacterial and histamine releasing activities of the active fragments of GNCP-1 (Arg-1 to Tyr-14 [Arg-1 Tyr-14] and Arg-15-Tyr-27 peptides) was examined by using peptides containing alkylated or nonalkylated sulfhydryl groups. Alkylation slightly increased the histamine-releasing activity of the Arg-15-Tyr-27 (RRLGTCIFQNRVY) peptide but abrogated the antibacterial activity. Alkylation of the Arg-1-Tyr-14 (RRCICTTRTCRFPY) peptide similarly reduced the antibacterial activity of this fragment but had minimal effect on the histamine-releasing activity. These findings suggest that cysteine residues with free sulfhydryl groups play an important role in the expression of the antibacterial activity of the active fragments of GNCP-1. PMID- 7539407 TI - A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa. AB - The lacto-N-neotetraose-containing lipooligosaccharide (LOS) present on the surface of most Neisseria gonorrhoeae organisms may serve many important functions in gonococcal pathogenesis. This surface glycolipid contains the cross reactive epitope to human paragloboside and can be sialylated by gonococci grown in the presence of CMP-N-acetylneuraminic acid. Another possible role for this glycolipid could be to mimic human asialocarbohydrates and act as a ligand for asialoglycoprotein receptors contained on numerous human cells. The most noted of this large family of receptors is that expressed on the surface of hepatic cells. In a model cell system, using the hepatoma tissue culture cell line HepG2, we wanted to investigate if the presence of this asialoglycoprotein receptor influenced the adherence and/or invasion of gonococci expressing the lacto-N neotetraose structure. Piliated variants of the gonococcal wild-type strain 1291 and its isogeneic LOS mutant 1291E were used in adherence-invasion assays. This gonococcal strain is somewhat unusual in that it expresses large amounts of predominantly one species of LOS, thus reducing the complexity of interpreting the data. The data from these assays suggested that the Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc carbohydrate structure on the wild-type LOS affected the adherence-invasion of gonococci into the HepG2 cells. In studies to determine whether the major hepatic asialoglycoprotein receptor was involved in these interactions, we found that the HepG2 cells contained two receptors which bound gonococcal LOS. One of these was the asialoglycoprotein receptor, and the data concerning this receptor will be reported elsewhere. The data on the second receptor are reported here. Purified, 125I-labeled gonococcal LOS was used to identify specific high-affinity LOS-binding sites. These binding experiments revealed one major binding site corresponding to a protein with a molecular mass of 70 kDa (p70). Several lines of evidence in this study suggested that the oligosaccharide region of LOS played an important role in LOS binding to the p70 of HepG2 cells. In addition, we show that this human LOS receptor has some similarities to the gonococcal Opa proteins. PMID- 7539406 TI - Cloning, characterization, and antigen specificity of T-lymphocyte subsets extracted from gingival tissue of chronic adult periodontitis patients. AB - Chronic periodontitis is characterized by dense infiltrations of B and T lymphocytes within the gingival connective tissue. Distinct anaerobic gram negative bacteria as well as autoimmunity to collagen have been reported to play a role in the etiology and the pathogenesis of this disease. Here we describe the cloning and characterization of CD4+ and CD8+ T lymphocytes isolated from inflamed gingival tissue obtained from four patients with chronic periodontitis. Clones were raised with phytohemagglutinin and interleukin-2 and tested for proliferation in response to whole-cell antigens of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, human collagen type I, and two bacterial heat shock proteins. CD4+ T-cell clones reactive with collagen type I were obtained from all four patients. Eighty percent of these clones had phenotypes resembling the mouse type 2 T helper (Th) phenotype, i.e., they produced high levels of interleukin-4 and low levels of gamma interferon. No collagen-type-I-reactive CD8+ clones were obtained. Bacterial-antigen-reactive CD4+ and/or CD8+ T-cell clones were also obtained from each patient, and the majority of the clones showed a Th0-like cytokine pattern and produced equal amounts of interleukin-4 and gamma interferon. Although most clones were reactive with P. intermedia, it seems that the immune response is not strictly directed against this particular microorganism, as clones reactive with one of the other bacteria were also obtained from two patients. We propose that collagen-specific CD4+ Th2-like T cells contribute to the chronicity of periodontitis but that their modes of activation might be controlled by Th0-like T cells specific for periodontitis-associated bacteria. PMID- 7539410 TI - Synthetic peptides representing two protective, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa elicit whole-cell-reactive antibodies that are functionally pseudomonad specific. AB - Peptide 9 (TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) represent surface exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Rats immunized with four intramuscular inoculations on days 0, 14, 28, and 42 with either peptide 9 or peptide 10 conjugated to keyhole limpet hemocyanin were afforded protection against pulmonary lesions when examined 7 days subsequent to challenge (day 56) via intratracheal inoculation of P. aeruginosa-containing agar beads. Peptide 9 shares considerable homology with other OmpA-related outer membrane proteins in various bacteria, whereas peptide 10 displays little homology with these other proteins. Antisera directed to peptide 9 reacted weakly with cell envelope proteins from the various other OmpA-associated bacteria upon immunoblot analysis. However, antisera directed to peptide 10 reacted only with Neisseria gonorrhoeae cell envelope proteins upon immunoblot analysis. Antisera to both peptides 9 and 10 reacted at minimal titers with whole cells of the various other bacteria in a whole-cell enzyme-linked immunosorbent assay (ELISA) but antisera to each of the peptides reacted at high titers when various strains of P. aeruginosa were used as the ELISA antigen. Antibodies to peptides 9 and 10 were protective, reactive to all strain of P. aeruginosa tested except for a protein F-deficient mutant, and functionally specific against pseudomonads. PMID- 7539411 TI - Differential expression of the H-ras mutated and normal alleles in rabbit DMBA induced keratoacanthomas. AB - Keratoacanthomas (KAs) are benign and self-regressing tumors in which a high incidence of the mutated H-ras oncogene has been observed both in humans and in experimental models. To determine the level of expression of the mutated H-ras allele with respect to its normal counterpart in 7,12-dimethylbenz(a)anthracene (DMBA)-induced KAs in rabbit skin, we have utilized a quantitative technique based on reverse transcription polymerase chain reaction (RT-PCR) and selective cleavage of the mutated molecules of the H-ras gene. Analysis of 16 KAs showed that the mutated H-ras transcripts were up to 3-fold more abundant than the non mutated H-ras transcript in the different tumors. This higher expression of the mutated allele appears to correlate with increased differentiation in the KAs and in turn may contribute to tumor regression. PMID- 7539412 TI - Evidence of a chromatin basis for increased mutagen sensitivity associated with multiple primary malignancies of the head and neck. AB - Individuals at increased risk for cancer development have been reported to exhibit increased mutagen sensitivity as detected by the G2 phase chromosome breakage assay. To better understand the basis for such chromosome sensitivity, we examined 4 lymphoblastoid cell lines derived from 4 head and neck cancer patients, 2 of whom were previously shown to have normal bleomycin sensitivity and 2 high bleomycin sensitivity (and multiple primary tumors). These cell lines were studied for cell survival and initial damage and repair at both the DNA and chromosome levels, and the results compared to a normal control cell line and 3 ataxia telangiectasia homozygote cell lines. While all cell lines exhibited nearly equal levels of initial DNA damage and repair throughout the cell cycle, both the ataxia telangiectasia homozygote cell lines and 2 cell lines derived from individuals with multiple head and neck primary tumors showed increased levels of initial chromosome damage (detected by premature chromosome condensation), a reduced fast repair component and higher residual chromosome damage. Our results suggest that one component of the enhanced mutagen sensitivity phenotype observed in cancer-prone individuals may involve an inherent chromatin alteration that allows a more efficient translation of DNA damage into chromosome damage following mutagen exposure. The degree of such an alteration might then be associated with an increased risk for second primary tumors in other carcinogen-exposed sites. PMID- 7539413 TI - Palliative operations for painful old Perthes' disease. AB - Thirty-one adults with painful old Perthes' disease were treated by subcutaneous adductor tenotomy and cheilectomy of the femoral head with the aim of increasing abduction and rotation of the hip. In addition, multiple drilling was carried out to decompress the femoral head, and muscle-pedicle bone grafting to improve the circulation and repair the necrotic bone. Follow up was from 38 to 120 months. Pain was relieved in every case and movement improved in most. There were 14 excellent, 12 good, 4 fair and one poor result. PMID- 7539414 TI - The role of nitric oxide in erectile function. PMID- 7539415 TI - Secretory leucocyte protease inhibitor in the male genital tract: PSA-induced proteolytic processing in human semen and tissue localization. AB - Secretory leucocyte protease inhibitor, SLPI, is a low-molecular-weight, acid stable protein present in the liquid part of fresh human ejaculate but not demonstrable in the gel structure. No fragmentation of SLPI occurred during gel dissolution, but a slow proteolytic cleavage of SLPI was seen on incubation of the liquified semen at 37 degrees C. The same pattern of degradation products was seen after incubation of SLPI with prostatic secretion and also with purified prostate-specific antigen, PSA. We could identify Arg 20-Tyr 21 and Met 73-Leu 74 to be the primary cleavage sites upon proteolytic modification of SLPI by purified PSA. However, we did not find any inhibition of the enzymatic activity of PSA by SLPI, even at a 100-fold molar excess of the inhibitor. The slow degradation of SLPI facilitated sampling and the reliable determination of the normal level of SLPI in seminal plasma, which was about 20 mg/L. We investigated the glandular origin of SLPI in the genital tract by immunocytochemistry. A strong immunostaining for SLPI was demonstrated in epithelial cells within the glandular lumina of the prostate gland, seminal vesicles, and epididymis but not in the stromal parts of these glands. In addition the immunostaining was also detected in the deferent ducts and the germinal epithelium of the testes. Taking into account that SLPI is a strong inhibitor of several proteases, including leukocyte elastase and cathepsin G, the results suggest that SLPI has a local protective function against proteolytic degradation of the male reproductive tract tissues during inflammation. PMID- 7539416 TI - Automated sperm morphometry analysis (ASMA) in the rabbit. AB - New methods of specimen preparation were developed and a new method of objective, automated sperm morphometry analysis (ASMA) was performed to reduce the technical variation in the rabbit sperm morphology assay. The optimal staining procedure was a modified GZIN stain, which allowed the ASMA instrument to accurately recognize the distal end of the sperm head and to achieve the highest sperm recognition rate (94%). Washing and resuspending sperm to a standard concentration increased the number of sperm per microscopic field in low concentration samples and reduced the field-to-field variation in all samples. Washing also decreased the number of sperm recognition errors by the ASMA instrument. Mean metric measurements for all sperm were: length, 7.38 microns; width, 3.91 microns; width/length, 0.53; area, 22.10 microns; and perimeter, 19.20 microns. Within-specimen coefficients of variation (CVs) ranged from 0.8% to 5.5% and between-animal CVs ranged from 2.7% to 7.5%. The use of standard specimen preparation techniques and ASMA technology can significantly reduce the technical variation in the rabbit sperm morphology assay. PMID- 7539418 TI - Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus. AB - A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus. PMID- 7539417 TI - Transcriptional analysis of the amidase operon from Pseudomonas aeruginosa. AB - The transcriptional start point for the amidase structural gene (amiE) of Pseudomonas aeruginosa has been identified, and the promoter (pE) has been shown to function constitutively, as predicted for a system regulated by transcription antitermination. Northern (RNA) analysis results show that in cells grown under inducing conditions, a major 1.3-kb amiE transcript arises from pE, and in addition, a larger transcript of approximately 5.0 kb in length has been shown to derive from the same promoter, encoding all of the genes of the operon. DNA sequencing and S1 nuclease mapping have located a transcription terminator downstream of amiE, which terminates approximately half of the pE transcripts. Previously, two RpoN-dependent promoter-like sequences (pN1 and pN2) were identified upstream of the negative regulator gene, amiC, and we have now constructed a promoter probe vector which shows weak constitutive promoter activity within this region. This promoter would be expected to provide basal levels of expression of the amiC and amiR regulatory genes to allow induction of the system. PMID- 7539420 TI - A polypurine sequence that acts as a 5' mRNA stabilizer in Bacillus subtilis. AB - A segment of early RNA from Bacillus subtilis bacteriophage SP82 was shown to function as a 5' stabilizer in B. subtilis. Several heterologous RNA sequences were stabilized by the presence of the SP82 sequence at the 5' end, and expression of downstream coding sequences was increased severalfold. The SP82 RNA segment encodes a B. subtilis RNase III cleavage site, but cleavage by B. subtilis RNase III was not required for stabilization. The sequence that specifies 5' stabilizer function was localized to a polypurine sequence that resembles a ribosome binding site. The ability of the SP82 sequence to stabilize downstream RNA was dependent on its position relative to the 5' end of the RNA. These results demonstrate the existence of a new type of 5' stabilizer in B. subtilis and indicate that attack at the 5' end is a principal mechanism for initiation of mRNA decay in B. subtilis. PMID- 7539419 TI - Pediococcus acidilactici ldhD gene: cloning, nucleotide sequence, and transcriptional analysis. AB - The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1 kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone. PMID- 7539421 TI - Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae. AB - Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA. PMID- 7539424 TI - How does the retinal rod Na-Ca+K exchanger regulate cytosolic free Ca2+? AB - The roles of 1) inactivation of Na-Ca+K exchange and 2) Ca2+ release from discs in regulation of cytosolic free Ca2+ were examined in intact rod outer segments (ROS) purified from bovine retinas. Measurements of cytosolic free Ca2+ (with fluo-3) were combined with Ca2+ flux measurements (45Ca) in ROS that contained about 600 microM total Ca2+. Na(+)-induced Ca2+ extrusion was measured in a Ca(2+)-free medium and did not lower cytosolic free Ca2+ to below 1 nM as expected from a coupling stoichiometry of 4Na+:(1Ca(2+) + 1K+). Instead, cytosolic free Ca2+ was rapidly (20 s) lowered from about 1300 nM to 100-150 nM, while at the same time about 35% of total ROS Ca2+ was removed. During the next 40 min cytosolic free Ca2+ remained virtually steady, but total ROS Ca2+ was reduced by a further 50% at a 100-fold lower rate than that observed for the initial fast phase. The steady cytosolic Ca2+ concentration resulted from Ca2+ release from discs and subsequent removal across the plasma membrane by Na-Ca+K exchange operating at a greatly reduced rate. Addition of the alkali cation channel ionophore gramicidin led to a persistent increase in cytosolic free Ca2+ concentration to about 400 nM, presumably caused by an increase in intracellular Na+. It is suggested that cytosolic free Ca2+ is not determined by the Na+:Ca2+ coupling ratio of the exchanger, but rather by a sensor on its cytoplasmic domain that controls inactivation of the Ca2+ extrusion mode and is sensitive to intracellular Ca2+, Na+, and K+. PMID- 7539423 TI - Activation of NADPH-oxidase and its associated whole-cell H+ current in human neutrophils by recombinant human tumor necrosis factor alpha and formyl-methionyl leucyl-phenylalanine. AB - Proton accumulation and efflux associated specifically with NADPH oxidation in neutrophils remains to be elucidated. Using confocal fluorescence and patch-clamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cytosolic acidification and whole-cell H+ current induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor alpha (rhTNF alpha). Intracellular pH changes were monitored utilizing the ratiometric imaging of the dual emission fluoroprobe, carboxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 units/ml rhTNF alpha or 0.1 microM fMLP changed the fluorescence of fluoroprobe-loaded cells, indicating generation of cytosolic H+ ions. In the absence of Ca2+ in the pipette solution, exposure of cells to rhTNF alpha or fMLP for 10 s activated voltage-dependent H+ currents. From tail current analysis, the threshold voltage for H+ current activation was approximately -50 mV. These fMLP- or rhTNF alpha activated voltage-dependent H+ currents were augmented further in the presence of 0.1 mM of NADPH in the pipette solution, and they were inhibited by bath application of 50 microM of apocynin, an NADPH oxidase inhibitor. These results indicate that rhTNF alpha- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H+ currents. PMID- 7539426 TI - Identification of amino acid residues essential for von Willebrand factor binding to platelet glycoprotein Ib. Charged-to-alanine scanning mutagenesis of the A1 domain of human von Willebrand factor. AB - At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). The VWF-GPIb interaction was investigated by clustered charged-to-alanine scanning mutagenesis of VWF domain A1 between His-473 and Gly-716. Recombinant variants of VWF were assayed for binding to conformation-dependent monoclonal antibody NMC-4, for ristocetin-induced and botrocetin-induced binding to platelets, and for direct binding to botrocetin. Substitutions at 32 amino acids had no effect on VWF function. The epitope of NMC-4 depended on charged residues between Asp-514 and Arg-632 and not on segments previously implicated by peptide inhibition studies, Cys-474-Pro-488 and Leu-694-Pro-708. Substitutions at Glu-626 and in the segment Asp-520-Lys-534 abolished ristocetin-induced binding of VWF to GPIb but did not affect botrocetin-induced binding, suggesting that these regions are required for modulation by ristocetin but not for binding of VWF to GPIb. Mutations at Glu-596 and Lys-599 decreased binding of VWF to GPIb without affecting its binding to botrocetin, suggesting that this segment interacts directly with GPIb. Alanine substitutions at Arg-545 and in the segments Glu-497-Arg-511 and Arg-687-Glu-689 caused increased binding of VWF to GPIb. These results, and the locations of von Willebrand disease type 2B mutations, suggest that two acidic regions containing the Cys-509-Cys-695 disulfide (Glu-497-Arg-511, Arg-687-Val-698) and one predominantly basic region (Met-540-Arg-578) cooperate to inhibit a distinct GPIb binding site in the VWF A1 domain. This inhibition is relieved by specific mutations, by the modulators ristocetin and botrocetin, or by binding to subendothelial connective tissue. PMID- 7539425 TI - Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway. AB - The 2-5A/RNase L system is considered as a central pathway of interferon (IFN) action and could possibly play a more general physiological role as for instance in the regulation of RNA stability in mammalian cells. We describe here the expression cloning and initial characterization of RLI (for RNase L inhibitor), a new type of endoribonuclease inhibitor. RLI cDNA codes for a 68-kDa polypeptide whose expression is not regulated by IFN. Its expression in reticulocyte extracts antagonizes the 2-5A binding ability and the nuclease activity of endogenous RNase L or the cloned 2DR polypeptide. The inhibition requires the association of RLI with the nuclease and is dependent on the ratio between the two proteins. Likewise RLI is coimmunoprecipitated with the RNase L complex by a nuclease specific antibody. RLI does not lead to 2-5A degradation or to irreversible modification of RNase L. The overexpression of RLI in stably transfected HeLa cells inhibits the antiviral activity of IFN on encephalomyocarditis virus but not on vesicular stomatitis virus. RLI therefore appears as the first described and potentially important mediator of the 2-5A/RNase L pathway. PMID- 7539422 TI - Expression of calcium-permeable cation channel CD20 accelerates progression through the G1 phase in Balb/c 3T3 cells. AB - CD20 is a transmembrane protein that functions as a Ca(2+)-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. D., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the cDNA encoding a Ca(2+)-permeable cation channel, CD20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing cDNA encoding CD20 expressed the CD20 protein, which was detected by assaying the binding of a monoclonal antibody against CD20. Calcium-permeable cation channel activity was detected in CD20-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of CD20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF I). IGF-I induced DNA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA synthesis by 30% of the quiescent CD20-expressing cells was initiated in response to IGF-I in the absence of priming with PDGF and EGF. When control quiescent cells were primed with PDGF and EGF, the addition of IGF-I led to the initiation of DNA synthesis after 14 h or more, whereas it induced DNA synthesis by CD20-expressing cells primed with PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent on extracellular Ca2+, and expression of CD20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, DNA synthesis by approximately 25% of the CD20-expressing cells was initiated after priming with PDGF and EGF, even in the absence of the progression factor IGF-I. These results indicate that CD20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca(2+)-permeable cation channel and that expression of CD20 accelerates G1 progression in a Ca(2+)-dependent manner. PMID- 7539428 TI - Alpha 2-macroglobulin-mediated clearance of proteases from the plasma of the American horseshoe crab, Limulus polyphemus. AB - Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma. In addition, mammals have alpha 2-macroglobulin (alpha 2M), which binds active proteases, and the alpha 2M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. alpha 2M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluorescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limulus alpha 2M, and that was precipitated by antibodies directed against Limulus alpha 2M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (< 10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t1/2 > 180 min). FITC-labeled, trypsin-reacted Limulus alpha 2M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus alpha 2M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus alpha 2M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of alpha 2M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and have shown that Limulus alpha 2M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physicological function for alpha 2M in invertebrates. PMID- 7539427 TI - Carbachol, substance P, and phorbol ester promote the tyrosine phosphorylation of protein kinase C delta in salivary gland epithelial cells. AB - The initiation of saliva formation by parotid acinar cells, which comprise the majority of cells in this salivary gland, is initiated by the release of neurotransmitters (acetylcholine, substance P) from parasympathetic nerves. In response to substance P and the muscarinic agonist carbachol, two ligands that activate phospholipase C-linked receptors, which stimulate fluid secretion, PKC delta was phosphorylated on tyrosine residues. The maximal agonist-dependent tyrosine phosphorylation occurred within seconds of the addition of either agonist and then returned rapidly to a smaller increased level. Phorbol ester also caused a rapid increase in tyrosine phosphorylation, which reached a maximal level 5 min after the addition of phorbol 12-myristate 13-acetate. The increase in tyrosine phosphorylation of PKC delta was blocked by tyrosine kinase inhibitors genistein and staurosporine. Ionophore-mediated elevation of [Ca2+]i or activation of the beta-adrenergic receptor, epidermal growth factor receptor, or insulin receptor did not promote the tyrosine phosphorylation of PKC delta. These results indicate that tyrosine phosphorylation plays a role in early signal transduction events promoted by the activation of muscarinic and substance P receptors and suggests that the tyrosine phosphorylation of PKC delta has a role in the activation of fluid secretion by neurotransmitters binding to phospholipase C-linked receptors. PMID- 7539429 TI - Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties. AB - MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant 5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo. PMID- 7539430 TI - Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. AB - MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides. By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH2 terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences. Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex. The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity. Sequencing revealed that the NH2 terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH2 terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence. Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH2-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region. PMID- 7539431 TI - The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases. AB - Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437 444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo. PMID- 7539432 TI - The role of human chorionic gonadotropin beta subunit elevation in small-cell lung cancer patients. AB - Human chorionic gonadotropin (HCG)-like immunoreactivity has been found in many non-trophoblastic tumours, but the biological behaviour of HCG-producing cells has not been clarified yet. The aim of the study was to estimate the frequency of serum HCG beta subunit (s beta HCG) elevation in patients with small-cell lung cancer (SCLC) and to assess its possible prognostic role in this type of tumour. An attempt was also made to reclassify the histology in selected cases to see whether the elevated (s beta HCG) level is connected with any special subtype of small-cell lung cancer. A total of 156 SCLC patients entered the study: 93 men, 63 women, median age 58 years. s beta HCG activity was measured by immunoenzyme assay (Abbott EIA beta HCG 15-15) before treatment. s beta HCG elevation (above 5 mIU/ml) was found in 21 of 156 patients (14%). Response to treatment after chemotherapy (complete and partial response) was obtained in only 48% of those patients in whom elevated s beta HCG was found, in comparison to the 73% response rate observed in the remaining patients. Only 5% of patients with elevated s beta HCG survived 2 years, in comparison to 21% surviving for 2 years among the remaining patients. The prognostic significance of elevated s beta HCG and extent of disease were independent of each other (Cox's proportional-hazard model). Thus s beta HCG elevation in SCLC seems to be a marker of more resistant tumours and of poor prognosis. We have not found any connection between the subtype of small cell lung cancer and elevated s beta HCG. Elevated s beta HCG was found in 2 out of 11 patients with oat-cell carcinoma, in 3 out of 10 patients with an intermediate cell type and in 5 out of 13 patients with small-cell lung cancer in which the assessment of the subtype was not possible. PMID- 7539433 TI - Bone cell function, regulation, and communication: a role for nitric oxide. AB - A large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post-translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology. PMID- 7539435 TI - The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein. AB - Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class. PMID- 7539434 TI - Evidence of redox-linked signaling for producing a giant signal complex. AB - Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen activated protein kinase. Finally, all of these signal elements and selected PTYR containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex. PMID- 7539436 TI - Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments. AB - The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix degrading enzymes and the induction of tenascin contribute to the loss of tissue specific gene expression and thus the involuting process. PMID- 7539437 TI - CDEBP, a site-specific DNA-binding protein of the 'APP-like' family, is required during the early development of the mouse. AB - A murine protein, termed CDEBP, was previously shown to bind the double-stranded DNA motif GTCACATG, identical to the yeast centromeric element CDEI. The cDNA sequence showed three domains with extensive similarities to the amyloid beta precursor protein (APP). The protein is homologous over its entire length to the human protein designated APPH. In situ immunofluorescence assays using antibodies raised against distinct parts of CDEBP detected discrete sites of accumulation inside the interphase nucleus, and the bulk of the protein was not associated with mitotic chromosomes. One of the complexes with double-stranded CDEI oligonucleotides detected by gel shift assay was not present when the protein had been selectively removed from nuclear extracts by immunoprecipitation. We reported previously that microinjection into one-cell mouse embryos of DNA fragments including the CDEI sequence results in an early arrest of development with abnormal nuclei containing variable amounts of DNA. The same characteristic figures were observed when embryos were treated with antisense oligonucleotides complementary to parts of the CDEBP coding region. Complexes between the CDEBP protein and CDEI sites in the mouse genome thus appear to play a critical role in the replication/segregation of the embryonic genome. PMID- 7539438 TI - Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm. AB - Acrosome-intact mammalian sperm can adhere to zona pellucida-free oocytes but are only capable of fusing if they have previously undergone the acrosome reaction. This suggests that the acrosome reaction results in presentation of at least one novel epitope which plays a role in sperm-oocyte fusion. Monoclonal antibodies were raised against unfixed acrosome-reacted guinea pig sperm and screened by indirect immunofluorescence for binding to the equatorial segment. They were back screened against unfixed acrosome-intact sperm for absence of binding. Using this approach, two antibodies, G11 and M13, were identified which detect equatorial segment epitopes presented de novo by sperm following an A23187-induced acrosome reaction. The localization of these epitopes to the equatorial segment was confirmed at the ultrastructural level by indirect immunogold-labelling. Fluorescein isothiocyanate-labelled Fab fragments of these two antibodies also localized to the equatorial segment. Affinity chromatography and western blotting established that the two mAbs recognize the same proteins, which have M(r)s of 34, 46, 48 and 51 x 10(3). When sperm were induced to undergo the acrosome reaction with A23187 and incubated with their discharged acrosomal contents, a further band was produced with an M(r) of 30 x 10(3). Production of this band was inhibited in the combined presence of 100 microM phenylmethylsulphonyl fluoride and 100 microM p-aminobenzamidine even though these compounds do not inhibit acrosomal exocytosis. Neuraminidase and O-glycosidase were without effect on the proteins detected by antibodies G11 and M13. Endoglycosidase F, however, eliminated the bands of M(r) 46, 48 and 51 x 10(3) and replaced them with a strong band of M(r) 44 x 10(3) and two minor bands of M(r) 43 and 45 x 10(3). Formaldehyde fixation of acrosome-intact sperm caused partial rupture of the acrosome with loss of the characteristic rouleaux (stacks) of guinea pig sperm. Indirect labelling of these formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539439 TI - Evidence that tenascin and thrombospondin-1 modulate sprouting of endothelial cells. AB - Cultured endothelial cells undergo a reversible transition from a resting (cobblestone) phenotype to an angiogenic (sprouting) phenotype. This transition mimics the early events of angiogenesis. We have previously reported that the addition of exogenous xylosides inhibits endothelial cel sprouting and modifies the extracellular matrix (ECM) synthesised by the cells. We have now investigated whether endothelial sprouting is mediated by the nature of the extracellular matrix in contact with the cells. Accordingly, cell-free matrices deposited by bovine aortic endothelial cells (BAEC) were isolated. These matrices were produced under conditions in which the formation of the sprouting phenotype was permitted (controls) or inhibited (by the addition of exogenous xylosides). BAEC were then plated on these matrices and grown under conditions which promote sprouting. Sprouting proceeded normally on control matrices, whereas it was inhibited when the cells were grown on matrices deposited in the presence of xylosides. The composition of the permissive and inhibitory matrices was then analysed. Inhibitory matrices contained reduced levels of tenascin and increased levels of thrombospondin-1 by comparison to the permissive matrices. In contrast, no differences were detected in the relative levels of laminin. The roles of tenascin and thrombospondin-1 in endothelial sprouting were confirmed using specific antibodies. Immunolocalisation studies revealed the presence of both proteins in sprouting cells. Antibodies to tenascin inhibited the formation of sprouting cells on permissive matrices and on gelatin-coated dishes without affecting cell growth. Tenascin synthesis was increased when sprouting cells were present in the cultures. Antibodies to thrombospondin-1 stimulated sprouting on inhibitory matrices. These results suggest that the transition from a resting to a sprouting phenotype is promoted by tenascin and inhibited by thrombospondin-1. PMID- 7539440 TI - Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice. AB - Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5' flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3' flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5' and 3' flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene. PMID- 7539441 TI - The role of alpha 4 beta 1 integrin in cell motility and fibronectin matrix assembly. AB - The alpha 4 beta 1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-alpha 4 beta 1 antibodies and fibronectin (Fn) fragments have suggested that alpha 4 beta 1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including alpha 5 beta 1 integrin, which is known to function in cell motility on Fn. To test whether alpha 4 beta 1 integrins mediate cell motility on Fn and VCAM 1 in the absence of alpha 5 beta 1 integrin, we expressed human alpha 4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in alpha 5 beta 1 integrin (CHO B2). The parental alpha 5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of alpha 4 beta 1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of alpha 4 beta 1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major alpha 4 beta 1 binding site on Fn. These findings demonstrate that alpha 4 beta 1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of alpha 5 beta 1. Moreover, they reveal important functional differences between Fn binding integrins. The alpha 4 positive, alpha 5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes. PMID- 7539442 TI - Overexpressing cell surface beta 1.4-galactosyltransferase in PC12 cells increases neurite outgrowth on laminin. AB - Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface beta 1.4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin. PMID- 7539444 TI - Complete removal of mycoplasma from viral preparations using solvent extraction. AB - Mycoplasma contamination of two alphavirus isolates and adenovirus 5 was completely removed using a very simple, rapid protocol based on solvent extraction. Treated virus preparations remained free of mycoplasma after 6 months in culture. PMID- 7539443 TI - ELISA for anti-HCV antibody employing a shorter synthetic core region peptide. AB - A new ELISA for anti-HCV antibody was developed employing a shorter synthetic N terminal peptide, 2-62aa, within the core region of 1-191aa. The basic performance of the assay was comparable to three other second-generation assays using longer HCV core antigens. To evaluate assay performance at the borderline level, 25 samples with indeterminate results were selected from 3000 routine serum samples. Only 5 of the 25 sera were found to be HCV-RNA-positive by a nested PCR assay and with apparent clinical evidence of HCV infection. The results of the new ELISA agreed with those of the PCR-RNA test in 23/25 (kappa statistics 0.75), whereas C22-3 of the RIBA II test using 2-120aa of the core agreed in 9/25 (0.09), the Abbott pHCV-34 EIA test using 1-150aa agreed in 10/25 (-0.12), and a neutralization inhibition assay for Abbott EIA II using 2-120aa agreed in 6/25 (0.02). These results indicate that the UBI CORE ELISA has greatly improved specificity and can be a useful indication of viremia in HCV infection. PMID- 7539445 TI - Immunohistochemical characterization of human endometrial microvascular basement membrane components during the normal menstrual cycle. AB - The expression of three basement membrane components [collagen IV (CIV), laminin and heparan sulphate proteoglycan (HSPG)] and platelet endothelial cell adhesion molecule (PECAM) were examined by immunohistochemistry in cryostat sections of normal human endometrium. Alkaline phosphatase (ALP) was detected using enzyme histochemistry. Endometrial biopsies from the menstrual (n = 4), mid-late proliferative (n = 5), early-mid secretory (n = 5) and late secretory (n = 5) stages were collected from women with a normal menstrual cycle. At all four stages of the menstrual cycle, CIV, laminin and HSPG were expressed on basement membranes of both vessels and glands whilst PECAM expression was localized specifically to endothelial cells. A similar number of vessels/mm2 stained for CIV and laminin, as well as for PECAM at each stage of the menstrual cycle, demonstrating that all vessels in endometrium stain for these two basement membrane components. By contrast, the number of vessels/mm2 that stained positively for HSPG and ALP was significantly lower, averaging approximately 55% of the total that stained positively for PECAM, CIV and laminin. During the menstrual stage, HSPG staining intensity remained strong in glandular basement membranes but decreased dramatically in vascular basement membranes. ALP activity was variable in both the vessels and glands throughout the four stages of the menstrual cycle studied. This study demonstrates heterogeneity in basement membrane components within the endometrial microvasculature. It is postulated that the disappearance of HSPG from vascular basement membranes may play a role in the process of vascular remodelling during the menstrual stage of the cycle. PMID- 7539446 TI - Site and menstrual cycle-dependent expression of proteins of the tumour necrosis factor (TNF) receptor family, and BCL-2 oncoprotein and phase-specific production of TNF alpha in human endometrium. AB - Apoptosis in human endometrial epithelium progressively increases from early to late secretory/menstrual phases and remains consistently more prominent in the basalis. It has been suggested that tumour necrosis factor (TNF) alpha secreted during the secretory/menstrual phases plays a role in induction of programmed cell death in these cells. In the present study, we characterized expression of receptors of TNF alpha, Fas antigen and BCL-2 in endometrial cells to gain insight as to whether this type of cell death in endometrium may be related to differential or preferential expression of these proteins at specific phases of the menstrual cycle. In addition, to relate production of TNF alpha to the development of apoptosis, the amount of TNF alpha released by human endometrium was measured. Immunostaining demonstrated that the TNF receptor (TNFr; p55/60)-I, TNFr-II (p75/80) as well as Fas protein were expressed in endometrial epithelium throughout the entire menstrual cycle. This expression was progressively diminished from the basalis towards the upper functionalis. In the proliferative phase, the expression of BCL-2 was prominent in the endometrial glands particularly in those residing in the basalis. This expression became weak as early as the third post-ovulatory day and remained low during the remaining phases of the menstrual cycle. The amount of TNF alpha released by endometrial fragments obtained from various phases of the menstrual cycle was determined. The amount of TNF alpha released into the culture medium by the endometrium was low in the proliferative phase. However, the amount of released TNF alpha progressively increased in the secretory phase and peaked in the menstrual phase. TNFr-I, TNFr-II, Fas, BCL-2 and TNF alpha could be identified by Western blot analysis of proteins extracted from endometrium. Therefore, endometrial epithelium by virtue of expression of receptors of TNF alpha as well as Fas protein is properly poised to respond to ligand signals that regulate apoptosis. Induction of apoptosis in endometrial epithelium and menstrual shedding may be related to loss of the protective effect of BCL-2 as well as to the amount of TNF alpha. PMID- 7539447 TI - Chlamydial immunoglobulin IgG and IgA antibodies in serum and semen are not associated with the presence of Chlamydia trachomatis DNA or rRNA in semen from male partners of infertile couples. AB - The role of Chlamydia (C.) trachomatis in male infertility is controversial. The objective of this study was to determine the prevalence of asymptomatic C. trachomatis infections in male partners of infertile couples, and to compare this result with the presence of chlamydial antibodies in serum and semen. C.trachomatis was detected in five of 50 semen specimens (10%) by either polymerase chain reaction for C. trachomatis DNA or direct DNA probing for C. trachomatis rRNA. There was no association between the detection of C. trachomatis in semen and the presence of chlamydial antibodies in serum or semen. Chlamydial serum antibodies were neither associated with antiserum serum antibodies nor with pathological standard semen parameters. These results indicate that the assessment of chlamydial immunoglobulin IgG and IgA antibodies in serum or semen is of limited use in male infertility work-up, in contrast to its significance in female tubal infertility. The presence of C. trachomatis in semen emphasizes the potential risk of transmission during artificial insemination and other assisted reproductive techniques, and underlines the importance of sensitive direct detection methods in this group of patients. PMID- 7539448 TI - Obstructive azoospermia with agenesis of vas deferens or with bronchiectasia (Young's syndrome): a genetic approach. AB - Two groups of infertile men with obstructive azoospermia were screened for cystic fibrosis (CF) gene mutations (delta F508, exons 3, 4, 7, 10, 11, 14a, 17b, 19, 20, 21). The first group was composed of 26 patients with congenital agenesis of vas deferens (CAVD). The second group was composed of 12 patients with obstructive azoospermia associated with chronic suppurating respiratory disease (Young's syndrome). Of the group with CAVD, 77% of patients showed at least one mutation in the CF transmembrane conductance regulator (CFTR) gene. The delta F508 mutation occurred most frequently (54%), and the second most frequent mutation to occur was R117H (27%). Six patients were double heterozygotes. In Young's syndrome, no CF mutations were detected. CAVD can be considered as an incomplete clinical form of CF. However, the differences observed in CF mutations between CF and CAVD suggest that they are different disorders resulting from mutations in the same gene. Young's syndrome is a very different clinical entity. PMID- 7539449 TI - CD44 is expressed throughout pre-implantation human embryo development. AB - The cell surface glycoprotein CD44 has been demonstrated in a variety of cell types in embryonic and adult tissues. We have established that CD44 is present on human oocytes, cumulus cells, early embryos and pre-hatched blastocysts by indirect immunofluorescence. We have also shown that CD44 is present on 8-11 week placental stroma cells, but not on the trophoblast. These findings demonstrate that CD44 is present throughout preimplantation development, and that down regulation occurs on the embryonic surface after implantation. PMID- 7539450 TI - Differential increase in the maternal serum concentrations of the placental proteins human chorionic gonadotrophin, pregnancy-specific beta 1-glycoprotein, human placental lactogen and pregnancy-associated plasma protein-A during the first half of normal pregnancy, elucidated by means of a mathematical model. AB - The present study was performed to compare the increase in maternal serum concentrations of four placental proteins during the first half of 240 normal pregnancies. The proteins were pregnancy-associated plasma protein-A (PAPP-A), human chorionic gonadotrophin (HCG), human placental lactogen (HPL) hormone, and pregnancy-specific beta 1-glycoprotein (PSG), all produced by trophoblast cells. The median increases were observed to be very close to exponential growth curves. Based on simple assumptions, these growth curves could be explained as being solely dependent on the growth of the placenta. The assumptions were that the proteins were produced in the placenta at a constant rate per gram of placental cell mass and secreted into the circulation shortly after synthesis. Our investigations showed that for two of the proteins, PSG and HPL, the rate constants were, in fact, close to the reported growth rate of the placenta, whereas the PAPP-A production rate constant was significantly higher than those of the others. The production curve for HCG was very different from that of the other proteins. PAPP-A and HCG must therefore have more complicated mechanisms for regulating the production. An equation was constructed that permitted estimation of the molar production of the placental proteins per gram of placental cell mass per day during the first half of normal pregnancy. The value was highest for HPL and lowest for PAPP-A. PMID- 7539451 TI - The CD28/CTLA-4:B7 receptor system in experimental autoimmune encephalomyelitis. PMID- 7539452 TI - Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion. AB - Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules. PMID- 7539453 TI - c-KIT expression enhances the leukemogenic potential of 32D cells. AB - The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells. PMID- 7539455 TI - Interactions between L-arginine and L-glutamine change endothelial NO production. An effect independent of NO synthase substrate availability. AB - The effect of extracellular L-arginine and L-glutamine on nitric oxide (NO) release was studied in cultured bovine aortic endothelial cells and in rabbit aortic rings. Increasing L-arginine (0.01 to 10 mM) did not alter NO release from cultured endothelial cells or modify endothelium-dependent relaxation to acetylcholine in isolated vessels. L-Glutamine (0.6 and 2 mM) inhibited NO release from cultured cells (in response to bradykinin) and from aortic rings (in response to acetylcholine or ADP). L-Arginine (0.1-10 mM) dose-dependently reversed the L-glutamine inhibition of receptor-stimulated NO release in both models. In contrast to its inhibitory response to receptor-mediated stimuli, glutamine alone slightly potentiated NO release in both models when the calcium ionophore, A23187, was added. Furthermore, cultured cells incubated with L arginine (0.01-10 mM), in the presence or absence of glutamine, released similar amounts of NO in response to A23187. L-Glutamine did not affect intracellular L arginine levels. Neither D-glutamine nor D-arginine affected NO release or endothelium-dependent vascular relaxation. L-Glutamine had no effect on the activity of endothelial NOS assessed by L-arginine to L-citrulline conversion. These findings show that in the absence of L-glutamine, manipulating intracellular L-arginine levels over a wide range does not affect NO release. L Glutamine in concentrations circulating in vivo may tonically inhibit receptor mediated NO release by interfering with signal transduction. One mechanism by which L-arginine may enhance NO release is via reversal of the inhibitory effect of L-glutamine, but apparently independently of enhancing NO synthase substrate. PMID- 7539454 TI - Specific binding of endocrine transforming growth factor-beta 1 to vascular endothelium. AB - The presentation of recombinant biologically active 125I-TGF-beta 1 via the bloodstream to potential target cells in mice and rats was evaluated by quantitative light and electron microscope radioautography. Specificity was evaluated by in vivo competition with excess unlabeled TGF-beta 1, and integrity of the ligand at the binding site was demonstrated by trichloroacetic acid precipitation after extraction from tissues. The distribution of radiolabel at 2.5, 15, 30, 45, and 60 min after 125I-TGF-beta 1 injection revealed radiolabel principally over microvasculature endothelium but at times > 2.5 min over endothelial endocytic components indicative of internalization. Nonspecific binding of 125I-TGF-beta 1 to the apex of the proximal convoluted tubule of the kidney indicated it as the likely site of rapid clearance of TGF-beta 1 from the circulation, while a comparison of the binding of 125I-TGF-beta 1 (endothelial) to that of 125I-TGF-beta 1 complexed with alpha 2-macroglobulin-methylamine (liver parenchyma) indicated that clearance of TGF-beta 1 complexed alpha 2 macroglobulin was likely via the hepatic alpha 2-macroglobulin receptor. The endothelial TGF-beta receptors uncovered here are likely involved in the local regulatory mechanism of leukocyte and monocyte adhesion and tissue infiltration regulated by endocrine TGF-beta 1. PMID- 7539456 TI - Blockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts. AB - Graft arteriopathy, a leading cause of cardiac allograft failure, is associated with increased intimal smooth muscle cells, inflammatory cells, and accumulation of extracellular matrix. We hypothesized that cellular fibronectin plays a pivotal role in the progression of the allograft arteriopathy by directing the transendothelial trafficking of inflammatory cells through interaction of the connecting segment-1 (CS1) motif with the very late antigen-4 (VLA-4) integrin, and tested this in vivo using a blocking peptide. Cholesterol-fed rabbits underwent heterotopic cardiac transplantation without immunosuppression. The treatment group (n = 7) received a synthetic CS1 peptide (1 mg/kg per d, subcutaneously), and the controls (n = 7) received an inactive peptide (1 mg/kg per d, subcutaneously). At 7-8 d after transplantation, hearts were harvested and sectioned for morphometric analysis and immunohistochemical studies. We observed a > 50% decrease in the incidence (P < 0.001) and severity (P < 0.001) of donor coronary artery intimal thickening in the CS1-treated compared with the control group. These findings correlated with reduced infiltration of T cells (P < 0.05), a trend toward decreased expression of adhesion molecules (P < 0.06), and less accumulation of fibronectin (P < 0.03). Our data suggest that the VLA-4 fibronectin interaction is critical to the progression of the allograft arteriopathy by perpetuating the immune-inflammatory response in the vessel wall. PMID- 7539457 TI - Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo. AB - Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1 deleted recombinant adenoviruses limit the duration of transgene expression. PMID- 7539459 TI - The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. AB - The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most specific serological markers of RA. Using indirect immunofluorescence, AKA label the stratum corneum of various cornified epithelia and APF the keratohyalin granules of human buccal mucosa epithelium. We recently demonstrated that AKA recognize human epidermal filaggrin. Here, we report the identification of the major APF antigen as a diffuse protein band of 200-400 kD. This protein is seen to be closely related to human epidermal (pro) filaggrin since it was recognized by four antifilaggrin mAbs specific for different epitopes, and since the APF titers of RA sera were found to be correlated to their AKA titers and to their immunoblotting reactivities to filaggrin. Immunoabsorption of RA sera on purified epidermal filaggrin abolished their reactivities to the granules of buccal epithelial cells and to the 200-400-kD antigen. Moreover, antifilaggrin autoantibodies, i.e., AKA, affinity purified from RA sera, were shown to immunodetect the 200-400-kD antigen and to stain these granules. These results indicate that AKA and APF are largely the same autoantibodies. They recognize human epidermal filaggrin and (pro) filaggrin related proteins of buccal epithelial cells. Identification of the epitopes recognized by these autoantibodies, which we propose to name antifilaggrin autoantibodies, will certainly open new paths of research into the pathophysiology of RA. PMID- 7539460 TI - 2'3'-Dideoxycytidine-induced thymic lymphoma correlates with species-specific suppression of a subpopulation of primitive hematopoietic progenitor cells in mouse but not rat or human bone marrow. AB - The nucleoside analogue, 2',3'-dideoxycytidine (ddC), is a potent inhibitor of HIV replication, and AIDS patients receiving ddC experience clinical improvement without significant hematologic toxicity. Repeated ddC administration (1,000 mg/kg per day) for 13 wk produces an increased incidence of thymic lymphoma in B6C3F1 mice. Previous studies reveal a common link between chemically induced and genetically associated models of mouse thymic lymphoma that involves a defect in a subpopulation of primitive hematopoietic progenitor cells. This defect is characterized by suppression of a subpopulation of IL-3-responsive cells and ablation of stem cell factor synergy with GM-CSF. The present study was undertaken to ascertain whether ddC produces the same pattern of bone marrow toxicity in mice, and whether this effect is observed in rat and human bone marrow. ddC exposure in vivo and in vitro produced a select suppression of murine CFU identical to that previously described for other models of mouse thymic lymphoma. In contrast, this selective CFU suppression was not observed in rat and human bone marrow or in CD34+ cells. These studies suggest that the mouse may not be a good predictive model for ddC hematotoxicity in humans and that susceptibility to the development of thymic lymphoma may be unique to the mouse. PMID- 7539461 TI - Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. AB - T cell activation involves not only recognition of antigen presented by the MHC, but also nonspecific interactions termed "costimulation." The costimulatory molecules B7-1 and B7-2 are ligands on antigen-presenting cells for the CD28 and CTLA-4 receptors on T cells. Previously, a fusion protein consisting of human CTLA-4 linked to human Fc was shown to bind B7-1 and B7-2 with high avidity and to prevent specific T cell activation. Here we investigated the effects of a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 bound to mouse IgG2a Fc (CTLA-4-Fc) upon experimental autoimmune encephalomyelitis, a T cell-mediated disease that serves as a model for multiple sclerosis. CTLA-4-Fc prevented experimental autoimmune encephalomyelitis in 26 of 28 CTLA-4-Fc-treated mice (median maximum score 0), whereas 28 of 30 mice treated with control mouse IgG2a developed disease (median maximum score 2.75). Less inflammation and virtually no demyelination or axonal loss occurred in CTLA-4-Fc treated compared with control-treated mice. Activated splenocytes from CTLA-4-Fc treated mice were able to transfer disease adoptively to naive recipients. These results indicate a key role for the B7/CD28 system in the development of actively induced murine experimental autoimmune encephalomyelitis, suggesting an area of investigation with therapeutic potential for multiple sclerosis. PMID- 7539458 TI - Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation. AB - The majority of human malignant glioma cells express Fas/APO-1 and are susceptible to Fas/APO-1 antibody-mediated apoptosis in vitro. The sensitivity of Fas/APO-1-positive glioma cell lines to Fas/APO-1 antibody-mediated killing correlates inversely with the constitutive expression of the antiapoptotic protooncogene bcl-2. Here we report that BCL-2 protein expression of human glial tumors in vivo correlates with malignant transformation in that BCL-2 immunoreactive glioma cells were more abundant in WHO grade III/IV gliomas than in grade I/II gliomas. Fas/APO-1 antibody-sensitive human glioma cell lines stably transfected with a murine bcl-2 cDNA acquired resistance to Fas/APO-1 antibody-mediated apoptosis. Forced expression of bcl-2 also attenuated TNF alpha mediated cytotoxicity of glioma cell lines in the presence of actinomycin D and cycloheximide and conferred partial protection from irradiation and the cancer chemotherapy drugs, cisplatin and BCNU. Preexposure of the glioma cell lines to the cytokines, IFN gamma and TNF alpha, which sensitize for Fas/APO-1-dependent killing, partially overcame bcl-2-mediated rescue from apoptosis, suggesting that multimodality immunotherapy involving cytokines and Fas/APO-1 targeting might eventually provide a promising approach to the treatment of human malignant gliomas. PMID- 7539463 TI - Retrovirus-like activity in an immunosuppressed dog: pathological and immunological findings. AB - A putative retrovirus was isolated from a dog with a severe, acquired immunodeficiency-like syndrome. The haematological abnormalities and immunological deficiencies included anaemia, leucopenia (lymphopenia and neutropenia), thrombocytopenia, decreased humoral immunity, and ineffective T cell responses in-vitro. The necropsy findings included generalized lymphoid depletion, severe bone marrow hypoplasia, plasmacytic infiltrates in lymphoid and non-lymphoid organs, and severe secondary infections. Supernates of peripheral blood mononuclear cell cultures from the affected dog contained an agent with manganese-dependent reverse transcriptase (RT) activity that sedimented at a density of 1.122 g/ml. RT activity was also found post-mortem in extracts prepared from the bone marrow, lymph nodes, and small intestine. The lymph nodes and small intestine expressed a 3.8 kb mRNA that was recognized by a bovine leukaemia virus (BLV) pol DNA probe by Northern blotting. DNA isolated from the lymph nodes and small intestine from the affected dog showed distinct band patterns by Southern analysis, suggesting an exogenous retrovirus. The retrovirus could be propagated in normal canine peripheral blood mononuclear cells or short term canine lymphocyte cell lines in-vitro, and was cytopathogenic for cells of canine, but not human, origin. These results suggest the existence of a pathogenic canine retrovirus capable of producing disease of the type associated with retroviruses in other species. PMID- 7539462 TI - Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia. AB - The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis. PMID- 7539464 TI - Nitric oxide synthase containing nerves in the atrioventricular node of the guinea pig heart. AB - Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in the guinea-pig atrioventricular node. Many small ganglia were found in the interatrial septum near the atrioventricular node. A small number of neurons in these ganglia were immunoreactive. NOS immunoreactive axons were observed in nerve bundles near or within the node. Very thick immunoreactive axons, 8-10 microns in diameter, were found between the conductive tissue and the ordinary cardiac muscles. Electron-microscopic examination confirmed that these axons were unmyelinated. Nerve bundles in this region contained both myelinated and unmyelinated axons; however, immunoreactivity was detected exclusively in unmyelinated axons. Fine NOS-immunoreactive nerve fibers with terminal varicosities were often seen in the atrioventricular node. Close contact between NOS-immunoreactive axon varicosity and specialized cardiac muscle cell was observed. Intranodal ganglia were observed among the specialized cardiac muscles. They were surrounded by numerous axons, some of them were immunoreactive. Direct axo-somatic synapses from NOS-immunoreactive terminals to the intranodal ganglion cells were observed. The present results indicate a possibility that nitric oxide plays a role in the neural control of the conductive tissue in the heart through direct neuromuscular contact. PMID- 7539465 TI - Gliomatosis cerebri presenting as intractable epilepsy during early childhood. AB - We review 160 cases of gliomatosis cerebri from the literature and report an additional three infants and young children who presented with intractable epilepsy, corticospinal tract deficits, and developmental delay in whom a pathologic diagnosis was made. The progressive nature of the encephalopathy in our cases was documented by serial clinical examination, electroencephalograms, magnetic resonance imaging, and positron emission tomographic scans. The natural history of gliomatosis cerebri was determined by a retrospective review of the literature of 160 cases in 85 reports. The most common neurologic symptoms and signs included corticospinal tract deficits (58%), dementia/mental retardation (44%), headache (39%), seizures (38%), cranioneuropathies (37%), increased intracranial pressure (34%), and spinocerebellar deficits (33%). The most commonly involved central nervous system structures were the centrum semiovale and cerebrum (76%), mesencephalon (52%), pons (52%), thalamus (43%), basal ganglia (34%), and the cerebellum (29%). Fifty-two percent of patients were dead within 12 months of onset. Different grades of glial neoplasm may also coexist within gliomatosis cerebri such as astrocytoma with anaplastic astrocytoma, atypical or anaplastic oligodendroglioma, and glioblastoma multiforme. Hypotheses regarding the pathogenesis of gliomatosis cerebri include blastomatous dysgenesis, diffuse infiltration, multicentric origin, in situ proliferation, and "field transformation." The biologic determinants of whether a transformed glial cell behaves as a relatively localized tumor mass or truly loses anchorage dependence to become migratory as well as proliferative are not understood. PMID- 7539466 TI - Differentiation of human trophoblast populations involves alterations in cytokeratin patterns. AB - Cytokeratins (CKs) are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villous and extravillous trophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravillous trophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CK13 showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblasts were also positive for CK7 and CK13. CK17 was found related to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblasts in cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti CK13 antibodies in the first trimester but was positive at term. CK4 and CK16 were not found in the placenta. Our study shows for the first time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specific patterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast. PMID- 7539468 TI - Pentoxifylline inhibits T-cell adherence to keratinocytes. AB - In many inflammatory dermatoses leukocyte function-associated antigen 1/intercellular adhesion molecule-1 mediated T-cell/keratinocyte adhesion is considered to play an important role. Pentoxifylline (PTX), a methylxanthine derivative widely used for the symptomatic treatment of various vascular disorders, was recently found to have anti-inflammatory effects. PTX can suppress tumor necrosis factor-alpha production and function, and inhibits leukocyte endothelial cell adherence. The aim of the present study was to investigate whether PTX also interferes with T-cell/keratinocyte binding. Peripheral blood T cells were activated with phorbol myristate acetate and co-incubated with interferon-gamma- or tumor necrosis factor-alpha-stimulated keratinocytes (SVK 14 cells) in the presence or absence of PTX. Using an enzyme-linked immuno cell adhesion assay PTX was found to inhibit T-cell/keratinocyte adhesion in a dose dependent manner. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methylxanthine derivative, or by a combination of two cyclic adenosine monophosphate analogues. No major effect on T cell/keratinocyte adherence was observed when PTX was present during the pre incubation of keratinocyte monolayers with tumor necrosis factor-alpha or interferon-gamma prior to the adhesion assay. In keratinocyte monolayers the interferon-gamma or tumor necrosis factor-alpha induced intercellular adhesion molecule-1 expression could not be inhibited by PTX. However, when PTX was added to short-term organ cultures of normal human skin biopsies, the lipopolysaccharide- and tumor necrosis factor-alpha-induced keratinocyte intercellular adhesion molecule-1 expression was blocked completely. The interferon-gamma-induced ICAM-1 expression was not blocked by PTX. The results presented herein suggest that impaired T-cell/keratinocyte binding may be one of the mechanisms by which PTX exerts a beneficial effect in certain inflammatory dermatoses. PMID- 7539467 TI - Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit. AB - Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity. PMID- 7539469 TI - Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus. AB - Patients with the autoimmune blistering disease pemphigus foliaceus (PF) have circulating autoantibodies directed against the desmosomal cadherin desmoglein 1 (Dsg1). Based on the fact that purified IgG fractions from PF patients induce loss of cell adhesion in organ culture and in a neonatal mouse model, it has been proposed that these anti-Dsg1 antibodies play a pathogenic role in blister formation. To directly address whether antibodies in PF sera specific for the Dsg1 extracellular domain are indeed pathogenic in the disease, PFIg, a chimeric protein containing the entire extracellular domain of human Dsg1 and the constant region of human IgG1, was produced by baculovirus expression. Incubation of PF patients' sera with the PFIg baculoprotein removed the immunoreactivity of autoantibodies against keratinocyte cell surfaces in all 20 PF and eight Brazilian PF patients' sera tested. This adsorption was conformation dependent, because PFIg protein denatured by low pH or heat was no longer able to adsorb the immunoreactivity of PF sera. Furthermore, the incubation with the PFIg baculoprotein eliminated the pathogenic activity of PF patients' sera and prevented gross blister formation in a neonatal mouse model of pemphigus. Anti Dsg1 antibodies eluted from the PFIg protein column were pathogenic as they resulted in the appearance of gross blisters in neonatal mice with typical histologic findings of PF. These observations indicate that the extracellular domain of Dsg1 expressed by baculovirus is capable of specifically immunoadsorbing pathogenic autoantibodies from PF patients' sera and provide direct evidence that the anti-Dsg1 autoantibodies in PF sera are indeed pathogenic. The availability of this Dsg1 recombinant protein may facilitate the development of antigen-specific plasmapheresis as a novel therapeutic strategy in pemphigus. PMID- 7539470 TI - Interstitial collagenase is expressed by keratinocytes that are actively involved in reepithelialization in blistering skin disease. AB - Migrating keratinocytes actively involved in reepithelialization in dermal wounds acquire a collagenolytic phenotype upon contact with the dermal matrix. To determine whether this phenotype is associated with repair in other forms of wounds, we assessed collagenase expression in 50 specimens representing a variety of blistering skin diseases, including subtypes of epidermolysis bullosa, porphyria cutanea tarda, bullous pemphigoid, pemphigus, transient acantholytic dermatosis, and suction blisters. Distinct from that seen in chronic ulcers or in normal healing by second intention, reepithelialization in these blistering conditions was not necessarily associated with a complete loss of basement membrane, as determined by immunostaining for type IV collagen. Collagenase mRNA was detected in the basal keratinocytes of several specimens of epidermolysis bullosa simplex (six of 10) and of pemphigus (three of seven), as well as in one quarter of transient acantholytic dermatosis samples in the presence of an intact basement membrane. In contrast, three of nine porphyria cutanea tarda, one third of epidermolysis bullosa acquisita, and one of 10 bullous pemphigoid samples had collagenase-positive basal keratinocytes with the basement membrane disrupted. The collagenase-positive lesions generally represented older blisters with evidence of epithelial regeneration. Collagenase was also expressed in suction blisters at 2 and 5 d after induction of the blister, but was shut off when the epidermis had healed. Other metalloproteinases were expressed occasionally, if at all. Our results suggest that keratinocyte migration is associated with collagenase expression and that contact of keratinocytes with the dermal matrix is not necessarily needed for collagenase induction. PMID- 7539471 TI - Intercellular adhesion molecule-3 (CD50) on human epidermal Langerhans cells participates in T-cell activation. AB - Three different intercellular adhesion molecules (ICAMs) have been identified acting as ligand for counter-receptor leukocyte-function-associated antigen-1 (LFA-1) (CD11a/CD18). We have recently shown that ICAM-1 (CD54) is present on cultured human epidermal Langerhans cells but not on freshly isolated Langerhans cells, and that this molecule participates in the generation of an antigen specific T-cell response. ICAM-2 (CD102) was not involved because this molecule is expressed by neither fresh nor cultured Langerhans cells. In this study, the presence of ICAM-3 (CD50) on Langerhans cells was examined. Flow cytofluorometric analysis demonstrated that ICAM-3 is strongly displayed by fresh Langerhans cells, and daily determinations showed that the level of this trypsin-resistant molecule remained nearly unchanged during in vitro culture for up to 4 d, indicating that Langerhans cells constitutively express this molecule. Analysis of RNA extracted from purified cultured Langerhans cells by means of reverse transcriptase-polymerase chain reaction demonstrated the presence of mRNA specific for ICAM-3. Antigen-specific T-cell responses triggered by Langerhans cells were dose-dependently inhibited by anti-ICAM-3 if the antibody was added within the first 16 h of T-cell stimulation. Simultaneous addition of anti-ICAM-1 and anti-ICAM-3 synergistically inhibited T-cell responses, although a total block was never achieved. Pretreatment of Langerhans cells with anti-ICAM-3 resulted in a reduced T-cell response, whereas pretreatment of T cells did not. These results suggest that ICAM-3 on Langerhans cells, like ICAM-1, is functionally involved in the initiation of antigen-specific activation of T cells, but the expression of these two ICAMs on Langerhans cells is differently regulated. PMID- 7539472 TI - Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). AB - The effect of the appearance of drug-resistant human immunodeficiency virus type 1 (HIV-1) on viral RNA load was studied in patients treated with the reverse transcriptase inhibitor lamivudine. During the first 12 weeks of treatment, HIV-1 RNA concentrations and amino acid changes in codon 184, causing high-level resistance to lamivudine, were determined in longitudinal serum samples from HIV 1 p24 antigen-positive and -negative patients. A marked decline in the amount of HIV-1 RNA (approximately 95% below baseline) and HIV-1 p24 antigen was observed within 2 weeks, followed by a rise that coincided with the appearance of lamivudine-resistant viruses in serum (isoleucine mutants initially, which were subsequently replaced by valine variants). After 12 weeks, a partial antiviral effect was observed despite the presence of a complete codon 184 mutant virus population in serum. This study shows that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load. PMID- 7539473 TI - Beneficial effect of recombinant human granulocyte colony-stimulating factor on fungicidal activity of polymorphonuclear leukocytes from patients with AIDS. AB - The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration on the functional status of polymorphonuclear leukocytes (PMNL) in neutropenic AIDS patients was investigated. PMNL destructive activity against Candida albicans or encapsulated or acapsular Cryptococcus neoformans was significantly impaired with respect to control subjects before rhG-CSF treatment. After subcutaneous administration of rhG-CSF (5 micrograms/kg), neutrophil counts increased 3- to 11-fold in 24 h and returned to baseline within 96 h. PMNL fungicidal activity showed significant enhancement 48-72 h after rhG-CSF administration that decreased to baseline within 96 h. Enhanced rhG-CSF-mediated destructive activity strictly correlated with augmented superoxide anion production by PMNL. These findings suggest that therapeutic use of rhG-CSF at appropriate schedules in neutropenic AIDS patients could decrease the risk of infection or, in association with antibiotic therapy, more rapidly resolve the occurring infections. PMID- 7539475 TI - Preliminary assessment of a human recombinant antibody fragment to hsp90 in murine invasive candidiasis. AB - Seroconversion to hsp90 is associated with recovery from systemic candidiasis in humans, and a murine monoclonal antibody to this hsp90 antigen (LKVIRK epitope) was protective in mice. A human recombinant antibody to the same epitope was assessed in acute and chronic models of murine invasive candidiasis. Lethal intravenous challenge with fluconazole-susceptible (strain 4) or fluconazole resistant (strain 019) Candida albicans, followed 2 h later by a single dose of recombinant antibody, was associated with a statistically significant drop in mortality of > or = 40% (two experiments in BALB/c mice given strain 4; one experiment in CD-1 mice given strain 019) or 23% (BALB/c mice, strain 019). In mice sublethally infected with strain 4, treatment with recombinant antibody was associated with improved renal clearance of infection. Antibody-mediated protection may involve neutralization of the protein-binding properties of circulating candidal hsp90, since LKVIRK strongly bound dexamethasone in vitro. PMID- 7539474 TI - Molecular characterization of a parasite antigen in sera from onchocerciasis patients that is immunologically cross-reactive with human keratin. AB - Onchocerca volvulus is a nematode that causes severe dermatitis and blindness in humans. Prior studies have shown that immune complexes in sera from onchocerciasis patients contain parasite antigens with M(r) of 23,000 and 65,000 70,000. Monoclonal antibody OV-1 binds to these antigens and to corresponding antigens in adult worm extracts and in vitro culture supernatants. OV-1 was used to immunoscreen an O. volvulus adult worm cDNA library. Clone OV1CF contains a 1632-bp open-reading frame that codes for a protein with a predicted M(r) of 63,000. The deduced protein sequence of OV1CF has 78% identity with Ascaris lumbricoides intermediate filament A and 40%-50% identity with several mammalian intermediate filaments. Antibodies raised to OV1CF bind to human keratin, and some sera from onchocerciasis patients contain antibodies to OV1CF and to keratin. Thus, immune complexes from onchocerciasis patients contain a parasite antigen that is immunologically cross-reactive with human intermediate filament proteins. PMID- 7539476 TI - Insulin-like growth factor-1 enhances epidermal growth factor receptor activation and renal tubular cell regeneration in postischemic acute renal failure. AB - Growth factors such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and hepatocyte growth factor have been shown to accelerate the recovery from postischemic acute renal failure (ARF) with a concomitant increase in DNA synthesis. Interactions between growth factors have been demonstrated in a number of in vitro studies. This study examined the effect of exogenous IGF-1 on the DNA synthesis and EGF receptor (EGF-R) activation in postischemic rat kidneys. Thirty minutes after the relief of 30-minute total occlusion of the left renal artery in anesthetized 225 to 300 gm Sprague-Dawley rats, either IGF-1 (75 micrograms/kg) or normal saline solution (NS, 0.2 ml) was given by intravenous bolus, followed by twice daily subcutaneous injections of IGF-1 (50 micrograms/kg) or 0.2 ml NS for 4 days, respectively, in IGF-1-Tx) and NS treated (NS-Tx) groups (n = 8 each). On the day after the completion of treatment, inulin clearance (ml/kg/min) of the postischemic kidneys in the IGF-1-Tx group was significantly higher (p < 0.01) than inulin clearance of kidneys in the NS-Tx group. This was associated with improved kidney morphology. IGF-1 treatment also enhanced the labeling index of 5-bromo-2'-deoxyuridine (percent of stained tubule cells), a marker for active DNA synthesis, in the outer medulla of postischemic kidneys at 1 day and 2 days after the injury. EGF-R tyrosine phosphorylation (which reflects receptor activation) increased in postischemic kidneys in both NS Tx (n = 5) and IGF-1-Tx (n = 3) groups 1 day after the injury as compared with nonischemic contralateral kidneys. In the IGF-1-Tx group there was also increased iodine 125-labeled EGF binding and EGF-R protein. Our results demonstrate a beneficial effect of IGF-1 on postischemic ARF. Furthermore, they suggest that EGF-R activation is involved in tubular regeneration and that IGF-1 may enhance EGF-R activation by increasing EGF-R expression. PMID- 7539478 TI - Dietary intake of pirfenidone ameliorates bleomycin-induced lung fibrosis in hamsters. AB - There are no clinically efficacious drugs available for preventing the development of pulmonary fibrosis (PF). In the present study, we tested the antifibrotic potential of pirfenidone (PD) in the bleomycin (BL) hamster model of PF. Hamsters were intratracheally instilled with isotonic saline solution or BL (7.5 U/kg/5 ml). The animals were fed control diet containing 0.5% PD or the same diet without the drug 2 days before and throughout the study. The four groups were as follows: saline-instilled and fed the control diet (SCD); saline instilled and fed the same diet containing PD (SPD); BL-instilled and fed the control diet (BCD); and BL-instilled and fed the same diet containing PD (BPD). The animals were killed at 21 days after intratracheal instillation and their lungs processed for various assays. The lung hydroxyproline levels, an index of PF, in SCD, SPD, BCD, and BPD groups were 949, 970, 1759, and 990 micrograms/lung, respectively. The SOD activity and malondialdehyde equivalent levels in the corresponding groups were 443, 524, 612, and 499 units/lung and 56, 49, 108, and 63 nmol/lung, respectively. The lung prolyl hydroxylase activities in the SPD, BCD, and BPD groups were 87%, 147%, and 93% of the control (SCD) group (4.2 x 10(4) dpm/lung/30 minutes), respectively. The lung myeloperoxidase activities were 97%, 236%, and 159% of the control group (0.73 units/lung), respectively. BL alone caused significant increases in all the biochemical markers of lung toxicity, and dietary intake of PD minimized the BL toxicity as reflected by significant decreases in all the above markers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539477 TI - Monocyte modulation of endothelial leukocyte adhesion molecules. AB - Leukocyte adhesion to endothelium is dependent on expression of specialized molecules. Several of these molecules are upregulated by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). We investigated the effect of medium conditioned by unstimulated (MCM) or stimulated monocytes and of recombinant cytokines on endothelial adhesion receptor expression. IL-1 beta, TNF-alpha, and MCM induced E-selectin similarly, whereas MCM induced VCAM-1 and ICAM-1 to a lesser extent than did TNF-alpha, and MCM induced VCAM-1 only weakly. The addition of pentoxifylline (10(-3) mol/L) to monocytes during MCM preparation blocked TNF-alpha production but not that of IL 1 beta or IL-6, and it reduced IL-1ra significantly (p < 0.05). When the MCM was devoid of TNF-alpha or when TNF-alpha was neutralized with a specific antibody, the action of MCM on E-selectin expression was significantly lower. Anti-IL-1 beta decreased the activity of MCM on endothelial E-selectin expression by about 50%. The effect of MCM on adhesion molecules was accompanied by an increase in monocyte adhesion. Inhibition of TNF-alpha production reduced monocytes adhesion slightly but significantly (18%, p < 0.05), whereas anti-IL-1 beta antibody decreased adhesion by 48% (p < 0.001). These results show that adherent monocytes released cytokines and antagonists that affect leukocyte adhesion receptors on endothelium differently from recombinant cytokines. E-selectin expression--and to a lesser extent ICAM expression--is modified, resulting in a modulation of leukocyte adhesion to endothelium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539479 TI - Behavior of nucleolar organizer regions in the different hepatic lobes after end to-side portacaval shunt in the rat. AB - Protein synthesis activity in the hepatic lobes in control and end-to-side portacaval shunt rats was studied by assaying one of the argyrophilic components (Ag-NOR) of the hepatocytic nucleolus. The differences found in the area and number of Ag-NOR for each nucleus and in the percentage of Ag-NOR area to nucleus area in each hepatic lobe in control rats as well as in the end-to-side portacaval shunt rats suggest that there are interlobular differences in the control rats related to this parameter that are indicative of protein synthesis and cell proliferation. Furthermore, the changes that occur in these parameters after the deprivation of portal flow produced by an end-to-side portacaval shunt make it possible to hypothesize on the existence of a hepatic lobular functional heterogeneity in the rat liver. PMID- 7539482 TI - Tenascin-C expression during wallerian degeneration in C57BL/Wlds mice: possible implications for axonal regeneration. AB - Schwann cells in the distal stumps of lesioned peripheral nerves strongly express the extracellular matrix glycoprotein tenascin-C. To gain insights into the relationship between Wallerian degeneration, lesion induced tenascin-C upregulation and regrowth of axons we have investigated C57BL/Wlds (C57BL/Ola) mice, a mutant in which Wallerian degeneration is considerably delayed. Since we found a distinct difference in the speed of Wallerian degeneration between muscle nerves and cutaneous nerves in 16-week-old C57BL/Wlds mice, as opposed to 6-week old animals in which Wallerian degeneration is delayed in both, we chose the older animals for closer investigation. Five days post lesion tenascin-C was upregulated in the muscle branch (quadriceps) but not in the cutaneous branch (saphenous) of the femoral nerve in 16-week-old animals. In addition myelomonocytic cells displaying endogenous peroxidase activity invaded the muscle branch readily whereas they were absent from the cutaneous branch at this time. We could further show that it is only a subpopulation of axon-Schwann cell units (mainly muscle efferents) in the muscle branch which undergo Wallerian degeneration and upregulate tenascin-C at normal speed and that the remaining axon-Schwann cell units (mainly afferents) displayed delayed Wallerian degeneration and no tenascin-C expression. Regrowing axons could only be found in the tenascin-C-positive muscle branch where they always grew in association with axon-Schwann cell units undergoing Wallerian degeneration. These observations indicate a tight relationship between Wallerian degeneration, upregulation of tenascin-C expression and regrowth of axons, suggesting an involvement of tenascin-C in peripheral nerve regeneration. PMID- 7539481 TI - Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise. AB - LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed. PMID- 7539480 TI - Regulation of CFTR Cl- channel gating by ADP and ATP analogues. AB - The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies. PMID- 7539483 TI - A novel omega-conopeptide for the presynaptic localization of calcium channels at the mammalian neuromuscular junction. AB - Voltage-sensitive Ca2+ channels are essential to transmitter release at the chemical synapse. To demonstrate the localization of voltage-sensitive Ca2+ channels in relation to the site of transmitter release, mouse neuromuscular junctions were double labelled with alpha-bungarotoxin and a novel voltage sensitive Ca2+ channel probe, SNX-260, a synthetic analog of omega-conopeptide MVIIC. Similar to omega-conopeptide MVIIC, biotinylated SNX-260 blocked nerve stimulated transmitter release at the mouse neuromuscular junction. Fluorescently tagged biotinylated SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen in en face view. This SNX-260 labelling was inhibited by preincubation with unconjugated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholine receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labelling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (omega conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel blocker. These findings suggest that SNX-260 is a novel probe for localizing non-N type voltage sensitive Ca2+ channels and that these voltage-sensitive Ca2+ channels are localized near the transmitter release sites at the mammalian motor nerve terminal membrane. The results are consistent with the suggestion that non-N, probably P/Q type voltage-sensitive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction. PMID- 7539484 TI - alpha-Aminoalkylphosphonate di(chlorophenyl) esters as inhibitors of serine proteases. AB - alpha-Aminoalkylphosphonate di(chlorophenyl) esters and (alpha aminoalkyl)phenylphosphinate phenylesters have been tested as irreversible inhibitors of human neutrophil elastase, porcine pancreatic elastase and chymotrypsin, serine proteases important in biochemical processes. Peptidyl derivatives of diphenyl (alpha-aminoalkyl) phosphonates have previously been shown to be potent and specific inhibitors of serine proteases at low concentrations. Addition of a halogen to the phenoxy group of the inhibitors should make the leaving group more electrophilic, and thus more reactive. Peptide phosphonate inhibitors with chlorine in the meta- or para-positions of the phenoxy ester moiety were synthesized and shown to be potent inhibitors of elastase. Tripeptide phosphonates are more potent inhibitors than dipeptide phosphonates, however, addition of the halogen did not increase the inhibitory potency of these phosphonates with elastase compared to the non-halogenated phosphonates. In the case of chymotrypsin, the halogenated phenoxy esters were more reactive, possibly due to an alternate binding mode. The novel (alpha aminoalkyl)phenylphosphinate phenylesters were poor inhibitors of serine proteases. PMID- 7539486 TI - Formation of pi, tau-dimethylhistidine on alkylation of trypsin with active-site directed sulfonic acid methyl esters. AB - The possibility of synthesizing stable alkyl analogues of acyl trypsins by introducing the alkyl residue by means of active-site-directed sulfonic acid esters was studied. Nine amidino- or guanidino-substituted sulfonic acids of different geometries and their methyl esters were prepared. The time-dependent inhibition of bovine trypsin by these esters, indicating modification at the active site of the enzyme, was followed. With the exception of p guanidinobenzenesulfonic acid methyl ester, all the esters acted as irreversible inhibitors. The site of methylation, Ser-195 or His-57 (chymotrypsinogen numbering), was determine by analyzing for O-methylserine and methylhistidines. With four of the esters indications of a possible formation of, at most, 0.1 residue of O-methylserine per inactivated trypsin molecule were obtained. tau Methylhistidine (but no pi-methylhistidine) was, however, always observed as the main product of the modification reaction. A further product, hitherto not yet described in active site methylations of serine proteinases, was pi, tau dimethylhistidine (1,3-dimethylhistidine). The failure of an attempted synthesis of the N-acetyl-ethanolamine ester of p-toluene-sulfonic acid reported in the literature is shown to be due to the high instability of this ester. PMID- 7539485 TI - Imidazolylbenzopyrane derivatives: a new class of acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors. AB - Inhibitors of the enzyme Acyl-CoA: Cholesterol Acyltransferase are regarded as potentially useful agents in the treatment of hypercholesterolemia and atherosclerosis. We report here a novel series of 2, 6-disubstituted-3 imidazolylbenzopyrane derivatives with significant in vitro ACAT inhibitory activity (IC50 range 0.05-0.5 microM). Compounds of this series such as 26 are examples of a new, structurally distinct class of potent ACAT inhibitors with high specificity for the aortic subtype of the enzyme. The structure-activity relationships of the 3-imidazolylbenzopyrane ACAT inhibitors were investigated by systematic manipulation of two regions of the parent compound 1 and the inhibitory activity resulted linked to the substituent in position 6 of the benzopyrane ring and modulated by the size of lipophilic substituents in position 2. Investigation of the mechanism of the inhibitory effect leads to the conclusion that these compounds act in a non-competitive fashion. PMID- 7539487 TI - Suppression of splenic enzyme activities by administration of aminopeptidase N (CD13) inhibitors: relationship between actions in vivo and in vitro. AB - The enzymatic changes in murine spleen caused by the administration for 20 successive days of various inhibitors of aminopeptidase N (leucocyte antigen CD13) have been compared. When compared with the control (saline), most of the inhibitors significantly suppressed splenic enzyme activities including those of ectoenzymes. A multivariate study indicated that the in vivo effects of the inhibitors were closely related to their inhibitory actions in vitro. PMID- 7539488 TI - Structure-activity relationship at the glycosidic moiety of digitalis compounds as found in tests with NA/K-ATPase isoforms from cardiac muscle of guinea-pig and man. PMID- 7539490 TI - Inhibition of a tumour protease with 3,4-dichloroisocoumarin, pentamidine isethionate and guanidino derivatives. AB - Guanidinobenzoatase (GB) is a cell surface proteolytic enzyme capable of degrading fibronectin, and is associated with tumour cells and cells capable of migration. The location of active GB in sections has been demonstrated with 9 aminoacridine (9-AA), a competitive inhibitor of GB. 3,4-Dichloroisocoumarin (3,4 DCI) and pentamidine isethionate (PI) are inhibitors of trypsin-like enzymes. It has now been demonstrated that 3,4-DCI, PI, and guanidino derivative compounds are significant inhibitors of GB, on the surfaces of lung squamous cell carcinoma cells in frozen sections and free GB in solution. Dexamethasone acetate (DMA) and medroxy-progesterone (MP) did not show any significant inhibition of GB activity. These molecules lack a reactive chloride or guanidino groups and are thought to react at the nuclear level, rather than directly on this cell surface protease. Kinetic studies have shown that 3,4-DCI, PI and guanidino derivatives are reversible competitive inhibitors of GB, as determined in vitro on the purified enzyme. The inhibition resulting with 3,4-DCI was a time-dependent process. It is suggested that these inhibitors interact with GB by binding to its active site, resulting in the formation of enzyme-inhibiter complexes (GB-I). The GB-I complexes can be dissociated with SDS treatment, resulting in the regain of GB activity. PMID- 7539489 TI - Tamoxifen is a Na(+)-antagonistic inhibitor of Na+/K(+)-transporting ATPase from tumour and normal cells. PMID- 7539491 TI - Cell biology of atherosclerosis. AB - FATTY STREAKS: The precursors of atherosclerotic plaques, fatty streaks, are subendothelial aggregations of lipid-filled macrophages which appear in human arteries within the first decade of life. Some fatty streaks disappear while others progress to fibrous plaques by about the fourth decade. PLAQUE FORMATION: The major cells comprising plaques are phenotypically modified, smooth-muscle, monocyte-derived, macrophages and T lymphocytes. The monocyte/macrophages and T lymphocytes are chemoattracted into the vessel wall by substances such as oxidized lipoprotein following their adhesion to a dysfunctional endothelium (caused for example by hyperlipidaemia, hypertension or diabetes). The macrophages and T lymphocytes produce specific matrix-degrading enzymes that initiate smooth muscle phenotypic change to a state in which they are responsive to a vast array of mitogens released by cells within the artery wall and by degranulating platelets. Cytokines also released are mediators of an immune response. These processes result in the formation of a thick fibrous cap of proliferated, phenotypically modified, smooth muscle cells and the extracellular matrix that they have produced, overlying a laterally placed cellular region of macrophages, T lymphocytes and smooth muscle cells and a central core of cell debris and cholesterol which has formed from necrotic, lipid-filled macrophages and smooth muscle cells. THROMBI: Ulceration and splitting of the fibrous cap exposes the highly thrombogenic necrotic core to flowing blood, resulting in thrombi which can travel distally to occlude smaller vessels and produce (dependent on the site of the plaque) myocardial infarction, stroke or gangrene of the extremities. PMID- 7539492 TI - Regulation of angiogenesis by extracellular matrix: the growth and the glue. AB - DEFINITION: Angiogenesis is broadly defined as the growth of new capillaries from extant vessels and constitutes a major part of developmental morphogenesis, response to injury and pathogenesis. Two regulatory pathways are proposed by which angiogenesis is thought to proceed. PROLIFERATIVE PATHWAY: The proliferative pathway depends on various cytokines and other factors that both stimulate and inhibit the proliferation of endothelial cells. One of these components, secreted protein acidic and rich in cysteine (SPARC), might function at several levels to control the progression of neovessels. Proteolysis of this component (e.g. by plasmin) results in the release of peptides containing the sequence Gly-His-Lys, which are angiogenic in vitro and in vivo. At later stages of angiogenesis when endothelial cell proliferation ceases, the intact protein is proposed to exert its known inhibitory effect on cell cycle progression. MORPHOGENETIC PATHWAY: The morphogenetic pathway depends on the synthesis and assembly of fibrillar type I collagen, which can be used as a template for endothelial cell migration and lumen formation. Endothelial cells interact with substrates of type I collagen and form networks based on the establishment of traction centers. These planar cellular networks, in some respects, resemble developing vasculature in vivo. CONCLUSION: An understanding of how proliferation and morphogenesis are controlled during vascular growth is likely to reconcile several models with respect to the factors that regulate this dynamic process. PMID- 7539493 TI - Nitric oxide. AB - PHYSIOLOGICAL EFFECTS OF NITRIC OXIDE: The generation of nitric oxide by the vascular endothelium maintains a vasodilator tone that is essential for the regulation of blood flow and pressure. In the brain, nitric oxide acts as a mediator of cell-cell signalling. In the peripheral nervous system nitric oxide is also released from many nerves previously classified as non-adrenergic and non cholinergic. Thus this simple gaseous molecule performs a wide variety of physiological functions. POTENTIAL FOR THERAPEUTIC MANIPULATIONS: Impaired production of nitric oxide can be countered by the administration of nitric oxide donors (in hypertension, atherosclerosis, gastrointestinal and genitourinary disorders) or by inhalation of nitric oxide gas (in chronic pulmonary hypertension or adult respiratory distress syndrome). The biggest challenge is to develop strategies that target the cytotoxic and damaging actions of nitric oxide without interfering with its essential protective functions. PMID- 7539494 TI - Relaxation technique for kinetic measurements of dye accumulation at the mitochondria of HeLa cells in situ. AB - The lipophilic cationic fluorescent dye ESI4 specifically stains the mitochondria in living cells. It was used to determine the kinetics of vital staining of mitochondria in single selected HeLa cells by quantitative microfluorometry in the concentration range CD = 6 x 10(-8) to 4 x 10(-6) M. The experiments were performed using the concentration jump method. They were evaluated with a quantitative staining model which considers dye binding as well as dye release at mitochondrial binding sites. The kinetic equations of the model are discussed in terms of the relaxation technique. The predictions of the theory agree well with the experimental results. The staining model was used to determine the rate constant k1 = 7.6 x 10(2)s-1 M-1 and k2 = 1.6 x 10(-3) s-1 for dye binding and dye release respectively, and the binding constant K = 4.8 x 10(5) M-1 of dye molecules at the mitochondria in HeLa cells (values according to relaxation experiments). The constants are discussed in detail. In addition, some experiments were performed on dye accumulation under the influence of antimycin, nigericin and oligomycin. PMID- 7539495 TI - Decidual-trophoblast interactions: decidual lymphoid cell populations in basal and parietal decidua. AB - Immunohistochemical analysis of tissue specimens from human pregnancy decidua basalis in contact with invasive trophoblast of chorion frondosum and decidua parietalis in contact with non-invasive chorion laeve do not differ in the frequency of lymphoid cells of the following phenotypes (CD2, CD4, CD8, CD14, CD21 and gamma/delta TCR). A practical implication of this observation is that the collection of lymphoid cells from whole decidua by curettage for functional studies is justified. PMID- 7539497 TI - Predictive evidence for a porin-type beta-barrel fold in CHIP28 and other members of the MIP family. A restricted-pore model common to water channels and facilitators. AB - Water channels are the subject of much current attention, as they may be central for cell functions in a host of tissues. We have analyzed the possible field of facilitators and water channels of the MIP family based on structural predictions, on findings about the topology of CHIP28, and on the biophysical characteristics of water channels. We developed predictions for the following proteins: MIP26, NOD26, GLP, BIB, gamma-TIP, FA-CHIP, CHIP28k, WCH-CD1, and CHIP28. We utilized Kyte Doolittle hydrophobicity, Eisenberg's amphiphilicity, Chou-Fasman-Prevelige propensities, and our own Union algorithm. We found that hydrophobic amphiphilic segments likely to be transmembrane were consistently shorter than required for alpha-helical segments, but of the correct length for beta-strands. Turn propensity was high at frequent intervals, consistent with transmembrane beta-strands. We propose that these proteins fold as porin-like 16 stranded antiparallel beta-barrels. In water channels, from the size of molecules excluded, an extramembrane loop(s) would enter the pore and restrict it to a bottleneck with a width 4 A < or = w < or = 5 A. A similar but more mobile loop(s) would act as gate and binding site for the facilitators of the MIP family. PMID- 7539496 TI - The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats. AB - Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats. PMID- 7539498 TI - The proximal straight tubule (PST) basolateral cell membrane water channel: selectivity characteristics. AB - Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mM mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 microns/sec (NaCl, M, g, E and U), 1 micron/sec (A), 84 microns/sec (F) and 0.02 micron/sec (G). The reflection coefficients sigma = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar sigma values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x 10(-5)). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4 x 8.2 x 12.0 (M), 3.6 x 5.2 x 5.4 (U), 3.8 x 5.2 x 5.4 (A) and (3.4 x 4.5 x 5.4 (F) A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539499 TI - Distribution of mRNA encoding the FA-CHIP water channel in amphibian tissues: effects of salt adaptation. AB - A water channel, the frog aquaporin-CHIP (FA-CHIP) was recently cloned from Rana esculenta urinary bladder. The 28.9 kDa encoded protein shows 78.8%, 77.4%, 42.4% and 35.6% identity with rat CHIP28, human CHIP28, rat WCH-CD and gamma-TIP, other members of the new transmembrane water channel family (Aquaporin-CHIP). We have now studied membranes from different frog (R. esculenta) organs employing semiquantitative PCR using FA-CHIP specific primers and an internal standard to quantify the PCR products. The FA-CHIP mRNA was abundantly expressed in the frog urinary bladder, skin, lung and gall bladder, while a lower expression was detected in the colon, liver and oviduct. FA-CHIP mRNA was not detected in the frog kidney, erythrocytes and brain but its expression was observed in the toad (Bufo arenarum) urinary bladder and skin, showing that FA-CHIP is probably a general amphibian water channel. Salt acclimation is known to increase the water permeability of frog and toad epithelia. We have now observed that salt acclimation for 1, 3, 4 or 5 days markedly increased skin and urinary bladder FA CHIP mRNA expression. It is generally accepted that water permeability is controlled in these tissues by the rate of water channel transfer from subapical vesicles (aggrephores) to the apical membrane. Our results indicate that water permeability is also regulated at the level of the FA-CHIP transcription. PMID- 7539500 TI - Ion binding constants for gramicidin A obtained from water permeability measurements. AB - Gramicidin A pores are permeable to water and small monovalent cations. For K, Rb, and Cs there is good evidence from conductances and permeability ratios that a second ion can enter a pore already occupied by another, but for Na this evidence is inconclusive and comparison of tracer fluxes and single channel conductances suggests that second ion entries are prohibited. Partly as a result of the complications of second ion entry there have been widely differing estimates for the dissociation constants for the first ion in the channel. Dani and Levitt (1981, Biophys. J. 35: 485-499) introduced a method for calculating ion binding constants from simultaneous measurements of water fluxes and membrane conductance. They found no evidence for second ion binding and calculated dissociation constants of 115 mM for Li, 69 mM for K, and 2 mM for Tl. It is shown here that the two-ion, four-state model predicts a dependence of water permeability on ion concentration that is difficult to distinguish from the predictions of block by a single ion. Using a modified technique that allows measurement of higher conductances, the first ion dissociation constants have been determined as 80 mM for Na, 40 mM for Rb and 15 mM for Cs. These values and those of Dani and Levitt fall in a smooth sequence. The dissociation constant for Cs is consistent with single channel conductances and flux ratios. There is a discrepancy between this constant for Na and the value, 370 mM, calculated from the single channel conductances and the assumption that a second ion cannot enter or affect an occupied pore. The dissociation constant for Rb is intermediate between those for K and Cs whereas tracer flux measurements (Schagina, Grinfeldt & Lev, 1983. J. Membrane Biol. 73: 203-216) have suggested that Rb interacts much more strongly with the channel than Cs. PMID- 7539501 TI - Aquaporin-CHIP-related protein in frog urinary bladder: localization by confocal microscopy. AB - Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP. PMID- 7539502 TI - The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells. AB - Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV. PMID- 7539504 TI - Synthesis of novel products in vitro by an RNA-dependent RNA polymerase. AB - RNA-dependent RNA polymerase from turnip crinkle virus-infected turnip transcribes both strands of a virus-associated satellite RNA, sat-RNA C (356 bases), in vitro. While both plus- and minus-strand sat-RNA C can direct the synthesis of full-length complementary-strand products, transcription of minus strand RNA also generates two non-template-sized products, L-RNA and S-RNA (C. Song and A. E. Simon, Proc. Natl. Acad. Sci. USA 91:8792-8796, 1994). Here we report that synthesis of L-RNA and S-RNA results from terminal elongation of the 3' end of the template. L-RNA has a panhandle structure and is composed of minus strand template covalently linked to newly synthesized RNA complementary to its 5' 190 bases. S-RNA is composed of template covalently linked to its full-length complementary strand. All minus-strand templates tested yielded S-RNA. However, synthesis of L-RNA was affected by deletion of the 3' end of the minus-strand template or several internal regions and base alterations near the 5' end or in an internal sequence immediately upstream from the template-product junction that could potentially form a heteroduplex with the 3' end. Furthermore, mutations that disrupted or restored a stem-loop involved in RNA recombination in vivo affected the level of L-RNA produced in vitro, suggesting that the mechanisms for intramolecular formation of panhandle RNAs and intermolecular RNA recombination involve similar features. PMID- 7539505 TI - Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. AB - We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle. PMID- 7539503 TI - Cellular CD44S as a determinant of human immunodeficiency virus type 1 infection and cellular tropism. AB - CD4 is the predominant cell membrane protein that binds human immunodeficiency virus type 1 (HIV-1) gp120 and facilitates HIV-1 infection, but other membrane associated molecules may be involved in determining HIV-1 cellular infection. Our prior work had suggested that CD44, the transmembrane receptor for hyaluronan, might play a role in the infection of mononuclear phagocytes with HIV-1. In the present work, we have used cells of the CD4-positive, CD44-negative human T lymphoblast cell line Jurkat to study the role of CD44 in HIV-1 infection and tropism. Cells were transfected with cDNA for the standard (S, or hematopoietic) CD44 isoform CD44S or the epithelial isoform CD44E. The resultant lines expressed appropriate CD44S or CD44E mRNA and protein. While the parent Jurkat cells, those transfected with vector alone, and those transfected with CD44E could be productively infected with only the lymphocytotropic strain HIV-1-LAI, cells transfected with CD44S were rendered susceptible to productive infection with the monocytotropic strains HIV-1-BaL and HIV-1-ADA. Also, CD44S-transfected cells displayed higher levels of infection with HIV-1-LAI than did the other transfected Jurkat cells. The transfected cell line cells all had comparable growth rates and expressed similar levels of the membrane antigens CD4, CD7, major histocompatibility complex (MHC) class I, MHC class II, and CD11a, while levels of CD3 were slightly higher in cells transfected with vector alone and in one of the clones transfected with CD44S. Hyaluronan binding was increased in cells transfected with either CD44S or CD44E. Mouse NIH 3T3 fibroblasts transfected with human CD4, human CD44S, or both human CD4 and CD44S displayed the appropriate antigens, but they could not be productively infected with lymphocytotropic or monocytotropic strains of HIV-1. The results indicate that in human leukocytes, CD44S is an important determinant of HIV-1 productive infection and may be involved in viral cellular tropism. PMID- 7539506 TI - Vesicular stomatitis virus infection induces a nuclear DNA-binding factor specific for the interferon-stimulated response element. AB - Vesicular stomatitis virus (VSV) has a broad host range. It replicates in the cytoplasm and causes rapid cytopathic effects. We show that following VSV infection, a nuclear factor that binds to a select set of interferon-stimulated responsive elements (ISRE) is induced in many cell types. This factor, tentatively called VSV-induced binding protein (VIBP), was estimated to have an approximate molecular mass of 50 kDa and was distinct from known members of the interferon regulatory factor family, that are known to bind to the ISRE. Induction of VIBP required tyrosine kinase activity but did not require cellular transcription. Treatment of cells with cycloheximide, which inhibits translation, only partially inhibited induction of VIBP. However, type I interferons and staurosporine, both of which inhibit VSV transcription, inhibited VIBP induction. Moreover, a double-stranded RNA analog, poly(I)-poly(C) also induced a DNA binding activity very similar to that of VIBP. These results indicate that a preexisting cellular protein is activated upon VSV infection and that this activation requires primary viral transcripts. The functional activity of VIBP was analyzed in cells stably transfected with a herpesvirus thymidine kinase luciferase reporter gene that is under control of the ISRE. While activity of the control promoter without ISRE was strongly inhibited following VSV infection (as a result of virus-mediated transcriptional shutdown of the host cell), the inhibition was reversed by the ISRE-containing promoter, albeit partially, which suggests that VSV infection differentially affects transcription of host genes. Although VIBP was induced in all other cells tested, it was not induced in embryonal carcinoma cells after VSV infection, suggesting developmental regulation of VIBP inducibility. PMID- 7539507 TI - Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo. AB - Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV 1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection. PMID- 7539508 TI - Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus infected patients. AB - The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed. PMID- 7539509 TI - Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells. AB - Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerases in heterologous systems. We have expressed human hepatitis B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. Analysis of immunoaffinity-purified pol indicated that (i) a portion of pol had initiated minus-strand DNA synthesis within infected insect cells; (ii) the pol mRNA appeared to be the template for reverse transcription; (iii) the products were small (100 to 500 nucleotides); (iv) only minus-strand DNA was synthesized; (v) the products were covalently bound to protein; and (vi) the 5' end of the minus-strand DNA mapped to DR1 by primer extension. The purified pol was also active in an in vitro polymerase assay. Analyses suggested that a different fraction of pol was active in the in vitro assays. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of the pol polypeptide in a reaction that appeared to represent in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of 32P-labeled phosphotyrosine on pol following in vitro reactions with 32P-labeled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will facilitate functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication. PMID- 7539511 TI - Medial neovascularization in abdominal aortic aneurysms: a histopathologic marker of aneurysmal degeneration with pathophysiologic implications. AB - PURPOSE: The purpose of this study was to characterize the distribution of aortic wall microvessels in normal aorta, atheroocclusive disease (AOD), and abdominal aortic aneurysms (AAA) and to evaluate whether medial neovascularization (MNV) is a reliable histopathologic marker of aneurysmal degeneration. METHODS: Aortic tissue specimens (9 normal, 10 AOD, and 10 AAA) were examined for elastin with Verhoeff-van Gieson stain and for Ulex europaeus type I lectin, an endothelial specific antigen, and laminin, a marker of basement membranes, by immunohistochemistry. The density of MNV was determined by morphometry of aortic sections stained for endothelium. The spatial distribution of aortic microvessels was compared with that of elastin destruction and chronic inflammation. RESULTS: Evidence of medial neovascularization was generally not observed in normal aorta or AOD, whereas AAAs showed strong spatial correlations between MNV, disruption and degradation of elastin, and chronic inflammation in the outer aortic wall. Several specimens of AOD had focal areas of MNV associated with localized elastin fragmentation and monocytic infiltration located at the interface between the atherosclerotic plaque and the inner media. The density of MNV was about fifteenfold higher in AAA compared with normal aorta and about threefold higher compared with AOD (microvessels per high-power field): normal aorta, 0.77 +/- 0.28; AOD, 3.40 +/- 0.51; AAA, 11.32 +/- 1.58 (ANOVA, p < 0.001). CONCLUSIONS: The presence and density of MNV in the abdominal aorta is a consistent histopathologic marker of aneurysmal degeneration that is spatially correlated with the destruction of elastin and chronic inflammation. The observation of focal MNV in some specimens of AOD, associated with partial elastin disruption, raises the possibility that early changes of aneurysm disease might develop by an extension of angiogenic/inflammatory processes from the atherosclerotic plaque into the aortic media. PMID- 7539512 TI - Induction of latent membrane protein expression in in vitro Epstein-Barr virus infected leukaemic B lymphocytes by interleukin 4 and antibodies to CD40. AB - Chronic lymphocytic leukaemia (CLL) B cells are clones representing the mature B cell phenotype. On infection with Epstein-Barr virus (EBV) CLL cells express the EB nuclear antigen (EBNA) complex but unlike EBV-infected normal B cells they do not express LMP nor do they proliferate or immortalize. Furthermore, EBV-CLL rapidly die by apoptosis in culture. In the present study we have used the B cell growth factors interleukin 4 and antibodies to CD40 to induce activation and proliferation of EBV-infected CLL cells. Although cell numbers did not significantly increase, apoptosis was partially inhibited in CLL cells which expressed increased levels of CD23 and were activated to immunoglobulin-secreting lymphoblasts. Expression of LMP was induced by interleukin (IL)-4 and anti-CD40 in all five EBV-infected CLL samples examined. However, this did not enhance cell proliferation or induce immortalization. Further analysis showed that LMP could be detected 4-5 days after EBV infection, and that both IL-4 and anti-CD40 could independently induce LMP but that their effect was additive. These results indicate that LMP expression is dependent on B cell activation processes and that in some circumstances full latent viral gene expression is not sufficient to cause B cell immortalization. PMID- 7539510 TI - Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase. AB - To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong stop DNA efficiently. PMID- 7539513 TI - Prognostic value of c-mpl expression in myelodysplastic syndromes. AB - The c-mpl proto-oncogene which encodes a member of the hematopoietic cytokine receptor superfamily has been recently shown to be the receptor for thrombopoietin (TPO), which stimulates megakaryocyte progenitor expansion and differentiation. We studied c-mpl expression by Northern blot analysis, in a large series of 58 MDS. No expression was found in 14 patients with refractory anemia (RA) or with refractory anemia with ring sideroblasts (RARS). In contrast 11/26 (42%) patients with refractory anemia with excess of blasts (RAEB), or with RAEB in transformation (RAEBt), and 8/18 (44%) patients with chronic myelomonocytic leukemia (CMML) expressed c-mpl. In CMML patients, no correlation was found between c-mpl expression and any prognostic factor tested, nor with the course of the disease. In contrast, in RAEB and RAEBt, expression of c-mpl was correlated with high Bournemouth scoring (P < 0.005) and poor survival (P = 0.02) due to leukemic transformation. Forty-five per cent (5/11) of the c-mpl positive patients evolved towards AML with a mean follow-up of 10.5 months, while 13% (2/15) of the c-mpl negative patients developed a secondary leukemia, with a mean follow-up of 21.1 months. Moreover, in RAEB and RAEBt, a significant correlation was observed between c-mpl, CD34, megakaryocyte glycoprotein IIb (GPIIb) expression, and the presence of dysmegakaryopoiesis. These results indicate that patients with RAEB and RAEBt, with high expression of the c-mpl, CD34, and GPIIb genes, may identify a subgroup of patients with particularly poor prognosis, due to an increased risk of secondary leukemia. More aggressive therapy could be justified in these patients. PMID- 7539514 TI - Resistance to APO-1 (CD95) induced apoptosis in T-ALL is determined by a BCL-2 independent anti-apoptotic program. AB - Selective induction of programmed cell death, apoptosis, may represent a new approach to the treatment of cancer. Apoptosis can be induced by the monoclonal antibody anti-APO-1 directed against the cell surface receptor APO-1, a member of the nerve growth factor (NGF) receptor/tumor necrosis factor (TNF) receptor superfamily. We determined APO-1 expression and sensitivity to anti-APO-1 mediated apoptosis in childhood acute lymphoblastic leukemia cells of T lymphocyte precursor phenotype (T-ALL). APO-1 was constitutively expressed by 21 of 30 T-ALL and by all T-ALL cell lines investigated. However, most APO-1 positive T-ALL were resistant to anti-APO-1 mediated apoptosis. Sensitivity to anti-APO-1 mediated apoptosis was independent of the density of APO-1 expression on the cell surface and independent of the amount of Bcl-2. Incubation of resistant T-ALL with the protein synthesis inhibitor cycloheximide reversed resistance and induced sensitivity to anti-APO-1 mediated apoptosis in most T ALL. These data suggest that resistance to anti-APO-1 mediated apoptosis in T-ALL is maintained by an active cellular program. Reversion of resistance to sensitivity towards induction of apoptosis in tumors may provide a new basis for successful therapeutic intervention. PMID- 7539515 TI - Differential expression of adhesion molecules in acute leukemia. AB - Interactions between hematopoietic cells and the stromal microenvironment are mediated by membrane-bound adhesion molecules. As the expression patterns of these molecules may alter the adhesive qualities of leukemic blasts, leukemic samples were investigated for the expression of beta 1-, beta 2-, beta 3 integrins, CD44, the three selectins and several members of the immunoglobulin family. CD44 (167/169), LFA-3 (158/169), the beta 1-integrins VLA-4 (120/123) and VLA-5 (45/51) and the beta 2-integrin LFA-1 (149/157) were found on > 70% of blasts in most cases of leukemias. Other molecules were restricted to specific differentiation stages and lineage. The beta 2-integrins Mac-1 (CD11b/CD18) and gp 150,95 (CD11c/CD18) were preferentially expressed on M4 and M5 subtypes, and NCAM (CD56) was only found on a subset of acute myeloid leukemias (17/113). Unexpectedly, the beta 1-integrins VLA-1 (1/51), VLA-2 (18/123), VLA-3 (5/43), VLA-6 (15/29) and the E-selectin (2/47) were expressed on > 70% blasts on a subset of leukemias of varied phenotype. These molecules were absent on normal CD34+ bone marrow precursors. The simultaneous analysis generally revealed a higher percentage of positive blasts in the blood than in bone marrow. Our observations therefore suggest that in leukemia these antigens are displayed on a non-adherent population that is defective and is unable to convert to an adherent, functionally active conformational state. PMID- 7539516 TI - Diversity and similarity in signaling pathways of hematopoietic growth factors in human leukemia cell lines. AB - Diversity and similarity in signaling pathways of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and stem cell factor (SCF) in five human factor-responsive leukemia cell lines were investigated by immunoblotting to detect tyrosine phosphorylation of intracellular proteins. G-CSF induced tyrosine phosphorylation of a set of proteins with few different components according to the cell lines. IL-3 also induced phosphorylation of several proteins. In a lymphoid cell line, phosphorylation patterns induced by IL-3 were somewhat different from that in myeloid cell lines. Phosphorylation patterns by G-CSF and those by IL-3 were similar in myeloid cell lines. In a cell line which responded to both IL-3 and SCF, almost similar sets of proteins were phosphorylated by each, although phosphorylation of a 92-KDa protein was specific to IL-3 and that of a 140-200 KDa protein was specific to SCF. Taken together, proliferative growth factors induced tyrosine phosphorylation of similar sets of proteins with little difference according to each growth factor and each target cell line. PMID- 7539518 TI - Long-term clinical and prostate-specific antigen follow-up in 500 patients treated with radiation therapy for localized prostate cancer. AB - During a 14-year period, 500 consecutive patients having localized prostate cancer were treated with external beam radiotherapy with curative intent. Patients were staged according to the Whitmore-Jewett and TNM systems. Median follow-up for all patients was 69 months. Local recurrence included failures detected by rectal examination and/or prostate biopsy. Progressively elevated prostate-specific antigen (PSA) values at follow-up with no evidence of systemic disease also was considered a local treatment failure. Overall 5- and 10-year survival rates were 65% and 37%, respectively. Cause-specific 5- and 10-year survival rates were 79% and 55%, respectively. Five- and 10-year local control rates for all patients were 72% and 52%, respectively. No patient with a T1 a tumor failed locally. When follow-up PSA data were included in control criteria, 10-year local control rates for patients with T1b, T2a, and T2b-T4 tumors were 66%, 55% and 44%, respectively. If only an abnormal rectal examination and/or a positive post-treatment biopsy was considered as evidence of local failure, control rates were significantly better. Approximately one third of the study patients had died of cancer or intercurrent disease before the PSA test was used routinely. Of the 244 patients (49%) for whom follow-up PSA values were available, 116 had a normal post-treatment PSA with no evidence of disease; 94 were alive and 22 died with no evidence of prostate cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539519 TI - RC3/neurogranin structure and expression in the caprine brain in relation to congenital hypothyroidism. AB - In view of the profound effects of thyroid hormone deficiency on the central nervous system (CNS), neuronal genes regulated by thyroid hormone could potentially be involved in the development of the CNS. Expression of the neuronal gene RC3/neurogranin was shown to be induced by thyroid hormone in the rat. No data are available on RC3 expression in mammals with prenatal brain development, like humans. To study RC3 mRNA expression in a genetic in vivo model of congenital hypothyroidism, which also resembles the human situation in the timing of brain development relative to birth, we used an inbred strain of congenitally hypothyroid goats. We isolated a cDNA for the caprine RC3 homolog. The deduced amino acid sequence had 99% similarity with the rat and bovine protein sequence. An analysis of the developmental expression of RC3 mRNA levels showed a 3-fold increase between E90 and P0. In situ hybridization analysis showed that in euthyroid goats, the RC3 expression pattern was region-specific and resembled that in rats. However, in contrast to rats, hypothyroid goats showed only a reduced RC3 mRNA expression in the striatum. Hypothyroidism had no effect on RC3 mRNA expression in all other brain regions. T4-treatment of the hypothyroid fetus increased RC3 mRNA expression in the striatum to euthyroid control levels. These data suggest that thyroid hormone is a regulator of RC3 gene expression in the caprine brain, and that the striatum is highly sensitive to thyroid hormone deficiency. PMID- 7539517 TI - Detection of minimal residual disease by reverse transcriptase polymerase chain reaction for the PML/RAR alpha fusion mRNA: a study in patients with acute promyelocytic leukemia following peripheral stem cell transplantation. AB - Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia (AML) that is characterized by the presence of a PML/RAR alpha fusion gene resulting from t(15;17). Peripheral stem cell transplantation (PSCT) has been used to treat patients with AML. To assess the presence of minimal residual disease (MRD) and the contamination of leukemic cells in peripheral stem cells (PSCs), we examined six patients with APL who were undergoing PSCT, using reverse transcriptase polymerase chain reaction analysis to detect the mRNA of the PML/RAR alpha fusion gene. The fusion gene was expressed in the bone marrow cells during the early phase of a complete remission and in some of the PSCs. Detection of the fusion gene can be useful in monitoring for leukemic cell contamination of PSCs and for predicting a relapse of APL. PMID- 7539520 TI - [The possible use of gramicidin for the treatment of zoonotic cutaneous leishmaniasis]. PMID- 7539522 TI - The effect of antioxidants on bleomycin treatment in in vitro and in vivo genotoxicity assays. AB - Antioxidants are thought to be important in protecting against damage from active oxygen species. The effects of the antioxidant nutrients vitamins C and E have been investigated after bleomycin treatment in the Salmonella typhimurium bacterial mutation assay, in the human peripheral lymphocyte chromosome aberration assay, and in the mouse micronucleus assay in peripheral blood and bone marrow cells. There were no protective effects from vitamins C and E in the bacterial mutation assay, but vitamin C and not vitamin E abolished chromosome damaging responses in human peripheral lymphocytes, and both vitamins reduced responses in micronuclei from peripheral blood cells in mice. This would suggest that in human cells in vitro and mouse cells in vivo these vitamins could have a protective role. PMID- 7539521 TI - Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues. AB - A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study. PMID- 7539523 TI - Time course of bromocriptine induced excitation in the rat: behavioural and biochemical studies. AB - The aim of the present study was to further investigate the behavioural and biochemical pharmacology of the directly acting dopamine (DA) receptor agonist bromocriptine (BRC). BRC produced an initial depression of locomotion followed after about an hour by a weak but significant locomotor stimulation. The stimulation was potentiated by concomitant administration of the D1 agonist SKF38393. Ex vivo biochemical determinations indicated that reductions in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels occurred in the striatum after BRC injection without a significant change in DA levels, indicating a reduced DA turnover. An increase in 5-hydroxytryptamine (5HT) and 5 hydroxyindoleacetic acid (5HIAA) levels occurred in the striatum leading to a significant increase in turnover (i.e. ratio of 5HIAA to 5HT). Noradrenaline concentrations increased in the striatum. In the cortex, sharp falls in HVA and DOPAC levels without a corresponding change in DA were observed. While there was no significant change in noradrenaline levels in this brain region, an increase in 5HIAA, but not in 5HT, levels occurred. These changes indicate an increase in 5HT turnover (ratio of 5HIAA to 5HT). In vivo dialysis indicated that extracellular levels of DA, DOPAC and HVA in the striata of freely moving rats were sharply reduced for at least 6 h after injection. In vitro binding studies showed that BRC exhibited high (Ki values in low nanomolar range) affinities for DA D2A, D2B, D3, alpha 1 and alpha 2 adrenergic receptors together with unexpectedly high affinity (about 1 nM) for 5HT1A receptors. The data indicate that the initial behavioural depression and later locomotor stimulation induced by BRC are accompanied by a sharp monophasic fall in striatal extracellular DA levels as indicated by dialysis studies. Since the behavioural stimulation was augmented by concomitant D1 receptor stimulation, the data suggest that the reduced DA turnover is influencing the amount of DA available to stimulate postsynaptic D1 receptors. However, the biochemical studies indicated that BRC has a high affinity for 5HT1A receptors and affects the turnover of 5HT in the brain. Thus, the behavioural effects of BRC may depend not only on effects on the DA system but also on 5HT systems. PMID- 7539524 TI - The effect of sample preparation and storage on maternal triple-marker screening. AB - OBJECTIVE: To evaluate the effect of different sample collection, storage, and preparation techniques on serum alpha-fetoprotein (AFP), beta-hCG, and unconjugated estriol (E3) concentrations. METHODS: A solution containing known concentrations of AFP, hCG, and unconjugated E3 was diluted in blood samples obtained from seven healthy male volunteers. Serum from each blood sample was removed immediately, and either assayed or frozen at -70C. Portions of the remaining blood were handled as follows: centrifuged and refrigerated, centrifuged and left at room temperature, not centrifuged and refrigerated, or not centrifuged and left at room temperature. Serum was removed from these samples for triple-marker analysis at 24, 48, 96, and 168 hours after the initial sample collection. RESULTS: Immediate freezing of serum and subsequent thawing resulted in a significant increase in beta-hCG and unconjugated E3 levels, but no change in AFP levels. There was a significant effect over time on AFP, hCG, and unconjugated E3 concentrations. The change in AFP levels was influenced by centrifugation status, whereas all three analytes were influenced by refrigeration status. CONCLUSION: Different sample collection, storage, and preparation techniques may affect maternal triple-marker screening. PMID- 7539526 TI - Screening for developmental disabilities: a suggestion! PMID- 7539527 TI - Advertisement by Medtronic, Inc. PMID- 7539525 TI - Ultrasound-adjusted risk and spectrum of fetal chromosomal abnormality in women with elevated maternal serum alpha-fetoprotein. AB - OBJECTIVE: To determine the incidence of chromosomal abnormality in sonographically normal fetuses after amniocentesis for elevated maternal serum alpha-fetoprotein (MSAFP), and to compare the spectrum of abnormality with that seen in women undergoing amniocentesis for advanced maternal age. METHODS: Cytogenetic and sonographic findings from women undergoing amniocentesis for elevated MSAFP (at least 2.0 multiples of median) between 1988-1992 were reviewed retrospectively. The literature was reviewed and data compiled regarding the risk of chromosomal abnormality for women with elevated MSAFP levels. RESULTS: The incidence of abnormal karyotype among all women with elevated MSAFP was 1.23% (nine of 733). The risk of an abnormal karyotype with a normal ultrasound examination was 1.01% (seven of 696). This included three fetuses with sex chromosome abnormalities, three with de novo, apparently balanced rearrangements, and one with an unbalanced structural rearrangement. CONCLUSIONS: The risk of chromosomal abnormality in nonselected patients, predominantly younger than 35 years of age, with elevated MSAFP and a sonographically normal fetus is 0.6%, based on a compilation of our data and reports published previously. The spectrum of abnormality in women with elevated MSAFP differs from that in women of advanced maternal age. Patients should be specifically counseled regarding this difference. PMID- 7539529 TI - Role of the clinical nurse coordinator in successful implementation of critical pathways in pediatric cardiovascular surgery patients. AB - Critical pathways reduce variations in clinical management and achieve quality patient outcomes within a defined time while reducing the average length of stay (ALOS). The critical path is a day-by-day plan that specifies the use and timing of procedures in relation to the stage in the patient's recovery. Variations from that plan are identified and aggressively approached. This article outlines the clinical nurse coordinator's role in utilizing critical pathways for the congenital heart surgery patients. Successful implementation of the pathways using the clinical nurse coordinator's role is supported by analysis of ALOS in 286 patients. PMID- 7539528 TI - Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei. AB - Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies. PMID- 7539530 TI - Targeting of phototoxic drugs to antigen-specific T lymphocytes in vitro using antigen-presenting cell membranes. AB - We have used the complex of antigen with class II major histocompatibility proteins (Ia) in membrane-bound form to target a phototoxic compound to antigen specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323-339 to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia+ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320-400 nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539531 TI - Cloning and characterization of polyphenol oxidase cDNAs of Phytolacca americana. AB - Two cDNA clones encoding polyphenol oxidases were isolated from a cDNA library constructed from a log-phase suspension culture of Phytolacca americana (pokeweed) producing betalains. The clones exhibit 93 and 86% sequence identity at the nucleotide and deduced amino acid levels, respectively. Both clones contain two copper-binding domains characterized by histidine-rich regions, which are found ubiquitously in all polyphenol oxidases/tyrosinases, and a putative third histidine-rich, copper-binding region, which is common to all plant polyphenol oxidases. One of the Phytolacca cDNA deduced amino acid sequences contains the ubiquitous transit peptide for all proteins targeted to the internal lumen of thylakoid membranes of plastids and is considered to be 98 residues in length based on a proposed sequence cleavage site motif. This would produce a processed peptide of approximately 54 kD. In addition to common features of transit peptides, it was found that an additional conserved region for polyphenol oxidases was located between the hydroxy amino acid-rich region and the thylakoid transfer domain. Spatial and temporal expression was investigated by northern blot analysis of total RNA from various organs of Phytolacca plants. Transcripts of the two clones were found to be 2.1 and 2.3 kb, respectively. Both transcripts were present only at substantial levels in ripening, betalain-containing fruit. PMID- 7539532 TI - Characterization of the promoter of the proto-oncogene c-kit. AB - In previous studies, we have characterized the nature and function of the proto oncogene c-kit, which encodes a receptor tyrosine kinase. This receptor together with its ligand, a stem cell growth factor, constitutes a cell signaling system which is crucial for the development of hematopoietic, melanocytic and germ cells. The expression of the gene correlates with its protein functions in specific cell lineages and is temporally and spatially regulated during fetal and adult life. As a start point to study the gene regulation, we have characterized the promoter of the c-kit gene. A single transcription initiation site located 58 bases upstream of the ATG start codon has been identified. The sequence upstream to the initiation site reveals a TATA-less, non-GC rich promoter. Several potential binding sites for transcription factors pertinent to c-kit expression, such as Sp-1, GATA-1, myb and Oct-4, have been identified. Promoter activities of different lengths of the 5' sequence have been analyzed in transient expression assay. The 2.7 kb of the 5' sequence facilitates the expression of the CAT gene in several cell lines while the sequence further upstream from 2.7 to 5.0 kb shows a negative regulatory activity. This study reveals a unique promoter of the c-kit gene and provides a basis for further elucidation of the regulatory mechanism of c-kit gene expression. PMID- 7539533 TI - Effects of ipsilateral denervation and contralateral ureteral obstruction on tubuloglomerular feedback. AB - The effects of acute ipsilateral denervation (AID) and contralateral ureteral obstruction on tubuloglomerular feedback (TGF) was studied by micropuncture in rats. Denervation alone was associated with an increase in the TGF turning point, the tubular flow rate required for a half-maximal TGF response, but other TGF characteristics were not affected. Contralateral ureteral obstruction alone was also associated with an increased turning point, but contralateral obstruction had no effect on turning point, when AID was done prior to the obstruction. We conclude that renal nerves have a tonic modulating effect on TGF in anesthetized rats, and that renorenal reflexes are involved in the TGF resetting associated with acute contralateral ureteral obstruction. PMID- 7539534 TI - Multiple pathways for ATP-induced intracellular calcium elevation in pheochromocytoma (PC12) cells. AB - Extracellular ATP excites neurons in both the peripheral and central nervous system. To elucidate the mechanisms involved, we used spectrofluorometric analysis to study the pathways by which extracellular ATP elevates the intracellular Ca2+ concentration ([Ca2+]i) in individual, fura-2-loaded, rat pheochromocytoma PC12 cells. ATP (> 1 microM) increased [Ca2+]i. The ATP effect on [Ca2+]i was completely abolished by a nominally Ca(2+)-free extracellular medium, which indicates that the ATP-induced increase in [Ca2+]i was due to an influx of extracellular Ca2+. We next applied specific blockers of voltage dependent Ca2+ channels and used experimental protocols with depolarizing external K(+)-rich solutions. Our results show that ATP induces influx of extracellular Ca2+ through (a) dihydropyridine-sensitive (Ln-type) Ca2+ channels, (b) Ca(2+)-permeable, voltage-independent, Cd(2+)-insensitive cation channels, and (c) an as yet unidentified, voltage-dependent, Cd(2+)-sensitive Ca2+ influx system. PMID- 7539535 TI - Corticosterone increases Na(+)-K(+)-ATPase activity in rat cortical collecting ducts with inhibition of 11 beta-hydroxysteroid dehydrogenase. AB - To examine a physiological role of 11 beta-hydroxysteroid dehydrogenase (11OHSD) in the aldosterone target tissue, we measured Na(+)-K(+)-ATPase activity in the cortical collecting ducts (CCD) from adrenalectomized rats, which were treated with a physiological dose of corticosterone and/or carbenoxolone (an inhibitor of 11OHSD). The Na(+)-K(+)-ATPase activity in adrenalectomized rats was not significantly changed by either corticosterone alone or carbenoxolone alone, whereas its activity showed a significant increase only in the rats that received both corticosterone and carbenoxolone. In these rats, the plasma concentration of corticosterone was within the physiological range (10(-6) M) and the plasma carbenoxolone concentration was about 10(-7) M. Furthermore, the direct effect of carbenoxolone was examined in the microdissected CCD because it has been reported that carbenoxolone per se had an affinity for the aldosterone receptor. Na+K(+) ATPase activity in the microdissected CCD was increased in a dose-dependent manner after a 3-hour incubation with carbenoxolone, and this effect was completely inhibited by canrenoic acid (an aldosterone antagonist). However, the minimal carbenoxolone concentration exerting a stimulatory effect was 10(-6) M, which is about 10 times higher than the plasma concentration of carbenoxolone in the in vivo treated rats. These results indicate that an inhibition of 11OHSD but not a direct action of carbenoxolone induces an increase in Na(+)-K(+)-ATPase activity, which is a well-known aldosterone effect in the CCD. PMID- 7539539 TI - L-arginine reduces kidney lipid peroxidation, glycoxidation and collagen accumulation in the aging NMRI mouse. AB - Collagen accumulation in organs is a main feature of the physiological aging process. There are two main theories for the responsible mechanisms, the free oxygen radical and the glycoxidation theory. Both lead to carbonyle-induced protein modification, increased collagen cross-linking and subsequently to the collagen accumulation. In previous studies we provided evidence for the blocking of reactive carbonyles by L-arginine in the diabetic state and present here data for this effect in the aging NMRI mouse. NMRI mice were fed L-arginine in tap water for a period of 6 months and compared to an untreated control group. Kidney collagen content of the treated group was significantly reduced (treated: 8.83 +/ 0.72 mg collagen/100 mg kidney weight; untreated: 11.95 +/- 0.98 mg collagen (100 mg kidney weight; p < 0.05). Using a colorimetric assay for lipid peroxidation products we found significantly reduced lipid peroxidation-derived aldehydes in the treated group (treated: 0.25 +/- 0.03 extinction; untreated: 0.36 +/- 0.04 extinction; p < 0.05). The parameter for glycoxidation, N-epsilon carboxymethyllysine (CML) was significantly lower in the experimental group as well (treated: 2.3 +/- 0.5 nM CML/microM hydroxyproline; untreated: 4.3 +/- 0.52 nM CML/microM hydroxyproline; p < 0.05). No differences were observed for the biomarker of hydroxyradical attack, o-tyrosine. L-Arginine could have reduced collagen accumulation by blocking collagen glyc(oxidation) which is known to lead to increased collagen cross-linking, solubility and degradation as a strong correlation between both CML and collagen content (r2 = 0.654, p < 0.05) as well as between lipid peroxidation products and collagen content (r2 = 0.539, p < 0.05) was observed. PMID- 7539537 TI - Angiotensin II induces in vivo c-fos expression from rat renal cortex and medulla. AB - In order to verify whether angiotensin II (Ang II) induced in vivo protooncogene, c-fos, expression in the rat renal cortex and medulla, we administered various concentrations of Ang II to Wistar rats and measured the c-fos expression from the renal cortex and medulla using the method of Northern hybridization. c-fos expression induced by 1 microgram (1.6 x 10(-6) M) of atrial natriuretic peptide (ANP) was also examined. The result was that the peak expression of c-fos mRNA was observed at approximately 10 min after Ang II administration in both rat renal cortex and medulla. This expression was reduced to the control level at 30 min. The measurement of the concentration of injected-Ang II and c-fos mRNA expression revealed that the peak expression of c-fos mRNA in the renal cortex and medulla was detected at the concentration of 1.0 x 10(-8) M and 1.0 x 10(-9) M Ang II, respectively. Nevertheless, ANP had no significant effect on the increase in c-fos mRNA expression. These data revealed that Ang II transiently increases the in vivo c-fos expression in both rat renal cortex and medulla but ANP does not. This protooncogene expression may induce vascular and mesangial proliferation in the kidney. PMID- 7539538 TI - Cathepsin B and L in isolated proximal tubular segments during acute and chronic proteinuria. AB - Acute and chronic proteinuria were studied in rats, using lysosomal cathepsin B and L as marker enzymes for tubular protein degradation. The activity of cathepsin B and L has been determined in microdissected segments S1, S2 and S3 of the proximal tubule by an ultramicroassay. Z-Phenylalanyl-arginine-7-amido-4 methylcoumarin served as a substrate. In normoproteinuric Sprague-Dawley rats, induction of acute unselective glomerular proteinuria with Adriamycin (5 mg/kg body weight) revealed a moderate activity increase of cathepsin B and L in the S2 segment, reaching 12.6 +/- 5.6 versus 8.6 +/- 4.2 pmol.mm-1.min-1 in controls. In contrast, Munich Wistar Fromter (MWF) rats, that are characterized by a genetically determined, chronically elevated glomerular protein excretion, showed a very high activity of cathepsin selectively in S2 of 25.0 +/- 12.1 pmol.mm 1.min-1. Acute proteinuria induced by Adriamycin in chronic proteinuric MWF rats could increase cathepsin activity in the S3 segment only, showing 12.0 +/- 8.3 versus 6.8 +/- 4.0 pmol.mm-1.min-1 in MWF control rats. In conclusion, chronically increased protein filtration changes the functional reserve capacity of the proximal tubule. While acutely induced glomerular proteinuria in normoproteinuric rats stimulates lysosomal proteolytic activity mainly in S2 segment, chronic proteinuric MWF rats may display already a maximally stimulated cathepsin activity in this segment probably due to long-term increased tubular protein load. In case of acute elevation of chronic proteinuria, the consecutive S3 segment shows increased lysosomal function for protein conservation. PMID- 7539536 TI - Altered extracellular matrix component gene expression in murine polycystic kidney. AB - The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. This study was designed to assess how the expression of extracellular matrix component genes is regulated in a model of murine polycystic kidney disease and control DBA/2 mice at 8, 16, and 30 weeks of age. The mRNA levels encoding for collagen IV, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, fibronectin, and collagens I and III increased with the progression of cystic lesions in the kidney of DBA/2FG-pcy mice. At 30 weeks of age, mRNA levels for collagen IV, laminin B1 and B2, heparan sulfate proteoglycan, fibronectin, and collagens I and III were increased 8.1-fold, 7.0 fold, 7.0-fold, 9.8-fold, 7.0-fold, 5.5-fold, and 5.4-fold, respectively, compared to those of control DBA/2 mice. An immunofluorescence study revealed the irregular staining for collagen IV, laminin, heparan sulfate proteoglycan, and collagens I and III around the cysts. These data suggest that changes in the expression of basement membrane components and interstitial collagens are associated with the development of polycystic kidney disease. PMID- 7539540 TI - Control of inositol phosphate turnover in human airways during histamine stimulation. AB - The control of inositol phosphate (IP) turnover was investigated in intact human airway smooth muscle cells (SMC) during a brief exposure to a bronchoconstrictor agonist. The pool of membrane phosphatidylinositol 4,5-biphosphate was labelled by incubating SMC with myo-[3H]inositol and the [3H]IPs synthetized ([3H]1,4-IP2, [3H]1,3,4,-IP3, [3H]1,4,5,-IP3 and [3H]1,3,4,5-IP4) were separated by HPLC. We examined the role of protein kinase C (PKC) and of Ca2+ on IP turnover during a 5 sec application of histamine. Activation of PKC with the phorbol ester PMA (0.2 microM) decreased, whereas inhibition of PKC with 1 microM staurosporine increased the production of the 4 IPs examined in unstimulated and in histamine stimulated SMC. Decreasing [Ca2+]i with 5 microM ionomycin in the absence of external Ca2+ diminished the IP production whereas in the presence of Ca2+, ionomycin exalted it and potentiated the response to histamine. Thapsigargin, 5 microM, which depletes the 1,4,5-IP3-sensitive Ca2+ stores, reduced the IP production due to histamine. The effects of PMA, staurosporine and thapsigargin were also tested on [Ca2+]i in fura-2-loaded single SMC. These results reveal that PKC exerts a negative and Ca2+ a positive feedback control on phospholipase C, that operate within 5 sec of agonist stimulation. PMID- 7539542 TI - Confocal laser scanning microscopy of small-intestinal mucosa in celiac disease. AB - BACKGROUND: Light microscopy of jejunal biopsy specimens is routinely used to diagnose celiac disease. Confocal laser scanning microscopy offers research advantages, since thin optical sectioning can be performed without mechanical damage to the specimen. Fixed as well as non-fixed specimens can be studied. METHODS: Confocal laser scanning microscopy was used to study the stanining of wheat germ agglutinin, which binds to glycoconjugates of the enterocyte, and rhodamine phalloidin, which binds to the F-actin of the cells. RESULTS: In healthy mucosa the wheat germ agglutinin labeling showed a strong and punctate staining of microvilli, which outlined a convoluted surface. Phalloidin-labeled actin formed a three-dimensional cage at the cell membrane. In the crypt hyperplastic mucosa, both staining patterns were irregular, and the cytoskeleton was disorganized. CONCLUSIONS: Confocal laser scanning microscopy offers the possibility to study the distribution of surface and cytoskeleton markers in thick, structurally intact specimens. PMID- 7539541 TI - Age-dependent mechanism in guinea pig bronchoconstriction induced by exsanguination. AB - Age-related mechanism in exsanguination-induced bronchoconstriction (EIB) was studied in guinea pigs. We used a total of 36 guinea pigs divided into three age categories (immature, adolescent, and mature). Each age category was separated into two groups: control and catalase (CAT). CAT is an antioxidant. Both saline (the control group) and CAT were instilled intratracheally 30 min before exsanguination. The animals were anesthetized, sternotomized and exsanguinated. Maximal expiratory flow (Vmax) was measured both before and 1-30 min after exsanguination. In the control group, exsanguination caused gradual decreases in both total lung capacity (TLC) and Vmax at 50% baseline TLC, and an increase in minimal lung volume, indicating EIB. EIB occurred faster in the immature than in the adolescent and mature guinea pigs, and it was significantly ameliorated by CAT. In 36 additional animals tested before exsanguination, there was no age related change in either airway neutral endopeptidase (NEP) activity or lung tissue substance P level. Thirty minutes after exsanguination in the control groups, airway NEP activity decreased significantly in all age categories, while lung tissue substance P level increased significantly only in the immature category. CAT prevented most of these NEP and substance P changes. These data suggest that exsanguination increases production of oxygen radicals, which inactivate NEP and enhance breakdown of substance P precursor; the increased tachykinin levels induce EIB. Furthermore, our data indicate that this chain reaction is age-dependent, with a faster and more severe reaction in immature guinea pigs. PMID- 7539544 TI - A randomized study comparing glucagon and hyoscine N-butyl bromide before endoscopic retrograde cholangiopancreatography. AB - BACKGROUND: This study tried to resolve whether glucagon is a better premedication for endoscopic retrograde cholangiopancreatography (ERCP). METHODS: We first measured the basal blood sugar and amylase levels. Then an endoscope was placed in the duodenum without premedication, and basal pulse and duodenal peristaltic rates were measured. ERCP began after studied subjects were randomly premedicated with either 1 mg glucagon (n = 38) or 40 mg hyoscine N-butyl bromide (n = 36) intravenously. Ten minutes later the variables were measured again. RESULTS: Glucagon elicited hyperglycemia whereas hyoscine N-butyl bromide manifested an anticholinergic effect. No difference was found between these two groups with regard to the necessary interval for ERCP (20.6 +/- 14.1 min versus 21.4 +/- 14.7 min; NS) or the success rate for cholangiopancreatography (92.1% versus 91.7%; NS). Neither hyperamylasemia nor pancreatitis was preventable when glucagon was used. CONCLUSIONS: The two premedications appear equally effective in the performance of ERCP. PMID- 7539543 TI - Effect of endothelin-1 on the development of hemorrhagic pancreatitis in rats. AB - BACKGROUND: It has been hypothesized that microcirculatory disturbance plays an important role in the development of severe pancreatitis. In this study we investigated the effects of exogenous endothelin-1 on the development of severe pancreatitis in rats. METHODS: Acute pancreatitis was induced by two intraperitoneal injections of cerulein (10 micrograms/kg body weight). Endothelin 1 was administered via an abdominal aortic catheter as a bolus of 250-750 pmol/kg BW every hour for 4 h. RESULTS: Remarkable morphologic changes in the pancreas, including hemorrhage, and increases in serum amylase level and active elastase content in pancreatic tissue were observed in rats treated with cerulein plus endothelin-1 in a dose-dependent manner 5 h after the first cerulein injection. Local pancreatic blood flow decreased significantly, and microcirculatory disturbances in the pancreas were demonstrated. CONCLUSIONS: These results suggest that endothelin-1 causes pancreatic microcirculatory disturbance and might be a contributing factor in the aggravation of acute pancreatitis. PMID- 7539545 TI - Cytokine production and serum proteins in depression. AB - One, as yet unemployed, approach to investigating immunology in depression is the assessment of the cytokine production by leucocytes, which would allow the determination of immune response under standardized conditions. Thus we measured the production of mitogen-induced cytokines (IL-1 beta, IL-2, IL-6, IL-10, interferon-gamma) and sIL-2R in a whole blood assay, and serum protein levels such as C-reactive protein (CRP), haptoglobin (Hp) and alpha 2-macroglobulin (alpha 2 M) in a longitudinal 6-week study in an attempt to assess leucocyte function during and after acute clinical stage of depression in 39 patients. Shortly after admission to hospital we found higher levels of all measured cytokines in the patients. Serum protein levels were significantly higher in the patients than in controls, and decreased over the study period. Whereas slightly elevated monokine levels in patients tended to reach control values, lymphokines showed a significant decrease over the 6 weeks as compared to baseline. These results suggest that the increase in immune activity seen at the beginning of the study may be followed by a suppressed cell-mediated immune function. PMID- 7539546 TI - Effects of anti-CD18 and LPS on CD14 expression on human monocytes. AB - In this study we show that the cytokine stimulatory effect of LPS on human monocytes is enhanced by addition of monoclonal antibodies against CD18 (alpha CD18 MoAbs). Incubation of monocytes with alpha CD18 MoAbs overnight increased the CD14 expression as detected by Leu-M3, but not with My-4. These results indicate that CD18 participates in LPS-induced TNF-alpha production as well as in regulating CD14 expression on monocytes. Addition of LPS to monocytes resulted in a reduction in the CD14 expression after 1/2, 1, 2 and 4 h, but increased CD14 expression was seen after LPS stimulation overnight. By doing double labelling of the monocyte population for CD14 and CD16 it was found that the reduction in CD14 expression occurred in the CD14+/CD16+ sub-population, while the increase in CD14 expression was seen in both the CD14+/CD16- and the CD14+/CD16+ cells. alpha CD14 MoAbs that were able to inhibit LPS-induced cytokine production from monocytes (3C10 and My-4) were considerably less able to detect the increase in CD14 expression after LPS stimulation than alpha CD14 MoAbs that did not inhibit LPS induced cytokine production (Leu-M3 and alpha CD14Serva). Our data indicate that My-4 and Leu-M3 define two populations of CD14+ cells on LPS stimulated human monocytes. PMID- 7539547 TI - The involvement of CD14 in stimulation of TNF production from peripheral mononuclear cells isolated from PNH patients. AB - Peripheral blood mononuclear cells (PBMC) from six patients with paroxysmal nocturnal haemoglobinuria (PNH) were analysed by flow cytometry for expression of CD14 and for ability to respond to bacterial lipopolysaccharide and beta 1-4 linked polymannuronic acid by TNF secretion. Expression of cell surface CD14 could not be detected on cells from the PNH patients, whereas the levels of expression of other monocyte antigens, e.g. CD33 and CD13, were comparable to that of cells from healthy subjects. The cells from the patients with PNH responded with secretion of significantly less TNF after stimulation with LPS and polymannuronic acid than mononuclear cells from healthy subjects, suggesting an impaired ability in PNH to respond to bacterial infection by TNF secretion from monocytes. Soluble CD14 appeared to be involved in the residual activation of CD14 negative PBMC, and the sera of these patients contained normal or slightly elevated levels of soluble CD14. After allogeneic bone marrow transplantation in one patient the monocytes expressed CD14 at normal levels and responded normally with respect to their ability to generate TNF upon stimulation. PMID- 7539548 TI - [Combination endobronchial and conventional therapy possibilities in inoperable central lung tumors]. AB - Up to 85% of patients with bronchogenic carcinoma are inoperable at the time of diagnosis and treatment remains largely palliative. Prognosis depends on the clinical tumor stage. In non-small cell carcinoma the clinical stages (I-IV) are defined according to the TNM classification, whereas in small cell carcinoma limited disease is distinguished from extensive disease. Neither classification accurately takes endobronchial tumor spread into account. At the time of diagnosis up to 30% of all lung cancer patients present with central airway obstruction and clinical signs of dyspnea, atelectasis and pneumonia. Most patients with central airway stenosis have inoperable tumors (stage IIIb and IV) and have until recently undergone conventional treatment consisting exclusively of chemo- and radiotherapy. Currently the best results are obtained with combined chemoradiotherapy. The rapid developments in the area of endobronchial treatment modalities enable us to relieve bronchial obstructions fast and safely. This achieves immediate symptomatic relief which in many cases is a precondition for starting chemo- or radiotherapy. Successful reopening of a major airway helps to prolong local tumor control and thus survival. Patients with inoperable lung cancer and obstruction of central airways should undergo initial endobronchial therapy followed by conventional chemo-radiotherapy. PMID- 7539549 TI - Bent helix formation between RNA hairpins with complementary loops. AB - The initial interaction between the ColE1 plasmid specific transcripts RNA I and RNA II, which function as antisense regulators of plasmid replication, comprises a transient complex between complementary loops found within the RNA secondary structures. Multidimensional heteronuclear magnetic resonance spectroscopy was used to characterize complexes formed between model RNA hairpins having seven nucleotide complementary loops. Seven base pairs are formed in the loop-loop helix, with continuous helical stacking of the loop residues on the 3' side of their helical stems. A sharp bend in the loop-loop helix, documented by gel electrophoresis, narrows the major groove and allows bridging of the phosphodiester backbones across the major groove in order to close the hairpin loops at their 5'-ends. The bend is further enhanced by the binding of Rom, a ColE1 encoded protein that regulates replication. PMID- 7539550 TI - Electronic states in gallium arsenide quantum wells probed by optically pumped NMR. AB - An optical pumping technique was used to enhance and localize nuclear magnetic resonance (NMR) signals from an n-doped GaAs/Al0.1Ga0.9As multiple quantum well structure, permitting direct radio-frequency measurements of gallium-71 NMR spectra and nuclear spin-lattice relaxation rates (1/T1) as functions of temperature (1.6 K < T < 4.2 K) and the Landau level filling factor (0.66 < v < 1.76). The measurements reveal effects of electron-electron interactions on the energy levels and spin states of the two-dimensional electron system confined in the GaAs wells. Minima in 1/T1 at v approximately 1 and v approximately 2/3 indicate energy gaps for electronic excitations in both integer and fractional quantum Hall states. Rapid, temperature-independent relaxation at intermediate v values indicates a manifold of low-lying electronic states with mixed spin polarizations. PMID- 7539551 TI - Identification of a graft versus host disease-associated human minor histocompatibility antigen. AB - Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1 positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation. PMID- 7539552 TI - [Sequential high-dose therapy in patients with low-malignity non-Hodgkin's lymphoma]. PMID- 7539553 TI - The enhanced immunosuppressive efficacy of newly developed liposomal FK506 in canine liver transplantation. AB - Local delivery of immunosuppressants to the graft and lymphatic tissue is a potential appraoch to enhance the immunosuppressive efficacy and to alleviate systemic adverse effects simultaneously. By taking advantage of this method, we developed liposomal FK506. Previous pharmacokinetic study of liposomal FK506 indicated increased FK506 levels in the liver and spleen. Because the liver is the site of the allograft in liver transplantation and the spleen is a major lymphoid tissue, we hypothesized that liposomal FK506 would increase immunosuppressive efficacy in liver transplantation. We evaluated this hypothesis in a canine model. Orthotopic liver transplantation was performed using beagle dogs, and the recipients were divided into the following groups: group I, no immunosuppression (n = 5); group II, 0.05 mg/kg/day of FK506 i.v. in a commercially available i.v. formulation for 14 days (n = 5); and group III, 0.05 mg/kg/day of FK506 i.v. in a liposomal formulation for 14 days (n = 5). All recipients in group I died within 2 weeks. Recipients in group II died within 33 days. In contrast, three recipients in group III survived for more than 200 days (P < 0.05 versus group I or group II). In DNA analysis, splenocyte proliferation activity in group III was significantly suppressed in comparison with group II. These results suggest that liposomal FK506 markedly increase the immunosuppressive efficacy of FK506 in liver transplantation. A local immunosuppressive effect in the grafted liver and significant suppression of splenocyte proliferation might contribute to enhancement of the immunosuppressive efficacy of liposomal FK506. PMID- 7539554 TI - In vivo studies of the maintenance of peripheral transplant tolerance after cyclosporine. Radiosensitive antigen-specific suppressor cells mediate lasting graft protection against primed effector cells. AB - Cellular mechanisms responsible for maintenance of peripheral transplant tolerance in a rodent model were evaluated. Donor-specific tolerance was established in ACI rats given a vascularized heterotopic cardiac allograft followed by a 10-day course of cyclosporine. Tolerance was associated with a reduction in donor-specific cytotoxic T lymphocyte precursors and the presence within the spleen of cells capable of transferring suppression in adoptive transfer assays. Experiments using thymectomized animals revealed that the establishment and maintenance of tolerance occurred peripherally, independently of the thymus. Adoptive transfer experiments demonstrated that ongoing graft tolerance was mediated by suppressor cells that were antigen-restricted, radiosensitive, and capable of preventing allograft rejection by naive as well as sensitized cells in vivo. Studies designed to disrupt tolerance demonstrated a remarkable durability of graft protection once established, and give insight into the identity and mechanism of action of suppressor cells generated in this model. PMID- 7539555 TI - Association of down-regulation of cytokine activity with rat hind limb allograft survival. AB - Cytokines are short-acting protein modulators of many physiologic processes including graft rejection. An understanding of the production, action, and interaction of cytokines may lead to better appreciation of the complex mechanism of graft rejection. The potential would then exist for more selective and less toxic means of modulating the immune response. A rat hind limb allograft model with major immunohistoincompatibility was used to study the local mRNA expression of IL-1 alpha, IL-2, IL-6, gamma interferon (gamma INF), platelet-derived growth factor-alpha (PDGF-alpha), basic fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta) during acute allograft rejection. A 14-day postoperative course of immunosuppressive therapy with FK506 or rapamycin was administered. In situ hybridization was performed on serial full-thickness skin punch biopsies of the untreated rejecting limb allograft and compared with tissue from treated allografts, isografts, and to normal limb tissue. A sequential pattern of cytokine mRNA expression was demonstrated which progressed in a time dependent manner and paralleled observed clinical rejection. Maximal cytokine mRNA expression correlated with peak graft rejection. Cellular expression of IL-1 alpha, IL-2, IL-6, gamma-INF, FGF, and TGF-beta mRNA was suppressed with FK506 to below isograft levels, and clinical rejection was not observed with the doses, routes, and schedules used. Rapamycin was ineffective in suppressing cytokine expression, and allograft rejection was not prevented. Isografts demonstrated no evidence of rejection. The in situ hybridization technique demonstrates a time dependent, selective expression of cytokines within rejecting allograft tissue, and the modification of this response with immunosuppressive therapy. Down regulation of cytokine expression is associated with clinical allograft survival. PMID- 7539556 TI - Induction of alloantigen-specific hyporesponsiveness in vitro by the short-chain fatty acid N-butyrate. PMID- 7539557 TI - Regulation of prostatic smooth muscle contractility by intracellular second messengers: implications for the conservative treatment of benign prostatic hyperplasia. AB - The increased sympathetic neurotransmission in benign prostatic hyperplasia (BPH) results in a alpha 1C-adrenoceptor-mediated increase in prostatic smooth muscle tone which seems to be responsible for the dynamic infravesical obstruction occurring in BPH. The prostatic smooth muscle contractions evoked by norepinephrine can be efficiently blocked by alpha 1-adrenoceptor blockers. Moreover, an impressive number of clinical trials illustrated the beneficial results of alpha 1-adrenoceptor blockers in the treatment of BPH. However, despite knowledge of alpha 1-adrenergic neurotransmission and the clinical application of its blockade by selective alpha 1-adrenoceptor antagonists, very little is known about the intracellular pathways involved in the regulation of prostatic smooth muscle contractility. To study the intracellular mechanism of the alpha 1C-adrenoceptor-induced prostatic smooth muscle contraction, the patch clamp technique in the whole-cell configuration mode combined with the Fura-II fluorescence technique was used in human, enzymatically isolated smooth muscle cells obtained from patients undergoing transurethral resection of the prostate because of symptomatic BPH. Furthermore changes in prostatic smooth muscle contractility were registered in organ bath experiments. Application of the selective alpha 1-adrenoceptor agonist phenylephrine (PE) increased the L-type Ca(2+)-channel current (ICa) dose dependently from 8 up to 18.5 microA/cm2, simultaneously elevating the free cytoplasmic Ca2+ concentration ([Ca2+]i) up to 1.9 microM. Pretreating the myocytes with pertussis toxin, an exotoxin of Bordetella pertussis which inactivates GTP-binding proteins (G proteins) of the Gi and G(o) family by ADP ribosylation, reduced the PE-induced ICa stimulation by 71.5 +/- 5.6% (n = 21). Dialysis of the cytosol with the second messenger inositol-1,4,5-trisphosphate (IP3), which releases Ca2+ from intracellular non mitochondrial, IP3-sensitive Ca2+ pools, imitated the PE-evoked responses. Pretreating the myocytes with the Ca(2+)-release blockers ryanodine (10-100 microM, n = 8), thapsigargin (0.1 microM, n = 11) or low-molecular weight heparin (n = 14) largely attenuated the PE-evoked responses. The experimental results suggest a coupling of alpha 1-adrenoceptors to phospholipase C-converting phosphoinositol-4,5-bisphosphate into diacylglycerol, an endogenous activator of the protein kinase C and IP3 which releases Ca2+ from intracellular stores stimulating ICa via Ca(2+)-calmodulin-dependent protein kinase induced phosphorylation of voltage-dependent Ca2+ channels. This knowledge could be of interest for conservative treatment in symptomatic BPH. PMID- 7539558 TI - Reference range for prostate-specific antigen levels after external beam radiation therapy for adenocarcinoma of the prostate. AB - OBJECTIVES: To determine a reference range for prostate-specific antigen (PSA) in patients who have had external beam radiation therapy for adenocarcinoma of the prostate. METHODS: Serum PSA levels were serially measured with the Hybritech Tandem-R assay in 35 patients who had completed radiation therapy for a mean of 51 +/- 32 months (range, 10 to 133). RESULTS: Serum PSA levels that were greater than 2.0 ng/mL (n = 20) continued to rise at a rate of more than 1 ng/mL per year, whereas levels between 0.0 and 2.0 ng/mL (n = 15) remained stable for an average of 71 +/- 32 months (range, 22 to 133). One of these latter 15 patients had a false-negative PSA level when he experienced a biopsy-positive prostate nodule and positive bone scan 43 months after therapy. CONCLUSIONS: Prostate cancer control after radiation therapy was indicated in most men by a PSA reference range of 0.0 to 2.0 ng/mL. Cancer activity was indicated in all men with levels above this range. PMID- 7539559 TI - Effect of radiation therapy after radical prostatectomy on serum prostate specific antigen measured by an ultrasensitive assay. AB - OBJECTIVES: To study prospectively the impact of adjuvant radiation therapy on the serum level of prostate-specific antigen (PSA), as measured by an ultrasensitive Yang Proscheck assay in patients with detectable serum PSA and a negative metastatic survey after radical prostatectomy for T1 or T2 prostate cancer. METHODS: Seventeen patients had a detectable serum PSA (2.40 +/- 2.1 ng/mL; range, 0.5 to 10) by the Yang polyclonal assay 2 to 71 months after radical prostatectomy for P2N0 (2 patients) or P3N0 (15 patients) prostate cancer. Metastatic workup (bone and computed tomography scan) was negative; 9 of 17 patients had a local recurrence documented by a positive biopsy of the vesicourethral anastomosis. All patients were treated by external radiotherapy, receiving 65 Gy on the prostate fossa over 5 weeks for an assumed low volume residual disease. Patients were followed up by determination of serum PSA every 3 months, using the Yang ultrasensitive assay for a mean duration of 14.4 months. RESULTS: In 17.6% of the patients (3 of 17) PSA became undetectable (less than 0.05 ng/mL) after radiotherapy. Radiotherapy had no impact on PSA in 35.3% (6 of 17). PSA decreased after radiation therapy within 6 months in 47.1% (8 of 17) and for up to 12 months in 2 patients, with a nadir of 0.28 ng/mL. All patients in this group experienced a secondary rise in PSA a mean of 10.6 months (range, 6 to 18 months) after radiotherapy. CONCLUSIONS: External radiotherapy has a limited impact on residual disease after radical prostatectomy, as assessed by its impact on PSA. PMID- 7539560 TI - Treatment of BPH by the TULIP procedure. PMID- 7539561 TI - Health status and quality of life of British men with lower urinary tract symptoms: results from the SF-36. AB - OBJECTIVES: To determine the extent to which urinary symptoms, and resulting bothersomeness interfere with daily activities and affect health status, as measured using the Medical Outcomes Study 36-item short form health survey (SF 36). METHODS: Postal population survey in a British health region of 217 men aged 55 years and over known to have reported mild, moderate, or severe lower urinary tract symptoms. Outcome measures are self-reported urinary symptoms, their bothersomeness, general health status, and quality of life (measured using the SF 36). RESULTS: Response rate among eligible subjects was 84%. Depending on the activity, between 9% and 49% of men with moderate or severe urinary symptoms reported interference with some of their daily activities. Increasing symptom severity was associated with worsening physical role, social functioning, vitality, mental health, and perception of general health, and increasing bothersomeness was associated with worsening of all dimensions of general health status and quality of life. The association between these measures and bothersomeness was stronger than with symptom score. Compared with the general population, men bothered by their symptoms to the extent that they were a medium or a large problem have worse health status for all dimensions except physical functioning. CONCLUSIONS: The SF-36 demonstrates a deterioration in general health status and quality of life with increasing lower urinary tract symptoms and the extent to which those symptoms are bothersome. As such, it provides a generic measure of the burden of ill health arising from these symptoms at a population level. There is, however, considerable individual variation in the way that men respond to their symptoms. PMID- 7539562 TI - Role of prostate-specific antigen density after applying age-specific prostate specific antigen reference ranges. AB - OBJECTIVES: To determine the potential role of prostate-specific antigen (PSA) density (PSAD) in the early detection of prostate carcinoma if we apply age specific PSA reference ranges (2.5 ng/mL or less for ages 40 to 49 years, 3.5 or less for ages 50 to 59, 4.5 or less for ages 60 to 69, and 6.5 or less for ages 70 to 79. METHODS: We retrospectively reviewed 3234 cases referred to us by urologists for transrectal ultrasound (TRUS) between January 1, 1991, and September 28, 1993. We included 2429 patients in the study, ages 40 to 79 years, with Hybritech or Abbott IMx serum PSA determinations and without previously diagnosed prostate cancer. We performed digital rectal examination (DRE) and TRUS in all cases, and TRUS-guided biopsies when indicated. We used stringent criteria to define 736 cases without clinical evidence of malignancy that were designated as a "benign group." RESULTS: In the benign group, we found serum PSA to increase with age in parallel with the increase in prostate volume with age (r = 0.25 and r = 0.26, respectively). The association between serum PSA and prostate volume was stronger (r = 0.46). Using multiple regression analysis, prostate volume accounted for 18% of the variation in serum PSA, whereas age accounted for only an additional 2%. PSAD, which directly relates serum PSA to prostate volume, showed a weak association with age (r = 0.1). In the entire study population of 2429 cases, 555 patients had negative DRE and TRUS results and a serum PSA level between the age-specific upper limit of normal and 10.0 ng/mL. According to the proposed age-specific algorithm, these patients would have required automatic biopsies. Of these, 315 cases (56.8%) still had a PSAD of less than 0.15. We performed biopsies in 108 of these 315 and detected only two cancers, for a positive biopsy rate (PBR) of 1.9%. The remaining 240 cases had a PSAD of 0.15 or higher, and we performed biopsies in 217 of these cases and detected 59 cancers, for a PBR of 27.2%. CONCLUSIONS: The use of age-specific PSA reference ranges does not totally account for the effect of prostate volume on serum PSA. Therefore PSAD can still be used to reduce safely the number of biopsies performed in patients with negative DRE and TRUS results and a serum PSA level 10.0 ng/mL or less and above the age-specific upper limit of normal. PMID- 7539563 TI - Utilization of high molecular weight cytokeratin on prostate needle biopsies in an independent laboratory. AB - OBJECTIVES: Immunoperoxidase staining of prostate tissues with antibodies to high molecular weight cytokeratin, which selectively labels basal cells, has recently been shown to be useful in the diagnosis of prostate cancer in academic centers. A growing sector in pathology is large independent laboratories, where little is known regarding practice patterns. The following study evaluated the use of high molecular weight cytokeratin in an independent laboratory specializing in prostate needle biopsies. METHODS: In a 2-month period (July 1, 1994 to August 31, 1994), 4047 prostate needle biopsies were evaluated. RESULTS: Without the use of ancillary studies, 2710 (67%) were diagnosed as benign, 978 (24.1%) were diagnosed as cancer, and 23 (0.6%) were diagnosed as high-grade prostate intraepithelial neoplasia. The remaining 336 atypical cases (8.3%) were further evaluated with antibodies to higher molecular weight keratin. Of the 336 cases, 253 (6.2% of total) were resolved as diagnostic for cancer, 68 (1.7% of total) were diagnosed as benign, and 15 (0.4% of total) remained atypical. The cost of performing high molecular weight cytokeratin was approximately $5.00 per case, which was not passed on to the patient. CONCLUSIONS: The use of high molecular weight cytokeratin decreased the rate of an atypical prostate biopsy from 8.3% to 0.4% at a negligible cost to the pathologist and patient. PMID- 7539564 TI - Birth expectations of women in the United States: 1973-88. PMID- 7539566 TI - [Status of the kallikrein-kinin systems and level of antiproteinase potential during postoperative treatment of various forms of peritonitis]. AB - Some parameters of the kallikrein-kinin system and the antiproteinase potential were studied in patients with diffuse peritonitis during the routine treatment course, after additional intramuscular administration of mexidol (inhibitor of free radical oxidation with antiproteinase activity) as well as after treatment of the patients with physiological solution containing ozone (4-7 mg/l used as a donor of free radicals), which was applied for washing of abdominal cavity in one group of patients during surgical operation and for intravenous administration in the other group. The kallikreinkinin system parameters were distinctly altered in the patients with peritonitis and the rate of the system activation correlated with the disease severity. Content of the alpha 1-inhibitor of proteinases (alpha 1-PI) was increased by the middle period of the treatment course and decreased as the inflammation lowered. alpha 2-Macroglobulin (alpha 2-MG) was altered less distinctly as compared with alpha 1-PI. Patterns of the antiproteinase potential in blood plasma (alpha 1-PI and alpha 2-MG) may be used for diagnostic and prognostic purposes in post-surgical treatment of patients with diffuse peritonitis; a decrease in the potential value proved to be a symptom of danger. Both mexidole and ozone exhibited positive clinical effect in treatment of the disease. Although these drugs had an opposing effect on the proteinases activity, their similar action on the antiproteinase potential appears to be responsible for the medicinal efficiency. Clinical effect of the drugs may be arranged as follows: mexidole, ozone administered intravenously and ozone used for intraabdominal washing. PMID- 7539565 TI - [The effect of phosphatidylcholine on repair processes in liver cells in acute CCl4 damage]. AB - Effect of phosphatidylcholine, isolated from soy beans and sunflower seeds for laboratory use, on synthesis of RNA, DNA, albumin and content of newly synthesized mRNA in polyribosomes was studied in hepatocytes after chemical hepatectomy produced by single administration of CCl4 into rats; histological and histochemical studies of liver tissue were also carried out. Phosphatidylcholine from these plants was found to prevent hepatocyte dystrophy and necrosis development, to activate the macrophage response and to stimulate reparation inducing synthesis and secretion of the tumor necrosis factor. The rate of highly labelled RNA synthesis, mainly mRNA, decreased in liver tissue of rats treated with CCl4, was increased approximately 2-fold after the phospholipid administration. The phospholipid from soy beans restored the albumin synthesis as well as the content of newly synthesized mRNA in rat polyribosomes, while phosphatidylcholine from sunflower seeds restored effectively the mitochondrial DNA synthesis. Promising application of these phosphatidylcholines as hepatoprotectors is discussed. PMID- 7539568 TI - Immunohistochemical and glycohistochemical localization of the beta-galactoside binding S-type lectin in human placenta. AB - The localization of the beta-galactoside binding lectin was studied immunohistochemically on acetone-fixed cryostat sections of full-term placental tissue using a biotinylated monoclonal antibody and glycohistochemically applying biotinylated asialofetuin and lactosylated bovine serum albumin. On blots of placental tissue lysates the lectin is recognized by the biotinylated lactosylated bovine serum albumin. The glycoconjugate recognition of the lectin on blots was inhibited in the presence of 0.1 M lactose showing the specificity of the interactions. The anti-lectin monoclonal antibody stained syncytiotrophoblast and trophoblastic cells. Both reagents applied for glycohistochemistry stained syncytiotrophoblast and trophoblastic cells of placental villi and the trophoblastic layer of extraplacental membranes. A strong uniform cytoplasmic staining was characteristic for syncytiotrophoblast and to a lower extent for cytotrophoblastic cells. The localization of the lectin is discussed with respect to a possible immunosuppressive function. PMID- 7539569 TI - Distribution and evaluation of complex carbohydrates in the quail collecting ducts. AB - The cell heterogeneity within the collecting duct epithelium of quail kidney was investigated using histochemical methods for complex carbohydrates. The selective distribution of acidic glycoconjugates allowed further discrimination between metachromatic, mucin-secreting cells and dark, proton-transporting cells. Enzymatic digestion by glycosidases was performed to characterize the carboxylated and sulphated glycoconjugates of mucin-secreting cells. The staining intensity of all histochemical reactions with and without prior treatment, was quantitated by means of scanning histophotometry and differences in absorbance values were evaluated by statistical analysis. A different distribution of sulphated components was found for the mucin-secreting cells of renal cortex and medulla, suggesting a morphofunctional heterogeneity within mucin-secreting cell population. PMID- 7539567 TI - Expression of cytokeratins, vimentin and basement membrane components in human fetal male mullerian duct and perimullerian mesenchyme. AB - The expression and distribution of cytokeratins 8, 18, 19, pan-cytokeratin, vimentin, type IV collagen, laminin and fibronectin were investigated immunohistochemically in the Wolffian and Mullerian ducts and perimullerian mesenchyme of human male fetuses ranging from 8 to 12 weeks of gestation. The epithelial cells of both genital ducts, the coelomic epithelium and the mesenchymal perimullerian cells coexpressed cytokeratins 18, 19, pancytokeratin and vimentin. Type IV collagen, laminin, and fibronectin were detected in the basement membranes of both genital ducts, the coelomic epithelium and in the interstitium of the perimullerian mesenchyme. The coelomic basement membrane adjacent to the mullerian duct showed interruptions and perimullerian cells were in contact with the coelomic epithelium. The cytoskeletal immunophenotype of perimullerian cells and their relationship with the coelomic epithelium suggested their coelomic origin. During the 12th week of gestation, mullerian duct began to regress with its basement membrane detaching from the surrounding mesenchyme. This coincided with the disappearance of vimentin from the ductal epithelial cells, as well as type IV collagen, laminin and fibronectin from perimullerian mesenchyme. Thus mullerian ductal basement membrane dissolved. These findings indicate that the human mullerian duct regression is associated with changes in the interactions between Mullerian ductal and perimullerian mesenchymal cells. PMID- 7539570 TI - [Photosensitization of dicoumarol on tumor cells and enhancement by sodium diethyldithiocarbamate]. AB - AIM: To study the photosensitization of dicoumarol (Dic) on tumor cells and any effect by sodium diethyldithiocarbamate (DDC). RESULTS: Dic, 40, 80, 120 mumol.L 1 concentration-dependently inhibited the DNA synthesis (81%-93%) and increased the mortality (50%-70.4%) of ascitic hepatoma (Hep A) cells exposed in light. These 2 indices were changed slightly by Dic alone even in non-illuminating group. DDC enhanced the photosensitization of Dic group. CONCLUSION: Photosensitization of Dic showed strong antitumor activities against Hep A cells. The mortality of Hep A and inhibition of the DNA synthesis in Dic-DDC-light group significantly stronger than in Dic-light group. Photosensitization of Dic was not due to 1O2 and OH., but closely related to O2.- and H2O2. PMID- 7539571 TI - [Treatment of acute urine retention caused by benign prostatic hypertrophy with an LH-RH agonist (Goserelin)]. AB - Nineteen patients seen for AUR secondary to benign prostate hypertrophy and with high surgical risk, were treated with an LH-RH agonist (GOSERELIN). After six months of treatment a 43.28% decrease in the prostate volume was seen, Improvement, i.e., spontaneous miction, was achieved in 46.6% patients, although post-mictional volumes remained high. PMID- 7539574 TI - [The IPPS questionnaire in patients and controls. Psychometric validation]. AB - The translation into Spanish of the AUA/International Prostatic Symptom (AUA/IPS) Score hadn't been validated in spanish. METHODS. AUA/IPS questionnaire was self administered by 103 consecutive patients and by 24 healthy volunteers. An alternative formulation to question 4 (ALT-4) was added. RESULTS. 51 patients (49%) and 17 controls (79%) completed al questions (Feasibility). Reliability was measured by: a) Crohnbach's alpha (0.68 and 0.70 with ALT-4) b) Kendall's coefficient of concordance (0.097, significance < 0.001) and c) retesting 17 patients within 2 months. The relation of the AUA/IPS scores with quality of life (Construct validity) showed a Spearman's correlation coefficient of 0.57 (confidence = 0.0001), and 0.52/0.0004 with ALT-4. The relation with uroflowmetry (Concurrent empirical validity) was poor as shown in ROC curves. CONCLUSIONS. The Spanish version of the AUA/IPS questionnaire given in an easy format improves feasibility without alteration of reliability or validity. The relation of the AUA/IPS score with uroflowmetry is poor, although the questionnaire can be considered valid and reliable. PMID- 7539573 TI - [TPS in prostate cancer]. AB - A study of the tumour proliferation marker TPS in prostate cancer has been carried out. The levels of this marker were determined in the following groups: controls (n = 51), prostate hyperplasia (n = 17), prostate cancer in clinical remission (n = 15), and progressive prostate cancer (n = 13). An upper normal limit of 129 U/l (percentile 95) was established. When the progressive disease group was compared with the other groups, a significant difference (p < 0.001) was found. Sensitivity, specificity and positive predictive value obtained in order to dismiss progression were 71%, 94% and 62% respectively. PMID- 7539572 TI - [Is it possible to do without bone gammagraphy in the staging of prostatic cancer?]. AB - Since the discovery of the prostate specific antigen, and its use as tumoral marker, not only this has been shown to have a significant application for the diagnosis and evaluation of prostate cancer management, but also to be an eventual help to predict occurrence of bone metastasis. A retrospective study was conducted in 50 patient with prostate cancer, with mean age of 74 years (range 56 90), where PSA levels were analyzed and then correlated with the results obtained with bone scintigraphy. Our work results were that, given a 40% prevalence of the metastatic disease, no patient had PSA values under 10 ng/ml and, at the same time, bone dissemination, i.e., a 100% negative predictive value. Thus, we can state that in our group, we could have given a PSA cut-off value over 10 ng/ml to perform bone scintigraphy, without making any staging mistake. PMID- 7539575 TI - Alkylation of purine bases by carbon-centered radicals. PMID- 7539576 TI - The role of eicosanoids and selectins in experimental asthma in monkeys. PMID- 7539577 TI - Human 15-lipoxygenase: a potential effector molecule for interleukin-4. PMID- 7539578 TI - Marked episodic elevations of alpha-fetoprotein without hepatocellular carcinoma in a patient with hepatitis B. PMID- 7539579 TI - Multiple false reactions in viral antibody screening assays after influenza vaccination. AB - In December 1991, US blood centers reported an unusual increase in donations that tested falsely reactive for antibodies to two or more (multiple false positive) of the following viruses: human immunodeficiency virus type 1 (HIV-1), human T cell lymphotrophic virus type I (HTLV-I), and hepatitis C virus. Many of these donations were from people who had recently received the 1991-1992 influenza vaccine, raising the possibility that this vaccine had somehow specifically caused the problem of multiple false reactivity. A case-control study of 101 affected donors and 191 matched controls found that recent receipt of any brand of influenza vaccine was significantly associated with testing multiple false positive (p < 0.05), as was a history of recent acute illness (p < 0.05) and of allergies (p < 0.05). Surveillance for monthly rates of multiple reactive donations from May 1990 through December 1992 linked the seasonal cluster of multiple false-positive donations to the use of viral screening test kits thought to react nonspecifically to donor immunoglobulin M. There was no similar increase in multiple false-positive donations during the 1992-1993 influenza vaccination season after the HIV-1 and hepatitis C virus tests were replaced; however, the number of donations that were falsely reactive for only HTLV-I almost doubled, indicating that false reactivity was not specifically associated with the 1991 1992 influenza vaccine. Retesting of affected donors found that the duration of HTLV-I and hepatitis C virus false reactivity was 3-6 months. The cluster of multiple false-positive donations in 1991 was most likely caused by the test kits used, rather than by the influenza vaccine. PMID- 7539581 TI - Prussian blue reaction and hemophagocytosis: a new use for an old test. PMID- 7539580 TI - Serum antibodies to distinct epitopes of the tissue-type plasminogen activator (t PA) in patients with systemic lupus erythematosus. AB - Defective fibrinolysis due to decreased tissue-type plasminogen activator (t-PA) activity is a well-established finding in patients with systemic lupus erythematosus (SLE). The possibility that this decrease in t-PA activity may be related to the presence of autoantibodies directed against t-PA, and the possible role of these autoantibodies in the pathophysiology of fibrinolysis in SLE, were investigated. Serum samples from 115 SLE patients and 63 normal volunteers were analyzed for the presence of such antibodies. The search for antibodies to t-PA was performed by means of several systems, allowing for the identification of epitopes presented in different conformational physical states of t-PA: free or associated to its inhibitor (PAI-1) in plasma, specifically bound to fibrin surface, or passively adsorbed to solid supports. Antibodies in variable amounts were detected by all systems used; however, t-PA activity was not inhibited by the IgG fraction of the positive sera in a fibrin-agar fibrinolysis system. Moreover, the demonstration of serum anti-t-PA antibodies was not associated with clinical or laboratory abnormalities related to vasoocclusive episodes. These results indicate that, as in the case of other autoantibodies, their detection in serum does not imply their direct participation in the pathophysiology of thrombosis in SLE. PMID- 7539583 TI - On a dramatic response of refractory anemia with excess blasts in transformation to 5-azacytidine. PMID- 7539582 TI - Etoposide, dexamethasone, and continuous-infusion cyclophosphamide with G-CSF for VAD-resistant multiple myeloma. PMID- 7539584 TI - Case report: finasteride-induced gynecomastia in a 62-year-old man. AB - The authors describe a case of bilateral (with left prevalence) gynecomastia in a 62-year-old man after finasteride treatment because of benign prostatic hypertrophy. Finasteride is an inhibitor of 5 alpha-reductase, the enzyme responsible for testosterone metabolism to dihydrotestosterone. In this patient, nonspecific endocrine alterations were found, except for a significant decrease in dihydrotestosterone levels. In addition, there were no pathologic conditions affecting other organs or pharmacologic treatments that could be responsible for gynecomastia. Drug withdrawal started a progressive reduction of the lumps until complete their disappearance. It is possible that gynecomastia was caused by alterations of estrogen/androgen ratio because of a finasteride-induced decrease in circulating dihydrotestosterone levels. In this article, the authors confirm finasteride antiandrogenic activity and recommend a close follow-up of long-term treatments with finasteride to find out other possible side effects. PMID- 7539585 TI - Subtotal but not unilateral nephrectomy induces hyperplasia and protooncogene expression. AB - It is generally accepted that renal compensatory growth after unilateral nephrectomy (Uni) is due to prominent hypertrophy with no involvement of protooncogenes. Neither the balance between hypertrophy and hyperplasia nor the expression of the early-growth-related genes has been studied after subtotal nephrectomy (Nx). The occurrence of cystic tubular dilatations after Nx may suggest an excessive cell proliferation in this model. We measured DNA, RNA, and protein content, number of nuclei per tubular section, as well as c-fos, c-jun, c myc, c-H-ras, c-sis, and c-erb-B2 protooncogene expression in kidneys taken at time of surgery and 2, 7, and 14 days after sham operation (control rats), Uni, or Nx. After Uni, hyperplasia was greater than expected (+79% for DNA at day 14) and was associated with moderate hypertrophy (+11% for protein/DNA ratio). After Nx, compensatory growth was due only to hyperplasia (+117% for DNA at day 14), with unchanged protein/DNA ratio (vs. Uni, P < 0.02). The greater hyperplasia after Nx was confirmed by nuclei counting. The protooncogene mRNA expression was constantly absent in control and Uni rats, whereas that of c-fos and c-jun genes was detected in Nx rats at day 14 with a 2- to 12-fold increment. The c-fos and c jun protein levels were also increased at that time in Nx rats. This suggests the following: 1) the cellular events following Uni and Nx are not the same, and 2) the late protooncogene expression in Nx exclusively could favor a particular type of cell proliferation possibly more related with cystic formation than with actual compensatory growth. PMID- 7539588 TI - Altered beta-adrenergic and muscarinic response of CFTR Cl- current in dialyzed cardiac myocytes. AB - Autonomic regulation of the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current was studied in isolated guinea pig ventricular myocytes using various configurations of the whole cell patch-clamp technique. When currents were recorded using the conventional patch-clamp technique, it was possible to continue to activate the Cl- current on repeated exposure to isoproterenol (Iso) for up to 60 min after initiating dialysis. However, there was significant rundown of the magnitude of the Cl- current response to the maximally stimulating concentrations of Iso. In addition, the concentration of Iso that produced half-maximal activation of the Cl- current (K1/2) increased with time. Conversely, the K1/2 for acetylcholine inhibition of the Iso-activated current decreased with time. When currents were recorded using the perforated patch-clamp technique, the sensitivity to both beta-adrenergic- and muscarinic receptor stimulation was stable. Immediately after initiation of dialysis with the conventional patch-clamp technique, the sensitivity to Iso was nearly identical to that determined using the perforated patch-clamp technique. However, the initial sensitivity to muscarinic-receptor activation was significantly greater. These results indicate that cell dialysis associated with conventional patch-clamp techniques not only results in a time-dependent rundown of current amplitude, but it also significantly alters the concentration dependence of beta adrenergic and muscarinic-receptor regulation of ion channel function. PMID- 7539587 TI - Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells. AB - In this study, we investigated the effects of endosomal alkalinization on kinetics of endocytotic uptake in intact proximal tubule-derived opossum kidney cells. We used fluorescein isothiocyanate (FITC)-labeled albumin and FITC-dextran as endocytotic substrates for receptor-mediated endocytosis and fluid-phase endocytosis, respectively. The pH in endosomes labeled with either FITC-albumin or FITC-dextran rose in the presence of the vacuolar-type ATPase inhibitor, bafilomycin A1, and in the presence of NH4Cl. Cytoplasmic pH, decreased in the presence of bafilomycin A1, but was not significantly different from control during prolonged exposure of the cells to NH4Cl. Endocytotic uptake of FITC dextran was not affected by endosomal pH changes. Endocytotic uptake of FITC albumin was reduced markedly by bafilomycin A1 (decrease of maximum transport rate and apparent affinity). Selective alkalinization of endosomes using NH4Cl (i.e., with the cytoplasmic pH not different from control) reduced FITC-albumin uptake in a similar way but to a lesser extent than did bafilomycin A1. Intracellular albumin degradation was impaired by bafilomycin A1 and NH4Cl. Prevention of endosome-lysosome fusion (lowering the temperature to 20 degrees C) abolished the effects of endosomal alkalinization. Furthermore, specific binding of albumin to the plasma membrane was reduced after incubation with bafilomycin A1, indicating an impairment of receptor recycling. These data show that endosomal pH is an important determinant for the kinetics of receptor-mediated endocytotic uptake of albumin in the proximal tubule but not for fluid-phase endocytosis. Endosomal alkalinization disturbs intracellular ligand handling and receptor trafficking, leading to a reduction of endocytotic capacity and affinity. PMID- 7539586 TI - Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. AB - Nitric oxide (NO) is generated from L-arginine by NO synthase (NOS). We have investigated the localization of constitutive NOS isoforms in rat, mouse, guinea pig, rabbit, pig, and human kidney. NADPH diaphorase (NADPH-d) reaction was used for histochemical detection of NOS enzyme activity, neuronal NOS (NOS I) and endothelial NOS (NOS III) were identified by specific antibody, and in situ hybridization was applied for NOS I mRNA detection. Strong presence of NOS I in macula densa (MD), previously detected in rat, was found in all species including humans. Additional NOS I-positive cells of the thick ascending limb (TALH) were defined. A clear-cut distinction between Tamm-Horsfall-protein-positive cells of the TALH and NOS I-positive cells of the TALH was shown. Ultrastructurally, NOS I was located in the cytosol. Intimate spatial relation between NOS I-positive cells and renin-containing preglomerular afferent arteriole suggests an effect of MD-derived NO on the juxtaglomerular granular cells. In the renal vasculature, both NADPH-d and NOS III were located in the endothelium of cortical and medullary vessels, whereas the muscle layer was unreactive. The glomerular arterioles showed stronger labeling in the efferent than in the afferent endothelium, and efferent endothelium selectively contained both NOS I and NOS III. The unique morphology of efferent endothelial cells indicates a particular role for NO in this vessel segment. At the capillary level, only the glomerular tuft showed NOS-positive endothelia. A subpopulation of renal nerves containing NADPH-d and NOS I was found in perivascular connective tissue and near pelvic epithelium. These results demonstrate a wide distribution of two constitutive NOS isoforms in the kidney of various animal species including humans. The distinct location of both isoforms in the cortex confirms that NO plays a crucial role in local glomerular signaling events. PMID- 7539589 TI - Capillary hydraulic conductivity is decreased by nitric oxide synthase inhibition. AB - Nitric oxide (NO) has been reported to modulate microvascular permeability to solutes in whole organs, venules, and cultured endothelial cell monolayers. NO derived from L-arginine via NO synthase activates soluble guanylate cyclase in vascular smooth muscle and endothelial cells. While the effects of NO on capillary water permeability have not been characterized, other activators of guanylate cyclase, such as sodium nitroprusside and atrial natriuretic peptide, increase capillary hydraulic conductivity (Lp). We hypothesized that inhibition of NO synthase with the arginine analogue, NG-monomethyl-L-arginine (L-NMMA), would decrease Lp from control levels. Lp was assessed in situ in single perfused frog mesenteric capillaries, first during control conditions (Lcontrolp) and then during superfusion (Ltestp) with either L-NMMA, NG-monomethyl-D-arginine (D NMMA), a biologically inert enantiomer, or L-NMMA and L-arginine. Superfusion with 1 microM L-NMMA caused a decrease in Lp (Ltestp/Lcontrolp = 0.6 +/- 0.1, P < 0.001), whereas 1 microM D-NMMA was without effect on Lp (Ltestp/Lcontrolp = 1.0 +/- 0.2). The decrease in Lp by 1 microM L-NMMA was not only prevented by the presence of excess L-arginine (100 microM), but Lp increased from control (Ltestp/Lcontrolp = 1.4 +/- 0.2, P < 0.05). Furthermore, superfusion of L arginine (100 microM) caused an increase in capillary Lp (Ltestp/Lcontrolp = 2.4 +/- 0.9, P < 0.05), whereas D-arginine had no effect on Lp (Ltestp/Lcontrolp = 1.2 +/- 0.3). The results of this study support our hypothesis that inhibition of NO synthase decreases capillary Lp in the intact circulation. In addition, L arginine increases capillary Lp in our model.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539590 TI - Altered subcellular Ca2+ regulation in papillary muscles from cardiomyopathic hamster hearts. AB - To investigate whether cardiac dysfunction in prefailure cardiomyopathic (CM) hamster hearts is due to Ca2+ overload or alternatively to decreased availability of Ca2+ in the sarcoplasmic reticulum (SR), the Ca2+ channel agonist, BAY K 8644, was used to compare the effects of increased Ca2+ influx on function and subcellular Ca2+ distribution in papillary muscles from hearts of 110-day-old CM and normal hamsters. A band, mitochondrial, and junctional SR Ca2+ were measured by electron probe microanalysis in CM and normal papillary muscles, which were either untreated or pretreated with BAY K 8644. Muscles were then rapidly frozen during contraction or relaxation. The results showed decreased tension development and decreased inotropic response to BAY K 8644 in CM muscles versus normals. There was no elevation of mitochondrial or A-band Ca2+ in BAY K 8644 treated or untreated CM muscles frozen during contraction or relaxation compared with similarly treated normals. In muscles frozen during relaxation, junctional SR Ca2+ was lower in both untreated and BAY K 8644-treated CM muscles versus comparably treated normals. These results do not support the hypothesis of an increased sensitivity to Ca2+ in hypertrophied, prefailure CM hearts but do indicate that less Ca2+ is available in the SR for activation of contraction. PMID- 7539591 TI - Inhibitory effect of indomethacin on prostacyclin receptor-mediated cerebral vascular responses. AB - The present study addresses the hypothesis that indomethacin, in addition to blocking prostaglandin synthesis, directly inhibits prostacyclin receptor mediated cerebral vascular responses. To test this hypothesis, the effects of indomethacin on pial arteriolar dilation in response to the prostacyclin receptor agonist iloprost were investigated using a cranial window technique in newborn pigs. Topically applied iloprost resulted in dose-dependent pial arteriolar dilation and concomitant increases in cortical adenosine 3',5'-cyclic monophosphate (cAMP). Indomethacin (5 mg/kg iv + 10(-4) M topically) greatly reduced both the vasodilation and the increase in cortical cAMP in response to iloprost. In contrast, indomethacin did not attenuate beta-adrenoreceptor mediated vasodilation and the increase in cortical cAMP in response to isoproterenol. Aspirin (50 mg/kg iv + 10(-3) M topically) did not affect pial arteriolar dilation or the increase in cortical cAMP in response to iloprost. Unlike indomethacin, aspirin was not effective in inhibiting prostanoid associated cerebral vasodilation and increase in cortical cAMP in response to hypercapnia. The present data suggest that indomethacin selectively inhibits prostacyclin receptor-mediated responses in the newborn pig cerebral circulation. The combination of highly effective inhibition of prostaglandin H synthase and prostacyclin receptor-mediated vasodilation may contribute to the increased efficacy of indomethacin compared with other prostaglandin H synthase inhibitors in blocking certain vasodilator responses associated with prostanoids. PMID- 7539592 TI - Quantitative evaluation of flow dynamics of erythrocytes in microvessels: influence of erythrocyte aggregation. AB - Effects of erythrocyte aggregation on the flow dynamics of erythrocytes in microvessels were examined quantitatively by perfusing human erythrocytes suspended in isotonic medium containing various concentrations of dextran (70,400 avg mol wt, Dx-70) into a part of the microvascular bed isolated from rabbit mesentery. Thickness of the marginal cell-free layer was measured with an image analyzer, total flow resistance was determined on the basis of the perfusion pressure-volume flow relationship, and homogeneity of erythrocyte flow was evaluated by the power spectrum obtained by the fast Fourier transform of the light intensity change monitored on single microvessels. With increasing dextran concentration, suspension viscosity of erythrocytes at high shear rates increased linearly and thickness of the cell-free layer increased in a sigmoidal fashion. Flow resistance increased relatively little over the range of dextran concentrations in which the cell-free layer increased most rapidly. Furthermore, the flow pattern of erythrocytes in microvessels became inhomogeneous. In conclusion, the present study shows that Dx-70-induced erythrocyte aggregation results in increased flow resistance in the circulatory system, even through the widening of the cell-free layer tends to reduce the resistance and also results in inhomogeneous flow of erythrocytes in microvessels. PMID- 7539594 TI - August Krogh Lecture. The renal concentrating mechanism in insects and mammals: a new hypothesis involving hydrostatic pressures. AB - Water moves from compartments of higher to compartments of lower water potential. Osmotically active solutes and negative hydrostatic pressure both lower water potential by stretching the hydrogen bonds between water molecules (Hammel Scholander hypothesis). In trees the negative hydrostatic pressure in the sap is balanced by the osmotic pressure of the leaves. In response to differences in water potential, water flows across biological membranes through water-filled pores. Protein molecules, aquaporins, forming hourglass-shaped pores have been identified, cloned, and located in plasma membranes in mammalian as well as other tissues. Water molecules flow single file through aquaporins. Insects concentrate the urine in the rectum. Mammals concentrate the urine in the collecting ducts in the inner medulla. In both, a compartment with a high osmotic concentration is created through ion transport. Both have a muscular coat surrounding the tissue, which shows peristaltic contractions. In insects it is the muscular layer around the rectum; in mammals it is the renal pelvic wall that surrounds the papilla. Mechanisms are proposed whereby these peristaltic contractions, through the creation of positive and negative hydrostatic pressures in the tissues, can lead to hyperosmotic excreta. PMID- 7539593 TI - Effects of nitric oxide synthase inhibitor on hemodynamic change and O2 delivery in septic dogs. AB - To elucidate the role of nitric oxide (NO) in septic shock, we measured hemodynamic and pulmonary gas changes in anesthetized dogs after intravenous administration of bacterial lipopolysaccharide (LPS) with or without NO synthase inhibitor, NG-nitro-L-arginine (L-NNA). Infusion of LPS (250 ng.kg-1.min-1) for 2 h decreased mean arterial pressure over 1-4 h. Although L-NNA (10 mg/kg) blocked LPS-induced hypotension, it decreased cardiac index, oxygen delivery index, arterial pH, and arterial PO2 and increased systemic vascular resistance index in the presence or absence of LPS. Administration of NG-nitro-D-arginine (D-NNA, 10 mg/kg) alone caused fewer hemodynamic effects (increased systemic vascular resistance index and decreased cardiac index) than L-NNA alone. Our study provides evidence that L-NNA prevents endotoxin-induced hypotension but decreases cardiac output and oxygen delivery, effects that may, in part, be due to a nonspecific NO synthase-independent event. Thus clinical use of NO synthase inhibitors for the treatment of septic shock should be cautiously considered. PMID- 7539596 TI - Short report: detection of 72-75-kD and 123-kD fractions of Leishmania antigen in urine of patients with visceral leishmaniasis. AB - Two polypeptide fractions of 72-75 kD were detected in the urine of 14 of 15 patients with visceral leishmaniasis (VL) and another fraction of 123 kD was found in 10 of the 15 patients by using a Western blot technique. None of these fractions was detected in the urine of 20 controls. These results suggest that antigen detection in urine could be a powerful, noninvasive method for VL diagnosis. PMID- 7539595 TI - Nitric oxide contributes to functional hyperemia in cerebellar cortex. AB - We used the parallel fibers (PF) system of the cerebellar cortex as a model to investigate the role of nitric oxide (NO) in the increases in blood flow elicited by neural activation. Rats were anesthetized with halothane and ventilated. The vermis was exposed, and the site was superfused with Ringer (37 degrees C; pH 7.3 7.4). PF were stimulated electrically (100 muA; 30 Hz), and the associated changes in cerebellar cortex blood flow (BFcrb) were monitored by laser-Doppler flowmetry. The field potentials evoked by PF stimulation were recorded using microelectrodes. During Ringer superfusion (n = 7), PF stimulation increased BFcrb (+ 52 +/- 4%). Topical application of the NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA; 0.1-1 mM) attenuated the increases in BFcrb dose dependently and by 50 +/- 4% at 1 mM (n = 9; P < 0.001; analysis of variance and Tukey's test). L-NNA (1 mM) inhibited NOS catalytic activity, assessed ex vivo using the citrulline assay, by 95 +/- 9% (P < 0.001). L-NNA did not influence the field potentials evoked by PF stimulation. D-NNA (1 mM; n = 6), the inactive stereoisomer of nitroarginine, did not attenuate the BFcrb response (P > 0.05). Methylene blue (1 mM; n = 7) reduced the response by 41 +/- 9% (P < 0.01) without affecting NOS catalytic activity (P < 0.05). The increases in BFcrb were not affected by lesioning the NOS-containing nerve fibers innervating cerebral vessels, indicating that these nerves are not the source of NO. Thus the increases in BFcrb elicited by activation of PF are, in part, mediated by NO produced in the molecular layer during neural activity. The results indicated that NO participates in the coupling of function activity to blood flow and support the hypothesis that NO is one of the mediators responsible for functional hyperemia in the central nervous system. PMID- 7539597 TI - Plasmodium falciparum: sera of individuals living in a malaria-endemic region recognize peptide motifs of the human erythrocyte anion transport protein. AB - Specific regions of the human erythrocyte anion transport protein, AE 1 or band 3, have been identified as being adhesive in Plasmodium falciparum-infected erythrocytes. In addition, synthetic peptides based on these sequences and murine monoclonal antibodies (Mabs) to these block cytoadherence/sequestration. These findings suggest the possibility that humans who are able to control P. falciparum infections may produce anti-adhesin (and thus anti-band 3) antibodies. To test this hypothesis, sera from individuals living in The Gambia and southern California were assayed for reactivity to decapeptides patterned on putative exofacial regions of the human anion transporter, band 3 protein. Sera from an area highly endemic for malaria, The Gambia, were found to have strong reactivity to well-defined regions of the band 3 protein (some of which contain the adhesin), whereas minimal reactivity was observed with sera from individuals living in a geographic area where malaria transmission is rare (California). Sera from The Gambia reacted strongly with residue blocks 534-560, 638-660, and 808 842. Gambian sera that reacted strongly with peptides patterned on band 3 failed to react with native band 3 on an immunoblot, but did react with fixed P. falciparum-infected erythrocytes. Using reactivity to decapeptides has allowed the determination of the epitopes of previously described murine MAbs that inhibit or promote cytoadherence; this reactivity with a specific region of a protein can be correlated with a specific effect on cytoadherence. A MAb (5H12) directed against amino acids 474-488 promoted cytoadherence, whereas those directed against amino acids 540-550 and 750-766 (Mabs 1C4 and 4A3, respectively) inhibited cytoadherence. PMID- 7539598 TI - Circulating antibodies directed against tryptophan-like epitopes in sera of patients with human African trypanosomiasis. AB - Human African trypanosomiasis is often associated with an intense proliferation of B lymphocytes, leading to polyclonal antibody synthesis. Using a modified enzyme-linked immunosorbent assay method, we have found highly significant levels of circulating anti-conjugated tryptophan-like epitope antibodies in sera of patients with sleeping sickness. These antibodies were immunoglobulins (Ig) of the M isotype. There was no correlation between immunologic binding and the Ig levels found in sera of patients with human African trypanosomiasis. Higher antibody levels in stage II of the disease than in stage I may be related to damage to the central nervous system. The specificity of this immunologic binding was evaluated by 1) comparison with that obtained with other related conjugates and 2) serum titration. Anti-conjugated tryptophan-like epitope antibodies were not found in other neurologic diseases tested. Their involvement in this pathology remains unknown. PMID- 7539599 TI - Effects of ethanol on cellular immunity to facultative intracellular bacteria. AB - Alcohol abuse has been associated with an increase in infectious diseases caused by pathogenic and opportunistic microorganisms. Study results obtained from this laboratory and other laboratories have shown that consumption of large amounts of ethanol is associated with numerous changes in the immune system. The purpose of this article is to report findings obtained from this laboratory, as well as review those obtained from other laboratories, from experiments designed to evaluate the effects of ethanol on various components of antimicrobial host defense mechanisms. The effects of ethanol on various aspects of immunity obtained with the use of in vivo and in vitro model systems are reviewed as they pertain to antimicrobial defenses. All current data would support the suggestion that ethanol affects both the development of an antigen-specific immune response and the effector mechanisms of the cellular immune response. Findings obtained from animal models show that ethanol prevents the formation of granulomas in infected tissues, perhaps by inhibiting the response of macrophages to T-cell cytokines. Data obtained from this laboratory also support the suggestion that the inability of the immune system to control the intracellular growth of microorganisms results in an exaggerated inflammatory response that is responsible for at least a part of the tissue damage. PMID- 7539602 TI - Ethanol inhibition of inducible nitric oxide synthase activity in C6 glioma cells. AB - Nitric oxide (NO.), a free radical gas, has been implicated in the CNS actions of ethanol. The brain contains several cell types that can produce NO., including neurons and glia. This study examined the effect of acute and chronic ethanol exposure on the activity of the inducible isoform of nitric oxide synthase (iNOS) found in neuroglia. Experiments were performed using intact rat C6 glioma cells, and NO. production was assessed by nitrite accumulation after iNOS induction by coadministration of phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Ethanol was inhibitory at high concentrations (IC50 approximately 150 mM) when acutely present during the 24-hr period subsequent to initiation of enzyme induction. In contrast, cells exposed to ethanol were inhibited chronically at clinically relevant lower concentrations (IC50 approximately 30 mM with 10 days exposure). Chronic inhibition was both time- and concentration-dependent. Inhibition by ethanol seems to be a consequence of interference with LPS signal transduction. Acutely, ethanol did not affect the ability of PMA to synergize with LPS to induce activity, but it attenuated the ability of LPS to synergize with the PMA. Ten days exposure to 50 mM ethanol decreased the LPS potency by 4 fold in the presence of a maximally activating concentration of PMA, although not significantly changing PMA potency. Inhibition by chronic ethanol exposure was long-lasting, being retained over 24 hr in cells returned to control conditions. Thus, chronic ethanol may downregulate key components needed for iNOS expression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539601 TI - Regulation of intracellular pH in periportal and perivenular hepatocytes isolated from ethanol-treated rats. AB - The aim of this study was to gain information on intracellular pH (pHi) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pHi was analyzed by the pH-sensitive dye BCECF in perfused subconfluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH4Cl and were alkali-loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHout. In cells from C rats: (a) steady-state pHi was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pHi recovery from an acid load was 35% higher in PP than in PV cells in the presence of HCO3-, whereas it was similar in HCO3( )-free experiments; and, on the contrary, (c) pHi recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pHi was always lower than in cells isolated from pair-fed animals; (b) steady-state pHi was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pHi recovery from an acid load was not significantly different in PP and PV cells either in the presence of HCO3- or in HCO3(-)-free experiments; and (d) pHi recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pHi by affecting the activity of ion transport mechanisms regulating pHi in PP and PV hepatocytes isolated from rat liver. PMID- 7539600 TI - Nitric oxide-related agents alter alcohol withdrawal in male rats. AB - Evidence has been reported supporting the hypothesis that nitric oxide (NO) partially mediates the expression of morphine dependence. To examine whether NO related agents also affect the expression of alcohol dependence, adult male rats were treated chronically with alcohol. Upon withdrawal of alcohol administration, abstinence signs were observed after treatment with a NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (NAME), or a NO donor, isosorbide dinitrate (ISDN). Withdrawal severity was based primarily on the presence and intensity of tremors, rigidity, hyperactivity, and spontaneous and audiogenic convulsions. The NOS inhibitor, NAME (10-100 mg/kg), injected during alcohol withdrawal significantly inhibited withdrawal severity decreasing the intensity of signs of hyperactivity, tremors, and rigidity, but not affecting the occurrence of convulsions. The NO donor, ISDN (30 mg/kg), administered during alcohol withdrawal significantly increased the severity of most withdrawal signs. These results suggest that NO mediates some aspects of the expression of alcohol dependence. PMID- 7539604 TI - Intramitochondrial protein synthesis is regulated by matrix adenine nucleotide content and requires calcium. AB - The hypothesis that fluctuations in matrix adenine nucleotide content (ATP + ADP + AMP) may regulate intramitochondrial protein synthesis was investigated in newborn and adult rat liver mitochondria. Protein synthesis in mitochondria from 0-h-old newborns, which contain 3.4 +/- 0.3 nmol adenine nucleotide/mg protein, was > 90% lower than protein synthesis in mitochondria from 4-h-old newborns, which contain 9.1 +/- 0.2 nmol adenine nucleotide/mg protein. If 0-h newborn mitochondria were preincubated to accumulate adenine nucleotides to 16.8 nmol/mg protein in vitro, the protein synthesis rate increased 25-fold compared to control. Adult rat liver mitochondria normally contain 12-14 nmol adenine nucleotide/mg protein and exhibit a brisk rate of protein synthesis. Following a preincubation to deplete adenine nucleotides in vitro down to 3 nmol/mg protein, protein synthesis in adult liver mitochondria was nearly abolished. Conversely, when adult mitochondria were preincubated to superload adenine nucleotides (to 29 nmol/mg protein), the rate of protein synthesis was doubled. Protein synthesis was also inhibited when the matrix ATP/ADP ratio was lowered by adding FCCP or by omitting phosphate. In adult mitochondria, protein synthesis was inhibited by 0.5 mM EGTA and was increased in proportion to buffered free calcium between 0 and 20 microM. The rate of intramitochondrial RNA synthesis was not inhibited by EGTA nor affected by variations in matrix adenine nucleotide content. The results show that intramitochondrial translation requires matrix calcium and is regulated by changes in the matrix adenine nucleotide content that affect the matrix ATP concentration. The matrix adenine nucleotide content is controlled by the ATP Mg/Pi carrier. In newborns, the matrix adenine nucleotide content increases 3 fold within 2-4 h after birth, stimulating mitochondrial translation 10-fold and probably contributing to the onset of postnatal mitochondrial biogenesis and adaptation to aerobic metabolism. PMID- 7539606 TI - Endobronchial management of benign, malignant, and lung transplantation airway stenoses. AB - Since 1991, we have managed 57 patients with benign (10), malignant (23), or lung transplantation (24) airway obstructions using silicone stenting and debridement (manual/neodymium:yttrium-aluminum garnet laser). Ten patients with benign lesions (4 intubation, 4 inflammatory, 1 malacia, 1 bronchial fistula) had 4 T tubes, 3 Y stents, 3 bronchial stents, and 1 straight tracheal stent placed. Eight of 10 patients (80%) received symptomatic relief with the stents in place for up to 43 months. Twenty-three patients with malignant strictures (18 lung, 5 metastatic) had 26 stents inserted (13 Y stents, 12 bronchial, 1 T tube) of which 16 required combined debridement and stenting. Four stents required repositioning. three hospital deaths were due to unrelated causes. Of 20 discharged patients, 6 remain alive at 2 to 10 months, whereas 14 patients who died of progressive disease obtained effective palliation for 10.5 +/- 5.6 months. Significant bronchial anastomotic complications developed in 24 of 212 lung transplants (11.3%). Thirty-one stents were placed in 19 of the patients; 5 patients were managed with laser debridement alone. Of the 19 patients receiving stents, 3 required only temporary stents (6 to 15 days), 11 patients needed long term stents (40 to 507 days), and 5 patients died with their stents in place functioning well. All patients received symptomatic relief with stenting. There were no procedure-related deaths and one bronchial laceration during attempted stent placement. Early, aggressive treatment of benign and malignant tracheobronchial strictures with endoscopic debridement and stenting is safe and well tolerated, and effectively palliates airway obstruction. Repositioning of stents frequently may be required in the transplant population.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539605 TI - Partial characterization of novel serine proteinase inhibitors from human umbilical vein endothelial cells. AB - Serine proteinase inhibitors play major roles in physiological and pathophysiological processes such as angiogenesis, intravascular fibrinolysis, wound healing, and tumor invasion, and metastasis. Here, we report that human umbilical vein endothelial cells (HUVEC) synthesize three inhibitors (33,000, 31,000, and 27,000 Da) which inhibit degradation of gelatin or casein by trypsin, elastase, plasmin, and alpha-chymotrypsin. The 33- and 31-kDa inhibitors were constitutively found in the cell lysate and extracellular matrix, but not in the conditioned medium of HUVEC. Following treatment with phorbol 12-myristate-13 acetate (PMA), all the three inhibitors were expressed in the CM and increased activity was found in cell lysates and extracellular matrix. The inhibitors were not detected in PMA-treated cells in the presence of cycloheximide or actinomycin D. The inhibitors specifically bound to trypsin and were recovered from trypsin affinity column as smaller-sized inhibitors without loss of antitrypsin activity. Polyclonal antibodies to inter-alpha-trypsin inhibitor did not cross-react with the 33-, 31-, and 27-kDa inhibitors. These results suggest that HUVEC synthesize and secrete novel 33-, 31-, and 27-kDa serine proteinase inhibitors. PMID- 7539603 TI - Developmental expression of KG-CAM in the rat neostriatum. AB - The present study examines the developmentally regulated expression pattern of an Ig superfamily member, KG-CAM, in the neostriatum of the rat. KG-CAM is a 90-kDa glycoprotein that is related to the DM-GRASP/Neurolin family of adhesion molecules. In the embryonic and early postnatal neostriatum, the distribution of KG-CAM correlates with the distribution of dopaminergic terminals. Early in neostriatal development, KG-CAM is found in the tyrosine hydroxylase-positive patches. In the maturing neostriatum, the levels of KG-CAM remain high within the patches, and KG-CAM upregulates in the matrix compartment. As the neostriatum is reaching its adult morphology, 5 weeks postnatal, the expression of KG-CAM in the matrix is approximately equal to that of the patches. When the distribution of KG CAM is examined at the ultrastructural level, the immunoreactivity is localized to the external surface of neuronal and glial profiles in the neuropil. KG-CAM does not appear to be associated with the guidance of dopaminergic axons from the substantia nigra to the striatum, for this pathway is not immunopositive for this member of the Ig superfamily. The present study identifies an Ig superfamily member, KG-CAM, that appears to play a major role in the development of the neostriatum. Furthermore, the high levels of KG-CAM in the adult neostriatum suggest that this Ig superfamily member may be involved in maintaining the integrity of this structure in the adult rat. PMID- 7539607 TI - Univentricular heart with systemic outflow obstruction: palliation by primary Damus procedure. AB - In 24 consecutive infants (19 male and 5 female) with complex forms of single ventricle physiology and systemic outflow obstruction, a modified Damus operation without the use of exogenous material was undertaken in conjunction with creation of an aortopulmonary shunt 3.5 mm in diameter. The median age at operation was 6 days (range, 1 to 170 days) and the median weight, 3.4 kg (range, 2.6 to 4.6 kg). There were nine early deaths. All 15 survivors (median follow-up, 6.5 months) were clinically well without major systemic ventricular dysfunction or atrioventricular or arterial valve regurgitation. Ten of them have undergone a superior vena cava-pulmonary shunt (one death), and 1 has required patch angioplasty of the aortic arch and innominate artery with revision of the aortopulmonary shunt. The 4 other survivors are awaiting a cavopulmonary shunt. Univariate analysis yielded the chronologic rank for an individual procedure (higher risk of death early in the series), presence of aortic arch atresia, and presence or absence of transposition of the great arteries as predictors of death. This aggressive surgical approach provides excellent early palliation, and because the operation prevents abnormal ventricular hypertrophy from pressure or volume overload, systemic ventricular function is optimally conserved for a future Fontan-type procedure. PMID- 7539608 TI - Nature of coarctation in hypoplastic left heart syndrome. AB - The study was designed to establish the underlying pathology of aortic coarctation in hypoplastic left heart syndrome. Aortic coarctation in patients with hypoplastic left heart syndrome is a significant problem when a Norwood procedure or heart transplantation is performed. Previous reports have claimed that the obstruction was merely a branch-point phenomenon and that ductal tissue was absent. Five heart specimens with hypoplastic left heart syndrome and aortic coarctation were examined histologically. The distal segment of the aortic arch, together with the arterial duct, were dissected and processed en bloc. The blocks were sectioned parallel to the longitudinal axis of the aortic arch. In 4 hearts the coarctation was in the preductal position. Ductal tissue encircled the aortic lumen and extended into the aorta both proximally and distally from the ductal orifice. The fifth case showed medial thickening of the aortic wall in the preductal position, but without ductal tissue. The arterial duct showed histologic characteristics of persistent patency. Our observations suggest that aortic coarctation in the preductal position in hypoplastic left heart syndrome is caused by the extension of ductal tissue. The findings may have an impact on clinical strategies for hypoplastic left heart syndrome. PMID- 7539610 TI - Heart transplantation for chyloptysis after Fontan operation. AB - Chyloptysis is a rare clinical problem, fewer than 10 patients having been reported in the literature. We report a patient with intractable chyloptysis associated with a Fontan procedure, who was palliated by heart transplantation. PMID- 7539609 TI - Half-dose aprotinin preserves hemostatic function in patients undergoing bypass operations. AB - High-dose aprotinin reduces bleeding in cardiac operations but with potential side-effects and increased cost. It is therefore mandatory that the effectiveness of a low dose be investigated. Half of the Hammersmith regimen was studied in cardiac surgical patients to find out how it could reduce bleeding. Blood fibrinolysis parameters were studied in 40 elective patients undergoing coronary artery bypass grafting in a double-blind, placebo-controlled study design. The plasma activities of tissue plasminogen activator, plasminogen activator inhibitor, alpha 2-antiplasmin, plasminogen, fibrinogen, and D-dimer as well as platelet number, bleeding times, activated clotting time, and aprotinin plasma concentrations were assessed before, during, and after the operation. Fibrinolysis was inhibited by aprotinin as evidenced by decreased D-dimer (p = 0.0001) and tissue plasminogen activator (p = 0.0432) levels and increased plasminogen activator inhibitor (p = 0.0105) and alpha 2-antiplasmin (p = 0.0002) levels during the operation. A postoperative abnormal bleeding time occurred 38% more frequently in the placebo group (p < 0.05). Aprotinin plasma concentrations reached adequate levels to inhibit plasmin and plasma kallikrein. This study showed that half-dose aprotinin significantly inhibits fibrinolysis and prevents postoperative abnormal bleeding time in cardiac surgical patients. PMID- 7539611 TI - Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3 kinase. AB - Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway. PMID- 7539613 TI - 9-cis-retinoic acid is more effective than all-trans-retinoic acid in upregulating expression of the alpha-fetoprotein gene. AB - In McA-RH 8994 rat hepatoma cells, all-trans-retinoic acid (t-RA) induces expression of the alpha-fetoprotein (AFP) and albumin genes and results in a phenotype similar to differentiated fetal hepatocytes. The present study elucidated the mechanism involved in AFP gene regulation mediated by retinoic acid. Northern blot analyses demonstrated that 9-cis-retinoic acid (c-RA), a ligand for retinoid x receptors (RXRs), also induced expression of the AFP gene in McA-RH 8994 cells. The induction was time- and dose-dependent. Northern blots and transfection assays using the 7.3 kb full-length regulatory region of the AFP gene demonstrated that c-RA was more effective than t-RA in regulating expression of the AFP gene. At 10(-7) M, c-RA increased AFP mRNA 5-fold and chloramphenicol acetyltransferase (CAT) activity 2.5-fold. In contrast, t-RA at a concentration of 10(-7) M exerted no significant effect; 10(-6) to 10(-5) M t-RA was needed to affect AFP gene expression. These data suggested that activation of RXRs is essential for the regulation of the AFP gene. Co-transfection experiments revealed that over-expression of RXR alpha in McA-RH 8994 cells further enhanced the CAT activity induced by c-RA. In addition, c-RA did not alter the half-life of AFP mRNA. Thus, RXR alpha may play a crucial role in transcriptional regulation of the AFP gene and in controlling hepatocyte phenotype. PMID- 7539612 TI - Mitochondrial gene expression in small intestinal epithelial cells. AB - Most mitochondrial genes are transcribed as a single large transcript from the heavy strand of mitochondrial DNA, and are subsequently processed into the proximal mitochondrial (mt) 12 S and 16 S rRNAs, and the more distal tRNAs and mRNAs. We have shown that in intestinal epithelial biopsies the steady-state levels of mt 12 S and 16 S rRNA are an order of magnitude greater than those of mt mRNAs. Fractionation of rat small intestinal epithelial cells on the basis of their maturity has shown that the greatest ratios of 12 S mt rRNA/cytochrome b mt mRNA or 12 S mt rRNA/cytochrome oxidase I mt mRNA are found in the surface mature enterocytes, with a progressive decrease towards the crypt immature enteroblasts. Cytochrome b and cytochrome oxidase I mt mRNA levels are relatively uniform along the crypt-villus axis, but fractionation experiments showed increased levels in the crypt base. The levels of human mitochondrial transcription factor A are also greater in immature crypt enteroblasts compared with mature villus enterocytes. These results show that the relative levels of mt rRNA and mRNA are distinctly regulated in intestinal epithelial cells according to the crypt-villus position and differentiation status of the cells, and that there are higher mt mRNA and mt TFA levels in the crypts, consistent with increased transcriptional activity during mitochondrial biogenesis in the immature enteroblasts. PMID- 7539614 TI - Localization of the response elements of a gene induced by intermittent growth hormone stimulation. AB - Three regions required for the expression of a mouse major urinary protein (MUP) transgene were identified by a deletion analysis. One of these was located upstream of the cap site between -2139 and -1800, another was the proximal promoter region downstream of -324 and the third lay within the 338 nucleotide intron 1. Both the proximal promoter and intron 1 are involved in sexually dimorphic expression of the transgene (male/female ratio 20), which is dictated by the different temporal profiles of circulating GH in the two sexes. The data also indicated that the region between exons 3 and 7 may contribute to full expression in males and that a region between -718 and -324 may contribute towards the low expression level that obtains in females, but compared with the three principal regions the effects of these regions are relatively minor. We propose (1) that full expression of the transgene requires the co-operation of transcription factors bindings to the three principal regions and (2) that the difference in expression between the sexes relates to interactions between transcription factors bound to the proximal promoter and to sites in intron 1. Our results complement earlier in vitro footprinting and gel-retardation studies of the homologous rat apha 2u-globulin genes. These identified a number of response elements, including putative C/EBP and AP1 sites in the proximal promoter and intron 1 respectively and three putative psi NF-1 sites, two in the proximal promoter and one in intron 1, but proof of the functionality of these sites in regulating transcription was lacking. The proximal promoter also contained a 34 nucleotide sequence that has 70% identity with the SPI GH response element. PMID- 7539616 TI - Antigen recognition. PMID- 7539615 TI - Execution and suicide: cytotoxic lymphocytes enforce Draconian laws through separate molecular pathways. AB - With the ability to analyze cytotoxicity in animals deficient in effector molecules, the debate over the biological significance of individual effector pathways is finally being settled. For CD8+ cytotoxic T lymphocytes, the two primary cytotoxic pathways are mediated by the Fas ligand and perforin. The dichotomy between the two killing pathways is mirrored in the dichotomy between their biological roles: the primary function of the Fas ligand is the control of normal cell renewal by inducing programmed cell death of actively proliferating cells via the Fas pathway. This type of cell death is part of the normal endogenous homeostatic mechanism responsible for maintaining rapidly changing cell populations such as clonally expanding and contracting T cells. In contrast, the biological function of perforin and associated granule proteins is the killing and elimination of parasitized, non-compliant cells that arise as part of pathophysiological processes and may resist lysis pathways signalled to induce apoptosis. Thus, the main function of perforin and granzymes is the maintenance of immune surveillance against both exogenous and endogenous hazards. PMID- 7539617 TI - Autolytic effect of the antibiotic produced by Myxococcus coralloides D. AB - Myxococcus coralloides D secretes an antibiotic, named corallolysin, when grown on a rich medium. When a critical concentration is reached, this antibiotic lyses the producer bacterium either during vegetative growth or during morphogenesis. Corallolysin has not effect on resting cells nor on myxospores. The autolytic effect is caused by the early inhibition of RNA synthesis. PMID- 7539619 TI - Effects of tumor necrosis factor alpha and vascular permeability factor on neovascularization of the rabbit ear flap. AB - OBJECTIVE: The survival of compromised skin flaps depends on neovascularization for their nutrition and metabolic waste removal. Our study investigated the effectiveness of angiogenic factors in accelerating peripheral neovascularization and in increasing skin flap viability. DESIGN: We elevated pedicled dorsal skin flaps on the ears of 23 New Zealand white rabbits, and the vascular pedicle was ligated to achieve partial flap necrosis. Fifteen flaps were treated with 0.1 micrograms/mL vascular permeability factor, and eight flaps were treated with 1.0 micrograms/mL tumor necrosis factor alpha. The ear flaps that were not treated with growth factor functioned in each rabbit as a normal saline control. MAIN OUTCOME MEASURES: The viability of skin flaps was observed visually and was measured by Cartesian planimetry using templates. Neovascularization was documented by microangiography and by histologic analysis of the flaps. RESULTS: Although the angiogenic factors accelerated neovascularization, increased flap survival was demonstrated only in those animals treated with vascular permeability factor that was supplied by an absorbable gelatin sponge. CONCLUSION: This experimental model, despite different levels of controls, contains multiple variables, including the use of an absorbable gelatin sponge, seroma formation, bioactivity of the angiogenic factors, optimal dosages and dosimetry, the need for a "blinded" format, and the validity of the histologic analysis. Additional investigation must be done and the experimental model itself must be improved before these apparently positive results may be accepted as clinically useful. PMID- 7539618 TI - Cytotoxic ent-kaurene diterpenoids from three Isodon species. AB - From Isodon loxothyrsa, one new diterpenoid, loxothyrin A, together with one known diterpenoid, adenolin B, from I.pleiophyllus, three known diterpenoids, coetsoidins A, B and G, and from I. adenoloma, one known diterpenoid, longikaurin F, were isolated. The structure determination of loxothyrin A, and the unambiguous NMR spectral assignments of the known compounds were made by a combination of 1D and 2D NMR techniques and computer modelling calculations. The isolates showed potent cytotoxic activities. PMID- 7539620 TI - Everyday life activities photo series. Jacqueline Stark. Vienna, Austria. PMID- 7539621 TI - Post-reperfusion coagulopathy. PMID- 7539622 TI - Changes of cytokeratin and involucrin expression in squamous cell carcinomas of the skin during progression to malignancy. AB - The detection of cytokeratins in neoplastic tissues by immunohistochemical methods has numerous diagnostic and investigative applications, because cytokeratins are usually conserved in tumour cells during malignant transformation. Recently, however, it has been reported that progression to malignancy is associated with commencement of expression of low-molecular-weight cytokeratins. In the present study, 42 specimens from 35 cases of squamous cell carcinoma (SCC) of the skin were analysed by immunohistochemical techniques, using polyclonal anti-involucrin antibody and a panel of monoclonal antikeratin antibodies, in order to investigate the nature and differentiation of SCCs. The expression of cytokeratins and involucrin in well-differentiated SCCs was similar to that in normal epidermis. In contrast with well-differentiated SCCs, the expression of differentiation-specific cytokeratins and involucrin was diminished in the immature tumour cells in proportion to the malignancy of the SCCs. Some antibodies, however, stained all tumour cells, irrespective of the degree of malignancy. Furthermore, expression of simple epithelial and non-cornifying stratified squamous epithelial cytokeratins was observed in atypical tumour cells of poorly differentiated SCCs. It is of interest that similar expression was noted in many tumour cells in the lymph node metastases and in some tumour cells in the primary cutaneous lesions. Cytokeratin expression similar to that in normal epidermal keratinocytes was conserved in well-differentiated SCCs, but the expression of cytokeratins changed during progression to malignant transformation. The expression of simple epithelial or non-cornifying stratified squamous epithelial cytokeratins in cutaneous SCCs may be a marker for their capability of invasion and metastatic potential. PMID- 7539623 TI - Adhesion of flowing leucocytes to immobilized platelets. AB - Adhesion of neutrophils, lymphocytes and promyelocytic HL60 cells was compared in a flow-based model in which a monolayer of activated platelets formed the adhesive substrate. Each type of leucocyte formed P-selectin-mediated rolling attachments on the platelet surface under physiologically relevant flow conditions. Lymphocytes adhered less, and HL60 in similar numbers, compared to neutrophils, whereas the lymphocytes and HL60 cells rolled much more rapidly. Sulphated, sialylated saccharide(s) were implicated as ligand(s) for P-selectin for all leucocytes, but L-selectin (borne by neutrophils and lymphocytes, but not HL60 cells) appears to be a major presenter of ligand for neutrophils alone. T cells enriched from peripheral blood lymphocytes adhered in greater numbers than B cells. Differentiation of HL60 cells to neutrophil-like cells (induced by DMSO) caused cell volume to decrease and surface expression of integrin adhesion molecules to increase, but only a small percentage of cells were converted to an L-selectin-bearing phenotype. Differentiated cells showed evidence of stabilization of adhesion with increasing stress and a marked reduction in rolling velocity. These studies indicate that cell differentiation may be accompanied by alteration of adhesive behaviour, resulting from changes in physical characteristics as well as surface properties. Moreover, results suggest that P-selectin could promote lymphocyte attachment to endothelium in acute inflammatory conditions and possibly mediate lymphocyte-platelet interaction during thrombosis. PMID- 7539624 TI - Absence of G-CSF receptors and absent response to G-CSF in childhood Burkitt's lymphoma and B-ALL cells. AB - The expression of the granulocyte colony-stimulating factor (G-CSF) receptor in childhood Burkitt's lymphoma (BL) cells, and the mitogenic effect of G-CSF on these cells, was studied in a panel of 13 Epstein-Barr virus (EBV) positive and negative BL cell lines derived from nine children. G-CSF receptor mRNA expression was investigated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Binding of G-CSF to BL cell lines was measured by chemical crosslinking of 125I-G-CSF, and proliferation by thymidine incorporation. Inducibility of the G-CSF receptor was studied by stimulation with interleukin-1 beta, tumour necrosis factor-alpha, Staphylococcus aureus Cowan A, anti-human IgM, phorbol myristate acetate, calcium ionophore A23187, and by infection in vitro by immortalizing and non-immortalizing strains of EBV. BL cell lines, unstimulated or stimulated by biological reagents or EBV infection, did not bind radioionated G-CSF in crosslinking experiments. No stimulation by recombinant human G-CSF was observed in 3H-thymidine incorporation assays. No G CSF receptor mRNA was detected by Northern blot analysis or RT-PCR in BL cell lines. It is concluded that G-CSF plays no direct stimulatory role in the growth of these malignant B-cells, making a deleterious influence of G-CSF in the clinical treatment situation unlikely. PMID- 7539625 TI - Mediastinal large B-cell lymphoma: clinical and immunohistological findings in 18 patients treated with different third-generation regimens. AB - We report on the immunophenotype, clinical findings and response to aggressive chemotherapy of 18 patients with mediastinal large B-cell lymphoma (MLCL). Cases were collected from a series of 286 high-grade non-Hodgkin's lymphomas (HG-NHL) which, in the period September 1988 to August 1991, were enrolled in a prospective multicentre trial designed to compare the MACOP-B and F-MACHOP regimens. Immunostaining on frozen sections revealed a previously unrecognized phenotype, i.e. co-expression of B-cell (CD19, CD20, CD22, Ig-associated dimer) and activation-associated antigens (CD30 and CDw70) in about 60% of MLCL cases; in contrast, the activation-associated antigens CD25 and Ki-27 (unclustered) were consistently negative. This peculiar phenotype may reflect a derivation of the tumour from a subset of thymic activated B cells. Clinically, the patients (median age 31 years; F/M ratio 2.6) presented with bulky mediastinal mass (72%) associated with mediastinal syndrome in > 50% cases; disease was stage IIA in most cases. All 18 patients received aggressive chemotherapy (F-MACHOP 11; MACOP B 7). Complete response (CR) was achieved in 57.1% of cases treated with MACOP-B. In contrast, the response of the 11 MLCL treated with F-MACHOP was poor (CR 18.2%) as compared to that of the 135 HG-NHL treated with the same regimen during the trial (CR 69.6%). This difference was still statistically significant after adjusting for negative prognostic factors (mediastinal mass > 10 cm plus increased LDH) and suggests that F-MACHOP might not be the most appropriate regimen for this kind of lymphoma. PMID- 7539627 TI - Osteoporosis in severe congenital neutropenia treated with granulocyte colony stimulating factor. AB - Recombinant human granulocyte colony-stimulating factor (G-CSF) has substantially improved life expectancy for children with severe congenital neutropenia (SCN). Severe osteoporosis, reported in this population, may relate to the disease process, or be a therapeutic side-effect. This report details bone loss, quantitated absorptiometrically and histomorphometrically, in a child with SCN and vertebral collapse, and the positive response to anabolic steroid and bisphosphonate therapy. PMID- 7539626 TI - Soluble c-kit levels in acute GVHD after allogeneic bone marrow transplantation. AB - Serum soluble c-kit concentrations were measured in 11 patients with or without acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. The serum soluble c-kit levels in the patients with moderate to severe acute GVHD (grade II-IV) were significantly lower than those in the patients with no or mild acute GVHD (grade 0-I) following the onset. The data from this study indicate that measurement of serum soluble c-kit concentration is a useful indicator of severe acute GVHD. PMID- 7539628 TI - Rheology of hydroxyethylated starch aqueous systems. Analysis of gel formation. AB - This paper reports dynamic viscoelastic and steady shear measurements of aqueous solutions of two commercial hydroxyethyl ethers of potato starch, allowing us to define three different viscoelastic regions, depending on the polymer concentrations. The fluid-like zone (I) corresponds to a homogeneous solution, free of associations between chains, where the linear viscoelastic model can be applied; the fluid-gel transition zone (II) is associated with the rheology of complex systems which possess intermolecular specific interactions; and the gel like zone (III) involves a network formation giving rise to a gel. The reversibility of the hydrogels was studied by means of steady/dynamic transient experiments allowing breakdown of the gel network and analysis of rehealing. Comparing the elastic moduli of the starch derivative gels leads us to assume that the functionality depends on the capacity to form hydrogen-bonding associations between polymer chains. PMID- 7539630 TI - Immunohistochemical evaluation of tenascin in benign eccrine gland tumors: comparison with laminin and fibronectin. AB - Surgically resected specimens of skin appendage tumors, spiradenoma, poroma of eccrine glands, syringoadenoma, syringoadenoma papilliferum and mixec tumor of skin were evaluated for tenascin, as well as the basement membrane-associated protein laminin, and the extracellular matrix protein fibronectin, using immunohistochemical methods. In normal sweat glands, tenascin was localized as a delicate, thin band in the immediate vicinity of the basement membrane and laminin was limited to the basement membrane. Tenascin in syringoadenoma was also expressed linearly adjacent to the basement membrane. Mixed tumors of skin showed a characteristic distribution of tenascin in the modified myoepithelial cell area adjacent to the tumor epithelia with a heterogeneous pattern, and particularly strong expression in the hyalinous and chondroid areas. In addition, the localization of laminin in the basement membranes of tumors, intracellular immunoreactivity was observed in syringoadenoma, syringocystadenoma papilliferum, and mixed tumor of skin. Fibronectin reactivity was visualized as a diffuse staining in the tumor stroma of eccrine poroma but its expression was restricted in eccrine spiradenoma. The present study suggests a heterogeneity in the distribution of basement membrane and extracellular matrix proteins in skin appendage tumors and elucidation of their functional roles would assist in understanding the changes in the supporting tumor stroma following neoplastic changes. PMID- 7539629 TI - In vitro and in vivo evaluation of progesterone implants based on new biodegradable poly(glutamic and glutamate esters) copolymers. AB - New biodegradable polypeptidic copolymers of glutamic acid and tert butyloxycarbonylmethyl glutamate were evaluated as implantable drug delivery systems. Three copolymers varying in degrees of esterification, which is inversely proportional to the number of glutamic residues, were selected for their range of hydrophilicity and biodegradation rates. Progesterone-containing implants were then prepared by means of an extrusion process and both in vitro and in vivo evaluations were performed. The effects of drug loading, nature, and pH of release medium were investigated. In vitro/in vivo correlations were established for some types of implants. Finally, mathematical fitting of the data, using published models, helped to better understand the mechanisms governing release. PMID- 7539632 TI - [Treatment of viral hepatitis]. PMID- 7539631 TI - Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing. AB - A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis. PMID- 7539633 TI - Modulation of postoperative immune response by enteral nutrition with a diet enriched with arginine, RNA, and omega-3 fatty acids in patients with upper gastrointestinal cancer. AB - OBJECTIVE: To find out whether an enteral diet supplemented with arginine, RNA, and omega-3 fatty acids modulated the production of interleukin-1 (IL-1), interleukin-2 (IL-2), IL-2 receptor, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha) after operations for upper gastrointestinal cancer. DESIGN: Prospective double blind clinical study. SETTING: University hospital, Germany. SUBJECTS: 42 patients randomised into two groups (n = 21 each), one of which was given an isocaloric and isonitrogenous placebo diet and one of which was fed the same diet supplemented with arginine, RNA, and omega-3 fatty acids. INTERVENTIONS: The cytokines were measured before operation and on postoperative days 1, 3, 7, 10, and 16. MAIN OUTCOME MEASURES: Comparison of concentrations of cytokines in the two groups. RESULTS: Among those receiving the placebo diet (after spontaneous stimulation) IL-6 concentrations were significantly higher on days 3 and 7 (p < 0.05) and TNF-alpha concentrations on day 7. In contrast (after stimulation with phytohaemagglutinin) mean concentrations of IL-2 receptor were significantly higher on days 3 and 7, and of IL-1 beta and IL-2 on day 16 (p < 0.05) in the group receiving the supplemented diet. CONCLUSION: Supplementation of an enteral diet with arginine, RNA and omega-3 fatty acids can modulate the acute phase reaction as indicated by the reduction in concentrations of TNF-alpha and IL-6 in the group fed the supplemented diet. Patients receiving the supplemented diet also showed accelerated recovery in the concentrations of IL-1 beta and IL-2 receptor. PMID- 7539634 TI - Functional aspects of vascular tenascin-C expression. AB - The arterial tenascin C expression in vivo and in vitro has been studied using immunohistochemistry. The functional relevance of localized tenascin C expression was assessed in vitro using various human cell types involved in the progression of vascular disease. Normotensive and hypertensive rats exhibited age-dependent patterns of vascular (aorta) tenascin expression, but the lumen-to-media-directed progression of tenascin induction was accelerated in hypertensive rats. Tenascin rich neointimal lesions (spontaneous) were observed at branching sites of aorta from aged (80 weeks) hypertensive rats. Subendothelial tenascin foci contained lipid-laden smooth muscle cells and monocytes/macrophages. Medial tenascin foci encaged smooth muscle cells which synthesized DNA. Tenascin was expressed both in vivo and in vitro by endothelial and smooth muscle cells but not by monocytes/macrophages; angiotensin II, oxidized-low density lipoprotein and transforming growth factor beta 1 induced expression of tenascin transcripts and glycoprotein in vitro. Endothelial and smooth muscle cells, but not monocytes, adhered to tenascin substrata. Tenascin reduced focal adhesion integrity in confluent endothelial and smooth muscle cell cultures. Angiotensin II-induced migration of endothelial and smooth muscle cells was accompanied by tenascin deposition within extracellular matrix migration trails. Tenascin may function both as a defense against monocyte invasion and medial smooth muscle replication, as well as a substratum for directed endothelial and smooth muscle cell migration. PMID- 7539636 TI - Effect of recombinant human granulocyte colony-stimulating factor on variations of morphologically identifiable bone marrow cells in myelosuppressed mice. AB - To examine the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophilic recovery after cytotoxic agents, the variations of marrow colony-forming units of granulocytes and macrophages (CFU-GM) and morphologically identifiable bone marrow cells were investigated in cyclophosphamide (CPA)-treated mice. In mice treated with CPA at 200mg/kg intraperitoneally (day 0), marked decreases in peripheral neutrophils and nucleated cells in the femur were observed. In the femur of mice treated with CPA, the greatest depression in number occurred firstly with CFU-GM and the most immature granulocytes, such as myeloblasts and promyelocytes, followed in turn by myelocytes, metamyelocytes and mature neutrophils. Administration of rhG-CSF for four successive days (days 1-4) after CPA treatment completely prevented the neutropenia. In the femur, rhG-CSF enhanced the recovery of progenitors and immature granulocytes from their depression in the order of their differentiation, and recovery of marrow neutrophils was also promoted. From these studies, we confirmed that rhG-CSF effects an increase in peripheral neutrophils by enhancing the proliferation and differentiation of CFU-GM and immature marrow granulocytes. PMID- 7539635 TI - Influence of FK 506 (tacrolimus) on circulating CD4+ T cells expressing CD25 and CD45RA antigens in 19 patients with chronic progressive multiple sclerosis participating in an open label drug safety trial. AB - We have taken the opportunity of a clinical trial of the potential efficacy and safety of FK 506 (tacrolimus) in chronic progressive multiple sclerosis (MS) to examine the influence of this potent new immunosuppressant on circulating T lymphocytes in an otherwise healthy non-transplant population. Peripheral blood levels of subsets of CD4+ T lymphocytes expressing the activation molecule interleukin-2 receptor (p55 alpha chain; CD25) or the CD45RA isoform were determined sequentially in 19 patients that were treated continuously with oral FK 506 (starting dose 0.15 mg/kg/day) for 12 months. No significant change in the proportion of circulating CD25+ CD4+ cells was observed over the study period in which the mean trough plasma FK 506 level rose from 0.3 +/- 0.2 to 0.5 +/- 0.4 ng/ml. There was also no significant effect of FK 506 on the percentage of CD45R+ CD4+ cells in the peripheral blood at 12 months compared with pretreatment values. Analysis of a subgroup of 7 patients, who showed a sustained reduction in CD25+ CD4+ cells and a reciprocal increase in CD45RA+ CD4+ cells for at least 6 months after start of treatment, did not reveal any difference in disability at one year compared with the treatment group as a whole. The side effects of FK 506 were mild and the overall degree of disability estimated by the mean Kurtzke expanded disability status scale (EDSS) score or the ambulation index did not deteriorate significantly in the 19 patients studied over the 12 months of FK 506 administration. PMID- 7539637 TI - Vascular endothelial growth factor (rhVEGF165) stimulates direct angiogenesis in the rabbit cornea. AB - The object of this study was to test vascular endothelial growth factor (VEGF) for angiogenic activity in the rabbit corneal assay. VEGF doses ranging from 20 ng to 1000 ng were incorporated into a slow release polymer and implanted into the avascular rabbit cornea. Capillary formation in the cornea was visually analyzed on a daily basis and examined with histology, transmission electron microscopy and scanning electron microscopy of vascular corrosion casts on days 2 and 7 post-implantation. VEGF implants (200ng to 1000ng) consistently stimulated angiogenesis. This neovascularization occurred in the absence of inflammation. We conclude that VEGF acts directly on endothelial cells, initiating and mediating the formation of capillaries. PMID- 7539638 TI - [Extraosseous Ewing's sarcoma]. PMID- 7539639 TI - Role of the phytotoxin coronatine in the infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. AB - The role of the phytotoxin coronatine in the virulence of Pseudomonas syringae pv. tomato in Arabidopsis thaliana was evaluated by comparing symptom development, in planta bacterial multiplication, and the induction of defense related genes in Arabidopsis plants inoculated with the coronatine-producing (Cor+) P. s. pv. tomato strain DC3000 and the coronatine-defective (Cor-) strain DC3661 by either infiltration or dipping methods. The Cor+ strain, P. s. pv. tomato DC3000, caused severe disease symptoms and multiplied by 4-6 logs after inoculation by either infiltration or dipping. P. s. pv. tomato DC3661 failed to produce any disease symptoms and multiplied by only 1-1.5 logs in dipped plants, whereas it caused mild symptoms and multiplied 6 logs over the 4-day experimental period in plants inoculated by infiltration. Parallel experiments using a natural host, tomato, yielded similar results. Analysis of the accumulation of mRNAs encoded by several distinct defense-related genes in Arabidopsis leaves infiltrated with either DC3000 or DC3661 demonstrated that the Cor- strain consistently induced higher levels of these transcripts. These results demonstrate that coronatine production is required under more natural inoculation conditions for the successful infection of Arabidopsis by DC3000, and that coronatine may play a critical role during the early stages of infection by suppressing the activation of defense-related genes. PMID- 7539641 TI - Future developments in endoscopic imaging. AB - Endoscopic imaging capabilities have significantly improved over the past 10 years. Improvements in fibreoptic technology have made possible the development of very thin endoscopes that can directly visualize the biliary and pancreatic ducts. The application of the CCD to endoscopy has made electronic endoscopy possible, and holds promise for stereoendoscopy. The ability to digitize endoscopic images can be developed to store, transmit, magnify, enhance and otherwise manipulate data obtained during endoscopy, and will probably be utilized routinely in the future. Laser and ultrasound technology are likely to enhance significantly our ability to examine ultrastructural aspects of gastrointestinal organs and surrounding tissues, and may play an important role in cancer surveillance programs. Vital staining techniques are likely to find widespread use in early cancer detection programmes, and may be useful to follow prospectively lesions observed or treated during endoscopy. Finally, the new developments in 'virtual imaging' may find applications in the field of gastrointestinal endoscopy and other 'minimally invasive' surgical procedures. PMID- 7539642 TI - Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. AB - The effect of theanine, one of the components of green tea, on the blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) was investigated by intraperitoneally administering theanine. The effect of glutamine, which is structurally similar to theanine, was also examined. When SHR were injected with various amounts of theanine (0, 500, 1000, 1500, and 2000 mg/kg), the change was dose-dependent, and a significant decrease in blood pressure was observed with the high doses (1500 and 2000 mg/kg). A dose of 2000 mg/kg of theanine did not alter the blood pressure of WKY, while the same dose to SHR decreased it significantly. On the other hand, glutamine administration to SHR did not change either the blood pressure or the heart rate. The brain 5-hydroxyindole level was significantly decreased by theanine administration to both WKY and SHR, the decrease being dose-dependent. PMID- 7539643 TI - Glycosidic aroma precursors of 2-phenylethyl and benzyl alcohols from Jasminum sambac flowers. AB - Benzyl 6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside (beta-primeveroside) (1), 2-phenylethyl beta-primeveroside (2), and 2-phenylethyl 6-O-alpha-L rhamnopyranosyl-beta-D-glucopyranoside (beta-rutinoside) (3) were isolated as aroma precursors of benzyl alcohol and 2-phenylethanol from flower buds of Jasminum sambac Ait. The isolation was guided by an enzymatic hydrolysis and GC and GC-MS analyses. PMID- 7539640 TI - GmN56, a novel nodule-specific cDNA from soybean root nodules encodes a protein homologous to isopropylmalate synthase and homocitrate synthase. AB - We have isolated a novel nodule-specific cDNA clone, GmN56, from soybean root nodules. The expression of GmN56 was induced almost concomitantly with the onset of nitrogen fixation, together with leghemoglobin and other late nodulin genes. In situ hybridization studies demonstrated the localization of GmN56 mRNA in the bacterial infected cells of mature nodules. The predicted amino acid sequence of the GmN56 protein exhibits significant homology to those of LeuA (isopropylmalate synthase) of several microorganisms and NifV (putative homocitrate synthase) of nitrogen-fixing bacteria, suggesting that GmN56 encodes an enzyme catalyzing a reaction involving acetyl-CoA and alpha-keto acid as substrates. PMID- 7539645 TI - Glycoprotein hormones: glycobiology of gonadotrophins, thyrotrophin and free alpha subunit. AB - Chorionic gonadotrophin and pituitary luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone comprise the family of glycoprotein hormones, which regulate major metabolic and reproductive functions of the body. These are heterodimeric glycoproteins composed of a common alpha subunit and a hormone-specific beta subunit. The N-linked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity. The free alpha subunit, which is shown to have a physiological function, is maintained in the uncombined form due to its glycan structures. The N-glycans of the glycoprotein hormones contain a variety of terminal residues, which are responsible for the differential targeting and clearance of the hormones. Glycosylation of these hormones is regulated by a variety of physiological and pathological conditions, leading to subtle alterations in their bioactivities. Recent studies on the structures and specific functions of different glycans of natural and recombinant glycoprotein hormones have provided valuable insight into the glycobiology of these hormones. This information will be useful in the development of diagnostic and therapeutic applications of the glycoprotein hormones. PMID- 7539644 TI - Further studies of the binding specificity of the leukocyte adhesion molecule, L selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems. AB - This communication is concerned with the binding specificity of the leukocyte adhesion molecule L-selectin (leukocyte homing receptor) towards structurally defined sulphated oligosaccharides of the blood group Le(a) and Le(x) series, and of the glycosaminoglycan series heparin, chondroitin sulphate and keratan sulphate. The recombinant soluble form of the rat L-selectin (L-selectin-IgG Fc chimera) investigated here was shown previously to bind to lipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated at galactose, such as sulphatides and a mixture of 3-sulphated Le(a)/Le(x) type tetrasaccharides isolated from ovarian cystadenoma, as well as to the HNK-1 glycolipid with 3-O sulphated glucuronic acid. In the present study, the L-selectin investigated in both chromatogram binding and plastic microwell binding experiments using neoglycolipids was found to bind to the individual 3-sulphated Le(a) and Le(x) sequences (penta-, tetra- and trisaccharides), and with somewhat lower intensities to their non-fucosylated analogues. Glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types were also bound by L-selectin in one or both assay systems, leading to the conclusion that clustered glycosaminoglycan oligosaccharides with 6-O sulphation of N-acetylgalactosamine, N acetylglucosamine or glucosamine, 4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronic acid, N-sulphation of glucosamine and, to a lesser extent, the non-sulphated uronic acid-containing disaccharides, can support L-selectin adhesion. As inflammatory chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation) are known to bind to endothelial glycosaminoglycans, we propose that the binding of the lymphocyte membrane L selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades. The possibility is also raised that lymphocyte L-selectin interactions with glycosaminoglycans may contribute to pathologies of glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoid arthritis and inflammatory lesions of the cornea. PMID- 7539646 TI - The effect of FK506 on warm ischemia and reperfusion injury in the rat liver. AB - The protective effect of FK506 on hepatocytes against ischemia and reperfusion injury was examined by evaluating the following: the high energy phosphorus metabolism obtained using 31P magnetic resonance spectroscopy (31P-MRS) and the tissue blood flow of the liver in ischemia and the reperfusion process, mitochondrial glutamic oxaloacetic transaminase (m-GOT) and glutamic pyruvic transaminase (GPT), the survival rates of the animals, a histological study and immunohistological staining for intercellular adhesion molecule-1 (ICAM-1) in the liver after ischemia. The rats were treated with FK506 1 mg/kg/day i.m. for 4 days before testing. Ischemia was induced by clamping the hepatoduodenal ligament for 30 min. In 31P-MRS, the recovery of the hepatic energy status after ischemia, evaluated by beta-ATP/inorganic phosphate (Pi), was significantly better in the FK506 group. It also coincided with the recovery of tissue blood flow monitored with a laser Doppler flowmeter. In the histological examination, the congestion observed in the periportal region of the control group was mild, while there was less induction of ICAM-1 in the endothelial cells of the portal veins and hepatic veins in the FK506 group. From these findings, we concluded that FK506 had a protective effect on hepatocytes against warm ischemia and reperfusion injury, and the mechanism for this could partially be attributed to improved tissue blood flow after ischemia by the modulation of immunological events. PMID- 7539647 TI - Changes in the mononuclear cell subpopulations of rat cardiac transplant recipients administered FK506 for the treatment of ongoing rejection. AB - The inhibitory effect of ongoing rejection and the changes that occurred in mononuclear cell subpopulations were compared between four groups of rats treated with FK506 or steroids. Group 1 was given no immunosuppressive drugs, group 2 was given FK506 from the day of grafting, group 3 was commenced on FK506 on the 4th day after grafting, and group 4 was commenced on methylprednisolone (MP) on the 4th day after grafting. The graft survival times in groups 2 and 3 were significantly longer than those in groups 1 and 4, and there were fewer CD3+ and CD4+ T lymphocytes in the peripheral blood in the groups treated with immunosuppressive drugs than in group 1. In group 4, the levels in both the peripheral blood and thymus were significantly lower than those in the groups treated with FK506 despite the fact that graft rejection occurred soon after the discontinuation of steroid administration. Moreover, the levels of interleukin-2 receptors and macrophages in groups 2, 3, and 4 were significantly lower than that in group 1 postoperatively; however, the number of macrophages in groups 2 and 3 was significantly lower than that in group 4 on the 10th day after transplantation. The findings of this study demonstrated that FK506, even if administered after rejection has begun, might inhibit the subsequent extensive allograft rejection more specifically and effectively than steroids, and that the measurement of a marker for macrophages in the peripheral blood could be useful for the detection of rejection following allograft transplantation in rats. PMID- 7539649 TI - Repulsive and inhibitory signals. AB - Repulsive or inhibitory interactions between growth cones and their environment are now widely implicated in neural development and regeneration. Over the past year, descriptive studies of the various neuronal systems in which repulsion may participate have clarified its biology. Molecular and genetic studies have also provided the necessary entry point for further experimental manipulations, and are beginning to yield important clues regarding the function of repulsion in vivo. Although candidate second messengers underlying the growth cone response have been identified, they have yet to be incorporated into a comprehensive mechanism. PMID- 7539648 TI - Anaplastic thyroid carcinoma producing the granulocyte colony stimulating factor (G-CSF): report of a case. AB - We report herein the unusual case of a 60-year-old woman with an anaplastic thyroid carcinoma which produced granulocyte colony stimulating factor (G-CSF). She presented with large neck masses, respiratory difficulty, and a high fever. Laboratory examinations revealed marked leukocytosis of 43,200/mm3 with 85% granulocytes and an elevated G-CSF level of 67 pg/dl. Total thyroidectomy with bilateral node dissection and tracheostomy was performed, and a histological diagnosis of large-cell anaplastic thyroid carcinoma was confirmed. Immunohistochemical examination with a polyclonal antibody against G-CSF stained the tumor cells. Although the respiratory difficulty, fever, and marked granulocytosis subsequently improved, she died 1 month after undergoing surgery due to metastatic mediastinal disease. PMID- 7539651 TI - Special issues in pain control during terminal illness. AB - Pain control is still a prime concern in managing patients with terminal illnesses, such as AIDS and cancer. I review some special issues that confront family physicians providing such care. Issues include common blocks to good pain management, understanding different types of pain, and the appropriate use of adjunct analgesic drugs and therapies. PMID- 7539652 TI - Talking with patients. Is it different when they are dying? AB - Talking with dying patients is different in some ways from talking with patients who do not have life-threatening illness; it can be frightening for physicians who have little experience. It is important to realize that, ultimately, the principles remain the same, and that the differences arise in the context and goals of the intervention. This article draws upon bedside experience to define several principles useful in talking with dying patients. Case examples illustrate each principle. PMID- 7539650 TI - The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells. AB - Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K 252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase. PMID- 7539653 TI - Preventing crises in palliative care in the home. Role of family physicians and nurses. AB - With the current shift to community care, the need for palliative care in the home involving the family physician has increased. Potential causes of crises in the home care of the dying are identified. Strategies to prevent crises are suggested that rely on a team's providing comprehensive and anticipatory care. PMID- 7539654 TI - Radioautographic study of the macromolecular synthesis of Leydig cells in aging mice testis. AB - The studies on DNA, RNA and protein synthesis of Leydig cells in aging mice testis of several groups were carried out by light microscopic radioautography. The cells labelled with 3H-thymidine, 3H-uridine and 3H-leucine were observed in various aging groups. The labeling index with 3H-thymidine was low at embryonic and early postnatal stages. It increased slightly at 6 months, reached its peak at 9 months and maintained a relatively high level at a later period. The number of the silver grains labelled with 3H-uridine over the nuclei and cytoplasm were observed from embryonic day 19 and increased from 3 months onwards. From adult to old ages, the activity of 3H-uridine incorporation was maintained at a high level in the nuclei and a relatively low level in the cytoplasm. The aging change of 3H leucine incorporation was found from this experiment. The labeling indices between embryonic and early postnatal stages showed no obvious differences while the number of the silver grains over both cytoplasm and nucleus increased from 6 months onwards and was maintained a high level until old age. PMID- 7539655 TI - Nucleic acid synthesis in the developing mouse ovary, uterus and oviduct studied by light and electron microscopic radioautography. AB - The DNA and RNA synthesis in the developing mouse ovary and oviduct were studied by 3H-thymidine and 3H-uridine radioautography. The results showed that both DNA and RNA syntheses were active in all surface epithelial cells, in stromal and follicular cells of the ovaries between postnatal days 1 to 7; then they decreased significantly from day 14 on. However, the DNA synthesis in the epithelial and in the stromal cells of the uteri and oviducts was active at days 1 and 3 and decreased from day 7. The RNA synthesis in the uteri and oviducts was active at postnatal day 1, increased from postnatal day 1 to day 14, decreasing between days 30 to 60. An unparalleled alternation of the DNA and RNA synthesis was shown between the ovary and uterus or oviduct. After postnatal day 3, the RNA synthesis was more important in the uterus and oviduct than in the ovary. PMID- 7539656 TI - Tracing the expression of CD7 and other antigens during T- and myeloid-cell differentiation in the human fetal liver and thymus. AB - During the last decade, the function/s of the cell membrane CD7 antigen have been investigated in human mature T and NK cells, showing the direct involvement of this molecule in multiple effector functions related with activation, proliferation, production of cytokines and modification of adhesion properties. The CD7 glycoprotein is not only expressed by mature lymphoid cells, but also by early hematopoietic progenitors and several types of leukemias, suggesting a role of CD7 during hematopoiesis. However, the function of CD7 in the early stages of hematopoietic development has not yet been elucidated. CD7 has been classically considered the earliest T-cell specific marker. This assumption was based on data indicating the presence of CD45+CD7+CD3-CD4-CD8- cells in the human embryonic/fetal liver at the gestational age at which the thymic rudiment is colonized by T-cell progenitors. In the present article, we review recent results obtained by several groups concerning the expression of CD7 and various other cell surface antigens by T-, B- and myeloid-cell progenitors generated in the adult bone marrow and fetal liver. In addition, we present an hypothetical model of hematopoiesis in the fetal liver and thymus. PMID- 7539657 TI - CD7 expression in acute myeloid leukemia. AB - The clinical significance of the expression of CD7 antigen on the blasts of 207 consecutive patients with de novo acute myeloid leukemia (AML) was evaluated. For this purpose, fifty-three CD7+ patients (23 females and 30 males; mean age 52 years) were analyzed and classified into the following subtypes according to French-American-British (FAB) classification: 7 M0, 13 M1, 9 M2, 1 M3, 9 M4, 14 M5. Immunophenotypic studies were carried out by flow cytometry and blast cells were selected on the basis of forward light scatter gating and pan-myeloid marker, either CD13 or CD33. All the CD7+ patients were negative for surface CD3 and T-cell-receptor (TCR) molecules. We found no correlation between CD7 expression and sex, age, hepatosplenomegaly and/or central nervous system involvement. The immaturity of CD7+ leukemic cells was supported by the high expression of CD34 (P = 0.001). CD7 positivity was significantly associated with a white blood cell count (WBC) greater than 100 x 10(9)/L (P = 0.003). P Glycoprotein (P-170) expression was also evaluated in 135 patients by a flow cytometric assay: there was a close relationship between CD7 and P-170 positivity (P < 0.001). For remission induction, all patients received therapeutic regimens routinely used for AML. The complete remission (CR) rate was significantly lower in CD7+ cases (32% vs 74%, P = 0.001). The overall survival and disease free survival rate of CD7+ AML was lower than those of CD7- patients (P < 0.001 and = 0.002, respectively). CD7+ AML with coexpression of CD14 had a particularly unfavourable response and prognosis in comparison with CD7+ patients without CD14.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539658 TI - Economic analysis of prophylactic G-CSF after mini-BEAM salvage chemotherapy for Hodgkin's and non-Hodgkin's lymphoma. AB - The objectives of this study were to compare the costs of managing lymphoma patients who underwent mini-BEAM salvage chemotherapy with G-CSF prophylactic support against a group of similar patients without growth factors. Methods used included: 1) A retrospective chart review was conducted to estimate the average length of hospitalization and resource consumption for the management of fever and neutropenia in the two groups of patients and 2) An economic analysis was then performed from a hospital perspective which considered only institutional resource utilization. Costs of antibiotic support and monitoring, lab tests as well as G-CSF were calculated. Results demonstrated that overall, patients who received prophylactic G-CSF after chemotherapy required 2 fewer hospital days compared to controls. The administration of G-CSF resulted in a savings of approximately $1580/patient relative to control. When the initial G-CSF expenditure was included in the analysis, the total net cost/patient was similar between the two groups. In conclusion, the results of the current study support the routine use of G-CSF in patients receiving salvage chemotherapy with mini BEAM. The initial G-CSF expenditure would be offset by reduced hospitalization. PMID- 7539659 TI - CD34-positive early stages of human T-cell differentiation. AB - Thymus, the main organ for T lymphopoiesis, requires a permanent influx of progenitors from bone marrow (BM) or fetal liver. An essential question relating to early T-cell development is the identification of the progenitor population which actually homes to the thymus. Recent findings have shown that human multipotent progenitor/stem cells expressing CD34 have the capacity to differentiate into T cells when introduced into a thymic environment. More mature CD34+ bone marrow cells coexpressing CD7 and having a poor myeloid differentiation capacity can also efficiently differentiate into T cells in vitro. These lymphoid committed precursors might be the true thymic repopulating cells. In the thymus, cells with a similar CD34+7+ phenotype include the most primitive thymocyte precursors. CD34+ thymocytes have no myeloid differentiation potential, but may include precursors for natural killer (NK) cells. Interleukin 7 (IL7) is a potent in vitro growth factor for CD34+ thymocytes. Whereas current data do not support a crucial role for IL2, patients with IL2 receptor gamma chain (IL2R gamma) deficiency lack T- and NK cells. The recent demonstration that IL2R gamma is part of the receptor for IL7 strongly suggests that this cytokine plays an essential role in in vivo T lymphocyte and NK development. PMID- 7539660 TI - Functional consequences of APO-1/Fas (CD95) antigen expression by normal and neoplastic hematopoietic cells. AB - Murine monoclonal antibody (mAb) 7C11 binds to the same cell surface epitope as anti-APO-1 and anti-Fas and reacts specifically with cells transfected with a cDNA encoding the human Fas antigen. Furthermore, incubation with 7C11 causes death of hematopoietic cell lines that express APO-1/Fas but not APO-1/Fas negative cell lines. 7C11 therefore recognizes the human APO-1/Fas (CD95) antigen, a 40 to 50 kDa cell surface glycoprotein that can trigger apoptosis or programmed cell death. Expression of APO-1/Fas antigen by normal and neoplastic hematopoietic cells was determined by flow cytometry using 7C11. APO-1/Fas is expressed by approximately 30 to 40% of resting peripheral blood T cells, B cells, and monocytes and by approximately 5% of resting NK cells and thymocytes. It was not detected on granulocytes, erythrocytes, or platelets. Approximately 80 to 90% of activated T cells, B cells, and thymocytes express APO-1/Fas, as do the majority of activated NK cells. Perturbation of APO-1/Fas by 7C11 does not affect the viability of resting lymphocytes or monocytes. In contrast, activated T cells and NK cells undergo apoptosis within 3 hours of exposure to 7C11. Other mAb that stimulate T cells or NK cells do not cause rapid induction of programmed cell death. APO-1/Fas antigen is expressed by many cell lines of lymphoid and myeloid lineage. However, this antigen was detected on neoplastic cells from only one of 69 patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or multiple myeloma. Only 3 out of 25 tumor samples from patients with non-Hodgkin's lymphoma were found to express APO-1/Fas. All three of these lymphomas harbored the bcl-2-Ig fusion gene associated with the chromosomal translocation t (14;18). Conversely, only 27% of lymphomas that possessed the bcl-2-Ig gene were found to express the APO-1/Fas antigen. Like normal activated lymphocytes, leukemia and lymphoma cells that expressed APO-1/Fas antigen were found to undergo apoptosis in vitro after incubation with 7C11. The APO-1/Fas antigen appears to regulate the growth of normal hematopoietic cells, and the marked upregulation of this antigen on activated normal lymphocytes contrasts sharply with the absence of APO-1/Fas on neoplastic cells of hematopoietic lineage. Defects in the apoptotic signal delivered through this antigen might contribute to the pathogenesis of hematopoietic neoplasms. Thus, the gene encoding APO-1/Fas can be considered a novel type of tumor suppressor gene, just as bcl-2 can be considered a cellular proto-oncogene. PMID- 7539661 TI - High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry. AB - We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied. PMID- 7539663 TI - Purification and characterization of a raw-starch digesting amylase from a soil bacterium--Cytophaga sp. AB - A newly isolated bacterium from soil, identified as Cytophaga sp. was found to produce raw-starch digesting amylase. The enzyme was purified from 24-hr cultured medium through ammonium sulfate fractionation, DEAE-Sepharose CL 6B ion exchange chromatography and Sephacryl S-200 gel filtration. The preparation was proved to be homogeneous by SDS-PAGE. The subunit molecular weight determined by SDS-PAGE was 59 KD. The optimum temperature was 50 degrees C on soluble starch and 60 degrees C on raw starch. The optimum pH was in the range of 4.5 to 6.5 on soluble starch and 6.5 to 9.5 on raw starch. In the presence of Mn+2, Cu+2 or Zn+2, the enzyme activity on either substrate was inhibited. Dinitrofluorobenzene, N bromosuccinimide and trinitrobenzene sulfonic acid all showed inhibitory effect on the enzyme acting on both substrates. PMID- 7539662 TI - Cisplatin stimulates protein tyrosine phosphorylation in macrophages. AB - Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID- 7539664 TI - Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells. AB - The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID- 7539666 TI - Bone marrow stromal function from patients after bone marrow transplantation. AB - A two-stage long-term bone marrow culture (LTBMC) has been used to assess haemopoietic supporting ability from patients following allogeneic bone marrow transplantation (BMT). Irradiated confluent LTBMCs derived from recipients following BMT were recharged with flow cytometry sorted CD34+/CD38- cells from normal BM donors. The output of granulocyte/macrophage colony-forming cells (GM CFC) in the non-adherent fraction of the cultures was monitored weekly until output ceased. Patient stromal cell function was determined by comparing the percentage of the total cumulative GM-CFC output to the output from normal control stroma. Neutrophil recovery was rapid and complete early in the post transplant period. However, in contrast, GM-CFC recovery was significantly reduced in all patients up to 5 years after BMT. In 58% of patients studied following BMT the BM stroma is considerably compromised in its ability to support early uncommitted haemopoietic cells contained within the CD34+/CD38- cell fraction of normal BM. Bone marrow stroma from BMT recipients exhibited severe deficits in their abilities to support primitive haemopoietic cells and in some patients this effect lasted for several years. While the growth of stroma from patients following BMT was severely delayed and often incomplete, characterisation of cells types using APAAP techniques did not show significant differences in proportion when compared with normal cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539665 TI - Phenotypic analysis of early hematopoietic progenitors in cord blood and determination of their correlation with clonogenic progenitors: relevance to cord blood stem cell transplantation. AB - Human cord blood (CB) can be utilized as a source of transplantable stem cells. In the clinical setting, the numbers of clonogenic progenitors and nucleated cells are used as indirect measures of the stem cell content of CB samples. In an attempt to define other parameters of engraftment potential, we have determined the numbers of CD34+ cells, and of CD34+ cells co-expressing CD38 and/or HLA-DR in 17 CB samples. At the same time, clonogenic progenitors were assayed in the presence of erythropoietin and different combinations of rhIL-3, rhG-CSF, rhGM CSF, rh-Steel Factor and medium conditioned by the PU34 primate cell line. Unseparated CB contained a mean of 14.8 x 10(4) CD34+ cells/mL. CD34+CD38-, CD34+DR+ and CD34+DR+ CD38- cells represented 12.2%, 91% and 10% of all CD34+ cells, respectively. Linear regression analysis demonstrated that the total number of CD34+ cells, as well as the different subsets, correlated positively with the numbers of total colonies and day 14 CFU-GM. Furthermore, the proportion of CD34+CD38- cells was significantly higher than has been reported for adult marrow. Cord blood CD34+CD38- and CD34+DR+ cells have a high proliferative potential and are enriched for primitive hematopoietic progenitors. Thus, we conclude that a single collection of CB could be sufficient to engraft an adult recipient, and that quantitation of CD34+ cells and their subsets may be useful in determining the engraftment potential of CB samples. PMID- 7539667 TI - Prophylaxis for acute graft-versus-host disease following unrelated donor bone marrow transplantation. AB - Despite the use of conventional chemoprophylaxis regimens, patients receiving unrelated-donor BMT are at high risk of developing severe acute GVHD. We evaluated a prophylactic regimen combining CsA, MTX and anti-CD5-ricin A chain immunotoxin (H65-RTA) in 31 patients; pentoxifylline was also given to reduce the anticipated nephrotoxicity of CsA. In most cases, planned doses of CsA, MTX and H65-RTA were given (i.e. to 77%, 77% and 93% of patients, respectively). Although fluid retention requiring diuretic therapy was frequent, only 1 patient had a > 10% unexplained increase in body weight during the first 21 days post-BMT. Also, while significant increase of the baseline serum creatinine was noted in 7 patients, none required dialysis. One patient suffered a reversible allergic reaction to the immunotoxin; no other side effects attributable to this regimen were observed. All but 2 patients engrafted (1 died of fungemia on d + 19 and the other had persistent leukemia) and no late graft failures were observed. Seventeen patients developed acute GVHD grade > or = II (probability, 58% [95% CI 41-76%]); 7 had grade > or = III (probability, 24% [95% CI 12-43%]). In the 27 patients who achieved stable engraftment and have survived beyond d + 100, the 3 year probability of developing chronic GVHD was 66% (95% CI 48-84%). As of the last follow-up prior to 01 May 1994, 13 patients are alive in CR and one in relapse; 9 of these patients are off all immunosuppressives and well. Four other patients relapsed and died, and 13 died of other transplant-related causes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539668 TI - Results of a randomised, controlled, multicentre study of recombinant human granulocyte colony-stimulating factor (filgrastim) in patients with Hodgkin's disease and non-Hodgkin's lymphoma undergoing autologous bone marrow transplantation. AB - In 54 patients with malignant lymphoma, haematopoietic recovery after high-dose chemotherapy and autologous bone marrow transplantation (BMT) was compared between patients randomised to receive 10 or 30 micrograms/kg/day of r-metHuG-CSF (filgrastim) or no growth factor. After standard high-dose chemotherapy with cyclophosphamide, etoposide and BCNU (CVB regimen for patients with Hodgkin's disease) or BCNU, etoposide, cytosine arabinoside and melphalan (BEAM regimen for patients suffering from non-Hodgkin's lymphoma) followed by autologous BMT, r metHuG-CSF was administered by continuous intravenous infusion from the first day after autologous BMT until neutrophil recovery. When the r-metHuG-CSF groups were compared with the control group the major findings were: the median time to reach an absolute neutrophil count (ANC) > or = 0.5 x 10(9)/L was 20 days in the control group and 12 and 14 days, respectively, in the r-metHuG-CSF groups (P = 0.0004). The duration of neutropenia (ANC < 0.5 x 10(9)/L) was reduced from 27 days in the control group to 11 and 13 days in the r-metHuG-CSF groups (P = 0.0001). In addition, fewer days of febrile neutropenia were observed in the r metHuG-CSF groups (5 and 6 days) than in the control group (10 days; P = 0.036). No significant effects of r-metHuG-CSF administration on the number of days with fever, the use of intravenous antibiotics and hospitalisation were detected. R metHuG-CSF was well tolerated without any serious side-effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539669 TI - Generation of rubella virus-neutralising antibodies by vaccination with synthetic peptides. AB - Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFN gamma in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used. PMID- 7539670 TI - Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: assessment of cross-reactivity with Pseudomonas aeruginosa. AB - Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa. The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa. The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. this study suggests that there is a specific anti-B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa. Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS. PMID- 7539672 TI - A mutation in the ceruloplasmin gene is associated with systemic hemosiderosis in humans. AB - We identified a mutation in the ceruloplasmin (Cp) gene in a Japanese family with aceruloplasminemia, some of whose members showed extrapyramidal disorders, cerebellar ataxia, and diabetes mellitus. A post-mortem study of the proband revealed excessive iron deposition mainly in the brain, liver and pancreas. The G to A transition at the splice acceptor site introduces a premature termination codon at the amino acid position 991 by defective splicing, thereby truncating the carboxyl terminus of Cp in affected individuals. We conclude that the mutation in the Cp gene is associated with systemic hemosiderosis in humans. PMID- 7539671 TI - Inhibition of heparan sulphate and other glycosaminoglycans binding to Helicobacter pylori by various polysulphated carbohydrates. AB - Heparan sulphate binding to Helicobacter pylori at pH 4 to 5 was inhibited with various sulphated polysaccharides (heparin and chondroitin sulphates, fucoidan, carrageenans and some others), but not by carboxylated or nonsulphated compounds. Heparin binding proteins are exposed on the cell surface. PMID- 7539674 TI - Benign prostatic hyperplasia, a common disease of the ageing male. PMID- 7539673 TI - Keratin 16 and keratin 17 mutations cause pachyonychia congenita. AB - Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC. PMID- 7539675 TI - Cystoscopy in the evaluation of benign prostatic hyperplasia. AB - Cystoscopy has a limited role in the evaluation of benign prostatic hyperplasia (BPH). The examination is recommended in the evaluation of BPH patients with hematuria or a history of risk factors for urethral stricture. Available data suggest that bladder trabeculation in BPH is a predictor of the treatment outcome. Cystoscopy might be performed prior to invasive therapy to guide the urologist in choosing an operative approach. PMID- 7539676 TI - Postvoid residual urine in the evaluation of men with benign prostatic hyperplasia. AB - Traditionally, the measurement of postvoid residual urinary volume (PVR) has played a prominent role in the evaluation of men with symptoms suggestive of benign prostatic hyperplasia (BPH). This article reviews current opinions about the cause of PVR, methods and accuracy of measurement, correlations between PVR and symptoms as well as physiologic measures of BPH and the role of PVR determination in the evaluation of BPH patients. Community-based data obtained on PVR in men aged between 55 and 74 years in the city of Rotterdam (The Netherlands) are reported. PMID- 7539678 TI - Bladder-outlet obstruction--assessment of symptoms. AB - Assessment of symptoms plays a central role in the evaluation of men with suspected bladder-outflow obstruction (BOO) secondary to benign prostatic hyperplasia (BPH). It is on the basis of symptoms that treatment designed to relieve outflow obstruction is recommended. However, a variety of studies have suggested that a considerable proportion of men presenting with suspected outflow obstruction are not obstructed as determined by pressure-flow criteria. The relationship between many so-called "obstructive" symptoms and BOO has not yet been defined. Furthermore, all previous studies have defined BOO on the basis of urethral resistance factors, which are now regarded as outdated. Using current concepts of urethral pressure-flow relationships, we studied in detail the relationship between the "obstructive" symptoms of BPH, objective evidence of abnormal voiding and BOO. Only the symptoms of hesitancy and poor flow were found to be significantly related to BOO. The symptoms of straining to void, intermittency, terminal dribbling and feeling of incomplete bladder emptying were not specific for BOO. Objective evidence of an intermittent flow pattern and of terminal dribbling do, however, have a high specificity and positive predictive value for BOO. The assessment of men with lower-urinary-tract symptoms must include objective tests of voiding function. A better understanding of the pathophysiology of lower-urinary-tract symptoms should lead to an improved symptomatic outcome of prostatectomy. PMID- 7539677 TI - Preoperative cystometrography in patients with clinical benign prostatic hypertrophy. AB - Preoperative water cystometrograms obtained from 437 patients with benign prostatic hyperplasia (BPH) were examined in a retrospective study. The cystometrographic results were analyzed regarding the preoperative clinical features: the patients' age, presence or absence of urinary incontinence, history of urinary retention, and rate of residual urine. Their prognostic value in terms of improvement in voiding difficulty and postoperative urinary incontinence was also analyzed at 1 and 6 months after elective transurethral resection of the prostate (TURP). Of these patients, 263 (60.2%) had detrusor instability (group I), whereas 174 did not (group II). Vesical denervation supersensitivity (Vds) to bethanecol chloride was noted in 47 (12.5%) of 375 patients. The observed difference in clinical features was significant between the two groups, with group I being older (P < 0.01) and showing a greater incidence of urinary incontinence (P < 0.001) and retention (P < 0.001). The difference seen between groups I and II in terms of mean bladder capacity (P < 0.01), compliance (P < 0.01), and a greater positive rate of Vds (P < 0.001) was also significant. The clinical and cystometrographic parameters studied worsened with advancing age of the patients. Although the majority of the patients (94.7%) were relieved of obstructive symptoms after TURP (6 months later), 113 (25.9%) showed no relief at 1 month.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539679 TI - The Abrams-Griffiths nomogram. AB - The treatment of benign prostatic hyperplasia and the definition of bladder outlet obstruction has preoccupied urologists and researchers in recent years. Bladder-outlet obstruction can be defined only by pressure-flow measurement. Various methods of analysis of pressure-flow data have been proposed. The Abrams Griffiths nomogram is an easy method of classifying these data to distinguish between the presence or absence of obstruction. Using the values for the maximal flow and the corresponding voiding detrusor pressure a point can be plotted on the nomogram that determines whether the bladder outlet is obstructed, unobstructed, or equivocally obstructed. For those that fall in the equivocal zone, further criteria for the mean slope of the pressure-flow plot and the minimal voiding detrusor pressure are used to determine whether there is obstruction or not. The nomogram's prognostic value in predicting the outcome of prostatectomy has been studied and found to be excellent. The Abrams-Griffiths nomogram can be modified by assigning an Abrams-Griffiths number to each set of pressure-flow data. This number is easy to calculate and use and gives a continuous variable that can be used to evaluate the effects of therapy. Although the Abrams-Griffiths nomogram and number are somewhat simplistic, none of the more complex methods of pressure-flow analysis have been shown to be better predictors of treatment outcome to date. PMID- 7539680 TI - CHESS classification of bladder-outflow obstruction. A consequence in the discussion of current concepts. AB - The present discussion on the natural history, growth rate pathophysiology, morbidity, and possible treatment of benign prostatic hyperplasia (BPH) reinforces the need to objectify mechanical BPH-induced obstruction with pressure flow study. Different methods for the quantification of BPH-induced obstruction exist and the grade of simplification depends on their working mechanism. To review the limitations of the current concepts, 118 BPH patients were selected for computerized pressure-flow data analysis according to quadratic passive urethral resistance relation (PURR) function. The CHESS classification differs from all other concepts due to its two-dimensional character and was developed as a result of the following way of thinking: (1) the whole individual pressure-flow plot is the most accurate basis for further analysis; (2) PURR as the true low pressure flank of the individual plot is the most favorable definition for mechanical obstruction; (3) PURR quantification requires at least two parameters, footpoint and curvature/slope, with regard to the documented missing significant correlation of both; and (4) a two-parameter-based classification is two dimensional (CHESS classification). PMID- 7539681 TI - Estimation of urethral resistance by curve fitting in the pressure-flow plot. Theory and experience in normal men and men with benign prostatic hypertrophy. AB - The curvature of the urethral resistance relation was estimated in 21 normal men, in 32 prostatism patients, in 60 men with a diagnosis of benign prostatic hypertrophy (BPH) and obstruction, and in 55 men after treatment of BPH. The curvature was determined by curve fitting in the pressure-flow plot using the method of least squares. In all, 58% of the normal men and prostatism patients had a urethral resistance relation with the convexity directed upward, demonstrating a low-compliant type of distensibility. After transurethral resection (TUR), significantly more men had the convexity of the urethral resistance relation directed downward, showing that the distensibility was more of a constrictive type. This change in the elastic properties of the flow controlling zone can be assessed only by using several curvatures to estimate the urethral resistance relation. Another advantage for the method of using different curvatures is that the effects of bladder contractility are minimized. However, should one use only one form of the urethral resistance relation, it is recommended that a linear relation be chosen. PMID- 7539685 TI - Staining methods in gel electrophoresis, including the use of multiple detection methods. AB - Polyacrylamide gel electrophoresis is a reliable and widely used technique for the separation, identification and characterization of proteins and protein mixtures. With the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis in 1975 upward to 2000 individual polypeptides spots are easily separated on a single electrophoretic gel thereby necessitating the availability of highly sensitive protein detection methods. Although a plethora of protein-staining and -visualization protocols have been described utilizing both radioactive and non-radioactive reagents, many times the use of mono dimensional detection procedures is insufficient to address the experimental questions asked. The present review highlights the utilization of combined protein-labeling and -staining methodologies in gel electrophoresis including selected applications in polyacrylamide gels and solid membrane matrixes. PMID- 7539682 TI - Sequence analysis and evolutionary aspects of piscine alpha-1 microglobulin/bikunin mRNA transcripts. AB - Studies on mammalian alpha-1-microglobulin/bikunin (AMBP) protein sequences suggest the ancestral AMBP gene arose between 270 and 80 million years ago. Here we report a similar study including recently reported piscine AMBP messenger RNA sequences. The analysis implies a far earlier data for the assembly of the ancestral AMBP gene, more than 450 million years ago. Similarly, a revised date for the origin of the ancestral Kunitz-type protein is estimated at approximately 875 million years ago. Evolutionary conservation is apparent among all the AMBP amino acid sequences. This implies a common functional significance in both teleosts and mammals. However, an unusually rapid rate of evolution is observed for domain II of the piscine bikunin sequences, suggesting that this domain is no longer truly orthologous in fish. PMID- 7539683 TI - Effects of acyclovir and vidarabin 5'-monophosphate on anti-duck hepatitis B virus in an in vitro culture system. AB - Various anti-viral agents, e.g., interferon, have recently been used for the treatment of viral hepatitis. In the present study, duck hepatitis B virus (DHBV) was cultured in vitro and the anti-DHBV effects of acyclovir (ACV) and vidarabin 5'-monophosphate (VMP) were studied. The portal perfusion method was applied to the livers of 7-day-old white ducks weighing 100 g, bred in Japan, and hepatocytes were infected with DHBV in vitro. Duck hepatocytes infected with DHBV were cultured in medium containing ACV or VMP, and the anti-DHBV effects of these drugs were assessed by determining DHBV-DNA and duck hepatitis B surface antigen in the medium. Both ACV and VMP had anti-DHBV effects when used immediately after infection; however, both drugs were ineffective in hepatocytes obtained from a DHBV carrier duck. In conclusion, the anti-DHBV effects of these drugs were very limited. However, this culture system appears to be useful for studies of hepatitis virus and anti-viral drugs. PMID- 7539686 TI - Immunochemical, genetic and morphological comparison of fucosylation mutants of Dictyostelium discoideum. AB - Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intact modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed accumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism. PMID- 7539684 TI - Effects of ethanol on the pancreas of disulfiram-treated rats. AB - To study the effects of ethanol on disulfiram-treated rats, we administered ethanol orally at a does of 2000 mg/kg, twice daily for 5 days. The administration of ethanol or disulfiram alone produced no recognizable changes in pancreatic acinar cells. Ethanol administration, in disulfiram-treated rats resulted in a decrease in the content of zymogen granules in acinar cells, and the appearance of intraplasmic vacuolization. Electron microscopically, these vacuoles appeared on the basal side of nuclei. In addition, similar vacuoles appeared in liver cells, and these vacuolizations seemed to show lipid inclusions. However, ethanol administration to disulfiram-treated rats did not cause inflammatory changes or edema in the pancreas. A comparison of blood ethanol levels in rats receiving ethanol alone and disulfiram plus ethanol showed no significant difference, but acetaldehyde levels in rats receiving ethanol plus disulfiram rats were significantly higher than those in rats receiving ethanol alone. These findings suggested that acetaldehyde caused a decrease of zymogen granules and the presence of lipid inclusions in pancreatic acinar cells. PMID- 7539687 TI - Effect of sialylation of lipopolysaccharide of Neisseria gonorrhoeae on recognition and complement-mediated killing by monoclonal antibodies directed against different outer-membrane antigens. AB - Growth of gonococci in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) has previously been shown to induce resistance to the bactericidal effect of normal human serum and is accompanied by sialylation of the gonococcal lipopolysaccharide (LPS). We have used monoclonal antibodies (mAbs) to compare the effect of LPS sialylation on recognition of gonococci and complement-mediated killing by antibodies directed either against LPS or against defined epitopes on outer-membrane protein PI. Despite differences in binding to sialylated LPS on Western blots, all three mAbs directed against LPS showed considerably reduced binding to gonococci grown in the presence of CMP-NANA and a concomitant reduction in ability to promote complement-mediated killing. In contrast, mAbs directed against previously defined epitopes on a surface exposed loop of PI showed little difference in binding between sialylated and non-sialylated gonococci and promoted killing of the sialylated gonococci. Similarly a mAb directed against an epitope on a loop of the outer-membrane Rmp protein, which had previously been shown to block killing by antibodies directed against other surface antigens, also exerted a blocking effect with sialylated gonococci. Thus in the present study the continued biological effect of mAbs was correlated with the ability of the antibody to recognize surface-exposed epitopes on sialylated gonococci. Despite the presence of the sialylation which is likely to occur in vivo, it should be possible to induce complement-mediated killing by focusing the immune response to those surface-exposed epitopes which are least susceptible to the potential inhibitory effect of LPS sialylation. PMID- 7539690 TI - Weighing waits and delays. PMID- 7539689 TI - A Clostridium acetobutylicum regulator gene (regA) affecting amylase production in Bacillus subtilis. AB - Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole. The nucleotide sequence of the C. acetobutylicum DNA controlling metronidazole sensitivity in E. coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated Mr of 35,000. The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E. coli encoded by the shl gene, and the GalR, Lacl and PurR repressors of E. coli. The C. acetobutylicum ORF, which was termed regA, complemented a B. subtilis ccpA mutant and an E. coli shl mutant, but was unable to complement E. coli galR, lacl or purR mutants. To determine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading enzyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The RegA protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli. PMID- 7539688 TI - A gene region in Dichelobacter nodosus encoding a lipopolysaccharide epitope. AB - Dichelobacter nodosus is a Gram-negative anaerobic bacterium that is the causative organism of footrot in sheep. A D. nodosus locus responsible for a modification of the host lipopolysaccharide (LPS) in Escherichia coli was cloned and sequenced. Genetic studies showed that the modification occurred within the inner-core region of the host LPS, most likely to one or more of the heptose molecules. Antibodies eluted from the modified LPS reacted preferentially with the lipid-A-core region of D. nodosus LPS, suggesting that the cloned epitope was present in this region of the D. nodosus LPS. The gene responsible for the modification, IpsA, potentially encoded a polypeptide of approximately 37 kDa which was highly basic, a characteristic of enzymes which interact with the acidic inner LPS core. The IpsA gene appeared to be arranged in a complex operon with a downstream gene, prfC, which encoded a protein with similarity to E. coli peptide-chain release factor 3. PMID- 7539691 TI - Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy. AB - Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I-IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae I-II. [125I]PYY binding was most strongly attenuated by NPY13-36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13-36 completely abolished PYY binding, which was also reduced by [Leu31,Pro34] NPY. However, the increase in PYY binding seen in laminae I-IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31,Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I-IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539692 TI - Regulation of rat sympathetic nerve density by target tissues and NGF in maturity and old age. AB - Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of approximately 50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density approximately 50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target derived neurotrophic factors. PMID- 7539693 TI - Clearance of myelin constituents and axonal sprouting in the transected postcommissural fornix of the adult rat. AB - Following transection of the postcommissural fornix in the adult rat, fibres retract from the lesion zone but then start to regrow within their former pathway up to the lesion site, where they terminate. The fibres neither penetrate nor bypass this region. In order to define the molecular mechanisms that cause regenerative failure at the lesion site, we analysed the spatiotemporal relationship between clearance/re-expression of myelin constituents and axon sprouting. Using immunocytochemical methods, we investigated the distribution of myelin-associated growth inhibitor (NI-35/250) and myelin basic protein after transection of the postcommissural fornix. In the studies described here we demonstrate the sequential removal of neurofilaments and myelin constituents in a perilesion zone and at the lesion site. The removal of myelin constituents was followed by the extensive regrowth of fornix fibres in the proximal segment. However, these fibres stopped at the lesion site, an area that lacked immunostaining for NI-35/250 and. In the distal stump we observed the disappearance of neurofilament along the entire fornix segment but spatial differences in the removal of myelin constituents. While both NI-35/250 and myelin basic protein disappeared in the perilesion zone, they persisted in the more distal segment for at least 28 months after lesion. In conclusion, our study indicates that the onset of axon sprouting is correlated with the removal of myelin basic protein and NI-35/250. Furthermore, we suggest that it seems unlikely that the myelin growth inhibitor NI-35/250 constitutes the stop signal of the axon growth barrier in the transected formix. PMID- 7539694 TI - Crucial role for the myelin-associated glycoprotein in the maintenance of axon myelin integrity. AB - It has recently been shown that mice deficient in the gene for myelin-associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon-myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination-induced Schwann cell proliferation and remyelination lead to the formation of so-called onion bulbs. Expression of tenascin-C, a molecule indicative of peripheral nerve degeneration, was up-regulated by axon-deprived Schwann cells and regenerating axons were occasionally seen. Myelin-associated glycoprotein thus appears to play a crucial role in the long-term maintenance of the integrity of both myelin and axons. PMID- 7539695 TI - Pre-operative estimation of complete resection for patients with oesophageal carcinoma. AB - Three hundred and seventy-nine patients were studied retrospectively regarding the possibility of a complete resection of the oesophageal carcinoma based on the combined findings of pre-operative oesophagogoraphy and computed tomography (CT). One hundred and four out of 129 patients (96.1%) having lesions which did not demonstrate all three of the aforementioned factors (a lesion shorter than 8 cm, a normal oesophageal axis, and normal contact of the lesion with neighboring organs in CT) underwent a complete resection of the oesophageal lesion. Fifty three percent of the patients (52/97) with a lesion showing only one of these factors had a complete resection. Whereas, on the other hand, a complete removal of the malignancy was only possible in 22% of the patients with two or all three of the findings. Moreover, as a result of further analysis limited for resected cases, the number of positive factors in these pre-operative findings correlated with the advancement of the surgical stage, which reflected a curability in surgery and a rate of postoperative complications. In order to make adequate plans for the treatment of patients with advanced oesophageal cancer, the finding of (i) the length of lesion, (ii) a deep ulceration and deformity of the oesophageal axis and (iii) any abnormal contact in CT, are considered to be very useful. PMID- 7539696 TI - Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle. AB - 1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3 like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only. PMID- 7539699 TI - A comparative study of preparation techniques for improving the viability of nigral grafts using vital stains, in vitro cultures, and in vivo grafts. AB - The intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells. Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation. We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies. For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts. The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported. In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension. The optimal preparation technique, therefore, uses E13-E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette. This may have important implications in improving intracerebral transplantation for Parkinson's disease. PMID- 7539697 TI - Differential effects of non-selective and selective phosphodiesterase inhibitors on human eosinophil functions. AB - 1. The effect of non-selective (3-isobutyl-1-methylxanthine, IBMX; theophylline) and type IV- or type III/IV-selective (rolipram, RP 73401; zardaverine, tolafentrine) phosphodiesterase (PDE) inhibitors on human eosinophil functions was investigated. 2. For this purpose human eosinophils were purified from blood of healthy donors by a magnetic cell separation (MACS) technique to a purity > or = 99%. From the stimuli investigated (complement C5a; N-formyl-methionyl-leucyl phenylalanine, fMLP; platelet activating factor, PAF; opsonized zymosan) C5a was selected to test the influence of the above mentioned compounds on secretion of granule constituents (eosinophil cationic protein, ECP; eosinophil-derived neurotoxin, EDN) as well as on formation of reactive oxygen species measured by luminol-enhanced chemiluminescence in intact cells. For comparison, inhibition of PDE IV activity in the cytosol of disrupted cells, which contains about 75% of total PDE IV activity, was determined. 3. Both theophylline and IBMX inhibited the two cell responses with IC50 values which were in the range of their IC50 values obtained for inhibition of PDE IV activity in the cell-free system. The beta 2-adrenoceptor agonist, salbutamol (1 mumol l-1), which by itself did not substantially influence the two cell responses, only marginally improved the potency of theophylline and IBMX in inhibiting ECP/EDN secretion. Only the IC50 value of IBMX for inhibition of chemiluminescence was lowered by about one order of magnitude in the presence of salbutamol. 4. In contrast, none of the selective PDE inhibitors tested substantially inhibited the two cell responses at concentrations up to 10 mumol l-1. This was surprising because all of the compounds investigated inhibited PDE IV activity in the cell-free system with IC50 values which were at least 30 fold lower than the highest concentration of the compounds used with intact cells. In combination with salbutamol, however, both ECP/EDN secretion and chemiluminescence was inhibited by rolipram and zardaverine with IC50 values similar to the IC50 values for inhibition of PDE IV activity. Although RP 73401 and tolafentrine also inhibited both cell responses in the presence of salbutamol, the potency of these two compounds in inhibiting eosinophil function in intact cells was at least two orders of magnitude lower than would have been expected from the inhibition of PDE IV activity in the cell free system. 5. These results indicate that (i) C5a-stimulated human eosinophils are sensitive to inhibition by then on-selective PDE inhibitors theophylline and IBMX, (ii) the inhibitory effect of these non-selective PDE inhibitors cannot be mimicked by selective PDE IV or PDE III/IV inhibitors although human eosinophils almost exclusively contain PDE IV; (iii) the selective PDE inhibitors need an additional cyclic AMP trigger like a beta 2-adrenoceptor agonist to be effective; but (iv) under the latter conditions inhibition of cell responses in intact cells does not correspond to inhibition of PDE IV activity in the cell-free system.6. We conclude that the non-selective PDE-inhibiting xanthines may inhibit C5a stimulated human eosinophil responses by other action(s) in addition to PDE IV inhibition, and that inhibition of PDE IV activity in the cell-free system by the selective inhibitors may not generally represent the potency of the compounds in intact cells. PMID- 7539700 TI - Tracer technique to measure in vivo chemical transport rates within an implantable cell transplantation device. AB - An in vivo tracer technique that uses radiolabeled insulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the host's circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device. PMID- 7539698 TI - Platelet-activating factor: the effector of protein-rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis. AB - 1. The involvement of platelet-activating factor (PAF) in immune complex induced/polymorphonuclear-mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats. 2. Extravasation of protein-rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed. 3. Treatment of rats with either UR-12460 or BB-823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein-rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN. 4. Inhibition of NO production with both NG-mono methyl-L-arginine and NG-nitro-L-arginine failed to prevent protein-rich plasma extravasation. 5. The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls. 6. Addition of 10 nM PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals. 7. Treatment with either UR-12460 or BB-823 prior to the induction of RPA produced an almost complete inhibition of NO production. 8. Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme. 9. Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR-12460-treated rats.10. These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA,and also that PAF is implicated in the triggering of long term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS. PMID- 7539701 TI - A randomized, crossover evaluation of methylphenidate in cancer patients receiving strong narcotics. AB - Sedation may be a dose-limiting side-effect of opioid therapy in some cancer patients. This study was designed to evaluate further the use of the psychostimulant, methylphenidate, an agent that has been reported to counter-act opioid-induced sedation, in patients with cancer-related pain. Patients receiving a stable dose of an opioid for cancer-related pain were recruited for this randomized, double-blind, crossover clinical trial. In addition to their regular dose of narcotics, they received 5 days of methylphenidate followed by 5 days of placebo, or vice versa. Our data did not definitively demonstrate any statistically significant benefit for methylphenidate, but did suggest that this drug could mildly decrease narcotic-induced drowsiness and could increase night time sleep. These data, in conjunction with other published data, suggest that methylphenidate can counteract narcotic-induced daytime sedation to a limited degree. PMID- 7539702 TI - Intrapleural mitoxantrone for the palliative treatment of malignant pleural effusions. AB - A group of 21 patients with malignant pleural effusions were treated with the instillation of 40 mg mitoxantrone intrapleurally, after complete fluid drainage. There was a 100% complete response rate with no toxic effects (0%). The mean survival of the patients was 57.1 weeks. PMID- 7539703 TI - Palliative care in Edmonton. AB - The Palliative Care Program at the Edmonton General Hospital, Edmonton, Canada provides a comprehensive multidisciplinary service to terminally ill patients in the area. The political, clinical, educational, and research developments impacting on this program are described. PMID- 7539705 TI - Demonstration of axon collateral projections from the substantia nigra pars reticulata to the superior colliculus and the parvicellular reticular formation in the rat. AB - It was revealed in the rat that single neurons in the substantia nigra pars reticulata (SNr) innervated both the superior colliculus (SC) and the parvicellular reticular formation (RFp) in the pons and medulla oblongata by way of axon collaterals. After injecting Fluoro-gold into the lateral part of the SC and Fluoro-ruby into the RFp on the same side, some SNr neurons were double labeled with both tracers. They were localized in the dorsolateral part of the caudal half of the SNr ipsilateral to the injection sites. PMID- 7539704 TI - Dermabrasive scar revision. Immunohistochemical and ultrastructural evaluation. AB - BACKGROUND: Dermabrasion of facial scars 4-8 weeks after injury frequently completely eliminates visible evidence of scar formation. However, efforts to define the cellular and structural mechanisms by which this phenomenon occurs have been limited in their success. OBJECTIVE: We investigated wound healing after dermabrasive scar revision. METHODS: The surgical scars of seven patients were abraded 6-8 weeks after injury. Comparative electron microscopic and immunohistochemical studies were performed on punch biopsy specimens taken before and after the dermabrasion. Ultrastructural changes in the basement membrane components and dermal structures were evaluated. Monoclonal antibody staining techniques were used to observe the presence, location, and temporal expression of tenascin, epiligrin, cadherins, and integrin subunits. RESULTS: We observed: 1) an increase in collagen bundle density and size with a tendency toward unidirectional orientation of fibers parallel to the epidermal surface, 2) an upregulation of tenascin expression throughout the papillary dermis, and 3) expression of alpha-6/beta-4 integrin subunit on the keratinocytes throughout the stratum spinosum. CONCLUSIONS: The mechanisms by which dermabrasive scar revision alters the events of primary cicatrix formation include modification of extracellular ligand expression, thereby influencing epithelial cell-cell interaction, and reorganization of connective tissue. PMID- 7539706 TI - Noxious peripheral stimulation produces antinociception mediated via substance P and opioid mechanisms in the rat tail-flick test. AB - Physiological experiments were run to examine the effects of noxious thermal stimulation of one hindpaw on the tail-flick reflex in the lightly anesthetized rat. Male Sprague-Dawley rats were anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, either a non-noxious or a noxious stimulus was applied which consisted of immersion of one hindpaw in water at 40, 45, 50 or 55 degrees C for 1.5 min. After immersion, tail-flick readings were taken at 3-min intervals for at least 16 min. Paw immersion in water at 55 degrees C induced an antinociceptive response, consisting of an increase in the reaction time, at 0.5 min after immersion. Recovery to baseline levels occurred over the next 3-6 min. Immersion at lower temperatures provoked smaller antinociceptive responses, except at 40 degrees C, where readings remained around the baseline values. The increase in reaction time in response to immersion at 55 degrees C was attenuated or blocked by the novel, nonpeptide substance P (NK-1) receptor antagonist, CP-96,345, administered s.c. 30 or 60 min, respectively, prior to paw immersion. Similar injection of CP-96,344, the inactive stereoisomer, had no effect on the response, while another NK-1 receptor antagonist, CP-99,994, also attenuated the antinociceptive effect of the immersion. The increase in reaction time induced by immersion at 55 degrees C was absent in animals treated neonatally with capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539707 TI - Protein-nucleic acid interactions. PMID- 7539708 TI - Structures of DNA and RNA polymerases and their interactions with nucleic acid substrates. AB - DNA and RNA polymerases are enzymes that are primarily responsible for copying genetic material in all living systems. The four polymerases whose structures have been determined by X-ray crystallographic methods have significant similarities at the polymerase active site that are indicative of common requirements for polynucleotide synthesis. Structural studies of complexes of the Klenow fragment of Escherichia coli DNA polymerase I, HIV type 1 reverse transcriptase, and rat DNA polymerase beta with DNA are leading to generalized models for catalysis. PMID- 7539709 TI - Nucleoside drug resistance in HIV-1 reverse transcriptase. PMID- 7539712 TI - Confusion of drug names signalled. PMID- 7539713 TI - Detection of sperm cells in the canine vagina. PMID- 7539711 TI - Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 poliovirus and its viral epitope. AB - The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the viral epitope has been determined at 3.0 A resolution. Although this antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I beta turns in series. At the carboxyl end, the peptide is bound snugly in the antibody combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between the peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization. PMID- 7539714 TI - Lewis Y antigen expression in hepatocellular carcinoma. An immunohistochemical study. AB - BACKGROUND: The altered expression of the Lewis blood group-related antigens during malignant transformation can be used clinically as a tumor marker or as a prognostic indicator. The Lewis Y (LeY) antigen, which is one of the Type 2 human blood group-related antigens, also is thought to behave as an oncodevelopmental cancer-associated antigen. In this study, the authors examined the association between human LeY antigen expression and the clinicopathologic features of HCC, including its proliferative activity. METHODS: Forty-six histologically confirmed cases of HCC were studied retrospectively. Liver biopsy specimens from the main tumor of each case were obtained under ultrasonic guidance before treatment was initiated. The formalin fixed, paraffin embedded serial sections were immunostained using a modification of the avidin-biotin-peroxidase complex method, with a primary monoclonal antibody (MoAb) directed against the LeY antigen (BM-1/JIMRO). The relationship between LeY antigen expression and the HCC's proliferative activity was analyzed similarly by immunohistochemical methods using a primary MoAb directed against the Ki-67 antigen (MIB 1). In addition, to clarify the relationship between LeY antigen expression and the histologic heterogeneity within HCC, seven cases of surgically resected HCC also were immunostained. RESULTS: The LeY antigen was detected on the membrane and in the cytoplasm of the cancer cells. Of the 46 HCC cases, 20 (43.5%) expressed the LeY antigen in the tumor cells. There was no correlation between LeY antigen expression and the maximum tumor dimension or the Stage. However, the incidence of LeY antigen-positive cases in poorly differentiated HCCs was found to be significantly higher than that in well or moderately differentiated HCCs (P < 0.01). In resected HCC cases, LeY antigen expression within HCC nodules was frequently greater in the less differentiated tumor than in adjacent differentiated tumor. Moreover, the incidence of LeY antigen expression in alpha fetoprotein (AFP)-positive (AFP > or = 200 ng/ml) HCC cases was significantly higher than that in AFP-negative (AFP < 200 ng/ml) HCC cases (P < 0.05). Furthermore, the mean value of the Ki-67 labeling index in LeY antigen-positive HCC cases (25.2 +/- 11.3%) was significantly higher than that in LeY antigen negative HCC cases (9.4 +/- 4.1%) (P < 0.001). CONCLUSIONS: These results suggest that LeY antigen expression correlated closely to the dedifferentiation and proliferative activity of HCC. PMID- 7539710 TI - Partially folded, molten globule and molten coil states of bovine pancreatic trypsin inhibitor. AB - Three denatured states of bovine pancreatic trypsin inhibitor have been characterized, using two chemically synthesized analogues designed for study of folding intermediates. One analogue, [14-38]Abu, retains only the 14-38 disulphide. At pH 4.5-6 and 1-7 degrees C, [14-38]Abu is a highly ordered beta sheet molten globule; it has the circular dichroism (CD), ANS-binding and folding kinetics of a molten globule; is partially folded by NMR analysis; and undergoes cooperative thermal denaturation. At low temperature [14-38]Abu also forms an acid state at pH 1.5, as well as a denatured state at pH 2.5. A second BPTI analogue with all three disulphide bridges eliminated, [R]Abu, lacks detectable secondary and tertiary structure but has stable hydrophobic surfaces and is collapsed. We term this species a 'molten coil'. PMID- 7539715 TI - Expression of tumor-associated polymorphic epithelial mucin and carcinoembryonic antigen in gastrointestinal carcinoid tumors. Implications for immunodiagnosis and immunotherapy. AB - BACKGROUND: Gastrointestinal neoplastic epithelium of glandular origin commonly produces N-linked and O-linked glycoproteins such as carcinoembryonic antigen (CEA) and tumor-associated polymorphic epithelial mucin (PEM). Antibodies to these glycoproteins increasingly are being used in immunodiagnosis and/or immunotherapy. Although GI carcinoid tumors have a neuroendocrine immunophenotype and generally have an indolent clinical course compared with their adenocarcinomatous counterparts, they also arise from undifferentiated crypt epithelium. The purpose of this study was to determine whether GI carcinoids similarly expressed epitopes for CEA and PEM. METHODS: Thirty-nine GI carcinoid tumors from various topographic sites were analyzed by immunohistochemistry using the murine monoclonal antibodies B72.3, ACT19, and T84. The former two antibodies recognize epitopes in PEM, whereas the latter is an anti-CEA antibody, which was confirmed using enzyme-linked immunosorbent assays. RESULTS: The majority of carcinoid tumors (74%), particularly jejunoileal carcinoids, reacted for ACT19 antibody, whereas considerably fewer reacted for B72.3 (31%) and T84 (26%). The extent of staining was also greatest with ACT19. The GI mucosa adjacent to or overlying the carcinoid tumors also stained for the various antibodies. CONCLUSIONS: The extent and degree of positivity of carcinoid tumors for ACT19 raises the possibility that this antibody may be used in the future for the radioimmunodiagnosis and/or immunotherapy of these tumors, particularly in cases that are multicentric, unresectable, or with metastatic disease. PMID- 7539716 TI - Biphasic synovial sarcoma in the small intestinal mesentery. AB - BACKGROUND: A 46-year-old man presented with recurrent anemia and polyarthralgia. Investigations revealed a mass in the ileal mesentery, which was resected. Results of routine histologic examination suggested a diagnosis of synovial sarcoma, a rare malignancy usually not reported at this site. METHODS: Tissue was examined immunohistochemically, ultrastructurally, and by fluorescent in situ hybridization to confirm the diagnosis. RESULTS: Immunohistochemical studies revealed widespread labeling for cytokeratins and focal labeling for desmin and vimentin in the epithelial component, with labeling for epithelial membrane antigen in the epithelial and spindle-cell components. Fluorescent in situ hybridization analysis showed the characteristic t(X;18) translocation of synovial sarcoma. CONCLUSIONS: This is a unique case of synovial sarcoma in the small intestinal mesentery. Immunohistochemical labeling confirmed the diagnosis, although, to the authors' knowledge, the pattern of desmin labeling has not been described previously. The clinical association with polyarthralgia, which resolved after removal of the neoplasm, also has not been described previously. PMID- 7539717 TI - Structural studies of the O-specific chain and a core hexasaccharide of Hafnia alvei strain 1192 lipopolysaccharide. AB - The structure of the O-specific side-chain and a core hexasaccharide of the Hafnia alvei strain 1192 lipopolysaccharide has been investigated. Methylation analysis, NMR spectroscopy, MALDI-TOF spectrometry, and various specific chemical degradations were the principal methods used. It is concluded that the polysaccharide is composed of hexasaccharide repeating-units having the following structure which is partially O-acetylated in the 2-position of the --> 4)-alpha-D Glc pA-(1-->(70%) and on different positions of the L-Rha residues (50%). [Formula: see text] The core hexasaccharide was found to have the following structure: [Formula: see text] PMID- 7539718 TI - The structure of the O-specific polysaccharide from Thiobacillus sp. IFO 14570, with three different diaminopyranoses forming the repeating unit. AB - The O-specific polysaccharide, liberated by mild acid hydrolysis of the lipopolysaccharide (LPS-P) from Thiobacillus sp. IFO14570 as isolated from the phenol phase after phenol-water extraction, is shown to have a linear trisaccharide repeating-unit containing three different diamino sugars, namely 2,4-diacetamido-2,4-dideoxyglucuronic acid, 2-acetamidino-4-acetamido-2,4,6 trideoxyglucopyranose, and 2,3-diacetamido-2,3-dideoxyglucuronic acid in the molar ratio of 1:1:1. On the basis of 1H and 13C spectroscopy, including 2D COSY and coherent transfer (RCT)COSY, 1D NOE in the difference mode, and 2D rotating frame NOE(ROESY) the sequence, the type of substitution, and the position of the acetamidino group could be determined. These experiments allowed formulation of the following structure for the O-specific polysaccharide, although the depicted D configuration of the three sugar residues is not yet proven: [structure: see text] PMID- 7539719 TI - Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. AB - Peripheral glia help ensure that motor and sensory axons are bathed in the appropriate ionic and biochemical environment. In Drosophila, peripheral glia help shield these axons against the high K+ concentration of the hemolymph, which would largely abolish their excitability. Here, we describe the molecular genetic analysis of gliotactin, a novel transmembrane protein that is transiently expressed on peripheral glia and that is required for the formation of the peripheral blood-nerve barrier. In gliotactin mutant embryos, the peripheral glia develop normally in many respects, except that ultrastructurally and physiologically they do not form a complete blood-nerve barrier. As a result, peripheral motor axons are exposed to the high K+ hemolymph, action potentials fail to propagate, and the embryos are nearly paralyzed. PMID- 7539720 TI - The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. AB - Axonal transport has been intensively examined as a good model for studying the mechanism of organelle transport in cells, but it is still unclear how different types of membrane organelles are transported through the nerve axon. To elucidate the function of this mechanism, we have cloned KIF1A, a novel neuron-specific kinesin superfamily motor that was discovered to be a monomeric, globular molecule and that had the fastest reported anterograde motor activity (1.2 microns/s). To identify its cargo, membranous organelles were isolated from the axon. KIF1A was associated with organelles that contained synaptic vesicle proteins such as synaptotagmin, synaptophysin, and Rab3A. However, this organelle did not contain SV2, another synaptic vesicle protein, nor did it contain presynaptic membrane proteins, such as syntaxin 1A or SNAP-25, or other known anterograde motor proteins, such as kinesin and KIF3. Thus, we suggest that the membrane proteins are sorted into different classes of transport organelles in the cell body and are transported by their specific motor proteins through the axon. PMID- 7539721 TI - Fungal metabolites. XVIII. New membrane-modifying peptides, trichorozins I-IV, from the fungus Trichoderma harzianum. AB - New membrane-modifying peptides, trichorozins I-IV, have been isolated from conidia of the fungus Trichoderma harzianum. Their amino acid sequences were clearly determined by spectrometric methods and, furthermore, they were synthesized by the solution-phase method. Trichorozins are a family of the class of peptaibols and are composed of 11 residues including an amino alcohol. Trichorozins exhibited voltage-dependent ion channel-like activity in lipid bilayers. PMID- 7539722 TI - Absolute stereostructures of hovenidulciosides A1 and A2, bioactive novel triterpene glycosides from hoveniae semen seu fructus, the seeds and fruit of Hovenia dulcis Thunb. AB - Two bioactive novel triterpene glycosides named hovenidulciosides A1 and A2 have been isolated from a Chinese natural medicine, Hoveniae Semen Seu Fructus, the seeds and fruit of Hovenia dulcis Thunb. (Rhamnaceae). The absolute stereostructures of hovenidulciosides A1 and A2 with a migrated 16,17-seco dammarane skeleton have been determined on the basis of chemical and physicochemical evidence which included the X-ray crystallographic analysis of the p-bromobenzoate of their common aglycone, hovenidulcigenin A. Hovenidulciosides A1 and A2 exhibited inhibitory activity on the histamine release from rat mast cells induced by compound 48/80 or calcium ionophore A 23187. PMID- 7539723 TI - Inhibition of IL-4 receptor up-regulation on B cells by antisense oligodeoxynucleotide suppresses IL-4-induced human IgE production. AB - IL-4 is shown to up-regulate its own receptor (IL-4R) on human lymphocytes, but the functional significance of up-regulated IL-4R is not clear regarding IgE production. This study investigated the possible role of IL-4-induced up regulation of IL-4R on B cells in the induction of human IgE synthesis by means of antisense strategy. Among three antisense oligodeoxynucleotides designed against the downstream of translation initiation site of IL-4R cDNA, S-oligo 1, complementary to nucleotide 1-24, showed the strongest inhibition of the constitutive expression of IL-4R on Daudi cells. Addition of S-oligo 1 together with IL-4 also decreased the up-regulated but not constitutive levels of IL-4R on peripheral blood B cells without affecting the concomitant enhancement of CD23, CD40, HLA-DR and surface IgM expression, indicating that its effect is specific for IL-4R up-regulation. When S-oligo 1 was added to B cells costimulated with IL 4 and anti-CD40 MoAb, it induced a dose-dependent inhibition of IgE production. This inhibition was accompanied by a decrease in the expression of mature C epsilon transcripts, whereas the accumulation of germ-line C epsilon transcripts was not affected by S-oligo 1. These data suggest that the signal transduction mediated by the up-regulated IL-4R on B cells may be intimately associated with the induction of isotype switching to IgE that leads to mature C epsilon transcription and IgE production. PMID- 7539724 TI - An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes. AB - Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4 associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes. PMID- 7539726 TI - Suppression of haematopoiesis by IgG autoantibodies from patients with systemic lupus erythematosus (SLE). AB - The inhibiting activity of serum on haematopoiesis has been described in patients with SLE. To explore further the features of serum inhibitor, we first examined the suppression of granulocytic and erythroid colony formation in vitro by serum from patients with SLE using methylcellulose culture. The potent inhibiting activity was demonstrated in six of 20 patients. All of these six patients were associated with leukocytopenia and/or anaemia. Five of 10 sera from patients with active SLE suppressed the colony formation of both burst-forming units of erythrocyte (BFU-E) and colony-forming units of granulocyte/macrophage (CFU-GM), and one serum suppressed BFU-E only. IgG fraction isolated from sera with inhibiting activity suppressed colony formation without complement involvement. The elimination of monocytes and lymphocytes from target mononuclear cells did not affect the suppression by the IgG fractions. The suppressive effect was completely eliminated after incubation of the IgG fractions with progenitor enriched mononuclear cells. Flow cytometric analysis showed these IgG bound to CD34+ haematopoietic progenitor cells, but not to CD33+ cells. These data suggest that (i) the inhibitor of colony formation in serum was observed in IgG fraction; (ii) its suppressive effect on colony formation was mediated by neither monocytes and lymphocytes nor complements; and (iii) IgG fraction could bind to primitive haematopoietic progenitor cells and suppress the growth of these cells. Thus, IgG autoantibodies to primitive haematopoietic progenitor cells are demonstrated to be present in the sera of a significant proportion of active SLE patients with anaemia and leukocytopenia and to suppress the progenitor cell growth. PMID- 7539725 TI - Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIV-infected subjects. AB - The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage-related fashion. CD28- CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL-2R alpha. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co-stimulation with cell-bound anti-CD3 and B7. Nevertheless, when stimulated with plate-fixed anti-CD3, CD8+ T cells from HIV infected subjects proliferated normally, and normal levels of IL-2R alpha and transferrin-receptor could be induced on CD28- CD8+ T cells from the patients. In addition, stimulation with plate-fixed anti-CD3 induced proliferative responses in highly purified CD28- CD8+ T cells from both HIV- and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti-CD3 redirected assay, was predominantly exerted by CD28- CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down-regulation and were slightly hyporesponsive to B7 co-stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co-stimulation via B7. CD28- CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7 independent stimulation. PMID- 7539727 TI - Ketotifen therapy in chronic graft-versus-host disease (cGVHD): effect on mast cells and fibroblasts. AB - Current treatment options for cGVHD are limited. Mast cells (MC) and fibroblasts have been shown to play a role in the murine model of cGVHD. Ketotifen is an anti H-1 antihistamine with MC-stabilizing properties. We therefore treated eight patients with cGVHD with ketotifen (6 micrograms/day for 3 months). Three additional age- and sex-matched cGVHD patients served as controls. MC count and activation state in cGVHD skin biopsies and the in vitro effect of peripheral blood mononuclear cell (PBMC) supernatants on (i) histamine release by MC; (ii) 3T3-fibroblast proliferation; and (iii) prostaglandin E2 (PGE2) production, were evaluated. Ketotifen therapy resulted in clinical improvement in 4/8 patients, stabilization of the disease in 2/8, while in 2/8 patients the cGVHD progressed and they died of bacterial sepsis. Side effects were minimal. In the skin biopsies the number of MC was found to be 0.58 +/- 0.17 (n = 8) (field x 400) and the MC looked degranulated (toluidine blue staining). Following ketotifen therapy MC number was increased to 1.2 +/- 0.28 (n = 8) (P < 0.05). PBMC supernatants of cGVHD patients stimulated histamine release from cultured rat MC (n = 8) (2.7 +/- 0.5 micrograms/ml; normal values are 2.1 +/- 0.4 micrograms/ml, n = 5). Ketotifen therapy reduced the histamine release level to the normal range (2.0 +/- 0.5 micrograms/ml, P < 0.05) (n = 8). Ketotifen therapy had no significant effect on: (i) 3T3 fibroblast proliferation which was suppressed by cGVHD PBMC supernatants; (ii) the elevated PGE2 production which we observed when fibroblasts were incubated with the PBMC supernatants. These results indicate that ketotifen may play a therapeutic role in cGVHD. PMID- 7539728 TI - [Human peroxisome-deficient disorders and pathogenic gene]. AB - Peroxisome is a model organelle to investigate the mechanism of protein translocation and organelle assembly. Human autosomal recessive peroxisomal disorders are of clinical consequence and a model system to study the biogenesis and physiological significance of peroxisomes. In patients with generalized peroxisomal disease such as cerebrohepatorenal Zellweger syndrome where peroxisomes are morphologically absent, all peroxisomal proteins appear to be normally synthesized but assembly of peroxisomes is impaired. Thus far, nine complementation groups have been reported for these peroxisome-deficient disorders including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. To investigate the molecular mechanism of peroxisome biogenesis and the primary defect of peroxisomal disorders, we have thus far isolated three different complementation groups of Chinese hamster ovary (CHO) cell mutants defective in biogenesis of peroxisomes. By genetic functional complementation analysis following the transfection of cDNA library to one of these cell mutants, Z65, we identified 35-kDa peroxisome assembly factor-1 (PAF 1) essential for peroxisome assembly. Moreover, we delineated the primary defect in a Zellweger patient who belonged to the same complementation group as Z65. The cause of this syndrome was a homozygous nonsense point mutation at 119Arg in PAF 1 gene. Comparison of PAF-1 sequences from rat, human, and Chinese hamster revealed that PAF-1 is highly conserved through the evolution and contains a novel cysteine-rich zinc finger, RING finger motif. PMID- 7539729 TI - Congenital cyclic neutropenia. PMID- 7539730 TI - Treatment options for hypoplastic left heart syndrome: a mother's perspective. AB - The congenital heart defect of HLHS is nearly uniformly fatal without intervention. As surgeons gain experience with the techniques the success rates are improving; more infants with HLHS are being offered hope for survival and quality of life. The critical nature of this diagnosis, the relatively new treatment options, and uncertainty of the surgical outcome impose tremendous stress on the parents. Choice of treatment is difficult and should be made in collaboration with the attending physician. Although the first year of my son's life was difficult, the past 5 years have been wonderful. He continues to thrive and enjoy most of the activities other 6-year-olds enjoy. In retrospect, palliative surgery was a good option for him. As both his mother and a cardiac nurse, I recognize that he may once again require surgical intervention, but I am encouraged that medical and surgical advances are being made for children with HLHS. PMID- 7539731 TI - Fas-based T cell-mediated cytotoxicity. PMID- 7539732 TI - Effect of sorbitan monolaurate polyoxyalkylene (Tween 20) on the ultrastructure of some bacteria. AB - The ultrastructure of Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa treated with 0.5 and 1% Tween 20 was studied by means of transmission and scanning electron microscopy. Specific changes in the cell wall and cytoplasmic membrane were observed. PMID- 7539733 TI - Reversal of refractory hypotension in septic shock by inhibitor of nitric oxide synthase. AB - Septic shock is a life-threatening condition that results from exposure to bacterial endotoxin. It is mediated by the release of cytokines. Some of these cytokines cause the release of vasoactive substances. We report the case of a 62 year-old male patient who received redo operation for replacement of the degenerative porcine aortic and mitral prostheses. High cardiac output shock developed on the seventh postoperative day with severe metabolic acidosis and oliguria. Systemic vascular resistance and mean arterial pressure elevated within 5 min and stabilized 60 min after the start of a single dose of intravenous administration of NG-monomethyl-L-arginine (50 mg), a potent and selective inhibitor of nitric oxide synthesis. These findings indicate that nitric oxide overproduction is an important contributor to refractory hypotension in high cardiac output septic shock. Our findings suggested the utilization of nitric oxide synthase inhibitor in the treatment of septic shock in humans. PMID- 7539734 TI - [The experimental study of anti-human AFP variant McAb in radioimmuno-detection and radioimmuno-therapy of human hepatocellular carcinoma model in nude mice]. AB - Anti-human AFP variant monoclonal antibody (AFP-R-LCA McAb) was labeled by 131I and injected intraperiton ealy. The 131I-AFP-R-LCA McAb was gathered in tumor of nude mice bearing human hepatocellular carcinoma (HCC). The tumor/liver radioactivity redio was 5.2, but normal mice IgG labeled by 131I-(131-mIgG) or free 131I was not gathered in tumor and distributed everywhere in average. The group of 131I-AFP-R-LCA McAb was positively visualized by gamma camea. The test showed that 131I-AFP-R-LCA McAb cured the nude mice bearing HCC. The tumor inhibition rate in 131I-AFP-R-LCA McAb group at the 4th week after treatment was significantly higher than those of the other groups (85%, 45%, 20%, 7% respectively, P < 0.05). The result showed that the AFP-R-LCA McAb have strong affinity and special combination to HCC cells and it can be recognized as a carrier for radioimmunodetection and radioimmunotherapy. PMID- 7539735 TI - [The prognostic value of focusylated alpha-fetoprotein measurement in liver cancer patients after surgery]. AB - Postoperative change of focusylation index (FI) of serum alpha-fetoprotein (F AFP) and the result of surgery were studied in 34 patients of hepatocellular carcinoma. In 23 patients, serum F-AFP was disappeared with serum AFP turning negative and no tumor recurrence heppaned within the first 6 months postoperatively. In 4 patients, owing to hepatitis or active cirrohosis in the remained liver, serum AFP didn't turn negative, but FI of AFP was obviously decreased, follow-up found no signs of tumor recurrence within the first 6 months. In 5 of 7 patients, with decreased postoperative serum AFP levels and unchanged FI, tumor recurred. These results showed that postoperative F-AFP monitoring is helpful in evaluating surgical efficacy in liver tumor producing AFP. PMID- 7539736 TI - [Established oneself the detection of serum prostate specific antigen and application in clinical practice]. AB - Serum prostate specific antigen (PSA)-the biotinavidin enzyme immunoassay (BA ELISA) was established and used in clinical practice. We assayed serum PSA in normal control and prostatic patients. If 10ng/ml was taken as the cutoff value, the positive rate of prostatic cancer was 62.96%. The results of determination showed: PSA serum levels of patients with complicated BPH (acute prostatitis and chronic prostatitis) were conspicuously higher than the noncomplicated BPH. In the BPH group, we simultaneously detected serum levels of PSA by BA-ELISA and Tandem-E PSA Immuno Enzy Metric Assay. The results suggested BA-ELISA technique is more sensitive and more economical than Tandem-E PSA Immuno Enzy Metric Assay for clinical use. PMID- 7539737 TI - [Clinical significance of urodynamic study in benign prostatic hyperplasia]. AB - Urodynamic study, including UFR, cystometry, UPP, and EMG was carried out in 74 patients with benign prostatic hyperplasia (BPH) during the past 5 years. The correlations of prostate volume (57.3 +/- 6.3 ml), prostate length (4.94 +/- 1.23 cm) and detrusor pressure (6.54 +/- 4.73 kPa) were analysed. The results showed that urodynamic study plays an important role in indicating the time and method of operative treatment and predicting the prognosis of BPH operation. The fistularization of the bladder should be performed on the patients with the bladder disfunction. The measurement of the urethral resistance is of value in the diagnosis of posterior urethra obstruction than that of UFR. PMID- 7539738 TI - Role of AgNOR in diagnosis of thyroid follicular neoplasms on fine-needle aspiration smears. AB - This is a retrospective study to examine the role of AgNOR in differentiating thyroid follicular carcinoma and adenoma preoperatively on fine-needle aspiration (FNA) smears. Nineteen histopathology-proven cases of follicular adenoma and carcinoma were selected for this study. May-Grunwald-Giemsa-stained smears were destined and AgNOR staining technique was employed. Significant differences of AgNOR values were observed among follicular carcinoma versus adenoma (P < .05) and follicular carcinoma versus control cases (P < .01). Thereby the AgNOR technique may have some role in differentiating between benign and malignant follicular neoplasms of thyroid. PMID- 7539739 TI - Non-small-cell lung carcinoma. Current and future therapeutic management. AB - Non-small-cell lung cancer (NSCLC) is one of the most common malignancies in developed countries and accounts for millions of deaths worldwide. The incidence of NSCLC correlates with smoking tobacco and is likely to increase in those countries with increasing per capita consumption of tobacco. NSCLC is particularly associated with morbidity--in the US several published clinical studies involving a variety of intensive surgical, chemotherapeutic and radiotherapeutic interventions have produced only a 10% 5-year survival. New technologies and breakthroughs in treatment notwithstanding, the current emphasis in clinical trials is to refine and optimise available therapies to maximise patient disease-free and overall survival. These accruing multi-institutional clinical trials may offer surgical resection with or without neoadjuvant therapy, multiagent chemotherapy, and/or standard or hyperfractionated radiation therapy. Since most NSCLC patients have inoperable disease, the most active current trial design entails chemoradiotherapy, since previous trials appear to suggest superior results to radiotherapy alone in these patients. PMID- 7539740 TI - Bombesin down modulates pulmonary fibrosis elicited in mice by bleomycin. AB - The role of bombesin was investigated in the course of the pulmonary inflammation and fibrosis (PF) elicited by the intratracheal (IT) or intravenous (i.v.) administration of bleomycin in mice. Bleomycin-induced alveolitis was associated with an accumulation of cells, presumably macrophages, containing bombesin, as evidenced by immunohistochemistry. Administration of bombesin by an osmotic minipump implanted IP, at a rate of 6 micrograms/h, decreased the lung hydroxyproline evident 15 days after IV or IT administration of bleomycin. In contrast, antibombesin monoclonal antibody 2A11 (mAb) increased the lung hydroxyproline content after IT administration of bleomycin. In addition, the mortality of bleomycin-injected mice was increased by the anti-bombesin mAb. Bombesin administration induced an increase, and anti-bombesin mAb induced a decrease, in the number of macrophages recovered from the bronchoalveolar lavage. Administration of bombesin did not change the mRNA levels of TNF-alpha, TGF-beta, and PDGF, as seen on Northern blots made with the lung RNA. Pulmonary platelet trapping, which was increased by a bleomycin injection, was decreased by an infusion of bombesin as demonstrated by the distribution of 111In-labeled platelets. This study indicates that bombesin act as an inhibitor of the development of pulmonary fibrosis, possibly by decreasing pulmonary platelet trapping. PMID- 7539741 TI - Amelioration of bleomycin-induced lung fibrosis by treatment with the platelet activating factor receptor antagonist WEB 2086 in hamsters. AB - Therapeutic use of bleomycin, an antineoplastic drug, is complicated by the development of a dose-dependent lung toxicity leading to fibrosis. This study tested the effectiveness of a platelet activating factor (PAF) receptor antagonist, WEB 2086, against bleomycin (BLEO)-induced lung fibrosis in hamsters. The animals were assigned to four groups: (1) saline (SA) + SA, (2) WEB 2086 (WEB) + SA, (3) SA + BLEO, and (4) WEB + BLEO. Sterile isotonic saline or WEB 2086 (10 mg/kg IP) was administered daily for the duration of the study starting 2 days prior to intratracheal (IT) instillation of saline or bleomycin (2.5, 2.0, and 1.5 units/kg 5 mL-1) in three consecutive doses at weekly intervals. The animals were killed at 21 days after the last IT instillation and their lungs were processed for various studies. The lung hydroxyproline levels in SA + SA, WEB + SA, SA + BLEO, and WEB + BLEO groups were 932 +/- 31, 943 +/- 48, 1302 +/- 72, and 964 +/- 63 micrograms/lung, respectively. The lung myeloperoxidase (MPO) activity and malondialdehyde equivalent, an index of lipid peroxidation, in the corresponding groups were 10 +/- 2, 8 +/- 2, 14 +/- 3, and 5 +/- 1 units/lung and 93 +/- 7, 77 +/- 5, 102 +/- 8, and 75 +/- 6 nmol/lung, respectively. The lung prolyl hydroxylase activity in the WEB + SA, SA + BLEO, and WEB + BLEO groups was 130.1 +/- 7.7, 236.2 +/- 12.8, and 138.1 +/- 7.0% of the SA + SA control group (8.3 x 10(4) dpm/lung 30 min-1), respectively. Daily treatment with WEB 2086 caused significant (p < or = .05) reductions in the BLEO-induced increases in the lung hydroxyproline content, prolyl hydroxylase and MPO activities, lipid peroxidation, and acid phosphatase activity of the BALF supernatant. Although daily treatment with WEB 2086 reduced the bleomycin-induced increases in the BALF total and neutrophil cell counts, BALF supernatant protein, and morphometric estimates of the lesions, these parameters were not significantly different from those of the SA-BLEO group. Histopathologic studies revealed that there were no lesions of alveolar consolidation and fibrosis in the lungs of WEB + BLEO group as compared with the SA + BLEO group. The results suggest that PAF is involved in the BLEO-induced lung fibrosis and that PAF-receptor antagonist may therefore be potentially useful in the attenuation of lung fibrosis caused by bleomycin. PMID- 7539742 TI - Misplaced iron in kwashiorkor. AB - OBJECTIVES: To determine the presence of radical promoting iron (non-protein bound or loosely bound or free iron) in the plasma of children with kwashiorkor. DESIGN: The bleomycin assay was employed for the quantitation of free or loosely bound iron. SETTING: The Red Cross War Memorial Children's Hospital, Cape Town, Tertiary Care. SUBJECTS: Fifty children on admission with kwashiorkor: six with marasmus and twelve healthy well-nourished controls. RESULTS: Non-protein-bound iron was detected in the plasma of 58% of children with kwashiorkor but was absent in marasmic and healthy well-nourished children. CONCLUSIONS: The presence of radical promoting iron supports the hypothesis that a free radical injury probably plays a role in the pathogenesis of kwashiorkor and its removal may improve mortality. PMID- 7539743 TI - Gene and genome scanning by two-dimensional DNA typing. AB - A major effort in the analysis of DNA currently focuses on identifying genes and their pathological variants underlying disease. Once such disease genes have been isolated a major task of molecular medicine is to identify the spectrum of DNA sequence variations responsible for the aberrant function of such genes. These efforts, however, are hindered by the vast amount of genetic information to scan for variations and the limited capacity of analytical techniques in terms of accuracy and speed. Recently, a number of techniques were developed, so-called "genome scanning" techniques, which allow complete genomes to be analyzed for sequence variation in parallel, i.e., at multiple sites or loci simultaneously rather than serially at predefined loci. Here we present the background and applications of a particular electrophoretic parallel processing approach, generically termed two-dimensional DNA typing. The approach is based on separating DNA fragments by two-dimensional electrophoresis [1], including denaturing gradient gel electrophoresis, thus allowing hundreds of fragments to be simultaneously assessed by comparative analysis for variations in size and sequence. The method is suitable for hybridization analysis with locus-specific and multilocus probes of genomic DNA restriction fragments derived from human and other DNA, and for analysis of polymerase chain reaction (PCR) fragments derived from large genes. Two-dimensional DNA typing has been applied, e.g., in linkage analysis of pedigrees, analysis of tumor genomes for rearrangements, and to scan the cystic fibrosis transmembrane regular gene for sequence variations such as point mutations. PMID- 7539745 TI - In young primary cultures of rabbit kidney cortical collecting ducts intercalated cells originate from principal or undifferentiated cells. AB - The evolution of a primary culture of rabbit kidney cortical collecting tubule was followed over a period of 10 to 11 days. The cell types of this segment were characterized by using monoclonal antibodies, specifically directed against principal (Mab 703) and intercalated (Mab 503) cells of the apical membrane. The activity of a H+ pump ATPase was revealed in Mab 503-labeled cells, confirming that these cultured cells present characteristics of intercalated cells. The primary culture was also stained with peanut agglutinin (PNA), a specific ligand of beta intercalated cells. During the first two days, some cells, mainly Mab 503 labeled cells, disappeared, and cell division did not occur. At 2 days, the culture showed 80% and 18% of Mab 703-labeled and Mab 503-labeled cells, respectively. The first mitoses were observed at 2 days. From two to four days, cell division was nestly in Mab 703-labeled cells and only rarely seen in Mab 503 labeled cells, although during this period the proportion of Mab 703-labeled cells decreased to 44% of cells and that of Mab 503-labeled cells increased to 30%. The labeling with PNA was curious. Up to 2 days, PNA stained Mab 503-labeled cells, but from 4 days it stained other cells, probably dedifferentiated ones. In our culture conditions types of cells other than Mab 703-labeled and Mab 503 labeled cells occurred. First, throughout the life of the culture, some cells were not recognized by any monoclonal antibody; their number varied between 10 and 28% of the total cell number.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539744 TI - Fusion of SYT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. AB - We demonstrate that the cytogenetically defined translocation t(X;18)(p11.2;q11.2) found in human synovial sarcoma results in the fusion of the chromosome 18 SYT gene to either of two distinct genes, SSX1 or SSX2, at Xp11.2. The SSX1 and SSX2 genes encode closely related proteins (81% identity) of 188 amino acids that are rich in charged amino acids. The N-terminal portion of each SSX protein exhibits homology to the Kruppel-associated box (KRAB), a transcriptional repressor domain previously found only in Kruppel-type zinc finger proteins. PCR analysis demonstrates the presence of SYT-SSX1 or SYT-SSX2 fusion transcripts in 29 of 32 of the synovial sarcomas examined, indicating that the detection of these hybrid transcripts by PCR may represent a very useful diagnostic method. Sequence analysis has demonstrated heterogeneity in the fusion transcripts with the formation of two distinct SYT-SSX1 fusion junctions and two distinct SYT-SSX2 fusion junctions. PMID- 7539746 TI - Effects of brefeldin A on the formation of the cell plate in tobacco BY-2 cells. AB - Treatment with 20 microM brefeldin A (BFA) for 60 min caused the disassembly of the Golgi apparatus in tobacco BY-2 cells, and the effect of BFA was reversible. Connections between Golgi cisternae and the ER were observed in cells that had been treated for 15 min with BFA. BFA applied to cells at metaphase allowed the cells to form aniline blue-positive cell plates but not to complete cytokinesis. BFA seems to inhibit cytokinesis by shutting off the supply of cell-plate materials by disassembling the Golgi apparatus. PMID- 7539748 TI - Co-expression of B7-1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response. AB - Although the transfection of B7-1 cDNA into a few mouse tumor cell lines can induce anti-tumor T cell immunity, its expression alone is ineffective in many other tumor cell lines tested. We were interested to study what factors limit B7 1 co-stimulatory activity, and decided to investigate whether B7-1 requires the cooperation of ICAM-1 to provide the minimal co-stimulatory signal for establishing an efficient anti-tumor immunity. We show that the transfection of B7-1 cDNA into three ICAM-1+ (plasmocytoma J558L, T lymphomas EL-4 and RMA), but not into two ICAM-1- tumors cell lines (adenocarcinoma TS/A and melanoma B16.F1), is sufficient to induce their complete rejection in syngeneic mice. The expression of ICAM-1 is necessary for the rejection of the B7 expressing tumors, since the primary response elicited by B7-1+ EL-4 and RMA clones expressing reduced levels of ICAM-1 is severely reduced. Furthermore, super-transfection of ICAM-1 cDNA into B7-1+ adenocarcinoma and melanoma clones optimizes their primary rejection. Histologic examination of transfected tumors reveals that B7-1 and ICAM-1 exert a potent pro-inflammatory activity. The intra-tumor infiltration is composed of both eosinophils and lymphomonocytes, and is already massive 5 days after the tumor challenge. The primary rejection of the B7-1+ ICAM-1+ tumors depends critically on CD8+ T cells, natural killer cells and granulocytes, but is independent of CD4+ T cells. Remarkably, in addition to its effects on the early phases of the immune response, the co-expression of ICAM-1 and B7-1 on tumors is also necessary for the efficient induction of a memory response. In fact, only the primary challenge with B7-1+, ICAM-1+ tumor cells protects the majority of the mice from a second injection of parental tumor cells. Collectively, our findings indicate that B7-1 and ICAM-1 are fundamental components for triggering the primary rejection of tumors and establishing a protective memory response. These findings may help to define new strategies for the rational application of co-stimulation in tumor immunotherapy. PMID- 7539747 TI - Changes in immunocytochemical detectability of proteasome epitopes depending on cell growth and fixation conditions of lung cancer cell lines. AB - The localization of proteasome epitopes in the lung cancer cell lines NCI-H82, derived from a small cell lung cancer, and MR65, derived from a squamous cell lung carcinoma, was studied in relation to cell growth conditions. For this purpose the proteasome monoclonal antibodies MCP34 and MCP20 were applied to the cells growing under different nutritional conditions, resulting in different proliferative states. Using indirect immunofluorescence microscopy with brief fixation in methanol (5 sec, -20 degrees C) followed by three dips in acetone (5 sec at room temperature), it became obvious that the intracellular detectability of the proteasomes changes depending on the nutritional and proliferative status of the tumor cells. Two types of experiments were carried out: (1) cells were grown for two days at different cell densities, with an excess of culture medium, and (2) cells were seeded in a low cell density and monitored for 6 days without change of medium. In cells grown at low density, the proteasomes can be detected mainly in the nuclei, while the nucleoli are almost devoid of staining, and the cytoplasm is only slightly stained. In cells grown at high density, the staining pattern changes with a much less pronounced nuclear staining than in the cells at low density, while the cytoplasm remains slightly stained. In the nutrient depletion experiment similar changes were seen. In cells growing under favorable conditions (1 or 2 days in fresh medium) proteasomes are detected mainly in the nuclei, whereas when the medium becomes depleted of nutrients (4 or 5-day-old medium) the staining pattern changes to one with a much less pronounced nuclear staining. However, in immunofluorescence studies on cells grown under similar conditions but fixed in ethanol (-20 degrees C) for 15 min, the changes in proteasome localization pattern were not detected during medium depletion. Using this fixation protocol the proteasomes are detected mainly in the nuclei at all stages of the medium exhaustion experiment. These apparently contrasting results suggest that upon nutrient depletion the proteasome epitopes become less accessible to the antibodies used. Apparently, the epitopes can regain accessibility if an extended ethanol fixation is used. This hypothesis was confirmed by flow cytometry and immunoblotting experiments. In flow cytometry of ethanol-fixed cells the fluorescence intensity of only a minor part of the cell population decreases to some extent with medium depletion, but in the majority of the cells fluorescence remains at its initial level. The immunoblotting experiments show no quantitative changes in proteasome content of the tumor cells at the different growth conditions. PMID- 7539751 TI - Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand. AB - Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells. PMID- 7539749 TI - Anergy induction in encephalitogenic T cells by brain microvessel endothelial cells is inhibited by interleukin-1. AB - Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) which can be induced, in susceptible strains like Lewis rats, by transfer of activated myelin basic protein (MBP)-specific CD4+ T lymphocytes. The role of cerebral endothelium in the onset of EAE, with regard to adhesion, activation and infiltration in the CNS of encephalitogenic T lymphocytes, is not fully understood. When pretreated by interferon-gamma, the immortalized Lewis rat brain microvessel endothelial (RBE4) cells expressed major histocompatibility complex class II molecules and stimulated MBP-specific proliferation and cytolytic activity of the syngeneic encephalitogenic T cell line, designated PAS. However, RBE4-stimulated PAS lymphocytes subsequently entered an unresponsive state, known as anergy. When inoculated in syngeneic animals, anergic PAS cells, although still cytotoxic, failed to induce EAE, and no cell infiltration was detectable within CNS. The addition of interleukin-1 beta (IL-1 beta) during MBP presentation by RBE4 cells prevented T cell anergy induction, and maintained T cell encephalitogenicity, although PAS cells stimulated in these conditions caused delayed and attenuated clinical signs of EAE, with only discrete inflammatory lesions in the CNS, compared with EAE induced by PAS cells fully activated by thymic cells. Altogether, our results indicate that MBP presentation by brain microvessel endothelial cells to encephalitogenic T cells induces T cell anergy and loss of pathogenicity. In addition, IL-1 beta co-stimulation of T cells prevents anergy induction in vitro and at least partially maintains encephalitogenicity in vivo. PMID- 7539752 TI - Interleukin-2 secretion by human B lymphocytes occurs as a late event and requires additional stimulation after CD40 cross-linking. AB - While Epstein-Barr virus (EBV)-immortalized B cell lines have been shown to secrete interleukin (IL)-2 after stimulation with either teleocidin or phorbol myristate acetate (PMA) and ionomycin, experimental conditions leading to IL-2 production by normal human B cells have not been reported. In the present study we investigated various B cell activating conditions, including--by analogy to EBV-immortalized B lymphocytes--stimulation of B cells that are already proliferating (in cultures with IL-4 and immobilized anti-CD40 monoclonal antibody; the anti-CD40 system). This approach showed that B lymphocytes secreted IL-2 in the culture medium, but only if they were first activated for more than 24 h in the anti-CD40 system before exposure to PMA plus ionomycin. The production rate of IL-2 by B lymphocytes reached a maximum after 6 days of priming in such cultures followed by 48 h of stimulation with PMA plus ionomycin, corresponding to 7% or 15% of that of fresh CD4+ T cells activated, respectively, with phytohemagglutinin plus PMA, or with PMA plus ionomycin for 48 h. This IL-2 production could not be attributed to T cell contamination nor to EBV-infected B cells according to flow cytometric and reverse transcriptase-polymerase chain reaction analysis of cultured B cells. Lower IL-2 expression (detected only as mRNA synthesis) was also induced in the cultured B lymphocytes after incubation with cross-linking anti-IgM antibodies instead of PMA plus ionomycin. The appearance of IL-13 mRNA, but not IL-4 mRNA, was detected under the same stimulation conditions as for IL-2 mRNA. These results show that the production of IL-2 by normal B lymphocytes occurs as a late event relative to their activation and proliferation, and is in this respect subject to regulation different to that found in T lymphocytes. PMID- 7539754 TI - Phagocyte-induced antigen-specific activation of unprimed CD8+ T cells in vitro. AB - The strict segregation of the major histocompatibility complex (MHC) class I and class II loading pathways has been challenged by recent reports indicating that MHC class I molecules can acquire antigen in the phagocytic pathway. We now show that this alternative peptide loading pathway can be used efficiently to generate macrophages able to activate unprimed antigen-specific cytotoxic T cells in vitro. Short peptides (8-11 residues), administered in the phagocytic pathway at nanomolar concentrations, were found to be effective in specifically activating naive cytotoxic T lymphocytes (CTL) in vitro, but longer peptides or whole protein antigen were not. Whole protein antigen coated on beads did, however, render macrophages susceptible to lysis by an antigen-specific CTL clone. This indicates that proteolysis in the phagocytic pathway has limited capability for class I-restricted presentation. We propose a model for class I loading in the phagocytic pathway consisting of direct trafficking of nascent MHC class I from the trans-Golgi network to the phagosome, prior to appearance at the cell surface, and the use of the narrow cavity between bead and phagosomal membrane as a peptide exchange/loading compartment. Targeting immunogenic class I-binding peptide to the phagocytic pathway of macrophages facilitates presentation in association with class I. This is a useful tool for CTL response induction in vitro. PMID- 7539755 TI - Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120. AB - CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4+ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120 induced CD4+ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59 CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26 and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4+ interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that th e associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components. PMID- 7539750 TI - Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies. AB - Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66 specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response. PMID- 7539753 TI - Activation of thymic B cells by signals of CD40 molecules plus interleukin-10. AB - We have previously found that thymic B cells, particularly thymic CD5+ B cells, show low responsiveness to the usual B cell stimulants such as lipopolysaccharide or anti-IgM plus interleukin (IL)-4, although they proliferate and produce antibodies after direct interaction with major histocompatibility complex class II-restricted T blasts. These findings raise the possibility that a CD40-CD40 ligand (L) interaction is involved in the activation of thymic B cells. In the present study, we therefore examine this possibility using CD40L-transfected Chinese hamster ovary (CHO) cells or anti-CD40 monoclonal antibody (mAb). When B cells in the spleen and peritoneal cavity were stimulated, they proliferated and produced immunoglobulin (Ig) in the presence of CD40L-CHO cells or anti-CD40 mAb alone. However, another signal delivered by IL-10 in addition to CD40L-CHO cells or anti-CD40 mAb was found to be necessary for thymic B cells to proliferate and secrete Ig. Other interleukins acting on B cells, such as IL-4, IL-5, and IL-6, had no effect on the activation of thymic B cells, which thus have unique characteristics not found in peripheral B cells. This report discusses the physiological significance of IL-10- and CD40-driven signals in the activation of thymic B cells. PMID- 7539756 TI - Abnormalities of B cells and dendritic cells in SAMP1 mice. AB - The age-related changes in the function of antigen-presenting cells (APC) were examined using a substrain of senescence-accelerated mouse (SAMP1). In the primary mixed lymphocyte reaction (MLR), dendritic cells (DC) from aged SAMP1 mice showed less stimulatory activity than those of age-matched BALB/c or young SAMP1 mice. In the secondary MLR, the stimulatory activity of B cells was found to be lower in aged SAMP1 mice but not in age-matched BALB/c or young SAMP1 mice. In addition, these age-related decreases in the stimulatory activity of APC were found to be related to changes in the surface density of major histocompatibility complex class II and intercellular adhesion molecule-1 (ICAM-1) (but not B7-1 or B7-2 molecule) on APC (DC and B cells). PMID- 7539757 TI - The role of bcl-XL in CD40-mediated rescue from anti-mu-induced apoptosis in WEHI 231 B lymphoma cells. AB - The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2 independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-XL) renders these cells refractory to anti-Ig induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-XL protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca(2+)-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-XL. We, therefore, conclude that bcl-XL plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype. PMID- 7539758 TI - Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2. AB - The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab. PMID- 7539760 TI - Both membrane-bound and soluble forms of CD14 bind to gram-negative bacteria. AB - Tissue macrophages and their precursors-the blood monocytes-respond rapidly to a bacterial infection with the release of inflammatory mediators. These mediators are involved in the recruitment of phagocytic cells, principally neutrophils, from the blood to the site of infection. To initiate this process macrophages and monocytes must be able to detect the presence of bacteria in a reliable, but nevertheless nonspecific, fashion. It is thought that this is achieved by means of receptors on the cell surface which recognize structures common to many different bacteria. One candidate for such a "pattern recognition element" is the cell surface glycoprotein CD14. CD14 has been shown to bind components of the Gram-positive cell wall and it also binds soluble lipopolysaccharide released from Gram-negative bacteria. In both cases the interaction with CD14 leads to an activation of the cell. Here we show that human peripheral blood monocytes can, in addition, bind intact Gram-negative bacteria in the presence of serum and this process involves CD14. When CD14 expression is induced on the myelomonocytic cell line U937 by treatment with vitamin D3 the cells concomittently acquire the capacity to bind bacteria. Furthermore, a non-monocytic cell line which does not bind bacteria acquires the capacity to do so when transfected with either the human or mouse CD14 gene. This binding can be inhibited by blocking the CD14 receptor with anti-CD14 antibody or by blocking the ligand on the bacteria with soluble CD14. Finally we demonstrate binding of sCD14 to Escherichia coli. We conclude that in the presence of serum both membrane-bound and soluble forms of CD14 can bind to Gram-negative bacteria. This suggests that CD14 may play a role in the detection and elimination of intact bacteria in vivo. PMID- 7539759 TI - Role of T helper cell precursor frequency on vesicular stomatitis virus neutralizing antibody responses in a T cell receptor beta chain transgenic mouse. AB - During most immune responses, T cells help antigen-specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the beta chain (V beta 2) of a major histocompatibility complex class II-restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV-IND). These mice exhibit an increased precursor frequency of VSV-specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching. PMID- 7539761 TI - CD28/CTLA-4 ligands: the gene encoding CD86 (B70/B7.2) maps to the same region as CD80 (B7/B7.1) gene in human chromosome 3q13-q23. AB - CD86 (B70/B7.2) is an antigen of the immunoglobulin superfamily expressed on monocytes, dendritic cells and activated B, T, and natural killer cells. CD86 was recently identified as a second ligand for the T cell antigens CD28 and CTLA-4, and plays an important role in the co-stimulation of T cells in a primary immune response. We report here the assignment of the CD86 gene to human chromosome 3 using Southern blot analysis on a panel of hamster x human somatic cell hybrid genomic DNA. Fluorescence hybridization in situ on metaphase chromosomes coupled with GTG banding (G-bands by trypsin using Giemsa staining) confirmed this assignment and localized the CD86 gene to 3q13-q23 region. The CD86 gene is, therefore, located in the proximity of the CD80 (B7/B7.1) gene, the first identified ligand for CD28 and CTLA-4, previously mapped to chromosome 3q13.3 q21. Deletions, inversions and insertions of chromosome 3q21-q26, as well as translocations of 3q21 with other chromosomes have been described in many cases of acute myeloid leukemia (AML), acute non-lymphocytic leukemia (ANLL), chronic myeloid leukemia (CML) and myelodisplastic syndromes (MDS), suggesting that this region contains several genes involved in the leukemic process. PMID- 7539762 TI - The role of gamma delta T cells in priming macrophages to produce tumor necrosis factor-alpha. AB - The secretion of tumor necrosis factor (TNF)-alpha from macrophages is regulated by both priming and triggering signals. We found that macrophages from mice lacking gamma delta T cells [T cell receptor (TCR) delta-/- mice], which lack the gene encoding the delta chain, produced only small amounts of TNF-alpha in response to lipopolysaccharide (LPS) and showed a reduced level of expression of CD14. Pre-incubation of macrophages from TCR delta-/- mice with gamma delta T cells from their TCR delta +/- littermates restored their capacity to produce TNF alpha in response to LPS. The priming activity of gamma delta T cells was in part inhibited by neutralizing anti-interferon (IFN)-gamma monoclonal antibodies. Collectively, these results suggest that gamma delta T cells play a role in priming macrophages to a steady state of activation via IFN-gamma secretion, which allows them to produce TNF-alpha when exposed to LPS. PMID- 7539764 TI - Increase in serum NG-hydroxy-L-arginine in rats treated with bacterial lipopolysaccharide. AB - Aortic rings isolated from rats 4 h after an injection i.p. of 30 mg/kg Escherichia coli lipopolysaccharide showed a marked hyporeactivity to noradrenaline. This effect was paralleled by an increase in the level of nitrite/nitrate in the serum of lipopolysaccharide-treated rats, indicative of an enhanced nitric oxide (NO) synthase activity. Most important, however, the serum concentration of the NO synthase intermediate, NG-hydroxy-L-arginine, was also markedly elevated from 3.7 to 15.8 microM. Circulating NG-hydroxy-L-arginine may thus represent a sensitive and specific marker of NO synthase activity in vivo. PMID- 7539765 TI - Nitric oxide synthase inhibitors block behavioral sensitization to methamphetamine in mice. AB - Repeated administration of methamphetamine (1.0 mg/kg) once daily for 7 consecutive days resulted in an augmentation of the locomotor-activating effect of methamphetamine (0.5 mg/kg) challenged 72 h after the last injection. Administration of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (10 and 30 mg/kg), before daily methamphetamine injections dose dependently prevented the development of behavioral sensitization to subsequent methamphetamine challenge. The mice given another NO synthase inhibitor, NG-nitro L-arginine methyl ester (100 mg/kg), before daily methamphetamine injections showed significantly less locomotor activity in response to 0.5 mg/kg methamphetamine challenge than the mice given daily methamphetamine alone. Such effects were not observed when the inactive isomer, NG-nitro-D-arginine methyl ester (100 mg/kg), was administered daily prior to methamphetamine. Both NO synthase inhibitors exerted the acute effect to reduce spontaneous and methamphetamine-stimulated locomotor activity, while neither spontaneous locomotion nor hyperlocomotion in response to 1.0 mg/kg methamphetamine was altered 72 h after repeated administration of NG-nitro-L-arginine (30 mg/kg) or NG-nitro-L-arginine methyl ester (100 mg/kg) alone once daily for 7 days. On the other hand, pretreatment with the NMDA receptor antagonist, MK-801 ((+)-5-methyl 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate), at 0.2 mg/kg also suppressed the development of sensitization to the locomotor activating effect of methamphetamine. These findings suggest that NO formation possibly mediated by NMDA receptors is involved in mechanisms underlying the development of behavioral sensitization to methamphetamine. PMID- 7539763 TI - Purification and characterization of endogenous peptides extracted from HLA-DR isolated from the spleen of a patient with rheumatoid arthritis. AB - We have purified HLA-DR from the spleen of a patient with rheumatoid arthritis. The patient had Felty's syndrome and was heterozygous for the DR4Dw4 antigen. We have isolated endogenous peptides from purified HLA-DR molecules. The peptides were purified by reverse phase HPLC and the major peaks were subjected to N terminal sequencing. The peptides were derived from a variety of proteins: human serum albumin, human erythroid protein 4.1, 60S ribosomal proteins L31 and L35, VCAM-1, human immunoglobulin lambda chain and cathepsin-S. A peptide corresponding to the sequence of human serum albumin (HSA) residues 106-120 was synthesized and shown to bind to HLA-DR4Dw4 (IC50 = 1.41 microM). We have confirmed and refined current ideas about the structural motif for the binding of peptides to HLA-DR and HLA-DR4Dw4. PMID- 7539766 TI - Cholinergic activation of Cl- secretion in rat colonic epithelia. AB - Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11 [[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3 b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor mediated Cl- secretion appear to belong to the M3 class. PMID- 7539767 TI - The blood flow in pancreatico-duodenal grafts in rats: inhibition of nitric oxide synthase preferentially decreases islet blood flow. AB - In this study normoglycemic inbred Wistar-Furth rats were implanted with a syngeneic pancreatico-duodenal graft, i.e. a denervated pancreas. The blood flow to the intact native pancreas and to the transplanted gland was measured with a microsphere technique in anesthetized rats 2 weeks after transplantation. The animals were given an intravenous injection with saline alone, NG-nitro-L arginine (25 mg/kg body weight) or sodium nitroprusside (10 micrograms/kg body weight) 10 min before blood flow measurements. Administration of NG-nitro-L arginine increased mean arterial blood pressure and caused a pronounced decrease in whole pancreatic blood flow in both the native and transplanted gland. The islet blood flow was more markedly decreased by NG-nitro-L-arginine in both the native and transplanted pancreas, and constituted about 4% of whole pancreatic blood flow compared with 10% in the control animals. Sodium nitroprusside markedly decreased mean arterial blood pressure, but did not affect pancreatic or islet blood flow in any of the glands. It is concluded that inhibition of nitric oxide synthase causes a preferential decrease in islet blood flow both in the native pancreas and in the transplanted pancreas. This suggests that nitric oxide which affects islet blood flow is mainly endothelial-derived, and does not emanate from external nervous fibers. PMID- 7539768 TI - Mechanism of histamine release by endothelin-1 distinct from that by antigen in mouse bone marrow-derived mast cells. AB - The mechanisms of endothelin-1-induced histamine release were examined and compared with those responsible for antigen-induced release by using passively sensitized mouse bone marrow-derived mast cells and various drugs that may influence histamine release. The following results were obtained: (1) Although islet-activating protein potently inhibited endothelin-1-induced histamine release, it did not affect the antigen-induced release. (2) Histamine release induced by endothelin-1 was relatively more sensitive to ethylenediaminetetraacetic acid than that induced by antigen, although extracellular Ca2+ is a requisite for both types of the release. (3) (8R*, 9S*, 11S*)-(-)-9- hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy 1H,8H, 11H-2, 7b,11a-triazadibenzo[a,g]cycloocta[c,d,e]trinden-1-one and (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tet rahydro - 8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta-[c ,d,e] trinden-1-one, which possibly inhibit protein kinases, strongly inhibited antigen induced histamine release, while these drugs alone did not inhibit endothelin-1 induced release. (4) Staurosporine, a non-selective protein kinase inhibitor, prevented the elevation of cytosolic Ca2+ concentrations induced by antigen, whereas that induced by endothelin-1 was not influenced; histamine release induced by either stimulus was greatly inhibited by the drug. These results indicate and/or suggest that some biological events induced by endothelin-1 leading to histamine release are different from those involved in the histamine release induced by antigen. PMID- 7539769 TI - Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost. AB - Human platelets suspended in plasma or buffer were incubated with low concentrations of the nitric oxide (NO)-donor 3-morpholino-syndnonime (SIN-1; 100 nM to 1 microM) and the stable prostacyclin analogue iloprost (50 or 100 pM) and analyzed for cyclic nucleotide levels and protein phosphorylation. SIN-1 and iloprost synergistically stimulated the phosphorylation of rap1B and the 50 kDa vasodilator-stimulated phosphoprotein. SIN-1 stimulated platelet cyclic GMP and cAMP-levels and enhanced the increase in cyclic AMP elicited by iloprost. It was found that the mechanism underlying the synergistic phosphorylation of the 50 kDa protein and rap1B was different: synergistic phosphorylation of the 50 kDa protein seemed to be mediated by activation of both protein kinases A and G, whereas the synergistic rap1B phosphorylation could be attributed entirely to activation of protein kinase A. Measurement of rap1B phosphorylation might be a useful tool to monitor the action of systemically applied prostacyclin-analogues and nitrovasodilators in pharmacological studies. PMID- 7539770 TI - 4-Hydroxyphenethyl alcohol--a new cytokinin-like substance from the phototrophic purple bacterium Rhodospirillum rubrum 1R. AB - Three compounds with cytokinin activity have been isolated from the medium of Rhodospirillum rubrum grown photosynthetically. Two N-6 aminopurine cytokinins revealed in the medium were identical with those obtained from R. rubrum cells previously. The third compound with cytokinin activity in Amaranthus caudatus bioassay proved to be a simple phenolic compound with elemental composition C8H10O2. This cytokinin-like substance according to absorption spectra, mass spectrometry and 1H NMR spectra data was identified as 4-hydroxyphenethyl alcohol. PMID- 7539772 TI - The prognostic significance of lymphovascular space invasion in endometrial cancer when conventional hemotoxylin and eosin staining is compared to immunohistochemical staining. AB - The current study was undertaken to compare the usefulness of hemotoxylin and eosin (H&E) staining and immunohistochemical staining to identify lymphovascular space invasion (LVSI) in endometrial cancer and to evaluate the presence of LVSI detected by either technique as an independent prognostic factor. Histologic sections from 92 patients with clinical stage I-II endometrial cancer were reviewed, and representative sections were stained immunohistochemically with antibodies for von Willebrand factor and blood group isoantigens. To compare the prognostic significance of LVSI detected by H&E staining with that detected by immunohistochemical staining, univariate and multivariate analyses were performed. Thirty (32.6%) of the 92 cases showed LVSI in H&E staining. In 8 of the 30 cases, LVSI was negative by immunohistochemical staining, while LVSI was positive by immunohistochemical stainings in 2 of 62 cases showing negative LVSI in H&E staining. In univariate analysis, LVSI detected by H&E and immunohistochemical staining was proved to be significant as a prognostic factor. In multivariate analysis by Cox's proportional hazards model, LVSI identified by H&E staining was selected as one of significant prognostic factors, but LVSI identified by immunohistochemical staining not selected. The results of this study indicate that LVSI is one of the independent prognostic factors in endometrial cancer, and that LVSI as detected by H&E is more prognostic of survival than immunohistochemical detection. PMID- 7539771 TI - Ca(2+)-triggered membrane permeability transition in deenergized mitochondria from rat liver. AB - The opening of the cyclosporin A-sensitive permeability transition pore (MTP) in deenergized mitochondria was induced only at millimolar Ca2+. Pretreatment of the mitochondria with 'inducers', such as duroquinone and phenylarsine oxide, allowed observing the pore opening at 0.01-0.1 mM Ca2+. Duroquinone caused a rapid (within 20 s) NAD(P)H oxidation which was followed by a slow (20 min) induction of the pore sensitive to low Ca2+. Phenylarsine oxide capable of cross-linking of vicinal SH-groups caused pore formation without the oxidation of NAD(P)H. The pore opening by both 'inducers' was prevented by N-ethylmaleimide. We propose that oxidation or cross-linking of critical dithiol(s) in membrane proteins increase the sensitivity of a putative 'Ca(2+)-sensor' that regulates the permeability transition pore opening. PMID- 7539773 TI - Progesterone treatment decreases sulfate carbohydrate antigen on endometrial carcinoma cells and inhibits the cell binding to laminin. AB - To investigate the effects of progesterone on endometrial carcinoma, sulfated carbohydrate antigen was studied using a monoclonal antibody, termed E8, that reacts with sulfatide. The reactivity of monoclonal antibody (MAb) E8 with endometrial carcinoma tissues decreased when patients were treated with medroxyprogesterone acetate (MPA). A reduction of reactivity of MAb E8 with endometrial carcinoma cells was also observed in in vitro examinations using an endometrial carcinoma cell line, Ishikawa cells incubated in MPA-containing medium. Sulfated carbohydrate, such as sulfatide, is the site of binding with laminin. The binding of cell surface components with laminin is thought to be an initial step in the invasion and metastasis of carcinoma cells. The MPA-treated Ishikawa cells bound to laminin-coated dishes less [corrected] efficiently than nontreated cells. MPA treatment of endometrial carcinoma reduces the sulfatide carbohydrate on the carcinoma cell surface and may be clinically more effective in reducing cell binding to laminin. PMID- 7539775 TI - Splice variants of CD44 in human cervical cancer stage IB to IIB. AB - Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumors and the expression of specific CD44 isoforms (splice variants) has been shown to be associated with metastasis and poor prognosis in human malignancies. We used three different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exon v5, exon v6, or exon v7-v8 of human variant CD44 to study the expression of CD44 splice variants by immunohistochemistry in human cervical cancer. One-hundred five patients with surgically treated squamous cell carcinomas of the cervix stages IB to IIB were included in the study. CD44 splice variants CD44v5, CD44v6, and CD44v7-8 were detected in 70, 67, and 26%, respectively. Tumors expressing exon v6 had significantly more often metastasized to the pelvic nodes (58 vs 79%, P = 0.04). Expression of exon v6 was significantly correlated with a greater probability of vascular space invasion (73 vs 50%, P = 0.04) and a significantly lower rate of inflammatory stromal reaction (48 vs 78%, P = 0.004). Patients suffering from tumors expressing splice variant CD44v6 showed poorer overall survival (P = 0.03). In cases with negative pelvic lymph nodes we found a poorer prognosis when tumors expressed CD44v6 (P = 0.01) or CD44v7-8 (P = 0.02). Among the investigated CD44 splice variants expression of exon v6 is the most promising prognostic marker in surgically treated cervical cancer. PMID- 7539776 TI - The CD36, CLA-1 (CD36L1), and LIMPII (CD36L2) gene family: cellular distribution, chromosomal location, and genetic evolution. AB - CD36, CLA-1, and LIMPII are single polypeptide membrane glycoproteins, and the genes encoding them constitute a recently described gene family (D. Calvo and M. A. Vega (1993) J. Biol. Chem. 268: 18929). In the present paper, a cDNA encoding the human lysosomal membrane protein LIMPII was used to determine its expression pattern in cells of various lineages. Like CLA-1, and in contrast with the restricted expression of CD36, the expression of LIMPII is widespread. Mapping of the human LIMPII and CLA-1 genes (gene symbols CD36L2 and CD36L1, respectively) to specific chromosomes revealed that CLA-1, LIMPII, and CD36 do not form a gene cluster, but are found dispersed on chromosomes 12, 4, and 7, respectively. These data, together with the phylogenetic analysis carried out for the members of this family, indicate that the LIMPII, CLA-1, and CD36 genes diverged early in evolution from an ancestor gene, possibly before the divergence between the arthropods and the vertebrates. PMID- 7539774 TI - Basement membranes in cervical cancer: relationship to pelvic lymph node metastasis and prognosis. AB - In this retrospective study, the pattern of basement membrane expressed by type IV collagen immunostaining was evaluated in 156 patients with cervical cancer FIGO stage IB-IV. Staining patterns were assessed semiquantitatively and divided into three patterns, according to intensity and continuity of staining: thick basement membranes with minimal discontinuity (pattern I), thin basement membranes with moderate discontinuity (pattern II), and fragmentary or absent basement membranes (pattern III). Correlations between basement membrane pattern, FIGO stage, and pelvic lymph node status were tested using chi 2 statistics. Kaplan-Meier estimates of disease-free survival were calculated for groups of patients with basement membrane pattern I, II, or III and compared using the log rank test. A fragmented or absent basement membrane, pattern III, was more frequently observed in advanced cervical cancer stage IIB/IV (P = 0.02). In patients with early cervical cancer stage Ib/IIA this pattern was associated with the presence of pelvic lymph node metastasis (P < 0.0001) and significantly associated with a decreased survival rate (P = 0.05). In advanced-stage disease no prognostic value was observed. These data indicate that in patients with early stage cervical cancer the basement membrane pattern, expressed as the type IV collagen staining pattern, may be a useful marker in predicting lymph node metastasis and survival. PMID- 7539777 TI - A new approach to phosphoserine, phosphothreonine and phosphotyrosine synthons and to thiophospho analogs. Stepwise synthesis of mono- and multiphosphorylated phosphopeptides related to src-protein kinase. AB - Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2 trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase. PMID- 7539778 TI - Role of ring size on the secondary structure and antibiotic activity of gramicidin S. AB - In order to investigate the contribution of ring size to the secondary structure and antibiotic activity of gramicidin S, many analogs consisting of 6, 7, 8, 9, 11, 12, 13, and 14 amino acid residues were synthesized. In the analogs with smaller ring sizes than that of gramicidin S, only des-Val1-, des-Leu3- and des Pro5-gramicidin S showed weak activity against the gram-positive micro-organisms tested. On the other hand, all analogs having larger rings, in which L- or D-Leu residues were inserted, were active. The activity of the analogs consisting of 10, 11 and 12 amino acid residues was stronger than those of the analogs with other ring sizes, and the activity of endo-Leu2a-gramicidin S against S. epidermidis SP-al-1 and B. subtilis ATCC 6633 was twice of that of gramicidin S. PMID- 7539779 TI - The effect of GM-CSF and G-CSF on human neutrophil function. AB - A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB4. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. albicans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (Fc gamma R1, CR-1 and CR-3), and adhesion signalling (ICAM-1), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article. PMID- 7539780 TI - Induction of acute phase proteins in mice and humans by treatment with AS101, an immunomodulator with radioprotective properties. AB - AS101 (ammonium trichloro(dioxyethylene-0,0')tellurate) is a new synthetic compound previously described by us as having immunomodulating properties and minimal toxicity. Phase II clinical trials are currently in progress with AS101 on cancer patients. AS101 has been recently found to have both radioprotective and chemoprotective effects on hemopoiesis of irradiated mice or mice treated with various chemotherapeutic drugs. The present research was designed to study the in vivo induction of liver acute phase proteins secretion in mice or patients treated with AS101. Induction of these proteins, some of which have the capacity to scavenge free radicals, may contribute to radioprotection. We present evidence that treatment with the immunomodulator AS101 increases production of a variety of acute phase proteins. We demonstrate a significant elevation of serum amyloid A (SAA) in sera of treated mice, as well as an increase in SAA, factor B and ceruloplasmin in sera of patients treated with AS101. The same AS101 treatment was shown to decrease the amount of the negative acute phase protein, albumin. In addition we show that IL-1, IL-6 and TNF-alpha are important mediators of changes in SAA concentrations induced by AS101. Abrogation of SAA production in AS101 treated mice by any one of the anti IL-1R, IL-6R or TNF-alpha antibodies indicates that at least in mice, SAA production is not controlled by a single extracellular signal, but rather it is regulated, at the least, by all three cytokines in various combinations. A better understanding of the mechanism by which AS101 confers radioprotection will enable us to use it more effectively in the restoration of hemopoiesis in patients following radiation or suffering from overdose or accidental radiation. PMID- 7539781 TI - Metabolism and menstrual cycle rhythmicity of serotonin in primary headaches. AB - We investigated the platelet and plasma levels of serotonin and its metabolite, 5 hydroxyindoleacetic acid, in patients suffering from episodic tension-type headache and migraine with and without aura, during a headache-free period. In female subjects, blood samples were drawn during the follicular, ovulatory, and late luteal phases of the menstrual cycle. In tension headache and migraine with aura, the group mean values of serotonin and 5-hydroxyindoleacetic acid in platelets and plasma were significantly increased, but migraine without aura patients' levels were normal. The pattern of menstrual cycle-related fluctuations in platelet serotonin levels were similar in female patients with tension headache and in controls, with a maximum value in the follicular phase. In both migraine groups, in contrast, the peak occurred in the ovulatory phase. The results are discussed in view of whether these aberrations in peripheral markers of the metabolism and menstrual cycle-related rhythmicity of serotonin may reflect similar alterations in the central nervous system. PMID- 7539783 TI - Inhibition of tumor-induced angiogenesis by a synthetic lipid A analogue with low endotoxicity, DT-5461. AB - We investigated the effect of a synthetic lipid A analogue with low endotoxicity, DT-5461, on the neovascularization induced by B16-BL6 melanoma in syngeneic mice. A systemic single administration of DT-5461 caused a marked decrease in the number of vessels oriented toward the tumor mass and in the tumor size during the early phase of vasculogenesis (on day 4 after tumor inoculation), with little or no inhibition in the following phases. Multiple i.v. administrations of DT-5461 at intervals of 4 days (an effective schedule for inhibiting tumor metastasis) significantly reduced the number of capillary vessels and tumor growth over a period of 14 days after the tumor implantation. Multiple systemic administrations of DT-5461 on days 1, 5 and 9 after tumor inoculation caused a high production of endogenous tumor necrosis factor-alpha (TNF-alpha) in tumor sites although this treatment modality induced a low production in serum of tumor-bearing mice. Tumor homogenate from mice treated with DT-5461 suppressed the proliferation of endothelium in vitro, whereas sera from animals given DT-5461 had little effect. Furthermore, the antiproliferative effect of the tumor homogenate from mice treated with DT-5461, was completely abrogated by anti-mTNF-alpha monoclonal antibody (mAb). The anti-angiogenic effect of DT-5461 was also completely abrogated by rabbit anti-mouse tumor necrosis factor-alpha (anti-mTNF-alpha) antiserum, whereas the inhibition of tumor growth by DT-5461 was only slightly diminished. Tumor homogenate from mice treated with DT-5461 suppressed the proliferation of endothelium in vitro, whereas sera from animals given DT-5461 had little effect. Furthermore, the antiproliferative effect of the tumor homogenate from mice treated with DT-5461 was completely neutralized by anti-mTNF alpha mAb. Multiple i.v. administrations of DT-5461 after s.c. implantation of B16-BL6 cells significantly inhibited the growth of primary tumors measured at the time of tumor excision on day 21, and the lung metastasis of melanoma cells as compared with the untreated control in the spontaneous metastasis model. These results suggested that the suppressive effect upon tumor-associated angiogenesis by DT-5461 contributes in part to the inhibition of tumor metastasis. PMID- 7539782 TI - Tyrosine phosphorylation of the proto-oncogene product Vav and its association with the adapter Grb2/Ash in a human leukemia cell line UT-7. AB - The vav proto-oncogene product (Vav) is expressed exclusively in hematopoietic cells and is reported to have guanine nucleotide exchange activity. Here we report that granulocyte-macrophage colony-stimulating factor, interleukin-3, and erythropoietin induce tyrosine phosphorylation of Vav in a human leukemia cell line UT-7. Tyrosine phosphorylation of Vav is rapid and transient; it occurs within 1 min of the stimulation and at physiological concentrations of the factors. Furthermore, we show that Vav is constitutively associated with the adapter molecule Grb2/Ash in UT-7. These data suggest that tyrosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav are members of a signaling pathway leading to Ras activation in hematopoietic cells. PMID- 7539784 TI - A method for measuring Cl efflux from dispersed cells of airway epithelium. AB - This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539785 TI - Risperidone in childhood schizophrenia. PMID- 7539786 TI - Filgrastim treatment of three patients with clozapine-induced agranulocytosis. AB - BACKGROUND: Agranulocytosis is the most serious side effect of clozapine therapy, occurring in approximately 1% of all treated patients. Despite careful blood monitoring, a significant number of cases of agranulocytosis and resulting fatalities have occurred. Strategies are needed to manage clozapine-induced agranulocytosis more safely. METHOD: This report describes the management of three state hospital inpatients who developed clozapine-induced agranulocytosis. All patients were diagnosed as having chronic paranoid schizophrenia according to DSM-III-R criteria and had previously failed to respond to treatment with standard antipsychotic medications. After onset of agranulocytosis, all patients were transferred to a medical service in a university hospital and treated with recombinant granulocyte colony-stimulating factor (filgrastim). RESULTS: White blood count and absolute neutrophil count returned to within normal limits in each patient after 5 to 8 days of treatment with filgrastim 300 micrograms/day subcutaneously. No side effects were observed during filgrastim treatment. CONCLUSION: Treatment with filgrastim appears to be safe and effective in decreasing the duration of clozapine-induced agranulocytosis. While further studies are necessary to establish the safety and effectiveness of this treatment, filgrastim should presently be considered a treatment of choice for clozapine-induced agranulocytosis. PMID- 7539787 TI - Synthesis and self-cleavage reaction of a chimeric molecule between RNase P-RNA and its model substrate. AB - We synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed. PMID- 7539788 TI - Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. AB - Recombinant human erythropoietin (EPO) produced by Chinese hamster ovary cells and distributed by two different pharmaceutical companies were confirmed to contain about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since chickens, like humans, do not synthesize Neu5Gc, they were used to determine the immunogenicity of Neu5Gc epitope in EPO. Chickens immunized with EPO did not produce significant titer of antibody that was specific to GM3(Neu5Gc) as compared to antibody titers produced in chickens immunized with fetuin containing 7% Neu5Gc or GM3(Neu5Gc) containing 100% Neu5Gc. Results obtained by an ELISA inhibition test showed that EPO, compared to GM3(Neu5Gc), reacted almost one thousand times less strongly with a human Hanganutziu-Deicher (HD) antibody. This study implies that an increase of Neu5Gc content in a molecule enhances its HD antigenicity. The response to Neu5Gc in patients receiving therapeutic injections of EPO is currently under investigation. PMID- 7539789 TI - A role for the distal CCAAT box of the gamma-globin gene in Hb switching. AB - Hereditary persistence of fetal hemoglobin (HPFH) is a condition manifested by continued expression of the gamma-globin gene in adult life. The four types of HPFH mutations (-117 G-A, -114 C-T, -114 C-G, and 13 bp del) clustered in the vicinity of the distal CCAAT box of the gamma-globin gene suggest the relevance of this region to the fetal-to-adult hemoglobin switching. Functional analysis in erythroid cell lines showed that these HPFH mutations reduced the activity of the gamma-globin promoter in K562 expressing embryonic and fetal globin chains, but not in KU812 expressing fetal and adult globin chains. These results suggest that the distal CCAAT box region has different functions in the fetus and the adult. In vitro, binding of NFE3 to the distal CCAAT box was commonly reduced by these HPFH mutations, suggesting that the binding of NFE3 to the distal CCAAT box may repress the gamma-globin gene in adults. Overlap of the binding sites of NFE3 and CP1, a potential activator, indicates the regulation of the gamma-globin gene by the competition between these factors. In addition, an unknown factor interacting with the -114 C-T HPFH mutant CCAAT box may be involved in elevation of the gamma globin expression in an individual with the -114 C-T mutation. PMID- 7539790 TI - Transforming growth factor-beta-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action. AB - Most estrogen receptor-negative breast cancer cells, including Hs578T cells, express mRNAs encoding insulin-like growth factor-binding protein (IGFBP)-3, as well as transforming growth factor (TGF)-beta receptors. Our previous studies (Oh, Y., Muller, H. L., Lamson, G., and Rosenfeld, R. G. (1993) J. Biol. Chem. 268, 14964-14971; Oh, Y., Muller, H. L., Pham, H. M., and Rosenfeld, R. G. (1993) J. Biol. Chem. 268, 26045-26048) have demonstrated a significant inhibitory effect of exogenous IGFBP-3 on Hs578T cell growth and existence of IGFBP-3 specific receptors that may mediate those direct inhibitory effect of IGFBP-3. TGF-beta is also a potent growth inhibitor in human breast cancer cells in vitro and regulates IGFBP-3 production in different cell systems, suggesting that IGFBP 3 is a major anti-proliferative factor and a key element for TGF-beta-induced growth inhibition in human breast cancer cells. In support of this hypothesis, we have demonstrated using Hs578T cells that: 1) TGF-beta stimulates IGFBP-3 gene expression and production prior to its inhibition of cell growth, 2) treatment with an IGFBP-3 antisense oligodeoxynucleotide selectively inhibits TGF-beta induced IGFBP-3 synthesis and cell growth inhibition, and 3) treatment with IGF II and IGF-II analogs diminish TGF-beta effects by blocking TGF-beta-induced binding of IGFBP-3 to the cell surface. These findings suggest that IGFBP-3 is a major anti-proliferative factor and a key element in TGF-beta-induced growth inhibition in human breast cancer cells. PMID- 7539791 TI - Identification of angiotensinogen and complement C3dg as novel proteins binding the proform of eosinophil major basic protein in human pregnancy serum and plasma. AB - In sera from pregnant women, pregnancy-associated plasma protein-A (PAPP-A) circulates as a disulfide-bound complex (approximately 474 kDa) with the proform of eosinophil major basic protein (proMBP) (Oxvig, C., Sand, O., Kristensen, T., Gleich, G. J., and Sottrup-Jensen, L. (1993) J. Biol. Chem. 268, 12243-12246). We have produced monoclonal antibodies (mAbs) against the PAPP-A.proMBP complex and established a radioimmunoassay utilizing a mAb recognizing the PAPP-A subunit. Surprisingly, serum levels of proMBP exceed those of PAPP-A four to 10-fold on a molar basis throughout pregnancy. This result prompted an investigation of the status of proMBP in pregnancy. Using a proMBP-specific mAb two novel proMBP complexes have been isolated by chromatographic techniques. Based on sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reaction with specific antibodies, one is shown to be a 2:2 disulfide-bound complex (approximately 200 kDa) between proMBP and angiotensinogen. The other is a 2:2:2 complex (approximately 300 kDa) between proMBP, angiotensinogen, and complement C3dg. Circulating proMBP in pregnancy is thus present in three types of complexes. These results suggest that specific interactions between the complexed proteins occur in pregnancy, and the possibility is raised that their interactions are important in the pathophysiology of pregnancies associated with hypertension. PMID- 7539792 TI - Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting. AB - Human immunodeficiency virus encodes the regulatory protein Rev, which is required for expression of viral structural proteins. It binds to an RNA element (RRE) in the viral transcript and up-regulates the cytoplasmic appearance of unspliced and singly spliced viral mRNA. We have studied the structure of Rev alone and complexed with the RRE and two monoclonal antibodies, using a protein footprinting approach. The method involves radioactive labeling at the C-terminal end of Rev fusion protein followed by limited proteolysis under native conditions, using 10 different proteinases. Rev protein was mainly cleaved within the basic domain and in the C-terminal part. The periodicity of the proteolytic cleavages within the basic domain strongly suggests that it forms an alpha helical structure with one side facing the solvent. In the presence of RRE, these cleavages became significantly reduced. In addition, strong protection was observed at position 66 outside the basic domain. As a control for the specificity of the footprinting reaction, we confirmed the position of the epitopes for two monoclonal antibodies. This protein footprinting methodology is generally applicable to other proteins for which terminal modifications are acceptable, and provides a useful tool for mapping structure, substrate binding, and conformational changes. PMID- 7539793 TI - HNK-1-reactive novel oligosaccharide, sulfate-O-3GlcA beta 1-4Xyl beta 1-(4 methylumbelliferone), synthesized by cultured human skin fibroblasts. AB - 4-Methylumbelliferyl-beta-D-xyloside (Xyl-MU) was added to the medium of cultured human skin fibroblasts. After incubation, the culture medium was pooled, and the Xyl-MU-induced oligosaccharides in the medium were purified by gel filtration chromatography. A novel Xyl-MU derivative was obtained, in addition to the previously reported Xyl-MU derivatives such as Gal-Gal-Xyl-MU, Gal-Xyl-MU, Sia Gal-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU. The novel Xyl-MU derivative was purified using gel-filtration chromatography and high performance liquid chromatography and then subjected to carbohydrate composition analysis, enzymic digestion, Smith degradation, and ion spray mass spectrometric analysis. The results indicated that it was sulfate-O-3GlcA beta 1-4Xyl beta 1-MU. The structure of the nonreducing terminal of this Xyl-MU-induced oligosaccharide was the same as that of the oligosaccharide chain of a human peripheral nerve-derived glycolipid, reactive with the mouse monoclonal antibody HNK-1, and this Xyl-MU-induced oligosaccharide also reacted with HNK-1. These results suggest that the oligosaccharide, which is structurally identical to that of human peripheral nerve-derived glycolipid synthesized by nervous tissue and related to cell adhesion, is synthesized also by mesenchymal cells. PMID- 7539795 TI - Subcellular localization of human voltage-dependent anion channel isoforms. AB - The voltage-dependent anion channel of the outer mitochondrial membrane, VDAC (also known as mitochondrial porin), is a small abundant protein which forms a voltage-gated pore when incorporated into planar lipid bilayers. This protein forms the primary pathway for movement of major metabolites through the outer membrane. Recently, it has been demonstrated that two human VDAC genes, HVDAC1 and HVDAC2, produce three proteins that differ most significantly at their amino termini. These results suggest that the distinct amino termini lead to the targeting of individual VDAC isoforms to different cellular compartments. Consistent with this hypothesis, recent reports suggest that HIV-DAC1 is found in the plasma membrane of mammalian cells. To define the subcellular location of HVDAC isoforms, HVDAC genes were modified so that the encoded proteins contain COOH-terminal epitopes recognized by either of two monoclonal antibodies. Introduction of these epitope tags had no effect on the function of modified VDAC proteins. Epitope-tagged proteins were then individually expressed in COS7 cells or rat astrocytes and the intracellular location of each isoform subsequently identified by subcellular fractionation, light level immunofluorescence, and immunoelectron microscopy. Our results demonstrate that each HVDAC protein is exclusively located in fractions or subcellular regions containing mitochondrial marker proteins. In addition, immunofluorescence and immunoelectron microscopy show that an individual mitochondrion can contain both HVDAC1 and HVDAC2. Our results call into question previous reports demonstrating VDAC molecules in the plasma membrane and suggest that functional differences between individual VDAC isoforms may result in distinct regulatory processes within a single mitochondrion. PMID- 7539794 TI - A comparison of the interaction of Shc and the tyrosine kinase ZAP-70 with the T cell antigen receptor zeta chain tyrosine-based activation motif. AB - Tyrosine-based activation motifs (TAMs) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that couples the T cell antigen receptor to protein tyrosine kinases and adapter molecules. The present study shows that phosphorylation of both tyrosines within the motif is required for high affinity binding of the tyrosine kinase ZAP-70 whereas phosphorylation of the single COOH-terminal tyrosine within the motif is optimal for the binding of the adapter Shc. There were also quantitative differences in the ZAP-70 and Shc association with the zeta 1-TAM since nM concentrations of the doubly phosphorylated zeta 1-TAM are sufficient for ZAP-70 recruitment whereas micromolar levels of singly phosphorylated TAMs are necessary for Shc binding. Shc is tyrosine phosphorylated in antigen receptor-activated T cells and can potentially form a complex with the adapter molecule Grb2 and could thus recruit the Ras guanine nucleotide exchange protein Sos into the antigen receptor complex. The present data show that Grb2 can bind to the phosphorylated TAM, but this binding is independent of Shc and there is no formation of zeta 1-TAM.Shc.Grb2.Sos complexes in antigen receptor activated cells. Accordingly, Shc function should not be considered in the context of Grb2/Sos recruitment to the T cell antigen receptor complex. PMID- 7539796 TI - Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines. AB - By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related. PMID- 7539797 TI - A single amino acid residue can determine the ligand specificity of E-selectin. AB - E-selectin (ELAM-1) is a member of the selectin family of cellular adhesion molecules. This family of proteins possesses an amino-terminal Ca(2+)-dependent lectin or carbohydrate recognition domain that is essential for ligand binding. A known E-selectin ligand is the carbohydrate antigen, sialyl Lewis(x) (sLe(x) (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc). We have developed a model of E-selectin binding to the sLe(x) tetrasaccharide, (Neu5Ac alpha 2-3Gal beta 1 4(Fuc alpha 1-3)GlcNAc), using the NMR-determined, E-selectin-bound solution conformation of sLe(x) (Cooke, R.M., Hale, R.S., Lister, S. G., Shah, G., and Weir, M. P. (1994) Biochemistry 33, 10591-10596) together with the x-ray crystallographic structures of E-selectin (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, s., Huang, K.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) (ligand unbound) and a related C-type animal lectin, the mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson, A. (1992) Nature 360, 127-134) (ligand bound). Analysis of this model indicated that the alteration of one E-selectin amino acid, alanine 77, to a lysine residue might shift binding specificity from sLe(x) to mannose. The results presented here show that an E-selectin mutant protein possessing this change displays preferential binding to mannose containing oligosaccharides and that further mutagenesis of this mannose-binding selectin confers galactose recognition in a predictable manner. These mutagenesis data support the presented model of the detailed interactions between E-selectin and the sLe(x) oligosaccharide. PMID- 7539798 TI - Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase. AB - Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src. PMID- 7539799 TI - A novel form of dipeptidylpeptidase IV found in human serum. Isolation, characterization, and comparison with T lymphocyte membrane dipeptidylpeptidase IV (CD26). AB - Human CD26, a Type II membrane glycoprotein with intrinsic dipeptidylpeptidase IV (DPPIV) activity and ability to bind adenosine deaminase type I (ADA-1), is expressed on epithelial cells constitutively, but on T lymphocytes its expression is regulated. A soluble form of CD26/DPPIV has been described in plasma and related to immunological status, but it has been defined by the presence of DPPIV activity rather than by isolation. Using nondenaturing chromatographic techniques followed by nondenaturing native preparative electrophoresis, we obtained a homogeneous preparation of soluble serum DPPIV and compared it with a recombinant soluble CD26/DPPIV (rsCD26). We show that serum DPPIV is a monomer of 175 kDa in contrast to rsCD26 of 105-110 kDa, that it exists as a trimer, and that it is probably a serine proteinase. Deglycosylation removed N-linked sugar from both serum DPPIV and rsCD26; no O-linked glycosylation was observed, revealing a protein core of 130 kDa for serum DPPIV. The large serum form expresses functional DPPIV activity with substrate and inhibitor specificities and pH activity profile similar to those of rsCD26. Epitope analysis showed that monoclonal antibodies against five epitopes expressed by rsCD26 also bound, but more weakly, with serum DPPIV. Analysis of peptides after limiting proteolysis and N-terminal sequences reveals no homology with rsCD26 but some identity with other peptidases. Unlike rsCD26, the serum form does not bind ADA-1 and has no ADA-1 already associated with it. Similarly to rsCD26, serum DPPIV is a potent T cell costimulator. We conclude that the serum form of DPPIV is unique and is not a breakdown product of membrane CD26. The conservation of DPPIV activity and five epitopes specific to rsCD26 suggest, however, a significant structural similarity. PMID- 7539800 TI - Autocrine transforming growth factor beta 1 modulates the expression of integrin alpha 5 beta 1 in human colon carcinoma FET cells. AB - Transforming growth factor beta (TGF-beta) has been extensively studied as an exogenous agent that stimulates the expression of extracellular matrix proteins and their cell-surface integrin receptors in a variety of cell types. However, the recent demonstration of autocrine TGF-beta growth effects in a number of cell types suggests that the steady-state expression of extracellular matrix and integrin proteins and their biological activity may also be under autocrine TGF beta control. Previously, we reported that repression of autocrine TGF-beta 1 activity by constitutive expression of a full-length TGF-beta 1 antisense cDNA led to abrogation of autocrine negative TGF-beta and, as a result, increased tumorigenicity and anchorage-independent growth of a poorly tumorigenic, well differentiated colon carcinoma cell line designated FET (Wu, S., Theodorescu, D., Kerbel, R. S., Willson, J. K. V., Mulder, K. M., Humphrey, L. E., and Brattain, M. G. (1992) J. Cell Biol. 116, 187-196). Consequently, we have used this model system to study the effects of repression of autocrine TGF-beta 1 activity on the expression of integrin alpha 5 beta 1 and integrin alpha 5 beta 1-mediated cell adhesion to fibronectin. The expression of the integrin alpha 5 subunit was reduced in TGF-beta 1 antisense transfected FET cells at both mRNA and protein levels as determined by RNase protection assays and immunoprecipitation, respectively. Autocrine TGF-beta 1 had no effect on the transcription of integrin alpha 5 and beta 1 subunits, indicating that autocrine TGF-beta 1 may regulate integrin alpha 5 beta 1 expression at the post-transcriptional level. The diminished expression of integrin alpha 5 beta 1 on the cell surface led to the reduced adhesion of TGF-beta 1 antisense transfected cells to fibronectin. This phenomenon could be reversed by treatment with exogenous TGF-beta 1. PMID- 7539801 TI - Alpha 2-macroglobulin bait region variants. A role for the bait region in tetramer formation. AB - To test the hypothesis that a large portion of the bait region of human alpha 2 macroglobulin (alpha 2M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human alpha 2M variants with truncated bait regions and examined whether these variants folded normally and functioned as proteinase inhibitors. Each variant contains sites that are normal bait region cleavage sites in wild-type alpha 2M, including the primary trypsin cleavage site. The truncated bait regions are shorter by 23 and 27 residues, respectively, and lack the C-terminal portion as well as different parts of the N-terminal section of the bait region. We found that such bait region truncation permitted normal folding of the monomers as well as formation of the thiol ester and dimerization by disulfide cross-linking, although the resulting species bound 6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more like thiol ester-cleaved alpha 2M than native alpha 2M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type alpha 2M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric alpha 2M, decoupled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to "trap" proteinase. This was despite apparent cleavage of the bait region by proteinase, albeit at very much reduced rates relative to wild type tetrameric alpha 2M. The kinetics of thiol ester cleavage-dependent protein conformational changes also changed from sigmoidal to exponential. These findings indicate that residues in the bait region appear to be necessary for noncovalent association of 360-kDa disulfide-linked dimers to give tetrameric alpha 2M and suggest a role for the bait region in normal alpha 2M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping. PMID- 7539803 TI - Total cavopulmonary connection (TCPC) for complicated congenital heart malformations. AB - We reviewed our experience with 40 patients who had undergone total cavopulmonary connection (TCPC) during the past three years. Thirty-one patients had functional single ventricle; only 8 of these with tricuspid atresia, five patients had complex forms of double outlet right ventricle (DORV), and four complex A-V canal. Previous palliative procedures, mostly systemic-pulmonic shunts, were performed in 34 patients. Concomitant procedures were required in 18 patients, mainly reconstruction of distorted pulmonary arteries. A subgroup of 14 high risk patients, that did not fulfil the classical Fontan criteria, underwent 4 mm fenestration of the intra-atrial baffle. There were three (7.5%) early postoperative deaths which occurred in the higher risk group (fenestrated group). However, the remaining patients were all in functional class I or II. Total cavopulmonary connection provides reasonably good definitive palliation for patients with single ventricle physiology. Fenestration of the intra-atrial baffle increases the number of candidates suitable for the Fontan procedure, although the exact inclusion criteria for these patients has yet to be defined. PMID- 7539802 TI - Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells. AB - The c-kit-encoded tyrosine kinase receptor for stem cell factor (Kit/SCFR) is crucial for the development of hematopoietic cells, melanoblasts, and germ cells. Ligand stimulation of Kit/SCFR leads to receptor dimerization and autophosphorylation on tyrosine residues. We recently showed, that protein kinase C (PKC) acts in an SCF-stimulated negative feedback loop, which controls Kit/SCFR tyrosine kinase activity and modulates the cellular responses to SCF (Blume Jensen, P., Siegbahn, A., Stabel, S., Heldin, C.-H., and Ronnstrand, L. (1993) EMBO J. 12, 4199-4209). We present here the identification of the major phosphorylation sites for PKC in Kit/SCFR. Two serine residues in the kinase insert, Ser-741 and Ser-746, are PKC-dependent phosphorylation sites in vivo and account for all phosphorylation by PKC in vitro. Together they comprise more than 60% of the total SCF-stimulated receptor phosphorylation in living cells and 85 90% of its phosphorylation in resting cells. Two additional serine residues, Ser 821 close to the major tyrosine autophosphorylation site in the kinase domain and Ser-959 in the carboxyl terminus are SCF-stimulated PKC-dependent phosphorylation sites. However, they are not phosphorylated directly by PKC-alpha in vitro. Both specific receptor tyrosine autophosphorylation and specific receptor-associated phosphatidylinositide 3'-kinase activity was increased approximately 2-fold in response to SCF in PAE cells stably expressing Kit/SCFR(S741A/S746A). Furthermore, the kinase activity of Kit/SCFR(S741A/S746A) toward an exogenous substrate was increased, which was reflected as a decreased Km and an increased Vmax, in accordance with the negative regulatory role of PKC on Kit/SCFR signaling. PMID- 7539804 TI - The use of Tachocomb in thoracic surgery. AB - Diffuse bleeding of the thoracic wall still remains a major problem in thoracic surgery. Especially after lysis of strong adhesions of the lung to the parietal pleura blood loss from multiple bleeding points can be quite extensive. The thoracic apex, poorly accessible to visual inspection and electrocoagulation for anatomic reasons, can be a major bleeding source too. The closure of multiple vascular leaks with fibrin sealant alone has not proven to be satisfactorily efficient, since fibrin often is washed away by the bleeding itself. With Tachocomb, a newly developed collagen patch, successful tamponade of large bleeding areas has now become possible. By local positioning of the patch the stopping of bleeding can be achieved easily and quickly. Another important use of Tachocomb in lung surgery is the closure of parenchymal leakage. PMID- 7539805 TI - Pulmonary resection in invasive pulmonary aspergillosis. PMID- 7539806 TI - The use of high doses of aprotinin to decrease blood loss in aortocoronary by pass surgery. AB - There were examined 100 patients who had been operated in extracorporeal circulation for ischemic heart disease. In the 1st group (50 patients), each patient intraoperatively received 6,000,000 KIE of aprotinin; in the 2nd group (50 patients) placebo. Both groups were similar as for the age, duration of extracorporeal circulation, and myocardium anoxia. The total intraoperative blood loss was higher in 2nd group, 638 +/- 5 ml and 952.4 +/- 15.5 ml p < 0.05 correspondingly. The rate of cases without the intraoperative use of donor blood was 42% in group 1 and 18% in group 2. The postoperative blood loss was significantly lower with the use of aprotinin, 392 +/- 6.6 ml and 689.7 +/- 9.6 ml, p < 0.05 correspondingly; and in that the Hb concentration in the drainage blood was higher in group 2: 31.3 +/- 0.5 g and 73.1 +/- 2.2 g, p < 0.05. In group 1 22% of the patients received donor blood in the early postoperative period, and group 2 twice as many -56%. Thus prolonged intraoperative use of large aprotinin dosages in aortocoronary by-pass surgery is an effective means for stabilization of homeostasis, and it allows to reduce intra- and postoperative blood loss and diminishes the requirements in donor blood. PMID- 7539807 TI - Consequences of a combined administration of different doses of aprotinin and autologous blood transfusions for coagulation and fluid replacement in cardiac surgery. PMID- 7539808 TI - Role of protease inhibition in myocardial preservation following ischemia and reperfusion. AB - Aprotinin, a naturally occurring protease inhibitor, in concentrations of 10(6) KIU/L was found to have no effect on myocardial performance in normally perfused isolated rat hearts, before ischemia. Given during the preischemic period, the drug had a significant protective effect on the reperfused hearts, following cardioplegic ischemia: better contractility upon reperfusion (p < 0.011), faster decline of the ischemic contracture, higher coronary flow (p < 0.025), lower AV difference (p < 0.05), and lower CPK levels (p < 0.01). PMID- 7539809 TI - A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. AB - We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed. PMID- 7539811 TI - Calcium signal induced by mechanical perturbation of osteoclasts. AB - Multinucleated osteoclasts from rabbit long bone, 1-6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin-induced [Ca2+]i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release. PMID- 7539810 TI - The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14. AB - Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer. PMID- 7539812 TI - Tyrosine kinases are involved with the expression of inducible nitric oxide synthase in human articular chondrocytes. AB - The present study characterizes mechanisms involved with the induction of nitric oxide (NO) production, nitric oxide synthase (NOS) enzymatic activity and mRNA expression in human articular chondrocytes. Activation of chondrocytes with lipopolysaccharide (LPS) or IL-1 resulted in time- and dose-dependent increases in iNOS mRNA followed by increased NOS enzymatic activity and NO release. The protein tyrosine kinase (PTK) inhibitors herbimycin A or genistein reduced IL-1 or LPS-induced NO release and NOS enzymatic activity. This was associated with inhibition of iNOS mRNA expression as determined by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. In contrast, inhibitors of protein kinase C (PKC) or protein kinase A (PKA) did not affect these responses. These results were confirmed in experiments with second messenger agonists where neither activation of PKC, nor increases in cyclic adenosine monophosphate (cAMP) or increased intracellular calcium levels were associated with the induction of iNOS mRNA or NO release. These results suggest that PKC, PKA and calcium-dependent signals are not required or sufficient for the stimulation of NO production. However, NO production is dependent on tyrosine kinases due to their role in the expression of iNOS mRNA. PMID- 7539813 TI - De novo synthesis of glutathione is required for both entry into and progression through the cell cycle. AB - To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH-3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose-dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit. PMID- 7539814 TI - Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. AB - Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems. PMID- 7539815 TI - Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line. AB - Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain incompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPIb expression. Moreover, although these agents used alone did not affect GPIIb/IIIa expression, they markedly reversed phorbol ester-induced GPIIb/IIIa expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPIb and GPIIb/IIIa expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis. PMID- 7539816 TI - Fibroblast growth factor-2 (FGF-2) is present in maternal and cord serum, and in the mother is associated with a binding protein immunologically related to the FGF receptor-1. AB - Fibroblast growth factor-2 (FGF-2) is expressed in human fetal tissues and placenta. We, therefore, determined whether FGF-2 appeared in either fetal or maternal circulations during normal pregnancies [fetuses appropriate for gestational age (AGA)] or those complicated by fetal growth restriction (small for gestational age). Cordocentesis was performed, and matched maternal blood was collected between 19-39 weeks gestation, whereas maternal and cord blood and amniotic fluid (AF) were collected at term. FGF-2 was extracted from maternal serum (MS), cord serum (CS), and AF by heparin-Sepharose affinity chromatography and subjected to Western blot analysis or quantified by specific RIA. Western blot analysis of MS, CS, and AF revealed, in each case, a single immunoreactive FGF-2 species of 18 kilodaltons (kDa), although this was not present in nonpregnant serum. In AGA pregnancies, immunoreactive FGF-2 was present in MS from at least 18 weeks gestation and rose to maximum values at the end of second trimester (weeks 28-31; mean +/- SEM, 342 +/- 62 pmol/L), but by term had declined (weeks 40-42; 104 +/- 24 pmol/L). In CS, FGF-2 immunoreactivity was highest at weeks 18-20 of gestation (662 +/- 144 pmol/L), but thereafter, slowly declined to term (weeks 40-42; 119 +/- 28 pmol/L). Immunoreactive FGF-2 levels in MS and CS of small for gestational age pregnancies in the second trimester tended to be lower than those in AGA pregnancies, but differences were not statistically significant. AF also contained immunoreactive FGF-2 at term (91 +/- 35 pmol/L). Neutral gel chromatography on Sephadex G-200 revealed that FGF-2 immunoreactivity eluted as a broad peak with an apparent molecular size of 55-160 kDa. These same fractions contained peptides of 55-60, 90-95, and 120-130 kDa, which were recognized by antisera against the extracellular domain of the high affinity FGF receptor, FGFR1, after Western blot. Ligand blot analysis of the same nitrocellulose filters using 125I-labeled FGF-2 revealed that the 55- to 60-kDa species specifically bound FGF-2. This binding species was not recognized during Western blot analysis using an antiserum raised against the intracellular tyrosine kinase domain of FGFR1, suggesting that it represents a truncated receptor form. Similar FGFR1 immunoreactive species were present in nonpregnant female and male sera, but were barely detectable in term CS or AF.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7539817 TI - The effects of recombinant human insulin-like growth factor-I and growth hormone on body composition in elderly women. AB - The purpose of this study was to determine the effects of recombinant human GH (rhGH; 0.025 mg/kg.day) and one of two doses of recombinant human insulin-like growth factor-I (rhIGF-I; 0.015 and 0.060 mg/kg, twice daily) on body composition in elderly women. Sixteen healthy elderly women (mean age +/- SEM, 71.9 +/- 1.3 yr) were randomly assigned to receive either rhGH (GH; n = 5), low dose rhIGF-I (n = 6), or high dose rhIGF-I (n = 5). A 2-week predrug baseline period was followed by 4 weeks of hormone treatment, with a standardized diet fed throughout. All groups experienced a significant increase in serum IGF-I and IGFBP-3 levels over the treatment period, accompanied by significant decreases in IGF-II (P < 0.05). Fat mass decreased in all groups, with significant increases in lean body mass and nitrogen retention occurring in the high dose IGF and GH groups. Total body water did not change, whereas increases observed in intracellular fluid approached significance (P = 0.06). These anabolic changes were accompanied by numerous negative side-effects in the GH and high dose IGF groups, including headaches, lethargy, joint swelling/pain, and bloatedness. The low IGF dose was well tolerated. These results demonstrate that the administration of rhGH and rhIGF-I for 4 weeks results in anabolic changes in body composition in elderly women. PMID- 7539818 TI - Human galanin reduces plasma norepinephrine levels in man. AB - The neuropeptide galanin (GAL) is widely distributed in the peripheral and central nervous systems, where it often coexists with catecholamines. To gain insight into the action of human GAL on sympathetic nervous system activity in man, we investigated the effects of a 60-min infusion of human (h) GAL (80 pmol/kg.min) or saline on peripheral norepinephrine (NE) and epinephrine concentrations, heart rate (HR), and systolic and diastolic blood pressure (BP) in the supine position as well as after assumption of the upright posture (UP) in eight healthy male volunteers. hGAL depressed supine plasma NE (0.84 +/- 0.06 vs. 0.33 +/- 0.02 nmol/L) and blunted the NE response to assumption of the UP (1.68 +/- 0.03 vs. 0.44 +/- 0.03 nmol/L), but caused a significant enhancement of the epinephrine response to assumption of the UP (0.22 +/- 0.02 vs. 0.65 +/- 0.06 nmol/L). hGAL significantly increased supine HR (70 +/- 2 vs. 99 +/- 4 beats/min) and potentiated the HR response to assumption of the UP (82 +/- 3 vs. 107 +/- 4 beats/min). hGAL did not alter supine systolic and diastolic BP, but caused a significant decrease in the systolic (121 +/- 3 vs. 98 +/- 2 mm Hg) and diastolic (74 +/- 2 vs. 62 +/- 2 mm Hg) BP responses to assumption of the UP. Our data show that hGAL decreases supine position- and UP-stimulated release of NE, suggesting an inhibitory modulation of hGAL on sympathetic outflow in man. The finding that hGAL induces an increase in HR, both in the supine position and after UP, and an inhibition of the systolic and diastolic BP response to UP provides further support for an involvement of hGAL in regulation of the cardiovascular and autonomic nervous systems in man. PMID- 7539819 TI - Human testicular aromatase: immunocytochemical and biochemical studies. AB - The location of the aromatase enzyme in the testes of human subjects ages 3 months to 72 yr was determined immunocytochemically using a monoclonal antibody specific to human aromatase. In addition, an assay was developed to measure aromatase activity in suspensions of cryosections in order to validate the presence and to quantitate the activity of aromatase in the sections. In mature testes, aromatase immunostain was always associated with Leydig cells and was absent from Sertoli cells. Aromatase activity ranged from 0.014-0.55 pmol estrogen per mg/h and was significantly correlated with the immunostain intensity (P < 0.02). Thus, the aromatase activity provided a semiquantitative estimate of the immunoreaction. Activity and immunostain intensity of aromatase did not correlate with increasing age. Rather, the highest levels were measured in four of six testes of men ages 18-20 yr, three of whom also had the strongest immunostain for aromatase. The Leydig cell clusters of these subjects were larger and more prominent than those in the other specimens. A low level of aromatase activity but no immunostain was detected in prepubertal testes. Aromatase was not detected by either method in normal tissue from the testis of a patient with testicular cancer. PMID- 7539820 TI - Islet amyloid polypeptide in pancreatic tissue of children with persistent hyperinsulinemic hypoglycemia caused by primary islet hyperplasia and nesidioblastosis. AB - Islet amyloid is a recognized characteristic finding in insulinoma and secondary islet hyperplasia resulting from severe insulin resistance. Little information is available about the presence of islet amyloid in primary islet hyperplasia (nesidioblastosis) of childhood. Here we report that islet amyloid was not present in 12 children with primary islet hyperplasia associated with hyperinsulinemic hypoglycemia. PMID- 7539821 TI - Differences between normal and developmentally disabled children in a first dental visit. AB - Twenty-eight three- and four-year-old normal children participated in a study where they were exposed to a tape-slide series before a first dental examination. The audiovisual product gives an explanation about a first examination, the children having no previous dental experience. The purposes of the study were to evaluate the behavior modification that took place and to see whether differences could be found with the results of the same study performed four years before, with developmentally disabled children. The results obtained with normal children were the opposite of those obtained with special children. The normal population subjected to the audiovisual technique behaved better and had a lower heart rate throughout the appointment. PMID- 7539822 TI - Treatment of ventricular arrhythmias in older adults. AB - OBJECTIVE: To review the prognosis and management of ventricular arrhythmias (VA) in persons with and without heart disease with emphasis on older adults. DATA SOURCES: A computer-assisted search of the English language literature (MEDLINE database) followed by a manual search of the bibliographies of pertinent articles. STUDY SELECTION: Studies on the prognosis and management of VA in persons with and without heart disease were screened for review. Studies in older people and recent studies were emphasized. DATA EXTRACTION: Pertinent data were extracted from the reviewed articles. Emphasis was on studies involving the older persons. Relevant articles were reviewed in depth. DATA SYNTHESIS: Available data about the prognosis and management of VA in persons with and without heart disease, with emphasis on studies involving older people, were summarized. CONCLUSIONS: VA in older persons without heart disease should not be treated with antiarrhythmic drugs. Class I antiarrhythmic drugs should not be used to treat VA in older persons with heart disease. Beta blockers should be used to treat complex VA in older persons with ischemic or nonischemic heart disease if there are no contraindications to beta blocker therapy. The use of amiodarone in treating complex VA should be reserved for life-threatening ventricular tachyarrhythmias in older persons who cannot tolerate or who do not respond to beta blockers. VA associated with congestive heart failure should be treated with angiotensin converting enzyme inhibitors. If older patients have life-threatening recurrent ventricular tachycardia or ventricular fibrillation resistant to antiarrhythmic drugs, invasive intervention should be performed. The automatic implantable cardioverter-defibrillator is recommended in older patients who have medically refractory sustained ventricular tachycardia or ventricular fibrillation. PMID- 7539823 TI - Probing T-cell signal transduction pathways with the immunosuppressive drugs, FK 506 and rapamycin. AB - In addition to their clinical utility in tissue transplantation the immunosuppressive agents FK-506 (Prograf) and rapamycin, have proven to be valuable tools for gaining insight into the biochemistry of T-cell activation. The findings that the protein phosphatase calcineurin and cell cycle control are key elements in T-cell activation and proliferation are the direct result of investigations into the mechanism of action of FK-506 and rapamycin and provide potentially novel therapeutic targets. PMID- 7539824 TI - Detection of T cells responsive to a vascular growth factor in rheumatoid arthritis. AB - The primary lesion in rheumatoid arthritis (RA) is a destructive synovitis characterized by proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells, and with perivascular lymphocyte aggregates. A nonhematopoietic growth factor, acidic fibroblast growth factor (aFGF), may induce many of the biological features found in rheumatoid synovium, including T cell activation. To determine if aFGF-responsive T cells are increased in RA, we developed an assay to measure the frequency of peripheral blood T cells that are costimulated by aFGF. The data indicate that the frequency of aFGF-responsive T cells is increased in RA and may change with disease activity and treatment. PMID- 7539826 TI - [Periodic changes in serum endogenous granulocyte colony stimulating factor concentration during the menstrual cycle]. PMID- 7539825 TI - Granulocyte-macrophage colony-stimulating factor mimicry and receptor interactions. AB - Development of small molecular mimics of larger polypeptide ligands is an important approach to pharmacophore design. One strategy for the development of such mimics is analysis of alternative ligands that bind to the same site as the native ligand. These allow examination of the structural and chemical constraints for binding within the setting of diverse backbone geometries. The use of antireceptor antibodies as alternative ligands has allowed the development of biologically active peptides in several ligand-receptor systems. This technology has been applied to the study of interactions between human granulocyte macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR). GM-CSF is one of a family of signal-transducing cytokines and growth factors characterized by a four-helix bundle core structure. The GM-CSFR is comprised of an alpha-chain (GM-CSFR alpha) specific for GM-CSF, and a beta-chain (beta c) shared with the interleukin-3 and interleukin-5 receptors. At least two sites on GM-CSF have been implicated in the GM-CSF-GM-CSFR alpha/beta c ternary complex. In studies summarized here, synthetic peptide analogs of GM-CSF sequences were designed and used to map neutralizing epitopes. One neutralizing epitope corresponded to the A helix of GM-CSF, and a synthetic analog displayed biological activity as a GM-CSF antagonist in vitro, suggesting interaction with the GM-CSFR alpha/beta c complex. A second peptide comprising the B and C helices was recognized by monoclonal neutralizing antibodies and similarly displayed antagonist activity. Recombinant antibody (rAb) technology was also employed. An expression library of rAbs from mice immunized with neutralizing anti-GM-CSF antibodies was developed and screened with a neutralizing anti-GM-CSF monoclonal antibody. One clone which displayed receptor binding activity exhibited structural similarity with epitopes on GM-CSF previously implicated as interaction sites with the neutralizing monoclonal antibody. A synthetic peptide analog of the rAb inhibited GM-CSF bioactivity. Critical contact residues were predicted on the basis of structural similarity of the rAb peptide and GM-CSF. These studies indicate the feasibility of using rAbs in bioactive peptide design, providing lead compounds and information regarding contact residues for drug design. PMID- 7539827 TI - Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans. AB - Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3' tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo. PMID- 7539828 TI - Clinical characteristics and antibody profiles of chronic hepatitis C patients: relation to hepatitis C virus genotypes. AB - Genotyping of 179 consecutive Japanese chronic hepatitis C patients was carried out based on the variation in the hepatitis C virus (HCV) core gene. The results were correlated with clinical features and antibody responses toward specific HCV proteins deduced from the nucleotide sequence of genotype I/1a. Genotypes II/1b, III/2a, and IV/2b were identified in 138 (77%), 24 (13%), and 12 (7%) patients, respectively. Five patients had double infections. Genotype dependence was observed only for antibody response toward the NS4 (5-1-1) protein, which was infrequent in genotype III/2a patients (33%) compared with genotype II/1b (81%; P < 0.01) and genotype IV/2b (75%; P < 0.05). Following interferon-alpha therapy, sustained amniotransferase normalisation was achieved by 89% (eight of nine) patients without antibody to the 5-1-1 protein and 33% (17 of 51) with it (P < 0.01). These findings indicate that absence of antibody response to the 5-1-1 protein is frequent in genotype III/2a HCV carriers and may serve to predict responses to interferon therapy. PMID- 7539830 TI - Dominant replication of either virus in dual infection with hepatitis viruses B and C. AB - To characterize the state of dual infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), HBV-DNA and HCV-RNA levels were determined by spot hybridization or polymerase chain reaction in the sera of patients who were positive for both hepatitis B surface antigen and HCV antibody. Among 27 patients who showed evidence of double infection, 21 (77.8%) had detectable levels of only either HBV or HCV genome in their sera, 2 (7.4%) showed none of the viral genomes, and 4 (14.8%) had both HBV-DNA and HCV-RNA. In the 4 patients with both HBV-DNA and HCV-RNA, the titers of HCV-RNA or HBV-DNA were lower than those in the patients with HCV-RNA or HBV-DNA alone. In some patients with chronic hepatitis, the viruses appeared to replicate in turn in the course of the disease. These results indicate that the viruses show alternating dominance in replication in most of the patients who have dual infection with HBV and HCV, probably due to interference of the viruses. PMID- 7539829 TI - Hepatitis B and D genomes in hepatitis B surface antigen negative patients with chronic hepatitis C. AB - Hepatitis B and hepatitis D viral genomes were tested by nested polymerase chain reaction in the serum and liver of 69 hepatitis B surface antigen (HBsAg) negative, anti-hepatitis C virus (HCV) positive patients (47 with HCV RNA and 22 without HCV RNA). Serum hepatitis B virus (HBV) DNA-was detected in 49% of the patients with HCV-RNA and in 64% of those without HCV-RNA. Furthermore, intrahepatic HBV-DNA was found in four of five (80%) of the biopsies analysed. Delta genome was found in 72% and 73%, respectively, of the anti-HCV positive patients with or without HCV-RNA. In addition, intrahepatic delta virus genome was detected in another four liver biopsies studied. In the group of patients with HCV-RNA, the simultaneous presence of hepatitis B and D genomes was statistically higher in transfused patients than in drug addicts, or in those with an unknown infection route (P < 0.001). These results show a high percentage of B and D genomes in HBsAg negative patients with anti-HCV, irrespective of the presence or absence of the HCV genome. However, the clinical implications of this finding should be examined in future studies. PMID- 7539831 TI - Hepatitis C infection and viremia in Dutch hemophilia patients. AB - Serum samples from 316 patients visiting the Dutch National Hemophilia Center were collected from 1979 to 1993 and stored at -30 degrees C. Patients were placed into three different groups: 1) patients ever treated with large pool non hepatitis C virus (HCV)-safe concentrate (n = 179); 2) patients treated with cryoprecipitate (n = 125); and 3) patients treated exclusively with HCV-save concentrate (n = 12). In order to examine the prevalence of HCV infection in the different treatment groups serum samples were tested retrospectively for anti-HCV antibody using second generation enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA-2). Significant differences in the prevalence of HCV infection were found between these 3 groups (group 1: 99%, group 2: 66%, group 3: 0%). The safety of currently administered clotting products is demonstrated in 57 patients who remained without HCV markers between 1989 and 1993. To examine the natural course of HCV infection fresh-frozen plasma samples were obtained recently from a subgroup of 277 hemophilia patients for HCV-RNA detection by a well-validated cDNA-PCR assay. In contrast to other reports, no evidence was found for seronegative HCV carriers. None of 52 patients without anti-HCV had detectable HCV-RNA. Of 225 patients with anti-HCV, 182 (81%) were HCV-RNA positive. None of 39 anti-HCV positive patients with a negative HCV-RNA reaction had serum alanine aminotransferase (ALT) levels above 50 U/l, whereas 44% of HCV-RNA positive patients had persistently elevated ATL levels above 50 U/l.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539832 TI - Prokaryotic expression and analysis of the antibody response to a Newcastle isolate of the core gene of hepatitis C. AB - The full length hepatitis C virus (HCV) core gene was isolated from a Newcastle strain and expressed in E. coli. A truncated HCV core gene which lacks the hydrophobic carboxyl-terminal sequence was also expressed. The truncated HCV core was expressed at higher levels with fewer cleavage products. Antibody reactivity to the recombinant HCV core antigen was analysed by ELISA and Western blotting in 60 HCV antibody-positive patients with a broad spectrum of liver disease. There was no significant difference between the presence of IgG to recombinant HCV core and reactivity to the core antigen in the RIBA-2 test. There was also no significant difference between the presence of IgG to recombinant core and diagnostic PCR as a marker for active liver inflammation. PMID- 7539834 TI - Screening a monoclonal antibody with a fusion-phage display library shows a discontinuity in a linear epitope within PreS1 of hepatitis B virus. AB - The epitope recognized by the monoclonal antibody MA18/7, specific for the PreS1 domain of the hepatitis B virus surface antigen, has been defined precisely by means of a library of fusion-phage carrying random hexapeptides on the tip of filamentous phage fd particles. Phage, isolated after only one round of affinity selection, displayed hexapeptides showing strong conservation of the PreS1 primary sequence in the region 19-23 with three noncontiguous residues, DP (20 and 21) and F (23) appearing in phage that bound the antibody. The importance of these core residues was supported by comparing the antibody binding of individual phage in solution, which provided relative dissociation constants for these interactions. Replacement of F (23) by Y was the only substitution observed in the three core residues, and resulted in somewhat weaker binding. Synthetic tetra and hexapeptides containing these key residues inhibited the reaction between the phage and the antibody. PMID- 7539833 TI - Localization and reactivity of an immunodominant domain in the NS3 region of hepatitis C virus. AB - Analysis of the amino acid sequences of the nonstructural region 3 (NS3) of the hepatitis C virus type 1 revealed four points with a high average hydrophilicity (Ah). Two of these potential antigenic sites were expressed in E. coli as short fragments. The first fragment of 91 residues (NS3f3: residues 1359-1449) harbors the hexapeptide K-K-K-C-D-E with an Ah of 2.33; the second fragment is 73 residues long (NS3f4: residues 1460-1532) and encompasses the heptapeptide R-S-N R-R-G-R with an Ah of 1.79. Both fragments were expressed with truncated hepatitis B core (tHBc) as a carrier protein. The fusion proteins were purified from the bacterial lysates by affinity chromatography on immobilized monoclonal antibodies against HBc, and evaluated as antigens in an enzyme immunoassay for the detection of HCV antibodies. In a specificity control panel, reactivity with NS3f3 was only found in proven HCV carriers, while reactivity with NS3f4 was weak in HCV carriers but accounted for some of the nonspecific serological reactions. In a group of 48 genotyped HCV-infected volunteer blood donors, antibodies against NS3f3 were detected in 90% (27/30) of HCV-type 1 infections and in all HCV-type 4 infections (5/5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539835 TI - How to develop a poster. 1989. PMID- 7539836 TI - Neonatal-onset propionic acidemia: neurologic and developmental profiles, and implications for management. AB - OBJECTIVES: To document the clinical and neurodevelopmental profiles of a cohort of patients with neonatal-onset propionic acidemia and to determine the efficacy of current therapy with respect to outcome. METHOD: The clinical, neurologic, and developmental status of six patients was prospectively evaluated during a 15 month period. Previous clinical and biochemical data were ascertained from hospital records to determine longitudinal nutritional status, number of episodes of hyperammonemia with ketoacidosis, and developmental performance with respect to age. RESULTS: No deaths resulted from propionic acidemia since the identification of the oldest patient in the series in 1980. Therapeutic intervention (e.g., gastrostomy tube feeding) resulted in improved nutritional status and possibly contributed to improved survival. All children had hypotonia, resulting in a significant effect on motor development; however, focal neurologic deficits and evidence of movement or seizure disorder were absent. Mild cortical atrophy was evident on cranial magnetic resonance imaging in four patients. All children, including two patients with no significant episodes of hyperammonemia and normal growth since the neonatal period, had a mild to moderate degree of intellectual impairment. CONCLUSIONS: The results of our study suggest that current therapy for neonatal-onset propionic acidemia is associated with improved survival and nutritional status, and an absence of focal neurologic deficits. However, hypotonia and cognitive delay were still present, even in children with "optimal" metabolic control. Additional therapeutic advances are required to improve the developmental and cognitive outcome. PMID- 7539837 TI - Lipopolysaccharides from periodontal pathogens prime neutrophils for enhanced respiratory burst: differential effect of a synthetic lipid a precursor IVA (LA 14-PP). AB - When neutrophils are incubated with bacterial lipopolysaccharide (LPS), they become primed for enhanced release of superoxide anion (O2-) in response to stimulation by FMLP. We investigated the human neutrophil-priming activity of LPS from the periodontal pathogens, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Actinobacillus actinomycetemcomitans (Aa) in comparison with that of LPS from Escherichia coli (E. coli). The optimum conditions for LPS to prime neutrophils were assessed for every LPS and found to be as follows: Neutrophils were incubated with LPS in the presence of 10% heat-inactivated plasma and 1 mM EDTA at 37 degrees C for 30 min and then stimulated with 1 microM FMLP at 37 degrees C for 7 min. Under these conditions, half-maximum priming was observed at 6.2 ng/ml Pg-LPS, 45 ng/ml Pi-LPS, 1.5 ng/ml Aa-LPS and 1.5 ng/ml E. coli-LPS. The priming activity of each LPS was neutralized by polymyxin B. Anti CD14 monoclonal antibody inhibited priming by all LPS. The priming by Aa-LPS and E. coli-LPS was inhibited by LA-14-PP, a synthetic lipid A precursor IVA, but that by Pg-LPS and Pi-LPS was not. Priming by tumor necrosis factor alpha was not affected by polymyxin B, anti-CD14 antibody or LA-14-PP. Gelation of Limulus amebocyte lysate occurred at 10 pg/ml Pg-LPS, 30 pg/ml Pi-LPS, 3 pg/ml Aa-LPS and 3 pg/ml E. coli-LPS. Thus LPS from different periodontal pathogens primed neutrophils with different efficacy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539838 TI - Multilayer culture of periodontal ligament epithelial cells: a model for junctional epithelium. AB - The unique features of junctional epithelium involve lack of keratinization, limited differentiation and a relatively permeable structure. In order to study the relationship between differentiation and permeability of stratified epithelium a model system was developed. Porcine periodontal ligament epithelial cells were cultured on the polycarbonate nucleopore membrane of the Transwell two compartment culture system. Within 5 days of culture the cells formed a confluent multilayered structure. Subsequently, maturation of the structure and differentiation of surface cells took place. Transmission electron microscopy showed that the cells were arranged into basal and suprabasal layers with sparse desmosomal attachments and wide intercellular spaces resembling the organization of junctional epithelium. The basal cells attached to a subepithelial basal lamina through numerous hemidesmosomes. The cytokeratin profile of the cultured epithelium (K5, 6, 14, 16, 19) resembled that of the cells of junctional epithelium attached to the tooth surface. The older cultures expressed differentiation markers, K4, K13 and involucrin, thereby resembling sulcular epithelium. The epithelial permeability, measured by diffusion of phenol red, radioactive dextran or methionine tracers, and as transepithelial electrical resistance, decreased with the increased cell number and maturation of the cultures. The new model provides an organotypic culture system which allows to control differentiation of a multilayered periodontal epithelium. It thus may serve as a valuable new tool for studies on the permeability and behaviour of periodontal epithelium under the influence of exogenous and endogenous factors. PMID- 7539839 TI - Angiotensin II activation of a chloride current in rabbit cardiac myocytes. AB - 1. The effects of angiotensin II (Ang II) on membrane currents were investigated in single ventricular myocytes from the rabbit heart by the whole-cell voltage clamp method. 2. In the presence of an inhibitor of Ca2+ currents (nifedipine at 3 microM or CdCl2 at 0.3 mM) and a beta-adrenoceptor blocker (bupranolol at 1 microM), 1 microM Ang II significantly increased the membrane conductance. 3. After elimination of K+ from external and internal solutions and its replacement by Cs+, Ang II at 0.1 microM increased an outwardly rectifying current that reached a maximum after about 40 min. The effect was concentration dependent (10( 9)-10(-6) M) and was inhibited by saralasin, an antagonist of Ang II receptors. 4. The reversal potential of the Ang II-induced current in the absence of K+ was compatible with the Cl- equilibrium potential at various external concentrations of Cl-. 5. A Cl- channel blocker, 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS, at 5 mM), reversibly decreased the Ang II-induced current. 6. The Ang II induced current developed when the internal solution contained Ca2+ (pCa 7.2 or 7.0) but not when it contained 10 mM EGTA without Ca2+. 7. Besides developing a Cl- current, Ang II at 1 microM increased the inwardly rectifying K+ current (IK1) and this effect reached maximum within 3 min. 8. The effect of Ang II on the action potential was biphasic: the duration of the action potential was initially reduced and then it was increased. 9. These results suggest that Ang II induces a Cl- current that appears likely to modulate the action potential in rabbit ventricular myocytes. PMID- 7539840 TI - The slow Ca(2+)-activated K+ current, IAHP, in the rat sympathetic neurone. AB - 1. Adult and intact sympathetic neurones of the rat superior cervical ganglion maintained in vitro at 37 degrees C were analysed using the two-electrode voltage clamp technique in order to investigate the slow component of the Ca(2+) dependent K+ current, IAHP. 2. The relationship between the after hyperpolarization (AHP) conductance, gAHP, and estimated Ca2+ influx resulting from short-duration calcium currents evoked at various voltages proved to be linear over a wide range of injected Ca2+ charge. An inflow of about 1.7 x 10(7) Ca2+ ions was required before significant activation of gAHP occurred. After priming, the gAHP sensitivity was about 0.3 nS pC-1 of Ca2+ inward charge. 3. IAHP was repeatedly measured at different membrane potentials; its amplitude decreased linearly with membrane hyperpolarization and was mostly abolished close to the K+ reversal potential, EK (-93 mV). The monoexponential decay rate of IAHP was a linear function of total Ca2+ entry and was not significantly altered by membrane potential in the -40 to -80 mV range. 4. Voltage-clamp tracings of IAHP could be modelled as a difference between two exponentials with tau on approximately 5 ms and tau off = 50-250 ms. 5. Sympathetic neurones discharged only once at the onset of a long-lasting depolarizing step. If IAHP was selectively blocked by apamin or D-tubocurarine treatments, accommodation was abolished and an unusual repetitive firing appeared. 6. Summation of IAHP was demonstrated under voltage-clamp conditions when the depolarizing steps were repeated sufficiently close to one another. Under current-clamp conditions the threshold depolarizing charge for action potential discharge significantly increased with progressive pulse numbers in the train, suggesting that an opposing conductance was accumulating with repetitive firing. This frequency dependent spike firing ability was eliminated by pharmacological inhibition of the slow IAHP. 7. The IAHP was significantly activated by a single action potential; it was turned on cumulatively by Ca2+ load during successive action potential discharge and acted to further limit cell excitability. PMID- 7539841 TI - Activation of ATP-dependent K+ channels by hypoxia in smooth muscle cells isolated from the pig coronary artery. AB - 1. The perforated patch technique with amphotericin B was used to record whole cell currents activated by hypoxia in smooth muscle cells, isolated enzymatically from pig coronary arteries. 2. Superfusion with hypoxic solution (O2 partial pressure, 25-40 mmHg) activated an inward current at -60 mV in 143 mM extracellular K+. The reversal potential of the current induced by hypoxia shifted with extracellular [K+] as expected for a K+ current, while its current voltage relation was consistent with the channels showing little voltage dependence. 3. The hypoxia-induced current was inhibited by glibenclamide (10 microM), but was unaffected by charybdotoxin (50 nM). 4. In whole-cell recordings at -60 mV in 143 mM K+ solution, openings of single channels passing a current close to -2 pA could sometimes be detected in normoxic solution. Openings became more frequent during the onset of the response to hypoxia, when several levels could be detected. Channels with a similar conductance were activated by hypoxia in cell-attached patches. 5. Our results suggest that hypoxia activates ATP dependent K+ channels. We discuss possible mechanisms by which this activation may occur. PMID- 7539842 TI - Ca2+ regulation in the presynaptic terminals of goldfish retinal bipolar cells. AB - 1. To investigate regulation of the intracellular free Ca2+ concentration ([Ca2+]i) in presynaptic terminals, the Ca2+ current (ICa) and [Ca2+]i in axon terminals were simultaneously monitored in acutely dissociated retinal bipolar cells under whole-cell voltage clamp. 2. The recovery phase of the Ca2+ transient, which was evoked by activation of ICa, became slower when the Na(+) Ca2+ exchanger was suppressed by removing extracellular Na+. 3. Inhibition of the plasma membrane Ca2+ pump produced by raising extracellular pH to 8.4 increased the basal [Ca2+]i and caused incomplete recovery from the Ca2+ transient. These effects were not observed in orthovanadate-loaded bipolar cells. 4. The Ca2+ transient was not significantly affected by ryanodine, caffeine, thapsigargin, Ruthenium Red or FCCP. Internal Ca2+ stores may not participate in shaping the Ca2+ transient. 5. The ratio of the peak amplitude of the Ca2+ transient to the total amount of Ca2+ influx became smaller as the size of the Ca2+ influx increased. This action was not affected by blockage of Ca2+ transporters in the plasma membrane, or by reduction of the rate of Ca2+ influx. The peak amplitude of the Ca2+ transient seemed to be determined by Ca2+ buffering substances with a positive co-operativity. PMID- 7539843 TI - Ca(2+)-activated K+ channels in isolated type I cells of the neonatal rat carotid body. AB - 1. Ca(2+)-activated K+ (K+Ca) channels in neonatal rat type I carotid body cells were studied using single channel patch clamp techniques. In outside-out patches, using symmetrical 120 mM [K+] solutions, channels were observed with a slope conductance of 190 pS and a reversal potential of 0 mV. Reducing [K+]o to 5 mM shifted the reversal potential as expected for a K(+)-selective channel. 2. With 100 nM Ca2+ bathing the cytosolic aspect of patches, channel activity (number of active channels in a patch x open probability, NPo) increased with depolarization. NPo also increased with increasing 'cytosolic' [Ca2+] at a fixed membrane potential (0 mV). Using outside-out patches, bath application of 20 or 100 nM charybdotoxin reduced NPo by > 85%. These data indicate the presence of K+Ca channels in type I cells. 3. At 0 mV, using solutions of identical composition (1 microM Ca2+ bathing the cytosolic aspect of the channels), NPo was higher in outside-out patches than in inside-out patches. NPo was greatest in recordings using the perforated-vesicle technique. 4. Hypoxia and anoxia were without effect on K+Ca channels in outside-out patches, but caused significant, reversible reductions of NPo in channels recorded in perforated vesicles. 5. The whole-cell perforated-patch technique was used to record membrane potential at 35 37 degrees C. Hypoxia, anoxia and charybdotoxin all depolarized type I cells. 6. Our results suggest an important role for K+Ca channels in type I carotid body cells, and their activity in relation to a model for chemotransduction is discussed. PMID- 7539844 TI - Conductance and kinetics of single cGMP-activated channels in salamander rod outer segments. AB - 1. The conductance and kinetics of single 3',5'-cyclic guanosine monophosphate (cGMP)-activated channels of retinal rod outer segments were studied in inside out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two-state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s-1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s-1 and was voltage insensitive. Assuming that the open state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady-state macroscopic current-voltage relation of multichannel patches, at saturating cGMP concentration. PMID- 7539846 TI - Oral manifestations of infantile systemic hyalinosis. AB - Oral manifestations of infantile systemic hyalinosis in a child of Asian origin are presented. Infantile systemic hyalinosis is a rare fatal condition with probably an autosomal recessive mode of inheritance. The symptoms become apparent soon after birth and death usually occurs before the age of two years. The systemic features are essentially due to widespread deposition of hyaline material in tissues. These include thickening and nodularity of skin, growth failure, joint contractures, osteoporosis, diarrhoea and recurrent infections. The oral changes in the case reported here included thickening of the oral mucosa, extensive overgrowth of gingival tissue, osteoporosis, marked curvature of the dental roots, and replacement of periodontal ligament by hyaline fibrous material. Immunohistochemistry revealed widespread presence of Type VI collagen in the connective tissue with particularly intense staining in the hyaline material. PMID- 7539845 TI - Substance P effects on blood flow, fluid transport and vasoactive intestinal polypeptide release in the feline small intestine. AB - 1. Substance P (SP) infusions were given close I.A. to the feline small intestine in vivo in a dose that produced plasma concentrations of 1-5 microM. This infusion regularly evoked a net fluid secretion measured with a gravimetric technique. Concomitantly, the release into blood of vasoactive intestinal polypeptide (VIP), a putative neurotransmitter of the enteric nervous system, increased. 2. The SP-induced fluid secretion was blocked by tetrodotoxin (7 micrograms close I.A.), a blocker of fast sodium channels in excitable tissues, and hexamethonium (10 mg (kg body wt)-1, I.V.), a nicotinic receptor antagonist, suggesting that the SP effect was mediated by the enteric nervous system. In line with this it was shown that the SP-evoked release of VIP was also significantly diminished by hexamethonium. 3. Close I.A. infusions of methionine enkephalin (Met-enkephalin; 7-23 nmol min-1) or electrical stimulation of the sympathetic nerve fibres (6 Hz) to the intestine markedly diminished net fluid secretion and the release of VIP caused by SP given close I.A. 4. The cyclo-oxygenase inhibitor diclofenac (5 mg (kg body wt)-1, I.V.) or the histamine-1 receptor antagonist pyrilamine (10 mg (kg body wt)-1, I.V.) did not influence the fluid secretion caused by SP, indicating that the effects of SP were not due to the actions of prostaglandins or histamine. 5. It is proposed that SP activates a nervous reflex arch that we have shown to be activated by various luminal stimuli, including cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539848 TI - Radical surgery for advanced and recurrent breast cancer. AB - Locally advanced or recurrent breast cancer is commonly seen in breast clinics. One method of palliation for ulcerating tumours is a radical mastectomy with replacement of the chest wall defect with a myocutaneous flap. We report on our experience of this technique in the management of 35 women (21 advanced disease, 14 recurrent disease). The perioperative complications were minimal and there are some longterm survivors. 15 women are alive and well (range 20-96 months postoperatively, mean 55 months). This radical surgery, therefore, has a significant role in the management of selected women with breast cancer. PMID- 7539847 TI - DNA content and alkaline phosphatase expression in cells of different gingival overgrowths. AB - The present study compared the alkaline phosphatase (ALPase) expression and DNA content at specific periods in cultured cells derived from non-inflamed enlarged gingivae of idiopathic gingivofibromatosis (IGF) and phenytoin-induced hyperplasia (PHG). Cultured cells from healthy gingiva or periodontal ligament (PDL) were used as controls. The DNA assay, ALPase assay and cytochemical staining for ALPase in cultured cells were performed at four, seven, and nine days. The presence of intense ALPase activity was a prominent feature in cultured IGF cells, whereas very low ALPase activity was detected in PHG cells. The cell lines tested showed no significant differences in DNA content. The expression of ALPase in these cells was population density-dependent. The observation that cells isolated from both types of gingival overgrowth exhibited a different ALPase profile at variance with normal gingival fibroblasts suggested that a distinct pathogenic mechanism may be involved in each type of gingival overgrowth. PMID- 7539849 TI - Pregnancies complicated by idiopathic intrauterine growth retardation. Severity of growth failure, neonatal morbidity and two-year infant neurodevelopmental outcome. AB - We evaluated the impact of the severity of intrauterine growth retardation (IUGR) measured as the proportion of expected birth weight (birth weight x 100/median birth weight) on short-term neonatal complications and two-year infant neurodevelopmental outcome. The study was carried out on 236 singleton pregnancies complicated by idiopathic IUGR. The rates of bradycardia, respiratory distress syndrome, hypocalcemia, ventilatory support, apneic crises, transient neurologic signs and poor neonatal outcome (neonatal death or cerebral palsy) significantly correlated with the increasing severity of IUGR. In logistic regression analysis more severely growth retarded infants (< 67.5% of expected birth weight) had higher rates of bradycardia, respiratory distress syndrome, hypocalcemia and bacterial sepsis when compared with those less severely affected (84-67.5% of expected birth weight). In pregnancies complicated by idiopathic IUGR, most short-term neonatal complications are inversely related to the severity of growth failure as evaluated by the proportion of expected birth weight. PMID- 7539850 TI - Methotrexate treatment of cervical pregnancies with different clinical parameters. A report of three cases. AB - Three cases of cervical pregnancy with different clinical parameters were successfully treated with methotrexate. Case 1 was a viable cervical pregnancy, case 2 was a cervical pregnancy after curettage that developed into a cervical hematoma, and case 3 was a cervical pregnancy at an early gestational age. The interval between diagnosis and treatment ranged from 45 to 76 days. The maximum quantitative beta-human chorionic gonadotropin (beta-hCG) levels in cases 1, 2 and 3 were 100,180, 19,093 and 956 mIU/mL, respectively. These patients showed a progressive decline in beta-hCG levels, and ultrasound showed a gradual decrease in the size of the pregnancies. The interval between treatment and beta-hCG resolution ranged from 14 to 59 days. Only in case 2 did side effects occur, including stomatitis and transient elevation of serum transaminase. In view of the risks of standard therapy and patients' desire for fertility, methotrexate treatment may be a therapeutic alternative for cervical pregnancy. PMID- 7539851 TI - Hyalin degeneration in the cardiomyopathy of overload. PMID- 7539852 TI - Effects of the luteinizing hormone-releasing hormone agonist leuprolide on lipoproteins, fibrinogen and plasminogen activator inhibitor in patients with benign prostatic hyperplasia. AB - The impact of chronic administration of the luteinizing hormone-releasing hormone agonist leuprolide depot on cardiovascular risk factors was investigated in a controlled double-blind study comprising 50 evaluable patients with benign prostatic hyperplasia. In the 26 patients receiving leuprolide the mean total cholesterol level increased by 10.6%, high density lipoprotein cholesterol by 8.2% and triglycerides by 26.9% (p = 0.003, 0.052 and 0.050, respectively). Low density lipoprotein cholesterol levels were unchanged. Apolipoprotein A1 increased by 13.2% (p = 0.001), while apolipoprotein B, fibrinogen, thrombocytes and plasminogen activator inhibitor were unchanged. Hemoglobin decreased by 1.2 gm./100 ml. without a concomitant decrease in serum erythropoietin concentration. These changes act in different directions with regard to cardiovascular risk and the overall effect is difficult to assess. PMID- 7539853 TI - Doxazosin in the treatment of benign prostatic hyperplasia in normotensive patients: a multicenter study. AB - A 16-week, double-blind, placebo controlled, dose titration study was done on 100 normotensive patients age 45 years or older to determine the efficacy and safety of doxazosin, a selective alpha 1-adrenoceptor antagonist, in the treatment of benign prostatic hyperplasia (BPH). Of the 41 efficacy evaluable patients 88% underwent dose titration to a maximum of 8 mg. doxazosin once daily. Maximum and average urinary flow rates increased significantly above baseline with doxazosin (2.9 ml. per second and 1.4 ml. per second, respectively) compared with placebo (0.7 ml. per second and 0.3 ml. per second, respectively). A significant effect on maximum flow rate was noted as early as week 2 of double-blind treatment at the initial efficacy evaluation. Doxazosin was superior to placebo in patient and investigator assessments of total, obstructive and irritative BPH symptoms. The onset of efficacy for total patient-assessed symptoms was significant for doxazosin compared to placebo 4 weeks after the start of the treatment regimen. Statistically significant decreases in mean blood pressure of 4 to 6 mm. Hg were noted with doxazosin compared with placebo. Adverse events, primarily mild to moderate in severity, were reported in 44% of patients given doxazosin and 30% of those given placebo. Our results strongly demonstrate that doxazosin is significantly superior to placebo in the treatment of BPH in normotensive patients, with the patient experiencing significant relief early after initiation of therapy. PMID- 7539854 TI - Doxazosin for the treatment of benign prostatic hyperplasia in patients with mild to moderate essential hypertension: a double-blind, placebo-controlled, dose response multicenter study. AB - A total of 248 hypertensive patients 45 years old or older with benign prostatic hyperplasia (BPH) was included in this 16-week, multicenter, double-blind, placebo-controlled, parallel-group dose-response study. Doxazosin, a selective alpha 1-adrenoceptor antagonist, produced a significant increase in maximum urinary flow rate (2.3 to 3.6 ml. per second) at doses of 4 mg., 8 mg. and 12 mg., and in average flow rate (8 mg. and 12 mg.) compared with placebo. The increase in maximum flow rate was significant with doxazosin versus placebo within 1 week of initiating double-blind therapy. Doxazosin compared to placebo significantly decreased patient-assessed total, obstructive and irritative BPH symptoms. Blood pressure was significantly lower with all doxazosin doses compared with placebo. Adverse events, primarily mild to moderate in severity, were reported in 48% of patients on doxazosin and 35% on placebo. Our results strongly support the use of doxazosin as a nonoperative therapeutic alternative in the management of uncomplicated BPH. Doxazosin would also be particularly useful in the management of patients who have BPH and hypertension. PMID- 7539855 TI - Prostatic histology in secondary transurethral resection of the prostate. AB - Human benign prostatic hyperplasia consists of 3 major components, stromal and glandular tissue, and glandular lumen. To our knowledge morphometric analysis of prostatic tissue from patients who have undergone a secondary transurethral resection of the prostate has never been reported. Quantification of these histological components might aid in selecting treatment for patients with recurrent urinary symptoms following transurethral resection of the prostate. Transurethral resection specimens from 13 consecutive patients who had undergone prostatectomy twice were stained by the Masson trichrome method. Quantitative morphometric analysis was performed using computer image analyzing software. The mean total area was 15.9%, 15.4% and 68.7% for glandular lumen, and glandular and stromal areas, respectively, in the initial transurethral resection specimens and 13.3%, 14.1% and 72.6%, respectively, for secondary transurethral resection of the prostate. There was no significant difference among the 3 morphometric components in the 2 prostatectomy specimens with respect to percent area, within subject variability and skewness (all p values > or = 0.38). Our study confirms previous findings that the majority of initial transurethral resection specimens are predominantly stroma. Furthermore, our study indicates that the percent distributions between initial and secondary transurethral resection of the prostate with respect to the 3 major histological components are not different, suggesting that the stromal, glandular and glandular lumen content of an initial transurethral resection specimen is representative of the recurrent prostatic tissue. PMID- 7539856 TI - Benign prostatic hyperplasia. PMID- 7539857 TI - A multivariate analysis of clinical and pathological factors that predict for prostate specific antigen failure after radical prostatectomy for prostate cancer. AB - A Cox regression multivariate analysis was done to determine the clinical and pathological indicators that predict for prostate specific antigen (PSA) failure in 347 patients who underwent radical prostatectomy for clinically localized prostate cancer between 1989 and 1993. In the patient subgroups (PSA less than 20 ng./ml. and biopsy Gleason sum 5 to 7 or PSA more than 10 to 20 ng./ml. and biopsy Gleason sum 2 to 4) not classifiable into those at high and low risk for postoperative PSA failure using PSA and biopsy Gleason sum, the status of the seminal vesicles and prostatic capsule on endo-rectal coil magnetic resonance imaging (MRI) allowed for this categorization. Specifically, 2-year actuarial PSA failure rates were 84% versus 23% in patients with and without seminal vesicle invasion, respectively, on MRI (p < 0.0001) and 58% versus 21% in those with and without extracapsular extension, respectively (p = 0.0001). In patients with extracapsular extension but without pathological involvement of the seminal vesicle(s) or poorly differentiated tumors (pathological Gleason sum 8 to 10), the 2-year actuarial PSA failure rates were 50% (margin positive), 28% (margin negative with established extracapsular disease) and 9% (margin negative with focal microscopic extracapsular disease). Therefore, endo-rectal coil MRI showing seminal vesicle invasion or extracapsular extension when the PSA level is less than 20 ng./ml. and the biopsy Gleason sum is 5 to 7 or the PSA level is more than 10 but less than 20 ng./ml. and the biopsy Gleason sum is 2 to 4 predicted for PSA failure. In patients with extracapsular extension who had pathological Gleason sum less than 8 disease with uninvolved seminal vesicles, the margin status and extent of extracapsular disease predicted for PSA failure. PMID- 7539858 TI - Prostate cancer. PMID- 7539859 TI - Relationship between lower urinary tract abnormalities and disease-related parameters in multiple sclerosis. AB - Multiple sclerosis affects the lower urinary tract in many patients. The relationship between lower urinary tract abnormalities and disease-related parameters of multiple sclerosis is not well described. We screened urologically and neurologically 212 patients according to a standard protocol. Micturition complaints were noted in 52% of the patients and urodynamic abnormalities were found in 64%. A statistical correlation was found between detrusor hyperactivity and detrusor hypoactivity with disease-related parameters, that is disease duration, disability status, myelin basic protein concentration in the cerebrospinal fluid and neurophysiological investigations. No relationship was found between detrusor hypersensibility or detrusor hyposensibility and the aforementioned disease-related parameters. In 1 patient upper urinary tract abnormalities were noted in combination with urodynamic abnormalities. We conclude that lower urinary tract abnormalities can be found in every patient with multiple sclerosis unrelated to the state of the disease. Severe upper urinary tract abnormalities are rare. PMID- 7539861 TI - Practice trends in the diagnosis and management of benign prostatic hyperplasia in the United States. AB - PURPOSE: The American Urological Association (AUA) first commissioned the Gallup Organization of Princeton, New Jersey to conduct a study to assess urologists' practice patterns in 1992. MATERIALS AND METHODS: In August 1994 a random sample of 514 American urologists was surveyed by the Gallup Organization regarding practice patterns for the diagnosis and management of benign prostatic hyperplasia (BPH). It asked questions regarding the practice patterns of respondents in their diagnosis and management of BPH. RESULTS: The survey revealed that 99% of the respondents were aware of and used the AUA symptom score index, and that 21% of these respondents had altered their diagnosis and management strategies because of its existence. CONCLUSIONS: Therapeutic recommendations of the respondents based upon AUA symptom score index severity parallel the recently announced federal Agency for Health Care Policy and Research BPH practice guidelines. PMID- 7539860 TI - Urodynamic results of laser treatment in patients with benign prostatic hyperplasia. Can outlet obstruction be relieved? AB - PURPOSE: A urodynamic study was done to judge the capability of laser treatment to relieve bladder outlet obstruction. MATERIALS AND METHODS: Advanced urodynamic studies with pressure-flow analysis were performed before and 6 months after laser treatment using 3 different laser devices. RESULTS: Forty patients showed significant improvement in all obstruction parameters (detrusor pressure at maximum flow rate, urethral resistance relation, theoretical cross-sectional urethral area, minimal detrusor pressure and linear passive urethral resistance relation) together with significant subjective improvement in international prostate symptom score. After treatment 82 to 92% of the patients could no longer be considered to have obstruction. No difference in outcome among the devices used was found. CONCLUSIONS: Laser prostatectomy is indeed capable of relieving bladder outlet obstruction. PMID- 7539862 TI - Practice trends in the diagnosis and management of prostate cancer in the United States. AB - PURPOSE: The American Urological Association first commissioned the Gallup Organization of Princeton, New Jersey to conduct a study to assess urologists' practice patterns in 1991. MATERIALS AND METHODS: A random sample of 514 American urologists was surveyed by the Gallup Organization regarding practice patterns used in the staging and treatment of prostate cancer. The third annual survey taken during August 1994 asked questions regarding the practice of respondents in their diagnosis and management of prostatic cancer. RESULTS: The survey revealed that 95% of the respondents would recommend radical prostatectomy for men younger than 70 years with confirmed clinically localized prostate cancer. CONCLUSIONS: In this patient age group, surprisingly, the survey resulted in some interesting facts: 1) 13% regard hormonal manipulation for early stage cancer appropriate, 2) the respondents used extensive numbers of diagnostic staging studies in the new prostate cancer patient with a prostate specific antigen of less than 10 and 3) despite the obvious increased costs, the majority of patients receiving hormone manipulation were being treated with luteinizing hormone-releasing hormone agonists rather than orchiectomy. PMID- 7539863 TI - Re: National patterns of prostate cancer treatment by radical prostatectomy: results of a survey by the American College of Surgeons Commission on Cancer. PMID- 7539865 TI - c-met proto-oncogene expression in benign and malignant human prostate tissues. AB - Previously, we demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is expressed by human bone stromal cells and is a powerful mitogen to prostatic epithelial cells in culture. Based on these observations, we hypothesized that, if prostate cancer cells in the prostate or bone environment respond to HGF/SF as a mitogen, then they must express the HGF/SF receptor, which is coded by the c met proto-oncogene. We used immunohistochemical techniques to: 1) assess the presence and localization of c-met protein in benign and malignant human prostate tissues and 2) correlate the presence of c-met protein with tumor stage, grade and androgen sensitivity. c-met protein immunostaining was consistently observed in the basal epithelial layer of normal prostate glands but was absent in luminal epithelial cells of the peripheral and transition zones. c-met protein immunostaining was detected in 10 of 11 foci (91%) of high grade prostatic intraepithelial neoplasia (PIN). Overall, c-met protein staining was noted in 36 of 43 (84%) primary prostate cancer samples versus 2 of 11 (18%) benign prostate hyperplasia samples (p < 0.0001) and in 4 of 4 (100%) lymph node metastases, 23 of 23 (100%) bone marrow metastases and 1 of 3 (33%) other metastatic sites. There was a clear relationship between c-met protein staining and higher grade adenocarcinomas (p < 0.001). c-met protein is frequently detected in PIN and higher grade prostate cancers; future studies should evaluate the biological significance of these findings. PMID- 7539864 TI - Tachykinin effects on bladder activity in conscious normal rats. AB - When instilled intravesically in normal, unanesthetized rats, neurokinin A (NKA), but not substance P (SP) and neurokinin B (NKB), stimulated micturition. The effect of NKA was inhibited by the NK2 receptor selective antagonists SR 48,968 and MEN 10,627, but not by the NK1 receptor selective antagonist RP 67,580, suggesting that the effect was mediated by stimulation of NK2 receptors. Given intra-arterially near the bladder, NKA produced an increase in basal intravesical pressure before initiating micturition, indicating that the tachykinin had a direct contractant effect on the detrusor smooth muscle. Such a contractile effect was not observed when NKA was given intravesically. The effect of intra arterial NKA could not be blocked by the NK1 receptor selective antagonist SR 140,333 or the NK2 receptor selective antagonist SR 48,968, but by their combination. Also intra-arterial NKB stimulated micturition, but was less potent than NKA. Intra-arterial SP had only weak stimulating effects. The results suggest that intravesically administered NKA can initiate micturition in the normal rat by stimulation of superficially located NK2 receptors in the urothelium. Intra-arterially administered NKA caused bladder hyperactivity via stimulation of both NK1 and NK2 receptors. PMID- 7539866 TI - A comparison of transurethral surgery with watchful waiting for moderate symptoms of benign prostatic hyperplasia. PMID- 7539867 TI - Prognostic factors for survival in patients with metastatic renal cancer treated with biological response modifiers. AB - PURPOSE: Clinical characteristics prognostic of survival in patients with metastatic renal cell carcinoma treated with biological response modifiers are poorly understood. Understanding these prognostic features may help with better stratification of patients in clinical trials and define further appropriate treatment for each prognostic subgroup. MATERIALS AND METHODS: A retrospective study of 84 patients with recurrent or metastatic renal cancer was conducted to identify prognostic factors for survival in patients who received biological response modifiers (alpha-interferon, beta-interferon, gamma-interferon and interleukin-2). RESULTS: Univariate analysis identified Eastern Cooperative Oncology Group (ECOG) performance status (1 versus 0, p < 0.001), bone metastasis (p = 0.008), recent weight loss (greater than 10% of total body weight versus no loss, p = 0.028), history of nephrectomy (no versus yes, p = 0.025), recurrence at the renal bed (p = 0.043) and sarcomatoid histology (yes versus no, p < 0.001) as important prognostic indicators. Multivariate analysis of prognostic factors in this patient population indicated that ECOG performance status, sarcomatoid histology and bone metastasis were most significant, while other factors were less significant (p > 0.05) after adjusting for ECOG performance status and sarcomatoid histology. Based on the total positive number of 5 risk factors defined previously the study population separates into 3 risk groups, with a median survival from the low to high risk groups of 14.4, 10.9 and 1.3 months, respectively. Prognostic scores based only on ECOG performance status, sarcomatoid histology and bone metastasis allowed for stratification of our patients into 3 distinct groups with median survivals of 18.6, 8.4 and 3.8 months, which were also predictive of survival (p < 0.05). CONCLUSIONS: Risk factors of ECOG performance status, sarcomatoid histology, bone metastasis, history of nephrectomy, recent weight loss and recurrence at the renal bed are predictive of survival in patients treated with biological response modifiers. In addition to previous findings of prognostic factors in renal cancer patients treated with chemotherapy, we identified sarcomatoid histology as an important risk factor in patients treated with biological response modifiers. PMID- 7539868 TI - Fluorescein angiography of the bladder: technique and relevance to bladder cancer and interstitial cystitis patients. AB - PURPOSE: Fluorescein angiography has been used in the study of bleeding vessels, neovascularity, tumors and ischemic tissues in a variety of disorders. This pilot study was designed to evaluate the feasibility, safety and relevance of this interesting technology for the evaluation of bladder wall vessels in patients with interstitial cystitis and bladder cancer. MATERIALS AND METHODS: Five patients with National Institutes of Health defined interstitial cystitis symptoms and 10 with bladder cancer were studied during cytoscopy while they were under general anesthesia. A yellow-green barrier filter (520 nm.) was placed over the cystoscope eyepiece and a blue exciter filter (465 nm.) was attached to the light source. Patients received a 5 ml. bolus of 10% fluorescein intravenously. RESULTS: After hydrodistension, glomerulations in interstitial cystitis patients were more prominent with fluorescein angiography and occurred in the venule phase. Areas of papillary transitional cell tumor and carcinoma in situ developed a brilliant yellow-green fluorescence. Adjacent normal urothelium was nonfluorescent and provided a contrasting dark background facilitating the detection of all lesions. No allergic reaction or other adverse effect related to the fluorescein injection was observed. CONCLUSIONS: These unique observations in a limited number of patients suggest that fluorescein angiography of the bladder is a safe and simple procedure. This preliminary report underscores the relevance of fluorescein angiography in the detection of bladder tumor and offers a new approach to the evaluation of bladder wall vessels in interstitial cystitis patients. PMID- 7539869 TI - Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. AB - Neovascularization of tumor tissue (tumor angiogenesis) is considered essential for tumor growth, proliferation and eventually metastasis. Microvessel density or count, a measure of tumor angiogenesis, correlates with clinical outcome in skin, breast, lung and prostate carcinomas. To determine whether an association of tumor angiogenesis and nodal metastasis exists in invasive bladder cancer, microvessel counts in 41 primary invasive stages (T2 to 4,NX,M0) bladder cancers were assessed. Microvessels were identified by immunostaining of endothelial cells for factor VIII-related antigen. Microvessels were scored in selected areas showing active neovascularization, either counting a 200 x field (0.74 mm.2) or by using a 10 x 10 square ocular grid (0.16 mm.2). The microvessel count correlated with the presence of occult lymph node metastases (p < 0.0001) by both techniques. The mean microvessel count in 27 patients without lymph node metastases was 56.2 microvessels per 200 x field (standard deviation [SD] 29.5, range 7 to 130) or 28.6 microvessels per grid (SD 14.4, range 4 to 65). The 14 patients with histologically proved lymph node metastases showed mean 138.1 microvessels per 200 x fields (SD 37.9, range 82 to 202) or 74.7 microvessels per grid (SD 14.4, range 43 to 115). Good correlation was noted between area and grid counting (r = 0.97). Tumor T stage, grade and the presence of vascular or lymphatic invasion did not correlate with the presence of lymph node metastases (p = 0.41, 0.59 and 0.26, respectively). Microvessel count may provide important information regarding the risk of occult metastasis in patients with invasive bladder carcinomas. PMID- 7539870 TI - The influence of patient education level on the International Prostatic Symptom Score. AB - The American Urological Association developed a questionnaire to quantify the severity of symptoms due to benign prostatic hyperplasia (BPH). An additional question relating to the impact of BPH on the quality of life was added, and this questionnaire became known as the International Prostate Symptom Score (I-PSS) and was adopted by the World Health Organization. The objective of our study was to determine the influence of education on the I-PSS and to analyze the relationship of the effect on the questionnaire when self-administered or administered by professional medical personnel. I-PSS was administered to 92 patients 50 to 81 years old (mean age 63) with BPH. Patients were assessed into 2 groups according to the level of education. Group 1 was composed of 68 patients considered literate and group 2 comprised 24 patients considered illiterate by UNICEF criteria. Patients received orientation about the questionnaire and the manner of completion. The symptom index was self-administered (subgroup P) and administered with physician help (subgroup D). Statistical analysis was done using Wilcoxon method for nonparametric samples and regression analysis. The difference between P and D subgroups in group 1 was not statistically significant (p = 0.55). However, the difference between subgroups P and D in group 2 was statistically significant (p < 0.001). There was no correlation between the mean I-PSS in subgroup D of literate and illiterate groups. Our study suggests that illiterate patients were not able to answer the questionnaire but with the help of professional medical personnel the I-PSS could be extended to this group of patients without impairing the quality. PMID- 7539871 TI - [Transurethral radiofrequency thermotherapy for benign prostatic hyperplasia]. AB - Transurethral radiofrequency thermotherapy was performed in 38 patients with benign prostatic hyperplasia using Thermex II (Direx) between December 1992 and June 1993, and its clinical efficacy was evaluated by analysing the subjective and objective responses following the treatment. Each patient was treated with an indwelling 16 Fr balloon catheter with electrode, in a single session of 90 minutes at 47-47.5 degrees C. The clinical efficiency was evaluated by total of AUA symptom score and objective findings in terms of maximum flow rate 12 weeks after treatment. The symptom score improved from 15.4 +/- 4.9 (mean +/- SD) to 7.3 +/- 5.2 25% or more improvement was found in 80% (20/25) of patients. No significant change was found in average of maximum flow rate before and after treatment, which were 8.9 +/- 4.1 (ml/sec) and 9.2 +/- 3.3, respectively. During the treatment no severe adverse effect except sense of heat and urgency was detected. Posttreatment urinary retension was found in 9 patients and catheters were indwelled in eight of them for a few days and prostatectomy was performed in one patients after 8 weeks of treatment. In four patients prostatectomy was performed between one day and 8 weeks after thermotherapy and histrogical effect on the resected specimen was examined. In the prostatic urethra, hemorrhage and inflammatory change were observed. However, in the prostatic tissue there was no necrotic region. Transurethral radiofrequency thermotherapy is safe and easy and it may be one of the useful treatments for benign prostatic hyperplasia. PMID- 7539872 TI - An easy-to-use preteaching program to assist vision screening for developmentally delayed preschoolers. PMID- 7539873 TI - Combination chemotherapy as induction therapy for advanced resectable head and neck cancer. AB - Fifty-four previously untreated patients with locally advanced resectable squamous cell carcinoma of the head and neck (SCCHN) were enrolled into a prospective randomized controlled trial to evaluate whether induction chemotherapy improves the disease-free survival compared to the standard treatment (surgery + radiation). Thirty patients received chemotherapy, which consisted of cisplatin 20 mg/m2 day 1-5, bleomycin 10 mg/m2, continuous infusion from day 3-7, and methotrexate 40 mg/m2 given on day 15 and day 22. The cycle was repeated on day 29 for two cycles. Twenty patients completed chemotherapy courses. Overall response rate was 77% (23 of 30). No survival improvement was observed. Kaplan-Meier analysis indicated survival (and 95% confidence interval) at 3 years was 57% (29%-84%) for the control group and 60% (34%-87%) for the chemotherapy group, and 57% (29%-84%) and 45% (12%-78%) at 4 years (P = 0.736). However, patients who had a complete response were significantly better in terms of long-term survivors (5 of 7 patients were still alive), in contrast to patients who had partial responses among whom only 4 of 16 were alive. Toxicities of this induction protocol are tolerable; one chemotherapy-related death occurred from profound thrombocytopenia. If efforts in determining a chemotherapy sensitive patient were successfully established, along with a better sequence and the discovery of new and safer drugs, survival of SCCHN should be much improved. PMID- 7539874 TI - Lower cardiac troponin T levels in patients undergoing cardiopulmonary bypass and receiving high-dose aprotinin therapy indicate reduction of perioperative myocardial damage. AB - Nowadays in many European heart centers the activation of the fibrinolytic system, always occurring during cardiopulmonary bypass, is routinely reduced by high-dose application of the proteinase inhibitor aprotinin (total of > 4 million KIU). In this study parameters of myocardial ischemic injury were investigated with the aim of identifying further benefits of aprotinin, particularly the protection of the myocardium during the ischemic period of aortic crossclamping. Forty patients with coronary artery disease who underwent aorta-coronary bypass grafting were randomly and in a double-blind fashion divided into two groups, one that received high-dose aprotinin therapy and one that received only saline solution. Markers such as troponin T, with high specificity for detection of myocardial ischemia and infarction, and markers with more general specificity such as creatine kinase, its isoenzyme, and lactate dehydrogenase showed significantly increased values after ischemia in both groups. In patients who received high-dose aprotinin therapy 3 days after cardiopulmonary bypass all parameters measured showed significantly lower levels compared with those in the control group. Therefore we can presume that the application of high-dose aprotinin provides myocardial protection from perioperative ischemic injury. PMID- 7539875 TI - Prospects for pharmacotherapy of schizophrenia. PMID- 7539876 TI - Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins. PMID- 7539877 TI - Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins. PMID- 7539878 TI - Birthmark due to cutaneous mosaicism for keratin 10 mutation. PMID- 7539879 TI - The Ottawa Prenatal Prospective Study (OPPS): methodological issues and findings- it's easy to throw the baby out with the bath water. AB - In the OPPS we have been studying the effects of marihuana used during pregnancy since 1978. The subjects are primarily middle-class, low risk women who entered the study early in their pregnancy. Extensive demographic and life-style information was gathered several times during pregnancy and postnatally. The offspring have been assessed repeatedly during the neonatal period, at least annually until the age of 6 and less frequently thereafter. The outcome measures include a variety of age appropriate standardized global measures as well as a large battery of neuropsychological tests attempting to assess discrete functioning within particular domains including language development, memory, visual/perceptual functioning, components of reading and sustained attention. The results suggest that in the neonate, state alterations and altered visual responsiveness may be associated with in utero exposure to marihuana. Global measures, particularly between the ages of 1 and 3 years, did not reveal an association with prenatal marihuana exposure. However, this initial, apparent absence of effect during early childhood should not be interpreted as in utero marihuana exposure having only transient effects for, as the children became older, aspects of neuropsychological functioning did discriminate between marihuana and control children. Domains associated with prenatal marihuana exposure at four years of age and older included increased behavioral problems and decreased performance on visual perceptual tasks, language comprehension, sustained attention and memory. The nature and the timing of the appearance of these deficits is congruent with the notion of prenatal marihuana exposure affecting 'executive functioning'--goal directed behavior that includes planning, organized search, and impulse control. Such an interpretation would be consistent with the extant literature with animals and non-pregnant adult users suggesting that chronic marihuana use may impact upon prefrontal lobe functioning. PMID- 7539880 TI - Macroregenerative nodules in cirrhosis are not associated with elevated serum or stainable tissue alpha-fetoprotein. AB - We have explored the relationship of serum alpha-fetoprotein and macroregenerative nodules (MRNs), possible precursor lesions of hepatocellular carcinoma (HCC), and sought to demonstrate alpha-fetoprotein (AFP) expression in these nodules. One hundred and sixty-eight sequential adult cirrhotic resected livers were examined and MRNs were identified by standard criteria. Pretransplant serum AFP was available for 158 of these patients (normal < 20 ng/ml). One hundred and seventy-two randomly selected lesions, including ordinary and atypical MRNs, some containing microfoci of HCC, and HCCs were stained for AFP by immunohistochemistry. In the series, 12 cases had grossly apparent HCCs, four associated with high serum alpha-fetoprotein (p < 0.006). Forty-four cases had MRNs, 32 without grossly apparent HCC. Five of these 32 cases were associated with high serum AFP (not significant). Immuno-staining for AFP was seen in three specimens of HCC and in a cirrhotic nodule from a patient without HCC, but not in MRNs. 1) Neither the presence of MRNs--whether ordinary, atypical, or containing micro-foci of HCC--nor that of gross HCC is ruled out by a normal serum AFP. 2) Elevated serum AFP is not associated with the presence of MRNs. 3) MRNs rarely stain for tissue AFP. PMID- 7539882 TI - Autoantibodies, mortality and ageing. AB - Immunological failure may be the cause of predisposition to certain infections, neoplasms, and vascular diseases in adulthood. Mortality risks through life may reflect an undetermined number of causes. This study describes the prevalence of positivity of autoantibodies through life, along with general and specific mortality causes in three countries with different socioeconomic development (Guatemala, Mexico and the United States). Prevalence of autoantibodies by age was obtained from previous reports. In spite of having involved different ethnic groups, the observed trends in prevalence of autoantibodies, as well as mortality through life, showed a similar behavior. Thus, both the increase in autoantibody production and death risk as age rises, may share physiopathological phenomena related to the ageing process. PMID- 7539883 TI - A hypothesis defining an objective end point for the relief of chronic pain. AB - A three-part hypothesis for an objective end point for pain is presented: 1) chronic pain results in a characteristic, but reproducible, pattern for the distribution of T lymphocytes in the various phases of their cell cycle; 2) Significant reduction or complete loss of chronic pain will cause a reproducible change in the distribution of T lymphocytes in their cell cycle; 3) The change in T lymphocytes cell cycle distribution will be a function of the degree of recovery from the pain experience. A preliminary test of the hypothesis is presented. The cell cycle distribution of T cell lymphocytes was determined in a group of 10 subjects (experiencing chronic pain) before and after participating in a 10-day educotherapy program given by a master teacher. Associated with a significant reduction of pain was a highly significant shift of the T cell lymphocytes into the S phase of the cell cycle. This observation is consistent with parts one and two of the hypothesis. PMID- 7539881 TI - An immunohistochemical analysis of 13 cases with combined hepatocellular and cholangiocellular carcinoma. AB - Thirteen cases of combined hepatocellular (HCC) and cholangiocellular carcinoma (CCC) were examined. In addition to routine pathology, immunoreactivities for carcinoembryonic antigen, alpha-fetoprotein (AFP), cytokeratin (Cam 5.2 and AE1), epithelial membrane antigen (EMA) and tumor-associated glycoprotein 72 (B72.3) were also examined. The average age of the 13 cases was 64.8 years, which lay between the average ages of pure HCC and CCC cases. They were categorized as separate type (2), collision type (6), and intermingled type (5). AE1 and EMA were the best markers to differentiate the CCC from the HCC area. B72.3 immunoreactivity was detected only in CCC (46%). There were no transitional features between HCC and CCC in two cases of the separate type and two cases of the collision type. However, focal transitional features from HCC to CCC were observed in all cases of the intermingled type and in four of six cases of the collision type. In one case of the intermingled type, many cancer cells contained both bile and mucus simultaneously, and revealed dual immunoreactivities. The conclusions are: 1) the combined type is generated from two sources; one is the intrahepatic double cancer (thoroughly separate type and a part of the collision type) and another is the stem cell origin with diverse phenotypes (intermingled type and a part of the collision tumor); and 2) AE1 was the most helpful marker to differentiate the CCC area from HCC, and other markers, e.g. AFP for HCC and EMA, CEA, and B72.3 for CCC, were also supportive but somewhat limited in the differential diagnosis. PMID- 7539884 TI - Prostate cancer: enthusiasm for screening. PMID- 7539885 TI - [Treatment of iodine-induced hyperthyroidism. Possibilities and limits of pharmacotherapy]. PMID- 7539886 TI - Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells. AB - In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells. PMID- 7539887 TI - Amino acid residues in the third alpha-helix of growth hormone involved in growth promoting activity. AB - The third amphiphilic alpha-helix of GH has been found to be an important motif in the biological activities of the molecule. To further characterize this growth promoting domain, three bovine (b) GH analogs were designed: one contained a scrambled third amphiphilic alpha-helix (SAH); a second contained a scrambled hydrophilic region of the helix (SAP); and a third contained a scrambled hydrophobic region of the helix (SAB). Transgenic mice that expressed these mutated bGH genes were produced. SAH transgenic mice displayed a phenotype identical to nontransgenic littermates. SAB transgenic mice grew slightly larger than nontransgenic littermates but remained smaller than bGH transgenic mice. On the other hand, SAP transgenic mice exhibited a dwarf phenotype. We subsequently generated individual amino acid substitutions in the hydrophilic region of the helix. The results from the growth rates of corresponding transgenic mice demonstrated that most bGH analogs with individual amino acid substitution within the third alpha-helix retained wild type-like growth-promoting activity except those with alterations at positions 115, 119, 122, and 123. Together these residues are predicted to form a cleft in the helix. To further substantiate the importance of the cleft, we deleted Gly 119 (delta 119). This resulting bGH analog was inactive in vivo as well as in in vitro assays. These results indicated that the primary structure of the third alpha-helix is critical for GH's growth-promoting activity and Gly 119 is a crucial amino acid in this region. Three adjacent amino acids, Asp 115, Ala 122, and Leu 123, also contribute to the growth-enhancing ability of the molecule. PMID- 7539888 TI - Amino acids of the human growth hormone receptor that are required for proliferation and Jak-STAT signaling. AB - Dimerization of the GH receptor after ligand binding leads to the rapid association and tyrosine phosphorylation of the intracellular kinase, Jak2, as well as to the tyrosine phosphorylation and activation of STAT protein(s). The tyrosine phosphorylation of these and other related proteins has been shown to be required for intracellular signaling by the interferon receptors. Carboxyl terminal truncations of the human GH receptor have been used to demonstrate that a 54-amino acid, intracellular region of the receptor is sufficient to signal cell proliferation in response to ligand binding. In this work, we examine 10 single and double amino acid mutations of this 54-amino acid region for their ability to signal the proliferation of stably transfected Ba/F3 cells, an interleukin-3 dependent pro-B cell line. The mutation of either of two proline residues or of a lysine residue abolished the GH-induced proliferation. These amino acids are located in or adjacent to a proline-rich sequence known as box 1 that is weakly conserved in other receptors of the GH/cytokine receptor family. The tyrosine phosphorylation of Jak2, as well as the activation of transcription factors (as judged by electrophoretic mobility shift assays), was also induced by GH in the transfected Ba/F3 cells. A consistent pattern of proliferation, Jak2 phosphorylation, and transcription factor activation was found for the 10 GH receptor mutants, a finding that is consistent with the hypothesis that the Jak STAT pathway is required for the signaling of proliferation in these cells. PMID- 7539890 TI - Amiodarone in patients with congestive heart failure and asymptomatic ventricular arrhythmia. Survival Trial of Antiarrhythmic Therapy in Congestive Heart Failure. AB - BACKGROUND: Asymptomatic ventricular arrhythmias in patients with congestive heart failure are associated with increased rates of overall mortality and sudden death. Amiodarone is now used widely to prevent ventricular tachycardia and fibrillation. We conducted a trial to determine whether amiodarone can reduce overall mortality in patients with congestive heart failure and asymptomatic ventricular arrhythmias. METHODS: We used a double-blind, placebo-controlled protocol in which 674 patients with symptoms of congestive heart failure, cardiac enlargement, 10 or more premature ventricular contractions per hour, and a left ventricular ejection fraction of 40 percent or less were randomly assigned to receive amiodarone (336 patients) or placebo (338 patients). The primary end point was overall mortality, and the median follow-up was 45 months (range, 0 to 54). RESULTS: There was no significant difference in overall mortality between the two treatment groups (P = 0.6). The two-year actuarial survival rate was 69.4 percent (95 percent confidence interval, 64.2 to 74.6) for the patients in the amiodarone group and 70.8 percent (95 percent confidence interval, 65.7 to 75.9) for those in the placebo group. At two years, the rate of sudden death was 15 percent in the amiodarone group and 19 percent in the placebo group (P = 0.43). There was a trend toward a reduction in overall mortality among the patients with nonischemic cardiomyopathy who received amiodarone (P = 0.07). Amiodarone was significantly more effective in suppressing ventricular arrhythmias and increased the left ventricular ejection fraction by 42 percent at two years. CONCLUSIONS: Although amiodarone was effective in suppressing ventricular arrhythmias and improving ventricular function, it did not reduce the incidence of sudden death or prolong survival among patients with heart failure, except for a trend toward reduced mortality among those with nonischemic cardiomyopathy. PMID- 7539889 TI - The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene. AB - The insulin-like growth factors (IGFs) are important mitogens that exert their proliferative effects on cells via the type I IGF receptors (IGF-R). The IGFs also associate with IGF binding proteins (IGFBPs). IGF-inhibitory, IGF stimulatory, and IGF-independent effects of IGFBPs on cell growth have been reported. We have asked whether the IGFBP-3 has an effect on cell growth, which is independent of its ability to bind IGF-I and thus inhibit the activation of the IGF-I receptor. For this purpose, we have used a fibroblast cell line (R- cells) derived from mouse embryos homozygous for a targeted disruption of the IGF R gene. When compared with wild type cells (W), which bind IGF-I with high affinity and specificity, R- cells transfected with a mammalian expression vector containing the human (h) IG-FBP-3 cDNA were selected (R-/BP3) and found to express hIGFBP-3 mRNA (detected by Northern blots) and to secrete hIGFBP-3 protein [detected by Western ligand blotting (WLB), immunoblotting, and immunoprecipitation as well as immunofluorescence confocal microscopy]. Growth of five different R- cells, and 10-fold slower compared with W cells, grown under identical conditions. Confluent R- cells. These experiments show that the overexpression of IGFBP-3 has an inhibitory effect on cell growth which does not involve IGF binding to, or signal transduction via, the type I IGF-R. We conclude that the cellular production of IGFBPs serves as a negative regulator of cell proliferation which involves a cellular signaling pathway separate from the IGF IGF-R system. PMID- 7539891 TI - A cystic fibrosis mutation associated with mild lung disease. AB - BACKGROUND: Cystic fibrosis is the most common lethal autosomal recessive disorder among whites. Among Dutch patients with cystic fibrosis, delta F508 is the most common mutation and A455E the second most common mutation of the cystic fibrosis transmembrane conductance regulator gene on chromosome 7. A455E is associated with preserved pancreatic function and residual secretion of chloride across membranes. We investigated whether it is also associated with less severe pulmonary disease in patients with cystic fibrosis. METHODS: A total of 33 patients with compound heterozygosity for the A455E mutation were matched according to age and sex with patients who were homozygous for the delta F508 mutation. The pairs were analyzed with respect to the following outcome variables: age at diagnosis, pulmonary-function values, and the frequency of pseudomonas colonization, pancreatic sufficiency, and diabetes mellitus. RESULTS: Cystic fibrosis was diagnosed at a later age in the patients with the A455E mutation than in the delta F508 homozygotes (mean age at diagnosis, 15.0 vs. 3.1 years; P < 0.001). Fewer patients with the A455E mutation had pancreatic insufficiency (21.2 percent vs. 93.9 percent, P < 0.001), and none had diabetes mellitus (0 percent vs. 27.3 percent, P = 0.004). Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were significantly higher in the patients with the A455E mutation (mean FEV1, 73.9 percent of the predicted value vs. 54.3 percent of the predicted value; P = 0.002; mean FVC, 88.7 percent of the predicted value vs. 76.3 percent of the predicted value; P = 0.04). Fewer patients with the A455E mutation were colonized with Pseudomonas aeruginosa (33.3 percent vs. 60.6 percent, P = 0.02). CONCLUSIONS: A455E is a common mutation causing cystic fibrosis in the Netherlands. Although several mutations are known to be associated with less severe pancreatic disease, our findings demonstrate a correlation between the A455E mutation and mild pulmonary disease. Because mortality in this disease depends primarily on the progression of pulmonary disease, patients with the A455E mutation have a better prognosis than patients who are homozygous for the delta F508 mutation. PMID- 7539893 TI - Comparison of second-generation screening and confirmatory assays with recombinant antigens and synthetic peptides against antibodies to hepatitis C virus: a study in renal patients. AB - The aim of this study was to compare some common tests which are nowadays routinely used to screen and to confirm anti-HCV antibodies in renal patients. There was agreement between Ortho 2 and Abbott 2 in 94% of samples; structural and nonstructural beads of the Abbott supplementary assay were in agreement with 4-RIBA in 98 and in 85% of samples, respectively; 61% of Ortho 2 samples and 65% of Abbott 2 samples were confirmed by 4-RIBA; there was a correlation between semiquantitative analysis of screening tests (Ortho 2 and Abbott 2) and positive results by 4-RIBA; 36 and 33% of Ortho-2- and Abbott-2-positive samples were 4 RIBA indeterminate: in these instances more sophisticated techniques (polymerase chain reaction) (PCR) could be useful as a third-level assay. The comparison between Ortho 2, based on recombinant antigens, and Innotest, based on synthetic peptides, showed agreement only in 44% of samples, but this preliminary comparison cannot afford definitive conclusions. These findings suggest that second-generation assays may sometimes yield conflicting results in renal patients. These contradictions will be resolved by new HCV tests or PCR. PMID- 7539892 TI - Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. AB - The depletion of CD4+ T cells in AIDS is correlated with high turnover of the human immunodeficiency virus HIV-1 and associated with apoptosis. The molecular mechanism of apoptosis in HIV infection, however, is largely unknown. T-cell apoptosis might be affected by viral proteins such as HIV-1 Tat and gp120 (refs 10, 11). T-cell-receptor (TCR)-induced apoptosis was recently shown to involve the CD95 (APO-1/Fas) receptor. We show here that HIV-1 Tat strongly sensitizes TCR- and CD4(gp120)-induced apoptosis by upregulation of CD95 ligand expression. Concentrations of Tat found to be effective in cultures of HIV-1-infected cells were also observed in sera from HIV-1-infected individuals. Taken together, our results indicate that HIV-1 Tat and gp120 accelerate CD95-mediated, activation induced T-cell apoptosis, a mechanism that may contribute to CD4+ T-cell depletion in AIDS. PMID- 7539894 TI - Neurons in the barosensory area of the caudal ventrolateral medulla project monosynaptically on to sympathoexcitatory bulbospinal neurons in the rostral ventrolateral medulla. AB - Neurons in the caudal ventrolateral medulla may function as interneurons in the baroreceptor reflex are by inhibiting sympathoexcitatory bulbospinal neurons in rostral ventrolateral medulla. While some caudal ventrolateral medullary neurons are excited orthodromically by baroreceptors and antidromically from the rostral ventrolateral medulla, there is no anatomical evidence to prove that these barosensory neurons of the caudal ventrolateral medulla monosynaptically innervate the bulbospinal neurons in the rostral ventrolateral medulla. To establish the presence of such a direct projection, barosensory neurons were identified in the rostral caudal ventrolateral medulla of anesthetized rats by criteria that they spontaneously discharged with a cardiac rhythm and were excited by baroreceptor stimulation. The anterograde tracer biocytin was iontophoresed onto these neurons and, in the same animal, the retrograde tracer wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles was injected by micropressure into the ipsilateral spinal (thoracic level 3) intermediolateral cell column to label bulbospinal neurons. After 18-24 h, rats were killed and sections through the rostral ventrolateral medulla were processed for both markers. By light microscopy, numerous biocytin-labeled varicose processes overlapped rostral ventrolateral medullary neurons containing wheatgerm agglutinated apo-horseradish peroxidase conjugated to gold particles. By electron microscopy, biocytin was found in axons and terminals. The terminals (n = 76) were large (0.6-1.2 microns in diameter), contained numerous small, clear vesicles and formed primarily symmetric synapses on perikarya and large (1.5-4.5 microns) dendrites within the rostral ventrolateral medulla. Some of these target neurons contained wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles associated with lysosomes and multivesicular bodies in the cytoplasm. The results indicate that: (i) neurons in the barosensory sympathoinhibitory region of the caudal ventrolateral medulla directly synapse on bulbospinal neurons in the rostral ventrolateral medulla; and (ii) the synaptic profile (symmetric synapse) and location (perikarya and large dendrites) is consistent with the conclusion that baroreceptor neurons of the caudal ventrolateral medulla potently and monosynaptically inhibit sympathoexcitatory neurons of the rostral ventrolateral medulla. The findings support the hypothesis that the barosensory region of the rostral caudal ventrolateral medulla is an intermediate relay in the baroreceptor reflex are. PMID- 7539895 TI - Excitatory innervation of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis in the rat. AB - We examined the possible innervation of the caudal hypoglossal nucleus by the nucleus reticularis gigantocellularis of the medulla oblongata, based on single neuron recording and retrograde tracing experiments in Sprague-Dawley rats. Under pentobarbital sodium (50 mg/kg, i.p.) anesthesia, electrical stimulation of the caudal portion of the nucleus reticularis gigantocellularis with repetitive 0.5 ms rectangular pulses increased (46 of 51 neurons) the basal discharge frequency of spontaneously active cells, or evoked spike activity in silent, hypoglossal neurons located at the level of the obex. This excitatory effect was related to the intensity (25-100 microA) and/or frequency (0.5-20 Hz) of the stimulating pulses to the nucleus reticularis gigantocellularis. Perikaryal activation of neurons by microinjection of L-glutamate (0.5 nmol, 25 nl) into the caudal portion of the nucleus reticularis gigantocellularis similarly produced an excitatory action on eight of 14 hypoglossal neurons. Retrogradely labeled neurons were found bilaterally within the confines of the nucleus reticularis gigantocellularis following unilateral microinjection of wheatgerm agglutinin conjugated horseradish peroxidase or Fast Blue into the corresponding hypoglossal recording sites. Furthermore, the distribution of labeled neurons in the nucleus reticularis gigantocellularis substantially overlapped with the loci of electrical or chemical stimulation. These complementary electrophysiological and neuroanatomical results support the conclusion that an excitatory link exists between the nucleus reticularis gigantocellularis and at least the caudal portion of the hypoglossal nucleus in the rat. PMID- 7539898 TI - Prostacyclin receptor in the brain and central terminals of the primary sensory neurons: an autoradiographic study using a stable prostacyclin analogue [3H]iloprost. AB - Presence and localization of the prostacyclin receptor in the rat brain and spinal cord were examined by in vitro autoradiography using [3H]iloprost, a highly specific and stable agonist for this receptor. Density of specific binding sites for iloprost was generally high in four regions of the lower brain stem, that is, the medial and commissural subnuclei of the nucleus tractus solitarius, the area postrema, superficial layers of the spinal trigeminal nucleus caudalis and dorsal horn. Moderate density was found in the thalamus, cerebral cortex, hippocampus, striatum and dorsal cochlear nucleus. The distribution pattern was distinct from those of other prostanoid binding sites previously studied except that prostaglandin E2 binding sites were also abundant in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn. Even in these regions, binding sites for iloprost had several features clearly different from those for prostaglandin E2. First, within the medial and commissural subnuclei of the nucleus tractus solitarius, iloprost binding sites were distributed preferentially in the dorsal part, while those for prostaglandin E2 were located more ventrolaterally. Second, on postnatal day 0, iloprost binding sites have already been expressed in large amounts in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn of rats, while prostaglandin E2 binding sites are negligible at this stage. Thirdly, the binding of 10 nM [3H]iloprost in these three regions was almost completely displaced by 10 microM unlabelled iloprost but only slightly by 10 microM unlabelled prostaglandin E2. Unilateral nodose ganglionectomy or dorsal rhizotomy decreased the density of iloprost binding sites in the nucleus tractus solitarius or dorsal horn, respectively, with a greater decrease in the operated side. Ligation of the vagus either central or peripheral to the nodose ganglion resulted in an accumulation of iloprost binding sites proximal to the ligation. These results suggest that specific binding sites for iloprost, presumably prostacyclin receptor, are present in the nervous system and, in particular, that the iloprost binding sites in the nucleus tractus solitarius, dorsal horn and possibly in the superficial layers of the spinal trigeminal nucleus caudalis are produced in their sensory ganglia and transported to central terminals of the primary sensory afferents as well as to their peripheral terminals. PMID- 7539897 TI - Chronic treatment with dizocilpine maleate increases the number of striatal neurons expressing the D2 receptor gene. AB - N-methyl-D-aspartate antagonists have been proposed as potential therapeutic agents in different neurological diseases, including Parkinson's disease. The effects of gene expression of a chronic treatment with the non-competitive N methyl-D-aspartate antagonist, dizocilpine maleate (0.8 mg/kg day, per os for 50 days) were analysed in rat striata. Using quantitative in situ hybridization, we measured the messenger RNA expression of the genes encoding D1, D2 dopamine receptors, N-methyl-D-aspartate receptor 1 subunit of N-methyl-D-aspartate receptor, preproenkephalin A and substance P. Chronic treatment with dizocilpine maleate induced a moderate but significant increase in messenger RNA of the N methyl-D-aspartate receptor 1 subunit in the striatum and the adjacent cortex, suggesting an action of dizocilpine maleate in these two regions. This treatment did not induce any change in D1 receptor, preproenkephalin A or substance P messenger RNA content in the striatum, whereas D2 receptor messenger RNA was increased in the striatum of treated rats. Microscopic analysis revealed that it was the number of medium-sized neurons expressing D2 receptor messenger RNA that was significantly enhanced, while the mean amount of message per cell remained unchanged. These results demonstrate that glutamate via N-methyl-D-aspartate receptors, regulates the D2 receptor gene in striatal neurons. A chronic treatment with dizocilpine maleate increases the number of striatal neurons expressing the D2 receptor gene, suggesting a recruiting phenomenon. PMID- 7539896 TI - Differential regulation by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of acetylcholine release from the rat striatum in vivo. AB - The modulation of striatal cholinergic neurons by glutamatergic inputs was studied by monitoring the output of acetylcholine collected via a transversal microdialysis probe implanted into the striatum of freely moving rats. A transversal microdialysis membrane was inserted in the striatum and acetylcholine or GABA levels in the dialysate were measured. Acetylcholine levels in the dialysate were quantified by a high-performance liquid chromatography method with an electrochemical detector, while GABA levels were measured by a high performance liquid chromatography method with a fluorescence detector. The dialysis membrane was perfused with Ringer solution containing 7 microM physostigmine sulphate and drugs, dissolved in the perfusion solution, were administered locally via the dialysis membrane. Local administration of the N methyl-D-aspartate antagonist 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1 phosphonic acid (25-100 microM) brought about a decrease in striatal acetylcholine output which was dose-dependent, reversible and partially antagonized by 100 microM N-methyl-D-aspartate. On the other hand, local administration of the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7 sulfamoil-benzo(F)quinoxaline was followed by an increase in acetylcholine output which reached a maximum of about +55% at 12.8 microM 2,3-dihydroxy-6-nitro-7 sulfamoil-benzo(F)quinoxaline and was readily reversed when the drug was withdrawn from the perfusion solution. Local administration of the non-N-methyl-D aspartate receptor agonist (S)-alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (50 and 200 microM) decreased acetylcholine output and this effect was reversed by simultaneous perfusion with the GABA antagonist bicuculline (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539899 TI - Nitric oxide synthase-containing neurons in sensory ganglia of the rat are susceptible to capsaicin-induced cytotoxicity. AB - Nitric oxide synthase in lumbar dorsal root ganglia of neonatal rat was studied by reduced nicotinamide adenine dinucleotide phosphate diaphorase and in situ hybridization histochemistry. Induction of nitric oxide synthase in neonatal capsaicin-treated rats after sciatic axotomy was compared with the axotomy induced nitric oxide synthase increase observed in vehicle-treated littermates. In neonatal capsaicin-treated animals, the number of neurons constitutively labeled by reduced nicotinamide adenine dinucleotide phosphate diaphorase was greatly reduced as compared to vehicle-treated littermates. Nitric oxide synthase messenger RNA was not readily identified constitutively in dorsal root ganglion neurons. Seven days after sciatic transection the induction of reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthase messenger RNA found in the vehicle-treated group was not observed in the capsaicin group. The presence of nitric oxide synthase in dorsal root ganglion neurons thus does not appear to protect against Ca(2+)-mediated capsaicin-induced cytotoxicity. However, since some nitric oxide synthase dorsal root ganglion neurons persist after the capsaicin neurotoxicity, nitric oxide synthase expression must occur in a neurochemically diverse subpopulation of small (< 1000 microns2) neurons. The capsaicin sensitivity of most nitric oxide synthase dorsal root ganglion neurons indicates that they have unmyelinated axons and are likely to be involved in nociception. PMID- 7539900 TI - Secretory cleavage of beta-amyloid precursor protein in the cerebral white matter produces amyloidogenic carboxyl-terminal fragments. AB - To elucidate the metabolic process generating amyloid-beta protein (A beta) from beta-amyloid precursor protein (APP) in human brain, we partially purified secretory forms and carboxyl-terminal fragments (CTFs) of APP from the white matter of a Down's syndrome brain. We obtained secretory forms of APP which lack the entire A beta sequence and CTFs which contain the full-length A beta from the cerebral white matter. Some A beta-lacking secretory APP isoforms in the white matter were derived from APP695. These results suggest that amyloidogenic CTFs can be produced by secretory cleavage of APP which is anterogradely transported through the axon in human brain. PMID- 7539901 TI - Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury. AB - Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury. PMID- 7539902 TI - Clinical and biochemical long-term toxicity after postoperative cisplatin-based chemotherapy in patients with low-stage testicular cancer. AB - In this case-control study the long-term chemotherapy-related morbidity was assessed in testicular cancer patients (> or = 5 years after treatment). The case group consisted of 47 patients with nonseminomatous testicular cancer who had undergone retroperitoneal surgery and 3-4 cycles of conventional-dose cisplatin based chemotherapy (accumulated cisplatin dose: 300-400 mg/m2), also containing vinblastine and bleomycin. The results of the clinical and biochemical investigations and the patients' responses to a questionnaire were compared to similar observations from a control group comprising 47 patients treated with surgery only. In the case group the serum magnesium values and the levels of FVIIvWF were significantly lower (but within the normal range) than in the control group. Serum cholesterol values were distributed similarly in both groups. About 40% of the patients from the chemotherapy group still suffered from Raynaud-like phenomena and/or sensoric neurotoxicity as compared to 10% of the patients from the control group. Chemotherapy resulted in a significant elevation of serum LH within the normal range, probably associated with a slight subclinical Leydig cell dysfunction. Serum FSH levels were similar in the case and control groups, indicating an absence of long-term disturbance of spermatogenesis after chemotherapy. No increased cardiovascular morbidity was observed as a result of chemotherapy. Raynaud-like phenomena and sensoric neurotoxicity represent the principal clinical long-term side effects after 3-4 cycles of cisplatin-based chemotherapy containing vinblastine and bleomycin. Serum LH is slightly elevated after chemotherapy, whereas long-term spermatogenesis seems to be unaffected. PMID- 7539903 TI - Expression of CD44 and its clinical implication in diffuse-type and intestinal type gastric adenocarcinomas. AB - Knowing the differential expression of CD44 isoforms in intestinal- and diffuse type gastric carcinomas, we used antibodies against the standard form of CD44 (CD44s) and the domain encoded by exon v6 (CD44v6) in 103 patients with primary gastric adenocarcinomas to explore the role of CD44 isoforms in metastases of both types of gastric cancer. Carcinomas of the intestinal type were more frequently CD44s and CD44v6 positive than carcinomas of the diffuse type (p = 0.034 for CD44s and p = 0.022 for CD44v6). The reactivity to these two antibodies did not correlate with histopathological and clinical prognostic factors in intestinal-type carcinoma. In contrast, expression of CD44v6 was associated with infiltrative tumor growth (p = 0.021), depth of invasion (p = 0.012), lymph node involvement (p = 0.005) and a higher incidence of distant metastasis (p = 0.069) in cancers of the diffuse type. CD44s-expressing diffuse-type tumors had a higher incidence of distant metastasis at presentation (p = 0.001), but expression of CD44s was not correlated with other clinicopathologic indices. For all cases, there was a nonsignificant association between CD44s expression and poor survival. Unexpectedly, there was also no significant difference in survival regarding expression of CD44v6 for all cases or the diffuse-type subset. This study showed the role of CD44v6 in invasion and metastases of diffuse-type gastric carcinoma and demonstrated the necessity of subclassifying tumor types when studying the clinical significance of CD44 in human cancers. PMID- 7539904 TI - [A comparative study of pregnancy-associated protein A and its analogs in animals]. AB - Glycoproteins similar in composition, structure, and functions were isolated from the blood serum of pregnant women, cows, dogs, and rats. They consist of four identical subunits, which form dimers due to covalent bonds, while dimers form tetramers due to hydrogen bonds. All these proteins are non-specific inhibitors of proteases and bind these enzymes in the same way as alpha-macroglobulins do, by capture in a "trap". Furthermore, they are capable of binding and transporting various cytokines. The results obtained indicate that these proteins belong to the family of macroglobulins. PMID- 7539905 TI - The natural history of idiopathic subfoveal choroidal neovascularization. AB - PURPOSE: To study the long-term natural history of idiopathic subfoveal choroidal neovascularization (CNV) in young patients. METHODS: A retrospective survey of 19 consecutive patients with idiopathic subfoveal CNV diagnosed in an urban eye hospital and a single practice. RESULTS: Twenty-three (26%) of 87 consecutive patients with idiopathic CNV demonstrated subfoveal CNV. Nineteen patients with subfoveal involvement were followed for a median of 87 months (range, 5-230 months). On initial examination, the median best-corrected Snellen visual acuity was 20/100 (range, 20/40-counting fingers); at final examination, the median visual acuity was 20/70 (range, 20/20-counting fingers). A total of 95% of patients had stable or significantly improved visual acuity, whereas only 5% had significant visual loss. Size of the CNV was the only variable associated with long-term final visual acuity. Lesions 1 disc area or smaller at the time of initial fluorescein angiography were more likely to be associated with a final visual acuity of 20/60 or better and less likely to be associated with a final visual acuity of 20/200 or worse (P = 0.038) as compared with larger lesions. These results were confirmed with multiple logistic regression analysis (P = 0.027). Fellow eyes remained unaffected during the follow-up period. CONCLUSIONS: The natural history of idiopathic subfoveal CNV is not necessarily associated with a profound loss of vision. Therapies for this type of subfoveal lesion must take into consideration the possibility of a favorable natural course. PMID- 7539906 TI - Rabies virus antigenicity: an overview. PMID- 7539907 TI - Molecular epidemiology of lyssaviruses: focus on the glycoprotein and pseudogenes. PMID- 7539909 TI - [Peptide nucleic acids -- a new group of DNA analogues]. PMID- 7539908 TI - Neurologic and developmental outcomes following pediatric cardiac surgery. AB - The successful early repair of congenital heart disease has placed the focus on the long-term outcome for surviving patients. This article reviews the neurologic risks of cardiopulmonary bypass, deep hypothermia, and circulatory arrest. Potential neurologic and developmental sequelae in both the immediate postoperative period and the long term are discussed, with a review of current, research-based literature. PMID- 7539911 TI - Sweat gland carcinoma in a human immunodeficiency virus-infected patient. AB - Eccrine (sweat gland) carcinoma is a rare form of skin cancer that may be locally destructive. It is known to recur after resection and can metastasize to regional or distant lymph nodes. There have been two reported cases in association with patients immunocompromised as the result of organ transplantation (I. Penn: Prog Allergy. 37: 259, 1986). We report here the first case of sweat gland carcinoma in a patient infected with the human immunodeficiency virus. PMID- 7539912 TI - Bacillary angiomatosis. PMID- 7539910 TI - [Biological properties of M. leprae passaged in laboratory animals]. PMID- 7539913 TI - Configurational effects in antibody-antigen interactions studied by microcalorimetry. AB - In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high-sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is -350 +/- 60 cal K-1 mol-1 while for E8 it is -165 +/- 40 cal K-1 mol-1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25 degrees C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (-77 +/- 15 vs. -34 +/- 11 cal K-1 mol-1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen-deuterium exchange data, detected using 2D NMR, on the two antibody-antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539914 TI - Local nitric oxide production in viral and autoimmune diseases of the central nervous system. AB - Because of the short half-life of NO, previous studies implicating NO in central nervous system pathology during infection had to rely on the demonstration of elevated levels of NO synthase mRNA or enzyme expression or NO metabolites such as nitrate and nitrite in the infected brain. To more definitively investigate the potential causative role of NO in lesions of the central nervous system in animals infected with neurotropic viruses or suffering from experimental allergic encephalitis, we have determined directly the levels of NO present in the central nervous system of such animals. Using spin trapping of NO and electron paramagnetic resonance spectroscopy, we confirm here that copious amounts of NO (up to 30-fold more than control) are elaborated in the brains of rats infected with rabies virus or borna disease virus, as well as in the spinal cords of rats that had received myelin basic protein-specific T cells. PMID- 7539915 TI - Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes. AB - We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity. PMID- 7539916 TI - Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer. AB - We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis. PMID- 7539917 TI - Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives). AB - Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3' spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development. PMID- 7539919 TI - Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide. AB - The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodon independent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a species-specific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals. PMID- 7539918 TI - Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. AB - Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia. PMID- 7539920 TI - Sticky ions in biological systems. AB - Aqueous gel sieving chromatography on Sephadex G-10 of the Group IA cations (Li+, Na+, K+, Rb+, Cs+) plus NH4+ as the Cl- salts, in combination with previous results for the halide anions (F-, Cl-, Br-, I-) as the Na+ salts [Washabaugh, M.W. & Collins, K.D. (1986) J. Biol. Chem. 261, 12477-12485], leads to the following conclusions. (i) The small monovalent ions (Li+, Na+, F-) flow through the gel with water molecules attached, whereas the large monovalent ions (K+, Rb+, Cs+, Cl-, Br-, I-) adsorb to the nonpolar surface of the gel, a process requiring partial dehydration of the ion and implying that these ions bind the immediately adjacent water molecules weakly. (ii) The transition from strong to weak hydration occurs at a radius of about 1.78 A for the monovalent anions, compared with a radius of about 1.06 A for the monovalent cations (using ionic radii), indicating that the anions are more strongly hydrated than the cations for a given charge density. (iii) The anions show larger deviations from ideal behavior (an elution position corresponding to the anhydrous molecular weight) than do the cations and dominate the chromatographic behavior of the neutral salts. These results are interpreted to mean that weakly hydrated ions (chaotropes) are "pushed" onto weakly hydrated surfaces by strong water-water interactions and that the transition from strong ionic hydration to weak ionic hydration occurs where the strength of ion-water interactions approximately equals the strength of water-water interactions in bulk solution. PMID- 7539921 TI - The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function. AB - In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N terminal region with transcription factor IIB. PMID- 7539923 TI - Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants. AB - To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development. PMID- 7539922 TI - Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses. AB - Genes containing the interferon-stimulated response element (ISRE) enhancer have been characterized as transcriptionally responsive primarily to type I interferons (IFN alpha/beta). Induction is due to activation of a multimeric transcription factor, interferon-stimulated gene factor 3 (ISGF3), which is activated by IFN alpha/beta but not by IFN gamma. We found that ISRE-containing genes were induced by IFN gamma as well as by IFN alpha in Vero cells. The IFN gamma response was dependent on the ISRE and was accentuated by preexposure of cells to IFN alpha, a treatment that increases the abundance of ISGF3 components. Overexpression of ISGF3 polypeptides showed that the IFN gamma response depended on the DNA-binding protein ISGF3 gamma (p48) as well as on the 91-kDa protein STAT91 (Stat1 alpha). The transcriptional response to IFN alpha required the 113 kDa protein STAT113 (Stat2) in addition to STAT91 and p48. Mutant fibrosarcoma cells deficient in each component of ISGF3 were used to confirm that IFN gamma induction of an ISRE reporter required p48 and STAT91, but not STAT113. A complex containing p48 and phosphorylated STAT91 but lacking STAT113 bound the ISRE in vitro. IFN gamma-induced activation of this complex, preferentially formed at high concentrations of p48 and STAT91, may explain some of the overlapping responses to IFN alpha and IFN gamma. PMID- 7539927 TI - Aging and insulin secretion. AB - Aging in mammals has often been associated with decreased insulin secretion and a subsequent deterioration in the ability to maintain glucose homeostasis. However, recent studies have demonstrated that factors such as disease, obesity, and physical activity more closely reflect diminished insulin secretion rather than aging per se. Thus, the purpose of this article is to review recent studies of how biological aging, i.e. the process independent of disease states such as type II diabetes, may affect insulin secretion. To this end, this review will address the impact of aging on insulin secretion in terms of in vivo and in vitro assessment, as well as possible age-related alterations in the hormonal and neural regulation of insulin secretion. Finally, this review describes some evidence that alterations in the functional heterogeneity of the beta-cell population may represent a means by which the endocrine pancreas is able to maintain appropriate insulin secretion during senescence. PMID- 7539925 TI - The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction. AB - Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function. PMID- 7539928 TI - Herbimycin A inhibits phorbol ester-induced morphologic changes, adhesion, and megakaryocytic differentiation of the leukemia cell line, MEG-01. AB - 12-O-Tetradecanoylphorbol 13-acetate (TPA) induces rapid changes in the morphology of the human megakaryoblastic leukemia cell line, MEG-01, as well as changes in adhesion and megakaryocytic differentiation. To investigate the signal transduction pathway of these three phenomena, we studied the effect of herbimycin A, an inhibitor of tyrosine kinase (TK) and the effects of calphostin C, a specific inhibitor protein kinase C (PKC) on TPA treated MEG-01 cells. Both herbimycin A and calphostin C inhibited all three TPA-induced phenomena, suggesting that both pathways are required for these phenomena. Herbimycin A but not calphostin C blocked the tyrosine phosphorylation of cellular proteins. Immunohistochemical staining of PKC using an anti-PKC monoclonal antibody showed that herbimycin A did not interfere with the translocation and subsequent down regulation of PKC induced by TPA, suggesting that the TPA-induced effect on PKC (translocation and probably its activation) is not dependent on TK. Induction of c-fos and c-jun expression by TPA was inhibited by both herbimycin A and calphostin C, suggesting that both PKC and TK pathways are necessary for the induction of the TPA-induced transcription factor AP1, which is a known TPA inducible early immediate gene product. Taken together, our results show that the tyrosine kinase signal transduction system as well as the PKC pathway is indispensable for the TPA-induced phenomena of morphologic change, cell attachment, early immediate gene expression, and lineage-specific phenotypic expression in the MEG-01 cell line. PMID- 7539926 TI - Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L fucosyltransferase is associated with human colon adenocarcinoma progression. AB - GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor associated allelic variations within the H beta-D-galactoside alpha-2-L fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma. PMID- 7539929 TI - Office care of the small, premature infant. AB - About 1% of infants are born before 32 weeks of pregnancy, and 0.9% have a birthweight of 1500 gm or less. More of these premature infants are surviving. Contributing factors include prenatal corticosteroid therapy and early surfactant therapy to reduce the incidence of respiratory distress syndrome. Because of this, more family physicians are providing office care for premature infants after discharge from neonatal intensive care centers. These infants require special care in the office as well as the neonatal intensive care unit. The goal of the family physician's care should be to minimize mortality and morbidity. This article summarizes recommendations for office care of the surviving premature infant. PMID- 7539930 TI - [Hydrolysis of RNA and protein by rare-earth metal ions]. PMID- 7539924 TI - Human immunodeficiency virus type 1 reverse transcriptase: enhancement of activity by interaction with cellular topoisomerase I. AB - A number of studies have suggested that topoisomerase I (topo I) activity may be important in human immunodeficiency virus type 1 (HIV-1) replication. Specifically it has been reported that purified virus particles have topo I activity and that inhibitors of this enzyme can inhibit virus replication in vitro. We have investigated a possible association of HIV-1 gag proteins with topo I activity. We found that whereas the gag-encoded proteins by themselves do not have activity, the nucleocapsid protein p15 can interact with and enhance the activity of cellular topo I. Furthermore it could be demonstrated that topo I markedly enhanced HIV-1 reverse transcriptase activity in vitro and that this could be inhibited by the topo I-specific inhibitor camptothecin. The findings suggest that cellular topo I plays an important role in the reverse transcription of HIV-1 RNA and that the recruitment of this enzyme may be an important step in virus replication. PMID- 7539931 TI - [Aquaporins: function and structure for facilitated water transport]. PMID- 7539933 TI - The effects of augmented information on motor learning: a multidimensional assessment. AB - Subjects performed trials of a two-hand coordination task involving the manipulation of a small, steel ball through a maze. During the acquisition phase, control subjects received no augmented information while subjects in two experimental groups were provided with visual aids or a cognitive strategy. One day later, subjects performed under control conditions on familiar (relearning phase) and unfamiliar (transfer phase) sections of the maze. A 3 x 3 (Group x Phase) repeated measures multivariate analysis of variance revealed significant phase differences for all dependent measures but no significant group effects. Chi-square analysis of ratings of cue usage as well as postexperimental interviews suggested, however, that practice conditions differentially influenced the search strategies of subjects. Implications of the findings for contemporary theories of motor learning are discussed. PMID- 7539932 TI - [Reduction of the need for blood derivatives in liver transplantation using aprotinin]. AB - OBJECTIVES: To evaluate the efficacy of aprotinin in reducing the need for blood products in orthotopic liver transplantation. PATIENTS AND METHODS: Blood product needs and coagulation test results were studied in 42 adults with cirrhosis of the liver who received orthotopic liver transplants. The first 16 liver transplants carried out without aprotinin (control group) were compared with the next 26 consecutive transplant patients who received aprotinin. Each of the first 9 received a loading dose of 2 million units that was followed by the infusion of half a million units per hour until the end of surgery. The next 17 received the same infusion dose at the same rate but no loading dose. RESULTS: Patients who received aprotinin required fewer transfusions of blood products (5.3 units of packed red blood cells as opposed to 13 units; 9 units of fresh frozen plasma versus 14.6 units; 1.7 units of platelets versus 4.2 units; and 3.8 units of cryoprecipitates versus 8.8 units). We observed a marked reduction of fibrinolysis (less increase in D dimers after removal of the liver when aprotinin was used. CONCLUSIONS: Prophylactic use of aprotinin during surgery has a beneficial effect on hemostatic mechanisms, reducing the need for blood products. A reduction in fibrinolysis seems to contribute to this effect. PMID- 7539934 TI - Capsaicin pretreatment attenuates chronic hypoxic pulmonary hypertension. AB - Capsaicin pretreatment was used to deplete tachykinins in order to study the role of tachykinins in chronic hypoxia-induced pulmonary hypertension. Forty three young Wistar rats weighing 235 +/- 4 g were randomly divided into four groups: control (n = 10); capsaicin pretreatment (n = 10); intermittent chronic hypoxia (n = 10); and capsaicin pretreatment + intermittent chronic hypoxia (n = 13). Control animals breathed room air. Rats in the capsaicin pretreatment groups were given capsaicin via subcutaneous injection over a three-day period. Hypobaric hypoxia was intermittently applied by placing animals into a hypobaric chamber with a barometric pressure of 380 Torr for two weeks. In the capsaicin pretreatment + intermittent chronic hypoxia group, rats were exposed to intermittent hypoxia for two weeks immediately after the last dose of capsaicin. Subsequently, pulmonary vascular function, as well as substance P (a tachykinin) level and neutral endopeptidase (NEP, the major degradation enzyme for tachykinins) activity in the lungs were measured. Chronic hypoxia caused significant increases in pulmonary artery pressure, right ventricle/(left ventricle + septum) weight ratio, hematocrit, and lung substance P level, as well as a significant decrease in lung NEP activity. All these chronic hypoxia-induced changes were significantly lessened by capsaicin pretreatment. Capsaicin pretreatment alone did not induce any significant alteration in vascular function. These results suggest that the chronic hypoxia causes an increase in lung tachykinin levels which, in turn, enhance the development of pulmonary hypertension. PMID- 7539935 TI - Metaphorical pain language among fibromyalgia patients. AB - Fourteen women with fibromyalgia described their pain experiences using words indicating a model of pain that presented pain as an aggressive physical deformation and a torture-like experience. Metaphorical expressions were used which was seen as a means for enabling the patients to disclose tacit knowledge. The pain experience was narrated as being steady and without any distinct bodily location. Only causes explaining the present aggravation of the pain and treatment leading to a temporary relief were related. PMID- 7539936 TI - Natural protection against HIV-1 infection provided by HIV-2. AB - Significant differences have been observed in the rates of transmission and disease development in human immunodeficiency virus (HIV) types 1 and 2. Because many HIV-2-infected people remain asymptomatic for prolonged periods, the hypothesis that HIV-2 might protect against subsequent infection by HIV-1 was considered. During a 9-year period in Dakar, Senegal, the seroincidence of both HIV types was measured in a cohort of commercial sex workers. Despite a higher incidence of other sexually transmitted diseases (STDs), HIV-2-infected women had a lower incidence of HIV-1 than did HIV-seronegative women, with a relative risk of 0.32 (P = 0.008). An understanding of the cross-protective mechanisms involved may be directly relevant to HIV-1 vaccine development. PMID- 7539937 TI - Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation. AB - In vivo somatosensory stimuli evoked the release of substance P from primary afferent neurons that terminate in the spinal cord and stimulated endocytosis of substance P receptors in rat spinal cord neurons. The distal dendrites that showed substance P receptor internalization underwent morphological reorganization, changing from a tubular structure to one characterized by swollen varicosities connected by thin segments. This internalization and dendritic structural reorganization provided a specific image of neurons activated by substance P. Thus receptor internalization can drive reversible structural changes in central nervous system neurons in vivo. Both of these processes may be involved in neuronal plasticity. PMID- 7539938 TI - Peripheral blood stem cell transplantation: from laboratory to clinical practice. PMID- 7539939 TI - Separation of antigenic glycoprotein fractions from cell-free homogenate of Pseudomonas pseudomallei and characterization as tyrosine phosphatase. AB - Cell-free extracts were prepared from Pseudomonas pseudomallei cells by freezing thawing, sonication, and differential ultracentrifugation. The extracts were subjected to column chromatography with DEAE-sepharose to obtain glycoprotein fractions. The fractions showed acid phosphatase activity to p-nitrophenyl phosphate, tyrosine phosphate, serine phosphate, but not to threonine phosphate. They were highly antigenic when tested by immunofluorescence assay with the sera of melioidosis patients. PMID- 7539940 TI - [Acute phase proteins in acute pancreatitis]. AB - The submitted investigation deals with the value of assessment of selected reactants of the acute stage in patients with acute pancreatitis and their follow up in the course of the disease. For the investigation C-reactive protein, alpha 1-antitrypsin and haptaglobin were selected. In addition to the prognostic impact the authors focused attention on assessment of the dynamics of individual proteins and their importance for the early detection of complications in acute pancreatitis with different etiologies. The high CRP level on admission of severe cases of pancreatitis--mean 166.9 mg/l (range from 100 to 320 mg/l)--correlated with other signs suggesting severe pancreatitis and the latter was confirmed by computed tomography (CT), surgery or post-mortem examination in 90% of the patients with a CRP level above 100 mg/l. This correlation was not confirmed for alpha-1-antitrypsin or haptaglobin. PMID- 7539941 TI - Enhanced efficacy of bleomycin adsorbed on silica particles against lymph node metastasis in patients with esophageal cancer: a pilot study. AB - BACKGROUND: Lymph node metastases occur very frequently and extensively in patients with esophageal cancer. The aim of this pilot study was to try the targeting chemotherapy for lymph node metastases by use of bleomycin adsorbed on silica particles (BLM-SI). METHODS: BLM-SI or bleomycin solution (BLM-SOL) was injected into the submucosa of the esophageal wall by means of endoscopy 3 days before operation in 16 patients with middle thoracic esophageal cancer. The distribution of bleomycin in the regional lymph nodes and surrounding connective tissues was studied. RESULTS: When BLM-SI was administered, bleomycin activity was found in both the regional lymph nodes and connective tissues, not only in the mediastinal region but also in the cervical and abdominal region. Bleomycin activity was significantly higher in all regions after BLM-SI administration than after BLM-SOL administration. Degenerative or necrotic changes were microscopically observed in 11 of 36 lymph nodes with metastatic foci. Bleomycin activity in the blood was significantly lower after BLM-SI was administered than after BLM-SOL. Serious systemic side effects except for fever were not observed in any patients. CONCLUSIONS: These results indicate that BLM-SI could be a useful treatment modality for targeting lymph node metastasis of esophageal cancer without serious side effects. PMID- 7539942 TI - Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. AB - BACKGROUND: Fulminant acute pancreatitis is a disease of complex origin that results in activation of several of the proinflammatory cytokines. Because interleukin-1 (IL-1) is an integral early component of the acute inflammatory process, the use of an IL-1 receptor antagonist (IL-1ra) was investigated in experimental acute pancreatitis to determine the therapeutic potential of proximal cytokine blockade and to further establish the role of inflammatory cytokines in the pathogenesis of acute pancreatitis. METHODS: IL-1ra was administered in escalating doses either before or after acute edematous, necrotizing pancreatitis was induced in adult male mice by injection of cerulein. The severity of pancreatitis was quantified by serum amylase, lipase, interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) levels, pancreatic wet weight, and blinded histologic grading. RESULTS: Administration of medium (10 mg/kg) and high (100 mg/kg) doses of IL-1ra either before or after the induction of pancreatitis significantly decreased the expected rise in pancreatic wet weight, lipase, IL-6, and TNF-alpha (all, p < 0.01). Serum amylase was significantly reduced when IL-1ra was administered in either dosage before (p < 0.05), but not after, induction of pancreatitis. Pancreatic edema, necrosis, and inflammatory cell infiltrate were significantly diminished (p < 0.05) by histologic grading in all animals receiving medium or high doses of IL-1ra. Low doses of IL-1ra (1.0 mg/kg) had modest effects if given before, but no effect if given after, induction of pancreatitis. CONCLUSIONS: The proinflammatory cytokines IL-6 and TNF-alpha are elevated during experimental acute pancreatitis and correlate well with the severity of local pancreatic destruction. Blockade of the cytokine cascade at the level of the IL-1 receptor before or soon after induction of pancreatitis significantly attenuates the rise in these cytokines and is associated with decreased severity of pancreatitis and reduced intrinsic pancreatic damage. PMID- 7539943 TI - Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents. AB - BACKGROUND: Peritoneal adhesions continue to be a significant cause of postoperative complications. Elucidating the origin of these adhesions has been hampered by the lack of a reproducible animal model. The purpose of this study was to create a standardized model in which a single, specific adhesion could be objectively measured. With this model the kinetics of adhesion formation were then evaluated. A variety of potential antiadhesive agents were then tested and compared. METHODS: In this study a reproducible, quantitative rat model was developed that used uniform defects on the peritoneal wall and cecal surface. The resulting adhesions were subsequently scored, and their strength was measured with a tensiometer. An evaluation of the kinetics of peritoneal adhesion formation was obtained by using a timed removal of silicone elastomer sheeting held between the two injured surfaces. The following antiadhesive agents were evaluated: Ringer's lactate solution; dextran 70 (32%); modified carboxymethylcellulose (1.0% and 2.0%); an absorbable barrier of specially knitted material composed of oxidized regenerated cellulose; fibrin sealant; silicone elastomer film; and expanded polytetrafluoroethylene membrane. RESULTS: Evaluation of the kinetics of peritoneal adhesion formation indicated that the susceptibility for adhesion formation was significantly decreased or eliminated after the first 36 hours. Evaluation of antiadhesion agents indicated that the magnitude of adhesion prevention was directly proportional to the agent's ability to remain at the site of injury during the critical period of adhesion formation. Permanent barriers (silicone elastomer film, expanded polytetrafluoroethylene membrane) provided the greatest antiadhesion effect but were not believed to be ideal agents because they remained at the site of injury well after the critical period of adhesion formation. The incidence of adhesion formation for the other agents was as follows: control (34 of 34), Ringer's lactate (12 of 12), absorbable barrier of knitted cellulose (10 of 10), 32% dextran 70 (8 of 12), 1% carboxymethylcellulose (6 of 12), fibrin sealant (4 of 9), and 2% carboxymethylcellulose (4 of 12). CONCLUSIONS: The efficacy of antiadhesion agents appears to be related to the agent's viscosity, ability to coat the wound surface, and residence time at the site of injury. In this rat model an agent that remained on the injured surfaces for at least 36 hours after injury appeared to be more effective in reducing adhesion formation than an agent with a shorter residence time. PMID- 7539944 TI - Des-gamma-carboxy prothrombin and proliferative activity of hepatocellular carcinoma. AB - BACKGROUND: Des-gamma-carboxy prothrombin (DCP) is a useful marker for the prognosis of hepatocellular carcinoma (HCC). In this report we investigated the relationship between the positivity of DCP and proliferative activity of HCC and discuss the cause of poor prognosis of DCP-positive HCC. METHODS: Immunohistochemical and clinicopathologic study was done in 114 patients with resected HCC measuring less than 6 cm in diameter by using monoclonal antibody for proliferating cell nuclear antigen (PCNA). RESULTS: PCNA labeling index (PCNA LI) was significantly higher in the patients with DCP-positive HCC than in those with DCP-negative HCC; also a positive correlation was noted between the PCNA-LI and the DCP level. We divided patients into two groups according to the PCNA-LI. In the high PCNA-LI group the patients with DCP-positive HCC exhibited a higher PCNA-LI than did the patients with DCP-negative HCC. As for pathologic prognostic factors, the DCP-positive high PCNA-LI group showed the highest incidence of tumor thrombus of the portal vein and intrahepatic metastasis while also exhibiting the lowest recurrence-freedom rate. From multivariate analysis we find that DCP, as well as PCNA-LI, is one of the risk factors for recurrence of HCC after hepatectomy. CONCLUSIONS: Our results thus suggest that DCP-positive HCC showed high PCNA-LI, and this might be the main cause for early intrahepatic spread and poor prognosis of DCP-positive HCC. PMID- 7539945 TI - Differentiation of the microvascular endothelium during early angiogenesis and respiratory onset in the chick chorioallantoic membrane. AB - The present study served to determine the extent of microvascular endothelial differentiation during early stages of morphogenesis (days 4.5-5.5 of the 21-day incubation) in the chick chorioallantoic membrane (CAM). CAM's, which serve as the embryonic lung, were prepared for intravital injections of a graded series of FITC-dextrans and subsequent ultrastructural morphometric analyses of the microvascular units. The precapillary, capillary, and postcapillary microvascular segments presented a continuous endothelium that was substantially thicker than that of adult lung endothelia (DeFouw, 1988). Further, plasmalemmal vesicles were uniformly sparse, while endothelial vacuoles, of variable diameters, were present continuously in the proliferating microvascular units. Average widths and depths of the interendothelial clefts were uniform and suggested complete structural differentiation from the onset of CAM morphogenesis. Based on our recent estimates of CAM microvascular permeability coefficients (Rizzo et al., 1995), the observed endothelial ultrastructure was associated with microvascular selectivity comparable to that of adult pulmonary microvessels (Lanken et al., 1985). Therefore, despite incomplete ultrastructural differentiation of the early CAM microvascular endothelium, these angiogenic microvessels presented adult-like barrier properties. Further they were less permeable than (Wu et al., 1993; Yuan et al., 1993) and ultrastructurally distinct from (Kohn et al., 1992) certain tumorigenic microvessels. Thus, angiogenesis is likely not a routinely homogeneous process, and CAM microvascular permeability characteristics may be teleologically significant. PMID- 7539946 TI - The cerebral neurons of Helix aspersa during hibernation. Changes in the cytochemical detection of calmodulin, cytoskeletal components and phosphatases. AB - Some markers of the intracellular systems that regulate neuronal activity and morphology were analyzed in the cerebral ganglion of hibernating snails (Helix aspersa), in comparison with active animals. The immunocytochemical expression of a calcium-binding protein, i.e. calmodulin, and some cytoskeletal components, i.e. 200 kDa phosphorylated neurofilament protein (pNFH), microtubule associated protein 2 (MAP2) and alpha-tubulin were analyzed by the use of a panel of antibodies raised against mammal antigens. Moreover, by enzymatic reactions the Ca(2+)-ATPase and alkaline phosphatase (AIPase) activities were demonstrated. In comparison with the active phase, the hibernation induced an increase in the immunopositivity for calmodulin in all the neurons. The increase may be linked to unmasking of immunoreactive epitopes due to conformational changes of the protein, which in turn may be a consequence of a reduction or absence of binding with calcium ions or of a real increase in the amount of calmodulin in the somata of neurons. In any event, both the hypotheses indicate that neurons have decreased or suppressed the Ca(2+)-dependent mechanisms as also shown by the lower Ca(2+)-ATPase activity. Nevertheless, the AIPase activity, which was localized in the epineural sheat, was not significantly changed during hibernation and this supports that some metabolic activities are preserved in the hibernated animals. Changes in the immunopositivity for cytoskeletal components were found. There was an increase in the epitopes recognized by the mammalian pNF antibody, that concerned both the positivity of the entire cytoplasm of some clusters of metacerebral neurons and the intensity of the reaction. This would be aimed to improve the stability of the somata and primary neurites. Moreover, the decrease of alpha-tubulin and MAP2 immunopositivity, suggests that a disassembly of microtubules have occurred. The findings indicate that the transport of vesicles in the axons is slowed down during hibernation. In fact, research in progress show that the patterns of neurotransmission and neuromodulation are also deeply modified. PMID- 7539947 TI - Antennal lobe projection patterns of olfactory receptor neurons involved in sex pheromone detection in Spodoptera littoralis (Lepidoptera: Noctuidae). AB - Pheromone-specific receptor neurons in male and female cotton leafworms, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) were typed physiologically and traced into the antennal lobe using cobalt lysine as a marker. In male S. littoralis, the macroglomerular complex (MGC), which is responsible for the initial integration of information concerning sex pheromone components, contains three glomerular compartments as revealed in a morphological study. No such specialized structure was seen in the female. In the male, olfactory receptor neurons that responded selectively to stimulation with the major sex pheromone component, (Z)9, (E)11-tetradecadienyl acetate (Z9E11 14:OAc), had arborizations that were restricted to a large glomerulus of the MGC (designated a), situated near the antennal nerve entrance into the antennal lobe. Neurons that were stimulated by (Z)9,(E)12-tetradecadienyl acetate (Z9E12 14:OAc), a second pheromone component, had arborizations in a lateral, smaller glomerulus of the MGC (designated c), while receptor neurons specifically tuned to a behavioural antagonist, (Z)-9-tetradecenol (Z9-14:OH), projected to a medial glomerulus of the MGC (b). In the female, receptor neurons tuned to the major pheromone component projected to a glomerulus situated at the entrance of the antennal nerve. This glomerulus did, however, not have the size or the structure of the male MGC. A second neuron housed in the same sensillum projected its axon to an ordinary glomerulus situated medially in the antennal lobe. PMID- 7539948 TI - Patient statement: a canary's tale. AB - I am Jacob B. Berkson, a 68-year-old resident of Hagerstown, Maryland. I was a trial lawyer for some 40 years. I am now retired and writing a book on Environmental Pollution and Environmental Illness, titled A Canary's Tale. I was invited to speak to you as a patient--one who was poisoned by an organophosphate pesticide and who subsequently developed Multiple Chemical Sensitivity (MCS, or sometimes referred to as Environmental Illness, EI). PMID- 7539949 TI - The phenomenology of limbic kindling. AB - Kindling is a model of epilepsy whereby repeated administration of brief low intensity trains of electrical stimulation come to elicit electrographic and behavioral manifestations of seizure. In the absence of overt tissue damage, an animal that has been kindled is rendered in a permanent state of increased susceptibility to seizures. A number of persistent biochemical and physiological alterations in function accompany kindling, some of which may impact upon behavior of the organism for a long period of time despite the absence of further seizure activation. The sensitivity of limbic structures to kindling may contribute to the behavioral categories of cognition and affect that are particularly impacted by the kindling process. The increased proclivity for seizure disorders that characterizes kindling is not restricted to the initial kindling stimulus, but generalizes to other agents with convulsive properties. This paper provides an overview of the phenomenology of kindling, describes some of the conditions necessary for its induction, and some of the functional alterations that accompany its development and endure when overt convulsive behavior has subsided. Finally, a series of studies in our laboratory is presented which provides evidence of chemically induced kindling by repeated low level exposure to some pesticides, namely those of the chlorinated hydrocarbon class. PMID- 7539950 TI - Olfaction and multiple chemical sensitivity. AB - In this paper, a description of olfactory anatomy is presented, followed by a brief review of modern procedures for testing olfactory function. Information from the sole study which has quantitatively examined olfactory function in patients with apparent multiple chemical sensitivity (MCS) is presented. In essence, this study suggests that MCS is associated with increased nasal airflow resistance, respiration rate, heart rate, and scores on the Beck Depression Inventory, but not with significant changes in odor detection threshold sensitivity to phenyl ethyl alcohol and methyl ethyl ketone, the two target stimuli evaluated. Whether MCS patients evidence hypersensitivity to other chemicals is unknown. PMID- 7539951 TI - Prevalence of hepatitis B and C viruses in healthy Indonesian blood donors. AB - Blood samples were collected from 7572 healthy volunteer blood donors from 21 of the 27 Indonesian provinces, and tested for antibodies to hepatitis C virus (anti HCV) using the new second-generation enzyme immunosorbent assay, and also tested for hepatitis B surface antigen (HBsAg). We detected anti-HCV in 2.1% of the blood donors. No statistically significant difference was found between males and females or between locations, but there was a statistically significant increasing likelihood of anti-HCV prevalence with increasing age. HBsAg was found in 8.8% of the 3839 tested donors. There was no statistically significant difference between sexes or age groups, but there was a statistically significant higher prevalence in the islands of Sulawesi and eastern Indonesia. Only 7 individuals, from 5 locations, were both anti-HCV and HBsAg positive. Based on responses to a questionnaire, a history of surgery, blood transfusion, intravenous medication, and acupuncture were identified as risk factors for the presence of anti-HCV. No such risk factor was identified for HBsAg prevalence. The combined data suggest separate modes of transmission for the 2 viruses, and indicate the need for continued surveillance for these agents in Indonesian blood banks. PMID- 7539952 TI - Hepatitis C virus infection and chronic liver disease in Ethiopia where hepatitis B infection is hyperendemic. AB - To assess the prevalence and possible aetiological association of hepatitis C virus (HCV) with chronic liver disease and hepatocellular carcinoma (HCC), antibodies to HCV (anti-HCV) were determined by enzyme-linked immunosorbent and recombinant immunoblot assays in 500 healthy volunteer blood donors, 14 patients with chronic hepatitis, 156 cirrhotics and 68 cases of hepatocellular carcinoma (HCC) in Ethiopia. The prevalences of anti-HCV were 1.4%, 21%, 36% and 46%, respectively. There was no apparent risk factor to suggest the mode of transmission of HCV. Of the 238 patients, 65 (27%) had circulating hepatitis B surface antigen (HBsAg) (denoting current infection), 131 (55%) had antibodies to hepatitis B surface (anti-HBs) and/or core (anti-HBc) antigens (past infection) and 42 (18%) had no hepatitis B virus (HBV) marker. Anti-HCV antibodies were present in only one patient with HBsAg, in 54% with past infection and in 68% of those without HBV markers. Thus, HCV infection was uncommon in HBsAg-positive patients but significantly more common in patients with chronic liver disease and HCC who had evidence of past HBV infection or no marker for HBV infection. HCV infection appeared to be a more common cause of chronic liver disease and HCC than HBV infection in this population. However, considering the high prevalence of overall exposure to HBV infection (68% in healthy blood donors and 82% in those with chronic liver disease, including HCC), HBV is significant in terms of national preventive strategies. PMID- 7539953 TI - QBC and Giemsa-stained thick blood films: diagnostic performance of laboratory technologists. AB - This study compared the proficiency of military medical technicians in the diagnosis of malaria using Giemsa-stained thick blood films (GSF) and the QBC system. Fourteen technicians, with no previous experience of the QBC and limited experience in interpreting GSF, were given training in the 2 techniques and then tested on their ability to make a malaria diagnosis when provided with previously prepared GSF and QBC specimens (9 positive and 10 negative). The students achieved a sensitivity of 75% and 84%, respectively, with the QBC and GSF; specificity was 84% and 76%. There was no difference in the concordance of the 2 techniques with the actual diagnosis (80% vs. 81%). PMID- 7539954 TI - Improved microscopical diagnosis of pulmonary tuberculosis in developing countries. AB - The diagnosis of pulmonary tuberculosis (TB) relies on the bacteriological examination of sputum. However, microscopy of smears made directly from sputum has a low sensitivity and there is an urgent need for improved methods. We have compared microscopy of smears made directly from sputum with microscopy after liquefaction of sputum with household bleach (NaOCl) and concentration of bacteria by centrifugation. In 3 studies performed in Ethiopia and India, the use of the NaOCl method increased the number of samples positive for acid-fast bacilli by more than 100%. The technique is appropriate for developing countries and its application would increase the efficiency of TB control programmes. As a potent disinfectant, NaOCl also has the advantage of lowering the risk of laboratory infection. PMID- 7539956 TI - A new concept for successful long-term pulmonary preservation in a dog model. AB - We developed a dextran-glucose-based extracellular perfusion solution (DGX) that supports limited aerobic metabolism to maintain cellular integrity of an inflated donor lung during long-term ischemia and a storage temperature of 10 degrees C. In a dog model, we compared respiratory and hemodynamic function of orthotopically transplanted left lungs preserved using this method (DGX, group I, n = 6) with function of those preserved with EuroCollins solution (EC) stored at a temperature of 4 degrees C (group II, n = 6). All lungs were inflated with room air and stored for 12 hr. Pulmonary function was monitored for 5 hr of reperfusion. Values expressed below are group means with standard deviation. Statistical significance was calculated using a two-tailed t test. For PO2 (mmHg) (FiO2 = 0.4), group I (EC): control = 193 +/- 8, 30 min p.o. = 87 +/- 20*, 300 min p.o. = 174 +/- 13*; and group II (DGX): control = 217 +/- 28, 30 min p.o. = 184 +/- 46*, 300 min p.o. = 248 +/- 5*. For pulmonary vascular resistance (dynes), group I: control = 389 +/- 22, 30 min p.o. = 1209 +/- 301, 300 min p.o. = 1025 +/- 204*; and group II: control = 401 +/- 31, 30 min p.o. = 522 +/- 129, 300 min p.o. = 458 +/- 137* (*P < 0.05 DGX vs. EC). Gas analysis performed on air samples taken from the ischemic donor lung immediately after harvest and after 12 hr storage showed (calculated as group means) a significant decrease of PO2 and a significant increase of PCO2, respectively. Histology of the lungs after 5 hr of reperfusion showed essentially normal-appearing lungs in the DGX group, whereas lungs in the EC group showed thickening of the intra-alveolar septi, marked cellular infiltration, and accumulation of protein-like material in the alveoli. In this study, preservation with DGX resulted in satisfactory respiratory and hemodynamic function of the transplanted lung even after 12 hr of ischemia. It does not cause an increase of pulmonary vascular resistance as seen after preservation with EC. Data from the intrabronchial air analysis of the donor lung suggest that aerobic metabolism continues even under preservation conditions. PMID- 7539955 TI - Thiacetazone reactions. PMID- 7539957 TI - Increased anti-Gal activity in diabetic patients transplanted with fetal porcine islet cell clusters. AB - The natural anti-Gal antibody seems to create a major obstacle for discordant xenotransplantation in humans. Anti-Gal, which is produced in large amounts in humans (1% of circulating IgG), interacts specifically with the carbohydrate structure Gal alpha 1-3Gal beta 1-4Glc-NAc-R (termed the alpha-galactosyl epitope). This epitope is present in large amounts on porcine cells, as well as on cells of other nonprimate mammals (1 x 10(6) to 35 x 10(6) epitopes/cell). The interaction of anti-Gal with alpha-galactosyl epitopes on the xenograft was found to mediate the immune destruction of discordant xenografts. In the present study, the human immune response to alpha-galactosyl epitopes on xenografts was assessed by measuring changes in anti-Gal titers and affinity in sera of diabetic patients transplanted with fetal porcine islet cell clusters. The activity of this antibody was assessed by a hemagglutination assay with RBC, by ELISA with mouse laminin as a solid-phase antigen, and by equilibrium dialysis with the radiolabeled free haptenic form of the alpha-galactosyl epitope, i.e. [3H]Gal alpha 1-3Gal beta 1-4GlcNAc. All assays revealed a marked increase in anti-Gal activity after transplantation. The increase in anti-Gal titers ranged between 8- and 64-fold. A similar increase was observed in the binding of free alpha galactosyl epitopes to anti-Gal, as assayed in equilibrium dialysis. Immunoglobulin concentration did not increase after transplantation, suggesting that the observed increase in anti-Gal activity is the result of a specific immune response against alpha-galactosyl epitopes on the xenograft. The elevation in anti-Gal activity was observed in all three immunoglobulin classes and the highest activity was found within the IgG class. Analysis of IgG binding to fixed porcine endothelial cells suggested that most of the observed increased activity against these cells in transplanted patients may be attributed to the elevation in anti-Gal activity. PMID- 7539958 TI - The use of granulocyte colony-stimulating factor after liver transplantation. AB - Granulocyte colony-stimulating factor (G-CSF) increases the number of circulating granulocytes and decreases TNF production while improving survival in sepsis models. To study the effects of G-CSF administration on sepsis and rejection, 37 primary liver allograft recipients received intravenous recombinant human G-CSF (rhG-CSF; 5-10 micrograms/kg/day) for the first 7-10 days following transplantation, targeting a blood absolute granulocyte count of between 10,000 and 20,000 cells/mm3. These recipients were monitored prospectively for sepsis and rejection, as were the previous 49 primary liver allograft recipients who did not receive G-CSF. Both groups utilized identical protocol immunosuppression and standardized diagnosis and treatment of sepsis and rejection. Univariate and logistic regression analysis of risk factors for sepsis and rejection revealed no difference between the two patient groups. G-CSF-treated patients developed an increased absolute granulocyte count over time (P < 0.0001, repeated-measures analysis of variance). G-CSF-treated patients had a decreased number of sepsis episodes per patient (0.92 +/- 1.5 vs. 2.18 +/- 2.8, P < 0.02, t test), and a lower percentage of sepsis-related deaths (8% vs. 22%, P < 0.04, chi-square test). The incidence of acute rejection was decreased in the G-CSF-treated group (22% vs. 51%, P < 0.01, chi-square test). These pilot data support further investigation into G-CSF's favorable effects on sepsis and rejection. PMID- 7539959 TI - A synergistic increase in transplantable peripheral blood stem cells in mice by co-administration of recombinant human interleukin 6 and recombinant human granulocyte colony-stimulating factor. AB - We examined the effects of co-administration of recombinant human (rh) IL-6 (10 micrograms/day) and rh granulocyte colony-stimulating factor (G-CSF) (0.35 micrograms/day) on the number of peripheral blood cells and peripheral progenitor cells in mice. Among blood cells counts, only white blood cells were synergistically enhanced by co-administration of rhIL-6 and rhG-CSF. Moreover, it was found that co-administration of rhIL-6 and rhG-CSF also caused a marked synergistic increase in the number of peripheral blood progenitor cells. Namely, in combination with rhG-CSF, which alone induced a 170-fold increase in peripheral granulocyte-macrophage colony-forming units (CFU-GM) on day 14, rhIL-6 synergistically increased the number of CFU-GM to more than 1600-fold higher than the number in control mice. Administration of rhIL-6 alone induced a 46-fold increase in CFU-GM. Similar synergistic increases of other hematopoietic progenitors, such as colony-forming units in spleen and megakaryocyte colony forming units in blood, were also observed in mice co-administered rhIL-6 and rhG CSF. The survival rate of lethally irradiated recipient mice transplanted with mononuclear cells from 100 microliters of blood from mice administered rhIL-6 and/or rhG-CSF was examined. When mononuclear cells from mice co-administered rhIL-6 and rhG-CSF were injected, survival rate at day 100 was 92%. In contrast, recipient mice transplanted with mononuclear cells from mice administered either rhIL-6 or rhG-CSF alone showed a survival rate of 31% or 46%, respectively, although transplantation of mononuclear cells from control mice failed to rescue any lethally irradiated recipient mice. These results suggest that co administration of rhIL-6 and rhG-CSF may be useful for peripheral blood stem cell transplantation. PMID- 7539960 TI - Transcriptional inhibition of insulin by FK506 and possible involvement of FK506 binding protein-12 in pancreatic beta-cell. AB - FK506 (tacrolimus) is a strong immunosuppressant: it has been approved as a drug for liver transplantation in Japan, the United States, and the United Kingdom. One of its main adverse effects is hyperglycemia. Thus, in this study, we investigated the mechanism and the reversibility of the hyperglycemia caused by FK506. FK506 did not affect the glucose uptake by insulin into rat strio-muscle cell line, but suppressed insulin production in rat insulinoma cells. Two-week oral administration of FK506 at 10 mg/kg/day suppressed insulin production time dependently at the transcriptional step in pancreatic beta-cells, while glucagon content in pancreatic alpha-cells was not affected. When FK506 administration was stopped in these rats, insulin mRNA transcription and insulin production returned to normal. This recovery indicates that the adverse effect of FK506 on the pancreas is reversible. A high content of FK506 binding protein-12 (FKBP-12) in the pancreatic beta-cells was confirmed by immunostaining with anti-human FKBP-12 mAb, but the content was less in the pancreatic alpha-cells and almost negligible in the acinar cells. In contrast, a high content of calcineurin in the pancreatic alpha-cells was confirmed by using anti-calcineurin polyclonal antibody, but this content was less in the pancreatic beta-cells and not found in the acinar cells. Thus, as in the case with NF-AT in T cells, these findings point to the reduction of unidentified nuclear factors for insulin mRNA transcription caused by the binding of FK506 to FKBP-12 and a subsequent inhibition of calcineurin in the beta-cells. PMID- 7539961 TI - FK506 hepatotoxicity in liver allograft recipients. PMID- 7539962 TI - Unraveling the modular design of glutamate-gated ion channels. AB - Glutamate receptors that function as ligand-gated ion channels are essential components of cell-cell communication in the nervous system. Despite a wealth of information concerning these receptors, details of their structure are just beginning to emerge. We propose that glutamate receptors comprise four modules: two modules that are related to bacterial periplasmic-binding proteins, one module that is related to the pore-forming region of K+ channels, and one regulatory module of unknown origin. A K(+)-channel-like domain inserted into a crucial region of a periplasmic-binding protein-like domain suggests a mechanism for transduction of binding energy to channel opening. This modular design also suggests an evolutionary link between a ligand-gated ion-channel family and voltage-gated ion channels. PMID- 7539963 TI - Glycans and the modulation of neural-recognition molecule function. AB - Neural-recognition molecules are carbohydrate-bearing glycoproteins, glycolipids or proteoglycans that are found at the cell surface or in the extracellular matrix that regulate cell interactions during development, modification of synaptic activity and regeneration of nerve connections after damage in the adult. The expression of the carbohydrates appears to be fine tuned to these functions. Among the identified carbohydrates are polysialic acid, a 3'-sulfated glucuronic acid, and oligomannosidic residues. They act not only between apposing partner cell surfaces (trans-interaction) but also between recognition molecules within the surface membrane of one cell (cis-interaction), thereby forming complexes that influence transduction of signals to the cell interior. PMID- 7539964 TI - Efficacy and toxicity of vinorelbine-carboplatin combination in the treatment of advanced adenocarcinoma or large-cell carcinoma of the lung. AB - AIMS AND BACKGROUND: The aim of the study was to assess the activity and toxicity of the vinorelbine-carboplatin combination in advanced adenocarcinoma or large cell carcinoma of the lung. The new vinca derivative, vinorelbine, shows promising activity when combined with cisplatin, but toxicity of the combination is substantial. METHODS: Accordingly, we substituted carboplatin for cisplatin in the combination in order to improve the therapeutic index. From March 1992 to March 1994, 55 untreated patients with undifferentiated unresectable or metastatic adenocarcinoma or large-cell carcinoma of the lung were recruited. The treatment consisted of a course of carboplatin (300 mg/m2) and vinorelbine (25 mg/m2) repeated every 4 weeks. The only grade 3 toxicity observed was 16 cases of grade 3 vomiting and 2 cases of grade 3 stomatitis. RESULTS: The positive response rate was 40% (partial response, 22 patients). In conclusion, the vinorelbine-carboplatin combination may be regarded as an active, safe regimen for the palliative treatment of advanced adenocarcinoma or large-cell carcinoma of the lung. PMID- 7539965 TI - Comparison of immunoassays for tumor markers CA 19-9, CA 15-3 and CA 125: data from an international quality assessment scheme. AB - Data collected in the 1993 and 1994 cycles of an international external quality assessment (EQA) program and in a national multicenter collaborative study were cumulatively analyzed to evaluate the standardization of the methods currently in use for the assay of mucinous tumor markers CA 19-9, CA 15-3 and CA 125. On average the between-laboratory variability was 15.2 and 16.0 CV% for CA 15-3 and CA 125 respectively; the between-laboratory variability found for CA 19-9 was markedly worse (mean 28.3 CV%). The variability component attributable to systematic differences between different methods/kits was relatively small for CA 15-3 and CA 125 (18% and 24% of the total variability) but markedly larger for CA 19-9 (48% of the total variability). The agreement of CA 19-9 results worsened in the last few years when new nonisotopic techniques became available. The precision of the methods/kits most used in the survey ranged from 9.9 to 13.3 CV% for CA 125 and from 11.6 to 13.9 CV% for CA 15-3. For these two tumor markers the precision of the traditional IRMAs does not appear different from that of the new fully automated nonisotopic techniques. The precision of CA 19-9 methods was on average worse (from 11.7 to 19.6 CV%) although two automated systems exhibited a precision better than that of IRMAs. In conclusion, the results of this study indicate that CA 15-3 and CA 125 are satisfactorily assayed whereas CA 19-9 assay appears affected by larger differences between methods and by poorer precision of laboratories and kits. PMID- 7539966 TI - Role of granulocyte colony-stimulating factor in relapsed/resistant intermediate and high-grade non-Hodgkin's lymphoma patients treated with the E-SHAP regimen. AB - AIMS AND BACKGROUND: The study assessed the role and potential benefit of rhG-CSF in reducing the frequency, duration and severity of neutropenia following cytotoxic chemotherapy according to the E-SHAP protocol and, at the same time in improving the response rate. METHODS: Twenty patients with resistant/relapsed intermediate or high-grade non-Hodgkin's lymphoma were treated with the E-SHAP regimen (etoposide+methyl prednisolone+high dose cytosine arabinoside and cisplatin), and in 15 of them, we administered rhG-CSF between chemotherapeutic courses. RESULTS: The 15 patients who received G-CSF after E-SHAP were neutropenic for a short time and experienced no febrile episodes or infective complications. In contrast, in the group (5 patients) who did not receive G-CSF, the WBC nadir was lower and the number of days with a neutrophil count below 1.0 x 10(9)/L was longer, with a greater risk of inferctious complications. Of the 15 patients, only one had a delay in chemotherapy administration, and the RDI was 95% in the 65% of patients who received G-CSF. Of 5 patients treated with chemotherapy alone, 4 had a delay and the RDI was over 95% in only one patient. We obtained a good overall response rate (70%) in the group who received G-CSF. In the historical group of 5 non-Hodgkin lymphoma patients, we observed only 1 partial response and 4 had progression of disease. CONCLUSIONS: Administration of G-CSF is associated with an acceleration of neutrophil recovery, indicating its potential to reduce the risk of infection. The use of G-CSF permitted us to administer intensive chemotherapy without delay and according to standard dosage, with an improved response rate. PMID- 7539967 TI - Activation of the apoptotic Fas antigen-encoding gene upon influenza virus infection involving spontaneously produced beta-interferon. AB - We previously demonstrated that influenza virus infection induces apoptosis in culture cells. Here, we examined the activation of the Fas antigen gene that encodes an apoptosis-mediating membrane protein in the virus-infected cells. The virus elicited a transient but marked increase in Fas antigen mRNA 3 to 4 hr after infection, followed by the expression of the antigen on the cell surface. Poly(I)-poly(C), a synthetic double-stranded RNA, similarly activated Fas antigen gene expression, and poly(I)-poly(C)-treated cells are highly susceptible to the cell killing effect of IgM isotype of anti-Fas monoclonal antibody. On the other hand, the IgG isotype of anti-Fas monoclonal antibody, which has an inhibitory effect on Fas Ag-mediated cell death, suppressed the virus-induced cell death. Prior exposure of the cells to anti-interferon-beta antibody decreased the degree of cell death as well as the amount of Fas mRNA. The autophosphorylation activity of double-stranded RNA-activated protein kinase was also decreased in the antibody-treated cells. Moreover, a protein kinase inhibitor, 2-aminopurine, blocked the Fas Ag gene activation by poly(I)-poly(C). These results suggested that the activation of Fas Ag gene in the early phase of infection is an important event for apoptosis, and that it is regulated by the double-stranded RNA/interferon system involving protein phosphorylation. PMID- 7539968 TI - Expression and characterization of proteins produced by mRNAs spliced into the X region of the human T-cell leukemia/lymphotropic virus type II. AB - In previous studies we showed that human T-cell leukemia/lymphotropic virus type I (HTLV-I) may produce novel proteins encoded in the X region. To investigate a possible correlation between expression of viral genes and different biologic properties of HTLV-I and HTLV-II, we analyzed expression of HTLV-II in the chronically infected cell line MoT. Reverse transcription-polymerase chain reaction analyses revealed that the virus produces several mRNAs singly or doubly spliced into the X region. Corresponding cDNAs were cloned and transfected into a HeLa cell line; resulting proteins were designated according to their sizes and coding open reading frames (ORFs). p10xI and p11xV were produced by a dicistronic doubly spliced mRNA. p10xI was generated by translation of the first exon of rex linked to the x-I ORF; p11xV was translated from the tax initiation codon linked to the x-V ORF. Two singly spliced polycistronic mRNAs produced p28xII, coded by the x-II ORF, and several isoforms generated by initiation within the x-III ORF. Studies of the proteins' subcellular localization revealed that they exhibited distinct targeting patterns. Comparison of these proteins with their HTLV-I counterparts indicated intriguing differences between these two viruses, suggesting that further study of the X region products may aid in defining genetic determinants of pathogenicity. PMID- 7539969 TI - Trace amount of satellite RNA associated with tobacco ringspot virus: increase stimulated by nonaccumulating satellite RNA mutants. AB - The small satellite RNA of tobacco ringspot virus (sTRSV RNA) is dependent on tobacco ringspot virus (TRSV) for replication and encapsidation. sTRSV RNA has appeared during serial passage of certain TRSV strains in some hosts. Co inoculation of bean with TRSV and either of two related, nonaccumulating mutants of sTRSV RNA induced the appearance of sTRSV RNA in a single passage (van Tol et al., 1991, Virology 180, 23-30). The sTRSV RNA obtained after serial passage and after co-inoculation have the same nucleotide sequence, designated the endogenous sequence. The endogenous sTRSV RNA nucleotide sequence differs from that of each of the nonaccumulating sTRSV RNA at three positions. In order to detect possible trace amounts of endogenous satellite RNA in virion RNA preparations, RNA from two TRSV isolates was subjected to reverse transcription and polymerase chain reaction of the transcript (RT-PCR), using primers with sTRSV RNA terminal sequences. The yield of RT-PCR product suggests that the virion RNA preparations contained approximately 0.1 fg of sTRSV RNA per microgram of virion RNA. The nucleotide sequence of the RT-PCR product corresponded to that of the endogenous sTRSV RNA. The endogenous sTRSV RNA of TRSV inocula appears to be latent, being maintained in very small amounts during serial passage of TRSV in some hosts but capable of dramatic increase during serial passage in other hosts or when TRSV was co-inoculated with either of two specific sTRSV RNA mutants. Ten other nonaccumulating sTRSV RNA mutants did not induce a detected increase in sTRSV RNA. PMID- 7539970 TI - Characterization of the leader papain-like proteinase of MHV-A59: identification of a new in vitro cleavage site. AB - Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a. PMID- 7539971 TI - The multimeric nonstructural NS2 proteins of bluetongue virus, African horsesickness virus, and epizootic hemorrhagic disease virus differ in their single-stranded RNA-binding ability. AB - The structure and single-stranded (ss) RNA-binding by the nonstructural protein NS2 of three different orbiviruses were studied and compared. African horsesickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) were analyzed in recombinant baculovirus-infected cells and in cells infected with BTV and AHSV. Sedimentation analysis and nonreducing SDS PAGE revealed that NS2 of all three orbiviruses is a 7S multimer with both inter- and intramolecular disulfide bonds, probably consisting of six or more NS2 molecules. The 7S NS2 multimer of all three viruses binds ssRNA but there is a marked disparity in the ssRNA-binding ability between the three proteins. At physiological salt concentration, BTV NS2 binds ssRNA very efficiently, whereas AHSV NS2 shows only a low efficiency for binding ssRNA. EHDV NS2 binds with intermediate efficiency. The result was the same irrespective of whether poly(U) Sepharose or viral mRNA was used, indicating that ssRNA-binding by NS2 is nonspecific. The difference in RNA-binding ability may be related to the alpha helix content of the respective proteins. NS2 of BTV has the highest predicted alpha-helix content followed by EHDV and AHSV. The ability of the NS2 proteins to form virus inclusion body-like structures in baculovirus-infected cells is not affected by the ssRNA-binding disparity. PMID- 7539972 TI - Mapping of a serotype specific epitope of the major capsid protein VP2 of infectious pancreatic necrosis virus. AB - A serotype-specific, conformational epitope of VP2 of infectious pancreatic necrosis virus (IPNV) (Jasper), has been mapped by restriction enzyme site specific deletions of cloned viral cDNA. Subclones encoding fragments of VP2 were expressed in Escherichia coli followed by polyacrylamide gel electrophoresis and Western blot analysis. The epitope (between amino acid residues 242 and 325) reacted with monoclonal anti-VP2 antibodies that neutralized the homologous (Jasper), but not the heterologous (Sp) serotype of IPNV. PMID- 7539973 TI - Teaching nursing research using two-way video technology. AB - Schools of nursing are meeting the needs of students by instituting distance education courses that use two-way video technology. This article describes the structural features of the classroom that are changed and provides strategies that can be effective for teaching nursing research. These strategies are related to: teaching and demonstrating use of statistical formulas; selecting and teaching use of software for data analysis; teaching about research design, sampling, and data collection procedures; and critiquing empirical articles. PMID- 7539976 TI - Palliative care at Killam General Hospital. PMID- 7539974 TI - [The antigen-specific activity of a multicomponent vaccine administered orally and subcutaneously]. AB - The antigen-specific activity of polycomponent vaccine VP-4 consisting of Klebsiella pneumoniae, Staphylococcus aureus, Proteus vulgaris and Escherichia coli antigens was studied. The same form of the vaccine was used for both subcutaneous injection and oral administration. The oral administration of the preparation to rabbits and guinea pigs induced a rise in the level of antibodies to all components of the vaccine in their blood sera, the optimum oral doses exceed only 2- to 4-fold the doses used for subcutaneous injection. The form of vaccine VP-4, developed for subcutaneous injection, may be recommended for wider trials with its oral administration. PMID- 7539975 TI - [A complex evaluation of the lytic potential of cellular cytotoxicity displayed by natural killers and K cells]. AB - The method of testing the lytic potential of cell cytotoxicity, shown by whole blood natural killer (NK) cells and K-cells, including the determination of the responsiveness of killers to interferon, is proposed. The method makes it possible to carry out mass survey of the population and to work with contaminated material. In this study the influence of different gene engineering preparations of interferon on the activity NK cells of humans and hamadryas monkeys is determined. The individual reaction of killers to their stimulation with interferon was confirmed. The study revealed that in volunteers immunized with yellow fever vaccine a decrease in the sensitivity of NK cells was observed. PMID- 7539977 TI - Elevated plasma thrombopoietic activity in patients with metastatic cancer related thrombocytosis. AB - BACKGROUND AND PURPOSE: High platelet counts are occasionally seen in patients suffering from progressive malignant disorders. While granulocyte colony stimulating factor (G-CSF) has been implicated in paraneoplastic leukemoid reactions, the stimulus for thrombocytosis is unknown. Our purpose in this study was to determine if plasma from cancer patients with thrombocytosis contains a factor or factors with thrombopoietic activity. METHODS: We tested the effects of plasma obtained from 5 individuals with advanced tumors and high platelet counts and from 4 patients with advanced cancer and normal platelet counts on megakaryocytic differentiation of two megakaryoblastic cell lines (Dami and HEL). Differentiation was evaluated by assessing the expression of the platelet specific cell-surface antigens CD41 (HUPL-mI) and glycoprotein IIb-IIIa using an immunocytochemical staining score. In addition, plasma samples from 7 of the 9 patients and from 5 additional cancer patients with thrombocytosis were assayed for the levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), G-CSF, and IL-1 beta protein using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Expression of platelet-specific cell surface antigen was increased in HEL cells after exposure to plasma from all 5 of the cancer patients with thrombocytosis, and in Dami cells after exposure to plasma from 4 of the 5. Similar, but less significant, results were found when these cells were incubated with control combinations of recombinant GM-CSF plus IL-6 or of IL-3 plus IL-6. Platelet-specific cell-surface-antigen expression was not increased in HEL or Dami cells after exposure to the plasma from the 4 cancer patients with normal platelet counts or to normal control plasma. ELISA revealed elevated levels of IL-6 in the plasma from 4 patients with thrombocytosis (38, 40, 63, and 99 pg/mL). In addition, GM-CSF concentration was high in 3 of these 4 patients (33, 47, and 127 pg/mL), and the G-CSF level was elevated in 1 (543 pg/mL). IL-1 beta and IL-3 levels were undetectable. CONCLUSIONS: Our data suggest that the thrombocytosis observed in individuals with advanced malignant disease is mediated by a humoral mechanism. Levels of IL-6, GM-CSF, and G-CSF are elevated in some of these patients, but the plasma concentrations are generally lower than those required for in vitro induction of megakaryocytic differentiation. Plasma from patients with paraneoplastic thrombocytosis may therefore contain thrombopoietins that have not yet been identified, and which might have clinical usefulness. PMID- 7539978 TI - Reactivation of rheumatoid arthritis and development of leukocytoclastic vasculitis in a patient receiving granulocyte colony-stimulating factor for Felty's syndrome. PMID- 7539979 TI - Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies. AB - Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas. Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain. In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha. Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein. The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15. The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha. PMID- 7539980 TI - Histogenesis and ultrastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial cells of host origin. AB - In previous studies we have demonstrated that syngeneic and xenogeneic pancreatic islet grafts are revascularized within a 10 to 14-day period after transplantation. With the combined use of intravital and electron microscopy, as well as immunohistochemistry using a set of species-specific or -crossreacting antibodies to endothelial cell antigens, we investigated 1) the origin of the endothelium of the newly formed capillaries in free pancreatic islet isografts (hamster-->hamster) and xenografts (rat-->hamster), and 2) the ultrastructural characteristics of these microvessels. Intravital microscopy demonstrated that newly formed microvessels grow from the vascular bed of the host muscle tissue into the islet grafts. Immunohistochemical analysis of host tissue and transplanted islets with antibodies against factor VIII (recognizing both hamster and rat factor VIII), bovine PECAM-1 (CD31; endoCAM, crossreacting with hamster but not rat PECAM-1), and rat ICAM-1 (CD54, non-crossreacting with hamster ICAM 1) showed that the transplanted rat islets were revascularized by endothelium of hamster (host) origin. At an ultrastructural level, the endothelial lining of the newly formed microvessels showed diaphragmatic fenestration, a characteristic feature of endothelial cells of pancreatic islets in situ. On the basis of these findings we suggest that pancreatic islet transplantation may take a unique position in the field of organ transplantation, since the generally proposed mechanisms of endothelial cell-dependent antigen recognition as a trigger of graft rejection may not be transferred to islet grafts, containing microvessels lined by endothelial cells of host origin. PMID- 7539982 TI - Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation. PMID- 7539983 TI - The extent to which ribonucleases cleave ribonucleic acid. PMID- 7539981 TI - Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. AB - Uterine myometrial tumors are the most commonly found gynecological neoplasm in women. The underlying causes of uterine leiomyomata are poorly understood, a result in part of the absence of a good animal model system in which to study these tumors. This report describes a novel rat model (Eker rat) in which spontaneous gynecological smooth muscle tumors arise with a high frequency. Leiomyomas are the predominant reproductive tract tumor that arise in these animals, although leiomyosarcomas have also been observed. Cell lines have been established from both the benign and malignant lesions. All of the lines express smooth muscle-specific actin, and leiomyoma-derived cell lines express desmin. Two of the cell lines are tumorigenic in nude mice, and the lines are variable for expression of estrogen and progesterone receptors. These lines are the first rodent tumor-derived lines to be established from leiomyomata and are the only lines available from a hereditary form of these tumors. Together with Eker rats that spontaneously develop leiomyomata, they constitute an in vitro/in vivo model system for gaining insights into the mechanism of transformation of uterine smooth muscle cells and the role of steroid hormones and hormone receptors in myometrial tumorigenesis. PMID- 7539985 TI - Enzyme immunometric assay for L-thyroxine using direct ultraviolet irradiation. AB - A new enzyme immunometric assay for L-thyroxine using uv irradiation as a cross linking procedure is described. L-Thyroxine in plasma samples is immunocaptured by a monoclonal anti-L-thyroxine antibody coated on 96-well microtiter plates. After uv irradiation and methanol treatment, the covalently linked L-thyroxine is measured using the same monoclonal anti-L-thyroxine antibody labeled with acetylcholinesterase. A minimal detectable concentration of 4.8 nmol/liter was observed with a coefficient of variation less than 16% in the 20-320 nmol/liter. Specificity of the assay was very satisfying and a good correlation (r = 0.959) was noted for 33 human plasma samples between this assay and a commercial competitive radioimmunoassay. PMID- 7539984 TI - Chemiluminescent detection of dextran bound to streptococcal glucan-binding lectin. PMID- 7539986 TI - Fluorometric measurement of reverse transcriptase activity with 4',6-diamidino-2 phenylindole. AB - We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A).oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA.DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the Klenow fragment using poly(dA).oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [alpha-32P]dTTP into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA] heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7539987 TI - Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence. AB - Reaction conditions were defined for the selective quantitative derivatization and fluorophore labeling of phosphoserine residues on peptides and proteins. Phosphoserine was derivatized with 1,2-ethanedithiol using a modification of the reaction conditions defined by R. C. Clark and J. Dijkstra (1967) Int. J. Biochem. 11, 577-585 and H. E. Meyer, E. Hoffman-Posorke, H. Korte, and M. G. Heilmeyer (1986) FEBS Lett. 204, 61-66 for stabilizing the phosphoamino acid during Edman degradation reactions. Following derivatization, the thiol-serine residues were coupled to fluorescence by iodoacetate reaction. Characterization by capillary zone electrophoresis and laser-induced fluorescence allowed quantitation of phosphoserine content of peptides and proteins at < 75 amol. In three separate experiments, the overall reaction efficiency for 1,2-ethanedithiol derivatization of phosphoserine was estimated at 89.27 +/- 2.44% (SDM). Subsequent coupling of the derivatized serine residue with 6 iodoacetamidofluoroscein was estimated at > 98% efficiency. Fluorescent probe tagging of phosphoamino acids on proteins and peptides offers direct quantitative evaluation of cellular phosphorylation states at the attomole level in tissue samples derived from plants, animals, and humans, without the use of radioisotopes, antibodies, or mass spectrometry. PMID- 7539988 TI - Mechanosensitive channels. PMID- 7539989 TI - The CFTR chloride channel of mammalian heart. PMID- 7539990 TI - The 5-HT3 receptor channel. PMID- 7539991 TI - Mechanisms of activation of muscle nicotinic acetylcholine receptors and the time course of endplate currents. PMID- 7539992 TI - Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission. PMID- 7539993 TI - Nitric oxide signaling in the central nervous system. PMID- 7539994 TI - Nitric oxide synthases: properties and catalytic mechanism. PMID- 7539995 TI - Nitric oxide: pathophysiological mechanisms. PMID- 7539996 TI - Nitric oxide in the regulation of blood flow and arterial pressure. PMID- 7539998 TI - Environmental transmission of hepatitis B and hepatitis C viruses within the hemodialysis unit. AB - The hepatitis B virus (HBV) can be transmitted in the dialysis setting through blood transfusions and environmental surfaces. Transfusion related hepatitis C virus (HCV) infection is very well known, but only recently the environmental transmission of this virus was postulated. In order to study the prevalence, mechanisms of transmission, and the ALT patterns of HBV and HCV infections in hemodialysis and CAPD patients before the implementation of HBV vaccination and HCV screening in the blood bank, we conducted a study from January 1987 to January 1990. Sera from 185 hemodialysis and 124 CAPD patients were stored in this period and later analyzed for HBsAg, anti-HBc, anti-HBs, and anti-HCV (second generation ELISA). The prevalence of any HBV marker was 55.7% (103/185) for hemodialysis patients and 31.5% (39/124) for CAPD patients (hemodialysis vs. CAPD, p < 0.001). The prevalence of positive anti-HCV was 35.1% (65/185) for hemodialysis and 33.9% (42/124) for CAPD patients (not significant). There was a significant association between HBV markers positivity and anti-HCV positivity. The multivariate analysis of risk factors revealed an association of the positivity of each virus with the duration of renal replacement therapy (RRT), number of previous blood transfusions, and past history of hemodialysis treatment. Thus, besides the transfusion-related transmission, hemodialysis environmental transmission may also occur for both viruses. The findings of a high prevalence of both viruses and evidence for environmental transmission in the dialysis setting are of major importance for the planning of future preventive measures. PMID- 7539997 TI - The nonribosomal peptide biosynthetic system--on the origins of structural diversity of peptides, cyclopeptides and related compounds. AB - A variety of peptides have been detected in microorganisms. Some have found applications in various fields, for example the classical beta-lactam antibiotics, immunosuppressors like cyclosporin, promising new antibacterials like teichoplanin or daptomycin and antifungals like echinocandin. For none of these has it been established how their complicated biosynthetic pathways have evolved or what functions they fulfill within or for their producers. So it is unclear what selection processes limit the range of their structural analogues within various groups of microorganisms. We here consider recent data in the field of biosynthesis and how they may suggest mechanisms of genetic diversity. These may illustrate the complexity of genetic and intracellular organization of biosynthetic pathways and indicate the cellular context of some metabolites related to the complex background of the production of each metabolite. Research focusing on various targets like the increase of productivity of fermentations or the spread of resistances to antibacterials is slowly being understood. PMID- 7540001 TI - The influence of RNA and DNA template structures during transcript elongation by RNA polymerases. AB - It was previously thought that elongating Escherichia coli transcription ternary complex consists of an RNA polymerase molecule enclosing 17 +/- 1 melted bases (bubble) of the template DNA and a 12-base-pair RNA-DNA hybrid ("transcription bubble paradigm"). Recent evidence suggests that ternary elongation complexes are heterogeneous and possibly vary in bubble size and length of RNA-DNA hybrid. We used a new type of assay to address the relative contributions of bubble size, secondary structure of RNA and RNA-DNA hybrid length during elongation. Synthetic RNA-DNA bubble duplexes are assembled in vitro. RNA structure 5' to the RNA-DNA hybrid, hybrid length and bubble size are systematically changed. The relative efficiency of E. coli and T7 RNA polymerases to elongate RNA primer is quantitated. RNA elongation was high (approximately 22-30%) when a stable hairpin was present towards the 5' end of the primer. Efficiency of elongation was lower for RNA primers without hairpins. Hairpin RNAs with presumed RNA-DNA hybrids of 3 7 bp were efficiently elongated compared to hairpins that presumably form 10bp hybrids. Preformed bubbles of different sizes (2,5 or 20 bases) were functional in all cases where elongation was moderate or high. We concluded that RNA secondary structure plays a dominant role compared to hybrid length or bubble size in determining efficient elongation by RNA polymerases. PMID- 7540000 TI - Peptides from the amino-terminus of RANTES cause chemotaxis of human T lymphocytes. AB - RANTES is a potent chemotactic and activating agent for a variety of leukocytes including T-lymphocytes. To identify the region of the molecule responsible for this activity, we have made overlapping 10 amino acid peptides scanning the protein. A micro-Boyden chamber chemotaxis assay showed the most efficacious peptides came from at the N-terminus. EC50 values of 8 nM (+/- 2.2 nM), 3.7 nM (+/- 2.0 nM) and 3.1 nM (+/- 2.0 nM) were calculated from dose response curves for the peptides (1-10), (3-12), and (5-14). Control peptides from other regions are not active. In THP-1 cells, none of the peptides give a Ca2+ response. The active peptides are shown to be principally chemotactic rather than chemokinetic by a checker board analysis. The results imply that the principal region of RANTES responsible for chemotaxis is located in the amino terminus. PMID- 7539999 TI - The effects of thimerosal, a sulfhydryl reagent, on phasic myometrial contractions. AB - Thimerosal inhibits calcium uptake and IP3-induced calcium release from IP3 sensitive endoplasmic reticulum; this study sought to evaluate the effects of thimerosal on agonist-stimulated phasic myometrial contractions. Thimerosal was found to significantly inhibit phasic contractions stimulated by oxytocin, aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium uptake and IP3 induced calcium release are important events during agonist-stimulated phasic myometrial contractions. PMID- 7540003 TI - Evidence against dephosphorylation of insulin-elicited phosphotyrosine proteins in vivo by the phosphatase PTP2C. AB - In order to determine whether the tyrosine phosphatase PTP2C dephosphorylates insulin-elicited phosphotyrosine proteins in vivo, we have compared the patterns of protein tyrosine phosphorylation and its reversal in the kidney 293 cell line with those in 293 cell lines overexpressing PTP2C and a catalytically inactive point mutant of PTP2C. In all three cell types insulin caused the rapid tyrosine phosphorylation of a 160 kD protein, which was shown not to be the insulin receptor substrate 1 (IRS-1) and may be the recently described IRS-2, as well as that of a 100 kD polypeptide, which is probably a mixture of the beta subunits of the insulin and insulin-like growth factor I receptors. There was no difference among the three cell lines in the extent of tyrosine phosphorylation or in the rate of its reversal upon insulin withdrawal. These results indicate that PTP2C does not function to dephosphorylate these proteins significantly in vivo. PMID- 7540002 TI - Coordinate up- and down-modulation of inducible nitric oxide synthase, nitric oxide production, and tumoricidal activity in rat bone-marrow-derived mononuclear phagocytes by lipopolysaccharide and gram-negative bacteria. AB - Simultaneous incubation of primary rat bone-marrow-derived mononuclear phagocytes (BMMo) and tumor cells with gram-negative agents triggers within 24 h interferon gamma (IFN gamma)- and tumor necrosis factor (TNF alpha)-independent tumoricidal activity. On the other hand, BMMo that had been incubated for 24 h with gram negative agents prior to re-exposure to the same agent had largely lost their ability to generate tumoricidal activity, although their ability to bind lipopolysaccharide (LPS) was not diminished. Parallel measurements of the kinetics of inducible nitric oxide synthase (iNOS), nitrite secretion, and tumoricidal activity triggered in primary BMMo by LPS revealed that these parameters take a coordinate course, reaching a peak within 24 h and then rapidly decaying. Down-regulation of expression of NOS protein and iNOS activity could be attributed neither to down-regulation of LPS receptors nor to L-arginine depletion. PMID- 7540004 TI - E-cadherin peptide sequence recognition by anti-E-cadherin antibody. AB - Cadherins are calcium dependent glycoproteins involved in homophilic cell-cell adhesion. To further elucidate the interaction between cadherins we have developed an immobilized peptide ELISA to quickly scan the extracellular domains of E-cadherin. Peptides displaying antigenic reactivity to anti-E-cadherin antibody are presumably on the surface and thus may be involved in cadherin cadherin interaction. We have found three peptides from the EC-1 domain which are recognized by the anti-E-cadherin antibody. These peptides are in the two important regions of the EC-1 domain that was deduced from its secondary structure. PMID- 7540005 TI - Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene. AB - The chromatin structure of 120kb of genomic DNA 5' to the CFTR gene has been analysed in a number of CFTR expressing and non-expressing cell types, including primary genital duct epithelial cells. Novel DNAse I hypersensitive sites have been observed at -79.5kb and -20.5kb 5' to the ATG translation start codon of the CFTR coding sequence. Neither of these sites appears to show strong correlation with CFTR expression in the cell types investigated, hence they are unlikely to reflect the sites of binding of the major CFTR tissue specific regulator(s). However, they may still play an important part in the complex series of events involved in the regulation of CFTR transcription. PMID- 7540006 TI - The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity. AB - Human cytomegalovirus encodes several proteins with high similarity to seven transmembrane domain receptors. We investigated the ability of one of these proteins, the product of the US28 open reading frame, to bind various chemoattractant ligands. When transfected into COS-7 cells, the US28 product conferred high affinity binding to the labeled chemokines monocyte chemoattractant protein-1 (MCP-1) (Kd = 6.0 x 10(-10) M) and RANTES (Kd = 2.7 x 10(-10) M). Binding of these labeled ligands could be competed by the unlabeled macrophage inflammatory proteins MIP-1 alpha and MIP-1 beta, with Kd values in the range 1.2 x 10(-9) to 7.5 x 10(-9) M. Comparisons of the sequences of US28 and other receptors that bind chemokines should help to define regions responsible for receptor-ligand interactions. PMID- 7540007 TI - Differential induction of mRNA for ICAM-1 and selectins in hepatocytes, Kupffer cells and endothelial cells during endotoxemia. AB - Intercellular adhesion molecule-1 (ICAM-1) and selectins (E- and P-selectin) mRNAs were determined in individual liver cell types by Northern blot analysis before and after injection of endotoxin. A constitutive expression of ICAM-1 mRNA was found in endothelial cells and Kupffer cells but not in hepatocytes. All three cell types showed upregulation of ICAM-1 mRNA after endotoxin. No constitutive selectin expression could be detected in any liver cell, but endotoxin induced massive synthesis of E- and P-selectin mRNA in endothelial cells and Kupffer cells. The differential expression of cellular adhesion molecules in the liver is consistent with the involvement of selectins in neutrophil rolling in the vasculature and ICAM-1 in transendothelial migration and adherence to parenchymal cells. PMID- 7540010 TI - Childhood aphasia. What is it? PMID- 7540008 TI - Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria. AB - The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC. PMID- 7540009 TI - Expression of E-selectin messenger RNA and protein in rheumatoid arthritis. AB - OBJECTIVE: To examine the de novo synthesis and cellular distribution of the E selectin adhesion molecule in synovial tissues obtained from patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry techniques combined with in situ hybridization were used to examine RA synovium. RESULTS: There were numerous endothelial cells positive for E-selectin and E-selectin messenger RNA in the RA synovial membranes. Moreover, E-selectin expression appeared to correlate with inflammatory activity. CONCLUSION: The strong vascular expression of E-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of RA. PMID- 7540011 TI - Outcome of pregnancies with elevation of both maternal serum alpha-fetoprotein and human chorionic gonadotropin. AB - We evaluated the pregnancy outcome of all patients with elevations of both maternal serum alpha-fetoprotein and human chorionic gonadotropin since institution of combined serum screening at our program. After analysis of 34,404 samples, 99 patients were found to have significant elevations of both maternal serum alpha-fetoprotein and human chorionic gonadotropin. The ultrasound findings, amniocentesis results, and pregnancy outcomes were determined in each case. Sixty-six patients with singleton gestations met entry criteria. Pregnancy outcome information is available for 63 of these patients, 60% of whom had at least one complication. Thirty-three patients with multiple gestations met inclusion criteria. Pregnancy outcome information is available for 31 of these, 81% of whom had at least one complication. These patients had a high incidence of pregnancy related complications. This group would appear to be at higher risk than women with elevation of either maternal serum alpha-fetoprotein or human chorionic gonadotropin alone. PMID- 7540012 TI - Thyrotropin-releasing hormone in treatment of intractable epilepsy: neurochemical analysis of CSF monoamine metabolites. AB - The efficacy of thyrotropin-releasing hormone in children with intractable epilepsy was investigated and changes in cerebrospinal fluid monoamine metabolites were analyzed. The 18 patients had either West syndrome (12 patients) or Lennox-Gastaut syndrome (6 patients), which was intractable to antiepileptic drug therapy and to adrenocorticotrophic hormone. Thyrotropin-releasing hormone tartrate was administered for 4 weeks. Before and after the thyrotropin-releasing hormone administration, cerebrospinal fluid was collected and analyzed for 5 hydroxyindoleacetic acid, kynurenine, homovanillic acid, and 3-methoxy-4 hydroxyphenyl glycol. The patients were classified into 3 groups, based on seizure frequency and electroencephalographic effects: cessation of seizures and seizure discharges (very effective; group A), reduction of seizures and/or seizure discharges (effective; group B), and no changes in frequency of seizures or discharges (not effective; group C). There were 6 patients in group A, 3 in group B, and 9 in group C. There were no significant differences in monoamine metabolites before and after the thyrotropin-releasing hormone therapy. A trial of thyrotropin-releasing hormone for the treatment of intractable epilepsy is warranted and further study is required on the mechanism of the antiepileptic action of thyrotropin-releasing hormone. PMID- 7540013 TI - Complementarity, specificity and the nature of epitopes and paratopes in multivalent interactions. AB - Structural elements of an antibody (Ab) or antigen (Ag) distant from the actual sites mediating contact between Ab and Ag can exert substantial influence on binding to, and discrimination among, multivalent targets. Consequently, multivalent molecules that express the same number of identical binding sites, but that differ in other structural features, can exhibit differences in their ability to discriminate between multivalent ligands. Here, Neil Greenspan and Laurence Cooper review evidence for these effects, and explore implications of the conclusion that effective specificity in multivalent interactions is not completely determined by the degree of complementarity between epitopes and paratopes. PMID- 7540014 TI - Fixation of various aldehydic dextrans onto human hemoglobin: study of conjugate stability. AB - Formation and stability of different aldehydic dextran-hemoglobin conjugates were studied. Two types of polymers were used: sulfated or unsulfated oxidized dextrans and 4-carboxamidobenzaldehyde dextran. Periodate-oxidized dextran forms imine and ketoamine linkages by reaction with hemoglobin and the obtained conjugates are not completely stable, as their molecular size increases with time or decreases after incubation with lysine. The sulfated conjugates are more sensitive to lysine action than the unsulfated ones, which is consistent with the decreased possibilities of Amadori rearrangement. Therefore, this proves the importance of ketoamines for ensuring the cohesion of oxidized dextran-based conjugates. Carboxamidobenzaldehyde dextran forms only imine linkages with hemoglobin and the corresponding conjugates possess a marked instability in the absence of reductive treatment. The different types of conjugates could be stabilized by a sodium borohydride treatment in a satisfying manner. PMID- 7540015 TI - Quantum chemical studies employing an ab initio combination approach on the binding of the bis-benzimidazole Hoechst 33258 to the minor groove of DNA. AB - Ab initio calculations (Hartree-Fock) using the 3-21G and the STO-3G Gaussian basis sets were performed on the sequence selective minor groove binding bis benzimidazole Hoechst 33258. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecule were derived. The binding of the optimized conformations of the drug to both alternating and non alternating (AT)n sequences was studied. PMID- 7540017 TI - Simple method to produce RNA size markers using cis ribozymes. PMID- 7540016 TI - Bivalent vaccines against bacterial enteropathogens: construction of live attenuated vaccine strains with two O-serotype specificities. AB - A considerable interest exists worldwide in the development of live attenuated oral vaccines against diarrhoeal diseases. In addition to vaccination against the corresponding pathogens, such vaccine strains can be used as carriers for the expression of protective antigens from other organisms. The antigenic repertoire of a given vaccine strain may thereby be extended, potentially leading to a bivalent vaccine. The lipopolysaccharide is known to be a major antigenic surface component of bacterial enteric pathogens. The feasibility of the development of combined vaccines based on live attenuated carriers expressing two O-serotype specificities is illustrated here by the development of candidate live oral vaccines against Shigella sonnei using Salmonella typhi and Vibrio cholerae as carriers. Various factors that may limit the potential of such hybrid strains as bivalent vaccines are discussed. PMID- 7540018 TI - Freezing of isolated cells provides free mRNA for RT-PCR amplification. PMID- 7540019 TI - Recovery of RNA from oral streptococci. PMID- 7540020 TI - Use of 33P-labeled primer increases the sensitivity and specificity of mRNA differential display. PMID- 7540023 TI - The influence of nitric oxide on tumour vascular tone. AB - Acetylcholine and sodium nitroprusside, which vasodilate via release of NO by endothelium-dependent and endothelium-independent mechanisms respectively, had little effect on tumour vascular resistance when administered to tissue-isolated tumours perfused in their normal state. However, under phenylephrine-induced vasoconstriction, sodium nitroprusside induced vasodilation whilst acetylcholine induced a small vasoconstriction. Phenylephrine itself induced an oscillatory change in tumour perfusion pressure. The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA) caused a dose-dependent increase in vascular resistance in ex vivo perfused tumours which was greater than that in normal perfused hindlimbs. Systemic administration of L-NNA caused a 50% decrease in tumour blood flow which was a larger effect than in any of the normal tissues studied except spleen and skeletal muscle. Modification of NOS activity in tumours is a promising means for selective tumour blood flow modification. Investigation of endothelium-dependent versus endothelium-independent methods for modifying tumour blood flow may provide methods for further selectivity. PMID- 7540021 TI - Rapid characterization of growth-arrest genes in transient transfection assays. AB - We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay. PMID- 7540024 TI - Prostaglandins and gangliosides of tumor microenvironment: their role in angiogenesis. AB - In the solid tumor the microenvironment is the space limited by the basement membrane of the microvessels and the neoplastic cells membrane. It includes the stroma and a liquid phase, the tumor interstitial fluid (TIF). We developed a method to sample TIF in vivo and found it rich in prostaglandins. In the rabbit cornea PGE1 induces neovascularization and acts as an angiogenesis factor. Before angiogenesis appears the ganglioside content of the cornea doubles with sharp reduction of the GM3/GD3 ratio. These gangliosides are not angiogenic but they influence the endothelial cell behavior. In particular when the PGE1 dose is insufficient to induce angiogenesis, enrichment of corneal tissue with GD3 or GM1 stimulates angiogenesis. However, when the corneal tissue is enriched with GM3, doses of PGE1, normally angiogenic fail to do so. The same was observed when bFGF substituted PGE1 as an angiogenesis trigger. The gangliosides tested acted as modulators of the angiogenic response by promoting the angiogenic capacity of molecules, such as PGE1 or bFGF, normally present in the tissue microenvironment. Several neoplastic cells, especially melanomas, shed gangliosides in the microenvironment. Their modulatory effect on angiogenesis may influence metastatic and/or primary tumor growth. PMID- 7540022 TI - Mesoglycan and sulodexide act as stabilizers and protectors of fibroblast growth factors (FGFs). AB - Heparin and heparan sulfate proteoglycans (HSPGs) stabilize FGFs which belong to heparin-binding growth factors (HBGFs) on active conformation. They also strongly potentiate their mitogenic activity on many cell types, and protect them against thermal denaturation and enzymatic degradation. In the present work we have tested two heparin-like substances named mesoglycan and sulodexide obtained from bovine intestinal mucosal extracts. These products are used as heparin, in various of therapeutic fields such as atherosclerosis or antithrombotic therapy. The compositions of mesoglycan and sulodexide are partially known and include chondroitin, dermatan and heparan sulfate. We have shown that mesoglycan and sulodexide potentiated the mitogenic activity of FGF1 and FGF2. The magnitude of this effect was identical with that of heparin used as a control substance but at double concentration. Mesoglycan and sulodexide also exerted stabilizing and protective effects on FGFs for heat denaturation and enzymatic degradation. The suppression of the protective properties after heparinase treatment of mesoglycan and sulodexide indirectly demonstrated the presence of heparan sulfate which was shown to represent about 60% of the commercial products. PMID- 7540025 TI - The ability of nicotinamide to inhibit the growth of a C3H mouse mammary carcinoma. AB - Experimental studies have suggested that nicotinamide and its analogs may inhibit the growth of murine tumours. We have now investigated this using a C3H mouse mammary carcinoma implanted into the right rear foot of female CDF1 mice. From days 1 to 30 after implantation mice were intraperitoneally (i.p.) injected with either 100, 200, 500 or 1,000 mg/kg nicotinamide. The tumour volume (+/- 1 S.E.) after 30 days in saline-treated mice had reached 1540 mm3 (+/- 260). No change in tumour growth was seen at that time with daily doses of up to 500 mg/kg nicotinamide, but at 1,000 mg/kg tumour volume was reduced to 904 mm3 (+/- 233). However, this large dose of nicotinamide was also toxic to the mice with some 16% of animals dying during the 30-day treatment period. A similar growth inhibition was seen with daily i.p. injections of 5 mg/kg fumagillin (tumour volume at 30 days = 821 +/- 191 mm3), a known inhibitor of angiogenesis, but whether this mechanism also explains the nicotinamide effect is not clear. PMID- 7540026 TI - Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase as a target for nitric oxide signaling. AB - Nitric oxide signaling is achieved through cGMP-dependent and -independent mechanisms. The latter are exemplified by the NAD(+)-dependent automodification of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The experimental post-translational, covalent modification of the enzyme by [32P]NAD+ is achieved using NO-releasing compounds and an active constitutive or inducible NO-synthase. Potential roles for NO in this covalent enzyme modification can be grouped as follows: S-Nitrosylation of GAPDH by NO+ NAD(+)-dependent, post translational covalent automodification of GAPDH. Oxidative modification of GAPDH by NO-related compounds, probably ONOO. GAPDH modification by one of the proposed mechanisms would lead to inhibition of enzyme catalysis. It is likely that the NAD(+)-dependent automodification process occurs in vitro, in intact cells, and in whole animals. Besides its normal function in glycolysis, GAPDH not only is a target for NO-mediated direct and indirect modifications but also is ADP ribosylated in the presence of brefeldin A (90). The relation of such ADP ribosylation to enzyme activity is so far unknown. GAPDH also may be involved in one of the following functions unrelated to its glycolytic activity (81 and refs. therein; 90): binding and transport of tRNA associated with nuclear localization of GAPDH. DNA-repair activity, i.e., uracil DNA glycosylase. Activation of transcription in neurons. Interaction with tubulin and microtubules. The transport of nitric oxide. Serves as a substrate for brefeldin A stimulated ADP ribosylation. Because some of these alternative functions of GAPDH, just like NO mediated modification of the enzyme, are related to the NAD+ binding site of the protein, we are interested in searching for the significance of these activities in relation to NO actions. In recent years, several functions of NO have been linked to direct, cGMP-independent actions. Modification of GAPDH is probably just one interesting target related to NO-redox chemistry and active-site thiol modification. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to the patho-physiological role of NO in medically related conditions. PMID- 7540027 TI - Indirect use of immobilized metal affinity chromatography for isolation and characterization of protein partners. PMID- 7540028 TI - Structure and function of RNA pseudoknots. PMID- 7540029 TI - Immunological monitoring and clinical trials of biological response modifiers. PMID- 7540030 TI - Growth and differentiation control. PMID- 7540032 TI - Bleomycin. PMID- 7540031 TI - Hematopoietic growth factors in cancer chemotherapy. PMID- 7540034 TI - Tenascin may not be required for intestinal villus development. AB - Tenascin, a large extracellular matrix protein, is subject to complex spatial and temporal patterns of expression in the course of various organogenetic processes including the formation of the small intestinal villus. In the present study, the presence of tenascin was analyzed in human fetal colonic villi, which are transient structures that are programmed to disappear at the time of colonic mucosal remodeling. While the labeling of muscles and peripheral mesenchyme was similar in both segments, surprisingly the colonic villus cores were mostly devoid of tenascin as opposed to those of the small intestine. Western blot analysis revealed that the 220 and 320 kDa forms of tenascin were detected in both segments. However, the 200 kDa form present in small intestinal villi was absent in colonic villi. These data suggest that tenascin under its 200 kDa form is not required for villus formation. PMID- 7540033 TI - Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians. AB - Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed. PMID- 7540035 TI - 5-Azacytidine changes gene expression and causes developmental arrest of early chick embryo. AB - Methylation of DNA appears to be an important maintenance mechanism for inhibiting gene expression during development in vertebrates. 5-azacytidine (5 azaC) is used as an experimental tool for demethylation and it induces differentiation in various systems. In the chick embryo, the first cellular migrations signal the onset of primitive streak and gastrula formation and result in neural induction and morphogenesis of the embryonic axis. In the present work with the early chick embryo, 5-azaC perturbs normal cellular migrations and the embryos produce an atypical short, thickened primitive streak. These embryos have the tendency to form neural tissue but the embryonic axis shows sparse identity of patterning along its length. A small percentage of embryos display formation of double embryonic axes. Blastula embryos show reduced expression of some polypeptides and express characteristic polypeptides which are not present in morula embryos normally. Under the influence of 5-azaC, blastula embryos expressed all the polypeptides which are characteristic of embryos at both the morula and the blastula stages. If 5-azaC perturbs DNA methylation as the chick embryo develops from the histologically simple blastula, then the wave of methylation which has been reported to start at the late blastula and continues during postgastrulation in vertebrate embryos does not seem to be important for the induction of mesodermal and of neural tissues, but is important for the patterning of these tissues. PMID- 7540036 TI - Nitric oxide synthase inhibition irreversibly decreases perfusion in the R3230Ac rat mammary adenocarcinoma. AB - We examined the microvascular effects of competitive nitric oxide synthase (NOS) inhibition with NG-monomethyl-L-arginine (MeArg), followed by L-arginine, on R3230Ac mammary adenocarcinoma perfusion. In window preparations containing tumours, superfusion of 50 microM MeArg reduced diameters of central tumour venules by 13%, of peripheral tumour venules by 17% and of normal venules near tumours by 16% from baseline. MeArg reduced red blood cell (RBC) velocity in central tumour venules by 25%, and increased intermittent flow and stasis frequency by 20% in central tumour venules. Subsequent superfusion of 200 microM L-arginine did not restore diameters or RBC velocity of any tumour preparation venules, and decreased length density in both central tumour venules and peripheral tumour venules. In contrast, MeArg reduced control preparation venule diameter by 30% and RBC velocity by 66%, but did not decrease length density or increase intermittent flow or stasis frequency. Unlike tumour preparation venules, L-arginine restored control venule diameters and velocities. NOS inhibition reduces both tumour and control venule perfusion, but the effect is blunted in the vicinity of tumours, possibly because of increased NOS levels. Perfusion can be subsequently restored in control, but not tumour, venules with L arginine. Tumour NOS inhibition, followed by normal tissue rescue with L arginine, may provide a novel means to achieve the goal of selective tumour hypoxia. PMID- 7540037 TI - Burnout and psychiatric disorder among cancer clinicians. AB - The prevalence and causes of 'burnout' and psychiatric disorder among senior oncologists and palliative care specialists have been measured in a national questionnaire-based survey. All consultant non-surgical oncologists in the UK were asked to participate. Sources of work-related stress and satisfaction were measured using study-specific questions which were aggregated into factors. Psychiatric disorder was estimated using the 12-item General Health Questionnaire. The three components of 'burnout'--emotional exhaustion, depersonalisation and low personal accomplishment--were assessed using the Maslach Burnout Inventory. Three hundred and ninety-three out of 476 (83%) consultants returned their questionnaires. The estimated prevalence of psychiatric disorder in cancer clinicians was 28%, and this is similar to the rate among British junior house officers. The study group had equivalent levels of emotional exhaustion and low personal accomplishment to those found in American doctors and nurses, but lower levels of depersonalisation. Among cancer clinicians, 'burnout' was more prevalent among clinical oncologists than among medical oncologists and palliative care specialists. Psychiatric disorder was independently associated with the stress of feeling overloaded (P < 0.0001), dealing with treatment toxicity/errors (P < 0.004) and deriving little satisfaction from professional status/esteem (P = 0.002). 'Burnout' was also related to these factors, and in addition was associated with high stress and low satisfaction from dealing with patients, and with low satisfaction from having adequate resources (each at a level of P < or = 0.002). Clinicians who felt insufficiently trained in communication and management skills had significantly higher levels of distress than those who felt sufficiently trained. If 'burnout' and psychiatric disorder among cancer clinicians are to be reduced, increased resources will be required to lessen overload and to improve training in communication and management skills. PMID- 7540038 TI - Phase I study of accelerated FEC with granulocyte colony-stimulating factor (Lenograstim) support. AB - With the aim of increasing the dose intensity of chemotherapy in breast cancer, 14 patients with stage II-IV breast cancer were treated with FEC chemotherapy at 2 week intervals together with granulocyte colony-stimulating factor (G-CSF) 5 micrograms kg-1 s.c. on days 2-14. Five of six patients completed six courses of 5-fluorouracil 600 mg m-2, epirubicin 60 mg m-2 and cylcophosphamide 600 mg m-2 within 11 weeks. Eight patients were treated with 5-fluorouracil 700 mg m-2, epirubicin 70 mg m-2 and cyclophosphamide 700 mg m-2 and four had dose-limiting toxicity with sepsis, thrombocytopenia or mucositis. All patients who received G CSF had satisfactory neutrophil counts by day 15 of each course. Cumulative anaemia and thrombocytopenia were observed, but treatment at the first dose was tolerable. Seven of eight patients with measurable disease had partial responses. This regimen permits a 50% increase in dose intensity compared with conventional treatment at 3 week intervals and warrants further evaluation. PMID- 7540039 TI - Four cycles of BEP versus an alternating regime of PVB and BEP in patients with poor-prognosis metastatic testicular non-seminoma; a randomised study of the EORTC Genitourinary Tract Cancer Cooperative Group. AB - We have investigated whether an alternating induction chemotherapy regimen of PVB/BEP is superior to BEP in patients with poor-prognosis testicular non seminoma. A total of 234 eligible patients were randomised to receive an alternating schedule of PVB/BEP for a total of four cycles or four cycles of BEP. Poor prognosis was defined as any of the following: lymph node metastases larger than 5 cm, lung metastases more than four in number or larger than 2 cm, haematogenic spread outside the lungs, such as in liver and bone, human chorionic gonadotrophin > 10,000 IU l-1 or alphafetoprotein > 1000 IU l-1. The complete response (CR) rates to PVB/BEP and BEP were similar, 76% and 72% respectively (P = 0.58). In addition, there was no significant difference in relapse rate, disease-free and overall survival at an average follow-up of 6 years. The 5-year progression-free and survival rates in both treatment groups were approximately 80%. The PVB/BEP regime was more toxic with regard to bone marrow function; the frequencies of leucocytes below 1000 microliters-1, leucocytopenic fever and platelets below 25,000 microliters-1, throughout four cycles were 28% vs 5% (P < 0.001), 16% vs 5% (P = 0.006), and 10% vs 1% (P = 0.001) respectively. Neuropathy also occurred more often in the PVB/BEP arm: 47% vs 25% (P = 0.001). This study shows that an alternating regimen of PVB/BEP is not superior to BEP and that it is more myelo- and neurotoxic. PMID- 7540040 TI - Potassium flux through gramicidin ion channels is augmented in vesicles comprised of plasmenylcholine: correlations between gramicidin conformation and function in chemically distinct host bilayer matrices. AB - The functional role of distinct phospholipid subclasses and molecular species in modulating gramicidin-mediated K+ flux was characterized through quantification of changes in the fluorescence intensity of ion specific fluorescent probes encapsulated inside vesicles comprised of individual molecular species of plasmenylcholine and phosphatidylcholine. The rate constant of gramicidin mediated K+ ion flux across bilayers comprised of 1-O-(Z)-hexadec-1'-enyl-2 octadec-9'-enoyl-sn-glycero-3-p hos phocholine (plasmenylcholine) was 18.9 +/- 1.7 s-1, while that present across bilayers comprised of 1-hexadecanoyl-2-octadec 9'-enoyl-sn-glycero-3-phosphocholine (phosphatidylcholine) was 12.3 +/- 1.5 s-1. The observed changes were not due to alterations in the nature of the sn-2 aliphatic chain or the net surface charge present at the membrane interface and were unaltered by the addition of several amphiphilic agents (including charged amphiphiles), suggesting that the observed alterations specifically reflect changes in channel function which result from the covalent alteration of host phospholipid in the proximal portion of the sn-1 aliphatic chain (i.e., phospholipid subclass-specific alterations). Addition of cholesterol to bilayer matrices comprised of plasmenylcholine resulted in dose-dependent attenuation of the rate of gramicidin-mediated K+ flux, but did not alter the rate of gramicidin mediated K+ flux in membranes comprised of phosphatidylcholine. Gramicidin ion channels experience distinct environments in membranes comprised of phosphatidylcholine and plasmenylcholine host lipids demonstrated by both the different fluorescence anisotropies of endogenous tryptophan residues and the different C=O stretching frequencies of intramonomer carbonyls in gramicidin incorporated into these two choline glycerophospholipid subclasses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540041 TI - Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron. AB - The splicing factor CBP2 is required to excise the yeast mitochondrial group I intron bI5 in vivo and at low magnesium ion concentrations in vitro. CBP2 binding is strengthened 20-fold by increasing Mg2+ concentrations from 5 to 40 mM, implying the protein binds, in part, to the same structure as that stabilized by the cation. The same transition is also observed as a cooperative increase in the rate of self-processing between 5 and 40 mM Mg2+, providing strong evidence for an RNA folding transition promoted by either Mg2+ or CBP2. The first step of splicing, guanosine addition at the 5' splice site, is rate limiting for exon ligation. At low (5 mM) magnesium ion, reaction (measured as kcat/Km or kcat) is accelerated 3 orders of magnitude by saturating CBP2. At near-saturating Mg2+ (40 mM), acceleration is 8- and 30-fold, for kcat and kcat/Km, respectively, so high magnesium ion concentrations fail to compensate completely for protein facilitation. Thus, self-splicing proceeds via two additional transitions as compared with reaction of the bI5-CBP2 complex, only the first of which is efficiently promoted by the cation. Guanosine 5'-monophosphate binds (Kd approximately 0.3 mM) with the same affinity to bI5 and the bI5-protein complex, supporting independent binding of the nucleophile and CBP2. Substitution of a phosphorothioate at the 5' splice site and pH profiles provide evidence that kcat is limited by chemistry at low pH and by a conformational step at high pH. Because binding by either Mg2+ or CBP2 increases the rate of chemistry more than the rate of the conformational step, in the physiological pH range (7-7.6) the protein-facilitated reaction is limited by a conformation step while self splicing reaction is limited by chemistry. We conclude that CBP2 makes manifold contributions to bI5 splicing: binding compensates for at least two structural defects and accelerates the rate of the chemistry. PMID- 7540042 TI - Use of aprotinin in pediatric lung transplantation. AB - BACKGROUND: Aprotinin has been shown to decrease perioperative bleeding in adults undergoing cardiac surgery. We evaluated its efficacy in reducing blood loss in pediatric lung transplantation. METHODS: Aprotinin was given to a group of pediatric lung transplant recipients (n = 24) identified as being at high risk for bleeding by virtue of preoperative diagnosis of cystic fibrosis or previous cardiothoracic operation (group 1). Comparison was made to a group of pediatric recipients (n = 19) believed to be at low risk for bleeding who did not receive aprotinin (group 2). All transplantations were accomplished with the use of cardiopulmonary bypass. RESULTS: No difference in intraoperative blood requirement was identified between groups (18 +/- 3 cc/kg [group 1] versus 30 +/- 8 cc/kg [group 2], p = 0.16). Neither postoperative blood transfusion requirement (12 +/- 5 cc/kg [group 1] versus 16 +/- 6 cc/kg [group 2], p = 0.55) nor chest tube output in the first 24 postoperative hours (43 +/- 9 cc/kg [group 1] versus 53 +/- 13 cc/kg [group 2], p = 0.55) was significantly different between groups. Reexploration for bleeding was required in 8% (2 of 25) in group 1 and 16% (3 of 19) in group 2 (p = 0.64). CONCLUSIONS: Aprotinin reduced the amount of perioperative hemorrhage in a group of pediatric patients at high risk for bleeding after lung transplantation. The magnitude of the effect could not be quantified but was sufficient to normalize the transfusion requirement to that of a low risk group of patients. PMID- 7540044 TI - Comparison of the effects of amino acid substitutions and beta-N- vs. alpha-O glycosylation on the T-cell stimulatory activity and conformation of an epitope on the rabies virus glycoprotein. AB - The first potential N-glycosylation site of the rabies virus glycoprotein, the antigen that carries epitopes for glycoprotein-specific T-cells and virus neutralizing antibodies, is glycosylated inefficiently. Recently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished the T-cell stimulatory activity and reduced the characteristic alpha-helicity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strains and appropriate tridecapeptide sequences and their analogs was investigated. The T-cell recognition pattern of the rabies and rabies-related viruses was identical to that of the synthetic peptides representing the respective epitope sequences. While the asparagine could be replaced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-galactosamine moiety at the serine, or galactosyl-N-acetyl galactosamine moieties at the threonines preceding or replacing the asparagine (all O-linked sugars in the natural alpha-anomeric configuration) resulted in epitopes that lowered rather than abolished the T-cell stimulatory activity. All non-glycosylated peptides assumed a low-to-medium helicity in trifluoroethanol. O glycosylation was more efficient than N-glycosylation in breaking the helical conformation of the peptides to result in the formation of reverse-turns or unordered structure. PMID- 7540043 TI - Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells. AB - The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation. PMID- 7540045 TI - Development of oxidase 'priming' in maturing HL60 cells: correlation with protein expression and tyrosine phosphorylation. AB - The mechanisms involved in 'priming' of the oxidase response of neutrophils are unknown. Two major problems are encountered in using circulating neutrophils; firstly, prior exposure to circulating 'priming' cytokines cannot be controlled and secondly, non-intentional 'priming' during cell separation can occur. In this study, these problems were avoided by differentiating the promyeloid leukaemic cell line, HL60, towards granulocytes using dibutyrl cyclic AMP, to produce a 'virgin cell' model system. We have demonstrated that the ability of substance P to both prime the oxidase response and induce tyrosine phosphorylation increased during differentiation. The major tyrosine-phosphorylated protein, with molecular weight of 74 kDa, was not recognised by anti-c-raf1 antibodies. Furthermore, c raf1 expression rapidly declined during HL60 cell granulocytic differentiation. This data shows that although there was no simple relationship between c-raf quantity and priming, the data were consistent with tyrosine phosphorylation of a 74 kDa protein being important for oxidase 'priming'. PMID- 7540047 TI - A selective stain for eosinophils using two oxazine dyes applied sequentially. AB - A methanolic solution of the oxazine textile dye, C.I. basic blue 122, followed by an aqueous alkaline solution of the oxazine dye, C.I. basic blue 141, and a brief rinse in an acetate buffer at pH 3.45 produces intense black staining of eosinophil granules. This staining was selective for eosinophils while other types of peripheral blood leukocytes showed little if any staining under the same conditions. This staining procedure may be useful for detecting eosinophils in samples of blood, bone marrow, or urine when eosinophiluria results from interstitial nephritis. PMID- 7540046 TI - Ethylenediaminetetraacetic acid in ammonium hydroxide for reducing decalcification time. AB - Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new decalcification fluid using concentrated ammonium hydroxide (NH4OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH4OH required an average of six days. Light microscopy indicated good retention of cellular detail. PMID- 7540048 TI - Improved lipid visualization with a modified osmium tetroxide method using ultrasonic treatment and intensification with imidazole or triazole. AB - Formalin fixed autopsy tissue containing lipids were cut into 1-5 mm thick blocks, washed well, then postfixed in 2% OsO4 in 0.03 M veronal acetate buffer for 30, 60, 90, 120, or 180 min with or without ultrasonic treatment. Tissues exposed to ultrasound for 90 min showed superior penetration of OsO4 and well preserved histological architecture. Tissues also were immersed for 1 hr in veronal acetate buffer (pH 7.4) containing 0.5% imidazole or triazole and compared with untreated controls. Paraffin sections, 4 microns thick, were examined under a light microscope with an image analyzer. Both intensity and percentage area of osmium blackening were significantly higher in samples immersed in imidazole or triazole than in untreated controls. No difference was observed between imidazole- and triazole-immersed samples. The OsO4 method, modified by ultrasound treatment and imidazole- or triazole-immersion, can be applied to routine formalin fixed autopsy materials for improved lipid visualization. PMID- 7540049 TI - Histochemical demonstration of DNA double strand breaks by in situ 3'-tailing reaction in apoptotic endometrium. AB - A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with lambda-exonuclease (lambdaEx) prior to the TdT reaction to generate 3'-protruding ends. The lambdaEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states. PMID- 7540050 TI - Loss of low-density lipoprotein utilization by regressing porcine luteal cells: effects of protein kinase C activation. AB - We recently reported a decline in low-density lipoprotein (LDL) uptake by regressing porcine luteal cells that correlated with diminished LDL stimulation of progesterone (P) production. The objectives of the present study were to determine 1) whether loss of LDL utilization is a specific lesion in the steroidogenic pathway in regressing luteal cells and 2) whether in vitro activation of protein kinase C (PKC) in mid-cycle luteal cells acutely suppresses LDL utilization. Dispersed cells (4 x 10(4)/0.2 ml) from mid-(Days 6-10, estrus = Day 0; n = 5) or late-(Days 15-18; n = 7) cycle porcine CL were cultured in Dulbecco's Modified Eagle/F-12 medium supplemented with insulin, transferrin, sodium selenite, and aprotinin for 24 h with human (h) LDL (0-100 micrograms/ml), 22(R)-hydroxycholesterol (22[OH]-C;-0-25 micrograms/ml), or pregnenolone (0-1000 nM). P production by mid-cycle luteal cells was dose-dependently increased (p < 0.05) by LDL (up to 2.8-fold), 22(OH)-C (up to 3.2-fold), and pregnenolone (> 3 fold). In contrast, LDL (10-100 micrograms/ml) failed to stimulate P production by late-cycle luteal cells. But 22(OH)-C (up to 4-fold) and pregnenolone (> 10 fold) were as effective in promoting (p < 0.05) steroidogenesis relative to basal levels in late luteal cells as in mid-cycle cultures. The PKC activator, TPA (12 O-tetradecanoyl-phorbol-13-acetate; 10 ng/ml), inhibited (approximately 20%; p < 0.01) basal and LDL-supported steroidogenesis, but did not suppress 22(OH)-C stimulated P secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540051 TI - Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. AB - During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility. PMID- 7540052 TI - High water permeability of human spermatozoa is mercury-resistant and not mediated by CHIP28. AB - A novel integral membrane protein with an apparent molecular mass of 28 kDa (CHIP28) was first isolated from human erythrocytes and is now recognized as a water channel protein. The expression of this protein has been found in several other cell types that all require high water permeability for their functions. Recent studies have shown that the water permeability (Lp) of human spermatozoa is among the highest reported for mammalian cells. Together with the low activation energy of human spermatozoa for Lp, this suggests that CHIP28 water channel may be present in the plasma membrane of human spermatozoa. However, our current studies do not support this hypothesis. Results from Western blot analysis on human sperm plasma membrane proteins, performed through use of an antibody against human erythrocyte CHIP28 protein, indicated that human spermatozoa do not express CHIP28 protein on their cell surface (n = 10). Consistent with the Western blot finding, mercuric chloride (HgCl2), a known water channel blocker, failed to reduce the osmotic water permeability of human spermatozoa. The calculated Lp values were 1.30 +/- 0.29 micron/min/atm (n = 16; mean +/- SEM) for the control group and 1.31 +/- 0.29 (n = 9; mean +/- SEM), 1.04 +/- 0.27 (n = 11; mean +/- SEM), and 1.34 +/- 0.19 (n = 6; mean +/- SEM), respectively, for the 10 microM, 30 microM, and 50 microM HgCl2-treated groups. These Lp values are not different (p > 0.05). In contrast, the same concentration of HgCl2 significantly blocked the osmotic water transport across the membrane of human erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540053 TI - Changes in epidermal growth factor receptor and the levels of its ligands during menstrual cycle in human endometrium. AB - We examined menstrual cycle-dependent changes in the expression of human endometrial epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and epidermal growth factor receptor (EGFR) and their mRNA using immunoblot analysis, 125I-EGF binding, and competitive reverse transcription and polymerase chain reaction (RT-PCR). We also studied their localization in the endometrial tissue by immunohistochemistry. Endometrial samples were obtained at three stages of menstruation: the early follicular stage, which exhibits low serum estradiol (E2) and progesterone (P) levels; the late follicular stage, which exhibits high E2 and low P levels; and the luteal stage, which exhibits high E2 and P levels. Immunohistochemical examination showed that EGF, TGF alpha, and EGFR were localized to the endometrial epithelium. Immunoblot analysis revealed that endometrial EGF, TGF alpha, and EGFR levels were significantly (p < 0.01) increased at the late follicular and luteal stages compared to the early follicular stage. 125I-EGF-specific binding levels at the late follicular and luteal stages were significantly (p < 0.01) higher than at the early follicular stage, consistent with the results of immunoblot analysis. Competitive RT-PCR revealed that EGF, TGF alpha, and EGFR mRNA levels were significantly (p < 0.01) higher at the late follicular and luteal stages than at the early follicular stage. Changes in EGF, TGF alpha, and EGFR mRNA levels were consistent with changes in protein levels. These findings suggest that synthesis and expression of human endometrial EGF, TGF alpha, and EGFR vary with the stage of the menstrual cycle and that their expression in the human endometrium is associated with the increase in the serum E2 but not with the increase in P levels. PMID- 7540054 TI - The conformational analysis of peptides using Fourier transform IR spectroscopy. AB - Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance-FTIR spectroscopy. Hydrogen-deuterium exchange can be monitored by the analysis of the amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and alpha-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3(10)-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a beta-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel beta-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular beta-sheet structure can also be detected. PMID- 7540055 TI - The importance of extended conformations and, in particular, the PII conformation for the molecular recognition of peptides. AB - Crystallographic, isotopic labeling nmr and transferred nuclear Overhauser effect studies have highlighted the extended conformation as a very important element of secondary structure at the binding site of many peptide/protein complexes including peptide inhibitors-enzymes, B-cell epitopes-antibodies, and T-cell epitopes-major histocompatibility complex (MHC) of class I and II complexes. This paper discusses the peptide ligand conformation consequences of these findings particularly in view of the identification of the PII conformation (left-handed extended polyproline II) in free solution. PMID- 7540057 TI - Serum granulocyte macrophage colony stimulating factor (GM-CSF) in gestational trophoblastic diseases. AB - GM-CSF was assayed by a solid phase enzyme immunoassay in sera of 42 cases of vesicular mole, 24 cases of choriocarcinoma and 23 cases of normal pregnant women at their first trimester (controls). Diagnosis of gestational trophoblastic disease (GTD) was based on histopathologic examination of biopsy specimens obtained by endometrial curettage in cases of choriocarcinoma and molar tissue in patients with vesicular mole. Serum concentration of chorionic gonadotrophin beta subunit (hCG beta) was determined by I125 solid phase radioimmunoassay before and after treatment at weekly intervals for 3 months and then monthly for one year. According to pathologic diagnosis and serial serum hCG beta assays, cases of GTD were subdivided into remission and progressive tumour groups. Results demonstrated that serum GM-CSF levels in cases of vesicular mole as well as remission cases of choriocarcinoma were not significantly different from those of controls. On the other hand, progressive tumour cases of choriocarcinoma which later showed an abnormal hCG beta regression curve had significantly elevated serum GM-CSF level, where the mean fold rise amounted to 2.55 of the mean control value. Moreover, the incidence of abnormally elevated serum GM-CSF in these cases was 80%. It seems that this rise reflects a possible mechanism of immune surveillance. These results indicate that serum GM-CSF assay may be considered as a valuable prognostic biomarker in cases of malignant trophoblastic tumours where the assay is clinically useful in identifying high risk choriocarcinoma cases, thus justifying the installation of early specific treatment as well as intensive follow up care. PMID- 7540059 TI - Control of nitric oxide synthase expression by transforming growth factor-beta: implications for homeostasis. AB - Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-beta s (TGF beta s) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-beta s counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-beta may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-beta production may lead to effective therapeutic strategies in various diseases. PMID- 7540060 TI - Humoral response to Mycobacterium tuberculosis-specific antigens in African tuberculosis patients with high prevalence of human immunodeficiency virus infection. AB - SETTING: The applicability of serodiagnosis of tuberculosis using Mycobacterium tuberculosis-complex-specific antigens in a Tanzanian population with high prevalence of HIV. OBJECTIVE: This study was performed to evaluate the usefulness, sensitivity and specificity of serology using M. tuberculosis specific antigens in the diagnosis of tuberculosis in patients with and without HIV co-infection. DESIGN: Patients with proven pulmonary and extrapulmonary tuberculosis at a major referral centre in Tanzania were enrolled in the study. The control group consisted of patients without a history of previous tuberculosis admitted to the trauma ward and of healthy volunteers. Sera were analysed by an enzyme linked immunoassay (ELISA) using two M. tuberculosis specific proteins as antigen: the 38 kDa protein [3T] and a 17 kDa protein. In addition was recorded presence or absence of BCG scar and tuberculin sensitivity and the sera were tested for HIV and analysed for beta-2-microglobulin content. RESULT: Sensitivity and specificity were markedly reduced in tuberculosis patients with HIV co-infection compared to patients without this disease (73% and 70% versus 52% and 50% respectively). CONCLUSION: Serology for diagnosis of tuberculosis is not feasible in an HIV endemic region. PMID- 7540058 TI - Magnesium substitution in elective coronary artery surgery: a double-blind clinical study. AB - Magnesium may be beneficial in the control of ventricular ectopy and supraventricular tachyarrhythmias after coronary artery bypass graft (CABG) surgery, but it is not known whether a high-dose magnesium regimen is superior to a regimen keeping the patient normomagnesemic. A prospective randomized and double-blind clinical comparison was performed in 81 elective CABG patients in order to assess the effects of two different magnesium infusion regimens on electrolyte balance and postoperative arrhythmias. Forty-one patients (high-dose group, H) received 4.2 +/- 0.7 g (mean +/- SD), of magnesium sulfate before cardiopulmonary bypass, followed by an infusion of 11.9 +/- 2.8 g of magnesium chloride until the first postoperative (PO) morning, and a further 5.5 +/- 1.0 g until the second PO morning. Forty patients (low-dose group, L) received magnesium sulfate only after bypass to a total of 2.9 +/- 0.5 g at the first, and 1.4 +/- 0.1 g at the second PO morning. A blood cardioplegia technique was used in both groups, including bolus doses of magnesium chloride to a total of 2.4 +/- 0.6 g and 2.3 +/- 0.6 g to H and L patients, respectively. Continuous Holter tape recording was used for 12 to 15 hours preoperatively, and for 48 hours postoperatively. Serum magnesium peaked in H patients on the first PO morning at 1.60 +/- 0.25 mmol/L, whereafter it declined to the normal level on the third PO morning. Patients in the L group were normomagnesemic, except after the start of bypass.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540056 TI - Differential effects of PKC inhibitors on gelatinase B and interleukin 6 production in the mouse macrophage. AB - Pretreatment of LPS-induced RAW 264.7 cells with three PKC inhibitors suggests that induction of TNF-alpha, nitric oxide (NO), gelatinase B (Gel B) and IL-6 involves at least three distinct signalling pathways. We confirmed the PKC dependence of TNF-alpha and NO productions and found that Gel B was inhibited by Calphostin C (CAL), but potentiated by staurosporine (STAR) and CGP 41 251. IL-6 production was stimulated by the three inhibitors. Our results indicate that up regulation of Gel B, TNF-alpha and NO seems to involve PKC at different levels, whereas up-regulation of IL-6 production appears to be PKC-independent. However, IL-6 production in RAW 264.7 cells seems to be down-regulated by a PKC-dependent feedback mechanism. PMID- 7540061 TI - Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein-1 alpha. AB - BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G-CSF) and the combination of G CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100 fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117 , and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB-10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation. PMID- 7540063 TI - Primitive human hematopoietic progenitors adhere to P-selectin (CD62P). AB - P-selectin was shown to bind committed human hematopoietic progenitors (colony forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX 1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells. PMID- 7540062 TI - Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony-stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte-CSF. AB - Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte-macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G-CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM-CSF: median, 28 days; GM CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7540066 TI - Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells. AB - Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T-cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL 4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40 signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL. PMID- 7540065 TI - Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells. AB - We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)- cells were purified using fluorescence activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation. PMID- 7540067 TI - Expression and function of Fas (APO-1/CD95) in patient myeloma cells and myeloma cell lines. AB - Cross-linkage of the Fas antigen induces programmed cell death in many normal and malignant lymphoid cells by a process known as apoptosis. In this study, we examined the sensitivity of myeloma cell lines and patient plasma cells to a cytolytic anti-Fas monoclonal antibody (MoAb). Eight of 10 myeloma cell lines were induced to undergo programmed cell death by anti-Fas MoAb as determined by DNA fragmentation and morphologic changes. Of the two myeloma cell lines that were resistant to anti-Fas treatment, one did not express the Fas antigen. Only the U266 cell line expressed Fas, but was not killed by the anti0Fas MoAb. To extend these studies, we have examined the expression and function of Fas in freshly isolated CD38hiCD45neg-int plasma cells from patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis (AL). By three-color flow cytometry, we found Fas expression in CD38hiCD45neg-int plasma cells from all patient groups to be variable, as Fas was expressed in 15 of 28 MM, 3 of 6 MGUS, and 2 of 7 AL patients. In morphologic studies of apoptosis, Fas-positive myeloma cells in patient bone marrow mononuclear cell (MNC) cultures appeared to be resistant to anti-Fas-mediated apoptosis. By contrast, purified myeloma cells from the same patient were sensitive to anti-Fas treatment, suggesting the presence of a protective factor(s) in unseparated MNC cultures that may inhibit Fas-induced apoptosis of plasma cells. Of interest, serum from normal individuals and myeloma patients also protected myeloma cell lines from undergoing Fas-mediated apoptosis. These studies show that Fas expression in myeloma cell lines and CD38hiCD45neg-int patient plasma cells is variable and may reflect a variance in the maturation status of the various plasma cell populations. Moreover, Fas-mediated killing of patient cells and myeloma cell lines was also variable, which may be influenced, in part, by the presence of a soluble protective factor. PMID- 7540068 TI - The functional characterization of interleukin-10 receptor expression on human natural killer cells. AB - Human natural killer (NK) cells are large granular lymphocytes that constitutively express functional forms of the interleukin-2 receptor (IL-2R) and lyse tumor and virally infected cells without prior sensitization. NK cells with high density expression of CD56 (CD56bright) express the high affinity IL-2R and proliferate in response to low (picomolar) concentrations of IL-2. CD56dim NK cells express the intermediate affinity IL-2R and demonstrate enhanced cytotoxic activity without proliferation in response to high (nanomolar) concentrations of IL-2. In the present study, we characterized IL-10R expression on human NK cells and the functional consequences of IL-10 binding directly to highly purified subsets of CD56bright and CD56dim NK cells. Binding studies using 125I-IL-10 indicated that resting human NK cells constitutively express the IL-10 receptor protein at a surface density of approximately 90 receptor sites per cell, with a kd of approximately 1 nmol/L. Alone, IL-10 did not induce proliferation of CD56bright or CD56dim NK cell subsets. However, at low concentrations (0.5 to 5 ng/mL), IL-10 significantly augmented IL-2-induced proliferation of the CD56bright NK cell subset mediated via the high-affinity IL-2R. In the absence of IL-2, IL-10 was able to induce significant NK cytotoxic activity against NK resistant tumor cell targets in both subsets of NK cells in a dose-dependent fashion. Furthermore, the combination of IL-10 and IL-2 had an additive effect on NK cytotoxic activity, whereas that of IL-10 and IL-12 did not. Production of interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony stimulating factor by IL-2-activated NK cells was also significantly enhanced by IL-10. Neither resting nor activated human NK cells appear to produce human IL-10 protein. In summary, NK cells constitutively express the IL-10R protein in low density, and the functional consequences of IL-10 binding directly to human NK cell subsets appear to be stimulatory and dose-dependent. In contrast to its direct effects on human T cells and monocytes/macrophages, IL-10 potentiates cytokine production by human NK cells. PMID- 7540064 TI - Transcriptional induction of pim-1 protein kinase gene expression by interferon gamma and posttranscriptional effects on costimulation with steel factor. AB - Steel factor (SLF) synergizes with interferon gamma (IFN gamma) to stimulate proliferation of the human factor-dependent cell line MO7e. We examined the effects of IFN gamma and SLF treatment, alone or in combination, on the expression of a 33-kD cytoplasmic protein serine/threonine kinase designated pim 1 whose expression has been closely associated with proliferation induced by related myeloid cytokines. IFN gamma alone, but not SLF, stimulated expression of pim-1 RNA and protein in MO7e cells; compared with IFN gamma alone, costimulation with IFN gamma and SLF resulted in a twofold to threefold increase in pim-1 message and protein expression, correlating with synergistic effects on cell proliferation. Both IFN gamma and IFN gamma/SLF induced pim-1 mRNA in the absence of de novo protein synthesis. Nuclear run-on assays showed that, although IFN gamma alone increased the rate of pim-1 gene transcription, costimulation with IFN gamma and SLF did not further potentiate this effect; however, the stability of pim-1 message was significantly enhanced in the presence of both cytokines. An IFN gamma-responsive element within the 5' flanking region of the pim-1 gene that could confer IFN gamma responsiveness on a heterologous promoter was identified. This sequence, designated PMGAS, formed a specific complex with Stat (signal transducers and activators of transcription) 1 alpha (the p91 subunit of the transcription factor ISGF3 [interferon-stimulated gene factor 3]) in IFN gamma treated cell extracts, suggesting that the transcriptional effects of IFN gamma on pim-1 expression may be mediated by Stat 1 alpha. PMID- 7540069 TI - Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients. AB - We have previously reported the presence of activated (HLA-DR+) T cells in multiple myeloma (MM) patients. These cells produce high amounts of interleukin (IL)-2 and interferon (IFN)-gamma and generate a potent antiplasma cell activity after appropriate in vitro stimulation, but they are unable in vivo to hold in check the disease. Activated T cells are highly susceptible to apoptosis, a form of programmed cell death involved in the modulation of immune responses and regulated by molecules such as Fas (CD95) and bcl-2. The aim of this study was to determine the expression of Fas and bcl-2 antigens and the susceptibility to apoptosis in T cells of MM patients. Fas+ cells were significantly higher, whereas bcl-2+ cells were significantly lower in MM patients than in the controls. MM patients with the highest number of HLA-DR+ T cells showed the highest Fas and the lowest bcl-2 expression. Two-color cytofluorometric analysis confirmed in individual cells that HLA-DR+ T cells coexpressed Fas and lacked bcl 2. Susceptibility to apoptosis was then investigated to evaluate the consequence of dysregulated Fas and bcl-2 expression. The percentage of apoptotic cells after incubation in medium alone (spontaneous apoptosis) or in the presence of methylprednisolone (MP) or anti-Fas monoclonal antibody (triggered apoptosis) was significantly higher in MM and mainly restricted to HLA-DR+ T cells. Spontaneous apoptotosis was reverted by exogenous IL-2. In conclusion, MM T cells have a dysregulated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis. These data may unravel a novel mechanism by which activated MM T cells are weakened in their ability to exert an effective antitumor activity in vivo. PMID- 7540070 TI - Sialylated O-glycans and L-selectin sequentially mediate myeloid cell rolling in vivo. AB - Leukocyte rolling precedes firm adhesion and emigration in inflammatory cell recruitment. Both P-selectin, an endothelial lectin that binds to sialylated O glycans containing sialyl-Lewisx (sLex) on the granulocyte surface, and leukocyte L-selectin have been shown to mediate leukocyte rolling in vivo. Here, we investigate rolling of isolated human neutrophils (PMN), HL-60 promyelocytes, and an L-selectin-transfected cell line (300.19-L) during trauma-induced inflammation in rat mesenteric venules. HL-60 cells, which express no L-selectin but abundant sLex, rolled effectively immediately after abdominal surgery. HL-60 cell rolling was almost completely abolished by pretreatment with sialidase or monoclonal antibody (MoAb) AM-3 recognizing sLex, and was reduced by about 80% by O sialoglycoprotein-endopeptidase (OSGP). By contrast, 300.19-L cells rolled poorly immediately after surgery but rolled well between 40 and 120 minutes after surgery. Their rolling was completely inhibited by the blocking L-selectin MoAb LAM1-3, but not by a binding control MoAb. PMN express both L-selectin and clustered, sialylated glycoproteins including P-selectin glycoprotein ligand-1 (PSGL-1). PMN showed effective rolling at all times, which was abolished by sialidase or MoAb AM-3 pretreatment during the first 30 minutes after surgery, but not later, when PMN rolling was largely L-selectin-dependent. We conclude that in trauma-induced inflammation, a two-step mechanism accounts for most of myeloid cell rolling, which initially requires O-glycans and subsequently depends on L-selectin function. PMID- 7540071 TI - Tacrolimus (FK506) alone or in combination with methotrexate or methylprednisolone for the prevention of acute graft-versus-host disease after marrow transplantation from HLA-matched siblings: a single-center study. AB - The pharmacokinetics, safety, and efficacy in marrow transplantation of FK506 based immunosuppression for graft-versus-host disease (GVHD) prophylaxis was evaluated in an open label pilot study of 18 patients. Patients more than 12 years of age (median, 35 years; range, 15 to 50 years) with advanced hematologic malignancies receiving HLA-matched sibling marrow grafts were randomized to receive FK506 alone, FK506 and methotrexate (MTX), or FK506 and methyl prednisolone. Of 17 evaluable patients, all had evidence of sustained marrow engraftment. The median time to an absolute neutrophil count of greater than 500/microL was 15 days for patients receiving FK506 alone or FK506 plus methylprednisolone and 23 days for FK506 plus short MTX. Pharmacokinetic studies did not show any significant difference in clearance of FK506 when administered alone or in combination with methylprednisolone or MTX. The mean bioavailability after oral administration in these same three groups was 0.49 +/- 0.1, 0.27 +/- 0.12, and 0.16 +/- 0.08, respectively (P = .003). The decrease in bioavailability may have resulted from an exacerbation of radiation-induced gastroenteritis by MTX. The most significant adverse effect associated with the administration of FK506 was nephrotoxicity, which occurred in 14 of 18 patients (78%). The mean glomerular filtration rate, determined by clearance of (99MTc)DTPA, decreased to 56% (+/- 18%) of the pretransplant baseline level by week 8 (P = .002). Eight of 18 patients (44%) developed grades II-IV acute GVHD, predominantly of the skin and gastrointestinal tract. The actuarial probability of transplant-related mortality during the first 100 days was 24%. The actuarial probability of 1-year disease-free survival was 39%. In conclusion, although bioavailability of FK506 may be affected in patients receiving MTX, this study suggests that FK506 may have a role in the management of patients after allogeneic marrow transplantation. PMID- 7540072 TI - CD3+, CD56+ large granular lymphocyte leukemia. PMID- 7540074 TI - Effect of acetate and erythropoietin on fetal hemoglobin in hemodialyzed uremic patients. PMID- 7540073 TI - Glycoprotein IIb-IIIa and glycoprotein IV expression on Bernard-Soulier syndrome platelets. PMID- 7540076 TI - [Health educational program focusing on supportive attitude toward HIV positives by emotional approach]. PMID- 7540075 TI - Relationship of CD4 and CD34 expression in acute leukemia. PMID- 7540077 TI - [Modulation of angiogenesis induced by lymphocytes by extracellular matrix proteins]. AB - The neovascularization is a known event in the de development of tumors. The progressive growth of solid tumors is strictly dependent on angiogenesis. Furthermore, shedding of tumor cells into the circulation is not observed in a prevascular phase of tumors. Therefore, the inhibition of angiogenesis could be a good target for cancer control. Lymphocytes from, tumor bearing-mice were capable of inducing neovascular response in the skin of syngeneic mice. This response was named syngeneic lymphocyte-induced angiogenesis (SLIA). This work was an attempt to study if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modulate lymphocyte-induced angiogenesis. The angiogenic response induced by lymphocytes from S13 tumor bearing-nice in the skin of BAL/c mice was blocked by treatment with FN and Gly. Arg. Gly. Asp. peptide. On the contrary, collagen and Gly. Arg. Gly. Asp. Ser did not modify SLIA response. PMID- 7540078 TI - Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification. AB - The developmental expression of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid containing glycosphingolipids (KDN-gangliosides) in rainbow trout testis during spermatogenesis was studied using a monoclonal antibody, mAb.kdn3G, which recognizes the KDN alpha 2-->3Gal beta 1-->epitope. A major KDN-ganglioside found in mature sperm, (KDN)GM3, KDN alpha 2-->3Gal beta 1-->4Glc beta 1-->Cer (where Cer is ceramide), was expressed in testis throughout all stages of its maturation. On the contrary, four new KDN-gangliosides which were reactive with mAb.kdn3G were not detected in mature sperm, although they were identified in immature testis and expressed during spermatogenesis. The structures of these KDN gangliosides were established by chemical, enzymatic and immunochemical methods as: (i) (KDN)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2- >3)Gal beta 1-->4Glc beta 1-->Cer; (ii) (KDN, Neu5Ac)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(Neu5Ac alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer and Neu5Ac alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1- >4Glc beta 1-->Cer; (iii) (KDN) GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(KDN alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer; and (iv) (KDN,Neu5Ac)GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer. (KDN)GD1a and (KDN,Neu5Ac)GD1a first appeared at an early stage of spermatogenesis, but (KDN)GD1 alpha and (KDN,Neu5Ac)GD1 alpha were not expressed until 2 months prior to spermiation. While (KDN)GM3 was previously shown to contain only 4-sphingenine (d18:1) acylated with a C16:0 fatty acid, the new KDN-gangliosides discovered in this study were composed of 4-hydroxysphinganine (t18:0) or 4-sphingenine (d18:1), and were acylated with a C24:1 or C16:0 fatty acid. A possible function of these KDN gangliosides is suggested. PMID- 7540079 TI - The granulocyte colony-stimulating factor produced in the human lung and its effect on liquid movement in the rabbit lung. AB - Levels of the granulocyte colony-stimulating factor (G-CSF) were determined in the plasma and resected lung tissue from patients who underwent pulmonary resection. Moreover, the effect of recombinant human (rh) G-CSF on the permeability of pulmonary endothelium and on liquid clearance from the alveolar spaces was investigated in rabbits. The plasma levels of G-CSF increased from 30 pg/ml preoperatively to 409 +/- 236 pg/ml 3 h postoperatively (P < 0.05), while the levels of G-CSF in the resected lung tissue were increased in the alveolar fluid, to 1,834 +/- 1,054 pg/ml, and in the pulmonary blood, to 5,466 +/- 2,019 pg/ml. It was found that rh G-CSF 25 micrograms administered into the subcutaneous tissue of rabbits increased extravascular lung water to 3.45 +/- 0.26 vs 2.98 +/- 0.20 in control experiments (P < 0.05); however, rhG-CSF 0.75 microgram/kg administered into the alveolar spaces did not affect liquid clearance from the alveolar spaces. The findings of this study led us to conclude that G-CSF is synthesized in the human lung and increases the permeability of pulmonary endothelium, but not liquid clearance across the alveolar epithelium. PMID- 7540080 TI - Mother-to-infant transmission of hepatitis C virus. AB - Anti-hepatitis C virus (HCV) antibodies and HCV-RNA were measured in the sera of 22 anti-HCV positive, HIV-1 negative mothers and their infants. ELISA and RIBA II were used for anti-HCV determination. HCV-RNA was measured by a nested polymerase chain reaction. HCV-RNA was found in 12 of 22 mothers. All 22 children were followed for 12 months. All were anti-HCV positive by the fourth month; 18 became anti-HCV negative between the 8th and 12th month. HCV-RNA was detected in 5 of 22 infants in the fourth month. They remained HCV-RNA positive. All children born to HCV-RNA negative mothers were HCV-RNA negative while 5 of 12 babies born to HCV RNA positive mothers were infected. All five infected babies were born to mothers infected through transfusions or drug use. ALT levels in mothers seemed to have no effect on mother-to-infant transmission. Hence evidence for perinatal transmission of HCV from HCV-RNA positive mothers was demonstrated in the present study. PMID- 7540081 TI - Influence of melatonin on bovine pulmonary vascular and bronchial airway smooth muscle tone. AB - The influence of melatonin on tension of isolated bovine pulmonary vascular and bronchial smooth muscle rings were examined in these experiments. Melatonin caused a dose-dependent relaxation of precontracted (30 mM KCl) pulmonary artery and vein, although the effect is greater in arterial smooth muscle. This relaxant response was blocked by preincubating vessels with antagonists of vasoactive intestinal peptide or Substance P. In bronchial smooth muscle, melatonin caused a small contractile response. These experiments demonstrate that in response to melatonin the pulmonary vasculature relaxes, while in airway smooth muscle the reverse, constriction, occurs. It is hypothesized that nocturnal exaggeration of asthma may, in part, be due to changes in circulating levels of melatonin. PMID- 7540082 TI - Identification of structural domains within the cauliflower mosaic virus movement protein by scanning deletion mutagenesis and epitope tagging. AB - Plant viruses encode proteins that mediate their movement from cell to cell through plasmodesmata. Currently, the mechanisms of action of these movement proteins (MPs) can be divided broadly into two types, requiring or not requiring the presence of viral capsid protein. Cauliflower mosaic virus encodes a multifunctional MP (P1) that modifies plasmodesmata through the formation of tubules that contain virus particles. To investigate the structure of P1, 26 small deletions (scanning deletions) were used to characterize regions of P1 essential for full biological activity. These deletions identified an N-terminal region and a region close to but not at the C terminus as domains that could tolerate manipulation, although gross deletions of either domain abolished infection. In sequence comparisons with other caulimovirus MPs, these regions coincided with the areas of least amino acid homology. Epitope tags inserted into either of these regions were stably maintained in systemic infections, and in extracts from infected plants, tagged P1 was detected on immunoblots. We predicted that, from the hypervariability of these regions, they would be located on the surface of the native P1 structure. Immunofluorescence of P1-specific tubules formed on the surface of infected protoplasts confirmed that the N terminal and C terminus-proximal regions were exposed on the surface of the P1 tubule subunit. These findings establish a structure for P1 that is likely to be applicable to other tubule-forming MPs. PMID- 7540083 TI - [Germ cell tumors of the testis: current status and future progress]. AB - The treatment of testicular cancer has undergone considerable evolution since the introduction of cisplatin and widespread recognition of its curative potentials at any stages of disease. This article provides an overview on statistical and epidemiological information, the latest developments in testicular cancer biology. Also, the results of treating 360 patients with nonseminomatous and 97 patients with seminomatous germ cell tumors are presented. A combined chemotherapy with cisplatin, etoposide and bleomycin demonstrates the highest rate of activity in nonseminomatous germ cell tumor patients. Surgical resection of residual masses after chemotherapy continues to be an important component of combined modality therapy in nonseminomatous testicular tumors. The needs for regular clinical examination during a follow-up have been underlined. PMID- 7540085 TI - Nursing care of children with developmental disabilities having surgery. AB - Children with developmental disabilities (CWDD) who undergo surgery have special needs. The nurse is required to make accurate assessments of the intellectual, psychosocial, and physical state of the child as well as the emotional functioning of the family. The nurse must be aware of the legal implications and his/her own attitudes regarding CWDD. Parents must be accepted as partners with the health care team and open communication should be established. Special concerns for the health care team are latex allergy and malignant hyperthermia. Nursing Care Plans specific to the child with pain, epilepsy, hydrocephalus, myopathy, Down syndrome, myelomeningocele, and cerebral palsy are provided. The material presented here should serve as a basis for nurses to deliver family centered compassionate care to children and their families who are living with the burdens and enduring the hardships brought about by developmental disabilities. PMID- 7540084 TI - Posters as a health education medium in a rural setting. PMID- 7540086 TI - Response of guinea pig smooth and striated urethral sphincter to cromakalim, prazosin, nifedipine, nitroprusside, and electrical stimulation. AB - Prazosin (an alpha-1-adrenergic blocker) and cromakalim (potassium channel opener), given alone, induced significant fatigue of the urethral sphincter at a concentration of 10(-4) M; both drugs combined achieved a significant sphincteric fatigue at a concentration of 10(-5) M each. To 10(-4) M hexamethonium (ganglionic smooth muscle blocker) and 10(-4) M decamethonium (nicotinic blocker of striated muscle) the striated urethral sphincter responded like striated muscle with no detectable function of its smooth muscle component. Therefore, the striated component seems to play a dominant role in sphincteric function. With calcium depletion or in the presence of a calcium channel blocker (10(-4) M nifedipine) the urethral sphincter showed a relative enhancement of response to electrical field stimulation when compared with smooth and skeletal muscle, whose responses were both significantly reduced. This phenomenon could not be explained with calcium-dependent, inhibitory, nitric oxide-releasing nerves, as the NO synthase blocker N-nitro-L-arginine (10(-5) M to 5 x 10(-5) M) failed to induce the enhancement of sphincter contraction during electrostimulation found with calcium depletion. Still, NO-releasing nerves might play a role in sphincteric relaxation because sodium nitroprusside (10(-5) M) induced a significant relaxation of the urethral sphincter precontracted with 80 mM potassium. The potential to weaken sphincteric closure with drugs, exemplified by the results obtained in response to prazosin and cromakalim, would represent a therapeutic advance in the patient with neurogenic bladder dysfunction. PMID- 7540087 TI - Cancer vaccines: the interferon analogy. PMID- 7540088 TI - Transcription in vitro using bacteriophage RNA polymerases. PMID- 7540089 TI - Subtraction hybridization cDNA libraries. PMID- 7540091 TI - An in vitro transcription assay for probing drug-DNA interactions during active transcription of DNA. PMID- 7540090 TI - An in vitro transcription system from BmN cells of the silkworm, Bombyx mori. PMID- 7540096 TI - RNA metabolism in situ at the 93D heat shock locus in polytene nuclei of Drosophila melanogaster after various treatments. AB - Quantitative in situ hybridization to RNA on polytene chromosome spreads, using the 93D exon-, intron- and repeat-specific 35S-labeled antisense RNA probes, revealed treatment- (heat shock, benzamide, colchicine, heat shock followed by benzamide and heat shock in the presence of colchicine) specific differences in the metabolism (synthesis and/or accumulation at the puff site) of the various hsr-omega transcripts, namely hsr-omega-nuclear (omega-n), omega-pre-cytoplasmic (omega-pre-c) and omega-cytoplasmic (omega-c). While heat shock increased the levels of all the three transcripts at the 93D puff site in a coordinated manner, benzamide led to a significant increase in the levels of hsr-omega-n and pre-c; on the other hand, colchicine caused increased levels of the omega-n and omega-c RNA species at 93D. The results also suggested splicing of hsr-omega-pre-c RNA at the site of synthesis with the spliced-out 'free' intron (hsr-omega-fi) accumulating at the puff site. The rate of splicing and/or turnover of the hsr omega-fi varied in a treatment-specific manner. Although a combined treatment to salivary glands with heat shock and benzamide or colchicine is known to inhibit puffing and [3H]uridine incorporation at 93D, the two treatments resulted in a treatment-specific increase in the in situ levels of different hsr-omega transcripts at the 93D site, suggesting a reduced turnover of specific transcripts from the site under these conditions. We suggest that the different 93D transcripts have roles in turnover and/or transport of RNA in nucleus as well as some role in cytoplasmic translation. PMID- 7540095 TI - 4,6-Dibromo-3-hydroxycarbazole (an analogue of caffeine-like Ca2+ releaser), a novel type of inhibitor of Ca(2+)-induced Ca2+ release in skeletal muscle sarcoplasmic reticulum. AB - 1. 4,6-Dibromo-3-hydroxycarbazole (DBHC) was synthesized as an analogue of bromoeudistomin D (BED), a powerful Ca2+ releaser, and its pharmacological properties were examined. 2. In Ca2+ electrode experiments, DBHC (100 microM) markedly inhibited Ca2+ release from the heavy fraction of sarcoplasmic reticulum (HSR) induced by caffeine (1 mM) and BED (10 microM). 3. DBHC (0.1 to 100 microM) inhibited 45Ca2+ release induced by Ca2+ from HSR in a concentration-dependent manner. 4. DBHC (100 microM) abolished 45Ca2+ release induced by caffeine (1 mM) and BED (10 microM) in HSR. 5. Inhibitory effects of calcium-induced calcium release (CICR) blockers such as procaine, ruthenium red and Mg2+ on 45Ca2+ release were clearly observed at Ca2+ concentrations from pCa 7 to pCa 5.5, and were decreased at Ca2+ concentrations higher than pCa 5.5 or lower than pCa 7. However, DBHC decreased Ca2+ release induced by Ca2+ over the wide range of extravesicular Ca2+ concentrations. 6. [3H]-ryanodine binding to HSR was suppressed by ruthenium red, Mg2+ and procaine, but was not affected by DBHC up to 100 microM. 7. [3H]-ryanodine binding to HSR was enhanced by caffeine and BED. DBHC antagonized the enhancement in a concentration-dependent manner. 8. 9-[3H] Methyl-7-bromo-eudistomin D, an 3H-labelled analogue of BED, specifically bound to HSR. Both DBHC and caffeine increased the KD value without affecting the Bmax value, indicating a competitive mode of inhibition. 9. These results suggest that DBHC binds to the caffeine binding site to block Ca2+ release from HSR. This drug is a novel type of inhibitor for the CICR channels in SR and may provide a useful tool for clarifying the Ca2+ releasing mechanisms in SR. PMID- 7540093 TI - Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells. AB - 1. Effects of atrial natriuretic peptide (ANP) on the L-type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole-cell and cell attached configurations of the patch clamp methods. ANP produced a concentration dependent decrease (10-100 nM) in amplitude of a basal Ca2+ channel current. 2. The inactive ANP (methionine-oxidized ANP, 30 nM) failed to decrease the current. 3. 8-Bromo-cyclic GMP (300 microM), a potent activator of cyclic GMP-dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP-induced inhibition of the Ca2+ channel current was still evoked in the presence of 1-isobutyl-3-methyl-xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8-bromo-cyclic GMP. 4. In the single channel recording, ANP and 8-bromo-cyclic GMP also inhibited the activities of the L-type Ca2+ channels. Both agents decreased the open probability (NPo) without affecting the unit amplitude. 5. The present results suggest that ANP inhibits the cardiac L-type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG. PMID- 7540098 TI - Expression of cell surface markers during differentiation of CD34+, CD38-/lo fetal and adult bone marrow cells. AB - Even though there has been considerable progress in the phenotypic characterization of CD34+ bone marrow cells, there is still limited knowledge about the cell phenotypes corresponding to functional terms such as colony forming cells, burst-forming cells, long-term culture-initiating cells, and high proliferative potential cells. In this study, we performed a detailed analysis of phenotypic characteristics of subsets of CD34+ cells. We compared cells from adult and fetal bone marrow to investigate whether reported functional differences are reflected in the cellular phenotypes. CD34+, CD38-/lo, HLA-DR+ cells, which have been shown to contain the most immature hematopoietic progenitor cells, stained as a homogeneous population with most monoclonal antibodies (mAbs). The antigens sLex, CD32, and CD7 were, however, heterogeneously expressed in the CD38-/lo population. Phenotypic differences in the CD34+, CD38-/lo population was found when comparing adult and fetal bone marrow cells. Adult bone marrow CD34+, CD38-/lo cells stained more brightly with CD4, Thy-1, and CD49b and more dimly with CD32 than the same population in fetal bone marrow. Certain antigens that have previously been regarded as lineage specific were found on the CD34+, CD38-/lo, HLA-DR+ cells in both fetal and adult bone marrow. These included CD52, CD13, and CD33. The markers that were found to be most useful in discriminating between subsets of lineage-committed cells within the CD34+, CD38+ population included the B cell marker CD19 and the granulomonocytic marker CD64. The phenotypic analysis presented here should provide a basis for establishing a better link between functional and phenotypic characteristics of hematopoietic progenitor cells. PMID- 7540092 TI - Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. AB - 1. The effect of extracellular nucleotides on the transepithelial ion transport of Madin Darby canine kidney cells (MDCK) was investigated. Cells were grown up to confluency on permeable supports and the short circuit current (ISC) was measured with an Ussing chamber-like mini-perfusion system. 2. Apical ATP stimulated a biphasic ISC increase consisting of a first rapid and transient peak followed by a broader one. 3. The first peak evoked by ATP was reversibly blocked by basilen blue (BB) in a concentration-dependent fashion, with an EC50 of 7.5 microM. 4. The P2 gamma receptor agonist, 2-methylthioATP (2-MeSATP) caused a single transient ISC increase that was completely blocked by pretreatment with BB. On the contrary, the P2x agonist, alpha, beta-methylene ATP (alpha, beta meATP) was almost completely ineffective on ISC. UTP essentially induced a monophasic response the time-course of which resembled that of the second peak stimulated by ATP. The agonist potency order was 2-MeSATP > or = ATP >> UTP, alpha, beta-meATP for the first peak and UTP > or = ATP > 2-MeSATP > alpha, beta meATP for the second peak. 5. Monolayer incubation with the membrane permeable calcium chelator [bis-o-aminophenoxy)-ethane-N,N,N',N',-tetraacetic acid, tetra(acetoximethyl)-ester] (BAPTA/AM) inhibited the ATP-evoked first peak. 6. The non-hydrolyzable ATP analogue, adenosine-5'-O-(3-thio)-trisphosphate (ATP gamma-S) elicited a biphasic response similar to that of ATP. The P1 receptor agonist, 2-chloroadenosine and CGS-21680, were almost unable to induce an ISC increase.2+ increase. The second induces prostaglandin synthesis probably through a P2U receptor activation. PMID- 7540094 TI - Impairment of nitrergic-mediated relaxation of rat isolated duodenum by experimental diabetes. AB - 1. Diabetes mellitus is associated with changes in gastrointestinal motility. The effects of experimental diabetes, induced by streptozotocin administration to rats 3-4 weeks previously, on the nitric oxide (NO)-mediated (nitrergic) relaxation of the duodenum have now been investigated. 2. The non-adrenergic, non cholinergic (NANC) relaxation of the isolated duodenum induced by nicotine (0.3 10 microM) or the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP; 10 microM) was inhibited by the NO synthase inhibitor, NG-nitro-L-arginine (3-100 microM). 3. This nitrergic relaxation induced by nicotine or DMPP of the duodenum from diabetic rats was substantially smaller than that of the tissue from control rats. 4. By contrast, the relaxation of the duodenum from diabetic rats to the NO donor, nitroprusside (0.3-10 microM) was similar to that of control tissue, whereas the relaxation to ATP (0.1-3 microM) was enhanced to a small but significant degree. 5. Incubation of duodenal tissue from control rats at 4 degrees C for 72 h, which leads to neuronal disruption, significantly attenuated the relaxation to nicotine or DMPP whereas the relaxation induced by nitroprusside or ATP was not affected. Comparable cold-storage did not affect the endothelium-dependent relaxation of rat aortic rings induced by acetylcholine (0.01-2 microM). 6. The calcium-dependent NO synthase activity in duodenal tissue, determined by the conversion of radiolabelled L-arginine to citrulline, was significantly reduced in cold-stored tissue and in tissue obtained from diabetic rats. 7. These findings in the rat duodenum indicate that a reduction in intestinal NO synthase activity is associated with an impairment of the NANC relaxation. A defect in the intestinal nitrergic innervation could thus contribute to the motility dysfunction observed in diabetes. PMID- 7540099 TI - Isolation of CD34-positive hematopoietic progenitor cells. PMID- 7540097 TI - Role of platelet activating factor in the inflammatory and secretory effects of Clostridium difficile toxin A. AB - Clostridium difficile is a major recognized cause of antibiotic-associated diarrhea, an effect mediated through its toxin A. Toxin A has been reported to disrupt epithelial tight junctions, attract neutrophils, and cause striking intestinal inflammation and secretion. Having demonstrated that phospholipase A2 inhibitors block the secretory effects of toxin A, we next wished to examine whether platelet activating factor (PAF) was involved in either the direct epithelial or secretory effects of toxin A. The effects of toxin A on net secretion in ligated rabbit ileal segments were significantly inhibited by the PAF antagonists 10(-4)-10(-5) M BN 52021, 10(-5) M WEB 2170, or 10(-5) M SR 27417 by 59-102%. SR 27417 also inhibited secretion induced by toxin A in loops adjacent to the drug (by 58%). Furthermore, the striking inflammation and epithelial disruption seen at 6 h and ligated ileal segments with toxin A was largely prevented by simultaneous treatment with the PAF antagonist SR 27417. In addition, we noted a significant synergistic effect of 10(-8) M PAF with 10 micrograms/ml toxin A in the ligated rabbit ileal segments. To examine direct effects of PAF antagonists on toxin A in T-84 epithelial cell monolayers, rhodamine-labeled phalloidin stained F-actin demonstrated significant disruption of F-actin by toxin A that was reduced by the PAF antagonist BN 52021 or WEB 2170. However, the PAF antagonists (10(-4) M WEB, 10(-5) M BN or 10(-4) M SR) failed to alter the disruption of T-84 cell tissue resistance by C. difficile toxin A (0.03 micrograms/ml). We conclude that PAF may be involved in the secretory effects of C. difficile toxin A, and that PAF antagonists deserve further study in C. difficile diarrhea. PMID- 7540100 TI - Ex vivo hematopoietic progenitor cell expansion. AB - The ability to culture and expand hematopoietic progenitor cells ex vivo has major implications for both bone marrow and stem cell support following marrow ablative or subablative high-dose therapy and for improving the efficiency of retroviral transfection in gene marking and gene therapy. This review focuses on methods for the generation of myeloid progenitor and post-progenitor cells from peripheral blood stem cell collections, with particular emphasis on the characterization of these cells and practical issues associated with their expansion. PMID- 7540101 TI - RNA-protein interactions. Diverse modes of recognition. AB - The structure of a complex between the RNA-binding domain of the small nuclear ribonucleoprotein U1A and an RNA hairpin stresses the diversity of solutions to the problem of sequence-specific RNA recognition. PMID- 7540102 TI - [Expression of cytokines and interferon-related genes in the mouse embryo]. AB - The expression of the cytokine and IFN-related genes was studied in mouse embryo using RT-PCR method capable of detecting low levels of mRNA. Total RNA was prepared from the days 7 embryos by acid guanidinium thiocyanate-phenol chloroform (AGPC) method. cDNA was synthesized by M-MLV reverse transcriptase, and amplified using the specific oligonucleotide primers for IL-1, IL-2, IL-3, IL 4, IL-5, IL-6, TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, IFN-alpha/beta receptor (IFN-alpha/beta R), IFN-gamma receptor (IFN-gamma R), interferon reguratory factor (IRF)-1, IRF-2 and 2'-5' oligoadenylate synthetase (2-5AS) by PCR method. Although the expressions of IL-1 beta, IL-4, IL-5, IL-6, TNF-alpha and IFN-gamma mRNA were detected in all the embryos tested, the expressions of IL-2, IL-3, IFN alpha and IFN-beta mRNA were not detected at all. On the other hand, the expressions of IFN-related genes such as IFN-alpha/beta R, IFN-gamma R, IRF-1, IRF-2 and 2-5AS mRNA, were also detected. These results suggest that these cytokine may play an important role in early embryonic development. PMID- 7540103 TI - [Effects of scopolamine on serotonin in the spinal cord and atria of morphine dependent rats]. AB - The effects of scopolamine on serotonin (5-HT) in the spinal cord and atria of morphine-dependent rats were studied. Levels of 5-HT and its metabolite, 5 hydroxyindolactic acid (5-HIAA) in the spinal cord and atria were analyzed by HPLC-ECD method. The results showed that contents of 5-HT, 5-HIAA in the spinal cord of morphine-dependent rats were statistically higher than those of controls (P < 0.01, n = 6), and that scopolamine inhibited morphine's effect by decreasing 5-HT, 5-HIAA in the spinal cord. After acute abstinence (1d), the contents of 5 HT in the spinal cord in natural withdrawal group were decreased, but increased in scopolamine group. After chronic abstinence (6d), the contents of 5-HT in the spinal cords in the two groups were all increased to above normal level. The results also showed that scopolamine did not affect the increasing of 5-HT in atria induced by morphine. The data support a role of muscarinic receptor on changes of 5-HT metabolism in the spinal cord in morphine-dependent rats. PMID- 7540105 TI - Neurodevelopmental outcome after neonatal extracorporeal membrane oxygenation. AB - OBJECTIVE: To determine the neurodevelopmental outcome of neonates who underwent extracorporeal membrane oxygenation (ECMO group) and similarly critically ill newborns with a lower Oxygenation Index who underwent conventional treatment (comparison group), and to determine whether factors such as the underlying diagnosis and the distance transported from outlying areas affect outcome. DESIGN: Multicentre prospective longitudinal comparative outcome study. SETTING: An ECMO centre providing services to all of western Canada and four tertiary care neonatal follow-up clinics. SUBJECTS: All neonates who received treatment between February 1989 and January 1992 at the Western Canadian Regional ECMO Center and who were alive at 2 years of age; 38 (95%) of the 40 surviving ECMO-treated subjects and 26 (87%) of the 30 surviving comparison subjects were available for follow-up. INTERVENTIONS: ECMO or conventional therapy for respiratory failure. OUTCOME MEASURES: Neurodevelopmental disability (one or more of cerebral palsy, visual or hearing loss, seizures, severe cognitive disability), and mental and performance developmental indexes of the Bayley Scales of Infant Development. RESULTS: Six (16%) of the ECMO-treated children had neurodevelopmental disabilities at 2 years of age, as compared with 1 (4%) of the comparison subjects; the difference was not statistically significant. The mean mental developmental index (91.8 [standard deviation (SD) 19.5] v. 100.5 [SD 25.4]) and the mean performance developmental index (87.2 [SD 20.0] v. 96.4 [SD 20.9]) did not differ significantly between the ECMO group and the comparison group respectively. Among the ECMO-treated subjects those whose underlying diagnosis was sepsis had the lowest Bayley indexes, significantly lower than those whose underlying diagnosis was meconium aspiration syndrome. The distance transported did not affect outcome. CONCLUSIONS: Neurodevelopmental disability and delay occurred in both groups. The underlying diagnosis appears to affect outcome, whereas distance transported does not. These findings support early transfer for ECMO of critically ill neonates with respiratory failure who do not respond to conventional treatment. Larger multicentre studies involving long-term follow-up are needed to confirm these findings. PMID- 7540104 TI - Analysis of the sequences of human beta-1,4-galactosyltransferase cDNA clones. AB - UDP-galactose:beta-1,4 N-acetyl glucosamine galactosyltransferase (4 beta GT) is a promising tumor marker for ovarian cancer. To study the role of 4 beta GT in malignant transformation at the molecular level human 4 beta GT cDNA and genomic clones were isolated and analyzed. For the isolation of 4 beta GT cDNA and genomic clones, a human fetal liver cDNA library in lambda gt11 and a human genomic library in EMBL-3B vectors respectively were screened using a 4 beta GT cDNA insert as the probe. Complete sequence of the cDNA clones were determined by subcloning in plasmid vectors, and compared with the published sequence of human liver 4 beta GT. Presence of various 4 beta GT exons in the genomic clones were determined by Southern blot analysis using specific oligodeoxynucleotide probes. Among the 5 cDNA clones isolated, 2 clones GTN 6 and GTN 17 were sibling clones and had a nucleotide sequence identical to the published 4 beta GT cDNA sequence, except at the 3'-end, where these clones had 7 unique nucleotide sequences. One cDNA clone, GTN2 also had a nucleotide sequence identical to that of 4 beta GT, except for 3 G residues at the 5'-end. One cDNA clone, GTN 1, had a unique sequence at the 5'-end comprising of 74 nucleotides. Another clone, GTN 20, was unrelated to 4 beta GT. Analysis of genomic clones showed that 4 beta GT exons 3, 4, 5 and 6 were present in a 14 kb genomic clone, EMGT-4. Exon 1 was present in a separate 16 kb clone, EMGT-6.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540106 TI - Eradication of large human B cell tumors in nude mice with unconjugated CD20 monoclonal antibodies and interleukin 2. AB - Since antibody-dependent cellular cytotoxicity is considered an important mechanism by which mAbs may exert their antitumor effects, it seems likely that these antitumor effects can be enhanced by the activation of the appropriate effector cell populations. We have used nude mice xenografted with human Daudi tumor cells as a model to compare the antilymphoma effects of unconjugated CD19 (CLB-CD19) and CD20 (BCA-B20) mAbs (IgG2a subclass) alone or in combination with recombinant human interleukin 2 (rhIL-2) or recombinant mouse granulocyte macrophage-colony-stimulating factor (rmGM-CSF). Treatment of established tumors with BCA-B20 or rhIL-2 or rmGM-CSF as a single agent, all resulted in highly significant decreases of tumor growth rates, but did not increase the number of complete regressions. The combination of CLB-CD19 or BCA-B20 mAbs with rhIL-2 or rmGM-CSF resulted in larger decreases of growth rates than either of the agents alone. Complete eradication of large Daudi tumors could be achieved when treatment with BCA-B20 mAbs was combined with rhIL-2, but not with the combination of CLB-CD19 mAbs and rhIL-2 nor with the combination of BCA-B20 mAbs and rmGM-CSF. Cured animals kept for 2-3 months after complete regression of the tumors were still tumor free. Regression of tumors was correlated with the infiltration of lymphocytes as well as macrophages into the tumor. This is the first report to show that unconjugated CD20 mAbs are to be preferred over unconjugated CD19 mAbs, and interleukin 2 over GM-CSF in the combinational treatment of large B cell tumors. PMID- 7540108 TI - Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases. AB - Prostate specific antigen (PSA) is generally believed to be expressed only by prostate epithelium. If this were true of PSA, RNA, then detecting PSA RNA in cells outside of the prostate would indicate metastasis. PCR can detect rare prostate cancer cells. To enhance sensitivity, we developed "nested primer" PCR to detect PSA RNA. With this method, PSA RNA is present in several non-prostate cell lines, including BG-1 (ovarian), SK-MES-1 (lung), and HL-60 (myeloid leukemia), and some normal blood. A low level of PSA RNA detectable by nested primer PCR is present in some cells of non-prostate origin and may interfere with sensitive methods to detect micrometastases. Transcripts of other genes thought to be organ specific may have similar limitations. PMID- 7540109 TI - Ion channel function in disease. PMID- 7540107 TI - Immunological responses in human papillomavirus 16 E6/E7-transgenic mice to E7 protein correlate with the presence of skin disease. AB - The human papillomavirus (HPV) oncogenes, E6 and E7, are believed to contribute to the development of cervical cancers in women infected with certain HPV genotypes, most notably HPV-16 and HPV-18. Given their expression in tumor tissue, E6 and E7 have been implicated as potential tumor-specific antigens. We have examined an HPV-16 E6- and E7-transgenic mouse lineage for immune responses to these viral oncoproteins. Mice in this lineage express the HPV-16 E6 and E7 genes in their skin and eyes, and on aging, these mice frequently develop squamous cell carcinomas and lenticular tumors. Young transgenic mice, which had measurable E7 protein in the eye but not in the skin, were immunologically naive to E7 protein. They mounted an immune response to E7 on immunization comparable to that of nontransgenic controls, suggesting a lack of immune tolerance to this protein. Older line 19 mice, which are susceptible to skin disease associated with transcription of the E6 and E7 open reading frames, had measurable E7 protein in their skin. These older transgenic mice spontaneously developed antibody responses to endogenous E7 protein, particularly in association with skin disease. Also detected in older mice were delayed-type hypersensitivity responses to E7. These finding parallel the humoral immune response to E7 protein in patients with HPV-associated cervical cancer and suggest that line 19 mice may provide a model for studying the immunobiology of HPV-associated cancers. PMID- 7540110 TI - ATPo but not cAMPi activates a chloride conductance in mouse ventricular myocytes. AB - OBJECTIVE: The aim was to test for the presence of cAMPi activated and ATPo activated chloride conductances (ICl(cAMP) and ICl(ATP)) in mouse ventricular myocytes. METHODS: Membrane currents were measured in enzymatically dissociated ventricular myocytes using the whole cell configuration of the patch clamp technique. RESULTS: After inhibiting the activation of sodium, calcium, and potassium currents, increasing [cAMP]i with isoprenaline (1 microM), forskolin (10 microM), or isobutylmethylxanthine (500 microM) had no effect on residual membrane current but extracellular ATP (100 microM) elicited a time independent background conductance. The ATP-activated conductance was observed during voltage steps ranging from +40 to -110 mV from a holding potential of -30 mV within 1 min after adding ATP to the bath. The reversal potential for the ATP activated conductance varied with changes in the chloride gradient and was within several mV of the predicted Nernst potential for chloride. Partially substituting external chloride with aspartate attenuated the ATP activated currents and shifted the reversal potential to values close to those predicted for ECl, suggesting the chloride selective nature of the conductance. The poorly hydrolysable ATP analogue ATP gamma S (100 microM) also elicited the chloride conductance, suggesting purinoceptor linked activation rather than a mechanism dependent on hydrolysis of ATP by ectoATPases. The conductance may be activated following P2 and not P1 purinoceptor stimulation, since the P1 purinoceptor agonists adenosine (500 microM) and adenosine monophosphate (500 microM) had no effect in seven and four cells, respectively. Activation of ICl(ATP) is not likely to be dependent on increased [Ca2+]i since ATP activated the current in cells dialysed with 10 mM EGTA or 20 mM BAPTA. CONCLUSIONS: (1) cAMPi does not activate a chloride conductance in mouse ventricular myocytes; (2) ATPo does activate a chloride conductance, through stimulation of a P2 purinoceptor; and (3) Ca2+i and cAMPi are not involved in activation of ICl(ATP). Also, macroscopic ICl(ATP) from mouse ventricle and ICl(cAMP) from other species appear to be indistinguishable in terms of time independence and rectification properties that depend on the concentration and permeability of intracellular anions. ICl(ATP) could be of physiological and pathophysiological significance considering the ability of chloride channels to modulate cardiac electrical activity and the fact that ATP may be an important neuromediator in the heart. PMID- 7540111 TI - Attenuation of the reflex responses to muscle contraction by the coadministration of antagonists to substance P and somatostatin into the dorsal horn. AB - OBJECTIVE: The aim was to determine if the coadministration of antagonists to substance P and somatostatin into the L7 dorsal horn blunts the reflex cardiovascular responses to static contraction to a greater extent than each antagonist alone. The possibility that this attenuation is mediated by blunting the contraction evoked increases in sympathetic outflow was also tested. METHODS: Using alpha chloralose anaesthetised cats (n = 8), static contraction and stretch of the triceps surae muscle were performed before and after microinjecting (1 microliter) 250 ng of the substance P antagonist, D-Pro2-D-Phe7-D-Trp9-substance P, and the somatostatin antagonist, cyclo(7-amino-heptanoyl-phenylalanyl-D tryptophyl-lysyl-threonyl-[ benzyl]). The muscle was contracted by electrically stimulating the peripheral end of the cut L7 ventral root. RESULTS: Before injecting the antagonists, static muscle contraction increased mean arterial blood pressure by 40(SEM 6) mm Hg, heart rate by 13(2) beats.min-1, and renal sympathetic nerve activity (RSNA) by 41(7)%. These changes were blunted by the antagonists since the increases in blood pressure, heart rate, and RSNA were reduced to 21(3) mm Hg, 8(1) beats.min-1, and 23(5)%, respectively. In contrast, antagonist administration did not affect the pressor [33(5) v 31(5) mm Hg], heart rate [9(2) v 10(2) beats.min-1], or RSNA [23(4)% v 25(5)%] responses to muscle stretch. Microinjection of 2% lignocaine into the dorsal horn virtually abolished the reflex changes elicited by muscle stretch. CONCLUSIONS: The release of substance P and somatostatin in the spinal cord plays a role in mediating the cardiovascular changes caused by static contraction, but the release of other neurotransmitters/neuromodulators is also involved. The attenuation produced by these antagonists is mediated, at least in part, by reducing sympathetic outflow. PMID- 7540112 TI - Insulin-like growth factor I is involved in inflammation linked angiogenic processes after microembolisation in porcine heart. AB - OBJECTIVE: Angiogenesis in the porcine heart can be induced by myocardial ischaemia following vascular occlusions. This process is characterised by increased numbers of monocytes/macrophages, known to be potent producers of various mitogens such as insulin-like growth factors (IGF) and interleukins (IL). The aim of the study was to examine gene expression of these factors by means of northern blot hybridisation, slot blot analysis, and in situ hybridisation in a porcine model of coronary angiogenesis. METHODS: Experimental ischaemia and subsequent focal necroses were induced by selective injection of 25 microns microspheres into the left circumflex artery. The hearts were excised after 3-168 h of microembolisation, and tissue was collected from a non-ischaemic control area and the circumflex region of the same heart for further analysis. RESULTS: IGF-I was constitutively transcribed in normal porcine myocardium mainly by myocytes. Following microembolisation, IGF-I mRNA expression was significantly increased in the experimental region (1.8-fold) after 72 h and to a lesser extent after 168 h. In the ischaemic region, characterised by capillary sprouting, numerous mononuclear cells contained IGF-I mRNA. In contrast, IGF-II mRNA levels, constitutively produced by porcine myocytes, were not altered by microembolisation. IL-1 alpha, IL-1 beta, and IL-4 mRNA expression was undetectable in our animal model, whereas IL-6 was constitutively transcribed in normal and ischaemic heart and remained insensitive to microembolisation and focal necrosis. CONCLUSION: After microembolisation, increased IGF-I mRNA expression occurred by infiltrating monocytes in areas of microsphere induced focal necrosis, where capillary sprouting can be detected, suggesting that IGF-I is involved in inflammation linked angiogenic processes. PMID- 7540114 TI - [A comparative study of primary and re-infection with hepatitis C]. AB - A nested polymerase chain reaction (PCR) was used to assess HCV re-infection and primary infection. In 10 hepatitis cases defined as primary infection, 9 showed clinical hepatitis, and 1 case was subclinical; the interval between transfusion and elevation of alanine aminotransferase (ALT) levels was 15-60 (37.9 + 13.9) days. 10/10 and 7/10 were persistently positive for anti-HCV and HCV RNA for more than 1 year respectively. Similarly, in 5 cases defined as re-infection, 4/5 showed clinical hepatitis, the interval between transfusion and elevation of ALT was 30-46 (34.8 +/- 6.4) days, and 5/5 and 3/5 were persistently positive for anti-HCV and HCV RNA for more than 1 year respectively. All 5 re-infected recipients showed abnormal ALT. In conclusion, there was no significant difference (P > 0.05) in the aspect of the clinical figure, ALT abnormality, the duration of anti-HCV and HCV RNA etc. Resulting from primary or re-infection with HCV, suggesting that primary infection fail to induce a Protective immune response. PMID- 7540115 TI - [Outcome of HCV infection in intravenous drug addicts and influential factors]. AB - A total of 154 intravenous drug addicts with antibody to hepatitis C virus (Anti HCV) were followed up for 6 and 15 months, respectively. The corresponding negative conversion rates of anti-HCV were 11.0% (17/154) and 33.3% (29/87), respectively. The data were analysed by logistic curve fitting. Results showed that marriage and income status have significant influence on negative conversion rate of anti-HCV. PMID- 7540113 TI - Localization of CD44, the hyaluronate receptor, on the plasma membrane of osteocytes and osteoclasts in rat tibiae. AB - CD44 is a multifunctional adhesion molecule that binds to hyaluronic acid, type I collagen, and fibronectin. We have studied the immunohistochemical localization of CD44 in bone cells by confocal laser scanning microscopy and transmission electron microscopy in order to clarify its role in the cell-cell and/or cell matrix interaction of bone cells. In round osteoblasts attached to bone surfaces, immunoreactivity is restricted to their cytoplasmic processes. On the other hand, osteocytes in bone matrices show intense immunoreactivity on their plasma membrane. Intense immunoreactivity for CD44 can be detected on the basolateral plasma membranes of osteoclasts. There is considerably less reactivity observed in the area of the plasma membrane that is in direct contact with bone. The pre embedding electron-microscopical method has revealed that CD44 is mainly localized on the basolateral plasma membrane of osteoclasts. However, the ruffled border and clear zone show little immunoreactivity. A CD44-positive reaction can be detected on both plasma membranes in the contact region between osteoclasts and osteocytes. These findings suggest that: 1) cells of the osteoblast lineage express CD44 in accordance with their morphological changes from osteoblasts into osteocytes; 2) osteoclasts express CD44 on their basolateral plasma membrane; 3) CD44 in osteoclasts and osteocytes may play an important role in cell-cell and/or cell-matrix attachment via extracellular matrices. PMID- 7540116 TI - A place in the world for RNA editing. PMID- 7540117 TI - Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. AB - Five unrelated children are described with a rare autoimmune lymphoproliferative syndrome (ALPS) characterized by massive nonmalignant lymphadenopathy, autoimmune phenomena, and expanded populations of TCR-CD3+CD4-CD8- lymphocytes. These findings, suggesting a genetic defect in the ability of T lymphocytes to respond to normal immunoregulatory mechanisms, prompted an evaluation of lymphocyte apoptosis. Each child had defective Fas-mediated T lymphocyte apoptosis associated with a unique, deleterious Fas gene mutation. One mutation appeared to cause a simple loss of function; however, four others had a dominant negative phenotype when coexpressed with normal Fas. Family studies demonstrated the inheritance of the mutant Fas alleles. The occurrence of Fas mutations together with abnormal T cell apoptosis in ALPS patients suggests an involvement of Fas in this recently recognized disorder of lymphocyte homeostasis and peripheral self tolerance. PMID- 7540118 TI - cornichon and the EGF receptor signaling process are necessary for both anterior posterior and dorsal-ventral pattern formation in Drosophila. AB - In Drosophila, the dorsal-ventral polarity of the egg chamber depends on the localization of the oocyte nucleus and the gurken RNA to the dorsal-anterior corner of the oocyte. Gurken protein presumably acts as a ligand for the Drosophila EGF receptor (torpedo/DER) expressed in the somatic follicle cells surrounding the oocyte. cornichon is a gene required in the germline for dorsal ventral signaling. cornichon, gurken, and torpedo also function in an earlier signaling event that establishes posterior follicle cell fates and specifies the anterior-posterior polarity of the egg chamber. Mutations in all three genes prevent the formation of a correctly polarized microtubule cytoskeleton required for proper localization of the anterior and posterior determinants bicoid and oskar and for the asymmetric positioning of the oocyte nucleus. PMID- 7540121 TI - The nephrotoxicity of immunosuppressive drugs. AB - Nephrotoxicity from immunosuppressive drugs can complicate otherwise successful therapy. This paper reviews the clinical and pathophysiologic aspects of nephrotoxicity. The chronic progressive nephropathy is emphasized based on a recently developed animal model. Experimental and clinical data regarding FK506, new cyclosporine analogs, and Rapamycin are also discussed. PMID- 7540119 TI - [Modifications of gene expression by tumor promoters]. AB - The modifications of gene expression by tumor promoters were analyzed in vitro and in vivo. The results of slot blot hybridizations showed that tumor promoter TPA induced c-fos and c-myc expressions in mouse fibroblast cell line BALB/3T3 and rat liver, decreased the levels of Rb RNA in BALB/3T3 cell line and of alpha 1-I3 RNA in rat liver. It was also demonstrated that tumor promoter phenobarbital influenced c-fos and c-myc expressions and decreased alpha 1I3 mRNA level in rat liver during a long term experiment. Phenobarbital was found to have no effect on c-fos and c-myc expressions in rat liver during a short experiment. Tumor promoters induced the expressions of c-fos and c-myc which were positively related to cancer formation and inhibited the expressions of Rb and alpha 1-I3 which were negatively-related to cancer formation. This implied that tumor promotion played an important role in cancer development and tumor promoters exerted their effects selectively according to the attributes of different genes. PMID- 7540120 TI - Phase I-II study of the somatostatin analogue lanreotide in hormone-refractory prostate cancer. AB - Lanreotide (BIM 32014), a somatulin analogue, was found to be as effective as castration in a rat prostate tumor model. Therapeutic benefit was also demonstrated in the hormone-resistant phase of this tumor model. The activity of lanreotide may be due to a reduction in the levels of growth factors such as insulin growth factor 1 (IGF1). A total of 30 patients with hormone-refractory prostate cancer were treated with a slow-release formulation of lanreotide. The mean age was 71 years. Patients were treated with one intramuscular injection of 30 mg BIM 23014 once a week and were followed for prostate-specific antigen (PSA) level evolution until disease progression or WHO grade 3 or 4 toxicity and for survival. The patients were treated for a mean duration of 12 weeks (range, 2-60 weeks). The performance status and bone pain were improved in 40% and 35% of patients respectively. In all, 20% of the patients had a decrease of > or = 50% in PSA levels and 16% showed a stabilization. The biological response was correlated with clinical improvement. The 1-year global survival rate was 72%, with the rate being 89% in the group of patients who were responders on PSA plasma level and 64% in patients with progressive disease. The response duration ranged from 16 to 60 weeks. Toxicity was minor, with transient grade I digestive side effects being noted in a few patients. Lanreotide given at 30 mg once a week to patients with metastatic hormone-refractory prostate cancer was well tolerated. The response rate was higher than that reported in recent published series. Higher doses of lanreotide should be evaluated. PMID- 7540122 TI - [Fas antigen gene in thymuses from patients with myasthenia gravis]. AB - The Fas antigen is a cell surface protein that can mediate apoptosis, and plays a major role in functional maturity in clonal deletion of autoreactive T cells in the thymus. Recently a cDNA encoding the human Fas antigen was isolated. It was suggested that a signal-transducing domain, along with inhibitory one, was presented in the cytoplasmic domain of the Fas antigen. We examined whether any abnormality in Fas antigen gene may breakdown the immunotolerance in patients with myasthenia gravis (MG). We used single-strand conformational polymorphism (SSCP) analysis, generally used to screen for unknown mutation, to examine the cytoplasmic cDNA of Fas antigen obtained by reverse transcription (rt)-PCR from mRNA of MG thymuses. Furthermore, we studied in situ expression of Fas antigen mRNA in thymuses from MG patients. Fragments from all the 12 (3 control subjects, and 9 MG patients) thymuses produced by rt-PCR showed equally stained two bands of the two single strands. In the thymuses from both controls and MG patients, the Fas antigen mRNA was mainly expressed in small thymic cells. These cells were located near clustered cells with relatively large cytoplasms in the cortex and sometimes surrounded them, but were also founded in clusters in the follicules. In situ expressions of Fas antigen mRNA were more remarkable in MG thymuses than in control subjects, and in hyperplastic thymuses than in normoplastic ones. These results suggest that mRNA for the cytoplasmic domain of the Fas antigen have no mutation, and its expression is not reduced in thymuses from patients with MG. PMID- 7540123 TI - Selective impairment in manipulating Arabic numerals. AB - This paper describes an acalculic patient (B.A.L.) with an unusual selective deficit in manipulating arabic numerals. The patient was unimpaired in reading aloud letters, words and written number names but unable to read aloud single arabic numerals. Furthermore, his ability to produce the next number in the sequence and his ability to produce answers to simple addition and subtraction was relatively spared when the stimuli were presented as number names but impaired when the stimuli were presented as arabic numerals. Using magnitude comparison tasks it was demonstrated that his knowledge of cardinal values of arabic numerals was preserved. His impairment in manipulating arabic numerals was interpreted in terms of a deficit in the connection between format specific number codes and the verbal numeral production system. PMID- 7540124 TI - Hematopoietic growth factors. Defining the appropriate clinical role in multimodality cancer therapy. AB - Laboratory investigations have begun to elucidate the regulatory molecules that control the processes of blood cell growth and differentiation. Recombinant human colony-stimulating factors are examples of biotechnology-produced molecules that have epitomized the translation of such basic scientific investigation into therapeutic advances. Small cell lung cancer, a malignancy that is overall highly sensitive to aggressive myelosuppressive chemotherapy at initial presentation, has been used as a clinical model in which the activity of human colony stimulating factors has been tested. In this article, the clinical applications of hematopoietic growth factors are reviewed in brief. The appropriate clinical use of these agents may allow novel therapeutic strategies to be developed in a research setting. Similarly, these agents have the potential to improve supportive care and improve certain clinical outcomes in the non-research clinical care of patients. Issues of cost of treatment are raised by these agents, but the true clinical value of hematopoietic growth factors needs to be studied more rigorously, with emphasis on quality of life and redistribution of care costs outside of hospitals before definitive statements can be made. PMID- 7540125 TI - New therapeutic strategies involving radiation therapy for patients with non small cell lung cancer. AB - Recent notable developments have occurred involving radiation therapy (RT) for patients with non-small cell lung cancer (NSCLC). For patients with good performance status with unresected thoracic tumors, induction cisplatin-based chemotherapy followed by RT has resulted in a significant survival advantage over RT alone in two North American trials. However, the best sequence of administration of these two modalities in NSCLC remains to be determined. For palliation of tumor-related symptoms, efforts under way to improve control of brain metastases include the use of twice-daily cranial RT to a higher total dose, the use of focused radiation boost techniques like stereotactic radiosurgery to small metastatic deposits, and increased use of neurosurgical extirpation. For patients with NSCLC with symptomatic bone metastases, use of wider-field irradiation may benefit selected patients. Metastases to the adrenal gland, liver, and subcutaneous tissues can be palliated successfully by brief courses of RT. Intrathoracic tumor symptoms are well palliated by brief courses of thoracic RT. As adjuvant therapy following curative surgery, RT reduces the intrathoracic tumor recurrence rate among patients with metastatic tumor foci in hilar or mediastinal lymph nodes. PMID- 7540127 TI - Protective effects of endothelin-1 on acute pancreatitis in rats. AB - Endothelin-1, a 21-residue peptide isolated from vascular endothelial cells, has a broad spectrum of actions. To clarify the involvement of endothelin-1 in acute pancreatitis, we examined the effects of endothelin-1 and its receptor antagonist BQ-123 on cerulein-induced pancreatitis in rats. Rats were infused intravenously with heparin-saline (control), endothelin-1 (100 pmol/kg/hr), cerulein (5 micrograms/kg/hr), or cerulein plus endothelin-1 for 3.5 hr. In another experiment, cerulein or cerulein plus BQ-123 (3 mg/kg/hr) was infused. Infusion of cerulein caused hyperamylasemia and pancreatic edema. Endothelin-1, when infused with cerulein, decreased the extent of pancreatic edema with a significant increase in the pancreatic dry- to wet-weight ratio. Histological changes induced by cerulein were markedly attenuated when endothelin-1 was given with cerulein. In contrast, endothelin-receptor blockade with BQ-123 further augmented pancreatic edema caused by cerulein. The extent of inflammatory cell infiltration was greater than BQ-123 was given with cerulein. Endothelin-1 or BQ 123 had no influence on hyperamylasemia. This study suggests that endothelin-1 has protective effects on experimental acute pancreatitis. PMID- 7540128 TI - [Risk of carcinoma in cystic fibrosis]. PMID- 7540126 TI - Endoscopic stenting for palliation of malignant biliary obstruction. A review of progress in the last 15 years. AB - Since the late 1970s, endoscopic biliary stenting has become a standard palliative treatment for obstructive jaundice due to malignancies of the pancreas and the hepatobiliary system. Despite the high initial success rate in achieving biliary drainage, endoscopic stenting therapy has been limited by the clogging of biliary stents, usually after four to five months, due to formation of adherent bacterial biofilm and accumulation of biliary sludge. Various methods for the prevention of bacterial adhesion and prolongation of stent patency have been investigated, including prophylactic antimicrobial agents and bile salts, new stent materials, and new stent designs. Recently, the introduction of self expandable metal stents has significantly improved the duration of stent patency but the cost is considerably higher. Each method has its own merits as well as specific problems. This article reviews the pathogenesis of biofilm formation on the biliary stents and the latest status of research in avoiding stent occlusion. PMID- 7540130 TI - Progress of myelination in the human fetal spinal nerve roots, spinal cord and brainstem with myelin basic protein immunohistochemistry. AB - The early progress of myelination was studied, by means of myelin basic protein (MBP) immunohistochemistry and luxol-fast-blue (LFB) staining, in the spinal cord, spinal nerve roots and brainstem of 66 fetuses and neonates. The degree of myelination was classified from 1 (slight) to 4 (mature). MBP immunoreactivity exhibited slight LFB positivity. Myelination first occurred in the medial longitudinal fasciculus at 20 weeks of age, reaching degree 4 at 34 weeks, but began at 23-24 weeks in the other sites. Myelination of spinal nerve roots progressed with gestation and attained degree 4 at 35-36 weeks. The cuneate fasciculus also reached degree 4 at 34-36 weeks, but corticospinal tracts and solitary tracts, which exhibited long myelinating phases, were slow and incomplete at 40 weeks. This normal development of MBP and LFB myelination can be used for the assessment of delayed myelination in fetal and neonatal diseases. PMID- 7540129 TI - Fetal haemoglobin concentration and mean red cell volume are not related to the maternal values at 18-25 weeks' gestation. AB - A cross-sectional study of 64 women and their fetuses undergoing cordocentesis at 18-25 weeks showed no significant correlation between maternal and fetal Hb or MCV and no relationship between these measurements and fetal abdominal circumference. We found no evidence for a nutritional or physiological association between maternal and fetal haematological status. PMID- 7540131 TI - Segmental myoclonus in Whipple's disease. AB - A patient suffering from Whipple's disease, presenting with segmental myoclonus in the muscular distribution of the muscles of the right facial nerve, which is one of the first neurological findings of the disease is described. Patients suffering from segmental myoclonus and Whipple's disease are reported and the possible anatomical sites of the injury, responsible for the presence of this symptom is discussed. PMID- 7540132 TI - Molecular and cellular aspects of the insulin-like growth factor I receptor. PMID- 7540133 TI - A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease. AB - The effects of the mild cystic fibrosis (CF) mutation P574H were analysed and compared with those of three severe ones (delta I507, delta F508 and R560T). Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H >> delta F508 >> R560T approximately 0. Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that of the wild type CFTR-Cl- channel. This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. The P574H mutation modifies the gating mode of the channel with a large increase (approximately x 7) in the mean channel open time. Proline 574 might play an important role in the process connecting ATP hydrolysis at the nucleotide binding domain and opening and closing events of the CFTR-Cl- channel. PMID- 7540134 TI - Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway. AB - Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain. PMID- 7540135 TI - Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection. AB - Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion. PMID- 7540138 TI - Histamine in the heart. PMID- 7540136 TI - Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET). AB - A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation. PMID- 7540137 TI - Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. AB - We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription. PMID- 7540139 TI - Phosphatidylinositol hydrolysis is involved in production of Ca(2+)-dependent currents, but not non-selective cation currents, by muscarine in chromaffin cells. AB - Whether phosphatidylinositol hydrolysis and a subsequent Ca2+ mobilization are responsible for muscarine-induced transient outward currents (IO) and non selective cation currents (INS) in the guinea-pig chromaffin cell was investigated using the perforated patch method. IO, but not INS, failed to be reproduced in Ca(2+)-free solution and was markedly reduced by prior exposure to caffeine under Ca(2+)-free conditions or by addition to normal solution of cyclopiazonic acid (CPA), a Ca2+ ATPase inhibitor. Application of CPA in Ca(2+) free solution, however, suppressed INS by about 50% in 73% of the cells tested. Bath application of 1.5 mM neomycin, a phospholipase C inhibitor, induced the time-dependent decline of IO with near abolition at 20 min or less, whereas it produced a time-independent decrease of INS and an inwardly rectifying K+ current. INS in the presence or absence of neomycin was well fitted to rectangular hyperbolas with the same ED50 of 2.17 microM, but with a 33% smaller maximum amplitude in the former, indicating a non-competitive inhibition by neomycin. We conclude that, while phosphatidylinositol hydrolysis mediates the production of IO, it does not mediate that of INS by muscarine. PMID- 7540140 TI - Relaxant influence of phosphodiesterase inhibitors in the cat gastric fundus. AB - The breakdown of the relaxation-inducing second messengers cAMP and cGMP is mediated by phosphodiesterases. Inhibitors of functionally present phosphodiesterases can be expected to induce relaxation by increasing the basic amount of cAMP and/or cGMP. In the cat gastric fundus, vinpocetine, which has some selectivity for phosphodiesterase type I, only induced contractions, but the inhibitors of type III [5-(4-acetimidophenyl)pyrazin-(1H)-one; SKF 94120], type IV (rolipram) and type V (zaprinast) phosphodiesterase all caused concentration dependent relaxation, as did the non-specific phosphodiesterase inhibitor 3 isobutyl-1-methylxanthine (IBMX). The most potent relaxant agent was rolipram (EC50 9 +/- 5 x 10(-7) M and 3 +/- 1 x 10(-7) M in longitudinal and circular smooth muscle strips, respectively). These results suggest that type III, IV and V phosphodiesterases are functionally present in the cat gastric fundus and are involved in the regulation of tone. The possible influence of the phosphodiesterase inhibitors on non-adrenergic non-cholinergic (NANC) relaxation induced by nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and train and sustained electrical field stimulation was then tested. Rolipram (3 x 10(-8) M), SKF 94120 (10(-5) M) and IBMX (10(-6) M) did not potentiate any of the relaxant stimuli studied. Zaprinast (10(-5) M), the cGMP specific type V phosphodiesterase inhibitor, caused a significant increase of the relaxation induced by exogenous NO and by train electrical field stimulation. These stimuli are thought to induce relaxation via an increase of intracellular cGMP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540141 TI - Effect of substance P and capsaicin on stomach fundus and ileum of streptozotocin diabetic rats. AB - The in vitro responses of longitudinal preparations of rat stomach fundus and ileum to capsaicin at 1, 8, 4, 16 and 26 weeks and to substance P at 1 and 8 weeks from diabetes induction were studied. The results were compared with those obtained in age-matched control rats. The contractile responses to exogenous substance P and capsaicin were not affected in the stomach fundus from diabetic rats. Atropine (1 microM) did not antagonize the substance P-induced response whereas it inhibited about 90% of the capsaicin-induced response in controls and about 60% of the response in diabetic rats. At the resting tone, capsaicin induced a relaxation followed by a contraction in stomach fundus of control rats. Only a contraction was evoked in diabetic rats. In carbachol (0.05-0.1 microM) pre-stimulated strips, a complete restoration of the biphasic response was obtained in the diabetic state. The contractile response elicited by exogenous substance P was not significantly increased in the ileum preparations from diabetic rats; nevertheless the EC50 value for substance P was reduced 8 weeks after the onset of diabetes. The response elicited by capsaicin in the ileum of control rats was also biphasic. The capsaicin-induced contraction was greater in tissue from diabetic rats as compared with controls and relaxation was not evident. An age-related decrease of the contraction was also evident in both groups. Atropine (1 microM) partially antagonized the responses to substance P and capsaicin. The inhibition of the responses with atropine was more evident in control than in diabetic rats. These results suggest that the myogenic actions of several agonists in these two tissues are differently modified in experimental diabetes. PMID- 7540142 TI - Nitric oxide modulates agonist-evoked Ca2+ release and influx responses in PC12 64 cells. AB - Nitric oxide (NO) is a signalling molecule involved in events crucial to neuronal cell function such as neurotransmitter release, gene transcription, and neurotoxicity. In these, as well as in many other neuronal processes, a key role may be played by the increases of the intracellular Ca2+ concentration ([Ca2+]i) occurring in response to activation of plasma membrane receptors coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis. Such a [Ca2+]i increases are sustained by release of the cation from intracellular stores and stimulation of influx through specific Ca2+ channels. We have investigated the role of NO in modulating the two above Ca2+ processes occurring subsequently to muscarinic receptor activation in a selected clone (PC12-64) of PC12 cells, a neurosecretory/neuronal cell model. Analysis of [Ca2+]i variations in fura-2 loaded cells, exposed to different NO synthase inhibitors or NO donors, showed that Ca2+ release from intracellular stores was moderately inhibited and stimulated by these two groups of drugs, respectively, while Ca2+ influx through the channels directly coupled to muscarinic receptors was found to be insensitive to NO action. In contrast, Ca2+ influx activated by muscarinic receptor-induced store depletion (investigated also by Mn2+ quenching of the fura-2 signal) was increased by NO generation and inhibited by NO synthase blockade. Incubation of the cells with 8-bromo cGMP did not mimick the action of NO, suggesting that the effect of the messenger on Ca2+ influx is exerted through a signalling pathway different from cGMP generation. PMID- 7540143 TI - Structural requirements for neuropeptide Y in mast cell and G protein activation. AB - Incubation of neuropeptide Y or its C-terminal fragments with rat peritoneal mast cells resulted in a dose-dependent histamine release. Fragment 18-36 of neuropeptide Y was the most biologically active peptide. EC25 value on rat mast cells was 7.2 +/- 2.2 nM. Neuropeptide Y was also able to induce a flare response after intradermal injection in humans. The histamine releasing effects of neuropeptide Y related peptides were greatly inhibited by pretreatment of rat mast cells with pertussis toxin or benzalkonium chloride. Neuropeptide Y and C terminal related peptides also stimulated the GTPase activity of purified heterotrimeric G proteins in a dose-dependent manner from 1 to 50 microM. Binding studies with [125I]neuropeptide Y were unable to provide evidence for the presence of specific binding sites on the surface of mast cells. The alpha helical conformation of neuropeptide Y fragments was studied by measuring the circular dichroism spectra. Neuropeptide Y-(18-36) was the smallest fragment having a strong helical conformation. Our results demonstrate that neuropeptide Y activates mast cells through a non-specific process leading to G protein activation. PMID- 7540144 TI - Enzymatic characterisation of recombinant murine inducible nitric oxide synthase. AB - A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme. PMID- 7540145 TI - Effects of Ca2+ channel antagonist subtypes on mitochondrial Ca2+ transport. AB - This study was carried out to define the effects of various Ca2+ channel modulatory drugs on mitochondrial Ca2+ movements. Bovine adrenal medulla mitochondria took up Ca2+ at an initial rate of 6.8 nmol mg protein-1 5 s-1, with a Km of 15 microM and a Bmax of 30 nmol mg protein-1. At 30 microM, neither verapamil, diltiazem, nitrendipine nor Bay K 8644 [methyl-1,4-dihydro-2,6 dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] affected the initial rate of Ca2+ uptake. Ca(2+)-loaded mitochondria retained their Ca2+ contents in the presence of ruthenium red for at least 30 min. Cinnarizine and flunarizine, but not verapamil, diltiazem, isradipine, Bay K 8644 or nitrendipine, caused a fast and dramatic Na(+)-independent Ca2+ loss. Other Ca2+ channel antagonists assayed such as penfluridol, R56865 [N-[1-(4-(4 fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2- benzothiazolamine], lidoflazine, R87926 [(+)-(S)-4-(2-benzothiazolyl-methylamino)-alpha-[(3,4-difluorophenoxy ) methyl] 1 piperidine] and sabeluzole, also had a mitochondrial Ca2+ depleting effect which seemed to be directly related to their octanol/water partition coefficient. The Na(+)-dependent Ca2+ efflux from mitochondria was completely inhibited by diltiazem and greatly blocked by nitrendipine. Isradipine caused a moderate blockade and Bay K 8644 and verapamil had no effect. All these data open the possibility of developing novel Ca2+ channel antagonists having selective actions on plasmalemmal Ca2+ channels, and others with additional and different effects on mitochondrial Ca2+ transport.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540146 TI - IFN-alpha induces MxA gene expression in cultured human corneal fibroblasts. AB - Treatment of certain human cells with Interferon-alpha (IFN-alpha) induces the synthesis of a 76 kDa protein designated MxA that is involved in resistance to viral infection. We have used a specific cDNA clone and monoclonal Ab to show that MxA is induced in IFN-alpha treated human corneal fibroblast cultures. Mx RNA was increased 23-fold and 45-fold after 5 and 9 h of IFN-alpha treatment, respectively. The MxA protein was detectable by immunoblotting at 5 hr after IFN treatment and peaked at 17 hr. Concentrations of IFN-alpha as low as 1 U/ml induced detectable amounts of MxA, and expression was maximal at 1 x 10(3) U ml 1. These results confirm that MxA synthesis is induced in human corneal fibroblasts treated with IFN-alpha. PMID- 7540147 TI - Priming and treatment with molgramostim (rhGM-CSF) in adult high-risk acute myeloid leukemia during induction chemotherapy: a prospective, randomized pilot study. AB - In a randomized study of 18 adult patients with high-risk or advanced acute myeloid leukemia (AML) we investigated the effect of supplementing conventional induction chemotherapy with recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF). For comparison, a historical control group of 90 patients treated for de novo AML with conventional chemotherapy during the previous period, 1984-1990, was also analyzed. Before induction chemotherapy, 10 patients were randomized to receiving rhGM-CSF, starting on day 1 to 3 before chemotherapy and continued for a maximum of 21 days after the start of induction treatment. Fatal complications and treatment outcome did not differ between the study groups and historical controls. Nor were there any differences between the groups in terms of hematological toxicity, e.g. time to three-lineage regeneration and need for supportive therapy. However, sequential weekly bone marrow examinations revealed a prolonged reduction of the relative number of myeloid (CD33-positive) marrow cells in the rhGM-CSF treated group. Although the small number of patients studied may not permit a definite conclusion, this randomized study did not demonstrate major beneficial effects of combining rhGM CSF with standard induction chemotherapy in high-risk patients with AML. PMID- 7540148 TI - Increase of monocytes predicts mobilization of peripheral stem and progenitor cells after chemotherapy followed by G-CSF administration. AB - Mobilization of primitive haematopoietic cells to the peripheral blood was studied in 25 patients with haematological malignancies. The optimal level of peripheral stem cells (PSC), defined by their surface expression of CD34, was significantly higher after mobilization with G-CSF, either following chemotherapy or alone (median: 123 x 10(6)/l and 143 x 10(6)/l of CD34+ cells respectively) than without administration of G-CSF subsequent to chemotherapy (median: 27 x 10(6)/l of CD34+ cells). An individual variation in when optimal mobilization of CD34+ cells and myeloid progenitors occurs after chemotherapy and G-CSF administration was noted (median: day 12, range 7-24 days), which makes it difficult to predict when PSC collections in a given patient should be performed. In this study, chemotherapy followed by G-CSF administration resulted in a short lasting (2-3 days) peak appearance of CD34+ cells that could predicted by a 2 fold increase in absolute numbers of monocytes, as compared to the previous day. After the peak level of CD34+ cells in the blood was reached, no further increase in monocytes was seen. The identification of an increase in monocytes, to be used as a predictive variable for when optimal mobilization of PSC will occur in a given patient, may be particularly useful in the individual timing of PSC collections from non-hospitalized patients. PMID- 7540149 TI - Nitric oxide synthase activity in molluscan hemocytes. AB - The hemocytes of the freshwater snail Viviparus ater have nitric oxide synthase (NOS) activity, as demonstrated by [3H]citrulline and nitrite + nitrate formation. The enzyme is NADPH dependent and is competitively inhibited by the mammalian NOS inhibitor NG-monomethyl-L-arginine (Ki = 4.7 microM). The Km for L arginine is 2.5 microM. 70% of the total activity is observed at very low free Ca2+ concentration (3 nM). LPS treatment increased total NOS activity 2.4 fold. The activity is partly present in the non-soluble fraction of hemocytes (24% and 8% in non-stimulated and LPS-stimulated snails, respectively). An antiserum to the C-terminal synthetic pentadecapeptide of the rat cerebellar NOS inhibited the enzyme activity in a concentration-dependent manner. This is the first biochemical demonstration of the existence of NOS activity in molluscan hemocytes, the cells responsible for defence mechanisms. PMID- 7540150 TI - Rev-dependency of expression of human immunodeficiency virus type 1 gag and env genes. AB - Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator. PMID- 7540152 TI - Angiogenesis in pre-eclampsia. AB - OBJECTIVE: To study the angiogenetic activity of decidua basalis from primigravid women with pre-eclampsia. STUDY DESIGN: Fresh fragments of decidua basalis from 10 primigravid women with pre-eclampsia and from 10 healthy, control primigravid women, were grafted onto the chick embryo chorioallantoic membrane (CAM) at the 6th incubation day. Four days later the CAMs were fixed and the angiogenic response of the CAMs was assessed on histologic sections by a planimetric point count method. RESULTS: Decidua from pre-eclamptic pregnancies induced angiogenesis to a greater extent than that from normotensive pregnancies. CONCLUSIONS: Angiogenesis might be stimulated in pre-eclampsia through endothelia and decidua changes induced by the pathological condition which, in turn, would lead to a greater expression of angiogenic factors also present in normal condition. PMID- 7540151 TI - Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel. AB - According to the 'shuttle hypothesis', vasopressin increases the water permeability of renal epithelial cells by exocytotic fusion of vesicles containing the water channel AQP-CD with the apical plasma membrane, whereas withdrawal of vasopressin results in endocytotic uptake of AQP-CD. The proteins involved in the redistribution of AQP-CD have not been identified. With a panel of monoclonal antibodies, we detected Rab3-, Rab5a- and synaptobrevin II-like proteins in a kidney preparation enriched in AQP-CD-containing vesicles. The synaptobrevin II-like proteins is not identical with the ubiquitous cellubrevin. Rab3- and synaptobrevin II- but not Rab5a-like proteins were co-enriched with AQP CD. The data suggest that the proteins involved in hormonal regulation of water permeability in kidney epithelial cells are identical or similar to those involved in regulated exocytosis in secretory cells. PMID- 7540153 TI - Insulin-related growth factor binding protein-1 levels in ovum donation pregnancies. AB - The finding that endometrial maturation may be delayed following hormone replacement therapy has suggested that a generalised endometrial dysfunction may exist in ovum recipients. In order to investigate this suggestion further, circulating levels of IGFBP-1 were measured in samples taken throughout pregnancies conceived either spontaneously or following ovum donation. When analysed at two-weekly intervals, the serum levels of IGFBP-1 in ovum donation pregnancies failed to show the expected peak towards the end of the first trimester and were significantly reduced at week ten (U = 364.5, p = 0.0002) and twelve (U = 138.0, p = 0.0047). For the remainder of pregnancy, circulating IGFBP 1 levels were similar in both groups. The birth weight of children born to the ovum donation group was not significantly different from a normal control group, suggesting that circulating levels of IGFBP-1 in early pregnancy do not reflect local function and that IGFBP-1 does not have an essential function (in relation to birth weight) in early pregnancy. PMID- 7540154 TI - A periodic network of G protein beta gamma subunit coexisting with cytokeratin filament in starfish oocytes. AB - Heterotrimeric G proteins are membrane-bound and carry signals from activated receptors on plasma membranes to cytoplasmic effector enzymes and channels. In starfish oocytes, the beta gamma subunit of G protein mediates 1-methyladenine stimulation of oocyte maturation. In order to investigate the localization of beta gamma subunits in starfish oocytes during oocyte maturation, we raised a monoclonal antibody against the beta subunit. By immunofluorescence microscopy using the antibody, immature oocytes show a network of fibers in the cytoplasm. The staining of fibers is beaded with a periodicity of 0.7 microns. The same staining pattern is obtained by anti-gamma subunit antibody. In addition, the fibers are stained by anti-cytokeratin antibody. These results indicate that the G protein beta gamma subunit coexists with cytokeratin filaments in starfish oocytes. Stimulation of oocyte maturation by 1-methyladenine causes the beta gamma subunit to be disassembled. PMID- 7540155 TI - Steel factor directs melanocyte development in vitro through selective regulation of the number of c-kit+ progenitors. AB - Studies of mice containing mutations in the genes for a receptor tyrosine kinase, c-kit, or its cognate ligand, Steel factor (SLF), establish that this signaling pathway is required for the development of melanocytes from their precursors in the embryonic neural crest (NC). In order to define the mechanism of this requirement, we have labeled cells expressing c-kit with an anti-c-kit antibody (ACK2) and studied the action of SLF on these cells in cultures of murine trunk NC. c-kit positive (c-kit+) cells first appeared after 2 days in culture and were morphologically indistinguishable from other NC cells. These cells subsequently expressed tyrosinase-related protein, an early marker for the melanocyte lineage, and became pigmented in the presence of a phorbol ester. Further, elimination of the c-kit+ population, by incubating the cultures in ACK2, resulted in the ablation of the melanocyte population, but had no effect on the generation of other neural crest derivatives. These data indicate that c-kit+ cells arising from the neural crest are melanocyte progenitors. The addition of SLF to these cultures stimulated an increase in the number of c-kit+ cells, and further studies indicated that SLF acts as both a survival and a proliferative factor for c-kit+ cells. These findings provide a mechanism of regulation of melanocyte development, whereby c-kit is exclusively expressed by melanocyte progenitors within the neural crest precursor population, and subsequent survival and proliferation of these progenitors is regulated by SLF. PMID- 7540156 TI - Free radicals and phagocytic cells. AB - Phagocytes mediate their innate immunological response by releasing products that damage invading microorganisms. These products include proteins such as lysozyme, peroxidases, and elastase as well as reactive oxygen species such as superoxide, hydrogen peroxide, hypohalous acid, and hydroxyl radical. Although it is clear that many phagocytic secretory products have direct cytotoxic potential, understanding is limited of how multiple products interact to generate and modulate the cytotoxic response. This review focuses on recent findings that elucidate the biochemical nature of secretory product interaction in the formation of free radicals, particularly the highly reactive hydroxyl radical. The possible role of these reactions in phagocyte microbicidal activity and inflammatory tissue injury is discussed. PMID- 7540157 TI - Independent suppression of nitric oxide and TNF alpha in the lung of conscious rats by ethanol. AB - Tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) mediate in part the microbicidal response of murine and rodent alveolar macrophages (AM) and recruited neutrophils (PMN) to airborne infections. Ethanol (ETOH) suppresses intrapulmonary TNF alpha and NO release and impairs pulmonary host defense mechanisms. We tested the concept that ETOH down-regulates NO by inhibiting production of TNF alpha. Male rats were given intratracheal (i.t.) saline (PBS), a polyclonal anti-TNF alpha antibody (TNFab) or nonimmune IgG (22 mg/kg, i.m.) 2 h before giving i.t. Escherichia coli endotoxin (LPS) to normal rats or rats pretreated with ETOM (5.5 g/kg, i.p.) 30 min before experimentation. AM and PMN were obtained from the bronchoalveolar lavage fluid (BAL) fluid of rats killed 2 and 4 h after administration of LPS. mRNA for inducible NO synthase (iNOS) and TNF alpha were measured in AM and PMN with competitor equalized RT-PCR techniques. The BAL fluid, AM, and PMN were assayed for TNF alpha and NO2-, and NO3- (RNI) with the L929 bioassay and chemiluminescence, respectively. TNFab abolished LPS-induced increases in TNF alpha but did not suppress the NO content of the BAL fluid or gene expression for iNOS by AM or PMN. ETOH suppressed LPS induced increases in mRNA for iNOS, production of RNI, and BAL fluid TNF alpha but did not affect LPS-induced increases in mRNA for TNF alpha. ETOH-induced attenuation of LPS-induced up-regulation of the iNOS system did not differ in rats pretreated with TNFab or IgG. Thus, ETOH down-regulates iNOS gene expression and RNI production independent of its effects on TNF alpha. Acute ETOH administration suppresses iNOS at the level of transcription and TNF alpha at the level of translation or release of the peptide. PMID- 7540158 TI - [Adenocarcinoma of the cardia: does the extent of gastric resection and lymph node excision influence survival?]. AB - OBJECTIVES: In the curative treatment of the adenocarcinoma of the cardia (AC), the extent of the esogastrectomy and the need for lymph node dissection are still debated. The palliative treatment of AC is now currently non-surgical. The aim of this study was: a) to assess early results of palliative surgery; b) to evaluate the results of curative resection with reference to the influence of the extent of gastrectomy and lymph node dissection on early results and long-term survival. METHODS: From 1979 to 1989, 179 patients (mean age = 60 +/- 12 years) with AC had 45 palliative resections (mean age = 56 +/- 15) and 134 curative resections (mean age = 61 +/- 12). Thirty-eight proximal subtotal esogastrectomies (PSOG) and 7 total esogastrectomies (TOG) were palliative; 72 PSOG and 62 TOG extended to the spleen were curative and associated with lymphadenectomy. RESULTS: The operative mortality rate was 8.9% regardless of the palliative or curative intent of resection. After palliative resection, the mortality rate was 2.6% (1 case out of 38) after PSOG and 42.9% (3 cases out of 7) after OGT (P = 0.01); the median survival was 8 months. After curative resection, the mortality rate was 12.5% (9 cases out of 72) after PSOG and 4.8% (3 cases out of 62) after extended TOG (P = 0.2); actuarial 5-year survival rate was 42% after PSOG and 39% after extended TOG. CONCLUSIONS: These results suggest that: a) palliative PSOG for AC can be performed with a low mortality; b) resection with extensive lymphadenectomy allows substantial survival regardless of the extent of gastrectomy. PMID- 7540159 TI - [Clinical and virological aspects of hepatitis C virus infection in liver transplantation]. PMID- 7540160 TI - Juvenile hormone-dependent LHPI and RNA synthesis in Melanoplus sanguinipes long hyaline tubule: events associated with the "insensitive period". AB - Juvenile hormone (JH) regulation of the synthesis of LHPI, the major secretory protein of the long hyaline tubule in the male accessory reproductive gland (MARG) of Melanoplus sanguinipes, was examined by in vitro radiolabeling and immunoprecipitation. In MARG taken from normal insects JH III immediately stimulates production of immunospecific LHPI. In contrast, JH III does not initially promote synthesis of LHPI in MARG of allatectomized insects. Only after prior exposure to the hormone [for 24 hr when applied in vivo (topically) or 16 hr under in vitro conditions] is LHPI synthesis enhanced by JH III in the MARG of allatectomized insects. These results suggest that in the prolonged absence of JH III the MARG are "switched off," that is, lose their sensitivity to the hormone. Sensitivity is regained during the 24- or 16-hr "lag phase." Use of the translational inhibitor cycloheximide confirmed the existence of the lag phase in JH III-mediated LHPI synthesis. JH III stimulates RNA synthesis in a dose dependent manner in the long hyaline tubule at concentrations < 64 nM. Above this level, RNA synthesis was depressed. Actinomycin D given simultaneously with JH III inhibited RNA synthesis, but not the synthesis of LHPI in the long hyaline tubule. It is suggested that understanding the nature of the lag phase will facilitate clarification of the mechanism of action of JH in the MARG. PMID- 7540161 TI - Differential expression of various T cell surface markers in young and elderly subjects. AB - The purpose of this study was to investigate the expression of lymphocyte markers in the peripheral blood of aged individuals and of young healthy blood donors. Results showed significant changes in the distribution of various T cell subpopulations. The CD3, CD4 and CD8 surface expression was not different in the elderly patients when compared to the young subjects. The number of CD4+ T cells expressing CD45 RO isoform (memory cells) was increased in aged people; CD45 RA+ CD4+ cells (naive cells) were decreased. The expression of the CD45 RB on the CD4+ cells was significantly lower in the elderly when compared to the young subjects. In contrast, the surface expression of CD45 RO and CD45 RB in the CD8+ cells was not statistically different in the young and aged subjects. The proportion of CD8+ cells coexpressing CD57 was significantly increased in the elderly subjects. These results indicate age-dependent alterations of various surface markers of T cells. These observations may be of importance in the investigation of immunoregulatory mechanisms in geriatric patients with infection, autoimmune disease or cancer. PMID- 7540162 TI - Effect of androgens on the germ cell-depleted testes of prenatally irradiated rats. AB - To study the effect of androgens on somatic testicular cells, rats were rendered germ cell depleted by prenatal irradiation (RX). Adult RX rats were treated with a desensitizing dose of a GnRH agonist (GnRHa; Zoladex), combined with an antiandrogen (Nilutamide) to preclude all androgen effects, or combined with testosterone or hCG to restore androgen action. The effect of these treatments for 3 weeks on the weight of testes and accessory sex glands, hormones (LH, FSH, testosterone, inhibin), testicular proteins, the pattern of incorporation of [35S]-methionine into testicular proteins (studied by two dimensional gel electrophoresis) and steady state mRNA levels for transferrin and androgen binding protein (ABP) were evaluated. Combined treatment with GnRHa and antiandrogen virtually eliminated gonadotrophins, androgens and androgen effects. Testicular weight was reduced to 50% of that observed in RX controls. Treatment with GnRHa and testosterone resulted in supraphysiological levels of testosterone and testicular weights comparable to those observed in RX controls. FSH levels in these animals, however, were in the normal range. A low dose of hCG also restored testicular weight in the presence of low concentrations of serum testosterone and low normal levels of FSH. Neither polyacrylamide gel electrophoresis of total testicular proteins nor two dimensional gel electrophoresis of [35S]-methionine labelled proteins revealed striking changes in distinct testicular proteins as a result of androgen withdrawal or androgen treatment. Dot blot hybridization showed a three-fold increase in the mRNA level for ABP (expressed per microgram total RNA) in the Sertoli cell enriched testes of RX rats. This level was barely influenced by androgen withdrawal or androgen administration. The mRNA level for transferrin was increased six-fold in RX rats. A 50% reduction of this level was observed after combined treatment with GnRHa and antiandrogen. It is concluded that, in the germ cell-depleted testis, the major effect of androgens is an overall increase in protein and RNA synthesis rather than a very important and selective increase of a few gene products. PMID- 7540163 TI - Conformational study of a synthetic analogue of alamethicin. Influence of the conformation on ion-channel lifetimes. AB - Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. A synthetic analogue (L2) in which all Aib were replaced by Leu shows a conductance behaviour similar to alamethicin, but channel lifetimes are drastically reduced. Among several hypotheses, a different conformation for L2 might be responsible for this phenomenon by increasing the alpha-helical content (alamethicin presents some 3.0(10)-helical parts) and thus decreasing the length of the transmembrane part. A conformational study of L2 was undertaken using FTIR, CD and NMR spectroscopy, and the secondary structure was compared with alamethicin. These techniques showed an enhanced predominant helical structure as compared to alamethicin. Moreover, the NOE pattern showed an exclusively alpha-helical conformation, resulting in a smaller length of the L2 peptide. This shortening somewhat impedes the complete crossing of the membrane, and could then explain the reduction of its ion-channel lifetimes. PMID- 7540164 TI - A sensitive novel staining agent for the resolved proteins on PAGE. AB - A more sensitive, stable and fast-reacting dye was isolated from the bark of Ratanjot by a simple solvent extraction procedure at high pH. The dye produced sharp, clear distinct stable bands of known proteins when resolved on polyacrylamide gel electrophoresis (PAGE) using sodium dodesyl sulfate (SDS), showing a rapid and strong affinity with protein molecules. Not only did proteins from peanut, separately stained with the new dye and Coomassie Brilliant Blue R 250, produce rapid comparable results, but also the protein bands were easily visualized and were less diffusible with clearer boundaries in the case of the new staining agent. PMID- 7540165 TI - Investigation of mechanisms influencing the accumulation of ultrasmall superparamagnetic iron oxide particles in lymph nodes. AB - RATIONALE AND OBJECTIVES: The current study was designed to investigate the lymph node accumulation mechanisms of dextran-coated ultrasmall superparamagnetic iron oxide particles (USPIO) in rats. METHODS: The iron deposition in the lymph nodes after intravenous administration of USPIO at a dose of 200 mumol Fe/kg was measured in vitro by inductively coupled plasma atomic emission spectrometer in control rats, in rats after depletion of complement C3, after induction of antidextran antibodies, and with prior ligation of afferent lymphatic vessels. The results were correlated with baseline iron concentration and histology. RESULTS: A significant increase in iron concentration but unequal distribution between central and peripheral nodes occurred after administration of USPIO in rats. Much less accumulation was observed in guinea pigs. In rats, the nodal uptake of USPIO was not impaired by depletion of complement C3 using cobra venom factor. The central lymph nodes (mesenteric nodes) showed significantly more accumulation of iron particles in the presence of antidextran antibodies induced by dextran preimmunization. Afferent lymphatic vessel ligation did not effect iron particle accumulation. CONCLUSIONS: Accumulation of USPIO in lymph nodes is largely species-dependent but is independent of afferent lymphatic flow and of C3 complement opsonization in plasma. However, regional distribution of particles can be influenced by preimmunization using dextran. PMID- 7540166 TI - Criticism or caricature? PMID- 7540167 TI - Immunolocalization of desmoglein and intermediate filaments in human oral squamous cell carcinomas. AB - BACKGROUND: Information is limited as to how to predict the malignant behavior of the oral squamous cell carcinomas. The invasive and metastatic phenotype of the carcinoma cells may be related to changes in the expression of desmosomal and cytoskeletal proteins. METHODS: To investigate the expression of desmoglein, cytokeratins, and vimentin, 22 biopsy specimens of oral squamous cell carcinomas were examined by immunohistochemistry. The data were analyzed by morphometry with reference to the cell differentiation, mode of invasion, and cervical lymph node metastasis. RESULTS: The expression of desmoglein, a major desmosomal glycoprotein, was remarkably reduced in the poorly differentiated, highly invasive, and metastatic carcinomas. Although the immunohistochemical expression of cytokeratins in the carcinoma cells was a good marker for the differentiation status of the carcinomas, the level of expression demonstrated no correlation with tumor invasiveness or lymph node metastasis. On the other hand, the vimentin expression was inversely correlated to the differentiation and mode of invasion, and carcinomas in the metastatic group had significantly higher levels of vimentin expression than the nonmetastatic group. CONCLUSION: The results suggest that immunohistochemical examination of desmoglein and vimentin in oral squamous cell carcinomas is valuable in evaluating the malignant behavior of tumors. PMID- 7540168 TI - Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster. AB - The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways. PMID- 7540169 TI - Slow calcium-dependent inactivation of depletion-activated calcium current. Store dependent and -independent mechanisms. AB - Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of approximately 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EFTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by approximately 50%, indicating that store refilling normally contributes significantly to this process. The store independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor generated Ca2+ signals. PMID- 7540170 TI - Strain-specific presence of two TGN38 isoforms and absence of TGN41 in mouse. AB - TGN38 and TGN41 are isoforms of an integral membrane protein that is predominantly localized to the trans-Golgi network (TGN) in rat cells. They have been proposed to form a heterodimer and to be involved in the budding of exocytic transport vesicles from the TGN. By cDNA cloning and analysis using polymerase chain reaction, we found that there were two TGN38 isoforms in a strain of mouse (ICR), whereas other strains examined (BALB/c, DBA/2, and C57BL/6) had only one TGN38. The major difference between the two isoforms was in the number of characteristic octapeptide repeats. Apart from this, there were several nucleotide substitutions between them. The two isoforms appeared to be derived from two distinct genes but not from one gene via alternative splicing. Furthermore, we failed to show the presence of TGN41 in all the strains examined. This result suggests that TGN38 may function as a monomer or a homodimer in mouse cells. PMID- 7540171 TI - Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. AB - The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484 508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387 444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population. PMID- 7540172 TI - Regulation of yeast Golgi glycosylation. Guanosine diphosphatase functions as a homodimer in the membrane. AB - The Golgi lumenal GDPase plays an important role in the mannosylation of proteins and lipids of Saccharomyces cerevisiae by regulating the amount of GDP-mannose available in the Golgi lumen. The enzyme makes available GMP as an antiporter to be coupled with entry of GDP-mannose into the Golgi lumen from the cytosol. Using radiation inactivation and target analysis, we have now determined the functional molecular mass of the GDPase within the Golgi membrane and whether or not the enzyme has functional associations with other Golgi membrane proteins, including mannosyltransferases and the GDP-mannose transporter. The functional size of the GDPase was found to be approximately twice the estimated structural target size of the protein; this strongly suggests that the GDPase protein in situ functions as homodimer and does not require association with other membrane proteins for its function. PMID- 7540173 TI - Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. AB - Nitric oxide synthase (NOS) isoforms are discovered in an increasing variety of cell types with different roles in signaling. The inducible NOS (i.e. iNOS or NOS II) is expressed in cardiac myocytes in response to specific cytokines. Independent of iNOS induction, however, receptor-dependent signaling is modulated by a constitutive nitric oxide (NO) synthase isoform in these cells (Balligand, J. L., Kelly, R.A., Marsden, P.A., Smith, T. W., and Michel, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 347-351). We now show that cardiac myocytes constitutively express the endothelial isoform of NO synthase (ecNOS or NOS III). Transcripts for NOS III were detected by Northern blot in myocyte extracts using as a probe a polymerase chain reaction-generated cDNA amplified with isoform and species-specific primers. In subcellular fractionation experiments, a calcium sensitive NO synthase activity was present primarily in the particulate fraction, coinciding with the distribution of NOS III analyzed by protein immunoblotting. The localization of NOS III within cardiac myocytes was further demonstrated by immunohistochemistry. The functional role of NOS III was explored by analyzing the effects of NOS inhibitors on single myocyte L-type calcium current and contractility. Inhibition of NOS blocked the attenuation by carbamylcholine of the increases in both parameters induced by beta-adrenergic stimulation. We conclude that NO-dependent parasympathetic signaling is mediated by NOS III in cardiac myocytes. PMID- 7540174 TI - Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1). AB - An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mammalian cells. Immunoblot analysis of transfected human embryonic kidney cells (HEK293) and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abundant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39.1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions. PMID- 7540175 TI - Identification of an endothelial cell binding site on kininogen domain D3. AB - High and low molecular mass kininogen, two multidomain plasma proteins, bind to endothelial cells, platelets, and neutrophils in the intravascular compartment. The specific cell attachment site on their common heavy chain is mediated by domain-3, a cystatin-like structure with inhibitory capacity for papain-like proteinases (Jiang, Y., Muller-Esterl, W., and Schmaier, A. H. (1992) J. Biol. Chem. 267, 3712-3717). In this report, the domain-3 cell binding site is determined by an antibody-directed strategy. The epitope of monoclonal antibody HKH15, which binds to domain-3 and blocks the binding of kininogens to platelets and endothelial cells, was mapped using seven synthetic peptides, which span the entire domain-3 sequence. One peptide, LDC27, specifically bound to HKH15. Fine mapping of the epitope of HKH15 revealed that a minimal 13-residue segment in LDC27, named CNA13, is the antibody binding site. LDC27 and CNA13 inhibited biotinylated high molecular mass kininogen binding to endothelial cells with apparent IC50 values of 60.3 +/- 12 and 113.3 +/- 63.7 microM, respectively. Carboxymethylated papain and affinity-purified anti-LDC27 polyclonal antibodies also inhibited the binding of biotinylated high molecular mass kininogen to endothelial cells with an apparent IC50 of 1.04 microM and 59 nM, respectively. Biotinylated LDC27 itself directly bound to endothelial cells, and domain-3 inhibited biotinylated LDC27 binding to human umbilical vein endothelial cells with an IC50 of 41 nM. Using the crystalline structure of cystatin to computer model domain-3, LDC27 and CNA13 were located in the second hairpin loop of the reactive site of cystatin-like proteins (Bode, W., Engh, R., Musil, D., Thiele, U. Huber, R., Karshikov, A., Brzin, J., Kos, J., and Turk, V. (1988) EMBO J. 7, 2593-2599). These results indicate that the major endothelial cell attachment site on kininogen domain-3 is located on its carboxyl-terminal portion and that it overlaps its cysteine protease inhibitory region. PMID- 7540177 TI - Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. AB - Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin. PMID- 7540176 TI - Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. AB - Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S 100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant. PMID- 7540178 TI - Regions of the JAK2 tyrosine kinase required for coupling to the growth hormone receptor. AB - Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2ATD), carboxyl terminus (JAK2CTD), or kinase-like domain (JAK2PKD) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2PKD allowed GH-induced signaling, whereas JAK2ATD and JAK2CTD did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2PKD, and JAK2CTD, but not JAK2ATD. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH2-terminal one fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain. PMID- 7540179 TI - Distribution of leukocyte subtypes in the sheep ovary after laser drilling. AB - The distribution of leukocyte subtypes in the sheep ovary following laser drilling of the ovarian capsule was examined to understand a possible mechanism by which this treatment promotes ovulation in polycystic ovarian syndrome. Ovaries were removed from sheep at different time-points following laparoscopic laser drilling and immunohistochemical detection of leukocyte subtypes, using specific monoclonal antibodies; standard histological staining was performed. Migration of leukocytes into the laser-drilled site was observed as early as 6 h after laser drilling and the total number of leukocytes in the site was found to increase up to the 12th day after surgery. In the earlier period, polymorphonuclear leukocytes were the dominant leukocyte subtypes, while macrophages and lymphocytes were the major cellular components on the 12th day and later. These results show that the tissue changes in the ovary following laser drilling are consistent with a local inflammatory reaction. The prolonged appearance of numerous macrophages following the acute inflammatory phase could lead to the secretion of cytokines and other substances suggested to be important in promoting ovulation. These data indicate that part of the effectiveness of the laser drilling in polycystic ovarian syndrome may be attributable to the secretory products of these leukocytes. PMID- 7540180 TI - Variations in concentrations of the major endometrial secretory proteins (placental protein 14 and insulin-like growth factor binding protein-1) in assisted conception regimes. AB - We have previously shown that placental protein 14 (PP14) concentrations were depressed in two pregnancies that followed down-regulation of the anterior pituitary and exogenous hormone support prior to a frozen-thawed embryo transfer. We now report on a more comprehensive series of pregnancies following this form of treatment, in-vitro fertilization (IVF) and natural cycle frozen-thawed embryo transfer. Serum specimens were analysed for PP14 and insulin-like growth factor binding protein-1 12 days after embryo transfer and at 7 weeks gestation. At 12 days after embryo transfer, the mean serum PP14 concentrations in the IVF and natural cycle were significantly higher in those who conceived than those who did not (82 versus 23 and 107 versus 39 micrograms/l respectively, P < 0.001). Although the mean PP14 concentration in the hormone-supported pregnant patients was higher than in the non-pregnant patients, this had not reached statistical significance 12 days after embryo transfer (49 versus 31 micrograms/l). By 7 weeks gestation the PP14 concentrations in the hormone-supported pregnant patients were significantly higher than in the non-pregnant patients (152 versus 31 micrograms/l, P < 0.001). However, the PP14 concentrations for hormone supported pregnant patients were significantly lower (P < 0.001) than those for pregnant IVF or natural cycle patients at 7 weeks gestation (152, 777 and 660 micrograms/l respectively). The PP14 concentrations in the pregnant patients, although lower than those in IVF and natural cycle pregnancies, were higher than those previously reported in ovarian failure and Turner's syndrome ovum donation cycles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540181 TI - Cell cycle genes c-mos and cyclin-B1 are expressed in a specific pattern in human oocytes and preimplantation embryos. AB - Little is known about the molecular mechanisms governing the development of human oocyte and pre-embryo. We characterized the expression pattern of c-mos, cyclin B1 and beta-actin mRNA in oocytes and granulosa cells from human and monkey, and in human early embryos, using both qualitative and semi-quantitative reverse transcriptase polymerase chain reaction. The proto-oncogene c-mos was expressed in an oocyte-specific manner and no mRNA for c-mos could be detected in the granulosa cells. Similarly, strong expression of cyclin-B1 was seen in the oocytes. In human pre-embryos, the expression of cyclin-B1 and beta-actin increased from the 6-cell stage onwards, indicating active transcription and thus activation of embryonic genome either at or before the 6-cell stage. The expression of c-mos was transient and very little c-mos mRNA could be detected in the human embryos beyond the 6-cell stage. Thus, both its time-specific and site specific expression suggest meiosis-specific functions for the proto-oncogene c mos in human oocytes. As judged by the disappearance of c-mos, the maternal pool of mRNA seems to be degraded towards the 6- to 8-cell stage. The transient expression of c-mos and high levels of cyclin-B1 mRNA suggest that mechanisms similar to those found in lower organisms govern the growth and development of the human oocyte and preimplantation embryo. PMID- 7540182 TI - Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells. AB - Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type 1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5 x 10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 micrograms/cm2) in 0.5 ml McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 ml serum-free medium with or without various hormones or other compounds. PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed. Monkey Sertoli cells were also capable of secreting PAI-1. Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540183 TI - A positive correlation between expression of beta 1-integrin cell adhesion molecules and fertilizing ability of human spermatozoa in vitro. AB - The purpose of this study was to investigate firstly whether beta 1-integrin cell adhesion molecules are expressed by human spermatozoa, and secondly whether there is any relationship between the expression of beta 1-integrin cell adhesion molecules and the fertilizing ability of human spermatozoa in vitro. A total of 50 semen samples were examined. The samples were obtained from the male partners of couples undergoing in-vitro fertilization (IVF) for either unexplained, tubal or male factor infertility. A panel of six monoclonal antibodies against beta 1 integrin cell adhesion molecules and immunohistochemical techniques were used to identify the presence of these molecules on the spermatozoa. The percentage of spermatozoa showing strong immunolabelling with each monoclonal antibody was assessed in each sample. The relationship between these results and the aetiology of infertility and incidence of fertilization was examined. beta 1-Integrins, and primarily the ones with alpha 4-, alpha 5- and alpha 6-chains, were expressed by human spermatozoa. Compared with semen samples from unexplained or male factor infertility patients, samples from tubal infertility patients had a significantly higher (P < 0.05) percentage of spermatozoa expressing adhesion molecules. There was a positive correlation between the expression of alpha 4, alpha 5 and alpha 6 adhesion molecules and the fertilizing ability of spermatozoa. The positive correlation between the presence of certain beta 1-integrin cell adhesion molecules and the fertilizing ability of human spermatozoa suggests that integrins may be putative determinants in egg-sperm recognition and interaction. PMID- 7540184 TI - Substance P, bombesin, and leucine-enkephalin immunoreactivities are restored in the frog tectum after optic nerve regeneration. AB - Extensive regeneration of the optic nerve takes place in adult Amphibia. In this study, we have determined whether one aspect of retinotectal organisation, namely immunoreactive laminae in the retinorecipient layers of the optic tectum, is restored after optic nerve regeneration. To do so, the distributions of substance P, bombesin, and leucine-enkephalin immunoreactivities were examined in the optic tectum of the frog Litoria (Hyla) moorei. Results of a normal series were compared with those at intervals up to 84 days and at 196 days after either unilateral deafferentation or optic nerve crush. In the normal series, distinct neuropeptide immunoreactive laminae were located within the retinorecipient tectal layers. There were two major laminae with substance-P, two with bombesin, and one with leucine-enkephalin immunoreactivities. Additional faint laminae of both substance-P and bombesin immunoreactivity were present in the tectal region that receives input from the visual streak. In addition, labelling of cell bodies and dendrites was seen elsewhere in the tectum. All except one immunoreactive lamina changed after deafferentation. The deeper of those with substance-P immunoreactivity, along with both bombesin laminae, were eventually lost; the lamina with leucine-enkephalin immunoreactivity was halved in intensity. We assume that these laminae are wholely or, in the case of the leucine-enkephalin lamina, partially associated with primary optic input. By contrast, the more superficial lamina with substance-P immunoreactivity remained unchanged and is presumably not directly related to visual input. During nerve regeneration, the intensity of all laminae associated with optic input initially fell as in the deafferentation series but, in the long term, recovered to approximately 80% of normal intensities. We conclude that ganglion cells associated with each of the immunoreactivities tested had successfully regenerated. The reduced intensity of immunoreactivities after regeneration is due presumably in part to the cell loss from the ganglion cell population. Furthermore, we discuss the findings of similar studies for Rana pipiens (Kuljis and Karten [1983] J. Comp. Neurol. 217:239-251 and [1985] 240:1-15) in light of the present findings. We argue that some of the previous observations can be reinterpreted to indicate that regeneration was not limited to ganglion cells associated with substance-P immunoreactivity as first thought. PMID- 7540185 TI - Macrophages, microglia, and astrocytes are rapidly activated after crush injury of the goldfish optic nerve: a light and electron microscopic analysis. AB - Several matrix and adhesion molecules in fish optic nerve, which are constitutively expressed, are increased during axonal regeneration and are primarily associated with nonneuronal cells (W.P. Battisti, Y. Shinar, M. Schwartz, P. Levitt, and M. Murray [1992] J. Neurocytol. 21:557-573). The current study examines the reactions of specific cell types to optic nerve crush and axonal regeneration. The goldfish optic nerve contains macroglia and microglia as well as a population of monocyte-derived cells (granular macrophages) unique to goldfish. Two cell types were OX-42 positive (granular macrophages and microglia), indicating monocyte lineage, each with a distinct morphology and distribution within the nerve. Within hours of the optic nerve crush, the number of OX-42-labeled cell profiles increased near the crush site, remained elevated during the time axons were elongating, and then declined. Microglia, but not granular macrophages, were phagocytically active. Astrocytes are readily identified in the normal optic nerve, but they exhibited marked morphologic changes within hours of injury, which is consistent with the contribution these cells make to the altered environment. Oligodendroglia could not be reliably identified in regenerating optic nerves until myelin was formed. A comparison of the distribution of OX-42-labeled cells with that of transforming growth factor beta-1 (TGF-beta 1) and tenascin suggests that these molecules are expressed by granular macrophages. Tenascin staining may be additionally associated with astrocytes and/or microglia. The rapid response of these nonneuronal cells to injury, their rapid phagocytic activity, and the secretion of growth-promoting factors by these cells likely contributes to the environment that supports robust regeneration by optic axons in the goldfish. PMID- 7540186 TI - Subcloning the MAC-T bovine mammary epithelial cell line: morphology, growth properties, and cytogenetic analysis of clonal cells. AB - The objectives of the present study were 1) to determine the heterogeneity of the MAC-T cell line; 2) to examine whether homogeneous clones could be derived from MAC-T cells; and 3) to examine cell morphology, cytoskeletal characteristics, size, colony-forming ability, growth characteristics, beta-casein production, response to oxytocin, and cytogenetic properties of the clones. Three clonal cells, designated CU-1, CU-2, and CU-3, were derived from MAC-T cells. CU-1 and CU-2 cells were morphologically homogeneous. CU-3 cells were heterogeneous and contained two distinct subtypes. All clones contained cytokeratin 14 and 18. CU-2 and CU-3 cells were 30 and 18% larger, respectively, than CU-1 cells. CU-1 cells did not grow in serum-free medium. Doubling times for MAC-T, CU-2, and CU-3 were 46, 48, and 78 h, respectively, in serum-free medium. MAC-T cells and clones constitutively expressed beta-casein in culture ranging from .1 to .3 micrograms/ml per 24 h. Cytogenetic analyses revealed Robertsonian translocations and isochromosomes in the clonal lines. We conclude that parental MAC-T cells are heterogeneous in morphology, growth, and cytogenetic characteristics. PMID- 7540187 TI - Additives containing bacteria and enzymes for alfalfa silage. AB - First-cutting alfalfa was wilted, harvested from alternate rows, left untreated or treated with additives containing lactic acid bacteria and enzymes (cellulase, amylase, and pectinase), and ensiled in bag silos. Inoculation increased lactic acid bacteria from 5 x 10(4) to 1 x 10(6) cfu/g of forage. Because treatments were bagged consecutively, the DM of treated silages was higher than that of untreated silage. However, after 4 d of ensiling, the pH of treated silage, about 4.3, was lower than that of untreated silage, 4.7, and remained lower throughout the ensiling period. After 177 d of ensiling, total lactate was about 25% higher, and ammonia N was about 40% lower, in treated silage. In addition, NDF and ADF contents were lower in treated than in untreated silage. Between 51 and 177 d of storage, glucose content increased in treated silage, but not in untreated silage, suggesting that some plant cell-wall hydrolysis occurred during prolonged storage. In vitro digestion of NDF did not differ among treatments during early incubation, but the extent of digestion after 36 and 48 h was lower in treated than in untreated silage. The microbial and enzyme silage additives used in this study improved fermentation characteristics and reduced fiber content of silage but decreased the in vitro digestibility of fiber. PMID- 7540188 TI - The effects of neuropeptides (calcitonin gene-related peptide and substance P) on cultured human pulp cells. AB - The sensory neuropeptides substance P and calcitonin gene-related peptide have been implicated in the mediation of pulpal inflammation. A possible role in healing following injury has also been suggested (Byers et al., 1990). This possibility has been investigated by an examination of a direct effect of substance P and calcitonin gene-related peptide in vitro on fibroblast-like cells derived from human dental pulp. Cells were cultured for 48 hr in Dulbecco's modified Eagle medium plus 20% fetal calf serum and antibiotics. Substance P and calcitonin gene-related peptide were added in the range from 10(-12) to 10(-4) mol/L. Fibroblast growth factor was used as a positive control. Effects on cell proliferation were assessed by cell counts (daily for 6 days) and [3H]-thymidine uptake (24 hr after the addition of peptides). An effect on cellular functional activity was measured by [35S]-sulfate incorporation into glycosaminoglycans, in confluent cell cultures. Both substance P and calcitonin gene-related peptide showed concentration-dependent stimulation of cell proliferation. The maximum stimulation of approximately 40% was achieved at substance P and calcitonin gene related peptide concentrations of 10(-6) mol/L, comparable with stimulation by fibroblast growth factor. By contrast, little increase in glycosaminoglycan synthesis by confluent cells could be detected. The direct effect on pulp cells is consistent with a role of the neuropeptides in pulp healing. This is exerted at the level of cell proliferation, rather than functional activity. PMID- 7540189 TI - Studies on nerves of the upper respiratory tract in the ferret and the mink. AB - The central localization of the superior laryngeal nerve (SLN), recurrent laryngeal nerve (RLN) and pharyngeal nerve (PHAR) in the ferret (Mustela putorius furo) and mink (Mustela vision) was determined by neuronal tract tracing techniques. The anterograde transport of horseradish peroxidase (HRP)/wheat germ aggluttinin conjugated HRP mixtures (WGA-HRP) revealed afferent fibres of the SLN projecting ipsilaterally in the tractus solitarius (TS) before terminating in the ipsilateral nucleus of the tractus solitarius (nTS). In both mustelids large concentrations of terminal reaction product were observed in the dorsal/dorsolateral and medial subnuclei of the nTS; however, at levels near obex significant projections of the SLN to the interstitial subnucleus were also observed. Caudal to obex, sparse terminal labelling was identified bilaterally in the nucleus commissuralis (n com). There were no labelled afferent projections of the SLN to the spinal trigeminal complex in either mustelid; neither was afferent terminal reaction product detected in the medulla oblongata following labelling of the RLN (in either the ferret or the mink) or PHAR (in the ferret). Projection of the SLN to areas of the nTS associated with upper airway functions, like swallowing and respiration, suggest a substantial role for this nerve in the initiation and control of airway reflexes. Extensive labelling of the nucleus ambiguus (nA) was revealed following HRP/WGA-HRP application to the PHAR, RLN and SLN and there was some evidence of a topographical arrangement of neuronal cell bodies in the nA, relative to the different nerves, in the ferret. In the case of the SLN, retrogradely labelled cells were also identified in the dorsal motor nucleus of the vagus (DmnX), nTS and reticular formation (rf). Electrical stimulation of the SLN produced periods of apnoea/ataxic breathing in both mustelids. As well as respiratory effects, stimulation of the SLN often caused bradycardia in the mink, but not in the ferret, in which heart rate generally remained unchanged. This difference did not reflect anatomical differences in the central localization of the SLN. PMID- 7540190 TI - Analysis of carotid chemoreceptor responses to substance P analogue in anaesthetized cats. AB - Analogues of Substance P (SP) have been shown to act as SP receptor antagonists in various physiological systems. We have previously shown that the carotid body sensory response to hypoxia could be attenuated by D-Pro2-D-Trp7,9-SP (DPDT-SP) and suggested that SP may be important for chemoreception. In the absence of detailed characterization of the antagonistic effects of DPDT-SP, the role of SP in carotid body chemoreception remains uncertain. The present study was undertaken to analyze the effects of DPDT-SP on carotid body activity in anaesthetized cats (n = 18). Intra-carotid infusion of DPDT-SP antagonized SP induced chemoreceptor stimulation. 90% blockade of SP responses was obtained at infusion rates of 15 micrograms/kg per min of DPDT-SP for 15 min. By contrast, infusions of either saline (controls) or at doses below 10 micrograms/kg per min had no effect on SP responses. The doses that effectively antagonized SP excitation (i.e., 15 micrograms/kg per min) also blocked or markedly attenuated the chemoreceptor responses to hypoxia, without affecting the carotid body stimulation caused by nicotine. The effects of DPDT-SP were associated with significant reduction in baseline activity in normoxia. The antagonistic effects were reversible after terminating the infusion of DPDT-SP. Increasing the dose to 25 micrograms/kg per min, however, abolished the carotid body excitation by any of the stimuli tested (i.e., SP, hypoxia and nicotine), indicating that at higher doses DPDT-SP is non-selective. These results demonstrate that DPDT-SP given in adequate doses to block SP response also attenuates or abolishes the carotid body excitation by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540191 TI - Delayed myelination in infants and young children: radiographic and clinical correlates. AB - Magnetic resonance imaging (MRI) is the best method for assessing myelination in infants and young children. Although delayed myelination is a common neuroradiologic diagnosis, there are few or no data regarding the reliability of this diagnosis or radiographic and clinical findings in cohorts of such patients. We evaluated the cranial MRI scans of 109 patients from age 0 to 36 months, without knowledge of any patient's age or previous clinical or radiologic diagnosis. For each cranial MRI, seven neuroradiologic landmarks were evaluated and established criteria used to assess the state of myelination. We found that in 12 of 109 patients, delayed myelination was misdiagnosed, whereas the diagnosis of delayed myelination was missed in four other patients. Lack of familiarity with the myelination milestones of infancy was the most common reason for a misdiagnosis of delayed myelination. Failure to recognize delayed myelination was due to a failure to appreciate the forceps minor as a landmark. Overall, the diagnosis of delayed myelination was inaccurately applied or missed in 15% of the patients in this series. Of the 14 patients identified as having delayed myelination, 10 had other central nervous system structural abnormalities seen on MRI, most commonly cortical atrophy. Developmental delay was the most common clinical correlate of delayed myelination and was documented in 12 of the 14 patients. To increase the reliability of neuroradiologic assessments in young children, we propose that central nervous system myelin maturation be evaluated and expressed as a myelination age equivalent, analogous to the assessment of pediatric bone age using conventional radiographs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540192 TI - The prognostic significance of moderate hyperamylasemia in the evaluation of the emergency department patient. AB - Hyperamylasemia of greater than five times the upper limit of the normal range (200 IU/L) is highly specific for the diagnosis of pancreatitis, but the meaning of lower values is unclear. The purpose of this study was to evaluate the prognostic significance of amylase values > 200 and < 1000 IU/L. A controlled historical cohort study was conducted to determine whether moderate hyperamylasemia is associated with an increased severity of outcome compared to patients with normal amylase values. Subjects met certain inclusion criteria and had a serum amylase of > 200 and < 1000 IU/L (normal < 200 IU/L). The case group consisted of 44 patients (medium serum amylase = 307.5 IU/L) and resembled the control group of 77 patients (median serum amylase = 117.5 IU/L) with regard to sex distribution and presenting complaint. However, the case group was older, was on more medications, and had a shorter duration of symptoms prior to the ED visit (< 72 h). Analysis of clinically important outcomes revealed that the groups were similar in terms of 6-month mortality, general admission rate, ICU admission rate, and rate of surgical intervention. The proportion of patients who had radiologically or endoscopically documented gastrointestinal pathology was also similar. The results demonstrate that patients with moderate hyperamylasemia (i.e. amylase < 1000 IU/L), notwithstanding the fact that they are older, are on more medications, and have more acute symptomatology, did not have a worse outcome than patients with the same complaints and normal amylases. PMID- 7540193 TI - [Substance P-like immunoreactivity in cerebrospinal fluid in lumbar disc herniation]. AB - Substance P-like immunoreactivity (SP-LI) of the cerebrospinal fluid was measured by radioimmunoassay in 40 patients with lumbar disc herniation (hernia group), and in 10 patients with no low back pain and no leg symptoms (control group). The SP-LI was significantly higher in the hernia group (5.49 +/- 3.01 pg/ml) than in the control group (2.05 +/- 0.52 pg/ml) (p < 0.01). In the hernia group, the SP LI was significantly higher in patients with severe pain in the lower extremities than in those with only mild pain. As the SP-LI was found to be correlated with the severity of pain, it was considered to be a useful index of pain. As for the correlation of SP-LI with the hernia type, the SP-LI was significantly higher in patients with transligamentous extrusion type hernia than in those with protrusion type hernia. This result suggested that the release of substance P was increased with marked compression on the dorsal root. PMID- 7540194 TI - In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase. AB - Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis. PMID- 7540195 TI - Intercellular adhesion molecule 3, a candidate human immunodeficiency virus type 1 co-receptor on lymphoid and monocytoid cells. AB - The CD4 molecule serves as the principal cell surface receptor common to both the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Since binding to CD4 is not sufficient to permit virus entry, HIV 'co-receptors' have been implicated in mediating the fusion of viral and cellular membranes necessary for completing the entry process. In order to identify candidate co-receptor molecules, a panel of monoclonal antibodies (MAbs) directed against adhesion molecules was tested for the ability of the MAbs to inhibit HIV-1-induced cell fusion (syncytium formation) and HIV-1 entry. Certain antibodies directed against CD18, CD11b and CD11c inhibited HIV-1-induced syncytium formation but not entry, in agreement with previous reports. Interestingly, certain antibodies to ICAM-3 (intercellular adhesion molecule 3) (CD50) significantly inhibited HIV-1-specific entry but not syncytium formation using human SupT1 cells. Only one antibody directed against ICAM-3 significantly inhibited HIV-1-induced syncytium formation, entry and infectivity. Our results suggest that certain epitopes of ICAM-3 may be involved in mediating HIV-1-specific entry into lymphoid and monocytoid cells. PMID- 7540197 TI - Binding of lanthanides to cell membranes in the presence of ligands. AB - The effect of a series of ligands on the binding of the lanthanide, europium (Eu), to rabbit intestinal cell membranes was investigated in vitro. When tested as Eu-ligand complexes (ratio of Eu:ligand, 1:2) of intermediate stability (log stability constant, log K1, for the reaction Eu + L = EuL, of about 7-12) such as Eu-citrate and Eu-nitrilotriacetate (NTA), Eu was available for uptake in a soluble form by intestinal brush-border membrane vesicles (BBMV) in phosphate- and bicarbonate-free solutions at pH 7.2. Ligands with lower log K1 did not maintain Eu in solution whilst those of higher affinity did not donate it to membranes. Generally, there was a clear relationship between log K1 of the Eu ligand complex and the binding of Eu to BBMV. This relationship identifies ligands that can effectively donate Eu to vesicles under these conditions. BBMV uptake of Eu was due to binding at two sites. Binding to the diethylenetriaminepentaacetate (DTPA)-sensitive site predominated at 20 degrees C and uptake by the DTPA-insensitive site was enhanced at 37 degrees C. Only trace amounts of the bound Eu appeared to be internalized within the vesicles. In the presence of physiological concentrations of phosphate and bicarbonate in cell culture medium, Eu was precipitated from most complexes (at 1:2 and 1:5 Eu:ligand ratio) except DTPA and albumin. Eu precipitation could be prevented by increasing the ligand:Eu ratio. When isolated hepatocytes in cell culture medium were incubated with EuCl3, about 60% of Eu was bound to the cells; Eu-albumin was not bound by hepatocytes. PMID- 7540196 TI - The disease associations of the antibody response against the Epstein-Barr virus transactivator protein ZEBRA can be separated into different epitopes. AB - The BamHI-Z-encoded Epstein-Barr virus (EBV) replication activator (ZEBRA) is a key mediator of the switch from latency to productive cycle in EBV virus. Antibodies against ZEBRA are a marker of EBV reactivation and are regularly found among patients with infectious mononucleosis (IM) or nasopharyngeal carcinoma (NPC), but are only rarely found among healthy EBV-seropositive donors. In order to define the serologically reactive epitopes in the ZEBRA protein, we synthesized a set of overlapping peptides and tested them for reactivity with serum samples from EBV-seronegative persons, patients with NPC, IM, chronic fatigue syndrome, lymphoma or from healthy donors. Three major EBV-specific epitopes were found. These epitopes were further defined and optimized using substitution or truncation analogues of the peptides. Reactivity with epitope number 22 was found in 63% of NPC patients' sera, with < 2% of healthy donors' sera being positive. Serological reactivity with epitope number 19 was associated with IM (57% positive, 5% healthy donors positive). Serum antibodies against epitope 1 were found among healthy donors, but were significantly elevated among patients with NPC, IM or lymphomas. In conclusion, different serologically reactive epitopes in the ZEBRA protein associate with different EBV-associated diseases. PMID- 7540198 TI - Decisions not to transplant: futility or rationing. AB - Since the 1980s, heart transplantation has become an acceptable treatment therapy for patients with end-stage congestive heart failure. In recent years, the demand for heart transplantation has exceeded the supply of available organs. Potential transplant candidates undergo rigorous screening to determine which patients will be offered transplantation as a treatment option. Heart transplant recipients are selected based on a determination of which patients will experience an improvement in symptomatology, functional class ability, and life expectancy after transplantation. Refusal of transplantation for an individual patient is usually framed in a futility argument: Either transplantation will not benefit the patient or the risks involved in undergoing the transplant are considered to outweigh the benefits. However, futility is an elusive and ambiguous concept. Furthermore, although authors, clinicians, and ethicists argue for the separation of futility and rationing issues, clearly it is not always possible to do so. The purpose of this article is to argue that many decisions to refuse heart transplantation are actually based on the rationing of organs and not on futility. PMID- 7540199 TI - Ethical issues in hospital-based nursing practice. AB - A professional nursing ethic is a living, dynamic set of standards for nurses' professional moral behavior. For a professional ethic to be adequate, it must address the ethical issues perceived as relevant to the profession. The purpose of this descriptive study was to identify the ethical issues hospital-based nurses face in their practice. The authors sought to determine whether there were any differences in these ethical issues over time or according to specialty practice, position, age, educational level, or years in practice. Different groups of nurses (N = 794) were surveyed on five separate occasions over 9 years. Data were analyzed using descriptive and chi 2 statistics. The most frequent ethical issue faced was pain relief and management. Over time, the percentage of nurses who reported being faced with ethical issues related to morphine drips increased. Implications for future research on development of a normative nursing ethic are discussed. PMID- 7540200 TI - Synergistic effect of basic fibroblast growth factor and methylprednisolone on neurological function after experimental spinal cord injury. AB - The authors evaluated the effects of exogenous basic fibroblast growth factor (bFGF) in combination with intravenous methylprednisolone on neurological function and cord angiogenesis in a model of spinal cord injury. Cord injury was produced by extradural clip compression through a T-1 laminectomy. Rats were randomized to one of six groups. Group A was given sham laminectomy without cord injury or treatment. The remaining animals were divided into five groups: untreated injury (Group B); injury treated with methylprednisolone (Group C); combined methylprednisolone and 1 microgram bFGF administered locally at the site of injury (Group D); methylprednisolone and 3 micrograms bFGF (Group E); or methylprednisolone and 3 micrograms heated bFGF (Group F). Groups C through F received treatment 1 hour after cord injury. At 1, 2, 3, and 4 weeks after surgery, neurological function of hindlimbs was assessed by blinded observers using an established multiple test method (toe spread, reflexes to extension, pain, and pressure as well as inclined plane and swim test) with tests graded and results expressed as a combined behavioral score. Animals were killed to study spinal cord angiogenesis in cord samples (2-mm sections proximal and distal to the injury site) by capillary density determination. Behavioral scores over time showed a significant difference among Groups B, C, D, E, and F (p = 0.0044), with Groups E and B maintaining highest and lowest scores, respectively. There was a linear dose effect of bFGF over time (p = 0.0187). At 4 weeks, scores showed a difference among the five groups (p = 0.006), with Group E showing higher scores than any other treatment group (for example, vs. group F: p = 0.035). There was a significant difference among the groups in gray matter capillary density counts: proximal (p = 0.0192) and distal (p = 0.024), whereas white matter capillary counts were similar across treatment groups. These results show: 1) possible synergism exists between methylprednisolone and bFGF, such that combinations of these drugs significantly enhance neurological recovery, 2) bFGF exhibits a dose response effect in function but not in capillary density, and 3) heated, inactivated bFGF is not therapeutically effective. PMID- 7540201 TI - Polynucleotides compensate for impaired T-dependent antibody production induced in C57B1/6 mice by a nucleotide-free diet both in vivo and in vitro, but a mononucleotide-nucleoside mixture is effective only in vivo. AB - Actions of nucleotides on in vitro humoral immune responses were studied in mice fed a nucleotide-free diet, a nucleotide-free diet plus a mononucleotide nucleoside mixture or a nucleotide-free diet plus yeast RNA (polynucleotides). Cultured spleen cells from mice fed a nucleotide-free diet produced fewer numbers of antibody-secreting cells in response to a T-dependent antigen, compared with those from controls fed nucleotide-supplemented diets. Immunoglobulin M concentrations in these supernatants were significantly lower in cultured cells from mice fed the nucleotide-free diet or the nucleotide-free diet plus the mononucleotide/nucleoside mixture compared with concentrations in cells from mice fed the nucleotide-free diet plus RNA. Concanavalin A-potentiated cytokine (interleukin-4 and interleukin-5) production by purified T helper cells was also lower in cultured cells from mice fed a nucleotide-free diet than in those from mice fed nucleotide-supplemented diets. In vivo supplementation with the mononucleotide/nucleoside mixture restored impaired in vitro antibody and concanavalin A-potentiated cytokine production in mice fed a nucleotide-free diet. However, addition of RNA to the culture enhanced antibody production in spleen cells from mice of all diet groups. Supplementing the culture with RNA did not enhance mitogen-potentiated cytokine production. This in vitro action of RNA was retained after the removal of oligonucleotides (molecular weight < 1000), but was reduced by modification of bases and cleavage of phosphodiester bonds of RNA. Thus the in vitro action of RNA is mainly attributed to polynucleotides, indicating their potential role in modulation of local humoral immune responses in the body. PMID- 7540202 TI - Methionine overcomes neural tube defects in rat embryos cultured on sera from laminin-immunized monkeys. AB - Sera from laminin-immunized monkeys were previously found to cause neural tube defects in cultures of whole rat embryos by unknown mechanisms. In the present study, adding L-methionine to either the culture media or to the diets of the monkeys overcame the toxicity of the serum from one of these monkeys (LAM3) but not the other (LAM4). The antilaminin antibody levels and avidities for isolated murine laminin of sera from the two monkeys were comparable. However, when yolk sac homogenates were tested on ELISA, antibodies from LAM4 had greater binding than LAM3, which was further supported by immunoelectron microscopy. These differences in antibody binding were explained by the findings that antibodies from LAM4 recognized more epitopes than LAM3 and that LAM4 recognized specific epitopes not recognized by LAM3. These antibodies caused reductions in the number of microvilli on the cells and the cell sizes of the yolk sac endoderm. In addition, uptake of [14C]methionine, [14C]sucrose and [14C]valine by yolk sacs from embryos cultured on serum from LAM4 was less than that for LAM3. We suggest that the neural tube defects caused by the antilaminin antibodies were a result of reduced nutrient flow caused by the reduction in the number of microvilli on the cells of the yolk sac endoderm. PMID- 7540204 TI - Altered expression of extracellular matrix proteins and integrins in oral lichen planus (OLP). AB - The immunohistochemical distribution of collagens type I, III, IV, V, VI, of undulin and tenascin, and of integrins alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 4, was studied in 14 biopsies of oral lichen planus (OLP), 5 biopsies of orthokeratinized gingiva and 4 biopsies of oral fibrous hyperplasia. The localization of extracellular matrix proteins showed altered expression in OLP when compared to normal oral mucosa, with two principal patterns corresponding to the reticular or atrophic type. Whereas in the reticular type a focal loss of immunoreactivity for collagen types I, III, V, VI and undulin was noted in areas with a cellular infiltrate, in the atrophic variant almost complete loss of immunoreactivity of the subepithelial extracellular matrix was found. There was no clear correlation between the distribution of extracellular matrix proteins and their integrin receptors. The present findings suggest that the autoimmune reaction in OLP might not be primarily targeted to oral keratinocytes but to an unknown antigen in the connective tissue stroma. The changes in the subepithelial extracellular matrix associated with the inflammatory reaction might, especially in the atrophic variant, impair the cross-talk between epithelium and mesenchyme and favour both the loss of barrier function and the development of erosions in the clinical course of the disease. PMID- 7540203 TI - [Study of monocyte chemotactic and activating factor in the human nasal mucosa]. AB - Recently monocyte chemotactic and activating factor (MCAF) has been reported to be a potent histamine releasing factor (HRF) for human basophils. In this study we investigated the localization and biological activity of MCAF in human nasal mucosa. To investigate the localization of MCAF within the nasal mucosa we performed specific immunohistochemical staining of sections of large pieces of the inferior turbinate obtained by conchotomy from patients with nasal allergy and without nasal allergy and compared them. Sections of nasal mucosa from allergic subjects stained with anti-MCAF mAb demonstrated the presence of many MCAF-positive cells in the lamina propria especially around small vessels and excretory gland, but only a few positive cells were found in the nasal mucosa of normal subjects. We cultured allergic nasal mucosa and sinus mucosa from patients with chronic inflammation for 2 days and detected MCAF by western blot analysis of the culture supernatants. One portion separated from sinus mucosa supernatant by column chromatography also released histamine from human basophils. Mean release was 3-4% and the difference from the control was statistically significant (p < 0.05). In the human nasal mucosa it seemed to exist IgE independent histamine releasing system sustained by MCAF producing cells. These results suggested that MCAF is one of the important mediators causing histamine release in the late phase reaction and in protracted inflammation of the nasal mucosa. PMID- 7540206 TI - Expression of estrogen receptor related protein (p29) and estradiol binding in human arthritic synovium. AB - OBJECTIVE: We investigated the expression of estrogen receptors (ER) and the presence of estradiol in human synovial tissues from subjects without arthritis and patients with traumatic arthritis, osteoarthritis, and rheumatoid arthritis. METHODS: Synovial tissues were immunostained using ER-D5 monoclonal antibody (Mab) directed toward an ER related protein (p29), ER-ICA Mab directed toward nuclear ER, and antiestradiol serum. Fluorescent estradiol was used to demonstrate specific estradiol binding to ER. RESULTS: (1) p29 was present predominantly in hyperplastic synovial lining cells and smooth muscles of blood vessels. (2) Exogenous estradiol bound to synoviocytes and sublining macrophage like cells in the inflamed synovium. (3) Endogenous estradiol was also detected in hyperplastic lining cells and macrophage-like cells in arthritic synovium. CONCLUSION: Our study demonstrated the expression of ER in arthritic synovium and suggested that estrogen modulates local inflammation in various joint diseases via synoviocytes as well as sublining macrophage-like cells. PMID- 7540205 TI - Exfoliative cheilitis. AB - Exfoliative cheilitis is an uncommon condition affecting the vermilion zone of the upper, lower or both lips. It is characterized by the continuous production and desquamation of unsightly, thick scales of keratin; when removed, these leave a normal appearing lip beneath. The etiology is unknown, although some cases may be factitious. Attempts at treatment by a wide variety of agents and techniques have been unsuccessful. Three patients with this disease are reported and its relationship to factitious cheilitis and candidal cheilitis is discussed. PMID- 7540207 TI - Novel inhibitors of potassium ion channels on human T lymphocytes. AB - The in vitro biological characterization of a series of 4-(alkylamino)-1,4 dihydroquinolines is reported. These compounds are novel inhibitors of voltage activated n-type potassium ion (K+) channels in human T lymphocytes. This series, identified from random screening, was found to inhibit [125I]charybdotoxin binding to n-type K+ channels with IC50 values ranging from 10(-6) to 10(-8) M. These analogs also inhibit whole cell n-type K+ currents with IC50 values from 10(-5) to 10(-7) M. The preparation of a series of new 4-(alkylamino)-1,4 dihydroquinolines is described. Structure-activity relationships are discussed. Naphthyl analog 7c, the best compound prepared, exhibited > 100-fold selectivity for inhibition of [125I]charybdotoxin binding to n-type K+ channels compared with inhibition of [3H]dofetilide binding to cardiac K+ channels. These compounds represent a potent and selective series of n-type K+ channel inhibitors that have the potential for further development as anti-inflammatory agents. PMID- 7540208 TI - Thiadiazole derivatives: highly potent and specific HIV-1 reverse transcriptase inhibitors. PMID- 7540210 TI - Thermodynamics of RNA unfolding: stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion. AB - RNAs with interesting secondary and tertiary structures tend to melt in several broad and overlapping transitions over a wide temperature range, and it has been consequently difficult to resolve the thermodynamics of individual unfolding steps. In the case that a ligand selectively binds a single folded state of the RNA, it is possible to obtain reliable thermodynamic parameters for both RNA unfolding and RNA-ligand binding simply from the hyperchromicity of RNA denaturation. The analysis procedure involves fitting a three-dimensional surface to absorbance data collected as a function of both temperature and ligand concentration. Analysis of the unfolding of a fragment of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1109) is presented; both an antibiotic (thiostrepton) and ammonium ion specifically stabilize a tertiary structure within this RNA. A consistent set of thermodynamic parameters (delta H and tm) for the first two sequentially linked unfolding transitions is obtained from the experiments, and the binding constants obtained for the two ligands are consistent with other independent measurements. The approach is applicable to a variety of RNAs that specifically bind proteins, antibiotics, ions or other ligands. PMID- 7540209 TI - Design and synthesis of novel inhibitors of HIV-1 reverse transcriptase. AB - A variety of N1-substituted pyrimido[5,4-f]benzo[1,4]thiazepines, 5, designed as conformationally constrained analogs of 1-[(2-hydroxyethoxy)methyl]-6 (phenylthio)thymidine HEPT (1), were synthesized and evaluated for their inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The preparation of these compounds was carried out based on a Mannich-type cyclization of 6-[(2-aminophenyl)thio]uracils followed by alkylation at N1 by a one-pot Vorbruggen reaction. The pyrimidobenzothiazepines were developed to give molecules with IC50 values in the micromolar range, as exemplified by [[(2 ethoxyethyl)oxy]methyl]-pyrimido[5,4- f]benzo[1,4]thiazepine, 25, (IC50 = 0.64 microM), the most active compound of this series. The structural and electronic features of this novel class of HIV-1 RT inhibitors are presented and compared with those of HEPT (1), TIBO (2), and nevirapine (3). PMID- 7540211 TI - Ranking potential binding peptides to MHC molecules by a computational threading approach. AB - In this paper, an approach developed to address the inverse protein folding problem is applied to prediction of potential binding peptides to a specific major histocompatibility complex (MHC) molecule. Overlapping peptides, spanning the entire protein sequence, are threaded through the backbone coordinates of a known peptide fold in the MHC groove, and their interaction energies are evaluated using statistical pairwise contact potentials. With currently available tables for pairwise potentials, promising results are obtained for MHC-peptide complexes where hydrophobic interactions predominate. By ranking the peptides in an ascending order according to their energy values, it is demonstrated that, in most cases, known antigenic peptides are highly ranked. Furthermore, predicted hierarchies are consistent with experimental binding results. Currently, predictions of potential binding peptides to a specific MHC molecule are based on the identification of allele-specific binding motifs. However, it has been demonstrated that these motifs are neither sufficient nor strictly required to ensure binding. The computational procedure presented here succeeds in determining the MHC binding potential of peptides along a protein amino acid sequence, without relying on binding motifs. The proposed scheme may significantly reduce the number of peptides to be tested, identify good binders that do not necessarily show the known allele-specific binding motifs, and identify the best candidates among those with the motifs. In general, when structural information about a protein-peptide complex is available, the current application of the threading approach can be used to screen a large library of peptides for selection of the best binders to the target protein. PMID- 7540212 TI - Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis. AB - Bovine pancreatic trypsin inhibitor (BPTI) serves as an important model system for the examination of almost all aspects of protein structure. Systematic studies of the effects of mutation on the thermodynamic stability of BPTI, however, have been limited by the extreme stability of the protein. A derivative of BPTI containing only the 5-55 disulfide bond, termed [5-55]Ala, has been shown previously to fold into a structure very similar to that of native BPTI and to be a functional trypsin inhibitor. [5-55]Ala undergoes a reversible thermal unfolding transition with a melting temperature of 39 degrees C, and is therefore well suited for stability studies. Using an alanine-scanning mutagenesis approach, we have examined the contribution to stability of each side-chain in the [5-55]Ala derivative of BPTI. These studies demonstrate the importance of the two hydrophobic cores composed largely of clusters of aromatic residues, as well as the internal hydrogen-bonding network, in stabilizing BPTI. Overall, there is a strong relationship between change in buried surface area and stability for both polar and hydrophobic residues, with proportionality constants of 50 and 20 cal/A2, respectively. None of the alanine substitutions substantially stabilized [5-55]Ala. Nonetheless, approximately 60% (28/46) of the alanine mutants were destabilized by less than 10 degrees C, suggesting that a form of BPTI with up to half of its residues being alanine could fold into a stable structure resembling the native one. PMID- 7540213 TI - Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. AB - In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. PMID- 7540214 TI - Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates. AB - The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations. PMID- 7540215 TI - Crystal structure of an RNA dodecamer containing the Escherichia coli Shine Dalgarno sequence. AB - The synthetic dodecameric RNA fragment rUAAGGAGGUGAU resembles a region upstream of the initiation site in prokaryotic mRNAs whereas the pyrimidine-rich complementary strand is identical to the last 12 nucleotides of Escherichia coli 16 S rRNA. The complex thus serves as a model for the Shine-Dalgarno interaction which is required for proper initiation of translation. The crystal structure of rUAAGGAGGUGUA.rAUCACCUCCUUA has been determined at 2.6 A resolution and refined against 2957 1 sigma(F) structure amplitudes to an R-value of 0.195. The unit cell of the triclinic crystals contains two double-stranded RNA molecules. The conformation of the two duplexes is similar, with a root-mean-square deviation of 0.683 A between equivalent atoms, and resembles calf thymus A-DNA as determined by X-ray fiber diffraction methods. Both molecules from continuous helices that penetrate the entire crystal, but the dinucleotide step in between dodecameric duplexes has an unusual geometry with a negative twist angle. The long helices cross over each other in a characteristic manner by inserting the backbone of one molecule into the minor groove of another. These contacts are stabilized by several direct intermolecular hydrogen bonds most of which are mediated by 2' hydroxyl groups of the ribose sugars suggesting a general mode for the interaction between RNA molecules which is different from DNA-DNA interactions. PMID- 7540216 TI - Evolution of tRNA recognition systems and tRNA gene sequences. AB - The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group. PMID- 7540218 TI - Astrocytic reaction after graded spinal cord compression in rats: immunohistochemical studies on glial fibrillary acidic protein and vimentin. AB - The relation between the degree of spinal cord compression and the extent of early posttraumatic reaction of astrocytes was investigated in rats using the blocking-weight technique to induce a spinal cord compression at the level of the Th8-9. Immunohistochemistry was used to detect changes in the expression of glial fibrillary acidic protein (GFAP) and vimentin up to 24 h after injury. A mild compression, which did not cause any measurable neurological deterioration, induced a mild increase of GFAP immunoreactivity at 4 h and a more marked and widespread immunoreactivity at 24 h. The greatest increase of GFAP immunoreactive astrocytes occurred in rats with moderate compression of the cord causing reversible paraparesis and in animals with severe compression leading to paraplegia. The increase of GFAP immunoreactivity was present already 4 h after injury in virtually all the segments investigated (Th5-6-Th11-12) and was most marked at 24 h. Vimentin immunoreactivity of control rats was present in the ependymal cells of the central canal, the leptomeninges, and walls of a few intramedullary vessels. Occasional astrocytes were stained. In rats surviving 24 h after moderate and severe compression vimentin immunoreactivity was increased in the walls of intramedullary blood vessels including capillaries of one rostral and one caudal segment. Many macrophages with immunoreactivity appeared and occasional glial cells with astrocyte shape were stained. This investigation shows that within 24 h after compression of the spinal cord a widespread astrocyte reaction occurs. Even a mild compression that does not produce any signs of motor dysfunction can induce widespread astrocyte alterations in the spinal cord. This astrocyte response is more marked in rats with more severe compression leading to more pronounced neurological deterioration. The increase in vimentin immunoreactivity of blood vessels is more localized and occurs in moderate and severe compression of the cord. PMID- 7540219 TI - [A combined consecutive therapy with fosfomycin and sulbactam/cefoperazone for bacterial infections associated with hematological diseases]. AB - A combination antibacterial therapy with fosfomycin (FOM) and sulbactam/cefoperazone (SBT/CPZ) was applied to 78 patients with severe infections associated with hematological diseases. In this protocol, FOM was followed by SBT/CPZ and each drug was administered for 1 hour intravenously and consecutively. Among 72 evaluable patients, 43 patients had acute leukemia, myeloblastic or lymphoblastic, 22 had malignant lymphoma, 3 had multiple myeloma, and 4 had other hematological diseases as underlying diseases. Bacterial infections diagnosed were sepsis in 21 patients, suspected sepsis in 47, and other infections in 4. The overall efficacy rate of this treatment was 72.2%, and those for individual infections were 66.7% for sepsis, 74.5% for suspected sepsis, and 75.0% for other infectious diseases. Among 22 bacteria separated from patients with sepsis, 78.6% (11/14 strains) were eradicated by this treatment. This protocol was also effective in 57.1% (8/14) of patients whose granulocyte count was less than 100/mm3 during the course of treatment as well as in 83.3% (15/18) of patients with granulocyte count over 500/mm3. There was no difference in effectiveness between those patients to whom G-CSF was administered and those to whom it was not (17/24, 70.8% vs 35/48, 72.9%). As an adverse reaction, a transient increase of GOT and/or GPT was observed in 2 patients (2.8%). The consecutive administration treatment of FOM and SBT/CPZ is thus an effective and safe regimen for the treatment of patients with hematological diseases complicated by severe infections. PMID- 7540221 TI - [Peripheral blood stem cell collection with high dose etoposide]. AB - Peripheral blood stem cells (PBSC) were collected from 24 patients who were treated with high dose etoposide. Studied patients included one with acute lymphoblastic leukemia, 4 with acute myeloid leukemia (AML), 1 with myelodysplastic syndrome, 13 with lymphoma, 1 with malignant histiocytosis, 2 with myeloma, and 4 with testicular tumor. Etoposide was infused at a dose of 500 mg/m2 for 4 days, followed by subcutaneous injection of recombinant human granulocyte-colony stimulating factor from the nadir of leukocyte. PBSC were collected by processing 15-20 liters of blood apheresis in the recovery phase of chemotherapy. In all patients, the number of CFU-GM collected per aphereresis ranged from 0.01 to 59.4 x 10(5)/kg, and more than 5 x 10(5)/kg CFU-GM were collected in 19 of the patients (73%). All leukemia patients treated along with our protocols have remained in complete remission, but one patient with AML relapsed within 1 month after the treatment. Ten lymphoma patients were assessable for antitumor effect, and complete response (CR) was observed in 2, partial response (PR) was 7, and no change (NC) in one patient. Two patients with myeloma were classified to be NC. Three of the 4 patients with testicular tumor were PR, and the other one was NC. Eleven patients subsequently underwent PBSCT. The number of days required to achieve an absolute granulocyte count of 0.5 x 10(9)/l was 7 to 11 days, with a mean of 8.6. PMID- 7540220 TI - [Comparison of low-dose cytosine arabinoside and aclarubicin in combination with granulocyte colony-stimulating factor to intermediate-dose cytosine arabinoside and mitoxantrone with or without etoposide in the treatment of relapsed acute myeloid leukemia]. AB - We used a new chemotherapy regimen for the treatment of 18 consecutive patients with relapsed AML. The regimen consisted of low-dose cytosine arabinoside (Ara C), low-dose aclarubicin and concurrent use of G-CSF (CAG regimen). Fifteen out of 18 patients (83%) achieved complete remission (CR). Median CR duration and median survival were 6 months and 15 months, respectively. These results were similar to those of previously reported salvage therapies for relapsed AML including intensive chemotherapy consisting of intermediate-dose Ara-C and sequential mitoxantrone with or without etoposide (MC/MEC), which we previously adopted. Myelosuppression and non-hematological toxicities were apparently lower and less frequent compared to MC/MEC. The CAG regimen seems promising for the treatment of relapsed AML with its low toxicity contributing to a high quality of life for the patient. PMID- 7540217 TI - Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code. AB - Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl tRNA synthetase and tRNA(ser) in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 aminoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3' end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence. PMID- 7540222 TI - [An enzyme-linked immunosorbent assay determining anti-principal neutralizing determinant antibodies using synthetic peptides deduced from cDNA sequences of HIV-1 V3 loop domain]. AB - The V3 domain, one of the hypervariable regions of gp 120 in HIV-1 possesses principal type-specific neutralizing determinant (PND). The V3 domain has the epitopes for class I major histocompatibility antigens of cytotoxic-T lymphocyte and helper-T-lymphocyte recognition sites, and also has major determinants for cell tropism towards macrophage, microglia, and human brain-derived fibroblast. Therefore, establishment for the assay of anti-PND antibodies in patients with HIV-1 infection appears to be important for the estimation on developing AIDS in these patients. We here describe a simple enzyme-linked immunosorbent assay (ELISA) for the measurement of anti-PND antibodies found in Japanese hemophiliacs. ELISA was performed using synthetic peptides, each 15 amino acid residues deduced from cDNA sequences of seven HIV-1 V3 domain mutants, which include 5 North-American and European strains (IIIB, MN, RF, SC, and WMJ-2) and 2 African strains (Af1. Con and Af2, Con). The specific antibody binding to each peptide was determined after subtraction of the non-specific binding from the total. In 49 control sera from healthy individuals with HIV-1 antibody negative, the absorbance (M +/- 1SD) at 405 nm was -0.002 +/- 0.064. Then, the cut off value was determined to be M + 4SD. In 48 hemophiliac sera with HIV-1 antibody negative, the absorbance was uniformly less than the cut off value. However, among 44 hemophiliac sera with HIV-1 antibody positive, the anti-PND antibodies were detected in the following frequencies; 2 for IIIB, 20 for MN, 1 for RF, 1 for Af1. Con, and 5 for Af2. Con.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540223 TI - [Long term remission following relapsed CNS lymphoma]. AB - A case of NHL which has been in long-term remission following vigorous treatment for CNS relapse with intrathecal administration of Ara-C and focal irradiation to the brain is presented. The patient was a 61-year-old man, who was successfully treated with CHOP followed by MACOP-B for diffuse large cell NHL in 1988. Five months later he was admitted to our hospital because of loss of visual acquity and numbness in the right upper and lower extremities. The presence of lymphoma cells in CSF, abnormal shadow in the left frontal lobe on a cranial CT scan and MRI scan, and positive Ga scintigraphy yielded a diagnosis of CNS relapse of NHL. Twenty one whole brain and additional 1.0Gy to the left frontal lobe of irradiation were performed. Eight days later the left tumor disappeared. Neurological remission was obtained and has continued until now. PMID- 7540225 TI - [Sweet's syndrome in a patient with chronic myeloproliferative disorder during recombinant human granulocyte colony stimulating factor therapy]. AB - A patient with chronic myeloproliferative disorder (CMPD) developed Sweet's syndrome during granulocyte colony-stimulating factor (G-CSF) therapy. A 61-year old man with essential thrombocythemia was treated with busulfan intermittently since April, 1991. In February, 1993, hepatosplenomegaly with leukoerythroblastosis arose and a diagnosis of myelofibrosis with extramedullary hematopoiesis in the spleen was established. For alleviation of left hypochondralgia due to splenomegaly, he received splenic irradiation in September, 1993. Soon after the irradiation, his peripheral blood revealed pancytopenia and then administration of rhG-CSF was begun on the 9th of October, 1993. One week after G-CSF therapy, he became feverish and painful eruptions on the face and the upper extremities appeared and enlarged. Skin biopsy resulted in a diagnosis of Sweet's syndrome. Treatment with oral prednisone, 30 mg daily, was begun, and rapid and significant improvement of the skin lesions was obtained. The pathogenesis of Sweet's syndrome remains obscure, but careful follow up is necessary for patients during G-CSF therapy with respect to development of Sweet's syndrome. PMID- 7540224 TI - [Low titer cold agglutinin disease due to anti-HI antibody and a review of this disease in Japan]. AB - A 79 year-old Japanese man was admitted because of brownish urine and Raynaud phenomenon. Laboratory data showed mild anemia with reticulocytosis, positive direct Coombs test against anti-C3, elevated level of lactate dehydrogenase, undetectable level of serum haptoglobin and low titer of cold agglutinin (1:256 at 4 degrees C), the immunoglobulin subclass of which was IgM. Cold agglutinin disease is usually associated with very high levels of cold agglutinins, the specificity of which is anti-I. This case had low titer cold agglutinin disease due to anti-HI cold agglutinin. PMID- 7540226 TI - [Neuron specific enolase-producing IgD multiple myeloma with high serum amylase activity]. AB - A 68-year-old man who was admitted to our hospital because of general fatigue and anorexia. He was diagnosed as suffering from neuron specific enolase (NSE) producing IgD-lambda type multiple myeloma with high activity of serum amylase, based on the detection of monoclonal IgD-lambda M-protein in the serum and urine, markedly high activities of salivary-type amylase and NSE in the serum, and immunohistochemical evidence of NSE and IgD in the myeloma cells. After two courses of alpha-IFN and VMCP chemotherapy, serum IgD, amylase and NSE decreased to normal levels. These observations indicate that NSE was ectopically produced by myeloma cells. PMID- 7540228 TI - [Detection of platelet activation]. AB - It is important to detect platelet activation in order to predict prethrombotic states. We investigated the method for detection of in vivo platelet activation. First, we compared the assay of CD62P and Glycocalicin (GC) in plasma with plasma beta-thromboglobulin (beta-TG) measurement. Plasma CD62P and GC assay using enzyme-immunoassay (EIA) was inferior to plasma beta-TG assay in precision. The concentration of CD62P and beta-TG in plasma prepared with anticoagulant mixture was lower than that prepared with Na citrate only. The level of plasma CD62P did not correlate with that of beta-TG and GC and platelet counts, although the level of plasma GC correlated with plasma beta-TG levels positively. These studies suggest that GC may be a useful marker of platelet damage or activation, but CD62P may reflect the activation of endothelial cells rather than platelets. Secondly, we assayed the appearance of activation-dependent granular protein on platelet plasma membrane using flow cytometry. Thrombin stimulated increase in surface expression of CD62P, CD63 and Factor XIII a-chain(XIII-a) on platelet membrane in vitro. The expression of CD63 was more sensitive than that of CD62P. The platelets of patients with chronic hemodialysis showed increased in expression of CD63 and XIII-a compared with control subjects. These data suggest that the surface expression of CD63 and XIII-a on platelets may be useful markers of in vivo platelet activation. PMID- 7540227 TI - [RAEB in T with monosomy 7 after treatment of severe aplastic anemia with long term G-CSF]. AB - A 19-year-old male who suffered from severe aplastic anemia had been treated with granulocyte colony stimulating factor (G-CSF) from September 1991. Marked increase of hematopoietic cells in his bone marrow was observed, and maintenance administration of G-CSF was continued. 15 months later, myeloblasts with nuclear abnormality increased, and 22 months later, myeloblasts with chromosomal abnormality presenting 46, XY, -7, +21 exceeded 20%, and aplastic anemia seemed to be transformed into refractory anemia with excess of blasts in transformation (RAEB in T). The usefulness of G-CSF in the treatment of aplastic anemia is now established, but there are some reports questioning the effect of long-term administration, especially transformation to MDS with monosomy 7. Leukemic transformation from aplastic anemia is very complex, but in some cases, long term administration of G-CSF may affect the natural course and may lead to the earlier development of leukemia. PMID- 7540229 TI - [Detection of CD62P (GMP-140) on peripheral blood platelet membrane in various heart diseases]. AB - The protein CD62P expressed on platelet surface membrane was measured by flow cytometry to evaluate its clinical significance. Whole blood contained 0.32% citrate from 64 patients with heart disease and 30 healthy adults were fixed with 0.1% paraformaldehyde. To 50 microliters of the fixed blood, 10 microliters of CD62 PE (Becton Dickinson) was added. After standing for 20 minutes at room temperature, the samples were washed and suspended in 1ml of PBS. Platelets were analyzed with Spectrum III (Ortho) flow cytometer. In healthy control, the percentage of platelets positive for anti-CD62P was 0.16 +/- 0.20 (mean +/- SD)%. Abnormal levels of CD62P were observed in 5 patients with unstable angina, 6 patients with acute myocardial infarction, 1 patient with old myocardial infarction, and 2 patients with mitral stenosis. These results show that activated platelets may play some roles in pathogenesis of heart disease though it is not fully clear yet. PMID- 7540230 TI - ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP 2 cascade. AB - We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of 125I labeled ECM (Matrigel). ECM degradation (release of 125I into the medium) was dependent on exogenous plasminogen, proportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. ECM degradation was completely blocked (P < 0.001) by two plasmin inhibitors, alpha-2-antiplasmin (40 micrograms/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 micrograms/ml), a specific inhibitor of matrix metalloproteinases. Zymography of medium obtained from cells cultured in the absence of plasminogen revealed the presence of latent matrix metalloproteinase-2 (MMP-2) which was converted to a lower molecular weight, active form in the presence of mesangial cells and plasminogen. Northern analysis of poly A+RNA prepared from cultured human mesangial cells revealed mRNA for tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR). The presence of uPA protein in medium obtained from cultured human mesangial cells was demonstrated by Western blotting and ELISA which revealed a large molar excess of PAI-1 (1.2 +/- 0.1 x 10(-9) M) over uPA (1.2 +/- 0.1 x 10(-12) M) and tPA (0.19 +/- 0.04 x 10(-9) M). ECM degradation was reduced by a monoclonal antibody (MAb) against human tPA (-54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated with identical amounts of non-specific monoclonal IgG (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540231 TI - Possible clinical effects of the interaction of hemodialysis membranes with adhesion proteins. AB - In this study data obtained in an in vitro hemodialysis model are related to various parameters measured in patients' plasma during hemodialysis in a clinical crossover study. In vitro, blood from healthy volunteers was dialyzed under standardized conditions using the capillaries Fresenius MTS C (Cuprophane) and Hospal AN 69 (Polyacrylonitrile). Following dialysis, surface bound proteins were eluted with saline, 1 M Tris and 2% SDS, and analyzed by immunoblotting. Depending on the capillary, different protein patterns could be identified in the eluates. Intact forms of adhesion proteins were predominantly (fibrinogen) or exclusively (vitronectin) found in PA eluates. In contrast, low molecular weight products of fibrinogen as well as high molecular weight components containing antithrombin III (AT III) were present in CP eluates. Their presence may reflect fibrinolytic and procoagulatory activity during dialysis with CP capillaries. A microscopic investigation of CP membranes demonstrated fibrin lined platelet conglomerates. In the plasmas of patients dialyzed with CP capillaries high D Dimer concentrations were found. We also noticed that during dialysis with PA membranes less heparin was consumed than during dialysis with CP membranes. Our study showed a good correlation of the observations in the vitro system and the measurements in patient samples. PMID- 7540232 TI - Vascular endothelial growth factor. The trigger for neovascularization in the eye. PMID- 7540233 TI - Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. AB - BACKGROUND: It is generally assumed that unwarranted, excessive neovascularization of the retina and iris is a direct response to a hypoxic retinal environment. Prompted by our previous findings that the potent angiogenic factor, vascular endothelial growth factor (VEGF), is hypoxia-inducible, we used in situ hybridization techniques to examine the thesis that VEGF functions as the link between retinal ischemia and a pathologic, intraocular, angiogenic response. EXPERIMENTAL DESIGN: To gain molecular access to human material representing progressive stages of angiogenic eye diseases, in situ hybridization analysis was carried out on sections of whole globes enucleated at the time of ongoing neovascularization. This methodology identified cells that have up-regulated VEGF expression during natural progression of the indicated diseases. A rabbit model was also used to determine whether experimentally induced retinal ischemia leads to up-regulation of VEGF expression. RESULTS: Proliferation of vascular elements in proliferative diabetic retinopathy and neovascularization of the retina and/or iris secondary to central retinal vein occlusion, retinal detachment, and intraocular tumors were always accompanied by induction of retinal VEGF expression. Furthermore, in each case, expression of VEGF was induced only in a particular layer of the retina (either the outer nuclear layer, the inner nuclear layer, or the ganglion cell layer), matching the zones affected by impaired perfusion. In a rabbit model simulating retinal vein occlusion, elevated levels of VEGF mRNA were detected within a few days of experimental induction of retinal ischemia, exclusively in the ischemic region. CONCLUSIONS: VEGF may be one of the long anticipated factors linking retinal ischemia and intraocular angiogenesis. Irrespective of the cause of retinal ischemia, sustained overproduction of VEGF by ischemic retinal cells may promote retinal and iris neovascularization in a number of neovascular eye diseases. PMID- 7540234 TI - Natural state of mutant p53 protein and heat shock protein 70 in breast cancer tissues. AB - BACKGROUND: Nuclear accumulation of p53 protein in breast cancer cells is generally accepted as a marker for the presence of p53 gene mutation and as an indicator of poor prognosis. Mutation in the p53 gene has been shown to cause conformational change in the p53 protein, which gives rise to a complex with heat shock protein 70 (hsp70). EXPERIMENTAL DESIGN: To clarify whether the p53 protein that accumulates in the nuclei of human cancer cells is bound to hsp70 in vivo, we compared the immunohistochemical localization of both p53 and hsp70 in 65 surgically resected cases of primary breast cancer. In four cases showing both nuclear accumulation of p53 protein and mutation of the p53 gene, identified by single-strand conformation polymorphism and direct sequence analysis, the presence of the p53-hsp70 complex was examined by coimmunoprecipitation assay. In 12 human cancer cell lines showing nuclear accumulation of the p53 protein, the presence of the p53-hsp70 complex was also examined by immunoprecipitation with two anti-p53 Ab after labeling with [35S]methionine. RESULTS: Among the 65 cases, p53 and hsp70 immunoreaction in cancer cell nuclei was detected immunohistochemically in 16 (25%) and 23 (35%) cases, respectively. The localizations of the two proteins were significantly correlated, but there was a discrepancy in the localization patterns in 23 cases. Although the p53-hsp70 complex was detected in all of the four cases in which the p53 mutation was identified, the expression levels of hsp70 detected by coimmunoprecipitation assay were much weaker than those of p53 protein. In all of the 12 cancer cell lines with nuclear accumulation of p53, no distinct 70-kDa bands were immunoprecipitated with the p53 protein. CONCLUSIONS: In human breast cancer tissue, only part of the pool of p53 is bound to hsp70. PMID- 7540235 TI - Establishment of a new human cell line, LI90, exhibiting characteristics of hepatic Ito (fat-storing) cells. AB - BACKGROUND: Thus far, human hepatic Ito (fat-storing) cell lines have not been established. Therefore, functional characteristics of Ito cells have not been fully investigated. EXPERIMENTAL DESIGN: We established a new cell line, LI90, that exhibited characteristics compatible with those of Ito cells from a human hepatic mesenchymal tumor. LI90 cells were examined with phase-contrast microscopy, immunohistochemistry, and cytogenetics, and their vitamin A-storing activity was analyzed. To obtain a marker specific for Ito cells for immunohistochemical analyses, we raised mAb against LI90 cells and clarified the molecular nature of the Ag recognized with the new Ab using an expression cloning approach. RESULTS: LI90 cells showed polygonal shape and had well developed alpha smooth muscle actin filaments in their cytoplasm. In an overconfluent culture condition, LI90 cells aggregated to form a typical hills-and-valleys structure, LI90 cells produced various connective tissue components, such as collagen types I, III, IV, V, and VI, laminin, and fibronectin. In culture media containing vitamin A, LI90 cells formed many fat droplets in their cytoplasm, and fluorescence characteristic of vitamin A was observed in the droplets. By immunizing mice with LI90 cells, three separate mAb specifically reacting with Ito cells in human liver sections were established, and the Ag recognized with all three Ab were identified as extracellular matrix tenascin. CONCLUSIONS: The above-described morphologic and functional characteristics, including vitamin A storage and biosynthesis of tenascin, are compatible with those of Ito cells. Therefore, LI90 cells will be useful for in vitro studies of functions of human Ito cells. PMID- 7540236 TI - Cytokeratin patterns of liver carcinomas induced by diethylnitrosamine in monkeys. AB - BACKGROUND: Although the general conception is that hepatocyte- and bile duct specific cytokeratin (CK) patterns are maintained throughout the neoplastic process, there is an increasing number of reports showing deviation from the rule. CK patterns have been found to be similar across species barriers, so it could be expected that studying the CK patterns of experimentally induced liver tumors may contribute to the elucidation of these controversies. EXPERIMENTAL DESIGN: A CK immunohistochemical study was carried out on histologic sections from hepatocellular carcinomas (HCCs) and preneoplastic lesions from 118 monkeys chronically dosed with diethylnitrosamine (DEN), using mAbs to CK 8, CK 18, CK 7, and CK 19. RESULTS: Normal monkey hepatocytes differed from human hepatocytes by displaying CK 19 in addition to the CK 8/CK 18 pairs, whereas the CK pattern of the bile duct epithelial cells was identical in monkey and human liver. In association with DEN-induced hepatocarcinogenesis, heterogeneity was observed in the CK expression, both in the HCCs and nontumorous parts of the livers. The majority of the HCC cases displayed one of the three CKs normally present in monkey hepatocytes, whereas positive expression of all three CKs (CK 8, CK 18, CK 19) and negative CK 7 was preserved in only 19.5% of the HCC cases. A so-called mixed staining pattern (negative and positive CK staining within the same tumor) was observed in approximately one-fourth of the cases. There was no correlation between the preservation of the hepatocyte-specific CK pattern and the degree of differentiation, tumor grade, or DNA ploidy of the HCCs. In approximately 10% of the primary tumors, CK 7 was expressed in the entire parenchymal cell compartment of the HCC nodules, whereas it was present in a mixed staining pattern in more than half of the cases. In lung metastases, CK 7 was less common, only expressed in approximately one-fourth of the cases. Alterations in the CK patterns were observed in the nonneoplastic hepatocytes of the tumor-bearing monkeys. These included mixed staining patterns in which the CKs appeared as positive and negative regenerating nodules side-by-side. As was observed in the HCCs, CK 7 was more commonly expressed in the nonneoplastic parenchyma in the form of mixed staining pattern than the other three CKs. Moreover, CK 7-negative HCCs occurred more frequently in CK 7-negative livers than in positive livers. Proliferation of CK 7- and CK 19-positive bile ductules and bile ductular-like (oval) cells was frequently associated with the DEN-induced liver injury and hepatocarcinogenesis. CONCLUSIONS: This is the first report on CK expression in monkey liver. The findings show that the hepatocyte specific pattern is not always preserved during DEN-induced hepatocarcinogenesis and may therefore not be useful in differentiating between HCCs and cholangiocarcinomas. PMID- 7540237 TI - Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates. AB - BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA. PMID- 7540238 TI - Dr. Gao Zhuofeng's experiences in the treatment of arrhythmia. PMID- 7540239 TI - [Thoracoscopic excision of the splanchnic nerve. An effective treatment in chronic pancreatic pain]. PMID- 7540240 TI - Immunosuppressive drugs and hypertrophic cardiomyopathy. PMID- 7540241 TI - Immunosuppressive drugs and hypertrophic cardiomyopathy. PMID- 7540242 TI - Immunosuppressive drugs and hypertrophic cardiomyopathy. PMID- 7540243 TI - Effects of the prostacyclin analogue iloprost on patency in small arteries and veins: an experimental study in the rabbit. AB - Arteriotomy/intimectomy and venotomy/intimectomy were performed in the ears of 43 rabbits. Twenty were treated with iloprost given as intravenous doses of 10 micrograms/kg body weight (bw) administered shortly before reperfusion followed by hourly infusions (3 micrograms/kg b w) until 12 hrs after reperfusion. At reperfusion venous and arterial bleeding times were noted. Patency was determined at 15-min intervals until 2 hrs after reperfusion and at 1 and 2 weeks postoperatively. As controls, 23 rabbits were given a single infusion of saline. Compared to controls, iloprost significantly prolonged arterial and venous bleeding times and significantly improved patency 2 hrs after reperfusion. One and two weeks later, however, virtually all vessels were occluded. Administered in this fashion, iloprost does not improve long-term patency in highly traumatized small veins and arteries. PMID- 7540244 TI - Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria? AB - Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenolpyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fructose 1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR. PMID- 7540245 TI - Regulation of gene expression by trans-encoded antisense RNAs. AB - Members of a class of antisense RNAs are encoded by genes that are located at loci other than those of their target genes. Three examples of antisense RNA genes are discussed here. micF is found in Escherichia coli and other bacteria and functions to control outer membrane protein F levels in response to environmental stimuli. dicF is also found in E. coli and is involved in the regulation of cell division. lin-4 is found in the nematode Caenorhabditis elegans and functions during larval development. Nucleotide sequences of at least two of these genes appear to be phylogenetically conserved. The trans-encoded antisense RNAs are small RNAs which display only partial complementarity to their target RNAs. Models for RNA/RNA interactions have been proposed. It is possible that currently known unlinked antisense RNA genes are part of a larger class of heretofore undiscovered regulatory RNA genes. Possible ways of detecting other unlinked antisense RNA genes are discussed. PMID- 7540246 TI - Phage-exclusion enzymes: a bonanza of biochemical and cell biology reagents? AB - Many parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of lambda prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA-protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF-Tu, and in the other case tRNALys.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540247 TI - Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis. AB - Bacillus subtilis contains at least three classes of heat-shock genes regulated by different mechanisms. We are studying class I heat-shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat-shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat-shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39-grpE-dnaK-dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37 degrees C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat shock genes. In the other mutant an internal 0.8 kb Bg/II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16-52 degrees C. At temperatures above 52 degrees C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540249 TI - Blood levels of cytokines in brain-dead patients: relationship with circulating hormones and acute-phase reactants. AB - We hypothesized that increased levels of blood cytokines occur in brain-dead patients, and that these cytokines are responsible for some of the endocrine and/or acute-phase reactant abnormalities found in these patients. We measured blood levels of cytokines, hormones, and acute-phase reactants in 18 brain-dead potential organ donors at the moment of establishing the legal diagnosis of brain death and compared them with levels found in a control group. Although interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) levels were within the normal range, interleukin-6 (IL-6) levels were clearly above the normal range in all patients (median, 1,444 pg/mL; range, 75 to 11,780). In the brain-dead group, total thyroxine (tT4), free T4 (fT4), triiodothyronine (T3), thyrotropin (TSH), dehydroepiandrosterone sulfate (DHEA-S), testosterone, albumin, Zn, and osteocalcin levels were decreased, T3 resin uptake index (T3 RUI), corticotropin (ACTH), cortisol, 11-deoxycortisol (11-DOC), 17 hydroxyprogesterone (17-OHPr), aldosterone, luteinizing hormone, and follicle stimulating hormone levels were normal, and reverse T3 (rT3), renin, and C reactive protein (CRP) levels were increased. Multiple regression analysis demonstrated significant interrelations between IL-6 and T4, T3, testosterone, and CRP. We also studied the evolution of some of these parameters in four patients with severe head injury who finally developed brain death. IL-6 levels on admission to the intensive care unit (ICU) were above the normal limits, as in other patients with cranial trauma, but when the patients developed brain death, there was a pronounced increase in IL-6 levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540248 TI - Increased transendothelial permeation of albumin by high glucose concentration. AB - Vascular endothelial cells, which are polyfunctional, play an important role in the pathogenesis of diabetic complications. The increase in vascular permeability, ie, regulated by vascular endothelial cells, has been reported in patients with diabetes mellitus complicated by angiopathy. To determine the role of hyperglycemia in endothelial cell permeability, we examined the effect of high concentrations of glucose on the permeability of cultured bovine aortic endothelial cells. The permeations of albumin and fluorescein-labeled dextran (FD) across endothelial cell monolayers were increased when cultured with a high concentration of glucose (400 mg/dL). This increased permeation of albumin but not FD was temperature-dependent and was partially reduced by adding 100 mumol/L ponalrestat (ICI 128,436, Statil; ICI, Cheshire, UK), which is an aldose reductase inhibitor. Stimulation or inhibition of Na,K-adenosine triphosphatase (ATPase) in bovine aortic endothelial cells failed to alter their permeability. These findings suggest that high concentrations of glucose enhance transendothelial permeability of albumin in part by activating the polyol pathway, but independently of Na,K-ATPase activity. PMID- 7540250 TI - Epitope analysis using anti-oligosaccharide (G4) monoclonal antibody. AB - A murine monoclonal antibody recognizing (1-->6)-beta-D-glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan. PMID- 7540251 TI - Cellular lipid and fatty acid compositions of Burkholderia pseudomallei strains isolated from human and environment in Viet Nam. AB - Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and -2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1:1. Since snake venom phospholipase A2 digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16:0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2 OH-C16:0), 2-hydroxy hexadecenoic (2-OH-C16:1), 2-hydroxy octadecenoic (2-OH C18:1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH C14:0) and 3-hydroxy palmitic (3-OH-C16:0) acids. There were significant differences in the concentration of hexadecenoic (C16:1), methylene hexadecanoic (C17CPA), octadecenoic (C18:1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins. PMID- 7540253 TI - [Reverse transcriptase and its biological role]. AB - Elements with open-reading translation frames homologous to retroviral RNA dependent DNA polymerases (reverse transcriptases) were found in eukaryotic cell genomes. Expression of endogenous reverse transcriptases in prokaryotic and eukaryotic cells is confirmed directly and indirectly. Evolution and function of reverse transcriptases are analyzed, and their phylogenetic and ontogenetic role is evaluated. PMID- 7540252 TI - Increased expression of adhesion molecules (CD54, CD29 and CD44) on fibroblasts infected with cytomegalovirus. AB - The expression of ICAM-1 (CD54), beta 1 integrin (CD29), and CD44 on cytomegalovirus (CMV)-infected human embryonic fibroblasts (HEF) was analyzed by flow cytometry. The expression of these adhesion molecules increased significantly on CMV-infected HEF, on days 2 and 5 after inoculation, compared to uninfected HEF. However, the expression of these adhesion molecules decreased on herpes simplex virus (HSV)-1 and varicella-zoster virus (VZV)-infected HEF. Increased expression was not observed on HEF treated either with inactivated CMV or with supernatant fluid of CMV-infected cells. The addition of anti-cytokine (TNF-alpha, IL-1 beta, or IFN-gamma) antibodies had no effect on the increase of these adhesion molecules. This suggests that the increase in CD54, CD29, and CD44 on CMV-infected cells requires active virus replication and was not mediated by a soluble factor released from CMV-infected cells. Changes in adhesion molecules on CMV-infected fibroblasts may contribute to inflammation induced by CMV infection. PMID- 7540254 TI - [Conformation of crystalline 3'-nitro-2',3'-dideoxythymidine and properties of its 5'-triphosphate as a terminator substrate of retroviral reverse transcriptase]. AB - The structure of new nucleoside--3'-nitro-2',3'-dideoxythymidine (NIT) possessing moderate anti HIV activity in MT-4 cell culture was investigated by X-ray analysis. These data showed that conformation of NIT in crystal is similar to that of one of crystallographically independent forms of 3'-azido-2',3' dideoxythymidine. 3'-Nitro-2',3'-dideoxythymidine 5'-triphosphate (NITTP) was synthesized and its ability to inhibit human and viral DNA polymerases was studied. NITTP proved to be effective and highly selective terminating substrate of DNA synthesis catalyzed by HIV and AMV reverse transcriptases. Human DNA polymerase alpha as well as DNA polymerase beta (rat liver), terminal deoxynucleotidyltransferase (calf thymus) or HSV-1 and CMV DNA polymerases did not incorporate NITTP into a growing DNA chain. PMID- 7540255 TI - [Study of reverse transcriptase selectivity. Substrate properties of new 2',3' unsaturated acyclic nucleotide analogs]. AB - A new series of nucleotide analogs, (Z)-pyrophosphoryl (phosphonyloxymethyl) but 2-enyl derivatives of pyrimidines and purines, was synthesized. Their substrate and inhibitory properties toward some DNA polymerases and reverse transcriptases were evaluated. They were shown to be selective inhibitors of HIV reverse transcriptase. The structure-substrate properties relationships for nucleotide analogs were discussed. PMID- 7540256 TI - A general route to fingerprint analyses of peptide-antibody interactions using a clustered amino acid peptide library: comparison with a phage display library. AB - We provide a general route to fingerprint analyses of peptide-antibody interactions using a novel chemically synthesized peptide library. A combinatorial clustered amino acid peptide library XO1O2O3O4X (O = one of six amino acid clusters [APG], [DE], [HKR], [NQST], [FYW] and [ILVM]; X = randomized position) bound to a continuous cellulose membrane support was designed to overcome the problem of combinatorial explosion in the synthesis of peptide libraries. This library served as the starting point for the identification and detailed characterization of a TGF alpha peptide epitope recognized by the antibody Tab2. By analysing 1728 hexapeptide mixtures and 1600 single hexapeptides we identified a large number of structurally different high affinity Tab2 binding molecules. Our data provide a detailed picture of the structural basis of this antibody-peptide interaction. In addition to the detection of key amino acids involved in Tab2 binding we observed a high variability of Tab2 binding sequences supporting an induced fit mechanism in antibody-peptide recognition. In contrast, a phage display hexapeptide library led to the detection of only one dominant Tab2 binding peptide. The data obtained also demonstrate the influence of phage proteins on the interaction between the antibody and the displayed peptide. Comparing both approaches with regard to ease of handling and identified sequences, the chemical libraries are clearly favored to study antibody-peptide interactions. PMID- 7540258 TI - Motor conduction parameters in neuropathies associated with anti-MAG antibodies and other types of demyelinating and axonal neuropathies. AB - We measured residual latency (RL), motor conduction velocity (MCV), and terminal latency index (TLI) in 15 patients with neuropathy and anti-MAG or SGPG antibodies and compared these to values obtained in 103 patients with other types of polyneuropathy (PN) and to 57 normal subjects. Ten patients had anti-MAG antibody titers of 25,600 or higher, and 5 had titers between 800 and 12,600. Patients with the highest titers had longer RL, slower MCV and shorter TLI than those with lower titers, acute or chronic inflammatory demyelinating PN, hereditary neuropathy, and metabolic or axonal neuropathy. In contrast F-wave latencies did not contribute to the differentiation between the groups of demyelinating neuropathies. RL and TLI correlated best with anti-MAG antibody titers, whereas there was a poor correlation with anti-SGPG titers suggesting that MAG more than SGPG may be the antigen in PN, and that the distal nerves are affected more than their proximal segments. The RL rather than TLI turned out to be the best variable to classify the demyelinating type of anti-MAG neuropathy. Sural nerve biopsy in 5 of the patients with the highest titer of anti-MAG antibodies showed deposits of IgM and C3 on the myelin sheaths, pronounced demyelination and widening of the myelin lamellae. In 4 of the patients with lower titers demyelination was absent or less pronounced. PMID- 7540257 TI - An idiotype D23-bearing polyspecific, murine anti-DNA monoclonal antibody forms glomerular immune deposits. Pathogenic role of natural autoantibodies? AB - Identification of the immunochemical and structural properties of pathogenic anti DNA antibodies is a major goal for understanding their origins and the mechanisms whereby they induce tissue lesions. Herein, we report on the production of an IgG2a,k anti-DNA monoclonal antibody (4B1), derived from a 12-month-old (NZB x NZW)F1 lupus mouse, able to form glomerular immune deposits. mAb 4B1 is a polyspecific antibody able to bind to ssDNA, actin, tubulin, cardiolipin and to laminin as shown by solid phase ELISAs. Indirect immunofluorescence labeling of HEp-2 cells gave a cytoplasmic staining pattern similar to that obtained with anti-cytoskeleton antibodies. Western blot analysis demonstrated that mAb 4B1 bore idiotype D23, previously shown to be characteristic of natural antibodies derived from normal mice. After injecting the 4B1-secreting hybridoma intraperitoneally into normal (NZW x BALB/c)F1 mice, glomerular immune deposits were observed along the capillary wall. These deposits contained mainly IgM, IgG2a and mAb 4B1, as demonstrated by direct immunofluorescence using a biotinylated-rat anti-4B1 idiotype mAb and kidney eluate analysis. Nucleotide sequence analysis of the VH and VL genes showed that mAb 4B1 is encoded by VH Q52, DSP2.9 and JH2 genes with minimal mutations and by VK8 very similar to the canonic D23 light chain, and JK1 germline genes. No arginine residues were observed in the VH CDR and both chains lacked N-segment addition. Thus, no structural characteristics deduced from the primary structure of mAb 4B1 could explain its pathogenic potential. However, the immunochemical and structural properties suggest that autoantibodies closely related to natural autoantibodies may be pathogenic. PMID- 7540259 TI - Reverse transcriptase-polymerase chain reaction assay for acetylcholinesterase mRNA in rat brain. AB - In order to examine the molecular basis for regional variation in expression of brain acetylcholinesterase (AChE), an assay using reverse transcription and polymerase chain reaction (RT-PCR) was developed to measure steady state levels of AChE mRNA. The amplification method was designed to be specific for templates derived from AChE mRNA and to avoid potential artifacts induced by the presence of genomic DNA. RT-PCR made it possible to assay AChE mRNA in milligram samples from different regions of the rat brain. Determinations by RT-PCR were faster and more sensitive than Northern blotting. The results, including a surprisingly low level of AChE mRNA in the caudate nucleus, agreed with earlier observations by Northern blot and in-situ hybridization. Quantitative RT-PCR may be useful in future studies on developmental and physiological regulation of AChE expression in the brain. PMID- 7540263 TI - Short-term effects of centrally administered galanin on the hyperosmotically stimulated expression of vasopressin in the rat hypothalamus. An in situ hybridization and immunohistochemistry study. AB - Galanin (GAL) is a 29-amino acid peptide that is present in the hypothalamic magnocellular neurons of the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN). Since previous studies revealed a possible role of GAL in the hydro-osmotic regulation, we have investigated the effects of GAL on the vasopressin (AVP) mRNA content in the hypothalamic magnocellular neurons. We demonstrated by in situ hybridization (ISH) and immunohistochemistry that 100 pmol of GAL or GAL fragment (1-16) injected into the lateral ventricle of anesthetized dehydrated male rats induced a rapid (10 min time interval) decrease of AVP mRNA and AVP content in the magnocellular cell bodies of SON and PVN. These effects were quantified on an autoradiographic film and were also obvious at the cellular level by using ISH with a radiolabeled or digoxigenin-labeled oligonucleotide probe. Oxytocin mRNA content is not altered by the same injection either in dehydrated male or lactating female rats. Under the same conditions of lactation, AVP mRNA content is not modified and the i.c.v. injection of GAL has no effect. GAL antagonist (M15) injection suppressed the GAL-induced decrease of AVP mRNA in the dehydrated rats and increased AVP mRNA level in control rats. The efficacy of M15 in antagonizing GAL effects on AVP mRNA suggests the involvement of GAL receptors in the regulation of the vasopressinergic cell bodies. Moreover, endogenous GAL seems to depress the AVP mRNA content of SON and PVN in vivo. The possible origin of endogenous GAL and the mechanisms by which GAL can regulate the AVP mRNA content are also discussed. PMID- 7540262 TI - Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes. AB - The association of [3H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25 degrees C follows a sigmoid path with a Hill coefficient h = 1.25 +/- 0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33 +/- 0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5 +/- 7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5 hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the synaptosomal membranes at 23.4 +/- 1.2 degrees C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8 +/- 0.8 degrees C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10(-10)-10(-4) M). These effects were reversed by naloxone (10(-4) M) which alone was ineffective in changing NO and cGMP production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540260 TI - Further characterization of axonally transported proteoglycans. AB - We report further analysis of axonally transported proteoglycans in soluble and membranous subfractions of goldfish optic tectum. Distribution of transported 35SO4 radioactivity was 35.2% soluble, 63.4% Triton-NaCl extractable and 1.4% unextracted. Proteoglycans isolated on DEAE cellulose were treated with chondroitinase AC or nitrous acid and remaining heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) were sized on Sepharose CL 6B. Kav values and estimated molecular weights were: Soluble CSPG-0.36 (160 kDa), Triton-NaCl extracted CSPG-.031 (200 kDa), Soluble HSPG-0.37 (150 kDa), Triton NaCl extracted HSPG-0.37 (150 kDa). For constituent CS and HS chains the Kav values and estimated molecular weights on CL-6B were: Soluble CS-0.55 (15 kDa), Triton-NaCl extracted CS-0.55 (15 kDa), Soluble HS-0.59 (13 kDa) and Triton-NaCl extracted HS-0.65 (9 kDa). CS was shown to be sulfated exclusively at carbon 4 for both soluble and Triton NaCl extracted fractions. PMID- 7540261 TI - 5-HT, dopamine, norepinephrine, and related metabolites in brain of low alcohol drinking (LAD) rats shift after chronic intra-hippocampal infusion of harman. AB - Harman (1-methyl-beta-carboline) has been shown to induce preference for alcohol in the genetically bred, low alcohol drinking (LAD) rat. This study was undertaken in the LAD rat to determine whether monoamines and their metabolites in different regions of the brain are altered by harman infused chronically into the dorsal hippocampus. For this purpose, a cannula was implanted stereotaxically into the dorsal hippocampus. The cannula was attached to an osmotic minipump implanted subcutaneously within the intrascapular space. The pump was filled with either an artificial cerebrospinal fluid (CSF) vehicle or harman, which was delivered at a rate of 1.0 or 3.0 micrograms/h (i.e., 5.5 or 16.5 nmol/h, respectively) for a period of 14 days. Four days after surgery, a standard preference test for ethyl alcohol was given to the rats over 10 days in which concentrations were increased daily from 3%-30%. The higher concentration of harman infused into the hippocampus elevated the level of serotonin (5-HT), both ipsilateral and contralateral to the hippocampal site of infusion, as well as in the midbrain, frontal cortex, striatum and nucleus accumbens. Similarly, this treatment resulted in a rise in the levels of norepinephrine in the hippocampus and midbrain but decreases in dopamine levels in the pons. The levels of 5 hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were diminished in the pons of rats given 3.0 micrograms/h harman, whereas both concentrations of the beta-carboline reduced the level of homovanillic acid (HVA) in the frontal cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540264 TI - Cytokine-producing cells in experimental autoimmune encephalomyelitis and multiple sclerosis. AB - In both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis, the regulation of the cytokine spectrum and production is likely to have a decisive influence on disease outcome. Studies of cytokines, however, are hampered by the autocrine or paracrine nature of cytokines. Studies of cellular production by messenger RNA detection or cellular secretion are therefore necessary. Collective data suggest that certain cytokines associated with the TH1 phenotype or lymphocytes, such as tumor necrosis factor alpha, lymphotoxin, interleukin-12, and interferon gamma, may promote disease, while cytokines produced by the TH2 subset, such as interleukin-10, may limit disease. In addition, transforming growth factor beta is a putative disease downregulator. Increased knowledge in this field will likely lead to improved therapy for MS patients. PMID- 7540266 TI - Nucleus reticularis thalami input to the anterior thalamic nuclei in the monkey: a light and electron microscopic study. AB - Three anterograde tracers (PHA-L, WGA-HRP, BDA) placed in the most dorsomedial segment of the anterior part of the nucleus reticularis thalami (NRT) in the Rhesus monkey combined with postembedding immunoctochemistry with anti-GABA antibody resulted in double labeling of a distinct population of synaptic boutons in the thalamic nuclei of the anterior group as well as laterodorsal and mediodorsal nuclei. EM analysis of the labeled terminals in the anteroventral and anteromedial parts of the anterior nuclear complex showed that the NRT input is distributed diffusely and in equal proportion to the dendrites of projection and local circuit neurons. PMID- 7540265 TI - Cytokine localization in multiple sclerosis lesions: correlation with adhesion molecule expression and reactive nitrogen species. AB - The relative levels and cellular distribution of proinflammatory and regulatory cytokines have been examined by immunohistochemistry in multiple sclerosis (MS) lesions of differing activity and compared with CNS tissue from other neurologic diseases with an inflammatory or noninflammatory component. Results show widespread distribution of cytokines in association with both perivascular inflammatory cells and glial cells in all types of inflammatory lesions. Although no obvious pattern of proinflammatory versus regulatory cytokines could be determined in MS lesions, proinflammatory cytokines were rarely noted in normal and noninflammatory conditions, whereas regulatory cytokines were readily detectable in the same diseases. The possible relevance of these cytokine patterns to adhesion molecule expression and the presence of reactive nitrogen species is also addressed. PMID- 7540268 TI - Neuroprotection by R(-)-deprenyl and N-2-hexyl-N-methylpropargylamine on DSP-4, a neurotoxin, induced degeneration of noradrenergic neurons in the rat locus coeruleus. AB - N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a neurotoxin and capable of selectively depleting noradrenergic axons and subsequently causing lesions of locus coeruleus (LC) noradrenergic neurons in the rat. R(-)-deprenyl and N-(2 hexyl)-N-methylpropargylamine (2-HxMP) have been previously shown to be quite effective in protecting NA nerve fibers in different brain tissues against DSP-4. The present report reveals the neuroprotective effect of these drugs on the LC noradrenergic cell bodies using a histochemical method. Neurons were quantitatively assessed using Nissl-stained sections. DSP-4 induced a 34% loss of LC perikarya neurons 3 months after a single systemic administration in comparison to control animals. Approximately 90% and 88% of neurons in the same regions survived against DSP-4 induced insult following multiple injections of R( )-deprenyl and 2-HxMP, respectively. The neuroprotective effect towards the LC neurons against DSP-4 is probably due to prevention of retrograde degeneration of NA axons. PMID- 7540267 TI - Lesions to terminals of noradrenergic locus coeruleus neurones do not inhibit opiate withdrawal behaviour in rats. AB - The involvement of neurones of the locus coeruleus (LC) in expression of opiate withdrawal behaviour was tested in morphine-dependent rats using N-2-chloroethyl N-ethyl-2-bromobenzylamine (DSP4), a neurotoxin selective for noradrenergic terminals arising from LC. Lesions were validated by determination of cortical noradrenaline concentrations using gas chromatography-mass spectrometry, inhibition of the post-decapitation hindpaw reflex and dopamine-beta-hydroxylase immunohistochemistry. Lesions did not inhibit the expression of any naloxone precipitated withdrawal signs. These results suggest no involvement of noradrenergic LC neurones in expression of the overt signs of opiate withdrawal, and raise the possibility that previous microinjection and electrolytic lesion studies were confounded by effects on nearby brain regions. PMID- 7540269 TI - Topical spinal administration of a nitric oxide synthase inhibitor prevents the hyper-reflexia associated with a rat model of persistent visceral pain. AB - The effects of a neuronal selective nitric oxide synthase (NOS) inhibitor, L-Ng nitro arginine p-nitroanilide (L-Napna), upon the hyper-reflexia of a rat model of persistent visceral pain was investigated. A baseline cystometrogram (CMG) was performed by measuring intravesical pressure during vesical inflation. L-Napna (125-1000 micrograms) or vehicle (control) was then administered topically onto the exposed spinal cord, followed by another CMG. The bladder was then inflamed with turpentine and a final CMG performed. Neither L-Napna nor vehicle affected vesical reflexes in the absence of inflammation. However, following inflammation, a vesical hyper-reflexia was demonstrated in the control animals, which was prevented by L-Napna. Therefore, spinal NOS does not have a role in the generation of normal bladder reflexes, however it does modulate them during vesical inflammation. PMID- 7540270 TI - Galanin is contained in GABAergic neurons in the rat spinal dorsal horn. AB - In order to determine which types of neuron in laminae I-III of the rat spinal dorsal horn contain the peptide galanin, pre-embedding immunocytochemistry with antiserum to galanin was combined with post-embedding detection of GABA- and glycine-like immunoreactivities. Sixty-eight galanin immunoreactive neurons in laminae I-III selected from four rats were examined, and in each case semi-thin sections through the cell body were tested with a monoclonal antibody to GABA and an antiserum to glycine. All of the 68 galanin-immunoreactive neurons tested were GABA-immunoreactive, while only one of them (in lamina III) was glycine immunoreactive. This suggests that galanin is contained in inhibitory interneurons, and that (like enkephalin, neuropeptide Y and thyrotropin-releasing hormone) it is mainly restricted to GABAergic neurons which do not use glycine as a co-transmitter. PMID- 7540271 TI - Contribution of L-type Ca2+ channels to long-term enhancement of high K(+)-evoked release of dopamine from rat striatal slices. AB - Pre-treatment with BAY K 8644, an L-type voltage-gated Ca2+ channels agonist, produced long-term enhancement (LTE) of over 2 h of high K(+)-evoked dopamine release from rat striatal slices. Exposure to BAY K 8644 under Ca(2+)-free conditions did not enhance this release. Thus, a transient activation of L-type voltage-gated Ca2+ channels followed by Ca2+ entry can trigger LTE of the evoked DA release from striatal tissues. This type of LTE in the striatum underlines the importance of presynaptic mechanisms, including L-type voltage-gated Ca2+ channels of 'long-term potentiation' expression. PMID- 7540272 TI - Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin. AB - Rat galanin inhibits basal as well as forskolin-stimulated adenylate cyclase activity in rat ventral and dorsal hippocampus. The inhibition of adenylate cyclase activity, both basal and forskolin-stimulated, is characterised by IC50 values being 250-fold lower in ventral hippocampus (IC50 = 1.1 nM) compared to the dorsal hippocampus (IC50 = 270 nM). The maximal inhibition of basal and forskolin-stimulated adenylate cyclase activity in both ventral and dorsal hippocampus in the presence of 10 microM rat galanin is 34-45%. The analysis of the binding data obtained with 125I-labelled Tyr26-porcine galanin as a tracer reveals similar binding constants for rat galanin in both ventral and dorsal hippocampus with 4.8-fold higher concentration of galanin receptors in the ventral hippocampus. Putative galanin receptor subtype differences between the ventral and dorsal hippocampus have been noted by Hedlund et al. (Eur. J. Pharmacol., 224 (1992) 203-205). This study yields further confirmation for the existence of different galanin receptor subtypes or for differential coupling of galanin receptors to the adenylate cyclase in the dorsal versus ventral hippohampus. PMID- 7540273 TI - Arachidonic acid may mediate the galanin-induced hyperpolarization in parasympathetic neurons from Necturus maculosus. AB - The effects of arachidonic acid (AA) and compounds that inhibit intracellular signalling pathways on membrane potential and galanin-induced hyperpolarizations were investigated in parasympathetic neurons from Necturus maculosus. Treatment for 10-90 min with 10-20 microM 4-bromophenacylbromide or 10 microM cyclosporin A caused a progressive decrease in the amplitude of galanin-induced hyperpolarizations without any change in resting membrane potential. The galanin induced hyperpolarization was not altered following a 10-120 min treatment with the protein kinase inhibitor H-7. These results indicated that phospholipase A2 activation, but not protein kinase activation, may be required for the galanin induced hyperpolarization. Arachidonic acid (20-100 microM) caused a concentration-dependent membrane hyperpolarization of the parasympathetic neurons and a decrease in the amplitude of the galanin-induced hyperpolarization. These data indicate that phospholipase A2-catalyzed liberation of AA may be involved in the galanin-induced membrane hyperpolarization observed in mudpuppy parasympathetic neurons. PMID- 7540274 TI - Carbon fibre micro-electrodes for concomitant in vivo electrophysiological and voltammetric measurements: no reciprocal influences. AB - Differential pulse voltammetry and more recently cyclic voltammetry have been successfully used to monitor basal levels of endogenous chemicals by means of treated carbon fibre microbiosensors inserted in specific brain regions. In this study, feasibility of concomitant in vivo recordings of stable electrophysiological signals and basal ascorbate, catecholaminergic and indolaminergic voltammetric peaks at the same cerebral site by means of a single electrically treated carbon fibre micro electrode (microbiosensor) is presented. The results indicate that these two independent techniques can be combined in vivo at a single electrode, and that voltammetric measurements of unstimulated levels of extracellular compounds do not alter concomitant basal cell firing for a period long enough (more than 6 h) to allow pharmacological manipulations. PMID- 7540275 TI - Intracellular injection of inositol 1,4,5-trisphosphate increases a conductance in membranes of turtle vomeronasal receptor neurons in the slice preparation. AB - Inositol 1,4,5-trisphosphate (IP3) was injected into turtle vomeronasal receptor neurons in the slice preparation under a whole-cell patch clamp, and the evoked current was measured. Application of 0.1 mM IP3 evoked a prolonged, inward current (52 of 98 neurons) with an average peak amplitude of 89.9 +/- 10.9 pA. The reversal potential of the response induced by IP3 was estimated to be -32.3 +/- 1.5 mV (6 neurons). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP3 evoked inward current to 18.0 +/- 4.6 pA (5 neurons). This is the first study to demonstrate that the membranes of the turtle vomeronasal neurons carry IP3-activated conductance. PMID- 7540276 TI - Advent of maternal serum markers for Down syndrome screening. AB - Trisomy 21, or Down syndrome, is the most common serious autosomal chromosome aberration in which affected individuals survive beyond infancy. The association between advancing maternal age and increased risk of trisomy 21 is well known, and pregnant women older than 35 years at delivery are routinely offered invasive prenatal diagnostic testing. More recently, the use of maternal serum markers in the second trimester of pregnancy to predict the risk of trisomy 21 for women under the age of 35 has received intensive analysis. Maternal serum alpha fetoprotein (MSAFP) was the first of these markers to be identified, and an inverse correlation between MSAFP level and risk of trisomy 21 was noted. A second marker, unconjugated estriol (uE3), has also been studied, and a correlation between low uE3 and trisomy 21 has been demonstrated, with a high level of correlation between AFP and uE3. The addition of uE3 to the screening protocol has not consistently improved detection rates, possibly because of its high correlation with AFP. A strong association of human chorionic gonadotropin (hCG) and Down syndrome was reported. This analyte is the most sensitive one in use today, although controversy exists regarding the best form of the analyte to use for trisomy 21 prediction. Several groups of investigators advocate measurement of total hCG, while others feel that measurement of the free-beta subunit of the molecule offers greater detection ability. The maximum detection rate that has been reported is 80 percent with a 5 percent false-positive rate using a combination of MSAFP, free-beta hCG, and maternal age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540277 TI - Association between the melanoma-inducing receptor tyrosine kinase Xmrk and src family tyrosine kinases in Xiphophorus. AB - Melanoma formation in the fish Xiphophorus is an in vivo model for the function of receptor tyrosine kinases (RTKs) in tumor development. The overexpression and high activity of the RTK Xmrk (Xiphophorus melanoma receptor kinase) is responsible for the formation of hereditary malignant melanoma in this fish, but the mechanism by which Xmrk signals cell proliferation has not been elucidated. Remarkably, in earlier experiments an elevated level of a pp60c-src related kinase activity was found in the melanomas. In order to evaluate the significance of src family SH2 domain interactions in the intracellular signalling of Xmrk, we determined its relative binding affinity to the ubiquitous general RTK substrate, PLC gamma, and to the Xiphophorus cytoplasmic kinases Xsrc, Xfyn and Xyes. Recombinant Xmrk purified from baculovirus infected Sf9 cells bound with high affinity to the SH2 domains of PLC gamma and Xfyn in vitro. The affinity of Xmrk to Xsrc and Xyes SH2 domains was 5- to 10-fold lower. Coprecipitation experiments revealed that the Xmrk/Xfyn interaction occurred also in melanoma cells. Moreover, stimulation of the Xmrk kinase activity was paralleled by an increase in Xfyn activity. These results suggest that in malignant melanoma of Xiphophorus the highly activated Xmrk may enhance the activity of Xfyn through direct interaction and that both kinases are linked in a signal transduction pathway. PMID- 7540278 TI - Bcl-x and Bcl-2 inhibit TNF and Fas-induced apoptosis and activation of phospholipase A2 in breast carcinoma cells. AB - Tumor necrosis factor (TNF) induces cel death in several tumor cell lines by undefined mechanisms. Using a cDNA expression cloning strategy we identified two cDNAs that completely inhibit the TNF-induced death pathway in MCF7 breast carcinoma cells. These cDNAs encoded for Bcl-2 and Bcl-x. To compare the cytotoxic signal transduction pathway induced by the TNF receptor versus that induced by Fas, we transfected MCF7 cells with a Fas expression construct. The resulting cell line, MCF-Fas, was highly sensitive to cytotoxicity induced by TNF or anti-Fas. Expression of either bcl-2 or bcl-x in these cells rendered them completely resistant to lysis induced by either TNF or Fas. Interestingly, exposure of MCF-Fas cells to anti-Fas or TNF induced activation of phospholipase A2 (PLA2), while only TNF activated NF-kappa B. Activation of PLA2 was completely blocked whereas activation of NF-kappa B was unaffected by overexpression of either bcl-x or bcl-2. Moreover, PLA2-inhibitors, quinacrine and dexamethasone, partially inhibited cytotoxicity induced by either TNF or anti-Fas. These data suggest an involvement of PLA2 in both TNF- and Fas-mediated cytotoxicity and a novel mechanism of action for bcl-2 and bcl-x, i.e. inhibition of arachidonic acid metabolism, by which they may, in addition of apoptosis, modulate inflammation. PMID- 7540279 TI - Increase in plasma calcitonin gene-related peptide from the extracerebral circulation during nitroglycerin-induced cluster headache attack. AB - In this study, changes in plasma levels of calcitonin gene-related peptide (CGRP) and substance P (SP) during a spontaneous-like cluster headache attack provoked by nitroglycerin were evaluated. Peptide variations after spontaneous or sumatriptan-induced remission were also assessed. Blood was collected from the external jugular vein homolateral to the pain side of 30 male cluster headache patients; 18 men were in an active and 12 in a remission one. Plasma levels of CGRP and SP were determined using sensitive radioimmunoassays for each peptide. CGRP-like immunoreactivity (CGRP-LI) was found to be augmented in patients in an active period and became elevated further at the peak of the provoked attack. A complete reversal occurred both after spontaneous and sumatriptan-induced remission. On the contrary, nitroglycerin neither provoked a cluster headache attack nor altered CGRP-LI in the patients in a remission period. The augmented levels of CGRP-LI measured before and after nitroglycerin administration, when the provoked attack reached the maximum intensity, suggest an activation of the trigeminovascular system during the active period of cluster headache. Moreover, the clinical and biochemical actions showed by sumatriptan stress the involvement of serotonin in cluster headache mechanisms. PMID- 7540280 TI - The neurokinin-1 receptor antagonist, sendide, exhibits antinociceptive activity in the formalin test. AB - Sendide is a selective and extremely potent antagonist of neurokinin-1 (NK1) receptors in the mouse spinal cord. The antinociceptive activities of sendide, an antagonist of NK1 receptors, and its analogue, [D-Trp7]sendide have been examined after intrathecal (i.t.) administrations in the mouse paw formalin test. Intrathecal administration of sendide (in pmol) reduced both the early and late phases of the formalin-induced licking response. [D-Trp7]sendide also produced a significant antinociceptive response with less potent activity than sendide. Even highest doses (4000 pmol sendide and 8000 pmol [D-Trp7]sendide) examined, there was no motor paralysis of the hindlimbs. Intrathecal morphine inhibited both the early and late phases of the formalin-induced licking response in a dose dependent manner. The results indicate that the antinociceptive effects of sendide and [D-Trp7]sendide may be mediated at NK1 receptors in the formalin induced nociception. PMID- 7540282 TI - The proportions of G gamma- and A gamma-globins in the fetal hemoglobin synthesized in preterm and term infants. AB - The change in G gamma to A gamma ratio in relation to the switchover of fetal HB (HbF) to adult Hb (alpha 2 beta 2) synthesis has not been well defined. The gamma globins of HbF (alpha 2 gamma 2) have either glycine (G gamma) or alanine (A gamma) at position 136. During fetal life the G gamma makes up 70% of the gamma globins, although they are 40% of the small amounts of HbF in the adult. To further the understanding of this switchover, globin chain synthesis was determined sequentially in eight preterm and 20 full-term infants postnatally. To complete the study, single analysis was also carried out in eight term infants at birth and six preterm infants at the postconceptional age equivalent to term. Blood samples were incubated in an amino acid mixture containing 3H-leucine and subjected to reversed-phase liquid chromatography. The results demonstrated that the fetal proportions of G gamma to A gamma are rigidly controlled according to postconceptional age and not affected by postnatal age after preterm birth. During the early postconceptional age switchover from HbF to adult Hb synthesis, an equal repression of the G gamma and A gamma chains was found. However, based on the values obtained from the term infants, after a postconceptional age of 44 wk, the levels of G gamma to total gamma synthesis begin to decrease and become more variable. PMID- 7540281 TI - Effects of maternal protein malnutrition on fetal growth, plasma insulin-like growth factors, insulin-like growth factor binding proteins, and liver insulin like growth factor gene expression in the rat. AB - We examined the effects of maternal dietary protein restriction on fetal growth and expression of IGF-I and -II, and IGF-binding proteins (IGFBP). We sought to dissociate the respective effects of maternal protein versus calorie restriction on growth indices and IGF synthesis by the neonates of protein-restricted dams. Pregnant Wistar rats (six to eight per group) fed a low (5%) protein diet throughout gestation had impaired body weight gain compared with controls fed a normal (20%) protein diet (by 45%, p < 0.001). Their serum and liver IGF-I concentrations and liver IGF-I mRNA concentrations were also reduced by 60, 80, and 50%, respectively. Serum IGFBP-3 was reduced by 60% in protein-restricted dams within 1 to 2 h after delivery (p < 0.001 versus controls), although IGFBP 1, -2, and -4 were not significantly affected by the dietary protein intake. In pups of protein-restricted dams, the mean body and liver weight at birth was 15 20% less than that observed in the progeny from normal protein-fed dams (p < 0.01). Their plasma and liver IGF-I concentrations were 30 and 60% lower, respectively, whereas liver IGF-I mRNA abundance was reduced by 50% (p < 0.01). In contrast, neonatal plasma IGF-II and liver IGF-II mRNA concentrations were not significantly affected by the maternal protein malnutrition. Also, the plasma levels of IGFBP were not altered in the growth-retarded pups. Maternal protein restriction did not affect fetal and placental growth, plasma and liver IGF-I levels, and liver IGF-I mRNA abundance in 20-d-old fetuses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540283 TI - alpha 2-Macroglobulin remains as important as antithrombin III for thrombin regulation in cord plasma in the presence of endothelial cell surfaces. AB - Infants and children rarely develop thrombotic complications compared with adults, suggesting that there are protective mechanisms in place for the young. Because endothelial cell surfaces regulate thrombin formation and inhibition, we compared thrombin regulation by human umbilical vein endothelial cell surfaces exposed to defibrinated cord and adult plasmas. After activation by either 10% activated partial thromboplastin reagent (strong activator) or coagulant phospholipids (weak activator) the following were measured: free thrombin, thrombin bound to antithrombin III (ATIII), heparin cofactor II, alpha 2 macroglobulin (alpha 2M), and prothrombin concentration. Free thrombin activity was expressed as remaining activity, after subtraction of thrombin-alpha 2M activity. After 10% activated partial thromboplastin reagent, 100% of prothrombin was consumed and significant amounts of thrombin generated by 2 min. Cord plasma generated significantly less thrombin than adult plasma, reflecting the lower initial plasma concentration of prothrombin. correspondingly, concentrations of thrombin inhibitor complexes were significantly greater in adult plasma than in cord plasma. After coagulant phospholipids, 50% of prothrombin was consumed and negligible thrombin activity measured for both adult and cord plasma. Similar amounts of thrombin inhibitor complexes were formed. ATIII was the predominant inhibitor of thrombin in adult plasma, whereas alpha 2M was as important as ATIII in cord plasma for both activators. When cord plasma concentrations of ATIII were increased to adult values, the proportion complexed to alpha 2M decreased. We conclude that on human umbilical vein endothelial cells, the capacity to generate thrombin is decreased in adult and cord plasmas.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540285 TI - RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColE1-type plasmids in Escherichia coli. AB - RNAII, an RNA species encoded by ColE1-type plasmids, serves as a primer for plasmid DNA replication. Previous work has shown that overproduction of RNAII transcribed by Escherichia coli RNA polymerase results in elevated plasmid copy number. To produce a plasmid in which the elevation of its copy number is inducible, we placed transcription of RNAII under the control of a bacteriophage T7 late promoter regulated by IPTG-inducible T7 RNA polymerase. During induction of T7 RNA polymerase by IPTG, we found that RNAII was overexpressed, but that, surprisingly, this increase in RNAII did not result in elevation of plasmid copy number. These results suggest that RNAII transcribed by T7 RNA polymerase does not function as a primer for plasmid DNA replication. Since RNAII function requires folding of its multiple stem-loop structures in a precise conformation and folding of RNAII can be influenced by its rate of transcription, the extremely rapid rate of travel of the T7 RNA polymerase may preclude proper folding of RNAII during its elongation. PMID- 7540286 TI - Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices. AB - Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied. PMID- 7540288 TI - Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2. AB - We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy 3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands. PMID- 7540287 TI - RNA polymerase III promoter and terminator elements affect Alu RNA expression. AB - Promoter elements derived from the 7SL RNA gene stimulate RNA polymerase III (Pol III) directed Alu transcription in vitro. These elements also stimulate expression of Alus transfected into 293 cells, but transcripts from these same constructs are undetectable in HeLa cells. A terminator resembling the terminator for the 7SL RNA gene has no effect on in vitro Alu template activity, but increases expression in vivo in a position independent manner. Alu transcripts generated from templates with and without this terminator have identical half lives, indicating that this terminator stimulates expression by increasing template activity. Together, these results show that Alu expression may be regulated at multiple levels and can respond to cis-acting elements. This new found ability to express Alu transcripts by transient transfection provides an opportunity to monitor their post-transcriptional fate. Primary Alu transcripts are not extensively adenylated or deadenylated following transcription, but are short-lived compared to 118 nt scAlu RNA. In addition to Alu RNA, transfected templates encode scAlu RNA, but very high levels of Alu RNA expression does not increase the abundance of scAluRNA. ScAluRNA is not merely a transient RNA degradation product, but is instead tightly regulated by factors other than the abundance of primary transcripts. PMID- 7540289 TI - Cardiac dysrhythmias in children with idiopathic dilated or hypertrophic cardiomyopathy. AB - To assess the incidence and prognostic significance of cardiac dysrhythmias in children with idiopathic dilated or hypertrophic cardiomyopathy, the clinical course of 59 patients was retrospectively reviewed over a period of 27 years. Dilated cardiomyopathy (DCM) was diagnosed in 28 patients and hypertrophic cardiomyopathy (HCM) in 31 patients. The mean age at the time of diagnosis was 2.8 +/- 0.7 years in DCM patients and 6.7 +/- 0.8 years in HCM patients. Mean follow-up time after diagnosis of cardiomyopathy was 4.1 +/- 1.0 years in DCM patients and 6.6 +/- 0.8 years in HCM patients. Clinically significant cardiac dysrhythmias were found in 17 of 59 patients (29%): 7 of 28 patients (25%) with DCM and 10 of 31 patients (32%) with HCM. The initial diagnosis of a cardiac dysrhythmia was made by standard electrocardiography in 12 of 17 patients (71%) and by 24-hour Holter monitoring in 5 of 17 patients (29%). Ventricular dysrhythmias were present in 5 of 7 patients with dilated cardiomyopathy and in 5 of 10 patients with hypertrophic cardiomyopathy. During the followup time, death occurred in 18 of 59 patients (31%): 8 of 59 patients (14%) died from congestive heart failure and 10 of 59 patients (17%) died suddenly. Among the sudden deaths were 4 of 28 patients (14%) with dilated cardiomyopathy and 6 of 31 patients (19%) with hypertrophic cardiomyopathy. Cardiac dysrhythmias had been documented in 6 of the 10 patients dying suddenly (3 of 4 patients with DCM and 3 of 6 patients with HCM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540290 TI - Pharmacological evidence for neurokinin receptors in murine neuroblastoma C1300 cells. AB - We found that neurokinin A (NKA) and neurokinin B (NKB) induce an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in murine neuroblastoma C1300 cells (EC50: NKA 87 +/- 13 nM, NKB 97 +/- 15 nM). Substance P (SP) also caused a transient Ca2+ increase, although the potency of SP was much less than that of NKA and NKB. The increase in [Ca2+]i induced by NKA and NKB was inhibited by SR 48,968, a selective antagonist for NK2, and [beta Ala8]NKA(4-10), a selective agonist for NK2, did not stimulate the increase in [Ca2+]i. NKA- and NKB-induced Ca2+ mobilization was not inhibited by CP-96,345 and [Trp7, beta Ala8]NKA(4-10), selective antagonists for NK1 and NK3, respectively. These results suggested that C1300 cells express endogenous NK2 neurokinin receptors that have different features from known NK2 receptors. PMID- 7540291 TI - Distribution of galanin-immunoreactive nerves in the canine gastrointestinal tract. AB - The distribution of nerves containing galanin immunoreactivity (GAL-IR) in the canine gastrointestinal tract was determined by immunohistochemistry. GAL-IR was observed in nerve cell bodies and nerve fibers in all layers of the lower esophagus, gastric antrum, pylorus, ileum, and colon, and in the sphincters of lower esophagus and pylorus. There were numerous GAL-IR-containing nerve cell bodies in the myenteric plexus of gastric antrum. GAL-IR nerve somata were also common in the myenteric plexus of lower esophagus, ileum, pylorus, and colon, and in the submucous plexus of stomach, ileum, and colon. GAL-IR-positive nerve fibers were also observed around submucous blood vessels of the stomach. GAL-IR nerves are distributed more widely in the canine enteric nervous system than previously recognized. PMID- 7540292 TI - Carboxyfluorescein and biotin neuromedin C analogues: synthesis and applications. AB - Two neuromedin C (NC) analogues were constructed by Fmoc synthesis and in situ coupling of 4(5)-carboxyfluorescein or biotin to the N-terminus. Both displayed full agonism in an amylase release assay and cross-reacted fully with a NC specific antiserum. Biotin NC functioned in a streptavidin-capture ELISA. Carboxyfluorescein NC was used to probe receptor localization in rat stomach. Specific NC binding sites, which did not interact with substance P, angiotensin I, or neurokinin A, were labeled in the antrum. Identity of NC binding sites was confirmed by microautoradiography. The specifically labeled cells were all found in the lamina propria and at least some of cells were identified as eosinophils. PMID- 7540294 TI - Effects of calcitonin gene-related peptide on cyclic AMP production and relaxation of longitudinal muscle of guinea pig ileum. AB - The effects of rat calcitonin gene-related peptide (rCGRP) on the content of cyclic nucleotides in the cells of plexus-free longitudinal muscle (LM) of guinea pig ileum relative to smooth muscle relaxation were investigated. In the absence of isobutyl-methyl-xanthine (IBMX), a nonspecific inhibitor of phosphodiesterases, rCGRP significantly increased cyclic 3'5'-adenosine monophosphate (cAMP) content that correlated temporally to the relaxation of the muscle. Cyclic 3'5'-guanosine monophosphate (cGMP) content was not affected by this peptide. IBMX potentiated both the increase in cAMP content and the muscle relaxation induced by rCGRP. rCGRP increased cAMP content in a concentration dependent manner, with an ED50 value of 7.5 x 10(-9) M. Both the rCGRP-stimulated increase in cAMP content and the rCGRP-induced muscle relaxation were blocked by hCGRP(8-37), a selective antagonist of CGRP receptors. The pA2 value of hCGRP(8 37) for rCGRP-induced inhibition of tension was calculated to be 6.48. These results suggest that CGRP relaxes the LM through its specific receptors and involves the generation of cAMP, but not the generation of cGMP. PMID- 7540284 TI - Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid binding proteins. PMID- 7540293 TI - The peptidergic innervation of the human superficial temporal artery: immunohistochemistry, ultrastructure, and vasomotility. AB - The peptidergic innervation of the human superficial temporal artery was investigated by means of immunohistochemical, ultrastructural, and in vitro pharmacological techniques. A dense network of nerve fibers was found in the adventitia. The majority of the nerve fibers displayed immunoreactivity for tyrosine hydroxylase and neuropeptide Y (NPY). A moderate supply of perivascular nerve fibers displayed either acetylcholinesterase activity or immunoreactivity for vasoactive intestinal peptide (VIP), peptide histidine methionine-27 (PHM), and calcitonin gene-related peptide (CGRP). Only a few nerve fibers displayed substance P (SP), neurokinin A (NKA), and neuropeptide K (NPK) immunoreactivity. In double immunostained preparations, SP immunoreactivity was co-localized with NPK and CGRP in the same nerve fibers. Ultrastructural studies revealed the presence of numerous axon variocosities at the adventitial--medial border. NPY, VIP, and CGRP immunoreactivities occurred in the same type of large granular vesicles, but in morphological distinct nerve profiles. NPY had, in general, no direct vasoconstrictor effect. However, at a low concentration of NPY contractile response induced by NA (10(-7)-10(-6)M) was 9-15 times enhanced. The NPY-induced potentiation of the NA-induced contraction was not dependent on the presence of an intact endothelium. No significant difference was found between acetylcholine, VIP, and PHM in either potency or degree of relaxation. SP, NKA, and CGRP also acted as vasodilatory agents, with CGRP being more potent than the tachykinins. The response to SP, but not CGRP, was dependent on an intact endothelium. Pretreatment of the vessels with a low concentration of NPY did not change the responses to ACh, VIP, SP, or CGRP. PMID- 7540295 TI - Adrenal medulla is involved in the aldosterone secretagogue effect of substance P. AB - Substance P (SP) increased aldosterone secretion of rat adrenal slices, but not of isolated zona glomerulosa cells, and this effect was annulled by two specific antagonist of SP (SP-A). Both tissue preparations displayed an aldosterone secretory response to isoprenaline (IP) that was blocked by l-alprenolol (AL). AL reversed the aldosterone response of adrenal slices to IP, SP, or IP plus SP, whereas SP-A only suppressed that to SP. Quarters of adrenocortical autotransplants, which are completely deprived of chromaffin cells, showed an aldosterone response to IP, but not to SP. These findings suggest that the mechanism underlying the aldosterone secretagogue action of SP probably involves the stimulation of catecholamine release by adrenal medulla chromaffin cells. PMID- 7540296 TI - Effects of RP 67580 on substance P-elicited responses and postsynaptic potentials of motoneurones of the rat isolated spinal cord. AB - The effect of RP 67580, a recently developed antagonist selective for the NK1 tachykinin receptors of peripheral tissues, was studied with intracellular recording from motoneurones of the rat isolated spinal cord. In the presence of RP 67580 (1-2 microM), membrane depolarization induced by the putative transmitter substance P (SP) was either unchanged or enhanced (an effect prevented by tetrodotoxin; TTX). Neither short nor long excitatory synaptic potentials (EPSPs) were antagonized by RP 67580. Sustained synaptically evoked depolarizations (mimicking noxious stimuli and thus presumed to be at least partly mediated by SP) were also insensitive to RP 67580. These data suggest the existence of a pharmacologically distinct NK1 receptor population insensitive to RP 67580) in the neonatal rat spinal cord. PMID- 7540300 TI - Secretory symptoms from metastatic adrenal cortical carcinoma responding to octreotide. AB - The prognosis of patients with metastatic adrenal cortical carcinoma is poor, and their disabling symptoms are usually unresponsive to conventional therapy. A patient with Cushing's syndrome secondary to a secretory adrenal cortical carcinoma was treated with octreotide, endocrine therapy and chemotherapy having failed. Treatment led to a dramatic relief of her symptoms with a fall in corticosteroid secretion. Somatostatin analogue therapy for this tumour should be encouraged in view of the lack of alternative palliative treatment. PMID- 7540301 TI - Symptomatic cardiomyopathy as a presentation in Whipple's disease. AB - A patient presenting with congestive cardiac failure and anaemia underwent investigation which led to the diagnosis of Whipple's disease, associated with dilated cardiomyopathy. Conventional antibiotic therapy for Whipple's disease resulted in resolution of the traditional features of Whipple's disease and a marked improvement in the patient's heart failure. PMID- 7540298 TI - Induction of benign prostatic hyperplasia in intact dogs by near-physiological levels of 5 alpha-dihydrotestosterone and 17 beta-estradiol. AB - Benign prostatic hyperplasia was induced in mongrel dogs treated for 60 days with one silastic implant containing 17 beta-estradiol and four containing 5 alpha dihydrotestosterone. The condition was characterized by (1) a marked increase of the stromal elements, particularly the stromal septa between the individual glands, (2) a slight increase in prostatic volume, and (3) a morphology that resembled spontaneous complex benign prostatic hyperplasia in the dog. Other groups of animals that remained untreated or received only 17 beta-estradiol or only 5 alpha-dihydrotestosterone did not develop this condition. Prostate volumes decreased by 14% in the estrogen-treated dogs, whereas they increased in the androgen-treated animals by 6% compared to pretreatment prostate volumes. The morphology of the epithelium of the prostates of androgen-treated animals was not different from that of controls despite the increase in prostate volume. The serum 17 beta-estradiol and 5 alpha-dihydrotestosterone concentrations were increased from 25 +/- 2 (mean +/- SEM) and 256 +/- 42 pg/mL, respectively, in control dogs to 52 +/- 37 and 562 +/- 37 pg/mL, respectively, in the dogs treated with the hormone combination. Thus, hormone concentrations were two- to three fold higher than control values, and the ratio of estradiol-17 beta to 5 alpha dihydrotestosterone was increased by up to 19%. These data demonstrate that treatment of dogs with low levels of estrogen and androgen may be an excellent model for the study of spontaneous complex benign prostatic hyperplasia in aging men. PMID- 7540299 TI - Clearance of serum PSA after open surgery for benign prostatic hypertrophy, radical cystectomy, and radical prostatectomy. AB - OBJECTIVE: To study the clearance of serum prostate-specific antigen (PSA) after several types of prostatic tissue ablation. METHODS: Serum PSA levels were measured (YANG Proscheck ultrasensitive assay) just before surgery, immediately after specimen removal, then twice weekly for 5 weeks or until it was undetectable (< 0.05 ng/ml) in patients undergoing radical cystoprostatectomy for bladder cancer (n = 10), or radical prostatectomy for T1 T2 prostate cancer (n = 18) and daily for 6 days after open surgery for benign prostatic hypertrophy (BPH) (n = 10). RESULTS: Open enucleation for BPH: the immediately postoperative PSA level was 6 times its preoperative value. It decreased following a monoexponential curve with a very short half-life of 0.55 +/- 0.39 days, range (0.14-1.3), reaching a value lower than the preoperative level in all cases, except one by day 3. After radical cystoprostatectomy: the decrease of serum PSA is monoexponential with a half life of 1.92 +/- 1.2 days (0.57-4.24) reaching undetectable level (< 0.05 ng/ml) in all patients by day 21. After radical prostatectomy: 11/18 patients (61%) showed a one-component exponential decrease in PSA with a half-life of 2.5 +/- 1.33 days (range 0.97-4.6 days), and 7/18 showed a two-component exponential decrease with a first half-life of 0.94 +/- 0.8 days and a second of 7.62 +/- 6.35 days); 100% of the patients reached undetectable serum PSA by day 28 in the first group compared to 14.2% of the patients with a two component exponential decrease (P < 0.01). There was no difference between these groups as far as preoperative PSA levels and specimen pathology were concerned. CONCLUSION: Serum clearance of PSA after extirpative prostatic surgery is closely related to the type and indication of procedure used. Radical cystoprostatectomy is probably the best model in which to study the pharmacokinetics of PSA. PMID- 7540304 TI - Cocrystallization of lysyl-tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys. AB - Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlys or Escherichia coli tRNAlys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18-22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 A, c = 119 A) and diffract to at least 2.7 A resolution. However the homocomplex is of space group P42(1)2 and the heterocomplex of space group I422. PMID- 7540303 TI - [Onset of language development in general developmental delay at age 5]. AB - The course of development of a 5-year-old boy with a serious developmental retardation and massive behaviour problems who spoke only a few words is documented and described with psychometric tests and spontaneous language samples up to the age of 8-9. Inspite of this developmental retardation and poor care, the child reached until then an average to above-average intelligence in nonverbal functions. The symptoms were reduced only to a specific language impairment. At the age of 11, this disorder as well had practically disappeared. Such a course of development after the age of 5 is not common, whereas a specific language impairment normally remains a deficit. Possible causes for this favourable development are discussed. PMID- 7540297 TI - Cost-effectiveness analysis in early detection of prostate cancer: an evaluation of six screening strategies in a randomly selected population of 2,400 men. AB - Based on the findings in an early detection study for prostate cancer [Gustafsson et al.: J Urol 148:1827-1831, 1992] using digital rectal examination (DRE), transrectal ultrasound (TRUS), and prostate-specific antigen (PSA), a cost effectiveness analysis was performed based on 6 screening strategies, namely: 1) DRE of all individuals; 2) TRUS of all individuals; 3) DRE, TRUS, and PSA analysis followed by reexamination of individuals with PSAs > or = 7 ng/ml; 4) DRE of individuals with PSAs of > or = 4 ng/ml; 5) TRUS of individuals with PSAs of > or = 4 ng/ml; 6) DRE and PSA analysis followed by TRUS on individuals with PSAs > or = 4 ng/ml. The detection rates of prostate cancer using these 6 strategies were 2.4%, 3.3%, 3.6%, 2.0%, 2.6%, and 3.2%, respectively. Except for costs per detected cancer, costs were also calculated per detected small cancer (< or = 1.5 cm) and per detected cancer treated for cure. The cost calculations were based on total costs, i.e., direct plus indirect costs. When the 6 strategies were compared, taking into account the detection rate of cancers treated for cure and cost-effectiveness with respect to cancers treated for cure, strategies 1), 2), 3), and 4) were ruled out as less favorable than the remaining 2 strategies. TRUS of individuals with PSAs > or = 4 ng/ml (strategy 5) was the most cost-effective strategy and detected 80% of the cancers actually treated for cure. Screening with DRE and PSA analysis followed by TRUS of individuals with PSAs > or = 4 ng/ml (strategy 6) had a somewhat lower cost-effectiveness, but detected 90% of the cancers treated for cure. PMID- 7540306 TI - Omega-3 fatty acids and endothelial leukocyte adhesion molecules. AB - Although dietary fatty acids can modulate atherogenesis and inflammation, the mechanisms by which this occurs are poorly understood. Induction in endothelial cells of adhesion molecules for circulating leukocytes and of inflammatory mediators by cytokines likely contributes to early phases of atherogenesis and inflammation. We report here that incorporation into cellular lipids of one specific fatty acid of the omega-3 family, docosahexaenoic acid (DHA), decreases cytokine-induced expression of endothelial leukocyte adhesion molecules, secretion of inflammatory mediators, and leukocyte adhesion to endothelial cells. These properties of DHA may contribute to antiatherogenic and antiinflammatory effects of omega-3 fatty acids. PMID- 7540305 TI - Fatty acids and steroid hormone action. PMID- 7540307 TI - Formation of a novel arachidonic acid metabolite in peroxisomes. AB - A new radiolabeled metabolite was released into the extracellular fluid by normal human skin fibroblasts that were labeled with [5,6,8,9,11,12,14,15-3H] arachidonic acid. This product continued to accumulate during a 24 h incubation, and its formation was not saturated at arachidonic acid concentrations up to 15 mumol/L. The compound, identified as hexadecatrienoic acid, was not produced by Zellweger fibroblasts which are deficient in peroxisomal fatty acid beta oxidation. By contrast, radiolabeled hexadecatrienoic acid was produced by mutant fibroblasts having other peroxisomal defects, including X-linked adrenoleukodystrophy, adult Refsum's disease, and rhizomelic chondrodysplasia punctata. This radiolabeled metabolite also was produced by mutant fibroblasts that cannot oxidize long-chain fatty acids in the mitochondria. These results indicate that hexadecatrienoic acid is synthesized from arachidonic acid by peroxisomal beta-oxidation. The absence of this pathway may account for some of the biochemical and functional abnormalities that occur in Zellweger's syndrome. PMID- 7540302 TI - Comparison of the antiviral activities of 3'-azido-3'-deoxythymidine (AZT) and gossylic iminolactone (GIL) against clinical isolates of HIV-1. AB - 3'-Azido-3'-deoxythymidine (AZT) and gossylic iminolactone (GIL) were compared for antiviral activities in vitro against fresh clinical isolates of HIV-1 obtained from 19 subjects on AZT therapy. IC50 values for AZT ranged from 0.015 to 6.7 microM (447-fold range) while IC50 values for GIL ranged from 0.40 to 6.6 microM (16-fold range). There was no correlation between IC50 values for AZT and GIL, suggesting that the anti-HIV activity of GIL does not involve inhibition of reverse transcriptase. PMID- 7540308 TI - Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1. AB - The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to 173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea. PMID- 7540310 TI - Malignant liver tumors treated with MR imaging-guided laser-induced thermotherapy: technique and prospective results. AB - PURPOSE: To evaluate magnetic resonance (MR) imaging-guided laser-induced thermotherapy (LITT) of liver metastases. MATERIALS AND METHODS: In a phase II study, 20 patients with 33 metastases from colorectal carcinoma (75%) or other primary tumors (25%) underwent LITT. MR thermometry performed with fast low-angle shot sequences was used to monitor therapy on-line, and dynamic and static contrast material-enhanced MR images enabled estimation of the degree of resultant necrosis. Follow-up studies were performed 3 months after thermotherapy. RESULTS: The thermosequences enabled accurate on-line monitoring in 85% of lesions. In 69% of lesions 20 mm in diameter or smaller, contrast enhanced MR images depicted substantial necrosis, with a local tumor control rate of 69% after 6 months and 44% after 12 months. Among lesions larger than 20 mm, necrosis was frequently incomplete, with a local control rate of only 41% after 6 months and 27% after 12 months. CONCLUSION: MR imaging-guided LITT of liver metastases is a safe and promising therapy for liver metastases. PMID- 7540311 TI - A method evaluating population risks from chemical exposure: a case study concerning prohibition of chlordane use in Japan. AB - The proposed method evaluating population risks from chemical exposure is based on estimation of the individual variabilities in the internal concentration of the chemical of concern and estimation of the magnitude of toxic effect caused at a given internal concentration. We assume a log-normal distribution for individual variability and propose loss of life expectancy (LLE) as a measure of the magnitude of toxic effects. We applied it to the evaluation of the governmental action of prohibiting the use of chlordane as a termiticide in Japan. Because the method is applicable to both carcinogenic and noncarcinogenic chemicals, the cancer risk due to chlordane and the noncancer risk due to chlorpyrifos, a major substitute, were evaluated and compared. The estimated values of risk represented in terms of LLE are 0.10 days for residents of untreated houses, 1.9 days for residents of treated houses, and 4.4 days for termite control workers when chlordane is used, and 0, 2.8, and 31 days when chlorpyrifos is used, respectively. From the result of the case study, we can see that the prohibition of chlordane is not necessarily effective in reducing risk. PMID- 7540313 TI - The airflow resistance profile of healthy nasal cavities. AB - Distribution of resistance to respiratory airflow in the nasal cavities was determined by digitized pressure/flow measurements of consecutive 2-cm airway segments between nostril and nasopharynx. Healthy adult subjects seated in a head out body plethysmograph breathed exclusively through a single nasal cavity while transnasal pressure and flow signals were transduced, digitized and processed by a programmed desk-top computer to provide resistance values. Mean total resistances of untreated and decongested single nasal cavities were 0.44 (n = 30; SD +/- 0.25) and 0.26 (n = 15; SD +/- 0.06) Pa/cm3/s, respectively. The proportion of total airway resistance of successive 2-cm segments from nostril to nasopharynx was 56%, 22%, 16%, and 6% in the untreated nose, and 88%, 5%, 2%, and 5% following decongestion. The findings from 45 nasal cavities are consistent with previous pressure/flow measurements from six nasal cavities and support recent acoustic reflection assessments of nasal cross-sectional areas of both untreated and decongested noses. PMID- 7540309 TI - The kinetics of voltage-gated ion channels. PMID- 7540312 TI - [Occupational exposure and malignant hemopathies: a case-control study in Lyon (France)]. AB - A hospital based case-control study was carried out in Lyon with the aim of assessing the association between haematologic malignancies and occupational exposures to 320 compounds. Job histories were obtained by questionnaire for 118 cases (52 non Hodgkin lymphomas, 48 acute myeloid leukemia, 18 others leukaemias), and 118 controls with diseases other than cancer from the same general hospital; controls were matched for sex, age and nationality. Systematic coding of exposures based on a blind analysis of job histories, was done by a team of experts in chemistry and occupational health. Mantel-Haenszel analysis was performed. Significantly elevated odds-ratio were observed for non Hodgkin lymphomas and exposure to mineral oils (14.86; 2.76-80.0), excavation dusts (3.91; 0.94-15.95), alkali compounds (2.90; 1.09-7.68), inks (2.47; 1.09-5.17). For inks, a dose-response relation was observed. Elevated odds-ratios appeared for acute myeloid leukaemias and arsenic compounds (3.02; 0.90-10.13) and lead compounds (3.70; 1.09-13.44). When regarding industrial activities, two of them are more frequently found: food industries (14 cases/5 controls), public works (12 cases/0 control). When regarding jobs, winding (6 cases/0 control), glass workers (8 cases/1 control) and warehousemen (10 cases and 4 controls) are more often seen among cases. PMID- 7540314 TI - Sustained use of xylometazoline nasal spray shortens the decongestive response and induces rebound swelling. AB - Long-term use of topical vasoconstrictors for the nose may result in rhinitis medicamentosa, drug addiction and tachyphylaxis. Some authors also believe that the severity of rebound swelling is proportional to the period during which the drug has been used, the frequency of its administration, and the amount of drug given. It has previously been reported that four-week use of the recommended dose of oxymetazoline induces rebound swelling, a sign of rhinitis medicamentosa. To study the effect of an increased amount of vasoconstrictor on rebound swelling and the decongestive effect of the drug, nine healthy subjects were given xylometazoline nasal spray in double the recommended dose (1.0 mg/ml; 0.28 ml in each nostril thrice daily) for 30 days. After 30 days on xylometazoline, the decongestive effect was the same 1 h after drug administration as before starting the medication. Similarly, after 30 days on xylometazoline, the decongestive effect was less 5 h after drug administration than it was 6 h after drug administration at the start of medication (p < 0.005). After 10 days no rebound swelling was recorded, but after 30 days rebound swelling occurred in eight out of nine subjects (p < 0.05). When comparing the results of this trial with the corresponding results of the oxymetazoline study, no further increase in rebound swelling was found. We conclude that long-term use of xylometazoline nasal spray shortens the decongestive response in healthy volunteers. Moreover, double the recommended dose of xylometazoline did not further increase the rebound swelling seen when using the recommended dose of oxymetazoline. PMID- 7540315 TI - Septal surgery and tubal function: early and late results. AB - The influence of nasal septal deviation and its surgical correction on the opening pressure of the Eustachian tube was studied. The Tuba Compliance Manometric test by the Valsalva manoeuvre was used. On the basis of early and late post-operative measurements, it was stated that surgical correction of a nasal septal deviation in order to improve the tubal opening pressure is justified, both on the deviated side and on the non-deviated side. Late post operative results confirmed this finding. PMID- 7540316 TI - Short- and long-term prognosis for middle-aged and elderly patients hospitalized with community-acquired pneumonia: impact of nutritional and inflammatory factors. AB - To investigate the impact of nutritional and inflammatory factors on short- and long-term prognosis for patients hospitalized for community-acquired pneumonia (CAP), 97 patients, 50-85 years old, admitted to the Department of Infectious Diseases at Danderyd Hospital were enrolled in a prospective study. 13 enrolment variables were examined for association with 6 outcome variables. Serum orosomucoid concentration, acute physiology and chronic health evaluation (APACHE) II score, body mass index (BMI) and triceps skinfold (TSF) were each associated with duration of hospital stay. A low TSF and BMI, and a high APACHE II score were all associated with death. A high APACHE II score and a high TSF were both associated with readmission within 6 months of discharge. The alfa-1 antitrypsin concentration was the most closely correlated with duration of fever. We conclude that the admission concentrations of alfa-1-antitrypsin and orosomucoid are better predictors of hospital morbidity than the more commonly used albumin and C-reactive protein (CRP) levels. Measurement of APACHE II and TSF on admission may give additional prognostic information on the interval from admission to 6 months after discharge. PMID- 7540317 TI - [Regulatory role of galanin on prolactin and beta-endorphin release from anterior pituitary lobe of rat]. AB - The role of brain-gut peptide galanin in the regulation of prolactin (PRL) and beta-endorphin (beta-EP) release from anterior pituitary lobe was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells of the rat. Galanin (1 microgram or 3 micrograms/rat) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of PRL resting secretion, but did not alter resting secretion of beta-EP and restraint stress-induced release of PRL and beta-EP. However, galanin (0.05, 0.5 and 1.0 micrograms/5 x 10(5) cells.ml-1) induced highly significant, dose-related increase of beta-EP secretion from dispersed anterior pituitary cells, but failed to alter significantly PRL secretion from the cultured cells. These results indicate that central galanin has a stimulatory role in pituitary PRL resting secretion via the hypothalamus, whereas peripheral galanin stimulates beta-EP secretion only via direct action of this peptide in anterior pituitary cells. PMID- 7540318 TI - [Changes of cytoplasmic calcium and magnesium concentration and calcium distribution in human platelets caused by thrombin]. AB - Changes of calcium and magnesium concentration in human platelets during thrombin induced agglutination and the distribution of the respective concentration in the platelet population were measured by fluorescence probe using fura 2, mag-fura 2 and fluo 3. The cytoplasmic free calcium concentration increases with addition 2 thrombin. When the thrombin concentration was increased to 0.5 U/ml, the calcium concentration increase showed two rising peaks. The popular cytoplasmic calcium concentration in platelets shows normal distribution. The cytoplasmic free magnesium concentration also increases with increase of calcium concentration, showing that magnesium also plays a significant effect in the condensation of platelets. PMID- 7540320 TI - NO news is good news. PMID- 7540319 TI - Lack of effect of chronic morphine treatment and naloxone-precipitated withdrawal on tyrosine hydroxylase, galanin, and neuropeptide Y mRNA levels in the rat locus coeruleus. AB - Morphine dependence was experimentally induced in rats by daily injection of increasing doses of morphine for seven days. Withdrawal was precipitated in half of the morphine-dependent rats by a single injection of naloxone on day 8. Behavioral signs of withdrawal were evident in the morphine/naloxone group. Gene expression in locus coeruleus (LC) neurons was investigated using quantitative in situ hybridization analysis. Messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for precursors to galanin (GAL) and neuropeptide Y (NPY), peptides that coexist with norepinephrine in LC neurons, were not altered by chronic morphine treatment or naloxone precipitated withdrawal. In contrast, mRNA levels for c-fos were dramatically elevated in the LC following naloxone-precipitated withdrawal. Chronic morphine treatment caused a small decrease in levels of mRNA encoding the precursor to corticotropin-releasing factor (CRF) in Barrington's nucleus. Although long-term adaptations of LC neurons have previously been implicated in the development of morphine tolerance, dependence, and withdrawal, alterations in the levels of TH, GAL, or NPY mRNA in the LC apparently do not underlie this process. PMID- 7540321 TI - Ca(2+)-independent nitric oxide synthase activity in human lung after cardiopulmonary bypass. AB - BACKGROUND: Because surgery involving cardiopulmonary bypass induces a systemic inflammatory response, the effect of cardiopulmonary bypass on nitric oxide (NO) generation was investigated in human lung tissue. METHODS: Nitric oxide synthase (NOS) activity was measured by the conversion of 14C-L-arginine to 14C-L citrulline in tissue biopsy samples obtained before and after cardiopulmonary bypass. RESULTS: The Ca(2+)-independent production of NO found before cardiopulmonary bypass was extremely low (1.5 (0.5) pmol citrulline/mg/min), but was increased after the bypass operation (23.6 (11) pmol/mg/min). CONCLUSIONS: Ca(2+)-independent NOS activity was detected in human lung after cardiopulmonary bypass. This finding may provide an important insight into the pathogenesis of the tissue damage and acute phase response observed after such surgery. PMID- 7540323 TI - Continuous blood density measurements and volume changes during extracorporeal circulation in patients undergoing cardiac surgery. AB - The changes of blood volume and transcapillary fluid shifts during extracorporeal circulation (ECC) was examined using continuous measurements of blood density by the mechanical oscillator technique. Sixteen patients (1 female, 15 male) with a mean age of 61.4 years (+/- 7.6 years, 47-70 years) undergoing coronary artery bypass surgery were included in this study. The equipment for continuous measurement of the blood density (DPRT by Paar/Austria) was installed at the arterial line of the heart-lung bypass. Higher-precision discrete measurements of some parameters used the DMA 55 equipment produced by the Paar company. Measurements were taken at 37 degrees C. In 11 patients the transcapillary volume loss (difference of total volume between beginning and end of ECC) during heart lung bypass (mean observation period of 55 min (+/- 16 min, 28-82 min) was found to be 870 ml/m2 body surface area (BSA) (+/- 360 ml, 290-1560 ml/m2) by the dilution method using the priming solution at the beginning and 500 ml lactated Ringer's solution added at the end of the ECC. The calculated volume shift using the "double density method", which takes into account blood density, plasma density, and hematocrit, amounted to 830 ml/m2 BSA (+/- 200 ml, 450-1210 ml/m2). The density of the transcapillary volume loss into the interstitial layer was calculated and found to be 1.0026 g/ml (+/- 0.0017 g/ml, 1.0003-1.0063 g/ml). A significant difference of transcapillary volume shift correlated with the administered catecholamine (dopamine, dobutamine) dosage postoperatively. Without catecholamine this difference was 260 ml/m2 BSA, with 3 microns/kg/min dopamine it rose to > 500 ml/m2 BSA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540324 TI - Comparison of porcine biological valves with pericardial valves--a 12-year clinical experience with 1123 bio-prostheses. AB - In order to evaluate selective differences of biological porcine valves versus pericardial valves and to analyze various valve models, 8 different bioprostheses (4 porcine valves, 4 pericardial valves) were studied in a 12-year follow-up. From 1978 to 1990, 476 porcine bioprostheses (Carpentier-Edwards: n = 104, Carpentier Edwards Supraanular: n = 59, Hancock I: n = 41, Hancock II: n = 272) and 647 pericardial valves (Hancock-Extracorporeal: n = 479, Ionescu-Shiley: n = 76, Carpentier-Edwards: n = 57, Mitroflow: n = 35) were implanted. At time of implantation, the patient age ranged from 21-85 years, mean 57.1 +/- 12.4 years. 831 patients were analyzed in the long-term follow-up (62.3 +/- 18.6 months, cumulative follow-up of 6632 patient-years). The incidences of thromboembolic complications (TE), endocarditis (E), primary tissue failure (PTF), rate of reoperation (ReOp), and late mortality due to prosthesis dysfunction were analyzed, calculated, and compared within the different valve models (actuarial data, chi 2 test, log rank analysis). The incidences of TE and E were lower for pericardial valves when compared with porcine bioprostheses (TE: 0.88 vs. 1.8%/patient year; E: 0.24 vs. 0.5%/patient year); within the 2 groups, the different valve models did not show any major differences. However, the incidence of PTF was significantly higher in the pericardial valve group, being 36 +/- 6.5%, 68 +/- 10% and 86 +/- 19.5% after 6, 8, and 10 years; the respective figures for the porcine valves were 6 +/- 3.5%, 18 +/- 7.1%, and 60 +/- 13.1% (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540325 TI - Peroperative hemodynamic study of left internal mammary artery grafts. AB - The present study was undertaken to analyse hemodynamic features of an arterial graft and to determine parameters which influence primarily flow and velocity. A total of 284 consecutive patients having isolated coronary bypass surgery underwent peroperative hemodynamic assessment by pulsed Doppler ultrasonics of a left internal mammary artery bypass graft implanted onto the left anterior descending artery. Internal mammary artery free flow was 109.2 +/- 3.7 ml/min and flow in the internal mammary artery measured after completion of the distal anastomosis was 70.9 +/- 2.7 ml/min. All values quoted are mean +/- standard error of the mean. Mean velocity was 21.2 +/- 0.6 cm/sec, internal diameter was 2.65 +/- 0.04 mm and pulsatility index was 2.24 +/- 0.12. Resistance was expressed as mmHg/(ml . min-1) and averaged 1.65 +/- 0.13 for total resistance, 0.87 +/- 0.05 for graft resistance, and 0.73 +/- 0.13 for coronary resistance. From a set of 35 variables, stepwise multiple regression analysis selected two parameters influencing independently flow in internal mammary artery (R2 = 0.8762): flow velocity (p < 10(-4)) and internal diameter (p < 10(-4)). Variables influencing velocity (R2 = 0.3071) were: pulsatility index, which is a dimensionless expression of peripheral resistance (p < 10(-4)), and free internal mammary artery flow (p = 0.0007). Furthermore, a significant correlation between internal diameter and total resistance was observed (R = -0.5363, p < 10(-4), Y = 1.676X-1.545), and the exponentially fitted regression line was characterized by a marked increase of resistance at diameters less than 2 mm.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540322 TI - The concept of "direct mechanical ventricular assistance" in the treatment of left-ventricular failure. Part 1: Idea, development and construction of an implantable multi-chamber pump system partially surrounding the heart. AB - In this paper we present a therapeutic concept for the treatment of heart failure due to muscular inability to pump properly. The basic principle of this concept triggered numerous studies, then with the aim of cardiopulmonary resuscitation, back in the 1970s. In general, it dealt with mechanical systems which led to an increase in stroke volume, systolic blood pressure, and cardiac output through the application of pressure directly to the left ventricle. After a critical appraisal of the relevant literature from technical, physical, and medical viewpoints, and our own preliminary studies on animal hearts within the framework of mock circulation experiments, we have conceived a new functional principle for direct mechanical ventricular assistance based on squeezing the left ventricle only. We have begun development of the system which has the advantages of ease of use, high biocompatibility due to lack of contact between blood and system components, the prevention of infection through complete intrathoracic implantation (long-range goal), and the fact that the patient's own heart can be supported by the system without being removed from the circulatory system (support of the residual myocardial function). Technical as well as medical prerequisites are indicated, and the materials selection and construction principles of the control, pressure generation, pressure transduction, and ventricular compression unit are described. It has proved possible to construct a prototype system to be used in animal experiments, which, through pneumatic inflation of a chamber system partially surrounding the left ventricle, should be able to augment or take over the pumping function of the left ventricle. PMID- 7540326 TI - Coronary artery bypass grafting after failed coronary angioplasty: risk factors and long-term results. AB - In this case-control study we investigated the preoperative risk factors and long term results in patients undergoing emergency coronary artery bypass grafting (CABG) after failed percutaneous transluminal coronary angioplasty (PTCA). From January 1990 to March 1993, 56 emergency CABGs (Group A) were performed in patients from 7 different cardiology centres--six of these located outside our hospital within a radius of 19 km-1 h to 114 h (phi 7.3 h) after failed PTCA. No special stand-by was given and patients were operated in the next available theatre. Criteria of anatomically unfavourable stenoses for PTCA were defined as follows: Long stenoses, occlusions of other major vessels, tandem stenoses, stenoses at or near a bend or branch and, finally, eccentric stenoses. Thirty-two percent met one criterion, 35.8% two, 6.9% three and 1.8% four criteria. All patients were examined echocardiographically 14.6 +/- 8.2 months postoperatively. Patients who had been operated on electively within the same period served as a control (Group B; n = 56; case-control study). The patients were matched for age, sex, EF, LVEDP, body surface area, reoperation status, and concomitant diseases. The mortality rate for Group A patients was 10.7% (n = 6); for Group B, 1.8% (n = 1). Five of the Group A patients who died came from our own hospital and only one from a hospital 1 km away (mortality: 14.3% vs. 4.8%). There were no deaths among patients with an anatomically favourable stenosis for a PTCA; in patients with more than one criterion for an unfavourable stenosis mortality increased to 19.0% (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540327 TI - Idiopathic isolated coronary ostial stenosis: a rare lesion with particular clinical and surgical implications. AB - Isolated coronary ostial stenosis is an unusual condition, most often atherosclerotic in origin. Only few cases have been reported as idiopathic. The clinical profile of the latter patients suggests that this group may represent a distinct clinical entity, with peculiar diagnostic and therapeutic features. We present our experience concerning six patients with isolated idiopathic coronary ostial stenosis, who successfully underwent transaortic left main ostioplasty with autologous pericardial patch fixed in glutaraldehyde. A review of previous reports in regard to the surgical management of coronary ostial disease is presented. PMID- 7540330 TI - Completion pneumonectomy: experience with 47 cases. AB - From 1982 through March 1994, fourty-seven patients underwent completion pneumonectomy for a reappearing lung cancer, lung metastases, late complications, benign lung diseases, and early complication of bronchial or pulmonary artery sleeve resections. Intraoperative bleeding was higher than in standard pneumonectomy; there was one intraoperative mortality (2.3%). Operative mortality was 14.9% overall but was 3.6% in completion pneumonectomy for lung cancer, 20% for late complications and benign disease, and 57% for the treatment of early complications of sleeve resections. Three and five-year survival in patients with lung cancer who survived the operation was 43.8% and 28.7% according to the Kaplan-Meier method; no significant difference in long-term survival was present between patients with a second primary lung cancer or recurrence. Completion pneumonectomy is indicated in reappearing lung cancer and should be considered in benign disease when a less invasive procedure is not available. Completion pneumonectomy for the treatment of early complications of bronchial or pulmonary artery sleeve resections has a very high mortality but no alternative is available. PMID- 7540329 TI - Surgical considerations in management of left hemianomalous pulmonary venous connections. AB - Anomalous pulmonary venous connection of the entire left lung into the left innominate vein is very rare. 5 patients with this anomaly were managed in the authors' institution over the last 12 years. Clinically they were diagnosed as cases of pretricuspid left-to-right shunt at atrial level. Cardiac catheterisation and angiography demonstrated the anomalous drainage of the entire left lung into the left innominate vein. Anastomosis between the vertical vein and the left atrium was established using conventional cardiopulmonary bypass, hypothermia, and cold cardioplegic diastolic arrest of the heart. Postoperative course was uneventful. 4 of the 5 patients presented for follow-up during 1-12 years (mean 6.5 years). All are asymptomatic and have been studied with echocardiography, angiography, and magnetic resonance imaging techniques. The anastomatic site was found to be non obstructive in all the patients. PMID- 7540328 TI - Relief of airway compression by enlarged pulmonary artery--modified palliative suspension of the artery after plication. AB - A new modification of the technique for plication and suspension of the pulmonary artery in critically ill infants and children is described. The technique consists of (1) left lobe thymectomy, (2) insertion of a polytetrafluoroethylene sheet to provide complete coverage of pericardium, (3) plication of dilated pulmonary artery and (4) suspension of plicated pulmonary artery to the left side of the anterior chest wall. We employed this technique in 7 patients with respiratory distress due to tracheal and/or broncheal compression with massive left-to-right shunt (5 patients) and with absent pulmonary valve (2 patients). Five of these patients (71%) could be weaned from the respirator in the postoperative phase with this surgery. Although this procedure is palliative, this simple technique might be one of the choices for critically ill infants and children. PMID- 7540331 TI - Surgical treatment for coronary and iliac arterial lesions in a case of Kawasaki disease. AB - We report a case of Kawasaki disease in a Japanese boy who developed combined aneurysms and stenoses in both the coronary and iliac arteries. The boy underwent coronary artery bypass grafting using the left internal thoracic artery at the age of 7 years. The lower abdominal aorta and both common iliac arteries were replaced with a Dacron graft when he was 11 years old. Recovery from both operations was uneventful. This is the first report of coexisting coronary and extracoronary arterial lesions in a patient with Kawasaki disease which required surgical treatment. PMID- 7540332 TI - Video-assisted thoracoscopy for closure of a bronchial stump fistula. AB - It is thought that thoracoscopic closure of a bronchial stump fistula is beyond the capabilities of current thoracoscopic techniques. We describe the successful use of thoracoscopy in the therapy of a late right main bronchial stump dehiscence after pneumonectomy and chemotherapy of a stage IIIA adenocarcinoma. We clipped the fistula with a Multifire Endo Hernia Stapler (Auto Suture) and we obtained intraoperative airtight closure of the fistula. PMID- 7540333 TI - Simultaneous operation for hydatid cyst of right lung and liver. AB - Hydatid disease is endemic in many regions of Turkey. We observed 23 cases of simultaneous liver hydatid cyst in 344 patients having pulmonary hydatid cyst referred to our center. The simultaneous hydatid cysts were diagnosed by ultrasonography. In all cases complete resection was possible in a single operation. We performed thoracophrenotomy in 21 cases and thoracolaparotomy in two cases. Empyema was observed in three patients postoperatively. No mortality has occurred. Recurrence of a pulmonary cyst was observed in one patient 20 months postoperatively. PMID- 7540334 TI - Effect of the benzene metabolite, hydroquinone, on interleukin-1 secretion by human monocytes in vitro. AB - Decreased interleukin-1 (IL-1) production by mononuclear phagocytes has been shown to contribute to benzene myelotoxicity in animals. The study presented here was designed to examine the relevance of this mechanism in humans. Fresh human blood monocytes were exposed to 0.001-10 microM hydroquinone (HQ) and assessed for their ability to release IL-1 alpha and IL-1 beta in response to a stimulation with endotoxin. Both cytokines were measured by specific ELISA. Exposure of human monocytes to micromolar concentrations of HQ for 2 hr resulted in a dose-dependent reduction of IL-1 secretion. For both IL-1 alpha and IL-1 beta, the decreases were statistically significant at concentrations of 5 microM and above. HQ also inhibited RNA and protein synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 21 +/- 11 and 10 +/- 9 microM, respectively. Furthermore, monocytes treated with 5 microM HQ also displayed a reduced total protein content when compared with control cells. These data suggest that the reduction of IL-1 production caused by HQ results from a global impairment of monocyte essential functions such as transcription or translation. Taken as a whole, our results support a mechanism whereby HQ may contribute to the myelotoxicity of benzene in humans by inhibiting the production by mononuclear phagocytes of cytokines involved in the regulation of hematopoiesis. PMID- 7540335 TI - Alterations in thyroid function in female Sprague-Dawley rats following chronic treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multisite carcinogen. Although the hepatocarcinogenic actions of TCDD have received the most attention, it has been demonstrated in several rodent carcinogenicity bioassays that TCDD causes a dose-related increase in thyroid follicular cell adenomas and carcinomas. The purpose of the present experiment was to investigate the dose-response relationship for thyroid function alterations in female Sprague-Dawley rats following chronic treatment with TCDD. TCDD was administered via oral gavage biweekly for 30 weeks at average daily equivalent doses of 0.1-125 ng/kg/day, thereby more than encompassing the dose range historically used in previous TCDD rodent bioassays. The endpoints examined include serum levels of thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In addition, the induction of the dioxin-responsive genes UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P450 1A1 (CYP1A1) in liver were measured using reverse-transcriptase polymerase chain reaction (RT-PCR). In agreement with previous hypotheses, TCDD appears to alter thyroid function via a secondary mechanism, namely increased excretion of T4-glucuronide resulting from TCDD induction of UGT1. The observed follicular cell hyperplasia and hypertrophy are consistent with the observed elevated TSH levels and may represent the early stages in the progression of thyroid carcinogenesis. Therefore, TCDD induces alterations in thyroid hormone function, probably as a result of chronic perturbations of liver-pituitary thyroid axis. PMID- 7540336 TI - Extrathyroidally mediated changes in circulating thyroid hormone concentrations in the male rat following administration of an experimental oxyacetamide (FOE 5043). AB - Evidence of increased hepatic metabolizing capacity coupled with reductions in serum thyroxine (T4) levels were noted in the rat during preliminary toxicity studies with FOE 5043, an oxyacetamide with herbicidal properties. These findings were consistent with reports in the literature suggesting that declines in T4 as a result of exposure to various classes of chemicals may be mediated extrathyroidally, such as through chemical induction of hepatic thyroid hormone metabolism. To examine this question with respect to FOE 5043, male rats were surgically thyroidectomized and provided thyroid hormone replacement therapy via implanted osmotic minipumps capable of maintaining a T4/triiodothyronine (T3) serum concentration for approximately 4 weeks at a level comparable to that of euthyroid controls. Seven days after minipump implantation, thyroidectomized + T4/T3 (TX + T4/T3) and nonthyroidectomized intact rats (NTX) were fed diets containing 0, 25, 1000, or 3000 ppm FOE 5043 for up to 3 weeks. Dose-related and equivalent declines in total and free serum T4 levels in both TX + T4/T3 and NTX rats were measured at Weeks 1, 2, and 3. Alterations in thyrotropin, total, free, and reverse serum T3 levels were also noted in both TX and NTX animals; however, a compound-related trend was difficult to discern and, when compared to the T4 response, the changes were markedly less consistent with respect to both time and dose. Additionally, dose-related increases in absolute and relative liver weights were measured in both TX + T4/T3 and NTX animals. As the only source of thyroid hormone in the TX + T4/T3 animals was that provided by the pump, these data suggest that FOE 5043-induced alterations in serum thyroid hormone levels, most notably T4, are being mediated indirectly, possibly as a result of increased hepatic metabolism, rather than through a direct effect on the thyroid gland. PMID- 7540337 TI - Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. AB - Modification of the genetic content of cultured cells or of whole animals is now a key strategy in both basic biological research and applied biotechnology. Yet obtaining the desired level and specificity of expression of an introduced gene remains highly problematic. One solution could be to couple expression of a transgene to that of an appropriate intact genomic locus. The identification and functional characterization of RNA sequences known as internal ribosome entry sites now offer the possibility of achieving precise control of transgene expression through the generation of dicistronic fusion mRNAs. PMID- 7540338 TI - [The potentials of local and intravascular laser irradiation for eliminating the immunological disorders in patients with chronic pyelonephritis]. AB - 61 patients suffering from chronic pyelonephritis in the stage of active inflammation associated with either urolithiasis (67.4%) or prostatic adenoma (32.6%) were divided into three groups. Group 1 received conventional antibacterial treatment, group 2 conventional treatment + local laser radiation (LLR), group 3 received combined therapy including intravascular laser irradiation of blood (IVLIB). The treatments efficacy was controlled by urine seeding for microflora and its sensitivity to antibiotics, by immunological supervision. The patients were found to have immunological abnormalities in the form of cellular and humoral immunity suppression, neutrophil phagocytic hypoactivity, elevated levels of medium-molecular peptides nonresponsive to standard antibacterial therapy. Adjuvant use of LLR improved humoral immunity and reduced total urine bacterial count. LLR replacement for IVLIB resulted in a complete regression of immunological shifts and more pronounced reduction of urinary bacterial contamination. PMID- 7540339 TI - [The relationship of postoperative thrombotic-hemorrhagic complications to the local acute inflammatory process in patients with prostatic adenoma]. AB - Clinical, laboratory and pathomorphological investigations evidence for close relationships between acute infectious inflammatory conditions of the prostate and thrombohemorrhagic complications arising both locally and systemically. All these postoperative complications manifesting clinically as acute prostatitis, epididymo-orchitis, urosepsis, bacteriotoxic shock, bleeding, thrombosis and embolism, latent or marked DIC syndrome have underlying local infectious inflammatory process (postoperative acute prostatitis). Preoperative detection of local infection (concomitant chronic prostatitis), monitoring of hemocoagulation, antibacterial and antiinflammatory therapy of chronic prostatitis and normalization of blood rheology improve surgical outcomes in prostatic adenoma and lead to less frequent occurrence of both acute inflammatory and thrombohemorrhagic complications. PMID- 7540340 TI - [The use of damping tension systems in urological practice]. PMID- 7540341 TI - Cell-free and cell-associated viremia in pigs after oronasal infection with Aujeszky's disease virus. AB - Nine pigs were examined for the presence of viremia during the first week after oronasal inoculation of 10(8.0) TCID50 Aujeszky's disease virus (ADV). Blood was taken at 1, 2, 3, 5 and 7 days post inoculation (PI) and the presence of cell free ADV in plasma and of ADV-infected mononuclear cells was examined by titration and by cocultivation with permissive cells, respectively. The mononuclear cells of 6 of the 9 pigs, collected at 3 and 5 days PI were further separated into subpopulations of enriched monocytes and enriched lymphocytes. Both subpopulations were cocultivated. Nasal secretions were collected from 4 of the 9 pigs for the determination of virus titers and interferon concentrations. Both infected mononuclear cells and cell-free ADV were demonstrated in 5 pigs, infected mononuclear cells only were found in 2 pigs, and neither cell-associated or cell-free ADV were detected in 2 pigs. Two of the 7 viremic animals were positive on one single day, 3 on 2 days, 1 on 3 days and 1 on 4 days. The number of infected cells was approximately 5 times higher in monocytes than in lymphocytes. The highest virus titers were present in those nasal fluids with the lowest alpha-interferon concentration. A correlation between the titer of locally produced ADV in the nose and the presence of a viremia was not found. In conclusion, we can state that a viremia regularly occurs under both cell-free and cell-associated form after an oronasal inoculation of ADV and that monocytes are the most susceptible mononuclear cells. PMID- 7540342 TI - [Therapeutic approaches in advanced breast cancer]. PMID- 7540343 TI - Serum prostate-specific antigen assay--an update. PMID- 7540345 TI - [Detection of enzyme activity in decontaminated spices in industrial use]. AB - A range of decontaminated species of industrial use have been examined for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material. PMID- 7540346 TI - [Differential development of 2 children with developmental dysphasia]. AB - The development of two boys with the diagnosis of specific language impairment each of whom spoke only five words at the age of three is described through the third grade (regular school). In spite of similarities in nonverbal abilities and in conditions of therapy and general care the two children developed quite differently. One achieved an average language level by about age five, whereas the other seemed to stagnate from about this age on even though he made good progress in other areas. The author discusses the role of auditory perception, learning strategies, memory and behavior in the latter child's development. PMID- 7540344 TI - Common antigenic epitopes are present on heat-labile oligomers of MDV glycoprotein B and on HSV glycoprotein B. AB - The antigenic cross-reactivity between the Marek's disease virus glycoprotein B (MDV gB) and glycoprotein B (gB) of herpes simplex virus type 1 and 2 (HSV1 and HSV2) was analysed by the immunoblotting method. We studied cell lysates in both denatured and in undenatured form (i.e., unheated) and reacted them with convalescent sera from chickens infected with the RBIB MDV strain and with human anti-HSV1 gB. Both sera detected the heat-labile MDV gB and the HSV gB oligomers. In addition, monospecific antibodies to the MDV gB 230 kDa oligomer (strain CVI988) were immunoaffinity purified from both the chicken and the human sera. The chicken and human monospecific antibodies detected the homologous and the heterologous gB oligomers in native MDV- and HSV1-infected cell lysates. 15 human sera were tested by immunoblotting and by immunofluorescence on HSV1-, CVI988-and herpes virus of turkeys (HVT)-infected cells. By both assays about half of the human sera reacted with MDV-infected cells. This study demonstrates that the MDV gB heat-labile oligomers possess conformational epitopes shared with the human alpha-herpes virus HSV1 and HSV2 gB heat-labile oligomers. PMID- 7540347 TI - Ca(2+)-dependent and Ca(2+)-independent NO-synthesizing activities of human primordial placenta. AB - In order to localize the site of production of nitrogen monoxide (NO) in first trimester human pregnancy, the cytosol and microsome fractions prepared from homogenized primordial placentas were tested for NO-synthase (NOS) activities by measuring the NADPH-dependent conversion of [3H]arginine to [3H]citrulline. Our results demonstrate that Ca(2+)-dependent enzyme activities are present in both fractions, whereas microsomes exhibit significant Ca(2+)-independent enzyme activity too. The highest specific activity is measurable in the presence of Ca2+ with microsomes, this activity is about 2-fold higher than the Ca(2+)-dependent specific activity of the cytosol. The Ca(2+)-independent specific NOS activity is about 30% of the Ca(2+)-dependent microsomal activity. The microsomal Ca(2+) dependent NOS activity is inhibited by 50% in the presence of 0.5 mM aminoguanidine (AG), whereas the Ca(2+)-independent activity does not respond to this concentration of AG, suggesting that it is not the inducible isoform of NOS. Our results indicate that (I) NOS activity is present from an early phase of placental development, (II) the NOS activity is of trophoblastic origin, since the primordial placenta is avascular and (III) NO-production by the primordial placenta can proceed in the absence of any Ca(2+)-mobilizing agonist. PMID- 7540348 TI - The effect of colchicine and vinblastine on bleomycin-induced lung fibrosis in rats. AB - The effect of colchicine and vinblastine on collagenase, lysyl oxidase and prolyl hydroxylase activities as well as on collagen cross-linking and types of this protein in bleomycin induced lung fibrosis in rats was investigated. Both colchicine and vinblastine diminish bleomycin-induced changes in the above mentioned enzyme activities as well as collagen cross-linking and normalize type I/type III collagen ratio. PMID- 7540349 TI - Expression and functional role of nitric oxide synthase in osteoblast-like cells. AB - Nitric oxide synthases (NOS) are enzymes that produce nitric oxide (NO) from L arginine in a reaction yielding citrulline as a coproduct. Nitric oxide modulates the activity of a wide variety of cells, but little is known about its effects on bone cells. In the present study we report that the NOS inhibitor NG-monomethyl-L arginine (NMMA) induced a dose-dependent inhibitory effect on the proliferation of the osteoblast-like cell lines MG63 and ROS 17/2.8. The inhibitory effect was prevented by increasing L-arginine concentrations in the medium and by the NO donor sodium nitroprusside. Likewise, NMMA inhibited interleukin-6 secretion, independently of its effect on cell number. NOS expression by MG63 cells was confirmed by measuring their ability to metabolize radiolabeled L-arginine to citrulline. NOS bioactivity was detected in unstimulated cells, but was markedly increased by stimulating the cells with cytokines, lipopolysaccharide, or 1,25 dihydroxyvitamin D3. NOS activity was partially dependent upon the presence of calcium in the medium. Furthermore, constitutive-type NOS (c-NOS) and inducible type NOS (i-NOS) mRNA expression was detected in ROS 17/2.8 cells after reverse transcription and polymerase chain reaction amplification. In conclusion, osteoblast-like cells express c-NOS and i-NOS, and NOS activity seems to play an important role in the regulation of cell proliferation and function. PMID- 7540350 TI - Nitric oxide: what role does it play in inflammation and tissue destruction? AB - Large amount of nitric oxide (NO) are produced at sites of inflammation through the action of inducible nitric oxide synthase (iNOS) present in both infiltrating leucocytes and activated, resident tissue cells. However, the role of NO in inflammation remains unclear. NO is a vasodilator, which inhibits the adhesion of neutrophils to the vascular endothelium; it reduces the production of IL-6 by Kupffer cells and chondrocytes, and the production of gamma-IFN and TNF-alpha by splenocytes. The literature provides contradictory information on the effect of NO on vascular leakiness, chemotaxis, prostaglandin production and tissue damage. Increasingly, data suggest that NO is immunosuppressive. Inhibitors of NOS have potent prophylactic activity in several but not all, animal models of inflammatory disease. However, in rat adjuvant arthritis, therapeutic activity is weak. Whether inhibitors of iNOS will be therapeutically useful in human inflammatory diseases cannot be predicted on the basis of present information. PMID- 7540351 TI - Regulation of function and expression of P-selectin. AB - P-selectin is an adhesion receptor for leukocytes that is expressed on activated platelets and endothelial cells. The surface expression of P-selectin is tightly regulated through signals in the cytoplasmic domain that mediate sorting into secretory granules, internalization into coated pits, and targeting to lysosomes for degradation. Like the other selectins, P-selectin binds sialylated, fucosylated carbohydrate ligands such as sialyl Lewis x. However, it binds with much higher affinity to PSGL-1, a sialomucin-like glycoprotein on myeloid cells. Binding of PSGL-1 to P-selectin may be essential for leukocytes to roll on P selectin-expressing cells under shear forces. PMID- 7540352 TI - Regulation of L-selectin expression by membrane proximal proteolysis. AB - L-selectin is a lectin cell adhesion molecule expressed on the cell surfaces of lymphocytes, monocytes and granulocytes. Upon leukocyte activation or L-selectin cross-linking the transmembrane-bound L-selectin is rapidly shed from the cell surface. Based on these observations, it has been proposed that L-selectin is proteolytically cleaved from the cell surface. However a panel of common protease inhibitors have no effect on L-selectin proteolysis. To further define the mechanism of L-selectin down-regulation we have produced reagents to study proteolytic fragments of L-selectin. We have developed a trapping ELISA for the detection of soluble L-selectin. In addition we have produced a high affinity polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L selectin from metabolically labeled PHA lymphoblasts and peripheral blood neutrophils. We review here our progress in defining a 6 kD L-selectin transmembrane peptide (L-STMP) from PMA activated lymphoblasts and fMLP-activated neutrophils. Radiochemical sequencing data indicate that the cleavage site occurs between Lys321 and Ser322 in a short membrane-proximal region of the extracellular domain. PMID- 7540353 TI - Transcriptional regulation of endothelial cell adhesion molecules: a dominant role for NF-kappa B. AB - A growing body of evidence demonstrates that elevated expression of the endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 at sites of inflammation in vivo is due in whole or part to the upregulation of transcription of their respective genes. Pharmacologic antagonism of transcription from these genes may therefore represent a novel approach to the development of anti inflammatory therapeutics. This paper reviews our current understanding of nuclear factors which act to regulate the transcriptional activity of the E selectin, VCAM-1 and ICAM-1 genes, and discusses that evidence which suggests that the nuclear transcription factor NF-kappa B acts as a dominant regulator of transcription from these genes. PMID- 7540354 TI - Gene targeting for inflammatory cell adhesion molecules. AB - Using gene targeting in mouse embryonic stem cells, it is possible to introduce diverse mutations into specific genes. Using these methods, various laboratories have reported mutations for a variety of inflammatory cell adhesion molecules including CD18, alpha 5 integrin, ICAM-1, P-selectin, and L-selectin; preliminary reports of other mutations are also available. Mutations in CD18 and ICAM-1 cause impaired inflammatory and immune responses, mutations in P-selectin and L selectin cause decreased leukocyte rolling and emigration, and a mutation in alpha 5 integrin causes embryonic lethality. Gene targeting complements other approaches for analyzing the function of inflammatory cell adhesion molecules. PMID- 7540355 TI - Nitric oxide poster discussion. PMID- 7540356 TI - Cytoplasmic transcription factors: mediators of cytokine signaling. AB - The distinct pattern of transcriptional responses of cells to different extracellular signals requires a signal transduction pathway that provides rapid, accurate, and faithful transmission of information from the cell surface to the nucleus. One mechanism exploited by many cytokines, exemplified by interferons (IFN) but also used by many interleukins and growth factors, uses a family of cytoplasmic transcription factors that are activated by tyrosine phosphorylation. Once phosphorylated by receptor-associated tyrosine kinases, these proteins assemble into multimeric transcription factors, translocate to the nucleus, and bind specific DNA sequence elements in the promoters of target genes. PMID- 7540357 TI - [Induction and properties of nitric oxide synthase in rat retinal pigment epithelial cells in culture]. AB - We investigated the production of nitric oxide (NO) and nitric oxide synthase (NOS) by rat retinal pigment epithelial cells (RPE) in culture. METHODS: RPE was isolated from the eyes of 7-10 day-old rats and cultured in Dulbecco's modified Eagle medium with 10% fetal calf serum. NO2- concentration in the culture medium was determined with the Griess reaction. NOS was assayed by measuring 3H citrulline as the reaction product. RT-PCR was performed on the isolated RNA fractions from RPE. RESULTS: Rat RPE produced NOS with the combined stimulation of the bacterial endotoxin and cytokines including interferon gamma. PCR experiments showed that stimulated RPE expressed a gene product corresponding to inducible-type NOS. The enzyme required arginine, NADPH, and tetrahydrobiopterin for the reaction. The specific activity of the crude RPE extract was approximately 270 pmol/min/mg. CONCLUSION: Like bovine RPE, rat RPE produced NOS by the immunological stimulation. Induced NOS and the product NO may play important roles in inflammation of the eye. PMID- 7540359 TI - [The effect of interferon-beta on experimental choroidal neovascularization]. AB - We studied histologically the effect of systemic administration of human interferon-beta (IFN-beta) on experimental choroidal neovascularization which was caused by intensive laser photocoagulation at the posterior pole of monkey eyes. The regression of choroidal neovascularization was observed in IFN-treated monkeys. Histologically, retinal pigment epithelium (RPE) was remarkably abundant around choroidal neovascularization in the subretinal space, and the activity of neovascularization was weaker in IFN-treated monkeys than in untreated monkeys. These results indicate that IFN-beta promotes the proliferation of RPE and suppresses the activity of endothelial cells of neovascularization to cause regression of choroidal neovascularization. The results suggest that IFN-beta is clinically useful to treat the choroidal neovascularization of age-related macular degeneration. PMID- 7540361 TI - Medical posters: lessons on the wall. PMID- 7540358 TI - [Effect of human interferon-beta on reconstruction of the choriocapillaris in monkeys following laser photocoagulation]. AB - We studied the effect of human interferon-beta on reconstruction of the choriocapillaris following laser photocoagulation in monkey eyes. Moderate dye laser photocoagulation caused the occlusion of the choriocapillaris by the intraluminal thrombus in the photocoagulated lesions on the retina. After 3 days, immature endothelial cells began to migrate towards the center from the edge of the lesions. After 7 days capillaries were newly formed, and after 14 days the choriocapillaris in the lesions was almost reconstructed. Systemic administration of interferon-beta after photocoagulation suppressed the reconstruction of the choriocapillaris remarkably. These results suggest that interferon-beta inhibits proliferation and migration of the capillary endothelium on the retina. Interferon-beta may be effective as medication for the treatment of age-related macular degeneration. PMID- 7540360 TI - [Clinical efficacy of the Porges Urospiral, a reversible intraprostatic spiral stent, in patients with benign prostatic hypertrophy]. AB - The Porges Urospiral, a urethral spiral stent made of stainless steel wire, is designed for patients with benign prostatic hypertrophy. To evaluate the safety and efficacy of the stent, a prospective uncontrolled study was performed on 60 patients who complained of urinary retention (48 patients) or severe dysuria (12 patients) due to prostatic hypertrophy. Four patients had neurogenic disorders resulting in bladder dysfunction. The average age of the patients was 77.4 years old, ranging from 61 to 91 years old. In 48 patients, the stent was used instead of transurethral resection of the prostate (TUR-P) because of the patients poor general conditions, while in 12 patients the stent was transiently used until TUR P. Subjective symptoms, according to an original scoring system, and objective signs, uroflowmetry measurements and residual urine volume, before and 3 months after stent insertion were compared. The stent was indwelt for more than 3 months in 40 of the 57 patients in whom the stent could be inserted. Out of 37 evaluable patients, subjective symptoms improved in all the patients and objective symptoms improved in 26 patients (70.3%). Complications included urinary incontinence, urination pain, urethral pain, gross hematuria or migration of the stent. Although these complications disappeared in a few days after the insertion of the stent in a majority of the patients, the stent was removed in 17 patients due to complications within 3 months after insertion. The overall clinical efficacy of this stent was 56% in 50 evaluable patients who had no neurogenic disorders.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540362 TI - Cyclin D1 expression in non-Hodgkin's lymphomas. Detection by immunohistochemistry. AB - Cyclin D1/PRAD1 (bcl-1) is a recently discovered proto-oncogene that is overexpressed in mantle cell lymphomas and several other human tumors. In a previous study, the authors demonstrated expression of cyclin D1 in 15 of 15 cases of mantle cell lymphoma and 1 of 8 cases of B-chronic lymphocytic leukemia (B-CLL) using a polyclonal antibody and microwave enhanced immunohistochemical staining method on paraffin-embedded tissue sections. In this study, 107 additional B- and T-cell neoplasms were studied, including 47 cases of high grade lymphoma (33 diffuse large B-cell type, 9 Burkitt and Burkitt-like, 4 precursor T lymphoblastic lymphoma, and 1 adult T-cell lymphoma/leukemia), 38 additional cases of low grade B-cell lymphoma (18 CLL, 15 hairy cell leukemia and 5 mantle cell lymphoma), and 22 plasmacytomas for expression of cyclin D1 using the same immunohistochemical staining technique. All cases of mantle cell lymphoma showed diffuse nuclear staining. No additional cases of CLL showed cyclin D1 expression. In contrast, 1 of 15 hairy cell leukemias and 1 of 22 plasmacytomas showed cyclin D1 staining. None of the high grade lymphomas demonstrated expression of cyclin D1 protein by immunostaining, including three cases of large B-cell lymphoma that coexpressed CD5. The authors conclude that cyclin D1 is expressed in all cases of mantle cell lymphoma, and only in very rare cases of B-CLL, hairy cell leukemia and plasmacytoma/myeloma. Cyclin D1 does not appear to play an important role in high grade lymphomas. In addition, most CD5 positive high grade B-cell lymphomas do not express cyclin D1, and are not likely to be derived from mantle cell lymphoma or other lymphomas with t(11;14). PMID- 7540364 TI - Therapeutic considerations: symptoms, cells and mediators. AB - Immuno-histopathological studies of conjunctival tissue biopsied from patients with non-sight-threatening allergic conjunctivitis or with sight-threatening allergic keratoconjunctivitis should lead to more effective management of these eye conditions, based on the specific cellular involvement. The major difference between these two categories of eye disease was the occurrence of T-lymphocytes, which were absent in the former but prominent in the sight-threatening disorders. Seasonal and perennial allergic conjunctivitis both showed a heavy mast cell increase, due to infiltration of mucosal type mast cells, and allergen challenge studies linked mast cell histamine release to the early phase reaction occurring within 20 minutes. A second histamine peak at six hours after challenge might implicate basophils (or refractory mast cells) and was accompanied by a rise in eosinophil cationic protein. In sight-threatening, chronic allergic keratoconjunctivitis the responses were clearly directed by T-cells, themselves the primary effector cell in atopic keratoconjunctivitis, whereas vernal keratoconjunctivitis displayed a T-cell driven eosinophilia, with increased expression of the adhesion molecules involved in tissue invasion by these cells. Appropriate therapies for each different category of conjunctivitis should be based on the specific immunopathology, and directed at the activated cell types that are primarily responsible for the disease process. PMID- 7540363 TI - The effect of povidone-iodine solution applied at the conclusion of ophthalmic surgery. AB - PURPOSE: Povidone-iodine 5% solution decreases the incidence of postoperative endophthalmitis when used on the eye for preoperative preparation. We sought to determine whether it also minimized conjunctival bacterial flora immediately after surgery by preventing bacteria present on the surface of the eye from entering surgical wounds. METHODS: In 42 eyes of 40 patients, at the conclusion of surgery, on an alternating basis, each patient received either a drop of a broad-spectrum antibiotic solution (polymyxin B sulfate-neomycin sulfate gramicidin) or a 5% povidone-iodine solution in the operated-on eye. Bacterial cultures were taken before and after surgery and 24 hours later. The 38 unoperated-on eyes in the unilateral cases served as control eyes. RESULTS: Relative to the control group, povidone-iodine was effective in preventing an increase in the number of colony-forming units (P = .035), while the antibiotic was not. At 24 hours after surgery, the species count was lower in the eyes receiving povidone-iodine than in the antibiotic-treated eyes (P = .034) and was increased in the antibiotic group since the completion of surgery (P = .013), but was lower in the povidone-iodine and antibiotic groups than in the control eyes for both groups (P < .01). CONCLUSIONS: Povidone-iodine 5% solution applied to the eye at the conclusion of surgery was more effective at minimizing the number of colony-forming units and species for the first postoperative day than was a broad-spectrum antibiotic. While not true for the antibiotic, the antimicrobial effect of povidone-iodine lasted for at least 24 hours after the completion of surgery. PMID- 7540365 TI - Analysis of phosphorylhydroxyamino acids present in hydrolyzed cell extracts using dabsyl derivatization. AB - We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell studies. PMID- 7540367 TI - Determination of sulfamethazine residues in milk by a surface plasmon resonance based biosensor assay. AB - The use of antibiotics and chemotherapeutics in animal husbandry has led to the occurrence of veterinary drug residues in all types of food of animal origin. Due to the specification of toxicologically based maximum residue levels for a large number of substances, existing control strategies need even faster and more sensitive methods to meet new and more rigorous regulations. The applicability of an immunosensor device for biospecific interaction analysis was investigated and the development of an assay for analysis of sulfamethazine (SMZ) in milk is described. SMZ was covalently immobilized to a carboxymethyldextran-modified gold film. Spiked samples with known concentrations of SMZ were prepared in HBS buffer and skim and raw milk for construction of standard curves. Polyclonal antibodies against SMZ were added to the sample and the immobilized surface was used to determine the amount of free antibodies by surface plasmon resonance detection. After each measurement the surface was regenerated by NaOH and HCl. In milk, the mean relative standard deviation of the assay was approximately 2% and the limit of detection less than 1 ppb. By introduction of a secondary sheep anti-rabbit antibody, the use of specific antibody could be reduced. Milk samples from the individual cow, herd, and tanker levels were analyzed and the relative standard deviations within each sample category were 4.4, 2.4, and 2.2%, respectively. The effect of some potential interferences, e.g., high somatic cells, bacterial contamination, and preservatives, was investigated. The results were not influenced in such a way that the risk for so-called false-positive findings was obvious. PMID- 7540366 TI - Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. AB - We propose a three-dimensional (3-D) sugar-mapping technique for pyridylaminated (PA) neutral and sialyl oligosaccharides as a powerful structural characterization of N-linked oligosaccharides using only picomoles of samples. The new map consists of the elution data from 42 different sialyl oligosaccharides, 26 of which are mono-, 7 of which are di-, 7 of which are tri-, and 2 of which are tetra-sialylated oligosaccharides. The 20 standard sialyl oligosaccharides were released from human serum and calf fetuin by digestion with glycoamidase A. The other 22 standard sialyl oligosaccharides were obtained by subsequent digestion of the above 20 sialyl oligosaccharides with beta galactosidase, beta-N-acetylhexosaminidase, alpha-fucosidase, and alpha 2-->3 specific sialidase. The present 3-D mapping method involves the following four steps: First, a neutral and sialyl PA-oligosaccharide mixture is separated by HPLC on the diethylaminoethyl (DEAE) column according to the sialic acid content, and the elution data are considered as one of the three dimensions (Z-axis). Then, neutral, mono-, di-, tri-, and tetra-sialyl oligosaccharides are individually separated on the octadecylsilyl (ODS)-silica (X-axis) and amide silica (Y-axis) columns. The fourth step is to plot the coordinates on a two dimensional (2-D) map. Thus, for each of the groups separated on the DEAE column, a 2-D map can be achieved. By repeating the whole process for each group of different sialylation, the layers of the 2-D map lined up on the Z-axis form a 3 D map.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540368 TI - Activity staining of xylanases in polyacrylamide gels containing xylan. PMID- 7540369 TI - Optimization of RNase A artificial hydrophobization in AOT reversed micelles. PMID- 7540370 TI - Osteopontin overview. PMID- 7540371 TI - Osteopontin-induced modifications of cellular functions. AB - Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the alpha v beta 3 integrin. We have investigated the action on mammalian cells of recombinant OPN made both in E. coli and in human cells. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosphorylation status of several proteins including pp60src and components of focal adhesion complexes. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell peroxide levels, promote the survival of cells exposed to hypoxia, and inhibit the killing of tumor cells by activated macrophages. PMID- 7540372 TI - Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity. PMID- 7540373 TI - Interaction of osteopontin with osteoclast integrins. AB - Mammalian osteoclasts express three integrin receptors--alpha v beta 3 (vitronectin receptor), alpha 2 beta 1 and alpha v beta 1. The vitronectin receptor recognizes bone matrix proteins, including bone sialoproteins, in an RGD dependent manner, whereas adhesion to collagen involves beta 1 integrins. Interference with integrin function, by anti-receptor antibodies or RGD-peptides, blocks bone resorption. Data on the mechanism of osteoclast adhesion to sialoproteins and the differential synthesis of osteopontin and bone sialoprotein by osteoclasts is presented. Thus, osteoclasts adhere to both osteopontin and bone sialoprotein with a characteristic irregular morphology with numerous, peripherally placed, actin-rich podosomes. Adhesion is predominantly RGD and beta 3 dependent, though alpha v beta 1 may also be involved in adhesion to bone sialoprotein. KQAGD and AGDV, but not H12, fibrinogen peptides induce osteoclast 'rounding' on osteopontin suggesting there is an alternative anti-adhesive signal to 'RGD.' However, adhesion is not completely inhibited and is not specific for osteopontin as equivalent effects are seen with adhesion to serum. The role of sialoproteins in osteoclast adhesion in situ in the skeleton is complicated by the finding of endogenous synthesis of osteopontin, but not bone sialoprotein, by osteoclasts. The disposition of osteoclast integrins during resorption and the role of integrins and sialoprotein-derived peptides in osteoclast adhesion and function is also reviewed. PMID- 7540374 TI - Roles of osteopontin in bone remodeling. PMID- 7540375 TI - Characterization of the interaction between integrins and recombinant human osteopontin. PMID- 7540376 TI - Osteopontin: a ligand for the alpha v beta 3 integrin of the osteoclast clear zone in osteopetrotic (ia/ia) rats. PMID- 7540377 TI - Activation of a signal transduction pathway by osteopontin. PMID- 7540378 TI - Divergent expressions of two cell adhesion molecules at the leading edge of a migrating cell sheet. PMID- 7540379 TI - Gene expression and phosphorylation of mouse osteopontin. AB - Osteopontin is expressed in many different cell types and has been proposed to play several functions. Distinct forms of the protein have been detected. Various tissues and cell lines from mouse, however, exhibit two classes of transcripts with different 5'-untranslated ends but with an identical coding region (exons II through VII). These transcripts do not arise from the alternative splicing of coding exons. These results suggest that posttranslational modifications of osteopontin, such as phosphorylation, are a major mechanism to generate different forms of the protein. Mouse osteopontin was expressed in E. coli and used as a model to study its phosphorylation. PMID- 7540380 TI - Osteopontin at the tumor/host interface. Functional regulation by thrombin cleavage and consequences for cell adhesion. PMID- 7540381 TI - Serum amyloid A protein in patients with acute myocardial infarction. AB - The concentrations of four acute phase proteins were measured in sera of 40 patients with acute myocardial infarction (AMI) to evaluate their behaviour from day-to-day and to find out if they can serve for early prediction of postinfarction complications and mortality rate. Peak levels of serum amyloid A protein (SAA) were increased up to 5000-fold above the normal value and those of C-reactive protein (CRP) about 100-fold, 3 days after AMI. alpha 1 antichymotrypsin (ACT) and alpha 1-acid glycoprotein (AGP) peak levels were increased up to eightfold above their normal values. Patients who developed postinfarction complications had significantly higher SAA values on admission than those without complications (mean values of 379 and 45 mg/L, respectively; P < 0.0001). Using a level of 100 mg/L on admission as a reference value gave a reasonable sensitivity and predictive value for complications (73%) and a very good sensitivity (80%) for early prediction of fatal outcome. Patients with SAA values above this limit had double the risk of complications and four times the risk of a fatal outcome. The correlation with CRP values was lower than it was with SAA values (P = 0.028) using a level of 15 mg/L on admission as reference value gave low sensitivity (55%) and predictive value (60%) for complications as well as low sensitivity for early prediction of fatal outcome (60%). The present study did not allow prediction of complications or mortality based on ACT or AGP values. PMID- 7540382 TI - First trimester biochemical screening for trisomy 21: the role of free beta-hCG and pregnancy associated plasma protein. PMID- 7540383 TI - Analysis of alpha-fetoprotein by a procedure combining crossed immunoaffinoelectrophoresis and immunoblotting. AB - A newly developed procedure combining crossed immunoaffinoelectrophoresis and an immunoblotting assay (CIAE-IBA) has been used to discriminate between and precisely quantitate lectin-bound and lectin-free forms of alpha-fetoprotein (AFP) derived from human serum at levels of AFP as low as 5.6 ng/ml (adult reference range, 0 to 15). Twenty-two serum specimens with elevated levels of AFP were examined by a reference CIAE method, using either of two lectins, concanavalin A (Con A) or Lens culinaris agglutinin (LCA); aliquots of the specimens were then diluted in AFP-free serum to normal or near-normal levels of AFP and examined by CIAE-IBA, which included a final protein transfer to nitrocellulose. The quantitative results produced by the two methods were essentially the same. Overall, the sensitivity of the CIAE-IBA procedure surpasses that of other CIAE methods of AFP fractionation. The CIAE-IBA procedure may be used for study of the heterogeneity of AFP and may be applicable as well to the study of other proteins. PMID- 7540385 TI - Hytrin treatment of hypertension and benign prostatic hyperplasia. AB - BPH is a diagnosis that needs to be considered and evaluated in older male renal disease/failure patients. Rather than proceeding to the invasive treatment options, Hytrin should be considered as the treatment of choice. Not only can it relieve the symptoms of BPH, but also be an effective treatment for hypertension. Ongoing assessment, observation for side effects, monitoring for lower urinary tract dysfunction, along with patient education and documentation are the responsibilities of nephrology nurses. PMID- 7540386 TI - Species specificity at the molecular level: the case of nitric oxide synthases. PMID- 7540384 TI - Differential activities of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives against different human immunodeficiency virus type 1 mutant strains. AB - A series of 23 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives that were highly potent inhibitors of wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1/IIIB) replication in CEM cells were evaluated against a panel of HIV-1 mutant strains containing the replacement of leucine by isoleucine at position 100 (100-Leu-->Ile), 103-Lys-->Asn, 106-Val-->Ala, 138-Glu-->Lys, 181 Tyr-->Cys, 181-Tyr-->Ile, or 188-Tyr-->His in their reverse transcriptase (RT). A different structure-antiviral activity relationship was found, depending on the nature of the mutated amino acid in the HIV-1 RT. The results show that 5-ethyl-1 ethoxymethyl-6-(3,5-dimethylbenzyl)uracil, 5-ethyl-1-ethoxymethyl-6-(3,5 dimethylphenylthio)uracil, and 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylphenylthio) 2-thiouracil remain active against the majority of viruses containing single mutations which confer resistance to nonnucleoside RT inhibitors. PMID- 7540387 TI - Acinic cell carcinoma of the lacrimal gland. AB - An 18-year-old woman underwent exenteration of the right orbit for tumor recurrence 3 years subsequent to external-beam irradiation for a lacrimal gland tumor diagnosed as an "adenocarcinoma." Light microscopy of the exenteration specimen revealed an acinic cell carcinoma of the lacrimal gland, with a predominant microcystic (latticelike) pattern of growth. Cytoplasmic vacuoles and the secretion within the microcysts stained positive with periodic acid-Schiff with and without alpha-amylase, alcian blue (at a pH of 2.5), mucicarmine, and colloidal iron with and without hyaluronidase. This histochemical staining for epithelial mucins supports the theory that the lacrimal gland, although serous in type, may also function as a modified mucus gland. There was cytoplasmic immunopositivity for keratin (CAM 5.2, KAE 1-3); immunostaining for vasoactive intestinal polypeptide was negative. Electron microscopy disclosed undifferentiated features of intercalated duct cells. We speculate that the lack of immunoreactivity for vasoactive intestinal polypeptide may be correlated with the predominantly undifferentiated intercalated duct cell features observed ultrastructurally. PMID- 7540388 TI - Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin. AB - OBJECTIVE: To investigate photodynamic therapy of experimental choroidal neovascularization using benzoporphyrin derivative monoacid (Verteporfin). METHODS: Photodynamic therapy using benzoporphyrin derivative monoacid was investigated in cynomolgus monkeys. Following intravenous injection of benzoporphyrin derivative monoacid (1 to 2 mg/kg) complexed with low-density lipoprotein, the eyes were irradiated with 692-nm light at a fluence of 50 to 150 J/cm2 and irradiance of 150 to 600 mW/cm2. Choroidal neovascularization was documented before photodynamic therapy and closure was demonstrated by fundus photography, fluorescein angiography, and light and electron microscopic examination. RESULTS: Following photodynamic therapy, vessels within choroidal neovascularization were occluded, and there was damage to the choroidal neovascularization endothelium and the subjacent choriocapillaris. Damage to the retinal pigment epithelium and photoreceptors was also observed. CONCLUSION: Photodynamic therapy with lipoprotein-delivered benzoporphyrin derivative monoacid was effective in this animal model of choroidal neovascularization and may be a promising, potentially selective, therapy for choroidal neovascularization. PMID- 7540390 TI - [Pathobiologic aspects of the limited extracranial metastasis capability of tumors of the central nervous system]. AB - Primary tumors of the central nervous system (CNS) very rarely metastasize to other parts of the body, both in animals and in man, irrespective of survival time. The only exception to this rule is observed in meningiomas in man with a metastasis incidence of 18.7%. Experimental studies have provided strong evidence for an important modulating role of the extracellular matrix upon the invasion capacity of tumors. Malignant progression of tumors depends upon accumulation of somatic mutations which impart a selective advantage for invasion into the surrounding connective stroma. Such mutations seem to result from an active confrontation and interaction between tumor cells and components of the stroma. As the parenchyma of the CNS is largely free of connective tissue no such selection interplay occurs in genuine CNS tumors, in contrast to tumors of the other parts of the body with an abundance of connective tissue surrounding them. This explains the rarity of extracranial metastases of CNS tumors. In contrast, other tumors of the body metastasize more frequently into the CNS. Obviously, this potency is correlated with the frequency of mutations at the CD44 gene locus. PMID- 7540389 TI - Cellular adhesion molecules on periodontal lymphocytes. AB - T cell induced differentiation of B cells has been shown to be dependent on the CD2/LFA-3 and LFA-1/ICAM-1 pathways. Flow cytometric analysis was used to examine these adhesion molecules on T and B cells extracted from gingival tissues before and after stimulation with the putative periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion molecule expression on peripheral blood cells from healthy adults was used as a control. Approximately 50 per cent of B cells extracted from gingival tissues expressed LFA-3 and ICAM-1 compared with 30 per cent positive peripheral blood B cells. Around 50 per cent of gingival T cells expressed CD2 relative to 76 per cent positive peripheral blood T cells. However, 40-50 per cent of both gingival and peripheral blood T cells expressed LFA-1. There was no difference in the expression of adhesion molecules on T and B cells extracted from health/marginal gingivitis or adult periodontitis lesions. After stimulation of gingival cells in vitro, the per cent CD2 positive T cells and LFA-3 and ICAM-1 positive B cells remained relatively stable over the six-day culture period, although P. gingivalis and F. nucleatum appeared to induce an increase in the percentage of gingival T cells expressing LFA-1. In contrast to the gingival lymphocytes, stimulation of peripheral blood cells resulted in an increase in the per cent CD2 positive T cells, LFA-3 and ICAM-1 positive B cells, with a decrease in LFA-1 positive T cells. The results therefore demonstrated that gingival T and B cells express adhesion molecules in vivo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540391 TI - Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enriched endothelial cell membranes. AB - Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in superoxide (O2-) and a 2.5-fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO-) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius plots of [(ro/r)-1] 1 indicated that the lipid phase separation of the membranes at 26.2 +/- 1.5 degrees was elevated to 34.4 +/- 1.9 degrees in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 +/- 1.6 degrees, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 +/- 1.8 degrees which rose to 35.6 +/- 2.1 degrees in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 +/- 1.4 degrees in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties of plasma membrane-bound NO synthase inhibited by Mn2+ (as reflected by changes in the Hill coefficient) were changed by cholesterol, consistent with modulations of the fluidity of the lipid microenvironment of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7540392 TI - Stimulation of placental prorenin secretion by selective inhibition of cyclic nucleotide phosphodiesterases. AB - Prorenin secretion by human villous placenta is known to be stimulated by activation of adenylate cyclase and enhanced cyclic AMP (cAMP) generation. Placental tissue contains predominantly type III (cGMP-inhibited) and type IV (cAMP-specific) phosphodiesterases (PDEs), which inactivate cAMP. To evaluate the role of PDE subtypes in the regulation of prorenin secretion by human placenta, explants were cultured in the presence of isobutylmethylxanthine (IBMX), a non selective PDE inhibitor, and selective inhibitors for various PDE subtypes. Inhibition of PDE subtypes with cilostamide (type III), Ro 20-1724 (type IV) and zardaverine (types III and IV) increased prorenin release. Inhibition of type I (Ca(2+)/calmodulin-dependent) PDE by 8-MeoM-IBMX and of type V (cGMP-specific) PDE by zaprinast or dipyridamole did not affect prorenin secretion. The stimulation of prorenin secretion by PDE inhibitors was attenuated by cAMP dependent protein kinase inhibition. The selective PDE inhibitors caused a parallel increase in media cAMP and prorenin and also increased tissue prorenin levels. These studies demonstrate that cAMP degradation by type III and IV PDE isoenzymes is a major regulatory mechanism for placental prorenin secretion. It is suggested that enhancers of adenylate cyclase activity are constitutively present in placenta and influence prorenin synthesis and release. PMID- 7540393 TI - Processing of the neuropeptide growth factor antagonist [Arg6, D-Trp7.9, NmePhe8] substance P (6-11) by a small cell lung cancer cell line (H69). AB - [Arg6, D-Trp7.9, NmePhe8]-substance P (6-11) (antagonist G) is a broad spectrum neuropeptide growth factor antagonist about to enter clinical trials as an anticancer drug. Its fate has been studied after incubation with two densities (5 x 10(4) cells/mL and 1 x 10(6) cells/mL) of the H69 small cell lung cancer cell line for up to 7 days at a concentration of 20 microM, corresponding to the IC50 for growth inhibition. HPLC analyses were conducted on cell pellets and media and in controls consisting of cell free media and water. Over 7 days in media containing cells a 70.4% reduction in parent peptide concentration occurred at the high density and a 44.1% reduction at low density. Despite this, there was a steady elevation in peptide associated with cells reaching a 189% increase by day 7. Oxidation of G at the C-terminal methionine residue occurred in all media studied indicative of a chemical process. The two major active metabolites of antagonist G (deamidated G and G minus Met11) were detected only in media in the presence of cells. These accumulated with time in media and cells together with oxidized products. These results reveal complex cellular pharmacology for antagonist G where H69 cells are increasingly exposed to 4 different peptide products rather than 1. PMID- 7540394 TI - Four diterpene esters from Euphorbia myrsinites. AB - The Turkish species Euphorbia myrsinites has yielded four new tetracyclic diterpene tetraesters from a cytotoxic acetone extract, in addition to the known cycloartane-type triterpenoids and betulin. The new compounds and their hydrolysis product have been extensively characterized by high field spectroscopic techniques, and were shown to be four new tetraesters of the parent alcohol, myrsinol. PMID- 7540395 TI - Biocatalysis in organic chemistry (Part I): Past and present. AB - Methods used in the manufacture of both ethanol and ascorbic acid have been developed over the past 150 years. The early stages of the development of both processes were characterized by the interaction between chemists, biologists and engineers, but social and economic influences also played their part. The history of these two examples illustrates the diversity of skills and influences needed for the application of biological catalysis to large-scale chemical manufacture. PMID- 7540396 TI - The effect of NG-nitro-L-arginine methyl ester upon hindlimb blood flow responses to muscle contraction in the anaesthetized cat. AB - The aim of the present experiment was to investigate the relative contribution of nitric oxide produced in endothelial cells to functional and reactive hyperaemia in the hindlimb of anaesthetized cats. Cats (2.5-3.4 kg) were anaesthetized with alphadalone-alphalaxone, and breathed spontaneously following tracheotomy. Left hindlimb blood flow was measured with a flow probe and hyperaemia responses were monitored following 10 s occlusion of the left external iliac artery and during 20 min stimulation of the sciatic and femoral nerves at 3 Hz. This was repeated following nitric oxide synthase inhibition with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg kg-1, I.V.). Following L-NAME administration, baseline hindlimb blood flow and arterial blood pressure were restored by infusion of sodium nitroprusside (range, 0.3-2.25 micrograms kg-1 min-1, I.V.). Following arterial occlusion, L-NAME reduced the peak reactive hyperaemia (6.5 +/- 0.8 vs. 4.5 +/- 1.0 ml min-1 kg-1, P < 0.05) and blood flow repayment (9.9 +/- 2.3 vs. 6.1 +/- 2.6 ml, P < 0.05) responses. In contrast, the total functional hyperaemia response during hindlimb contraction was not altered (264.7 +/- 68.2 vs. 264.4 +/ 62.8 ml kg-1, n.s.). The results of the study suggest that the production of nitric oxide from endothelial cells does not contribute to functional hyperaemia in contracting skeletal muscle, but plays a large role in reactive hyperaemia. The results imply that flow-dependent dilatation of feed arteries is mediated by NO in reactive hyperaemia. PMID- 7540397 TI - Suppressed fibrinolysis after administration of low-dose aprotinin: reduced level of plasmin-alpha2-plasmin inhibitor complexes and postoperative blood loss. AB - Various clinical investigation have shown that aprotinin therapy reduces bleeding after open-heart operations. In this study low-dose aprotinin, 30,000 KIU/kg in the cardiopulmonary bypass (CPB) priming volume and 7,500 KIU/kg intravenously each hour during CPB, was used in ten patients undergoing primary myocardial revascularization or surgery for valvular diseases. Another ten patients served as controls. Blood loss, platelet count and plasma levels of hemoglobin, antithrombin III, fibrinogen, fibrinogen degradation products (FDP), total plasmin inhibitor and alpha2-plasmin inhibitor-plasmin complexes were evaluated at nine preoperative, intraoperative and postoperative points. Intraoperative and postoperative blood loss was significantly reduced in the aprotinin group. There was no significant difference between the two groups in platelet count and levels of hemoglobin and antithrombin III. A significant increase in FDP during CPB in the control group indicated hyperfibrinolysis. The levels of plasmin inhibitor were significantly reduced during CPB in the control group. The alpha 2-plasmin inhibitor-plasma complex levels, indicating the plasmin activity, were significantly reduced in the aprotinin group. These results confirmed that low dose aprotinin reduced blood loss with the prevention of hyperfibrinolysis during CPB and demonstrated improved hemostasis. PMID- 7540398 TI - Asthma and the motorways--one District's experience. AB - BACKGROUND: Proposals to widen the section of the M25 motorway that passes through East Surrey caused considerable concern and claims that the traffic on the road was responsible for an increasing prevalence of asthma. This study was designed to ascertain the prevalence of asthma-related symptoms among schoolchildren in the District and determine whether there was an association with proximity to a motorway. METHODS: A total of 2387 children aged between 13 and 14 from 17 schools in East Surrey were invited to participate in a survey of the prevalence of asthma-related respiratory symptoms. The survey instrument used was the core asthma questionnaire of the International Survey on Asthma and Allergies in Childhood (ISAAC). The children were grouped according to electoral ward of residence and comparisons were made between urban and rural wards, and the presence or absence of a section of motorway. RESULTS: The prevalence of reported symptoms varied from 2.2 per cent reporting sleep disturbance on more than one night each week to 40 per cent reporting a wheezing episode at some time in their lives; 16 per cent reported a diagnosis of asthma. The overall prevalence of symptoms did not differ greatly from that reported in other similar studies. There was a decrease in the prevalence of asthma-related symptoms in areas close to motorways, which was statistically significant for two questions. This is unlikely to be due to the rural location of the motorways. CONCLUSION: This study suggests that the motorways in East Surrey, in their present configuration, are not responsible for an increased prevalence of respiratory symptoms associated with asthma. PMID- 7540399 TI - Lymphocytotropic strains of HIV type 1 when complexed with enhancing antibodies can infect macrophages via Fc gamma RIII, independently of CD4. AB - Antipeptide sera raised against the gp120/gp41 sequences of human immunodeficiency virus type 1 (HIV-1) were used to determine their capacity to enhance infection. Antisera to the five variable regions (V1 to V5) of gp120 and conserved parts of gp120 and gp41 facilitated infection of primary human macrophages with the homologous virus HIV-1 SF2mc. In contrast, heterologous virus infection with HTLV-IIIB was mediated only by antisera to the conserved regions, predominantly C4 and C5. Heterologous virus infection occurred more rapidly and was consistent between different cell donors. The neutralizing monoclonal antibody (MAb) SC258 (murine IgG2a) but not MAb 684-238 (mIgG1) against conformational epitopes of the V2 region also induced antibody-dependent infection enhancement (ADE). Therefore, preincubation with certain antibodies can cause altered tropism of the lymphocytotropic viruses mentioned above. Viral infection was completely abolished by preincubation with the F(ab)2 fragment of MAb 3G8 against the Fc gamma receptor III (CD16). A MAb (7.3F11) against the gp120-binding site of CD4 had no effect on viral infectivity. Possible mechanisms and their implications for disease progression are discussed. PMID- 7540400 TI - Testicular cancer treated in a minor general oncology department. AB - The question of where to treat testicular cancer and by whom has been debated in several medical journals over the last few year. Here we present data from 98 patients (47 seminomas, 51 non-seminomas) treated between January 1985 and March 1993 at the Department of Oncology, University Hospital of Tromso, Norway. During a 4-year median follow-up period 2 patients died of progressive disease. Our results are similar to those of major specialised oncology centres. We argue that within the context of multicentre cooperative studies or treatment protocols, patients with testicular cancer can be treated in a small general oncology centre with the same expectations of cure and treatment-related mortality and morbidity as achieved in major centres. PMID- 7540402 TI - Levels of circulating intercellular adhesion molecule-1 in patients with metastatic cancer of the prostate and benign prostatic hyperplasia. AB - Current reports suggest a role for intercellular adhesion molecule-1 (ICAM-1) in the progression of malignancy. The availability of a new antibody makes it possible to measure circulating ICAM-1 (cICAM-1) in human body fluids including serum; this might help in monitoring tumour burden and in providing additional prognostic information. In this study, serum levels of cICAM-1 were measured by an ELISA assay in patients with benign prostatic hyperplasia (BPH; n = 20) and metastatic cancer of the prostate (CaP; n = 25). Serum ICAM-1 concentrations were also measured in a group of healthy men (n = 8). The mean +/- S.E.M. cICAM-1 level for BPH was 339.52 +/- 15.30 ng/ml compared with 263.55 +/- 18.54 ng/ml for CaP. Even though the difference between the two groups was significant (P < 0.005), there was a marked overlap between the individual values in both groups, thus minimising the prognostic value of these measurements in prostate cancer. Endocrine therapy had no notable effect on the serum levels of cICAM-1. The mean +/- S.E.M. cICAM-1 concentrations in serum from a younger group of healthy volunteers was 204.1 +/- 10.38 ng/ml, and this value was significantly lower than that measured in serum from either BPH or CaP. We also undertook some immunohistochemical studies to examine the distribution of ICAM-1 in prostate tissue. We observed focal epithelial cell membrane staining which was exceedingly patchy in both the BPH and cancer specimens. On the basis of these studies, we suggest that cICAM-1 levels do not provide additional information on patients with metastatic CaP. PMID- 7540401 TI - Value of granulocyte colony stimulating factor in radiotherapy induced neutropenia: clinical and laboratory studies. AB - We report the effect of granulocyte colony stimulating factor (G-CSF) on neutropenia occurring during extended field radiotherapy in two groups of patients. The first group comprised 8 patients receiving craniospinal irradiation for a variety of central nervous system (CNS) neoplasms. None of these patients received cytotoxic chemotherapy. G-CSF was administered when the absolute neutrophil count (ANC) approached 1.5 x 10(9)/l. Neutropenia was promptly corrected in all cases, thereby avoiding unscheduled interruptions in radiotherapy. Following each G-CSF administration, ANC reached a peak on the following day and then declined steadily. Mean ANC rose from 1.33 x 10(9)/l on the day of G-CSF treatment to 7.07 x 10(9)/l the next day. Patients received 2-6 G-CSF injections during radiotherapy. Experiments were carried out in vitro to assess the risk of G-CSF causing increased CNS tumour cell proliferation. 11 human CNS tumour cultures (2 medulloblastomas, 2 primitive neuroectodermal tumours and 7 astrocytic tumours) were cultured in the presence of G-CSF at a range of concentrations up to 100 ng/ml. Their proliferation was compared with that of a G-CSF dependent murine leukemia cell line (NFS-60). None of the human tumour cultures demonstrated a significant increase in proliferation in response to G-CSF. 4 patients undergoing "mantle" type radiotherapy for Hodgkin's Disease or Non-Hodgkin's Lymphoma also received G-CSF treatment for neutropenia. All 4 had previously received cytotoxic chemotherapy. The number of G-CSF injections given per patient during radiotherapy ranged from 3-6. Mean ANC rose from 1.76 x 10(9)/l to 10.8 x 10(9)/l the next day. These results suggest that G-CSF is a reliable treatment for radiotherapy induced neutropenia and that an intermittent dosage schedule is effective. PMID- 7540404 TI - Renal cell carcinoma and interleukin-2: a review. PMID- 7540405 TI - CSF-1 treatment promotes angiogenesis in the metaphysis of osteopetrotic (toothless, tl) rats. AB - It has recently been shown that following treatment with colony-stimulating factor-1 (CSF-1) the osteopetrotic condition in toothless (tl) rats greatly improves and growth is accelerated. We have examined the effects of such treatment on the microvasculature of the distal femoral chondro-osseous junction, a site where bone growth in length is coordinated with angiogenesis. Vascular casts and ultrastructural analyses of this region showed that, compared to untreated normal rats, untreated mutants showed little bone growth or angiogenesis. When mutants were treated with CSF-1 angiogenesis was markedly accelerated. These data show a remarkable effect of this growth factor on angiogenesis in this osteopetrotic mutation. Whether this effect of CSF-1 on angiogenesis is direct or indirect is not known and indicates that its effects on the normal microvasculature deserve further study. PMID- 7540403 TI - Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines. AB - The influence of an artificial basement membrane (BM), Matrigel, and four individual extracellular matrix proteins, fibronectin, laminin, collagen I and vitronectin, on cell proliferation, morphology and migration was assessed in four glioma cell lines. Matrigel and individual BM proteins differentially inhibited cell proliferation of all cell lines studied. In addition, Matrigel was found to induce extensive morphological changes in glioma cells. Polycarbonate filters, of 8-microns porosity in modified Boyden chambers, were used to assess the chemoattraction activity of Matrigel and the individual proteins on glioma cells. All these components were found to stimulate cell migration, albeit to different extents but laminin proved to be the most effective chemoattractant for glioma cells in vitro. These data suggest that basement membrane proteins may inhibit proliferation and stimulate migration in order to facilitate invasion. PMID- 7540406 TI - Surgery for infants with a hypoplastic systemic ventricle and severe outflow obstruction: early results with a modified Norwood procedure. AB - OBJECTIVE: Prospective audit of the first year of implementation of a modified approach to palliation for infants with hypoplastic systemic ventricle and severe systemic outflow obstruction. SETTING: Tertiary referral centre for neonatal and infant cardiac surgery. PATIENTS AND METHODS: 17 of 19 infants (aged < 35 days) presenting to Birmingham Children's Hospital in 1993 with hypoplastic systemic ventricle and severe outflow obstruction underwent surgery. This was performed using a new modification of the Norwood-type arch repair, without the use of exogenous material, and a 3.5 mm Gore-tex shunt between the innominate and right pulmonary arteries. The Gore-tex shunt was replaced by a cavopulmonary shunt between 3 and 5 months later. Clinical, morphological, and functional determinants of outcome were examined. RESULTS: 10 (59%) infants survived initial surgery. All proceeded to cavopulmonary shunt without further loss. Significant atrioventricular valve regurgitation seemed to be the main risk factor for poor outcome. If this was excluded, the morphology of the dominant ventricle seemed to have little effect on the outcome of initial surgery. CONCLUSIONS: Early survival was achieved in 59% of patients in the first year of implementation of a protocol for surgery in infants with hypoplastic systemic ventricle and severe outflow obstruction. The construction of a neoaorta without the use of exogenous material may allow improved later growth of the neoaorta. Early cavopulmonary shunt can be performed safely and should reduce mid-term complications from cyanosis and systemic ventricular volume loading. PMID- 7540407 TI - Possible involvement of a magnesium dependent mitochondrial alkaline phosphatase in the regulation of the 25-hydroxyvitamin D3-1 alpha-and 25-hydroxyvitamin D3 24R-hydroxylases in LLC-PK1 cells. AB - It has been demonstrated that dephosphorylation of the ferredoxin component of the mitochondrial 25-hydroxyvitamin D3-1-hydroxylase, as a result of a PTH-cAMP mediated activation, involves a protein phosphatase activity. However, the nature and properties of this phosphatase are uncertain. It has been proved that alkaline phosphatase, a magnesium dependent enzyme, could dephosphorylate in vitro the ferredoxin component of the 25-hydroxyvitamin D3-1-hydroxylase. Moreover, some evidence of mitochondrial localization of some alkaline phosphatases has been published. Although the existence of a levamisole inhibitable alkaline phosphatase activity has been described in renal cells, its role remains to be elucidated. In the present work, the existence of an alkaline phosphatase in mitochondrial membrane preparations from LLC-PK1 cells has been described. This alkaline phosphatase is magnesium dependent and levamisole inhibitable. Preparations of mitochondrial membrane from LLC-PK1 cells also showed 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin D3-24R-hydroxylase (24-hydroxylase) activities being both enzymes responsive to the 8Br-cAMP mediated regulation. The 8Br-cAMP not only stimulated the 1 hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial alkaline phosphatase activity. In the same way, the levamisole (specific inhibitor of some alkaline phosphatases) inhibited the mitochondrial alkaline phosphatase and also the 1-hydroxylase activity. In addition, the inhibition of mitochondrial alkaline phosphatase by levamisole avoids the effect of 8Br-cAMP on the 1-hydroxylase and 24-hydroxylase activities. On the other hand, the mitochondrial alkaline phosphatase and the 1-hydroxylase activities showed similar behaviour with respect to the magnesium concentrations in the incubation medium. Taking these results together it could be possible to suggest the implication of the Mg(2+)-dependent mitochondrial alkaline phosphatase activity found in LLC-PK1 cells in the regulation of the 1,25(OH)2D3 and 24,25(OH)2D3 synthesis. PMID- 7540409 TI - Neurectomy for palliation of primary erythermalgia. AB - Ankle nerve crushing followed 6 months later by neurectomy was used for relief of a burning sensation and pain in a 32-year-old woman who had symptoms of unilateral primary erythermalgia. The patient continues to experience considerable improvement of the preceding symptoms and no complications from the insensate foot 1 year after neurectomy, although redness and tenseness of the ankle and the foot have persisted. PMID- 7540408 TI - Patency of a small vessel prosthesis bonded to tissue plasminogen activator and iloprost. AB - To test the thrombosis resistance of a vascular prosthesis coated with antithrombogenic agents, we evaluated a small vessel prosthesis of expanded polytetrafluoroethylene (ePTFE) implanted in the rat aorta and removed 1 week following surgery. Control grafts consisted of 1 mm internal diameter ePTFE. Experimental grafts consisted of 1 mm internal diameter ePTFE noncovalently bonded to tissue plasminogen activator (tPA) and iloprost using the surfactant tridodecylmethylammonium chloride. After 1 week the grafts were harvested, patency was determined, and histologic specimens were prepared for electron microscopy. Six of 10 control grafts were thrombosed, whereas 9 of 10 tPA iloprost-bonded grafts were patent (p < 0.03). Of concern, there was an unexpectedly high mortality rate in the tPA-iloprost group compared to the control group among animals that died before completion of the study. Evaluation of the safety of these drugs must, therefore, be an early component of future experiments. Nevertheless, these studies indicate that a small vessel prosthesis bonded to tPA and iloprost may ameliorate some of the complications associated with early graft failure. PMID- 7540410 TI - PREP: a portfolio to be proud of. PMID- 7540412 TI - Evidence of a dual role of endogenous histamine in angiogenesis. AB - The specific activation of mast cells in situ causes vigorous local mast-cell mediated angiogenesis (MCMA). The mast cell is a major source of histamine and, as recently reported, specific histamine H1- and H2-membrane receptor antagonists are able individually to significantly suppress MCMA in rats, as assessed using the mesenteric window angiogenesis assay (MWAA). In addition to membrane receptors for histamine, a type of intracellular histamine receptors, designated Hic, has been described. It is now demonstrated that the potent Hic-receptor antagonist DPPE (N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl), administered parenterally, stimulates MCMA significantly in rats, as quantified by the MWAA. Although the target cell(s) are not known, there are several ways by which their Hic receptors could be activated: uptake of histamine released from mast cells, mobilization from preformed cytoplasmic and nuclear stores, and production of de novo histamine by histidine decarboxylase activity. The fact that the occupancy by histamine of H1- and H2-membrane receptors stimulates MCMA and the occupancy by histamine of Hic inhibits MCMA suggests that endogenous histamine is capable of regulating angiogenesis by a dual mode of action. This is apparently the first report ascribing a dual role of this type in angiogenesis to a single molecule. PMID- 7540413 TI - A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the A1 disulphide loop (Cys509-Cys695) discriminates between type 2A and type 2B von Willebrand disease. AB - Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin-induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin-treated platelets nor on vWF-dependent platelet aggregation induced with ristocetin and asialo-vWF mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512-673 sequence of the A1 disulphide loop of vWF, MoAb B724 was used as second antibody in a two-site ELISA to test a series of patients with type 1, 2A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the A1 disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region. PMID- 7540414 TI - G-CSF and neutrophil chemotaxis. PMID- 7540415 TI - Administration of rHuGM-CSF activates monocyte reactive oxygen species secretion and adhesion molecule expression in vivo in patients following high-dose chemotherapy. AB - Microbicidal and cytocidal products of the respiratory burst and integrin adhesion molecule expression have been studied in monocytes from patients who received rHuGM-CSF during regeneration after high-dose chemotherapy. In this study, administration of rHuGM-CSF after high-dose chemotherapy significantly augmented the secretion of inducible products of the monocyte respiratory burst. Monocyte activation persisted for several weeks after the cessation of GM-CSF therapy. Under in vitro conditions that mimicked gram-negative (LPS) and gram positive (opsonized Staphylococcus aureus) sepsis, the monocyte responded to such stimulation by exhibiting an enhanced release of hydrogen peroxide at both regeneration and several weeks later (P < 0.001). Similarly, GM-CSF administration significantly augmented the phenotypic expression of the beta 2 integrin adhesion molecules and allowed the leucocyte-specific selectin, LAM-1, and the beta 2-integrins to respond normally to inflammatory stimulation by LPS. We further present evidence that GM-CSF therapy restored the otherwise refractory status of monocytes to inflammatory stimulation that existed in those patients given chemotherapy alone. The restoration of monocyte responsiveness by GM-CSF following high-dose chemotherapy could be a potentially valuable and hitherto not described action of rHuGM-CSF on monocyte function. We conclude that administration of GM-CSF may have the potential for restoring as well as augmenting the anti-microbial and anti-tumour function of the monocyte after high dose chemotherapy. PMID- 7540416 TI - The effect of continuous G-CSF application in human cyclic neutropenia: a model analysis. AB - Human cyclic neutropenia (CN) is a rare haematological disorder characterized by oscillations of blood neutrophils at subnormal levels with a stable period of approximately 21 d. During the phase of severe neutropenia (neutrophils < 250 cells/microliters), which last 4-10 d, the patients are endangered by serious infections. Several authors report that continuous G-CSF application can elevate the blood neutrophils to such a level that the risk of infections is significantly reduced. Although the characteristic cycles are not eliminated by G CSF, the period of the oscillations is shortened to 12-14 d. Based on a previously proposed computer-simulation model of human CN, the effects of continuous G-CSF application on CN are studied. It is shown how the known different cell-kinetic effects of G-CSF on granulopoiesis explain the clinical data in CN. The reduced length of the cycles emerges as a result of the transit time reduction of the post-mitotic granulopoietic cells by G-CSF. The measured increase of the neutrophil maxima is reproduced by the additional mitoses of the immature granulopoietic bone marrow cells induced by G-CSF. The slight elevation of the neutrophil nadirs can be attributed to a weak effect of G-CSF on the assumed underlying defect in CN (an abnormally small variance of the granulopoietic bone-marrow transit time). PMID- 7540417 TI - Invariance of textural features in image cytometry under variation of size and pixel magnitude. AB - Textural features of the granular structure of stained cell nuclei and nuclear sections derived from co-occurrence and run length matrices are often used for correlation with external (clinical) parameters. The representation of cell nuclei and nuclear sections vary considerably under changes of preparation and fixation conditions. Most obvious are changes in size e.g. by fixation as well as changes in the amount of bound stain. Computer-simulated variations in size and pixel magnitude of a set of images of cell nuclei stained with Feulgen were featured and compared. Resulting feature dependencies are used for the setting of parameters of feature extraction procedures as well as for the selection of invariant features for texture quantification of material with variations examined. PMID- 7540418 TI - Combination 5-fluorouracil (FU), folinic acid (FA), and alpha-interferon 2B in advanced gastric cancer: results of a phase II trial. AB - BACKGROUND: Based on encouraging treatment results with FU/FA or FU/IFN in gastrointestinal tract cancer, a phase II study was conducted to evaluate the effects and toxicity of combination FU/FA/IFN in patients (pts) with inoperable/metastatic gastric cancer. PATIENTS AND METHODS: IFN 6 M.U. s.c. 1 x/week, FU 500 mg/m2 bolus i.v. 1 x/week and FA 500 mg/m2 1 x/week as a 2-hour infusion. Of 72 treated pts, 72 (22 females, 50 males) are evaluable for response and toxicity. Median age was 55.6 years (28-80), and median Karnofsky performance status was 80% (70-100). Sites of measurable disease were inoperable primary tumors/local recurrence (18), liver metastasis (22), lymph nodes (31) and peritoneum (20). One pt had bone marrow metastasis and another had paraneoplastic hyperfibrinolytic coagulopathy. RESULTS: 10/72 pts had complete response, 20/72 pts partial response, 40/72 pts tumor stabilization and 2/72 pts progressive disease. The median duration of response (CR/PR) was 9 months, the median progression-free interval 6 months, the median survival time was 9 months, and for responding patients (CR/PR) 12.5 months. TOXICITY: 1/72 pts had WHO grade 4 toxicity (diarrhea), 5/72 pts had WHO grade 3 toxicity (nausea 1, diarrhea 4). Except for 1 treatment-limiting grade 4 toxicity, no modifications of dose or schedule due to toxicity were required. Thirty-six of 44 pts experienced a significant reduction in tumor-related pain under treatment. CONCLUSION: Biochemical modulation of FU with FA and IFN is effective in advanced gastric cancer. Moderate toxicity, outpatient treatment setting and high rates of amelioration tumor-related pain contribute to an effective palliation. PMID- 7540419 TI - Hybrid MOPP/ABVD and radiotherapy in advanced Hodgkin's disease. AB - BACKGROUND: In patients with advanced Hodgkin's disease (HD), the alternation of MOPP with ABVD or hybrid MOPP/ABVD are associated with a high CR rate and a high probability of 5-year survival. However, even after effective chemotherapy the risk of nodal relapse is not negligible, and not only in initial bulky site(s) of disease. For this reason, in an attempt to prevent relapses after combination chemotherapy alone, we performed a prospective study to evaluate the efficacy and toxic effects of 6 courses of hybrid MOPP/ABVD followed by radiotherapy (RT) in stages II A bulky, II B, III and also in stage IV with bulky disease of residual after chemotherapy. PATIENTS AND METHODS: From January 1985 to August 1993, 133 patients with HD (128 newly diagnosed, stage II A bulky-IV, 5 in first relapse after RT) were treated according to the following program: 6 courses of the hybrid MOPP/ABVD regimen followed by RT (STNI + spleen in stages II A, II B, III without pelvic lymph node involvement, TNI + spleen in stage III with pelvic lymph node involvement, involved field in stage IV with bulky disease or residual after chemotherapy). The total dose of RT was 4000 cGy to the sites of bulky or residual disease and 2000 cGy to the other sites. RESULTS: After hybrid MOPP/ABVD, 107 of 130 (82.3%) fully evaluable patients were classified as in CR or CR(U). After completion of RT, 108 patients were in CR and 3 were in PR, for an overall response rate of 85%. With a median follow-up duration of 45 months, the actuarial 5-year survival is 76% and the progression-free survival 68.6%. So far, only 14 patients have relapsed (6 within the irradiation field) and the 5 year relapse-free survival is 82.5%. CONCLUSION: Six courses of hybrid MOPP/ABVD followed by RT in stages II A bulky, II B, III and in stage IV with bulky disease or residual after chemotherapy produced a high CR rate with low risk of relapse. However, a longer follow-up is necessary to evaluate the late effects of combined therapy. PMID- 7540421 TI - Ambulatory 4-day continuous-infusion schedule of high-dose ifosfamide with mesna uroprotection and granulocyte colony-stimulating factor in advanced solid tumours: a phase I study. AB - BACKGROUND: There is evidence that continuous infusion allows the delivery of higher doses of a drug while reducing the incidence of neurologic and renal toxicity. To verify prior to phase II testing the feasibility of ambulatory treatment with high-dose ifosfamide/mesna by continuous infusion in adult solid tumours, the maximum tolerated dose (MTD) and the non-haematological dose limiting toxicities for the achievement of 2 courses of therapy were determined. PATIENTS AND METHODS: Thirty-two patients with advanced solid tumours were given continuous-infusion ifosfamide, from 9 to 16 g/m2, over 4 consecutive days, with equidose mesna uroprotection and granulocyte colony-stimulating-factor support; courses were repeated every 3 weeks. Total dose/course was escalated by 1 g/m2, in cohorts of 3 to 5 patients until the MTD was determined. RESULTS: At 16 g/m2, the dose-limiting toxicity was renal, with 2 of the 5 patients treated developing renal failure. Haematological toxicity was dose-related and significant at higher dosages, but generally surmountable. Ifosfamide at 15 g/m2/course with mesna uroprotection was identified as the MTD. CONCLUSIONS: The present study shows that high-dose continuous-infusion ifosfamide, administered by portable infusion pumps, is feasible in an ambulatory regimen, with acceptable non-haematological toxicity and good patient compliance. PMID- 7540420 TI - A complicated case of metastatic teratoma. Growing teratoma syndrome and cerebral metastasis. AB - All patients presenting with metastatic teratoma should be regarded as potentially curable and this case demonstrates the multiple treatment modalities which are often needed in the management of such patients. Increasing experience with cisplatin based combination chemotherapy has led to the development of prognostic factors which are used to determine the intensity of treatment given to individual patients. Surgical intervention plays a very important role in the management of residual disease at the completion of chemotherapy. Recognition of the growing teratoma syndrome and the importance of early surgical excision is illustrated by this case. Isolated CNS relapse may occur because the CNS may act as a sanctuary site in patients receiving systemic chemotherapy, but does not preclude long term disease free survival. PMID- 7540422 TI - High-dose chemotherapy and autologous peripheral stem cell transplantation in patients with relapsing malignant lymphomas--first experience in the Slovak Republic. PMID- 7540423 TI - Do antioxidant vitamins reduce infarct size following acute myocardial ischemia/reperfusion? AB - There is controversy concerning the ability of antioxidant vitamins to reduce myocardial infarct size. We sought to determine whether a brief prophylactic treatment of vitamin C or vitamin C plus Trolox (a water-soluble form of vitamin E) could reduce myocardial infarct size in an experimental model. We used an anesthetized open-chest rabbit model in which a branch of the circumflex coronary artery was ligated for 30 minutes followed by 4 hours of reperfusion. Experiments were performed in a randomized and blinded fashion. An IV injection of normal saline pH balanced to 7.4 (control group n = 15), vitamin C (150 mg/kg, n = 14), or vitamin C plus Trolox (150 mg/kg plus 100 mg/kg, respectively, n = 15) was administered prior to coronary occlusion. Collateral blood flow during coronary occlusion was measured by radioactive microspheres, myocardial risk zone (AR) was assessed by blue dye injection, and myocardial infarct size (AN) was assessed by triphenyltetrazolium chloride staining. All rabbits received comparable ischemic insult: Collateral blood flow and AR were similar among all three groups. Infarct size, measured as a percent of AR, did not differ significantly among the controls (21%), vitamin C (29%), or the vitamin C plus Trolox (18%) groups. Therefore, in this ischemia/reperfusion model, antioxidant vitamins did not alter myocardial infarct size. PMID- 7540425 TI - The development of MK-801, kainate, AMPA, and muscimol binding sites in cat visual cortex. AB - Previous work using homogenate binding has shown that the development of (+)-5 methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e maleate (MK-801) binding in cat visual cortex increases from 21 days to 42 days, the height of the plastic period, and decreases in adulthood. We have studied the generality of this finding by examining the development of NMDA binding sites in several brain regions and by examining the development of other binding sites in the visual cortex. After confirming the original finding, we extended it by showing that the sensitivity of MK-801 binding sites to glutamate and glycine decreases when the cat becomes an adult. We then examined the regional specificity of MK-801 binding. Retinal binding did not change significantly with age. Binding in both visual cortex and hippocampus increased significantly from 7 days to 42 days regardless of whether binding was measured per milligram wet weight or per milligram protein. The decline from 42 days to adulthood was less dramatic in the hippocampus than in the visual cortex and was statistically significant only when binding was measured per milligram protein. Saturation analyses also showed a difference in the two structures. Bmax in the visual cortex, but not in the hippocampus, decreased from 42 days to adulthood. To determine whether these developmental changes were specific to MK-801 binding sites, we compared the age dependent binding of MK-801, kainate, alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA), and muscimol. Like MK-801, kainate binding increased from 7 days to 42 days and decreased from 42 days to adulthood. AMPA and muscimol binding showed a similar increase in binding from 7 days to 42 days but did not decrease significantly from 42 days to adulthood. Displacement experiments suggest that AMPA and kainate bind to separate sites. The 42-day peak in NMDA and kainate binding suggests that their associated receptors may have a role in determining the plastic period of visual cortex. PMID- 7540424 TI - Preventive treatment of postinfarction heart rupture. PMID- 7540426 TI - A path-analytical approach to differentiate between direct and indirect drug effects on negative symptoms in schizophrenic patients. A re-evaluation of the North American risperidone study. AB - The hypothesis that differences in drug effects of risperidone and haloperidol on negative symptoms in schizophrenia are secondary to effects on positive, extrapyramidal, and depressive symptoms was investigated by means of an analysis of the data from the USA-Canada risperidone double-blind randomized clinical trial of 523 chronic schizophrenic patients. Regression analyses in the total sample and within treatment groups confirmed a strong relationship between changes in negative symptoms and the other variables studied (R2 = 0.50-0.51, p < 0.001). Only depressive symptoms did not contribute significantly to these results (p > 0.10). Path analysis showed that the greater mean change (p < 0.05) of negative symptoms with risperidone compared to haloperidol could not be fully explained by correlations with favourable effects on positive and extrapyramidal symptoms. The relationship between shift in extrapyramidal symptoms and shift in negative symptoms failed to reach statistical significance; however, there was a clear tendency in the expected direction in both treatment groups. PMID- 7540427 TI - Charge selectivity of the designed uncharged peptide ion channel Ac-(LSSLLSL)3 CONH2. AB - Charge selectivity in ion channel proteins is not fully understood. We have studied charge selectivity in a simple model system without charged groups, in which an amphiphilic helical peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, forms ion channels across an uncharged phospholipid membrane. We find these channels to conduct both K+ and Cl-, with a permeability ratio (based on reversal potentials) that depends on the direction of the KCl concentration gradient across the membrane. The channel shows high selectivity for K+ when [KCl] is lowered on the side of the membrane that is held at a positive potential (the putative C-terminal side), but only modest K+ selectivity when [KCl] is lowered on the opposite side (the putative N-terminal side). Neither a simple Nernst Planck electrodiffusion model including screening of the helix dipole potential, nor a multi-ion, state transition model allowing simultaneous cation and anion occupancy of the channel can satisfactorily fit the current-voltage curves over the full range of experimental conditions. However, the C-side/N-side dilution asymmetry in reversal potentials can be simulated with either type of model. PMID- 7540428 TI - Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance. AB - We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels. PMID- 7540431 TI - First trimester fetal nuchal translucency. AB - This review examines the development of a new method of screening for Down's syndrome based on the combination of fetal nuchal translucency thickness, maternal age and maternal serum biochemistry at 10-14 weeks of gestation. This method can potentially identify more than 80% of affected fetuses for a false positive rate of less than 5%. PMID- 7540430 TI - Cell volume measured by total internal reflection microfluorimetry: application to water and solute transport in cells transfected with water channel homologs. AB - Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size. PMID- 7540429 TI - Modeling success and failure of Langmuir-Blodgett transfer of phospholipid bilayers to silicon dioxide. AB - Formation of planar phospholipid bilayers on solid and porous substrates by Langmuir-Blodgett transfer of monolayers from the air-water interface could be of much greater utility if the process were not irreproducible and poorly understood. To that end the energetics of transferring two phospholipid monolayers to a hydrophilic surface has been examined. An approximate mathematical relationship is formulated that relates the surface pressure of the precursor monolayers to the tension within the bilayer created. Data are presented that demonstrate that bilayer transfer can be carried out reproducibly even with refractory phospholipids such as phosphatidylcholine, but only over a very narrow range of precursor monolayer surface pressures. This range is related to the lysis tension of the bilayer. The morphology of films formed within and below the successful range of surface pressures are examined by fluorescence microscopy, and the observed features are discussed in terms of the relationship above. These results provide practical guidelines for successful formation of lipid bilayers on hydrophilic surfaces; these guidelines should prove useful for research into the properties of biomembranes and for development of bilayer-based biosensors. PMID- 7540432 TI - Insulin-like growth factor-1 (IGF-1) enhances recovery from HgCl2-induced acute renal failure: the effects on renal IGF-1, IGF-1 receptor, and IGF-binding protein-1 mRNA. AB - Several growth factors have been found to play an important role in the recovery from acute renal failure (ARF). The effect of the continuous subcutaneous infusion of human recombinant insulin-like growth factor (IGF)-1 (125 micrograms daily by osmotic minipumps) in a rat model of mercuric chloride (HgCl2)-induced ARF was examined. HgCl2 (4 mg/kg) induced ARF with a mortality that was unaffected by IGF-1. However, IGF-1 significantly enhanced functional and histologic recovery in the survivors, as measured by serum creatinine and creatinine clearance and by histologic scoring. Solution hybridization RNAase protection assays showed that renal IGF-1 mRNA, IGF-1 receptor (IGF-1R) mRNA, and IGF-binding protein-1 (IGFBP-1) mRNA were unaffected by exogenous IGF-1, but this treatment significantly increased renal IGF-1 in ARF rats compared with normal rats and ARF rats not receiving IGF-1. After ARF renal mRNA for IGF-1 was decreased, IGF-1R was unchanged and IGFBP-1 was increased. Similar changes occurred in IGF-1-infused ARF rats. Thus, (1) IGF-1 enhances recovery from nephrotoxic ARF both functionally and histologically; (2) in nephrotoxic ARF, there is (a) a reduction in IGF-1 mRNA expression that is not prevented by IGF-1 infusion, and (b) an increase in renal IGFBP-1 mRNA. This may allow a significant increase in renal IGF-1 levels in IGF-1-infused ARF rats, despite the decrease in renal IGF-1 mRNA. A local increase in renal IGFBP-1 and IGF-1 may explain the accelerated recovery from ATN in this model. It was concluded that HgCl2-induced ARF is amenable to improvement by IGF-1 infusion and that the increase in renal IGFBP-1 mRNA may be an important modulator in the recovery of the kidney. PMID- 7540434 TI - Single-tube reverse transcription and heminested polymerase chain reaction of hepatitis C virus RNA to detect viremia in serologically negative hemodialysis patients. AB - Detection of hepatitis C virus genome is the only method currently available for detecting viremia in infected individuals. We describe a substantial simplification of current methods for viral RNA reverse transcription and polymerase chain reaction amplification, which allows cDNA synthesis and nested amplification in a single tube; this is achieved through compartmentalization of the reagents for the two reactions with a wax barrier. This procedure retains a high sensitivity: analysis of serum samples from hemodialysis patients, a high risk group for hepatitis C virus infection, confirmed the presence of the virus in 88% of antibody-positive patients and detected viremia in patients without serological or biochemical evidence of hepatitis C virus infection. PMID- 7540433 TI - Loss of heterozygosity and analysis of mutation of p53 in hepatocellular carcinoma. AB - Thirty-six hepatocellular carcinoma (HCC) tissues obtained from 34 patients were classified according to histological diagnosis into six well-differentiated HCC, 20 moderately differentiated HCC and 10 poorly differentiated HCC. High molecular weight DNA was prepared from each tumour and the corresponding non-tumour tissue. Loss of heterozygosity (LOH) on chromosomes 4q, 5q, 10q, 11p, 16q, 17p, mutation of the p53 gene and polymorphism of intron 25 of the retinoblastoma (RB) gene were simultaneously analysed. The patients were composed of three cases of small HCC (the diameter of which was < 3 cm) and 31 cases of advanced HCC. Twenty-nine of 34 (85.3%) patients analysed had been exposed to hepatitis B virus and/or hepatitis C virus. The frequencies of LOH on seven chromosomes were 57.9% in 17p13.3, 45.1% in 17p, 45.1% in 11p, 41.9% in 5q, 41.9% in 16q24, 29.0% in 4q, 25.8% in 10q in advanced HCC (four of well differentiated, 18 of moderately differentiated and nine of poorly differentiated carcinoma). In contrast, LOH was observed on 4q, 5q, 16q and 17p in 33% (1/3) of the small HCC (two of well differentiated and one of moderately differentiated carcinoma). The mutation of the p53 genes and polymorphism of the RB gene were present in 25.8% (8/31) and 12.9% (4/31) of the advanced tumours, respectively, but the mutation was not found in small HCC. LOH on every chromosome and the p53 mutation were observed more frequently in more advanced tumours, and the genetic changes accumulated with the increase of the histopathological grade.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540436 TI - Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant. AB - Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other. PMID- 7540438 TI - What's up, doc? PMID- 7540437 TI - Isolation of immortal human endothelial cells. AB - To get immortalized endothelial cells, human endothelial cells were transfected with a temperature-sensitive simian virus 40 DNA. The majority of transfected cells showed a prolonged life span and temperature-dependent cell growth. One of the clones, #5-1, grew either at 33 or 37 degrees C and could be cultured more than 110 generations. The cells expressed several endothelial cell specific characteristics. PMID- 7540435 TI - Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants. AB - Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin. PMID- 7540439 TI - Complications of intrathecal opioids and bupivacaine in the treatment of "refractory" cancer pain. AB - OBJECTIVE: To test the concept that externalized tunneled intrathecal catheters lead to a high risk of complications, such as meningitis and epidural abscess, and therefore should not be used for durations of intrathecal pain treatment of > 1 week. DESIGN: Prospective, cohort, nonrandomized, consecutive, historical control trial. SETTING: Tertiary care center, institutional practice, hospitalized and ambulatory care. PATIENTS: Two hundred adults (107 women, 93 men) with refractory cancer pain treated for 1-575 (median, 33; total, 14,485) days; 79 patients were treated at home for 2-226 (median, 36; total, 4,711) days. All patients had died by the close of the study. INTERVENTIONS: Insertion of intrathecal tunneled nylon (Portex) catheters (223 in 200 patients) with Millipore filters. The catheter hubs were securely fixed to the skin with steel sutures. Standardized care after insertion: (a) daily phone contact with the patients, their families, or the nurses in charge; (b) weekly dressing change at the tunnel outlet by the nurses; (c) refilling of the infusion containers by the nurses; (d) exchange of the infusion systems when empty (within 1 month) and of the antibacterial filter once a month by specially instructed Pain Department nurses. All contact between the connections of the syringes, cassettes, and needles with the operator's hands was carefully avoided during filling and refilling of the infusion containers and exchange of the antibacterial filters; no other aseptic precautions were taken. MAIN OUTCOME MEASURES: We recorded the rates of perfect function and complications of the systems. The rates of complications recorded in this study with externalized tunneled intrathecal catheters are discussed and compared with the rates reported in the literature with externalized (tunneled and non-tunneled) epidural and intrathecal catheters, as well as with internalized (both epidural and intrathecal) catheters connected to subcutaneous ports, reservoirs, and pumps. RESULTS: The following rates (as a percentage of number of patients) of perfect function and complications of the systems were recorded (the ranges of rates reported in the literature are given in parentheses): perfect function of the system, 93% (31-90%); accidental injury of an unknown epidural tumor followed by an epidural hematoma, 0.5% (0-6%); skin breakdown at the insertion site, 2% (2-50%); postdural puncture headache, 15.5% (10%); external leakage of CSF, 3.5% (4-27%); CSF hygroma ("pseudomeningocele"), 1.5% (4-6.25%); hearing loss and Meniere-like syndrome, 0% (12%); pain on injection, 0% with continuous infusion and 4.5% with intermittent injections (3 36% with intermittent injections); catheter tip dislodgement, 1.5% (6-33%); catheter (system) occlusion, 1% (3-12%); accidental catheter withdrawal, 4% (3 22%); catheter (system) leakage, 1.5% (2.1-26.6%); all mechanical complications, 8.5% (10-44%); local (catheter entry site) infection, 0.5% (2-33%); catheter track infection, 0% (6-25%); epidural abscess, 0% (0.6-25%); meningitis, 0.5% (1 25%); systemic infection, 0% (3%); incidence of all infections (n/treatment days), 1/7,242 (1/168-1/2,446). CONCLUSIONS: In our population and with the technique of insertion and care reported here, the use of externalized tunneled intrathecal catheters has not been associated with higher rates of complications when compared with earlier reported rates of externalized epidural catheters and internalized (both epidural and intrathecal) catheters connected to subcutaneously implanted ports, reservoirs, and pumps. The opinion that the use of externalized tunneled intrathecal catheters should be restricted only to patients who need pain treatment for < 1 week (because of the potential risk of infection, particularly meningitis and epidural abscess) is unfounded. PMID- 7540440 TI - Intractable pain and suffering in a cancer patient. PMID- 7540442 TI - Treatment of malignant esophagorespiratory fistulas with silicone-covered metallic Z stents. AB - PURPOSE: To prospectively evaluate the clinical efficacy of covered metallic Z stents in the treatment of esophagorespiratory fistulas (ERFs). PATIENTS AND METHODS: Twelve patients with severe aspiration symptoms from malignant ERFs were treated with silicone-covered, metallic, self-expanding Gianturco-Rosch Z (GRZ) stents. RESULTS: Fluoroscopically guided stent placement was successful and well tolerated in all patients. Immediate postprocedural endoscopy and esophagography showed excellent coverage of the fistulas in all cases. Aspiration symptoms were completely relieved in eight of 12 patients (67%). Four of 12 patients (33%) were improved and able to eat a soft diet. There were no stent-related deaths. Nine patients have died and three patients are alive. Mean follow-up for the entire group was 3.9 months (range, 1 week to 10.5 months). Nonfatal complications occurred in three of 12 patients (25%). Complications included one membrane disruption and one granulomatous reaction with a slight upward stent migration. CONCLUSION: GRZ stents are an effective and safe means of palliation in patients with malignant esophagorespiratory fistulas. PMID- 7540441 TI - An analysis of platelet activation and aggregation produced by three classes of contrast media. AB - PURPOSE: To evaluate the platelet activation and aggregation produced by ionic high-osmolality contrast media (HOCM) and both ionic and nonionic low-osmolality contrast media (LOCM). MATERIALS AND METHODS: After each agent was mixed with heparinized blood, sequential platelet counts were used to monitor platelet aggregation, and flow cytometry was used to monitor both aggregation and activation. Aggregation was measured with CD41a-FITC (specific for glycoprotein IIb-IIIa) and activation was measured with CD62-PE (specific for P-selectin). RESULTS: High concentrations of the nonionic LOCM (> 31% by volume in whole blood) induced more than 90% platelet activation within 2 minutes of exposure to freshly drawn heparinized whole blood. Aggregation followed immediately after activation and was somewhat reversible. High concentrations of the ionic HOCM (> 31%) induced prominent activation, although it occurred at a much slower rate and to a lesser degree than with the nonionic media. There was approximately 70% activation after 45 minutes of exposure. Ionic HOCM inhibited platelet aggregation, however. The ionic LOCM ioxaglate produced minimal or no platelet aggregation or activation. CONCLUSION: There is likely to be an agent-related difference in the risk of platelet activation and aggregation in the catheter lumen and in the immediate environment of the catheter tip, where high concentrations of contrast media are known to exist. PMID- 7540443 TI - Malignant esophageal stricture and fistula: palliative treatment with polyurethane-covered Gianturco stent. AB - PURPOSE: To evaluate the effectiveness of a polyurethane-covered Gianturco stent in the palliative treatment of malignant esophageal stricture and fistula. PATIENTS AND METHODS: Twenty-seven patients with recurrent stricture (n = 24), fistula formation (n = 8), or both (n = 5) underwent palliative treatment for aphagia (n = 15) or dysphagia (n = 12). Eight patients had fistulas to the respiratory tract or mediastinum. A 15-F delivery sheath system was passed through the stricture; the inner dilator was removed, and the stent was compressed into the sheath and advanced with a pusher catheter. Follow-up included chest radiography for 3 days and monthly esophagography or endoscopy. RESULTS: Covered stents occluded fistulas and opened strictures in 100% of patients. Food intake was upgraded to liquids in 7% of patients, to soft foods in 37% and to regular foods in 56%. New stricture or fistula occurred in 4% and 7% of patients, respectively. Stent migration occurred in 15% of patients. Twenty one patients died after stent placement, and average life expectancy was 11 weeks. CONCLUSION: A polyurethane-covered Gianturco stent is effective in the palliation of advanced malignant esophageal strictures and fistulas. PMID- 7540444 TI - Outcome of patients with advanced neoplastic disease receiving vena caval filters. AB - PURPOSE: The authors examined the appropriateness and outcome of inferior vena caval (IVC) filter placements in patients with advanced malignancies and limited expected survival. PATIENTS AND METHODS: Over a 35-month period, 35 IVC filters were inserted in 34 adult patients with advanced neoplasms. Follow-up was as long as 28 months (mean, 5.2 months). Filter effectiveness, complications, recurrent pulmonary emboli, patient survival, and hospital discharge status were recorded. RESULTS: Twenty-eight patients (82%) were discharged home (n = 21) or to nursing facilities (n = 7) between 1 and 193 days (mean, 23 days) after filter insertion. Six patient (18%) died during hospitalization 1-95 days after filter insertion. The overall mean survival was 6.6 months; for patients with stage III and IV tumors mean survival was 8.0 and 5.5 months, respectively. Even among patients with stage IV disease, 59% survived longer than 3 months. There were no complications related to filter insertion and no clinical evidence of recurrent pulmonary emboli. In 14% of patients, filters enabled invasive therapeutic and palliative procedures to be performed. CONCLUSION: The presence of advanced neoplastic disease by itself should not be a deterrent to insertion of IVC filters, as most patients survived well beyond initial hospitalization. PMID- 7540446 TI - Novel three-dimensional 1H-13C-31P triple resonance experiments for sequential backbone correlations in nucleic acids. AB - Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C 31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination. PMID- 7540445 TI - Isovolemic hemodilution alters the ratio of whole-body to large-vessel hematocrit (F-cell ratio). A prospective, randomized study comparing the volume effects of hydroxyethyl starch 200,000/0.62 and albumin. AB - OBJECTIVE: To evaluate potential changes in the ratio of whole-body/large-vessel hematocrit (f-cell ratio) during isovolemic hemodilution and to compare the volume effects of 2 different plasma exchange solutions (hydroxyethyl starch 200,000/0.62 6% and human albumin 5%). DESIGN: Prospective, randomized, controlled trial. SETTING: Operating theater in a university hospital. PATIENTS: 24 gynecological patients scheduled for elective surgery. INTERVENTIONS: Isovolemic hemodilution was performed using 2 different plasma exchange solutions. Plasma volume was determined using dye dilution technique before and after hemodilution. The volume of withdrawn blood was measured from the change in weight of the blood bags taking into account the specific gravity of blood. RESULTS: The volume of administered plasma exchange solutions exceeded the amount of withdrawn blood by 80 +/- 47 ml (p < 0.001). Plasma volume was 3,067 +/- 327 ml before and 3,517 +/- 458 ml after hemodilution. Using red cell volumes calculated from measured plasma volumes and peripheral hematocrit, a deficit of 249 +/- 133 ml (p < 0.0001) in red cells after hemodilution appeared with the measured withdrawn red cell volumes taken into account. This finding can be explained by a change in the f-cell ratio during isovolemic hemodilution. The volume effect of the exchange solutions was 1.05 for hydroxyethyl starch and 0.95 for albumin. CONCLUSIONS: The results demonstrate that a change in the f-cell ratio occurs during isovolemic hemodilution. The estimation of red cell volume or plasma volume changes by using either the hematocrit or plasma or red cell volume determinations together with the hematocrit may lead to erroneous results. PMID- 7540448 TI - [Immunochemical studies of the system of specific proteins of the human placenta]. AB - The levels of placental lactogen, (PL), trophoblastic beta-globulin (TBG), placental alpha-microglobulin (PAMG-1), alpha 2-microglobulin of fertility (AMGF) in the blood serum of females, amniotic liquid and tissue of the placenta during pregnancy were comparatively studied and the protein-synthesizing function in the placenta in gestosis was evaluated. An original approach to differential assessment of the placental function was developed, by using the comprehensive serological immunochemical assay for placental proteins, which was based on concurrent immunodiffuse determination of PL, TBG, and enzyme immunoassay for AMGF and PAMG-1. PMID- 7540447 TI - [Computed morpho-densitometry and its possibilities in experimental and clinical studies]. AB - The paper deals with the automatic morphodensitometric analysis of various biomedical objects (such as interphase chromatin in the cellular nuclei of chondroblasts and lymphocytes or human peripheral blood, opacities in the lens of chick embryos. A complex algorhythm of computational image processing was created for submolecular restructuring of interphase chromatin cell nuclear examinations. It includes specific methods of staining and unifies different quantitative analytical methods: photometrical, geometrical, gradiental, informational, and statistical. The blood cells of the children who had received low-dose radiation were detected by employing a new method of TV image computational processing of DNA-stained lymphocytes. The approach demonstrated by this study provides a sensitive and meaningful mean for diagnosing human diseases and can be useful in the clinical setting. PMID- 7540449 TI - [Immunochemical studies of several proteins in angina pectoris]. AB - A comprehensive immunochemical assay was developed for quantification of apoproteins of the atherogenic lipoproteins B, H, Lp(a) and the "acute phase proteins" with unstable angina pectoris that had varied increases in the parameters studied in comparison with their physiological measures. The significance of the proteins under test was discussed in terms of pathogenesis of atherosclerosis. The asset of a complex immunochemical assay as part of the procedure involved simplicity and accessibility for wide medical practice, providing valid information. PMID- 7540450 TI - [Psoriasis and the problem of gene expression regulation]. AB - During the epidermis maturation different keratin genes are expressed in strictly definite sequences according to the level of epidermis. In psoriasis this sequence in deranged (permutation) Perhaps this is due to implication of homeobox system similar to what occurs in (Ftz) gene. This problem is reviewed and discussed in this paper. All keratin as well as involukrine genes have been sequenced. Regions of K5 gene have been found which play regulatory role in transcription. It has been achieved using transfection K5 into keratinocytes. Psoriatic injury reversed by yEPP peptide of chick Y. sack cells. It is well established that protooncogenes are involved in transcription regulation. Expression of c-myc is increased in psoriatic plagues whereas that of c-fos and c jun is decreased. This fact may be regarded as an indirect evidence for abnormal functioning c-myc in regulation. It is not ruled out that the system implicated in shedding in reptiles recapitulate in psoriasis. PMID- 7540451 TI - Palliative care in terminal cardiac failure. Hospices cannot fulfil such a vast and diverse role. PMID- 7540452 TI - Palliative care in terminal cardiac failure. Small numbers of patients with terminal cardiac failure may make considerable demands on services. PMID- 7540453 TI - Effect of albumin on diuretic treatment in the nephrotic syndrome. PMID- 7540454 TI - Vitamin C and vascular disease. PMID- 7540455 TI - Dutch doctor convicted of murdering disabled infant. PMID- 7540456 TI - [Data on the pharmacology of a new enzyme complex for replacement therapy in children]. AB - A model of experimental pancreatitis was used to study the enzymatic compensatory properties of a new polyenzymatic complex. When given in a dose of 70 mg/kg, the complex was found to normalize amylolytic, lipolytic and partially proteolytic activities in the small intestinal cavity of rats. The complex had some advantages in exhibiting its pharmacological effect over some foreign drugs. PMID- 7540458 TI - A scoring system for the classification of CD5-B CLL versus CD5+ B CLL and B PLL. AB - B CLL is a monoclonal proliferation of lymphocytes which express the CD5 antigen (CD5+ CLL). Rare exceptions (less than 10%) are CD5-, as are the majority of B PLL. We have studied the clinical, cytological and immunophenotypic characteristics of a series of 12 CD5-CLL and have established a score which allows the distinction between CD5+ CLL, CD5- CLL and PLL. Among the CD5- CLL, there were significantly more cases with advanced stage (Rai and Binet) and splenomegaly. The cytological study found more mixed CLL according to FAB classification (more prolymphocytes). There were significantly more CD23-, FMC7+, SIg strong positive cases. A score from 0 to 6 was established based on clinical, cytological and immunophenotypic criteria. Typical CD5+ CLL was scored 0, score 6 corresponded to typical PLL. There were significantly more higher scores amongst CD5- CLL. It therefore appears that CD5- CLLs share certain features with B PLL. The use of this scoring system will allow determination of prognosis within these different categories, thus identifying groups which require specific therapy. PMID- 7540457 TI - Expression of MDR1 by normal bone marrow cells and its implication for leukemic hematopoiesis. AB - Expression of MDR1 is a well-characterized mechanism leading to resistance of tumor cells to drugs like vinca-alkaloids, anthracyclines, and epipodophyllotoxins. In hematopoiesis, recent data indicate that not only leukemic cells, but also some populations of normal hematopoietic cells, particularly CD34+ progenitor cells as well as peripheral blood lymphocytes, express a functional multidrug-resistant phenotype. Among CD34+ cells, we found evidence that myeloid committed precursor cells (CD34+/CD33+) have lower levels of MDR1 expression than earlier CD34+ cell populations, but there was no difference in MDR1 expression between CD34+/HLA-DR- and CD34+/HLA-DR+ subpopulations. During normal myeloid differentiation, MDR1 expression is down regulated, which is similar to our observations in acute myelogenous leukemia (AML): MDR1 expression was only rarely detected in acute promyelocytic leukemia, which was in contrast to other subtypes of AML; also, within leukemic subpopulations of the same patient, higher MDR1 levels were correlated with a more immature immunophenotype. Regarding regulation of MDR1 expression, we did not observe changes of MDR1 expression in normal CD34+ cells in response to various cytokines. However, in 2 patients with AML treated with interleukin-3 and granulocyte-colony stimulating factor, respectively, a significant down regulation of MDR1 expression was found after 24 hours. In conclusion, there is evidence that the pattern of MDR1 expression observed in leukemias reflects the distribution of MDR1 in normal hematopoiesis. In contrast to normal CD34+ cells, leukemic cells from some AML patients can respond to cytokines with a down regulation of MDR1, which may contribute to response to cytokine/chemotherapy combinations. PMID- 7540459 TI - Human immunodeficiency virus associated Hodgkin's disease: report of 45 cases from the French Registry of HIV-Associated Tumors. AB - Clinical and pathological characteristics as well as outcome of 45 Hodgkin's disease (HD) cases collected by the French registry of HIV-associated tumors between January 1987 and December 1989 (all clinically staged according to the Ann Arbor system) were analyzed and compared with those of a cohort of 407 patients with clinical stages (CS) IA to IVB enrolled between September 1981 and August 1988 in a multicentric clinical trial. The route of HIV infection, initial CD4 cell count at the time of HD diagnosis and CDC class of HIV infection were studied as well as the progression to AIDS onset were recorded. HIV-HD is characterized by a predominance of advanced CS (75%), B symptoms (80%) and mixed cellularity histology (49%), as well as by early bone marrow involvement (24%); a specific feature is the rare occurrence of mediastinal involvement (13% in HIV-HD versus 71% in primary HD). With standard therapies, 79% of the patients achieved a complete remission, but hematological and infectious complications were very frequent. The proportion of intravenous drug abusers (IVDA) in HIV-HD (38%) is higher than in French HIV-infected population as a whole (20.8%). Median CD4 cell count was 306/microliters at the time of HD diagnosis, while only 5 cases (11%) were preceded by an AIDS manifestation; progression to AIDS rate was 94% at 2 years. Overall 2-year survival was 41%, with 71% for patients with an initial CD4 cell count over 300/microliters and 0% for those with CD4 cell count lower than 300/microliters (p < 0.01); opportunistic infections were the most frequent cause of death. HIV-HD seems to occur preferentially in.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540460 TI - MACOP-B vs F-MACHOP regimen in the treatment of high-grade non-Hodgkin's lymphomas. AB - A prospective randomized study on aggressive non-Hodgkin's lymphomas was conducted by investigators at several Italian institutions with the intent of comparing two third-generation conceptually different regimens: the regimen containing methotrexate with leucovorin rescue, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B), a short-term continuous twelve week therapy, and F-MACHOP (5-fluorouracil, methotrexate with leucovorin rescue, cytarabine, cyclophosphamide, doxorubicin, vincristine, and prednisone), a monthly intensive cyclic treatment combining prednisone with six active non-cross resistant cytotoxic drugs. The goals of this study were the response rate, relapse-free survival, and incidence of hematologic and nonhematologic toxicities. Two hundred-eighty-six patients included between 15 and 60 years fulfilled the criteria for entry to the study; 140 patients were treated with MACOP-B and 146 with F-MACHOP. The minimum follow-up was 24 months. Clinical characteristics of all patients were similar and known prognostic factors were equally distributed between the two groups. Complete remission (CR) was achieved by 61% and 67% of the patients treated with MACOP-B and F-MACHOP, respectively; 4% and 6% were primarily resistant, 2% and 5%, respectively, died of causes directly related to therapy. At 50 months, 74% of all CR patients were alive without disease and there were no significant differences in relapse-free survival between the two groups: 75% in the F-MACHOP group and 73% in the MACOP-B group at 50 months. There was a higher incidence of mucositis among patients treated with MACHOP-B than among those given F-MACHOP (11% vs 3.5%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540461 TI - Granulocyte colony-stimulating factor (G-CSF) allows the delivery of effective doses of CHOP and CVP regimens in non-Hodgkin lymphomas. AB - The aim of this study was to evaluate the role and potential benefit of G-CSF administered following standard regimen chemotherapy (CHT) in non-Hodgkin lymphomas. Twenty patients with NHL were given CHOP or CHOP/CVP CHT every 21 days. None was given G-CSF after the first cycle. After each cycle, G-CSF was administered only for: 1) ANC < 1 x 10(9)/L between cycles; or 2) delay in cycle schedule due to ANC < 1 x 10(9)/L on the planned day of treatment; or 3) development of a febrile syndrome or a documented infection, regardless the ANC. Once administered, G-CSF was maintained in the following cycles. Nineteen patients required administration of G-CSF (5 micrograms/Kg B.W.), but for different reasons only 16 were treated (a mean of 10 +/- 3 doses/cycle). Comparing 48 cycles where G-CSF was not administered, with 50 where it was, in this last group we observed: 1) a ANC significantly higher at day 7 (p < 0.0001), day 14 (p < 0.0001) and day 21 (p = 0.0030); 2) a significantly lower (p = 0.0001) incidence of neutropenias (6 vs 29); 3) a trend (p = 0.1040) in favour of lower incidence of febrile neutropenias of infections (1 vs 6); 4) a significantly lower (p < 0.0001) incidence of cycle delays (1 vs 22) with a median of 8 days (1 to 20); and 5) a significantly higher (p < 0.0001) dose intensity (99.5% vs 87.8%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540462 TI - Effects of rhG-CSF on infection complications and impaired function of neutrophils secondary to chemotherapy for non-Hodgkin's lymphoma. Hokkaido Study Group of Malignant Lymphoma, and rhG-CSF, Japan. AB - It has been previously demonstrated that the administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF) ameliorates the decrease of the polymorphonuclear neutrophils (PMNs) count after the cytotoxic chemotherapies, thereby reducing the infection complications associated with neutropenia. In this multi-center study, we studied the prophylaxtic effect of rhG-CSF administration on infection complications in patients with non-Hodgkin malignant lymphoma, who received cytotoxic chemotherapies (CHOP or ProMACE/CytaBOM). rhG-CSF administration reduced the frequency of infection complications, and there was no obvious difference in it's frequency between the CHOP-treated and the ProMACE/CytaBOM-treated groups when administered with rhG-CSF, thereby indicating that third generation therapy for NHL may be safely completed in Japanese in combination with rhG-CSF administration. Furthermore, we investigated both the in vitro and the in vivo effects of rhG-CSF on the function of PMNs in patients with NHL and healthy donors, and revealed that the administration of rhG-CSF for NHL patients receiving cytotoxic chemotherapy brought on an improvement of the production of active oxygen but did not affect serum levels of IFNs, IL-1-beta, and IL-6, inspite of a slight elevation of TNF-alpha. Consistent with these results, in vitro treatment of PMNs with rhG-CSF induced no significant production of these inflammatory cytokines and their mRNA expressions. Furthermore, rhG-CSF administration showed no significant effects in vivo on the expression of CD11a, CD11b and LECAM-1 on PMNs and integrins on platelets. PMID- 7540463 TI - Characterization of CD34+ human hemopoietic progenitor cells from the peripheral blood: enzyme-, carbohydrate- and immunocytochemistry, morphometry, and ultrastructure. AB - Following an immunomagnetic isolation and enrichment procedure, CD34+ cells were harvested from the peripheral blood of about 50 healthy donors. A battery of cytochemical staining reactions, monoclonal and carbohydrate-specific antibodies, proliferation markers and lectins was applied on smears and sections from paraffin-embedded pellets. Additionally, a morphometric analysis and ultrastructural investigation was carried out. More than 95% of the total yield of progenitor cells expressed CD34 and CD43 (MT1) and of these about 90% CD45 (LCA) and 25% CD45A (MT2). The CD34+/CD45RA-population was thought to represent very primitive, probably not lineage-restricted stem cells. On the other hand, reactivity with ANAE, CD11c, CD15, CD20, Ret40f, KiM1P, and CD61 (ranging between 1 to 20%) was considered to indicate a transition into more differentiated elements of hemopoiesis. The failure to detect any staining with proliferation markers (Ki-67/MIB 1, PCNA, KiS1) was in keeping with a quiescent status. Carbohydrate antigens revealed a pattern which underlines the fact that the CD34 and CD43 antigens belong to the family of heavily O-glycosylated sialomucins. Blood group antigens which are located at the peripheral regions of mucin oligosaccharides (H type 2, Lewis, Lewis) could be demonstrated, but not A, B, Sialyl-Lewis and Lewis. Morphometric analysis revealed that CD34+ progenitors were larger than small lymphocytes. Electron microscopy showed a relatively primitive cytoplasmic organization and numerous tiny magnetic beads clustered at the plasma membrane. PMID- 7540465 TI - Potentiation by calcium ions of [3H]MK-801 binding to an ion channel associated with the N-methyl-D-aspartate receptor complex in rat brain. AB - In vitro addition of Ca2+ ions was effective in almost doubling binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) before equilibrium to an ion channel associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. The addition of inhibitors for phospholipase (PLase) A2 markedly reduced binding in the absence of added Ca2+ ions, while Ca2+ ions restored the reduction to the level found in the absence of the inhibitors. Pretreatment with PLases A2 and C but not D was effective in potentiating [3H]MK-801 binding in a biphasic manner at a concentration range of 5 mU/ml-10 U/ml. Moreover, PLases A2 and C at low concentrations not only suppressed the abilities of 3 different agonists to potentiate [3H]MK-801 binding before equilibrium, but also reduced that of Ca2+ ions. These results suggest that Ca2+ ions may potentiate [3H]MK-801 binding to the NMDA channel highly permeable to Ca2+ ions through a mechanism common to that underlying potentiation by exogenous PLase A2 and/or C. PMID- 7540464 TI - Factor VIII inhibitor prior to and during secondary acute nonlymphocytic leukemia in a patient with cured Hodgkin's disease. AB - We report a 54-year-old patient with Hodgkin's disease who achieved a complete remission after combined modality treatment. Three years later the patient developed a severe hemorrhagic syndrome, concomitant with the onset of a factor VIII inhibitor in plasma. The control of very proteiform bleedings was extremely difficult, even with plasmaphereses, as well as with immunosuppressive and substitutive therapies. Two years later, a secondary acute nonlymphocytic leukemia (ANLL) was diagnosed. Two courses of chemotherapy with fludarabine, cytosine arabinoside and G-CSF (FLAG) were able to obtain a complete remission. Hemorrhagic complications were mainly linked to thrombocytopenia and continued until recovery of thrombopoiesis. Factor VIII inhibitor levels and related clinical symptoms decreased progressively. In conclusion, we suggest that FLAG succeeded in inhibiting an abnormal lymphoid clone responsible for factor VIII inhibitor production, suggesting a possible role for intensive chemotherapy in similar situations, which are often refractory to conventional immunosuppressive and depletive therapy. PMID- 7540467 TI - Dizocilpine (MK-801) elicits a tetrodotoxin-sensitive increase in extracellular release of dopamine in rat medial frontal cortex. AB - We have examined in the rat the effects of a selective non-competitive antagonist for the N-methyl-D-aspartate (NMDA) type excitatory amino acid receptor, dizocilpine (MK-801), on cortical dopamine (DA) metabolism using an in vivo dialysis technique. An acute intraperitoneal injection of MK-801 (0.4-1.25 mg/kg) dramatically increased the concentrations of dopamine, 3,4-dihydroxy-phenylacetic acid and homovanillic acid in the dialysates from the medial frontal cortex in a dose-dependent fashion. Moreover, MK-801 caused a delayed and small augmentation of the cortical extracellular release of 5-hydroxyindoleacetic acid. Continuous infusion of tetrodotoxin into the prefrontal region via the microdialysis tube completely blocked the ability of MK-801 (1.25 mg/kg, intraperitoneally) to augment the extracellular release of DA, its metabolites and the serotonin metabolite in the frontal cortex. The present results suggest that MK-801 facilitates DA release in the medial frontal cortex by increasing impulse flow in the DA neurons projecting to the cortical area adding further support to the view that the NMDA receptor may be involved in the tonic inhibition of frontal cortical DA neurons. It is also proposed that frontal serotonin neurons might be under regulation by excitatory amino acidergic transmission via the NMDA receptor. PMID- 7540466 TI - AMPA receptor activation regulates the glutamate metabotropic receptor stimulated phosphatidylinositol turnover in human cerebral cortex slices. AB - The effect of excitatory amino acids (EAA) on phosphatidylinositol (PI) turnover in human cerebral cortical slices was investigated. Trans-1-aminocyclopentane-1,3 dicarboxylic acid (ACPD) increased inositol phosphate (IP) formation in the 1 1000 microM range. Quisqualic acid (QA) was maximally effective at 10-100 microM, showing an inverse correlation between concentration and effect in the 100-1000 microM range. The glutamate metabotropic receptor antagonist 2-amino-3 phosphonopropionic acid (AP3), the ionotropic non-NMDA receptor antagonist 6 cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA channel blocker dizolcipine (MK-801) failed to prevent the PI response to ACPD (1000 microM). However, CNQX (100 microM) modified the concentration-response curve of QA reducing the effect of QA 10 microM by approx. 50% and enhancing that of QA 1000 microM by 2-fold. In addition, CNQX (100 microM) together with MK-801 (100 microM) unmasked the ability of L-glutamate (L-GLU) 3000 microM to stimulate PI turnover. The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on the EAA-induced PI turnover was also studied. AMPA (0.1-1 microM) potentiated the response to submaximal (30 microM) ACPD and (1 microM) QA concentrations. However, higher AMPA concentrations (10 microM) failed to synergize with ACPD 30 microM and, in addition, inhibited the PI turnover maximally stimulated by QA 10 microM. These results further support the presence of the glutamate metabotropic receptor in the human neocortex. In addition, they show the occurrence of a concentration-related dual interaction between AMPA and glutamate metabotropic receptor activation in the IP formation in this brain area. PMID- 7540468 TI - Immunotherapy of multiple myeloma. AB - In 1994, an estimated 12,700 new cases of multiple myeloma (MM) will be diagnosed in the USA and 9,800 patients will die from this disease. At present, a cure for MM has not been achieved with any chemotherapeutic regimen. Therefore, it is important to develop novel therapeutic approaches to treat this fatal disease. This review focuses on new concepts in the immunotherapy of MM. Thus far, interferons and anti-human interleukin (IL)-6 monoclonal anti-bodies (MAbs) have been used to treat patients with this disease. Bone marrow transplantation using autologous marrow purged with MAbs and complement, with anti-myeloma immunotoxins (ITs), or MAb-magnetic bead conjugates has been reported. Adoptive cellular therapy, in vivo with anti-CD3 and IL-2, as well as transplantation of purified autologous CD34+ peripheral blood stem cells, is now being evaluated in clinical trials. Anti-human IL-6 receptor (IL-6R) and anti-CD54 (ICAM-1) MAbs have shown promising results in the therapy of human myeloma cell lines in SCID mice, while an IL-6 antagonist protein, anti-gp130 MAbs, recombinant soluble gp130, anti-B7, anti-HLA-DR, and recombinant soluble CD16 also inhibit the growth of myeloma cell lines in vitro. These experimental therapeutic modalities hold promise for use in humans and may also provide further insights into the pathogenesis of MM. PMID- 7540469 TI - A unique population of CD34+ cells in cord blood. AB - Human umbilical cord blood (CB) is a rich source of hematopoietic stem cells for both research and stem cell transplantation. In clinical studies, it appears that recovery from myeloablative therapy using CB requires significantly fewer cells than a typical allogeneic marrow transplant. This suggests that CB may be enriched for early hematopoietic progenitors. The present studies were undertaken to determine the presence of CD34+ cells in CB with the phenotypic characteristics of multipotential stem cells. In 22 CB harvests, the average percentage of CD34+ cells was 1.33 +/- 0.21% (SE), a value similar to that in adult normal bone marrows (BM). However, the distribution of CD34+ cells was distinctly different from either BM or granulocyte colony-stimulating factor (G CSF) mobilized peripheral blood stem cell harvests. CB contained a defined population of brightly staining CD34+ cells with low side scatter. These CD34 (bright) cells comprised a mean of 14.5 +/- 2.5% of the CB CD34+ cells, whereas < 1% of BM CD34+ cells has been shown to be CD34- bright. Eighty-five to ninety percent were negative for three antigens expressed at an early stage of stem cell maturation: CD38, HLA-DR and LFA-1. Fifty-five percent of these CD34 (bright) cells did not express the CD45RA isoform, an additional marker of immaturity. The antigen-bright cells also lacked lineage-specific antigens including CD33, CD56, CD19, CD10 and CD7 as well as CD71. Approximately 46% were Thy-1+, and 40% expressed c-kit receptors. These data suggest that, by phenotypic criteria, CB may be a particularly enriched source of primitive hematopoietic precursors. PMID- 7540470 TI - CD34+ cell expansion and expression of lineage markers during liquid culture of human progenitor cells. AB - A 96-well-based suspension culture system for human hematopoietic progenitor cells has been developed to monitor the commitment and differentiation of CD34+ cells to specific lineages and the maintenance and expansion of CD34+ cells in vitro. The expression of maturation and lineage markers on the cells in culture was measured by enzyme-linked immunosorbent assay (ELISA). CD34+ cells were isolated from umbilical cord blood and fetal liver (90% purity) and were grown in liquid culture in 96-well plates for 10 days. The cells were then fixed with a glutaraldehyde-paraformaldehyde mixture, attaching the cells firmly to the plastic. An ELISA was performed, using appropriate primary antibodies directed against cell surface markers. The expression of four different lineage markers was measured: CD14 (monocyte), CD15 (neutrophil), platelet glycoprotein (GP) IIb/IIIa (CD41a, megakaryocyte) and glycophorin A (erythroid). The two-growth factor combination of interleukin 3 (IL-3) and stem cell factor (SCF) stimulated expression of CD14, CD15 and GP IIb/IIIa. Lineage-restricted growth factors such as erythropoietin (EPO), in combination with SCF, stimulated expression of glycophorin A. The three-factor combination of IL-3, SCF and EPO stimulated expression of all four lineage markers. Other multiple growth factor combinations all stimulated myeloid and megakaryocyte growth, as measured by ELISA and flow cytometry, but erythroid growth was present only when EPO was included in the growth factor mixture. In serum-free medium or plasma-containing medium, CD14 expression was markedly reduced, whereas glycophorin A expression was greatly elevated in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540471 TI - AM5, a protein-associated polysaccharide, stimulates hematopoiesis and modulates the expression of endogenous hematopoietic growth factors in murine long-term bone marrow cultures. AB - We have investigated the mechanism behind the hematopoietic stimulation mediated by AM5, a protein-associated polysaccharide that stimulates in vivo the murine hematopoietic system. A dose-dependent increase in hematopoietic progenitors was observed in long-term bone marrow cultures (LTBMCs) treated in vitro with AM5. The stimulatory effect was more marked in colony-forming units-granulocyte macrophage (CFU-GM) than in CFU-spleen (CFU-S) progenitors and also more significant in the supernatant than in the adherent layer. This stimulatory effect was reversible, and continuous stimulation with high doses of AM5 was conductive to a progressive exhaustion of the culture. The analysis of the CFU-GM stimulating activity present in the supernatant of AM5-treated cultures revealed a dose-dependent induction of granulocyte-macrophage colony-stimulating activity (GM-CSA), in contrast with control cultures in which no CSA was detected. Northern blots of LTBMC-adherent layers obtained after in vitro treatment with AM5 revealed a significant mRNA expression for interleukin 1 alpha (IL-1 alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (M-CSF) and granulocyte CSF (G-CSF), in contrast with adherent layers from untreated cultures which only expressed, in detectable levels, M-CSF and stem cell factor (SCF). The SCF expression was down-modulated in AM5-treated cultures. Our results strongly suggest that the hematopoietic stimulation induced by AM5 is mediated by the modulated expression of endogenous hematopoietic growth factors. PMID- 7540472 TI - Stem cell factor augments tumor necrosis factor-granulocyte-macrophage colony stimulating factor-mediated dendritic cell hematopoiesis. AB - We describe the effects of stem cell factor (SCF) on the dendritic cell (DC) pathway and provide evidence for the existence of a post granulocyte-macrophage colony-forming unit (GM-CFU) DC progenitor. When employed with cytokines regulating DC development (tumor necrosis factor [TNF] + GM colony-stimulating factor [GM-CSF]), SCF increased the size of monocyte (mono) and mono-DC colonies arising from cord blood CD34+ progenitor cells. The overall plating efficiency of these colonies increased approximately threefold, as compared with growth in TNF + GM-CSF. Most (approximately 70%) of the CFUs were mono-DC CFU, and SCF did not alter the proportion of mono-DC CFU to mono-CFU obtained with TNF + GM-CSF alone. Proliferation, as measured by thymidine uptake and manual cell counts, at least doubled and occurred earlier (by day 4). In long-term cultures established with TNF + GM-CSF + SCF, high levels of proliferation were prolonged for up to three weeks. These were associated with extended DC development and the capacity to form 2 degree mono-DC colonies. There was no induction of polymorphonuclear (PMN) cells in 2 degree cultures treated with either GM-CSF, GM-CSF + SCF or GM-CSF + granulocyte CSF (G-CSF), implying that the DC progenitor being replated was post GM-CFU. DC progeny arising in the presence of SCF exhibited typical DC features including: the lack of nonspecific esterase and phagocytic activity, the presence of class II major histocompatibility complex (MHC) antigens, the absence of CD14 antigens, and the ability to induce a potent mixed leukocyte reaction. Thus, SCF augments DC growth from progenitor cells without altering the developmental commitment instituted by TNF + GM-CSF. This enhancement follows the same general mechanisms previously reported for SCF-mediated lineage enhancement, i.e., increased colony size, number and plating capacity. PMID- 7540473 TI - [Topography of a binding site for bis-cationic ganglioblockers in the neuronal nicotinic cholinergic receptor ion channel]. PMID- 7540474 TI - Production and characterization of monoclonal antibodies to shark cartilage proteoglycan. AB - 1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10(6) Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller than PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and further characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteoglycans react with MST1, indicating that the antibody does not recognize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does not recognize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 and PG2 aggregate with hyaluronic acid. PMID- 7540475 TI - CD44 expression in T-cell lymphoblastic leukemia. AB - 1. CD44 is an adhesion molecule expressed by B and T lymphocytes that mediates cell attachment to extracellular matrix components and specific cell surface ligands. In normal process of T-cell development, CD44 can be implicated in homing of bone marrow-derived T-cell precursors into the thymus. In hematopoietic malignancies, CD44 and other adhesion molecules can mediate the behavior of neoplastic cells such as metastatic migration. In the leukemic process, CD44 expression is correlated with increased numbers of circulating blasts and it is present at other sites such as the central nervous system. 2. In the present study, CD44 was investigated in lymphoblasts from 30 patients with T-cell lymphoblastic leukemia (T-ALL) and in peripheral lymphocytes from 10 healthy individuals. CD44 expression was detected in 23 (77%) of T-ALL cases studied and was correlated with clinical features such as mediastinal mass, adenomegaly, and infiltration of the central nervous system and other organs. Interestingly, CD44 expression in patients with tumor infiltration was higher than in patients with no tumor infiltration. 3. These data suggest that CD44 may be regarded as an additional marker for tumor expansion in T-cell leukemias. PMID- 7540477 TI - Evaluation of cervico-urethral obstruction with ambulatory pressure/flow studies: an alternative to conventional pressure/flow studies? AB - Most of the urodynamic studies are conducted in the laboratory during a brief recording time and under nonphysiological conditions and, thus, may fail to unfold the nature of existing pathological conditions of the lower urinary tract; false positives and false negatives are possible. To overcome some of the difficulties associated with conventional P/F studies we have developed, with the cooperation of Medical Measurement System company, Entschede, The Netherlands, a portable system (UDS 2000) for ambulatory monitoring of intravesical pressure, abdominal pressure and EMG connectable with a weight-transducer flowmeter that allows to perform Holter P/F measurements. We compared the results obtained with conventional P/F studies and with Holter P/F studies in 58 BPH patients. During the filling phase we observed a slightly increased number of stable detrusors with Holter P/F recording (46 vs 42); conversely, the number of patients suffering from urge incontinence was the same (7 pts). During the voiding phase, out of 45 patients considered obstructed at conventional P/F study, only 42 were really urodynamically obstructed (93.3%), while other 3 patients had bordeline obstruction. 4 patients with bordeline obstruction at conventional P/F study were considered non-obstructed after Holter P/F. In conclusion, Holter P/F proved to be reliable in the assessment of bladder function during storage and emptying in BPH patients and to be more specific in defining outlet obstruction than the conventional study because of the physiologic filling, the reduction of urethral irritation, the lack of a urethral catheter either during filling or voiding, and the reduction of emotional stress.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540476 TI - Forskolin enhanced off-response in the turtle olfactory system. AB - Olfactory responses appear not only at the onset of odor stimulation, but also at the termination of the stimulation. It is widely considered that the cAMP-pathway is involved in the generation of odor responses. We examined how cAMP affects the generation of off-responses. To explore the role of the cAMP-pathway, odorants were applied to the turtle olfactory epithelium after forskolin at high concentrations which saturated the olfactory response to forskolin. Various odorants induced off-responses after 50 microM forskolin, indicating that off responses are not induced via the cAMP-dependent pathway. However, the magnitude of the off-response after forskolin varied from 100 to 1400% of those of off responses in its absence, indicating that forskolin greatly enhanced the off responses to some odorants. The off-response after 0.1 mM citralva was also enhanced by 3 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cpt cAMP). These results suggested that cAMP modulates off-responses in the turtle olfactory system. PMID- 7540478 TI - Growth hormone treatment during pregnancy in a growth hormone-deficient woman. AB - Information on the course and outcome of pregnancies in growth hormone (GH) deficient patients is sparse, and GH treatment during pregnancy in such women has not been described previously. We have studied fetal growth and serum levels of GH, insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) during pregnancy, as well as birth weight and hormone levels after delivery in a 25-year-old woman with idiopathic, isolated GH deficiency diagnosed at the age of 7 years. As part of a clinical trial, the patient was treated with 2 IU/M2 GH for a period of 5 years. At this time she became pregnant after donor insemination. The GH treatment was continued until variant GH production from the placenta was evident. Serum levels of GH, IGF-I and IGFBP-3 were measured monthly during pregnancy after 3 days off GH therapy. Abdominal ultrasound was performed five times. Hormonal levels were measured immediately after delivery and during the following days. Serum GH and IGF-I levels increased during the second half of pregnancy; serum IGFBP-3 remained constant throughout pregnancy at a normal level. Serum levels of GH fell within 1 h after delivery, and levels of IGF-I and IGFBP-3 decreased into the range of GH-deficient women 4 days after. The fetal biparietal diameter increased normally, and birthweight was 3.564 kg, length 52 cm. No adverse events were recorded. We conclude that the role of GH replacement during pregnancy of GH-deficient women should be investigated further. PMID- 7540479 TI - Levels of circulating intercellular adhesion molecule-1 and -3 but not circulating endothelial leucocyte adhesion molecule are increased in patients with rheumatoid vasculitis. AB - The aim of this study was to investigate whether levels of circulating adhesion molecules reflect vascular inflammation in rheumatoid vasculitis (RV). Levels of circulating intercellular adhesion molecule-1 (cICAM-1), c-ICAM-3 and circulating endothelial leucocyte adhesion molecule (cE-selectin) were determined in 14 patients with RV and compared to 47 patients with rheumatoid arthritis (RA) and 100 healthy donors (HD). Enzyme-linked immunosorbent assays were used to quantify cICAM-1, cICAM-3 and cE-selectin. We found that in RV significantly (P < 0.0001) elevated levels of cICAM-1 and cICAM3, but not cE-selectin, were found when compared with RA patients. Levels > 2 S.D. above the mean level of HD were present for cICAM-1, cICAM-3 and cE-selectin in 57, 71 and 21%, respectively of patients with RV and 2, 21 and 44%, respectively of the RA patients. Increased levels of both cICAM-1 and cICAM-3 were found in 43% of the RV patients and in none of the RA patients. Comparison of the serum levels of patients studied in an active and inactive phase of RV revealed significantly lower levels of cICAM-3 levels in the inactive phase. In conclusion we find that determination of cICAM-1 and cICAM-3 may be useful as a marker of vascular inflammation in patients with RV. PMID- 7540480 TI - Antigen specificity of ANCA in systemic vasculitis. PMID- 7540481 TI - Influence of end-stage renal disease and renal transplantation on serum prostate specific antigen. AB - OBJECTIVE: To evaluate the effect of dialysis and kidney transplantation on serum prostate-specific antigen (PSA) levels, and to compare these results with those from normal age-matched controls. In addition, to evaluate the efficacy of PSA and digital rectal examination (DRE) for detection of prostate cancer in patients on dialysis and following kidney transplantation. PATIENTS AND METHODS: Between January 1990 and July 1993 all men in the transplant programme over the age of 40 years who were undergoing pre-transplant evaluation or post-transplant follow-up underwent a yearly DRE and PSA evaluation. Twelve patients were evaluated pre transplant and 70 patients were evaluated post-transplant. A total of 136 PSA levels were obtained (1.7 per patient). Patients with suspicious findings underwent further evaluation with transrectal ultrasound and biopsies if indicated. Controls were patients without known prostate cancer who had been evaluated in a Prostate Cancer Awareness Clinic. RESULTS: The average PSA values in the study groups were unaffected by either dialysis or transplantation when compared with age-matched controls. In addition, 12 patients who had been on dialysis at the time of their initial evaluation and who subsequently underwent transplantation did not show any difference in their average PSA values pre- or post-transplant. Three patients (4%) were found to have prostate cancer and two underwent radical retropubic prostatectomy. They are free of disease 24 and 36 months post-operatively and neither has experienced any decline in renal function. Immunosuppression was not modified. CONCLUSION: Transplantation and dialytic therapy do not appear to affect clinical serum PSA levels. PSA and DRE appear to be equally valid for detection of prostate cancer in patients on dialysis and post-transplant when compared with the general population. Finally, radical prostatectomy appears to be a safe and feasible treatment option in this group of patients. PMID- 7540482 TI - Increase of serum prostatic specific antigen and clinical progression in pN + MO prostate cancer. AB - OBJECTIVE: To analyse the increase of serum prostatic specific antigen (PSA) as a means of early detection of progression in hormonally untreated or androgen deprived patients with T1-T4, pN+ and MO prostate cancer. PATIENTS AND METHODS: From 1986 to 1992 40 patients with T1-T4 pN+ MO prostate cancer were either deprived of androgen at diagnosis (Group 1, 19 patients) or had no immediate hormone manipulation (Group 2, 21 patients) and were followed at 3-6-monthly intervals when determinations of PSA and routine clinical/radiological examinations were performed. A significant increase in PSA was defined as a > or = 50% increase of the baseline PSA value which was the either the lowest PSA value within 6 months from the start of androgen deprivation (Group 1) or the initial PSA value (Group 2). RESULTS: By June 1993 22 of the 40 patients had clinically progressed. In 12 patients the progression was preceded by a significant increase in PSA (Group 1, three of four progressing patients; Group 2, nine of 18 progressing patients). A PSA increase of > or = 50% was observed simultaneously with clinical progression in six patients, whereas clinical progression occurred in four patients with no previous or simultaneous significant increase in PSA. In four of nine hormonally untreated patients > or = 1 year elapsed between antecedent PSA increase and clinical progression. CONCLUSION: In routine clinical practice PSA does not significantly increase (> or = 50% of baseline value and > 10 micrograms/L) before disease progression in about one third of patients with pN+ MO prostate cancer managed with or with no hormone manipulation. Future studies should be carried out to determine whether a lower rate of increase in PSA during follow-up has any clinical significance. PMID- 7540483 TI - Prostate-specific antigen and prognosis in patients with metastatic prostate cancer--a multivariable analysis of prostate cancer mortality. AB - OBJECTIVE: To analyse the prognostic significance of pre- and post-treatment serum prostate-specific antigen (PSA) levels, together with a variety of other factors, in a multivariable analysis of survival in men with stage D2 prostate cancer. PATIENTS AND METHODS: Cox's proportional hazards model was used to compare survival in 134 men with stage D2 metastatic prostate cancer followed prospectively over a 4 year period, using both univariable and multivariable models. The influence of the following factors on survival was analysed: pre- and post-treatment PSA (both absolute and percentage values), age, treatment, testosterone, pre- and post-treatment alkaline phosphatase (absolute and percentage values), acid phosphatase, haemoglobin, symptom score and performance status and extent of disease on bone scan. RESULTS: Pre-treatment PSA levels did not significantly influence survival. Similarly, a low absolute post-treatment PSA level at 3 months after the start of treatment conferred no survival advantage relative to patients with a high PSA level at this time. A post treatment percentage PSA of < 10% at 2, 3 and 6 months after commencement of treatment was associated with prolonged survival. Low pre-treatment alkaline phosphatase (less than the upper limit of normal) and high pre-treatment testosterone levels (> or = 10 nmol/L) were similarly associated with prolonged survival. CONCLUSIONS: The strong influence of post-treatment percentage PSA on survival in patients with stage D2 prostate cancer suggests that the percentage change in bulk of metastatic deposits is more important in determining survival than the absolute volume of tumour. Pre-treatment alkaline phosphatase seems to be a better indicator of tumour activity than pre-treatment PSA. These findings have important implications for the design and analysis of clinical trials of new therapies in men with stage D2 prostate cancer and for the future selection of alternative treatments for patients with this stage of the disease. PMID- 7540484 TI - A congenital pelvic arteriovenous malformation adjacent to the prostate. PMID- 7540486 TI - How useful are tests for rheumatoid factors, antiperinuclear factors, antikeratin antibody, and the HLA DR4 antigen for the diagnosis of rheumatoid arthritis? AB - To evaluate the usefulness for the diagnosis of rheumatoid arthritis of tests for rheumatoid factors, antiperinuclear factors, antikeratin antibodies, and the HLA DR4 antigen, we retrospectively reviewed the medical records of 138 patients consecutively admitted to our rheumatology department between January 1, 1988 and December 31, 1990 for evaluation of peripheral inflammatory joint manifestations. Each patient had a standard work-up including a physical examination, laboratory tests, and roentgenograms. In 1994, after a follow-up of three to six years, the final diagnosis was rheumatoid arthritis in 39 patients and another well-defined disorder in 63; no diagnosis was established in 36 patients, among whom nine were lost to follow-up. The decreasing order of diagnostic usefulness was antiperinuclear factors, HLA DR4, rheumatoid factors (latex test), and antikeratin antibody. The likelihood of rheumatoid arthritis was greatest in those patients with positivity of two of the three following markers: rheumatoid factors, antiperinuclear factors, and the HLA DR4 antigen. PMID- 7540485 TI - Activity of the MAP kinase ERK2 is controlled by a flexible surface loop. AB - BACKGROUND: The mitogen-activated protein (MAP) kinase, ERK2, is a tightly regulated enzyme in the ubiquitous Ras-activated protein kinase cascade. ERK2 is activated by phosphorylation at two sites, Y185 and T183, that lie in the phosphorylation lip at the mouth of the catalytic site. To ascertain the role of these two residues in securing the low-activity conformation of the enzymes we have carried out crystallographic analyses and assays of phosphorylation-site mutants of ERK2. RESULTS: The crystal structures of four mutants, T183E (threonine at residue 183 is replaced by glutamate), Y185E, Y185F and the double mutant T183E/Y185E, were determined. When T183 is replaced by glutamate, few conformational changes are observed. By contrast, when Y185 is replaced by glutamate, 19 residues become disordered, including the entire phosphorylation lip and an adjacent loop. The conservative substitution of phenylalanine for Y185 also induces relatively large conformational changes. A binding site for phosphotyrosine in the active enzyme is putatively identified on the basis of the high-resolution refinement of the structure of wild-type ERK2. CONCLUSIONS: The remarkable disorder observed throughout the phosphorylation lip when Y185 is mutated shows that the stability of the phosphorylation lip is rather low. Therefore, only modest amounts of binding energy will be required to dislodge the lip for phosphorylation, and it is likely that these residues will be involved in conformational changes associated with both with binding to kinases and phosphatases and with activation. Furthermore, the low-activity structure is specifically dependent on Y185, whereas there is no such dependency on T183. Both residues, however, participate in forming the active enzyme, contributing to its tight control. PMID- 7540487 TI - Bone metastases from prostate cancer without elevated prostate-specific antigen levels: diagnosis by anti-PSA immunostaining. PMID- 7540488 TI - Human CD4+ T cells can discriminate the molecular and structural context of T epitopes of HIV gp120 and HIV p66. AB - CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66 specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells. PMID- 7540489 TI - Diminished expression of cell-surface complement regulatory proteins in HIV infected children and with HIV infection of peripheral blood mononuclear cells in vitro. AB - Experimental data have established that HIV-infected lymphocytes activate the complement system. However, because mammalian lymphocytes possess a series of cell-surface complement regulators that inhibit amplification on autologous cells, complement-mediated destruction of host cells is usually inhibited. These studies were performed to examine whether alterations in the cell-surface complement regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) may occur during HIV infection in vitro or in vivo. The physiologic significance of these alterations were assessed by radiolabeled chromium release experiments. We show that MCP fluorescent intensity is significantly lessened in HIV-infected children and that DAF intensity is similarly lessened in infected children with advanced disease. These findings could be duplicated with HIV infection of peripheral blood mononuclear cells in vitro. PMID- 7540490 TI - Comparison of selective polymerase chain reaction primers and differential probe hybridization of polymerase chain reaction products for determination of relative amounts of codon 215 mutant and wild-type HIV-1 populations. AB - A mutation at codon 215 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene results in decreased sensitivity to zidovudine (ZDV). In order to follow changes in codon 215 mutant (MUT) and wild-type (WT) populations in the plasma of patients during therapy, two polymerase chain reaction (PCR) procedures were investigated. The first was a nested, selective PCR, wherein a first round with viral-specific primers was followed by a second round with allele-specific primers. Although the procedure is relatively sensitive, some samples in the first round of PCR could not be amplified. In mixing experiments, mispriming of the MUT primer made relative determination of quantities subjective and difficult. Differential hybridization of PCR product with probes specific for codon 215 MUT or WT sequences was also investigated. A probe directed to a highly conserved region of the RT gene in the amplified PCR product was used to determine the total amount of PCR product analyzed. Differential hybridization was linear and reproducible over several logs of MUT:WT ratios, and determination of a 1:100 ratio of MUT:WT was readily achieved. When applied to longitudinal samples from three patients, dramatic changes in each population were readily apparent. These changes were evaluated with regard to viral load. PMID- 7540491 TI - HTLV-I "indeterminate" western blot patterns observed in sera from tropical regions: the situation revisited. PMID- 7540493 TI - Tumor vascularization, Doppler measurements and chaos: what to do? PMID- 7540492 TI - Audit of a screening service for fetal abnormalities using early ultrasound scanning and maternal serum alpha-fetoprotein estimation combined with selective detailed scanning. AB - The objective of the study was the evaluation of a screening service for fetal abnormality using early ultrasound scanning to date and establish viability of the pregnancy combined with maternal serum alpha-fetoprotein estimation at 16 weeks and thereafter selective detailed scanning at 18-20 weeks based on clinical indication. The study was designed to be retrospective, emphasizing the possibility of diagnosis. Early scans were performed by radiographers and radiologists, detailed ones by radiologists and obstetricians with subspecialty training in prenatal diagnosis. The study was carried out in the ultrasound department of a large University Hospital. The subjects studied were all cases of abnormality identified pre- or postnatally in women delivering in the hospital over 4 years and to whom the screening service applied. Information was obtained from ultrasound and maternity records, birth notifications, pathology and autopsy reports and a special needs register. Cases were classified as not detectable, potentially detectable and usually detectable. Major fetal anomalies numbered 255 (total deliveries 19,497), a prevalence of 1.3%; 130 (51%) were diagnosed by ultrasound with 64% before 24 weeks and 36% later. Eleven chromosomal anomalies (4%) were diagnosed by genetic methods and 114 (45%) anomalies were not diagnosed antenatally. The sensitivity of the program was 37% before 24 weeks. Chromosomal anomalies, lesions of the central nervous system and cardiac defects were most common, followed by lesions of the gastrointestinal and urinary tracts. A large proportion of the detected and detectable anomalies were lethal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540494 TI - [Molecular basis and clinical significance of HIV-1 resistance to nucleoside compounds]. AB - The prolonged use of anti-viral nucleosides (ZDV, ddI, ddC) in HIV-infected patients has given rise to the isolation of viral variants that display resistance against these compounds. Tissue culture selection experiments, involving increasing concentrations of anti-viral compounds, have likewise been shown to select for drug-resistant strains of HIV. Cloning, sequencing and site directed mutagenesis have shown that a series of point mutations in the viral reverse transcriptase (RT) are responsible for this phenomenon. A different series of mutations in RT are responsible for resistance against non-nucleoside inhibitors of this enzyme. These mutations are due to the error-prone nature of RT during viral replication. Mutated forms of recombinant RT, that derive from drug-resistant viruses, have reduced affinity for relevant triphosphorylated nucleosides. PMID- 7540495 TI - Functional human platelet generation in vitro and regulation of cytoplasmic process formation. AB - A recent report from this laboratory described an in vitro system in which CD34+ cells are stimulated to form mature megakaryocytes and, subsequently, cytoplasmic processes also known as pro-platelets that give rise to functional platelets. Thrombin, an important regulator of hemostasis, has been demonstrated to have an inhibitory role in cytoplasmic process formation from both human and guinea pig megakaryocytes. This inhibition can be reversed by antithrombin III (ATIII), an inhibitor of thrombin, in combination with heparin, a cofactor of ATIII and a glycosaminoglycan. Using the described human in vitro system, the role of thrombin and of glycosaminoglycans are investigated. Thrombin receptors are expressed on megakaryocytes, suggesting that the inhibition by thrombin may be direct. Matrigel, a basement membrane matrix containing glycosaminoglycans, is used to compare the frequency and the rate of cytoplasmic process formation. A possible role of glycosaminoglycans in platelet production is discussed. PMID- 7540496 TI - [Genetic diagnosis of a subtype diabetes mellitus with mitochondrial tRNA Leu(UUR) gene mutation]. AB - The A-G point mutation of mitochondrial tRNALeu(UUR) gene at nucleotide 3243 caused clinical NIDDM in combination with or without deafness and was inherited through maternal transmission. An ApaI restriction site was resulted by this mutation, which could be detected by polymerase chain reaction (PCR) with ApaI digestion and be used in clinical genetic diagnosis. The first Chinese family with this mutation found through our screening of NIDDM patients was reported. Genetic diagnosis was done in 12 of 15 members in this three-generation pedigree. Positive results (present of ApaI restriction site) were observed in all 5 NIDDM patients (four with deafness). In 6 of 7 non-diabetic members, negative genetic diagnosis was obtained. The presymptomatic diagnosis of this disease was made in a 13-year old non-diabetic boy with positive genetic diagnosis in conjunction with the maternal segregation pattern of the mutation in this family. The finding of this disease in Chinese provides firm evidence of genetic heterogeneity in Chinese NIDDM. The techniques of the genetic diagnosis of this disease can be used in clinical laboratory, which indicates that in the diagnosis of NIDDN, the molecular etiologic level can be reached in daily clinical practice. PMID- 7540497 TI - Galanin--10 years with a neuroendocrine peptide. AB - Galanin is a 29/30 amino acids long neuropeptide which does not belong to any known peptide family. The N-terminal first 16 amino acids of the molecule are both necessary and sufficient for receptor recognition and receptor activation. The main pharmacophores of galanin in its central and pancreatic actions are Gly1, Trp2, Asn5 and Tyr9, respectively. The neuropeptide galanin has multiple effects in both the central and peripheral nervous systems. Centrally, galanin potently stimulates fat intake and impairs cognitive performance. Anoxic glutamate release in the hippocampus is inhibited by galanin and the noradrenergic tonus in the brain is influenced by a hyperpolarizing action of galanin in the locus coeruleus. In the spinal cord galanin inhibits spinal excitability and potentiates the analgesic effect of morphine. In the neuroendocrine system galanin acts in a stimulatory manner on the release of growth hormone and prolactin, and peripherally galanin inhibits glucose induced insulin release. Galanin also causes contraction of the jejunum. The galanin receptor is a Gi-protein-coupled, membrane-bound glycoprotein with an estimated molecular mass of 53 kDa. Several putative tissue specific galanin receptor subtypes have been proposed on a pharmacological basis. The distribution of galanin receptors and of galanin like immunoreactivity are overlapping in the CNS, both being high in areas such as the locus coeruleus, raphe nucleus and hypothalamus. Galanin receptor activation leads to a reduced intracellular Ca(2+) concentration, either by direct action on voltage sensitive Ca(2+)-channels or indirectly via opening of K(+)-channels or via inhibition of adenylyl cyclase activity. The lowered intracellular Ca2+ level subsequently leads to a reduced PLC activity. Galanin also inhibits cGMP synthesis induced by depolarization. A number of synthetic high affinity galanin receptor antagonists of the peptide type were developed recently, which have enabled the elucidation of functional roles of endogenous galanin in several systems. Furthermore, putative subtypes of galanin receptors can be distinguished by the use of these new galanin receptor ligands. PMID- 7540498 TI - Separation of lipoproteins, albumin and gamma-globulin by single-step ultracentrifugation of human serum. Application. I: Binding of hematoporphyrin to human serum and to albumin. AB - Previous studies of the serum binding of the photosensitizer hematoporphyrin (Hp) have given widely different results. The serum binding of Hp is therefore further illuminated by experiment and discussion. Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classes and the two major high density proteins (HDP), albumin and gamma-globulin, were separated in a NaCl-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (10(3)/M) was much lower than earlier published ones (10(6)/M) found by other techniques. The association of Hp with HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreased with increasing Hp concentration. The serum binding of Hp is the same as that found previously using another rotor and another salt gradient (70.1 Ti, 70,000 rpm, NaCl-CsCl). LDL has high-affinity-low-capacity binding sites for Hp. HSA is the major HDP protein that binds Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presence of other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp. PMID- 7540499 TI - Purification and characterization of the serotype-specific polysaccharide antigen of Trichosporon cutaneum serotype II: a disease-related antigen of Japanese summer-type hypersensitivity pneumonitis. AB - Summer-type hypersensitivity pneumonitis (SHP) is a unique type of hypersensitivity pneumonitis and the most prevalent in Japan. Our previous study clarified that the causative agent of the disease is Trichosporon cutaneum, and that the patients with SHP have high titres of antibodies against the serotype specific antigen of polysaccharide nature which exist in the high molecular weight fraction of the culture supernatant of the yeast. In this study, we purified the serotype-specific antigen of serotype II T. cutaneum by gel filtration and affinity chromatography using a monoclonal antibody, D-8, specific for a high molecular weight antigen of serotype II T. cutaneum, and elucidated the structure of the antigen. This affinity-purified antigen was shown to be an essentially acidic polysaccharide comprising mannose, xylose, and glucuronic acid (6:44:4.7). Chemical analysis showed that this polysaccharide antigen contains a (1-3)-linked mannan backbone attached with short side chains of (1-4)-linked mannose and a small proportion of (1-2)-linked xylose residues by substituting the 2- or 4-positions of the (1-3)-linked mannose residues of the main chain. Approximately one-fifth of the side chains were terminated with glucuronic acid residues. The antigenic epitope of the serotype-specific antigen was shown to involve the terminal glucoronic acid residues as revealed by immunodiffusion test and sandwich enzyme-linked immunosorbent assay using monoclonal antibody D-8. PMID- 7540500 TI - Horse gammaglobulin-induced thrombocytopenia in anaphylaxis involving sequestration and activation of platelets. AB - Thrombocytopenia as well as hemoconcentration and leukopenia followed by leukocytosis were induced after HoGG challenge on HoGG-sensitized mice. Thrombocytopenia was induced within 2 min and sustained for 1 day. HoGG-induced thrombocytopenia was not observed until day 10 after sensitization; mice challenged with HoGG dose > or = 10 micrograms developed thrombocytopenia. Two types of thrombocytopenia were observed in appropriately sensitized mice. HoGG induced thrombocytopenia at 2 min and 60 min, whereas, alpha-macroglobulin induced thrombocytopenia at 2 min, the platelet count of which returned to normal within 60 min. Poly (Glu60Ala30Tyr10) did not induce thrombocytopenia at 2 min or 60 min. The tracing study by 3H-serotonin labelled platelets demonstrated the 2 min-sequestration of platelets in lungs or livers. The HoGG-induced sequestration of platelets at 2 min was blocked by high dose heparin or Cobra Venom factor. Platelet activation at 60 min was partially inhibited by dexamethasone, rhodostomin synthetic peptide 45-59, or platelet activation factor antagonist (WEB 2086). Furthermore, the thrombocytopenia could be transferred by heat (56 degrees C, 4h) treated immune sera. This suggests that HoGG-induced, non-IgE mediated thrombocytopenia in anaphylaxis involves sequestration and activation of platelets. The sequestion in lungs occurs within 2 min and can be inhibited by high dose heparin or Cobra Venom factor. The activation of platelets involves platelet activation factor, and fibrinogen receptor. PMID- 7540501 TI - [Fetomaternal macrotransfusion--a cause for decreased fetal movements. 2 cases]. AB - Two cases of fetomaternal macrotransfusion in otherwise normal pregnancies are reported. In both cases the main symptoms observed by the patients were decreasing fetal movements. Cardiotocography revealed a highly pathologic pattern an immediate cesarean section was performed. The cause of massive fetomaternal hemorrhage (390 and 635 ml fetal blood) remains unclear. Delayed treatment leads to severe anemia followed by hypovolemic shock and ultimately to stillbirth. Pregnancies complicated by fetomaternal hemorrhage develop normally until signs of fetal decompensation begin to appear. Decreasing fetal movements in the most common symptom reported by the patients. In addition to a contraction-stress test, blood from the pregnant women should be looked at for fetal erythrocytes, a test that can be performed very quickly and easily. If the fetus is viable, immediate delivery should be performed and blood transfusions to the newborn should be administered. In preterm pregnancies cordocentesis and intrauterine blood transfusion may be considered. PMID- 7540502 TI - Protective effects of prophylaxis with a protease inhibitor and a free radical scavenger against a temporary ischemia model of pancreatitis. AB - OBJECTIVE: To evaluate the protective effects of combined prophylaxis with 4 sulfamoylphenyl, 4-guanidinobenzoate methanesulfonate (E3123), potent new protease inhibitor, and superoxide dismutase (SOD), a free radical scavenger, against the multifactor-related pancreatic injuries that occur in acute pancreatitis. DESIGN: Controlled experimental study in rats with pancreatitis induced by a temporary ischemic model of pancreaticobiliary-duct (PBD) obstruction. SETTING: A university-affiliated hospital. EXPERIMENTAL SUBJECTS: Seventy-eight male Wistar rats, weighing approximately 300 g each. INTERVENTIONS: In 18 rats the PBD was occluded with a metal clip, and cerulein was infused for 30 minutes at an hourly rate of 0.2 microgram/kg. After 1 hour the metal clip was removed and the abdomen closed (PBD obstruction group). In 21 rats the same protocol was followed, but ischemia was induced by occlusion of celiac and caudate mesenteric arteries for 30 minutes (ischemia group). In 24 rats the same protocol was followed as for the ischemia group, but 1 hour before and throughout the experiment, E3123 was infused at an hourly rate of 5 mg/kg, and before occlusion of the PBD and immediately after release of the occlusions of the PBD and arteries, SOD, 1 mg/kg, was injected intravenously (treatment group). Fifteen rats underwent laparotomy and gentle manipulation of the pancreatobiliary duct and celiac and caudate mesenteric arteries only (control group). MAIN OUTCOME MEASURES: Serum amylase levels and pancreatic water and amylase levels, histologic changes in pancreas, subcellular amylase and cathepsin-B activities and in-vivo amylase and cathepsin-B outputs. RESULTS: Serum amylase levels and pancreatic water and amylase content were significantly (p < 0.05) reduced in the treatment group (mean [+/- standard error] 13 [2] U/mL, 77 [2] % of wet weight and 503 [56] U/mg of DNA respectively) compared with the ischemia group (25 [3] U/mL, 83 [2] % of wet weight and 731 [52] U/mg of DNA respectively). PBD obstruction and ischemia also caused a significant (p < 0.05) subcellular redistribution of cathepsin B from the lysosomal to the zymogen fraction and impaired output of amylase and cathepsin B into pancreatic juice, which was compensated for in the treatment group. CONCLUSIONS: Temporary pancreatic ischemia and oxygen-derived free radicals appear to play an important role in the pathogenesis of acute pancreatitis. E3123 and SOD may be useful in the prophylaxis of clinical pancreatitis. PMID- 7540503 TI - Internal mammary artery embolization for hepatic tumors. AB - PURPOSE: To prospectively identify patients with collateral internal mammary artery (IMA) supply to hepatic tumors, and to embolize the IMA as part of palliative hepatic artery embolization (HAE). METHODS: Over a 4-year period, 222 patients were enrolled in an HAE protocol for inoperable liver tumors. All underwent abdominal computed tomography (CT) prior to preembolization diagnostic hepatic arteriography. When anterior subcapsular hepatic tumor was seen on the CT scan, patients underwent IMA arteriography as well. All arteries supplying tumor were embolized. RESULTS: Six patients had anterior subcapsular lesions identified on CT. IMA catheterization revealed that these lesions drew blood supply directly or indirectly from one or both IMAs in all six patients. All IMAs were embolized. CONCLUSION: When an hepatic tumor is identified in an anterior subcapsular location prior to HAE, the IMA should be examined and, if significant tumor supply is demonstrated, these branches should be embolized. PMID- 7540505 TI - Melanosis of the prostate in an elderly patient--a case report. AB - A case of melanosis of the prostate in an 85 year old Libyan patient is reported. This rare lesion is the first incidence from our area. The occurrence of melanin pigment in the prostate is extremely rare. After the initial report by Nigogosyan and co-workers in 1963, Ro et al in their review have analysed 23 documented lesions in which melanin pigment was confined to melanocytes in the stroma of the prostate only (blue nevus of the prostate), melanin pigment was present in both the stromal melanocytes and glandular epithelium (melanosis of the prostate) and lessions in which the pigment was observed in both normal and neoplastic epithelium. Later Lew et al reported another case of blue nevus of the prostate. However, reports of melanosis of the prostate are less frequent than blue nevus of the prostate. Melanosis of the prostate has not been reported from the African continent and from the Arab population according to the accessible English literature. Here we present the first case of melanosis of the prostate in an 85 year old Libyan Arab bringing the total number of melanosis of the prostate to nine. PMID- 7540506 TI - Divergent differentiation in choroid plexus papilloma. An immunohistochemical study of five cases. AB - Intermediate filament and S100 expression was studied in five cases of choroid plexus papilloma. All the cases showed positivity for S100 and Nimentin. Cytokeratin and glial fibrillary protein (GFAP) intermediate filaments were present in 80 pc of the cases. None of the cases expressed epithelial membrane antigen. These results confirm the divergent differentiation of choroid plexus tumours. PMID- 7540504 TI - Combined transjugular intrahepatic portosystemic shunt and segmental Lipiodol hepatic artery embolization for the treatment of esophagogastric varices and hepatocellular carcinoma in patients with cirrhosis: preliminary report. AB - PURPOSE: To evaluate the feasibility of combining placement of a transjugular intrahepatic portosystemic shunt (TIPS) and transcatheter hepatic segmental artery chemoembolization with Lipiodol (Seg-Lp-TAE) in patients with cirrhosis, esophagogastric varices, and hepatocellular carcinoma (HCC). METHODS: Five patients with bleeding or large, high-flow esophagogastric varices and HCC were treated by TIPS and Seg-Lp-TAE. RESULTS: The mean portosystemic pressure gradient decreased from 20.8 cm H2O to 7.8 cm H2O after TIPS. The direct portogram and endoscopic examination revealed reduction of varices. At 6 months, one shunt had functionally occluded and could not be reopened; the other TIPS remained functional. Follow-up CT and the changes of alpha fetoprotein indicated effective therapy of Seg-Lp-TAE for HCC. Four patients are in stabile clinical condition at 9, 6, 1, and 1 months after the combined therapy; one died after 14 months due to decompensated liver cirrhosis. CONCLUSION: The combined therapy of TIPS and Seg Lp-TAE will become a new interventional approach for patients with HCC and esophagogastric varices. PMID- 7540508 TI - [Study on serum anti-HCV antibodies in hemodialysis patients]. PMID- 7540507 TI - [Basaloid squamous carcinoma of the oesophagus: a distinctive clinico pathological entity]. AB - 5 cases of basaloid-squamous carcinoma (BSC) of oesophagus were reported. Their pathological features were: 1. The main component of the tumors were basaloid carcinoma cells. 2. Concomitant squamous cell differentiation. 3. Comedo-like necrosis in the basaloid carcinoma component of the tumor. 4. Hyaline degeneration within the stroma of the basaloid carcinoma nests (PAS+). The immunohistochemistry of keratin 10.11, CEA and EMA in the basaloid carcinoma component of BSC were negative or weak positive, while actin and S-100 were positive in some parts of the tumor sections. This suggested that the carcinoma component was poorly differentiated and somewhat tended to differentiate toward myoepithelia or other directions. We therefore consider that the origin of BSC may be the primitive totipotential cell. BSC occurred more frequently in elderly males. The biological behavior of BSC was highly malignant. Regional lymph nodes or distant organ metastasis were usually found at the first operation. The mean survival period after operation was very short, BSC was therefore considered to be a specific clinicopathological entity. PMID- 7540509 TI - [Exploration of combining textbooks and teaching of medical nursing with surgical nursing in China]. PMID- 7540511 TI - [Care of radical surgery of Budd-Chiari syndrome]. PMID- 7540510 TI - [Postoperative care of cerebellar ataxia]. PMID- 7540512 TI - Acute normovolaemic haemodilution does not reduce the inflammatory process induced by facial surgery. AB - The place of preoperative acute normovolaemic haemodilution (haematocrit = 28% 32%) in reducing postoperative inflammation was evaluated after facial surgery. Thirty-two patients scheduled for mandibular osteotomy were randomized to a nonhaemodiluted group or to a haemodiluted group. The degree of postoperative inflammation was evaluated: first by an x-ray technique (radiotelemetry) providing measurements of the tissue thickness (quantitation of facial oedema), and second by the measurement of four acute phase protein plasma concentrations during the first postoperative week. Throughout the study, no changes in facial oedema or in variation of acute phase proteins were detected after haemodilution. It is concluded that acute normovolaemic haemodilution has no effect on the intensity of facial oedema and the biological inflammation process after facial surgery. PMID- 7540513 TI - Formation of an oxidative DNA damage, 8-hydroxydeoxyguanosine, in mouse lung DNA after intratracheal instillation of diesel exhaust particles and effects of high dietary fat and beta-carotene on this process. AB - Diesel exhaust particles (DEP) cause tumors in the respiratory tracts of experimental animals. It was previously shown that DEP produced superoxide and hydroxyl radical. To examine whether oxygen radicals are involved in mouse lung tumorigenesis induced by DEP, formation of an oxidative DNA damage, 8 hydroxydeoxyguanosine (oh8dG), by DEP was investigated. Furthermore, the role of high dietary fat and beta-carotene on this process was studied. After intratracheal instillation of DEP, a significant increase of oh8dG in mouse lung DNA was observed. High dietary fat enhanced the formation of oh8dG in lung DNA. Intake of beta-carotene suppressed the formation of oh8dG in lung DNA, but the protective effect of beta-carotene against this process was not statistically significant. These results suggest that formation of oh8dG in lung DNA was induced by oxygen free radicals produced by DEP. Thus, it is possible that oh8dG is a promutagenic lesion in DEP-induced lung tumorigenesis in mice and high dietary fat enhances this process through the generation of oh8dG in mouse lung DNA. PMID- 7540514 TI - Ribozyme-mediated inhibition of expression of leukocyte-type 12-lipoxygenase in porcine aortic vascular smooth muscle cells. AB - Activation of a leukocyte-type 12-lipoxygenase (12-LO) has been proposed to be an important mechanism for angiotensin II- and glucose-induced vascular smooth muscle cell growth. Currently, no specific pharmacological inhibitors for the leukocyte-type 12-LO are available to test this hypothesis. We have therefore designed a chimeric DNA-RNA hammerhead ribozyme to produce cleavage at the first GUC sequence at nucleotide 7 of porcine leukocyte 12-LO mRNA. The ribozyme was tested in vitro with a 206-base 12-LO mRNA as substrate. We observed that the ribozyme specifically and dose-dependently cleaved porcine leukocyte 12-LO mRNA at the predicted site under physiological temperature. Furthermore, we also efficiently delivered the ribozyme into porcine aortic vascular smooth muscle cells by transfection with cationic liposomes. The ribozyme caused a dose dependent decrease in levels of porcine leukocyte-type 12-LO mRNA in these cells and was more potent than an antisense oligonucleotide directed against porcine leukocyte 12-LO. The 12-LO ribozyme also attenuated 12-LO protein levels in the cells. The action of the ribozyme was primarily a result of its catalytic activity, since a modified ribozyme that lacks catalytic activity showed reduced effects. This represents the first ribozyme directed against a mammalian LO pathway. These results demonstrate the potential utility of new ribozyme technology to generate novel agents for gene modulation experiments to modify the development or progression of vascular disease in humans. PMID- 7540515 TI - Effect of graded hypoxia on the induction and function of inducible nitric oxide synthase in rat mesangial cells. AB - Inducible nitric oxide synthase (iNOS) catalyzes the formation of nitric oxide (NO) from L-arginine and O2. Although some O2 is required for this reaction, it is uncertain whether biologically relevant levels of hypoxia alter this pathway. We examined the effects of graded hypoxia on several steps in the iNOS pathway in lipopolysaccharide (LPS)-stimulated rat glomerular mesangial cells: induction of iNOS mRNA, NO synthesis, NO oxidation to nitrite (NO2-) and nitrate (NO3-), and accumulation of cGMP. Cultured cells were incubated for 24 hours in airtight flasks containing O2 (21%, 10%, 2.5%, and 0%), CO2 (5%), and N2 (balance), resulting in media PO2 levels of 140 +/- 3, 85 +/- 1, 46 +/- 3 (moderate hypoxia), and 32 +/- 5 (severe hypoxia) mm Hg, respectively. During normoxia (PO2, 85 to 140 mm Hg) LPS increased iNOS mRNA with associated increases in NO synthesis, NO2- and NO3- accumulation, and intracellular cGMP levels. In the absence of LPS, there was minimal NO synthesis and no detectable iNOS mRNA. Even during severe hypoxia, LPS elevated NO2- and NO3- relative to levels in unstimulated cells (P < .05), although to a lesser extent than during normoxia (P < .05). The induction of iNOS mRNA by LPS was preserved in hypoxia, and intracellular cGMP levels were similar at all levels of oxygen tension, indicating that iNOS induction and function were not altered by moderate or severe hypoxia. However, moderate hypoxia did alter the partitioning and oxidation of NO, favoring the appearance of NO in the "headspace" (defined as the gas overlying the cells) and NO3- in the media.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540517 TI - Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue. AB - Gene transfer to blood vessels in vivo generally requires interruption of blood flow. Thus, gene transduction to cerebral blood vessels in vivo has not yet been achieved. In this study, we injected replication-deficient adenovirus into cerebrospinal fluid in an attempt to transduce genes to cerebral blood vessels. Recombinant adenovirus (1 x 10(9) infectious units) expressing nuclear-targeted bacterial beta-galactosidase driven by the cytomegalovirus promoter was injected into the cisterna magna of Sprague-Dawley rats. The brains were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-beta-D galactopyranoside 1 to 7 days after injection of adenovirus. Leptomeningeal cells overlying the major arteries were efficiently transduced, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. beta-Galactosidase was expressed on days 1 and 3 after injection but was undetectable by day 7. Expression of the gene was 'targeted' by altering the position of the head. When viral suspension was injected while the rat was in a nose-down position, the reporter gene was expressed extensively on the ventral surface of the brain, especially along the circle of Willis. When the position was changed to the nose-up or lateral position, the inferior or lateral region of the brain was stained primarily. Administration of the virus into the lateral ventricle provided extensive expression in ependymal cells and leptomeninges with some transduction to cerebral blood vessels. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, cisternal delivery may target specific brain regions by positioning of the head.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540516 TI - Differential activation of mitogen-activated protein kinases by H2O2 and O2- in vascular smooth muscle cells. AB - Increased generation of active oxygen species such as H2O2 and O2- may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H2O2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H2O2 and O2- on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O2- was generated in a concentration dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [3H]thymidine incorporation, was stimulated by 200 mumol/L H2O2 (110% increase versus 0.1% serum) and 1 mumol/L LY83583 (175% increase) to levels comparable to 10 ng/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 mumol/L) but not by H2O2. The time course for activation of MAP kinase by LY83583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H2O2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H2O2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540518 TI - [A preliminary report on diagnostic complementarity of gut-associated and egg associated antigenemia in schistosomiasis japonica]. AB - With Hyroxylapatite purified preparations and BACH (biotin aminocapryl hydrazide) biotinylated McAbs, 274-2H10 and 273-2H1, recognizing different egg-associated epitopes, biotin-avidin (BA) involved alkaline phosphatase (AP) ELISA with detecting sensitivities reaching nanogram levels (10(-9), were set up. The detectable limit for crude preparations of Schistosoma japonicum SEAJ-TCA in 2H10 ELISA achieved 1. 0.3. 2 ng/ml, in which only S. japonicum specific egg antigens were efficiently detected, whereas with 2H1-ELISA, which could detect SEA-TCA of both S. japonicum and S. mansoni species, an end point of detecting 3.2 ng/ml was obtained. Repeated tests with human serum groups revealed very significant differences of extinction OD readings between patients and normal individuals. For detection combinations, a previously established anti-CAA homologous AP-ELISA system was parallelly used for gut-associated antigenemia determinations. Taking the mean extinction OD reading of a parallel normal serum group plus 3 SD as corresponding cut off values, 3 patient groups (n = 82, 52, 39) from different areas of transmission intensity were subjected to accumulating determinations for egg- and gut-associated antigenemia. Improved detectabilities to variable extent were achieved in either of the 2 or 3 combinations. The study thus demonstrated that the diagnostic efficiency for human schistosomiasis could be improved by multi-epitope detections for more than one target molecule using corresponding McAbs, especially in areas where the transmission intensity of the disease is comparatively lower. PMID- 7540520 TI - The effect of nasal mucosal vasoconstriction on nasal airflow sensation. AB - The correlation between the objective measurement of nasal resistance and nasal airflow sensation is usually regarded as poor. To investigate the relationship between these two parameters 20 healthy volunteers had nasal resistance to airflow measured by rhinomanometry compared with nasal sensation by visual analogue scoring before and after nasal mucosal vasoconstriction using topical 0.1% xylometazoline. The median change in nasal resistance was 0.2 kPal-1s (95% CI 0.08-0.28 kPal-1s) and in nasal sensation 24 mm (95% CI 17 mm-35 mm). A significant relationship between nasal sensation and nasal resistance to airflow was found (Kendall's Rank correlation (P < 0.05). This function can be described by the linear regression equation: dS = 13.2 + 70.dNR where dS = change in nasal sensation and dNR = change in nasal resistance. There may be a much closer relationship between subjective and objective measures of nasal patency than has previously been thought. PMID- 7540519 TI - [Study on adjuvant effect of Schistosoma japonicum iRNA]. AB - The adjuvant effect of schistosome iRNA on the schistosome antigen was studied. The results by using ELISA showed that the antibody level in mice immunized with S iRNA + SWA was significantly higher than in those only immunized with S iRNA or SWA. After cercariae challenge infection, mice immunized with S iRNA + SWA or S iRNA had higher worm reduction rate. However, there were no significant differences between the two groups of mice in terms of worm reduction rate. These results suggested that the host humoral response can be enhanced by S iRNA and that the host immunity against S. japonicum failed to be enhanced by S iRNA. PMID- 7540521 TI - Autoimmune mechanisms in the pathogenesis of paraneoplastic nervous system disease. AB - There is compelling circumstantial evidence that humoral autoimmune response may be involved in the generation of paraneoplastic neurologic syndromes (PNS). However, still it is unproven whether the autoantibody is pathogenic or represents an epiphenomenon to the actual disease process. Paraneoplastic antibodies have been identified in patients with PNS which react with similar antigenic epitopes in tumor and neurons. However, the antigens identified by the paraneoplastic antibodies are primarily intracellular in location, questioning the pathogenetic role of the antibody. The molecular events of antigen processing and presentation, T-cell receptor restriction, polyclonal B-cell proliferation, cellular immunity, and the role of cytokines in mediation of neuronal injury must be further defined in order to elucidate the role of autoimmunity in the pathogenesis of these disorders. PMID- 7540522 TI - Autoimmune paraneoplastic cerebellar degeneration: immunohistological localization of antibody-binding sites. AB - Sera from 3 patients with breast or ovarian tumors and paraneoplastic cerebellar degeneration (PCD) contained anti-Purkinje cell antibodies (PCAbs) which also bind to other neurons on frozen sections of adult rodent brain. PCAbs tested on new-born rodent (rabbit, rat, mouse) brain tissue detected only oligodendrocyte like cells (ODLC) in the white matter and allowed us to speculate on the nature of the antigenic structure in the neuronal cytoplasm. All these PCAbs appear histochemically identical and recognize antigens which belong to so-called "Yo" proteins. PMID- 7540523 TI - Developmental delay caused by a supernumerary chromosome, inv dup (15), identified by fluorescent in situ hybridization. PMID- 7540525 TI - Cigarette smoking influences cytokine production and antioxidant defences. AB - 1. Smoking exerts an inflammatory stimulus on lung macrophages, and smokers generally have low intakes of antioxidant micronutrients. This study was performed to investigate the relationship between whole-blood tumour necrosis factor production, plasma interleukin-6 and acute-phase protein concentration and antioxidant vitamins in smokers and non-smokers. 2. Measurement of tumour necrosis factor was conducted in whole blood stimulated with endotoxin (lipopolysaccharide), and interleukin-6 concentrations were measured in the plasma of smokers and non-smokers. Enzyme and dietary antioxidant concentrations and acute-phase proteins were determined in the two groups. 3. Tumour necrosis factor production and plasma interleukin-6 concentrations were 38% (P = 0.01) and 16% (P = 0.07) greater, respectively, in smokers than in non-smokers. Plasma vitamin A and E concentrations were unaffected by smoking; however, a 21% lower plasma vitamin C (P = 0.04) concentration was observed in smokers, than in non smokers despite a similar intake of this vitamin by the two groups. 4. Concentrations of the acute-phase proteins alpha 1-acid glycoprotein, caeruloplasmin and alpha 2-macroglobulin were increased in the plasma of smokers compared with non-smokers by 39%, 28% and 12% respectively (P < 0.01). Our studies indicate that smokers have a compromised antioxidant status and elevated concentrations of tumour necrosis factor and interleukin-6 as a consequence of smoking. 5. These observations may provide some insight into the biological mechanisms underlying the pathology associated with smoking. PMID- 7540524 TI - Human skeletal muscle protein: effect of malnutrition, elective surgery and total parenteral nutrition. AB - 1. The concentration of alkali-soluble protein, DNA and RNA in percutaneous muscle biopsy specimens was analysed. Tissue alkali-soluble protein/DNA ratio is a measure of muscle protein concentration, while tissue RNA/DNA ratio may reflect the capacity for protein synthesis. 2. Patients with weight loss due to cancer (n = 6) were compared with metabolically healthy patients before elective surgery (n = 7). Alkali-soluble protein/DNA and RNA/DNA ratios in the weight loss group were 248 (14) g/g and 1.3 (0.1) g/g respectively as compared with 404 (13) g/g and 2.1 (0.1) g/g in otherwise healthy patients. All of the alkali-soluble protein/DNA ratios and 5/6 of the RNA/DNA ratios in the weight loss group were below the 95% confidence interval for the healthy control subjects. 3. Patients undergoing elective open cholecystectomy (n = 7) were studied preoperatively and on days 3, 10, 20 and 30 post-operatively. The alkali-soluble protein/DNA ratio remained unchanged on post-operative day 3 but decreased by 8.7% (P < 0.01), 9.6% (P < 0.05) and 20.4% (P < 0.01) on days 10, 20 and 30 respectively in patients eating at will after the operation. No significant post-operative changes in alkali soluble protein/DNA ratio were seen in patients given post-operative total parenteral nutrition with (n = 9) or without (n = 7) glycyl-glutamine supplementation for 3 days after surgery. 4. In conclusion, patients with weight loss due to malignant disease have a low muscle protein concentration. Elective surgery of medium magnitude results in a decrease in muscle protein lasting for more than 30 days.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540526 TI - Treatment of vasculitic leg ulcers in connective tissue disease with iloprost. AB - Leg ulcers are a recognised manifestation of cutaneous vasculitis in connective tissue diseases (CTDs) including rheumatoid arthritis (RA). Iloprost a stable prostacyclin analogue has been successfully used to treat Raynaud's phenomenon and digital ulcers associated with CTD's. Our aim was to assess iloprost in the treatment of vasculitic leg ulcers in CTD. In this paper we describe eight cases of vasculitic leg ulceration in association with RA and CTD, treated with intravenous iloprost. The iloprost was administered for 6-8 hours daily and continued for 21-28 days. Immunosuppressive therapy was required in three patients with severe necrotising vasculitis (RAnv). Complete ulcer healing was achieved in four patients within 6 weeks of commencing therapy while rapid improvement occurred in the other four patients. This suggests that iloprost may be useful as an adjunct to therapy for vasculitic leg ulcers. A double-blind placebo controlled study of iloprost therapy for RA leg ulcers is under way. PMID- 7540527 TI - Felty's syndrome treated with rhG-CSF associated with flare of arthritis and skin rash. AB - A patient with Felty's syndrome and rheumatoid arthritis was treated with recombinant granulocyte stimulating factor rhG-CSF (Neupogen) in view of severe neutropenia. He had a prompt rise in his neutrophil count and associated with this a severe flare of his arthritis and a skin rash. rhG-CSF was stopped, his neutrophil count fell rapidly and his symptoms resolved. rhG-CSF and the resulting rise in neutrophil count may be associated with flare of autoimmune disease in susceptible individuals. PMID- 7540528 TI - Flare of arthritis with successful treatment of Felty's syndrome with granulocyte colony stimulating factor (GCSF). AB - A 58-year-old white male with Felty's syndrome was successfully treated with granulocyte colony stimulating factor (GCSF). GCSF can correct the granulocytopenia of Felty's syndrome and may be a beneficial therapeutic adjunct in patients who have serious infections associated with neutropenia. The patient developed a flare of arthritis concomitant with increased circulating neutrophils following GCSF therapy. PMID- 7540529 TI - Combination effects of gamma-interferon with pinyanmycin or psoralen on tongue cancer HSC3 cell line in vitro and in vivo. AB - The inhibitory effects of gamma-interferon (gamma-IFN), pinyanmycin (PYM), and psoralen (PSO) on human tongue cancer HSC3 cells were tested with MTT assay (tetrazolium-based colorimetric assay). The IC50 (drug concentration which causes 50% inhibition of the cell line) values of gamma-IFN, PYM and PSO were 371,535 U/ml, 224 ng/ml and 25,119 ng/ml, respectively. The CI50 (combination index) values of the combinations of PYM with gamma-IFN at the concentrations of 10(2), 10(3), 10(4) and 10(5) U/ml were 0.12, 0.04, 0.05, and 0.29, respectively; and those of PSO with gamma-IFN at the concentrations of 10(2), 10(3), 10(4) and 10(5) U/ml were 1.00, 0.66, 0.30 and 0.28, respectively. The growth inhibition (%) of the HSC3 cells transplanted tumor in nude mice by gamma-IFN, PYM, gamma IFN plus PYM, PSO, and gamma-IFN plus PSO were 67.6, 103.5, 110.0, 83.1 and 102.9, respectively; the body weight loss (%) of the treated mice were 1.2, 6.9, 0.8, 5.0 and 1.8 respectively, and that of the control was 12.5. The results indicated that the combination effects of gamma-IFN with PYM or gamma-IFN with PSO are synergic and beneficial to the general health of the victim mice. PMID- 7540530 TI - Predictors of human papilloma virus in patients with keratinization. AB - Given the prevalence of human papilloma virus (HPV) infection, an attempt was made to determine whether certain factors such as keratinization and/or squamous atypia are associated with its development. Review of our gynecologic cytology files from 1989 yielded 1,615 specimens showing parakeratosis and/or hyperkeratosis, without cytologic evidence of HPV. Concomitant diagnoses included no atypia [keratinization only (KO)], inflammatory squamous atypia (ISA), and squamous atypia (SA). Morphologic follow-up including repeat cytology or biopsy was available for 916 cases, 92 (10.0%) of which possessed changes of HPV. For any case with both cytologic and biopsy evidence of HPV, only the biopsy result was tabulated. HPV on follow-up examination was detected in 52 (6.7%) of the 764 cases with KO; in 20 (20.8%) of the 96 cases with keratinization and ISA (KISA); and in 20 (35.7%) of the 56 cases with keratinization and SA (KSA). The definitive diagnosis of HPV was based on previously described features (Gupta, In: Comprehensive Cytopathology, Philadelphia: WB Saunders, 1991:133-140) including nuclear enlargement with nuclear membrane irregularities in combination with sharply demarcated paranuclear cytoplasmic clearing. Affected cells have rounded borders. Binucleated cells are not uncommon. The increasing percentage of HPV from KO to KISA to KSA is not necessarily surprising. However, mathematical analysis revealed statistically significant differences in the development of HPV in each of the 3 groups: KISA vs. KO (P < 0.001), KSA vs. KO (P < 0.001), and KSA vs. KISA (P < 0.05). Therefore, a cytologic diagnosis of keratinization with ISA or especially SA should warrant closer follow-up than that of KO. PMID- 7540531 TI - Neural crest cell-cell adhesion controlled by sequential and subpopulation specific expression of novel cadherins. AB - We identified two cadherins, c-cad6B and c-cad7, expressed by neural crest cells at their premigratory and migratory stages, respectively, in chicken embryos. cDNA transfection experiments showed that both were homophilic adhesion molecules, endowing cells with specific adhesiveness. During development, c-cad6B appeared in the neural fold, localizing at the future neural crest area. This expression was maintained during neural tube closure, but disappeared after neural crest cells had left the neural tube, suggesting its role in neural fold fusion and/or in the formation and maintenance of the presumptive neural crest domain in the neural plate/tube. Crest cells emerging from the neural tube lost c cad6B, and a subpopulation of them began to express c-cad7. This subpopulation specific expression of c-cad7 persisted during their migration. The migrating c cad7-positive cells clustered together, and eventually populated restricted regions including the dorsal and ventral roots but very little ganglia. The latter was populated with N-cadherin-positive crest cells. Migrating neural crest cells expressed alpha- and beta-catenin at cell-cell contacts, indicating that their cadherins are functioning. These results suggest that the migrating crest cells are grouped into subpopulations expressing different cadherins. The cadherin-mediated specific interaction between crest cells likely plays a role in intercellular signaling between homotypic cells as well as in sorting of heterotypic cells. PMID- 7540532 TI - A cloned, immortal line of murine melanoblasts inducible to differentiate to melanocytes. AB - Cultures of differentiated melanocytes can readily be grown from the dissociated epidermis of neonatal mice, and immortal cell lines often develop from these. However, the first cells that grow and transiently dominate the cultures, while similar to melanocytes, are unpigmented. These have been shown to be precursors of melanocytes and may be termed melanoblasts. Under our previous standard culture conditions, involving the use of keratinocyte feeder cells, foetal calf serum, the phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) and cholera toxin, all the melanoblasts spontaneously differentiated to pigmented melanocytes within about 3 weeks. We now describe some factors affecting the proliferation and differentiation of diploid murine melanoblasts in the presence of serum. Murine stem cell factor/steel factor (SCF), basic fibroblast growth factor (bFGF) and murine leukaemia inhibitory factor/differentiation-inhibiting activity (LIF/DIA) all increased melanoblast numbers. SCF and LIF also slightly inhibited melanoblast differentiation, while cholera toxin and TPA promoted differentiation. Using some of these findings, and by regular replacement of keratinocyte or fibroblastoid feeder cells, we have established a clonal line of immortal murine melanoblasts, 'melb-a'. These cells express tyrosinase-related protein-2 but not, in general, tyrosinase. They can be induced to differentiate irreversibly to functional melanocytes (also proliferative and immortal) by plating in the absence of feeder cells. Thus a new immortal melanocyte line, 'melan-a2', has also been produced. PMID- 7540533 TI - The effects of a chronic application of chlorpyrifos on the macroinvertebrate fauna in an outdoor artificial stream system: species responses. AB - An outdoor artificial stream system was used to examine the effects of a chronic application of the organophosphate pesticide chlorpyrifos on the invertebrate fauna of the system. Two replicate streams received chlorpyrifos for 21 days at the high dose (5 micrograms.liter-1) or low dose (0.1 microgram.liter-1) or only the carrier solution with which chlorpyrifos is mixed for commercial sale (1,1,1 trichloroethane and xylene). Streams behaved as replicates with respect to five water quality parameters. Seventy-four nonchironomid and 24 chironomid taxa were recorded during the study. The number of taxa and total invertebrate abundance were significantly reduced by both high and low doses of the pesticide. Shannon Weaver diversity was also reduced by both high and low doses of the pesticide whereas evenness increased in high dose streams. The individual abundances of 9/36 nonchironomid and 13/19 chironomid taxa were significantly reduced by pesticide application; the abundance of one taxon, the gastropod Physastra, increased. The biomass of periphyton in the streams was affected by changes in the abundance of chironomid grazers and Physastra, and the slow recovery of grazers from mortality due to chlorpyrifos appeared to result in a higher biomass of periphyton in the high dose streams than in controls and low dose streams 21 days after dosing ceased. Inverse relationships between the amounts of fine particulate organic matter (FPOM) and the sum of the numbers of all taxa of collector-gatherers (as well as numbers of four individual taxa) were interpreted as disruption of the processing of FPOM by members of this trophic group following toxic effects of chlorpyrifos. The implications of the study for biological monitoring of the direct and indirect effects of chronic doses of chlorpyrifos on streams are discussed, and the use of chironomid larvae and periphyton in biomonitoring to detect pesticide impacts is particularly recommended. PMID- 7540534 TI - Exploratory analysis of population genetic assessment as a water quality indicator. I. Pimephales notatus. AB - Biological sampling has been implemented to assess water quality in many states. The purpose of this study was to examine whether genetic diversity and structure of Pimephales notatus could serve as effective biomarkers of exposure to anthropogenic stressors by comparing genetic measures with other biological indicators of water quality. Fish were collected from 15 sites on eight streams by the Ohio Environmental Protection Agency as part of their stream water quality evaluation program. Values for the Index of Biotic Integrity (IBI) and the Invertebrate Community Index (ICI) were determined for these 15 sites. Starch gel electrophoresis was used to collect genetic data for seven variable enzyme loci. Genetic diversity measures were not associated with site IBI or ICI values. However, the range of site IBI and ICI values was limited. The proportion of individuals not expressing esterase locus 3 could be used to predict IBI; IBI decreased as the proportion of nonexpression increased. Allele and genotype frequency differences were observed between sites on the Little Scioto River, the one stream with a large difference in IBI and ICI values between sites. This study suggested that allele and genotype frequencies may have been impacted without affecting overall species diversity. PMID- 7540536 TI - Rapid toxicity assessment using an in vivo enzyme test for Brachionus plicatilis (Rotifera). AB - A 1-hr in vivo enzyme inhibition assay based on esterase activity has good potential for marine toxicity assessment. A test was developed for the rotifer Brachionus plicatilis based on the nonfluorescent substrate fluorescein diacetate (FDA), which is metabolized by esterases to a fluorescent product. Enzyme inhibition, as determined by reduced fluorescence, can be scored visually or quantified using a fluorometer. Quantification of fluorescence permits the calculation of NOEC, LOEC, chronic value, and IC20. The 1-hr esterase inhibition test has sensitivity comparable to that of 24-hr rotifer acute tests for several compounds. The toxicity of six compounds was examined using the quantified assay. The resulting IC20s were within a factor of 3 of the 24-hour LC50s. IC20 values ranged from 0.017 mg/l for tributyltin to 3.1 mg/l for zinc, with an average coefficient of variation of 17.8%. Electrophoretic analysis of rotifer homogenates suggested that a single C esterase (acetylesterase) was responsible for FDA metabolism in B. plicatilis. Several other aquatic species are capable of metabolizing FDA, including Brachionus calyciflorus, Mysidopsis bahia, Menidia beryllina, Pimephales promelas, Ceriodaphnia dubia, Daphnia pulex, Artemia salina, and Ophryotrocha sp. The esterase inhibition test is an attractive tool for assessing aquatic toxicity because of its speed, simplicity, sensitivity, and applicability to a broad range of aquatic species. PMID- 7540535 TI - Exploratory analysis of population genetic assessment as a water quality indicator. II. Campostoma anomalum. AB - Biological monitoring programs to assess contaminant-induced impacts in aquatic systems are being developed and implemented by several federal agencies and many states. Genetic diversity and allozyme frequency may be valuable indicators of such impact because they are both sensitive to exposure and ecologically relevant in populations. The purpose of this study was to examine whether genetic diversity and structure of Campostoma anomalum populations could serve as effective biomarkers of exposure to anthropogenic stress by comparing genetic measures with other biological indicators of water quality. Fish were collected from 14 sites on seven streams by the Ohio Environmental Protection Agency as part of their stream water quality evaluation program. Values for the Index of Biological Integrity (IBI) and the Invertebrate Community Index (ICI) were determined for these 14 sites. Starch gel electrophoresis was used to collect genetic data for eight variable enzyme loci. Genetic diversity measures were not associated with site IBI or ICI values. However, the range of site IBI and ICI values was limited. Allele and genotype frequencies were significantly different at the point source compared to sites upstream and downstream indicating that genetic structure may be an indicator of water quality that is sensitive enough to detect change prior to species loss. PMID- 7540538 TI - Influence of 3,4-dichloroaniline (3,4-DCA) on benthic invertebrates in indoor experimental streams. AB - The influence of 3,4-dichloroaniline (3,4-DCA) on benthic invertebrates has been examined. Acute toxicity tests were carried out with the following species: Pristina longiseta, Aelosoma variegatum (Oligochaeta), Hydrozetes lacustris (Acarina), Planorbarius corneus, and Gyraulus albus (Planorbidae). LC50 values (96 hr) were obtained for Pri. longiseta (2.5 mg/liter) and for Hy. lacustris (4.7 mg/liter). For all other species ranges of toxicity (maximal concentration with 0% dead to minimum concentration with 100% dead) were determined. These ranges were 0.8-20 mg/liter 3,4-DCA for Pri. longiseta, 1.6-20 mg/liter, 3,4-DCA for Hy. lacustris, 10-20 mg/liter 3,4-DCA for G. albus, 50-100 mg/liter 3,4-DCA for Pl. corneus, and 10 mg/liter 3,4-DCA (maximal concentration with 0% dead; minimum concentration with 100% dead was not determined) for Ae. variegatum. In two experimental streams, the recolonization of benthic organisms into defined sample areas was studied. Therefore, a phase without chemical treatment was compared with a following exposure phase. Test concentrations were 0.2 and 1.4 mg/liter 3,4-DCA (nominal concentrations). Significant effects were the complete extinction of Pri. longiseta in 0.2 mg/liter 3,4-DCA within the first 3 weeks of exposure, as well as the reduction of immigrating individuals of another species of Pristina in both test concentrations, and of Hy. lacustris and Stentor sp. in 1.4 mg/liter 3,4-DCA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540537 TI - Influence of the sample preservation mode to assess the chronic toxicity of an industrial effluent. AB - To improve the preservation of effluent samples, the contribution of refrigeration, freezing, and freeze-drying techniques to maintain the toxic characteristics of the samples was evaluated. To achieve this evaluation one acute test, three long-term toxicity tests, a biochemical test (P450 induction), and a mutagenicity test (Ames test) were used on the same industrial effluent. Refrigeration and freezing demonstrated a similar capacity of preservation. However, freezing should be recommended when the delay between sampling and the start of the bioassay exceeds 48 hr. Freeze-drying did not prove to be useful in that case but this could be due to methodological problems. Nevertheless, this technique allowed the detection of mutagenic compounds because of its aptitude to concentrate effluent samples. For the toxic evaluation of the samples the advantages of the use of a battery of biotests were confirmed and it was noted that the cladoceran (Ceriodaphnia dubia) reproduction test and the algal (Selenastrum capricornutum) growth test had a higher sensitivity within the three tests used. PMID- 7540539 TI - The kinetics of cadmium in Daphnia magna as affected by humic substances and water hardness. AB - In order to investigate mechanisms causing enhanced toxicity of cadmium in humic lake waters the kinetics of cadmium in Daphnia magna was studied. The uptake rate of cadmium was two times faster in humic lake water than in the humic-free reference water. On the other hand, the depuration rate was equal in humic and reference waters. Thus the acutely lethal concentration in animal is reached faster in humic than in humic-free water. However, altered kinetics of cadmium in humic lake water was also affected by lower water hardness compared to the reference. PMID- 7540541 TI - Sources of pollution and sediment contamination in Newark Bay, New Jersey. AB - A review of historical water quality and sediment quality data and chemical body burdens in biota reveals significant levels of a wide range of chemicals and water quality stressors throughout the Newark Bay, New Jersey estuary. The estuary, which encompasses the Passaic River, Hackensack River, Kill van Kull, and the Arthur Kill, has been heavily impacted by both industrial and urban expansion for more than 200 years. The available information was sufficient to identify the major sources of pollution and the contributions of various metals and organic chemicals to the aquatic environment. Mass loadings for several chemicals and water quality parameters were estimated for publicly owned treatment works (POTWs) and combined sewer overflows (CSOs), industrial waste discharged either directly to the estuary or through POTWs, stormwater runoff, and accidental spills of petroleum products and hazardous chemicals. The recent trend toward regionalized waste treatment systems to provide secondary levels of treatment have provided some reductions of overall contaminant mass loadings. However, CSOs remain a significant source of untreated waste in the estuary. Additionally, the magnitude of the major sources relative to the ability of the estuary to absorb and dilute the various waste streams suggests that much more must be done to reduce chemical inputs at the source. PMID- 7540540 TI - Possible side effects of airborne pesticides on fungi and vascular plants in The Netherlands. AB - A survey of the potential side effects of volatilized pesticides on fungi and vascular plants in The Netherlands has been made for two herbicides (atrazine and MCPA), a fungicide (captan) and a soil fumigant (metham sodium). By applying existing models, a prediction is presented of post-treatment dispersal and deposition due to volatilization, both at short and long ranges, and of the potential side effects on fungi and vascular plants. The general conclusion is that side effects (stunting and growth anomalies) are to be expected outside the target area, especially close (< 500 m) to treated plots with atrazine and metham sodium. In view of agricultural acreage in The Netherlands, these short-range effects may impinge on a relatively large scale. There may also be an impact at greater distances from treated plots, especially in the case of compounds that are slow to degrade such as MITC (methyl isothiocyanate), the active toxic moiety generated in soil from metham sodium. PMID- 7540542 TI - Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding. AB - Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG2a, and IgG2b. The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20%, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39% and 43%, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR. PMID- 7540543 TI - Novel expression of luteinizing hormone subunit genes in the rat testis. AB - The two gonadotropins, LH and FSH, are thought to be synthesized and secreted solely by the anterior pituitary. We present here evidence for expression of the LH beta and common alpha-subunit (C alpha) genes in the rat testis. The LH beta and C alpha-subunit messenger RNAs (mRNAs) were detected by reverse transcriptase polymerase chain reaction in the rat testis and pituitary with primer pairs producing 247- and 199-base pair complementary DNA (cDNA) fragments, corresponding to nucleotides 154-400 of LH beta and nucleotides 250-448 of C alpha cDNA, respectively. The specificity of the cDNA species generated was verified by Southern hybridization using nested [32P]cDNA or oligonucleotide probes, and identity with the published rat LH beta and C alpha-subunit gene structures was determined by sequencing. The mRNA bands with specific hybridization to complementary RNA (cRNA) probes corresponding to nucleotides 154 368 of the rat LH beta cDNA and nucleotides 250-448 of the rat C alpha cDNA were found in the rat pituitary and testis by Northern hybridization. The major C alpha mRNA had a size of 0.8 kilobases (kb) in the pituitary and testis. The major LH beta transcripts were 0.8 and 2.7 kb in the pituitary and testis, respectively. To further characterize the larger testicular LH beta-subunit transcript, rapid amplification of the 3'-end of cDNA (3'-RACE) was performed using an oligo(deoxythymidine-17) adapter and a specific 5'-primer. Southern hybridization of the 3'-RACE product of rat testicular RNA with a LH beta [32P]cDNA probe had the same size as the 3'-RACE product of pituitary RNA. The pituitary and testicular RNAs were then cut into two segments using oligonucleotide-directed ribonuclease H digestion and subjected to Northern hybridization using a cRNA probe specific to the 5'-end segment. The digested 5' end segments of the pituitary and testicular mRNAs were 0.4 and 2.3 kb, respectively, indicating that the testicular LH beta mRNA has a 1.9-kb 5' extension, compared to the cognate pituitary mRNA. This was further verified by Northern hybridization using a cRNA probe corresponding to nucleotides -790 to 10 upstream of the pituitary initiation site of LH beta gene transcription. Specific hybridization of a 2.7-kb mRNA transcript was found in the rat testis, but none in the pituitary. Hence, the 3'-end polyadenalytion site of the LH beta mRNA is the same in rat pituitary and testis, and the different transcript sizes are due to a difference at the 5'-end.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7540544 TI - Oxytocin receptor messenger ribonucleic acid: characterization, regulation, and cellular localization in the rat pituitary gland. AB - The hypothalamic neuropeptide oxytocin (OT) stimulates the release of several pituitary hormones, including ACTH, LH, and PRL. Although specific OT receptors have been identified in anterior pituitary membranes, the structure and cellular localization of these binding sites have not been elucidated. We previously cloned a rat OT receptor (OTR) gene and showed that its expression in rat uterus results in several transcripts ranging in size from 2.9-6.7 kilobases. In this study we show, by using Northern blot analysis, reverse transcriptase-polymerase chain reaction, and ultrastructural in situ hybridization that the same OTR gene is also expressed in the pituitary, where it gives rise to a 6.7- and a 4.8 kilobase messenger RNA. Ultrastructural in situ hybridization combined with immunogold labeling indicated that pituitary OTR gene expression is highly cell specific and restricted to lactotrophs. In accordance with this finding, only the lactotroph-derived cell line MMQ expressed the OTR gene among several pituitary cell lines tested. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis indicated a dramatic increase in pituitary OTR gene expression at the end of gestation and after estrogen treatment. Our results suggest that the OT effect on lactotrophs is direct, whereas OT actions on gonadotrophs and corticotrophs are either indirect or mediated via different receptors. Moreover, our findings imply that OT exerts its full potential as a physiological PRL-releasing factor only towards the end of gestation, and that therefore the role of OT as a hypothalamic PRL-releasing factor may so far have been underestimated. PMID- 7540545 TI - Lys91 and His90 of the alpha-subunit are crucial for receptor binding and hormone action of follicle-stimulating hormone (FSH) and play hormone-specific roles in FSH and human chorionic gonadotropin. AB - Glycoprotein hormones, FSH, LH, CG, and TSH, consist of a common alpha-subunit and a hormone-specific beta-subunit. Both subunits are thought to interact with the hormone receptors. Although several C-terminal residues of hCG alpha are known to contact the LH/CG receptor, little is known about the roles of individual C-terminal residues of FSH alpha. In this report, substitutions of various amino acids for the penultimate Lys91 and the upstream His90 of the alpha subunit demonstrate that these two residues of FSH alpha are important for high affinity receptor binding and hormone action to induce cAMP production. In contrast, the same residues of hCG alpha are more important for cAMP induction than for high affinity receptor binding. Some substitutions significantly improved receptor binding of FSH and hCG, whereas others were detrimental. Some had the same effect on both hormones, and others impacted differently. Particularly, the substitution of Val for alpha Lys91 resulted in an improved receptor binding of and a loss of cAMP induction by FSH and hCG. On the other hand, the substitution of Arg or Pro for alpha His90 abolished receptor binding of FSH, but not of hCG. These results allowed us to generate an antagonist to FSH. Our results indicate that alpha His90 and alpha Lys91 play roles in receptor binding and cAMP induction of FSH and hCG in strikingly different ways. They will be useful to elucidate the underlying mechanisms for the interaction of FSH and hCG with their complementary receptors as well as for receptor activation. PMID- 7540546 TI - Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity. AB - Osteoclast adhesion via integrin alpha v beta 3 to the bone matrix protein osteopontin (OPN) results in stimulation of bone resorption. We characterized an immediate signaling event that takes place upon osteoclast interaction with OPN. OPN binding to alpha v beta 3 results in the rapid production of the phosphoinositides (PtdInsP), including phosphatidylinositol triphosphate (PtdIns 3,4,5P3). Stimulation of 3,4,5-PtdInsP3 production by OPN was produced by increased activity of PtdInsP 3-OH kinase, which was found in immunoprecipitates formed by antibodies to integrin alpha v beta 3. The association of PtdIns 3-OH kinase with integrin may have been through association with c-src. The latter was present in immunoprecipitates formed by the antibodies to integrin alpha v beta 3, and src kinase was activated by OPN. These findings demonstrate a mechanism for rapid generation of cell signals upon matrix protein binding to alpha v beta 3, which resembles the mechanisms used by tyrosine kinase growth factor receptors. PMID- 7540547 TI - Frog vasoactive intestinal polypeptide and galanin: primary structures and effects on pituitary adenylate cyclase. AB - Vasoactive intestinal polypeptide (VIP) and galanin were isolated in pure form from the stomach of the European green frog, Rana ridibunda. Frog VIP is identical to the previously characterized VIP from chicken and alligator. The primary structure of frog galanin contains only two amino acid substitutions (asparagine for histidine at position 23 and histidine for tyrosine at position 26) compared with porcine galanin. The data indicate that evolutionary pressure to conserve the amino acid sequence of both peptides during the evolution of amphibia to mammals has been strong. Synthetic frog VIP produced a dose-dependent increase in cAMP concentration in frog anterior pituitary fragments. The potency of the peptide (ED50 = 1.2 x 10(-6) M; mean +/- SE; n = 8) was comparable to that of porcine VIP (EC50 = 1.3 x 10(-6) M), but was approximately 10-fold less than that of frog pituitary adenylate cyclase-activating polypeptide [PACAP-(1-38); ED50 = 1.1 x 10(-7) M] in the same system. The increases in cAMP concentrations produced by maximal doses of PACAP (10(-5) M) and VIP (10(-5) M) were not additive. The data suggest that the effects of both peptides are mediated through a common PACAP-preferring receptor that is pharmacologically different from the mammalian PACAP type I receptor. Synthetic frog galanin also produced a dose dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 9.3 x 10(-8) M) consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians. PMID- 7540549 TI - Functional nuclear epidermal growth factor receptors in human choriocarcinoma JEG 3 cells and normal human placenta. AB - Immunocytochemistry with a monoclonal antiepidermal growth factor (anti-EGF) receptor antibody directed against the extracellular domain which can inhibit ligand binding to the receptors showed that nuclei of choriocarcinoma JEG-3 cells and normal placental trophoblasts were distinctly immunostained for EGF receptors. This finding led us to investigate the structure and function of nuclear EGF receptors. Western immunoblotting revealed that cell membranes, isolated intact pure nuclei, and nuclear membranes contain a 170-kilodalton EGF receptor protein. Covalent receptor cross-linking demonstrated that the 170 kilodalton receptor protein in nuclei and nuclear membranes can bind [125I]EGF just as in cell membranes, and that this binding is inhibited by excess unlabeled EGF. As in cell membranes, the addition of EGF resulted in an increased receptor autophosphorylation in the nuclei and nuclear membranes. In addition, the activated receptor kinase stimulated, and in some cases inhibited, tyrosine phosphorylation of a number of lower molecular size proteins, especially in nuclei and nuclear membranes. Although the identity of these proteins is not known, none of them could bind [125I]EGF. The addition of EGF to isolated nuclei resulted in a time-dependent specific transcriptional inhibition of hCG/LH receptor gene. In summary, our data demonstrating the presence of functional nuclear EGF receptors are novel, potentially important, and go against the traditional concepts of growth factors action. The nuclear receptors have the capacity to transduce signals from EGF and may mediate intracrine and paracrine actions of EGF in the regulation of trophoblast functions. PMID- 7540548 TI - Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. AB - A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta. PMID- 7540550 TI - Tropheryma whippelii endocarditis confirmed by polymerase chain reaction. AB - This report concerns a patient with cardiac manifestation of Whipple's disease. For the first time, the gene that encodes the 16s rRNA of Tropheryma whippelii was identified in a native aortic valve by means of a polymerase chain reaction technique. DNA amplification gives evidence of Tropheryma whippelii as a causative organism in infective endocarditis. PMID- 7540553 TI - Impaired vasoreactivity to nitric oxide in experimental diabetic neuropathy. AB - Nerve blood flow (NBF) is reduced in experimental diabetic neuropathy (EDN), but the mechanism of its reduction is uncertain. We tested the hypothesis that reduced NBF might be due to alterations of nitric oxide synthase (NOS) and endothelin of microvascular endothelial cells of sciatic nerve. We evaluated epineurial arteriolar vasoreactivity in response to superfused test agents. NBF was measured using microelectrode H2 polarography. Vasoconstrictor responses to endothelin-1 (ET-1; 10(-6), 10(-7), 10(-8), 10(-9), 10(-10) M) showed dose response curves with similar EC50 values, indicating no change in potency. We applied the NOS inhibitor NG-nitro-L-arginine and observed reduced inhibition of NBF in EDN, correctable with insulin treatment and also with infused L-arginine. We conclude that vasoreactivity is disturbed in EDN, and is due to a combination of an impairment of NOS activity with reduced NO and increased endothelin effect (normal receptor sensitivity and increased plasma values) in EDN. Hyperglycemia is likely to be the mechanism of NOS inhibition since insulin treatment reversed this abnormality. PMID- 7540551 TI - The clinical value of prostate-specific antigen and bone scintigraphy in the staging of patients with newly diagnosed, pathologically proven prostate cancer. AB - Recent reports suggest that radionuclide bone scan (BS) may not be necessary in the standard staging evaluation of patients with prostate cancer when serum prostate-specific antigen (PSA) levels are normal. To evaluate the ability of PSA to predict BS findings, we retrospectively reviewed the case records of 118 consecutive patients (median age 73 years, range 50-90 years) with newly diagnosed, untreated, pathologically proven prostate cancer who underwent BS and serum PSA sampling within a period of no more than 3 months. Fifty-four out of 118 BSs demonstrated metastatic bone disease. A PSA value of less than 10 ng/ml excluded bone metastasis; of 35 patients with a serum PSA level of 20 ng/ml or less, seven had a positive BS (negative predictive value of 80%). These findings provide additional confirmation of the value of low serum PSA concentrations in excluding the need for a staging BS, although the threshold for a high value of negative predictive accuracy is lower than previously reported. PMID- 7540555 TI - Olfactory bulb transplants establish afferent and efferent connections with host forebrain in rat. AB - We are using wheat germ agglutinin-conjugated horseradish peroxidase (WHRP) to study reconnectivity in the transplanted (TX) olfactory bulb (OB) in Sprague Dawley rats. Tritium-labeled OBs from fetal rat donors of Embryonic Days 14-15 were immediately grafted into neonatal rats in the site from which the host OB had been removed. Following survival times of 7 weeks and longer, WHRP solution was injected into the TX OB, and subjects were perfused after 24 h. The WHRP transport is seen in fibers from the TX OB into layer I of the host olfactory peduncle (OP) and olfactory cortex (OC) and in cell bodies in layers II and III of the OP and OC, the lateral hypothalamus, and the contralateral anterior olfactory nucleus (AON). These findings reaffirm that the axons from a TX OB make connections with some appropriate areas of the host brain and also indicate that axons from cells in the target areas of the host brain, including contralateral AON, reinnervate the TX OB. PMID- 7540554 TI - Development of kainic acid and N-methyl-D-aspartic acid toxicity in organotypic hippocampal cultures. AB - The excitotoxic effects of N-methyl-D-aspartic acid (NMDA) and kainic acid (KA) were studied in organotypic hippocampal slices maintained in vitro for various periods of time. Cultures aged to equivalent Postnatal Day (EPD) 10-12, 15-17, and 23-26 were exposed to 50 microM KA or 50 microM NMDA and were analyzed at 0, 3, 6, 9, 12, 24, 48 h, or 5 days after the initiation of the excitotoxin exposure. Neuronal injury was determined by: (1) propidium iodide (PI) uptake; (2) lactate dehydrogenase (LDH) release; (3) morphological damage in hematoxylin and eosin (H/E) stained sections; (4) loss of Nissl stain. Changes in PI uptake and LDH release after KA or NMDA treatment indicated that there was a developmental shift towards increasing sensitivity to KA toxicity during in vitro development, whereas cultures of all ages were equally sensitive to NMDA toxicity. The profile of damage in H/E-stained sections after treatment with KA or NMDA indicated a transient phase of damaged morphology at 12 and 24 h that was not evident after 5 days. To determine whether the disappearance of morphological manifestations of neuronal damage 5 days after treatment was due to recovery of morphology or to neuronal death, neuronal loss in Nissl-stained sections was also quantified. KA treatment did not cause significant neuronal loss in any hippocampal region in EPD 10-12 cultures, indicating that the neurons were able to successfully recover from the damage demonstrated in H/E sections at 12 and 24 h in these cultures. KA treatment in mature cultures (EPD 23-26) and NMDA treatment in all cultures produced a marked loss of identifiable Nissl-stained neurons at 5 days, indicating neuronal death and disintegration. The results provide further support for the similarities between the organotypic hippocampal culture model and in vivo excitotoxic models and also confirm that excitotoxic neuronal injury can be reversible under some conditions. PMID- 7540552 TI - Apolipoprotein E immunoreactivity within neurofibrillary tangles: relationship to Tau and PHF in Alzheimer's disease. AB - The present immunohistochemical study determined the relationship between ApoE and the expression of the cytoskeletal protein tau (Tau2) and paired helical filaments (PHF), within the magnocellular neurons of the nucleus basalis of Meynert and layer II stellate neurons of the entorhinal cortex in Alzheimer's disease (AD). Although nearly all ApoE immunoreactive perikarya within these two brain regions were PHF immunoreactive, not all PHF and Tau2 containing neurons stained for ApoE in AD. Moreover, more Tau2-immunostained neurons, as compared to PHF, were ApoE immunonegative. This was particularly evident in a population of control subjects which exhibited AD-like pathology intermediate between the AD and normal aged individuals. Thus, neurons within the nucleus basalis of Meynert and entorhinal cortex layer II stellate exhibit evidence of cytoskeletal pathology prior to displaying ApoE. These observations suggest that (1) ApoE plays a secondary role in NFT formation or (2) this protein is accumulated within these neurons in response to reparative process(es) induced by NFT-associated neuronal damage. PMID- 7540556 TI - Evidence for a lymphotropic nature of circulating plasmacytoid monocytes: findings from a case of CD56+ chronic myelomonocytic leukemia. AB - Because the cells previously designated plasmacytoid T cells share major immunophenotypic features with cells of the mononuclear-phagocyte system, they have been re-named and are now known as plasmacytoid monocytes (PM). We describe a unique case of chronic myelomonocytic leukemia with circulating PM. The patient, a 48-year-old man, presented initially with refractory anemia. Four years later his general condition deteriorated, accompanied by an increase in leukocytes to 200,000/microliters blood. The bone marrow histology was interpreted as compatible with a diagnosis of chronic myelomonocytic leukemia. Two months before he died, the patient developed generalized lymphadenopathy clinically simulating malignant lymphoma. Histologic examination of an axillary lymph node revealed diffuse infiltration by PM. The PM in the lymph node and some circulating cells closely resembling PM expressed L-selectin, a finding that could be interpreted as a morphologic correlate of their marked lymphotropism. The detection of large numbers of CD56/CD33 double-positive circulating blast cells by FACS analysis strongly supported the diagnosis of a leukemia of myelogenous origin. The patient died of tumor cachexia. Autopsy revealed widespread leukemic infiltrates (always containing clusters of PM) in bone marrow, spleen, liver, lymph nodes, and mucosa-associated lymphoid tissue of the oropharynx. The final diagnosis was one of chronic myelomonocytic leukemia with marked lymphotropism and partial differentiation towards PM. We consider that the rare instances of a hematologic tumor with differentiation towards PM should be classified amongst the myelogenous leukemias. PMID- 7540557 TI - Hematologic effects of stem cell factor (SCF) and leukemia inhibitory factor (LIF) in vivo: LIF-induced thrombocytosis in SCF-primed mice. AB - Stem cell factor (SCF) administered as daily bolus injections in dose-response experiments in mice causes a progressive and dramatic dose-dependent panleukocytosis characterized by neutrophilia, eosinophilia, monocytosis, and lymphocytosis. SCF causes circulating platelet numbers to be dose-dependently increased after 2 weeks of daily injections. Leukemia inhibitory factor (LIF) administered as daily bolus injections in mice causes a peripheral leukopenia that is largely due to peripheral lymphopenia. LIF causes thrombocytosis peaking after approximately 1 w. Coinjection of SCF and LIF for 1 to 2 wk in mice does not cause a much greater thrombocytosis than the maximum thrombocytosis achievable with SCF or LIF alone. On the other hand, daily injection of SCF for 5 days followed by daily injection of LIF for 5 to 6 d in mice causes a very substantial increase in platelets that was lineage-specific in terms of not being accompanied by a generalized leukocytosis. In contrast, only a very modest thrombocytosis was noted in SCF-primed LIF-treated rats. LIF causes a large increase in the cytoplasmic volume of splenic megakaryocytes in mice, but not in rats. In conclusion, SCF-induced priming followed by LIF-induced maturation of megakaryocytes causes a substantial selective increase in the numbers of circulating platelets in mice. PMID- 7540558 TI - Stem-cell factor in aplastic anemia: in vitro expression in bone marrow stroma and fibroblast cultures. AB - In vitro expression of stem-cell factor (SCF) by bone marrow (BM) cells of 30 patients with aplastic anemia (AA) has been analyzed at the mRNA and protein levels. While no deficiencies were found in SCF mRNA expression, low levels of soluble SCF protein were measured in poorly growing AA stroma cultures. The SCF protein concentration in the supernatant and the confluence of AA stroma growth were found to correlate (R = 0.70). Defective proliferation was observed in the majority (20/30) of AA stroma cultures and was paralleled by poor growth of homogeneous cultures of fibroblasts from the same marrow sample. AA stroma growth was enhanced by addition of exogenous SCF in combination with interleukin-11 (IL 11), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Our results demonstrate that deficient growth of stroma cells results in decreased production of SCF. Therefore, SCF and other stroma-derived cytokines may be of therapeutic value in AA patients with documented defects within the BM microenvironment. PMID- 7540559 TI - Effects of simultaneous rhG-CSF and methylprednisolone "pulse" therapy on hepatitis A virus-associated haemophagocytic syndrome. PMID- 7540560 TI - Cytoplasmic abnormalities of erythroblasts as a marker for ringed sideroblasts in myelodysplastic syndromes. PMID- 7540561 TI - CYFRA 21-1 as a tumour marker for bronchogenic carcinoma. AB - Despite extensive research, the role of the commonly employed tumour markers in the diagnosis of lung carcinoma is yet to be clarified. The utility of a new marker, CYFRA 21-1, in the preoperative evaluation of patients with bronchogenic carcinoma was investigated. CYFRA 21-1 was determined with a radiometric assay in serum of 280 patients with lung cancer and 208 patients with various nonmalignant lung diseases. The levels of the marker were significantly higher in lung cancer patients. Among benign lung diseases, elevated CYFRA 21-1 levels were found in pulmonary fibrosis. Using a cut-off of 3.2 ng.ml-1 (95th percentile of levels obtained in benign lung disease), the total sensitivity of the marker was 48%. The best sensitivity was obtained in squamous cell lung cancer (60%). The highest values of CYFRA 21-1 were found in metastatic lung cancer, and the marker sensitivity was more elevated in stage IIIb and IV. On the other hand, 40% of patients with surgically resectable lung cancer had CYFRA 21-1 levels above the cut-off. We conclude that CYFRA 21-1 may be satisfactorily employed in the differential diagnosis between malignant and benign lung diseases in association with other clinical and radiological data. PMID- 7540562 TI - The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine alpha-amidating enzyme and dopamine beta-monooxygenase. AB - Peptidylglycine alpha-amidating enzyme (alpha-AE) and dopamine beta-monooxygenase (D beta M), two copper-dependent monooxygenases that have catalytic and structural similarities, are irreversibly inactivated by sodium sulfite in a time and concentration-dependent manner. Studies with alpha-AE show that the sulfite mediated inactivation is dependent on the presence of redox active transition metals free in solution, with Cu(II) being the most effective in supporting the inactivation reaction. Sulfite inactivation of alpha-AE is specific for the monooxygenase reaction of this bifunctional enzyme and amidated peptides provide protection against the inactivation. Consequently, the sulfite-mediated inactivation of alpha-AE and D beta M most likely results from the transition metal-catalyzed oxidation of sulfite to the sulfite radical, SO3-. PMID- 7540563 TI - Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similarities. AB - Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different. PMID- 7540564 TI - A time course study of capacitation and the acrosome reaction in human spermatozoa using a revised chlortetracycline pattern classification. AB - OBJECTIVE: To study the time course of capacitation, spontaneous, and A23187 induced acrosome reaction of human spermatozoa during 8 hours incubation in vitro using the chlortetracycline (CTC) assay with a revised fluorescent pattern classification. DESIGN: Fertile donor spermatozoa were isolated by direct swim-up and incubated in Earle's balanced salt solution for up to 8 hours. At hourly intervals, spermatozoa were stained with CTC before and after the addition of A23187 to induce the acrosome reaction. SETTING: The University Clinic, Jessop Hospital for Women, Sheffield, United Kingdom. PATIENTS: Donors participating in the Donor Insemination Program. MAIN OUTCOME MEASURES: Eight fluorescent patterns identified by the CTC assay and acrosome-reacted spermatozoa detected by indirect immunofluorescence using 18.6 monoclonal antibody. RESULTS: Using a statistical model defined by analysis of deviance allowed rationalization of the CTC pattern classification by grouping together patterns that showed a similar and significant change over time. In addition, spontaneous and A23187-induced acrosome-reacted spermatozoa identified by the CTC assay were shown to be correlated significantly to those identified by indirect immunofluorescence. CONCLUSION: The CTC assay using a revised pattern classification offers a more precise description of human spermatozoa capacitation in vitro. Also, CTC identified acrosome reaction (both spontaneous and A23187 induced) was confirmed independently by indirect immunofluorescence. PMID- 7540565 TI - Coexpression of cytokeratin and vimentin in eutopic endometrium and endometriosis throughout the menstrual cycle: evaluation by a computerized method. AB - OBJECTIVE: To evaluate quantitatively cytokeratin and vimentin staining in glandular and stromal cells of eutopic and ectopic endometrium. DESIGN: The investigation of cytokeratin and vimentin was carried out using the new computerized technology of image analysis. SETTING: University Hospital, Department of Gynecology. PATIENTS: Biopsies were taken from patients undergoing a laparoscopy for infertility (29 endometrial biopsies and 31 biopsies of peritoneal endometriosis). None of them were treated. RESULTS: Cyclic variations of cytokeratin and vimentin staining were noted in eutopic endometrium. The cytokeratin and vimentin staining pattern consistently was lower in ectopic endometrium than in eutopic endometrium. CONCLUSIONS: Endometriotic epithelial and stromal cells undergo a complex program of differentiation giving them histochemical characteristics similar to those observed in endometrium. Such a concomitant expression of antigenicity indicates their close relationship with their mesodermal mullerian origin. PMID- 7540566 TI - Intraumbilical oxytocin for management of retained placenta. AB - OBJECTIVE: The purpose of the study was to investigate the removal of a retained placenta. METHOD: Oxytocin was injected into the vein of the umbilical cord. A total of 109 patients with retention of the placenta were randomized into two groups. Active management of the third stage of labor was carried out by giving oxytocin 5 IU intravenously and ergometrine maleate 0.2 mg intramuscularly after delivery of the fetus. Group 1, which comprised 68 patients, was allocated to receive 50 IU oxytocin diluted in 10 ml 0.9% sodium chloride solution, and the 41 patients in group 2 were given 20 ml plasma expander (dextran 70) into the umbilical vein. RESULTS: Forty-nine cases (72%) in the oxytocin group and 22 cases (54%) in the dextran 70 group required manual removal of the retained placenta. No significant differences were found between group 1 (oxytocin) and group 2 (dextran 70). CONCLUSION: Our results indicate that intraumbilical vein injection of oxytocin is not effective for removal of a retained placenta. PMID- 7540568 TI - ACOG committee opinion. Statement on surgical assistants. Number 145--November 1994 (replaces no. 92, March 1991). Committee on Gynecologic Practice. Committee on Obstetric Practice. American College of Obstetricians and Gynecologists. PMID- 7540567 TI - CA 125, CA 15.3, CA 27.29, CEA, beta-hCG and alpha-fetoprotein levels in cyst fluid of breast macrocysts. AB - OBJECTIVE: To determine the tumoral markers CA 125, CA 15.3, CA 27.29, CEA, beta hCG and alpha-fetoprotein in serum and breast cystic fluid in women with fibrocystic breast disease. METHODS: The study comprised 50 women diagnosed with fibrocystic breast disease but without breast macrocysts, and 60 women with macrocysts. RESULTS: Significantly higher levels of CA 125, CA 27.29, beta-hCG and CEA were observed in the cyst fluid than in the serum of the same patients. According to their K+/Na+ quotient we found that when K+/Na+ was < 3, cyst fluid CA 125, CA 27.29, beta-hCG and CEA levels were significantly higher than serum levels. However, when K+/Na+ was > 3, only CA 27.29 and beta-hCG levels were significantly higher. Comparing cyst fluid marker levels with respect to the K+/Na+ ratio, the only difference observed was in CA 125 which was significantly greater when K+/Na+ was < 3. A negative correlation was found between CA 125 and the K+/Na+ quotient. CONCLUSION: These results suggest that CA 125 could be used as a marker to identify cyst type. PMID- 7540569 TI - Triiodothyronine modulates growth, secretory function and androgen receptor concentration in the prostatic carcinoma cell line LNCaP. AB - There is increasing evidence that the course of prostatic carcinoma is determined by a complex interplay between genetic events, paracrine interactions, and hormonal and dietary factors. These latter factors include several ligands of the nuclear receptor family such as androgens, vitamin D3 and retinoids. To test whether thyroid hormones also influence the growth and differentiated function of prostatic carcinoma cells, LNCaP cells were treated with or without triiodothyronine (T3) in the absence or in the presence of other regulatory factors. Exposure of LNCaP cells to T3 for 6 days in the absence of androgens caused a dose-dependent increase in [3H]-thymidine incorporation with a maximal stimulation of 2.5-fold at 10(-9) M T3. Secretion of prostate-specific antigen (PSA) was also stimulated 2-3-fold. The observed effects may well be mediated by a nuclear T3 receptor as evidenced by displaceable T3 binding studies. Combined treatment of LNCaP cells with androgens and T3 revealed intriguing interactions between these two pathways. Below and up to 10(-10) M of the synthetic androgen R1881, the concentration that evokes optimal proliferative responses, T3 stimulated [3H] thymidine incorporation. At higher concentrations of androgens, T3 displayed antiproliferative effects. No androgen-dependent effects on T3 receptor levels were observed. Conversely, T3 increased androgen receptor levels up to twofold. Androgen as well as T3 stimulation of proliferation was abolished by high concentrations of the retinoid 9-cis-retinoic acid. These data add T3 to the list of factors that influence growth and differentiation of prostatic tumor cells and contribute to our understanding of the intricate pathways that ultimately determine the course of prostatic carcinoma. PMID- 7540570 TI - Inhibition of proteoglycan synthesis induces an increase in follicle stimulating hormone (FSH)-stimulated estradiol production by immature rat Sertoli cells. AB - In order to define the possible involvement of proteoglycans (PG) in the regulation of Sertoli cell functions, we have examined the effect of para nitrophenyl-beta-D-xyloside (PNPX), a specific inhibitor of PG synthesis, on follicle stimulating hormone (FSH)-dependent estradiol production by immature rat Sertoli cells. Addition of PNPX to the culture medium induced a dose-dependent inhibition of 35S-labeled PG synthesis in Sertoli cells both in the medium and the cell layer. Simultaneously there was a drastic increase in 35S-labeled secreted glycosaminoglycans. By 1 mM PNPX, syntheses of chondroitin sulfate proteoglycans released into culture medium and of heparan sulfate proteoglycans associated with the cell layer were 35% of values from untreated cells. Simultaneously, PNPX induced a twofold (mean of seven experiments, range 17-250%) enhancement of FSH (100 ng/ml)-stimulated estradiol production. In each individual experiment, there was an inverse relationship between the amplitude of PNPX-induced increase in FSH responsiveness and the FSH capability to stimulate basal estradiol production in cultured rat Sertoli cells. The effect of PNPX on FSH-stimulated aromatase activity was not mimicked by para-nitrophenyl-beta-D galactoside, a structural analog of PNPX that has no effect on PG synthesis. The (Bu)2cAMP-stimulated estradiol synthesis was not modified in the presence of PNPX. Moreover, PNPX enhancement of FSH-stimulated estradiol synthesis disappeared when Sertoli cells were cultured in the presence of 1-methyl-3 isobutylxanthine, an inhibitor of phosphodiesterase activity. These findings suggest that inhibition of PG synthesis under PNPX conditions did not affect signal transduction steps distal to cAMP but rather decreased the phosphodiesterase activity in Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540571 TI - Interferon expression in the pancreases of patients with type I diabetes. AB - We have used a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-alpha in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-beta was detected at comparable levels in both groups, while IFN-gamma was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN alpha cDNAs generated by the RT-PCR were cloned and sequenced to determine which alpha-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-alpha or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN alpha was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes. PMID- 7540572 TI - Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells. AB - Nitric oxide (NO) is believed to be an effector molecule that mediates interleukin (IL)-1 beta-induced destruction and dysfunction of pancreatic beta cells. We have demonstrated that both exogenous NO and NO generated endogenously by IL-1 beta brought about apoptosis of isolated rat pancreatic islet cells as well as pancreatic beta-cell tumor-derived cell line HIT. This apoptosis was characterized by cleavage of DNA into nucleosomal fragments of 180-200 bp and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. The IL-1 beta-induced internucleosomal DNA cleavage occurred in a time and dose-dependent manner. Actinomycin D, cycloheximide, and nitric oxide synthase inhibitors inhibited the DNA cleavage, which was correlated with the amount of NO produced, indicating that NO produced by HIT cells themselves could mediate the apoptosis. Furthermore, in the presence of tumor necrosis factor (TNF)-alpha, large amounts of NO were produced by IL-1 beta and DNA cleavage occurred more noticeably, although TNF-alpha alone did not generate NO. Streptozotocin (STZ), a diabetogenic reagent containing a nitroso moiety, also released NO and induced internucleosomal DNA cleavage in HIT cells. These results suggest that NO-induced internucleosomal DNA cleavage is an important initial step in the destruction and dysfunction of pancreatic beta-cells induced by inflammatory stimulation or treatment with STZ. PMID- 7540574 TI - Glucose-induced insulin receptor tyrosine phosphorylation in insulin-secreting beta-cells. AB - In the beta TC3 insulin-secreting beta-cell line, glucose rapidly induces the tyrosine phosphorylation of the 97-kDa insulin receptor beta-subunit. Phosphorylation is transient, with fourfold stimulation by 2 min and subsequent dephosphorylation to basal levels by 10-15 min. Elevating the extracellular KCl concentration equipotently initiates receptor phosphorylation. Preventing insulin secretion with 1 mumol/l epinephrine or by removing extracellular Ca2+ blocks the effect. In the absence of glucose-induced secretion, exogenous insulin also stimulated insulin receptor autophosphorylation transiently and with an ED50 of 4 x 10(-9) mol/l. In addition, functional insulin-like growth factor I (IGF-I) receptors are also expressed by these beta-cells, as indicated by IGF-I-induced receptor tyrosine phosphorylation (ED50 = 5 x 10(-9) mol/l) and also by detection of hybrid insulin/IGF-I receptor autophosphorylation at 10(-7) mol/l IGF-I. Both glucose and insulin stimulate the tyrosine phosphorylation of the insulin receptor substrate (IRS) IRS-1 and increase by two- to fivefold the rapid association of IRS-1 with the 85-kDa alpha-subunit of the phosphatidylinositol-3 kinase, as determined by co-immunoprecipitation assays. These results demonstrate that in these beta-cells, glucose-induced insulin secretion activates the beta cell surface insulin receptor tyrosine kinase and its intracellular signal transduction pathway, suggesting a new autocrine mechanism for the regulation of beta-cell function. PMID- 7540573 TI - Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans. AB - An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN 5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types. PMID- 7540575 TI - Very-low-dose streptozotocin induces diabetes in insulin promoter-mB7-1 transgenic mice. AB - Transgenic mice that express mouse B7-1 (mB7-1, recently designated CD80) on their pancreatic beta-cells maintain normal islet architecture, have normal pancreatic insulin content, and only rarely spontaneously develop insulitis and diabetes. Nevertheless, these mice display an extreme sensitivity to streptozotocin (STZ)-induced diabetes. Female mice were administered two STZ doses intraperitoneally, 20 and 40 mg/kg body wt, each for five consecutive days. Nontransgenic but otherwise syngeneic mice responded to the STZ with a moderate diminution in pancreatic insulin content but not with persistent glycosuria. In striking contrast, STZ administered to transgenic mice resulted in a severe diminution of pancreatic insulin content and in diabetes. Notably, the lower STZ dose resulted in diabetes only after a prolonged (26- to 100-day) latency. STZ induced diabetes appears to be T-cell dependent, since treatment with T-cell depleting (and in particular CD8+ subset-depleting) antibodies ameliorated the response. Anti-mB7-1 monoclonal antibody administration also prevented STZ induced diabetes. Thus, unmasked mB7-1 is a required component in the pathway resulting in beta-cell killing. Immunohistological analysis revealed that early after STZ administration, both mB7-1 transgenic and nontransgenic mice developed insulitis. While this insulitis resolved in the nontransgenic mice, the islet infiltrating CD4+ and CD8+ T-cells in the transgenic mice were associated with complete beta-cell destruction. These data suggest that STZ-induced diabetes in mB7-1 transgenic mice is an immune-mediated process with distinct potential advantages over existing insulin-dependent diabetes models. PMID- 7540576 TI - Endoscopic Nd:YAG laser palliation of malignant duodenal tumors. AB - This study assesses the outcome of 20 patients referred for neodymium: yttrium aluminum-garnet laser therapy of malignant duodenal tumors between 1984 and 1992. Almost all (95%) of these patients required palliative therapy for gastrointestinal hemorrhage, and nearly half (45%) also had obstructive symptoms. A mean of 3 (range, 1 to 6) laser treatment sessions were required for palliation. Laser therapy eliminated the need for blood transfusions in only 38% of patients. Obstructive symptoms were improved in all patients after laser treatment. Treatment failure could not be predicted on the basis of demographic factors (other than age), tumor characteristics, or transfusion requirements. Survival after laser therapy was 30% at 6 months and 15% at 12 months. Endoscopic neodymium:yttrium-aluminum-garnet laser therapy is a reasonable approach for palliation of malignant tumor obstruction or hemorrhage in selected cases; however, hemorrhage often continues. PMID- 7540577 TI - Palliation of malignant oesophageal obstruction by endoscopic alcohol injection. PMID- 7540578 TI - Danazol decreases transcription of estrogen receptor gene in human monocytes. AB - 1. Administration of danazol for over one month reduced the levels of estrogen receptor (ER) and its mRNA to approximately 50 and 20%, respectively in monocytes. 2. Danazol did not alter the degradation rate of ER mRNA in monocytes. 3. Danazol decreased the transcription rate of ER gene to approximately 50% in monocytes in a run-on assay. 4. Danazol may release estrogen predominance via the reduction of transcription for ER gene, which leads to the reduction of ER mRNA and ER expressions in monocytes. PMID- 7540579 TI - Contribution of activation of K+ channels to glyceryl trinitrate-induced relaxation of rabbit aorta. AB - 1. Possible contribution of K+ channel opening to the relaxation by glyceryl trinitrate (GTN) was examined using isolated rabbit aorta. 2. While glibenclamide and apamin failed to affect relaxation by GTN, both charybdotoxin (ChTx) and iberiotoxin (IbTx) effectively attenuated GTN-induced relaxation. 3. The increase in cGMP produced by GTN was not attenuated by ChTx and IbTx. 4. The inhibitory effect of ChTx on GTN-induced relaxation was not reduced in the presence of zaprinast, indicating that cGMP but not GMP was responsible for activation of the K+ channel. 5. Okadaic acid, a selective inhibitor of protein phosphatase 2A, had no effect on the relaxation by GTN. These results indicate that, though small in degree, activation of a ChTx-sensitive K+ channel (large conductance Ca(2+) activated K+ channel) is involved in the GTN-induced relaxation in rabbit aorta. PMID- 7540580 TI - On the relation of calcium channel blockers to rat parotid and submandibular glands function in vivo. AB - 1. The effects of nifedipine, verapamil and diltiazem on rat parotid and submandibular glands function were studied. 2. Nifedipine (5 mg/kg), verapamil (5 mg/kg) and diltiazem (10 mg/kg) were injected intraperitoneally 15 min before saliva collection. 3. Animals were anesthetized with 50 mg/kg of sodium pentobarbital and 8 mg/kg of pilocarpine was used as secretagogue. 4. Submandibular saliva was analyzed for flow rate, protein and calcium concentrations; and parotid saliva for calcium and amylase contents. 5. In treated groups, flow rate and calcium of submandibular saliva were significantly lower than controls. Parotid calcium in the nifedipine group was decreased and in verapamil and diltiazem groups was increased. Parotid amylase was significantly decreased in both the nifedipine and diltiazem groups. 6. It is concluded that a blockade of calcium channels in salivary glands acinar cells by CCBs causes some alterations in salivary secretions. PMID- 7540581 TI - Stability of tandem repeats in the Drosophila melanogaster Hsr-omega nuclear RNA. AB - The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing > 5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is approximately 280 bp. Sequences of 19 1/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than approximately 5 kb nor more than approximately 16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA. PMID- 7540582 TI - Putative O-antigen transport genes within the rfb region of Vibrio cholerae O1 are homologous to those for capsule transport. AB - The nucleotide sequence of that part of the Vibrio cholerae (Vc) O1 rfb region encompassing rfbG, rfbH and rfbI is presented. Expression of these genes has enabled the products for rfbG and rfbI to be confirmed, but the rfbH product has not been detected. Comparisons with the sequences of known proteins reveals that RfbH and RfbI are likely to be involved in the export of lipopolysaccharide (LPS). RfbH shows considerable homology to a number of integral membrane proteins, some of which have been identified as possibly having a role as an export channel for capsular polysaccharides. RfbI corresponds to an ATP-binding protein usually found linked to the membrane protein and is thought to be required for energizing this export process. Thus, we propose that RfbH and RfbI form a complex for the export of Vc O1 LPS. The function of RfbG is unknown, but it would appear to be a relatively hydrophilic protein and we can only speculate that it may be either a specific transferase or possibly the O-antigen polymerase. PMID- 7540583 TI - Pseudomonas aeruginosa outer membrane protein OprF as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope. AB - OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been proposed to be comprised of a series of beta-strands separated by periplasmic or surface-exposed loop regions. In this study, a simple malarial epitope was used to demonstrate that OprF can be used as an expression vector to present foreign peptide sequences, namely, the 4-amino-acid (aa) repeating epitope (Asn-Ala-Asn Pro = NANP) of the circumsporozoite protein of the human malarial parasite Plasmodium falciparum. Eight permissive sites, that allowed the expression and surface exposure of the malarial epitope, were identified throughout OprF. Using a monoclonal antibody (mAb) specific for the malarial epitope, we investigated the effects of positioning and length of the epitope on its antigenicity in the OprF expression vector system. It was demonstrated that the malarial epitope inserted at aa26 was significantly more reactive with the epitope-specific mAb (i.e., more antigenic) when assayed in the context of whole cells whereas those at aa213 and aa290 were more antigenic when assayed in the OM. The malarial epitope inserted at aa188 and aa196 was moderately antigenic, while this epitope inserted at aa215 and aa310 showed low antigenicity with the same mAb in both whole cell and OM assays. For two insertion sites, aa26 and aa213, we demonstrated that the insertion of multiple copies of the epitope enhanced reactivity with the malarial epitope-specific mAb. These data are discussed with respect to the local OprF sequences into which the epitope was inserted. PMID- 7540584 TI - [Clinical follow up in a case of fetal arrhythmia--review of the literature]. AB - Fetal heart arrhythmias are divided into 3 categories: 1. extrasystoles 2. tachycardia and 3. bradycardia. In each of these fetal echocardiography follow-up is necessary during the pregnancy. Extrasystoles are usually evaluated every 2 weeks, are mostly benign and no pharmacological treatment is necessary. Vaginal delivery is recommended. Tachycardia should be monitored every week for any evidence of incoming fetal heart failure. In such circumstances pregnant women should be in hospitalized and pharmacological treatment is strongly recommended, usually with digoxin or digoxin and verapamil. Furthermore, other drugs were reported. In a case of bradycardia fetal echocardiography should be performed every week. Fetal heart failure usually leads to cesarean sections, similar like in cases of tachycardia. PMID- 7540585 TI - High-dose cisplatinum, vinblastine, bleomycin, and etoposide in the therapy of malignant nondysgerminomatous germ cell tumors of the ovary. PMID- 7540586 TI - Concanavalin A variants of alpha-fetoprotein in first trimester fetuses with trisomy 21 and with normal karyotypes. AB - Total alpha-fetoprotein (AFP) concentrations and proportions of AFP non-reactive with the lectin concanavalin A (Con A) were studied in extracellular fluid of 22 first-trimester fetuses. Total AFP concentrations were significantly lower in fetuses with Down's syndrome than in those with Mendelian-inherited diseases and normal karyotypes. The proportion of non-Con-A-reactive AFP was low in the fetal compartment (< or = 3.5%) irrespective of the fetal karyotype. So the AFP production in the fetal liver as well as in the yolk sac seems to be reduced in Down's syndrome. The fetus itself contributes only marginally to the non-Con-A reactive AFP pool of amniotic fluid, which is therefore almost entirely of yolk sac origin. This pool is preserved well into the second trimester of pregnancy, despite cessation of yolk sac AFP production at the end of the first trimester, indicating a very slow rate of disappearance of proteins out of amniotic fluid. PMID- 7540587 TI - Three novel sequence variations in the 5' upstream region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: two polymorphisms and one putative molecular defect. AB - More than 400 sequence alterations have been identified in the whole coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene corresponding to the 27 exons and their exon-intron boundaries. However, in some CF chromosomes, no mutation has yet been detected. In such cases, we have explored the promoter and the sequence up to position -1000 from the cap site, by using denaturing gradient gel electrophoresis. This study concerning 35 CF chromosomes has allowed us to identify three novel sequence variations located in the 5' upstream region of the gene. The T to G substitution located at position 895 from the cap site could be considered as a polymorphic variation. The second substitution (C to T at position -816) has been detected on only one CF chromosome, but does not concern a regulatory DNA element previously described. Conversely, the third substitution (a T to G substitution at position -741 from the cap site) is located at a potential AP-1 binding site. We have investigated, by electrophoretic mobility shift assay, the ability of this region to bind nuclear factors. We have found that the normal sequence between -740/-745 does not bind either the AP-1 transcription factor or AP-1 related proteins, and that the T to G-741 mutated sequence exhibits an abnormal binding pattern suggesting the possible deleterious effect of still unknown negative trans-acting factors. PMID- 7540588 TI - A 1.8-Mb YAC contig spanning three members of the receptor tyrosine kinase gene family (Pdgfra, Kit, and Flk1) on mouse chromosome 5. AB - We constructed a yeast artificial chromosome (YAC) contig spanning the genes encoding Kit (Kit), the platelet-derived growth factor alpha receptor (Pdgfra), and fetal liver kinase 1 (Flk1), three members of a receptor tyrosine kinase gene family located in the central portion of mouse chromosome 5. The orientation of YAC clones and the extent of their overlap was determined by "probe content mapping," that is, hybridization analysis of YAC clones using the available gene probes and YAC end sequences. For four YAC clones, which constitute a minimal set spanning 1.8 Mb, a detailed restriction map was constructed. This map, in conjunction with the previously published long-range restriction map, indicates the order, the physical distances, and the relative transcriptional orientations of the Pdgfra, Kit, and Flk1 genes. The YAC clones and corresponding YAC end probes presented here provide an important resource for the molecular analysis of a cluster of developmental mutations, namely dominant white spotting (W), patch (Ph), recessive spotting (rs), and rump-white (Rw), associated with this chromosomal region. PMID- 7540589 TI - Localization of the aquaporin 1 (AQP1) gene within a YAC contig containing the polymorphic markers D7S632 and D7S526. PMID- 7540590 TI - Collagen-induced arthritis in B10.RIII mice (H-2r): identification of an arthritogenic T-cell determinant. AB - Susceptibility to collagen-induced arthritis (CIA), a murine model of autoimmune arthritis, is strongly linked to only two major histocompatibility complex (MHC) haplotypes, H-2q and H-2r. In order to identify the determinants of type II collagen (CII) required to induce arthritis in H-2r-bearing mice, B10.RIII mice were immunized with bovine, chick or human CII. Only bovine CII induced significant arthritis and autoantibodies. When the major CNBr peptides of bovine collagen were isolated and used for immunization, only mice immunized with CB8, representing CII 403-551, developed arthritis. To identify immunogenic epitope(s) within CB8, a panel of synthetic peptides representing overlapping sequences of the bovine peptide was generated. When each peptide was cultured with T cells from B10.RIII mice immunized with CII, one peptide, representing CII 430-466, contained a major T-cell epitope. By using an in vitro lymphokine production assay, the T-cell epitope was further narrowed to CII 442-456. These findings suggest that a T-cell determinant important for the initiation of arthritis in B10.RIII (H-2r) mice is located within a 15 amino acid sequence, residues 442-456 of bovine CII. PMID- 7540591 TI - The role of lymphocytes in the neutrophil migration induced by ovalbumin in immunized rats. AB - In the present study, we investigated the role of resident cells in the neutrophil migration induced by ovalbumin (OVA) in immunized rats. OVA administration induced dose-dependent neutrophil migration, which was inhibited by pretreating the animals with dexamethasone, but not with indomethacin or BW 70C. Lymphocytes, but not macrophages or mast cells, obtained from sensitized animals and stimulated in vitro with OVA released a factor that induced neutrophil migration in vivo and in vitro. Both the release of this factor in vitro and the neutrophil migration induced in vivo were inhibited by dexamethasone, thus explaining the inhibitory effect of glucocorticoids on the neutrophil migration induced by OVA in immunized animals. Neither indomethacin nor BW 70C had any such effect. The fact that actinomycin D also inhibited the release of the factor from OVA-stimulated lymphocytes suggests that this substance is of a proteinaceous nature. The importance of lymphocytes for neutrophil recruitment in OVA-immunized rats was supported by the fact that homologous lymphocyte transfer into air pouches rendered these cavities responsive to OVA. Lymphocytes obtained from naive rats and stimulated with the lectins concanavalin A (Con A) or phytohaemagglutinin (PHA) were also able to release a factor that induced neutrophil migration in vivo. In vitro incubation of the supernatant from OVA-stimulated lymphocytes with antisera to interleukin-1 beta (IL-1 beta), IL-8 and tumour necrosis factor-alpha (TNF-alpha) did not inhibit the neutrophil chemotactic activity. These data suggest that IL-1 beta, IL-8 and TNF-alpha are not involved in the neutrophil chemotactic activity of the supernatant. Overall, these results indicate the importance of lymphocyte participation in neutrophil recruitment during inflammatory immune reaction, through the release of a neutrophil chemotactic factor different from IL-1 beta, IL-8 and TNF-alpha. PMID- 7540593 TI - Analysis of perforin expression in human peripheral blood lymphocytes, CD56+ natural killer cell subsets and its induction by interleukin-2. AB - In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin positive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56bright+ subset, while 70-75% represents the CD56dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin positive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2. PMID- 7540594 TI - Bone marrow stroma-dependent modulation of CD45R isoform expression on Abelson virus transformed pre-B cells. AB - The CD45 glycoprotein family exhibits cell-lineage-associated structural heterogeneity which is due, in part, to alternative pre-mRNA splicing. The Abelson murine leukemia (A-MuLV) preferentially transforms immature B cells that express a B-cell-specific high molecular weight CD45 isoform, called B220. However, we observed that A-MuLV-transformed cell lines are often B220- while maintaining high levels of "pan" CD45 expression. In vitro transformation of murine bone marrow revealed that the stromal microenvironment over which A-MuLV transformed lymphoblasts are grown affected the B220 phenotype of the pre-B cells. Over a period of a few weeks, B220+ populations grown over a clonal stromal cell line gradually became B220-. However, the transition from a B220+ to B220- phenotype was dependent on the lot of fetal calf serum used. In contrast, cells grown over a heterogeneous bone marrow stroma maintained B220+ expression for long periods of time. The appearance of B220- cells in clonal B220+ populations indicated that the change in phenotype resulted in part from modulation of B220 expression. B220- B-cell lines did not express the high molecular weight CD45 RNA species indicating that the B220- phenotype was due to alternative pre-mRNA splicing. Finally, the shift from B220+ to B220- was not accompanied by changes in the stage of development of the cultures. These observations demonstrate that expression of B220 is not required for the continued proliferation of Abelson-transformed pre-B cells and is regulated by unknown environmental factors. PMID- 7540592 TI - Activation of effector functions by immune complexes of mouse IgG2a with isotype specific autoantibodies. AB - Analysis of five monoclonal autoantibodies, rheumatoid factors produced by hybridomas generated from spleen cells of BALB/c mice repeatedly infected with A/PR/8/34 human influenza A virus, revealed that they recognized distinct but spatially related epitopes. The differing isoallotypic specificity of the IgM and IgA monoclonal antibodies correlated with the presence of Ile258 and Ala305, respectively. Although these data suggest that the epitopes recognized are within the CH2 domain, all antibodies failed to inhibit IgG antigen reactivity with Staphylococcus aureus protein A (SpA), C1q, mouse C3, human Fc gamma RI or mouse Fc gamma RII, activities known to be predominantly determined by CH2 domain structures. Reactivity of the IgA antibody, Z34, with IgG2b allowed further specificity studies using a panel of 26 mutant IgG2b proteins, each having single amino acid replacements over the surface of the CH2 domain. The only substitution that affected Z34 reactivity was Asn/Ala297, which destroyed the glycosylation sequon, resulting in secretion of an aglycosylated IgG molecule. The epitope recognized by Z34 therefore seems to be located outside of the Fc gamma R and C1q binding sites, but to be dependent on the presence of carbohydrate for expression. In contrast to the binding studies, complement activation by aggregated IgG2a, through classical or alternative pathways, was inhibited by the presence of autoantibodies. The functional significance of isotype-specific autoantibody in immune regulation is discussed. PMID- 7540596 TI - Binding of mannose-binding protein to Klebsiella O3 lipopolysaccharide possessing the mannose homopolysaccharide as the O-specific polysaccharide and its relation to complement activation. AB - Lipopolysaccharide from Klebsiella pneumoniae O3, which possesses the mannose homopolysaccharide as the O-specific polysaccharide, exhibits an extraordinarily high ability to activate the human complement system. We isolated the mannose binding protein with a Klebsiella O3 lipopolysaccharide affinity column. The protein isolated had a molecular mass of much higher than 200 kDa, and it consisted of subunits with an apparent molecular mass of 32 kDa. The NH2-terminal sequence of the 32-kDa subunits was completely consistent with a part of the amino acid sequence of human serum mannose-binding protein. In immunoblotting, an anti-mannose-binding protein monoclonal antibody was definitely reactive with the isolated protein with the higher molecular mass. The protein isolated was bound exclusively to lipopolysaccharides possessing the mannose homopolysaccharide, not to lipopolysaccharide possessing the heteropolysaccharides. Klebsiella O3 lipopolysaccharide did not exhibit a high anticomplement activity in the serum from which the mannose-binding protein was depleted. It was concluded that the serum factor that bound to Klebsiella O3 lipopolysaccharide may be mannose binding protein and that it may play a crucial role in the strong complement activation by Klebsiella O3 lipopolysaccharide. PMID- 7540595 TI - Expression of adhesion molecules on human granulocytes after stimulation with Helicobacter pylori membrane proteins: comparison with membrane proteins from other bacteria. AB - Type B gastritis in its active form is characterized by a dense infiltration of the lamina propria with granulocytes. Since the bacterium Helicobacter pylori does not invade the epithelial barrier, a signaling pathway chemoattractive for granulocytes must exist across this mucosal boarder. One possible mechanism tested was whether granulocytes are directly activated by water-soluble membrane proteins (WSP) from H. pylori. These findings were compared with the effects of WSP from other bacteria (Helicobacter felis, Campylobacter jejuni, Escherichia coli, and Staphylococcus aureus). A unique activation pattern by H. pylori WSP was found. Like all other WSP tested, they induced an upregulation of CD11b but had no influence on CD11c and, most strikingly, CD62L expression. In contrast, E. coli WSP, e.g., not only induce a strong CD11b and CD11c expression but also lead to a loss in surface CD62L. The lack of CD62L shedding conserves rolling of granulocytes along the endothelium, creating a favorable precondition for granulocytes to stick more readily to activated endothelium after H. pylori stimulation via CD11b-CD54 receptor-counterreceptor interaction. This may explain why H. pylori infection is a very strong stimulus for granulocyte infiltration. The active fraction for the induction of CD11b on granulocytes is a heat- and protease-sensitive protein with a molecular mass between 30 and 100 kDa. One activation step involved may be the binding of WSP to CD15 determinants on granulocytes with subsequent induction of CD11b. PMID- 7540599 TI - Mouse embryo fibroblasts transformed by activated ras or dominant-negative p53 express cross-reactive tumor rejection antigens. AB - To study the immune response against oncogene-transformed tumors, C3H/HcN mouse embryo fibroblasts (MEF) were transfected with an activated allele of the H-ras proto-oncogene VaII2 and a dominant-negative allele of the murine p53 tumor suppressor gene VaII35. Transformed cell lines were derived and found to be tumorigenic in syngeneic mice. Immunization with irradiated p53 + ras-transformed MEF, but not primary MEF or unrelated syngeneic cells, protected mice from subsequent challenge with live tumor cells. The role of different immune cell subsets in the effector phase of anti-tumor immunity induced by immunization with p53 + ras-transformed MEF was investigated by in vivo antibody depletion experiments. Immunized mice depleted of CD8+ T, NK or B cells were resistant, but depletion of CD4+ T cells rendered mice susceptible to tumorigenic challenge. In contrast to the tumor-specific immune responses mounted against most chemically or UV-induced tumors, a series of independently derived p53 + ras-transformed MEF were cross-reactive in tumor rejection assays. In addition, immunization with C3H derived L-929 cell lines expressing single gene products H-ras or p53 did not protect mice against tumorigenic challenge with p53 + ras-transformed tumors. However, MEF transformed by expression of either H-ras or p53 were cross protective in vivo. Our data suggest that the p53 + ras-transformed MEF share tumor rejection antigens which are also induced by single gene transformation of the parental primary cell but are not the products of oncogenic ras or p53 protein. PMID- 7540598 TI - Chemoprevention by galanin against colon carcinogenesis induced by azoxymethane in Wistar rats. AB - The effects of the neuropeptide galanin on the development of colon tumors induced by azoxymethane (AOM) and on the labeling index of colon epithelial cells were investigated in Wistar rats. Treatment with galanin significantly decreased the incidence of colon tumors at week 45. Galanin did not influence the histological appearance of the colon tumors, but it slightly increased the frequency of sub-mucosal adenocarcinomas. Furthermore, it significantly decreased the labeling index of colon mucosa during and after AOM treatment. These findings indicate that galanin inhibited the development of colon tumors and that this effect may be related to its suppression of colon-epithelial-cell proliferation. PMID- 7540600 TI - Clinical application of nitric oxide synthase inhibitor for atopic dermatitis. PMID- 7540597 TI - Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein. AB - Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction. PMID- 7540601 TI - Quality assessment instruments in nursing: towards validation. AB - The aim of this study was to explore the validity of the nursing quality assessment instruments, Monitor, Senior Monitor and Qualpacs. This follows recommendations in the literature for the need for more comprehensive validation of instruments than has been the case hitherto. A multiple triangulation research design was used which included observation of and interviews with nurses and patients as well as administration of the instruments with the same patients and a questionnaire completed by the nurses in charge on ward organisation and their approach to nursing care. Results reported here focus on our experiences of using the instruments, their inter-rater reliability and comparisons of instrument scores within medical, surgical and elderly care wards. Difficulties were encountered in using the instruments but most of these can be overcome given sufficient time for preliminary discussions. Inter-rater reliability of all three instruments taken as a whole reached acceptable levels, although some of the section score correlation coefficients were low, especially for Qualpacs. Convergent validity was achieved for the Senior Monitor-Qualpacs comparisons in four elderly care wards. The results were less clear for the Monitor-Qualpacs comparisons in seven medical and surgical wards. Explanations for the equivocal results are suggested and subsequent hypotheses were tested which supported these explanations. PMID- 7540602 TI - A comparison of the patient-borne costs of therapy with gamma globulin given at the hospital or at home. AB - The major aim of this study was to estimate and compare the patient-borne costs of lifelong subcutaneous gamma globulin therapy at the hospital and at home. Thirty patients were included and the data were collected with a questionnaire. The introduction of self-therapy at home reduced the total yearly costs by approximately 50% and the out-of-pocket expenses for the patients by 85%. PMID- 7540604 TI - [The role of basophilic leukocytes in inflammatory skin diseases]. AB - Basophilic leukocytes are effector cells of the peripheral blood. They have several morphological and functional characteristics in common with tissue mast cells, such as expression of the high-affinity IgE receptor (Fc epsilon RI) and a high content of histamine within their granules. Functional comparison of human basophils and human mast cells isolated from different tissues revealed marked heterogeneity of mediator release after incubation with different secretagogues. Owing to their wide range of surface receptors, their (pro)inflammatory mediators released after activation, their mobility, and their rapid turnover, basophils appear basically to be potent effector cells, which migrate transiently into the skin during IgE-mediated (and/or IgE-independent) inflammatory reactions. The present paper reviews recent findings on the possible role of basophils for different immune reactions of the skin. Inhibition of basophil (and/or mast cell) activity seems to be necessary for both effective prophylaxis and therapy of allergic and inflammatory skin diseases. To date, however, the pharmacological modulation of mediator release from basophils lacks potent clinically useful compounds that can suppress the cellular response, since mast cell stabilizers such as cromoglycate and nedocromil are not effective. With a view to the development of active compounds, further in vitro studies should focus on the mechanisms of cell activation. PMID- 7540605 TI - [Posturographic registration of body sway after odor stimulation]. AB - The methods of "reflex olfactometry" are rarely able to detect simulated anosmia because of the lack of reproducible correlations between stimulus and intensity of reaction. Interactions between the olfactory and vestibulospinal systems have not yet been investigated. Thirty persons with normosmia and 21 patients with anosmia were studied using a posturographic platform. Only in subjects with normosmia were body sway increases statistically significant in an anterior posterior direction after the application of an olfactory nerve stimulant. Stimulations of the trigeminal nerve cause both in subjects with normosmia, and anosmia an increase in body sway occurred in a sagittal and lateral direction. The recording of vestibulospinal reactions during platform posturography after application of an olfactory stimulations enables quantification of body sway. These responses can also be useful in differentiating real from stimulated anosmia. PMID- 7540603 TI - [Autoimmune-associated MHC class II molecules]. AB - The major histocompatibility complex (MHC) is a large region of highly polymorphic genes. MHC-encoded molecules bind foreign-protein antigens and form complexes that are recognized by antigen-specific T lymphocytes. Interactions between MHC-peptide complexes and T-cell receptors play a central role in the selection of the T-cell repertoire and in the elicitation of an immune response. The majority of disease associations with the MHC have been with class-II molecules. Despite the awareness of such associations, the pathophysiological role of MHC class-II molecules in conferring susceptibility to autoimmune disease is poorly understood. The recent demonstration that human class-II molecules can function as antigen-presenting molecules in vivo in a transgenic murine system paves the way to investigate the role of HLA class II molecules in regulating immune responses and disease susceptibility. These mice should be useful for studying the mechanisms by which particular human leukocyte antigen (HLA) alleles are associated with specific human-autoimmune diseases. PMID- 7540607 TI - Treatment of nonregenerative anemia with human gamma-globulin in dogs. AB - Five dogs with nonregenerative anemia were treated with human immunoglobulin as a 12-hour IV infusion, at dosages ranging from 0.5 to 1.5 g/kg of body weight. All dogs had a rapid response to treatment, with reticulocytosis within 1 to 4 days and a substantial increase in hematocrit within 3 to 8 days of treatment. In 2 of 5 dogs, the hematocrit returned to values within reference range and remained in the reference range for 8 to 14 months after treatment, despite discontinuing or tapering prednisone treatment to a low dose. In 3 of 5 dogs, the hematocrit did not return to the reference range. In 1 of these 3 dogs, the hematocrit remained at the new, increased value (26 to 28%) for 248 days after treatment, at which time the dog was euthanatized. In the other 2 dogs, the hematocrit had decreased to pretreatment values by 52 days after treatment. Retreatment of these 2 dogs resulted in a similar, but blunted, response to human immunoglobulin. Human immunoglobulin may be an effective treatment for some dogs with immune-mediated anemia that fail to respond to conventional treatment. PMID- 7540606 TI - Radiotherapy, combined with simultaneous chemotherapy with mitomycin C and bleomycin for inoperable head and neck cancer--preliminary report. AB - PURPOSE: Prospectively designed randomized clinical study was undertaken to assess the efficacy of simultaneous application of irradiation, Mitomycin C, and Bleomycin in treatment of patients with inoperable head and neck carcinoma. METHODS AND MATERIALS: Between March 1991 and October 1993, 49 patients with inoperable head and neck carcinoma were randomly assigned to receive either radiation therapy alone (group A) or radiotherapy combined with simultaneous application of Mitomycin C and Bleomycin (group B). Patients in both groups were irradiated five times weekly with 2 Gy to the total dose of 66-70 Gy. Chemotherapy regimen included intramuscular application of Bleomycin 5 units twice a week, with the planned dose being 70 units and Mitomycin C 15 mg/m2 applied intravenously after delivery of 9-10 Gy of irradiation. The application of Mitomycin C was planned to be repeated on last day of radiotherapy in the dose of 10 mg/m2. In attempt to enhance the effect of chemotherapeutic drugs, patients in group B received also Nicotinamide, Chlorpromazine, and Dicoumarol. RESULTS: The difference in complete response rate between both treatment groups (24% in group A and 63% in group B) was statistically significant (p = 0.015). The difference in response rate was much more pronounced in patients with oropharyngeal carcinoma only (18% in group A compared to 81% in group B; p = 0.0003), while for all other subgroups added together, there was observed no benefit of multidrug therapy. Median follow-up was 18 months. Disease-free survival of patients in group A (9%) was significantly lower then in group B (48%) (p = 0.001). The difference between both treatment groups was even greater in patients with oropharyngeal carcinoma only: disease-free survival of these patients in group B was 66%, while in group A, all recurred (p = 0.00001). CONCLUSION: From results of our prospective randomized study it seems that the group of patients that received multidrug treatment with Mytomycin C, Bleomycin, Nicotinamide, Chlorpromazine, and Dicoumarol as enhancers of radiotherapy fared better than patients treated by radiotherapy alone. PMID- 7540608 TI - Direct transplantation of chronic myelogenous leukemia cells into nude mice and establishment of a leukemic stem cell (Ph1+, CD34+) line dependent on mouse bone marrow stromal cells in vitro. AB - Peripheral blood cells from a female patient with Ph1-positive chronic myelogenous leukemia (CML) in blast crisis were serially transplanted in BALB/c nude mice for 16 passages. This in vivo cell line, designated CML-N-1, had Ph1 chromosome abnormality and BCR gene rearrangement. The cells expressed CD11b, CD13, CD33, CD34, CD38, and HLA-DR antigens until the 11th passage and subcutaneous tumors produced by these passages were composed of admixtures of immature and maturing cells that differentiated to basophils when cultured in vitro. From the 12th passage on, the tumors became composed mainly of immature cells expressing CD13, CD34, and HLA-DR, and no longer differentiated to basophils even upon in vitro culture. In contrast to the vigorous proliferation in vivo, CML-N-1 cells from any passage failed to proliferate in vitro under standard liquid culture conditions with or without growth factors, such as granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, monocyte colony-stimulating factor, interleukin 3, interleukin 6 and stem cell factor. However, a continuously growing cell line, designated CML-C-1, was established by culturing CML-N-1 cells on feeder layers of mouse bone marrow stromal cells. This mouse bone marrow stromal cell-dependent cell line showed immature cell morphology and expressed early myeloid phenotype positive for CD13, CD34, and HLA-DR. These results indicate that mouse bone marrow stromal cells provide a certain growth factor(s) active on human leukemia cells. PMID- 7540610 TI - A new extracellular type solution for lung preservation: "in vitro" comparison with Beltzer, low potassium Dextran and Euro-Collins solutions by means of human lung fibroblasts. AB - The attempt to synthesise efficacious solution to prolong lung preservation is, at present one of the most interesting challenges in transplant research. Recently, several issues emphasise the central role of ionic composition of lung flush solutions, underlining, however, that colloid-free solutions are clearly detrimental. We have been studying a complex extracellular type solution (SPAL UP) synthesised to minimise the pathological events that occur during both preservation and reperfusion period. We report the results of toxicity of SPAL UP on normal human fibroblasts obtained from foetal lung (WI-38). WI-38 cells were seeded at 1.4 x 10(4)/cm2 in disposable plastic 12-well plates. After 3 days, cells were incubated in SPAL UP, Beltzer (UWS), Low Potassium Dextran (LPD) and Eurocollins (ECS) solutions for 6 hours at 10 degrees C. Cellular viability was evaluated by the rate of protein synthesis exploiting the incorporation of 35S Methionine (2 microCi/ml) in growth medium with 10 mM unlabelled Methionine during 30 minutes incubation at 37 degrees C. The results were expressed as nmol. 35S-Methionine/mg of proteins/minute, and presented as means +/- SD of data of three (n = 3) well for each solution studied. RESULTS: the viability at time 0 before incubation (considered as control) was 1.65 +/- 0.1; after hypothermic preservation the data were respectively as follow: SPAL UP 0.51 +/- 0.09; UW 0.24 +/- 0.02; ECS 0.19 +/- 0.01; LDP 0.19 +/- 0.05. CONCLUSIONS: in this "in vitro" model SPAL UP solution provides a significantly (p < 0.05) better cell preservation than regular UW, ECS and LPD solutions. PMID- 7540609 TI - Osteoporosis-like changes in Walker carcinoma 256-bearing rats, not accompanied with hypercalcemia or parathyroid hormone-related protein production. AB - Walker carcinoma 256 (W256) was reported to induce hypercalcemia dependent on bone metastasis and/or parathyroid hormone-related protein (PTHrP) in the rat, providing a model of the humoral hypercalcemia of malignancy. In this study, after the subcutaneous inoculation of cells of the W256/S line, which is maintained in this laboratory, into young female Wistar Imamichi rats (6 weeks old), serum calcium and phosphorus levels changed only within the control range, whereas serum alkaline phosphatase activity and urinary calcium level significantly increased and urinary phosphorus decreased during the tumor growth, resulting in hypercalciuria and hypophosphaturia. W256/S did not express PTHrP mRNA, whereas LLC-W256 cells did express it. Serum PTHrP level was not changed in W256/S-bearing rats. Osteoporosis-like changes, bone weight loss, low contents of bone calcium and phosphorus, and a decrease in the bone mineral density (BMD), were observed in the femur 14 days after the tumor inoculation. There was a pronounced decrease in the serum 17 beta-estradiol level during the tumor growth. The reduction of BMD of femurs in W256/S-bearing rats was significantly inhibited by treatment with salmon calcitonin or 17 beta-estradiol. On the basis of these results, W256/S carcinoma-bearing rats seem to be a useful model for osteoporosis of hypoovarianism. PMID- 7540611 TI - Insulin stimulates the tyrosine phosphorylation of caveolin. AB - The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin-sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin-associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton-insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin dependent tyrosine phosphorylation of caveolin represents a novel, insulin specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor. PMID- 7540612 TI - Increased intracellular Ca2+ selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability. AB - This study addresses the role of intracellular calcium (Ca2+) in the expression of iNOS, an IL-1 inducible gene in human articular chondrocytes. The calcium ionophore A23187 and ionomycin did not induce NO release or iNOS expression but inhibited dose dependently IL-1-induced NO release with IC50 of 200 nM and 100 nM, respectively. Increased intracellular Ca2+ induced by thapsigargin or cyclopiazonic acid, inhibitors of the endoplasmic reticulum Ca2+ ATPase, had similar inhibitory effects with IC50 of 1 nM and 3 microM, respectively. LPS and TNF alpha induced NO production were also suppressed by these Ca2+ elevating drugs. Levels of IL-1-induced iNOS protein were reduced by A23187, thapsigargin, and cyclopiazonic acid. These drugs as well as Bay K 8644 and KCl inhibited IL-1 induced iNOS mRNA expression. To analyze the role of Ca2+ in the expression of other IL-1 responsive genes in chondrocytes, these Ca2+ modulating drugs were tested for effects on COXII. In contrast to the inhibitory effects on iNOS mRNA, these drugs induced COXII mRNA expression and in combination with IL-1, enhanced COXII mRNA levels. Ca2+ mediated increases in COXII mRNA expression were associated with an increase in COXII protein. The kinetics of Ca2+ effects on IL 1-induced iNOS mRNA levels suggested a posttranscriptional mechanism. Analysis of iNOS mRNA half life showed that it was 6-7 h in IL-1-stimulated cells and decreased by A23187 to 2-3 h. In conclusion, these results show that Ca2+ inhibits IL-1-induced NO release, iNOS protein, and mRNA expression in human articular chondrocytes by reducing iNOS mRNA stability. Under identical conditions increased Ca2+ enhances IL-1-induced COXII gene and protein expression. PMID- 7540614 TI - ARIA can be released from extracellular matrix through cleavage of a heparin binding domain. AB - ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. Here we demonstrate that the ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2 terminal portion, but preserves the active, EGF-like domain. We also show that ARIA can be released from freshly dissociated cells from embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. We present a model of how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis. PMID- 7540613 TI - Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment. AB - The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus-host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle. PMID- 7540616 TI - Epidermal growth factor promotes a neural phenotype in thymic epithelial cells and enhances neuropoietic cytokine expression. AB - Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus dependent immune functions by promoting neuronal gene expression in neural crest derived cells. PMID- 7540615 TI - NGF-stimulated retrograde transport of trkA in the mammalian nervous system. AB - The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo. PMID- 7540618 TI - Induction of bovine bronchial epithelial cell filopodia by tetradecanoyl phorbol acetate, calcium ionophore, and lysophosphatidic acid. AB - The morphological responses of primary bovine bronchial epithelial cells (BBECs) cultured in serum-free medium to protein activators have been examined. When attached to type I collagen-coated tissue culture dishes, the cells responded to tetradecanoyl phorbol acetate (TPA), calcium ionophore A23187, and lysophosphatidic acid (LPA) by extruding filopodia. In contrast, no morphological changes were elicited by exposures to either epinephrine or dibutyryl-cAMP. Formation of filopodia was accompanied by actin filament reorganization as demonstrated by staining with labeled phalloidin. Exposures to varied TPA concentrations for 2 h showed maximal stimulation of filopodial extrusions at 10 nM TPA with half-maximal stimulation at 1 nM. Time-course measurements with 10 nM TPA showed filopodia formation within 30 min of exposure, with 85% of the BBECs being filopodia positive after 5 h. Filopodia induction in 20-30% of the cells could be achieved by 1-100 microM LPA concentrations. BBECs acquired increasing resistance to TPA-induced filopodia during the initial 5 days in culture; however, responsiveness to TPA was regenerated by mild treatment with trypsin. Inclusion of fibronectin or vitronectin into the attachment matrix had no effects on the rates or extent of TPA-induced filopodia formation. PMID- 7540617 TI - Fibrin II induces endothelial cell capillary tube formation. AB - We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2-terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation. PMID- 7540619 TI - Regulation of insulin-like growth factor (IGF) binding protein-2 synthesis and degradation by platelet-derived growth factor and the IGFs is enhanced by serum deprivation in vascular smooth muscle cells. AB - Growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) stimulated proliferation and migration of vascular smooth muscle cells (SMC). IGF-I bioactivity is modulated by high-affinity binding proteins (IGFBP) which are important regulators of these processes. Porcine vascular SMC synthesize IGFBP-2 and IGFBP-4 in vitro. In the present study, levels of IGFBP-2 in conditioned media (CM) were increased approximately 1.6 to 2.2-fold when cells were exposed to PDGF (20 ng/ml) or insulin (5 micrograms/ml) for 24 hr following a 24 hr incubation in serum-free media, or following a 72 hr exposure to either growth factor. Similar increases in IGFBP-2 mRNA levels were observed. Exposure of cells to PDGF for 24 hr without prior serum deprivation resulted in smaller (47 +/- 11%) increases in IGFBP-2 protein levels but failed to alter mRNA levels. IGF-I, FGF, TGF-beta and EGF failed to increase IGFBP-2 using either experimental paradigm. In contrast, IGFBP-2 protein levels were consistently decreased (75 +/- 14%) after 72 hr of exposure to IGF-II without corresponding decreases in IGFBP-2 mRNA levels. Immunoprecipitation of [35S]methionine-labeled IGFBP-2 indicated that this decrease was not due to a decrease in synthesis of IGFBP-2. Immunoblot analysis of CM from cells treated with IGF-II indicated that the decrease in intact protein corresponded with an increase in two non-IGF binding IGFBP-2 fragments of 22 and 14 kD. Increased abundance of these fragments was also observed following IGF-I exposure, although corresponding decreases in intact IGFBP-2 were not usually observed. The relative abundance of these fragments did not appear to be affected by treatment with PDGF or insulin. In contrast to IGFBP-2, regulation of the levels of IGFBP-4 in CM did not appear to be altered by serum deprivation. Insulin consistently increased IGFBP-4 mRNA and protein levels under all situations. PDGF tended to increase IGFBP-4 protein levels, although this effect was less consistent and not as great as the increase observed with insulin. Treatment with IGF-I or -II consistently decreased IGFBP-4 levels in CM but tended to increase their mRNA levels under all situations. These data indicate that insulin, PDGF, and the IGFs regulate both IGFBP-2 and IGFBP-4. While PDGF and insulin stimulate IGFBP-2 and 4 synthesis, the IGFs appear to activate protease(s) which regulate IGFBP-2 and -4 levels post translationally.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7540620 TI - Ion channel involvement in anoxic depolarization induced by cardiac arrest in rat brain. AB - Anoxic depolarization (AD) and failure of ion homeostasis play an important role in ischemia-induced neuronal injury. In the present study, different drugs with known ion-channel-modulating properties were examined for their ability to interfere with cardiac-arrest-elicited AD and with the changes in the extracellular ion activity in rat brain. Our results indicate that only drugs primarily blocking membrane Na+ permeability (NBQX, R56865, and flunarizine) delayed the occurrence of AD, while compounds affecting cellular Ca2+ load (MK 801 and nimodipine) did not influence the latency time. The ischemia-induced [Na+]e reduction was attenuated by R56865. Blockade of the ATP-sensitive K+ channels with glibenclamide reduced the [K+]e increase upon ischemia, indicating an involvement of the KATP channels in ischemia-induced K+ efflux. The KATP channel opener cromakalim did not affect the AD or the [K+]e concentration. The ischemia-induced rapid decline of extracellular calcium was attenuated by receptor-operated Ca2+ channel blockers MK-801 and NBQX, but not by the voltage operated Ca2+ channel blocker nimodipine, R56865, and flunarizine. PMID- 7540621 TI - Time dependence of effect of nitric oxide synthase inhibition on cerebral ischemic damage. AB - Nitric oxide, a potent vasodilator and an inhibitor of platelet aggregation, may be beneficial in the early stages of focal cerebral ischemia as it may facilitate collateral blood flow to the ischemic territory. Accordingly, the effect of inhibition of nitric oxide synthesis on cerebral ischemic damage may vary depending on the timing of the inhibition relative to the induction of ischemia. We therefore studied the time course of the effect of nitric oxide synthesis inhibition on focal cerebral ischemic damage. The middle cerebral artery was permanently occluded in spontaneously hypertensive rats and the nitric oxide synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME) was administered systemically (3 mg/kg) < 5 min or 2, 3, or 6 h later. Arterial pressure, rectal temperature, plasma glucose, and hematocrit were monitored. Infarct volume was determined on thionin-stained sections 24 h after induction of ischemia. NOS activity was determined in cerebellum from the conversion of L-[3H]arginine to L [3H]citrulline. Administration of L-NAME < 5 min after arterial occlusion increased the infarct volume by 23 +/- 14% (mean +/- SD; p < 0.05, analysis of variance), while administration of L-NAME at 2 or 6 h did not affect the size of the infarct (p > 0.05). L-NAME administration 3 h after induction of ischemia reduced neocortical infarct size by 14 +/- 11% (p < 0.05). L-NAME decreased cerebellar NOS activity comparably in all groups (range 16-25%). We conclude that the effects of inhibition of nitric oxide synthesis on focal cerebral ischemic damage are time dependent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540622 TI - Endothelin-3-induced relaxation of isolated rat basilar artery is mediated by an endothelial ETB-type endothelin receptor. AB - The endothelin (ET) receptor mediating relaxation of cerebral arteries was characterized using ring segments obtained from the rat basilar artery. Under resting tension, ET-3 (> 10(-8) M) but not the specific ETB receptor agonist IRL 1620 induced contraction. In ring segments precontracted with 3 x 10(-6) M prostaglandin (PG) F2 alpha, ET-3 (10(-12) - 10(-8) M) and IRL 1620 (10(-14) - 10(-6) M) induced concentration-related relaxation. IRL 1620 was more potent than ET-3, the pD2 (-log10EC50) values being 10.002 +/- 0.751 (mean +/- SD) for IRL 1620 and 8.836 +/- 0.415 for ET-3. Relaxation was abolished after preincubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (10(-5) M) as well as in segments devoid of a functionally intact endothelium. At a concentration above 10(-8) M, ET-3 resulted in a further increase of PGF2 alpha induced contraction that was not observed with IRL 1620. The presumably specific ETB receptor antagonist IRL 1038 (10(-7) - 3 x 10(-6) M) diminished or even abolished (3 x 10(-6) M) the relaxation induced by ET-3 or IRL 1620. IRL 1038 did not exert any vasomotor effect by itself, and it did not significantly affect ET 3-induced contraction. These results indicate that in the rat isolated basilar artery, the ET-3-induced relaxation is probably due to activation of an ETB-type receptor located on the endothelial cells and mediated by release of nitric oxide. PMID- 7540624 TI - Evaluation of lacto-phenol cotton blue for wet mount preparation of feces. AB - The use of lacto-phenol cotton blue (LPCB) stain for wet mount preparation of stools to demonstrate intestinal parasites by routine microscopy was evaluated in this study. LPCB-stained trophozoites and cysts and helminthic ova could easily be detected and identified in LPCB wet mounts of stools. The stain is recommended for routine use in the wet mount preparation of stools in a parasitology laboratory. PMID- 7540623 TI - Burkholderia cepacia and cystic fibrosis: do natural environments present a potential hazard? AB - An environmental survey of 55 sites yielded only 12 Burkholderia cepacia isolates, none of which displayed the phenotypic properties of a multiresistant epidemic strain associated with pulmonary colonization in patients with cystic fibrosis. Although the environment probably poses a low risk for patients with cystic fibrosis as a source of B. cepacia, the pathogenic potential of individual environmental strains remains unclear. We advise caution in the development of B. cepacia as a biocontrol agent. PMID- 7540625 TI - Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid. AB - A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible. PMID- 7540627 TI - Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis. AB - Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis. PMID- 7540626 TI - Diagnosis of intestinal microsporidiosis by examination of stool and duodenal aspirate with Weber's modified trichrome and Uvitex 2B strains. AB - Severe, chronic diarrhea is a frequent complication of human immunodeficiency virus disease, and intestinal microsporidiosis is being recognized with increasing frequency in patients with AIDS. Noninvasive, cost-effective techniques are needed to optimize its diagnosis. Weber's modified trichrome stain (MTS) and the fluorochrome Uvitex 2B stain were used to detect microsporidial spores in smears of stool and duodenal aspirate (DA) samples received from human immunodeficiency virus-infected patients for examination for ova and parasites. Of 305 samples (292 stool and 13 DA samples) from 140 patients examined by MTS, 83 samples from 26 (18.6%) of the patients were positive for microsporidia (23 patients diagnosed initially and 3 diagnosed upon review). A subset of the samples studied by MTS consisting of 108 smears of stool and DA specimens from 60 patients was examined by Uvitex 2B. All 44 samples positive by MTS were also positive by Uvitex 2B. In addition, seven specimens and three patients were initially detected as positive by Uvitex 2B only (all three patients were positive also by MTS upon review). Confirmation of the diagnosis was obtained for 24 of 26 smear-positive patients by duodenal biopsy and/or stool transmission electron microscopy. Of 114 patients with stained smears negative for microsporidia, 23 had duodenal biopsies which showed no microsporidia. For the 43 patients who underwent duodenal biopsy, the sensitivity of both the MTS and the Uvitex 2B methods compared with biopsy results was 100%. Of six patients with negative duodenal biopsies and positive stained smears, four had microsporidia demonstrated by stool transmission electron microscopy. The examination of stool and DA smears stained by Uvitex 2B and/or MTS is a sensitive, noninvasive test for diagnosis of intestinal microsporidiosis which can be successfully implemented in a clinical laboratory. Strict adherence to precise diagnostic criteria is necessary to avoid incorrect results. The simultaneous use of both staining methods enhances performance and may provide greater accuracy, especially for patients with light infections. PMID- 7540628 TI - Molecular subtyping scheme for serotypes HS1 and HS4 of Campylobacter jejuni. AB - We describe a molecular subtyping scheme for two principal O (heat-stable [HS]) serotypes of Campylobacter jejuni, HS1 and the HS4 complex. A 16S rRNA gene specific probe confirmed that almost all the C. jejuni strains had three copies of this gene, and strains could be assigned with complete typeability to 1 of 16 combined (Pst1 and HaeIII) 16S ribotypes. Macrorestriction profiles (mrps) consisting of up to 10 SmaI fragments from approximately 40 to approximately 480 kbp were resolved by pulsed-field gel electrophoresis (PFGE). There were 11 mrps among the HS1 strains and 9 mrps among HS4 strains which corresponded to valid types--they occurred in multiple isolates, hosts, places, and times. There were 14 additional single-strain mrp fingerprints in HS1 and 20 in HS4. PFGE exhibited complete typeability when formaldehyde fixation of cells was employed, and PFGE was generally more differential than ribotyping. The data presented elucidate a high-resolution genotypic subtyping scheme for these common subspecific phenotypes of C. jejuni, which is both coherent and efficient for epidemiological purposes. PMID- 7540629 TI - Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene. AB - We report the use of PCR to detect DNA from Mycoplasma hyopneumoniae, the etiological agent of enzootic porcine pneumonia. A primer set was designed for the amplification of a 649-bp fragment of the 16S rRNA gene from M hyopneumoniae. The PCR product was identified by ethidium bromide staining after gel electrophoresis and by Southern hybridization with an M. hyopneumoniae-specific oligonucleotide probe. No amplification was observed from any other porcine bacteria tested, including several closely related mycoplasmas. It was also possible to demonstrate the presence of M. hyopneumoniae in bronchial lavage samples and lung tissue samples from experimentally infected pigs. Furthermore, the PCR system was used for analysis of nasal samples obtained from pigs in a fattening herd. By this method, we were able to detect M. hyopneumoniae in nose swabs from naturally infected pigs. However, our results suggest that M. hyopneumoniae can be detected in the nasal cavities only during a limited time period. PMID- 7540631 TI - Midgut carcinoid tumors: CT findings and biochemical profiles. AB - OBJECTIVE: Our goal was to describe the abdominal CT findings in 52 patients with midgut carcinoid tumors and correlate these findings with their biochemical profiles. MATERIALS AND METHODS: Abdominal/pelvic CTs of 52 patients with midgut carcinoid tumors were reviewed retrospectively for the presence of liver metastases, mesenteric and peritoneal disease, bowel changes, lymphadenopathy, and the presence of the primary tumor. Logistic regression models were used to evaluate the association between these findings and the serum and platelet serotonin and urine 5-hydroxyindolacetic acid levels. RESULTS: The most common finding was hepatic metastases (34/52). Nonspecific mesenteric soft tissue stranding was present in 26 of 52 and a discrete mesenteric mass was present in 25 of 52. These masses had linear, radiating soft tissue spokes in 16 of 25 and contained calcification in 10 of 25. Retroperitoneal and mesenteric lymphadenopathy was present in 14 of 52 and 11 of 52 cases, respectively. Carcinomatosis was present in 11 of 52. Bowel wall thickening was seen in 9 of 52. Six patients had a small bowel obstruction. Elevated serum serotonin, platelet serotonin, and urine 5-hydroxyindolacetic acid levels were significantly associated with the presence of liver metastases (p = 0.0032, 0.0098, and 0.0450, respectively). Elevated platelet serotonin levels were also significantly associated with the presence of a mesenteric mass (p = 0.0101). CONCLUSION: In our population, the most common findings of a midgut carcinoid tumor are liver metastases, nonspecific mesenteric soft tissue changes, a discrete mesenteric mass with radiating soft tissue spokes, often containing calcification, and lymphadenopathy. As expected, liver metastases correlate strongly with the presence of elevated biochemical levels. A new observation is the correlation of elevated platelet serotonin levels and mesenteric masses. We hypothesize that platelet serotonin may be the factor that stimulates stromal cells to produce mesenteric fibrosis and mass formation. PMID- 7540630 TI - Detection of genomic heterogeneity in Streptococcus suis isolates by DNA restriction fragment length polymorphisms of rRNA genes (ribotyping). AB - Whole-cell chromosomal digests of 54 isolates of Streptococcus suis encompassing all known serotypes from a geographically varied collection were examined by PstI restriction fragment length polymorphisms and then hybridized with a digoxigenin 11-dUTP-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli MRE600. The hybridization patterns showed genetic heterogeneity within and between S. suis serotypes. Most isolates (87%) representing 28 serotypes contained a common band at approximately 1.8 kb. However, 13% of the isolates representing seven serotypes lacked the 1.8-kb band, indicating that the species as currently defined is diverse. Nonetheless, the 1.8-kb band may be a useful genotypic marker for identification of most S. suis isolates. We tested the ability of this technique to discriminate between virulent and avirulent S. suis type 2 isolates. A virulent strain of S. suis type 2 could be distinguished from avirulent strains by the presence of specific bands. No correlation was obvious between band pattern and hemolysin production. PMID- 7540632 TI - The effect of feed intake and growth hormone-releasing factor on lactating dairy cows. AB - Twenty-four lactating dairy cows, averaging 30.0 kg/d of milk and 159 d of lactation, were used to study the effect of feed intake and growth hormone releasing factor in a 2 x 2 factorial arrangement. For the first 10-d period, cows had free access to a TMR and received a fixed amount of high moisture corn, protein supplement, and hay. In the second 10-d period, 12 cows were maintained on this high intake, and 12 cows received 70% of their previous intake (low intake). During the following 10-d period, each intake group was divided, and each of two subgroups (n = 6) received twice daily s.c. injections of saline or growth hormone-releasing factor (10 micrograms/kg of BW per injection). Feed restriction decreased milk production by 24%. Milk production increase was not different following growth hormone-releasing factor treatment for cows maintained at high intake (4.6 kg/d) or low intake (3.4 kg/d). Feed restriction increased concentration of growth hormone but did not affect IGF-I concentration. Growth hormone-releasing factor increased IGF-I concentration similarly for both intake groups but increased concentrations of insulin and IGF-binding proteins-1 and -3 only in the high intake group. Low intake did not affect growth hormone, IGF-I, or milk responses to growth hormone-releasing factor, but suppressed the increase in concentrations of insulin and IGF-binding proteins-1 and -3 following treatment with growth hormone-releasing factor for cows on high intake. PMID- 7540633 TI - Effect of dietary energy restriction on the expression of insulin-like growth factor-I in liver and corpus luteum of heifers. AB - The effects of energy balance on the corpus luteum may be mediated by IGF-I. Our objective was to determine whether negative energy balance decreases expression of IGF-I mRNA in the liver or corpus luteum of heifers. For four consecutive estrous cycles, 14 Holstein heifers were maintained in negative or positive energy balance to lose 510 g/d of BW or gain 560 g/d of BW, respectively. The liver was biopsied and the corpus luteum was collected 7 d after fourth estrus. Heifers fed below maintenance had a smaller corpus luteum than did control heifers. Compared with that of controls, negative energy balance increased growth hormone in serum but decreased IGF-I in serum and the abundance of mRNA for growth hormone receptor and IGF-I in liver. In contrast, diet did not affect the abundance of mRNA for the growth hormone receptor or IGF-I in luteal tissue. Negative energy balance increased IGF binding protein-2 in serum but did not affect IGF binding protein-3. We conclude that negative energy balance of cattle decreases IGF-I mRNA abundance in the liver but not in the 7-d corpus luteum. If decreased IGF-I mediates the adverse effect of negative energy balance on luteal growth, the mode of action is likely endocrine, not autocrine or paracrine. PMID- 7540634 TI - Marginal ditching and staining as a predictor of secondary caries around amalgam restorations: a clinical and microbiological study. AB - Caries at the margins of restorations is difficult to diagnose. In particular, the relevance of both marginal ditching and staining around amalgam restorations is unclear. This clinical study questions the relevance of marginal ditching and color change to the level of infection of the dentin beneath the margins of amalgam restorations. Clinically visible sites (330) on the tooth/restoration margin were selected on 175 teeth. The enamel adjacent to each site was noted as stained (a grey discoloration) or stain-free. One hundred and seventy-eight sites were clinically intact, 83 sites had narrow ditches (< 0.4 mm), and at 49 sites, wide ditches were present (> 0.4 mm). Twenty sites with frankly carious lesions were also included. Plaque was sampled at the tooth-restoration margin, and the dentin was sampled at the enamel-dentin junction below each site. Samples were vortexed, diluted, and cultured for total anaerobic counts, mutans streptococci, lactobacilli, and yeasts. Plaque samples showed that margins with wide ditches (> 0.4 mm) harbored significantly more bacteria, mutans streptococci, and lactobacilli than did clinically intact margins and margins with narrow ditches. There were no significant differences in the degree of infection of the dentin beneath clinically intact restorations and those with narrow ditches, but samples associated with wide ditches and carious lesions yielded significantly more bacteria, mutans streptococci, and lactobacilli. The color of the enamel adjacent to the sample site was irrelevant to the level of infection of the dentin beneath the filling margin, provided a frankly carious lesion was not present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540636 TI - Overexpression of P-glycoprotein in adenomatous hyperplasia of human liver with cirrhosis. AB - Adenomatous hyperplasia in the human liver with cirrhosis is similar to the hyperplastic nodule in rat hepato-carcinogenesis in that the mdr gene or its product P-glycoprotein is overexpressed. We immunohistochemically stained archival formalin-fixed, paraffin-embedded sections of 15 adenomatous hyperplasias with or without hepatocellular carcinoma in livers with cirrhosis, using the avidin-biotin-complex method and the JSB-1 monoclonal antibody which specifically binds the cytoplasmic epitope of P-glycoprotein. Of 15 cases with adenomatous hyperplasia, four were found solely in livers with cirrhosis. In six cases, adenomatous hyperplasia and hepatocellular carcinoma were found in the same liver separately. Hepatocellular carcinoma was discovered within adenomatous hyperplasia in five cases. All 15 livers with cirrhosis and those with adenomatous hyperplasia were positively stained for P-glycoprotein. When the grade of staining was compared between adenomatous hyperplasia and the surrounding liver, P-glycoprotein was overexpressed in 12 of 15 cases with adenomatous hyperplasia. P-glycoprotein was also stained more strongly in well differentiated hepatocellular carcinoma than in the liver, but the staining grade of hepatocellular carcinoma was weaker than that of adenomatous hyperplasia. Moreover, the glycoprotein expression was less when the tumor was less differentiated. PMID- 7540635 TI - Treatment of immune cell-mediated liver damage by nonpeptidic mimetics of the extracellular matrix-associated Arg-Gly-Asp epitope. AB - The etiology of T-cell-mediated liver injury involves the migration of immune cells, notably CD4+ T lymphocytes, into liver tissues. This process is mediated primarily by integrin-recognition of the sub-endothelium basement membranes and the extracellular matrix. The Arg-Gly-Asp-containing peptide, a major cell adhesive ligand of extracellular matrix, is present in various plasma- and matrix glycoproteins, such as fibronectin. Recently, we have described the design and usage of nonpeptide mimetics of Arg-Gly-Asp which bind specifically to integrins, and thereby, inhibit T cell immunity in vivo. We examined the efficacy of Arg-Gly Asp-mimetics as potential therapeutic compounds for the treatment of experimental T-cell-mediated liver injury induced in mice by injection of Concanavalin-A. We now report that the Arg-Gly-Asp-mimetics specifically inhibited the binding of murine T cells to fibronectin, but did not affect the proliferative response of these cells in vitro. Intraperitoneal or oral administration of the Arg-Gly-Asp mimetics but not the Arg-Gly-Asp-containing peptide, inhibited liver damage in mice if given before their inoculation with Con-A, as manifested by a lesser elevation in their serum levels of hepatic enzymes. The inhibitory effect of the Arg-Gly-Asp-mimetics was dose-dependent, the ED50 of the tested molecules being in the range of 100 micrograms per mouse and reaching maximal effect, e.g. approximately 95% inhibition, at 500 micrograms per mice. Thus, the Arg-Gly-Asp mimetics described here may be used therapeutically to prevent immune-cell mediated acute or chronic pathological reactions in the liver. PMID- 7540637 TI - Lack of vascular hyporesponsiveness to the L-type calcium channel activator, Bay K 8644, in rats with cirrhosis. AB - BACKGROUND/AIMS: In cirrhosis, the mechanism(s) for vascular hyporesponsiveness to vasoconstrictors such as, alpha 1-adrenoceptor agonists, vasopressin and angiotensin II, are unclear. Moreover, vascular reactivity to substances such as L-type calcium channel activators is unknown. METHODS: Thus, pressor dose response curves to vasoconstrictors [phenylephrine (an alpha 1-agonist, 0.1-500 micrograms/kg) angiotensin II (10-500 ng/kg), vasopressin (0.01-5 IU/kg), and Bay K 8644 (an L-type calcium channel activator, 0.5-50 micrograms/kg)] were obtained in normal rats and in rats with secondary biliary cirrhosis. All experiments were performed in ganglionic-blocked animals to limit the influence of cardiovascular reflexes. Doses of vasoconstrictor necessary to obtain a 40 mmHg increase in arterial pressure (D40) were calculated. RESULTS: Compared to normal animals, rats with cirrhosis had significantly higher D40 values for angiotensin II (171 +/- 57 vs. 344 +/- 41 ng/kg), phenylephrine (2.6 +/- 0.2 vs. 26.4 +/- 10.7 micrograms/kg) and vasopressin (73 +/- 19 vs. 401 +/- 150 mU/kg). Pressor responses to Bay K 8644 did not differ between normal rats and rats with cirrhosis (8.8 +/- 0.9 vs. 10.5 +/- 2.1 micrograms/kg). CONCLUSIONS: In conclusion, this study shows that cirrhosis produces vascular hyporeactivity to phenylephrine, vasopressin and angiotensin II but not to Bay K 8644. Therefore, cirrhosis impairs certain constrictor mechanisms which are shared by phenylephrine, angiotensin II and vasopressin but which do not contribute to the vascular response to Bay K 8644. PMID- 7540638 TI - Normalization of urinary porphyrin level and disappearance of skin lesions after successful interferon therapy in a case of chronic hepatitis C complicated with porphyria cutanea tarda. PMID- 7540639 TI - Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y. AB - We developed an improved technique that permits simultaneous DNA and RNA quantitation by a flow cytofluorometry using 7-amino-actinomycin D (7AAD) and pyronin Y (PY), respectively. Detailed cell cycle analyses based upon the cellular DNA/RNA levels were performed using cells suspended in a buffer containing 0.004% saponin. This method preserved the light scattering properties of human peripheral blood cells, thus lymphocyte, monocyte and granulocyte populations could be evaluated. In addition, since 7AAD and PY exhibit red (> 650 nm) and orange fluorescence (570 nm) respectively, the green fluorescence channel of the flow cytometer was reserved for surface phenotyping using FITC-conjugated antibodies. The 7AAD/PY method is applicable to the simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation when combined with FITC-conjugated surface markers in heterogeneous samples. To demonstrate the three-color analysis, PHA-activated human peripheral blood lymphocytes were stained for cell surface markers with monoclonal antibodies. The cells were suspended in buffer containing 0.004% saponin, then stained with 7AAD and PY. The DNA and RNA were analyzed in indivisual CD4+, CD8+ and CD20+ cells, and the characteristic cell cycle status was found. Cell activation was further analyzed using antibodies against interleukin-2 (IL-2) receptors (CD25), transferrin receptors (CD71) or HLA-DR molecules. Transferrin receptors were expressed in late G1 phase (G1B) just before the initiation of DNA synthesis, whereas IL-2 receptors and HLA-DR were expressed very early in the G1 phase (G1T). Since this technique preserves both light scatter properties as well as cell surface proteins, it is ideally suited for detailed cell cycle analyses of heterogeneous samples such as peripheral blood or bone marrow cells. PMID- 7540640 TI - Monopalmitic acid-peptide conjugates induce cytotoxic T cell responses against malarial epitopes: importance of spacer amino acids. AB - Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2-RS) propyl]-N-palmitoyl-(R)-cysteine (tripam-C). Inbred mice [(BALB/c x C57BL/6)F1] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freund's complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates. PMID- 7540641 TI - Grafting peptides onto polystyrene microplates for ELISA. AB - Three peptides corresponding respectively to two Epstein-Barr viral epitopes and to the c-erbB-2 oncogene product were synthesized with the aim of developing an immunoenzymatic assay. Preliminary experiments indicated that the efficiency of the assay was profoundly affected by the nature of the solid phase for each peptide. In order to optimize the assay the three peptides were covalently coupled to functionalized polystyrene microplates which were used to immobilize both haptens and nucleic acids in a previous study. The results obtained indicate that the use of the carboxylated surfaces permits the linking strategy to be adapted to each peptide. Moreover, high sensitivities (5 x 10(-10)-1 x 10(-13) M) were obtained using amounts of the peptides much lower than those used in the standard system. PMID- 7540644 TI - Modulation of human G-CSF receptor mRNA and protein in normal and leukemic myeloid cells by G-CSF and retinoic acid. AB - Granulocyte colony-stimulating factor (G-CSF) is produced by several cell types throughout the body and has a variety of effects on neutrophils and their precursors, which are mediated by binding to its receptor. It is not yet known what physiologic factors modulate G-CSF receptor mRNA expression in these cells. We studied the effect of G-CSF on freshly isolated neutrophils and bone marrow cells from normal human subjects and on myeloid leukemic cell lines. We found that G-CSF receptor mRNA levels were maintained by G-CSF in neutrophils but not in bone marrow cells. Of the leukemic cell lines tested, K562 and BV173, both of which contain the bcr-abl translocation, neither expressed G-CSF receptor mRNA. Whereas G-CSF did not affect mRNA levels for its receptor in myeloid leukemic cell lines, exposure of the acute promyelocytic cell line, NB4, to all-trans retinoic acid induced a striking increase in G-CSF receptor mRNA expression and resulted in increased G-CSF receptor surface expression. The effect of retinoic acid on G-CSF receptor mRNA on NB4 cells occurred early, before morphologic evidence of differentiation, and required protein synthesis. All-trans retinoic acid also upregulated G-CSF receptor mRNA in the myeloid leukemia cell line HL 60. Thus, maintenance of G-CSF receptor on neutrophils by G-CSF may extend the duration of ligand responsiveness. Furthermore, the ability of retinoic acid to up-regulate G-CSF receptor may account for the synergistic effect of G-CSF and retinoic acid in differentiation induction of acute promyelocytic leukemia. PMID- 7540643 TI - Negative regulators may mediate some of the inhibitory effects of HIV-1 infected stromal cell layers on erythropoiesis and myelopoiesis in human bone marrow long term cultures. AB - This report presents results concerning the potential role of negative regulators in hematopoietic suppression observed in human immunodeficiency virus (HIV) infected long-term cultures (LTC) of human bone marrow cells. Confluent stromal cell layers established from human bone marrow cells were exposed to HIV-1ADA, a monocytotropic strain of HIV-1. A progressive increase in the concentration of HIV-1 p24 antigen in cultures exposed to HIV-1ADA demonstrated that there was a productive infection. Cells from both noninfected and HIV-infected stromal cell layers produced factors that stimulated the proliferation of colony-forming units for granulocytes and macrophages (CFU-GM) from non-infected CD34+ cells. In contrast, when noninfected CD34+ cells were directly cocultured on intact stromal cell layers fewer CFU-GM and burst-forming units for erythroid cells (BFU-E) were detected in HIV-infected LTC than in noninfected LTC. One week after the addition of CD34+ cells, the number of CFU-GM in HIV-infected LTC in six of nine experiments was reduced compared to noninfected control LTC. In those six experiments, the number of CFU-GM was only 53 +/- 5% (SEM) of the number in noninfected LTC. The number of BFU-E in HIV-1-infected LTC was only 46 +/- 5% of the number in noninfected LTC (n = 5). There were fewer BFU-E in HIV-1-infected LTC, whether or not there was a reduced number of CFU-GM. Neutralizing antibody to tumor necrosis factor alpha (TNF-alpha) had no effect on the number of BFU-E in HIV-infected LTC. The number of BFU-E, however, was 2.1 +/- 0.2-fold greater (n = 3) in HIV-infected LTC incubated with neutralizing antibody to interferon alpha. In HIV-infected LTC with decreased numbers of CFU-GM, the number of CFU-GM was approximately 2-fold greater after incubation of HIV-infected LTC with anti interleukin-4 (IL-4). The effect of anti-TNF-alpha was variable, and anti transforming growth factor-beta had no effect on the number of CFU-GM in HIV infected LTC. After 2 weeks, the number of CFU-GM in HIV-infected LTC incubated with anti-IL-4 and anti-TNF-alpha was 2- to 4-fold greater than in untreated HIV infected LTC. Antibody treatment did not promote an increase in the number of CFU GM in noninfected LTC or in LTC in which CFU-GM numbers were not reduced after HIV infection.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7540642 TI - Monocytes cultured in cytokine-defined environments differ from freshly isolated monocytes in their responses to IL-4 and IL-10. AB - To investigate the regulatory effects of the prototypic Th2 lymphocyte products and potential immunotherapeutic agents interleukin-4 (IL-4) and IL-10 on macrophages differentiated in vitro under different cytokine-defined environments, blood monocytes were incubated for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony stimulating factor or IL-4. The effect of monocyte culture in the presence or absence of serum was also investigated. Functional responses by 7-day-cultured cells to IL-4, quantified as decreased CD14 expression and suppression of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta production, and as a positive response, increased CD23 expression, were compared directly with the responses by monocytes from which they were derived. In response to IL-10, decreases in LPS-induced TNF-alpha and IL-1 beta production and reduction in the expression of major histocompatibility complex (MHC) class II antigens were examined. Seven-day cultured monocytes/macrophages showed (1) diminished TNF-alpha production in response to IL-10 but not IL-4 (2), diminished IL-1 beta production in response to both IL-4 and IL-10, and compared with fresh monocytes (3), diminished CD14 expression in response to IL-4, and (4) a lesser increase in CD23 expression in response to IL-4. This was the case regardless of the cytokine in the presence of which the cells had been cultured for 7 days. Monocytes cultured for 7 days in GM-CSF expressed increased levels of MHC class II and LPS-induced TNF-alpha and responded inefficiently to IL-10 for decreased MHC class II. The responses by monocytes cultured for 7 days with GM-CSF resemble the published properties of synovial fluid macrophages from patients with chronic inflammatory arthritis. The study highlights the complexity of monocyte/macrophage responses to the immunoregulatory cytokines IL-4 and IL-10 and concludes that responses to IL-4 and IL-10 by blood monocytes may not be representative of responses by their differentiated or activated counterparts. PMID- 7540645 TI - Molecular alterations in the aging immune system. PMID- 7540646 TI - Prevention of age-related T cell apoptosis defect in CD2-fas-transgenic mice. AB - T cell dysfunction and thymic involution are major immunologic abnormalities associated with aging. Fas (CD95) is a bifunctional molecule that is critical for apoptosis and stimulation during T cell development, but the role of Fas during aging has not been determined. Fas expression and function on T cells from old (22-26-mo-old) mice was compared with young (2-mo-old) mice and old CD2-fas transgenic mice. Fas expression and ligand-induced apoptosis were decreased on T cells from old mice compared with young mice. This correlated with an age-related increase in CD44+Fas- T cells. There was a marked decrease in the proliferation of T cells from old mice after anti-CD3 stimulation compared with young mice. Anti-CD3-stimulated T cells from young mice exhibited increased production of interleukin (IL)-2 and decreased production of interferon-gamma and IL-10 compared with old mice. There was an age-related decrease in the total thymocyte count from 127 +/- 10 cells in young mice compared with 26 +/- 8 x 10(6) in old mice. In 26-mo-old CD2-fas-transgenic mice, Fas and CD44 expression, Fas-induced apoptosis, T cell proliferation, and cytokine production were comparable to that of the young mice. These results suggest that T cell senescence with age is associated with defective apoptosis, and that the CD2-fas transgene allows maintenance of Fas apoptosis function and T cell function in aged mice comparable to that of young mice. PMID- 7540648 TI - Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer. AB - The major target of the neutralizing antibody response to infection by the human immunodeficiency virus type 1 (HIV-1) is the outer envelope glycoprotein, gp120. The spectrum of HIV-1 neutralization specificity is currently represented by monoclonal antibodies (mAbs) that can be divided broadly into five groups. We have studied the binding of these mAbs to functional oligomeric and soluble monomeric gp120 derived from the molecular clone of a cell line-adapted isolate of HIV-1, and compared these binding properties with virus neutralization. Binding of all mAbs except those reactive with the V3 loop was much weaker to oligomeric than to monomeric gp120. This reduction in binding to oligomeric gp120 was determined mostly by a slower relative rate of association, although the dissociation rate also had some influence on relative variation in mAb affinity. Virus neutralization correlated broadly with mAb binding to the oligomeric rather than to the monomeric form of gp120, and neutralization potency was related to the estimated association rate. Thus, with the exception of the hypervariable V3 loop, regions of HIV-1 gp120 with the potential to induce a neutralization response are likely to be poorly presented for antibody recognition on the surface of cell line-adapted virions. PMID- 7540647 TI - Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. AB - Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis. PMID- 7540649 TI - The immediate phase of c-kit ligand stimulation of mouse bone marrow-derived mast cells elicits rapid leukotriene C4 generation through posttranslational activation of cytosolic phospholipase A2 and 5-lipoxygenase. AB - c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5 lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2. PMID- 7540651 TI - T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus. AB - Changes in T cell receptor (TCR) V beta repertoire and their correlation with virologic events were investigated in rhesus monkeys after acute infection with the simian immunodeficiency virus (SIV). 11 genetically defined rhesus monkeys were experimentally infected with SIVmac or a chimeric simian-human immunodeficiency virus (SHIV), and their peripheral blood lymphocytes (PBL) and lymph nodes were prospectively assessed for TCR V beta gene expression. PBL and lymph nodes of the acutely infected monkeys demonstrated an expansion of selected V beta-expressing T lymphocyte subpopulations as early as 3 d after infection. These expanded V beta-expressing lymphocyte subpopulations were comprised predominantly of CD8+ cells. Six of seven infected monkeys sharing a single electrophoretically defined major histocompatibility complex class I allele exhibited a similar expansion of V beta 14-expressing PBL. Sequence analyses of V D-J segments of TCR-beta cDNA indicated that the V beta-expressing T cell subpopulation expansion can be oligoclonal. SIVmac-specific CD8+ cytotoxic T lymphocytes were demonstrated in both PBL and lymph nodes of the infected monkeys at the time expansion of the selected V beta-expressing cell subpopulations was seen. Finally, the expansion of the selected V beta-expressing lymphocytes in PBL coincided with the emergence and clearance of SIV p27 from the plasma of the infected monkeys. These results demonstrate that acute infection of rhesus monkeys with SIVmac or SHIV results in an expansion of CD8+ lymphocyte subpopulations expressing selected V beta gene families. The selectively expanded T lymphocytes may contribute to early viral clearance after acute SIVmac or SHIV infection. PMID- 7540652 TI - T cells with gamma/delta T cell receptors (TCR) of intestinal type are preferentially expanded in TCR-alpha-deficient lpr mice. AB - Fas-mediated apoptosis is essential for activation-induced cell death of alpha/beta T cells, but it is not clear what role, if any, it plays in regulating other components of the immune system. To study the role of Fas in gamma/delta T cell development, Fas-deficient lpr mice were bred with T cell receptor alpha gene-ablated (TCR-alpha-/-) mice to generate mice deficient in one or both genes. The TCR-alpha-/-, lpr/lpr mice had a nearly 10-fold increase in total lymph node cell (LNC) number compared with Fas-intact TCR-alpha-/- mice, because of expansion of TCR-gamma/delta+ and TCR-beta+ cells. In Fas-intact TCR-alpha-/- mice, approximately one third of the LNCs expressed TCR-gamma/delta. These were evenly divided between the CD4-, CD8-alpha+ and the CD4-, CD8- subsets, and rarely expressed the B220 epitope of CD45. In contrast, in TCR-alpha-/-, lpr/lpr mice, TCR-gamma/delta+ cells comprised half of the LNCs and were primarily CD4-, CD8-, and B220+. Moreover, Fas deficiency in TCR-alpha-/- mice caused a preferential expansion of gamma/delta T cells expressing variable region genes characteristic of intestinal intraepithelial lymphocytes. These results demonstrate a role for Fas in regulating the gamma/delta T cell contribution to peripheral lymph nodes. This mechanism may be most important in limiting the access of activated intestinal intraepithelial lymphocytes to the peripheral lymphoid system. PMID- 7540653 TI - Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes. AB - It has previously been shown that bone marrow-generated dendritic cells (DC) are potent stimulators in allogeneic mixed leukocyte reactions and are capable of activating naive CD4+ T cells in situ in an antigen-specific manner. In this study we have investigated whether bone marrow-generated DC are capable of inducing antigen-specific CD8+ T cell responses in vivo. Initial attempts to induce specific cytotoxic T lymphocyte (CTL) responses in mice injected with bone marrow-generated DC pulsed with ovalbumin (OVA) peptide were frustrated by the presence of high levels of nonspecific lytic activity, which obscured, though not completely, the presence of Ag-specific CTL. Using conditions that effectively differentiate between antigen-specific and nonspecific lytic activity, we have shown that bone marrow-generated DC pulsed with OVA peptide are potent inducers of OVA-specific CTL responses in vivo, compared with splenocytes or RMA-S cells pulsed with OVA peptide, or compared with immunization with free OVA peptide mixed with adjuvant. Antibody-mediated depletion experiments have shown that the cytotoxic effector cells consist primarily of CD8+ cells, and that induction of CTL in vivo is dependent on CD4+ as well as on CD8+ T cells. These results provide the basis for exploring the role of bone marrow-generated DC in major histocompatibility class I-restricted immune responses, and they provide the rationale for using bone marrow-generated DC in CTL-mediated immunotherapy of cancer and infectious diseases. PMID- 7540650 TI - N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation. AB - Although the classical chemotactic receptor for complement anaphylatoxin C5a has been associated with polymorphonuclear and mononuclear phagocytes, several recent studies have indicated that this receptor is expressed on nonmyeloid cells including human endothelial cells, vascular smooth muscle cells, bronchial and alveolar epithelial cells, hepatocytes, and in the human hepatoma cell line HepG2. In this study, we examined the possibility that other members of the chemotactic receptor family are expressed in HepG2 cells and human liver, and the possibility that such receptors mediate changes in acute phase gene expression in HepG2 cells. Using polymerase chain reaction (PCR) amplification of HepG2 mRNA with primers based on highly conserved regions of the chemotactic subgroup of the G protein-coupled receptor family, we identified a PCR fragment from the formyl methionyl-leucyl-phenylalanine (FMLP) receptor, as well as one from the C5a receptor. Immunostaining with antipeptide antisera to FMLPR confirmed the presence of this receptor in HepG2 cells. Receptor binding studies showed specific saturable binding of a radioiodinated FMLP analogue to HepG2 cells (Kd approximately 2.47 nM; R approximately 6 x 10(3) plasma membrane receptors per cell). In situ hybridization analysis showed the presence of FMLPR mRNA in parenchymal cells of the human liver in vivo. Both C5a and FMLP mediated concentration- and time-dependent changes in synthesis of acute phase proteins in HepG2 cells including increases in complement C3, factor B, and alpha 1 antichymotrypsin, as well as concomitant decreases in albumin and transferrin synthesis. The effects of C5a and FMLP on the synthesis of these acute phase proteins was evident at concentrations as low as 1 nM, and they were specifically blocked by antipeptide antisera for the corresponding receptor. In contrast to the effect of other mediators of hepatic acute phase gene regulation, such as interleukin 6, the effects of C5a and FMLP were reversed by increased concentrations well above the saturation point of the respective receptor. These results suggest that acute phase gene regulation by C5a and FMLP is desensitized at high concentrations, a property that is unique among the several known mechanisms for hepatic acute phase gene regulation. PMID- 7540655 TI - Expression of functional CD40 by vascular endothelial cells. AB - The interaction between activated vascular endothelium and T cells has been shown to play an important role in the recruitment and activation of T cells at sites of inflammation. Here we report the expression of CD40 by vascular endothelial cells and its regulation by inflammatory agents. Using the soluble recombinant CD40 ligand, sgp39, we show that the interaction of CD40 with its ligand can lead to endothelial cell activation, which in turn leads to leukocyte adhesion. This adhesion is partly mediated by the expression of E-selectin. In addition to E selectin expression, sgp39 induces the expression of intercellular adhesion molecule 1 and augments the tumor necrosis factor alpha-induced expression of vascular cell adhesion molecule 1. The effects of sgp39 on endothelial cells can be blocked with anti-gp39 monoclonal antibody (mAb), anti-CD40 mAb, or soluble CD40. Staining of tissues from healthy human skin using anti-CD40 mAb showed very weak expression of CD40 by the endothelium, while skin involved in inflammatory disease showed marked upregulation of CD40 expression. These studies suggest that interactions between cell surface proteins expressed by activated T cells with their receptors on vascular endothelium can stimulate the vasculature at sites of inflammation and may be involved in normal inflammatory responses and in inflammatory disease. PMID- 7540654 TI - T cell priming versus T cell tolerance induced by synthetic peptides. AB - It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33 41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases. PMID- 7540656 TI - Differentiation in vitro of hybrid eosinophil/basophil granulocytes: autocrine function of an eosinophil developmental intermediate. AB - Granulocytes with the hybrid characteristics of eosinophils and basophils have been identified in the bone marrow and peripheral blood of humans with myeloid leukemias. We now describe a technique by which such hybrid granulocytes can be developed in vitro from normal cord blood precursors cultured in the presence of recombinant human interleukin (rhIL) 3 (350 pM) and rhIL-5 (200 pM) in a plastic vessel coated with Matrigel. After 14 d in culture, 90 +/- 3% (mean +/- standard error of the mean) of the nonadherent cells cultured in the Matrigel-coated flasks contained both eosinophil and basophil granules, as indicated by staining with Wright's and Giemsa stains. Of the nonadherent cells, 93 +/- 1% contained cyanide-resistant peroxidase, and 88 +/- 2% were toluidine blue-positive, characteristic of eosinophil and basophil granules, respectively. Transmission electron micrographs showed hybrid cells containing ultrastructurally distinct eosinophil granules with developing crystalline cores and basophil granules with reticular structures. These 14-d cord blood-derived cell cultures showed strong hybridization signals for eosinophil-derived neurotoxin by RNA blot analysis and contained 78 ng histamine per 10(6) cells. When the granulocytes were removed from cytokine-containing medium and suspended without Matrigel in RPMI 1640 medium containing 10% fetal calf serum (FCS), more than 80% of the granulocytes excluded trypan blue for as long as 5 d, and 93% had developed into eosinophils at 6 d. Conditioned medium prepared over 48 h from the 14-d cell cultures (hybrid granulocytes) sustained the 4-d viability in vitro of 78% of peripheral blood eosinophils from atopic donors. In comparison, 13% survived in RPMI 1640 containing 10% FCS alone. This viability-sustaining activity was nearly completely neutralized by an anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody and was only minimally reduced by anti-IL-3 or IL-5. Thus, cells possessing both eosinophil and basophil granules by both histochemical and ultrastructural analysis can be developed from normal progenitors in vitro in response to eosinophilopoietic cytokines and Matrigel. Their subsequent spontaneous development into mature eosinophils suggests that hybrid granulocytes are part of a normal developmental sequence during eosinophilopoiesis. Furthermore, these hybrid granulocytes are capable of autoregulation through elaboration of GM-CSF, which sustains their viability. PMID- 7540657 TI - GD2 oligosaccharide: target for cytotoxic T lymphocytes. AB - Carbohydrate antigens rarely provide target epitopes for cytotoxic T lymphocytes (CTL). Disialoganglioside GD2 is a glycolipid expressed at high levels in human tumors and a small group of murine lymphomas (EL4, RBL5, RMA, RMA-S, A13, and BALBRVE). Immunization of C57B1/6 mice with irradiated EL4 cells stimulated a specific CTL response and protected these animals from engraftment of EL4 lymphoma. The CTL activity resided in the CD4-CD8+ population, was dependent on T cell receptor alpha/beta, and was not removed by anti-natural killer cell immunoabsorption, but was restricted to GD2 and H-2b bearing targets. CTL activity could be completely inhibited by GD2-oligosaccharide-specific monoclonal antibodies and their F(ab')2 fragments, but not by immunoglobulin G3 myelomas or antibodies against GD3 or GM2. Soluble GD2 did not inhibit specific tumor lysis. RMA-S lymphoma cells (GD2+H-2b-TAP2 deficient) were resistant to GD2-specific CTL. Sialic acid-containing peptides eluted from EL4 lymphoma cells could (a) stabilize H-2 molecules on RMA-S cells and (b) sensitize them for GD2-specific CTL. Control peptides (derived from vesicular stomatitis virus nucleoprotein peptide and GD2-negative lymphomas) could also stabilize H-2 on RMA-S, but were resistant to GD2-specific CTL. These H-2-binding peptides could be purified by anti-GD2 affinity chromatography. We postulate a new class of naturally occurring epitopes for T cells where branched-chain oligosaccharides are linked to peptides with anchoring motifs for the major histocompatibility complex class I pocket. While analogous to the haptens trinitrophenyl and O-beta-linked acetyl glucosamine, the potential implications of natural carbohydrates as antigenic epitopes for CTL in biology are considerable. PMID- 7540659 TI - Anomalous increase in nitric oxide synthase activity by certain nitric oxide generating compounds in intact neuronal cells. AB - It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated L-[3H]citrulline formation in a time- and concentration dependent manner when cells were incubated with the compounds for > 1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca2+, suggesting that it might be due to increased Ca2+ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540658 TI - Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. AB - The role of epitope spreading in the pathology of relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) was examined. Using peripherally induced immunologic tolerance as a probe to analyze the neuropathologic T cell repertoire, we show that the majority of the immunopathologic reactivity during the acute phase of R-EAE in SJL/J mice induced by active immunization with the intact proteolipid (PLP) molecule is directed at the PLP139-151 epitope and that responses to secondary encephalitogenic PLP epitopes may contribute to the later relapsing phases of disease. Intermolecular epitope spreading was demonstrated by showing the development of T cell responses to PLP139-151 after acute disease in mice in which R-EAE was initiated by the transfer of T cells specific for the non-cross-reactive MBP84-104 determinant. Intramolecular epitope spreading was demonstrated by showing that endogenous host T cells specific for a secondary encephalitogenic PLP epitope (PLP178-191) are demonstrable by both splenic T cell proliferative and in vivo delayed-type hypersensitivity responses in mice in which acute central nervous system damage was initiated by T cells reactive with the immunodominant, non-cross-reactive PLP139-151 sequence. The PLP178-191-specific responses are activated as a result of and correlate with the degree of acute tissue damage, since they do not develop in mice tolerized to the initiating epitope before expression of acute disease. Most importantly, we show that the PLP178-191-specific responses are capable of mediating R-EAE upon adoptive secondary transfer to naive recipient mice. Furthermore, induction of tolerance to intact PLP (which inhibits responses to both the initiating PLP139-151 epitope and to the PLP178-191 epitope) after the acute disease episode is sufficient to prevent relapsing disease. These results strongly support a contributory role of T cell responses to epitopes released as a result of acute tissue damage to the immunopathogenesis of relapsing clinical episodes and have important implications for the design of antigen-specific immunotherapies for the treatment of chronic autoimmune disorders in humans. PMID- 7540660 TI - Ethanol inhibition of recombinant heteromeric NMDA channels in the presence and absence of modulators. AB - The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25-100 mM. We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady-state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg2+ (3 and 12.5 microM), Zn2+ (1 and 10 microM), or the glycine antagonist 7-chlorokynurenic acid (1.25 or 5 microM), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 mM) or 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor. PMID- 7540661 TI - The second messenger, cyclic AMP, is not sufficient for myelin gene induction in the peripheral nervous system. AB - The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8-10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P0 mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P0 gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath. PMID- 7540662 TI - Phospholipase A2 down-regulates the affinity of [3H]AMPA binding to rat cortical membranes. AB - The effects of exogenous phospholipase A2 on the binding of alpha-[3H]amino-3 hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A2 (sPLA2) elicited a concentration dependent decrease in specific [3H]AMPA binding due mainly to a decrease in affinity (KD). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA2 effect is not due to proteolysis. The sPLA2 evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA2 inhibition. These results suggest that the effects of sPLA2 arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA2 action, did not cause a similar decrease in the affinity of [3H]AMPA binding. In contrast to the effects on [3H]AMPA binding, sPLA2 caused an increase in [3H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA2 may play a role in the physiological and pathophysiological regulation of AMPA receptors. PMID- 7540665 TI - Sympathetic sprouting: time course of changes of noradrenergic, cholinergic, and serotonergic markers in the denervated rat hippocampus. AB - As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria-fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high-affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5-hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1-2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5-hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near-normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear-cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high-affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540663 TI - Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats. AB - The effects of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-[[(S)-2 (hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl) - 1 pyrrolidinecarboxamide (JTP-4819), on the PEP activity in the brain and on the contents of substance P (SP)- and arginine-vasopressin (AVP)-like immunoreactivity (LI) in the cerebral cortex and hippocampus of young and aged rats were investigated using enzyme immunoassay. JTP-4819 exhibited a concentration-dependent in vitro inhibitory action on PEP activity in the brains of both young and aged rats, with IC50 values of approximately 0.7 and 0.8 nM, respectively. A single dose of JTP-4819 (3 mg/kg, p.o.) increased the SPLI content in the cerebral cortex but not the hippocampus of aged rats (23-24 months old). In addition, repeated administration of JTP-4819 (1 mg/kg, p.o., for 21 days) increased the SPLI content in the cerebral cortex and restored the SPLI content in the hippocampus, which had decreased with aging. In contrast, single (1 mg/kg, p.o.) and repeated (1 mg/kg, p.o., for 21 days) administration of JTP 4819 only tended to increase the AVPLI content of the hippocampus and cerebral cortex in aged rats, respectively. These results indicate that JTP-4819 increases the cerebral and hippocampal SPLI content in aged rats by inhibiting the action of PEP. PMID- 7540666 TI - Neurokinin receptors could be differentiated by their capacity to respond to the transglutaminase-synthesized gamma-(glutamyl5)spermine derivative of substance P. AB - Four different gamma-(glutamyl5)amine derivatives of substance P (SP) were synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The 1,3-diaminopropane, spermidine, spermine (Spm), and monodansylcadaverine adducts of the neuropeptide were purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The gamma-(glutamyl5)Spm derivative of SP (Spm-SP) was found to be able, like the parent neuropeptide, to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure, and to inhibit collagen-induced platelet aggregation. Unlike SP, only a weak inflammatory response was observed when Spm SP was injected in the rat hind limb. All these effects were found to be prevented by N omega-nitro-L-arginine methyl ester, a well-known nitric oxide synthesis inhibitor. In contrast, Spm-SP was completely ineffective in contracting guinea pig ileal segments, thus confirming our preliminary observations indicating that Spm-SP does not evoke SP-like spasmogenic effects on isolated smooth muscle preparations. The specificity of the effects due to the selective introduction of a Spm moiety at the glutamine5 level was demonstrated by the SP agonist pharmacological profile of the other gamma-(glutamyl5)amine derivatives tested. These results suggest that neurokinin receptors could be differentiated by their capacity to respond to Spm-SP. PMID- 7540667 TI - Characterization of nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies. AB - Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila. PMID- 7540664 TI - Characterization of pharmacologically active anti-peptide antibodies directed against the first and second extracellular loops of the serotonin 5-HT1A receptor. AB - The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96-111), Ab-2 (loop II; sequence G-W-R T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173-193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT1A receptor (KDH = 0.54 +/- 0.09 nM and KDL = 13.74 +/- 4.9 nM) for the agonist 8-hydroxy-2-(di-n [3H]propylamino) tetralin ([3H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (KD of approximately 2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the Bmax of the [3H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket. PMID- 7540669 TI - R-cadherin expression during nucleus formation in chicken forebrain neuromeres. AB - The primordial neuroepithelium of the vertebrate forebrain consists of transverse and longitudinal morphogenetic compartments ("neuromeres"). During development, neurons born in the ventricular zone of each neuromere migrate outward to the mantle zone. Here, neuroblasts gradually accumulate and aggregate either into sheets ("laminae") or into roundish structures ("nuclei"). As brain architecture matures, sets of nuclei and laminae derived from several neuromeres become connected by fiber tracts to form functional circuits. We show by immunostaining and in situ hybridization techniques that, in the E3-E5 chicken embryo, the cell adhesion molecule R-cadherin is expressed in several stripes and patches in the forebrain neuroepithelium. This expression pattern reflects, at least in part, the neuromeric organization of the forebrain. For example, in both the ventral and dorsal thalamus, R-cadherin expression has a sharp border at the respective caudal neuromere boundary. Moreover, focusing on the mid-hypothalamic region, we demonstrate that a subset of postmitotic neuroblasts in the ventricular zone express R-cadherin during their migration to the mantle zone, where they aggregate into particular nuclei. In the mantle zone, R-cadherin-expressing neuroblasts accumulate in parallel with neuroblasts expressing another cadherin, N-cadherin. The two types of cells segregate from each other to form adjacent nuclei. Some of the R- and the N-cadherin-positive nuclei form parts of particular functional circuits in the mature brain. In conclusion, our results suggest that cadherins play a role in the formation of brain nuclei and in the developmental transformation from neuromeric to functional organization in the vertebrate forebrain. PMID- 7540670 TI - Extent of intracortical arborization of thalamocortical axons as a determinant of representational plasticity in monkey somatic sensory cortex. AB - The extent of intracortical arborization of individual thalamocortical axons in area 3b of the somatic sensory cortex and the degree of overlap in the cortical projections of relay cells in the ventral posterior nucleus of the thalamus were examined in macaque monkeys. Paired intracortical deposits of Fast blue (FB) and Diamidino yellow (DY) separated by 100-1500 microns were made by inserting crystals of dye into the tracks of tungsten microelectrodes used to record receptive field data on area 3b cells. Each injection gave retrograde labeling of one or more clusters of cells extending in elongated anteroposterior arrays through the ventral posterior medial (VPM) or ventral posterior lateral (VPL) nucleus. Double-labeled cells were only found when the distance between the centers of the dye deposits was less than 600 microns. With interdeposit distances greater than 600 microns, most clusters of retrogradely labeled cells had a majority of cells labeled by FB or DY. However, even with interdeposit distances of 1-1.5 mm the labeled clusters also contained significant numbers of cells labeled with the other dye. These results and an accompanying regression analysis indicate that the extent of intracortical arborization of single thalamocortical axons in area 3b is no greater than 600 microns. However, adjoining cells in the same part of the thalamic body representation can project to cortical targets as discrepant as 1.5 mm. It is proposed that the fine grain of the cortical representation depends upon inputs from the majority population of each thalamic cell cluster.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540668 TI - Characterization of L-arginine and aminoguanidine uptake into isolated rat choroid plexus: differences in uptake mechanisms and inhibition by nitric oxide synthase inhibitors. AB - Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N-methyl-D-aspartate mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of L-[14C]arginine and the NOS inhibitor [14C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of L-[14C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors NG-methyl-L-arginine, NG-amino-L-arginine, and N5-imidoethyl-L ornithine. L-[14C]Arginine uptake was not inhibited by aminoguanidine or NG-nitro L-arginine. Because aminoguanidine is an organic cation that bears some structural similarity to L-arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [14C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for L-[14C]arginine. PMID- 7540671 TI - mu-Opioid receptor activation reduces multiple components of high-threshold calcium current in rat sensory neurons. AB - Whole-cell patch-clamp recordings were used to characterize calcium channel types that are modulated by mu-opioid receptor activation in rat dorsal root ganglion (DRG) neurons. Five distinct components of high-threshold calcium current were isolated on the basis of their sensitivity to the selective channel blockers omega-conotoxin GVIA, nifedipine, omega-conotoxin MVIIC, or omega-agatoxin IVA. The mu-opioid selective agonist Tyr-Pro-NMePhe-D-Pro-NH2 (PLO17) routinely suppressed high-threshold currents and this effect was always reduced by omega conotoxin GVIA. A fraction of PLO17-sensitive current remained after omega conotoxin GVIA that was eliminated by application of omega-agatoxin IVA alone or in combination with omega-conotoxin MVIIC. Nifedipine had no effect on mu-opioid responses nor did PLO17 affect the slow component of tail current induced by Bay K 8644. These data suggest that mu-opioid receptors are negatively coupled to three types of calcium channels in rat DRG neurons, including an omega-conotoxin GVIA-sensitive (N-type) channel, an omega-agatoxin IVA-sensitive (P-type) channel and an omega-conotoxin MVIIC-sensitive, nifedipine/GVIA/omega-Aga IVA-resistant (presumptive Q-type) channel. PMID- 7540672 TI - Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. AB - Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse. PMID- 7540673 TI - Effects of proctolin on contractions, membrane resistance, and non-voltage dependent sarcolemmal ion channels in crustacean muscle fibers. AB - The neuropeptide proctolin in nanomolar concentrations enhances the contraction of crustacean muscle fibers manyfold. The cellular mechanisms underlying this potentiation were investigated in single, isolated, fast-contracting abdominal extensor muscle fibers of a small crustacean, the marine isopod Idotea baltica. Force measurements and current-clamp experiments revealed two actions of proctolin on the muscle fibers. In half of the preparations, proctolin (10(-9) 10(-6) M) increased the fiber's input resistance by up to 25%. In about one fourth of the preparations, proctolin induced all-or-none action potentials in response to depolarizing current pulses in muscle fibers that showed graded electric responses under control conditions. In both cases, proctolin potentiated the peak force of muscle contractions (between 1.5- and 18-fold for 5 x 10(-9) M proctolin). Proctolin affected neither the membrane resting potential nor the threshold for excitation-contraction coupling. Using cell-attached patches on the sarcolemmal membrane, we identified non-voltage-dependent ion channels which contribute to the passive membrane properties of the muscle fibers. A 53 +/- 6 pS channel had its reversal potential near rest and carried outward current at depolarized potentials with physiological saline in the recording pipette. With isotonic K+ saline in the patch pipette, the reversal potential was +85 +/- 12 mV depolarized from the resting potential and single-channel conductances ranged from 36 to 166 pS. Proctolin modulated the activity of all these putative K+ channels by reducing the number of functionally active channels. The effects of proctolin on force of contraction, input resistance, and single-channel activity were mimicked by a membrane-permeating analog of cAMP. Conversely, a monothio analog of cAMP (Rp-cAMPS), a blocker of protein kinase A activity, substantially decreased the membrane input resistance of the muscle fibers. The results suggest that activation of the cAMP signal pathway and phosphorylation of non-voltage dependent K+ channels by protein kinase A are involved in the potentiation of contractions by proctolin in the muscle fibers of this crustacean. PMID- 7540674 TI - Androgen alters the dendritic arbors of SNB motoneurons by acting upon their target muscles. AB - In adult male rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) have been shown to retract and reextend their dendritic branches in response to systemic androgen deprivation and readministration. Furthermore, other studies have suggested that the dendritic complexity of neurons can be regulated by their targets. To assess whether androgens might act upon the target muscles to mediate changes in SNB dendrites, adult male rats were castrated and implanted with a small capsule filled with testosterone (T) next to the bulbocavernosus and levator ani muscle complex (BC/LA) on one side, while the muscles on the contralateral side were implanted with another capsule containing hydroxyflutamide (hFl), an anti-androgen. We have previously shown that after 30 d of this focused, lateralized androgen treatment the BC/LA complex is significantly larger on the T-treated side. We now report that the total dendritic lengths of SNB motoneurons innervating muscles given androgen blockade are reduced by 44% compared to SNB motoneurons innervating muscles given androgen stimulation. Dendrite lengths within three regions of the spinal cord were altered in a nonuniform manner: large changes occurred in the dorsal and contralateral dendritic fields while there was no difference in the ipsilateral dendritic field. These results suggest that BC/LA muscles, in response to androgen stimulation, produce a trophic substance which regulates the dendritic organization of SNB motoneurons in adulthood. PMID- 7540675 TI - Topography of visual cortex connections with frontal eye field in macaque: convergence and segregation of processing streams. AB - The primate visual system consists of at least two processing streams, one passing ventrally into temporal cortex that is responsible for object vision, and the other running dorsally into parietal cortex that is responsible for spatial vision. How information from these two streams is combined for perception and action is not understood. Visually guided eye movements require information about both feature identity and location, so we investigated the topographic organization of visual cortex connections with frontal eye field (FEF), the final stage of cortical processing for saccadic eye movements. Multiple anatomical tracers were placed either in parietal and temporal cortex or in different parts of FEF in individual macaque monkeys. Convergence from the dorsal and ventral processing streams occurred in lateral FEF but not in medial FEF. Certain extrastriate areas with retinotopic visual field organizations projected topographically onto FEF. The dorsal bank of the superior temporal sulcus projected to medial FEF; the ventral bank, to lateral FEF, and the fundus, throughout FEF. Thus, lateral FEF, which is responsible for generating short saccades, receives visual afferents from the foveal representation in retinotopically organized areas, from areas that represent central vision in inferotemporal cortex and from other areas having no retinotopic order. In contrast, medial FEF, which is responsible for generating longer saccades, is innervated by the peripheral representation of retinotopically organized areas, from areas that emphasize peripheral vision or are multimodal and from other areas that have no retinotopic order or are auditory. PMID- 7540676 TI - Mice doubly deficient in the genes for P0 and myelin basic protein show that both proteins contribute to the formation of the major dense line in peripheral nerve myelin. AB - In search for the molecular mechanisms underlying the formation of the major dense line in peripheral nerve myelin we investigated mice deficient in the myelin proteins P0 and MBP. In mice lacking both molecules axons were enwrapped by myelin-like processes devoid of the major dense line, while mice deficient in either protein showed, respectively, partial and normal compaction. Mice heterozygous for P0 but devoid of MBP showed myelin of reduced thickness around axons of normal caliber. Both molecules thus contribute to the formation of the major dense line and to the determination of myelin thickness. Furthermore, our observations modify the view that axon caliber is dependent on normal myelin. PMID- 7540677 TI - On the linkage between AMPA and NMDA receptor-mediated EPSPs in homosynaptic long term depression in the hippocampal CA1 region of young rats. AB - Homosynaptic long-term depression (LTD) was studied in hippocampal slices from 12 18-d-old rats using field EPSP recording in the apical dendritic layer of CA1 pyramidal cells. Independent estimates of the alpha-amino-3-hydroxy-5 methylisoxazolepropionic acid (AMPA) and the N-methyl-D-aspartic acid (NMDA) receptor-mediated components of the field EPSP were obtained in parallel using early and late measurements of a dual-component EPSP in a low-magnesium solution. LTD was induced by low-frequency stimulation (LFS; 2 Hz for 10 min), resulting in equal relative changes of the AMPA and NMDA receptor-mediated components. Under conditions when the AMPA receptor-mediated component was fully blocked, a similarly sized LTD was observed for the pure NMDA receptor-mediated EPSP (measured as initial slope or peak amplitude). Equal changes in AMPA and NMDA receptor-mediated components occurred also upon application of the adenosine agonist N6-cyclohexyladenosine (CHA), known to act by decreasing transmitter release. On the other hand, LTD was found to interact in a multiplicative manner with the presynaptic release changes induced by CHA and by paired-pulse facilitation. The induction of the LTDs of both AMPA and NMDA receptor-mediated EPSPs was blocked by the NMDA receptor antagonist D(-)-2-amino-5 phosphonopentanoic acid and by the phosphatase inhibitor okadaic acid. Partial blockade of LTD by okadaic acid resulted in equal partial blockade of the LTDs of the AMPA and NMDA receptor-mediated components. On the other hand, the L-type calcium channel blocker nifedipine, the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine, the nitric oxide synthase inhibitor N omega-nitro-L-arginine, and the heme oxygenase inhibitor protoporphyrin IX zinc(II) had no effect on LTD of either the AMPA or the NMDA receptor-mediated component. These results of equal changes of AMPA and NMDA receptor-mediated components of the field EPSP in association with LTD, and the consistent parallelism of effects or noneffects on these components by various receptor antagonists and enzyme inhibitors, seem more easily explained by a presynaptic locus for LTD than by a postsynaptic one. PMID- 7540679 TI - Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity. AB - We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity. PMID- 7540680 TI - The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin. AB - Dynorphin is one of the endogenous opioids that modulates the excitability of nociceptive (pain-sensing) neurons. We have shown recently that dynorphin blocks NMDA-activated currents directly without the participation of kappa-opioid receptors. In order to understand the mechanism underlying this novel action of dynorphin, we examined, in detail, the interactions between dynorphin and NMDA receptors in isolated trigeminal neurons. Dynorphin reversibly blocks NMDA activated current (INMDA). The onset and recovery of the block were determined with concentration jump experiments. The association rate (k+) of dynorphin(1-17) is 4.9 x 10(6) sec-1 M-1 and the dissociation rate (k-) is 7.5 sec-1. The apparent dissociation constant (KD) of dynorphin, calculated from these rate constants, is 1.6 microM. Dynorphin does not change the EC50 of NMDA, nor the potentiating action of glycine. The binding site for dynorphin is distinct from that of Zn2+ or H+. Upon treatment with the disulfide reducing agent dithiothreitol (DTT), NMDA receptors become less susceptible to dynorphin block. The affinity of dynorphin for the modified NMDA receptors is reduced by 2.7-fold. In analyses of single NMDA channels in cell-free patches, we found that dynorphin shortens the mean open time, decreases the probability of opening of NMDA channels, but has no effect on the single channel conductance. These results suggest that dynorphin interacts with a site conformationally linked with the redox site(s) on the NMDA receptor, thus altering the gating properties of the channel. PMID- 7540678 TI - AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity. AB - Cerebellar Purkinje cells are selectively vulnerable to ischemia, although the reasons for this are unknown. In cultured embryonic rat cerebellar neurons, the steady state responses to the desensitizing agonist AMPA relative to responses to the nondesensitizing agonist kainate were greater in Purkinje cells compared to other cells, as measured by whole cell voltage clamp studies. Fluorimetric [Ca2+]i imaging experiments similarly found greater responses to AMPA relative to kainate in Purkinje cells than in other cerebellar neurons. In toxicity experiments measuring cell survival 24 hr following agonist exposure, AMPA and glutamate produced Ca(2+)-dependent toxicity which was selective for the Purkinje cell fraction of the neurons, whereas kainate produced nonselective toxicity, and NMDA selectively spared the mature Purkinje cells. Cyclothiazide, which inhibits AMPA receptor desensitization, enhanced steady state current responses to AMPA and increased the toxicity of AMPA. We conclude that the vulnerability of cerebellar neurons in culture to glutamate agonist-induced toxicity parallels the magnitude of the steady state currents produced, and that Purkinje cells may be selectively vulnerable because they express AMPA receptors which undergo less complete desensitization. PMID- 7540682 TI - Inputs to combination-sensitive neurons in the medial geniculate body of the mustached bat: the missing fundamental. AB - This study examined projections to combination-sensitive neurons in the medial geniculate body of the mustached bat. These specialized neurons respond to the combination of two temporally and spectrally distinct components of the bat's sonar pulse and echo, encoding target information. Combination-sensitive neurons respond to the bat's sonar fundamental, between 24-31 kHz, in conjunction with a higher harmonic signal. They are thought to be formed in the medial geniculate body (MGB) by convergent input from inferior colliculus representations of 24-31 kHz and higher frequencies. This study used anterograde and retrograde tract tracing methods in conjunction with physiological recording to test this MGB convergence hypothesis. In anterograde tracing experiments, multiple deposits of two different tracers were placed in the central nucleus of the inferior colliculus (ICC), one tracer in the 24-31 kHz region and another in an ICC representation responding to a higher sonar harmonic. We found only limited overlap in the MGB labeling patterns of the two tracers, and little in many areas where combination-sensitive neurons are common. In retrograde tracing experiments, a single deposit of tracer was placed at a combination-sensitive recording site in the MGB. With the deposit mostly limited to combination sensitive MGB areas, labeling in 24-31 kHz representations of the ICC was absent or minor. These results suggest that many combination-sensitive neurons in the MGB do not receive 24-31 kHz ICC input. The strongest inputs to combination sensitive MGB regions originate in high-frequency representations of the ICC and combination-sensitive regions of auditory cortex. Additional projections arrive from the thalamic reticular nucleus, external nucleus of the inferior colliculus, and pericollicular tegmentum. Each projection may contribute to the 24-31 kHz sensitivity of combination-sensitive neurons in the medial geniculate body. PMID- 7540681 TI - The optic tract and tectal ablation influence the composition of neurofilaments in regenerating optic axons of Xenopus laevis. AB - Neurofilaments have been proposed to regulate axonal stability and diameter through changes in number and subunit composition. We have found that pathway and target innervation directly influence the molecular composition of neurofilaments within regenerating optic axons of Xenopus laevis. Immunocytochemistry was used to examine neurofilaments within two abnormal visual pathways. The first was an aberrant, transient retinoretinal projection, which formed when some axons entered the contralateral optic nerve at the chiasm. The second was formed by regenerating axons deprived of their normal targets by surgical ablation of both optic tecta. Distal to an orbital nerve crush, the neurofilament proteins NF-L, NF-M, NF-H, and XNIF disappear from degenerating fibers. In normally regenerating axons, these neurofilament proteins emerge in a progression reminiscent of development. In the aberrant retinoretinal projection, levels of XNIF, NF-L, and M remained lower than in normally regenerating axons, whereas NF-H and a phosphorylated form of NF-M were undetectable for at least 35 d after nerve crush. Normally, these two latter forms reappear between 15 and 21 d after surgery. Thus, this transient, incorrect axonal projection expressed neurofilaments in a very different pattern from correctly regenerating axons. In tecta-ablated frogs, staining of phosphorylation independent epitopes of XNIF, NF L, and -M increased normally after axons entered the tract, but that of NF-H and phosphorylated NF-M remained low for at least 42 d after axotomy. Thus, separate parts of the visual pathway influence the complexity of neurofilaments. PMID- 7540683 TI - Analysis of synaptic distribution within single retinal axonal arbors after chronic NMDA treatment. AB - Activation of the NMDA subtype of glutamate receptor has been implicated in structural synaptic plasticity in many developing sensory systems. In the frog retinotectal system, chronic exposure of the optic tectum to NMDA, which decreases the effectiveness of NMDA receptors (Debski et al., 1991), results in the pruning of the branches of retinal terminal arbors (Cline and Constantine Paton, 1990). However, it is difficult from these studies to relate the involvement of NMDA receptors to changes in synapse distribution. In this study, we have developed an EM sampling procedure to quantitatively compare the amount and the distribution of synaptic contact within single retinal arbors. We report that within each retinal arbor, synaptic contact gradually increases from the main branches to the end branches of the arbor. Chronic NMDA treatment, however, significantly reduces the total amount of synaptic contact within each arbor. This reduced synaptic contact appears to be due to the pruning of the end branches, and the synapses these branches bear. The results are consistent with the hypothesis that NMDA receptor is an integral part of the mechanism that stabilizes coactive synapses, and that maintenance of an axonal branch requires a minimum density of synapses that are correlated with converging neighbors. PMID- 7540685 TI - An evaluation of the staining of lymphomas and normal tissues by the rabbit polyclonal antibody to protein gene product 9.5 following non-enzymatic retrieval of antigen. AB - We have previously described the staining of normal follicle centre lymphoid cells by the rabbit polyclonal antibody to protein gene product 9.5 (PGP9.5), following an antigen unmasking step employing heat pretreatment. Applying this finding to a range of lymphomas to determine whether this phenomenon could have a role in identifying lymphomas of follicle centre origin revealed no relationship between type or grade of lymphoma and staining of neoplastic cells. Testing a range of normal tissues following antigen retrieval displayed increased sensitivity and a greater range of tissue positivity. Immunoblots of gel electrophoresed tonsil and brain extract were performed. Although subjecting these blots to antigen unmasking increased sensitivity, no novel epitopes, in terms of additional bands, appeared. This suggested that antibody specificity remained unaltered. PMID- 7540684 TI - Tenascin immunoreactivity in cryptogenic fibrosing alveolitis. AB - Tenascin is a hexameric extracellular matrix (ECM) glycoprotein which has been demonstrated to have a temporal relationship with active scar formation in adult tissues. We hypothesized that this ECM protein might therefore serve to identify areas of active scarring in lung biopsies from patients with cryptogenic fibrosing alveolitis (CFA). The distribution of tenascin was examined in open lung biopsies from ten patients with CFA, six patients with sarcoidosis, and six pulmonary resection specimens from patients with no evidence of interstitial lung disease, using an immunohistochemical technique. Immunoreactive tenascin was not identified in histologically normal control lung parenchyma and was only focally found around large aggregates of granulomas in sarcoidosis. In the CFA, tenascin production was demonstrated in minimally damaged alveolar walls and areas of active disease but not in end-stage scarred lung. There was considerable local heterogeneity of staining within cases, which did not appear to relate to the density of the local inflammatory infiltrate. Large plaques of tenascin were noted to be particularly associated with hyperplastic type II alveolar epithelial lining cells, which are recognized to produce fibrogenic cytokines. The examination of tenascin expression in open lung biopsies from patients with CFA may be useful in assessing fibrogenic activity and may thus provide additional prognostic information. PMID- 7540686 TI - A sensitive staining method for NORs. AB - A sensitive staining method for nucleolar organizer regions (NORs) is described, using blue toning of AgNORs. NORs are loops of DNA which are transcribed into ribosomal RNA. NORs can be demonstrated by staining with silver nitrate, since NOR-associated proteins are argyrophilic, producing structures termed AgNORs. Normal blood lymphocytes were stained with both methods. The number and resolution of NORs increased 2-3 times by blue toning (30 mmol/l FeCl3, 11 mmol/l potassium hexacyanoferrate(III), and 33 mmol/l oxalic acid) compared with silver staining. A significant difference in the number of NORs was noticed between silver-stained and blue-toned cells (P < 0.001). The blue toning technique thus appears to be more sensitive in detecting NORs than the AgNOR method and may prove a useful alternative for applications in histopathology. PMID- 7540687 TI - Saturable uptake of a recombinant human granulocyte colony-stimulating factor derivative, nartograstim, by the bone marrow and spleen of rats in vivo. AB - Kinetic analysis of the in vivo tissue distribution of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivative, nartograstim (NTG; nartograstim is an international nonproprietary name of KW-2228, Kyowa Hakko Kogyo, Tokyo, Japan) was studied in rats to determine the mechanism of the growth factor's nonlinear pharmacokinetic properties. The early-phase tissue uptake clearance (CL(uptake)) by each tissue was determined within 5 min after the i.v. administration of 125I-NTG. Coadministration of various amounts (0.6-150 microgram/kg) of unlabeled NTG did not produce any significant change in CL(uptake) by kidneys and liver. The hepatic and renal extraction ratios of NTG were low, 0.10 to 0.15, which suggests that there is no saturable uptake system in either of these organs. CL(uptake) by the kidneys was comparable to the glomerular filtration rate of unbound NTG. By contrast, a dose-dependent reduction in CL(uptake) by bone marrow and spleen was clearly observed with increasing doses of unlabeled NTG, i.e., at the maximal dose, these values were 14% and 20%, respectively, of those after a tracer dose. A saturable process was, therefore, involved in the tissue uptake of 125I-NTG by bone marrow and spleen. The intrinsic clearance (Vmax/Km) of this saturable uptake by bone marrow, 1.4 ml min-1 kg-1, was greater than that by the spleen, 0.09 ml min-1 kg-1. The sum of the intrinsic clearances of the saturable process was comparable with the value for the clearance of the saturable elimination process of NTG obtained previously by nonlinear pharmacokinetic analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540688 TI - Action potentials, ionic currents and cell water in guinea pig ventricular preparations exposed to dimethyl sulfoxide. AB - Superfusion of guinea pig papillary muscles with Tyrode's solution that contained 0.3% to 10% dimethyl sulfoxide (DMSO) caused a small hyperpolarization, a prolongation of the action potential (e.g., 4%, 15% and 33% prolongation with 1%, 5% and 10% DMSO, respectively) and a reduction (< or = 14%) in the maximal upstroke velocity (Vmax). Action potential prolongation was also induced by DMSO in muscles treated with the inorganic Ca++ channel blocker Co++ (5 mM) and in those depolarized with 24 mM K(+)-containing Tyrode's solution. In the latter case, there was no significant change in Vmax. In voltage-clamp experiments, superfusion of guinea pig ventricular myocytes with 0.1% to 10% DMSO solution had little effect on Ca++ and inward-rectifier K+ currents, moderately inhibited Cl- and Na+ currents, partially inhibited Na+ pump current and markedly depressed delayed-rectifier K+ current. The cell water shrinkages measured in muscles and myocytes superfused with DMSO solution appear to be only partially responsible for the solvent's effects. Other mechanisms that may be of greater importance in explaining the action of DMSO include direct block of channels by DMSO molecules and hindrance of channel hydration and opening related to osmotic stress. PMID- 7540689 TI - The nitric oxide synthase inhibitor, L-NG-monomethylarginine, reduces carrageenan induced pleurisy in the rat. AB - Nitric oxide (NO) is a biological mediator that, when produced by the type II (inducible) nitric oxide synthase (NOS), has been implicated in the pathophysiology of inflammatory diseases. To examine this putative role of NO, pleural inflammation was elicited in rats by the intrapleural injection of carrageenan (1 mg). A pleural exudate and cellular influx developed, which peaked at 24 h and generally resolved by 72 h. The cellular influx was primarily composed of polymorphonuclear cells during the first 24 h, followed by macrophages during the subsequent 24 h. Inflammatory cell-associated NOS activity and pleural exudate nitrite (NO2-) + nitrate (NO3-) (NOx) also increased, peaking at 6 h and 24 h, respectively. Cell-associated NOS activity was calcium independent, indicating the presence of the type II NOS isoform; NOS activity in the pleural cavity and polymorphonuclear cells influx were temporally correlated. Administration of L-NG-monomethylarginine (L-NMA) (200 mg/kg/day) attenuated the pleural exudation, cellular influx, pleural exudate NOx, and cell-associated NOS activity. The relative composition of the pleural cavity cellular infiltrate was not changed by L-NMA, indicating the influx of individual cell types were affected equally. L-Arginine (500 mg/kg/day) completely prevented the effects of L-NMA on pleural exudation and cellular influx and partially prevented the inhibition of pleural exudate NOx accumulation by L-NMA. These data implicate NO as a modulator of the pleural inflammatory response and support a future clinical role for NOS inhibitors in the treatment of inflammatory disease. PMID- 7540690 TI - Peripheral blood and synovial fluid monocyte expression of interleukin 1 alpha and 1 beta during active rheumatoid arthritis. AB - OBJECTIVE: To investigate the mononuclear cell (MNC) types from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) expressing interleukin-1 beta and -1 alpha (IL-1 beta, IL-1 alpha). METHODS: PB and SF MNC from 16 patients with classical or definite RA and controls were analyzed by flow cytometry for IL-1 beta and IL-1 alpha staining. RESULTS: MNC from normal individuals do not stain for either IL-1 beta or IL-1 alpha. By contrast, PB and SF CD14+ monocytes from patients with active RA show significant staining for surface and intracellular IL-1 beta and IL-1 alpha. Furthermore, the erythrocyte sedimentation rate (ESR) correlates with the surface expression of IL 1 alpha (r = 0.52, p < 0.05) while the number of active joints (i.e., tender and/or swollen) correlate with the intracellular expression of IL-1 beta (r = 0.58, p < 0.03) in CD14+ monocytes. CONCLUSION: The analysis of IL-1 beta and IL 1 alpha expression on CD14+ monocytes from peripheral blood by flow cytometry could be a potential immunological marker of disease activity in RA and its variation could be used to monitor the effects of therapy. PMID- 7540692 TI - The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis. PMID- 7540693 TI - Modulatory action of Helicobacter pylori on histamine release from mast cells and basophils in vitro. AB - Helicobacter pylori is important in the aetiology of peptic ulceration. Despite inducing an inflammatory response in the mucosa, the organism persists, suggesting that it has efficient protective mechanisms. Some bacterial and viral products modulate histamine secretion from inflammatory cells. Therefore, this study examined the modulatory effects of H. pylori preparations on histamine release from rat peritoneal mast cells and human basophils. Eleven clinical isolates of H. pylori were prepared in different ways: as whole washed bacteria, washed sonicated bacteria, and formalin-killed bacteria, and as outermembrane and lipopolysaccharide (LPS) extracts. Histamine release from mast cells or basophils was not elicited by any of these bacterial preparations alone. However, when mixed with various secretory stimulants, the bacterial preparations caused inhibition of histamine release from rat mast cells (calcium ionophore A23187, compound 48/80, concanavalin A, anti-rat IgE) and human basophils (A23187, N formyl Met-Leu-Phe). The degree of inhibition ranged from 48% to 97%. These results indicate that H. pylori exerts an inhibitory effect on cells of the immune system that contributes to its persistence within the gastric mucosa. PMID- 7540691 TI - HLA-DRB1 alleles associated with rheumatoid arthritis in southern France. Absence of extraarticular disease despite expression of the shared epitope. AB - OBJECTIVE: Extraarticular rheumatoid arthritis (RA) is almost unknown in Marseille in southern France. We investigated whether this was due to rare expression of the HLA-DRB1 shared epitope. METHODS: HLA-DRB1 alleles were characterized in 73 patients with RA and 108 controls by polymerase chain reaction amplification and oligonucleotide hybridization. RESULTS: In patients with RA, 76% expressed the shared epitope (46% DR1, 45% DR4). Four HLA-DRB1 alleles were positively associated with disease: DRB1*0101, DRB1*0401, DRB1*0404, DRB1*0405. Patients with double dose shared epitope had the most severe articular damage, but no extraarticular disease. CONCLUSION: In Marseille, 76% of patients with RA are shared epitope positive. Still, most do not develop extraarticular RA. This may be caused by the low frequency of HLA-DRB1*0401 in this population. PMID- 7540694 TI - Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. AB - Three genes called rocD, rocE and rocF were found near the rocR gene in B. subtilis. The product of rocD is similar to eukaryotic ornithine aminotransferases. The product of rocE shares similarity with the product of B. subtilis rocC and with the product of E. coli lysP. The rocE gene may encode an arginine permease. The rocF gene encodes a polypeptide similar to several arginases. Heterologous expression in E. coli indicated that rocD encodes an ornithine aminotransferase and that rocF encodes an arginase. Arginine utilization was abolished in both rocD and rocF mutants of B. subtilis confirming the role of these genes in arginine catabolism. The rocDEF genes form an operon transcribed from a -12, -24 promoter almost identical to the -12, -24 promoter of the rocABC operon. The expression of the rocDEF operon was induced by the presence of arginine, ornithine or proline in the growth medium and depended on the presence of the sigma factor SigL. Transcription of this operon was also abolished in a B. subtilis strain containing a null mutation in the regulatory gene rocR. Two tandemly repeated upstream activating sequences very similar to those previously identified in the rocABC system were found centered at positions -120 and -70, respectively, upstream from the transcription start site of rocDEF. Deletion analysis showed that at least one upstream activating sequence is involved in the expression of the rocDEF operon. These sequences are probably the target of RocR. Analysis of a rocR'-'lacZ fusion strain showed that the expression of rocR is not induced by arginine and is negatively autoregulated. PMID- 7540695 TI - The volume of atoms on the protein surface: calculated from simulation, using Voronoi polyhedra. AB - We analyze the volume of atoms on the protein surface during a molecular-dynamics simulation of a small protein (pancreatic trypsin inhibitor). To calculate volumes, we use a particular geometric construction, called Voronoi polyhedra, that divides the total volume of the simulation box amongst the atoms, rendering them relatively larger or smaller depending on how tightly they are packed. We find that most of the atoms on the protein surface are larger than those buried in the core (by approximately 6%), except for the charged atoms, which decrease in size, presumably due to electroconstriction. We also find that water molecules are larger near apolar atoms on the protein surface and smaller near charged atoms, in comparison to "bulk" water molecules far from the protein. Taken together, these findings necessarily imply that apolar atoms on the protein surface and their associated water molecules are less tightly packed (than corresponding atoms in the protein core and bulk water) and the opposite is the case for charged atoms. This looser apolar packing and tighter charged packing fundamentally reflects protein-water distances that are larger or smaller than those expected from van der Waals radii. In addition to the calculation of mean volumes, simulations allow us to investigate the volume fluctuations and hence compressibilities of the protein and solvent atoms. The relatively large volume fluctuations of atoms at the protein-water interface indicates that they have a more variable packing than corresponding atoms in the protein core or in bulk water. We try to adhere to traditional conventions throughout our calculations. Nevertheless, we are aware of and discuss three complexities that significantly qualify our calculations: the positioning of the dividing plane between atoms, the problem of vertex error, and the choice of atom radii. In particular, our results highlight how poor a "compromise" the commonly accepted value of 1.4 A is for the radius of a water molecule. PMID- 7540696 TI - Improving treatment of chemotherapy-induced neutropenic fever by administration of colony-stimulating factors. AB - BACKGROUND: Several randomized trials have tested the use of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in relieving chemotherapy-induced bone marrow suppression. However, the use of CSFs in the treatment of neutropenic fever remains virtually unexplored. PURPOSE: This study evaluated the benefits of adding CSF therapy to the standard antibiotic treatments given to cancer patients for chemotherapy-induced neutropenic fever. The usefulness of CSFs was quantified in terms of reducing the following: (a) the duration of neutropenia, (b) the length of hospitalization, and (c) the overall cost of the treatment. METHODS: A randomized trial was conducted to test whether the administration of either G-CSF or GM-CSF improved the outcome of standard antibiotic therapy (ceftazidime plus amikacin) in nonleukemic cancer patients with fever (> 38 degrees C) and grade IV neutropenia (absolute neutrophil count [ANC] < 500/mm3) induced by standard-dose chemotherapy. Of 121 patients who entered the trial, 39 received G-CSF (5 micrograms/kg body weight per day), 39 received GM-CSF (5 micrograms/kg body weight per day), and 43 received a placebo beginning just after the first dose of antibiotics. Treatments were continued for at least 5 days (7 days with clinically or microbiologically documented infections) or until 2 days after fever subsided and ANCs rose above 1000/mm3. RESULTS: The median duration of grade IV neutropenia (ANC of < 500/mm3) was 2 days in both CSF arms and 3 days in the placebo arm (P < .001). The median duration of neutropenia with an ANC of less than 1000/mm3 was also significantly shorter in patients receiving G-CSF or GM-CSF (P < .001). The median duration of fever was similar in the three arms. The median hospital stay was 5 days (range, 5-14 days) in the G-CSF arm, 5 days (range, 5-10 days) in the GM-CSF arm, and 7 days (range, 5-34 days) in the placebo arm (P < .001). The median time on CSF was 4 days in both treatment arms. The mean cost of overall treatment was reduced by $1300-$1400 in the CSF arms compared with the placebo arm (P = .11 for G-CSF versus placebo; P = .06 for GM CSF versus placebo; P = .7 for G-CSF versus GM-CSF). CONCLUSIONS: Adding G-CSF or GM-CSF therapy to antibiotic treatment shortens the duration of neutropenia and the duration of hospitalization in patients with neutropenic fever. A statistically nonsignificant trend toward lower cost was observed in the CSF arms as compared with the placebo arm. IMPLICATIONS: The benefits of CSFs to cancer patients with chemotherapy-induced neutropenic fever merit further evaluation in large randomized trials. PMID- 7540697 TI - Leukocyte activation in the peripheral blood of patients with cirrhosis of the liver and SIRS. Correlation with serum interleukin-6 levels and organ dysfunction. AB - OBJECTIVE: Leukocyte adhesion plays an important role in inflammation. Adhesion molecules such as CD11b on polymorphonuclear neutrophil leukocytes (PMNs) up regulate in response to tumor necrosis factor-alpha, interleukin-8 (IL-8), and other mediators that are involved in systemic inflammatory response syndrome (SIRS). This study examined the behavior of CD11b and other membrane molecules in SIRS in relation to serum cytokines and the severity of illness. DESIGN: Survey study. SETTING: Liver transplantation intensive care unit at a tertiary care center. PATIENTS: A consecutive sample of 22 patients admitted to the liver transplantation intensive care unit for complications related to cirrhosis of the liver in the absence of other disease. Sixteen of the patients developed SIRS and multiple organ dysfunction syndrome with suspected bacterial infections. Seven control subjects were also studied. MAIN OUTCOME MEASURES: Modified Goris organ failure score and Acute Physiology and Chronic Health Evaluation II score. RESULTS: Mean serum IL-6 levels, but not IL-1 beta or tumor necrosis factor-alpha levels, correlated with organ failure (r = 0.79, P < .001). Leukocyte cell surface markers fluctuated from day to day. The mean of several values was more stable. Mean CD11b and CD35 on PMNs correlated with serum IL-6 level (r = 0.75, P < .001, and r = 0.77, P < .005, respectively). Up-regulation of both CD11b and CD35 display on PMNs correlated with organ failure (r = 0.74, P < .001, and r = 0.71, P < .01, respectively). Polymorphonuclear neutrophil leukocyte L-selectin, CD31, and CD16 were simultaneously decreased, consistent with PMN activation. Monocytes appeared to be activated, but the pattern of surface molecule display was different. CONCLUSIONS: In human SIRS, the circulating monocyte and PMN pools undergo alterations suggestive of leukocyte activation, including up-regulation of PMN CD11b in correlation with the serum IL-6 level and severity of organ dysfunction. PMID- 7540698 TI - [A possible mechanism of interstitial pneumonia during interferon therapy with sho-saiko-to]. AB - Interstitial pneumonia has been reported to be a side effect of treatment with interferon, and Sho-saiko-to (Xiao-Chai-Hu-Tang) may enhance this side effect. It is well known that activated neutrophils are important mediators of pulmonary fibrosis, so we studied the effects of interferon and Sho-saiko-to on neutrophil activation. Homogenized lung myeloperoxidase (MPO) activity was assayed after intraperitoneal injection of interferon with or without pretreatment with Sho saiko-to. Although Sho-saiko-to alone did not change the lung MPO content, MPO in the lung was significantly increased by interferon administration. The increase was enhanced further by pretreatment with Sho-saiko-to. When the accumulated neutrophils are activated by some cytokines, such as TNF alpha or IL-1 beta from monocytes/macrophages, they may damage lung tissue. We therefore studied the effects of Sho-saiko-to and interferon on TNF alpha production in freshly isolated human monocytes. Sho-saiko-to increased the production of TNF alpha, but interferon did not. In addition, Sho-saiko-to significantly increased the production of TNF alpha by monocytes stimulated by lipopolysaccharide. Taken together, these data indicate that interferon causes neutrophils to accumulate in the lung. Sho-saiko-to alone may not injure lung tissue, but it increases the effect of interferon. When stimulated by some antigen, Sho-saiko-to may overstimulate the neutrophils. Granulocytes elastase and oxygen radicals released from activated neutrophils may damage lung tissue. The fibroblasts that repair the damaged tissue may increase the risk of pulmonary fibrosis. PMID- 7540699 TI - Endothelial cell response to hypoxia-reoxygenation is mediated by IL-1. AB - Reperfusion injury involves the adhesion and activation of neutrophils (PMN) both in affected tissues and distant organs. Cell adhesion molecules (CAM) such as endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) are known to mediate, at least in part, the adherence of activated PMN to the endothelium. To characterize the cellular mechanisms of this phenomenon, we exposed cultured human umbilical vein endothelial cells (HU-VEC) to hypoxia and reoxygenation (H/R) using an incubator chamber purged of oxygen with 100% nitrogen. Confluent monolayers of HUVEC were subjected to 60 min of hypoxia followed by variable periods of reoxygenation (120 and 240 min). Flow cytometry was utilized to assess the expression of ELAM-1 and ICAM-1, expressed as percent shift from baseline expression. To determine what role endothelium derived cytokines such as IL-1 play in the expression of CAM after H/R, we performed additional experiments in the presence of recombinant IL-1 receptor antagonist (IL-1RA). ICAM-1 was present on unstimulated HUVEC while ELAM-1 was not constitutively expressed. Following exposure of cells to hypoxia and reoxygenation, significant increases in ELAM expression were seen (8.4 +/- 2.4% at 120 min; 19.1 +/- 7.4%, P < 0.05). While there was similar trend in ICAM expression, this did not achieve statistical significance (0.10 < P < 0.05). The addition of IL-1RA (10 ng/ml) to hypoxic HUVEC consistently attenuated ELAM-1 upregulation during reoxygenation (0.8 +/- 0.7% at 120 min and 5.9 +/- 4.1% at 240 min) such that expression was not significantly greater than baseline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540701 TI - Constitutive nitric oxide synthase is expressed and nitric oxide-mediated relaxation is preserved in retrieved human aortocoronary vein grafts. AB - Although little is known about the endothelial cell function of human saphenous vein coronary artery bypass grafts, there is evidence to suggest that receptor activated, endothelial-dependent relaxation mediated by nitric oxide is impaired. This study examines the expression and function of endothelial cell constitutive nitric oxide synthase (cNOS) of aortocoronary vein bypass grafts and human saphenous veins obtained from 10 patients undergoing repeat coronary artery bypass grafting for recurrent ischemic symptoms. Following precontraction with norepinephrine (10(-5) M), responses to acetylcholine (receptor-mediated, endothelium-dependent), calcium ionophore (A23187; receptor-independent, endothelium-dependent), and sodium nitroprusside (endothelium-independent) were assessed. Following total RNA extraction using phenol/guanidinium isothiocyanate from specimens of human saphenous vein and vein graft, a quantitative RNase Protection Assay (RPA) was performed using a cRNA riboprobe corresponding to a fragment of the human endothelial cell cNOS gene. Histologically, the vein grafts showed both intimal hyperplasia development and focal atherosclerosis formation compared to the saphenous veins. Scanning electron microscopy of the saphenous veins and the vein grafts showed an intact endothelium. Precontracted vein grafts did not relax in response to acetylcholine; in contrast, the saphenous vein relaxed in a dose-dependent manner to reach a maximal relaxation of 19 +/- 4% precontracted tension. Saphenous veins and vein grafts relaxed in response to A23187 with maximal relaxation of 92 +/- 5 and 73 +/- 13%, respectively. Both vessels relaxed in a dose dependent manner to sodium nitroprusside. RPA normalized to beta-actin showed similar levels of expression of endothelial cell cNOS equivalent to 1 pg of sense RNA in both the saphenous vein and vein graft.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540700 TI - Vasoactive intestinal peptide and substance P receptor antagonists improve postoperative ileus. AB - Octreotide, a somatostatin analogue that inhibits the release of most gut peptides, hastens the resolution of experimental postoperative ileus, suggesting that gut peptides mediate this process. We studied the role of two gut peptides involved in the control of normal gut motility, vasoactive intestinal peptide (VIP), and substance P (SP), in the initiation and maintenance of postoperative small bowel ileus in rats by preoperative administration of VIP and SP receptor antagonists, (VIP-ra and SP-ra). Thirty male Sprague-Dawley rats (300-350 g) underwent laparotomy. One half underwent placement of a duodenal catheter for transit studies while the other half had serosal electrodes placed on the proximal jejunum for myoelectric recordings. Six days later, animals were separated into three treatment groups of five each. Control animals were pretreated with ip saline, while the others received either VIP-ra or SP-ra prior to standardized laparotomy. Following abdominal closure, [Na51]CrO4 was injected into the duodenum and the animals were sacrificed 25 min later. The small bowel was then excised and divided into 10 equal segments. Small bowel transit was calculated as the geometric center of [Na51]CrO4 distribution. The interval until the return of migrating myoelectric complexes (MMCs) was determined in animals with intestinal electrodes. VIP-ra-treated rats demonstrated a 67% improvement in the geometric center of radiolabel relative to controls and SP-ra-treated rats had a 23% improvement (3.67 +/- 0.06 VIP-ra vs 2.69 +/- 0.09 SP-ra vs 2.20 +/- 0.09 control, P < 0.01). MMCs returned 180 +/- 17 min in controls vs 99 +/- 14 min in VIP-ra-treated rats (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540702 TI - Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. AB - The antibiotic gramicidin, when incorporated into lipid membranes, forms pores that are exclusively permeable to monovalent cations and small unchanged molecules. We report the use of gramicidin for perforated patch-clamp recordings in the whole-cell mode. Recordings were performed in cultured rat spinal cord dorsal horn neurons. Cells had stable resting potentials and series resistances for times routinely exceeding 60 min. To test if intracellular chloride concentration ([Cl]i) remains stable with this technique, we measured responses to agonists of glycine and GABAA receptors, both of which gate chloride conductances. The driving force for these responses remained stable at values that differed significantly from values that would be expected if [Cl-]i were biased towards pipette [Cl-]. We conclude that gramicidin perforated-patch recording, in addition to other properties of the perforated-patch recording technique, has the advantage of not altering [Cl-]i. It is, therefore, an electrophysiological method particularly suitable for studies of anionic channels when [Cl-]i is a variable of interest, as well as for studies of homeostatic [Cl ]i regulation. PMID- 7540703 TI - A modification of biotinylated dextran amine histochemistry for labeling the developing mammalian brain. AB - Biotinylated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine sodium tungstate (TMB-ST) method for horseradish peroxidase (HRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled exons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled. The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contracts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system. PMID- 7540704 TI - Dietary calorie restriction in the Emory mouse: effects on lifespan, eye lens cataract prevalence and progression, levels of ascorbate, glutathione, glucose, and glycohemoglobin, tail collagen breaktime, DNA and RNA oxidation, skin integrity, fecundity, and cancer. AB - The Emory mouse is the best model for age-related cataract. In this work we compare the effects of feeding a control diet (C) with a diet restricted (R) by 40% relative to C animals. In the R animals, median lifespan was extended by 40%. The proportion of R mice with advanced cataract was lower than C mice as early as 5 months of age. The mean grade of cataract was lower in R animals, beginning at 11 months and continuing until the end of the study. Ascorbate levels in R plasma and liver were 41-56% of C animals. There was no difference between diet groups with respect to lens ascorbate. Aging was associated with a decrease in ascorbate in lenses and kidneys in C and R mice. By 22 months, R animals had 48% higher liver glutathione levels than C mice. Liver glutathione levels were maximal at 12 months. Plasma glucose levels were > 27% lower in R animals at 6.5 and 22 months, and there was a 14% increase in glucose levels upon aging for both diet groups. In R mice, glycohemoglobin levels were 51% lower and tail collagen breaktime was decreased by 40%, even in younger animals. Collagen breaktime increased > 360% upon aging for both diet groups. Rates of production of urinary oxo8dG and oxo8G were higher in R animals compared with C animals, and increased upon aging. C animals exhibited more cancer and dermatological lesions, but less tail tip necrosis and inflamed genitals than R mice. These data allow evaluation of several theories of aging. PMID- 7540706 TI - Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia. PMID- 7540705 TI - Randomised, placebo-controlled, double-blind clinical trial of beta-sitosterol in patients with benign prostatic hyperplasia. Beta-sitosterol Study Group. AB - Medical treatments have become available for benign hypertrophy of the prostate, including alpha-receptor blocking agents and 5-alpha-reductase inhibitors. Drugs derived from plants, for which no precise mechanism of action has been described, are widely used for this purpose in Europe. In a randomised, double-blind, placebo-controlled multicentre study, 200 patients (recruited between April and October 1993) with symptomatic benign prostatic hyperplasia were treated with either 20 mg beta-sitosterol (which contains a mixture of phytosterols) three times per day or placebo. Primary end-point was a difference of modified Boyarsky score between treatment groups after 6 months; secondary end-points were changes in International Prostate Symptom Score (IPSS), urine flow, and prostate volume. Modified Boyarsky score decreased significantly with a mean of -6.7 (SD 4.0) points in the beta-sitosterol-treated group versus -2.1 (3.2) points in the placebo group p < 0.01. There was a decrease in IPSS (-7.4 [3.8] points in the beta-sitosterol-treated group vs -2.1 [3.8] points in the placebo group) and changes in urine flow parameters: beta-sitosterol treatment resulted in increasing peak flow (15.2 [5.7] mL/s from 9.9 [2.5] mL/s), and decrease of mean residual urinary volume (30.4 [39.9] mL from 65.8 [20.8] mL). These parameters did not change in the placebo group (p < 0.01). No relevant reduction of prostatic volume was observed in either group. Significant improvement in symptoms and urinary flow parameters show the effectiveness of beta-sitosterol in the treatment of benign prostatic hyperplasia. PMID- 7540707 TI - Modulation of nitric oxide-dependent relaxation of pig tracheal smooth muscle by inhibitors of guanylyl cyclase and calcium activated potassium channels. AB - Nitric oxide is released from intrinsic nonadrenergic, noncholinergic (NANC) nerves of pig tracheal smooth muscle (TSM) in response to electrical field stimulation (EFS). In this study, we investigated the role of guanylyl cyclase in the NANC relaxation by using guanylyl cyclase inhibitors, LY83583 and methylene blue (MB). The role of large conductance calcium-activated potassium (KCa) channels in mediating NANC relaxation was studied by using inhibitors of this channel, charybdotoxin and iberiotoxin. In carbachol-contracted TSM strips, LY83583 (10-20 microM) and MB (10-100 microM) resulted in inhibition of EFS induced relaxations at all frequencies studied. Relaxations induced by exogenous 8-Bromo-cyclic 3',5'-guanosine monophosphate (8-Br-cGMP) were unaffected by LY83583. The concentration-relaxation curves to isoproterenol, which acts by elevating adenosine-3',5'-cyclic monophosphate (cAMP), and the nitric oxide donors sodium nitroprusside (SNP) or S-nitroso-n-acetylpenicillamine (SNAP) were unaffected by LY83583. Both charybdotoxin (240 nM) and iberiotoxin (180 nM) attenuated relaxations induced by EFS and SNAP. The role of guanylyl cyclase activation in the relaxation to EFS of pig TSM is suggested by the sensitivity of the responses to MB. The selective inhibitory effects of LY83583 on relaxation to neurally released, but not to the nitric oxide donors, suggests that it acts by inhibiting nitric oxide release. The lack of any effect of LY83583 on isoproterenol- or guanosine, 3'5'-cyclic monophosphate (cGMP)-mediated relaxation suggests a mechanism that does not involve elevation of cAMP but lies proximal to the generation of cGMP. The susceptibility of the relaxations to EFS and SNAP to charybdotoxin and iberiotoxin suggests a mechanism that involves the selective activation of KCa channels in airway smooth muscle cells. PMID- 7540708 TI - Soluble CD14 in serum mediates LPS-induced increase in permeability of bovine pulmonary arterial endothelial cell monolayers in vitro. AB - Lipopolysaccharide (LPS) is a mediator of septic shock and acute respiratory distress syndrome (ARDS), conditions which are characterized by high-permeability pulmonary edema. LPS increases endothelial permeability both directly and indirectly via the pro-inflammatory cytokines produced by monocytes and macrophages. We investigated the role of soluble CD14 in serum in the increased endothelial permeability induced by LPS. Bovine pulmonary artery endothelial cells were grown to confluence on a microporous filter and the 125I-albumin clearance rate across the monolayer was determined. Even a high concentration of LPS (1 microgram/ml) did not increase endothelial permeability under a serum-free condition. In the presence of more than 3% normal human serum, LPS increased endothelial permeability. The presence of neutralizing anti-CD14 monoclonal antibody eliminated the serum-dependent effect of LPS. The addition of recombinant sCD14 completely replaced the requirement for serum. LPS-binding protein (LBP) did not enhance the rsCD14-mediated LPS effect, and anti-LBP antibody did not attenuate the serum-dependent LPS effect. These findings suggest that sCD14 in serum mediates the permeability-increasing effect on LPS on endothelial cells but that LBP is not necessary for this effect. PMID- 7540709 TI - Dopaminergic modulation of serotonin metabolism in rat striatum: a study with dopamine uptake inhibitor GBR-12909. AB - The dopamine (DA) uptake inhibitor, GBR 12909 (GBR) and a neonatal dopaminergic denervated rat model were used as tools to study the influence of DA on the serotonin (5HT) system in the striatum. The striatal levels of the amines and their acid metabolites (dihydroxyphenylacetic acid, DOPAC; 5-hydroxyindoleacetic acid, 5HIAA) were determined by HPLC. The administration of a single dose (20 mg/kg) of GBR failed to affect the steady-state levels of the amines or metabolites. Repeated administration of GBR (20 mg/kg/day) for 2 or 4 days decreased DA and DOPAC; only the 4-day regimen decreased 5HT and increased 5HIAA levels. The neonatal dopaminergic lesion with 6-hydroxydopamine (6OHDA) depleted (> 95%) DA and DOPAC and increased 5HT and 5HIAA levels in the striatum. Administration of GBR (20 mg/kg/day for 4 days) to lesioned animals failed to influence the lesion-induced increases in 5HT and 5HIAA levels. The results suggest GBR decreases the steady-state levels of DA, resulting in a compensatory increase in the turnover of 5HT that is dependent on the presence of intact dopaminergic terminals. Thus, the effect of GBR on 5HT turnover is indirect. The studies provide further support for a prominent dopaminergic influence on striatal 5HT metabolism. PMID- 7540710 TI - Trophic effects of substance P and beta-amyloid peptide on dibutyryl cyclic AMP differentiated human leukemic (HL-60) cells. AB - The neuropeptide substance P (SP) is a mediator of neurogenic inflammation. Also, beta-amyloid protein (beta AP) can directly activate the cell types involved in inflammatory processes. The relationship between SP and beta AP on their biological actions has attracted much interest. SP is trophic in neuronal cells and protected them against the death induced by beta AP. In this study, we examined the effects of SP and beta-amyloid peptide on cell viability in neutrophil-like HL-60 cells, by means of the WST-1 tetrazolium and lactate dehydrogenase (LDH) release assays. The results showed that SP promoted the cell survival on neutrophil-like HL-60 cells in serum-free conditions. Also, beta amyloid peptide showed trophic effects rather than toxic in these cells in WST-1 assay, though it is reportedly toxic in neuronal cells. PMID- 7540711 TI - Lack of involvement of protein kinase C in ethanol-induced inhibition of metabotropic-glutamate receptor function in primary cultures of astrocytes. AB - The stimulation of [3H] inositol phosphate (InP) formation by the selective metabotropic-glutamate receptor agonist, 1S, 3R-ACPD, was significantly reduced in rat cortical astrocytes chronically exposed to 100 mM ethanol for 4 days. Under the same conditions, chronic ethanol either increased or did not affect the InP responses to norepinephrine and carbachol, respectively. The InP responses to all three agonists were sensitive to phorbol 12-myristate 13-acetate. Although the protein kinase C inhibitors, calphostin C and staurosporine, significantly relieved the ethanol induced inhibtion of the InP response to 1S, 3R-ACPD, these responses were still significantly less than corresponding values obtained from control cells treated with these inhibitors. The data suggests that mechanisms in addition to protein kinase C are responsible for the ethanol induced inhibition of metabotropic-glutamate function. PMID- 7540712 TI - Predictive factors in the response to interferon therapy in chronic hepatitis C. AB - Factors predicting the efficacy of interferon therapy were statistically analyzed on 111 patients with chronic hepatitis C. Of the treated patients (total doses of interferon; 96-468 MU), 35 (31.5%) had a long-term remission. On multivariate analysis, hepatitis C virus genotype (p < 0.0001), histological diagnosis (p < 0.05), fibrosis score of histological activity index (p < 0.01) and source of infection (p < 0.05) were found useful for predicting the response to interferon therapy. Our findings suggest that the outcome of interferon therapy can be predicted to some degree from pretreatment data, and that a new therapeutic strategy is necessary for the group of patients who are predicted to be nonresponders. PMID- 7540713 TI - Hepatitis B virus precore mutations and HBeAg negative reactivation of chronic hepatitis B after interferon therapy. AB - The objective of this study was to look for HBV precore mutations in three patients with chronic active hepatitis B who developed HBV-DNA-positive/HBeAg negative reactivation after HBe seroconversion induced by interferon therapy. Direct sequencing of polymerase chain reaction products was performed on serum collected before and after HBe seroconversion. In two patients precore sequence showed only wild-type HBV before and after interferon therapy. In one patient, precore sequence showed only wild-type HBV before interferon therapy and a mixed infection by wild-type HBV and precore mutant viruses (1858 and 1896 nucleotide mutations) after treatment. The presence of HBeAg/anti-HBe immune complexes was found after HBe seroconversion in all cases. Our results suggest that: 1) precore mutations are not always found in patients with chronic hepatitis B who develop HBV DNA-positive/HBeAg-negative reactivation; and 2) HBeAg negativity, despite the presence of wild-type HBV, might be due to HBeAg/anti-HBe immune complexes. We speculate that the production of these immune complexes may be favored by interferon therapy. PMID- 7540714 TI - Lipopolysaccharide recovery restores susceptibility levels towards beta-lactams in Serratia marcescens. AB - O-side chain-defective spontaneous mutants of Serratia marcescens, selected by phage resistance, showed lower MICs against various beta-lactams than did their parental strains. The recovery of their ability to produce O-antigen restored the original MIC values, as well as phage susceptibility. The permeability coefficients of wild-type, O- mutants, and revertants, demonstrated that O antigen modifies the permeability of antibiotics in S. marcescens. PMID- 7540716 TI - The euthanasia controversy. Decision-making in extreme cases. PMID- 7540715 TI - Medicine and the community--the euthanasia debate. PMID- 7540717 TI - [Interferons in pediatric therapy]. PMID- 7540719 TI - Rapid mRNA degradation mediated by the c-fos 3' AU-rich element and that mediated by the granulocyte-macrophage colony-stimulating factor 3' AU-rich element occur through similar polysome-associated mechanisms. AB - The different 3' noncoding AU-rich elements (ARE) that mediate the degradation of many short-lived mRNAs may function through distinct decay pathways; translation dependent and -independent mechanisms have been proposed. To investigate the cotranslational model, we designed an expression system that exploits the properties of the ferritin iron-responsive element to shuttle chimeric mRNAs from ribonucleoproteins to polyribosomes. The iron-responsive element was introduced in the 5' untranslated regions of alpha-globin mRNAs that harbored in their 3' untranslated regions either the c-fos ARE or the granulocyte-macrophage colony stimulating factor ARE as prototypes of the different ARE subsets. The cytoplasmic location of the transcripts was controlled by intracellular iron availability and monitored by polysomal profile analysis. We report that these two mRNA subsets behaved identically in this system. Iron deprivation by desferrioxamine treatment stabilized both transcripts by sequestering them away from polyribosomes. Sequential treatments with desferrioxamine, followed by hemin to concentrate the mRNAs in the ribonucleoprotein pool prior to translation, showed that rapid degradation occurred only upon redistribution of the transcripts to polyribosomes. Deletion of a critical cytosine in the iron responsive element abolished targeted sequestration and restored high-level constitutive mRNA instability. These observations demonstrate that the c-fos and granulocyte-macrophage colony-stimulating factor ARE subsets mediate selective mRNA degradation through similar polysome-associated mechanisms coupled with ongoing translation. PMID- 7540721 TI - RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element. AB - R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested. The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat retrotransposable elements. PMID- 7540718 TI - Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation. AB - When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway. PMID- 7540720 TI - Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene. AB - The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo. PMID- 7540722 TI - Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. AB - BACKGROUND: Pulmonary hypertension is characterized by abnormal thickening of the pulmonary arteries and increased pulmonary vascular resistance. Nitric oxide is a potent endothelium-derived vasorelaxant substance and an inhibitor of smooth muscle-cell growth. Nitric oxide is produced in various cell types by the action of an enzyme, nitric oxide synthase. We compared the expression of endothelial nitric oxide synthase in the lungs of control subjects with that in the lungs of patients with pulmonary hypertension. METHODS: We investigated the expression of endothelial nitric oxide synthase by histochemical and immunohistochemical analysis, in situ hybridization, and Northern blot analysis in the lungs of 22 patients with plexogenic pulmonary arteriopathy (arteriopathy of grades 4 through 6), 24 patients with secondary pulmonary hypertension (arteriopathy of grades 1 through 3), and 23 control subjects. RESULTS: In the lungs of the control subjects, nitric oxide synthase was expressed at a high level in the vascular endothelium of all types of vessels and in the pulmonary epithelium. In contrast, little or no expression of the enzyme was found in the vascular endothelium of pulmonary arteries with severe histologic abnormalities (i.e., plexiform lesions) in patients with pulmonary hypertension. The intensity of the enzyme immunoreactivity correlated inversely with the severity of histologic changes. There was an inverse correlation between the arterial expression of the enzyme and total pulmonary resistance in patients with plexogenic pulmonary arteriopathy (r = -0.766, P = 0.004). CONCLUSIONS: Pulmonary hypertension is associated with diminished expression of endothelial nitric oxide synthase. It is possible that decreased expression of nitric oxide synthase may contribute to pulmonary vasoconstriction and to the excessive growth of the tunica media observed in this disease. PMID- 7540723 TI - Nitric oxide and vascular disease. PMID- 7540724 TI - HIV pathogenesis. Lines drawn in epitope wars. PMID- 7540725 TI - Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. AB - Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development. Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor). We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression. Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light. We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction. Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes. Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction. VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn. Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis. PMID- 7540726 TI - Antigen presentation mediated by recycling of surface HLA-DR molecules. AB - Class II histocompatibility molecules associate with peptides derived from antigens that are processed in endocytic compartments. Antigen presentation to class II-restricted T cells generally requires newly synthesized class II molecules, associated invariant chain, and HLA-DM. Exceptions to these rules have been reported, but without description of an underlying mechanism. Here we show that presentation of immunodominant epitopes in the haemagglutinin protein of influenza virus and in myelin basic protein correlates with recycling of surface HLA-DR molecules. Truncation of either one of the alpha or beta cytoplasmic tails virtually eliminated internalization of HLA-DR molecules and presentation of haemagglutinin from inactive virus particles. In contrast, the invariant chain dependent presentation of matrix antigen from the same virus particles was unaffected by these truncations. Thus HLA-DR cytoplasmic tails are not required for the conventional presentation pathway, but jointly contribute a signal for an alternative pathway involving internalization of HLA-DR molecules. PMID- 7540727 TI - Inhibition of antigen processing by the internal repeat region of the Epstein Barr virus nuclear antigen-1. AB - The Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA1) is expressed in latently EBV-infected B lymphocytes that persist for life in healthy virus carriers, and is the only viral protein regularly detected in all malignancies associated with EBV. Major histocompatibility complex (MHC) class I-restricted, EBNA1-specific cytotoxic T lymphocyte (CTL) responses have not been demonstrated. Using recombinant vaccinia viruses encoding chimaeric proteins containing an immunodominant human leukocyte antigen A11-restricted CTL epitope, amino acids 416-424 of the EBNA4 protein, inserted within the intact EBNA1, or within an EBNA1 deletion mutant devoid of the internal Gly-Ala repetitive sequence, we demonstrate that the Gly-Ala repeats generate a cis-acting inhibitory signal that interferes with antigen processing and MHC class I-restricted presentation. Insertion of the Gly-Ala repeats downstream of the 416-424 epitope inhibited CTL recognition of a chimaeric EBNA4 protein. The results highlight a previously unknown mechanism of viral escape from CTL surveillance, and support the view that the resistance of cells expressing EBNA1 to rejection mediated by CTL is a critical requirement for EBV persistence and pathogenesis. PMID- 7540728 TI - [Autoimmune diseases of the thyroid in the mother; consequences for fetus and newborn infant]. PMID- 7540729 TI - [Biliary tract carcinoma: surgery or irradiation?]. PMID- 7540730 TI - [Hepatitis-C virus antibody conversion in 3 hemodialysis patients in various dialysis departments]. AB - In three haemodialysis patients, two women of 72 and 35 years and a man of 65 years, who were dialysed in three different dialysis departments, recent seroconversion of hepatitis C was observed. The source of the infection and the transmission route remained unknown. PMID- 7540731 TI - [Treatment of hyperthyroidism and indications for radioactive iodine]. PMID- 7540732 TI - Sleep EEG and developmental dysphasia: lack of a consistent relationship with paroxysmal EEG activity during sleep. AB - In order to clarify the relationship between developmental dysphasia and EEG abnormalities, paroxysmal activities during sleep were studied in a series of 24 children with expressive developmental dysphasia (mean age 8 years) and compared to a control group of 39 children (mean age 9 years). The children of both groups were selected excluding cases with prior history of neurological disease or epilepsy. In the control group, 37 children had normal sleep EEG while 2 children had paroxysmal abnormalities. In the dysphasic group, epileptic abnormalities were observed in 9 cases, rare in 4 cases and frequent in 5 cases (density: 2.5 to 66.2% of total sleep time). Nevertheless, paroxysmal abnormalities did not reach the frequency described in the Landau-Kleffner syndrome, and it is unlikely that EEG abnormalities could have produced dysphasia. PMID- 7540733 TI - The effects of pretreatment with tachykinin antagonists and galanin on the development of spinal cord hyperexcitability following sciatic nerve section in the rat. AB - The effects of acute section of the sciatic nerve on the excitability of the flexor reflex was examined in decerebrate, spinalized, unanaesthetized rats. In control experiments without drugs, the excitability of the flexor reflex was dramatically increased in two phases following axotomy. An early intense, brief reflex hyperexcitability was followed by a less intense, prolonged period of facilitation. The selective NK1 tachykinin receptor antagonist CP-96,345 injected intrathecally at lower (1.2-2.4 nmol) and higher (12 nmol) doses blocked both components of spinal sensitization. The selective NK2 tachykinin receptor antagonist Men 10376 at a dose of 2.4 nmol also reduced both response components, as did the same dose of the inhibitory neuropeptide galanin. Thus, antagonists of excitatory neuropeptides released during and after nerve section, such as substance P and neurokinin A, can block the spinal response to peripheral nerve injury. Furthermore, the inhibitory neuropeptide galanin also reduced spinal cord sensitization. PMID- 7540735 TI - Transcranial magnetic coil stimulation of motor cortex in patients with central pain. AB - We report two patients with deafferentation pain secondary to central nervous system lesions who were evaluated by noninvasive magnetic coil stimulation of the motor cortex followed by electrical motor cortex stimulation with epidural electrode array implantation. Magnetic coil stimulation was very useful to estimate the effect of electrical stimulation. Our first patient was a 52-year old man who experienced a left putamenal hemorrhage at the age of 48. Two years later, he had paresthesias and intractable pain in the extremities and face on the right side. Pain was resistant to barbiturates but responded to magnetic coil stimulation of the motor cortex. Electrical motor cortex stimulation provided excellent relief from the pain. The second patient was a 43-year-old man who was suffering from congenital cerebral palsy for which left thalamotomy was performed two times, at the ages of 9 and 13. He began to experience intractable pain on the right side 20 years later. Although barbiturate administration was effective for pain relief, neither magnetic coil stimulation nor electrical stimulation of the motor cortex gave relief from pain. PMID- 7540734 TI - Factors regulating the production of vasopressin-associated human neurophysin by small-cell carcinoma of the lung: evaluation by computer-enhanced quantitative immunocytochemistry. AB - Expression of the vasopressin gene appears to be a property common to all small cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production. PMID- 7540736 TI - [Retrospective study of the efficacy of aprotinin in heart valve surgery in adults]. AB - Aprotinin effects on postoperative bleeding, redo operations due to excessive bleeding and postoperative blood products use have been retrospectively evaluated on a population of 894 consecutive patients operated upon between 1987 and 1991 for valvular surgery. In this period, aprotinin has been routinely used following the "High Dose" protocol starting from January 1990. To analyse results, all the patients considered (Group I) were divided into subgroups, following the heart valve operated upon, in "mitral" patients (391 patients, 43.7%, Group II), "aortic" patients (375 patients, 41.9%, Group III) and "mitro-aortic" patients (128 patients, 14.3%, Group IV). Each of these Groups has been subsequently split depending on whether or not they received aprotinin (Subgroups IA, IIA, IIIA, IVA with aprotinin, subgroups IB, IIB, IIIB, IVB without aprotinin). Considering the whole population (Group I), aprotinin determined a significant reduction of post operative bleeding (499 +/- 634 ml Group IA versus 713 +/- 572 Group IB, p = 0.000), redo operations for bleeding (15/410, 3.7% Group IA versus 45/484, 9.3% Group IB, p = 0.0000) and consequently the percentage of patients exposed to blood products transfusion (37/410, 9% Group IA versus 163/484, 54.3% Group IB p = 0.0000). When the other Groups (II, III and IV) were considered, aprotinin determined a significant reduction of postoperative bleeding and of donor blood transfusions, while redo operations for bleeding, although less in total number, were not significantly reduced. Moreover, aprotinin has been effective in reducing postoperative blood losses, redo-operation for bleeding and blood use independently of the kind of oxygenator used: bubble vs hollow fibers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540737 TI - Nephrotoxicity of FK-506 in the rat. Studies on glomerular and tubular function, and on the relationship between efficacy and toxicity. AB - Recent studies in liver and kidney transplant recipients revealed a nephrotoxic adverse effect of the new macrolide immunosuppressant FK-506. Therefore the effect of FK-506 0.1 to 0.8 mg per kg per day was investigated in rats using clearance methods including lithium clearance. In rats given FK-506 or placebo during 1 week the nephrotoxicity of FK-506 was characterized by a slight reduction of inulin clearance. The end proximal delivery as measured by the lithium clearance was decreased by FK-506. In rats treated for 4 weeks with FK 506 0.8 mg/kg/day the glomerular filtration rate (GFR) had decreased to 23% of the GFR found in controls (P < 0.001), while end proximal delivery was only 8% of normal. Renal histopathological investigation showed a slight but statistically significant increase of tubular basophilia and atrophy in FK-506-treated rats. Skin transplantation studies in the same rat strain showed a dose-dependent immunosuppressive effect of FK-506. FK-506 0.8 mg/kg was significantly more immunosuppressive than 0.2 or 0.4 mg/kg, so it was concluded that the lower doses of FK-506 did not fully exploit the drug's immunosuppressive potential. Thus in a dosage inside the therapeutic range defined from skin transplantations, FK-506 generated a number of toxic effects including a considerable nephrotoxic effect. The FK-506 induced changes in glomerular and tubular function was a close match to the changes found in cyclosporin A nephrotoxicity. The present study suggests that FK-506 nephrotoxicity is caused by constriction of preglomerular vessels. PMID- 7540738 TI - The sodium-potassium ATPase inhibitor ouabain is neurotoxic in the rat substantia nigra and striatum. AB - Injection of ouabain, a potent and selective inhibitor of Na+/K+ ATPase, into the rat striatum and substantia nigra caused a selective neuronal loss in up to 70% of the lesioned area. However, there was a glial cell/macrophage proliferation in the core of the lesion. Neuronal loss was demonstrated by staining in vivo with Trypan Blue, a marker for dead cells. Cholinesterase staining was also lost, but in the striatum this was revealed only after pre-existing enzyme was irreversibly inhibited in vivo. PMID- 7540739 TI - Direct action of 4-beta-phorbol-12,13-dibutyrate (PDBu) on nicotinic acetylcholine receptor channel independent of protein kinase C activation. AB - Torpedo acetylcholine receptor (AChR) has a protein kinase C (PKC) phosphorylation site, which modulates channel properties, on the alpha and delta subunit. The effect of a potent PKC activator, PDBu on AChR expressed into Xenopus oocytes was examined by whole cell voltage clamp recordings. The pretreatment with 4-beta-PDBu reversely accelerated desensitization of ACh elicited membrane currents and the same effect was shown by co-application of 4 beta-PDBu and ACh without pre-incubation. Treatment with the inactive stereoisomer of phorbol ester, 4-alpha-PDBu also demonstrated an acceleration of desensitization. Furthermore, 4-beta-PDBu enhanced the rate of desensitization in mutant AChR deleting PKC phosphorylation sites on the alpha and delta subunit. These results indicate that phorbol ester directly acts on the AChR channel independent of PKC activation. PMID- 7540741 TI - Colocalization of vasoactive intestinal polypeptide and nitric oxide in penis innervating neurons in the major pelvic ganglion of the rat. AB - By using a combination of fluorescent retrograde labeling and double immunofluorescence histochemistry, the present study revealed in the rat that the vast majority of major pelvic ganglion neurons innervating penile erectile tissue contained both vasoactive intestinal polypeptide (VIP) and nitric oxide synthase. The result suggests that VIP and nitric oxide may exert a cooperative action in penile erection. PMID- 7540740 TI - Blockade of nitric oxide synthase differentially influences background activity and electrical excitability in rat dorsal horn neurones. AB - A previous study has shown that inflammation of the gastrocnemius-soleus muscle in rats leads to an increase in excitability of dorsal horn neurones particularly in the spinal segment L3. Here, we have blocked the nitric oxide synthase (NOS) in L3 by spinal cord superfusion with NG-monomethyl-L-arginine (L-NMMA) to find out if this effect is due to a release of nitric oxide (NO). L-NMMA had no influence on the excitability of L3 neurones but caused a marked increase in background activity. The L-NMMA effect on background activity was also present in rats with intact muscle. The data show that the myositis-induced increase in spinal excitability is not mediated by NO. The background activity, however, appears to be strongly dependent on NO production. PMID- 7540742 TI - Cerebellotectal projection in the rat: anterograde and retrograde WGA-HRP study of individual cerebellar nuclei. AB - Cerebellotectal projections were studied in the rat by the anterograde and retrograde tracing methods using wheat-germ-agglutinin-conjugated horseradish peroxidase. The pathway arises from all four cerebellar nuclei on the contralateral side; mainly from the posterior interpositus nucleus and lateral nucleus and to a lesser extent from the medial nucleus and anterior interpositus nucleus. The fibers arising from the medial nucleus and the posterior interpositus nucleus terminate mainly in the deeper zone of layer IV and in layer VI throughout the entire rostrocaudal extent of the contralateral superior colliculus. Those arising from the anterior interpositus nucleus and the lateral nucleus terminate mainly in the superficial zone of layer IV in the rostral three fourths of the contralateral superior colliculus. In addition, the fibers from the lateral nucleus terminate densely in a zone extending from the deep part of layer III through layer VII in the lateral portion of the rostral half of the superior colliculus. In comparison with data on other species the present findings are discussed with respect to the evolutional changes from monocular to binocular vision. PMID- 7540743 TI - Effect of chemotherapy on immune response to egg antigens of Schistosoma mansoni in chronically infected children from areas of low transmission. AB - In an attempt to identify antigenic molecules from Schistosoma mansoni eggs, a serological study was performed on children of a Venezuelan town (Caraballeda) in which the transmission of schistosomiasis had been interrupted two years prior to sera sampling. Infected children received treatment with Praziquantel and, based on the disappearance of eggs in the stools plus negativization of the circumoval precipitin test (COPT) one year after treatment, they were classified as either responders or non-responders to chemotherapy. Western blots of soluble egg antigen (SEA) with a very sensitive chemiluminescent substrate were performed. Sera from responder children recognized a 25 kDa band of SEA which diminished significantly after treatment. This was less frequent in non-responder children. When the sera of responder and non-responder children were compared before treatment, we found that the recognition of the 40 and 41 kDa proteins could be predictive of response to chemotherapy. All these antigens, used in ELISA-type techniques, might be of importance in the evaluation and follow-up of large scale schistosome control programmes. PMID- 7540745 TI - Cl- channel inhibition by glibenclamide is not specific for the CFTR-type Cl- channel. AB - As long as the question of which channels are responsible for cAMP-mediated epithelial Cl- secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl- currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl- channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl- channel which, potentially, may contribute to transepithelial Cl- secretion. PMID- 7540744 TI - Characterization of epitopes on excretory-secretory antigens of Taenia saginata metacestodes recognized by monoclonal antibodies with immunodiagnostic potential. AB - Four monoclonal antibodies (MoAbs) produced against the excretory-secretory (ES) antigens of Taenia saginata metacestodes are currently used for the detection of circulating antigen in the sera of cattle with bovine cysticercosis. The epitopes recognised by these MoAb were characterized by periodate oxidation, alkaline borohydride and heat treatment, trichloracetic acid precipitation, enzymatic proteolysis and deglycosylation of the ES-products. These results together with those of the homologous sandwich, inhibition and competition ELISAs showed that three repetitive epitopes are recognized, which are present on the tegument and on the ES-products of T. saginata metacestodes. They are different from phosphorylcholine and seem to be either of carbohydrate or partly carbohydrate/partly protein nature. PMID- 7540746 TI - Actin-dependent activation of ion conductances in bronchial epithelial cells. AB - Activation of Cl- and K+ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in 36Cl- efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K+ conductance whilst histamine and forskolin induced mainly a Cl- conductance. HBS activated both K+ and Cl- conductances. Incubation of the cells with brefeldin A (up to 100 mumol/l) did not inhibit WC current activation and 36Cl- efflux. Nocodazole (up to 170 mumol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mumol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also 36Cl- efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540747 TI - Nonselective cationic currents elicited by extracellular ATP in human B lymphocytes. AB - Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor dependent ion channels of about 3 pS was determined by noise analysis of Ip. PMID- 7540749 TI - A frog, a rock, a ritual: myth, mystery, and metaphors for an ecocaring cosmology in a universe that is turning over. PMID- 7540748 TI - Enhancement of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by phorbol esters with and without activation of protein kinase C. AB - The effects of phorbol esters on Ca2+ channel currents in human neuroblastoma SH SY5Y cells were studied using whole-cell patch-clamp recordings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu; 100 nM to 1 microM), known activators of protein kinase C (PKC), enhanced Ca2+ channel currents in a voltage-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca2+ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19-36), and in cells which had been pre-incubated with 500 nM staurosporine, and which were exposed to staurosporine during recordings. Application of 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD; 100 nM), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca2+ channel currents. Residual Ca2+ channel currents recorded after exposure to 1 microM omega-conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 microM) or nifedipine (5 microM), TPA was without effect. When cells were pre-incubated for 10 min at 37 degrees C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 microM nifedipine still inhibited currents to the same degree. This enhancement was not mimicked by 4 alpha-PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at concentrations commonly used to activate PKC) enhance L-type Ca2+ channel currents in SH-SY5Y cells via a PKC-independent mechanism which appears similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC. PMID- 7540750 TI - Princesses and princes from far and wide. PMID- 7540751 TI - Face painting as metaphor. PMID- 7540752 TI - Overexpression of the p53 tumor suppressor gene product in esophageal and gastric carcinomas. AB - PURPOSE: p53 protein has been reported as frequently overexpressed in esophageal and gastric carcinomas. However, the correlation between p53 protein expression and clinico-pathological features of the tumors is debated in this heterogeneous group of cancers. The aim of this study was to establish the prevalence of p53 protein overexpression in a series of resected esophageal squamous carcinomas (n = 78), adenocarcinomas developed on Barrett's esophagus (n = 20), adenocarcinomas of the cardia (n = 36), and adenocarcinomas of the antrum (n = 30), and to correlate this expression with the clinico-pathological and flow-cytometric characteristics of the tumors. METHODS: Immunohistochemical staining was performed on frozen sections with a monoclonal antibody directed against wild type and mutated p53 protein (Pab 1801). An adjacent frozen specimen was used for flow cytometric determination of the DNA-ploidy and S phase fraction. RESULTS: p53 protein nuclear expression was detected in 76% of esophageal squamous carcinomas, in 75% of adenocarcinomas developed in Barretts esophagus, in 56% of adenocarcinomas of the cardia, and in 27% of adenocarcinomas of the antrum. Only the number of positive adenocarcinomas of the antrum was significantly lower when compared to the other three types of tumors (p = 0.001). No significant correlation was observed between p53 protein expression and most of the clinico pathological and flow-cytometric parameters (sex, age, tobacco smoking, chronic alcohol consumption, size of the tumor, grade of differentiation, depth of infiltration, presence of lymph node metastases, UICC stage, DNA-ploidy, S phase fraction). p53 protein expression was more frequent in Lauren's intestinal adenocarcinomas (67%) when compared to the diffuse type tumors (24%) (p = 0.002). CONCLUSIONS: Our results confirm that overexpression of p53 protein is a common feature of esophageal and gastric carcinomas. The high prevalence of p53 protein overexpression found in cardiac adenocarcinoma when compared to antral adenocarcinoma reinforces the hypothesis of distinct carcinogenetic mechanisms in these two cancers. In particular the lack of correlation between p53 expression and tumor stage suggests that p53 protein overexpression is an early event in these tumors. PMID- 7540753 TI - Bone marrow one step fixation-decalcification in Lowy FMA solution: an immunohistological and in situ hybridization study. AB - The immunoreactivity of paraffin embedded bone marrow biopsies (BMB) was studied following a one step 20-hour-fixation-decalcification in Lowy formalin mercuric chlorid acid solution which permits excellent histological stainings. Antibodies reactive with myeloid, megakaryocytic, erythroid cells, T and B lymphocytes, mastocytes and metastatic cells were compared. Nearly all antibodies working on paraffin sections were demonstrated on Lowy FMA fixed BMB. Special care was taken to define an optimal working dilution. Trypsinization was not necessary. A slide microwave pre-treatment appeared essential before testing CD20 L26, CD8, CD3, CD34, MB1 Kappa and Lambda antibodies. It was suitable for UCHL1, LN2, CD30 antibodies. The same fixative allowed an m RNA Kappa or Lambda in myeloma and EBER 1 EBV RNAs in HIV lymphoma visualization by in situ hybridization. The safety handling of the toxic mercuric chloride component is discussed. PMID- 7540754 TI - Cathepsin-D and tumor associated antigen DF3 in salivary gland neoplasia. Differential diagnostic and prognostic applications. AB - Salivary gland tumors pose considerable difficulty in diagnostic and prognostic assessment based on the histopathologic features alone. Cathepsin-D is overexpressed in cancer cells where its concentration in the primary tumor is correlated with increased risk of metastasis. DF3 antigen is a tumor associated glycoprotein that is specific for malignant cells of glandular origin. We examined the distribution patterns of cathepsin-D and DF3 antigens in benign (n = 11) and malignant (n = 44) salivary gland tumors of various histologic types. The frequency of cathepsin-D expression is significantly increased (p < 0.001) in salivary gland carcinomas compared to benign mixed tumors (BMT). High levels of cathepsin-D expression was frequent in carcinomas ex BMT, mucoepidermoid carcinomas, poorly differentiated adenocarcinomas-NOS and adenoid cystic carcinomas (ACC). Acinic cell carcinomas and polymorphous low-grade adenocarcinomas were mostly negative. Intense cytoplasmic staining for DF3 antigen was noted in the tumor cells of mucoepidermoid carcinomas, carcinomas ex BMT and poorly differentiated adenocarcinomas-NOS whereas other types of salivary gland carcinomas exhibited either negative or only focal membrane staining. The noted differences in the reactive patterns of cathepsin-D and DF3 antigen among various histologic types of salivary gland carcinomas may have differential diagnostic and prognostic applications. PMID- 7540756 TI - Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro. AB - We have examined the distribution of the endothelial isoform of nitric oxide synthase (eNOS) in villous and extravillous trophoblast populations by immunohistochemistry and have further studied expression of eNOS during differentiation of cytotrophoblast into syncytiotrophoblast in culture. In first trimester villous tissue, NADPH diaphorase activity and eNOS immunostaining were present in syncytiotrophoblast but not the progenitor cytotrophoblast layer. Extravillous trophoblast in the basal plate of the placenta was identified by anticytokeratin immunostaining and displayed NADPH diaphorase activity, but not eNOS immunostaining. Both amnion epithelial cells and chorion cytotrophoblast had NADPH diaphorase activity but no eNOS immunostaining, whereas eNOS immunostaining was seen in the fibroblast layer of amnion. Purified villous cytotrophoblast cells from term placentae aggregated and fused to form a syncytium with increasing time in culture as assessed by antidesmosomal protein and antinuclear antibody immunostaining. Following 24 h in culture, the majority of cells were still mononucleate cytotrophoblast which did not display eNOS immunostaining, whereas a few syncytial aggregates had formed which were both eNOS positive and hPL positive. By 3 to 5 days in culture, the majority of cells were present as syncytiotrophoblast. However, eNOS and hPL immunostaining was more diffuse and not all syncytial aggregates were positive. Of the trophoblast populations, only syncytiotrophoblast appears to express eNOS. Differentiation of cytotrophoblast into syncytiotrophoblast is associated with eNOS expression. PMID- 7540757 TI - Basal and interferon-induced 2',5'-oligoadenylate synthetase activity in human placental trophoblast and trophoblast-derived malignant cell lines. AB - Human placental trophoblasts produce interferon (tro-IFNs) when stimulated with viral inducers. Since the antiviral and cellular functions of IFNs are partly mediated by the 2',5'-oligoadenylate synthetase (2-5A synthetase) pathway, the aim of the present study was to determine the basal and IFN-induced levels of 2 5A synthetase in villous trophoblast cultures. A considerable basal level of 2-5A synthetase was observed in syncytiotrophoblast cultures from both first and third trimester. In contrast no basal activity was detectable in placental fibroblast- and trophoblast-derived malignant cell lines (Far, FEG-3, and BeWo). Stimulation with tro-IFN-beta, -alpha and leucocyte-IFN (leu-IFN)-alpha increased the enzyme activity in first and third trimester human syncytiotrophoblast cultures. Treatment with recombinant-IFN (rec-IFN)-gamma significantly enhanced 2-5A synthetase activity in first trimester syncytiotrophoblast, but had no effect on third trimester syncytiotrophoblast. Tro-IFN-beta, -alpha and leu-IFN-alpha induced high levels of 2-5A synthetase activity in placental fibroblast, BeWo and FEG-3 cell-lines, whereas rec-IFN-gamma treatment did not induce 2-5A synthetase activity in any of these cells. No detectable 2-5A synthetase activity was found in the Far cell line. The capability of cells deriving from the fetoplacental unit to raise an antiviral response by the induction of 2-5A synthetase may be important in defending the fetus against viral infection from the mother. Furthermore 2-5A synthetase in cells of the fetoplacental unit may play a role in their normal growth and development. PMID- 7540755 TI - Stimulation of cultured melanocytes in medium containing a serum substitute: Ultroser-G. AB - Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP, LTC4 and a purified form of alpha melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPA/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/alpha-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype. PMID- 7540759 TI - Trypsinogen and other pancreatic enzymes in patients with renal disease: a comparison of high-efficiency hemodialysis and continuous ambulatory peritoneal dialysis. AB - Although serum amylase and lipase levels have been studied extensively in patients with renal disease, there are fewer data regarding trypsinogen levels in patients with end-stage renal disease (ESRD) treated with different dialytic modalities. We therefore evaluated the blood concentrations of trypsinogen, amylase, and lipase in asymptomatic patients with chronic renal insufficiency (CRI) and ESRD, to determine whether treatment modality or renal handling of these enzymes is important in determining steady-state levels in asymptomatic patients with chronic renal disease. Mean trypsinogen concentration levels were higher in hemodialysis (HD) patients and patients with CRI compared with normal subjects when values in the different groups were compared. There was no difference in the mean trypsinogen levels between patients treated with HD and those with CRI, between patients treated with chronic ambulatory peritoneal dialysis (CAPD) and those treated with HD, or between CAPD patients and patients with CRI. The mean circulating trypsinogen concentration was elevated more frequently and to a higher level than amylase or lipase in patients with CRI and ESRD. HD treatment did not result in a lowering of mean circulating pancreatic enzyme levels. We propose that decreased peripheral clearance, pancreatic overproduction, increased release from the pancreas, or a combination of these mechanisms is responsible, at least in part, for the increased plasma concentration of trypsinogen in patients with CRI, rather than simply a decrease in renal clearance. PMID- 7540758 TI - Trophoblast basement membrane haemosiderosis in the placental lesion of fetal artery thrombosis: a marker for disturbance of maternofetal transfer? AB - The placental lesion of fetal artery thromboses is characterized by collapse and obliteration of chorionic vasculature, an increase in stromal connective tissue and syncytial knots, with a thickening of trophoblast basement membrane. An additional feature, not previously described in association with the lesion, is linear trophoblast basement membrane haemosiderosis. Thirty-five such lesions were examined for this feature which was identified in 32. Random tissue sections of placentae from cases of intrauterine death showed a similar basement membrane haemosiderosis and were used as positive controls. None of 20 normal control cases examined demonstrated the feature. Electron microscopy demonstrated electron-dense bodies within the basement membrane. Spectrographic analyses confirmed the presence of iron within these deposits. The significance of this finding lies not so much in the fact that it is an additional finding in fetal artery thrombosis but rather in the underlying pathophysiology. PMID- 7540760 TI - The relationship between pancreatic enzyme release and activation and the acute phase protein response in patients with acute pancreatitis. AB - It has been suggested that the severity of an attack of acute pancreatitis is related to the presence of intraglandular trypsinogen activation and that disease severity is also reflected by the degree of the acute-phase protein response. In this study we examine the relationships among amylase release, the degree of trypsinogen and prophospholipase A2 activation [as measured by urinary trypsinogen activation peptide (TAP) and prophospholipase A2 activation peptide (PLAP) concentrations], and the serum concentrations of the acute phase-protein C reactive protein (CRP) and the principal mediator of the acute-phase protein response, interleukin-6 (IL-6). Twenty-four patients (14 mild and 10 severe attacks) were studied. Peak serum amylase concentrations were seen within 12 h and peak urinary TAP/creatinine (Cr) and PLAP/Cr ratios between 12 and 24 h after the onset of symptoms, preceding those of IL-6 and CRP. The integrated TAP/Cr and PLAP/Cr responses were significantly greater in those with severe disease [95% confidence internal (CI) = 106-259.6 pmol/mmol/h, p < 0.0008; and 95.1% CI = 462.2-3887 pmol/mmol/h, p < 0.003, respectively]. The integrated amylase response was not significantly greater in those with severe disease (95.6% CI = -415 to 832 IU/L/h, p < 0.14). There was a strong correlation among the integrated IL-6, TAP/Cr (r = 0.63, p < 0.01), and PLAP/Cr (r = 0.64, p < 0.01) responses but a poor correlation with the integrated amylase response (r = 0.19, NS).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540762 TI - Cytotoxicity of neutrophil-induced superoxide to isolated pancreatic acinar cells. PMID- 7540761 TI - Cholinergic stimulatory effect of intragastric administration of a prostaglandin E2 analogue on pancreatic exocrine secretion in conscious rats. AB - The effect of a long-acting, potent synthetic analogue of prostaglandin E2, enprostil, on pancreatic exocrine secretion was examined in conscious rats. Rats were prepared with cannulae draining bile and pancreatic juice separately. Pancreatic exocrine secretion was increased by intragastric administration of enprostil but inhibited by its intravenous administration. The pancreatic response to intragastric administration of enprostil was not inhibited by the administration of cholecystokinin antagonist or secretin antibody, or by bilateral vagotomy, but was completely abolished by atropine. Therefore, intragastric administration of enprostil seemed to stimulate pancreatic exocrine secretion via a peripheral gastro-(entero)-pancreatic reflex. PMID- 7540763 TI - [Development and characterization of water soluble dextran fatty acid esters as excipients for colon-targeting]. AB - In this paper production methods of certain colon-degradable dextran fatty acid esters and their relevant properties as excipients for colon-targeting dosage forms are reported. Such dextran fatty acid esters must be insoluble in, resp. resistant against, gastric and small intestinal fluids. But to a certain degree they must be swellable in aqueous liquids in order to allow an enzymatic attack of the colonic microflora. Furthermore, they must be adequately soluble in pharmaceutically accepted solvents to permit the technological application of film coatings. Otherwise, they can only be used as embedding excipients or matrices. As the most suitable excipients for film coatings, biodegradable by the colonic microflora, but resistant against gastric and small intestinal fluids, the dextran lauroyl esters were found. Their molecular weight have to be between 60,000 and 250,000, and their average degree of substitution (DS) between 0.06 and 0.2. Are the molecular weights higher the DS must be correspondingly lower. PMID- 7540764 TI - Effect of hydrocortisone and disodium cromoglycate on mast cell-mediator release induced by substance P. AB - Release of inflammatory mediators from mast cells following immunoglobulin bridging by specific allergens triggers episodes of asthma and bronchial hyperreactivity. Recent evidence has shown that neuropeptides, such as substance P (SP), may modulate the pulmonary inflammatory response in these airway diseases. This suggests that SP may affect secretory events of mast cells. To investigate these effects, resident peritoneal mast cells were collected from Sprague-Dawley male rats and stimulated with Con A (utilizes surface-bound immunoglobulin), compound 48/80 (acts in a peptide-like manner) and SP. Secretion was assessed as the release of preloaded [14C]serotonin. All secretagogues induced dose-dependent release. Pharmacologic modulation of release was then studied with two drugs employed for treatment of airway disease, hydrocortisone, a classical anti-inflammatory steroid, and disodium cromoglycate (DSCG). Following pretreatment with 5 mumol/l hydrocortisone, serotonin release induced by Con A was inhibited by 59%. No inhibition was noted with compound 48/80 or SP release. Similarly, following DSCG (300 mumol/l) pretreatment, 40% inhibition of release was noted with Con A, but no inhibition occurred following compound 48/80 or SP-stimulated release. Collectively, these results suggest that mast cells possess multiple activation-secretion coupling pathways which respond differently to clinically used pharmacologic agents. Diseases involving SP modulation of mast cell mediator release may not be successfully treated with anti-inflammatory steroids or DSCG. PMID- 7540765 TI - Intracellular translocation of endothelial nitric oxide synthase by lysophosphatidylcholine. AB - The amphiphile lysophosphatidylcholine (LPC) modulates the activity of membrane associated enzymes such as phospholipase A2, adenylate and guanylate cyclases and ATPase. LPC also relaxes vascular smooth muscle through production of nitric oxide. On the basis of reports that bradykinin translocates nitric oxide synthase (NOS) from the membrane to the cytosol, we investigated whether a similar translocation occurs with LPC. It was found that LPC translocated NOS from the membrane to the cytosolic fraction. Total NOS activity remained at the control level. PMID- 7540766 TI - Transcription factors in interferon signaling. AB - Interferons (IFNs) comprise a family of polypeptides that exhibit diverse biological effects such as inhibition of cell growth and protection against viral infection. These activities are based mainly on the transcriptional induction of cellular genes by both type I (IFN-alpha and IFN-beta) and type II (IFN-gamma) interferons. Several of these IFN-induced early response genes have been cloned and common elements within their promoters defined. Transcription factors, such as interferon-stimulated gene factor-3, IFN-gamma activation factor and FcRF gamma, that bind to these enhancers subsequently have been isolated and their components identified. This review shall provide an overview of the DNA response elements, the components of the IFN-induced transcription factors and their mechanism of action. PMID- 7540767 TI - Cellular interactions and metabolism of aflatoxin: an update. AB - The aflatoxins are a group of closely related mycotoxins that are widely distributed in nature. The most important of the group is aflatoxin B1 (AFB1), which has a range of biological activities, including acute toxicity, teratogenicity, mutagenicity and carcinogenicity. In order for AFB1 to exert its effects, it must be converted to its reactive epoxide by the action of the mixed function mono-oxygenase enzyme systems (cytochrome P450-dependent) in the tissues (in particular, the liver) of the affected animal. This epoxide is highly reactive and can form derivatives with several cellular macromolecules, including DNA, RNA and protein. Cytochrome P450 enzymes may additionally catalyse the hydroxylation (to AFQ1 and AFM1) and demethylation (to AFP1) of the parent AFB1 molecule, resulting in products less toxic than AFB1. Conjugation of AFB1 to glutathione (mediated by glutathione S-transferase) and its subsequent excretion is regarded as an important detoxification pathway in animals. Resistance to AFB1 toxicity has been interpreted in terms of levels and activities of these detoxifying pathways. This article reviews the multiple reactions and effects attributed to aflatoxin, with particular reference to the interaction of aflatoxin with nucleic acids and proteins, and the contribution this mycotoxin has in disease development and in the promotion of hepatocellular carcinoma (HCC). The anti-mutagenic properties of several dietary factors are also considered in this article. Undoubtedly, the most important aspect of aflatoxin action is its putative role in the development of human cancer, in particular, HCC. Recently, there has been a renewed interest in this aspect and experimental evidence is rapidly accumulating at the molecular level, indicating aflatoxin as an important consideration in the aetiology of human HCC. PMID- 7540768 TI - Mapping and geographic display of data. PMID- 7540769 TI - [Hormone treatment of patients with breast carcinoma]. AB - Endocrine treatments of breast cancer patients play an important role in the management of this common tumor of women. In this short review the most frequently applied drugs are discussed; thereafter, their use is shown in the curative (adjuvant) and in the palliative situation. In premenopausal women who suffer from a hormone-sensitive cancer, adjuvant treatments include either ovarian ablation or adjuvant chemotherapy with cyclophosphamide, methotrexate, and 5-fluoro-uracil. In post-menopausal women, the adjuvant treatment of choice is tamoxifen. The currently active protocols for adjuvant treatments of hormone sensitive breast cancers of the International Breast Cancer Study Group (IBCSG) and the Swiss Group for Clinical Cancer Research (SAKK) should not be followed because of the anthracyclines administered to a high percentage of the involved patients. These drugs, known for their cardiotoxic side effects, have for the time being no place in the adjuvant treatment of hormone-sensitive breast cancers; therefore, the duty of the primary-care physician is to protect his patients from these protocols. In the palliative treatment, tamoxifen is the first choice. The success of this treatment is shown by a few examples. PMID- 7540770 TI - The culture of credit. AB - Scientific ideals call for collaboration and sharing. But in today's competitive scientific enterprise, a tremendous premium is placed on individual credit, setting the stage for conflict. PMID- 7540771 TI - Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. AB - The crystal structures of a cysteine-215-->serine mutant of protein tyrosine phosphatase 1B complexed with high-affinity peptide substrates corresponding to an autophosphorylation site of the epidermal growth factor receptor were determined. Peptide binding to the protein phosphatase was accompanied by a conformational change of a surface loop that created a phosphotyrosine recognition pocket and induced a catalytically competent form of the enzyme. The phosphotyrosine side chain is buried within the period and anchors the peptide substrate to its binding site. Hydrogen bonds between peptide main-chain atoms and the protein contribute to binding affinity, and specific interactions of acidic residues of the peptide with basic residues on the surface of the enzyme confer sequence specificity. PMID- 7540772 TI - Impact of brain tumor biology on the evolution of therapy. PMID- 7540773 TI - Diagnostic significance of serum neuron-specific enolase and myelin basic protein assay in patients with acute head injury. AB - BACKGROUND: Neuron-specific enolase (NSE) and myelin basic protein (MBP) in the peripheral venous blood (PVB) have been reported to be sensitive markers for judging the prognosis of patients with head injury. However, to our knowledge, the levels of NSE and MBP in the internal jugular venous blood (IJVB) have never been studied. METHODS: In 25 patients with acute head injury, blood samples were taken from the internal jugular vein and the peripheral vein at the same time before any medical or surgical procedure was performed. The levels of NSE and MBP in the both venous blood samples were measured. The time interval between injury and sampling was 1.5-8.0 hours (mean 4.3 hours). The levels of NSE and MBP in the IJVB were compared to those in the PVB. The relationship between the clinical outcome and the serum levels of those was evaluated. RESULTS: The levels of NSE and MBP in the IJVB were almost equal to those in the PVB. The levels of NSE and MBP were significantly higher in the patients who died than in those who survived. In the survivors, the levels of NSE and MBP in the IJVB were 17.6 +/- 11.4 ng/ml and 1.4 +/- 1.5 ng/ml, whereas in the patients who died, both levels were elevated to 51.3 +/- 27.3 ng/ml (p < 0.005) and to 11.3 +/- 9.5 ng/ml (p < 0.01), respectively. CONCLUSIONS: The assay of serum NSE and MBP levels provides a reliable laboratory indicator of the degree of brain damage and allows early prediction of the prognosis in patients with acute head injury. PMID- 7540775 TI - First experience with FK 506 for treatment of chronic pulmonary rejection. PMID- 7540774 TI - Factors that contribute to spontaneous platelet aggregation and streptokinase induced aggregation in whole blood. AB - When whole blood is stirred there is a "spontaneous" platelet aggregation (SPA) which is presumed to be caused by proaggregatory factors released from platelets and other blood cells. Adding streptokinase (SK) to stirred whole blood frequently increases the rate and extent of the platelet aggregation that occurs; this is likely to be via immune complex formation between SK and natural anti-SK antibodies leading to increased release of pro-aggregatory factors. In this investigation we have examined the effects of several inhibitors and antagonists in an attempt to identify the proaggregatory factors that contribute to both SPA and SK-induced aggregation (SKA) and to evaluate different means of inhibiting both processes. The effects of the inhibitors/antagonists were determined in vitro after adding them to citrated whole blood obtained from healthy volunteers. Platelet aggregation was measured using a platelet counting technique. Inhibition of both SPA and SKA by apyrase and by FPL 66096 (a P2T receptor antagonist) demonstrated the involvement of ADP in both processes. Inhibition by chlorpromazine indicated that the most likely source of the ADP is red cells. The effects of sulotroban (a TXA2 antagonist) indicated involvement of TXA2 in SKA but not in SPA. The lack of effect of specific antagonists at S2, alpha 2 and PAF receptors suggested lack of involvement of serotonin, catecholamines and platelet activating factor in either SPA or SKA. Both SPA and SKA were potently inhibited by low concentrations of iloprost (a PGI2 analogue), but a high concentration of SIN-1 (a NO donor) was much less effective.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540776 TI - Nitric oxide synthase induction and chronic cardiac allograft rejection. PMID- 7540779 TI - Isolation of a small monocyte subset with high accessory function: a dendritic cell precursor? PMID- 7540778 TI - Expression of alternatively spliced CD44 mRNA in sheep renal allografts. PMID- 7540777 TI - Quantitation of cytokine mRNA expression in biopsies from human liver allografts by a novel reverse transcriptase/polymerase chain reaction method. PMID- 7540781 TI - The two-state model of receptor activation. AB - Over the past few years, the concept that the activation of G protein-coupled receptors and transmitter-gated ion channels depends on a conformational change has received increasingly widespread acceptance. As a result, these two structurally distinct families of receptors can now be considered to obey a similar two-state mechanism. However, traditional receptor theory has largely overlooked this concept. In this article, Paul Leff explains and illustrates the predictions of the two-state model of receptor activation and discusses its impact on the analysis and interpretation of agonist-receptor interactions. PMID- 7540780 TI - Immunosuppressive and diabetogenic effect of FK 506 on pancreatic islet xenotransplantation. PMID- 7540783 TI - Prevalence and activation phenotype of Sendai virus-specific CD4+ T cells. AB - The relative prevalence of Sendai virus-specific CD4+ T helper precursors (Thp) has been assessed by limiting dilution analysis (LDA). Within 10 days after intranasal inoculation of C57BL/6 mice, Thp prevalence increased from < 1 in 10(5) to approximately 1 in 200 in CD4+ T-cell-enriched spleen and mediastinal lymph node populations. These frequencies remained elevated relative to naive animals, with the majority of the Thp recovered at 2-3 months after infection being found in the spleen. The "memory" Thp express an "activated" L-selectin low, CD44-high, alpha 4-integrin-high phenotype, comparable to that described previously for CD8+ cytotoxic T lymphocyte precursors (CTLp) specific for Sendai virus. Background effects for the IL-2-based Thp analysis are, however, less predictable than those found previously for the 51Cr release CTLp LDA, as the extent of non-virus-specific lymphokine production varies. However, provided the analysis is appropriately controlled, the approach does allow useful comparisons between phenotypically different subsets of antigen-specific CD4+ T cells. PMID- 7540782 TI - Expression of adhesion molecules in the host response to colon carcinoma. AB - Expression of the adhesion molecules LFA-1, ICAM-1, and VCAM-1 was studied in five resected colon carcinomas, one villous adenoma, and normal colon mucosa to determine whether expression of these markers is increased in response to tumor. Frozen tissue samples were stained by a labeled avidin-biotin technique using primary antibodies to LFA-1 (CD11a), CD2, CD4, CD8, CD20, CD68, HLA-DR, ICAM-1 (CD54), and VCAM-1. For each marker, the number of positive mononuclear cells was graded semiquantitatively, and stromal and endothelial cells were scored as either positive or negative. Overall grade of inflammation was increased in tumor compared with normal mucosa in five cases. Cells positive for LFA-1, CD2, CD4, CD8, and CD20 were increased in the tumors in the same cases. ICAM-1 was expressed in vessels, inflammatory cells, and stromal cells in normal mucosa. It was markedly increased in tumor stroma in all six cases. VCAM-1 was negative in normal mucosa, and focally expressed in tumor vessels and stroma. We conclude that increased expression of adhesion molecules occurs around colon neoplasms, particularly in stromal cells, and may be a mechanism for the recruitment of activated leukocytes as part of the inflammatory response to colon carcinomas. PMID- 7540784 TI - Resistance to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives is generated by mutations at multiple sites in the HIV-1 reverse transcriptase. AB - Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400 resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds. PMID- 7540786 TI - [Immunomodulating therapy of rheumatoid arthritis (chronic polyarthritis): use of monoclonal antibodies]. AB - Rheumatoid arthritis is a chronic inflammatory disease mainly affecting the joints. The etiology is still unknown, but it shows several clinical and laboratory features of an autoimmune process. The conventional medical therapy consisting of a combination of analgetic and antiphlogistic agents with so called slow acting drugs is often not satisfactory and fails to persistently suppress disease activity. Therefore, therapeutic alternatives are necessary which are provided by a new generation of immunosuppressing agents including monoclonal antibodies. The paper will summarize some therapeutic approaches using monoclonal antibodies towards cell surface antigens or soluble mediators of inflammation for therapy of rheumatoid arthritis. PMID- 7540785 TI - A highly divergent retroviral sequence in the tuatara (Sphenodon). AB - Vertebrate retroviruses have been classified into a number of different genera, and although many mammalian and avian examples have been characterized, less information exists about the retroviruses present within other vertebrate classes. We designed oligonucleotide primers against two highly conserved motifs within retroviral protease and reverse transcriptase genes and used them to isolate a retroviral fragment from the reptile tuatara (Sphenodon). Sequence analysis of this element, termed SpeV, demonstrated that it is substantially different from previously reported viruses, and that it cannot easily be placed into any known retroviral genus. Furthermore SpeV suggests that there may be some major differences between the retroviral populations found in reptiles and those present in mammals and birds. PMID- 7540787 TI - [Gestational trophoblastic tumors--a report of experiences]. AB - The paper reports on clinical experiences of treatment of 58 patients with Gestational Trophoblastic Tumors (GTT), collected between 1978 and 1991. According to the Bagshawe-Score, 29 patients were at low-risk, 10 patients were assigned to the high-risk category. Among 29 metastatic cases, 5 patients had brain metastasis. In 33 patients, treatment started from the time of diagnosis. In 25 cases, treatment was initiated at other hospitals and patients were referred only after various unsatisfactory treatment measures. Low-risk patients were mainly subjected to methotrexate and folinic acid. Patients at medium-risk received a sequential chemotherapy. In high-risk patients we preferred the CHA MOCA- or the EMA/CO-regimen. Treatment was successful in 91.4% of patients including all cases of low- and medium-risk. Five patients with brain metastases received systemic chemotherapy combined with intrathecal application of methotrexate and radiotherapy. Three of them could be cured. Patients taken from other hospitals more often underwent primary hysterectomies prior to systemic chemotherapy (40% versus 3%) and more often developed drug resistant tumors due to inadequate primary treatment. Five patients (8.6%) died from their disease, but only one of them received primary treatment in our department. Thus, the outcome (1/33 compared to 4/25) was significantly better for patients treated primarily at specialized centers. PMID- 7540788 TI - Effect of dextran on plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) during surgery. AB - Dextran is known to increase the plasminogen activation rate in vitro and to decrease the alpha2-antiplasmin activity. We decided to explore the effect of dextran on plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) during surgical trauma. Thirty-one patients undergoing elective surgery were given 500 ml of 6% dextran 70. Another nine patients serving as controls were given 500 ml of a glucose-electrolyte solution. The activities of t-PA and PAI-1 during surgery were determined, as was the concentration of t-PA antigen. PAI-1 activity was decreased by 19% after infusion of 250 ml of dextran. After 500 ml, the activity was reduced by 22% (both P < 0.05). The activity of t-PA was increased by 43% and 29% (both P < 0.05) and the antigenic amount of t-PA was increased by 18% and 15% (both P < 0.05) after infusion of 250 ml and 500 ml of dextran, respectively. No changes in these variables were observed in the control patients. It is concluded that infusion of dextran promotes fibrinolysis by enhancing plasminogen activation in patients subjected to trauma. Since elevated levels of PAI-1 prior to surgery are known to predispose to deep vein thrombosis, which may form already during the operation, the effect of dextran on PAI-1 described here may explain its clot preventing properties. PMID- 7540789 TI - Replacement of major surgical blood loss by hypo-oncotic or conventional plasma substitutes. AB - The purpose of the study was to examine the effects of hypooncotic 4% hydroxyethyl starch 120/0.7, 3% dextran 70, 5% albumin and hyperoncotic 6% hydroxyethyl starch 120/0.7 on the perioperative colloid osmotic pressure (COP), albumin and protein concentrations and fluid balance. The plasma substitutes were used with red cell concentrates to replace blood loss with equal volume in sixty major abdominal or urological surgeries. A special effort was made to keep replacements and losses at even volumes constantly and to avoid fluctuation of blood volume. The blood specimen were obtained before induction, after each 20% blood loss, at the end of the recovery room phase and on the three following postoperative mornings. There were significant differences in the peroperative and immediate postoperative COPs. However, these differences had vanished by the first postoperative morning. COP was preserved above 16 mmHg in all groups throughout the study. The identical peroperative albumin and protein concentrations of the synthetic colloid groups suggests that their volume effect was the same, regardless of the varying COP. During the observation period there were no significant differences among the groups concerning the diuresis and the fluid balances. We conclude, that the hypooncotic 4% HES 120 and 3% dextran 70 solutions provide the same clinical effect as 6% HES 120 solution. Consequently less colloid is needed, which allows the use of greater volumes of the dilute colloid solutions in replacement therapy. PMID- 7540790 TI - Does colloid-induced plasma hyperviscosity in haemodilution jeopardize perfusion and oxygenation of vital organs? AB - BACKGROUND AND METHODS: The infusion of dextran solutions is associated with haemodilution and, under some conditions, with a slight increase in plasma viscosity. To clarify the compound effects of simultaneous haemodilution and plasma viscosity increases on macro- and microhaemodynamics, we investigated the changes in arterial perfusion (radiolabelled microspheres, 15 microns o) and oxygenation (tissue Po2) of vital organs using an animal model of plasma hyperviscosity. In nine splenectomized beagles plasma viscosity was increased step by step from 1.06 (baseline) to 2.14, and 2.99 mPa.s by infusion of small amounts (4% of total blood volume) of an ultra-high-molecular-weight dextran (50% w/v, mw: 500,000). RESULTS: Despite the significant increase in plasma viscosity, cardiac output as well as specific organ blood flows in heart, brain, liver, and muscle rose steadily with each step of viscosity, while the haematocrit declined from 0.31 to 0.24 and 0.20, respectively. Medians of tissue Po2 in liver peaked at a viscosity of 2 mPa.s and returned to baseline values at 3 mPa.s, whereas in non-working skeletal muscle Po2 values were maximal at 3 mPa.s. CONCLUSION: These results indicate that the impact of plasma viscosity on the rheological properties of whole blood is completely offset by the concomitant reduction of haematocrit. Thus, the comparatively minor changes in plasma viscosity observed after prolonged use of clinical dextrans and other colloids in no way compromise the perfusion and oxygenation of vital organs. PMID- 7540791 TI - Oral tolerance: 'of mice and men'. PMID- 7540793 TI - Granulocyte-colony stimulating factor levels in cord blood and neonatal peripheral blood. AB - We measured the granulocyte-colony stimulating factor (G-CSF) levels in cord blood and peripheral blood obtained from full-term or pre-term infants during the first 3 days of birth. The mean G-CSF level among cord blood (17.2 pg/mL) was similar to that of peripheral blood on day 0 (18.3 pg/mL) and day 1 (13.6 pg/mL), while that of peripheral blood on day 0 was significantly higher than on day 2 (10.9 pg/mL) and day 3 (8.8 pg/mL; both P < 0.05). There was no correlation between neutrophil counts and G-CSF levels. No difference was found in neutrophil counts or G-CSF levels between infants who weighed more or less than 2500 g at birth. These results suggest that the neonatal neutrophil count depends on regulatory factors other than G-CSF. PMID- 7540792 TI - Ex vivo expansion of umbilical cord blood hematopoietic progenitor cells by combinations of cytokines. AB - Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-gamma). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38- and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38- cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus IFN-gamma were significantly effective in maintaining CD34+CD38- cells than the other combinations (P < 0.05). After 4 weeks of culture. CD34+CD38- cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 +/- 3.3-fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 +/- 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34+CD38- cells (15.8 +/- 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-gamma, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines. PMID- 7540794 TI - Belgian multicenter clinical study of alfuzosin, a selective alpha 1-blocker, in the treatment of benign prostatic hyperplasia. The Alfuzosin Belgian Group. AB - The effects of alfuzosin, a potent alpha 1-blocker, were assessed in patients with benign prostatic hyperplasia, in a double-blind, multicenter, placebo controlled, cross-over study. Treatment duration was 4 weeks for both alfuzosin and placebo. A significant beneficial effect of alfuzosin (7.5 to 10 mg a day) on clinical subjective and objective criteria was observed as compared to placebo: total Boyarsky score decreased by 1.63 points, peak urinary flow improved by 2.03 ml/sec. Alfuzosin was well tolerated, the observed adverse events corresponded to the pharmacological properties of this compound, and resolved rapidly. PMID- 7540795 TI - [5-alpha-reductase inhibitors]. AB - A reflection is made, on the one hand, on the lack of correlation between the intensity of micturition problems and the volume of the prostate and, on the other hand, on the different therapeutic approaches of irritative or obstructive voiding problems, and finally on the insufficiently convincing activity of Finasteride. PMID- 7540796 TI - Transurethral needle ablation (TUNA): histopathological, radiological and clinical studies of a new office procedure for treatment of benign prostatic hyperplasia. AB - Many attempts have been made to develop a method for treating benign prostatic hyperplasia (BPH) that is minimally invasive, efficacious, and low cost. The transurethral needle ablation (TUNA) device has recently been developed to treat BPH by selectively ablating hyperplastic prostatic tissue. A special catheter incorporates needles that deliver low-level radiofrequency power directly to a very localized area of the prostate. The needles have adjustable shields to protect the urethra if desired or necessary. It is positioned via transrectal ultrasound or direct vision. A pilot study was performed in patients to evaluate TUNA feasibility via histopathological measurement of thermal lesion size and TUNA safety. Fifty patients have been treated, twenty-five patients were treated using TUNA prior to scheduled retropubic prostatectomy. The surgical prostatic specimens were recovered from 1 day to 1 month after TUNA, were stepsectioned, and examined histologically. Patients were 69-years-old on average with prostate weight varying from 14 to 88 g. The TUNA procedure averaged 30 minutes, 4 lesion treatments per prostate, and 4-15 W of power applied for 3 to 5 minutes. Proximal lesion temperature was about 40-70 degrees C with central lesion temperatures of about 110 degrees C with central lesion temperatures of about 110 degrees C. Urethral temperature averaged 37-42 degrees C and rectal temperature remained unchanged. Macroscopic examination of the specimens demonstrated localized lesions averaging 12 x 7 mm for 3 min and 10 x 17 for 5 min treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540797 TI - Characterization of monoclonal antibodies against broad bean stain and red clover mottle viruses. AB - Seven monoclonal antibodies (MoAbs) against red clover mottle comovirus (RCMV) and/or broad bean stain comovirus (BBSV) were characterized and used for epitope comparison of both viruses. All tested MoAbs, exclusively of IgG class, were directed to detergent-stable epitopes (metatopes and cryptotopes). Two of them were species-specific, while five others cross-reacted with both viruses to different extent. On the basis of the results of competitive ELISA, we assume different mutual position of common linear epitopes in RCMV and BBSV. They are more closely "packed up" on BBSV, while the BBSV-specific metatope is markedly dominant. The investigation of other RCMV and BBSV isolates by use of our MoAbs confirmed close relationship between both viruses and even showed that it is difficult to determine unambiguously the species of these isolates using immunochemical methods only. PMID- 7540798 TI - Effects of concomitant risperidone and lithium treatment. PMID- 7540800 TI - Prolonged erection associated with risperidone treatment. PMID- 7540799 TI - Behavioral stimulation associated with risperidone initiation. PMID- 7540801 TI - Risperidone treatment for a Tourette's disorder patient with comorbid obsessive compulsive disorder. PMID- 7540802 TI - Switching patients from clozapine to risperidone therapy. PMID- 7540803 TI - Risperidone-induced prolactin elevations in premenopausal women with schizophrenia. PMID- 7540804 TI - Papillary renal cell carcinoma. Histology and immunohistochemistry. AB - Papillary renal cell carcinoma (RCC) is an uncommon subtype of RCC that has distinctive gross, histologic, and cytogenetic features, but for which only limited immunohistochemistry data have been reported. We compared 36 papillary RCCs and five renal cell adenomas with 19 non-papillary (clear cell and granular) RCCs using a variety of antibodies to keratin and carcinoembryonic antigen (CEA). Papillary tumors were often multifocal and associated with coexistent adenomas, whereas nonpapillary tumors generally lacked these features. Low-grade papillary RCCs demonstrated three occasionally overlapping histologic patterns (typical, trabecular, and sclerotic), whereas high-grade tumors were characterized by an admixture of many patterns. Immunohistochemically, 100% (36 of 36 cases) of the papillary tumors were positive for AE1/AE3, and 92% (33 of 36 cases) were positive for callus keratins; only 3% (one of 36 cases) stained for 34BE12, and 11% (four of 36 cases) weakly stained for CEA. The five renal cell adenomas were likewise positive for AE1/AE3 (five of five cases) and callus (five of five cases) keratins. In contrast, 85% (16 of 19 cases) of the nonpapillary tumors stained for AE1/AE3, but only 5% (one of 19 cases) stained for callus; none (0/19) stained for 34BE12, and 10% (2/19) weakly stained for CEA. The consistent expression of callus keratins by papillary RCCs and renal cell adenomas underscores the close relation of these lesions, providing additional evidence for their oncological distinction from nonpapillary RCCs. PMID- 7540805 TI - Prognostic significance of keratinization in nasopharyngeal carcinoma. AB - INTRODUCTION: Keratinization may be a vulnerable aid in predicting response to therapy for nasopharyngeal carcinoma. METHODS: The presence or absence of keratin in biopsy specimens was correlated with tumor behavior, locoregional control, patterns of failure, and survival of patients irradiated for nasopharyngeal carcinoma. RESULTS: Patients with keratinizing squamous-cell cancers (World Health Organization [WHO] type 1) had a higher incidence (76%) of locally advanced tumors than those with nonkeratinizing (WHO type 2) and undifferentiated (WHO type 3) cancers (55%). The former group of patients had a lower incidence (29%) of lymph node metastases than the later group (70%). Primary tumor was controlled in 62% and neck nodes were controlled in 82% of all patients. Primary tumor control rates were 29% in patients with keratinizing squamous-cell cancers and 79% in those with nonkeratinizing and undifferentiated cancers (P = .001). Nodal control rates were 76% for keratinizing squamous-cell cancer and 85% for nonkeratinizing and undifferentiated cancers (P = .001). The incidence of distant metastases was 6% in patients with keratinizing squamous-cell cancer and 33% in those with nonkeratinizing and undifferentiated cancers (P = .001). Patients with keratinizing squamous-cell cancers, even though they had a lower incidence of lymphatic and distant metastases, had a poorer survival rate because of a higher incidence of deaths from uncontrolled primary tumors and nodal metastases. The 5 year survival rates were 35% for all patients, 6% for those with keratinizing squamous-cell cancers, and 51% for nonkeratinizing and undifferentiated cancers respectively (P = .001). CONCLUSION: Higher doses of external beam radiotherapy with or without brachytherapy boost may be needed to improve local control and survival of patients with keratinizing squamous-cell carcinoma of the nasopharynx, whereas an effective systemic therapy is needed for nonkeratinizing and undifferentiated cancers, which tend to metastasize. PMID- 7540806 TI - [The role of serum beta-HCG levels for the diagnosis of ectopic pregnancy]. AB - The study includes 56 cases of women admitted at Second gynaecology clinic of the University Maternity Hospital Sofia with evidence or suspicion of having an ectopic pregnancy who had their serum beta-HCG levels determined quantitatively. 27 of them showed no beta-HCG in their sera and none turned out to have pregnancy, neither intrauterine nor ectopic. All cases of ectopic pregnancies (a total of 20) were associated with detectable beta-HCG levels in the serum. The great diagnostic value of serum beta-HCG is emphasized in the cases of old disturbed ectopic pregnancies accompanied by mild and uncommon symptoms and very low beta-HCG levels. In case of unruptured pregnancies or tubal rupture the diagnosis is verified before the result is available by the clinical and sonographic data. On the other hand the high sensitivity of the method leads to an increased number of cases with elevated beta-HCG in which the location of the pregnancy cannot be proven. The precise quantitative evaluation of serum beta-HCG enables us to follow the tendency of beta-HCG which may according to the clinical manifestations warrant invasive diagnostic procedures or just observation without active interference. PMID- 7540807 TI - A method for measuring leukocyte rolling on the selectins. AB - An inexpensive, high-throughput method to simulate leukocyte rolling in the microvasculature has been developed. The method utilizes a 0.22-mm-inner diameter, fused silica capillary tube, coated with E- or P-selectin. Fluorescently labeled HL-60 cells are delivered to the capillary tube at a constant flow rate, exposing the cells to wall stresses approximating those found in postcapillary venules. Cells that physically associate with the inner walls of the tube and whose rate of movement through the tube is retarded, i.e., rolling cells, are monitored by fluorescence microscopy. Images are recorded on a time lapse videocassette recorder. Both rolling incidence and velocity were shown to be related to the concentration of selectin utilized to coat the tube. Due to the extremely small volume (50 microliters) required to fill the capillary tube, this technique is useful for testing the effect of limited quantities of potential antagonists on cell rolling. Using this technique, sLex(Glc) tetrasaccharide was shown to prevent the rolling of HL-60 cells on immobilized E-selectin while fucoidan and dextran sulfate were shown to inhibit rolling of HL-60 cells on P selectin. PMID- 7540808 TI - The use of preparative polyacrylamide gel electrophoresis and electroelution for purification of mucus glycoproteins. AB - This paper describes a novel technique for purifying glycoproteins from porcine gastric mucus by preparative polyacrylamide gel electrophoresis and electroelution. The method is based on the observation that the high-molecular weight buffer/SDS-soluble mucins do not penetrate through the polyacrylamide gel, but remain on the gel surface. Mucus solution extracted with 6 M urea was fractionated on Sepharose CL-2B column and Vo peak mucin was submitted to purification by preparative polyacrylamide gel electrophoresis (22 h). Nonpenetrated mucin layer was electroeluted from the gel after the reversing of electrode polarity (3 h). A comparison of mucin preparations purified by our method and by CsCl density gradient centrifugation indicated that the GalNAc/protein and GalNAc/DNA ratios were three times higher than those of the first method. The method is a relatively short and efficient procedure and yields pure mucin preparation free of contaminating proteins and nucleic acids. PMID- 7540809 TI - Preparation of anti-mucin polypeptide antisera to study mucin biosynthesis. AB - Mucins are very heavily O-glycosylated glycoproteins. For in depth studies on the cell biological aspects of mucins, anti-polypeptide antibodies are essential. We therefore developed a method for the preparation and screening of polyclonal antisera against mucin peptide epitopes. Mucins from five different tissues were isolated using CsCl/guanidinium.HCl density gradient centrifugation, and polyclonal antisera were prepared. Specificity for mucin peptide epitopes was determined by Western blotting, immunohistochemistry, and immunoprecipitation. The versatility of each anti-mucin antiserum for the study of mucin biosynthesis was tested in metabolic labeling experiments on tissue explants. All polyclonal antisera were directed primarily against peptide epitopes of mucin precursors as well as of fully glycosylated mucins. Each of the polyclonal antisera enabled us to study the mucin biosynthesis in the organ where the mucin was isolated from originally. Our mucin isolation method yields very pure mucins with sufficiently intact polypeptides to reproducibly elicit polyclonal anti-polypeptide antisera. As the sera recognized the polypeptides, primarily independent of the state of O glycosylation, the intermediate steps in the biosynthesis of the mucins could be identified. PMID- 7540810 TI - Quantitative measurement of rat uncoupling protein-mRNA by competitive polymerase chain reaction. PMID- 7540811 TI - Modifications to the differential display technique reduce background and increase sensitivity. PMID- 7540812 TI - Effect of 7.2% hypertonic saline/6% hetastarch on left ventricular contractility in anesthetized humans. AB - BACKGROUND: Although a positive inotropic effect of hypertonic saline has been demonstrated in isolated cardiac tissue as well as in animal preparations, no information exists about a possible positive inotropic action of hypertonic saline in humans. The aim of this investigation was to determine whether a clinically relevant positive inotropic effect can be demonstrated in humans. METHODS: Twenty-six patients without cardiovascular disease were randomized to receive 4 ml/kg of either 7.2% hypertonic saline/6% hetastarch or 6% hetastarch (control) at a rate of 1 ml.kg-1.min-1 while under general endotracheal anesthesia. Transesophageal echocardiography was used to evaluate left ventricular function. Arterial pressure, heart rate, and left ventricular end systolic and end-diastolic diameter, area, and wall thickness were measured immediately before and after administration of either solution. Fractional area change, end-systolic wall stress, and the area under the end-systolic pressure length relationship curve (ESPLRarea) were calculated. ESPLRarea was used to assess left ventricular contractility. RESULTS: Administration of hypertonic saline/hetastarch resulted in a significant decrease of mean arterial pressure and end-systolic wall stress from 77 +/- 14 (mean +/- SD) to 64 +/- 17 mmHg (P < 0.01) and from 52 +/- 14 to 32 +/- 11 10(3) dyne/cm2 (P > 0.01), respectively. End-diastolic area and fractional area change increased from 16.5 +/- 2.9 to 21.7 +/- 3.3 cm2 (P < 0.01) and from 0.53 +/- 0.07 to 0.70 +/- 0.06 (P < 0.01), respectively, whereas there was only a minor change of ESPLRarea from 38 +/- 13 to 44 +/- 13 mmHg.cm (P < 0.05). CONCLUSIONS: The apparent improvement of left ventricular systolic function in response to hypertonic saline/hetastarch is caused mainly by the combined effect of increased left ventricular preload and reduced left ventricular afterload. A possible positive inotropic action of hypertonic saline/hetastarch is not likely to be clinically relevant. PMID- 7540813 TI - A MspI polymorphism at the bovine alpha-lactalbumin gene. PMID- 7540814 TI - [Scleroderma of the shoulders after treatment with combined bleomycin and radiotherapy]. AB - INTRODUCTION: There are many causes of scleroderma. We observed a case of scleroderma involving the shoulder and neck area after combined bleomycin and radiotherapy. CASE REPORT: After six cycles of a CHOP-bleo protocol followed by radiotherapy for lymph node lymphoma, a female patient presented scleroderma involving the shoulders and neck area. Total bleomycin dose was 252 mg. DISCUSSION: Among the imputable factors, particularly extrinsic factors, in a series of 33 cases of different chemotherapy protocols published by Peters et al. in 1988, we retained the bleomycin radiotherapy administration sequence as the cause of this complication. Given after bleomycin, radiotherapy could increase the risk of scleroderma. PMID- 7540815 TI - Does subclinical pancreatic inflammation occur after parathyroidectomy? AB - Pancreatitis is accepted as an uncommon complication of parathyroid surgery, but it has been suggested that up to 35% of patients may experience hyperamylasaemia after parathyroidectomy indicating subclinical inflammation of the pancreas. A series of 26 patients undergoing parathyroidectomy were studied by preoperative biochemical analyses repeated 24 and 48 h postoperatively allowing changes in calcium metabolism and serum and urinary amylase levels to be documented. Of the patients, 21 also underwent a CT scan of the pancreas between 24 and 48 h after operation. Despite highly significant changes in serum parathormone, calcium and phosphate levels postoperatively, there was no evidence in any patient of acute pancreatic inflammation or hyperamylasaemia. Twenty-one patients underwent unilateral neck exploration, and we suggest that the absence of any detectable amylase elevation supports the suggestion that such elevation may reflect an increase in salivary isoamylase as a result of extensive neck dissection, rather than reflecting a subclinical pancreatitis. The development of postparathyroidectomy pancreatitis appears to be an all or nothing phenomenon of unknown aetiology. PMID- 7540816 TI - Controversial issues in the management of pancreatic cancer: Part one. A debate held at St Mary's Hospital, London on 18 November 1993. PMID- 7540817 TI - [Palliative treatment of cancers of the head of the pancreas. Surgery versus endoscopy]. AB - Cancers of the head of the pancreas are only resectable in 10 to 25% of cases. The majority of patients are therefore candidates for a palliative treatment jaundice, pain or upper gastro-intestinal obstruction. Palliative surgical bypasses offer a durable efficiency for patients with a relatively short-term life expectancy. Endoscopic placement of biliary stents for jaundice is an alternative but leads to frequent complications and does not act on other clinical symptoms. Controlled trials did not show any statistical difference between surgical or endoscopic treatments. We only recommend endoscopic procedures for patients unfit for surgery with poor general status or distant metastases. PMID- 7540818 TI - [Arrhythmogenic right ventricular dysplasia disclosed by ventricular extrasystole]. AB - Arrhythmogenic right ventricular dysplasia is one of the principal causes of sudden cardiac death in young subjects. In the absence of ventricular tachycardia, the disease can be revealed by simple ventricular extrasystole. In the light of such a case, the authors discuss the value of various complementary investigations for the diagnosis of these clinical forms with little or no symptoms. Detailed examination of the electrocardiogram followed by a search for late ventricular potentials are decisive steps before proceeding to invasive investigations which often remain essential to confirm the diagnosis. The prognosis remains uncertain, and is always dominated by the risk of sudden death, even in these apparently minor forms of the disease. This point further emphasizes the need for detection, which should be facilitated by the recent establishment of a list of diagnostic criteria. PMID- 7540819 TI - Novel mutation in 16S rRNA associated with streptomycin dependence in Mycobacterium tuberculosis. AB - Molecular characterization of a streptomycin-dependent mutant of Mycobacterium tuberculosis revealed the presence of a novel mutation in the rrs gene encoding 16S rRNA. Insertion of an additional cytosine in the 530 loop of 16S rRNA, a region known to be involved in streptomycin susceptibility and resistance, was associated with streptomycin dependence. PMID- 7540820 TI - A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin. AB - A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required. PMID- 7540822 TI - N-nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation toward the three nitric oxide synthases. AB - The ability of NG-nitro-L-arginine (NNA) and NG-methyl-L-arginine (NMMA) to inactivate native neuronal, endothelial cell, and macrophage nitric oxide synthases (nNOS, eNOS, and iNOS, respectively) was investigated. Each NOS isozyme (plus cofactors) was preincubated with either NNA or NMMA and then assayed for remaining activity by measuring the conversion of labeled L-arginine to labeled L citrulline. Consistent with previous reports (Olken, N. M., et al., Biochem. Biophys. Res. Commun. 177, 828-833, 1991), NMMA was a mechanism-based irreversible inhibitor of iNOS, exhibiting time- and concentration-dependent inactivation of iNOS with a KI equal to 2.6 microM and a kinact equal to 0.042 min-1. When assayed without a preincubation period, NMMA exhibited typical reversible inhibition of iNOS (Ki = 3.9 microM). NMMA also reversibly inhibited nNOS and the eNOS with Ki equal to 0.65 and 0.7 microM, respectively. However, NMMA did not inactivate eNOS at concentrations up to 10 microM. In the presence, but not the absence, of 4 microM tetrahydrobiopterin, NMMA inactivated nNOS with a kinact equal to 0.022 min-1 and a KI equal to 2.0 microM. Since NNA did not inactivate iNOS at concentrations up to 25 microM, NNA is strictly a reversible inhibitor of iNOS (Ki = 8.1 microM). Neuronal NOS and eNOS, however, were rapidly inactivated by NNA with kintact equal to 0.083 and 0.047 min-1 and KI equal to 0.09 and 0.02 microM, respectively, when preincubated with NNA. Tetrahydrobiopterin did not affect the rate of inactivation of nNOS by NNA. In all cases, L-arginine protected against inactivation, suggesting that inactivation occurs at or near the active site. Thus, inactivation of the three NOS isozymes with NMMA and NNA reveals active-site differences between the isoforms. PMID- 7540821 TI - Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia. AB - We have recently shown that the ability of some gram-negative bacteria to dissolve poorly soluble calcium phosphates (Mps+ phenotype) is the result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct oxidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline quinone (PQQ) essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, these strains do not produce gluconic acid and are Mps . Evidence is presented to show that expression of a single 396-base Pseudomonas cepacia open reading frame (designated gabY) in E. coli JM109 (a K-12 derivative) was sufficient to induce the Mps+ phenotype and production of gluconic acid. We present the nucleotide sequence of this open reading frame which coded for a protein (GabY) with a deduced M(r) of 14,235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) carrying gabY resulted in production of a protein with an M(r) of 14,750. Disruption of the open reading frame of gabY via site directed mutagenesis changed the phenotype to Mps- and eliminated gluconic acid production. The deduced amino acid sequence of gabY has no apparent homology with those of previously cloned direct oxidation pathway genes but does share regions highly homologous with the histidine permease system membrane-bound protein HisQ as well as other proteins in this family. In the presence of 1 microM exogenous PQQ, both JM109(pSLY4) and JM109(pGAB1) produced 10 times as much gluconic acid as was seen with either the plasmid or exogenous PQQ alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540823 TI - [Chemotherapy and multimodality therapy in the treatment of esophageal cancer]. AB - Many single-agent phase II studies for patients with esophageal cancer had been performed, and at least five agents with modest activity have been indentified, BLM, MMC, CDDP, VDS and MTX, that revealed no CR. Even in the phase II study of 254-S, a new anticancer platinum complex, with a response rate of 43%, CR could not be recognized. Only two regimens of combination chemotherapy have been studied extensively, CDDP/BLM/VDS and CDDP/5-FU. The combination of CDDP/5-FU has gained popularity and JEOG phase II study revealed a response rate of 36%. As surgical adjuvant therapy, postoperative adjuvant therapy has been common in Japan. The 4th JEOG phase III clinical trial, comparing surgery alone and postoperative chemotherapy of CDDP/VDS, showed no additive effect to surgery by postoperative chemotherapy with such a regimen. In western countries, neoadjuvant chemotherapy is frequent and most of the regimens include CDDP/5-FU. Pathological CR rates were less than 10%, and median survival terms were from eight to 28 months. The rationale for the concurrent use of chemotherapy and radiotherapy is to combine an agent that has an effect upon systemic micrometastases with a modality that enhances local tumor control. In addition, a number of chemotherapeutic agents have radiosensitizing effects. In western countries, more than 35 trials studying over 1400 patients have been reported. The majority of trials have employed CDDP/5-FU combined with RT to a total dose of 30Gy. Pathological CR rates were from 20 to 40% and median survival terms were from 12 to 29 months. Both neoadjuvant chemotherapy and chemoradiotherapy did not change operative morbidity or mortality, and there was a statistically significant increase in survival in complete responders. However, early and median survival seems improved but cure rates are not. Both therapies require further investigation. PMID- 7540824 TI - [Combined effect of bleomycin and SDB-ethylenediamine, a synthetic isoprenoid, on oral squamous carcinoma cell lines and transplantable nude mouse tumors]. AB - Combined effect of bleomycin (BLM) and N-solanyl-N, N'-bis (3, 4-di-methoxy benzyl)-ethylenediamine (SDB-ethylenediamine), a synthetic isoprenoid was studied on 3 oral squamous carcinoma cell lines (SCCTF, SCCKN, SCCRY) and clonal cell lines from SCCRY in vitro and on transplantable nude mouse tumors. The cytotoxicity of SDB-ethylenediamine varied among those cell lines in vitro. SDB ethylenediamine potentiated more than 2-fold the cytotoxic effect of BLM in 6 cell lines, but no distinct correlation was found between cytotoxicity of SDB ethylenediamine and potentiation by BLM. Compared with verapamil, the cytotoxic effect of BLM was potentiated 3.9-fold by SDB-ethylenediamine in SCCTF. The growth of the transplantable nude mouse tumors (SCCRY and SCCTF) was strongly suppressed when BLM was combined with SDB-ethylenediamine. Correlation between BLM resistance and potentiation of BLM by SDB-ethylenediamine was not observed in this experiment. PMID- 7540825 TI - [Development of quality of life (QOL) questionnaire for use of lung cancer patients in palliative therapy--study of validity and reliability. No.1]. AB - We investigated the validity and reliability of QOL questionnaire for lung cancer patients in palliative therapy. The questionnaire covered twelve items: appetite, feelings, sleep, mental and physical fatigue, pain, anxiety, daily activity, abdominal and respiratory conditions, linear and face scales. The data were collected from 65 patients and analyzed with principal component analysis and correlation analysis. 1) The percentage of complete answers was 81.5%. 2) Appetite, feelings, sleep, mental fatigue, anxiety and mental scale, pain, respiratory condition, abdominal condition, physical fatigue and physical scale, and satisfied internal consistency. 3) The test-retest reliability was satisfied 4) The inquiry items were grouped into physical, mental and activity scales, and these scales belonged to different dimension. 5) There were correlations between a linear scale, face scale, total score and items. 6) In concurrent validity, there were correlations between performance status and the activity scale, SDS, STAI and the mental scale. 7) In sensitivity, the total score and face score were worst within one week after chemotherapy, and then recovered. This questionnaire was indeed valid and reliable for use as a QOL questionnaire for lung cancer patients in palliative therapy. PMID- 7540826 TI - Possible role of HLA-B27 associated cytotoxic T lymphocyte activity in the pathogenesis of the seronegative arthropathies. PMID- 7540827 TI - Innervation of the synovium. PMID- 7540828 TI - Escherichia coli and other species of the Enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins. AB - A newly identified gene in Escherichia coli, fkpA, encodes a protein with extensive similarity to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. The FkpA protein may be a new member of the family of FK506-binding proteins (FKBPs) because its carboxyl domain includes a sequence that matches the consensus FK506-binding motif in 40 of 48 positions, including those amino acids at the active site that form hydrogen bonds with the drug FK506. The amino acid sequence of the 29 kDa FkpA protein is 30-35% identical to the Mip proteins of L. pneumophila, L. micdadei, and C. trachomatis. Of the 270 amino acids of FkpA, 113 (42%) are identical to the sequence of one or another of these Mip proteins. Overexpression of FkpA or deletion of fkpA from the E. coli chromosome had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots revealed that similar genes exist in several representatives of the Enterobacteriaceae. Thus, mip-like genes are not found exclusively in bacteria having a predominately intracellular life style, but instead appear to be a new FKBP subfamily that is a common constituent of many bacteria. PMID- 7540829 TI - Synthetic peptides representing sequences within gp41 of HIV as immunogens for murine T- and B-cell responses. AB - Within the gp41 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) there is a relatively conserved region which appears accessible to the immune system during the course of HIV infection and is recognised by antibody from virtually all patients with AIDS. This region has also been shown to function as a target for human T cells. We have examined synthetic peptides spanning this sequence, between residues 572 and 604, with a view to evaluating their potential as immunogens. Peptides 572GIKQLQARILAVERYLKDQQ591 and 579RILAVERYLKDQQLLGGIWGCSGK601 were good immunogens in two different strains of mice while peptide 576LQARILAVERYLKDQQ591 was an inferior immunogen, and peptide 593LGIWGCSGKLIC604 was non-immunogenic unless coupled to a carrier protein. For both antibody and T cell responses it was apparent that sequences that could function as determinants within one peptide could not do so in the context of a different peptide immunogen. It follows that by judicious choice of immunogen sequence it may be possible to direct the immune response towards a desired fine specificity. Unwanted responses by CD4+ T cells isolated from certain peptide primed animals were also observed. These T cells showed an unusual reactivity in that they were incapable of recognising their determinant AVERYLKDQQ if it was extended at the C-terminal end with the native sequence and as such would not be expected to recognise the native molecule unless processing created the identical C-terminus. PMID- 7540830 TI - Retroviral properties inherent to viral erythrocytic infection in sea bass. AB - The characterization of the aetiological agent of viral erythrocytic infection (VEI) of sea bass suggests a retroviral origin of the disease. RNA from viral erythrocytic infection virus (VEIV) and DNA from blood and organs of VEI-affected fish hybridized to a specific retrovirus cDNA probe. Sequences homologous to retrovirus genome were also detected in non-infected SBL cells (a sea bass cell line), however, Southern blot analysis showed that the DNA restriction patterns in VEI-affected erythrocytes differed from those of SBL cells. RNA-dependent DNA polymerase activity was detected in VEI-affected sea bass blood. This reverse transcription was strongly Mn(2+)-dependent and is the first report of its occurrence in a marine fish and in fish blood samples. Nucleic acid sequences homologous to retrovirus RNA were detected in chromatographic fractions exhibiting reverse transcriptase activity and the presence of virus-like particles, 125-150 mm in diameter. The density of VEIV in sucrose was 1.17-1.18 g/cm3. The symptomatology of VEI is not far from those described for some retroviral diseases. PMID- 7540831 TI - Mapping of cross-reacting and serotype-specific epitopes on the VP3 structural protein of the infectious bursal disease virus (IBDV). AB - The binding sites of a panel of monoclonal antibodies cross-reacting with the structural protein VP3 of the two serotypes of the infectious bursal disease virus (IBDV) could be mapped to four segments of the VP3 gene. Two of these antigenic domains also carry epitopes which are specific for one serotype only. Formation of the common or type-specific epitopes is in agreement with homologous or mismatching amino acid sequences yielding hydrophilic segments on the VP3 polypeptide. These antigenic patterns obtained by immunoblotting could be verified by a competitive ELISA. PMID- 7540832 TI - Co-variation of neuropeptide Y, calcitonin gene-related peptide, substance P and neurokinin A in joint fluid from patients with temporomandibular joint arthritis. AB - Forty-one patients (37 female and four male) with signs and symptoms of temporomandibular joint arthritis, were separated into two diagnostic groups (group I: inflammatory; group II: degenerative/non-specific joint disease). They were examined clinically, fluid was aspirated from the joint with saline and venous blood samples were collected at the same time. The joint fluid and plasma samples were analysed for neuropeptide-like immunoreactivity, i.e. neuropeptide Y (NPY-LI), calcitonin gene-related peptide (CGRP-LI), substance P (SP-LI) and neurokinin A (NKA-LI), using competitive radioimmunoassays. The aim was to investigate any co-variation of the peptides in the joint fluid and plasma. In group I, the median values of peptide concentrations in joint fluid were SP-LI = 129, CGRP-LI = 75, NKA-LI = 36 and NPY-LI = 676 pmol/l and in group II, SP-LI = 52, CGRP-LI = 64, NKA-LI = 45 and NPY-LI = 318 pmol/l. There were no significant differences between the groups for peptide concentrations. In group I, all the neuropeptides were strongly correlated. In group II, SP-LI and NKA-LI were strongly correlated while CGRP-LI was weakly correlated with NPY-LI and NKA-LI. Multiple step-wise regression analysis showed that most of the variation in NPY LI, CGRP-LI and SP-LI in group I was explained by NKA-LI, but the regression did not reach statistical significance in group II. PMID- 7540833 TI - Induction of the CD1a Langerhans cell marker on human monocytes. AB - Monocytes have recently been recognized as a precursor of Langerhans cells. This study examined the regulatory influence of the epithelial environment on the putative first step of the transition towards a Langerhans cell phenotype--the induction of CD1a antigen. The keratinocyte-derived cytokines granulocyte macrophage-colony-stimulating factor, tumour necrosis factor-alpha, interleukin 6, and interleukin-1 beta induced CD1a expression, as did supernatants of keratinocytes extracted from inflammatory sites (periodontitis). Induction was abrogated by transforming growth factor-beta and a keratinocyte-derived interleukin-1 inhibitor. The optimal temperature for induction was 34 degrees C, not 37 degrees C. These results demonstrate that the components of the epithelial environment (cytokines and lower temperature) exert important influences, which may be part of local regulation of Langerhans cell development. PMID- 7540835 TI - Spirituality and palliative care. AB - A terminal illness represents a spiritual crisis for many, but not all, patients. As the general practitioner confronts the illness with the patient a relationship develops which can itself be therapeutic. I believe that general practitioners can become attuned to the spiritual needs of their patients by having a commitment to the patient and focusing on the illness rather than the disease. Medical school did not prepare me for these lessons, rather they have been taught since by my patients. PMID- 7540834 TI - Histological, histocytochemical and ultrastructural study on the effects of surface charge on bone formation in the rabbit mandible. AB - The osteogenic potential of different surface charges was studied by implanting uncharged, negatively changed and positively charged Sephadex beads subperiosteally on the buccal aspect of the mandible. The implant sites were examined 1, 2, and 4 weeks later. New bone formation was observed around the positively charged beads at 2 weeks. An amorphous collagen-free layer seen around the beads consisted of anionic substances and contained needle-like crystals. Tartrate-resistant acid phosphatase-positive, multinucleated giant cells, which had typical fine structural characteristics of osteoclast-like ruffled borders and a clear zone, were observed on the surface of positively charged beads, with osteoblastic cells juxtaposed to them. Bone formation was still going on at 4 weeks in this group. Around the uncharged or negatively charged beads, new bone formation and osteoclast-like cells were not observed at any time. The findings suggest that the surface charge of implant materials has a definite influence on the differentiation of osteoclastic cells and subsequent occurrence of osteoblastic cells leading to formation of bone. PMID- 7540836 TI - Epicardial keratinaceous cyst in a tawny frogmouth (Podargus strigoides plumiferus). AB - Tissues from a tawny frogmouth that died acutely in a zoo collection were submitted for histopathologic examination. A small yellow nodule was observed grossly on the ventricular wall by the zoo veterinarian during collection of tissues. Histologically, the epicardial nodule consisted of a well-circumscribed, thinly encapsulated cyst, 4 mm in diameter. The cyst was composed of gradually keratinizing, attenuated, stratified squamous epithelium, one to three cell layers in thickness. The cyst was filled with laminated keratin and a few keratinized epithelial cells. The cyst was separated from the myocardium by a thin layer of adipose tissue. Based on these observations, the structure was diagnosed as an epicardial keratinaceous cyst. PMID- 7540837 TI - Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. AB - Immunohistochemistry shows that skeletal muscle fibers express endothelial-type (ec)-NOS in a heterogeneous pattern distinct from that of neuronal (nc)-NOS. Analysis of adjacent serial sections reveals: 1) a strong correlation of ec-NOS expression to mitochondrial content (visualized histochemically by succinate dehydrogenase); 2) lack of correlation of ec-NOS to fiber ATPase type (in contrast to nc-NOS); and 3) constitutive co-expression of ec- and nc-NOS in some fibers. Preparations of mitochondria from diaphragm exhibited calcium-dependent NOS activity, which functioned to inhibit in vitro oxygen consumption. The results establish that normal skeletal muscle cells can express two constitutive isoforms of NOS (nc- and ec-) and suggest a functional role for ec-NOS in oxidative, mitochondria-rich fibers. PMID- 7540838 TI - 12(S)-HETE is a mitogenic factor for microvascular endothelial cells: its potential role in angiogenesis. AB - 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid] is a lipoxygenase metabolite of arachidonic acid. Treatment of murine-lung-derived microvascular endothelial cells (CD clone 4) with exogenous 12(S)-HETE promoted wound healing of injured endothelial cell monolayers. 12(S)-HETE, in a time- and dose-dependent manner, enhanced the growth of CD clone 4 cells. Thymidine incorporation assays demonstrated that 12(S)-HETE increased the DNA synthesis by > 4 fold. In addition, normal endothelial cell growth stimulated by serum could be dose dependently inhibited by a select 12-lipoxygenase inhibitor (BHPP), suggesting that 12(S)-HETE is a physiological mitogenic factor for microvascular endothelial cells. PMID- 7540839 TI - Modulation by bovine angiogenin of tubular morphogenesis and expression of plasminogen activator in bovine endothelial cells. AB - Angiogenin is a potent angiogenic molecule in the chick chorioallantoic membrane assay and rabbit corneal assay. However, no angiogenic activity has been reported in in vitro system. In this study, we isolated and purified angiogenin from bovine milk, and the biological effect of the bovine angiogenin on bovine endothelial cells was examined in in vitro angiogenesis models. Bovine angiogenin significantly stimulated both cell migration and formation of tube-like structures in the collagen gel by bovine aortic endothelial cells. Angiogenin at 10-100 ng/ml stimulated about 2-fold higher the tube formation when basic fibroblast growth factor (bFGF) at 10 ng/ml stimulated about 3-fold over the control. Tubular morphogenesis stimulated by bFGF or angiogenin was almost completely blocked in the presence of aprotinin, an inhibitor of serine proteases. Angiogenin up-regulated both mRNA level and activity of urokinase type plasminogen activator, a key mediator of angiogenesis. Moreover, both bFGF and bovine angiogenin induced a marked increase of c-fos mRNA level at 30 min after stimulation. These novel effects of bovine angiogenin are to be discussed in relation to its structure specificity. PMID- 7540841 TI - The regulatory site of functional GTP binding protein coupled to the high affinity cholecystokinin receptor and phospholipase A2 pathway is on the G beta subunit of Gq protein in pancreatic acini. AB - A non-hydrolysable guanosine nucleotide analog, GTP[S] at 200 microM, stimulated amylase secretion which was inhibited by an anti-phospholipase A2 (PLA2) antibody in permeabilized pancreatic acini, indicating that the PLA2 pathway is linked to the GTP binding protein. A high affinity cholecystokinin (CCK) receptor agonist, CCK-OPE (10 microM), and a low affinity receptor agonist, CCK-8 (0.1 microM), both caused amylase secretion in permeabilized cells. The action of CCK-OPE was abolished by the G beta antibody but not by the G alpha-q,11 antibody, whereas the opposite was true of the CCK-8 response. Biscoclaurine alkaloid isotetrandrine (10 microM), a specific inhibitor of PLA2-coupled G proteins, abolished Ca2+ oscillations and amylase secretion induced by CCK-OPE (0.1-100 nM), but not by CCK-8 (10 pM) in intact acini. Gp antagonist-2A (10 microM), which inhibits the activation of Gq, also inhibited the actions of CCK-OPE (10 pM 1 microM) in intact acini. These observations indicate that the functional unit of the heterotrimeric G protein coupled to the high affinity CCK receptor appears to be different from that linked to the low affinity CCK receptor/Gq-alpha pathway. The regulatory site of this G protein coupled to the high affinity CCK receptor is on the beta subunit of Gq protein which elicits Ca2+ oscillations and monophasic amylase secretion via the PLA2 pathway. PMID- 7540840 TI - High D-glucose stimulates the cell cycle from the G1 to the S and M phases, but has no competent effect on the G0 phase, in vascular smooth muscle cells. AB - The effects of high (28mM) D-glucose (HG) on the cell cycle progression were investigated in rat aorta vascular smooth muscle cells (VSMCs) in primary culture, using an immunocytochemical analysis of cell-cycle-specific nuclear antigens. HG had no effect on the cell cycle of the serum-deprived G0 cells, whereas platelet-derived growth factor (PDGF) stimulated the entry of the G0 cells to the G1 phase without a further progression to the S and M phases. HG, but neither mannitol nor L-glucose, stimulated the progression of the PDGF pretreated G1 cells to the S and M phases, which was blocked by calphostin-C, a protein kinase C (PKC) blocker. HG did not affect the cytosolic Ca2+ concentration ([Ca2+]i). These data suggest that HG has no competent effect on the G0 cells and acts as a progression growth factor (to stimulate the cell cycle from the G1 to the S/M) in VSMCs through the mechanism, which may be insensitive to [Ca2+]i and mediated by PKC. PMID- 7540842 TI - Tyrosine phosphorylation of vav protooncogene product in primary human myelogenous leukemic cells stimulated by granulocyte colony-stimulating factor. AB - Granulocyte colony-stimulating factor(G-CSF), but not granulocyte-macrophage CSF(GM-CSF), induced tyrosine phosphorylation of 95-kDa protein in 13 cases of primary human acute myelogenous leukemic cells. Electrophoretic mobility of 95 kDa phosphoprotein and the protooncogene product p95vav was identical. In addition, p95vav was tyrosine-phosphorylated only in G-CSF-stimulated cells. In contrast to primary leukemic cells, amount of p95vav was under detectable level and G-CSF did not induce tyrosine phosphorylation of 90-100-kDa proteins in human neutrophils. These results indicate specific involvement of vav product in signaling pathway of G-CSF in primary human leukemic cells. PMID- 7540843 TI - Effects of small-volume bolus treatment with intravenous normal saline and 7.5 per cent saline in combination with 6 per cent dextran-40 on metabolic acidosis and survival in burned mice. AB - Standard murine burn models include the administration of intraperitoneal (i.p.) saline solutions which are intended to resuscitate the animals during subsequent burn shock. Prehospital administration of small volumes of concentrated salt solutions has been recommended for the early treatment of haemorrhagic shock, and have also been utilized for burn shock. We studied the effects of bolus intravenous (i.v.) hypertonic saline (HS) or hypertonic saline/dextran-40 (HS + DEX) on animal survival and acid-base balance following 25 per cent total body surface area, full-thickness burn injury in mice. I.v. injections were administered via a tail vein immediately prior to burn injury. Some mice received 1 ml i.p. normal saline (NS) immediately after burn injury. Acid-base balance of vena caval blood was measured during the period of maximal metabolic acidosis following burn injury (12 h postburn). Preburn i.v. administration of 5 ml/kg of HS or HS+DEX, followed by 1 ml i.p. NS, only slightly decreased the degree of metabolic acidosis compared to animals receiving i.p. fluid alone, the standard resuscitation regimen for burned mice. Preburn i.v. administration of 0.2 ml volumes of HS or HS + DEX, without i.p. fluid administration, resulted in extremely high mortality. Immediate preburn i.v. administration of HS or HS + DEX did not eliminate metabolic acidosis in this murine burn model, and markedly increased the mortality when subsequent i.p. fluids were not administered. The degree of metabolic acidosis in the murine experimental burn model has not previously been clearly described. Furthermore, adequate fluid resuscitation of these animals may be difficult to achieve without indwelling vascular catheters which could deliver continuous i.v. fluids following burn injury. PMID- 7540844 TI - The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis. AB - In the last decade a considerable amount of research has been dedicated to studying the process of angiogenesis. In the field of tumor biology angiogenesis is a relevant subject of investigation as well, since newly formed blood vessels are required for the growth of tumors and provide an exit route for metastasizing tumor cells. In this review we discuss some aspects of tumor angiogenesis with emphasis on the role that growth factors bFGF and VPF play in this process. A number of biochemical characteristics and biological properties of the two factors and their receptors are reviewed, and the expression of bFGF and VPF in both normal tissues and in tumors is discussed. Finally, we speculate on the use of bFGF and VPF expression as a diagnostic parameter and on possible clinical applications. PMID- 7540845 TI - Adhesion co-receptor expression and intracellular signalling in HIV disease: implications for immunotherapy. AB - OBJECTIVES: To investigate, in lymphocytes from HIV-1-infected individuals, the phenotypic expression of various adhesion co- or counter-receptors [lymphocyte function-associated antigen (LFA)-3, LFA-1 and intercellular adhesion molecule (ICAM)-1] involved in providing the co-stimulatory signal through the phospholipase C-gamma pathway in relation to inositol polyphosphate metabolism. DESIGN AND METHODS: Cell adhesion molecule profiles of peripheral blood lymphocytes (PBL) from 39 HIV-1-infected individuals at various stages of infection and 20 healthy laboratory controls were studied using flow cytometry. These were studied in 14 patients with late-stage disease in conjunction with their inositol polyphosphate metabolic profiles measured by high performance liquid chromatography. Levels of HIV-1 present in cell lysates were concurrently measured by a p24 antigen capture assay. In addition, the effects of a specific anti-ICAM-1 antisense oligonucleotide on the intracellular phosphatase activities of lymphocytes from a separate group of eight HIV-1-infected individuals were examined. RESULTS: The expression of LFA-1, a beta 2 integrin, was upregulated among patient PBL in parallel with disease progression, whereas that of LFA-3 (CD58) was found to be significantly reduced among the CD4+ lymphocyte subset in all stages of infection. The 5-phosphatase activity, which we previously observed to be defective in HIV disease, was found to correlate linearly with the expression of both LFA-1 and its ligand, ICAM-1. Treatment of patient lymphocytes with an antisense oligonucleotide, which reduced the cell surface expression of ICAM-1 by blocking the translation of its mRNA, resulted in further reduction of intracellular phosphatase activities. CONCLUSIONS: Our results suggest a pivotal role for adhesion co- and counter-receptors in influencing lymphocyte signalling and hence cellular response to recall antigens in HIV-1-infected individuals. PMID- 7540846 TI - Development of HIV-1 resistance to (-)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex. AB - OBJECTIVE: To determine the rate of development of in vitro HIV resistance to ( )2'-deoxy-3'-thiacytidine (3TC) and relate the effect of dose to emergence of resistance. METHODS: HIV-infected men and non-pregnant women, aged > or = 18 years, with a CD4 count < or = 300 x 10(6)/l cells were followed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma-associated HIV RNA. RESULTS: Phenotypic resistance was detected in approximately one-third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV-1 variants appeared reduced at high 3TC doses. Amino-acid changes at codon 184 in HIV-1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184. CONCLUSIONS: In vitro resistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild-type virus, with regard to codon 184, in both patient plasma and PBMC. PMID- 7540847 TI - Dissociation of cardiac hypertrophy, myosin heavy chain isoform expression, and natriuretic peptide production in DOCA-salt rats. AB - We examined the relationship between cardiac hypertrophy, myosin heavy chain (MHC) isoform expression, and production of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) before and after the development of DOCA-salt hypertension. DOCA-salt rats exhibited significant left ventricular hypertrophy at the prehypertensive stage (1 week of treatment), without MHC isoform switch or change in natriuretic peptide gene expression. In the hypertensive stage (5 weeks of treatment), pronounced left ventricular hypertrophy was observed, and this was characterized by an increase in beta-MHC protein, resulting in a switch from 90% alpha-MHC to 51% alpha-MHC and 49% beta-MHC. ANF and BNP mRNA levels and peptide content were significantly increased at this stage. Unexpectedly, the MHC isoform switch was evident in the non-hypertrophied right ventricle to the same degree as in the left ventricle. Natriuretic peptide production was also increased in the right ventricle at 5 weeks of treatment, but to a lesser degree than in the left ventricle. In contrast, in the hypertrophied left atrium there was no MHC isoform switch, while ANF and BNP mRNA levels were augmented. Plasma ANF was significantly increased in the prehypertensive stage; this was accompanied by a partial depletion of atrial ANF stores. Plasma BNP was increased only in the hypertensive stage, reflecting an increase in ventricular BNP synthesis and secretion. These results suggest that 1) cardiac hypertrophy, MHC isoform expression, and stimulation of natriuretic peptide production are processes that may be dissociated from each other; 2) increases in plasma ANF without a concomitant increase in plasma BNP reflect atrial hemodynamic overload, while increases in both ANF and BNP in plasma are associated with ventricular hypertrophy; and 3) there exist differences in the storage, secretion, and processing patterns of ANF and BNP in the atria. PMID- 7540848 TI - [Macroamylasemia: a clinical study of 10 cases]. PMID- 7540849 TI - Human rabies in the Americas. PMID- 7540851 TI - A reliable marker of endothelial cell dysfunction: does it exist? PMID- 7540850 TI - Discovery of aquaporins: a breakthrough in research on renal water transport. AB - Several membranes of the kidney are highly water permeable, thereby enabling this organ to retain large quantities of water. Recently, the molecular identification of water channels responsible for this high water permeability has finally been accomplished. At present, four distinct renal water channels have been identified, all members of the family of major intrinsic proteins. Aquaporin 1 (AQP1), aquaporin 2 (AQP2) and the mercury-insensitive water channel (MIWC) are water-selective channel proteins, whereas the fourth, referred to as aquaporin 3 (AQP3), permits transport of urea and glycerol as well. Furthermore, a putative renal water channel (WCH3) has been found. AQP1 is expressed in apical and basolateral membranes of proximal tubules and descending limbs of Henle, AQP2 predominantly in apical membranes of principal and inner medullary collecting duct cells and AQP3 in basolateral membranes of kidney collecting duct cells. MIWC is expressed in the inner medulla of the kidney and has been suggested to be localised in the vasa recta. The human genes encoding AQP1 and AQP2 have been cloned, permitting deduction of their amino acid sequence, prediction of their two-dimensional structure by hydropathy analysis, speculations on their way of functioning and DNA analysis in patients with diseases possibly caused by mutant aquaporins. Mutations in the AQP1 gene were recently detected in clinically normal individuals, a finding which contradicts the presumed vital importance of this protein. Mutations in the AQP2 gene were shown to cause autosomal recessive nephrogenic diabetes insipidus. The renal unresponsiveness to arginine vasopressin, which characterises this disease, is in accordance with the assumption that AQP2 is the effector protein of the renal vasopressin pathway.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540852 TI - Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells. AB - Bone marrow stromal cells synthesize and secrete insulin-like growth factor (IGF) I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [125I]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal-derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC 1 cells. Forskolin (10(-5) M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment. PMID- 7540853 TI - Transcriptional and post-transcriptional regulation of granulocyte-macrophage colony-stimulating factor production in human growth factor dependent M-07e cells. AB - To elucidate the regulatory mechanisms of granulocyte-macrophage colony stimulating factor (GM-CSF) production in human myeloid leukaemic cells we studied GM-CSF gene transcription, mRNA expression and GM-CSF secretion in human growth factor dependent M-07e cells. GM-CSF transcript was detected in cells cultured in the presence of interleukin-3 (IL-3). GM-CSF or mast cell growth factor (MGF), whereas it was undetectable in growth factor deprived cells. Growth factor re-addition induced, within 2 h, the appearance of GM-CSF mRNA. Nuclear run-on experiments demonstrated that the increase of GM-CSF mRNA levels depends on GM-CSF gene transcription. The simultaneous addition, to deprived cells, of the growth factor, and of cycloheximide (CHX) for 2 h inhibited GM-CSF mRNA expression, suggesting the requirement for newly made proteins for GM-CSF gene transcription. By means of the M-07e bioassay, which allows the detection of GM CSF, IL-3 and MGF activities, and neutralizing antibodies to each of these factors, GM-CSF activity was detected in the cell-free extract of both IL-3- and MGF-sustained cells and of cells deprived for 24 h. This finding demonstrates that M-07e cells produce and store biologically active GM-CSF in response to both IL-3 and MGF. In contrast, analysis of the growth stimulatory activity present in the culture supernatants revealed that MGF, unlike IL-3, is able to induce the secretion of consistent amounts of GM-CSF. Taken together, our results suggest that, in M-07e cells, GM-CSF gene transcription and GM-CSF production are mediated, unlike its secretion, by mechanisms shared by IL-3 and MGF. PMID- 7540854 TI - All-trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34+ haemopoietic progenitor cells. AB - To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we performed serum-free liquid and semisolid cultures using CD34+ cells purified from midtrimester human fetal blood samples. RA, at both physiological (10(-11) and 10(-12)M) and pharmacological (10(-6) and 10(-7)M) concentrations, significantly (P < 0.01) promoted the survival of fetal CD34+ cells in liquid cultures from day 3 onwards, by suppressing apoptosis induced by serum and growth factor deprivation. On the other hand, RA alone had no significant effect on the proliferation and differentiation of fetal haemopoietic progenitors. In the presence of optimal concentrations of recombinant interleukin-3 (IL-3), stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), and erythropoietin (Epo), low and high doses of RA induced striking differential effects on CD34+ cell proliferation in liquid cultures and colony formation in semisolid assays. In fact, 10(-11)M and 10(-12)M RA were able to: (i) significantly (P < 0.05) increase 3H-thymidine uptake by fetal CD34+ cells in liquid cultures, and (ii) variably promote the growth of pluripotent (CFU-GEMM, P < 0.05), early (BFU-meg) and late (CFU-meg, P < 0.01) megakaryocyte, granulocyte/macrophage (CFU-GM, P < 0.01) and erythroid (BFU-E) progenitors in semisolid cultures. On the contrary, 10(-6) and 10(-7)M RA induced: (i) an overall inhibition (P < 0.01) of CD34+ cell growth in liquid cultures; (ii) a marked suppression of BFU-E colony formation (P < 0.01) at all Epo concentrations examined (0.002-4 IU/ml); and (iii) a significant (P < 0.01) stimulation of CFU GM with a shift from mixed granulocyte/macrophage to pure granulocyte colonies, whereas it had little effect on the growth of CFU-GEMM, BFU-meg and CFU-meg. Our data, as a whole, demonstrate that RA has direct complex effects on the survival, growth and clonal expansion of fetal haemopoietic progenitor cells, mainly depending on the presence of recombinant cytokines, the type of progenitor and the concentrations of RA. PMID- 7540855 TI - Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection. AB - Peripheral blood lymphocytes of 46 recipients of lymphocyte-depleted bone marrow allografts were phenotypically analysed over a period of 1 year. We investigated the repopulation of lymphocyte subpopulations and their relation with clinical parameters such as graft-versus-host disease (GVHD), graft-versus-leukaemia and cytomegalovirus (CMV) infection. The number of repopulated T cells varied strongly between the blood samples of the recipients. In 45% of the recipients the number of T cells recovered to or above normal levels within 3 months after bone marrow transplantation (BMT), whereas the other recipients remained below normal up to 1 year after BMT. In recipients with a high repopulation, the CD8+ T cell subset contributed more to this high repopulation than the CD4+ T-cell subset. We showed that the majority of T cells of these recipients expressed the alpha beta T-cell receptor, CD8, CD57 and CD11b. HLA-DR was also highly expressed reflecting the activation stage of T cells in these recipients. BMT recipients with a high repopulation of CD8+ T cells showed a lower incidence of leukaemic relapse than recipients with a low repopulation. The 3-year probability of relapse was 19% versus 64% (P = 0.03), respectively. The relative high number of CD8+ T cells at 3 months after BMT was not associated with the incidence of GVHD. In contrast, occurrence of CMV infection after BMT was significantly higher in these recipients. Our results indicate that CD8+ T cells, predominantly CD57+, of BMT recipients with an expansion of these cells represent an in vivo activated cell population. This CD8+ T-cell population may consist partially of cytotoxic cells with anti-leukaemic activity as suggested by a low relapse rate. The signal for the strong expansion of these CD8+CD57+ T cells after BMT is still unclear, but association with CMV infection suggests that viral antigens are involved. PMID- 7540856 TI - Comparative study of peripheral blood progenitor cell collection in patients with multiple myeloma after single-dose cyclophosphamide combined with rhGM-CSF or rhG CSF. AB - Patients suffering from high-risk multiple myeloma (MM) were randomized to receive single high-dose cyclophosphamide followed by either rhGM-CSF or rhG-CSF in order to harvest circulating peripheral blood progenitor cells. The safety of the procedure, the mobilization kinetics, the relative efficacy of rhGM-CSF and rhG-CSF to mobilize progenitor cells and their relative toxicity were studied. Special attention was paid to the antigenic profile of CD34+ progenitor cells. Group I patients (n = 11) were treated with cyclophosphamide 4 g/m2 i.v. followed by rhGM-CSF at 10 micrograms/kg/d by subcutaneous administration. Group II (n = 11) patients received rhG-CSF s.c. at 10 micrograms/kg/d after the same dose cyclophosphamide. Both mobilization regimens appeared to be equally effective. No significant differences in absolute numbers of circulating progenitors, determined by CD34 expression or in yields of MNC, CFU-GM, BFU-E and CD34 subsets were observed. rhGM-CSF administration resulted however in delayed haemopoietic recovery and an increased complication rate. We conclude that rhG-CSF may be preferred because of its markedly lower toxicity and lower in-hospital costs. PMID- 7540857 TI - Clinicopathological correlates of CD56 expression in multiple myeloma: a unique entity? AB - Prior studies have suggested that loss of plasma cell CD56 expression in multiple myeloma defines a unique patient subset and that CD56 expression reliably discriminates between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). We conducted a study of 68 untreated patients with MM from a single institution to define the clinicopathological correlates of CD56 expression. We find CD56 expression in 55% of MM. Lack of CD56 expression does not define a unique clinicopathological or prognostic entity in MM. Strong CD56 expression can also be found in MGUS and does not help to distinguish from MM. PMID- 7540858 TI - C-myc amplification, double minutes and homogenous staining regions in a case of AML. AB - c-myc amplification is usually associated with lymphoid malignancies and Burkitt's lymphoma in particular. We present a case of AML with c-myc amplification which was associated with homogenous staining regions (hsr) and double minutes (dmin). The administration of GCSF following induction chemotherapy resulted in a marked increase in blast numbers. The GCSF was stopped and further courses of chemotherapy given, which resulted in complete remission. The patient relapsed 7 months after diagnosis and failed to go into a second remission with reinduction therapy. We conclude that c-myc amplification is a rare event in AML, but may be associated with chemotherapy resistance and a poor prognosis, are as dmins and hsr. Growth factors should be used with caution in these patients. PMID- 7540859 TI - Identification of p90RSK as the probable CREB-Ser133 kinase in human melanocytes. AB - Normal human melanocytes proliferate in vitro only in response to costimulation by at least two selected peptide growth factors. In the presence of only one mitogen, melanocytes become quiescent or die. These mitogens also enhance expression of differentiated functions, since in their presence the proliferating melanocytes become progressively more pigmented. To assess the intermediates participating in this dual response, we have determined the activated state of several known ligand-induced signal transducers. We demonstrate that hepatocyte growth factor/scatter factor, mast/stem-cell growth factor, basic fibroblast growth factor, and endothelin-1 induce phosphorylation of Ser133 within the KID domain of the cAMP-responsive element binding protein, a modification necessary for transcriptional activation of all members of this family of transcription factors, including also cAMP-responsive element modulator tau and activating transcription factor 1. The costimulation with synergistic growth factors prolonged the phosphorylated state and activity of the mitogen-activated protein kinase 2 cascade. cAMP-responsive element binding protein Ser133 phosphorylation in response to synergistic growth factors was due probably to the activation of p90RSK and, to a lesser extent, to p70S6K. Our findings support the concept that signals initiated at the cell surface converge on regulatory proteins that sustain both cell division and differentiation. PMID- 7540860 TI - CD40 signalling in ileal Peyer's patch B cells: implications for T cell-dependent antigen selection. AB - The ileal Peyer's patch (PP) plays a central role in B cell development in young sheep and it is hypothesized that this B cell development occurs independent of extrinsic antigen and T cells. Therefore, it was of interest to examine ileal PP follicular (iPf) B cell responses to CD40 ligand, a molecule integral to T cell dependent B cell development. A variable level of CD40 expression was detected on a subpopulation of iPfB cells and J558L cells, expressing a membrane form of mouse CD40 ligand (mCD40L), interacted specifically with the CD40 molecule on iPfB cells. In response to mCD40L the non-S phase iPfB cells were rescued from apoptotic cell death and there was a marked proliferative response but viable cell number remained relatively constant. The mCD40L also induced decreased cytoplasmic cAMP levels, blocked anti-Ig-induced iPfB cell death and induced functional IL-2 receptor expression on a subpopulation of iPfB cells. Many of the mCD40L-induced responses of iPfB cells were similar to those reported for germinal centre and immature B cells, and indicated that cognate T cell-B cell interaction could influence iPfB cell proliferation and differentiation. Finally, that mCD40L induced iPfB cell activation and differentiation was evident as increased expression of CD5, the BAQ44A molecule, the CACT65A molecule and the expansion of surface IgG1+ B cells. These mCD40L-induced phenotypic changes were also observed on subpopulations of freshly isolated iPfB cells and jejunal PP follicular B cells. However, few iPfB cells had a phenotype similar to that observed in co-culture with mCD40L and this suggested that T cell-dependent B cell development may play a minor role in ileal PP B cell development. The possible significance of CD40 signalling is discussed in terms of the selection of iPfB cells during development. PMID- 7540861 TI - Induction of CD5 on B and T cells is suppressed by cyclosporin A, FK-520 and rapamycin. AB - The expression of CD5 can be induced on murine B-2 cells by anti-IgM, a recognized analog of thymus-independent 2 type (TI-2) antigen. Given that cyclosporin A (CsA) sensitivity is a distinguishing feature of TI-2 type B cell activation, we asked whether the in vitro induction of CD5 on B cells by anti-mu is CsA sensitive. We report that anti-mu induced CD5 expression on B-2 cells was inhibited by CsA as well as FK-520 and rapamycin. When L-685,818, a FK-520 and rapamycin antagonist, was added to anti-mu stimulated B cell cultures containing FK-520 or rapamycin, but not CsA, suppression was abrogated and complete induction of CD5 was seen. When we used either CD4+CD8+ thymocytes or peripheral T cells activated by phorbol ester and ionomycin, the cell surface induction of CD5 was also partially blocked by CsA, FK-520 and rapamycin. Moreover, in both B and T cells, the same immunosuppressive drugs did not affect constitutive CD5 expression but only blocked de novo induction. To determine the level of CD5 regulation, we activated T cells using phorbol myristate acetate (PMA)/ionomycin and report that CD5 induction was sensitive to actinomycin D (AcD). Similarly, the induction of CD5 on anti-mu activated B cells was blocked by AcD. In addition, T cells that were activated by PMA/ionomycin expressed more abundant CD5 mRNA than CsA or FK-520 treated cells. Based on the CsA-sensitive regulation of CD5 we thought that the CsA-sensitive nuclear factor of activated T cells (NFAT) might be involved in CD5 regulation. We report evidence by Western blot analysis that NFATp is expressed by both resting and TI activated B cells but apparently not CD4+CD8+CD5+ thymocytes. We conclude that in both B and T cells the induction of CD5 requires transcriptional regulation, and that the inhibition of CD5 expression by the immunosuppressive drugs CsA, FK-520 and rapamycin requires drug-immunophilin complex formation. PMID- 7540862 TI - IL-5 production by CD4+ T cells of asthmatic patients is suppressed by glucocorticoids and the immunosuppressants FK506 and cyclosporin A. AB - IL-5 was produced in vitro by peripheral blood mononuclear cells (PBMC) of mite sensitive atopic patients upon challenge with specific allergen, while PBMC of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester and Ca2+ ionophore induced marked IL-5 production by PBMC obtained from atopic and non-atopic asthmatics, suggesting that both protein kinase C and Ca2+ influx are required for IL-5 production. CD2- or CD4-bearing cell depletion almost completely removed IL-5-producing cells while CD8-bearing cell depletion rather enriched them. These findings indicate that CD4+ T cells are the principal source of IL-5 in PBMC. The capacity of PBMC of atopic asthmatics, non-atopic asthmatics and healthy controls to produce IL-2, IL-4, IL 5 and IFN-gamma was compared, to find that cytokine-producing capacities other than that of IL-5 (IL-2, IL-4 and IFN-gamma) were not significantly different among the three groups. Dexamethasone, FK506 and cyclosporin A suppressed IL-5 production in vitro in a dose-dependent manner. Clear dose-dependent suppression of IL-5 gene expression by FK506 was also observed. Treatment of asthmatic patients with inhaled glucocorticoid (beclomethasone dipropionate) ameliorated clinical symptoms, improved lung function and markedly suppressed IL-5 production by PBMC, suggesting the essential role of IL-5 in the pathogenesis of bronchial asthma and the clinical importance of its regulation. PMID- 7540863 TI - Human vascular endothelial cells process and present autoantigen to human T cell lines. AB - The effectiveness of cultured human umbilical vein endothelial cells as accessory cells for T cell activation has been investigated using T cell clones and lines derived from patients with myasthenia gravis which were specific for different epitopes on the alpha subunit of the human acetylcholine receptor. The endothelial cells were induced with IFN-gamma to express HLA-DR and -DQ at high and low levels respectively. They could then efficiently present specific peptides of the alpha subunit to an HLA-DR- and an HLA-DQw5-restricted T cell line. They could also process epitopes for both T cell lines from the full-length recombinant alpha subunit (r1-437) of the human acetylcholine receptor, where the known epitopes are 80 amino acid residues apart. The endothelial presentation of r1-437, but not of the peptides, was sensitive to chloroquine inhibition. Presentation appeared slightly less efficient (by 1.5- to 3.0-fold) with endothelial cells than with presenting cells from peripheral blood. This may reflect differences in accessory signalling since mAb blocking studies suggested that ligands for CD28 provided important accessory signalling by peripheral blood presenting cells while LFA-3 was used by endothelial cells. PMID- 7540865 TI - Role of afferent sensory neurones in gastric injury/protection. AB - The role of afferent sensory neurones in gastric mucosal protection is discussed. The principal effects of substance P and capsaicin on gastric motility and mucosal blood flow are taken in correlation with gastric mucosal injury. It seems likely that the protective effect of sensory neuropeptides is dependent on gastric mucosal blood flow and is mediated through the nitric oxide-generating system and partly the prostaglandins. The interaction between these two systems and the primordial effect of one of them on gastric mucosal blood flow and mucosal integrity after neuropeptide release is still not clear. PMID- 7540867 TI - The effects of nitrous oxide on a glutamate-gated ion channel and their reversal by high pressure; a single channel analysis. AB - Nitrous oxide reversibly affects the kinetics, but not the conductance, of the qGluR channel of locust muscle. 0.5 atm N2O at 20.5 degrees C was without effect but both 1.5 and 2.7 atm significantly reduced the probability of the channel opening, the frequency of opening and the mean open time, and prolonged the mean closed time. 100 atm helium was without effect on these parameters, but when 98.5 atm He was combined with 1.5 atm N2O they, and the associated dwell time distributions, were restored to normal. 100 atm similarly combined with 2.7 atm N2O exerted a comparable trend which fell short of significance. The results are consistent with nitrous oxide binding to the channel with a significant molar volume increase, which pressure opposes. This suggests that nitrous oxide may cause conformational changes in the channel, and that the pressure reversal of nitrous oxide anaesthesia in animals could be caused by molecular antagonism. PMID- 7540866 TI - Signal transduction pathways leading to insulin-induced early gene induction. AB - We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC50 = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat 1 fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C terminal truncated receptors (delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in delta CT cells, whereas stimulation of MAP kinase activity was almost absent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540864 TI - Lymphocytes infiltrating the CNS during inflammation display a distinctive phenotype and bind to VCAM-1 but not to MAdCAM-1. AB - The nature of inflammatory lymphocytes recruited to the CNS has been studied in a model of chronic inflammation. Injection of killed Corynebacterium parvum into the cortex of the mouse brain produces a circumscribed inflammatory cellular infiltrate around the injection site, and recruited mononuclear inflammatory cells (IC) can be isolated for flow cytometric analysis. The majority of IC were T cells. In comparison with the predominant naive population of mesenteric lymph node T cells, IC T cells express much higher levels of CD44, LFA-1 and ICAM-1, and lower levels of CD45RB, features commonly associated with memory (previously activated) cells. In addition, in contrast to the L-selectin+ alpha 6-integrinlow phenotype of naive lymph node T cells, IC T cells lacked L-selectin and were alpha 6-integrin-. Mac-1, recently proposed as another marker of memory T cell differentiation, was not displayed by IC T cells, suggesting that Mac-1 expression may be heterogeneous among memory T cell subsets. A subset of mesenteric lymph node (MLN) T cells, probably representing activated T cells undergoing the naive to memory transition, but not of IC T cells, expressed high levels of alpha 6-, beta 7- and alpha E-integrin. IC and MLN naive T cells expressed comparable levels of alpha 4-integrin, but IC T cells stain poorly with anti-beta 7 mAbs and with mAb DATK 32, specific for the alpha 4 beta 7 heterodimeric lymphocyte homing receptor for the mucosal addressin MAdCAM-1, suggesting that these inflammatory cells express more alpha 4 beta 1 than alpha 4 beta 7. Consistent with this, in in vitro adhesion assays, brain IC bound better than MLN cells to the alpha 4 beta 1 integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adhered very poorly to the alpha 4 beta 7 ligand MAdCAM-1. These findings are consistent with and extend previous immunohistological studies of T cells in murine experimental autoimmune encephalomyelitis, and demonstrate a distinctive phenotype for lymphocytes being present in the chronically inflamed brain. PMID- 7540868 TI - Cloning of a cGMP-gated cation channel from mouse kidney inner medullary collecting duct. AB - The cDNA sequence coding for the cGMP-gated cation channel expressed in the mouse kidney inner medullary collecting duct has been determined. The kidney cGMP-gated cation channel cDNA has an open reading frame of 2055 nucleotides and encodes a 685 amino acid protein. One cDNA clone is alternatively spliced thereby producing a deletion of 107 bp. Two differentially spliced 5' untranslated regions were determined by 5' RACE. PMID- 7540869 TI - Non-selective cation channels in cerebrovascular smooth muscle cells of adult rats. AB - Inside-out patch clamp recordings have revealed the presence of a novel, large conductance channel of the non-selective, cation permeable type in smooth muscle cells dispersed from the cerebral arteries of adult rats. In physiologically appropriate ionic gradients, current flow in these channels reversed polarity at a membrane potential of about -42 mV. Single channel conductance in symmetrical 140 mM K+ salines was 211 pS. The channel was permeable to both K+ and Na+, with a ratio PNa/PK = 0.15, while Cl- was effectively impermeant. Calcium ions were weakly permeant (PCa/PK = 0.03, PCa/PNa = 0.20). Channel open probability increased with membrane depolarization and was weakly dependent on the concentration of free intracellular Ca2+. This channel would contribute outward membrane current at potentials more positive than about -42 mV. In concert with outward potassium currents, it may serve to limit membrane depolarization during action potential activity in cerebrovascular smooth muscle cells. PMID- 7540870 TI - Ion channel formation by synthetic analogues of staphylococcal delta-toxin. AB - Ion channel formation by three analogues of staphylococcal delta-toxin, an amphipathic and alpha-helical channel-forming peptide, has been evaluated by measurement of ionic currents across planar lipid bilayers. Replacement of beta branched, hydrophobic residues by leucine and movement of a tryptophan residue from the hydrophilic to the hydrophobic face of the helix does not significantly alter ion channel activity. Removal of the N-terminal blocking group combined with the substitution of glycine-10 by leucine changes the single channel properties of delta-toxin, without altering macroscopic conductance/voltage behaviour. Truncation of the N-terminus by three residues results in complete loss of channel-forming activity. These changes in channel-forming properties upon altering the peptide sequence do not mirror changes in haemolytic activity. The results lend support to the proposal that channel formation and haemolysis are distinct events. Channel properties are discussed in the context of a model in which the pore is formed by a bundle of approximately parallel transbilayer helices. PMID- 7540871 TI - Solvent stabilized solution structures of galanin and galanin analogs, studied by circular dichroism spectroscopy. AB - Circular dichroism spectroscopy has been used to study how different solvents stabilize secondary structure in the neuropeptide galanin (rat), two N-terminal fragments of galanin, galanin(1-12) and galanin(1-16), and six other differently charged analogs. Among these analogs, the peptide M40, galanin(1-13)-Pro-Pro-Ala Leu-Ala-Leu-Ala amide, is a high affinity, receptor subtype specific galanin receptor antagonist. The different solvents include sodium dodecyl sulfate (SDS) micelle solutions, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2 dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) vesicle solutions. 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFP) and 100% 2,2,2-trifluoroethanol (TFE). DOPC vesicles did not change the structure of the peptides as compared to aqueous solvent. The negatively charged DOPG vesicles and SDS micelles induced similar changes towards alpha-helical structures in all peptides. The HFP and TFE solvents have an even stronger tendency to stabilize alpha-helical conformations in these peptides. Since DOPG vesicles can be considered as a model system for negatively charged biological membranes, the solution structures observed in the presence of DOPG or SDS may be the most relevant for the in vivo situation. Correlations between the binding affinity of the peptides to hippocampal galanin receptors and their observed structures in the DOPG solvent were investigated. PMID- 7540873 TI - [Phenomenological exploration of suicidal ideation]. AB - Death fuels the idea of suicide with a strong emotional charge that often turns suicidal tendencies and suicide into one and the same. The idea of suicide, at times linked with depression and personality disorders, is nonetheless full of a meaning which deserves further research. Suicide can appear as a solution to a problem which the idea of suicide helps to identify and resolve. When it is a conscious realization, the idea of suicide can lead the patient to consult. As a danger signal, it allows to determine whether the individual's defense mechanism functions. Also, it attracts attention to a situation, reveals a form of repressed self-hate and even helps in passing from one life state to another, as in transition rituals described by anthropology. PMID- 7540872 TI - Horizontal 'solvent-free' lipid bimolecular membranes with two-sided access can be formed and facilitate ion channel reconstitution. AB - We present here an easily used method and apparatus for formation of horizontal 'solvent-free' lipid bilayer membranes affording two-sided access. These horizontal bilayers allow direct delivery of submicroliter volumes of samples onto the membrane upper surface increasing the efficacy of reconstitution by several orders of magnitude, as demonstrated using Staphylococcus aureus alpha toxin. Also, they permit creation of locally high and transient transbilayer osmotic gradients to initiate fusion of ion-channel containing liposomes with planar membrane, which, following fusion, leaves the membrane and channel in essentially symmetric bathing solutions. This method is especially advantageous for cases where thickness of the membrane, absence of hydrocarbon solvent, or presence of differing lipid compositions in the two monolayers is critical. PMID- 7540874 TI - Presumed central nervous system Whipple's disease in a child: case report. AB - Whipple's disease is a rare, chronic, multisystem illness that is pathologically characterized by the accumulation of macrophages in the involved tissue that have a positive periodic acid-Schiff reaction. It is typically seen in middle-aged white men, and only four cases involving persons younger than 15 years of age have been reported. CNS Whipple's disease without intestinal manifestations is rare; only six cases have been reported in the literature, all involving adults. We report the case of a young boy with clinical, laboratory, radiographic, and pathological signs and symptoms consistent with CNS Whipple's disease who responded to therapy with trimethoprimsulfamethoxazole. PMID- 7540875 TI - Treatment of ectoparasitic infections: review of the English-language literature, 1982-1992. AB - We reviewed the English-language literature of 1982-1992 for papers concerning scabies, pediculosis, and their treatments; this information was supplemented with opinions of workers in the field of ectoparasitic disease. Six treatment trials for scabies, one study of the treatment of pediculosis pubis, and additional studies on the treatment of pediculosis capitis were included. Both lindane 1% and permethrin 5% are effective treatments for scabies, although resistance to lindane does exist. Lindane 1%, permethrin 1%, and pyrethrins with piperonyl butoxide are all effective treatments for pediculosis. Severe reactions to lindane therapy may occur when increased skin absorption of lindane occurs. Permethrin 5% or lindane 1% is recommended for the treatment of scabies. Lindane should be used with precautions to avoid excess skin penetration and should not be used to treat infants, young children, or pregnant or lactating women. Permethrin 1%, lindane 1%, or pyrethrins with piperonyl butoxide is recommended for the treatment of pediculosis pubis. Pregnant and lactating women should be treated with either permethrin or with pyrethrins containing piperonyl butoxide. PMID- 7540876 TI - Human papillomavirus infection and genital warts: update on epidemiology and treatment. AB - The treatment of genital warts remains frustrating since it is often painful, expensive, and unsuccessful. Moreover, little is known about the infectivity and natural history of exophytic genital warts or subclinical genital infection with human papillomavirus. The traditional goals of therapy for sexually transmitted diseases--eradication of infection, elimination of symptoms, prevention of long term sequelae, and interruption of transmission--are currently not attainable for or applicable to genital warts. The medical literature from January 1988 to August 1993 was reviewed for recent studies on the treatment of exophytic warts. The following treatments were included in the reviewed studies: podofilox (which was recently approved by the Food and Drug Administration), podophyllin, cryotherapy, topical 5-fluorouracil, intralesional interferon, systemic interferon, and laser surgery. No single treatment modality was superior to another, and recurrence rates associated with all modalities were high. Treatment of genital warts should be guided by preferences of the patient, and a specific therapeutic regimen should be chosen with consideration of expense, efficacy, convenience, and potential for adverse effects. PMID- 7540877 TI - Cyclic adenosine monophosphate-dependent mechanisms induce von Willebrand factor expression in the Dami megakaryoblastic cell line. AB - It has been proposed that cyclic adenosine monophosphate (cAMP) is involved in the differentiation of several cell types and this study analysed whether von Willebrand factor (vWf) synthesis, which is a marker of the megakaryocyte maturation of these cells, would be enhanced by agents acting on cAMP formation. Different compounds known to stimulate cAMP accumulation in cells were used: dibutyryl cAMP (db-cAMP), isobutyl-methylxanthine (IBMX) or pentoxifylline (PTX) and forskolin. Treatments with db-cAMP or IBMX (10-1,000 microM) induced a dose dependent increase in vWf synthesis. Associations of IBMX with forskolin produced a synergistic enhancement in vWf synthesis. PTX alone did not enhance vWf synthesis but a latent effect was revealed in the presence of forskolin or db cAMP. The increase in vWf mRNA shown by Northern blot analysis demonstrates that the protein synthesis correlates with the transcript expression after db-cAMP or IBMX treatments. vWf synthesis paralleled the accumulation of cAMP in the cells. Moreover vWf expression induced by combination of IBMX with forskolin was associated with a moderate increase in the percentage of GPIIb/IIIa positive cells and in the ploidy level related to an important inhibition of cell growth. These data provide evidence that agents acting on cAMP metabolism induce vWf synthesis in the Dami megakaryoblastic cells. PMID- 7540878 TI - Recombinant hirudin for the treatment of disseminated intravascular coagulation in patients with haematological malignancy. AB - The purpose of this pilot study was to determine the effect of recombinant hirudin (r-hirudin) on coagulopathy and the relationship between concentrations of thrombin-antithrombin III (ATIII) complex (TAT) and thrombin-hirudin complex (THC) in patients with disseminated intravascular coagulation (DIC). Five patients with haematological malignancy associated with DIC were studied. r Hirudin was administered by continuous intravenous infusion at a dose of 0.005 mg/kg/h for 4-9 days to each patient. Fibrin/fibrinogen degradation products (FDP), D-dimer, TAT and plasmin-alpha 2 antiplasmin complex (PAP) concentrations decreased after treatment with r-hirudin in four patients studied. However, in one patient, serum creatinine increased to 1.7 mg/dl and aPTT was prolonged to 74.4s. Statistical analysis disclosed significant positive correlations between plasma concentrations of hirudin and THC, and between concentrations of THC and TAT. The concentrations of THC were much higher than those of TAT. In conclusion, these findings indicate that r-hirudin more strongly inhibited thrombin than did ATIII without heparin, and that administration of r-hirudin to renal insufficiency required individual adjustment of dosage. The present findings also suggest that r-hirudin can be considered a new agent for the treatment of DIC. PMID- 7540880 TI - Cytology of a benign phyllodes tumour with keratin cyst formation; a spectrum of diagnostic pitfalls. PMID- 7540879 TI - Coagulopathy following bites by the Papuan taipan (Oxyuranus scutellatus canni). AB - The mechanisms of haemostatic failure were studied in 87 patients bitten by the Papuan taipan (Oxyuranus scutellatus canni). Eighty (92%) had evidence of a coagulopathy on laboratory testing; 36 (41.4%) developed spontaneous systemic bleeding, although this was rarely of clinical significance. Coagulation assays in 48 completely defibrinated patients showed marked reductions in factors V and VIII and reductions in factors II, IX, XI, XII and XIIIA. There was a reduction in plasminogen and alpha 2-antiplasmin levels and both total and cross-linked fibrin(ogen) degradation products (FDP) levels were elevated. The mean platelet count was initially decreased and fell further during admission. Similar but less severe changes were seen in patients who were mildly defibrinated. Following treatment with antivenom, fibrinogen levels rose rapidly and coagulability was restored within 6-12 h in 93% of patients. These abnormalities may be primarily attributable to the prothrombin activator present in taipan venom, but it is likely that other uncharacterized venom components contributed. PMID- 7540881 TI - Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients. AB - We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC-ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients. PMID- 7540882 TI - Coordinate expression and developmental role of Id2 protein and TAL1/E2A heterodimer in erythroid progenitor differentiation. AB - The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Id and TAL1/E2A bHLH genes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2 and TAL1/E2A genes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2 mRNA is detected by reverse transcriptase polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mRNAs are not. At the onset of erythroid differentiation, Id2 mRNA is downregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2 is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1 and E2A are only transiently induced in the first week of granulopoietic differentiation. The expression pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, analyzed by Western blot, shows a pattern consistent with gelshift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA. The results are strictly consistent with the expression studies: anti-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increase of erythroid colony formation, whereas alpha TAL1 induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-Id2 polypeptides compete the TAL1/E2A-specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation. PMID- 7540883 TI - Interferon-gamma upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production. AB - The human interleukin-3 (IL-3) receptor is constitutively expressed on certain hematopoietic cells where it mediates proliferation and differentiation, or functional activation. We have recently found that human umbilical vein endothelial cells (HUVECs) also express IL-3 receptors and that the expression is enhanced by stimulation with the monokine tumor necrosis factor alpha. In this report we show that the lymphokine interferon gamma (IFN gamma) is a powerful stimulator of the IL-3 receptor of HUVECs and that the combination of IL-3 and IFN gamma has a synergistic effect on major histocompatibility complex (MHC) class II expression and on the production of the early-acting hematopoietic cytokines IL-6 and granulocyte colony-stimulating factor (G-CSF). IFN gamma caused a time- and dose-dependent up-regulation of mRNA for both the alpha and beta chains of the IL-3 receptor, with maximal effects occurring 12 to 24 hours after stimulation with IFN gamma at 100 U/mL. Induction of mRNA correlated with protein expression on the cell surface, as judged by monoclonal antibody staining of both receptor chains and by the ability of HUVEC to specifically bind 125I labeled IL-3 (125I-IL-3). Scatchard analysis of HUVECs stimulated with IFN gamma at 100 U/mL for 24 hours showed approximately 6,300 IL-3 receptors per cell that were of a high affinity class (dissociation constant [kd] = 500 pmol/L) only. The addition of IL-3 to IFN gamma-treated HUVECs strongly enhanced the expression of MHC class II antigen. Importantly, IFN gamma and IL-3 also exhibited a synergistic effect in the induction of the mRNA for G-CSF and IL-6. This was reflected in increased amounts of G-CSF and IL-6 protein in HUVEC supernatants. In contrast, IFN gamma and IL-3 did not stimulate granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-8 production in HUVECs. These results show that IFN gamma is a strong stimulator of IL-3 receptor expression in HUVECs and suggest that in vivo T-cell activation, causing the concomitant production of IFN gamma and IL-3, may lead to enhanced endothelial MHC class II expression and to the selective production of early-acting hematopoietic cytokines. Thus, IL-3 could influence immunity and hematopoiesis by acting not only on hematopoietic cells, but also on vascular endothelium. PMID- 7540884 TI - The impact of three-dimensional structure on the expression of PlA alloantigens on human integrin beta 3. AB - We have compared the binding of affinity-purified anti-PlA1 IgG from seven nonrelated donors with chimeric integrin subunit beta 3 molecules expressed in the baculovirus-Spodoptera frugiperda insect cell system. Beta 3 chimeras were engineered to include segments of antigenic human beta 3 sequences spliced to intervening segments of nonantigenic Xenopus beta 3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human beta 3 Cys26-Cys38 loop only when it is presented in a correct orientation that must be maintained by noncontiguous human sequences. Key downstream sequences are located within the region beta 3(288 490), flanking either side of the putative long-range disulfide at Cys435. Although our results confirm unambiguously that the Leu/Pro polymorphism at position 33 in human beta 3 is necessary for the expression of PlA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human beta 3 sequence transcends the need to maintain a correct orientation within the Cys26-Cys38 loop itself, because the murine monoclonal antibody SZ21, which recognizes the sequence beta 3(28-35) contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PlA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288-490 either directly contribute to the composition of the PlA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the beta 3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop. PMID- 7540885 TI - Serum levels of stem cell factor are increased in asymptomatic human immunodeficiency virus-infected patients and are associated with prolonged survival. AB - Cytopenia is a common complication of human immunodeficiency virus (HIV) infection and can affect different hematopoietic lineages, including erythropoiesis, lymphopoiesis, thrombopoiesis, and granulopoiesis. Stem cell factor (SCF), a cytokine expressed by bone marrow stromal cells, is a multipotential growth factor acting on early progenitor cells of most hematopoietic lineages. Therefore, we investigated the serum SCF levels in 74 HIV infected persons without active secondary infection at different stages of HIV infection [Centers for Disease Control (CDC) stages A through C]. Circulating SCF levels were determined by enzyme-linked immunosorbent assay and were found to be significantly elevated in CDC stage A as compared with normal controls (7.18 +/- 1.94 ng/mL v 3.95 +/- 0.91 ng/mL, P = .04). However, in CDC groups B and C, SCF levels were lower than in CDC group A (3.29 +/- 0.75, P = .162, and 1.95 +/- 0.39, P = .005, respectively). Serum levels greater than 1.8 ng/mL were associated with a longer survival (P = .0037) in 74 HIV type 1 (HIV-1) seropositive patients monitored for up to 114 weeks, suggesting that this cytokine may be directly associated with the disease course. A Cox proportional hazards model showed SCF to be an independent prognostic factor for survival (risk ratio for death, 0.73; 95% confidence interval, 0.56 to 0.95; P = .019). Serum SCF levels decreased on follow up in 24 of 38 patients or remained below 0.4 ng/mL in 10 of 38 patients from whom a second blood sample was collected after a mean interval of 44 weeks. To determine potential regulatory factors of SCF expression by stromal cells, we exposed cultured fibroblasts to various cytokines. Only interleukin-4 (IL-4) upregulated SCF mRNA. As IL-4 is modulated during early HIV disease, it may be a key regulator of SCF secretion. Further studies are required to elucidate the mechanism of SCF action and regulation in patients with HIV infection. PMID- 7540886 TI - cDNA structure, tissue-specific expression, and chromosomal localization of the murine band 7.2b gene. AB - Band 7.2b is an integral phosphoprotein absent from the erythrocyte membranes of patients with hydrocytosis, an autosomal, dominantly inherited, hemolytic anemia characterized by stomatocytic red blood cells with abnormal permeability to Na+ and K+. The role of this protein in the erythrocyte membrane is not well understood. To gain additional insight into the structure and function of this protein, we have cloned the murine band 7.2b cDNA and studied its tissue-specific expression. 2,873 bp of cDNA with an open reading frame of 852 bp were isolated. This fragment encodes a protein of 284 amino acids with a predicted molecular weight of 31 kD. The band 7.2b gene had a wide pattern of expression, with high levels of mRNA in heart, liver, skeletal muscle, and testis and low levels in lung, brain, and spleen. Using fluorescent in situ hybridization, the murine band 7.2b gene was mapped to chromosome 2, at the border of the distal region of 2B and proximal region of C1, syntenic to 9q33-q34, the location of the human homologue. Models of the predicted protein structure showed a short NH2-terminal head, a strongly hydrophobic 28-amino acid stretch presumably encoding a single membrane-spanning domain, and a large domain composed of beta sheet and alpha helix. Database searching showed no significant homology of other known proteins to murine or human band 7.2b. PMID- 7540888 TI - Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. AB - A major potential problem of autologous transplantation in the treatment of advanced malignancy is the infusion of tumor cells. A multi-institutional study of purified CD34-selected peripheral blood progenitor cell (PBPC) transplantation was conducted in 37 patients with advanced multiple myeloma receiving myeloablative chemotherapy. Fourteen days after intermediate-dose cyclophosphamide, prednisone, and granulocyte colony-stimulating factor (G-CSF), a median of 3 (range, 2 to 5) 10-L leukaphereses yielded 9.8 x 10(8)/kg (range, 3.7 to 28.3) mononuclear cells. The adsorbed (column-bound) fraction contained 5.9 x 10(6) cells/kg (range, 1.6 to 25.5) with 4.65 x 10(6) CD34 cells/kg (range, 1.2 to 23.3). Using Poisson distribution analysis of positive polymerase chain reactions with patient-specific complementarity-determining region 1 (CDR1) and CDR3 Ig-gene primers, tumor was detected in leukapheresis products from 8 to 14 unselected patients and ranged from 1.13 x 10(4) to 2.14 x 10(6) malignant cells/kg. After CD34 selection, residual tumor was detected in only three patients' products. Overall, a greater than 2.7- to 4.5-log reduction in contaminating multiple myeloma cells was achieved. CD34 PBPCs were infused 1 day after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg), and granulocyte macrophage colony-stimulating factor was used until hematologic recovery. The median time to both neutrophil and platelet recovery was 12 days (range, 11 to 16 days and 9 to 52 days, respectively). The median number of erythrocyte and platelet transfusions was 7 (range, 2 to 37) and 3 (range, 0 to 85), respectively. Patients receiving fewer than 2 x 10(6) CD34 cells/kg had significantly prolonged neutropenia, thrombocytopenia, and an increased red blood cell and platelet transfusion requirement. Thus, CD34 selection of PBPCs markedly reduces tumor contamination in multiple myeloma and provides effective hematopoietic support for patients receiving myeloablative therapy. PMID- 7540887 TI - Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells. AB - High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly. PMID- 7540889 TI - Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia. AB - Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low-density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation. PMID- 7540890 TI - Accelerated cell-cycling of hematopoietic progenitor cells by growth factors. AB - Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the proliferation of hematopoietic progenitor cells in vitro and in vivo. Although these factors have been extensively studied, little is known of their role in the regulation of cell cycling of hematopoietic progenitors, especially in the early stage of hematopoiesis. In the present study, we examined the effects of early acting growth factors on proliferative kinetics of hematopoietic progenitors by monitoring the number of cells in individual developing colonies, using an in vitro clonal assay. Interleukin-11 (IL-11) or steel factor (SF), alone or in combination, shortened the time for the size of IL-3-dependent colonies to double. Consecutive replating experiments provided evidence for direct action of growth factors on the growth rate of hematopoietic progenitor cells. Shortening of the time for the total cell number in the colonies to double was due to a reduction in time for each single cell within the respective colonies to become two daughter cells, and there was no alteration in the incidence of cells with a proliferative capacity. Cell-cycle analysis demonstrated that IL-11 has the potential to induce a shortened time for cell-cycle of hematopoietic progenitor cells without affecting distribution of each fraction of the cell-cycle, whereas SF has the potential to reduce cell-cycle time mainly by decreasing the time required for hematopoietic progenitor cells to go through the G1 phase. These results suggest that growth factors may modulate cell-cycling of hematopoietic progenitor cells. PMID- 7540891 TI - The severe combined immunodeficient-human peripheral blood stem cell (SCID huPBSC) mouse: a xenotransplant model for huPBSC-initiated hematopoiesis. AB - Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose intensified anticancer chemotherapy and were transplanted into unconditioned "nonleaky" young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL 3), IL-6, and granulocyte-macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment. PMID- 7540892 TI - The pattern of emesis following high-dose cyclophosphamide and the anti-emetic efficacy of ondansetron. AB - Two randomized, double-blind placebo-controlled ondansetron dose ranging studies in patients receiving high-dose cyclophosphamide (with or without doxorubicin) were completed in the US. These studies enable the pattern of emesis and nausea for 3 days following high-dose cyclophosphamide to be described and give some insight into the mechanisms of emesis which may be operating. Nausea and vomiting induced by cyclophosphamide-based chemotherapy has a long latency of onset (8-13 h) and continues for at least 3 days. These findings are of particular importance as many of these patients receive chemotherapy as outpatients and emphasize the need for appropriate anti-emetic prophylaxis for patients at home. Ondansetron was extremely effective over this time in the control of emesis and nausea. These results suggest that high-dose cyclophosphamide-induced emesis over days 1-3 is largely mediated via 5-hydroxytryptamine (5-HT) and 5-HT3 receptors. PMID- 7540894 TI - Bryostatin 1 enhances lymphokine activated killer sensitivity and modulates the beta 1 integrin profile of cultured human tumor cells. AB - Bryostatin 1 interferes with protein kinase C (PKC) signaling which is involved in the activation of human and murine cytotoxic T lymphocytes, and in the growth and differentiation of tumor cells. Bryostatin 1 has immunomodulating and antitumor properties as demonstrated by preclinical and clinical studies. Here we report that bryostatin 1 increases the susceptibility to lymphokine activated killers and modifies the pattern of beta 1 integrin expression of human tumor cells. On the basis of these results the use of bryostatin 1 in combination with immunostimulating cytokines such as interleukin-2 in the treatment of human cancer is suggested. PMID- 7540893 TI - Colorectal cell line suppression of lymphokine activated killer cell generation is reversed by suramin. AB - Progressive tumor growth is associated with a state of immunosuppression. One mode of immunosuppression is thought to be mediated by immunosuppressive factors of tumour origin. We have investigated in vitro the possibility that suramin could be used to blockade tumor derived suppressor factors and enhance the effectiveness of rhIL-2 therapy. These data show that factors derived from cultures of the colorectal carcinoma cell line LoVo suppressed the percentage of cells expressing the natural killer cell antigen CD56 in 92% of individuals and the cytotoxic T cell antigen CD8 in 77% of individuals tested from a panel of 13 normal healthy volunteers. Suramin at 200 micrograms/ml restored the percentage of cells expressing CD56 to levels higher than the control cultures and reduced the suppression in those expressing CD8 to non-significant levels. LoVo produced factors also suppressed the expression of the activation associated antigens CD25, CD71 and HLA-Dr with suramin restoring CD25 expression but not CD71 or HLA Dr. Functional studies using 51Cr-release assays showed that LoVo produced factors could suppress cytotoxicity in 46% of individuals tested, and of these a reduction in suppression by suramin was demonstrated in 50% of individuals against Daudi target cells and 33% against K562 target cells. PMID- 7540895 TI - Biochemical pharmacology and DNA methylation studies of arabinosyl 5-azacytidine and 5,6-dihydro-5-azacytidine in two human leukemia cell lines PER-145 and PER 163. AB - 1-beta-D-arabinofuranosyl-5-azacytosine (ara-AC) and 5,6-dihydro-5-azacytidine (DHAC) are two new antitumor agents under clinical investigations, which exhibit the chemical similarities found in the tumoricidal drug cytosine arabinoside (ara C) and the nitrogen substitution in the 5 position of the pyrimidine ring found in 5-azacytidine (5-aza-C). The cellular anabolism of ara-AC and DHAC and their effect on DNA methylation have been examined in two new human leukemia cell lines, which are sensitive (PER-145) and resistant (PER-163) to ara-C. The triphos-phate anabolite of ara-AC, ara-ACTP, was the major cellular anabolite in the cellular extracts of the PER-145 cells, reaching a cellular saturation concentration of 64.1 +/- 3.2 microM using 25 microM of the drug. Only trace levels of ara-ACTP were detected in the PER-163 cell line, which lacks deoxycytidine kinase, after exposure to a similar concentration. Notably, after 1 mM, the ara-ACTP concentration averaged 12 +/- 3 microM. DHAC was anabolized by both cell lines to a similar degree but required much higher nucleoside concentrations (100 microM or higher) to achieve similar cellular concentrations of its triphosphate, DHACTP. Although the deoxy-derivative, DHAdCTP, was detected in both cell lines, it was detected at 1-2 log10 lower concentrations than DHACTP. DNA methylation studies showed that DHAC had a profound effect in inducing DNA hypomethylation in both cell lines, with nadir values of 27.3 and 29.2% of control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540897 TI - The effect of forskolin on the teratogenicity of methylxanthines in the chick embryo heart. AB - Interactions between forskolin and methylxanthines, including caffeine and isobutylmethylxanthine (IBMX), in the developing chick embryo heart were investigated. Forskolin, a potent activator of adenylate cyclase, was administered to young chick embryos (Hamburger-Hamilton stage 24) together with caffeine or IBMX at doses where each agent alone caused minimal embryotoxicity. The incidence of malformation in the embryonic chick heart or aorta induced by caffeine (5 x 10(-7) or 5 x 10(-6) mol) and IBMX (1 or 2.5 x 10(-6) mol) significantly increased with coadministration of forskolin (1 x 10(-9) mol). Cardiovascular malformations included ventricular septal defect, double outlet right ventricle, and aortic arch anomalies. These results indicate that forskolin potentiates the teratogenicity of caffeine or IBMX on the cardiovascular system in the chick embryo and suggest that this potentiation may be related to increase intracellular cAMP due to stimulation of adenylate cyclase (forskolin) and inhibition of phosphodiesterase (methylxanthines). PMID- 7540896 TI - Prevalence of anti-HCV among spouses and offspring of anti-HCV positive subjects: an Italian multicentre study. AB - The role of familial environment in the spreading of hepatitis C virus (HCV) infection is not well established. We studied 1670 family members for 578 anti HCV+ subjects enrolled in 8 centres distributed throughout Italy. The prevalence of anti-HCV positivity was significantly higher in spouses than in offspring (15.6% and 2.1% respectively; p < 0.01), with no difference between northern and central-southern regions of Italy. Anti-HCV positivity was found almost exclusively in adults; among offspring, during the first two decades of life, the prevalence of anti-HCV positivity was significantly lower than in subjects over 20 years old (0.6% vs 3.1%, respectively). PMID- 7540899 TI - Neuropsychological deficits in very young bone marrow transplant recipients. AB - Bone marrow transplantation (BMT) involves conditioning with cyclophosphamide and, for patients with malignant disease, total body irradiation (TBI). This study describes the neuropsychological development of 10 children treated for leukemia (n = 7), neuroblastoma (n = 1) or severe aplastic anemia (SAA; n = 2) at 3 years of age or younger. A moderate general developmental delay, with pronounced motor deficits and varying degrees of perceptual and cognitive problems, was observed in all children treated for malignant disease. Children treated for SAA had normal development. We conclude that BMT, including TBI, can be directly associated with long-term neuropsychological impairment in children treated at a very young age. Continued medical and psychological follow-up procedures are needed. PMID- 7540898 TI - The insulin-like growth factor axis and collagen turnover in asthmatic children treated with inhaled budesonide. AB - Serum concentrations of growth hormone-dependent insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3), the carboxy terminal propeptide of type I procollagen (PICP), the carboxy terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) and the amino terminal propeptide of type III procollagen (PIIINP) were studied in 14 prepubertal children with asthma (mean age 9.7 years) during treatment with inhaled budesonide. The study design was a randomized, crossover trial with two double-blind treatment periods (200 and 800 micrograms) and one open, non randomized treatment period (400 micrograms). All periods were 18 days' duration. Budesonide treatment was associated with a dose-related suppressive trend in serum concentrations of PIIINP when the 400 micrograms period was included (p < 0.01; z = -2.7) and when it was excluded from the calculations (p < 0.01; z = 2.6), indicating reduced synthesis of type III collagen. A similar trend was observed in ICTP levels when the 400 micrograms period was excluded from the calculations (p = 0.05; z = -1.9). No other statistically significant variations were seen. PMID- 7540900 TI - In vivo angioscopic detection of the damaged endothelium on the atheromatous plaque: application with vital staining by methylene blue. AB - We attempted to detect the lesion of the damaged endothelium on the atherosclerotic plaque in vivo with dye and angioscope. The plaques were induced on the aortae of rabbits by the continuous mechanical injury with the insertion of the polyethylene tube and feeding with 2% cholesterol-added diet. We observed the some plaques could be stained with 5 x 10(-2) mol/L methylene blue by angioscope. Control aorta, without any stimulations on the aorta, could not be stained. One month after injury and cholesterol feeding the plaques were stained. In such plaques, the lack of the endothelium and the existence of the lipid containing cell in the intima was observed. However, the plaques of the aorta 6 months after finishing these procedures could not be stained. In these plaques, the morphological characteristics known as the regression of the atheromatous plaque, ie, the regeneration of the endothelium, the thick collagen layer on the plaque and the massive calcification of the plaque were observed. The results obtained were as follows. (1) The atheromatous lesion with the damaged endothelium could be detected in vivo by our vital staining method. (2) The atheromatous plaques stained in vivo are the lesion with complication and enhanced permeability. (3) The slightly stained plaques indicate the lesions with regression and lost high permeability. (4) Our method would be able to add important information on the atheroscrelotic lesions in the clinical situations than that now we perform. PMID- 7540901 TI - Localization of peanut (Arachis hypogaea) root lectin (PRA II) on root surface and its biological significance. AB - The glucose-specific peanut root lectin, PRA II, is localized on the surface of 7 day-old peanut seedling root and in root cortical parenchymatous cells. The lectin is eluted from intact roots upon washing with buffer containing glucose. Rabbit erythrocytes bind to the root surface and the cortical cells; the binding is inhibited by antibodies raised against PRA II, peanut-specific Rhizobium cells and by glucose. Lipopolysaccharides isolated from host-specific Rhizobium strain inhibit the haemagglutinating activity of PRA II and are precipitated by the lectin. Our results suggest that PRA II might be involved in recognition of Rhizobium by peanut roots. PMID- 7540902 TI - Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. AB - The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate. PMID- 7540903 TI - Modulation of multidrug resistance in de novo adult acute myeloid leukemia: variable efficacy of reverting agents in vitro. Eastern Cooperative Oncology Group. AB - The efficacy of verapamil and cyclosporine A as modulators of P-glycoprotein, the multidrug resistance (MDR1) gene product, was studied in leukemic blast cells from 56 patients with de novo acute myeloid leukemia (AML) in vitro. Rhodamine123 dye-efflux was measured flow cytometrically as a cellular parameter reflecting P glycoprotein activity. While dye-efflux was measurable in 3/4 of the cases, the capacity of the P-glycoprotein inhibitors varied substantially among patients. In 23 patients, P-glycoprotein function was completely inhibited by the resistance modulators, whereas in 17 patients neither verapamil nor cyclosporine had any reverting effect on dye-efflux at concentrations even 10-times higher than achievable in vivo. Cells with a drug-sensitive rhodamine123-pump effluxed more efficiently (p = 0.0016) and contained significantly higher levels of MDR1 specific RNA transcripts (p = 0.0002), as determined by quantitative PCR, than cells exhibiting an efflux process that could not be inhibited. However, flow cytometric evaluation of the staining of gated blast cells with the anti-P glycoprotein antibody, 4E3.16, revealed no difference in P-glycoprotein expression between modulator-sensitive and -insensitive cases (p = 0.86), indicating disproportionate translation of MDR1 mRNA. In leukemic cell populations with increased P-glycoprotein function that could be inhibited, significantly more blasts expressed the progenitor cell antigen, CD34 (median 83%), than was the case in leukemias with P-glycoprotein activity that could not be inhibited (median 7%) (p = 0.0001). The present study demonstrates that a substantial fraction of AML patients constitutively display a drug-efflux mechanism suggestive of P-glycoprotein activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540904 TI - CD40 and its ligand: central players in B lymphocyte survival, growth, and differentiation. AB - This article brings together information garnered over the last 10 years on the B cell-associated CD40 surface glycoprotein. The discovery in 1992 of a natural counterstructure, the CD40 ligand (CD40L), which can be rapidly induced on T cells has hastened research and increased interest in this area dramatically. It has become clear that CD40 and its ligand are central players in B cell responses to T-dependent antigens. The capacity of CD40 to deliver signals for B cell growth, differentiation and survival is covered together with discussion of the signal transduction pathways which operate to bring about these changes. Future issues surrounding this important receptor-ligand pair are discussed. PMID- 7540905 TI - Indocyanine green angiographically well-defined choroidal neovascularization: angiographic patterns obtained using the scanning laser ophthalmoscope. AB - Several studies have shown that indocyanine green (ICG) angiography improves the detection of occult choroidal neovascularization (CNV). However, uncertainty remains about the angiographic patterns of CNV in ICG angiography. We examined the ICG angiographic findings of 104 patients (average age, 72.4 years) with newly diagnosed neovascular age-related macular degeneration and well-defined CNV in ICG angiography using the scanning laser ophthalmoscope. CNVs were grouped into one of three categories according to their appearance on biomicroscopy and fluorescein angiography: I, early CNV measuring less than half a disc diameter in size (7 eyes); II-1, advanced nonhemorrhagic CNV (44 eyes); II-2, advanced hemorrhagic CNV (29 eyes); and III, fibrovascular CNV (24 eyes). Of the 104 CNV, 36 demonstrated well-defined CNV and 68 ill-defined or occult CNV in fluorescein angiography. CNV in patients suffering from advanced nonhemorrhagic CNV (group II 1) demonstrated a thick, branching, early hyperfluorescent vascular pattern (27 of 44 eyes). In group III, CNV was depicted as thin, slightly branching vascular structures (16 of 24 eyes). In group I, dye leakage was found in all affected eyes, and in group II-2 it was present in 20 of 29 eyes. However, dye exudation was rare in group II-1 and group III. Feeder vessels were detected in 16 of 104 eyes. The structure and permeability of CNV can vary substantially in ICG angiography, depending on the stage of development or regression. PMID- 7540907 TI - Simultaneous modeling of multiple loops in proteins. AB - The most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by "threading" through a set of predefined protein folds ("inverse folding"). Both sets of methods provide a model for the core of the protein--the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop-closure algorithms are aimed at predicting loop structures, given their end-to-end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond-scaling-relaxation loop-closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5-7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6-1.7 A for backbone atoms and 1.1-3.3 A for all atoms. PMID- 7540908 TI - Psychoanalytic psychotherapy and the tragic sense of life and death. AB - The authors discuss the role of psychotherapy with dying patients and explore the psychological impact of imminent death. The psychotherapy case they present highlights the symbolic meaning of fatal symptoms and the light this meaning sheds on the relationship between how we live and how we die. These features are addressed in the context of larger unresolved clinical issues: Is there a "healthy" way to die? And is there a unique role for psychoanalytic psychotherapy with dying patients? PMID- 7540906 TI - Comparative modeling of the three-dimensional structure of type II antifreeze protein. AB - Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540909 TI - Symptoms in a 'leaky' mouse. PMID- 7540910 TI - Lung disease in the cystic fibrosis mouse exposed to bacterial pathogens. AB - Lung disease is the major cause of death in cystic fibrosis (CF), but there is no evidence for overt lung involvement at birth. We show here that the same is true for the gene targeted cftrm1HGU mutant mouse. Furthermore, this CF mouse model demonstrates an impaired capacity to clear Staphylococcus aureus and Burkholderia (Pseudomonas) cepacia, two opportunistic lung pathogens closely associated with lung disease in CF subjects. The cftrm1HGU homozygotes display mucus retention and frank lung disease in response to repeated microbial exposure. Thus, lung disease in the cftrm1HGU mouse develops in response to bacterial infection, establishing a model to dissect the pathogenesis of CF pulmonary disease and providing a clinically relevant end point to assess the efficacy of pharmacologic or genetic interventions. PMID- 7540911 TI - The raccoon spinocervical and spinothalamic tracts: a horseradish peroxidase study. AB - The locations of the cells of origin of the spinocervical tract (SCT) and spinothalamic tract (STT) were examined in relation to the somatotopic and laminar organization of the cervical enlargement of the raccoon dorsal horn (DH). In different animals, either the lateral cervical nucleus or the lateral thalamus was injected with a 2% solution of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Following 24 h or 4 days, respectively, the animals were sacrificed and both injection and target sites (spinal cord segments C6-T2) were processed using the TMB method. All labelled cells were counted in every fifth 50 microns section. Following injection of WGA-HRP into the lateral cervical nucleus, all labelled SCT cells were located ipsilateral to the injection sites. Most (84%) were in laminae III and IV, laminae known from other studies to contain cells preferentially responsive to light tactile stimulation, with very few (3%) in lamina I. Nearly 50% of labelled cells were located in the medial 1/3 of the DH, the region of representation of the glabrous surfaces of the raccoon forepaw. The mean number of labelled SCT cells per section was 4.19. After tracer injections of the lateral thalamus, more than 75% of STT cells were located contralateral to the injection sites. Forty-three percent were located in lamina I and 24% were in lamina V, laminae whose cells have been shown to be responsive to more intense forms of stimulation, as well as to light touch. Only 22% were located in the medial 1/3 of the DH. The mean number of labelled STT cells per section was 0.83. The results suggest that the SCT may play a more critical role in relaying discriminative light tactile and nociceptive information from the glabrous surfaces of the forepaw, but that there may be a greater role for the STT in relaying nociceptive information from the forelimb as a whole. PMID- 7540912 TI - Surface bacterial antigen CS31A as a tool to design new recombinant vaccines displaying heterologous antigenic determinants. PMID- 7540913 TI - Evaluation of the evidence for ion channels in synaptic vesicles. AB - Synaptic vesicles (SVs) have been the focus of much research for many years, however only recently have ion channels from SV membranes been reported. There is now convincing evidence that SVs contain ion channels. This conclusion is based on direct experimental results from several different laboratories using the patch clamp or planar lipid bilayer technique on SVs and neurosecretory granules (NSG). Some limitations of patch clamping and of fusing synamptic vesicles to a bilayer are described and the advantages of the nystatin/ergosterol fusion method are presented. Six different channels appear to exist in SV (or NSG) membranes. Two large channels (250 and 154 pS) have been observed in SVs isolated from mammalian brain, two channels (180 and 13 pS) from Torpedo electric organ, and two channels (130 and 30-40 pS) from NSG. The three larger channels from each set (250, 180 and 130 pS7) are novel in that they have a subconductance state. The 154 pS channel has been identified as synaptophysin but the identity and function of the other channels is unknown. Although some of the channels are gated by voltage, only the 130 pS channel is modulated by Ca2+. Further knowledge of what regulates these channels is mandatory if we are to determine the physiological significance of these channels. PMID- 7540915 TI - Pathological case of the month. Systemic cat-scratch disease. PMID- 7540914 TI - The surf-4 gene encodes a novel 30 kDa integral membrane protein. AB - The mouse surfeit locus is a tight cluster of at least six genes (surf-1 to -6), unrelated by sequence homology, whose unique organization is conserved in vertebrates. We show that the surf-4 coding sequence is conserved between mouse and human. Primary sequence analysis predicts that the mouse surf-4 protein contains seven transmembrane domains and a double lysine endoplasmic reticulum (ER) retrieval motif on the carboxyl terminus. Translation of the mouse surf-4 cDNA in vitro resulted in the production of a 30 kDa membrane protein. Salt and detergent extraction procedures showed that the surf-4 protein associated tightly with the microsomal membranes. Proteolysis protection of 14 and 3 kDa fragments indicates that the surf-4 protein contains at least two membrane spanning domains: this is consistent with the proposed topology. Addition of the c-Myc epitope into three different regions of the surf-4 protein resulted in transfectants that expressed a myc-tagged protein. Immunofluorescence analysis of the three surf-4 myc chimeras yielded a cytoplasmic staining pattern. Consistent with the presence of the ER retrieval motif, the surf-4 myc protein was not detected at the plasma membrane. A model for the proposed structure of the surf-4 protein is presented. PMID- 7540916 TI - Sample preparation, high-performance liquid chromatographic separation and determination of substance P-related peptides. AB - This review deals with the determination of low levels of substance P and peptide fragments derived from the undecapeptide, i.e. covers the whole amount of so called substance P-like immunoreactivity (SPLI) in biological samples. First an overview of the most currently used sample pretreatment procedures is given, followed by a description of the most effective high-performance liquid chromatographic (HPLC) separation methods. Special attention is paid to the choice of the appropriate column and the possible pitfalls encountered in separation of fmol amounts of peptide material. Subsequently the most important techniques of detection are discussed. This section primarily focuses on the coupling of HPLC with radioimmunoassay (RIA), which is indispensable for detection of components in the fmol range at present. Finally, some aspects of preparation and chromatographic separation of radiolabelled antigens for use in RIA are discussed. PMID- 7540917 TI - Effect of anti-GMP antibodies on the growth of transformed cells. PMID- 7540919 TI - Monitoring for engraftment following rat orthotopic liver transplantation by in vitro amplification of Y-chromosome gene using polymerase chain reaction. AB - The polymerase chain reaction (PCR) using primers specific for the rat Y chromosome gene made it possible to distinguish a very small number of male rat cells from a large excess of female rat cells. In nonimmunosuppressed LEW recipients of ACI liver allografts, the donor cells in the bloodstream disappeared rapidly by day 3, earlier than the biochemical changes indicative of liver dysfunction. In immunosuppressed LEW recipients of ACI liver allografts, the donor cells were detected for a longer time. Moreover, in LEW recipients surviving for long period, the PCR revealed mixed-microchimerism. Our results indicated that this Y chromosomal gene-specific PCR method is useful for assessing engraftment following rat liver transplantation. PMID- 7540920 TI - Cell transfer therapy of cancer metastasis with autologous lymphocytes. PMID- 7540918 TI - Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: a model for endochondral ossification. AB - Chondrocytes isolated from the proliferative and differentiating zones of 3-wk old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification. PMID- 7540921 TI - Structure-activity relationships in a series of 3-sulfonylamino-2-(1H) quinolones, as new AMPA/kainate and glycine antagonists. AB - This paper describes the design and synthesis of a new class of molecules, the 3 sulfonylamino-2-(1H)-quinolones, which are potent and selective antagonists at both the AMPA/kainate site as well as at the NMDA-associated glycine site. The molecules were characterized by their binding affinities to rat cortical membranes and by electrophysiology on Xenopus oocytes injected with mRNA isolated from rat cerebral cortex. The most potent compound 61 has an IC50 of 0.09 microM for binding at the AMPA/kainate site, and 0.16 microM in oocyte electrophysiology. PMID- 7540922 TI - Facial responsiveness to odours in normal and pervasively developmentally disordered children. AB - The facial responsiveness of 10 mutic children with pervasive developmental disorder (PDD) and 10 normal (N) children matched for sex and chronological age were covertly videotaped while presented with a set of odours contrasted in hedonic valence. Hedonic ratings of the stimuli were obtained both from the group of N subjects and a panel of adults. Two methods were used to measure facial responses in the same subjects. The first method consisted in an analysis of facial movements with the Facial Action Coding System. Results show that PDD and N subjects displayed distinct action units in response to unpleasant odours. PDD subjects typically displayed muscular actions indexing negative experience, while N subjects showed more smiles. With the second method, odour-elicited facial behaviour was rated by a panel of observers, who were asked to judge whether the subjects were exposed a pleasant, neutral or unpleasant smell. The facial responses to unpleasant odours were classified more accurately in PDD than in N subjects. These findings suggest a functional ability to sense the hedonics attached to odours, but a deficit of socialization of hedonic facial displays in developmentally disordered subjects. PMID- 7540923 TI - Cystic fibrosis. PMID- 7540924 TI - Tumour markers. AB - Tumour markers are now used in the diagnosis, anatomical localization, staging, monitoring and definition of prognosis of a wide range of malignancies. The last decade has seen an expansion of our definition of these markers to include oncogenes and oncogene products, and provided us with new possibilities of developing more specific tests to help in the management of cancer. PMID- 7540925 TI - Lactate accumulation following concussive brain injury: the role of ionic fluxes induced by excitatory amino acids. AB - During the first few minutes following traumatic brain injury, cells are exposed to an indiscriminate release of glutamate from nerve terminals resulting in a massive ionic flux (e.g., K+ efflux) via stimulation of excitatory amino acid (EAA)-coupled ion channels. The present study was undertaken to elucidate the causal relationship between these ionic shifts and lactate accumulation in the injured brain, by examining the effects of ouabain (an inhibitor of Na+/K+ ATPase), Ba2+ (an inhibitor or non-energy-dependent glial K+ uptake) and kynurenic acid (KYN; a broad-spectrum EAA antagonist) on lactate accumulation. Two microdialysis probes were placed bilaterally in the rat parietal cortex. One was perfused with a test drug (1.0 mM ouabain, 2.0 mM Ba2+ or 10 mM KYN) and the other with Ringer's solution (control) for 30 min prior to injury. Following a 2.2-2.7 atm fluid-percussion injury, lactate levels in the dialysate increased (up to 116.6% above baseline) for the first 16 min and returned to baseline levels within 20 min after injury. This lactate accumulation was attenuated by preinjury administration of ouabain and KYN and was prolonged by Ba2+ administration. These findings indicate that lactate accumulations following concussive brain injury is a result of increased glycolysis which supports ion pumping mechanisms, thereby, restoring the ionic balance which was disrupted by stimulation of EAA-coupled ion channels. PMID- 7540926 TI - Peptidergic neurons of subcortical white matter in aging and Alzheimer's brain. AB - Most of the neurons in the subcortical white matter of the adult cerebrum are remnants of the transient subplate cortex which appears during early cortical development. The peptidergic neurons in the subcortical white matter, beneath the striate cortex were examined qualitatively and qualitatively with immunohistochemistry for substance P, cholecystokinin, somatostatin and neuropeptide Y in seven control patients and eight patients with Alzheimer's disease. The different peptidergic subcortical neurons still persisted in normal aging. In Alzheimer's disease, however, the substance P- and somatostatin immunoreactive neurons were decreased in numbers and showed degenerative changes. PMID- 7540928 TI - Changes in preprotachykinin mRNA expression and substance P levels in dorsal root ganglia of monoarthritic rats: comparison with changes in synovial substance P levels. AB - We have measured changes in the expression of gamma-preprotachykinin mRNA and levels of the neuropeptide substance P in the lumbar 4 and 5 dorsal root ganglia at various time points following the induction of an antigenic monoarthritis in the rat knee. The results were compared with changes in substances P levels in the knee joint synovium during acute and chronic phases of the disease. On day 3 post-induction, there was a significant increase in the expression of gamma preprotachykinin mRNA in the dorsal root ganglia. Concomitant with this increase in message was a rise in the levels of substance P in the dorsal root ganglia. On days 7, 10 and 21, mRNA expression had returned to control values whereas ganglion peptide levels were significantly below controls. In contrast there was little change in the total substance P levels in the synovium on days 1 and 3 despite the observed changes in the ganglia. By day 10, however, synovial levels had risen significantly above control values and remained elevated thereafter. Our results show a transitory increase in substance P synthesis after induction of an antigenic monoarthritis. This response is not mirrored in the periphery where there is no initial change in total substance P levels perhaps reflecting increased degradation be enzymes known to be present within inflamed tissue. Paradoxically synovial substance P levels are increased in the latter phases of the model which may serve to modify the inflammatory response. PMID- 7540927 TI - Neurochemical correlates of sexual exhaustion and recovery as assessed by in vivo microdialysis. AB - The extracellular levels of the dopamine (DA) metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the serotonin metabolite 5 hydroxyindoleacetic acid (5-HIAA) in the medial preoptic area (MPOA) of male rats were monitored during unrestricted copulation, the ensuing state of sexual refractoriness and the resumption of mating activity. MPOA dialysates were collected from the same animal during four consecutive days. In the first day the subjects were allowed to copulate until reaching a satiation criterion. That was associated with a marked increase in the dialysate levels of the three metabolites assessed. During the next two days the animals remained sexually inactive when exposed to receptive females. Their basal levels of DOPAC and HVA were elevated, whereas those of 5-HIAA remained as low as in the first session. During the non-mating exposure to receptive females there were only minor changes in the three metabolites. By the fourth day, just before the animals resumed copulation, the basal levels of the DA metabolites, especially HVA, had decreased to values closer to those found in the first day. When they mated again to exhaustion the levels of DOPAC, HVA, and 5-HIAA increased as in the first session. The neurochemical changes found during the intervening state of sexual inactivity (i.e. increased levels of DA metabolites) are reminiscent of the effects of DA receptor blockers, which suggests a possible neurochemical mechanism for sexual refractoriness. PMID- 7540929 TI - Galanin neurons exhibit estrogen receptor immunoreactivity in the female rat mediobasal hypothalamus. AB - Galanin has been shown to augment the hypothalamic luteinizing hormone releasing hormone and pituitary luteinizing hormone release and to play an important role in the feedback effects of ovarian steroids on pituitary hormone secretion. To further characterize estrogen effects on galanin, we tested for the existence of estrogen receptors in arcuate nucleus galanin-producing cells. Hypothalamic vibratome sections from colchicine-pretreated female rats were double immunolabeled for estrogen receptor and galanin. Neurons which exhibited immunoreactivity for either estrogen receptor or galanin were distributed throughout the hypothalamus; and a population of neurons, located predominantly in the mediobasal hypothalamus, displayed immunoreactivity for both galanin and estrogen receptor. These results raise the possibility that estrogen may act directly on galanin-producing arcuate nucleus neurons to regulate pituitary hormone secretion. PMID- 7540930 TI - Effects of combined or separate 5,7-dihydroxytryptamine lesions of the dorsal and median raphe nuclei on responding maintained by a DRL 20s schedule of food reinforcement. AB - Previous studies have shown that long-term 5-hydroxytryptamine (5-HT) depletion induced by combined dorsal and median raphe injections of 5,7-dihydroxytryptamine (5,7-DHT) leads to impairments in the acquisition and performance of behaviour maintained under a differential-reinforcement-of-low-rate (DRL) schedule of reinforcement. The present studies examined the relative importance of dorsal versus median raphe 5-HT projections in mediating these effects by investigating the impact of separate versus combined infusions of 5,7-DHT into these sites on DRL responding. Adult male rats received injections of 3 micrograms 5,7-DHT into both dorsal raphe and median raphe, dorsal raphe only, or median raphe only. Sham operated controls received vehicle injections. Animals were then trained to respond on a DRL 20s schedule for 36 days. Combined dorsal raphe and median raphe 5,7-DHT infusions depleted striatal and hippocampal 5-HT by > 90%, increased responding, decreased the number of reinforcers earned, lowered the mean inter response time (IRT), and shifted the frequency distribution of IRTs to the left. Median raphe 5,7-DHT infusions induced similar behavioural effects and reduced hippocampal 5-HT by 64%. Dorsal raphe 5,7-DHT lesions, that depleted striatal 5 HT by 56%, and hippocampal 5-HT by 30%, had no effect on behaviour. In a final experiment it was shown that providing an external cue light to signal food availability, and that removed the need to rely on internal timing processes, resulted in rapid acquisition of responding, and prevented the behavioural deficits in 5,7-DHT-lesioned rats. Following removal of this cue, responding deteriorated, and the 5,7-DHT treated animals performed worse than controls. These results suggest that destruction of 5-HT neurons arising from the median raphe is sufficient to disrupt behaviour maintained by a DRL 20s schedule, and that the behavioural changes observed may involve a deficit in timing. PMID- 7540931 TI - Excitotoxic action of NMDA agonists on nigrostriatal dopaminergic neurons: modulation by inhibition of nitric oxide synthesis. AB - Focal infusions of N-methyl-D-aspartate (NMDA) or an endogenous NMDA agonist, quinolinic acid (QUIN), into the substantia nigra pars compacta (SNc) of adult Sprague-Dawley rats resulted in a dose-dependent depletion of ipsilateral striatal tyrosine hydroxylase (TH) activity, a biochemical marker for dopaminergic neurons. To assess the intermediary role of nitric oxide in the neurotoxicity elicited by these toxins, their action was tested in animals treated with N omega-nitro-L-arginine methyl ester (L-NAME). Systemic injections (2 injections; 8 h apart) of L-NAME (100, 150 and 250 mg/kg) produced a dose related inhibition of cerebellar nitric oxide synthase (NOS) activity. The time course of cerebellar NOS inhibition following L-NAME (250 mg/kg) was rapid in onset and lasted for at least 24 h following the second injection. An L-NAME treatment regimen of 250 mg/kg, with the second injection given 24 h prior to assessment of NOS activity, produced an 87 and 91% inhibition of cerebellar and nigral NOS activity, respectively. Intranigral infusion of 40 and 60 nmol QUIN reduced ipsilateral striatal TH activity by 62 and 75%, respectively. However, 40 and 60 nmol QUIN infusions into animals pretreated with L-NAME (250 mg/kg) reduced striatal TH activity by 83 and 96%, respectively. Intranigral infusion of 15 and 30 nmol NMDA produced a 48 and 77% decrease in striatal TH activity, respectively, whereas the same doses of NMDA given to animals pretreated with L NAME (250 mg/kg) resulted in a 59 and 88% decrease in TH activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540932 TI - Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling. AB - The present study investigated the effect of daily handling and gentling between postnatal days 1 and 28 on experimental allergic encephalomyelitis (EAE) in 8 week old DA rats. Handling consisted of removing pups from the mother, and placing them in the novel cage for 15 min. The gentling procedure included handling accompanied by 3 min of dorsal tactile stimulation before returning the pups to the nest cage. Adult rats of both sexes handled in infancy showed increased susceptibility to EAE, as revealed by higher incidence of the disease, and more severe clinical signs. Anti-myelin basic protein (MBP) autoantibodies were increased in handled males, and decreased in handled females, compared to controls. Gentling induced aggravation of clinical signs and histopathological lesions of EAE in males, while in gentled females suppression was observed. These results indicated that both neonatal handling and gentling aggravated EAE induced in adult male rats. In female rats handling exacerbated, and gentling suppressed clinical EAE. The overall effect of neonatal manipulations was more pronounced in males. Furthermore, in mothers separated from their offspring due to handling and gentling, and immunized for EAE at day 28 postpartum, earlier appearance of clinical signs, and increased frequency of relapses compared to control dams was recorded. PMID- 7540933 TI - Prescribing risperidone. PMID- 7540934 TI - High resolution structures of HIV-1 RT from four RT-inhibitor complexes. AB - We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 A for all data to 2.2 A. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/configurational rearrangement of the compounds. PMID- 7540935 TI - Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. AB - The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit. PMID- 7540936 TI - RNA-d2: a computer program for editing and display of RNA secondary structures. AB - RNA-d2 is a user-friendly program developed for interactively generating aesthetic and non-overlapping drawings of RNA secondary structures. It designed so that the drawings can be edited in a very natural and intuitive way, in order to emphasize structural homologies between several molecules, as well as the foldings themselves to update the base-pair sets according to new data. The program automatically produces a polygonal display in which the unpaired nucleotides are regularly positioned on circles and the stems harmoniously distributed on their periphery. RNA secondary structures can be encoded via the keyboard, but the program also automatically draws output files from thermodynamic prediction programs. The user interacts directly on different screen displays according to the editing functions. Rotation/translation of any subdomain and deletion of stems are performed on a coloured backbone view to make easier the identification of structural features, whereas addition of new base pairings and numbering manipulation are realized on a complete polygonal view. Each modification is displayed in real time on the screen. When the display is obscured by numerous overlaps despite the colour code of the backbone view, an automatic function progressively straightens the subdomains which are highly compacted by very dissymmetric internal loops. RNA-d2 allows easy untangling and editing of RNA molecules > 1000 nucleotides long. PMID- 7540937 TI - PROANAL version 2: multifunctional program for analysis of multiple protein sequence alignments and for studying the structure--activity relationships in protein families. AB - A new version of the program PROANAL is described. A multiple linear regression analysis of the protein structure--activity relationship allows one to investigate the combinations of protein sites and factors influencing the activity. The program also provides the possibility to seek out protein sites, conservative or variable in variations of physicochemical characteristics, and regions with high or low values of these characteristics. PROANAL2 may be useful in the simulation of protein-engineering experiments and in the search of a number of protein regions such as functional sites, secondary structures, solvent exposed regions, T- and B-cell antigenic determinants, etc. PMID- 7540938 TI - Prostate cancer screening. Why the controversy? AB - The development of new technologies has stimulated a crusade of screening for earlier detection of prostate cancer. This effort has resulted in controversy regarding its clinical use in this format. Until randomized clinical screening trials define the value of prostate cancer screening, clinicians who use screening approaches must gain a strong understanding of the natural history of the disease, the strengths and weaknesses of available screening methodologies, and epidemiologic considerations of prostate cancer risk. PMID- 7540939 TI - The impact of PSA on prostate cancer management. Can we abandon routine staging pelvic lymphadenectomy? AB - Despite a negative metastatic evaluation, up to 100% of patients with high-grade tumors are found to harbor metastatic disease within the pelvic lymph nodes at the time of surgery. This article reviews the literature regarding efforts to refine noninvasive preoperative staging through the use of serum tumor markers, improved radiologic imaging, and histologic analysis of the biopsy specimens. A discussion of the renewed controversy regarding the role of pelvic lymphadenectomy in the management of adenocarcinoma of the prostate follows. PMID- 7540940 TI - New perspectives of CD28-B7-mediated T cell costimulation. PMID- 7540941 TI - Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. AB - Understanding the immunological mechanisms of protection and pathogenesis in tuberculosis remains problematic. We have examined the extent to which tumor necrosis factor-alpha (TNF alpha) contributes to this disease using murine models in which the action of TNF alpha is inhibited. TNF alpha was neutralized in vivo by monoclonal antibody; in addition, a mouse strain with a disruption in the gene for the 55 kDa TNF receptor was used. The data from both models established that TNF alpha and the 55 kDa TNF receptor are essential for protection against tuberculosis in mice, and for reactive nitrogen production by macrophages early in infection. Granulomas were formed in equal numbers in control and experimental mice, but necrosis was observed only in mice deficient in TNF alpha or TNF receptor. TNF alpha and the 55 kDa TNF receptor are necessary conditions for protection against murine M. tuberculosis infection, but are not solely responsible for the tissue damage observed. PMID- 7540942 TI - Cross-linking of CD30 induces HIV expression in chronically infected T cells. AB - CD30, a member of the tumor necrosis factor (TNF) receptor family, is expressed constitutively on the surface of the human T cell line ACH-2, which is chronically infected with human immunodeficiency virus type-1 (HIV)-1. We demonstrate that cross-linking CD30 with an anti-CD30-specific monoclonal antibody, which mimics the described biological activities of the CD30 ligand (CD30L), results in HIV expression. CD30 cross-linking does not alter proliferation of ACH-2 cells and the induction of HIV expression is not mediated by endogenous TNF alpha/beta. Furthermore, cross-linking of CD30 leads to NF kappa B activation and enhanced HIV transcription. Thus, CD30-CD30L interactions mediate the induction of HIV expression by a kappa B-dependent pathway that is independent of TNF. This mechanism may be important in the activation of HIV expression from latently infected CD4+ T cells, especially in lymphoid organs where cell to cell contact is conducive to receptor-ligand interactions. PMID- 7540944 TI - Specific T cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands. AB - The T cell receptor (TCR) can interact with a spectrum of peptides as part of its ligand, including the immunogenic peptide, variants of this peptide,and apparently unrelated peptides. The basis of this broad specificity for ligand was investigated by substitution analysis of a peptide antigen and functional testing using a B cell apoptosis assay. A peptide containing as few as 1 aa in common with this peptide could stimulate a specific T cell response. Two endogenous ligands, an agonist and a partial agonist, were readily identified from a search of the SwissProt database, indicating that multiple endogenous ligands likely exist for a given T cell. These findings strongly support the concept that one TCR has the ability to interact productively with multiple different ligands, and provide evidence that such ligands exist in the endogenous peptide repertoire. PMID- 7540945 TI - Toxic effects of gallium arsenide on sperm in rats by repeated intratracheal instillations. PMID- 7540943 TI - In the absence of a CD40 signal, B cells are tolerogenic. AB - When B cells are deprived of signaling through CD40, they exhibit the ability to induce T cell tolerance. The in vivo administration of anti-gp39 and allogeneic B cells diminished the ability of mice to mount an allogeneic response. Tolerance induction was specific for the haplotype expressed on the allogeneic B cells. Selective allospecific unresponsiveness was induced in the CD8 and CD4 compartments by the administration of anti-gp39 and class II-deficient B cells or class I-deficient B cells, respectively. As predicted by studies with anti-gp39 treatment, diminished allospecific responsiveness was induced by the administration of B cells to mice genetically deficient in gp39. Taken together, these data are consistent with the premise that deprivation of CD40 signaling engenders B cells with enhanced tolerogenicity. These studies provide insights into the tolerogenic capacity of resting B cells and outlines a practical approach to exploit this function. PMID- 7540946 TI - [Developmental mechanisms of neural network in the cerebellar system]. AB - Various cell adhesion molecules and neurotrophic factors are involved in cell migration, axonal elongation and synaptogenesis during the development of neural networks. The developmental mechanisms of neural networks were investigated in the mouse cerebellar system in relation to the neuron-glia interactions and the expression of cell adhesion molecules. Radial migration of Purkinje cells in the cerebellar primordium is guided by contact with radial glial processes, and expression of the neuron-glia cell adhesion molecule tenascin is involved in this process. Purkinje cell migration is obstructed in the reeler mutant mouse, and the disorganized arrangement of radial glial processes is considered to be responsible for this defect. Pontine nuclei and inferior olivary nuclei constitute the cerebellar afferent system, and these neurons are generated from the rhombic lip which is originated from the alar plate of the metencephalon in common with the cerebellar primordium. Pontine nuclei migrate tangentially from the rhombic lip under the pia mater, and the binding sites for peanut lectin are specifically expressed on the migratory route. Thy-1 molecule, a member of the immunoglobulin superfamily is expressed transiently in the synaptogenesis between the cerebellar afferent fibers and their target neurons in the developing cerebellar cortex. The homophilic binding activity of this molecule is considered to be involved in the recognition between presynaptic terminals and postsynaptic structures. PMID- 7540947 TI - Comparative studies on the uridine-5'-diphosphate-galactose: glycoprotein galactosyltransferase activity in rat liver and Zajdela ascitic hepatoma. AB - We report here data on the galactosyltransferase activity in serum, liver and Zajdela ascitic hempatoma cells and about the rate of galactose attachment to appropriate acceptors by beta(1-4, beta(1-3) and alpha(1-3) linkages in liver and hepatoma cell homogenates. It was established that in serum of Zajdela bearing rats, in host liver and in Zajdela ascitic cells, galactosyltransferase activity towards ovomucoid was elevated in comparison with control serum and liver. The activity towards low molecular acceptor (N-acetylglucosamine) in liver and hepatoma was nearly the same. The predominant isomer synthesized in both tissues was beta 1-4-galactose-N-acetylglucosamine. When asialofetuin was used as acceptor the activity of alpha(1-3) galactosyltransferase was measured. In Zajdela ascitic hepatoma cells alpha(1-3) galactosyltransferase activity was 3 times higher than that in liver. The elevated ability of the tumor cells to attach galactose residues with alpha(1-3) linkage to Gal beta 1-4GlcNAc-R sequence should be considered as a characteristic feature of these malignant cells. PMID- 7540948 TI - Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity. AB - The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H 2Dk molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7-1. Expression of B7-1 rendered BW Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK-mediated elimination of metastasizing, NK-resistant tumor cells. PMID- 7540949 TI - Mouse skin tumor progression results in differential expression of retinoic acid and retinoid X receptors. AB - Retinoids are powerful regulators of epidermal cell growth and differentiation and are widely used in the prevention and treatment of skin disorders and cancers in humans. Since many of the effects of retinoids on cell growth and differentiation are mediated by nuclear retinoid receptors (RARs and RXRs), we were interested in determining RAR and RXR gene expression during mouse skin tumor progression. The two-stage system of mouse skin carcinogenesis was used to generate papillomas and carcinomas, and the different stages of malignant progression (papillomas, differentiated squamous cell carcinomas, undifferentiated squamous cell carcinomas, and spindle cell carcinomas) were characterized in each tumor by specific keratin expression prior to receptor characterization. Using in situ hybridization analysis, we show that the two major RAR isoforms (alpha 1 and gamma 1), which account for most of RARs in the skin, were expressed in both the basal and suprabasal layers in mouse epidermis. In contrast, RXR alpha transcripts were compartmentalized to the basal cell layers and concentrated in hair follicles. During skin tumor progression, RAR (alpha 1 and gamma 1) transcripts were down-modulated in malignant tumor cells, whereas RXR (alpha and beta) transcript expression was expanded in papillomas and carcinomas as the number of undifferentiated cells also increased. RXR gamma was not detected in the skin or at any stage during skin tumor progression. Spindle cell tumors lacked markers of the keratinocyte phenotype and lost RAR expression, yet retained expression of RXR alpha and beta. The increased abundance of transcripts for RXRs and decreased presence of RARs in skin tumor progression may favor other nuclear signal transduction pathways requiring RXR for heterodimer formation and contribute to phenotypic progression of cancer cells. PMID- 7540950 TI - Metabolism and actions of 2-chloro-9-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl) adenine in human lymphoblastoid cells. AB - 2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (Cl-F-ara-A) is a new deoxyadenosine analogue that is resistant to phosphorolytic cleavage and deamination. Studies with a variety of cell lines demonstrated that Cl-F-ara-A is a potent cytotoxic agent; in cell-free systems, its triphosphate (Cl-F-ara-ATP) inhibited DNA polymerase alpha and ribonucleotide reductase. To further characterize its mechanism of cytotoxicity, the present study investigated the cellular metabolism of Cl-F-ara-A and the actions of its nucleotide metabolites in human T-lymphoblast leukemia CCRF-CEM cells. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, with the monophosphate as its major metabolite. After washing cells into drug-free medium, the elimination of each Cl F-ara-A nucleotide was nonlinear with a prolonged terminal phase. Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA; a much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. At low Cl-F-ara-ATP:dATP values, incorporation was mainly in phosphodiester linkages at internal sites, whereas at higher Cl-F-ara-ATP:dATP values, Cl-F-ara-AMP was principally detected at terminal sites. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA. These results suggest that the incorporation of Cl-F-ara A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A. PMID- 7540952 TI - The alpha-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis, and inhibits tumor growth. AB - The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Experiments in vivo with castanospermine, an inhibitor of the glucosidases that convert protein N-linked high mannose carbohydrates to complex oligosaccharides, resulted in significant inhibition of tumor growth in nude mice. Angiogenesis to basic fibroblast growth factor in castanospermine treated C57/BL mice was similarly reduced. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. In vitro differentiation models using Matrigel and postconfluent cultures of endothelial cells were used to study the effects of glycosidase inhibitors on endothelial cell behavior. FACS analysis of cell surface oligosaccharides using either Concanavalin A or L-phytohemagglutinin lectins confirmed an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-N-acetylglucosamine on mannose residues of Asn-linked oligosaccharides upon drug treatment. Castanospermine and the glucosidase inhibitor N-methyldeoxynojirimycin prevented the morphological differentiation of endothelial cells in vitro. These compounds did not alter the proliferation of cultured endothelial cells or their ability to attach to various extracellular matrix molecules. However, the cells showed a reduced ability to migrate and to invade basement membrane gels in vitro and an increased tendency to form aggregates that was inhibitable by D-mannose. These studies suggest that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth. PMID- 7540951 TI - Expression of a deletion mutant of the E2F1 transcription factor in fibroblasts lengthens S phase and increases sensitivity to S phase-specific toxins. AB - To better understand how the E2F1 transcription factor contributes to the process of cell proliferation, NIH-3T3 cell lines were generated that constitutively express either the wild-type E2F1 protein or an amino terminal deletion mutant, termed E2F1d87. Proliferating E2F1d87-expressing cells exhibit a significant lengthening of S phase relative to control and E2F1 cell lines and are hypersensitive to the cytotoxic effects of the S phase-specific antitumor drug camptothecin. This sensitivity is associated with an increase in drug-induced p53 and WAF1 levels. The E2F1 and E2F1d87 cell lines are both able to initiate, but not complete, S phase under conditions of serum starvation. However, quantitation of DNA synthesis, during culture in serum-deprived media, indicates that the E2F1d87 cell line synthesizes more DNA/cell as compared to the E2F1 cell line. Consistent with this relative increase in DNA synthesis, the E2F1d87 cell line undergoes camptothecin-induced apoptosis when cultured under conditions of serum starvation, while the control and E2F1 cell lines are unaffected by drug treatment under the same conditions. Thus, the sensitivity of the E2F1d87 cell line to camptothecin is not dependent on cell proliferation. The data presented here suggest that cell cycle parameters can be manipulated in order to enhance sensitivity of a cell to the toxic effects of specific chemotherapeutic agents. PMID- 7540954 TI - The onset and autonomic regulation of cardiac pacemaker activity: relevance of the f current. PMID- 7540953 TI - Fas/APO-1 gene transfer for human malignant glioma. AB - Human malignant glioma cells are susceptible to apoptosis induced by antibodies to Fas/APO-1, a cytokine receptor protein of the nerve growth factor/tumor necrosis factor receptor superfamily. Here we show that a critical level of cell surface expression of Fas/APO-1 is a prerequisite for induction of glioma cell apoptosis via Fas/APO-1. Although Fas/APO-1 mRNA was expressed in three Fas/APO-1 antibody-resistant glioma cell lines, these cells expressed either little Fas/APO 1 protein (LN-319 and LN-405) or an abnormal Fas/APO-1 protein that was not translocated to the cell membrane and therefore functionally inactive (LN-308). Although all glioma cell lines expressed mRNA for Fas/APO-1-delta TM, a soluble form of Fas/APO-1 lacking the transmembrane domain, none of the cell lines released detectable amounts of soluble Fas/APO-1, a potential endogenous antagonist of Fas/APO-1-mediated glioma cell apoptosis. Stable transfection of three resistant glioma cell lines with a human Fas/APO-1 cDNA expression vector dramatically enhanced cell surface expression of Fas/APO-1 and induced susceptibility to Fas/APO-1 antibody-mediated apoptosis. These data indicate that malignant glioma cells, unlike other tumor cells, uniformly harbor the intracellular cascade required for Fas/APO-1-mediated apoptosis. Low level of Fas/APO-1 expression results from inefficient transcription and translation of the Fas/APO-1 gene or the synthesis of mutant Fas/APO-1 proteins. gamma Interferon, tumor necrosis factor-alpha, and interleukin 1 beta augmented Fas/APO 1-mediated apoptosis of Fas/APO-1-transfected glioma cells by acting on the subcellular suicidal cascade triggered by Fas/APO-1 activation. Dexamethasone attenuated Fas/APO-1 antibody-induced apoptosis, not only of constitutively Fas/APO-1-positive glioma cells, but also of Fas/APO-1-transfected glioma cells. The antiapoptotic effect of dexamethasone could be overcome by preexposure of the glioma cells to gamma-interferon or by coexposure to Fas/APO-1 antibodies and cycloheximide. Thus, Fas/APO-1 gene transfer and combined immunotherapy using Fas/APO-1 antibodies and cytokines may overcome Fas/APO-1 antibody resistance of Fas/APO-1-negative human malignant glioma cells, which may represent subpopulations within single gliomas or form a separate subgroup of human malignant gliomas. PMID- 7540955 TI - Innervation of human epicardial coronary veins: immunohistochemistry and vasomotility. AB - OBJECTIVE: The aim was to investigate the innervation and vasomotor responses to classical and putative transmitters of the coronary venous bed. METHODS: The innervation of human epicardial coronary veins was investigated using acetylcholinesterase histochemistry and immunofluorescence staining, together with antisera against the general neuronal marker protein gene product 9.5 (PGP 9.5), the catecholamine synthesising enzyme tyrosine hydroxylase, and neuropeptides [neuropeptide Y, vasoactive intestinal peptide (VIP), substance P, and calcitonin gene related peptide (CGRP)]. The vasomotor responses to noradrenaline, acetylcholine, neuropeptide Y, substance P, human alpha calcitonin gene related peptide (alpha CGRP), and VIP were tested on isolated circular human epicardial coronary vein segments. RESULTS: A network of nerve fibres was shown in the major epicardial coronary veins by means of an antiserum to PGP 9.5. The majority of the perivascular nerve fibres possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Only a few nerve fibres displayed substance P, CGRP, and VIP immunoreactivity and acetylcholinesterase activity. Noradrenaline and acetylcholine induced powerful contractions of all the tested segments, whereas no contraction was induced by neuropeptide Y, alpha CGRP, substance P, or VIP. All segments precontracted with U46619 responded with potent relaxation to alpha CGRP, substance P, and VIP, whereas noradrenaline and acetylcholine only in low concentrations induced weak relaxation of a few of the segments. No relaxation was induced by neuropeptide Y. CONCLUSIONS: This is the first study to demonstrate comprehensively the perivascular innervation of human coronary veins and corresponding vasomotor effects, suggesting a role in regulation of the coronary venous circulation. PMID- 7540957 TI - Enhanced adrenergic neurotransmission in diabetic rabbit carotid artery. AB - OBJECTIVE: The aim was to examine the effects of diabetes mellitus on adrenergic neurotransmission and smooth muscle responsiveness in the densely innervated carotid artery from six-week alloxan diabetic rabbits. METHODS: Rings of carotid arteries were isolated from normal and diabetic rabbits and isometric tension was measured in response to stimulation of adrenergic nerves, alpha adrenoceptors, and activation by calcium. RESULTS: Basal content and stimulated overflow of endogenous noradrenaline were reduced by approximately 25% in arteries from diabetic as compared to normal rabbits. In contrast, responses to endogenous noradrenaline released from adrenergic nerves by electrical stimulation or tyramine displacement were not different between arteries from normal and diabetic groups. Neuronal uptake blockade using cocaine caused a significantly smaller leftward shift in the contractions produced by electrical stimulation and exogenously applied noradrenaline in arteries from diabetic rabbits. The tonic, but not phasic, contractions caused by phenylephrine were larger in arteries from diabetic rabbits. Calcium-induced contractions caused by readdition of calcium to a calcium-free medium containing potassium (15 mmol.litre-1) were also significantly larger in arteries from diabetic rabbits. BAY K 8644, a calcium channel activator, caused an increase in calcium induced contractions and abolished the difference between the two groups. CONCLUSIONS: Although neurogenic contractions of diabetic carotid artery are normal, there is inefficient or reduced neuronal uptake as well as increased activity of calcium channels in the smooth muscle which increase contractions to alpha adrenoceptor agonists. PMID- 7540959 TI - [Emphasizing demonstration teaching in surgical nursing courses]. PMID- 7540958 TI - Human fibroblasts (KMST-6/RAS cell line) transformed with 60Co gamma-rays and c Ha-ras oncogene produce a large amount of granulocyte colony-stimulating factor (G-CSF); production is enhanced by cAMP, theophylline, and butyrate. AB - Human fibroblasts (KMST-6/RAS cell line), which was malignantly transformed in vitro with 60Co gamma-rays and the c-Ha-ras oncogene, produced a large amount of granulocyte colony-stimulating factor (G-CSF). The production was greater during the logarithmic growth phase than during the stationary phase. cAMP and theophylline, alone or in combination, and butyrate significantly enhanced G-CSF production, but dexamethasone or 5-azacytidine did not. Enhanced production of G CSF by these agents was regulated at the posttranscriptional level. Neither the expression of the ras oncogene nor the tumorigenicity of the cells correlated with the production of G-CSF. PMID- 7540956 TI - Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction. AB - OBJECTIVE: Inducible nitric oxide synthase (iNOS) activity, as measured by conversion of L-14C-arginine to L-14C-citrulline is significantly increased in infarcted rabbit myocardium as compared to healthy myocardium 48 h after coronary occlusion. The aim of this study was to localise the nitric oxide synthase (NOS) isoforms in normal myocardium and compare these findings with NOS activity in cells of the infarcted region. METHODS: Activities of NOS isoforms were enzymatically assayed in normal and infarcted myocardium. Localisation of NOS was performed on identical sections using specific monoclonal IgG antibodies against endothelial constitutive (cNOS) and macrophage inducible (iNOS) nitric oxide synthase. In addition, macrophages were identified using fluorescein conjugated ChromPure rabbit IgG, Fc fragment. RESULTS: iNOS activity increased significantly in infarcted as compared to normal myocardium [0.42(SEM 0.03) v 0.85(0.08) fmol.microgram-1.min-1, P = 0.02, respectively]. However, cNOS did not increase significantly in infarcted regions [0.18(0.04) v 0.24(0.06) fmol.microgram-1.min 1; P = 0.16, respectively]. cNOS was immunohistochemically localised in endothelial and endocardial cells in normal and infarcted tissues. The presence of iNOS activity in macrophages in infarcted myocardium was identified immunohistochemically. Cardiomyocytes and neutrophils did not label with the antibodies to cNOS and iNOS. CONCLUSIONS: (1) Infiltrating macrophages are the main site of increased iNOS activity in infarcted rabbit myocardium. (2) cNOS activity is not significantly increased in infarcted tissues as compared to normal myocardium. (3) Neutrophils and cardiomyocytes do not express NOS immunoreactivity in infarcted and normal rabbit myocardium. PMID- 7540961 TI - Reassembly of the alpha 6 beta 4 integrin and laminin in rabbit corneal basement membrane after excimer laser surgery: a 12-month follow-up. AB - We investigated the reassembly of hemidesmosomes and epithelial basement membrane (BM) following experimental excimer laser photorefractive keratectomy (PRK) immunohistochemically using monoclonal antibodies against integrin subunits alpha 6 and beta 4 (hemidesmosome components) or laminin (a BM component). The rabbits were killed 3 days, 1, 3, 6, or 12 months after a -5.0 D PRK. For untreated corneas, integrin subunits alpha 6 and beta 4 and laminin showed normal distribution at the basal aspect of the basal epithelial cells, appearing as a distinct fluorescent line. The alpha 6 subunit was also found at the basolateral membranes of the basal epithelial cells. The average healing time for the PRK wound was 6 days, but secondary epithelial defects occurred in 17 of 22 corneas. The subepithelial labeling for all the antibodies investigated showed focal discontinuities at the level of the BM in the wound area up to 12 months after PRK. After 6 months, secondary epithelial defects were very rare. In addition to the patchy subepithelial labeling, lamellar anterior stromal immunoreaction for alpha 6 and beta 4 integrin subunits was also present 1-12 months after PRK. Laminin was also observed in the anterior stroma 1-6 months after PRK. Our results suggest that focal discontinuities of hemidesmosomes or BM observed immunohistochemically in the unexpectedly slow-healing PRK wound areas might correlate with the epithelial healing problems observed in most rabbits. PMID- 7540960 TI - Histopathological and immunohistochemical analysis of adenomatous hyperplasia and hepatocellular carcinoma: cellularity, thickness of cell cord, and Ki-67 proliferative activity. AB - To characterize adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC), and to establish their histopathological differences, morphometrical and immunohistochemical analyses, namely, cellularity, thickness of cell cord, and Ki 67 labeling index (Ki-67 LI) were done on surgically obtained hepatic lesions from patients with positive serum antibody against HCV. The hepatic lesions analyzed include chronic active hepatitis (CAH) (11 specimens), regenerative nodules of liver cirrhosis (LC) (29), AH (11), small HCC Edmondson's Grade I (GI) (19), GII (26), GIII (14). The results showed that AH has relatively high cellularity, and significantly greater thickness of cell cord than LC; whereas, HCC GI has significantly higher cellularity and Ki-67 LI than AH. From the data of these markers, and from the absence of conspicuous structural atypism, AH is considered to be in a different category from HCC GI. The premalignant potential of AH is supported only by its high incidence of coexistence adjacent to HCC GI or GII(6/11). Most lesions of HCC seem to develop from the liver tissue having a background of CAH or LC without passing through AH. Focal fatty changes are frequently observed within lesions of both AH and HCC GI (5/11, 8/19). When non fatty regions of AH and HCC GI are compared, with respect to their markers, particularly Ki-67 LI, as well as the structural atypism, such as microacinus formation and pseudoglandular structure, and invasive growth into the surrounding liver parenchyma, HCC GI can be diagnosed as an early or well-differentiated malignant lesion. PMID- 7540963 TI - Palliative therapy for carcinoma of the esophagus. AB - Despite improvements in medical and surgical care, the prognosis of patients with cancer of the esophagus has remained poor. The majority of patients are candidates for palliative rather than curative therapy. Surgery probably provides the most effective palliation of dysphagia, but it is associated with a high morbidity, and many patients are not candidates for surgery because of underlying medical problems. There are several endoscopic modalities for palliation. The choice of therapy depends on the location of the tumor, the presence or absence of a tracheo-esophageal fistula, and the projected prognosis for the patient. Expandable stents will probably become the treatment of choice for the inoperable patient because of ease of insertion and relative safety. Nevertheless, other endoscopic techniques will be a useful complement prior to stent insertion in order to debulk the tumor. With adequate palliation, attention can be given to treatment that may serve to prolong survival. Future trials on radiation therapy and chemotherapy are necessary so that we can improve survival without sacrificing the patient's quality of life. On the horizon is the administration of chemotherapy through such vehicles as tumor-specific photosensitizing agents, or intratumoral injection of cytotoxic agents. PMID- 7540964 TI - Ion channel events simulated with the program SIMSTATE. AB - Ion channel transitions between conducting (open) and non-conducting (closed) states are often described in terms of a chemical kinetic model, where the rate constants describing the transitions between states can be derived by analysing a data record and measuring channel dwell times. In this paper, a menu-driven program for IBM-compatible microcomputers, SIMSTATE, is described which permits simulations of one or more channels according to a user-specified set of transition rates. Rates can be constant, voltage- or ligand-activated, exhibit co operativity, or dependent on the calcium concentration resulting from the opening of one or more nearby Ca channels. To illustrate the functionality of SIMSTATE, open and closed events are simulated for a well-known Ca channel model and an example of co-operative gating is examined. Furthermore, the control of a Ca dependent K channel by a nearby Ca channel, which also opens and closes according to a user-specified transition rates, is described. Potential uses for SIMSTATE as a tool for theoretical analysis, education and experimental design are discussed. PMID- 7540962 TI - Conservative treatment of urinary incontinence. PMID- 7540965 TI - Design of the Prostate Cancer Prevention Trial (PCPT). AB - The PCPT is a chemoprevention trial of finasteride with a primary endpoint of biopsy-proven presence or absence of prostate cancer. A total of 18,000 healthy men, aged 55 years and older, will be randomized. Half will receive finasteride (5 mg/day) and half will receive placebo (one matching tablet per day) for 7 years. The trial is designed to have 92% power to detect a 25% reduction in period prevalence of biopsy-proven disease using a two-sided test with alpha = 0.05. The trial is complicated by the known impact of finasteride on the major screening test for prostate cancer, prostate specific antigen (PSA). This paper describes the PCPT design with reference to alternatives that were considered. The chosen design depends on five critical assumptions that must be monitored closely throughout the 9-year trial. PMID- 7540966 TI - Pattern of axonal loss in longstanding papilledema due to idiopathic intracranial hypertension. AB - There is both clinical and histopathologic evidence for peripheral visual field loss and optic nerve degeneration in longstanding papilledema due to idiopathic intracranial hypertension (IIH). The purpose of this study was to look at the extent and distribution of axonal dropout in secondary optic atrophy due to IIH. Both optic nerves from a 29-year-old man with a two year history of IIH were examined histologically and morphometrically. A high-contrast lipid (myelin) stain, paraphenylenediamine (PPD), and a semiautomated image analysis system were employed to resolve sufficiently the optic nerve fiber images for counts and for measurement. There were 80% and 90% losses of axons, respectively, in the right and left optic nerves consequent to IIH. The axonal loss in the peripheral area of each optic nerve was much more severe than that in inner sectors (= 0.001 for the right optic nerve and = 0.005 for the left). This pattern of axonal dropout is consistent with the preservation of good central visual acuity despite devastating optic nerve atrophy, and with the severe peripheral visual field loss noted in this patient. PMID- 7540967 TI - Effects of topical FK506 on endotoxin-induced uveitis (EIU) in the Lewis rat. AB - FK506 is a macrolide antibiotic and a potent immunosuppressant. To investigate the effect of topical FK506 on acute ocular inflammation, we evaluated its action on the development of endotoxin-induced uveitis (EIU). At two different concentrations of 0.05% and 0.3%, topical FK506 was applied to Lewis rats with EIU. In aqueous, the mean number of inflammatory cells per microliter +/- SEM was 2,389 +/- 1,277, 1,571 +/- 1,562, 898 +/- 882, and 69 +/- 152 for rats treated with vehicle alone, 0.05%, 0.3% FK506, and 1% prednisolone acetate. The median of histological grades was 2, 1.5, 0.8, and 0.5 for animals treated with these 4 different regimens respectively. Analysis of aqueous protein showed a small reduction in FK506-treated animals. Mean blood levels of FK506 were low in rats treated with topical FK506 (0.05%, 0.84 ng/ml; 0.3%, 2.0 ng/ml) suggesting that its therapeutic effect was not secondary to the systemic absorption of the drug. Although FK506 is not as effective as prednisolone, 0.3% FK506 produced significant decreases in the mean aqueous inflammatory cell number and histological inflammatory score as compared to control vehicle alone. We conclude that topical FK506 can suppress EIU in a dose-dependent fashion and may be an alternative medication for patients with anterior uveitis and contra-indication to topical steroid. PMID- 7540968 TI - Acidic fibroblast growth factor reduces rat skeletal muscle damage caused by ischemia and reperfusion. AB - Acute interruption of arterial blood flow to the extremities is often associated with significant morbidity and mortality. Broad-spectrum mitogenic and non mitogenic activities of FGFs inspired us to study its protecting effects on tissue injuries in ischemia reperfusion condition. We found that systemic administration of aFGF after reperfusion onset prevented severe skeletal muscle injuries. In rats treated with aFGF, the tissue edema was reduced significantly, the tissue viability was increased, and the muscle fibers contained more succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase). The pathological results supported the concept of improved prevention with aFGF treatment. The possible tissue protection by aFGF may come from its ability to regulate the concentration of extra- and intracellular calcium ion. Besides, it may moderate other Ca2+ dependent enzyme conversion processes. Also, it may take part in the vascular tone regulation under ischemia and reperfusion conditions. These results suggest further study of tissue ischemia prevention with FGF and its possible mechanisms in the future. PMID- 7540970 TI - Immunity to the HER-2/neu oncogenic protein. AB - The study of oncogenic viruses led to the discovery that transforming retroviruses contain oncogenes homologous with and/or derived from cellular proto oncogenes. In humans malignant transformation is often the result of the activation of proto-oncogenes. Normal proto-oncogenes can be activated to transforming proto-oncogenes by a variety of mechanisms including point mutation, translocation and amplification. Development of successful strategies for the immunotherapy of human cancers is an area of intense investigation. Part of the problem in developing cancer-specific immunotherapy has been the lack of well defined tumour antigens. Our laboratory has focused on the question of whether oncogenic proteins expressed by transforming proto-oncogenes can serve as targets for immune attack. Some patients with HER-2/Neu-positive breast cancer have an existent immune response to the HER-2/neu protein with no clinical signs of autoimmunity, supporting the idea that overexpressed oncogenic proteins can be targeted in therapy without fear of destructive autoimmunity. The identification of candidate cytotoxic T lymphocyte epitopes might allow the generation of tumour specific cytotoxic T lymphocytes for use in therapy and identify potential epitopes for peptide vaccines. PMID- 7540969 TI - Propagation of mouse and human T cells with defined antigen specificity and function. AB - Difficulties maintaining fully functional CD4+ T cells in culture have historically limited the study of their role in tumour rejection as well as other clinical applications. As the therapeutic value of current antitumour CD8+ T cell adoptive therapy becomes better defined, a strong impetus exists to determine optimal conditions for culturing antitumour CD4+ T cells. Our goal is to promote broadly polyclonal, antigen-specific CD4+ T cell responses of either Th1 or Th2 character for use in antitumour therapy or allograft facilitation, respectively. Similar obstacles exist in murine and human cultures: (1) during even brief periods of culture CD4+ T cells develop high 'background' reactivity to class II positive antigen-presenting cells; (2) maintenance of antigen specificity as evidenced by cytokine secretion and short-term proliferation assays is insufficient to ensure bulk numerical expansion; (3) Th1-type CD4+ T cells often lose their potential for antigen-specific secretion of interleukin 2 on re stimulation (though remain inducible by 12-O-tetradecanoylphorbol 13 acetate/ionomycin); (4) during prolonged culture selection pressure favours CD4+ subpopulations that recognize artifactual antigens such as culture medium proteins; (5) even with optimal culture conditions, cultured CD4+ T cells may function differently in vivo to uncultured CD4+ T cells. We have devised various strategies to surmount these obstacles by use of selected cytokines, antigen presenting cells and timely culture manoeuvres. PMID- 7540971 TI - Strategies for studying mouse and human immune responses to human papillomavirus type 16. AB - Cytotoxic T lymphocytes (CTL) are an important protective mechanism in viral infection and can be effective against tumours. We have investigated the tumour associated E6 and E7 genes of human papillomavirus type 16 as CTL targets. In H 2b mice we have defined epitopes in E6 and E7 which can readily generate CTL in vivo and we have shown that HLA-A2.1 transgenic mice can generate an HLA-A2.1 restricted response. We have been unable to reveal a primed CTL response in humans. These paradoxical findings imply that human papillomavirus may fail to stimulate a systemic CTL response and/or employ strategies for evading or down regulating such a response. PMID- 7540972 TI - Topical application of 12-O-tetradecanoylphorbol-13-acetate induces dyssynchronous expression of keratins K1 and K10 in mouse epidermis. AB - 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter that causes severe alterations in the biosynthesis of epidermal keratins. This study shows that TPA induces a dyssynchronous effect on keratin expression in stratified squamous epithelium. The effect of TPA on the separate expression of the maturation-associated keratins K1 and K10 was studied by immunohistochemistry in an unperturbed replicative keratinocyte population of hairless mice epidermis in relation to changes in the cell cycle time during regeneration. Keratinocytes in DNA synthesis were pulse-labeled by intraperitoneal injection of the thymidine analogue 5-bromodeoxyuridine (BrdUrd) 1 h before a single topical application of TPA. The BrdUrd-labeled cell cohort, representing an originally unperturbed replicative keratinocyte population exposed to TPA mainly in the postreplicative period, was followed for up to 97 h. The results suggested unaltered timing of the onset of K1 and K10 expression compared with normal epidermis (18 and 24 h, respectively, following DNA synthesis). This indicates that the synthesis of both keratins was programmed before the keratinocytes entered their last DNA synthesis. A reduction in K10 expression from about 30 h compared with that of K1 expression was observed. Mathematical modeling suggested a delay in K10 expression related to the second and third rounds of cell divisions after pulse labeling. How TPA induces such dyssynchrony in K1 and K10 regulation remains unknown. PMID- 7540973 TI - Enumeration of viable Candida albicans blastospores using tetrabromofluorescein (eosin Y) and flow cytometry. AB - A rapid assay was developed for determination of the viability of blastospores of Candida albicans utilizing flow cytometry to detect the accumulation of tetrabromofluorescein in non-viable yeast cells. Eosin Y was shown to stain non viable C. albicans blastospores selectively without affecting the cellular viability of competent yeast cells or resulting in non-specific staining of viable cells. Results of these studies show that this flow cytometric method of determining cell viability of C. albicans is more accurate and more precise than the more common method of enumerating colony forming units. PMID- 7540974 TI - Is routine urography necessary in all patients undergoing suprapubic transvesical prostatectomy? AB - One hundred and forty consecutive patients were offered transvesical prostatectomy after haematological and urinary investigations but without urography. The results of treatment are comparable to those in similar setting where urography had been performed as a routine procedure. While the renal function was generally poor in the earlier period, improving later in the study period, no urinary abnormality which would have affected outcome was missed by clinical assessment and other investigations. In view of the high cost of urography and possible adverse effects we believe that unless specifically indicated by presence of haematuria or loin pain urography is unnecessary in the diagnostic work of patients for transvesical prostatectomy. PMID- 7540976 TI - Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A. AB - The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12 FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit. PMID- 7540975 TI - Activation of CFTR chloride current by nitric oxide in human T lymphocytes. AB - Nitric oxide, which is produced by cytokine-activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole-cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI- currents in normal human cloned T cells by a cGMP-dependent mechanism. This pathway is defective in cystic fibrosis-derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis. PMID- 7540979 TI - Serotonin in rat oral tissues: role of 5-HT1 receptors in sympathetic vascular control. AB - In this study we examined whether the indoleamine, serotonin (5 hydroxytryptamine, 5-HT), is contained in the rat incisor pulp and gingiva as well as its possible role in regulation of blood flow in these tissues. Tissue biochemical analysis, by means of high performance liquid chromatography coupled to electrochemical detection, revealed the presence of 5-HT and its metabolite, 5 hydroxyindoleacetic acid (5-HIAA), as well as the catecholamine, dopamine, in both pulp and gingiva. Unilateral surgical sympathectomy or resection of the inferior alveolar nerve failed to affect 5-HT levels in either tissue while dopamine contents in the pulp and gingiva were diminished following sympathectomy. Electrical stimulation of the sympathetic trunk induced a frequency-dependent vasoconstriction in the pulp and gingiva as measured by laser Doppler flowmetry. This vasoconstriction was unaffected by infusion of 5-HT2 or 5 HT3 receptor antagonists or dopamine receptor antagonists but it was significantly reduced in both tissues after alpha 1-adrenoceptor blockade. During this blockade the remaining vasoconstriction induced by high frequency stimulation (16 Hz) was reduced in gingiva by the 5-HT1 receptor blocker, methiothepin. The results indicate an involvement of 5-HT1 receptors and alpha 1 adrenoceptors in the sympathetic vascular control in the gingiva. PMID- 7540980 TI - Electrophysiologic alterations in the rabbit nodal cells induced by membrane lipid peroxidation. AB - To investigate cellular electrophysiologic alterations due to lipid peroxidation of the cell membrane by free radicals as a possible cause of coronary reperfusion arrhythmias, we studied the effects of t-butyl hydroperoxide on the spontaneous action potential and membrane currents of the rabbit sinoatrial and atrioventricular node preparations (0.2 x 0.2 x 0.1 mm). 1-5 min of superfusion with t-butyl hydroperoxide (100-500 microM) caused a transient increase in the spontaneous firing frequency by 9%, accompanied by a 4% increase in the action potential amplitude and a 33% increase in the maximal rate of depolarization (P < 0.05, n = 6). t-Butyl hydroperoxide then gradually suppressed physiological automaticity, but induced abnormal repetitive firing due to early and delayed after-depolarizations. 15 min of superfusion with t-butyl hydroperoxide caused a complete standstill of nodal cells at a resting potential of -46 +/- 3 mV (n = 12). Such effects of t-butyl hydroperoxide on the spontaneous action potential were attenuated by pretreating the cells with butylated hydroxytoluene, a lipid peroxidation inhibitor. Voltage clamp experiments using double microelectrode methods revealed that t-butyl hydroperoxide transiently increased the Ca2+ current by 22% after 5 min of superfusion but subsequently reduced it to 46% of the control value after 15 min (P < 0.05, n = 6). Similar biphasic changes were observed in the delayed rectifying K+ current and hyperpolarization-activated inward current (n = 6). Background current was progressively increased without any change in its reversal potential (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7540981 TI - Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta. AB - The application of capsaicin (1 microM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K+ or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba2+ inward current (IBa) through the voltage-dependent L-type Ca2+ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in IBa. Capsaicin did not alter the overall shape of current-voltage relationship of IBa. However, capsaicin (3 microM) shifted the quasi-steady-state inactivation curve of IBa to more negative membrane potential by about 5 mV. These effects of capsaicin on IBa were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K+ currents. These results suggest that inhibition of the voltage dependent L-type Ca2+ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage dependent K+ channels might produce an increase in cell excitability. PMID- 7540978 TI - Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding. AB - We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex. PMID- 7540982 TI - Up-regulation of integrin alpha 5 beta 1 expression by interleukin-6 in rabbit corneal epithelial cells. AB - Interleukin-6 (IL-6) has been shown to promote the attachment of rabbit corneal epithelial cells to fibronectin-coated substratum and ex vivo migration of the cells on the corneal stroma. To examine whether IL-6 promotes cell attachment through up-regulation of expression of integrin alpha 5 beta 1, i.e., the major cell surface fibronectin receptor, we quantified the levels of both alpha 5 and beta 1 subunit transcripts by reverse transcription-polymerase chain reaction in cultured rabbit corneal epithelial cells pretreated with various concentrations of IL-6. The levels of both alpha 5 and beta 1 mRNAs were dose-dependently elevated by IL-6, attaining 1.5- and 1.8-fold increases, respectively, at 10 ng/ml. The stimulatory effect of IL-6 was transient; the levels of both subunit mRNAs reached a maximum 1 h after the addition of IL-6 and returned to the basal levels after 6 h. The IL-6-induced up-regulation of integrin alpha 5 and beta 1 mRNAs was also confirmed by Northern blot analysis. These results indicate that the increased attachment of corneal epithelial cells to fibronectin and enhanced ex vivo migration on corneal stroma by IL-6 is, at least in part, due to the temporal up-regulation of integrin alpha 5 beta 1 expression in corneal epithelial cells. PMID- 7540983 TI - Adhesive substrates influence acid-productive activities of cultured rabbit osteoclasts: cultured osteoclasts with large vacuoles have enhanced acid productive activities. AB - Since acid secretion of polarized osteoclasts on the bone surface has an important role in bone resorption, we examined the acid-productive activities of osteoclasts by measuring the pH of acidic organelles such as endosomes, lysosomes, and vacuoles in cultured rabbit osteoclasts with FITC-dextran as a fluorescent pH probe. The average pH value of acidic organelles of osteoclasts cultured on plain glass coverslips was 5.3 +/- 0.2. The pH of the acidic organelles correlated well with the size and amount of the vacuoles in the cells. Using this FITC-dextran method, we also examined the effects of adhesive substrates, such as type I collagen, vitronectin, and dentine, on the acid productive activities of osteoclasts and found that the pH value of acidic organelles of osteoclasts cultured on dentine slices was significantly lower than that of osteoclasts cultured on plain glass coverslips. Likewise, in the case of the osteoclasts cultured on type I collagen- or vitronectin-coated glass coverslips, the pH values of acidic organelles were slightly lower and the proportion of osteoclasts having large vacuoles was increased compared with the cells cultured on the plain glass coverslips. These results indicate that osteoclasts containing large vacuoles have high acid-productive activities, and adhesive substrates such as type I collagen and vitronectin influence the formation of large vacuoles in cultured osteoclasts. PMID- 7540977 TI - v-Myb DNA binding is required to block thrombocytic differentiation of Myb-Ets transformed multipotent haematopoietic progenitors. AB - The E26 avian leukaemia virus encodes a fusion oncoprotein consisting of truncated versions of the c-Myb and c-Ets-1 transcription factors. When used to infect embryonic chicken haematopoietic cells two types of self-renewing progenitors are obtained, namely myeloblasts and 'MEPs' (Myb-Ets progenitors). In earlier work we have shown that myeloblasts transformed by the ts21 mutant of E26, which has a lesion in v-Myb, can be induced to differentiate into macrophages following shift to the non-permissive temperature. Here we show that the ts21 v-Myb is temperature sensitive for DNA binding in band shift experiments and that its inactivation in transformed MEPs induces their maturation into thrombocytes. The MEP transforming capacity of v-Myb is not confined to its fusion with v-Ets, as it is also seen with a virus that co-expresses tsMyb with v ErbB. As with wild-type E26-transformed MEPs, ts21-transformed MEPs are multipotent, differentiating into eosinophils and myeloblasts following treatment with 12-O-tetradecanoylphorbol-13-acetate. In addition, ts21-transformed myeloblasts differentiate into macrophages when shifted to the non-permissive temperature. This shows that v-Myb blocks haematopoietic differentiation at two distinct stages. In contrast, v-Ets inactivation in MEPs transformed by a ts E26 mutant with a lesion in the corresponding oncoprotein leads to their differentiation into erythrocytes, myeloblasts and probably eosinophils. These data show that the two domains of Myb-Ets selectively affect decision making processes in different types and stages of haematopoietic cells. PMID- 7540984 TI - Mechanical induction of beta 1-integrin-mediated calcium signaling in a hepatocyte cell line. AB - Mechanical stress influences growth, differentiation, and gene expression in a variety of cell types. It is believed that via extracellular matrix the mechanical stimulus is transmitted to integrin receptors which thus play a key role in transducing signals into the cell interior. Here we demonstrate that incubation of suspended hepatocytes with specific antibodies to beta 1-integrin subunits followed by a short-term mechanical stimulation is sufficient to induce a rise in intracellular Ca2+. The results indicate that mechanical loading of individual integrin subunits activates Ca(2+)-specific signal pathways. PMID- 7540985 TI - Beta 1 integrins in epithelial tissues: a unique distribution in the lens. AB - Integrins have been shown to play a role in directing and maintaining cell differentiation and polarization. The embryonic lens provides a good system in which to examine their role in epithelial cell differentiation, because all stages of lens development are represented in an individual embryonic lens. Therefore, we examined the expression and distribution of beta 1 integrin heterodimers in both the lens epithelium and the differentiated lens fiber cells. In lens epithelial cells beta 1 integrin was found to be localized to all membrane surfaces. Lens fiber cells contained beta 1 integrin all along their lateral borders as well as at the site of their attachment to the lens capsule and at their interface with lens epithelial cells. The distribution of beta 1 integrin in the lens was distinct from that observed in simple epithelia, the retinal pigment epithelium (RPE), kidney, and intestine, where it was limited to a basal lateral localization. We examined the specific beta 1 integrin heterodimers expressed in the lens by Western blot analysis for the integrin alpha subunits following beta 1 immunoprecipitation and compared them with those expressed in RPE cells. In the lens we detected alpha 3 and alpha 6 subunits but not alpha 1, alpha 5, or alpha v. When the lens was separated into epithelial and fiber cells, we found that alpha 3 was expressed at a higher level in the epithelial cells, while alpha 6 was primarily associated with the fiber cells. In the RPE the primary beta 1 integrin detected was alpha 3. Unlike in lens and kidney, alpha 6 beta 1 integrin in RPE cells was expressed only at a low level. alpha v was also expressed in RPE cells but not as a beta 1 heterodimer. As in the lens, neither alpha 5 beta 1 nor alpha 1 beta 1 integrin was detected in RPE. Both lens and RPE cells express a specific subset of beta 1 integrin heterodimers which are likely to be important to the initiation and maintenance of their differentiated phenotype. PMID- 7540986 TI - Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. AB - To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 microM) or cytochalasin D (10 microM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against alpha- and beta tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 microM), a putative protein kinase inhibitory flavonoid, offered complete protection against the autophagy inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 microM) and prunin (100 microM), as well as KN-62 (10 microM), a specific inhibitor of Ca2+/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 microM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 microM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process. PMID- 7540987 TI - Upregulation of interleukin-8 receptor in human polymorphonuclear neutrophils by formyl peptide and lipopolysaccharide. AB - Interleukin-8 (IL-8) is implicated in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. Although the cytokine activates neutrophils through a receptor, no information is available regarding the regulation of IL-8 receptor (IL-8R) expression. The present study shows that, compared to control, the bacterial products--formylpeptide and LPS (serum activated) upregulate IL-8 receptor by 54% and 115%, respectively, the former by degranulation of the secretory vesicle and the latter by de novo protein synthesis. The newly expressed IL-8R could be demonstrated with anti-IL-8R antibody and by autoradiogram of the receptor crosslinked with [125I]IL-8. The study may be useful for understanding the potential role of IL-8 during neutrophil mediated inflammatory response. PMID- 7540988 TI - Reverse transcriptase-polymerase chain reaction detects induction of cardiac-like alpha myosin heavy chain mRNA in low frequency stimulated rabbit fast-twitch muscle. AB - Using reverse transcriptase-polymerase chain reaction we quantified in rabbit skeletal muscles expression levels of the highly homologous cardiac alpha and beta myosin heavy chain (alpha MHC, beta MHC) mRNA isoforms. Masseter muscle displayed highest levels of a cardiac-like alpha MHC mRNA. This isoform was present at 20-fold lower amounts in slow soleus and at 200-fold lower levels in several fast-twitch muscles. Low-frequency stimulation periods exceeding 20 days drastically induced the alpha MHC mRNA in fast tibialis anterior. The alpha MHC mRNA was 140-fold elevated after 60 days when beta MHC mRNA had increased 50 fold. Our results demonstrate the wide distribution of a cardiac-like alpha MHC mRNA in skeletal muscle and its marked induction during fast-to-slow transition as induced by low-frequency stimulation. PMID- 7540990 TI - Amino acids 225-235** of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex. AB - Protein C (PC) is a vitamin K-dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin-thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin-thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225-235 in an Elisa assay. The peptide inhibited PC activation by the thrombin-thrombomodulin complex. These data confirm that the calcium-binding loop of the serine-protease domain is involved in the interaction of PC with the thrombin-thrombomodulin complex. PMID- 7540991 TI - N,N,N-trimethylsphingosine inhibits interleukin-1 beta-induced NF-kappa B activation and consequent E-selectin expression in human umbilical vein endothelial cells. AB - We examined the effect of N,N,N-trimethylsphingosine (TMS) on the interleukin-1 beta (IL-1 beta)-induced E-selectin expression in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with TMS (0.1-10 microM) resulted in a concentration-dependent inhibition of IL-1 beta-induced E-selectin expression. Sphingosine or N,N-dimethylsphingosine had no effects on the expression. Electrophoretic mobility shift assay revealed that TMS inhibited IL-1 beta-induced NF-kappa B activation, which is essential for E-selectin expression. This inhibitory effect of TMS on IL-1 beta-dependent endothelial cell activation may partly explain the known anti-inflammatory or anti-metastatic effect of TMS in vivo. PMID- 7540989 TI - Bait region-thiol ester mapping in human alpha 2-macroglobulin. AB - The separations between aromatic residues in the bait region and nitroxide spin labels attached to the thiol ester-forming residues (Cys949 and Gln952) in human alpha 2-macroglobulin (alpha 2M) have been determined from paramagnetic broadening effects of the spin labels on bait region 1H NMR signals. We found that both the Cys949 and Gln952 residues are within 11-17 A of the aromatic residues in the bait region, with closer approach of some residues to the Gln952 spin label than to the spin label attached to Cys949. A model of the location of bait regions and thiol esters within an alpha 2M half-molecule is proposed that places the bait regions in the central region of alpha 2M at the interface between the subunits. PMID- 7540992 TI - Characterization of the human Y1 neuropeptide Y receptor expressed in insect cells. AB - We have expressed the human Y1 NPY receptor in insect cells using a recombinant baculovirus (BacY1). Non-linear curve fitting of competition binding data indicates the presence of 500,000-750,000 saturable NPY binding sites per cell. The affinity of the recombinant Y1 receptor for NPY (Kd = 0.38 +/- 0.8 nM) was identical to the natural receptor. We used a foreign epitope to characterize, immunopurify, and localize the recombinant protein. Cross-linking experiments identified a 65 kDa band as the major NPY binding species. Confocal microscopy indicated that although some recombinant proteins are detectable as early as 12 h post-infection, significant expression at the cell surface is only seen 24-48 h post-infection. We also describe a procedure to treat infected Sf21 cells in such a way that they can be frozen and stored at -80 degrees C for many months before being used for binding studies. PMID- 7540993 TI - Mechanisms controlling nitric oxide synthesis in osteoblasts. AB - Nitric oxide (NO) modulates the activity of a number of cell types, but little is known about its possible role in bone metabolism. In the present study we demonstrate that freshly isolated murine osteoblasts and an osteoblastic cell line express NO-synthase mRNA and release NO when stimulated with IL-1 or LPS, thus confirming the results of some recent reports using human and rat osteoblast like cells. Synergistic effects were found between IL-1 and LPS or TNF. Enzyme induction was blocked by dexamethasone and IL-4. 1,25-dihydroxyvitamin D3 did not modify basal NO synthesis, but it markedly increased the cytokine-induced NO release. M-CSF, GM-CSF, IL-3, LIF, PTH, estradiol and calcitonin did not show significant effects on NO synthesis. NOS induction was blocked by various tyrosine-kinase inhibitors, geldanamycin and herbimycin A being the most potent. These results suggest that endogenous NO might participate in the regulation of bone remodeling at the local level, and may mediate some effects of vitamin D on bone. NO has recently been reported to inhibit osteoclastic bone resorption. The release of NO induced by bone-stimulating factors such as IL-1 may represent a protective mechanism helping to avoid excess resorption and preserve bone integrity in inflammatory conditions. PMID- 7540995 TI - Accuracy of invasive and noninvasive tests to diagnose Helicobacter pylori infection. AB - BACKGROUND & AIMS: Multiple tests are available for determining Helicobacter pylori infection. Our aim was to compare the sensitivity, specificity, and negative and positive predictive value of the most widely available tests for diagnosis of H. pylori. METHODS: A total of 268 patients (mean age, 53.7 +/- 15.8 years; 142 male and 126 female; 125 white and 143 nonwhite) was tested for H. pylori infection by [13C]urea breath test (UBT), measurement of serum immunoglobulin (Ig) G and IgA antibody levels, and antral biopsy specimens for CLO test, histology, and Warthin-Starry stain. No patient received specific treatment for H. pylori before testing. The infection status for each patient was established by a concordance of test results. RESULTS: Warthin-Starry staining had the best sensitivity and specificity, although CLO test, UBT, and IgG levels were not statistically different in determining the correct diagnosis. The absence of chronic antral inflammation was the best method to exclude infection. Stratification of results by clinical characteristics showed that UBT and chronic inflammation were the best predictors of H. pylori status in patients older than 60 years of age. IgA was a better predictor in white patients. CONCLUSIONS: The noninvasive UBT and IgG serology test are as accurate in predicting H. pylori status in untreated patients as the invasive tests of CLO and Warthin-Starry. PMID- 7540994 TI - FK506 increases permeability in rat intestine by inhibiting mitochondrial function. AB - BACKGROUND & AIMS: Under normal physiological conditions, the intestine presents an adenosine triphosphate (ATP)-dependent barrier to luminal contents. Disruption of this barrier function can occur when cellular metabolism is compromised. This study examined the effects of FK506 on intestinal permeability and enterocyte metabolic function in Lewis rats. METHODS: Rats were administered FK506 at a dose of 0.1, 0.5, or 2 mg/kg on alternate days for 6 weeks. Intestinal permeability was assessed by measuring urinary recovery of 99mTc-diethylenetriamine pentacetate, and electrophysiological conductance measurements were performed in Ussing chambers. Metabolic function was assessed in isolated enterocytes by measuring total ATP and CO2 release from [14C]pyruvate and [14C]glucose. RESULTS: Rats treated with FK506 showed a dose-dependent reduction in weight gain as well as increased in vivo and in vitro intestinal permeability. There was no difference in plasma creatinine or urinary output. Changes in permeability correlated with reduced ATP levels and CO2 release because of diminished mitochondrial function. Lactate production, as a measure of glycolytic activity, was not altered by FK506. CONCLUSIONS: In a dose-dependent manner, FK506 treatment in rats reduces weight gain, increases intestinal permeability, and decreases the ability of the small intestine to use glucose as an energy source. PMID- 7540997 TI - Interleukin 1-induced production of nitric oxide inhibits benzenetriol-mediated oxidative injury in rat hepatocytes. AB - BACKGROUND & AIMS: Nitric oxide modifies free radical-mediated cell processes in multiple in vivo and in vitro systems. The aim of this study was to determine the role of hepatocyte production of NO in oxidative injury. METHODS: Rat hepatocytes in primary culture were incubated with 1,2,3-benzenetriol, a source of superoxide. Interleukin (IL) 1 was added to induce NO synthesis. Injury was determined by aspartate aminotransferase (AST), malondialdehyde (MDA), and glutathione (GSH) levels. RESULTS: Benzenetriol-induced injury increased AST and MDA levels and decreased GSH levels in control and IL-1-treated cells. Inhibition of NO synthesis in IL-1-treated cells significantly increased AST and MDA production while enhancing GSH depletion. In the presence of superoxide dismutase or S-nitroso-albumin, an exogenous source of NO, injury was decreased or abolished. NO production was significantly increased with oxidative stress. In benzenetriol-induced injury in IL-1-stimulated hepatocytes, reverse-transcription polymerase chain reaction showed significantly increased levels of inducible NO synthase messenger RNA, whereas immunoblot analysis showed similarly increased levels of inducible NO synthase protein. CONCLUSIONS: In this rat hepatocyte model of IL-1/benzenetriol-mediated injury, NO, derived from endogenous synthesis or an exogenous donor, is protective. Oxidative stress may have a role in the transcriptional control of NO synthesis. PMID- 7540999 TI - Acute hypercalcemia causes acute pancreatitis and ectopic trypsinogen activation in the rat. AB - BACKGROUND & AIMS: Clinical and experimental observations have associated acute and chronic hypercalcemia with pancreatitis. The aim of this study was to determine whether acute hypercalcemia can induce acute pancreatitis and, if so, whether the pathogenesis involves premature protease activation. METHODS: Rats given bolus infusions of CaCl2 (200 mg/kg; n = 76) were compared with saline treated controls (n = 40). Serum [Ca2+], serum amylase activity, trypsinogen activation peptide (TAP) concentration in serum and pancreatic tissue, pancreatic wet/dry weight ratio, and histology were assessed for 24 hours. For dose-response analysis, CaCl2 was injected at a dose of 50-200 mg/kg, and the aforementioned indices were assayed for 1 hour (n = 5 each). RESULTS: There were no significant changes in the controls. Calcium infusion increased serum [Ca2+] 3-fold after 5 minutes (P < 0.001). Within 1 hour, serum amylase (2.5-fold) and tissue TAP (3 fold) levels increased along with macroscopic and microscopic edema formation and leukocytic infiltration. The extent of the changes at 1 hour correlated with the calcium dose. Amylase and tissue TAP concentrations remained elevated until 24 hours when serum TAP concentration had increased (P < 0.001) and focal acinar necrosis became evident. CONCLUSIONS: Acute experimental hypercalcemia induces dose-dependent morphological alterations characteristic of acute pancreatitis, acute hyperamylasemia, and early ectopic trypsinogen activation. This supports the pathophysiological relevance of excess calcium and offers a possible pathogenetic mechanism for its association with clinical pancreatitis. PMID- 7541000 TI - Peptidergic and nitrinergic denervation in congenital esophageal stenosis. AB - Congenital esophageal stenosis (CES) is a rare disorder with narrowed esophageal lumen that presents as dysphagia from childhood and that is often associated with tracheobronchial remnants or webs. The pathogenesis of CES is unknown. The aim of this study was to examine the histological and immunohistochemical features of CES. Esophagi from 2 young adults with CES and 3 controls with no motility disorders underwent routine H&E staining, trichrome staining for collagen, and detailed immunocytochemical studies for general neuronal markers (protein gene product 9.5, neuron-specific enolase, and S-100) and neurotransmitters (vasoactive intestinal polypeptide, substance P, and galanin) and nitric oxide synthase by beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and a specific NO synthase antibody. Quantitative experiments compared the numbers of myenteric neurons and amounts of fibers at the circular muscle. CES esophagi showed infiltration of neutrophils in the myenteric plane, without any increase in collagen. NADPH-diaphorase histochemistry showed a significant reduction of myenteric nitrinergic neurons (7 +/- 3.4 vs. 2.7 +/- 1.8 neurons per high-power field) and fibers at the circular muscle. Other peptidergic neurons studied were not significantly reduced in CES. The specific total lack of NO inhibitory innervation may be an important mechanism in the pathogenesis of stenosis and aperistalsis of the esophagus in this disorder. PMID- 7540998 TI - Interdigestive cycling in chronic pancreatitis: altered coordination among pancreatic secretion, motility, and hormones. AB - BACKGROUND & AIMS: Secretions from the exocrine and endocrine pancreas may modulate interdigestive motility. To test this hypothesis in humans, we investigated interdigestive cycling in patients with chronic pancreatitis (CP) as a model of impaired pancreatic function. METHODS: Antroduodenal motility, pancreatic enzyme output, and pancreatic polypeptide release were monitored for two consecutive interdigestive cycles in 13 controls and 9 patients with CP. RESULTS: Interdigestive enzyme output was severely impaired in patients with CP (> 80% decrease); however, secretory cycling was still evident in most patients. All parameters describing interdigestive motility were similar in controls and patients with CP (duration of the migrating motor complex [MMC] was 107 +/- 19 minutes in patients with CP vs. 114 +/- 15 minutes in controls). The time between cyclic peaks of enzyme secretion (76 +/- 4 minutes vs. 101 +/- 4 minutes in controls) and pancreatic polypeptide (63 +/- 4 minutes vs. 106 +/- 7 minutes in controls) was shortened in patients with CP, and peaks were no longer temporally related to the MMC. Only 56% of phase III activity fronts were associated with a concomitant secretory peak in patients with CP compared with 92% in healthy subjects. CONCLUSIONS: CP not only decreases pancreatic secretion but interrupts the coordination among interdigestive cyclic phenomena. Our findings in several animal and human models refute the concept that pancreatic mechanisms exert a major regulatory influence on interdigestive motor activity. PMID- 7540996 TI - Lack of benefit of escalating dosage of interferon alfa in patients with chronic hepatitis C. AB - BACKGROUND & AIMS: In patients with chronic hepatitis C treated with interferon alfa, sustained normalization of alanine aminotransferase was observed in about 20%, and no predictive factor of response could be clearly identified. The aims of this study were to assess the efficacy of an escalating dose of interferon and to determine the predictive factors of response. METHODS: Seventy-five patients were randomly assigned to two groups. Twenty-five patients received a dosage of 3 million units of recombinant interferon alfa-2b three times weekly for 24 weeks, and 50 patients received a dose that was increased to 5 million units at 8 weeks in nonresponders and to 10 million units 8 weeks later in persistent nonresponders. Multivariate analysis was performed to determine the features associated with response. RESULTS: A sustained response was observed in 17% of the patients with constant dosage and in 19% of patients with an escalating dosage. Low pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype were found to be independent predictive factors of sustained response. CONCLUSIONS: In patients with chronic hepatitis C, an escalating dosage of interferon did not improve the overall rate of response. Low pretreatment serum hepatitis C virus RNA levels and genotype other than 1b were the only predictive factors of sustained response. PMID- 7541001 TI - STa receptors: physiological and pathophysiological regulation of intestinal secretion by 5'-cyclic guanosine monophosphate. PMID- 7541002 TI - Boosted systemic immune and local responsiveness after intestinal inflammation in orally sensitized guinea pigs. AB - BACKGROUND & AIMS: Intestinal inflammation resulting in disruption of the mucosal barrier function has been proposed as a cause of increased incidence of allergic diseases. This study was designed to evaluate whether intestinal inflammation is able to change the immune responsiveness to sensitization and antigen challenge responses. METHODS: Guinea pigs orally sensitized to cow's milk proteins were either treated or not treated with trinitrobenzenesulfonic acid (TNBS) to induce intestinal inflammation and compared with control animals (not sensitized). Systemic immune and local responsiveness to antigen challenge were assessed by measuring antibody serum titers, colonic fluid secretion, mucosal histamine level, and mucus depletion. Intestinal permeability was evaluated from 51Cr ethylenediaminetetraacetic acid (EDTA) recovery and beta-lactoglobulin serum level. RESULTS: Immunoglobulin E titers were higher in TNBS-treated animals than in non-TNBS-treated sensitized animals. Antigen challenge in TNBS-treated animals induced a fourfold increase of colonic secretion and greater histamine and mucus depletion than in non-TNBS-treated animals. Permeability to 51Cr-EDTA increased 5 days after TNBS treatment but was unchanged after antigen challenge. In contrast to controls, beta-lactoglobulin was not detected in the sera of challenged sensitized and TNBS-treated animals. CONCLUSIONS: Intestinal inflammation increasing gut permeability enhances the sensitization process. Therefore, local anaphylactic reactions are exacerbated after antigen challenge. PMID- 7541003 TI - Photodynamic therapy for obstructing esophageal cancer: light dosimetry and randomized comparison with Nd:YAG laser therapy. AB - BACKGROUND & AIMS: Light dosimetry analysis to achieve predictable tumor necrosis has not been performed for photodynamic therapy (PDT) in the gastrointestinal tract. We evaluated dihematoporphyrin ethers for sensitizing esophageal carcinomas to 630 nm light and compared PDT with neodymium:yttrium-aluminum garnet (Nd:YAG) laser therapy in a randomized trial. METHODS: Of 52 patients with dysphagia, 32 received palliative PDT. Ten patients treated with PDT participated in a preliminary trial using various doses of 630-nm light, and 22 patients treated with PDT participated in a randomized trial using a derived standardized light dose for comparison with 20 patients treated with the Nd:YAG laser. RESULTS: Light dosimetry correlated with depth of tumor necrosis (r = 0.664; P < 0.001). PDT activity was similar for squamous cell and adenocarcinoma. Among randomized patients, both PDT and Nd:YAG therapy relieved dysphagia, but PDT resulted in improved Karnofsky performance status at 1 month (+7 vs. -7; P < 0.001) and longer duration of response (84 vs. 57 days; P = 0.008). Skin photoreactions were unique to PDT. CONCLUSIONS: The extent of PDT tumor ablation correlates with light dosimetry, enabling selection of a standardized light dose. PDT can relieve esophageal obstruction from squamous cell and adenocarcinoma and is an alternative to Nd:YAG thermal necrosis with a longer duration of response. However, PDT requires patient precautions to minimize skin photoreactions. PMID- 7541004 TI - Protection by gastrin in the rat stomach involves afferent neurons, calcitonin gene-related peptide, and nitric oxide. AB - BACKGROUND & AIMS: Certain gut peptides exert gastroprotective effects. However, the underlying mechanism is not fully understood. This study examines the contribution of afferent neurons, calcitonin gene-related peptide, and nitric oxide to the protection conferred by gastrin 17 in the rat stomach. METHODS: Gastroprotection by gastrin 17 against ethanol-induced gross and histological damage was studied after capsaicin-induced defunctionalization of afferent neurons, pretreatment with the calcitonin gene-related peptide receptor antagonist human calcitonin gene-related peptide8-37, anti-calcitonin gene related peptide antibodies, and the NO synthase inhibitor NG-nitro-L-arginine. RESULTS: Gastrin 17 (1-25 pmol/kg) dose-dependently prevented mucosal damage caused by ethanol. Protection was inhibited by functional ablation of afferent neurons or pretreatment with human calcitonin gene-related peptide8-37 (50% inhibitory dose, 86 pmol.kg-1.min-1), anticalcitonin gene-related peptide antibodies, or NG-nitro-L-arginine (50% inhibitory dose, 1 mg/kg). L-Arginine but not D-arginine reversed the effect of NG-nitro-L-arginine. Effects on gross damage were paralleled by histology. Protective doses of gastrin 17 increased gastric mucosal blood flow and, in addition, elevated plasma gastrin concentrations to the same extent as intragastric peptone perfusion. CONCLUSIONS: Gastrin 17 has potent gastroprotective activity that involves afferent neurons, calcitonin gene-related peptide, and NO. PMID- 7541005 TI - Sulfhydryl blocker-induced rat colonic inflammation is ameliorated by inhibition of nitric oxide synthase. AB - BACKGROUND & AIMS: Sulfhydryl compounds are essential in maintaining mucosal integrity, and nitric oxide may contribute to tissue injury. The aim of this study was to characterize experimental colitis induced by a sulfhydryl blocker. METHODS: Colitis was induced in rats by intracolonic administration of 0.1 mL 3% iodoacetamide with and without addition of 0.1 mg/mL NG-nitro-L-arginine methyl ester (L-NAME) to the drinking water. After death, the distal colonic segment was resected and weighed, and mucosal inflammatory mediator, myeloperoxidase, and NO synthase activities were determined. RESULTS: Iodoacetamide induced multifocal mucosal erosions and ulceration that were present for up to 1 week. At 3 weeks, the mucosa was almost intact. Colonic wet weight was maximal at 7 days. Myeloperoxidase activity and NO generation were increased in the first 72 hours, and NO synthase activity and prostaglandin E2 generation were increased up to 21 days. Leukotriene B4 and leukotriene C4 generation were increased up to 14 days. One week after iodoacetamide plus L-NAME treatment, lesion area was reduced by 85% and NO synthase activity by 52%. CONCLUSIONS: Inflammatory mediators have an important contribution to the pathogenesis of colonic injury induced by a sulfhydryl alkylator. The protective effect of L-NAME indicates that NO contributes to tissue injury and that its modulation may be a novel approach to treat inflammatory bowel disease. PMID- 7541006 TI - The relationship between HTLV-I-infected cell lines and uveitis. AB - BACKGROUND: Recently it has been revealed that human T-lymphotropic virus type I (HTLV-I) infection causes uveitis in human. We previously reported HTLV-I uveitis in a rabbit. To investigate the relationship between HTLV-I infection and uveitis, we established an HTLV-I-infected T-cell clone from the cells infiltrated in the anterior chamber of this rabbit and compared the viral production with that in other HTLV-I-infected cell lines. METHODS: The clonality was determined by Southern blot hybridization with various restriction enzymes. Flow-cytometric analysis was used for investigating the expression of cell surface antigens. To compare viral production, we performed reverse transcriptase assay of the culture media and inhibition enzyme-linked immunosorbent assay to determine the quantity of intracellular HTLV-I antigens. RESULTS: The established clone was Ia (MHC class II) positive T cell. This T-cell clone was able to produce about three times more HTLV-I antigens than other HTLV-I-infected cell lines tested. CONCLUSION: A T-cell clone established from anterior aqueous of an HTLV-I uveitis rabbit can produce more HTLV-1 antigen than other HTLV-I-infected cell lines tested and it can be recognized easily by the immune system. Therefore, this high virus production may have a causal relation to uveitis. PMID- 7541007 TI - Effect of capsaicin and chilli on ethanol induced gastric mucosal injury in the rat. AB - Capsaicin, the pungent ingredient of chilli, is gastroprotective against experimental gastric injury when given intragastrically. Epidemiological and clinical data suggest that chilli ingestion may have a beneficial effect on human peptic ulcer disease. This study showed a gastroprotective effect of intragastric capsaicin, in doses of 2 and 5 mg, on ethanol induced gastric mucosal injury using macroscopic, histological, scanning electron microscopic, and biochemical indices. Subcutaneous administration of 2 mg of capsaicin had the same gastroprotective effect as intragastric administration. Acute intragastric administration and chronic ingestion of chilli powder in doses comparable with that consumed in humans (up to 200 mg in single doses or 200 mg daily for four weeks) likewise protected the gastric mucosa. Both the mucosa and gastric juice had higher mucus contents when capsaicin or chilli rather than saline or solvent was used before ethanol challenge. In control animals capsaicin also increased gastric juice mucus content although the mucosal content was unaffected. Increased gastric mucus production may therefore be one mechanism by which capsaicin and chilli exert their gastroprotective effect although an alternative explanation is that the reduction in mucosal mucus depletion is secondary to the protective effect of capsaicin and chilli. PMID- 7541008 TI - Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. AB - Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1 fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury. PMID- 7541011 TI - Colorectal tumorigenesis. PMID- 7541010 TI - Laparoscopic biliary and gastric bypass: a useful adjunct in the treatment of carcinoma of the pancreas. AB - Over 90% of patients with inoperable carcinoma of the pancreas are successfully palliated by endoscopic retrograde cholangiopancreatography and stent insertion. Treatment of the residual 10% of patients often entails a laparotomy, which is difficult to justify when median survival of these patients is only 150 days. Laparoscopic biliary and gastric bypass offers a less invasive alternative than open surgery with shorter hospital stay and more rapid return to normal activity. Between August 1991 and March 1994, 16 patients (median age 69 years, range 31 85) had laparoscopic bypass surgery. The indications for surgery were gastric outlet obstruction at initial presentation (n = 4), blocked biliary stent (n = 8), and metastatic tumour at laparoscopy (n = 4). Surgery took the form of cholecystjejunostomy (n = 7), gastroenterostomy (n = 5), both procedures (n = 3), and failed operation (n = 1). Operative duration was 75 minutes (range 45-190) and hospital stay four days (range 3-33) and all apart from two patients were discharged from hospital in seven days or less. Morbidity occurred in two patients (13%) in the form of a cerebrovascular accident and delayed gastric emptying. Median survival in 10 patients who have died is 201 days (range 20 525). Laparoscopic biliary and gastric bypass is possible in most patients in whom endoscopic stenting has failed and in those who subsequently develop gastric outlet obstruction. Hospital stay is shorter than after open surgery and recovery more rapid. PMID- 7541009 TI - Adhesion molecules in inflammatory bowel disease. AB - The ability of leucocytes to adhere to endothelium is essential for leucocyte migration into inflammatory sites. Some of these adhesion molecules are released from the cell surface and can be detected in serum. The soluble adhesion molecules intercellular adhesion molecule 1 (ICAM-1), E selectin, and vascular cell adhesion molecule 1 (VCAM-1) were studied in the serum of patients with Crohn's disease, ulcerative colitis, and healthy controls. A second blood sample was taken from patients with active disease after one month of treatment and a third two months after remission was achieved. Tissue expression of the same adhesion molecules was studied by immunohistology. Circulating VCAM-1 concentrations were significantly higher in patients with active ulcerative colitis (n = 11, median = 165 U/ml) compared with patients with inactive ulcerative colitis (n = 10, median = 117 U/ml, p < 0.005), active Crohn's disease (n = 12, median = 124 U/ml, p < 0.02), and controls (n = 90, median = 50 U/ml, p < 0.0001). Within each disease group there were no significant differences in E selectin or ICAM-1 concentrations between the active and inactive states, however, patients with active Crohn's disease had significantly higher ICAM-1 concentrations (n = 12, median = 273 ng/ml) than controls (n = 28, median = 168, p < 0.003). VCAM-1 concentrations fell significantly from pretreatment values to remission in active ulcerative colitis (p < 0.01). In Crohn's disease there was a significant fall in ICAM-1 both during treatment (p < 0.01) and two months after remission (p < 0.02). Vascular expression of ICAM-1 occurred more often and was more intense in inflamed tissue sections from patients with ulcerative colitis and Crohn's disease than from controls. Vascular labelling with antibody to E selectin also occurred more often in patients with active inflammatory bowel disease. In conclusion, increased circulating concentrations of selected adhesion molecules are associated with inflammatory bowel disease. There is also evidence of local upregulation, particularly of ICAM-1. Differential expression of adhesion molecules in tissue may play a part in the initiation of leucocyte migration and local inflammation; the function of circulating adhesion molecules is unknown, but may play a physiological part in blocking adhesion. PMID- 7541012 TI - Serum levels of bleomycin released from the lymphatic system. AB - Endolymphatic injection Oil-Bleo (Nippon Kayaku, Japan) was performed on 24 patients for treatment of residual retroperitoneal malignant lymphoma detected after intravenous chemotherapy. The drug was administered once only in a dosage of 60 mg/30 mg per foot. Serum levels of the drug were monitored. The results were compared with data obtained after routine intravenous infusion of aqueous bleomycin to patients with advanced cervical cancer. The results revealed that bleomycin administered intralymphatically reaches maximum concentration after 15 minutes, and bleomycin after intravenous administration reaches maximum concentration at 36 hours. PMID- 7541013 TI - [Should pancreatic lipase dethrone amylase as a pancreatic marker?]. PMID- 7541014 TI - Characterization of kappa and lambda light immunoglobulin chains in adult Macaca fascicularis. PMID- 7541015 TI - Large cell calcifying Sertoli cell tumour of the testis. AB - Five cases of large cell calcifying Sertoli cell tumour of the testis not associated with complex dysplastic syndromes are reported. The age of the patients ranged from 13 to 34 years and all the tumours were histologically similar, having large, isomorphic, non-mitotic, eosinophilic Sertoli cells with foci of calcification. Flow cytometry demonstrated the cells to be diploid or hypodiploid. All cases were positive for vimentin and focally positive for low molecular weight keratin. The present cases, together with a review of the 22 previously reported tumours, demonstrate that there are two clear cut types of large cell calcifying Sertoli cell tumour; those which are associated with complex dysplastic syndromes and which are bilateral and multifocal, and those which are not associated and are unilateral and focal. Prognosis in all of our cases was uniformly good despite invasion of the rete testis in two cases. It is considered that conservative resection of the tumour is the treatment of choice in cases not associated with complex dysplastic syndromes, since the malignancy rate is low. PMID- 7541017 TI - Pancreatic mucinous cystadenocarcinoma with a mural nodule of anaplastic carcinoma. PMID- 7541016 TI - Synovial sarcoma lack synovial differentiation. PMID- 7541018 TI - Do autoantibodies to IgE play a role in IgE-mediated events? AB - Elevated blood levels of IgG anti-IgE are detectable in individuals exhibiting enhanced IgE production, namely those with allergic conditions and helminthic parasite infections. The fact that there are epitope-specific subpopulations of autoanti-IgE suggests that this autoantibody could potentially have multiple effects in immunological events involving IgE. PMID- 7541020 TI - Fine specificity analysis indicates that the primary and secondary fluorescein specific cytotoxic T cell receptor repertoires are indistinguishable. AB - Previous data from this laboratory have shown that fluorescein-specific cytotoxic T cells derived from naive mice are capable of distinguishing between different isomeric forms of the fluorescein hapten, as well as between the iodoacetamido-, isothiocyanate- and dichlorotriazinyl amino-fluorescein derivatives. In this report, we show that T cells derived from previously immunized animals, while demonstrating a stronger and more cross-reactive response overall, display identical fine specificity patterns to those of primary T cells for a panel of fluorescein homologues. We interpret this finding to indicate that the antigen receptor repertoires utilized by primary and secondary cytotoxic T cells are indistinguishable and that the enhanced response of memory as compared with naive CTL results from factors such as a higher density of cell surface receptors or receptor-associated molecules and/or a higher frequency of antigen-responsive T cells. PMID- 7541019 TI - Production of monoclonal antibodies specific to the carboxyl terminal region of the 85 kDa subunit of phosphatidylinositol 3-kinase: use of the antibodies in recognition of mutant p85. AB - We have established two hybridomas producing mAb to the carboxyl terminal region of phosphatidylinositol-3 kinase 85 kDa subunit type alpha (p85 alpha). Analysis using deletion mutants of p85 revealed that epitopes for the two mAb were located on the border of the src homology 2 (SH2) sequence located at the carboxyl end of p85. They immunoprecipitated free p85 efficiently, but reactivity to p85 bound to p110 was very weak. Together with the mAb which we have reported previously, a panel of mAb that covered the various parts of p85 alpha was obtained. Using this panel, we characterized two mutants of p85 (70 and 50 kDa) expressed in the human colon carcinoma cell line, HCC2998. No wild-type p85 was detected in these cells. A mAb specific to the carboxyl terminal region detected p70 but not p50, suggesting that this region is missing in p50. The panel of mAb is a useful tool to use to analyse mutant forms of p85. PMID- 7541021 TI - Feedback suppression of B cell colony formation by supernatants of B colony cells: role of immunoglobulin. AB - We have reported previously that CD5+ B cells from mature B cell colonies provide a negative feedback signal to the growth of autologous B cell colonies. Now we have observed that supernatants from mature B cell colonies also provide a negative feedback signal to the growth of autologous B cell colonies. We investigated the mechanism of this effect by growing B cell colonies physically separated by a 0.45 micron filter from T cells in millicell-CM chambers. Addition of colony supernatants to the T cell compartment reduced the number of B cell colonies by 28 +/- 6%. Colony numbers were reduced by 11 +/- 2 and 17 +/- 5% when the supernatants were added to the B cell or to both compartments, respectively. Pulsing T cells with the B cell colony supernatants before adding them to the colonies also decreased colony numbers by 33 +/- 13%. The addition of exogenous Ig classes and IgG subclasses to B cells decreased B cell colony numbers, although the effect was variable. In the presence of T cells, IgG had the greatest suppressive activity and the subclass IgG4 was most suppressive. In the absence of T cells, high concentrations of IgG almost abolished B cell colony formation. We conclude that these supernatants provide a negative feedback signal either directly to B cells, or via T cells which may be mediated at least in part by Ig. PMID- 7541022 TI - Potential role of Fc gamma R in early development of murine lymphoid cells: evidence for functional interaction between Fc gamma R on pre-thymocytes and an alternative, non-Ig ligand on thymic stromal cells. AB - During early development of the murine fetal thymus a fraction of Thy+ thymocytes express Fc gamma receptors. Concurrently, a small fraction of Thy- CD44- thymocytes bind recombinant soluble Fc gamma R. These findings suggested the possibility that Fc gamma R+ pre-T cells interact with a non-Ig ligand present on fetal thymic stromal cells. Evidence in support of this concept was the finding that experimental manipulation of this putative receptor-ligand pair by anti receptor antibody or recombinant soluble Fc gamma R influenced the developmental pattern of alpha/beta-TCR+ T cells. The restricted expression of CD16 on some pre B cells raises the possibility that an Fc gamma R-dependent step may play a similar role in B-cell development. PMID- 7541024 TI - Interplay between the human TCR/CD3 epsilon and the B-cell antigen receptor associated Ig-beta (B29). AB - T and B lymphocytes are characterized by the surface expression of highly variable antigen receptors called the T-cell (TCR) and B-cell (BCR) receptor. In both B and T cells, binding of antigen to their respective surface receptors results in transmembrane signaling which leads either to programmed cell death or to proliferation and differentiation. The human and murine TCR consist of the highly variable TCR alpha and beta or gamma and delta chains recognizing antigen and the non-covalently associated invariable CD3 complex (gamma, delta, epsilon) and zeta. The antigen-recognizing surface membrane-bound immunoglobulin (Ig) molecules on the surface of human and murine B cells are non-covalently associated with a heterodimeric protein complex of Ig-alpha (MB-1) and Ig-beta (B29). Like the CD3 complex associated with TCR the Ig-associated proteins are predicted to regulate the assembly and transport of the Ig complex to the cell surface and to couple membrane-bound Ig to intracellular signal transduction pathways. To gain more insight into structure/function relationships between CD3 proteins and Ig-alpha and Ig-beta, we transiently co-transfected pairs of expression vectors encoding either TCR/CD3 chains on the one hand and Ig-alpha or Ig-beta on the other into Cos cells. Thus we found a very strong interaction between CD3 epsilon and Ig-beta mediated by the extracellular domains. Experiments in which we could stain Jurkat T cells with soluble Ig-beta but not Ig-alpha protein indicated the recognition of CD3 epsilon by Ig-beta even in the context of the whole TCR/CD3 complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541025 TI - Idiotypic replica of a Toxocara canis excretory/secretory antigen epitope. AB - This study describes the production, characterization and use of an anti-idiotype serum raised against the monoclonal antibody TC-1 which recognizes a T. canis excretory/secretory antigen (ES Ag) epitope. Anti-idiotypic (anti-Id or Ab2) antibodies were produced in rabbits using TC-1 F(ab')2 fragments; these anti-Id inhibited ES Ag binding to biotinylated TC-1, and also inhibited a larval microprecipitation assay using TC-1. Assays show that the Ab2 beta or "internal image" of a T. canis ES Ag epitope was obtained. The antibodies have been used as an idiotypic copy of ES Ag in a diagnostic ELISA for murine toxocariosis. Affinity-purified anti-Id antibodies were used to raise a homologous anti-anti-Id (Ab3) response in rabbits. Antibody formation was followed in the sera of BALB/c mice inoculated with embryonated eggs of T. canis during a 12-month infestation. A 3-week latency period was observed before specific anti-TC-1 epitope antibodies were detected. High levels were reached at 7 weeks post-inoculation with a maximum at the ninth month, and were then maintained until the end of the experiment. The results show the possible utility of anti-Id antibodies as an ES Ag molecular replica. PMID- 7541023 TI - Rapid desensitization of B-cell receptor by a dithiol-reactive protein tyrosine phosphatase inhibitor: uncoupling of membrane IgM from syk inhibits signals leading to Ca2+ mobilization. AB - B-cell antigen receptor (BCR)-mediated calcium response can be blocked by phenylarsine oxide (PAO), a dithiol group-reactive protein tyrosine phosphatase inhibitor. We have examined the mechanism of this inhibition in BL41 Burkitt lymphoma cells. PAO-dependent inhibition is not restricted to the BCR-mediated functions, as evidenced by the failure of the same cells to mobilize Ca2+ in response to CD19 cross-linking. In contrast, calcium response induced by a putative syk activator, H2O2, exhibited only a moderate sensitivity to PAO, demonstrating that PAO did not cause general suppression of all the functions leading to Ca2+ mobilization. BCR cross-linking or H2O2 treatment leads to the induction of almost complete non-responsiveness for the reciprocal stimulation. Since BCR cross-linking did not generate non-responsiveness to H2O2 in the presence of PAO, and PAO-treated cells remained responsive to syk activation by H2O2, we suppose that PAO may inhibit BCR-mediated signal transduction events upstream of syk activation. This assumption was supported by additional data, indicating that PAO was able to modulate functions of at least 2 different protein tyrosine kinase enzymes involved in BCR-mediated signaling. PAO induced rapid and dose-dependent tyrosine phosphorylation of lyn and selectively inhibited BCR-mediated tyrosine phosphorylation of syk. The results presented in this paper demonstrate that PAO may provoke cellular desensitization process by alteration of the signal transducer functions of lyn and syk tyrosine kinase enzymes. PMID- 7541026 TI - Identification and characterization of Schistosoma mansoni antigens recognized by T and B lymphocytes of humans with early active intestinal and/or urinary schistosomiasis. AB - Schistosome antigens selected as vaccine candidates should induce in the majority of humans T and B cell-mediated immunity that results in protection against infection. As a first step towards the identification of such antigens, we attempted to define and characterize the soluble adult Schistosoma mansoni worm antigen (SAWA) bands that are recognized by serum antibodies and/or peripheral blood mononuclear cells of Egyptian children with early active S. mansoni and/or S. haematobium infection. Considerable inter-subject variation was observed in the SAWA bands recognized by antibodies and T lymphocytes, as demonstrated by Western blotting and T cell Western assays, respectively. The humoral response rate for the separated SAWA bands varied between 0% and 88% of infected subjects. The bands of 153, 144, 38 and 32 kDa reacted with the sera of 60 to 88% of infected subjects but not with the sera of uninfected controls. The bands of 144, 38, 32 and 18 kDa elicited proliferative responses in the lymphocytes of 42-63% of infected subjects. It was thus concluded that the SAWA bands of 144, 38 and 32 kDa are likely to carry T and B cell epitopes that could stimulate immune responses in a majority of individuals. The selected bands (144, 38 and 32 kDa) were found to include glycoproteins containing D-mannopyranosyl or glycosyl residues, and respectively 62.5, 46 and 55% amino acids by weight. The amino acid molar ratios of these bands were completely different. PMID- 7541027 TI - Bilateral radial chorioretinal folds. AB - Chorioretinal folds are most commonly idiopathic. Radial chorioretinal folds can be a sign of choroidal neovascularization. A case of bilateral radial chorioretinal folds as a presenting sign of age-related macular degeneration is reported. This patient has maintained good visual acuity in one eye in which the only sign of occult choroidal neovascularization is the presence of chorioretinal folds and minimal leakage on fluorescein angiography. PMID- 7541028 TI - An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique. AB - A simple procedure is described for removing Epon resin from semi-thin 1 micron sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results. PMID- 7541029 TI - Immunoreactive atrial and brain natriuretic peptides are co-localized in Purkinje fibres but not in the innervation of the bovine heart conduction system. AB - Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity was present in Purkinje fibres and nerve fibre varicosities in the conduction system of the bovine heart. In order to elucidate further the morphological correlation between natriuretic peptides and the conduction system, the distribution of brain natriuretic peptide (BNP) was examined. The different parts of the conduction system in the bovine heart were dissected out and processed for immunohistochemistry with antisera against BNP and ANP. BNP immunoreactivity was frequently observed in Purkinje fibres of the atrioventricular bundle, whereas only a few Purkinje fibres in the ventricular part of the conduction system showed immunoreaction. BNP immunoreactivity was detected in regions of the Purkinje fibres which also showed ANP immunoreactivity. BNP immunoreactivity was not observed in nerve fibre varicosities. Methodologically, a larger number of small BNP immunofluorescent granular structures was observed by using an elution restaining technique instead of conventional immunohistochemistry. The present study shows that BNP and ANP immunoreactivities frequently occur in the atrioventricular bundle and that they are co-localized in Purkinje fibres, but not in nerve fibre varicosities, in the conduction system. As previously has been proposed for ANP, the present observations suggest that also BNP may act in an autocrine and/or paracrine way in the conduction cells. PMID- 7541030 TI - The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif. AB - Reversible assembly of intracellular signaling complexes is, in some cases, mediated by direct binding of a Src homology 2 (SH2) domain of one protein to a phosphotyrosine moiety of another protein (Cantley, L. C., Auger, K. R., Carpenter, C. L., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302). Using a degenerate phosphotyrosine-containing peptide library, we showed that individual SH2 domains recognize phosphotyrosine in a specific sequence context to provide fidelity in signaling (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778). Recently a second type of phosphotyrosine interaction domain (PID) or phosphotyrosine-binding domain (PTB) was discovered in the amino terminus of the SHC proto-oncoprotein (Kavanaugh, W. M., and Williams, L. (1994) Science 266, 1862-1865; Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B. (1994) J. Biol. Chem. 269, 32031-32034). Here we demonstrate, using a phosphotyrosine peptide library, that the SHC PID domain preferentially binds to the sequence Asn-Pro-Xaa-phosphotyrosine. This motif is in agreement with sequences at sites implicated in in vivo SHC binding. These results indicate that while SH2 domains predominantly interact with specific residues carboxyl terminal of phosphotyrosine, the PID domain has high specificity for residues amino-terminal of phosphotyrosine. PMID- 7541031 TI - Epidermal growth factor-induced association of the SHPTP2 protein tyrosine phosphatase with a 115-kDa phosphotyrosine protein. AB - Epidermal growth factor (EGF) stimulation of HepG2 and NIH 3T3 cells expressing high levels of the human EGF receptor (3T3/ER) resulted in the tyrosine phosphorylation of a 115-kDa protein that was co-immunoprecipitated with the Src homology 2 (SH2) domain containing protein tyrosine phosphatase, SHPTP2. In contrast, activation of the EGF receptor resulted in a relatively low level (< 1%) of the total SHPTP2 pool associated with the tyrosine-autophosphorylated EGF receptor itself. Similarly, quantitative immunoprecipitations also demonstrated that only trace amounts of the total EGF receptor pool were associated with SHPTP2. Further, activation of the EGF receptor did not result in any significant tyrosine phosphorylation of SHPTP2 and/or the association of the 115-kDa protein with Grb2. In comparison, activation of Jurkat cells with a T cell receptor agonist monoclonal antibody resulted in the co-immunoprecipitation of a 120-kDa tyrosine-phosphorylated protein with Grb2 and a 105-kDa protein with SHPTP2. Thus, these data have identified the 115- and 105-kDa proteins as the predominant SHPTP2-associated phosphotyrosine proteins in EGF- and T cell receptor-activated cells, respectively. PMID- 7541032 TI - T cell-targeted immunofusion proteins from Escherichia coli. AB - Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome inactivating protein. PMID- 7541033 TI - Influence of human immunodeficiency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro. AB - Human immunodeficiency virus (HIV) nucleocapsid protein (NC) influences HIV reverse transcriptase (RT) catalyzed strand transfer synthesis from internal regions of natural sequence RNA. In the strand transfer assay reaction in vitro, primer synthesis initiated on a donor template can transfer and be completed on an acceptor template. NC was added at concentrations up to twice that needed for 100% template coating. As the concentration of NC was increased, primer extension was stimulated until NC coated approximately 50% of the template. Stimulation was caused in part by an increase in the number of primers that sustained synthesis. Subsequent increments of NC decreased synthesis. The presence of NC also increased the efficiency of the strand transfer reaction, allowing a greater proportion of extended primers to transfer from donor to acceptor templates. Processivity of the RT on the donor template was measured using both challenged and enzyme dilution assays. NC did not alter the proportion of synthesis products that reached the end of the template, indicating little effect on processivity. This result suggests that the increase in full-length product synthesis, observed in reactions where the RT repeatedly bound the primer-template, resulted from promotion of RT reassociation by NC. Consequently, since the RT could not reassociate with the template in the processivity assay, NC could not stimulate the amount of full-length synthesis. No strand transfer was observed in dilution processivity assays, suggesting that the RT must dissociate and rebind during the transfer reaction. Stimulation of synthesis, e.g. by increased dNTP concentration, normally inhibits strand transfer. Stimulation of both synthesis and transfer by NC indicates that properties of NC that improve the transfer event prevail over the negative effects of rapid synthesis on transfer efficiency. PMID- 7541034 TI - Anti-CD9 monoclonal antibody activates p72syk in human platelets. AB - NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab')2 fragments of NNKY 1-19 did not, suggesting that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I2 (PGI2). The activity of p72syk was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72syk associated tyrosine kinase activity that peaked at 10 s and subsided to the original level 2 min after stimulation. Coinciding with this time course, p60c src transiently associated with p72syk. In platelets preexposed to GRGDS peptides or PGI2, NNKY 1-19 also increased the p72syk-associated tyrosine kinase activity and led to the association of p60c-src with p72syk. However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation. F(ab')2 fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI2 treatment on intact IgG NNKY 1-19 stimulation. F(ab')2 fragments of another CD9 MoAb, PMA2, had effects on p72syk essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CD9 MoAb to CD9 on the platelet membrane per se induces an increase in the p72syk-associated tyrosine kinase activity but that Fc gamma II receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI2 acts at a step distal to the activation of p72syk and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72syk activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72syk activity induced by anti-CD9 MoAb. PMID- 7541035 TI - Shc binding to nerve growth factor receptor is mediated by the phosphotyrosine interaction domain. AB - Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2.Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXN-PXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways. PMID- 7541036 TI - Diversity of sites for measles virus binding and for inactivation of complement C3b and C4b on membrane cofactor protein CD46. AB - The complement system membrane cofactor protein (MCP) CD46 serves as a C3b/C4b inactivating factor for the protection of host cells from autologous complement attack and as a receptor for measles virus (MV). MCP consists of four short consensus repeats (SCR) which are the predominant extracellular structural motif. In the present study, we determined which of the four SCR of MCP contribute to its function using Chinese hamster ovary cell clones expressing each SCR deletion mutants. The results were as follows: 1) SCR1 and SCR2 are mainly involved in MV binding and infection; 2) SCR2, SCR3, and SCR4 contribute to protect Chinese hamster ovary cells from human alternative complement pathway-mediated cytolysis; and 3) SCR2 and SCR3 are essential for protection of host cells from the classical complement pathway. These results on cell protective activity of the mutants against the human classical and the alternative complement pathways were compatible with factor I-mediated inactivation profiles of C4b and C3b, respectively, in the fluid-phase assay using solubilized mutants and factor I; the results were mostly consistent with those reported by Adams et al. (Adams, E. M., Brown, M. C., Nunge, M., Krych, M., and Atkinson, J. P. (1991) J. Immunol. 147, 3005-3011). SCR2 and SCR3 were required for C3b and C4b inactivation, and SCR4-deleted MCP showed weak cofactor activity for C4b cleavage but virtually no cofactor activity for C3b cleavage. The functional domains of MCP for the three natural ligands C3b, C4b, and MV, therefore, map to different, although partly overlapping, SCR domains. PMID- 7541037 TI - Protein hydration during generation of coagulation factor Xa in aqueous phase and on phospholipid membranes. AB - The energetic contribution of protein solvation-desolvation reactions to generation of coagulation activated factor X (FXa) by the extrinsic pathway protease complex was determined using the technique of osmotic stress. The initial rate of FXa generation by limited proteolysis of human FX was measured in reaction mixtures with human tissue factor (TF) and factor VIIa (FVIIa) assembled either in aqueous phase or on phospholipid membranes. Osmotic stress was induced on the surface of reacting proteins with either polyethylene glycol, or dextran of 6000 and 500,000 molecular weight, respectively. These inert polymers are sterically excluded from the solvation shells of proteins and thus increase the water activity in the excluded spaces. The volume of water transferred either to or from the excluded spaces during formation of reaction intermediates was calculated from the ratio of change in free energy of activation with change in osmotic pressure, delta G*/delta II. For aqueous phase-assembled reactions, delta G* values decreased with delta II at ratios of -2.36 +/- 0.38 and -2.26 +/- 0.26 kcal/mol/atm for polyethylene glycol and dextran, respectively. These values correspond to 5488 +/- 883 and 5255 +/- 604 mol of water transferred from the reacting protein surfaces per mol of FXa generated. At a physiologic osmotic pressure of 7 atm the work of transfer corresponded to 16 kcal/mol, approximately 70% of delta G*. The observed osmotic effects were independent of the viscosity, temperature, and ionic strength of solutions. For reactions assembled on phospholipid membranes, delta G* increased with delta II at a ratio of 0.35 +/- 0.05 kcal/mol/atm, corresponding to 814 +/- 116 mol of water tansferred from bulk solution to protein surfaces. At physiologic osmotic pressure the work of transfer is 2.45 kcal/mol, approximately 12% of delta G*. Results indicate that for factor Xa generation in aqueous phase the work of desolvation is a significant component of the free energy of activation. Results also suggest that phospholipid membranes catalyze the reaction by reducing the desolvation component of the free energy of activation. PMID- 7541038 TI - Cyclophilin A and FKBP12 interact with YY1 and alter its transcriptional activity. AB - YY1 is a zinc finger transcription factor with unusual structural and functional features. In a yeast two-hybrid screen, two cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12 (FKBP12), interacted with YY1. These interactions are specific and also occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA and FKBP12 have different effects on YY1-regulated transcription with these effects being promoter-dependent. These results suggest that immunophilins may be mediators in the functional role of YY1. PMID- 7541039 TI - Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells. AB - To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from -1033 to -779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90% reduction in normalized luciferase activity. Gel shift and antibody supershift analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein complexes binding to the eNOS Sp-1 site, with the slowest migrating form composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1 site is an important cis-element in the core eNOS promoter. PMID- 7541041 TI - Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase. AB - L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band comigrated with mitogen-activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab')2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion. PMID- 7541040 TI - Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs. AB - p130Cas (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47v-crk (v Crk) and p60v-src (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125FAK (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s). PMID- 7541042 TI - Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA 4). A disulfide-linked homodimer binds two CD86 molecules. AB - CD28 and CTLA-4 are homologous T cell receptors of the immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and CD86) on antigen-presenting cells and transmit important costimulatory signals during T cell activation. Here we have investigated the subunit structure of CTLA-4 and the stoichiometry of its binding to B7 molecules. We demonstrate CTLA-4 is a homodimer interconnected by one disulfide bond in the extracellular domain at cysteine residue 120. Each monomeric polypeptide chain of CTLA-4 contains a high affinity binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a receptor structure consisting of preexisting covalent homodimers with two binding sites. Dimerization of CTLA-4 and CD28 is not required for B7 binding, nor is it sufficient to trigger signaling. PMID- 7541043 TI - Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian cells. AB - Iron regulatory proteins (IRPs) are iron-sensing proteins that bind to RNA stem loop sequences known as iron-responsive elements (IREs) when cells are depleted of iron. Although IRPs have been shown to bind to IREs derived from ferritin and transferrin receptor (TfR) mRNAs in vitro, there has not been a direct demonstration of the impact of a recombinant IRP on the expression of endogenous IRE-containing transcripts. In this study, we evaluate the impact of expression of C437S, a mutant of IRP1 that binds IREs regardless of cellular iron status, on the regulation of biosynthesis of ferritin and TfR. Despite being made iron replete, cells expressing C437S continue to synthesize and express high amounts of TfR, while the synthesis of ferritin is repressed. Thus, a single mutant IRP can prevent the usual homeostatic changes in ferritin and TfR biosynthesis. Cells expressing the mutant protein would therefore be predicted to be unable to defend against iron overload. Preliminary results show that cells treated with iron have diminished cell survival when C437S is expressed, and we have thus created a tissue culture model system for the study of iron toxicity. PMID- 7541045 TI - Non-SH2 domains within insulin receptor substrate-1 and SHC mediate their phosphotyrosine-dependent interaction with the NPEY motif of the insulin-like growth factor I receptor. AB - Insulin receptor substrate-1 (IRS-1) and SHC become rapidly phosphorylated upon tyrosines after insulin-like growth factor I receptor (IGFIR) activation. In this study we demonstrate that IRS-1, SHC, and the p85 subunit of phosphatidylinositol 3-kinase interact directly and specifically with the IGFIR. The interaction of all three proteins is dependent upon IGFIR kinase activity and, furthermore, substitution of Tyr-950 with Phe within the NPEY motif of the IGFIR eliminated interaction with both SHC and IRS-1 but had no effect upon p85 interaction. We show that residues 160-516 of IRS-1 and 1-238 of SHC are sufficient and necessary for receptor interaction in the yeast two-hybrid assay. We also demonstrate a direct in vitro interaction between the IGFIR and a fusion protein containing SHC amino acids 1-238. No interaction was observed with a SHC protein containing only the SH2 domain. We conclude that SHC and IRS-1 interact with the tyrosine phosphorylated NPEY motif of the IGFIR, and that both proteins interact via related motifs located in their amino termini. We conclude that the interactions of SHC and IRS-1 with the IGFIR are similar to those which we have previously defined with the insulin receptor. PMID- 7541044 TI - Interaction of FKBP12-FK506 with calcineurin A at the B subunit-binding domain. AB - Calcineurin is a calcium-dependent protein phosphatase that plays a pivotal role in antigen-stimulated T cell activation. The complexes formed between the immunosuppressants cyclosporin A and FK506 and their respective intracellular binding proteins (immunophilins) block T cell activation by binding to calcineurin. Recent studies have shown that the immunophilin-immunosuppressant complexes interact with the latch region of the calcineurin B subunit (Milan, D., Griffith, J., Su, M., Price, E. R., and McKeon, F. (1994) Cell 79, 437-447). Mutations in the B subunit-binding domain of the calcineurin A subunit result in a reduction of calcineurin activity that correlates with B binding affinity. Calcineurin A subunit mutants D348A, F350A, W352A, S353A, and E359A lost greater than 90% of their activity to activate the transcription factor NF kappa B in Jurkat T cells. Furthermore, calcineurin A subunit mutants of residues Thr351, Leu354, and Lys360 showed NF kappa B transactivation activity and phosphatase activity with increased resistance to FKBP12-FK506 but displayed no or minimal increase in resistance for cyclosporin A inhibition. Together, these results strongly suggest that the B subunit-binding domain is required for calcineurin activity intracellulary and interacts with the FKBP12-FK506 complex. PMID- 7541046 TI - Studies of the functional topography of the catalytic center of Escherichia coli primase. AB - The catalytic center of E. coli primase (581 amino acids) was identified by using, in the G4oric single-strand binding protein (SSB) primer RNA (pRNA) synthesis system, ATP and AMP derivatives, which were modified on the 5' side with reactive groups that can be cross-linked to the ATP binding site plus [alpha 32P]GTP. The position of the covalently attached 32P-labeled dinucleotide was mapped by chemical and enzymatic cleavage of labeled wild type and deletion mutants of primase. The catalytic center involves one of the Lys residues Lys 211, Lys-229, and Lys-241. The ATP binding site is preformed in primase, and the cross-linked ATP residue can be elongated to a 5-nucleotide limit, which implies significant stretching of the catalytic center during pRNA synthesis. His-43 close to the N terminus in a proposed zinc finger and Lys-528 near the C terminus were also cross-linked to ATP residues in the primase ATP binding site, suggesting that these regions are topographically close to the catalytic center during pRNA synthesis. When cross-linking was performed on the preformed primase/SSB/G4oric complex with long arm reagents (12-15 A), SSB was also labeled, indicating a close proximity to the site of pRNA synthesis. PMID- 7541047 TI - Electroporation of pp60c-src antibodies inhibits the angiotensin II activation of phospholipase C-gamma 1 in rat aortic smooth muscle cells. AB - Our previous study has shown that angiotensin II induces the rapid tyrosine phosphorylation and activation of phospholipase C-gamma 1 in cultured rat aortic smooth muscle (RASM) cells (Marrero, M.B., Paxton, W.G., Duff, J. L., Berk, B. C., and Bernstein, K. E. (1994) J. Biol. Chem, 269, 10935-10939). This signaling pathway is initiated by ligand binding to the AT1 receptor, a cell surface G protein-coupled receptor. Antibodies to pp60c-src were introduced into RASM cells by electroporation. Angiotensin II-stimulated tyrosine phosphorylation of phospholipase C-gamma 1 was eliminated by the anti-pp60c-src antibodies but not by anti-mouse IgG or bovine serum albumin. Angiotensin II also induced the rapid tyrosine phosphorylation of pp120, a known pp60c-src kinase substrate, and this phosphorylation was also specifically inhibited by anti-pp60c-src antibodies. Electroporation of RASM cells with anti-pp60c-src antibodies had no effect on platelet-derived growth factor-stimulated tyrosine phosphorylation of PLC-gamma 1. Anti-pp60c-src also reduced the angiotensin II-stimulated inositol 1,4,5 trisphosphate production by 78%, while it had no effect on the platelet-derived growth factor-stimulated inositol 1,4,5-trisphosphate production. These data provide the first evidence for a direct involvement of pp60c-src kinase in angiotensin II-mediated PLC-gamma 1 phosphorylation and activation. Furthermore, it also describes a pathway in which a seven-transmembrane receptor can stimulate an intracellular tyrosine kinase. PMID- 7541048 TI - Regulation of glucagon receptor mRNA in cultured primary rat hepatocytes by glucose and cAMP. AB - Glucagon, the pancreatic hormone secreted in response to hypoglycemia, is a key regulator of hepatic glucose production. Since the number of specific glucagon receptors expressed on the cell surface affects the sensitivity of the liver to glucagon, we have examined the regulation of glucagon receptor mRNA levels in cultured primary rat hepatocytes. By ribonuclease protection assay we have identified glucose and intracellular cAMP as regulators of glucagon receptor mRNA expression in cultured rat hepatocytes. We observed a concentration-dependent increase in glucagon receptor mRNA expression when hepatocytes were cultured in the presence of increasing glucose. A 2-fold induction in glucagon receptor mRNA levels was obtained in hepatocytes cultured for 24 h with 22.5 mM glucose as compared with 5.5 mM glucose. Factors such as 3-isobutyl-1-methylxanthine (IBMX), isoproterenol, and forskolin, which are known to raise intracellular cAMP levels, all caused a reduction in glucagon receptor mRNA expression. IBMX alone, IBMX together with isoproterenol, and forskolin reduced glucagon receptor mRNA expression to approximately 25, 10, and 50%, respectively. Glucagon was found to dose dependently decrease glucagon receptor mRNA expression in the hepatocytes with an approximately 70% reduction in response to 100 nM glucagon. Finally, we observed a marked reduction in the number of glucagon binding sites (35% of control) after hepatocytes were cultured with the combination of IBMX and isoproterenol. These results indicate that hepatic glucagon receptor mRNA levels can be regulated by glucose and intracellular cAMP and that this is also reflected at the protein level. Furthermore, the observed effects of cAMP and glucagon suggest that this may be a means by which glucagon can down-regulate its own receptor expression. PMID- 7541050 TI - The G protein gamma subunit. Requirements for dimerization with beta subunits. AB - Guanine nucleotide-binding protein beta and gamma subunits form a tightly bound complex that can only be separated by denaturation. Assembly of beta and gamma subunits is a complicated process. The beta 1 and gamma 2 subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but beta 1 cannot form beta gamma dimers when synthesized in a wheat germ extract. In contrast, gamma 2 translated in either system can dimerize with beta 1, suggesting that dimerization-competent gamma 2 can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent gamma 2 in solution forms an asymmetric particle with a Stokes radius of about 21 +/- 0.4 A (n = 4), s20,w of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration). To define the part of gamma 2 that is needed for native beta gamma dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with beta 1. Dimerization was assessed by stabilization of beta 1 to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with beta 1, whereas removal of 27 amino acids prevented it. Therefore, a region between residues 45 and 59 of gamma 2 is important for dimerization. Truncation of 15 amino acids from the N terminus greatly diminished the formation of beta gamma dimers, while removal of 25 amino acids entirely blocked it. Thus, another region important for forming native beta gamma is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent beta gamma dimerization. Furthermore, in intact 35S-labeled COS cells, epitope-tagged gamma 2 coimmunoprecipitates with beta and alpha subunits. The N terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody. PMID- 7541049 TI - Identification of heterogeneous ribonucleoprotein A1 as a novel substrate for protein kinase C zeta. AB - The zeta isoform of protein kinase C (zeta PKC) has been shown to be an important step in mitogenic signal transduction. Using a yeast interaction screen to search for potential novel substrates of zeta PKC, we identified the heterogeneous ribonucleoprotein A1 (hnRNPA1). This protein specifically interacts with the catalytic domain of zeta PKC but not with its regulatory region or with the full length protein, or with a kinase-defective mutant of the zeta PKC catalytic domain. In addition, no interaction was detected with other kinases such as Raf-1 or Mos, that, like zeta PKC, are critically involved in signal transduction, or with the catalytic domain of epsilon PKC, which is the PKC isotype with the highest homology to zeta PKC. hnRNPA1 is directly phosphorylated by both recombinant and native zeta PKC, and this phosphorylation is increased when zeta PKC is immunoprecipitated from mitogen-activated fibroblasts. As an additional control, hnRNPA1 is not phosphorylated appreciably by catalytic epsilon PKC or by a mixture of highly purified classical PKC isotypes maximally activated by phosphatidylserine and Ca2+. Treatment of quiescent cell cultures with a potent mitogen such as platelet-derived growth factor promotes a significant phosphorylation of hnRNPA1 in vivo that is impaired by expression of a dominant negative mutant of zeta PKC. Furthermore, expression of a catalytically active zeta PKC mutant phosphorylates hnRNPA1 in vivo. These findings suggest that zeta PKC could be critically involved in a novel pathway that connects membrane signaling to nuclear regulatory events, at the level of RNA transport and processing. Results also shown here by using different zeta PKC mutants suggesting the control of the cytoplasmic localization of hnRNPA1 by zeta PKC. Also of potential functional relevance are the results demonstrating that the phosphorylation by zeta PKC severely impairs both hnRNPA1 RNA binding and its ability to promote strand annealing in vitro. PMID- 7541051 TI - Randomized study of chemoembolization as an adjuvant therapy for primary liver carcinoma after hepatectomy. AB - From April 1990 to December 1993, 140 patients were recruited to a randomized study to evaluate transcatheter hepatic arterial chemoembolization (TACE) as an adjuvant therapy for primary liver carcinoma after hepatectomy. This study investigated the principle, techniques and results of TACE. The results showed that the intrahepatic recurrence rate was 48.9% in the patients who underwent radical resection only, but only 21.3% in the patients who also underwent TACE 3 4 weeks after hepatectomy (P < 0.01). The 1-, 2-, 3-, and 4-year survival rates were 72.3%, 52.7%, 35.1%, and 35.1% respectively for the patients who underwent radical resection only, and were 97.9%, 85.5%, 69.5%, and 56.9% for the patients who also underwent TACE 3-4 weeks after radical resection (P < 0.001). The 1-, 2 , 3-, and 4-year survival rates were 38.9%, 0%, 0%, and 0% for the patients who underwent palliative resection only, and were 68.3%, 32.3%, 21.5%, and 21.5% respectively for the patients undergoing TACE 3-4 weeks after palliative hepatectomy (P < 0.001). PMID- 7541052 TI - Interleukin-6 production by U373 MG, a human astrocytoma cell line: different pathways involved in substance P and lipopolysaccharide activation. AB - Substance P (SP) and lipopolysaccharide (LPS) stimulated interleukin-6 (IL-6) gene expression, as well as IL-6 protein secretion in the human astrocytoma cell line U373 MG. Staurosporine, an inhibitor of protein kinase C (PKC), entirely blocked SP- but not LPS-induced IL-6 release. In addition, the down regulation of PKC inhibited the SP response and only marginally altered LPS activation. Differently from SP, LPS-induced IL-6 release was markedly reduced by W7, a calmodulin antagonist. Moreover, SP interacted in a synergistic manner with LPS. Thus, neural (SP) and bacterial (LPS) mediators stimulate U373 MG IL-6 release via distinct, though not antagonistic, activation pathways. PMID- 7541053 TI - In vivo clonal expression of T lymphocytes specific for an immunodominant N terminal myelin basic protein epitope in healthy individuals. AB - The role of myelin basic protein (MBP) T cell recognition in the induction of experimental allergic encephalomyelitis (EAE) has been well established in mice and rats. A remarkable restriction has been observed in T cell receptor (TCR) genes utilized by encephalitogenic T cell lines (TCLs) specific for immunodominant epitopes in these species. Pathological similarities between this animal model and multiple sclerosis (MS) has led to consider MBP as a major candidate autoantigen in this human disorder. Unlike in inbred strains of animals, the T cell response to MBP in humans is quite heterogenous with regard to fine epitope specificity. The existence of V alpha and/or V beta restriction in MBP-specific T cells, from MS patients and healthy controls, is still a matter of debate. In this study we generated 77 MBP-specific TCLs from nine healthy donors and showed that peptide 7-27 is one of the most frequently recognized epitopes. 37% of all epitope-specific TCLs recognized this peptide and p7-27 specific TCLs were generated from seven out of the nine subjects studied. A high level of in vivo clonal expansion was observed in p7-27-specific TCLs in several subjects, which however is not specific of this epitope since this phenomenon was also observed in p85-104- and 149-162-specific TCLs. PMID- 7541054 TI - Cloning of myelin basic protein-reactive T cells from the experimental allergic encephalomyelitis-resistant rat strain, LER. AB - Rats of the LER inbred strain are resistant to the active induction of experimental allergic encephalomyelitis (EAE), although they are susceptible to adoptively transferred EAE when they are injected with encephalitogenic T cells from EAE-susceptible Lewis rats. The mechanism of resistance remains to be elucidated. We report here that myelin basic protein (MBP)-specific T cells can be cloned from LER rats immunized with MBP, that these CD4+ LER T cells can recognize the encephalitogenic peptide (MBP-EP) and will divide vigorously when it is presented to them, and that these T cells bear V beta 8 + TCR chains. Nevertheless, in contrast to Lewis T cells with the same specificity and TCR beta chains, LER T cells from MBP-EP-specific clones cannot induce EAE when adoptively transferred into naive rats of either strain. Thus, LER T cells can assemble and use a TCR with the canonical encephalitogenic V beta 8.2-D beta-J beta region in response to immunization with MBP, yet they continue to display resistance to EAE. PMID- 7541055 TI - Expression of cell adhesion molecules in inflammatory myopathies. AB - We examined the expression of cell adhesion molecules in 25 cases of inflammatory myopathies. Inflammatory myopathies showed upregulation of adhesion molecules. ICAM-1 was strongly expressed on endothelial cells as well as on fibroblasts and infiltrating leukocytes while the expression of VCAM-1, similar in its distribution, was much weaker. A few muscle fibers in polymyositis revealed sarcolemmal labeling for ICAM-1. ELAM-1 showed only weak expression on vessels. The inflammatory cellular infiltrates contained varying amounts of cells bearing the VCAM-1 ligand VLA-4 and the ELAM-1 ligand SLeX as well as large amounts of cells expressing LFA-1 alpha and beta, ligands of ICAM-1. PMID- 7541056 TI - Junctional region of the myelin basic protein-specific T cell receptor beta chain in mice. AB - Evidence for the existence of both conserved and diverse amino acid sequences in the junctional regions of the myelin basic protein (MBP)-specific T cell receptors (TCR) in mice is presented. The junctional region of the Nac1-11 MBP peptide-specific, H-2u-restricted TCR beta-chains is characterized by the utilization of similar amino acid sequences. In contrast, diverse junctional sequences within the TCR beta-chains of the p89-101 MBP peptide and H-2s restricted T cell clones are reported. These findings demonstrate that a limited heterogeneity of the MBP-specific T cell clones does exist. However, it may not be universal even in inbred mouse strains. PMID- 7541057 TI - Increased levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in the cerebrospinal fluid and sera of patients with multiple sclerosis and human T lymphotropic virus type-1-associated myelopathy. AB - We evaluated the relationship between the soluble form of vascular cell adhesion molecule-1 (sVCAM-1) and disease activity in patients with multiple sclerosis (MS) or with human T lymphotropic virus type 1-associated myelopathy (HAM), and measured levels of sVCAM-1 in their cerebrospinal fluid (CSF) and sera. Serum and CSF levels of sVCAM-1 were significantly increased in patients with acute relapsing MS during an exacerbation (P < 0.01 and P < 0.001), as well as in chronic progressive MS (P < 0.05 and P < 0.001), compared with healthy individuals and patients with other neurological diseases, respectively. Patients with acute relapsing MS during an exacerbation also exhibited significantly higher serum and CSF levels of sVCAM-1 vs. patients with acute relapsing MS in remission (P < 0.001). Significantly higher serum levels of sVCAM-1 were observed in patients with HAM vs. either healthy individuals (P < 0.01) or non-HAM carriers (P < 0.01). These results suggest that the determination of sVCAM-1 in the sera and CSF may be useful in monitoring the activity of MS and HAM. PMID- 7541059 TI - Natural and recombinant enzymatically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with Hymenoptera allergy. AB - BACKGROUND: A complementary DNA encoding the major bee venom allergen phospholipase A2 (PLA) has been characterized recently. Recombinant PLA was produced in Escherichia coli and purified to apparent homogeneity. Natural PLA was compared with recombinant PLA in its ability to release histamine from blood basophils. METHODS: A synthetic gene encoding the mature form of PLA was expressed in E. coli, and the polypeptide was purified to homogeneity by affinity chromatography and refolded, yielding fully enzymatically active PLA. In addition, we have produced a genetically engineered enzymatically inactive variant by substitution of a single amino acid residue in the catalytic center. A standard histamine release assay was used to compare the potency of natural PLA with correctly folded enzymatically active and inactive recombinant PLA to release histamine from blood basophils of nine patients with bee venom allergy. RESULTS: Recombinant enzymatically active PLA and purified natural protein were equally effective in releasing histamine from sensitized basophils. By comparing the histamine-releasing capacity of enzymatically active and inactive recombinant allergen, we further demonstrate that catalytic activity is not a requirement for allergenicity in the effector phase. Denaturation of natural PLA or incorrect folding of recombinant protein resulted in a total loss of allergenic potency. CONCLUSION: We demonstrate the feasibility of producing native-like recombinant allergens with or without enzymatic activity. We also provide evidence for the requirement of correct three-dimensional structure of PLA to induce histamine release from basophils and thus evidence for its recognition by IgE. PMID- 7541058 TI - Abnormalities in intramyocardial arteries detected in cardiac transplant biopsy specimens and lack of correlation with abnormal intracoronary ultrasound or endothelial dysfunction in large epicardial coronary arteries. AB - OBJECTIVES: We sought to determine whether abnormalities in small intramyocardial vessels could be detected on routine cardiac transplant biopsy specimens and whether these features correlate with intimal thickening by intracoronary ultrasound and endothelial dysfunction in large epicardial vessels. BACKGROUND: Variability in clinical presentation of allograft vasculopathy suggests differential involvement of large and small vessels. Intracoronary ultrasound and endothelial function studies detect large-vessel abnormalities but may not reflect changes in small intramyocardial arteries. The latter could be detected in routine cardiac biopsy specimens by histologic and immunohistochemical studies. METHODS: Thirty-nine cardiac transplant recipients underwent intracoronary ultrasound and acetylcholine studies 5 to 7 days after endomyocardial biopsy. Biopsy tissue was evaluated for coronary artery endothelial plumping and intimal thickening and increased immunostaining for fibronectin, tumor necrosis factor-alpha and receptor for hyaluronan-mediated motility. Large-vessel disease was assessed by calculating an average intimal index from intracoronary ultrasound of the left anterior descending coronary artery. Endothelial function was determined by quantitative coronary analysis after acetylcholine challenge. RESULTS: Coronary arteries were found in the biopsy tissue of 30 (76%) of the 39 patients who formed the study group. Fourteen of 30 patients had abnormal histologic findings. Immunohistochemical analysis for fibronectin, possible in 20 of 30 patients, was positive in 14 (70%) of 20 and correlated with abnormal histologic findings (p = 0.01). Immunostaining was positive for tumor necrosis factor-alpha and receptor for hyaluronan-mediated motility in 12 (40%) and 13 (43%) of 30 patients, respectively. All patients had intimal thickening by intracoronary ultrasound, but intimal index did not correlate significantly with small-artery disease by histologic or immunohistochemical analysis. Large-vessel endothelial dysfunction in 13 patients (43%) did not correlate with either abnormal ultrasound findings or small-vessel disease. CONCLUSIONS: Intramyocardial arteries are readily observed in biopsy specimens from cardiac transplant recipients and provide useful information about allograft vasculopathy. Lack of correlation between intramyocardial and epicardial vessel disease suggests discordant progression of allograft vasculopathy. PMID- 7541060 TI - Reduction of FK-506 requirements by combination with polyethylene glycol superoxide dismutase in orthotopic rat liver transplantation. AB - Reperfusion after ischemia results in endothelial cell injury and Kupffer cell activation. Inflammatory cytokines thus released can induce major histocompatibility complex antigens and increase the immunogenecity of the graft. An orthotopic rat liver allotransplant model was used to test the hypothesis that prevention of reperfusion injury by infusion of polyethylene glycol superoxide dismutase (PEG-SOD) would result in long-term allograft survival in the presence of subthreshold immunosuppressive dosages. ACI rats were used as donors, and Lewis strain rats as recipients. Orthotopic liver transplantation was initially performed to identify a subthreshold dose of the immunosuppressant FK-506, which would be unable to extend survival longer than control untreated rats with this strain combination. After testing three intramuscular FK-506 doses of 0.04, 0.08, and 0.16 mg/kg, it was observed that an FK-506 dose of 0.04 mg/kg/day for 14 days was unable to extend survival longer than in untreated recipients. This dose of FK-506 was used in combination with PEG-SOD at doses of 1000, 3000, 10,000, or 30,000 units. Recipient animals were treated intravenously with PEG-SOD as a loading dose to facilitate tissue penetration on day 1, and beginning on the day of transplantation, every 2 days for the duration of the study. Results of histologic studies and mean survival time were compared in untreated recipients and in rats treated with PEG-SOD plus 0.04 mg/kg/day FK-506.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541061 TI - Increased adherence of Escherichia coli RDEC-1 to human tissue culture cells results in the activation of host signaling pathways. AB - Attaching and effacing (AE) adhesion is associated with the pathogenesis of enteropathogenic Escherichia coli (EPEC) and rabbit diarrheogenic E. coli (RDEC 1). Although RDEC-1 provides an animal model for investigating pathophysiology of EPEC infection, RDEC-1 does not adhere to human cell lines, thereby limiting in vitro investigation. Therefore, transformed RDEC-1 strains expressing adhesins derived from human diarrheogenic E. coli were studied. These adhesins promoted AE adhesion of RDEC-1 and led to the accumulation of alpha-actinin aggregates in the cytoplasm of infected cells. Furthermore, these strains induced host signal transduction pathways, resulting in tyrosine phosphorylation of host proteins and an intracellular elevation of calcium. These results demonstrate that RDEC-1 and EPEC stimulate similar signal transduction pathways in infected epithelial cells, thus lending additional support for the use of RDEC-1 as a model for the study of human EPEC infection. PMID- 7541063 TI - Insertional sequence primers for Bordetella pertussis diagnostic polymerase chain reaction differentiate strains of Pseudomonas cepacia. AB - Insertion sequence primers originally intended to amplify a singular specific product for the rapid diagnosis of Bordetella pertussis respiratory infection were used to differentiate strains of Pseudomonas (Burkholderia) cepacia. A modified sample preparation of proteinase K treatment and boiling was used in lieu of DNA extraction. The method was simple, rapid, and reproducible. This scheme identified 10 variations among 35 strains. Repeat strains from patients with cystic fibrosis and epidemiologically linked strains from an infection associated with a jet gun injection device were homologous in each setting. PMID- 7541062 TI - Nitric oxide enhances resistance of SCID mice to mucosal candidiasis. AB - The capacity of macrophages from SCID and C.B-17 mice to kill Candida albicans via a nitric oxide (NO)-dependent pathway and the contribution of NO in resistance to mucosal candidiasis were assessed. In vitro, an inhibitor of NO synthase (NOS) reduced the candidacidal activity and nitrite-producing capacity of activated resident peritoneal macrophages from immunocompetent C.B-17 and immunodeficient SCID mice. In vivo, stomachs from gnotobiotic SCID mice that were colonized with a pure culture of C. albicans had low-grade infections and expressed inducible NOS (iNOS) mRNA. C. albicans-monoassociated SCID mice treated with an inhibitor of NOS had more severe orogastric candidiasis than controls. These data suggest that NO contributes to the candidacidal capacity of activated macrophages from C.B-17 and SCID mice and that NO synthesized by iNOS may contribute to the resistance of SCID mice to mucosal candidiasis. PMID- 7541064 TI - Drug resistance and heterogeneous long-term virologic responses of human immunodeficiency virus type 1-infected subjects to zidovudine and didanosine combination therapy. The AIDS Clinical Trials Group 143 Virology Team. AB - Plasma human immunodeficiency virus (HIV) type 1 RNA levels, CD4 lymphocyte changes, and drug resistance were studied in HIV-infected patients with 200-500 CD4 lymphocytes/microL who received zidovudine and didanosine combination therapy for 2 years. Among 35 patients, 10 had sustained and 16 had transient > 10-fold reductions in HIV RNA: 9 did not have 10-fold HIV RNA reductions. Only patients with sustained HIV suppression maintained increased CD4 cell counts for 2 years (370 to 501 cells/microL; P = .006). Patients with transient HIV suppression were more likely to develop drug-resistant HIV strains (12/16 vs. 5/19, P = .01) and reverse transcriptase (RT) mutations (4.5 vs. 2.5/strain; P = .02) than were patients with sustained or no HIV suppression. Zidovudine resistance occurred with RT mutations at codons 41, 67, 70, 215, and 219. Multidrug resistance occurred with mutations at codons 62, 75, 77, 116, and 151. Mutations occurred at codons 60, 68, 118, 210, and 228 in > or = 4 patients each. Heterogeneity exists among individual virologic responses to zidovudine and didanosine combination therapy. HIV resistance mechanisms during combination therapy appear more complex than reported with monotherapy. PMID- 7541066 TI - Peak nasal inspiratory flow--the plateau effect. AB - To study the efficacy of peak nasal inspiration flow (PNIF) as a means of recording change in nasal patency 20 subjects were given increasing doses of intranasal xylometazoline. Nasal resistance (NR) and peak nasal inspiratory flow (PNIF) were measured in the resting state and after each xylometazoline administration. Successive increases in dose caused a progressive decrease in nasal resistance and an increase in PNIF but the change in peak nasal inspiratory flow (PNIF) was much less. Peak nasal inspiratory flow shows a plateau effect as nasal resistance decreases. The reasons for this plateau are discussed in terms of respiratory flow mechanics. PMID- 7541067 TI - Proteins in tissue extracts which bind insulin-like growth factor binding protein 3 (IGFBP-3). AB - Membrane associated IGFBP-3 is now known to play a role in the modulation of IGF at the cellular level, but mechanisms involved in cell membrane binding are far from certain. In this study we report the identification and initial structural characterisation of proteins in a range of sheep and rat tissues which specifically bind recombinant human non-glycosylated IGFBP-3. Tissues were homogenised in Tris HCl (0.1 M, pH 7.4), containing proteolytic enzyme inhibitors and the residues re-extracted in buffer containing SDS and Triton X-100 (both 1% w/v) prior to analysis. These were subjected to SDS-PAGE, electro-blotted onto nitrocellulose and subjected to ligand blot analysis (LBA) using radioiodinated IGFBP-3 as ligand. LBA revealed a major band of binding activity migrating at 60 kDa in extracts of rat muscle while sheep muscle contained forms of 52 and 40 kDa as the principal species and ovine pancreatic extracts an abundance of the 40 kDa variant alone. Distribution of the binding activity appears tissue specific. Apart from skeletal muscle, pancreas and a small amount of the 52 kDa form in pituitary, analysis revealed no evidence of IGFBP-3 binding in a range of other sheep tissues including liver, kidney, spleen, intestine, adrenal, brain, mammary, uterus, ovary and plasma. The binding activity is TCA precipitable, trypsin digestible, dose responsive and relatively specific for IGFBP-3 since the related proteins recombinant human IGFBP-2 and purified caprine IGFBP-4 failed to bind.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541065 TI - Ex vivo expansion of CD4 lymphocytes from human immunodeficiency virus type 1 infected persons in the presence of combination antiretroviral agents. AB - Expansion of CD4 lymphocytes from human immunodeficiency virus type 1 (HIV-1) infected persons ex vivo has been limited by enhanced virus replication and cell death. The successful expansion of functional CD4 lymphocytes from HIV-1-infected persons has now been accomplished using a bispecific monoclonal antibody to CD3 and CD8 in combination with three antiretroviral agents. CD4 lymphocytes were polyclonally expanded by a factor of 10(3)-10(7) during 4-8 weeks in culture. Supernatants from most cultures were persistently HIV-1 p24 antigen-negative by day 14 and remained negative despite removal of antiretroviral agents at day 28. In such cultures, HIV-1 could not be recovered by cocultivation, and amounts of HIV-1 DNA declined or remained stable at low levels, eventually becoming undetectable in 2 cases. This approach establishes the feasibility of CD4 lymphocyte expansion in persons with HIV disease and may be useful for immune based or gene therapies. PMID- 7541068 TI - Effect of sodium selenite on growth, insulin-like growth factor-binding proteins and insulin-like growth factor-I in rats. AB - Selenium is an essential trace element although at higher doses it is also known to be a toxic agent causing a wide range of symptoms including growth retardation. In order to investigate the effect of sodium selenite on growth, insulin-like growth factor-binding proteins (IGFBPs) and insulin-like growth factor-I (IGF-I), 30 male Wistar rats were randomized into three groups. Group A was treated with sodium selenite in the drinking water (3.3 mg selenium/l). Group B was ad libitum fed with free access to standard fodder and tap water and group C was pair fed relative to the selenium-treated rats. Serum IGF-I and IGFBPs were determined on days 0, 14 and at the end of the study on day 35. Selenium-treated rats had significantly lower body weights compared with group B rats on day 9 and group C rats on day 14 (P < 0.05). Tibia length was measured at the end of the study and no difference was observed between groups B and C (3.77 +/- 0.04 cm vs 3.60 +/- 0.02 cm); however, selenium-treated rats had significantly shorter tibia lengths (3.46 +/- 0.03 cm) compared with rats in groups B (P < 0.001) and C (P < 0.05). Selenium treatment induced a significant reduction in circulating IGF-I by the end of the study compared with ad libitum and pair fed rats (P < 0.05). Serum subjected to Western ligand blots showed four distinct IGFBP bands with apparent relative molecular weights of 38-47 kDa (doublet) (IGFBP-3), 30 kDa (IGFBP-1 and/or IGFBP-2) and 24 kDa (IGFBP-4).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541069 TI - Colocalization of neuronal nitric oxide synthase with GABA in rat cuneate nucleus. AB - Pre- and post-embedding immunocytochemistry were employed in this electron microscopic investigation of cuneate neurons that are enriched in GABA and in nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide. GABAergic neurons are local circuit interneurons; 10-20% of them also contain nitric oxide synthase. These are among the smallest GABA-positive perikarya. We describe a network of processes in the rat cuneate nucleus that are immunopositive for nitric oxide synthase. Axon terminals positive for nitric oxide synthase are small and make synapses mainly onto dendrites; they make only occasional axo-axonic contacts. Double-labelling immunocytochemistry verified that the large majority of terminals positive for nitric oxide synthase also contained GABA. However, most GABA-positive profiles were negative for nitric oxide synthase and GABA-positive terminals that are negative for nitric oxide synthase frequently made axo-axonic contacts. These results suggest that nitric oxide synthase is within a specialized subpopulation of interneurons in the cuneate nucleus. PMID- 7541070 TI - Interferon for hepatitis? PMID- 7541071 TI - AIDS palliative care--challenging the palliative paradigm. PMID- 7541072 TI - Sharing the challenge: the integration of cancer and AIDS. PMID- 7541073 TI - Dancing across the lines: people in pain. PMID- 7541075 TI - Palliative care in the home? AB - Finally, in conclusion, I return to the question: Palliative care at home? Indeed, it is an option, one that more and more PLWH/As are choosing. And to some extent, the choice to die at home is a testimony to the significant progress we have made in our ability and commitment to provide palliative care at home. The challenge before us is to find solutions to the problem when the option to die at home, based on existing services, is not viable. As is the case with every aspect of care along the HIV continuum, the difficulties we encounter in AIDS palliative care should be viewed as opportunities to expand our understanding of the HIV illness experience; the meaning of quality of life from the consumer's perspective and how it can be achieved and maintained; and how we as health professionals can work together in teams that honor and support the role of families, social networks, and community-based AIDS service organizations involved in AIDS palliative care. Despite the trend to shift part of our focus from institutional to community settings of care, we must never lose sight of the fact that when we enter the homes of our clients, regardless of our role, we are guests. And it is in that role that we must, when necessary, put aside our loyalty to traditional approaches to care and open our hearts and minds to the direction and needs of those who choose to die at home with AIDS. PMID- 7541076 TI - Palliative care for women with AIDS: identifying the issues. PMID- 7541074 TI - Dementia in HIV disease--a challenge for palliative care? PMID- 7541077 TI - Palliative care for HIV disease. PMID- 7541079 TI - Working on the edge: palliative care for substance users with AIDS. PMID- 7541078 TI - AIDS and palliative medicine: medical treatment issues. AB - Caring for patients with AIDS in hospice programs presents numerous challenges. These go beyond the strictly medical decisions discussed in this article to include unique problems with social support, emotional support, and bereavement. This discussion centers on medical issues as they are commonly encountered. They can be stumbling blocks, or even surrogates, for addressing the other important issues at the end of life. Communication between all people involved in the multidisciplinary treatment of these patients is essential. Common goals should be identified and general approaches agreed upon among the physicians, nurses (clinic, hospice, home care), therapists, clergy, and volunteers. In this way we feel that the principles of hospice and palliative care can be effectively applied to most patients with endstage AIDS. PMID- 7541080 TI - Palliative care for HIV disease: the desirable vs the possible. PMID- 7541082 TI - Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells. AB - To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-) freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes. PMID- 7541081 TI - The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers. AB - We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37 degrees C either with HEPES-buffered solutions containing 137 mM Cl or bicarbonate/CO2-buffered solutions containing 127 mM Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Cl-insensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERF-dextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mM (HEPES buffer) or between 0 and 127 mM (bicarbonate/CO2 buffer). LIS Cl in HEPES-buffered solutions averaged 176 +/- 19 mM (n = 12), calibrated with digitonin, and 170 +/- 9 mM (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO2-buffered solutions averaged 174 +/- 10 mM (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Cl-sensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 +/- 0.1 min (n = 21) for Cl decrease and 0.3 +/- 0.04 min (n = 21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high. PMID- 7541083 TI - Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC. AB - Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative beta strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel(s). This raises the prospect of an auto directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor. PMID- 7541084 TI - Ion selectivity of colicin E1: III. Anion permeability. AB - The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 A. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N',N'-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 M NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions. PMID- 7541085 TI - Calcium entry in rabbit corneal epithelial cells: evidence for a nonvoltage dependent pathway. AB - We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca++ channels, Na+/Ca++ exchange, and nonvoltage dependent Ca(++)-permeable channels. Whole cell inward currents carrying either Ca++ or Ba++ were not detected using voltage clamp techniques. We also used imaging technology and the Ca(++)-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca++ concentration ([Ca]i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 microM), or Ni++ (40 microM), a blocker of many voltage-dependent calcium channels, did not affect [Ca++]i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca++]i. These results are inconsistent with the presence of voltage gated Ca++ channels. Nonvoltage gated Ca++ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K+ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn++ and bath perfusion with 5 mM Ni++ decreased [Ca++]i suggesting that the Ca++ conductance was blocked. These results are most consistent with a nonvoltage gated Ca++ influx pathway. Finally, replacing extracellular Na+ with Li+ resulted in an increase in [Ca++]i if the cells were first Na(+)-loaded using the Na+ ionophore monensin and ouabain, a Na(+)-K(+) ATPase inhibitor. These results suggest that Na+/Ca++ exchange may also regulate [Ca++]i in this cell type. PMID- 7541086 TI - Jaw muscles of New World squirrels. AB - The jaw, suprahyoid, and extrinsic tongue muscles are described for eight species of New World squirrels, spanning more than an order of magnitude in body mass. Anatomical differences are discussed in the light of body size, natural history, and phylogeny. The relative sizes of different muscles, their orientations, and the shapes and positions of their areas of attachment vary but show few trends in relation to body size. The anatomical differences are likewise not readily explained by the mechanical requirements of the animals' diets, which are similar. The most marked anatomical differences occur in Sciurillus (the pygmy tree squirrel), as well as those genera--Glaucomys (the flying squirrel) and Tamias (the chipmunk)--that are taxonomically most distinct from the tree squirrels. Sciurillus is noteworthy for its unusually small temporalis and an anterior deep masseter that is oriented to assist in retraction of the jaw. Tamias has a more vertically oriented temporalis and greater inclination in the anterior masseter muscles than the other squirrels, features that may be associated with its large diastema and relatively posteriorly situated cheek teeth, which in turn may relate to its having cheek pouches. Our results form a valuable database of information to be used in further studies of functional morphology and phylogeny. PMID- 7541088 TI - [Studies on the mechanisms of action of fibroblast growth factors in stromal cells in hyperplastic human prostate]. AB - Stromal-epithelial cell interaction is very important for the development of human benign prostatic hyperplasia (BPH). Growth factors and their receptors in the prostate are thought to mediate the cell communication and play some roles in the development of BPH. Among many growth factors, fibroblast growth factor (FGF) family members have received the most intensive study, and mRNAs for acidic FGF (aFGF), basic FGF (bFGF) and keratinocyte growth factor (KGF) have been identified in rat or human prostate. However, synthesis sites and roles of them in stromal-epithelial cell interaction remain to be undefined. In the present study, to define the mechanisms for the regulation of prostatic cell growth by stromal cells in human BPH, we established the method for isolation and culture of epithelial cells as well as stromal cells from human BPH tissue. Using these primary cultured prostatic cells, we evaluated the effects of stromal cell conditioned medium (SCM) and stromal cell extract (SCE) on the growth of stromal cells and epithelial cells by [3H]-thymidine incorporation assay. We also examined the expression of mRNAs for aFGF, bFGF, KGF, FGF receptor 1 (FGFR1) and FGFR2 in epithelial and stromal cells using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis. As the results, both SCM and SCE stimulated the growth of stromal cells, and the growth promoting effects of them to stromal cells were completely suppressed by anti-bFGF neutralizing antibody, but not by anti-aFGF neutralizing antibody at all. SCM also stimulated the growth of epithelial cells. The growth promoting effect of it to epithelial cells was not completely suppressed by anti-bFGF neutralizing antibody, and not by anti-aFGF neutralizing antibody at all. Furthermore, RT-PCR analysis demonstrated the expression of mRNAs for bFGF, KGF and FGFR1 in stromal cells and that of FGFR2 in epithelial cells. These findings suggest that bFGF produced by stromal cells acts on stromal cells through FGFR1 by an autocrine mechanism, KGF produced by stromal cells acts on epithelial cells through FGFR2 by a paracrine manner in human BPH. These mechanisms for the regulation of cell growth by stromal cells were thought to contribute to the development of human BPH. PMID- 7541089 TI - [The preventive effect of indeloxazine hydrochloride to the sexual dysfunction caused by anti-androgenergic agent (allylestrenol)]. AB - Anti-androgenergic agents are usually used for patients with benign prostatic hypertrophy (BPH). However steroidal anti-androgenergic agents tend to suppress the sexual function. This side effect is very significant in middle-aged men. Therefore we studied the preventive effect of indeloxazine hydrochloride (INDX), which induces an increase of the dopamine level in the brain, on the sexual dysfunction induced by an anti-androgenergic agent (allylestrenol: ALE). Thirty six patients with BPH were classified into two groups, one used ALE only, and the other ALE with INDX. For the subjective evaluation of the sexual function, a self assessment questionnaire method was employed before and after administration. We especially studied 3 questions, "morning erection", "erectile capacity" and "frequency of sex". For the objective evaluation of the sexual function, nocturnal penile tumescence (NPT) was measured using an erectometer. NPT occurs in healthy males as a physiological phenomenon and it shows the erectile capacity objectively. The levels of LH, total testosterone and free testosterone were also determined. In the ALE only group, sexual dysfunction was found subjectively and objectively, but in the ALE with INDX group, it was not found. Levels of LH, total testosterone and free testosterone were decreased in the both groups. There was no significant difference between the two groups. We hypothesized that the sexual dysfunction due to ALE is related with not only to the decrease of androgen, but also to suppression of the central nervous system; for example, the suppression of the area of the brain mediating sexual behavior.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541087 TI - Restricted pulmonary diffusion capacity after exercise is not an ARDS-like injury. AB - Pulmonary diffusion capacity (DLCO) is reduced 2 h after various types of exercise, such as rowing, treadmill running, arm cranking and marathon running. The decrease in DLCO may involve alterations in the alveolar-capillary membrane as well as depletion of the central blood volume. We hypothesized that the reduction in DLCO might also be influenced by oxygen free radicals, acute phase proteins and endotoxin, which are also involved in the adult respiratory distress syndrome (ARDS). Ten competitive male oarsmen performed a 6 min 'all-out' ergometer row. Single breath DLCO was determined before and 2 h after rowing and venous blood samples were also obtained during the row. Absolute DLCO decreased by 11% (range 0-20%) 2 h after rowing, whereas the concentration of endotoxin did not change significantly and interleukin (IL)-1-alpha, IL-8 and tumour necrosis factor (TNF)-alpha were below the levels of detection before, during and 2 h after rowing. Oxygen free radicals were evaluated by oxidative modification of amino acids and DNA. Corrected for creatinine in urine voided 3 h post-exercise, the DNA repair product 8-oxo-7,8-dehydro-2-deoxyguanosine (8-oxodG) did not change significantly. The ratio of fluorescence due to dityrosine to that due to tryptophan in plasma proteins increased after exercise. This might reflect an effect of oxygen free radicals, but it might also indicate an altered relative composition of plasma proteins. These results suggest that the reduced pulmonary diffusion capacity following exercise is unrelated to factors typically associated with ARDS. PMID- 7541091 TI - [Molecular aspects of urological cancers]. PMID- 7541090 TI - [A case of recto-urethral fistula following transrectal hyperthermia treated by anterior anorectotomy approach]. AB - We report a case of 73 years old male with recto-urethral fistula developing after transrectal hyperthermia for prostatic hypertrophy. This is the first case report of recto-urethral fistula probably caused by transrectal hyperthermia. Anterior anorectotomy approach for fistula closure provided a wide operative field and lead to a successful result. PMID- 7541092 TI - Galanin causes presynaptic inhibition of cholinergic transmission in rabbit parasympathetic ganglia. PMID- 7541094 TI - Poster art as women's rhetoric: raising awareness about breast cancer. PMID- 7541093 TI - Effects of CP-99,994, an NK1-receptor antagonist, on the inward current produced by substance P. PMID- 7541095 TI - Multidrug resistant cells with high proliferative capacity determine response to therapy in acute myeloid leukemia. AB - High spontaneous proliferation of acute myeloid leukemia (AML) in vitro is an unfavorable, tumor-specific prognostic factor. We investigated the frequency of drug-resistant tumor cells with high proliferating capacity in de novo AML and analyzed the expression of multiple resistance parameters in relation to the response to chemotherapy and overall survival. Thirty-eight patients were included in this study. P-glycoprotein (P-gp) expression was found in 28/38 patients and was associated with lower intracellular accumulation of DNR (P = 0.0001). Thirty-five out of 38 patients were treated with 1-2 regimens of daunorubicin (DNR)/cytarabine (Ara-C), and 57% attained a complete remission (CR). Failure to achieve a CR correlated with autonomous growth (P = 0.0064), CD34 and P-gp expression alone (P = 0.0005 and P = 0.048 respectively), and with simultaneous expression of P-gp and CD34 (P = 0.0001), but not with expression of the non-P-gp drug resistance associated-protein (p110), the multidrug resistance associated protein (MRP), Ara-CTP formation or Ara-C incorporation, respectively. AML cells with CD34/P-gp double expression were more frequently observed in samples with high autonomous growth (P = 0.003). The median survival was 6 months in CD34+/P-gp+ patients as compared with 15 months in other AML patients (P = 0.003). In patients with de novo AML who fail on chemotherapy, a population of autonomously proliferating, immature AML cells with a multidrug resistant phenotype can be recognized. These cells thus show primary resistance to chemotherapy and have the potential for rapid regrowth, leading to resistant disease. PMID- 7541097 TI - Synergistic action of interleukin-2 and Steel factor (SLF) on a human T lymphoblastoid cell line. AB - Ten cell lines recently established from paediatric patients with acute lymphoblastic leukaemia (ALL) were examined for expression of P145c-kit, the growth factor receptor encoded by the c-kit proto-oncogene, by immunofluorescence and flow cytometry using monoclonal antibody YB5.B8. Three of five T-ALL cell lines, but none of five B lineage ALL cell lines displayed significant binding of the antibody. The cell line with the highest level of binding was PER-423 (Kees et al, Leukemia Res 1993; 17: 51-59 which has the phenotype CD7+, CD56bright, CD2 , CD4-, CD5-, CD8-, CD16-, has rearranged T cell receptor beta-chain genes, expresses cytoplasmic CD3 and is strictly dependent on interleukin 2 (IL-2) for proliferation. Recombinant to act in synergy with IL-2 to promote proliferation of PER-423 cells. In five experiments, SLF increased the maximal amount of proliferation by 105 +/- 15%, and decreased the level of IL-2 required for a half maximal response by 43 +/- 7%. The cells constitutively express the intermediate affinity IL-2 receptor (beta/gamma), but can be induced in the presence of phorbol ester to express the alpha chain (CD25, Tac) which confers high affinity binding of IL-2. In contrast, the alpha chain was not induced by SLF. The enhancement of proliferation of PER-423 cells by SLF could be prevented by inclusion in the assay of a blocking monoclonal antibody to P145c-kit. These experiments demonstrate that SLF/P145c-kit can provide a significant growth stimulus for ALL cells, and PER-423 cells may be a useful system for investigating the mechanism of synergy between SLF and IL-2. PMID- 7541096 TI - De novo induced mutations in the deoxycytidine kinase (dck) gene in rat leukemic clonal cell lines confer resistance to cytarabine (AraC) and 5-aza-2' deoxycytidine (DAC). AB - We have investigated whether cytarabine (AraC) or decitabine (DAC) induce deficiency of deoxycytidine kinase (DCK) through different mutations of the dck gene, related to their distinct interference with DNA replication. Also, it is not known whether mutations of the dck gene are the result of selection of mutants or de novo induction. To address these issues, three subclones of a rat leukemic cell line (RCL/O), sensitive to cytotoxicity mediated by AraC and DAC, were exposed to gradually increasing concentrations (from 0.1 to 10 microM) of either AraC or DAC over a 140 days vs a 180 days period. During the course of resistance induction DCK activity was monitored. We found that all clones acquired irreversible cross-resistance, at marginally cytotoxic AraC or DAC concentrations of 0.1 to 0.4 times the IC50 for the parental clones. Furthermore, all resistant cell lines were DCK deficient and harbored different mutations in the dck gene. AraC induced both rearrangements and point mutations in the dck gene when administered over 140 days and 180 days, respectively. 140 days DAC induction yielded point mutations only. All point mutations detected were nonrandomly distributed within the dck coding region. SSCP analysis showed that in the majority of resistant clones more than one bandshift was present. The data suggest the presence of multiple resistant clones, originating from one sensitive clone and thus arguing against selection of mutants as a mechanism for the development of AraC and DAC resistance. PMID- 7541099 TI - Human tumour necrosis factor-alpha inhibits glucose transport in cultured Ehrlich ascites tumour cells. AB - Human recombinant tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-alpha treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-alpha on the inhibition of glucose transport of tumour cells is discussed. PMID- 7541098 TI - Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat. AB - The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2 5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals. PMID- 7541101 TI - Regional 5-hydroxyindoleacetic acid production in humans. AB - Veno-arterial plasma concentration differences and regional organ plasma flows were used to quantify the relative amounts of 5-hydroxyindoleacetic acid (5-HIAA) contributed by various sites into the peripheral circulation. Positive venoarterial concentration gradients were found in the hepatosplanchnic, forearm, cardiac and jugular vessels in the healthy subjects. The renal circulation was determined to be the principal site of 5-HIAA clearance, extracting 18 +/- 2 nmol/min. The gut was the greatest contributor to the total 5-HIAA plasma pool with the relative contributions of the various organs being as follows: hepatosplanchnic organs 58%, skeletal muscle 26%, brain 6% and the heart 3%. The source of 5-HIAA stemming from these regional beds remains unknown, it may derive from serotonin taken up by and deaminated in ubiquitous endothelial cells, enterochromaffin cells of the gut, peripheral serotonergic nerves, serotonin turnover in platelets or perhaps the metabolism of serotonin taken up by sympathetic nerves. To test the latter hypothesis we examined 23 patients with chronic congestive heart failure and 9 patients with pure autonomic failure to investigate the possible effects of sympathetic nervous system overactivity and underactivity on peripheral 5-HIAA production and plasma 5-HIAA concentration. The resting arterial plasma 5-HIAA concentration in the heart failure patients was increased three-fold. This elevated plasma 5-HIAA concentration was attributable to an increased rate of whole body 5-HIAA production. The arterial 5 HIAA plasma concentration in the autonomic failure patients was paradoxically elevated, being 70% greater than that of the healthy subjects. The increased 5 HIAA plasma concentration in these patients was accounted for by a reduction in 5 HIAA plasma clearance. In all subjects studied there was a weak relationship only between total body norepinephrine spillover to plasma and the arterial 5-HIAA plasma concentration. We found that in healthy subjects the overflow of 5-HIAA into the hepatic vein was significantly related to the underlying degree of sympathetic activity. It can be concluded that 5-HIAA is produced at a number of sites throughout the body with the arterial plasma concentration being dependent on both the level of production and plasma clearance. By far the majority of 5 HIAA in plasma is derived from the gut with only minimal contribution from the brain. PMID- 7541100 TI - Effect of endothelin-1 on arterial response to Bay K 8644. A comparison of ischemic and nonischemic arteries. AB - Endothelin-1 (ET-1) has been proposed as one of the possible mediators of the vasoconstriction seen following ischemia and reperfusion. We investigated the effect of ischemia and reperfusion on the contractile response of canine renal and iliac arteries to the dihydropyridine-type calcium channel agonist (+/-)Bay K 8644, following subthreshold doses of ET-1. No significant difference in the maximum tension was observed between the ischemic and nonischemic arteries in response to Bay K 8644 in the absence of ET-1. The addition of subthreshold dose of ET-1 (10(-10) M) resulted in a significant increase in sensitivity to Bay K 8644 in both the ischemic-reperfused and non-ischemic-reperfused arteries, with a 38 fold increase in iliac arteries and about 8 fold increase in the renal arteries. However, the ET-1 potentiated response was enhanced in the ischemic reperfused in comparison to the non-ischemic-reperfused vessels in the iliac artery. These data suggest that the potentiating mechanism of ET-1 is not only intact, but enhanced in ischemic-reperfused vessels. Since the enhanced release of ET-1 in vivo is preceded by ischemia and reperfusion, the vasospastic phenomenon observed following these events could well be mediated by ET-1. PMID- 7541103 TI - Retrograde axonal cytokine transport: a pathway for immunostimulation in the brain inducing hypoxia and sudden infant death? AB - The etiology and pathogenesis of sudden infant death syndrome (SIDS) remain unknown. A hypothesis for SIDS should explain three characteristic findings: (a) an age distribution peaking at 2-4 months; (b) frequent association with respiratory tract infections; and (c) occurrence during sleep. The diagnosis of SIDS is applied when death cannot be explained, and this syndrome therefore probably includes various underlying causes. Based on recent observations, however, we suggest a pathogenic pathway that might be common to most SIDS victims. PMID- 7541102 TI - Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat. AB - Neurokinin A and B are present in neurons situated in lung and NK-1 receptors have been described on tracheal submucosal gland cells. In the present study we compared the ability of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) to stimulate airway mucus secretion. Furthermore, we characterized the interaction of NKA and NKB with galanin and somatostatin. The rank order of the tachykinins to stimulate airway mucus secretion was SP > NKA > NKB suggesting that NK-1 receptors mediate these effects(EC50:SP: 50 nmol/l, NKA: 200 nmol/l, NKB: 400 nmol/l). Galanin and somatostatin were equally potent to inhibit NK-A and NK-B stimulated airway mucus release. These results suggest that NK-A and NK B are potent stimulators of airway macromolecule secretion. Galanin and somatostatin potently inhibit these actions of the tachykinins. Therefore, airway mucus secretion is controlled by a complex network of several different mediators. PMID- 7541104 TI - Ethylene oxide as a major factor in DNA and RNA evolution. AB - Gaseous ethylene oxide (EO) is genotoxic in a wide variety of biological systems and carcinogenic in rats and mice. The major product of EO reaction with DNA is 7 (2-hydroxyethyl)guanine. In the organism, EO originates through biotransformation of ethylene. Ethylene in higher plants has been found to be a key factor in the control of a number of developmental processes including senescence, abscission and ripening. Ethylene oxide can participate in the transition of pyrimidine bases from C to T or U and from U to C. Thus, EO may provide a key approach to the study of DNA and RNA evolution. The studies here suggest a role for EO in modulating multiple messenger pathways in DNA and RNA functions. Perhaps surprisingly, it exerts two quite different activities, expressed in diverse cells. On the one hand, EO causes mutagenesis and carcinogenesis if available in excess. On the other, at low concentrations it can mediate repair of DNA at replication and RNA at transcription and translation. The report provides substantial evidence that EO can also play a very important role in the DNA and RNA evolution of whole biological systems. This important effect of EO needs to be understood more completely. PMID- 7541106 TI - Use of polymerase chain reaction and specific monoclonal antibodies as rapid method to recognize Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii among Italian isolates of B. burgdorferi. AB - We previously classified locally isolated strains of Borrelia burgdorferi by a restriction fragment length polymorphism analysis of total DNA, by DNA/DNA Southern Blot hybridization and by a hybridization with rRNA 16 + 23S from Escherichia coli [Cinco et al. (1993) Microbiologica 16:323-332] into three genetic groups which, according to the reference strains used, should correspond to the three species so far described as B. burgdorferi sensu stricto, B. garinii and B. afzelii. To find a simpler method for strain identification, in this study we analyzed the Italian strains and some strains identification, in this study we analyzed the Italian strains and some strains originating from other European countries, employing the species-specific 16S rRNA primers in the polymerase chain reaction technique (PCR) and some phenotypic markers like the B. afzelii specific monoclonal antibodies and the battery of OspA-specific monoclonal antibodies which were reported to give a reactivity pattern correlated to the species [Wilske et al. (1993) J Clin Microbiol 31:340-350]. The PCR results confirmed those obtained previously by identifying the three groups as B. burgdorferi sensu stricto, B. garinii and B. afzelii; the reactivity patterns obtained with the monoclonal antibodies (mAb) also corresponded to those described as typical of the three species. We standardized the PCR technique to amplify a sample of crude template DNA obtained from a culture of 10(5) spirochetes. PMID- 7541107 TI - Taxonomic classification of 29 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis: a comparison of five different phenotypic and genotypic typing schemes. AB - Twenty-nine European and North American Borrelia burgdorferi strains isolated from patients with Lyme borreliosis, were investigated by restriction fragment length polymorphism (RFLP) of two phylogenetically highly conserved chromosomal genes encoding flagellin (fla) and the p60 common antigen (CA), as well as of the plasmid-borne outer surface protein A (ospA) gene. RFLP of the ospA, fla and CA gene revealed five, two and four distinct subspecies-specific patterns, respectively. RFLP classification of the B. burgdorferi strains was compared with four different classification schemes proposed by others: (i) molecular mass profile of OspA and OspB (Adam et al. [1]); (ii) OspA serotyping (Wilske et al. [34]); (iii) genomic fingerprinting on the central region of the B. burgdorferi fla gene (Picken [24]) and (iv) 16S rRNA signature nucleotide analysis (Marconi and Garon [19]). Results obtained with the different methods correlated highly. All strains classified as B. burgdorferi sensu stricto and B. afzelii could be unequivocally identified as one distinct group by all five typing methods. B. garinii isolates, however, were more heterogeneous and according to RFLP of the CA and ospA gene fell into either two or three subgroups. The agreement of the different approaches supports the recent concept that B. burgdorferi sensu lato strains should be delineated to three genomic groups and that B. burgdorferi sensu lato is clonal. All 12 US strains were B. burgdorferi sensu stricto, whereas the 17 European isolates belonged to any of three genospecies. Among European B. burgdorferi isolates there was an association between B. burgforferi genospecies and the clinical manifestation of Lyme borreliosis. B. afzelii strains were found to predominate in 11 skin isolates (75%), whereas all 6 cerebrospinal fluid isolates from patients with neuroborreliosis were B. garinii. These findings support the concept of a strain-dependent organotropism of B. burgdorferi. PMID- 7541105 TI - The role of CD14 and lipopolysaccharide-binding protein (LBP) in the activation of different cell types by endotoxin. PMID- 7541108 TI - Effect of hypothyroidism on the expression of cytochrome c and cytochrome c oxidase in heart and muscle during development. AB - The effect of thyroid hormone on the expression of mitochondrial proteins was evaluated during development by measuring cytochrome c oxidase (CYTOX) activity and cytochrome c protein and mRNA levels in heart and skeletal muscle of control and hypothyroid rats. Animals were killed at the late fetal, early, and late postnatal stages up to 56 days of age. In heart, CYTOX activity increased 2.3 fold above the fetal level throughout development, most of which occurred prior to 2 days of age. No increase was observed in muscle. CYTOX activity was reduced in hypothyroid animals throughout development in heart compared to controls (by 50% at 56 days), but in muscle no effect of hypothyroidism was observed. In muscle and heart 4- and 1.5-fold increases in cytochrome c above the fetal level were evident by 1 day of age, with further increases to 8.5- and 2.7-fold by 56 days, respectively. The increase in cytochrome c differed from the increase in CYTOX, indicating changes in mitochondrial composition. Hypothyroidism reduced cytochrome c in muscle by 30-35% at 56 days, but had no effect in heart, indicating a muscle type-specific effect of thyroid hormone on cytochrome c protein expression. Cytochrome c mRNA increased rapidly to 4-5 fold above the fetal level in both heart and muscle by 6 h post-partum. Between 7 and 56 days of age, further increases to 6- and 25-fold were observed in muscle and heart, respectively. In muscle, the 6-fold developmental increase in mRNA paralleled that of the protein, suggesting transcriptional regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541109 TI - Repair of mitochondrial DNA damage induced by bleomycin in human cells. AB - Damage to mitochondrial DNA (mtDNA) has recently been associated with a variety of human diseases including cancer, diabetes mellitus, and aging. The mechanisms by which the mitochondria respond to DNA damage are of prime importance in understanding how damage can persist and cause disease. Here we demonstrate the repair of mitochondrial DNA damage induced by the naturally occurring, radiomimetic drug bleomycin. WI-38 cells were first permeabilized using 20 micrograms/ml lysophosphatidylcholine in order to increase the intracellular concentration of bleomycin. Dose response studies with the permeabilized cells showed that a concentration of 5 micrograms/ml bleomycin given for 30 min caused sufficient DNA damage for repair studies. Following treatment with this concentration of bleomycin, repair of mtDNA damage was found to be about 80% by 2 h. However, after 4 h no additional repair was observed. The results indicate that there is an efficient DNA repair system in human mitochondria for some types of damage caused by bleomycin. However, there is a component of damage caused by this agent that either is not repaired or is removed at a much slower rate. PMID- 7541110 TI - Starvation-associated mutation in E. coli strains with and without reverse transcriptase. AB - Starvation-associated mutation to Tyr+ in E. coli WU3610 is identical to that in an isogenic derivative lacking reverse transcriptase encoded by a retron phage. PMID- 7541111 TI - Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo. AB - BACKGROUND: Autologous peripheral-blood progenitor cells can restore hematopoiesis after high-dose chemotherapy in patients with solid tumors or hematologic cancers. We investigated the ability of peripheral-blood progenitor cells generated ex vivo to restore hematopoiesis in patients with cancer who have undergone high-dose chemotherapy. METHODS: Ten patients who had received high dose chemotherapy were given transplants of autologous progenitor cells that had been generated ex vivo. We used 11 million CD34+ hematopoietic progenitor cells as the starting population for the cell growth. This number corresponds to less than 10 percent of the usual preparation of peripheral-blood CD34+ mononuclear cells used in leukapheresis. The CD34+ cells were grown in medium containing autologous plasma, recombinant human stem-cell factor, interleukin-1 beta, interleukin-3, interleukin-6, and erythropoietin. RESULTS: No toxic effects were observed with the infusion of the generated cells. The cells promoted a rapid and sustained hemopoietic recovery when transplanted after treatment with high-dose etoposide (1500 mg per square meter of body-surface area), ifosfamide (12 g per square meter), carboplatin (750 mg per square meter), and epirubicin (150 mg per square meter). The pattern of hematopoietic reconstitution was identical to that in historical controls treated with unseparated mononuclear cells or positively selected CD34+ cells. CONCLUSIONS: A small number of peripheral-blood CD34+ cells, when grown ex vivo, can supply a population of hematopoietic precursors that have the ability to restore blood formation in patients treated with high doses of chemotherapy. This method, which requires only a small volume of the patient's blood, may reduce the risk of tumor-cell contamination, circumvent the need for leukapheresis, and allow repeated cycles of high-dose chemotherapy. PMID- 7541112 TI - Multiple sclerosis. Presenting an odd autoantigen. PMID- 7541113 TI - Life at extremely low pH. PMID- 7541114 TI - Powering the flagellar motor of Escherichia coli with an external voltage source. AB - Rotary motors of bacterial flagella are driven by ions that move across the cytoplasmic membrane down an electrochemical gradient. For Escherichia coli, the ions are protons, and the maximum work per unit charge that they can do is the protonmotive force. To test whether motor efficiency is limited by proton leakage or mechanical nonlinearities, we measured torque as a function of protonmotive force. Filamentous cells were drawn into micropipettes and energized with an external voltage source. Torque was proportional to protonmotive force up to -150 mV, twice the span accessible by earlier techniques. This is consistent with a mechanism in which a fixed number of protons, working at unit efficiency, carry the motor through each revolution. We also found that individual torque generating elements inactivate at low potentials or potentials of reverse sign. When normal potentials are restored, they reactivate sequentially. PMID- 7541115 TI - Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis. AB - A variety of hyperthermophilic bacteria and archaea have been isolated from high temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities. PMID- 7541117 TI - A cyclic-nucleotide-suppressible conductance activated by transducin in taste cells. AB - Taste can be divided into four primary sensations: salty, sour, sweet and bitter. Salty and sour are directly transduced by apical channels, whereas sweet and bitter utilize cyclic nucleotide second messengers. We have shown that rod transducin is present in mammalian taste receptor cells, where it is activated by a bitter receptor and in turn activates a phosphodiesterase. Here we introduce into frog taste cells peptides derived from transducin's phosphodiesterase interaction region, which cause an inward whole-cell current in a subset of cells. We find that the peptides' effects are reversibly suppressed by IBMX and forskolin, indicative of a transducin-activated phosphodiesterase. Cyclic nucleotides suppress the whole-cell current, indicating that cyclic nucleotides may regulate taste-cell conductance. IBMX modifies taste-cell responses to two taste stimuli, implicating phosphodiesterase in taste transduction. Submicromolar cyclic nucleotides directly suppress the conductance of inside-out patches derived from the taste-cell plasma membrane, independently of protein phosphorylation. The channels are unusual in that they are suppressed, rather than activated by cyclic nucleotides. We propose that transducin, via phosphodiesterase, decreases cyclic nucleotide levels to activate the cyclic nucleotide-suppressible conductance, leading to Ca2+ influx and taste-cell depolarization. PMID- 7541118 TI - Angiogenesis in microvascular endothelial cells induced by glioma cells and inhibited by tumor necrosis factor in vitro. AB - Neovascularization is a prerequisite for glioma growth, so inhibition of angiogenesis may achieve control of glioma growth. We examined whether glioma cells induce angiogenesis and proliferation in microvascular endothelial cells from Fisher 344 rat brains by co-culture in a physical separation system with rat C6 glioma cells or rat T9 gliosarcoma cells. Endothelial cells cultured on type 1 collagen formed capillary-like structures. C6 glioma cells co-cultured with endothelial cells promoted the formation of these capillary-like structures. However, conditioned medium from C6 cells inhibited the proliferation of endothelial cells. T9 cells had little effect on the formation of capillary-like structures and no effect on the proliferation of endothelial cells. We also examined the effects of human tumor necrosis factor (TNF)-alpha on the formation of the capillary-like structures and on the proliferation of endothelial cells. Human TNF-alpha inhibited the formation of capillary-like structures induced by C6 glioma cells at a concentration of 100 U/ml, as well as the proliferation of endothelial cells at a concentration of 1000 U/ml. These results indicate that induction of angiogenesis varies with glioma cell lines and angiogenesis does not correspond with proliferation of endothelial cells. TNF-alpha can inhibit angiogenesis in gliomas in vitro. PMID- 7541116 TI - Mesoderm induction in Xenopus caused by activation of MAP kinase. AB - Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras (ref. 5). Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction, and the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. Here we show that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation. PMID- 7541119 TI - Effects of three alpha 2-adrenoceptor agonists, rilmenidine, UK 14304 and clonidine on bradykinin- and substance P-induced airway microvascular leakage in guinea-pigs. AB - The effects of three alpha 2-adrenoceptor agonists, clonidine, rilmenidine and UK 14304 on the increase of microvascular permeability induced by bradykinin or substance P in guinea-pigs airways have been studied in vivo. Extravasation of intravenously (i.v.) injected Evans blue dye was used as index of permeability. The effects of the three alpha 2-adrenoceptor agonists on the contraction induced by bradykinin (0.3 micrograms.kg-1, i.v.) and substance P (0.3 micrograms.kg-1, i.v.) were also studied on the isolated guinea-pig trachea. The increase of plasma exudation induced by bradykinin (0.3 micrograms.kg-1, i.v.) was inhibited partially by rilmenidine and UK 14304 (20 micrograms and 100 micrograms, intratracheally) respectively. These two substances had no action on the effects of substance P. The effects of rilmenidine and UK 14304 were abolished by alpha 2 blockers (idazoxan 1mg.kg-1 i.v. and RX 821001 100 micrograms.kg-1, i.v.), but they were not altered by the alpha 1-blocker prazosin (30 micrograms.kg-1, i.v.). Under similar conditions, clonidine or the alpha 1-adrenoceptor agonist methoxamine were without significant effects. In vitro, rilmenidine and UK 14304 inhibited partially the contractile effects of bradykinin but not those of substance P. To conclude, both rilmenidine and UK 14304 inhibit the bradykinin induced increase of vascular permeability in the airways, and they probably do so on peptidergic nerve endings at the prejunctional level since these substances are without effect on substance P. The absence of activity of clonidine in our study might be due to a difference in spectrum of action on the several types of alpha 2-adrenergic or imidazole receptors. PMID- 7541120 TI - Neurokinin A and substance P vary independently in different regions of rat sensory neurons. AB - Neurokinin A (NKA) and substance P (SP) are often co-localized in small sensory neurons and have been suggested to subserve similar roles. We have now compared tissue levels of NKA and SP in rat cervical dorsal root ganglia with those in the central and peripheral terminations of the same neurons. We found that NKA content was less than that of SP in dorsal root neuron perikarya and in two peripheral tissues (superior cervical ganglion and ear skin) containing SP axon terminals from cervical spinal ganglia; in a third peripheral tissue, trachea, equal amounts of NKA and SP were present. By contrast, in the spinal cord containing the central terminals of these sensory neurons there was almost twice as much NKA as SP. Our results indicate that, although NKA and SP are co localized in sensory neurons, their levels vary independently, suggesting distinct functional roles. PMID- 7541121 TI - Caruncular malignant melanoma in a black patient. AB - An 84-year-old black woman had a right caruncular lesion that had progressively enlarged over the past 3 months. Surgical excision and cryotherapy were performed. A biopsy confirmed the diagnosis of caruncular malignant melanoma, a rare lesion which, to our knowledge, has not been previously reported in a black patient. PMID- 7541123 TI - Application of immunoperoxidase techniques to formalin-fixed brain tissue for the diagnosis of rabies in southern Africa. AB - Two immunoperoxidase techniques, viz. avidin-biotin complex (ABC) and peroxidase anti-peroxidase (PAP) procedures, were applied to paraffin-wax-embedded brain tissue sections, from brains which had been fixed in 10% formalin, to demonstrate the presence of rabies-virus antigen by light microscopy. These techniques positively identified both "viverrid" and "canid" rabies-virus antigen in tissues sections of species commonly infected with rabies virus in southern Africa, viz. the domestic dog (Canis familiaris), yellow mongoose (Cynictus penicillata), black-backed jackal (Canis mesomelas), bat-eared fox (Otocyon megalotus), cattle (Bos taurus), sheep (Ovis aries) and humans. With both of these techniques rabies virus antigen stained as sharply demarcated, brown precipitates within the cytoplasm of neurons. The virtual absence of background staining enabled identification of fine granules of viral antigen, often referred to as "virus dust", within axons, dendrites and cytoplasm of the nerve cell body. Staining with the ABC procedure clearer, more deeply-coloured precipitates than the PAP method. PMID- 7541122 TI - Immunolocalization of calcitriol receptor, plasma membrane calcium pump and calbindin-D28k in the cornea and ciliary body of the rat eye. AB - Epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump (PMCA) and the 28-kD vitamin-D-dependent Ca-binding protein (calbindin-D28k) were detected in sections of the albino rat eye using light microscopy and immunohistochemistry of paraffin-embedded tissues. Calcitriol receptor and calbindin-D28k epitopes were detected in both the inner and outer layers of the ciliary body epithelium. PMCA was present in both epithelial cell layers of the ciliary body but was most prominent in the inner layer. Corneal epithelium and endothelium also contained epitopes for calcitriol receptor, PMCA and calbindin D28k. These Ca-regulatory proteins may play a role in the cellular physiology of the albino rat eye by maintaining appropriate intracellular and aqueous humor Ca concentrations. PMID- 7541124 TI - The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH. AB - Following cell invasion, Trypanosoma cruzi trypomastigotes transform into amastigotes, which are the mammalian replicative forms of the parasite. Although amastigotes represent a critical stage in the life-cycle of T. cruzi, little is known of the factors controlling trypomastigote to amastigote transformation. Kanbera et al. (1990) observed that exposure of trypomastigotes to acidic pH induced their transformation into rounded forms resembling amastigotes. We confirm their observation and, using two strains of T. cruzi, establish that these transformants are ultrastructurally and biochemically indistinguishable from natural amastigotes. Incubation of trypomastigotes in medium at pH 5.0 for 2 h was sufficient to trigger their transformation into forms resembling amastigotes. Electron microscopical analysis confirmed that the kinetoplast structure, and general morphological features of the acid-induced, extracellular amastigotes were indistinguishable from those of intracellular-derived amastigotes. The extracellular transformation was accompanied by the acquisition of the stage-specific surface antigen of the naturally transformed amastigotes (Ssp-4), and loss of a stage-specific trypomastigote antigen (Ssp-3). Trypomastigotes incubated at neutral pH did not transform into amastigotes, and did not acquire the Ssp-4 epitope or lose the Ssp-3 epitope. Finally, acid induced amastigotes subsequently incorporated [3H]thymidine into their DNA, indicating that the important replicative property of intracellular amastigotes is also exhibited by these in vitro transformants. This effect of low pH appears to be of physiological relevance, and acid-induced extracellular transformation appears to represent a valid experimental technique for studies of the molecular mechanisms involved in the differentiation process. PMID- 7541125 TI - Haemangiomatosis in children: value of MRI during therapy. AB - MRI findings of 18 examinations of six children with haemangiomatosis and one with extended arteriovenous malformation are reported. Structures involved were the liver, liver and lung, periorbital area and the thigh. Response to interferon therapy, in particular, was assessed. With MRI the disease can be characterized and the extent of the lesions and size of the haemangiomas measured. Coronal views provide excellent demonstration of the involved structures in liver and lung haemangiomatosis. Two children showed response to interferon therapy with a reduction in lesion size and subsequently in number. Signal intensity decreased slightly on T2-weighted images. During treatment, however, no definitive fibrotic zones were seen. Following complete regression, signal intensity of the liver parenchyma was homogeneous in both weightings, that is, no fibrotic areas were visible 18 months after the beginning of treatment. Two children showed no response and one child died from congestive cardiac failure. The periorbital haemangioma was reduced in size and the lesion in the thigh might be classified as an arteriovenous malformation. In children MRI can replace CT as it is a reliable imaging modality for diagnosing haemangiomatosis and monitoring therapy. PMID- 7541126 TI - Fetal hemoglobin expression in transplant recipients of placental blood hematopoietic progenitor cells. AB - Patients who achieved bone marrow engraftment of cord blood-derived progenitor cells provided an opportunity to examine the expression of fetal Hb by neonatal hematopoietic progenitors in a postneonatal host. Cord blood cells from histocompatible siblings were successfully transplanted in two children with the Fanconi anemia syndrome. One of the transplant donors had heterocellular hereditary persistence of fetal Hb, apparently due to gamma-globin gene triplication; the other donor was hematologically normal. The G gamma/A gamma ratio of the patient who received his transplant from the donor with hereditary persistence of fetal Hb was markedly elevated, similar to that of the transplant donor's cord blood, and this ratio remained elevated in subsequent months. In the other child, the G gamma/A gamma ratio immediately after her transplant was typical of the normal newborn, and over the next several months it reverted to the adult pattern. Globin synthesis studies performed shortly after engraftment demonstrated ratios of fetal Hb/adult Hb synthesis in both patients that were typical of those of normal newborns. Over the next several months, both patients converted to the adult pattern. Fetal Hb to adult Hb switching in these patients seemed to follow a temporal sequence intrinsic to the transplanted neonatal progenitor cells, without discernible influence of postneonatal environmental factors. The program for Hb switching seems to be an inherent feature of neonatal hematopoietic progenitor cells. PMID- 7541127 TI - Fetal cytokine expression in utero detected by reverse transcriptase polymerase chain reaction. AB - Cytokine expression at the maternal-fetal interface is well documented. Some authors have postulated the existence of a bidirectional cytokine signaling mechanism that is critical to the maintenance of normal pregnancy. The role of the fetus (versus fetally derived placental tissues) in this process is unknown. Using reverse transcription polymerase chain reaction techniques, we studied paired maternal and fetal samples from 16 pregnancies (including two twin pregnancies) for the presence of tumor necrosis factor-alpha and IL-1 beta mRNA. We demonstrate that mRNA for both of these cytokines can be detected in both maternal and fetal blood as early as the 21st wk of gestation. These results support a potential role for the fetus in the bidirectional cytokine signaling of pregnancy. PMID- 7541129 TI - Metaphors in the practice of the human becoming theory. PMID- 7541128 TI - The house-garden-wilderness metaphor: caring frameworks and the human becoming theory. PMID- 7541130 TI - Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. AB - For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O methyl-NTPs, and the products contained up to 58% 2'-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATP alpha S, 2'-O-methyl-GTP alpha S and 2'-O-methyl-CTP alpha S, whereas they were found with dATP, 2'-O-methyl-ATP alpha S and 2'-O-methyl-UTP alpha S. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented. PMID- 7541131 TI - An upstream U-snRNA gene-like promoter is required for transcription of the Arabidopsis thaliana 7SL RNA gene. AB - The genes transcribed by RNA polymerase (pol) III can be placed into four distinct groups based on the nature and position of their promoter elements. In the higher eukaryotes equivalent genes usually belong to the same sub-type of pol III promoters and there are few examples of genes which have changed promoter type during evolution. In this work we demonstrate that the promoter of the Arabidopsis thaliana 7SL RNA gene is located upstream of the coding region and is identical to the promoters of pol III-specific plant U-small nuclear RNA (U snRNA) genes. Sequence analysis of two different 7SL genes from A. thaliana revealed that both genes contain two sequence elements in their 5' flanking regions identical in sequence and position to the highly conserved USE and TATA elements of the pol III-transcribed plant U-snRNA genes. Mutational analysis of these elements in the At7SL-2 gene indicates that the USE and TATA elements are both necessary and account for > or = 90% of the transcriptional activity of this gene in transfected plant protoplasts. Within the coding region of both genes there is a sequence element which is a 10/11 nt match to the consensus B-box element of tRNA genes, however, this element is not important for gene activity. These findings distinguish the plant genes from the human 7SL gene, which has both internal and upstream promoter elements and its upstream elements are different from those found in the human U-snRNA genes. PMID- 7541132 TI - Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. AB - The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2' fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides. PMID- 7541134 TI - The in vitro characterization and biostability of 99mTc-dextran and its accumulation within the inflamed paws of adjuvant-induced arthritic rats. AB - The in vitro and in vivo stability in normal and adjuvant-induced arthritic rats of 99mTc-dextrans (10, 40 and 500 kDa) have been investigated. The circulation half-lives were molecular weight dependent, with 10 and 40 kDa fractions being cleared relatively rapidly due to their ability to cross the glomerular basement membrane. The 500 kDa dextran was eliminated more slowly although 79% had been removed from the circulation 4 h post injection which probably was due to its degradation by dextranases and subsequent glomerular excretion. Dextran accumulation by the RES was found to be similar for all molecular weight preparations with no significant differences found. The sequestration of the dextrans by tissues of the RES (liver, spleen and lung) was independent of clearance rate. No differences were seen between normal and arthritic groups. Accumulation of the polymers by inflamed paws greatly exceeded that of normal paws for the 10 kDa (5-fold) and 500 kDa (6-fold) although no differences were seen with the 40 kDa dextran. PMID- 7541133 TI - Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer: comparison with isolated rat jejunum and colon. AB - Drug transport through the tight-junctional pathway in Caco-2 monolayer was studied by examining the relationship between its permeability to hydrophilic drugs and membrane conductance. Compared with the rat isolated jejunum or colon, Caco-2 monolayer displayed high electrical resistance and low conductance, as well as low permeability to sulfanilic acid and FITC-dextran (M.W. 4000). However, there was a linear relationship between the drug permeability and partial Cl- ion conductance for Caco-2 monolayer, rat jejunum and colon. Hence, the permeability to those drugs per unit of Cl- conductance is similar in the three membranes, suggesting that the size (radius) of the tight-junctional pathway in the three membranes is similar. In addition, when the electrical resistance of Caco-2 monolayer was reduced to the same level as that of the jejunum or colon by pretreatment with disodium ethylenediaminetetraacetate, its permeability to FITC-dextran became significantly higher than that of other membranes. Accordingly, the high resistance and the low permeability of Caco-2 monolayer compared with rat intestinal membrane may be due to structural differences between the membranes, rather than a difference in the tightness of the junction. PMID- 7541135 TI - A molecular sensor system based on genetically engineered alkaline phosphatase. AB - Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination. PMID- 7541136 TI - Oligonucleotide N3'-->P5' phosphoramidates. AB - Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 3' NHP(O)(O-)O-5' internucleoside linkages and the study of their hybridization properties. Thermal dissociation experiments show that these compounds form very stable duplexes with single-stranded DNA, RNA, and with themselves following Watson-Crick base pairing. The duplex thermal stability was enhanced by 2.2-2.6 degrees C per modified linkage compared with phosphodiesters. The structure of complexes formed by phosphoramidates closely resembles that of RNA oligomers and corresponds to an A form, as judged by CD spectroscopy. N3'-->P5' phosphoramidates also form stable triplexes with double-stranded DNA under near physiological conditions when natural phosphodiesters fail to do so. Physicochemical characteristics of the amidates are similar to those of RNA oligomers, even though they are composed of 2'-deoxyfuranose-based nucleosides. PMID- 7541138 TI - Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design. AB - The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines. PMID- 7541137 TI - Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells. AB - The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection. PMID- 7541139 TI - Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase. AB - Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program. PMID- 7541140 TI - Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a non-V beta 8-specific superantigen. AB - Superantigens such as the staphylococcal enterotoxins can play an important role in exacerbation of autoimmune disorders such as experimental allergic encephalomyelitis (EAE) in mice. In fact, superantigens can reactivate EAE in PL/J mice that have been sensitized to rat myelin basic protein (MBP). The T-cell subset predominantly responsible for disease in PL/J mice bears the V beta 8+ T cell antigen receptor (TCR). The question arises as to whether T cells bearing other V beta specificities are involved in induction or reactivation of EAE with superantigen. Thus, we have investigated the ability of a non-V beta 8-specific superantigen, staphylococcal enterotoxin A (SEA) (V beta specificities 1, 3, 10, 11, and 17), to induce EAE in PL/J mice that have been previously protected from disease by anergy and deletion of V beta 8+ T cells. PL/J mice were first pretreated with the V beta 8-specific superantigen staphylococcal enterotoxin B (SEB) and then immunized with MBP. These mice exhibited V beta 8-specific anergy and depletion and did not develop EAE, even when further treated with SEB. However, administration of SEA to these same mice induced an initial episode of EAE which was characterized by severe hindleg paralysis and accelerated onset of disease. In contrast to SEB pretreatment, PL/J mice pretreated with SEA did develop EAE when immunized with MBP, and after resolution of clinical signs of disease these mice were susceptible to relapse of EAE induced by SEB but not by SEA. Thus, superantigens can activate encephalitogenic MBP-specific non-V beta 8+ T cells to cause EAE in PL/J mice. These data suggest that superantigens can play a central role in autoimmune disorders and that they introduce a profound complexity to autoimmune diseases such as EAE, akin to the complexity seen in multiple sclerosis. PMID- 7541141 TI - Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells. AB - Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5 lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL 10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively. PMID- 7541144 TI - [Advances in the research of viral hepatitis]. PMID- 7541142 TI - The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up regulates Bcl-2 expression. AB - Anchorage-dependent cells that are prevented from attaching to an extracellular matrix substrate stop proliferating and may undergo apoptosis. Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors, which are known to elicit intracellular signals upon cell adhesion. We show here that Chinese hamster ovary cells expressing the alpha 5 beta 1 integrin, which is a fibronectin receptor, do not undergo apoptosis upon serum withdrawal when the cells are plated on fibronectin. However, the alpha v beta 1 integrin, which is also a fibronectin receptor and binds fibronectin on the same RGD motif as alpha 5 beta 1, did not prevent apoptosis on fibronectin of the same cells. The cytoplasmic domain of the integrin alpha 5 subunit was required for the alpha 5 beta 1-mediated cell survival on fibronectin. The fibronectin-mediated survival effect appeared to be independent of the level of tyrosine phosphorylation of the focal adhesion kinase, which is induced by integrin-mediated cell attachment. The expression of the Bcl-2 protein, which counteracts apoptosis, was elevated in cells attaching to fibronectin through alpha 5 beta 1; cells attaching through alpha v beta 1 survived only if exogenous Bcl-2 was provided. Thus, alpha 5 beta 1, but not the closely related alpha v beta 1 integrin, appears to suppress apoptotic cell death through the Bcl-2 pathway. PMID- 7541143 TI - Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis. AB - To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis. PMID- 7541145 TI - Differentiation and tumor progression. AB - Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy. PMID- 7541146 TI - Kaposi's sarcoma: a reevaluation. AB - Kaposi's sarcoma (KS) is a multicentric neoplasia of microvascular origin arising during development of immunodeficiency in human immunodeficiency virus (HIV) infected individuals. More than 130 patients with HIV-associated KS (98% male homosexuals; median age, 35 years) have been diagnosed at the Department of Dermatology, University Medical Center Steglitz, Berlin, during the years 1982 1992. Mucocutaneous and visceral involvement was a common finding in patients with HIV-associated KS, increasing from 39% at the first visit to 65% at the last observation. In 90% of the patients significant immunosuppression was found (75% had a CD4+ count < 200/mm3) at the time of first diagnosis. However, immunosuppression was not a prerequisite for the development of KS, since the tumor had been diagnosed before severe immunosuppression was present in about 10% of the patients. Significant prognostic predictors for the final outcome were: (a) the degree of immunosuppression, (b) the presence of mucosal and visceral manifestation, and (c) the past history of opportunistic infections. The median survival time was 28 months in KS patients with more than 300 CD4+ lymphocytes (n = 18), but only 14 months in immunosuppressed (less than 300 CD4+ lymphocytes) individuals with KS (n = 70). The median survival time in the entire group evaluated (n = 89 patients) was 17 months after first diagnosis. In 71 HIV infected individuals who died at the Berlin Department during the last 8 years, disseminated KS was the major direct or indirect cause of death (49% of cases). Therapeutic benefit for KS patients was observed after long-term administration of recombinant interferon alpha (rIFN-alpha; 9-18 million IU s.c. every 2 days) alone or combined with antiretroviral drugs such as zidovudine over several months. Prolongation of survival was found after such treatment modalities in 30% 40% of treated patients. Bleomycin and vincristine and other systemically used cytostatics have also been applied with moderate results. The etiology of HIV associated KS is still unknown and coinfection with herpes simplex virus (HSV), cytomegalovirus (CMV), or human papillomavirus (HPV) as well as certain growth stimulating cytokines (transforming growth factors, TGF; tumor necrosis factor alpha, TNF-alpha; interleukin-6, IL-6; tat; vascular endothelial growth factors, VEGF; oncostatin M) produced by HIV-infected cells may be cofactors. Overall, KS was found to be a tumor with high malignant potential, and the median survival times were short.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541147 TI - Immunohistochemistry of eccrine poroma and porocarcinoma--more than acrosyringeal tumors? AB - Sweat gland tumors have been classified according to their presumed physiological counterpart of the sweat apparatus. Both benign poroma and malignant porocarcinoma are thought to be acrosyringeal tumors. In order to specify this general assumption, we performed histochemistry and immunohistochemistry on paraffin sections of 29 poromas and eight porocarcinomas. In detail, we used Lapham's stain, Masson's silver impregnation, and immunoperoxidase staining with glandular marker antibodies against glycoproteins (CEA, LS59, NKI/C-3) and intermediate filament proteins (wide spectrum keratin, Cam 5.2, Vim 9(1)). Poromas disclosed some scattered S100-positive dendritic cells, red-stained cells in Lapham's method, several silver impregnated dendritic cells, and numerous cells surrounding poromas which were positive for LS59 and NKI/C-3. The labeling with wide spectrum keratin antiserum was low compared to epidermal keratinocytes. Porocarcinomas made some difference. CEA-positive single vacuolated cells could be observed, and S100-positive cells failed to show dendrites as in poromas. Some tumor cell clusters were stained weakly with LS59 and NKI/C-3 in addition to surrounding cells in both tumor entities. Three out of eight porocarcinomas disclosed sparsely distributed scattered cells weakly reactive with antibodies Cam 5.2 or Vim 9(1). In general, malignant porocarcinomas expressed a greater variety of cellular markers than benign poromas. The differentiation of both tumors, however, was directed toward inner duct cells and myoepithelium. Since myoepithelial cells are missing in normal acrosyringium, poromas and porocarcinomas are thought to be sweat gland tumors related to the distal portion of the dermal duct. PMID- 7541149 TI - An inside job. IL-12 attacks tumors on two fronts, but can it win the battle? PMID- 7541148 TI - Effect of NGF antibodies on mast cell distribution, histamine and substance P levels in the knee joint of TNF-arthritic transgenic mice. AB - We have previously shown an increase in nerve growth factor (NGF) levels and in mast cell (MC) distribution in the synovium of patients affected by rheumatoid arthritis. We now report that purified NGF antibodies injected into arthritic transgenic mice carrying the human tumour necrosis factor-alpha (TNF-alpha) gene caused reduction in the number of MCs, as well as a decrease in histamine and substance P levels within the synovium. These observations suggest that NGF antibody might be useful in studying the role of these pro-inflammatory markers in joint arthritis. PMID- 7541151 TI - Paclitaxel in the treatment of metastatic breast cancer: M.D. Anderson Cancer Center experience. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) 250 mg/m2 by 24 hour infusion at 21-day intervals was evaluated at M.D. Anderson Cancer Center as a single agent in patients who had received one prior chemotherapy regimen either as adjuvant therapy or for metastatic disease. Of 25 patients treated, 12% had a complete remission and 44% had a partial response. The median time to progression was 9 months (range, 1 to 20 months). In the next phase of development, a phase I trial evaluated sequentially administered paclitaxel and doxorubicin as initial therapy for metastatic disease. Granulocyte colony-stimulating factor also was administered in each cycle. The dose-limiting toxicity was either stomatitis or neutropenic fever. The maximum tolerated dose (MTD) was 125 mg/m2 for paclitaxel and 48 mg/m2 for doxorubicin. Because of much lower than anticipated MTDs of both drugs in this schedule, it was hypothesized that there may be a schedule dependent toxicity; therefore, in the second phase I study the schedule of administration was reversed (ie, doxorubicin followed by paclitaxel infusion). The MTDs of this schedule were 60 mg/m2 and 150 mg/m2 for doxorubicin and paclitaxel, respectively. Pharmacokinetic studies subsequently have confirmed that administration of paclitaxel before doxorubicin impairs the elimination of doxorubicin by some unknown mechanism. In an ongoing phase II study, paclitaxel is being evaluated in patients who have received three or more treatments with chemotherapy. Paclitaxel is administered at doses of 135 and 150 mg/m2 (for poor- and good-risk patients, respectively) without granulocyte colony-stimulating factor. Six patients (19%) have shown objective partial responses. Our initial phase II study showed significant antitumor activity for paclitaxel in patients who had received limited prior chemotherapy. Our phase I studies established that initial administration of paclitaxel alters the pharmacokinetics of doxorubicin and increases morbidity. The reverse sequence of administration was associated with better tolerance and a higher MTD. In heavily treated patients this drug also has significant antitumor activity. PMID- 7541150 TI - Preliminary experience with paclitaxel in advanced bladder cancer. AB - In a phase II Eastern Cooperative Oncology Group trial, single-agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) was administered to 26 patients with advanced urothelial cancer who had not received prior systemic chemotherapy. Paclitaxel was given at a dose of 250 mg/m2 by 24-hour continuous infusion along with prophylactic granulocyte colony-stimulating factor. Despite a 23% incidence of grade 3 or 4 neutropenia, only two patients developed febrile neutropenia; other hematologic toxicity was mild and not dose limiting. Nonhematologic toxicity included grade 3 neurologic toxicity in three patients, grade 3 mucositis in three patients, and grade 4 diarrhea in one patient. Eleven of 26 (42%) patients had an objective response (seven clinical complete responses, four partial responses), two had stable disease as their best response, and 13 patients progressed while on therapy. Preliminary response data suggest significant single-agent activity for paclitaxel in transitional cell carcinoma of the bladder. Future studies will evaluate paclitaxel-containing combination regimens as first-line therapy for advanced disease and define the role of paclitaxel in salvage therapy following conventional chemotherapy. PMID- 7541152 TI - Dose-finding study of paclitaxel and cyclophosphamide in patients with advanced breast cancer. AB - Escalating doses of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) (administered as a 3-hour infusion) and cyclophosphamide have been given to patients with advanced breast cancer every 3 weeks to determine the maximum tolerated dose of the two drugs in combination without granulocyte colony stimulating factor (G-CSF) support. The maximum tolerated dose of the same regimen with G-CSF support will then be determined in a subsequent group of patients. Patients who had received no more than one prior chemotherapy regimen for advanced disease were eligible for this study. The need for G-CSF support was established by an absolute neutrophil count (ANC) less than 0.5 x 10(9)/L for more than 7 days, an ANC count less than 0.1 x 10(9)/L for more than 3 days, febrile neutropenia requiring intravenous antibiotics, World Health Organization grade 3 or greater mucositis for more than 7 days, or the failure of the ANC to recover by day 28. The maximum tolerated dose was defined as the dose level at which more than two of six patients treated needed G-CSF support during the second treatment cycle. To date, 20 patients have been entered at the following dose levels: paclitaxel 135 mg/m2 and cyclophosphamide 750 mg/m2 (level 1), paclitaxel 155 mg/m2 and cyclophosphamide 750 mg/m2 (level 2), paclitaxel 175 mg/m2 and cyclophosphamide 600 mg/m2 (level 3), and paclitaxel 175 mg/m2 and cyclophosphamide 750 mg/m2 (level 4). Only one patient at level 2 needed G-CSF support because of an ANC below 0.1 x 10(9)/L for more than 3 days after the first cycle. Neither febrile neutropenia nor treatment delay for more than 1 week was reported. Antitumor activity has been observed from level 3. The evaluation of the toxicity of paclitaxel 200 mg/m2 and cyclophosphamide 175 mg/m2 without G CSF support is ongoing. PMID- 7541153 TI - A phase I trial of paclitaxel and etoposide for metastatic or recurrent malignancies. AB - Etoposide and paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) each exhibit substantial activity against a variety of solid tumors. Etoposide promotes accumulation of cells in late S phase and in G2. Paclitaxel causes cell arrest in G2 and M. In this phase I trial, an empiric combination of a fixed dose of etoposide daily for 3 days followed by a 3-hour intravenous infusion of escalating doses of paclitaxel on day 4 is being tested. Cycles are repeated every 3 weeks. Dose level I, etoposide 100 mg/m2 intravenously days 1 to 3 and paclitaxel 80 mg/m2 intravenously day 4, was well tolerated. Dose level 2, with paclitaxel at 120 mg/m2, is near completion and appears tolerable as well. Further escalation of the paclitaxel dose is anticipated. Once the maximum tolerated dose of this combination is defined, growth factor will be added and further escalation of the paclitaxel dose will be attempted. PMID- 7541154 TI - Combined modalities in the treatment of head and neck cancers. AB - The higher the T and N stages at diagnosis of head and neck cancer, the lower the proportion of patients who achieve complete, durable local control and the lower the survival. These cancers and their treatments often produce considerable anatomic distortion, affecting function, nutritional status, and appearance. New treatment approaches for locally and regionally advanced head and neck cancers are thus needed to improve survival, quality of life, or both. Combined-modality approaches show promise. Induction chemotherapy and subsequent radiotherapy produce results equivalent to aggressive surgery but allow for better organ function and speech. Induction chemotherapy and radiotherapy are superior to radiotherapy alone. Concurrent chemotherapy and radiotherapy may produce additive or synergistic interactions but increase toxicities. Some studies suggest that concurrent chemotherapy and radiotherapy significantly improves survival over radiotherapy alone in regionally advanced disease. Drug selection criteria have included enhancement of radiation cytotoxicity, effect on cellular kinetics, and, possibly, single-agent antitumor activity. The platinum compounds are of interest, especially in combination with other chemotherapy agents, like 5 fluorouracil and paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ). Concurrent carboplatin and radiotherapy produced a 77-week duration of survival in responders in a University of Maryland Cancer Center study. A study of concurrent carboplatin/bleomycin/radiotherapy had to be halted because of severe bleomycin-induced mucositis. The results in this small group suggest that attenuating mucositis would be desirable. In a subsequent trial, paclitaxel, which shows considerable activity against head and neck cancers, was substituted for bleomycin. Data from the seven patients accrued thus far are too immature to define response. The study continues to accrue patients. PMID- 7541155 TI - Paclitaxel in the treatment of carcinoma of the esophagus. AB - Systemic chemotherapeutic agents, given either singly or in combination, have not affected the dismal prognosis of patients with metastatic or locoregional unresectable carcinoma of the esophagus. New active agents are needed to improve the outcome for these patients. A phase II study evaluated paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) administered by 24-hour intravenous infusion with granulocyte colony-stimulating factor support to patients with unresectable locoregional or metastatic carcinoma of the esophagus. Response rate, duration of response, and toxicity were assessed. No prior chemotherapy or biologic therapy was allowed, but radiotherapy to a limited field that did not compromise signal lesion evaluation could have been given. The starting paclitaxel dose was 250 mg/m2 repeated every 21 days. The study group included 44 men and seven women with a median age of 57 years and a median Zubrod performance status of I. Among 51 evaluable patients, one complete response and 15 partial responses (PRs) occurred, for a total response rate of 31%. In addition, 11 (22%) achieved a minor response. Among 33 patients with adenocarcinoma, 12 achieved either a complete response (one patient) or a PR (11 patients), and six patients had a minor response. Four of 18 patients with squamous cell carcinoma had a PR and five (28%) had a minor response. The median duration of PR was 17 weeks (range, 7 to 58 weeks). At a median follow-up of 12+ months, 28 patients are alive, with an actuarial median survival duration of 10.2 months (range, 2 to 20+ months). Paclitaxel was well tolerated. Granulocytopenia was frequent, resulting in 11 hospitalizations in nine patients. Grade 3 neuropathy was observed in three patients. Our data suggest that paclitaxel is active against both adenocarcinoma and squamous cell carcinoma of the esophagus. Plans to study paclitaxel in combination with cisplatin and 5-fluorouracil in this group of patients are under way. PMID- 7541156 TI - Paclitaxel and carboplatin in the treatment of advanced non-small cell lung cancer. AB - Based on the superior response rates (21% to 24%) of patients treated with single agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in Eastern Cooperative Oncology Group and M.D. Anderson Cancer Center trials in non-small cell lung cancer (NSCLC) and on the superior 1-year survival rates of NSCLC patients treated with carboplatin in a randomized study of cisplatin combination and analogues, we initiated a phase II trial of paclitaxel/carboplatin in patients with stage IV or effusion-positive stage III NSCLC. Eligibility stipulated chemotherapy-naive patients with measurable disease, good performance status, and adequate hematologic, hepatic, and renal function. Previous radiotherapy was restricted to < or = 30% of marrow-bearing bone. Paclitaxel was initially given at 135 mg/m2 over 24 hours followed by carboplatin dosed to a targeted area under the concentration versus time curve (AUC) of 7.5, with treatment repeated at 3-week intervals for six cycles. Granulocyte colony stimulating factor was introduced during the second and subsequent cycles, with the paclitaxel dose sequentially escalated in 40 mg/m2 increments to a maximum dose of 215 mg/m2 in patients with less than grade 4 granulocytopenia and less than grade 3 thrombocytopenia. Of 54 patients enrolled, 30 currently are evaluable for response, 23 for toxicity. Myelosuppression has been the principal toxicity, with grade 3 or 4 granulocytopenia occurring in 70% of patients after the first cycle. After the introduction of granulocyte colony-stimulating factor, granulocytopenia decreased to 37% during the second cycle and then consistently to 20% or lower during subsequent cycles. Only 22% of cycles have been delayed for 1 week or more. Neutropenic fever has occurred in five (5%) of 100 evaluable cycles. Other grade 3 or 4 toxicities include thrombocytopenia (13%), anemia (9%), fatigue (9%), and hemorrhagic cystitis (1%). The paclitaxel dose was boosted to 215 mg/m2 in 12 (70%) of 17 patients by cycle 3 or 4. At an AUC of 7.5, the median first-cycle carboplatin dose was 434 mg/m2 (range, 293 to 709 mg/m2). The objective response rate is 50%, with three complete, 12 partial, and five minor responses. We conclude that the paclitaxel/carboplatin combination is active in advanced NSCLC and, with AUC-based dosing of carboplatin, can be given at 3-week intervals. Although dose limiting at a paclitaxel dose of 135 mg/m2, granulocytopenia can be reduced substantially with granulocyte colony-stimulating factor, allowing sequential dose escalation of paclitaxel to 175 mg/m2 and 215 mg/m2 in 70% of patients receiving three or more cycles. PMID- 7541157 TI - A phase II study of paclitaxel and granulocyte colony-stimulating factor in previously untreated patients with extensive-stage small cell lung cancer: a study of the North Central Cancer Treatment Group. AB - In 1994, approximately 34,000 to 43,000 Americans will be diagnosed with small cell lung cancer, and 60% to 70% of these individuals will have extensive-stage disease. The median survival time of patients with extensive-stage small cell lung cancer is 8 to 10 months and 10% or less will survive 2 years. There have been no major advances in the treatment of this stage of disease in the past decade. Phase II trials with promising new single-agent chemotherapeutic drugs are justifiable. We report the design and toxicity of a phase II trial with single-agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in previously untreated patients with extensive-stage small cell lung cancer. PMID- 7541158 TI - Carboplatin and paclitaxel in ovarian cancer. AB - A phase I trial of carboplatin plus paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) therapy for untreated patients with advanced ovarian cancer has been completed, demonstrating that these drugs can be combined in full doses with acceptable toxicity. This combination has now gone forward to be evaluated in prospective randomized trials. The Gynecologic Oncology Group will be doing a randomized trial comparing therapy with cisplatin plus paclitaxel versus carboplatin plus paclitaxel in patients with optimal stage III ovarian cancer. Patients with limited-stage disease but with poor prognostic features will be randomly assigned to receive either three or six cycles of carboplatin plus paclitaxel. Carboplatin will be dosed using the Calvert formula at an area under the plasma concentration versus time curve of 7.5 and paclitaxel will be administered at a dose of 175 mg/m2 by 3-hour continuous infusion. Cycles are planned to be administered every 21 days without granulocyte-colony stimulating factor. PMID- 7541160 TI - Hepatitis C: diagnostic assays. PMID- 7541159 TI - Paclitaxel, cisplatin, and cyclophosphamide in human ovarian cancer: molecular rationale and early clinical results. AB - The three most active types of agents in the treatment of cancer of the ovary are platinum compounds (cisplatin or carboplatin), bifunctional alkylating agents (cyclophosphamide, melphalan, etc), and the recently developed natural product paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ). In an effort to improve long-term disease-free survival in patients with advanced disease, we have developed a three-drug regimen consisting of cisplatin, paclitaxel, and cyclophosphamide. Granulocyte-colony stimulating factor is given as bone marrow support using a flexible dosing approach. The molecular basis for this approach is founded in the positive molecular interactions between cisplatin and bifunctional alkylating agents and between paclitaxel and DNA-damaging agents. Cisplatin and cyclophosphamide damage DNA by two very different mechanisms; the respective importance of DNA strand "kinking" versus DNA strand "cross-linking" may explain the positive cell kill interaction between these two drugs. Paclitaxel appears to markedly slow the repair of DNA lesions caused by DNA damaging agents, which may include radiation-induced lesions, cisplatin adducts, and cyclophosphamide cross-links. Clinical data to date from several different groups strongly suggest that these molecular interactions translate into positive clinical benefit in human ovarian cancer. Preliminary data from our clinical trial show that these three agents are well tolerated in the doses administered and that this combination shows exceptional promise as a possible therapeutic advance in this disease. PMID- 7541161 TI - The medication minute: terazosin. PMID- 7541162 TI - Myonecrosis due to Clostridium septicum in a patient with unexplained neutropenia: successful treatment with granulocyte colony-stimulating factor. AB - We report a case of sepsis due to Clostridium septicum successfully treated with granulocyte colony-stimulating factor (GCSF). This case prompted our review of clostridial sepsis and considerations regarding the use of GCSF in cases of drug induced neutropenia. PMID- 7541163 TI - Biochemical screening tests for Down syndrome. PMID- 7541164 TI - Modes and means of channel behavior: an introduction. AB - A flush of technical developments and the resultant burgeoning research on channel molecules in biological membranes has, over the past two decades, led to clear operational distinctions between channel behavior and carrier behavior. These have evinced the tacit assumption that the two types of behavior are associated with distinctly different structural motifs (though certainly having some elements in common). Although a full survey of the physiological situation is hampered by low activation probabilities for most channels and by the small currents for most carrier systems (necessitating study of large ensembles of molecules), it is nevertheless clear that many intermediate types of behavior do exist, either in comparison of different channel and carrier molecules or in the responses of a given (class of) molecule at different times and in different membranes. Then in order really to understand the molecular mechanisms of transport, we must explicitly design experiments to isolate and quantify these unconventional modes of behavior. PMID- 7541165 TI - Coordination of plasma membrane and vacuolar membrane ion channels during stomatal movement. AB - Transduction of extracellular signals in guard cells requires coupling of ion fluxes across the vacuolar and plasma membrane. Changes in cytoplasmic proton and Ca2+ concentrations are important messages during stomatal movement. Here we summarize the different biophysical properties of voltage- and ligand-dependent ion channels in both the vacuolar and plasma membranes of guard cells, focusing on how cytoplasmic Ca2+ and proton concentrations direct ion fluxes towards stomatal opening or closure. Particular attention is given to the patch clamp analysis of plasma membrane cation channels (GCKCin, GCKCout and Ca2+ channels), the anion channel (GCAC1) and the vacuolar SV-type channels. A working model is presented of how the ion channels of both membrane systems could communicate to synchronize potassium salt release during stomatal closure. PMID- 7541166 TI - Flow cytometric detection of abnormal fetal erythropoiesis: application to 5 fluorouracil-induced anemia. AB - We sought to determine whether flow cytometric analysis of circulating fetal blood cells could be used to rapidly detect perturbations of fetal erythropoiesis. In addition, we wanted to determine whether this approach would allow sample collection by exsanguination instead of fetal cardiac puncture, a difficult technique used to prevent contamination of samples with maternal erythrocytes. To monitor fetal erythropoiesis from gestational day (GD) 14-20, we analyzed the cell size, RNA content, and percentage of circulating liver-derived reticulocytes relative to yolk-sac-derived erythroblasts. As a model toxicant, we chose 5-fluorouracil (5-FU), since we previously observed that maternal administration at 20-40 mg/kg on gestational day (GD) 14 produced fetal anemia on GD 16-17, as evidenced by dose-dependent decreases in the cell counts, hematocrit, and hemoglobin content of fetal blood obtained by cardiac puncture. We report herein that 48 hr after maternal 5-FU administration, both cardiac and peripheral blood samples exhibited a dose-dependent decrease in the relative percentage of reticulocytes, indicating a reduced rate of reticulocyte release from the fetal liver. Moreover, at 30 and 40 mg/kg, reticulocytes exhibited increased size and reduced RNA content on GD 16, but elevated RNA content (indicative of premature release) by GD 18. These data suggest that 5-FU inhibits both erythroid cell proliferation and RNA synthesis reversibly, resulting in an anemia that triggers compensatory release of immature reticulocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541168 TI - Epididymal spermatozoa: recovery and subsequent improvements of mouse epididymal spermatozoa via the SpermPrep filtration method. AB - This study was designed to determine the effects of Sephadex filtration (SpermPrep method) on the separation of viable motile, morphologically normal mouse epididymal spermatozoa and to study the viability of the recovered spermatozoa over a 3 hr incubation period. Spermatozoa were harvested from the caudae epididymides (5 animals per run or replication; n = 10) following bilateral testicular excision and incubated in 2-ml of Test-Yolk buffer (TYB) at 37 degrees C for 15 min. The specimen was then split into 2 aliquots, with Alquot 1 as the control and Aliquot 2 used for filtration. SpermPrep I column was employed according to the manufacturer's specifications (ZBL, Inc., Lexington, KY, USA) using TYB. During filtration (10 min), different fractions were obtained: first 5 min (Sample 1) and second 5 min (Sample 2). The filtered fractions were evaluated and incubated at 37 degrees C and assessed for percentage and grade of motility (0-4) every 30 min for 3 hr. Filtration resulted in a significant improvement in percentage and grade of motility (91.5% and 3.0 vs. 76.5% and 2.5, respectively). The results point out very clearly that the filtration via the SpermPrep method improved the percentage and grade of motility (p < 0.05) but not the percentage normal morphology of the spermatozoa. Also the SpermPrep I enabled the recovery of 45% (8.3 x 10(6) spermatozoa) of the total spermatozoa processed in the control aliquot (18.4 x 10(6) spermatozoa) which is consistent with previous observations. Most importantly, filtered spermatozoa incubated for 3 hr showed greater percentage and grade of motility than the control spermatozoa (63% and 1.66 vs. 39% and 0.82, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541169 TI - Cadmium-induced expression of metallothionein and suppression of RNA to DNA ratios during molluscan development. AB - One-week-old larvae of the mollusc Crassostrea virginica, an oyster, were exposed to Cd concentrations ranging from the control with no added Cd to 0.2 microM CdCl2 for 24 hr. Concentration-dependent increases in total cadmium accumulation, cytosolic and metallothionein-bound Cd concentrations, and levels of the MT mRNA were detectable over this range of exposure concentrations. Increases in these measures were apparent at the lowest exposure concentration of 0.005 microM, with dramatic increases observed between 0.04 and 0.2 microM Cd. Although the concentrations of MT-bound Cd increased, the fraction of total Cd that was bound to MTs declined in larvae treated with 0.04 and 0.2 microM Cd, presumably due to the inability of MT biosynthesis to keep up with the higher rates of Cd uptake at the higher concentrations. This decreased effectiveness of MTs in sequestering Cd in relation to the total Cd load and the associated increase in the nonthionein Cd fraction at these higher treatment conditions coincided with a decline in the RNA to DNA ratio and an increase in a nonacetylated variant of the MT. The former is indicative of an inhibition of larval metabolism and growth. The latter is an unexpected form for this MT and is believed to signify disruption of normal cotranslational modification of the N-terminal amino acid sequence. PMID- 7541167 TI - Changes in neuroendocrine elements in bronchial mucosa in chronic lung disease in adults. AB - BACKGROUND: It is not clear whether there is any association between metaplasia of the bronchial epithelium and changes in the distribution of neuroendocrine cells. This study examined, by immunohistological techniques, the distribution of neuroendocrine cells and juxtamucoscal nerve fibres in bronchial biopsies showing metaplastic changes. METHODS: Bronchial biopsies from 12 subjects with epithelial metaplasia associated with bronchiectasis and diffuse pulmonary fibrosis were examined by conventional light microscopy and immunohistological techniques for protein gene product 9.5 (PGP), chromogranin A and B (CAB), serotonin, vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), calcitonin (CT), and gastrin releasing peptide (GRP). RESULTS: Regions of non-metaplastic epithelium contained numerous PGP and serotonin immunoreactive cells. Sub-populations of these cells displayed CAB, CGRP, CT, and GRP immunoreactivity. Metaplastic epithelium contained only a few weakly stained PGP, serotonin, CAB, GRP, CT and CGRP immunoreactive cells in six cases. Metaplastic epithelium was characterised by a high number of CAB-containing cells in six cases and in these biopsies prominent PGP-containing nerve bundles were seen in the subepithelial layer beneath the metaplastic epithelium. CONCLUSIONS: The distribution patterns of neuroendocrine cells and neuronal elements vary between areas of normal and metaplastic epithelium and within areas of metaplastic epithelium. Neuronal hyperplasia was associated with an increase in the number of CAB-containing cells within the metaplastic epithelium. PMID- 7541171 TI - Onchocerciasis and its control. Report of a WHO Expert Committee on Onchocerciasis Control. PMID- 7541170 TI - Effect of dietary acid or aluminum on growth and growth-related hormones in young chickens. AB - The effect of two concentrations of dietary acid (sulfuric acid) or aluminum (aluminum sulfate) on growth and growth-related hormones was examined in a heavy (broiler) strain of chicken between 4 and 18 days old. Growth (body weight, average daily gain, and tibial length) in chicks receiving either dietary acid or aluminum-containing diets were compared to chicks fed a control diet and to chicks fed diets containing sodium sulfate. Despite the reduced growth in acid fed chicks, there were no changes in the plasma concentrations of growth hormone, insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding proteins (IGF-BP). However, in chicks receiving the high aluminum diet where growth was markedly depressed (by 57%), plasma concentrations of IGF-I were depressed, while those of the 36-kDa IGF-BP were elevated. The effects of acid and aluminum on growth were mediated at least partially by reductions in feed intake. Pair feeding depressed growth and tended to decrease plasma concentrations of IGF-I. Some differences were observed between acid or aluminum groups and their respective pair-fed controls. For the groups receiving the low aluminum and both the high- and low-acid diets, values were increased compared to corresponding pair-fed controls for average daily gain, the level of the 36-kDa IGF-BP, and skeletal growth. On the other hand, high aluminum had a toxic effect in addition to reducing feed intake, with skeletal growth being reduced more in chicks receiving the aluminum diet than in the pair-fed controls. PMID- 7541172 TI - Cerebellovestibular projection from the posterior lobe cortex in the rabbit: an experimental study with the retrograde HRP method. I. Topographical relationships. AB - The sources of corticovestibular projections from the cerebellar posterior lobe vermis and hemisphere in the rabbit were investigated by the retrograde horseradish peroxidase (HRP) tracing method. Following iontophoretic injections into various subdivisions of the vestibular nuclear complex (VNC), labelled Purkinje neurones were identified ipsilaterally in all vermal lobules (VI through IX) and some regions of the hemisphere. The results indicate that projection is profuse and directed to all four nuclei of VNC. The labelling suggests some topographical relationships between lobules and especially sublobules of the posterior lobe and individual nuclei of VNC. The major projection originates from lobules VI and IX. A moderate projection derives from lobule VIII whereas that from lobule VII is small. Projections from the hemisphere arise mainly from crus I and crus II of the ansiform lobule. Those from the copula pyramidis, lobule HV and the ventral paraflocculus are sparse. In the present study a topographical relationships were more precisely established than before because the technique revealed VNC afferents from individual sublobules of the cerebellar posterior lobe cortex. PMID- 7541173 TI - Cerebellovestibular projection from the posterior lobe cortex in the rabbit: an experimental study with the retrograde HRP method. II. Zonal organization. AB - The mediolateral distribution of neurones of origin of the corticovestibular projection from the cerebellar posterior lobe in the rabbit was studied with the retrograde horseradish peroxidase (HRP) tracing method. After iontophoretic injections confined to various subdivisions of the vestibular nuclear complex (VNC) labelled Purkinje neurones were found ipsilaterally in widespread mediolateral cortical regions of vermal lobules from VI to IX, as well as in crus I and crus II of the ansiform lobule, the copula pyramidis, lobule HV and the ventral paraflocculus. However, when projections from individual sublobules were examined, a clearly visible zonal pattern was found. Thus, Purkinje neurones projecting to VNC were arranged in longitudinal bands with medial and lateral boundaries quite well demarcated. These bands had a different width and were found to be located at different distances from the midline. The present findings constitute the first indication of a zonal organization of corticovestibular projections from individual sublobules of the cerebellar posterior lobe in the rabbit. This zonal arrangement of VNC afferents is discussed with the special emphasis on the cerebellar longitudinal zones and related to previous studies on other species. PMID- 7541175 TI - Critical oxygen extraction in dog hindlimb after inhibition of nitric oxide synthase and cyclooxygenase systems. PMID- 7541174 TI - The role of endothelium-derived relaxing factor (EDRF) in the whole body and hindlimb vascular responses during hypoxic hypoxia. PMID- 7541176 TI - The Haldane effect under different acid-base conditions in premature and adult humans. AB - The Haldane effect (HE) was investigated in human adults and prematures under normal metabolic acid-base conditions but at different levels of PCO2. Venous blood samples were equilibrated with low and high PCO2 in either O2 or N2. The change in plasma pH of oxygenated blood by deoxygenation did not differ between both groups. Thus, ontogenetic differences of human hemoglobin structure do not influence the net proton Haldane effect measured in terms of whole blood pH changes. Since the present data quantitatively agree with those we reported earlier for rabbits, cats and dogs (Kiwull-Schone et al., 1992), phylogenetic differences in hemoglobin structure of these mammalian species do not either seem to play a role in this respect. The influence of the Haldane effect on plasma pH has to be considered in blood-gas and acid-base analysis of samples with incomplete oxygenation. This is important for the indirect determination of PCO2 through pH by the equilibration method (Astrup and Schroder, 1956), serving as reference method for determination of metabolic acid-base status and CO2 buffering capacity. Likewise, HE-correction is important for indirect estimation of metabolic acid-base status (BE and HCO-3st) from clinical routine PCO2- and pH measurement. In spite of the vaste amount of literature on the Haldane effect in human blood, quantitative data for practical purpose are less available and still equivocal. By the present study, a strong inverse linear correlation between the HE-induced delta pH and 1g[HCO3-] could be shown over a wide range of acid-base changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541177 TI - A new approach for quantitative evaluation of coronary capillaries in longitudinal sections. PMID- 7541178 TI - Perfused rat liver responses to crude venoms from marine snails. PMID- 7541179 TI - Dietary effects on DNA methylation: do they account for the hepatocarcinogenic properties of lipotrope deficient diets? PMID- 7541181 TI - [A case of bladder cancer producing alpha-fetoprotein (AFP)]. AB - The high incidence of increased level of AFP in association with primary hepatoma and embryonic tumors of the ovary and testis is well recognized, but elevated AFP is very uncommon in bladder cancer. A 74-year-old man, diagnosed with transitional cell carcinoma of the bladder, showed an increased level (410 ng/ml) of circulating AFP. After partial cystectomy, the high level of AFP returned to the normal value. The AFP level remained normal for 4.5 years after surgery and no recurrence of bladder cancer was seen by the cystoscopy. X-ray and ultrasound examination has revealed no evidence of neoplasm in the other organs. AFP producing localization was detected in bladder cancer cells by the use of immunocytochemical technique with the avidin-biotin method. PMID- 7541180 TI - [Fine needle aspiration cytology in screening of prostatic cancer]. AB - Between October 1990 and June 1993, 112 patients underwent fine needle aspiration cytology and core needle biopsy of the prostate under transrectal ultrasonographic guidance. They were suspected of having prostatic cancer from the prostatic antigen level, digital rectal examination and/or transrectal ultrasonography. Twenty seven of the 112 cases (24%) were diagnosed with prostatic cancer. Their cytological diagnoses showed 22 class IV or V, 2 class III, and 3 class I or II. Efficiency, false negative rate and false positive rate were 86%, 11% and 0% in fine needle aspiration cytology. We could obtain sufficient samples for fine needle aspiration cytology in all cases. No severe complication was observed. However, we missed 3 patients, in fine needle aspiration cytology, who were strongly suspected of having prostatic cancer, and reexamination or additional core needle biopsy would have been necessary on 15 of the 112 patients (13%) who showed false negative or class III cytological results, if we had screened prostatic cancer only by fine needle aspiration cytology. In conclusion, fine needle aspiration cytology may not be as useful as core needle biopsy to screen prostatic cancer. PMID- 7541182 TI - [Two cases of cavity formation in prostatic urethra after transurethral microwave thermotherapy (TUMT) with Prostatron]. AB - The Prostatron device, developed by Dr. Devonec, provides microwave heating of the prostate and simultaneous conductive cooling of the urethra. Treatment with Prostatron (transurethral microwave thermotherapy: TUMT) of BPH can be performed in a single session, and the urethra, the rectum and the surrounding tissue are protected with the temperature monitoring system. TUMT with Prostatron may be more suitable for a small adenoma with mild symptoms than a large adenoma with severe symptoms, because objective improvement is rarely observed compared with subjective improvement. We have treated 40 cases of BPH with Prostatron (Prostasoft 2, max. energy: 50 watt, max. urethral temperature: 44.5 degrees C, max. rectal temperature: 42.5 degrees C) since March 1993. Among them, there were 2 cases of large cavity formation involving the prostatic urethra. With cavity formation, TUMT may have its place in the treatment of a large adenoma with severe urinary symptoms, but loses its advantage in the prevention of persistent urinary infection and retrograde ejaculation, especially in the treatment of a small adenoma in a younger patient with mild urinary symptoms. It is unknown why such cavity formation occurred only in 2 patients among 40 equally treated cases, but the causes of cavity formation and safety of TUMT should be evaluated again, even though no injury to the rectum and the external urethral sphincter were detected. We report these 2 cases, and causes of cavity formation and discuss the prospects of TUMT. PMID- 7541183 TI - Quantitative investigations into the histostructural nature of the human putamen. I. Staining, cell classification and morphometry. AB - Combined staining with aldehyde and cresyl violet allows a reliable morphological distinction to be made between seven different types of neurons in the human putamen. We examined the age distribution of nearly 42,000 neurons in 27 normal putamina, using a semiautomatic morphometric procedure on defined tissue blocks. For morphometric evaluation and stereological calculations a section thickness of 20 microns is recommended. We modified routine aldehyde fuchsin cresyl violet combination staining for nervous tissue, since Braak's original method (Braak 1978, 1980) was developed for thick sections. The results show that neuronal density varies with age for the different types of neurons. PMID- 7541184 TI - Morphological analysis of the fish heart ventricle: myocardial and connective tissue architecture in teleost species. AB - Light and scanning electron microscopy were used to study the structure of the heart ventricle in three species of marine teleost fishes: the hake (Merluccius merluccius), the angler fish (Lophius piscatorius) and the sea bream (Pagellus centrodontus). Our findings show the ventricle to be shaped differently in each species: tubular in the hake, saccular in the angler fish and pyramidal in the sea bream. From a structural viewpoint, interest was centered on two aspects: organization of the myocardial fibres and arrangement of connective tissue. In hake and angler fish ventricles, the myocardium was exclusively trabecular in nature, whereas the bream ventricle, in addition to trabecular myocardium, presented a thin compact layer. Muscle fibres showed precise patterns of organization at the level of the ventricular orifices. With the techniques used the intramyocardial connective tissue was detected in the following ventricular zones: i) at the level of subepicardial and subendocardial spaces, ii) surrounding the myocardial fascicles, and iii) surrounding individual myocardial cells. According to this structural study, the pyramidal ventricle of the fish should be considered as a ventricular pump with greater efficiency. PMID- 7541185 TI - Mechanism of systemic vasodilation during normovolemic hemodilution. AB - In the nonfailing heart, normovolemic hemodilution increases cardiac output and decreases total peripheral resistance (TPR). Putative mechanisms mediating the decrease in TPR include reflex vasodilation and changes in the local regulation of blood flow. Our objectives were to determine whether ablation of reflex neural mechanisms or the inhibition of nitric oxide (NO) synthase, the enzyme responsible for the synthesis of the endothelium-derived relaxing factor (EDRF NO), modulates the systemic vasodilator response to normovolemic hemodilution. Three groups of male Sprague-Dawley rats were subjected to acute normovolemic hemodilution, which was achieved by exchanging a volume of blood equivalent to 3.8% of body weight with hydroxyethyl starch. Hemodilution increased cardiac output and decreased TPR. Subsequent administration of the NO synthase inhibitor, L-nitroarginine (LNA), returned both cardiac output and TPR to control values. Pretreatment with LNA prior to hemodilution increased TPR, an effect that was partially reversed by the NO donor, sodium nitroprusside. In this setting, hemodilution failed to decrease TPR. After spinal cord destruction by "pithing," hemodilution decreased TPR to the same extent as that observed in intact rats. This hemodilution-induced decrease in TPR was abolished by the subsequent administration of LNA. These results indicate that neural reflexes do not modulate the systemic vascular response to hemodilution. Moreover, the systemic vasodilator response to hemodilution is abolished after inhibition of endogenous NO synthesis. PMID- 7541186 TI - Astemizole in the treatment of granulocyte colony-stimulating factor-induced bone pain. PMID- 7541187 TI - Molecular biology of diabetes insipidus. AB - The identification, characterization, and mutational analysis of three different genes, namely the prepro-arginine-vasopressin-neurophysin II gene (prepro-AVP NPII), the arginine-vasopressin receptor 2 gene (AVPR2), and the vasopressin sensitive water channel gene (aquaporin-2, AQP2), provide the basis for our understanding of three different hereditary forms of diabetes insipidus: autosomal dominant neurogenic diabetes insipidus, X-linked nephrogenic diabetes insipidus, and autosomal recessive nephrogenic diabetes insipidus, respectively. These advances provide diagnostic tools for physicians caring for these patients. PMID- 7541188 TI - Inhibition of constitutive and inducible nitric oxide synthase: potential selective inhibition. AB - Nitric oxide (NO) is a molecule that has been shown to be involved in a diverse array of physiological events. A variety of disease states and disorders are, in fact, due to either an over- or an underproduction of NO. As a result of the ubiquity and diversity of NO-mediated phenomenon, pharmacological manipulation is difficult. NO biosynthesis is the result of an oxidation of a terminal nitrogen on the amino acid arginine by a class of enzymes generally referred to as the nitric oxide synthases (NOSs). Since the various isoforms of NOS are distributed in cells and tissues according to their function, there is the possibility that manipulation of NO levels can be accomplished by designing specific pharmacological agents targeted at a single NOS isoform. Thus, this review discusses general inhibition of the NOSs by a variety of agents and then focuses on the possibility of developing agents for specific isoform inhibition. PMID- 7541189 TI - Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas. AB - In order to identify possible cellular abnormalities in human mastocytosis, sections from 13 urticaria pigmentosa lesions and 5 mastocytomas were compared with 5 normal skin specimens using histochemical, enzyme histochemical and immunohistochemical techniques. All toluidine blue-positive mast cells also reacted with Fc epsilon RI and c-kit antibodies, almost all stained for tryptase, many for chymase and the myeloid workshop mast cell antibodies, few for Fc epsilon RII and none for the proliferation marker Ki-67. Urticaria pigmentosa lesions contained fewer epidermal Langerhans cells and a lower percentage of avidin-positive mast cells than mastocytomas and normal skin. Mastocytomas exhibited generally weaker staining for mast cell markers and mostly lacked Fc epsilon RI-bound IgE on mast cells and Langerhans cells, although the receptor was able to bind IgE in tissue sections. Most of the mast cell antibodies also reacted with other cell types. Only toluidine blue, avidin, tryptase and chymase stains were mast cell specific. Mast cells in mastocytosis thus differed only to a minor degree from normal mast cells, although distinct pathomechanisms may play a role in urticaria pigmentosa and mastocytosis. PMID- 7541190 TI - The effects of topical corticosteroids on delayed pressure urticaria. AB - Six patients with delayed pressure urticaria (DPU) applied clobetasol propionate (0.05%) ointment or its base to predetermined test sites on the right and left thigh as part of a randomized, double-blind study. A pressure challenge was administered to each test site at the initial visit and repeated after 3 days and 6 weeks of treatment and at between 4 and 8 weeks after treatment. The areas of pressure-induced weals were measured 6 h after each challenge. At the 6-week visit, a 4-mm punch biopsy was taken from pressure-challenged skin on each test site. Sections were stained for mast cells and immunohistochemical labelling was used to demonstrate neutrophils (neutrophil elastase), eosinophils (eosinophil cationic protein), monocytes/macrophages (EBM 11), cells expressing the beta-2 integrins (CD11/18) and the vascular adhesion molecules, E selectin and intercellular adhesion molecule-1 (ICAM-1). In the steroid-treated sites, there was a significant decrease (P < 0.05, Wilcoxon's matched-pairs test) in the size of the pressure weals compared with baseline at 3 days, 6 weeks and at follow-up. Demonstrable mast cells were significantly decreased (P = 0.059) in the pressure challenged areas in the steroid-treated sites compared with the base-treated sites. The histological response to pressure was minimal in both sites perhaps demonstrating an active pharmacological effect of the ointment base. In conclusion, the application of potent topical steroids significantly reduced the clinical response to pressure in patients with DPU, possibly through a reduction in mast cells. PMID- 7541191 TI - Cyclosporin A, FK506 and dithranol after tyrosine-specific protein phosphorylation in HaCaT keratinocytes. AB - Protein tyrosine kinases (PTKs) are closely related to cell growth, proliferation and differentiation. In keratinocytes, various growth factor receptors and cytosolic proteins, including the EGF and IGF receptors, the proteins of the src family and others, exhibit PTK activity. In psoriatic epidermis an increased level of EGF receptors and their ligand TGF-alpha has been found, and this is thought to be one reason for the pathological hyperproliferation of keratinocytes in this disease. Oral treatment with cyclosporin A (CsA) and FK506 or topical treatment with dithranol lead to an improvement in psoriasis. In the present study we examined the effect of these three drugs on the cellular content of phosphorylated tyrosines in highly proliferative HaCaT keratinocytes. HaCaT keratinocytes can be used as a model for highly proliferative epidermis, e.g. psoriatic epidermis. CsA had no effect whereas FK506 and dithranol reduced the phosphorylation of tyrosine residues in HaCaT keratinocytes. The activation of serine/threonine protein kinase C (PKC) is known to downregulate PTKs. Therefore we incubated keratinocytes with the selective PKC inhibitor Ro 31-8220 in addition to the other drugs. Only after the addition of Ro 31-8220 to FK506 treated keratinocytes was the phosphotyrosine (p-tyr) level elevated, but this was only one-third of the increase measured without additional therapeutic drugs. We assume that an induction of PKC alone is not responsible for the reduced p-tyr level after treatment with dithranol and FK506.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541193 TI - [Segment resection of the ureter in transitional cell carcinoma]. AB - OBJECTIVE: Although it is not the treatment of choice, conservative surgery by segmental resection of the ureter has achieved very good results in a patient with transitional cell carcinoma of the ureter. These results prompted us to report on the present case. METHODS/RESULTS: Diagnosis was fundamentally by IVP. Treatment by partial resection of the ureter achieved good results as shown by the postoperative control evaluation and follow up. CONCLUSIONS: Although nephroureterectomy continues to be the treatment of choice in transitional cell carcinoma of the urinary tract, conservative treatment (endoscopic resection of the tumor) may achieve good results in some selected cases. If conservative surgery is performed, the patient must be followed very closely using urine cytology, IVP or ureteroscopy. PMID- 7541194 TI - The importance of the prostate weight in transurethral microwave thermotherapy. AB - OBJECTIVE: Transurethral resection of the prostate (TURP) is still the gold standard in the treatment of benign prostatic hyperplasia (BPH). Prostate weight could be of importance on the result of transurethral microwave thermotherapy (TUMT). The present study attempted to elucidate this point. MATERIAL AND METHODS: Transurethral microwave thermotherapy (TUMT) was administered to 106 men with BPH. The patients were classified into two groups according to the weight of the prostate. Group 1 comprised 69 patients, aged 52 to 84 years (mean 65 years), whose prostate weighed up to 50 gm. Group 2 comprised 37 men, aged 55 to 87 years (mean 68 years) whose prostate weighed more than 50 gm. Prostate weight was determined by transrectal ultrasound. The protocol included history and physical examination (particularly digital rectal examination), laboratory evaluation (particularly measurement of PSA), transrectal ultrasound and uroflowmetry. The post-void residual urine was measured by urethral catheterization and ultrasound examination. RESULTS: There were 5/106 (4.7%) failure; therefore, a total of 101 patients were followed from 3 to 27 months, mean 7.7 months. Postoperatively, both groups showed improvement of all the parameters analyzed, except PSA (p < 0.01). However, comparison of the two groups for irritative and obstructive symptoms score and uroflow showed no significant difference. The decrease of the post-void residual urine was statistically greater in group 1 (p < 0.01). Group 2 showed a larger reduction in weight in comparison to group 1 (p < 0.01). Complications were observed in 12/101 (11.9%) cases, with no statistical difference between groups. Of a total of 7 patients with ejaculatory disorders, 5 patients with smaller prostate were observed. Recovery was not seen at more than 6 months follow up. CONCLUSION: The results showed no correlation between the prostatic weight and the efficiency of TUMT in the treatment of BPH. PMID- 7541195 TI - [Effect of rantes (regulated on activation, normal T expressed and secreted) on eosinophil adhesion to plasma coated glass using eosinophilic cell line (EoL-1)]. AB - Recently, adhesion molecules expressed on various inflammatory cells, especially on eosinophils are considered to play an important role in allergic inflammation such as bronchial asthma. RANTES (Regulated on activation normal T expressed and secreted) seems to be the most important chemokine which belongs to Platelet factor 4 (PF4) superfamily, since RANTES is revealed as a chemotactic factor for eosinophils as well as memory T cells or monocytes. In this study, we examined the effect of RANTES on eosinophil adhesion to plasma coated glass using eosinophilic cell line named EoL-1. RANTES augmented EoL-1 adherence to Plasma coated glass and EoL-1 adherence to plasma coated glass was inhibited by anti-VLA 4 antibody, anti-CR3 antibody. On the other hand, RANTES did not augment expression of adhesion molecules (VLA-4, CR3) on EoL-1. These findings indicate that RANTES intensify the adhesion of eosinophils without augmentation of their expression, and suggest that it induce qualitative, but not quantitative changes in their adherence. Taken together, our study suggest that RANTES up-regulates the inflammatory reaction through augmentative effect on adhesion of eosinophils in bronchial asthma. PMID- 7541192 TI - Keratinocytes constitutively express the Fas antigen that mediates apoptosis in IFN gamma-treated cultured keratinocytes. AB - The Fas antigen is a cell surface protein that can mediate apoptosis in many cell types. Although its physiological function is still unclear, recent evidence indicates that this surface molecule is involved in apoptosis in the immune system and the liver. The epidermis is an organ that undergoes terminal differentiation with the eventual death of keratinocytes, and it has been suggested that this is a specialized form of apoptosis. In the present study, we examined whether or not the Fas antigen is involved in keratinocyte apoptosis. Immunoreactivity for the Fas antigen was found throughout the epidermis in normal human skin sections and cultured normal human keratinocytes, and mRNA for the Fas antigen was found to be constitutively expressed in normal epidermis and cultured normal keratinocytes by RT-PCR analysis. To determine whether the Fas antigen in keratinocytes is functional, we used a cytotoxic monoclonal antibody (mAb) against the Fas antigen to induce apoptosis. This antibody did not induce apoptosis of cultured keratinocytes even though they expressed the Fas antigen. We then tested the ability of several cytokines (TGF beta, TNF alpha and IFN gamma) to induce Fas-mediated keratinocyte apoptosis. Only pretreatment with IFN gamma followed by the addition of the anti-Fas mAb induced apoptosis, as assessed by cell viability, morphological changes and ultrastructural characteristics, suggesting that constitutive expression of the Fas antigen is not sufficient to induce apoptosis in keratinocytes and that keratinocyte apoptosis via the Fas antigen-mediated mechanism may require the activation of keratinocytes by IFN gamma, which is thought to be produced by activated T cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541196 TI - Phosphorothioate oligonucleotides block reverse transcription by the Rnase H activity associated with the HIV-1 polymerase. AB - We demonstrate the degradation of RNA bound to an antisense oligonucleotide by a reverse transcriptase enzyme-associated RNase H activity. We found that phosphorothioate oligonucleotides inhibit the RNase H activity by binding to AMV RT, rather than to the template RNA, whereas the RNase H activity of HIV-1 RT is not affected by the antisense phosphorothioate oligonucleotide. Selective inhibition of HIV-1 gene expression involves the degradation of the template RNA bound to the antisense phosphorothioate oligonucleotide by the RNase H activity associated with the HIV-1 polymerase. PMID- 7541197 TI - Growth stimulation by transfection of intestinal epithelial cells with an antisense insulin-like growth factor binding protein-2 construct. AB - IEC-6 cells, an intestinal epithelial cell line, produce insulin-like growth factor (IGF)-II and IGF-binding protein-2 (IGFBP-2). Exogenous IGFs stimulate and IGFBP-2 attenuates DNA synthesis. To investigate whether endogenously secreted IGFBP-2 inhibits proliferation, IEC-6 cells were transfected with a full-length rat IGFBP-2 cDNA antisense expression construct. The steady-state level of IGFBP 2 mRNA decreased by 54% in the antisense cDNA-transfected cells (denoted revBP2) compared with vector-transfected controls. Moreover, revBP2 cells secreted less IGFBP-2 than controls (maximally a 68% decrease). In serum-containing medium, revBP2 cells exhibited a 35% increase in log-phase proliferation rate and growth arrested at a maximum density 14% higher than controls. We conclude that endogenous IGFBP-2 inhibits proliferation of IEC-6 cells, probably by sequestering IGFs. PMID- 7541198 TI - SARs do not impair position-dependent expression of a kit/lacZ transgene. AB - We have cloned and sequenced 4.2 kb of the murine c-kit promoter region. Analysis of transgenic embryos for a kit/lacZ fusion gene showed (i) an unexpected expression in the neural retina, and (ii) an unusual sensitivity to position effects. This indicates that major c-kit tissue specific regulating elements lie outside the proximal promoter. We took advantage to the intrinsic properties of the kit/lacZ transgene to investigated the potential of scaffold attachment regions (SARs) to confer position-independence on gene regulation in an in vivo context. No SAR-dependent isolation of the kit/lacZ transgene was found in the transgenic embryos, indicating a limited capacity of SAR sequences to buffer the effect of flanking sequences on transgene expression. PMID- 7541199 TI - Binding of sequence-specific proteins to the 3'-untranslated region of vasoactive intestinal peptide mRNA. AB - Multimeric AUUUA elements in an AU-rich 3'-untranslated region (3'-UTR) have been shown to confer message instability to numerous ephemeral transcripts through the formation of RNA-protein complexes. We show here that the 3'-UTR of VIP mRNA, which contains 3 AUUUA motifs in an AU-rich context, forms specific complexes with cytoplasmic proteins in a concentration-dependent, tissue-specific manner. We also demonstrate that an AU-rich segment of c-fos mRNA can successfully compete with VIP mRNA for binding with cytoplasmic proteins. These studies provide the first evidence for a mechanism by which VIP is post-transcriptionally regulated through specific sequences in its 3'-UTR. PMID- 7541200 TI - Sensitivity/resistance profile of a simian immunodeficiency virus containing the reverse transcriptase gene of human immunodeficiency virus type 1 (HIV-1) toward the HIV-1-specific non-nucleoside reverse transcriptase inhibitors. AB - To develop an animal model for the therapy of AIDS with human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors, we recently constructed a hybrid simian immunodeficiency virus (SIV)/HIV-1 in which the RT gene of SIV was replaced by the RT gene of HIV-1. This chimaeric virus, designated RT-SHIV, was found to be markedly sensitive to the inhibitory effects of both nucleoside (ddN) and non-nucleoside RT inhibitors (NNRTIs). In contrast, SIV was inhibited only by ddNs (i.e., 3TC and AZT), but not NNRTIs. When RT-SHIV was grown in the presence of 3TC, nevirapine, TSAO-m3T or the thiocarboxanilide UC-42 drug-resistant mutant virus strains emerged in cell culture as rapid as for HIV-1(IIIB). The antiviral sensitivity/resistance spectrum of the mutant RT-SHIV strains against NNRTIs and ddNs, and the nature of the mutations that appeared in their RT were similar to those of the mutant HIV-1 strains that were selected under identical experimental conditions. Infection of macaques with RT-SHIV may be a useful tool for studying the mechanism of NNRTI-resistance development and the therapy of NNRTI-resistant viruses in an animal model. PMID- 7541201 TI - Evidence for close in vivo association of protein with mitochondrial ribosomal RNA in a trypanosome. AB - Riboprotein particles containing the ribosomal RNA-like 9S and 12S RNAs in the trypanosome mitochondrion have never been isolated. Using the formaldehyde cross linking procedure I show here that one or more of three proteins, q2 (16.5 kDa), r (15 kDa) and s (13 kDa), are closely associated in vivo with the 9S and 12S RNAs of the trypanosomatid parasite, Crithidia fasciculata. These proteins were also found to be associated with the parasite's cytosolic ribosomal RNA and to have strong immunological cross reactivity with riboprotein S11 of the bacterium Escherichia coli. These data provide the first evidence for the association of riboprotein-like proteins with the 9S and 12S mitochondrial RNA in a trypanosome, possibly as components of a mitochondrial ribosome. PMID- 7541202 TI - Mechanisms involved in the uptake of D-glucose into the milk producing cells of rat mammary tissue. AB - There is a rapid stimulation of the rate of efflux of 3-O-methyl D-glucose from intact lactating rat mammary tissue slices in response to a change in the Na(+) gradient across the basolateral plasma membrane. RNA extracted from lactating mammary glands contains a 4 kb transcript which hybridises with the cDNA for rabbit intestinal SGLT1. A fraction of the mammary gland cells which is enriched in endoplasmic, Golgi and plasma membranes contains a protein that is immunologically similar to the Na(+)-dependent D-glucose symporter present in the plasma membrane of polarised epithelial cells, e.g., rabbit enterocytes, ovine enterocytes and parotid acinar cells. PMID- 7541203 TI - Selective delivery to the liver of antiviral nucleoside analogs coupled to a high molecular mass lactosaminated poly-L-lysine and administered to mice by intramuscular route. AB - In order to obtain hepatotropic conjugates of antiviral drugs suitable for intramuscular administration, three nucleoside analogs (adenine arabinoside monophosphate, ribavirin and azidothymidine) were coupled to a high molecular mass lactosaminated poly-L-lysine. The conjugates had a high molar ratio drug/conjugate and after intramuscular administration to mice, were selectively taken up by the liver and eliminated by the kidney only in minute quantities. The high molar ratio and low renal elimination are important properties not possessed by conjugates previously prepared by using a small molecular mass lactosaminated poly-L-lysine. The conjugate with adenine arabinoside monophosphate (ara-AMP) was found to be devoid of acute toxicity for mice and in spite of its high molecular dimension (Mn = ca. 72,500) did not induce antibodies in this animal after repeated intramuscular injections. This conjugate could have two advantages over a similar complex of ara-AMP with lactosaminated human albumin currently under clinical trials for the treatment of chronic type B hepatitis which must be injected intravenously: it might provide better patient compliance since it is injectable intramuscularly and could introduce larger amounts of ara-AMP into hepatocytes due to its higher drug/carrier molar ratio. PMID- 7541204 TI - Research & writing basics: poster presentations. PMID- 7541205 TI - Localization of T helper cell epitopes in the vesicular stomatitis virus: the nucleoprotein is responsible for serotype cross-reactive T help. AB - The glycoprotein (G) of vesicular stomatitis virus (VSV) is known to contain the biologically relevant sites for virus-neutralizing antibodies as well as T helper (Th) cell epitopes. The capacity of other VSV proteins to elicit potent Th cell responses has not yet been investigated. Additionally, a short-lived cross reactivity between the two serologically distinct VSV serotypes Indiana (IND) and New Jersey (NJ) on the T helper cell level has been reported. Here we address the question of whether the VSV nucleoprotein (N) or matrix protein (M) can elicit T help to VSV-G-specific B cells and which of the VSV proteins contains the elements responsible for the IND/NJ cross-reactivity. The N, G, and M of the VSV Indiana serotype produced in a recombinant baculovirus system were assayed for the ability to activate VSV-specific Th cells to induce immunoglobulin class switch of neutralizing antibody responses in vivo in C57BL/6 (H-2b) mice. All three VSV-IND proteins helped in the production of neutralizing IgG antibodies to the homologous VSV-Indiana serotype but only VSV-IND N was able to trigger an IgG response to the heterologous VSV-New Jersey serotype. This data suggest that Th epitope(s) in the VSV-IND N are responsible for the observed cross-reactivity of T helper cells. PMID- 7541206 TI - Transplantation in utero of fetal human hematopoietic stem cells into mice results in hematopoietic chimerism. AB - Allogeneic and xenogeneic hematolymphoid chimerism has been achieved in large and small animals using varied techniques to circumvent immune mediated graft rejection by the recipient. We show here the establishment of long-term chimerism in normal mice transplanted in utero with human fetal hematopoietic stem cells (HSC). HSCs from fetal (13-20 weeks' gestation) human livers were injected into fetal mouse peritoneal cavities on days 11-13 of gestation. Histologic examination demonstrated human chimerism in 29% of 38 live born mice using fluorescein conjugated antibodies to both the CD45 and CD14 antigens present on human peripheral blood (PB) cells. Further investigation using flow cytometric analysis of cells from 70 mice transplanted in utero revealed 28% of mice greater than 16 weeks of age contained human cells in at least one organ at the following frequencies: 14% PB, 8% bone marrow, 8% spleen and 12% thymus. These data indicate that human fetal HSC can be engrafted into mouse fetuses. Additionally, the identification of circulating human cells 18 months following transplantation supports the engraftment and proliferation of a primitive hematopoietic progenitor. PMID- 7541207 TI - Mapping of neurochemical markers in quail central nervous system: VIP- and SP like immunoreactivity. AB - The distribution of cells and fibres containing vasoactive intestinal polypeptide (VIP) and substance P (SP) was investigated in the brain of Japanese quail focussing on the centers involved in reproductive activities. SP-immunoreactive (ir) structures were chiefly present within the ventral telencephalic regions, the periventricular hypothalamus and the dorsal aspects of thalamus. VIP immunopositive structures were rarely associated with recognizable nuclei and they were observed in the organum septi laterale (LSO), the lobus paraolfactorius (LPO), the eminentia mediana (ME), the nucleus striae terminalis (nST) and the area ventralis of Tsai (AVT). SP- and VIP-ir structures were both associated with regions implicated in the control of reproduction. SP was mainly distributed within regions that control male copulatory behavior (the preoptic region, the anterior hypothalamus and the central gray), whereas VIP was prevalently located in the mediobasal hypothalamus that is implicated in the control of female reproductive activities. PMID- 7541208 TI - Nitric oxide synthase in the hypothalamo-pituitary pathways. AB - Several histochemical and physiological studies in the literature suggest that nitric oxide (NO) is involved in the posterior pituitary regulation. This study was set out to demonstrate the distribution of the pituitary projecting nitric oxide containing hypothalamic pathways. The fluorescent retrograde tracer fluorogold (FG) was injected into the pituitary gland in order to reveal the hypothalamic nuclei projecting to the pituitary gland. Nitric oxide synthase (NOS) was subsequently visualized from the same sections by NADPH-diaphorase histochemistry. More than 70% of the FG labelled neurons in the preoptic area, paraventricular, supraoptic, retrochiasmatic, anterior commissural and circular nuclei as well as in many other small neurosecretory centers in the hypothalamus contained also NADPH-diaphorase activity (NADPH-DA). In addition, some of the neurons in the periventricular nucleus as well as occasional neurons in the lateral hypothalamus projecting to the pituitary gland contained NADPH-DA. The present results give the first systematic neuroanatomical description of the hypothalamic NO synthase containing neurons projecting into the pituitary gland. They indicate that the pituitary receives a widely distributed NO innervation, which originates mostly in the magnocellular neurosecretory hypothalamo-pituitary system. In addition, the finding that some presumably non-neurosecretory pituitary-projecting neurons contain NO as well suggests that NO might be a more widely used regulator of the posterior pituitary secretion than previously expected. PMID- 7541209 TI - Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem. AB - The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed. PMID- 7541210 TI - Functional effects of phosphoramidon and captopril on exogenous neuropeptides in human nasal mucosa. AB - The functional effects of the intranasal application of exogenous vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were evaluated in 12 healthy volunteers before and after neutral endopeptidase (NEP) inhibition with phosphoramidon (PA) and angiotensin converting enzyme (ACE) inhibition with captopril. The three neuropeptides increased nasal airway resistance (NAR) measured by anterior rhinomanometry and superficial capillary blood flow measured by laser Doppler flowmetry (LDF). After pretreatment of the nasal mucosa with PA, the effects of VIP, SP and CGRP on the LDF signal, NAR and mucus production were potentiated, whereas local pretreatment with captopril did not modify these functional effects. These observations suggest that NEP, but not ACE, may participate in the catabolism of neuropeptides when applied directly to the human nasal mucosa. Furthermore, since these neuropeptides induced nasal obstruction, increased blood flow and rhinorrhea, a decreased activity of the enzymes involved in their degradation could be involved in the physiopathology of rhinitis symptoms. PMID- 7541212 TI - Modified method for RNA isolation from bacteria by guanidinium thiocyanate-cesium chloride centrifugation. PMID- 7541213 TI - Proteoglycans partially co-purify with RNA in TRI Reagent and can be transferred to filters by northern blotting. PMID- 7541211 TI - Immunohistochemical studies of sensory neurons in rat olfactory epithelium. AB - The olfactory sensory neurons undergo continuous turnover under normal physiological conditions. Injured olfactory sensory neurons are also replaceable. In this study, we investigated cellular differentiation and growth of sensory neurons in the rat's olfactory epithelium after nerve transection by using immunohistochemical staining with polyclonal or monoclonal anti-olfactory marker protein (OMP), anti-c-jun protein and anti-p53 protein antibodies. OMP is found exclusively in olfactory sensory neurons, while c-jun functions as a transcription factor. p53 protein functions as a negative regulator of cellular proliferation related to the apoptotic pathway induced by DNA damage. The olfactory epithelium sections incubated with anti-OMP antibody showed staining of mature neurons and axons in the epithelium. Nerve transection resulted in a significant reduction in neurons labelled with OMP. On the 9th day after operation, our study indicated some recovery with an increasing number of neurons expressing OMP. In control animals without nerve lesions, c-jun protein immunoreactive neurons were present in the olfactory epithelium adjacent to the basal region. Following days 1 and 3 after nerve transection, no expression of c jun protein was seen in neurons of the epithelium. On day 9 after transection, neurons in some basal areas indicated expression of c-jun protein. The immunolocalization demonstrated that p53 protein was present in some neurons located on the upper part of the olfactory epithelium. In contrast, an abundance of neurons expressing p53 protein was evident in the olfactory epithelium 1 and 3 days after nerve transection, indicating more cell deaths.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541214 TI - Rapid and efficient transfer of high molecular weight RNA onto positively charged nylon membranes. PMID- 7541215 TI - Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. AB - A continuous reverse transcription polymerase chain reaction (RT-PCR) procedure was designed with all reaction components included in a single tube prior to thermal cycling. This procedure was compared to uncoupled RT-PCR procedures wherein the addition of reagents was separated. In the latter, in particular, conditions for reverse-primer annealing and cDNA synthesis were investigated. The two RT-PCR approaches were compared in the detection of singly spliced and multiply spliced human immunodeficiency virus type 1 (HIV-1) mRNs. The avian myeloblastosis virus reverse transcriptase and Taq DNA Polymerase were used in the continuous procedure under the compromised condition wherein the two enzymes were active in the same buffer. Reverse transcription was carried out at an elevated temperature of 50 degrees C to overcome problems of mRNA secondary structures that could inhibit the reaction. The continuous procedure was found to be as specific and efficient as the best uncoupled procedure. The procedure was shown to be reliable and to have the sensitivity to detect one HTLV-IIIB-infected H9 cell in a million uninfected H9 cells. PMID- 7541216 TI - Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP. AB - We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein 15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity. PMID- 7541217 TI - Recovery of DNA, RNA and protein from gels with microconcentrators. AB - The use of a new product, Microcon/Micropure (a centrifugal ultrafiltration device combined with a microporus insert), for the purification of DNA, RNA, peptides and proteins from gels is described. Using this system, DNA can be recovered from agarose gel in concentrated, contamination-free form in only 15 min. Results of studies on the effects of fragment size and various pretreatment of the gel slice on DNA recovery are presented. The Microcan/Micropure combination can also be used for the recovery of macromolecules from polyacrylamide gels. Optimized protocols for the recovery of RNA, oligonucleotides and proteins from polyacrylamide gels using a crush and elute method, along with a study of critical parameters, are presented. PMID- 7541218 TI - Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes. AB - It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267-270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA "masking" proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation. PMID- 7541219 TI - Modulation of fibronectin and tenascin production in human bronchial epithelial cells by inflammatory cytokines in vitro. AB - Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix glycoproteins participating in tissue morphogenesis and repair. The regulation of their synthesis and deposition during airway inflammation and their possible contribution in asthma are poorly understood. In this study, modulation of Fn and Tn production was investigated in transformed human bronchial epithelial cells in culture. The cells were treated with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), a combination of these cytokines, interleukins 3 and 6 (IL-3 and IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), and a combination of IL-3 and IL-6 for 48 h. Immunofluorescence and immunoblotting methods with monoclonal antibodies to Fn and Tn antibodies suggested the production of some Fn and Tn in the untreated cells. Fn was minimally induced in response to IFN-gamma and TNF-alpha, when compared with the untreated cells, whereas TNF-alpha and especially the IFN-gamma plus TNF-alpha combination resulted in a prominent Tn induction. Interleukins and GM-CSF did not induce Fn or Tn in any case. These results show that human bronchial epithelial cells are capable of producing Fn and Tn. The modulation of Fn and Tn may have an important impact on the pathology of epithelial cells during airway inflammation in vivo. PMID- 7541220 TI - Expression of immunoreactive and bioactive activin A protein in adult murine lung after bleomycin treatment. AB - Activin A is a homodimeric protein structurally and functionally related to transforming growth factor beta (TGF-beta), and the expression of activin A is modulated by TGF-beta. Here, we demonstrate the expression of activin A in normal and bleomycin (BLM)-treated murine lungs. ICR mice were treated with BLM intraperitoneally for 10 days, whereas saline vehicle was injected into control mice. Intra-alveolar fibrotic changes were observed in the lung tissue obtained from the mice at day 14 after the final BLM administration. Immunohistochemical studies using a polyclonal antibody to activin A revealed the presence of activin A in the bronchiolar epithelium and smooth muscle cells of veins in both control and BLM-treated mice. In the BLM-treated mice at days 7 and 14, the marked infiltration of immunoreactive alveolar macrophages was observed in the area of fibrotic changes. Bioactivity of activin A measured by erythroid differentiation factor assay in the conditioned medium of alveolar macrophages obtained from BLM treated mice at day 14 was significantly increased. These findings indicate that alveolar macrophages are a potent source of activin A after BLM treatment. The present study demonstrates for the first time the abundant expression of activin A in murine lung tissues after BLM administration, suggesting that activin A may play a role in the pathogenesis of BLM-induced pulmonary fibrosis. PMID- 7541221 TI - Increased expression of transforming growth factor beta isoforms (beta 1, beta 2, beta 3) in bleomycin-induced pulmonary fibrosis. AB - Evidence suggests that transforming growth factor beta (TGF-beta) may play a central role in a variety of fibroproliferative disorders via the induction of extracellular matrix accumulation. The three mammalian TGF-beta isoforms are present in the normal lung, but very little is known about their expression during lung injury and repair. To more fully understand the role of TGF-beta in lung repair, we investigated the expression of the TGF-beta 1, TGF-beta 2, and TGF-beta 3 isoforms in a bleomycin-induced model of pulmonary fibrosis using immunohistochemical and in situ hybridization techniques. We found expression of the three TGF-beta isoforms, in an identical pattern, widely distributed throughout the normal rat lung: in airways, blood vessels, lung parenchyma, and alveolar macrophages. In general, the distribution of TGF-beta mRNA and protein coincided; however, bronchial epithelial cells were a notable exception, exhibiting immunoreactivity but no mRNA expression. During the "inflammatory" phase (days 1 and 3) of bleomycin-induced injury there was an increase in the mRNA and protein expression of all three TGF-beta isoforms in the injured areas, most prominently in parenchymal cells and alveolar macrophages. There was a further increase in TGF-beta isoform expression in the areas of developing fibrosis during the later reparative phase (days 7 and 14), and the bronchial epithelium, previously not expressing TGF-beta mRNA, showed strong expression of mRNA for the three isoforms concomitant with increased immunoreactivity. These findings implicate the three mammalian TGF-beta isoforms in the dysregulated repair process that results in pulmonary fibrosis. Furthermore, the pattern of TGF-beta mRNA and protein expression by the bronchial epithelium suggests that a transition may occur at this site from a paracrine mode of action in the normal lung to an autocrine mode of action during the "reparative" phase of fibrosis. PMID- 7541222 TI - Regulation of nitric oxide release by macrophages after intratracheal lipopolysaccharide. AB - We investigated the effect of intratracheal (i.t.) lipopolysaccharide (LPS) on alveolar macrophage release of nitric oxide. Mice received i.t. LPS at doses ranging from 1 to 100 micrograms/100 g body weight and were killed at serial intervals for bronchoalveolar lavage. Control mice received i.t. phosphate buffered saline. We found that after i.t. LPS, there was an early (1 to 3 days) influx of neutrophils followed by a later (5 to 7 days) influx of macrophages into the lungs. Alveolar macrophages lavaged from mice given i.t. LPS did not spontaneously release nitric oxide (measured as nitrite), but the capacity of these cells to release nitric oxide in vitro in response to interferon-gamma (IFN gamma) or LPS was markedly upregulated. Alveolar macrophages lavaged from mice given i.t. LPS but not i.t. phosphate-buffered saline also expressed mRNA for inducible nitric oxide synthase as measured by semiquantitative reverse transcription polymerase chain reaction. To investigate possible mechanisms for cellular priming for increased nitric oxide release after i.t. LPS, mice were depleted of CD4+ lymphocytes with an anti-CD4 antibody. Alveolar macrophages from CD4-depleted mice given i.t. LPS released significantly less nitric oxide in vitro in comparison to macrophages from nondepleted mice. Additional mice were treated with neutralizing doses of anti-tumor necrosis factor or anti-IFN-gamma antibody before i.t. LPS. Pretreatment with each cytokine antibody decreased but did not eliminate macrophage priming for nitric oxide release after i.t. LPS. We conclude that intratracheal LPS induces mRNA for nitric oxide synthase in alveolar macrophages, priming the cells for increased release of nitric oxide in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541223 TI - Neutrophils activated by granulocyte colony-stimulating factor suppress tumor necrosis factor-alpha release from monocytes stimulated by endotoxin. AB - We investigated the in vitro effects of granulocyte colony-stimulating factor (G CSF) on tumor necrosis factor-alpha (TNF-alpha) release from monocytes. Peripheral blood monocytes and neutrophils were obtained from healthy donors (n = 8). Neutrophils alone, neutrophils plus monocytes, and monocytes alone were incubated with and without G-CSF (10 ng/ml) and were studied for TNF-alpha release; monocytes subsequently were stimulated by endotoxin (lipopolysaccharide [LPS] at 10 and 1,000 ng/ml). Neutrophils alone did not produce TNF-alpha after LPS stimulation irrespective of G-CSF treatment. TNF-alpha release from monocytes was suppressed significantly by pretreatment with G-CSF in the presence of neutrophils (P < 0.01). Suppression of TNF-alpha release after LPS was not observed when monocytes were preincubated with G-CSF in the absence of neutrophils. TNF-alpha release from monocytes stimulated by LPS was not inhibited when monocytes were incubated with the supernatant from G-CSF-activated neutrophils. Pretreatment with G-CSF inhibited intracellular TNF-alpha production, as measured by flow cytometry, of monocytes stimulated by LPS (P < 0.05). These data suggest that neutrophils activated by G-CSF directly suppress TNF-alpha release from monocytes stimulated by LPS. PMID- 7541225 TI - Specific hemoglobin (poly)saccharide recognition. AB - Hemoglobin (Hb) is not a glycoprotein but can easily be glycosylated by a non enzymatic mechanism. Based on some kinetic particularities, consisting in a faster glycosylation of the amine terminal group of the beta-chain located on the allosteric site, a hypothesis of a recognition center for specific sugars has been advanced. Affinity chromatography materials based on epichlorohydrin crosslinked amylose, agarose and dextran (Sephadex), were used. A specific interaction with the CL-amylose (alpha-1,4-glycosidic links) was found, while for Sephadex (alpha-1,6-glycosidic links), no Hb retention was observed. An affinity retention of hemoglobins (human and bovine) on agarose (a galactose containing carbohydrate) has also been established. Chromatographic studies of Hb competitive elution with several monosaccharides (glucose, fructose, galactose) and disaccharides (saccharose, cellobiose, maltose, lactose) indicated that Hb retained by affinity onto CL-amylose columns, can be eluted only with galactose and lactose; with the other tested saccharides, very poor or no desorption at all, has been observed. These new data suggest: (1) a selective recognition of a limited number of (poly)saccharidic materials; and (2) a different behavior towards various sugars, the best interaction being observed with galactose containing sugars (lactose). These aspects fit well with the hypothesis of a hemoglobin recognition center for sugars. PMID- 7541224 TI - A novel class of embryonic cell adhesion glycan epitopes is expressed in human colon carcinomas. AB - A new class of acidic glycans isolated from marine sponges and sea urchin embryos was shown to mediate cell adhesion via carbohydrate-carbohydrate interactions. Cell aggregation blocking monoclonal antibodies (Block 1 and Block 2 mAbs) directed against these polysaccharides localized functional epitopes in embryonic sea urchin gut. Immunofluorescence light microscopy of human colon carcinomas and healthy colon samples with Block 1 and Block 2 mAbs established that the carbohydrate structures similar to the invertebrate acidic glycan adhesion molecules are also expressed in humans. The Block 1 mAb labeled basal and apical lamina of tumor cells, whereas the Block 2 bound exclusively to the apical part of the epithelium. In normal tissue whole goblet cell membrane was stained with both antibodies indicating that transformation leads to spatial rearrangement of glycan antigens. Acidic glycans from human colon carcinomas and normal colon were isolated after delipidation, and complete pronase and DNase digestion, by gel filtration and adsorption to anion exchange membranes. Immunodot assay with Block 1 and Block 2 mAbs revealed that tumor cells have elevated expression of both carbohydrate structures. These results suggest that the acidic glycan adhesion molecules, originally found in sponges and sea urchin embryos, may represent a new class of carbohydrate carcino-embryonal antigens involved in cellular interactions associated with morphogenesis, metastasis and renewal of adult tissue. PMID- 7541226 TI - Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies. AB - The authors have recently described the development of a carboxymethyl dextran based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin-avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e.g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a 'site directed' immobilization strategy for monoclonal antibodies has no advantage. In general the specific activity for optimized systems was approximately 75% for the binding of beta 2 mu-globulin to an immobilized monoclonal antibody directed against beta 2 mu-globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioration of the active site of the antibody. PMID- 7541227 TI - Study of cell-free protein synthesis in aqueous two-phase systems. AB - Protein synthesis in an aqueous two-phase system is reported here as a novel type of extractive bioconversion. Translation of BMV RNA was studied using either rabbit reticulocyte lysate or wheat-germ extract in aqueous dextran-PEG systems. Both polymers inhibited protein synthesis and the translation system partitioned into both phases. In the rabbit reticulocyte two-phase system, protein synthesis reached 30% of that in free solution, while in wheat-germ extract it was 60% of that in free solution. Protein was found mainly in the dextran (lower) phase but its partitioning was related to the polymer concentration, and molecular weight, as well as the ionic strength of the system. Protein synthesis was highly influenced by PEG concentration, potassium chloride concentration, and dextran molecular weight. PMID- 7541228 TI - In search of the 'bio-active conformation'--is it induced by the target cell membrane? AB - Conformations of regulatory peptides interacting with artificial lipid membranes were compared with those of chemically constrained molecules that react selectively with different receptor classes. Striking similarities in the topochemistry of molecules with similar activity were observed. The membrane induced topomers were almost congruent with the artificial topomers that are selectively recognized by the same receptors. Finally, the ideas developed in the membrane compartments theory which allow a quantitative prediction of receptor preference are compatible with our present knowledge of receptor structure. PMID- 7541229 TI - (Patho)physiologic pathways to drug targeting: artificial viral envelopes. AB - The goal of this study was to exploit molecular recognition of cell surface receptors by viral surface glycoproteins as a means for the selective intracellular delivery of macromolecules. To accomplish this, artificial viral envelopes (AVE) resembling the human immunodeficiency virus-1 (HIV-1) were designed as a model system. Recombinant HIV-1 surface glycoprotein gp160 (HIV-1 rgp160) was inserted in the artificial envelope by a two-step detergent dialysis process. The artificial HIV-1 envelope recognized the CD4 cell surface receptor. FITC-dextran and ricin A were employed as model macromolecules as they cannot passively diffuse across cell membranes. Selective transfer of FITC-dextran encapsulated in HIV-1 rgp160 AVE into a CD4-positive cell line (REX-1B) versus a CD4-negative cell line (KG-1) was demonstrated. Ricin A at concentrations as low as 2 ng/ml arrested cell growth of CD4-positive MOLT-4 cells, whereas 8 ng/ml ricin A in solution had no effect on cell growth. The arrest of cell growth was reverted in the presence of excess anti-gp120 monoclonal antibody. Naked envelopes (without HIV-1 rgp160 inserted) were also found to interact with cells and transfer material, although less efficiently and in a non-specific manner. Viral mimicry using AVE may be a means for targeted intracellular delivery of peptides, proteins, enzymes, toxins, oligodeoxynucleotides, gene constructs, and other non-diffusive, labile or toxic macromolecules. PMID- 7541230 TI - Functional conformations of calmodulin: I. Preparation and characterization of a conformational specific anti-bovine calmodulin monoclonal antibody. AB - Calmodulin, similarly to many other Ca(2+)-activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to its high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAM1, CAM2 and CAM4) were purified and characterized. MAb CAM1 was identified as an IgG1 while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAM1 binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAM1 was found to be especially sensitive to the conformational state of calmodulin in the presence of Ca2+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca2+ removal. In addition mAb CAM1 failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM1 and CAM4 share common epitopes with the above molecules. PMID- 7541231 TI - Chaperone-like effect of monoclonal antibodies on refolding of heat-denatured carboxypeptidase A. AB - The availability of monoclonal antibodies which bind to a specific antigen at distinct and well-defined sites has led to a better understanding of the effects of highly specific enzyme-antibody interactions on enzyme behaviour. By appropriate selection it has been possible to isolate those antibodies that are non-inhibitory to biological activity of the enzyme and bind at strategic locations on the antigen molecule, resulting in a considerable stabilization effect on the enzyme conformation. Moreover, such monoclonal antibodies proved to have a chaperone activity leading to a considerable refolding effect on the enzyme which was already partially heat denatured. Renaturation of carboxypeptidase A after heat denaturation in the presence of selected monoclonal antibodies, was followed by recovery of its enzymatic activity. The refolding effect of anti-CPA monoclonal antibodies on heat-denatured enzyme depends on the degree of denaturation of the enzyme and on the location of the antigenic site of each antibody. The additivity effect of the pairs of monoclonal antibodies on the refolding process of CPA proved to be dependent on the localization of the antigenic sites of the monoclonal antibodies studied. PMID- 7541232 TI - Determination of the interactions between lectins and glycoproteins by surface plasmon resonance. AB - A simple and rapid analytical method for detecting interactions between oligosaccharides of glycoproteins and different lectins was studied by surface plasmon resonance using a biosensor (BIAcore). The interactions of three lectins, Sambucus sieboldiana agglutinin (SSA), Ricinus communis agglutinin I (RCA I) and Concanavalin A (Con A) for fetuin and digested fetuins with glycosidases, asialo , agalacto-, and aglucosamino-fetuin, were investigated as a model system. These fetuins were immobilized to the matrix of of the sensor chip and the lectins were injected into the sensor chip cartridge. The association and dissociation reactions could be monitored as resonance signals in real time. The interactions with lectins significantly changed as the oligosaccharides of fetuin were trimmed. The interactions between fetuin and SSA, asialofetuin and RCA I, and aglucosaminofetuin and Con A show the highest affinity properties, respectively. The association constants of these lectins were estimated to be 1.4 x 10(7), 1.9 x 10(8) and 5.3 x 10(7) (M-1), respectively. These results suggested that the interactions between lectins and glycoproteins could be well defined in real time and kinetically, and that the partial structure of oligosaccharides of glycoproteins can be estimated by determination of the interactions with various lectins after glycosidase digestions using the biosensor. PMID- 7541233 TI - Differential CD44 expression patterns in primary brain tumours and brain metastases. AB - Splicing variants of CD44 (CD44v) are increasingly recognised as metastasis promoting factors in rodent and some human cancers. However, the frequency for CD44v expression in human cancers and their metastases and the status of CD44v expression in low or non-metastatic tumours is still uncertain. To address this issue, we investigated CD44 expression patterns in brain metastases (BMTs) spread from more than ten organs and five types of primary brain tumours (PBTs) by Northern blot, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical analysis. The results demonstrated that all of the 56 PBTs examined express standard form of CD44 (CD44s) but none of them express CD44v. In contrast, 22 of 26 BMTs studied were found with CD44v expression. Our data thus present direct evidence of a general distribution of CD44 in BMTs but suggest that such expression is an extremely rare event in PBTs. Therefore, the presence or absence of CD44v expression may be related to high or low metastatic potential of human malignancies. PMID- 7541235 TI - Intensification of chemotherapy for the treatment of solid tumours: feasibility of a 3-fold increase in dose intensity with peripheral blood progenitor cells and granulocyte colony-stimulating factor. AB - Dose intensity may be an important determinant of the outcome in cancer chemotherapy, but is often limited by cumulative haematological toxicity. The availability of haematopoietic growth factors such as granulocyte colony stimulating factor (G-CSF) and of peripheral blood progenitor cell (PBPC) transplantation has allowed the development of a new treatment strategy in which several courses of high-dose combination chemotherapy are administered for the treatment of solid tumours. PBPCs were mobilised before chemotherapy using 12 or 30 micrograms kg-1 day-1 G-CSF (Filgrastim) for 10 days, and were collected by 2 5 leucaphereses. The yields of mononuclear cells, colony-forming units and CD34 positive cells were similar at the two dose levels of Filgrastim, and the numbers of PBPCs were sufficient for rescue following multiple cycles of chemotherapy. High-dose chemotherapy (cyclophosphamide 2.5 g m-2 for 2 days, etoposide 300 mg m 2 for 3 days and cisplatin 50 mg m-2 for 3 days) was administered sequentially for a median of three cycles (range 1-4) to ten patients. Following the 30 evaluable cycles, the median duration of leucopenia < or = 0.5 x 10(9) l-1 and < or = 1.0 x 10(9) l-1 was 7 and 8 days respectively. The median time of thrombopenia < or = 20 x 10(9) l-1 was 6 days. There was no cumulative haematological toxicity. The duration of leucopenia, but not of thrombopenia, was inversely related to the number of reinfused CFU-GM (granulocyte-macrophage colony-forming units). In the majority of patients, neurotoxicity and ototoxicity became dose limiting after three cycles of therapy. However, the average dose intensity delivered was about three times higher than in a standard regimen. The complete response rate in patients with small-cell lung cancers was 66% (95% CI 30-92%) and the median progression-free survival and overall survival were 13 months and 17 months respectively. These results are encouraging and should be compared, in a randomised fashion, with standard dose chemotherapy. PMID- 7541234 TI - Clinical evaluation of seven tumour markers in lung cancer diagnosis: can any combination improve the results? AB - In this study we compared the diagnostic utility of: (1) neuron-specific enolase (NSE); (2) squamous cell carcinoma antigen (SCC); (3) carcinoembryonic antigen (CEA); and (4) cytokeratin markers (CYFRA 21-1, TPA, TPM, TPS) in patients with small-cell lung cancer (SCLC) (21 cases) and non-small-cell lung cancer (94 cases). For comparison we also studied 66 patients with benign lung diseases and nine with pleural mesothelioma. NSE levels in SCLC patients were significantly higher than those in all the other groups studied. No significant variations were found among the SCC levels in all groups. CEA levels in patients with adenocarcinoma were significantly higher than those in all other groups studied. CYFRA 21-1 serum levels significantly increased in patients with squamous cell carcinoma and mesothelioma, while TPA, TPS and TPM increased in patients with lung cancer irrespective of the histological type. In patients with SCLC, high levels of all markers except SCC were found when the disease was extensive. In patients with non-SCLC, the highest levels of all tumour markers were usually found in those with advanced disease, although CYFRA 21-1 gave a sensitivity of 44% when a specificity of 95% was fixed in stage I non-SCLC patients. An analysis of receiver operating characteristic curves revealed that the highest diagnostic accuracies in distinguishing benign from malignant lung diseases were achieved with TPM (81%), CYFRA 21-1 (72%), CEA (78%) or TPA (78%) when using cut-off values of 46 Ul-1, 3.0 micrograms l-1, 2.0 micrograms l-1 and 75 Ul-1 respectively. When all patients were considered, the combined evaluation of more than one marker did not significantly improve the results obtained with TPM alone. However, taking into consideration the fact that CYFRA 21-1 is the most sensitive index of early lung tumours and that its combined determination with TPM did not worsen the overall sensitivity and specificity of the latter, the combined use of these two markers may be suggested as a useful took for the diagnosis of lung tumours. PMID- 7541236 TI - Ibuprofen reduces energy expenditure and acute-phase protein production compared with placebo in pancreatic cancer patients. AB - The aim of this study was to investigate the effect of the cyclo-oxygenase inhibitor ibuprofen on the acute-phase protein response and resting energy expenditure (REE) of weight-losing patients with pancreatic cancer. Patients with irresectable pancreatic cancer (n = 16) were treated with either ibuprofen (1200 mg day-1 for 7 days (n = 10) or placebo (n = 6). A group of 17 age-related non cancer subjects were also studied. Indirect calorimetry, anthropometry, multifrequency bioelectrical impedence analysis and serum C-reactive protein (CRP) estimation were performed immediately before and after treatment. Before treatment, total REE was significantly elevated in the pancreatic cancer patients compared with healthy controls (1499 +/- 71 vs 1377 +/- 58 kcal) (P < 0.02). Following treatment the mean REE of the ibuprofen group fell significantly (1386 +/- 89 kcal) compared with pretreatment values (1468 +/- 99 kcal) (P < 0.02), whereas no change was observed in the placebo group. Serum CRP concentration was also reduced in the ibuprofen-treated group (pre-ibuprofen, 51 mg l-1; post ibuprofen, 29 mg l-1; P < 0.05). These results suggest that ibuprofen may have a role in abrogating the catabolic processes which contribute to weight loss in patients with pancreatic cancer. PMID- 7541238 TI - Nitric oxide synthase activity in human breast cancer. AB - Nitric oxide (NO) is generated by a family of isoenzymes (NO synthases) expressed in a wide range of mammalian cells. We have recently reported NO synthase expression in human gynaecological cancers. In this study we have assessed the activity and distribution of NO synthase in a series of human breast tumours and in normal breast tissue. Calcium-dependent (constitutive) and -independent (inducible) NO synthase activity, as well as NO biosynthesis, was high in invasive tumours compared with benign or normal tissue. Furthermore, for invasive ductal carcinomas, NO biosynthesis was significantly greater for grade III compared with grade II tumours. Immunohistochemical investigations revealed immunolabelling with a monoclonal antibody to murine inducible NO synthase predominantly within tumour-associated macrophages. Immunolabelling with a polyclonal antiserum raised against rat brain NO synthase was also observed in vascular endothelial and myoepithelial cells. Thus NO synthase is expressed in human breast tumours, where its presence correlates with tumour grade. PMID- 7541237 TI - Immunohistochemical expression of tenascin in normal stomach tissue, gastric carcinomas and gastric carcinoma in lymph nodes. AB - The immunohistochemical expression of tenascin was examined in the normal adult mucosa of the stomach, primary tumours and lymph node metastases of gastric cancer patients. In normal gastric tissue tenascin was expressed in the muscularis mucosae, muscularis propria and vessel walls, however it was not expressed in either the mucosal connective tissue or the stromal tissue in the submucosal layer. In gastric cancer, tenascin was expressed in 35 of 85 primary tumours, and in 8 of 25 metastases in lymph nodes. Tenascin was located in the fibrous stroma surrounding foci of cancer. The expression of tenascin in the primary tumour did not correlate with the depth of invasion, lymph node metastasis or prognosis. Tenascin appears during the process of either malignant transformation or tumour progression in gastric cancer, and the positive expression of tenascin may be useful as a stromal marker for the early detection of gastric cancer. PMID- 7541239 TI - Distinct biogenesis mechanisms for the water channels MIWC and CHIP28 at the endoplasmic reticulum. AB - MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 2097, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell free (rabbit reticulocyte lysate+ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with N- and C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]-[HR2]-P.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541241 TI - Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels. AB - Ion channels from retinal rods and a variety of other cells are directly gated by cyclic nucleotides. The rod channel is known to contain a 63-kDa subunit, and there is molecular genetic evidence for the existence, in human retina, of a second subunit with a deduced molecular mass of about 100 kDa. When purified from bovine rods, the channel consists of the 63-kDa subunit and a 240-kDa associated protein that has been shown recently to contain a version of the cloned second subunit as part of a larger complex. We had previously shown that a photoaffinity analog of cGMP, 8-(p-azidophenacylthio)-[32P]cGMP, specifically labels both the 63- and 240-kDa proteins. Here the analog was used to identify cGMP-binding regions and amino acid contact points within these proteins. The specific labeling of the 63-kDa subunit was localized to a 66 amino acid fragment (Tyr-515 Met-580) that is contained entirely within a 110 amino acid region proposed to be the cGMP-binding site on the basis of homology with other cyclic nucleotide binding proteins. Within this fragment, amino acid residues Val-524, Val-525, and Ala-526 were found to contain label. These residues are part of a larger hydrophobic cluster that appears to line the binding pocket. The results also indicate that the 240-kDa protein contains a similar cGMP-binding site. Sequencing of a specifically labeled 8-kDa fragment through 16 amino acid residues indicated that the fragment was derived from the portion of the 240-kDa complex that contains the second subunit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541240 TI - SNX-325, a novel calcium antagonist from the spider Segestria florentina. AB - A novel selective calcium channel antagonist peptide, SNX-325, has been isolated from the venom of the spider Segestria florentina. The peptide was isolated using as bioassays the displacement of radioiodinated omega-conopeptide SNX-230 (MVIIC) from rat brain synaptosomal membranes, as well as the inhibition of the barium current through cloned expressed calcium channels in oocytes. The primary sequence of SNX-325 is GSCIESGKSCTHSRSMKNGLCCPKSRCNCRQIQHRHDYLGKRKYSCRCS, which is a novel amino acid sequence. Solid-phase synthesis resulted in a peptide that is chromatographically identical with the native peptide and which has the same configuration of cysteine residues as the spider venom peptide omega-Aga-IVa [Mintz, I. M., et al., (1992) Nature 355, 827-829]. At micromolar concentrations, SNX-325 is an inhibitor of most calcium, but not sodium or potassium, currents. At nanomolar concentrations, SNX-325 is a selective blocker of the cloned expressed class B (N-type), but not class C (cardiac L), A, or E, calcium channels. SNX-325 is approximately equipotent with the N-channel selective omega conopeptides (GVIA and MVIIA as well as closely related synthetic derivatives) in blocking the potassium induced release of tritiated norepinephrine from hippocampal slices (IC50s, 0.1-0.5 nM) and in blocking the barium current through cloned expressed N-channels in oocytes (IC50s 3-30 nM). By contrast, SNX-325 is 4 5 orders of magnitude less potent than is SNX-111 (synthetic MVIIA) at displacing radioiodinated SNX-111 from rat brain synaptosomal membranes. SNX-325 will be a useful comparative tool in further defining the function and pharmacology of the N- and possibly other types of high-voltage activated calcium channels. PMID- 7541243 TI - Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P identified by in vitro selection. AB - Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P (P RNA) have been obtained by in vitro selection. The selection method involves cleavage of a circular RNA library by the P RNA, isolation of the linear cleavage product, and regeneration of circular RNA to allow amplification and multiple cycles of selection. The use of circular RNA ensures that potential substrates can be selected without restricted location of the cleavage site. Such a selection method has been used previously to isolate RNA motifs that undergo autolytic cleavage with Pb2+ [Pan, T., & Uhlenbeck, O. (1992) Biochemistry 31, 3887-3895]. The circular RNA pool after eight cycles of selection was cleaved by the B. subtilis P RNA as efficiently as a pre-tRNA(Phe) substrate, estimated to be more than 10 orders of magnitude better than the unselected RNA library. Kinetic analysis of individual variants showed that the kcat/KM of the selected RNA was up to 4-fold higher than that of the pre-tRNA(Phe). When cleavage was carried out with Escherichia coli P RNA, the selected RNA was 10-60-fold less reactive than the reaction of the pre-tRNA(Phe). Two distinct classes of variants are selected, both of which appear to differ significantly from the known P RNA substrates. Terminal truncation experiments suggest that a large number of nucleotides in the class I variants can be deleted without affecting the cleavage activity. The resulting minimal class I substrates contain a short stem-loop with no other apparent helical structures. The class II substrates are cleaved within a putative helical stem that is formed entirely by the primer sequences.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541244 TI - Structural, physical, and biological characteristics of RNA.DNA binding agent N8 actinomycin D. AB - The crystal structure of the self-complementary DNA octamer d(GAAGCTTC)2 complexed with N8-actinomycin D (N8AMD) has been determined at 3.0 A resolution (space group: P3(1)21; unit cell: a = 62.30, b = 62.30, c = 42.97 A; R = 0.173 for 1845 reflections). The DNA structure was severely distorted by the N8AMD bound intercalatively into the middle dinucleotide, 5'-GC-3'. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base--peptide and chromophore--backbone hydrogen bonds. The complexes are stacked together to form a pseudocontinuous helix running through the crystals. The structure of d(GAAGCTTC)2-actinomycin D (AMD) crystallized in the space group C2 [Kamitori S., & Takusagawa, F. (1992) J. Mol. Biol. 225, 445-456] was re-refined in order to compare it directly to the N8AMD complex structure. The asymmetrical binding mode of AMD has been confirmed on the basis of the two complex structures. The crystal structures of the N8AMD and AMD complexes bound to the same d(GAAGCTTC)2 differed by a root-mean-square deviation on all atom positions of 1.77 A, but most of the structural differences can be attributed to molecular packing in two different crystal forms, and not to structural differences induced by the interaction with the intercalating agents. However, the DNA binding and biological characteristics of N8AMD and AMD are quite different from each other. The DNA association constant of N8AMD is 33-fold less than that of AMD in an aqueous solution. N8AMD required a concentration > 10.0 microM to inhibit RNA synthesis activity in HeLa cells by 50%, whereas AMD reached to the same inhibitory level at only 35 nM. The structure of the DNA-N8AMD complex suggested that substitution of the N-methyl-L valine residue in the cyclic depsipeptide with a N-methyl-D-valine residue might increase the hydrophobic interaction with the minor groove of the DNA. Thus the DNA association constant and RNA synthesis inhibitory activities of 5,5'-N-methyl D-valine AMD (D-MeVal-AMD) have also been determined. The DNA association constant of D-MeVal-AMD is more than 2-fold greater than that of AMD, and the RNA synthesis inhibitory activity is about 20-fold greater. PMID- 7541242 TI - Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C. AB - A series of recombinant (r) chimeric mutants of human coagulation protein C (PC) and activated protein C (APC) containing replacements of homologous PC domains by those of human coagulation factor IX (fIX) were generated, with the intention of determining whether the specific bovine aortic endothelial cell (BAEC) receptor binding characteristics of fIX could be incorporated into the chimeric r-PC while maintaining the essential properties of PC and APC. Using a competitive BAEC displacement assay with [125I]fIX, we found that a chimeric r-PC (r[delta PC1 46/delta fIX1-47]PC), consisting of the entire gamma-carboxyglutamic domain ([GDIX], residues 1-38) and helical stack ([HSIX], residues 38-47) of fIX as replacements for these same domains of PC, provided an IC50 for fIX-related BAEC binding of 13 nM, as compared to 10 nM for that of unlabeled fIX. This showed that all of the BAEC tight binding determinants for fIX existed within the [GDIX/HSIX]. Additionally, this chimera reacted to the same extent as fIX with the Ca(2+)-dependent, [GDIX]-specific monoclonal antibody H5B7 and lost its reactivity to a similar antibody specific for the [GDPC], JTC1. A synthetic peptide containing residues 1-47 of fIX also competed effectively (IC50 = 16 nM) with intact fIX for BAEC binding. Displacement of [125I]fIX from BAEC did not occur with a chimera containing the [HSIX] alone or with another mutant protein possessing a replacement of the two epidermal growth factor (EGF) homology regions of r-PC (residues 47-137) with those same domains of fIX.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541246 TI - Mitochondrial antigens, molecular mimicry and autoimmune disease. AB - The immune system is normally tolerant to mitochondrial self-antigens, but responsive against bacteria. Low-titre anti-mitochondrial antibodies (AMA) might be involved in this discrimination. Tolerance is broken in diseases characterised by high titre AMA. Some of these AMA, against cardiolipin, cross-react with DNA. The best studied AMA are those characterising primary biliary cirrhosis (PBC). These are directed against E2 subunits of the oxo-acid dehydrogenase complexes, and also against subunits E1 alpha, E1 beta and X of the pyruvate dehydrogenase complex. AMA of PBC patients also react with bacterial E2s. Reactivities are primarily peptide-specific but with cross-reactivity between mitochondrial and microbial antigens and between E2s of respective complexes. Immunodominant epitopes, for anti E2 AMA, include the conserved sequence flanking the site of lipoyl attachment. It is proposed that the initial stimulus for antibody production is chronic urinary tract infection. AMA themselves are not pathogenic, but CD4+ T-cells would be primed, recognising the lipoyl domain epitope in association with class II HLA. Inappropriate expression of class II antigens on bile duct epithelia, (as found in PBC), might lead to presentation of a particular fragment of HLA-DR alpha, known to be a major MHC presented self peptide in the mouse. That sequence strongly mimics the lipoyl domain and might be recognised by primed T-cells, initiating the autoimmune cascade. In the mouse, a peptide of ND1 of Complex I is presented in association with class I MHC. Cells exhibiting somatic mutation of such a peptide might thus be subject to attack by CD8+ T-cells. If such peptides were presented by class II HLA, autoimmune diseases might arise, related to mimicry between such peptides and microbial sequences and/or self-antigens. These considerations might apply in Leber's disease and in age-related pathology. PMID- 7541247 TI - A desktop, low-cost video fluorometer for quantitation of macromolecules after gel electrophoresis. AB - After gel electrophoresis, quantification of in-gel fluorescence is sometimes used to assay the macromolecules fractionated. Both procedures and equipment that have the following improvements are presented here for direct video fluorometry of gels used for electrophoresis: comparatively low cost, high ease of use and low consumption of space. This equipment has a linear response to the amount of ethidium-stained DNA that forms a band in an agarose gel. PMID- 7541248 TI - Versatile procedure of multiple introduction of 8-aminomethylene blue into oligonucleotides. AB - The coupling of 8-aminomethylene blue to oligonucleotides via poly-L-glutamic acid linker using carboxy-anchor groups will be described. The introduction of carboxy-anchor groups into oligonucleotides proceeds both during automated synthesis using 6-(ethoxycarbonyl)hexyl 1-O-phosphoramidite and by reaction of 5' amino-functionalized oligonucleotides with succinic anhydride. O-(N-Succinimidyl) 1,1,3,3-tetramethyluronium tetrafluoroborate was used as activating reagent for binding of poly-L-glutamic acid to the carboxylated oligonucleotides. The successful 5'-carboxylation and poly-L-glutamic acid coupling were proven both by polyacrylamide gel electrophoreses and HPLC. 8-Aminomethylene blue in its leucoform was covalently coupled to the oligonucleotides in the presence of water soluble carbodiimide. PMID- 7541245 TI - Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats. AB - Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decarboxylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt) to 3-4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2'-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140-240%) in the relative activity of Tyr k of three mucosal proteins with MI of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF R. Daily injection of tyrphostin (300 micrograms/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-alpha in isolated colonocytes. In addition, the levels of TGF-alpha and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF alpha through an autocrine mechanism. PMID- 7541250 TI - tRNAs as primer of reverse transcriptases. AB - Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I). PMID- 7541249 TI - Conjugation of adenine arabinoside 5'-monophosphate to arabinogalactan: synthesis, characterization, and antiviral activity. AB - A conjugate consisting of the antiviral nucleotide analogue adenine arabinoside 5'-monophosphate (araAMP, vidarabine monophosphate) and the naturally occurring polysaccharide arabinogalactan was synthesized. The conjugate consisted of 7.9 araAMP residues per molecule of arabinogalactan. The proposed structure of the conjugate was consistent with 13C NMR spectroscopic studies. Daily injections of the conjugate, at a dose of 3 mg of araAMP/kg, into woodchuck carriers of woodchuck hepatitis virus (WHV) decreased serum levels of WHV DNA. A dose of 3 mg/kg of unconjugated araAMP was ineffective, while a higher dose of araAMP (15 mg/kg, 14 days) produced a drop in WHV DNA. After cessation of dosing with the conjugate, serum viral DNA levels remained depressed for 42 days. In contrast, after cessation of dosing with araAMP, WHV DNA rapidly returned to original levels. PMID- 7541252 TI - Posttranscriptionally modified nucleosides in transfer RNA: their locations and frequencies. PMID- 7541253 TI - Nucleotide modification and base conversion of RNA. Part II. Proceedings of a EMBO-CNRS workshop. Aussois, 4-8 May 1994. PMID- 7541251 TI - Structures of posttranscriptionally modified nucleosides from RNA. PMID- 7541254 TI - Implications of a functional large ribosomal RNA with only three modified nucleotides. AB - The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both. PMID- 7541255 TI - Aminoacylation of transfer RNAs with 2-thiouridine derivatives in the wobble position of the anticodon. AB - The first position or 'wobble base' in the anticodon of tRNAs is frequently the site of post-transcriptional modification. In Escherichia coli, glutamine, glutamate, and lysine tRNAs contain 2-thiouridine derivatives in this position, and the significance of these modifications has been under investigation since their discovery. Here we describe the investigations to link 2-thiouridine derivatives to aminoacylation of these tRNAs. The implications of these findings on the evolution of specificity of aminoacyl-tRNA synthetases and on translational regulation are also discussed. PMID- 7541258 TI - Controlled trial of different dosages of octreotide in the prevention of hyperamylasemia induced by endoscopic papillosphincterotomy. AB - Pancreatic reaction after endoscopic papillosphincterotomy (EPT) is a common event occurring in about 70% of cases. Acute pancreatitis may also develop in 1% 6% of cases. Previous attempts to prevent this reaction with an inhibitor of exocrine pancreatic secretion such as somatostatin provided conflicting results. The somatostatin long-acting analogue octreotide has recently proposed for the prevention of ERCP/EPT-induced pancreatic reaction. Therefore we tested the prophylactic effect of a subcutaneous administration of octreotide in two different dosages in 60 consecutive patients undergoing EPT for common bile duct stones and benign papillary stenosis. They were given either octreotide 0.2 mg (20 cases), or octreotide 0.1 mg (20 cases), or placebo (20 cases) before the procedure. Serum amylase levels were determined at baseline and 2, 4, 8 and 24 hours thereafter. The differences were statistically significant at 2 hours between subjects pretreated with octreotide 0.2 mg and control subjects (p = 0.01); at 4 and 8 hours after the procedure between both octreotide-treated groups and control subjects (octreotide 0.1 mg: p < 0.05, at 4 and 8 hrs; octreotide 0.2 mg: p = 0.01, at 4 hrs, and p < 0.01, at 8 hrs). In patients with previous episodes of relapsing pancreatitis, the increase in serum amylase was significantly reduced in the octreotide 0.2 mg group vs control group, at 4 hrs (p < 0.05) and 8 hrs (p < 0.05). Our data suggest that octreotide 0.2 mg has a greater prophylactic efficacy than 0.1 mg in reducing pancreatic reaction after EPT. PMID- 7541257 TI - Effect of continuous subcutaneous administration of a small dose of granulocyte colony stimulating factor (G-CSF) by the use of a portable infusion pump in patients with non-Hodgkin's lymphoma receiving chemotherapy. AB - The effects of continuous subcutaneous infusion (CSI) of human granulocyte-colony stimulating factor (G-CSF) on the absolute neutrophil count (ANC) and serum G-CSF level were examined in 11 patients with non-Hodgkin's lymphoma (NHL) during cytotoxic chemotherapy. Recombinant G-CSF (rG-CSF) was subcutaneously infused using a portable infusion pump at a constant flow rate of 1 microgram/20 microliters/h for 14 days starting 2 days after the end of the second course of chemotherapy. The ANC was lowered after the chemotherapy without rG-CSF infusion whereas the duration of neutropenia and the nadir level of the ANC after the chemotherapy were ameliorated by the combined administration of rG-CSF (mean +/- S.E., 0.6 +/- 0.5 days vs. 4.7 +/- 1.9 days, P < 0.05; 455 +/- 135/microliter vs. 1906 +/- 598/microliter, P < 0.05). Serum G-CSF levels increased after the start of rG-CSF infusion, reaching a mean peak value of 418.5 +/- 128.5 pg/ml at the 8th day, and then returned to the basal level (35.6 +/- 13.5 pg/ml) immediately after the end of continuous infusion of rG-CSF. Although a slight increase in serum G-CSF was obtained in the patients after the chemotherapy without rG-CSF administration, the mean serum level was much lower than that in the patients after the chemotherapy with rG-CSF administration (88.2 +/- 24.8 pg/ml vs. 199.6 +/- 20.6 pg/ml, P < 0.01). No notable side effects of the CSI of rG-CSF were noted.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541256 TI - Differential response of human basophils and mast cells to recombinant chemokines. AB - Chemokines are proinflammatory peptides regulating the functions of various hematopoietic cells. We have analyzed the effects of seven recombinant human (rh) chemokines (MCAF, RANTES, MIP-1 alpha, MIP-1 beta, IL-8, GRO, and IP-10) on the growth and function of human basophils and mast cells. We found that MCAF, but not RANTES, MIP-1 alpha, MIP-1 beta, IL-8, GRO, or IP-10, causes direct and dose dependent histamine release from basophils (MCAF, 5 micrograms/ml: 26.9 +/- 3.4%; other chemokines: < 5% of total histamine). An increased (2.1 to 3.5-fold) response to MCAF was obtained when basophils were preincubated with rh interleukin-3 (100 units/ml). Moreover, IL-3-primed basophils became responsive to physiologic concentrations (< 1 microgram/ml) of MCAF, IL-8, and RANTES. None of the chemokines tested was able to induce histamine secretion in mast cells obtained from lung (n = 2), skin (n = 1), uterus (n = 3), or tonsils (n = 3), even when cells had been preincubated with the mast cell agonist SCF. The chemokines also failed to modulate the expression of activation antigens (CD11b/C3biR, CD25/IL-2R beta, CD63, IL-3R alpha, CD117/c-kit) on the mast cell line HMC-1 or the basophil cell line KU-812 and were unable to induce differentiation of basophils or mast cells in culture. Together, our results show that basophils respond to rhIL-8, rhMCAF, and rhRANTES and that, unlike human basophils, human mast cells are unresponsive to recombinant chemokines. PMID- 7541260 TI - Confocal microscopy of thick sections from acrylamide gel embedded embryos. AB - The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10-15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 microns. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water-soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water-miscible media. PMID- 7541261 TI - Nerve growth factor receptor and neurochemical markers in human oral mucosa: an immunohistochemical study. AB - BACKGROUND: The innervation of the oral mucosa has so far been studied mainly by histochemical and ultrastructural techniques. Only few studies have investigated the presence of neural proteins and neurotransmitters in human gingival mucosa. OBJECTIVE: The purpose of the present study was to evaluate the presence and distribution of neural structural and transmitter proteins in different areas of normal human oral mucosa. METHOD: Indirect immunofluorescence was employed on specimens taken from different mucosal regions (gingiva, lips, gums, palate). Both structural (low-affinity nerve growth factor receptor, NGFr; protein gene product 9.5, PGP 9.5) and neuropeptide markers (substance P; calcitonin gene related peptide; vasoactive intestinal peptide, neuropeptide Y) were used. RESULTS: NGFr and PGP 9.5 intensely labelled both nerve fibres and selected epithelial cells, while neuropeptide immunoreactivity was scarcely expressed and exclusively localized in nerve fibres. CONCLUSIONS: Similarly in the distribution pattern and neurochemistry between oral and cutaneous innervation is apparent. Expression of NGFr could be relevant to the trophism of both the oral innervation and epithelium. PMID- 7541262 TI - 'Flagellate' erythema in dermatomyositis. AB - We report a case presenting with 'flagellate' erythema which is a characteristic skin lesion of dermatomyositis seen in Japan. A possible pathological mechanism is proposed in comparison with the 'flagellate' erythema caused by bleomycin. PMID- 7541259 TI - Subclinical exocrine pancreatic derangement in human diabetic patients evaluated from pure pancreatic juice. AB - To elucidate the mechanism responsible for histological derangement of pancreas in diabetic patients, pure pancreatic juice (PPJ) aspirated directly from pancreatic duct was analyzed before and after strict glycemic control on poorly controlled diabetic patients without clinical exocrine pancreatic dysfunction. PPJ obtained from 18 diabetics showed a significantly decreased amylase activity compared with ten healthy subjects (109 +/- 4 versus 168 +/- 9 U/mg protein, p < 0.005) in spite of negligible changes in lipase activity, protein concentration, bicarbonate concentration, and its volume. PPJ showed higher concentrations of prostanoids in diabetic patients than in healthy subjects (TXB2, 259 +/- 48 versus 118 +/- 30 pg/mL, p < 0.05; 6-keto-PGF1 alpha, 52.6 +/- 11.5 versus 38.5 +/- 7.5 pg/mL, p > 0.05). Ratio of 6-keto-PGF1 alpha/TXB2 of PPJ in diabetic patients was significantly lower than in healthy subjects (0.195 +/- 0.016 versus 0.422 +/- 0.041, p < 0.005). After strict glycemic control for 1-3 months on nine of 18 diabetic patients, amylase activity of PPJ was significantly higher than that before the control (112 +/- 4 versus 128 +/- 7 U/mg protein, p < 0.01), but still significantly lower than that of healthy subjects (p < 0.005). Lipase activity showed no significant difference between before and after the control. TXB2 and 6-keto-PGF1 alpha concentrations were decreased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541263 TI - Immunohistochemical study on the distribution of galanin-containing nerves in the chicken pancreas. AB - The distribution of galanin-containing nervous elements in the chicken pancreas was investigated by use of immunohistochemical method. Galanin-immunoreactive nerve fibres formed a perivascular plexus and a dense network in the subepithelial layer of secretory ducts. The muscle layers of secretory ducts were also supplied with galanin-immunoreactive nerve fibres. The exocrine pancreas received a supply of varicose nerve fibres showing galanin immunoreactivity. In the endocrine part, B-islets were innervated by galanin-immunoreactive nerve fibres, whereas A-islets received fewer nervous elements. Double staining combined with the immunofluorescence method for galanin and acetylcholinesterase histochemistry showed at least two types of ganglion cells in the interlobular connective tissue; one showing both acetylcholinesterase activity and galanin immunoreactivity, and the other showing acetylcholinesterase activity only. The present results demonstrate that the chicken pancreas is innervated by galanin containing nerves of intrinsic origin and suggest that galanin coexists with acetylcholine in the chicken pancreas. PMID- 7541264 TI - Mosaic lectin labelling in the quail collecting ducts. AB - Morphological and histoenzymological differences have been observed between intercalated and principal cells of the quail Coturnix coturnix japonica collecting ducts. The present study was designed to shed light on the lectin affinity of the collecting duct cells within cortex and medulla by the use of HRP labelled lectins combined with glycosidase degradation. Binding of PNA and RCA-I lectins consequent to enzymatic release of sialic acid revealed abundant sialylated carbohydrate moieties within the principal cell cytoplasm. This characteristic binding pattern differed considerably from the staining observed in the intercalated cells. Interesting information also emerged about the presence of sialoglycoconjugates having the terminal disaccharide sialic acid beta-N-acetylgalactosamine originating from the increased SBA binding and the unmodified DBA labelling after removal of sialic acid. Sequential degradation by sialidase/beta-galactosidase followed by incubation with DBA offered the possibility to suspect that the receptor sugar for the penultimate beta-galactose may be N-acetylgalactosamine. Conversely, we were not able to define the accept sugar for penultimate beta-GalNAc owing to the lack of availability of beta-N acetylgalactosaminidase enzyme. When although further studies are clearly needed to elucidate the physiological role of the cellular sialoglycoconjugates detected, the present results already provide valuable insight into the carbohydrate composition of intercalated and principal cells in the quail collecting ducts. PMID- 7541265 TI - A morphometric and statistical study of the effects of soft laser (He-Ne) irradiation on the pineal gland. AB - Thirty-six male Wistar rats (225g average b.w.) were studied after direct He-Ne laser irradiation (632.8 nm, 5 mW) of the surgically-exposed pineal gland. Total irradiation time was 5 minutes, with rest intervals of 1 minute for every minute of irradiation. The animals were sacrificed in groups of 4 (controls, irradiated animals and sham-operated rats--i.e., craniotomy without irradiation) on days 3, 7 and 10 postirradiation. A significant increase was seen (p < 0.0001) in the medullary and cortical karyometric indices of the pineal body in all experimental, control and sham-operated animals. The increase was greatest (over 50%) on day 3 postirradiation among the experimental animals, with respect to the control and sham-operated groups. A progressive and significant decrease in karyometric index (p < 0.05) was in turn observed on days 7 and 10 postirradiation, although the values remained higher than among the controls. Ultrastructurally, there were cytoplasmic signs of an increase in metabolic activity in the experimental animals on days 3 and 7, followed by a decrease in activity by day 10 with the appearance of numerous lipid droplets, pericanallicular dark cells and mesoglial cells. We suggest that laser irradiation stimulates cortical and medullary pinealocytes, followed by a decreased effect at day 10 postirradiation. The effect of laser light is in turn determined by experimental action and the duration of exposure. PMID- 7541267 TI - [A mutant form of bacteriophage T7 RNA polymerase capable of catalyzing RNA and DNA synthesis]. PMID- 7541266 TI - Effects of L-trans-pyrrolidine-2,4-dicarboxylate and L-threo-3-hydroxyaspartate on the binding of [3H]L-aspartate, [3H]alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionate (AMPA), [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainate studied by autoradiography in rat forebrain. AB - L-trans-Pyrrolidine-2,4-dicarboxylate (L-t-PDC) and L-threo-3- hydroxyaspartate (L-t-3OHA), compounds known to interact strongly with the Na(+)-dependent high affinity uptake of excitatory amino acids in central nervous tissue, were tested as potential inhibitors of binding to glutamate receptors and transport sites in frozen sections of rat brain. [3H] alpha-amino-3-hydroxy- 5-methyl-4 isoxazolepropionate (AMPA), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and [3H] kainate were used as ligands for the binding sites on the "non-NMDA" classes of glutamate receptors and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653) was used to label NMDA receptor binding sites. The Na(+)-dependent glutamate-uptake site was marked by [3H]L-aspartate. The autoradiograms, obtained by exposing 3H-sensitive film to sections of rat forebrain preincubated with 3H labelled ligands, were scanned by laser beam and quantified. Distribution patterns of the receptor and transporter sites visualized by the 3H-labelled ligands were compatible with previously published results. [3H]CNQX binding, however, was found to be significantly decreased by Na+.L-t-3OHA was about an order of magnitude stronger than L-t-PDC as an inhibitor of [3H]L-aspartate binding. Neither of the compounds had any important effect at the "non-NMDA" receptor binding sites but L-t-3OHA was a weak inhibitor of [3H]CGP 39653 binding (< 40% at 100 microM). The results suggest that, at low nanomolar concentrations, both compounds are likely to be selective for Na(+)-dependent high affinity glutamate transporter sites. Moreover, L-t-3OHA seems to have a sufficiently high affinity for the site to be almost certainly useful, if available in a 3H labelled form, as a ligand in autoradiographic studies. PMID- 7541268 TI - G-CSF after autologous bone marrow transplantation for malignant diseases in children. Spanish Working Party for Bone Marrow Transplantation in Children. AB - The use of recombinant human granulocyte-stimulating factor (G-CSF) has been shown to effectively accelerate granulocytic recovery after autologous bone marrow transplantation (BMT) in adults. The experience, however, is limited in children. We evaluated the hematopoietic reconstitution in 41 consecutive children undergoing autologous BMT for hematologic malignancies (21 acute lymphoblastic leukemia, five non-Hodgkin's lymphoma) and solid tumours (seven neuroblastoma, two brain tumor, three Ewing's sarcoma, two Wilms' tumor, one rhabdomyosarcoma). Their ages ranged from 2 to 16 years (mean 7.2 years). rhG-CSF was given at a dose of 10 micrograms/kg/day i.v. in a 2h infusion from day +1 until +28 or until the absolute neutrophil count (ANC) was > 1 x 10(9)/L. These patients were compared with a similar historical control group of 38 children who did not receive rhG-CSF after autologous BMT. The number of cells infused was similar in both groups. At the dose and schedule used in the present study, rhG CSF was well tolerated and no side-effects were observed. The number of cell infused was similar in both groups. At the dose and schedule used in the present study, rhG-CSF was well tolerated and no side-effects were observed. Our data show that rhG-CSF accelerates engraftment and reduces the number of febrile days and antibiotic use. Furthermore, patients who were treated had less infections. PMID- 7541269 TI - Quantitative CD34 analysis may be used to guide peripheral blood stem cell harvests. AB - The duration of neutropenia and thrombocytopenia after high-dose chemotherapy has improved since the introduction of myeloid growth factors and peripheral blood progenitor cells (PBPC), yet there remains a subset of patients who have delayed hematopoietic recovery. Currently, there ar no established, reliable parameters which may be used to guide stem cell harvests. We investigated the utility of measuring harvested CD34 positive cell populations by flow cytometry. From March 1990 to July 1993, 30 women with advanced breast cancer underwent therapy with high-dose cyclophosphamide and thiotepa and stem cell rescue. Patients received either cyclophosphamide (CY) mobilized PBPC or CY/G-CSF mobilized PBPC. The number of harvested CD34+ cell and CFU-GM (colony forming units-granulocyte macrophage) were quantitated for each stem cell produce. There ar complete CD34 data for 21 patients and complete CFU-GM data for 20 patients. There was a significantly delayed neutrophil recovery in those patients reinfused with < 0.75 x 10(6) CD34+ cells/kg body weight (median days 22) compared with patients reinfused with > 0.75 x 10(6)/kg (median days 12, P = 0.0004); a similar trend was seen with platelet recovery (median 135 days vs 18 days, respectively, P = 0.002). With neutrophil recovery, there was no improvement in time to engraftment with a large number of reinfused CD34+ cells, but there was a trend towards shortened platelet recovery when the number of reinfused CD34+ cells exceeded 2.0 x 10(6)/kg (median 15 days) compared with CD34+ < 2.0 x 10(6)/kg (median 75 days, P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541270 TI - Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever. AB - Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii) were studied by immunoblot to investigate cross reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus OX 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis. PMID- 7541272 TI - A Combined golgi and postembedding GABA and glutamate electron microscopic study of the nucleus dorsomedialis thalami of the rat. AB - The synaptic organisation of neurons in the nucleus medialis dorsalis (MD) was investigate by combined Golgi, and post-embedding gamma-aminobutyric acid (GABA) and/or glutamate (GLU) immunogold methods. The morphological features of the impregnated neurons in the MD (detailed by Kuroda et al. 1992a) were similar to thalamocortical relay cells present in other thalamic nuclei. GABAergic cell bodies could not be found. Distal dendritic branches of gold toned neurons established synaptic contacts with small and medium size glutamate as well as GABA-immunoreactive axonal endings. Large glutamate and GABA-positive axon terminals formed synaptic contacts with proximal dendrites. GABAergic axon terminals exhibited symmetric synaptic contacts and contained pleomorphic vesicles. Glutamate immunoreactive terminals formed asymmetric synapses and contained spheroid vesicles, although, some large glutamate immunoreactive endings contained pleomorphic vesicles. Synaptic glomerulus-like complexes could also be observed with the participation of glutamate-, and GABA-positive large axon terminals synapsing with Golgi impregnated proximal dendrites. PMID- 7541271 TI - Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates. AB - Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method. PMID- 7541273 TI - Complete screening of mutations in the coding sequence of the CFTR gene in a sample of CF patients from Russia: identification of three novel alleles. AB - To date, a large number of mutations causing the disease, cystic fibrosis, have been reported worldwide. Having analysed the coding sequence of a sample of cystic fibrosis (CF) patients from Russia, we have identified three novel CF mutations. Two of them, 175 del C in exon 1 and 624 del T in exon 5, are frameshift mutations, predicted to result in premature termination of the CFTR transcript. The third mutation is missense and occurs in exon 12 (D572N). The profile of mutations in this sample of Russian CF patients is particular, with two mutations in exon 13 (2143 del T and 2184 ins A), accounting for 12% of the non-delta F508 alleles. PMID- 7541274 TI - Transcript analysis of CFTR nonsense mutations in lymphocytes and nasal epithelial cells from cystic fibrosis patients. AB - The mutational effects at the mRNA level were investigated by RT-PCR analysis of nine different nonsense mutations (Q39X, E60X, R75X, G542X, L719X, Y1092X, R1162X, S1196X, W1282X) and one frameshift mutation (1078delT) within the CFTR gene. With the exception of mutation R1162X, reduced mRNA levels ranging from 30% to less than 5% of the wild type have been observed. In case of the R75X and E60X mutations, the mRNA reduction was accompanied by the appearance of atypical CFTR isoforms. Single exon 3 skipping, as well as joint exon 2 and 3 skipping, was observed in lymphocyte and nasal epithelial mRNA derived from R75X alleles. The analysis of mRNA transcribed from E60X alleles revealed skipping of exon 3 (lymphocytes and nasal epithelial cells) or skipping of exons 3 and 4 (nasal epithelial cells). With the exception of the E60X mutation, no obvious tissue specific differences in the splicing pattern and ratios of mutation to wild-type transcripts were detected between lymphocytes and nasal epithelial cells. In addition to aberrant splicing, the reduction of transcripts is the most common effect of nonsense and frameshift mutations within the CFTR gene. PMID- 7541275 TI - Cytokeratin expression in non-neoplastic oesophageal epithelium and squamous cell carcinoma of the oesophagus. AB - The expression of cytokeratins (CK) 19, 8, 18, 13, 10 and 7 was examined in 35 cases of squamous cell carcinomas of the oesophagus (10 well-differentiated, 13 moderately-differentiated, and 12 poorly-differentiated) and the adjacent mucosa by means of a panel of monoclonal antibodies on frozen sections. The study was undertaken to assess the pattern of expression of these keratins in oesophageal tumours and its relation to the degree of differentiation. The normal oesophageal epithelia expressed CK19 in 86%, CK18 in 17% and CK13 in 14% of cases. CK8, CK10 and CK7 immunoreactivity was not observed. The tumours expressed CK19 in 86%, CK8 in 46%, CK18 in 97%, CK13 in 83%, CK10 in 34% and CK7 in 29% of cases. Thus, the so-called simple epithelial markers CK18 and CK19 occurred in the majority of oesophageal squamous cell carcinomas. CK13 (the so-called non-keratinizing squamous epithelial marker) was only infrequently demonstrated in the non neoplastic oesophageal mucosa, and its expression was more frequent in carcinomas. CK10 was not demonstrated in non-neoplastic mucosa, but was mostly associated with well-differentiated carcinomas. We therefore conclude that the pattern of expression of cytokeratins in oesophageal carcinomas is different from that in normal oesophageal epithelia and varies with differentiation. PMID- 7541276 TI - Alpha fetoprotein-producing acinar cell carcinoma of the pancreas showing multiple lines of differentiation. AB - An alpha fetoprotein (AFP)-producing tumour occurring in the head of the pancreas of a 30-year-old woman is reported. Histological examination revealed a markedly solid proliferation of tumour cells with prominent nucleoli and occasional luminal structures, some of which contained mucinous material stained with mucicarmine and alcian blue. No squamoid corpuscles were recognized. Immunohistochemistry showed intense positivity for lipase trypsin, and AFP basically, and single cells were also positive for carcino-embryonic antigen, CA19-9, synaptophysin and neuron-specific enolase. Pancreatic hormone-positive cells were absent. Electron microscopical examination revealed numerous granules of variable sizes in the tumour cells, which were considered to be zymogen. The tumour is an acinar cell carcinoma with multi-directional differentiation including the ability to produce AFP. Among AFP-positive pancreatic tumours, acinar cell carcinoma and pancreatoblastoma seem to be the most frequent. PMID- 7541277 TI - A dual-binding antibody to E- and L-selectin attenuates sepsis-induced lung injury. AB - Many studies indicate a pivotal role for neutrophil adhesion in sepsis-associated lung injury. Neutrophil adhesion to endothelium depends on activation and expression of selectin and integrin adhesion receptors. We studied the effects of pretreatment with a dual-binding porcine anti-E- and anti-L-selectin monoclonal antibody (EL-246) on a porcine model of sepsis-induced lung injury. Four groups were studied for 5 h. Group 1 (control animals) received intravenous saline only. Group 2 (septic) received a 1-h infusion of Pseudomonas aeruginosa. Group 3 (EL 246 pretreatment) received EL-246 (1 mg/kg) prior to Pseudomonas infusion. Group 4 (EL-246 controls) received EL-246 infusion only. Group 2 animals showed rapid, significant decline in arterial pH and oxygen tension whereas, in Group 3, physiologic deterioration was significantly attenuated. Bronchoalveolar lavage at 5 h showed a significant increase in neutrophil count and protein content in Group 2. Group 3, however, showed no significant differences in these parameters compared with control animals. Despite severe neutropenia, lung myeloperoxidase content at 5 h was significantly reduced in Group 3 compared with Group 2. There was no significant difference in pulmonary and systemic hemodynamics between Groups 2 and 3. Group 4 animals exhibited a transient neutropenia, but otherwise no other differences in measured parameters were found compared with Group 1 control animals. In conclusion, EL-246 significantly reduced neutrophil accumulation in lung and attenuated sepsis-induced lung injury, but failed to attenuate deranged pulmonary and systemic hemodynamics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541278 TI - Clara cell protein (CC-16) induces a phospholipase A2-mediated inhibition of fibroblast migration in vitro. AB - Clara cell protein (CC-16, also designated CC-10) is synthesized by the bronchiolar epithelium and has been suggested as an inhibitor of phospholipase A2 (PLA2) activity. Therefore, CC-16 is a candidate for controlling inflammatory events in the lung. Because CC-16 amounts and function may be altered in fibrosing lung diseases in which bronchiolar injury has been reported, it was measured in alveolar fluids and sera. Secretory PLA2 activity in alveolar fluids and the influence of CC-16 on platelet-derived growth factor-induced human fibroblast chemotaxis and cytosolic PLA2 activity were also explored. CC-16 content was decreased in alveolar fluids from idiopathic pulmonary fibrosis (IPF: 1.3 +/- 0.1 mg/L) and bleomycin lung (1.1 +/- 0.2 versus 2.1 +/- 0.2 mg/L in controls, p < 0.05), whereas there was a three- to ninefold increase in secretory PLA2 activity (p < 0.05 versus controls). CC-16 inhibited fibroblast chemotaxis in a dose-dependent manner (90% inhibition at 30 micrograms/ml CC-16). This inhibition was reversed by reducing CC-16. CC-16 was also able to lower fibroblastic cytosolic PLA2 activity by 50% in vitro. In summary, CC-16 is able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to a blockage of cytosolic PLA2 activity. It can be postulated that CC-16 deficiency may contribute to fibroblast burden activity in fibrosing lung diseases. PMID- 7541279 TI - Interaction of clozapine with the histamine H3 receptor in rat brain. AB - We examined possible interactions between neuroleptics and the histamine H3 receptor and found an interaction of clozapine with this receptor. In competition binding experiments, using the H3 antagonist, [125I]-iodophenpropit, we observed a Ki of 236 +/- 87 nM. Functionally, clozapine was studied on the H3-mediated inhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from rat brain cortex slices. Clozapine acts as an antagonist with an apparent KB value of 79.5 nM. PMID- 7541280 TI - Functional interaction between losartan and central tachykinin NK3 receptors in the conscious rat. AB - 1. The cardiovascular and behavioural effects elicted by the intracerebroventricular (i.c.v.) injection of substance P (SP), neurokinin A (NKA), [MePhe7]neurokinin B ([MePhe7]NKB) or angiotensin II (AII) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for angiotensin AT1 (losartan and its active metabolite EXP 3174), angiotensin AT2 (PD 123,319) or tachykinin NK3 (R 486) receptors. 2. I.c.v. administration of 25 pmol AII evoked an increase in mean arterial blood pressure (MAP) and water intake behaviour, accompanied by a transient bradycardia, whereas 25 pmol [MePhe7]NKB caused a transient increase in MAP and heart rate (HR) concurrently with marked wet dog shake behaviour. At the same dose, SP and NKA were more potent than [MePhe7]NKB in increasing MAP and HR, but did not produce water intake or wet dog shake behaviours. 3. Losartan (650 pmol, i.c.v.) reduced significantly the cardiovascular and behavioural responses to AII or [MePhe7]NKB, but not to SP or NKA. While 65 pmol losartan was inactive, 260 pmol inhibited selectively the central effects of AII. Whereas EXP 3174 (6.5 nmol) blocked both AII and [MePhe7]NKB-mediated responses, the dose of 650 pmol blocked only the responses to AII. 4. The central responses to AII and [MePhe7]NKB were not affected by PD 123,319 (650 pmol). On the other hand, the [MePhe7]NKB-induced central effects were significantly reduced by R 486 (650 pmol). The NK3-selective antagonist had no effect against AII. 5. This study provides functional evidence, to support earlier binding data, that losartan (and to some extent its active metabolite EXP 3174) interact with the tachykinin NK3 receptor in rat brain. However,the cardiovascular and behavioural responses induced by central tachykinin agonists (SP, NKA and[MePhe7]NKB) and All are mediated by unrelated mechanisms. PMID- 7541281 TI - The interaction of trichloroethanol with murine recombinant 5-HT3 receptors. AB - 1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2 methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5 HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H] granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM). 6 Unexpectedly, the competition for [3H]-granisetron binding by the 5-HT3 receptor antagonist,quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM 0.3 microM) antagonized 5-HT evoked currents recorded from oocytes expressing the 5-HT3R-A subunit with an IC50 of 18 +/- 3 nM(n = 4). Additionally, quipazine (30 nM-0.3 microM) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist.7. The demonstration that a recombinant homo-oligomeric receptor, expressed in a foreign membrane,retains a sensitivity to alcohols, together with the sequencing of alcohol-insensitive 5-HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s). PMID- 7541283 TI - Comparison of effects of chronic and acute administration of NG-nitro-L-arginine methyl ester to the rat on inhibition of nitric oxide-mediated responses. AB - 1. Vascular responses to acetylcholine and sodium nitroprusside in vivo and in vitro, in the isolated perfused kidney and in rings of rat thoracic aorta, were measured in rats treated chronically with NG-nitro-L-arginine methyl ester (L NAME; approx, 70 mg kg-1) and compared to responses in age-matched control animals, and age-matched animals after the acute administration of L-NAME (3-100 mumol kg-1). Parallel experiments examined alterations in responsiveness in rings of trachea and anococcygeus muscles taken from the same animals. 2. Chronic oral administration of L-NAME elevated the blood pressure in anaesthetized animals from 114 +/- 5 mmHg to 153 +/- 11 mmHg (n = 5). The hypotensive responses to both acetylcholine (1 nmol kg-1) and sodium nitroprusside (10 nmol kg-1) were enhanced by chronic L-NAME treatment (n = 5-7) whereas acute L-NAME administration enhanced only the response to sodium nitroprusside (n = 5). 3. After chronic treatment with L-NAME, the basal perfusion pressure in the isolated perfused kidney was elevated. However, vasodilator responses to either acetylcholine (1 nmol) or sodium nitroprusside (3 nmol) were unaltered (n = 5-7). The vasodilatation induced by acetylcholine was inhibited in a concentration dependent manner by the administration of acute L-NAME (0.1 - 100 microM; n = 5), such that significant inhibition was seen at 10 microM L-NAME. The response to sodium nitroprusside was unaffected by L-NAME. 4. The relaxations of isolated rings of rat thoracic aorta induced by acetylcholine were inhibited in tissues prepared from rats treated chronically with L-NAME (n = 5-7). Acute administration of L-NAME (0.1-100 microM) concentration-dependently inhibited the relaxations induced by acetylcholine in this preparation, with significant inhibition occurring at 1 microM L-NAME (n = 5). Responses to sodium nitroprusside were unaffected by either chronic or acute exposure to L-NAME (n = 5-7).5. Relaxations of precontracted anococcygeus muscles induced by electrical field stimulation, or contractions of rings of trachea induced by carbachol or endothelin-1, were unaffected by chronic oral administration of L-NAME (n = 4-6). Acute addition of L-NAME (0.1-100 microM) to the organ baths inhibited in a concentration-dependent manner the relaxations of anococcygeus muscles taken from control animals, with a significant effect being seen at a concentration of 10 micro.M (n = 4-6).6. Our cardiovascular data are consistent with chronic oral administration of L-NAME inhibiting the production of nitric oxide (NO) within the vasculature, although the pattern of inhibition is not uniform between different tissues. Despite the inhibition of endothelial NO production, chronic L NAME does not alter the vasodepressor activity of acetylcholine in vivo or in the isolated perfused kidney. This maybe explained by an enhanced responsiveness of guanylyl cyclase pathways, the increased release of vasodilators other than nitric oxide or a decreased importance of nitric oxide in resistance vessels compared with conductance vessels. The resistance of peripheral neuronal NO responses to chronic treatment with L-NAME indicates that selective inhibition of different isoforms of NOS may be achieved in vivo. PMID- 7541282 TI - Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock. AB - 1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P < 0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS rats. 3. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10-9- 10-6 M). Pretreatment with aminoguanidine (15 mg kg- 1, i.v.,at 20 min prior to LPS) significantly prevented this LPS-induced hyporeactivity to NA ex vivo.4. Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 +/- 0.2 pmol mg-1 min-1 (control, n = 4) to 4.8 +/- 0.3 pmol mg 1 min-1 (P<0.05, n = 6). In LPS-rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44+/- 5% (n = 6, P <0.05).Moreover, when added in vitro to lung homogenates obtained from LPS-rats, aminoguanidine and N omega nitro-L-arginine methyl ester (L-NAME; 10-8 to 10-3 M) caused a concentration dependent inhibition of iNOS activity (n = 3-6, IC50: 30 +/- 12 and 11 +/- 6pEM, respectively P>0.05). In contrast,aminoguanidine was a less potent inhibitor than L-NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3 6, IC50 is 140 +/- 10 and 0.6 +/- 0.1 I1M, respectively, P<0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L-NAME(maximal inhibition at 90 min and 30 min, respectively).5. Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg-1, i.p.)resulted in a survival rate of only 8% at 24 h (n = 12). However, therapeutic application of aminoguanidine (15 mg kg-1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75%(n = 8), whereas L-NAME (3 mg kg-1, i.p. at 2 h and 6 h after LPS) did not affect the survival rate(11%, n=9).6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine,or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock. PMID- 7541284 TI - Vascular actions of thrombin receptor-derived polypeptides: structure-activity profiles for contractile and relaxant effects in rat aorta. AB - 1. Using endothelium-denuded and intact rat aortic rings, we have determined the contractile and relaxant structure-activity profile for a series of thrombin receptor-derived polypeptides (TRPs) based on the human and rat receptor sequences: SFLLR (P5), SFLLR-NH2 (P5-NH2) SFFLR (Rat P5), SFFLR-NH2 (Rat P5-NH2), SFLLRNP (P7), SFLLRNP-NH2 (P7-NH2), SFFLRNP (Rat P7), SFFLRNP-NH2 (Rat P7-NH2), and SFLLRNPNDKYEPF (P14). 2. A contractile response to thrombin and the TRPs in the endothelium-denuded aortic tissue was minimal or absent in preparations obtained from animal weighing less than 180 g (< 6 weeks of age), but increased with animal size, plateauing in tissues derived from animals weighing between 320 and 420 g (about 9 to 14 weeks of age). In contrast, the contractile responses to KCl and noradrenaline did not differ in the tissues and relaxant responses to the TRPs in endothelium-intact aortic preparations were comparable for tissues obtained from either young (< or = 180 g) or older (> or = 320 g) animals. 3. The contractile response of the endothelium-denuded preparation to thrombin and the TRPs showed marked cross-desensitization: the relaxation response of the intact rings did not desensitize to the TRPs. 4. The relative potencies for the TRPs in the aortic contraction assay were comparable to those for the relaxation assay, but were distinct from the relative potencies we measured previously in a rat gastric longitudinal muscle contraction assay. Further, P5 behaved as a partial agonist in the aortic contraction assay, whereas it had been observed to be a full agonist in the gastric contraction assay. 5. The contractile activity of P5 NH2 in endothelium intact aortic rings was low or absent, but in the presence of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L NAME), the contractions in the intact preparation were equivalent to the response of the endothelium-denuded preparation in the absence of L-NAME.6. The contractile response of the endothelium-denuded aortic preparation to P5-NH2 was inhibited by nifedipine and the kinase C antagonist, chelerythrine, but was resistant to the action of indomethacin,tetrodotoxin and the tyrosine kinase inhibitor, genistein.7 We conclude that the receptor system for the TRPs in the aortic smooth muscle elements, responsible for the contractile response, is similar to the aortic endothelial cell receptor responsible for the relaxation response, but is distinct from the receptor that we have previously characterized in gastric longitudinal smooth muscle, results pointing to the presence of receptor subtypes in the vascular and gastric smooth muscle elements. PMID- 7541286 TI - Expression of a serotonin-gated ion channel in embryonic neural and nonneural tissues. AB - The neurotransmitter serotonin (5HT) has been implicated in morphogenesis of central nervous system and craniofacial structures. The actions of serotonin are mediated by multiple receptor subtypes, one of which, the 5HT3 receptor, is a ligand-gated ion channel. To determine whether this channel may contribute to the proposed morphogenic actions of serotonin, the expression of 5HT3 receptor transcripts was examined during mouse embryogenesis and correlated with the distribution of serotonin transporter mRNA and serotonin immunoreactivity. The pattern of 5HT3 receptor mRNA expression within the brain suggests possible roles for this receptor in the proliferation, differentiation, or migration of CNS neurons. In the peripheral nervous system, 5HT3 receptor transcripts were observed within cranial nerve sensory ganglia, olfactory neuroepithelia, and sympathoadrenal and enteric nervous systems during the initial stages of their formation. Striking expression of 5HT3 receptor transcripts occurred outside the nervous system, in association with regions of active chondrogenesis in the vertebral column, limbs, and craniofacial region, suggesting a possible involvement of this receptor subtype in the morphogenesis of olfactory receptor neurons, teeth, and genitalia. PMID- 7541285 TI - Mechanism of action of the inhibitory effect of nifedipine on the growth of cultured aortic cells from spontaneously hypertensive and normotensive rats. AB - 1. To gain insight into the parameters which control vascular structure, we investigated the mechanisms whereby nifedipine, and other dihydropyridines, inhibit the growth of cultured fibroblasts isolated from the adventitia of the aorta of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. 2. The effects of nifedipine on cell proliferation and on serum-induced DNA synthesis were determined by measuring the cell number and the incorporation of [3H]-thymidine, respectively. The mechanism of action of nifedipine was studied by adding the drug either to randomly growing cells or to quiescent, G0/G1 arrested and synchronized cells. The effects of varying the duration of drug treatment were also examined. 3. In randomly growing cultures nifedipine, like other dihydropyridines concentration-dependently inhibited cell proliferation; the rank order of effect (measured at a concentration of 10 microM) was nifedipine > nisoldipine > nitrendipine approximately nimodipine. 4. In G0/G1 arrested cell cultures, nifedipine concentration-dependently inhibited serum induced [3H]-thymidine incorporation. In this respect it had similar effects in cell cultures from WKY and SHR. In both SHR and WKY cultures, nifedipine delayed the transition from G0/G1 to S phase, and inhibited serum-induced DNA synthesis possibly by acting on the early G1 phase. 5. In cell cultures from both SHR and WKY, serum-induced DNA synthesis was similarly (approximately 40%) inhibited after a 1 day treatment with 10 microM nifedipine. In contrast, after 5 days treatment with the drug, the inhibition of DNA synthesis was approximately 65% and approximately 10% in SHR and WKY cultures, respectively. The inhibitory effects of nifedipine against proliferation of fibroblasts were 25% and 60%, respectively,after 1 and 5 days of treatment, and were similar in cells derived from SHR and WKY. This indicates that 5 days treatment with nifedipine inhibited the proliferation of SHR and WKY fibro blasts by acting mostly on the early G1 phase and the M phase, respectively.6. Irrespective of the duration of treatment (1 or 5 days) with 10 microM nifedipine, the inhibition of DNA synthesis could be abolished and partially reduced by Bay K 8644 (1 microM) in WKY and SHR fibroblasts,respectively. In cell cultures from both SHR and WKY the inhibitory effects of a short term and of along term treatment with nifedipine against cell proliferation were reduced and unaffected, respectively by Bay K 8644.7. These results indicate that nifedipine inhibited cell proliferation and serum-induced DNA synthesis by altering the cell cycle through different mechanisms in SHR and WKY fibroblasts. They also suggest the existence in aortic fibroblasts of interactions between calcium channel blockers of the dihydropyridine series and the mitogenic signalling pathways of growth factors contained in serum. PMID- 7541287 TI - Insecticides: their effect on microorganisms and persistence in rice soil. AB - A field experiment was conducted to investigate the effect of four insecticides, HCH, phorate, carbofuran and fenvalerate, at recommended doses on the preponderance of bacteria, actinomycetes and fungi. We also measured the persistence of the insecticides in the rhizosphere soil of rice. HCH and fenvalerate stimulated the proliferation of all of the microorganisms significantly. Phorate increased the population of bacteria and actinomycetes. Carbofuran accentuated the preponderance of actinomycetes in soil. Insecticides, in general, did not have marked influence on the proliferation of Bacillus, Streptomyces, Aspergillus and Fusarium in soil. However, we observed a stimulation of growth of Staphylococcus, Proteus and Sarcina with HCH, Pseudomonas, Corynebacterium, Erysipelothrix and Rhizopus with phorate, Serratia, Corynebacterium, Klebsiella, Escherichia, Rhizopus and Humicola with carbofuran, and Staphylococcus, Sarcina, Klebsiella and Nocardia with fenvalerate. On the other hand, there was an inhibition in growth of Pseudomonas, Micrococcus, Nocardia and Penicillium with HCH, of Pseudomonas, Micrococcus and Penicillium with carbofuran, and of Pseudomonas, Micrococcus and Micromonospora with fenvalerate. Different types of insecticides exhibited differential patterns of dissipation in soil. HCH had the highest persistence followed by phorate, carbofuran and fenvalerate, respectively. PMID- 7541288 TI - Mechanisms of vascular remodeling in hypertension: role of autocrine-paracrine vasoactive factors. AB - The vasculature is an integrated organ capable of modulating its structure in accordance with hemodynamic conditions and locally generated mediators. Vasoactive substances produced within the vasculature are important modulators of vessel structure and vessel tone. This review focuses on the pathogenic role of vasoactive substances in vascular remodeling in hypertension and the potential clinical implications of this pathobiological process. It is speculated that therapeutic strategies that influence vascular remodeling may be clinically more effective. PMID- 7541289 TI - Quantitative polymerase chain reaction: a new approach to the evaluation of cytokine expression. PMID- 7541290 TI - From the syndrome of Charcot, Marie and Tooth to disorders of peripheral myelin proteins. AB - The description of the peroneal muscular atrophy syndrome in 1886 by Charcot, Marie and Tooth was followed by an era of nosological confusion. This was partly clarified by the advent of nerve conduction studies and the definition of the most common, but heterogeneous, disorders underlying this syndrome, hereditary motor and sensory neuropathies (HMSN) types I and II. The classification of HMSN is now changing as a result of the identification of underlying mutations in genes encoding myelin proteins. Abnormalities of peripheral myelin protein 22 (PMP-22) account for dominantly inherited HMSN type I in approximately 90% of families. The commonest genetic defect is a duplication of this gene and the surrounding region of chromosome 17, although point mutations also occur. A deletion of the same region causes hereditary neuropathy with liability to pressure palsies. Point mutations of the P0 gene cause HMSN I in a small number of families. The X-linked type of HMSN is associated with defects of the connexin 32 gene, which encodes a gap junction protein. These molecular genetic advances can be translated into clinical practice, leading to improved diagnosis and genetic counselling. PMID- 7541291 TI - Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule. AB - Adenovirus VA1 gene is efficiently transcribed by RNA polymerase III and gives rise to a small highly ordered RNA. To inhibit replication of human immunodeficiency virus (HIV), a chimeric VA1 RNA molecule was designed that contained a short antisense RNA sequence complementary to a conserved region of the HIV-1 rev encoding mRNA (28 nucleotides). This sequence, which was inserted into a projecting loop of the VA1 RNA central domain, was mainly single stranded and available for binding with its complementary sequence. The chimeric VA1 antisense was abundantly expressed in human cells constituting 3% of mRNA and promoted strong and specific inhibition of HIV-1 gene replication. The stable expression of antisense RNA in human T cells (CEM) protected these cells from HIV 1 multiplication for at least 3 months. No side effects were detected because of the lack of antisense effect upon replication of the closely related HIV-2. The VA1 gene may provide a suitably compact gene cassette for the intracellular expression of short antisense RNA directed against HIV. PMID- 7541292 TI - In vivo LDL receptor and HMG-CoA reductase regulation in human lymphocytes and its alterations during aging. AB - The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase-polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (P = .030) and HMG-CoA reductase mRNA (P = .062). The age related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo. PMID- 7541293 TI - The biologic behavior of balloon hyperinflation-induced arterial lesions in hypercholesterolemic pigs depends on the presence of foam cells. AB - Lack of a large-animal model of accelerated atherosclerosis has limited study of the biologic behavior of atherosclerotic lesions. We hypothesized that mechanical vascular trauma combined with diet-induced hypercholesterolemia would result in rapid development of complex atherosclerosis-like lesions. Accordingly, we induced deep injury to a carotid artery by repetitive balloon hyperinflations in minipigs that were fed either an atherogenic diet (n = 30) or a standard diet (controls, n = 4) and examined the resultant lesions 1 month later. The neointimal lesions that evolved in 23 patent vessels from cholesterol-fed animals were complex, exhibiting infiltration of smooth muscle and foam cells and evidence of organized thrombus, recent thrombus, hemorrhage, and calcification. Lesions were separable histologically into two groups: foam-cell rich (n = 12), with 33 +/- 10 foam cells per high-power field, and foam-cell poor (n = 11), with 4 +/- 1 foam cells per high-power field. Minipigs with foam cell-rich lesions had higher serum cholesterol levels than those with foam cell-poor lesions (712 +/- 178 vs 468 +/- 240 mg/dL, P < .02). The incidence of intralesional thrombus was also significantly greater in foam cell-rich than in foam cell-poor lesions (50% vs 9%, P < .04). In addition, the degree of luminal stenosis was greater in the presence of lesions containing thrombus compared with those without thrombus (60 +/- 38% vs 30 +/- 29%, P = .05). Lesions in the control animals were fibrocellular and lacked foam cells and thrombus. Thus, hypercholesterolemia appeared to affect lesion composition and behavior.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541294 TI - TGF-beta and endothelial cells inhibit VCAM-1 expression on human vascular smooth muscle cells. AB - Vascular smooth muscle cells (VSMCs) are normally devoid of the adhesion protein vascular cell adhesion molecule-1 (VCAM-1), which has, however, been observed on human VSMCs in atheroma. We now show that cultured human saphenous vein VSMCs express small amounts of VCAM-1 and that the cytokine tumor necrosis factor-alpha (TNF-alpha) induces, in a time- and dose-dependent fashion, a significant increase in its expression. Interleukin (IL)-4, IL-1, and to a lesser extent interferon gamma have similar effects. TNF-alpha-stimulated human VSMCs demonstrate increased binding of T lymphocytes that is totally VCAM-1 mediated. The cytokine transforming growth factor-beta (TGF-beta) at 2.0 ng/mL inhibited basal VCAM-1 expression by 84 +/- 8% and the induction by TNF-alpha by between 56 +/- 16% and 77 +/- 15% depending on the dose of TNF. Furthermore, coculture on opposing sides of a polycarbonate filter of human VSMCs with human umbilical vein endothelial cells also inhibited the induction of VCAM-1 by 47 +/- 6%. As active TGF-beta is produced upon the coculture of VSMCs and endothelial cells, we suggest that the close physical proximity of these cells in vivo is responsible for the lack of expression of VCAM-1 on VSMCs and that the interruption of this contact in atheroma is an important pathogenic event. As VCAM-1 not only serves as an adhesion molecule but also as a costimulator of immune cells, its expression may be crucial in the propagation of vascular lesions. PMID- 7541296 TI - Immunologic detection and measurement of hypochlorite-modified LDL with specific monoclonal antibodies. AB - Oxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal anti human HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynonenal , malondialdehyde-, and Cu(2+)-oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- and Cu(2+)-modified LDL cross-reacted with HOCl LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross-react with either native LDL or aldehyde- or Cu(2+)-modified LDL. MoAb-A (clone 1B10A11, subtype IgG1 kappa) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype IgG2b kappa), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl-mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases. PMID- 7541295 TI - Uptake of radiolabeled and colloidal gold-labeled chyle chylomicrons and chylomicron remnants by rat platelets in vitro. AB - This study examined the uptake of chyle chylomicrons (CMs) and chylomicron remnants (CMRs) by rat platelets in vitro. CMs and CMRs were doubly labeled with [3H]arachidonate ([3H]-20:4) and [14C]cholesterol and were incubated with platelets for up to 4 hours. A significant uptake (binding and/or internalization) of CMs by the platelets occurred, as indicated by the parallel increase of [3H]20:4 and [14C]cholesterol in platelets with incubation time. Addition of unlabeled CMs, VLDLs, LDLs, and HDLs decreased the uptake of labeled CMs. The competition experiments suggested that there is both a saturable binding and a nonspecific uptake of CMs. During incubation with CMs, the proportion of [3H]20:4 in phospholipids decreased and that in 1,2-x-diacylglycerol increased. The data indicated that a phospholipase C-mediated degradation of phosphatidylcholine and phosphatidylethanolamine occurred, whereas [3H]20:4 in triglyceride and 14C in cholesterol ester did not change. Electron microscopic studies after incubation with colloidal gold-labeled CMs (CM-Au's) demonstrated an accumulation of CM-Au particles in the open canalicular system of the platelets. Some CM-Au particles were localized in cytoplasmic vacuoles that were not stained by ruthenium red. Some CM-Au's or free gold particles were in vacuoles that showed acid phosphatase activity, indicating that some true endocytosis of CM occurred. The uptake of [3H]-20:4- and [14C]cholesterol-labeled CMRs was low compared with the uptake of CMs. After incubation with colloidal gold-labeled CMRs (CMR-Au's), only a few platelets contained CMR-Au in their open canalicular systems, and no CMR-Au particles were seen in the cytoplasm or in acid phosphatase-positive vacuoles. Rat platelets can thus interact with CMs by a process that leads to a sequestration in the open canalicular system and endocytosis and a net degradation of CM phospholipids. The conversion of CMs to CMRs counteracts this interaction. PMID- 7541298 TI - Modulation of tau neuronal expression induced by NMDA, non-NMDA and metabotropic glutamate receptor agonists. AB - We have analysed changes in tau protein immunoreactivity in rat embryonic neurons degenerating in response to treatment with N-methyl-D-aspartate (NMDA), non-NMDA and metabotropic agonists. Glutamate agonists were applied in Mg(++)-free and glycine-supplemented medium 8 days after initial plating. Cell viability was assessed by fluorescein diacetate staining and neuronal survival was evaluated by cell counting. Immunocytochemical and confocal laser microscopic studies used a tau2 monoclonal antibody. Acute and chronic NMDA treatment induced a concentration-dependent increase in intraneuronal tau immunoreactivity. Increased tau immunolabelling during chronic NMDA toxicity was dramatically attenuated by tetrodotoxin and also by 6-cyano-7-nitroquinoxaline-2,3-dione. Non-NMDA and metabotropic receptor agonist treatment produced a weaker augmentation in tau2 immunoreactivity. These findings suggest that, in this model, glutamate-receptor and sodium-channel coactivation are together needed to produce changes in tau immunoreactivity. PMID- 7541297 TI - Host and viral characteristics affecting the response to interferon therapy in chronic hepatitis C. AB - OBJECTIVE: To define parameters determined before and after 4 weeks of interferon therapy (3 MU three times per week for 24 weeks) which could be reliable predictors of a response to therapy. PATIENTS: Thirty-four patients with chronic hepatitis C virus (HCV) infection were investigated prospectively. METHODS: A complete response was defined as the normalization of serum alanine aminotransferase levels (ALT) at the end of treatment. The genotype of HCV was determined and the level of HCV-RNA was quantitated both before and after 4 weeks of treatment. RESULTS: After 4 weeks, 16 out of 20 responders [95% confidence interval (CI) 54-94%] and two out of 14 non-responders (95% CI 2-44%) normalized their ALT levels (P = 0.0002). The prevalence of genotype 1b was significantly (P < 0.04) higher among non-responders (eight out of 10; 95% CI 44-92%) than in responders (four out of 18; 95% CI 4-40%). Before treatment, the viraemia determined by branched DNA was significantly lower in responders than in non responders (46.4 versus 116 x 10(5) eq virus/ml). After 4 weeks of treatment, the level of viraemia in responders was still significantly lower than that in non responders (22.8 versus 66 x 10(5) eq virus/ml). In responders, a significant decrease in the level of viraemia was observed after 4 weeks of treatment. CONCLUSION: In a stepwise regression analysis only age and the normalization of ALT levels after 4 weeks of treatment were predictive of response to interferon at the end of treatment. PMID- 7541300 TI - The mRNA expression and in vitro production of cytokines and other proteins by hepatocytes and Kupffer cells following thermal injury. AB - Inflammatory cytokines and acute-phase proteins are closely interrelated, and their levels of production by various cells are increased by thermal injury. It was hypothesized that burn-mediated increases in the production of TNF and IL-6 by Kupffer cells and the production of acute phase proteins by hepatocytes are paralleled by increases in the corresponding message RNA levels in these cells. The mRNA expression of the cytokines, IL-6 and TNF alpha, and acute phase proteins, alpha-1 acid glycoprotein (alpha-1 AGP), and albumin in liver tissue were determined in rats 24 h after thermal injury. Also IL-6 and TNF alpha from in vitro cultured Kupffer cells, and alpha-1 AGP and albumin from in vitro cultured hepatocytes, and the serum levels of these proteins, were determined. An increased expression of IL-6 mRNA in liver tissue from animals of the burned group was accompanied by an elevation of IL-6 released from cultured Kupffer cells and by increased serum levels of this cytokine. Thermal injury caused a decrease in TNF mRNA but no change in the production of this cytokine by Kupffer cells, and TNF could not be found in the serum. Also, an increase in alpha-1 AGP mRNA expression following thermal injury was consistent with the increase of alpha-1 AGP production by hepatocytes and with the elevated serum level of this acute phase protein. Thermal injury caused no change in albumin mRNA expression or in the in vitro production of this negative acute phase protein, however, the serum level of albumin increased. The results suggest that thermal injury may cause alterations in the cytokine and acute phase protein mRNA levels in liver, which may cause alterations in the cellular production and serum levels of the corresponding proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541299 TI - Lack of involvement of nitric oxide in the mechanisms of seizures and hippocampal damage produced by kainate and ouabain in rats. AB - The gross behavioural, electrocortical and neuropathological effects of kainate (10 mg/kg i.p,) and ouabain (1 micrograms, given into one dorsal hippocampus) were studied in rats. The effects of these treatments on nitric oxide synthase (NOS) activity in homogenates of hippocampus and cortex were also studied. Administration of kainate or ouabain produced motor and electrocortical seizures similar for latency to onset (approximately 15 min) and intensity (in all instances 80-100% of the treated rats showed behavioural and electrographic seizures). These effects were accompanied at 24 h by severe damage to all subsectors of the hippocampal formation and this concerned a similar proportion of the treated rats (n = 4-8 per treatment). No significant changes in nitric oxide synthase (NOS) activity were noted in the cerebral cortex and hippocampus of rats receiving injections of kainate and ouabain. In addition, pretreatment with N omega-Nitro-L-arginine methyl ester (300 micrograms, given into one lateral cerebral ventricle 15 min previously) was ineffective in preventing the effects of kainate and ouabain. In conclusion, present data suggest that excessive production of NO is not involved in the mechanisms triggering seizures and neurodegeneration produced by kainate or ouabain. PMID- 7541301 TI - The effect of hypertonic fluid resuscitation on brain edema in rabbits subjected to brain injury and hemorrhagic shock. AB - Small-volume resuscitation with 7.2% NaCl/10% dextran 60 (HHS) restores cardiovascular stability faster than all other therapeutic modalities currently known. This study was undertaken to elucidate the effects of HHS on the brain, specifically on the formation of posttraumatic brain edema. HHS was administered to anesthetized albino rabbits with or without a focal cryogenic brain lesion and hemorrhagic shock. Specific gravity of small tissue samples was determined 4 h after injury and values were topographically assembled to form a color-coded map of both hemispheres, allowing for a high resolution mapping of brain edema. Cerebral blood flow on the side of the lesion, as assessed by the H2 clearance method, increased transiently after injury but remained unchanged from baseline during shock and after infusion of HHS, indicating intact cerebrovascular autoregulation. The cryogenic lesion without subsequent HHS infusion resulted in significant brain edema formation in grey and white matter of the exposed hemisphere. In injured animals, resuscitation with HHS led to a global reduction of brain water content in both hemispheres. We conclude that small-volume resuscitation with HHS does not worsen posttraumatic brain edema. To the contrary, our results show that it decreases cerebral water content even in regions close to the injury. This makes it worthwhile to investigate the benefits of HHS for the treatment of intracranial hypertension. PMID- 7541302 TI - Diagnosis of early ectopic pregnancy by measurement of the maternal serum to cul de-sac fluid beta-hCG ratio. AB - In patients suspected of having an ectopic pregnancy, because of a positive beta human chorionic gonadotropin (beta-hCG) test and a sonographically undetectable gestational sac, even laparoscopy cannot always achieve a precise diagnosis, when the gestational age is very early. This means that the patient has to be followed by repeated beta-hCG measurements and repeated laparoscopy, which are expensive and inconvenient for the patient. This paper reports on a method that is simple to perform and that will provide the clinician with valuable information about the strong likelihood of a tubal pregnancy when ultrasound and beta-hCG measurements raise the suspicion but are not diagnostic. A prerequisite for this approach is the availability of a minimal volume (1-2 ml) of peritoneal fluid in the pouch of Douglas. A total of 131 patients who had a positive beta-hCG test and an undetectable gestational sac within the uterine cavity and who had a small volume of cul-de-sac fluid were studied. The fluid was obtained transvaginally by means of an ultrasound-guided automatic puncture system. The beta-hCG level was determined in the cul-de-sac fluid and in the maternal serum, for comparison. In two cases with tubal pregnancy, concentrated viscous blood samples were aspirated and beta-hCG concentrations could not be measured for technical reasons. In 129 cases, the ratio of levels of beta-hCG between serum and cul-de-sac fluid could be measured. In 42 out of 44 patients with intrauterine pregnancy, the ratio was greater than unity; in 82 out of 85 patients with a tubal pregnancy, the ratio was less than unity. With this ratio used for detection of tubal pregnancy, the diagnostic sensitivity of the test was 95.4% and the specificity was 95.2%. The positive predictive value was 97.6% and the negative predictive value 91.3%. We conclude from these findings that the measurement of the maternal serum to cul-de sac fluid beta-hCG ratio facilitated the early diagnosis of an ectopic pregnancy in the Fallopian tube. PMID- 7541303 TI - Low beta-hCG is associated with poor prognosis in association with an embryo with positive cardiac activity. AB - Seven pregnancies with positive fetal heart activity and low or very low serum beta-human chorionic gonadotropin (beta-hCG) levels were investigated. The gestational age by dates was between 6 weeks 3 days and 8 weeks 4 days at the time of the first ultrasound scan and beta-hCG ranged between 282 and 10,000 mIU/ml of the Second International Standard. The crown-rump lengths ranged between 6 and 13 mm and heart activity was always detected. Four cases were scanned because beta-hCG was lower than that expected for the gestational age; in these cases the yolk sac was present but the gestational sacs were small, with a trophoblastic ring that looked thin and subjectively seemed abnormal. The beta hCG level was measured in the other three cases, because the gestational sacs were small and had a thin trophoblastic ring. In these cases, fetal heart activity was clearly imaged. All cases were scheduled for a repeat ultrasound scan 2 weeks later. Three cases aborted spontaneously the following week, whereas in the other four no heart activity was detected in a repeat sonogram 2 weeks later. Low serum beta-hCG levels indicate a poor prognosis even when associated with an embryo with positive cardiac activity detected ultrasonographically. PMID- 7541304 TI - Presence of anti-hepatitis C virus serum markers in a dental school patient population. AB - Hepatitis C virus (HCV) has joined the list of infections liver diseases of which the practicing dentist must be aware. This recently described RNA virus causes between 20% to 40% of reported cases of viral hepatitis in the United States. Patients with HCV show symptoms only one quarter of the time, but chronicity is maintained longer than with the other liver infections. The major mode of transmission is percutaneous, so dentists are potentially at risk to contact the virus. To assess the presence of exposure to HCV, a total of 500 dental school patients were screened for serum antibody to this virus (second-generation anti HCV). In addition, participants were required to fill out a special screening questionnaire designed to indicate high-risk groups. Their serum was also analyzed for serum chemistries known to be associated with acute and chronic liver disease. Analysis of the 15 response questionnaires revealed statistical significance relating to questions inquiring about previous exposure to blood (transfusions, surgical procedures, etc) but little significance relating to lifestyle. In either case responses were not of practical predictive value. More than 5% of our sample patients were found to be positive reactors to anti-HCV, indicating previous HCV exposure. A significant number of those positive for anti HCV also showed elevation of serum chemistries associated with hepatitis. PMID- 7541305 TI - The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. AB - The Thy-1.1loSca-1hiLin-/lo population, representing 0.05% of C57BL/Ka-Thy-1.1 bone marrow, is highly enriched for hematopoietic stem cells and includes all multipotent progenitors in this mouse strain; however, the functional reconstituting activity of this fraction is heterogeneous. Only around 25% of clonal reconstitutions by cells from this population are long term; remaining clones yield transient multilineage reconstitutions. By fractionating based on lineage marker expression, the Thy-1.1loSca-1hiLin-/lo population has been resolved into three subpopulations: Lin-Mac-1-CD4-; Lin-Mac-1loCD4-; and Mac 1loCD4lo. Of these, only the Lin-Mac-1-CD4- population is highly enriched for long-term reconstituting hematopoietic stem cells. A comparison of transient and long-term multipotent progenitors indicates that long-term progenitors have less CFU-S activity, are equally radioprotective, and are less frequently in cell cycle. The ability to predict the longevity of reconstitution based on lineage marker expression indicates that reconstitution potential is deterministic, not stochastic. PMID- 7541306 TI - Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins. AB - The initial rolling interaction of leukocytes with the blood vessel wall during leukocyte trafficking has been postulated to rely on members of the selectin family of adhesion molecules. Two selectins, E-selectin and P-selectin, have been identified that are expressed on activated endothelial cells. Mice deficient in E selectin expression have been produced in order to examine the role of this selectin in leukocyte trafficking. Mice homozygous for an E-selectin null mutation were viable and exhibited no obvious developmental alterations. E selectin-deficient mice displayed no significant change in the trafficking of neutrophils in several models of inflammation. However, blocking both endothelial selectins by treatment of the E-selectin-deficient animals with an anti-murine P selectin antibody, 5H1, significantly inhibited neutrophil emigration in two distinct models of inflammation. While neutrophil accumulation at early times during thioglycollate-induced peritonitis was dependent on P-selectin, neutrophil accumulation at later time points was blocked by 5H1 only in E-selectin-deficient mice but not in wild-type mice. Similarly, edema as well as leukocyte accumulation in a model of delayed-type hypersensitivity in the skin was almost completely prevented by blockade of P-selectin function with 5H1 in the E selectin-deficient mice while the same treatment had no effect in wild-type mice. These data demonstrate that the majority of neutrophil migration in both models requires an endothelial selectin but that E-selectin and P-selectin are functionally redundant. These data have important implications in the use of selectin antagonists in the treatment of inflammatory disease. PMID- 7541308 TI - The palliative management of fungating malignant wounds. PMID- 7541307 TI - Binding of diverse peptides to MHC class I molecules inhibits target cell lysis by activated natural killer cells. AB - Class I MHC expression by target cells inhibits lysis mediated by natural killer (NK) cells, often in an allele-specific fashion. It has been proposed that NK cell inhibitory receptors recognize complexes of class I molecules with specific cellular peptides that define self, displacement of which would render cells NK sensitive. By loading the mostly empty Dd class I molecules of cell lines deficient in peptide transporter molecules with synthetic or natural Dd-bound peptides, we have demonstrated specific dose-dependent inhibition of the Ly49+ subset of activated NK cells by class I-peptide complexes. Inhibition occurred with most if not all Dd-binding peptides, suggesting that Ly49+ NK cells recognize class I-peptide complexes largely independently of peptide composition. The results suggest a primary role of NK cells in the destruction of cells that have down-regulated or extinguished cell surface expression of some or all class I molecules. PMID- 7541309 TI - Antiallergic activity of loratadine: inhibition of leukotriene C4 release from human leucocytes. AB - The H1 antagonist loratadine has the capacity to inhibit histamine release from human basophils. The aim of this study was to investigate whether loratadine can also inhibit leukotriene C4 (LTC4) release from human leucocytes. Basophil enriched mononuclear cell suspensions were prepared by centrifugation of peripheral venous blood (n = 10) on discontinuous Percoll gradients. Leucocytes were stimulated with anti-IgE, N-formylmethionyl-leucyl-phenylalanine (FMLP) and Ca2+ ionophore A23187; immunoreactive (i) LTC4 release in the cell supernatant was measured by a competitive radioimmunoassay and histamine release was evaluated by an automated fluorometric technique. Loratadine, in the concentration range of 1-50 microM, exerted a dose-dependent inhibitory effect on IgE-mediated and IgE-independent histamine and iLTC4 release. The concentrations inhibiting 50% of histamine release were 30 microM (anti-IgE), 27 microM (FMLP) and 19 microM (Ca2+ ionophore A23187). The concentrations inhibiting 50% of iLTC4 release were 2.3 microM (anti-IgE). 11 microM (FMLP) and 1.7 microM (Ca2+ ionophore A23187). The inhibitory activity on iLTC4 release was optimal after preincubation for 2 h at 37 degrees C, and was no longer evident when leucocytes were stimulated 2 h after cell washing. Increased extracellular Ca2+ concentrations reduced the inhibitory activity of loratadine. These results indicate that loratadine has the capacity to inhibit the release of preformed and newly generated mediators from human basophil-enriched mononuclear cell suspensions. PMID- 7541310 TI - Comparison of hydromorphone and meperidine for ureteral colic. AB - OBJECTIVE: To compare the efficacies of meperidine and hydromorphone in the treatment for ureteral colic in the emergency department (ED). METHODS: A prospective, double-blind, randomized clinical trial was conducted over six months at a tertiary referral center with 93,000 annual ED visits. Seventy-three patients completed the study. The patients received either 1 mg of hydromorphone or 50 mg of meperidine IV at t = 0. Pain intensity was determined using a 10-cm visual analog scale at t = 0, 15, 30, 60, and 120 minutes. A second dose of the study drug could be given between t = 15 and t = 120 minutes when the clinician believed the initial dose was ineffective. Patients requiring more than one additional dose of analgesia were treated as nonresponders and were removed from the study. RESULTS: Thirty-six patients received hydromorphone and 37 received meperidine. The initial pain intensities (hydromorphone group = 8.4 +/- 1.5; meperidine group = 8.5 +/- 2.1), age distributions, sex distributions, and side effects of the two groups were comparable. Pain relief was better (p < 0.05) with hydromorphone at t = 15, 30, 60, and 120 minutes. The hydromorphone group required rescue analgesia less often (31% vs 68%, p < 0.01), had fewer IV pyelographies (IVPs) (28% vs 54%, p < 0.05), and had a lower proportion of hospital admissions (25% vs 49%, p = 0.08). CONCLUSIONS: For the fixed doses used in this study, the adult ureteral colic patients receiving hydromorphone achieved more pain relief, required less rescue medication, underwent fewer IVPs, and avoided hospital admission more frequently than did those receiving meperidine. PMID- 7541311 TI - Presence and actions of vasoactive intestinal peptide in the isolated rabbit heart. AB - The objectives of these experiments were to localize vasoactive intestinal peptide immunoreactivity (VIP-IR) in the intracardiac ganglia of the interatrial septum of the rabbit heart and to examine the effects of vasoactive intestinal peptide (VIP) on the isolated perfused rabbit heart. Cell bodies of neurones containing VIP-IR were located in the interatrial septa of rabbit hearts by using immunocytochemistry. In addition, the effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on the isolated rabbit heart were compared with those of VIP. Bolus injections of SP, NKA, VIP, and NKB caused dose-dependent decreases in the perfusion pressure of hearts perfused at constant flow with ED50 values of 2.73 +/- 0.10 fmol (n = 6), 0.18 +/- 0.01 pmol (n = 8), 93.75 +/- 1.88 pmol (n = 6), and 75.00 +/- 3.06 pmol (n = 6), respectively. The slope of the dose-response curve of VIP was much greater than those of SP and NKA, suggesting the presence of one receptor subtype for VIP and multiple receptor subtypes for the neurokinins on rabbit coronary vessels. Differences in the slopes of the dose response curves and potency may reflect differences in the mechanism of vasodilatation. The maximal values of vasodilatation of all of the peptides did not differ. None of the peptides produced significant changes in heart rate, left ventricular pressure, or contractility. These results suggest that VIP found in the intracardiac neurones of the rabbit heart can mediate coronary vasodilatation. PMID- 7541312 TI - Deregulated c-Fos/AP-1 modulates expression of the cyclin and the cdk gene in splenic B cells stimulated with lipopolysaccharide. AB - Overexpression of c-Fos/AP-1 augments proliferation of splenic B cells stimulated with lipopolysaccharide (LPS). To elucidate mechanisms of the augmentation by c Fos/AP-1, a cell cycle of the LPS-activated B cells from c-fos transgenic mice was analyzed. Cell cycle progression into the S phase was accelerated in the c fos B cells. Expression of genes related to the cell cycle progression was examined in these B cells. Amount of cyclin D3 and cdk4 mRNA increased in the c fos B cells at 6 h earlier than that in the control B cells, indicating that the kinetics of these mRNA expressions correlate with the acceleration of cell cycle progression. Furthermore, cyclin D1 and cyclin E mRNA were detected in the c-fos B cells but not in the control B cells. These results indicate that deregulated c Fos/AP-1 modulates expression of the cyclin and the cdk gene in splenic B cells stimulated with LPS. These modulations may accelerate cell cycle progression and augment proliferation of the B cells. PMID- 7541313 TI - CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl secretory pathways in addition to itself conducting Cl-. PMID- 7541314 TI - Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation? PMID- 7541316 TI - Extraction of water-soluble vitamins from pharmaceutical preparations using AOT (sodium di-2-ethylhexyl sulfosuccinate)/pentane reversed micelles. AB - Reversed micelles can be used to concentrate water-soluble materials in the water pool. In this study, the extraction of water-soluble vitamins into reversed micelles was attempted, and a flow system was used to determine the time-course of the vitamin extraction. The efficiency of extraction was strongly affected by the extraction temperature and the concentration of reversed micelles, and the selectivity depended on the size of micelles. Water-soluble vitamins could be efficiently and rapidly extracted. The selective extraction of a model mixture of vitamins from pharmaceutical preparations was also attempted. Moreover, the usefulness of the proposed method for the determination of vitamins in various commercial tablets was also demonstrated. Using of this method, the surfactant remains mixed with the extracted compounds, and so we attempted to remove the surfactant from the extract by supercritical fluid extraction. Supercritical carbon dioxide containing 7.5% ethanol as entrainer was found to be the most efficient solvent for removing residual surfactant from the extract. PMID- 7541315 TI - The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. AB - We have solved the crystal structure of an all-RNA hammerhead ribozyme having a single 2'-O-methyl cytosine incorporated at the active site to prevent cleavage. The conditions used differ from those in another recent solution in four significant ways: first, it is an all-RNA ribozyme rather than a DNA-RNA hybrid; second, the connectivity of the ribozyme backbone strands is different; third, the crystals were grown in the presence of a much lower concentration of salt; and fourth, the crystal packing scheme is very different. Nevertheless, the three dimensional structure of the all-RNA hammerhead ribozyme is similar to the previous structure. Five potential Mg(II)-binding sites are identified, including one positioned near the ribozyme catalytic pocket. Upon this basis, as well as upon comparisons with the metal-binding sites in the structurally homologous uridine turn of tRNAPhe, we propose a mechanism for RNA catalytic cleavage. PMID- 7541317 TI - Inhibitory effects of Egyptian folk medicines on human immunodeficiency virus (HIV) reverse transcriptase. AB - Extracts of 41 medicinal plants used in Egyptian folk medicine were screened for their inhibitory effects on human immunodeficiency virus-1 reverse transcriptase. The extracts of fruits of Phyllanthus emblica, Quercus pedunculata, Rumex cyprius, Terminalia bellerica, Terminalia chebula and Terminalia horrida showed significant inhibitory activity with IC50 < or = 50 micrograms/ml. Through a bioassay guided-fractionation of the methanol extract of the fruit of P. emblica, putranjivain A (1) was isolated as a potent inhibitory substance with IC50 = 3.9 microM, together with 1,6-di-O-galloyl-beta-D-glucose (2), 1-O-galloyl-beta-D glucose (3), kaempferol-3-O-beta-D-glucoside (4), quercetin-3-O-beta-D-glucoside (5) and digallic acid (6). The inhibitory mode of action by 1, 2 and 6 was non competitive with respect to the substrate but competitive with respect to a template-primer. Furthermore, the stereochemistry of 1 was established in this paper by nuclear magnetic resonance spectroscopy. PMID- 7541318 TI - Accumulation of an endogenous inhibitor of nitric oxide synthase during graded hemorrhagic shock. AB - Asymmetric dimethylarginine (ADMA) represents an endogenous inhibitor of nitric oxide (NO) production. The production of ADMA has been shown to increase during cellular stress, e.g., hypoxia. Furthermore, ADMA has recently been reported to accumulate in plasma during terminal renal failure as a consequence of diminished urinary excretion. Since tissue hypoxia and oliguria are both characteristics of severe hemorrhagic shock, this study was performed in order to establish whether plasma concentrations of ADMA increase during hemorrhagic shock. Six pigs were subjected to graded hemorrhage (20% and 40% of the calculated blood volume), resulting in significant (P < 0.05) reductions in blood pressure and cardiac output (from 98 +/- 4 to 36 +/- 5 mm Hg and from 3.0 +/- 0.2 to 1.4 +/- 0.2 L/min, respectively). Plasma ADMA concentrations as determined by high performance liquid chromatography (HPLC) increased from a pre-hemorrhage value of 3.4 +/- 0.3 microM to 3.9 +/- 0.4 microM (ns) and 5.2 +/- 0.4 microM (P < 0.05), respectively. The present study demonstrates that plasma ADMA concentrations increase significantly during hemorrhagic shock. Thus, inhibition of the arginine nitric oxide pathway as a result of ADMA accumulation, may represent an additional physiological mechanism to maintain systemic blood pressure in response to acute hypovolemia. PMID- 7541319 TI - Sudden death and tetralogy of Fallot. Risks, markers, and causes. PMID- 7541320 TI - Characterization of c-kit-positive neurons in the dorsal root ganglion of mouse. AB - Previously, we showed that c-kit receptor tyrosine kinase is expressed by a subpopulation of dorsal root ganglion (DRG) neurons, and that the ligand for the c-kit receptor, stem cell factor (SCF), induces the neurite outgrowth and supports the survival of these neurons in culture [16]. However, it is unknown which class of DRG neurons express c-kit receptor and which factor regulates differentiation and survival of c-kit-positive neurons. In the present study, we attempted to characterize c-kit positive neurons in the mouse DRG. The c-kit positive neurons were small or medium in size, and 44% of these neurons contained substance P. Central fibers of the c-kit-positive neurons terminated in laminae I and II of the gray matter of the spinal cord. These results suggest that c-kit positive neurons in the DRG belong to a functional subpopulation. The c-kit receptor protein was presented on the membrane of processes and growth cones in neurons. When DRG cells of embryonic day 15.5 or 17.5 were cultured, the survival of c-kit-positive neurons was supported by SCF, nerve growth factor (NGF) or leukemia inhibitory factor. SCF and NGF synergistically supported the survival of c-kit-positive neurons at submaximal concentrations. c-kit-positive DRG neurons from neonatal mice survived without addition of any factor in culture, suggesting that the requirement for trophic support in c-kit-positive neurons changes during development. PMID- 7541321 TI - Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture. AB - Cerebellar granule neurons obtained from 9-day-old rats were grown in vitro for 4 days in high K+ (26 mM) medium. The culture medium was then replaced with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determine whether the granule neurons respond directly to BDNF, we analyzed the induction of the Fos protein in these neurons. Individual cells that synthesize Fos protein after exposure to neurotrophin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF, but few of them were immunoreactive in the absence of BDNF or in the presence of NGF. Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+. PMID- 7541324 TI - [Progress in the diagnosis and treatment of small hepatocellular carcinoma]. PMID- 7541323 TI - Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial monolayers. AB - OBJECTIVE: Incubation with interferon-gamma has been shown to increase the permeability of cultured monolayers of intestinal epithelial cells. We sought to determine whether this phenomenon is mediated, at least in part, by increased production of nitric oxide. DESIGN: Prospective, controlled, laboratory study. Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports mounted in bicameral chambers. Permeability was assessed by adding fluorescein sulfonic acid (molecular weight = 478 daltons) to the apical compartment and determining the apical-to-basolateral clearance of the probe over a 24-hr period of incubation. SETTING: Basic science laboratory. MEASUREMENTS AND MAIN RESULTS: The permeability of monolayers to fluorescein sulfonic acid was significantly increased after incubation in the presence of interferon-gamma (250 to 1000 U/mL). The effect of interferon-gamma on permeability was dependent on both the concentration of the cytokine and the duration of exposure to it. Concentrations of nitric oxide oxidation products, nitrite and nitrate, in incubation media were increased after exposure of cells to interferon-gamma. When intestinal epithelial (Caco-2BBe) monolayers were incubated with interferon-gamma in the presence of inhibitors of nitric oxide synthase (NG-monomethyl-L-arginine NG-nitro-L-arginine-methyl ester, or NG-nitro-L-arginine), both of the effects of the cytokine (i.e., increased epithelial permeability and increased production of nitrite/nitrate) were attenuated. CONCLUSIONS: These results suggest that upregulation of nitric oxide biosynthesis plays a pivotal role in the increase in permeability of intestinal epithelial (Caco-2BBe) monolayers induced by interferon-gamma. Increased production of nitric oxide induced by proinflammatory cytokines, such as interferon-gamma, may be an important factor contributing to gut mucosal hyperpermeability in sepsis. PMID- 7541322 TI - Plasminogen activator activity and plasma-coagulum lysis measured by use of optimized fibrin gel structure preformed in microtiter plates. AB - We introduce a new fibrin plate assay performed in microtiter plates. By means of spectroscopic studies we optimized the structure of the fibrin gel and then used the optimized fibrin gel to determine plasminogen activator activity. Plasminogen activator solutions were applied on top of the fibrin gel, and the absorbance of the gel was recorded at 405 nm. After incubation for 17 h at 25 degrees C, the absorbance was measured again. The difference in absorbance was proportional to the concentration of plasminogen activator, such that the dose-response curves were linear when the difference in absorbance was plotted as a function of the logarithmic concentration of plasminogen activator. We assayed both tissue-type and urokinase-type plasminogen activator activity. The intraassay CV was < 4.7% (n = 20); the interassay CV was < 3.1% (n = 15). Using the optimized procedure, we modified the assay for determination of plasma-coagulum lysis time in human plasma. We established a reference interval for lysis time in apparently healthy subjects of 75 to 201 ks. Patients with deep vein thrombosis showed significantly (P = 0.013) higher values. PMID- 7541325 TI - [Clinical study of recombinant human granulocyte colony-stimulating factor (RHG CSF) on leucopenia induced by chemotherapy with CE regimen on lung cancer patients]. AB - The authors have studied the efficacy and safety of recombinant human granulocyte colony-stimulating factor (rhG-CSF, Granulocyte Injection) on leucopenia and neutropenia induced by chemotherapy with regimen CE (Carboplatin and Etoposide) in lung cancer patients in a randomized, matched and cross-over clinical trial. The total enrolled patients were twenty-two. They were randomized into A and B groups (11 patients in each group). Each patient received two cycles treatment. In group A chemotherapy and rhG-CSF were used in the first cycle and chemotherapy alone was used in the second cycle, while in group B chemotherapy alone was used in the first cycle and chemotherapy and rhG-CSF were used in the second cycle. The results showed that rhG-CSF significantly increased the number of white blood cell (WBC) and absolute neutrophil count (ANC) at the nadir, decreased incidence of leucopenia and neutropenia, and reduced the number of days with WBC < 4.0 x 10(9)/L, ANC < 2.5 x 10(9)/L as well as the number of days with WBC > 4.0 x 10(9)/L and ANC > 2.5 x 10(9)/L. rhG-CSF ensures the completion of chemotherapy and its side-effects were slight. PMID- 7541327 TI - [The change of platelet number in patient with acute myocardial infarction and its clinical significance]. AB - Platelet number and alpha-granule membrane protein (GMP-140) level on platelet surface were determined in 20 patients with acute myocardial infarction (AMI) and 20 normal subjects. The results showed that platelet number decreased significantly after AMI, especially on the second day after onset and that the level of GMP-140 increased rapidly after AMI and reached a peak on the second after the attack. Both returned to the normal on the 5-7th day after the heart attack. Correlative analysis between increase of serum CK-MB and platelet number was negative (r = -0.6123, P < 0.005), and positive between that and GMP-140 level (r = 0.5895, P < 0.01). Platelet number decreased markedly in cases with heart failure and cardiac death. The results indicate that platelets are involved in the pathological process of AMI. PMID- 7541326 TI - [The relationship between P-selectin on platelet membrane and platelet function in chronic cor pulmonale]. AB - Platelet plays an important role in thromboembolic diseases. It is demonstrated that recurrent pulmonary embolism may cause pulmonary hypertention. In recent years, P-selectin (granule membrane protein, GMP140) was found to be a surface marker for platelet activation. In order to investigate the role of platelet in chronic cor pulmonale, the changes of P-selectin on platelet membrane and its effect on platelet adhesiveness (PAdT) and platelet aggregation (PAgT) were studied. The results showed that the changes of P-selectin PAdT and PAgT in chronic cor pulmonale were significant than those in chronic pulmonary diseases and controls (P < 0.01). There was positive correlation between P-selectin and PAdT (r = 0.6831, P < 0.01) as well as PAgT (r = 0.7142, P < 0.01). The levels of von Willebrand factor (vWF) and fibrinogen (Fg) were markedly increased in chronic cor pulmonale as compared with chronic pulmonary disease and control group (P < 0.01). There was also positive correlation between vWF and PAgT (r = 0.4143, P < 0.05) as well as between Fg and PAgT (r = 0.4392, P < 0.05). It is concluded that platelet plays an important role in the pathogenesis of chronic cor pulmonale. PMID- 7541328 TI - [Clinical and immunophenotyping features of CD34-positive acute nonlymphoblastic leukemia]. AB - Antigen CD34 and other markers of cell membrane were investigated in cells from 43 patients with primary acute nonlymphoblastic leukemia (ANLL) by immunofluorescence test. The blast cells of 13 patients (30.2%) expressed antigen CD34. The patients with positive CD34 were no significantly different from the remaining 30 patients with negative CD34 with respect to age, serum lactate dehydrogenase (LDH), hemoglobin, white blood cell count, platelet count and the proportion of blast cells in blood and bone marrow, but their blasts were more likely to express HLA-DR, CD38, CD7 and lack of CD15 antigen. These patients had FAB M1 or M5a morphologic characteristics and lower complete remission (CR) rate. This result demonstrated that CD34 positive ANLL is poorly differentiated. PMID- 7541329 TI - [The diagnostic significance of anti-HCV IgM in chronic hepatitis C viral infection]. AB - The significance of anti-HCV IgM testing in the diagnosis and evaluation of treatment of chronic hepatitis C was studied. The serum samples from 82 patients with chronic hepatitis C were tested for ALT, anti-HCV IgG and IgM and HCV RNA. All the 82 cases were positive for anti-HCV IgG and 51 (62.19%) positive for anti HCV IgM. The emergence of anti-HCV IgM might be of four types: (1) persistent positive type, 24 (29.26%) cases; (2) intermittent positive type, 8 (9.75%) cases; (3) transient positive type, 19 (23.17%) cases; (4) persistent negative type, 31 (37.80%) cases. Usually there was progressive liver damage in type 1 and 2. If the patients were of transient positive type, they would usually recover. There was a significant correlation between the levels of serum anti-HCV IgM and ALT, while no correlation existed between the levels of serum ALT anti-HCV IgG. It is suggested that in patients with chronic hepatitis C, detection of anti-HCV IgM may be useful for estimating the extent of progressive liver damage and guiding the antiviral therapy. PMID- 7541330 TI - [Epidemiological studies on endemic fluorosis and arsenism in Xinjiang]. AB - Epidemiological and clinical studies on the water of 102 wells containing high level fluoride and arsenic and a population of 50,760 drinking the water in Kuitun Area, Xinjiang were conducted. Results indicated fluoride and arsenic contents in the well water were associated with geochemical environment, and drinking the water containing high-level fluoride and arsenic for a long-term was the cause of fluorosis and arsenism. Also, diagnostic criteria and major clinical manifestations for fluorosis and arsenism were put forward in the paper. Relationship between fluoride and arsenic contents in the well water and prevalence of fluorosis and arsenism in local residents was analyzed. It suggested fluorosis and arsenism was a systematic disorder and further studies on its potential hazard were merited. PMID- 7541331 TI - Specific staining of iododeoxyuridine and bromodeoxyuridine in tumors double labelled in vivo: a cell kinetic analysis. AB - The simultaneous and specific staining of iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) allows for more accurate estimates of potential doubling time (Tpot). Because CldUrd is not approved for human use, the procedure was adapted for the staining of IdUrd and bromodeoxy-uridine (BrdUrd). The fluorescein isothiocyanate-conjugated B44 antibody (B44-FITC) stained both IdUrd and BrdUrd in tumor nuclei labelled singly with one or the other pyrimidine analogue. However, when MCaK tumors in exponential growth in vivo were pulse labelled with both IdUrd and BrdUrd, the staining of BrdUrd was not seen, and the labelling pattern reflected specificity to IdUrd. These observations were confirmed using tumors pulse labelled with IdUrd and/or BrdUrd at 6 h and/or 0.3 h prior to tumor removal in all possible combinations. Simultaneous specific staining of BrdUrd by Br3 and of IdUrd by B44-FITC was documented by quantification of labelling indices (LIs) from double-labelled tumors. The specificity of B44-FITC for IdUrd in double-labelled tumors was due to a greater affinity of this antibody for IdUrd than for BrdUrd. This technique allowed for two independent estimates of LI and Tpot when tumors were double labelled for 3.0 and 5.5 h. Both IdUrd and BrdUrd are approved for clinical use, and this double labelling technique should prove to be valuable for measuring the cell kinetics of solid tumors in vivo. PMID- 7541332 TI - Use of the biotinylated antibody DAKO-ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells. AB - A method for the use of a biotinylated antibody (DAKO-ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating inflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. PMID- 7541333 TI - Benign prostatic hyperplasia. AB - Benign prostatic hyperplasia (BPH) is a pathologic disorder that develops in response to the action of dihydrotestosterone on the aging prostate and to changes in stromal and epithelial cells in this exocrine gland. The current therapies for this disorder are chosen after other causes for irritative and obstructive symptoms have been excluded and the status of the urinary tract has been assessed. This evaluation includes a detailed medical history, a thorough genitourinary and neurological examination, assessment of serum prostate specific antigen and creatinine levels, as well as a urinalysis. A urodynamic evaluation consisting of a combined pressure-flow study is required if the diagnosis of obstruction is to be made. Patients with minimal symptoms and normal test results require no therapy. Mild to moderate symptoms can be controlled, at least temporarily, with alpha-adrenergic blockers such as terazosin or doxazosin. A subset of BPH patients with obstructive symptoms respond to the 5 alpha-reductase inhibitor finasteride. Early results with minimally invasive treatments such as laser prostatectomies, hyperthermia, and ultrasonic and radiofrequency ablation appear encouraging for those with moderate symptoms of prostatism. Severe symptoms, urinary retention, gross hematuria, recurrent urinary tract infections, bladder calculi, and hydronephrosis or renal insufficiency warrant transurethral incision, resection, vaporization, or open prostatectomy (for very large neoplasms). Although the morbidities of these latter surgical therapies are not insignificant, these treatments offer the best and most durable results for relief of obstruction and amelioration of symptoms. PMID- 7541334 TI - A maternal factor affecting mouse blastocyst formation. AB - Normal development of the mouse embryo requires the presence of both paternal and maternal genomes. This is due to functional differences having their origin in a differential imprinting of parental genomes. Furthermore, several lines of evidence show that the very early interactions between egg cytoplasm and pronuclei may influence the programming of the embryonic genome and modulate the functional inequality of the parental contribution even during preimplantation stages. In this paper, we show that a factor present in ovulated oocytes of the mouse mutant strain DDK and therefore of maternal origin prevents the formation of the blastocyst. This factor, which acts via an interaction with the paternal genome, is present in oocytes as an RNA and is still active in preimplantation embryos. This is the first direct evidence of such a maternal control in the mouse. PMID- 7541336 TI - Follow-up by questionnaire? AB - Recently, proposals have been made to include larger numbers of infants and reduce the cost of obtaining follow-up information pertaining to modern perinatal management. These proposals have been made in response to requests from purchasers and providers of health care as well as the obstetricians and neonatologists actively engaged in delivery of the service. These initiatives are welcome, but care must be taken to provide objective, meaningful data. In addition to standardised recording including by questionnaire, standardised data collection designed to identify relevant impairments must be the primary objective; the nature and extent of disability at particular ages can then be assigned but it is misleading to regard disability as the principle outcome measure. PMID- 7541335 TI - Lenograstim. A review of its pharmacological properties and therapeutic efficacy in neutropenia and related clinical settings. AB - Lenograstim is a recombinant glycosylated human granulocyte colony-stimulating factor (rHuG-CSF) which principally regulates the formation and function of neutrophils. Like other colony-stimulating factors (CSFs), lenograstim has been developed for the prevention and treatment of iatrogenic and disease-related neutropenic conditions. In phase III clinical studies, prophylactic administration of lenograstim shortened the duration of chemotherapy-induced neutropenia in patients with nonmyelogenous cancers who received standard-dose chemotherapy or myeloablative regimens followed by bone marrow transplantation (BMT). A decrease in the incidence of infection after standard regimens and fewer days with infectious and febrile neutropenic episodes during recovery from BMT occurred concomitantly with the amelioration of neutropenia. In each setting, the decrease in morbidity was associated with shorter hospitalisation times and reduced administration of parenteral antibacterial agents. As with another rHuG CSF, filgrastim, bone pain (non-serious) was the most common adverse reaction to lenograstim therapy. This occurred in 13% of lenograstim recipients and 5% of placebo recipients treated for chemotherapy-induced neutropenia with standard regimens. Lenograstim may facilitate dose optimisation and permit limited dose intensification of standard chemotherapy. Furthermore, the drug, used alone or in combination with chemotherapy, is effective in mobilising peripheral blood progenitor cells (PBPCs) for subsequent reinfusion. The latter is a promising technique which may supplement or ultimately replace BMT for stem cell rescue after myeloablative chemotherapy. However, it has yet to be established whether the dose intensification achievable with lenograstim and/or stem cell rescue has a material effect on relapse-free and survival times. Preliminary data suggest that lenograstim is effective in increasing the neutrophil count in patients with severe chronic neutropenia (Kostmann's syndrome), as well as patients with AIDS or AIDS-related complex with zidovudine-induced neutropenia. Thus, lenograstim, like other CSFs, is a valuable adjunct to cytotoxic chemotherapy for the treatment of nonmyelogenous cancers, including myeloablative regimens followed by stem cell rescue with BMT and/or PBPC infusion. Future clinical experience is likely to confirm the usefulness of the drug in the management of disease-related neutropenia, myeloid disorders and neutropenia in patients with AIDS. PMID- 7541337 TI - Review of whole-organism bioassays: soil, freshwater sediment, and freshwater assessment in Canada. AB - Whole organism bioassays for the assessment of soil, freshwater sediment, and freshwater quality were evaluated for their application in the assessment and remediation of contaminated sites in Canada under the National Contaminated Sites Remediation Program. Using 3 essential and 12 desirable methodological criteria, bioassays were categorized as currently usable, prototype, or under development. Based on further considerations related to bioassay application, a battery of usable screening and definitive tests was recommended (with suggestions for augmentation) for each medium. Of the 18 bioassays reviewed for soil quality assessment, 6 were usable, 5 were prototypes, and 7 were under development. Battery screening and definitive tests included 14-day Eisenia andrei survival, 120-hr lettuce and radish seedling emergence, and 72-hr Selenastrum capricornutum growth inhibition. Augmentation with the following bioassays was recommended: soil/freshwater bacterial growth, arthropod reproduction, earthworm reproduction, and reproduction of other soil-dependent organisms. Of the 9 bioassays reviewed for freshwater sediment quality assessment, 1 was usable, 2 were prototypes, and 6 were under development. Three bioassays in the latter two groups were considered usable with the imminent completion of research underway. Screening tests selected included 10-day Chironomus tentans survival, 10-day Hyalella azteca survival, 10-day Hexagenia spp. survival, and 72-hr S. capricornutum growth inhibition. Definitive tests included screening tests, substituting 28-day H. azteca sexual maturation for 10-day survival. Augmentation with the following bioassays was recommended: sediment/freshwater bacterial test, 28-day Tubifex tubifex reproduction, and rooted aquatic plant growth. Of the 25 bioassays considered for freshwater quality assessment, 8 were usable, 7 were prototypes, and 10 were under development. Screening tests selected included 72-hr S. capricornutum growth inhibition; 48-hr Daphnia sp. survival, and 5- and 15-min Photobacterium phosphoreum bioluminescence. Definitive tests included first screening test, 7-day Ceriodaphnia dubia, 7-day fathead minnow larval survival, or 96-hr rainbow trout survival. Augmentation with the following bioassays was recommended: Brachionus calyciflorus 24-hr survival, 48-hr reproduction; freshwater bacterial growth; and aquatic vascular plant growth. PMID- 7541340 TI - The toxicity of substituted phenols in the nitrification inhibition test and luminescent bacteria test. AB - The nitrification inhibition test and the luminescent bacteria test were used to determine the inhibiting effect of different phenolic compounds. The chlorinated phenols demonstrated an increased toxicity in the nitrification inhibition test, which was much more sensitive to these compounds than other bacterial toxicity tests. In practice, the nitrification inhibition test was very useful for estimating the toxicity of chemical compounds to nitrifying bacteria, but the test is not recommended for the assessment of general bacterial toxicity. In comparison to other bacterial toxicity tests, the luminescent bacteria test also proved to be very sensitive. Due to its character, which measures a certain physiological parameter, this test system could be used as a screening tool for possible inhibitory effects to bacteria, although positive results should be confirmed by other bacterial toxicity tests. PMID- 7541338 TI - Effects of a heterogeneous set of xenobiotics on RNA synthesis of yeast cells. AB - Previously the toxicity of 45 heterogeneous environmental chemicals on growth and membrane functions in Saccharomyces cerevisiae and Chinese hamster ovary (CHO) cells (Cascorbi et al., 1993) was examined. In this study the inhibitory effects of the same set of chemicals on yeast RNA synthesis rate, measuring [2-14C]uracil uptake during cell proliferation are presented. The sensitivity of this test system was three to six times higher than that of the proliferation rate assay. In addition, the range of the EC20 values were similar to the range from the mammalian CHO cell proliferation test. The relatively good correlation with the yeast cell growth rate (r = 0.78, P = 0.0001) suggests that yeast RNA synthesis is a simple and rapid method of detecting a wide range of toxic compounds without the disadvantage of the insensitivity of the growth rate assay. PMID- 7541341 TI - Bacterial inoculum density and probability of para-nitrophenol biodegradability test response. AB - This study has been carried out to establish a model linking probability of positive response in para-nitrophenol biodegradability test to controlled variables of the test (suspended solids, SS; total bacteria, AODC; cultivable bacteria, CFU; specific biodegraders, MPN). Series of dilution of 11 raw inocula (6 activated sludges, 5 river waters) were tested. They reveal very dispersed values of biomass measured as SS, AODC, and CFU and quite comparable values of specific biodegraders for each category of inoculum (river or sludge). The proposed model fits well the empirical distribution of the experimental frequency of positive results versus inoculum density for each controlled variable. The constants k of the model, representing the fraction of biodegraders for each inoculum, were tested by the likelihood ratio test and were proven to be different from one another according to the biomass descriptor and the origin of the inoculum. The probabilistic model, in the case of para-nitrophenol biodegradation, indicates that standardized official tests (closed bottle, AFNOR, Sturm, and MITI I) are seldom optimal under those conditions. It allows the determination of which inoculum concentration can lead to a high probability (e.g., 99.9%) of observing paranitrophenol biodegradation by raw inocula. PMID- 7541339 TI - Effect of lindane exposure on rainbow trout (Oncorhynchus mykiss) immunity. IV. Prevention of nonspecific and specific immunosuppression by dietary vitamin C (ascorbate-2-polyphosphate). AB - The effect of the organochlorine insecticide lindane (gamma hexachlorocyclohexane) administered intraperitoneally at 10 or 50 mg/kg body wt on some major immune functions of rainbow trout (Oncorhynchus mykiss) was examined. Fish were fed vitamin C as ascorbate-2-polyphosphate at a basal level (60 mg ascorbic acid-(AA)-equivalent/kg of feed) or a high level (2000 mg AA equivalent/kg) 1 month before lindane exposure and during the whole experiment. The aim of the experiment was to determine whether dietary vitamin C is able to prevent immunosuppression due to lindane. The concentration of ascorbic acid in organs and the circulation was controlled, and the number of lindane residues in whole body was measured by gas chromatography. Nonspecific immune response was investigated through the determination of sera lysozyme and ceruloplasmin; both were significantly modified by lindane exposure while the immediate stimulating effects of vitamin C were observed. Cellular immunity was investigated by determining the number of B lymphocytes (analyzed by cytofluorometry) and their ability to proliferate with mitogens. One month after exposure to lindane (10 mg/kg) the proportion of Ig+ lymphocytes in head kidney was significantly decreased by the insecticide. Higher levels of vitamin C (2000 mg/kg) led to a significant increase in this parameter. Thus, vitamin C had a compensating effect on the number of Ig+ lymphocytes in exposed fish. Lindane at 10 mg/kg decreased the proliferation of B lymphocytes, but this was not confirmed at 50 mg/kg. Vitamin C stimulated the proliferation for the latter concentration after 2 months of intake. In lindane-exposed fish, the PMA-induced chemiluminescent response of head kidney phagocytic cells was variable from one assay to another, while most of the time vitamin C acted as a stimulant. The humoral response to Yersinia ruckeri was not modified by lindane but was significantly increased by vitamin C for 1 month after the antigen injection and thus 2 months after vitamin intake. PMID- 7541343 TI - The use of plants for environmental monitoring and assessment. AB - This paper presents a critical review on phytotoxicity tests for environmental monitoring and assessment. Vascular macrophytes used in the laboratory testing are emphasized; algae are mentioned only for comparison. Several issues are discussed, including the rationale for and misconceptions about phytotoxicity tests, relation to regulation, status of phytotoxicity test protocols, advantages and disadvantages of phytotoxicity tests, and possible research directions. Aquatic and terrestrial macrophytes, along with algae, are essential components of ecosystems. Macrophytes are becoming more important for the monitoring and assessment of herbicides, effluents, and industrial chemicals. In the United States, Canada, and international organizations, phytotoxicity tests can be required for environmental monitoring and assessment in statutes such as Federal Insecticide, Fungicide, and Rodenticide Act; Toxic Substances Control Act; Water Quality Act; Canadian Pest Control Products Act; and Canadian Environmental Protection Act. Possible research directions for phytotoxicity tests are discussed relative to the role in regulations of industrial chemicals, effluents, hazardous waste sites, and pesticides. PMID- 7541342 TI - Acute aquatic toxicity of protolyzing substances studied as the microtox effect. AB - In tests of acute aquatic toxicity, the effects of protolyzing substances varies with pH. The Microtox toxicity of six chlorophenols was studied over a pH interval and the relation of effects to dissociation was examined. The pKa values of the chlorophenols under the optimal test conditions are presented. The results of the Microtox toxicity tests supported a simple model of two species, phenol and phenate, with different specific toxicity. In five of the six chlorophenols only the effects of the phenol form were observed. The toxicity of the phenol form was 20 times or more higher than that of the phenate. PMID- 7541344 TI - HPLC determination of plasma thiocyanate concentrations in fish blood: application to laboratory pharmacokinetic and field-monitoring studies. AB - The pharmacokinetics of thiocyanate (SCN-) in the blood plasma of 35-g rainbow trout (Oncorhynchus mykiss) were followed during a 20-day exposure to 39.8 mg SCN liter-1 and the subsequent 16-day depuration period. SCN- concentrations were determined by reverse-phase HPLC. Kinetic constants were estimated using a one compartment first-order kinetic model fitted to the data by the computer programs BIOFAC and SYSTAT. The respective BIOFAC and SYSTAT estimates for the uptake rate constant (k1, 0.55 and 0.49 day-1), the depuration rate constant (k2, 0.34 and 0.29 day-1), and the bioconcentration factor (BCF, 1.61 and 1.66) were similar for both methods of calculation. Field studies were conducted to determine the impact of SCN- on white sucker (Catostomus commersoni) populations in waters receiving SCN(-)-bearing effluents. The assessment was based on SCN- concentrations in water and fish plasma, and the thyroid histology of the fish. Although SCN- was detected in the water at one site, no SCN- was detected in fish plasma and none of the thyroid pathology characteristic of chronic SCN- exposure was present. The results suggest that SCN- was not a hazard to the white sucker populations studied. PMID- 7541345 TI - A role for airborne particulates in high mercury levels of some cetaceans. AB - In a study of 25 Tursiops truncatus and two Globicephala macrorhynchus examined by necropsy, abundant HgSe was found in both the liver and in the respiratory system (lung and hilar lymph nodes). In the liver HgSe was consistently associated with the cell-breakdown pigment lipofuscin, whereas in lung and hilar lymph nodes it was consistently associated with particulates consisting of partially graphitic soot and silicates. This supports earlier suggestions that in the liver HgSe may be a storage end product of Hg metabolism, while adding the new suggestion that in the respiratory system HgSe may be inhaled, preformed in combustion emissions. PMID- 7541346 TI - Endoscopic palliation of a biliocutaneous fistula with expandable metal stents. PMID- 7541347 TI - Relief of malignant external gastric obstruction by endoscopic implantation of a self-expanding metal stent. AB - We report on the case of a 63-year-old woman with a gastric stenosis caused by peritoneal carcinomatosis. Vomiting and nausea disappeared after the implantation of a self-expanding metal stent in the 5 cm-long stenosis, and the patient was able to maintain a normal diet. She was discharged from hospital and remained in good condition for 14 weeks. Then, 16 weeks after stent implantation, the patient died of metastatic tumor spread, having remained free of nausea and vomiting. An autopsy showed complete patency of the stent, and no signs of ulceration or tumor ingrowth. The success of the treatment in this patient suggests the importance of further investigations. PMID- 7541348 TI - High-affinity ligand binding to subunit H1 of the asialoglycoprotein receptor in the absence of subunit H2. AB - The hepatic asialoglycoprotein receptor is a hetero-oligomer composed of two homologous subunits. The specificity and affinity of ligand binding depends on the number and spatial arrangement of several galactose-binding sites within the receptor complex. Previous studies indicated that both subunits are required for high-affinity ligand binding, i.e. for the simultaneous interaction with three galactose residues within an N-linked glycan. However, we found that asialoorosomucoid (ASOR) and asialofetuin (ASF) bind to transfected COS-7 cells expressing subunit H1 in the absence of the second subunit H2. ASOR binding occurred with a dissociation constant of approximately 40 nM, approximately four times higher than the Kd of ASOR binding to the hetero-oligomeric receptor. Normalized to the amount of H1 expressed, approximately 10-times fewer binding sites were produced by H1 alone. A glycopeptide with a single tri-antennary N linked glycan purified from ASF bound to the hetero-oligomeric receptor, but did not bind detectably to H1-expressing COS-7 cells. H1 is thus unable to simultaneously recognize all three galactose residues in a glycan. From this, we conclude that, at a sufficiently high density of H1 on the cell surface, high affinity binding of ASOR and ASF is the result of two or more glycans interacting with H1 oligomers with low affinity in a bivalent manner. PMID- 7541349 TI - Presence of an extended duplication in the putative low-density-lipoprotein receptor-binding domain of apolipoprotein B. Cloning and characterization of the domain in salmon. AB - The sequence of the C-terminal 1058 amino acids of atlantic salmon (Salmo salar) apolipoprotein (apo) B was deduced from the nucleotide sequence of cloned cDNA. In comparison with chicken or mammals apoB-100, salmon apoB is C-terminally truncated and extended gaps are found. The two clusters of positively charged residues, previously identified as part of the putative low-density-lipoprotein (LDL) receptor-binding domain of apoB, are brought into close proximity in salmon apoB. This is achieved by the absence between the two clusters of the proline rich area with the potential to form an amphipathic beta sheet, present in higher vertebrates. In addition, analysis of apoB amino acid sequences currently available in vertebrates revealed the presence of an extended internal duplication in the putative LDL receptor-binding domain. Thus, the two basic clusters would have been duplicated resulting in the presence, except for salmon apoB, of two homologous sites in the C-terminal part of the molecule. The results described here together with earlier biochemical and genetic evidence support the view that Arg3500, a residue mutated in familial defective apoB-100, could be included in a folded critical region of the putative LDL receptor-binding domain of human apoB-100. This region possibly brings the two sub-domains that arise from the duplication close to each other. PMID- 7541350 TI - Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase. AB - We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg 1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min 1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis. PMID- 7541351 TI - Regulation of the interferon-inducible protein kinase PKR and (2' 5')oligo(adenylate) synthetase by a catalytically inactive PKR mutant through competition for double-stranded RNA binding. AB - The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild-type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another double-stranded RNA dependent enzyme, the 40-kDa form of (2'-5')oligo(adenylate) synthetase. Inhibition of both wild-type PKR and (2'-5')oligo(adenylate) synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between [K296R]PKR and wild-type PKR or (2' 5')oligo(adenylate) synthetase for limiting amounts of double-stranded RNA. Moreover, the data imply that the tumorigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-stranded RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself. PMID- 7541353 TI - Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. AB - The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period. PMID- 7541354 TI - Structures of the N-linked oligosaccharides on human plasma vitronectin. AB - The structures of N-linked oligosaccharides present on human plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N glycosidase F digestion and tagged with 2-aminopyridine; the pyridylamino oligosaccharides were then fractionated by anion-exchange and reverse-phase HPLC. Ten major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N-acetyllactosamine type, with a lesser amount of the tri- and a small amount of the mono-antennary type, to which 1-3 mol sialic acid residues were linked, mostly through alpha 2-6 linkages, although alpha 2-3 linkages were also present. The possibility that several binding activities of vitronectin can be ascribed to its glycan moiety was discussed, based on the specific features of the N-linked oligosaccharides on human vitronectin revealed here. PMID- 7541352 TI - Fibroblast growth factors decrease inducible nitric oxide synthase mRNA accumulation in bovine retinal pigmented epithelial cells. AB - Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage. PMID- 7541355 TI - Flurbiprofen local action transcutaneous (LAT): clinical evaluation in the treatment of acute ankle sprains. AB - One hundred and thirty-one male and female outpatients, aged 18-70 yr, with acute pain in the ankle joint caused by a post-traumatic sprain, entered a multicentre, randomised, double-blind, parallel-group, study. The patients were assigned to a 40 mg flurbiprofen patch (n = 65) or a non-medicated (but otherwise identical) control (n = 66), 12-hourly over 7 days, and were assessed at entry and after 3 and 7 days treatment. On day 7, spontaneous pain (the prime efficacy parameter), as evaluated by the patient on a visual analogue scale in the physician's office, showed significant improvement in the 40 mg flurbiprofen patch group compared to control (change from baseline) (p = 0.039), a result corroborated by the evaluation of the periarticular oedema: a reduction of 77.4% was observed in the 40 mg flurbiprofen patch group, compared with 63.8% in the control group (p = 0.025). The other selected efficacy criteria showed changes with a trend in favour of the 40 mg flurbiprofen patch but without statistical significance. Two mild and local adverse events were reported by two flurbiprofen patch patients, but neither patients discontinued the treatment prematurely. Physicians and patients found the flurbiprofen patch to be efficacious and well tolerated. Compliance was excellent in both groups. The efficacy and tolerability of the 40 mg flurbiprofen patch are therefore confirmed in the treatment of acute ankle sprains. PMID- 7541356 TI - Mass screening for prostate carcinoma: a study in Hokkaido, Japan. AB - Although there has been controversy about the early detection of prostate carcinoma by mass screening, we carried out an investigational study of such screening, using digital rectal examination (DRE) and serum prostate-specific antigen (PSA) determination in Hokkaido, Japan. We determined the detection rate and some profiles of the carcinoma detected by screening in Japanese men 50 years old and older. The detection rate of carcinoma was 1.10% in 1,639 participants. The rate linearly increased with aging from 0.25% for men in their fifties to 2.29% for those in their seventies. Two thirds of the carcinomas were T1(c) or T2, the frequency of which seemed to be higher than that of patients who visited medical institutions. The simultaneous use of both DRE and serum PSA determination showed a higher positive predictive value than either alone. Our current results indicated that the detection rate of carcinoma was higher than we previously anticipated for Japanese men, and that simultaneous use of DRE and serum PSA determination yielded a higher positive predictive value than either alone. PMID- 7541357 TI - Potential effects of age-specific reference ranges for serum prostate-specific antigen. AB - Age-specific reference ranges for serum prostate-specific antigen (PSA) may improve this test for detecting prostate cancer. We have analyzed PSA levels from 10,024 men to determine the potential effects of these reference ranges. PSA levels (ng/ml) were grouped by patient age for comparison between standard (all ages: PSA < or = 4.0) and age-specific (< or = 49 years: PSA < or = 2.5; 50-59 years: PSA < or = 3.5; 60-69 years: PSA < or = 4.5; > or = 70 years: PSA < or = 6.5) reference ranges. Serum PSA correlated significantly with age (r = 0.33; p < 0.001). Fewer men > or = 60 years had elevated levels when age-specific reference ranges were applied (1,373 vs. 1,967; p < 0.001). Prostate biopsies and prebiopsy PSA levels from 865 men were reviewed. Sensitivities and specificities were calculated using both reference ranges. A significant increase in specificity with the age-specific reference ranges was seen for men > or = 70 years (58.6 vs. 34.2%; p < 0.001). There was, however, a concomitant decrease in sensitivity (77.6 vs. 91.7%; p < 0.001). We conclude serum PSA increases with age and we support the concept of age-specific reference ranges. However, the specificity of this test remains low, illustrating its limitations for prostate cancer detection. PMID- 7541358 TI - Pretreatment with chlormadinone acetate eliminates testosterone surge induced by a luteinizing-hormone-releasing hormone analogue and the risk of disease flare in patients with metastatic carcinoma of the prostate. AB - We investigated the efficacy of 2-week and 4-week pretreatments with 100 mg/day chlormadinone acetate (CMA) to prevent the flare reaction induced by luteinizing hormone-releasing hormone (LHRH) in patients with metastatic carcinoma of the prostate. CMA lead-in therapy suppressed bone pain and serum levels of luteinizing hormone, testosterone and prostate-specific antigen levels. CMA therapy also suppressed the transient increases in these levels associated with the initiation of therapy. The 4-week regimen appeared to be more effective than the 2-week regimen. PMID- 7541359 TI - Estramustine phosphate for preventing flare-up in luteinizing hormone-releasing hormone analogue depot therapy. AB - The usefulness of estramustine phosphate (ECT) for preventing flare-up in goserelin acetate depot therapy for advanced prostate cancer was studied. Pretreatment with ECT 560 mg daily for 3 weeks almost completely prevented the rise in testosterone level seen in goserelin acetate depot therapy and no signs or symptoms of tumor flare were observed. Long-term ECT completely blocked the rise in luteinizing hormone and testosterone level, but ECT at this dosage was likely to cause complications. The administration of ECT 560 mg daily for 3 weeks prior to goserelin acetate depot therapy was considered sufficient to prevent tumor flare, and its effect was considered to be more marked than that of short term treatment with antiandrogens. PMID- 7541360 TI - Tumor marker doubling time in patients with prostate cancer: determination of prostate-specific antigen and prostatic acid phosphatase doubling time. AB - To estimate the growth rate of prostate cancer, the doubling times of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) were determined in 51 patients: 44 were refractory to endocrine therapy, and 7 were in an untreated state. Since an exponential increase in PSA and PAP was observed in all patients, the doubling time was calculated from a semilogarithmic plot of the respective markers. PSA doubling time was almost identical with that of PAP. The tumor marker doubling time in untreated patients was approximately 10 times greater than that in the patients who were refractory to endocrine therapy. In endocrine refractory patients, the tumor marker doubling time in patients who showed deterioration of bone lesions was less than that in patients with local regrowth and/or lymph node metastasis. The prognosis of endocrine refractory patients from the time showing tumor marker failure was examined. The group showing the longest time (> 80 days) had better prognosis than that shown by the other groups with shorter doubling times. It is concluded that the determination of tumor marker doubling time is of value for measuring the growth rates of prostate cancer, and for assessing prognosis after relapse. PMID- 7541361 TI - Relationship of actin filament assembly to clearance of fibrinogen gold, GPIIb IIIa complexes on spread platelets. AB - The present study has evaluated the influence of high concentrations of cytochalasins B and E on the detergent-resistant actin levels in fully spread platelets by PAGE gel electrophoresis, and the effects of the two inhibitors of new actin filament assembly on translocation of fibrinogen gold (Fgn/Au) labelled GPIIb-IIIa receptors on the surface-activated cells. Concentrations of 10(-4) M and 10(-5) M cytochalasin B and E reduced detergent-resistant actin in fully spread platelets to levels present in resting discoid platelets in suspension. Despite reduction of actin filaments to levels in resting cells, cytochalasin B did not prevent translocation of Fgn/Au from platelet margins into channels of the open canalicular system (OCS). Similar concentrations of cytochalasin E completely blocked translocation of receptor-ligand complexes and produced a patching phenomenon not observed in previous studies. Rinsing of the spread cells to remove cytochalasin, followed by incubation of the treated platelets in Hank's buffered salt solution (HBSS) restored levels of detergent-resistant actin to those found in untreated, spread platelets. Resting grids of 10(-5) M cytochalasin E-treated platelets on drops of HBSS for 15 min restored their ability to clear FGN/Au linked to GPIIb-IIIa from exposed surfaces to the OCS, but 10(-4) M cytochalasin E-treated cells remained anergic after incubation on drops of HBSS. Thus a fully assembled cytoplasmic actin filament cytoskeleton does not appear to be essential for translocating receptor-ligand complexes on the platelet surface to the OCS, nor does its presence guarantee that the ability to clear GPIIb-IIIa receptors will be restored. PMID- 7541363 TI - [Genotoxic damage in subjects exposed to automobile exhaust: preliminary results]. AB - The associations between selected biomarkers of biologically effective dose (32P post-labelling-DNA adducts), early biological effects (Micronuclei, MN) and sister chromatid exchanges (SCE) and individual measurements of exposure to benzo(a)pyrene (a surrogate index of exposure to polynuclear aromatic hydrocarbons, PAHs) are investigated in a study being conducted among traffic police workers and referent subjects in the City of Genova, Italy. Preliminary findings show B(a)P mean values of 1.33 ng/m3, SD = 1.35 ng/m3 and 0.06, SD = 0.17 in traffic police workers and referent subjects, respectively. Micronuclei mean levels in peripheral blood lymphocytes of 26 traffic police workers and 25 referents were 3.30, DS = 1.24 e 3.90, DS = 1.50 (per 1,000 binucleated cells). Eleven subjects (61%) out of 18 had detectable levels of DNA adducts (32P-post labelling technique, range 1.8-9.0 Relative Adduct Level (RAL) per 10(9) nucleotides). PMID- 7541364 TI - [Air pollutants: legislation in Italy and EEC]. PMID- 7541362 TI - Studies on the protective effect of azepexole on ouabain-induced cardiac arrhythmias and lethality in guinea-pig. AB - Azepexole, an alpha 2-adrenoceptor agonist (125, 250 and 500 micrograms/kg i.v.), was examined for its effect on ouabain-induced ventricular premature beats, ventricular tachyarrhythmias and lethality in guinea-pigs. The doses of ouabain required to cause ventricular arrhythmias and lethality were significantly higher in azepexole-treated animals. However, it did not offer any protection in reserpinised guinea-pigs. Idazoxan, the alpha 2-adrenoceptor antagonist (100 micrograms/kg i.v.) inhibited the protective action of azepexole while corynanthine, the alpha 1-adrenoceptor antagonist (1 mg/kg i.v.), potentiated the effect. Azepexole inhibited the rate of the ouabain-induced rise in mean arterial blood pressure and the peak pressor response. In isolated paced left atria of guinea-pig, azepexole (2.76 x 10(-3) M) did not offer any protection against extrasystolic contractions induced by ouabain. Therefore the protective effect of azepexole may be mediated through the stimulation of alpha 2-adrenoceptors and the resultant suppression of the indirect neural components of ouabain toxicity. PMID- 7541365 TI - [Carcinogenic effects of exposure to automobile exhaust]. PMID- 7541366 TI - [Interactions between genetics and the environment: the example of tumors]. PMID- 7541367 TI - The granulocyte-macrophage regulators: reappraisal by gene inactivation. PMID- 7541368 TI - Modulation of adhesion molecule CD54 expression on CD34+ hematopoietic progenitors. AB - Multiple cellular adhesion molecules are constitutively expressed on hematopoietic progenitors. CD54 (ICAM-1), a member of the immunoglobulin supergene family, has recently been shown to be present on a subset of hematopoietic progenitors. To demonstrate that this adhesion molecule is expressed on the surface of the hematopoietic progenitor following induction rather than in its basal state, we studied the expression of cell surface CD54 on flow-cytometric (FACS) gated progenitors (CD34+; lymph plus blast light scatter). A mean +/- standard deviation (SD) of 2.75 +/- 2.10% was seen for FACS-gated progenitors from freshly isolated bone marrow mononuclear cells (BMMNC) stained with anti-CD54 MAb. Following incubation at 37 degrees C for 20 to 22 hours in media supplemented with heat-inactivated bovine fetal serum (FBS), the percentage of FACS-gated progenitors staining with anti-CD54 MAb increased to 46.28 +/- 18.38. This increase was temperature and time dependent. The percentage of FACS gated progenitors stained with anti-CD54 MAb did not increase following 20 to 22 hours of incubation at 4 degrees C. Furthermore, a subpopulation of FACS-gated progenitors staining with anti-CD54 MAb was detectable by 5 hours of incubation at 37 degrees C; the percentage of these progenitors staining with anti-CD54 MAb plateaued after 10 hours of timed incubation. Once induced, progenitor cell surface CD54 antigen density remained stable, as evidenced by a constant mean fluorescence intensity of anti-CD54 MAb staining. Three-color immunofluorescent studies showed that induction of CD54 occurred on CD45+ FACS-gated progenitors and that CD54 was induced on a subset of BMMNC expressing CD34 and CD11a, a counter receptor for CD54 that is limited in distribution to leukocytes. CD54 expression was induced to the same degree on FACS-gated progenitors positive or negative for CD19, CD36, and HLA-DR, respectively. These data indicate that CD54 is a unique adhesion molecule as its expression can be induced on CD34+ hematopoietic progenitors, and this induction does not appear to be related to hematopoietic progenitor lineage commitment or maturity. PMID- 7541369 TI - Neurotoxicity of acute glutamate transport blockade depends on coactivation of both NMDA and AMPA/Kainate receptors in organotypic hippocampal cultures. AB - Excessive activation of glutamate receptors is neurotoxic, contributing to brain injury caused by cerebral ischemia. The pharmacology of glutamate neurotoxicity is difficult to study in animals because it is efficiently cleared from the extracellular space by a family of glutamate transporters. We have investigated the receptor specificity of endogenous glutamate's toxic effects in organotypic cultures of the hippocampus by acute blockade of these transporters. The organotypic cultures used in these transporters. The organotypic cultures used in these experiments preserve the intrinsic connections and regional differentiation of the hippocampus in long term culture and may more closely reproduce the pharmacology of the mature brain region. Membrane injury was measured with digital fluorescence imaging of the vital dye, propidium iodide, 24 h after a 30 min exposure to glutamate receptor agonists or to antagonists of glutamate transport. Confirming our previous results, bath-applied, exogenous glutamate caused dose-dependent neuronal injury. Glutamate was less potent than the selective agonists NMDA, AMPA, and quisqualate. Blockade of glutamate transport with the selective antagonists threo-hydroxy-aspartate and pyrrolidine dicarboxylic acid also caused dose-dependent neuronal injury. Endogenous or exogenous glutamate toxicity was caused by a coactivation of both NMDA and AMPA/kainate receptors; blockade of either was sufficient to substantially prevent neuronal injury. Protective effects of combined application of antagonists were generally less than additive. We conclude that AMPA/kainate receptors play a more prominent role in glutamate neurotoxicity in organotypic cultures than in dissociated cortical or hippocampal cultures, acting together with NMDA receptors to cause neuronal injury. PMID- 7541370 TI - Preferential expression of the cell adhesion molecule CD44 in papillary thyroid carcinoma. AB - CD44 is a polymorphic family of immunologically related integral membrane glycoproteins associated with cell matrix adhesion, lymphocyte activation and targeting, and tumor growth and metastasis. We studied CD44 expression in 114 formalin-fixed paraffin-embedded thyroid tumors using the A3D8 anti-human CD44 monoclonal antibody. Sixty-five of 67 papillary carcinomas (97%) strongly expressed CD44 with an intense plasma membrane pattern. Thirty-seven of these cases originated from Albany, New York, and 30 cases from Russia. Immunoreactivity was also observed in 9 of 16 follicular adenomas (56%); 4 of 8 Hurthle cell neoplasms (50%); 5 of 15 medullary carcinomas (33%); and 3 of 8 follicular carcinomas (38%). These results show that among thyroid neoplasms, papillary carcinomas preferentially display the CD44 antigen (P < or = 0.001). Nonneoplastic follicular epithelium exhibited a low to moderate level of staining. To further characterize the CD44 isoform, we tested a subset of cases with the 2F10 anti-human CD44 variant 6 monoclonal antibody, which recognizes a CD44 variant exon (CD44v6) implicated in tumor metastasis. Eleven of 11 papillary carcinomas tested were 2F10 positive, and 1 of the follicular carcinomas was positive. These results suggest the hypothesis that deregulated CD44v6 expression on the plasma membrane of papillary carcinoma cells contributes to the ability of those cells to metastasize to regional lymph nodes and then to remain dormant for years. Our results suggest that human papillary thyroid cancer will be an interesting model in which to further study the role of CD44 isoforms, particularly those containing CD44v6, in tumor metastasis and lymphatic invasion. PMID- 7541371 TI - A method for distinguishing human and mouse cells in solid tumors using in situ hybridization. AB - A common technique used in the study of human malignancies involves the inoculation of nude mice with human neoplastic cells. It is usually assumed that the tumor arising is composed predominantly of human cells with mouse tissue present only to provide minimal stromal support. Several reports, however, have shown evidence of host cell neoplastic transformation. Therefore, in order to effectively study and characterize such xenografts, it is important to establish the relative involvement of human and mouse cells. In the present study, a method for easily distinguishing human and mouse cells is described. The method involves in situ hybridization of formalin-fixed tissues using DIG-labeled oligomer probes which correspond to species-specific portions of Alu sequences. This method can be applied to archival material either as a means of confirming that the tissue taken from nude mice xenografts is predominantly human or as a vehicle for studying the mechanisms of host cell neoplastic transformation and their relevance to human malignant spread. The proposed technique may also serve as a basis for other in situ applications, particularly those involving formalin-fixed tissues and oligomer probes. PMID- 7541372 TI - Basic properties of a novel ryanodine-sensitive, caffeine-insensitive calcium induced calcium release mechanism in permeabilised human vascular smooth muscle cells. AB - The efflux of 45Ca2+ from preloaded intracellular stores of saponin-permeabilised human uterine artery smooth muscle cultured cells was used to study the mechanisms underlying Ca2+ release from the sarcoplasmic reticulum (SR). The present paper demonstrates directly a functional Ca2+ release mechanism that is dependent on an increase in free Ca2+ (100 nM-30 microM) and is completely inhibited by 20 microM Ruthenium red. The amount of Ca2+ released at 30 microM free Ca2+ was reduced by approximately 50% compared to the release at 10 microM. This Ca(2+)-induced Ca2+ release (CICR) mechanism was not sensitive to caffeine. Exposure of cells to low free Ca(2+)-containing solutions (10 nM) indicated that a component of the CICR mechanism may be functional at basal free Ca2+ levels of 100 nM. Application of ryanodine (0.1-100 microM) induced 45Ca2+ efflux from the sarcoplasmic reticulum and this release was also inhibited by 20 microM Ruthenium red. PMID- 7541373 TI - Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine containing peptide, to HIV TAR RNA in vitro. AB - The ability of a small molecule, 2-methyl,4-carboxy,5-hydroxy-3,4,5,6 tetrahydropyrimidine (THP(A)), which accumulates intracellularly in various streptomyces, to inhibit the interaction of Tat peptide (R52) with TAR RNA is presented. Using gel-shift assay, we found that the inhibition constant Ki of THP(A) is 50-100 nM, which is in the range of the binding constants of Tat peptide and protein. THP(A) is approximately 10(6) times more tightly bound than the free L-arginine. The high binding affinity may be attributed to the special delocalized positive charge on the NCN group and the hydroxyl group at the 5 position of this molecule. A model for THP(A)-TAR interaction, analogous to the arginine guanidinum group-TAR interaction, is presented. The relatively high uptake of THP(A) by mammalian cells warrants in vivo Tat/TAR inhibition studies. PMID- 7541375 TI - Fibronectin attachment is permissive for IL-1 mediated gene regulation. AB - This study examines the effect of cell-matrix interaction on IL-1 induced gene regulation. In fibroblasts and smooth muscle cells attached to fibronectin, IL-1 caused a pronounced reduction in proteoglycan synthesis, while no reduction occurred in cells plated on bare plastic. Further, fibronectin attachment was permissive for IL-1 mediated suppression of both versican and collagen mRNA levels, initially noted after 4-6 h of IL-1 incubation. Attachment to vitronectin was less potent in influencing regulation, and collagen had no effect, suggesting specificity of the matrix modulation of the IL-1 induced response. Similar fibronectin induced dependence was demonstrated for IL-1 regulation of IL-6 gene expression, supporting the notion of a general effect of fibronectin receptor engagement on IL-1 induced signal transduction. PMID- 7541374 TI - Localization of a sequential B-epitope in the VP2 protein of hepatitis A virus. AB - A set of synthetic peptides derived from the capsid protein of hepatitis A virus was used to search for B-epitopes. Peptides from the 115-139 region of the VP1 protein, from the 69-99 region of the VP2 protein and peptide 137-150 from the VP3 protein were found to react with monoclonal and polyclonal anti-HAV antibodies. MAPs based on 64-80 and 66-80 fragments of VP3 were reactive as well. Peptides, their conjugates with protein carriers and MAPs were used for antipeptide antibody production. Only free peptide 69-99 from the VP2 protein caused formation of HAV binding antibodies. PMID- 7541376 TI - Brefeldin A provokes indirect activation of cdc2 kinase (MPF) in Xenopus oocytes, resulting in meiotic cell division. AB - Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements. PMID- 7541377 TI - Localized calcium signals in early zebrafish development. AB - Activation of the phosphoinositide (PI) pathway has been shown to be involved in the compaction of blastomeres in mouse embryos and in embryonic axis formation in Xenopus and in zebrafish embryos. Here we investigate Ca2+ signals in individual blastomeres of zebrafish embryos with the goal to better understand the role of PI and Ca2+ signaling for early vertebrate embryogenesis. Initial studies showed that the inositol 1,4,5-trisphosphate (IP3) concentration increases after the 32 cell stage of development, suggesting that IP3-mediated Ca2+ signals may be present during the blastula stage. Ca2+ signals were measured by identifying individual cells using confocal imaging of a nuclear localized Ca2+ indicator. Using this in situ indicator, changes in Ca2+ concentration were measured over several hours in each cell of a series of sections through the developing embryo. Transient increases in Ca2+ concentration that lasted 20-50 sec (Ca2+ spikes) were first triggered during the 32- to 128-cell stage in cells of the outer embryonic cell layer. These cells develop epithelial characteristics and specialize into the enveloping layer (EVL). No Ca2+ activity was observed during the earlier cleavage cycles or in deep blastomeres. Ca2+ spikes remained restricted to the EVL until the end of the blastula stage. Ca2+ spikes in neighboring EVL cells often occurred in the same short time interval, indicating that small groups of EVL cells can synchronize their activity. When averaged over several cell cycles, Ca2+ activity showed an even distribution in the EVL and did not indicate future polarities. PMID- 7541379 TI - Development of a mechanism-based dosimetry model for 2,4,4-trimethyl-2-pentanol induced alpha 2u-globulin nephropathy in male Fischer 344 rats. AB - A mechanism-based dosimetry model was developed to describe 2,4,4-trimethyl-2 pentanol (TMP-2-OH) dosimetry and renal alpha 2u-globulin (alpha 2u) nephropathy in the male Fischer 344 rat. Experimental data were collected to estimate the chemical-specific parameters (metabolic constants, tissue solubility, and oral absorption rate) necessary to describe TMP-2-OH dosimetry in male rats. The concentrations of alpha 2u and TMP-2-OH were measured in male rats up to 64 hr after a single oral dose of TMP-2-OH (6, 60, or 600 mg/kg). The model predicted the time course behavior of TMP-2-OH and alpha 2u in the kidney, but overestimated their renal concentrations by two or threefold. Simulations of renal alpha 2u concentration were sensitive to changes in TMP-2-OH-alpha 2u binding affinity and degradation rate of the TMP-2-OH-protein complex. In contrast, simulation of the concentration of TMP-2-OH in the kidney was most sensitive to the amount of protein present. Oral absorption of TMP-2-OH was dose dependent. The model predicted that alpha 2u and TMP-2-OH concentration in the kidney is sensitive to changes in the rate of TMP-2-OH absorbed after oral administration. This model permitted a more rigorous evaluation than has previously been possible of the combination of protein characteristics and chemical dosimetry required for the accumulation of alpha 2u in the kidney of male rats. The behavior of the model is consistent with the qualitative aspects of the alpha 2u hypothesis. However, further characterization of alpha 2u distribution and renal hydrolysis will be required in order to fully characterize the hypothesis at the quantitative level. PMID- 7541378 TI - Control of epitheliomesenchymal transformation. I. Events in the onset of neural crest cell migration are separable and inducible by protein kinase inhibitors. AB - Neural tubes isolated from quail embryos prior to epitheliomesenchymal transformation (EMT) and neural crest (NC) cell migration, when explanted onto fibronectin surfaces, replicated properties of normal NC morphogenesis such as (i) cell outgrowth, (ii) loss of A-CAM (N-cadherin) junctions and adoption of mesenchymal form, and (iii) development of HNK-1 immunoreactivity. The timetable of property (i) was essentially normal but the outgrowing cells were initially mainly epithelial, unlike NC outgrowth in vivo and in cultures of older neural tubes. Mesenchymal properties (ii) and (iii) were progressively and variably retarded relative to the in vivo timetable. Achievement of these properties by EMT was principally related to proximity to the neural tube and to preexisting mesenchymal cells, rather than being related temporally to the outgrowth timetable. This EMT, combined with a higher mitotic rate in the mesenchyme cells, resulted in the outgrowth passing from mainly epithelial at 16 hr to mainly mesenchymal at 48 hr in vitro. Immediate precocious EMT and outgrowth of A-CAM negative mesenchymal cells from pre-EMT neural tubes was stimulated by the protein kinase inhibitors staurosporine and bisindolymaleimide in a cycloheximide independent manner. EMT could be induced not only on the dorsal (i.e., NC) side, but also on the ventral side of the neural tube, but the ventral cells were less sensitive than the dorsal cells, and with developmental age became still less sensitive while the dorsal cells became more sensitive. The results suggest that the complex events of EMT in the NC system are not obligatorily coregulated, can be triggered by epigenetic events involving differential protein phosphorylation, and may be controlled via intraneural signaling. PMID- 7541380 TI - Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. AB - Diesel exhaust (DE) is a known pulmonary carcinogen in rats, and the carcinogenic response is known to require the presence of soot. Many estimates of human lung cancer risk from inhaled DE have been developed from rat bioassay data or from the comparative mutagenic potencies of DE soot extract and known human chemical carcinogens. To explore the importance of the DE soot-associated organic compounds in the lung tumor response of rats, male and female F344 rats were exposed chronically to diluted whole DE or aerosolized carbon black (CB) 16 hr/day, 5 days/week at target particle concentrations of 2.5 mg/m3 (LDE, LCB) or 6.5 mg/m3 (HDE, HCB) or to filtered air. The CB served as a surrogate for the elemental carbon matrix of DE soot. Considering both the mass fraction of solvent extractable matter and its mutagenicity in the Ames Salmonella assay, the mutagenicity in revertants per unit particle mass of the CB was three orders of magnitude less than that of the DE soot. Both DE soot and CB particles accumulated progressively in the lungs of exposed rats, but the rate of accumulation was higher for DE soot. In general, DE and CB caused similar, dose related, nonneoplastic lesions. CB and DE caused significant, exposure concentration-related increases, of similar magnitudes, in the incidences and prevalences of the same types of malignant and benign lung neoplasms in female rats. The incidences of neoplasms were much lower in males than females, and the incidences were slightly higher among DE- than CB-exposed males. Survival was shortened in the CB-exposed males, and the shortened survival may have suppressed the expression of carcinogenicity as measured by crude incidence. Logistic regression modeling did not demonstrate significant differences between the carcinogenic potencies of CB and DE in either gender. The results suggest that the organic fraction of DE may not play an important role in the carcinogenicity of DE in rats. PMID- 7541381 TI - Induction and variants of neuronal nitric oxide synthase type I during synaptogenesis. AB - In the adult central nervous system, nitric oxide (NO) is formed from L-arginine by the so-called constitutive or type I NO synthase (NOS-I155). However, expression of NOS-I155 immunoreactivity and activity was low or not detectable in developing mouse and rat brain. NOS-I155 was sharply induced coincident with the onset of synaptogenesis in specific brain regions. This was followed by a second phase in which total NOS-I155 expression decreased both in specific cell populations and in the total synaptosomal subcellular fraction.Furthermore, two putative variants of NOS-I were transiently observed: an NOS-I-immunoreactive protein with increased electrophoretic mobility (NOS-I144) and a transient hypersensitivity of NOS-I155 to the competitive substrate inhibitor N omega-nitro L-arginine. It is concluded that NOS-I expression is not constitutive but locally induced. In the central nervous system, this regionally specific, biphasic pattern of postnatal NOS-I induction is consistent with a role for NO in synaptogenesis and synaptic plasticity. PMID- 7541382 TI - c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase independent mechanism. AB - We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-/- mice (src-/- fibroblasts) exhibit a reduced rate of spreading on fibronectin. These defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/- fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of c-Src is followed by its redistribution to newly formed focal adhesions. These results suggest that the enzymatic activity and subcellular distribution of c-Src are coordinately regulated during cellular adhesion and that c-Src can affect adhesion by a kinase independent mechanism. PMID- 7541383 TI - When there is no cure: palliative care for the dying patient. AB - Acknowledging that death is a normal and inevitable process is often difficult for both physicians and patients. This article highlights by case example the type of care typically received by the terminally ill in acute care hospitals in the United States. The lack of familiarity with the core concepts of palliative care demonstrated by the medical team in this case reflects the pressing need for death education for health care professionals. We discuss the major principles of palliative care, including breaking bad news, developing a palliative care plan, planning for death, and withdrawal of artificial nutrition and hydration. PMID- 7541384 TI - [89-Strontium for painful osteoblastic metastases]. PMID- 7541385 TI - Experiments in transgenic mice show that hepatocytes are the source for postnatal liver growth and do not stream. AB - One hypothesis is that postnatal liver growth involves replication of mature hepatocytes, which have an unlimited proliferative potential. An alternative viewpoint is that only certain periportal cells can replicate extensively and that daughter cells stream slowly from the periportal to the pericentral region of the liver. Transgenic mice expressing the beta-galactosidase (beta-gal) gene from the human alpha 1 antitrypsin promoter were used to examine the proliferative potential of hepatocytes. Surprisingly, only 10% of hepatocytes in two different transgenic lines stain blue with X-gal. In neonatal animals, singlets or doublets of expressing cells are randomly scattered throughout the liver. Although the overall frequency of blue cells is similar in older animals, these cells are present in much larger clusters, suggesting that individual expressing cells have replicated to form a clonally derived cluster. Expression patterns are not altered by the administration of an acute phase stimulus or by the performance a partial hepatectomy, suggesting that the expression state cannot be easily altered, and making it more likely that the expression state is indeed fixed. These results suggest that the clusters of blue cells are clonally derived in the transgenic mice. They argue that the parenchymal hepatocyte is responsible for growth in the postnatal liver and that streaming of liver cells does not occur. PMID- 7541387 TI - Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. AB - Hepatitis C virus (HCV) populations in vivo exist as a mixture of heterogeneous viruses called quasispecies, which have variations in the hypervariable region (HRV). However, the relationship between the diversity of HVR quasispecies, the disease stage, or the interferon (IFN) responsiveness remains to be elucidated. To study these, serum samples were obtained from 42 patients with chronic hepatitis C virus infection; 24 with chronic active hepatitis (CAH) treated with IFN, 9 with cirrhosis, 9 with hepatocellular carcinoma (HCC). HCV quasispecies populations were separated by the single-strand conformation polymorphism (SSCP) method targeted to the HVR. The patients were classified into two groups; a single-band group (n = 12) in which HVR quasispecies was homogeneous and a multiple-band group (n = 30) in which HVR quasispecies was heterogeneous. Patients with multiple bands had significantly more advanced liver disease than those with a single-band group (P = .0082). The percentage of patients with single band were 41% in CAH, 22% in cirrhosis, and 0% in HCC. Multivariate analyses showed that viral diversity was independently related to the progression of liver disease and was not correlated with the duration of infection. We also found that in CAH, the patients who had multiple bands (n = 14) were more resistant to IFN therapy than those who had a single band (n = 10) (P = .002). These results indicate that the diversity of HCV quasispecies becomes more complex as the disease stage progresses and that CAH with more complex diversity shows less IFN effectiveness. PMID- 7541386 TI - Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression. AB - In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin. PMID- 7541390 TI - Humoral response to linear B cell epitopes in the amino terminus of the hepatitis C virus envelope glycoprotein gp72 (E2): role in protective immunity still unknown. PMID- 7541389 TI - Immune mechanisms of alcohol-induced liver disease. PMID- 7541388 TI - Fc receptors in liver sinusoidal endothelial cells in NZB/W F1 lupus mice: a histological analysis using soluble immunoglobulin G-immune complexes and a monoclonal antibody (2.4G2). AB - In systemic lupus erythematosus accompanied by the abnormal appearance of circulating immune complexes (ICs), Fc gamma receptor (FcR)-mediated IC handling in macrophages including Kupffer cells has been shown previously. However, sinusoidal endothelial cells (SECs) largely ingest soluble immunoglobulin (Ig) G ICs through FcRs. In this study, the character, antigenic expression, and activity (i.e., ligand-binding capacity of SEC FcRs in NZB/NZW F1 lupus and NZW nonautoimmune mice) were immunohistochemically analyzed using monoclonal antibody (MAb) 2.4G2 to FcRs and peroxidase-antiperoxidase IgG as a ligand on cryosections. MAb 2.4G2 stained SECs and blocked the ligand binding of SEC FcRs in both mice strains. The staining intensities with MAb 2.4G2 in SECs and the FcR activities in SECs alone and all sinusoidal cells in both mice strains reached their maximum values at the age of 5 months. Staining intensities in NZB/W F1 were significantly higher at 1 and 2 months and lower at 9 months than those in NZW. The number of Kupffer cells detected by MAb F4/80 to macrophages in both mice strains gradually increased until 5 months, but their number in NZB/W F1 at 9 months was twice as large as that in NZW. In conclusion, SEC FcRs in mice are low-affinity FcRs that react with MAb 2.4G2. The data of FcR activity suggest no impairment of the FcR-mediated IgG-IC binding on SECs in NZB/W F1 in early life.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541391 TI - Epitope specific monoclonal antibodies from heterologous antigen for immunodiagnosis of filariasis. AB - The excretory secretory (ES) antigens produced from Setaria digitata were studied by PAGE and Western blot and compared with human filarial systems. Monoclonal antibodies (K3AE7 and K3BD5) developed against the ES antigen were evaluated by ELISA for the diagnosis of human filariasis. Cent per cent positive reaction was found with the circulating filarial antigen (CFA) of microfilaria carriers, occult and symptomatic patients and clear negative reaction with both the endemic and nonendemic controls. The clones offer promise for the development of highly sensitive diagnostic reagents for the detection of filariasis. PMID- 7541394 TI - Expression of acid cysteine proteinase inhibitor (ACPI) in the normal human prostate, benign prostatic hyperplasia and adenocarcinoma. AB - Acid cysteine proteinase inhibitor (ACPI or cystatin A) is a protein (12 kDa) which inhibits the action of several cysteine proteinases, e.g. cathepsins B, H, L and S. In this study the cellular location of ACPI has been immunohistochemically investigated in the normal human prostate, in benign prostatic hyperplasia (BPH) and in adenocarcinoma. ACPI was found in the basal epithelial cells of the normal prostate. The secretory epithelial cells did not express ACPI. In the hyperplastic prostate, the expression of ACPI was decreased and it was also expressed more focally in the basal cells. Hyperplastic basal cells also expressed ACPI. In prostatic adenocarcinoma, no ACPI expression was found. The absence of ACPI expression was obvious and if the sections contained both benign and malignant cells, only the benign glandular structures always expressed ACPI. The results suggest that expression of ACPI might be related to prostatic epithelial cell proliferation and differentiation. Possibly the detection of ACPI in tissue sections might be helpful in identifying prostatic adenocarcinoma, especially in cases with small carcinomatous foci. PMID- 7541392 TI - Effect of tumor necrosis factor receptor binding protein on cell infiltration induced by lipopolysaccharide and Sephadex beads in guinea pig lung. AB - In the present study, the effect of a tumor necrosis factor receptor binding protein (TNFbp) on the cell infiltration induced by lipopolysaccharide (LPS) and Sephadex beads in guinea pig lung was examined. The intratracheal injection of LPS (2.5 micrograms) induced a six-fold increase in total cell number recovered in bronchoalveolar lavage (BAL) fluid at 24 hr. This increase in bronchopulmonary inflammation was mainly due to a neutrophil and macrophage infiltration, representing 60% and 35% of the total cells, respectively. The intravenous or intratracheal injection of Sephadex beads to guinea pigs induced a three-fold increase in total cell number recovered in BAL at 24 h and was characterized by a prominent eosinophil, macrophage, and neutrophil infiltration representing 36%, 42%, and 16% of the total cells, respectively. In addition, bronchial tissues isolated from Sephadex-treated guinea pigs showed an increased in vitro reactivity to both histamine and acetylcholine. TNFbp (1-50 micrograms) induced a dose-dependent inhibition of cell infiltration induced by LPS. In contrast TNFbp neither attenuated the bronchopulmonary cell infiltration observed 24 h following intravenous or intratracheal administration of Sephadex beads nor inhibited the increase in bronchial reactivity. These results show that TNF plays an important role in cell infiltration induced by LPS, but not that induced by Sephadex, in the guinea pig lung. PMID- 7541395 TI - Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti melanoma CTL line. AB - Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp 100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp 100 protein. Apart from the peptide corresponding to gp 100 amino acids 457-466, we identified the gp 100 peptide 154 162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp 100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp 100 epitope for TIL 1200. Together with the recently described gp 100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp 100, all presented in the context of HLA-A2.1. Therefore, gp 100 is an attractive target antigen in the development of immuno-therapeutic protocols against melanoma. PMID- 7541393 TI - Colitis reduces short-circuit current response to inflammatory mediators in rat colonic mucosa. AB - Inflammatory mediators may contribute to the diarrhea associated with colitis. Although the secretory action of such mediators is reported in normal tissue, there is little information regarding their effects on inflamed tissue. We examined the short-circuit current response (Isc) to these mediators, in mitomycin-C (MC)-induced colitis, a model with histological similarities to colitis in man. Rats were injected once with MC (3.25 mg/kg, intraperitoneally) or vehicle. The colons were removed three and seven days later and mounted, devoid of muscularis, in Ussing chambers for measurement of Isc, potential difference (PD), and resistance (Rt). MC-treated rats had diarrhea after three days, and microscopic studies revealed colonic inflammation. There were no significant differences in Rt, PD, and Isc between control and MC-treated tissues at three and seven days. Maximal increases in Isc to bradykinin, prostaglandin E1, carbachol, substance P, and serotonin were depressed at three and/or seven days after MC. The Isc response to theophylline was not affected. Theophylline activates secretion through an intracellular mechanism; the other agonists act by interaction with epithelial cell membranes. Therefore, the mechanism for the decreased Isc may result from uncoupling of receptors to second-messenger systems or desensitization of receptor-linked secretory mechanisms. PMID- 7541396 TI - Expression of muscarinic receptor subtype mRNA in the human ciliary muscle. AB - PURPOSE: To investigate the expression of the muscarinic receptor (mAChR) subtypes (m1, m2, m3, m4, and m5) in the human ciliary muscle. METHODS: cDNA probes specific for the m1 to m5 subtypes were used to characterize the expression of subtype-specific mRNAs by in situ hybridization and Northern blot analysis on a cell line derived from human ciliary muscle tissue (H7CM), sectioned tissue from four donors, and fresh human ciliary muscle tissue from three donors. RESULTS: Messenger RNA for m2, m3, and m5 was detected in situ hybridization and Northern blot analysis in the H7CM cell line and in human ciliary muscle tissue from the donors. In human ciliary muscle tissues, the expression of mRNA of the m2 and m3 subtypes was more prominent in the circular portion than in the longitudinal portion. In contrast, mRNA for the m5 subtype was greater in the longitudinal portion than in the circular portion. Additionally, Northern blot analysis showed that m1 and m4 were expressed in ciliary muscle tissue. CONCLUSION: These studies show that the human ciliary muscle definitely expresses the mRNA of subtypes m2, m3, and m5, and may also express the mRNA of m1 and m4. Although the expression of mRNA for the m2, m3, and m5 subtypes differed between the circular and longitudinal portions of the ciliary muscle, the strong expression of all three subtypes of muscarinic receptors by both portions demonstrates that the differential distribution of muscarinic subtypes probably is not responsible for the dissociation between outflow facility and accommodation that can occur under certain conditions. PMID- 7541397 TI - The effect of caffeine on mammary gland development and milk yield in primiparous sows. AB - Pregnant Yorkshire gilts (n = 42) were fed caffeine (6 g/d) or served as controls from d 60 of pregnancy until d 4 postpartum to test the effect of caffeine on mammary gland development, milk yield, and feed consumption. Caffeine reduced voluntary feed intake (P = .001) and body weight gain (P = .001) of gilts from d 60 to 109 of gestation. Pig birth weight in the treated group was less than (P = .01) that in the control group. However, pig viability score at birth was not affected by maternal caffeine ingestion. For assessing mammary gland DNA, RNA, dry fat-free tissue (DFFT), fat, and protein content, four sows from the caffeine group and three controls were slaughtered on the 1st d of lactation. Immediately after slaughter, mammary systems were removed, separated by gland, and dissected free of skin, muscle, and fatty pad, which had not been invaded by glandular tissue. The DNA and RNA content were evaluated in DFFT. Caffeine increased mammary RNA content (P = .023) and milk yield (P = .001) on d 1 of lactation. However, DNA, DFFT, fat, and protein were not significantly increased, although values were somewhat greater in the treatment group (approximately 82%). On d 21 of lactation, milk yield of treated sows did not differ from that of controls. The increased milk yield on d 1 of lactation was due to increased mammary epithelial cell activity and cell numbers. These results indicate that caffeine feeding can have a positive effect on porcine mammary gland development as well as milk yield. PMID- 7541398 TI - Reinforcer assessment for children with developmental disabilities and visual impairments. AB - We assessed the applicability of two previously developed reinforcer assessment procedures to children with developmental disabilities and visual impairments. Greater differentiation between stimuli was observed with a choice procedure than with a preference procedure. Measurement of compliance and rate of responding in adaptive skill training confirmed that the choice procedure accurately identified reinforcing stimuli. The preference procedure produced false positive predictions of reinforcer efficacy. PMID- 7541399 TI - Localization of penicillin-binding proteins to the splitting system of Staphylococcus aureus septa by using a mercury-penicillin V derivative. AB - Precise localization of penicillin-binding protein (PBP)-antibiotic complexes in a methicillin-sensitive Staphylococcus aureus strain (BB255), its isogenic heterogeneous methicillin-resistant transductant (BB270), and a homogeneous methicillin-resistant strain (Col) was investigated by high-resolution electron microscopy. A mercury-penicillin V (Hg-pen V) derivative was used as a heavy metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the presence of an abnormally large (17 to 24 nm in width) splitting system within the thick cross walls or septa of Hg-pen V-treated bacteria of all strains. Untreated control cells possessed a thin, condensed splitting system, 7 to 9 nm in width. A thick splitting system was also distinguishable in unstained thin sections, thereby confirming that the electron contrast of this structure was not attributed to binding of bulky heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membranes as well as sodium dodecyl sulfate-treated cell walls and that two or more PBPs in each strain bound to this antibiotic. In contrast, the splitting system in penicillin V-treated bacteria was rarely visible after 30 min in the presence of antibiotic. These findings suggest that while most PBPs were associated with the plasma membrane, a proportion of PBPs were located within the fabric of the cell wall, in particular, in the splitting system. Inhibition of one or more high-M(r) PBPs by beta-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesis and architectural design of these structures in S. aureus. PMID- 7541401 TI - Molecular analyses of the phase-2 antigen complex 1,2,.. of Salmonella spp. AB - The nucleotide sequences of the structural genes (fljB) for salmonellar flagellins representative of the phase-2 flagellar antigens 1,2.., 1,5.., 1,6.., and 1,7.. were determined. The results did not indicate linear epitopes for the antigen 1 subfactors, suggesting that conformational aspects are involved in determining these antigenic specificities. PMID- 7541400 TI - Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans. AB - Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific beta-1,6 glucan antiserum but not with a beta-1,3-glucan antiserum. They lost their beta 1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that beta-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure beta-1,3-glucanase, several mannoproteins that were recognized by both the beta-1,6-glucan antiserum and the beta-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that beta-1,3-glucan and beta-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of beta-glucans in the retention of cell wall proteins is discussed. PMID- 7541402 TI - Optimal growth of human blood hematopoietic progenitor cells collected by apheresis for autografts. AB - For safe autografts with peripheral blood hematopoietic cells (PBSCT), better methods for determining the kinetics of stem cell populations and predicting engraftment speed after PBSCT need to be established. Current methods include culture in semi-solid medium and measurement of CD34 cell surface antigen. In this study with only partially purified blood cells obtained from children with cancer in remission, we compared the effects of phytohemagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM) and recombinant human cytokines on the growth of progenitor cells in a methylcellulose culture system. Interleukin-3 (IL 3) alone supported more progenitor growth than standard PHA-LCM by a factor of 1.54 for colony-forming unit granulocyte/macrophages (CFU-GM) and by a factor of 1.84 for burst-forming unit/erythroids (BFU-E). No significant change, in terms of the number of growing colonies, was observed by adding granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), or IL-1 to IL-3. However, the addition of G-CSF resulted in increased colony size. A further increase in CFU-GM growth was observed by the addition of IFN-gamma to the combination of cytokines. No significant effect was observed when stem cell factor (SCF) was added to the combination of cytokines containing IL-3, G-CSF, and IFN-gamma. This analysis suggests that the combination of IL-3, G-CSF, and IFN-gamma may provide sufficient stimulation for the growth of human blood cells. The effects of different oxygen tensions on progenitor growth in the presence of IL-3, G-CSF, and IFN-gamma were also evaluated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541403 TI - Effects of whole blood flow rates on mononuclear cell yields during peripheral blood stem cell collection using Fenwal CS 3000 Plus. AB - It is generally assumed that using high whole blood flow rates (WBFR), 80 ml/min, during peripheral blood stem cell (PBSC) collection on the Fenwal CS 3000 Plus blood cell processor will result in higher yields of mononuclear cells (MNC) than using lower WBFR (50 ml/min). To test this assumption, we retrospectively studied 129 PBSC procedures on 17 patients in a multiple myeloma protocol comparing MNC yield, as well as red blood cell (RBC), granulocyte, and platelet (Plt) content, of four average WBFR groups. Standard PBSC procedures were performed using modified procedure 1, interface offset 100, anticoagulant (AC) ratio of 11:1, small volume collection chamber, and a processing time of 4 hours. After correcting for AC volume used, the volume processed was divided by 240 minutes to obtain the average WBFR. WBFRs were separated into 4 groups of 40-49, 50-59, 60 69, and 70-79 ml/min. When compared to the highest flow rate group (70-79 ml/min), the three lower flow rate groups had significantly higher MNC yields of 16.2 +/- 6.9, 13.1 +/- 5.1, and 11.5 +/- 4.7 x 10(9), respectively, as compared to 8.9 +/- 6.1 x 10(9) MNC for the 70-79 ml/min group. There was no significant difference in granulocyte yield which ranged from 1.6 +/- 2.1 to 4.5 +/- 4.8 x 10(9).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541404 TI - Quantitative analysis of HIV-1 RNA in plasma preparations. AB - HIV-1 RNA extraction methodology, stability and cellular location in plasma were studied by quantitative analysis using reverse transcriptase (RT) and polymerase chain reaction (PCR). HIV-1 RNA as intact virus was stable in plasma at room temperature for at least for 24 h, or stable in RNAzol (Tel-Test, Inc. Texas) at 70 degrees C for at least 6 months. The HIV-1 RNA PCR signal did not decline significantly after freezing and thawing of the virus in plasma or in RNAzol. To assess the effect of plasma constituents from different individuals upon quantitative PCR, identical copy members of HIV LAI were spiked into plasma from 9 different, normal individuals. PCR detection of HIV-1 RNA did not show any significant variation in quantitative signals. Additionally, platelet-rich plasma from three seropositive subjects was fractionated into a platelet-free plasma fraction and a platelet pellet fraction. The quantitative analysis of HIV-1 RNA in these fractions, and in the corresponding peripheral blood lymphocytes (PBLs) from each patient, demonstrated that the majority of the HIV-1 RNA was distributed in the plasma, and the HIV-1 RNA in the plasma of these patients seemed not to be strongly platelet associated. PMID- 7541405 TI - Detection of all serotypes of human astrovirus by the polymerase chain reaction. AB - A reverse transcription (RT) and polymerase chain reaction (PCR) was designed for the detection of astroviruses based on a conserved nucleotide sequence in the 3' end of the genome of the 7 known serotypes of human astrovirus. Thirty-eight samples found to contain astrovirus by electron microscopy (EM) were used for evaluation of the assay. The samples were dialyzed for 1 h to remove potential low molecular weight inhibitors of the RT-PCR. Immediately before RT, 1 microliters of the samples were incubated at 94 degrees C for 2 min to disrupt the viral particles. Thirty-six of the samples were positive by PCR, including samples of all 7 serotypes. The two samples that were negative, could hve been false positive by EM, or the viral RNA could have been degraded. All other viruses examined, including calici-, rota- and enteroviruses, were negative. PMID- 7541406 TI - Variational expression of functionally different macrophage markers (27E10, 25F9, RM3/1) in normal gingiva and inflammatory periodontal disease. AB - Keratinocytes and macrophages share under immunologically activated conditions several surface proteins. We investigated immunohistochemically with monoclonal antibodies and the APAAP technique the expression pattern of 27E10 antigen (inflammatory macrophages), 25F9 antigen (resident macrophages) and RM3/1 antigen (intermediate macrophages in healing tissue) in 29 specimen biopsies of different stages of gingivitis and periodontitis. Macrophages of each subtype exhibited a different localization pattern depending on the stage of inflammation. Furthermore, suprabasal oral gingival epithelia showed a constant 27E10 expression, independent of the stage of inflammation. In contrast, all layers of the sulcus and pocket epithelia in gingivitis and periodontitis were strongly 27E10-positive, indicating immunological activation. 25F9 antigen was expressed on basal keratinocytes independent of the stage of inflammation, whereas RM3/1 was constantly negative on keratinocytes. The expression pattern of these functionally different macrophage markers on lesional macrophages and keratinocytes indicates varying differentiation and activation and suggests a participation of these cells in the local immune response in periodontal infection. PMID- 7541408 TI - Biologic response modifiers in chronic hepatitis C. AB - Biologic response modifiers include various colony-stimulating factors and interleukins such as granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and interleukin-2. Their use as therapy has proven to be beneficial in the treatment of various diseases, and in preventing hematologic disorders. Their biologic effects and the rationale for their use in chronic viral hepatitis, alone or in combination with interferon alpha, are reviewed with respect to the treatment of chronic hepatitis C. PMID- 7541407 TI - Lifelong treatment with gammaglobulin for primary antibody deficiencies: the patients' experiences of subcutaneous self-infusions and home therapy. AB - Primary antibody deficiencies are chronic conditions and the patients usually need lifelong replacement therapy with gammaglobulin to prevent or reduce infections. It has been shown that the gammaglobulin can be given safely as subcutaneous infusions, instead of intramuscular injections or intravenous infusions. The major aim of this multi-centre study was to investigate the perceptions of the subcutaneous method among patients using it, both in hospital settings and as self-infusions at home. The study included 152 patients: 89 women, 63 men, mean age 44 years (range 18-76). Data were collected by using questionnaires. The patients were found to have a strongly positive attitude towards receiving the replacement therapy as subcutaneous infusions, perceived the method as effective in preventing infections and wished to retain the treatment. However, the younger patients found the subcutaneous infusions more uncomfortable and were less determined to continue with the therapy as compared with the older individuals. The responsibility for self-infusions at home was accepted by the patients, leading to an increased independence from the health care personnel and to a feeling of flexibility and freedom. As these patients have a chronic disease and are in need of lifelong treatment, it is important to discuss the development of structured education and training programmes in which special emphasis is placed on the support of the younger patients. It is suggested that Orem's nursing model of self-care may serve as a conceptual framework for nurses working in this specific area of nursing care. PMID- 7541409 TI - Antigen-presenting T cells induce the development of cytotoxic CD4+ T cells. I. Involvement of the CD80-CD28 adhesion molecules. AB - The development of cytotoxic CD4+ T lymphocytes that can kill target cells in a MHC class II-restricted manner was evaluated by comparing different APCs. B lymphoblasts (B-LCL) pulsed with the superantigen staphylococcus enterotoxin B or allogeneic B-lymphoblasts induce CD4+ T cells without cytotoxic activity. In contrast, superantigen-pulsed, MHC class II+ T cell blasts or allogeneic T cell blasts preferentially induce the development of specific, MHC class II-restricted CD4+ cytotoxic effector cells. CD4+ T cell clones generated with T or B cell blasts as APCs (T- or B-APCs) differ in their cytolytic potential, but secrete a similar cytokine pattern. Our data implicate that activated T-APCs preferentially induce a cytotoxic, CD8+ and CD4+ T cell response. Because the density of CD80 expression is lower on activated T-APCs than on B-APCs, we studied the involvement of CD28 and CD80 adhesion molecules in the generation of CD4+ CTLs. Partial blockade of the CD80 molecule with a CTLA4-Ig fusion protein and with specific anti-CD80 mAbs on B-APCs enhanced the generation of CD4+ CTLs. Specific anti-CD86 mAbs, on the contrary, had no effect on the generation of CD4+ CTLs. In contrast, stimulation of CD28, the CD80 counter-receptor, with a cross-linked B7 Ig fusion protein or with an anti-CD28 mAb, inhibited the generation of CD4+ CTLs. Thus, a reduced interaction between CD80 and CD28 may be relevant for the induction of CD4+ CTLs. This shows a new and not yet described function of these adhesion molecules. This induction of a cytotoxic immune response by T cells as APCs may be relevant for the anticlonotypic regulation of T cells and for the depletion of CD4+ T cells in HIV infection. PMID- 7541410 TI - Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. AB - The effect of progesterone (P) on the cytokine production profile of Ag-specific human CD4+ T cell lines and clones was investigated. T cell lines specific for purified protein derivative or streptokinase (SK) derived in the presence of P exhibited significant increased ability to produce IL-5 in comparison with T cell lines derived in the absence of P. Moreover, IL-4 was significantly increased in SK-specific T cell lines derived in the presence of P in comparison with SK specific T cell lines derived in the absence of this hormone. In addition, SK specific T cell lines generated in the presence of P developed into T cell clones showing a Th0-, instead of Th1-like, cytokine profile. Furthermore, SK-specific T cell clones with an established Th1 profile of cytokine secretion did express mRNA for, and produced detectable amounts of, IL-4 when stimulated with P in combination with insoluble anti-CD3 mAb. Combined stimulation with P and insoluble anti-CD3 mAb also enabled Th1 clones to express CD30 on their surface membrane. These results indicate that P can favor the development of Th cells producing Th2-type cytokines and is an inducer of both transient IL-4 production and CD30 expression in established Th1 cells. Thus, P production at the placental level may be responsible, at least in part, for increased production of Th2-type cytokines which have been implied in fetal allograft survival and maintenance of successful pregnancy. PMID- 7541411 TI - B cell superstimulatory influenza virus activates peritoneal B cells. AB - We evaluated the potential of B cell "superstimulatory" influenza viruses to activate peritoneal B cells (PBC) from BALB/c mice containing both CD5+ and CD5- "sister" cells. Like conventional B cells, PBCs responded to influenza viruses in a hemagglutinin glycoprotein (HA) subtype-specific manner with proliferation and vigorous Ig synthesis. However, a number of HA subtypes that are highly stimulatory for conventional B cells failed to induce significant responses of PBC. Isotype-determination revealed a high predominance of IgM and only very low production of IgA and IgG. HA-activated CD5+ B cells showed a hyperexpression of various activation markers, including MHC class II, intercellular adhesion molecule 1 (CD54), and B7-1 molecules. In contrast to conventional B cells, where activation by HA is antagonized by phorbol esters (PMA), HA and PMA acted synergistically on PBC, suggesting differential activation requirements of B-2 cells vs PBC in response to HA. Like HA stimulation of B-2 cells, virus-triggered proliferation of PBC was abrogated by a simultaneous treatment with F(ab')2 fragments of anti-Ig Ab and exhibited synergistic effects with LPS stimulation. HA-mediated proliferative responses of PBC, but not of B-2 cells, were positively controlled by various cytokines, including IL-4 and IL-10, and to a lesser extent by IL-6. In conclusion, our data present the first example of a stimulation of peritoneal B cells by a polyclonal-activating virus, findings that call for considering infections with polyclonal B cell-stimulatory viruses as a means of expanding the pool of potentially autoreactive CD5+ B cells. PMID- 7541413 TI - Involvement of nitric oxide in bone marrow-derived natural suppressor activity. Its dependence on IFN-gamma. AB - Bone marrow (BM)-derived natural suppressor (NS) cells are strong inhibitors of lymphoproliferative responses. In this study we have assessed the involvement of nitric oxide (NO) in BM-derived NS activity, as detected in cocultures of BM and spleen cells stimulated with B cell (LPS) or T cell (Con A) mitogens. The results indicate that NS activity is readily inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or N-acetylcysteine, a free radical scavenging thiol compound. High amounts of nitrite, a stable end product of NO, are detected only in supernatants of Con A- or LPS-stimulated spleen cells cocultured with BM cells enriched in NS activity (Fr3 cells). These amounts (15 to 55 microM) are strongly antiproliferative for both Con A and LPS responses, as was established with a nitrite curve made with a NO donor (sodium nitroprusside). Fr3 cells cultured alone release large quantities of NO and express inducible NO synthase (iNOS) mRNA upon LPS stimulation, but require spleen cells in cultures stimulated with Con A. Anti-IFN-gamma-neutralizing Abs blocked both NO production and NS activity, irrespective of the mitogen used; yet, only exogenous IFN-gamma is unable to promote successful NO production by Fr3 cells, but does induce detectable iNOS mRNA expression in these cells. Taken together the results indicate that: 1) NO is the major mediator of BM-derived NS activity; 2) BM cells enriched in NS activity produce large amounts of NO through an IFN-gamma dependent iNOS induction. PMID- 7541412 TI - Expression of CD45RB and CD27 identifies subsets of CD4+ memory T cells with different capacities to induce B cell differentiation. AB - The capacity of four subsets of CD4+ memory T cells, defined by expression of CD45RB and CD27, to provide help for B cells was examined. Larger amounts of Ig were induced by CD45RBdimCD27- cells compared with the CD45RBdimCD27+ population, whereas CD45RBbrightCD27+ or CD27- cells were poor inducers of Ig synthesis. Mitomycin C treatment, which prevents suppressive activity, markedly enhanced Ig production supported by each subset except for CD45RBbrightCD27- cells. Mitomycin C-treated CD45RBdim cells remained the most efficient inducers of Ig production, but no difference was detected between CD27+ and CD27- cells. The subsets also differed in their ability to proliferate and secrete cytokines, but these differences did not explain variations in the capacity to provide help for B cells. Both CD27- subsets exhibited decreased proliferation and uniquely secreted IL-4, with the CD45RBdimCD27- subset producing the greatest quantities of IL-4. No differences in IL-2 and IFN-gamma production were found. IL-10 secretion increased with the acquisition of the CD45RBdim phenotype and, within the CD45RBdim cells, with the loss of CD27. Staining for cytoplasmic cytokines indicated that individual populations of CD27-CD4+ helper T cells produced either IL-4 or IFN-gamma, whereas more than half of the IL-4 producers also synthesized IL-2. Finally, the different abilities of CD4+ memory T cell subsets to support B cell differentiation did not relate to variations in the expression of CD40 ligand. These results indicate that within the CD4+ memory T cell population an increase of helper activity associates with the shift from a CD45RBbright to a CD45RBdim phenotype. Within the CD45RBdim subset, the loss of CD27 is associated with a reduction of suppressive activity. PMID- 7541414 TI - Flow cytometric analysis of T-independent antigen binding to dinitrophenyl specific cells. AB - Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation. PMID- 7541415 TI - Immunogenicity of T epitope-containing cyclic peptides. Increasing neutralizing antibody responses by introducing fine chemical changes. AB - We showed previously that the disulfide-containing T peptide 24-41 C from a highly structured snake toxin elicits, in a free state, Abs that neutralize the toxin, and only a turn structure commonly exists in 24-41 C and the corresponding toxin region. To tentatively increase the neutralizing capacity of antipeptide Abs, we 1) replaced Gly-40 by an aminoisobutyric moiety (24-41 Aib), 2) substituted the half cystines 24 and 41 by penicillamine moieties (24-41 Pen), and 3) introduced an amide bond between the epsilon NH2 of Lys-27 and the gamma COOH of Glu-38 (24-41 K-E). A solution ELISA made with antitoxin Abs revealed that 24-41 Pen is more antigenic than 24-41 Aib and 24-41 C, which are more antigenic than 24-41 K-E, suggesting that the conformation of 24-41 Pen is most closely related to the corresponding region in the native toxin. The peptides 24 41 Pen, 24-41 Aib, and 24-41 C stimulate T cells from BALB/c mice, whereas 24-41 K-E has lost this property and thereby fails to elicit Abs. Finally, anti-24-41 Pen Abs are more potent at neutralizing the native toxin than anti-24-41 C Abs, which are more potent than anti-24-41 Aib Abs. The efficacy of anti-24-41 Pen Abs was similar to that of a toxin specific mAb. Therefore, introduction of appropriate constraints makes it possible to improve the neutralizing Ab response raised by a synthetic peptide. Such observations should be of interest for the design of efficient synthetic vaccines. PMID- 7541416 TI - Subverting lymph node trafficking by treatment with the Mel-14 monoclonal antibody to L-selectin does not prevent an effective host response to Sendai virus. AB - A single 250-micrograms dose of the Mel-14 mAb to L-selectin greatly diminished the extent of L-selectin expression on lymphocytes and decreased (60 to 90%) the massive cellular recruitment to the cervical and mediastinal lymph nodes that follows intranasal infection of naive C57BL/6 mice with Sendai virus. The numbers of CD8+ CTL precursors in the mediastinal lymph nodes were considerably reduced on day 7, when compared with virus-infected mice given a control rat IgG2a, but potent CTL effectors were present in the lungs of both groups by day 10 after infection, and the overall magnitude of CTL precursor generation was not obviously compromised. The early dominance of Sendai virus-specific IgM Ab forming cells was prolonged in the Mel-14-treated mice, whereas plasma cells producing virus-specific IgA were abnormally prominent in the lymph nodes but not in the spleen. The kinetics of virus-specific Ab-forming cells generation and the serum Ab response for the various IgG isotypes were also delayed. Thus, though L selectin is clearly important for the localization of naive lymphocytes to regional lymph nodes, the Mel-14-treated mouse can still deal effectively with a virus that causes productive infection only in the respiratory tract. The spleen, where L-selectin does not determine lymphocyte trafficking, is a major site for the compensatory T cell and B cell responses. PMID- 7541417 TI - Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. Stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin. AB - Acute inflammation is characterized by increased production of acute phase proteins in the liver. The induction of the hepatocytic response is primarily mediated through soluble cytokines such as IL-1, IL-6, TNF-alpha, and transforming growth factor beta, which bind to specific cell surface receptors and regulate gene expression of acute-phase proteins. Hepatoma cell lines, such as HepG2, represent a model system for studying acute-phase protein synthesis. HepG2 is induced to produce a variety of acute-phase proteins, including alpha 1 antitrypsin, alpha 1-antichymotrypsin, fibrinogen, alpha 1-acid glycoprotein, and haptoglobin, upon stimulation with cytokines. Analysis of HepG2 by reverse transcriptase PCR indicated that this cell line synthesized mRNA specific for the human C5a receptor (CD88). Flow cytometric analysis of HepG2 cells indicated that these cells bound anti-CD88 Ab, thus confirming our RT-PCR data by demonstrating that these cells also express the C5a receptor. Because C5a has been shown to be a potent mediator of inflammation and HepG2 cells express CD88, we assessed the possibility that C5a was capable of stimulating acute-phase protein synthesis by HepG2 cells. The results indicate that binding of human C5a to CD88 on HepG2 cells resulted in an increased production of alpha 1-antitrypsin- and alpha 1 antichymotrypsin-specific mRNA as assayed by RT-PCR. Analysis of culture supernatants derived from C5a-stimulated HepG2 cells showed an increased production of alpha 1-antitrypsin as measured by solid-phase ELISA. alpha 1 antitrypsin production by HepG2 cells was a direct result of C5a stimulation as evidenced by the fact that anti-C5a receptor Ab inhibited the response. These results suggest that C5a may be an important mediator of APP production in the regulation of the inflammatory response. PMID- 7541419 TI - Identification of complement regulatory domains in human factor H. AB - Factor H, a regulator of complement activation, contains 20 short consensus repeat (SCR) domains common among the family of C3b/C4b-binding proteins. Chinese hamster ovary cells transfected with cDNA corresponding to the N-terminal tryptic fragment of factor H (containing SCR 1-5 and part of SCR 6) secreted protein with cofactor activity for factor I-dependent cleavage of C3b. A series of deletion mutants, each lacking one of the first five SCR, were constructed, and the supernatants of transfected Chinese hamster ovary cells were tested for cofactor activity. Supernatants of Chinese hamster ovary cells transfected with SCR 1, SCR 4, and SCR 5 deletion mutants retained cofactor function, although the SCR 1 deletion had reduced cofactor activity. Deletion of SCR 2 or 3 totally abolished cofactor activity. Expression and functional analysis of SCR units 1-3, 2-3, and 2-4 demonstrated that the SCR 1-3 unit is sufficient for cofactor activity, but SCR 1-4 is required for full activity. For assays involving cell protection, a construct linking SCR 1-5 to the glycosyl-phosphatidylinositol anchor of decay accelerating factor was prepared, and stable transfectants were obtained. These cells were protected against complement-mediated cytotoxicity, similarly to decay accelerating factor- and membrane cofactor protein-transfected cells. These studies define the complement regulatory domains in factor H and suggest that the general complement functional unit for C3 convertase regulation involves three or four consecutive SCR units. PMID- 7541418 TI - Lipopolysaccharide binding protein and CD14 modulate the synthesis of platelet activating factor by human monocytes and mesangial and endothelial cells stimulated with lipopolysaccharide. AB - The biosynthesis of platelet-activating factor (PAF) during Gram-negative involves the interaction of LPS with the cells of the host. We have investigated the molecular mechanism that controls cell recognition and PAF biosynthetic response to LPS in human monocytes (MO), glomerular mesangial cells (MC), and HUVEC in culture. The synthesis of PAF by MO and MC involves two proteins, plasma LPS binding protein (LBP) and cell membrane CD14 (mCD14). As MO, MC were shown to express the mCD14 molecule by several mAbs. MO and mCD14-positive MC were stimulated to synthesize PAF either by the 63D3 and IOM-2 mAbs or by the natural ligand LBP-LPS complex. Moreover, LeuM3, 28C5, and 18E12 mAbs that were themselves unable to stimulate the synthesis of PAF blocked PAF synthesis initiated by LBP-LPS complex. LBP was required for synthesis of PAF by MO. In MC, which synthesize PAF also after stimulation by LPS alone, the LBP was shown to speed and significantly enhance the synthesis of PAF. The soluble form of CD14 (sCD14), when added to MO stimulated with LBP-LPS complexes, inhibited the synthesis of PAF possibly by competing with mCD14. In contrast, sCD14 was shown to be required for LPS-induced synthesis of PAF by HUVEC, which did not express mCD14. Therefore, membrane receptors (mCD14) and plasma soluble proteins (LBP and sCD14) may enable different human cell types to synthesize PAF after LPS stimulation. PMID- 7541420 TI - A novel canine leukointegrin, alpha d beta 2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood. AB - The beta 2 or leukointegrin family is comprised of three structurally related leukocyte surface heterodimers: LFA-1 (CD11a/CD18), Mac-1/Mo-1 (CD11b/CD18), and p150,95 (CD11c/CD18). In this work, we describe a novel canine beta 2 (CD18) associated leukointegrin, designated alpha d. Expression of alpha d in tissues was prominent in macrophages in splenic red pulp, lymph node medullary regions, and bone marrow. In peripheral blood, alpha d expression was limited to a minor subpopulation of CD8+ T cells, which included small lymphocytes and large granular lymphocytes. A minor subpopulation of either CD8+ or CD4-CD8- splenic red pulp lymphocytes also expressed alpha d. Immunoprecipitation of alpha d from canine splenocytes revealed a heterodimer of 155 kDa and 95 kDa. Prior clearance of splenocyte extracts with an anti-CD18 mAb resulted in complete removal of alpha d. In addition, prior clearance of canine splenocyte extracts with anti CD11a, anti-CD11b, or anti-CD11c mAb failed to clear alpha d. These immunoclearance data indicated that canine alpha d was antigenically distinct from the three known CD11 molecules, and occurred as an alpha d beta 2 heterodimer. Amino acid sequencing of canine alpha d affinity isolated from spleen further suggested that canine alpha d beta 2 probably represented a fourth member of the canine leukointegrin family via its homology to a subsequently discovered, novel human leukointegrin, alpha d beta 2, which further supported the uniqueness of the canine protein. The discovery of canine alpha d, and the demonstration of its highly restricted cell and tissue distribution, support a re evaluation of leukointegrin-dependent inflammatory and immunologic interactions that involve cells now known to express alpha d. PMID- 7541421 TI - Lung macrophage uptake of unopsonized environmental particulates. Role of scavenger-type receptors. AB - The receptors responsible for avid alveolar macrophage (AM) phagocytosis of unopsonized environmental particulates have not been well defined. This study used flow cytometry to quantitate the effects of a panel of soluble ligands for macrophage adhesion receptors on AM binding of unopsonized environmental dusts (titanium dioxide, TiO2; iron oxide, Fe2O3; alpha-quartz, SiO2; diesel engine exhaust dust) or fluorescent latex beads. Polyanionic ligands of the macrophage scavenger receptor (SR) for acetylated-LDL caused marked inhibition of AM binding of the oxide particles and latex beads (e.g., TiO2 binding; polyinosinic acid (polyl), 10 micrograms/ml: 70.2 +/- 1.5% inhibition, mean +/- SE, n = 11). In contrast, no inhibition was seen with the polyanions heparin and chondroitin sulfate (chond-S), or dextran, consistent with the known inhibitor profile of macrophage SRs for acetylated-LDL AM uptake of latex or SiO2 beads instilled into lungs of hamsters was inhibited by administration of polyl but not chondroitin sulfate (AM beads per cell: control, 6.1 +/- 0.7; polyl, 3.5 +/- 0.2; chond-S, 5.1 +/- 0.7, n > or = 4, p < 0.05 for control vs polyl) indicating macrophages SRs operate in vivo as well as in vitro. In contrast, AM binding of the carbonaceous diesel dust particles was not inhibited by any ligand tested. AM uptake of unopsonized TiO2, SR ligands or acetylated LDL caused no significant activation of AM respiratory burst or TNF production, consistent with past observations that opsonin-independent phagocytosis of inert particles by normal AMs is not accompanied by pro-inflammatory activation. These data implicate macrophage-type SRs in AM binding of charged environmental particles and indicate that distinct mechanisms mediate binding of carbonaceous dusts. PMID- 7541422 TI - Effects of complement activation on the expression of CD59 by human mesangial cells. AB - Human mesangial cells (HMC) were analyzed for their expression of 20-kDa homologous restriction factor (CD59), a glycophospholipid-anchored regulatory protein of the complement cascade. Flow cytometric analysis showed that CD59 was expressed on the HMC membrane and, following the activation of the terminal pathway complement components, CD59 expression on the HMC membrane increased. Northern blot analysis showed that CD59 mRNA levels increased by complement activation. Of interest, CD59 mRNA levels increased by soluble complement activation product, zymosan-activated C8-depleted serum, but not zymosan activated C5-depleted serum. This effect of soluble complement activation product was due to the presence of the action of C5a. Thus, recombinant C5a increased CD59 mRNA levels. The capacity of CD59 to reinsert into rat E and inhibit C mediated lysis was inhibited by mAb against CD59 (1F5). Metabolic labeling using [35S]cysteine showed that the molecular mass of CD59 in HMC was 20 kDa. In conclusion, CD59 is present on HMC and the expression of CD59 is controlled by at least two steps of complement activation pathway, C5 and C8. PMID- 7541424 TI - Chimeric horse/human recombinant C9 proteins identify the amino acid sequence in horse C9 responsible for restriction of hemolysis. AB - Equine C9, in contrast to human C9, has extremely low hemolytic activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting with its hinge region. In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and horse C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could be achieved by insertion of the human C9 hinge region into horse C9. PMID- 7541423 TI - Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids. AB - The chemokine RANTES is a potent chemoattractant for eosinophils, lymphocytes, and monocytes, and has been detected recently in the epithelium of human airways mucosa. We have studied, therefore, the expression of RANTES mRNA and protein in the human bronchial epithelial cell line BEAS-2B. Using Northern blot analysis, RANTES mRNA was not detectable in unstimulated BEAS-2B cells. Incubation of cells with TNF-alpha or IFN-gamma induced the expression of RANTES mRNA and protein within 16 h. The combination of TNF-alpha and IFN-gamma displayed a marked synergism in inducing RANTES expression. Pretreatment of cells with the glucocorticoid budesonide (10(-10)-10(-7) M) for 24 h inhibited expression of RANTES mRNA and protein stimulated by either TNF-alpha or TNF-alpha plus IFN gamma in a concentration- and time-dependent manner. Nonglucocorticoid steroids did not inhibit RANTES mRNA expression. Production of RANTES by epithelium could contribute to the mechanism of selective cellular recruitment occurring in the airways during inflammation, thus playing a relevant role in the pathogenesis of diseases such as asthma, rhinitis, and polyposis. The down-regulation of RANTES production by glucocorticoids in epithelial cells may contribute to the efficacy of these compounds in reducing cellular infiltration and, ultimately, to their anti-inflammatory properties. PMID- 7541425 TI - Inhibitors of protein tyrosine kinase suppress TNF-stimulated induction of endothelial cell adhesion molecules. AB - We studied the role of tyrosine phosphorylation in the induction of vascular cell adhesion molecule 1 (VCAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), and intercellular adhesion molecule 1 (ICAM-1) in HUVEC. Induction of VCAM-1 and ELAM-1 surface expression by TNF was dose-dependently reduced by pretreatment with the protein tyrosine kinase inhibitors herbimycin A (HMA, IC50 300 nM) or genistein (IC50 30 microM). Only genistein attenuated ICAM-1 induction. Genistein or HMA did not affect adhesion molecule up-regulation by PMA. U937 monocyte adhesion to TNF-stimulated HUVEC was markedly inhibited by a combination of anti VCAM-1 and anti-ELAM-1 mAb, as well as by HMA or genistein, probably due to suppression of VCAM-1 and ELAM-1 up-regulation. HMA appeared to prevent VCAM-1 transcription, since it reduced induction of VCAM-1 mRNA by TNF. Gelshift analysis demonstrated inhibition of TNF-induced nuclear factor-kappa B (NF-kappa B) mobilization by HMA. TNF rapidly enhanced tyrosine phosphorylation of a protein migrating with an apparent molecular mass of 35 kDa. HMA and genistein suppressed constitutive tyrosine phosphorylation of all detectable proteins and prevented TNF-induced tyrosine phosphorylation of the 35 kDa protein with an IC50 and dose range, similar to inhibition of VCAM-1 and ELAM-1 induction. Our data suggest that specific phosphorylation following protein tyrosine kinase activation may be required for NF-kappa B mobilization and induction of VCAM-1 and ELAM-1 by TNF. PMID- 7541426 TI - Costimulatory requirements of naive CD4+ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response. AB - Efficient initiation of a CD4 T cell response requires both activation through the TCR and costimulation provided by molecules on APC with counterreceptors on the T cell. We investigated the relative contribution of the ICAM-1:LFA-1 and B7:CD28/CTLA-4 costimulatory pathways in naive T cell activation, using either anti-CD28 Ab or fibroblast cell lines transfected with I-Ek, which express either no costimulatory molecules, ICAM-1 alone, B7-1 alone, or ICAM-1 and B7-1 together. Peptide Ag or immobilized anti-CD3 was used to provide the TCR signal. CD4 T cells from mice transgenic for the V beta 3/V alpha 11 TCR, which recognize a peptide of pigeon cytochrome c complexed to I-Ek, were used as a source of naive T cells. Naive T cells stimulated with Ag or anti-CD3 responded well to high numbers of APC expressing either ICAM-1 alone or B7-1 alone. However, APC expressing both ICAM-1 and B7-1 were much better stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers, indicating a synergy between the two pathways. Stimulation provided by ICAM-1 could not be solely attributed to adhesive strengthening of other pathways, since costimulation was seen when immobilized anti-CD3 was used and when ICAM-1 only APC were added, indicating that ICAM-1 was in fact acting as a classic costimulatory molecule. Both the magnitude of the response and the amount of costimulation required for response were dependent on the intensity of TCR interaction. These results suggest that an efficient naive T cell response requires both a strong TCR signal and more than one costimulatory signal that will synergize with the TCR signal. This offers an explanation as to why APC such as dendritic cells and activated B cells, which express high levels of multiple costimulatory/adhesion molecules, are the only APC that elicit naive T cell responses. PMID- 7541427 TI - CD14 and innate recognition of bacteria. PMID- 7541428 TI - Monoclonal antibodies to human heart fatty acid-binding protein. AB - Heart-type fatty acid-binding protein (H-FABP), a 15 kDa non-enzymatic protein, which is abundantly present in heart and some skeletal muscles, was recently found to be a useful plasma marker for acute myocardial infarction. The BIAcore biosensor technology was used to raise and characterize a panel of 13 monoclonal antibodies against human H-FABP which did not crossreact with other FABP types of human origin. The kinetics of association and dissociation between FABP and the monoclonal antibodies was studied. By pairwise mapping several distinct epitopes could be identified. PMID- 7541429 TI - In vivo and in vitro interactions between idiotypic and antiidiotypic monoclonal antibodies against placental alkaline phosphatase. AB - A monoclonal antiidiotypic antibody alpha H7, was generated against a monoclonal antibody H7 with specificity towards placental alkaline phosphatase. The in vitro and in vivo effects of alpha H7 were investigated. The antiidiotypic antibody was found to generate stable complexes with the radiolabeled idiotypic antibody, visualized both in vivo and in vitro, as revealed by PAGE and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate, dissociation rate, and affinity constants between the reactants were determined (KA H7/PLAP 6.7 x 10(9) M-1, KA H7/alpha H7 3.2 x 10(9) M-1). By in vivo injection of the antiidiotype, a rapid dose dependent clearance of circulating radiolabeled idiotypes was demonstrated and a decrease in total body radioactivity was recorded with a concomitant dramatic increase in non-protein-bound 125I excreted in the urine. It is concluded that idiotypic-antiidiotypic interactions offer advantages in the regulation of antibody levels in vivo. PMID- 7541430 TI - Epitope mapping and binding analysis of insulin-specific monoclonal antibodies using a biosensor approach. AB - Molecular interactions between the human insulin molecule and a panel of seven monoclonal antibodies (mAbs) against insulin were investigated by surface plasmon resonance. Using real-time analysis (BIAcore technology), the antigenic sites recognized on the human insulin molecule by these mAbs were mapped. Three overlapping regions could be defined on the insulin molecule coupled to the dextran matrix on the basis of its recognition by three mAbs groups: group I (mAbs 19, 10), group II (mAbs 13, 5, 25, 15) and group III (mAb P10). The antigenic determinants of the molecule in solution were mapped using a two-site assay in which insulin was presented to each mAb, bound to a rabbit anti-mouse IgG Fc gamma 1 coupled to the dextran. Four antigenic regions were defined: regions I and III were defined by the same antibodies as above whereas region II was recognized differently by mAbs 13, 5 (region IIa) and mAbs 25,15 (region IIb). The Ka value of the mAbs determined using the BIAcore were in the same range (from 3 x 10(7) M-1 to 6.6 x 10(9) M-1) whether insulin was coupled to the dextran matrix or in solution. Furthermore, mAbs 19 and P10, which recognize two distinct regions on the insulin molecule, were selected to develop a highly sensitive immunoassay for measurement of insulin in serum. PMID- 7541432 TI - An automated ELISA system using a pipette tip as a solid phase and a pH-sensitive field effect transistor as a detector. AB - A fully automated ELISA system was constructed using a pipette tip as a solid phase, urease as a detecting enzyme, and a pH-FET as a detector of urease activity. The inner wall of the end part of a pipette tip was used as a solid phase, and the urease activity of the conjugate, captured after a two-step immunoreaction, was measured by coupling the pipette tip with the pH-FET in a pH measuring cell. Full automation of the ELISA system was achieved by using a disposable reagent cartridge and three pipetters for all mechanical operations, including sample dilution and B/F separation. This system can treat 60 samples per hour with an assay time of 21 min for all assay configurations. The system was applied to two-step sandwich assays for AFP, CEA, HBsAg, and HBsAb, a two step competition assay for HBcAb, and a second antibody assay for HTLV-I Ab. PMID- 7541433 TI - Comparative study on the levels of alpha-fetoprotein (AFP) in different pathological cases. AB - The research represents a trial to demonstrate the changes which probably took place in AFP levels as a tumor marker among pregnant egyptian women suffering from bilharzial and cancerous infestations. It was found that combination of schistosomal infection with pregnancy leads to obvious high significant increase in AFP in late pregnancy (6-8 months) compared to unpregnant patients. Although the combination between bilharzial infestation and early pregnancy leads to increase in AFP level, the differences were non significant compared to unpregnant ones. There is an extremely significant increase in AFP levels among urinary and intestinal schistosomal pregnant patients compared with healthy pregnant women which was higher in intestinal infection and more pronounced in early pregnancy than late ones. Malignancy in combination with pregnancy caused markable greatly significant increase compared to unpregnant patients, and the increases were slightly higher in bladder than colonic cancerous cases and their AFP levels were gradually increased with progress of both pregnancy and malignancy. In combination of malignancy with pregnant schistosomal infested women to study the effect of schistosomal infection the detected increase in AFP was statistically non significant at early pregnancy and malignancy, turned to significancy at late ones either in bladder or colonic cases. PMID- 7541431 TI - Enzymatically mediated, glycosidic conjugation of immunoglobulins with viral epitopes. AB - We developed a novel enzymatic procedure to couple a peptide to the sugar moieties of immunoglobulins (Igs). The synthesis of the conjugates consists in galactose (Gal) oxidation of desialylated Igs followed by covalent attachment of the peptides with concurrent stabilization of the Schiff bases upon mild reduction. The peptide used in this study, corresponds to the amino acid residues 110-120 of hemagglutinin (HA) of PR8 A virus and is recognized by CD4 T helper cells in association with I-Ed class II major histocompatibility complex (MHC). The degree of coupling as determined by competitive inhibition of radioimmunoassay (IRIA) using FPLC purified conjugates was estimated at 11.4 peptides per IgG molecule. Coupling of HA110-120 peptide to the sugar moiety of various mouse and human Igs was confirmed by Western blot analysis developed with anti-HA110-120 antibodies. Complete detachment of the peptide from the conjugates by N-deglycosylation with PGNase F indicated a defined specificity of coupling HA peptide to the N-linked oligosaccharides of Igs. To facilitate quick release of the peptides from the conjugates into the lysosomal compartment of the antigen processing cells (APC) we introduced at the alpha amino terminus of the peptide (HAc110-120), a cleavage site for cathepsins (AAAL). The immunoglobulin-galactose HAc110-120 conjugates (IGP) were able to activate HA110-120 specific T hybridoma cells as efficient as influenza PR8 A virus and 40-100-fold higher than the synthetic peptide itself. PMID- 7541434 TI - Morphine and the "lytic cocktail" for terminally ill patients in a French general hospital: evidence for an inverse relationship. AB - Undertreatment of cancer pain with analgesic drugs is still a frequent problem in French hospitals. In the absence of good analgesic practices, the use of a so called lytic cocktail, which combines a neuroleptic (chlorpromazine), an opioid (meperidine), and an antihistamine (promethazine) has become common during the terminal phase of the disease. The lytic cocktail (LC) has been subsequently denounced as a type of disguised euthanasia. The aim of our study was to examine the prescription of morphine and lytic cocktail for terminally ill patients in a 427-bed French general hospital during a 3-year period (1989-1991) that coincided with the beginning of a pain relief service. The study was performed in two steps: a chart review of the 841 deceased patients during the observation period and an examination of morphine and parenteral promethazine consumption from the hospital pharmacy. Data from both the charts and the pharmacy showed an inverse relationship between these treatments. Morphine consumption increased while LC consumption decreased. The number of deceased patients who received LC were 24.4% in 1989, 19.9% in 1990, and 6.6% in 1991 (P < 0.001 between 1990 and 1991). The number of deceased who received morphine were 13.6% in 1989, 20.6% in 1990, and 23.9% in 1991 (P < 0.01 between 1989 and 1990). During the same period, the annual hospital morphine consumption increased by 191%, and the annual hospital parenteral promethazine consumption decreased by 62.5%. Our results suggest that, when pain is more correctly treated, the use of an inappropriate method of symptom control decreases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541436 TI - Update on the cancer pain role model education program. AB - Application of traditional educational methods has done little to improve cancer pain management in the United States. This report details the results of the first year of the expanded Wisconsin Cancer Pain Initiative Role Model Program, a novel approach to cancer pain education. One hundred and ninety-six physicians and nurse educators together with their clinical partners attended one of three role model conferences in 1992-1993 and developed Action Plans detailing their proposed educational goals. Results indicate that participants demonstrated significant improvement in cancer pain knowledge as a result of the 1-day conference. Within 12 months of the conference, 64% of Role Model teams completely or partially met their Action Plan goals. In total, 227 educational or clinical practice projects were completed. The Cancer Pain Role Model Program represents an excellent educational program for disseminating cancer pain information and instituting positive changes in clinical practice. PMID- 7541435 TI - The morphine-sparing effect of propacetamol in orthopedic postoperative pain. AB - The analgesic efficacy and safety of propacetamol (Pro-Dafalgan), an injectable prodrug of acetaminophen, in combination with morphine administered by patient controlled analgesia (PCA) were studied in 60 patients (56 men, 4 women; age 18 40 years; mean age, 26 years) after knee ligamentoplasty. Using a double-blind, randomized, parallel-group design, the effects of four (every 6 hr) intravenous injections of 2 g propacetamol (= 1 g acetaminophen) were compared with four injections of placebo (PL) in the recovery room immediately after surgery. Efficacy was assessed over 24 hr by automatic recording on the PCA device of the cumulative dose of morphine and the number of boluses requested. It was also assessed on pain scores rated on a five-point verbal scale and a visual analogue scale before administration, at 1, 2, 3, and 4 hr, and then every 2 hr until the 24th hr after administration. A five-point global efficacy scale was also administered. Any side effects were recorded throughout the duration of the study, and the ability to tolerate the drug was assessed by recording arterial pressure, cardiac and respiratory frequency, and sedation at the same assessment times as the pain scores. The 24-hr morphine consumption was significantly decreased in the propacetamol group (number of 1 mg boluses: 14.7 +/- 11.3 versus 23.2 +/- 13.8, P = 0.01; PCA usage: 26.4 +/- 12.3 mg versus 34.6 +/- 15.4 mg, P = 0.03; PCA usage + titration: 34.5 +/- 12.7 mg versus 43.1 +/- 15.9 mg, P = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541437 TI - Effective treatment of severe cancer pain of the head using low-dose ketamine in an opioid-tolerant patient. AB - We report the case of a 39-year-old man with severe pain due to unresectable squamous-cell carcinoma of the maxillary sinus that had invaded cranial bone and had metastasized to the cervical spine. Tolerance to opioids had developed, and high doses of transdermal, continuous intravenous, and epidural opioids did not control his pain. An acute episode of extremely severe head pain was immediately controlled with a subanesthetic dose of ketamine after failure of a stress dose of corticosteroid and intravenous lidocaine. Because the patient was terminally ill and invasive procedures were not options, we controlled his pain using a low dose ketamine infusion until his death 13 days later. Ketamine may be a good co analgesic for breakthrough pain and for severe pain caused by terminal cancer when invasive techniques are inappropriate. Its mechanism of action may include reversal of opioid tolerance in addition to an inherent analgesic effect. PMID- 7541438 TI - Use of methadone in a highly tolerant patient receiving parenteral hydromorphone. AB - A 59-year-old woman with metastatic breast cancer experienced a poor response to increasing doses of hydromorphone, possibly related to the neuropathic nature of her pain. Ultimately, the cost of this treatment was more than $1000 per day. Administration of methadone, initially as an adjuvant and eventually as the sole analgesic, at a much smaller dose than expected resulted in very satisfactory analgesia, without the development of serious side effects. In addition, the cost of treatment decreased to less than $25 per day. This case illustrates the rationale and advantages for the use of methadone. Intraindividual variability in the response to various opioids is a factor to be considered when selecting analgesics. Methadone is a useful second-line or third-line opioid for the patient who is highly tolerant to other opioids, as it may demonstrate incomplete cross-tolerance with other agonist opioids. Switching of the opioid to methadone may be a worthwhile option to consider in managing patients who are highly tolerant to other opioids. PMID- 7541439 TI - Anileridine-induced delirium. AB - Delirium is a common complication in hospitalized patients and is often associated with significant morbidity. It is important to recognize this syndrome early so that potential causes can be identified and properly managed. Although the etiology of delirium in critically or terminally ill patients is often multifactorial, opioid analgesics are often implicated as a potential underlying cause. Certain opioids, such as meperidine, have been identified as having a greater potential for causing delirium than others. There have been no published reports of anileridine-induced delirium and an illustrative case is presented. PMID- 7541440 TI - Celiac plexus blockade in a 7-year-old child with neuroblastoma. AB - Celiac plexus blockade with ethanol is a widely accepted modality of pain control for adults with cancer pain. The role of interventional strategies in children is less well established. A 7-year-old child with abdominal pain secondary to a Wilms tumor was treated with neurolytic celiac plexus blockade. This resulted in control of abdominal pain for close to three months. This modality is underutilized and should be considered for children with pain due to upper abdominal malignancy. PMID- 7541442 TI - [Functional molecules expressed in hematopoietic stem cells]. PMID- 7541441 TI - [Molecular and genetic approaches to germ cell formation in Drosophila embryogenesis]. PMID- 7541443 TI - [Structure and signal transduction of granulocyte colony-stimulating factor receptor]. PMID- 7541444 TI - Retention of long-term repopulating ability of xenogeneic transplanted purified adult human bone marrow hematopoietic stem cells in sheep. AB - We have previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human adult marrow CD34+ HLA-DR- cells in sheep after transplantation in utero. The persistence in these animals of CD34+ HLA-DR- cells, the phenotype of the original graft, and the presence of assayable high proliferative potential colony-forming cells (HPP CFCs), a primitive progenitor cell that has self-renewal capacity and multilineage differentiative capacity, suggested that these chimeric animals continued to contain human pluripotent hematopoietic stem cells (HSCs). To test this hypothesis, CD45+ cells from the bone marrow of such an animal were isolated by panning at 19 and 24 months after birth, were pooled, and were transplanted into 4 preimmune sheep fetuses (0.6 x 10(6) viable cells per fetus). Three of the sheep were born alive. Bone marrow from one of these secondary recipients (lamb 58) exhibited significant donor (human) cell presence at 3, 9, 12, and 15 months (last evaluation period) after transplant. The engraftment was sustained along with multilineage expression involving lymphoid, erythroid, and myeloid cells of human origin. These results indicated that human CD34+ HLA-DR- cells are capable of long-term marrow repopulation and that this cell population is enriched for human long-term repopulating HSCs. PMID- 7541445 TI - Genetic influence in rheumatoid arthritis. AB - Development of rheumatoid arthritis is influenced by genetic and environmental factors. Over the years intense interest has been focused on analysis of genetic factors contributing to pathogenesis and it appears that the major histocompatibility complex class II genes make a major contribution. Utilizing epidemiological data as well as molecular techniques, an epitope located on a number of HLA-DR molecules (class II) seems to be associated with an increased susceptibility to development of the condition. Furthermore, the disease may be more severe among individuals homozygous for the disease-associated DR types. The specific immunological mechanisms accounting for these findings are not known but are likely related to the ability of class II molecules to bind autoantigens and stimulate the appropriate T cells which can lead to joint damage. A search for additional genetic factors contributing to disease development should be high on the list of objectives and would help to further elucidate the pathogenesis of this complex disorder. PMID- 7541447 TI - Cortical architectural abnormalities and MIB1 immunoreactivity in gangliogliomas: a study of 60 patients with intracranial tumors. AB - Gangliogliomas are generally low grade neoplasms composed of mixtures of neoplastic glial and neuronal elements whose origin and exact nature are still controversial. We studied a series of 60 intracranial gangliogliomas looking for coexistent cortical architectural abnormalities (cortical dysplasia, microdysgenesis) and to determine if tumor behavior correlates with MIB1 (marker of cellular proliferation) labeling. The patients included 34 males and 26 females who ranged in age from 6 months to 55 years (mean 20 years). Thirty-eight tumors (63%) were located in the temporal lobe and 6 (10%) in the frontal lobe. Fifty-four patients (90%) presented with seizures (most with intractable epilepsy) and the duration of seizures ranged from 1 to 38 years (mean 14 years). In all cases, the predominant glioma component resembled a low grade fibrillary astrocytoma. In 14 tumors (23%), an oligodendroglial component was present. In one case, the glial component resembled an anaplastic astrocytoma. The tumors were characterized variously by perivascular chronic inflammation (N = 45, 75%), vascular proliferation (N = 36, 60%), granular bodies (N = 54, 90%), binucleated neurons (N = 36, 60%), calcification (N = 28, 47%), and cystic degeneration (N = 26, 43%). Meningeal involvement by tumor was observed in five (8%) cases. In 38 patients, sufficient tissue was resected to evaluate for the presence of concomitant cortical architectural abnormalities. Cortical architectural abnormalities were identified near to but clearly separate from the tumor in 19 (50%) patients. Only four patients including the anaplastic tumor died with tumor progression. MIB1 indices (positive tumor cells/1,000 tumor cells counted) in 54 cases ranged from 0 to 10.2 (mean 1.1 +/- 1.0).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541448 TI - Lenograstim prevents morbidity from intensive induction chemotherapy in the treatment of inflammatory breast cancer. AB - PURPOSE: To compare the efficacy and safety of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) versus its inert vehicle in patients with unilateral nonmetastatic inflammatory breast cancer treated with fluorouracil, epirubicin, and cyclophosphamide high-dose (FEC-HD) neoadjuvant chemotherapy. PATIENTS AND METHODS: One hundred twenty patients have been enrolled by nine French centers in this double-blind, parallel-group, vehicle-controlled study to compare at each cycle subcutaneous lenograstim (5 micrograms/kg/d) with placebo given from day 6 to day 15 after the induction chemotherapy (day 1 to day 4, fluorouracil 750 mg/m2 continuous intravenous [IV] infusion; day 2 to day 4, epirubicin 35 mg/m2 and cyclophosphamide 400 mg/m2 both IV push). Four cycles were planned every 3 weeks before locoregional treatment. Patients with febrile neutropenia remained blinded for the subsequent cycles. RESULTS: Lenograstim significantly reduced the duration of neutropenia at less than 0.5 x 10(9)/L and less than 1 x 10(9)/L to a median duration of 2 and 3 days, respectively, as compared with 5 and 7 days in the placebo group. This translated into a statistically significant reduced incidence of microbiologically documented infections, and a decreased need for rehospitalizations for infectious events and antibiotic use. Clinical objective tumor response rate observed after four cycles was 89.6% and 93%, respectively, in the placebo and treated groups. Mild transient bone and injection-site pain, myelemia, and hyperleukocytosis were the most frequently reported adverse events associated with lenograstim. CONCLUSION: Lenograstim is safe and effective to reduce morbidity associated with FEC-HD neoadjuvant chemotherapy in inflammatory breast cancer. Response rate is not affected. PMID- 7541446 TI - Cytokines and growth factors induce HSP27 phosphorylation in human astrocytes. AB - In previous studies, an enhanced expression with abnormal phosphorylation of a low molecular weight heat shock protein, HSP27, was identified in reactive astrocytes and Rosenthal fibers in Alexander disease brain. To investigate the relevance of HSP27 to the pathological change of astrocytes, phosphorylation of HSP27 was examined in astrocyte cultures isolated from fetal human brains. In unstimulated human astrocytes, HSP27 was identified in the cytoplasm and was comprised of a major unphosphorylated "a" isoform and a minor monophosphorylated "b" isoform. The level of HSP27 phosphorylation was elevated greatly after a 30 minute exposure to heat shock, sodium arsenite, interleukin-1 (IL-1 alpha and IL 1 beta), and tumor necrosis factor-alpha (TNF-alpha) with an increased expression of a diphosphorylated "c" isoform. Treatment with interferon-beta (IFN-beta), platelet-derived growth factor-AA, leukemia inhibitory factor, phorbol 12 myristate 13-acetate, and dibutyryl cyclic AMP stimulated phosphorylation of HSP27 moderately, while IFN-gamma, TNF-beta, basic fibroblast growth factor, epidermal growth factor, or fetal bovine serum did not significantly alter the level of HSP27 phosphorylation. Total amount of the HSP27 protein and its cytoplasmic localization were unaffected by any of these reagents. These results indicate that HSP27 is a constitutive protein in human astrocytes. The induction of HSP27 phosphorylation by a specific set of cytokines and growth factors suggests that HSP27 is a key cellular substrate by which signaling events are mediated in human astrocytes under normal and pathological conditions. PMID- 7541449 TI - High-dose ifosfamide: circumvention of resistance to standard-dose ifosfamide in advanced soft tissue sarcomas. AB - PURPOSE: The study was designed to assess the toxicity and activity of high-dose ifosfamide (HDI) administered by continuous infusion at a dose of 4 g/m2/d over 3 days every 4 weeks in adult patients with advanced soft tissue sarcomas (ASTS) pretreated with doxorubicin and/or a standard-dose ifosfamide (SDI)-containing regimen. PATIENTS AND METHODS: Between January 1991 and November 1993, 40 patients with progressive ASTS were entered onto the study. Twenty-eight patients had been pretreated with a multidrug regimen that contained SDI and were classified as follows: SDI-refractory (n = 21), SDI-resistant (n = 2), and indeterminate SDI-sensitive (n = 5). Patients were treated until progression or major toxicity. RESULTS: One hundred forty-seven cycles of HDI were administered. Neutropenia was dose-limiting, with 100% of patients experiencing grade 3 to 4 toxicity and 12 admissions for febrile neutropenia (30% of patients). Neurotoxicity (17% of patients) was significantly associated with acute renal failure (n = 4) (P < .001), grade 4 thrombocytopenia (P < .01) and febrile neutropenia (P = .048). Chronic renal toxicity (n = 4) was significantly associated with retroperitoneal masses and/or prior nephrectomy (P = .008). Partial responses (PRs) were observed in 12 of 36 assessable patients (33%) and eight patients (22%) experienced disease stabilization. All but one response occurred in patients pretreated with SDI, with five PRs among SDI-refractory patients. Leiomyosarcomas appear resistant to HDI. The median response duration was 8 months (range, 6 to 13+) and the median overall survival time was 12 months. CONCLUSION: The activity of HDI in these pretreated ASTS patients and the apparent circumvention of SDI resistance suggest a real dose-response relationship for ifosfamide and deserve further evaluation. Although toxic, this treatment appears feasible and manageable using routine clinical support. Since prophylaxis of ifosfamide-induced renal damage remains unknown, frequent monitoring of renal and tubular functions during therapy is highly recommended. PMID- 7541450 TI - Peripheral-blood stem-cell collections after paclitaxel, cyclophosphamide, and recombinant human granulocyte colony-stimulating factor in patients with breast and ovarian cancer. AB - PURPOSE: Here we evaluate Taxol (paclitaxel; Bristol-Myers Squibb, Princeton, NJ) and cyclophosphamide (CY) with recombinant human granulocyte colony-stimulating factor (rhG-CSF) for mobilization of peripheral-blood stem cells (PBSCs) for autologous stem-cell transplantation in patients with breast and ovarian cancer. PATIENTS AND METHODS: PBSCs were collected from 17 patients with breast (n = 11), ovarian (n = 5), and gastric (n = 1) cancer after administration of Taxol (170 mg/m2 x 1) and CY (4 g/m2 x 1) followed by rhG-CSF (10 micrograms/kg/d). PBSC collections after Taxol and CY were compared with PBSC collections from nine patients with stage IV breast (n = 8) or stage III ovarian (n = 1) cancer who had received CY (4 g/m2 x 1) followed by rhG-CSF (16 micrograms/kg/d) for mobilization. RESULTS: Mean WBC and platelet counts on the first day of apheresis were 6.3 x 10(9)/L (range, 1.9 to 22.1) and 35 x 10(9)/L (range, 19 to 77), respectively. The median numbers of CD34+ cells, peripheral-blood mononuclear cells (PBMNC), and peripheral-blood total nucleated cells (PBTNC) collected were 13.02 x 10(6)/kg (range, 5.4 to 57.8; mean, 16.02), 6.86 x 10(8)/kg (range, 1.9 to 51.2), and 17.41 x 10(8)/kg (range, 2.4 to 106.6), respectively. In the comparison group, the median yield of CD34+ cells was 6.39 x 10(6)/kg (range, 0.2 to 28; mean, 10.01; P = .01). The mean daily yield of CD34+ cells/kg/collection was 3.5 (range, 0.8 to 28.9) after Taxol and CY, as compared with 1.3 (range, 0.1 to 7.0) for patients who received CY alone (P = .01). All patients who received CY and Taxol reached a target level of 5 x 10(6) CD34+ cells/kg, as compared with five of nine patients (55.5%) who received CY alone (P = .03). CONCLUSION: These data suggest that Taxol and CY followed by rhG-CSF mobilizes PBSCs in patients with advanced breast and ovarian cancer more effectively than this regimen without Taxol. PMID- 7541451 TI - Simple prognostic model to predict survival in patients with undifferentiated carcinoma of unknown primary site. AB - PURPOSE: We performed this study to identify prognostic factors in a subgroup of patients with carcinoma of unknown primary site treated with cisplatin combination chemotherapy. PATIENTS AND METHODS: Seventy-nine patients with poorly differentiated adenocarcinoma or undifferentiated carcinoma of unknown primary site were treated on two consecutive phase II chemotherapy protocols. The first protocol consisted of treatment with 3-week courses of cisplatin, etoposide, and bleomycin (BEP). In the second protocol, cisplatin was administered weekly combined with oral administration of etoposide (DDP/VP). To identify prognostic factors, univariate and multivariate analyses were conducted. RESULTS: In the univariate analysis, performance status, histology, liver or bone metastases, and serum levels of alkaline phosphatase and AST were significant variables to predict survival. In the multivariate analysis, performance status and alkaline phosphatase were the most important prognostic factors. CONCLUSION: Good prognosis patients had a performance score of 0 (World Health Organization [WHO]) and an alkaline phosphatase serum level less than 1.25 times the upper limit of normal (N). These patients had a median survival duration greater than 4 years. Intermediate-prognosis patients were characterized by either a WHO performance status < or = 1 or an alkaline phosphatase level > or = 1.25 N. These patients had a median survival duration of 10 months and a 4-year survival rate of only 15%. The poor-prognosis group had both a WHO performance status > or = 1 and an alkaline phosphatase level > or = 1.25 N. These patients had a median survival duration of only 4 months and none survived beyond 14 months. Treatment strategies for these three groups are discussed. It is suggested that this prognostic model be validated in other patients series. PMID- 7541453 TI - p53 and angiogenesis. PMID- 7541452 TI - Hodgkin's disease and human immunodeficiency virus infection: clinicopathologic and virologic features of 114 patients from the Italian Cooperative Group on AIDS and Tumors. AB - PURPOSE: To describe virologic, clinicopathologic, and therapeutic features of a large series of Italian patients with Hodgkin's disease (HD) and human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS: From November 1986 to March 1994, 114 cases were observed. The relationship between Epstein-Barr virus (EBV) and HD was determined by an in situ hybridization technique, immunostaining for EBV-encoded latent membrane protein-1 (LMP-1) expression, and Southern blotting. Twenty-six patients were included in a prospective study evaluating the combination of chemotherapy (CT) with zidovudine. RESULTS: Combined approach on EBV study revealed that 14 (78%) of 18 patients were EBV associated. An almost equivalent distribution of EBV subtypes was observed in EBV carrying cases, indicating that in the HIV setting, type 2 EBV also may be pathogenetically involved in HD development. In comparing these 114 patients with our single-institutional series of 104 HIV-negative patients with HD, we observed at presentation a younger median age (29 v 38 years); a prevalence of males (90% v 56%); and a higher percentage of stage IV disease (52% v 15%), presence of B symptoms (77% v 35%), and extranodal disease (63% v 29%). The complete remission (CR) rate (58%) and median survival (13 months) of patients treated prospectively were similar to that of patients treated with standard CT regimens. The statistically significant favorable prognostic factors for survival being the following: achievement of CR, CD4+ count greater than 250/microL, and no prior diagnosis of AIDS at onset of HD. CONCLUSION: Our virologic findings indicate that HIV-related HD is more closely associated with EBV than HD in the general population. The peculiar clinicopathologic findings, the role of some prognostic factors, and the possibility of cure of HIV-related HD have been demonstrated. PMID- 7541454 TI - Osteoarthritis in cynomolgus macaques. II. Detection of modulated proteoglycan epitopes in cartilage and synovial fluid. AB - The purpose of the present study was to determine the usefulness of the monoclonal antibodies 7-D-4 and 3-B-3 as biomarkers of severity of naturally occurring osteoarthritis in the knee joints of adult cynomolgus macaques. The antibodies were used to immunolocate chondroitin sulfate proteoglycan epitopes in articular cartilage or synovial fluid from knee joints with a range in severity of osteoarthritis. The joints were examined radiographically, grossly, microradiographically, and histologically to characterize the severity of disease, and the results of three different methods of proteoglycan analysis (immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot analysis) were compared. Subjectively, the degree of positive immunostaining for 7-D-4 was minimal in normal sites and increased as damage to articular cartilage increased. The scores for 7-D-4 immunostaining in the medial tibial plateau (the site most severely involved in this model) were correlated significantly with severity of damage to articular cartilage (p < 0.05, r2 = 0.50), thus supporting the subjective observations. The ratio of 7-D-4 to sulfated glycosaminoglycans in synovial fluid also was correlated with the score for 7-D-4 immunostaining in the medial tibial plateau (p < 0.05, r2 = 0.54) and with the score for 3-B-3 immunostaining in the medial femoral condyle (p < 0.05, r2 = 0.65). There were no significant correlations among scores for 3-B-3 immunostaining, severity scores, and the ratios of 3-B-3 to sulfated glycosaminoglycans in the synovial fluid. By Western blot analysis, both epitopes were sensitive markers of early cartilage damage in young adult monkeys but were less sensitive in older monkeys. This work provides evidence that measurement of the epitope recognized by 7-D-4 in synovial fluid or, by immunohistochemical or Western blot methods, in articular cartilage has potential use as a marker of severity of naturally occurring osteoarthritis. PMID- 7541455 TI - Measurement of c-erbB-2 proteins in sera from patients with carcinomas and in breast tumor tissue cytosols: correlation with serum tumor markers and membrane bound oncoprotein. AB - Using a commercial kit with antibodies against the ectodomain of c-erbB-2 protein, we detected c-erbB-2 immunoreactivity in human serum. We found that the percentages of patients with elevated serum c-erbB-2 immunoreactivities were 35, 21, and 9% in breast, prostate, and ovarian carcinoma, respectively. The majority of the elevated immunoreactivities were associated with sera containing highly elevated tumor markers with the highest in breast carcinoma (35%) and lowest in ovarian cancer (9%). Excellent correlations were also observed between the serum levels of c-erbB-2 immunoreactivity and the dominant tumor markers in serial specimens from individual cancer patients. We could also detect the c-erbB-2 immunoreactivity in the cytosols prepared from the breast tumor tissue for estrogen and progesterone receptor (ER & PgR) measurements using the same commercial kit for serum studies, and the intact c-erbB-2 oncoprotein (p185) in the extracts of the tissue membrane fractions with a different kit designed for tissue extract. The level of c-erbB-2 immunoreactivity in the cytosol from 124 human breast tumor specimens had an excellent correlation with the cell membrane concentrations of p185 (gamma = 0.89). Most of the elevated cytosol c-erbB-2 immunoreactivities were also found to associate with breast tumor specimens containing low concentrations of ER & PgR. It appears that measuring the c-erbB-2 immunoreactivity potentially could be used as a prognostic marker without performing tissue biopsies and also as a serum tumor marker for managing cancer patients. PMID- 7541456 TI - Aggregation of proteins and its prevention by carbohydrate excipients: albumins and gamma-globulin. AB - Moisture-induced (2-10 microL added to 10 mg) aggregation of solid-state albumin and gamma-globulin was investigated by incubation at 37 degrees C for 24 h. The insoluble aggregates were centrifuged from a reconstituted solution, dissolved in a solution containing denaturant and reducing agent, and analysed by a Bio-Rad protein assay kit. Of the three albumins used, maximum aggregation (8.2%) was observed with bovine serum albumin that was essentially fatty-acid free. The maximum aggregation observed with gamma-globulin was 7.0%. A bell-shaped curve for percent aggregation was observed with increasing moisture content and was especially prominent for bovine serum albumin. When mixed with carbohydrate excipients in a 1:1 ratio, aggregation was reduced for both bovine serum albumin and gamma-globulin by all four of the following excipients used: Emdex, dextrose, trehalose and hydroxypropyl beta-cyclodextrin. For bovine serum albumin, the aggregation was reduced about sixfold, with Emdex being the most effective excipient. The likely mechanism of the resulting aggregation was covalent linkages formed due to intermolecular thiol disulphide interchange. PMID- 7541457 TI - Some anti-allergic and anti-inflammatory actions of 2-N-carboxamidinonormianserin (FCC5). AB - The aims of these studies were to examine the effects of FCC5 (2-carboxamidino 1,2,3,4,10,14b-hexahydrodibenzo (c,f) pyrazino (1,2,-a) azepine HCl), an analogue of mianserin, on immediate type hypersensitivity reactions in-vitro. The actions of FCC5 were examined on the Schultz-Dale reaction of guinea-pig ileum and on histamine and leukotriene release from human- and guinea-pig-sensitized lung fragments. FCC5 (applied topically) was assessed for anti-inflammatory activity in-vivo against phorbol-12-myristate-13-acetate (PMA)-induced oedema in the mouse ear. FCC5 (IC50 = 0.17 microM) was a potent inhibitor of the Schultz-Dale reaction in-vitro, as assessed by a concentration-dependent attenuation of egg albumin-induced contractions of sensitized guinea-pig isolated ileum. Using human and guinea-pig isolated sensitized lung fragments, FCC5 (1-100 microM) attenuated antigen-induced release of sulphidopeptidoleukotrienes and histamine. FCC5 (50 micrograms topically) resembled mianserin and indomethacin in attenuating PMA induced mouse ear inflammation. These properties together with previously published evidence of long lasting antihistamine properties in-vivo, suggest that FCC5 has therapeutic potential as an anti-allergic agent, especially in pathological conditions where an inflammatory component is present. PMID- 7541459 TI - Electronmicroscopic diagnosis of human papilloma virus in verruca plantaris. AB - The purpose of this study was to identify the human papilloma virus in verruca plantaris by transmission electron microscopy and study the cellular effect of human papilloma virus at both the transmission electron microscopy and light histochemistry level. The authors discuss the cellular pathology in relation to early studies and keratin disorders. Features identified by transmission electron microscopy are consistent with characteristics of human papilloma virus-1. PMID- 7541458 TI - [Immunochemical study on mannan, the antigenic polysaccharide of pathogenic yeasts in man of genus Candida]. AB - This article accounts for the development of immunochemical studies on the antigenic polysaccharide, mannan, a major antigen of pathogenic yeast in man, genus Candida, in order to determine the chemical structures dominating the serological specificities of the parent cells as follows. 1. The serological classification system of 7 medically important Candida species by detecting 10 antigenic factors, 1, 4, 5, 6, 8, 9, 11, 13, 13b, and 34 the corresponding antisera, established by Tsuchiya and his coworkers is documented. 2. The structural studies of Candida mannans until early 1980s, which did not include any evidence for the presence of beta-1,2-linked Man unit, the common constituent of antigenic factors, 5, and 6, are also reviewed. 3. The process of structural identification of antigenic factor 5 residing in the mannans of C. albicans, C. tropicalis, and C. stellatoidea, in the phosphate-bound form of beta-1,2 linked mannooligosaccharides, is summarized. 4. The results of structural identification of antigenic factors 4 and 6 in the mannans of the acid-stable domains of C. albicans are summarized as follows: In order to isolate oligosaccharides containing beta-1,2 and alpha-1,6 linkages, a modified acetolysis method under mild conditions was established. By means of this procedure, oligosaccharides corresponding to antigenic factors 4 and 6 were successfully isolated, and their structures determined, subsequently. 5. Furthermore, effects of the alteration of cultivation conditions, carbon source, pH and temperature, on the chemical structure of mannans, especially of decrease and/or loss of densities of antigenic factors, 4, 5 and 6, are documented, because of the significance of these findings as basic concepts for in situ assay of Candida cells by antibody staining technique in patients' foci. PMID- 7541460 TI - Relative degree of stimulation-evoked glycogen degradation in muscle fibres of different type in rat gastrocnemius. AB - 1. The relative degree of glycogen degradation, caused in different fibre types by supramaximal electrical activation of the muscle nerve, was investigated in m. gastrocnemius medialis of young adult rats under general pentobarbitone anaesthesia. Four different protocols of intermittent maximal tetanic activation were used, each lasting 6 s (33% duty cycle; fast and slow isovelocity concentric (shortening) contractions, brief- and long-burst isometric contractions; 6 rats per group). All contractions were evoked under ischaemic conditions. 2. Work output finally dropped to 29% of the initial value for the fast concentric and to 87% for the slow concentric contractions. In isometric protocols evoked by the same stimulation patterns, the force x time area rose to 110% for brief-burst contractions and dropped to 95% for the long-burst contractions. 3. Following the physiological procedures, the experimental muscle and its contralateral control were removed and prepared for histochemical analysis. Serial sections were stained for glycogen (periodic acid-Schiff (PAS) method) and myofibrillar ATPase (mATPase), the latter reactions being used for classifying the fibres as types I, IIA, IIBd and IIBm. 4. For deep 'red' regions of non-stimulated contralateral control muscles the optical density of PAS staining was ranked between fibre types such that I < IIA < IIBd < IIBm. In superficial 'white' regions of the same muscles, no significant difference in PAS staining density was found between IIBd and IIBm fibres (types I and IIA not present). 5. All contractile protocols produced a significant glycogen degradation in IIBm fibres, and the fast concentric activation procedure was associated with a significant decline of PAS staining in all fibre types. For all activation protocols, the relative degree of glycogen degradation within a given region was ranked such that IIBm > IIBd > IIA > I. For IIBm vs. IIBd fibres, the differences in relative degradation were greater and more consistently significant for superficial white regions than in the deeper red muscle portions. 6. The results are discussed in relation to glycogen degradation measurements in studies of motor unit recruitment. Furthermore, the results from red vs. white muscle regions underline that fibres of seemingly the same mATPase type may differ considerably in other properties. PMID- 7541461 TI - Hyperpolarization induced by vasoactive substances in intact guinea-pig endocardial endothelial cells. AB - 1. The responses of guinea-pig endocardial endothelial (EE) cells to various vasoactive substances were investigated in either the small tissue preparation or freshly isolated cells using the patch clamp technique. 2. The mean resting potential of the EE cell was -44 mV in the small tissue preparation, and applications of ATP, ADP, AMP, adenosine, histamine and substance P induced transient hyperpolarizations of -22, -21, -9, -10, -23 and -15 mV, respectively. The membrane potential of EE cells failed to respond to acetylcholine, bradykinin, thrombin, atrial natriuretic peptide, vasopressin and serotonin. 3. The whole-cell voltage clamp of dissociated cells revealed a transient increase of K+ conductance underlying the ATP and histamine responses. The agonist-induced current showed no time-dependent change during voltage steps. The response was showed no time-dependent change during voltage steps. The response was prevented by adding 10 mM EGTA to the pipette solution. 4. In the cell-attached single channel recordings, ATP induced transient K+ channel activities having a slope conductance of 34 pS. In inside-out patches, similar K+ channels were activated by applying Ca2+ of more than 0.1 microM. 5. These findings are consistent with the idea that the Ca(2+)-dependent K+ channel is involved in the hyperpolarizing response of EE cells, as described in vascular endothelial cells. PMID- 7541462 TI - Classification of ion channels in the luminal and abluminal membranes of guinea pig endocardial endothelial cells. AB - 1. The ion channels on both the luminal and abluminal membranes of endocardial endothelial (EE) cells were separately recorded using the patch clamp technique in the guinea-pig heart. 2. The major population consisted of two types of non selective cation channels, which showed open probabilities of 0.21 and 0.33 at the resting potential, and conductances of 36 and 11 pS, respectively. 3. The next major class was Cl- channels with an ohmic conductance of 409 pS. The channel was quiescent in the cell-attached mode but was activated by strong depolarization after excising the patch membrane. 4. The channels activated by intracellular Ca2+ were mainly K+ channels showing a 34 pS slope conductance and, less frequently, Ca(2+)-dependent K+ channels having a large conductance (210 pS). The inward rectifier K+ channel (32 pS) was also observed. 5. The non selective cation channels were recorded on the luminal membrane, but scarcely on the abluminal membrane, suggesting an active transport of K+ and Na+ across the endocardium. 6. The resting membrane conductance of the EE cells may be provided mostly by non-selective cation channels and 34 pS Ca(2+)-dependent K+ channels. PMID- 7541463 TI - Ca2+ modulation of the cGMP-gated channel of bullfrog retinal rod photoreceptors. AB - 1. The outer segment of an isolated rod photoreceptor from the bullfrog retina was drawn into a pipette containing choline solution for recording membrane current. The rest of the cell was sheared off with a glass probe to allow internal dialysis of the outer segment with a bath potassium solution ('truncated rod outer segment' preparation). The potential between the inside and the outside of the pipette was held at 0 mV. 2. Application of bath cGMP, in the presence of 3-isobutyl-1-methylxanthine (IBMX), gave rise to an outward membrane current. At saturating cGMP concentrations, this current was insensitive to intracellular Ca2+ at concentrations between 0 and 10 microM. At subsaturating cGMP concentrations, however, this current was inhibited by intracellular Ca2+. This sensitivity to Ca2+ declined after dialysis with a low-Ca2+ solution, suggesting the involvement of a soluble factor. 3. At low (nominally 0) Ca2+, the half maximal activation constant and Hill coefficient for the activation of the cGMP gated current by cGMP were 27 microM and 2.0, respectively. At high (ca 10 microM) Ca2+, the corresponding values were 40 microM cGMP and 2.4. 4. The inhibition of the current by Ca2+ was characterized at 20 microM cGMP. Ca2+ inhibited the current by up to 60%, with half-maximal inhibition at 48 nM Ca2+ and a Hill coefficient of 1.6. PMID- 7541464 TI - Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. AB - 1. By the development of a new perforated patch method using gramicidin, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intracellular chloride concentration. 2. Using the patch pipette solution containing gramicidin (100 micrograms ml-1), the access resistance dropped to less than 20 M omega within 40 min after making the gigaohm seal. 3. Under current-clamp conditions, GABA caused a hyperpolarization accompanied by a blockade of spontaneous firing. Under voltage clamp at a holding potential (Vh) of -50 mV, GABA evoked an outward current by way of bicuculline- and picrotoxin-sensitive GABAA receptors. 4. A 10 fold change of extracellular chloride concentration resulted in a 58 mV shift of the reversal potential of GABA-induced outward current (EGABA), indicating that the membrane behaves like a chloride electrode in the presence of GABA. 5. The intracellular chloride activities (aCli), calculated with the Nernst equation using both extracellular chloride activity and EGABA values, ranged from 2.8 to 19.7 mM with a mean value of 9.5 mM. The aCli was not affected either by different pipette solutions or by different holding potentials more hyperpolarized than -40 mV. 6. In the recording from SNR neurones in brain slice using the gramicidin-perforated patch-clamp technique, the inhibitory and excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline. 7. In conclusion, the gramicidin perforated patch method will disclose previously unknown aspects of biological responses involving Cl-. PMID- 7541465 TI - Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula. AB - 1. Neuronal nicotinic acetylcholine receptors in slices of rat medial habenula were studied using patch clamp recording techniques. 2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expected for nicotinic receptors. The whole cell current-voltage relations were linear at negative membrane potentials, but showed strong inward rectification when chloride currents were minimized. 3. When 1 mM Ca2+ (0 mM Mg2+) was present in the external recording solution, the single channel conductances elicited by acetylcholine or nicotine in twenty patches were in the range 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups of conductances. 4. In the open point amplitude distributions of three patches, the most common amplitude corresponded to 41 pS (81% of the area). In another four patches the most common amplitude corresponded to a mean conductance of 51 pS (83% of the area). Direct transitions between open levels were rare. 5. Channel closed times were not significantly different for the two conductance groups. However, for the four patches with predominantly 51 pS openings, the means of the distributions of open times longer than two filter rise times averaged 5.8 ms. Those patches with predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly 51 pS openings the overall mean burst length was 5.8 ms, whereas for patches with predominantly 41 pS openings it was 16.1 ms. 6. These observations suggest that 51 and 41 pS openings result from the activity of at least two, but possibly more, different receptor subtypes. We conclude that nicotinic receptors in the rat ventral medial habenula are heterogeneous. PMID- 7541466 TI - Effects of lowered cytoplasmic calcium concentration and light on the responses of salamander rod photoreceptors. AB - 1. In order to study the interactions between cytoplasmic calcium concentration ([Ca2+]i) and light in modulating the responses of rod photoreceptors, [Ca2+]i was held at different levels by manipulating Ca2+ fluxes across the outer segment membrane. 2. If [Ca2+]i was reduced by the removal of external Ca2+ in the continued presence of Na+, and then held near this reduced level by exposure to 0 Ca(2+)-0 Na+ solution, the onset of the recovery phase of the response to a bright flash delivered just before the return to Ringer solution was accelerated, much as is the case during light adaptation, provided that precautions were taken to minimize Na+ influx. 3. If the rod was first allowed to adapt to steady light, [Ca2+]i held near the appropriate light-adapted level by superfusion with 0 Ca(2+)-0 Mg(2+)-0 Na+ solution and the light extinguished, the onset of the recovery phase of the bright flash response varied with the original background intensity in the same way as in the continued presence of steady light. These results indicate that reduction of [Ca2+]i is sufficient to induce this manifestation of light adaptation in darkness. 4. When [Ca2+]i was held at a reduced level in darkness, not only was the sensitivity to dim flashes reduced, but the response rising phase was also delayed and its amplitude increased supralinearly with flash intensity, neither of which changes is seen during light adaptation. However, similar changes in response kinetics resulted when [Ca2+]i was held near its normal dark level and the phosphodiesterase was partially inhibited by 3-isobuty-1-methylxanthine (IBMX), suggesting that they arose indirectly from an elevated cyclic GMP concentration rather than from a direct effect of Ca2+. 5. If [Ca2+]i was held near the normal dark level and bright steady light presented, the circulating current was completely suppressed. Partial inhibition of the phosphodiesterase by superfusion with 0 Ca(2+)-0 Na+ solution including IBMX resulted in restoration of the circulating current. Dim flash responses recorded under these conditions exhibited kinetics similar to those recorded in 0 Ca(2+)-0 Na+ solution in darkness, in contrast to the response acceleration seen when [Ca2+]i was held near the appropriate light adapted level. These results indicate that the kinetics of the flash response depend on [Ca2+]i rather than on the steady light intensity. PMID- 7541467 TI - Efficacy of peak Ca2+ currents (ICa) as trigger of sarcoplasmic reticulum Ca2+ release in myocytes from the guinea-pig coronary artery. AB - 1. Increments in cytosolic Ca2+ concentration (delta[Ca2+]c) were measured in single smooth muscle cells from guinea-pig coronary artery together with the density of peak Ca2+ currents (ICa) in response to clamp steps from -50 to 0 mV. The comparison of depolarization- with caffeine-induced delta[Ca2+]c was used to define the efficacy by which ICa can trigger Ca2+ release from the sarcoplasmic reticulum (SR). 2. At 2.5 mM extracellular calcium concentration ([Ca2+]o), depolarization induced a rapid rise of delta[Ca2+]c followed by a slow creep. Peak [Ca2+]c occurred within ca 30 s and could be followed by an undershoot and a second rise in [Ca2+]c. The creep was blocked by ryanodine but was insensitive to block of InsP3 receptors with heparin. The creep was not observed in Cs(+)-filled cells. After disappearance of the creep, a tonic delta[Ca2+]c became unmasked. 3. At 2.5 mM [Ca2+]o, peak ICa was -0.80 +/- 0.17 microA cm-2. delta[Ca2+] peaked at the end of the 6 s pulse at 202 +/- 98 nM while caffeine-induced delta[Ca2+]c peaked at 1330 +/- 410 nM. The ratio of depolarization- to caffeine-induced delta[Ca2+]c was 10 +/- 6%. 4. In media containing 10 mM [Ca2+]o plus 1 microM Bay K 8644, peak ICa was -2.6 +/- 1.1 microA cm-2 and delta[Ca2+]c peaked within 2.5 s at 451 +/- 194 nM. Paired measurements yielded the ratio of depolarization- to caffeine induced delta[Ca2+]c as 30 +/- 10%. Depolarization-induced delta[Ca2+]c was nearly blocked by caffeine and reduced by ryanodine to 30%, suggesting the contribution of Ca2+ release from caffeine- and ryanodine sensitive Ca2+ stores. 5. Trypsin (1 mg ml-1) in the electrode solution (10 mM [Ca2+]o plus 1 microM Bay K 8644) increased peak ICa up to 12.5 microA cm-2. ICa induced a delta[Ca2+]c of 990 +/- 210 nM and was accompanied by a 'hump' of IK,Ca. When applied briefly after peak delta[Ca2+]c, caffeine increased [Ca2+]c only moderately. The results suggest that a peak ICa can trigger a synchronized whole-cell Ca2+ release only if ICa is strongly augmented. 6. Amplitude and rate of rise of delta[Ca2+]c were graded by test step potentials along a bell-shaped voltage-dependent curve, similar to that of L-type ICa. Steps to +80 mV induced no delta[Ca2+]c when the electrode solution contained 10 mM Na+. However, with 150 mM intrapipette Na+, pulses to +80 mV induced delta[Ca2+]c.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541468 TI - Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors. AB - 1. Vasopressin-secreting neurones in the rat hypothalamic supraoptic nucleus display patterned spontaneous phasic activity, which is apparently maintained in vivo through yet unidentified neurotransmitter system(s). The present investigation used extracellular recording techniques in anaesthetized Long-Evans rats to evaluate whether the neurotransmitter mechanism underlying phasic firing is provided via a family of ionotropic glutamate receptors. 2. N-Methyl-D aspartate (NMDA) reliably evoked bursts of activity in twenty-seven of twenty eight phasic neurones. Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainate also elicited pronounced excitations in twenty-one of twenty-one and and fourteen of fifteen phasic cells, respectively. 3. A rapid blockade of on going phasic activity was consistently induced following brief applications of both NMDA and non-NMDA receptor antagonists; extended application of antagonists resulted in prolonged silent periods, during which phasic activity failed to recur for minutes. Neither saline nor a cholecystokinin receptor antagonist influenced cell firing. 4. In contrast to putative vasopressin cells, application of NMDA receptor ligands did not affect the spontaneous activity in most putative oxytocin-secreting neurones, whereas kainate and AMPA potently excited seven of nine and four of five putative oxytocin cells, respectively. 5. These results imply that the maintenance of spontaneous phasic discharges in vivo in supraoptic vasopressin-secreting neurones requires tonic synaptic activation involving both NMDA and non-NMDA glutamate receptors. In putative oxytocin-secreting neurones, spontaneous firing appears to be predominantly regulated by non-NMDA receptors. Glutamatergic innervations may be in a unique position to influence the genesis of patterned electrical activity in supraoptic vasopressin neurones. PMID- 7541469 TI - Role of membrane potential in endothelium-dependent relaxation of guinea-pig coronary arterial smooth muscle. AB - 1. Membrane potential and tension were measured simultaneously in ring segments of main coronary artery of guinea-pigs. The synthetic thromboxane A2 analogue U46619 depolarized the tissues from -58 +/- 2 to -40 +/- 1 mV and increased tension by 12 +/- 1 mN mm-1. Nitric oxide (NO) and Iloprost, the stable analogue of prostacyclin, evoked hyperpolarization and relaxation. 2. The concentration of NO required to evoke half-maximal hyperpolarization (EC50 of 2 x 10(-5) M) was 40 fold higher than that which was required to induce relaxation (EC50 of 5 x 10(-7) M). The EC50 for Iloprost-induced hyperpolarization (3 x 10(-8) M) was similar to that for relaxation (4 x 10(-8) M). 3. Glibenclamide (10(-6) M) abolished the hyperpolarization in response to both NO and Iloprost but was without effect on the amplitudes of the relaxations over the complete concentration-response curves. 4. Acetylcholine evoked concentration-dependent hyperpolarization and relaxation in the presence of N omega-nitro-L-arginine methyl ester (NAME; 10(-5) M) and indomethacin (10(-6) M), and these responses were attributed to endothelium-derived hyperpolarizing factor (EDHF). The hyperpolarization produced by EDHF always preceded relaxation, and relaxation never occurred at concentrations of acetylcholine that were insufficient to evoke hyperpolarization. 5. The concentration-hyperpolarization and concentration relaxation curves in response to acetylcholine were not affected by glibenclamide or barium (1-3 mM) but were shifted to the right 4- and 5-fold, respectively, by 1 mM tetraethylammonium. The hyperpolarization and relaxation evoked by acetylcholine were also reduced in a parallel manner when the potassium concentration in the superfusate was increased. 6. Hyperpolarizing current steps, applied to spiral strips of coronary artery denuded of endothelium and depolarized and constricted with U46619, caused relaxation. The relationship between hyperpolarization and relaxation evoked electronically was similar to that which was due to EDHF in intact tissues stimulated with acetylcholine. 7. It is concluded that the ability of NO or Iloprost to relax guinea-pig coronary artery does not depend upon hyperpolarization of the smooth muscle. In contrast, hyperpolarization is likely to play a major, if not the only, role in the relaxation in response to EDHF in this tissue. PMID- 7541471 TI - The computer simulation of RNA folding pathways using a genetic algorithm. AB - A procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as disruption of temporarily formed structures, the folding of a molecule during its synthesis and pseudoknot formation. The simulations are able to predict functional metastable foldings and kinetically driven transitions to more stable structures. The analysis of free energies for intermediate foldings allows estimation of the ranges of kinetic refolding barriers and suggests that in some RNAs the selective evolutionary pressure suppresses the possibilities for alternative structures that could form in the course of transcription. It is shown that the folding pathway simulation can result in structure predictions that are more consistent with phylogenetically proven structures than minimum energy solutions. This suggest that RNA folding kinetics is very important for the formation of functional RNA structures. Therefore, apart form its value for predictions of RNA structures, the proposed computer simulations can be a powerful tool in the studies of RNA folding features. PMID- 7541472 TI - Catalase and hydrogen peroxide cytotoxicity in cultured cardiac myocytes. AB - We examined the role of intracellular catalase activity in modulating hydrogen peroxide (H2O2)-induced cytotoxicity in cultured chick embryo cardiac myocytes. Injury was quantitated by release of lactate dehydrogenase (LDH). Application of 1.5 mM H2O2 to myocytes caused LDH release beginning at 2 h. Inactivation or inhibition of catalase with aminotriazole or sodium azide increased LDH release but did not cause earlier release. Free catalase which entered or became associated with myocytes, but not catalase bound to agarose beads, which did not enter or become associated with myocytes, was protective. Separate experiments demonstrated that myocyte catalase activity decreased by 27% between 1 and 4 h of H2O2 exposure. Treatment with aprotinin, a protease inhibitor, prevented the H2O2 induced fall in catalase activity at 4 h but treatment with deferoxamine, an iron chelator, had no effect on catalase activity. Thus, with exposure of cardiac myocytes to H2O2, the magnitude of the cytotoxicity is modulated by endogenous or cell associated exogenous catalase. It is proposed that in addition to excessive accumulation of H2O2, a reduction intracellular catalase activity may be required before substantial cell injury occurs during H2O2 exposure. Activation of proteases may cause the reduction in catalase activity in this setting. PMID- 7541470 TI - Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis. AB - The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A. PMID- 7541473 TI - Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels. AB - Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca++ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K+ currents, both delayed outward and inward rectifying. The inwardly rectifying K+ currents were often as large as the outward delayed rectifying K+ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl2. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K+ currents. Both Ca++ and Na+ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABAA receptors and kainate receptors that are Ca(++)-permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. PMID- 7541474 TI - PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin. AB - Possible differentiation mechanisms were investigated in a glioblastoma multiform cell line (GL15) presenting an undifferentiated phenotype with weak glial fibrillary acidic protein (GFAP) and strong vimentin (VIM) expression. Serum-free conditions induced time-dependent increases of GFAP-mRNA and GFAP protein levels, associated with a process-bearing astrocytic morphology. Activation of protein kinase C (PKC) by tumor promoter phorbol 12-myrystate 13-acetate (PMA) induced a rapid morphological differentiation and a decrease in GFAP mRNA, whereas the GFAP level remained unchanged. Such parameters were shown to characterize a physiological differentiation stage in astroglial cultures. Treatment of process bearing GL15 cells with dibutyryl cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, induced a time-dependent decrease in the GFAP mRNA and GFAP protein levels and reverted morphological changes induced by serum-free conditions. Neither PMA nor dbcAMP influenced the VIM mRNA expression. In GL15 cells, PKC and PKA activation have opposite effects. Understanding the role of these kinases in malignant transformation and in the in vitro differentiation process is of both basic and clinical interest. PMID- 7541475 TI - Glutamate-activated ionic currents in cultured astrocytes from trout: evidence for the occurrence of non-N-methyl-D-aspartate receptors. AB - Glutamate-induced currents were recorded from cultured trout astrocytes with the whole-cell variation of the patch-clamp technique. Ninety percent of the tested cells were directly depolarized by the amino acid neurotransmitter in a concentration-dependent manner. The depolarizing effect was due to an inward current that reversed near 0 mV and was accompanied by a noise increase, indicating the opening of an ion channel. Ion substitution experiments revealed that the glutamate-induced current was mainly carried by sodium ions but not chloride or calcium ions. The glutamate-induced response could be mimicked by the neuronal glutamate receptor subtype agonists kainate and quisqualate, while N methyl-D-aspartate was without detectable effect. PMID- 7541476 TI - Cryogenic spinal cord injury induces astrocytic gene expression of insulin-like growth factor I and insulin-like growth factor binding protein 2 during myelin regeneration. AB - To study injury-induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45: 742-757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin-like growth factor I (IGF-I), IGF-I receptor (IGFR-I), IGF binding protein 2 (IGFBP-2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3-56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin-positive, GFAP negative astrocyte-like cells in the lesion expressed IGF-I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP-positive, hypertrophic astrocytes, many of which also were vimentin-positive. Levels of IGF-I, IGFBP-2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21-28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7-14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin-positive, oligodendroglia-like cells also were immunostained by anti-IGFR-I. Our findings suggest that early astrocytic production of IGF-I and IGFBP-2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury. PMID- 7541479 TI - [Usefulness of amylase isoenzyme determination for the diagnosis of pancreatic diseases]. AB - Serum amylase shows the greatest increase among the various pancreatic enzymes that increase at the onset of acute pancreatitis. However, the diagnostic value of the total serum amylase activity has been questioned due to its lack of specificity. To differentiate hyperamylasemia due to pancreatic disease from that due to other causes, the activity of pancreatic amylase should be determined by using a monoclonal antibody that specifically binds to pancreatic or salivary amylase, or by electrophoresis. The most useful and accurate method for distinguishing pancreatic from salivary-type hyperamylasemia is isoamylase analysis by electrophoresis. In patients with acute pancreatitis, increase of Amylase-1 and -2 is accompanied by the appearance of Amylase-4, a minor component of the pancreatic-type isoamylases, and by disappearance of the salivary-type isoenzymes, thereby leaving a pattern of the pancreatic isoenzymes alone. This pancreatitis pattern persists for about 10 days after the onset of illness. Therefore, if such a pattern is found in a patient with clinical findings suggesting acute pancreatitis despite a normal serum amylase level, the patient can be diagnosed as having acute pancreatitis or a recent attack of the disease. However, the existence of an inherited trait of the pancreatitis pattern in some healthy individuals must be borne in mind. Patients with recurrent chronic pancreatitis also show pancreatic-type hyperamylasemia, whereas the pancreatic amylase activity decreases when pancreatic exocrine insufficiency progresses. Hyperamylasemia due to elevated salivary amylase activity is also common in patients with diabetic ketosis or malignancies such as lung cancer (adenocarcinoma). Hyperamylasemia is also found following various types of operation. In most cases, it is salivary-type hyperamylasemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541480 TI - [Vitronectin in children with renal disease--1. Immunofluorescence study of vitronectin and C5b-9 in childhood IgA nephropathy]. AB - Renal biopsy specimens from 54 children (33 boys, 21 girls) with IgA nephropathy were investigated by immunofluorescence microscopy to determine the localization of vitronectin and C5b-9. vitronectin and C5b-9 deposits were found within the glomeruli of all cases and the intensity of vitronectin and C5b-9 deposits was as follows: vitronectin: (3+) 17 cases, (2+) 21 cases, (1+) 16 cases and C5b-9: (3+) 28 cases, (2+) 18 cases, (1+) 8 cases. In 19 cases and C5b-9 deposits were shown to be more predominant than vitronectin (Group C) and the same or less intense in 35 cases (Group V). In 33 cases, vitronectin deposits were demonstrated in the mesangial area only (M type), while in 21 cases deposits were presented both the mesangial area and the capillary wall (MC type). Linear staining of vitronectin was noted in Bowman's basement membrane and in the tubular basement membrane. Granular staining of vitronectin was observed in Bowman's basement membrane (29 cases) and tubular basement membrane (46 cases). M type C5b-9 deposits were demonstrated in 34 cases and MC type in 20 cases. C5b-9 deposits were observed in Bowman's basement membrane (19 cases) and in tubular basement membrane (37 cases). Intensely stained vitronectin was found in the sclerotic glomeruli and segmental sclerotic portion of the tissues. In serial biopsy, the intensity of both vitronectin and C5b-9 deposits became less intense. It was found that many patients of Group C had heavy proteinuria (P < 0.05) and that nephrotic patients were more frequent in Group C than in Group V (p < 0.05). In the light microscopic examination, most cases with intense C5b-9 deposits had mesangial proliferation (p < 0.10) and an increase in mesangial matrix (p < 0.10). Mesangial sclerosis index was correlated with the intensity of vitronectin deposits (p < 0.05). These findings suggest that vitronectin and the terminal complement system may play a critical role as a mediator of immunological tissue injury, especially mesangial sclerotic lesion in childhood IgA nephropathy. PMID- 7541478 TI - Effect of dietary restriction on the response of alpha 2-macroglobulin during an acute phase response. AB - The effects of aseptic abscesses induced by subcutaneous injections of turpentine (5 mL/kg body weight) on the acute-phase protein response (alpha 2-macroglobulin, alpha 2-M) and on the circulating albumin and total protein concentrations were assessed in young rats that were made malnourished by restricting dietary intake to an extent that either impaired growth (60% of normal intake) or caused weight loss (0% to 45% of normal intake). The measurements were obtained daily during a period of 4 days in malnourished rats that had lost about 25% body weight but had maintained a stable weight thereafter, or in rats that had lost about 12% body weight and were continuing to lose weight at various rates at the time of the turpentine injection. In animals that were injected while they maintained a stable weight after losing about 25% body weight, the alpha 2-M response was attenuated fourfold to eightfold compared with control animals (the area under the 4-day alpha 2-M curve in control rats was 23.3 +/- 2.3 g/L/d). In the depleted animals that were injected while they were actively losing weight (approximately 12.5% weight loss), the attenuation of the alpha 2-M response was related to the rate of weight loss or to the extent of dietary restriction (area under the 4-day alpha 2-M curve ranged from 5.7 +/- 1.4 g/L/d in animals receiving 15% restricted diet to 15.6 +/- 1.5 g/L/d in animals receiving the 45% restricted diet).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541481 TI - [Vitronectin in children with renal disease--2. Examination of urinary vitronectin excretion]. AB - Urinary vitronectin excretion was measured by ELISA in healthy and various renal diseases children. In 36 healthy children, urinary vitronectin was excreted at low levels in the range of 0-2.71 (0.187 +/- 0.655) micrograms/g.Cre. Urinary Vitronectin and the positive ratio in various renal diseases were as follows: IgA nephropathy: 0-59.22 (3.420 +/- 9.180) micrograms/g.Cre, 41%; Henoch-Schonlein purpura nephritis: 0-118.40 (19.619 +/- 31.713) micrograms/g.Cre, 53%; membranous nephropathy: 0-2.76 (0.595 +/- 0.885) micrograms/g.Cre, 18%; membrano proliferative glomerulonephritis: 0-11.00 (1.888 +/- 3.078) micrograms/g.Cre, 33%; acute glomerulonephritis: 0.07-0.84 (0.456 +/- 0.317) microgram/g.Cre, 0%; Alport's syndrome: 0.81-112.12 (32.378 +/- 44.038) micrograms/g.Cre 67%; focal segmental glomerulosclerosis: 0.30-31.86 (12.070 +/- 17.241) micrograms/g.Cre, 67%; idiopathic nephrotic syndrome with proteinuria: 0-2.89 (0.551 +/- 1.064) micrograms/g.Cre 17%; idiopathic nephrotic syndrome in remission: 0-0.97 (0.258 +/- 0.372) microgram/g.Cre 0%, respectively. In patients with chronic renal failure, urinary vitronectin was excreted at high levels in the range of 2.15 446.08 (118.696 +/- 144.338) micrograms/g.Cre and the positive ratio of urinary vitronectin was 100%. In IgA nephropathy, Henoch-Schonlein purpura nephritis, membrano-proliferative glomerulonephritis, Alport's syndrome, focal segmental glomerulosclerosis and chronic renal failure, the positive ratio of urinary vitronectin was higher than in healthy children, but in membranous nephropathy, acute glomerulonephritis, idiopathic nephrotic syndrome with proteinuria and in remission, the positive ratio of urinary vitronectin was not significantly different from that of healthy children.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541477 TI - Myelinogenic potential of an immortalized oligodendrocyte cell line. AB - The myelinogenic potential of an oligodendrocyte cell line (N20.1) immortalized by transformation with a temperature-sensitive retrovirus (Verity et al., J Neurochem 60:577-587, 1993) has been evaluated in a co-culture system utilizing dorsal root ganglion neurons. When N20.1 cells were placed in co-culture with dorsal root ganglion neurons at 39 degrees C, the temperature at which TAg expression is decreased relative to that in cells maintained at 34 degrees C, there was a dramatic decrease in the N20.1 proliferation rate compared to cells maintained in the absence of neurons at either temperature. This decrease in proliferation was observed within 3 days of co-culture and appeared to precede a further decrease in TAg expression that occurred with time in response to the neurons. In co-cultures the immunoreactivity of N20.1 cells for galactocerebroside increased with time, and the cells appeared to establish contact with neurites and initiate formation of membranous sheets. When the duration of co-culture was extended to 52 days, myelin-like figures were noted by electron microscopy. Thus, the extent of N20.1 differentiation is dependent on the presence of neurons and the duration of co-culture. This culture system represents a potentially powerful tool for the study of neuronal-glial interactions influencing myelinogenesis and remyelination. PMID- 7541483 TI - [Importance of the IGF system in metabolic regulation]. PMID- 7541482 TI - [Cytokine production by human airway epithelial cells and its modulation]. AB - There is increasing evidence that airway epithelial cells play an active role in allergic inflammation, including bronchial asthma. We showed that human airway epithelial cells in culture release GM-CSF, G-CSF, M-CSF, IL-6, and IL-8, using a serum-free culture system. These cytokines are known to modulate the bioactivities of inflammatory cells that accumulate at the site of inflammation. Among them, GM-CSF, IL-8, or both may be important because they influence the bioactivities of eosinophils, which are characteristic of allergic inflammation. Here we report on the effects of air pollutants such as suspended particulate matter and diesel exhaust particulates on release of cytokines from airway epithelial cells. All air pollutants we tested stimulated epithelial cells to release GM-CSF. These results suggest that one cause of the recent increase in the prevalence of allergic disorders is direct stimulation of airway epithelial cells by air-pollutants. Furthermore, anti-inflammatory agents such as steroids and anti-allergic drugs were found to suppress the release of GM-CSF from airway epithelial cells in vitro. PMID- 7541484 TI - [Insulin-like growth factors (IGF) and their binding proteins (IGFBP). Physiological and pathological regulation]. PMID- 7541485 TI - [First experience in the use of endoprosthesis in the treatment of respiratory tract stenosis of tumorous genesis]. AB - The experience of the main bronchi endoprosthesis in treatment of 15 patients with the respiratory tract stenosis of tumoral genesis was analyzed. The endoprosthesis of original construction was proposed. An operation was done with the help of rigid bronchoscope after preliminary laser recanalization of the stenosed segment. The immediate and late follow-up results permit to recommend the method for palliative treatment of pulmonary cancer. PMID- 7541487 TI - [Combined intrathoracic surgery in an aged patient]. PMID- 7541489 TI - [Possibilities of internal quality assurance at the nursing service of a department of surgery]. PMID- 7541486 TI - [Laser photodestruction of previously stained bronchial tumors]. PMID- 7541490 TI - Serum levels of selected liver proteins following partial hepatectomy in the female rat. AB - Liver regeneration in the rat following partial hepatectomy (PH) is a frequently used model to study regulatory mechanisms in relation to cell growth and differentiation. In the present study, we analysed quantitative changes in the peripheral circulation of a number of important serum proteins following PH and laparotomy. Alpha-fetoprotein synthesis was induced in the PH rats and remained at very low levels in non-operated controls. Pregnancy-associated murine protein 1 levels and fibronectin levels were lower in the circulation of all operated animals compared with the intact controls. The serum levels of alpha-2 macroglobulin were elevated in all operated animals compared with the non operated controls. Circulating complement factors C3c and C4 were present at significantly lower levels in PH animals than in rats following laparotomy. Small growth stimulating molecules (< 10 kDa) synthesized by different organs during the regenerative process have been described in the literature, but dialysates of the spleen and liver from non-operated rats and PH rats showed no significant impact on serum levels of the proteins in the present study. PMID- 7541488 TI - [Prolonged regional thermo- and chemotherapy in multiple metastases of colorectal cancer to the liver following a palliative operation]. PMID- 7541492 TI - Human oligodendrocytes are not sensitive to complement. A study of CD59 expression in the human central nervous system. AB - BACKGROUND: One or more components of the oligodendrocyte-myelin unit are the target of immune attack in multiple sclerosis. The role of complement in this process has been suggested by the demonstration in vitro that rat oligodendrocytes are sensitive to lysis by Ab-independent complement attack, partly because of a lack of the complement regulatory protein molecule, CD59. EXPERIMENTAL DESIGN: This study assessed the sensitivity in vitro of human oligodendrocytes derived from neurosurgical specimens to complement attack and analyzed CD59 expression on their surface. The presence of CD59 was also examined in the human central nervous system during myelination and in both the normal and diseased adult brain. RESULTS: Human oligodendrocytes are insensitive in vitro to complement attack in the absence of Ab and using the Ab YTH 53.1 and were shown to possess CD59 on their surface. CD59 is absent from the human central nervous system before myelination, at which stage strong expression occurs in areas of myelin production. CD59 expression is then normally down-regulated but is particularly strong in reactive astrocytes in diseases such as multiple sclerosis. CONCLUSIONS: The findings suggest that the previous demonstration of rat oligodendrocyte complement sensitivity and lack of CD59 expression do not extend to the human central nervous system. There may be a role for CD59 in normal human myelination. PMID- 7541491 TI - Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology. AB - The purpose of this study was to characterize a spontaneous disease condition causing hyperkeratosis in nude mice and to explore the etiologic role of a particular species of coryneform bacteria in this disease, colloquially known as "scaly skin disease." The study was divided into two parts. In the first phase, a series of inoculation experiments was conducted with a field isolate of the coryneform species used to study the clinical and histopathologic development of the disease syndrome. Athymic nude mice (4 to 5 weeks old) were inoculated on the skin of the back with a suspension of a pure culture of the coryneform bacterium that had been isolated from a field case. The culture was applied with a sterile cotton swab in concentrations varying from 6.1 x 10(4)/ml to 5.0 x 10(7)/ml. All inoculated mice became persistently infected throughout the 33 days of the experiment. Clinically evident hyperkeratosis in inoculated animals developed more frequently in mice housed in a microisolator cage than in a semi-rigid isolator and more frequently in mice inoculated with higher numbers of organisms. In all animals in which hyperkeratosis developed, it was first noted on day 7 after inoculation. The second series of experiments was designed to determine the success of various housing methods in excluding the infection, mechanisms of transmission, susceptibility of other stocks and strains of mice to the organism, and whether the other strains might serve as a source of the organism. Results of the study in various strains indicated that both immunocompetent and immunodeficient mice, whether glabrous or hirsute, could be infected with the organism, but only glabrous animals developed hyperkeratosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541495 TI - Anaphylaxis due to hydroxyethyl-starch-reactive antibodies. PMID- 7541494 TI - [Antigenic determination of human anti-rabies vaccine against viral street strains common in the wild animal population in Poland]. AB - The aim of the study was to compare the antigen properties of a vaccine strain with street strains isolated from various animal hosts throughout the country. Investigation was carried out using monoclonal antibodies against NC protein. Also, two tests were carried out: the modified NIH test for potency and the neutralization test using the sera of people vaccinated against rabies (PM vaccine strain). The investigated street strains were used in both tests as the challenge viruses. A suspension of these strains diluted five times made it possible to avoid extreme values of animal survival (0% or 100%) what, consequently, made calculation of the LD50 value easier. A different rabies virus serotype (EBLI virus) in the population of insectivore bats Eptesicus serotinus and antigen variants within the first serotype, having common epitopes with strains of the vaccine virus SAD B19 and the polar rabies virus, were found to be present throughout the country. The concentrated and purified vaccine containing the PM virus did not protect mice against infection with strains of viruses isolated from bats (protection index 10 and lower). For the remaining strains, depending on the animal source of their isolation, the protection index ranged from 10 to 1000 and higher. The properties neutralizing a dose of 5 i.u./ml of serum from the subject inoculated with the vaccine containing the PM strain were similar for all the investigated strains; 0,5 i.u./ml did not neutralize the strain isolated from a racoon dog. PMID- 7541497 TI - Where now for colony-stimulating factors? PMID- 7541493 TI - ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. AB - BACKGROUND: Since the introduction of mAb, immunohistochemistry has become an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and it has become the gold standard for morphologic tissue preservation. Although the molecular mechanism underlying the tissue fixation is not well understood, it has become clear that available immunoreactive Ag are progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroyed during the fixation process. Although monoclonal anti-Ig Ab frequently worked inadequately, polyclonal anti-Ig Ab were shown to produce reproducible staining results. It thus appeared possible that most cellular Ag might not be irreversibly destroyed but only masked. EXPERIMENTAL DESIGN: Although some Ag may be retrieved under appropriate conditions, there might still be many for which available antigenic epitopes are still too sparse to be visualized, as observed for a large number of leukocyte differentiation Ag. One reliable approach to resolve this dilemma is the use of a combination of an optimized Ag retrieval system and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplished by covalent deposition of biotin molecules. RESULTS: Cryostat and paraffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical staining protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Although pretreatment with detergent, protease, a chaotropic substance, or microwave heating resulted in only moderately improved immunostaining, the biotinylated tyramine enhancement step proved to be the most efficient one, although the latter is not sufficient for many Ag when used without pretreatment steps. The combination of an Ag retrieval step with the biotinylated tyramine enhancement step resulted in a 100 to 10,000-fold boost in sensitivity without loss of specificity. CONCLUSIONS: With the ImmunoMax method, defined Ag can be reproducibly detected in formalin-fixed, paraffin-embedded tissues, and the sensitivity of the method is tremendously enhanced. Moreover, it also allows many previously unreactive or unsatisfactorily reactive Ag to be detected, as shown here for IgD, IgM, and CD7 with the use of mAb. PMID- 7541496 TI - Gene transfer to primary chronic granulomatous disease monocytes. AB - For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential. PMID- 7541499 TI - Risperidone for psychotic and behavioural symptoms in Lewy body dementia. PMID- 7541500 TI - Passive immunization with an anti-substance P antibody prevents substance P- and neurokinin A-induced bronchospasm in anesthetized guinea-pigs. AB - In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs. PMID- 7541498 TI - Decisions and care at the end of life. PMID- 7541502 TI - Effects of nicotine and electric footshock on peripheral serotonergic measures and on platelet aggregation in whole blood of rats. AB - The effects of nicotine and electric foot shock as well as of their combination on blood serotonergic measures and on whole blood aggregation have been analyzed. In rats subjected to electric footshock a rise (p < 0.05) in plasma but not in whole blood serotonin was observed, whereas this parameter was not influenced in nicotine-treated rats when compared to the control group. The combination of nicotine with electric footshock only slightly increased plasma serotonin and showed no effect on whole blood serotonin, but 5-hydroxyindole acetic acid (5 HIAA), the major metabolite of serotonin (5-HT), as well as the 5-HIAA/5-HT ratio were markedly increased (p < 0.01) suggesting an enhanced turnover of 5-HT under these conditions. The collagen-induced aggregation in whole blood was not influenced in nicotine-, in footshock- nor in combined-treated rats when compared to the controls. Our data indicate that stress as well as the combination of stress with nicotine may affect the serotonergic system which is in contrast to the exposure to nicotine alone. PMID- 7541503 TI - Electron paramagnetic resonance investigations of nitrosyl complex formation during endotoxin tolerance. AB - The prior administration of low dose endotoxin induces a state of hyporesponsiveness or tolerance to the lethal effects of endotoxin. It is generally accepted that macrophages are main cellular components in the development of tolerance, hence, nitric oxide (.NO) as one of the macrophage mediators may play a role in host defense mechanisms during tolerance. In this study, we utilized EPR spectroscopy to directly detect nitrosyl complexes as products of .NO in whole blood, livers and intestines of lipopolysaccharide (LPS) tolerant rats. Male Sprague-Dawley rats were injected with a "low dose" LPS (0.5 mg/kg) 12-168 h prior to a "high dose" LPS (3 mg/kg), then sacrificed 6 h later. EPR signals of nitrosyl hemoprotein complexes were detected in specimens after high dose LPS. The post-LPS EPR signals of nitrosyl complexes from all samples were attenuated by a prior injection of low dose LPS. The signals of dinitrosyl iron-dithiolate became apparent in samples from tolerant rats as signals of nitrosyl hemoprotein decreased. The maximal tolerance in terms of diminished .NO production was observed when low dose LPS was given 48-96 h prior to high dose LPS. Hemoglobin concentrations in the intestine used as biomarkers of hemorrhagic damage, were concomitantly attenuated in the jejunum of tolerant rats. These results together with our previous studies indicate that suppression of .NO production may contribute to the amelioration of hepatic and intestinal injury during endotoxin tolerance. PMID- 7541504 TI - Liability for failure to offer prenatal AFP testing. PMID- 7541501 TI - Diethyldithiocarbamate ameliorates the effect of lipopolysaccharide on both increased nitrite production by vascular smooth muscle cells and decreased contractile response of aortic rings. AB - The depression of vasoconstrictor responsiveness caused by bacterial lipopolysaccharide (LPS) is mediated, in part, by the induction of nitric oxide synthase (NOS) and the resultant increase in nitric oxide production by vascular smooth muscle. The present study evaluated the ability of the antioxidant, diethyldithiocarbamate (DDTC), to attenuate the LPS-stimulated induction of NOS in cultured vascular smooth muscle cells (VSMC) and the depression of in vitro vascular reactivity caused by LPS administration to rats. The LPS-stimulated increase in nitrite production by cultured VSMC was inhibited 85% by DDTC (100 microM). When VSMC were stimulated with a combination of LPS, interferon-gamma (INF) and tumor necrosis factor (TNF) nitrite production was 5-fold greater than with LPS alone. DDTC inhibited 49% of the increase caused by LPS plus INF and TNF. Aortic rings taken from animals injected with LPS showed a depression of maximum force in response to phenylephrine which was reversed by inhibition of NOS activity. Pretreatment of animals with DDTC attenuated this depression of vascular reactivity. The DDTC treatment did not reduce the increase in serum TNF levels caused by LPS. These results suggest that DDTC can attenuate the LPS stimulated induction of NOS in vascular smooth muscle and may thereby ameliorate the impairment of vascular reactivity. PMID- 7541505 TI - Differential lectin binding to microvascular endothelial glycoconjugates during normal angiogenesis in the chick chorioallantoic membrane. AB - The ontogenesis of endothelial glycoconjugate expression during normal angiogenesis and microvascular development remains unknown. Using intravital fluorescent microscopy, we studied temporal and spatial lectin binding to carbohydrate moieties of luminal microvascular endothelia of the chick embryo chorioallantoic membrane (CAM) during Days 4.5 to 6.0 of the 21-day incubation. We used a battery of eight FITC-lectins (100-200 micrograms/ml). Fluorescent images from precapillary, capillary, and postcapillary segments of the lectin perfused microvascular units were analyzed by image analysis software to quantitate differences in lectin binding. Results served to indicate a significant decrease in lectin binding to terminal N-acetyl glucosamine, N-acetyl galactosamine, and the N-acetyl galactosamine disaccharide in the glycocalyx of pre- and postcapillary vessels from Day 4.5 to Day 5.0. Lectin binding to N acetyl glucosamine and N-acetyl galactosamine subsequently increased at Days 5.0 and 6.0. In the capillaries, lectin binding to endothelial galactose, fucose, and sialic acid increased significantly from Day 5.5 to Day 6.0. That these temporal changes in lectin binding to endothelial luminal glycoconjugates coincide with concomitant changes in CAM microvascular permeability (Rizzo et al., in press) serves to suggest a possible association between expression of endothelial glycoconjugates and the ontogeny of microvascular perm-selectivity during normal angiogenesis. PMID- 7541506 TI - Quantitative angiogenesis in a syngeneic tumor spheroid model. AB - The aim of this work was to develop a system for noninvasive, in vivo, and in situ study of tumor angiogenesis in awake mice. Tumor spheroids were prepared from Lewis lung carcinoma cells prelabeled with methylrhodamine. A transparent chamber consisting of two titanium frames was implanted into the dorsal skin of CB6 mice. One layer of the skin was removed in a 15-mm area and covered with a coverslip. A few days later, the coverslip was removed and one to three tumor spheroids (diameters, 500-900 microns) were placed over the upper tissue layer. The selected fields were recorded under trans- and epi-illumination using video microscopy. Separate fluorescence filter sets were used to visualize the FITC labeled plasma and the rhodamine-labeled tumor spheroids. The dual labeling technique allowed precise identification of the tumors and the study of tumor and microvessel growth for up to 14 days. The tumor area and morphometric parameters of tumor vessels were measured from recorded images. After implantation, tumor cells formed well-defined tumor foci. Venular and capillary dilation and tortuosity were observed in the surrounding tissue 1-2 days after implantation, followed by the appearance of buds and sprouts. After that, vascular networks developed around and within the spheroid. During the first week, angiogenesis was very intense: at Day 6, vascular density and tumor area reached 81 and 19% of their respective maximum values. Vascular densities at Days 3, 6, 10, and 14 were 106 +/- 59, 147 +/- 62, 183 +/- 108, and 173 +/- 38 cm-1, respectively. Tumor volume increased exponentially, with a doubling time of 2 days. Similar results were obtained in nude mice. The model allows detailed repeated observations of angiogenesis and permits quantitative evaluation of tumor growth and angiogenesis in vivo. It is applicable for mechanistic studies as well as therapeutic and pharmacokinetic studies of angiostatic and cytotoxic anti-tumor agents. PMID- 7541508 TI - Use of granulocyte colony stimulating factor to reduce the toxicity of super-VAC chemotherapy in advanced solid tumours in childhood. AB - Children with advanced solid tumours at the Royal Alexandra Hospital for Children (RAHC) receive an intensive four drug chemotherapy combination, Super-VAC (cyclophosphamide, 500 mg/m2, adriamycin, 30 mg/m2, actinomycin-D, 0.5 mg/m2, all daily for 3 days, and vincristine, 1.5 mg/m2 weekly). The majority of patients respond well to three courses of such therapy, but with considerable morbidity, including fever, neutropenia, and mucositis. In an attempt to reduce the morbidity of Super-VAC, G-CSF was added. We documented various parameters in 12 patients who received 28 cycles with G-CSF and compared them to an historical control group of 37 cycles in the preceding 14 patients who received Super-VAC. The median duration of each cycle was 23 days with G-CSF and 28 days without G CSF (P = 0.004). However, differences in requirements for inpatient care (median 16 v. 20 days), intravenous antibiotics (median 9 v. 10 days), amphotericin (median 5 v. 3 days), morphine (median 8.5 v. 7 days), or TPN (median 6.5 v. 8 days) did not reach statistical significance. As expected, a significant difference in neutrophil recovery was demonstrated between the two groups (median 11 v. 16 days, P < 0.0001) but not in platelet recovery (median 13 v. 13 days). The use of G-CSF with Super-VAC resulted in a shorter cycle length, so increasing the dose intensity. A reduction in morbidity could not be demonstrated. No toxic side effects from G-CSF were noted. PMID- 7541509 TI - Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine. AB - The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI. PMID- 7541507 TI - Effect of substitution for arginine residues near position 146 of the A subunit of Escherichia coli heat-labile enterotoxin on the holotoxin assembly. AB - Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized D-galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement. PMID- 7541510 TI - Identification of six novel CFTR mutations in a sample of Italian cystic fibrosis patients. AB - The spectrum of cystic fibrosis (CF) mutations has been determined in many populations of different ethnic and geographic origins. However, in the south of Europe, the commonest mutation, delta F508, accounts for only about 50% of CF chromosomes, while identification of most of the other mutant alleles has not been achieved. In an ongoing effort to identify these alleles, we have scanned the entire coding sequences of the CF gene using a GC clamp denaturing gradient gel electrophoresis assay in a sample of 57 chromosomes from patients of italian origin. We have identified six novel mutations (C276X, H139R, R117L, S42F, A1006E and 3121-2A---> T). Each has only been found once in this sample of CF patients. PMID- 7541511 TI - Homozygosity for a novel splice site mutation (2790-2 A--->G) preceding exon 15 of the CFTR gene in a cystic fibrosis patient of North-East Italian descent. AB - We have been screening a cohort of 225 chromosomes from cystic fibrosis patients for mutations in the cystic fibrosis transmembrane regulator gene using a combination of DGGE,RNA-SSCP and DNA sequencing. A novel splice site mutation was detected by multiplex DGGE in a homozygous patient. Restriction-site generating PCR (RG-PCR) analysis demonstrated that both parents carried the same mutation. The molecular haplotype was the same. All the known ancestors came from the same (Veneto) region, and no consanguinity was documented up to the sixth generation. PMID- 7541512 TI - [Pseudomonas mallei and Pseudomonas pseudomallei: introduction and maintenance of natural and recombinant plasmid replicons]. AB - Comparative analysis of recipient activity of Pseudomonas mallei, Pseudomonas pseudomallei, and Pseudomonas cepacia strains towards naturally occurring and recombinant plasmid replicons was carried out. Autonomic broad host range vector plasmids based on RSF1010(IncQ) and pSa(IncW) replicons as well as integrative vectors based on pSUP202(Co1E1) replicon have been constructed. The study has shown that naturally occurring plasmids RSF1010(IncQ), pSa(IncW), R15(IncN), and RP4(IncP) are being efficiently transferred and stably maintained in investigated Pseudomonas strains. However, recombinant plasmids with the mini-replicon pSa which are stable in Escherichia coli have shown segregative instability in Pseudomonas strains, whereas derivatives of plasmid RSF1010 demonstrated different stability depending on the type of insertion. Plasmid pSUP202 derivative integrative vector pSM525 is efficiently introduced and stably maintained in P. mallei C-5 strain. Two vector systems for genetic manipulations in P.mallei and P.pseudomallei cells have been developed. PMID- 7541513 TI - Phenotypic expression of mitochondrial genotypes in cultured skin fibroblasts and in Epstein-Barr virus-transformed lymphocytes in Pearson syndrome. AB - Pearson syndrome is a fatal disorder involving the hematopoietic system and exocrine pancreas. Mitochondrial respiratory chain deficiencies and/or rearrangements of the mitochondrial DNA were consistently observed in all patients. We report here on the variant phenotypic expression of mitochondrial genotypes in cultured cells from a patient with Pearson syndrome. Skin fibroblasts and lymphocytes harbored, respectively, 60% and 80% of deleted mtDNA molecules initially and displayed defective respiratory chain activities. In both cases, there was a progressive recovery of respiratory chain activities during in vitro cell proliferation due to the loss of deleted mtDNA molecules in cultured skin fibroblasts and to an increase in the mtRNA translation efficiency in Epstein-Barr virus-transformed lymphocytes. The present study suggests that various cellular responses to abnormal mitochondrial genotypes might contribute to the tissue-specific expression of mitochondrial disorders in vivo. PMID- 7541514 TI - Cancer pain management in North Carolina. Physician knowledge, attitude, and behavior. PMID- 7541515 TI - Altered neuropeptide content and cholinergic enzymatic activity in the inflamed guinea pig jejunum during parasitism. AB - We investigated the effects of an enteric infection with the parasitic nematode, Trichinella spiralis, on peptidergic and cholinergic neural pathways of the guinea pig jejunum. The content of the enteric neuropeptides, substance P (SP) and vasoactive intestinal peptide (VIP), and the activities of the key cholinergic enzymes, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT), were measured and compared in extracts of jejunal muscularis externa (ME) obtained from uninfected jejunum and T. spiralis-inflamed jejunum. Significant decreases were detected in both SP immunoreactivity and AChE activity on days 6 and 10 postinfection (PI) in nematode-infected guinea pig jejunum compared to uninfected controls. The maximum changes observed for SP and AChE both occurred on day 10 PI and were evident as decreases of 37% and 48%, respectively, from the mean uninfected control values for SP and AChE. In contrast, VIP immunoreactivity and ChAT activity showed no significant changes during the enteric phase of T. spiralis infection. Nematode-evoked histopathological changes in jejunal tissues from infected animals were associated with significant increases in myeloperoxidase (MPO) activity, an index of inflammation intensity, which occurred on day 6 PI (885% of mean control) and day 10 PI (469% of mean control) coinciding temporally with the significant decrease in SP content and AChE activity during infection. Thus, intestinal motor disturbances observed in mammalian hosts during enteric nematode infections involve inflammation-generated changes in the neurohumoral control of smooth muscle function. PMID- 7541516 TI - Tachykinin-induced nasal fluid secretion and plasma exudation in the rat: effects of peptidase inhibition. AB - Substance P (SP) evokes fluid secretion and plasma extravasation when applied to the nasal mucosa of rats. SP and another tachykinin, neurokinin A (NKA), are degraded in vitro by neutral endopeptidase (NEP) and angiotensin-1-converting enzyme (ACE). In this study, NKA or SP were applied locally to the nasal mucosa of rats. Subsequent fluid secretion was measured by a filter paper technique. Plasma exudation was derived as the recovery of intravenous (i.v.) administered 125I-albumin from the fluid-containing filter papers. In order to inhibit enzymatic degradation of the tachykinins by NEP and ACE, the rats were treated with i.v. administered phosphoramidon or captopril respectively or their combination. SP evoked fluid secretion that was augmented by phosphoramidon and further enhanced by adding captopril. NKA evoked nasal fluid secretion less effectively than SP and the effect was unaffected by peptidase inhibition. SP, but not NKA, evoked increased plasma exudation but only after pre-treatment with phosphoramidon. PMID- 7541517 TI - Voltage- and use-dependent block by 1-methyl-4-phenylpyridinium ion (MPP+) of N methyl-D-aspartate-activated currents in rat hippocampal neurons. AB - The effects of 1-methyl-4-phenylpyridinium ion (MPP+) on N-methyl-D-aspartate (NMDA) receptor-channel complex were studied in rat hippocampal neurons using intracellular- and whole-cell voltage clamp-recording techniques. Intracellular recordings were made from CA1 pyramidal cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and picrotoxin (PTX; 50 microM) which block non-NMDA and GABA receptors, respectively. Superfusion of of MPP+ reversibly decreases the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSP(NMDA)) in a concentration-dependent manner. In other experiments, we observed that MPP+ attenuated NMDA-evoked whole-cell currents in a voltage- and use-dependent manner and was not dependent on the extracellular glycine or spermine concentration on neurons freshly dissociated from rat hippocampi CA1 region. These results suggest that MPP+ applied at micromolar concentrations, is non-competitive NMDA receptor antagonist in rat hippocampal neurons. PMID- 7541518 TI - The effects of cyanide on the extracellular levels of dopamine, 3,4 dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyindoleacetic acid and inositol phospholipid breakdown in the brain. AB - The effects of sodium cyanide on the extracellular levels of dopamine and the main brain metabolites of dopamine and 5-hydroxytryptamine were studied in awake, freely moving rats using microdialysis technique. Already 20 min after the administration of cyanide (2 mg/kg; ip) the extracellular level of striatal dopamine was increased and after 40 min an enhancement of the homovanillic acid level was observed. Slight but significant decreases of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid were also observed. The increased extracellular levels of dopamine and homovanillic acid could indicate an increased release of dopamine. An alternative explanation could be an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Cyanide has also been reported to induce marked changes in cytosolic brain Ca2+. We examined the effect of cyanide on inositol phospholipid breakdown in rat cerebral cortex and pig striatum in vitro. This breakdown was not affected by sodium cyanide and appears therefore not to be responsible for the reported changes of cytosolic Ca2+ induced by sodium cyanide. PMID- 7541519 TI - Recent advances in the management of tracheobronchial obstruction. PMID- 7541520 TI - Effect of dextran on gentamicin-induced ototoxicity. AB - The effect of low-molecular-weight dextran (DX) on gentamicin (GM)-induced ototoxicity was examined in guinea pigs. GM (100 mg/kg) was administered intramuscularly for 10, 12 and 14 days with or without DX. Cochlear damage was evaluated by compound action potential thresholds and surface preparation after fixation with osmium tetroxide. The percentage of missing outer hair cells was significantly increased by combined administrations of GM with DX. However, there was no significant difference in blood urea nitrogen levels in guinea pigs with or without DX. When aminoglycoside antibiotics and DX are administered together, attention should be paid not only to nephrotoxicity but also to ototoxicity. PMID- 7541521 TI - Basal cell carcinoma with hyaline inclusions. AB - A 69 yr old man had a 4 mm basal cell carcinoma completely excised from the chin. Numerous hyaline cytoplasmic inclusions were contained within the tumor cells. The inclusions stained intensely red with Masson's trichrome, and immunocytochemically there was prominent rim labelling for keratins (bovine, callus and AE1/3) and muscle-specific actin, the latter more faintly decorating the centre of some inclusions. The inclusions were negative for antibodies to cytokeratin Cam5.2, epithelial membrane antigen (EMA), vimentin, S100, neurofilaments, glial fibrillary acidic protein (GFAP) and carcinoembryonic antigen (CEA) and there was no post Congo red apple green birefringence to indicate amyloid. Ultrastructure indicated the inclusions were composed of proteinaceous material surrounded by a defined rim of tonofilaments in cells showing no degenerative features. The findings suggested aberrant tumor cell keratinization. Familiarization with this rare variant of a common cutaneous carcinoma will alleviate diagnostic difficulties that may arise, particularly in superficial tumor curettage. PMID- 7541522 TI - Insulin-like growth factor (IGF)-I and IGF-binding protein 3 during the first year in term and preterm infants. AB - Plasma IGF-I and IGF-binding protein 3 (IGFBP-3) were determined by radioimmunoassay in term infants (n = 51) at 0, 2, 4, 6, and 12 mo and preterm infants (n = 51) at -3 (28.4 wk postconceptional age), -2, -1.5, -1, 0, 2, 4, 6, 9, and 12 mo from expected term. The effects of gestational age at birth (term or preterm) and study age were determined by repeated measures analysis of variance and Fisher's least squares difference. In preterm infants, IGF-I increased between -3 and 2 mo from 0.75 to 10.4 nM, decreased between 2 and 9 mo to 7.3 nM, and increased again between 9 and 12 mo to 10.1 nM (p < 0.0001), whereas IGFBP-3 increased relatively little from -3 to 0 mo (14.2 to 30.2 nM, p < 0.05) and plateaued from 2 to 12 mo (49.8 to 62.3 nM). At 0 mo, IGF-I and IGFBP-3 were the same in term and preterm infants, but preterm infants had higher IGF-I from 2 through 12 mo (p < 0.05), higher IGFBP-3 at 2 and 4 mo (p < 0.05), and lower IGFBP-3 at 12 mo (p < 0.05). IGF-I and IGFBP-3 were correlated at most ages. First year IGF-I and IGFBP-3 are influenced by study age and by gestational age, even after adjusting for early birth. PMID- 7541523 TI - The in vitro effects of granulocyte and granulocyte-macrophage colony-stimulating factor on interleukin-3-dependent proliferation of human neonatal circulating progenitor cells. AB - Recombinant human granulocyte (rhG) colony-stimulating factor (CSF) and recombinant human granulocyte-macrophage (rhGM) CSF have been used to enhance neonatal neutrophil host defense. We aimed to determine the comparative efficacy of rhG-CSF and rhGM-CSF in increasing numbers of granulocyte colony-forming unit (CFU-G) and granulocyte-macrophage colony-forming unit (CFU-GM) in recombinant human (rh) IL-3-dependent cultures of human neonatal circulating hematopoietic progenitor cells, including cells from infants born to hypertensive mothers. We also investigated the relationship between fractional increase in CFU-G and endogenous plasma concentration of G-CSF. Circulating mononuclear cells were harvested from 25 neonates, and standard short-term assays in semisolid agar were established in the presence of rhIL-3 alone, rhIL-3 with rhG-CSF and rhGM-CSF, and both rhG-CSF and rhGM-CSF. CFU-G and CFU-GM were counted on d 14. Total colony number and CFU-G were significantly greater in cultures supplemented with rhG-CSF, with or without rhGM-CSF (p < 0.001 and p < 0.0005 for total colony number and CFU-G, respectively), when compared with cultures with rhIL-3 alone. Progenitor cells from three infants born to hypertensive mothers responded similarly. Total colony numbers and CFU-G were not increased by rhGM-CSF alone or by addition of rhGM-CSF to rhG-CSF; however, the proportions of CFU-GM were (p < 0.05 and p < 0.001, respectively, compared with rhIL-3 alone).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541524 TI - GABAergic inhibition of crayfish deep extensor abdominal muscle exhibits a steep dose-response relationship and a high degree of cooperativity. AB - A patch-clamp study was done to characterize the recently found GABAergic (i.e. gamma-aminobutyric acid) inhibitory synaptic channels of crayfish deep extensor abdominal muscle. Outside-out patches were rapidly activated by GABA to measure the dose/response curves for the open probability of the channels, Po, and the rise time, tr, (time from Po = 0.1 to Po = 0.9). In some of the patches the GABA activated currents decayed due to desensitization and such patches were not studied further. Rare channel openings were elicited with 0.1 mM GABA. The Po at this low concentration of GABA was 0.0005 to 0.01. Application of 10 mM GABA was necessary to reach the maximal Po of 0.9. The slope of the dose/response relationship in the double logarithmic plot was 5.4 +/- 1.1 (mean +/- SD; n = 9) between 0.1 mM and 0.2 mM GABA. The plot of tr versus GABA concentration had a peculiar shape, recently found to be characteristic for positive cooperativity of the binding sites. tr increased from a minimum at 10 mM GABA with declining concentrations of GABA and reached a peak at 0.4 mM GABA. Below 0.4 mM GABA, tr decreased again. With 0.2 mM GABA tr was 0.40 +/- 0.1 (mean +/- SD; n = 4) of the peak value measured at 0.4 mM GABA. Simulations were compared with the experimental results and a linear reaction scheme with five binding sites for GABA was established to describe the dose/response curves for Po and tr. PMID- 7541526 TI - Wound care. Easing the pressure. PMID- 7541525 TI - Mechanisms of dopamine effects on Na-K-ATPase activity in Madin-Darby canine kidney (MDCK) epithelial cells. AB - Dopamine decreases tubular sodium reabsorption, attributed in part to Na-K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where specific dopamine DA1 binding sites have been demonstrated, we examined the effects of dopamine, as well as of DA1 and DA2 receptor agonists on Na-K-ATPase activity and on the number of units in Madin-Darby canine kidney (MDCK) cells, which retain differentiated properties of the renal cortical collecting tubule epithelium. Dopamine (10(-5) M) inhibited pump activity (by 50%) and reduced the number of units. This effect was reproduced by the DA1 agonist SKF 38393, which inhibited pump activity in a dose- and time-dependent manner (maximum, 10(-5) M). The DA2 agonist quinpirole hydrochloride was without effect, either alone or in combination with SKF 38393. Inhibition of pump activity by dopamine was totally abolished by H7 (100 microM), an inhibitor of protein kinase (PK), but partially by 2',5'-dideoxy-adenosine (DDA) and H4, respective inhibitors of cAMP production and PKA, which suggests that the dopamine effect on Na-K-ATPase activity may be linked to activation of both PKC and PKA. In these cells, amiloride addition during preincubation did not alter the effect of dopamine on Na-K-ATPase activity; in contrast, furosemide increased further the inhibitory effect of dopamine on the enzyme activity. Monensin addition (10(-3) M) reversed the inhibitory effect of dopamine after a 30-min preincubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541527 TI - Assay variability in serum prostate-specific antigen determination. AB - Consistency and reproducibility of serum prostate-specific antigen (PSA) measurement are essential in the application of this analyte to early detection or screening programs. In the present investigation, we sought to compare serum PSA levels determined by the IMx assay (Abbott Laboratories, North Chicago, IL) and the Tandem E (Hybritech Inc., San Diego, CA) to determine whether there were differences. Two hundred twenty-eight random sera from our archival bank were investigated. One hundred-eight specimens were in the Tandem range of 2.0-10.0 ng/ml, and prostatic histology was known based on either systematic sector needle biopsy or transurethral resection. PSA was measured with three different lost of the IMx and Tandem assays. Over the entire range, there was a good correlation (r2 = 0.985); however, in the more useful clinical range of 2.0-10.0 ng/ml, the correlation was reduced to 0.923; in the 2.0-6.0 ng/ml range, it was further reduced to 0.852. The slope for the entire range was 0.948; however, in the 2.0- to 10.0-ng/ml range, it was 0.894; in the 2.0- to 6.0-ng/ml range, the slope was 0.815. Using PSA cutoffs of 4.0, 5.0, and 6.0 ng/ml, significant decrease in abnormal PSA values in men with cancer was observed with the IMx compared with Tandem. These data suggest that the IMx and the Tandem PSA assays are not equivalent, and in most patients a lower value is realized with the IMx assay. This bias appears to be greater in men with prostate cancer. Clinicians must be aware which assay their patients are being tested with, and laboratory technicians should run internal standards to ensure lack of significant intralot variability and consistency over time. PMID- 7541529 TI - Survival of human prostate carcinoma, benign hyperplastic prostate tissues, and IL-2-activated lymphocytes in scid mice. AB - Mice, homozygous for the mutation severe combined immunodeficiency (scid) and also segregating for the mutation hypogonadal (hpg), were tested for their potential use as an in vivo model system for studying the growth of human prostate cancer and benign hyperplastic prostate tissue grafts. Fresh human prostate cancer or benign hyperplastic prostate tissue was implanted subcutaneously into androgen-replete C.B. 17 scid/scid males, and into androgen deficient hpg/hpg scid/scid or androgen-replete +/? scid scid males. The tissue grafts grew in both androgen-replete and androgen-deficient host mice. When dihydrotestosterone (DHT) was administered at tissue grafting, both the incidence and size of the tissue grafts increased. Histology of tissue from tumors in the androgen-deficient hpg/hpg scid/scid host showed either undifferentiated tumors or adenocarcinomas with few glandular structures. These data suggest the androgen deficient environment selected for growth of androgen-independent tumor tissue. Finally, when interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes were injected into scid/scid hosts, the cells were found to survive and could be identified in the spleen of the recipient mice. These results indicate that growth of human prostate tissues and IL-2-activated lymphocytes in scid/scid mice is a viable model system for in vivo studies of prostatic disease. PMID- 7541530 TI - Immunocytochemical characterization of explant cultures of human prostatic stromal cells. AB - The study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human prostatic stromal cells and their immunocytochemical characterization. As determined by antibodies to keratin and prostate specific acid phosphatase, only small numbers (< 5%) of epithelial cells were present in primary cultures; subsequent passaging further reduced epithelial cell contamination. Antibodies against intermediate filament proteins (keratins, vimentin, and desmin) and smooth muscle actin microfilaments demonstrated that stromal cells from benign prostatic hyperplasia and prostate carcinoma differed in regard to their differentiation markers. Two contrasting phenotypes were identified in cultures derived from these two different lesions: One exhibiting fibroblastic features, was predominant in cultures derived from benign lesions and a second, showing varying degrees of smooth muscle differentiation, was more abundant in carcinoma-derived cultures. These findings are indicative of a remarkable divergence in the stromal-epithelial relationships associated with these pathological conditions and may provide us with a potential tool for studying these processes. PMID- 7541531 TI - Pathology review in an early prostate cancer detection program: results from the American Cancer Society-National Prostate Cancer Detection Project. AB - Biopsy materials obtained in the American Cancer Society National Prostate Cancer Detection Project were reviewed at the Central Pathology Laboratory at the Armed Forces Institute of Pathology. Of 265 cases submitted, 177 were diagnosed as prostatic carcinoma, 7 as prostatic intraepithelial neoplasia (PIN), 13 as atypical glands or atypical hyperplasia, and the remaining 68 were benign hyperplasias. Irrespective of the means of detectin or the grading system used (Gleason or WHO-Mostofi), a large majority of the cancers were detected as low grade tumors. Of 27 cases of PIN reported, 20 were associated with cancer, leaving 7 cases with the sole diagnosis of PIN. These data may indicate the increased use of prostate-specific antigen (PSA), digital rectal examination (DRE), and transrectal ultrasound (TRUS) in the United States is shifting the spectrum of prostate cancer pathology toward early low-grade tumors. PMID- 7541528 TI - Longitudinal evaluation of serum androgen levels in men with and without prostate cancer. AB - Androgens are thought to play a role in the pathogenesis of prostate cancer. We evaluated androgen levels in 3 age-matched groups of men who were part of the Baltimore Longitudinal Study of Aging: 1) 16 men with no prostatic disease by urologic history and exam (control group); 2) 20 men with a histologic diagnosis of benign prostatic hyperplasia (BPH) who had undergone simple prostatectomy; and 3) 20 men with a histologic diagnosis of prostate cancer (16 with local/regional cancer, and 4 with metastatic cancer). Luteinizing hormone (LH), total testosterone (T), and free T were measured on stored AM sera by radioimmunoassay (RIA). Free T was also calculated from the measured concentrations of total T and sex hormone binding globulin (SHBG). The median number of repeated sex steroid measurements ranged from 6-9 over a period from 7-25 years prior to the diagnosis of prostate disease. There were no significant differences in age-adjusted LH, total T, SHBG, or calculated free T levels among the groups at 0-5, 5-10, and 10 15 years before diagnosis. These data suggest that there are no measurable differences in serum testosterone levels among men who are destined to develop prostate cancer and those without the disease. PMID- 7541532 TI - Cotranslational folding and calnexin binding during glycoprotein synthesis. AB - To analyze cotranslational folding of influenza hemagglutinin in the endoplasmic reticulum of live cells, we used short pulses of radiolabeling followed by immunoprecipitation and analysis with a two-dimensional SDS/polyacrylamide gel system which was nonreducing in the first dimension and reducing in the second. It separated nascent glycopolypeptides of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epitopes, N-linked glycosylation, and oligosaccharide-dependent binding of calnexin, a membrane-bound chaperone that binds to incompletely folded glycoproteins via partially glucose-trimmed oligosaccharides. When glycosylation or oligosaccharide trimming was inhibited, the folding pathway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin. PMID- 7541533 TI - Use of an oocyte expression assay to reconstitute inductive signaling. AB - We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors. PMID- 7541534 TI - Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit. AB - The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans acting stimulators of RecBC-dependent recombination. PMID- 7541535 TI - Presynaptic modulation of cortical synaptic activity by calcineurin. AB - Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism. PMID- 7541536 TI - Overexpression of csk inhibits acid-induced activation of NHE-3. AB - Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE 3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+) antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3. PMID- 7541537 TI - The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex. AB - The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA. PMID- 7541540 TI - Scorpion toxins as natural scaffolds for protein engineering. AB - A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins. PMID- 7541538 TI - Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. AB - The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information. PMID- 7541539 TI - Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin. AB - A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis. PMID- 7541541 TI - Liquid-phase combinatorial synthesis. AB - A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained. PMID- 7541543 TI - [Whipple disease. Report of a case with unusual therapeutic aspects]. AB - Whipple's disease is a chronic systemic syndrome which typically involves small intestinal mucosa. There is no doubt today about its bacterial etiology and its good response to antibiotic therapy. A peculiar case is reported, in which the failure of a protracted corticosteroidal treatment is demonstrated and a particularly prompt endoscopic healing after therapy with cotrimoxazole is obtained. The value of duodenal biopsy in course of upper gastrointestinal endoscopy in comparison with classic intraluminal jejunal biopsy is confirmed for the diagnosis of Whipple's disease. PMID- 7541542 TI - Isolation of small polarized bile duct units. AB - Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes." PMID- 7541546 TI - The changing therapy of benign prostatic hyperplasia. AB - The diagnosis and treatment of benign prostatic hyperplasia (BPH) is increasingly under review and discussion. The diagnosis of BPH can be based on microscopic, clinical, or urodynamic criteria, and has important implications for treatment. Recommendations for treatment regimens are regularly updated, as new therapies enter the market and the cost effectiveness of new and established regimens are reviewed. Surgery, the mainstay of treatment for BPH, is declining, and medical therapy is assuming a greater role. Pharmacological regimens, for example alpha adrenoceptor blockers and 5-alpha reductase inhibitors, are becoming established for the treatment of mild and moderate symptoms of BPH. This paper will review the spectrum of BPH treatment modalities facing the urologist, and the factors that influence individual treatment choice. PMID- 7541544 TI - L-arginine enhances injury in the isolated rabbit lung during hyperoxia. AB - L-Arginine is the substrate for synthesis of nitric oxide (NO.) by NO synthase which physiologically produces vasodilation. The reaction of NO. or its metabolites with O2 or its metabolites, however, can produce toxic reactive species which may cause cellular injury. We hypothesized that excessive NO. production in isolated perfused rabbit lungs at elevated PO2 could support the production of toxic nitrogen metabolites. In isolated perfused rabbit lungs ventilated with 95% O2, 1.0 mM L-arginine caused significant pulmonary hypertension and edema. These effects of L-arginine were attenuated by the NO. synthase inhibitor, L-NAME (0.5 mM), not affected by SOD pretreatment (100 u/ml) and reversed by pretreatment with catalase (200 u/ml), suggesting a mechanism involving H2O2. This mechanism was supported by producing L-arginine mediated injury in normoxic lungs in the presence of a H2O2 generating system. This injury also was attenuated by L-NAME. On the basis of these results, we conclude that H2O2 interacts with NO. or one of its oxidized metabolites to contribute to acute lung injury during hyperoxia. Such a mechanism may involve peroxynitrite anion, although direct proof of its formation is lacking under these conditions. PMID- 7541547 TI - Alpha1 adrenergic receptor blockade in the male lower urinary tract and other body systems. AB - Recent developments in pharmacology and molecular cloning techniques have led to an increased interest in the therapeutic potential of drugs which act via alpha adrenoceptors. Of particular interest are the alpha 1 adrenoceptors. Pharmacologically, two subtypes of the alpha 1 adrenoceptor have been characterised, namely alpha 1A and alpha 1B. Three subtypes with high affinity for selective alpha 1 adrenoceptor antagonists have also been cloned, that is the alpha 1b, alpha 1c and alpha 1a/d subtypes. Selective alpha 1 adrenoceptor antagonists have become an accepted treatment alternative in the management of both benign prostatic hyperplasia and hypertension, based on their ability to inhibit alpha 1 adrenoceptors of the lower urinary tract and cardiovascular system. Central actions may also be apparent. While cloned receptors of the prostatic stroma seem to be of the alpha 1c subtype, none of the adrenoceptor antagonists available today is subtype selective. This paper will discuss the characterization and localization of the different alpha 1 adrenoceptors and the therapeutic implications of inhibition. PMID- 7541548 TI - Profile of doxazosin in patients with benign prostatic hyperplasia. AB - Alpha adrenergic receptors in prostatic smooth muscle play a major role in the development of bladder outlet obstruction in patients with benign prostatic hyperplasia (BPH). As a result, alpha adrenoceptor antagonists have been used increasingly in the treatment of this condition. Doxazosin is a selective alpha 1 antagonist with a long half-life and gradual onset of action, which make it suitable for once-daily treatment. In placebo-controlled trials, doxazosin has been shown to improve maximum and mean urine flow rates, reduce urethral resistance and bladder contractility, and improve obstructive and irritative symptoms. These changes develop within a few weeks of starting treatment and are maintained during long-term treatment over periods of years. Doxazosin is also well tolerated; adverse events are generally mild or moderate and do not interfere with treatment. These features combine to make doxazosin an effective, convenient and well-tolerated treatment for the management of patients with BPH. PMID- 7541549 TI - Efficacy of doxazosin in normotensive and hypertensive patients with benign prostatic hyperplasia. AB - Benign prostatic hyperplasia (BPH) and hypertension are both common conditions in older men. It is estimated that 20 to 41% of middle-aged to elderly men may have both. Alpha-1 adrenoceptor antagonists, such doxazosin, produce significant improvements in urine flow rate and symptoms in patients with BPH. The effect of doxazosin on urine flow rate and symptoms in hypertensive men with BPH is similar to that seen in normotensive men. By contrast, doxazosin produces a significant fall in blood pressure in hypertensive men, but has minimal effect in normotensives. Doxazosin is well tolerated, irrespective of blood pressure status. Thus, it can be concluded doxazosin is an effective and well tolerated treatment for BPH which appears to have additional benefits in the management of patients with concomitant hypertension. PMID- 7541545 TI - Neutrophil elastase inhibitor reduces asthmatic responses in allergic sheep. AB - To determine the role of neutrophil elastase in asthmatic responses, we studied the effect of ONO-5046, a specific neutrophil elastase inhibitor, on antigen induced asthmatic responses in allergic sheep. Pulmonary resistance (RL) was measured for 8 h after antigen challenge. Measurements of airway responsiveness to methacholine and bronchoalveolar lavage fluid (BALF) were obtained 8 h after challenge. Antigen challenge caused early and late increases in RL, airway hyperresponsiveness (AHR), and recruitment of neutrophils and eosinophils along with increases in TXB2 and LTB4 in BALF. ONO-5046 treatment significantly reduced both early and late bronchoconstriction, neutrophil recruitment, increases in LTB4 in BALF, and AHR. ONO-5046 post-treatment significantly reduced the increase in RL 8 h after antigen challenge. Another neutrophil elastase inhibitor, FR 134043, significantly reduced both early and late bronchoconstriction. ONO-5046 had little effect on calcium ionophore-induced LTB4 release from isolated neutrophils and whole blood obtained from drug-treated sheep. These findings suggest that neutrophil elastase is involved in antigen-induced bronchoconstriction and AHR mediated by neutrophil accumulation and 5 lipoxygenase products in allergic sheep. PMID- 7541550 TI - Perspectives on the management of benign prostatic hyperplasia. PMID- 7541551 TI - The epidemiology of benign prostatic hyperplasia and observations on concomitant hypertension. AB - Benign prostatic hyperplasia (BPH) and hypertension are highly prevalent, age related disorders in men which place a considerable burden on healthcare resources worldwide. Evidence of pathological BPH can be found in over 80% of autopsies conducted in men over 70 years of age; around 40% of men aged 50-64 years have symptoms compatible with the disease. Population studies suggest that over 50% of people aged 65-74 years may be hypertensive, and that cardiovascular disease is a major cause of death worldwide. The continued ageing of the population means that more people are living to older ages; life expectancy for men is likely to exceed 80 years by the end of this century in some developed countries. While BPH and hypertension are apparently diverse disease processes, they have some features in common (e.g. underlying aetiology of the sympathetic nervous system). However, little else is known about any associations between the two or the frequency of concomitant disease. There are clearly opportunities for further epidemiologic studies and the development of preventive strategies aimed at the early detection and treatment of concomitant BPH and hypertension. PMID- 7541553 TI - Risperidone for the treatment of schizophrenia: does it have a niche? PMID- 7541552 TI - [Trisomy-21 screening using AFPplus in the eastern part of Switzerland]. AB - Two years after introduction of maternal serum screening for Down's syndrome in German-speaking Switzerland, based on measurements of alpha-fetoprotein, unconjugated estriol and total beta-HCG, results were analyzed of the two cytogenetic laboratories in Zurich and of a separate collective of the Department of Obstetrics at the University of Zurich. In a total of 489 cases with increased risk for Down's syndrome (> or = 1:380 at term) 19 (1:26; approximately 4%) had an abnormal fetal karyotype from which 16 had a trisomy 21. 13 out of these 16 mothers were aged below 35 years. Thus, after ultrasound, maternal serum screening detects the highest percentage of fetuses with chromosomal abnormalities. At the Department of Obstetrics 2962 serum screening tests were performed during a period of 2 1/2 years. 14.6% of the women were > or = 35 years old, 7.6% showed increased risk and 14 fetuses had an abnormal karyotype including 10 with Down's syndrome. 7 of these 10 were detected by the serum test. Nevertheless, the limited sensitivity of serum screening, its limitation predominantly to detection of fetuses with Down's syndrome and the rather late stage of screening, make effective first trimester screening mandatory. PMID- 7541554 TI - Requirement for TNF-alpha and IL-1 alpha in fetal thymocyte commitment and differentiation. AB - CD25 expression occurs early in thymocyte differentiation. The mechanism of induction of CD25 before T cell receptor rearrangement and the importance of this mechanism for T cell development are unknown. In a thymus reconstitution assay, tumor necrosis factor alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), two cytokines produced within the thymic microenvironment, induced CD25 expression on early immature thymocytes. Either TNF-alpha or IL-1 alpha was necessary for further thymocyte maturation and CD4+CD8+ differentiation. In irradiated mice reconstituted with CD117+CD25+ thymocytes, commitment to the T cell lineage was marked by the loss of precursor multipotency. PMID- 7541555 TI - Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. AB - Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src. PMID- 7541558 TI - Disruption of retinal axon ingrowth by ablation of embryonic mouse optic chiasm neurons. AB - Mouse retinal ganglion cell axons growing from the eye encounter embryonic neurons at the future site of the optic chiasm. After in vivo ablation of these chiasm neurons with a monoclonal antibody and complement, retinal axons did not cross the midline and stalled at approximately the entry site into the chiasm region. Thus, in the mouse, the presence of early-generated neurons that reside at the site of the future chiasm is required for formation of the optic chiasm by retinal ganglion cell axons. PMID- 7541557 TI - In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium. AB - Many proteins are associated with the outer layer of the cell membrane through a posttranslationally added glycosyl phosphatidylinositol (GPI) anchor. The functional significance of this type of protein linkage is unclear, although it results in increased lateral mobility, sorting to the apical surface of the cell, reinsertion into cell membranes, and possibly cell signaling. Here evidence is presented that GPI-linked proteins can undergo intermembrane transfer in vivo. GPI-linked proteins expressed on the surface of transgenic mouse red blood cells were transferred in a functional form to endothelial cells in vivo. This feature of GPI linkage may be potentially useful for the delivery of therapeutic proteins to vascular endothelium. PMID- 7541560 TI - Neuropeptide changes in compressed spinal nerve roots. AB - STUDY DESIGN: Compression-induced changes in the concentration of substance P and VIP (vasoactive intestinal polypeptide), in spinal nerve roots and dorsal root ganglia were studied in an experimental nerve root compression model in pigs. OBJECTIVES: To analyze by radioimmunoassay the concentration of the neuropeptides substance P and VIP in a model for experimental chronic nerve root compression. SUMMARY OF BACKGROUND DATA: Neuropeptides such as substance P and VIP seem to be involved in the transmission of pain and changes in the levels of these neuropeptides have been described in models where peripheral or spinal nerve injury was induced. METHODS: An ameroid constrictor was applied on a spinal nerve root just cranial to the dorsal root ganglion. The inner diameter of this constrictor is gradually reduced. After 1 or 4 weeks, tissue samples were taken from the nerve root cranial to the constrictor and from the dorsal root ganglion for measurement of substance P and VIP concentrations. RESULTS: There was a statistically significant increase in substance P concentrations in the compressed dorsal root ganglia when compared to the noncompressed dorsal root ganglia at both 1 and 4 weeks. Substance P concentration was also significantly increased in the nerve root after 1 but not after 4 weeks. The VIP levels were not significantly changed in either tissue. CONCLUSIONS: The results of the study indicates an increase in substance P levels in the dorsal root ganglion (after 1 and 4 weeks) and in the nerve root (after 1 week) in a model for chronic nerve root compression in pigs. There were no significant differences in the VIP concentrations. The study thus indicates that changes in substance P are related to experimental chronic nerve root compression. PMID- 7541559 TI - Anti-GPIIb/IIIa antibodies: powerful tools to investigate function and regulation of an integrin. PMID- 7541556 TI - Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. AB - A 95-kilodalton mouse sperm protein with characteristics of a protein tyrosine kinase has been identified as a receptor for ZP3, a glycoprotein in the egg's extracellular matrix. The structure of the human homolog was determined by screening an expression library from human testis; a testis-specific complementary DNA was isolated that encodes a protein similar to receptor tyrosine kinases and appears to be expressed only in testicular germ cells. Antibodies against a synthetic peptide from the intracellular domain recognized a 95-kilodalton human sperm protein that contains phosphotyrosine; human ZP3 stimulates the kinase activity of this sperm protein. Synthetic peptides corresponding to regions of the predicted extracellular domain inhibited sperm binding to human zona pellucida. Availability of the primary sequence of a receptor for ZP3 provides a rational starting point for sperm-targeted contraceptive development. PMID- 7541562 TI - Modulation of APRT transcription by altering spacing between cis-regulatory elements. AB - Three regulatory regions on the promoter of CHO adenine phosphoribosyltransferase gene (APRT) were identified. Spacing constraints between these regions were analyzed. With normal spacing, region II (-33 to +19), which separates regions I (-101 to -53) and III (+56 to +85), is critical for APRT transcription. However, when regions I and III are artificially placed in proximity to each other (region II deleted), they are able to drive transcription as efficiently as the wild-type APRT promoter. Neither region I nor III alone is sufficient for efficient transcription. As the spacing between the two regions increases, the transcription decreases. Region I may activate transcription in two ways: through a stringent sequence-specific manner (as in the transcription mediated by regions I and III) and through a manner with relaxed requirement for sequence specificity (as in the transcription from the wild-type APRT promoter). PMID- 7541561 TI - Endogenous and transfected mouse alpha-fetoprotein genes in undifferentiated F9 cells are activated in transient heterokaryons. AB - Mouse F9 teratocarcinoma cells provide a system to study developmentally regulated alpha-fetoprotein (AFP) gene expression. AFP is not expressed in undifferentiated F9 cells but is induced when cells differentiate as cell aggregates in the presence of retinoic acid. Previous studies have led to the suggestion that undifferentiated F9 cells contain negative regulators of AFP expression. To test this, we have used transient heterokaryons to ask whether inactive AFP genes in undifferentiated F9 cells are responsive to positively acting trans-acting factors. Our results indicate that silent endogenous and transfected AFP genes are activated when undifferentiated F9 cells are fused to human hepatoma HepG2 cells. This suggests that the lack of AFP expression in undifferentiated F9 cells is due to the absence or insufficient level of positive acting transcription factors, rather than the presence of dominant negative regulators. We also demonstrate that stably transfected AFP genes, although unmethylated, are properly regulated in F9 cells. PMID- 7541563 TI - Characterization of stem cells in human airway capable of reconstituting a fully differentiated bronchial epithelium. AB - The epithelia of the lung are complex structures that play an important role in normal lung physiology and are often involved in the pathophysiology of pulmonary diseases. The dynamics of cell turnover, lineage, and differentiation within these epithelia are complex and poorly understood. We have coupled the technique of retrovirus-mediated gene transfer with a xenograft model of proximal human airway to evaluate pathways of cellular proliferation and differentiation in human bronchial epithelia. Primary isolates of human bronchial epithelial cells (HBECs) were infected with mixtures of recombinant retroviruses expressing different reporter genes and seeded into denuded rat trachea, which were implanted subcutaneously into athymic mice. The HBECs were allowed to regenerate for four weeks in xenografts, which were then explanted. Clonal expansion of individual retrovirus-marked cells in the regenerated human bronchial epithelium was detected as clusters of transgene-expressing cells. Clone size varied with seeding density, resulting in the largest clones comprising 10(3)-10(4) cells. A substantial number of clones showed transgene expression in basal as well as differentiated columnar cells, a finding that appeared independent of clone size. These studies demonstrate the existence of a cell type within the human bronchial epithelium that is capable of extensive self-renewal and pluripotent development. Further characterization of these potential stem cells will be important in defining pathogenesis of pulmonary diseases and in developing novel approaches to treatment such as gene therapy. PMID- 7541565 TI - [Benign prostatic hyperplasia: anamnesis and indications for surgery]. AB - The benign prostatic hyperplasia is a very common disease we are confronted with in the urological clinical practice. An exact anamnesis, a careful interpretation and evaluation of the symptoms and of the (initial) findings are indispensable for an obstructive prostatic hyperplasia to be diagnosed. The diagnostic effort to make an appropriate diagnosis considering as well the pathological process exists mainly of noninvasive methods. Though once the operation is indicated, the spectrum of exploration must be enlarged. Provided that the prostatic hyperplasia is identified as being the clear reason for miction difficulties, an adequate therapy may be applied. PMID- 7541564 TI - Peripherin gene is linked to keratin 18 gene on human chromosome 12. AB - Peripherin is a neuron-specific intermediate filament (IF) protein, found primarily in phylogenetically old regions of the nervous system. Whereas other neuronal IF genes have only two to three introns and are scattered in the genome, the peripherin gene (PRPH) has a complex intron-exon structure like nonneuronal IF genes that are clustered in tandem arrays, e.g., those encoding the keratins. We used a cosmid containing the human peripherin gene (PRPH) to determine its chromosomal location in relationship to nonneuronal IF genes. Using a rodent human mapping panel, we localized the PRPH gene to human chromosome 12. Since a cluster of keratin genes maps to 12q12-13, polymorphic markers were developed for PRPH and for one of the keratin genes presumed to be in the cluster, keratin 18 (KRT18). Both markers were typed in CEPH reference families. Pairwise and multipoint analyses of the CEPH data revealed that KRT18 is tightly linked to DNA markers D12S4, D12S22, D12S90, D12S96 and D12S103, which lie between D12S18 and D12S8, with odds greater than 1000:1. These markers are physically located at 12q11-13, thus supporting the fine localization of KRT18 in or near the group of type II keratins in this region. Furthermore, linkage analysis showed that the peripherin gene (PRPH) is tightly linked to KRT18 (Z = 15.73, theta = 0.013), and therefore appears to be in close proximity to the cluster. PMID- 7541567 TI - [Benign prostatic hyperplasia: prostatectomy and alternatives]. AB - Benign prostatic hyperplasia is a common disease of men and will lead in most cases to micturition difficulties. Up to now 2 or 4 of 10 men in their sixties are operated for BPH. As standard treatment we know the transurethral resection of the prostate and in some cases of very big adenomas the open adenomectomy carried out in a suprapubic or retropubic way. However, in view of a rare, but as a result of these treatments appearing morbidity we have to ask ourselves whether the surgical resection of the obstructive prostatic tissue remains the only way of treatment. For several years now apart from drug therapy there have also been applied less invasive alternatives. Whether these alternatives of little side effect and further complications are equally effective for the treatment of the obstructive BPH is to verify. This paper provides a general view of the possibilities of the surgical interventions as well as of their alternatives. Because of a lack of dates as to the efficiency of particular procedures a final judgement is outstanding in most cases. Where possible we hear of particular alternatives, though fully promoted, being nothing but a passing tendency. This is certainly true of the hyperthermy, the balloon dilatation and the urethral implants. These by now various conspicuous necrotising procedures (transurethral microwave thermotherapy, laser-ablation, high intensity focused ultrasound, transurethral needle-ablation) are by now in prospective random studies still subject to extensive clinical tests. The question whether one of these alternatives will one day be apt to exceed the TURP as "golden standard" of BPH treatment, that remains as far as now the question to be put. The advantages of the necrotising procedures, at present highly recommended, consist in the first place of a rare intra- and postoperative morbidity with a simultaneous lack of mortality. These procedures can be performed as a outpatient or as a short stationary treatment and sometime even under local anaesthesia.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541568 TI - [Screening of prostate carcinoma]. AB - Taking into consideration the height costs of screening healthy men from general population at risk for prostate cancer, we do not recommend prostate cancer screening except for scientific reasons in prospective random trials. To diagnose prostate cancer we know the following methods for patients with prostatic problems: DRE = (digital rectal examination) PSA = (prostate-specific antigen) TRUS = (transrectal ultrasonography) The possible validity of DRE, PSA and TRUS are discussed. The best and most economic way of diagnosing prostate cancer is a combination of DRE and PSA. PMID- 7541566 TI - [Pharmacotherapy in benign prostatic hyperplasia]. AB - To avoid an operation, there are innumerable pharmaceutics on sale for the treatment of the benign prostatic hyperplasia. For this reason we have to distinguish between pharmaceutics without effect and those with proved action on the hyperplastic metaplasia. The symptoms can often be lessened at the initial stage by way of drugs with ineffective action (phytopharmaceutics); there could not yet be proved any effect on the biology of the prostatic hyperplasia. We make use of this therapeutic at the initial stage. Some of the substances with a real effect (alpha-receptor blockers, alpha-reductase inhibitors) do not necessarily provide an effective result in all cases. In addition to that, these therapeutics of partially serious side effects have to be taken for life. Here immediately arises the question of how it could be managed financially. PMID- 7541570 TI - [Radiotherapy in prostatic carcinoma]. AB - Treatment of patients with prostate cancer has become one of the most frequent indications in radiation oncology. Reasons for this fact may be the increasing number of elderly patients, early diagnosis and urologists who are familiar with the possibility of tumor control by radiation. The treatment results and side effects of modern techniques are presented. Many questions concerning treatment policy remain unanswered. Due to the long natural history of prostate cancer we will have to endorse clinical trials and wait many years for their results. PMID- 7541569 TI - [Possibilities of surgical therapy in prostatic carcinoma]. AB - Radical surgical treatment of localized prostate cancer is performed to heal the patient of his malignant disease. Surgery for advanced prostate cancer, e.g. transurethral resection for obstruction or hemorrhage, is always a palliative modality and has to be seen in the context of systemic treatment. Radical prostatectomy is a major intervention which allows complete removal of the prostate gland and the seminal vesicles at the same time. Due to resection at the distal end of the prostatic urethra, the bladder sphincter system is partially destroyed. In general, to guarantee radicality, the erectile nerves and vessels are sectioned, resulting in erectile impotency. Preservation of these structures to maintain potency is indicated only exceptionally. Radical prostatectomy is today a routine operation with minimal mortality (0.5-1%) and minor complication rate. Average hospitalization is about 10 days. Long-term postoperative morbidity is characterized by urinary incontinence and erectile impotency. Incontinence takes a long time to heal. A certain number of patients will keep a generally not very bothersome stress dribbling; however, some may show complete leakage, necessitating an anti-incontinence operation. For an indication of radical prostatectomy, two essential factors have to be considered: the usually extremely slow growth of prostate cancer and the high prevalence of clinically insignificant latent carcinomas. It is for these reasons and for the consequences on live quality that radical prostatectomy should not be performed on patients with a life expectancy of less than 10 years. PMID- 7541571 TI - [Possibilities in drug therapy in prostatic carcinoma]. AB - At the time of diagnosis, about two thirds of all prostate cancers are not organ confined anymore and, thus, are not eligible for local therapy with curative intention. Drug treatment of these tumors is purely palliative; so far, no survival advantage could be demonstrated. Therefore, the goal of systemic therapy is prevention or palliation of complications such as obstructive symptoms and pain. The first step in drug therapy of advanced prostate cancer is always withdrawal of male sex hormones; 70 to 80% of the patients respond favorably. Orchiectomy and LH-RH analogues are considered equal with regard to effectiveness and side effects. However, the surgical approach is less expensive and does not depend on the patient's compliance. Estrogens are rarely used anymore, because they can cause cardiovascular complications. In asymptomatic patients, the question remains to be answered if androgen withdrawal should be performed immediately at the time of diagnosis or delayed in case of possible symptoms. Antiandrogen agents block directly the androgen receptors in the prostatic cell. However, monotherapy with antiandrogens is not yet an established procedure. Instead, since 5% of circulating androgens are of adrenal origin, antiandrogens are combined with either orchiectomy or LH-RH analogues for total androgen suppression. The benefit of maximal androgen ablation is a highly controversial issue nowadays. In most cases, tumor progression after hormonal therapy is due to hormone-refractory cells. Cytotoxic chemotherapy is largely ineffective in treating prostate cancer. Commonly used chemotherapeutic substances lead to temporary remission in 10 to 20% of patients at most. PMID- 7541573 TI - Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. AB - BACKGROUND AND PURPOSE: The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat. METHODS: The MCA of male Wistar rats (n = 24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n = 4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls for apoptosis detection. RESULTS: Within 5-microns-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, > 95% neurons) were primarily localized to the inner boundary zone of the infarct. CONCLUSIONS: A range of mild to severe ischemia reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis. PMID- 7541574 TI - Delayed antagonism of calpain reduces excitotoxicity in cultured neurons. AB - BACKGROUND AND PURPOSE: Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment. METHODS: Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval. RESULTS: The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170. This inhibitor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E-64, also blocked the proteolysis and toxicity produced by kainate in cerebellar neurons. Blocking nitric oxide synthase activity after 1 hour with the antagonist NG-nitro-L arginine was also protective of cerebellar and hippocampal neurons, as was the combination of MDL-28170 and NG-nitro-L-arginine. CONCLUSIONS: The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutamate receptor stimulation, and blocking these postreceptor mechanisms can be effective in protecting neurons from excitotoxicity at delayed time points. PMID- 7541572 TI - A randomized, double-blind, placebo-controlled pilot trial of intravenous magnesium sulfate in acute stroke. AB - BACKGROUND AND PURPOSE: Magnesium ions act as endogenous vasodilators of the cerebral circulation and act pharmacologically as noncompetitive antagonists of the N-methyl-D-aspartate receptor by virtue of their role as endogenous voltage sensitive blockers of the ion channel. The preclinical efficacy of magnesium has been demonstrated in standard models of stroke. METHODS: Sixty patients were randomized to magnesium sulfate (8 mmol IV over 15 minutes and 65 mmol over 24 hours) or placebo within 12 hours of clinically diagnosed middle cerebral artery stroke. Pulse, blood pressure, and serum magnesium levels were monitored. Primary outcome was death or significant functional impairment (Barthel Index score < 60) at 3 months. RESULTS: Magnesium was well tolerated, with no significant adverse effects and no change in blood pressure or pulse rate. Laboratory and electrocardiographic variables did not differ significantly between placebo- and magnesium-treated groups. Serum magnesium rose from 0.76 mmol/L to 1.42 mmol/L over 24 hours and remained significantly higher than in the placebo group at 48 hours. Thirty percent of magnesium-treated and 40% of placebo-treated patients were dead or disabled (Barthel Index score < 60) at 3 months (P = .42). There was a decrease in the number of early deaths in the magnesium-treated group (P = .066, log-rank test). CONCLUSIONS: Magnesium sulfate is well tolerated after acute stroke and has no deleterious hemodynamic effects at this dose. Further trials are required to determine efficacy. PMID- 7541576 TI - Circulating adhesion molecules during kidney allograft rejection. AB - Adhesion molecules appear on leukocytes and endothelial cells mediating the localization and migration of leukocytes to sites of inflammation. Rejecting kidney grafts have shown an increased expression of these molecules. Recent reports have detected in serum soluble forms of adhesion molecules that could play a role in regulating inflammation. We have measured by ELISA the circulating serum levels of ICAM-1, VCAM-1 and E-selectin in: 23 controls, 33 chronic renal failure patients (CRF), 20 hemodialysis patients (HD), 17 samples from 6 patients with stable kidney graft function (STx), 25 samples from 8 patients with steroid responsive rejection proven by biopsy, and 28 samples from 9 patients with steroid-resistant rejection and good response to OKT3. There was not a rise in cICAM-1 or cE-selectin levels during rejection compared with the steady phase before and after rejection. In the case of cVCAM-1, only the OKT3 group showed increased rejection levels (P < 0.05) that were maintained after rejection. For ICAM-1, CRF and HD groups had higher levels than the remaining groups. cVCAM-1 levels were elevated in all groups when compared with control, furthermore, OKT3 and HD groups had higher levels than the STx, CRF, or steroid-responsive groups. For cE-selectin, we only found differences between the CRF and both rejection groups. Serum creatinine correlated significantly with c-ICAM-1 and cVCAM-1 R = 0.30 and R = 0.22), but not with cE-selectin. We conclude that soluble adhesion molecules levels are not valuable markers for rejection. Patients with chronic renal failure have increased levels of adhesion molecules, which could reflect an impaired elimination. PMID- 7541575 TI - De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients. AB - Hepatitis C virus (HCV) is the leading cause of non-A, non-B hepatitis among renal allograft recipients. We sought to identify and describe a proteinuric renal disease occurring in our HCV-infected renal transplant patients. Patients with proteinuria exceeding 1 g/day were identified from a cohort of 98 HCV infected kidney recipients. Qualitative and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism of the amplified RT-PCR product was performed to detect circulating HCV RNA, viral titer, and strain type, respectively. An immune complex nephritis (ICN) of the membranoproliferative pattern (MPGN) was found on five of eight biopsies. Two patients infected with the Hutch strain-type developed nephrotic-range proteinuria within three months posttransplant while the remaining three MPGN patients had been transplanted greater than 5 years prior to the onset of proteinuria. Testing for rheumatoid factors, cryoglobulins, hypocomplementemia, and circulating immune complexes failed to show a consistent pattern. Sucrose density gradient (SDG) equilibrium centrifugation was used to determine the buoyant-density of HCV virions from control (HCV-infected nonproteinuric recipients; n = 5) and nephrotic patients (n = 5). Whereas HCV virions from the control patients had a low buoyant density on sucrose gradients, a substantial percentage of the circulating HCV RNA from the MPGN patients was present in the high-density fractions in association with IgM and IgG. Treatment of the pooled high-density layers with NP40 followed by recentrifugation resulted in a shift of the HCV RNA to the medium-density layers. In conclusion, MPGN developed in five HCV-infected kidney recipients despite pharmacologic immunosuppression. Both the physicochemical properties of the HCV virions on SDG and their association with IgG and IgM in the high-density layers provide indirect evidence for the presence of circulating complexes of anti-HCV antibody and HCV antigen(s). PMID- 7541578 TI - No evidence of an influence of the noninherited maternal HLA antigens on the alloreactive T cell repertoire in healthy individuals. AB - In order to test whether a selective T cell nonresponsiveness to noninherited maternal human leukocyte antigens (NIM) exists, we measured the frequencies of alloreactive T cells of healthy individuals to their NIM HLA antigens and to their noninherited paternal (NIP) HLA antigens by limiting dilution assays. Both the frequencies of cytotoxic T cell precursors (CTLp) and IL-2-producing helper T cell precursors (HTLp) were determined. Similar frequencies were observed for NIM class I-reactive CTLp and NIP class I-reactive CTLp. This was the case when frequencies were determined against total NIM and NIP haplotypes but also when CTLp frequencies against individual NIM and NIP antigens were measured. A positive finding of this study was the confirmation of our earlier observation that CTLp frequencies against individual HLA-A antigens are generally lower than CTLp frequencies against HLA-B. This was found both for the maternal and the paternal HLA-A and -B antigens. Similarly, comparable frequencies of IL-2 producing helper T cell precursors directed against NIM HLA class II antigens and NIP HLA class II antigens were found. When breast-feeding in the neonatal period was considered, no differences in the frequencies of CTLp and HTLp were observed between children who had been breast-fed and children who had not. Therefore the present data do not support the hypothesis that confrontation with noninherited maternal HLA in neonatal life leads to down-regulation of alloreactive T cell responses to the mother. PMID- 7541580 TI - 1995 Receptor and ion channel nomenclature supplement. PMID- 7541577 TI - Implication of cyclosporine in up-regulation of Bcl-2 expression and maintenance of CD8 lymphocytosis in cytomegalovirus-infected allograft recipients. AB - T cell homeostasis and CD4/CD8 ratios are normally reestablished by apoptotic clearance of activated T cells after immune stimulation. In allograft recipients with cytomegalovirus infection, CD8 lymphocytosis persists after negativation of viral cultures, contrary to immunocompetent hosts. We investigated the expression of Bcl-2 protein, an intracellular suppressor of apoptosis, and of CD95 (APO 1/Fas), a membrane inducer of apoptosis, in peripheral blood lymphocytes from 45 solid organ recipients. During the viremic phase of CMV infection, we found absence or diminished expression of Bcl-2 protein and increased expression of CD95 antigen in activated CD8+ T cells. Opposite evolution of these molecular regulators of apoptosis was reflected by the presence of 10-25% of apoptotic lymphocytes with fragmented DNA, as shown by both in situ nick translation and electrophoresis. Normalization of Bcl-2 expression was progressive over several months but still lower than in uninfected allograft recipients. These results suggest that the initial evolution of CMV infection in allograft recipients resembles acute viral infection in immunocompetent hosts. Conversely, we showed that overexpression of Bcl-2 protein in lymphocytes from uninfected allograft recipients, and culture of unstimulated normal lymphocytes with 0.5 micrograms/ml cyclosporine led to an increase in the expression of intracellular Bcl-2. This up regulation of Bcl-2 protein by cyclosporine suggests the acquisition of resistance to apoptosis. Thus, the reversion of balance between T cell death and survival after acute CMV infection might be impeded by cyclosporine. Combination of CMV latent infection and cyclosporine therapy appears therefore critical to shift the homeostatic maintenance of the peripheral lymphocyte compartment toward persistingly high numbers of CD8+ T cells. PMID- 7541579 TI - The successful use of tacrolimus (FK506) in a pancreas/kidney transplant recipient with recurrent cyclosporine-associated hemolytic uremic syndrome. PMID- 7541581 TI - Primary squamous cell carcinoma of the prostate after radiation seed implantation for adenocarcinoma. AB - We describe a 67-year-old man with a history of Stage B1 adenocarcinoma of the prostate who was treated with iodine-125 implantation in 1985. His prostate specific antigen values subsequently increased in conjunction with an intraprostatic recurrence. Biopsy revealed primary squamous cell carcinoma arising in the prostate; no adenocarcinomatous elements were noted. PMID- 7541582 TI - Reinventing the ball. PMID- 7541584 TI - The outcome of suprapubic prostatectomy: a contemporary series in the developing world. AB - OBJECTIVES: To assess the appropriateness of the technique of suprapubic prostatectomy using a removable bladder neck partition suture for use in a developing world hospital and to provide contemporary open prostatectomy outcome data currently lacking in the world's literature. METHODS: From 1984 to 1994, 240 consecutive patients presenting to a developing world hospital with acute urinary retention underwent suprapubic prostatectomy using a removable bladder neck partition suture. The average length of time from bladder decompression until operation was 2.5 months. The outcome of these cases was retrospectively analyzed. RESULTS: The overall early complication rate was 19.6%. There were no deaths. The transfusion rate was 4.6%. Clot retention occurred in 6.7%, and 2.9% required return to the operating room for evaluation. For the second half of the series, the early complication rate decreased to 8.3%, the clot retention rate to 0.8%, and the transfusion rate to 1.7%. Other early and late complications were minimal. The length of delay from decompression until operation did not affect outcome. CONCLUSIONS: The technique of suprapubic prostatectomy using a removable bladder neck partition suture is appropriate for use in developing world hospitals because of its low morbidity and mortality rates. The outcome in this contemporary series of open prostatectomy cases compares favorably with the outcome from reported contemporary transurethral resection of the prostate (TURP) series. These data demonstrate that suprapubic prostatectomy is an acceptable option when the patient's anatomy or the state of local medical facilities precludes TURP. PMID- 7541583 TI - Should Medicare provide reimbursement for prostate-specific antigen testing for early detection of prostate cancer? Part I: Framing the debate. PMID- 7541585 TI - Temporal (circadian) and functional relationship between prostate-specific antigen and testosterone in healthy men. AB - OBJECTIVES: To study the circadian relationship between serum prostate-specific antigen (PSA) and total testosterone in men without clinically evident prostate disease. METHODS: Blood samples were collected every 3 hours for 24 hours (eight per subject) from 11 clinically healthy men, ages 46 to 72 years. PSA was also monitored once a week for 6 weeks in 16 additional healthy men. PSA, testosterone, and age were correlated by linear regression, and 3-hourly PSA and testosterone values normalized to percent of individual mean were analyzed for circadian rhythm by the least squares fit of a 24-hour cosine. RESULTS: Mean PSA correlated positively (P < 0.001) and testosterone correlated negatively (P = 0.014) with age and inversely with each other (P < 0.001). The mean circadian range of change (ROC) from lowest to highest values for PSA was 0.37 +/- 0.07 ng/mL (28 +/- 9%), and for testosterone it was 202 +/- 23 ng/dL (53 +/- 7%). The mean ROC over 6 weeks was 0.32 +/- 0.04 ng/mL. A significant circadian rhythm was found for PSA (P = 0.011, amplitude = 5.4 +/- 1.8%, acrophase = 5:02 AM; 95% limits, 2:40 to 7:24 PM) and testosterone (P < 0.001, amplitude = 9.4 +/- 1.8%, acrophase = 8:38 AM; 95% limits, 7:12 to 10:04 AM). CONCLUSIONS: The temporal relationship between circadian rhythms in PSA and testosterone suggests different physiologic states over the 24 hours, which may be of chronopharmacologic interest with regard to dosing time of drugs or hormonal treatments intended to affect prostate growth and function. Within-day variation in PSA is of little diagnostic significance and does not prevent accurate clinical classification when a single specimen is used. PMID- 7541586 TI - Age-specific reference ranges for serum prostate-specific antigen. AB - OBJECTIVES: To assess the relationship of the distribution of serum prostate specific antigen (PSA) to age in a population of subjects for whom PSA levels were determined as part of a health fair. METHODS: Between March 19 and March 28, 1993, 1716 men aged 40 to 79 years from eastern Nebraska who participated in "The Health Fair of the Midlands" provided blood for serum PSA determination. RESULTS: Serum PSA concentration was correlated with patient age, with the observed 95th percentile increasing from 1.5 ng/mL for subjects 40 to 44 years of age to 7.7 ng/mL for subjects 75 to 79 years of age. Variability in serum PSA concentrations increased with increasing age. Taking this heteroscedasticity into account provided the following upper limits of normal (95th percentiles) for serum PSA: age 40 to 49, 1.5; age 50 to 59, 2.6; age 60 to 69, 4.4; age 70 to 79, 7.5. CONCLUSIONS: Previously published age-specific reference ranges did not consider the increasing variability of PSA concentration with age and have upper limits of normal that are too high for subjects under age 60 and may be too low for subjects aged 70 to 79. Upper reference ranges of 1.5 ng/mL for subjects aged 40 to 49, 2.5 ng/mL for subjects aged 50 to 59, 4.5 ng/mL for subjects aged 60 to 69, and 7.5 ng/mL for subjects aged 70 to 79 years provide specificity near 95%. PMID- 7541587 TI - Physiologic variations of serum testosterone within the normal range do not affect serum prostate-specific antigen. AB - OBJECTIVES: To determine the relationship between endogenous total serum testosterone levels and serum prostate-specific antigen (PSA) concentrations. If a correlation exists between these two parameters, then use of testosterone specific reference ranges may enhance the utility of PSA as a marker for prostate cancer. METHODS: Data were obtained from 150 men without previous history of prostate cancer. PSA was measured by the Abbott IMX microparticle enzyme immunoassay and total testosterone determined by the Coat-A-Count radioimmunoassay. RESULTS: No correlation was found between testosterone and PSA, even when corrected for age and weight. CONCLUSIONS: The data suggest that determination of the total serum testosterone level does not improve the sensitivity or specificity of PSA as a tumor marker. PMID- 7541588 TI - The correlation between serum prostate-specific antigen and prostate cancer is not influenced by the serum testosterone concentration. AB - OBJECTIVES: To determine if the serum testosterone (T) concentration influences the ability of prostate-specific antigen (PSA) to predict prostate cancer volume and stage. METHODS: One hundred consecutive patients with clinically localized prostate cancer who underwent radical prostatectomy were examined prospectively. Each patient was evaluated preoperatively with a serum PSA, total T, free T, and percent free T. All surgical specimens were evaluated using the whole mount, step section technique for Gleason score, tumor volume, and extraprostatic disease. RESULTS: Serum total T, free T, and percent free T did not correlate with the serum PSA level (r = .03, .08, and .07, respectively), tumor volume (r = .11, .08, and .11, respectively), prostate weight (r = .00, -.08, and .11, respectively), or Gleason score (r = .11, .08, and .11, respectively). Serum PSA correlated with tumor volume (r = .51, P < 0.0001). Extraprostatic disease was significantly associated with a higher percent free T value (r = .26, P = 0.02) but not with either the total or the free T level. Linear regression analysis showed that neither the total nor the free T concentration was a significant predictor of extraprostatic disease in the presence of PSA (P = 0.30 and 0.24, respectively); percent free T contributed only slightly to PSA in the prediction of extraprostatic disease (P = 0.05). However, neither total T, free T, nor percent free T was a significant predictor of tumor volume; in essence, the association between PSA and tumor volume was independent of the serum T concentration (P = 0.30, 0.24, and 0.60, respectively). CONCLUSIONS: Serum total T, free T, and percent free T values do not enhance the ability of PSA to predict the tumor volume or pathologic stage in patients with clinically localized prostate cancer. PMID- 7541591 TI - Disease outbreaks in pigs in Great Britain due to an influenza A virus of H1N2 subtype. PMID- 7541589 TI - Serum prostate-specific antigen and the biologic progression of prostate cancer. AB - OBJECTIVES: We have previously shown that serum prostate-specific antigen (PSA) is proportional to prostate cancer volume and that progression of prostate cancer is proportional to volume, but other investigators have not found serum PSA to be as useful in predicting pathologic stage at the time of radical prostatectomy. Because our series is the only study to examine prospectively all radical specimens at 3-mm intervals, we have examined the relationship between serum PSA and the morphologic indicators of cancer progression in our first 350 radical prostatectomies. METHODS: Preoperative serum PSA level was tabulated in 350 consecutive patients with prostate adenocarcinoma and compared with morphologic variables in the radical prostatectomy specimen. Morphologic variables included cancer volume, histologic grade, capsular penetration, seminal vesicle invasion, and lymph node metastasis. RESULTS: Serum PSA showed strong correlation with all morphologic variables, which were highly intercorrelated. Serum PSA level was strongly correlated with cancer volume, histologic grade, and frequency of regional spread to lymph nodes. Close intercorrelations found between all variables were translated into a scale relating each level of serum PSA elevation to stage of disease in morphologic terms. Using this scale, serum PSA level can contribute to patient evaluation and treatment decisions in men with prostate cancer. CONCLUSIONS: Serum PSA is primarily determined by prostate cancer volume and secondarily by the percentage of high-grade cancer (Gleason grades 4 and 5) in the prostate. Because of this basic relationship, serum levels of PSA provide a clinically useful estimate of morphologic findings in the prostate. Serial PSA determinations should reflect the growth of the cancer as well as the gradual evolution of more malignant cells with the passage of time. The use of a serum PSA-based rating scale can contribute to patient evaluation and treatment decisions in men with prostate cancer. PMID- 7541590 TI - P-selectin- and CD18-mediated recruitment of canine neutrophils under conditions of shear stress. AB - Neutrophil mobilization at sites of inflammation or thrombosis involves the participation of several adhesion molecules expressed on neutrophils and vascular endothelial cells. Local vascular damage with disruption of the endothelium results in adhesion of platelets to the exposed subendothelium, and these platelets could also participate in neutrophil recruitment. This initial phase of mobilization could be followed by heterotypic aggregation to recruit more leukocytes in the area. The present study first examined the interactions of adherent canine platelets and flowing canine neutrophils using an in vitro system that simulates vascular flow conditions. Results showed that collagen-adherent platelets express the adhesion molecule P-selectin on their surface and can support neutrophil arrest (612 +/- 43 neutrophils/mm2) at shear stresses of approximately 2.5 dynes/cm2. Both transient adhesion (manifested by a rolling type behavior) and complete arrest were observed. These interactions could be totally inhibited by a monoclonal antibody directed against platelet P-selectin (24 +/- 18 neutrophils/mm2) but not by a monoclonal antibody against neutrophil CD18 (625 +/- 46 neutrophils/mm2). Additionally, under shear mixing conditions (700 RPM), canine blood leukocytes exhibited aggregation (> 80% singlets recruited into aggregates after 5 minutes), and this process does not involve P selectin but is dependent on the neutrophil integrin CD18. However, stimulation of the blood with platelet-activating factor (5-20 ng/ml) induced a rapid aggregation with a significantly greater number of aggregates when compared with stirring alone (68.3% +/- 3.2% versus 35.2% +/- 6.3% at 1 minute, P < 0.05), and this aggregation was both P-selectin and CD18 dependent. Overall, these two mechanisms of leukocyte recruitment (neutrophil arrest on adherent platelets and aggregation) could act sequentially and in a cooperative manner to bring into close contact platelets and neutrophils at sites of inflammation and thrombosis in pathologic conditions in the dog. PMID- 7541592 TI - Characterization of ovine lentivirus envelope glycoprotein expressed in Escherichia coli cell and baculovirus systems. AB - The ovine lentivirus (OLV) envelope protein NH2- and COOH-terminal subunits gp70 and the NH2-terminal subunit gp40 were expressed in Escherichia coli cell. The entire gp70 envelope protein was also expressed in insect cells by the recombinant baculovirus. Guinea pigs were immunized with each bacterially expressed recombinant protein, and a serum neutralization assay was used to determine their capacity to neutralize OLV. These results showed that the major neutralization epitopes are located in the NH2-terminal half of the gp70. The baculovirus expressed gp70 was found on the surface of insect cells and was immunobiologically active. Virus neutralization activity was also produced in sheep immunized with the baculovirus expressed recombinant protein. PMID- 7541594 TI - Andrological implications of visual laser ablation of prostate (VLAP). AB - 300 VLAP procedures are reported with no mortality and very little morbidity. The results are comparable to TURP with lower incidence of impotence, preservation of ejaculation, little blood loss, less chance of scarring and higher acceptance of the public undergo a laser procedure. PMID- 7541593 TI - [The immunological and cytogenetic changes in patients with pulmonary tuberculosis]. AB - The performed studies showed that patients with active phase pulmonary tuberculosis have their T-system function suppressed, that of B-system activated, this phenomenon being the most marked in those patients with fibrocavernous tuberculosis. Increase in the activity of phagocytes was more common in patients with infiltrative tuberculosis. The immunity system state in subjects with non active post tuberculous alterations did not depart from normal values. DNA and RNA concentrations were decreased in those patients having more pronounced immunologic disturbances (infiltrative and fibrocavernous tuberculosis), reparative capacity of RNA being altered only in patients with focal tuberculosis. Strong correlation was established between immunologic and cytogenetic indices. PMID- 7541596 TI - Is MRI helpful in evaluating the response of cervical cancer to neoadjuvant chemotherapy? AB - BACKGROUND: To assess the potential role of magnetic resonance imaging (MRI) in evaluating the response of cervical tumor to neoadjuvant chemotherapy. METHODS: We studied 14 patients with locally advanced carcinoma of the cervix. MRI was performed before and after each cycle. All patients underwent laparotomy four weeks after the last cycle. MR images after chemotherapy were compared with surgical-pathologic findings. RESULTS: Related to parametrial state after chemotherapy, surgical examination correlated with MRI in nine patients (nine true negative). In three cases parametrial invasion was over-estimated (three false-positive) and in two it was underestimated, so that tumor resection was not possible once laparotomy was performed (two false-negative). MRI estimation of tumor size was confirmed at pathologic examination in only eight patients, whilst it was over-estimated in the other four ones. CONCLUSIONS: These initial results suggest that MRI could not be as precise for cervix cancer staging and evaluation of response to chemotherapy as has recently been postulated. This is especially important since a false negative result may induce the surgeon to perform laparotomy in error in cases of unresectable tumors. PMID- 7541599 TI - Long-term treatment of mood disorders in schizophrenia. AB - Mood disorders in schizophrenia are common and are associated with a poor outcome, an increased risk of relapse and a high rate of suicide. Consequently, treatment strategies need to take mood disorders into account. In depressed and actively psychotic schizophrenic and schizoaffective patients, treatment with neuroleptic plus antidepressant may be less effective than neuroleptic alone. However, patients with post-psychotic depression on maintenance neuroleptics respond well to tricyclic antidepressants. Mood disorders can be caused by neuroleptics and if so will often improve if the dose is reduced or if the drug is changed. Anticholinergics may also help. In schizoaffective disorder, lithium is usually beneficial, especially for patients with classical affective disorder. Carbamazepine may be more effective in patients with schizoaffective and schizophreniform disorders. At doses comparable with those effective in schizophrenia, clozapine may be as good or better than conventional neuroleptics in schizophrenic patients with psychotic mood disorder or schizoaffective disorder. In patients with high BPRS anxiety/depression scores, risperidone (8 mg/day) was more effective than haloperidol (10 mg/day). Risperidone at a mean dose of 8.6 mg/day was also more effective than haloperidol (mean dose 9.2 mg/day) or levomepromazine (methotrimeprazine -- mean dose 125 mg/day) on the Psychotic Anxiety Scale. Mood-related symptoms are therefore amenable to treatment. Risperidone and clozapine appear to be good candidates for the long term treatment of mood disorders in schizophrenia, although long-term, double blind, controlled studies are needed to confirm this. PMID- 7541598 TI - New pharmacotherapeutic modalities for negative symptoms in psychosis. AB - Negative symptoms in schizophrenia comprise a psychopathologic and pathophysiologic syndrome which is absent from normal mental function. Renewed interest in negative symptoms has led to the development of better measuring instruments, among which is the Positive And Negative Syndrome Scale (PANSS), which provides a way of measuring and reporting positive and negative symptoms in a balanced and convenient form. A number of strategies are being investigated for treating negative symptoms. Dopamine agonists such as levodopa, amphetamines and bromocriptine have been shown to produce improvements in negative symptoms, although good, well-controlled clinical trials are lacking. Partial dopamine agonists, such as MAR 327, are also currently under investigation and results are expected soon. Tricyclic, selective serotonin reuptake inhibitors and monoamine oxidase antidepressants appear to be able to modify negative symptoms in schizophrenia, although, once again, carefully designed trials are needed. Modification of GABAergic transmission has shown little promise, but the use of glycine to augment transmission at N-methyl-D-aspartate (NMDA) synapses suggests that the strategy may be beneficial. These results also imply that altered glutamate receptor function may be partly responsible for negative symptoms. One strategy that has been shown to have a beneficial effect against negative symptoms is combined serotonin/dopamine antagonism. Clozapine was found to have this profile after its introduction, and the recently introduced antipsychotic, risperidone was developed intentionally to be a combined 5-HT2/D2 antagonist. Both risperidone and clozapine have been shown to be effective against negative symptoms. One problem associated with the assessment of drug effects on negative symptoms, however, is that drugs can act on both primary and secondary negative symptoms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541597 TI - The negative component in schizophrenia. AB - Many conflicts in the literature about negative symptoms arise because different authors use different definitions of negative symptoms. If narrow definitions are used, it appears that drugs have little effect against negative symptoms, although those who use broader definitions tend to conclude that negative symptoms do respond to drugs. Another complication is that negative symptoms may be secondary to other causes, such as psychotic symptoms, the side effects of drugs, depression and understimulation as a result of hospitalisation. Attempting to measure drug effects on negative symptoms can be complicated by such factors as different co-medication in the test and control groups and different pharmacological profiles of test and standard drugs, in particular in terms of extrapyramidal side effects or antidepressant effects. Several approaches to dissecting the direct effect of drugs from their indirect effects on negative symptoms have included analyses of covariance, regression analyses and path analyses. Applying path analysis to the data from the North American clinical trial of risperidone suggests that a significant component of the advantageous effect of risperidone on negative symptoms, compared with haloperidol, is due to a direct effect that cannot be accounted for by the effect of risperidone on positive symptoms and its superior side-effect profile. Although it is possible to analyse studies retrospectively in this way, the best approach is probably to design future clinical trials so that the effects of drugs on negative symptoms are more easily determined than has been the case in the past. PMID- 7541600 TI - Principal components and further possibilities with the PANSS. AB - At the end of the last century, Hughlings-Jackson suggested that positive and negative syndromes should be kept apart in psychotic disorders. When the concepts of dementia praecox and schizophrenia were introduced by Kraepelin and Bleuler, emphasis was laid on the negative symptoms, regarded as fundamental. After the introduction of the "first rank symptoms" by Schneider emphasis switched to the positive symptoms in schizophrenia and these symptoms were included in most diagnostic criteria. In the 1980s Andreasen and Crow suggested a dichotomy into positive and negative syndromes in schizophrenia. Kay and co-workers introduced a Positive And Negative Syndrome Scale (PANSS) for schizophrenia. In the original studies satisfactory construct validity and inter-rater reliability were demonstrated. However, in studies outside the USA a high construct validity was found for the negative scales but not for the positive and general psychopathology scales. Furthermore, the inter-rater reliability of the negative scale was a problem. After introduction of the Structured Clinical Interview for the PANSS (SCI-PANSS) the inter-rater reliability increased for all three scales. In an early study Kay and Sevy found seven factors in a principal component analysis of the PANSS and suggested a four factor pyramidical model. Later principal component analyses by Lepine, Peralta et al. and Kawasaki et al. suggested that the four factor model was an oversimplification and Lindstrom and von Knorring suggested a five factor pyramidical model. A similar model was later suggested by Bell et al. after a reanalysis of the original series of Kay and Sevy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541595 TI - Expression of mRNA for keratinocyte growth factor and its receptor in human endometrium. AB - BACKGROUND: The aim of the present study was to evaluate the expression of mRNA for the keratinocyte growth factor and the keratinocyte growth factor receptor in human endometrium at different stages of the menstrual cycle. The role of estrogen and progesterone in regulating the expression of the mRNAs encoding keratinocyte growth factor and its receptor was further examined by studying the effect of continuous progestin (endometrium exposed to levonorgestrel releasing intrauterine contraceptive device), and continuous estrogen (endometrium hyperplasia) on the endometrium. METHODS: The expression of mRNA in endometrial samples was evaluated using reverse transcriptase polymerase chain reaction. RESULTS: The expression of KGF mRNA was found to vary during the menstrual cycle, with the highest levels in the progesterone-dominated late-secretory stage endometrium. Keratinocyte growth factor mRNA expression was low in both the endometrium that had been under the influence of continuous progestin (atrophic endometrium) and continuous estrogen (hyperplastic endometrium). The highest level of keratinocyte growth factor receptor mRNA expression was seen in late proliferative stage of the menstrual cycle and in hyperplasia when the estrogen exposure to endometrium is high. A low receptor mRNA level was found in endometrium exposed to continuous progestin. CONCLUSION: The results suggest that keratinocyte growth factor mRNA expression is progesterone dependent, whereas keratinocyte growth factor receptor mRNA expression seems to be more estrogen than progesterone dependent. PMID- 7541601 TI - Managing patients with benign prostatic hyperplasia. AB - Benign prostatic hyperplasia is the usual cause of prostatism--irritative and obstructive urinary symptoms. Steps in making the diagnosis include a focused neurologic examination, urinalysis, serum creatinine determination and, possibly, catheterization to detect urinary retention. It is difficult to predict the likelihood of future complications, such as complete urinary obstruction, even for patients with severe symptoms. The natural course of prostatism is a waxing and waning of symptoms. Treatment options are watchful waiting, medication, transurethral prostatectomy, and newer surgical treatments such as microwave thermopathy and laser ablation. The family physician can counsel patients about the potential side effects of these treatments as well as the problems incurred by simply adjusting to the disabilities associated with benign prostatic hyperplasia. PMID- 7541602 TI - Establishment and characteristics of a gastric cancer cell line (HuGC-OOHIRA) producing high levels of G-CSF, GM-CSF, and IL-6: the presence of autocrine growth control by G-CSF. AB - We successfully established a human gastric cancer cell line, HuGC-OOHIRA, from the ascites of a 60-year-old patient with advanced gastric cancer (poorly differentiated adenocarcinoma) complicated by peritonitis carcinomatosa and leukocytosis of unknown origin. Morphologically, the cells were polygonal and adhered weakly to the culture flask. They tended to pile up upon reaching confluence. Chromosome analysis revealed that the cell line has two modes of chromosome number, namely near diploidy and tetraploidy. Double minutes (DMs) were present in abundance in each cell. The doubling time was 25-30 hr. The cell line was successfully transplanted into nude mice, and their peripheral leukocyte counts increased in proportion to the growth of the tumors. At 2 weeks after the transplantation, the serum rG-CSF level was elevated to 2,893 pg/ml. The concentration of human G-CSF in the culture supernatants was an extraordinary high level of 145,380 pg/ml/day. Secretion of GM-CSF and IL-6 was also detected. The intracellular localization of the G-CSF was identified for the first time by immunofluorescence. Moreover, Northern blot analysis detected G-CSF mRNA in this cell line. Anti-recombinant human G-CSF serum suppressed the propagation of HuGC OOHIRA cell line. Therefore, it is likely that the autocrine growth loop by G-CSF is present in this cell line. This cell line would be very useful for understanding both the cellular and molecular basis for the production of various cytokines such as G-CSF as well as cytokine-dependent tumor proliferation. PMID- 7541605 TI - Use of 2-chlorodeoxyadenosine, granulocyte-colony-stimulating factor, and erythropoietin in a Jehovah's Witness with hairy cell leukemia. PMID- 7541604 TI - Isobutyramide therapy in patients with sickle cell anemia. AB - We have administered Isobutyramide as a suspension over a period of 3 months, from a starting dose of 50 mg/kg/day up to 150 mg/kg/day, to four adult sickle cell (SS) anemia patients. The maximum dose was maintained for 3 weeks. The blood counts remained stable and the Hb F levels decreased slightly. The G gamma levels increased at the end of the trial, suggesting activation of the G gamma gene at the highest dose of Isobutyramide. Three patients showed a stable rate of hemolysis, while in one patient, an increase of lactate dehydrogenase occurred. None of the patients experienced pain crisis or organ-specific crisis, but all four complained about mild epigastric burning and a bitter taste. After the first month of treatment one patient complained about intolerable epigastric discomfort which was relieved by Omeprazole. Another patient complained about increasing dyspepsia in the 12th week leading to the termination of the trial. Oral Isobutyramide administration does not qualify as an effective treatment of SS patients. PMID- 7541603 TI - High grade malignant lymphoma with clinical characteristics and immunophenotype of natural killer cells. AB - The malignant proliferation of natural killer (NK) cells which are morphologically characterized as large granular lymphocytes (LGL) is a well known clinical entity which was named after its morphological appearance as LGL leukemia/lymphoma. Similar to non-malignant NK-cells, these tumors can be divided into those which express the CD3-T-cell receptor complex and those which do not. The CD3-positive type of LGL-leukemia is immunophenotypologically characterized by the expression of CD16, and variably CD 56 and CD 57, and generally follows a more indolent course. In contrast, malignant proliferations of CD3-negative LGL express either CD16 or CD 56, and only occasionally CD 57 on their cell surface. Clinically, CD3-negative NK-lymphomas tend to progress rapidly. We report here the case of a high grade malignant lymphoma which was characterized by an immunophenotype typical for CD3-negative NK-cells (CD2+, CD3-, CD16+, CD56(+), CD57-). The disease proved to be rapidly fatal despite aggressive chemotherapy. Interestingly, the patient suffered from a high turn over pancytopenia, which also characterizes NK-cell leukemias/lymphomas of the LGL-type. However, our patient's lymphatic cells appeared highly immature, and cytoplasmic granules, characteristic for LGL-cells, could not be discerned either microscopically or electronmicroscopically. Furthermore, the malignant lymphatic population had the T-cell receptor beta-chain rearranged. We therefore concluded that our patient might have suffered from a malignant proliferation of a putative precursor cell intermediate between T-cells and NK-cells. PMID- 7541606 TI - Pseudoleukemia following the use of G-CSF. PMID- 7541608 TI - Longitudinal observations on 15 children with Wiedemann-Beckwith syndrome. AB - We conducted a follow-up study on 15 patients with Wiedemann-Beckwith syndrome (WBS) to further clarify major and minor diagnostic clinical characteristics and longterm expectations for growth and development. We found patients with WBS tended to have polyhydramnios with large placentas which were almost twice normal placental weight. The large fetal size and polyhydramnios often resulted in early delivery with occasional perinatal mortality (three cases). Increased placental size, with associated polyhydramnios resulting in excessive umbilical cord length, may be useful in suspecting WBS prior to delivery, thereby facilitating perinatal management. The presence of abdominal wall defects and/or macroglossia may help to confirm the diagnosis. At birth, patients were almost 2 standard deviations above the expected mean for gestational age, length, and weight. This trend continued through early childhood and then excessive size became less dramatic with increasing age. We detected no cytogenetic variations in nine patients who had studies done and, to date, no tumors have been detected other than a gastric teratoma that was evident in one infant at birth. Longitudinally, the children have not had an unusual incidence of medical problems, and long-term ultrasound monitoring was not burdensome to the families. In comparison, mental and social development to unaffected siblings and cousins appeared normal. PMID- 7541607 TI - Effect of interferon therapy on human papillomavirus copy number in patients with Condyloma acuminatum. AB - Interferons exert antiviral, antiproliferative, and immunomodulatory effects on target cells. The effectiveness of interferon treatment can be followed by measuring parameters involved in interferon action. The clinical effectiveness of interferons has been proved in the human papillomavirus (HPV)-associated disease condyloma acuminatum. Because one of the most important goals in the treatment of this condition is the elimination of recurrences, it was asked whether clinically effective interferon therapy was associated with elimination of HPV DNA. The authors used a polymerase chain reaction-based method to detect the HPV from minute amounts of clinical biopsies. Human papillomavirus-transformed cell lines or cloned HPV genomes served as different copy number controls. The intensity ratio of L1 HPV and the human beta-globin (which served as an internal control) fragments was determined and used for estimation of HPV copy number in different biopsies. A direct effect of interferon treatment on HPV viral genome copies was observed in a group of responder patients, but not in the clinically resistant group. With these data, a correlation was found between virologic data and clinical findings. This method also can be used in other lesions to determine the HPV copy number and HPV type and may have value in determining the antiviral activities of other agents used in the treatment of HPV-related lesions. PMID- 7541609 TI - Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Expression of allograft endothelial VCAM-1 can be manipulated with antagonist of IFN-alpha or IL-4 and is not required for allograft rejection. AB - This report provides evidence to support the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and IL-4 promote the expression of new endothelial surface molecules in rejecting murine heterotopic cardiac allografts. The microvascular endothelia of these cardiac allografts all develop strong reactivity with the monoclonal antibodies (mAbs) YN1.1/74 (anti-ICAM-1), M/K-2 (anti-VCAM-1) and MECA-32 (undefined molecule) within 3 to 5 days of graft implantation. Daily treatment of the allograft recipients with pentoxifylline (PTX), soluble TNF receptor (TNFR:Fc), anti-interleukin-4 (IL-4) mAb (11B11), or soluble IL-4 receptor, each abrogate the expression of endothelial VCAM-1 and reduce the endothelial reactivity with the mAbs YN1.1/74 and MECA-32 to levels found in cardiac isografts. This is accompanied by a reduction, but not an elimination, of interstitial leukocytic infiltration. Despite this, cardiac allograft recipients treated with PTX or the mAb 11B11 rejected allografts at the same rate as untreated allograft recipients, ie, within 10 to 12 days after graft implantation. These rejected grafts contained mRNAs for TNF-alpha and IL-4, as well as for all other cytokines that have been associated with rejecting allografts. This suggests that endothelial VCAM-1 expression, which is characteristic of rejection, is not an essential element of the rejection process. Interestingly, the grafts from the PTX-treated recipients continued to display rare, isolated VCAM-1 positive cells in the interstitium, which may be dendritic cells. In general, these studies demonstrate a role for IL-4 and TNF alpha in the alterations of vascular endothelial phenotype observed in rejecting cardiac allografts. They also demonstrate that endothelial VCAM-1 expression is not essential for the allograft rejection process, and that the role of VCAM-1 in this process may be more subtle than was initially suspected. PMID- 7541610 TI - Expression of epithelial adhesion proteins and integrins in chronic inflammation. AB - Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. PMID- 7541611 TI - De novo CD5-positive and Richter's syndrome-associated diffuse large B cell lymphomas are genotypically distinct. AB - Diffuse large B cell lymphomas (DLBLs) represent a heterogeneous collection of aggressive non-Hodgkin's lymphomas that can arise either de novo or as a result of transformation from chronic lymphocytic leukemia, small lymphocytic lymphoma, follicular lymphomas, or lymphomas of mucosa-associated lymphoid tissue. A small percentage of DLBLs express the CD5 antigen. The majority of these cases have evolved from a pre-existing low grade non-Hodgkin's lymphoma (Richter's syndrome). However, we identified and characterized nine CD5-positive DLBLs in which the patients did not have a previous history or concomitant evidence of chronic lymphocytic leukemia, small lymphocytic lymphoma, follicular lymphoma, or mucosa-associated lymphoid tissue-associated non-Hodgkin's lymphoma, suggesting that they arose de novo. All nine cases expressed CD20 and monotypic immunoglobulin, all eight cases examined expressed CD19, CD22 and CD43, eight of the nine cases expressed HLA-DR, and two of eight cases expressed CD11c. None of the cases expressed CD3, CD10, CD11b, CD21, CD23 or CD30. CD5 expression by these cells was found to be identical to that of CD5-positive B cell chronic lymphocytic leukemia by quantitative polymerase chain reaction analysis of CD5 mRNA. These nine de novo CD5-positive DLBLs exhibited clonal immunoglobulin heavy and light chain gene rearrangements but lacked integration of the Epstein-Barr virus genome and structural alterations of the bcl-1, bcl-2, c-myc, H-ras, K-ras, and N-ras proto-oncogenes and the p53 tumor suppressor gene. However, bcl-6 proto oncogene rearrangement, which is involved in chromosome band 3q27 aberrations, was found in four cases (44.4%). This is comparable with the frequency of bcl-6 gene rearrangement in CD5-negative DLBL. In contrast, bcl-6 gene rearrangement was absent in six cases of DLBL associated with Richter's syndrome. These findings suggest that de novo CD5-positive DLBLs are genotypically similar to CD5 negative DLBLs and may be pathogenetically distinct from the DLBLs associated with Richter's syndrome. PMID- 7541612 TI - Tumor angiogenesis in advanced stage ovarian carcinoma. AB - Tumor angiogenesis has been found to have prognostic significance in many tumor types for predicting an increased risk of metastasis. We assessed tumor vascularity in 43 cases of advanced stage (International Federation of Gynecologists and Obstetricians stages III and IV) ovarian cancer by using the highly specific endothelial cell marker CD34. Microvessel counts and stage were associated with disease-free survival and with overall survival by Kaplan-Meier analysis. The plots show that higher stage, higher average vessel count at 200x (200x avg) and 400x (400x avg) magnification and highest vessel count at 400x (400x high) magnification confer a worse prognosis for disease-free survival. Average vessel count of less than 16 (400x avg, P2 = 0.01) and less than 45 (200x avg, P2 = 0.026) suggested a better survival. Similarly, a high vessel count of less than 20 (400x high, P2 = 0.019) conferred a better survival as well. The plots suggest that higher stage, higher average vessel count at 200x and 400x, and highest vessel count at 200x and 400x show a trend to worse overall survival as well. With the Cox proportional hazards model, stage was the best predictor of overall survival, however, the average microvessel count at 400x was found to be the best predictor of disease-free survival. These results suggest that analysis of neovascularization in advanced stage ovarian cancer may be a useful prognostic factor. PMID- 7541613 TI - Intratumor microvessel density as a prognostic factor in cancer. PMID- 7541614 TI - Parasympathetic preganglionic neurons in the spinal cord involved in uterine innervation are cholinergic and nitric oxide-containing. AB - BACKGROUND: The purpose of this study was to elucidate parasympathetic preganglionic neurons in the spinal cord that project axons in pathways to the uterus and to reveal their neurotransmitter phenotype. METHODS: "Uterine-related" neurons were identified by using a combination of retrograde axonal tracers: (1) Fluorogold injected into the ganglia of termination of preganglionic fibers, and (2) a transganglionic axonal tracer (pseudorabies virus) injected into the uterus. Immunohistochemistry was used to reveal virus-labeled neurons and their neurotransmitter marker. RESULTS: Double-labeled (Fluorogold+pseudorabies virus) "uterine" preganglionic neurons were identified in the sacral parasympathetic nucleus of the rat lumbosacral spinal cord. Subpopulations of neurons in the sacral parasympathetic nucleus were shown to be immunoreactive for choline acetyltransferase or nitric oxide synthase. Double-staining immunohistochemistry (for pseudorabies virus+neurotransmitter enzyme) revealed that some of the uterine-related preganglionic neurons were cholinergic and some nitric oxide synthase-containing. CONCLUSIONS: These results demonstrate a subpopulation of preganglionic parasympathetic neurons in the sacral parasympathetic nucleus that are involved in uterine innervation. In addition, both acetylcholine and nitric oxide could be used to modify activity in the postganglionic neurons, which directly innervate the uterus. PMID- 7541615 TI - Effects of targeted neuronal nitric oxide synthase gene disruption and nitroG-L arginine methylester on the threshold for isoflurane anesthesia. AB - BACKGROUND: Considerable evidence suggests that nitric oxide plays a role in synaptic transmission in the central and peripheral nervous system. Nonselective inhibition of nitric oxide synthase by nitroG-L-arginine methylester (L-NAME) reduces the minimum alveolar concentration of halothane anesthesia. The effects of selective neuronal nitric oxide synthase inhibition on the anesthetic requirements in mice congenitally deficient in neuronal nitric oxide synthase (knockout mice) were examined. METHODS: Isoflurane minimum alveolar concentration and righting reflex ED50 (RRED50) were determined in knockout and wild-type mice. Subsequently, the effects of intraperitoneal L-NAME on minimum alveolar concentration and RRED50 of knockout and wild-type mice were examined. In a separate experiment, the effects of week-long administration of L-NAME were examined in wild-type mice. Isoflurane minimum alveolar concentration and RRED50 were measured on the 8th day and were repeated after an acute intraperitoneal dose of L-NAME. RESULTS: Targeted disruption of the neuronal nitric oxide synthase gene did not modify isoflurane minimum alveolar concentration and RRED50 of knockout mice. Acute intraperitoneal L-NAME decreased the minimum alveolar concentration and RRED50 of wild-type but did not alter those values in knockout mice. The wild-type mice, when given L-NAME for a week, showed a minimum alveolar concentration and RRED50 identical to that of untreated wild-type mice. CONCLUSIONS: Although acute nonselective inhibition of nitric oxide synthase reduces the anesthetic requirements of wild-type mice, a chronic congenital deficiency of neuronal nitric oxide synthase or a week of L-NAME treatment of wild-type mice does not produce a state of greater sensitivity to the effects of isoflurane anesthesia. PMID- 7541616 TI - Nitric oxide and minimum alveolar concentration. TKO or knockout? PMID- 7541617 TI - [Conservative treatment of gastroesophageal hemorrhage in portal hypertension syndrome in children]. AB - Intraoperative examinations of 15 patients with extrahepatic portal hypertension carried out to elucidate the effect of pituitrin infusion on the central and portal hemodynamics and analysis of the clinical results of treatment of gastroesophageal hemorrhages with pituitrin infusions in 17 patients showed that infusions of pituitrin solution were sufficiently effective in this patient population. The drug exerted the minimal effect on the central hemodynamics and appreciably improved the portal pressure and bloodflow. PMID- 7541618 TI - Comparative anti-HIV evaluation of diverse HIV-1-specific reverse transcriptase inhibitor-resistant virus isolates demonstrates the existence of distinct phenotypic subgroups. AB - We have biologically and biochemically evaluated a structurally diverse group of HIV-1-specific reverse transcriptase (RT) inhibitors and determined that the members of this class share many common properties. These include reproducible and selective antiviral activity against a panel of biologically distinct laboratory and clinical strains of HIV-1, activity against HIV-1 in a wide variety of cultured and fresh human cells, and potent inhibition of HIV-1 RT when evaluated using a heteropolymeric ribosomal RNA template assay. Each of the HIV-1 specific compounds was capable of inhibiting HIV replication when challenged at high m.o.i., further distinguishing them from the nucleoside analogs 3'-azido-3' deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC). When tested in combination with AZT, each of the HIV-1-specific compounds synergistically inhibited the replication of HIV-1. HIV-1 isolates resistant to different HIV-1-specific inhibitors exhibited heterogeneous patterns of cross-resistance to other members of this pharmacologic class. Four distinct phenotypic classes have been defined through the use of drug-resistant virus isolates which derive from distinct mutations in the RT. These results indicate that the various subgroups of HIV-1 specific inhibitors interact differently with HIV-1 RT, suggesting important potential implications for drug combination therapeutic strategies. PMID- 7541619 TI - The inhibition of human immunodeficiency virus type 1 in vitro by a non nucleoside reverse transcriptase inhibitor MKC-442, alone and in combination with other anti-HIV compounds. AB - MKC-442, a derivative of the non-nucleoside reverse transcriptase (RT) inhibitor 1-[(2-hydroxyethoxy)methyl)-6-(phenylthio)thymidine (HEPT), showed potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) replication in vitro, using a range of host-cell/virus systems including human peripheral blood mononuclear cells infected with primary clinical isolates. MKC-442 was evaluated in combination with the nucleoside analogues AZT, ddI and ddC, the non nucleoside RT inhibitor nevirapine, the HIV-1 proteinase inhibitor Ro-31-8959, and the alpha-glucosidase 1 inhibitor, MDL-28,574, using a cell viability assay. Drug interactions were evaluated by the isobologram technique and by calculating combination indices. Notable synergistic inhibition of HIV-1 replication was observed when MKC-442 was combined with AZT and MDL-28,574 and moderate synergy with ddI. In combination with ddC, nevirapine or Ro-31-8959, only a slightly better than additive effect was observed. Impressive synergy was seen using the three-drug combinations of MKC-442, AZT and MDL-28,574 or MKC-442, AZT and Ro-31 8959. No additional cytotoxicity was observed as measured by [3H]thymidine incorporation by concanavalin A-stimulated peripheral blood mononuclear cells, when MKC-442 was combined with any of the above-mentioned compounds. The use of MKC-442 in a two- or three-drug combination regimen with other RT inhibitors, a proteinase inhibitor or an alpha-glucosidase 1 inhibitor should be considered for HIV-1-related chemotherapy. PMID- 7541620 TI - [Laboratory control of effectiveness of chemotherapy in melioidosis]. AB - An undoubtable role of the melioidosis germ persistence in the form of L-variants with the development of DTH in the relapses of the disease was stated. Serological reactions such as CFT and PHAR were shown to be of the diagnostic value but it was by far lower than the allergotest. No correlation between the antibody titers in the tested sera and the isolation of the bacterial or L variants of the melioidosis germ was observed. The criteria of the recovery from melioidosis were: stable normalization of the patient temperature, positive time course of the body weight changes, negative tests for the type and morphologically changed forms of the melioidosis germ and no allergic reactions. PMID- 7541621 TI - Analysis of functional epitopes on the dengue 2 envelope (E) protein using monoclonal IgM antibodies. AB - Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions. PMID- 7541622 TI - The effects of long-term treatment with salmeterol and salbutamol on the flow rate and composition of whole saliva in the rat. AB - The effect of long-acting beta 2-adrenoceptor agonists on salivary glands and saliva secretion has not been studied before. Sprague-Dawley rats were given either the long-acting beta 2-agonist salmeterol, 1 mg/kg body wt per day or the short-acting agonist salbutamol, 5 mg/kg per day. Saline solution was used as control. After 18 days pilocarpine-stimulated saliva was collected, and after 21 days saliva was collected after stimulation with isoproterol and pilocarpine in combination. The saliva was analysed for total protein, amylase, hexosamine, sialic acid, sodium, potassium and calcium. At day 25 the salivary glands were extirpated and weighed. The weight of the parotid glands increased significantly after both salmeterol and salbutamol treatment, approx. 40%; the submandibular gland weights were not affected by either beta 2-agonist treatment. Pilocarpine stimulated salivary flow rate was increased in the salbutamol, but not in the salmeterol, group. In the salmeterol group the concentration of sialic acid was increased and that of calcium was decreased. In saliva stimulated with pilocarpine and isoproterenol in combination, the concentrations of total protein, amylase and calcium were decreased after salmeterol. In the salbutamol group, total protein and potassium were decreased. The ratio sialic acid: total protein was increased at both saliva collections in both beta 2-agonist groups. It is concluded that rats treated chronically with the long-acting beta 2 adrenoceptor agonist salmeterol have an impaired secretion of salivary proteins and calcium and that the effect resembles that of salbutamol. PMID- 7541624 TI - The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells. AB - Mechanical stimulation of the skeleton alters the metabolism of bone cells, but the effects of mechanical strain on the cytoskeleton of osteoblasts are poorly understood. While changes in the distribution of the cytoskeleton in mechanically strained cells have been reported, little is known about the pathways by which these changes are transduced into cell functions. Human osteosarcoma (HOS) TE-85 cells were cultured in Dubelcco's modified Eagle's medium/F-12 and grown to confluency in Flexercell type I dishes in a humidified incubator with 5% CO2 and 95% air. Intermittent strain (3 cycles/min) was applied to the cells for periods of 15 and 30 min, 2, 4 and 24 h, and 3, 5, 7, 10, 14, 20 and 28 days. Unstrained cells were used as controls. The distribution of beta 1 integrin was studied immunocytochemically. Total RNA was isolated at every period of time and Northern blots were used to study the effects of strain on the levels of beta 1-integrin expression. The results indicated that mechanical strain increased the synthesis of beta 1 integrin. Northern blots showed that beta 1 mRNA expression was increased significantly (p < 0.005) at 30 min and 3 days of strain application. Strain also affected beta 1 distribution markedly in 24-h cultures. The response of HOS cells to mechanical strain demonstrates that the cytoskeleton of the osteoblast adapts to strain through the stimulation of specific cytoskeletal and receptor proteins. These results suggest a pathway through which mechanical strain is transmitted to the osteoblastic-like cells. PMID- 7541623 TI - Induction of cytoskeletal rearrangements and loss of volume regulation in epithelial cells by Treponema denticola. AB - The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in alpha-MEM (minimal essential medium) for periods of 1-4 h at 37 degrees C without serum. Control cultures were exposed to bacteria conditioned alpha-MEM without serum or bacteria or to alpha-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4 h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter (p < 0.05), a 60% decrease of F-actin (p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II (p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant (p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541625 TI - Choroidal neovascularization with retinal pigment epithelial folds. PMID- 7541626 TI - Effects of adenosine receptor agonists on nitric oxide release in mouse during endotoxemia. AB - The effects of adenosine receptor agonists on plasma NOx- (NO2- and NO3-) production in mice treated with lipopolysaccharide (LPS) were investigated. NOx- is the stable decomposition product of nitric oxide (NO), which can be measured as a marker of NO production. Injection of the mice with LPS resulted in increased plasma NOx- concentration, reaching a peak after 8 hr (38 times basal level) and then declining slowly. Pretreatment with the adenosine agonists R phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamidoadenosine (NECA), 5'-(N cyclopropyl)carboxamidoadenosine (CPCA) and N6-cyclohexyladenosine (CHA) 1 hr before LPS administration caused a dose-dependent reduction of plasma NOx- concentration. The rank order of inhibitory potency was NECA > or = R-PIA > CPCA > CHA, which is characteristic of neither A1 nor A2 receptors. PMID- 7541627 TI - Cases in electrocardiography. PMID- 7541628 TI - The Jeanne Manery Fisher Memorial Lecture 1994. Molecular biology of rubella virus structural proteins. AB - Rubella virus is a small, enveloped, positive-stranded RNA virus in the Togaviridae family and bears similarities to the prototype alphaviruses in terms of its genome organization and strategy for viral gene expression. Despite being an important human pathogen, the cell biology of rubella virus remains poorly characterized. This review focuses on the molecular biology of rubella virus structural proteins, with emphasis on the proteolytic processing and maturation of virus structural proteins, the glycosylation requirement for intracellular transport and function of glycoproteins, and the localization of hemagglutinin- and virus-neutralizing epitopes. A number of significant differences between rubella virus and alphavirus structural protein expression and maturation were discovered. PMID- 7541629 TI - The intricacies of beta-globin gene expression. AB - Gene expression is an extremely complicated process in which several mechanisms are involved. Owing to its developmental and tissue-specific expression, the beta globin gene is an excellent model for studying gene expression. beta-Globin gene expression involves an interplay between several different mechanisms. Chromatin structure is thought to be altered by the locus control region (LCR) located far upstream of the beta-globin gene locus. As well, multiple transcription factors come into play both in the LCR and in the individual promoters and enhancers of the beta-globin genes. The interaction between these then allows for delicate regulation of beta-globin gene expression. In the following review the elaborate system of beta-globin gene expression will briefly be examined. PMID- 7541631 TI - Chemorepulsion of developing motor axons by the floor plate. AB - In the developing nervous system, motor axons grow away from the ventral midline floor plate, suggesting that the latter might be a source of repulsive axonal guidance cues. In donor to host transplantation experiments, ectopic pieces of floor plate were positioned between chick hindbrain motor neurons and their exit points. Immunohistochemistry and retrograde axonal labeling techniques demonstrated that motor axons diverted from their normal pathways to avoid grafted floor plate, often traversing abnormally long circuitous trajectories to reach exit points. When ventral explants of rat hindbrain and spinal cord were cocultured at a distance from floor plate explants within collagen gel matrices, the outgrowth of motor axons was dramatically reduced from explant borders that faced the floor plate. Thus, the floor plate secretes diffusible repulsive cues in vitro that may exclude motor axons from the midline during development. PMID- 7541630 TI - PCR and patch-clamp analysis of single neurons. AB - The combination of patch-clamp and molecular biology techniques has made it possible to characterize the pharmacological and biophysical properties of ion channels in single neurons and to screen for expression of specific mRNAs in the same cell. Following whole-cell recording, the cytoplasm of the cell is harvested, and RNA is reverse transcribed into cDNA and amplified in PCR with primers specific for individual ion channel subunits. Additional experiments can then be designed to relate structure and function at the protein level more directly, since cells appear to regulate the composition of ion channels at least partly at the posttranscriptional stage. PMID- 7541633 TI - Differential role of the low affinity neurotrophin receptor (p75) in retrograde axonal transport of the neurotrophins. AB - The receptor mechanisms mediating the retrograde axonal transport of the neurotrophins have been investigated in adult rats. We show that transport of the TrkB ligands NT-4 and BDNF to peripheral neurons is dependent on the low affinity neurotrophin receptor (LNR). Pharmacological manipulation of LNR in vivo using either an anti-LNR antibody or a soluble recombinant LNR extracellular domain completely blocked retrograde transport of NT-4 and BDNF to sensory neurons, while having minimal effects on the transport of NGF in either sensory or sympathetic neurons. Furthermore, in mice with a null mutation of LNR, the transport of NT-4 and BDNF, but not NGF, was dramatically reduced. These observations demonstrate a selective role for LNR in retrograde transport of the various neurotrophins from distinct target regions in vivo. PMID- 7541632 TI - Axonin-1, Nr-CAM, and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons. AB - Immunoglobulin/fibronectin type III-like cell adhesion molecules have been implicated in axon pathfinding based on their expression pattern in the developing nervous system and on their complex interactions described in vitro. The present in vivo study demonstrates that interactions by two of these molecules, axonin-1 on commissural growth cones and Nr-CAM on floor plate cells, are required for accurate pathfinding at the midline. When axonin-1 or Nr-CAM interactions were perturbed, many commissural axons failed to cross the midline and turned instead along the ipsilateral floor plate border. In contrast, though perturbation of Ng-CAM produced a defasciculation of the commissural neurites, it did not affect their guidance across the floor plate. PMID- 7541635 TI - Axonal transport of tubulin in Ti1 pioneer neurons in situ. AB - In neurons, tubulin is synthesized only in the cell body or dendrites, yet the growing axon requires a steady supply of this protein at the growth cone. Hence, some mechanism must exist to move tubulin from the cell body to the growth cone. Transport could conceivably occur by simple diffusion, translocation of polymer, or some form of monomer or oligomer transport. Evidence for all these has been presented in a variety of experimental systems. We have directly studied the movement of microtubules in 12 growing axons in live grasshopper Ti1 neurons in their natural environment by labeling the polymer with a caged fluorophore, biscaged fluorescein. No evidence of polymer transport was found. Hence, tubulin movement in these neurons must occur by movement of monomeric tubulin, either by transport or diffusion. To resolve these conflicting views, we discuss the conditions under which diffusion is feasible as a transport mechanism. PMID- 7541634 TI - The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C. AB - This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8. PMID- 7541638 TI - Deletion mapping of chromosome 11 in carcinoma of the bladder. AB - Deletions of the short arm of chromosome 11 have been identified by both cytogenetic and molecular criteria in bladder and other types of solid tumor, indicating the presence of one or more suppressor loci in this region. To localize the 11p deletion target(s) more precisely and to screen for loss of heterozygosity (LOH) on the long arm of the chromosome, 100 bladder tumors were analyzed for LOH on chromosome 11 using restriction fragment length polymorphisms (RFLPs) and microsatellite markers mapped to both 11p and 11q. Thirty-four tumors were found to have LOH at 1 or more loci. Of these, 17 had LOH restricted to 11p, 13 had LOH of both 11p and 11q, and 4 had LOH of 11q only. Eight tumors showed LOH at all informative loci indicating probable loss of an entire copy of chromosome 11. A common region of deletion was defined on 11p between D11S922 (11p15.5) and D11S569 (11p15.1-15.2). This region does not include the HRAS or WT1 loci (at 11p15.5 and 11p13, respectively). Seventeen tumors had LOH on 11q, 4 of which had LOH on 11q only. The common region of deletion on 11q was between FGF3 and D11S490 (11q13-q23.2). Two tumors showed LOH on both 11p and 11q with a clear region of retention of heterozygosity between, indicating the existence of two deletion targets on chromosome 11. PMID- 7541636 TI - Tubulin dynamics in neuronal axons of living zebrafish embryos. AB - The mechanism of cytoskeletal protein transport, especially the question of what kind of form the cytoskeletal proteins assume during transport in neurons in situ, has been an important, as yet unsettled issue. To clear up this matter, we adopted the embryonic zebrafish as a living animal model and applied the fluorescence recovery after photobleaching (FRAP) method. The zebrafish embryo is appropriate for this kind of study because of its transparency during the early developmental stage, allowing the observation of neurons that incorporate the microinjected fluorescent tubulin directly under fluorescence microscopy. FRAP revealed no movement of the bleached zone proximodistally, where fluorescence recovered gradually (recovery half-time, 44.2 +/- 11.2 min; n = 36), suggesting that the polymers are stationary but dynamic and that the true moving form could be small oligomers or heterodimers. PMID- 7541637 TI - Aprotinin in elective primary bypass surgery. Graft patency and clinical efficacy. AB - The proteinase inhibitor aprotinin is used in open heart surgery to reduce intraoperative and postoperative blood loss and transfusion requirements. To investigate a possible influence on graft patency, a randomized double-blind group comparison study was carried out in male patients elected for primary bypass surgery. One hundred ten (55/55) patients received either placebo treatment or aprotinin according to the Hammersmith scheme (2 Mio KIU as loading dose before sternotomy, followed by an infusion of 0.5 Mio KIU/h until the end of surgery; 2 Mio KIU added to the priming volume additionally). Graft patency was evaluated by angiography in 44 aprotinin and 35 placebo patients between the 18th and 35th days postoperatively. There was no difference in the overall graft occlusion: in the aprotinin group 89.5% (111/124) grafts were found patent compared to 87.2% (89/102) in the placebo group. Of the aprotinin patients 72.7% (32/44) and 71.4% (25/35) of the placebo patients had all grafts patent. Venous grafts were occluded in 16% (7/44) of aprotinin patients and in 29% (10/35) of placebo patients. On the other hand 5/27 patients in the aprotinin group vs 0/27 in the placebo group had occluded internal mammary artery (IMA) grafts (P = 0.0511%). Graft occlusions were not accompanied by signs of myocardial infarction in any case. Fifty-one patients in the aprotinin group and 47 patients in the placebo group were valid for parameters of clinical efficacy: blood loss within 6 h postoperatively was reduced by 58.5% in the aprotinin group (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541639 TI - Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18. AB - Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome 18 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome 18 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing and extra copy of chromosome 18 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the Ishikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome 18 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene. PMID- 7541641 TI - Characterization of add(1)(p36) in non-Hodgkin lymphomas by fluorescence in situ hybridization. AB - Chromosome rearrangements involving chromosome I, band p36, are among the most common aberrations in non-Hodgkin lymphomas (NHL). We have studied nine cases of NHL with add(1)(p36) using fluorescence in situ hybridization (FISH) from a series of 205 cases. Five were follicular low-grade NHL and four were follicular or diffuse high-grade NHL. Three of the five cases with follicular low-grade NHL did not contain the 14;18 translocation. The extra material on the add(1)(p36) in these three cases was derived from chromosome segment 2q31-qter; in one it was observed as a sole clonal rearrangement. In the two remaining cases, with t(14;18), the add(1)(p36) consisted of material from chromosome arms 3q and 17q, respectively. In the four cases of high-grade NHL, the material added on to Ip36 was derived from chromosomes 6, 9, 17, and 19, respectively. Using a Ip36 specific probe, DIS94, we showed a deletion on the add(1) in one of the cases with low-grade NHL, whereas no loss was observed in one of the cases with high grade NHL. Our study indicates that cytogenetically similar add(1)(p36) are found in both high- and low-grade NHL, and the breakpoint on Ip36 as well as the origin of translocated material may vary. PMID- 7541640 TI - Identification of two transcripts of AML1/ETO-fused gene in t(8;21) leukemic cells and expression of wild-type ETO gene in hematopoietic cells. AB - The t(8;21) is a common chromosomal abnormality, preferentially associated with acute leukemia showing features of myeloid differentiation. Recently, two genes- AML1, which has a unique runt domain, and ETO (MTG8)--have been isolated from the chromosomal breakpoint. In this study, we isolated and identified two fused transcripts from a leukemic cell line carrying t(8;21). AML1 and ETO were fused at the same position in these transcripts. One of the transcripts codes a unique domain, including two zinc finger domains and three proline- and one leucine-rich region. The other transcript codes only for one proline- and leucine-rich region but lacks zinc finger domains. We demonstrated by polymerase chain reaction (PCR) analysis that 1) these two transcripts are consistently expressed in leukemic cells with t(8;21) obtained from patients and 2) expression of AML1 was not restricted to the particular stage of hematopoietic differentiation but was present in all hematopoietic cells investigated. We also provide evidence that two wild types of ETO transcripts containing the region of the ETO gene in fused transcripts are expressed in hematopoietic cells from different lineages. The widespread expression of AML1 and ETO in hematopoietic cells suggests a fundamental role of these proteins in hematopoiesis. Furthermore, the differences in the carboxy termini of ETO may modulate the activity of fused proteins resulting from the chromosomal translocation t(8;21). PMID- 7541642 TI - Allelic losses in human chromosome 11 in lung cancers. AB - The relatively frequent loss of heterozygosity at loci on the short arm of chromosome 11 in human lung cancers has suggested the presence of a putative tumor suppressor gene. For location of the gene, a fine deletion map of human chromosome 11 was constructed by analysis of DNAs from 79 lung cancers with 31 sequence-tagged-site markers that dotted chromosome 11 and detected polymorphic changes in nucleotide sequences. The results showed that three regions, 11p12 p15, 11q12, and 11q14-q24, were commonly deleted in a considerable number of cancers, indicating the possible presence of more than one tumor suppressor gene. The range of deletion in the 11p15 region was estimated to be 4.5 megabases. That in the 11q24-q24 region was divided into two portions: one was 3 cM in length, and the other was longer and could not be specified because of lack of appropriate markers. The deletion in the 11q12 region was so short that two markers flanking the region could not be identified by genetic analysis. PMID- 7541643 TI - Human chromosome 11 inhibits tumorigenicity of a murine squamous cell carcinoma cell line. AB - Loss of heterozygosity (LOH) of mouse chromosome 7 has been consistently demonstrated in chemically induced murine squamous cell carcinomas (SCCs). The region of this chromosome presenting LOH in the mouse tumors is syntenic to human chromosome segments 11p15 and 11q. To determine whether the introduction of human chromosome (Hchr) 11 can suppress the growth of murine SCC, we injected four clones of a chemically induced murine SCC cell line bearing an Hchr 11 into athymic BALB/c nude mice. All microcell hybrid clones with Hchr 11 (CH72/Hchr 11) had latency periods twice as long as those of the parental CH72 cells and control hybrids containing a Hchr 12. Tumor-derived cells from CH72/Hchr 11 hybrids had lost centromeric and telomeric sequences from Hchr 11. All repressed cell lines grew significantly more slowly in vitro than did the controls. These results suggest that Hchr 11 contains a tumor-suppressor gene capable of inhibiting tumorigenicity in chemically induced SCC, confirming common pathways in the development of human neoplasias and the murine model. PMID- 7541647 TI - Genetic mosaicism at the insulin locus in liver associated with childhood hepatoblastoma. AB - Hepatoblastoma is commonly associated with loss of heterozygosity (LOH) involving chromosome region 11p15.5. This region may contain an as yet unidentified tumor suppressor gene relevant to this and other tumors associated with the Beckwith Wiedemann syndrome. Using the insulin (INS) locus as a marker for this region we have identified two of three patients with hepatoblastoma whose livers demonstrated genetic mosaicism. One tumor arose from the clone demonstrating LOH in the liver while the other arose from the clone without LOH. PMID- 7541645 TI - Involvement of chromosomes 17 and 22 in dermatofibrosarcoma protuberans. AB - Literature on the cytogenetics of dermatofibrosarcoma protuberans (DFSP) is limited; only 10 cases with chromosome aberrations have been reported. They are karyotypically characterized by the presence of supernumerary ring(s), either as the sole cytogenetic abnormality or together with a few additional structural or numerical changes. We report the cytogenetic and fluorescence in situ hybridization (FISH) analysis of three new DFSP, one primary and two recurrent tumors. In two cases we found a supernumerary ring as the sole change, whereas the third had two copies of a marker chromosome and monosomy of chromosome 22. Sequences of chromosomes 17 and 22 were identified by FISH in the supernumerary rings and in the markers. The fluorescence pattern suggested that additional sequences were present in the two rings, but showed that the marker chromosomes were entirely painted by chromosome 17 and 22 probes. The findings indicate that juxtaposition and/or amplification of chromosome 17 and 22 sequences could be crucial in the pathogenesis of DFSP. PMID- 7541646 TI - Cytogenetic and molecular studies on the neuroblastoma cell line NGP: identification of a reciprocal t(1;15) involving the "consensus region" 1p36.1. AB - A reciprocal t(1;15)(p36.1-36.3;q25-26) has been identified in an established neuroblastoma cell line (NGP) that earlier studies had shown to carry, among others, a rearrangement at the 1p subtelomeric region. Though it has not been possible to establish whether this translocation was constitutional, it is of interest to note that one of the breakpoints is located within the well-known 1p consensus site of tumor-associated chromosomal rearrangements where, as a result of the reciprocal translocation, the FES oncogene has been transferred from autosome 15. It is to be expected that the molecular cloning of the 1p and 15q translocation breakpoints may yield crucial data for understanding the association between specific chromosomal rearrangements and malignant tumor progression. PMID- 7541644 TI - dic(9;20): a new recurrent chromosome abnormality in adult acute lymphoblastic leukemia. AB - Loss of chromosome 20 and rearrangement of the short arm of chromosome 9 were identified by banding analysis of three adult patients with acute lymphoblastic leukemia (ALL). The G-banding pattern suggested an identical deletion of 9p, but, also, an unbalanced translocation with chromosome 20 was taken into consideration. Dual-color chromosome painting with probes for chromosomes 9 and 20 revealed the presence of material from chromosome 20 at the short arm of the abnormal chromosome 9 in all three cases. Centromeric alpha-satellite DNA of both chromosome 9 and chromosome 20 was demonstrated by fluorescence in situ hybridization and indicated the presence of a dicentric chromosome. The hybridization of a YAC clone of the short arm of chromosome 20 proved that the dicentric chromosome contained the short arm of chromosome 20, which had been suspected from the G-banding pattern. Thus, the rearrangement was interpreted as dic(9;20)(p11;q11.?1). Because this was the sole chromosome abnormality in two patients, dic(9;20) may be a primary chromosome aberration in ALL. In one case, a 9q+ chromosome derived from a Philadelphia (Ph) translocation was involved in the formation of the dicentric chromosome. Immunophenotyping revealed CD10+ B-cell precursor ALL in all three cases. Whereas the two patients in whom dic(9;20) was the sole cytogenetically detectable change are in continuous complete remission for 10 and 45 months, respectively, the Ph+ patient relapsed with leukemia and died 8 months after diagnosis. PMID- 7541648 TI - Frequent loss of chromosome arm 1p DNA in parathyroid adenomas. AB - Two molecular defects have been described in parathyroid adenomas: rearrangement and overexpression of the PRAD1/cyclin D1 oncogene and allelic loss of chromosome 11 DNA, often including the multiple endocrine neoplasia type 1 (MEN1) putative tumor suppressor gene region. In an effort to identify additional parathyroid tumor suppressor genes, we examined 25 parathyroid adenomas for tumor-specific allelic loss of polymorphic DNA loci located near known or candidate tumor suppressor genes. Control leukocyte DNA from all 25 patients was heterozygous for 1 or more of the 9 chromosome 1 markers examined. Allelic loss at 1 or more of these informative loci on chromosome 1 was observed in 10 of 25 (40%) adenomas. Although many tumors lost extensive regions on chromosome 1, all but one of these tumors had allelic loss of distal 1p (1p32-pter); four tumors also lost loci on 1q. Allelic loss at 11q13, the site of the MEN1 gene, was detected in 5 of 21 (24%) informative cases, including 3 with 1p loss. In contrast, allelic loss was rarely observed at loci on 9q and 10p and was not observed at loci on 3p, 3q, 4p, 5q, 12q, 14q, 18q, 22q, or Xp. In summary, clonal allelic loss of loci on chromosome arm 1p is a frequent feature of parathyroid adenomas, implying that inactivation of a tumor suppressor gene(s) on 1p commonly contributes to their pathogenesis. PMID- 7541649 TI - cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma derived cell line, pANS6. AB - Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR. PMID- 7541650 TI - Systematic screening for RNA with skipped exons--splicing mutations of the ferrochelatase gene. AB - A systematic method was designed to screen a large population of patients with erythropoietic protoporphyria (EPP) for aberrant ferrochelatase RNA with skipped exons. The method utilizes the new junction sequence created by exon skipping as the probe to detect such RNA species. In 7 of 17 EPP families, an aberrant ferrochelatase RNA with one exon missing was observed. Two previously unreported splicing mutations were also identified in 2 EPP families. One was a G >> T transversion at the +1 position of the acceptor site of intron 8, causing exon 9 to be skipped during RNA splicing. Both the patient and her father were found to be heterozygous for this mutation. In another family, an A >> G transition at the +3 position of the donor site of intron 10 was identified, associated with exon 10 skipping during RNA splicing. Both the patient and her father were heterozygous for this mutation. PMID- 7541652 TI - HCV RNA monitoring for tailored regimens of interferon therapy in hepatitis C. AB - Sustained non-viraemia is prerequisite for a virological cure of hepatitis C virus (HCV) disease. We monitored serum HCV RNA in our patients during interferon therapy and for 6 months of follow-up. The rate of sustained responders (RNA negative at the end of follow-up) differed significantly by whether or not RNA became sero-negative within 24 weeks of the initiation of therapy (71% vs 7%); and also by whether or not the RNA-seronegativity lasted for 12 weeks during therapy (88% vs 11%). Thus, by monitoring viraemia in individual patients, we can tailor the duration of therapy and minimize waste of interferon. PMID- 7541651 TI - IgG3 reactive rheumatoid factor in rheumatoid arthritis: etiologic and pathogenic considerations. AB - Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3 reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA. PMID- 7541653 TI - Spinal projections to the parabrachial nucleus are substance P-immunoreactive. AB - Substance P plays an important role in nociceptive processing in the spinal cord. Substance P is also present in several supraspinal regions, such as the pontine parabrachial nucleus, a major relay for autonomic regulation. In this study we examined in the cat with an immunogold method the presence of substance P-like immunoreactivity in spinoparabrachial terminals that were labelled by anterograde transport of unconjugated and lectin-conjugated horseradish peroxidase. We found that dense core vesicles in anterogradely labelled terminals were substance P immunoreactive. Taken together with previous observations that noxious stimuli increase preprotachykinin expression in ascending nociceptive pathways from the spinal dorsal horn, the present finding provides evidence that substance P is involved in nociceptive processing also in the brain stem. PMID- 7541654 TI - Ca2+ and in vitro kainate damage to cortical and hippocampal SMI-32(+) neurons. AB - SMI-32 is a monoclonal antibody to non-phosphorylated neurofilament epitopes, which labels subsets of pyramidal neurons prone to degeneration in Alzheimer's disease. We found SMI-32 to identify a small minority of neurons in dissociated cultures of murine cortex and hippocampus (SMI-32(+) neurons). Labeled neurons, which were larger than average and were often immunoreactive for GABA, were preferentially destroyed by brief kainate exposures. This rapidly triggered kainate damage to SMI-32(+) neurons was dependent upon the presence of Ca2+ in the media during the toxic exposure. Furthermore, most SMI-32(+) neurons in both cortex and hippocampus were subject to kainate-activated cobalt uptake, a histochemical procedure that marks cells with Ca2+ permeable AMPA/kainate channels. The unusual vulnerability of cortical and hippocampal SMI-32(+) neurons to AMPA/kainate receptor-mediated injury may result from rapid Ca2+ entry through Ca2+ permeable AMPA/kainate receptor-gated channels. PMID- 7541655 TI - Inhibitors of PLA2 and NO synthase cooperate in producing amnesia of a spatial task. AB - Both nitric oxide and arachidonic acid have been suggested to play a role as a retrograde messenger in synaptic plastic changes which underlie memory formation. However, inhibitors of nitric oxide (NO) synthesis or of arachidonic acid release have produced contradictory results. We suggest a model which involves simultaneous release of both messenger types which can compensate for the loss of one type. To test this theory, rats were injected either with a drug that inhibits release of arachidonic acid, or a drug that inhibits synthesis of NO, or with both drugs. Acquisition of a water maze task was not different between groups. In the test sessions, however, animals injected with both drugs showed marked amnesic symptoms, while the groups injected with a low dose of nordihydroguaiaretic acid (NDGA) or with nitro-L-arginine (L-NARG) showed a trend towards amnesia. The effect of both drugs appears to be additive. The results support the proposed theory. PMID- 7541656 TI - Jun-B expression in Purkinje cells by conjunctive stimulation of climbing fibre and AMPA. AB - Co-application of alpha-amino-3-hydro-5-methyl-4-isoxazole-propionate (AMPA) and 8-bromo cGMP (8-Br-cGMP) which cause long-term desensitization also induces c-Fos and Jun-B expression in Purkinje cells of cerebellar slices [Nakazawa K, Karachot L, Nakabeppu Y et al. NeuroReport 4, 1275-1278 (1993)]. Here, we report an increased local induction of Jun-B immunoreactivity in Purkinje cells in vivo when electrical stimulation of the inferior olive nucleus (IOn) was conjunctively applied with AMPA on the vermis. The present data further supports the idea that conjunctive heterosynaptic inputs to cerebellar Purkinje cells can trigger active gene transcription thus possibly contributing to cerebellar long-term plasticity. They also demonstrate that Jun-B may be a useful transcriptional marker to study cerebellar coincidence phenomena. PMID- 7541658 TI - Effects of sodium on PKC translocation; relationship to neurotransmitter release. AB - Our previous work using Na+ channel activators such tityustoxin (TsTX), indicated that local increases in Na+ modulate glutamate release from synaptosomes. We have now investigated the role of the Ca2+/phospholipid-dependent protein kinase (PKC) in mediating this effect. TsTX and KCl stimulate 'fast' glutamate release to the same extent but TsTX is more effective than KCl in enhancing the 'slow' phase of release. KCl greatly stimulates PKC translocation. However, TsTX inhibits basal and phorbol ester-induced translocation while the Na(+)-ionophore, gramicidin D, has no effect. Taken together, these data suggest TsTX mediated localized Na+ entry inhibits PKC translocation and that this effect may be associated with recruitment of vesicles to the readily releasable pool. PMID- 7541657 TI - Direct retinal communication with the peri-amygdaloid area. AB - Retinal projections to the basal forebrain in male Syrian hamsters were examined at the ultrastructural level following bilateral intraocular injections of horseradish peroxidase conjugated to either cholera toxin (CT-HRP) or wheat germ agglutinin (WGA-HRP). Light level microscopic analysis confirmed retinal projections along basal telencephalon, and examination on the electron microscope of individual fibers from the peri-amygdaloid area revealed en passant synaptic profiles. Sections from animals treated with WGA-HRP showed evidence of transsynaptic communication in the form of labeled dendrites in the peri amygdaloid area. Taken together, these data show that the retina communicates directly with the periamygdaloid area, where photic and chemosensory information may be integrated to modulate reproductive behavior. PMID- 7541659 TI - [Stent implantation as a palliative therapeutic measure in tumor-induced stenoses of the large veins of the body]. AB - PURPOSE: Self-expandable metallic stents were used to treat patients with malignant venous obstructions to determine their effectiveness in producing symptomatic palliation. METHODS: 20 patients with a total of 28 tumour-induced obstructions of the superior vena cava (n = 13), the inferior vena cava (n = 4), the subclavian (n = 4), the innominate (n = 5) and the iliac veins (n = 2) were treated with self-expandable metallic stents. 38 Gianturco stents and 21 Wallstents were applied. Patients were heparinised during the procedure and up to three days afterwards. RESULTS: In all patients correct positioning of the stents was achieved. In 16 patients stent placement resulted in relief of their symptoms. In 8 patients the symptoms completely disappeared without recurrence until death as a result of tumour progression. The follow-up ranged from 10 days to 14 months. In 8 still living patients no re-obstruction occurred (follow-up average three months). In one patient thrombotic stent occlusion occurred 6 days after the procedure; the v. cava superior was reopened again by local urokinase therapy. Three patients finally developed re-occlusion due to tumour progression. In one patient stent placement was complicated by migration of the Gianturco stent one day after stent implantation and reocclusion of the subclavian vein. CONCLUSION: The application of self-expandable metallic stents in patients with malignant venous obstruction is a useful palliative therapy. PMID- 7541660 TI - Concomitant granulocyte colony-stimulating factor and induction chemoradiotherapy in adult acute lymphoblastic leukemia: a randomized phase III trial. AB - This prospective multicenter study examined whether simultaneous administration of granulocyte colony-stimulating factor (G-CSF; Filgrastim) and induction chemotherapy for adult acute lymphoblastic leukemia (ALL) could prevent treatment related neutropenia, infections, and resulting treatment delays. Seventy-six patients were randomly assigned to receive either G-CSF (n = 37) or no growth factor (n = 39) in conjunction with a uniform chemotherapy consisting of cyclophosphamide, cytarabine, mercaptopurine, intrathecal methotrexate, and cranial irradiation. The median duration of neutropenia (absolute neutrophil count < 1 x 10(9)/L) during chemotherapy was 8 days in patients receiving C-CSF, compared with 12.5 days in the control group (P < .002). A similar reduction from 11.5 to 7 days was observed in patients with T-ALL receiving additional mediastinal irradiation (P = .13). Infections occurred in 43% and 56% of patients in the G-CSF and control arm, respectively (P = .25); the incidence of nonviral infections was reduced by 50%, from 32 episodes in the control arm to 16 episodes in the G-CSF arm. Prolonged interruptions of chemotherapy administration were less frequent, with delays of 2 weeks or more occurring in only 24% of patients receiving G-CSF as opposed to 46% in the control arm (P = .01). Accordingly, chemotherapy was completed significantly earlier with the use of G-CSF (39 v 44 days, P = .008). With a median follow-up of 20 months, the probability of disease free survival was 0.45 in the G-CSF group and 0.43 in the control group (P = .34). In conclusion, adult ALL patients appear to benefit by the simultaneous administration of G-CSF with induction chemotherapy because of a significant reduction in the duration of neutropenia, a trend to fewer infections, and a more rapid completion of chemotherapy. PMID- 7541661 TI - High-dose cyclophosphamide, carmustine (BCNU), and etoposide (VP16-213) with or without cisplatin (CBV +/- P) and autologous transplantation for patients with Hodgkin's disease who fail to enter a complete remission after combination chemotherapy. AB - Patients with Hodgkin's disease (HD) who fail to enter a complete remission after an initial course of combination chemotherapy are usually considered to have an induction failure (IF); this subset of patients has an extremely poor outcome with further conventional therapy. Since 1985, we have entered 30 IF patients into protocols using conditioning with high-dose cyclophosphamide, carmustine (BCNU), and etoposide (VP16-213) with or without cisplatin (CBV +/- P) followed by autologous stem cell transplantation (ASCT) with bone marrow (19 patients), peripheral blood stem cells (PBSCs; 8 patients), or both (3 patients). All except 2 patients had previously received chemotherapy regimens for HD that contained at least 7 drugs, and 9 had received prior radiotherapy (RT). After documentation of IF, the majority of patients received some cytoreductive therapy as specified by protocol (local RT in 9, two cycles of conventional chemotherapy in 2, both modalities in 2, or high-dose cyclophosphamide to enhance PBSC collection in 11) before CBV +/- P. Five treatment-related deaths occurred, all before day 150 posttransplant. Eleven patients have had progressive HD at a median of 6 months (range, 0.1 to 45 months) after ASCT. The actuarial progression-free survival (PFS) at a median follow-up of 3.6 years (range, 0.2 to 8.2 years) is 42% (95% confidence intervals, 21% to 61%). The statistical analysis identified only prior clinical bleomycin lung toxicity as an adverse risk factor for PFS, mainly because of the increased nonrelapse mortality seen in these patients. CBV +/- P and ASCT can produce durable remission in a substantial proportion of IF HD patients who otherwise have a poor survival, and we believed ASCT approaches represent the best therapy currently available for these patients. Additional measures are needed to reduce the primary problem of disease progression despite high-dose chemotherapy and stem cell transplantation. PMID- 7541662 TI - Primordial germ cells are capable of producing cells of the hematopoietic system in vitro. AB - The identity of the cells giving rise to the hematopoietic system in the mouse embryo are unknown. The results presented here strongly suggest that hematopoietic cells are derived from a nonhematopoietic cell population that has been previously thought to give rise to the germ cells. These cells are called primordial germ cells (PGCs) and can be recognized as large cells showing blebbing and pseudopodial extrusions on their surface. They are alkaline phosphatase (AP) positive and possess a stage-specific embryonic antigen (SSEA-1) on their surface. They represent a small pool of cells in the extraembryonic mesoderm at the base of the allantois in late day-6 embryos. Primordial germ cells from 7.5- and 8.5-day visceral yolk sac and embryo proper form AP+ and SSEA 1+ colonies within 5 days when grown on an embryonic fibroblast feeder cell layer in the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and interleukin-3 (IL-3). Individual colonies taken from day-5 cultures can be shown to differentiate into erythroid lineage cells in secondary methyl cellulose culture and produce secondary and tertiary PGCs in the presence of LIF, SCF, and IL-3. Cells taken from the region of the allantois and primitive streak can form colonies on hydrophilic Teflon (DuPont, Wilmington, DE) foils precoated with collagen and fibronectin. The cells from these colonies were then shown to form cobblestone areas on irradiated adult bone marrow stromal layers, indicating that the most primitive in vitro hematopoietic stem cell, the cobblestone-area forming cell (CAFC), was present. PGC colonies were grown in methyl cellulose in the presence of LIF, SCF, and IL-3 for 5 days, and the colonies were removed and passaged 3 times on pretreated extracellular matrix hydrophilic Teflon foils. After each passage, the cells were assayed for their differentiation capacity and PGC content. After the last passage, the number of CAFCs was also determined. It was found that, under these conditions, the PGC population expanded more than 400 fold and also contained CAFCs. It is postulated that the PGC represents a totipotent stem cell population capable of producing a variety of different cell types including cells of the hematopoietic system. PMID- 7541663 TI - Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus infected mice. AB - We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV-infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages. PMID- 7541664 TI - Substance P (SP) mediates production of stem cell factor and interleukin-1 in bone marrow stroma: potential autoregulatory role for these cytokines in SP receptor expression and induction. AB - Substance P (SP) is a neuropeptide widely distributed in the nervous system. Its release within the bone marrow (BM) can mediate bidirectional neurohematopoietic communication via specific receptors: neurokinin-1R (NK-1R), NK-2R, or NK-3R. We have previously reported that SP effects on hematopoiesis are mediated by an NK-1 type receptor, the BM stroma, and growth factors. Here, we have studied the induction of stem cell factor (SCF) and interleukin-1 (IL-1) by SP in stroma. At 10(-9) mol/L SP, cytokine levels in supernatants were IL-1 alpha, 20 +/- 5 ng/mL; IL-1 beta, 40 +/- 10 ng/mL; and SCF, nondetectable; and the cell-associated levels were SCF, 21 +/- 2 ng/mL; IL-1 alpha, 90 +/- 6 ng/mL; and IL-1 beta, 45 +/ 3 ng/mL. Reverse transcriptase-polymerase chain reaction and ligand-binding studies with stroma stimulated by these two cytokines resulted in (1) NK-1-like receptor mRNA accumulation and (2) downregulation of SP binding sites (day 1) followed by an upregulation (day 3). Low numbers of high-affinity receptors were expressed by day 1 but not by day 3. The results indicate that SP induces IL-1 and SCF in stroma and that these cytokines have the potential to autoregulate NK R. PMID- 7541665 TI - Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice. AB - Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions. PMID- 7541666 TI - Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor. AB - The isolation and cloning of the ligand for the cytokine receptor, Mpl, have been recently described. In this report we present details of the purification of this novel cytokine (megakaryocyte growth and development factor [MGDF]) from aplastic canine plasma. Two forms of canine MGDF, with apparent molecular weights of 25 kD and 31 kD and sharing a common N-terminal amino acid sequence, were isolated. The sole contaminant detected in purified 25-kD or 31-kD MGDF was canine Ig. Canine MGDF is characterized as a human megakaryocyte colony-stimulating factor that acts synergistically with human recombinant stem cell factor but not interleukin 3. MGDF also appears to be physiologically regulated in response to platelet demand. In canine and murine models, serum levels of MGDF activity peak during the thrombocytopenic periods after irradiation, 5-fluorouracil, or antiplatelet antisera injections. These data indicate that the megakaryocyte-stimulating activity that accumulates in plasma in response to platelet losses is a novel cytokine that functions through an interaction with the Mpl cytokine receptor. PMID- 7541667 TI - Identification of a novel DNA sequence differentially expressed between normal human CD34+CD38hi and CD34+CD38lo marrow cells. AB - We have applied the recently developed differential display method to extend the molecular characterization of the less mature CD34+CD38lo bone marrow progenitors in comparison with the CD34+CD38hi cells to better understand their functional differences. Immunomagnetic enrichment of CD34+ cells followed by flow cytometry was used to isolate CD34+CD38lo and CD34+CD38hi cells from human organ donor bone marrow. A limited set of the poly A+ RNA sequences present in these two cell populations was amplified by a combination of reverse transcription with an anchored oligo dT-based primer and polymerase chain reaction with the same oligo dT primer and arbitrary decamers. A radioactive tracer allowed these sequences to be displayed as a series of bands on a denaturing polyacrylamide gel. Eight bands were chosen that appeared in multiple displays to represent gene sequences differentially expressed between CD34+CD38hi and CD34+CD38lo cells. Comparison of the sequences with public DNA sequence databases available identified one sequence as myeloperoxidase. Two other clones matched sequence fragments of unknown function, whereas the remaining five are novel sequences not present in existing databases. The relative level of expression of all of the sequences was tested by an independent reverse transcriptase-polymerase chain reaction with sequence-specific oligonucleotide primers. The lower level of myeloperoxidase mRNA in CD34+CD38lo cells was confirmed, as was the higher expression of the novel sequence 345. Sequence 345 expression is highest in CD34+CD38- cells and decreases with increased CD38 expression. It is expressed in negligible amounts in hematopoietic cell lines and other sources of human tissue, suggesting it may have a functional role in normal hematopoiesis. PMID- 7541668 TI - Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation. AB - Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death. PMID- 7541669 TI - Differential expression of receptors for interleukin-3 on subsets of CD34 expressing hematopoietic cells of rhesus monkeys. AB - The target cell specificity of interleukin-3 (IL-3) was examined by flow cytometric analysis of IL-3 receptor (IL-3R) expression on rhesus monkey bone marrow (BM) cells using biotinylated IL-3. Only 2% to 5% of unfractionated cells stained specifically with the biotinylated IL-3 and most of these cells were present within the CD34+ subset. IL-3Rs were detected on small CD34dull/RhLA DRbright/CD10+/CD27+/CD2-/++ +CD20- cells, which probably represent B-cell precursors. IL-3R+ CD34- BM cells, which were detected at low frequencies, consisted of small CD20dull/surface-IgM+/RhLA-DR+ cells. These cells represented immature B lymphocytes, whereas CD20bright mature B cells were IL-3R-. The highest IL-3R levels were detected on CD34dull/RhLA-DRbright blast-like cells. These cells differentiated into monocytes, neutrophils, and basophils after IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in vitro. The CD34bright/IL-3R- subset contained all clonogenic erythroid and myeloid progenitors (burst-forming unit-erythroid and colony-forming unit culture), whereas CD34bright/IL-3Rdull cells differentiated into monocytes, neutrophils, and erythroid cells after shorter culture periods. This finding showed that IL-3R expression increases during monocyte and granulocyte differentiation. Results of three-color experiments indicated that IL-3Rs are expressed on CD34bright/RhLA-DRbright cells as well as on CD34bright/RhLA-DRdull cells, with the latter population expression approximately twofold to threefold lower IL-3R levels. A large fraction (> 30%) of single-cell/well-sorted CD34bright/RhLA-DRdull cells formed multilineage colonies after 2 to 4 weeks of stimulation with IL-3, GM-CSF, Kit ligand, and IL-6. Individual colonies contained cells that still expressed CD34 as well as differentiated monocytes, granulocytes, and erythroid cells. These results confirmed that the CD34bright/RhLA-DRdull subset was enriched for immature, multipotent progenitor cells, whereas the CD34bright/RhLA-DRbright population mainly contained lineage committed precursors. The results are consistent with the concept that IL-3Rs are induced at very early stages of hematopoiesis, as identified by high expression of CD34 and low expression of RhLA-DR. IL-3R expression continues to be low during differentiation into lineage-committed progenitors; gradually increases on differentiating progenitor cells for B cells, granulocytes, monocytes, and, possibly also, erythrocytes; but finally declines to undetectable levels during terminal differentiation into mature cells of all lineages in peripheral blood, with the exception of basophils.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541670 TI - Interleukin-3 treatment of rhesus monkeys leads to increased production of histamine-releasing cells that express interleukin-3 receptors at high levels. AB - To understand the hematopoietic and nonhematopoietic responses to interleukin-3 (IL-3), expression of cell-surface IL-3 receptors (IL-3R) was examined on bone marrow (BM) cells and peripheral blood (PB) cells of rhesus monkeys during the course of in vivo IL-3 treatment. Whereas IL-3R expression is low in untreated monkeys, IL-3 administration led to a gradual increase in both low- and high affinity binding sites for IL-3. This increase reflected the total number of cells expressing IL-3Rs, as detected by flow cytometry using biotinylated IL-3. Most of these IL-3R+ cells in both BM and PB could be characterized as basophilic granulocytes that contained high levels of histamine. In contrast to the effect on these differentiated cells, IL-3 administration did not significantly alter the low level IL-3R expression on immature, CD34+ cells. Further flow cytometric analysis using biotinylated growth factors showed that the IL-3R+ basophils also expressed receptors for granulocyte-macrophage colony-stimulating factor (GM CSF), but not for IL-6 or Kit ligand. These findings indicated that the IL-3R+ cells included neither monocytes, which express GM-CSFRs and IL-6Rs abundantly, nor mast cells, which express c-kit. By combining flow cytometric and Scatchard data, it was calculated that the basophils contain as many as 1 to 2 x 10(3) high affinity IL-3Rs and 15 to 30 x 10(3) low-affinity sites. The finding that in vivo IL-3 treatment leads to the production of large numbers of cells that express high levels of IL-3R and are capable of producing histamine provides an explanation for the often severe allergic reactions that occur during prolonged IL-3 administration. It also indicates that IL-3, in addition to its direct effects on hematopoietic cells, may also stimulate hematopoiesis through the release of secondary mediators such as histamine by IL-3-responsive mature cells. PMID- 7541671 TI - Tyrosine phosphorylation of the erythropoietin receptor: role for differentiation and mitogenic signal transduction. AB - The erythropoietin (Epo) receptor belongs to the cytokine receptor superfamily. Although the cytokine receptors do not possess a tyrosine kinase consensus sequence in the intracellular domain, rapid stimulation of a tyrosine kinase activity occurs after activation by the ligand. We and others have shown that Epo induces the tyrosine phosphorylation of its cognate receptor as well as phosphorylation of other proteins. In this report, we examined the role of the receptor tyrosine residues in signal transduction. Eight tyrosine residues are located within the intracellular domain of the murine Epo receptor. A single tyrosine residue is present in the region previously shown to be sufficient for proliferative signal transduction. This tyrosine (Tyr 343) was mutated to phenylalanine. Moreover, mutant receptors were also generated with either a tyrosine residue or a phenylalanine residue at position 343 and with a COOH terminal truncation that removed the 7 other tyrosine residues. Expression vectors carrying these mutated receptors were transfected into the interleukin-3 dependent murine cell line Ba/F3. Epo-induced growth was sustained efficiently by all these receptors, although receptors without any tyrosine residues conferred a significantly reduced mitogenic activity. Moreover, all receptors were able to mediate Epo-dependant accumulation of beta-globin mRNA. The mutated receptors all induced the tyrosine phosphorylation of several cellular proteins after Epo stimulation. However, the truncated receptors induced the phosphorylation of a reduced number of proteins, suggesting that phosphorylated tyrosines of the receptor could have a role in the recruitment either of a tyrosine kinase or of tyrosine kinase substrate proteins. The receptors were all able to mediate Epo induced activation of phosphatidylinositol 3-kinase, although truncated receptors no longer bound phosphatidylinositol 3-kinase. PMID- 7541672 TI - Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin. AB - Thrombopoietin (TPO) is a newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto-oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c-mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL 3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors. PMID- 7541673 TI - Changes in the growth properties of CD34+, CD38- bone marrow progenitors during human fetal development. AB - We have previously described the isolation of separate populations of CD34+, CD38 stromal and hematopoietic progenitors cells within fetal bone marrow. The CD34+, CD38-, CD50+, HLA-DR+ population contained the majority of primitive hematopoietic progenitor cells, whereas stromal progenitors were contained within the CD34+, CD38-, CD50-, HLA-DR- population. In this study, we compared the frequencies and total numbers of clonogenic CD34+, CD38- stromal and hematopoietic cells as a function of fetal gestational age using single-cell fluorescent-activated cell sorting (FACS). At 14 weeks of gestation, 1/500 fetal bone marrow mononuclear cells were primitive hematopoietic CD34+, CD38-, HLA-DR+ progenitor cells, whereas 1/1,000 were stromal progenitors with the CD34+, CD38-, HLA-DR- phenotype. During fetal ontogeny there was a continuous, age-dependent decrease in the frequency of stromal progenitors, such that, at 24 weeks of gestation, only 1/100,000 of bone marrow cells had the CD34+, CD38-, HLA-DR- phenotype and were clonogenic stromal cells when isolated by FACS. In contrast, 1/250 bone marrow cells in a 24-week fetus had the CD34+, CD38-, HLA-DR+ phenotype and were clonogenic hematopoietic progenitors. The decrease in the frequency of stromal progenitors was a function of both a decreased frequency of cells with the CD34+, CD38-, HLA-DR- phenotype and a decrease in the growth potential of individual with this phenotype. The total numbers of mononuclear cells and the total numbers of hematopoietic progenitors in two fetal femurs increased in parallel, 100-fold, between 14 and 24 weeks of gestation. In contrast, the total numbers of clonogenic CD34+, CD38-, HLA-DR- stromal progenitor cells remained constant during this period. Although adult bone marrow samples contained stromal progenitor cells at a frequency of approximately 1/7,000 mononuclear cells, clonogenic stromal cells with the CD34+, CD38-, HLA-DR phenotype could not be isolated by single-cell FACS from these samples. Thus, there are significant differences between the frequencies and biologic characteristics of stromal and hematopoietic stem cells during fetal and postnatal ontogeny. PMID- 7541674 TI - Benign marrow progenitors are enriched in the CD34+/HLA-DRlo population but not in the CD34+/CD38lo population in chronic myeloid leukemia: an analysis using interphase fluorescence in situ hybridization. AB - Fluorescence in situ hybridization (FISH) was used to discriminate between benign and malignant cells in sorted populations of chronic myelogenous leukemia (CML) marrow. FISH has the advantage of allowing for a cell by cell analysis of the breakpoint cluster region (BCR) gene rearrangement immediately after flow sorting in nondividing G0/G1 cells that are potentially transcriptionally inactive. We initially selected CD34+ cells with very low expression of the activation antigen CD38 as a candidate phenotype for an immature and hypothetically more benign cell population, but found no enrichment for Ph negativity in that subtype. In five CML samples, 55% +/- 3.3% (mean +/- SE) of CD34+/CD38hi cells had the BCR gene rearrangement, similar to 57% +/- 3.7% seen in the CD34+/CD38lo population. In contrast, subsequent experiments (n = 4) determined that the CD34+/HLA-DRlo population in CML marrow does contain an increased proportion of benign cells: 15% +/- 1% of the CD34+/DRlo cells were BCR rearranged, compared with 52% +/- 5.8% of the CD34+/DRhi cells (P = .001). Our results indicate that benign progenitors in CML are enriched within the CD34+ cells with low DR antigen expression, but not low CD38 expression. One possible interpretation of these observations is that low CD38 antigen expression is not as useful as low HLA-DR expression for isolating immature cells. PMID- 7541675 TI - Organ culture of benign, aging, and hyperplastic human prostate. AB - Organ culture of the human prostate began in the 1970s and was modeled after the work of Lasnitzki and her collaborators in the mouse two decades earlier. In organ culture of human prostates, one sees a rapid increase in epithelial cells and decrease in stromal cells during the first 3-5 days of culture. While modulation of many phenotypic properties occurs, these cultures provide a simple and rapid way to achieve large numbers of human prostatic epithelial cells in cultured tissues that are markedly depleted of stromal cells. There is some evidence that organ cultures are maintained in slightly better functional states in the presence of androgens; however, most of this evidence is less than quantitative. Most organ culture of prostates has been accomplished with tissues from unspecified locations within the prostate; interpretation of cultures carried out in this fashion has been less complete than would have been possible if they had been carried out from specific anatomic locations within the prostate. Careful pathological characterization of locations contiguous to the cultured tissue is mandatory if cultures are to be interpreted meaningfully. PMID- 7541676 TI - Androgen, estrogen, and progesterone receptors in normal and aging prostates. AB - Testicular hormones regulate the growth and development of the prostate. The presence of androgen receptors in prostatic tissue and their importance in the normal development of the prostate has been established. Age-related changes in the hormonal milieu, and perhaps steroid hormone receptor profile, could set in motion pathological changes leading to the onset of benign prostatic hyperplasia (BPH) or prostate cancer which primarily affect older men. The accumulation of dihydrotestosterone with age, the reawakening of the inductive potential of the prostatic stroma, the altered rate of apoptosis with age, and the age-related changes in the ratio of testosterone:estrogen have all been implicated in the etiology of BPH. In addition to androgen receptors, several studies have documented the presence of estrogen and progesterone receptors in BPH and prostate cancer. So far, most studies have focussed on the correlation between the presence/absence of steroid hormone receptors and response to hormonal therapy. The molecular mechanisms by which these steroid hormone receptors regulate the onset or progression of BPH and prostate cancer are not yet clear. The chronological changes in the levels and distribution of steroid hormone receptors in normal prostatic tissue and the effect of such changes on the synthesis of growth factors, growth factor receptors, and oncogenes should be investigated. PMID- 7541677 TI - Mesenchymal-epithelial interactions and transforming growth factor-beta 1 expression during normal and abnormal prostatic growth. AB - Mesenchymal-epithelial interactions are associated with growth and morphogenesis of the prostate. We have detected three isoforms of transforming growth factor beta (TGF-beta) in the developing mouse prostate that may mediate some of these interactions. Separation of the fetal urogenital sinus (UGS) tissue into mesenchymal and epithelial components indicated that mRNA expression of TGF-beta 1, 2, and 3 was more abundant in the mesenchyme compared to the epithelium. Immunohistochemical analysis revealed accumulation of TGF-beta 1 in the mesenchyme surrounding ductules in the UGS and neonatal prostate. Further analysis of TGF-beta 1 localization in surgically removed adult human prostate tissues revealed more intense staining associated with regions of abnormal growth compared to normally appearing tissue. The percent of the total stained area with extracellular accumulation of TGF-beta 1 was 59% in prostate cancer, 26% in benign prostatic hyperplasia (BPH), and 8.6% in normal tissue. In additional immunohistochemical studies we observed that intracellular TGF-beta 1 was predominantly associated with the epithelial cells in prostate cancer (epithelial cells = 33.5% of the total stained area, stromal cells = 13.3%, and unstained = 53.2%), whereas in BPH intracellular TGF-beta 1 was predominantly associated with stromal cells (stromal cells = 32.2% of the total stained area, epithelial cells = 12.3%, and unstained = 55.5%). Although additional experimental and clinical studies are needed to better understand the relationships between TGF-beta 1 and abnormal prostatic growth, our observations thus far suggest a role for TGF-beta 1 in the development of benign and malignant growth abnormalities in the prostate. One approach to establishing the pathobiological significance of TGF beta 1 accumulation in the prostate is by introducing and overexpressing the TGF beta 1 cDNA in prostate tissue using the mouse prostate reconstitution model system. We discuss applicability of transgenic animal and organ reconstitution models for experimental studies concerning TGF-beta-induced prostate growth abnormalities. PMID- 7541678 TI - Clinical use of pentastarch in cardiac surgery without homologous blood transfusion. AB - In an attempt to avoid the use of homologous blood transfusion, we used pentastarch 6% or 10% in a consecutive series of open heart surgery. After induction of anesthesia, whole blood 500 cc was withdrawn from the patient via an artery line, and 6% or 10% pentastarch 500 cc was infused simultaneously via a central venous line. A further 500 cc pentastarch was placed in the heart-lung machine as a priming solution. After the operation the autologous blood was transfused. Pentastarch 500cc was also infused in the intensive care unit as a plasma expander. Significant improvements in cardiac output with decrease in hematocrit, plasma viscosity, systemic and pulmonary vascular resistance were noted after pentastarch infusion, due to its hemodiluting effects. Patients treated with pentastarch did not require transfusion of homologous blood, and they also displayed no abnormal coagulopathy, or increased amounts of postoperative bleeding during hospitalization. All patients were discharged without additional hospital stay. With the aid of pentastarch, acute normovolemic hemodilution can be performed safely in cardiac surgery with the advantages of saving stored blood, reducing the risks associated with homologous blood transfusion and profiting from hemodilution. It is therefore concluded that cardiac surgery can be performed safely with 6% or 10% pentastarch without any homologous blood transfusion. PMID- 7541680 TI - The 'dice' game: a new test of pragmatic language skills after closed-head injury. AB - There are few clinical tools available to assess communication skills following closed-head injury. This paper describes one such task in which the subject is required to explain a board game to a naive listener. The explanation is taped, transcribed and the content is quantified. Reliability studies demonstrated that the test can be consistently scored. A group of 43 normal subjects was investigated, and some variation in performance according to age and educational background was revealed. A group of 20 brain-injured male adults with executive type deficits were then compared to a matched subgroup of the controls. The clinical group produced less essential information and relatively more unnecessary information than their non-brain-damaged counterparts. Qualitative features of discourse disorganization were also revealed. PMID- 7541679 TI - Correlations between mutagen sensitivity and plasma nutrient levels of healthy individuals. AB - Increased mutagen sensitivity and decreased intake of antioxidant-rich fruits and vegetables have been associated with an increased risk of upper aerodigestive tract cancers. The objective of this study was to investigate the intraindividual variation in mutagen sensitivity and its possible correlation with plasma nutrient levels in a group of 25 healthy individuals in Hawaii. Mutagen sensitivity, as assessed by bleomycin-induced chromosomal breaks in cultured peripheral blood lymphocytes and plasma nutrient levels were measured monthly for 11 months. The monthly numbers of chromosomal breaks/cell ranged from 0.04 to 0.80 and showed considerable intraindividual variation. Based on individual means, significant inverse correlations were found between mutagen sensitivity scores and the plasma levels of alpha-carotene (r = -0.64), total carotenoids (r = -0.41), and ascorbic acid (r = -0.40). There were also significant inverse associations between monthly mean plasma levels of alpha-carotene (r = -0.58), beta-carotene (r = -0.76) and total carotenoids (r = -0.72) and monthly mean chromosomal breaks. In contrast, there was a significant positive correlation between monthly mean plasma triglyceride level (r = 0.60) and monthly mean mutagen sensitivity. These results suggest that mutagen sensitivity as assessed by the bleomycin assay may be influenced by plasma levels of certain nutrients and could potentially be modified by dietary interventions or micronutrient supplementation. PMID- 7541681 TI - Recovery of function following severe traumatic brain injury: a retrospective 10 year follow-up. AB - It has been widely assumed that most of the recovery following severe traumatic brain injury (TBI) occurs within the first 6 months, and that virtually all of the recovery occurs within the first 1-2 years post-injury. In an effort to evaluate the long-term recovery of patients who had sustained severe TBI, we interviewed the relatives and significant others of 20 patients who had sustained TBI at least 5 years earlier, using a modified version of the Portland Adaptability Inventory. Retrospective ratings were collected to evaluate the patients' psychosocial, cognitive, physical, and emotional status prior to their injury, and at 1, 2, 5, and an average of 10.3 years post-injury. The results indicated that TBI patients exhibit significant improvements in their social, cognitive, physical, and emotional functioning after 2 years post-injury regardless of the severity of their initial brain trauma. These data suggest that patients who sustain severe TBI continue to make gradual improvements in their functioning for at least 10 years post-injury. Our findings contradict the widely held assumption that the recovery process ends after 1 or 2 years post-injury. PMID- 7541682 TI - Trends in mortality from nonneoplastic gallbladder disease. AB - Trends in mortality rates from gallstones and other nonneoplastic gallbladder and biliary tract diseases between 1955 and 1990 for 38 countries (8 from America, 3 from Asia, 25 from Europe, Australia, and New Zealand) were analyzed. Age adjusted mortality rates standardized on the world population were computed from official death certifications derived from the World Health Organization database. There were generalized and substantial declines in the rates in both sexes and all countries considered, except for males in Czechoslovakia and Poland. Over the calendar period considered, the average declines were over 70% for males and over 80% for females in North America, over 60% for males and 70% for females in Latin America, although mortality remained relatively high in Chile. The declines were 80% for both sexes in Japan and over 70% for males and 80% for females in Australia. The pattern was more heterogeneous in Europe, with decreases of approximately 70 to 80% in northern Europe, but more modest in central and southern Europe, with particularly moderate downward trends for males. In several countries the decreases were rather steady over the calendar period considered, but in a few others the decline was restricted or larger during the most recent calendar period. The trends in gallstone and other gallbladder disease mortality in various areas are affected by differences and potential biases in death certification reliability, and by underlying variations and changes in the prevalence of gallstones and gallbladder surgical removal. A likely interpretation for the generalized decline in mortality over the last calendar period is, however, improved diagnosis and treatment of gallstone disease. PMID- 7541683 TI - The WHO objectives for palliative care: to what extent are we achieving them? AB - This paper examines recent research in palliative care in the light of the guiding principles set out by the World Health Organization. It outlines the gaps in the literature and suggests priorities for future research. Areas of unmet need are documented and it is argued that research comparing outcomes across care settings and relating particular care practices to outcome measures would help to set care targets. Further definition of the expected outcomes of psychological and spiritual care, as well as care for carers, is recommended. Available measures are reviewed and suggestions made for the development of additional measures. Finally, some key methodological problems are discussed, including making cross-setting comparisons, identifying appropriate outcome measures, prioritizing patients' own identification of outcomes, using different methodologies as death approaches, and combining different perspectives offered by patients, lay carers and professional carers. The role of qualitative data as an indicator of rating scale validity is discussed in this context. PMID- 7541684 TI - Assessing the effectiveness of a hospital palliative care team. AB - A prospective study was carried out of 125 hospital inpatients with malignant disease, referred to the King's College Hospital advisory palliative care team. A palliative care assessment (PACA) tool was developed in order to assess the outcome of interventions made within two weeks of referral with regard to: symptom control, change in the patients' and their relatives' insight regarding diagnosis and prognosis, and facilitation of patient placement. Reliability was assessed by cross-observer analysis, and validity by comparison with data obtained using the McCorckle symptom distress scale in a separate group of hospice inpatients. At initial assessment, the commonest symptom was pain, as reported by 74% of patients. One-third of the patients were unsure of their diagnosis and placement had not been decided in 61%. In total, the team undertook 245 pharmacological interventions for symptom control, 165 interventions regarding insight and 114 interventions concerning placement. Analysis of the data showed statistically significant improvements in pain (p < 0.001), nausea (p < 0.009), insomnia (p < 0.004), anorexia (p < 0.001) and constipation (p < 0.02). Discussion regarding diagnosis significantly changed the insight of patients (p < 0.001) and relatives (p < 0.02). Appropriate placement was assisted by interventions undertaken by the team. This study shows that a hospital palliative care team is effective at improving symptom control, facilitates understanding of the diagnosis and prognosis, and contributes to the appropriate placement of patients. PMID- 7541686 TI - Will the doors open? Multicultural issues in palliative care. PMID- 7541685 TI - Ethical issues in palliative care research revisited. PMID- 7541687 TI - Staff stress in hospice/palliative care: a review. AB - A review of the research in the area of staff stress in hospice/palliative care since the start of the modern hospice movement shows that, while high stress was identified as a problem in the early days of the movement, later studies have shown that stress and burnout in palliative care are by no means universal. Staff stress and burnout in hospice/palliative care has been demonstrated to be less than in professionals in many other settings. However, other studies have noted suicidal ideation, increased alcohol and drug usage, anxiety, depression, and difficulty in dealing with issues of death and dying. It is hypothesized that part of the reason that stress may be lower than expected in some settings was the early recognition of the potential stress inherent in this field and the development of appropriate organizational and personal coping strategies to deal with the identified stressors. Staff in hospice/palliative care have been found to have increased stress when mechanisms such as social support, involvement in work and decision-making, and a realistic work-load are not available. The stress that exists in palliative care is due in large measure to organizational and societal issues, although personal variables were also found to have an influence. Suggestions are given for the direction of future research in the field. PMID- 7541688 TI - Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. AB - 1. Lipopolysaccharide (LPS) co-induces nitric oxide synthase (iNOS) and cyclo oxygenase (COX-2) in J774.2 macrophages. Here we have used LPS-activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX-2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX 2 activity was assessed by measuring the formation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Western blot analysis was used to determine the expression of COX-2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS-treated rats. 2. Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 microgram ml-1) for 24 h caused a time-dependent accumulation of nitrite and 6-keto-PGF1 alpha in the cell culture medium which was first significant after 6 h. The formation of both 6-keto-PGF1 alpha and nitrite elicited by LPS was inhibited by cycloheximide (1 microM) or dexamethasone (1 microM). Western blot analysis showed that J774.2 macrophages contained COX-2 protein after LPS administration, whereas untreated cells contained no COX-2. 3. The accumulation of 6-keto-PGF1 alpha in the medium of LPS-activated J774.2 macrophages was concentration-dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1-1000 microM). Sodium nitroprusside (1-1000 microM) also acutely (30 min) inhibited COX-2 activity in broken cell preparations of LPS-activated (12 h) J774.2 macrophages, in a similar concentration dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX-2 in broken cell preparations. In the presence of these co-factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 microg ml-1) and NG-monomethyl-L-arginine (L-NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX-2 in cell homogenates with co-factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 microg ml-1)and L-NMMA (1 mM), the addition of SNP (0.001-1I000 microM) increased in a concentration dependent manner the accumulation of 6-keto-PGF1a in intact cells (measured at 24 h) and COX-2 activity in cell homogenates in the presence of co-factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h)decreased the amount of COX-2 protein induced by LPS in J774.2 macrophages.4. Indomethacin (30 1AM) abolished the formation of 6-keto-PGFa by LPS-activated macrophages, but had no effect on the release of nitrite. Conversely, L-NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6-keto-PGFIa an effect which was reversed by excess L-arginine (3 mM)but not by D-arginine. Similarly, the decrease in nitrite formation caused by L-NMMA was partially reversed by L-arginine (3 mM), but not by D-arginine. L-NMMA (10 mM; together with LPS for 12 h)increased the amount of COX-2 protein induced by LPS in J774.2 macrophages.5. In separate experiments, J774.2 macrophages were activated with LPS (1 microg ml-1), and L-NMMA(10 mM) was added for various times (0.5-24 h) before the collection of mediun at 24 h. L-NMMAenhanced the release of 6-keto PGFI,, in a time-dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h. 6. In experiments on male Wistar rats, we investigated the effect of L-NMMA on the release of prostanoids (6-keto PGF1a prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA,30nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS-treated rats was unaffected by L-NMMA intraperitoneally (30 mg kg-1) for 6 h together with LPS(5 mg kg-1) or LPS vehicle. Similarly, acute (5 min) in vitro exposure to L-NMMA (1 mM) of the perfused organs from control and LPS treated animals did not change the release of prostanoids elicited by AA (30 nmol).7. These results show that LPS causes the induction of iNOS and COX-2 in J774.2 macrophages. The co-release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag-time of 6-12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX-2 activity and induction. These results demonstrate that NOS and COX can be co-induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity.In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX-2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX. PMID- 7541689 TI - Substance P-induced inflammatory responses in guinea-pig skin: the effect of specific NK1 receptor antagonists and the role of endogenous mediators. AB - 1. The sensory neuropeptide substance P (SP), when released from sensory nerves, has been implicated in the development of neurogenic inflammation. In the present study, using an in vivo model system, we have characterized and investigated the mechanisms underlying SP-induced leukocyte accumulation and oedema formation in the guinea-pig. 2. Intradermally injected SP (i.d., 10(-13) - 10(-9) mol per site), induced a dose- and time-dependent accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation as measured by the local accumulation of i.v. injected 125I-albumin. The leukocyte accumulation evoked by SP was significant at 10(-10) and 10(-9) mol per site, whereas oedema formation was significant at the lowest dose tested (10(-13) mol per site). 3. The NK1 receptor antagonists, CP-96,345 (1 mg kg-1, i.v.) and RP-67,580 (10 micrograms per site, i.d.), significantly attenuated the oedema formation induced by the lower doses of SP. Oedema formation and leukocyte accumulation induced by 10(-9) mol per site SP were unaffected by either antagonist. 4. SP-elicited responses were not significantly affected by the platelet activating factor (PAF) receptor antagonist, UK-74,505 (2.5 mg kg-1, i.v.) or the H1 histamine receptor antagonist, chlorpheniramine (10(-8) mol per site, i.d.). However, the 111In eosinophil accumulation, but not the 111In-neutrophil accumulation or oedema formation, induced by SP was significantly inhibited by the specific 5 lipoxygenase (5-LO) inhibitor, ZM-230,487 (10(-8) mol per site, i.d.). 5. The accumulation of both 111 In-neutrophils and 111 In-eosinophils induced by SP was abolished in guinea-pigs treated i.v. with an anti-CD18 monoclonal antibody 6.5E F(ab')2 (2.5 mg kg-1). The oedema response was unaffected in these animals.6. These results suggest that SP-induced inflammatory events may be mediated via two mechanisms involving NK1 receptor-dependent and independent pathways. Oedema formation induced by the lower doses of SP may be mediated via the direct activation of NK1 receptors whilst, at higher doses, oedema formation and leukocyte accumulation may be mediated via the release of secondary mediators, possibly mast cell derived, with 5-LO products playing an important role in the leukocyte infiltration. The leukocyte accumulation, but not the oedema induced by SP, is dependent on the expression of the CD18antigen on leukocytes. PMID- 7541690 TI - Assessment of renal dopaminergic system activity in the nitric oxide-deprived hypertensive rat model. AB - 1. The present paper reports changes in the urinary excretion of dopamine, 5 hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long-term administration of NG-nitro-L-arginine methyl ester (L NAME). Aromatic L-amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly-formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined. 2. Twenty four hours after exposure to L-NAME, both systolic (SBP) and diastolic (DBP) blood pressure were increased by 20 mmHg; heart rate was slightly decreased. During the next 13 days both SBP and DBP increased progressively reaching 170 +/- 3 and 116 +/- 3 mmHg, respectively. 3. Baseline urinary excretion of L-DOPA, dopamine, 3,4 dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) during the 4 day period of stabilization averaged 4.4 +/- 0.5, 13.8 +/ 0.3, 37.4 +/- 0.8, 180.0 +/- 2.7 and 206.1 +/- 6.7 nmol day-1, respectively. The urinary excretion of L-DOPA, dopamine and DOPAC, but not that of 3-MT and HVA, were increased from day 6-8 of L-NAME administration onwards (L-DOPA, up to 13.4 +/- 2.1; dopamine, up to 23.0 +/- 1.6; DOPAC, up to 62.8 +/- 3.7 nmol day-1). Baseline daily urinary excretion of 5-hydroxytryptamine and 5-hydroxyindolacetic acid (5-HIAA) averaged 73.5 +/- 1.1 and 241.7 +/- 5.4 nmol day-1, respectively. During the first week of L-NAME administration, the urinary excretion of both 5 hydroxytryptamine and 5-HIAA did not change significantly; however, as was found with dopamine and DOPAC, changes in the urinary excretion of 5-hydroxytryptamine were evident during the second week of L-NAME administration. 4. In experiments performed on homogenates of isolated renal tubules, the decarboxylation of L-DOPA to dopamine was dependent on the concentration of L-DOPA used (10 to 5000 microM) and saturable at 1000 microM. AAAD activity as determined in homogenates (Vmax, in nmol mg-1 protein h-1; Km in microM) was significantly (P < 0.01) higher in rats given L-NAME for 14 days (Vmax = 25 +/- 2; Km = 72 +/- 10) than in control rats (Vmax = 14 +/- 1; Km = 63 +/- 7), rats given L-NAME for 7 days (Vmax = 15 +/ 1; Km = 69 +/- 5) and rats given L-NAME plus L-arginine (Vmax = 13 +/- 1; Km = 60 +/- 3) for 14 days. 5. A considerable amount of the total dopamine formed from added L-DOPA in kidney slices escaped into the incubation medium. The application of the Michaelis-Menten equation to the net transport of newly-formed dopamine allowed the identification of a saturable (carrier-mediated transfer) and a non saturable component (diffusion). No significant differences in the diffusional rate of transfer(0.14 +/- 0.02 micro mol-1) were observed between the four experimental groups. However, the saturable outward transfer of dopamine (Vmax, in micromol mg-1 protein h-1; Km in microM) was higher in control animals(Vmax= 2.3 +/- 0.2; Km = 568 +/- 67) than that in rats treated with L-NAME for 14 days (Vmax = 0.8 +/- 0.02;Km = 241 +/- 21), but similar to that observed in rats receiving L-NAME plus L-arginine (Vmax= 2.4+/- 0.2; Km= 618 +/- 61); the saturable dopamine outward rate of transfer in rats given L-NAME for 7days (Vmax = 3.9 +/- 0.2; Km = 1006 +/- 32) was higher than in controls.6. In conclusion, the present studies show that the hypertensive response resulting from the long term administration of L-NAME is accompanied by an increased urinary excretion of dopamine and 5-hydroxytryptamine, which appears to follow an enhanced activity of renal AAAD. The observation that the increased AAAD activity can be reversed by the administration of L-arginine to L-NAME treated rats favours the view that the adaptational response which results in an enhanced AAAD activity probably involves a decrease in the generation of nitric oxide. PMID- 7541691 TI - Effects of acute and chronic ethanol on cyclic AMP accumulation in NG108-15 cells: differential dependence of changes on extracellular adenosine. AB - 1. This study investigated the effects of acute and chronic ethanol on basal, agonist- and forskolin-stimulated cyclic AMP formation in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, and examined the role of changes in extracellular adenosine concentrations on the effects observed. 2. NG108-15 cells incubated acutely with ethanol (1-200 mM) displayed concentration-dependent increases in basal and iloprost-stimulated (300 nM; a prostanoid IP receptor agonist) cyclic AMP accumulation but a concentration-dependent decrease in forskolin-stimulated (10 microM) accumulation. 3. Cells treated chronically with ethanol (200 mM) for 48 h displayed increases over control in basal, iloprost- (0.001-10 microM) and forskolin (0.01-100 microM)-stimulated cyclic AMP formation. However, chronic ethanol did not affect [3H]-iloprost binding to cell membranes. 4. Inclusion of adenosine deaminase (ADA; 1 unit ml-1) during the incubation period to measure cyclic AMP accumulation completely abolished the increase in basal accumulation following chronic ethanol, but did not affect the increase in iloprost stimulation. On the other hand ADA partially reversed the increase in forskolin stimulation following chronic ethanol, but even in the presence of high concentrations of ADA (5 units ml-1) the forskolin stimulation remained elevated above control. 5. Cells treated chronically with the adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA; 10 microM for 48 h) displayed a reduction in subsequent NECA- and forskolin-stimulated cyclic AMP accumulation, but iloprost stimulation was not affected. ADA included acutely during the incubation period to measure cyclic AMP accumulation abolished the reduction in forskolin but not NECA stimulation produced by the chronic NECA pretreatment. 6. We have previously noted that ethanol inhibits NG108-15 cell proliferation and alters cell morphology.To mimic this, cells were incubated in the absence of foetal calf serum for 48 h. Following this time, basal, iloprost- and forskolin-stimulated cyclic AMP formation was enhanced over that in cells grown in the presence of serum.7. These results indicate that chronic ethanol enhances cyclic AMP formation in intact NG108-15 cells by more than one mechanism: one involves increased extracellular adenosine concentrations and the other a change in the transduction system beyond the receptor, possibly involving the adenylyl cyclase enzyme. Furthermore the ethanol-induced changes in cyclic AMP accumulation may relate to alterations in NG108-15 cell growth and development. PMID- 7541692 TI - Inhibition of serum-induced proliferation of bovine tracheal smooth muscle cells in culture by heparin and related glycosaminoglycans. AB - 1. The effect of heparin and related glycosaminoglycans on bovine airway smooth muscle proliferation has been investigated. 2. Foetal bovine serum stimulated division of bovine trachealis smooth muscle cells in a concentration-dependent fashion at concentrations between 1 and 30%. 3. Heparin (0.1-100 micrograms ml 1), heparan sulphate (0.1-100 micrograms ml-1) and fragmin (0.1-100 micrograms ml 1) inhibited smooth muscle division in a concentration-dependent fashion between 0.1-100 micrograms ml-1. A heparin disaccharide did not exhibit inhibition of division at 100 micrograms ml-1. 4. Dextran sulphate at molecular weights of 5 x 10(3) and 5 x 10(5) concentration-dependently inhibited division between 0.1-100 micrograms ml-1. Dextran without sulphation did not exhibit inhibition of division at 100 micrograms ml-1. 5. The magnitude of inhibition of proliferation did not reach 100% for any compounds examined at concentrations up to 100 micrograms ml-1 during incubations for 5 and 14 days. IC50 values for inhibition of proliferation ranged between 1-5 micrograms ml-1. 6. These findings suggest that heparin and related glycosaminoglycans inhibit bovine airway smooth muscle cell division. PMID- 7541693 TI - Increase by NG-nitro-L-arginine methyl ester (L-NAME) of resistance to venous return in rats. AB - 1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on mean circulatory filling pressure (MCFP), total peripheral resistance (TPR), cardiac output (CO) and resistance to venous return (Rv) were studied in rats. 2. In conscious, unrestrained rats, L-NAME (0.5-16 mg kg-1) dose-dependently increased mean arterial pressure (MAP) but not MCFP, an inverse index of venous compliance, either in the absence or presence of the ganglionic blocker mecamylamine (10 mg kg-1). 3. In pentobarbitone-anaesthetized rats, L-NAME (2, 4, 8 mg kg-1) increased MAP and reduced CO in a dose-related manner but did not change MCFP, TPR (+84, +140 and +192%) as well as Rv (+62, +72, +110%) were dose-dependently increased by L-NAME. 4. Our results show that L NAME reduces CO by increasing arterial as well as venous resistances. L-NAME does not affect MCFP. PMID- 7541696 TI - Molecular mechanics and dynamics studies on two structurally related amide modified DNA backbones for antisense technology. AB - The effect of the replacement of the natural phosphodiester linkage -C3'-O-PO2-O CH2-C4'- in the DNA strand of RNA.DNA hybrid duplexes by either of the two amide linkages -C3'-CH2-CO-NH-CH2-C4'- or -C3'-CH2-NH-CO-CH2-C4' has been investigated by molecular mechanics (MM) and molecular dynamics (MD) simulations. Conformational analysis has been used to assess various low-energy conformers of the amide-modified backbones. MD simulations have been carried out to study the dynamic behavior of the modified duplexes. The modified RNA.DNA hybrid double helices kept a conservative base pairing scheme during the MD simulations. Although the general behavior has been found to be similar to that of the corresponding wild-type hybrid duplexes, some notable differences, especially regarding the sugar puckering in the amide-modified DNA strands, have been observed. The behavior of the RNA strands in the hybrid duplexes has not been affected by the modified DNA strands and is similar to that in wild-type RNA.DNA duplexes. PMID- 7541694 TI - Blockade of mast cell histamine secretion in response to neurotensin by SR 48692, a nonpeptide antagonist of the neurotensin brain receptor. AB - 1. Pretreatment of rat isolated mast cells with SR 48692, a nonpeptide antagonist of the neurotensin (NT) receptor, prevented histamine secretion in response to NT. 2. This inhibition was rapid in onset (approximately 1 min) and dependent upon the concentration of SR 48692 (IC50 approximately 1-10 nM). 3. SR 48692 (1 1000 nM) did not inhibit histamine secretion elicited by substance P, bradykinin or compound 48/80, or by anti-IgE stimulation of sensitized mast cells. 4. When SR 48692 was injected intradermally (5 pmol in 50 microliters) into anaesthetized rats, 15 min before the intradermal injection of NT, it reduced the effect of NT on vascular permeability. 5. When injected intravenously, SR 48692 attenuated the effects of NT on haematocrit and blood stasis. 6. These results demonstrate that SR 48692 selectively antagonizes the actions of NT on rat isolated mast cells as well as mast cells in vivo. Given the demonstrated specific interaction of SR 48692 with receptors for NT in brain, our results suggest the presence of specific NT receptors on mast cells. PMID- 7541695 TI - Modulation of acetylcholine release at mouse neuromuscular junctions by interaction of three homologous scorpion toxins with K+ channels. AB - 1. The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 125I-labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes. 2. On chick biventer cervicis preparations, CTX and IbTX (125 nM) augmented the twitch responses to indirect muscle stimulation. Further, the increase (about 70 80% of control twitch height) was fast in onset, reaching a maximum within 25-30 min. NTX at 125 nM produced a slower augmentation of the twitch responses to indirect muscle stimulation, with the maximum response being seen after 40-50 min. 3. On mouse triangularis sterni preparations, CTX (300 nM after 35-40 min) and IbTX (100 nM after 15 min) increased quantal content of the evoked endplate potentials (e.p.p.) by about two fold. However, NTX (300 nM) caused only a small increase in e.p.p. amplitude, which was followed by repetitive e.p.ps in response to single shock nerve stimulation after 40-50 min. 4. Extracellular recording of nerve terminal current waveforms in triangularis sterni preparations revealed that CTX and IbTX (3-100 nM), but not NTX (100 nM), blocked the Ca(2+)-activated K+ current, IK-Ca. However, there was no major change in the portion of the nerve terminal waveform associated with voltage-dependent K+ currents, IKv. 5. In the radioligand binding assay, NTX potently displaced labelled [125I]-DpI, whereas CTX produced only partial displacement. However, IbTX did not displace [125I]-DpI from its binding sites on rat brain synaptosomal membranes.6. We conclude that these three structurally homologous scorpion toxins act on different K+ channels and that this leads to different patterns of facilitation of acetylcholine release. IbTX acts selectively on high conductance Ca2+-activated K+ channels, leading to an increase in the amplitude of e.p.ps without any other changes. NTX acts on voltage-dependent K+ channels that are sensitive to dendrotoxin and causes repetitive e.p.ps. CTX shares amino acid residues that exist in the structures of IbTX and NTX;CTX acts on both Ca2+- and voltage-dependent K+ channels. PMID- 7541697 TI - Substance P markedly ameliorates scopolamine-induced impairment of spontaneous alternation performance in the mouse. AB - We investigated the effects of intracerebroventricular injection of substance P (SP) on the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance in the mouse. SP (0.001-3 micrograms) alone did not influence either spontaneous alternation performance or total arm entries. Scopolamine (1 mg/kg) impaired spontaneous alternation performance accompanied by an increment in total arm entries. In contrast, SP (0.01-1 micrograms) significantly improved the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance without influencing the scopolamine (1 mg/kg)-induced increase in total arm entries. The effects of SP (0.1 micrograms) on the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance were almost completely reversed by pretreatment with WIN 62577 (1 mg/kg), a tachykinin NK-1 receptor antagonist. These results suggest that SP improves the scopolamine-induced impairment of spontaneous alternation performance through the mediation of tachykinin NK-1 receptors. PMID- 7541699 TI - The projections to the medulla of neurons innervating the carotid sinus in the dog. AB - The localization and brain stem projections of neurons innervating the carotid sinus of the dog were studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Within the brain stem, labeled afferent fibers and presumptive terminals were found bilaterally in the caudal nucleus tractus solitarius (nTS), the area postrema (AP), and the lateral tegmental field (LTF), reaching the area of the nucleus ambiguus (nA). Sparse labeling was also seen in the ipsilateral spinal trigeminal nucleus (SpV) and lateral cuneatus nucleus (LCn). These findings suggest the existence of multiple pathways by which peripheral baroreceptor inputs may influence central cardiovascular-related neurons. In addition to classically defined relay in the nTS, carotid sinus afferents may also interact more directly with these neurons in other brain stem regions. PMID- 7541698 TI - Congenital brain damage spares the basic patterns of parental behavior in affected mice. AB - Pregnant albino mice were treated with 5-azacytidine so that the embryonic brains were affected late in their morphological ontogeny. The offspring showed retarded body growth and a conspicuous reduction in the size of the cerebral hemispheres as measured at the end of development. Histological alterations were found in the hippocampus and the cingulate cortex. No behavioral alterations were detected during development, with the exception of the hyperactivity which probably caused the better performance of treated offspring observed in a self-feeding test. This functional abnormality, attributed by previous authors to retardation in telencephalic development, persisted into adulthood. The parental behavior of virgin females towards a weak stimulus-object was robust. Treated subjects were non-neophobic, seldom aggressive and showed clearcut parental responses. In addition, although the frequency of overall parental tendency was lower in the treated subjects, it gradually approached that of the controls across repeated trials. The brain structures affected by this treatment seem influential on behavioral organization and habituation to novelty, not on basic patterns of behavior, which are probably rooted in phylogenetically more ancient structures. PMID- 7541700 TI - Calmodulin regulates fast axonal transport of squid axoplasm organelles. AB - The role of calmodulin (CaM) in organelle motility (fast axonal transport) in the axoplasm of the squid giant axon was evaluated directly using video-enhanced microscopy. Addition of 6 microM CaM to extruded squid axoplasm produced a 2.6 fold increase in the number of organelles moving per minute per unit area of axoplasm. When lower concentrations of CaM, including physiological concentration (2 micrograms/ml), were added to extruded axoplasm, the number of organelles moving was equally increased. CaM had no significant effect on the mean velocity of organelle translocations. The stimulatory effect of CaM was reduced significantly by the CaM inhibitors melittin (36 microM) and trifluoperazine (50 microM). Parvalbumin, a high-affinity calcium binding protein, did not stimulate motile activity. These results suggest that CaM is a positive regulator of fast axonal transport. At the molecular level, this regulation may involve microtubule and/or actin-based motor proteins. Several possible molecular mechanisms are proposed. PMID- 7541701 TI - Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: a comparison to 5-HT neurons. AB - Anti-nitric oxide synthase antibody was used to study the distribution, cytoarchitecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudal length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventral cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoreactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoreactive axon terminals that were in this area formed synapses. PMID- 7541702 TI - Cytochemical characteristics of cat spinal neurons activated during fictive locomotion. AB - Using standard immunohistochemical and histochemical techniques, we have examined the neurochemical characteristics of a subpopulation of locomotor-related neurons as labeled by the activity-dependent marker c-fos. Results were compared to those obtained from a small sample of intracellularly labeled locomotor-related neurons. In the paralyzed, decerebrate cat, fictive locomotion was evoked by electrical stimulation of the mesencephalic locomotor region. Most c-fos immunoreactive neurons were distributed in medial lamina VI and VII and in lamina VIII and X. Double labeling of c-fos with various cytochemical markers revealed that about one-third of the c-fos-immunoreactive neurons were choline acetyltransferase immunoreactive, about one-third were glutamate immunoreactive, and about one-third were aspartate immunoreactive. In addition, approximately 15% of the c-fos-labeled neurons contained NADPH-diaphrorase reaction product, while almost 40% appeared to receive close contacts from calcitonin gene-related peptide-immunoreactive fibers and boutons. Choline acetyltransferase- or aspartate immunoreactivity was observed in some intracellularly labeled neurons. These findings have implications regarding the putative neurotransmitters utilized by subpopulations of locomotor-related neurons in the cat spinal cord. PMID- 7541704 TI - Differential age-related influences on memory for verbal-symbolic information and visual-spatial information? AB - Adults from a wide range of ages were administered parallel versions of three memory tasks designed to involve verbal-symbolic information or visual-spatial information. Several analytical procedures were used to determine whether there were selective age-related effects on measures reflecting spatial information processing compared to those reflecting verbal-symbolic information processing. The results provided little support for this hypothesis and instead were consistent with the existence of a common age-related factor that contributes to the age differences in many cognitive measures. PMID- 7541705 TI - Morphine for pain control, not death. PMID- 7541703 TI - Analysis of asparagine-linked oligosaccharides by sequential lectin affinity chromatography. AB - Lectins are proteins that specifically bind to a particular carbohydrate structure. Affinity chromatography with immobilized lectins is a quite effective technique not only for the fractionation of glycoproteins or oligosaccharides but also their structural assessment. In this article, we focus on the separation of glycopeptides and oligosaccharides derived from glycoproteins by affinity chromatography on immobilized lectin columns. PMID- 7541707 TI - The "issue" around this "piece". PMID- 7541706 TI - Oxidized lipoproteins induce long-lasting inhibition of nitric oxide synthase from a murine endothelioma cell line (bEnd.4). AB - BACKGROUND: The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood. METHODS: We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO4. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using Greiss reagent. RESULTS: The synthesis of nitric oxide by the bEnd.4 cell line was calcium dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 micrograms/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins. CONCLUSION: These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular disease. PMID- 7541708 TI - A novel p16INK4A transcript. AB - p16INK4A and p15INK4B were initially identified as potent inhibitors of activated cyclin/cyclin-dependent kinase complexes. These genes were colocalized to chromosome 9p21, and p16 was subsequently found to be mutated in familial melanoma and deleted in a wide variety of sporadic cancers. We recently found that de novo methylation of a 5' CpG island led to transcriptional block of full length p16 in many neoplasms. However, the presence of a truncated p16 transcript in methylated cell lines led us to investigate the presence of an alternative promoter or initiation site. We have now identified an abundant alternative p16 transcript in both methylated and unmethylated cell lines generated from a novel sequence (exon 1 beta) potentially involved in the complex regulation of these critical cell cycle genes. PMID- 7541709 TI - Distant metastases from prostatic carcinoma express androgen receptor protein. AB - Nearly all primary prostatic carcinomas have been found to express the androgen receptor (AR) protein, which is the intracellular mediator of androgen action. To gain a better insight into the mechanisms of androgen independence of advanced prostatic carcinoma, it is important to know whether the AR is also present in metastases of androgen-independent tumors. We have assessed the status of the AR and the prostate-specific antigen in 22 metastases of 18 patients with progressive prostate cancer. In 18 cases, the metastases were localized in bone, in 3 cases in the epidural space, and in 1 case in the periosteum. All but one patient had received some kind of endocrine treatment for prostatic carcinoma. Paraffin-embedded tissue sections were stained for the AR following a streptavidinbiotin-peroxidase protocol with the polyclonal antibody PG-21, which is directed against amino acids 1 through 21 of the rat and the human AR. The percentage of AR-positive cells was evaluated on the basis of an arbitrary 4 point scale. All 22 tumor metastases displayed AR positivity. One AR-positive metastatic lesion did not stain for prostate-specific antigen, but in all other metastases, this protein was detected by means of immunohistochemistry. The present study provides evidence that, unlike androgen-independent prostatic carcinoma cell lines, distant prostatic carcinoma metastases do express the AR. These findings indicate that the AR may be involved in the progression of prostate cancer. PMID- 7541710 TI - Induction of apoptosis by 5-azacytidine: drug concentration-dependent differences in cell cycle specificity. AB - There are conflicting data in the literature as to whether cytotoxicity of the cytidine antimetabolite 5'-azacytidine (AZC) is a consequence of its incorporation into RNA, DNA, or both. Because apoptosis appears to be the predominant mode of tumor cell death after treatment with most antitumor drugs, and in the case of some drugs, the proclivity of the cell to undergo apoptosis varies depending on the cell cycle position, this study was aimed toward elucidating whether induction of apoptosis by AZC is cell cycle phase specific. Human promyelocytic leukemic HL-60 cells were treated with varying concentrations of AZC, and flow cytometric methods that identify apoptotic cels and provide information about the cell cycle distribution of the apoptotic and nonapoptotic cell populations were used. At 2-6 microM concentrations of AZC, the cells in the G1 phase preferentially underwent apoptosis, whereas the cells in G2-M were particularly resistant. Although incorporation of bromouridine into RNA was suppressed at that low AZC concentration, the rate of 5'-bromo-2-deoxyuridine incorporation into DNA was not significantly affected. At an AZC concentration of 8-40 microM, no cell cycle phase specificity in induction of apoptosis was apparent, but both the rate of 5'-bromo-2-deoxyuridine incorporation into DNA and bromouridine into RNA were reduced in proportion to drug concentration. The data suggest that the mechanism of cell killing by AZC may be different, depending on its concentration. Namely, whereas incorporation of AZC into RNA may play a predominant role in the induction of cytotoxicity of G1 cells at low drug concentrations, the perturbation of both RNA and DNA metabolism may be responsible for triggering cell death in the G1 and S phases, as is seen at higher concentrations of this antimetabolite. PMID- 7541711 TI - Activity of a single-chain immunotoxin that selectively kills lymphoma and other B-lineage cells expressing the CD40 antigen. AB - We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas. Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) [G28-5 sFv(VL-VH)-PE40] and the second with the sFv in the opposite orientation [G28-5 sFv(VH-VL)-PE40]. Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher. A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40. All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin. The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40. PMID- 7541712 TI - Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter. AB - The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but is reactivated in hepatocellular carcinoma. It has been shown that the positive and negative transcriptionally regulatory elements of the human AFP gene, which play an important role in its developmental regulation, exist over the quite extended region (4 kb). We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of the 0.3-kb human AFP gene promoter and inserted it into a retroviral vector. When AFP-producing hepatoma cells were infected with this recombinant retrovirus (LNAF0.3TK virus), the cells expressed HSV-tk gene and exhibited increased sensitivity to ganciclovir parallel with the ability of AFP production. On the other hand, the retroviral infection had little effect on ganciclovir-mediated cytotoxicity in AFP-nonproducing hepatoma or non hepatoma cells. Moreover, the addition of dexamethasone increased the cytotoxicity of aciclovir to the virus-infected, AFP-producing cells through a glucocorticoid-responsive element in the AFP promoter, although aciclovir, by itself, had little cytotoxicity. These results demonstrate that the AFP promoter sequence alone can provide enough tumor-specific activity for therapeutic gene expression and induce selective growth inhibition by ganciclovir in the virus infected, AFP-producing human hepatoma cells. In addition, it is possible that expression of the therapeutic gene is modulated by administration of dexamethasone or other agents that alter AFP promoter activity after gene transduction. PMID- 7541713 TI - Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362. AB - The purpose of this study was to determine whether the activation of inducible nitric oxide synthase (iNOS) can serve as a target for immunotherapeutic agents for treatment of murine reticulum cell sarcoma metastases. Liver metastases were established by the i.v. injection of M5076 cells into syngeneic C57BL/6 mice. Multiple systemic administrations of multilamellar vesicle-liposomes (MLV) containing the lipopeptide CGP 31362 (MLV-31362) or MLV-31362 combined with murine IFN-gamma eradicated the metastases. Tumor regression correlated with iNOS expression within the tumor lesions detected by Northern blot and immunohistochemistry techniques and with increased production of nitric oxide (NO). The administration of a specific iNOS inhibitor, NG-methyl-L-arginine, significantly decreased NO production and diminished the antitumor activities of the immunomodulators. Consistent with the regression of hepatic metastases, the combination of MLV-31362 and IFN-gamma synergistically induced iNOS gene expression, NO production, and apoptosis in the tumor cells under in vitro and in vivo conditions. The addition of NMA prevented the production of NO and apoptosis. These data imply that multiple systemic administrations of MLV-31362 plus IFN-gamma activate endogenous iNOS in sarcoma cells, which then undergo apoptosis, leading in turn to the regression of M5076 sarcoma hepatic metastases. PMID- 7541715 TI - Retinoid-induced suppression of squamous cell differentiation in human oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice. AB - Retinoids are promising agents for therapy of squamous cancers. In vitro, retinoids decrease expression of differentiation markers in head and neck squamous carcinoma cells. Little information is available on effects of retinoids on head and neck squamous carcinoma cell xenograft growth in vivo. To address this issue, head and neck squamous carcinoma cells (line 1483) were established as xenografts in nude mice. Control tumors grew rapidly with doubling times of 4 6 days to mean volumes of 1696 mm3 after 24 days. Histological analyses indicated the formation of well-differentiated squamous carcinoma cells exhibiting pronounced stratification (basal and suprabasal cells) and keratinization (keratin pearls) with abundant stroma. Cytokeratin 19 expression was restricted to the basal cell layers, and cytokeratin 4 expression was abundant in suprabasal cells. Mice were treated daily with 30 mg/kg 9-cis retinoic acid, 20 mg/kg all trans-retinoic acid, or 60 mg/kg 13-cis retinoic acid by p.o. gavage on a schedule of 5 days/week over 4 weeks. Low micromolar (1.48-3.67 microM) and nanomolar (200-490 nM) concentrations of 9-cis retinoic acid and all-trans retinoic acid were measured in plasmas and xenografts, respectively, 30 min after dosing. Retinoid treatment produced a marked suppression of the squamous cell differentiation of tumor cells manifest by decreased keratinization, loss of stratification, and accumulation of basal cells. This was accompanied by large decreases in the number of CK4-positive cells and concomitant increases of CK19 positive cells. REtinoic acid receptor-beta expression was also increased by 2.9 9.7-fold after chronic retinoid treatment. 9-cis retinoic acid and all-trans retinoic acid decreased tumor volumes by 23 +/- 5 (SE) and 19 +/- 3%, respectively (P < or = 0.05); 13-cis retinoic acid was inactive. These retinoids did not decrease the rate of exponential tumor growth but increased the latent period until exponential growth began. These studies demonstrate that retinoids do not universally decrease tumor growth but profoundly suppress squamous cell differentiation in vivo in this xenograft model. PMID- 7541716 TI - Myocardial performance is modulated by interaction of cardiac endothelium derived nitric oxide and prostaglandins. AB - OBJECTIVE: The endocardial endothelium modulates the performance of the subjacent myocardium and plays an important role in regulating cardiac function. The aim of this study was to investigate the effects on cardiac function of the mutual interaction of nitric oxide (NO) and prostaglandins released from the endocardial endothelium. METHODS: The effects of NO and prostaglandin interaction were investigated in isolated cat papillary muscle (Krebs Ringer; 1.25 mM Ca2+; 35 degrees C), using substance P (1 mumol.litre-1) to release NO, L-nitroarginine (L NOARG:30 mumol.litre-1) to inhibit NO synthase, arachidonic acid (10 mumol.litre 1) to stimulate endogenous prostaglandin (mainly prostacyclin) release, and indomethacin (1 mumol.litre-1), a cyclooxygenase inhibitor. RESULTS: Administration of substance P resulted in a negative inotropic effect. This response to substance P was abolished by arachidonic acid, while it was preserved in the presence of indomethacin. In the presence of L-NOARG, arachidonic acid induced a positive inotropic effect with prolongation of the twitch duration, while indomethacin reduced the twitch duration. CONCLUSIONS: Activation of prostaglandins abolishes the effect of NO, while prostaglandin-induced positive inotropic effect requires NO synthesis inhibition. Accordingly NO and prostaglandins interact to regulate myocardial performance. PMID- 7541714 TI - Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes. AB - MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART 1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART 1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27 35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC. PMID- 7541717 TI - Insulin-like growth factor II is an experimental stress inducible gene in a porcine model of brief coronary occlusions. AB - OBJECTIVE: Previous observations have shown that myocardium activates many adaptive processes after brief ischaemia. The aim of this study was to determine whether insulin-like growth factors (IGF) as well as their receptors and binding proteins (IGFBP), which control the activity of the IGF, may play an important role during these processes. METHODS: Ischaemia was induced in anaesthetised open chest pigs by two 10 min occlusions of the left anterior descending coronary artery, separated by 30 min of reperfusion, and followed by reperfusion up to 210 min. Tissue from the ischaemic area and from a non-ischaemic control region of the same heart was examined by means of northern blot, slot blot, and in situ hybridisation. RESULTS: IGF-I, IGF-II, the type I receptor, the insulin receptor, and IGFBP-2-6 are constitutively expressed in porcine myocardium. In situ hybridisation showed that IGF-I and IGF-II are mainly transcribed by myocytes. Ischaemia/reperfusion led to an early and significant increase in IGF-II mRNA compared to non-sham controls but not in comparison with sham operated animals, which already showed a (not significantly) enhanced IGF-II expression. In each case the IGF-II mRNA levels are equal in the control and the experimental region of the same heart. Whereas IGF-II expression was already increased by experimental stress, IGFBP-5 mRNA was enhanced only by ischaemia/reperfusion. The expression of IGF-I, the receptors, and IGFBP-2, 3, 4, and 6 remained unchanged during the experimental protocol. IGFBP-1 was neither expressed nor induced in our model. CONCLUSIONS: IGF-II acts like a stress-response gene activated by the experimental conditions (surgery, anaesthesia) and remains induced during following episodes of ischaemia/reperfusion. A possible interaction of IGFBP-5 with other components of the IGF system may contribute to the preconditioning response. PMID- 7541718 TI - Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig. AB - Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electron-dense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed. PMID- 7541719 TI - Substance P-, F8Famide-, and A18Famide-like immunoreactivity in the nervus terminalis and retina of the goldfish Carassius auratus. AB - We re-investigated the occurrence of substance P-like immunoreactivity in the retina of the goldfish Carassius auratus using antisera to substance P and other tachykinins. Most antisera labelled a previously described single class of mono stratified amacrine cells arborizing in layer 3 of the inner plexiform layer. Preabsorption experiments showed that these amacrine cells contained at least one tachykinin-like peptide. One antiserum (INC 353) to substance P labelled not only these amacrine cells but also fibres in layer 1 of the inner plexiform layer and fibres in the optic nerve. These fibres were identified as retinopetal projections of the nervus terminalis, in part because of colocalized labelling with antisera against gonadotropin-releasing hormone and FMRFamide. Preabsorption experiments showed that the substance P-immunoreactive material in the nervus terminalis was not substance P or any other typical tachykinin. Labelling of the nervus terminalis with INC 353 was blocked by preabsorption with two bovine FMRF amide-like peptides, F8Famide and A18Famide, which contain a substance P(4-7) like region. Antisera to F8Famide and A18Famide strongly labelled ganglia of the nervus terminalis and retinopetal fibres. We suggest that labelling of the nervus terminalis by antisera to substance P and FMRFamide occurs because of homologies between these antigens and a non-tachykinin, endogenous peptide that is similar to F8Famide and A18Famide. PMID- 7541720 TI - Quadruple colocalization of calretinin, calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in fibers within the villi of the rat intestine. AB - Double-labeling immunofluorescent histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi. PMID- 7541721 TI - Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. AB - Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene. PMID- 7541722 TI - Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. AB - Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued. PMID- 7541723 TI - Migration of human polymorphonuclear leukocytes through a synovial fibroblast barrier is mediated by both beta 2 (CD11/CD18) integrins and the beta 1 (CD29) integrins VLA-5 and VLA-6. AB - Polymorphonuclear leukocytes (PMNL) accumulate in joint fluid in inflammatory arthritides. We investigated the molecular mechanisms required for PMNL migration through a barrier of human synovial fibroblasts (HSF) grown on microporous filters, as a model of PMNL migration through synovial connective tissue and compared this process with PMNL migration through human dermal fibroblast (HDF) barriers and through human umbilical vein endothelium (HUVE). A small amount of PMNL migration occurred spontaneously only through the synovial fibroblast/filter unit (6-10%). Migration markedly increased through all cell monolayers when the chemotactic factors C5a, IL-8, or zymosan-activated plasma (containing C5adesArg) were added to form a chemotactic gradient. The migration induced by C5a, IL-8, or C5adesArg across HSF was partially inhibited (25-76% depending on stimulus) by mAb to CD18 (beta 2 integrin). The CD18-independent migration induced by IL-8 or C5adesArg was almost completely inhibited by mAbs to beta 1 integrin, but with C5a, inhibition by mAb to beta 1 integrin was only partial (40-50%). Inhibition by mAb to beta 1 integrin required treatment of the PMNL, but not the HSF and was only observed when the function of CD11/CD18 on PMNL was also blocked by a mAb. Treatment of PMNL with mAb to alpha 5 (VLA-5) plus alpha 6 (VLA-6) in combination, was required to inhibit CD18-independent migration through HSF to the degree observed with mAb to beta 1 integrin. There was no qualitative difference in the mechanisms utilized by PMNL for migration through HSF or HDF in response to chemotactic factors. In contrast, PMNL migration across HUVE was almost completely CD18-dependent (85%) with no role for beta 1 integrins. The results suggest that (a) PMNL migration through HSF in response to chemotactic factors utilizes both CD11/CD18 and beta 1 (CD29) integrins; (b) the VLA-5 and VLA-6 members of beta 1 integrins are involved in mediating migration; and (c) PMNL utilize similar mechanisms for migration through HSF and HDF, which are distinct from migration through HUVE. PMID- 7541724 TI - Germinal center T cells exhibit properties of memory helper T cells. AB - Potential roles of germinal center (GC) T cells in immune memory were investigated by examining T cells in GCs lymph nodes (LNs) following immunization with myelin basic protein (MBP). MBP preferentially stimulates T cells expressing the V beta 8 TCR family in B10.PL mice. By staining lymph nodes with peanut agglutinin, GCs were first identified 8 days after immunization and could still be detected 50 days later. When T cells within GCs were examined at Day 8, the majority (> 90%) were CD45RB-negative and a large percentage (20%) had already proliferated several days before as determined by bromodeoxyuridine (BrdU) incorporation. When BrdU+ V beta 8+ cells were examined in various areas of LNs 3 and 8 weeks after immunization, BrdU+ V beta 8+ cells were approximately fourfold more frequent in GCs than other areas of LNs. When GCs were examined for IL-4 mRNA+ cells using an in situ hybridization method, only 0.01% of GC cells expressed IL-4 mRNA, corresponding to about 1% of GC T cells. Shortly following restimulation with MBP, the frequency of cells expressing IL-4 mRNA increased fivefold. Taken together, these findings indicate that GC T cells possess characteristics associated with memory T cells. PMID- 7541725 TI - Synthetic copolymer 1 and myelin basic protein do not require processing prior to binding to class II major histocompatibility complex molecules on living antigen presenting cells. AB - In the present study we attempted to examine whether copolymer 1 (Cop 1), a synthetic basic random copolymer of amino acids (a candidate drug for multiple sclerosis (MS)), and myelin basic protein (MBP) undergo processing prior to their binding to MHC class II molecules on antigen-presenting cells (APC). The direct binding of biotinylated Cop 1 and MBP to living APC was monitored by flow cytometry using phycoerythrin (PE)-streptavidin. The time course for either Cop 1 or MBP binding was similar at 37 degrees C and on ice. Both Cop 1 and MBP bound to glutaraldehyde-fixed APC. Furthermore, these biotinylated antigens bound also in the presence of protease inhibitors and lysosomotropic agents, suggesting that proteolysis is not required prior to their interaction with the MHC determinants. Finally, short fragments of Cop 1 molecule did not bind to most of the APC, suggesting that the polymeric nature of Cop 1 is important for its efficient and promiscuous binding. PMID- 7541726 TI - The molecular mechanism of FasL-mediated cytotoxicity by CD4+ Th1 clones. AB - Murine CD4+ Th1 clones require de novo synthesis of proteins to express a cytotoxicity that is mediated by de novo synthesized Fas ligand (FasL). The cytotoxic process of the CD4+ Th1 effectors can be separated into four stages, namely conjugate formation, activation, lethal hit, and effector-independent target cell death. The present study describes the cytotoxic process in terms of FasL induction and Fas/FasL molecular interactions for death signal transduction. Fas-Ig fusion proteins, cycloheximide, actinomycin D, and EGTA+MgCl2 were used to analyze each stage of the cytotoxic process in terms of FasL/Fas participation. The results demonstrate that the activation-induced de novo mRNA and protein synthesis were for FasL, which provided the predominant cytotoxic activity of CD4+ Th1 effectors. Once activated, Th1 effectors express cytotoxic activity in the presence of EGTA+MgCl2, an experimental [Ca2+]ext-independent condition characteristic of FasL-mediated cytotoxicity. The ability of Fas-Ig to inhibit target lysis declined rapidly after conjugate formation, indicating that FasL mediated lethal hit is critically dependent on conjugate formation and, once delivered, the effector-independent target lysis proceeds. After the lethal hit stage, transduction of Fas-mediated death signal was independent of de novo synthesis of macro-molecules in targets because treatments that inhibited more than 98% of the macromolecule synthesis had little effect on target lysis. Our study provides the first molecular view in terms of FasL/Fas of the cytotoxic process of CD+ Th1 cells. PMID- 7541727 TI - CD4+CD57+ T cells derived from peripheral blood do not support immunoglobulin production by B cells. AB - A small subpopulation of CD4+ T cells found in peripheral blood coexpresses the CD57+ marker normally found on, e.g., NK cells. It is known that this population occurs in a higher frequency in certain diseases. The same antigen has also been shown to be expressed on CD4+ T cells derived from germinal centers. The localization of this cell population to specialized lymphoid structures suggests that it may play a role in the evolution of the antibody response following antigenic stimulation in vivo. We have examined the ability of peripheral blood helper T cells coexpressing CD57 to participate in B cell activation/differentiation and evaluated their responses to polyclonal stimulation. The CD4+CD57+ T cells do not express mRNA for a number of different cytokines or for the CD40 ligand after activation in vitro. Furthermore these cells do not induce differentiation of B cells into immunoglobulin-producing cells. Consequently, despite their CD4 phenotype and their ability to be activated, to express the IL-2 receptor, and to enter into the cell cycle, they do not act as T helper cells under conditions where CD4+/CD57- cells normally do so. The findings suggest that this peripheral blood helper T cell population is functionally different from regular CD4+ T cells. The basis for the lack of proper costimulatory signals for immunoglobulin production might be related to the low expression of CD28. PMID- 7541728 TI - Differential expression of Fas antigen and Bcl-2 protein on CD4+ T cells, CD8+ T cells, and monocytes. AB - Fas antigen and Bcl-2 protein are considered to be involved in cellular homeostasis. We precisely analyzed the expression of human Fas antigen and Bcl-2 protein on CD4+ T cells, CD8+ T cells, and monocytes. The positivities of Fas antigen on CD4+ and CD8+ T cells increased with aging. In CD4+ T cells, the Fas /CD45RO+ subpopulation was dominant compared with the Fas+/CD45RO- subpopulation. Conversely, in CD8+ T cells, the Fas+/CD45RO- subpopulation was dominant compared with the Fas-/CD45RO+ subpopulation. Monocytes exhibited high positivity of Fas antigen and low fluorescence intensity of Bcl-2 protein compared with T cells. These results suggest that Fas antigen acts in different processes of cellular homeostasis between CD4+ and CD8+ T cells and that expression of Fas antigen and Bcl-2 protein is involved in homeostasis of monocytes as well as lymphocytes. PMID- 7541729 TI - IL-5 enhances in vitro and in vivo antigen-specific IgA production in MHC genetically determined low IL-5 responder mice. AB - Lymphonode cells from BALB/k mice, but not from BALB/c mice, immunized with picryl chloride (PCl) produce IL-5 when stimulated with the specific antigen in vitro and this correlates with picryl-specific IgA levels in vivo, which are 6 to 10 times higher in BALB/k mice. B lymphocytes from BALB/k mice cultured with PCl immune T cells from BALB/k produce in vivo anti-PCl-IgA, while B lymphocytes from BALB/c mice, cultured with T cells from BALB/c mice, fail to produce appreciable amounts of anti-PCl IgA, unless IL-5 is added to cultures. B lymphocytes from both strains of mice produce similar amounts of total IgA antibodies when stimulated in vitro with lipopolysaccharide. In vivo administration of IL-5 to BALB/c mice increases significantly PCl-specific IgA levels to those observed in BALB/k mice and a dose-response analysis reveals that 500 units of IL-5 was the minimal effective dose, although a small increase in PCl-specific IgA levels was observed with 100 units of IL-5. Total IgA levels were increased in both strains of mice following in vivo injection of IL-5, but no significant difference in the values was observed. Our results therefore indicate that IL-5 in vivo enhances antigen-specific IgA production in MHC-determined low IL-5 responder mice and suggest an explanation for IgA deficiency in humans. PMID- 7541730 TI - The activity of xenobiotic enzymes and the cytotoxicity of mitoxantrone in MCF 7 human breast cancer cells treated with inducing agents. AB - This study investigated the effect of inducers on the major enzymes responsible for metabolising the quinone antitumor agent mitoxantrone, and on its cytotoxicity in MCF 7 human breast cancer cells. Four inducers were used: 1,2 benzanthracene (BA), phenobarbitone (PB); rifampicin (R) and dexamethasone (DEX). Of these, BA was the most effective, increasing cytochrome P450 dependent metabolism 64-fold and DT-diaphorase activity 1.6-fold. R did not cause an increase in any of the enzyme activities measured and, in fact inhibited glutathione peroxidase activity. PB and DEX increased NADPH cytochrome c reductase activity but had no effect on either DT-diaphorase or cytochrome P450 dependent activities. BA potentiated the cytotoxicity of mitoxantrone in terms of leakage of lactate dehydrogenase (LDH) activity and loss of reduced glutathione (GSH) and protein from cultures. PB had a smaller potentiating effect on cytotoxicity and DEX had no effect. Studies with the enzyme inhibitors, dicoumarol (inhibits DT-diaphorase) and metyrapone (inhibits cytochrome P450), indicate that at least two reactive species are involved in mitoxantrone cytotoxicity. One intermediate, formed by cytochrome P450, caused LDH leakage and GSH depletion. Formation of the second intermediate was catalysed by DT diaphorase and this hydroquinone caused loss of intracellular protein and GSH. We propose that autooxidation of the hydroquinone resulting in generation of reactive oxygen species contributes to mitoxantrone cytotoxicity. Concomitant exposure to inducing agents may alter the cytotoxicity associated with many cytotoxic drugs, not just mitoxantrone, and this is an important consideration as many cytotoxics have a narrow therapeutic index. PMID- 7541731 TI - Pelizaeus-Merzbacher disease: a point mutation in exon 6 of the proteolipid protein (PLP) gene. AB - Pelizaeus-Merzbacher disease has been known since 1885. It is characterized by severe dysmyelination of the central nervous system. We describe a new mutation in exon 6 of the proteolipid protein gene in a 9-year-old boy with severe connatal Pelizaeus-Merzbacher disease. PMID- 7541732 TI - The diversity of perinuclear antineutrophil cytoplasmic antibodies (pANCA) antigens. PMID- 7541733 TI - The treatment of Wegener's granulomatosis. PMID- 7541734 TI - Role of nitric oxide in autoimmunity. PMID- 7541735 TI - High-affinity monoclonal antibodies against P-glycoprotein. AB - Cancer patients treated with one anticancer agent often develop resistance to a broad spectrum of chemotherapeutic agents. This type of multiple drug resistance (MDR) is often accompanied by a decrease in drug accumulation and an increase in expression of a 170,000-Da plasma membrane glycoprotein (P-170) that can effectively pump various anticancer agents out of cytoplasm. A panel of 12 IgG1, IgG2a, or IgG2b monoclonal antibodies was generated against the extracellular portion of P-glycoprotein by immunizing mice with a human MDR1 gene-transfected BA3T3 fibroblast line. We have characterized two of the anti-P-glycoprotein monoclonal antibodies, 15D3 and 17F9, in some detail. Both antibodies immunoprecipitate a 170- to 180-kDa protein from MDR cells, but do not block binding of the known anti-P-glycoprotein antibody MRK16, suggesting that 15D3 and 17F9 bind to a different epitope on the extracellular domain of P-glycoprotein than MRK16. Scatchard analysis revealed that 15D3 and 17F9 had association constants of 1.3 and 1.1 x 10(8) M-1, respectively. 15D3 and 17F9 had little effect on MDR cell growth except for a minor inhibition of KB-V1 cells when the cells were incubated in the presence of vinblastine. Neither antibody inhibited the efflux of P-glycoprotein substrates from MDR cells. Because of their strong binding activity, these antibodies may be useful for diagnostic detection of MDR in patients undergoing chemotherapy or as targeting components of immunotherapeutic agents. PMID- 7541736 TI - Mapping of B-cell epitopes recognized by antibodies to histones in subsets of juvenile chronic arthritis. AB - The sera of 138 patients with juvenile chronic arthritis (JCA) were tested in ELISA with the five individual histones, 34 histone peptides covering the full length of the four core histones, and two peptides corresponding to the N- and C terminal domains of H1. The occurrence of IgG antibodies was examined regarding the different subsets of JCA (pauciarticular, polyarticular, and systemic onset) and regarding clinical features (chronic anterior uveitis, CAU) and other serological features (antinuclear antibodies, ANA). Seventy-two percent of the 138 sera reacted with at least 1 histone peptide. The peptides 204-218 of H1, 1 25 of H2B, and 111-130 of H3 were recognized by 22-28% of JCA sera, and 42% of JCA sera reacted with one or both peptides 1-25 of H2B and 111-130 of H3. The frequency of occurrence of anti-histone antibodies (AHA) regarding the type of histone fraction (H1, H2A, H2B, H3 and H4) or the regions of histones was not significantly different in the three subsets of JCA. No obvious association was found between IgG antibodies to histone peptides and uveitis. In the subset of pauciarticular JCA, 13/31 patients (41.9%) with CAU against 14/41 patients (34.1%) without CAU possessed IgG antibodies reacting with peptides of the C terminal domain 83-135 of H3. This difference is not statistically significant. Finally, the presence of antibodies to histones and histone peptides cannot completely explain ANA reactivity found in patients' sera. Although antibodies to histone peptides occur frequently in the serum of children with JCA, the antibody profiles seem to be highly individual and do not correlate with disease subtype or activity. Identification of AHA present not only in the circulation but also in tissue deposits may provide better insight into the identification of pathogenic antibodies. PMID- 7541737 TI - Serum anti-dextran antibodies in IgA nephropathy. AB - Abnormally high humoral responses have been described in IgA nephropathy (IgAN), towards antigens commonly involved in infectious events or food intolerance. Here we report a comparative analysis of humoral responses to a common environmental antigen, dextran B 512, present both in nonpathogenic microorganisms and normal diet. Dextran-specific IgG, IgA and IgM were assayed using an ELISA method in serum samples from 121 patients with IgAN, 40 controls and 32 patients with biopsy-proven renal diseases different from IgAN. Among the latter, 17 were transplant recipients. Anti-dextran IgG antibodies were found significantly (p < 0.05) more frequently and at higher levels in IgAN patients than in patients with other renal diseases. Anti-dextran IgA were at significantly (p < 0.05) higher levels in IgAN patients than in controls or untransplanted NIgAN patients. However, similarly elevated levels of anti-dextran IgA were found in IgAN patients and transplanted patients with other renal diseases. These data confirm that IgA and IgG responses towards common antigens are elevated in IgAN, possibly as a consequence of the pathogenesis of this disease rather than because of renal impairment. Moreover, since the same degree of dysregulation was noted in the control group of transplanted patients whose initial diagnosis was not IgAN, these data favour the hypothesis of a global dysregulation of the IgA system in IgAN, yielding enhanced humoral immune responses to possible pathogens and diet antigens. PMID- 7541738 TI - Characterization of a synthetic peptide antibody recognizing rat kidney renin and prorenin. AB - A fourteen amino acid peptide from the cDNA predicted rat renin amino acid sequence was synthesized and used to produce a polyclonal antibody. The ability of this antibody to recognize rat renin was characterized and compared to that of a previously tested polyclonal anti-rat renin antibody raised against purified rat renin to verify its usefulness as a reagent in renin processing. The anti renin peptide antibody and anti-rat renin antibody were evaluated for specificity of recognition of rat renin by immunohistochemical staining and immunoblotting. The ability of the anti-renin peptide antibody to inhibit renin enzymatic activity and to immunoprecipitate rat recombinant protein was also tested. Anti renin peptide antibody and anti-rat renin antibody had identical recognition of rat renin and prorenin in immunohistochemical stains of rat kidney section and immunoblots. However the anti-renin peptide antibody does not inhibit renin enzymatic activity nor does it immunoprecipitate recombinant rat prorenin. We have produced and characterized an antirenin peptide antibody which recognizes denaturated rat renin and prorenin in a way that is identical to that of an antibody raised against native rat renin. The anti-renin peptide antibody does not recognize nondenatured renin because it did not inhibit renin enzymatic activity nor was it able to immunoprecipitate renin or prorenin. The technique of using synthetic peptides to produce antibody recognizing at renin is useful because low yields of native renin purified from rat kidney are generally insufficient for antibody production. This limits the widespread availability of antibodies that recognize rat renin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541739 TI - Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry. AB - The lens epithelial cells are a primary site of involvement in galactosemia. Changes in their size, shape and proliferative capacity have been observed upon exposure to high galactose. In this report, changes in the cell cycle pattern of normal and galactosemic lens epithelial cells were examined by use of flow cytometry. Both changes in DNA and RNA were observed using the fluorochrome, acridine orange. Under the appropriate conditions acridine orange can be used to differentiate double-stranded DNA from single-stranded RNA. Using this approach, the DNA and RNA of normal and galactosemic (1, 4, or 7 days) lens epithelial cells can be compared. The results indicate that lens epithelial cells, when exposed to 40 mM galactose media or 30 mM glucose for 7 days, are induced to enter mitosis. Mannitol did not mimic these results. Changes in the cell cycle pattern were not observed when the cells were treated for 1 or 4 days. Although higher numbers of cells in mitosis were observed after 7 days exposure to 40 mM galactose, a correlation between proliferation, as measured by 3H-thymidine uptake, and mitosis was not possible. Apoptosis was evaluated as a possible explanation for these results. The changes in the DNA staining pattern could be use to monitor lens epithelial cells during galactosemia. PMID- 7541740 TI - Poor mixing of microdrops with the tear fluid reduces the accuracy of tear flow estimates by fluorometry. AB - The initial rapid fall in tear film fluorescence after instilling a 1 microliter drop of fluorophore could be a result of stimulated tear flow or of distribution of the dye throughout the tear fluid present in the conjunctival sac. An attempt was made to decide this by comparing the fall in fluorescence of two different fluorophores instilled at an interval of several minutes. In fact, very few patterns of initial rapid loss were found, but it was noted that there was usually a strong gradient of fluorescence from side to side across the cornea that took several minutes to dissipate. It seems that lateral mixing of a microdrop with the tear fluid is not instantaneous and that caution should be exercised in interpreting such measurements to estimate the total volume of tear fluid. PMID- 7541741 TI - Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes. AB - The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina. PMID- 7541743 TI - Expression of cell adhesion molecules in the lungs of patients with idiopathic pulmonary fibrosis. AB - Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory disorder restricted to the lungs. Leukocyte entry into the area of inflammation is regulated, at least partly, by endothelial expression of leukocyte-selective cell adhesion molecules (CAMs). To investigate the relevance of these CAMs to the accumulation of leukocytes in IPF, we examined the expression of E-selectin (endothelial leukocyte adhesion molecule-1; ELAM-1), intercellular adhesion molecule-1 (ICAM 1), and vascular cell adhesion molecule-1 (VCAM-1) by immunohistochemistry in lung tissue from nine patients with IPF and five nonsmoking normal subjects. The results demonstrated that in normal lungs, ICAM-1 was weakly expressed on endothelial cells, but neither E-selectin nor VCAM-1 was detected. In the lungs of patients with IPF, E-selectin expression on endothelial cells was restricted to honeycombing regions. Endothelial expression of ICAM-1 was increased throughout the tissue, but VCAM-1 was not detected in IPF. The distribution of leukocytes in lungs with IPF consisted of mostly lymphocyte accumulation in the interstitium and neutrophil accumulation within the airspaces of honeycomb regions. These results suggest that E-selectin may play a role in the recruitment of neutrophils in regions of honeycombing and that ICAM-1 may play a role in lymphocyte recruitment into the interstitium in addition to contributing to neutrophil recruitment in regions of honeycombing in patients with IPF. PMID- 7541742 TI - Cyfra 21-1 as a biologic marker of non-small cell lung cancer. Evaluation of sensitivity, specificity, and prognostic role. AB - BACKGROUND: Cytokeratins are epithelial markers whose expression is not lost during malignant transformation. Cyfra 21-1 is a cytokeratin-19 fragment that is soluble in serum and may be a useful circulating tumor marker. STUDY OBJECTIVE: The aims of this study were (1) to confirm sensitivity and specificity of Cyfra 21-1 in detecting non-small cell lung cancer (NSCLC) and especially the squamous cell subtype, (2) to assess the potential relationship between Cyfra 21-1 and disease stage of the disease in NSCLC, and (3) to evaluate prognostic effect of Cyfra 21-1 in NSCLC. METHODS: An immunoradiometric assay of serum Cyfra 21-1 was performed in 161 patients with lung cancers and 71 others with benign lung diseases. The ability of Cyfra 21-1 to detect different histologic subtypes of lung cancer vs benign lung diseases was assessed through receiver operating characteristic (ROC) curves and comparisons with other tumor markers such as carcinoembryonic antigen, neuron-specific enolase, and squamous cell carcinoma antigen. Comparisons of Cyfra 21-1 levels according to histologic subtype and disease stage were done using Kruskal-Wallis test. Independent prognostic value of Cyfra 21-1 was studied with a multivariate analysis of survival (Cox's model). RESULTS: Using a threshold of 3.3 ng/mL for Cyfra 21-1, sensitivity and specificity were, respectively, 0.59 and 0.94 in NSCLC, 0.68 and 0.94 in the subgroup of the squamous cell carcinoma, and 0.19 and 0.94 in small cell lung cancer. Cyfra 21-1 levels were significantly higher in advanced NSCLC than in early-stage disease. All 29 patients with serum concentrations > 32 ng/mL had stage IIIB-IV and only one of 14 patients with stage I-II disease had Cyfra 21-1 level > 18 ng/mL. In the multivariate analysis of survival, Cyfra 21-1 was an independent prognostic factor along with performance status and disease stage in NSCLC. CONCLUSIONS: Cyfra 21-1 is a sensitive and specific tumor marker of NSCLC, especially of squamous cell subtype. It also reflects the extent of the disease and has an independent prognostic role along with performance status and disease stage in NSCLC. IMPLICATIONS: A high level of Cyfra 21-1 in apparently early stage NSCLC should be an indication for more extensive workup before thoracotomy. The independent prognostic role of Cyfra 21-1 level may be useful in stratifying populations with advanced NSCLC or early-stage resected NSCLC as elevated Cyfra 21-1 levels might identify those patients at high risk for treatment failure. PMID- 7541744 TI - [Surgical and drug therapy of metastatic gastrinoma. Successful palliation over the course of 2 1/2 years]. AB - A midaged lady suffering from ulcer disease for about seven years had undergone several surgical and drug therapies without any satisfying success. Then the diagnosis of a gastrinoma was made. By this time there were already multiple liver metastases. First the large supposed primary tumor of the pancreas was resected and within six months later the resection of the progressing liver metastases became necessary because of local tumor complications. The additional drug therapy with octreotide (Sandostatin) given three times daily subcutaneously prevented further tumor progress. Until now this combination of surgical and medical treatment has proved to be a successful palliative method of treating the malignant gastrinoma for 2.5 years. PMID- 7541746 TI - Congenital corneal vascularisation in a neonatal Thoroughbred foal. PMID- 7541745 TI - Risk factors for a first febrile seizure: a matched case-control study. AB - We conducted a matched case-control study to identify risk factors for first febrile seizures, with special emphasis on characteristics of the acute illness episode. Cases were identified through hospital emergency departments; controls were identified through outpatient clinics and emergency departments. Sixty-nine children with first febrile seizures and no history of previous unprovoked seizures were matched for age (+/- 6 months), site of routine pediatric care, and date of visit (+/- 2 weeks) with 1 or 2 febrile controls who had no history of previous febrile or unprovoked seizures. Medical records for the index visit were reviewed, and parents were interviewed by telephone. Illness characteristics examined included height of temperature, type of underlying illness, contact with a physician during the illness but before the index visit, and use of acetaminophen or decongestants. Family history of febrile and of unprovoked seizures, sociodemographic characteristics, daycare use, and selected pre- and perinatal variables were also studied. On multivariable analysis, significant independent risk factors were height of temperature, history of febrile seizures in a first- or in a higher degree relative. Gastroenteritis as the underlying illness had a significant inverse (i.e., protective) association with febrile seizures. Maternal smoking during pregnancy was a marginally significant predictor of febrile seizures. PMID- 7541747 TI - Molecular epidemiology of respiratory syncytial virus: a review of the use of reverse transcription-polymerase chain reaction in the analysis of genetic variability. AB - Respiratory syncytial virus (RSV) is the major viral cause of lower respiratory tract disease (bronchiolitis and pneumonia) in babies and infants. Infections with the virus occur as annual winter epidemics in temperate climates, placing considerable pressure on the provision of hospital beds. The virus is unusual in that it can reinfect individuals and it can infect babies despite the presence of maternal antibody. RSV has a negative sense nonsegmented RNA genome and as such is liable to high levels of mutation. This paper describes methods developed to determine the degree of genetic variability of the virus both during individual epidemics and worldwide. It is necessary for these methods to be quick, easy and cheap so that large numbers of samples can be analysed readily. They are based on extraction of viral RNA directly from clinical samples or from viral cultures, reverse transcription of the viral RNA, and then amplification of selected regions of the genome by the polymerase chain reaction (PCR). PCR products are then analysed by restriction mapping, or, if necessary, direct nucleotide sequencing. In this way isolates of RSV have been shown to fall into a number of genotypes, with epidemics being made up of cocirculating genotypes whose relative proportions vary with each epidemic. An understanding of the molecular epidemiology of this important human pathogen will be of significance in the search for an effective vaccine. PMID- 7541748 TI - Detection of picornavirus genomic and template RNA strands by a novel semi-nested polymerase chain reaction-technique and agarose-gel electrophoresis. AB - Attempts to evaluate the relative levels of enteroviral genomic and template RNA strands in small biopsy tissue samples from patients have yielded ambiguous data, largely due to the limited amount of RNA available. A novel semi-nested polymerase chain reaction (PCR) technique was developed to enable RNA levels to be examined more accurately. PCR products were visualised by horizontal agarose gel electrophoresis. This technique was demonstrated on linear single-stranded plasmid DNA; viral RNA isolated from a human rhabdomyosarcoma (RD) cell line persistently infected with a mutated coxsackie B5 virus (piRD) and two cell lines, RD and HEp2 cells, acutely infected with a wild-type clinical isolate of coxsackie B5 virus. PMID- 7541749 TI - Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with sodium chloride solutions. AB - A novel method for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R-250, is described, based on the use of 0.5 M NaCl in water as the destainer, requiring only 2-3 h. Concentrated (> 2 M) or dilute (< 0.1 M) salt solutions were unsuitable. The method affords the advantage that expensive organic solvents, such as methanol, acetic acid or trichloroacetic acid, are not needed. Furthermore, salt destaining results in darker purple-blue protein bands as compared to the pale blue color with e.g. methanol/acetic acid destaining. PMID- 7541751 TI - Aneugen-induced micronuclei (MN) in human lymphocytes may be discerned using image analysis techniques when cell-cycle stage is taken into account. AB - We show that for the in vitro cytochalasin-B human lymphocyte micronucleus (MN) test, the quantification of the DNA content of MN and the difference in DNA content between the two macronuclei in the binucleate cells without MN, as measured by image analysis, gives a first estimation of the aneugenic potential of a test compound. Cultures of isolated human lymphocytes were exposed either to gamma-rays as a clastogen or to carbendazim (MBC) as an aneugen. The lymphocytes were stained with Feulgen stain and the MN were analyzed for DNA content with a Magiscan 2A image analyzer. The mean DNA content of MN induced by MBC were statistically higher than gamma-irradiation-induced MN. It was demonstrated that in culture the lymphocytes, as well as the MN, are in different stages of the cell cycle, but this will not affect the discriminating power of the MN DNA content when only G1 cells are considered, or when DNA content of the MN is expressed relative to the total genome. The identification of G1 and G2 cell populations from image analysis data was performed by extrapolation of DNA content data from G1- and G2-sorted lymphocytes with a FacStar plus flow sorter. It was demonstrated that in MBC-treated cells the DNA rearrangement between the macronuclei in binucleates without MN was on the average higher than in gamma irradiated and untreated cells, which points to aneugenic effects of MBC without the formation of MN. In contrast to DNA content measurements, the area of the MN is not a reliable measure for discriminating clastogens from aneugens. PMID- 7541750 TI - On-line capillary electrophoresis-electrospray ionization mass spectrometry of nucleotides. AB - Capillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on-line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP-tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleotides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA. PMID- 7541752 TI - Induction of micronuclei in murine lymphocytes by morphine. AB - Although individuals who abuse drugs are prone to an increased risk of malignancy, the mutagenic and carcinogenic potential of these agents has received relatively little attention. We report here on the potential of morphine to induce micronuclei in murine lymphocytes. Following a single intraperitoneal injection of 20 mg/kg morphine, the frequency of micronucleated binuclear (cytochalasin-blocked) murine T- and B-splenocytes was elevated from 12-36 hr after treatment. The maximum frequencies seen 24 hr after injection were 6.3- and 4.9-fold greater than the respective controls. A dose-dependent induction of micronuclei was observed from 5-20 mg/kg morphine, with no further increases in frequency produced by higher doses. In contrast, incubation of mitogen-stimulated splenocytes with 10(-7)-10(-4) M morphine in vitro produced no change in frequency of micronucleated cells relative to controls. Treatment with the narcotic antagonist naloxone (5 mg/kg) alone had no effect on the frequency of micronuclei, but reduced the clastogenic response of a subsequently administered dose of morphine (20 mg/kg). Thus, in murine lymphocytes morphine indirectly produces genetic damage, which is at least in part opioid receptor-mediated. PMID- 7541753 TI - Structure and epitope characterisation of the O-specific polysaccharide of Proteus mirabilis O28 containing amides of D-galacturonic acid with L-serine and L-lysine. AB - The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P. mirabilis O28 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2 acrylamidoethyl glycosides of amides of alpha-D-GalA with amino acids copolymerised with acrylamide) showed the importance of D-GalA(L-Lys) for manifesting serological specificity of the O-antigen. Serological cross-reactions between P. mirabilis O28, S1959, and R14/S1959 (a transient-like form) are discussed. PMID- 7541754 TI - Structure and epitope specificity of the O-specific polysaccharide of Proteus penneri strain 12 (ATCC 33519) containing the amide of D-galacturonic acid with L threonine. AB - O-specific polysaccharide was isolated from Proteus penneri strain 12 (ATCC 33519) lipopolysaccharide (LPS) and studied using NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear correlation spectroscopy, 13C,1H heteronuclear correlation spectroscopy and chemical methods (O-deacetylation, Smith degradation, partial acid hydrolysis followed by borohydride reduction and methylation). The amide of D-galacturonic acid with L-threonine [D-GalA(L-Thr)] was identified as a constituent of the polysaccharide and the following structure of the tetrasaccharide repeating unit was established: [formula: see text] where the degree of O-acetylation at either position varies over 20-40%. Serological study with LPS, its degradation products and related synthetic glycoconjugates (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with L-amino acids copolymerised with acrylamide) showed that D GalA(L-Thr) plays an important role in manifesting the serological specificity of the P. penneri 12 O-antigen. Serological cross reactions between LPSs of P. penneri 12 and Proteus mirabilis S1959, R14/S1959 (transient-like form), O23 and O28 are discussed. PMID- 7541755 TI - Three-phase bone scintigraphy of hydroxyapatite ocular implants. AB - Hydroxyapatite ocular implants are replicas of lamellar bone tissue derived from the exoskeleton of a reef-building coral by a hydrothermal chemical exchange reaction. Attached to the eye muscles, they act as a passive framework for fibrovascular ingrowth and can be drilled to hold the visible part of the artificial eye and allow synchronous eye movement. Fibrovascular ingrowth has to be confirmed by bone scintigraphy before the drilling procedure. This study monitored the vascular ingrowth into the implant in ten patients over 12 months to establish a clinically feasible imaging protocol. Tracer accumulation was monitored visually and quantitatively in dynamic and single-photon emission tomography (SPET) scans after the intravenous administration of 600 MBq of 99mTc DPD. The implants showed no tracer accumulation in the arterial or blood pool phase. Accordingly, dynamic scintigraphy can be omitted from the imaging protocol. Delayed tracer accumulation appeared no earlier than 2 and no later than 6 months after surgery. Planar scintigraphy is not recommended as high resolution SPET is necessary to separate the implant from the surrounding bone. We conclude that imaging can be confined to high-resolution SPET 3 h after tracer injection, no earlier than 3 months after surgery. The vascularized hydroxyapatite orbital implant is an important in vivo model for bone-seeking agents to study their uptake kinetics independently of any soft tissue and bone disease. Our results provide evidence that in normal bones the chemical absorption of 99mTc-DPD into the crystalline structure of hydroxyapatite is the only quantitatively relevant uptake mechanism. PMID- 7541757 TI - Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns. AB - Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low Km with ethanol and a branched chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala-141 and a higher Km). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding. PMID- 7541758 TI - Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes. AB - Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes DdeI and MnlI was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other. PMID- 7541756 TI - Characterization of the 3' untranslated region of the mouse homeobox gene HoxB5. AB - The mouse pre-B cell line, 70Z/3, expresses multiple transcripts of the homeobox gene, HoxB5. We show here that this heterogeneity is due, at least in part, to the usage of alternative poly-A addition sites in the 3' untranslated region (UT) of the primary HoxB5 transcript. Furthermore, upon analysis of the subcellular distribution of the different HoxB5 RNA species, we found that the transcripts are present mainly in the nucleus, with two-to-five-fold less RNA present in the cytoplasm. These studies suggest that multiple post-transcriptional regulatory mechanisms are involved in the expression of HoxB5 RNA. PMID- 7541759 TI - Biological and immunological comparisons of Enterobacter cloacae and Escherichia coli porins. AB - Bacteriocin susceptibilities indicate that during cloacin DF13 uptake the F porin of Enterobacter cloacae plays a similar role to that reported for the OmpF porin of Escherichia coli during colicin A entry. The translocatory activities of these two porins during the bacteriocin uptake can be substituted by the porins D and OmpC, respectively, under conditions not requiring the receptor binding step. Using anti-peptide antibodies, a peptide located in the internal loop L3 of the Escherichia coli OmpF porin was identified in the D and F porins of Enterobacter cloacae. The results demonstrated the existence of a close relationship between porins in terms of both antigenic determinants and bacteriocin susceptibilities. PMID- 7541760 TI - Expression of the mcrA gene of Escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region. AB - The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA+/RglA+ phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA+/RglA- phenotype and directed production of a 23-kDa polypeptide. Smaller inserts displayed McrA-/RglA- phenotypes, though a 0.7-kb insert did direct production of a 24-kDa polypeptide. A construct carrying the 1.0-kb mcrA insert yielded a single 1.3-kb transcript. The mcrA transcript was found to start from C, G, T and G, namely the fourth, fifth, sixth and seventh nucleotides (nt), respectively, downstream from the last nt of the putative -10 region. Two mcrA transcriptional/transational fusions were made in the pT7-7 expression vector and the protein encoded by these constructs were analysed. Regulation of mcrA expression was studied by quantitative Northern analysis of RNA from various mcrA clones. Together with a computer analysis of the translation initiation region in these mRNAs, the results suggest that the expression of mcrA may be regulated at the translational level. PMID- 7541761 TI - Possible regulation of DNA methyltransferase expression by RNA processing in Streptococcus pneumoniae. AB - Atypical ribosome-binding sites lacking Shine-Dalgarno sequences appear to be used for translation of the DpnM and DpnA DNA methyltransferases of the DpnII restriction system. Preliminary results indicate that the 5'-endpoints of DpnII system mRNAs result from degradation of the original transcript. These tentative findings serve as the basis for a possible regulatory model that would accommodate the DpnII cassette either as a single copy in the chromosome or on a multicopy plasmid. PMID- 7541762 TI - Effect of 5-azacytidine and sinefungin on Streptomyces development. AB - The effect of two DNA-methyltransferase inhibitors, 5-azacytidine (5azaC) and sinefungin (Sf), on the development of Streptomyces antibioticus ETH7451 (Sa) was studied. Pulse labeling experiments and SDS-PAGE analysis of proteins from cells grown in sporulation synthetic medium showed that both inhibitors affect a limited number of systems. Synthesis of the antibiotic rhodomycin was increased in the presence of 5azaC. 5azaC also stimulated the production of actinorhodin in cultures of S. coelicolor A3(2) grown in minimal medium. The analog did not affect the expression of whiB and whiG, two sporulation genes from S. coelicolor A3(2) whose homologues are present in Sa. Overall results indicated that 5azaC and Sf affect specific events associated with differentiation and secondary metabolism in Streptomyces. PMID- 7541763 TI - A classical enhancer element responsive to both lipopolysaccharide and interferon gamma augments induction of the iNOS gene in mouse macrophages. AB - The ability of macrophages to kill some kinds of tumor cells is dependent upon the production of the free radical nitric oxide (NO) by the inducible enzyme NO synthase (iNOS; EC 1.14.13.39). Expression of the iNOS gene is induced by lipopolysaccharide (LPS) and augmented by interferon-gamma (IFN-gamma). Two regions of the iNOS promoter are known to regulate induction, a promoter proximal region I (RI) and a more distal region II (RII). Reconfiguration of RI within the iNOS regulatory region revealed its dependence upon native position and orientation for maximal activity, suggesting that it is a core promoter module, and further implicated the putative octamer element as a contributor to promoter activity. RII, however, functioned in a relatively orientation- and position independent manner. Therefore, it had the characteristics of a classical enhancer element. PMID- 7541764 TI - Cloning and sequence of the cDNA encoding the rat oligodendrocyte integrin beta 1 subunit. AB - We have isolated a cDNA clone encoding an integrin (Itg) beta-subunit polypeptide from a rat cerebral cortex oligodendroglia cDNA library. This beta-subunit was previously designated beta OL (OL for oligodendroglia), since it had an M(r) that differed from that of the rat fibroblast beta 1 protein. The complete nucleotide sequence encoding the signal sequence and mature protein was determined. Comparison of the deduced amino acid (aa) sequence of beta OL to those of integrin beta subunits from other species revealed that beta OL is a member of the integrin (Itg) beta 1 class. The aa sequence of beta OL shows 78.5-97.9% similarity to beta 1 chains from four other species and displays all the features characteristic of the Itg beta subunits, indicating the high degree of structural conservation seen in this subunit of the Itg receptors. PMID- 7541765 TI - Purification and cDNA sequence of a murine protein homologous to the human p62 tyrosine phosphoprotein that associates with the Ras GTPase-activating protein p120 GAP. AB - A 68-kDa protein that binds to the murine dendritic cell-specific monoclonal antibody M342 was purified and its amino acid sequence was partially determined. Corresponding cDNA clones code for a protein that is closely related to a 62-kDa human protein (p62) that associates with the Ras GTPase-activating protein, p120 GAP, suggesting that p62 may have a dendritic cell-specific function or that an M342 cross-reactive epitope may exist on the murine p62 molecule. PMID- 7541766 TI - Interferon-alpha directly inhibits DNA polymerase activity in isolated chromatin nucleoprotein complexes: correlation with IFN-alpha treatment outcome in patients with chronic myelogenous leukemia. AB - We have developed an in vitro assay to assess and predict the potential efficacy of in vivo interferon-alpha (IFN-alpha) treatment (5 x 10(6) units/m2 per day) for patients with chronic myelogenous leukemia (CML). Although determining the numbers and affinities of IFN-alpha receptors on CML cells has been developed as a method for predicting treatment response to IFN-alpha, it fails to predict response in CML. Previously, we and others observed that mitogens, toxins and lectins that bind to cell-surface receptors are endocytosed, escaping endosomes in order to act directly on cellular targets. Therefore, we tested the ability of low concentrations of IFN-alpha (5-10 units) to act directly on DNA polymerase (Pol) in purified chromatin nucleoprotein complexes (NPC). NPC were prepared by a methodology that uses direct treatment of leukocyte nuclei with MspI to generate six NPC-containing fractions (S1, M1, S2, M2, 0.1K and R). We found three general categories of in vitro DNA synthesis response for the six different NPC fractions isolated from the white blood cells of patients with CML (n = 19) before their treatment with IFN-alpha. IFN-alpha induced either stimulation, inhibition or had no apparent effect on Pol activity in the six different NPC fractions in a blind assay. In most of the NPC fractions isolated from the leukocytes of patients with progressive CML and in those from CML patients who failed to show a clinical response to IFN-alpha, this cytokine stimulated or had no effect on Pol activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541768 TI - [Diagnostic follow-up of alveolitis. Inflammatory activity can be determined by alveolar macrophages and their products]. PMID- 7541767 TI - Diagnosis of micrometastases by the amplification of tissue-specific genes. AB - Micrometastases of solid tumors are most commonly detected by immunocytochemistry using monoclonal antibodies directed against tissue-specific gene products like cytokeratin-18 (CK-18) and the carcinoembryonic antigen (CEA). While CK-18 is a marker for epithelia in general, CEA is mainly employed in the detection of gastrointestinal and breast carcinomas. To improve the sensitivity and specificity of micrometastasis detection, we planned to establish polymerase chain reaction (PCR) assays for both markers. Here we provide strong evidence for the existence of a CK-18 pseudogene, since specific amplification (i) was readily obtained from healthy bone marrow donors, (ii) did not require reverse transcription of CK-18 mRNA and (iii) was not abolished by RNase treatment. Using a CK-18-specific probe, Southern blot analyses revealed identical-size fragments for both genomic DNA and a CK-18 cDNA after digestion with appropriate restriction enzymes. On the other hand, the amplification of CEA mRNA (i) was never observed in bone marrow samples of healthy donors or patients without solid tumors, (ii) required intact mRNA and the reverse transcriptase reaction, and (iii) could not be obtained after RNase treatment. In reconstitution experiments, single CEA-expressing tumor cells were reliably detected among 2 x 10(7) normal bone marrow cells. We conclude that, due to the presence of pseudogene(s), PCR based detection systems are not readily suitable for CK-18, while the CEA mRNA amplification should provide a sensitive and specific test for the presence of ectopic, and hence presumed malignant, CEA-expressing cells in body fluids. PMID- 7541769 TI - Dose-dependent responses in insulin-like growth factors, insulin-like growth factor-binding protein-3 and parameters of bone metabolism to growth hormone therapy in young adults with growth hormone deficiency. AB - There is increasing awareness that growth hormone (GH) replacement therapy is also essential in adult patients with growth hormone deficiency (GHD). There are little data available on the dose requirements for replacement therapy in this age group. In childhood, the growth response to GH therapy can serve as an indicator for correct replacement dose. Because this indicator does not exist in adults, we analyzed growth factors and biochemical markers of bone metabolism by specific radioimmunoassays in a group (n = 12) of adult patients (age, 20.0-31.6 years) with GHD with childhood onset before and after a 4-week treatment period (daily, s.c.) with recombinant, human GH at different doses (0.125, 0.25 and 0.5 IU/kg body weight/week). Comparing the basal levels to those on low-dose GH (0.125 IU/kg/week) and on a high dose (0.5 IU/kg/week), the following results were obtained. Insulin-like growth factor-I (IGF-I) in serum: basal, 68.6 +/- 37 ng/ml; low dose, 176.9 +/- 65 ng/ml (p < or = 0.05); high dose, 380.6 +/- 200 ng/ml (p < or = 0.01). IGF-binding protein-3 in serum: basal, 2.13 +/- 0.58 mg/l; low dose, 3.23 +/- 0.84 mg/l (p < or = 0.01); high dose, 3.97 +/- 0.82 mg/l. Osteocalcin in serum: basal, 3.88 +/- 1.27 ng/ml; low dose, 7.01 +/- 2.20 ng/ml (p < or = 0.01); no further increase. Procollagen-I peptide in serum: basal, 113.6 +/- 36.7 microgram/l; low dose, 211.6 +/- 90.4 microgram/l (p < or = 0.01); no further increase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541770 TI - Effects of the gonadotropin-releasing-hormone agonist, D-Trp-6-GnRH, on prolactin secretion in healthy young men. AB - The objective of this study was to examine the changes in basal plasma gonadotropin, alpha-subunit, sex steroids, and prolactin levels and the prolactin and luteinizing hormone (LH) secretion pattern before, during and 161 days after treatment with a depot preparation of D-Trp-6-GnRH in young men. Gonadotropins, alpha-subunit, sex steroids, and prolactin were measured in pooled plasma samples. Additionally, before treatment, several times during its course and on day 161 after treatment, blood samples were drawn for 8 h every 15 min for prolactin and LH measurements. After initial stimulation of the pituitary, administration of a depot preparation of D-Trp-6-GnRH resulted in a constant decrease in gonadotropin and sex steroid concentrations with LH and testosterone concentrations remaining within the limits of prepubertal levels from days 16 to 48. Alpha-Subunit concentrations (0.4 +/- 0.09 IU/l; mean +/- SE) increased after application of D-Trp-6-GnRH, and remained elevated until day 48. Basal prolactin levels (3.5 +/- 0.25 microgram/l) did not change significantly during treatment but afterwards increased consistently with maximal levels at day 141 (15.3 +/- 3.8 microgram/l); they had decreased at day 161 to 10.3 +/- 1.8 microgram/l which is significantly higher than before treatment (p < 0.05). On day 161, prolactin pulse amplitude was significantly higher than before and during treatment (p<0.05), while no significant changes in pulse frequency occurred. No significant temporal coupling between LH and prolactin release could be detected.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541771 TI - Carcinosarcoma of skin. PMID- 7541772 TI - Prostate in ovarian mature cystic teratoma. PMID- 7541774 TI - Relationships between Eskimos, Amerindians, and Aleuts: old data, new perspectives. AB - Eskimos and Aleuts have long shown uncertain anthropological relationships to each other and to Amerindians. Various researchers, using different samples, data, and methods, have derived diverse relationships among them. Despite supervising the collection of anthropometric data during various expeditions around the turn of the century, Franz Boas did not use these data to formulate or support his Eskimo wedge theory. The perception of Eskimo biological uniqueness has persisted (Greenberg et al. 1986; Laughlin et al. 1979; Laughlin and Harper 1988), although it has been challenged by Szathmary and Ossenberg (1978), Szathmary (1979), and Ossenberg (1992). Genetic analyses, whether using nuclear or mitochondrial DNA, have not revealed consistent relationships for various reasons. A widely known multidisciplinary study (Greenberg et al. 1986) has failed to show agreement among data sets. As a result, Boas's theory has not been adequately tested to this day. A modern analysis of anthropometric data collected under Boas casts doubt on an Eskimo wedge and the assumed close relationship between language and biology. Compared with other north Pacific populations, the Siberian, Labrador, and MacKenzie Delta Eskimo samples are anthropometrically closest to northeast Siberians, whereas the Aleuts are closest to some Northwest Coast Amerindians. An analysis with additional data collected by Hrdlicka (1930) reflects ethnohistorical relationships and a geographic pattern of population affinities: The Eskimos of southwestern Alaska are similar to Aleuts and selected Amerindian tribes on the Northwest Coast. Despite linguistic similarities, Eskimo and Aleut populations are diverse, with some being more similar to populations from opposite sides of the north Pacific. PMID- 7541773 TI - T-cell rich B-cell lymphoma: a morphological variant of a variety of non Hodgkin's lymphomas or a clinicopathological entity? PMID- 7541775 TI - Multivariate quantitative genetics of anthropometric traits from the Boas data. AB - The use of multivariate quantitative trait information to address questions of population relationships and evolutionary issues has a long-standing history in human anthropometry. Previous analyses have usually rested on a number of explicit or implicit assumptions that allow phenotypic information to be used as a proxy for quantitative genetic information. One (usually implicit) assumption is that the additive genetic variance-covariance matrix (G) among traits is proportional to the phenotypic variance-covariance matrix (P). In this study we discuss the implications of this assumption, demonstrating that if it is true that G = h2P, where h2 is some constant of proportionality, then (1) the biological (phenotypic) Mahalanobis distance will be proportional to genetic distance, (2) phenotypic and genetic allometry coefficients will be equal, and (3) evolutionary models will become simplified. We then use a multivariate quantitative genetic analysis of 12 anthropometric traits in 5 tribes to demonstrate that G = h2P for at least a portion of the Boas data. PMID- 7541776 TI - B-cell-specific demethylation of BTK, the defective gene in X-linked agammaglobulinemia. AB - BTK, the gene that is defective in X-linked agammaglobulinemia, encodes a cytoplasmic tyrosine kinase that is critical for B-cell proliferation, or survival. To identify regulatory elements that control the expression of BTK we evaluated the methylation pattern of this gene in cell lines and in freshly isolated cells. An Hpa II site that was specifically demethylated in mature B cells but not in pre-B cells, T cells, neutrophils, or nonhematopoietic cells was identified in the tenth intron of BTK. In a 40 kilobase (kb) segment of DNA spanning the entire coding region of BTK plus 3 kb upstream of the first exon there were no other sites that demonstrated lineage-specific demethylation. The B cell-specific demethylation site in intron 10, which falls within the SH2 domain, 26 kb distal to the first exon, occurs in a region rich in regulatory elements including two E2 boxes, two AP-2 sites, and a cAMP response element. It is likely that this site plays a role in maintaining BTK transcription in mature B cells. PMID- 7541777 TI - Genomic organization of the gene coding for the costimulatory human B-lymphocyte antigen B7-2 (CD86). AB - The generation of an antigen-specific T-cell response requires that the T lymphocyte receive two signals from the antigen presenting cell. The specificity of this response is provided by antigen presented to the T lymphocyte and involves stimulation of the T lymphocyte via the T-cell receptor (TCR)/CD3 complex. The second, or costimulatory signal, can be provided by ligation of the B-lymphocyte activation antigens B7-1 (CD80) and B7.2 (CD86) to TCR antigen CD28. The cDNAs for both CD80 and CD86 have been isolated and are predicted to encode type 1 membrane proteins of the immunoglobulin (Ig) superfamily. The predicted protein is composed of a signal peptide followed by two Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail. Here we report that the genomic organization of CD86 reflects its functional structure, and is similar to that found for CD80. The gene is composed of eight exons which span more than 22 kilobases. The predicted protein functional domains of signal peptide, extracellular IgV- and IgC-like regions, and transmembrane domain coincide with the genomic structure. Two independent sequences had been reported for CD86 cDNA which differed in their 5'untranslated (UT) regions. We find CD86 exons 1 and 2 correspond to these alternate 5'UT sequences. Splicing of exon 1 or 2 with the signal peptide encoding exon 3 would produce mRNA transcripts complementary to the reported cDNA clones. Exons 4 and 5 correspond to IgV- and IgC-like extracellular domains, respectively. Exon 6 encodes the transmembrane region and beginning of the cytoplasmic tail. Exons 7 and 8 encode the remainder of the cytoplasmic tail and 3'UT sequences. PMID- 7541778 TI - Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen. AB - The renin-angiotensin system plays an important role in blood pressure regulation. Angiotensinogen, which is mainly produced in the liver, is a unique component of the renin-angiotensin system, because angiotensinogen is only known as a substrate for angiotensin I generation. It is unclear whether circulating angiotensinogen is a rate-limiting step in blood pressure regulation. Recent findings of genetic studies and analyses suggest that the angiotensinogen gene may be a candidate as a determinant of hypertension. To test the hypothesis that angiotensinogen may modulate blood pressure, we transfected antisense oligonucleotides against rat angiotensinogen into the rat liver via the portal vein using liposomes that contain viral agglutinins to promote fusion with target cells, a technique that has been reported to be highly efficient. Transfection of antisense oligonucleotides resulted in a transient decrease in plasma angiotensinogen levels in spontaneously hypertensive rats from day 1 to day 7 after the injection, consistent with the reduction of hepatic angiotensinogen mRNA. Plasma angiotensin II concentration was also decreased in rats transfected with antisense oligonucleotides. Moreover, a transient decrease in blood pressure from day 1 to day 4 was observed, whereas transfection of sense and scrambled oligonucleotides did not result in any changes in plasma angiotensinogen level, blood pressure, or angiotensinogen mRNA level. Overall, our results demonstrate that transfection of antisense oligonucleotides against rat angiotensinogen resulted in a transient decrease in the high blood pressure of spontaneously hypertensive rats, accompanied by a decrease in angiotensinogen and angiotensin II levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541779 TI - Nifedipine prevents renal injury in rats with chronic nitric oxide inhibition. AB - Chronic nitric oxide inhibition promotes hypertension, renal dysfunction, and renal injury by unclear mechanisms. We examined the effects in this model of concomitant treatment with the calcium channel blocker nifedipine. Six adult male Munich-Wistar rats received 0.025% nifedipine in chow. Six untreated rats served as controls. Fifteen days later, renal function was evaluated in anesthetized rats before and after a bolus injection of the nitric oxide inhibitor N omega nitro-L-arginine methyl ester at 3 mg/kg IV. Renal vasoconstriction and systemic hypertension induced by the inhibitor were similar in untreated and nifedipine treated rats. In a second protocol, eight rats received the nitric oxide inhibitor in their drinking water at 2.6 mmol/L. Eight additional rats also received nifedipine as above. At day 15, rats given the nitric oxide inhibitor exhibited systemic hypertension and renal vasoconstriction. Simultaneous nifedipine lowered blood pressure slightly without ameliorating renal hemodynamics. Tail-cuff pressure rose continuously in rats receiving the nitric oxide blocker, reaching 171 +/- 7 mm Hg at 30 days, but remained at 143 +/- 3 mm Hg in rats also given nifedipine. At this stage, rats treated with the nitric oxide inhibitor exhibited extremely variable plasma renin activity, tuft collapse in 10.1 +/- 2.2% of the glomeruli, and renal interstitial fibrosis. Simultaneous nifedipine treatment normalized the dispersion of plasma renin levels, while preventing renal morphological abnormalities. These results suggest that in the chronic nitric oxide inhibition model, sustained operation of voltage-sensitive calcium channels is not essential for renal vasoconstriction but contributes to systemic hypertension and plays a pivotal role in the development of renal structural injury. PMID- 7541780 TI - Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase in hypothalamus and medulla of two-kidney, one clip hypertensive rats. AB - Nitric oxide may act at autonomic sites in the brain to regulate arterial blood pressure. Our goal was to determine whether gene expressions of the brain isoform of nitric oxide synthase and of the beta subunit of soluble guanylyl cyclase, the target of nitric oxide, were altered in discrete autonomic brain regions after induction of hypertension in rats. The two-kidney, one clip model was used to induce hypertension, and measurements were made 3 and 6 weeks after the left renal artery was clipped. Only experimental rats with blood pressures elevated by at least 25 mm Hg were used. Total RNA was purified from microdissected tissue blocks containing hypothalamus, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nitric oxide synthase and guanylyl cyclase mRNA were semiquantified in each region by use of reverse transcription polymerase chain reactions in which known concentrations of deletion mutants of the two genes were coamplified as internal standards. Compared with controls, significant decreases and increases in nitric oxide synthase mRNA were found in the hypothalamus (x 2.2) and caudal ventrolateral medulla (x 6.4), respectively, of hypertensive rats 3 weeks after clipping. These alterations were reversed in hypertensive rats at 6 weeks; levels increased (x 4.6) in the hypothalamus and decreased (x 5.5) in the caudal ventrolateral medulla. Changes in guanylyl cyclase expression paralleled those for nitric oxide synthase in some but not all areas at both time points.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541781 TI - Transforming growth factor-beta 1 expression and phenotypic modulation in the kidney of hypertensive rats. AB - We have previously reported that renal mRNA levels for transforming growth factor beta 1, fibronectin, and collagens were increased in 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) with severe nephrosclerosis. To elucidate the mechanism of hypertension-induced nephrosclerosis, we examined gene expression and localization of transforming growth factor-beta 1 and cellular phenotype in the kidney of 25-week-old SHRSP with moderate renal damage. Renal mRNA was measured by Northern blot analysis. The localization of transforming growth factor-beta 1 and cellular phenotype was determined by immunohistochemistry. In the kidney of 25-week-old SHRSP, renal transforming growth factor-beta 1 mRNA was elevated compared with Wistar-Kyoto rats (WKY), whereas renal collagen mRNAs of SHRSP were not increased. Immunoreactive transforming growth factor-beta 1 in SHRSP was mainly localized in glomerular cells. Furthermore, alpha-smooth muscle actin and desmin were significantly expressed in SHRSP glomerular cells, in contrast to negligible expression of these proteins in WKY. alpha-Smooth muscle actin staining was also observed in interstitial cells, and vimentin, another phenotypic marker, was expressed in atrophic tubular cells of SHRSP, despite no staining of these proteins in WKY. Furthermore, all these phenotypic changes in SHRSP were associated with increased cell proliferation, as shown by the increased number of proliferating cell nuclear antigen-positive cells. Treatment of SHRSP with cilazapril and nifedipine (from the age of 13 to 25 weeks) prevented the increase in transforming growth factor-beta 1 expression and the cellular phenotypic modulation and was accompanied by a reduction of urinary albumin excretion and inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541783 TI - Condyloma acuminatum. PMID- 7541782 TI - Attentional difficulties and the effectiveness of a visual representation strategy for counseling drug-addicted clients. AB - Three hundred thirty-one opioid-addicted clients in methadone treatment were randomly assigned to counselors who used either mapping-enhanced or standard counseling. Those counseled with mapping had lower percentages of during treatment urine specimens that were positive for either cocaine or opiates, and they were rated by their counselors as having higher rapport, motivation, and self-confidence. Clients were further classified into those exhibiting either good or poor attention during an intake interview. The results suggest that clients with poor attention are more likely to have positive urines, but that this tendency appears to be reduced under mapping-enhanced counseling. PMID- 7541785 TI - [Hamartoma of the sebaceous follicle. An immunohistologic analysis with cytokeratins]. AB - A 62-year-old man presented with a nodule 2 cm in diameter on his left cheek, which he had had since childhood. Histological examination showed a circumscribed dermal nodule composed of sebaceous lobules attached to sebaceous ducts and to an infundibular cyst-like structure connected to the epidermis. The wall was lined with squamous epithelium with a stratum granulosum. This structure was reminiscent of a sebaceous follicle. In addition, a melanocytic naevus of the compound type was found. Immunohistological investigations of the tumour with various cytokeratins revealed a pattern of expression characteristic for the mature sebaceous follicle. PMID- 7541784 TI - Stenting and superior vena caval syndrome. AB - The use of intravascular stents in the management of Superior Vena Caval Obstruction is a relatively recent development. We describe our experience with six patients, aged between 64 and 85 years who developed SVC obstruction secondary to malignant disease and who had one or more intravascular stents inserted. Five of the patients had lung cancer and one metastatic breast carcinoma. Positive histology was available in three cases. A partial or complete clinical response was seen in all patients and two patients developed recurrent symptoms. Stenting appears to provide effective palliation of SVC obstruction symptoms. PMID- 7541786 TI - Ion channels in basolateral membrane of marginal cells dissociated from gerbil stria vascularis. AB - The basolateral membrane of isolated strial marginal cells has been probed for conductive pathways by the patch-clamp technique. Two types of voltage insensitive channels were identified in both cell-attached and excised patches. Of these, frequently (69% of excised patches) observed was a Ca(2+)-activated nonselective cation channel having a unit conductance of 24.9 +/- 0.5 pS (N = 16). Other characteristics of this type in excised patches include: 1) linear I-V relations with 150 mM K+ (pipette)/150 mM Na+ (bath), 2) a permeability sequence of NH4+ > Na+ = K+ = Rb+ > Li+, 3) a flickering block by quinine or quinidine (both 1 mM), and 4) a dose dependent block of its activity by ADP or ATP (IC50,ATP/IC50,ADP = 20-35), both from the cytosolic side. Channels with similar characteristics were found in the apical membrane of the same cell; however, the basolateral channels were 2-4 times more densely distributed than the apical counterparts. Also frequently (57%) detected was a Cl- channel of 80.0 +/- 0.5 pS (N = 6), whose activity was Ca2+ independent. Additionally, this Cl- channel had: 1) linear I-V relations with symmetric Cl-, 2) a permeability sequence of Cl- > Br- > I- > or = NO3- > or = gluconate-, and 3) a complete and reversible block by 1 mM diphenylamine-2-carboxylate. In contrast to the apical Cl- channels, the basolateral ones had a much higher density (57% vs. < 1%) as well as a higher unit conductance (80 pS vs. 50 pS) than the apical counterpart. The relative abundance of these two types as the major conductive pathways for Na+, K+, and Cl in the basolateral region must be taken into account when addressing the role of strial marginal cells in generating the positive endocochlear potential. The Cl- channel may facilitate Cl- distribution across the basolateral membrane. PMID- 7541787 TI - Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2. AB - The O antigen is a polymer of oligosaccharide units. O antigens differ in their sugar composition and glycosidic linkages, and genes responsible for O-antigen specific biosynthesis are grouped in the rfb gene cluster. In this study, we identified two abequosyltransferase genes and an acetyltransferase gene in Salmonella enterica groups B and C2 by in vitro assay and identified paratosyl-, tyvelosyl-, and abequosyltransferase genes from S. enterica groups A and D and Yersinia pseudotuberculosis serovar IIA, respectively, by comparison. PMID- 7541788 TI - Effects of growth rate and nutrient limitation on virulence factor production in Burkholderia cepacia. AB - The influence of growth rate and oxygen availability on siderophore, protease, and lipase production in Burkholderia cepacia was assessed for cells grown in a chemostat under iron limitation. Whereas siderophore and protease production increased with growth rate and oxygen yet decreased under oxygen depletion, lipase production demonstrated the opposite trend. PMID- 7541789 TI - Effect of KCl concentration on gating properties of calcium release channels in sarcoplasmic reticulum vesicles. AB - The effect of KCl concentration on the gating properties of the Ca2+ release channel in sarcoplasmic reticulum vesicles was studied through measurement of the permeation of choline and glucose by means of the light scattering method. The calcium concentration which activates the channel at micromolar range was shifted to higher concentration in the presence of KCl. This phenomenon can be explained by competition between Ca2+ and K+ at the Ca(2+)-binding sites. Five millimolar caffeine increased the apparent affinity of Ca2+ about 10 times but the effect of caffeine was not modified by K+. Inhibition of the channel by millimolar Ca2+ was also competed with by K+. These effects of K+ were not duplicated by choline, suggesting that the binding of choline to these sites is weak. Blocking of the channel by ruthenium red and Mg2+ was also competed with by K+, but blocking by procaine or tetracaine was not. After the opening of the channel by ryanodine, higher concentrations of these blockers were required to close the channel, and further, in the presence of K+ much higher concentrations of blockers were required. These results suggest that these drugs do not block the channel by plugging the pore directly but do so by regulating the gate, and that the Ca2+ release channel has many binding sites for activators and inhibitor to the gate. PMID- 7541791 TI - The structural loop II of cobrotoxin is the main binding region for nAChR and epitope in the region is conformation-dependent. AB - Modification of positively charged residues, Lys and Arg, in cobrotoxin revealed that Lys-27, Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 of cobrotoxin were essential for the lethality and binding activity to nicotinic acetylcholine receptor (nAChR). The antigenicity of cobrotoxin was drastically diminished when Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 were modified, while that of the Lys 27-modified derivative was not significantly changed. The CD spectra of cobrotoxin displayed similar patterns after modification of Lys-27, Lys-47, and Arg-28. These findings suggest that Lys-27, Lys-47, and Arg-28 residues may be related to the direct binding to nAChR, and that there is no involvement of Lys 27 in the antigenic determinants of cobrotoxin. Extending the modification to Arg 30, Arg-33, and Arg-36 caused progressive conformational changes of cobrotoxin and resulted in decreased binding activity to antibody and nAChR. This indicates that Arg-30, Arg-33, and Arg-36 may be of structural importance for maintaining the active conformation of cobrotoxin. These results, together with the facts that Tyr-25, Tyr-35, and Trp-29 of cobrotoxin are important in nAChR binding activity, but Trp-29 and Tyr-35 residues are not essential for the antigenicity, suggest that the structural loop II of cobrotoxin is the main binding region for nAChR and the epitope in that region is conformation-dependent. PMID- 7541790 TI - Purification and characterization of a novel glycoprotein which has significant homology to heavy chains of inter-alpha-trypsin inhibitor family from human plasma. AB - Plasmapheresis with a dextran sulfate column is a treatment for patients with hypercholesteremia. When proteins bound to the column during the treatment were fractionated to prepare some known proteins, we found a 57 kDa glycoprotein designated GP57 which showed a new N-terminal amino acid sequence. Western-blot analysis of human plasma revealed that only a 120 kDa protein, GP120, reacted with anti-GP57 antibody. Since GP120 and GP57 had an identical N-terminal amino acid sequence, GP120 is probably the intact form of GP57. The isoelectric point of GP120 was 6.8. N-Glycanase treatment decreased the molecular weight of GP120 by 15 kDa. Neuraminidase and O-glycanase, however, did not affect the molecular weight. Amino acid sequence analyses of the lysylendopeptidase digest of GP120 revealed significant homology to the heavy chains of inter-alpha-trypsin inhibitor (ITI) family. Since GP120 showed no bikunin sequence, and chondroitinase treatment and alkaline treatment of GP120 did not affect its molecular weight, we concluded that GP120 was not a complex with bikunin. We designated GP120 as IHRP (ITI heavy chain-related protein). PMID- 7541792 TI - Are protein-tyrosine phosphatases specific for phosphotyrosine? AB - Protein-tyrosine phosphatases (PTPases) are believed to exhibit restricted specificity toward phosphotyrosine. I demonstrate here that both the Yersinia PTPase and rat PTP1 can dephosphorylate alkyl phosphates such as flavin mononucleotide, pyridoxal 5'-phosphate, D-glucose 6-phosphate, DL-alpha glycerophosphate, O-phospho-L-serine, and O-phospho-L-threonine. The kcat values for alkyl phosphates are orders of magnitude slower than those for aryl phosphates such as p-nitrophenyl phosphate and O-phospho-L-tyrosine, reflecting the intrinsic lower chemical reactivity of the alkyl phosphates. In addition, the kcat values for the PTPase-catalyzed hydrolysis of alkyl phosphates are similar to the kcat values for the PTPase-catalyzed 18O exchange reaction between inorganic phosphate and water. I conclude that the rate-limiting step for the hydrolysis of alkyl phosphates has changed to the phosphorylation of the PTPases, i.e. the formation of the phosphoenzyme intermediate. The implications of the results described in this report in terms of studying the PTPase catalytic mechanism and their potential application in developing selective PTPase inactivators are discussed. PMID- 7541793 TI - Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium dependent epitope. AB - We established monoclonal antibodies (mAbs) against the mouse macrophage galactose/N-acetylgalactosamine-specific lectin (MMGL) that is a 42-kDa calcium dependent lectin, using a solid phase carbohydrate binding assay as a novel strategy for screening mAbs. The specificity of six mAbs were investigated by antibody binding to native or recombinant forms (rML) of MMGL, flow cytometry, and immunoprecipitation using a macrophage cell line RAW264.7. Four of these mAbs strongly inhibited the binding of fluorescein 5-isothiocyanate-labeled galactosylated polylysine to immobilized rML, one inhibited moderately, and one did not inhibit binding. The competitive binding study revealed that the binding sites of these four blocking mAbs were closely related to each other but were different from the rest of these mAbs. A non-blocking mAb having a unique binding specificity (LOM-11) exhibited calcium-dependent binding to rML, suggesting that calcium-dependent epitope was not situated in the vicinity of the ligand binding site. Furthermore, pretreatment of rML with the mAb LOM-11 preserved ligand binding activity, especially in a low calcium environment. The four blocking mAbs mentioned above facilitated the binding of the mAb LOM-11 to rML. These results indicate that there is a positive cooperativity between the lectin's ligand binding site and its physically distinct calcium-dependent epitope. PMID- 7541794 TI - Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. A possible signaling role for the Syk tyrosine kinase. AB - Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages. PMID- 7541795 TI - The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids. AB - The specific recognition of anionic phospholipids in the outer leaflets of cell membranes and lipoproteins by cell surface receptors may play an important role in a variety of physiologic and pathophysiologic processes (e.g. recognition of damaged or senescent cells by the reticuloendothelial system or lipoprotein homeostasis). Several investigators have described anionic phospholipid binding to cells, and phosphatidylserine (PS) binding to a partially purified approximately 95-kDa membrane protein has recently been reported (Sambrano, G.R., and Steinberg, D. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 1396-1400). Using both direct binding and ligand competition assays in transfected cells, we have found that two class B scavenger receptors, SR-BI and CD36, can tightly bind PS and phosphatidylinositol (PI)-containing liposomes (Kd for PS liposome binding to SR BI is approximately 15 micrograms phospholipid/ml or 0.18 nM (mol PS liposomes/l), but not phosphatidylcholine, phosphatidylethanolamine, or sphingomyelin liposomes. PS and PI liposomes, but not the others, could effectively compete with PS liposomes and modified or native lipoproteins for binding to these receptors. Phosphatidic acid, another anionic phospholipid, could also compete, but was not as effective as PS or PI. Class B scavenger receptors are the first molecularly well-defined, specific cell surface receptors for anionic phospholipids to be described. PMID- 7541796 TI - Differential binding and regulation of platelet-derived growth factor A and B chain isoforms by alpha 2-macroglobulin. AB - Alpha 2-Macroglobulin (alpha 2M) is a multifunctional secreted glycoprotein that serves as a ubiquitous proteinase inhibitor and as a binding protein for platelet derived growth factor (PDGF) BB and homologues of PDGF-BB secreted in culture by macrophages. The interaction of alpha 2M with PDGF-A chain molecules has not been addressed. This is a potentially important issue because fibroblasts and smooth muscle cells produce PDGF-AA, whereas macrophages produce mainly PDGF-BB. Recombinant human 125I-PDGF-B chain molecules (AB and BB) bound to plasma derived, native human, or bovine alpha 2M and trypsin-activated alpha 2M on Superose 6 fast protein liquid chromatography gel filtration and on nondenaturing polyacrylamide gel electrophoresis, whereas 125I-PDGF-AA did not. Similar results were obtained with 125I-PDGF isoforms binding to immobilized bovine alpha 2M and alpha 2M-methylamine. The same differential pattern of unlabeled PDGF isoforms binding to alpha 2M was observed by Western blotting of PDGF. Human lung fibroblasts secreted alpha 2M as measured by Western blotting, and fibroblast derived alpha 2M possessed the same differential binding pattern for PDGF isoforms as did plasma-derived alpha 2M. The specific binding of PDGF-AB and -BB to these fibroblasts was inhibited by native bovine alpha 2M, although PDGF-AA binding was not affected. Native alpha 2M preferentially blocked fibroblast chemotaxis to the PDGF-B chain dimers. These data suggest that only PDGF-B chain dimers, such as those produced by macrophages or released from platelets, are regulated by alpha 2M and that PDGF-AA produced by fibroblasts and smooth muscle cells is not controlled by this cytokine-binding protein. PMID- 7541797 TI - Calbindin expression in renal tubular epithelial cells. Altered sodium phosphate co-transport in association with cytoskeletal rearrangement. AB - Sodium-phosphate transport in the opossum kidney (OK) cell line was studied in an OK clonal cell line that was transfected with an episomal vector expressing high levels of rat calbindin (28 kDa). High level expression of calbindin buffered the influx of calcium induced by ionomycin by 53% and raised the basal intracellular calcium from 100 +/- 6 to 150 +/- 8 nM. The decrement in sodium phosphate uptake induced by parathyroid hormone or forskolin was identical in the two cell lines. However, phorbol esters (10(-10)-10(-7) M), which decreased sodium phosphate uptake in the parental OK line, increased it in the calbindin-expressing line. Similarly, the parental clone did not respond to phosphate deprivation, while the calbindin-expressing clone did increase phosphate uptake in response to phosphate deprivation. In the calbindin-expressing cells, phorbol 12-myristate 13-acetate or low phosphate medium, which increased phosphate transport, produced actin filament aggregation, dissociation of the myristoylated alanine-rich C kinase substrate protein from sub-apical actin, and membrane-associated tyrosine phosphate staining. Agonists that reduced sodium phosphate uptake (cAMP, parathyroid hormone) did not affect these cellular features. The cytoskeletal rearrangement, redistribution of the myristoylated alanine-rich C kinase substrate protein, and membrane tyrosine phosphorylation are suggested to be involved in the events by which phosphate transport is increased in this cell line. PMID- 7541798 TI - Interaction between lck and syk family tyrosine kinases in Fc gamma receptor initiated activation of natural killer cells. AB - Ligation of the Fc gamma R on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that Fc gamma R ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early Fc gamma R-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the zeta chain of the Fc gamma R complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-gamma 2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-gamma 2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling Fc gamma R stimulation to critical downstream signaling events. Taken together, our results suggest not only that Fc gamma R stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade. PMID- 7541799 TI - Genomic organization and chromosomal localization of the gene encoding human P selectin glycoprotein ligand. AB - The gene for P-selectin glycoprotein ligand (PSGL-1) has been cloned from a human placenta genomic DNA library. A single intron of approximately 9 kilobases was found in the 5'-untranslated region and the complete coding region resides in exon 2. The genomic clone differs from the cDNA clone isolated from HL-60 cells in that it encodes an extra copy of the decameric repeat located in the extracellular domain of PSGL-1. Further analysis indicated that the PSGL-1 genes of HL-60 and U-937 cells contain 15 repeats, whereas the PSGL-1 genes of polymorphonuclear leukocytes, monocytes, and several other cell lines contain 16 repeats. Transfection experiments did not indicate a functional difference between these two variants of PSGL-1. The two previously observed PSGL-1 mRNA species of 2.5 and 4 kilobases most likely arise from differential utilization of polyadenylation signal sequences. The organization of the PSGL-1 gene closely resembles those of CD43 and human platelet glycoprotein GPIb alpha, both of which have an intron in the 5'-noncoding region, a long second exon containing the complete coding region, and TATA-less promoters. The gene for human PSGL-1, which has been designated SELPLG by the Human Gene Nomenclature Committee, was mapped to chromosome 12q24 using Southern blot analysis of DNA from a set of human-mouse cell hybrids, and fluorescent in situ hybridization on metaphase chromosome spreads. PMID- 7541800 TI - Combination of screening and preoperative endocrine therapy: the potential for an important decrease in prostate cancer mortality. AB - Prostate cancer is the second cause of cancer death in men in the Western world; its medical and social impact is comparable to that of breast cancer in women. Although it is well recognized that early treatment is the only possibility for reducing the high rate of death from prostate cancer, screening and even early treatment are controversial issues due mainly to arguments based upon old literature and lack of awareness of the significant advances recently made in this field. As it is well known that surgical removal of organ-confined prostate cancer cures the disease, and it has been demonstrated that annual screening with prostate-specific antigen coupled with digital rectal examination followed, when indicated, by transrectal ultrasonography of the prostate more than doubles the proportion of organ-confined disease, screening alone offers the possibility of at least doubling the number of patients curable from prostate cancer or the potential for a cure to an estimated 45% of prostate cancer patients compared to a maximum of 20% in the absence of screening. It is important to mention that screening does not detect small and insignificant cancers, especially when random biopsies are not performed routinely. The critical volume of prostate cancer is estimated at 0.3 cm or a tumor 7.5 mm in diameter, if spherical. Such a tumor should increase serum prostate-specific antigen by 0.5 ng/mL. Contrary to the belief that screening detects cancers that are too small, the fact is that screening detects prostate cancer too late or nonorgan- or nonspecimen-confined cancer in 35-50% of cases. There is, thus, a narrow window when prostate cancer can be detected at a curable stage, and even the best available screening techniques cannot succeed in all cases. It should be mentioned that the recent improvements of the technique of radical prostatectomy have markedly improved the acceptability of surgery. Concerning the recent publicity related to watchful waiting, it is essential to indicate that all such reports support the notion that prostate cancer grows slowly, but steadily and irremediably, with increasing malignancy and risk of distant metastases and death if sufficient time is allowed. Another serious limitation of watchful waiting is that the available prognostic factors have a large margin of error and cannot predict with certainty the rate of progression of the tumor.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541801 TI - Behavior of soluble intercellular adhesion molecule-1 and endothelial-leukocyte adhesion molecule-1 concentrations in patients with Graves' disease with or without ophthalmopathy and in patients with toxic adenoma. AB - Expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelium can be considered a critical early step for leukocyte migration from blood to tissues during inflammatory processes. Increased circulating soluble ICAM-1 (sICAM-1) levels have been found in sera from patients with Graves' disease (GD) with or without ophthalmopathy. Serum soluble ELAM-1 (sELAM-1) levels have not been measured in these patients. The aim of this study was to clarify the behavior of sICAM-1 and sELAM-1 levels in patients with hyperthyroidism due to GD with or with or without ophthalmopathy and in hyperthyroid patients with toxic thyroid adenoma. We studied sICAM-1 and sELAM-1 levels in 130 subjects (age 23-54 yr), grouped as follows: group 1, 30 untreated hyperthyroid GD patients (21 females and 9 males) with active ophthalmopathy; group 2, 26 euthyroid GD patients (16 females and 10 males) with active ophthalmopathy; group 3, 33 hyperthyroid GD patients (22 females and 11 males) without ophthalmopathy; group 4, 11 untreated hyperthyroid patients (7 females and 4 males) with single toxic adenoma; and a control group of 30 healthy subjects (21 females and 9 males). sICAM-1 and sELAM-1 concentrations were measured by a sandwich enzyme linked immunosorbent assay (ELISA) method. Groups 1, 2, and 3 (P < 0.001 for all 3 groups) but not group 4 showed increased sICAM-1 levels compared with the control group. However, groups 1 and 2 (P < 0.001 for both) showed higher values of sICAM-1 than group 3, and group 1 showed higher sICAM-1 levels than group 2 (P < 0.002). Groups 1 and 2 (P < 0.001 for both) but not groups 3 and 4 showed sELAM-1 levels significantly higher than the control group and positively correlated to the severity score of Graves' ophthalmopathy (GO) (P < 0.002 for group 1 and < 0.01 for group 2). Our results confirm that increased sICAM levels in GD patients with or without ophthalmopathy (with higher levels in patients with GO) but not in hyperthyroid nonautoimmune patients may be the consequence of orbital and thyroid inflammation, and they also suggest that sICAM concentrations could reflect the degree of inflammatory activity. Increased sELAM-1 concentrations only, in patients with ophthalmopathy with or without hyperthyroidism significantly correlated to severity score of GO, suggest the measurement of sELAM-1 levels as a specific marker of endothelium activation in GO. PMID- 7541802 TI - Circulating selectins, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in hyperthyroidism. AB - Serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE selectin), soluble P-selectin, and soluble L-selectin (sL-selectin), tumor necrosis factor-alpha, and interleukin-6 were measured in patients with Graves' disease (GD) (n = 33), in patients with toxic nodular goiter (n = 34), and in a group of healthy controls (n = 36). The serum levels of sICAM-1, sVCAM-1, sE selectin, and sL-selectin were markedly elevated in patients with GD and in patients with toxic nodular goiter before treatment with methimazole (P < 0.05 for all). After 8 weeks of therapy, serum concentrations of sVCAM-1 and sE selectin normalized, whereas serum levels of sL-selectin and sICAM-1 remained elevated. Hormone concentrations normalized after 2 weeks, clearly preceding falling levels of circulating adhesion molecules. Serum concentrations of soluble P-selectin, TNF-alpha, and interleukin-6 did not differ among patients with GD and toxic nodular goiter and healthy subjects. Serum levels of sVCAM-1 and sICAM 1 correlated with the serum concentrations of TSH receptor antibodies (n = 33; r = 0.921 and r = 0.792, respectively) and thyroid peroxidase antibodies (n = 33; r = 0.682 and r = 0.761, respectively) but not thyroglobulin antibodies. However, no correlation between serum levels of sE-selectin, sL-selectin, and soluble P selectin or cytokines and serum levels of thyroid peroxidase antibodies, TSH receptor antibodies, or thyroglobulin antibodies, respectively, was found. In addition, no correlation between serum levels of adhesion molecules or cytokines and thyroid hormones was seen. We conclude that both the action of thyroid hormones and the autoimmune process in GD may contribute to elevated levels of soluble adhesion molecules. PMID- 7541804 TI - Decrease and long-term recovery of choline acetyltransferase immunoreactivity in adult cat somatosensory cortex after peripheral nerve transections. AB - The functional reorganization of cerebral cortex following peripheral deafferentation is associated with changes in a number of neurotransmitters and related molecules. Acetylcholine (ACh) enhances neuronal responsiveness and could play a role in activity-dependent cortical plasticity. In this study, choline acetyltransferase (ChAT) immunohistochemistry was used to investigate ACh innervation of the primary somatosensory cortex in cats sustaining complete unilateral forearm and paw denervations. Survival times of 2-52 weeks were examined. The deafferented contralateral cortex was defined electrophysiologically, and quantitative estimates of ChAT-immunoreactive fiber density were obtained from the forelimb and hindlimb sectors of area 3b in both hemispheres. In the 3b forelimb sector contralateral to the deafferentation, a decrease in density of ChAT-positive fibers relative to the ipsilateral hemisphere was apparent at 2 weeks and most pronounced at 13 weeks, involving all cortical layers except layer I. There was no such decrease in the hindlimb sector, but the loss of ChAT immunoreactivity extended to sectors representing proximal forelimb and trunk. Changes in ChAT immunoreactivity were no longer found after 1 year of survival. This long-lasting but reversible lowering of ChAT immunoreactivity could result from a loss of afferent activity in basalis neurons and/or trophic influences retrogradely exerted by cortex on these cells. Reduced ACh transmission might then contribute to the loss of gamma aminobutyric acid (GABA) inhibition in the deafferented cortex by decreasing the activation of inhibitory interneurons. The long-term recovery of a normal ChAT immunoreactivity in cortex could be a consequence of its functional reorganization. PMID- 7541803 TI - 5 alpha-Androstan-3 alpha,17 beta-diol is a hormone: stimulation of cAMP accumulation in human and dog prostate. AB - 5 alpha-Androstan-3 alpha, 17 beta-diol (3 alpha-diol) is a well known metabolite of dihydrostestosterone (DHT) and the concentration of its glucuronide in serum is looked upon as a marker of DHT activity in skin and prostate. The data in this communication show that 3 alpha-diol is a potent agonist of the human and canine prostatic SHBG-[SHBG receptor]. It causes a robust rise in intracellular cAMP within minutes; the 1/2 max. response occurs at a concentration equal to its concentration in male plasma. Estradiol is the only other agonist in this system. All other steroids that bind to SHBG are antagonists. PMID- 7541805 TI - Somatostatin-like immunoreactivity in primary afferents of the medial articular nerve and colocalization with substance P in the cat. AB - The proportion of somatostatin-containing dorsal root ganglion cells innervating the knee joint of the cat via the medial articular nerve was determined by using retrograde labeling with fast blue and immunohistochemistry. Immunoreactivity was found in 8.6% of labeled cell bodies. In colchicine-treated ganglia, the proportion increased to 16.8%. Only small and intermediate-sized perikarya showed somatostatin-like immunoreactivity, indicating that this neuropeptide is synthesized predominantly in primary afferent units with unmyelinated sensory axons but may also be present in primary afferents with thinly myelinated sensory fibers. Colchicine treatment had no influence on the cell size distribution. Colocalization of somatostatin with substance P was determined by comparing the proportions of immunopositive dorsal root ganglion cells after incubation with antibodies against substance P or somatostatin or with a mixture of both. Substance P-like immunoreactivity was found in 18.1% (untreated ganglia) and 19.6% (colchicine treated ganglia) of the labeled neurons. After incubation with a mixed antibody solution, 18.2% of joint afferents in untreated and 19.9% of the cells in colchicine-treated ganglia were immunopositive. Comparing this result with the results obtained using somatostatin and substance P antibodies alone, one can calculate that both neuropeptides are colocalized in about 17% of the cat's knee joint afferents. About 3% of the neurons contain only substance P, whereas almost none of the neurons contain only somatostatin. Based on this fact, one can assume that both neuropeptides are coreleased in peripheral tissue as well as in the central nervous system. PMID- 7541807 TI - Patterning of local intracortical projections within the vibrissae representation of rat primary somatosensory cortex. AB - Anterograde and retrograde tracing with biotinylated dextran amine and Phaseolus vulgaris leukoagglutinin was used to assess projection patterns within the vibrissae representation of the rat's primary somatosensory cortex (S-I). Large and small injections of either tracer into the center of the vibrissae representation yielded dense anterograde and retrograde labelling throughout much of the tangential extent of the vibrissae representation within S-I. In all layers, the pattern and extent of retrograde and anterograde label was in rough congruence. The organization of this labelling varied across cortical layers. In layers II and III, labelled fibers extended away from injection sites in all directions and yielded a uniform pattern, which decreased in density with increasing distance from the tracer injection. There was a tendency for labelling to be more extensive along the representation of the row of vibrissae follicles that included the injection site than across rows. There was also a tendency for anterograde labelling to be more extensive in the direction of the representation of follicles more rostral on the face than that injected. In lamina IV, both labelled fibers and cells were restricted for the most part to the septa regions between the barrels. However, a small number of retrogradely labelled neurons were also located in the barrels (approximately one-ninth of the number found in the septa). The pattern observed in laminae II-III was repeated in layers V and VI. In these laminae, there was no evidence of a pattern of intracortical connections related to the vibrissae representation in overlying lamina IV. PMID- 7541806 TI - Differential distribution of six calcium-binding proteins in the rat olfactory epithelium during postnatal development and adulthood. AB - Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. Ca2+ acceptors, such as calmodulin, may mediate between the change in intracellular calcium and the conductance mechanism underlying the initial electrical event. Ca2+ acceptors also may participate in subsequent processing of olfactory information. The identification and characterization of these molecules, therefore, should provide important information about the complex signal transduction pathway involving calcium in olfaction as well as other sensory systems. The present study describes the distribution of six calcium-binding proteins in the rat main olfactory epithelium during postnatal development to determine when different Ca2+ acceptors can be detected and whether they segregate into different layers or portions of the epithelium. Calmodulin, calretinin, calbindin-D28k, neurocalcin, and recoverin were detected immunohistochemically in olfactory receptors but not in basal cells. S-100 immunoreactivity was restricted to glial cells primarily around the cribriform plate. During postnatal development (from P1 to P20), calmodulin, calretinin, calbindin-D28k, and neurocalcin formed a gradient of immunoreactivity descending from the central to the lateral areas in the nasal cavity, whereas recoverin was expressed only in sporadic, mature receptors in the proximal region of the mucosa. At P20, the immunoreactivity pattern for each calcium-binding protein was identical to the adult profile, indicating that the olfactory epithelium had reached maturity by this stage. Olfactory nerve fiber bundles displayed a differential staining pattern from P1 until adulthood for calbindin-D28k and calretinin (internal portions of bundles). Differential calmodulin immunoreactivity of olfactory nerves (large external portions of bundles) appeared at P10. The immunoreactivity of the nerve fiber bundles may reflect a further degree of organization relevant to odor discrimination. PMID- 7541810 TI - Synaptic organization of the interstitial subdivision of the nucleus tractus solitarii and of its laryngeal afferents in the rat. AB - The nucleus tractus solitarii, the first central relay for gustatory and a variety of visceral afferents, is also an integrative center for numerous functions. Its interstitial subdivision is involved in swallowing and respiratory reflexes. The ultrastructural characteristics of this subdivision and of its laryngeal afferents were investigated in adult rat by a serial-section study and by application of wheat germ agglutinin-horseradish peroxidase conjugate to the peripheral afferent fibers. The interstitial subnucleus contained scattered small neuronal cell bodies with such ultrastructural features as a large nucleus with deep indentations and an organelle-poor cytoplasm. On the basis of their size and vesicular content, the axon terminals were classified into three categories. Group I and group II terminals were small or large, respectively, and contained mainly small, round, and clear synaptic vesicles. Group III terminals were also small but contained small, pleomorphic, and clear vesicles. Axodendritic synapses were the most numerous. They were either asymmetrical, comprised of group I and II terminals, or symmetrical, comprised of group III terminals. More than 50% were part of complex synaptic arrangements in the form of rosettes or glomeruli. Axosomatic contacts involved both group I and group III terminals and were always symmetrical. A high frequency of axoaxonic synapses was found. They were symmetrical, comprised of group III terminals on group I or II terminals. Different types of symmetrical synaptic contacts made by dendrites were also found. This study indicates also that the ipsilateral interstitial subdivision constitutes the preferential site of termination for superior laryngeal afferents. The labeled axon terminals belonged exclusively to groups I and II and were involved in both axodendritic and axoaxonic synapses. Some of the axodendritic synapses were part of rosettes or glomeruli. All these synaptic arrangements may be considered a morphological substrate for important processing of afferent information in the nucleus tractus solitarii. They may account for some of the integrative functions of the interstitial subnucleus such as physiological processes triggered from the superior laryngeal nerve. PMID- 7541809 TI - Glial cells of the lamprey nervous system contain keratin-like proteins. AB - Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate. PMID- 7541808 TI - The human entorhinal cortex: a cytoarchitectonic analysis. AB - The entorhinal cortex of man is in the medial aspect of the temporal lobe. As in other mammalian species, it constitutes an essential component of the hippocampal formation and the route through which the neocortex interacts with the hippocampus. The importance of knowing its architecture in detail arises from the possibility of extrapolating it to experimental findings, notably in the nonhuman primate. We have investigated the cytoarchitectonic features of the human entorhinal cortex by using as a base our previous study (D.G. Amaral, R. Insausti, and W.M. Cowan [1987] J. Comp. Neurol. 264:326-355) of the nonhuman primate entorhinal cortex. We prepared serial sections of the temporal lobe from 35 normal brains. Thionin- and myelin-stained series were made of all cases. Sections spaced 500 microns apart through the full rostrocaudal extent of the entorhinal cortex were analyzed. The human entorhinal cortex is made up of six layers, of which layer IV does not appear throughout all subfields of the entorhinal cortex. The overall appearance resembles that of the adjacent neocortex in lateral and caudal portions. In harmony with general structural principles in the nonhuman primate entorhinal cortex, our analysis supports the partitioning of the human entorhinal cortex into eight different subfields. (1) The olfactory subfield (EO), the rostralmost field, is little laminated. (2) The lateral rostral subfield (ELr), laterally located, merges with the laterally adjacent perirhinal cortex. (3) The rostral subfield (ER) is between EO and ELr, with better differentiation of layers II and III than EO. (4) The medial intermediate subfield (EMI) is located at the medial border. (5) The intermediate field (EI) is a lateral continuation of EMI; lamina dissecans (layer IV) can be best appreciated in this field. (6) The lateral caudal subfield (ELc) laterally borders on EI as a continuation of ELr. (7) The caudal subfield (EC) lies caudal to the beginning of the hippocampal fissure, with a distinctive, clear space (Vc) between layers V and VI. (8) The caudal limiting field (ECL) forms the caudal termination of the entorhinal cortex. Thus our parcellation of the entorhinal cortex in man is largely parallel to that arrived at in the monkey. This close homology provides a rational basis for the application to clinical problems of anatomical and functional information obtained in experimental work in nonhuman primates. PMID- 7541811 TI - Primary Sjogren's syndrome and psoriasis vulgaris in a case of OKT4 epitope deficiency. AB - We report a 29-year-old female OKT4 epitope deficiency patient with primary Sjogren's syndrome and psoriasis vulgaris. Immunological investigations during the prolonged clinical course of her herpes zoster revealed that she has OKT4 epitope deficiency and primary Sjogren's syndrome. She had been treated for psoriasis vulgaris for 17 years without systemic immunosuppressive therapy. Flow cytometric study revealed that her OKT4 deficiency is heterogeneous and excluded interference with the OKT4 epitope by anti OKT4 autoantibodies. The rare coexistence of primary Sjogren's syndrome and psoriasis implicates an immune disturbance due to an unusual phenotype of CD4. PMID- 7541812 TI - Nodular scleroderma: focally increased tenascin expression differing from that in the surrounding scleroderma skin. AB - Nodular scleroderma is a rare variant of the disease, whose pathogenesis is uncertain. Tenascin is a recently cloned extracellular matrix protein which is thought to be a marker for tissue remodelling. To further investigate the pathogenesis of nodular scleroderma, we have followed up a case of this disease and studied tenascin expression in the nodular lesions and surrounding progressive systemic sclerosis skin. Previously, we demonstrated a long-lasting intermediate level of dermal tenascin expression in progressive systemic sclerosis; morphea and hypertrophic scar lesions showed strong but short-lived tenascin expression. In our current patient, high levels of tenascin were found in the nodules, which rapidly resolved. Thus, the time course of the clinical and histopathological findings together with the tenascin expression were more suggestive of hypertrophic scar than progressive systemic sclerosis. These findings imply that nodular scleroderma has a supplementary pathogenesis, such as itching, in addition to the proceeding systemic sclerosis. PMID- 7541813 TI - Spontaneous sustained ventricular tachycardia in the Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) Trial. AB - OBJECTIVES: We compared the QRS waveforms of the initial and subsequent complexes of spontaneous sustained monomorphic ventricular tachycardia and the rhythm induced at electrophysiologic study to test the theory that premature ventricular complexes "trigger" spontaneous ventricular tachycardia and that a stable substrate exists such that the spontaneous arrhythmia can be reproduced at electrophysiologic study. BACKGROUND: Failure rates have been high in several recent studies in which prevention of ventricular tachyarrhythmias was guided by suppression of premature ventricular complexes or induced ventricular tachycardias. METHODS: Digital waveform analysis was used to distinguish events of ventricular tachycardia initiated by configurationally distinct, possibly triggering, complexes (type 1) from events in which the initial QRS waveforms were identical to subsequent complexes, suggesting no requirement for premature ventricular beats (type 2). RESULTS: Of 1,102 episodes of spontaneous ventricular tachycardia, 73 (6.6%) were type 1; 1,012 were type 2 (91.8%); and 17 (1.5%) were uncertain. Of 59 patients only 14 (24%) had only type 1 episodes (group 1), whereas 37 patients (63%) had predominantly type 2 events (group 2) (p < 0.0001). Sustained ventricular tachycardia was inducible in all group 1 patients, and in most (57%) the induced rhythm was similar to the spontaneous rhythm. Ventricular tachycardia could not be induced in 7 patients from group 2 (19%), and in 18 patients (49%) the induced and spontaneous rhythms were dissimilar. Recurrence of arrhythmia rates differed according to the guidance method in group 2. CONCLUSIONS: Discrepancies between observed and predicted modes of initiation of ventricular tachycardia and between spontaneous and induced rhythms could result in inappropriate guidance and subsequent failure of antiarrhythmic treatment. PMID- 7541815 TI - Fluorescent staining for proteins on polyacrylamide gels with 5-dimethylamino-1 naphthalenesulfonyl chloride (dansyl chloride). AB - A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 micrograms protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins including proteoglycans. PMID- 7541814 TI - A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates. AB - In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription. PMID- 7541816 TI - Comparison of the sensitivity of different India inks staining of electro-blotted proteins on filter membranes. AB - The sensitivity of different brands of India ink for staining proteins blotted onto filter membranes is described. Proteins that are electro-blotted onto filter membranes show better retention than those dot-blotted alone. Higgins engrossing waterproof black ink No. 893 can detect protein at a concentration as low as 5 ng which is much more sensitive than Coomassie blue. The nitrocellulose membrane from S&S is ideal for blotting proteins and gives low levels of background staining. The sensitivity of protein staining is however, affected by the types of India ink, ink concentrations, staining times and membrane lots. India ink is also found to be useful for staining circulating immune complexes separated by SDS-PAGE and electro-blotted onto filter membranes. PMID- 7541817 TI - Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy. AB - We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH. PMID- 7541818 TI - Leukocyte interactions with vascular endothelium. New insights into selectin mediated attachment and rolling. AB - Among the earliest signs of inflammation is the capture of leukocytes from the blood stream and their subsequent rolling along the endothelium of postcapillary venules. This commentary summarizes recent insight into the molecular basis of leukocyte rolling gained from gene-targeted mice, Ab blocking studies, and in vitro and in vivo reconstitution assays. These data reveal how the selectins individually and collectively contribute to the process of leukocyte capture and subsequent rolling on vascular endothelium. PMID- 7541819 TI - Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers. AB - Costimulatory interactions between T and B lymphocytes are crucial for T cell activation and B cell proliferation and differentiation. We have compared the roles of CD40L and B7-2 in the initiation and maturation of humoral immunity by administering anti-CD40 ligand (L) or anti-B7-2 Ab during the early (days -1 to 3) or late (days 6-10) phases of primary responses to thymus-dependent (Td) and independent (Ti) Ags. Germinal center (GC) formation in response to a Td Ag was inhibited completely by the early administration of anti-CD40L or anti-B7-2 Abs. Later in the response, established GCs remained sensitive to anti-CD40L but were resistant to treatment with anti-B7-2. However, Ig hypermutation was reduced dramatically in GCs of anti-B7-2-treated mice and humoral memory was impaired. Early administration of anti-CD40L reduced serum Ab levels to approximately 10% of controls, whereas early treatment with anti-B7-2 reduced Ab production by only 50%. Later treatments with either Ab had no effect on Ab production. Response to a type II Ti Ag was more resistant than Td responses to interruption of costimulatory interactions. Our findings suggest that the costimulatory roles of CD40:CD40L and B7-2:CD28/CTLA-4 differ in the GC; administration of anti-CD40L abrogates an established GC reaction, whereas Ab to B7-2 suppresses Ig hypermutation and entry into the B cell memory compartment. Once B cells have entered the differentiation pathway to Ab production, neither CD40L nor B7-2 is necessary for their continued differentiation and persistence. PMID- 7541820 TI - Requirement for natural killer cells in the induction of cytotoxic T cells. AB - Cell-mediated immunity involves the participation of both regulatory and cytotoxic cells. The conversion of precursors to effector CD8+ cytotoxic T (Tc) cells requires cell-cell collaboration in which CD4+ T cells are traditionally viewed as helper cells. An in vitro system was used here to demonstrate that the generation of human alloantigen-specific CD8+ Tc cells requires the participation of CD3-CD16+CD56+ NK cells but not CD4+ T helper cells. Depletion of NK cells from responders abolished the induction of alloantigen-specific Tc cells in mixed lymphocyte cultures (MLC). Purified CD5+CD8+ T cells stimulated with alloantigen proliferated but did not differentiate into fully functional effector Tc cells. Coculture of responder CD5+CD8+ T cells with NK cells promoted the conversion of CD8+ Tc cell precursors (pTc) into effector Tc cells. Anti-CD56 mAbs blocked Tc cell induction in MLC, suggesting a role for CD56 molecules expressed on NK cells in either alloantigen recognition or delivery of accessory signals to pTc cells. These findings suggest a novel critical link between the natural and specific immune responses. PMID- 7541821 TI - The role of self peptides in the allogeneic cross-reactivity of CTLs. AB - This study presents data relevant to understanding the molecular and structural basis for the cross-reactivity of many CTLs on multiple MHC targets. Five anti-H 2Kb alloreactive CTL clones derived from B6.C-H-2bm1 (bm1), B6.C-H-2bm8 (bm8), and B6.C-H-2bm11 (bm11) mice and a Sendai virus-specific H-2Kb-restricted CTL clone were studied. Self peptides extracted from Kb molecules were fractionated by HPLC and tested for their ability to be recognized on RMA/s (H-2b) cells by those clones. For each alloreactive clone, a single dominant peptide peak was found to sensitize target cells. In addition to recognizing peptides presented by the Kb molecule, the five alloreactive clones and the one Sendai virus-specific clone all showed cross-reactivities on a panel of Kbm mutant cells in a peptide dependent manner. Two CTL clones, one alloreactive and one virus specific, cross recognized Kbm targets by each responding to a unique self peptide in the context of the mutant MHC molecules. Our data underscore the prevalent idea that TCR alpha beta have an inherent structural capability to react with several peptide/MHC structural patterns other than the original peptide/MHC pattern that might have been used to select that TCR. The high incidence of cross-reactivity seems to reflect a feature of the mechanism of positive selection in the thymus and the need for T cells in the repertoire to have an expanded capability for responding to a wide variety of foreign Ags. PMID- 7541822 TI - Lack of strict correlation of functional sensitization with the apparent affinity of MHC/peptide complexes for the TCR. AB - We describe a comprehensive analysis of the effect of avidity of TCR-MHC/peptide interaction on activation of the (p2Ca). In study, monosubstituted variants of p2Ca were used and assessed for binding to purified H-2Ld, binding of H 2Ld/peptide complexes to sTCR, and ability to activate 2C cells to two independent effector functions. Among the > 20 variants analyzed, functional activity of most peptides that bound the MHC well correlated with the strength of interaction of MHC/peptide complexes with sTCR. However, with some variants, a clear discordance between the apparent TCR-MHC/peptide affinity and biologic function was observed, demonstrating that the former cannot always be gauged by the latter. In the case of L4 peptide (phenylalanine at position 4 substituted with leucine), peptide/MHC complexes showed no detectable binding to sTCR, indicating a 10-fold or greater decrease in affinity. Nevertheless, this peptide sensitized target cells for lysis at a level equivalent to the parental peptide. A clearer understanding was revealed by studying the extent to which activation by variant peptides was dependent on CD8. Our data indicate that resistance to anti-CD8 mAb blocking correlates with strong binding affinity between sTCR and MHC/peptide complexes. These data suggest that, for the activation of CTL function, the absolute level of intrinsic affinity of TCR for MHC/peptide ligand is not a single critical determinant, but rather, that activation is governed by the compound influence of several factors, which ensures a minimum threshold of intracellular triggering is reached to elicit the response. PMID- 7541823 TI - Immunization with a single T helper cell epitope abrogates Friend virus-induced early erythroid proliferation and prevents late leukemia development. AB - Synthetic peptide vaccines containing a single Th cell epitope identified in the gp70 envelope glycoprotein of Friend murine leukemia helper virus induced potent protective immunity against Friend virus infection. H-2a/b mice immunized by a single s.c. injection of the CFA emulsion containing a peptide that represented the N-terminal gp70 epitope recovered slowly from initial development of splenomegaly, and most did not develop late leukemia, whereas most of the control mice given an injection of CFA alone showed sustained leukemic splenomegaly after the challenge with Friend virus. The mice of the same genetic background immunized with the C-terminal Th cell epitope by a single injection of a separate synthetic peptide eliminated virus-producing cells from the spleen within 12 days after inoculation of Friend virus complex, and did not develop early splenomegaly or polycythemia. H-2a/a mice were not protected by immunization with either one of the two synthetic peptides. Earlier production and more rapid class switching of virus-neutralizing Abs were observed in H-2a/b mice immunized with the peptide vaccines after the challenge with Friend virus, compared with the responses of the control mice. Detailed kinetic and immunohistopathologic analyses suggested that Th cells might be directly involved in the growth inhibition and elimination of virus-infected erythroid precursor cells. PMID- 7541824 TI - HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. AB - CD8+ CTL specific for the Plasmodium yoelii sporozoite surface protein 2 (PySSP2) protect mice against malaria. For this reason, vaccines designed to induce CTL against P. falciparum SSP2 (PfSSP2) are under development. Optimal development of PfSSP2 as a component of human malaria vaccines requires characterization of HLA class I-restricted CTL against this Ag. For this purpose, PBMC from four HLA-A2+ human volunteers immunized with P. falciparum irradiated sporozoites were stimulated with a recombinant vaccinia virus expressing PfSSP2 and with 35 PfSSP2 derived 15-amino acid peptides containing sequences conforming to HLA-A2 binding motifs. Ag-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity against autologous target cells transfected with the PfSSP2 gene was demonstrated in the four volunteers. Twelve of the 35 peptides sensitized HLA-A2 matched target cells for lysis by peptide-stimulated effectors. Three volunteers had CTL against 9 of the 12 peptides, and one had no peptide-specific CTL. HLA A*0201 restriction was confirmed by demonstrating that effectors from the three responders could be stimulated with six different peptides to lyse HLA-A*0201+ T2 cells incubated with the homologous peptides. Peptide-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity was also demonstrated against two peptides using unstimulated PBMC as effectors. Available data indicate that the motif-bearing sequences in 6 of the 12 positive peptides are conserved among P. falciparum isolates and clones. Demonstration of HLA-A2 restricted CTL responses to multiple PfSSP2-derived peptides, and of circulating activated CTL against PfSSP2 in immune volunteers provide important information for optimal design and evaluation of vaccines containing this pre-erythrocytic stage Ag. PMID- 7541825 TI - Exogenous nitric oxide regulates IFN-gamma plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages. AB - This study was performed to determine the effects of nitric oxide (NO) on the expression of inducible NO synthase (iNOS) in mouse macrophages. We used the NO donor diethylamine dinitric oxide (DEA/NO) and the mouse macrophage cell line ANA 1 in these experiments. ANA-1 macrophages did not express iNOS mRNA either constitutively or following exposure to 100 U/ml IFN-gamma alone, to 10 ng/ml LPS alone, or to 200 microM DEA/NO alone. Similarly, ANA-1 macrophages did not express detectable levels of iNOS mRNA following treatment with 100 U/ml IFN gamma plus 200 microM DEA/NO. However, IFN-gamma (100 U/ml) plus LPS (10 ng/ml) induced high levels of iNOS mRNA in ANA-1 macrophages after 6 h. Low concentrations of DEA/NO (approximately 1 to 12 microM) caused up to a 2.5-fold augmentation of IFN-gamma plus LPS-induced iNOS mRNA expression. In contrast, 200 microM DEA/NO suppressed IFN-gamma plus LPS-induced iNOS mRNA expression (60% decrease). The effects of DEA/NO were gene-specific because DEA/NO did not affect the IFN-gamma plus LPS-induced expression of TNF-alpha mRNA. Moreover, the biphasic effects of DEA/NO were specifically due to released NO. Diethylamine and nitrite were unable to regulate IFN-gamma plus LPS-induced gene expression in ANA 1 macrophages. Time-response experiments suggested that the effects of NO were short-lived and occurred early during the induction of iNOS gene expression. The effects of NO were not limited to iNOS mRNA expression but were apparent at the level of iNOS protein expression and enzymatic activity. Overall, these results suggest that NO has immunoregulatory effects and may control the extent and duration of cytokine- and/or endotoxin-induced iNOS expression in macrophages. PMID- 7541826 TI - Induction of oral tolerance to myelin basic protein in CD8-depleted mice: both CD4+ and CD8+ cells mediate active suppression. AB - We have previously shown that orally administered myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis in both the Lewis rat and the SJL mouse. In the Lewis rat fed low doses of MBP, we found that protection can be adoptively transferred by CD8+ cells and that these cells inhibit immune responses via the secretion of TGF-beta after Ag-specific triggering. In the present study, we investigated the cellular requirements for the generation of active suppression following oral administration of MBP in SJL and (PLJ x SJL)F1 mice. We first determined the frequency of MBP cells secreting Th1 (IFN-gamma) and Th2 (IL-4/IL-10) cytokines or TGF-beta after oral administration of MBP. We found that in SJL mice, orally administered MBP (0.5 mg/feeding) led to an increased frequency of TGF-beta-, IL-4-, and IL-10-secreting cells and a decreased frequency of IFN-gamma-producing cells. This pattern was observed in both CD4+ and CD8+ populations; adoptive transfer of either CD4+ or CD8+ cells from orally tolerized mice suppressed autoimmune encephalomyelitis in recipient animals. We then studied the role of CD8+ cells on the generation of oral tolerance to MBP by depleting CD8+ cells in vivo with anti-CD8 mAb. Oral tolerance was successfully induced in such animals, as demonstrated by a decrease in clinical disease and T cell proliferative responses, although there was less TGF-beta production in vitro and less disease protection on days 20 to 22 in CD8 depleted animals. These studies demonstrate that CD4+ cells in the absence of CD8+ cells can mediate the active suppression component of oral tolerance in mice and that there is a reciprocal relationship between Th1- and Th2-type cytokine production associated with oral tolerization. PMID- 7541827 TI - HIV gp120 inhibits T cell activation by interfering with expression of costimulatory molecules CD40 ligand and CD80 (B71). AB - One mechanism of the immune suppression in HIV infection has been postulated as being caused by the interaction of HIV envelope glycoprotein gp120 with CD4 molecules. Thus, pretreatment of purified peripheral blood T cells or CD4+ T cell clones with gp120 (or an anti-CD4 mAb) results in inhibition of anti-CD3 mAb induced proliferative responses. In this study, we have analyzed the role of the interacting pairs of costimulatory molecules, CD28-B71 (CD80) and CD40 ligand (CD40L)-CD40, to elucidate further the mechanism of HIV gp120-induced inhibitory effects on T cell functions. Interactions between CD28-B71 and CD40L-CD40 were found to be essential for the anti-CD3 mAb-induced T cell proliferation, as demonstrated by up-regulation of B71 and CD40L and the ability of anti-B71 and anti-CD40L mAbs to inhibit this response. Pretreatment of CD4+ T cells with gp120 before CD3 ligation with anti-CD3 mAb resulted in failure of up-regulation of CD40L on T cells and B71 on APC. Exogenous addition of anti-CD28 mAb overcame the inhibitory effect of gp120 on anti-CD3 mAb-induced T cell proliferation. We conclude that binding of gp120 to CD4 molecules on T cells may interrupt the sequential cascade of intercellular interaction involving 1) Ag/MHC class II TCR/CD4, 2) CD40L-CD40, and 3) B71-CD28. These studies indicate that the CD4 gp120 interaction results in dysregulation of expression of costimulatory molecules, CD40L, and B71 expression on T cells and APC, respectively, thereby contributing to the T cell hyporesponsiveness in HIV infection. PMID- 7541828 TI - T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans. AB - We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short term T cell lines were generated from 49 individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes. PMID- 7541829 TI - Primary haemostasis in thyroid disease. AB - OBJECTIVES: There have been reports on a bleeding tendency in hypothyroidism resembling von Willebrand's disease. The aim of the present study was to investigate whether altered primary haemostasis is a general phenomenon in thyroid disease. DESIGN/SETTING: A total of 10 patients with hyperthyroidism and nine patients with hypothyroidism were studied at diagnosis, and during treatment with carbimazole or L-thyroxine, respectively, when euthyroidism had been achieved. RESULTS: In untreated hypothyroidism, template bleeding time was prolonged (median 9.3 min, range 3.8-20.0 min) compared to that in controls (median 4.0 min, range 3.0-6.0 min; P < 0.05), whereas maximal agglutination velocity induced by ristocetin was decreased (38% min-1, range 4-52% min-1 vs. 70% min-1, range 60-81% min-1, P < 0.05). The level of von Willebrand factor antigen in plasma from hypothyroid patients was less than half of the value in hyperthyroid patients. This difference disappeared after euthyroidism was achieved. CONCLUSIONS: We found that changed primary haemostasis is a general feature of hypothyroidism, and that it is resolved after levothyroxine treatment. PMID- 7541830 TI - Low lumbar CSF concentrations of homovanillic acid in the autosomal dominant ataxias. PMID- 7541832 TI - Serotonergic modulation of locust motor neurons. AB - 1. The effects of the putative endogenous neuromodulator serotonin (5-HT) on the fast extensor and flexor tibiae motor neurons in the locust (Schistocerca gregaria) metathoracic ganglion, were analyzed. 2. 5-HT consistently increased the duration of the fast extensor spike and usually reduced the afterhyperpolarization, although this effect was less consistent. The spike broadening in the fast extensor was associated with an increase in the amplitude of the excitatory postsynaptic potential (EPSP) evoked monosynaptically in the flexor motor neurons by fast extensor stimulation. 5-HT also increased the membrane resistance of the fast extensor and flexor tibiae motor neurons. 3. The effects of 5-HT were mimicked by bath application of the 5-HT uptake inhibitor imipramine, and blocked by the 5-HT receptor antagonist ketanserin. The effects were also mimicked by dibutryl cyclic AMP, a membrane permeant analogue of cyclic AMP, and by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine, but not by dibutryl cyclic GMP. The 5-HT-dependent modulation was blocked by the protein kinase A inhibitor H8. In addition, injection of cyclic AMP into the fast extensor or a flexor motor neuron could mimic the effects of 5-HT on these neurons. 4. 5-HT probably broadened the FETi action potential by modulating potassium conductances responsible for spike repolarization. 5. These results show that 5-HT modulates both the fast extensor and flexor tibiae motor neurons, resulting in potentiation of synaptic transmission between these neurons. In addition, the increase in flexor membrane resistance will potentiate other inputs onto these cells, which will affect the output of the motor neurons during locomotion. PMID- 7541831 TI - Innervation of motoneurons based on dendritic orientation. AB - 1. The distribution of terminals from vestibulospinal (VS) axons on the dendritic trees of neck motoneurons was determined by combining the anterograde transport of Phaseolus Vulgaris Leucoagglutinin (PHA-L) with intracellular staining techniques and three-dimensional reconstruction methods. 2. Contacts between VS axon terminals and dendrites were arranged in a nonuniform pattern that depended on the orientation (i.e., rostro-caudal vs. dorsolateral) of the dendrites. 3. This innervation pattern satisfies a critical structural condition necessary for selective nonlinear interactions between pairs of simultaneously active inputs to motoneurons. PMID- 7541833 TI - Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups. AB - OBJECTIVE: To use the technique of complementation analysis to help define genotype and classify patients with clinical manifestations consistent with those of the disorders of peroxisome assembly, namely the Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). STUDY DESIGN: Clinical findings, peroxisomal function, and complementation groups were examined in 173 patients with the clinical manifestations of these disorders. RESULTS: In 37 patients (21%), peroxisome assembly was intact and isolated deficiencies of one of five peroxisomal enzymes involved in the beta-oxidation of fatty acids or plasmalogen biosynthesis were demonstrated. Ten complementation groups were identified among 93 patients (54%) with impaired peroxisome assembly and one of three phenotypes (ZS, NALD, or IRD) without correlation between complementation group and phenotype. Forty-three patients (25%) had impaired peroxisome assembly associated with the RCDP phenotype and belonged to a single complementation group. Of the 173 patients, 10 had unusually mild clinical manifestations, including survival to the fifth decade or deficits limited to congenital cataracts. CONCLUSIONS: At least 16 complementation groups, and hence genotypes, are associated with clinical manifestations of disorders of peroxisome assembly. The range of phenotype is wide, and some patients have mild involvement. PMID- 7541835 TI - Immunochemical characterization of neural cell adhesion molecule (NCAM), nitric oxide synthase, and neurofilament protein expression in pyloric muscle of patients with pyloric stenosis. AB - The etiology of hypertrophic pyloric stenosis (HPS) is not known. We made an immunocytochemical examination of pyloric muscle from 18 patients with HPS and 10 controls using specific monoclonal antibodies to neural cell adhesion molecule (NCAM) as well as neurofilament protein and NADPH-diaphorase histochemistry. In HPS, bundles of hypertrophic muscle fibers expanded the circular muscle layer. The longitudinal muscle also appeared hypertrophic but to a less marked degree. The most striking difference between HPS and the control tissues was that NCAM, NADPH-diaphorase, and neurofilament protein immunoreactive fibers were absent or markedly reduced within the hypertrophied circular and longitudinal musculature. In contrast, NCAM, NADPH-diaphorase, and neurofilament protein immunoreactivity was preserved in the myenteric plexus where nerve fibers and ganglion cells were stained. The lack of expression of NCAM, NADPH-diaphorase, and neurofilament protein on nerve fibers within the circular and longitudinal muscle in patients with pyloric stenosis suggests that the smooth muscle is not innervated in this condition. PMID- 7541837 TI - Resistance to organophosphorous insecticides in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Martinique. AB - Before beginning a widespread control program against Culex pipiens quinquefasciatus in Martinique, resistance to temephos, chlorpyrifos, and two organophosphorous insecticides, was investigated at seven breeding sites. At LC95, populations exhibited resistance ratios between 6.9 and 11.6 for temephos and between 6.4 and 51.4 for chlorpyrifos. Overproduced esterases A2-B2 and B1, known to be involved in organophosphorous-resistance, were present at all breeding sites; esterases A2-B2 frequency was > 50% at all sites but one; and esterase B1 frequency was < 7%. Experimental treatment of three breeding sites with temephos induced no significant increase in resistance, but our esterase studies indicated a significant increase in the frequencies of esterase B1 and of a new highly active esterase C2. These results indicate that a large-scale C. p. quinquefasciatus control program with organophosphorous insecticides will induce a rapid increase of these resistance genes throughout Martinique. However, this may not necessarily result in high levels of resistance, because, at present, the level of gene amplification of esterase B still appears to be low. PMID- 7541836 TI - Use of beta-methylphenylalanine (beta MeF) residues to probe the nature of the interaction of substance P with its receptor: effects of beta MeF-containing substance P analogs on rabbit iris smooth muscle contraction. AB - The effects of substituting (2S,3S)-beta-methylphenylalanine (S-beta MeF) or (2S,3R)-beta-methylphenylalanine (R-beta MeF) for the Phe7 and/or Phe8 residues of the tachykinin substance P (SP, RPKPQQFFGLM-NH2) upon the ability of SP to stimulate contraction of the rabbit iris smooth muscle were investigated. The eight beta MeF-containing SP analogs (four monosubstituted analogs, four disubstituted analogs) 1-8 were synthesized and found to be agonsts of SP in the smooth muscle contraction assay, having EC50 values ranging from 0.15 to 10.0 nM. Three analogs are significantly more active than SP [8R-(beta MeF)SP (4), 7S,8S (beta MeF)2SP (5), and 7R,8S-(beta MeF)2SP (6)], three analogs are approximately equipotent with SP [7S-(beta MeF)SP (1), 7R-(beta MeF)SP (2), and 7S,8R-(beta MeF)2SP (8)], and two analogs are significantly less active than SP [8S-(beta MeF)SP (3) and 7R,8R-(beta MeF)2SP (7)]. The effects of the beta MeF substitutions upon the activity of SP are not additive and cannot be explained using simple conformational models which focus only on the side chain conformations of the beta MeF residues. It is postulated that the beta MeF residues induce minor distortions in the peptide backbone with resultant consequences upon peptide-receptor binding which are not dictated soley by the side chain conformations. This idea is consistent with 1H-NMR data for the monosubstituted analogs 1-4, which imply that the beta MeF substitutions cause slight distortions in the peptide backbone and that the beta MeF side chains are assuming trans or gauche(-) conformations. PMID- 7541834 TI - The treatment of intractable rejection with tacrolimus (FK506) in pediatric liver transplant recipients. AB - We report our experience in 17 pediatric orthotopic liver transplant (OLT) patients converted from cyclosporine (CsA) to FK506 for intractable acute and chronic rejection. FK506 was initiated orally at a dose of 0.3 mg/kg/day in most patients; the dose was then adjusted to achieve serum levels of 0.5-1.5 ng/ml. Azathioprine was discontinued and low-dose prednisone maintained. The median time between liver transplantation and FK506 conversion was 41 months. Patients have been treated for an average of 14.8 +/- 9.6 months. Six patients were converted for acute rejection and 11 for chronic rejection, i.e., vanishing bile duct syndrome (VBDS). After FK506 conversion, the actual patient and graft survival was 88% and 82%, respectively, in the group as a whole. Two patients died, one of chronic active hepatitis C and the other of lymphoma. Three patients, all with VBDS, did not respond to FK506 and therefore required retransplantation. The serum bilirubin is currently normal in 14 patients and the serum transaminases < 100 IU/ml in 12. The mean bilirubin pre-FK506 of patients successfully converted to FK506 was 4.2 mg/dl compared to 11.8 mg/dl in patients who failed conversion. Major complications included nephrotoxicity, neurotoxicity, and lymphoma. The mean glomerular filtration rate (GFR) of 97 +/- 29 mls/min/1.73m2 prior to FK506 conversion dropped to 51 +/- 20 mls/min/1.73m2 (p = 0.0001) after a mean of 13.6 months of FK506 therapy. Three patients have developed B-cell lymphomas; two of them responded to decreased immunosuppression and one died. We conclude that intractable liver graft rejection in children is most successfully reversed if FK506 is instituted before cholestasis becomes pronounced.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541838 TI - An extended -10 promoter alone directs transcription of the DpnII operon of Streptococcus pneumoniae. AB - The genetic cassette encoding the DpnII restriction-modification system of Streptococcus pneumoniae gave transcription products of approximately 2.7 and 1.8 kilobases. The larger, mRNA1, covered both of the methylase genes, dpnM and dpnA, and the endonuclease gene dpnB; the smaller, mRNA2, covered only the dpnA and dpnB genes. Transcription of mRNA1 was shown to begin at the translation start site for dpnM, thereby producing an mRNA without any apparent ribosome-binding site for translation of the DpnM methylase. The promoter for mRNA1 was shown by base substitution and deletion analysis to consist of an extended -10 site, TaTGgTATAAT, with no required -35 site. A possible promoter further upstream with close matches to a -35 site and a nonextended -10 site was not used. A survey of 36 proven and putative promoters used by S. pneumoniae revealed that 61% of them contained the full -10 extension, although, other than the dpnM promoter, they matched at a -35 site, as well. It appears that, unlike those found in Escherichia coli, S. pneumoniae promoters frequently require an extended -10 site, and such a site can function naturally without a -35 site. PMID- 7541839 TI - The archaeal dnaK-dnaJ gene cluster: organization and expression in the methanogen Methanosarcina mazei. AB - The organization and expression of the first archeael dnaK-dnaJ gene cluster cloned and sequenced have been elucidated. The work focused on the methanogen Methanosarcina mazei strain S-6, but a survey of two other strains (JC3 and LYC) and species (Methanosarcina sp. JCV and Methanosarcina acetivorans) showed that the findings are pertinent to other mesophilic methanosarcinas as well. The organization and some expression features of the archaeal genes resemble eubacterial equivalents for which comparable sequence information is available. However, the archaeal genes also display characteristics that are distinct from those of eubacterial and eucaryotic homologs. dnaK and dnaJ are transcribed into monocistronic messages. The initiation site is the same for transcription under optimal cell-growth conditions, and under stress due to a temperature upshift. The two genes are expressed constitutively at lower levels than those observed after heat shock. The constitutive and post-heat-shock expression levels are higher for dnaK than for dnaJ. Both genes withstand heat shocks of at least one and a half hours without a decline in transcript levels. While the transcription termination signals are to some extent reminiscent of those of eubacteria, the initiation signals are not. These have archaeal characteristics, which resemble those of eukaryotes. The intergenic dnaK-dnaJ region contains inverted repeats. These have the potential to build firm stem-loops in the transcript and in single stranded DNA. PMID- 7541840 TI - A continuum model for protein-protein interactions: application to the docking problem. AB - The prediction of protein-protein interactions in solution is a major goal of theoretical structural biology. Here, we implement a continuum description of the thermodynamic processes involved. The model differs considerably from previous models in its use of "molecular surface" area to describe the hydrophobic component to the free energy of conformational change in solution. We have applied this model to a data set of alternative docked conformations of protein protein complexes which were generated independently of this work. It was found previously that commonly used energy evaluation techniques fail to distinguish between near-native and certain non-native complexes in this data set. Here, we found that an energy function that takes into account (1) total electrostatic free energy, (2) hydrophobic free energy and (3) loss in side-chain conformational energy was able to reliably discriminate between near-native and non-native configurations but only when molecular surface is used as a descriptor of the hydrophobic effect. It is shown that the molecular surface and the more conventional surface descriptor "solvent accessible surface" give very different quantitative measures of hydrophobicity. In terms of the contribution of different energy components to the free energy of complex formation it was found that loss in side-chain conformational entropy is a second order effect. Electrostatic interaction energy (which is commonly used to score docked conformations) was a poor indicator of complementarity when starting from unbound conformations. It was found that electrostatic desolvation energy and the hydrophobic contribution (based on a molecular surface area descriptor) are much less sensitive to local fluctuations in atomic structure than point-to-point interaction energies and thus may be more suited for use as a scoring function when docking unbound conformations, where atomic complementarity is much less apparent. Whilst a combined energy function was able to distinguish near-native from non-native conformations in the six systems studied here, it remains to be determined to what extent more sizeable conformational changes would influence the results. PMID- 7541841 TI - Inhibition and aging of channel catfish brain acetylcholinesterase following exposure to two phosphorothionate insecticides and their active metabolites. AB - The inhibition and aging of acetylcholinesterase (AChE) in fingerling channel catfish (lctalurus punctatus) brain tissue was studied after single in vivo exposures to high levels of chlorpyrifos (0.25 mg/L), chlorpyrifos-oxon (7 micrograms/L), parathion (2.5 mg/L), or paraoxon (30 micrograms/L). Exposure to both parent compounds produced identical initial inhibition (95%), but in the later sampling times there was significantly more inhibited AChE in the chlorpyrifos-treated fish than in the parathion-treated fish (47% and 28%, respectively, on d 16). There were higher levels of aged AChE following chlorpyrifos exposure than following parathion exposure, but differences were not significant. Exposure to both oxons produced initial inhibition greater than 90%, and patterns of recovery and aging were statistically similar between both compounds; no significant inhibition was observed after d 11. The similar patterns of inhibition, recovery, and aging between the two oxon treatments, which have similar lipophilicities, suggest that the greater amount of AChE inhibition and aging observed in the chlorpyrifos-treated fish compared with the parathion-treated fish probably results from the higher lipophilicity of chlorpyrifos than of parathion. Overall, the prolonged brain AChE inhibition exhibited in catfish exposed to phosphorothionates is not the result of aging of the inhibited enzyme but is the result of either a slow rate or a lack of spontaneous reactivation. PMID- 7541843 TI - Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120. AB - On the basis of previous studies of dicistronic (dc) polioviruses that carried two internal ribosomal entry sites (L. Alexander, H.-H. Lu, and E. Wimmer, Proc. Natl. Acad. Sci. USA 91:1406-1410, 1994; A. Molla, S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer, Nature [London] 356:255-257, 1992), we have constructed a variety of dc polioviruses which express foreign genetic elements that were inserted either between two internal ribosomal entry site elements upstream of the poliovirus open reading frame (pPNENPO derivatives) or upstream of the open reading frame for the poliovirus proteinase 2Apro (pDI-E2A derivatives). Surprisingly, the addition of an N-terminal secretory pathway signal sequence to the open reading frame of the inserted foreign sequences (specifying either truncated versions of human immunodeficiency virus type 1 [HIV-1] gp120 or chloramphenicol acetyltransferase) resulted in a null phenotype, whereas removal of the signal sequence led to the production of viable viruses. Constructs that carried a foreign gene with a signal sequence were negative in RNA synthesis, an observation that suggested a very early block in viral replication. The insertion of transmembrane sequences downstream of the leader sequence did not reverse the replication block. Studies of dc polioviruses that encoded the truncated versions of HIV-1 gp120 showed an increase in genetic stability that correlated with a decrease in the size of the insert. A dc construct that contained a minigene encoding the principal neutralization determinant of HIV-1 produced a stable virus that retained the foreign sequence through multiple passages in cultured cells. These data indicate that dc polioviruses have potential as vaccines for the expression of small foreign epitopes. PMID- 7541842 TI - Antibody to adhesion molecule LFA-1 enhances plasma neutralization of human immunodeficiency virus type 1. AB - We have shown that a monoclonal antibody to the cell surface adhesion molecule LFA-1 (CD18/CD11a) enhances plasma neutralization of a laboratory isolate (HIVMN) and a primary isolate (HIV28R) of human immunodeficiency virus type 1. Human phytohemagglutinin blasts were infected with HIVMN or HIV28R in the presence of plasma pooled from HIV-positive individuals (AIDS plasma) or immunoglobulin G from AIDS plasma alone or combined with a monoclonal antibody (MAb) to LFA-1. While AIDS plasma alone at a dilution of 1:1,250 neutralized HIVMN and HIV28R infection by 15 and 0%, respectively, in the presence of a saturating concentration of the MAb to LFA-1 the plasma neutralized both viruses by more than 80% at this dilution. Immunoglobulin G purified from AIDS plasma, when used in combination with the MAb to LFA-1, showed the same synergistic effect in HIV neutralization as seen with the AIDS plasma and anti-LFA-1. The MAb against LFA-1 partially neutralized both viral isolates (45 to 55%) on its own. These results demonstrate significant synergy between the plasma and antibody against LFA-1 in the neutralization of HIV. The observations therefore suggest an important role for adhesion molecules in HIV infectivity and transmission. The results have implications for the recently observed host effect on HIV susceptibility to antibody neutralization. PMID- 7541844 TI - Steps in maturation of influenza A virus neuraminidase. AB - We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers. PMID- 7541845 TI - Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. AB - The Nef protein of human immunodeficiency virus type 1 (HIV-1) stimulates viral infectivity. The mechanism of this phenotype was investigated. Viruses containing disrupted nef genes were 4 to 40 times less infectious than wild-type HIV-1 in a single-round infection. The Nef-mediated stimulation HIV-1 infectivity was dependent on the association of Nef with the plasma membrane and could be observed when Nef was provided in trans in the virus producer but not target cells. The impaired infectiousness of nef-defective (delta Nef) virions was observed whether or not CD4 was present in either of these cells. Furthermore, it was independent of the mode of viral entry, since it was not rescued by pseudotyping Env- HIV-1 virions with the amphotropic murine leukemia virus envelope glycoproteins. As predicted from this result, wild-type and delta Nef virions entered cells with equal efficiencies. However, despite their normal content in viral genomic RNA and reverse transcriptase activity, delta Nef viruses were limited in their ability to perform reverse transcription once internalized in several cell types, including peripheral blood lymphocytes. Since Nef does not appear to be abundant in virions, these results suggest that Nef acts in producer cells to allow the generation of particles fully competent for completing steps that follow entry, leading to efficient reverse transcription of the HIV-1 genome. Using a trans complementation assay, we found that Nef proteins from a number of primary HIV-1 isolates as well as, to a milder degree, those from HIV-2ST and SIVMAC239 could enhance the infectivity of delta Nef HIV-1. This indicates that the Nef-mediated stimulation of proviral DNA synthesis is highly conserved and likely plays an important role in vivo. PMID- 7541846 TI - Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. AB - The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection. PMID- 7541847 TI - Reversion of a Moloney murine leukemia virus RNase H mutant at a second site restores enzyme function and infectivity. AB - The reverse transcriptase of retroviruses contains an RNase H activity essential for the proper synthesis of the viral DNA copy of the RNA genome. We have previously characterized a number of point mutations altering the RNase domain of the Moloney murine leukemia virus reverse transcriptase (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). One such mutation, Y586F (a Y-to-F change at position 586), reduced RNase H activity, as assayed by in situ gel analysis, to about 5% of the wild-type level and prevented viral replication. We have now recovered a revertant virus with near-normal infectivity and in vitro enzymatic activity. The revertant contains a single substitution, N613H, distant in the primary sequence of the protein, but modeling with the Escherichia coli RNase H structure suggests that the reverted residue is close in space to the original substituted residue. Examination of the structure permits some suggestions as to how this second-site revertant restores enzyme activity. PMID- 7541849 TI - Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase. AB - The interferon-inducible, RNA-dependent protein kinase (PKR) is activated by autophosphorylation, a process mediated by double-stranded RNA. A catalytically deficient, histidine-tagged mutant PKR protein [His-PKR(K296R)] was used as the substrate for characterization of the intermolecular phosphorylation catalyzed by purified wild-type PKR [PKR(Wt)]. The intermolecular autophosphorylation of His PKR(K296R) by PKR(Wt) was RNA dependent. Excess His-PKR(K296R) substrate inhibited both the auto- and the trans-phosphorylation activities of PKR(Wt). Inhibition of PKR(Wt) by His-PKR(K296R) was relieved by higher concentrations of activator double-stranded RNA. Phosphopeptide analysis revealed that the sites of intermolecular autophosphorylation in His-PKR(K296R) were very similar, if not identical, to the sites that were autophosphorylated in PKR(Wt) and suggest a multiple of four major phosphorylation sites per PKR molecule. PMID- 7541850 TI - Morphometric analysis and clinical followup of isolated prostatic intraepithelial neoplasia in needle biopsy of the prostate. AB - PURPOSE: We evaluate the significance of grade and extent of isolated prostatic intraepithelial neoplasia in prostate needle biopsies as a predictor of cancer on repeat biopsy. MATERIALS AND METHODS: We reviewed our experience with 58 men 50 years or older who had isolated prostatic intraepithelial neoplasia on initial prostate needle biopsy during a prostate specific antigen (PSA) based screening trial for prostate cancer. All 58 men underwent repeat biopsy to follow the initial findings of prostatic intraepithelial neoplasia. We assessed the relationship of patient age, digital rectal examination, serum PSA concentration, PSA density, prostatic intraepithelial neoplasia grade, number of foci of neoplasia and linear extent of prostatic intraepithelial neoplasia in the initial biopsy specimen to the finding of cancer on the repeat biopsy. We also compared the cancer detection rate in the 58 men with and 427 without prostatic intraepithelial neoplasia in the same screening trial. RESULTS: Of 21 men with low grade and 37 with high grade prostatic intraepithelial neoplasia 4 (19%) and 19 (51%), respectively, had cancer on repeat biopsy (p < 0.02), compared to 82 of 427 (19%) without cancer or prostatic intraepithelial neoplasia on the initial biopsy. High grade prostatic intraepithelial neoplasia was a significant predictor of malignancy on repeat biopsy (p < 0.05). The number of foci of neoplasia and the linear extent of prostatic intraepithelial neoplasia on initial biopsy were not predictive of cancer on repeat biopsy. CONCLUSIONS: Our results demonstrate that the presence of high grade prostatic intraepithelial neoplasia is a strong predictor of prostate cancer in men with elevated serum PSA concentrations and they should be followed with repeat biopsy. PMID- 7541851 TI - Subcutaneous urinary diversions for palliative treatment of pelvic malignancies. AB - PURPOSE: We developed 2 subcutaneous urinary diversion techniques, pyelovesical bypass and anterior cutaneous nephrostomy, to improve the quality of life of patients undergoing permanent palliative percutaneous nephrostomy. MATERIALS AND METHODS: A total of 21 patients underwent either pyelovesical bypass (19) or anterior cutaneous nephrostomy (13). Technically, the percutaneous nephrostomy tube is replaced by a self-retaining expanded polytetrafluoroethylene-silicone tube tunneled underneath the skin. The distal extremity can be either introduced into the bladder, creating a pyelovesical bypass, or brought out directly through a cutaneous orifice, creating an anterior cutaneous nephrostomy. RESULTS: No tube was dislodged and none became obstructed due to incrustation or angulation. Standard percutaneous nephrostomy was necessary in 1 patient in each group. Improvement in the quality of life was stressed by all patients. Pyelovesical bypass eliminates external drainage and anterior cutaneous nephrostomy performed when the bladder is no longer functional allows for the creation of a single, easily dressed anterior stoma. CONCLUSIONS: Pyelovesical bypass and anterior cutaneous nephrostomy constitute valuable alternatives to standard permanent palliative percutaneous nephrostomy. PMID- 7541848 TI - Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor. AB - Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination. PMID- 7541852 TI - Prevalence of prostatism in Japanese men in a community-based study with comparison to a similar American study. AB - PURPOSE: We estimate the prevalence of urinary symptoms in Japanese men. MATERIALS AND METHODS: A total of 289 eligible residents 40 to 79 years old completed a questionnaire with questions worded similarly to those of the international prostate symptom score (response rate 42%). RESULTS: The ratio of moderate-to-severe symptoms was 41%, 29%, 31% and 56% for each age decade from ages 40 to 79 years, respectively, after adjusting for nonresponse. Within each age decade the median international prostatic symptom score was higher for Japanese men than for United States men with little difference in rates of increase with participant age or bother. CONCLUSION: Lower urinary tract symptoms were common in Japanese men, with age-related increases similar to those of United States men. PMID- 7541853 TI - The correlation of multichannel urodynamic pressure-flow studies and American Urological Association symptom index in the evaluation of benign prostatic hyperplasia. AB - PURPOSE: We correlated multichannel pressure-flow urodynamics and the American Urological Association (AUA) symptom index in the evaluation of benign prostatic hyperplasia. MATERIALS AND METHODS: We evaluated 121 consecutive, symptomatic patients older than 55 years with the AUA symptom score and multichannel pressure flow urodynamic studies. Testing was performed during a single session and the data obtained from 103 patients were plotted on the Schafer nomogram for assessment of outflow obstruction. Linear regression statistical analysis was used to determine correlations. RESULTS: There was no significant correlation between uroflowmetry and Schafer curves (r = 0.173 to 0.326), uroflowmetry and AUA symptom scores (r = 0.134 to 0.153) and, most importantly, AUA symptom scores and Schafer curves (r = 0.025 to 0.137). CONCLUSIONS: We conclude that these modalities measure independent variables, and should not be linked in the evaluation and treatment decision of the patient with prostatism. PMID- 7541855 TI - Benign prostatic hyperplasia. PMID- 7541854 TI - Placebo controlled double-blind study to test the efficacy of the aromatase inhibitor atamestane in patients with benign prostatic hyperplasia not requiring operation. The Schering 90.062 Study Group. AB - PURPOSE: We tested the theoretical concept that a selective decrease in estrogens has a beneficial therapeutic effect on established benign prostatic hyperplasia. MATERIALS AND METHODS: In a double-blind study 160 patients from 14 centers were randomized between 2 groups to receive either placebo or the aromatase inhibitor atamestane (1-methyl-androsin-1,4 diene-3 17-dione, 400 mg. daily for 48 weeks). RESULTS: The aromatase inhibitor decreased the mean estradiol level by approximately 40% and estrone by 60%. The testosterone concentration increased by more than 40% and dihydrotestosterone increased to 30%. Analysis of clinical parameters showed no difference between placebo and atamestane. CONCLUSIONS: The counter regulatory increase in androgens may counterbalance any positive effect of the decrease in estrogens to preserve intraprostatic homeostasis. PMID- 7541856 TI - Family history and the risk of prostatic carcinoma in a high risk group of urological patients. AB - PURPOSE: We examine the association of family history and prostatic carcinoma. MATERIALS AND METHODS: A total of 2,968 consecutive patients referred for prostate cancer detection responded to a questionnaire and underwent transrectal ultrasound examination with or without biopsy. RESULTS: Of the men 329 (11.1%) had a family history of prostate cancer. No differences were observed between groups with and without a family history with respect to mean patient age, serum prostate specific antigen level or biopsy rate. Prostate cancer was detected in 133 of 329 patients (40.4%) with a family history and 769 of 2,639 (29.1) with no family history (p < 0.001, odds ratio 1.7). No significant differences were observed between cancer patients with or without a family history with respect to mean Gleason score (6.0 versus 6.2), patient age at diagnosis (65.8 versus 66.7) and prostate specific antigen level (16.8 versus 17.1). CONCLUSIONS: Patients with a family history of prostate cancer have a greater risk of the disease. In this select group of patients a positive family history was not associated with an earlier age at cancer diagnosis or a different histological grade of tumor. PMID- 7541858 TI - Prostate cancer. PMID- 7541857 TI - Effect of inflammation and benign prostatic hyperplasia on elevated serum prostate specific antigen levels. AB - PURPOSE: We quantify the causes of elevated serum prostate specific antigen (PSA) concentrations in men whose prostate biopsies repeatedly showed no cancer. MATERIALS AND METHODS: The effects of prostate volume, inflammation, echogenicity on ultrasound and calculi were examined in a large PSA-based screening population of 148 men with serum PSA concentrations greater than 4.0 ng./ml., findings suspicious for cancer on digital rectal examination and multiple negative biopsies. These men were selected and compared to 64 men with suspicious rectal examinations, multiple negative biopsies and serum PSA concentrations of 4.0 ng./ml. or less. RESULTS: The high PSA group had larger prostates (68 versus 33 cc, p = 0.0001) and significantly more subclinical prostatic inflammation. Acute and chronic inflammation was more prevalent in the high PSA group (63% versus 27%, p = 0.0001 and 99% versus 77%, p = 0.0001, respectively). A simultaneous regression analysis showed that prostatic size accounted for 23%, inflammation 7%, prostatic calculi 3% and nonisoechoic ultrasound lesions 1% of the serum PSA variance. CONCLUSIONS: Prostate volume and inflammation are the most important factors contributing to serum PSA elevation in men without clinically detectable prostate cancer. PMID- 7541859 TI - Randomized prospective study comparing radical prostatectomy alone versus radical prostatectomy preceded by androgen blockade in clinical stage B2 (T2bNxM0) prostate cancer. The Lupron Depot Neoadjuvant Prostate Cancer Study Group. AB - PURPOSE: Nonrandomized clinical trials have suggested that preoperative androgen deprivation can decrease the likelihood of positive surgical margins in patients with clinically localized prostate cancer. A multicenter prospective randomized trial compared radical prostatectomy alone to radical prostatectomy after 3 months of leuprolide acetate depot and flutamide in patients with stage cT2bNxM0 prostate cancer and a serum prostate specific antigen level less than 50 ng./ml. MATERIALS AND METHODS: We randomized 149 patients to undergo androgen deprivation and 138 to undergo lymphadenectomy with (137) or without (1) prostatectomy. Of the 154 patients randomized to the surgery alone group 144 underwent pelvic node dissection with (138) or without (6) prostatectomy. RESULTS: There was no statistically significant difference between the 2 groups in operating time, blood loss, need for transfusion, postoperative morbidity or length of hospital stay. There were 4 rectal and 2 ureteral injuries in the surgery alone group and none in the pretreatment group (p < 0.05). Patients who received androgen deprivation preoperatively had a significantly lower rate of capsule penetration (47% versus 78%, p < 0.001), positive surgical margins (18% versus 48%, p < 0.001) and tumor at the urethral margin (6% versus 17%, p < 0.01). CONCLUSIONS: Long-term followup data will be needed to determine whether there will be a lower incidence of biochemical relapse as determined by prostate specific antigen, local recurrence or metastasis, with an improvement in patient survival. PMID- 7541861 TI - Transrectal ultrasound-guided transperineal cryoablation in the treatment of prostate carcinoma: preliminary results. AB - PURPOSE: We studied ultrasound-guided percutaneous cryoablation for treatment of prostate carcinoma. MATERIALS AND METHODS: Our series includes 83 individuals who underwent transrectal ultrasound-guided transperineal percutaneous cryoablation of the prostate. Prostate specific antigen levels, biopsy results and complications were assessed at 3 months. RESULTS: Of 61 biopsies 8 (13.1%) were positive for carcinoma (half showed stage D disease). Of patients with stages T1 to T3 cancer 92.6% were free of disease at 3 months. Prostate specific antigen levels were significantly decreased by an average of 1.90 ng./ml. (p < 0.05). Major complications were infrequent, including bladder perforation in 1 patient, urethral strictures in 3, bladder outlet obstruction in 2 and partial incontinence in 2. Impotence was frequent but transient. CONCLUSIONS: Transrectal ultrasound-guided transperineal percutaneous cryoablation of the prostate produces few major complications and appears at 3 months to be effective in eradicating local prostate tumors. Longer followup is required to test the original hypothesis. PMID- 7541860 TI - Neoadjuvant hormonal therapy before radical prostatectomy decreases the number of positive surgical margins in stage T2 prostate cancer: interim results of a prospective randomized trial. The Belgian Uro-Oncological Study Group. AB - PURPOSE: We investigated the effect of neoadjuvant treatment before radical prostatectomy for clinically localized prostate cancer. MATERIALS AND METHODS: A total of 130 patients with stages T2b and T3 prostate cancer was randomized in a multicenter study: 62 underwent immediate radical prostatectomy and 65 received 560 mg. estramustine phosphate daily for 6 weeks preoperatively. RESULTS: For clinical stage T2b tumors the neoadjuvant treatment resulted in a significant decrease in positive surgical margins compared to the nonpretreated group. This difference was not found for clinical stage T3 tumors. The impact on progression and survival still must be analyzed. CONCLUSIONS: Neoadjuvant treatment can be beneficial for clinical stage T2 prostate cancer. Optimal treatment for stage T3 tumors remains controversial. PMID- 7541862 TI - Prostate specific antigen after gonadal androgen withdrawal and deferred flutamide treatment. AB - PURPOSE: We assess the impact of deferred flutamide treatment on the serum prostate specific antigen (PSA) level in patients with localized or metastatic cancer. MATERIALS AND METHODS: The study included 45 patients with localized cancer and 50 with metastatic cancer with an increasing (87) or stable (8) PSA level after gonadal androgen withdrawal. RESULTS: Of 40 evaluable patients with localized cancer and 50 with metastatic cancer 32 (80%) and 27 (54%), respectively, had a PSA decrease of 50% or more of baseline during flutamide treatment (p = 0.014). Among patients with localized cancer actuarial analysis of freedom from PSA elevation during flutamide treatment favored those with a 50% or greater PSA decrease (p = 0.006) but in patients with metastatic cancer the analysis revealed no significant difference. CONCLUSIONS: The relative density of tumor cells that are dependent on adrenal androgen after gonadal androgen withdrawal may be greater in patients with localized cancer and deferred flutamide treatment may enhance cancer control in those with localized disease. PMID- 7541863 TI - Clinical and biochemical evidence of control of prostate cancer at 5 years after external beam radiation. AB - PURPOSE: We demonstrate the 5-year survival rate for patients with prostate cancer treated by irradiation, the value of the conformal technique and prostate specific antigen (PSA) doubling times after irradiation. MATERIALS AND METHODS: The outcome of 502 consecutive patients with stages T1 to T3 prostate cancer treated by irradiation alone is reported. PSA doubling times before and after failure are reported for 13 patients and posttreatment PSA doubling times are reported for 93 consecutive patients in whom radiation failed. RESULTS: The actuarial survival with biochemical freedom from disease (PSA nadir 1.5 or less not increasing) at 5 years was 44% for all patients, 50% for the conformal treatment group and 39% for the conventional therapy group. PSA doubling times after radiation failure were variable, with 42% greater than 12 months. CONCLUSIONS: The 5-year survival rate for patients with prostate cancer treated by irradiation is excellent. The conformal technique is superior to conventional therapy and there is no evidence that irradiation accelerates the growth rate of prostate cancer. PMID- 7541864 TI - Long-term survival and mortality in prostate cancer treated with noncurative intent. AB - PURPOSE: We investigate the long-term outcome of patients with prostate cancer treated with noncurative intent. MATERIALS AND METHODS: All 514 prostate cancer patients who died between 1988 and 1991 and who underwent either deferred or immediate hormonal treatment were followed from death until diagnosis. RESULTS: In all patients with stage M0 disease at diagnosis the ultimate cancer mortality rate was 50%. Among the 65 patients who survived at least 10 years the mortality rate due to prostate cancer was 63%. CONCLUSIONS: Mortality in patients with stage M0 prostate cancer was surprisingly high when followup exceeded 10 years. PMID- 7541867 TI - Re: Salvage radical prostatectomy: outcome measured by serum prostate specific antigen levels. PMID- 7541866 TI - Correlation between micturitional urethral pressure profile and pressure-flow criteria in bladder outlet obstruction. AB - PURPOSE: We correlate micturitional urethral pressure profilometry with pressure flow diagnoses of outlet obstruction. MATERIALS AND METHODS: Urodynamic evaluation was done of 86 consecutive men with voiding symptoms. Obstruction criteria were a micturitional urethral pressure profile (MUPP) gradient greater than 5 cm. water, Schafer's linear passive urethral resistance relation (PURR) greater than grade 1 and Abrams-Griffiths nomogram. RESULTS: Interpretable results were completed in 99% of the patients undergoing MUPP and 60% undergoing pressure-flow studies (p < 0.00001). MUPP diagnosis agreed with PURR (p = 0.0015) and Abrams-Griffiths nomogram results (p = 0.00004). MUPP gradients correlated well with PURR (r = 0.70, p < 0.00001). Using optimum cutoff values (11 cm. water), the sensitivity of MUPP was 83%, specificity 82% and positive predictive value 94%. CONCLUSIONS: MUPP correlates well with and yields interpretable results more often than pressure-flow studies. PMID- 7541865 TI - Need for hospital care and palliative treatment for prostate cancer treated with noncurative intent. AB - PURPOSE: We investigate the ultimate need for palliative treatments and hospital care in prostate cancer patients treated with noncurative intent. MATERIALS AND METHODS: A retrospective analysis was done of 514 prostate cancer patients who died between 1988 and 1990. RESULTS: Of the patients who later died of prostate cancer 61% needed 1 or more palliative treatments (transurethral resection of the prostate, radiation treatment or upper urinary tract diversion) before death. An average of 5 weeks was spent in the hospital due to prostate cancer. CONCLUSIONS: Patients who failed deferred treatment (thus dying of prostate cancer) needed a considerable amount of hospital care and palliative therapy before they ultimately died of the disease. PMID- 7541868 TI - Differential expression of apolipoprotein-D and prostate specific antigen in benign and malignant prostate tissues. AB - PURPOSE: To investigate Apolipoprotein-D (Apo-D) and prostate specific antigen (PSA) immunohistochemical staining of nonmalignant and malignant human prostate tissues. MATERIALS AND METHODS: Apolipoprotein-D and PSA immunoreactivity were evaluated by video image analysis in nonmalignant prostates and in 30 stage D2 prostate cancers. RESULTS: Apolipoprotein-D was detected in all 30 tumors, and the level of staining was elevated in comparison to age-matched nonmalignant prostates (p < 0.05). In contrast, the level of PSA staining in tumors was less than that detected in nonmalignant prostates. CONCLUSIONS: Apolipoprotein-D is expressed in normal human prostate. Elevated Apo-D staining is associated with advanced prostate cancer. PMID- 7541869 TI - A new bioimplant for the endoscopic treatment of vesicoureteral reflux: experimental and short-term clinical results. AB - We investigated the safety and clinical effects of a new biocompatible, biodegradable treatment, the Deflux system (dextranomer microspheres in sodium hyaluronan solution), for the endoscopic treatment of grades III and IV vesicoureteral reflux. In preclinical safety studies in pigs histopathological examination demonstrated excellent tolerance. Two weeks after submucous implantation in the pig bladder early ingrowth of fibroblasts and recently generated collagen were noted at the implantation sites. At 14 weeks of followup this ingrowth had slightly increased. Long-term followup in rats showed that the volume of subcutaneous implants was slightly reduced (23%) 1 year after implantation. In a clinical study we investigated the implantation technique and the short (3 months) and long-term (1 year) effects of Deflux implantation in 75 children (101 ureters) with grades III and IV vesicoureteral reflux. We report data from up to 3 months of followup. Implant volumes of 0.4 to 1.0 ml. were sufficient to create distinct boluses and crescent-like ureteral orifices. Although viscous, due to its viscoelastic properties the substance was easy to inject in a well controlled manner. At cystography 3 months later reflux had resolved in 68% of implants, was reduced to grades I and II in 13% of implants and was unchanged in 19% of treated ureters (grades III and IV reflux). No signs of ureteral obstruction or adverse reactions were noted. Results from 1 year of followup will be reported later. Our results indicate that the dextranomer microspheres act as micro-carriers that promote ingrowth of fibroblasts and generate new collagen. We conclude that the Deflux system may represent a new, safe, simple alternative to endoscopic treatment of vesicoureteral reflux in children. PMID- 7541870 TI - Physician-assisted suicide. PMID- 7541874 TI - [Clinicopathological study of nonpalpable and nonvisible (stage T1c) prostate cancer]. AB - Clinical and pathologic characteristics of stage T1c disease (nonpalpable and nonvisible cancer) was studied retrospectively in men who underwent radical prostatectomy in order to better understand this disease entity. Findings in stage T1c disease (16 patients) were directly compared with those in stage T2b disease (11 patients). No significant difference was observed between these groups with regard to age, preoperative serum prostatic acid phosphatase level, prostatic weight, numbers of tumor foci, total tumor volume, volume of index cancer and tumor grade (p > 0.05). Preoperative prostate specific antigen concentration was significantly lower in stage T1c group (p < 0.05). Substantially larger number of patients with stage T1c disease had pathologically organ confined disease when compared with T2b group (86.7% versus 45.5%, p < 0.05). Seventy-five percent (12/16) of stage T1c and 90.9% (10/11) of stage T2b disease were considered clinically significant. Most of stage T1c disease is organ confined and clinically significant. Clinical and pathologic features of these tumors are similar to those in T2b disease. Twenty-five percent of stage T1c disease, however, are small and thus may potentially be overtreated. Enhanced detection of prostate cancer achieved with modern technology can lead to undesirable treatment of clinically insignificant tumors. Preoperative diagnostic modalities which can reliably distinguish groups of tumor with different biological potential are needed to overcome this contradiction. PMID- 7541873 TI - [Cases of acute hepatitis C and positive antibodies to hepatitis C virus after tattooing by the same tattooer]. PMID- 7541872 TI - [A case of retroperitoneal NHL with direct invasion to the liver, pancreas and gastric wall]. PMID- 7541875 TI - [Drug therapy and prevention of hemorrhage in esophageal and gastric varices]. PMID- 7541871 TI - Combination drug therapy for benign prostatic hyperplasia. PMID- 7541876 TI - Stable isotope dilution analysis of GABA in CSF using simple solvent extraction and electron-capture negative-ion mass fragmentography. PMID- 7541877 TI - Dihydropyrimidinase deficiency presenting in infancy with severe developmental delay. PMID- 7541878 TI - Accumulation of phytanic acid alpha-oxidation intermediates in Zellweger fibroblasts. PMID- 7541879 TI - Role of nitric oxide in human esophageal circular smooth muscle in vitro. AB - The role of nitric oxide in human esophageal smooth muscle was examined. Immunostaining for constitutive nitric oxide synthase labeled nerve fibers and bundles within longitudinal and circular smooth muscle layers of resected tissue samples. Strips of circular muscle mounted in organ baths exhibited spontaneous contractions and active tone. When exposed to 5-second trains of electric field stimulation at 20 Hz, most strips exhibited intrastimulus "on" and poststimulus "off" contractions. Exposure to a 0.1 mumol/L (or greater) concentration of atropine converted "on" contractions to "on" relaxations and reduced "off" contractions by 63%. Exposure to NG-nitro-L-arginine resulted in concentration dependent enhancement of "on" contractions and abolition of "off" contractions. Excess L-arginine enhanced the reversal of these effects. Sodium nitroprusside inhibited both spontaneous and evoked contractions. These results suggest that nitric oxide synthesis is a mediator of neural inhibition of human esophageal circular smooth muscle and is necessary for the occurrence of "off" contractions. PMID- 7541880 TI - Immediate-early gene expression in human saphenous veins harvested during coronary artery bypass graft operations. AB - Saphenous vein graft occlusion is a common late complication of coronary bypass grafting. Intimal smooth muscle cell hyperplasia is a component of this pathobiology, but the underlying molecular events are poorly understood. Immediate-early genes are activated shortly after growth stimulation and subserve cellular functions, which may contribute to intimal smooth muscle cell accumulation. In the present study, human saphenous vein grafts were harvested with minimal manipulation during coronary bypass and processed for isolation of total ribonucleic acid to examine change in immediate-early gene expression of messenger ribonucleic acid by Northern blotting techniques. Thirty saphenous vein grafts were incubated at 4 degrees C in Dulbecco's modified Eagle media from 30 minutes to 10 hours. The messenger ribonucleic acids for immediate-early genes c fos and c-myc were weak or undetectable in controls but were increased (> 10 times controls) within 1 hour (c-fos) and persisted for at least 6 hours (c-myc) after harvest. Our results demonstrate, for the first time in human vascular tissue, incipient immediate-early gene induction. This information may lead to molecular therapies to control saphenous vein graft disease. PMID- 7541882 TI - Cloning cell death genes. PMID- 7541881 TI - Pleural complications in lung transplant recipients. AB - Pleural complications occurred in 30 (22%) of 138 patients after 53 single and 91 double lung transplants between September 1986 and February 1993. These were defined for the purpose of this study as pneumothorax persisting beyond the first 14 postoperative days, recurrent pneumothorax, or any other pleural process that necessitated diagnostic or therapeutic intervention. Overall, a higher pleural complication rate was seen in double lung transplantation (25 of 30) than in single lung transplantation (5 of 30) with no differences noted in the frequency among preoperative diagnostic groups (p > 0.05). Pneumothorax was the most frequent complication, affecting 14 of 30 patients, with 6 of 14 cases occurring after transbronchial biopsy. All pneumothoraces in single (n = 4) and double lung transplantation (n = 10) resolved spontaneously or with chest tube thoracostomy. One patient required placement of a Clagett window after open lung biopsy and another required thoracotomy and pleural abrasion after transbronchial biopsy. Parapneumonic effusion was observed in 4 of 30 double lung transplantations with spontaneous resolution in all cases. Empyema affected 7 of 30 patients and occurred exclusively in the double lung transplant group. Sepsis developed in three of the patients with this complication and they subsequently died. The risk of empyema was independent of preoperative diagnosis (p > 0.05). Of interest, all patients with cystic fibrosis (n = 3) with complicating empyema had Pseudomonas cepacia in the pleural fluid. Other miscellaneous complications included subpleural hematoma, chylothorax, and hemothorax. The latter two necessitated thoracic duct and bronchial artery ligation, respectively. In summary, a significant proportion of lung transplant recipients will have pleural space complications. The vast majority of these will resolve spontaneously or with conservative procedures. These complications were not related to preoperative diagnosis nor associated with a significant prolongation of hospital stay (p > 0.05). Empyema is the only pleural space complication associated with increased patient mortality and, as such, is an important clinical marker for those at risk for sepsis and death. PMID- 7541883 TI - The end of the (cell) line: methods for the study of apoptosis in vitro. PMID- 7541885 TI - Methods for studying cell death and viability in primary neuronal cultures. PMID- 7541886 TI - Identification of dying cells--in situ staining. PMID- 7541884 TI - Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. PMID- 7541887 TI - Transient transfection assays to examine the requirement of putative cell death genes. AB - In conclusion, this chapter provides a convenient and efficient method for the detection and analysis of transiently transfected cells. Such strategies allow a fast and simple analysis of the requirement for particular genes that have been identified as being induced during apoptosis. Our experience has been that, when screening for "cell death genes," it is easy to isolate genes induced during apoptosis but far more difficult to determine the requirement for any given gene. These protocols have rendered such determinations much simpler to perform. PMID- 7541889 TI - Outcome to five years of age of children born at 24-26 weeks' gestational age in Victoria. The Victorian Infant Collaborative Study Group. AB - OBJECTIVE: To determine the outcome to five years of age of liveborn children born at 24-26 weeks' gestation. DESIGN: Regional cohort study of preterm children. SUBJECTS: Consecutive children liveborn at 24-26 weeks' gestation in Victoria from 1 January 1985 to 31 December 1987. MAIN OUTCOME MEASURES: Survival rates and rates of sensorineural impairment and disability at five years of age. RESULTS: 95 of 316 (30.1%) children survived to five years; survival rates increased with gestational age. 94 children (99%) were assessed at five or more years of age, corrected for prematurity. Twelve children had some form of cerebral palsy, causing a severe disability in only one. Two children required hearing aids for sensorineural deafness, five had bilateral blindness, and four were too disabled for intelligence quotient measurement. Overall, sensorineural disability was severe in seven (7.4%), moderate in seven (7.4%), mild in 23 (24.5%), and nil in 57 (60.6%) of children assessed. There was no trend to increasing disability with lower gestational age. CONCLUSIONS: The sensorineural outcome for this cohort is mostly favourable, and is better than that reported from some contemporaneous regional cohorts born in other parts of the world. The rates of sensorineural impairments, such as blindness and cerebral palsy, are higher than in non-preterm children but are not inordinately high, and most have no sensorineural impairment or disability. PMID- 7541888 TI - Bacterial contamination of aerosol solutions containing antibiotics. AB - In an investigation of microbial contamination of aerosol solutions containing antibiotics (hospital pharmaceutical preparations, no preservatives added), five of six residual solutions after multiple use for 7 days were contaminated at a concentration of 10(6) viable counts/ml. Contaminants were glucose-nonfermenting Gram-negative bacilli such as Pseudomonas cepacia and Flavobacterium meningosepticum. The major contaminant, P. cepacia, multiplied rapidly in the aerosol solution under simulated actual-use conditions. The contamination seemed to have been caused by storage, at room temperature instead of in a refrigerator, of the multiple-dose solutions and by frequent re-use of syringes to measure the solutions. Prompt refrigerator storage after each use of the solutions and abandonment of the syringes within 24 h eliminated bacterial contamination of the solutions. Sufficient attention is not paid to prevent bacterial contamination of aerosol solutions containing antibiotics because of the fact that they contain antibiotics. Thus aerosol solutions containing antibiotics should be handled with great care to prevent bacterial contamination. PMID- 7541891 TI - Cytotoxic T cell recognition of a human melanoma derived peptide with a carboxyl terminal alanine-proline sequence. AB - Recently, we defined the antigenic epitope recognized by the human monoclonal antibody L94 to be a protein with a C-terminal sequence of alanine-proline (AP). An antigenic peptide no. 707 (RVAALARDAP), which was identified by the use of cDNA libraries of an antigen positive melanoma cell line M14, was evaluated for cellular immune responses in melanoma patients. PBMC from 16 of 19 melanoma patients were shown to lyse autologous B lymphoblastoid cell lines (BCL) pulsed with synthetic peptide no. 707 (hereafter no. 707). This specific cytotoxicity to the peptide significantly increased in 84% of melanoma patients after in vivo immunization with a melanoma cell vaccine (MCV). In contrast, peptide specific cytotoxicity was observed in only one of 19 normal volunteer donors. In vitro restimulation of MCV treated patients' PBMC with no. 707 augmented cytotoxicity against autologous no. 707-pulsed BCL. This cytotoxicity was specific to the C terminal sequence AP, since the removal of C-terminal AP completely abolished the specific lysis. no. 707 restimulation of PBMC enhanced cytotoxicity against autologous melanomas. Autologous melanoma and peptide-pulsed BCL targets were lysed by CD8+CTL in a HLA class I-restricted manner. The strong cytotoxicity was obtained from patients of HLA A24. CTL lysis of autologous no. 707-pulsed BCL was partially blocked by unlabeled autologous melanomas in a cold target inhibition test. This suggested that the epitope identical or cross-reactive to no. 707 may be presented on the melanoma cell surface by HLA class I antigens. Our findings suggest that peptide no. 707 presented on human melanoma cells is recognized by CTL and that C-terminal AP plays a critical role in both antibody and T cell recognition. PMID- 7541890 TI - pH dependent binding of high and low affinity myelin basic protein peptides to purified HLA-DR2. AB - Major histocompatibility complex (MHC) class II molecules are cell surface glycoproteins and are known to display processed antigens on the surface of antigen presenting cells (APC). Within the APC, the loading of processed antigenic peptides to MHC class II molecules is known to take place in the endosomal compartment at acidic pH environment. The present study describes the in vitro effect of pH on binding of four biotinylated myelin basic protein (MBP) peptides to affinity purified HLA-DR2 containing a mixture of DRB1*1501 and DRB5*0101 beta chain. The binding affinity of the selected peptides are in the order of MBP(83-102)Y83 > MBP(124-143) > MBP(143-168) > MBP(1-14). Most of these peptides in association with HLA-DR2 are considered as immunodominant epitopes for human multiple sclerosis autoimmune disorder. One epitope, MBP(1-14), had almost no affinity to purified HLA-DR2 and was used as a control peptide in all binding assays. The quantitation of the bound peptide at various pH was carried out by antibody capture of complexes followed by avidin-alkaline phosphatase detection system. Among four peptides tested, only the highest affinity MBP(83 102)Y83 peptide showed maximum binding to purified HLA-DR2 at acidic pH. Two other epitopes, MBP(124-143) and MBP(143-168), showed maximum binding at basic and neutral pH values, respectively. The binding of only high affinity peptides, MBP(83-102)Y83 and MBP(124-143), was significantly affected by changing the pH of the binding buffer. Such alteration in pH of the binding buffer resulted in 100% occupancy of DR2 with both high affinity MBP peptides. In contrast, no significant increase in binding of the low affinity MBP(143-168) peptide was observed at altered pH values. The specificity of the increased binding of high affinity peptides to HLA-DR2 at optimum pH was demonstrated by competitive binding assays using non-biotinylated peptides. Finally, the stability of various MBP peptide bound complexes was tested at 4 degrees, 25 degrees and 37 degrees C which correlates well with their affinity to HLA-DR2. These results suggest that pH plays an important role in in vitro binding of antigenic peptides and such manipulation of binding conditions can be utilized in generating 100% loaded MHC class II with high affinity antigenic peptides. Since high affinity peptides are generally considered as major immunodominant epitopes, the in vitro pH dependent binding can be utilized in screening immunodominant epitopes of various autoantigens and generating complexes of defined composition. PMID- 7541892 TI - [Prophylaxis against mycoses in neutropenic patients]. AB - During the last years, the proportion of cancer patients who develop systemic fungal infections has increased steadily. These infections are characterised by high mortality, especially in patients with persistent granulocytopenia and in those receiving allogeneic bone marrow transplants. The most important pathogens in neutropenic patients are Candida and Aspergillus spp. Usually, Candida infections arise from overgrowth in the gastrointestinal tract, while Aspergillus infections are acquired by inhalation of spores. Prophylaxis of systemic fungal infections seems mandatory since optimal strategies for diagnosis and treatment of these infections are lacking. Treatment with the non-absorbable polyenes nystatin and amphotericin B is useful for prophylaxis of superficial fungal infections, provided that compliance of the patients is optimal. The imidazoles ketoconazole and miconazole can reduce the incidence of superficial fungal infections, but there are conflicting data regarding their value for prevention of systemic mycoses. There are several studies indicating that prophylactic use of fluconazole reduces the incidence of mucosal and systemic fungal infections, especially in patients receiving allogeneic bone marrow transplants. Fluconazole shows reduced activity against several Non-albicans spp. and is not active against Aspergillus spp. Itraconazole has in vitro and in vivo activity against several Aspergillus spp. but high serum and tissue levels are necessary. However, bioavailability of itraconazole is reduced in patients with raised gastric pH and no i.v. formulation is available. Although there is some evidence for its prophylactic activity against Aspergillus infections in neutropenic patients, more studies are necessary to confirm these findings. Intravenous amphotericin B cannot be recommended for routine prophylactic use because of its toxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541894 TI - New revelations about the long-QT syndrome. PMID- 7541893 TI - [Future directions of antimycotic therapy]. AB - There are presently numerous developments in the field of antifungal armamentarium. Amphotericin B has been considered as the standard treatment since the fifties for most invasive fungal infections. However, emergence of resistant strains and failures despite adequate treatment with amphotericin B are numerous and toxicity as well as side effects are limiting factors in many patients. Alternative modalities to administer amphotericin B using liposomal or lipid formulations are presently evaluated. Tremendous research has been performed in the development of new azoles including ketoconazole, fluconazole and itraconazole. The emergence of resistant strains of yeasts in patients treated with fluconazole requires attention. The problem of possible cross resistance for all azoles should be a matter of further investigation. Itraconazole is a new broad-spectrum antifungal agent. But due to its pharmacokinetic properties monitoring of serum concentrations is recommended in life-threatening situations. New formulations of itraconazole are currently under development and may be extremely useful. The role of azole antifungals for prophylactical administration or therapeutic purposes must be balanced carefully. Studies designed to evaluate adjuvant antifungal treatment based on G-CSF, M-CSF or GM-CSF in combination to antifungals would be useful. Future directions of antifungal treatments should include evaluation of combination of antifungal agents to each other. There is a tremendous need for pivotal clinical studies in the field of antifungal treatments which need international collaboration in order to collect rapidly significant numbers of data. Such comprehensive approaches are initiated by the EORTC Invasive Fungal Infections Cooperative Group, and all European clinicians taking care of mycosis patients are invited to cooperate. PMID- 7541896 TI - [A fair trial? The European randomized prostatic cancer study]. PMID- 7541895 TI - Effects of lead on cloned voltage-operated neuronal potassium channels. AB - The action of lead (Pb2+) on cloned voltage-operated potassium channels of the rat brain was investigated in oocytes of Xenopus laevis. Pb2+ was found to decrease the potassium currents. This effect was due to a shift of the current voltage relation in a positive direction (up to 30 mV). The Pb2+ effect appeared at a threshold concentration of about 0.1 mumol/l and was maximal at a concentration of about 30 mumol/l. At a potential of -30 mV, the concentration needed for a 50% reduction of the potassium current was 1.0 mumol/l. The depressant effect of Pb2+ was obtained with all potassium channels tested (Kv1.1, Kv1.2, Kv1.4, Kv2.1, Kv3.4). It was minimal for the Kv2.1 channel and maximal for the Kv1.1 channel at potentials negative to 0 mV. An effect comparable with that of Pb2+ could not be induced by the application of magnesium or calcium. The external application of Pb2+ led to a decrease of potassium currents in outside out but not in inside-out membrane patches. Overall, Pb2+ had a significant effect on the potassium channels which may contribute to the mechanisms of Pb2+ neurotoxicity. PMID- 7541897 TI - Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve. AB - Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I and or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species. PMID- 7541899 TI - [Successful treatment with combined chemotherapy (carboplatin, etoposide, adriamycin, bleomycin) and radiotherapy for recurrent intracranial malignant germ cell tumor: case report]. AB - We report a case of a patient with recurrent malignant germ cell tumor who was successfully treated with carboplatin combination followed by radiation therapy. A 13 year-old female presented with diabetes insipidus six years ago. From the homogeneous tumor intensity on CT scan and negative titer of AFP and HCG, we made a diagnosis of suprasellar germinoma, and treated her with radiation therapy (40Gy), which resulted in almost complete disappearance of the tumor. The tumor recurred six years later in the primary region and in the cervical spinal cord with elevated AFP titer (160ng/ml) and HCG titer (404mlU/ml). She was treated with multi-agents chemotherapy consisting of carboplatin (450mg/m2, day 1), etoposide (150mg/m2, days 1-3), adriamycin (30mg/m2, day 1), and bleomycin (20mg/m2, days 1 and 8), followed by radiation therapy to the whole brain (20Gy), the whole spine (30Gy) and the cervical spine (20Gy). The tumors completely disappeared. We discuss treatment failures of clinically diagnosed germinoma without histological verification, and effective aggressive chemotherapy for recurrent malignant germ cell tumors. PMID- 7541898 TI - Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: evidence for feedback autoregulation. AB - Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N omega-nitro-L-arginine methyl ester-sensitive conversion of L [3H]arginine into L-[3H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC50 approximately 100 microM) and prevented by the NO scavenger oxyhemoglobin. L-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells. PMID- 7541900 TI - A case of Pelizaeus-Merzbacher disease showing increased dosage of the proteolipid protein gene. AB - Clinical, neuropathological and molecular genetic studies in a 9 month old boy with Pelizaeus-Merzbacher disease are described. The principal clinical features were developmental delay, nystagmus, stridor and seizures. Both brain and spinal cord showed almost complete absence of stainable central myelin, while cranial and spinal root myelin was preserved. Probes for cDNA in the boy and his asymptomatic mother indicated an increase in the dosage of proteolipid protein gene (of at least twofold) compared with controls. PMID- 7541901 TI - The proteolipid protein gene. AB - Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform, DM-20, produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM-20 is also present in myelin-forming and non-myelin-forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM-20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus-Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X-linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM-20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot-Marie-Tooth type in which the peripheral myelin-22 gene is mutated. PMID- 7541902 TI - Expression of the proteolipid protein gene in glial cells of the post-natal peripheral nervous system of rodents. AB - The proteolipid protein (PLP) gene encodes for two proteins, PLP and DM-20, which are produced by alternative splicing of exon 3B. PLP is the major CNS myelin protein and is postulated to play a structural role at the intraperiod line. Its developmental expression mirrors that of CNS myelination. DM-20 predominates in the embryo and prior to myelination of the CNS and may be involved in glial cell development. The PLP gene is expressed in the PNS in which DM-20 is the predominant isoform at all ages. In this study we describe the localization of the two isoforms in the post-natal rodent PNS using immunostaining and reverse transcriptase PCR. DM-20 is present in relatively high abundance in non-myelin forming Schwann cells and within cytoplasmic regions of myelinated internodes, particularly the paranodes and Schmidt-Lanterman incisures and also the outer Schwann cytoplasm and perinuclear cytoplasm. DM-20 is also located in the perineuronal satellite cells of spinal, cranial and autonomic ganglia and in the ensheathing cells of the olfactory nerve layer of the olfactory bulb. PLP was detected by immunocytochemistry in the perinuclear region of myelinated internodes; PCR analysis indicated small amounts of PLP mRNA in the other locations but protein was not detected by immunostaining. Neither protein was identified in compact myelin of the PNS. DM-20 is the predominant product of the PLP gene expressed in a wide variety of peripheral glia. Its presence is not correlated to a myelin-forming state. Other studies have demonstrated early embryonic expression of the PLP gene throughout the PNS and all these features support the hypothesis that any putative role for DM-20 is unrelated to myelination but may involve glial cell development. PMID- 7541903 TI - The dorsal midbrain anticonvulsant zone--II. Efferent connections revealed by the anterograde transport of wheatgerm agglutinin-horseradish peroxidase from injections centred on the intercollicular area in the rat. AB - Activation of the dorsal midbrain has a powerful anticonvulsant effect in the maximal electroshock model of epilepsy. The suppression of tonic seizures can be obtained most reliably from an area centred on the intercollicular nucleus overlapping into the deep layers of the superior colliculus and adjacent mesencephalic reticular formation. As part of a series of investigations to identify neural mechanisms responsible for mediating the anticonvulsant properties of the dorsal midbrain, the present study provides an anatomical description of the efferent projections of this region. Small amounts of wheatgerm agglutinin-horseradish peroxidase (10-30 nl of a 1% solution) were injected into the intercollicular nucleus and surrounding tissue. The resulting anterograde transport of the tracer was plotted on a set of standard atlas sections. Four major output pathways were identified: (i) an ipsilateral descending projection which had terminations in the microcellular tegmental nucleus, lateral and ventral pontine reticular nucleus pars oralis, ventrolateral tegmental nucleus, ventral and caudal pontine reticular nucleus pars caudalis, raphe magnus nucleus and the gigantocellular nucleus; (ii) a contralateral descending projection which for the most part targeted the same brainstem structures but with weaker terminal labelling; (iii) a projection to the contralateral dorsal midbrain with comparatively weak terminal label in the contralateral superior colliculus, intercollicular nucleus, periaqueductal gray, mesencephalic reticular formation and cuneiform area; (iv) ipsilateral ascending pathway with terminations in the red nucleus, zona incerta, peripeduncular area, parafascicular nucleus, lateral hypothalamus, parts of the pretectum and caudal thalamus. At a general level the dorsal midbrain anticonvulsant zone shares its major output projections and efferent targets with at least one of its near neighbours, including the superior colliculus, periaqueductal gray, the cuneiform nucleus and pedunculopontine nucleus. The possibility that anticonvulsant properties of the intercollicular area can simply be attributed to a unique set of efferent projections is therefore not supported by the anatomy. PMID- 7541905 TI - Galanin-immunoreactive nerve terminals innervating the striated muscle fibers of the rat esophagus. AB - Galanin (GAL) immunohistochemistry combined with acetylcholinesterase (AChE) histochemistry was applied to demonstrate the innervation of the rat esophageal muscle coats. GAL immunoreactivity was found in a number of nerve cell bodies in the myenteric ganglia and in numerous varicose and non-varicose nerve fibers in the myenteric plexus and around blood vessels. Many GAL-positive varicose fibers ran in the internodal strands and along the striated muscle fibers. They often ramified and terminated on the muscle fibers to form arborizing structures, which were most abundant in the thoracic portion of the esophagus. Such GAL-positive terminals were localized in most (87.7%) of AChE-reactive motor endplates on the esophageal striated muscles. Left supranodose vagotomy caused a significant decrease of the GAL-arborizing terminals on the striated muscles of the esophagus. This suggests that they are terminals of efferent fibers in the vagus nerve. PMID- 7541906 TI - Crossed thalamocortical connections in the Madagascan hedgehog tenrec: dissimilarities to erinaceous hedgehog, similarities to mammals with more differentiated brains. AB - The adult erinaceous hedgehog, unlike other mammals, has recently been shown to have prominent crossed projections from the thalamus to the motor cortex. There are suggestions relating this unique pattern of connectivity to the overall degree of brain differentiation and/or the poorly developed corpus callosum. The present tracing study demonstrates that the Madagascan lesser hedgehog tenrec, with its tiny corpus callosum and one of the lowest neocorticalization indices among insectivores, has extensive crossed cortico-thalamic projections, but essentially the same sparse thalamic projections to the contralateral cortex as have placental mammals with more differentiated brains. The implications of the findings and the relevance of extracallosal pathways are discussed. PMID- 7541904 TI - Afferent volley patterns and the spinal release of immunoreactive substance P in the dorsal horn of the anaesthetized spinal cat. AB - Microprobes bearing immobilized antibodies to the C-terminus of substance P were used to measure release of this neuropeptide in the spinal cord of the anaesthetized spinal cat in response to peripheral nerve stimulation. Release of substance P was just detectable in laminae I, II with 150 stimuli (0.5 Hz, 5 min) and was near maximal with 300 stimuli. Using two periods of stimulation of 10 min separated by 15 min, greater levels of substance P were detected during the second period. Fifteen to 25 min after two periods of peripheral nerve stimulation levels of substance P detected by microprobes were still elevated above those present prior to stimulation. Stimulation with bursts of three impulses when delivering a fixed number of stimuli resulted in detection of increased levels of substance P at sites adjacent to the areas of maximal release. The results suggest that maximal release of substance P from the central terminals of primary afferent fibres occurs with relatively few impulses and at low frequencies in agreement with what is known of release from the peripheral terminals of these fibres. PMID- 7541907 TI - Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). AB - Interferon regulatory factor (IRF) genes encode a family of DNA-binding proteins that are involved in the transcriptional regulation of type-I interferon and/or interferon-inducible genes. We report here the characterization of LSIRF, a new member of the IRF gene family cloned from mouse spleen by the polymerase chain reaction using degenerate primers. LSIRF was found to encode a 51 kDa protein that shares a high degree of amino acid sequence homology in the DNA-binding domain with other IRF family members. LSIRF expression was detectable only in lymphoid cells. In contrast to other IRF genes, LSIRF expression was not induced by interferons, but rather by antigen-receptor mediated stimuli such as plant lectins, CD3 or IgM crosslinking. In in vitro DNA binding studies, LSIRF was able to bind to the interferon-stimulated response element (ISRE) of the MHC class I promoter. The expression pattern and DNA binding activities suggest that LSIRF plays a role in ISRE-targeted signal transduction mechanisms specific to lymphoid cells. PMID- 7541908 TI - cIRF-3, a new member of the interferon regulatory factor (IRF) family that is rapidly and transiently induced by dsRNA. AB - In mammals, some of the effects of interferon (IFN) on gene transcription are known to be mediated by a family of IFN-inducible DNA-binding proteins, the IFN regulatory factor (IRF) family, which includes both activators and repressors of transcription. Although IFN activities have been described in many vertebrates, little is known about regulation of IFN- or IFN-stimulated genes in species other than human and mouse. Here, we report the cloning of a chicken cDNA, cIRF-3, encoding a protein with a DNA-binding domain similar to that found in the mammalian IRF family of proteins. Similarity between cIRF-3 and the mammalian IRFs is comparable with that between known members of the family. It is most similar to the IRF proteins ICSBP and ISGF3 gamma but is equally divergent from both. Gel mobility shift assays indicate that cIRF-3 is capable of binding a known IFN-stimulated response element that is conserved between the mammalian and chicken Mx genes. Expression of the cIRF-3 gene can be induced to high levels by poly(I).poly(C). Induction is rapid and transient with no requirement for protein synthesis. Co-treatment of cells with cycloheximide results in superinduction of cIRF-3 mRNA. The structural and regulatory characteristics of cIRF-3 indicate that it is the first example of a non-mammalian IRF protein. PMID- 7541909 TI - A family of cold-regulated RNA-binding protein genes in the cyanobacterium Anabaena variabilis M3. AB - I previously found a cold-regulated RNA-binding protein gene rbpA (now named rbpA1) in Anabaena variabilis M3 [Sato, N. (1994) Plant Mol. Biol. 24, 819-823]. I show here that this gene is a member of a gene family containing at least eight members as evidenced by Southern blot and immunoblot analyses. I have isolated three additional genes (rbpB, rbpC and rbpD) in this family. Of these, rbpB was 100% identical to the rbpB gene of Anabaena 7120 reported previously. Another gene named rbpA in Anabaena 7120 was also found to exist in A.variabilis M3 with identical sequence and named rbpA2. The amino acid sequences of these gene products were highly conserved, except that the RbpD protein lacked glycine-rich C-terminal domain present in all other known members of the gene family. RNA blot and immunoblot analyses showed that the expression of rbpA1, rbpA2, rbpB, rbpC and rbpD, as well as uncloned rbp genes was regulated by cold, though the exact time-course and extent of response to cold were different among these genes. Gel filtration assay showed that all of the Rbp proteins have higher affinities to poly(G) and poly(U) than to poly(A) and poly(C). PMID- 7541910 TI - Transcription and processing of the rodent ID repeat family in germline and somatic cells. AB - ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification. PMID- 7541911 TI - The Xenopus 9 bp ribosomal terminator (T3 box) is a pause signal for the RNA polymerase I elongation complex. AB - In Xenopus, termination by RNA polymerase I (pol I) is mediated by the 9 bp sequence GACTTGCNC and RNA 3'-ends are formed -15-20 nt upstream of this terminator element. Here I show that this 9 bp element, also called the 'T3 box', is a pause signal for the elongating transcription complex. The two major transcripts in the paused complex have 3'-ends mapping to 15 and 21 nt upstream of the T3 box, remain bound to the template in 0.25% Sarkosyl, are subject to pyrophosphorolysis and can be chased into longer transcripts. Mutations that reduce overall termination also affect pausing, indicating that pausing is a limiting step in the termination process. Oligonucleotide competition experiments, furthermore, suggest that pausing requires a DNA binding factor. The data support a model in which the first step leading to transcription termination by pol I in Xenopus is pausing of the elongation complex upstream of the T3 box. PMID- 7541913 TI - [Horizontal transmission of hepatitis C virus to family members of infected polytransfused pediatric patients]. AB - The aim of this study is to evaluate the prevalence of the HCV antibodies in the relatives of polytransfused patients with different haemoglobinopathies. Our results have shown that 4.7% of relatives are aHCV positive. This incidence is much higher than that one reported in scientific publications with regard to non haemoglobinopathis aHCV positive patients. Therefore a careful prevention and surveillance is needed in order to avoid the risk of infection in these subjects. PMID- 7541915 TI - Differential inhibitory effects of serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion. AB - BACKGROUND: Protein phosphorylation and dephosphorylation events are considered to be key steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of serine/threonine phosphatases in phosphoinositol/calcium- and cyclic adenosine monophosphate (cAMP)-mediated stimulus-secretion coupling in rat pancreatic acini. METHODS: Isolated rat pancreatic acini were incubated with either the serine/threonine phosphatase inhibitors okadaic acid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8, secretin, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase activating polypeptide (PACAP), and the intracellular second messengers calcium and cAMP were determined. RESULTS: Okadaic acid or calyculin A reduced secretagogue-stimulated amylase release to near-basal levels. Inhibition of cAMP mediated secretion (by VIP, secretin, or PACAP) occurred at lower concentrations than with inositol triphosphate (IP3)/Ca(2+)-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to cAMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP-or IP3/Ca(2+)-mediated secretagogues. Binding of 125I-CCK-8- or 125I-PACAP-(1-27) to acini was not influenced by the phosphatase inhibitors or W-7. Okadaic acid and calyculin A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27) stimulated cAMP synthesis, whereas W-7 inhibited by 50% and 40%, respectively. CONCLUSIONS: The inhibitory profiles of okadaic acid, calyculin A, cyclosporin A, and W-7 indicate that phosphatases 1 and 2A play a relevant role in cAMP-mediated enzyme release, whereas phosphatases 1 and 2B are predominantly involved in IP3/Ca(2+)-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways. PMID- 7541914 TI - Role of histamine and calcitonin gene-related peptide in the hyperemic response to hypertonic saline and H+ back-diffusion in the gastric mucosa of cats. AB - BACKGROUND: The present study was undertaken to measure the output of histamine and calcitonin gene-related peptide (CGRP) from injured and restituting gastric mucosa into venous blood and to study the effect of acid back-diffusion on the release of these mediators and their role in the hyperemic response to injury. METHODS: Stomachs of cats were perfused with saline at pH 1.0 or 7.4. Gastric mucosal blood flow (GMBF) was determined with radioactive microspheres, and blood flow in the portal vein and celiac artery was determined by transit-time flowmetry. H+ back-diffusion/secretion was measured by pH-stat titration and by measuring the arteriovenous base excess difference. Mucosal injury was produced by exposure to 2 M NaCl. Histamine and CGRP in portal venous blood were measured by radioimmunoassay. RESULTS: During mucosal exposure to 2 M NaCl GMBF increased, and histamine (0.23 nmol/min) and CGRP (1.2 pmol/min) were released from the mucosa into blood. The hyperemic response was reduced by pretreatment with H1 and H2 blockers and still further by addition of the blocker CGRP8-37. After mucosal damage and luminal perfusion at pH 7.4, GMBF and output of CGRP and histamine decreased towards base-line levels within 30 min. During luminal perfusion at pH 1.0 associated with acid back-diffusion, GMBF and histamine output remained high, whereas the output of CGRP decreased to base-line level. Pretreatment with H1 and H2 blockers reduced the hyperemic response as measured 30 min after damage. CONCLUSIONS: The hyperemic response caused by 2 M NaCl is most likely mediated by histamine and CGRP and maintained by histamine released by back-diffusion of H+ through the superficially damaged gastric mucosa. PMID- 7541912 TI - Structural and functional characterization of the promoter regions of the NFKB2 gene. AB - In order to clarify the transcriptional regulation of the NFKB2 gene (lyt-10, NF kappa Bp100), we have characterized the structure and function of its promoter regions. Based on the nucleotide sequence of cDNA clones and the 5' flanking genomic region of the NFKB2 gene, RT-PCR analysis in a number of human cell lines demonstrated the presence of two alternative noncoding first exons (1a and 1b). Two distinct promoter regions, P1 and P2, were identified upstream of each exon, containing multiple sites of transcription initiation, as shown by RNase protection analysis. Sequence analysis of these regions showed a CAAT box upstream of exon 1a and high G-C content regions within both P1 and P2. Consensus binding sites for transcription factors, including SP1, AP1 and putative NF-kappa B (kappa B sites), were found upstream of each exon. In particular, six kappa B sites were identified, all but one of them capable of binding NF-kappa B complexes in vitro. Transfection in HeLa cells of plasmids containing P1 and P2 sequences linked to a chloramphenicol acetyltransferase reporter gene indicated that both P1 and P2 can act independently as promoters. Co-transfection of NF kappa B effector plasmids (NF-kappa Bp52 and RelA) with a reporter gene linked to P1 and P2 showed that the NFKB2 promoter regions are regulated by NF-kappa B factors. RelA transactivates the NFKB2 promoter in a dose-dependent manner, whereas NF-kappa Bp52 acts as a repressor, indicating that the NFKB2 gene may be under the control of a negative feedback regulatory circuit. PMID- 7541916 TI - The role of hematopoietic growth factors in support of ifosfamide/carboplatin/etoposide chemotherapy. AB - The ifosfamide/carboplatin/etoposide (ICE) combination represents an active chemotherapy regimen across a wide variety of disease types. The most common limiting toxicity for all three of these agents individually and in combination is myelosuppression. Thus, this regimen represents an ideal model to evaluate the role of hematopoietic growth factor support in amelioration of hematologic toxicity, maintenance of dose intensity, and dose escalation. While chemotherapy strategies using colony-stimulating factors have abrogated neutropenia, cumulative thrombocytopenia is common with many chemotherapy regimens, including ICE chemotherapy. In preclinical and phase II trials, monotherapy with recombinant human interleukin-6 (IL-6) has demonstrated substantial thrombopoietic activity, but with little enhancement of neutrophil recovery. Thus, this study was designed to evaluate combination cytokine therapy with both recombinant IL-6 and granulocyte colony-stimulating factor (G-CSF) after ICE chemotherapy. Previously untreated patients with inoperable non-small cell lung cancer are eligible. Treatment includes two monthly cycles of ifosfamide 2,000 mg/m2 with mesna 1,600 mg/m2 intravenously on days 1, 2, and 3, carboplatin 350 mg/m2 intravenously on day 1 only, and etoposide 75 mg/m2 intravenously on days 1, 2, and 3. All patients then receive G-CSF at a dose of 5 micrograms/kg/d subcutaneously beginning on day 4 until a postnadir absolute neutrophil count of more than 10 x 10(9)/L. Cohorts of patients (n = 15) are randomized to receive 0, 1, 2.5, or 5 micrograms/kg/d of IL-6 subcutaneously on days 4 to 13 in successive cohorts. This study has now reached its target accrual in all cohorts. The final data analysis is in progress. It is hoped that this trial will define the safety and tolerability of the simultaneous administration of IL-6 and G-CSF following ICE chemotherapy in patients with non-small cell lung cancer. In addition, this trial should determine the biologic activity and hematopoietic recovery observed during the simultaneous administration of these two cytokines in this setting. PMID- 7541917 TI - Ultrasound of femoral head cartilage: a new method of assessing bone age. AB - This paper analyses the relationship between the thickness of the anterior femoral head cartilage (FHC), as measured by ultrasound, and some anthropometric parameters, such as height, weight, skeletal and chronological age. In addition, it provides standard norms for FHC thickness in a paediatric population. Both hips were examined in 213 consecutive subjects (99 boys and 114 girls), aged 1.9 14 years. Seventy-four subjects underwent hand and wrist X-rays for skeletal maturation: 32 of these were dropped from the study because a discrepancy as high as two standard deviations was found between their skeletal and their chronological age. The thickness of FHC correlated strongly with skeletal and chronological age, standing height and body weight. A side difference of 0.2 mm in FHC was considered to be abnormal. The study population was divided into 13 groups according to chronological and values of FHC for boys and girls are provided for each group. It is suggested that the magnitude of hyaline FHC is valuable feature in the evaluation of skeletal maturation in children. PMID- 7541918 TI - Contracting for palliative care. AB - In this paper the role of purchasing authorities, as set out in the current U.K. policy literature, is reviewed in the context of recent developments in palliative care. The notion of the purchasing cycle, from need assessment and strategic planning through to contracting and monitoring is outlined, along with core features of recent health and social care legislation in Britain: quality assurance, consumer choice and value for money. The role of providers and service users in the purchasing process is examined and the relationship between purchasers across the health/social care divide is explored. The implications of contracting for the voluntary sector provision of palliative care are also assessed. The review highlights the difficulties under the new legislation in reconciling the aim of efficiency with that of quality assurance, and of separating out the roles of purchasers and providers in a context where palliative care service developments have been provider-led. These factors reinforce the requirement to turn the purchaser-provider split into an effective partnership. Finally, the review highlights the need to address the current imbalances in the purchasing cycle by giving adequate attention to aggregate need assessment and strategic planning. PMID- 7541920 TI - P2X receptors bring new structure to ligand-gated ion channels. AB - P2X receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode P2X receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial Na+ channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of P2X RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of P2X RNA that is found in PC12 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function. PMID- 7541919 TI - Biological effects of diesel exhaust particles (DEP). II. Acute toxicity of DEP introduced into lung by intratracheal instillation. AB - Histopathological examination and cytological analyses in bronchial alveolar lavage fluids (BALF) were performed to clarify the acute toxicity of diesel exhaust particles (DEP) introduced into the lung of ICR mice by intratracheal instillation. Activated charcoal (Norit) was intratracheally administered as a control for non-oedemagenic carbon particles. After administration of two doses (0.4 mg or 0.8 mg per mouse) of DEP, lung water contents increased with instillation dose and with time and increased 1.9 and 2.7-fold, respectively, compared to control animals 24 h after the administration of DEP. In contrast, the instillation of Norit had no effect on the increase in water contents. An inflammatory response in lungs was observed by an increase of inflammatory cells in BALF from mice instilled with DEP. The degree of increase in neutrophils of BALF from mice treated with DEP was much greater than in mice treated with Norit. An intense color of MB-pigment, which showed the extent and degree of endothelial cell injury, was found up to 4 h after administration of DEP. Histopathologically, the disruption of capillary endothelial cells, the detachment from their basement membrane and necrosis, disruption and desquamation of type I pneumocytes were observed, 6 h after the injection of DEP, by electron microscopy. An influx of neutrophils into alveoli, intra-alveolar hemorrhage, perivascular oedema and bronchiolar cell hypertrophy were detected between 18 and 24 h after DEP administration. However, the magnitude of these appearances was greater in mice treated with 0.8 mg of DEP than in mice treated with 0.4 mg. The administration of Norit caused an increase of alveolar macrophages and slight infiltration of neutrophils into the alveolar air spaces and alveolar septa in the animals and had no effects on the bronchioles. These results may suggest that damage of capillary endothelial cells and type I pneumocytes are the earliest changes of lung toxicities by DEP and these cell injuries lead to alveolar oedema and the subsequent inflammatory response. PMID- 7541921 TI - Epithelial differentiation antigens and epidermal growth factor receptors in transitional cell bladder carcinoma: correlation with prognosis. AB - Epithelial differentiation antigens have been correlated with morphologic differentiation of neoplastic urothelium. Moreover, epidermal growth factor, which is a polypeptide regulating growth and differentiation of normal and neoplastic cells, is found in high concentrations in the urine while its receptors (EGFR) have been identified in bladder tumors. The aim of this study was to investigate the immunohistochemical expression of cytokeratin, epithelial membrane antigen (EMA), CEA and EGFR in transitional cell bladder carcinomas (TCC) and to define any correlation of their expression with tumor grade, stage and patient survival. Twenty-four biopsy specimens obtained from patients with TCC were studied retrospectively. There were 23 men and 1 woman with a mean follow-up of 64 months. Eight biopsy specimens, which represented tumor recurrences of 4 patients, were also included in our material. The immunohistochemical avidin-biotin complex method was performed on paraffin sections for the detection of cytokeratin and EGFR with monoclonal antibodies as well as CEA with a polyclonal antibody. Cytokeratin was detected in 83.5% of the TCC, EMA in 62% and CEA in 70%. The expression of the epithelial differentiation antigens in TCCs was heterogenous, showing an increased incidence in high-grade and high-stage TCC. The CEA expression in TCC demonstrated a statistically significant correlation with patient survival (p < 0.02). EGFR was detected in 50% of the TCC. Although not statistically significant, a trend was found for a higher percentage of EGFR detection in high-grade TCC. EGFR expression was significantly associated with tumor stage and patient survival (p < 0.01 and p < 0.04, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541922 TI - Benign prostatic hypertrophy affects the endothelin receptor density in the human urinary bladder and prostate. AB - The effects of benign prostatic hypertrophy (BPH) on the endothelin (ET) receptor density in the lower urinary tract tissues were studied using radioligand binding techniques. Saturation experiments revealed that there were significant amounts of the binding sites for the ET isoforms (ET-1, -2, -3) in the human bladder and prostate in both prostate hypertrophy (PH) and nonhypertrophy (NPH) patients. Autoradiograms of hypertrophic adenoma showed that ET-1 receptors were localized in both the stromal and glandular tissue. In the bladder and the prostate, the KD values were not significantly different between the PH and NPH groups. In the bladder dome, the Bmax values of 125I-ET-1, -2 and -3 decreased significantly in the PH groups, while, in the adenoma, they increased significantly in the PH group. BPH was found to affect the ET receptor density in both the bladder and the prostate. These data suggest that ETs are involved in the pathophysiology of BPH. PMID- 7541923 TI - Effects of magnesium ions on detrusor contraction in rat. AB - Contraction of smooth muscle including detrusor is initiated by an increase in the concentration of intracellular free calcium ions. Magnesium affects the translocation of calcium ions through the smooth muscle cell membrane. The effect of magnesium ions on detrusor contraction was investigated in vitro using the rat detrusor muscle. Detrusor contractility caused by intramural nerve stimulation increased in a magnesium-free solution but decreased in a high-magnesium solution (MgCl2 5 mM). Cumulative addition of MgCl2 suppressed the detrusor response induced by field stimulation dose-dependently, and 30 mM MgCl2 completely eliminated it. Mg ion also suppressed atropine-resistant detrusor contraction in a dose-dependent manner. After blocking nerve-mediated detrusor contractility with tetrodotoxin, Mg ions inhibited spontaneous detrusor contractions and reduced tonus of the muscle strips. Mg ions suppressed the detrusor contraction induced by carbachol and potassium. The effect of Mg ions on detrusor contraction was enhanced by verapamil but inhibited by Bay-K8644. These results suggest that Mg ions suppress detrusor muscle contractility through an inhibitory mechanism acting on calcium channels in the smooth muscle cell membrane. PMID- 7541925 TI - [Status of PSA determination for early detection of prostate carcinoma]. AB - Prostate Specific Antigen (PSA) appears to be useful in the early detection of prostate cancer when used in conjunction with digital rectal examination (DRE) and transrectal ultrasonography (TRUS). Recent studies suggest that the addition of PSA to the routine digital rectal examination leads to detection of prostate cancer at an earlier stage than when digital rectal examination is used alone. In patients with elevated PSA or abnormal DRE prostate biopsies are recommended. However, the routine use of PSA to detect early prostate cancer is limited due to the PSA elevation in patients with prostatic hyperplasia. Possibilities to distinguish BPH from prostate cancer among men who have minimal PSA elevation (4.0 to 10.0 ng/ml) are the use of PSA velocity, PSA density, age adjusted PSA, and analysis of free and complexed PSA. Screening programs for prostate cancer will increase the percentage of localized prostate cancer, which can be cured by radical prostatectomy. PMID- 7541924 TI - The importance of dose intensity in chemotherapy of advanced testicular cancer. AB - PVB regimen, the combination of cisplatin (CDDP), vinblastine (VLB) and bleomycin (BLM), has improved the treatment results of testicular cancer patients. However, modification of doses and schedules were strongly related with the treatment results. We analyzed the relative dose intensity (RDI) of each patient undergoing PVB regimen, in relation to the chemotherapeutic effect. In addition, a role of granulocyte colony-stimulating factor (G-CSF) on dose intensity of PVB regimen was analyzed. During the period 1981-1992, 33 patients with testicular cancer were treated with PVB regimen. Among the 25 evaluable patients, 5 (20%) achieved a complete response (CR), 15 (60%) had a partial response (PR), and the overall response rate was 80%. The average 5-year survival rate of all cases was 78.4%. The RDI in CR and PR cases were significantly higher than nonresponders in BLM (0.89 for CR and 0.91 for PR against 0.32 for NC + PD) in the combination of the three drugs (1.05 for CR and 0.97 for PR against 0.69 for NC + PD). The standard dose group (RDI > or = 0.9) had a higher survival rate than the modified-dose group (RDI < 0.9). The RDI of PVB regimen with administration of G-CSF was 0.98, whereas in the PVB-alone group it was 0.87 (p < 0.01). Our findings indicate that the RDI of BLM may be one of the most important factors in achieving a chemotherapeutic effect from the PVB regimen. G-CSF is useful for the completion of the schedule through the increase in dose intensification, and the prevention of leukocytopenia due to chemotherapy. PMID- 7541926 TI - [Laparoscopy and laparoscopic endosonography as staging examination of tumors of the upper gastrointestinal tract]. AB - Accurate staging of gastric and oesophageal cancer is of major importance for the therapeutic strategy. However conventional imaging procedures are often not sensitive enough for precise assessment of resectability and curability. In this study laparoscopy and laparoscopic ultrasound were evaluated for preoperative staging. PATIENTS AND METHODS: Laparoscopy was performed on 21 patients for staging of intraabdominal malignancy using a standard equipment. Laparoscopic ultrasound was done with a flexible echoendoscope equipped with a curved array transducer (5/7,5 MHz). The ultrasound probe was introduced via a 15 mm trocar. RESULTS: By laparoscopy and laparoscopic ultrasound additional informations compared to conventional staging were obtained and revealed a disseminated disease in 15 out of 21 (71%) patients. These informations induced a change of the initially planned treatment concept in 14 of these patients (67%). A definitive palliative treatment was performed in 5 patients after diagnosis of incurable disease. Laparoscopic ultrasound proved to be useful for the detection of small non superficial lesions followed by biopsy and can improve laparoscopic staging of intraabdominal tumor spread. PMID- 7541927 TI - [Laparoscopic staging of gastrointestinal tumors]. AB - Laparoscopic staging was carried out in 28 cases with carcinomas of the distal esophagus, cardia, stomach or pancreas. The intention was first to rule out peritoneal carcinomatosis and secondly to see, whether RO-resection would be possible, mainly for the reason of therapeutic splitting. The diagnostic accuracy can be raised to 95% using laparoscopic ultrasound, exploration of the lesser sac of the peritoneum and tissue sampling in combination with preoperative endoscopy and radiography. In 39.3% of the investigated patients the staging must be corrected after laparoscopic staging compared with the preoperative findings. 35.7% of the patients did not need explorative laparotomy. PMID- 7541928 TI - [Indications for laparoscopic surgery of the large intestine]. AB - Laparoscopic colon resection is feasible but not standardized regarding indication and surgical procedure. Indications are mainly benign diseases (diverticulitis, Crohn's disease, ulcerative colitis and sessile dysplastic polyps or adenomas). Rare indications are benign tumors, angiodysplasia of the colon and dolichocolon. Laparoscopic rectopexy and sutural closure of iatrogenic colon perforation have minimal invasive indication, too. Indication for laparoscopic colon resection for malign tumors in T1-stage is still unclear. Use of minimal invasive procedures for palliative reasons (construction of colostomy or bypass anastomoses in malign tumors) is appropriate. In 32 laparoscopic bowel operations we found no lethality. In 5 cases early conversions to laparotomy were necessary. Postoperatively following complications occurred: 1 intraabdominal bleeding, 1 stenosis of anastomosis and 1 wound infection. PMID- 7541929 TI - Effect of endotoxin on the advancing front between cultured middle ear mucosa and epidermis. A preliminary study. AB - The formation of Cholesteatoma is often accompanied by infection, and is very likely to be influenced by inflammatory mediators. Endotoxin, a strong inflammatory mediator, may play an important role in cholesteatoma pathogenesis. It has been demonstrated in middle ear effusions, and it causes marked reactions in middle ear epithelium and epidermis, both in vivo and in vitro. The effect of endotoxin on a simulation of the advancing front of cholesteatoma was investigated in this study. We developed a co-culture model composed of a simultaneous culture of rat middle ear mucosa and rat meatal epidermis in one culture dish, to which endotoxin was added. During the culture period the addition of endotoxin to the co-culture delayed take-over of the meatal epidermis by the middle ear mucosa, in comparison with control cultures. Morphological changes included the clustering of microvilli in co-cultures exposed to endotoxin. Although these results are preliminary, they suggest that endotoxin disturbs the normal healing process of the middle ear cavity. PMID- 7541930 TI - Expression of substance P, CGRP, and GABA in the vestibular periphery, with special reference to species differences. AB - The present study was conducted to elucidate species differences in the distribution of neuroactive substances, including substance P (SP), calcitonin gene-related peptide (CGRP) and gamma-aminobutyric acid (GABA), in the vestibular periphery of various animals (chicken, pigeon, rat, guinea pig and squirrel monkey). SP-like immunoreactivity was found in a subset of the primary vestibular afferents with no marked species differences in the staining pattern. In contrast, the localization of CGRP- and GABA-like immunoreactivities in the efferent nerve fibers varied according to species. This difference in the distribution pattern of neuroactive substances, found in the efferent system, may indicate that each species has a chemically (and probably functionally) distinct efferent system which is related to its specific environment and/or evolution. PMID- 7541931 TI - Ototoxic antibiotics: a review. AB - Fifty years of experience with aminoglycoside antibiotics has confirmed their usefulness in many infections with Gram-negative bacteria. The ototoxic action has been investigated clinically in patients and in numerous animal studies. The increased knowledge about molecular structure, pharmacology and pharmacokinetics as well as information of the site of action in the labyrinth has resulted in reduced risks for severe toxic damage in kidneys and in the ear. Nevertheless, a number of cases of severe hearing loss and a few cases of deafness still occur every year. Further studies on modified molecular structure as well as site of action at the molecular level and ways of avoiding toxicity will make this group of antibiotics still more useful. PMID- 7541932 TI - The tympanic membrane: a biochemical updating of structural components. AB - A cross-section of the tympanic membrane is discussed at the molecular level. Actually, the tympanic membrane can be described as a continuation of a vast number of structural proteins and molecular interactions. The epidermal layer is subject to soft keratinization, a differentiation process implying the genesis of a chemically resistant cell matrix (cytokeratins, filaggrin) and cornified membrane (involucrin, keratolinin, loricrin, transglutaminase). The epidermis is anchored in the connective tissue by means of hemidesmosomes whose biochemical nature begun to be elucidated rapidly in recent years. Of the first importance are the HD1 inner plaque protein, supposed to be associated with intermediate filaments, and the transmembrane components in the dense plaque (BPAG2 and alpha 6 beta 4 integrin), whose extracellular domains directly interact with basement membrane components. Though the complete characterization of the basement membrane has not yet been achieved, the molecular catalogue of its three distinct layers is really impressive (laminin, fibronectin, nidogen, kalinin, K-laminin, type IV collagen, heparan sulphate proteoglycan, type III and VII collagen, ...). Besides numerous mutual interactions, it appears that mainly laminin and kalinin (anchoring filaments) promote binding of epidermal cells. Furthermore, a continuation may exist between anchoring fibrils in the deepest layer and anchoring filaments in the upper layer of the basement membrane. Finally, the lamina propria, a specific type of extracellular matrix, is perhaps the most difficult to dissect, though substantial progress has been made in the last few years (fibronectin, type I, III, V and VI collagen, fibrillin, ...). PMID- 7541933 TI - [Expression of fibroblast growth factor receptor 1 in experimental choroidal neovascularization with in situ hybridization]. AB - Fibroblast growth factor (FGF) is an important factor for neovascularization in vivo. In order to clarify the role of FGF in experimentally produced choroidal neovascularization, we demonstrated mRNA for FGF receptor 1 in situ hybridization. Krypton laser photocoagulation was applied to the posterior retina of colored rats to produce choroidal neovascularization experimentally. These eyes were removed at several different intervals after photocoagulation. Chorioretinal section were used for in situ hybridization. FGF receptor 1 cDNA fragment was used to make antisense and sense probes for in situ hybridization. In normal chorioretinal tissue, staining indicating the existence of FGF receptor 1 mRNA was seen in the ganglion cell layer and inner nuclear layer. After the photocoagulation, the staining was seen in the retinal pigment epithelial cells, melanocytes in the choroid, and choroidal blood vessel wall in the photocoagulated lesions. FGF receptor 1 mRNA was expressed through the development of choroidal neovascularization, and it appears that FGF is necessary for development of choroidal neovascularization. Previous workers showed that the capillary endothelial cells and retinal pigment epithelial cells produce basic FGF in vitro. It seems that FGF effects those cells in an autocrine or paracrine manner in vivo. PMID- 7541934 TI - Quantitative immunohistochemical determination of cathepsin D levels in prostatic carcinoma biopsies. Correlation with tumor grade, stage, PSA level, and DNA ploidy status. AB - National screening programs resulting in an increased detection rate of prostatic adenocarcinoma have prompted the search for new methods of predicting disease outcome that can be applied to the initial narrow bore needle biopsy specimens. Cathepsin D, a lysosomal aspartyl protease and autocrine mitogen, has been studied in a wide variety of human neoplasms as an invasion and metastasis marker. Prostatic carcinoma needle biopsy tumor cell cathepsin D content was measured in 61 men using a semiquantitative image analysis assisted immunohistochemical procedure. Results were compared with preoperative serum prostatic specific antigen levels, tumor grade, DNA ploidy status, pathologic stage after radical prostatectomy and disease recurrence during a median 2.6 year follow-up. Biopsy cathepsin D levels significantly correlated with tumor grade (P = .022) and DNA ploidy status (P = .028) by logistic regression analysis. Post prostatectomy pathologic stage and disease recurrence did not correlate with tumor cathepsin D levels. Final prostatectomy grade and DNA ploidy status independently predicted metastasis and post-operative disease recurrence (P < .001). Although this study did not find independent prognostic status for cathepsin D in prostate cancer, the correlation with tumor grade and DNA ploidy status is noteworthy and the inter-relationship of outcome variables may prove of interest and warrant further evaluation of this potential predictor or CO predictor of disease outcome. PMID- 7541935 TI - Small bowel resection for relief of chronic intestinal pseudo-obstruction. AB - Chronic intestinal pseudo-obstruction refractory to medical therapy is a debilitating problem for patients and a challenge for clinicians. We report a case of chronic idiopathic intestinal pseudo-obstruction with giant upper intestinal diverticula, complicated by hypersecretion refractory to medical therapy and requiring 10 yr of home parenteral nutrition. Resection of the chronically dilated small bowel with giant diverticula and construction of improved gastric and duodenal drainage was performed. This not only relieved the hypersecretory state and improved gastric drainage, but allowed the patient to stop parenteral nutrition and eat regular food for the first time in 10 yr. Select patients with chronic intestinal pseudo-obstruction will respond favorably to palliative surgical intervention. PMID- 7541936 TI - Report of a case of benign cystic mesothelioma. AB - A 32-yr-old man was admitted to Anjo Kosei Hospital for the examination of a cystic peritoneal lesion. This patient had a past medical history significant for an appendectomy. Ultrasonography and computed tomography revealed multiple cysts in the abdominal and pelvic cavities. An exploratory laparotomy was performed on July 1, 1993, revealing numerous thin-walled and translucent cysts in the serosal tissues of the parietal and visceral abdominal peritoneum. These cysts were excised surgically because of clinical suspicion of pseudo-myxoma peritonei. Microscopic examination revealed cysts within the thin cyst wall that were composed of fibrous connective tissue and lined by a single layer of cuboidal or flattened epithelium. Immunohistochemical and ultrastructural studies demonstrated that the lining cells were of mesothelial origin, and the diagnosis was benign cystic mesothelioma. In this paper, we present appropriate diagnostic and treatment procedures for benign cystic mesothelioma and discuss the particular usefulness of using immunohistochemical methods to achieve a histological diagnosis. PMID- 7541938 TI - FRAXE expansion is not a common etiological factor among developmentally delayed males. AB - Expansion of a (CGG)n trinucleotide repeat unit at FRAXE, a newly defined fragile site distal to FRAXA, at Xq28, is reported to be associated with mild mental retardation. Three hundred developmentally delayed male patients referred for fragile X testing but negative for the FMR-1 gene trinucleotide expansion were screened for the FRAXE expansion. This group of patients had a wide range of intellectual or behavioral problems and included 19 patients who had low-level fragile site expression detected cytogenetically at Xq27-q28. None of the patients tested positive for the FRAXE expansion. These results suggest that FRAXE is not a common etiological factor among this group of patients. The data support the hypothesis that FRAXE is either very rare or a benign fragile site that is not associated with any clinical phenotype, similar to the FRAXF and FRA16A sites. PMID- 7541937 TI - Renal actions of endothelin-1 and endothelin-3: interactions with the prostaglandin system and nitric oxide. AB - Endothelins (ET) possess both vasodilatory and vasoconstrictive properties. The renal actions of ET-1 and ET-3, as well as in vivo interactions of these two isopeptides with the prostaglandin and endothelium-derived relaxation factor/nitric oxide systems were studied in anesthetized dogs. The ETs were infused intrarenally at doses not affecting systemic hemodynamics. Both ET-1 and ET-3 induced an early transient renal vasodilation, followed by a prolonged vasoconstriction. Inhibition of nitric oxide synthase with NG-monomethyl-L arginine completely abolished the renal vasodilation induced by either ET-1 or ET 3 and enhanced the vasoconstriction. Endothelin-1 was associated with an increase in the renal release of prostacyclin, while urinary thromboxane A2 was increased after ET-3 administration. Inhibition of cyclooxygenase (with indomethacin) augmented the renal vasoconstriction induced by ET-1, but inhibition of cyclooxygenase (with meclofenamate) abolished the ET-3-evoked vasoconstriction. Endothelin-1 showed little effects on urinary water and sodium excretion; however, ET-3 displayed significant diuretic and natriuretic effects, which were inhibited by nitric oxide synthase inhibition. These findings suggest that these two isopeptides activate the endothelial endothelium-derived relaxation factor/nitric oxide system, which elicits early renal vasodilation, whereas direct effects on the vascular smooth muscle leads to vasoconstriction. Endothelin-3 causes diuresis and natriuresis, possibly by inducing release of nitric oxide in medullary collecting duct cells. PMID- 7541939 TI - Selective use of indocyanine green angiography for occult choroidal neovascularization. AB - PURPOSE: Indocyanine green angiography is useful in situations where fluorescein angiography shows occult choroidal neovascularization or pigment epithelial detachment. We sought to determine how often the selective application of indocyanine green angiography results in useful information for eyes with occult choroidal neovascularization. METHODS: We reviewed 153 consecutive indocyanine green angiograms and identified 77 in which corresponding fluorescein angiograms showed occult choroidal neovascularization or pigment epithelial detachment. We examined the indocyanine green angiograms to detect areas of hyperfluorescence and to classify the margins of hyperfluorescence as well demarcated or poorly demarcated. RESULTS: Of 77 eyes, 42 (55%) eyes had occult choroidal neovascularization by fluorescein angiography, seven (9%) eyes had both classic and occult choroidal neovascularization by fluorescein angiography, and 28 (36%) eyes had pigment epithelial detachments. Of 42 eyes with occult choroidal neovascularization by fluorescein angiography, 21 (50%) had well-demarcated margins, 13 (31%) had poorly demarcated margins, and eight (19%) had no detectable hyperfluorescence by indocyanine green angiography. Of seven eyes with both classic and occult choroidal neovascularization by fluorescein angiography, two had both poorly demarcated and well-demarcated borders, two had only poorly demarcated borders, and three had well-demarcated borders by indocyanine green. The indocyanine green angiogram showed 23 (82%) of 28 pigment epithelial detachments to have well-demarcated borders of hyperfluorescence; 13 (57%) of 23 were treated. CONCLUSION: Indocyanine green angiography adds clinically useful information to fluorescein angiography by demonstrating well-demarcated areas of hyperfluorescence in 50% of eyes selected because of diagnosis of occult choroidal neovascularization and in 82% of eyes selected because of pigment epithelial detachment. PMID- 7541940 TI - Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. AB - Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13 fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism. PMID- 7541941 TI - Vasopressin increases AQP-CD water channel in apical membrane of collecting duct cells in Brattleboro rats. AB - The effect of vasopressin on subcellular localization of AQP-CD and AQP3 water channels was examined in thirsted Brattleboro rats by immunohistochemistry and immunoelectron microscopy. AQP-CD was mainly present in the cytoplasm of the collecting duct cells in association with cytoplasmic vesicles but was sparse in the apical membrane in control vehicle-injected rats. In rats given vasopressin 15 min before death, the number of immunogold particles for AQP-CD in the apical membrane increased significantly (P < 0.002) from 1.8 +/- 0.2 to 10.0 +/- 0.4/microns with a significant decrease (P < 0.05) of cytoplasmic labeling from 32.6 +/- 6.4 to 24.6 +/- 5.6/microns 2, indicating that AQP-CD is the vasopressin regulated water channel predicted by the "shuttle" hypothesis. In contrast, AQP3 was restricted to the basolateral membrane of the collecting duct cells, and the labeling density of AQP3 was unchanged by vasopressin treatment, indicating that AQP3 is constitutively expressed and may maintain high water permeability of the basolateral membrane. PMID- 7541942 TI - cAMP-independent regulation of CFTR by the actin cytoskeleton. AB - Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8 bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway. PMID- 7541943 TI - Insulin-like growth factor binding protein-3 inhibits porcine granulosa cell function in vitro. AB - Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) effects on basal, insulin-like growth factor I (IGF-I)-, and follicle-stimulating hormone (FSH)-stimulated progesterone (P4) secretion and [3H]aminoisobutyric acid (AIB) uptake by primary porcine granulosa cells (MDGs) and MDGs that have been passaged once (MDGp1) were assessed. Cells were treated concurrently or were preincubated with rhIGFBP-3 followed by treatment. rhIGFBP-3 had no effect on MDG or MDGp1 cell numbers after 24 h. Cotreatment with rhIGFBP-3 inhibited P4 secretion after treatment with FSH, IGF-I, and FSH plus IGF-I. FSH did not stimulate [3H]AIB uptake. However, the IGF-I-stimulated increase in [3H]AIB uptake was completely prevented by concurrent treatment with IGFBP-3. Preincubation of MDGp1 cells with IGFBP-3 dose dependently inhibited FSH- and IGF I-stimulated P4 secretion. This inhibition was associated with increased cell association of the binding protein and increased IGF-I binding to the cells. These results indicate that IGFBP-3 is inhibitory to a variety of crucial functions in porcine granulosa cells, supporting a role for it in the regulation of granulosa cell function. PMID- 7541944 TI - Differential regulation by TGF-beta 1 and insulin of insulin-like growth factor binding protein-2 in IEC-6 cells. AB - The purposes of this study were to determine the regulation of insulin-like growth factor binding protein-2 (IGFBP-2) in IEC-6 cells by transforming growth factor-beta 1 (TGF-beta 1) and insulin and to determine whether IGFBP-2 mediated the growth-inhibitory action on the cells. Utilizing Western ligand blot analysis, we found that TGF-beta 1 at concentrations of 0.5, 1.0, and 2 ng/ml significantly increased levels of 32-kDa IGFBP in the conditioned medium (CM) of IEC-6 cells in a dose-dependent fashion and that low doses of insulin (1.0 and 5.0 microgram/ml) also increased IGFBP levels in the CM of IEC-6 cells, but a high dose of insulin (10 micrograms/ml) depressed IGFBP release in the CM. Immunoblotting has shown that the IGFBP of 32 kDa was IGFBP-2 and further confirmed the above results. IGFBP-2 mRNA levels were stimulated by TGF-beta 1 (2.0 ng/ml) and suppressed by insulin (5.0 micrograms/ml). In addition, des (1-3) IGF-I (50 ng/ml) and insulin stimulated the proliferation of IEC-6 cells. Anti IGFBP-2 antibodies partially blocked the inhibitory role in IEC-6 cell growth evoked by des (1-3) IGF-I. These findings suggest that the upregulation of IGFBP 2 by TGF-beta 1 occurs, at least in part, at the level of mRNA, whereas the regulation by insulin appears to be at a posttranslational level, and that the TGF-beta 1-stimulated production of IGFBP may contribute to the growth-inhibitory action in intestinal epithelial cells. PMID- 7541945 TI - Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats. AB - In the stomach nitric oxide (NO) appears to be involved in vagally induced cholinergic vasodilation and nonadrenergic, noncholinergic relaxation of the fundus. We investigated whether central vagal activation by intracisternal injection of a thyrotropin-releasing hormone (TRH) analogue stimulates gastric NO release in anesthetized rats. To quantitate gastric NO production, the luminal release of NO breakdown products, nitrite (NO2-) and nitrate (NO3-), were measured by the Griess method. Intracisternal injection of RX-77368 (30-300 ng) dose dependently stimulated gastric NO2- and NO3- release (P < 0.05) along with a significant acid secretory response (P < 0.05). The specific inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (10 mg/kg ip), completely blocked gastric luminal NO2- and NO3- release without affecting the acid secretory response to the highest dose of RX-77368. Either bilateral cervical vagotomy, hexamethonium (15 mg/kg ip), or atropine (1 mg/kg ip) abolished both gastric luminal release of NO-derived metabolites and the acid secretory responses to RX 77368. These results indicate that intracisternal injection of RX-77368 stimulates gastric release of NO through vagal nicotinic pathways and peripheral activation of muscarinic receptors. These findings provide evidence for central nervous system regulation of NO-mediated functions in the rat stomach through TRH sensitive vagal pathways. PMID- 7541946 TI - Dual regulation of cGMP formation by calcium in pancreatic acinar cells. AB - Regulation of guanosine 3',5'-cyclic monophosphate (cGMP) formation by calcium and calcium-binding proteins was studied at the levels of nitric oxide synthase (NOS) and guanylyl cyclase (GC) in dispersed pancreatic acini isolated from guinea pig. In intact cells, in the cytosol, and on diethylaminoethyl fractions from cytosolic proteins, GC activity was negatively regulated by Ca2+. An increase in Ca2+ concentration ([Ca2+]) from 25 to 950 nM suppressed cGMP formation by 85%. On the other hand, NOS was stimulated by agents increasing cytosolic [Ca2+] and inhibited by intracellular Ca2+ chelators. Thus Ca2+ regulates cGMP production in opposite directions by activating NOS and inhibiting GC. Calmodulin antagonists W-7, trifluoperazine, and R-24571 inhibited NOS, suggesting that the enzyme is regulated by calmodulin as in other cell types. Calmodulin antagonists appeared to inhibit GC. In particular, 200 microM W-7 completely abolished the cGMP rise evoked by the nitric oxide donor, nitroprusside. The effect was not reversed by addition of excess calmodulin. The findings suggest that the negative regulation of GC by Ca2+ is due to factors other than calmodulin but affected by calmodulin antagonists. PMID- 7541947 TI - VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. AB - Airway smooth muscle (ASM) cell proliferation contributes to increased airway resistance in bronchial asthma. We have examined the modulation of ASM proliferation by vasoactive intestinal peptide (VIP), a cotransmitter of airway relaxation. Human ASM cells were grown in culture as a monolayer. VIP (1.0 nM-1.0 microM) inhibited proliferation in a dose-dependent manner by up to 82% on day 2, but the related peptide glucagon had no effect. Histamine (100 nM-100 microM) increased cell counts by 66%, but in the presence of VIP, cell counts and [3H]thymidine incorporation were reduced by up to 55%. Adenosine 3',5'-cyclic monophosphate (cAMP)-promoting agents, including 3-isobutyl-1-methylxanthine, forskolin, and 8-bromo-adenosine 3',5'-cyclic monophosphate, alone and especially combined with VIP, reduced cell counts and [3H]thymidine incorporation, in correlation with cAMP levels. KT-5720 (1.0 nM-1.0 microM), a selective inhibitor of cAMP-dependent protein kinase A (PKA), abolished the inhibitory effect of VIP. The results show that VIP selectively and potently inhibits human ASM cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens. PMID- 7541948 TI - Cytokine-binding proteins in the lung. AB - Numerous cytokines and growth factors signal the normal processes of tissue maintenance and remodeling in the lung, yet the aberrant expression of these peptide mediators is involved in a variety of pulmonary diseases. Furthermore, several different binding proteins function in controlling the extracellular levels of many of these cytokines in the lung. For example, a variety of cytokines and growth factors bind to and are regulated by the ubiquitous proteinase inhibitor, alpha 2-macroglobulin. The insulin-like growth factors are controlled by a specific class of six different insulin-like growth factor binding proteins. The transforming growth factor-beta family and fibroblast growth factors interact with extracellular matrix proteins. Several growth factor receptors are shed into the extracellular milieu where they retain a functional binding domain and thereby act as specific binding proteins. Cytokine-binding proteins appear to have a diversity of functions and may serve as extracellular cytokine reservoirs, protective shields against proteolytic degradation of cytokines, modifiers of cytokine-induced biological activity, or as clearance avenues for cytokines. The wide spectrum of cytokine-regulating molecules is important in cell-cell communications under normal conditions, whereas cytokine binding protein dysfunction could contribute to a number of pulmonary diseases. PMID- 7541949 TI - TNF-alpha and IL-1 beta upregulate nitric oxide-dependent ciliary motility in bovine airway epithelium. AB - Airway epithelial cells can be modulated by cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta that are released from inflammatory cells. Since ciliary motility is an important host defense function of airway epithelium, we hypothesized that cytokines, released from lung macrophages, upregulate ciliary motility. To test this hypothesis, ciliary beat frequency (CBF) was measured by video microscopy in cultured ciliated bovine bronchial epithelial cells (BBECs) incubated for 24 h with bovine alveolar macrophage-conditioned medium (AM-CM). Exposure to AM-CM resulted in a delayed (> or = 2 h) increase in CBF that was maximal after 24 h exposure (13.70 +/- 0.43 for AM-CM vs. 9.44 +/- 0.24 Hz for medium; P < 0.0001) and which was largely blocked by either anti-TNF-alpha or anti-IL-1 beta antibodies. rTNF-alpha or rIL 1 beta similarly increased CBF, which could be blocked by preincubation with either anti-rTNF-alpha or anti-rIL-1 beta antibodies. Preincubation of BBECs with actinomycin D or dexamethasone also blocked rTNF-alpha- and rIL-1 beta-induced cilia stimulation, suggesting that new protein synthesis is required for cytokine induced upregulation of CBF. Since NO is known to upregulate ciliary motility and cytokines can induce NO synthase (NOS), we hypothesized that TNF-alpha and IL-1 beta increase CBF by inducing NOS in BBECs. The cilia stimulatory effects of TNF alpha or IL-1 beta were inhibited by NG-monomethyl-L-arginine, a competitive NOS inhibitor, and restored by the addition of either L-arginine, an NOS substrate, or sodium nitroprusside, an NO donor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541950 TI - Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers. AB - We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction. PMID- 7541953 TI - Expression of mRNAs for different types of IP3 receptors in rat kidneys. AB - Cloning studies have extensively characterized two types of inositol 1,4,5 trisphosphate (IP3) receptors from the rat. An IP3 receptor from the cerebellum is referred to as type 1, and a second, recently described, receptor is referred to as the type 2 IP3 receptor. The significance of different types of IP3 receptors, especially in vivo in the kidney, is not fully understood. We investigated the localization of mRNAs encoding these two types of IP3 receptors in microdissected nephron segments of rats using reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Type 1 IP3 receptor mRNA displayed a widespread, although not uniform, distribution along the nephron. In contrast, type 2 IP3 receptor mRNA was confined almost exclusively to collecting ducts, suggesting specific expression of type 2 IP3 receptor in collecting ducts. We then detected mRNAs for the two types of IP3 receptors in collecting ducts in dehydrated rats. Dehydration downregulated type 2 IP3 receptor mRNA in cortical collecting duct, outer medullary collecting duct, and the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney. the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney. PMID- 7541951 TI - Differential effects of in vivo ethanol on LPS-induced TNF and nitric oxide production in the lung. AB - Alcohol (EtOH) has been shown to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) generation and tumor necrosis factor (TNF) production in the lung in vivo. We have previously reported that EtOH suppressed gene expression for inducible nitric oxide synthase (iNOS) with a subsequent decrease in release of reactive nitrogen intermediates by alveolar macrophages and recruited lung neutrophils. We hypothesized that a similar mechanism may be involved in EtOH induced suppression of LPS-stimulated TNF production. In contrast to what we found with iNOS, EtOH had no effect on TNF mRNA in alveolar macrophages or recruited lung neutrophils. However, immunoreactive and bioactive TNF was reduced by 72%. EtOH treatment resulted in an increased level of the membrane-bound 26 kDa form of TNF, which suggested that proteolytic cleavage of this prohormone was affected by EtOH. Experiments with t-butyl alcohol, a tertiary alcohol that is not metabolized to acetaldehyde, yielded similar results. Thus EtOH appears to be the active substance in suppression of TNF in the lung in vivo. Pretreatment with intratracheal interferon-gamma 24 h before intratracheal LPS increased TNF bioactivity partly due to increased TNF mRNA and by increasing TNF processing, as evidenced by a decrease in the 26-kDa TNF prohormone and an increase in immunoreactive and bioactive TNF. PMID- 7541952 TI - Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney. AB - The localization of aquaporin-1 water channels (AQP-1) in nephron and vascular structures in rat kidney were characterized, because vascular bundles are known to play a key role in urinary concentration. Immunohistochemistry and immunoelectron microscopy were applied on thin cryosections or ultrathin Lowicryl sections, using an optimized freeze-substitution method. Within the vascular bundles, AQP-1 is localized in descending thin limbs (DTL) of short nephrons in apical and basolateral membranes. The expression in DTL of short nephrons is considerably lower compared with the expression in long nephrons, consistent with the known lower osmotic water permeability of this segment. Furthermore, DTL of short nephrons expressing AQP-1 continue abruptly into a thin limb segment without AQP-1. This suggests the existence of a novel thin limb epithelium in the outer medulla. Extensive expression of AQP-1 is observed in apical and basolateral membranes of DTL of long nephrons, which are localized in the periphery of the vascular bundles. The expression decreases along the axis of long nephron DTLs in correlation with the known water permeability characteristics of thin limb segments. DTLs of both short and long nephrons continue abruptly into thin limb segments without AQP-1 expression, revealing an abrupt cell-to-cell transition. In vasa recta, AQP-1 is selectively localized in the nonfenestrated endothelium of descending vasa recta, whereas the fenestrated endothelium of ascending vesa recta and peritubular capillaries do not express AQP-1. AQP-1 is localized in both apical and basolateral plasma membranes, which is logical for transendothelial water transport. Isolated perfused descending vasa recta display high water permeability, and, unlike sodium permeability, diffusional water permeability is partly inhibited by mercurials, thus substantiating the presence of mercurial-sensitive water channels in descending vasa recta. Thus AQP-1 is localized in DTL and descending vasa recta within vascular bundles, and AQP-1 expression in DTL segments is in exact concordance with the known water permeability characteristics, strongly supporting that AQP-1 is the major constitutive water channel of the nephron. PMID- 7541955 TI - Expression of transmembrane-type protein tyrosine phosphatase mRNA along rat nephron segments. AB - Protein phosphorylation on tyrosine residues is one of the main cell signaling mechanisms. Cellular phosphotyrosyl levels are regulated by the activities of protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPase). We have previously reported cDNA cloning of several types of PTPase from rat kidney, including LRP (leukocyte common antigen-related protein; also known as the transmembrane-type tyrosine phosphatase, i.e., RPTP alpha). LRP mRNA was shown to be abundant in the kidney; however, our understanding of the functional role of LRP in the kidney is very limited. To gain keener insight into the function of LRP in the kidney, our first approach was to reveal its mRNA distribution along rat nephron segments. Large signals were found in inner medulla by Northern blot analysis. By using a reverse transcription and polymerase chain reaction assay of individual microdissected tubule segments along the nephron [proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD)] and glomeruli, we revealed intrarenal localization of LRP mRNA. LRP mRNA was detected in all nephron segments tested but was relatively rich in the IMCD. Rank order of the signal intensity was IMCD > PCT = OMCD > CCD > MTAL = glomeruli. Immunohistochemistry also revealed that LRP was abundant in IMCD. This pattern of expression gives rise to an interesting possibility that LRP might be involved in the specific renal tubule function, such as urinary concentrating mechanism; however, further study is required to describe the function of LRP in more detail. PMID- 7541954 TI - Nitric oxide alters cytosolic potassium in cultured glomerular mesangial cells. AB - Endothelium-derived relaxing factor is believed to be nitric oxide (NO). Evidence suggests that it has important functions in the regulation of mesangial cell (MC) tone and possibly in inflammation. As a vasodilator, its vasorelaxant effect depends, in part, on changes in cell membrane potential. We therefore tested the hypothesis that MCs in culture produce NO that results in cell relaxation through the opening of K+ conductance pathways. The K(+)-sensitive fluorescent dye, potassium-binding benzofuran isophthalate, was used to detect rapid changes in intracellular K+ concentration ([K+]i). The membrane potential-sensitive dye, 1,3 (sulfonatopropyl)-4-([beta-di-n- butylamino)-6-naphthyl]vinyl)pyridinium betaine (Di-4-AN-EPPS), was used to detect changes in membrane potential. Basal [K+]i was 92 +/- 9 mM (n = 46). In response to sodium nitroprusside (SNP), acetylcholine (ACh), and bradykinin (BK), [K+]i decreased to 72 +/- 7 (n = 5, P < 0.05), 70 +/- 8 (n = 7, P < 0.05), and to 69 +/- 13 mM (n = 6, P < 0.05), respectively. [K+]i rapidly returned to basal level in the continued presence of all three agonists. The SNP-, ACh-, and BK-induced decrease in [K+]i was significantly blunted by Ba2+ by 85, 92, and 89%, respectively (n > 4 for each agonist examined, P < 0.0001). NG-monomethyl-L-arginine (L-NMMA) and methylene blue inhibited the [K+]i lowering effect of ACh and BK by 94 and 85% for L-NMMA (n = 5 for each agonist, P < 0.0001) and by 67 and 72% for methylene blue (n = 5 for each agonist, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541956 TI - Ontogeny of NO synthase and renin in juxtaglomerular apparatus of rat kidneys. AB - The presence of NO synthase (NOS) in cells of the macula densa (MD) suggests a role for arginine-derived NO in tubulovascular information transfer. To investigate the postnatal development of the neuronal isoform of NOS and of renin in the kidney, the cellular distribution of these enzymes was examined in perfusion-fixed kidneys of 2-, 6-, and 15-day-old rats at both the protein and mRNA level (n = 4 rats/group). NOS and renin and their mRNAs were localized by immunohistochemical and in situ hybridization methods. In addition, NOS levels were assessed by using NADPH diaphorase (NADPH-d) histochemistry. For quantification, the fraction of NOS- and renin-positive glomeruli as well as the number of NOS-positive MD cells was evaluated at all stages. Presence of NOS in single cells of the developing distal tubule was encountered already in the S shaped body. Full expression of a NOS signal in MD cells was seen as soon as a glomerular urinary space was developed. Double labeling with NADPH-d and antibody to Tamm-Horsfall protein (THP) indicated mutual exclusiveness of NADPH-d-positive MD cells and neighboring THP-positive distal tubule cells at all levels of development. The relative intensity of renin status was 2 day > 6 day > 15 day, whereas NOS expression was maximal on postnatal day 6. Our data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells. PMID- 7541957 TI - Mechanisms of oxidized chylomicron-induced leukocyte-endothelial cell adhesion. AB - The objectives of this study were to determine whether oxidatively modified chylomicrons (oxCM) can elicit leukocyte-endothelial cell adhesion in the mesenteric microcirculation and to define the mechanisms underlying the oxCM induced adhesive interactions. Oxidation of chylomicrons (CM) with the peroxyl radical generator 2,2'-azobis(2-amidinopropane)hydrochloride was associated with the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides. Leukocyte rolling, adherence, and emigration as well as erythrocyte velocity were monitored in rat mesenteric venules infused with either native CM or oxCM. oxCM, but not native CM, increased the numbers of rolling, adherent, and emigrated leukocytes. The oxCM-induced leukocyte adherence was significantly blunted by pretreating the animals with either superoxide dismutase, a platelet-activating factor (PAF) receptor antagonist, or monoclonal antibodies (MAb) directed against either CD11/CD18 or intracellular adhesion molecule 1. A MAb against P-selectin reduced oxCM-induced leukocyte rolling but not adherence. These findings suggest that the increased plasma oxCM levels associated with ingestion of oxidized lipids may promote leukocyte adhesion through a mechanism that involves the superoxide anion, PAF, and adhesion receptors on leukocytes and endothelial cells. PMID- 7541958 TI - Direct and reversible inhibition of endothelial nitric oxide synthase by nitric oxide. AB - The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo. PMID- 7541959 TI - Effects of an endogenous inhibitor of nitric oxide synthesis on postcapillary venules. AB - The objective of this study was to determine whether an inhibitor of nitric oxide (NO) synthase (NG,NG'-dimethyl-L-arginine; L-DMA) that is produced by vascular endothelium elicits the inflammatory responses induced by synthetic analogues of L-arginine such as NG-nitro-L-arginine methyl ester (L-NAME). Leukocyte adherence and emigration, leukocyte-platelet aggregation, and albumin leakage were monitored in rat mesenteric venules exposed to different concentrations of either L-DMA or L-NAME. Increases in leukocyte adherence (7- to 9-fold) and emigration (3- to 5-fold), platelet-leukocyte aggregation, mast cell degranulation, and an enhanced albumin leakage (30-50%) were observed within 30 min after exposing the microvascular bed to either inhibitor; however, leukocyte emigration and albumin leakage responded more intensely to L-NAME than to L-DMA. The microvascular alterations and mast cell degranulation were attenuated by addition of L-arginine to the superfusate. These results suggest that the L-DMA is capable of eliciting an inflammatory response at concentrations detected in plasma under certain pathological conditions. PMID- 7541960 TI - Effects of chronic NO synthase inhibition in rats on renin-angiotensin system and sympathetic nervous system. AB - This study was designed to assess the role of renin and of the sympathoadrenal system in the maintenance of the hypertension induced by chronic nitric oxide synthase (NOS) inhibition in rats kept on a normal (RS) or a low-sodium (LS) diet. With the administration of NG-nitro-L-arginine methyl ester (L-NAME) in drinking water (0.4 milligrams) for 6 wk, mean intra-arterial blood pressure rose to a similar extent to 201 mmHg in the RS and 184 mmHg in the LS animals. Simultaneously, plasma norepinephrine was increased to 838 and 527 pg/ml and epinephrine to 2,041 and 1,341 pg/ml in RS and LS, respectively. Plasma neuropeptide Y levels did not change. Plasma renin activity rose to 21 ng.ml-1.h 1 in RS but remained at 44 ng.ml-1.h-1 in the LS. Both losartan (10 mg/kg) and phentolamine (0.1 mg/kg) intravenous bolus injections reduced blood pressure considerably in the L-NAME hypertensive animals. Whole brain NOS activity was reduced by 84%. Hypertension induced by chronic NOS inhibition in LS as well as in RS fed rats seems to be sustained by an interaction of several mechanisms, including the activation of the sympathetic nervous system and the renin angiotensin system. PMID- 7541961 TI - Endotoxin-induced contractile dysfunction in guinea pig hearts is not mediated by nitric oxide. AB - The decreased contraction amplitude of isolated cardiac myocytes from guinea pigs exposed to lipopolysaccharide (LPS) was reported to be partially reversed by nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) [Brady, et al., Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1963-H1966, 1992]. We have tested the potential involvement of NO formation in LPS-induced cardiac depression in the intact heart. Isolated perfused hearts of LPS-treated guinea pigs (4 mg/kg 4 h before organ removal) displayed a greatly decreased left ventricular pressure (LVP) when compared with untreated controls (48 +/- 11 vs. 93 +/- 18 mmHg, n = 6 hearts each), whereas heart rate and coronary flow were similar. Perfusion of LPS-treated hearts with L-NMMA or L-NAME (100 microM each) at constant flow did not increase LVP (50 +/- 14 and 44 +/- 11, respectively, vs. 52 +/- 14 mmHg). However, coronary resistance increased significantly. There was no difference between LPS-treated and control hearts in venous adenosine release (104 +/- 58 vs. 133 +/- 86 pmol.min-1.g-1). Measurement of the activities of the induced (iNOS) and constitutive forms of NOS revealed that there was no difference in total NOS activity (237 +/- 82 vs. 181 +/- 97 fmol.min-1.mg protein 1. There was no measurable induction of iNOS in the LPS-treated hearts either. Finally, cardiac energy status was studied by 31P nuclear magnetic resonance spectroscopy. There was no difference between LPS-treated and control hearts in myocardial ATP, creatine phosphate, pH, and free ADP (59 +/- 20 vs. 50 +/- 27 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541962 TI - Altered contractile and ion channel function in rabbit portal vein with dietary atherosclerosis. AB - This study was performed to determine the effects of dietary atherosclerosis on the pharmacology and ion channel properties of rabbit portal vein (PV). New Zealand White rabbits were fed normal rabbit chow +/- 2% cholesterol for 10 wk. Contractions to norepinephrine (NE) and serotonin were studied under isometric conditions with longitudinal strips. Ca2+ and K+ currents (ICa and IK, respectively) were recorded in freshly dispersed myocytes by whole cell voltage clamp methods. Cholesterol feeding increased total plasma cholesterol levels from 28.4 +/- 5.2 to 1,387 +/- 172 mg/dl as well as the cholesterol-to-phospholipid molar ratio of the PV from 0.34 +/- 0.02 to 0.66 +/- 0.08. Only maximum contractile responses to serotonin were larger in atherosclerotic PV when normalized to the maximum KCl response. Concentration-active stress curves of the atherosclerotic PV to NE and serotonin were shifted to the left. Maximum values of ICa were larger in myocytes from atherosclerotic compared with control animals (4.4 +/- 0.4 vs. 3.1 +/- 0.2 pA/pF, P < 0.05). The voltage dependence of activation and availability of ICa was shifted toward more negative potentials by approximately 10 mV. Whole cell K+ currents were smaller in atherosclerotic myocytes. At a test voltage of +20 mV, IK averaged 14.9 +/- 2.8 pA/pF in control compared with 7.7 +/- 0.8 pA/pF in atherosclerotic myocytes from a holding potential of -80 mV with external Ca2+ concentration of 5 mM. The reversal potential for IK tail currents was significantly less negative in atherosclerotic myocytes (-70 +/- 1 vs. -64 +/- 1 mV).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7541963 TI - Characterization of the endogenous intestinal peptide that stimulates the rectal gland of Scyliorhinus canicula. AB - It has been postulated that gut peptides play a major role in the regulation of rectal gland secretion in elasmobranchs. An isolated perfused rectal gland secretion in elasmobranchs. An isolated perfused rectal gland preparation was developed for Scyliorhinus canicula that responded to dibutyryl 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, increasing chloride clearance rates threefold over basal levels. Activity was stimulated by an endogenous peptide, isolated in pur form by reverse-phase high-performance liquid chromatography from the intestine of S. canicula. The primary structure was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly Leu-Met- NH2. This is a sequence identical to that of the tachykinin scyliorhinin II. Perfusion of synthetic scyliorhinin II increased secretion rate in the rectal gland of S. canicula in a dose-dependent manner with a maximal response at 10(-6) M, whereas vasoactive intestinal peptide, a stimulator in the spiny dogfish, Squalus acanthias, had no effect. We propose that scyliorhinin II is the uncharacterized peptide rectin, previously identified from the intestine of S. canicula. PMID- 7541964 TI - Sensitization of nociceptive C-fibers in zinc-deficient rats. AB - A marked decrease in zinc concentration was observed in plasma (P < 0.001), hindpaw skin (P < 0.01), and dorsal skin (P < 0.01) in zinc-deficient rats (rats fed a zinc-deficient diet for 3 wk), compared with the control rats fed the same zinc-deficient diet supplemented with ZnCO3 (50 mg/kg diet). The threshold intensity needed to elicit vasodilatation in the hindpaw skin of the zinc deficient rats on electrical stimulation of the saphenous nerve in a peripheral direction was markedly lower (P < 0.01) than that in the control rats. No difference was observed between control (n = 5) and zinc-deficient rats (n = 5) in the magnitude of the plasma extravasation evoked by either histamine or substance P. There was no difference between control and zinc-deficient rats in terms of the dose-response curve for release of histamine by substance P. Prostaglandin E2 (PGE2) concentration in the hindpaw skin of the zinc-deficient rats was nearly fourfold higher (P < 0.01) than that of the control rats, whereas no difference in the leukotriene B4 level in the hindpaw skin was observed between control and zinc-deficient rats. From the present study, it seems likely that an increased level of PGE2 in the vicinity of the nociceptive C-fiber terminals in the hindpaw skin of zinc-deficient rats may sensitize the terminals of the nociceptive C-fibers of the saphenous afferent nerve in the hindpaw and thus facilitate the production of antidromic vasodilatation. PMID- 7541966 TI - Hyaline-cell rich chondroid syringoma. A tumor mimicking malignancy. AB - We report three cases of chondroid syringoma composed almost entirely of hyaline cells and occurring in the soft tissues. These lesions were confused with a variety of malignant neoplasms, including malignant melanoma, myxoid chondrosarcoma, and alveolar soft part sarcoma. The tumors occurred in two men and one woman aged 37, 75, and 71 years, respectively, and involved the heel, great toe, and face. All cases arose in the subcutaneous tissue or fascial layer and ranged in size from 0.8 to 2.0 cm. The neoplasms were composed of plasmacytoid hyaline cells with variable amounts of myxoid stroma. Two of the chondroid syringomas contained rare duct structures. One contained cartilage lobules but no ducts. All were well circumscribed. Immunohistochemistry showed positive staining for keratin, S-100, and vimentin in all cases. Muscle markers, including desmin, smooth-muscle actin, and muscle-specific actin, were negative. Follow-up has showed no evidence of recurrence or metastasis. PMID- 7541967 TI - Epithelioid hemangioendothelioma. PMID- 7541965 TI - alpha 2-Macroglobulin gene expression during hibernation in ground squirrels is independent of acute phase response. AB - alpha 2-Macroglobulin, a major acute phase reactant in many species, increases in the Richardson's ground squirrel (Spermophilus richarsonii) during hibernation at the level of both mRNA and protein. To determine if the pattern of liver gene expression known as the acute phase response is elicited as a normal part of the physiological adaptation of hibernation, acute phase reactants were identified in the Richardson's ground squirrel and were then assayed for changes in mRNA expression in the livers of active and hibernating ground squirrels. Our data demonstrate that alpha 1-antitrypsin, alpha 2-macroglobulin, ceruloplasmin, C reactive protein, and serum amyloid A are acute phase reactants in the Richardson's ground squirrel. Of these, only alpha 2-macroglobulin (alpha 2M) mRNA increases during hibernation, demonstrating that the entire acute phase response is not elicited as a part of the adaptation for hibernation. Alternatively, data from blood clotting assays of serum from active and hibernating animals support a role for the increase in alpha 2M protein during hibernation in decreasing the coagulative properties of the blood. PMID- 7541968 TI - A high risk of hepatitis C infection among Egyptian blood donors: the role of parenteral drug abuse. AB - To determine the prevalence and risk factors of hepatitis C virus (HCV) infection among Egyptian blood donors, 188 consecutive adult blood donors from four hospitals and one temporary donor center located in Cairo, Egypt were evaluated. Sera were tested for HCV antibodies (anti-HCV) using second-generation enzyme linked immunosorbent assay (ELISA) test kits. Sera that were repeatedly reactive by ELISA were further verified by a second-generation recombinant immunoblot assay (RIBA). Antibodies to HCV were detected by RIBA in 26.6% of the blood donors, which is higher than the 10-19% prevalence of antibody found in other studies of Egyptian blood donors. A history of selling blood (odds ratio [OR] = 12.1) and the use of illicit parenteral drugs (OR = 2.5) were significantly associated with anti-HCV seropositivity after controlling for age and gender. These data indicate that the use of illicit drugs may be one reason for high levels of reported HCV infection among Egyptian blood donors. These findings also indicate that Egyptian blood donors should be screened for anti-HCV and individuals who have a history of drug abuse should be deferred from donating blood. PMID- 7541969 TI - CFTR expression and organ damage in cystic fibrosis. AB - The cloning of the defective gene in cystic fibrosis (CFTR) is the most important step to date toward understanding the pathogenesis of the disease and developing novel therapeutic strategies. Although many studies have provided insights into the molecular defects and knowledge of the expression and role of the gene, the basic defect and its pathogenesis are still unclear. We hypothesize that organ damage in cystic fibrosis is the result of a combination of at least three main factors: the genotype (the type of mutation that alters the function of the cystic fibrosis transmembrane regulator [CFTR]), the rate of CFTR-mediated chloride secretion in the epithelium of each organ (inferred from the level of expression of the gene), and the anatomical and physiologic characteristics of the affected organs (the size and contents of the ducts). Confirmation of this hypothesis should allow a better understanding of the pathogenesis of the disease and help prevent organ damage. PMID- 7541970 TI - Post-polio syndrome spinal cord pathology. Case report with immunopathology. AB - Post-polio syndrome (PPS) describes the clinical onset of progressive muscular weakness many years after survival of acute paralytic poliomyelitis, often in muscle groups clinically unaffected by the original disease process. Prior pathologic descriptions of this often disabling, but not usually fatal, syndrome have been scanty. These have emphasized the presence of persistent or new inflammation in the meninges, spinal cord, and muscles of affected patients. The inflammation suggests several pathogenetic hypotheses, including persistent active poliovirus infection, autoimmune attack on central and peripheral nervous system elements, or increased vulnerability of poliovirus-damaged tissue to new infections. We have recently examined the central nervous system from a PPS patient. The cord showed focal perivascular intraparenchymal chronic inflammatory infiltrates. Immunoperoxidase staining demonstrated that the infiltrates were virtually pure populations of B lymphocytes (immunopositive with antibody L26, and immunonegative with the T cell marker UCHL1 as well as the macrophage marker HAM56). There were rare macrophages (HAM56 immunopositive) and no T cells. The character of the infiltrates suggests that PPS could be an autoimmune disorder mediated by antibodies produced in situ, and not by a cell-mediated process. Additional important pathological features were the presence in the anterior horns of axonal spheroids and of moderate Wallerian degeneration in the lateral columns. The brain was entirely unremarkable, with no detectable cell loss of gliosis in the internal capsules or precentral gyri. PMID- 7541971 TI - Studies on the diversity of muscarinic receptors in the autoregulation of acetylcholine release in the rodent cerebrum using furan analogs of muscarine. PMID- 7541972 TI - Phosphorylation of the nicotinic acetylcholine receptor by protein tyrosine kinases. AB - Most neurotransmitter receptors examined to date are either regulated by phosphorylation or contain consensus sequences for phosphorylation by protein kinases. The nicotinic acetylcholine receptor (AChR), which mediates depolarization at the neuromuscular junction, has served as a model for the study of the structure, function, and regulation of ligand-gated ion channels. The AChR is phosphorylated by protein kinase A, protein kinase C, and an unidentified protein tyrosine kinase. Tyrosine phosphorylation of the AChR is correlated with a modulation of the rate of receptor desensitization and is associated with AChR clustering. We showed that agrin, a neuronally derived extracellular matrix protein, induces AChR clustering and tyrosine phosphorylation. In addition, we identified two protein tyrosine kinases, Fyn and Fyk, that appear to be involved in the regulation of synaptic transmission at the neuromuscular junction by phosphorylating the AChR. The two kinases are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. As demonstrated by coimmunoprecipitation, Fyn and Fyk associate with the AChR. Furthermore, the AChR is phosphorylated in Fyn and Fyk immunoprecipitates. We investigated the molecular basis for the association of the AChR with Fyn and Fyk using fusion proteins derived from the kinases. The AChR bound specifically to the SH2 domain fusion proteins of Fyn and Fyk. The association of the AChR with the SH2 domains is dependent on the state of AChR tyrosine phosphorylation and is mediated by the delta subunit of the receptor. These data provide evidence that the protein tyrosine kinases Fyn and Fyk may act to phosphorylate the AChR in vivo. PMID- 7541974 TI - Effects of peptide YY on CCK/CCK antagonist interactions in cerulein-induced pancreatic injury. PMID- 7541973 TI - Localization of the peptide binding domain of the NK-1 tachykinin receptor using photoreactive analogues of substance P. PMID- 7541975 TI - Characterization of voltage-sensitive Ca2+ channels activated by presynaptic glutamate receptor stimulation in hippocampus. PMID- 7541976 TI - Hu antigens: reactivity with Hu antibodies, tumor expression, and major immunogenic sites. AB - HuD, a human neuronal RNA-binding protein, was the first identified member of a family of antigens that also includes HuC and Hel-N1 (Hu antigens). The serum of all patients with anti-Hu-associated paraneoplastic encephalomyelitis and sensory neuronopathy react with HuD and Hel-N1, but the reactivity with HuC is unknown. In the current study we examined (1) the reactivity of anti-Hu sera with HuD, HuC, and Hel-N1; (2) the expression of HuD, HuC, and Hel-N1 messenger RNA in small-cell lung cancer of patients with and those without paraneoplastic encephalomyelitis/sensory neuronopathy; (3) the correlation between anti-Hu serum reactivity with these three Hu antigens and the type of neurological symptoms; and (4) the major immunogenic sites of HuD. Our findings indicate that all anti Hu sera react with HuD, HuC, and Hel-N1. However, only HuD is expressed in the small-cell lung cancer tumors, indicating that among the three Hu antigens, HuD appears to play a central role in triggering the anti-Hu immune response. No differences were identified regarding the reactivity of the anti-Hu antibodies with HuD, HuC, and Hel-N1 and the spectrum of neurological symptoms presented by the patients. Study of the major immunogenic sites of HuD resulted in the identification of two major immunodominant regions with at least two distinct epitopes recognized by the serum of all patients with anti-Hu-associated paraneoplastic encephalomyelitis/sensory neuronopathy. PMID- 7541978 TI - [Multiparametric cytometric analysis of hepatocellular carcinoma and its allied lesions combining DNA ploidy analysis with morphometry using DAPI/HP double staining]. AB - Hepatocellular carcinomas (HCC) of various differentiation, Edmondson (Edm) grade I-IV, often based upon viral infectious hepatitis transformed through regeneration, nodular and/or dysplastic adenomatous hyperplasia. Aspiration cytology, even needle biopsy for such lesions, is very difficult to interpret. We have been studying multiparametric cytologic analysis using DAPI/Hematoporphyrin (HP) staining for the cece samples provided from exactly trimmed 50-microns thick paraffin sections, employing a new software program which we developed, combining DNA ploidy and cell protein content analysis with morphometry. DAPI indicates DNA content and nuclear size and HP indicates cell protein content and cell size. From 21 cases of HCC, 43 nodules or masses including multiple lesions and nodule in nodule were analyzed. Surrounding non-neoplastic hepatic tissues were examined as the control specimens. On DNA histogram, cases with the peak over 2.4 c are identified as aneuploid (Aneupl). Cases of Edm I, or II, 6 of 8 samples showed Aneupl., all of 11 cases of Edm II, 11 cases of Edm III, and one of Edm IV showed Aneupl. Multinodular foci in the same cases and also nodule in nodule often showed different modes of Aneupl. This suggested heterogenetic carcinogeneity. Noncarcinomatous foci often showed Aneupl. modes, which may indicate malignant transformation or malignant potential. PMID- 7541977 TI - [Development of quality of life (QOL) questionnaire for use of lung cancer patients in palliative therapy--study of validity and reliability no. 2, the effects of chemotherapeutics in QOL]. AB - The effects of cisplatin and carboplatin in QOL were studied using a QOL questionnaire seeking to conform their validity and reliability in lung cancer patients. The questionnaire was composed of eleven items; appetite, feeling, sleep, mental fatigue, pain, anxiety, daily activity, abdominal and respiratory conditions, linear analog and face scale as global scale. The data were collected from 21 patients treated with cisplatin (Cis group) and 9 patients administered carboplatin (Carbo group). Chronological changes of QOL were measured by AUC (area under the curve) method. 1) The total score of 9 items, linear analog and face scales rose immediately to the highest levels (worse) after treatment and maintained this level for 1 week in the Cis group. The Carbo group levels rose from 3 days and returned to the control level at 9 days after treatment. 2) AUC of the total score, linear analog and face scales in the Cis group increased significantly when compared with those of the Carbo group. 3) When compared with the Cis and Carbo groups the physiological and active scales were not different, but the psychological scale showed a significant difference between the two groups. 4) The total score of the psychological scale correlated the abdominal score in the Cis group, but not in the Carbo group. 5) Sleep and mental fatigue were related to the aggravation of QOL at 5-6 days after Carboplatin treatment. These results suggested that this QOL questionnaire had sufficient sensitivity to reflect any chemotherapeutic side-effects. 6) AUC is useful method in chronological evaluation of QOL. PMID- 7541979 TI - Analysis of the polymerase chain reaction in the detection of herpesvirus DNA from fixed and stained tissue sections. AB - BACKGROUND AND DESIGN: The polymerase chain reaction (PCR) is a molecular diagnostic technique that has been applied to many infectious processes. Stained and unstained Tzanck smears, vesicle fluid swabs, and crusts have all been used as the source for template DNA for the PCR to document evidence of herpes simplex virus and varicella-zoster virus infection. Thirty-five cases with histologic evidence of acute herpesvirus infection were retrieved from archival tissue blocks that were up to 5 years old. Paraffin and hematoxylin-eosin-stained tissue sections obtained from routinely prepared glass slides from these cases were then examined for herpesvirus DNA using the PCR. RESULTS: The PCR-detected herpesvirus DNA from 34 (97.1%) of 35 paraffin tissue samples. Herpes simplex virus and varicella-zoster virus DNA were detected in eight and 26 of these cases, respectively. For hematoxylin-eosin-stained tissue samples, PCR detected herpesvirus DNA sequences in 16 (45.7%) of 35 cases. Herpesvirus DNA was isolated from paraffin tissue sections and recently prepared hematoxylin-eosin-stained tissue samples obtained from archival tissue blocks that were up to 5 and 2 years old, respectively. CONCLUSIONS: The PCR can detect herpesvirus DNA in extremely high yield from unstained paraffin-embedded tissue samples with histologic evidence of acute herpesvirus infection that are up to 5 years old. Herpesvirus DNA can also be identified in approximately 50% of these cases from hematoxylin eosin-stained tissue sections obtained from routinely prepared glass slides. PMID- 7541980 TI - Rapid diagnosis of premature rupture of membranes using a new kit employing anti AFP monoclonal antibody. AB - A kit using mouse monoclonal antibody against human alpha-fetoprotein (anti-AFP kit) has been developed. Cervical secretion (n = 34) and vaginal fluid (n = 15) was obtained from 30 pregnant volunteers. All 31 specimens obtained from women with intact membranes tested negative while all 18 specimens from women with premature rupture of membranes (PROM) tested positive. The newly developed kit allowed rapid and accurate diagnosis. We compared the kit results with those obtained by pH determination (nitrazine, bromthymol blue test) and measurement of AFP concentrations by enzyme-linked immunoassay (EIA). PMID- 7541981 TI - The relation between depth of trophoblastic invasion and beta-HCG levels in tubal pregnancies. AB - beta-HCG (human chorionic gonadotropin) values of over 2500 I.U./l are associated with higher failure rates for therapy with prostaglandin F2 alpha in tubal pregnancies. The purpose of our study was to ascertain if the 2500 I.U./l limit correlates with histopathology. We therefore compared the pre-operative beta-HCG values and intraluminal and extraluminal trophoblast growth in tubal pregnancy. Purely intraluminal trophoblast was significantly more frequent in patients of group I (beta-HCG < 2500 I.U./l), while group II patients (beta-HCG > 2500 I.U./l) almost exclusively had extraluminal growth (P = 0.0045). Since the efficacy of prostaglandin F2 alpha therapy depends on intact tubal musculature the correlation of the beta-HCG threshold level with histopathologic findings may explain the high failure rate in patients with beta-HCG values above 2500 I.U./l. PMID- 7541982 TI - Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus. AB - Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits. PMID- 7541983 TI - Antigenic analysis of the F protein of the bovine respiratory syncytial virus: identification of two distinct antigenic sites involved in fusion inhibition. AB - From two independent fusions, fifteen MAbs directed to the F protein of the bovine respiratory syncytial virus (BRSV) were characterized by radio immunoprecipitation assays. Competition binding assays among these MAbs identified two distinct antigenic sites (A and B) and one overlapping site (AB). All of the MAbs specific to epitopes belonging to site A neutralized the infectivity of the virus in vitro and recognized human and bovine RSV strains. Only two out of the five MAbs directed to epitopes of site B were neutralizing and three reacted with all of the RSV strains tested, suggesting that the epitopes constituting this domain present heterogeneous characteristics. In each of sites A and B, one of the neutralizing MAbs also inhibited cell fusion. The biological relevance of these domains was established by competing representative MAbs and sera from BRSV-infected calves. PMID- 7541985 TI - Effects of insulin-like growth factor (IGF) binding proteins (BPs) -3 and -6 on DNA synthesis of rat osteoblasts: further evidence for a role of auto-/paracrine IGF I but not IGF II in stimulating osteoblast growth. AB - IGFBP-3 and IGFBP-6 were used to study whether both IGF I and IGF II play a role in auto-/paracrine stimulation of rat osteoblast growth. Both IGFBPs decreased basal DNA synthesis in neonatal rat calvaria cells but with different potencies. Consistent with their IGF binding affinities, IGFBP-3 blocked both IGF I- and IGF II-stimulated DNA synthesis, whereas IGFBP-6 preferentially blocked IGF II stimulated DNA synthesis. These inhibitory effects of the two IGFBPs can be fully explained by the sequestration of IGFs. Because IGFBP-6 preferentially binds IGF II and is much less potent than IGFBP-3 in decreasing basal DNA synthesis in calvaria cells, IGF I but not IGF II appears to be an important auto-/paracrine stimulator of DNA synthesis. PMID- 7541984 TI - The inter-relationship between polyamines and the L-arginine nitric oxide pathway in the human placenta. AB - Nitric oxide (NO) and polyamines are both products of L-arginine metabolism. In placental villous tissue NO and polyamines have been shown to be synthesized although the physiological significance is not known. We have measured polyamine (putrescine, spermidine and spermine) concentrations and nitric oxide synthase activities (NOS) in first trimester and term placentae from normal and abnormal pregnancies, but no difference was observed in polyamine concentrations between normal term and placentae from growth-retarded and pre-eclamptic pregnancies. Significantly higher levels of polyamines were found in first trimester when compared to normal term placentae and there was a significant correlation between NOS activity and the cellular polyamine levels. Cultures of a trophoblast cell line, BeWo, have been used to study the interaction of added polyamines on NOS activity. Although there was a general tendency for all the polyamines to inhibit NOS activity only putrescine was able to significantly inhibit NO production by these cells. It is thought that the L-arginine-NO-polyamine pathway may have a physiological role during pregnancy. PMID- 7541986 TI - Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits adipocytic differentiation of 3T3-L1 cells. AB - The effect of wortmannin, which inhibits phosphatidylinositol 3-kinase (PI 3 kinase), on adipocytic differentiation of 3T3-L1 cells was examined. The extent of differentiation was evaluated by the staining of adipocytes with Oil Red O and measurement of glycerol 3-phosphate dehydrogenase (GPDH) activity. Wortmannin, at over 100 nM, significantly inhibited adipogenesis of cells treated with isobutyl methylxanthine, dexamethasone, and insulin with an IC50 value about 50 nM. These results suggest that PI 3-kinase plays a role in adipocytic differentiation of 3T3-L1 cells. PMID- 7541988 TI - Potential interaction between annexin VI and a 56-kDa protein kinase in T cells. AB - Annexins belong to a large family of calcium-dependent phospholipid binding proteins known to undergo post-translational modifications such as phosphorylation. Physiological function of each annexin is still unclear since they may participate in signal transduction. We have tested the presence of annexins in a T cell line (Jurkat) and studied their phosphorylation by protein tyrosine kinases of the src family. Among annexins I, II, V and VI found in Jurkat cells, annexin VI was shown to be phosphorylated in vitro by p56lck and annexins I and II by p60src. We could not detect the phosphorylation of A-VI in vivo, even after cell stimulation. However, a 56-kDa phosphoprotein was found to be associated with A-VI after T cell activation. This 56-kDa protein shares some characteristics with p56lck. PMID- 7541987 TI - The Ah receptor recognizes DNA binding sites for the B cell transcription factor, BSAP: a possible mechanism for dioxin-mediated alteration of CD19 gene expression in human B lymphocytes. AB - 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits murine and human B lymphocyte immunoglobulin production through an unknown mechanism. This study investigated the effect of TCDD on expression of the CD19 gene in a human B lymphocyte cell line. Northern blot analysis showed that TCDD treatment decreased steady state levels of CD19 mRNA by 67% in the IM-9 cell line. Using a gel mobility shift assay, we identified a DNA-binding complex in IM-9 nuclear extracts that by several criteria appears to be the Ah receptor. In addition, the Ah receptor complex recognized a DNA binding site for B cell lineage-specific activator protein (BSAP) in the promoter region of the human CD19 gene which is similar to the consensus Ah receptor DNA binding site. These results suggest that the AhR could interfere with BSAP-stimulated CD19 gene transcription by competition for a common DNA binding site. PMID- 7541989 TI - In the rat pineal gland, but not in the suprachiasmatic nucleus, the amount of constitutive neuronal nitric oxide synthase is regulated by environmental lighting conditions. AB - To investigate whether the expression of neuronal isoform of nitric oxide synthase is photoneurally regulated, we examined the amount of the enzyme by means of Western Blot analysis under different environmental lighting conditions in two photoneuronally regulated tissues, the pineal gland and the suprachiasmatic nucleus of the rat. In the pineal gland nitric oxide synthase immunoreactivity was strikingly decreased when rats had been exposed for 8 days to light:dark 20:4 conditions or to constant light. The decline in nitric oxide synthase immunoreactivity following LL reversed after 4 days under light:dark 12:12. We conclude that in the rat pineal the amount of neuronal nitric oxide synthase is controlled by environmental lighting conditions. That it is not justified to extrapolate from the pineal to other photoneuronally regulated centers is illustrated by the present finding that the suprachiasmatic nucleus did not reveal changes in the amount of neuronal nitric oxide synthase. PMID- 7541991 TI - Comments on "Effects of 60 Hz electric and magnetic fields on the development of the rat cerebellum," by Gona et al. PMID- 7541990 TI - Millimeter microwave effect on ion transport across lipid bilayer membranes. AB - The effects of millimeter microwaves in the frequency range of 54-76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB-). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% +/- 0.5%. At the same time, membrane current induced by TPhB- transport increased by 5% +/- 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% +/- 0.4%). No "resonance-like" effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB- transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 degrees C. PMID- 7541992 TI - Induction of early response genes by interferons, interleukins, and growth factors by the tyrosine phosphorylation of latent transcription factors. Implications for chronic inflammatory diseases. PMID- 7541993 TI - Changes in glycosaminoglycan epitope levels in knee joint fluid following injury. AB - OBJECTIVE: To measure the levels of epitope on the chondroitin sulfate (CS) and keratan sulfate (KS) chains of proteoglycan fragments in synovial fluids from injured and contralateral uninjured knees of patients with traumatic cruciate ligament and/or meniscus damage. METHODS: Enzyme-linked immunosorbent assays were used to determine the levels of monoclonal antibody epitopes 3-B-3 and 7-D-4 (CS), and 5-D-4 (KS), in paired joint fluids from the injured and uninjured knees of trauma patients. RESULTS: Levels of the CS epitopes were increased in the trauma joint fluids from most patients, with higher levels of 3-B-3 epitope in 12 of the 16 patients, but the difference did not achieve significance; however, 7-D 4 levels were higher in 15 patients, and the difference was highly significant (P = 0.0005). In contrast, the KS epitope detected by 5-D-4 was decreased in 13 of 15 patients, and the difference was significant (P = 0.0074). CONCLUSION: The increased level of 7-D-4 epitope on proteoglycans in joint fluid from injured knees may reflect the response of the articular cartilage to acute trauma resulting in altered expression of specific CS epitopes on cartilage proteoglycans. The fall in KS epitope levels may reflect the synthesis of proteoglycans that have lower KS content. PMID- 7541995 TI - Anti-anaphylactic activity of the novel selective histamine H1 receptor antagonist mizolastine in the rodent. AB - The anti-anaphylactic/anti-histamine activity of mizolastine (CAS 108612-45-9, SL 85.0324), a novel histamine H1 receptor antagonist devoid of sedative properties, has been evaluated in the rat, mouse and guinea pig. Mizolastine inhibited the passive cutaneous anaphylactic reduction caused by ovalbumin challenge in the rat (ED50 = 0.7 mg/kg i.v., 1.6 mg/kg p.o.) and effectively protected rats from the lethal shock induced by compound 48/80 (ED50 = 0.07 mg/kg p.o.). Mizolastine protected actively sensitized guinea pigs from anaphylactic mortality, bronchospasm and respiratory difficulties (increase in pulmonary resistance) preceding this event and from morphological modifications at doses from 0.05 mg/kg i.v. The pharmacological activity of mizolastine is linked to a selective blockade of histamine H1 receptors as indicated by the ability of this compound to antagonize rat paw edema induced by the subplantar injection of histamine (ED50 = 0.5 mg/kg p.o.) but not that induced by the injection of serotonin or bradykinin. Mizolastine also antagonized the increase in cutaneous capillary permeability caused by the intradermal injection of histamine (-80% at 0.3 mg/kg p.o.) and compound 48/80 (ED50 = 1.1 mg/kg p.o.) but not that induced by serotonin in the rat. In the guinea pig, mizolastine antagonized i.v. histamine induced bronchoconstriction (ED50 = 0.03 mg/kg p.o.) and histamine-induced vascular permeability and edema in trachea and bronchi (ED50 < or = 0.05 mg/kg i.v.). Moreover, at higher doses, mizolastine antagonized the bronchospasm caused by systemic injection of platelet-activating factor (PAF) and leukotriene D4 (LTD4) (ED50's = 0.30 and 3.0 mg/kg p.o., respectively). However, mizolastine only weakly antagonized bronchospasm induced by aerosolized PAF (-67% at 50 mg/kg p.o.), failed to antagonize (up to 3 mg/kg i.v.) PAF-induced microvascular permeability of the tracheal mucosa in the guinea pig and was a weak inhibitor of PAF-induced platelet aggregation in the rabbit (IC50 = 74 mumol/l). In addition to antagonizing histamine H1 receptors, mizolastine also inhibits the release of histamine during allergic reactions in tissues. Thus, mizolastine antagonizes the antigen-induced in vivo release of histamine from mast cells in bronchoalveolar lavages of actively sensitized guinea pigs (minimal effective dose 0.3 mg/kg p.o.) and the release of histamine from mast cells in the peritoneal fluid of passively sensitized rats (ED50 = 0.9 mg/kg i.v.). In these various models, mizolastine was more potent than loratadine and terfenadine but less potent than ketotifen. The apparent half-life for the pharmacological actions of mizolastine ranged from 6 to 8 h.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7541994 TI - Autoepitopes of the 52-kd SS-A/Ro molecule. AB - OBJECTIVE: This study was undertaken to clarify the mechanisms responsible for the generation of anti-52-kd SS-A/Ro autoantibodies and to elucidate why, as has recently been reported, anti-52-kd autoantibodies preferentially recognize the denatured form rather than the native 52-kd molecule. METHODS: Using a series of truncated 52-kd autoantigens, produced as beta-galactosidase fusion proteins in Escherichia coli, the B cell epitope distribution was probed with 18 anti-Ro positive sera by immunoblotting and by enzyme-linked immunosorbent assay. RESULTS: Nearly all the antigenicity of the molecule was found to be linked to its leucine zipper region. In a further study using 9 of the 18 sera, the antigenicity of the molecule was found to be mainly formed by multiple conformational epitopes, and one of these epitopes appeared to be universally recognized by all the sera tested. CONCLUSION: The recognition of multiple epitopes indicates that the Ro 52-kd antigen itself drives the autoimmunity to this molecule. Further, the concentration of the antigenicity at the leucine zipper region may explain why anti-52-kd antibodies preferentially recognize the denatured protein rather than its native form. PMID- 7541996 TI - Effects of a new foam formulation of ketoprofen lysine salt in experimental models of inflammation and hyperalgesia. AB - The anti-inflammatory and analgesic profile of a new topical foam formulation of ketoprofen lysine salt (CAS 57469-78-0, Artrosilene Schiuma, KLS-foam) was characterized in comparison with marketed gel formulations containing KLS (KLS gel) or diclofenac diethylammonium salt (DCF-gel). KLS-foam dose-dependently inhibited oedema formation and hyperalgesia induced by subplantar injection of carrageenan or substance P, being more potent than KLS-gel. At equieffective anti inflammatory doses, KLS-foam provided a more pronounced analgesic effect than DCF gel. KLS-foam also markedly inhibited exudate formation and prostaglandin production induced by subcutaneous implantation of carrageenan soaked sponges. In carrageenan induced paw inflammation, KLS-foam provided the same anti inflammatory effect as orally administered KLS, but induced significantly less gastric damages. Oral administration of KLS resulted in sustained systemic absorption of ketoprofen, whereas after topical application of KLS-foam no appreciable ketoprofen plasma levels were detected. These data support the anti inflammatory and particularly the analgesic effectiveness of the new foam formulation of KLS, a finding that, together with the high gastric tolerability, further emphasizes the usefulness of KLS-foam in the treatment of localized flogistic diseases and associated pain. PMID- 7541997 TI - Maternal serum levels of alpha-fetoprotein and fetal fibronectin as markers of labor in the third trimester. AB - Unexplained elevations of maternal serum alpha-fetoprotein (MSAFP) in the second trimester have been found to be associated with a two- to fourfold increase in the rate of preterm delivery, but the sensitivity is low. Therefore, we reasoned that MSAFP levels in the third trimester could prove to be a more useful biochemical marker to predict labor. The presence of placental and membrane derived fetal fibronectin (FFN) in cervicovaginal secretions has recently been shown to predict preterm delivery with a sensitivity of 81.7% and specificity of 82.5%. We postulated that damage to membranes and microscopic breakdown of fetomaternal blood barrier during labor might result in release of AFP or FFN into maternal serum. Maternal serum alpha fetoprotein and fetal fibronectin levels were measured prospectively in 29 patients in active labor at term and in 25 controls undergoing elective cesarean section. Neither MSAFP nor serum FFN levels were associated with labor at term. We did, however, note significantly higher MSAFP levels in mothers bearing male fetuses versus female fetuses (p < 0.01). Since the current literature supports a sex difference in the MSAFP levels in the second trimester, this does not appear to change as gestation advances. Further studies are needed to determine if, in addition to maternal weight and race, MSAFP levels should be also adjusted for fetal sex diagnosed on sonography. PMID- 7541998 TI - Does preeclamptic pregnancy increase fetal-maternal hemorrhage? AB - Since pathologic changes of the placenta have been found in preeclamptic pregnancies, a relationship between fetal-maternal hemorrhage and preeclampsia was investigated. Sixty-two women with singleton pregnancies in the third trimester were studied. Thirty-one of these women with preeclampsia were matched with 31 normotensive healthy pregnant women. Maternal serum levels of alpha fetoprotein (MSAFP) and Kleihauer-Betke (KB) test were determined to reflect the degree of fetal-maternal hemorrhage. There were no significant differences in MSAFP levels, MSAFP to creatinine ratio, or the appearance of fetal cells as measured by the KB test between preeclamptic pregnancies and matched controls. Our data do not suggest that fetal-maternal hemorrhage is increased in pregnant women with preeclampsia. PMID- 7541999 TI - Computerized interactive morphometry in the differential diagnosis of irradiated prostates. AB - Radiation therapy is becoming a treatment of choice for many patients with prostatic carcinoma. Distinguishing radiation change in prostate glands from carcinoma may be difficult. In this study we objectively assessed, by morphometric methods, the nuclear characteristics of benign and malignant prostates with a history of radiation treatment (125I implant with or without prior external beam radiation). This is part of our continuing efforts to achieve difficult differential diagnoses by analyzing perimeter, diameter and nuclear profile area of cells or interest and applying methods of statistical classification. Biopsies were performed 18-36 months following implant therapy. Eleven cases with residual prostate tumor constituted the malignant group. These were compared to 20 benign cases (benign glands in the 11 carcinoma cases plus 9 other cases with no residual carcinoma). Immunohistochemical staining with keratin 903 was performed on all cases. Differences in the nuclear parameters were most evident in the average nuclear profile areas (32.5 microns 2 for the malignant groups vs. 39.6 for the benign) and in the mean maximal cord length (diameter) (7.4 microns for the malignant group vs. 9.0 for the benign). Classification, however, is based on the size distribution plots of nuclear profile areas, which, in the malignant cases, had a sharper peak at lower value, while the benign cases had higher value and a broader peak with a trailing off into the larger values. This study emphasized the marked nuclear alterations that occur in irradiated prostates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542000 TI - DNA tetraploidy in Feulgen-stained bladder washings assessed by image cytometry. AB - The prognostic utility of DNA cytometry has been demonstrated for irrigation specimens from bladder neoplasms. While the traditional method of measuring the DNA content of cells recovered by bladder irrigation is flow cytometry, image analysis has been applied increasingly, with successful results. In some cases, image analysis has been shown to detect DNA aneuploid populations missed by flow cytometry. The DNA aneuploid population most frequently missed by flow cytometry is in the DNA tetraploid range. The purpose of the present study was to review image cytometry data on bladder washings analyzed at the University of Florida Diagnostic Referral Laboratories during a one-year period, with special emphasis on the subset with DNA tetraploid histograms. Of the 205 cases reviewed, 127 (62%) were DNA diploid, 36 (18%) DNA aneuploid and 42 (20%) DNA tetraploid. Corresponding cytology was negative in 113/127 (89%) of DNA diploid, 3/36 (8%) of DNA aneuploid and 29/42 (69%) of DNA tetraploid cases. Within the DNA tetraploid group, 45% of cases had no clinical (cystoscopic) or pathologic (cytologic and histologic) evidence of neoplasia. None of these patients developed tumors during follow-up. The presence of DNA tetraploidy in cytologically negative cases should be interpreted cautiously. PMID- 7542001 TI - Expression of lectin, interleukin-2 and histopathologic blood group binding sites in prostate cancer and its correlation with integrated optical density and syntactic structure analysis. AB - The binding of several biotinylated biologic probes was determined in sections of 20 surgical specimens of prostate cancer and of 21 biopsy specimens of hyperplastic prostate. Whereas neither the immunomodulatory, galactoside-specific lectin from Viscum album nor the human beta-galactoside-specific lectin (M(r) 14 kd) or its specific antibody discerned any remarkable differences, the lectin from Urtica dioica (UDA) and interleukin-2, the in vitro production of which is enhanced by this lectin, exhibited obvious preference for hyperplastic cells. In addition, the presence of binding sites for chemically synthesized blood group determinants was tested. Carcinoma cases revealed a higher percentage of binding of synthetic blood group trisaccharide H than hyperplasia cases. Due to these differences, diverse parameters, derived from measurement of integrated optical density (IOD) and from syntactic structure analysis, were correlated with the extent of binding of these biologic probes for the tumor cases. Primarily, parameters that are related to computation of a minimum spanning tree were significantly different in positive and negative cases for both UDA and interleukin-2. For the binding of blood group trisaccharide H the 5C exceeding rate, the 2CV deviation index and the distance of neighboring tumor cells with an IOD > 5 were clearly dissimilar. Our results thus suggest an extension of the panel of biologic probes for prostate cancer and substantiate the usefulness of correlations of binding of selected biologic probes to features derived from the assessment of IOD and syntactic structure analysis. PMID- 7542002 TI - Comparison of fluorescence in situ hybridization and flow cytometric DNA ploidy analysis in paraffin-embedded prostatic adenocarcinoma specimens. AB - Cancer cell nuclei extracted from 15 paraffin-embedded radical prostatectomy specimens were examined with fluorescent in situ hybridization (FISH) and flow cytometry (FCM) for DNA ploidy. Repetitive chromosome-specific alpha-satellite DNA probes to centromeres 7 and 10 were used in the FISH assay. Results from FISH and FCM were compared and viewed in relation to clinical experience. Of the 15 tumors examined, 9 were hyperdiploid for at least one chromosome by FISH assay. Seven were aneuploid by FCM, showing a good correlation between the two methods (P = .006). Using either evidence of clinical progression or a postoperative prostate-specific antigen (PSA) level > 0.5 ng/mL by Hybritech assay to indicate the risk of clinical progression, 56% of FISH hyperdiploid tumors had a risk of progression of carcinoma versus 17% of FISH diploid tumors. A preoperative PSA level > 30 ng/mL had a marginal correlation with risk of progression (P = .05). We demonstrate that FISH is a useful tool for chromosomal analysis in paraffin embedded tumor specimens. PMID- 7542003 TI - Antibody reactivity to mycobacterial 65 kDa heat shock protein: relevance to autoimmunity. AB - Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule. PMID- 7542005 TI - Comparative expression of TGF-alpha and EGF genes in the ovine conceptus and uterine endometrium in the peri-implantation period. AB - Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors. With the use of a very sensitive method, the reverse transcription-polymerase chain reaction, expression of some growth factor transcripts was examined during ovine peri-implantation development. Transcripts for transforming growth factor-alpha (TGF-alpha) were found in day 15 to day 30 conceptuses, and in uterine tissues. Epidermal growth factor (EGF) was not detectable in any ovine stages or tissues studied. TGF-alpha could be the normal physiological ligand for the epidermal growth factor receptor present on the trophoblastic tissues, and its expression pattern suggests an autocrine and paracrine role in the growth and differentiation of ovine embryos. PMID- 7542004 TI - Autoantibodies to cryptic epitopes of C-reactive protein and other acute phase proteins in the toxic oil syndrome. AB - Toxic oil syndrome (TOS) was caused by the consumption of rapeseed oil contaminated with derivatives of aniline. Many persons who survived the acute phase developed a puzzling, multi-year chronic disease considered to be inflammatory or autoimmune in nature. In attempting to characterize their autoantibodies, we found that 74% of TOS patients with chronic disease had IgG antibodies to C-reactive protein (CRP). This activity was detectable only when CRP was chemically or physically denatured and behaved like a previously described antibody produced by immunization with the CRP monomer. Significant antibody reactivities to other acute phase proteins, especially alpha 1 antitrypsin and fibrinogen (P < 0.025) and ceruloplasmin (P < 0.05) were also observed. IgG antibodies to cryptic epitopes in CRP and other major serum proteins that increase during the acute phase response may reflect an earlier toxin-mediated insult to the liver that included abnormal biosynthesis of and/or damage to acute phase proteins. PMID- 7542006 TI - Diagnosis and treatment of primary amyloidosis. PMID- 7542007 TI - cAMP-dependent phosphorylation of cytokeratin in hepatic inner mitochondrial membrane. AB - The phosphorylation pattern in mitochondrial fractions isolated from hepatocytes, preincubated with 32P-phosphate and stimulated with glucagon and calcium mobilizing hormones, was studied. Only in mitochondria from glucagon treated hepatocytes two phosphorylated protein bands were observed, one with a molecular weight (MW) of 54 kDa in the outer membrane fraction which, according to the literature, is suggested to represent protein kinase A; one with a MW of 20 kDa in the inner membrane fraction which has not been described earlier. Electroelution and digestion of the 20 kDa protein band yielded two tryptic peptides which were identified as fragments homologous to human cytokeratin type II (the sequence of rat cytokeratin type II is not known). From the amino acid composition and sequence, and from the known structure of type II cytokeratins, it is concluded that the 20 kDa phosphoprotein is composed of amino- and carboxylterminal proteolytic fragments of rat cytokeratin C8 which are tightly anchored in the inner mitochondrial membrane. The physiological significance of the possible interaction of cytoskeletal proteins with the mitochondrial inner membrane and its hormonal regulation are discussed. PMID- 7542008 TI - Nitric oxide generation during cellular metabolization of the diabetogenic N methyl-N-nitroso-urea streptozotozin contributes to islet cell DNA damage. AB - The N-methyl-N-nitroso-urea streptozotocin is an antibiotic with diabetogenic, carcinogenic and antitumor activity thought to act via alkylation of DNA and proteins. Evidence points to a release of bioactive nitric oxide (NO) from streptozotocin as an additional cytotoxic activity of this drug. Here we show by EPR spectroscopy, that NO is not generated during spontaneous decay of streptozotocin but that its metabolization in rat hepatocytes and pancreatic islet cells yields NO. This NO formation is not due to a NO synthase (NOS) activity since NO formation in hepatocytes in the presence of streptozotocin is not blocked by the NOS inhibitor NG-methyl-L-arginine. By iNOS-specific RT-PCR no positive signal for specific mRNA presence was obtained in streptozotocin-treated cells, proving that iNOS activity was not induced during cell isolation procedures and did not account for the NO release. Furthermore, early DNA-strand breaks induced either by SZ or by the NO donor nitroprusside were both significantly reduced in the presence of an intracellular NO scavenger. In contrast, DNA damage found after incubation with the purely alkylating agent methylmethanesulfonate was not inhibited by the NO trap. These results prove that intracellular formation of NO occurs during degradation of SZ within cells. This NO appears to contribute significantly to streptozotocin-induced cytotoxicity. PMID- 7542009 TI - The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. AB - B lymphocytes must respond to low concentrations of antigen despite having low affinity antigen receptors during the primary immune response. CD19, a B cell restricted membrane protein of the immunoglobulin superfamily that associates with the antigen receptor complex, may help the B cell meet this requirement. Cross-linking CD19 to membrane immunoglobulin (mIg) lowers, by two orders of magnitude, the number of mIg that must be ligated to activate phospholipase C (PLC) or to induce DNA synthesis. CD19 is coupled, via protein tyrosine kinases (PTKs), to PLC and phosphatidylinositol 3' kinase (PI3' kinase), and it interacts with the Src-type nonreceptor PTK lyn. It also associates with two other membrane proteins, CR2 (complement receptor type 2, CD21), which permits nonimmunologic ligation of CD19, and TAPA-1, a member of the tetraspan family of membrane proteins. CR2 binds fragments of C3 that are covalently attached to glycoconjugates. This indirectly enables CD19 to be cross-linked to mIg after preimmune recognition of an immunogen by the complement system. CR2 also can be ligated by CD23, a lectin-like membrane protein that resides on cells that may present antigen to B cells. TAPA-1 associates with several other membrane proteins on B and T cells, including MHC class II, CD4, and CD8, and it promotes Ca2(+)- and LFA-1-independent homotypic aggregation when ligated directly or indirectly through CD19 or CR2. This may facilitate interaction of the B cell with other cells essential for cellular activation. The formation of this membrane protein complex by representatives of three different protein families helps the B cell resolve its dilemma of combining broad specificity with high sensitivity. PMID- 7542011 TI - Histology of chronic pilonidal sinus. AB - Many theories concerning the development of chronic pilonidal sinus have been proposed. A histologic study of primary pilonidal sinus in 53 patients is presented. Subcutaneous tissue contained sinuses surrounded by chronic inflammation. Hair in sinuses was found in three quarters of the specimens examined. Examination showed that hair entered via one of the sinus openings created. Pits (defined as darker spots of varying width in the midline of the internatal cleft) were found to be indentations of the skin containing keratin plugs and debris. They may be isolated or connected with hair follicles. Pilonidal sinuses are chronic inflammatory processes of the skin caused by keratin plugs and debris clinically observed as pits, having penetrated the dermis. PMID- 7542010 TI - Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. AB - In humans and experimental animals the presence of bacterial lipopolysaccharide (endotoxin, LPS) signals the presence of gram-negative bacteria. Recognition of LPS triggers gene induction by myeloid and nonmyeloid lineage cells. These inducible genes encode proteins that include cytokines, adhesive proteins, and enzymes that produce low molecular weight proinflammatory mediators. Together the products of these inducible genes upregulate host defense systems that participate in eliminating the bacterial infection. Unfortunately, these same mediators contribute to a serious human disease known as septic shock. Considerable progress has been made during the past decade in determining the sources, identities, and sequence of release of these mediators. In contrast, until recently, marked gaps in our knowledge existed regarding the identity of the LPS receptor and intracellular signaling pathways responsible for LPS-induced cell activation. The discovery in 1986 of a plasma protein termed LPS binding protein (LBP) led to the discovery of unanticipated mechanisms of LPS-induced cell activation. CD14 was found as a soluble serum protein or as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells; it now occupies a key role in LPS-induced cell activation as we understand it today. Here we discuss how LBP enables LPS binding to CD14 and how complexes of LPS and soluble or GPI-anchored CD14 participate in cell activation. We also review the evidence supporting a model for a functional LPS receptor of myeloid cells, which is multimeric, comprised of GPI-anchored CD14 and a presently unidentified transmembrane protein that together bind LPS and initiate cell activation via kinase cascades. PMID- 7542012 TI - The identification of proliferating cell nuclear antigen (PCNA) on rat tissue cryosections and its application to double immunostaining with other markers. AB - Proliferating cell nuclear antigen (PCNA) in a useful marker for monitoring cell proliferation in most species. The immunostaining of PCNA on tissue cryosections, however, has been hampered by a loss of PCNA immunoreactivity during the staining process. The need for both identifying the actual phenotypes of proliferating cells and differentiating them from other tissue components prompted us to establish reliable techniques for PCNA immunostaining on cryosections and to apply these to double immunostainings with other markers. We tested various fixing conditions for rat tissue cryosections and the effect derived from unmasking with pepsin digestion to restore PCNA immunoreactivity after fixation. For single immunostainings, the unmasking was effective in most fixing conditions tested. Particularly, 4% paraformaldehyde/0.05% glutaraldehyde fixation followed by 0.001% pepsin digestion resulted in the strongest immunoreactivity for PCNA and the best morphology, and was the first choice for double immunostainings with relatively stable second antigens. Alternatively, periodate-lysine paraformaldehyde fixation was also applicable to other second antigens which are labile to the former treatment. These techniques can serve in the collection of important information from frozen tissues regarding the relationship between PCNA and other markers of interest which are usually susceptible to routine formalin and/or paraffin embedding. PMID- 7542013 TI - Nitric oxide synthase (NOS-I) in Leydig cells of the human testis. AB - By means of immunocytochemical methods, immunoreactivity for the brain isoform of nitric oxide synthase (NOS-I) was recognized in numerous Leydig cells of the human testis as well as in MA-10 tumor and TM3 non-tumor mouse Leydig cell lines. Within the Leydig cell cytoplasm, immunocytochemical results suggested the occurrence of factors known to activate NOS-I such as glutamate and aspartate, as well as molecules involved in the regulation of the NOS-I activity such as calmodulin and Ca2+/calmodulin-dependent protein kinase II. Leydig cells, Sertoli cells, some endothelial cells of the testis, MA-10- and TM3 mouse Leydig cell lines exhibited a relatively strong NADPH-diaphorase enzyme activity as well. Double sequential immunostainings provided evidence that NOS-like immunoreactivity of the testicular Leydig cells is colocalized with testosterone, calmodulin, aspartate, glutamate, and Ca2+/calmodulin-dependent protein kinase II. Sodium nitro-prusside treatment did not result in increased cGMP formation by MA-10- or TM3 mouse Leydig cells, suggesting that NO produced by these cells acts primarily in a paracrine fashion. The NO produced by NOS-I immunoreactive Leydig cells may act as a messenger: 1) between neighbouring NOS-I positive and/or negative Leydig cells as well as to mediate the action of numerous intracellular and extracellular neuroactive substances and growth factors; 2) between Leydig cells and the muscle cells or pericytes of blood vessels to regulate local blood flow and permeability; and 3) between Leydig cells and pertibular myofibroblasts to influence their contraction and the permeability of the lamina propria. PMID- 7542014 TI - Cytochemical localization of hyaluronic acid in human synovium with special reference to its possible process of degradation. AB - Fibroblast-like type B cells are known to produce hyaluronic acid (HA), but the process of its degradation remains unknown. In order to examine the possible route for the degradation of HA in normal human synovium, histochemical and immunohistochemical techniques were applied to the synovial tissue, using biotinylated HA binding region (HABR) and antibodies against CD44 and cathepsin B. Reaction products for HA and CD44 were detected on the cell surface of all synovial lining cells, while half of these lining cells contained intracellular stainings of HA and CD44. Electron microscopically, the lining cells containing intracellularly stained HA and CD44 extended cytoplasmic processes (type A cells), while the other lining cells possessed a smooth cell surface (type B cells). By light microscopic double staining, the intracellular stainings of HA and CD44 appeared co-localized in the cells immunopositive for cystatin beta, an endogenous cysteine proteinase inhibitor which has been shown to be localized in alveolar macrophages and osteoclasts. Moreover, these intracellular stainings of HA and CD44 were co-localized with immunodeposits for cathepsin B, a representative cysteine proteinase in lysosomes. In the extracellular staining of HA, dot-like reaction products appeared on fibrous structures with a periodicity of 41.7 nm. These results suggest that Type A cells in the normal human synovium participate in the degradation of HA by its CD44 mediated intake. Furthermore, HA may be closely associated with fibrous structures, probably type III collagen molecules. PMID- 7542015 TI - How the developing septo-preoptic medical basal hypothalamus stimulates the development of placode-derived LHRH neurons. AB - We examined the effects of the developing cerebral cortex (CC) and septo-preoptic medial basal hypothalamus (S-MBH) on the development of LHRH neurons in vitro. The serum-free basal culture medium (BCM) was supplemented with CC or S-MBH extracts prepared from 18.5-day-old embryos or from 2-day-old newborns, and the olfactory placode (NAP) of 12-day-old embryos was cultured. The migration of LHRH neurons was found on Day 3 in the cultures supplemented with the embryonic S-MBH extract (Group 3), where the cell development proceeded showing a numerical increase of the cells and the elongation of neurites. In cultures supplemented with the newborn S-MBH extract (Group 5), the cell development was less intensive in comparison with that of Group 3, while in cultures which had no brain extracts (Group 1), the neurons failed to survive a long term culture. The effects of the CC were less than of S-MBH extracts. Analysis of the protein composition of the extracts by electrophoretic and immunoblotting examinations demonstrated a protein spot of 70-kD in the embryonic S-MBH extract. Because the protein spot was identified to be alpha-fetoprotein (AFP), we further examined the effects of AFP. When the anti-AFP immunoglobulin was added to the Group 3 culture, the stimulative effects of the embryonal extract were inhibited, and the addition of AFP to Group 1 cultures did not show stimulative effects. We conclude that the developing S-MBH, the migrating target of LHRH neurons, contains some essential factors for the development of LHRH neurons, but further analysis is needed to determine the chemical natures of these factors. PMID- 7542016 TI - [Interferon therapy for demyelinating disease]. PMID- 7542017 TI - The methylation-acetylation method: an ultrastructural cytochemistry for nucleic acids compatible with immunogold studies. AB - Methylation-acetylation (MA) is an easy and reproducible ultrastructural cytochemical method which gives preferential contrast to nucleic acid containing structures. When performed en bloc before Lowicryl embedding it does not affect the main antigenic and chemical properties of the sample and is compatible with a large variety of modern immunogold methods permitting a better assignment of the labelling to the well-defined nuclear structures. DNA, RNA, and nuclear proteins, with different chemical nature, nuclear localization, and amount, can be immunolocalized on MA-treated samples. Ultrastructural in situ hybridization and other approaches for studying the functional regions of chromatin, the terminal deoxynucleotidyl transferase and the bromodeoxyuridine methods, are also compatible with the MA procedure. It can be also performed on ultrathin cryosections and Lowicryl sections. A much better visualization of the nuclear structures is obtained, enhancing the distinction between the nucleolar granular and dense fibrillar components. Moreover, the combination of the MA procedure with EDTA regressive staining gives preferential contrast to the RNA-rich structures. It is proposed as a useful approach which can be regularly used for in situ studies of the functional organization of the nucleus in both plant and animal cells. PMID- 7542018 TI - An ultrastructural characterization of in vitro-assembled hnRNP C protein-RNA complexes. AB - In mammalian cells approximately 700 nucleotide lengths of pre-mRNA are packaged during transcription by a unique group of abundant nuclear proteins to form a repeating array of regular ribonucleoprotein complexes termed 30-40S heterogeneous nuclear ribonucleoprotein particles (hnRNP particles). We have used electron microscopy to examine complexes that form when in vitro-transcribed RNA is bound by one of the purified native core-particle proteins which comprise the 40S monoparticle (the C protein tetramer). Negatively stained images of the C protein tetramer bound to particle-length RNA (700 nt) demonstrate that three tetramers bind each RNA molecule to form a stable closed triangular complex. The triangular complexes have an isosceles shape with a base of 18.0 nm and sides of 23.0 nm. When RNA molecules of 230 nt are used as substrates single tetramers bind to form complexes that appear as small rounded structures with an average diameter of 9.7 nm. Twice this length of RNA (456 nt) supports the assembly of mostly bilobed complexes that are 20.4 nm long and 11.8 nm wide. Images of the C protein-RNA complexes which assemble on 1400-nucleotide lengths of RNA (two particle lengths of RNA) clearly show complexes composed of two triangles while three-triangle complexes are seen when 2100-nt lengths of RNA are used as the assembly substrate. These ultrastructural results demonstrate that groups of three C protein tetramers combine with the length of RNA packaged in monoparticles to form a discreet triad structure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542019 TI - Cost analysis of palliative chemotherapy for patients with gastrointestinal malignancy. PMID- 7542020 TI - Granulocyte growth factors: achieving a consensus. AB - A consensus meeting held under the auspices of the European School of Oncology concluded that the use of granulocyte growth factors is definitely indicated, or acceptable given existing evidence, in the following circumstances: to alleviate congenital neutropenia; in the mobilisation of peripheral blood progenitor cells for autotransfusion; to encourage engraftment following bone marrow transplantation and in cases of failed engraftment; to support continuation of ganciclovir anti-CMV therapy in certain patients with AIDS, where the switch to foscarnet is contraindicated or where toxicity to foscarnet develops. It was also agreed that there is an overwhelming need for carefully controlled clinical trials in a wide range of indications in which growth factor use may improve outcome. In the majority of tumours, the possible benefit of dose optimisation and intensification, and therefore the role of growth factors in support of such measures has still to be defined. Extramedullary toxicities may in these instances become dose limiting. PMID- 7542021 TI - Cost-effectiveness of palliative chemotherapy in advanced gastrointestinal cancer. AB - BACKGROUND: Chemotherapy may relieve tumor-related symptoms, may improve quality of life and prolong survival in advanced gastrointestinal cancer. The extent of such improvements is unclear despite the extensive use of this treatment modality, and there are no studies concerning the economic cost of any gain achieved in the quantity and quality of life by chemotherapy. PATIENTS AND METHODS: Between January 1991 and May 1992, 61 patients with inoperable cancer (18 gastric, 22 pancreatic or biliary, and 21 colorectal) were randomized to either primary chemotherapy in addition to best supportive care or to best supportive care. Chemotherapy was allowed in the latter group if the supportive measures did not achieve palliation. All economic costs for medical care were prospectively recorded, and marginal cost-effectiveness analyses were performed. RESULTS: More patients in the primary chemotherapy group (19/33, 58%) had improved/prolonged high quality of life (QoL-patient, minimum duration 4 months) than in the best supportive care group (8/28, 29%, p < 0.05). Overall survival and quality-adjusted survival were significantly longer in the primary chemotherapy group (median 9 vs. 4 months, p < 0.05), and median 7 vs. 2 months, p < 0.05, respectively). When analysed by cancer site, survival was significantly prolonged in gastric cancer patients (median 10 vs. 4 months, p < 0.02), but not in colorectal (median 12 vs. 6 months, p = 0.1) and pancreatic-biliary cancer patients (median 8 vs. 5 months, p = 0.8). The average cost for all medical care was approximately 50% higher in the primary chemotherapy group, but the average cost per day was the same in the two groups. Hospitalization accounted for most of the costs in both groups. The incremental costs per gained year of life was SEK 166,400 ($21,300), per gained quality-adjusted year of life SEK 157,200 ($20,200), and per QoL-patient SEK 160,300 ($20,600). These costs were lower for gastric and colorectal cancer patients, and much higher for pancreatic-biliary cancer patients. CONCLUSIONS: The results of this study suggest that palliative chemotherapy is cost-effective in patients with advanced gastric and colorectal cancer. Knowledge about survival and quality of life benefits is still limited in patients suffering from gastric and pancreatic-biliary cancer. PMID- 7542022 TI - Doubts using family history, age, alpha-fetoprotein and total human chorionic gonadotrophin in screening for Down's syndrome. PMID- 7542023 TI - The sensitivity of ultrasound and serum alpha-fetoprotein in population-based antenatal screening for neural tube defects. South Australia 1986-1991. AB - OBJECTIVE: To determine the sensitivity of antenatal screening methods for neural tube defects in population-based screening in South Australia in 1986-1991, and whether ultrasound can replace serum alpha-fetoprotein screening in terms of achieving an equivalent level of sensitivity. DESIGN AND SETTING: Ascertainment of all births and terminations of pregnancy with neural tube defects from multiple sources for 1986-1991 in South Australia. Serum and amniotic fluid alpha fetoprotein results were obtained from the only laboratory performing the tests as a Statewide antenatal screening programme, and information on ultrasound screening from case notifications, hospital case records and medical practitioners who cared for the women. SUBJECTS: All 243 births and terminations of pregnancy with neural tube defects in South Australia in 1986-1991. MAIN OUTCOME MEASURES: The sensitivity of individual screening methods and of all methods used, particularly for spina bifida. RESULTS: For pregnancies with neural tube defects screened by any method (serum alpha-fetoprotein, ultrasound or amniocentesis), 86% sensitivity was achieved. Ultrasound screening for anencephaly achieved 100% sensitivity even in low risk pregnancies, compared with 92% for serum alpha-fetoprotein. For spina bifida, the sensitivity of ultrasound screening increased with the level of risk in pregnancy: it was 60% in low risk pregnancies, which was equivalent to that of serum alpha-fetoprotein screening (64%); 89% in high risk pregnancies and 100% for women referred for confirmation of a suspected spina bifida by another ultrasonographer (chi 1(2) for trend = 23.49, P < 0.0001). Ultrasound screening in high risk pregnancies for spina bifida achieved higher sensitivity in teaching hospitals compared with other ultrasound services in the State (97% vs 65%), but sensitivity was equivalent for low risk pregnancies. It is estimated that, had the serum screening programme not been in place, the level of sensitivity achieved for spina bifida by ultrasound and amniocentesis would have been 62% compared with the actual situation of 76% with the programme in existence, a difference of nearly 15% (95% CI 2.5 to 26.7) (chi 1(2) = 5.45, P = 0.02). CONCLUSIONS: Antenatal screening for neural tube defects in South Australia achieved a higher level of sensitivity with the maternal serum alpha-fetoprotein programme in place. We conclude that the serum screening programme should continue in South Australia pending a significant improvement in the sensitivity of routine ultrasound screening for spina bifida. PMID- 7542025 TI - Liver tumors in neonates and very young infants: diagnostic pitfalls and therapeutic problems. AB - We report on 26 infants under 3 months of age with various liver tumors, who were treated in our hospital since 1977 and/or registered in the German Cooperative Pediatric Liver Tumor Study HB-89. 17 of these had an infantile hemangioendothelioma (inf HE), 7 a hepatoblastoma (HB), one a mesenchymal hamartoma (mes H) and one a neuroblastoma stage IV-S (Nbl). Polyhydramnios occurred in 5 cases of inf HE, in one associated with hydrops fetalis. This led to a preterm delivery of 4 infants. 7 infants with a large inf HE suffered from high output congestive heart insufficiency, 2 additionally from a Kasabach Merritt-syndrome. Serum alpha-fetoprotein was within normal range of age in all infants except in one HB patient. Neuron-specific enolase was clearly elevated in the Nbl patient and slightly above normal range in one inf HE and 3 HB patients. Other tumor markers and platelet counts were not indicative of diagnosis. Also imaging techniques as ultrasonography, CT and angiography were not always reliable in differentiating the tumors. In 2 cases the initial histological diagnosis of an HB had to be corrected for an inf HE. In 6 children the tumors were initially treated under a wrong assumption: 4 tumors as an HB instead of an inf HE, one as an inf HE instead of a Nbl IV-S and one as a focal nodular hyperplasia of the liver instead of an HB. 7 not resected inf HEs (Dehner type I) showed spontaneous regression after 6 months to 3 years. One inf HE type II relapsed twice after resection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542026 TI - An osmolyte effect on the heat capacity change for protein folding. AB - We have carried out a differential scanning calorimetry study into the pH effect on the thermal denaturation of ribonuclease A at several concentrations of the osmolyte sarcosine. In order to properly analyze these data, we have elaborated the thermodynamic theory of the linkage between temperature, cosolvent, and pH effects. The denaturation heat capacity increases with sarcosine concentration. The effects of temperature and sarcosine concentration on the denaturation enthalpy and entropy values are well described by convergence equations, with convergence temperatures of around 100 degrees C for the enthalpy and around 112 degrees C for the entropy; we suggest that these effects might be related to a solvent-induced alteration of the apolar-group-hydration contribution to the folding thermodynamics. From our data, we estimate that about 70 extra molecules of water are thermodynamically bound upon ribonuclease denaturation in diluted aqueous solutions of sarcosine; this number is 6-9 times smaller than that predicted on the basis of the following two premises: (a) the osmolyte is strongly excluded from the surface of both the native and the denatured protein and (b) the denatured state is a fully solvated chain. We suggest that at least one of these two premises does not hold. We briefly comment on the potential use of cosolvent effects on thermal denaturation to evaluate the degree of hydration of denatured proteins (thus providing an independent measure of the consequence of their possible residual structure) and, also, on the possibility of finding substances that are more efficient protein stabilizers than known osmolytes are. PMID- 7542027 TI - Use of resonance energy transfer to determine the proximity of the guanine nucleotide binding site of transducin relative to a conformationally-sensitive site on the gamma subunit of the cyclic GMP phosphodiesterase. AB - In this work, we have used resonance energy transfer to determine the relative positions of a reactive cysteine residue on the gamma subunit of the retinal cyclic GMP phosphodiesterase (gamma PDE) and a reactive lysine residue on the alpha subunit of transducin (alpha T). The single cysteine residue on gamma PDE (residue 68) is located at a site that is sensitive to the binding of both the inactive and active forms of alpha T. This is demonstrated by the finding that the addition of an alpha T-GDP complex to a gamma PDE subunit labeled with the environmentally-sensitive probe 2-(4-maleimidoanilino)naphthalene-6-sulfonate (MIANS) results in an enhancement in the MIANS fluorescence. The alpha TGDP induced fluorescence enhancement is dose-dependent and yields an apparent Kd value of approximately 3 microM. Activation of alpha TGDP by aluminum fluoride, when bound to the MIANS-labeled gamma PDE (M-gamma PDE), then results in a quenching of the MIANS fluorescence. The aluminum fluoride-induced change in M gamma PDE fluorescence occurs on a time scale identical to that observed for changes in the intrinsic alpha T fluorescence that correspond to activating conformational changes in the alpha T "switch II" region. These results suggest that the induction of the activated state of the alpha T subunit results in a change in conformation close to cysteine 68 in gamma PDE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542024 TI - Lectin and immunohistochemical comparison of glycoconjugates in the conjunctiva of patients with and without exfoliation syndrome. AB - AIMS--The study was carried out to search for labelling similar to that of intraocular exfoliation material in the conjunctiva by light microscopy using lectin and immunohistochemistry. METHODS--Ten formalin fixed and paraffin embedded conjunctival biopsy specimens both from patients with and without exfoliation syndrome were studied with a panel of 11 lectins and with three monoclonal antibodies to the HNK-1 carbohydrate epitope, all of which react with intraocular exfoliation material. RESULTS--The lectin binding profile was essentially the same in specimens from patients with and without exfoliation syndrome. The superficial epithelium reacted similarly with Phaseolus vulgaris (PHA-E), Caragana arborescens (CAA), Helix pomatia (HPA), concanavalin A (ConA), and wheat germ (WGA) agglutinins. Binding was also detected with peanut (PNA) and Bauhinia purpurea (BPA) agglutinins, particularly in patients with exfoliation. The basement membrane generally reacted with Ricinus communis (RCA-I), PHA-E, Vicia villosa (VVA), ConA, and Lens culinaris (LCA) agglutinins. The stroma was weakly labelled with RCA-I, PHA-E, ConA, and LCA. Lectin binding to the vascular endothelium was moderate with RCA-I, PHA-E, CAA, ConA, LCA, and WGA. Inconsistent labelling was also detected with PNA, BPA, and Erythrina cristagalli agglutinin (ECA). The subendothelial region reacted weakly but consistently with PHA-E, ConA, and LCA, and inconsistently with PNA. Pretreatment with neuraminidase did not change that pattern. Antibodies to the HNK-1 epitope reacted only with myelinated stromal nerve branches. CONCLUSION--No evidence of abnormal deposits in any specimen was found. The carbohydrate composition of intraocular exfoliation material may differ from that of exfoliation-like fibres often detected in the conjunctiva by electron microscopy. PMID- 7542028 TI - Alteration of the substrate and inhibitor specificities of blood coagulation factor VIIa: importance of amino acid residue K192. AB - Initiation of blood coagulation occurs when the plasma serine protease factor VIIa (fVIIa) binds to its cell-surface receptor/cofactor, tissue factor (TF). This binding interaction mediates a large enhancement in both the proteolytic activity and the amidolytic activity (hydrolysis of small peptidylamide substrates) of fVIIa. This necessitates local changes in the catalytic center of fVIIa of which little is understood. Studies with thrombin and activated protein C have demonstrated that residue E192 (chymotrypsinogen numbering system) near the active site of these proteases is an important determinant for substrate and inhibitor specificity. By homology, residue 192 in fVIIa is K, bringing into question the potential role of this residue in fVIIa. We have prepared two mutants of fVIIa in which K192 has been replaced by either Q (as in factors IX and X) or E (as in thrombin). Both mutants were found to be defective in clotting: fVIIK 192Q was 44% active, while fVIIK192E was completely ineffective. This defect was attributable to proportional decreases in specificity constants for activation of factor X. Although both mutant enzymes were catalytically competent with respect to amidolytic activity, the selectivity of fVIIaK192E was greatly altered. Inhibition of both mutants by the TF pathway inhibitor (TFPI) and bovine pancreatic trypsin inhibitor (BPTI) was also drastically altered. Neither mutant was inhibited by TFPI, while fVIIaK192Q was inhibited by BPTI better than wild-type fVIIa. In contrast, fVIIaK192E was poorly inhibited by BPTI and made more refractory to inhibition when bound to TF. These results suggest a potential role for K192 in governing the substrate and inhibitor specificities of fVIIa. PMID- 7542029 TI - Adolescent readers' responses to the booklet on sex. AB - PURPOSE: To investigate the reception and the readers' reactions to a booklet about sex, which is mailed annually to all 16-year old Finnish adolescents. METHODS: Eight hundred youngsters were randomly selected to the study. A questionnaire was sent to them six weeks after the mailing of the booklet, The Sextiin. After three rounds, a response rate of 65% (N = 521) was achieved. RESULTS: The booklet reached 97% of the respondents, of whom 72% read all of it. The respondents' attitudes towards and reactions to the Sextiin were mainly positive. They reacted least positively to the articles on homosexuality and masturbation. Twelve percent of the respondents indicated that they opened the condom package that was attached to the Sextiin. CONCLUSIONS: The study shows that the campaign reached its target group well. As the adolescents' reactions to the Sextiin proved to be mainly positive, a claim for further use of the mass media for educational purposes is justified. PMID- 7542030 TI - Dose-dependent elevation of cyclic AMP, activation of glycogen phosphorylase, and release of lactate by amylin in rat skeletal muscle. AB - We report here our investigation of the role of cyclic AMP (cAMP) in amylin signal transduction in isolated strips of soleus muscle. Rat amylin, at 100 nM, increased cAMP levels, from 0.431 +/- 0.047 to a peak of 1.24 +/- 0.01 pmol cAMP/mg wet wt. after 5 min, in the absence of added phosphodiesterase inhibitor. The EC50 of the response was 0.48 nM (+/- 0.12 log units) in the absence of insulin and 0.3 nM (+/- 0.18 log units) in the presence of 7.1 nM insulin. The response seen with a maximally effective concentration of amylin (10 nM) was similar to that seen with a maximally effective concentration of epinephrine (1 microM) under the same conditions. Consistent with the observed rise in cAMP there was an increase in glycogen phosphorylase a (EC50 2.2 nM +/- 0.25 log units), decreased glycogen content (EC50 0.9 nM +/- 0.22 log units) and enhanced production of lactate (EC50 1.5 nM +/- 0.33 log units). These data support the concept that amylin promotes glycogenolysis in skeletal muscle and enhances production of lactate through glycolysis as a result of activation of Gs coupled receptors, stimulation of adenylate cyclase, elevation of cAMP levels and activation of glycogen phosphorylase. PMID- 7542032 TI - Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation. AB - The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor IX (FIX) and other cDNAs (100 and 1,000-fold lower, respectively), when produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII cDNA has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state RNA levels from a retroviral vector by 30- to 100-fold. An analysis of RNA degradation indicated that the FVIII INS vector RNA is relatively stable. Nuclear run-on experiments with the FVIII INS vector demonstrated a low signal for FVIII, in contrast to the high signal for a FIX vector. The low signal for FVIII INS was not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. We show that the inhibitory effect of FVIII INS is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII INS sequence increased when it was moved 1 kb further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the CFTR cDNA. The CFTR retroviral vector produced 1,000-fold decreased steady-state RNA levels, compared to the parent vector. Nuclear run-on analysis with the CFTR vector revealed a block to transcriptional elongation within the CFTR cDNA. The presence of blocks to transcriptional elongation within the FVIII and CFTR cDNAs complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for human gene therapy. PMID- 7542031 TI - In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. AB - Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl- secretion and an accelerated rate of basal Na+ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl secretion, and the rate of Na+ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients. PMID- 7542033 TI - Prostate cancer screening (United States). AB - In 1995, there will be 244,000 new cases of prostate cancer, and 40,400 deaths from prostate cancer, among men in the United States. The American Cancer Society reports that the incidence rate of prostate cancer is increasing at an accelerated pace, and was 21 percent higher in 1994 than in 1993. The major reason for this steep rise is likely to be due to increased popularity of prostate cancer screening which, by identifying latent, asymptomatic cases, may convert them into clinical cases. Is screening--an important means of cancer control for many sites--a reasonable approach for prostate cancer control? The answer is not straightforward because prostate cancer is not one, but three diseases: a latent form which will cause no harm; a progressive form which will become symptomatic and can kill; and a rapidly progressive form so malignant that it is likely to kill, whether detected early or late. Screen-detection may be worthwhile only for the second form, as tumors of the first form need never be detected, and tumors of the third form progress so rapidly that timely screen detection is nearly impossible and, if accomplished, may be valueless. As there is no way to differentiate among the three diseases when screening, the possible deleterious effects of screen-detection must be weighed against the benefits. PMID- 7542034 TI - Hydration of nucleic acid fragments: comparison of theory and experiment for high resolution crystal structures of RNA, DNA, and DNA-drug complexes. AB - A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution. PMID- 7542036 TI - CGRP in peripherally axotomized mesencephalic trigeminal neurones of the rat. AB - Immunoreactivities for substance P (SP-ir) and calcitonin gene-related peptide (CGRP-ir) were examined in neurones in the mesencephalic trigeminal tract nucleus of nerve-injured rats. One week following unilateral transection of the trigeminal root, the masseteric nerve (MN) or the inferior alveolar nerve (IAN), CGRP-ir but not SP-ir was detected in these neurones bilaterally with ipsilateral dominance. Most of the CGRP-ir (immunoreactive) neurones were retrogradely labelled when fluorogold (FG) had been applied to the proximal stump of the MN or the IAN. Sham surgery including insult to the masseter muscle induced CGRP-ir while skin incision did not. PMID- 7542037 TI - Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors. AB - During development, the regulation of expression of the Trk family of tyrosine kinase receptors plays an important role in defining the cellular responses to neurotrophin action. We report here that neurotrophin receptors are differentially expressed in distinct populations of retinoic acid-differentiated P19 cells. TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor, and LNGFR, the low-affinity receptor for all neutrophins, are preferentially expressed in P19-derived neurones. In contrast, retinoic acid induces the expression of TrkA, the high-affinity receptor for nerve growth factor, and of a non-catalytic form of TrkB, in non-neural subsets of differentiated cells. We propose P19 cells as a model system to study the mechanisms controlling the expression of neurotrophin receptors and the responsiveness of developing neurones to a specific neurotrophin. PMID- 7542035 TI - Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers. AB - The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions. PMID- 7542039 TI - The role of nitric oxide in modulating brain activity and blood flow during seizure. AB - The role played by nitric oxide (NO) in modulating seizure activity and cerebral blood flow (CBF) during seizures was investigated in rats. Seizures were induced with bicuculline (a GABA antagonist, 1.2 mg kg-1, i.v.). Each animal was subjected to an initial bicuculline-induced seizure followed by treatment with either L-nitroarginine (L-NA, a NO synthase inhibitor) or its less active enantiomer D-NA as a 50 mg kg-1 bolus followed by an infusion of 1 mg kg-1 min-1. The animals then received a second bicuculline treatment. Seizure duration was monitored using EEG and CBF was measured with laser-Doppler. There was no difference in seizure duration before or after D-NA administration. Seizure duration doubled from (6 +/- 1 to 12 +/- 2 min p < 0.05) following inhibition of NO synthase with L-NA. The increase in CBF that accompanied the seizure activity paralleled the seizure duration. Our data support the concept that (1) NO acts as an endogenous anticonvulsant, with seizure duration doubling when NO synthase is acutely inhibited, and (2) that NO is not the messenger that couples CBF to metabolism during bicuculline-induced seizures. PMID- 7542038 TI - Hyperekplexia mutations of the glycine receptor unmask the inhibitory subsite for beta-amino-acids. AB - beta-Alanine and taurine are agonists of the glycine receptor (GlyR) which, at low concentrations, antagonize the action of the principal agonist glycine. We analysed the potency of these ligands on alpha 1 subunits mutated at residue R271. GlyRs formed from alpha 1R271K subunits showed a reduction of beta-alanine and taurine affinities and maximal inducible currents; the mutants alpha 1R271Q and alpha 1R271L associated with human hyperekplexia gave no responses to these ligands. Inhibition of glycine-evoked currents by beta-alanine and taurine, however, was similar for all mutant GlyRs. These data are consistent with the existence of two subdomains within the ligand binding region of the GlyR, an agonistic one, which depends on arginine 271, and an antagonistic subsite, which is not connected to this residue. PMID- 7542040 TI - Characterization of Japanese families with early-onset type 2 (non-insulin dependent) diabetes mellitus and screening for mutations in the glucokinase and mitochondrial tRNA(Leu(UUR)) genes. AB - Genetic linkage studies of families with early-onset type 2 diabetes have facilitated the identification of diabetes-susceptibility genes. In order to assess the feasibility of using linkage approaches to identify genes responsible for the development of type 2 diabetes in Japanese subjects, we examined our clinical records for multigenerational families suitable for genetic studies. We identified 16 families in which at least one subject was diagnosed with type 2 diabetes before 25 years of age. Seven of these families had a pattern of inheritance consistent with a diagnosis of maturity-onset diabetes of the young (MODY) and nine families showed a complex pattern of inheritance of type 2 diabetes with transmission of diabetes-susceptibility genes from both parents. The glucokinase and mitochondrial tRNA(Leu(UUR)) genes were screened for mutations in at least one affected subject from each family in order to assess the contribution of mutations in these genes to the development of the diabetes. No mutations were found, which suggests that the diabetes in these families resulted from mutations in other genes. PMID- 7542041 TI - Impairment of the priming effect of glucose on insulin secretion from isolated islets of aging rats. AB - The time-dependent potentiation (TDP) of insulin release or priming effect exerted by glucose was evaluated in the islets of Langerhans of mature and old rats. Islets isolated from 12- and 26-month-old male Sprague-Dawley rats and incubated for two consecutive 60-min periods in the presence of various stimulating agents were unable to enhance their insulin responsiveness significantly during the second incubation period and showed other abnormalities in their sensitivity to secretagogues compared with islets from 3-month-old animals. The priming action of glucose plus arginine or isobutylmethylxanthine (IBMX) was not observed in islets from 12-month-old rats, but surprisingly, islets from senescent rats showed a restoration of the beta-cell memory in the presence of IBMX. Interestingly, the islets isolated from 2-month-old animals previously exposed to an intravenous glucose load in vivo released approximately twice as much insulin as the islets taken from fed rats not subjected to the load. This potentiation exerted by the intravenous glucose administration was reduced but not abolished in the islets of glucose-intolerant, 12-month-old rats. In conclusion, the glucose TDP of insulin secretion is impaired in islets of mature and old rats, confirming an early loss of sensitivity of beta-cells to secretagogues during aging. PMID- 7542042 TI - Lectin binding properties of bovine resting cartilage. AB - The aim of this study was to evaluate the differential localisation of glycoconjugates of bovine hyaline cartilage matrix by lectin histochemistry, to compare the results of lectin histochemistry with those that can be obtained in the same tissue with PAS and alcian blue. Frozen and paraffin sections were stained with HE, PAS and alcian blue (pH 1.8). Alcian blue staining was carried out also after 1 and 24 hour digestion with bovine testicular hyaluronidase. Peroxidase conjugated WGA, PNA and RS lectins were tested on all sections before and after 1 hour digestion with bovine testicular hyaluronidase. The results show that all the lectins used in this study react with sugars linked to proteoglycans of territorial matrix, the reaction being increased in territorial, and induced in interterritorial matrix by 1 hour hyaluronidase digestion. Alcian blue at pH 1.8 and PAS were complementary, the former staining territorial, and the latter interterritorial matrix. After 1 hour hyaluronidase digestion, alcian blue stained also the interterritorial matrix. These results suggest that lectins react with low molecular weight proteoglycans and that short hyaluronidase digestion causes depolymerization of high molecular weight proteoglycans without loss of their glucidic components, allowing: a) penetration of alcian blue molecules into the macromolecular proteoglycan network; b) an increase of sugar residuals available for lectin histochemistry. Lectin histochemistry can be useful for differential localisation of glycoconjugates in bovine cartilage, especially if associated with short hyaluronidase digestion and conventional histochemical techniques. PMID- 7542043 TI - The endocrine pancreas of lacertids: an immunocytochemical study of the genera Pedioplanis and Meroles. AB - The endocrine pancreas from 2 genera of lacertid lizards (Pedioplanis and Meroles) was investigated immunocytochemically for the presence of immunoreactivity to mammalian antisera to insulin (I), glucagon (G), pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide tyrosine (NPY), somatostatin 14 (SRIF 14) and somatostatin 28 (SRIF 28), pancreastatin (Pst), galanin (Gl), oxytocin (OT). Cells immunoreactive (IR) to all the antisera used, and nerve fibers IR only to anti-galanin were found. Moreover, three types of colocalized immunoreactivities were detected: type 1 (PP/PYY/NPY), type 2 (G/PP/PYY/NPY), and type 3 (G/PYY/NPY/Pst). PMID- 7542045 TI - Detection of ribonuclease H-generated mRNA fragments in human leukemia cells following reversible membrane permeabilization in the presence of antisense oligodeoxynucleotides. AB - The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjected Xenopus systems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells. PMID- 7542044 TI - Linkage of LHB and MAG to GPI on sheep chromosome 14. PMID- 7542046 TI - (2'-5')-Oligo-3'-deoxynucleotides: selective binding to single-stranded RNA but not DNA. AB - Oligodeoxynucleotides with (2'-5') internucleotide linkages have been synthesized on a solid support via standard cyanoethyl phosphoramidite chemistry. This simple change in the oligonucleotide bond connectivity led to unique properties. UV melting temperature experiments indicate that the (2'-5')-oligo-3' deoxyadenylates, (2'-5')-3'-dA8 and (2'-5')-3'-dA8(s) phosphorothioate, hybridize selectively to single-stranded RNA but not DNA. The complex (2'-5')-3'-dA8:poly (U) (Tm = 32 degrees C) was nearly as stable as the natural (3'-5')-2'-dA8 and poly (U) (Tm = 33 degrees C) in 130 mM NaCl, and 10 mM phosphate buffer (pH 7.5). However, no association was observed upon mixing (2'-5')-3'-dA8 and poly (dT). The (2'-5') linkages also confer greater resistance to exo- and endonucleolytic degradation compared with (3'-5')-linked oligomers. The rate of degradation of (2'-5')-3'-dA8 was almost four times less than that of (3'-5')-2'-dA8 in cell culture medium containing 10% heat-inactivated fetal calf serum. An increase in stability for (2'-5')-3'-dA8 against endonuclease activity was observed in both cytoplasmic and nuclear extracts. The nucleic acid selectivity of (2'-5')-oligo 3'-deoxynucleotides may represent an important design feature to improve the efficacy of antisense oligonucleotides. PMID- 7542047 TI - Inhibition of protein-tyrosine kinase activity in intact cells by the aptameric action of oligodeoxynucleotides. AB - Direct interaction of oligodeoxynucleotides (ODNs) with proteins represents one of the nonantisense-mediated effects of ODNs. Phosphorothioate-capped ODNs have been shown to inhibit directly the in vitro kinase activity of the chronic myelogenous leukemia-associated protein-tyrosine kinase p210bcr-abl. In this study we have determined the efficacy of this aptameric ODN in a cellular system using the K562 chronic myelogenous leukemia-derived cell line. Significant effects upon cellular phosphotyrosine content, as well as cellular growth in soft agar, are observed. These effects are sequence specific and are not mediated through changes in p210bcr-abl protein levels. Additional ODNs are described that also reduce cellular phosphotyrosine levels and inhibit growth in soft agar but do not inhibit p210bcr-abl kinase activity in vitro. PMID- 7542049 TI - The ins and outs of RNA nucleocytoplasmic transport. AB - Nucleocytoplasmic transport of RNA is an obligatory step in gene expression and may also be a target for regulation. The cellular machinery has the capacity to export a myriad of RNA transcripts that differ significantly in sequence and structure. The molecular mechanisms of RNA transport are (as yet) largely unknown. Thus, biochemical and genetic approaches are being used to identify cellular factors that mediate this process. Major advances over the past year include the cloning of genes for nuclear pore complex components and isolation of yeast mutants that harbor specific defects in RNA export. PMID- 7542048 TI - Helix-stabilizing compounds CC-1065 and U-71,184 bind to RNA-DNA and DNA-DNA duplexes containing modified internucleotide linkages and stabilize duplexes against thermal melting. AB - CC-1065 and U-71,184 bind and hyperstabilize DNA duplexes, but little is known about their effects on nucleic acid duplexes of different structure. A 20 mer DNA sequence (5'-TTACTTCAGTTATGAGACCA) containing a drug binding sequence (5'-AGTTA) was selected as the target sequence, and this was duplexed with complementary antisense sequences containing phosphodiester (PO), phosphorothioate (PS), and methylphosphonate (MP) bonds. The duplexes containing PO or PS bound 2 CC-1065 molecules per duplex, presumably at both the target site and at a lower affinity site (5'-AGTAA) on the antisense strand. The duplex containing MP bound only 1 CC 1065, and all duplexes bound only 1 U-71,184. Both CC-1065 and U-71,184 bound to 20 mer duplexes comprised of oligo(dA)-oligo(dT) (2.5 and 2 drugs per duplex, respectively) and poly(rA)-oligo(dT) (1 drug per 20 base pairs). CC-1065 also bound to duplexes between the PO- or PS-based antisense structures and a complementary synthetic 20 mer RNA sequence, with about 1 drug per duplex in each case. CC-1065 increased the Tm for the 20 mer DNA duplexes 17 to 29 degrees C, and the corresponding values for U-71,184 ranged from 7 to 19 degrees C. CC-1065 raised the Tm of oligo(dA)-oligo(dT) and poly(rA)-oligo(dT) 29 degrees C. U71,184 increased the Tm for oligo(dA)-oligo(dT) 30 degrees C but did not significantly elevate the Tm for the corresponding RNA-DNA duplex. The results show that CC 1065 and U-71,184 are capable of binding and stabilizing a variety of nucleic acid duplexes. These agents or their analogs may become useful ligands for antisense oligonucleotide applications. PMID- 7542050 TI - Reversible neutropenia during a cold: possible involvement of risperidone? A case report. AB - After 9 days of risperidone therapy (2-6 mg/day), a 24-year-old schizophrenic female patient developed a leucopenia with neutropenia < 1000/mm3. A few days after starting the neuroleptic treatment, she suffered from a cold, without fever. All immunological tests performed yielded negative results except for influenza B virus. The cessation of the risperidone therapy was followed within 48 h by a normalisation of the WBC differential count. Therefore, in the absence of a rechallenge with risperidone for ethical reasons, it cannot be excluded that risperidone represents at least a partial cause of the observed neutropenia. PMID- 7542052 TI - Fluvoxamine treatment of alcoholic amnestic disorder. AB - The serotonin uptake inhibitor fluvoxamine was assessed in treatment of alcohol induced Korsakoff's syndrome (KS) using fixed (4 weeks, 200 mg/day) or individualized (6 weeks, plasma concentration > or = 400 ng/ml) dosing in randomized placebo-controlled double-blind crossover studies. Cognitive functions and concentrations of the major cerebrospinal fluid (CSF) metabolites of serotonin (5-HIAA), norepinephrine (MHPG), and dopamine (HVA) were determined in abstinent, nondepressed KS patients (aged 45-75), at baseline and placebo (3-4 weeks), and after 3-4 (n = 10) or 6 (n = 4) weeks of fluvoxamine administration. Fluvoxamine decreased CSF 5-HIAA compared to placebo (P < 0.003) without consistent changes in HVA or MHPG. Reductions in 5-HIAA correlated with improvements on the Wechsler Memory Scale Memory Quotient (P < 0.05), independent of effects on attention/vigilance or Beck Depression Inventory scores. Reductions in 5-HIAA correlated with plasma fluvoxamine (P < 0.03) only for fluvoxamine concentrations below 450 ng/ml. These findings suggest improvement of memory consolidation and/or retrieval in patients with Korsakoff's syndrome by fluvoxamine via serotonergic mechanisms. PMID- 7542051 TI - Effect of clozapine and risperidone on 5-HT2 and D2-dopamine receptor binding in the post-mortem human brain. An autoradiographic study. AB - Effects of the antipsychotics risperidone and clozapine on 5-HT2 and D2-dopamine receptor binding were examined using [3H]N-methylspiperone ([3H]NMSP) and in vitro receptor autoradiography on human whole hemisphere cryosections. The 5-HT2 receptor antagonist ketanserin and the D2-dopamine receptor antagonist raclopride were used as references. [3H]NMSP binding was observed in caudate nucleus, putamen, and cerebral cortex indicating binding to D2-dopamine and 5-HT2 receptors. Risperidone and clozapine counteracted the binding to both receptor types. This was in contrast to raclopride, which selectively blocked the D2 dopamine receptors in the basal ganglia, and ketanserin, which selectively blocked the 5-HT2 receptors in the cerebral cortex. Risperidone (100 nM and 10 microM) blocked up to 90% of [3H]NMSP binding to both receptor types, whereas the blocking capacity of clozapine (10 microM) was lower (approximately 60%). The lack of total blockade of D2-dopamine receptors is in line with results obtained in with [11C]raclopride and positron emission tomography studies of clozapine treated human subjects. However, autoradiographic studies of clozapine competition of [3H]raclopride binding show total displacement of the binding at high clozapine concentrations, thus contradicting the PET results with [11C]raclopride, as well as the autoradiographic results obtained with [3H]NMSP. In conclusion it can be stated that pharmacological concentrations of the two drugs clozapine and risperidone block a large proportion of D2-dopamine receptors and 5-HT2 receptors in the human brain. Moreover, the study shows the usefulness of human whole hemisphere autoradiography for the study of interaction of drugs with different central neurotransmitter receptors. PMID- 7542053 TI - Hypofrontality does not occur with 6-hydroxydopamine lesions of the medial prefrontal cortex in rat brain. AB - This study examined the effect of lesions of dopamine (DA) nerve terminals the medial prefrontal cortex on local cerebral glucose utilization (LCGU) and dopamine metabolism in the rat brain. Bilateral 6-hydroxydopamine lesions were stereotaxically placed in the medial prefrontal cortex. Twenty-eight days after the lesion, concentrations of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were determined in eight brain regions with a high-performance liquid chromatographic assay. LCGU was assessed by [14C]2 deoxy-D-glucose autoradiography. The lesion produced a striking reduction in DA (to 6% of the control value), and a moderate reduction in DOPAC and HVA in the medial prefrontal cortex. The ratio of DOPAC to DA in the medial prefrontal cortex was significantly elevated in the 6-OHDA lesioned animals. In contrast to DA depletion, LCGU in the medial prefrontal cortex of the lesioned rats was unaltered when compared with the control. These findings suggest that decreased energy metabolism in the frontal cortex, i.e., hypofrontality, does not occur with decreased DA innervation of that site. PMID- 7542055 TI - Substance-P-induced histamine release from human nasal mucosa in vitro. AB - There is growing evidence that substance P (SP) is one of the main neuropeptides involved in neurogenic inflammation of the airways. However, a number of studies were not able to demonstrate histamine release from human mucosal mast cells after SP administration. Since cultures of isolated cell systems provide only a partial picture of the inflammatory processes in vivo, organ culture models promise to offer physiologically relevant assay systems for studying tissue interactions. Thus, we examined the influence of SP on histamine release and its morphological effects on human nasal mucosa using a previously described histoculture technique. Compared to controls, the histamine content in the culture bath was significantly elevated after SP stimulation (p < 0.05). Histomorphometry showed a decreased density of mast cells and an increased percentage of degranulated mast cells. These findings were dose and time dependent and support the hypothesis of a close interaction between C-sensory nerves and mast cells implying that these interactions are of pathogenetic importance in nasal mucosa inflammation. PMID- 7542054 TI - Proinflammatory cytokines: measurement in nasal secretion and induction of adhesion receptor expression. AB - We recently demonstrated that interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6 and IL-8 can be found in nasal secretions from allergic rhinitis patients under artificial and natural conditions. By ELISA measurements, significantly elevated baseline levels for IL-1 beta, IL-6 and IL-8 were found in seasonal allergic compared to control subjects. Within the first 2 h after nasal allergen challenge, IL-1 beta and TNF are secreted, whereas IL-6 and IL-8 showed a slow increase over 6-8 h. All cytokine levels returned to baseline within 24 h after exposure. Repeated measurements at 4-week intervals in perennial allergic rhinitis subjects (n = 27) showed significant correlations between IL-1 and IL-8, IL-6 and IL-8 and IL-6 and the symptom score (visual analogue scale). The IL-1 receptor antagonist IL-1ra was found in great molar excess in the secretions and correlated significantly with IL-8, but not IL-1 beta. In an in vitro assay using fresh nasal mucosa of grass-pollen-allergic subjects, we were able to demonstrate a strong and rapid induction of E-selectin adhesion receptor expression on endothelial cells by allergen, IL-1 beta and TNF. The adhesion receptor expression was markedly inhibited by soluble IL-1 receptors, sTNF-R and IL-1ra. These data indicate a key role for inflammatory cytokines in the regulation of allergic inflammation. PMID- 7542056 TI - Human peripheral blood eosinophils express and release interleukin-8. AB - Eosinophils can synthesize various cytokines. We demonstrate that interleukin-8 (IL-8) mRNA and protein are constitutively expressed by freshly isolated resting human eosinophils. Most of the patients with bronchial asthma or atopic dermatitis show evidence for up-regulated IL-8 protein expression in eosinophils but not in neutrophils, suggesting that an eosinophil-specific cytokine may act in these patients. To investigate whether the intracellular IL-8 can be released, eosinophils were stimulated by different cytokines and platelet-activating factor (PAF). Priming with granulocyte/macrophage-colory-stimulating factor and a subsequent 25-min stimulation with RANTES or PAF resulted in release of IL-8 from highly purified human eosinophils in vitro. PMID- 7542057 TI - Role of heat shock protein 65/60 in the pathogenesis of atherosclerosis. AB - Investigations in rabbits and humans have provided experimental evidence that autoimmune reactions play a major role in the initial stages of the development of atherosclerosis. These involve the infiltration of the arterial intima with T cells reacting with heat shock protein (hsp) 65/60 and the occurrence of anti-hsp 65/60 antibodies. This early immunologically mediated stage of atherosclerosis is still reversible but if additional risk factors, such as high cholesterol levels, come into effect, severe mostly irreversible lesions develop. PMID- 7542058 TI - Surface expression of IgE receptors, IgE occupancy and histamine releasability of mast cells: influence of genetic and T cell factors. AB - IgE receptor density and IgE occupancy were quantified on individual peritoneal mast cells using cytofluorometry. The secretory capacity of the mast cells was analysed in terms of histamine release as a function of anti-IgE challenge. Histamine releasability was found to be largely a function of the number of IgE molecules on the mast cells. The genetic background influenced IgE receptor expression but also, independently, histamine releasability. Both IgE receptor density and IgE occupancy on the mast cells appeared to be T cell independent under normal conditions. Up-regulated IgE receptor expression and an increased IgE occupancy on the mast cells could be obtained in athymic rats in response to a Nippostrongylus brasiliensis infection, although it was not as pronounced as in euthymic rats. The secretory response was diminished in athymic rats, suggesting that T cell factor(s) enhance secretory signal transduction in the mast cells in vivo. PMID- 7542060 TI - Human keratinocytes release mast cell differentiation factors other than stem cell factor. AB - Besides stem cell factor (SCF), additional fibroblast-derived mast cell growth factors have previously been described. Since keratinocytes have also been shown to produce SCF, we have studied the ability of culture supernatants from the human HaCaT keratinocyte cell line to induce SCF-independent mast cell differentiation. The immature human mast cells of the HMC-1 line which express a mutant continuously activated SCF receptor were used as model target cells. Culture supernatants from differentiating keratinocytes (at day 11 of culture), and far less so those from proliferating keratinocytes (day 4 of culture), caused a marked, dose-dependent increase of histamine and tryptase in HMC-1 cells. This suggests that human HaCaT keratinocytes release mast cell differentiation factors other than SCF, to a degree related to their state of differentiation. PMID- 7542059 TI - The role of c-Kit and its ligand, stem cell factor, in mast cell apoptosis. AB - The regulation of tissue mast cell number depends both on the rate of production of mast cell precursors and the length of survival of mature mast cells within tissues. Once mast cell precursors target to tissues, their survival may largely be dependent upon the local production of stem cell factor (SCF). Withdrawal of interleukin (IL)-3 results in mast cell apoptosis. The apoptotic changes following IL-3 deprivation are prevented by the addition of SCF which exerts its rescue effect upon interaction with its c-Kit tyrosine kinase receptor. Mast cells undergo apoptosis on withdrawal of IL-3 coincident with a decrease in endogenous bcl-2 mRNA; however, SCF does not induce expression of bcl-2 when added to these cells. When overexpressed, bcl-2 prolongs survival of bcl-2 transfected mast cells following IL-3 deprivation. Transforming growth factor beta was found to specifically prevent this SCF-mediated rescue from apoptosis, probably by down-regulating the expression of c-Kit. Thus, microenvironmental factors play an important role in regulating mast cell numbers by effecting survival in the periphery. PMID- 7542061 TI - Basophil and eosinophil activation by CC chemokines. AB - Recent studies have shown that CC chemokines act on monocytes, lymphocytes, eosinophils and basophils, but not neutrophils, with distinct target cell selectivities, possibly explaining the selective attraction and activation of these cell types in different types of chronic inflammation and allergic disease. Functional and desensitization studies on basophils and eosinophils indicate the expression of at least three distinct G-protein-coupled CC chemokine receptors (three on basophils and two on eosinophils), which exert partially selective and partially overlapping ligand specificities and also appear to mediate distinct functions despite similar signal transduction pathways. Functional studies with all known six human CC chemokines show that each cytokine has a distinct spectrum of bioactivities and target cell profile. Interestingly, overall sequence homologies between the chemokines are not predictive for the cell function or cell type that a particular chemokine will preferentially activate, and thus discrete sequence motifs may be important for activating the different CC chemokine receptors. Using different chemokine mutants and hybrids between chemokines, the functional importance of selected individual amino acids and short motifs are now being analysed. These structure-function studies could also lead to antagonists that have more disease-selective anti-inflammatory properties than currently available drugs. PMID- 7542062 TI - Regulation of integrin-dependent release in human lung mast cells and basophils. AB - The interaction of cells with surfaces or components of the extracellular matrix alters cell responses and is regulated by integrins on the cell surface. We have used monoclonal antibodies to CD29 and CD49d followed by an F(ab)2 fragment of rabbit anti-mouse IgG1 to cross-link the integrins on the surface of human lung mast cells and basophils. We found that cross-linking either CD29 or CD49d failed to initiate mediator release from the basophils of non-atopic and atopic donors [histamine release (HR) = 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, NS]. In contrast we found that clustering CD29 caused significant HR from the basophils of asthmatic donors (HR = 21 +/- 5%, n = 10, p < 0.005). Clustering of CD49d also caused significant degranulation in the same donors (HR = 9 +/- 3%, n = 10, p < 0.11). Incubating the basophils of these asthmatic donors with a synthetic RGD peptide significantly reduced CD29- and CD49d-induced histamine release. CS-1 peptide was also found to inhibit CD29-induced histamine release but had no significant effect on CD49d-induced histamine release. The tyrosine kinase inhibitors, genistein and piceatannol, completely ablated CD29- and CD49d induced degranulation. In summary, we have shown that cross-linking integrins can initiate mediator release from the basophils of asthmatic patients and that this appears to involve recognition of RGD and activation of tyrosine kinase. PMID- 7542063 TI - Effects of interleukin-4 or stem cell factor on mast cell mediator release and cytokine gene expression. AB - We have investigated the capacity of interleukin (IL)-4 or stem cell factor (SCF) to induce direct mediator release from rodent peritoneal mast cells, and also to induce or regulate cytokine gene expression in the human HMC-1 mast cell line. SCF, but not IL-4, induced low levels of serotonin release from mouse or rat peritoneal mast cells; rat mast cells acquired enhanced responsiveness to SCF during culture. IL-4, but not SCF, enhanced ionomycin-induced transcription and secretion of several genes, including the cytokines IL-3, IL-4, granulocyte/macrophage-colony-stimulating factor, IL-8 and the receptor for IL-6 in the human HMC-1 mast cell line. PMID- 7542064 TI - Interferon and antiallergic drug regulation of histamine and tumor necrosis factor-alpha in rat mast cell subsets. AB - We have further characterized the heterogeneity of mast cells (MCs) by comparing the ability of rat peritoneal MCs (PMCs) and intestinal mucosal MCs (IMMCs) to produce tumor necrosis factor (TNF)-alpha and by investigating its regulation by interferon (IFN) and the antiallergic drugs nedocromil sodium (NED) and sodium cromoglycate (SCG). Although IMMCs store less TNF-alpha than PMCs, they produced comparable amounts of TNF-alpha in cytotoxic assays. Just as SCG and NED inhibit histamine secretion from PMCs but not IMMCs, IFN exhibited a similar differential effect on histamine release from these cells. However, SCG, NED, and IFN inhibit TNF-alpha-dependent cytotoxicity by both PMCs and IMMCs and reduce the steady state levels of mRNA for TNF-alpha in PMCs. Thus, the modulation of MC mediator release depends upon the MC population and mediator studied. The inhibitory effect of SCG and NED on TNF-alpha release from MCs may explain some of their anti-inflammatory and therapeutic effects. PMID- 7542065 TI - Multiple cytokine mRNA expression in human mast cells stimulated via Fc epsilon RI. AB - Cross-linkage of Fc epsilon RI on human lung mast cells purified by affinity magnetic selection with monoclonal antibody YB5.B8 against c-kit (purity > 90%) expressed mRNA for multiple cytokines. There was no constitutive expression of interleukin (IL)-4 mRNA. Mast cell stimulation with anti-IgE induced IL-4 mRNA expression which appeared maximal at 2 h and waned slowly over the next 24 h. IL 5, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha mRNA were constitutively expressed. Mast cell activation with anti-IgE led to an increase of IL-5 and TNF alpha mRNA signals within 2 h and which persisted for at least 24-48 h. On the other hand, IL-6 and IL-8 mRNA expression were not affected by anti-IgE challenge. PMID- 7542066 TI - Role of tryptase, eosinophil cationic protein and histamine in immediate allergic reactions to drugs. AB - 20 subjects (8 men, 12 women) with anaphylactic or urticarial reactions after drug intake were evaluated. Tryptase (TRY) and eosinophil cationic protein (ECP) were determined in serum 2 and 24 h after the allergic episode. Histamine was determined in three sequential urine samples. Results indicated that in the group of subjects evaluated, TRY and histamine were elevated, although in not all cases for both markers. With ECP, no clear association was found either at 2 or 24 h after the allergic episode. These results suggested that mast cells participated in the adverse reactions, but a role for eosinophils could not be established. Further studies need to be undertaken to establish the participation of these cells in immediate reactions to drugs. PMID- 7542067 TI - Prevention by monovalent haptens of IgE-dependent leucocyte histamine release to muscle relaxants. AB - The cross-reactivity between muscle relaxants (MRs) is a risk for inducing anaphylaxis in sensitized patients. The preventive use of monovalent haptens (MHs) was studied in 21 cases. Inhibition of the skin reactivity by mixing MRs and MHs was observed, as was the inhibition of leucocyte histamine release to MRs, up to 3 h after infusion of MHs. These results argue for the use of such a protocol, if surgery requires the use of a MR in patients at risk for anaphylaxis. PMID- 7542068 TI - Interleukin-13 regulates the phenotype and function of human monocytes. AB - The monocyte glycosylphosphatidylinositol (GPI)-linked protein CD14 serves as the receptor for lipopolysaccharide (LPS), and regulates monocyte-lymphocyte interactions. We investigated whether CD14 expression is regulated by interleukin (IL)-13, a member of the chromosome 5 cytokine family. IL-13 inhibited CD14 expression on human monocytes. CD14 down-regulation resulted in the inhibition of LPS-induced release of tumor necrosis factor alpha, and involved neither shedding nor activation of endogenous GPI-anchor-cleaving enzymes. The CD14/actin RNA ratio was decreased 7-fold in IL-13-treated monocytes. Our results suggest that IL-13 down-regulates CD14 by suppressing CD14 RNA expression. Down-regulation of CD14, the LPS receptor, may play a major role in the anti-inflammatory effects of IL-13. PMID- 7542069 TI - Characterization of allergen (Bet v 1)-specific T cell lines and clones from non allergic individuals. AB - The immune response towards allergens in non-allergic healthy individuals was investigated. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analysed for epitope specificity. 49 T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. All TCCs revealed the Th phenotype. Cytokine production in response to specific stimulation revealed a majority of Th clones producing interleukin (IL)-4 and interferon (IFN)-gamma; however, most TCCs revealed a low IL-4/IFN-gamma ratio. Immunoblot revealed Bet v 1-specific IgG in non-allergic individuals whereas no IgE could be detected. Our results indicate that T cells from allergic and non-allergic individuals recognize the same epitopes on allergenic molecules, leading to activation, which then results in a differential production of cytokines and consequently to differential isotype switching in allergen-specific B cells. PMID- 7542070 TI - Comparison of histamine release from human blood monocytes, lymphocytes, adenoidal and skin mast cells. AB - Monocytes and lymphocytes from human blood contain 0.043 +/- 0.007 and 0.053 +/- 0.014 pg histamine/cell, respectively, which can be released by a number of stimulants (A 23187, C5a, substance P, specific allergen). The release process takes 60-120 min to reach its end point, in contrast to tissue mast cells which complete the release within 1-3 min. Both, ketotifen (10(-7) - 10(-5) M) and disodium cromoglycate (10(-5) - 10(-3) M) inhibited histamine release dose dependently up to 40-45%, which might be particularly relevant during the later stages of acute allergic or pseudoallergic reactions. PMID- 7542072 TI - Eosinophil-associated inflammation in bronchial asthma: a connection to the nervous system. AB - Evidence exists that eosinophil cationic proteins damage respiratory epithelium in bronchial asthma. Furthermore, the degree of eosinophilia in the blood and the lung is related to bronchial hyperreactivity. The eosinophil might increase airway irritability by increasing vagal responsiveness. Sensitized challenged guinea pigs develop M2 muscarinic receptor cholinergic dysfunction which is abolished by injection of heparin or polyglutamate and both the eosinophil granule major basic protein and the eosinophil peroxidase act as allosteric M2 receptor antagonists. Thus, eosinophil-associated pulmonary inflammation in asthma may enhance vagally mediated bronchoconstriction. PMID- 7542071 TI - Neurophysiology of mast cell-nerve interactions in the airways. AB - The sensitized guinea pig was employed as a model to study the effect of immunological activation of resident mast cells on neuronal activity in the airways. The trachea was isolated with the vagus nerves and vagal sensory ganglia intact. Using conventional electrophysiological recording techniques, we noted that antigenic stimulation led to an increase in the sensitivity of sensory nerve endings located in the airway wall. Moreover, previous work has revealed that antigen challenge potentiates action-potential-driven tachykinin release from afferent fibers in the guinea pig isolated airway. Anatomical and electrophysiological studies indicate that tachykinin-containing sensory fibers directly innervate the local parasympathetic ganglion neurons in the airway. Therefore, antigen-induced increases in the excitability of sensory fibers in the airways can increase parasympathetic tone in the airway by increasing central and peripheral reflex arcs. In addition, we have found that antigen stimulation has direct effects on the excitability of the parasympathetic ganglion neurons. Considered together, the data demonstrate that increased vagal sensory and parasympathetic activity may be a consequence of the allergic reaction in the airways. PMID- 7542073 TI - Epithelially derived endothelin and nitric oxide in asthma. AB - Immunohistochemical staining of endobronchial biopsies has identified increased expression of the 21-amino-acid peptide endothelin (ET) and the inducible form of the enzyme nitric oxide synthase (iNOS) within the airway epithelium in asthma. Elevated concentrations of ET are also recovered in bronchoalveolar lavage fluid from asthma patients. iNOS generates the gas nitric oxide from L-arginine, and elevated levels of NO in exhaled air have been described in asthma. ET is a potent bronchoconstrictor and levels of ET in lavage and resting airflow obstruction are correlated. The effects of ET on bronchomotor tone may be modified by NO as this is a bronchodilator. The relative balance between ET and NOS may thus contribute to resting bronchomotor tone. ET also stimulates fibroblast proliferation, collagen gene expression and through its inhibitory actions on collagenase will promote airway wall collagen deposition and contributes to airway wall thickening which underlies bronchial hyperresponsiveness. The regulation of these epithelial events may thus be important to the control of asthma. PMID- 7542074 TI - Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. AB - Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine that is overexpressed in many transplantable animal and autochtonous human cancers, in healing wounds, and in chronic inflammatory disorders such as psoriasis and rheumatoid arthritis. All of these entities are characterized by angiogenesis, altered extracellular matrix, and variable degrees of hypoxia. In addition, two VPF/VEGF receptors, flt-1 and kdr, are overexpressed by endothelial cells that line the microvessels that supply these tumors/inflammatory reactions. On the basis of these and other data, we have proposed a model of angiogenesis in which VPF/VEGF plays a central role: this model is applicable to tumors and also to the angiogenesis that occurs in non-neoplastic processes. PMID- 7542076 TI - Antigenic analysis (IgE and monoclonal antibodies) of the major shrimp allergen Pen a 1 (Tropomyosin) from Penaeus aztecus. AB - Pen a 1, the major shrimp allergen from the brown shrimp Penaeus aztecus was purified by preparative SDS-PAGE. Peptides were generated from Pen a 1 by CNBr cleavage and endoproteinase (Lys-C, Glu-C, trypsin, alkaline protease, Arg-C, chymotrypsin) digestion. The molecular weights of the resulting CNBr cleavage and enzymatic digestion products, separated by peptide SDS-PAGE, ranged from 1.5 to 20 kD. Following SDS-PAGE and semidry blotting, the analysis of monoclonal antibody (mAb) and subjects' IgE reactivities demonstrated that with the exception of alkaline protease, all cleavage procedures yielded IgE-binding peptides. However, since not all peptides of every digest bind IgE, it appears that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule. mAbs bound to CNBr, Lys-C, trypsin, Glu-C and Arg-C peptides. Since mAbs reacted to several peptides from the same digest, Pen a 1 may have several similar epitopes. The comparison of IgE and mAb reactivities demonstrated similar but not identical binding patterns. PMID- 7542075 TI - Molecular characterization of timothy grass pollen group V allergens. AB - Phl p V is the dominant allergen of timothy grass (Phleum pratense) with two isoforms having the apparent molecular weights of 38 (Phl p Va) and 32 kD (Phl p Vb) under Western blot conditions. Two-dimensional electrophoresis/immunoblotting reveals that each isoform is split into at least four isoallergens. Structural differences in the isoforms are shown by N-terminal sequencing (only 60% identity), by reaction patterns of monoclonal antibodies and, more convincingly, by enzymic degradation of purified isoforms followed by immunologic fingerprinting. These findings are confirmed by the deduced primary protein structure of cloned Phl p Va and Phl p Vb. Experiments with IgE--affinity purified by immobilized recombinant allergens or their fragments--reveal identical epitopes and at least one different epitope between the isoforms. Furthermore, on Phl p Va we can localize different IgE-reactive epitopes at the C terminus as well as the N terminus. By probing serum from 11 patients on recombinant C- or N-terminal fragments, an individual reaction pattern was found. Testing the histamine liberation potency of the fragments, we found the N terminal fragment of Phl p Va to be superior to that of the C-terminal fragment or the whole molecule. These results give insights into the variability of allergens, the individuality of human reaction patterns to epitopes and the alteration of allergenicity to higher or lower levels by fragmentation. PMID- 7542077 TI - Enhanced production and gene expression of interleukin-5 in patients with bronchial asthma: possible management of atopic diseases by down-regulation of interleukin-5 gene transcription. AB - Interleukin (IL)-5 was produced in vitro by peripheral blood mononuclear cells (PBMCs) of mite-sensitive atopic patients upon challenge with specific allergen, while PBMCs of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester (PMA) and CA2+ ionophore (ionomycin) induced marked IL-5 production by PBMCs obtained from atopic as well as nonatopic asthmatics. CD2- or CD4-bearing-cell depletion almost completely removed IL-5 production and gene transcription while CD8 depletion did not. These findings indicated that CD4+ T cells are the principal source of IL-5 in asthmatic PBMCs. The capacity of PBMCs of atopic asthmatics, nonatopic asthmatics and healthy controls to produce IL-2, IL-3, IL-4, interferon-gamma and granulocyte/macrophage colony-stimulating factor was comparable among the three groups. FK506 suppressed IL-5 production and gene expression in vitro in a dose-dependent manner. PMID- 7542078 TI - Effect of limonene and sobrerol on monocrotaline-induced lung alterations and pulmonary hypertension. AB - The effects of two monocyclic monoterpenes, limonene and sobrerol, known as inhibitors of farnesyltransferase activity, were studied on monocrotaline (MCT) induced lung injury, pulmonary hypertension and right ventricular hypertrophy in male Wistar rats. After 14 days, pulmonary arterial pressure values and the right ventricle to left ventricle plus septum weight ratios, RV/(LV + S), were markedly increased in rats subcutaneously injected with MCT (60 mg/kg). Limonene and sobrerol, administered daily at the oral dose of 400 mg/rat, markedly decreased the MCT-induced alterations. After treatment for 21 days, limonene still prevented pulmonary hypertension and the increase in RV/(LV + S). Both monoterpenes also reduced the increase in pulmonary arterial media thickness, the development of interstitial fibrosis and the increase in the number of macrophages in intra-alveolar spaces and of lymphocytes around the pulmonary veins. The present data indicate that treatment of rats with inhibitors of farnesyltransferase, like limonene and sobrerol, regulate the development of pulmonary hypertension. PMID- 7542080 TI - Genetic and environmental influences on airway inflammation in asthma. AB - The clear recognition that asthma has an inflammatory basis has led to a search for genetic and environmental causes. Genetic linkage studies provide evidence for allelic association between polymorphic markers of interleukin-9 on chromosome 5 and a principal component of the allergic phenotype, log IgE. Further studies that intrauterine environmental factors are an important determinant of the allergic phenotype in addition to allergen exposure during the first year of life. Once sensitised, the respiratory mucosa provides the allergic interface between the environment and internal milieux involving autacoid and cytokine release from mast cells. Up-regulation of endothelial cell adhesion molecules, specifically E-selectin and ICAM-1, is responsible for the leucocyte recruitment of the late-phase asthmatic response, while recruitment of T cells and their subsequent activation contribute to the ongoing inflammatory response of asthma. The epithelium is the origin and target tissue for the inflammatory response, resulting in detachment of suprabasal cells, up-regulation of adhesion molecules and enhanced mediator and cytokine secretion. These latter findings, being more evident in severe chronic disease, suggest that the 'formed' airway elements are also important in mediating the ongoing inflammation characteristic of asthma. PMID- 7542079 TI - The significance of isoallergenic variations in present and future specific immunotherapy. AB - The isoallergenic variation of the tree pollen major allergens has been studied by 2D gel electrophoresis, and by analysis of several recombinant clones. The studies have included both antibody-based and T cell stimulation assays. Bet v 1, the major allergen of birch, forms at least 24 spots when conventional extracts are analyzed by 2D gel electrophoresis. Comparison of Bet v 1-encoding DNA sequences reveals a considerable number of amino acid substitutions. This sequence variation can theoretically account for the number of spots observed in 2D gels. Whereas pools of serum from allergic individuals and monospecific antibodies raised in rabbits bind to most if not all spots in 2D gels, analyses of individual serum and/or murine monoclonal antibodies show individual patterns of reactivity with various subsets of spots. These observations point to a model in which amino acid substitutions induce local perturbations of the allergen surface, causing differences in epitope structure. Furthermore, analysis of pollen from individual trees shows that each tree produces individual subsets of Bet v 1 spots. When analyzed in stimulation assays, T cell clones also display differences in reactivity to different isoallergens. In conclusion, we have shown that Bet v 1 is heterogeneous, and that individual trees produce various subsets of isoallergens which display differences in reactivity both towards antibodies and T cells. A careful selection of isoform may therefore be of major importance if recombinant allergens or synthetic peptides are to be used for conventional immunotherapy. PMID- 7542081 TI - Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin. AB - Basidiospores are a prevalent and frequent cause of respiratory allergies, yet their allergens remain poorly defined; thus, we have attempted a molecular characterization of representative basidiomycete allergens. A Psilocybe cubensis mycelial cDNA library was immunoscreened with patient serum. A clone was isolated that expressed a 23-kD recombinant allergen as a fusion protein and inhibited a 16-kD band (Psi c 2) in immunoprints of P. cubenis extract, indicating antigenic identity. Sequence (cDNA) analysis of the clone indicates homology with cyclophilin and the deduced amino acid sequence of Psi c 2 showed 78% identity and 4% similarity with the amino acid sequence of Schizosaccharomyces pombe cyclophilin. This recombinant allergen is a useful model for epitope analysis of basidiospore allergens and fungal allergen cross-reactivity, and may provide an improved reagent for basidiospore allergy diagnosis and treatment. PMID- 7542082 TI - Epitope-specific down-regulation of anti-allergen antibodies following injection of allergen-antibody complexes in hypersensitive patients. AB - Administration to allergic patients of complexes made of allergen and anti allergen antibodies results in a reduction in the levels of specific IgE and IgG antibodies that is limited to antibodies present in the complexes. The epitope specific nature of this reduction is demonstrated by taking advantage of the cross-reactivity between Der p I and Der f I. In addition, an increased production of anti-idiotypic antibodies is demonstrated. As such treatment significantly improves patients with allergic asthma or atopic dermatitis, it may represent a valuable alternative to conventional immunotherapy. PMID- 7542083 TI - Development of tryptase-positive KU812 cells cultured in the presence of Steel factor. PMID- 7542084 TI - Human mast cells adhere to extracellular matrix proteins through their selective expression of beta 1 integrins. PMID- 7542085 TI - Tyrosine phosphorylation regulates activation and inhibition of apoptosis in human eosinophils and neutrophils. PMID- 7542086 TI - Human eosinophils release the lymphocyte and eosinophil active cytokines, RANTES and lymphocyte chemoattractant factor. PMID- 7542087 TI - Subcellular mechanisms of eosinophil degranulation: the role of RANTES, interleukin-5 and tumor necrosis factor-alpha. PMID- 7542088 TI - Molds modulate the release of histamine and sulfidoleukotrienes from human basophils. PMID- 7542089 TI - Activation of tumor necrosis factor-alpha and lymphotoxin-alpha via anti-CD40 in human B cells. PMID- 7542090 TI - CD40 ligand/CD40 deficiency. AB - CD40 is a surface antigen expressed on B cells. The CD40 ligand (CD40L) is expressed on activated T cells. Interaction between CD40 and CD40L is critical for proliferation and isotype switching in the context of a response to a T-cell dependent antigen. Patients with X-linked hyper-IgM syndrome (HIGMX-1) in their CD40L gene are unable to switch from IgM to IgG, IgA and IgE. Mice with a disrupted CD40 gene fail to undergo isotype switching to T-cell-dependent antigens but respond normally to T-independent antigens. PMID- 7542091 TI - Expression of adhesion molecules (ICAM-1, LFA-3) on human epithelial cells (A549) after respiratory syncytial virus infection. PMID- 7542092 TI - Establishment of a human fetal small intestinal epithelial cell line. PMID- 7542094 TI - Anti-IgE monoclonal antibodies that inhibit allergen-specific histamine release. PMID- 7542093 TI - Regulation of IgE synthesis by CD23/CD21 interaction. AB - At least two cell-derived signals have been shown to be necessary for the induction of immunoglobulin isotype switching in B cells. The first signal is given by either of the soluble lymphokines interleukin (IL)-4 or IL-13 which induce germline epsilon transcript expression, but alone is insufficient to trigger secretion of IgE. The second signal is provided by a physical interaction between B cells and activated T cells, basophils and mast cells, and it has been shown that the CD40/CD40L pairing is crucial for mediating IgE synthesis. In HIGM1 syndrome, which is characterized by greatly decreased levels of IgG, IgA and IgE, there are mutations in CD40L resulting in a completely non-functional extracellular domain. CD40L is therefore playing a central role in Ig switching. Amongst the numerous pairs of surface adhesion molecules, the CD23/CD21 pair seems to play a key role in the generation of IgE. The CD23 molecule is positively and negatively regulated by factors which increase or decrease IgE production, respectively. Antibodies to CD23 have been shown to inhibit IL-4 induced human IgE production in vitro and to inhibit antigen-specific IgE responses in a rat model, in an isotype-selective manner. CD23 interacts with CD21 on B cells, preferentially driving IgE production. CD23 recognises two main epitopes on the CD21 molecule. One region consists of short consensus repeat sequences (SCRs) 1-2 and the other of SCRs 5-8. In the latter region ASn370 and Asn295 are critical in the interaction with the lectin CD23. Therefore, a restricted number of cytokines and surface molecules seems to selectively regulate human IgE synthesis. PMID- 7542095 TI - Inhibitory effects of nedocromil sodium on cytokine production from mast and epithelial cells. PMID- 7542096 TI - Bradykinin-induced immediate skin reactions and H1-blockade. PMID- 7542097 TI - Expression of granulocyte/macrophage-colony-stimulating factor, interleukin-8 and RANTES in the bronchial epithelium of mild asthmatics is down-regulated by inhaled beclomethasone dipropionate. PMID- 7542099 TI - Effect of fluticasone propionate on acute and chronic inflammation. PMID- 7542098 TI - Expression of the CD40 ligand in T lymphocytes and induction of IgE isotype switching. AB - CD40 ligand (CD40L) delivers a contact-dependent signal to B cells which, in the presence of interleukin (IL)-4, drives immunoglobulin isotype switching to IgE. CD40L expression in T cells is transient, requires activation of protein kinase C and a rise in intracellular calcium concentration ([Ca2+]i), and is inhibited by cyclosporin A (CsA). CsA also inhibited T-cell-dependent IL-4-driven IgE synthesis. We have found that expression of CD40L is developmentally regulated. Expression of CD40L was restricted to mature single-positive thymocytes which, in the presence of IL-4, were capable of inducing B cells to undergo IgE isotype switching. CD40L expression was severely decreased in cord blood lymphocytes and was associated with a severely decreased ability to undergo T-cell-dependent IgE isotype switching. PMID- 7542100 TI - Immunological analyses of Phl p V. PMID- 7542101 TI - Regulation of mouse and human mast cell development, survival and function by stem cell factor, the ligand for the c-kit receptor. AB - Stem cell factor (SCF), the ligand for the receptor (SCFR) that is encoded by the c-kit proto-oncogene, has many important effects in mouse and human mast cell development, survival, and function. SCF can promote mast cell survival by suppressing apoptosis, induce mast cell hyperplasia in murine rodents, experimental primates and humans, directly induce SCFR-dependent mast cell mediator release, and significantly modulate the extent of mast cell activation by Fc epsilon RI-dependent mechanisms. These findings raise several clinical issues and, in some cases, point to potentially significant therapeutic opportunities. PMID- 7542102 TI - Regulation of development, survival and neoplastic growth of mast cells through the c-kit receptor. AB - Signaling through the c-kit receptor tyrosine kinase (Kit) is essential for development and survival of mast cells but not of basophils. Moreover, we recently found an activation mutation of Kit in several tumor mast cell lines. PMID- 7542103 TI - Interaction of mast cells with extracellular matrix proteins. AB - It is possible to divide surface receptors on mast cells conceptually into three groups. The first consists of immune response receptors. The index receptor for this group is Fc epsilon RI, now joined by Fc gamma receptors and receptors for complement products. The second group of receptors are those that are involved in growth and differentiation, such as those for interleukin-3 and stem cell factor. The third group consists of receptors regulating mast cell trafficking and distribution. Principle among the latter group of receptors are those that engage extracellular matrix components, including the classical integrin receptors. The engagement of mast cells to matrix components not only has relevance in determining the tissue distribution of mast cells, but also appears to have a major influence on the biologic responsiveness of mast cells to immune- and growth-factor-receptor-mediated signals. PMID- 7542104 TI - Characterization of cord-blood-derived human mast cells cultured in the presence of Steel factor and interleukin-6. AB - We generated > 10(7) mast cells by culturing 10(7) cord blood mononuclear cells for > 10 weeks in the presence of Steel factor, interleukin-6 and prostaglandin E2. 99% of the cultured cells had tryptase-positive granules, while 18% had chymase-positive granules. Cultured mast cells contained 3.6 micrograms histamine and 3.5 micrograms tryptase per 10(6) cells. Cells sensitized with 1 microgram/ml human IgE released 58.5% histamine and 1.55 ng tumor necrosis factor (TNF)-alpha per 10(6) cells when challenged with 1 microgram/ml antihuman IgE, whereas the control cells spontaneously released 3.7% histamine and 0.18 ng TNF-alpha. Analysis for surface antigens revealed that cultured mast cells expressed the following CD molecules: 9, 13, 14, 29, 33, 38, 43, 44, 45RA, 45RB, 46, 47, 48, 49d, 50, 51, 53, 54, 55, 58, 59, 60, 61 and 117 (c-Kit). Taken together, these cultured cells seem to be functionally mature mast cells. PMID- 7542105 TI - Functional characterization of the signal transduction events mediated by Fc epsilon RI alpha and gamma chimeric receptors. AB - Chimeric receptors containing the Fc epsilon RI alpha and gamma subunit domains were constructed, stably transfected into RBL-2H3 cells, and characterized for the biochemical events which are elicited upon receptor aggregation. Chimeric receptors containing the extracellular (EC) domain of the human Fc epsilon RI alpha subunit, or the EC domain of the p55 subunit of the interleukin-2 receptor were fused to the human Fc epsilon RI gamma subunit transmembrane and cytoplasmic (CT) domains or only the CT domain. The chimeras generated included alpha/gamma/gamma, I/gamma/gamma, alpha/I/gamma or I/I/gamma. The results indicate that both the Fc epsilon RI alpha EC domain and the Fc epsilon RI alpha CT domain are essential for signalling. PMID- 7542106 TI - Human heart mast cells in anaphylaxis and cardiovascular disease. AB - All sections of human heart tissue demonstrate tryptase- and chymase-containing mast cells (HHMCs) which have for the first time been isolated, partially purified and studied in vitro. HHMCs contain similar histamine levels as lung and skin mast cells, but tryptase levels are lower than in skin and higher than in lung mast cells. Complement C5a causes rapid dose-dependent release of histamine from HHMCs, but they are refractory to substance P and fMLP. Cross-linking IgE receptors on HHMCs leads to arachidonic acid metabolism through both the cyclooxygenase and 5-lipoxygenase pathways. HHMCs and their vasoactive mediators may be involved in anaphylactic/anaphylactoid reactions in humans and in the pathogenesis of some cardiovascular diseases. PMID- 7542107 TI - The interaction of cytokines with human basophils and mast cells. AB - Human mast cells and basophils participate in allergic inflammation by releasing preformed and newly synthesized mediators upon cross-linking of cell surface IgE/receptor complexes with specific antigen. It is becoming increasingly apparent, however, that the basophil response is up-regulated by a far greater number of cytokines than is the mast cell, making the basophil more sensitive to products that are commonly generated during immune reactions. As a result, basophils may have a greater role in the chronic allergic inflammation that characterizes diseases such as asthma. This belief is supported further by the fact that the basophil has recently been shown to generate interleukin-4, and possibly other cytokines, indicating a role for this cell in directing the immune response of other cell participating in allergic lesions. PMID- 7542109 TI - Therapeutic effects of finasteride in benign prostatic hyperplasia: a randomized double-blind controlled trial. AB - The clinical effects of finasteride, a 5 alpha-reductase inhibitor, in patients with benign prostatic hyperplasia (BPH) were evaluated in a double-blind, placebo controlled study. Forty-six patients with symptomatic BPH were randomly assigned to 2 groups, the finasteride group and the placebo group. The finasteride group received 5 mg of finasteride daily for 6 months. Prostate volume, urinary flow, urinary symptoms, serum prostate-specific antigen (PSA) and adverse events were determined before and after treatment. After 6 months of treatment the patients treated with 5 mg of finasteride per day had a 30% decrease in their total urinary symptom score, a 14% decrease in prostate volume and a 0.9 ng/dL decrease of PSA. Their maximal urinary flow rate increased by 1.42 mL per second and the mean urinary flow rate increased by 0.64 mL per second. The patients given placebo showed no significant changes in their prostate volume, serum PSA and maximal and mean urinary flow rate. However, the symptom scores in the placebo group also decreased significantly. When compared with the placebo group, those in the finasteride group had significantly lower prostate volume, serum PSA, maximal urinary flow rate and urinary symptoms, but not mean urinary flow rate. The frequency of adverse events was low in both the finasteride and placebo groups. These results show that finasteride may be an effective and safe alternative for the treatment of patients with BPH. PMID- 7542110 TI - Euthanasia debate. Doctors may be ignorant of treatments for intractable symptoms. PMID- 7542108 TI - Analysis of mast cell activation using diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry. AB - We review a new technique--diamine oxidase (DAO)-gold ultrastructural enzyme affinity labeling--which we developed to localize histamine in subcellular sites of mast cells. The DAO-gold method showed that isolated human lung mast cells contained abundant histamine in their cytoplasmic granules, a conclusion which was verified by a large number of specificity controls. We also studied mast-cell rich eyelid lesions which developed in interleukin-4 transgenic mice. The DAO gold method demonstrated histamine in the electron-dense granules of mast cells in these lesions, but little or no histamine was detected in the swollen, empty granules of mast cells undergoing piecemeal degranulation. This new enzyme affinity-gold method has permitted the first ultrastructural localization of histamine in subcellular sites of routinely prepared electron microscopy samples. The method has also permitted the first morphological studies of histamine secretion in vivo and has demonstrated that such secretion can be associated with the ultrastructural changes of piecemeal degranulation. PMID- 7542111 TI - Euthanasia debate. Death from motor neurone disease can be peaceful. PMID- 7542112 TI - Euthanasia debate. The choice to live or die should remain to the end. PMID- 7542114 TI - Prophylactic administration of granulocyte colony-stimulating factor (filgrastim) after conventional chemotherapy in children with cancer. AB - We evaluated granulocyte colony-stimulating factor (G-CSF) as an adjunct to courses of conventional chemotherapy in 16 children with cancer. One course followed by G-CSF (20 episodes) was compared to identical courses without G-CSF (20 episodes) in the same patients. The mean duration of G-CSF therapy was 8.8 (5 13) days. The periods of neutropenia (4.8 days versus 16.5 days; p < 0.0001), days of hospitalization for febrile neutropenia (13 days versus 65 days; p = 0.02) and days on broad-spectrum antibiotics (13 days versus 95 days; p = 0.003) were significantly reduced. With the use of G-CSF the profound neutropenia could be prevented in 11 (55%) episodes. There were two episodes of fever and neutropenia in the G-CSF group as compared to 10 febrile neutropenias in the control group (p = 0.04). G-CSF was well tolerated and did not cause additional expenses when compared to the expenses needed for the treatment of febrile neutropenias. The cost benefit analyses showed that through using G-CSF a savings was realized in the amount of U.S. $20,650 for 20 cycles of chemotherapy, i.e., U.S. $1,033/chemotherapy cycle. We conclude that the use of G-CSF was efficacious and did not increase the total costs of therapy. PMID- 7542113 TI - Characterization of enriched CD34+ cells from healthy volunteers and those from patients treated with chemotherapy plus granulocyte colony-stimulating factor (G CSF). AB - CD34+ cells were enriched, using a panning method, from peripheral blood (PB) and bone marrow (BM) of healthy volunteers and of patients treated with chemotherapy plus granulocyte colony-stimulating factor (G-CSF). In healthy volunteers, PB CD34+ cells expressed CD33 and CD13 at a higher frequency than BM CD34+ cells, and PB CD34+ cells contained a greater number of burst-forming units-erythroid (BFU-E) than colony-forming units granulocyte-macrophage (CFU-GM). Administration of G-CSF to healthy volunteers induced a marked increase in the number of PB CD34+ cells, although the proportions of those expressing CD33, CD13, and c-kit among these cells as well as colony-forming ability were not changed before and after G-CSF administration. There were no significant differences in surface antigens on PB CD34+ cells between healthy volunteers and patients after chemotherapy plus G-CSF, except for low expression of c-kit in the PB of patients. However, PB CD34+ cells from patients contained almost the same number of CFU-GM as BFU-E. These results indicate that there were clear differences in the features of CD34+ cells from BM and from PB, and between healthy volunteers and patients after chemotherapy plus G-CSF. Enriched CD34+ cells are useful for analyzing the characteristics of hematopoietic progenitor cells, and such analysis may predict the usefulness of autologous or allogeneic peripheral blood stem cell transplantation. PMID- 7542115 TI - Benzene and its metabolite, hydroquinone, induce granulocytic differentiation in myeloblasts by interacting with cellular signaling pathways activated by granulocyte colony-stimulating factor. AB - Chronic exposure of humans to benzene (BZ) causes acute myelogenous leukemia. These studies determined whether BZ, or its reactive metabolite, hydroquinone (HQ), affect differentiation of myeloblasts. BZ or HQ administered to C57BL/6J mice specifically induced terminal granulocytic differentiation of myeloblasts. The ability of the compounds to induce differentiation of the myeloblast was tested directly using the murine interleukin 3 (IL-3)-dependent myeloblastic cell line, 32D.3 (G) and the human HL-60 promyelocytic leukemic cell line. Treatment of HL-60 myeloblasts with BZ activated protein kinase C and upregulated the 5 lipoxygenase (LPO) pathway for the production of leukotriene D4 (LTD4), an essential effector of granulocytic differentiation. Differentiation was prevented by sphinganine, a kinase C inhibitor, as well as by LPO inhibitors and LTD4 receptor antagonists. BZ and HQ also induced differentiation in 32D.3 (G) myeloblasts. Both compounds interact with cellular signaling pathways activated by granulocyte colony-stimulating factor (G-CSF) and thus replace the requirement for G-CSF. IL-3 induces a growth response, whereas G-CSF provides both growth and differentiation signals. BZ does not induce growth in the absence of IL-3, but provides a differentiation signal. Both HQ and LTD4 induce differentiation and synergize with IL-3 for growth, however, neither support growth in the absence of IL-3. BZ-induced 32D cells showed a gradual progression of progenitor differentiation to granulocytes similar to that seen with G-CSF or LTD4. HQ blocks differentiation at the myelocyte stage; only a small percentage of progenitors proceed to granulocytes. BZ, like G-CSF, upregulates LTD4 production, whereas HQ obviates the requirement for LTD4 by activating the LTD4 receptor. PMID- 7542116 TI - Differential expression of cell adhesion molecules by human hematopoietic progenitor cells from bone marrow and mobilized adult peripheral blood. AB - The mechanisms responsible for mobilization of hematopoietic progenitor cells (HPC) from the bone marrow into the circulation are unknown. One possibility is that HPC downregulate cell adhesion molecule expression. We studied normal human bone marrow and adult peripheral blood following 4 g/m2 cyclophosphamide and recombinant human granulocyte colony-stimulating factor (rHuG-CSF). Each sample was studied for the coexpression of CD34 and a panel of cell adhesion molecules by dual immunocytometry. Bone marrow HPC express the immunoglobulin gene superfamily members of ICAM-1 (CD54), PECAM-1 (CD31) and LFA-3 (CD58), the integrins VLA-4 (CD49d/CD29), VLA-5 (CD49e/CD29) and LFA-1 (CD11a/CD18), L Selectin (CD62L), HCAM (CD44) and CD36. Mobilized peripheral blood HPC display less expression of LFA-3 (CD54) and VLA-5 (CD49e). Significant differences in cell adhesion molecule expression do exist between sessile and circulating HPC, but the biological relevance of these observations is currently unclear. PMID- 7542117 TI - Recombinant human megakaryocyte growth and development factor (rHuMGDF), a ligand for c-Mpl, produces functional human platelets in vitro. AB - Platelet formation, occurring from bone marrow or lung megakaryocytes, has been difficult to study mechanistically. Recombinant human megakaryocyte growth and development factor (rHuMGDF), a recently described cytokine, has now been used to establish an in vitro system in which this important and little understood process occurs. CD34+ cells cultured with rHuMGDF develop into megakaryocytes which form long cytoplasmic extensions (proplatelets) that fragment into platelet sized particles (in vitro platelets). Morphologically, in vitro and human plasma derived platelets (control platelets) are virtually identical with respect to size, dense granule distribution and ultrastructural features. Functionally, in vitro and control platelets have similar aggregation and activation responses, and similarly incorporate mepacrine into dense granules. These findings suggest that rHuMGDF is sufficient to generate platelet-synthesizing megakaryocytes from CD34+ cells and provide an experimental setting in which the study of human platelet formation can be adequately performed. PMID- 7542118 TI - Nuclear actin and RNA export in conidial cells of Neurospora crassa. PMID- 7542120 TI - Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates. AB - The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract. PMID- 7542119 TI - Calcium-activated chloride fluxes in cultured NCL-SG3 sweat gland cells. AB - The dependence of chloride permeability of the human sweat gland cell line NCL SG3 cell line on cytosolic free calcium ([Ca2+]i) was investigated. X-ray microanalysis, fura-2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na+ but was blocked by chloride channel blockers. Lowering bath Ca2+ decreased ic. On raising bath Ca2+ ic and [Ca2+]i responded with a transient rise which was not blocked by La3+ or D-600. La3+, but not D-600, blocked the entry of Mn2+. K(+) depolarization and Bay-K-8644 had little effect on [Ca2+]i. The rise in [Ca2+]i may be mediated primarily via depletion operated Ca(2+)-channels. Irrespective of substrate NCL-SG3 cells have a chloride permeability which depends on [Ca2+]i. PMID- 7542121 TI - New techniques in diagnosing tuberculosis. PMID- 7542122 TI - Hepatitis C virus and intravenous anti-D immunoglobulin. PMID- 7542123 TI - AMPA-induced lesions of the basal forebrain differentially affect cholinergic and non-cholinergic neurons: lesion assessment using quantitative in situ hybridization histochemistry. AB - The direct and transynaptic effects of lesions of the basal forebrain induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid were investigated using quantitative in situ hybridization histochemistry. Probes complementary to the sequences of choline acetyltransferase mRNA, glutamate decarboxylase mRNA and preproenkephalin mRNA were used to assess direct lesion effects within the basal forebrain and probes for postsynaptic M-1 and M-3 muscarinic receptors were used to assess long-term changes in neocortical muscarinic receptor mRNA expression following cholinergic deafferentation. AMPA induced basal forebrain lesions destroyed significantly more neurons that expressed choline acetyltransferase mRNA than ibotenic acid-induced lesions (90 versus 60%), but significantly fewer neurons which expressed either glutamate decarboxylase or preproenkephalin mRNA (61 versus 83% reduction in glutamate decarboxylase mRNA and 56 versus 79% reduction in preproenkephalin mRNA). AMPA induced lesions did, however, destroy a significant proportion of the neurons which expressed glutamate decarboxylase and preproenkephalin mRNA (approximately 60%). The neurons spared following AMPA-induced lesions were typically situated dorsolaterally within the dorsal pallidum, although neurons expressing glutamate decarboxylase or preproenkephalin mRNA were frequently observed within the areas of greatest cholinergic neuronal loss, i.e. the region of the nucleus basalis magnocellularis. These findings suggest that there is a population of non cholinergic pallidal neurons which are insensitive to AMPA but not to ibotenic acid, reflecting a possibly heterogeneous distribution of NMDA and non-NMDA subtypes of glutamate receptors within the rat basal forebrain. AMPA-induced lesions of the basal forebrain were, however, without significant effect on the levels of expression of M-1 and M-3 muscarinic receptor mRNAs in the cerebral neocortex. PMID- 7542124 TI - The effects of AMPA-induced lesions of the medial septum and vertical limb nucleus of the diagonal band of Broca on spatial delayed non-matching to sample and spatial learning in the water maze. AB - These experiments investigated in the rat the impact on spatial delayed non matching to sample and on acquisition of the Morris water maze of (i) AMPA induced lesions of the medial septal nucleus, which produced a marked reduction of hippocampal choline acetyltransferase activity and acetylcholine levels (measured using in vivo dialysis) together with lesser reductions in cholinergic markers in the cingulate cortex and (ii) similar AMPA-induced lesions of the vertical limb nucleus of the diagonal band of Broca (vDB), which produced more marked reductions in cholinergic markers in the cingulate cortex than in the hippocampus. Medial septal lesions produced a delay-dependent deficit in spatial working memory, while lesions of the vDB resulted in a delay-independent performance deficit. In addition, rats with vDB lesions adopted biased response strategies during the imposition of long delays. Neither lesion significantly affected the acquisition of a spatial reference memory task, the Morris water maze. The results are discussed in terms of cholinergic- and GABAergic-dependent functions of the hippocampal formation and cingulate cortex in spatial short-term and reference memory. PMID- 7542125 TI - Acidic FGF and FGF receptors are specifically expressed in neurons of developing and adult rat dorsal root ganglia. AB - Employing complementary technical approaches, we have studied the expression of acidic fibroblast growth factor (aFGF) and FGF receptors in rat dorsal root ganglia. The results clearly showed that within spinal nerves aFGF and two high affinity FGF receptors, FGFR-1 and FGFR-2, were prominently expressed in neurons, while expression in Schwann cells was undetectable. FGFR-3 and FGFR-4 were not expressed in dorsal root ganglia. Acidic FGF mRNA was detected in the majority of dorsal root ganglion neurons, including all size classes: FGFR-1 and FGFR-2 transcripts were only detected in subpopulations of mainly large and medium size neurons. In subcellular fractionation studies on dorsal root ganglion and spinal root tissue, aFGF was recovered in the soluble fraction and was thus not tightly associated with neuronal membranes. During development FGFR-1 and FGFR-2 mRNAs were found to be present at all stages examined (embryonic days 15-21 and postnatal days 1-120). Acidic FGF mRNA and protein were first detected at embryonic day 18, and their expression then increased progressively up to postnatal levels. In cultures of dorsal root ganglion neurons derived from day 15 embryos, aFGF expression was first detected 3 days after plating. The resulting neuron cultures continued to express aFGF in a Schwann cell-independent manner. In combination, these results indicate that aFGF expression in dorsal root ganglia is initiated and maintained in postmitotic neurons. Furthermore, the data suggest that the physiological function of aFGF in the peripheral nervous system is connected to processes specific to the mature sensory (and motor) system, such as the maintenance and survival of peripheral nerve neurons. PMID- 7542126 TI - The extracellular matrix molecule tenascin: expression in the developing chick retinotectal system and substrate properties for retinal ganglion cell neurites in vitro. AB - To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization. Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum. In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age. At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway. In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres. At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells. In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells. To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro. When retinal ganglion cell suspensions from 6-day-old chick embryos were maintained on homogeneous mouse or chick tenascin/polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 micrograms/ml. Higher coating concentrations (35 or 70 micrograms/ml) resulted in neurite lengths comparable to control values. Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a performed pathway to support growth of retinal ganglion cell axons in the tectum. PMID- 7542127 TI - Gene expression and in vitro release of galanin in rat hypothalamus during development. AB - The expression and distribution of the mRNA coding for galanin precursor, preprogalanin (ppGAL), were analysed in several rat hypothalamic nuclei (periventricular, paraventricular, supraoptic, dorsomedial and arcuate nuclei and the lateral hypothalamic area) during development by an in situ hybridization technique and computer-assisted grain counting over individual cells. ppGAL mRNA (expressed as number of grains/100 microns2) was detectable from postnatal day (PD) 1 in all the nuclei considered, and the amount of transcript per cell was 6 11 times less than in the adult. ppGAL mRNA progressively increased from PD8 to 14 to 21. The level of ppGAL mRNA in all the nuclei at PD21 was about half that in adulthood, except in the dorsomedial nucleus, where the difference was no more than 20%. As an index of the activity of galanin-containing neurons, we measured the basal and K(+)-evoked in vitro release of galanin-like immunoreactivity from hypothalamic slices of PD14, 21 and 90 rats by radioimmunoassay. Basal release of galanin-like immunoreactivity remained at the same level from PD14 to PD90, but the response to KCl (50 mM) stimulation was lower at PD14 (approximately 90%) and PD21 (> 200%) than at PD90 (350%). Basal and K(+)-evoked release was sensitive to tetrodotoxin, indicating a neuronal origin. This study provides the first evidence that the increase in ppGAL mRNA during the ontogeny of hypothalamic nuclei is associated with an increase in galaninergic neuronal function. PMID- 7542128 TI - Intratumoral production of tumor necrosis factor augmented by endogenous interferons results in potent antitumor effects of DT-5461, a synthetic lipid A analog. AB - We previously reported that DT-5461 exhibits potent antitumor effects on various murine syngeneic tumors, probably via activation of host immune systems. Of the various systemic administration routes, intravenous (i.v.) administration gave the best antitumor effects. When the total dose was fixed, multiple and intermittent applications resulted in greater therapeutic efficacy than single and daily applications, respectively. The therapeutically effective applications of DT-5461 induced endogenous tumor necrosis factor (TNF) activity in serum and tumor tissue. The TNF activity peaked at 1-2 h after the administration. Although TNF activity in the serum declined to an undetectable level by 4 h, intratumoral TNF activity persisted even at 16 h. TNF-alpha messenger RNA (mRNA) was clearly expressed in the tumor tissues as early as 0.5 h after the DT-5461 administration. DT-5461 also caused increases in interferon activity in tumor bearing mice. In vivo treatment with anti-interferon-alpha/beta serum or anti interferon-gamma serum, as well as with anti-TNF-alpha serum, significantly reduced the antitumor effect of DT-5461. DT-5461-induced endogenous TNF production was also inhibited by treatment with either of these anti-interferon antisera alone. These results suggest that intermittent i.v. administration is optimal for cancer treatment with DT-5461, and that the optimal application of DT 5461 causes a long-lasting production of intratumoral TNF-alpha that may play a crucial role in the antitumor mechanisms of this compound. Furthermore, endogenous interferons induced by DT-5461 are involved in the antitumor mechanisms of this compound, probably by regulating the intratumoral TNF induction. PMID- 7542129 TI - Fusaric acid in Fusarium moniliforme cultures, corn, and feeds toxic to livestock and the neurochemical effects in the brain and pineal gland of rats. AB - Fusaric acid is produced by several species of Fusarium, which commonly infect corn and other agricultural commodities. Since this mycotoxin may augment the effects of other Fusarium toxins, a gas chromatography/mass spectrometry method of analysis in feeds was developed. Fusaric acid was analyzed as the trimethylsilyl-ester from F. moniliforme-cultures, -contaminated corn screenings, and feeds toxic to livestock. The mycotoxin was found in all samples and ranged from 0.43 to 12.39 micrograms/g sample. Also, fusaric acid was tested for its neurochemical effects in the brain and pineal gland of rats. Animals were dosed intraperitoneally (100 mg/kg body weight) 30 min prior to the onset of the dark phase (lights out) and the effects were studied at 1.5, 3.5, and 5.5 h after treatment. Brain serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), tyrosine (TYRO), and dopamine (DA) were increased (P < 0.05) by fusaric acid, and norepinephrine (NEpi) was decreased (P < 0.05). Analogously, DA in the pineal gland increased and NEpi decreased (P < 0.05). Pineal N-acetylserotonin (NAc5HT) was increased (P < 0.05), whereas pineal 5HT and its two major metabolites 5HIAA and 5-hydroxytryptophol (5HTOL) decreased (P < 0.05). Elevated brain TYRO and brain and pineal DA, with decreased NEpi, may be consistent with fusaric acid's partial inhibitory effect on tyrosine-hydroxylase and its inhibitory effect on dopamine-beta-hydroxylase, respectively. Elevated pineal Nac5HT is consistent with decreased pineal 5HT and the increased pineal DA, and support the dopaminergic stimulatory activity of the enzyme responsible for the conversion of 5HT to NAc5HT. This is the first report of fusaric acid's in vivo effect on pineal DA, NEpi, 5HT, and NAc5HT in rats, and a relation for the effects on TYRO, 5HT, and 5HIAA in brain tissue. The results indicate fusaric acid alters brain and pineal neurotransmitters and may contribute to the toxic effects of Fusarium contaminated feeds. PMID- 7542130 TI - Zonal biochemical and morphological characteristics in BPH. AB - OBJECTIVE: To compare androgen, oestrogen, progesterone and epidermal growth factor receptor concentrations in the transition zone and peripheral zone of the prostate in benign prostatic hyperplasia (BPH), and to relate these findings to epithelial and stromal composition. PATIENTS AND METHODS: Tissue from both the transition and peripheral zone of the prostate was obtained from 26 patients undergoing transurethral prostatectomy for benign prostatic obstruction and used for both receptor binding studies and morphometric analysis. Androgen receptor (AR), oestrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor (EGFR) concentrations were assayed by saturation binding with a competitive inhibitor. The epithelial, stromal and luminal composition of the tissue was determined using a Zeiss AxioHOME microscope workstation. RESULTS: The epithelial content was significantly greater in the transition zone than in the peripheral zone. No overall zonal difference in AR concentration was detected; however, when values were expressed relative to the epithelial component, the AR content was significantly higher in the peripheral zone. Conversely, overall EGFR concentrations were significantly greater in the transition zone, although not when expressed per unit epithelium. Higher concentrations of oestrogen receptor were measured in the transition zone per unit stroma. No zonal difference in PR was detected. However, there was a significant correlation between AR and PR in the peripheral zone and between EGFR and AR in the transition zone. CONCLUSION: These data demonstrate that receptor concentrations should be related to tissue composition. Concentrations of AR were higher in the peripheral zone epithelium than in transition zone epithelium, suggesting greater androgen dependence. This may be important in determining its greater propensity for malignancy. Although EGFR concentrations were greater in the transition zone, there was no zonal difference after correction for the amount of epithelium. Finally, higher concentrations of ER were detected in the transition zone stroma which may reflect important zonal differences in regulating growth and provides further evidence of a role for oestrogens in BPH. PMID- 7542131 TI - Is follow-up necessary after transurethral resection of the prostate? AB - OBJECTIVE: To assess the necessity for routine out-patient review following transurethral resection of the prostate (TURP) for benign prostatic hypertrophy. PATIENTS AND METHODS: One-hundred and two patients with histologically proven benign prostatic hypertrophy were prospectively reviewed as out-patients. The views of their general practitioners were established by postal questionnaire. RESULTS: At their first out-patient review 94 of 102 (92%) patients were discharged and only 2% (2/102) patients remained under review after 1 year. Apart from routine urine analysis, only four additional investigations were initiated at the routine clinical assessment. One patient was listed to undergo prostatic surgery again following the post-operative out-patient review. Over the next 16 months only three patients (3%) were re-referred with further urological problems. Confidence in a system without routine hospital out-patient review was expressed by 90% of general practitioners and 78% of patients. CONCLUSION: Follow up after TURP cannot be justified in terms of the perceived therapeutic benefit to patients. Patients should be discharged to the community and be assessed rapidly in out-patients if and when problems arise. PMID- 7542132 TI - The International Prostate Symptom Score in a community-based sample of men between 55 and 74 years of age: prevalence and correlation of symptoms with age, prostate volume, flow rate and residual urine volume. AB - OBJECTIVES: To study the prevalence of symptoms of prostatism in the community and the correlation between these symptoms and age, prostate volume, flow rate and residual urine volume. SUBJECTS AND METHODS: The International Prostate Symptom Score (IPSS) was administered to a community-based population of 502 men aged between 55 and 74 years with no prostate cancer and no history of a prostate operation. Prostate volume parameters, flow rate variables and post-void residual urine volume were measured. RESULTS: Overall, 6 and 24% of the men were severely and moderately symptomatic, respectively. The results of a detailed questionnaire such as the IPSS (only 12% of the men scored 0) contrast with the men's global perception of their voiding function (82% claimed to have 'no voiding complaints'). A good correlation was found between the total symptom score and the single disease-specific quality of life question that is included in the IPSS (r = 0.74, P = 0.001). There was a weak correlation between the IPSS and total prostate volume (r = 0.19, P < 0.001), and between the IPSS and physiological measures such as peak flow rate (r = -0.18, P < 0.001) and post-void residual urine volume (r = 0.25, P < 0.001). There was a very weak correlation between the IPSS and age (r = 0.09, P = 0.04). CONCLUSIONS: The parameters used to characterize benign prostatic hyperplasia (BPH) should be considered independently because no predictions about the value of a certain parameter can be made by knowing one of the other parameter values. Symptom scores should therefore not be used as a pre-selection criterion in the determination of the prevalence of clinical BPH without taking other measures into account. The interpretation of the parameter values in a clinical setting should take the lack of correlation and the variability of the parameter values into account. PMID- 7542133 TI - The value of prostatic specific antigen in the early diagnosis of prostatic cancer: a Greek view. AB - OBJECTIVE: To assess whether it is worthwhile to screen asymptomatic men for prostate cancer using serum prostate specific antigen (PSA) and to determine how many patients could be cured of prostatic carcinoma if detected by screening. PATIENTS AND METHODS: Between June 1992 and January 1994 the serum PSA level of 1400 asymptomatic men over 50-years-old was assessed. Those men with PSA levels < 4 ng/mL were not evaluated further. Those men with PSA levels of 4-10 ng/mL underwent digital rectal examination (DRE) and transrectal ultrasonography (TRUS) and biopsies were taken when there were significant findings on DRE and/or TRUS. If the PSA levels were > 10 ng/mL patients were submitted for DRE and TRUS and, even if both examinations were negative, random biopsies were taken. Where cancer was detected the tumour was staged and if it was a clinically confined tumour a radical retropubic prostatectomy was performed. The pathological and clinical stages of the disease were then compared. RESULTS: The majority of patients (95%) had PSA levels of < 4 ng/mL. Forty-nine men had PSA levels of 4-10 ng/mL and of these 28 were biopsied, which detected 12 (24.5%) carcinomas. There were 20 men with PSA levels > 10 ng/mL and among them 11 (55%) were found to have carcinomas. Combining these figures, among the 1400 men there were 69 cases with PSA levels > 4 ng/mL and, using DRE and TRUS, 23 patients (33%) were diagnosed as having prostatic adenocarcinomas. Among these, one had metastatic disease, three had lymph node micrometastases during surgical exploration and 19 underwent radical prostatectomies. The pathological and clinical stages agreed in only eight patients. CONCLUSION: Only eight patients can be considered as cured because of the screening protocol and even this result is overoptimistic, as the future biological behaviour of these tumours is unknown. Therefore we cannot recommend screening for prostatic carcinoma among asymptomatic men in Greece. PMID- 7542134 TI - Differential suppression of serum prostatic acid phosphatase and prostate specific antigen by 5-alpha-reductase inhibitor. AB - OBJECTIVE: To evaluate the effect of finasteride (Proscar) on the serum levels of prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in patients with benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: Thirty patients on finasteride therapy for BPH formed the study group. Serum PSA and PAP levels were monitored for 2 years while the patients were receiving finasteride. RESULTS: During 12 months of finasteride therapy the serum PSA was suppressed but serum PAP was unaffected. The baseline mean PAP value was 1.303 ng/mL prior to finasteride therapy; this changed to 1.510 ng/mL (P = 0.195) at 6 months and 1.166 ng/mL (P = 0.383) at 12 months. The serum PSA was 2.630 ng/mL at baseline, 1.757 ng/mL (P < 0.001) at 6 months and 1.545 ng/mL (P = 0.001) at 12 months. CONCLUSIONS: Further studies are warranted to determine if PAP has a role as a tumour marker in patients whose PSA is suppressed as a result of finasteride therapy. PMID- 7542135 TI - A urological diagnostic conundrum: schwannoma masquerading as an enlarged prostate. PMID- 7542137 TI - High failure rate associated with long-term follow-up of neoadjuvant androgen deprivation followed by radical prostatectomy for stage C prostatic cancer. AB - OBJECTIVE: To evaluate whether neoadjuvant androgen deprivation before radical prostatectomy decreases tumour stage in patients with stage C prostatic cancer and to estimate the efficacy of cancer control achieved with this form of treatment. PATIENTS AND METHODS: Thirty men (mean age 65 years, range 52-74) with clinical stage C adenocarcinoma of the prostate were included in a phase II trial of neoadjuvant androgen deprivation (luteinizing hormone-releasing hormone agonist and an antiandrogen) before radical prostatectomy. The timing and extent of the changes in serum prostate specific antigen (PSA) levels and both prostate and cancer volume were recorded. Twenty-six men underwent radical prostatectomy with pelvic lymphadenectomy, two had pelvic lymphadenectomy alone, one had pelvic lymphadenectomy with radiotherapy and one refused additional treatment despite significant reductions in tumour volume and PSA while undergoing androgen deprivation. RESULTS: The toxicity of the treatment was low. Significant reductions in prostatic volume (mean 35%), tumour volume (mean 50%) and PSA concentrations (mean 96%) occurred in all patients, with the maximum reductions recorded during the first 2 months of androgen deprivation. However, despite significant physiological changes in prostate and tumour volume, tumour stage was reduced in only four patients. Of the patients who were surgically staged 41% were ultimately identified as having more advanced disease, including lymph node metastases in 21%. Overall, with a mean follow-up of 32.7 months, 72% of patients had evidence of disease recurrence, including detectable PSA. Of 26 patients who underwent radical prostatectomy, local recurrence occurred in five (19%), distant recurrence in one (4%) and both local and distant recurrence in one (4%). CONCLUSIONS: This study suggests that tumour stage reduction is uncommon in patients with stage C prostatic cancer treated with neoadjuvant androgen deprivation followed by radical prostatectomy. Furthermore, local and distant recurrences, as well as detectable levels of PSA, are common after such treatment. PMID- 7542136 TI - Increased number of substance P positive nerve fibres in interstitial cystitis. AB - OBJECTIVE: To explore the presence of the neuropeptide substance P (SP) in the bladders of rats and humans and to investigate its relationship to mast cells (MCs) in interstitial cystitis (IC), a bladder disorder which occurs mostly in women and is characterized by frequency of voiding, nocturia and debilitating suprapubic pain. PATIENTS, MATERIALS AND METHODS: Bladder biopsies from eight women with untreated IC (mean age 36 years, range 29-58) and five control patients with no IC were analysed and compared with each other and with bladder tissue from 12 rats. Immunohistochemistry and image analysis were used to examine the density of SP-positive nerve fibres and their relationship with MCs. RESULTS: SP-containing nerve fibres were present in the bladder of both rats and humans. They were increased only in the submucosa, but not in the detrusor, of IC patients and were frequently seen in juxtaposition to MCs. CONCLUSION: SP, a neuropeptide secreted from sensory nerve endings, has been implicated in the pathophysiology of pain and has been shown to trigger MC secretion. Moreover, MC secretion by SP is augmented by oestradiol and bladder MCs have been shown to express high affinity oestrogen receptors. A functional relationship between SP and MCs may explain the pathophysiology of the neuro-inflammatory and painful nature of IC. PMID- 7542138 TI - Are pelvic computed tomography, bone scan and pelvic lymphadenectomy necessary in the staging of prostatic cancer? AB - OBJECTIVE: To investigate the effectiveness and economy of pelvic computed tomography (CT), bone scan and pelvic lymphadenectomy as staging modalities in patients undergoing radical prostatectomy. The use of prostate specific antigen (PSA) and Gleason's score as adjuncts to predict extracapsular disease were also evaluated and their economic implications examined. PATIENTS AND METHODS: Between January 1990 and June 1993, 861 men were newly diagnosed with prostate cancer, of whom 409 underwent surgery. All patients underwent pelvic CT scans and PSA analysis. Patients undergoing surgery had pre-operative bone scans and Gleason's scoring of their pathological tissue. RESULTS: Only 13 (1.5%) of 861 men had positive pelvic CT scans. Of the 409 patients who underwent surgery, all had negative pelvic CT and bone scans, and all underwent a modified pelvic lymphadenectomy; 192 (47%) had extracapsular disease. Only 15 (3.7%) patients who underwent surgery were found to have positive nodes. CONCLUSIONS: The use of pelvic CT and bone scans for clinical staging in patients with a PSA level of < or = 20 ng/mL should not be advocated because they have a very low yield and are not cost effective. We question the role of a modified pelvic lymphadenectomy for staging purposes, either by an open or laparoscopic procedure, because the yield of positive diagnoses is very low. PMID- 7542139 TI - The current status of the use of lasers in the treatment of benign prostatic hyperplasia. PMID- 7542140 TI - Structure of HIV-1 reverse transcriptase in a complex with the non-nucleoside inhibitor alpha-APA R 95845 at 2.8 A resolution. AB - BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that copies the RNA genome of HIV-1 into DNA. It is a heterodimer composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. HIV-1 RT is a crucial target for structure based drug design, and potent inhibitors have been identified, whose efficacy, however, is limited by drug resistance. RESULTS: The crystal structure of HIV-1 RT in complex with the non-nucleoside inhibitor alpha-anilinophenyl-acetamide (alpha-APA) R95845 has been determined at 2.8 A resolution. The inhibitor binds in a hydrophobic pocket near the polymerase active site. The pocket contains five aromatic amino acid residues and the interactions of the side chains of these residues with the aromatic rings of non-nucleoside inhibitors appear to be important for inhibitor binding. Most of the amino acid residues where mutations have been correlated with high levels of resistance to non-nucleoside inhibitors of HIV-1 RT are located close to alpha-APA. The overall fold of HIV-1 RT in complex with alpha-APA is similar to that found when in complex with nevirapine, another non-nucleoside inhibitor, but there are significant conformational changes relative to an HIV-1 RT/DNA/Fab complex. CONCLUSIONS: The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS. PMID- 7542141 TI - In vivo storage of XR family interspersed RNA in Xenopus oocytes. AB - Interspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNA (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. In small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentation constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentation constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis. PMID- 7542143 TI - Combination chemotherapy with epirubicin, cisplatin and 5-fluorouracil for the palliation of advanced gastric and oesophageal adenocarcinoma. The MRC Gastric Cancer Working Party and the British Stomach Cancer Group. PMID- 7542142 TI - Definition of local recurrence after surgery for rectal carcinoma. AB - Local recurrence rates of rectal carcinoma have been analysed among 284 patients in a prospective randomized multicentre trial of adjuvant preoperative radiotherapy for locally advanced rectal carcinoma. Wide variations in local recurrence rates are demonstrated depending on the definition of local recurrence employed and the subgroup studied. Thus after surgical operation alone, rates as high as 43.3 per cent or as low as 12.7 per cent can be calculated. After both adjuvant preoperative radiotherapy and operation the overall local recurrence rate is 12.8 per cent, although the local recurrence rate inside the radiotherapy field (true recurrence) may be as low as 2.3 per cent. It is recommended that local recurrence after operation for rectal carcinoma be defined as any detectable local disease at follow-up, occurring either alone or in conjunction with generalized recurrence, in patients who have undergone resection. A rate should be given both for all patients and for those operated on for cure, but not for the latter group alone as this could introduce bias. PMID- 7542144 TI - The effect of hetastarch on the stability of L-asparaginase during freeze-thaw cycling. AB - L-asparaginase, a therapeutic agent for the treatment of acute lymphoblastic leukemia, was evaluated for its susceptibility to cold denaturation. It was found that the enzyme derived from Erwinia chrysanthemi loses its activity when exposed to freeze-thaw cycling. When it was frozen at -40 degrees C and thawed, the enzyme lost 67.3% of its activity; whereas, when frozen in liquid nitrogen (-190 degrees C), it lost almost all of its activity. Rheological studies of hetastarch showed that its viscosity dramatically increases with decreasing temperature, suggesting that at sub-zero temperatures it will create a highly viscous environment around the enzyme. It is proposed that this highly viscous environment retards the rate of conformational changes leading to losses in activity. Hetastarch solutions of various concentrations and degrees of hydroxyethylation were evaluated for their protective ability against the freeze thaw denaturation of L-asparaginase. It was found that the cryoprotective effect of hetastarch with 0.8 degree of substitution at a concentration of 0.2% was sustained over many freeze-thaw cycles while that of the lesser substituted starch was not. The cryoprotective effect of hetastarch was compared to that of other commonly used additives such as glucose and lactose, which failed to protect the enzyme from freeze-thaw denaturation. In addition, the protective effect of a monomer of hetastarch was evaluated in order to distinguish whether the protective effect of hetastarch was due to physicochemical interactions with the individual monomer units or to its polymeric nature. The monomer showed significant cryoprotection through the first freeze-thaw cycle which was not sustained over additional freeze-thaw cycles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542145 TI - Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla. AB - In urethane anesthetized rats, iontophoretic application of morphine or alpha methylnoradrenaline (alpha-MNE) inhibited (80-100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of alpha-MNE was blocked selectively by the alpha 2-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75-100%) by low i.v. doses of clonidine (10-15 micrograms/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 +/- 0.9 spikes/s to 12.3 +/- 1.5 spikes/s; n = 8). During withdrawal these cells could still be inhibited (80-100%) by i.v. injection of clonidine (15 micrograms/kg). C-Fos expression induced by naltrexone precipitated withdrawal was examined in the brainstem of freely moving morphine dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenergic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive for alpha 2A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate and alpha 2-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal. Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal. PMID- 7542146 TI - Folic acid requirements of broilers. AB - 1. Dietary folic acid requirements of broilers were studied in three experiments using wheat- and maize-based practical diets. Requirements were assessed on the basis of performance and metabolic criteria. 2. Growth and food conversion efficiencies were optimised with supplements of 1.5 mg folic acid/kg added to basal mash starter diets. The dietary folic acid requirement of broilers was estimated to be in the range of 1.7 to 2.0 mg/kg. 3. Red blood cell phosphoribosylpyrophosphate concentrations and dihydrofolate reductase activities did not show consistent changes over the range of dietary folate concentrations studied but plasma folate concentrations responded markedly to dietary folate supplementation. 4. Adding choline to diets in amounts greater than the normal requirement did not spare the requirement for folic acid. 5. It is suggested that minimum folic acid supplements for pelleted practical diets should be in the order of 2.5 to 3 mg/kg. PMID- 7542147 TI - Antenatal screening for neural tube defects. PMID- 7542148 TI - Soluble adhesion molecules and interleukin-2 receptor concentrations in patients with autoimmune chronic hepatitis. AB - OBJECTIVE: To test the hypothesis that elevated serum levels of intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), (s)E selectin, (s)P-selectin and soluble interleukin-2 receptor (sIL-2R) occur in patients with biochemically inactive autoimmune hepatitis (AICH) compared with controls. Such a finding would suggest continued immune activation, which in the long-term would contribute to the development of cirrhosis in such patients. METHODS: sICAM-1, sVCAM-1, sE-selectin, sP-selectin and sIL-2R were measured in serum from 50 patients with AICH, divided into two groups according to whether their serum alanine aminotransferase (ALT) levels were normal (below 40 IU/l) or increased (above 40 IU/l). Fourteen healthy subjects served as controls. Eight patients were followed after the induction of remission with increased immunosuppressive therapy. RESULTS: Elevated levels of sICAM-1, sVCAM-1 and sE selectin were found in patients with AICH compared with controls, independent of their ALT concentration. Only sICAM-1 was significantly elevated in the patients with active disease compared with patients with inactive disease. sIL-2R concentrations were significantly higher in patients with active disease (ALT above 40 IU/l) compared with controls. sP-selectin concentrations were similar in controls and patients with AICH. In patients with clinical and biochemical relapse, increased steroid therapy was followed by a significant reduction in sICAM-1, sVCAM-1 and sIL-2R. The response of sE-selectin was more variable. sICAM 1 remained elevated in five of the eight patients treated with increased steroid therapy despite normalization of their transaminase levels, sIL-2R, sE-selectin and sVCAM-1 levels. In 52% of patients with biochemically inactive AICH (ALT below 40 IU/l), sICAM-1 was above the control range. CONCLUSIONS: These data suggest that patients with AICH have ongoing immune activation despite biochemically inactive disease. This may result in low grade hepatic inflammation and may explain the progression to cirrhosis in patients with AICH despite immunosuppressive therapy. PMID- 7542149 TI - Ectopic pregnancy: what's new? PMID- 7542151 TI - Variations in activity of the human natural anti-Gal antibody in young and elderly populations. AB - BACKGROUND: Anti-Gal is a natural antibody which constitutes approximately 1% of circulating IgG in humans and which interacts specifically with the mammalian carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha galactosyl epitope). The purpose of this study was to determine whether there are age-associated changes in the affinity of anti-Gal in elderly individuals. METHODS: The age-associated variations in affinity of anti-Gal were determined by the extent of this antibody binding to solid-phase alpha-galactosyl epitopes (mouse laminin) in ELISA, performed at various serum dilutions with serum samples from 140 elderly and 127 young individuals. The data were expressed as the mean of optical density (O.D.) values measured at serum dilutions of 1:40, 1:80, and 1:160, and correlated with the affinity of anti-Gal as measured in equilibrium dialysis. RESULTS: The proportion of individuals with low-affinity anti-Gal is sixfold higher in the elderly than in the young population. Whereas low-affinity binding of anti-Gal (mean O.D. value < or = 1.0) was observed in only 6% of the young population, as many as 35% of the elderly population displayed such low affinity anti-Gal. Anti-Gal affinity in ELISA assay correlated with the affinity of this antibody as measured in equilibrium dialysis. CONCLUSION: The proportion of individuals with low affinity anti-Gal increases in the elderly population in comparison with the young population. The possible molecular mechanisms which may result in the age-associated decrease in the antibody affinity are discussed. PMID- 7542150 TI - Alteration of cytokeratin expression in oral lichen planus. AB - The purpose of this investigation is to examine the possible biochemical and topographic cytokeratin alterations in lichen planus of oral mucosa. Biopsy samples of clinically normal buccal mucosa (n = 5), normal gingiva (n = 5), lichen planus from buccal mucosa (n = 5), and lichen planus from gingiva (n = 5) were obtained from patients of both sexes. Cytokeratin expression was determined by means of immunohistochemical labeling with use of a battery of monoclonal antibodies against cytokeratins and filaggrin and two-dimensional gel electrophoresis. In buccal mucosa, which is not keratinized cytokeratins 4 and 13 are expressed in the majority. In buccal mucosa lichen planus, the appearance of cytokeratins 1, 2, 10, and 11 coincides with a decrease in cytokeratins 4 and 13 and a moderate increase in cytokeratins 6, 16, 17, and 19. In normal gingiva, which is normally keratinized, the main cytokeratins are 1, 2, 10, and 11. In gingival lichen planus, a slight decrease in these cytokeratins and in cytokeratin 13 expression was noted. Finally, alterations in cytokeratins 5 and 14, explained by marked alterations of basal cells, were observed. The battery of antibodies used in this study, in correlation with two-dimensional gel electrophoresis, could represent useful diagnostic tools that enable the distinction between inflammatory keratosis and so-called quiescent lichen planus. Moreover, this work showed that cytokeratins 1, 2, 10, and 11 and filaggrin are sensitive tools that may help detect early relapse before clinical exacerbation. Finally, these biochemical techniques may be useful to follow the evolution of lichen planus under treatment. PMID- 7542152 TI - Rationale for the use of alpha-blockers in the treatment of benign prostatic hyperplasia (BPH). PMID- 7542153 TI - Isolation and purification of a high molecular weight substance from human urine which inhibits calcium oxalate crystal growth. AB - A high molecular weight inhibitor of calcium oxalate crystal growth in human urine was investigated. Three inhibitors were isolated by DEAE-Sephacel ion exchange chromatography and, of these, the substance we named "Peak 3 protein" seemed to be the main inhibitor in human urine. Peak 3 protein and several was purified by fast protein liquid chromatography and polyacrylamide gel electrophoresis. This substance, with a molecular weight of 30 kDa, did not contain uronic acid and its inhibitory activity decreased after digestion with proteinase. The difference between Peak 3 protein and several inhibitors previously reported was investigated but no clear difference could be found. The fact that it was the protein structure which was responsible for the inhibitory activity and the fact that Peak 3 protein probably possessed many side-chains which did not contribute to the inhibitory activity influenced the outcome of the investigation. PMID- 7542154 TI - M-VEC (methotrexate, vinblastine, 4'-epirubicin and cisplatin) combined with glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the treatment of transitional cell carcinoma of urothelium: reduction in toxicity produced by rhG-CSF. AB - Forty-six patients with urothelial cancer were treated with a systemic chemotherapeutic regimen consisting of methotrexate, vinblastine, 4'-epirubicin and cisplatin (M-VEC) in conjunction with glycosylated recombinant human granulocyte colony stimulating factor (rhG-CSF); then 33 were evaluated for response. Complete response was observed in 7 patients (21%) and partial response in 13 (39%). As far as the toxic effects of this treatment are concerned, mucositis of a minimum grade and leukopenia greater than grade 3 occurred in 5% and 10% of the patients, respectively; there were no cases of nadir sepsis and drug-related death. Minor toxicity such as nausea vomiting occurred in 81% of patients, and no patient required either dose-reduction or a delay of more than 5 d before starting of the second cycle. Thus, it may be concluded that M-VEC chemotherapy combined with rhG-CSF is useful in the treatment of urothelial cancer, especially when used as a neoadjuvant. PMID- 7542155 TI - Neodymium:YAG laser ablation of the prostate gland--acute perioperative morbidity and short-term outcome. AB - Twenty-three patients with symptomatic bladder outlet obstruction due to benign prostatic hyperplasia were successfully treated with laser prostatectomy using the Urolase right-angle firing neodymium: Yttrium Aluminum Garnet laser fiber. Most of the patients had health problems of moderate severity. Their conditions were compared preoperatively and 3 mo postoperatively. Symptom scores decreased from an average of 23.9 to 7.0. Symptomatic relief was achieved in 95.7% of patients. Peak urinary flow rates increased from an average of 6.4 to 17.0 ml/s. The residual urine decreased from an average of 45.2 to 22.9 ml. The volume of the prostate gland measured by sonography decreased from an average of 32.8 to 25.7 ml. Virtually no blood loss was noted intraoperatively. Acute urinary retention occurred immediately following removal of the catheter in 3 out of 17 (17.6%) patients without a percutaneous cystostomy tube. Acute epididymitis developed in 5 out of 20 (25.0%) patients without bilateral vasectomy. Based on these results, laser prostatectomy may be concluded to be a safe and effective alternative to standard transurethral resection of the prostate gland, particularly in the case of high-risk patients. PMID- 7542156 TI - Transurethral microwave thermotherapy for benign prostatic hyperplasia: patient characteristics in good and poor responders. AB - Transurethral microwave thermotherapy (TUMT) has been shown to produce a clinical benefit in patients with symptomatic benign prostatic hyperplasia. In order to identify the features of the ideal candidate, a retrospective analysis was conducted in 32 patients who were followed for 2 mo or more. Good responders (GR) were defined as having their Siroky peak flow rate (PFR) standard deviation (SD) increase by < 0.5 or a decrease in the International Prostatic Symptom Score (I PSS) of > 10 (22 patients). Poor responders (PR) were defined as having their PFR SD increase by < or = 0.5 and their I-PSS decrease by < or = 10 (10 patients). The prostate volume, pre-TUMT I-PSS and intravesical opening pressure were significantly greater in the GR group, while there were no significant differences between the 2 groups for the other baseline patient characteristics: age, prostate length, PFR, PFR SD, post-voiding residual volume and quality of life. Concerning the operational parameters, significantly more total energy was delivered to the prostate in the GR group (mean 131 kJ) than in the PR group (mean 101 kJ). Moreover, the 7 patients with anti-androgen therapy pre-TUMT received less total energy and 5 of the 7 were poor responders. These results suggest that patients with apparent obstructive symptoms and with moderate enlargement of prostate could benefit more from this less invasive therapy. Clinical response seems to be dose-dependent and patients with a history of recent anti-androgen treatment may have a less favorable response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542158 TI - Prostate specific antigen and prostate acid phosphatase declines after estramustine phosphate withdrawal: a case report. AB - We report a case of advanced prostate cancer in which an initial response to hormonal therapy with surgical castration and estramustine phosphate (EMP) was followed by disease progression, as shown by sequential elevations in serum prostate specific antigen (PSA) and prostate acid phosphatase (PAP) and the development of new symptoms, during maintenance endocrine and anti-cancer chemotherapy. Discontinuation of EMP resulted in sustained reductions in serum PSA and PAP levels and a sustained improvement in symptoms. PMID- 7542157 TI - Diurnal rhythm of serum gamma-seminoprotein in patients with untreated prostate cancer: comparison of the original and revised assay kits. AB - To compare levels of gamma-seminoprotein (gamma-Sm) assayed by original and revised assay systems, blood was obtained every 4 h over a 32-h period from 8 untreated prostate cancer patients. Serum levels of prostate specific antigen (PSA) were also examined. In 6 patients, the coefficient of variation (CV) of the serum levels assayed by the revised assay was significantly different from that of the intra-assay samples. In contrast, the CV of the gamma-Sm serum levels assayed by the original assay differed significantly from that of the intra-assay samples in only 2 patients. The fluctuations in gamma-Sm assayed by the revised assay were, at least in part, similar to those of the PSA serum levels in all patients. The mean CV of the gamma-Sm serum levels assayed by the revised assay was significantly larger than that for levels measured by the original assay. After treatment, the rate of decrease in gamma-Sm serum levels determined by the original assay differed from that in the serum levels of PSA and prostatic acid phosphatase. These results indicate that the original assay for gamma-Sm do not detect diurnal differences in serum gamma-Sm levels, even at levels below 20 ng/ml. These observations indicate that the analysis of data obtained using the original gamma-Sm kit should be interpreted with caution. PMID- 7542159 TI - Transurethral microwave thermotherapy for benign prostatic hyperplasia: clinical results after a 1-year follow-up. AB - Since September 1992, 63 patients with symptomatic benign prostatic hyperplasia (BPH) have been treated with transurethral microwave thermotherapy (TUMT) using the Prostatron device. The International Prostate Symptom Score (I-PSS) and quality of life (QOL) score were used to evaluate subjective symptoms. The mean I PSS (total, irritative and obstructive scores) and QOL scores had decreased by 40, 38, 45 and 40%, respectively, at 12 months (p < 0.0001). While the mean peak flow rate had increased by 72% (p < 0.001). The clinical efficacy at 12 months was 42%, using a modification of the response criteria proposed at the 2nd International Consultation on Benign Prostatic Hyperplasia. There were no significant differences in the baseline and treatment parameters between those who responded favorably to TUMT and those who did not. The total thermal dose delivered to the prostate did not predict clinical response. However, there was a positive correlation between I-PSS or QOL at baseline and % reduction at 3, 6 and 12 months, and a negative correlation between peak flow rate at baseline and % increase at 3 and 6 months. There were no major complications associated with TUMT during the follow-up period. In summary, our 1-year clinical results are compatible with previous reports, suggesting that TUMT is a safe, effective and lasting non-surgical treatment for BPH. However, evaluation of efficacy should be based on uniform criteria to facilitate comparisons of different clinical trials. The most suitable patient profiles for TUMT could not be identified by retrospective analysis. PMID- 7542161 TI - Response criteria for the therapeutic efficacy of treatment for benign prostatic hyperplasia. AB - Response criteria for the therapeutic efficacy of treatments for benign prostatic hyperplasia (BPH) were proposed following the International Consultation on BPH held in 1993. In the present study, we validated the criteria and proposed a simplified form, which consists of the responses of three parameters: symptom score and quality of life assessed by the international prostate symptom score and the maximum flow rate. Each of the three individual parameters is evaluated as one of four response grades: excellent, good, fair or poor, and the number of response grade of the three parameters determines the overall response. Excellent and Good responses are regarded as effective, and fair and poor are regarded as not effective. The validity of the response criteria was assessed by comparing the responses determined by these criteria with those made by physicians in charge using a group of 225 patients receiving various treatments. The agreement rates on effectiveness of overall responses between the response criteria and physicians were 77% for a multicenter trial of medical treatment (n = 94), 100% for TURP (n = 23), 92% for laser treatment (n = 47), 80% for thermal treatment (n = 26) and 78% for alpha-blockers (n = 35), respectively. Altogether, 88% (198 of 225 cases) were evaluated accurately regarding effectiveness. Addition of prostate volume to the battery of response parameters made little contribution to diagnostic accuracy. Deletion of any of the other three parameters, however, significantly compromised the quality of assessment. These results suggest that the proposed criteria may be useful as the standard method for the assessment of the clinical efficacy of BPH treatment. PMID- 7542160 TI - Transurethral balloon laser hyperthermia for chronic non-bacterial prostatitis: a clinical trial. AB - Transurethral balloon laser hyperthermia (TUBAL-H) was performed on five patients with chronic non-bacterial prostatitis who failed to respond to conventional treatment administered for more than two years. The prostatic interstitial temperature during treatment was measured and the safety and efficacy of this treatment was assessed. TUBAL-H was performed at a target temperature of 43 degrees C (5 mm depth) and at a laser power of 30 watts with urethral cooling for 20 min in the first three patients and for 30 min in the remaining two patients. The prostatic interstitial temperature at a depth of about 5 mm from the urethral surface ranged from 40.5 to 43.0 degrees C during treatment. The temperature of the urethra ranged from 31.5 to 39 degrees C and that of the rectum remained below 39.5 degrees C. After treatment, no abnormal findings were noted in any of the hematological and biological tests carried out, including prostatic specific antigen. The leukocyte count in expressed prostatic secretions fell to less than five cells per high-power field in four of the five patients after three months. A complete improvement in symptoms was observed in one patient, partial improvement in three, and no improvement in one. Based on these results, TUBAL-H was considered to be safe and a suitable treatment for patients with chronic prostatitis. PMID- 7542162 TI - Disease progression in stage A prostate cancer. AB - To study the disease progression in stage A prostate cancer, 212 patients with stage A from a group of 3370 patients who underwent transurethral resection of the prostate, or subcapsular prostatectomy during the period 1972-1991 were followed. The stage A cases were subdivided into 103 A1 patients and 109 A2 patients and their subsequent course was followed for an average of 41.7 months (6-174 months). Progression to clinical cancer was found in 12 patients, 2 from A1 and 10 from A2 groups. This progression was evident 40.5 months (14-130 months) after prostatectomy. Eight (67%) of these cases responded to endocrine therapy. The rate of expression of ras p21, and the number of argyrophilic nucleolar organizer regions in stage A cancer cells were greater in progressing than in non-progressing cases. These results indicate that stage A cancer with progression arises mainly from the A2 subgroup and exhibits a distinctly proliferative potential even at small foci. PMID- 7542163 TI - Management of chronic orchialgia of unknown etiology. AB - The treatment of patients with chronic unilateral or bilateral orchialgia, defined as intermittent or constant testicular pain of greater than 3 months and of unclear cause, is difficult. This pain significantly interferes with the daily activities of the patient. We have seen 12 patients with chronic orchialgia of unknown etiology and each had a normal history, physical examination and normal scrotal sonogram. Three patients were treated with nonsteroidal anti-inflammatory drugs and obtained partial pain relief. Three patients underwent spermatic cord nerve blockade using a combination of 1% lidocaine and 40 mg methylprednisolone and experienced partial pain relief. Four patients underwent inguinal orchiectomy after failing conservative management: three reported complete relief of pain, and the other partial relief. Two patients had bilateral transrectal injections of local anesthetic (5 ml bupivacaine) and methylprednisolone into the region of the pelvic plexus under transrectal ultrasound guidance. They were successfully treated with this injection technique. On the basis of our results, we recommend transrectal blockade of nerves from the pelvic plexus or inguinal orchiectomy as the procedure of choice for patients in whom medical treatment fails. PMID- 7542164 TI - Volunteers, doctors take palliative care into the community. AB - The face of palliative care is changing. In Ontario's St. Catharines region there has been a concerted effort to make it more of a community-based procedure. A local college even teaches a 2-year course in palliative care. The trend is expected to continue because Canadians are living longer, and more frail elderly people will be dying at home. Dr. Sandra Hartman, a palliative-care consultant, says physicians interested in palliative care must remember that there is more to it than providing medical assistance. She considers bereavement counseling for the patient's family a necessary part of follow-up preventive care. PMID- 7542165 TI - Dr. Balfour Mount and the cruel irony of our care for the dying. AB - Dr. Balfour Mount of Montreal thinks that the health care system, because of its fixation on disease processes, may have forgotten that it also has a mandate to alleviate suffering. "We need to recapture that vision," says Mount, who describes palliative medicine as a "rich combination" of clinical pharmacology, rehabilitation medicine and internal medicine. Mount says there is a cruel irony in our care of the dying. "Although these are the sickest people in our health care system, when medical technology doesn't know what to do, the quality and quantity of care falls away. How can we justify that?" PMID- 7542166 TI - Suppression of tumor growth in vivo by local and systemic 90K level increase. AB - Expression levels of the immunostimulatory 90K antigen in mammary carcinoma, glioblastoma, and other tumor-derived cell lines inversely correlate with their tumorigenicity in athymic mice. Engineered enhancement of 90K expression results in significant (> 80%) tumor growth inhibition, not by direct action on the tumor cell, but by stimulation of the residual cell-mediated immune defense of the nude mouse. Enhanced 90K level effects are both localized and systemic and involve induction of ICAM-1 and VCAM-1 in the tumor endothelium. The findings presented suggest a role for 90K as a molecular alarm signal for the body's cellular defense against pathogens, which in a subset of tumors is suppressed to allow cancer progression. PMID- 7542167 TI - Functional evidence that cell surface galectin-3 mediates homotypic cell adhesion. AB - Galectin-3 (Gal-3) is a beta-galactoside-binding protein with M(r) approximately 30,000. Cell surface Gal-3 is postulated to be involved in homotypic aggregation of tumor cells in the circulation during metastasis through attachment to a complementary serum glycoprotein(s), which serves as a cross-linking bridge between adjacent cells. To test this hypothesis a recombinant strain of baculovirus encoding Gal-3 was used to infect Sf9 insect cells, which lack endogenous Gal-3. Immunoblotting and indirect immunofluorescence studies revealed that the infection with recombinant virus conferred Gal-3 expression on Sf9 cells, and the Gal-3 was localized on the cell surface as well as in the cytoplasm. Sf9 cells infected with recombinant virus underwent homotypic aggregation in the presence of exogenous glycoprotein (i.e., asialofetuin), whereas control cells uninfected or infected with wild-type virus did not. Lactose and Fab' fragments of anti-Gal-3 antibodies markedly inhibited the cell cell aggregation. Moreover, cosuspension of Sf9 cells infected with the recombinant virus with uninfected cells in the presence of asialofetuin resulted in a preferential cell-cell adhesion of the Gal-3-expressing cells. These results directly demonstrate the ability of cell surface Gal-3 molecules to mediate homotypic cell adhesion by bridging through branched, soluble complementary glycoconjugates. PMID- 7542168 TI - Effect of high-intensity focused ultrasound on human prostate cancer in vivo. AB - Transrectal high-intensity focused ultrasound (HIFU) was recently established as a highly effective means of inducing contact and irradiation-free intraprostatic coagulative necrosis. This technique, therefore, appears potentially useful for treating localized prostate cancer (PC). To evaluate this issue, a total of 29 human prostates with localized cancer was subjected to HIFU treatment in vivo before radical retropubic prostatectomy. HIFU therapy was performed with the use of HIFU transducers with focal lengths of 3.0 cm (n = 3), 3.5 cm (n = 19), and 4.0 cm (n = 7), and the site intensity was varied from 1260 to 2000 W/cm2. The extent of intraprostatic necrosis was determined by planimetrical analysis of whole mount prostatic sections. Transrectal HIFU consistently induced sharply delineated intraprostatic coagulative necrosis within the target area, whereas alterations of perioprostatic structures were never observed. The cross-sectional area of necrosis increased from 1.1 +/- 0.7 cm2 (SD; n = 3; 3.0-cm focal length; 1428 W/cm2) to 1.2 +/- 0.7 cm2 (n = 2; 3.5-cm focal length; 1428 W/cm2), 1.8 +/- 0.17 cm2 (n = 8; 3.5-cm focal length; 1680 W/cm2), 2.8 +/- 0.32 cm2 (n = 9; 3.5 cm focal length; 2000 W/cm2) and 3.8 +/- 0.4 cm2 (n = 7; 4.0 cm focal length; 1260 W/cm2). HIFU beam transmission and the therapeutic effect were comparable in benign and malignant prostatic tissue. Interstitial thermometry (n = 6) revealed maximum intraprostatic temperatures in the focal zone up to 98.6 degrees C. Outside the focal zone and on the rectal wall, no significant temperature rises were noted. Subsequently, HIFU was applied to unilateral histologically proven T2a/T2b PC (n = 10) in an attempt to destroy all cancer before radical retropubic prostatectomy. PC was always correctly targeted. In 7 individuals, PC was partially (mean, 53%; range, 38-77%) destroyed; in the remaining 3 cases the entire tumor was ablated. Although these histological data permit no definitive conclusion on the clinical efficacy of this approach, transrectal HIFU seems to be an attractive novel minimally invasive treatment option for localized PC. PMID- 7542169 TI - Detection of cancer micrometastases in lymph nodes by reverse transcriptase polymerase chain reaction. AB - There are few DNA-based studies that detect cancer micrometastases in lymph nodes. We have assayed for the specific detection of carcinoembryonic antigen (CEA)-expressing carcinoma cells in the lymph nodes of patients with gastrointestinal or breast carcinomas. A CEA-specific nested reverse transcriptase (RT)-PCR assay was optimized using limiting dilutions of a CEA positive cancer cell line mixed with normal lymphocytes. The expression of CEA mRNA was studied in 100 carcinoma tissues, 75 normal mucosal tissues, and 15 lymph nodes from patients with cholelithiasis. Each of 117 lymph nodes from 13 patients with carcinoma was divided into two pieces: one was used for histological examination and the other for RT-PCR, and the results were compared. The sensitivity ratio was one CEA-expressing cancer cell detected in 1 x 10(5) normal lymphocytes. All carcinoma tissues and normal mucosal tissues expressed CEA mRNA, while no amplification was detected in any control lymph nodes. Thirty of 117 lymph nodes were histologically involved by carcinoma cells, and all of these yielded the expected product by RT-PCR. Of the remaining 87 histologically negative nodes, CEA mRNA was detected in 47 lymph nodes by RT-PCR. The positive rate increased from 26% by histological examination to 66% by RT-PCR. The assay by CEA-specific nested RT-PCR is not only sensitive but widely applicable for the detection of cancer micrometastases in lymph nodes. This method may lead to an earlier diagnosis and treatment of patients with subclinical lymph node metastasis. PMID- 7542170 TI - Effects of stem cell factor on the growth and radiation survival of tumor cells. AB - Recombinant human stem cell factor (SCF) binds to the c-kit receptor on human bone marrow progenitor cells and enhances their survival following irradiation. Since the c-kit receptor has also been detected on malignant cells, experiments were performed to study the effect of SCF on the proliferation and radiation survival of a variety of both c-kit-positive and -negative human tumor cell lines using [3H]thymidine incorporation and colony formation assays. The addition of SCF to both c-kit-positive and -negative cell line cultures had no significant effect on the stimulation index (in [3H]thymidine assay). In contrast, colony formation by H69 (small cell lung cancer cell line), H128 (small cell lung cancer cell line), and HEL (erythroid leukemia cell line) cells was enhanced by SCF in a dose-dependent manner, but SCF did not promote the in vivo growth of H128 xenograft tumors in terms of graft rate, time from implantation to tumor detection, or tumor size. Furthermore, SCF did not significantly increase the surviving fraction of either c-kit-positive or -negative cell lines following radiation, and there were no statistically significant differences between D0 [defined by the slope of the terminal exponential region of the two-component (single-hit multitarget model) survival curve where slope = 1/D0], Dq (quasithreshold dose), n (extrapolation number), alpha, and beta values for any of the cell lines studied that were irradiated with and without SCF. Finally, nude mice with transplanted human LG425 cutaneous T-cell lymphoma (c-kit positive) were treated with 10 Gy with or without SCF (100 micrograms/kg i.p. 20 h before, 2 h before, and 4 h after irradiation). There were no significant differences in the median tumor quadrupling time between groups that received either no treatment or SCF alone, or between groups treated with 10 Gy and SCF or 10 Gy alone (P > 0.05). These results are encouraging and suggest that SCF does not stimulate tumor cell proliferation in vivo or enhance the survival of tumor cells following irradiation. PMID- 7542172 TI - [Care of tuberculosis of the thoracic and lumbar spine before and after surgery]. PMID- 7542173 TI - [Study of two methods of catheterization after radio frequency hyperthermia treatment of BPH]. PMID- 7542171 TI - Autocrine growth of transitional cell carcinoma of the bladder induced by granulocyte-colony stimulating factor. AB - Granulocyte-colony stimulating factor (G-CSF) produced by nonhematopoietic malignant cells has been reported to be capable of inducing a leukemoid reaction in the host through intense stimulation of leukocyte production. Furthermore, this is frequently associated with aggressive tumor cell growth and a detrimental clinical outcome. In this study, we identified bladder cancer cells producing G CSF with the expression of the functional receptor, which provides direct evidence of autocrine growth of bladder cancer cells induced by G-CSF. The cancer cells used in this study were obtained from a 76-year-old man who had a metastatic transitional cell carcinoma of the bladder and who demonstrated marked leukocytosis, his peripheral blood leukocyte count was 94,900 leukocytes/mm3, and his serum G-CSF level was 103 pg/ml. The culture medium in which the cancer cells were grown exclusively contained a significant amount of G-CSF (5560 pg/ml). Significant G-CSF mRNA expression and G-CSF receptor mRNA expression in the cultured cells were demonstrated by the reverse transcription-PCR method. In addition, binding studies with the use of radiolabeled recombinant G-CSF demonstrated the presence of high-affinity G-CSF binding receptors on the cultured cancer cells. Finally, the proliferation of the cultured cancer cells was stimulated by exogenous G-CSF administration, and this stimulation was inhibited by adding anti-G-CSF antibody, as demonstrated by both the flow cytometric bromodeoxyuridine incorporation technique and the [3H]thymidine incorporation assay. These results strongly suggest that G-CSF production by the bladder cancer cells studied augments autocrine growth. Therefore, we recommend exercising caution in the clinical use of G-CSF for bladder cancer patients. PMID- 7542174 TI - DNA-damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver. AB - The synthetic progestin cyproterone acetate (CPA) has been recently shown to elicit DNA repair synthesis in cultured rat hepatocytes and to form adducts with rat hepatocyte DNA in vitro and in vivo. In the present study we have examined the genotoxic potential of the structural analogues of CPA, chlormadinone acetate (CMA) and megestrol acetate (MGA) in rat liver cells. CPA strongly induced DNA repair synthesis in hepatocyte cultures from females but not from males. In contrast, CMA and MGA (2-50 microM) did not detectably increase repair synthesis in cultured hepatocytes from either gender. CMA and MGA, however, caused the formation of DNA adducts detectable by the 32P-postlabelling technique. At a concentration of 30 microM, between 30 and 50 adducts/10(9) nucleotides were found with MGA and CMA in cultured hepatocytes of female rats, and between 5 and 20 adducts/10(9) nucleotides were found in hepatocytes of males. By comparison, 30 microM CPA has been found to produce 1670 adducts/10(9) nucleotides in hepatocytes from female rats. CMA and MGA also induced low levels of DNA adducts in vivo. When female rats were treated with 100 mg/kg of CMA or MGA per os, the adduct levels were 2 and 19 adducts/10(9) nucleotides respectively. The results indicate that both CMA and MGA show some genotoxicity in rat liver cells, which is, however, much lower than that for CPA. Our findings further suggest that the high genotoxicity of CPA is associated with the presence of the 1,2 alpha methylene group, which is absent in CMA and MGA. PMID- 7542176 TI - Pigmented subcutaneous spindle cell tumors in native gizzard shad (Dorosoma cepedianum). AB - An epizootic of pigmented subcutaneous spindle cell tumors affected nearly 25% of the adult gizzard shad (Dorosoma cepedianum) sampled from Lake of the Arbuckles in central Oklahoma over a 2 year period. Grossly, the tumors were primarily distributed over the head, trunk and fins as superficial raised masses that were almost always darkly pigmented. Histologically, they were located in the dermis, had a variable amount of connective tissue, and consisted of cells in a variety of forms and arrangements. Most tumors were composed of fusiform or spindle cells arranged in wavy bundles, whirling patterns or interwoven fascicles. Pigmentation was attributed to large dense deposits of melanin or to scattered individual melanin-containing cells. Immunohistochemical detection of proliferating cell nuclear antigen revealed a high proliferative activity in the spindle cells. Electron microscopy showed that the tumors were composed of several cell types, including host reactive cells, melanocytes in stages of maturity, and fibroblast like cells. Tumor cells had neither cell-to-cell junctions nor an external lamina. Although the cell of origin of the tumors was not identified, evidence points toward melanocytes or, possibly, nerve sheath cells. However, an origin from fibroblasts or some other poorly differentiated cell cannot be ruled out. The etiology of the tumors was not determined. Fractionation of lake water and sediment samples followed by GC-MS analysis revealed no carcinogenic compounds. A retroviral etiology is unlikely because assays for reverse transcriptase in tumor homogenates were negative, and no evidence of viral particles was found in specimens examined by electron microscopy. PMID- 7542175 TI - Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated. AB - Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n-6 and n-3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 microM arachidonic acid (20:4 n-6) resulted in a rapid and transient loss of communication competence. The response to 20 microM 20:4 (n-6) was prolonged (> 210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20:4 (n-6). The fatty acids, 18:3 (n-6), 20:5 (n-3), 22:6 (n-3) and the 15-hydroxy- and the 15 hydroperoxy-derivatives of 20:4 (n-6) were also powerful inhibitors of GJC, while 23:4 (n-6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20:0, and the methyl ester of 20:4 (n-6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20:4 (n-6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n-6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20:4 (n-6) was antagonized by preincubating WB cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethyl xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20:4 (n-6). As expected, other inhibitors of gap junctional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancement in the phosphorylation of Cx43 protein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542177 TI - Positive immunohistochemical staining of p53 and cyclin D in advanced mouse skin tumors, but not in precancerous lesions produced by benzo[a]pyrene. AB - Squamous cell carcinomas (SCCs) of the mouse skin, as well as several types of preinvasive carcinoma precursor lesions, were produced by complete carcinogenesis protocols with benzo[a]pyrene (B[a]P). Groups of mice were studied histologically at several time points. Tumors and precursor lesions were systematically counted on microscope slides. The main feature of tumor development using this ubiquitous human carcinogen was the sequential appearance of in situ flat lesions with progressive degrees of dysplasia. These changes, preceding the development of SCCs, were observed 20 weeks after beginning the carcinogen treatments. At this time point, in situ lesions outnumbered SCC approximately 10:1 at the higher total carcinogen dose examined. Ten weeks later, this ratio was approximately 1:1. With the lower total carcinogen dose protocol, progression was delayed since at 27 weeks preinvasive lesions outnumbered SCCs approximately 8:1. In addition to the in situ lesions, papillomas and keratoacanthomas were noted with the high B[a]P dose protocol, but tended to disappear at the end of the experiment, also indicating their probable role as SCC precursors. A study of histochemical markers showed that gamma-glutamyltranspeptidase (GGT) and keratin 13, although good markers of malignant changes in early papillomas produced by two-stage carcinogenesis protocols, were mainly negative in dysplastic lesions produced by complete carcinogenesis with B[a]P. Immunohistochemical detection of p53 showed that 50% of SCCs were positively stained, whereas only 3% of in situ lesions were p53 immunoreactive. Similarly, 62% of SCCs were immunohistochemically positive for cyclin D, but no precursor lesions were positive. Molecular analysis of the tumors showed the absence of H-ras mutations. No amplification of the cyclin-D-1 gene was detected in eight SCCs examined. Collectively, these findings indicate that preinvasive in situ lesions are frequent during early stages of carcinogenesis when B[a]P is used in a complete carcinogenesis protocol. Although the absence of p53 immunoreactivity in this mouse model differs from the observed changes in human premalignant squamous lesions, the sequence of morphological changes and the final incidence of p53 and cyclin D staining abnormalities are very similar to the well-known alterations that take place during human squamous carcinogenesis. PMID- 7542178 TI - Micronuclei induced by modulators of methylation: analogs of 5-azacytidine. AB - Jones and coworkers demonstrated a qualitative correlation between 5-azacytidine and some of its analogs in inducing changes in cell morphology and their ability in preventing DNA methylation. Previously, we evaluated the same compounds to determine their ability to induce trifluorothymidine (TFT) resistance in L5178Y mouse cells and found that their mutagenic potency also correlated with their reported ability to induce morphological changes in C3H10T1/2 cells. Here, we examine four of the same analogs, 5-fluoro-2'-deoxycytidine, 5-azacytidine, 5,6 dihydro-5-azacytidine and 6-azacytidine, to find out if micronuclei induced by these compounds correlated with these effects. The most cytotoxic analog was 5 fluoro-2'-deoxycytidine, followed by 5-azacytidine. 5,6-Dihydro-5-azacytidine and 6-azacytidine were substantially less cytotoxic. All four compounds induced micronuclei. The lowest dose ranges at which responses were observed for micronucleus induction were -0.04 microM for 5-fluoro-2'-deoxycytidine, 0.2 microM for 5-azacytidine and 10-20 microM for 5,6-dihydro-5-azacytidine and 6 azacytidine. Lack of kinetochore staining in most of the micronuclei indicated that all four compounds were clastogenic. We note a general trend in the biological activity of these analogs: compounds that are specifically blocked at the 5 position such as 5-azacytidine and 5-fluoro-2'-deoxycytidine effect changes in cell morphology, cytotoxicity, TFT resistance and the induction of micronuclei at very low doses. 5-Azacytidine analogs that possess more chemically accessible 5 positions such as 5,6-dihydro-5-azacytidine and 6-azacytidine either require doses that are orders of magnitude greater to induce these effects or are unable to induce changes in cell morphology and TFT resistance at doses below which the compound is lethal to the cells. PMID- 7542179 TI - Requirement for protein kinase C activation in basic fibroblast growth factor induced human endothelial cell proliferation. AB - The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)-induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca2+ increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca2+ and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca2+, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca2+). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-delta, -eta, theta, and -zeta) as well as small amounts of the conventional (Ca(2+)-regulated) isoenzymes PKC-alpha and -beta. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca2+, suggests that one or more of these Ca(2+) independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF. PMID- 7542180 TI - Attenuation of the exercise pressor reflex. Effect of opioid agonist on substance P release in L-7 dorsal horn of cats. AB - Using alpha-chloralose-anesthetized cats, we studied blood pressure and heart rate responses to static contraction and passive stretch of the triceps surae muscle before and after microdialyzing the mu-opioid agonist [D-Ala2]-methionine enkephalinamide (DAME, 200 mumol/L) into the L-7 dorsal horn of the spinal cord. In addition, we measured contraction-induced substance P release in the dorsal horn before and after drug delivery. After 92 +/- 3 minutes of dialyzing the opioid agonist, contraction-induced increases in mean arterial pressure and heart rate were attenuated from control values of 58 +/- 7 mm Hg and 17 +/- 3 beats per minute to postdrug values of 27 +/- 7 mm Hg and 10 +/- 2 beats per minute, respectively. A similar attenuation was observed for the passive muscle stretches after 97 +/- 5 minutes of dialysis (control, 38 +/- 4 mm Hg and 8 +/- 2 beats per minute; after drug, 23 +/- 4 mm Hg and 5 +/- 1 beats per minute). Prior microdialysis of naloxone (300 mumol/L), a mu-antagonist, blocked this effect, suggesting that the opioid agonist has a specific receptor action. Naloxone alone had no effect on the pressor or tachycardiac responses. The contraction-induced increase in substance P-like immunoreactivity was reduced from a control value of 0.119 +/- 0.024 to 0.047 +/- 0.010 fmol/100 microL by DAME. Time-control experiments revealed no decrease in the release of substance P-like immunoreactivity. Thus, activation of opioid receptors modulates the transmission of group III and IV muscle afferent nerve activity through the L-7 dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542181 TI - Differential phospholamban gene expression in murine cardiac compartments. Molecular and physiological analyses. AB - Phospholamban, the regulator of the Ca2+ pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to alpha-myosin heavy chain (alpha-MHC) revealed a 3 fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca(2+)-ATPase (ratio of sarcoplasmic reticulum Ca(2+)-ATPase [SERCA2] to alpha-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40 +/- 0.71 milliseconds for atrium versus 30.58 +/- 2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36 +/- 0.82 milliseconds) compared with ventricular muscle (44.60 +/- 2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments. PMID- 7542182 TI - Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes. AB - The membrane potential (Em) of pulmonary arterial smooth muscle cells (PASMCs) regulates pulmonary arterial tone by controlling voltage-gated Ca2+ channel activity, which is a major contributor to [Ca2+]i. The resting membrane is mainly permeable to K+; thus, the resting Em is controlled by K+ permeability through sarcolemmal K+ channels. At least three K+ currents, voltage-gated K+ (KV) currents, Ca(2+)-activated K+ (KCa) currents, and ATP-sensitive (KATP) currents, have been identified in PASMCs. In this study, both patch-clamp and quantitative fluorescent microscopy techniques were used to determine which kind(s) of K+ channels (KV, KCa, and/or KATP) is responsible for controlling Em and [Ca2+]i under resting conditions in rat PASMCs. When the bath solution contained 1.8 mmol/L Ca2+ and the pipette solution included 0.1 mmol/L EGTA, depolarizations ( 40 to +80 mV) elicited both KCa and KV currents. Removal of extracellular Ca2+ and increase of intracellular EGTA concentration (to 10 mmol/L) eliminated the Ca2+ influx-dependent KCa current. 4-Aminopyridine (4-AP, 5 to 10 mmol/L) but not charybdotoxin (ChTX, 10 to 20 nmol/L) significantly reduced KV current under these conditions. In current-clamp experiments, 4-AP decreased Em (depolarization) and induced Ca(2+)-dependent action potentials; this depolarization increased [Ca2+]i in intact PASMCs. Neither ChTX nor the specific blocker of KATP channels, glibenclamide (2 to 10 mumol/L), caused membrane depolarization and the increase in [Ca2+]i. However, pretreatment of PASMCs with ChTX enhanced the 4-AP-induced increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542183 TI - Alpha-adrenergic control of volume-regulated Cl- currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism. AB - alpha-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K+ currents. The effects of alpha-adrenergic stimulation on Cl- currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl- current induced by cell swelling (ICl.swell). The present experiments were designed to assess the potential alpha-adrenergic modulation of ICl.swell in rabbit atrial myocytes. ICl.swell was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K+, Na+, and Ca2+ ions. A basal Cl- current (ICl.b) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl-]o as ICl.swell, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that ICl.b is carried by the same volume-sensitive Cl- conductance as ICl.swell. Phenylephrine produced a concentration-dependent and near-complete inhibition of ICl.b and ICl.swell, with EC50 values of 86 +/- 5 and 72 +/- 7 (mean +/- SEM) mumol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 mumol/L propranolol) also inhibited ICl.b and ICl.swell, with EC50 values of 2.6 +/- 0.1 and 2.8 +/- 0.4 mumol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (P < .001) to the right by the alpha 1-adrenoceptor antagonist prazosin and by the alpha 1A-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the alpha 1B-receptor antagonist chloroethylclonidine (100 mumol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on ICl.swell, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on ICl.swell and ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited ICl.swell in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced ICl.swell by 91 +/- 3%; its inactive analogue 4 alpha-PDD had no effect (mean change, 3 +/- 1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on ICl.swell, indicating a role for a PTX-sensitive guanine nucleotide-binding (G) protein. We conclude that alpha-adrenergic agonists inhibit volume-sensitive Cl- currents in rabbit atrial cells by interacting with an alpha 1A-adrenoceptor mechanism that is coupled to PKC via a PTX-sensitive G protein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542184 TI - Autoantibodies in sera from patients with L-tryptophan-associated eosinophilia myalgia syndrome. Demonstration of unique antigen-antibody specificities. AB - The purpose of this study was to determine whether specific autoantibodies could be identified that are associated with eosinophilia-myalgia syndrome (EMS). Sera from 44 patients with EMS were tested by indirect immunofluorescence, immunodiffusion against calf thymus extract, and immunoprecipitation from HeLa cell extract. Antinuclear antibodies were detected in the sera of 24/39 patients with EMS (61.5%) by indirect immunofluorescence against HEp-2 cells. Seven patients (16%) were demonstrated to have specific autoantibodies by immunoprecipitation in which at least two shared patterns were noted. In three sera immunoprecipitation identified a similar 63-kDa band (Ab-1). An additional four sera shared a pattern of bands consisting of a strong 110-kDa protein and a weak 95-kDa protein (Ab-2). Absorption of HeLa extract with a strongly positive Ab-2 serum confirmed that the four patients shared the same antibody. In conclusion, the detection of these autoantibodies provides evidence of autoimmunity in EMS, and may distinguish this syndrome from clinically related conditions. PMID- 7542186 TI - CD31, CD62E, and CD62P identify a specific pattern of endothelial activation in Graves' disease. AB - The different degrees of lymphocytic infiltration observed in Graves' disease, Hashimoto's disease, and De Quervain thyroiditis suggest that the regulation of adhesion molecules expressed on endothelial cells could be different in these autoimmune disorders of the thyroid. Using immunohistological techniques, we observed that thyroid samples from patients with Graves' disease displayed a characteristic pattern of capillary proliferation, with CD62P, CD62E, and CD31 expression on endothelial cells. This was different from the pattern and size of endothelial cells expressing adhesion molecules in the two other types of thyroiditis where larger vessels and high endothelial venules were stained. Almost no signs of endothelial cell activation could be seen in a comparative series of non-autoimmune disorders. PMID- 7542185 TI - Serum levels of soluble cell adhesion molecules (ICAM-1, VCAM-1, E-selectin) and of cytokine TNF-alpha increase during interleukin-2 therapy. AB - Circulating levels of soluble intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin were measured in 20 advanced cancer patients at different times during recombinant interleukin-2 immunotherapy. The concentration of all three molecules progressively increased as did the levels of circulating tumor necrosis factor-alpha (TNF-alpha), which is known to induce endothelial cell activation. A fair direct relationship (but not statistically significant) between the raised concentration of soluble cell adhesion molecules and TNF-alpha was observed. We suggest that elevated levels of soluble adhesion molecules and TNF-alpha in the blood of IL-2-treated patients may arise from a systemic inflammatory reaction producing endothelial cell activation. PMID- 7542187 TI - [Autotopagnosia ameliorated by looking at the image reflected in a mirror]. AB - A 52-year-old right-handed man presented progressive dystonia and apraxia of his right hand of five years' duration. He also suffered from parkinsonian features such as rigidity or impaired postural reflexes. Serial investigation of brain MRI revealed progressive cerebral atrophy, which started in the left parietal lobe, and subsequently extended to both hemispheres. He was clinically diagnosed as corticobasal degeneration. He could not point at any part of his own body in response to verbal or visual commands. On the other hand, he could point at every part of the examiner's body or of the illustrated body image. Deep sensations and linguistic functions were not involved. This cognitive impairment was regarded as autotopagnosia. In contrast with inability to recognize any part of the own body in response to the commands, he could name every part of his body as soon as the examiner touched there. Moreover, his symptoms of autotopagnosia were ameliorated by looking at himself in a mirror; he could point at any part of his own body. Disconnection between primary proprioceptive sensory area and the center of body schema was thought to be the mechanism of autotopagnosia in this patient, because the impairment improved with the aid of visual or tactile informations. We speculated the lesion was the left parietal lobe. PMID- 7542188 TI - [Expression of Fas-antigen on T cells in multiple sclerosis]. AB - We reported the expression of Fas antigen and activation markers (HLA-DR,CD69) on T cells in 14 patients with multiple sclerosis (MS) and healthy controls. The percentage of Fas antigen positive T cells was 29.7 +/- 7.8% in patients with untreated MS. This value was statistically higher than 17.7 +/- 10.5% of healthy controls (p = 0.03). But there was no significant difference in the rate of activation markers in peripheral blood between two groups. Fas antigen and activation markers in cerebrospinal fluid (CSF) in patients with MS tend to be higher than those in peripheral blood. High expression rate of Fas antigen and activation markers on T cells in CSF may be reflecting increased induction of apoptosis on activated T cells. PMID- 7542189 TI - Urinary beta-HCG in benign and malignant urinary tract diseases. AB - Detectable levels of HCG have been reported in conditions other than normal pregnancy, including threatened abortion, ectopic pregnancy, trophoblastic tumors, carcinomas of the stomach, liver, pancreas and breast as well as multiple myeloma and melanoma. The present study was conducted to estimate urinary beta HCG in bladder cancer and benign urinary tract disorders. 163 individuals were included, 68 with bladder cancer (60 males and 8 females), 64 with benign urinary tract diseases (55 males and 9 females) and 31 normal healthy controls (26 males and 5 females). Urinary beta-HCG was estimated by the ELISA technique using the reagents supplied by DRG International Inc., Germany. Results of the study revealed an overexpression of beta-HCG in malignant and benign urinary tract diseases. 60.3% of the cancer patients and 29.7% of patients with benign diseases showed urinary beta-HCG values above the upper limit of the control group (2mIU/ml). PMID- 7542190 TI - Stimulation of DNA and RNA synthesis in cultured rabbit cardiac fibroblasts by angiotensin IV. AB - 1. Although angiotensin II (ANG II) has been identified as a key factor in the development of cardiac hypertrophy and remodelling, the role of its degradation fragment ANG II (3-8), angiotensin IV (ANG IV), is unknown. The presence of ANG IV in the blood circulation as well as the identification of ANG IV receptors in the heart and other organs indicates that ANG IV may act as a peptide hormone. 2. ANG IV receptors were characterized by binding of 125I-ANG IV to membranes of cultured rabbit cardiac fibroblasts. Incorporation of [3H]thymidine, [3H]uridine and [3H]leucine into DNA, RNA and proteins, respectively, was determined to analyse the growth effects of ANG IV, ANG II and the combination of both peptides. 3. ANG IV displaces 125I-ANG IV bound to membranes of rabbit cardiac fibroblasts with high affinity, whereas ANG II receptor-specific ligands ([Sar1,Ile8]ANG II, losartan, CGP 42 112 A) do not. 125I-ANG IV binds to a single class of binding site with a dissociation constant (Kd) of 4.87 +/- 0.11 nmol/l. The density of ANG IV receptors (Bmax) is 371 +/- 8.3 fmol/mg of protein. 125I ANG IV binding is not markedly affected in the presence of the non-hydrolysable GTP analogue GTP gamma S, whereas binding of 125I-ANG II is reduced. 4. In quiescent cells, a 24 h exposure of ANG IV (100 nmol/l) increased rates of thymidine and uridine incorporation by 127% and 246%, respectively. A small but statistically insignificant increase in leucine incorporation was observed under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542191 TI - A method for predicting common structures of homologous RNAs. AB - We have developed a procedure, composed of a set of computer programs, for predicting common RNA structures of homologous sequences. Given a set of homologous RNAs, these programs perform a multiple sequence alignment, generate a list of possible helical stems that are thermodynamically favored in RNA folding from a selected individual sequence, establish a conserved stem list by inspecting the equivalent base pairings and/or conserved helical stems from the derived alignment of homologous RNAs, and build common RNA secondary structures with the maximum scores (i.e., compensatory base changes and number of base pairs, etc.). The approach is a combination of phylogenetic and thermodynamic methods and has been applied to the prediction of common folding structures of the 5' untranslated regions in a number of positive RNA viruses. PMID- 7542193 TI - Rational design of anticonvulsants: a quantum pharmacologic study of the ion channel-modulating FMRFamide tetrapeptide as an endogenous anticonvulsant. AB - We applied the computational techniques of quantum pharmacology to examine molecular conformations (shapes and geometries) of the tetrapeptide FMR-Famide (L Phe-L-Met-L-Arg-L-Phe-NH2), determining the geometric features necessary for anticonvulsant activity. The rigorous tiered hierarchical approach used molecular mechanics, molecular dynamics, and semiempirical quantum mechanics calculational methods. Low-energy conformations showed pertinent conformational information to be considered in the rational design of novel anticonvulsants. The FMRFamide peptide backbone assumes a bent but primary planar geometry. Distinct polar and nonpolar regions are created as the two Phe residues occupy one "face" of the bent conformation, while the Met and Arg residues occupy the opposite face. The aromatic rings point away from each other along the backbone, and this separation is consistent among the low-energy conformations at approximately 11-12 A. The Met side chain interacts with neither the peptide backbone nor the side chains of other residues. Molecular mechanics and semiempirical quantum mechanics calculations predict limited variation in the orientation of the Arg side chain. PMID- 7542192 TI - Distribution and origin of the peripheral innervation of rat cervical esophagus. AB - Several neurotransmitters, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), galanin, enkephalin, calcitonin-gene related peptide (GGRP), substance P, as well as nitric oxide synthase (NOS), and the noradrenergic marker tyrosine hydroxylase (TH) were localized by immunocytochemistry in the cervical esophagus of rat. Nerve fibers containing the neuropeptides, NOS, and TH were distributed in the myenteric plexus, around muscle bundles and small blood vessels. Injection of the retrograde tracer True Blue (TB) into the cervical esophagus resulted in the appearance of labeled nerve cell bodies in the superior cervical, the stellate, the nodose, the sphenopalatine, the dorsal root ganglia at levels C2 C7, and in local ganglia close to the thyroid. Most of the TB-labeled nerve cell bodies in the superior cervical ganglia contained NPY. In the stellate ganglion, a few labeled nerve cell bodies contained VIP whereas an additional few cell bodies stored VIP. In local ganglia, the majority of labeled cell bodies contained VIP. In the nodose ganglion and cervical dorsal root ganglia, the majority of the labeled nerve cell bodies stored CGRP. The results indicate that the cervical esophagus has a dense innervation with multiple neurotransmitters emanating from several ganglia. As judged by the pattern of nerve fiber distribution, they may regulate esophageal peristalsis and blood flow, some of them possibly in a cooperative manner. PMID- 7542194 TI - Lack of L-selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by Mel-14 antibody treatment. AB - The process of mononuclear cell extravasation from the blood into the islets of Langerhans in nonobese diabetic (NOD) mice is dependent on the expression of a set of molecules, most of which remain to be defined. The observation that vascular addressins are expressed in inflamed islets raises the issue of the involvement of one of their ligands, L-selectin, in the pathogenesis of autoimmune diabetes. Treatment of NOD females with Mel-14, an antibody specific for L-selectin, reduced the spontaneous development of both insulitis and diabetes. Pretreatment of diabetic donors with Mel-14 decreased the capacity of their splenocytes to transfer the disease. However, the treatment of recipients had no effect on the transfer of diabetes by untreated diabetogenic splenocytes. To reconcile these apparently conflicting results, we fractionated spleen T cells from diabetic mice according to L-selectin expression. Diabetogenic cells were found only in the L-selectin subpopulation. Thus, diabetogenic cells in adult mice share phenotypic characteristics with activated/memory cells, and enter the pancreas using L-selectin-independent migratory pathways. PMID- 7542195 TI - Expression of CD34 in endothelial cells, hematopoietic progenitors and nervous cells in fetal and adult mouse tissues. AB - The human cell surface molecule CD34 is selectively expressed on uncommitted and committed hematopoietic progenitor cells and on vascular endothelial cells. It has been suggested that CD34 regulates early events in blood cell migration and differentiation, possibly as a cell adhesion molecule. To characterize the patterns of expression of CD34 in the mouse embryo and in the adult, as well as to dissect the function of different portions of the extracellular domain of this molecule, we have generated the first monoclonal antibodies (mAb) specific for mouse CD34. The epitope(s) recognized by these mAb are not carbohydrate moieties, and are comprised either within the immunoglobulin-like domain or within a portion of the mucin domain, containing approximately half of the predicted O- and N-linked carbohydrate attachment sites. The specificity of the antibodies was established by ELISA and Western blotting. Western analysis revealed that these mAb recognize a protein of approximately 110 kDa in PA6 stromal cell lysates, which can be specifically blocked by the recombinant CD34 protein. To establish the reactivity of these mAb on different cell lineages, a panel of cell lines was stained. This analysis showed strong reactivities with 3T3 fibroblasts, stromal cell lines from fetal liver and with the endothelial cell line D10. Bone marrow hematopoietic progenitors were also stained by these mAb. Immunostaining of frozen sections from embryonic and adult tissues revealed a strong reactivity against vascular endothelial cells at different stages of development, including sinusoidal cells in the fetal liver, yolk sac, and in the fetal bone marrow, endothelial cells from adult lung and kidney, and neural cells, including those of the neural tube of midgestation embryos and neuronal bodies in adult brain. PMID- 7542196 TI - Non-exclusive Fas control and age dependence of viral superantigen-induced clonal deletion in lupus-prone mice. AB - To investigate the role of Fas in the induction of tolerance by viral superantigen (SAG), we infected MRL-+/+ and MRL-lpr (Fas mutant) mice with mouse mammary tumor virus (MMTV) (SW), a virus encoding an SAG with the same specificity as endogenous Mtv-7-SAG. In normal mice, this infection has two distinct consequences on specific V beta 6+CD4+ T cells, consisting of activation followed by clonal deletion. MMTV (SW)-SAG-induced activation in vivo was identical in MRL-+/+ and MRL-lpr mice. In contrast, clonal deletion showed age dependent impairment. Early infection (5 weeks) led to identical clonal deletion of specific T cells in blood lymphocytes from MRL-+/+ and MRL-lpr mice, although clonal deletion was slightly impaired in the MRL-lpr lymph nodes. Late infection (10 weeks) of MRL-lpr mice led to markedly delayed and reduced clonal deletion. V beta 6+CD4+ T cells which escaped clonal deletion in aging MRL-lpr mice were not anergized by interaction with SAG. These results show that peripheral clonal deletion induced by viral SAG in adult mice is controlled by Fas, but not exclusively so. PMID- 7542197 TI - Phosphotyrosine-dependent association between CD22 and protein tyrosine phosphatase 1C. AB - CD22 is a B lymphocyte-specific cell surface glycoprotein that becomes tyrosine phosphorylated upon B cell activation. To determine if tyrosine phosphorylated CD22 couples signaling through membrane immunoglobulin (mIg) to down-stream elements, we looked for molecules coprecipitating with CD22 after anti-Ig stimulation. We found that a 60-kDa molecule was stably associated with CD22 following cross-linking of mIg and have identified this molecule as protein tyrosine phosphatase 1C (PTP1C). The association between PTP1C and CD22 is dependent upon tyrosine phosphorylation of CD22, but does not appear to require tyrosine phosphorylation of PTP1C. PMID- 7542198 TI - Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules. AB - In many cancer cells, the p53 gene displays point mutations that result in stabilization and accumulation of the p53 protein. Therefore, p53 peptides could be presented to the immune system by tumor cells; thus, p53 might be a suitable target antigen for developing an immunotherapy against tumors using cytotoxic T lymphocytes (CTL). To map candidate CTL epitopes, we synthesized 150 peptides of 8-11 residues that contained putative anchor motifs required for binding to common HLA class I molecules. They were tested for their capacity to promote the assembly of purified and refolded HLA-A1, A2, B7 and B8 molecules. The following wild-type p53 peptides were found to be reactive with the HLA molecules tested: 196-205 and 226-234 bound moderately to HLA-A1; 25-35, 65-73, 129-137, 187-197, 263-272 and 264-272 bound strongly, and 187-195 and 256-264 moderately to HLA-A2; 26-35, 63-73, 189-197, 249-257 and 321-330 bound strongly to HLA-B7; and 135-143, 210-218 and 375-383 bound weakly to HLA-B8. We also analyzed the effects of p53 mutations occurring naturally in tumors on peptide/HLA assembly. We found substitutions that enhanced, diminished or had no effect on the peptide binding to HLA molecules. Polymorphism at position 72 mainly affected peptide/HLA-B7 binding, the proline allele P72 giving a less-reactive peptide (63-73) than the arginine allele R72. We have ranked potential p53 epitopes according to their reactivity for purified HLA molecules, allowing the selection of appropriate peptides and HLA molecules to attempt CTL induction in vitro. PMID- 7542200 TI - Heterogeneity of rat encephalitogenic T cells elicited by variants of the myelin basic protein (68-86) peptide. AB - By immunizing Lewis rats with myelin basic protein (MBP) peptide variants derived from the major encephalitogenic epitope of guinea pig (MBP(68-88) and then isolating encephalitogenic T cells from these animals, we demonstrated that the variant peptides do not elicit the same encephalitogenic T cell subsets as those induced by the wild-type peptide or by intact MBP. Rather, the pathogenic T cells differed in clonal composition as reflected by their heterogeneous responses to a panel of variant peptides and by their T cell receptor usage. Thus, molecules mimicking the MBP(68-88) autoantigen can elicit pathogenic T cell subsets without necessarily cross-reacting with T cells specific for the original autoantigen. This suggests that a more clonally diverse group of pathogenic T cells might be involved in EAE than has been apparent from studies with intact MBP or its unaltered peptides. PMID- 7542199 TI - Characterization of self-reactive T cells that evade tolerance in hepatitis B e antigen transgenic mice. AB - Previous studies of hepatitis B e antigen (HBeAg)-expressing transgenic (Tg31e) mice have indicated that the degree of T cell tolerance was epitope specific. For example, T cells specific for residues 120-131 of HBeAg are profoundly tolerant, whereas a proportion of T cells specific for residues 129-140 escape tolerance induction in B10. S x B10-Tg31e mice. To understand the basis for differential tolerance towards two T cell sites on the same self antigen, we characterized T cell recognition of HBeAg by primary T cells and T cell hybridomas derived from HBeAg-Tg and non-Tg mice. The self-reactive T cells surviving in B10-Tg31e mice exhibited a unique fine specificity, albeit still focussed on HBeAg residues 129 140, which could be distinguished from the HBeAg-specific T cell repertoire in non-Tg B10 mice. Further, self-reactive T cells were comprised predominantly of Th2-type cells that preferentially evaded tolerance induction as compared to their Th1 counterparts. Because HBeAg may act as a tolerogen during the vertical transmission of chronic hepatitis B virus (HBV) infection, these results suggest that a predominance of HBeAg-specific Th2 cells expressing a limited repertoire may influence the initiation or the maintenance of the HBV chronic carrier state. PMID- 7542201 TI - Expression and functional significance of an activation-dependent epitope of the beta 1 integrins in chronic inflammatory diseases. AB - The avidity of VLA integrins for their ligands can be increased by their transition to an active conformational state. This conformational change can be detected with a novel monoclonal antibody (mAb), termed 15/7, that recognizes an activation-dependent conformational epitope on the common beta 1 polypeptide of different VLA alpha beta 1 integrins. In an attempt to understand the possible role of the active conformational state of beta 1 integrins in vivo, we first investigated the expression of 15/7 epitope on T lymphocytes from patients with chronic inflammatory joint diseases. An enhanced expression of the 15/7 epitope was found in the synovial fluid (SF) T lymphocytes from these patients as compared to their peripheral blood (PB) T cells. The effect of different cytokines on the appearance of the 15/7 activation epitope in PB T lymphocytes was subsequently analyzed; interferon-gamma, interleukin-2 and, to a lower extent, tumor necrosis factor-alpha were able to induce an increased expression of the 15/7 epitope. This enhanced 15/7 expression correlated with a higher binding ability to fibronectin of cytokine-activated T cells. The presence of this activation epitope was detected in a small proportion of T lymphocytes scattered within inflammatory foci of synovial membrane from rheumatoid arthritis and thyroid glands from Hashimoto's chronic thyroiditis. We then analyzed the possible role of 15/7 epitope expression on cell adhesion in vitro. Immunofluorescence studies showed that the 15/7 epitope displayed a spot-like distribution, selectively decorating adhesive contacts of U-937 myelomonocytic cells attached to the 80 kDa proteolytic fragment of fibronectin (FN80). Furthermore, the anti-beta 1 15/7 mAb was able to induce both T lymphocyte, Jurkat and U-937 cellular binding and spreading on FN80. Altogether these results indicate that an activated conformation of beta 1 integrins is detected in vivo in lymphocyte infiltrates from chronic inflammatory conditions. The active conformations of beta 1 integrins are regulated by physiologic mediators such as cytokines, play an important role in cellular attachment and spreading, and appear to be involved in the development of inflammatory processes. PMID- 7542204 TI - Evidence that conformational changes upon the transition of the native to the modified form of vitronectin are not limited to the heparin binding domain. AB - Vitronectin (Vn) exists in vivo in at least two different conformational states, the native and the modified form, and these forms have different ligand binding properties. To characterize the molecular events associated with this conformational flexibility, modified Vn was analyzed by competitive ELISA using a panel of conformationally sensitive antibodies with known epitopes. These studies provided evidence for major molecular rearrangements upon the transition from the native to the modified form that are not limited to the C-terminal heparin binding domain, but also occur in the N-terminal part of the molecule. PMID- 7542203 TI - Treatment with granulocyte colony-stimulating factor for pneumonia in a patient with a variant form of X-linked chronic granulomatous disease. PMID- 7542205 TI - Epidermal growth factor inhibits cytokine-dependent nitric oxide synthase expression in hepatocytes. AB - Liver cells express a wide range of extracellular receptors involved in the control of cell growth and arrest that can be studied ex vivo. Incubation of primary cultures of hepatocytes with IL-1 beta, TNF-alpha or lipopolysaccharide promotes the expression of the inducible form of nitric oxide synthase and NO release, a process that is inhibited to a different extent by incubation of the cells with EGF. In addition to this growth factor, IL-6 and TGF-beta also inhibited NO synthesis. Therefore, EGF by itself or in combination with other cytokines may be involved in the down-regulation of the NO synthesis that occurs in the early steps of liver regeneration. PMID- 7542206 TI - Studies on human porin: XIII. The type-1 VDAC 'porin 31HL' biotinylated at the plasmalemma of trypan blue excluding human B lymphocytes. AB - In 1989, we demonstrated for the first time the expression of the VDAC 'Porin 31HL' in the plasmalemma of human B lymphocytes, then giving first evidence of a multi-topological expression of VDAC. Meanwhile, data from this and other laboratories support our proposal of a multi-compartment distribution of the channel in mammalian tissues. Here, by biotinylation of plasmalemma-integrated proteins of proven living B lymphocytes, followed by two-dimensional electrophoresis, immuno- and streptavidin affinity blotting, we show that part of the channel molecules can be labelled at the outer membrane of the cells. Thus, by a relevant approach our results invalidate objections concerning putative cross-reactivity of anti-human Type-1 porin antibodies with non-VDAC proteins at the outer cell membrane. PMID- 7542207 TI - The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3. AB - The histidine at position 1106 of the C4B isotype of human complement is involved in catalyzing the covalent binding of the thioester to glycerol and water. By replacing the histidine with other residues, it was found that tyrosine is also capable of mediating the reaction. We propose that they act as nucleophiles by first attacking the thioester, upon activation, to form acyl intermediates, which subsequently react with the hydroxyl groups of glycerol or water. The monomeric alpha-macroglobulin, alpha 1I3 of the rat, was also studied. Unlike alpha 2 macroglobulin, which is a tetramer, alpha 1I3 has binding properties similar to those of C4A. PMID- 7542202 TI - Expression in vivo of CD45RA, CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization. AB - We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No down-regulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA- or CD45RB- subset. PMID- 7542208 TI - Cell proliferation is increased in the endometrium of women with endometriosis. AB - OBJECTIVE: To compare the proliferation of endothelial, epithelial, and stromal cells in the endometrium of women with endometriosis and normal controls. DESIGN: Proliferating cells were identified using the monoclonal antibody antiproliferating cell nuclear antigen. A second antibody (CD34) was used to identify endothelial cells (ECs). SETTING: University Department of Obstetrics and Gynaecology. PATIENTS: Women with laparoscopically proven endometriosis, n = 30. Controls were women with a normal pelvis at laparoscopy performed for tubal sterilization or for infertility due to a male factor, n = 27. MAIN OUTCOME MEASURES: Endothelial cells: proliferative index. Epithelial and stromal cells: semi-quantitative immunostaining score. RESULTS: The mean EC proliferative index was significantly greater in those with endometriosis compared with controls. This difference was most marked during the proliferative phase of the menstrual cycle. Proliferative phase epithelial and stromal cells demonstrated significantly higher immunostaining scores in endometriosis patients than in controls. CONCLUSIONS: We have demonstrated increased numbers of proliferating ECs as well as epithelial and stromal cells in proliferative phase endometrium of women with endometriosis. This suggests that the endometrium of these women might have an enhanced ability to implant and survive in ectopic locations. PMID- 7542210 TI - Vineland Adaptive Behavior Scales as a summary of functional outcome of extremely low-birthweight children. AB - This study reports moderate to high Pearson correlations between Vineland Adaptive Behavior Scale (VABS) subscale and total scores and a variety of cognitive, academic and motor performance tests on a population of extremely low birthweight infants assessed at eight years of age. The subscales describe adaptive behaviour in daily living, communication, motor function and socialization, as well as an adaptive behaviour composite score. Because it can provide a norm-referenced description of functional outcomes and can be used to assess all children regardless of disability, the authors believe that the VABS should be applied uniformly by all groups reporting school-age outcome of neonatal intensive-care populations. PMID- 7542211 TI - Savant characteristics in a child with developmental delay and deletion in the short arm of chromosome 20. AB - The development outcome of a four-year-old boy with a deletion of the short arm of chromosome 20 is described. Despite a number of early medical problems, including infantile hypoglycemic convulsions secondary to growth hormone deficiency and delayed motor and language development, he has been reading (self taught) since 2.5 years and currently has computer proficiency, and exceptional memory for maps and spatial locations, an extremely rich and active fantasy life, good diction, and an extensive spoken vocabulary. Neuropsychological evaluation revealed low-average intelligence with normal language, memory and attention functions, and impaired visuomotor and graphomotor ability and motor skills. He showed extremely advanced decoding and reading comprehension skills while mathematics, spelling and general knowledge abilities were average. PMID- 7542209 TI - Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micromanipulation for men with bilateral congenital absence of vas deferens. AB - OBJECTIVE: To assess the effects of cystic fibrosis transmembrane-conductance regulator (CFTR) gene mutations on sperm function and fertility in men with bilateral congenital absence of the vas deferens. DESIGN: Prospective. SETTING: Division of urologic microsurgery and associated hospital-based IVF unit. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates. PATIENTS: Men referred to our fertility unit for treatment of bilateral congenital absence of the vas deferens, using sperm surgically retrieved from the epididymis with IVF and micromanipulation. RESULTS: Of 45 men with bilateral congenital absence of the vas, 54% (19/35) tested were found to be carriers of CFTR gene mutations, with one compound heterozygote. Epididymal sperm from men affected with CFTR mutations fertilized 19% (29/152) of oocytes, whereas men without mutations fertilized 22% (44/204) of oocytes. Pregnancy rates (PRs) were 36% (5/14) for cycles involving men with CFTR mutations and 33% (5/15) for other patients with congenital absence of the vas deferens but without detectable CFTR mutations. CONCLUSIONS: The presence of detectable CFTR mutations does not affect fertilization rates or PRs for men with bilateral congenital absence of the vas deferens when IVF and micromanipulation are applied. PMID- 7542212 TI - Limb growth in children with disabilities. PMID- 7542213 TI - The selectins: vascular adhesion molecules. AB - The selectin family of adhesion molecules mediates the initial attachment of leukocytes to venular endothelial cells before their firm adhesion and diapedesis at sites of tissue injury and inflammation. The selectin family consists of three closely related cell-surface molecules with differential expression by leukocytes (L-selectin), platelets (P-selectin), and vascular endothelium (E- and P selectin). The selectins have characteristic extracellular regions composed of an amino-terminal lectin domain that binds a carbohydrate ligand, an epidermal growth factor-like domain, and two to nine short repeat units homologous to domains found in complement binding proteins. In contrast to most other adhesion molecules, selectin function is restricted to leukocyte interactions with vascular endothelium. Multiple studies indicate that the selectins mediate neutrophil, monocyte, and lymphocyte rolling along the venular wall. The generation of selectin-deficient mice has confirmed these findings and provided further insight into how the overlapping functions of these receptors regulate inflammatory processes. Selectin-directed therapeutic agents are now proven to be effective in blocking many of the pathological effects resulting from leukocyte entry into sites of inflammation. Future studies are focused on how the selectins interact with the increasing array of other adhesion molecules and inflammatory mediators. PMID- 7542214 TI - Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. AB - Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM 1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules. PMID- 7542215 TI - Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. AB - The fibroblast growth factors are a family of structurally related polypeptides that are mitogenic for a broad range of cell types as well as mediators of a wide spectrum of developmental and pathophysiological processes in vivo and in vitro. The fibroblast growth factor family presently consists of nine distinct members. Indeed, the FGF prototypes FGF-1 (acidic) and FGF-2 (basic) are well described as modifiers of angiogenesis. The absence of a signal sequence to direct their secretion and their ability to traffic to the nucleus are unique structural features that may be relevant to the regulation of their activities. The FGF receptor family consists of four transmembrane receptor tyrosine kinases. Each of these receptors give rise to multiple isoforms as a result of alternative splicing of their mRNAs. The significance of these multiple isoforms is not fully understood; however it is known that alternative splicing in the extracellular domain of these receptors results in altered ligand binding specificities. In addition, alternative splicing in the cytoplasmic domain results in isoforms with increased oncogenic potential. This review will describe recent insights into the pathways used for the regulation of FGF secretion and cellular trafficking as well as signaling by FGFRs. PMID- 7542217 TI - Copresence of prostaglandin EP2 and EP3 receptors on gastric enterochromaffin like cell carcinoid in African rodents. AB - BACKGROUND & AIMS: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis, which might be a useful model of normal ECL cells. METHODS: A [3H]PGE2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP2 and EP3 receptor complementary DNAs was conducted. RESULTS: [3H]PGE2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE1 = PGE2 > enprostil > PGF2 alpha. Although PGE1 and PGE2 stimulated 5'-cyclic adenosine monophosphate (cAMP) production, neither PGF2 alpha nor enprostil had any effect. On the other hand, all of PGE1, PGE2, PGF2 alpha, and enprostil attenuated the forskolin induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP2 and EP3 receptor messenger RNAs was confirmed by RNA blot analysis. CONCLUSIONS: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP2 linked to cAMP production and EP3 coupled with inhibitory guanosine 5'-triphosphate-binding proteins mediating the inhibition of cAMP production. PMID- 7542219 TI - Diagnosis of Echinococcus multilocularis infection by reverse-transcription polymerase chain reaction. AB - Alveolar echinococcosis is a life-threatening parasitosis occurring in countries in the Northern hemisphere. The diagnosis of an Echinococcus multilocularis infection is routinely performed by radiological techniques and by the detection of specific antibodies in the sera of infected patients. However, because serodiagnosis fails in 5%-10% and radiological techniques are difficult to interpret in some cases, we developed a polymerase chain reaction for the detection of echinococcal-specific messenger RNA from fine-needle biopsy specimens. This technique was applied to the diagnosis of alveolar echinococcosis in a 20-year-old seronegative woman. Detection of messenger RNA not only allowed the diagnosis of echinococcal disease but also proved to be a reliable measure for determining the efficacy of an antiparasitic therapy. PMID- 7542218 TI - Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c kit-expressing cells in Ws/Ws mutant rats. AB - BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats. METHODS: The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach. RESULTS: The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats. CONCLUSIONS: The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells. PMID- 7542221 TI - [Specific and substitute markers of hepatitis C in screening donor blood]. AB - The efficacy of specific and substitute markers of hepatitis C was compared in donor blood screening. 2615 blood donors were examined using two EIA kits for detection of HCV antibodies. The presence of nonspecific markers of non-A non-B hepatitis was also checked. 1.3% of the donors were found to carry HCV antibodies. This necessitates donor blood testing for anti-HCV antibodies using EIA. Such measure reduces three-fold the risk of the virus transmission with blood components and preparations. The preference should be given to more sensitive test systems of the second generation, whereas less sensitive, but more specific, test systems are more valuable for accurate diagnosis and treatment effects assessment in HCV infection. It would be appropriate to perform compulsory examinations for HCV infection in medical staff. This will entail efforts on development of the infection diagnostic verification criteria and preventive measures. PMID- 7542216 TI - Expression and inducibility of vascular adhesion receptors in development. AB - ICAM-1 and VCAM are cell adhesion receptors expressed on vascular endothelial cells, and their expression is up-regulated as part of the inflammatory response. Tumor-derived Py-4-1 cells were a source of murine endothelial cells, and they showed increased lymphocyte binding when incubated with TNF alpha. Py-4-1 cells stimulated with TNF alpha or LPS also had elevated levels of ICAM-1 and VCAM RNAs, with maximum levels at 2 h. Developmental expression of adhesion receptors was assayed in murine yolk sacs and in cystic embryoid bodies (CEBs) that differentiate in vitro from murine embryonic stem cells. ICAM-1 and VCAM RNAs were expressed in unstimulated yolk sacs and CEBs. Expression was inducible in CEBs by exposure to LPS at all developmental stages, beginning at day 6. The time course of RNA induction in CEBs was similar to that of the Py-4-1 endothelial cell line. ICAM-1 protein expression was localized to vascular and hematopoietic blood islands in the CEBs. These results show that embryonic cells respond to inflammatory mediators by up-regulating expression of vascular adhesion receptors, and they imply an early ontogeny for the endothelial cell signal transduction pathway necessary for leukocyte recruitment. PMID- 7542220 TI - Immune regulation of cystic fibrosis transmembrane regulator. PMID- 7542222 TI - Nitric oxide synthase in gastric mucosa. PMID- 7542223 TI - Study of 12 mutations in Turkish cystic fibrosis patients. AB - 67 unrelated cystic fibrosis (CF) patients were screened for some of the most common mutations of the CFTR gene. This analysis resulted in the identification of 34.6% of all CF alleles. The most common mutation is delta F508 (28.4%). Two other mutations account for a further 6.7% of the alleles (R347H: 3.0%; N1303K: 3.7%). 1677delTA, G542X, G551D, S549N/I, R553X, L558S, R334W, and R297Q were not detected. PMID- 7542224 TI - Behaviour of macroglial cells, as identified by their intermediate filament complement, during optic nerve regeneration of Xenopus tadpole. AB - Assessment of glial cell behaviour during optic nerve (ON) regeneration in Xenopus tadpoles is hampered by the lack of classical cellular markers that distinguish different glial cells in mammals. We thus have characterized the intermediate filament (IF) complement of tadpole glial cells and used it to follow the fate of glial cell subsets during the first 10 days after ON crush. Glial cells synthesize a restricted number of cytokeratin (CK) species and vimentin. This pattern remains essentially unchanged during metamorphosis and regeneration. However, vimentin turnover is specifically enhanced after injury. The expression of CKs and vimentin has been followed immunocytochemically in situ and in isolated cells recovered from dissociated ON segments. In the normal nerve, 79% of ramified glial cells express both CK and vimentin, 1% CK and 4% vimentin only, whereas 16% express neither IF protein. We tentatively classified CK expressing cells as mature astrocytes and those without IF proteins as oligodendrocytes. In the regenerating ON, the relative number of oligodendrocytes is decreased, while the astrocytic subset becomes accordingly larger but is decreased by day 10 already in favour of cells expressing vimentin only. Astrocytes invade the lesion site soon after crush, arrange into a central core within the distal nerve segment and establish a peripheral scaffold that is readily crossed by axons. Unlike mammalian astrocytes that remain absent from the lesion site but form a scar at some distance to it, amphibian astrocytes appear to provide active guidance to axons growing through the lesion site. PMID- 7542226 TI - ras, p53 and HPV status in benign and malignant prostate tumors. AB - To study the role of ras, p53 genes and HPV virus (16 and 18) in the development of prostate cancer, we analyzed tissue sections from 27 patients affected with carcinomas (stages A to D) and from 24 patients with adenomas. Mutations of H, K and N-ras and p53 (exons 2-9) were studied by SSCP and DNA sequencing. Accumulation of p53 protein was studied by immunohistochemistry on tissue sections. Tumors were also analyzed for the presence of HPV16 and -18 sequences by PCR and DNA hybridization with sequence-specific oligonucleotides. No mutation was found in the three ras genes studied, either in carcinomas or adenomas. By SSCP analysis we identified p53 mutations in only 2 of 19 carcinomas studied, both in exon 7. Immunohistochemical results strongly correlate with the SSCP results: p53 protein was positive in tumors with p53 mutation but not in others; 32% of studied adenomas had detectable HPV16 DNA, while 53% of carcinomas were HPV16+. Among these I presented a p53 mutation. No HPV18 E6 sequence could be detected. Our data show that in prostate tumors from France, mutations of p53 and ras are rare events but that these tumors display detectable HPV16 DNA at a high frequency. The low incidence of p53 mutation, associated to a significant proportion of tumors showing HPV16 DNA, could suggest that in prostate cancer HPV16 infection could participate in p53 inactivation by E6. PMID- 7542227 TI - Detection of tumour cells in peripheral blood and bone marrow from Ewing tumour patients by RT-PCR. AB - The Ewing family of tumours (ET) is characterised at the cytogenetic level by unique chromosome 22 rearrangements. The breakpoints have been cloned and were shown to fuse the EWS gene on chromosome 22 to one of two closely related ETS proto-oncogenes, FLI-I or ERG, which reside on chromosomes II and 21, respectively. The rearrangement results in the expression of chimaeric transcripts, which can be identified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). We applied this method for the monitoring of ET cells circulating in the peripheral blood or infiltrating the bone marrow. The presence of tumour cells could be detected with a sensitivity of I in I x 10(6) nucleated cells. When samples were kept at 4 degrees C, tumour cells could still be identified after 48 hr of storage. Positive RT-PCR signals originated from intact ET cells rather than from free RNA released by ruptured tumour cells. We analysed peripheral blood, bone marrow samples and peripheral blood stem cell collections from 16 ET patients. At diagnosis, bone marrow specimens collected from 6 patients and I peripheral blood specimen tested positive for EWS chimaeric RNA. During therapy tumour cells were detected in bone marrow aspirations obtained from 2 patients. Our results show that ET cells infiltrating the bone marrow or circulating in peripheral blood can be identified by RT-PCR. The clinical implications for the presence of ET cells in samples detected by RT-PCR at diagnosis and during therapy requires further evaluation. PMID- 7542225 TI - High nm23-H1/NDPK-A expression in Ewing tumors: paradoxical immunohistochemical reactivity and lack of prognostic significance. AB - Expression of nm23-H1/NDPK-A has been reported to correlate inversely with metastasizing potential of rodent experimental cells and some human tumors. In the search for reliable molecular prognostic indicators for Ewing tumors (ET), a group of aggressive presumably neuroectodermal malignancies in children and adolescents, we studied nm23-H1/NDPK-A expression. Northern-blot and RT-PCR analyses were employed to semi-quantificatively measure nm23-H1 mRNA levels in ET cell lines and tissue extracts. A panel of monoclonal antibodies (MAbs) were used to evaluate protein abundance by Western blotting and immunohistochemistry. The nm23-H1/NDPK-A gene was also investigated on the DNA level to define possible genomic alterations. Our results revealed neither nm23-H1 allelic loss nor gene amplification and failed to show any significant variation in nm23-H1 mRNA or NDPK-A protein levels of primary or metastatic ET. NDPK-A protein levels were high and comparable to those of MCF-7 breast-cancer cells and to aggressive stage IV neuroblastoma cell lines. nm23-H2/NDPK-B expression in ET was slightly more variable but generally lower than in MCF-7 cells. In the immunohistochemical analysis, however, discrepancies in the reactivity patterns with different antibodies were observed. Differential sensitivity to various fixation methods and heat treatment pointed to a structurally polymorphic NDPK-A protein. nm23-H1 expression studies using immunohistochemistry for prognostic counselling should thus be interpreted with caution. PMID- 7542228 TI - Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast cancer. AB - We have investigated the expression of platelet-derived endothelial-cell growth factor/thymidine phosphorylase (PD-ECGF/dThdPase) in human breast-cancer tissues by the immunocytochemical method using anti-PD-ECGF/dThdPase monoclonal antibody. Out of 100 invasive-ductal-carcinoma tissue samples, 39 (39%) were evaluated as PD-ECGF/dThdPase-positive. The expression of PD-ECGF/dThdPase was identified mainly in the cytoplasma of tumor cells. The expression of PD-ECGF/dThdPase was significantly associated with the microvessel density assessed by immunostaining to factor-VIII-related-antigen (p < 0.05). However, there was no correlation between expression of PD-ECGF/dThdPase and menopausal status, tumor size, axillary lymph-node metastases, hormone-receptor status, epidermal-growth-factor receptor, or erb-B-2-protein and p53-protein expression. We suggest that expression of PD-ECGF/dThdPase plays an important role in the promotion of angiogenesis in human breast cancer. PMID- 7542229 TI - Clinical and retrospective evaluation of Eviprostat: a non-hormonal and non neuropharmacological agent for benign prostatic hyperplasia. AB - Eviprostat has been used as a non-hormonal and non-neuropharmacological treatment for benign prostatic hyperplasia (BPH) in Japan. We evaluated the clinical efficacy of Eviprostat in patients with symptomatic BPH and the anti-inflammatory effect of this drug was investigated by retrospective evaluation of TUR specimens. Clinically, Eviprostat subjectively relieved obstructive symptoms of BPH. Objective improvements were also demonstrated by ultrasonographic evaluation of prostatic volume and urinary flow rates. Histologically, preoperative administration of Eviprostat improved the degree of prostatic inflammation. This drug probably can have a place in the treatment of patients with mild or moderate symptoms of outflow obstruction. PMID- 7542230 TI - Diaminofluorene is more sensitive than benzidine for detecting hemoglobin in erythropoietin responsive J2E cells. AB - We have used the diaminofluorene stain to detect hemoglobin production in J2E cells following erythropoietin-induced differentiation. The pseudo-peroxidase activity of hemoglobin produces a colored product, fluorene blue, which can be measured spectrophotometrically. We found that the absorbance varied with time and concentration of hemoglobin, making it unsuitable for rapid, routine use. However, hemoglobin content could be determined from the initial reaction rate and this correlated extremely well with the number of benzidine positive cells. When used as a direct cytochemical stain diaminofluorene was shown to be more sensitive than benzidine in detecting hemoglobin-producing J2E cells. PMID- 7542231 TI - Substance P-induced histamine release in tracheally perfused guinea pig lungs. AB - The capacity of substance P (SP) and endogenously released tachykinins to liberate histamine was examined in isolated tracheally perfused guinea pig lungs. Increasing doses of tracheally injected SP were associated with the recovery of increasing amounts of histamine from lung effluent. The mechanism of SP-induced histamine liberation was explored in studies with neurokinin-(NK) receptor agonists and antagonists. Tracheal injection of either the NK1 agonist [Sar9,Met(O2)11]SP or the NK2 agonist [beta-Ala8]-neurokinin A-(4-10) was associated with a significant increase in histamine recovery from lung effluent. In addition, both the NK1 antagonist CP-99994 and the NK2 antagonist SR-48968 significantly inhibited SP-induced histamine release. These findings support the hypothesis that SP can liberate histamine from guinea pigs lungs by a mechanism that depends predominantly on NK1- and NK2-receptor activation. The liberation of endogenous tachykinins by acute tracheal injection of capsaicin was also associated with augmented histamine recovery, which was inhibited by combined NK1 and NK2-receptor blockade. Tracheal injection of SP was associated with an increase in the percentage of airway mast cells exhibiting histological evidence of degranulation. This study demonstrates that exogenous SP, as well as endogenous tachykinins released from capsaicin-sensitive neurons, can liberate histamine, most likely from airway mast cells, by a mechanism that depends predominantly on the activation of NK1 and NK2 receptors. PMID- 7542232 TI - Autonomic regulation of tissue resistance in the guinea pig lung. AB - Although the autonomic nervous system is known to influence airway tone and resistance in both the normal and inflamed lung, its effects on tissue resistance (Rti) have not been defined. To characterize autonomic neural control of Rti in the lung, we measured airway resistance (Raw) and Rti, lung elastance, and lung hysteresivity after adrenergic, cholinergic, and nonadrenergic noncholinergic (NANC) blockade in anesthetized mechanically ventilated guinea pigs by using the alveolar capsule technique. Five animals received 1 mg/kg i.v. phentolamine (alpha-blocker), eight received 1 mg/kg i.v. propranolol (beta-blocker), six received 0.1 mg/kg i.v. atropine (parasympatholytic), six had surgical bilateral vagotomy, and five were treated 14 days before study with 50 mg/kg sc capsaicin (peptidergic neurotransmitter depleter). Measurements were recorded at 5- to 10 min intervals for 120 min after pharmacological induction of autonomic blockade. Capsaicin treatment resulted in decreases in lung resistance (RL), Raw, and dynamic elastance (Edyn) compared with controls but Rti was not significantly affected. beta-Blockade resulted in significant increases in RL, Raw, and Edyn but did not affect Rti. alpha-Blockade and vagolytic maneuvers had no effect on RL, Raw, Rti, or Edyn. Furthermore, there was no effect of cholinergic, adrenergic, or NANC blockade on the relationship between Rti or elastance and breathing frequency or mean lung volume during tidal breathing. Our results suggest that, in the normal guinea pig lung, baseline Raw is modulated by both the beta-adrenergic and NANC nervous systems but Rti tone is largely independent of nervous system regulation. PMID- 7542234 TI - Generation of amyloidogenic C-terminal fragments during rapid axonal transport in vivo of beta-amyloid precursor protein in the optic nerve. AB - The amyloid beta-protein (A beta) is a major component of extracellular deposits that are characteristic features of Alzheimer's disease. A beta is derived from the large transmembrane beta-amyloid precursor protein (beta APP). In the rabbit optic nerve/optic tract (ON), beta APP is synthesized in vivo in retinal ganglion cell perikarya, rapidly transported into the ON axons in small transport vesicles and is subsequently transferred to the axonal plasma membrane as well as to the presynaptic nerve terminals (Morin, P. J., Abraham, C. R., Amaratunga, A., Johnson, R.J., Huber, G., Sandell, J. H., and Fine, R. E. (1993) J. Neurochem. 61, 464-473). Present results indicate that there is rapid processing of beta APP in the ON to generate a 14-kDa C-terminal membrane-associated fragment that contains the A beta sequence. By using equilibrium sucrose density gradient fractionation, this fragment, as well as non-amyloidogenic C-terminal fragments and intact beta APP, are detected in at least two classes of transport vesicles destined for the plasma membrane and the presynaptic nerve terminal. The two classes of transported vesicles are distinguished by labeling kinetics as well as by density. In contrast to the ON, only nonamyloidogenic C-terminal fragments are generated in the retina, which contains the perikarya of retinal ganglion cells and glial (Muller) cells which also produce beta APP. PMID- 7542233 TI - Identification of a domain in soluble CD14 essential for lipopolysaccharide (LPS) signaling but not LPS binding. AB - CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide (LPS) and enables LPS dependent responses in a variety of cells. Monoclonal antibodies of CD14 such as 3C10 and MEM-18 are known to neutralize biological activity of CD14. Recently, it has been demonstrated that MEM-18 recognizes the LPS-binding site of CD14, between amino acids 57 and 64. It has also been shown that 3C10 recognizes a distinct epitope from that of MEM-18, indicating that 3C10 may yet define another functional domain of CD14. In order to identify the epitope for 3C10, we constructed a series of alanine substitution mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions between amino acids 7 and 10 and between amino acids 11 and 14 are required for 3C10 binding. To assess the effect of altering the 3C10 epitope in CD14, we generated a stable cell line expressing a mutant sCD14 containing alanine substitutions in the region between amino acids 7 and 10, sCD14(7-10)A, and purified this protein to homogeneity. sCD14(7-10)A has impaired ability to mediate LPS-dependent IL-6 up-regulation in U373 cells, integrin activation in neutrophils, and NF-kappa B activation in U373 cells. Purified sCD14(7-10)A was, however, capable of forming a stable complex with LPS in an LPS binding protein-facilitated and LPS binding protein-independent fashion. The ability of sCD14(7-10)A to bind LPS was also demonstrated in assays in which excess sCD14(7-10)A inhibited LPS-mediated tumor necrosis factor-alpha production in whole blood and adhesion of polymorphonuclear leukocytes to fibrinogen. These data strongly suggest that a region recognized by neutralizing monoclonal antibody 3C10 contains a domain required for cellular signaling but not for LPS binding. PMID- 7542235 TI - Accurate transcription of the Trypanosoma brucei U2 small nuclear RNA gene in a homologous extract. AB - In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element. PMID- 7542236 TI - Integrin alpha IIb beta 3-mediated translocation of CDC42Hs to the cytoskeleton in stimulated human platelets. AB - To investigate the function of the human Ras-related CDC42 GTP-binding protein (CDC42Hs) we studied its subcellular redistribution in platelets stimulated by thrombin-receptor activating peptide (TRAP) or ADP. In resting platelets CDC42Hs was detected exclusively in the membrane skeleton (9.6 +/- 1.5% of total) and the detergent soluble fraction (90 +/- 4%). When platelets were aggregated with TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and decreased in the membrane skeleton, whereas RhoGDI (guanine-nucleotide dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein) remained exclusively in the detergent soluble fraction. Upon prolonged platelet stimulation CDC42Hs disappeared from the cytoskeleton and reappeared in the membrane skeleton. Rac translocated to the cytoskeleton with a similar time course as CDC42Hs. When platelets were stimulated under conditions that precluded the activation of the alpha IIb beta 3 integrin and platelet aggregation, cytoskeletal association of CDC42Hs was abolished. Translocation of CDC42Hs to the cytoskeleton but not aggregation was also prevented by cytochalasins B or D or the protein tyrosine kinase inhibitor genistein. Platelet secretion and thromboxane formation were not required but facilitated the cytoskeletal association of CDC42Hs. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton. This process is reversible and is mediated by activation of the alpha IIb beta 3 integrin and subsequent actin polymerization and protein-tyrosine kinase stimulation. CDC42Hs might be a new component of a signaling complex containing specific cytoskeletal proteins and protein-tyrosine kinases that forms after activation of the alpha IIb beta 3 integrin in platelets. PMID- 7542237 TI - A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase. AB - Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5 phosphate (cPRPP). Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation. First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme. PMID- 7542239 TI - The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. AB - Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF 1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma. PMID- 7542238 TI - Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis. AB - Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes. Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and gamma-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family. We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes. PMID- 7542240 TI - A study of the intracellular routing of cytotoxic ribonucleases. AB - Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility. PMID- 7542241 TI - Cloning and differential tissue-specific expression of three mouse beta chemokine receptor-like genes, including the gene for a functional macrophage inflammatory protein-1 alpha receptor. AB - Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and RANTES, members of the beta chemokine family of leukocyte chemoattractants, bind to a common seven transmembrane-domain human receptor. We have now cloned three related mouse genes: one for a selective MIP-1 alpha receptor (MIP-1 alpha R) and two for orphan receptors provisionally designated MIP-1 alpha receptor-like 1 and 2 (MIP 1 alpha RL1 and 2). Their deduced sequences are 80, 62, and 63% identical to the human MIP-1 alpha/RANTES receptor, respectively. K562 cells stably transfected with MIP-1 alpha R specifically bound 125I-human MIP-1 alpha and 125I-human RANTES with high affinity. The rank order of beta chemokine competition for 125I human MIP-1 alpha binding was human MIP-1 alpha > mouse MIP-1 alpha approximately RANTES approximately MIP-1 beta > MCP-1. However, human RANTES was approximately 100-fold less potent as a calcium-mobilizing agonist for MIP-1 alpha R than either human or mouse MIP-1 alpha, which matched the selectively of mouse leukocytes for calcium mobilization by MIP-1 alpha and RANTES. No other beta or alpha chemokines tested were agonists for MIP-1 alpha R. RNA for all three genes was detected in mouse leukocytes, but unique patterns of expression were identified in solid organs: MIP-1 alpha R, heart, spleen, and lung; MIP-1 alpha RL1, skeletal muscle; and MIP-1 alpha RL2, spleen and liver. These data identify potentially important new targets for beta chemokine action in the mouse. PMID- 7542242 TI - The membrane of leaf peroxisomes contains a porin-like channel. AB - Spinach leaf peroxisomes were purified by Percoll density gradient centrifugation. After several freeze-thaw cycles, the peroxisomal membranes were separated from the matrix enzymes by sucrose density gradient centrifugation. The purity of the peroxisomal membranes was checked by measuring the activities of marker enzymes and by using antibodies. Lipid bilayer membrane experiments with the purified peroxisomal membranes, solubilized with a detergent, demonstrated that the membranes contain a channel-forming component, which may represent the major permeability pathway of these membranes. Control experiments with membranes of other cell organelles showed that the peroxisomal channel was not caused by the contamination of the peroxisomes with mitochondria or chloroplasts. The peroxisomal channel had a comparatively small single channel conductance of 350 pS in 1 M KCl as compared with channels from other cell organelles. The channel is slightly anion selective, which is in accordance with its physiological function. The single channel conductance was found to be only moderately dependent on the salt concentration in the aqueous phase. This may be explained by the presence of positive point net charges in or near the channel or by the presence of a saturable binding site inside the channel. The possible role of the channel in peroxisomal metabolism is discussed. PMID- 7542243 TI - Nitric oxide is involved in non-adrenergic, non-cholinergic inhibitory neurotransmission in rat duodenum. AB - 1. In rat duodenum, electrical field stimulation (EFS) induced a relaxation due to activation of non-adrenergic, non-cholinergic (NANC) inhibitory intramural neurones. 2. Nitric oxide synthase (NOS) inhibitors, N omega-nitro-L-arginine (L NNA) and N omega-nitro-L-arginine methyl ester (L-NAME), caused a dose-dependent reduction in amplitude of the NANC relaxation. Responses to low frequencies of stimulation were more sensitive to NOS inhibitors than those to high frequencies. 3. Effects induced by NOS inhibitors were stereospecific since D-NNA and D-NAME did not affect NANC relaxation. L-arginine, but not D-arginine, partially prevented the effects induced by NOS inhibitors on NANC relaxation. 4. The nitrovasodilator drug, sodium nitroprusside, caused muscle relaxation which was not affected by preincubation with either tetrodotoxin (TTX), L-NNA or L-NAME. 5. alpha-Chymotrypsin reduced relaxations elicited by stimulation of NANC nerves, especially when high frequencies of stimulation were used. The residual NANC relaxation was further reduced by NOS inhibitors. In the same way, alpha chymotrypsin was able to further reduce the relaxation observed after NOS inhibitors. 6. These results suggest that nitric oxide (NO) and a peptide are involved in NANC relaxation of rat duodenal smooth muscle. NO and peptidergic pathways act in parallel to produce muscle relaxation and they are preferentially activated by stimuli at low and high frequencies, respectively. PMID- 7542244 TI - Oxidative biodegradation mechanisms of biaxially strained poly(etherurethane urea) elastomers. AB - As part of ongoing studies in polyurethane biostability and biodegradation, we have investigated an in vitro system to test strained poly(etherurethane urea) (PEUU). Recently, we utilized this system to reproduce in vivo stress cracking in strained Pellethane. In this study, strained PEUU was tested to determine whether it degrades through a common mechanism with Pellethane and to further examine the steps involved in this degradation. Biaxially strained PEUU elastomers were treated with an alpha 2-macroglobulin (alpha 2-Mac) protein solution followed by an oxidative H2O2/CoCl2 treatment. Characterization of the strained PEUU specimens was performed with attenuated total reflectance-Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), electron spectroscopy for chemical analysis, and contact angle analysis. The results from these characterization techniques provide conclusive evidence that biodegradation of PEUU and Pellethane occurs through a common mechanism. Chemical changes to the PEUU include cleavage of the polyether soft segments and urethane linkages, leaving the hard segment domains unaffected. SEM analysis shows that this chain cleavage leads to the development of severe pitting and cracking of the PEUU surface. In addition, the in vitro degradation accurately reproduces the in vivo degradation chemically and physically. This result verifies that the primary species responsible for biodegradation of PEUUs, in vivo, are hydroxyl and/or hydroperoxide radicals. alpha 2-Mac pretreatment increases the rate of degradation compared to direct treatment in H2O2/CoCl2. As the PEUU soft segment chains are cleaved, the degradation products are extracted into the treatment solution or environment. Finally, a new biodegradation mechanism of PEUUs is presented that involves crosslinking of the polyether soft segments. PMID- 7542245 TI - Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). AB - The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo. PMID- 7542247 TI - Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells. AB - Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions. PMID- 7542248 TI - Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo. AB - The NPXY sequence is highly conserved among integrin beta subunit cytoplasmic tails, suggesting that it plays a fundamental role in regulating integrin mediated function. Evidence is provided that the NPXY structural motif within the beta 3 subunit, comprising residues 744-747, is essential for cell morphological and migratory responses mediated by integrin alpha v beta 3 in vitro and in vivo. Transfection of CS-1 melanoma cells with a cDNA encoding the wild-type integrin beta 3 subunit, results in de novo alpha v beta 3 expression and cell attachment, spreading, and migration on vitronectin. CS-1 cells expressing alpha v beta 3 with mutations that disrupt the NPXY sequence interact with soluble vitronectin or an RGD peptide, yet fail to attach, spread, or migrate on immobilized ligand. The biological consequences of these observations are underscored by the finding that CS-1 cells expressing wild-type alpha v beta 3 acquire the capacity to form spontaneous pulmonary metastases in the chick embryo when grown on the chorioallantoic membrane. However, migration-deficient CS-1 cells expressing alpha v beta 3 with mutations in the NPXY sequence lose this ability to metastasize. These findings demonstrate that the NPXY motif within the integrin beta 3 cytoplasmic tail is essential for alpha v beta 3-dependent post-ligand binding events involved in cell migration and the metastatic phenotype of melanoma cells. PMID- 7542246 TI - c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. AB - Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190. PMID- 7542250 TI - Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia. AB - Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta catenin is found in association with E-cadherin, p120 Cas is not. In the ras transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia. PMID- 7542249 TI - CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium. AB - To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM 1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation. PMID- 7542252 TI - Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells. AB - We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the beta-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase. PMID- 7542253 TI - Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions. AB - Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins ("delRGD" or "RGE") supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions. PMID- 7542254 TI - Heritable formation of neuroectodermal tumor in transgenic mice carrying the combined E1 region gene of adenovirus type 12 with the deregulated human renin promoter. AB - Adenovirus early 1 (E1) region gene products, including E1A and E1B, are required for transcriptional regulation of viral and cellular promoters in infected and transfected culture cells and for transformation of primary rodent cells. Here, we established a line of transgenic mice carrying the E1 region gene of human adenovirus type 12 under the control of the human renin promoter, in which a neuroectodermal tumor derived from retroperitoneal, olfactory, and/or pelvic regions was heritably developed with varying degrees of incidence and the phenotype was successfully passed through six generations. The transgenes were located in the region E2-E3 bands of chromosome 7 with which no genetic linkage to neuroectodermal tumors was previously demonstrated, and expressed only in the tumors but not in another tissue examined. Notably, in addition to the expression of a neural marker gene N-CAM, the three nuclear oncogenes, c-, L-, and N-myc, were coexpressed in the tumors. These results suggest that E1A and E1B are cooperatively involved in the heritable formation of neuroectodermal tumors associated with co-expression of the three sets of myc family genes. PMID- 7542251 TI - Monoclonal antibodies to CD44 and their influence on hyaluronan recognition. AB - Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand. PMID- 7542256 TI - Heparin-binding vitronectin up-regulates latent TGF-beta production by bovine aortic endothelial cells. AB - Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-beta, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-beta. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-beta by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-beta activity observed when vitronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent TGF-beta with vitronectin in a cell-free system does not result in increased TGF-beta activity. Assays of total TGF-beta levels in heat treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-beta in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with antibodies specific for latent TGF-beta. These data suggest that vitronectin regulates expression and/or secretion of TGF-beta by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by alpha v beta 3 integrins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542255 TI - Adhesion-induced tyrosine phosphorylation of the p130 src substrate. AB - Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of which have now been identified as the focal adhesion proteins pp125FAK, paxillin and tensin. In addition, we have previously noted the adhesion-induced tyrosine phosphorylation of a fourth protein, with an apparent molecular mass of 130. As in the case of FAK, paxillin and tensin, a 130 kDa protein is also found to be highly tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed cells. This protein forms a stable complex with pp60src and is directly phosphorylated by activated forms of c-src. Using a monoclonal antibody (mAb 4F4) specific for the src-associated p130 we show that p130 is also phosphorylated in response to cell adhesion. Immunoprecipitation of p130 followed by an anti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fibroblasts (REF52) to fibronectin (FN) led to a significant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cell lysates before and after immunoprecipitation confirmed the absence of tyrosine phosphorylated p130 from lysates immunoprecipitated with mAb 4F4. Immunofluorescence staining of REF52s revealed that p130 is found in focal adhesions as well as along stress fibers in a pattern reminiscent of that exhibited by alpha-actinin. In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130. Finally, unlike pp125FAK, p130 does not appear to be itself a kinase as evidence by immune-complex kinase assays carried out in the presence or absence of exogenous substrates. PMID- 7542257 TI - The role of the microtubular system in the cell response to HGF/SF. AB - The effects of the microtubular drugs colcemid and taxol on the morphological changes induced by hepatocyte growth factor/scatter factor (HGF/SF) in MDCK cells were studied. Dynamic changes in the area and shape of individual cells were assessed by morphometric methods whereas alterations of the cytoskeleton were assessed by immunomorphological methods. The results suggest that there are two components in the response to HGF/SF: (a) activation of the extension of lamellae leading to cell spreading; and (b) reorganization of microtubules leading to polarization of cell shape. The latter response is highly sensitive to microtubular drugs, especially taxol. HGF/SF induced spreading in taxol-treated MDCK cells but these cells retained a non-polarized discoid shape and a pattern of actin microfilament bundles characteristic of the untreated cells. Colcemid and taxol did not prevent HGF/SF-induced migration of cells in Boyden chambers but completely inhibited the outgrowth of multicellular strands and tubules from cell aggregates in collagen gels. These results show that enhanced lamella formation in response to HGF/SF without polarization of cell shape is sufficient to induce cell motility. In contrast, microtubule-dependent polarization is essential for complex morphogenetic responses such as tubulogenesis in collagen gels. PMID- 7542258 TI - Regulation of growth and dissemination of a human lymphoma by CD44 splice variants. AB - CD44 is a polymorphic cell surface glycoprotein, currently proposed to be the principal cell surface receptor for hyaluronan. However, different isoforms of CD44, expressed in human lymphoid tumor cells, appear to have distinct effects on the ability of the cells to attach to hyaluronan-coated surfaces and on their capacity to form tumors in vivo. In the present study, we address the mechanisms that may regulate CD44 isoform-dependent adhesion to hyaluronan. We use a human Burkitt lymphoma, stably transfected with six different alternatively spliced human CD44 isoforms, to determine their potential hyaluronan binding and tumor growth promoting roles. We show that transfectants expressing CD44 splice variants that contain variable exons 6-10, 7-10 and 8-10 adhere to hyaluronan coated surfaces weakly and that corresponding tumor formation in vivo is delayed with respect to CD44-negative parental cell-derived tumors. Abundant shedding of these three isoforms may play a significant role in determining the rate of tumor development. Transfectants expressing variable exon 3, on the other hand, fail to display CD44-mediated adhesion to hyaluronan, but form bone marrow tumors rapidly following intravenous injection. These observations suggest that different mechanisms regulate CD44-mediated adhesion and tumor growth, and provide evidence that expression of exon v3 may confer novel ligand-binding properties. PMID- 7542259 TI - Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells. AB - Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells. PMID- 7542260 TI - Rapid intracellular assembly of tenascin hexabrachions suggests a novel cotranslational process. AB - Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino terminal location of the assembly domain. PMID- 7542261 TI - Maturation of early endosomes and vesicular traffic to lysosomes in relation to membrane recycling. AB - The controversy whether endocytic processing occurs by organellar maturation or by vesicular traffic has not been resolved. It is also not clear whether maturation continues to the stage of lysosomes, to what extent it involves a decrease in organellar fusogenicity, and how it relates to membrane recycling. Maturation and vesicular traffic imply distinct kinetics for the intermingling of endocytic markers after sequential endocytic uptake. We have studied the kinetics of intermingling of fluid-phase markers (fluorescein-labelled dextran and horseradish peroxidase) and cell surface-derived membrane (labelled by galactosylation) in organelles at early and late stages of the endocytic pathway in macrophage-like P388D1 cells. Intermingling declined by sigmoid kinetics, indicating that endosomes matured within about 3 minutes to become non-fusogenic towards early endosomes. During maturation about 60% of internalized membrane was recycled with T1/2 approximately 2 minutes. Whereas matured endosomes were non fusogenic towards early endosomes and towards each other, a second phase of intermingling was observed upon delivery to lysosomes. This intermingling occurred by a first-order process (T1/2 approximately 4 minutes), concurrent with recycling of the remaining 40% of internalized membrane marker. These kinetic observations suggest a model for endocytic processing which reconciles maturation of early endosomes with the known function of carrier vesicles: Endocytic carrier vesicles do not bud off from permanent early endosomes as proposed for vesicular traffic, but are derived, together with recycling vesicles, from the maturation of early endosomes which are consumed by this process; these carrier vesicles subsequently mediate delivery to lysosomes by vesicular traffic during which the remaining surface-derived membrane is recycled. PMID- 7542263 TI - Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences. AB - Members of the genus Acinetobacter are reported to be involved in hospital acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested. This study thus proposes the detection of restriction length polymorphism in the spacer sequences between the 16S and 23S rRNA genes as a method for the identification of isolates in the A. calcoaceticus-A. baumannii complex. PMID- 7542262 TI - Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia. AB - Homology in the 16S rDNAs shows that the agent of human granulocytic ehrlichiosis (HGE) is closely related to the veterinary pathogens Erlichia equi and Erlichia phagocytophila. After HGE, patients develop antibodies reactive with E. equi and E. phagocytophila; thus, we hypothesized that these species are closely related and share significant antigenicity. Antisera from humans, horses, dogs, and cattle were tested by indirect fluorescent-antibody assay (IFA) for antibodies reactive with E. equi and other ehrlichiae and tested by immunoblot to identify the specific reactions with E. equi. All convalescent-phase sera from human patients with HGE and from animals infected or immunized with E. equi or E. phagocytophila had antibodies reactive with E. equi by IFA; no reactions with Ehrlichia chaffeensis occurred with these sera, and only one horse naturally infected with E. equi had a serologic reaction against Ehrlichia sennetsu. Human and animal sera obtained after infection or immunization with other Ehrlichia, Rickettsia, and Bartonella species did not react with E. equi by IFA. E. equi immunoblots revealed as many as 19 bands with equine anti-E. equi serum. All HGE agent, E. equi, and E. phagocytophila antisera tested reacted with a 44-kDa antigen of E. equi, while other anti-Ehrlichia spp. sera reacted with this antigen rarely or not at all. HGE agent, E. equi, and E. phagocytophila antisera but not other sera also reacted occasionally with 25-, 42-, and 100-kDa antigens. Most sera reacted with antigens between approximately 56 and 75 kDa, probably heat shock proteins. The HGE agent, E. equi, and E. phagocytophila share significant antigenicity by IFA and immunoblot. Coupled with the nearly identical nucleotide sequences of 16S rRNA genes, these data indicate that E. equi, E. phagocytophila, and the human granulocytic ehrlichia are closely related or identical species. PMID- 7542264 TI - Long PCR-ribotyping of nontypeable Haemophilus influenzae. AB - PCR-ribotyping, a new typing method based on long PCR, has been developed for nontypeable Haemophilus influenzae (NTHi). Ribosomal operons of NTHi were amplified by long PCR and were found to be highly polymorphic for internal HaeIII sites. The technique was applied to 49 isolates previously subjected to conventional ribotyping, and the two methods showed a high level of concordance for serial isolates from individual subjects. PCR-ribotyping provides a powerful new typing tool for strain characterization in epidemiological investigations of NTHi. PMID- 7542266 TI - Enzyme-linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoalveolar fluid. AB - A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5' biotinylated. The amplification product is immobilized on streptavidin-coated microtiter plates. Because of the high binding capacity, no removal of nonincorporated biotin from the PCR product is required. After alkaline denaturation, the single-stranded PCR product is hybridized with a 5' digoxigenin-labeled probing oligomer. The amplification product is then detected by using peroxidase-labeled anti-digoxigenin antibodies in a luminescence or colorimetric reaction. The assay detects as few as 10 legionellae in 1-ml bronchoalveolar lavage fluid specimens. It is specific for medically relevant Legionella species, including Legionella pneumophila, L. bozemanii, and L. longbeachae. Of over 250 clinical specimens examined, 8 were positive for legionellae by both culture and the PCR assay. Six further specimens were culture negative but PCR positive for legionellae; of these, five specimens were from patients receiving high-dose erythromycin therapy for suspected or previously diagnosed legionella pneumonia. None of the remaining 240 specimens that were culture negative for legionellae yielded a positive PCR test, although a total of over 30 different bacterial species were cultured from these specimens. The PCR assay therefore appears to exhibit high sensitivity and specificity and thus could prove suitable for use in the routine microbiological diagnostic laboratory. PMID- 7542265 TI - Molecular subtyping scheme for Salmonella panama. AB - We describe a genotyping scheme for Salmonella panama. Defined probes specific for the 16S rRNA gene and the DNA insertion element IS200 were generated by PCR from S. panama and were used to probe genomic Southern blots made with enzymes selected to cut within and outside the probed sequences. Plasmid profiles were determined. The typeability and discriminatory power of the individual methods were compared. Ribotyping with 16S rRNA gene probe alone was slightly more discriminatory than phage typing, but unlike the latter, ribotyping was able to type all strains. IS200 profiling was the single most discriminatory method for S. panama, having an index of discrimination (D) of 0.8 and 100% typeability. Plasmid profiling, which had moderate discriminatory power but only 50% typeability, was valuable as an adjunct technique. The use of all three methods together or simply the combination of IS200 profiling with the two most discriminatory enzymes and plasmid profiling yielded a molecular typing scheme whose discriminatory power (D = 0.97) approached the maximum theoretical value. This should prove both useful and robust for epidemiological investigations of S. panama. PMID- 7542267 TI - Development of PCR-based hybridization protocol for identification of streptococcal species. AB - 16S rRNA of Streptococcus agalactiae, S. uberis, and S. parauberis was bound to streptavidin-coated magnetic beads by using a biotinylated oligonucleotide probe complementary to a highly conserved region of the molecule. In-solution hybridization of radiolabelled oligonucleotide probes to immobilized 16S rRNA allowed the specific identification of S. agalactiae and S. parauberis but not S. uberis. PCR was used to amplify a species-specific region of the 16S rRNA gene from these species. One of the PCR primers was biotinylated at the 5' end to allow purification of the amplified product on streptavidin-coated magnetic beads and subsequent denaturation to yield immobilized single-stranded DNA. Radiolabelled oligonucleotide probes were hybridized in solution to the single stranded target molecule and enabled species-specific identification of the target organism. This protocol overcame problems associated with hybridization of the S. uberis-specific probe to 16S rRNA in solution. A similar procedure may enable the specific detection of other streptococci which exhibit a species specific sequence in this region of the gene. PMID- 7542268 TI - Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay. AB - Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junin virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junin virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT PCR-based assay was compared with traditional methodologies, including enzyme linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junin virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%. PMID- 7542269 TI - Detection of the turkey pathogens Mycoplasma meleagridis and M. iowae by amplification of genes coding for rRNA. AB - PCR-based diagnostic tests using oligonucleotides specific to 16S rRNA were designed for the specific detection of the turkey pathogens Mycoplasma meleagridis and M. iowae. This method of detection was shown to be rapid, species specific, and unaffected by strain variation or the presence of other organisms. Detection of M. meleagridis in clinical samples by PCR was achieved and later confirmed by culture and growth inhibition. Definitive identification by culture and growth inhibition required up to 3 weeks, whereas positive results from PCR testing were obtained within a day and negative samples were confirmed within 4 days. PMID- 7542270 TI - Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene. AB - Conserved primers were used to PCR amplify 95% of the Helicobacter hepaticus 16S rRNA gene. Its sequence was determined and aligned to those of related bacteria, enabling the selection of primers to highly diverged regions of the 16S rRNA gene and an oligonucleotide probe for the development of a PCR-liquid hybridization assay. This assay was shown to be both sensitive and specific for H. hepaticus 16S rRNA gene sequences. PMID- 7542271 TI - Detection of Tropheryma whippelii DNA in a patient with AIDS. AB - A case of an AIDS patient infected with the Whipple's disease bacterium, Tropheryma whippelii, is reported. A DNA fragment with sequence specificity for the 16S rRNA gene of the bacterium was detected by PCR in a duodenal biopsy specimen from a 55-year-old male patient with AIDS and diarrhea. The biopsy specimen contained periodic acid-Schiff stain-positive macrophages which did not, however, resemble the sickleform-particle-containing cells characteristic of Whipple's disease. This observation raises two possibilities: either the patient had a coincidence of AIDS and Whipple's disease or Tropheryma whippelii acted as an opportunistic pathogen in this immunodeficient patient. The latter explanation is of interest in light of the ongoing discussion of immunologic abnormalities as predisposing factors for Whipple's disease. PMID- 7542273 TI - Identification of Campylobacter fetus by PCR-DNA probe method. AB - A PCR method for rapid identification of Campylobacter fetus subsp. fetus was evaluated. A fragment of the gene coding for 16S rRNA was amplified from crude cell lysates of 18 C. fetus strains and 30 strains representing other Campylobacter species and subspecies. The amplicons were probed by dot blot hybridization with a digoxigenin-labeled C. fetus-specific oligonucleotide probe. The probe reacted only with C. fetus subsp. fetus and C. fetus subsp. venerealis and may be useful for rapid identification in clinical laboratories. PMID- 7542272 TI - Influence of hepatitis C virus (HCV) genotypes on HCV recombinant immunoblot assay patterns. AB - Ninety-six patients with chronic hepatitis C were studied. A second-generation recombinant immunoblot assay detected anti-NS4 antibodies significantly more often in patients infected by hepatitis C virus genotype 1 than in patients infected by other types. By a third-generation recombinant immunoblot assay, the prevalences of the four antibodies measured did not differ according to the hepatitis C virus genotype. PMID- 7542274 TI - Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates. AB - Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates. PMID- 7542275 TI - Efficient discrimination of Mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified polymorphic DNA analysis. AB - Amplification of the region separating the genes coding for 16S and 23S rRNA was performed with 15 Mycobacterium tuberculosis isolates and the type strain, ATCC 27294. Reproducible amplification patterns were obtained. PCR products were then used as target DNA for random amplified polymorphic DNA (RAPD) analysis. The discriminatory power was higher than when whole genomic DNA was used as a RAPD template. 16S-23S spacer region-based RAPD analysis was a simple and efficient method of differentiation. Consequently, it may be a useful tool for epidemiologic studies of tuberculosis. PMID- 7542277 TI - Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells. AB - In this study, we have used enterocyte-like differentiated HT29-D4 human colonic carcinoma cells cultured in a glucose-free medium (HT29-D4-GAL cells) on semi permeable supports in order to investigate the polarity of the insulin-like growth factor (IGF) system. We report that these cells secrete endogenous IGF-II predominantly (66%) from the basolateral cell surface where type I IGF receptors are almost all (> 96%) localized. HT29-D4-GAL cells also secrete IGF-binding protein (IGFBP) -2, -4, and -6 as evidenced by Western ligand and immunoblot analyses of conditioned medium. IGFBP-2 and IGFBP-4 are secreted primarily into the basolateral side (71 and 87%, respectively), whereas IGFBP-6 is targeted to the apical surface (76%) as a possible consequence of an active sorting. Finally, HT29-D4-GAL cells are found to display responses to IGF-II added to the basolateral but not the apical membrane side in terms of intracellular tyrosine phosphorylation and long-term stimulation of amino acid uptake. This study indicates (a) that IGF-II is potentially capable of autocrine regulation on the basolateral side of HT29-D4-GAL cell, and (b) that IGFBP-6 has a unique pattern of secretory polarity. It supports the concept that a differential sorting of the various forms of IGFBPs might play a modulatory role in the maintenance of a functional polarity in the differentiated HT29-D4-GAL cells. PMID- 7542276 TI - Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces. AB - In acute inflammatory responses, selectins mediate initial rolling of neutrophils (PMNs) along the endothelial surface. This is followed by tight adhesion that requires activation-dependent up-regulation of CD11/CD18 integrins on PMNs. For emigration to occur, the initial bonds that are established at the endothelial surface must be disengaged. We show that activation of PMNs results in their detachment from P-selectin, a glycoprotein expressed at the surface of inflamed endothelium that mediates initial tethering of PMNs. Loosening of the bond occurs when PMNs are activated by platelet-activating factor, which is coexpressed with P-selectin, or by other signaling molecules. The time course of reduced adhesion to P-selectin, when compared to up-regulation of CD11/CD18 integrins, suggests that "bond trading" may occur as activated PMNs transmigrate in vivo. Activation of PMNs did not alter binding of fluid-phase P-selectin, indicating that the ligand(s) for P-selectin is not shed or internalized. Using microspheres coated with P-selectin, we found that ligands for P-selectin were randomly distributed over the surfaces of rounded, unactivated PMNs. An antibody against P-selectin glycoprotein ligand-1 (PSGL-1) completely inhibited binding of P-selectin-coated beads suggesting that P-selectin glycoprotein ligand-1 is the critical binding site in this assay. In contrast to the dispersed pattern on unactivated PMNs, the ligands for P-selectin were localized on the uropods of activated, polarized cells. Pretreating PMNs with cytochalasin D before activation prevented the change in cell shape, the redistribution of binding sites for P-selectin-coated beads, and the decrease in cellular adhesiveness for P-selectin. These experiments indicate that the distribution of ligands for P-selectin is influenced by cellular activation and by cytoskeletal interactions, and that redistribution of these ligands may influence adhesive interactions. Activation of PMNs may cause loosening or disengagement of bonds between P-selectin and its ligands, facilitating transendothelial migration. PMID- 7542278 TI - Expression of cryptantigen Th on paroxysmal nocturnal hemoglobinuria erythrocytes in association with a hemolytic exacerbation. AB - Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes lack complement regulatory membrane proteins and are susceptible to complement. Although the critical role of complement in intravascular hemolysis in PNH is accepted, the precise mechanism of complement activation in vivo is unknown. Accordingly, in a PNH patient who was suffering from a hemolytic precipitation soon after a common cold like upper respiratory infection, we analyzed the erythrocytes with lectins and by flow cytometry to detect membrane alteration that lead to complement activation. The lectin reactivity of erythrocytes showed the expression of cryptantigen Th. The patient serum at the time of the hemolysis induced the expression of Th on erythrocytes from PNH patients and from healthy volunteers in vitro, whereas neither the patient serum after recovery from the hemolysis nor blood type-matched control serum from healthy donor showed this activity. Moreover, autologous serum selectively hemolyzed Th+ PNH erythrocytes, but not Th PNH erythrocytes, or Th+ control erythrocytes. Hemolysis was not observed either in complement-inactivated serum or in blood type-matched cord blood serum, which lacks natural antibodies to cryptantigens. These findings indicate that the immunoreaction of infection-induced Th with natural antibody on PNH erythrocytes is a trigger of the complement activation, leading to intravascular hemolysis. PMID- 7542279 TI - Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. AB - We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype interferon sensitivity determining region appears to be interferon resistant strains. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment. PMID- 7542281 TI - Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. AB - We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced. PMID- 7542282 TI - Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells. AB - Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell permeant analogue of cAMP synergistically enhanced secretory responses to calcium mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses. PMID- 7542280 TI - Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis. AB - The murine TNF-alpha gene was expressed under the control of the human surfactant protein SP-C promoter in transgenic mice. A number of the SP-C TNF-alpha mice died at birth or after a few weeks with very severe lung lesions. Surviving mice transmitted a pulmonary disease to their offspring, the severity and evolution of which was related to the level of TNF-alpha mRNA in the lung; TNF-alpha RNA was detected in alveolar epithelium, presumably in type II epithelial cells. In a longitudinal study of two independent mouse lines, pulmonary pathology, at 1-2 mo of age, consisted of a leukocytic alveolitis with a predominance of T lymphocytes. Leukocyte infiltration was associated with endothelial changes and increased levels of mRNA for the endothelial adhesion molecule VCAM-1. In the following months, alveolar spaces enlarged in association with thickening of the alveolar walls due to an accumulation of desmin-containing fibroblasts, collagen fibers, and lymphocytes. Alveolar surfaces were lined by regenerating type II epithelial cells, and alveolar spaces contained desquamating epithelial cells in places. Platelet trapping in the damaged alveolar capillaries was observed. Pulmonary pathology in the SP-C TNF-alpha mice bears a striking resemblance to human idiopathic pulmonary fibrosis, in which increased expression of TNF-alpha in type II epithelial cells has also been noted. These mice provide a valuable animal model for understanding the pathogenesis of pulmonary fibrosis and exploring possible therapeutic approaches. PMID- 7542283 TI - The renal adaptation to pregnancy is now "NOS"-talgic. PMID- 7542284 TI - Acute blockade of nitric oxide synthase inhibits renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats. AB - Because the kidneys are vasodilated and the endogenous production of nitric oxide is increased in gravid rats, we tested whether nitric oxide mediates the renal vasodilatory response to pregnancy. Chronically instrumented, conscious rats of gestational days 12-14 were studied concurrently with age-matched virgin control animals. GFR and effective renal plasma flow (ERPF) were determined by the renal clearances of inulin and para-aminohippurate before and during acute infusion of N omega-nitro-L-arginine methyl ester (NAME; 2, 20, and 50 micrograms/min) or NG monomethyl-L-arginine (100 micrograms/min). Baseline GFR and ERPF were significantly increased, and effective renal vascular resistance was decreased by 30-40% in gravid rats compared with virgin controls. During infusion of all three dosages of NAME and NG-monomethyl-L-arginine, effective renal vascular resistance, GFR, and ERPF were equalized in the pregnant and virgin rats (the only exception being GFR during the 20 micrograms/min NAME infusion). When compared with virgin rats, the gravid animals were more responsive to nitric oxide synthase inhibition, showing a significantly greater decline in GFR and ERPF and rise in effective renal vascular resistance at each timepoint during the infusion of inhibitor. To exclude the possibility that nonspecific renal vasoconstriction per se led to equalization of renal function in the two groups of rats, we investigated angiotensin II. In contrast to the results observed with nitric oxide synthase inhibitors, pregnant rats were less responsive to the renal vasoconstrictory effects of angiotensin II, such that the baseline differences in renal parameters measured before infusion of the hormone were increased during the infusion. To determine whether nitric oxide synthase was inhibited to a similar extent in gravid and virgin rats, aortic and renal cortical cGMP content was assayed ex vivo at the end of inhibitor infusion. The lower 2-micrograms/min dose of NAME consistently reduced cGMP content of these tissues to comparable levels in the two groups of rats. In conclusion, we suggest that nitric oxide mediates reduced renal vascular resistance and hyperfiltration during pregnancy in conscious rats. PMID- 7542285 TI - Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation. AB - In long-term bone marrow cultures, stroma-adherent progenitors proliferate significantly less than nonadherent progenitors. Thus, close progenitor-stroma interactions may serve to regulate or restrict rather than promote hematopoietic progenitor proliferation. We hypothesized that signaling through adhesion receptors on hematopoietic cells may contribute to the inhibition of proliferation observed when progenitors are in contact with stroma. We demonstrate that progenitors cultured physically separated from stroma in a transwell proliferate significantly more than progenitors adherent to stroma. Furthermore, proliferation of colony forming cells (CFC) is reduced after specific adhesion to stroma, metabolically inactivated glutaraldehyde-fixed stroma, stromal-extracellular matrix, or the COOH-terminal heparin-binding domain of fibronectin. Nonspecific adhesion to poly-L-lysine fails to inhibit CFC proliferation. That the VLA-4 integrin is one of the receptors that transfers proliferation inhibitory signals was shown using blocking anti-alpha 4 monomeric F(ab) fragments. Furthermore, when synthetic peptides representing specific cell attachment sites within the heparin-binding domain of fibronectin were added to Dexter-type marrow cultures, significantly increased recovery and proliferation of CFC was observed, suggesting that these peptides disrupt adhesion-mediated proliferation inhibitory events. Thus, negative regulation of hematopoiesis may not only depend on the action of growth inhibitory cytokines but also on growth inhibitory signals resulting from direct adhesive interactions between progenitors and marrow stroma. PMID- 7542286 TI - Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. AB - To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL 8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall. PMID- 7542287 TI - Increased nitric oxide synthase activity despite lack of response to endothelium dependent vasodilators in postischemic acute renal failure in rats. AB - Lack of response to endothelium-dependent vasodilators generally has been considered to be evidence for decreased nitric oxide synthase (NOS) activity and NO generation after ischemic or hypoxic injury to vital organs including the kidney. In this study, renal blood flow (RBF) responses to endothelium-dependent vasodilators acetylcholine and bradykinin and the endothelium-independent vasodilator prostacyclin, the nonselective NOS inhibitor L-NAME (without and with L-arginine), the inducible NOS inhibitor aminoguanidine, and the NO-donor sodium nitroprusside were examined in 1-wk norepinephrine-induced (NE) and sham-induced acute renal failure (ARF) rats. Compared with sham-ARF, there was no increase in RBF to intrarenal acetylcholine and bradykinin, but a comparable RBF increase to prostacyclin in NE-ARF kidneys. However, there was a significantly greater decline in RBF to intravenous L-NAME in NE- than sham-ARF rats (-65 +/- 8 vs. -37 +/- 5%, P < 0.001) which was completely blocked by prior L-arginine infusion. There was no change in RBF to the inducible NOS specific inhibitor aminoguanidine. Unlike sham-ARF, there was no increase in RBF to intrarenal sodium nitroprusside in NE-ARF. Immunohistochemistry and immunofluorescence detection of constitutive (c) NOS using mouse monoclonal antibody were carried out to positively determine the presence of cNOS in NE-ARF. 90% of renal resistance vessels showed evidence of endothelial cNOS in both sham- and NE-ARF. Taken together, results of these experiments are consistent with the conclusion that NOS/NO activity is, in fact, maximal at baseline in 1-wk NE-ARF and cannot be increased further by exogenous stimuli of NOS activity. The increased NOS is likely of the constitutive form and of endothelial origin. It is suggested that the increased NOS activity is in response to ischemia-induced renal vasoconstrictor activity. Attenuated response to endothelium-dependent vasodilators cannot be interpreted only as evidence for decreased NOS activity. PMID- 7542288 TI - Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain. AB - In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis. PMID- 7542290 TI - Organization of thalamic projections to the ventral striatum in the primate. AB - Although thalamic projections to the dorsal striatum are well described in primates and other species, little is known about thalamic projections to the ventral or "limbic" striatum in the primate. This study explores the organization of the thalamic projections to the ventral striatum in the primate brain by means of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and Lucifer yellow (LY) retrograde tracer techniques. In addition, because functional and connective differences have been described for the core and shell components of the nucleus accumbens in the rat and are thought to be similar in the primate, this study also explores whether these regions of the nucleus accumbens can be distinguished by their thalamic input. Tracer injections are placed in different portions of the ventral striatum, including the medial and lateral regions of the ventral striatum; the central region of the ventral striatum, including the dorsal part of the core of the nucleus accumbens; and the shell region of the nucleus accumbens. Retrogradely labeled neurons are located mainly in the midline nuclear group (anterior and posterior paraventricular, paratenial, rhomboid, and reuniens thalamic nuclei) and in the parafascicular thalamic nucleus. Additional labeled cells are found in other portions of the intralaminar nuclear group as well as in other thalamic nuclei in the ventral, anterior, medial, lateral, and posterior thalamic nuclear groups. The distribution of labeled cells varies depending on the area of the ventral striatum injected. All regions of the ventral striatum receive strong projections from the midline thalamic nuclei and from the parafascicular nucleus. In addition, the medial region of the ventral striatum receives numerous projections from the central superior lateral nucleus, the magnocellular subdivision of the ventral anterior nucleus, and parts of the mediodorsal nucleus. After injection into the lateral region of the ventral striatum, few labeled neurons are seen scattered in nuclei of the intralaminar and ventral thalamic groups and occasional labeled cells in the mediodorsal nucleus. The central region of the ventral striatum, including the dorsal part of the core of the nucleus accumbens, receives a limited projection from the midline thalamic, predominantly from the rhomboid nucleus. It receives much smaller projections from the central medial nucleus and the ventral, anterior, and medial thalamic groups. The shell of the nucleus accumbens receives the most limited projection from the thalamus and is innervated almost exclusively by the midline thalamic nuclei and the central medial and parafascicular nuclei. The shell is distinguished from the rest of the ventral striatum in that it receives the fewest projections from the ventral, anterior, medial, and lateral thalamic nuclei. PMID- 7542289 TI - QBEND10 for the diagnosis of myelodysplastic syndromes in routinely processed bone marrow biopsy specimens. AB - AIM: The assessment of the value of the antibody QBEND10, which is directed against the haemopoietic stem cell related antigen CD34, in the immunohistochemical diagnosis of myelodysplastic syndrome in routinely processed bone marrow biopsy specimens. METHODS: 581 formalin fixed, paraffin embedded trephine biopsy specimens of the iliac crest were immunostained with QBEND10 (avidin-biotin complex/ABC method). The number of CD34+ haemopoietic stem cells/blast cells (referred to hereafter as CD34+ cells) was determined in each case. The Wilcoxon test was used for statistical analysis. RESULTS: The following diagnostic categories were defined: (1) normal or reactive bone marrow (n = 356), (2) lymphoproliferative disorders, usually non-Hodgkin's lymphoma of low grade malignancy or multiple myeloma (n = 118), (3) myelodysplastic syndrome (n = 22), (4) acute leukaemia (n = 44), and (5) myeloproliferative diseases (n = 41). The average number of CD34+ cells was very low (0.2/HPF) in normal and reactive bone marrow, in lymphoproliferative disorders and in the myelodysplastic syndrome subtypes RA and RARS. Myeloproliferative diseases showed an average of three CD34+ cells/HPF. However, the average number of CD34+ cells was significantly higher (p < 0.05) in the myelodysplastic syndrome subtypes RAEB and RAEB-T (8.7/HPF) and in acute leukaemia (including both myeloid and lymphoblastic leukaemia; 111.7/HPF). CONCLUSIONS: QBEND10 is of value for the identification of RAEB and RAEB-T in routinely processed bone marrow biopsy specimens because it enables the detection of even small increases in the number of CD34+ cells. PMID- 7542291 TI - Thalidomide-resistant HIV-associated aphthae successfully treated with granulocyte colony-stimulating factor. AB - Thalidomide has been advocated as the treatment of choice for recalcitrant aphthae. We describe the case of patient with HIV infection and extensive aphthae whose condition failed to respond to corticosteroids, cyclosporine, and thalidomide. The patient's course was complicated by colonic aphthae. Rapid and sustained resolution was achieved through treatment with granulocyte colony stimulating factor, a previously unreported therapeutic option. PMID- 7542292 TI - Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia. AB - Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40 70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons. PMID- 7542293 TI - NADPH-diaphorase-positive nerve fibers in smooth muscle layers of opossum esophagus: gradients in density. AB - Nitric-oxide-releasing nerves regulate esophageal smooth muscle function. The density of such nerve fibers may differ in the different functional parts of the esophagus. We used both inspection and gray-scale analysis of digitized images to seek differences in density of such nerve fibers, stained for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), between esophageal body and esophago-gastric sphincter and between smooth muscle layers in the opossum esophagus. Sections of Swiss roll preparations of the entire organ were stained for NADPH-diaphorase and for immunoreactivity to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP) and constitutive nitric oxide synthase (cNOS). In the circular muscle layer, NADPH-diaphorase-positive fibers were most abundant at the cephalic end of the esophageal body with a significant decline toward and through the esophago-gastric sphincter. In the longitudinal muscle layer and the longitudinally-oriented muscularis mucosae, NADPH-diaphorase-positive nerve fibers were most abundant at the esophago-gastric sphincter with a significant decline toward and through the striated-smooth muscle junction. cNOS immunoreactivity co-localized with NADPH-diaphorase activity. Fibers stained for CGRP immunoreactivity were distributed like the NADPH-diaphorase-positive fibers. Fibers stained for immunoreactivity to the other peptides (VIP, GAL, SP) showed no clear differences in distribution along the esophagus in any of the muscle layers. PMID- 7542295 TI - Up-regulation of CD44 expression by tumor necrosis factor-alpha is neutralized by interleukin-10 in Langerhans cells. AB - CD44 is a principal cell-surface receptor for hyaluronate and is found on a wide variety of cells. CD44 plays an important role in lymphocyte homing, lymphohemopoiesis, and T-cell activation as well as in cell motility and migration. CD44 is expressed on the cell surface of epidermal Langerhans cells (LC), and is one of the candidates for molecules that are involved in the migratory capability of LC, but little is known about its regulatory properties. We examined the modulatory effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 on the CD44 expression in LC. We found 1) that TNF-alpha significantly up-regulated the expression of CD44 in a concentration-dependent manner, 2) that IL-10 down-regulated the expression of CD44 in a concentration dependent manner, 3) that the effect of TNF-alpha or IL-10 was readily detectable as early as 24 h after the initiation of culture, and 4) that the simultaneous addition of TNF-alpha and IL-10 mutually neutralized the effect of each other. These data suggest that in the epidermal microenvironment the expression of CD44 in LC may be reciprocally regulated by TNF-alpha and IL-10, both of which are known to be produced by surrounding keratinocytes. PMID- 7542294 TI - Interleukin-4 as a bone regulatory factor: effects on murine osteoblast-like cells. AB - Bone remodelling is regulated at the local level by an incompletely elucidated cytokine network. In the present study we have determined the effect of interleukin-4 (IL-4), a cytokine produced by T lymphocytes and other cells, on the activity of murine osteoblasts. IL-4 (0.1-10 ng/ml) did not influence the proliferation rate of the osteoblast-like cell line MC3T3, but inhibited the expression of alkaline phosphatase. In long-term cultures supplemented with ascorbic acid and glycerophosphate such an effect was accompanied by a retardation of matrix mineralization. IL-4 also stimulated M-CSF expression by MC3T3 cells, both at the RNA and bioactivity levels. However, no stimulation of IL-1, IL-6, GM-CSF or PGE2 production was observed. An IL-4-induced inhibition of alkaline phosphatase expression and retardation of mineralization was also found in cultures of primary osteoblast-like cells isolated from neonatal mice calvariae. These results suggest that IL-4, probably released by cells within the bone marrow, may locally influence the activity of bone-forming cells. PMID- 7542296 TI - Proliferative potential of different keratinocytes of plucked human hair follicles. AB - We have examined colony-forming ability, localization of colony-forming cells, and in vitro life spans of outer root sheath keratinocytes of different fragments of adult human plucked hair follicles. These were shown by immunohistochemical staining for cytokeratins and integrins to contain a preserved basal cell layer. By microdissection, five fragments of the outer root sheath (B1, B2, B3-1, B3-2, B4) were separated, dispersed by trypsin into single cell suspensions, and grown on human feeder fibroblasts. All fragments gave rise to at least some colonies, but colony-forming ability was mostly marked in the intermediate part (B2) and the lower half of the central part (B3-1); approximately 60% of colony-forming cells of a hair follicle localized to the fragment B3-1 and 28% to the fragment B3-2 (upper half of the central part, including bulge). To compare the in vitro life spans of cells from the various fragments, we subcultured isolated keratinocytes under identical conditions. The longest was found in the fragment B3-2 and the shortest in the fragment B1 (bulb). Moreover, the differentiation state of the native cells and the cells of all cultures were studied during their whole life spans by immunocytochemical analysis of various proliferation and differentiation markers. Surprisingly, keratinocytes of all fragments, as shown by expression of high-molecular-weight cytokeratins and filaggrin, were capable of terminal differentiation. These data indicate that cells with long life spans are localized in central parts of the outer root sheath close to the bulge area and that cells with high colony-forming ability are localized in the lower central parts. The latter are usually removed by plucking and may therefore not represent stem cells but rather cells important for hair growth during a single cycle. Cells with long life spans--also included in plucked hair follicles--may be immediate progeny of stem cells that will be segregated in the bulge area. Finally, our results are important for gene transfer and stem cell gene therapy in genodermatoses, because plucked hair follicles are easily available and keratinocytes close to the bulge area should be used selectively. PMID- 7542297 TI - Expression of V beta gene segments by Sezary cells. AB - The T-cell receptor V beta repertoire expressed by Sezary cells was determined in a series of 16 patients whose samples have been shown to contain a majority of tumor cells. By using anti-V beta monoclonal antibodies, polymerase chain reaction analysis of expressed V beta, and, in selected cases, nucleotide sequencing, we have shown that the expressed V beta segments belong to five V beta families (V beta 5, V beta 6, V beta 8, V beta 13, and V beta 18), which contain a large fraction of the T-cell receptor V beta repertoire and do not share significant similarities in complementary determining region 4. V beta segments from these five families were also found to be strongly expressed by CD4 + CD7- peripheral blood cells obtained by fluorescence-activated cell sorting from two healthy donors. The diversity of the V beta repertoire expressed by Sezary cells appears to be similar to that expressed by circulating non neoplastic T cells. These data do not support the hypothesis that a common superantigen is involved in the initiation of this form of cutaneous T-cell lymphoma. PMID- 7542298 TI - Direct and indirect control of T-cell activation by keratinocytes. AB - Keratinocytes can function as antigen-presenting cells/accessory cells and regulate T cells with three distinct outcomes, depending on the nature of the stimulus. In the presence of alloantigen, it appears that a "null" event takes place between T cells and keratinocytes, with neither activation nor induction of tolerance. Using nominal antigen, keratinocytes induce antigen-specific tolerance. In contrast, with bacterial-derived superantigens, phytohemagglutinin, or immobilized CD3 monoclonal antibody, keratinocytes can significantly activate resting autologous T-cell proliferation and cytokine release. To understand these highly divergent responses, we focused on the two-signal model of T-cell activation, with particular emphasis on costimulatory molecules expressed by keratinocytes. Such second signals, as highlighted by the B7 and CD28 receptor families, provide useful insights into the complex interactions involving keratinocytes and T cells. In this review, we summarize recent evidence indicating that keratinocytes regulate T-cell activation in a direct and indirect manner by their mutual expression and responsiveness involving adhesion molecules, cytokines, and costimulatory signals. As investigative momentum continues to grow in the fields of immunology and keratinocyte biology, it is likely that manipulation of CD28:B7 interactions will not only provide a useful model to understand further the complexities of skin immune reactions, but will also serve as the basis for new therapeutic opportunities for numerous T-cell mediated diseases that involve aberrant reactions with keratinocytes. PMID- 7542301 TI - [Efficacy of combination chemotherapy with miconazole and G-CSF in deep mycosis accompanying hematological diseases]. AB - In order to examine the efficacy of the combination chemotherapy with miconazole and G-CSF, patients with deep mycosis and suspected deep mycosis were divided into 3 groups. Group I: miconazole and G-CSF were administered simultaneously. Group II: miconazole was administered later during G-CSF administration. Group III: only miconazole was administered. Of a total of 117 cases 105 cases were analyzed including group I 37 cases, group II 39 cases and group III 29 cases, excluding 12 dropout and inadequate cases. Of the 105 cases, deep mycosis were 31 and suspected deep mycosis were 74, and underlying diseases were hematological malignancies such as leukemias. Efficacy judged mainly by the change of fever was 62.2% (23/37) in group I, 43.6% (17/39) in group II, and 41.4% (12/29) in group III, respectively. Efficacy was better in the patients whose neutrophil counts increased from less than 500/microliters to more than 500/microliters (group I 75.0%, group II 72.7%) than in the patients whose neutrophil counts were less than 500/microliters throughout the time of miconazole administration (group I 33.3%, group II 33.3%). Adverse effects were minimal in 3 groups (group I 15.4%, group II 17.4%, group III 15.6%). It is concluded that the combination with miconazole and G-CSF is effective in the treatment of deep fungal infections. PMID- 7542299 TI - Phenotypic changes that TCR V gamma 3+ fetal thymocytes undergo during their maturation into dendritic epidermal T cells. AB - Murine Thy-1+, TCR V gamma 3/V delta 1+ dendritic epidermal T cells (DETC) express CD2 antigens, but differ from most other T-cell subsets in their absence of CD4, CD5, and CD8 antigens. To determine whether negativity for those antigens is an intrinsic feature of a given T-cell population or whether such triple negative T cells go through a maturational stage during which they express these antigens, we determined the phenotype of TCR V gamma 3+ fetal thymocytes, which are the precursor cells of DETC. We found that TCR V gamma 3+ fetal thymocytes at day 17 of gestation are CD2+, CD5+, mostly CD8+, and partly CD4+. The expression of CD5 is highest on early TCR V gamma 3+ thymocytes; these cells express intermediate levels of CD5 when they leave the thymus and lose CD5 expression until or shortly after arrival in the epidermis. A similar loss of CD5 expression by TCR V gamma 3+ cells was observed in vitro under various culture conditions. To determine whether expression of CD5 is important for the maturation of DETC, we searched for these cells in the epidermis of CD5-deficient mice. There was no alteration in the number of Thy-1+/TCR V gamma 3+ dendritic cells in the epidermis of CD5-/- mice. Even though the latter finding speaks against a pivotal role of CD5 during the maturation of DETC, the described cell system may serve as a useful tool in further experiments aimed to clarify the function of the CD5 glycoprotein as well as the mechanism(s) regulating its expression. PMID- 7542300 TI - [Therapeutic evaluation of combination therapy using human native immunoglobulin preparation for i.v. administration, with antibiotics and G-CSF in severe infections in the field of internal medicine]. AB - A human native immunoglobulin liquid preparation for intravenous injection, was used in combination with antibiotics and G-CSF to study its efficacy and safety in 49 patients with severe infections (Granulocyte counts were or= 38 degrees C) which had not responded to antibiotic and G-CSF therapy of a 3-day or more duration. As a result of the Committee judgment, 49 patients were included in this study; 30 (61.7%) were included in efficacy and safety analysis. The analysis of 30 patients consisted of 9 patients (30.0%) with suspected septicemia, 5 (16.7%) with pneumonia, and 4 (13.3%) with septicemia. All patients had severe underlying diseases such as leukemia and malignant lymphoma. Clinical efficacy of Immunoglobulin preparation was judged by the doctors in charge to be "excellent" and "good" in 70.0% of the total cases. The rate of effectiveness was calculated from the results of the Committee judgment was 83.3% when "excellent" and "good" cases were included. No side effects were observed in all cases. Our results suggest that the immunoglobulin in combination with G-CSF is very effective on patients with severe infections. PMID- 7542302 TI - [Successful surgical treatment of total cavopulmonary connection on a 4-year-old boy with complete transposition of the great arteries and hypoplastic left ventricle]. AB - A four-year-old boy with complete transposition of the great arteries, intact ventricular septum and hypoplastic left ventricle, underwent total cavopulmonary connection after two palliative operations; B-T shunt and central shunt. He had undergone cardiac catheterization three times; four months after birth, and at two and four years of age. LVEDV (% normal) were calculated 31%, 26%, 27%, and RVEDV (% normal) were 226%, 115%, 105% respectively. PA index increased from 178 to 230 and further to 380. This case indicates that intracardiac repair is possible, if appropriate palliative operations suitable for patient's cardiac function and pulmonary artery morphology are applied. PMID- 7542304 TI - Stem cell factor. AB - Stem cell factor (SCF) is the ligand for the tyrosine kinase receptor c-kit, which is expressed on both primitive and mature hematopoietic progenitor cells. In vitro, SCF synergizes with other growth factors, such as granulocyte colony stimulating factor (G-CSF), granulocyte macrophage-colony-stimulating factor, and interleukin-3 to stimulate the proliferation and differentiation of cells of the lymphoid, myeloid, erythroid, and megakaryocytic lineages. In vivo, SCF also synergizes with other growth factors and has been shown to enhance the mobilization of peripheral blood progenitor cells in combination with G-CSF. In phase I/II clinical studies administration of the combination of SCF and G-CSF resulted in a two- to threefold increase in cells that express the CD34 antigen compared with G-CSF alone. Other potential clinical uses include ex vivo expansion protocols and in vitro culture for gene therapy. PMID- 7542305 TI - Role of macrophage-derived nitric oxide in suppression of lymphocyte proliferation during blood-stage malaria. AB - Examination of the proliferative responses in vitro to mitogens (concanavalin A, phytohemagglutinin, lipopolysaccharide) of spleen cells recovered from C57BL/6 mice during blood-stage Plasmodium chabaudi AS infection revealed that the most severe suppression occurred during the first 14 days post infection, that is, during the acute phase of infection. Coincidently, inducible nitric oxide synthase gene expression was found to be up-regulated in the spleens of infected mice, and both splenic and peritoneal macrophages produced high levels of NO in vitro in response to stimulation with lipopolysaccharide (LPS). The roles of NO, a molecule recently found to mediate immunosuppression during parasitic infections, and of the well-recognized immunosuppressive molecule prostaglandin were, therefore, investigated in the suppression of proliferation to mitogens and specific antigen of spleen cells from 7- and 14-day P. chabaudi AS-infected mice. Addition of either 0.5 mM NG-monomethyl-L-arginine (L-NMMA) or 0.5 mM aminoguanidine (AG), inhibitors of NO synthase, or 10 micrograms/ml indomethacin (INDO), a prostaglandin inhibitor, partially but significantly abrogated the suppression in response to concanavalin A (Con A) and phytohemagglutinin (PHA). Only the addition of INDO significantly increased the responses to LPS. Addition of L-NMMA or AG in combination with INDO partially but significantly abrogated the suppression in response to Con A and completely abrogated the suppression in response to PHA. The addition of L-NMMA or AG also significantly increased proliferation in response to parasite antigen. The contribution of NO to suppression of lymphoproliferation was confirmed by adding 3-morpholino sydnonimine-hydrochloride (SIN-1), a chemical generator of NO, to mitogen stimulated splenocyte cultures prepared from normal mice. The mechanism of NO mediated suppression was investigated in coculture experiments using spleen cells from normal mice and peritoneal macrophages from either normal or day 7 infected mice. The addition of 5-10 x 10(4) peritoneal macrophages from infected mice significantly and consistently suppressed Con A- or PHA-stimulated proliferation of normal splenocytes. Moreover, suppression correlated with production of NO and could be reversed by the addition of L-NMMA or AG. These results suggest that, in addition to prostaglandin, increased NO production by macrophages within the first 2 weeks after infection with P. chabaudi AS contributes to immunosuppression associated with blood-stage malaria. PMID- 7542306 TI - CD66-dependent neutrophil activation: a possible mechanism for vascular selectin mediated regulation of neutrophil adhesion. AB - We have examined the role of CD66 in the modulation of neutrophil adhesion and effector function. Engagement of neutrophil CD66 with anti-carcinoembryonic antigen (anti-CEA) Ig results in activation-associated phenomena including shape change, activation of beta 2-integrins, and priming of the respiratory burst. Anti-CEA Ig-treated neutrophils underwent transient shape change distinct from that induced by formyl-Met-Leu-Phe (fMLP). fMLP stimulated beta 2-integrin up regulation and 70% loss of L-selectin, whereas only low-level up-regulation of the beta 2-integrins, without loss of L-selectin, occurred with anti-CEA Ig. Anti CEA F(ab')2 fragments and whole Ig augmented beta 2-integrin-dependent adhesion. Anti-CEA Ig-induced beta 2-integrin activation was transient, whereas fMLP induced activation persisted longer. Although they did not cause a significant increase in respiratory burst activity, CEA Ig and F(ab')2 fragments of antibody primed neutrophils so that the subsequent fMLP-induced respiratory burst was significantly increased. PMID- 7542303 TI - The B cell antigen receptor complex: mechanisms and implications of tyrosine kinase activation. AB - The B cell receptor is a multimeric receptor complex whose constituent chains appear to mediate distinct and possibly interrelated functions. In this review we have focused on how one chain, immunoglobulin (Ig)-alpha, may function to activate tyrosine kinases and the consequences of that activation. The cytoplasmic domain of Ig-alpha contains a consensus sequence, the antigen recognition homology 1 (ARH 1) motif, which is found in Ig-beta and other antigen recognition receptor associated chains. We argue that this conserved structure reflects an underlying conserved mechanism of secondary effector activation. Our data also indicates that the specificity of each motif (i.e., the elements which restrict secondary effector binding to particular motifs) is encoded within divergent sequences found in each ARH 1 motif. In the particular case of kinase activation by Ig-alpha, the subsequent phosphorylation of multiple tyrosines on Ig-alpha, Ig-beta, CD19, CD22 and possibly other functionally related chains form recruitment sites for a myriad of secondary signal transducers. In this model, proximal tyrosine kinases and phosphatases do not function so much to mediate the linear transfer of information as to establish and modulate an interrelated network of signal transducers capable of driving complicated cellular responses. PMID- 7542307 TI - IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1 alpha, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1. AB - We investigated the effect of interleukin-1 (IL-1) activation of human umbilical vein endothelium (HUVE) on human monocyte transendothelial migration induced by chemotactic factors. Monocyte migration across unactivated endothelium in response to macrophage inflammatory protein-1 alpha (MIP-1 alpha), RANTES, platelet-activating factor (PAF), or monocyte chemoattractant protein-1 (MCP-1) was completely inhibited (90%) by monoclonal antibodies (mAbs; 60.3) to CD18 of the CD11/CD18 complex on the monocyte and partially inhibited (by 75%) in response to C5a. When the HUVE was stimulated with IL-1 alpha (5 h, 0.1 ng/ml), monocyte migration in response to C5a, MIP-1 alpha, RANTES, or PAF was no longer inhibited by mAb to CD18. However, migration was blocked by the combination of mAb to the alpha 4-integrin (CD49d) chain of very late antigen-4 (CD49d/CD29) with the mAb to CD18. In contrast to the above stimuli, activation of the HUVE with IL-1 alpha inhibited the transendothelial migration of monocytes in response to MCP-1. mAbs to the adhesion molecules up-regulated on HUVE by IL-1, i.e., E selectin (CD62E), intercellular adhesion molecule-1 (CD54) or vascular cell adhesion molecule-1 (CD106), did not reverse the inhibitory effect. Transendothelial migration in response to MCP-1 but not to C5a was inhibited by the treatment of monocytes with culture supernatant from IL-1 alpha-stimulated (but not from unstimulated) HUVE. Such supernatant contained chemotactic activity for monocytes, and a mAb to MCP-1 blocked the migration inhibitory effect of IL-1 activation of the HUVE monolayer, as well as the chemotactic activity in the supernatant from IL-1-stimulated HUVE. The inhibitory effect on migration of IL-1 stimulated HUVE was specific for monocytes because polymorphonuclear leukocyte transendothelial migration in response to IL-8 (a related chemokine) was not inhibited by IL-1 activation of HUVE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542308 TI - Lipid organization in pig stratum corneum. AB - The lipid and keratin structure of pig stratum corneum has been elucidated by small- and wide-angle X-ray diffraction. The measurements were carried out as a function of hydration and temperature. In addition, the stratum corneum was measured after recrystallization of the lipids at various temperatures. The results led us to conclude that the intercellular lipids in the stratum corneum are organized in at least two different lamellar structures with repeat distances of 6 and 13.2 nm. There is an indication for the presence of a third phase with a periodicity of 9 nm. The wide-angle pattern revealed a hexagonal (0.414 nm spacing) and liquid lateral packing (approximately 0.46 nm spacing). The 0.414 nm reflection started to decrease in intensity between 60 and 66 degrees C and disappeared between 72 and 95 degrees C. Furthermore, crystalline cholesterol has been indicated by both, wide- and small-angle X-ray diffraction, while the reflections of alpha-keratin were observed in the wide-angle X-ray diffraction pattern. PMID- 7542310 TI - New and old trends for treating organ-specific autoimmune endocrinopathies. PMID- 7542311 TI - Amphibian oocyte: a model of a possible regulatory mechanism for prostaglandin E2 and prostaglandin F2 alpha synthesis. AB - To clarify the possible mechanisms regulating prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) synthesis, the effects of gonadotropin releasing hormone (GnRH) and substance P (SP) on the release of these two prostaglandins were studied in the oocytes of the crested newt, Triturus carnifex. Full-grown oocytes of T. carnifex, freed from follicular cells, were incubated in the presence of GnRH or SP and of the inhibitors of several enzymes involved in the release of arachidonic acid (AA) and in the conversion of AA into PGE2 and PGF2 alpha. In parallel, the same experiments were performed on oocytes with membrane phospholipids labelled with [3H]AA. In addition, the PGE2-9 ketoreductase activity was evaluated through the conversion of [3H]PGE2 into [3H]PGF2 alpha. The results showed that GnRH and SP could regulate prostaglandin synthesis through the activation of phospholipase C and diacylglycerol lipase, and through the modulation of PGE2-9-ketoreductase in the oocytes of T. carnifex. In particular, GnRH enhances the activity of PGE2-9-ketoreductase with a consequent increase in PGF2 alpha, while SP inhibits the enzyme which leads to an increase in PGE2. A similar mechanism could also be hypothesized for other vertebrate species. PMID- 7542309 TI - Kringle 4 of human apolipoprotein[a] shares a linear antigenic site with human catalase. AB - Monoclonal antibody (mab) 1A2, directed against human apolipoprotein[a] (apo[a]), revealed a strong reaction with peroxisomes as shown by immuno-gold labeled cryosections of human liver biopsies. This reactivity was not due to the presence of apo[a] in peroxisomes but to a cross-reactivity of mab 1A2. Immunoblot analysis of peroxisomal fractions and purified human catalase demonstrated that mab 1A2 reacts with catalase. Conversely, an anti-catalase antibody also recognized apo[a]. By sequence comparison we identified a 4-amino acid motif (Y-Y P-N) that is shared between the highly repetitive kringle 4 motif of apo[a] and the carboxy-terminal third of the peroxisomal marker enzyme catalase. No other identical sequences were identified in these proteins. Results from the following experiments indicated that 1A2 recognizes this short linear epitope. i) Mab 1A2 reacted only with the 4 amino acid peptide sequence in a pin-ELISA using immobilized overlapping peptides. ii) A synthetic peptide including this sequence completely inhibited the 1A2 immunoreactivity to apo[a] and catalase. iii) A recombinant fusion protein tagged with the putative epitope was recognized by mab 1A2. Our findings demonstrate that unknown linear epitopes in native proteins can be identified by sequence comparison between known proteins. The practical implication is that antibodies against apo[a] must be controlled for this cross reactivity before using them for immunohistochemical studies of intracellular apo[a] in tissues or cells. PMID- 7542312 TI - Resistance in some Caribbean populations of Aedes aegypti to several insecticides. AB - Thirty-four strains of Aedes aegypti larvae from 17 Caribbean countries were bioassayed for sensitivity to temephos, malathion, fenitrothion, fenthion, and chlorpyrifos. There were fairly high levels of resistance in Tortola (10-12-fold resistance) and Antigua (6-9-fold resistance) strains to temephos and to fenthion (Tortola, 7-10-fold; Antigua, 6-10-fold resistance). Most other strains showed some resistance to malathion, fenitrothion, and chlorpyrifos, but only moderate levels. Adult populations of Ae. aegypti--Aruba, Jamaica, Trinidad, Puerto Rico, St. Lucia, and Antigua strains--also showed moderate resistance to malathion. Mosquito control field data supported the laboratory findings. Doubling the diagnostic dosage of temephos for larval Ae. aegypti was only partially effective against a more resistant strain, and even so, the chemical lost its limited efficacy over a short period of time. Integrated strategies for Ae. aegypti control to mitigate the negative effects of insecticide resistance in the Caribbean strains are suggested. PMID- 7542313 TI - Increased nitric oxide synthase activities and L-[3H]arginine uptake in brain following portacaval anastomosis. AB - Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS) in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As L-arginine transport is a prerequisite for nitric oxide production, we also studied L-[3H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. L-[3H]Arginine uptake was significantly increased (by approximately 50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting. PMID- 7542314 TI - Resuscitation of brain neurons in the presence of Ca2+ after toxic NMDA-receptor activity. AB - Cultured cerebellar granule cells were subjected to toxic activation of the NMDA receptor that was terminated by MK-801. Subsequent resuscitation experiments were mostly conducted in the presence of a physiological concentration of Ca2+. Addition of pyruvate and inorganic phosphate, in addition to glucose, which was always present, rescued approximately 40% of the dying neurons. La3+ and ruthenium red were also effective resuscitating agents. The combination of pyruvate, inorganic phosphate, and ruthenium red rescued 65% of the dying neurons. Parallel studies with 45Ca indicated that La3+ and ruthenium red facilitated the decrease of 45Ca in the neurons, whereas inorganic phosphate, supported by energy-yielding pyruvate, formed perhaps, a less harmful Ca complex inside the neurons. PMID- 7542315 TI - Brain histaminergic system in mast cell-deficient (Ws/Ws) rats: histamine content, histidine decarboxylase activity, and effects of (S) alpha fluoromethylhistidine. AB - The mast cell-deficient [Ws/Ws (White spotting in the skin)] rat was investigated with regard to the origin of histamine in the brain. No mast cells were detected in the pia mater and the perivascular region of the thalamus of Ws/Ws rats by Alcian Blue staining. The histamine contents and histidine decarboxylase (HDC) activities of various brain regions of Ws/Ws rats were similar to those of +/+ rats except the histamine contents of the cerebral cortex and cerebellum. As the cerebral cortex and cerebellum have meninges that are difficult to remove completely, the histamine contents of these two regions may be different between Ws/Ws and +/+ rats. We assume that the histamine content of whole brain with meninges in Ws/Ws rats is < 60% of that in +/+ rats. So we conclude that approximately half of the histamine content of rat brain is derived from mast cells. Next, the effects of (S) alpha-fluoromethylhistidine (FMH), a specific inhibitor of HDC, on the histamine contents and HDC activities of various regions of the brain were examined in Ws/Ws rats. In the whole brain of Ws/Ws rats, 51 and 37% of the histamine content of the control group remained 2 and 6 h, respectively, after FMH administration (100 mg/kg of body weight). Therefore, we suggest that there might be other histamine pools including histaminergic neurons in rat brain. PMID- 7542316 TI - Adrenergic control of rat pineal NO synthase. AB - We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by beta-adrenoceptors, because it was not mimicked by the alpha-agonist phenylephrine. Pineal NOS activity was reduced in 16-h light/8-h dark animals treated for 4 days with the beta-adrenergic antagonist propranolol but not with the alpha 1-antagonist prazosin, indicating again an involvement of beta-adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through beta-adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under Vmax conditions. PMID- 7542317 TI - Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis. AB - The poor prognosis of patients with malignant gliomas is at least partially due to the invasive nature of these tumors. In this study, the authors investigated the possibility that the cysteine protease cathepsin B (CB) is a participant in the process of glial tumor cell invasion. To accomplish this, an immunohistochemical analysis was made of the localization of antibodies to CB in biopsies of five specimens of normal brain, 16 astrocytomas, 33 anaplastic astrocytomas, and 33 glioblastomas multiforme. Staining was scored according to the percentage of positive cells and the intensity of the stain, graded from 0 to 3+. Staining for CB was not seen in any of five samples of normal brain except for occasional neuronal cell bodies and microglia. Only five (31%) of 16 astrocytomas showed a small percentage of positive cells (0.01%-3%) that were stained in a light, diffuse cytoplasmic pattern (1+). Twenty-nine (87.8%) of 33 anaplastic astrocytomas showed positive light, granular staining in 2% to 40% of cells. In anaplastic astrocytoma, the staining within a tumor was heterogeneous with intensities of 1+ (17%), 1+ to 2+ (29%), or 2+ (55%). In contrast, all 33 (100%) glioblastomas were positive in 10% to 90% of cells. The staining was present in a coarse, granular pattern with an intensity of 2+ (12%) or 3+ (88%). Tumor cells infiltrating into brain adjacent to malignant gliomas stained positively in 26 cases that could be evaluated for glioblastoma multiforme; these invading cells frequently followed penetrating blood vessels as typical "secondary structures of Scherer." Moderate to intense CB staining associated with endothelial proliferation in high-grade tumors was also observed, especially in regions of tumor infiltration into adjacent normal brain. These results provide evidence consistent with the hypothesis that CB is functionally significant in the process of tumor invasion and angiogenesis in the clinical progression of the malignant phenotype in astrocytes. PMID- 7542318 TI - Hetastarch complication. PMID- 7542319 TI - Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase, produced by Streptomyces sp. SA-3501. AB - Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase (GlcNAc ase), have been purified from the culture broth of Streptomyces sp. SA-3501 isolated from a marine environment. They were purified by chromatography on Dowex 50W, silica gel and Capcell Pak C18 (HPLC) followed treatment with active carbon and then isolated as white powders. The structures of pyrostatins A and B were determined by NMR studies to be 4-hydroxy-2-imino-1-methylpyrrolidine-5 carboxylic acid and 2-imino-1-methylpyrrolidine-5-carboxylic acid, respectively. They were competitive with the substrate, and the inhibition constants (Ki) of pyrostatins A and B were 1.7 x 10(-6) M and 2.0 x 10(-6) M respectively. PMID- 7542320 TI - Regulation of arylsulfate sulfotransferase from a human intestinal bacterium by nucleotides and magnesium ion. AB - Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group from phenylsulfate esters to phenolic compounds. Pentachlorophenol, one of the selective inhibitors of phenol sulfoconjugation in mammalian tissues, inhibited both phenol and tyramine sulfation by ASST. Nucleotide triphosphates such as ATP, GTP, UTP and CTP, and pyrophosphate inhibited the ASST activity, whereas Mg2+ and Mn2+ activated the enzyme and prevented its inhibition by ATP and pyrophosphate. Equimolar binding of [alpha-] and [gamma-32P]ATP to the enzyme showed that the enzyme protein was not phosphorylated, but bound ATP. These results suggest that nucleotide triphosphates and divalent cations are important modulators in the control of ASST activity. PMID- 7542322 TI - Enolase and the arsonomethyl analogue of 2-phosphoglycerate. AB - (RS)-3-Arsono-2-(hydroxymethyl)propionic acid was synthesized by the action of alkaline arsenite on 3-bromo-2-(bromomethyl)propionic acid. It is a substrate for yeast enolase (EC 4.2.1.11) with a Km of 6.5 mM (for 2-phospho-D-glycerate Km = 0.08 mM). The catalytic constant of the enzyme with the arsonomethyl analogue is 230 times lower than with 2-phosphoglycerate. PMID- 7542321 TI - Comparison of the inhibition of type A and type B S-adenosylhomocysteine hydrolase: effects of cofactor content on inhibition behavior and nucleoside binding. AB - The enzyme S-adenosylhomocysteine hydrolase (E.C.3.3.1.1) occurs in two forms in bovine liver: Type A, which carries four moles of NAD+ per mole of enzyme tetramer, and Type B, which carries two moles of NAD+ per mole of tetramer. The inhibition of these two forms of the enzyme with 2',2'-difluoro-2'-deoxyadenosine has been investigated. The studies examined the binding stoichiometry and stability of the enzyme-inhibitor complexes formed from each type of the enzyme, the degree of NAD+ reduction and NAD+ release, and the possibility of covalent bond formation between the enzyme and the inhibitor. Significant differences in the behavior of the two forms of the enzyme were encountered which may have important implications for the design of S-adenosylhomocysteine hydrolase inhibitors as therapeutic agents. PMID- 7542324 TI - Identification by gas chromatography-mass spectrometry of an adduct between pure pig plasma benzylamine oxidase and the inhibitor 3,5-diethoxy-4 aminomethylpyridine. AB - 3,5-Diethoxy-4-aminomethylpyridine (B24) interacts with pure pig plasma benzylamine oxidase (BAO), giving a Schiff base with the carbonyl active site. This Schiff base was reduced, isolated by chemical hydrolysis of the enzyme, purified by HPLC and identified by gas chromatography-mass spectrometry (GC-MS) after derivatization. The isolated B24 adduct had the same absorption spectrum, retention time on HPLC and GC and the same mass spectrum as B24-pyridoxamine. B24, which is a reversible enzyme inhibitor, is also a weak substrate and competes with benzylamine, which is the best substrate, for the active site. These results further indicate the presence of pyridoxal-phosphate covalently linked to the pig plasma benzylamine oxidase and involved in the active site of this enzyme. PMID- 7542323 TI - 2'-Fluoropolynucleotide-directed reverse transcriptase reactions. Effect of homologous polynucleotides. AB - Several homologous polynucleotides have been tested as inhibitors on the reactions catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyribonucleotides and 2'-fluorinated polynucleotides as templates. Polynucleotides differentially inhibited the reactions catalyzed by reverse transcriptase in the presence of these synthetic templates. Polyriboadenylic acid (poly(rA), poly(2'-O-methyladenylic acid) (poly(Am)), poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl), polyinosinic acid (poly(rI)) and polyuridylic acid poly(rU)) inhibited the polyribonucleotide-, but not the 2' fluorinated polynucleotide-directed reverse transcriptase activity. PMID- 7542325 TI - The mode of inhibition of human erythrocyte membrane-bound acetylcholinesterase by cisplatin in vitro. AB - The effect of the well known anticancer drug "cisplatin" on the human erythrocyte membrane-bound acetylcholinesterase (AChE) has been investigated. It was found that cisplatin has inhibitory activity against AChE. Cisplatin (0.5-7.0 mM) inhibited AChE activity (27-82%) in a concentration dependent manner. The nature of the inhibition of AChE by cisplatin was complex and involved a partial reversible stage followed by a slow acting irreversible step. The inhibition was independent of a preincubation period up to 90 min while becoming dependent after this period. By diluting the preincubated AChE-cisplatin mixture for different times (0-24 h), the inhibition was partially reversed but again increased progressively with incubation time, ultimately reaching complete inhibition. The effect of increasing substrate concentration had the same behaviour as the dilution effect. PMID- 7542326 TI - Inactivation of Leuconostoc mesenteroids NRRL B-512F dextransucrase by specific modification of lysine residues with pyridoxal-5'-phosphate. AB - Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was inactivated by pyridoxal-5'-phosphate (PLP). The inactivation was reversible in as much as the loss of enzyme activity was completely reversed by prolonged dialysis. PLP modified dextransucrase after reduction with sodium borohydride showed a characteristic fluorescence emission maximum at 397 nm when excited at 325 nm. The stoichiometric results indicated that four lysine residues are modified by PLP under the experimental conditions. These results established for the first time that lysine residues are essential for the activity of dextransucrase. PMID- 7542327 TI - Purification and properties of branched chain amino acid aminotransferase from gramicidin S-producing Bacillus brevis. AB - The branched chain amino acid aminotransferase [EC 2.6.1.42] was purified to a homogeneous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme had a molecular weight of about 93,000 and consisted of two identical subunits, each with a molecular weight of about 47,000. One pyridoxal phosphate is bound per subunit. In addition to branched chain amino acids, the enzyme uses L-phenylalanine and L-tryptophan as the amino donor, indicating that B. brevis branched chain amino acid aminotransferase has a broad substrate specificity for the amino donor. The enzyme utilized 2-oxoglutarate as the amino acceptor. The purified enzyme exhibits its absorption maxima at 332 and 427 nm at neutral pH. PMID- 7542328 TI - The mammography and prostate-specific antigen controversies: implications for patient-physician encounters and public policy. PMID- 7542330 TI - To screen or not to screen: What is the question? PMID- 7542331 TI - Demonstration of DNA damage/repair in individual cells using in situ end labelling: association of p53 with sites of DNA damage. AB - We describe the development and application of in situ end labelling (ISEL) to identity sites of damaged DNA in the nuclei of individual cells. In cell culture, exposure to a variety of genotoxic agents induced a dose and time-dependent increase in nuclear labelling. In addition, examination of histological sections of human skin exposed to solar-stimulated UV light showed ISEL in both keratinocytes and superficial dermal cells, with the same spatial and temporal distribution as that of a marker of DNA repair, PCNA (proliferating cell nuclear antigen). Using co-localization techniques and confocal microscopy, we found increased levels of p53 in many ISEL-positive cells in vitro, with a similar distribution of labelling in the nucleus. This observation provides further evidence for a direct role of p53 in the recognition of damaged DNA. Thus, ISEL should prove a convenient method for demonstrating genotoxic insult in individual cells and in histological material, and may have value in toxicological screening. This high-resolution microscopy technique can also be used to compare the spatial distribution of various proteins implicated in the response to DNA damage with the sites of the lesion. PMID- 7542329 TI - Hepatitis C: diagnosis and treatment. PMID- 7542332 TI - Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques. AB - Recently developed techniques based on 'in situ end-labelling' (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM-ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly 'pre apoptotic' cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material. PMID- 7542334 TI - Pharmacological characterization of neuronal acetylcholine gated ion channel receptor-mediated hippocampal norepinephrine and striatal dopamine release from rat brain slices. AB - Neuronal acetylcholine-gated ion channel receptor-mediated [3H]-norepinephrine ([3H]-NE) and [3H]-dopamine ([3H]-DA) release from rat hippocampal and striatal slices, respectively, were compared. The nicotinic receptor agonists (-) nicotine, (-)-cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) increased both [3H]-NE and [3H]-DA release in a concentration-dependent manner. The rank order of potency for the three agonists was DMPP > (-)-cytisine > (-) nicotine for evoking [3H]-NE release and (-)-cytisine > DMPP = (-)-nicotine for releasing [3H]-DA. (-)-Cytisine acted as a partial agonist in stimulating DA release as it displayed lower efficacy and inhibited the agonistic effect of (-) nicotine. (-)-Cytisine and (-)-nicotine were equally effective in stimulating NE release. The responses to a maximally effective concentration of (-)-nicotine, ( )-cytisine or DMPP on [3H]-NE release were blocked by 1 microM tetrodotoxin (TTX). In contrast, the effects of the various agonists on [3H]-DA release were not blocked by tetrodotoxin. The nicotinic receptor antagonists, d-tubocurarine (3-100 microM) and mecamylamine (1.0-10 microM) blocked the 3H-NE release induced by (-)-nicotine and DMPP in the rat hippocampal slice, whereas dihydro beta erythroidine (3-300 microM) was without effect. In the striatum, mecamylamine (0.3-10 microM) and dihydro beta-erythroidine (3-100 microM) blocked the responses mediated by both agonists whereas d-tubocurarine (3-100 microM) was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542333 TI - Mechanisms of acylcarnitine-mediated enhancement of calcium transport in the Caco 2 cell monolayer model. AB - The overall objective of this study was to determine the mechanisms of acylcarnitine-mediated enhancement of calcium transport across Caco-2 cells. The different mechanisms of enhancement postulated are (a) loosening of tight junctions, thereby promoting paracellular transport; (b) opening of calcium channels, thus increasing calcium entry; and (c) stimulation of the basolateral Ca-ATPase pump, thereby aiding calcium extrusion. Although the existence of calcium channels and the reversal of verapamil-mediated inhibition of calcium uptake by acylcarnitines were demonstrated for the first time in Caco-2 cells, the channels do not appear to be a major contributing factor to the enhancement of calcium transport by acylcarnitines. Calmidazolium, a potent Ca-ATPase pump inhibitor in tissues such as rat intestinal segments, failed to inhibit this pump in Caco-2 cells. Thus, the predominant mechanism of enhancement of calcium transport by acylcarnitines in the Caco-2 model appears to be via promotion of paracellular transport. PMID- 7542335 TI - Substance P stimulates and inhibits intestinal peristalsis via distinct receptors. AB - The tachykinins substance P (SP) and neurokinin A participate in the neural control of intestinal peristalsis. This study aimed at elucidating the types of tachykinin receptors involved in SP's ability first to stimulate and then to inhibit propulsive activity. Peristalsis in the guinea pig isolated ileum was triggered by fluid-induced distension of the intestinal wall. Unlike SP, the neurokinin (NK)-1 receptor-selective agonist SP methyl ester (1-100 nM) failed to facilitate peristalsis but caused a delayed inhibition of peristaltic activity. In contrast, the NK-2 receptor-selective agonist [beta-Ala8]-NKA-(4-10) (BANKA, 1 100 nM) stimulated, but did not inhibit, peristalsis. The NK-3 receptor-selective agonist succinyl-[Asp6,N-MePhe8]-substance P-(6-11) (SENKTIDE, 0.1-10 nM) was most potent in facilitating propulsive activity, and only with 10 nM SENKTIDE was a delayed inhibition of peristalsis seen. The receptors responsible for the tachykinin-evoked stimulation and inhibition of peristaltic activity were further characterized by use of the NK-1 receptor-selective antagonist (+)-(2S,3S)-3-(2 methoxybenzylamino)-2-phenylpiperidine (CP-99,994, 300 nM) and the NK-2 selective antagonist (-)-N-methyl-N[4-acetylamino-4-phenyl-piperidino-2 (3,4 dichlorophenyl)butyl]-benzamide (SR-48,968, 100 nM). CP-99,994 antagonized the inhibitory effects of SP (100 nM) and SP methyl ester (100 nM) on peristalsis but did not alter the facilitation of propulsive motility brought about by SP or BANKA (100 nM). Conversely, SR-48,968 (100 nM) suppressed the ability of SP and BANKA to stimulate persitaltic activity but did not attenuate the inhibitory motor effects of SP and SP methyl ester.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542336 TI - Acute administration of buprenorphine in humans: partial agonist and blockade effects. AB - Buprenorphine, a mixed opioid agonist-antagonist, is being investigated as a treatment for opioid dependence. This study compared the acute subjective and physiological effects of sublingual buprenorphine to those of p.o. methadone over a wide range of doses and compared the ability of both drugs to alter the effects of an opioid challenge. Male inpatient volunteers (n = 9) with histories of opioid abuse participated in this double-blind, double-dummy study. Sublingual buprenorphine (0, 0.5, 2, 8, 16 and 32 mg) and p.o. methadone (3.75, 15 and 60 mg) were administered once weekly according to a Latin-square design, and subjects were monitored on a variety of physiological and subjective measures. Twenty-four hours later, subjects were tested with ascending doses of i.m. hydromorphone (0, 1 and 4 mg) given 45 min apart. Buprenorphine and methadone produced typical opioid agonist effects of long duration, including pupillary constriction, respiratory depression and elevations on subject-rated and observer rated indices of euphoria, sedation and opioid-like symptoms. The buprenorphine dose-effect curves were nonlinear and maximal effects for most physiological and subjective measures were observed between 4 and 8 mg, with no greater effects observed at higher doses. The methadone dose-effect curves were linear across the range of doses tested. High doses of buprenorphine and methadone both attenuated the response to hydromorphone challenge 24 hr later. These data indicate that there is a ceiling on the effects of buprenorphine in humans that may reduce its abuse liability and increase its safety, and indicate that opioid blockade occurs after acute administration of buprenorphine or methadone. PMID- 7542337 TI - Asparagine stimulates piglet intestinal Cl- secretion by a mechanism requiring a submucosal glutamate receptor and nitric oxide. AB - Amino acids are potential components of oral rehydration solutions for infants, which could combine with glucose to further stimulate intestinal Na+ and water absorption. L-Glutamine, the principal fuel of the intestine, stimulates neutral NaCl absorption and enhances enterocyte DNA synthesis, but is unstable in solution. L-Asparagine (ASN), a more stable amino acid with similar structure to L-glutamine, also stimulates enterocyte proliferation. We determined the effects of ASN on electrolyte transport across piglet jejunum in Ussing chambers. Mucosal but not serosal ASN produced electrogenic Cl- secretion (delta JClnet = -1.8 +/- 0.3 microEq/cm2.hr-1). ASN, when added at 0.1 to 30 mM, increased short-circuit current in a dose-dependent manner with a K1/2 of approximately 5 mM and maximal effect at approximately 10 mM. The stimulation of Cl- secretion by ASN was blocked by pretreatment with serosal tetrodotoxin and bumetanide and was inhibited by preincubation with capsaicin (8-methyl-N-vanillyl-6-nonenamide) or substance P. Inhibition of nitric oxide synthesis with the structural analog of L arginine, NG-monomethyl-L-arginine, reduced ASN-stimulated secretion by > 70%. Additionally, serosal 6-cyanonitro-quinoxaline 2-3-dione, which is a nonspecific blocker of neural non-N-methyl D-aspartate (NMDA) glutamate receptors, fully inhibited the ASN response (IC50 = 10(-6) M). Inhibition was specific for neurally mediated secretion. We found no inhibition of ASN-stimulated secretion by atropine, ketanserin, indomethacin or L-2-amino-5-phosphonovalerate (specific for NMDA receptors). When compared to ASN, L-glutamate was a weaker stimulator of jejunal Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542338 TI - Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits. AB - The role of nitric oxide (NO) in myocardial ischemia-reperfusion injury is still controversial. To determine the role of NO in the propagation of myocardial injury in a coronary artery occlusion-reperfusion model, we examined the effect of a competitive NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L NAME), with and without L-arginine, on the size of the infarct resulting from coronary artery occlusion (30 min) followed by reperfusion (48 hr) in rabbits. L NAME (300 micrograms/kg, as a bolus, and 100 micrograms/kg/min, i.v.) with and without L-arginine (30 mg/kg, as a bolus, and 10 mg/kg/min, i.v.) was administered immediately before coronary occlusion to 60 min after reperfusion. The infarct size in the L-NAME-treated rabbits (75.1% +/- 5.0%, n = 7), assessed as a percentage of infarcted region/ischemic region, was significantly larger than that of control rabbits (51.2% +/- 7.4%, n = 7; P < .05). The increase in infarct size was significantly attenuated by the treatment with L-NAME and L arginine (62.0% +/- 4.0%, n = 7). However, the infarct size for the treatment with L-NAME and D-arginine (76.7% +/- 5.7%, n = 6) did not differ from that in the L-NAME-treated rabbits. There was no significant difference in the infarct size between L-arginine-treated (60.1% +/- 7.3%, n = 6) and control rabbits. Rate pressure products, as an index of myocardial oxygen consumption, were comparable in all the groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542340 TI - Experimental diabetic neuropathy. Inhibition of protein mono-ADP-ribosylation prevents reduction of substance P axonal transport. AB - The extranuclear endogenous mono-ADP-ribosylation of proteins in cellular fractions from retinas of control and diabetic rats was studied. At least six proteins were ADP-ribosylated in the crude extract, membrane and cytosolic fractions from control preparations, whereas in diabetic rats the number of labeled proteins and the extent of labeling were highly reduced. Treatment of diabetic animals with silybin, a flavonoid with ADP-ribosyltransferase inhibitory activity, did not affect hyperglycemia, but prevented the alterations of the extent of ADP-ribosylation of the 38 K cytosolic, 39 K, 40 K membrane and 39 K, 41 K and 42 K crude extract proteins. These data suggest a hyperactivity of extranuclear endogenous protein mono-ADP-ribosylation in the diabetic rat retina, and that treatment with silybin inhibits such enzyme activity, thus improving the extent of ADP-ribosylation. Sciatic nerve axonal transport of substance P was reduced markedly in diabetic rats, and inhibition of mono-ADP-ribosylation with silybin prevented such a loss in spite of high blood glucose levels. These results suggest that the abnormal endogenous ADP-ribosylation of proteins might play a role in the onset of diabetic peripheral neuropathy and its inhibition may represent a novel pharmacological approach to the treatment of diabetes complications. PMID- 7542339 TI - Synergistic effects of tacrolimus and human interferon-alpha A/D in murine viral myocarditis. AB - The effects of interferon-alpha A/D (IFN) therapy in combination with various immunosuppressants were investigated in a murine model of viral myocarditis. Viral infection is an important cause of morbidity in immunocompromised hosts and transplant recipients. Human IFN therapy reduces viral replication, reducing the virus-induced myocardial destruction. Groups consisting of 25 C3H/He mice received i.p. injections of prednisolone, azathioprine, 15-deoxyspergualin, cyclosporine or tacrolimus (FK506), for 16 days beginning 2 days before inoculation with 500 plaque-forming units of encephalomyocarditis virus (EMCV). IFN, 10(4) U/g daily, was administered i.p. alone or in combination with immunosuppressants to separate groups of mice beginning on the day of viral inoculation. Animals were sacrificed at random at 4 or 10 days after inoculation with EMCV. The survival rate was significantly higher in mice treated with azathioprine, 15-deoxyspergualin, cyclosporine or FK506 in combination with IFN than in infected controls (P < .01) and was similar to the rate in the IFN monotherapy group. Survival in mice treated with prednisolone resembled that in infected controls and was significantly lower than in mice treated with IFN (P < .01). Heart weight was lower and cellular infiltration in the myocardium was reduced in mice treated with both FK506 and IFN compared with mice given IFN monotherapy. The results suggest that the effect of IFN therapy in viral myocarditis differs depending on which immunosuppressants is used. The findings suggest that the combination of FK506 and IFN may have beneficial effects in hosts with viral myocarditis by reducing cellular infiltration of heart. PMID- 7542342 TI - [Planar lipid bilayer system for study of ion channels]. PMID- 7542341 TI - Epithelial cell proliferation in odontogenic keratocysts: a comparative immunocytochemical study of Ki67 in simple, recurrent and basal cell naevus syndrome (BCNS)-associated lesions. AB - The aim of the present study was to investigate epithelial cell proliferation in the linings of odontogenic cysts, including three different subtypes of odontogenic keratocyst (OKC), namely simple (non-recurrent), recurrent and basal cell naevus syndrome (BCNS)-associated lesions. Ki67 immunoreactivity in OKC (simple, n = 10; recurrent, n = 8; syndrome, n = 9), dentigerous cysts (DC, n = 5), radicular cysts (RC, n = 5) and normal oral mucosa (n = 7) was studied using a biotin-streptavidin-peroxidase method on paraffin sections after microwave treatment. Ki67+ epithelial cells were counted manually and related to the length of basement membrane (BM) as determined by TV image analysis. Data were analysed by the Mann-Whitney U test. The number of Ki67+ cells in simple OKC linings (53.1 +/- 17.8 cells/mmBM) was similar to that in oral epithelium (42.5 +/- 12.7 cells/mmBM; P > 0.2). However, both contained significantly more Ki67+ cells than DC (3.9 +/- 1.3 cells/mmBM) and RC linings (6.7 +/- 4.8 cells/mmBM; P < 0.006). The epithelial distribution of Ki67+ cells differed between groups, with the percentage of positive cells in basal layers in OKC linings (7.0 +/- 2.1%) being significantly lower than that of oral epithelia (35.9 +/- 5.6%), DC (78.4 +/- 8.4%) and RC (80.6 +/- 7.7%) linings respectively (P < 0.003). Comparison of Ki67 expression within the OKC group revealed no significant difference between simple and recurrent lesions (44.0 +/- 13.8 cells/mmBM; P > 0.3). However, OKC associated with BCNS contained significantly higher numbers of Ki67+ cells (91.8 +/- 35.6 cells/mmBM; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542343 TI - Effect of immunization against synthetic peptide sequences of the alpha N-subunit of bovine inhibin on ovulation rate, gonadotrophin concentrations and fertility in heifers. AB - The effects of immunizing cattle against either of two peptides from the amino terminal peptide (alpha N) of the alpha 43-subunit of bovine inhibin on ovulation rate, gonadotrophin concentration and fertility were investigated. Two peptide sequences from the alpha N-subunit of bovine inhibin (P1N, bI alpha-(8-20) and P2N, bI alpha-(153-167)) were synthesized and conjugated to human serum albumin (HSA). Hereford-cross heifers (n = 5 per group) were given an initial injection of 3 mg of one of the peptide conjugates, followed by three booster injections (1.5 mg) at intervals of 11 weeks. Control heifers (n = 5) were injected with HSA only. Blood samples were taken once a week to measure antibody titre and every hour at about the time of the first oestrus and during the mid-luteal phase after the second booster injection, to measure FSH and LH concentrations. Ovulation rate was measured by ultrasonography. Gonadotrophin concentrations were analysed for four periods relative to the peak (time = 0 h) of the preovulatory LH surge as follows: pre-surge: -16 to -5 h; surge: -4 to 4 h; post-surge: 5 to 16 h and a period of 12 h during the mid-luteal (days 10-12) phase. Antibodies that bound to the individual peptides were generated and the ovulation rate increased (P < 0.05) in immunized heifers. Control heifers had one ovulation at all ovulatory cycles monitored. In group P1N, one heifer had two ovulations at each of the six cycles monitored, while another heifer had two ovulations at one cycle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542344 TI - Conceptus interferon in uterine flush, endometrial concentrations of oxytocin receptors and prostaglandin F2 alpha release in vitro after transfer of conceptuses to ewes induced to ovulate at 28 days postpartum. AB - We examined the key events underlying maternal recognition of pregnancy and the prevention of luteolysis in early postpartum ewes by synchronously transferring single expanded blastocysts recovered from control ewes on day 11 of pregnancy into the uterus of either postpartum recipients that had been induced to ovulate 28 days after lambing (n = 12) or control recipients (n = 11). Conceptus development uterine flush interferon (oTP-1) concentrations, endometrial oxytocin receptor concentrations and endometrial prostaglandin F2 alpha (PGF2 alpha) release in vitro were determined 5 days later (corresponding to day 16 of the ovarian cycle). By this stage, both conceptus mass and oTP-1 content of total uterine flush in the eight postpartum recipients that remained pregnant were significantly lower (P < 0.01) than in the eight pregnant control ewes (524 +/- 116.6 versus 959 +/- 80.6 mg and 968 +/- 16.9 versus 1512 +/- 106.2 ng oTP-1 for postpartum and control recipients, respectively). These effects were independent of ovulation rate and daily peripheral progesterone concentrations after blastocyst transfer, which were similar between groups. Endometrial oxytocin receptor density was variable in both groups when they were killed, and was generally higher in pregnant postpartum than in control recipients, and was significantly different (P < 0.05) when the values for the transfer but not the contralateral uterine horns were compared. Similarly, basal and oxytocin stimulated endometrial PGF2 alpha release during a 4 h incubation were higher (P < 0.01) in pregnant postpartum versus control recipients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542346 TI - Rare variants of chromosome 9 with extra G positive band within the qh region are not alike. PMID- 7542345 TI - Expression of gonadotrophin subunit genes in sheep that were homozygous carriers and non-carriers of the Booroola fecundity gene FecB. AB - Homozygous carriers (BB) of the Booroola fecundity gene FecB are characterized by high plasma concentrations of immunoreactive or biologically active FSH and, to a lesser extent, of immunoreactive LH, relative to non-carriers (++). Bovine cDNA probes for the alpha gonadotrophin, FSH beta and LH beta genes were used to investigate FecB-specific differences in the mRNA species for the gonadotrophin subunits in pituitaries obtained from ++ and BB mid-luteal phase ovary-intact ewes, ovariectomized ewes and ovary-intact or ovariectomized ewes with hypothalamic-pituitary disconnection (HPD) given the same regimen of pulsatile GnRH. No FecB-specific differences in the number or size of mRNA transcripts detected by northern blotting were noted for any of these genes. Densitometry of the northern blots revealed no significant FecB-specific differences in the relative amounts of mRNA encoding alpha gonadotrophin, FSH beta or LH beta in the pituitaries from any of the experimental groups of ++ and BB sheep. Furthermore, there were no significant FecB-specific differences in the pituitary content of FSH or LH in these animals, despite significantly higher plasma concentrations of FSH in the ovary-intact and ovariectomized HPD groups. These data show that whereas the FecB gene causes increased plasma concentrations of FSH, no consistent effects can be demonstrated on pituitary gonadotrophin content or on gonadotrophin subunit gene transcription, using northern analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542347 TI - Mild cystic fibrosis phenotype in patients with the 3272-26A > G mutation. PMID- 7542348 TI - Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding. AB - Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921 922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity. PMID- 7542349 TI - Tendamistat as a scaffold for conformationally constrained phage peptide libraries. AB - The alpha-amylase inhibitor Tendamistat (Hoe-467), a 74 amino acid beta-sheet protein from Streptomyces tendae has been expressed on the surface of the filamentous bacteriophage M13. Phage displaying Tendamistat inhibit the hydrolysis of starch by alpha-amylase, indicating that the displayed protein is functional. The displayed Tendamistat has been used as a molecular scaffold for the presentation of constrained random peptides. Two loops, comprising residues 38 to 40 and 60 to 65 of Tendamistat, were randomized using PCR mutagenesis. Libraries of approximately 10(8) different mutant Tendamistat molecules were tested for binding to monoclonal antibody A8, which recognizes endothelin. After three cycles of biopanning, phage were isolated that specifically bound the monoclonal antibody. Loop swapping and alanine replacement mutagenesis indicated that residues in the 60 to 65 loop are responsible for binding to the monoclonal antibody. This work demonstrates the use of relatively small non-antibody protein scaffolds for the presentation of constrained random peptide sequences to select for novel binding molecules. PMID- 7542350 TI - Conformational transitions in peptides containing two putative alpha-helices of the prion protein. AB - Prions are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPSc). Conversion of the cellular isoform (PrPC) to PrPSc is accompanied by a diminution in the alpha-helical content and an increase in the beta-sheet structure. To investigate the structural basis of this transition, peptide fragments corresponding to Syrian hamster PrP residues 90 to 145 and 109 to 141, which contain the most conserved residues of the prion protein and the first two putative alpha-helical regions in a PrPC model, were studied using infrared spectroscopy and circular dichroism. The peptides could be induced to form alpha-helical structures in aqueous solutions in the presence of organic solvents, such as trifluoroethanol and hexafluoroisopropanol, or detergents, such as sodium dodecyl sulfate and dodecyl phosphocholine. NaCl at physiological concentration or acetonitrile induced the peptides to acquire substantial beta sheet. The intermolecular nature of the beta-sheet was evident in the formation of rod-shaped polymers as detected by electron microscopy. Resistance to hydrolysis by proteinase K and epitope mapping argue that the beta-sheet structures were formed by the interaction of residues lying between 109 and 141. A similar range of residues was shown by nuclear magnetic resonance spectroscopy to be capable of forming alpha-helices. The alpha-helical structures seem to require a hydrophobic support from either intermolecular interactions or the hydrophobic environment provided by micelles, in agreement with the predicted hydrophobic nature of the packing surface among the four putative helices of PrPC and the outer surfaces of the first two helices. Our results suggest that perturbation of the packing environment of the highly conserved residues is a possible mechanism for triggering the conversion of PrPC to PrPSc where alpha helices appear to be converted into beta-sheets. PMID- 7542351 TI - Recognition molecules myelin-associated glycoprotein and tenascin-C inhibit integrin-mediated adhesion of neural cells to collagen. AB - Because of the importance of collagens in mediating cell-substrate interactions and the association of collagens with neural recognition molecules in the peripheral nervous system, the ability of neural recognition molecules to modify the substrate properties of collagens, in particular collagen type I, for cell adhesion was determined. Two cell lines, the N2A neuroblastoma and PC12 pheochromocytoma, were investigated for their capacity to adhere to different collagen types in the absence or presence of several neural recognition molecules. Adhesion of N2A or PC12 cells and membrane vesicles from PC12 cells to collagen type I was reduced when the collagen had been preincubated prior to its application as substrate with the extracellular domain of myelin-associated glycoprotein (s-MAG) or, as control, fibroblast tenascin-C (F-tenascin). In mixture with other collagen types, s-MAG was only able to reduce the adhesiveness of collagen types III and V, but not of collagen types II and IV. F-tenascin reduced the adhesiveness of all collagen types tested. In contrast to F-tenascin, s-MAG had to be present during fibrillogenesis to exert its effect, indicating that it must be coassembled into the collagen fibril to block the binding site. Cell adhesion to collagen type I was dependent on Mg2+ or Mn2+ and inhibited by a monoclonal antibody to the alpha 1 integrin subunit. The combined observations indicate that s-MAG and F-tenascin interfere with cell binding, most probably by modifying the integrin binding site, and that the two molecules act by different mechanisms, both leading to reduction of adhesion. PMID- 7542352 TI - Strontium 89 therapy for the palliation of pain due to osseous metastases. AB - OBJECTIVE: To present the current state of systemic radiopharmaceutical therapy for the palliation of pain in individuals with metastatic cancer and to evaluate the palliative effect and degree of hemotoxicity of strontium chloride 89 (89Sr) in patients with painful osteoblastic metastases primarily from prostate and breast cancer. DATA SOURCES AND STUDY SELECTION: A MEDLINE search through December 1994 was performed to identify English-language studies that met the following criteria. All eligible studies reported treatment of patients with painful osteoblastic bony metastases primarily from prostate or breast cancer treated with intravenous 89Sr. For study eligibility, evaluation of clinical response as assessed by the Karnofsky index, need for pain medication, or changes in mobility or sleep patterns was required. Hemotoxicity data were a requirement. A minimum of 10 prostate cancer cases was necessary for study inclusion. Only those studies assessing clinical response following one injection of 89Sr were included. Preliminary reports of cooperative studies were not included. Doses of 89Sr ranged from 0.6 MBq/kg (16 microCi/kg) to 400 MBq (10.8 mCi) per patient. Evaluation of patients for at least 3 months following 89Sr treatment was required. In addition, two studies examining issues of cost with regard to 89Sr treatment were identified. DATA EXTRACTION: Baseline pain assessment and periodic pain estimates as measured by the Karnofsky index, medication diaries, changes in mobility, sleep patterns, and/or ability to work were the basis for assessment of response. Baseline and periodic complete blood cell counts were the basis for hemotoxicity evaluation. DATA SYNTHESIS: Palliation and hemotoxicity data were analyzed separately for each study. Some improvement occurred in as many as approximately 80% of patients. Several studies demonstrated complete relief of pain in at least 10% of patients The nadir of platelet and white blood cell counts appears at approximately 4 to 8 weeks following injection, with a partial return to baseline by 12 weeks. As many as 10 injections spaced 3 months apart have been given to some patients with repeated palliative effect and without serious hemotoxicity. Reinjection may be limited by a platelet count below 60 x 10(9)/L, a white blood cell count below 2.4 x 10(9)/L, or the absence of osteoblastic skeletal metastasis as seen on bone scan. Studies examining treatment costs suggest that 89Sr may decrease costs associated with palliation of pain due to metastatic disease. CONCLUSIONS: As many as 80% of selected patients with painful osteoblastic bony metastases from prostate or breast cancer may experience some pain relief following 89Sr administration. In addition, as many as 10% or more may become pain free. Duration of clinical response may average 3 to 6 months in some cases. Hemotoxicity is mild. A decrease in treatment costs with administration of 89Sr to patients with painful osteoblastic bony metastases from prostate cancer may occur. These observations reflect the preliminary nature of knowledge in this field and point to the need for larger clinical trials of the use of 89Sr palliation. PMID- 7542353 TI - [Treatment of the porphyrias]. AB - There are seven porphyrias which are caused by defective functions of the enzymes in the haem biosynthesis. The clinical classification of porphyrias are divided into three types which are neuroporphyria, neurocutaneous porphyria and cutaneous porphyria. For acute porphyric attack of neuroporphyria and neurocutaneous porphyria, the treatments of choice are administration of glucose and/or hematin, haem arginate and tinprotoporphyrin. Porphyria cutanea tarda in cutaneous porphyria is controlled by removal of iron by phrebotomies or low-dose chloriquine. A novel approach of intravenous administration of interferon may be useful to control the associated case of chronic hepatitis type C. Skin symptoms in erythropoietic protoporphyria can be alleviated with beta carotene. Hepatic failure due to protoporphyria may need a liver transplantation. Cimetidine, a H2 receptor antagonist, may be useful in the treatment of acute porphyric attack and in remission stage in neuroporphyrias, neurocutaneous porphyrias and cutaneous porphyrias such as porphyria cutanea tarda and protoporphyria. PMID- 7542354 TI - [Porphyria cutanea tarda accompanying hepatitis C]. AB - Porphyria cutanea tarda (PCT) is due to reduced activity of the enzyme uroporphyrinogen decarboxylase in hepatocyte. Hepatic dysfunction is found in almost all cases of PCT. Recently, high prevalence of hepatitis C virus (HCV) infection in patients with PCT was reported in Europe. Is HCV infection a trigger of PCT? Several cases of PCT associated with hepatitis C were reported in Japan. The frequency of anti-HCV increased with the severity of liver histology. These findings implicate HCV in the etiology of PCT-associated liver disease. Treatment with interferon should be considered for PCT with HCV infection. PMID- 7542355 TI - Studies on anti-allergic action of AH 21-132, a novel isozyme-selective phosphodiesterase inhibitor in airways. AB - The effects of AH 21-132, a type III and IV phosphodiesterase (PDE) inhibitor, on allergic reactions in the airway were studied by comparing them with the effects of rolipram, a type IV PDE inhibitor, and aminophylline, a non-selective PDE inhibitor. The following results were obtained: 1) AH 21-132, rolipram and aminophylline inhibited the antigen-induced contraction of isolated guinea pig tracheal muscle in vitro. 2) AH 21-132 and aminophylline inhibited antigen induced histamine release from human lung tissue fragments. 3) Antigen-induced accumulation of inflammatory cells including eosinophils and macrophages in mice bronchoalveolar lavage fluid was clearly inhibited by AH 21-132 and rolipram, but not by aminophylline. 4) AH 21-132, rolipram and aminophylline inhibited immediate phase bronchocostriction induced by either an intravenous or an aerosol challenge of antigen in guinea pigs. 5) AH 21-132 and rolipram inhibited the aeroantigen challenge-induced late phase increase in the airway resistance in guinea pigs, but aminophylline did not. These results suggest that AH 21-132 has an anti-allergic effect in the airway and that these actions may be beneficial for the treatment of allergic bronchial asthma. PMID- 7542356 TI - Selective repression of growth-regulating cdk2, cyclin E and E2F1 genes in human cell senescence. AB - Normal human diploid TIG-1 fibroblasts underwent replicative senescence around 64 68 population doubling levels (PDL) by the irreversible serum-unresponsive G1 growth arrest. Repression of growth-promoting genes was searched in this study. The RT-PCR and Western blot analyses have shown that in senescent TIG-1 cells at PDL64-67, cdk2 and cyclin E were selectively repressed at the mRNA and protein levels even after serum stimulation, and cdc2 and cyclin A were less repressed than cdk2 and cyclin E. Such a specific lack of cdk2 and cyclin E proteins correlated with unphosphorylation of the retinoblastoma gene product (pRB) in senescent cells. Transcription factor E2F1 was also completely repressed at the mRNA and protein levels in senescent TIG-1 cells. Middle-passage cells exhibited active expressions of all the above genes and pRB phosphorylation. Therefore, the present results have indicated the selective repressions of cdk2, cyclin E and E2F1 in senescent cells. PMID- 7542357 TI - Treatment of B-cell lymphomas with combination of bispecific antibodies and saporin. AB - We report the use of a bispecific F(ab')2 antibody to target the ribosome inactivating protein saporin to the surface antigen CD22 in the treatment of low grade, end-stage, B-cell lymphoma. Four patients were treated. Toxic effects were minimal (grade I), with mild fever, weakness, and myalgia for 1-2 days after treatment. One patient showed an antibody response to mouse Fab' and saporin. All patients showed rapid and beneficial responses to treatment with improvements in most disease sites and in peripheral blood cytopenia. The responses were short lived (less than 28 days) but further study of this targeting system is warranted. PMID- 7542358 TI - CD44v6 and CD44v3 expression in ulcerative colitis and Crohn's disease. PMID- 7542359 TI - Post-transfusion hepatitis revisited. AB - OBJECTIVE: To evaluate the risk of post-transfusion and postoperative non-A non-B hepatitis in Australia immediately before the introduction of screening for hepatitis C. DESIGN: Retrospective testing of blood samples from a prospective study of cardiac surgery patients. Samples were taken from transfusion recipients and non-transfused controls at regular intervals for 12 months after surgery during 1987-1989. For all donor, recipient and control samples, alanine aminotransferase (ALT) levels were measured and tests for antibody to hepatitis B (anti-HBc, anti-HBs) and, when available, to hepatitis C (anti-HCV) were performed. SETTING: Cardiac surgery units. PARTICIPANTS: Participants were included if they lived in the metropolitan area, and had not had a transfusion in the past year. MAIN OUTCOME MEASURES: Post-transfusion hepatitis (two consecutive samples showing raised ALT levels, > 90 IU/L with no other known cause); hepatitis C infection and carriage (antibody to hepatitis C). RESULTS: Post transfusion hepatitis occurred in 1.1% of 736 recipients of blood not screened for hepatitis C (i.e., two cases per 1000 unscreened units given). No hepatitis occurred in 514 controls. Seven of the eight patients with post-transfusion hepatitis seroconverted to hepatitis C virus infection. Seven of the 26 anti-HCV positive donations transmitted hepatitis C, six of these were positive by recombinant immunoblot assay (RIBA) (one by second generation testing only) and one was RIBA indeterminate. Nineteen were RIBA non-reactive; one transmitted hepatitis but the recipient did not develop anti-HCV, although hepatitis C RNA was detected in the donation. Serum ALT was raised in four of the six infective donations. CONCLUSIONS: Hepatitis C virus infection accounted for almost all cases of non-A non-B post-transfusion hepatitis. First generation anti-HCV tests detected about 85% of infective donations. Surrogate testing of donations by ALT or anti-HBc offers no additional advantage. PMID- 7542360 TI - [Noninvasive ventilation before lung transplantation]. PMID- 7542361 TI - Brief report: correction of X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation. PMID- 7542363 TI - [Scripta manent? On close reading and written wills]. PMID- 7542362 TI - Failure of B-cell differentiation in mice lacking the transcription factor EBF. AB - Early B-cell factor (EBF) is a cell type-specific transcription factor that is expressed at all antigen-independent stages of B-lymphocyte differentiation and participates in the regulation of the mb-1 gene. Here we show, by targeted gene disruption in mice, that EBF is necessary for the generation of immunoglobulin expressing B cells. EBF-deficient mice lack B cells that have rearranged their immunoglobulin D and JH gene segments, but contain B220+CD43+ progenitor cells that express germline mu and IL-7 receptor transcripts. Various non-lymphoid tissues that express EBF are apparently normal in homozygous mutant mice, including olfactory neurons in which EBF was identified as Olf-1 (refs 5, 6). Together, these data suggest that EBF plays a specific and important role in the transcriptional control of B-cell differentiation at a stage before Ig (immunoglobulin) gene rearrangement but after commitment of cells to the B lymphoid lineage. PMID- 7542365 TI - Influence of liposomal muramyl tripeptide phosphatidylethanolamine on the changes of nucleic acids in the blood and hemopoietic tissues of irradiated mice. AB - Effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV) on nucleic acid content, and on DNA synthesis and cellularity in the blood and haemopoietic tissues of mice irradiated with a dose of 6 Gy gamma radiation were studied. It was found that injection of MTP-PE/MLV alone (200 micrograms per mouse; i.p.) resulted in a mild decrease in DNA content in the bone marrow as consequence of cellularity diminution in the femur. At the same time, incorporation of 3H-thymidine into bone marrow DNA increased in comparison to the non-stimulated mice. On the basis of simultaneous increase in RNA and DNA concentration in the blood we assume that the changes of nucleic acids and cellularity in the bone marrow of MTP-PE/MLV treated mice are related to the higher release of nuclear cells into the circulation. In mice irradiated 24 h after MTP-PE/MLV injection the decrease in nucleic acid content was similar to that in unprotected mice within the first three days after irradiation. However, during following days, the recovery of RNA and DNA content and cellularity in the bone marrow and blood proceeded at a much faster rate in mice protected by MTP PE/MLV as compared to unprotected animals. The increase in DNA synthesis in the bone marrow of protected mice preceded the DNA content recovery. In the spleen and the thymus, no effect of liposomal MTP-PE was found. PMID- 7542364 TI - Sialylated Lewis(x) and Lewis(a) determinants expression on human neoplastic cell lines: immunocytometric study with the 5th workshop monoclonal antibodies. AB - The reactivity of monoclonal antibodies from the 5th workshop Selectins/Selectin Ligands panel, directed to the sialylated Lewis(x) and sialylated Lewis(a) determinants, with the human breast carcinoma (BT-20, ZR-75-1 and MDA-MB-468) cell lines, human ovarian carcinoma cell line A2780, fibrosarcoma (HT-1080, B 6FS) and hematopoietic neoplastic (U-937, HL-60, K-562) cell lines were determined with the aid of flow immunocytofluorometry. The examined monoclonal antibodies to sialylated Lewis determinants reacted with examined breast carcinoma, but not with the examined ovarian carcinoma and fibrosarcoma cell lines. PMID- 7542366 TI - Multiple myeloma. Part II: Treatment. PMID- 7542367 TI - Human glomerular epithelial cells synthesize and secrete the third component of complement. AB - Complement proteins in serum are mainly synthesized by hepatocytes. Recently, many cell types have been reported to synthesize complement in various tissues. In this study, we report the synthesis and secretion of the third component of complement (C3) by cultured glomerular epithelial cells (GEC). Using reverse transcriptase polymerase reaction, we have found that GEC and whole kidney expressed the C3 mRNA for C3. By ELISA, we have found that C3 was secreted in culture supernatants harvested from cultured GEC. The secretion of C3 is regulated by proinflammatory cytokines (IL-1 beta, TNF-alpha and IL-6). IL-1 beta is shown to be the most potent stimulator of C3 secretion from GEC. The exact significance of C3 produced at glomerular site is not clear, but its upregulation by proinflammatory cytokines may suspect a role in local activation of complement which may lead to glomerular injury. We further studied the expression of C3 step regulatory proteins (membrane cofactor protein (MCP), decay-accelerating factor (DAF), CR-1 and CD59 (a terminal step regulatory protein) by cultured GEC. Treatment of GEC by proinflammatory cytokines IL-1 beta, TGF-beta, TNF-alpha and IL-6 did not modify the expression of MCP, DAF and CR-1 whereas an increase in the expression of CD59 could be observed after treatment with IL-1 beta and TGF beta. These results indicate that the expression of these regulatory proteins is tissue specific and may be implicated in inflammatory processes. PMID- 7542368 TI - Review: neurotransmitter receptors. II. AMPA and kainate receptors. PMID- 7542369 TI - 1-BCP, a memory-enhancing agent, selectively potentiates AMPA-induced [3H]norepinephrine release in rat hippocampal slices. AB - It is now clear that the AMPA subtype of ionotropic glutamate receptors (iGluRs) undergoes a rapid desensitization in response to activation by AMPA receptor agonists. This desensitization is inhibited by compounds such as aniracetam and cyclothiazide, which act at a distinct site on the AMPA receptor complex. In particular, cyclothiazide greatly potentiates AMPA receptor-mediated depolarizing responses in the hippocampus. We have recently shown cyclothiazide also increases AMPA-induced release of [3H]norepinephrine ([3H]NE). More, recently, a benzamide compound, 1-(1,3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), has been reported to enhance AMPA-induced currents and to facilitate memory retention in rats in a number of memory tasks. In this study, the effects of 1-BCP on excitatory amino acid agonist-induced [3H]NE release in rat hippocampal slices were determined. We report that 1-BCP, like cyclothiazide, selectively potentiates AMPA-induced [3H]NE release. However, cyclothiazide was more potent and efficacious than 1 BCP. Nevertheless, these data suggest a role for AMPA receptor-mediated enhancement of norepinephrine release as a mechanism of action for nootropic compounds such as 1-BCP. PMID- 7542370 TI - Inhibition of potassium-stimulated dopamine release by the nitric oxide generator isosorbide dinitrate. AB - In PC12 cells, isosorbide dinitrate (ISDN) and S-nitrosol-acetyl-penicillamine (SNAP), both nitric oxide (NO) generators, attenuated K+ (56 mM)-stimulated release of dopamine. The attenuation was not observed with isosorbide, an ISDN analog lacking NO generating capacity. In this model, A23187 (Ca2+ ionophore), Bay K8644 (Ca2+ slow channel agonist) and veratridine (Na+ channel agonist) stimulated dopamine release. Treatment with ISDN enhanced Bay K8644 and veratridine-evoked dopamine release, while ISDN had no significant effect on the A23187 response. Incubation with 8-bromo-cGMP (membrane permeable cGMP analog) had no effect on basal or stimulated dopamine release in these cells, suggesting NO's response was not mediated by cGMP. In additional studies, K+ (56 mM), Bay K8644 and veratridine elevated cytosolic free calcium levels ([Ca2+]i). ISDN reduced K(+)-stimulated increase in [Ca2+]i, but enhanced the increases of [Ca2+]i induced by Bay K8644 or veratridine. These results suggest NO interacts with K(+)-induced membrane depolarization (possibly by inhibiting membrane conductance to K+) to attenuate Ca2+ influx and Ca(2+)-mediated dopamine secretion stimulated by K+. PMID- 7542371 TI - Expression in mouse embryos and in adult mouse brain of three members of the amyloid precursor protein family, of the alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein and of its ligands apolipoprotein E, lipoprotein lipase, alpha-2-macroglobulin and the 40,000 molecular weight receptor-associated protein. AB - We have analysed by northern blotting and by in situ hybridization the expression patterns of eight different genes during the second half of mouse embryonic development and in adult mouse brain: we compared the messenger RNA levels of amyloid precursor protein and of the two amyloid precursor protein-like proteins 1 and 2 and we have analysed expression of apolipoprotein E and of its main receptor in brain, the alpha-2-macroglobulin/low density lipoprotein receptor related protein and three other ligands: the proteinase inhibitor alpha-2 macroglobulin, the modifying enzyme lipoprotein lipase and the 44,000 molecular weight heparin binding protein, a ligand of unknown function. During embryogenesis the temporal expression pattern differs considerably for the three members of the amyloid precursor proteins. Total embryo messenger RNA levels of amyloid precursor protein and amyloid precursor protein-like protein 2 increased progressively, while amyloid precursor protein-like protein 1 messenger RNA showed a burst of synthesis between days 10 and 13 post-coitum. Significantly, expression of the alpha-2-macroglobulin/low density lipoprotein receptor-related protein and of its associated protein, the 44,000 molecular weight heparin binding protein, exhibited their most important increase very similar to that of amyloid precursor protein-like protein 1, between days 10 and 13 post-coitum. Apolipoprotein E, lipoprotein lipase and alpha-2-macroglobulin messenger RNA levels in total embryos increased progressively, beginning most pronounced at days 13, 15 and 17, respectively. In mouse embryos, in situ hybridization established amyloid precursor protein, amyloid precursor protein-like protein 2 and alpha-2-macroglobulin/low density lipoprotein receptor-related protein messenger RNA to be expressed in most organs, with the notable exception of the liver, while expression of the other studied proteins was much more restricted. Among adult mouse tissues, the genes investigated were expressed very prominently in brain, except for lipoprotein lipase and for the complete absence of alpha-2 macroglobulin messenger RNA. In adult mouse brain, the cortex and hippocampus exhibited strong signals for most genes analysed. Exceptions are lipoprotein lipase and apolipoprotein E messenger RNAs, and the absent alpha-2-macroglobulin messenger RNA. Several interesting features, similarities as well as differences, between brain tissue sections hybridized with probes for amyloid precursor protein, amyloid precursor protein-like proteins 1 and 2 and between alpha-2 macroglobulin/low density lipoprotein receptor-related protein and heparin binding protein-44 were observed and are described. The results are further discussed in view of the known or anticipated physiological functions of the proteins examined and of their possible role in the etiology of Alzheimer's disease. PMID- 7542372 TI - Galanin expression in sympathetic ganglia after partial axotomy is highly localized to those neurons that are axotomized. AB - The neuropeptide phenotype of adult sympathetic neurons changes dramatically after postganglionic nerve transection. Studies, thus far, have been done on the superior cervical ganglion; however, one limitation of this preparation is that it is necessary to transect the postganglionic axons quite close to the ganglion. In the present study, we examined the effects of axonal damage on galanin-like immunoreactivity in the middle and inferior cervical ganglion complex. With these ganglia, it is possible to transect postganglionic axons at a considerable distance from their cell bodies and, therefore, to examine the extent to which local tissue damage, rather than specific axonal transection, is required for these changes in neuropeptide phenotype to occur. The anatomy of this system also allowed us to determine the extent to which the changes in galanin expression are restricted to those neurons that have been axotomized. The axons of a small population of the neurons in the middle and inferior cervical ganglia complex project into the cervical sympathetic trunk. Within two days after this trunk was transected, there was an increase in the level of galanin-like immunoreactivity in the complex and in the number of immunostained principal neurons. These neurons were concentrated primarily in the most rostral part of the complex. An increase in galanin-like immunoreactivity also occurred in response to the systemic administration of the sympathetic neurotoxin 6-hydroxydopamine. In that case, many more neurons were affected than after transection of the cervical sympathetic trunk, and the neurons were distributed evenly throughout the ganglion complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542374 TI - Astrocyte transplants alleviate lesion induced memory deficits independently of cholinergic recovery. AB - Basal forebrain tissue fragments taken from embryonic day 15 were separated into primary astrocytes and primary neurons in culture and grafted to rats with alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid lesions to the nucleus basalis and medial septal regions. The two cell types were compared in two experimental paradigms for their behavioural, biochemical and histochemical effects; standard transplants of whole basal forebrain and sham transplants served as positive and negative controls, respectively. Each transplant cell type was characterised by in vitro immunocytochemistry to assess content and purity. Memory deficits produced by the lesions in a spatial win-stay T-maze task (Experiment 1) and a spatial plus associative radial maze task (Experiment 2) were significantly improved by the astrocyte, but not by the neuronal, primary cell transplants. The astrocyte graft groups performed as well as standard cholinergic rich basal forebrain groups, reaching control levels on both tasks, while the neuronal transplant groups were not significantly different to lesioned (sham transplanted) rats. There was no recovery in choline acetyltransferase activity in brain regions containing astrocyte grafts whereas activity in the neuronal graft regions was increased (often to control levels), similar to recovery produced by basal forebrain grafts. Grafts in all groups survived, transplanted neurons displaying similar morphology and placement in the host brain to unseparated basal forebrain grafts, while astrocytes showed evidence of migration. The cultured astrocytes were estimated to be > 95% pure, showing positive staining for all astrocyte markers and an absence of staining for neuronal markers. The results indicate that the restoration of cognitive function following fetal grafting is not dependent upon a restoration of cholinergic neuronal activity but is more likely mediated via diffuse graft-host communication, with trophic secretion a probable factor. This study emphasizes the usefulness of astrocytes in the repair of central nervous system injury and has implications for therapeutic potential. PMID- 7542373 TI - A1 adenosine receptors differentially regulate the N-methyl-D-aspartate and non-N methyl-D-aspartate receptor-mediated components of hippocampal excitatory postsynaptic current in a Ca2+/Mg(2+)-dependent manner. AB - A1 adenosine receptors efficiently modulate the excitatory synaptic transmission in hippocampus. Here we report that in addition to previously known modulatory action on the synaptic efficacy, A1 adenosine receptors are also capable of regulating the relative contribution of N-methyl-D-aspartate receptor-mediated component of the excitatory postsynaptic current in CA3-CA1 excitatory synapses, in the rat. When applied externally, a selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine, increases not only the amplitude of excitatory postsynaptic current but also the relative contribution of the N methyl-D-aspartate receptor-mediated component of postsynaptic current recorded by in situ voltage clamp. This effect develops only at increased external Ca2+ concentration and also depends on the external Ca2+/Mg2+ ratio. The increased ratio of N-methyl-D-aspartate/non-N-methyl-D-aspartate components of excitatory postsynaptic current remains at a new level after the removal of 8-cyclopentyl 1,3-dimethylxanthine, even though the amplitude of excitatory postsynaptic current returns close to control value. These results indicate the existence of a mechanism that preferentially enhances the N-methyl-D-aspartate component of excitatory postsynaptic current when the A1 adenosine receptors are blocked and imprints the newly acquired ratio of corresponding excitatory postsynaptic current components. PMID- 7542376 TI - Risperidone in Gilles de la Tourette syndrome. PMID- 7542375 TI - Isolation and characterization of autoreactive proteolipid protein-peptide specific T-cell clones from multiple sclerosis patients. AB - During the course of multiple sclerosis (MS), myelin proteins are likely antigenic targets for autoreactive T cells. Although most studies have implicated myelin basic protein as a potent encephalitogenic myelin component, proteolipid protein (PLP) appears also to be a possible target antigen in the autoimmune response in MS. In this report, we investigated the human T-cell responses to PLP by using PLP104-117 and PLP142-153 synthetic peptides as target antigens in limiting dilution. One hundred twenty-five CD4+, T-cell receptor (TCR) alpha beta+ T-cell clones (TCCs) were established from the peripheral blood of seven MS patients and five control subjects. Despite the use of enriched cultures no gamma delta TCCs were obtained. Recognition of both PLP epitopes occurred in the context of multiple HLA-DR alleles. We found no differences in restriction element usage between MS patients and control subjects. TCR variable beta-region (V beta) usage was assessed by flow cytometry using a panel of monoclonal antibodies defining different V beta elements. In both MS patients and control subjects, there was a marked heterogeneity in the TCR V beta repertoire. Furthermore, sequential evaluation of MS patients during acute attacks and clinical remissions showed even more broadening of the TCR V beta repertoire. These data demonstrate that a heterogeneous T-cell response to PLP concerning HLA restriction and TCR usage is present in both MS patients and normal subjects. PMID- 7542377 TI - Cytokines in fetal blood and amniotic fluid in Rh-immunized pregnancies. AB - OBJECTIVE: To determine fetal serum and amniotic fluid (AF) levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin, and to explore the relationship between cytokines and hemoglobin concentration, white blood cell count (WBC), and platelet count in fetuses affected by Rh immunization. METHODS: Thirty-four consecutive Rh immunized patients in gestational weeks 19-33 were included. All patients were investigated by funipuncture and 13 by amniocentesis. The levels of IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin were estimated using commercially available immunoassays. RESULTS: There was a significant correlation between erythropoietin concentrations in fetal serum and AF (r = 0.54, P < .05), whereas none of the other cytokines showed a positive correlation between these two compartments. Fetal serum contained higher concentrations of IL-3, granulocyte-macrophage colony stimulating factor, stem cell factor, and erythropoietin compared with AF. In contrast, the IL-6 level was significantly higher in AF compared with fetal serum (P = .002). Erythropoietin and IL-3 levels were both negatively correlated with fetal hemoglobin concentrations (r = -0.75, P = .02, and r = -0.67, P = .045). The fetal WBC correlated significantly with the fetal serum concentration of granulocyte-macrophage colony-stimulating factor (r = 0.38, P = .04). CONCLUSION: Human fetuses with anemia due to erythrocyte immunization exhibit an increased production of erythropoietin and IL-3. Other studied cytokines (such as stem cell factor, granulocyte-macrophage colony-stimulating factor, and IL-6) did not correlate with the degree of fetal anemia. Among the studied cytokines, only erythropoietin showed a positive correlation between fetal serum and AF. PMID- 7542378 TI - Fetal karyotyping for chromosome abnormalities after an unexplained elevated maternal serum alpha-fetoprotein screening. AB - OBJECTIVE: To study the chromosome abnormality rate among women with elevated levels of maternal serum alpha-fetoprotein (MSAFP) and the types of chromosome abnormalities in this population, and to compare this rate with reports in the literature and the rate observed in the general population. METHODS: We studied 8097 women who chose to undergo amniocentesis and fetal karyotyping after having an elevated MSAFP test of 2.5 multiples of the median (MOM) or higher. All abnormal karyotypes were reviewed and grouped according to whether the elevated MSAFP value could be explained by a ventral wall or neural tube defect. RESULTS: The overall chromosome abnormality rate was 13.83 per 1000 amniocenteses. The rate in the "unexplained" group was 10.92 per 1000 amniocenteses. Just over half (53%) of the abnormal karyotypes were autosomal anomalies, and 47% were sex chromosome abnormalities. The autosomal aneuploidies observed most frequently were triploidy and trisomy 13. The sex chromosome abnormalities observed most frequently were the XXY and XYY karyotypes. CONCLUSION: Women who have unexplained elevated MSAFP values of 2.5 MOM or greater have a twofold increase in the rate of chromosome abnormalities in their fetuses compared with the general population (P < or = .001). This rate is consistent with other studies that used a 2.5 MOM cutoff. Studies that used a 2.0 MOM cutoff have reported chromosome abnormality rates that do not vary from general population estimates. PMID- 7542379 TI - Adverse pregnancy outcome after a false-positive screen for Down syndrome using multiple markers. AB - OBJECTIVE: To assess the relative risk of an adverse pregnancy outcome in women whose multiple-marker screening (maternal serum alpha-fetoprotein [MSAFP], unconjugated estriol [E3], and hCG levels, and age) indicating an increased risk for Down syndrome (more than 1:250) was not confirmed by amniocentesis. METHODS: Fifty-eight women with false-positive screens for Down syndrome were matched with a control group of 116 women whose screens indicated a risk for Down syndrome of less than 1:250. The risk for adverse pregnancy outcome was compared for the two groups, and the roles of MSAFP, unconjugated E3, and hCG as predictors of adverse pregnancy outcome were determined. RESULTS: Women with false-positive screens for Down syndrome were significantly different from their matched controls in the incidence of preterm delivery (20.6 versus 8.6%, respectively), preeclampsia (6.9 versus 0%), small for gestational age newborns (5.2 versus 0%), and fetal demise after 20 weeks' gestation (5.2 versus 0%). An adverse outcome occurred in 19 of 58 pregnancies (32.8%) in the study group and in 14 of 116 matched control pregnancies (12%) (odds ratio [OR] 3.5, 95% confidence interval [CI] 1.6-7.8; P < .01). Unconjugated E3 of 0.75 multiples of the mean (MoM) or less was significantly associated with adverse pregnancy outcome after controlling for the effects of MSAFP and hCG (OR 2.5, 95% CI 1.13-5.55; P < .02). CONCLUSION: One in three women with a false-positive screen for Down syndrome may experience an adverse pregnancy outcome. In this study, unconjugated E3 of 0.75 MoM or less appeared to be a better predictor of adverse pregnancy outcome than were MSAFP and hCG levels. PMID- 7542380 TI - Detection of fetomaternal haemorrhage associated with cordocentesis using serum alpha-fetoprotein and the Kleihauer technique. AB - Fetomaternal haemorrhage (FMH) was studied after 46 cordocenteses. alpha Fetoprotein (AFP) concentration and Kleihauer staining of maternal blood, taken both before and after the procedure, revealed increases in AFP values of more than 40 per cent in 30 per cent of the patients examined; fetal haemorrhage of more than 0.25 ml was detected in 46 per cent of the cases by the Kleihauer test. In the second trimester of pregnancy both techniques are comparable, while in the third trimester the Kleihauer technique appears to be more sensitive in detecting FMH after cordocentesis. An anterior position of the placenta is a risk factor for FMH. PMID- 7542381 TI - Neutrophil and monocyte beta 2-integrin expression in trisomic fetuses. AB - Flow cytometry was used to measure neutrophil and monocyte beta 2-integrin expression in fetuses with trisomy 18 (n = 7) and trisomy 21 (n = 7) at 20-25 weeks' gestation. The values were compared with those of 112 chromosomally normal fetuses. There were no significant differences in beta 2-integrin expression between normal and aneuploid fetuses. These findings demonstrate that in trisomies 21 and 18, alteration in beta 2-integrin expression is unlikely to contribute to the pathogenesis of immunological deficiencies that have been observed in these aneuploidies both prenatally and postnatally. PMID- 7542382 TI - Detecting neural tube defects by amniocentesis between 11 and 15 weeks' gestation. AB - Forty-two open neural tube defects (NTDs) were identified in our series of 7440 amniocenteses tested between 11 and 15 weeks of gestation. Using a cut-off of > or = 2.0 MOM, the detection rate for open NTDs was 95 per cent; 100 per cent each for anencephaly and spina bifida; and 78 per cent for encephalocele. Two encephaloceles had AFP levels less than 2.0 MOM and negative AChEs. Thirty-four (81 per cent) of these NTDs were tested between 13 and 15 weeks and 8 (19 per cent) before 13 weeks. There were 0.6 per cent false positives by AFP (excluding serious abnormalities and fetal death) and 0.1 per cent after AChE. The likelihood of an open NTD after an elevated AFP (> or = 2.0 MOM) was 24 and 77 per cent for any serious abnormality. These results, when combined with an earlier study, indicate that amniotic fluid AFP appears to be as sensitive a test for open NTDs between 13 and 15 weeks as between 16 and 20 weeks. Additional experience is necessary to determine this before 13 weeks. PMID- 7542383 TI - 'Faint-positive' or 'false-positive' amniotic fluid acetylcholinesterase. A diagnostic dilemma. PMID- 7542384 TI - In vitro and ex vivo expression of nucleolar proteins B23 and p120 in benign and malignant epithelial lesions of the prostate. AB - The expression of two specific nucleolar antigens, p120 and B23, has been investigated in the prostatic carcinoma cell line LNCaP as well as in 40 frozen and 40 formalin-fixed tissue samples of benign and malignant prostatic lesions (15 benign hyperplasias, 5 grade 1, 15 grade 2, and 5 grade 3 carcinomas). In vitro, immunoreactivity of p120 was confined to nucleoli of proliferating cells, with virtually no negative staining during S and G2/M phases. Unlike p120, B23 was expressed in the nucleoli of all LNCaP cells independently of growth and cell cycle phases. Hence, B23 was detectable in all stromal as well as in normal and malignant epithelial prostatic cells, both in fresh and in formalin-fixed tissue sections after microwave treatment. In contrast, the immunoreactivity of p120 was almost completely restricted to the nucleoli of prostate carcinoma cells: frozen sections of benign prostatic hyperplasia (n = 15) were either totally negative for p120 (n = 13) or had a low percentage of positively stained cells (labeling index = 3.3% in 3 cases). In the carcinoma group 76% (19/25) of the specimens were p120 positive, and there was a significant rise of labeling index from 18.1% in grade 1 to 82.2% in grade 3 carcinomas (P < 0.001). In contrast to B23, p120 could not be reliably demonstrated in formalin-fixed and paraffin-embedded tissue. We therefore conclude that anti-B23 is a general marker of nucleoli, whereas expression of p120 appears to correlate with "hyperactivity" of the nucleolus and provides a new tool for flow cytometrical and immunohistochemical assessment of nucleolar activity in tumor pathology. PMID- 7542385 TI - A detailed analysis of the Knodell score and other histologic parameters as predictors of response to interferon therapy in chronic hepatitis C. AB - The Knodell score is inaccurate at predicting response to interferon alpha (IFN alpha) therapy in patients with hepatitis C. Our aim was to see if specific histologic parameters, including iron deposition in liver biopsies, are better predictors of response to IFN-alpha than the total Knodell score. Thirty-five unselected patients were studied who had hepatitis C treated with IFN-alpha between 1990 and 1993, and pretreatment serum iron indices and liver needle biopsies performed. Biopsies were divided for light microscopy and quantitative iron determination. H&E-stained slides were graded for components I, II, III, IV, and total Knodell score. Quantitative determinations were percentage of portal triads with inflammation, piecemeal necrosis, lymphoid aggregates, and inflamed bile ducts; percentage of lobules with inflammation or acidophilic bodies; and percentage of triads with positive iron stain. Complete responders (CR) to IFN alpha were defined by normalization of serum alanine aminotransferase (< or = 40 IU/liter), and noncomplete responders (NCR) by partial or no response. Data were analyzed statistically. CR had < 40% of triads positive for iron (P = 0.02) and lower serum ferritin (P = 0.05) and higher scores for lobular necrosis (P = 0.04). The percentage of iron-positive triads correlated only with cirrhosis. Addition of cirrhosis to percentage of iron-positive triads did not improve the predictive power of the portal iron. CR and NCR did not differ with respect to total Knodell score or any of the other individual parameters except Knodell II. CONCLUSIONS: (a) Individual features of lobular necrosis and iron staining in portal triads are better predictors of response to IFN-alpha than the total Knodell score.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542386 TI - Polypoid pulmonary endodermal tumor resembling fetal lung: report of a case. AB - Pulmonary blastoma is a rare malignant neoplasm that histologically resembles airway structures and mesenchymal supporting tissues seen in the early phases of fetal lung development. The tumor either is biphasic with immature epithelial and stromal components or consists solely of an epithelial component. The preferred terminology for the latter is pulmonary endodermal tumor resembling fetal lung. Pulmonary blastoma is usually peripheral in location, and rarely does this tumor present intrabronchially. We report a case of pulmonary endodermal tumor resembling fetal lung presenting as a polypoid intrabronchial mass. PMID- 7542387 TI - Advantages of application of a yellow filter and simple half-gram staining for detection of Helicobacter pylori infection in gastric biopsy specimens. PMID- 7542388 TI - Correspondence re: Kaiserling E, Horny HP, Geerts M-L, Schmid U: Skin involvement in myelogenous leukemia: morphologic and immunophenotypic heterogeneity of skin infiltrates. Mod Pathol 7:771, 1994. PMID- 7542389 TI - Nitric oxide synthase inhibition and food intake: effects on motivation to eat and in female mice. AB - Recent studies have demonstrated that nitric oxide may play an important role in the regulation of food intake. The studies reported here extend these findings by demonstrating that NG-nitro-arginine-methylester, N-Arg(ME), a nitric oxide synthase inhibitor, decreased intake of a highly palatable substance (i.e., milk), though at a higher dose than necessary for decreasing consumption of food pellets. N-Arg(ME) failed to inhibit lever press for milk reward in nonprefed mice, but decreased lever pressing in prefed mice. N-Arg(ME) decreased food intake in female mice, being most potent in proestrus. These studies suggest that nitric oxide synthase inhibition decreases food intake without inducing aversion or illness. PMID- 7542390 TI - Association between brain indole levels and severity of posthypoxic myoclonus in rats. AB - We have previously reported the presence of posthypoxic, audiogenic myoclonus in rats after cardiac arrest and the ability of the 5-HT precursor, 5-HTP, to attenuate these muscle jerks. In addition, we have recently shown that 5-HT2 and 5-HT3 agonists can reduce the severity of myoclonus in these animals, suggesting a deficiency in serotonergic neurotransmission. In the present study, the levels of 5-HTP, 5-HT, and 5-HIAA were measured in seven regions of the brain in myoclonic and normal rats to identify the areas of the brain in which a serotonergic dysfunction resides. Similar to previous studies, we observed pronounced posthypoxic, audiogenic myoclonus 3 and 14 days after resuscitation from cardiac arrest, with a resolution of the abnormal movements by 45 days postarrest. HPLC measurements revealed significant changes in indole levels in the following areas of the brain: cortical 5-HIAA, striatal 5-HT, striatal 5 HIAA, hippocampal 5-HT, mesencephalic 5-HIAA, myelencephalic 5-HT, myelencephalic 5-HIAA, cerebellar 5-HTP, and cerebellar 5-HT. The changes in striatal 5-HT, cortical 5-HIAA, and mesencephalic 5-HIAA appear most relevant to the pathophysiology of posthypoxic myoclonus because regression analyses showed significant correlations between the myoclonus scores of the animals and the levels of these indoles. Based on the observed pattern of results, we postulate a dysfunction in serotonergic lateral (cortical) and far lateral (extrapyramidal) ascending pathways in posthypoxic myoclonus. PMID- 7542391 TI - Chronic social stress increases levels of preprogalanin mRNA in the rat locus coeruleus. AB - Galanin is a 29 amino acid neuropeptide that coexists with norepinephrine in approximately 80% of locus coeruleus (LC) neurons in the rat. The effects of chronic, naturalistic stress on preprogalanin mRNA in the LC was studied. The visible burrow system (VBS) served as the stress paradigm. Long-Evans rats (three males and two females) were housed together in the VBS for 13 days. The males established dominance hierarchies during this period. On day 14, preprogalanin mRNA in the LC was significantly elevated in subordinate rats compared to dominant and control rats, as measured by quantitative in situ hybridization. Levels of mRNA were positively correlated with the number of wounds by day 7 and day 14 and negatively correlated with body weight gain by day 14. These results suggest that the neuropeptide galanin may be overexpressed during chronic stress in rats. PMID- 7542392 TI - Low responsiveness to agents evoking 5-HT2 receptor-mediated behaviors in Sardinian alcohol-preferring rats. AB - The selective NK3 tachykinin agonist senktide evokes in rodents 5-HT mediated behaviors, including 5-HT2 receptor-mediated wet dog shakes (WDS) and head shakes (HS). It was observed previously that genetically selected Sardinian alcohol preferring (sP) rats show a small number of WDS and HS following intracerebroventricular (ICV) injection of senktide. The present study was aimed at confirming these observations and at providing information on the reasons accounting for the anomalous response of sP rats. Senktide (500-2000 ng/rat, ICV) produced a much lower number of WDS and HS in sP rats than in nonselected Wistar (nsW) rats. Both behaviors were suppressed by the 5-HT2 antagonist ritanserin (1 mg/kg, subcutaneously), confirming that 5-HT2 receptors mediate the response. HS induced by the ICV injection of 5-HT agonists endowed with marked activity at 5 HT2 receptors, such as quipazine (1500-6000 ng/rat) or DOI (500-3500 ng/rat), were much less pronounced in sP rats than in nsW rats. Moreover, WDS following peripheral injection of 5-hydroxytryptophan, 25-100 mg/kg, and carbidopa, 12.5 mg/kg, were less intense in sP and in ethanol-naive sP rats than in nsW and in Sardinian alcohol-nonpreferring rats. These findings suggest that sP rats have an inherent different regulation of central 5-HT2 mechanisms. PMID- 7542393 TI - Retarded acquisition of a temporal discrimination following destruction of noradrenergic neurones by systemic treatment with DSP4. AB - This experiment examined the effect of destroying central noradrenergic neurones, using the selective neurotoxin DSP4 (N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine) on the acquisition and performance of discrimination between two time intervals. Rats that had received systemic treatment with DSP4 and vehicle-treated control rats were trained in a series of discrete trials to press lever A following a 2-s presentation of a light stimulus and lever B following an 8-s presentation of the same stimulus. Both groups acquired the discrimination (> 90% correct choices) within 15 sessions; however, the DSP4-treated group showed significantly slower acquisition than the control group. When stable performance had been attained, 'probe' trials were introduced in which the light was presented for intermediate durations. Both groups showed sigmoid functions relating percent choice of lever B to log stimulus duration. Neither the bisection point (duration corresponding to 50% choice of lever B) nor the Weber fraction differed significantly between the DSP4-treated and control groups. The levels of noradrenaline were markedly reduced in the neocortex and hippocampus of the DSP4-treated group, but the levels of dopamine and 5-hydroxytryptamine were not altered. The results indicate that noradrenaline depletion induced by DSP4 retarded the acquisition of temporal discrimination, but did not impair steady state discriminative precision. PMID- 7542394 TI - Inhibition of MAO-B protects against MDMA-induced neurotoxicity in the striatum. AB - The effects of the MAO-B inhibitors, L-deprenyl and MDL-72974 on MDMA-induced serotonergic neurotoxicity in rats were examined. MDMA alone produced a significant decrease in the number of 5-HT uptake sites, measured as a decrease in the Bmax for binding of [3H]paroxetine, and in 5-HT and 5-HIAA levels in the striatum. L-Deprenyl and MDL-72974 attenuated this MDMA-induced decrease in serotonergic markers. The data suggest a key role for MAO-B in the expression of the neurotoxicity produced by MDMA in the striatum. PMID- 7542395 TI - Malignant duodenal stenosis: palliation with peroral implantation of a self expanding nitinol stent. AB - The authors implanted a duodenal stent in a woman with a high-grade duodenal stenosis due to inoperable tumor compression. A flexible self-expanding knitted nitinol stent that conformed to the shape of the duodenum was introduced orally to keep the duodenal passage patent. After stent implantation, repeated vomiting stopped and the patient was able to eat and drink. This technique resolved the patient's duodenal stenosis. PMID- 7542396 TI - [Oncologic pain]. PMID- 7542398 TI - Elevated plasma levels of acute phase proteins in mesangioproliferative glomerulonephritis, membranous nephropathy and IgA nephropathy. AB - In order to study the possible role of active inflammatory processes in clinical indolent primary chronic glomerulonephritides, plasma concentrations of the acute phase proteins: alpha 1-antitrypsin, haptoglobin, orosomucoid and C-reactive protein were measured in 166 glomerulonephritis patients. The patients had a diagnosis of either mesangioproliferative glomerulonephritis, membranous nephropathy or immunoglobulin A nephropathy and were divided in two groups, one with heavy urinary albumin losses and one with moderate to slight urinary albumin excretion. The median plasma concentration values for alpha 1-antitrypsin, haptoglobin and orosomucoid were increased in all three kinds of the investigated glomerulonephritides with exception for orosomucoid in patients with heavy urinary albumin losses and in the membranous nephropathy group. The plasma concentration values for C-reactive protein were not elevated at all in the material. The increase of plasma levels of acute phase proteins could be the result of persistent inflammatory stimuli that occur in primary chronic glomerulonephritides. The finding of unchanging plasma levels of C-reactive protein in contrast to increased concentrations of the other acute phase proteins could be of significance in diagnosing infections or other inflammatory diseases in patients with chronic glomerulonephritis. PMID- 7542397 TI - Prostate cancer. Mortality and morbidity after non-curative treatment with aspects on diagnosis and treatment. AB - AIMS OF THE STUDY: To investigate the mortality, need for hospital care and palliative treatments in patients with prostate cancer (PC) treated with non curative intention (i.e. deferred or hormonal treatment). To evaluate acceptance by patients and complications of a new diagnostic procedure for PC -- transrectal ultrasound (TRUS) and core biopsies. To investigate if knowledge of prostate volume enhances the accuracy of prostate specific antigen (PSA) to indicate non palpable PC. Finally, to investigate how neo-adjuvant hormonal treatment before radical prostatectomy affected PSA and tumour volume. METHODS: In a retrospective analysis of all 536 patients with a known diagnosis of PC who died in the city of Goteborg during the years 1988-90, age at diagnosis, survival time, need for hospital care and cause of death were registered (I and II). A questionnaire was sent to 511 patients who underwent TRUS with or without prostatic biopsies (III). In 120 consecutive patients admitted for TURP due to presumed benign prostatic hyperplasia, a comparison was made between PSA and prostate-volume-adjusted (measured via TRUS) PSA (PSADensity) to indicate the presence of non-palpable PC (IV). Of 56 patients who underwent radical prostatectomy, 28 received 3 months' pretreatment with a GnRH-agonist. The effects on tumour volumes (assessed by the planimetric method on whole mount slides) and PSA were studied (V). RESULTS: Overall, 62% of patients with a known diagnosis of PC died of the disease when all patients were followed from diagnosis until death (up to 25 years). Of patients in stage M0 at diagnosis, 50% died of PC. However, in patients who survived for more than 10 years the mortality reached 63% (I). The average PC patient needed 27 days of hospital stay (geriatric wards excluded) and 185 patients needed at least one palliative TURP, 103 patients palliative radiation therapy and 55 patients procedures due to upper urinary tract obstruction. The lion's share of these resources was consumed by patients who later succumbed to PC (II). Ninety-five per cent of patients reported none or minor discomfort after TRUS of the prostate and 92% if TRUS was combined with transrectal core biopsies of the prostate. Haematuria for > 2 days occurred in 13%, haematospermia > 2 days in 9% and blood in stool > 2 days in 3% among patients who underwent core biopsies but none of these patients needed active treatment. Overall, 4.1% of biopsied patients experienced urinary tract infection (III). The use of PSADensity with a cut-off value of 0.10 ng/ml/cc rendered both higher sensitivity (75 vs 50%) and positive predictive value (0.33 vs 0.15) for indicating non palpable PC in symptomatic patients with benign findings on digital rectal examination (IV). Pretreatment with a GnRH-agonist resulted in a significant PSA decrease not explained by changes in tumour volume. Tumour volume reduction was found in 36% of the patients. CONCLUSIONS: According to these studies, PC is a progressive disease with considerable mortality and morbidity when managed by non curative intention. Since new diagnostic and therapeutic methods described in this thesis are well accepted by patients and may increase the chance of radical surgery, it is reasonable to offer younger patients with long life expectancy the chance of early detection and treatment with curative intention. PMID- 7542399 TI - Estimated total costs of treating benign prostatic hyperplasia in Sweden. AB - New surgical methods and safe effective drugs for treating benign prostatic hyperplasia (BPH) have been introduced in recent years. The long-term effects of these measures and the patient categories for which each is best suited are not as yet known. Amid rapid changes in treatment policy, our study aims to establish a frame of reference in Sweden for the costs of treating BPH. In 1988 most treatment of BPH was surgical, with "watchful waiting" or indwelling urethral catheter as alternatives. Transurethral resection was the main surgical method. The costs of treating BPH in 1988 were calculated with a "prevalence" and an "incidence" approach. Direct costs accounted for 80-85% of the total were mainly hospital costs. The total economic burden of BPH treatment in Sweden was estimated as 305-390 million SEK, equivalent to 49-62 million US$ in 1988 prices. Because of the impending changes in management of BPH, follow-up of the financial consequences of these changes is important in order to secure effective use of health care resources. PMID- 7542400 TI - Endocrine environment of benign prostatic hyperplasia: prostate size and volume are correlated with serum estrogen concentration. AB - Estrogen plays an important role in the development of benign prostatic hyperplasia (BPH), as has been shown in both experimental and clinical studies. T determine the endocrine environment of BPH, serum total testosterone (Total-T), free testosterone (Free-T), and estradiol (E2) conceptions were measured, and the relationship between these levels and prostate size and volume was analyzed. Blood samples were collected from subjects who attended the mass screening for prostate disease performed by our institute. No significant correlations were found between Total-T levels, Free-T levels, and prostate size, as determined by digital rectal examination. However, E2 levels and the ratios for E2 levels and the ratios for E2/Total-T and E2/Free-T were significantly correlated with prostate size. To confirm these relationships, prostate volume was calculated from transrectal ultrasonographic images. E2 levels and these two ratios were, indeed, highly correlated with prostate volume. These results suggest that an estrogen-dominant environment plays an important role in the development of BPH. PMID- 7542401 TI - The spontaneous course of urodynamically verified sphincter lesion after surgery for benign prostatic hyperplasia. AB - Assuming that post-prostatectomy incontinence frequently resolves to various extents, forty patients selected from a background material of 105 consecutively referred patients with this complaint were reanalysed 3 to 106 months (median 55 months) after first examination. During the time interval between first examination and the revisit only 4 patients had some improvement of their incontinence. Eleven patients were evaluated urodynamically at both occasions showing no change in MUCP or other urodynamic parameters. Consequently we could not confirm the dogma that sphincter lesion due to prostate surgery may gradually resolve. Our investigation indicates a poor continence prognosis in patients incontinent after transurethral or transvesical surgery of the prostate and a more active treatment attitude. The patients should be offered an operation after evaluation of their incontinence, when a sphincter lesion is first diagnosed. PMID- 7542402 TI - CD1: presenting unusual antigens to unusual T lymphocytes. PMID- 7542403 TI - Peptide binding and presentation by mouse CD1. AB - CD1 molecules are distantly related to the major histocompatibility complex (MHC) class I proteins. They are of unknown function. Screening random peptide phage display libraries with soluble empty mouse CD1 (mCD1) identified a peptide binding motif. It consists of three anchor positions occupied by aromatic or bulky hydrophobic amino acids. Equilibrium binding studies demonstrated that mCD1 binds peptides containing the appropriate motif with relatively high affinity. However, in contrast to classical MHC class I molecules, strong binding to mCD1 required relatively long peptides. Peptide-specific, mCD1-restricted T cell responses can be raised, which suggests that the findings are of immunological significance. PMID- 7542404 TI - CD1-restricted T cell recognition of microbial lipoglycan antigens. AB - It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells. PMID- 7542406 TI - Interindividual variability in the urinary excretion of inorganic arsenic metabolites by C57 BL/6J mice: possible involvement of a thiol/disulfide exchange mechanism. AB - The 24-h urine of 75 C57 BL/6J mice injected s.c. with 0.5 mg/kg arsenic as sodium arsenite were examined for creatinine, S-adenosylmethionine (SAM), urea and inorganic arsenic metabolites including inorganic arsenic (ASi), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). There was interindividual variability of about a 10-fold range in the 24-h urinary excretion of creatinine (80-642 micrograms/24 h, variability (cv) of 33%), SAM (7.5-67.2 micrograms/24 h, cv of 38%), urea (9.5-89.7 mg/24 h, cv of 36%), ASi (0.1-1.6 microgram/24 h, cv of 48%), MMA (0.17-2.1 micrograms/24 h, cv of 50%), DMA (0.73-8.13 micrograms/24 h, cv of 32%) and total arsenic metabolites (1.0 10.4 micrograms/24 h, cv of 31%). Interindividual differences, varying by as much as 5-7-fold, were also found in the urinary proportion of ASi (3-23%, cv of 41%) and MMA (5-22%, cv of 37%), but not in the urinary proportion of DMA (64-90%, cv of 7%). The 24-h urinary excretion of all arsenic metabolites correlated with the 24-h urinary excretion of urea (r = 0.81), creatinine (r = 0.88) and SAM (r = 0.83) as did the 24-h urinary excretion of urea with creatinine (r = 0.94) and SAM (r = 0.86), and the 24-h urinary excretion of creatinine with SAM (r = 0.94). Taken together, these results suggest that the overall intracellular glutathione (GSH)-dependent redox state, as reflected by the 24-h urinary excretion of SAM and creatinine, is involved in the interindividual variability in total arsenic metabolite excretion by C57 BL/6J mice. These preliminary results were also discussed with regard to the involvement of intracellular GSH-dependent redox state in the regulation of the reduction and of the methylation steps of arsenic, and to interindividual variability in the urinary excretion of total arsenic metabolites as a possible complicating factor in the biological monitoring of occupational exposure to arsenic. PMID- 7542405 TI - Glomerular function of lead-exposed workers. AB - Among lead-exposed workers, there is evidence of increased mortality from chronic renal diseases (nephritis and nephrosis). Epidemiological studies using early markers of nephropathy among lead-exposed workers failed to demonstrate early renal changes. This study is aimed at assessing the glomerular function of 137 lead-exposed subjects and at evaluating whether changes in markers of glomerular function are related to exposure indices derived from longitudinal blood lead data. A control group of 153 postal workers was also investigated. Several exposure indices were derived for the exposed workers, including a time integrated index Pb in blood (PbB)int and the number of times the PbB was above critical values (PbB400, PbB500, PbB600). Through multiple linear regression analysis, PbBint was the best predictor of variation in serum beta 2 microglobulin (S beta 2m) and alpha 1-microglobulin (S alpha 1m) and urinary albumin (UA1b). A small but statistically significant difference in the mean beta 2m was found. S beta 2m was also the only marker showing a significantly higher prevalence rate ratio (PRR) of abnormalities among lead-exposed workers. Though there was no clear dose-response relationship with PbBint as the index of dose, all the 15 subjects with abnormal S beta 2m in the older age group were found in the highest PbBint group. Furthermore, of the 8 subjects with low 4-h creatinine clearance (CrCl4h), 6 had abnormal levels of beta 2m. Two subjects with CrCl4h of less than 75 ml/min/1.74 m2 had high PbBint values, thus suggesting that high blood lead levels over a prolonged time may be associated with decreased CrCl4h. Though the long-term significance of elevated S beta 2m and UA1b is unclear, their association with high PbBint and decreasing CrCl4h indicate a potentially adverse effect. Their relationship with PbB400 and PbB600 suggests that the threshold of 700 micrograms/l for PbB may not prevent the occurrence of lead nephropathy. PMID- 7542409 TI - Interferon therapy in chronic hepatitis B: more information but clarity eludes. PMID- 7542408 TI - Enhancement of the activity of bleomycin by cysteamine in a micronucleus assay in G0 human lymphocytes. AB - The aminothiol cysteamine enhances the induction of micronuclei by bleomycin in G0 human lymphocytes. The potentiation of bleomycin (12.5, 25, 50, or 100 micrograms/ml) increased with cysteamine concentration from 5 to 20 mM in a 2-h treatment before culturing the cells for the cytokinesis-block assay. The maximum clastogenic activity of bleomycin in the presence of cysteamine was more than 10 fold greater than that of the same dosage of bleomycin alone. Both the thiol and amine functions of aminothiols seem to contribute to the potentiation of bleomycin. PMID- 7542407 TI - Effects of postnatal aluminum exposure on biological parameters in the rat plasma. AB - Young rats were treated by gastric intubation with aluminum chloride (100 mg Al/kg/day) and aluminum lactate (100 and 200 mg Al/kg/day) from postnatal days 5 to 14. This treatment lead to a reduction in body weight. The plasma concentrations of total proteins and albumin decreased whereas the alpha 1 globulins increased in the treated rats. The aluminum concentrations in plasma and hepatic homogenates increased particularly at 200 mg Al lactate. The reduction in average body weight could be attributed to various causes: a decreased food consumption, a transient undernutrition, a reduction of the protein synthesis in the liver. The increase of the plasma concentration of the alpha 1 globulins revealed an inflammation process. PMID- 7542410 TI - Therapy of chronic hepatitis due to HCV. PMID- 7542411 TI - Dehydroepiandrosterone inhibits replication of feline immunodeficiency virus in chronically infected cells. AB - Dehydroepiandrosterone (DHEA) is a steroid hormone produced by the adrenal cortex that serves as an intermediary in sex steroid synthesis. DHEA is produced in abundance by humans and most other warm-blooded animals. Based upon previous reports demonstrating the antiviral and immunostimulatory activities of DHEA and DHEA-sulfate (DHEAS) we sought to determine whether introduction of these compounds would affect replication of feline immunodeficiency virus (FIV) in chronically infected cells. When cell number, cell viability, cellular DNA synthesis, and levels of FIV reverse-transcriptase (RT) were measured in cell cultures treated with various dilutions of DHEA or DHEAS it was found that the production of FIV RT was inhibited by DHEA at levels where cellular viability and DNA synthesis were not affected. At the concentrations tested DHEAS did not inhibit FIV replication or impact on cellular viability or proliferation. PMID- 7542412 TI - Two different mutations in the envelope protein of feline immunodeficiency virus allow the virus to escape from neutralization by feline serum antibodies. AB - Viral progeny of two molecular clones of feline immunodeficiency virus (FIV), 19k1 and 19k32, were tested in a virus neutralization assay. In this assay the infection of thymocytes with FIV19k1 was neutralized by serum S1422, derived from an SPF cat 22 weeks after infection with FIV19k1. We previously reported that a point mutation at position 560 in hypervariable region-5 (HV-5) of 19k1 confers resistance to virus neutralization (Siebelink et al., 1993, J. Virol. 67:2202 2208). Viral progeny of the other molecular clone, FIV19k32, which differs in the envelope protein in only six amino acids from 19k1, was not neutralized. In order to map sites involved in virus neutralization we constructed chimeric clones by reciprocal exchange of 19k1 and 19k32 envelope gene fragments. Reciprocal exchange of a 1662 bp fragment, encoding almost the whole surface protein, which differs in five amino acids between these two clones, resulted in exchange of the phenotype. Amino acids of the envelope protein of 19k1 and 19k32, in which these clones differ, were substituted by point mutation. We demonstrated that one of these mutations, a substitution of leucine to serine at position 483 in HV-4, also conferred resistance of 19k1 to neutralization by serum S1422. PMID- 7542413 TI - A determinant of feline immunodeficiency virus involved in Crandell feline kidney cell tropism. AB - Viral progeny of the molecular clone 19k1 of feline immunodeficiency virus (FIV) can infect feline T-cells but not Crandell feline kidney (CrFK) cells. In contrast, the biological isolate FIV-AM6c, which was CrFK adapted by co cultivation of FIV-AM6 infected thymocytes with CrFK cells, can infect both thymocytes and CrFK cells. The envelope gene of FIV-AM6c was amplified by polymerase chain reaction using DNA from infected CrFK cells, and subsequently cloned and sequenced. To map viral determinants of CrFK cell tropism, chimeric viruses with a 19k1 background containing envelope gene fragments of FIV-AM6c were constructed. CrFK cells were transfected with DNA of these chimeric clones and co-cultivated with thymocytes. After 3 days the CrFK cells and the thymocytes were cultured separately. FIV antigen could be detected in most of the thymocyte cultures within 14 days and in one of the CrFK cultures after 52 days. The resulting virus from this CrFK culture can infect both CrFK cells and thymocytes. The results of this study indicate that the envelope region contains determinants of CrFK tropism. The delay in replication indicates that also determinants other than those identified here are involved in CrFK cell tropism. More chimeric clones are being studied at present to map these determinants. PMID- 7542414 TI - Distinct patterns of IFN sensitivity observed in cells infected with vaccinia K3L and E3L- mutant viruses. AB - Recent results have implicated a role for both the VV K3L- and E3L-encoded gene products in conferring VV with an IFN-resistant phenotype (Beattie et al., Virology 183, 419-422, 1991; Beattie et al., J. Virol. 69, 499-505, 1995). As a means of further establishing the mechanisms by which these functions mediate this process in VV-infected cells, we have further assessed the IFN phenotype in K3L- (vP872) and E3L- (vP1080) virus-infected cells. Biochemical and molecular biological analyses were performed comparing the effects of IFN on wild-type as well as K3L- and E3L- virus-infected cells. Expression analyses of the K3L and E3L gene products revealed that both are evidenced in virus-infected cells as early as 0.5 hr postinfection. E3L expression, however, appears more prolonged, in that it was detectable between 3 to 4 hr postinfection while K3L was undetectable after 3 hr postinfection. Despite having similar expression profiles at early times postinfection, a pronounced sensitivity of protein synthesis to IFN was observed by 30 min postinfection in VV K3L- virus-infected cells, whereas IFN sensitivity was not observed in VV E3L(-)-infected cells until 2 hr postinfection. Subsequent analyses of the IFN-induced antiviral pathways in VV infected cells demonstrated that the K3L gene product does not contribute to the previously identified specific kinase inhibitory factor (SKIF) activity but does reduce the level of phosphorylated eIF-2 alpha in VV-infected cells. Interestingly, the IFN-induced 2',5'-oligoadenylate synthetase-mediated antiviral pathway was active in VV K3L(-)-infected cells and not in wild-type virus infected cells. Collectively these results suggest that the K3L(-)- and E3L(-) encoded products abrogate the antiviral effect of IFN at distinct levels. PMID- 7542415 TI - Induction of neutralizing antibodies to papillomaviruses by anti-idiotypic antibodies. AB - Anti-idiotypic antibodies (anti-Ids) were generated against three mouse monoclonal antibodies (MAbs) which neutralized three different papillomaviruses. The neutralizing MAbs (N-MAbs) were generated against infectious human papillomavirus type 11 (HPV-11), cottontail rabbit papillomavirus (CRPV), and bovine papillomavirus type 1 (BPV-1), and all recognized surface conformational epitopes that were lost upon denaturation of the virions. The polyclonal anti-Ids were screened in an ELISA using a panel of 10 N-MAbs (4 HPV-11 neutralizing, 5 CRPV neutralizing, and 1 BPV-1 neutralizing), and cross-reactive idiotypic reactivity was observed. Affinity-purified anti-H11.B2 (HPV-11 N-MAb) cross reacted with the 3 other HPV-11 N-MAbs, was unreactive to a nonneutralizing HPV 11 MAb and all 5 CRPV N-MAbs, but surprisingly was reactive to the BPV-1 N-MAb. In contrast, affinity-purified anti-CRPV-1A (CRPV N-MAb) reacted with all 5 CRPV N-MAbs, but not with any other MAb. Affinity-purified anti-B1.A1 (BPV-1 N-MAb) also showed unexpected cross-reactivity with the 4 HPV-11 N-MAbs, but not with any of the CRPV N-MAbs. Two of the three polyclonal rabbit anti-Ids induced strong anti-viral antibody responses (including virus-neutralizing antibodies) in BALB/c mice following immunization. The results indicate that within a mouse strain, papillomavirus-neutralizing antibodies share cross-reactive idiotopes that group the N-MAbs into several subsets. One group includes the CRPV N-MAbs, and another group includes N-MAbs directed to apparently unrelated papillomaviruses, including HPV-11, BPV-1, and possibly HPV-6. PMID- 7542416 TI - Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages. AB - The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full length viral DNA by 24 hr postinfection. The level of integrated delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between viral DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from delta DU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into viral DNA. PMID- 7542417 TI - Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus. AB - Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli. Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions. The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic. All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies. The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain. However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV. This information might be useful in designing a subunit vaccine against AHSV infection. PMID- 7542418 TI - Monoclonal antibodies which recognize the acidic configuration of the rabies glycoprotein at the surface of the virion can be neutralizing. AB - Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody. PMID- 7542419 TI - In vivo cellular tropism of human T-lymphotropic virus type II is not restricted to CD8+ cells. AB - We have examined the in vivo and in vitro susceptibility of lymphocyte subpopulations to human T-lymphotropic virus type II (HTLV-II) to determine the cellular tropism for this virus. Monoclonal antibodies to T-cell subsets were used to separate highly purified CD4+ and CD8+ cells from peripheral blood lymphocytes of 35 individuals previously shown to be infected with HTLV-II. The purified T-cell subsets were analyzed for HTLV-II provirus (pol and tax gene sequences) by polymerase chain reaction (PCR) and cultured to determine virus expression by p24gag antigen detection. On the basis of PCR amplification in the pol and tax gene regions, both CD8+ subsets (89 to 91%) and CD4+ subsets (54 to 80%) from most infected subjects demonstrated HTLV-II provirus, irrespective of the viral genotype. Analysis of cultured lymphocytes demonstrated a higher spontaneous lymphocyte proliferation (17,986 +/- 4675 cpm) and p24gag antigen production (median 115 pg/ml; range 14-1360 pg/ml) in CD8+ cells compared to CD4+ cells (2333 +/- 826 cpm; p24gag antigen; 9 pg/ml; 2-250 pg/ml), suggesting a higher proviral load in CD8 cells. Limiting cell-dilution PCR analysis indicated that the CD8+ subset carried a higher HTLV-II provirus burden than the CD4+ subset. In vitro infection of purified CD4+ and CD8+ lymphocytes with irradiated HTLV-II cell lines resulted in productive infection of both subsets. Cell sorting and PCR analysis of lymphocyte subsets from 4 HTLV-II-infected subjects further demonstrated that in addition to CD4+ and CD8+ subsets, both CD45RO+ and CD45RO- and non-T-cells (CD14, CD16, and CD19) carried HTLV-II provirus. Taken together, these data suggest that HTLV-II possesses a broad tropism for peripheral blood mononuclear cells. PMID- 7542420 TI - Effects of epithelium on the mechanism of mediator release from guinea pig tracheal tissues sensitized by IgG1 versus IgE antibody. AB - In the present work, we have examined the effect of PAF, removal of epithelium, the mechanism of desensitization, and the substances that increases the level of intracellular c-AMP on the differences of mediator release from superfused tracheal strips after passive sensitization with IgG1 versus IgE Ab. In the passive sensitized tracheal tissues, the effect of PAF and the mechanism of desensitization have been examined by PAF antagonist, CV 3988 and DFP, respectively. The epithelium was stripped from one-half of each trachea by mechanical means. Both superfused tracheal tissues were challenged with Ox-Ag. Inhibitors of mediator release were added into a superfused buffer. Hist released was determined by spectrophotofluorometer, and LT by radioimmunoassay. PAF known to mediate the allergic reaction was not released by Ag after both Ab sensitization. Epithelium removal resulted in similar contraction, Hist and LT release after IgG1 Ab activation, but in the IgE Ab activation, epithelium removal resulted in smaller contraction and Hist release. In the L-cysteine and indomethacin pretreatment after two Ab sensitization, epithelium removal decreased the release of Hist and LT. The compound 48/80 pre-challenge and epithelium removal resulted in the increase of Hist release, but in the decrease of LT release after IgG1 or IgE sensitization. The Amount of LT released by Ag after compound 48/80 pre-challenge increased in the absence or presence of epithelium after both Ab sensitization. Mediator release from tissues sensitized with both Abs was not changed by DFP. The responses of inhibitors to prevent the mediator release were more effective on the IgE Ab than on the IgG1 Ab sensitization. These studies suggest that the tracheal epithelium can act to inhibit immune- and non-immune-induced airway responses. Non-immunological responses may in part reflect the role of epithelium as a diffusion barrier and modulator of mediator release. These data also suggest that immunological responses are related to the localization and functional heterogeneity of tissue mast cells. PMID- 7542421 TI - Influence of the epididymis on integrins and matrix proteins of human spermatozoa. AB - Spermatozoa alter their antigenity and develop the ability of binding to zona pellucida and to vitellus during the epididymal transit. The involvement of spermatozoal adhesion molecules (AM), especially beta 1-integrins in this binding process between the gametes is very likely. Therefore, the influence of epididymis on the binding capacity of AM was indirectly investigated by comparison of AM on ejaculated and on testicular human spermatozoa and spermatogenic cells, respectively. The AM-expression was detected on ejaculated spermatozoa by flow cytometry (EPICS PROFILE II, Coulter Corp., Krefeld, Germany) and on spermatogenic cells by immunohistology using monoclonal antibodies against. beta 1-integrins and matrix proteins. The alpha-chains 3, 5, 6 of the beta 1-integrins and fibronectin were found on ejaculated spermatozoa as well as on spermatogenic cells except spermatogonia. The alpha-4-chain and laminin could only be detected after the epididymal transit of spermatozoa, whereas, the beta chain of beta 1-integrins was missing on the ejaculated spermatozoa. These findings suggest that AM are predominantly expressed on spermatogenic cells before the epididymal transit but an epididymis-mediated modulation of AM, i.e. masking, demasking or activating of cryptodeterminants is possible. On this way dysfunction of epididymis could be followed by an impairment of AM-mediated binding capacity of spermatozoa. PMID- 7542422 TI - A study of dementia with argyrophilic grains. Possible cytoskeletal abnormality in dendrospinal portion of neurons and oligodendroglia. AB - Two mildly demented patients with emotional disorder presented massive cortical argyrophilic grains (ArG) in the limbic area and coiled bodies mainly in the white matter. Immunohistochemically, the ArG consisted of partially ubiquitinated full-length phosphorylated tau. A modified Gallyas-Braak method, used after potassium permanganate and oxalic acid pretreatment, revealed many filiform, knobby or pleomorphic spine-like appendages on the ArG. Double immunohistochemical staining using anti-tau with either anti-microtubule associated protein 2 or anti-neurofilament 200K revealed that ArG were intimately associated with dendrites rather than with axons. Electron microscopic studies with tau immunohistochemistry and the Gallyas-Braak method revealed that the ArG were composed of bundles of smooth tubules of 25-nm diameter. The coiled body bearing cells had the morphological characteristic of oligodendroglia. These results indicate that the presence of ArG is a cytoskeletal abnormality affecting predominantly the dendrospinal portions of neurons. A survey of diseases with cytoskeletal disorders revealed that a small number of similar argyrophilic granular structures are sometimes observed in progressive supranuclear palsy, Pick's disease with Pick bodies and corticobasal degeneration. Dementia with ArG is thought to be a unique cytoskeletal abnormality associated predominantly with the dendrospinal portions of neurons and oligodendroglia. PMID- 7542423 TI - Loss of hilar somatostatin neurons following tetanus toxin-induced seizures. AB - A loss of inhibitory interneurons has been reported in the hippocampus following seizure activity in various animal models of epilepsy and in human epileptic tissue. The question of whether particular populations of inhibitory neurons are similarly affected by the chronic block of inhibition that results after tetanus toxin injections directly into the brain has not previously been addressed. In the present study a unilateral intrahippocampal injection of tetanus toxin into the ventral hippocampus was used to produce a chronic epileptic syndrome characterised by brief seizures that recurred intermittently for 6-8 weeks. The results reveal, for the first time, the morphological changes in somatostatin interneurons following tetanus toxin-induced seizures in the rat. A bilateral short-term increase in immunoreactivity of somatostatin neurons is present 1 week after injection. This is accompanied by an increased intensity of somatostatin immunoreactive axon terminals in the outer molecular layer of the dentate gyrus, which is more marked on the contralateral side. A chronic and significant loss of somatostatin-immunoreactive neurons was noted in the hilus of the dentate gyrus 2 months later. The significance of the chronic loss of the hilar somatostatin neurons in the control of excitatory activity in the dentate gyrus and whether the acute morphological changes are due to a direct action of the toxin on release mechanisms or as a result of seizure activity are discussed. PMID- 7542424 TI - Myelin basic protein does not have a mitogenic effect on adult oligodendrocytes. AB - Increased numbers of oligodendrocytes and remyelination are frequently observed in multiple sclerosis plaques. It is presumed the increased numbers of oligodendrocytes are due to cell division, but this has not been proven. The mitogens within the lesion which might be responsible for this are unknown. Since oligodendrocyte proliferation occurs in areas in which there is myelin breakdown, we undertook the present study to determine if myelin basic protein (MBP) or its breakdown products could induce oligodendrocyte proliferation. MBP, or MBP digested by the neutral proteinase plasmin, was added in three concentrations to the media of adult bovine oligodendrocytes in culture. Oligodendrocytes were identified by staining for galactocerebroside. Bromodeoxyuridine incorporation was used as a measure of cell division. Oligodendrocytes were found to divide only rarely in regular culture media, in the presence of MBP, plasmin, or MBP digested by plasmin. The results indicate that MBP is not a significant mitogen for the mature oligodendrocyte. PMID- 7542425 TI - Ganglioglioma with neurofibrillary tangles (NFTs): neoplastic NFTs share antigenic determinants with NFTs of Alzheimer's disease. AB - A cerebral ganglioglioma contained abundant neurofibrillary tangles (NFTs) of the paired helical filament (PHF) type. The NFTs in the tumor were argyrophilic and Congo red and thioflavin-S positive. Immunohistochemically, the NFTs were reactive with antibodies to phosphorylated neurofilament protein, PHF/tau and ubiquitin. The demonstration in the neoplasm of abnormally phosphorylated and ubiquitinated cytoskeletal components, similar in morphology and in immunoreactivity to those seen in NFTs of Alzheimer's disease, suggest that similar pathogenetic mechanisms may operate in both conditions. PMID- 7542426 TI - Human placental lactogen and pregnancy-associated plasma protein A in first trimester and subsequent fetal growth. AB - OBJECTIVE: To study in an optimized design the possible relation between serum levels in weeks 8-14 of human placental lactogen and pregnancy-associated plasma protein A and fetal size at delivery. METHODS: Analysis of data from 93 normal singleton pregnancies. Gestational age was assessed from a sonographic crown-rump length measurement. Serum levels of human placental lactogen and pregnancy associated plasma protein A were determined by radioimmunoassay, and were expressed in multiples of mean. The relative birth weight was used as an index of fetal growth. RESULTS: Serum levels of human placental lactogen and pregnancy associated plasma protein A showed a negative correlation to gestational age at delivery (p < 0.01 and p < 0.05, respectively), and there was a positive correlation between the serum level of pregnancy-associated plasma protein A and relative birth weight (p < 0.02). CONCLUSIONS: Higher levels of human placental lactogen and pregnancy-associated plasma protein A predicted earlier delivery, maybe because of better fetal growth, and higher levels of pregnancy-associated plasma protein A predicted better fetal growth. PMID- 7542427 TI - Induction of calcium-dependent nitric oxide synthase by sex hormones in the guinea-pig urinary bladder. PMID- 7542428 TI - Detection of reverse transcriptase activity by enzyme-linked immunosorbent assay in human immunodeficiency virus type 1. AB - An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP) conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1 positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors. PMID- 7542429 TI - Perspective of neurochemistry in neurological disorders. PMID- 7542430 TI - Hepatic artery embolotherapy of hepatic metastases from carcinoid tumors: value of using a mixture of cyanoacrylate and ethiodized oil. AB - OBJECTIVE: Transcatheter embolization of the hepatic arterial supply is a well known palliative treatment of tumor deposits in the liver. We performed a prospective study to evaluate the use of a mixture of N-butyl-2-cyanoacrylate and ethiodized oil with which a permanent vascular occlusion can be obtained, as an embolizing agent for transcatheter hepatic artery embolization for treatment of carcinoid hepatic metastases. SUBJECTS AND METHODS: Six patients had clinical symptoms from hormonal release by carcinoid hepatic metastases as well as elevated levels of 5-hydroxyindole acetic acid (5-HIAA) in the urine. Unilobar sequential transcatheter embolization of both the hepatic artery and the segmental hepatic arteries of both lobes of the liver was performed with a mixture of N-butyl-2-cyanoacrylate and ethiodized oil. CT and CT arterial portography (CTAP) were done to assess hepatic metastases and were used to monitor follow-up. Each patient had three CTAP studies; the third CTAP, performed 3 months after complete arterial devascularization, was compared with the first CTAP to evaluate tumor size. CT studies were performed routinely every 3 months thereafter and were compared with the initial CT scan to evaluate further tumor regression or progression. Tumor decrease and biochemical and symptomatic response rates were defined according to World Health Organization criteria. All complications and side effects of the treatment were documented. RESULTS: All patients showed complete symptomatic relief after embolization. The previously elevated levels of 5-HIAA in the urine returned to normal in three patients and in the other three patients were reduced by a mean of 89% of preembolization values. A decrease in tumor size by more than 50% was demonstrable in one patient; in five patients, hepatic lesions decreased in size by 25-50%. No new sites of metastatic liver disease were demonstrable in any patient during follow up. No deaths or serious complications were directly attributable to the embolization procedure. All patients are alive after 12, 17, 18, 19, 19, and 19 months (mean, 17.3 months), respectively, with permanent relief of symptoms so far. CONCLUSION: Transcatheter embolization of both the hepatic artery and the segmental hepatic arteries with a mixture of N-butyl-2-cyanoacrylate and ethiodized oil provided excellent palliation in patients with carcinoid hepatic metastases. Complete and long-lasting relief of symptoms, a significant decrease or normalization of levels of 5-HIAA in the urine, and a reduction of metastatic tumor in the liver seem most likely to be the effect of sustained ischemia obtained with this permanent embolizing agent. PMID- 7542431 TI - Decrease in hematocrit after coronary stent placement and dextran therapy. AB - In summary, dextran 40, when given after coronary stent placement, results in a marked decrease in hematocrit within 24 hours. Hematocrit often returns to near baseline levels within 48 hours of stopping dextran. This phenomenon most likely reflects dextran-related hemodilution. This hemodilutional decrease in hematocrit is often misinterpreted as acute blood loss and may result in blood transfusion in patients with low baseline hematocrit. However, far less aggressive anticoagulation regimens, which do not include dextran, are under investigation in patients undergoing coronary stent placement. PMID- 7542432 TI - Hemostatic factors and replacement of major blood loss with plasma-poor red cell concentrates. AB - The purpose of this study was to assess the change of platelet and fibrinogen concentrations and the change of activities of prothrombin and factors V and VII when major surgical blood loss was replaced with plasma-poor red cell concentrates (RCCs) and colloid plasma substitutes. Sixty patients were studied. The average blood loss was 65% +/- 41% of the calculated blood volume (CBV). Blood loss was monitored carefully and replaced without delay to ensure stable blood volume. Blood samples were obtained at the induction of anesthesia and at the end of the recovery room period, or before the patient was given fresh frozen plasma. In addition, a platelet count was determined after each 20% blood loss. The results were converted to relative values, and simple linear regression with logarithmic transformation was applied. The initial platelet concentration was 257 +/- 89 x 10(3)/mm3 and the extrapolation of the regression line intercepted the critical level of 50 x 10(3)/mm3 at 230% (confidence interval 169%-294%) blood loss. The initial fibrinogen concentration was 3.7 +/- 1.1 g/L and the hemostatically significant level of 1.0 g/L was already reached at 142% (117% 169%) blood loss (r2 = 0.90). Activities of prothrombin and coagulation factors V and VII reached their critical levels at 201% (160%-244%), 229% (167%-300%), and 236% (198%-277%) blood loss, respectively. We conclude that deficiency of fibrinogen develops earlier than any other hemostatic abnormality when plasma poor RCCs are used for the replacement of major blood loss. PMID- 7542433 TI - Epidural anesthesia for cesarean section in a patient with Romano-Ward syndrome. PMID- 7542434 TI - Effects of pentastarch on global cerebral ischemia and reperfusion. PMID- 7542435 TI - [Palliative chemotherapy in colorectal cancer: new molecular drugs]. PMID- 7542436 TI - [The contribution of immunohistochemistry in the diagnosis of neuroendocrine tumors]. PMID- 7542437 TI - [Helicobacter pylori infection and cancer of the stomach]. PMID- 7542438 TI - [Therapeutic strategies for inoperable cancer of the esophagus. Radiotherapy]. PMID- 7542439 TI - The Landau-Kleffner syndrome. PMID- 7542440 TI - Investigation of developmental delay. PMID- 7542441 TI - Respiratory illnesses in children and air pollution in Copenhagen. AB - The relationship between air pollution and the daily number of contacts (i.e., telephone calls and home visits) with or at Copenhagen Emergency Medical Service for children with and without respiratory illnesses was studied during a 91-d period (i.e., January 14, 1991, to April 14, 1991). A total of 12,132 contacts occurred. Diagnoses, which were recorded on the invoices for 5,307 contacts, revealed that 3,974 contacts were the result of respiratory illnesses. Regression analysis was used to investigate the short-term relationship between pollutants (i.e., carbon monoxide, nitric oxide, nitrogen dioxide, NOx, sulfur dioxide, ozone, and black smoke), measured at monitoring stations, and both the number of all contacts for children and the number of contacts for children with respiratory illnesses. Temperature and systematic effects that were the result of holidays and weekends were controlled for, after which only nitric oxide and NOx were associated significantly with the number of contacts for children who had respiratory illnesses. Nitric oxide and NOx, as indicators of traffic pollution, appeared, at low levels, to slightly exacerbate respiratory illnesses among children. PMID- 7542442 TI - Traffic-related air pollution: exposure and health effects in Copenhagen street cleaners and cemetery workers. AB - This questionnaire-based study found a significantly higher prevalence of chronic bronchitis, asthma, and several other symptoms in 116 Copenhagen street cleaners who were exposed to traffic-related air pollution at levels that were slightly lower than the 1987 World Health Organization-recommended threshold values, compared with 115 Copenhagen cemetery workers exposed to lower pollution levels. Logistic regression analysis, controlling for age and smoking, was conducted, and odds ratios and 95% confidence intervals were calculated to be 2.5 for chronic bronchitis (95% confidence interval = 1.2-5.1), 2.3 for asthma (95% confidence interval = 1.0-5.1), and 1.8-7.9 for other symptoms (95% confidence interval = 1.0-28.2). Except for exposure to air pollution, the two groups were comparable, i.e., they had similar terms of employment and working conditions. The exposure ranges during an 8-h work day, averaged from readings taken at five monitored street positions, were: 41-257 ppb nitric oxide (1-h max: 865 ppb); 23-43 ppb nitrogen dioxide (1-h max: 208 ppb); 1.0-4.3 ppm carbon monoxide (8-h max: 7.1 ppm); 14-28 ppb sulfur dioxide (1-h max: 112 ppb); and 10-38 ppb ozone (1-h max: 72 ppb). PMID- 7542443 TI - Use of Bartonella antigens for serologic diagnosis of cat-scratch disease at a national referral center. AB - BACKGROUND: Bartonella henselae (formerly the genus Rochalimaea) has recently been isolated from patients with cat-scratch disease and their cats, and since September 1992 the Centers for Disease Control and Prevention has offered an indirect fluorescent antibody assay for Bartonella-specific antibody. METHODS: Physicians submitted serum samples from patients suspected of having cat-scratch disease or other Bartonella-associated illness and completed a questionnaire that recorded clinical information. Indirect fluorescent antibody assay was performed with the use of antigen derived from three Bartonella species: B henselae, Bartonella quintana, and Bartonella elizabethae. RESULTS: During 16 months, 3088 serum samples were received. The largest numbers of specimens and the highest percentages positive (titer, > or = 64) were observed in the fall and winter. Clinical histories of the first 600 patients for whom serum samples and completed information forms were received were examined in detail; seropositivity was significantly associated with cat contact, cat age of less than 1 year, cat scratch, presence of an inoculation papule, and regional adenopathy. Of 91 patients whose illness met a strict clinical definition of cat-scratch disease, 86 (95%) had titers of 64 or greater to either B henselae or B quintana. A fourfold rise or fall in titer was observed in 87 of 132 patients with paired serum samples. CONCLUSIONS: The indirect fluorescent antibody assay for Bartonella-specific antibody is sensitive for the diagnosis of cat-scratch disease. Redefinition of cat-scratch disease on the basis of cause and use of this assay as a diagnostic criterion is recommended. PMID- 7542444 TI - [Inhibitory effect of histamine-added mouse gamma-globulin on eosinophil accumulation induced by allergen in BALB/c mice]. AB - Histamine-added human gamma-globulin (HG) has been clinically used as an anti allergic drug for asthma, allergic rhinitis and atopic dermatitis. Retrospective analysis of clinical data have indicated that s.c. administration with HG not only improves clinical symptoms but also suppresses the number of eosinophils of nasal secretion or peripheral blood in allergic patients. Thus, the possibility was explored that HG may actively suppress eosinophil accumulation in allergic inflammation. The eosinophil accumulation in peritoneal cavity was induced by i.p. injection with ragweed pollen extract in BALB/c mice which had been repeatedly sensitized with the allergen for three weeks. Histamine-added mouse gamma-globulin (Mouse HG) at 150 mg/kg/day markedly inhibited the allergen induced eosinophil accumulation when administered s.c. two times a week for three weeks. The inhibitory effect was almost the same as that of cyclosporin A at 100 mg/kg/day. Interestingly, equivalent dose of histamine or mouse gamma-globulin alone had no inhibitory effect in the same system. These results suggest that HG suppresses chronic allergic inflammation through the inhibition of eosinophil accumulation. PMID- 7542446 TI - Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes. AB - Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and cAMP-independent mechanism. PMID- 7542445 TI - Ligation of the alpha 2-macroglobulin signalling receptor on macrophages induces protein phosphorylation and an increase in cytosolic pH. AB - We have recently described an alpha 2-macroglobulin (alpha 2M) signalling receptor which is distinct from the low-density lipoprotein-related protein/alpha 2M receptor (LRP/alpha 2MR). Ligation of the macrophage signalling receptor by alpha 2M-methylamine stimulates production of several second messengers and involves a pertussis toxin-insensitive G-protein. We now report that binding of alpha 2M-methylamine, or the cloned M(r) = 20,000 receptor-binding fragment from rat alpha 1M, to macrophage alpha 2M signalling receptors induces protein phosphorylation. By use of a monoclonal antibody to phospholipase C gamma l (PLC gamma l) we were able to identify it as one target for protein phosphorylation. Phosphorylation was time and concentration dependent, being optimal at about 60 s of incubation and a 100-200 nM ligand concentration. By use of a second monoclonal antibody directed against phosphotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma l phosphorylation was then studied in membrane preparations at 4 degrees C in order to minimize serine or threonine modification. Preincubation of macrophage membranes with genistein, a tyrosine kinase inhibitor, drastically reduced phosphorylation of PLC gamma l. Receptor associated protein, which blocks alpha 2M binding to LRP/alpha 2MR but not to the alpha 2M signalling receptor, had no effect on alpha 2M-methylamine-induced tyrosine phosphorylation of PLC gamma l. Binding of lactoferrin to LRP/alpha 2MR failed to induce phosphorylation of PLC gamma l, further supporting the hypothesis that the alpha 2M signalling receptor and LRP/alpha 2MR are distinct entities. Growth factors which induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of a2M-methylamine to macrophages also gradually increased the intracellular pH in a concentration-dependent manner, being optimal at a 200 nM ligand concentration. The increase in pH was amiloride sensitive. We propose that receptor-recognized forms of a2M may function like growth factors with regard to macrophage regulation. PMID- 7542448 TI - Arachidonic acid mobilization is suppressed during mitosis: role of cytosolic phospholipase A2 activation. AB - In interphase HeLa cells, incubation with histamine or thapsigargin led to the rapid release of arachidonic acid. The release was absolutely dependent on Ca2+, consistent with the activation of an 85 kDa cytosolic phospholipase A2 (cPLA2). In metaphase-arrested HeLa cells, by contrast, the stimulation of arachidonate release by these agents was inhibited by more than 90%. The lack of arachidonic acid release by mitotic cells was at least partly expected, since histamine- or thapsigargin-induced Ca2+ influx and elevations of cytosolic free Ca2+ are known to be strongly inhibited during mitosis [Preston, Sha'afi and Berlin (1991) Cell Regul. 2, 915-925]. Indeed, incubation of interphase cells with the Ca2+ ionophore A23187 alone induced a high level of arachidonate release. However, even A23187 failed to elicit release from mitotic cells. Since the Ca(2+) dependent release of arachidonate by many cell types is promoted by preincubation with ligands that activate receptors of the tyrosine kinase class, and tumour promoters that lead to the phosphorylation of cPLA2, we determined if the responses of mitotic HeLa cells could be modified by this 'priming' process. We first established that epidermal growth factor and phorbol 12-myristate 13 acetate were effective priming agents in interphase cells: cells preincubated with the hormone or tumour promoter showed a 2-fold stimulation of thapsigargin- or A23187-induced arachidonic acid release. However, none of the priming agents reversed the lack of mitotic cell response. This refractoriness was not caused by destruction of cPLA2 during mitosis: by Western blotting, cPLA2 of interphase and mitotic cells was shown to be present in comparable amounts. Moreover, cPLA2 activities measured in extracts of interphase and mitotic cells were also comparable. Surprisingly, mitotic cPLA2 appeared to be constitutively phosphorylated in non-hormone-treated (control) cells. The results indicate a novel mechanism of regulation by cPLA2 activity in mitotic cells. PMID- 7542447 TI - Mechanism of action of gonadotropin-releasing hormone upon gonadotropin alpha subunit mRNA levels in the alpha T3-1 cell line: role of Ca2+ and protein kinase C. AB - Addition of [D-Trp6]gonadotropin-releasing hormone (GnRHa) to alpha T3-1 cells induced a very rapid response upon gonadotropin alpha-subunit mRNA which was detected after 30-60 min and was abolished by pretreatment with actinomycin D. A similar response was obtained with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), or the Ca2+ ionophore, ionomycin. GnRHa (10 nM) also stimulated a secondary rise in alpha-subunit mRNA levels between 12 and 24 h of incubation. No additivity was obtained (at 60 min) upon the combined addition of GnRHa and PMA, GnRHa and ionomycin, or PMA and ionomycin. The effect of GnRHa upon alpha-subunit mRNA was blocked by the PKC inhibitors staurosporine or GF 109203X. Down-regulation of endogenous PKC activity resulted in inhibition of the stimulatory effect of gonadotropin-releasing hormone (GnRH), PMA and ionomycin. Removal of extra-cellular Ca2+ abolished the effect of GnRHa and PMA upon alpha subunit mRNA levels. Interestingly PMA and ionomycin had no effect on alpha subunit mRNA levels at 24 h of incubation; however, the combined addition of the drugs mimicked the late phase of GnRHa (10 nM) action. The data provide evidence that PKC and Ca2+ are involved in mediating the early and the late responses of GnRHa upon alpha-subunit mRNA elevation and that differential cross-talk exists between the messengers. PMID- 7542449 TI - Muscle involvement in congenital insensitivity to pain with anhidrosis. AB - A patient with congenital insensitivity to pain with anhidrosis, who had characteristic clinical features and biopsied sural nerve, is presented. Nerve pathology findings indicated a loss of the small myelinated and unmyelinated fibers. Biopsied muscle disclosed a marked variation in fiber size, some small fibers with central nuclei, and a small number of small angulated fibers, consistent with neurogenic and myogenic changes. Many patients with congenital insensitivity to pain with anhidrosis had muscle weakness and absent or decreased deep tendon reflexes with normal nerve conduction velocity. We confirmed that lack of small myelinated fibers in motor neurons resulted in a striking change of muscle in our patient. PMID- 7542450 TI - [Significance for the health of the general population of exposure to benzene in traffic]. AB - Significant air pollution can be present in the interior of vehicles due to emissions from materials forming part of the interior fittings and to combustion and evaporation emissions, both when the motor is on and when the vehicle is stationary. In the case of new vehicles, escaping volatile organic compounds (VOC) in concentrations of 10-12 mg/m3 (partly up to 65 mg/m3) have been found. Benzene pollution of the interior of the vehicle is of particular significance to health. Benzene can be present in the interior of cars in average concentrations of about 50 micrograms/m3. Application of an overall risk assessment shows that, even under conditions of only a one-hour car ride per day, this pollution represents a risk of 30% of the total benzene risk (6 cases of cancer per 100.000 person exposed). PMID- 7542453 TI - Systemic CD56+ cells can predict pregnancy outcome. AB - PROBLEM: To evaluate differences in circulating CD56+ cells between successful and unsuccessful pregnancies, 114 pregnant women were studied prospectively. METHOD: Seventy women had a history of infertility (INF) and 44 had two or more previous spontaneous abortions (RSA). Among the infertile women, 12 were donor egg recipients (DER) and 15 underwent intracytoplasmic sperm injection (ICSI) for treatment of male factor infertility. Nineteen women were carrying multiple gestations (MG) and 55 had singleton gestations (SG). Thirteen additional women were receiving intravenous immunoglobulin (IVIg). RESULTS: The percentage of CD56+ cells was determined in 310 blood samples from 114 pregnant women by flow cytometry. The prevalence of women with persistent elevation of percent of 56+ cells (> 12%) was 58% among DER, 73% among ICSI, 37% among MG, 22% among SG, 18% among RSA, and 39% among INF. Thirteen women with SG received IVIG, 10 had CD56+ cells greater than 12% and all 13 experienced live births. Women with percentage CD56+ cells persistently greater than 12% who were not DER, not ICSI, not receiving IVIg, and not carrying MG had a live birth rate of 11%. Women with greater than 12% CD56+ cells had normal karyotype in 78% of concepti studied in contrast to women less than 12% CD56+ cells who had 68% abnormal karyotypes (P = 0.04). CONCLUSION: Elevated CD56+ cells in pregnant women who are not DER, not ICSI, not receiving IVIg, and not carrying MG predicts loss of a karyotypically normal conceptus with a specificity of 87% and positive predictive value of 78%. While the specificity value of this test is high in both infertile and RSA populations, the sensitivity is 86% in RSA and only 54% in INF suggesting this test does not identify all losses among INF. It may identify a subset of pregnancies at risk for loss of a karyotypically normal embryo that may respond to treatment with IVIg. PMID- 7542452 TI - Standardization of flow cytometric crossmatch (FCXM) for investigation of unexplained habitual abortion. AB - PROBLEM: To define a positive flow cytometric crossmatch (FCXM), in terms of channel shift, for maternal IgG and IgM (n = 28) against paternal T and B lymphocytes. METHOD: A reference range study. Mononuclear cells were obtained from 28 healthy volunteers using density gradient separation of heparinized blood, followed by pre-incubation with goat immunoglobulin. A total of twelve tubes were prepared for each volunteer. Primary incubation was with negative control serum, positive control sera (either IgG or IgM) and individual AB sera. Secondary incubation was with four combinations of fluorochromes: CD3 PE/IgG-Fc F(ab')2FITC, CD3 PE/IgM F(ab')2FITC, CD20 PE/IgG-Fc F(ab')2FITC and CD20 PE/IgM F(ab')2FITC. The cells were then analyzed with an EPICS Profile flow cytometer, using 256-channels and a four decade log scale. RESULTS: The linear mean channel fluorescence of the negative control serum was subtracted from the individual AB sera (channel shift) for each of the four combinations of fluorochromes. By determining the 95% one-sided upper reference limits of the negative control serum for each of the four trimmed data sets, we clinically defined a positive FCXM for bound IgG or IgM to T lymphocytes as a shift of 10 or more channels, and for bound IgG or IgM to B lymphocytes as a shift of 25 or more channels, above the linear mean channel shift of the negative control serum. CONCLUSION: Positive FCXMs were defined for maternal IgG and IgM against T and B lymphocytes, in terms of channel shift above the linear mean channel fluorescence of the negative control serum. By standardizing the dual-color FCXM methodology, the clinical significance of alloantibodies in the maintenance of pregnancy could be addressed in a collaborative manner. PMID- 7542451 TI - Actions of 3,5,3'-tri-iodothyronine on the synthesis and secretion of major plasma proteins by a human hepatoblastoma cell line (Hep G2). AB - We have elucidated the action of tri-iodothyronine (T3) on the synthesis and secretion of seven major plasma proteins in a human hepatoblastoma cell line, Hep G2, and established an in vitro experimental model of human liver cells for the study of the mechanism of the action of thyroid hormone. Hep G2 cells cultured in serum-free medium were treated with various concentrations of T3. During the first 24 h of T3 treatment, accumulation of alpha-fetoprotein in the medium was decreased in a dose-dependent manner (10(-11)-10(-8) M), and the inhibitory effect was enhanced during the second 24 h of T3 treatment. On the other hand, alpha 1-antitrypsin accumulation in the medium during the second 24 h of hormone treatment was decreased by T3 (10(-9)-10(-8) M), although no change in accumulation was observed during the first 24 h of T3 treatment. The newly synthesized [35S]Met-labelled alpha 1-acid glycoprotein was increased by T3 and reached 3.4-fold within 37 h of 10(-8) M T3 treatment. The stimulatory effect increased time-dependently (4.6-fold after 61 h). In contrast, the synthesis of alpha-fetoprotein was reduced to half of that of the control after T3 treatment for 37 h. Although the content of newly synthesized [35S]alpha 1-antitrypsin was not affected by 10(-8) M T3 treatment during 3 days of hormone treatment, the accumulation of alpha 1-antitrypsin in the medium decreased to 87%; in contrast, total cellular newly synthesized alpha 1-antitrypsin increased to 105-130% of that of the control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542454 TI - Immune status of newborns in perinatal chlamydial infection. AB - PROBLEM: Thirty newborns, delivered by mothers with urogenital chlamydiosis, diagnosed on the 35th-36th weeks of gestation, and 10 newborns of healthy mothers were studied immunologically. CD3 (T-lymphocytes), CD19 (B-lymphocytes), CD4 (T helpers-inductors), CD8 (T-suppressor-cytotoxic), T-lymphocyte response to PHA, levels of Ig G, A, M in umbilical blood were detected. METHOD: Chlamydia trachomatis antibody's titers in umbilical blood were determined by indirect immunofluorescent method (with antigen C. trachomatis, serovar L2). Antibody titers found in umbilical blood were in 50% of cases higher than antibody level in mother's blood serum, and they later varied from 1:64 to 1:256. It was established that newborns delivered by mothers with urogenital chlamydiosis had imbalance of T-lymphocyte subsets, decrease in the numbers of T- and B- lymphocytes, and increased level of Ig M, while the level of Ig G did not change significantly. The exposed disproportion in immune status neonatal, born from mothers with urogenital chlamydiosis, in combination with high C. trachomatis antibody titers in umbilical blood may be prognostic of the development of neonatal chlamydial infections. PMID- 7542456 TI - Lessons from our lives: breastfeeding in a personal context. PMID- 7542455 TI - Monoclonal IgM antiphosphatidylserine antibody reacts against cytoskeleton-like structures in cultured human umbilical cord endothelial cells. AB - PROBLEM: It has been proposed that antibodies against phospholipid-dependent antigens (aPLs), induce recurrent pregnancy loss and thrombosis through modulation of endothelial cell function, yet aPLs have not been conclusively shown to bind with endothelial cells. METHOD: Using indirect immunofluorescence we investigated the anti-endothelial cell reactivity of three monoclonal antibodies that differentiate between the phospholipids cardiolipin (CL) and phosphatidylserine (PS): BA3B5C4 (CL+/PS+); 3SB9b (CL-/PS+); and D11A4 (CL+/PS-). Cultured umbilical cord endothelial cells were prepared without fixation or with cold acetone fixation. RESULTS: None of the aPLs reacted with endothelial cells prepared without fixation. 3SB9B reacted strongly with cytoskeletal-like components in acetone-fixed cells, whereas BA3B5C4 and D11A4 were unreactive. The cytoskeletal-like binding of 3SB9b was completely blocked by a monoclonal antibody against vimentin, whereas antibodies against tubulin or actin were not inhibitory. Lipid extraction of the cells destroyed the 3SB9b reactive antigen without affecting the reactivity of anti-vimentin. CONCLUSION: These results suggest that phospholipid-dependent antigenic determinants are not expressed on the surface of resting endothelial cells but that a PS-dependent antigenic determinant is associated with endothelial cell intermediate filaments. PMID- 7542457 TI - Characterization of prokaryotic mRNAs by RT-PCR. PMID- 7542458 TI - Direct analysis of FACS-sorted hemopoietic cell fractions using FISH. PMID- 7542459 TI - Single, antigen-specific B cells used to generate Fab fragments using CD40 mediated amplification or direct PCR cloning. AB - A simple procedure for the generation of human antibody fragments directly from single B cells or B-cell clones is described here. The procedure is based on antigen-specific selection of single human B cells, using antigen-coated magnetic beads and a cellular amplification step based on a culture system involving both EL-4 thymoma cells and anti-CD40 antibodies, presented by CD32-expressing fibroblasts. Nested PCR was applied to rescue V-regions from both single B cells and B-cell clones obtained using the cellular amplification step. This amplification step both increased the cell number as well as activated the cells that amplified mRNA levels, thereby facilitating immortalization by cloning. The V-regions were cloned and expressed as Fab fragments and characterized by biosensor analysis. This approach allowed us to bypass cumbersome hybridoma technology and to obtain human antibody fragments that retained the original VH/VL pairing, a feature of importance when studying, e.g., the V-gene usage in various human diseases and in normal B-cell repertoires. PMID- 7542460 TI - Quantitation of PCR products with chemiluminescence. AB - Quantitative PCR and reverse transcription PCR (RT-PCR) are widely used in biomedical, industrial and other research applications to determine the number of RNA or DNA molecules of a specific type and/or sequence in a sample of interest. We have developed an assay system to accurately quantitate PCR products that utilizes solid-phase capture and an enzyme-linked chemiluminescent detection method. The entire assay is performed in a single tube or microplate well. Biotinylated PCR products are quantitated by capture onto a streptavidin-coated surface, followed by hybridization of an internal fluorescein-labeled oligonucleotide probe and subsequent detection with an anti-fluorescein-alkaline phosphatase conjugate and CSPD chemiluminescent substrate. Light signal is measured in a luminometer. The assay sensitivity enables accurate quantitation of target DNA because the measurement is performed on product generated during the exponential phase of amplification. The broad dynamic range of the assay, which is greater than three orders of magnitude of PCR product concentration, simplifies the determination of the number of amplification cycles necessary for accurate quantitation of target molecules. The PCR-Light system is an ultrasensitive, non-isotopic and rapid assay for PCR product detection that also has general application to solution hybridization assays and other quantitation methods. PMID- 7542461 TI - Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. AB - Sensory transduction in vertebrate photoreceptors and olfactory sensory neurons is mediated by cyclic nucleotide-gated (CNG) channels that conduct mono- and divalent cations. Ca2+ entering the cell through CNG channels intimately controls signaling pathways by regulating several key enzymes. Cloned CNG channels from photoreceptors and olfactory sensory neurons profoundly differ in their relative Ca2+ permeability, their blockage by external divalent cations, and the fraction of current carried by Ca2+. In particular, CNG channels from cone photoreceptors conduct significantly more Ca2+ than those from rod photoreceptors. Furthermore, the current through the olfactory CNG channel is entirely carried by Ca2+ at approximately 3 mM extracellular Ca2+. These results suggest that a major function of CNG channels is to provide a pathway for Ca2+ entry. PMID- 7542464 TI - Distance learning package for eye disease. PMID- 7542462 TI - Desensitized states prolong GABAA channel responses to brief agonist pulses. AB - We studied the role of desensitization at inhibitory synapses by comparing nonequilibrium GABAA channel gating with inhibitory postsynaptic currents (IPSCs). Currents activated by brief pulses of 1-10 mM GABA to outside-out patches from cultured hippocampal neurons mimicked GABA-mediated IPSCs. Although the average open time of single GABAA channels following brief pulses was less than 10 ms, channels entered long (tau = 38-69 ms) closed states and subsequently reopened. Movement through these states resulted in paired-pulse desensitization. The time required for deactivation after removal of agonist also increased in proportion to the extent of desensitization. These results suggest that visits to desensitized states buffer the channel in bound conformations and underlie the expression of long-lasting components of the IPSC. Reopening after GABAA receptor desensitization may thus enhance inhibitory synaptic transmission by prolonging the response to a brief synaptic GABA transient. PMID- 7542463 TI - The charybdotoxin family of K+ channel-blocking peptides. PMID- 7542465 TI - Culturally sensitive AIDS education and perceived AIDS risk knowledge: reaching the "know-it-all" teenager. AB - Video education is the most popular and effective medium for informing the adolescent population. This study investigated the impact of a culturally relevant HIV/AIDS video education. One hundred and ninety-four African-American teenagers were assigned to either a culturally sensitive or culturally dissimilar video education intervention. Results indicate that both interventions were effective in increasing AIDS knowledge scores. An interaction effect was found between levels of perceived AIDS risk knowledge and participation in the culturally sensitive intervention (CSV). Only the CSV intervention was effective with adolescents who claimed to "know a lot" about AIDS (e.g., "Know-It-All" subgroup). Students in both conditions who were worried about getting AIDS demonstrated higher AIDS risk knowledge at post-assessment. This study provides further evidence of within-ethnicity diversity among African-American youth and for developing culture- and subgroup-specific HIV/AIDS education. PMID- 7542466 TI - [Heterogeneity of antimicrosomal autoantibodies in chronic hepatitis C virus infection and delta hepatitis]. AB - Microsomal antigen autoantibodies are typical of type 2 autoimmune hepatitis, and a strong association with chronic hepatitis C virus (HCV) infection has been reported in certain geographical areas. These autoantibodies have been denominated LKM-1 to differentiate them from those associated with thienylic acid induced hepatitis (LKM-2) and from those seen in patients with chronic delta hepatitis (LKM-3). To investigate the antigenic specificity of autoantibodies associated with chronic hepatitis C and delta, we analyzed 52 LKM-1 positive serum samples from patients with chronic hepatitis C and 17 LKM-3 positive serum samples from patients with chronic delta hepatitis by indirect immunofluorescence and Western blotting (immunoblotting). Reactivity of subjects with chronic hepatitis C was heterogeneous: only 5 out of 52 LKM-1 positive patients, tested by Western blot, recognized a single protein of 50 kD, previously identified by Manns et al. with an immunogenic epitope of cytochrome P450IID6. Thirteen of the 52 patients also reacted with a 70 kD microsomal protein, and 12 out of 52 reacted only with a 59 kD protein. Twenty-two sera, notwithstanding the high titer in immunofluorescence, did not evidence any reactivity when tested by Western blot. The same sera tested positive in LKM-1 ELISA when solubilized human microsomal proteins were used. Fourteen out of 17 LKM-3 positive sera from patients with chronic hepatitis delta recognized a 55 kD microsomal protein in Western blot; three sera, HCV and HIV positive, did not react with any protein by Western blot. None of these sera was positive in ELISA LKM-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542467 TI - Dynamic and differential Oct-1 expression during early Xenopus embryogenesis: persistence of Oct-1 protein following down-regulation of the RNA. AB - As a first step towards the elucidation of the role of the transcription factor Oct-1 in development, we prepared a monoclonal antibody to study the spatio temporal distribution of Oct-1 protein in vivo. Here we report differential expression of the Oct-1 gene in the Xenopus embryo both at the RNA and the protein level. Transcripts and protein are detected in ectodermal and mesodermal cell lineages, in which the expression exhibits a pattern of progressive spatial restriction in the course of development. The Oct-1 expression as reported here is not correlated with cell density or cell proliferation in the embryo. Our results suggest a role of Oct-1 in the specification and differentiation of neuronal and neural crest cells. In many other cells, the developmental decision to down regulate Oct-1 is delayed, probably due to a high stability of the protein. PMID- 7542468 TI - Serological diagnosis of chronic viral hepatitis. AB - Chronic viral hepatitis must be distinguished from other chronic liver disorders. The clinical picture associated with typical biochemical findings provides no definite information about the causative agent. An important factor, both for the patient and for the individuals in contact with him, is the evaluation of his infectivity. From 172 chronic hepatitis disorders the serological diagnosis confirmed the viral etiology in 120 patients (69.7%). HBsAg was present in 33.7% (with HBeAg in 5%), anti-HCV antibodies 22.6% and markers for both viruses in 13.4% of cases. The HCV etiology was more frequently found in chronic hepatitis (44.9%) in liver cirrhosis (50.7%). The smaller number of patients displaying simultaneously anti-HBs and anti HCV antibodies did not confirm the recent suggestions that HCV would be the most important hepatotropic virus which enhances HBsAg clearance in chronic hepatitis. PMID- 7542469 TI - The distribution of nucleic acid chromophores at cellulose-water interfaces. 2. Thermodynamic parameters. AB - A Previous theoretical' model (O. L. Horer, Adriana Grigorescu Rev. roum. Virol, 1992, 43, 33) allowed us to appraise the "absorbed" mole fraction chi of chromophores, using the Multiple Light Scattering Enhanced Absorbance (MLSEA) bichromatic photometry. In the present paper the data were used to express thermodynamic parameters, based on the Gibbs free enthalpy G. In terms of the ion exchange capacity of several chromatography papers used in the former paper, the RNA chromophore distribution at the interface was correlated with thermodynamic parameter, showing characteristic patterns. The thermally denatured DNA was studied in the same way and a new insight of the helix-coil transition in terms of ionic strength was achieved. PMID- 7542470 TI - Intraluminal brachytherapy using the high dose rate microSelectron in the palliation of carcinoma of the oesophagus. AB - A total of 197 patients, who presented to the Christie Hospital with advanced carcinoma of the oesophagus, were treated with the high dose rate microSelectron between June 1988 and June 1992. In 54%, a single intraluminal brachytherapy treatment resulted in useful palliation, which was sustained for a substantial part of the patient's remaining life. The simplicity of the treatment, which could be completed as a day case procedure and did not cause significant morbidity, commends itself in the palliation of these patients who have poor overall survival and quality of life. Approaches that might improve the response rate in those patients who did not gain significant palliation after a single treatment are discussed. PMID- 7542471 TI - Combined chemotherapy and radiotherapy in diffuse large cell immunoblastic lymphoma: a phase II study of CHOP/bleomycin/methotrexate alternating with ifosfamide/methotrexate/etoposide. AB - The clinical outcome of 23 patients with high grade diffuse large cell immunoblastic lymphoma (Working Formulation, category H) treated by an intensive shortened schedule regimen of chemotherapy is described. Alternating cycles of cyclophosphamide, doxorubicin, vincristine, bleomycin and prednisolone, and ifosfamide, etoposide and methotrexate were given over an 18-week (range 16.0 20.8) period. External beam radiotherapy was administered as consolidation therapy to sites of original bulky disease in 17 patients. Treatment was well tolerated, though there were two toxic deaths. A 90% response rate was obtained. Sixteen of 18 patients followed for a minimum of 36 months are alive and in complete remission, representing a disease free survival of 69.5%; two further patients are alive following autologous bone marrow transplant. The 3-year disease free survival was 73% (+/- 9%) and the overall 3-year survival 78% (+/- 9%). PMID- 7542472 TI - The Barthel Index in assessing the response to palliative radiotherapy in malignant spinal cord compression: a prospective audit. AB - A prospective study of functional assessment of response to palliative radiotherapy (RT) in malignant spinal cord compression (SCC) is reported. The relevant components of the Barthel Activities of Daily Living Index (ADLI: walking, transfer, and bladder and bowel control) were used to assess functional capacity, and compared with a standard neurological assessment. Fifty-one consecutive patients (38 men, 13 women; mean age 63.9 years) were assessed, all of whom were treated by primary or postoperative radiotherapy. Five of the 51 patients had had decompressive laminectomy prior to RT. Median survival was 34 days (range 2-570). Walking improved in only five of 12 patients who had an improvement in motor power. The ability to transfer from bed to chair improved in 11 patients. Ambulation at presentation was the single most important determinant of outcome. Of the eight patients ambulatory at presentation, all were alive at 1 month and seven remained ambulatory. Of the 43 non-ambulatory patients, only four were able to walk following treatment (all prostatic carcinomas). Only four patients regained urinary continence, and none regained bowel control after this was lost. Functional assessment using ADLI and standard neurological examination provide a more useful guide to outcome in malignant SCC than neurological examination alone. PMID- 7542473 TI - Alternating combination chemotherapy and interferon improves survival in poor prognosis multiple myeloma. AB - To assess the efficiency and toxicity of alternating combination chemotherapy plus interferon-alpha-2b (IFN) in the treatment of poor prognosis multiple myeloma, we began a prospective clinical trial. The study involved 103 previously untreated patients with poor prognosis: Stage III, haemoglobin below 8.5 g/dl, beta 2-microglobulin > 5.0 micrograms/ml and multiple lytic lesions. All patients were treated with an alternating combined regimen given monthly for 2 years. After randomization, 52 patients also received IFN at a dosage of 5.0 MU three times weekly during the first year of the therapy. The remaining 51 patients received chemotherapy alone. Compared with patients treated with chemotherapy alone, those treated with chemotherapy plus IFN had a higher overall rate of response: (80% versus 47%), a longer duration of remission (36 months versus 18.5 months) and a higher rate of survival at 5 years (74% versus 39%; P < 0.001). Toxicity was similar in both arms. All patients received the planned dose of IFN. There were no deaths related to treatment. The addition of IFN to a regimen of alternating chemotherapy increased the rate of response, duration of remission and survival in patients with poor prognosis multiple myeloma, without serious side effects. PMID- 7542474 TI - Quantification and visualization of molecular surface flexibility. AB - Two new methods for the quantification and visualization of the flexibility of molecular surfaces are presented. Both methods rely on results of molecular dynamics (MD) simulations. Whereas method I is based on a simple but fast grid counting algorithm, method II uses a mapping function that allows for a sharp and clear visualization of atomic RMS fluctuations on a molecular surface. To demonstrate the scope of the methods, MD simulations of two proteins, PTI and ubiquitin, were performed. The flexibility data are mapped onto the molecular surfaces of the proteins and visualized using texture mapping technology available on modern workstations. PMID- 7542475 TI - Residence times of the buried water molecules in bovine pancreatic trypsin inhibitor and its G36S mutant. AB - The three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) contains 4 internal water molecules, denoted W111, W112, W113, and W122, the latter being replaced by a seryl side chain in the BPTI(G36S) analogue. To investigate the effect of the exchange between these explicit water sites and the bulk solvent, we have measured water 17O and 2H nuclear magnetic relaxation in solutions of BPTI and the G36S mutant over the Larmor frequency range 2.6-49 MHz. A comparison of the data from the two nuclei shows unequivocally that the isolated buried water molecule, W122, of BPTI contributes only to 2H, but not to 17O relaxation, while the other 3 waters contribute fully to the relaxation of both nuclei. This demonstrates that the residence time of W122 is in the range 10 200 microseconds, while the residence times of W111-W113 are in the range 15 ns-1 microseconds. The slower exchange of W122 indicates that the functionally active region of BPTI, near the Cys14-Cys38 disulfide bond, is less flexible than the central region of BPTI, where the other 3 buried waters are located. PMID- 7542476 TI - A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation. AB - Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl 1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3 dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl 1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542478 TI - Formation of membrane channels by chrysospermins, new peptaibol antibiotics. AB - The four homologous chrysospermins (Ia-d) are new 19 amino acid peptaibols which form cation selective ion channels in artificial lipid bilayer membranes. Conductance of channels formed by chrysospermins B (Ib) and D (Id) was twice as high (640 pS in 100 mM KCl) as found with chrysospermins A (Ia) and C (Ic). Single channel current traces were recorded for each of the four peptides even at very low (even zero) membrane voltages suggesting that non-gated channels are formed. PMID- 7542479 TI - Determination of ternary mixtures of antibiotics, by ratio-spectra zero-crossing first- and third-derivative spectrophotometry. AB - The ratio-spectra zero-crossing first- and third-derivative spectrophotometry have been used for determining ternary mixtures of penicillin-G sodium, penicillin-G procain and dihydrostreptomycin sulphate salts. The procedures are accurate, nondestructive and do not require resolutions of equations. In both methods, calibration graphs are linear, with zero-intercept, up to 30 micrograms ml-1 of penicillin-G sodium and penicillin-G procain, and up to 42 micrograms ml 1 of dihydrostreptomycin sulphate. r = 0.9999 in each instance. Working wavelengths, 218.5, 211 and 236 nm, respectively, in the first-derivative mode, and 222.5, 311.5 and 242 nm in the third-derivative mode. Detection limits for each drug at p = 0.01 level of significance were calculated to be 0.058, 0.010 and 0.014 micrograms ml-1 and 0.14, 0.012 and 0.34 micrograms ml-1, in the first- and third-derivative methods, respectively. Both methods apply favorably to either laboratory mixtures or commercial injections. PMID- 7542477 TI - Stimulation by alkylxanthines of chloride efflux in CFPAC-1 cells does not involve A1 adenosine receptors. AB - A series of 8-substituted derivatives of 1,3,7-alkylxanthines was synthesized as potential activators of chloride efflux from a human epithelial cell line (CFPAC) expressing the cystic fibrosis transmembrane regulator (CFTR) delta F508 mutation. Their interactions with rat brain A1 and A2a receptors were also studied in radioligand binding experiments. Substitution was varied at the xanthine 1-, 3-, 7- and 8-positions. 1,3-Dipropyl-8-cyclopentylxanthine (CPX) stimulated Cl- efflux in the 10(-8) M range, with a maximal effect reaching 200% of control and diminishing at higher concentrations. The potent adenosine antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3 dipropylxanthine, nonselective at human A1 and A2a receptors, was inactive in Cl- efflux. 1,3-Diallyl-8-cyclohexylxanthine (DAX) was highly efficacious in stimulating chloride efflux with levels reaching > 300% of control, although micromolar concentrations were required. 1,3,7-Trimethyl-8-(3 chlorostyryl)xanthine, an A2a-selective adenosine antagonist, was only weakly active. Caffeine, which acts as an nonselective adenosine antagonist in the range of 10(-5) M, was active in Cl- efflux in the low nanomolar range but with low efficacy. Thus, among the xanthine derivatives of diverse structure, there was no correlation between potency in Cl- efflux and adenosine antagonism. Poly(A)+ RNA isolated from CFPAC-1 cells showed no hybridization to a human A1 receptor cDNA probe, excluding this receptor as a mediator of CPX-elicited Cl- efflux. Thus, this action of xanthines in stimulating Cl- efflux in CFPAC cells, which express a defective CFTR, represents a novel site of action apparently unrelated to adenosine receptors. PMID- 7542481 TI - Comparison of histochemical autometallography (Danscher's stain) to chemical analysis for detection of selenium in tissues. PMID- 7542482 TI - [The natural history of HCV infection: the role of interferon therapy]. PMID- 7542480 TI - Evaluation of laboratory tests for confirming the diagnosis of encephalitic listeriosis in ruminants. AB - Retrospective analysis of 93 bovine, ovine, and caprine cases diagnosed as listerial encephalitis revealed positive bacterial isolations in only 63% of 59 cases in which bacterial culture was attempted. Only 42% of 41 attempted bovine brain cultures were successful, compared with 67% from 6 sheep brains and 92% from 12 goat brains. Gram stains and Listeria-specific immunohistochemistry were evaluated as tools for verifying the presence of bacteria or listerial antigens in 38 animals. Sixteen of 17 animals in this group with positive bacterial isolations were immunochemically positive for listerial antigens (including 5/6 cattle), but Gram stains detected only 9/17 positive animals (including 1/6 cattle). Antigen was also detected in 15 of 21 animals (including 5/9 cattle) with unsuccessful or unattempted bacterial isolations. Of all 38 animals, the histologic diagnosis could be verified in 82% by immunohistochemistry, compared to 47% verified by Gram stains. Immunohistochemical testing was especially beneficial in locating antigen in lesions with few bacteria or bacterial antigens and is a rapid method of confirming the diagnosis of encephalitic listeriosis where inappropriate material is submitted for bacterial isolation or in culture negative cases. PMID- 7542483 TI - [The cost-benefit of interferon therapy in chronic viral liver diseases]. PMID- 7542484 TI - [Chronic viral hepatitis: when interferon does not work]. PMID- 7542486 TI - [The goals of therapy]. PMID- 7542485 TI - [The role of liver biopsy in chronic viral hepatitis]. PMID- 7542488 TI - [To treat or not to treat: the indications and contraindications for the use of interferon in chronic viral hepatitis]. PMID- 7542487 TI - [The drug of first choice: interferon. Clinical pharmacological principles]. PMID- 7542490 TI - Role of macrophages and colony-stimulating factor-1 in murine antiglomerular basement membrane glomerulonephritis. AB - Macrophages have been shown to mediate glomerular injury in antiglomerular basement membrane (anti-GBM) glomerulonephritis in rats and rabbits. To evaluate the role of macrophages and the macrophage-related cytokines, colony stimulating factor-1 (CSF-1), monocyte chemoattractant protein-1 (MCP-1) and RANTES, accelerated anti-GBM nephritis was studied in op/op mutant mice, which lack CSF-1 and are severely macrophage deficient, and in heterozygous op/+ control mice. Observations were made 24 h and 3 days after the injection of rabbit anti-mouse GBM antibody in mice preimmunized with rabbit immunoglobulin G. Proteinuria rose progressively in both groups but did not differ between them (urine protein/creatinine ratio at 3 days: 1.01 +/- 0.38 in op/op versus 1.45 +/- 0.43 in op/+; P, not significant). In both op/op and op/+ mice, anti-GBM nephritis was associated with renal expression of mRNA for RANTES and MCP-1 and barely detectable levels of mRNA for CSF-1. In contrast, these cytokines were not expressed in sham-injected mice. Morphologic lesions appeared earlier in op/op mice but were comparable by Day 3. Glomerular injury consisted of capillary thrombosis and endothelial cell damage associated with mild to moderate leukocyte infiltration. Despite enhanced expression of mRNA for RANTES and MCP-1, glomerular macrophage infiltration was not increased in op/+ mice. It was concluded that, in mice, in contrast to rats and rabbits, accelerated anti-GBM nephritis may develop in the absence of both CSF-1 and macrophage infiltration. PMID- 7542489 TI - Cerebrospinal fluid levels of kynurenine pathway metabolites in patients with eating disorders: relation to clinical and biochemical variable. AB - In brain, most L-tryptophan is metabolized to indoleamines, whereas in systemic tissues L-tryptophan is catabolized to kynurenine pathway metabolites. Among these latter compounds are: quinolinic acid, an N-methyl-D-aspartate receptor agonist; kynurenic acid, an antagonist of excitatory amino acid receptors that also reduces quinolinic acid-mediated neurotoxicity; and L-kynurenine, a possible convulsant. Because the metabolism of L-tryptophan through the kynurenine pathway is dependent upon adequate nutrition, we sought to determine whether the impaired nutrition characteristic of eating-disordered patients might be associated with specific disturbances in this metabolic pathway. Cerebrospinal fluid levels of L tryptophan, quinolinic acid, kynurenic acid, L-kynurenine, and 5 hydroxyindoleacetic acid were measured in medication-free female patients meeting DSM-III-R criteria for either anorexia nervosa (n = 10) or normal-weight bulimia nervosa (n = 22), studied at varying stages of nutritional recovery. Eight healthy, normal-weight females served as a comparison group. Cerebrospinal fluid levels of kynurenic acid were significantly reduced in underweight anorectics, compared to normal females, but returned to normal values with restoration of normal body weight. Although cerebrospinal fluid quinolinic acid levels were not different from controls, the ratio of quinolinic acid to kynurenic acid was significantly increased during the underweight phase of anorexia nervosa. Furthermore, in the eating-disordered patients, kynurenic acid levels in cerebrospinal fluid correlated positively with percent-of-population average body weight.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542491 TI - Renal sodium handling with cyclosporin A and FK506 after orthotopic liver transplantation. AB - Hypertension is common after orthotopic liver transplantation and may be due, in part, to cyclosporin A-induced renal dysfunction and/or enhanced proximal tubular sodium reabsorption. To determine whether enhanced proximal tubular sodium reabsorption is central to the development of posttransplant hypertension, measurements of renal hemodynamics and fractional clearances of lithium and sodium were compared 1 month after orthotopic liver transplantation in previously normotensive patients receiving either cyclosporin A (N = 24) or FK506 (N = 18), an immunosuppressive agent that is structurally unlike cyclosporin A and that has a lower reported incidence of hypertension. Median prednisone doses were 20 and 13 mg/day in the cyclosporin A and FK506 groups, respectively (P < 0.05). At 1 month, mean arterial blood pressure was higher in the cyclosporin A versus the FK506 group: 108 +/- 2 versus 95 +/- 3 mm Hg (P < 0.05). GFR, RBF, and renal vascular resistance were not different between the two groups: 59 +/- 4 and 53 +/ 5 mL/min per 1.73 m2, 439 +/- 28 and 440 +/- 41 mL/min per 1.73 m2, and 22,429 +/- 1,822 and 22,977 +/- 3,506 dyne s/cm5 per 1.73 m2, respectively. Fractional lithium excretion was similar in the cyclosporin A and FK506 groups: 19.9 +/- 2.2 and 19.4 +/- 2.0% (P = not significant) although both values were lower than those of normal controls (25.5 +/- 1.1%) (P < 0.05). Fractional sodium excretion was 2.7 +/- 0.3 and 2.3 +/- 0.4% in the cyclosporin A and FK506 groups, respectively (P = not significant).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542492 TI - Endoscopic palliation of oesophageal carcinoma with Atkinson prosthesis. AB - To assess the degree of palliation, the associated morbidity and mortality and to compare our results with other published series, we reviewed our use of the Atkinson prosthesis in 100 consecutive patients for the palliation of unresectable oesophageal carcinoma. The group had a mean age of 71.2 +/- 2.3 years. All prostheses were placed by the pulsion method. Intubation was successful in 91%. Improvement in swallowing was seen in 82.1%. Major early procedure-related morbidity was high at 23% with 11 perforations (11%). Procedure related mortality was 12%. Those aged 70 years or more had a 34.5% risk of morbidity and 15.5% risk of dying from the procedure. Late procedure-related complications requiring further endoscopic procedures occurred in 27%. Our 7 day mortality was 14.7% (14 patients) and 31 patients (32.6%) had died within 30 days, usually from the disease itself. Those surviving the procedure (> 7 days) had a mean survival of 105 (range 9-735) days. We obtained acceptable palliation but with a significant morbidity and mortality. Endoscopic intubation remains a useful palliative treatment for patients with unresectable carcinoma of the oesophagus and cardia. PMID- 7542493 TI - A clinical study on pseudomyxoma peritonei. AB - To elucidate the clinical entity of pseudomyxoma peritonei, nine patients (male: female = 6:3) who had been treated in Chang Gung Memorial Hospital in the past 13 years were reviewed. The male patients with original appendiceal tumour were older than the male patients with original colon cancer or indefinite tumour (70, 67 and 67 years vs 42, 27 and 50 years). In addition, the former group survived in a disease-free status for 28 months on average, while the latter group died within 2 years. Echogenic ascites and diffuse low-attenuation intra-abdominal masses with scalloping on the surface of liver detected by ultrasonography and by computerized tomography, respectively, were found in most of the patients. Elevation of the serum carcinoembryonic antigen (CEA) during recurrence of the disease was also noted. This series suggested that: (i) the pre-operative diagnosis could be made with careful physical examination in conjunction with sonography or computerized tomography; (ii) the prognosis was better in patients with tumour of appendiceal origin; and (iii) serum CEA might be valuable for early detection of recurrence. PMID- 7542494 TI - [Stimulation of the proliferative activity of human natural killers (CD16+CD56+ cells) by recombinant interleukin-3 in vitro]. PMID- 7542495 TI - Blood cell dynamics in P-selectin-deficient mice. AB - P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin-deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury. PMID- 7542496 TI - In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti disialoganglioside/anti-Fc gamma RI bispecific antibody. AB - Neutrophils isolated from cancer patients treated with granulocyte colony stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo. PMID- 7542497 TI - Cytogenetically aberrant cells in the stem cell compartment (CD34+lin-) in acute myeloid leukemia. AB - Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs. PMID- 7542499 TI - Primitive human hematopoietic precursors express Bcl-x but not Bcl-2. AB - Bcl-2 and its homologue, bcl-xL, encode membrane-associated proteins that suppress programmed cell death of hematopoietic cell lines after growth factor withdrawal, and are expressed in hematopoietic precursor cells. To better understand the maintenance of long-term survival in the hematopoietic stem cell population, we evaluated the expression patterns of Bcl-2 and Bcl-x in primitive hematopoietic precursor populations. Hematopoietic precursor cells expressing CD34 (CD34+) and lacking maturation-linked surface antigens (lin-) were isolated from adult human bone marrow using two-color immunofluorescence cell sorting and fractionated on the basis of forward light scatter characteristics into blast sized and small to medium lymphocyte-sized cell populations. Bcl-2 expression was shown in 78% to 90% of CD34+ lin- blast-sized cells versus less than 10% of small to medium lymphocyte-sized CD34+ lin- cells by immunohistochemical analysis. Small to medium lymphocyte-sized CD34+ lin- cells were further enriched for primitive precursors by selecting cells that lacked expression of CD38 (CD34+ lin CD38-). In parallel experiments, only 1% to 4% of CD34+ lin- CD38- cells expressed Bcl-2, whereas 45% to 56% of these cells generated colony-forming cells. In contrast, > or = 94% of cells in all bone marrow subpopulations studied expressed Bcl-x protein. Both alternatively spliced bcl-x transcripts, bcl-xL and bcl-xs, were present. Our data show that the most primitive hematopoietic precursors express Bcl-x but not Bcl-2. Thus, the functional bcl-2 homologue, bcl xL, may be essential for the long-term survival of the hematopoietic stem cell population. PMID- 7542498 TI - Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages. AB - Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low. PMID- 7542500 TI - Increased basal and induced tyrosine phosphorylation of the insulin-like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera. AB - We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum-free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV. PMID- 7542501 TI - Functional expression of Fas antigen (CD95) on hematopoietic progenitor cells. AB - We investigated the expression of an apoptosis-associated antigen (Fas) (CD95) on hematopoietic progenitor cells in the presence or absence of interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha). CD34+ cells freshly isolated from bone marrow did not express Fas. However, IFN-gamma and/or TNF alpha induced the expression of both the mRNA of Fas and Fas itself in a dose dependent fashion on the surface of CD34+ cells after 48 hours of serum-free culture. IFN-gamma and TNF-alpha had a synergistic effect on the induction of Fas, when both cytokines were added to the culture. The TNF-alpha-induced Fas expression is mediated by p55 TNF-alpha receptor. CD34+ cells cultured in medium alone or with stem cell factor (SCF) showed some slight expression of Fas. When anti-Fas antibody (IgM) was added to CD34+ cells after the induction of Fas expression, CD34+ cells underwent apoptosis, as shown by a decrease in the number of viable cells, morphologic changes, the induction of DNA fragmentation, and a decrease in the number of colony-forming cells (CFC) including colony-forming unit granulocytes/macrophages (CFU-GM) and burst-forming unit erythroids (BFU-E). These observations indicate that IFN-gamma and/or TNF-alpha, well known as negative hematopoietic regulators, induce functional Fas on hematopoietic progenitor cells. The suppression of hematopoiesis by negative hematopoietic regulators may be mediated in part by Fas induction. PMID- 7542502 TI - Recombinant human interleukin-6 induces a rapid and reversible anemia in cancer patients. AB - Initial studies have shown that recombinant human interleukin-6 (rhIL-6) induces anemia. Until now, the pathophysiologic mechanism of this induced anemia has been unknown. To unravel the underlying mechanism, we examined 15 cancer patients receiving rhIL-6 as an antitumor immunotherapy in a phase II study. rhIL-6 was administered subcutaneously at 150 micrograms once daily for 6 consecutive weeks. Various hematologic and biochemical parameters were measured weekly during rhIL-6 treatment and 4 weeks after rhIL-6 discontinuation. To determine plasma volume and red blood cell (RBC) volume, radioisotope dilution assays with labeled autologous RBCs and with human serum albumin were performed before rhIL-6 administration and on day 8 of rhIL-6 therapy. Hemoglobin levels decreased (mean change +/- SE) 7% +/- 1.5% within 3 days after the start of rhIL-6 therapy (P < .0001) and 19% +/- 2% at week 4. Levels had normalized at follow-up. The plasma volume increased 18% +/- 5% during the first week of rhIL-6 administration (P < .003), whereas RBC volume remained unaffected. The mean RBC corpuscular volume remained unchanged for 2 weeks and then began to decrease slowly, reaching its nadir at week 6 (5% +/- 1%; P < .01). Serum iron levels decreased 65% +/- 12% at week 4 (P < .002) and then returned to initial baseline values. Erythropoietin levels increased rapidly up to 68% at week 3 (P < .0001) and had normalized 4 weeks after rhIL-6 therapy. Levels of serum albumin, prealbumin, and transferrin decreased (P < .0001, P < .003, and P < .0001, respectively), whereas levels of serum amyloid A (P < .003), C-reactive protein, haptoglobin, and alpha-1 antitrypsin (P < .0001) increased during rhIL-6 treatment. All levels returned to pretreatment values after discontinuation of rhIL-6. No alterations in reticulocyte counts, serum lactic dehydrogenase levels, and bilirubin levels were observed. A 6-week regimen of subcutaneous rhIL-6 results in a rapid dilution anemia, caused by an acute and significant increase in plasma volume and followed by hypoferremia. This anemia is reversible after the cessation of rhIL-6 treatment. PMID- 7542503 TI - Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants. AB - A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)-dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation. PMID- 7542504 TI - Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alpha v beta 3) integrin. AB - Human fetal livers contain progenitor cells that become mast cells after 4 weeks of culture with recombinant human stem cell factor. Expression of cell surface CD29 (beta 1), CD18 (beta 2), CD61 (beta 3), and beta 5 integrins was investigated on such cells by flow cytometry and adhesion measurements. High surface expression of CD49e, CD51, and CD61 along with kit was apparent by 4 weeks of culture, whereas expression of each at day 0 was low to undetectable. CD29 and CD49d were detected on cells from day 0 to 4 weeks of culture; CD49b, CD49c, CD49f, CD18, and CD54 expression was negligible. The fetal liver-derived mast cells spontaneously adhered to vitronectin. No evidence for degranulation was found during vitronectin-dependent adhesion. Adhesion occurred in part through the CD61/CD51 receptor. No evidence for adhesion to vitronectin through CD29 and beta 5 integrins was obtained. Almost all of the vitronectin-adherent cells expressed CD51, CD61, kit, and tryptase, and exhibited metachromasia with toluidine blue. Thus, among the fetal liver-derived cells, developing mast cells were selectively adherent to vitronectin. These mast cells and the other cell types present also adhere spontaneously to fibronectin and to laminin, this adhesion being partially inhibited by antibodies against CD61 and CD29 integrins. In conclusion, human mast cells acquire functional vitronectin receptors as they develop from fetal liver progenitors under the influence of rhSCF. This may be important for the recruitment, localization, and retention of developing mast cells. PMID- 7542505 TI - Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst promoting activity. AB - Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF-beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4-, and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF-beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF-beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors. PMID- 7542506 TI - Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells. AB - Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony forming units granulocyte-monocyte (CFU-GM) and long-term culture-initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony-forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells. PMID- 7542507 TI - Maternal deprivation and early weaning modulate experimental allergic encephalomyelitis in the rat. AB - The present experiment deals with the effect of maternal deprivation (MD) and early weaning (EW) on the development and course of experimental allergic encephalomyelitis (EAE) in Dark August (DA) rats. Five litters (five to nine pups per liter) were subjected to MD (4 h daily) from Day 1 until Day 28. EW rats were weaned on Day 15 (EW-15, five litters) or Day 21 (EW-21, four litters). Control rats and MD rats were weaned on Day 28. At the age of 8 weeks, rats were immunized with guinea pig spinal cord in complete Freund's adjuvant and clinical signs of EAE were recorded daily. On Day 18 after immunization, rats were bled and sacrificed. Brain and spinal cord were examined histologically for EAE lesions. Serum anti-rat myelin basic protein (MBP) antibodies were detected by ELISA. MD female rats exhibited suppression of neurological and histological signs of EAE in comparison with control rats. MD and control females showed elevated anti-MBP antibody level compared to MD and control males. EW-15 female rats demonstrated potentiation of neurological signs of EAE compared to control females. EW-21 females developed more severe clinical signs and histological lesions compared to control females. These results show that neonatal experiences, such as maternal deprivation and early weaning, influence the development of EAE in adult DA rats. PMID- 7542509 TI - Effect of theophylline administered intratracheally as a dry powder formulation on bronchospasm and airway microvascular leakage in the anesthetized guinea-pig. AB - The effect of theophylline (a non-selective phosphodiesterase (PDE) inhibitor), dosed intratracheally (it) as a dry powder, on histamine- and platelet activating factor (Paf)-induced bronchospasm and antigen (ovalbumin, OA)-, histamine- and Paf-induced microvascular leakage (MVL) in the airways, was studied in the anaesthetized guinea-pig. Bronchospasm was measured as the increase in pulmonary inflation pressure (PIP). MVL was assessed by fluorometric assay of fluorescein isothiocyanate dextran (FITC-dextran) content in airway tissues and tracheobronchial lavage fluid. OA (200 micrograms), histamine (60 nmol) and Paf (4 nmol), all given it, significantly increased MVL by up to 350% over levels in undosed unchallenged animals. Theophylline (50-500 micrograms it, n = 5-6) inhibited histamine-induced bronchospasm (30% inhibitory dose, ID30: 258 +/- 30 micrograms), and Paf-induced bronchospasm (ID30: 190 +/- 80 micrograms). An inhibition of 40-50% of maximal bronchospasm was the largest attained. Theophylline, at approximately the bronchospasm ID30 dose (200 micrograms it, n = 4-8), inhibited MVL induced by all agents by 30-80% in airway tissues and in lavage fluid samples. Theophylline (50-500 micrograms it, n = 3) produced plasma drug levels of 0.13 +/- 0.07 to 0.83 +/- 0.39 microgram/ml 10 min after dosing. Plasma levels were the same 60 min after dosing, suggesting retention of theophylline in the airways. The local concentration of theophylline retained in the airways should be sufficient to inhibit PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542511 TI - CD44 in differentiated embryonic stem cells: surface expression and transcripts encoding multiple variants. AB - Expression of the surface-adhesion molecule CD44 was investigated during the in vitro differentiation of the embryonic stem (ES) cell line D3. By immunofluorescence analysis, totipotent, undifferentiated ES cells did not show surface expression of CD44, although two transcripts of approximately 1.6 and 3.3 kb were detected on Northern blots. Following 1 week of differentiation in either suspension or substrate-attached cultures, CD44 appeared on the surface of some D3 cells, and synthesis of an additional 4.5 kb mRNA species was detected on Northern blots. At this stage, at least three distinct transcripts encoding CD44 variants were induced within the cultures, resulting from alternative splicing of additional exons in the variable domains of CD44. From PCR analysis, they all appeared to contain the variable exon v10, and two of them in addition contained v6. Taken together, these results suggest that CD44 may play a role in cell migration and adhesion in the early development of the mouse embryo. PMID- 7542510 TI - Regulation of airway surface liquid on the isolated guinea-pig trachea. AB - Isolated guinea-pig tracheae exposed to conditioned air mucosally and to circulating saline solutions serosally have been subjected to an osmotic gradient between saline and airway surface and to a range of drugs. Experiments were conducted to examine the mechanisms that regulate airway surface liquid during condensation and evaporation. Measurements were made of the depth of airway surface liquid, its sodium activity, the transepithelial potential difference, and of net water and sodium transport across the airway. Results suggest that osmotic gradients have profound effects on airway surface liquid but drugs that influence secretory activity (methacholine, substance P) or ion transport activity (amiloride, bumetanide, oubain) do not. PMID- 7542508 TI - Palliative therapies in elderly cancer patients. AB - Palliative therapy in elderly cancer patients deserves special attention because of differences between young and elderly cancer patients. In elderly patients, the treatment produces more adverse effects and the disease is cured less frequently, but the prognosis is usually better than in young patients with cancer. In the elderly population, cancer is seen more frequently than in the young population, yet elderly patients are less frequently included in trials. It is obvious that the results obtained from the treatment of young cancer patients do not necessarily apply to elderly cancer patients. The existing lack of formal data for the management of elderly cancer patients justifies 5 distinctive steps: collection of individual information; review of the available and appropriate knowledge; definition and specification of treatment goals; selection of treatment tools; and assessment of outcome. Following these steps will lead to coordination of oncology and palliative medicine. PMID- 7542512 TI - Role of beta 2 integrins in the binding of thymocytes to rat thymic macrophages. AB - A role of beta 2 integrins and one of their ligands, ICAM-1, in thymic macrophage (TMF)/thymocyte interactions was studied. TMF were isolated as adherent cells from 4-day old culture of thymic-cell suspensions either from normal or hydrocortisone-treated rats. Adherent cells were 94-98% positive with ED1 (a pan macrophage marker). The majority of them (75-95%) expressed the CD11b and CD18 molecules, and 60-70% expressed CD54 (ICAM-1). A low proportion of TMF (10-20%) expressed CD11a (LFA-1). The expression of all these antigens was upregulated by IFN-gamma and TNF-alpha. The effect of these mAbs on TMF/thymocyte binding was studied using a simple rosette assay by incubating unstimulated or IFN-gamma or TNF-alpha stimulated TMF, grown on microscopic slides with resting or ConA+IL-2 activated thymocytes. It was found that LFA-1/CD18 and ICAM-1 play a significant role in the TMF/thymocyte adhesion. In addition, a LFA-1-dependent/ICAM-1 independent adhesion pathway was observed, suggesting that LFA-1 might use another ligand. The inhibitory effect of anti-CD18 mAb (WT-3) was higher than the effect of anti-LFA-1 mAb (WT-1) and was a consequence of blocking the CD18 chain both on thymocytes and TMF. No significant difference in the expression and function of adhesion molecules was found between TMF obtained from normal or hydrocortisone-treated rats. The involvement of CD11b in these processes was of lesser importance than the role of the CD11a molecule. By using mAbs to different epitopes of the CD11b molecule, such as OX-42 (anti-CD11b/CD11c), ED7, and ED8 (anti-CD11b), it was found that they were either slightly or moderately inhibitory under certain experimental conditions or did not significantly modulate TMF/thymocyte binding. OX-42 was slightly stimulatory in some experiments. Cumulatively, these results show that beta 2 integrins play a significant role in TMF/thymocyte interactions and probably contribute to T-cell development in vivo. PMID- 7542513 TI - Streptococcal glucan-binding lectins do not recognize methylated alpha-1,6 glucans. AB - The glucan-binding lectin (GBL) of Streptococcus sobrinus is cell associated, enabling the bacteria to be aggregated by alpha-1,6 glucans. Glucans, such as amylose, pullulan, laminarin and nigeran, have no affinity for the lectin. High molecular weight alpha-1,6 glucans (dextrans) readily aggregate the bacteria, whereas low molecular weight glucans inhibit the aggregation brought about by the high molecular weight species. Methylated glucan T-2000 (an alpha-1,6 glucan with an average molecular weight of 2 x 10(6) Da) aggregated the bacteria very poorly when the extent of methylation (DS, or degree of substitution) was high, and less poorly when the DS was low. Similarly, methylated low molecular weight alpha-1,6 glucan was a poor inhibitor of aggregation induced by the high molecular weight glucan T-2000. Because the methylation occurred primarily on the hydroxyl of C-2, it is suggested that the hydroxyl is needed for formation of the lectin-glucan complex. It appears that the GBL is not only stereospecific in interaction with glucans, but also regio-specific, interacting only with the underivatized alpha 1,6-glucan. PMID- 7542515 TI - Role of vascular cell adhesion molecule-1 and platelet-activating factor in selective eosinophil migration across vascular endothelial cells. AB - To determine the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in mediating selective eosinophil migration across vascular endothelial cells, we examined the effect of anti-ICAM-1 monoclonal antibody (mAb) and anti-VCAM-1 mAb on transendothelial migration of human eosinophils and neutrophils across IL-1- and IL-1-stimulated cultured vascular endothelial cells. We also studied the effect of a selective antagonist for platelet-activating factor (PAF) on the transendothelial migration of eosinophils and neutrophils. Transendothelial migration of eosinophils was increased by stimulation of the endothelial cells with IL-1 and IL-4, the latter of which has been shown to selectively induce VCAM-1 expression on the endothelial cells. On the other hand, neutrophil transendothelial migration was increased only by IL-1. Eosinophil migration across IL-4-stimulated endothelial cells was decreased by anti-ICAM-1 mAb and anti-VCAM-1 mAb. A selective PAF antagonist E6123 also decreased eosinophil migration across IL-4-stimulated endothelial cells. Eosinophil migration across IL-1-stimulated endothelial cells was also decreased by anti-ICAM-1 mAb and anti-VCAM-1 mAb in the presence of PAF antagonist. In contrast, neutrophil migration across IL-1-stimulated endothelial cells was decreased by anti-ICAM-1 mAb but not anti-VCAM-1 mAb. PAF antagonist alone or together with anti-VCAM-1 had no significant effect on IL-1-induced neutrophil transendothelial migration. These results indicate that VCAM-1 and PAF play an important role in mediating selective eosinophil migration across vascular endothelial cells. PMID- 7542516 TI - Immunocytochemical localization of the chemokines RANTES and MIP-1 alpha within human platelets and their release during storage. AB - The cytokines RANTES and MIP-1 alpha are 8-kD peptides which belong to the C-C subfamily of chemokines. They are both potent chemotactic factors for basophils and eosinophils. Apart from lymphocytes, the RANTES peptides was recently shown to be released from human platelets stimulated with thrombin [Kameyoshi et al: J Exp Med 1992;176:587-592]. Employing postembedding immunocytochemistry, we could detect RANTES and for the first time also MIP-1 alpha within the alpha-granules of human platelets. To date, MIP-1 alpha was not reported to occur in platelets. In slightly activated platelets, as found in stored platelet concentrates (PC), label for RANTES and MIP-1 alpha could also be observed within cisterns of the open canalicular system and on the plasma membrane, indicating a release of both peptides. These findings were confirmed by in vitro studies in PC, by investigation of RANTES and MIP-1 alpha release into the suspending medium. Over a period of 8 days, RANTES was steadily released in relatively high amounts, whereas MIP-1 alpha was measured in rather small amounts in the suspending medium. As RANTES and MIP-1 alpha, besides their chemotactic activity on eosinophils and basophils, are able to mediate the release of histamine, it is tempting to speculate about a participation of platelets in inflammatory reactions in which eosinophils and basophils are involved. PMID- 7542514 TI - The antiperinuclear factor and antikeratin antibody systems. AB - Antiperinuclear factor (APF) and antikeratin antibody (AKA) have long been known to be associated with rheumatoid arthritis. Human buccal mucosa epithelial cells have hitherto been required as the substrate in the APF test, while AKAs are detected on rat esophagus sections, using an indirect immunofluorescence technique. These two autoantibodies proved to be interrelated. Cytoplasmic inclusions in buccal cells have presumptively been termed keratohyalin granules and the APF target antigen colocalizes exactly with that of antiprofilaggrin antibody within the perinuclear organelles. The latter protein has convincingly been identified as the genuine specificity of the so-called AKA. PMID- 7542517 TI - Effect of oxatomide nasal spray on experimental allergic rhinitis in guinea pigs and rats. AB - We investigated the effect of topically applied oxatomide, an antiallergic agent, on the assault of allergic rhinitis in actively sensitized guinea pigs. Topical application of oxatomide nasal spray (0.025%) reduced the severity of allergic rhinitis which was assessed by determining dye leakage and histamine released to the nasal cavity of guinea pigs. Furthermore, oxatomide nasal spray treatment significantly prevented the increase in dye leakage induced by histamine administration in guinea pigs and rats. These results indicate that the topical application of oxatomide inhibits both the release and the action of histamine. Therefore, oxatomide nasal spray may be beneficial for treatment of allergic rhinitis. PMID- 7542518 TI - Relationship between histamine release and leukotrienes production from human basophils derived from atopic dermatitis donors. AB - Basophils are known to release histamine and to produce leukotrienes (LTs) following both IgE-dependent and -independent stimuli. Although there exist a few reports which examined the relationship between histamine release and LTs production, their conclusions were not always in agreement with each other. In the present study, we examined the relationship between histamine release and LTs production from basophils in the presence or absence of 1 ng/ml of interleukin-3 (IL-3). Normal basophils released a smaller amount of histamine and LTs than atopic determatitis (AD) basophils, when basophils were stimulated with an optimal concentration of anti-IgE antibody. When we examined the relationship of histamine release and LTs production from AD donors induced through Fc epsilon RI, we found a significant exponential correlation between these two mediators (R2 = 0.58 in the absence of IL-3, R2 = 0.83 in the presence of IL-3). Although IL-3 enhanced both histamine release and LTs production from AD donors, the relationship between these two mediators was not affected. In conclusion, there was an exponential correlation between histamine release and LTs production from AD basophils, which was not affected by the pretreatment with IL-3. PMID- 7542519 TI - [Doppler ultrasound of the endometrium during hormone replacement]. PMID- 7542520 TI - Pharmacology of mesoionic oxatriazole derivatives in blood, cardiovascular and respiratory systems. AB - Mesoionic oxatriazole derivatives were synthetized by GEA LTD1. The GEA compounds (GEAC) constitute a new class of NO-donors, some of which stimulate selectively guanylate cyclase abiding either platelets or leukocytes or lung tissues. In consequence, some of GEAC are potent anti-platelet, fibrinolytic, thrombolytic or broncholytic agents, both in vitro and in vivo. GEAC synergize with prostacyclin in their thrombolytic actions. They also suppress the release of histamine and leukotriene B4, and prevent degranulation of granulocytes. Methylene blue reduces, and zaprinast augments their pharmacological effects. It is suggested that within a series of the newly synthetized GEA compounds there are likely to be found potential candidates for treating either thrombotic or asthmatic disorders. PMID- 7542521 TI - Relationship between different isoforms of nitric oxide synthase and cyclooxygenase in various cell types. AB - Nitric oxide (NO) and prostacyclin (PGI2), formed by NO synthase (NOS) and cyclooxygenase (COX), respectively, are two potent anti-aggregatory vasodilators released from endothelial cells. Both NOS and COX exist as constitutive and inducible isoforms. We have shown that NOS and COX are co-induced in vitro and in vivo by bacterial endotoxin and that low amounts of NO increase whereas high amounts inhibit the activity and expression of inducible COX in vitro. PMID- 7542522 TI - Role of nitric oxide in myocardial reactive hyperemia in a dog. AB - Reactive hyperemia (RH) was produced after short-term (5-30 s) cessation of blood flow in experiments on anesthetized dogs following catheterization of the circumflex branch of the left coronary artery (with the chest intact) autoperfused with blood from the subclavian artery. The increase of the coronary blood flow observed after occlusion was shown to depend strictly on its duration. Deendothelization of coronary vessels decreased the RH significantly. In coronary bed the infusion of L-arginine was accompanied by an increase of RH and endothelium-dependent acetylcholine-induced vasodilation. L-arginine produced its effect only when the endothelium was intact, whereas NO synthase inhibition substantially decreased the RH and vasodilating response to intracoronary administration of acetylcholine. It was concluded that the RH was endothelium dependent and that nitric oxide of endothelial origin was the active humoral component of this reaction. PMID- 7542524 TI - Purification of recombinant protein derived from the baculovirus expression system using glutathione affinity agarose. PMID- 7542523 TI - Molecular approaches to AIDS vaccine development using baculovirus expression vectors. PMID- 7542525 TI - Expression and secretion of a soluble form of myelin-associated glycoprotein (MAG). PMID- 7542526 TI - Developmental disorders. PMID- 7542527 TI - Cerebellar influence on olivary excitability in the cat. AB - This study examines the influence of the cerebellum on the excitability of inferior olivary neurons in the cat. Two major pathways from the cerebellar nuclei to the inferior olive have been investigated by electrophysiological and anatomical techniques. The first, excitatory pathway connects the cerebellar nuclei through nuclei at the mesodiencephalic junction with the inferior olive. The second is the direct, GABAergic, nucleo-olivary pathway. Intra- as well as extracellular recordings obtained in the rostral part of the medial accessory and principal olives revealed that electrical stimulation with a short burst of three pulses delivered at the mesodiencephalic junction results in short-latency activation (4-8 ms) of most olivary neurons. More than half of the units showed, in addition to the short-latency activation, a consistent response with a much longer latency (approximately 180 ms). Many units (66%) that responded to mesodiencephalic stimulation could also be activated by superior cerebellar peduncle stimulation with a similar stimulation paradigm (latency 9-15 ms). However, in such cases consistent long-latency responses were only rarely recorded (7%). To distinguish between the effect of the two pathways, both of which are activated by superior cerebellar peduncle stimulation, an electrolytic lesion of the nucleo-olivary fibres was made in the brainstem in six experiments. The effect of this lesion was verified in three cases by retrograde horseradish peroxidase tracing from the rostral inferior olive at the end of the experiment. This time only extracellular recordings were made. Stimulation of the mesodiencephalic junction still resulted in easily activated olivary units which showed an increased probability of firing a long-latency action potential. Stimulation of the superior cerebellar peduncle now resulted in a 50% decrease in probability of activating olivary units in the short-latency range. However, a five-fold increase in the chance of triggering action potentials in the long latency interval was noted, implying that many units reacted only with a long latency action potential. The results obtained with our experimental paradigm appear enigmatic since it is well established that the nucleo-olivary pathway is GABAergic and thus, by convention, should be inhibitory to the olivary neurons. However, it is possible to explain these results in terms of dynamic coupling of olivary neurons. This concept ascribes an important role to the nucleo-olivary pathway in regulating the degree of electrotonic coupling between olivary neurons (probably by a shunting mechanism) and as such may be an important instrument in the regulation of synchronous and rhythmic olivary discharges.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542528 TI - Age-related changes in the NMDA receptor/nitric oxide/cGMP pathway in the hippocampus and cerebellum of freely moving rats subjected to transcerebral microdialysis. AB - The N-methyl-D-aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3-month-old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S-nitroso-N-penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12-24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA-evoked cGMP response seen in the aged hippocampus were not compensated for by supplying L-arginine. Infusion of D-serine (1 mM) enhanced (150-200%) extracellular cGMP in the cerebellum with no age-related differences. The activity in vitro of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age-related decay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542529 TI - Up-regulation of a chondroitin sulphate epitope during regeneration of mouse sciatic nerve: evidence that the immunoreactive molecules are related to the chondroitin sulphate proteoglycans decorin and versican. AB - After transection of adult mouse sciatic nerve, the expression of a chondroitin sulphate epitope recognized by the monoclonal antibody 473-HD (mAb 473-HD) was found to be up-regulated. The epitope was localized immunocytochemically mainly in Schwann cell basal laminae and, more weakly, also in the endoneurium. In cultures of mouse dorsal root ganglion cells, Schwann cells expressed high levels but fibroblasts only low levels of the epitope. To identify the molecule(s) carrying this chondroitin sulphate epitope, human sciatic nerves were extracted with phosphate-buffered saline and shown to contain two chondroitin sulphate proteoglycans of apparent molecular weights of 130 and 900 kDa. The 900 kDa and, more weakly, the 130 kDa proteoglycan were reactive with mAb 473-HD, which was found to recognize chondroitin-6-sulphate as epitope. Following chondroitinase ABC treatment of the 130 kDa proteoglycan, a core protein of approximately 45 kDa was seen and shown to react with polyclonal antibodies against the chondroitin dermatan sulphate proteoglycan decorin from human fibroblasts. Chondroitinase ABC treatment of the 900 kDa proteoglycan yielded a core protein with a molecular weight of approximately 400 kDa that was recognized by polyclonal antibodies against recombinantly expressed fusion proteins from human versican. After transection of adult mouse sciatic nerves, the distal nerve stumps showed up regulation of the chondroitin-6-sulphate epitope of the 900 kDa proteoglycan, whereas the core protein of this proteoglycan did not show any detectable change in the level of expression. In contrast, the core protein of the 130 kDa proteoglycan was up-regulated in expression. These observations suggest that versican- and decorin-like molecules may contribute to successful regeneration in the peripheral nervous system of mammals. PMID- 7542530 TI - Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils. AB - A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils. PMID- 7542531 TI - Regulation of prostaglandin production by nitric oxide; an in vivo analysis. AB - 1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6 keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively. 3. The intravenous infusion of the NO donors sodium nitroprusside (SNP) or glyceryl trinitrate (GTN)increased prostaglandin production in normal animals (for instance urinary PGE2 excretion was increased from 96 +/- 10 to 576 +/- 12 pg min 1 and 400 +/- 24 pg min-1 in the presence of GTN or SNP respectively).4. Proteinuria was measured in order to evaluate the roles of NO and PG in renal damage associated with the in vivo injection of LPS. Interestingly, dexamethasone and the NOS inhibitors attenuated proteinuria in the LPS-treated rats. The COX inhibitors had no effect. It therefore appears that NO and not PG contributes to the LPS-induced renal damage; these findings support the potential use of NOS inhibitors in the treatment of renal inflammation.5. This study demonstrates the regulatory contribution of NO on the in vivo production of prostanoids and suggests that in inflammatory diseases that are driven by both NO and the prostaglandins, NOS inhibitors may act to reduce inflammation by the dual inhibition of cytotoxic NO and pro-inflammatory PG. PMID- 7542532 TI - Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat. AB - 1. We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock. 2. Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite caused by LPS (1 microgram ml-1). In contrast, pretreatment of macrophages with tetraethylammonium (10(-4) to 10(-2) M for 30 min), a non-selective inhibitor of potassium channels, did not affect the rise in nitrite caused by LPS. At the highest concentration (10(-5) M) used, cromakalim, an opener of ATP-sensitive potassium channels, caused a small, but significant inhibition of nitrite formation in macrophages activated with LPS, while lower concentrations (10(-7) to 3 x 10(-6) M) were without effect. 3. The inhibition by glibenclamide (3 microM) of the increase in nitrite induced by LPS in J774.2 macrophages was weaker when glibenclamide was given several hours after LPS, indicating that glibenclamide inhibits the induction, but not the activity, of iNOS. In contrast, the degree of inhibition of nitrite formation caused by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L NAME) was similar when this agent was given up to 10 h after LPS. 4.In anaesthetized rats, LPS caused a fall in mean arterial blood pressure (MAP) from 120 +/-(time 0)to 98 +/- mmHg at 180 min (P<0.05, n = 6). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v.at 60 min after LPS) caused a rapid and sustained rise in MAP (e.g. MAP at 180 min after LPS:122 +/-4 mmHg; n =6, P <0.05 when compared to LPS-rats). The maximum of the rise in MAP produced by glibenclamide (1 mg kg-1 , i.v.) was similar when the drug was given either at 60 or 180 min after LPS. However, the duration of the pressor response was significantly longer when glibenclamide was given at 60 min, rather than at 180 min after LPS.5. LPS-treatment caused a significant reduction of the pressor responses elicited by noradrenaline (NA,1 microg kg-1, i.v.) from 35 +/- 2 to 19 +/- 1 mmHg at 60 min and 20 +/- 2 mmHg at 180 min (P<0.05).Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 60 min) caused a significant restoration of the pressor responses elicited by NA from 19 +/- 1 mmHg at 60 min (prior to glibenclamide injection) to 29 +/- 3 mmHg at 180 min (P<0.05).6. Endotoxaemia for 180 min resulted in a significant increase in a calcium-independent NOS activity(which was taken to represent iNOS activity) in the lung from 0.17 +/- 0.1 (control, n =4) to 6.21 +/- 0.48 pmol mg-1 min-1 (n =6, P<0.05). Injection of glibenclamide (1 mg kg-1, i.v.) at 60 min after LPS attenuated the increase in iNOS activity caused by endotoxaemia in the lung by 43 +/- 7%(n = 6, P <0.05). In contrast, injection of glibenclamide at 180 min after LPS did not result in a significant inhibition of iNOS activity (n = 6, P <0.05. 7. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractions elicited by noradrenaline (NA, 10-9 to 10-6 M). Treatment of LPS rats with glibenclamide(1 mg kg-1, i.v. at 60 min after LPS) significantly alleviated this LPS-induced hyporeactivity to NA ex vivo. In contrast, when aortic rings from LPS-rats were incubated in vitro with glibenclamide (10 microM for 20 min), glibenclamide did not reverse the vascular hyporeactivity to NA. However, L-NAME (300 microM for 20 min) significantly enhanced the contractile response to NA in aortic rings obtained from LPS-rats(P<0.05, n=6).8. No significant amounts of tumour necrosis factor-alpha (TNF alpha) were detectable in the plasma before the injection of LPS. Endotoxaemia for 90 min resulted in a significant rise in plasma TNFalpha levels(0.05 +/- 0.05 ng ml-1 at time 0, 3.78 +/- 0.24 ng ml-1 at 90 min, n = 6, P < 0.05). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 15 min prior to LPS, n = 5) did not significantly reduce the rise in plasma TNF alpha levels caused by endotoxin.9. Thus, glibenclamide inhibits the induction, but not the activity, of iNOS in vitro and in vivo. This inhibition of iNOS induction may contribute to the beneficial haemodynamic effects of glibenclamide in endotoxic shock. PMID- 7542533 TI - Effects of the tachykinin NK1 receptor antagonist, RP 67580, on central cardiovascular and behavioural effects of substance P, neurokinin A and neurokinin B. AB - 1. We have investigated the effects of the non-peptide NK1 tachykinin receptor antagonist, RP 67580, and its inactive enantiomer, RP 68651, on the cardiovascular and behavioural responses to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) injected intracerebroventricularly (i.c.v.) in conscious rats. 2. The SP and NKA (25 pmol)-induced increases in blood pressure (BP) and heart rate (HR) were of the same magnitude. The cardiovascular responses to both peptides were associated with excessive grooming behaviour and wet dog shakes (WDS). Relative to SP, NKA was weaker in inducing hindquarter grooming (HG), but more effective in eliciting WDS. The cardiovascular response to NKB (50 pmol) comprised an increase in BP and HR, while the behavioural response was weak. 3. RP 67580 (100 pmol), injected 10 or 30 min prior to SP, effectively inhibited the cardiovascular and behavioural responses to the peptide whereas lower doses were ineffective. Pretreatment with 500 pmol of RP 67580, 10 or 30 min prior to SP, reduced the BP response. Of the behavioural manifestations, only face washing was attenuated when the antagonist was injected 10 min before SP. At 2500 pmol, the antagonist exaggerated the BP response to the peptide without affecting the behavioural response. RP 68651 (100 or 2500 pmol) did not modify the central responses to SP. 4. Neither RP 67580 nor RP 68651 (100 pmol), affected the cardiovascular and behavioural responses to NKA or NKB. 5. Our results indicate that RP 67580 is a selective and high affinity antagonist at central NK1 tachykinin receptors in the rat. PMID- 7542534 TI - Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats. AB - 1. This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro-organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat. 2. Intravenous injection of LTA (10 mg kg-1) resulted in a decrease in blood pressure from 123 +/- 1 mmHg to 83 +/- 7 mmHg after 270 min (P < 0.001) and a reduction of the pressor response to noradrenaline (1 microgram kg-1) from 33 +/- 1 mmHg.min to 23 +/- 3 mmHg.min after 270 min (P < 0.05). 3. The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg-1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg kg-1 h-1, i.v. infusion starting 30 min prior to LTA). 4. In contrast, treatment of rats with polymyxin B (0.05 mg kg-1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg-1 LPS, i.v.) for 270 min. 5. The delayed circulatory failure caused by LTA was associated with a time-dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA-rats with dexamethasone (10 mg kg-1). 6. Intravenous injection of LTA resulted in an increase in serum tumour necrosis factor (TNF)-alpha(maximum at 90 min after LTA), which was attenuated by pretreatment of rats with dexamethasone(10 mg kg-1, 60 min prior to LTA). The magnitude of the rise in TNF-alpha caused by LTA was similar to the one elicited by LPS (10mgkg-', i.v.).7. Thus, an enhanced formation of nitric oxide following the induction of iNOS contributes importantly to the delayed vascular failure (hypotension and vascular hyporeactivity) caused by LTA in the anaesthetized rat. We suggest that the endogenous release of TNF-alpha contributes to the induction ofiNOS caused by LTA in vivo. PMID- 7542535 TI - A simple and rapid semiquantitative method for measuring cellulase activity in agar media. AB - A simple and rapid semiquantitative technique for the determination of fungal cellulytic activities in solid (agar slant) media has been developed. This method is a combination of Congo-red staining widely used for qualitative cellulase detection and common cellulase activity tests. Previous investigation on the adsorptive effect of cellulose content of media showed that the real enzyme activity values can be measured with minimum loss by means of agar discs cut from the most active zones of slants visualized by Congo-red staining. Different cellulase activity tests (FPase, CMCase and beta-glucosidase by PNPG-method) of seven cellulolytic fungal strains were investigated by this technique. Data give information on the different enzyme profiles of the species. The method can be regarded as very simple and suitable for simultaneous rapid comparison of cellulase components of greater series of fungal strains from agar slant cultures. It can also be used in the case of fungi unable to grow in liquid cultures. PMID- 7542536 TI - Changes in serum granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6) after surgical intervention. AB - To evaluate the physiological role of G-CSF following surgery, we measured the serum levels of immunoreactive IL-6 and G-CSF sequentially in nine patients after major elective thoracoabdominal surgery for esophageal carcinoma. Both G-CSF and IL-6 levels reached their maxima at the first postoperative day and decreased thereafter. There was a significant correlation between serum G-CSF (y) and IL-6 (x) levels (y = 3.273x + 3.991; r = 0.787, n = 78, p < 0.001). In the case that developed aspiration pneumonia and ARDS at the second postoperative day, the measured G-CSF level was less than half the predicted value. The relationship between serum G-CSF and IL-6 levels supports the central role of G-CSF as the host defense response modifier and, thus, low G-CSF levels in the circulation is one reason for the immunodeficient state after major surgery. PMID- 7542537 TI - Cholecystokinin alterations and effects of levodopa administration in the MPTP treated mouse brain. AB - To clarify the effects of levodopa administration on MPTP-induced alterations in neuropeptides, we examined the effects of repeated levodopa injections (200 mg/kg i.p.) for 2 weeks starting 4 weeks after 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) treatment (30 mg/kg i.p. twice/day for 5 days) on cholecystokinin-octapeptide (CCK-8), substance P (SP) and thyrotropin-releasing hormone (TRH) concentrations at 6 weeks after the MPTP treatment. In the striatum, CCK-8 significantly but slightly decreased in the MPTP-treated mice, coinciding with the MPTP-induced marked reduction of dopamine (DA). This considerable reduction of striatal CCK-8 may result from the selectivity of MPTP since the mesolimbic DA neurons coexisting with CCK-8 are intact with the MPTP treatment. Furthermore, this MPTP-induced decrease in CCK-8 persisted with repeated levodopa administration; therefore, the ineffectiveness of the levodopa treatment may have been be due to the degeneration of the nigrostriatal DA neurons. SP and TRH contents showed little or no change with levodopa treatment in the MPTP-treated mouse brain. The CCK-8 level decreased in the thalamus+midbrain, hippocampus and hindbrain of the MPTP+levodopa-treated group, although there were no changes in the MPTP-treated controls. These results suggest that DAergic neurons, except those in the nigrostriatum, strongly interact with the CCK neurons in these brain regions. PMID- 7542538 TI - Biphasic expression of IGF-I following subtotal renal ablation. AB - Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of experimental renal growth. This study was designed to investigate the quantitative and qualitative changes in renal IGF-I which occur during the course of progressive renal scarring in rats submitted to extensive renal ablation. Analyses were carried out at 7, 15, 21, 30, 90 and 150 days after subtotal nephrectomy. Compensatory renal growth occurred over the first 30 days, amounting to a 161% increase in the protein content per remnant kidney (272 +/- 22 vs. 104 +/- 3 mg at the outset, p < 0.001, means +/- SD, n = 6) but DNA per remnant kidney continued to rise, reaching a 285% increase by day 90 (5.12 +/- 0.06 vs. 1.33 +/- 0.06 mg at the outset, p < 0.001), probably reflecting the continued recruitment of inflammatory cells. Changes in extractable renal IGF-I (eIGF-I) were measured by radioimmunoassay. Seven days after subtotal nephrectomy eIGF-I had increased by 70% compared with controls (2.37 +/- 0.22 ng/mg protein vs. 1.39 +/- 0.2 ng/mg at the outset, p < 0.001) but had returned to normal by 30 days. This coincided with a transient increase in the immunostainable IGF-I (iIGF-I) within the cortical collecting ducts which, again, had subsided by 30 days. However, at 90 and 150 days, as severe scarring was detected, a different pattern of immunostaining (iIGF-I) was noted. Strong staining was evident in the injured cells of the distal tubules and within the fibrosed interstitium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542539 TI - Localization and expression of a collecting duct water channel, aquaporin, in hydrated and dehydrated rats. AB - The cellular and subcellular localization and expression of a kidney collecting duct water channel, aquaporin (AQP)-CD, were examined in the kidneys of hydrated and dehydrated rats by immunostaining, Northern blot analysis, and radioimmunoassay. In hydrated rat kidneys, AQP-CD was selectively found in the collecting duct principal cells and inner medullary collecting duct cells, but not in the intercalated cells. At a light microscopic level, AQP-CD was diffusely present in a granular pattern throughout the cytoplasm of the collecting duct cells with a preferential accumulation in subapical regions. By immunoelectron microscopy, AQP-CD was frequently demonstrated along membranes of small vesicles in the subapical cytoplasm and occasionally along the basolateral membranes of these cells. However, immunolabeling was sparse on the apical membranes. In dehydrated rats, AQP-CD immunostaining was intensified in the subapical cytoplasm of the collecting duct cells, along with increases in the number and size of AQP CD-bearing vesicles in the subapical regions and with increment of labeling along the apical membranes. The increase in the amount of AQP-CD in the collecting duct cells of dehydrated rat kidneys was quantitatively confirmed by elevation of AQP CD at mRNA and protein levels. The AQP-CD localization is consistent with the predicted site of the antidiuretic hormone (ADH)-regulated water channel in the collecting ducts and the increase in AQP-CD at mRNA and protein levels by dehydration may account for high concentration of urine in dehydrated subjects. PMID- 7542540 TI - Reduction of AMPA-selective glutamate receptor subunits in the entorhinal cortex of patients with Alzheimer's disease pathology: a biochemical study. AB - Using biochemical techniques we determined concentrations of the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3 in the entorhinal cortex of patients with Alzheimer's disease pathology and age-matched controls. Tangle density was also determined in anatomically matched samples and correlated with GluR1 and GluR2/3 receptor concentration. In Alzheimer's disease brain, Western blot analysis revealed average reductions of 43% and 38% for GluR1 and GluR2/3, respectively. Based on previous immunohistochemical studies, we infer that the majority of protein reduction was due to decreases in GluR1 and GluR2/3 immunolabeled elements in the more superficial layers of the entorhinal cortex (layers II and III). These layers of the entorhinal cortex contained numerous neurofibrillary tangles in Alzheimer's disease, but neither GluR1 nor GluR2/3 protein concentration correlated significantly with tangle density. We hypothesize that the decrease in specific glutamate receptor subunits, particularly GluR2/3, may contribute to the vulnerability of neurons in the entorhinal cortex via mechanisms involving calcium conductance through AMPA selective channels. PMID- 7542541 TI - Postnatal maturation of layer V pyramidal neurons in the human prefrontal cortex. A quantitative Golgi analysis. AB - The pattern of dendritic development of layer V pyramidal neurons in the human prefrontal cortex was studied quantitatively. The main objective was to determine how dendritic growth and differentiation continued postnatally. In order to do this, we analyzed the three-dimensional branching pattern of the basal dendritic field of Golgi-Cox impregnated neurons. The subjects ranged in age from 7.5 months after birth up to 27 years. The results revealed a maturational time course with a rapid dendritic growth phase during the first postnatal year. The dendritic length and radial extension of the basal field increased. The growth continued, at a reduced rate, up to around 5 years of age by a lengthening of the terminal segments and a slight increase in soma size. The morphological maturation of the layer V pyramidal neurons was completed between one and five years. After this age the dendritic values appeared to level off and reach a stable level that lasted up to at least 27 years. This paper gives no indications for overgrowth or regressive events of the layer V basal dendritic trees during development. PMID- 7542542 TI - The influence of Bay K 8644 treatment on (+/-)-epibatidine-induced analgesia. AB - The purpose of this investigation was to determine if analogous to (-)-nicotine's analgesic effect, the analgesic effect of the recently characterized potent nicotinic acetylcholine receptor (nAChR) agonist (+/-)-epibatidine was altered in response to treatment with the calcium channel agonist (+/-)-Bay K 8644. In addition, the effects of the enantiomers, (+)-Bay K 8644, reported to be a calcium channel antagonist, and (-)-Bay K 8644, reported to be a calcium channel agonist were examined. (+/-)-Bay K 8644 (2.8 mumol/kg; i.p.) produced a large analgesic response in mice in the hot-plate paradigm that rapidly dissipated by 30 min after treatment. This analgesic effect of (+/-)-Bay K 8644 was not prevented by pre-treatment with the nicotinic antagonist mecamylamine (5 mumol/kg; i.p.). Treatment with non-analgesic doses of the calcium channel agonists (+/-)- and (-)-Bay K 8644 (1.4 mumol/kg; i.p.) significantly potentiated the analgesic effect of (+/-)-epibatidine (0.05 mumol/kg; i.p.). Potentiation of (+/-)-epibatidine's analgesic effect occurred when the agonists were administered prior to (+/-)-epibatidine or after (+/-)-epibatidine as long as analgesia testing was conducted 15 to 30 min after Bay K 8644 treatment. Pre-treatment with the calcium channel antagonist (+)-Bay K 8644 was found to attenuate (+/-) epibatidine-induced analgesia. When given after (+/-)-epibatidine, (+)-Bay K 8644 had no effect on (+/-)-epibatidine's analgesic effect. These data provide additional in vivo evidence that altering calcium dynamics can modulate neuronal nAChR function. PMID- 7542543 TI - A new cutaneous nerve fiber connection with the frontal nerve in the frog Rana esculenta: a morphological study. AB - The frontal nerve arises from the frontal organ, which represents the extracranial component of the pineal complex in some lower vertebrates, and interconnects the frontal organ with the epiphysis and the brain. The existence of a previously unreported nerve branch of the frontal nerve is described here in the frog Rana esculenta and called the lateral nerve. The course of the lateral nerve and its junction with the frontal nerve have been consistently detected by means of different techniques: toluidine blue staining in semithin sections, the Landau-Ignesti method for myelinated nerve fibers, the methylene blue intravital staining for peripheral nerves, and in vitro tracing with the carbocyanine Dil. A method to preserve intact the delicate lateral nerve during dissection is also described. The lateral nerve was consistently found to be unilateral, and to join the frontal nerve at one end (either on the left or the right side), whereas the other extremity was found to be dispersed in the dermis. Thus, the lateral nerve could represent a new pathway interconnecting the skin and the brain and/or the frontal organ in the frog. PMID- 7542544 TI - Effect of dextran and enoxaparin on early ePTFE graft thrombogenicity in sheep. AB - OBJECTIVES: To evaluate the effect of low molecular weight heparin (LMWH), dextran 70 and their combination on platelet adhesion and fibrinogen uptake in ePTFE grafts in an experimental sheep model. DESIGN: Prospective open study. SETTING: Animal Laboratory of a University Hospital. MATERIALS: Early thrombogenicity of ePTFE grafts was studied after interposition in the two common carotid arteries of 40 adult sheep. The animals received one of four different treatment regimens in a double blind randomised way: enoxaparin and polygeline, saline and dextran 70, enoxaparin and dextran 70 or saline and polygeline (control). The substances were administered i.v. with a total dose of 73 antifactor-Xa U/kg for enoxaparin and 1.0 g/kg for dextran 70. Polygeline and saline were used as placebo substances in equivalent volumes. On one side (random allocation) the carotid blood flow was restricted to 25 ml/min, on the other side it was left unrestricted. CHIEF OUTCOME MEASURES: The following variables were studied: 1) fibrinogen uptake; 2) platelet uptake; 3) early graft patency; 4) blood flow in patient grafts; 5) visible presence of graft thrombus; 6) thrombus weight. MAIN RESULTS: The results verified the importance of adequate blood flow as only 30% of grafts with restricted blood flow in the control group were patent compared with 80% of those with unrestricted blood flow (p = 0.038). Dextran 70, enoxaparin and the combination of the two increased early graft patency (p < 0.05) and reduced thrombus weights (p < 0.05) in grafts with restricted blood flow. The relative number of grafts with thrombus free surface was increased in the unrestricted blood flow situation. CONCLUSIONS: Dextran 70 and enoxaparin appeared to be equally effective in decreasing fibrinogen and platelet uptake in the grafts. Their combination was not significantly more effective although there was a favourable trend. PMID- 7542545 TI - A computer system for analysis and integrated description of regulation of the molecular-genetic system of interferon induction and action. AB - A new theoretical approach to elaboration of an information-analytical integrated knowledge base containing data on regulation and function of biological systems is presented. The knowledge base incorporates: (i) a reference database containing experimental data on the structural-functional organization of a biological system; (ii) a dynamic mathematical model for analysis of the evolution of the system over time; and (iii) an interpretation module of simulation results. Application of this approach to theoretical investigation of the interferon system in the case of viral infection is discussed. The approach is specific in that it uses mathematical modeling technology, which allows one to generate mathematical models of different degrees of complexity in the analysis of the diverse aspects of biological system behavior. This approach allows one not only to store and to treat available experimental data, but also to acquire new knowledge about the behavior of a biological system. The proposed approach is implemented as a computer system for the IBM PC and compatibles. PMID- 7542546 TI - Diagnostic criteria for neoplastic myoepithelial cells in pleomorphic adenomas and myoepitheliomas. Immunocytochemical detection of muscle-specific actin, cytokeratin 14, vimentin, and glial fibrillary acidic protein. AB - OBJECTIVE: Markers for normal salivary gland myoepithelium were used to determine the extent of their expression in the neoplastic myoepithelial (nonluminal) cells of pleomorphic adenomas and then in the tumor cells in myoepitheliomas and to gather information necessary to establish diagnostic criteria, especially muscle actin expression, for myoepitheliomas. STUDY DESIGN: Methanol/acetic acid-fixed and paraffin-embedded tissue was used to immunohistochemically study expression of intermediate and smooth-muscle actin filaments in nonluminal cells in 14 pleomorphic adenomas and to compare this to their expression in five myoepitheliomas. RESULTS: In routine histologic sections, the morphologic variants of nonluminal tumor cells--spindle, stellate, polygonal, angular, and plasmacytoid--in pleomorphic adenoma mirror the spectrum of tumor cells in myoepitheliomas. Immunocytochemical similarities are also apparent. Two specific markers for myoepithelial cells in the normal salivary gland, muscle-specific actin and cytokeratin 14, were both variably, independently, and never uniformly expressed in nonluminal cells of pleomorphic adenoma and tumor cells in myoepitheliomas regardless of their morphology. Cytokeratin 14 in addition labels basal cells of excretory ducts. Both muscle-specific actin and cytokeratin 14 preferentially localized to single layers of periductal cells in pleomorphic adenomas, angular, polygonal, and plasmacytoid cells preferentially expressed cytokeratin 14. Similar patterns were noted in the three myoepitheliomas with reasonable expression of the two markers. Only isolated single cells or small groups of plasmacytoid cells in four pleomorphic adenomas with a significant component of these cells and the two plasmacytoid myoepitheliomas immunostained for muscle-specific actin and cytokeratin 14. In both tumor types, vimentin was nearly uniformly expressed in nonluminal tumor cells of all morphologic types, including plasmacytoid cells. CONCLUSIONS: The range and transition of morphology of nonluminal cells in pleomorphic adenomas is reflected in myoepitheliomas. Incomplete or absent expression of the myoepithelial/basal cell markers, muscle specific actin, and cytokeratin 14, and the general expression of vimentin is common to both tumors. Because these findings apply to the majority of plasmacytoid cells in pleomorphic adenomas, tumor cells with a similar morphology and immunoprofile are to be expected in myoepitheliomas; the term plasmacytoid myoepitheliomas is thus appropriate regardless of the presence or absence of muscle-specific actin. PMID- 7542547 TI - Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV. AB - The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expression of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate V beta independent of CD40L expression. Nevertheless, CD40L-mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L- T cells to SAG in vitro is also impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step in the life cycle of MMTV. The initial step in SAG dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells. PMID- 7542548 TI - Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. AB - CD19-deficient mice were generated to examine the role of CD19 in B cell growth regulation in vivo. Deletion of CD19 had no deleterious effects on the generation of B cells in the bone marrow, but there was a significant reduction in the number of B cells in peripheral lymphoid tissues. B cells from CD19-deficient mice exhibited markedly decreased proliferative responses to mitogens, and serum immunoglobulin levels were also significantly decreased. In contrast, mice that overexpressed CD19 had significant defects in early B cell development in the bone marrow, augmented mitogenic responses, and increased serum immunoglobulin levels. These experiments indicate that CD19 functions to define signaling thresholds for cell surface receptors that regulate B lymphocyte selection, activation, and differentiation. PMID- 7542549 TI - Different MHC class I alleles compete for presentation of overlapping viral epitopes. AB - We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza specific CTL response to an epitope in the nucleoprotein that overlaps the B8 restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8. PMID- 7542551 TI - Recovery from severe cyclic antidepressant overdose with hypertonic saline/dextran in a swine model. AB - OBJECTIVE: To determine the effect of a hypertonic saline and dextran (HSD) solution on blood pressure and QS duration during severe cyclic antidepressant (CA) toxicity in swine. METHODS: Ten domestic swine weighing 20-24 kg were anesthetized and placed on mechanical ventilation. Nortriptyline solution was infused intravenously to achieve hypotension (systolic blood pressure equal to 50% of baseline) and a QRS duration of 120 msec. After reaching toxicity, the animals received in a randomized fashion either 10 mL/kg of a 7.5% saline/6% dextran solution or an equal volume of 0.9% saline as a rapid intravenous bolus. The animals were observed for one hour or until they died. Blood pressure and ECG were recorded continuously. Arterial pH was maintained in the physiologic range by controlled ventilation. RESULTS: Mean systolic blood pressure 10 minutes after treatment was 45 +/- 8 torr in the normal- saline group compared with 115 +/- 12 torr in the HSD group (p < 0.05). Mean QRS duration 10 minutes after treatment was 180 +/- 8 msec in the normal-saline group; it was 88 +/- 13 msec in the HSD group (p < 0.05). All normal-saline--group animals died within 20 minutes, and four of the five animals in the HSD group survived to 60 minutes (p < 0.05). The mean peak sodium concentration was 157 mmol/dL (mEq/dL) in the HSD group, and this was transient. CONCLUSION: In this swine model of severe CA toxicity, a solution of 7.5% saline/6% dextran significantly reversed hypotension and QRS prolongation. HSD also improved survival to 60 minutes. PMID- 7542550 TI - Distinct roles of L-selectin and integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer's patch-HEV in situ: the multistep model confirmed and refined. AB - Circulating lymphocytes home to the mucosal lymphoid organs, Peyer's patches (PP), through high endothelial venules (HEV). In situ analyses revealed that transfused lymph node cells (LNCs) interact with PP-HEV in a series of overlapping adhesion events: L-selectin (CD62L) > alpha 4 beta 7 initiates interaction, L-selectin and alpha 4 beta 7 both participate in rolling, and G alpha i-linked activation triggers arrest that requires both alpha 4 beta 7 and LFA-1. alpha 4 beta 7 dramatically reduces rolling velocity, and appears to be required for engagement of LFA-1. In contrast with resting LNC, preactivated LNC or alpha 4 beta 7hi lymphoma cells require only alpha 4 beta 7 for arrest in PP HEV. The predominant PP-HEV ligand for alpha 4 beta 7 but also apparently for L selectin is the mucosal addressin MAd-CAM-1. These results validate the concept of multimolecular adhesion/decision cascades in physiologic lymphocyte endothelial recognition, define a novel role for alpha 4 integrins as a "bridge" between selectin and beta 2 integrin-dependent events, and reemphasize the potential for direct adhesion through preactivated alpha 4 integrins alone. PMID- 7542552 TI - Functional defects are associated with abnormal signal transduction in T cells of mice inoculated with parental but not IL-2 secreting tumor cells. AB - CMS5, a murine fibrosarcoma, was transduced with the IL-2 gene to stimulate the host antitumor response. Previously, we described that splenocytes isolated from parental tumor bearing (PTB) mice and IL-2-secreting tumor bearing (ITB) mice differed significantly in their proliferative responses when restimulated with IL 2-secreting tumor cells. In this report we extend these results by showing that the inability of PTB cells to proliferate when stimulated with parental CMS5 cells is not corrected by providing a source of costimulation. Furthermore, we demonstrate that T cells from PTB animals exhibit defective signaling through the T-cell receptor, defined by decreased protein tyrosine phosphorylation, that correlates with the impairment of functions attributed to both CD4+ and CD8+ T cells. In contrast, T cells from ITB animals are normal in this regard. The results suggest that immunosuppression underlies functional differences between PTB and ITB splenocytes and that defects in signal transduction may be responsible for the lack of normal functional responses. PMID- 7542554 TI - [Report on interferon treatment in chronic hepatitis. Office of the General Deputy Director of Benefits and Evaluation of Health Technologies. Office of the General Director of Insurance and Health Planning]. PMID- 7542553 TI - B7-1 expression decreases tumorigenicity and induces partial systemic immunity to murine neuroblastoma deficient in major histocompatibility complex and costimulatory molecules. AB - Neuroblastoma may escape an immune attack by virtue of its low expression of surface accessory molecules essential in the antitumor response. Murine neuroblastoma, neuro-2a, was transduced with the retroviral vector LB7-1SN to examine the influence of B7-1 expression on the immune response directed against a low major histocompatibility class (MHC) I and class II negative, B7-2, and ICAM-1 negative tumor. Using a retroperitoneal model for implantation of neuroblastoma in its natural site, we demonstrated that expression of B7-1 by neuro-2a reduces its tumorigenicity. Coinjection of B7-1-positive and -negative cells improved survival compared with mice receiving B7-1-negative cells alone. This was dependent on the ratio of B7-1+ to B7-1- neuro-2a cells injected. CD8+ and not CD4+ T-cell depletion significantly increased tumor-induced mortality in syngeneic A/J mice, indicating that B7-1 decreases tumorigenicity primarily by direct constimulation of CD8+ T cells. Rejection of N-2a/B7-1 tumors or preimmunization with irradiated N-2a/B7-1 cells die not increase protection to challenge with unmodified neuro-2a cells over mice vaccinated with N-2a/neo. Furthermore, cytotoxic T lymphocyte (CTL) precursor frequencies were not significantly higher after in vivo priming and in vitro stimulation with irradiated N-2a/B7-1 compared with N-2a/neo, indicating that B7-1 costimulation by the tumor, in the absence of adequate antigen presentation by MHC molecules, may limit the generation of effective CTLs. PMID- 7542555 TI - [Peritoneal mesothelioma with elevated amylase in the ascitic fluid]. AB - The case of a 42-year-old woman with no previous disease admitted for abdominal pain and ascites is presented. Analysis of the ascitic fluid demonstrated high concentrations of amylase with normal lipase. The diagnosis of peritoneal mesothelioma was obtained by laparotomy. This association has not been previously described. The authors suggest that this diagnostic possibility should be considered in patients without pancreatic disease and high amylase levels in ascitic fluid. PMID- 7542556 TI - Differential effects of various oil diets on the risk of cardiac arrhythmias in rats. AB - BACKGROUND: Independently of the problem of atherogenesis, the amount and type of fat intake influences the risk of cardiac arrhythmias. However, the relative effectiveness of different fats and the underlying mechanisms are controversial. The aim of the present study was to compare the effects of various oil-enriched diets on the risk of ventricular arrhythmias in rat hearts under conditions of ischaemia and reperfusion and to help clarify the mechanisms underlying the differing effects of the oils on the occurrence of arrhythmias. METHODS: Over a 10-week period, we studied five groups of young male Wistar rats given a low-fat chow diet or one enriched with 10% hydrogenated coconut oil, corn oil, linseed oil or sardine oil. Electrocardiograms were recorded from the isolated hearts (Langendorff preparation) perfused with a modified Krebs-Henseleit solution. Ischaemia was induced by a 20 min occlusion of the left anterior descending coronary artery. In another series of experiments, a 10 min occlusion was followed by a 20 min reperfusion period. The times between the first occurrence of extrasystole and the incidence of ventricular tachycardia and fibrillation were determined. The size of the ischaemic zone was assessed using malachite green. The fatty acid composition of the myocardial tissue was analysed using gas chromatography. RESULTS: An increase in the risk of ventricular arrhythmias under conditions of both ischaemia and reperfusion was obvious in the rats that consumed large quantities of saturated fatty acids (coconut oil) and in the group with a very low intake of fat. Polyunsaturated fatty acids (PUFAs), particularly fish oil, exerted a protective effect. The incidence of ventricular fibrillation was 75% in the low-fat group, 67% in the coconut-oil group, 44% in the corn-oil group, 40% in the linseed-oil group and 10% in the fish-oil group. The time until the first occurrence of extrasystole, the incidence of ventricular tachycardia and the incidence of reperfusion-induced ventricular fibrillation were influenced in a similar manner. The size of the ischaemic zone was significantly reduced in the groups given diets enriched with PUFAs. All protective effects were abolished, however, by cyclooxygenase inhibition with aspirin. The fatty acid composition of myocardial tissue, the ratio of n-3 to n-6 fatty acids and the double-bond index were significantly affected by the various diets. CONCLUSION: Whereas saturated fatty acids are obviously proarrhythmic, diets enriched with n 6 or n-3 PUFAs both exert antiarrhythmic effects. Although n-3 fatty acids seem to be more effective, cardioprotection cannot simply be related to the replacement of n-6 by n-3 fatty acids in cardiac membrane phospholipids, given the beneficial effects of corn oil. In any case, replacement of n-3 by n-6 fatty acids is not the underlying mechanism. The overall reduction of prostaglandin formation cannot be the primary mechanism because the beneficial effects of diets rich in PUFAs were abolished by cyclooxygenase inhibition. We conlcude that, besides prostacyclin (PGI2 or PGI3), membrane fluidity and accompanying alterations in functional membrane proteins (e.g. protection from calcium overload) are key factors apart from vascular effects that influence the size of the ischaemic zone. PMID- 7542557 TI - Effects of nutrient solutions on concentration analysis of non-absorbable dilution markers--implications for studies of gastric emptying. AB - Gastrointestinal luminal contents may interfere with concentration analysis of non-absorbable dyes. However, non-absorbable markers are broadly used for studies of gastric emptying rates of nutrient solutions. This prompted us to evaluate the properties of non-absorbable markers to mark such nutrient solutions. In vitro concentrations of polyethylene glycol, phenol red, dextran blue, two anthroquinone dyes and inulin were determined spectrophotometrically in the presence or absence of a formula diet, single compounds of the diet or an oligo peptide diet, and the reproducibility and validity of the analyses were evaluated. The presence of the formula diet or the oligopeptide diet seriously impaired the analyses of marker concentrations, whereas single nutrient compounds did not uniformly interfere. The analysis of polyethylene glycol and phenol red concentrations was impaired by proteins, while the analysis of inulin concentration was impaired by carbohydrates. Dextran blue and the anthroquinones were completely eliminated by protein-precipitation procedures. In conclusion, phenol red and polyethylene glycol should only be used as marker substances for protein-free meals or nutrient solutions, while inulin should not be used with meals or nutrient solutions containing carbohydrates. Marker dilution techniques cannot be recommended for measurements of gastric emptying rates of complete meals. PMID- 7542559 TI - Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis? AB - Fas/APO-1, a member of the NGF/TNF receptor superfamily expressed on the cell surface of normal and malignant cells, is known to induce cell death by apoptosis. In the present study, we have investigated Fas/APO-1 gene defects in a human osteosarcoma cell line resistant to the apoptosis-inducing effects of anti Fas. cDNA cloning and sequencing revealed that these cells contained both 'authentic' and mutant Fas/APO-1 containing a 63 base pair in-frame deletion spanning the transmembrane domain, designated DFas/APO-1. Direct evidence for the existence of a soluble Fas/APO-1 protein was obtained by immunoprecipitation and Western blotting. Taken together with prior studies demonstrating a role for Fas/APO-1 and Fas ligand, respectively, in tumor target cell killing by cytotoxic T-lymphocytes, production of soluble Fas/APO-1 might have significant implications in malignant disease pathogenesis. PMID- 7542561 TI - Screening in general practice. AB - Screening for prostate cancer in a general practice setting seems to be technically feasible and generally acceptable. In our study, 14 cancers were diagnosed among the 568 men screened, giving an overall detection rate of 2%; five of these were either locally advanced or associated with metastatic disease. Although these results may reflect our somewhat conservative biopsy rate, there is a considerable false positive rate in the PSA range of 4-10 ng/ml, particularly among men with clinical evidence of benign prostatic hyperplasia. A large scale prospective controlled study will be necessary to establish the true benefit of screening. PMID- 7542560 TI - Alterations of cell surface antigens induced by placental isoform of ferritin in human carcinoma cell lines. AB - AIM: the ability of the breast cancer-associated placental acid isoform of ferritin (p43-PLF) to modulate cell surface expression of HLA, CD59 (protectin) and CD66 antigen (adhesion antigen related to CEA) was examined. METHODS: the expression of these antigens in human breast carcinoma cell lines BT-20, T47D and MDA-MB-468 was determined with the aid of flow cytometry and monoclonal antibodies. RESULTS: PLF induced a transient up-regulation followed by a down regulation of cell surface protectin (CD59 antigen) on the cell surface of T47D and to a lesser extent, BT-20 human breast carcinoma cell lines. Furthermore, PLF down-regulated cell surface expression of CEA-related CD66 antigen on both these cell lines. No PLF-induced alterations of protectin, CD66 antigen and HLA class I antigen were found on the MDA-MB-468 breast cancer cell line. CONCLUSIONS: breast cancer-associated p43 induces alterations of the expression of cell surface molecules in breast cancer cells which could have an effect on the modulation of cancer cell adhesive interactions. PMID- 7542562 TI - Tumour markers in patients on deferred treatment: prostate specific antigen doubling times. AB - Evidence is presented that cancer which is clinically confined to the prostate follows a predictable natural course. Biological aggressiveness of prostate cancer is directly related to tumour volume, and tumour volume is proportional to serum prostate specific antigen (PSA). Thus, the increase in PSA with time in patients on deferred treatment should reflect the growth rate (doubling time) of prostate cancer. In 43 untreated patients with prostatic carcinomas, serum PSA was serially determined over an average time span of 30 months. Log-PSA values were plotted versus time, tested for linearity and compared between different clinical stages and histological grades. The increase in serum PSA was exponential (log-linear) throughout the measured interval. This linearity allowed us to calculate a PSA doubling time. For clinically localized cancers, the median doubling time was 4 years. Doubling times were faster in patients with higher clinical stages and worse histological grades. Tumour doubling times were overestimated in patients with large volume benign prostatic hyperplasia, since hyperplasia also increases serum PSA, albeit 12 times less than cancer. We conclude that prostate cancer follows a constant (log-linear) growth pattern with a doubling time that is very slow. This extraordinarily long doubling time may explain the favourable outcome of patients with low grade tumours on deferred treatment. Furthermore, this slow doubling time has important implications for early detection of prostate cancer. PMID- 7542563 TI - Immediate versus deferred treatment for advanced disease. AB - The timing of hormonal treatment has been controversial for the last 50 years. The VACURG studies suggested that deferring treatment in symptom free patients might not compromise survival. Recent studies of maximal androgen blockade have revived an old observation that disease presenting earlier may be more hormone sensitive. The arguments in favour of immediate versus deferred treatment depend on the balance of advantages against side effects and costs, but a clear advantage in terms of survival from immediate treatment has yet to be demonstrated. An MRC study of this issue has recruited 1938 patients and should make it first report in 1996. Meanwhile, in many men, deferred treatment remains a valid management option, provided it is used with caution and careful follow up. PMID- 7542564 TI - Gene therapy: principles and potential. AB - Gene therapy is a new and provocative means of treating human malignancy. Insight into the mechanisms of growth and growth regulation within cancer cells has offered multiple potential methods for genetic intervention. The application of gene therapy to prostate cancer is in its infancy. The development of both adenoviral and retroviral replication deficient vectors has provided the ability consistently to transfer genes into cancer cells. Although the ideal gene for transfer has not yet been clearly identified, many genes capable of altering the biological behaviour of prostate cancer exist. Selection of the appropriate gene is highly dependent on the desired therapeutic outcome. A gene therapy strategy, whether dependent on ex vivo or in vivo transfection, must ultimately be tailored to meet the specific needs of the disease to be treated. The potential to treat locally confined disease, preventing subsequent metastasis, or widespread metastatic disease exists. The ultimate success of a prostate cancer gene therapy strategy will rely on comprehensive investigation of the biology of the tumour and careful planning of an effective intervention. PMID- 7542565 TI - Screening strategies: a clinical perspective. AB - Prostate cancer is increasing in incidence, particularly because of the control of competing causes of death and the lengthening life span of western men. Screening and early detection initiatives among symptom free men show that the disease can be detected at earlier stages when hope for cure is reasonable. Prostate specific antigen is the most useful marker for prostate cancer. Analytical methods to improve its sensitivity and specificity include PSA density, age specific PSA reference ranges and PSA velocity or the rate of change in PSA. Older paradigms of the natural history of prostate cancer have been heavily influenced by characteristics of clinical disease. However, improved detection of the disease through better diagnostic tools and heightened public awareness may be changing the dynamics of the disease. The prevalence of advanced disease is falling, and more knowledge is accumulating on the identification of clinically significant disease. The most appropriate definition of clinically significant disease today must be based on a new paradigm, one that can build on but not be bound by older models. PMID- 7542566 TI - Localization of integrin subunits alpha 6 and beta 1 during somitogenesis in the long-tailed macaque (M. fascicularis). AB - The distribution of integrin subunits alpha 6 and beta 1, and the alpha 6 beta 1 integrin ligand, laminin, was examined during somitogenesis in developmental stages 11, 13, and 16 in the long-tailed macaque, using peroxidase immunocytochemistry. Within differentiating somites in stage 11, alpha 6 expression was observed in the sclerotome, basal surface of dermamyotomal cells adjacent to the basal lamina and on scattered cells throughout the dermamyotome. In further advanced somites in stages 13 and 16, alpha 6 immunoreactivity became restricted to the myotome. alpha 6 was expressed on mesenchymal core cells within the myocele of undifferentiated epithelioid somites and the ventromedial wall of somites commencing differentiation at each stage. beta 1 distribution resembled that of alpha 6 in stage 11 somitic tissue, however, it remained present on myotome and sclerotome cells in the later stages, and was also expressed on dermatomal cells in stage 16. Laminin immunoreactivity, while more intense and prevalent than alpha 6 and beta 1 in each stage examined, occurred on the same somite cell populations as the 2 integrin subunits. These results show a defined distribution of alpha 6 on somitic tissue, and suggest this integrin is involved in somite differentiation. They also support a possible role for alpha 6 in myoblast formation and migration. Overlapping of beta 1 and laminin immunoreactivity with that of alpha 6 further suggests that alpha 6 pairs with beta 1 as a functional heterodimer for laminin in defined somitic regions. PMID- 7542568 TI - Protein synthesis in tissues cultured from the bovine hoof. AB - The mechanical strength of the bovine hoof depends on keratinization of cells in the germinal layers of the epidermis. Histological examination of hoof tissues in calves and young heifers has identified disturbances in this keratinization process which would result in ineffective hoof development and could precipitate lameness. Short-term culture of bovine hoof tissue was used to investigate epidermal keratinization. Cell function remains viable in these cultures. The rate of protein synthesis, measured by [35S]-methionine incorporation, continued for at least 3 h in culture. Radiolabelled proteins in tissue homogenates were separated by SDS-polyacrylamide gel electrophoresis and characterised by fluorography and were representative of the proteins found in hoof tissue. Three prominent radiolabelled bands were identified as keratins and actin by Western blotting. Immunohistochemistry showed that keratin was localised principally in the epidermal layers, and microautoradiography indicated that this was the major site of protein synthesis. Hoof tissues cultured under these conditions provide a useful system for studying the acute regulation of epidermal keratinization. PMID- 7542567 TI - Distribution of galanin-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana: Comparison with substance P-immunoreactive fibers. AB - Immunoreactivity of galanin (GAL) was detected in the nerve fibers distributed within the intervascular stroma of the bullfrog carotid labyrinth. GAL immunoreactive fibers are numerous, and some are close to the sinusoidal plexus. Most GAL fibers appear as thin processes with some varicosities. A combination of indirect double immunofluorescence labelling and image processing clearly demonstrated that the distribution pattern of GAL fibers is different from that of SP fibers. This indicates that GAL and SP do not coexist in the same nerve fibers. The role of GAL fibers may be different from that of previously reported neuropeptides (substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and others) as a neuromodulator in controlling vascular tone of the labyrinth. PMID- 7542570 TI - In vivo labelling of L-type Ca2+ channels by fluorescent dihydropyridine: correlation between ontogenesis of the channels and the acquisition of neural competence in ecotderm cells from Pleurodeles waltl embryos. AB - The ability of the ectodermal cells to be induced and to differentiate toward neural tissue, called neural competence, is acquired shortly before gastrulation and lost during late gastrula stages in Pleurodeles waltl embryos. We have examined ectodermal cells' neural competence in relation to the evolution of the density of L-type calcium channels using the fluorescent labelled dihydropyridine probe (STBodipy-DHP). We find that the appearance of dihydropyridine sensitive calcium channels (L-type Ca2+ channels) is correlated with the acquisition of neural competence by the ectoderm cells. The highest density of these channels is reached when competence of the ectoderm is optimal. Conversely, the decrease of L type Ca2+ channel density occurs simultaneously with the normal loss of competence. In addition, we show that these channels are functional since stimulation by S(-)-Bay K 8644 triggered an increase in [Ca2+]i revealed by fluorescence measurements using Fluo-3. This increase in [Ca2+]i is a function of the L-type Ca2+ channels' density. We propose that the molecular basis of the gain and loss of neural competence is linked to the presence of L-type Ca2+ channels in ectodermal cell membranes of Pleurodeles waltl embryos. PMID- 7542569 TI - IBMX induces calcium release from intracellular stores in rat sensory neurones. AB - The intracellular free calcium concentration ([Ca2+]i) was recorded from freshly isolated rat dorsal root ganglia (DRG) neurones by means of Fura-2 or Indo-1 based microfluorimetry. Extracellular application of IBMX at millimolar concentrations evoked transient elevations in [Ca2+]i. The amplitude and rate of rise of the [Ca2+]i transient increased with increasing IBMX concentration. The effects of IBMX on [Ca2+]i were not mimicked by direct manipulation of the intracellular level of cyclic nucleotides, nor incubation with dibutyryl-cAMP, cGMP or 8-bromo-cGMP; neither did treatment with forskolin induce similar [Ca2+]i transients. The IBMX-evoked [Ca2+]i elevation persisted in Ca(2+)-free extracellular solutions, suggesting its origination was from intracellular stores. Caffeine-induced depletion of internal Ca2+ pool prevented IBMX-evoked [Ca2+]i responses, and vice versa, IBMX treatment strongly reduced the amplitude of subsequent caffeine-induced [Ca2+]i elevation. Both ryanodine and procaine, agents known to interact with Ca(2+)-gated Ca2+ release channels of the intracellular endoplasmic reticulum calcium stores, effectively inhibited IBMX induced [Ca2+]i transients. We suggest that, in addition to its known phosphodiesterase-inhibiting activity, IBMX is an effective liberator of Ca2+ from ryanodine/caffeine-sensitive internal calcium stores in neural cells. PMID- 7542571 TI - Soluble cell adhesion molecules and von Willebrand factor in children with Kawasaki disease. AB - Fifty-nine children with acute Kawasaki disease (KD), a childhood vasculitis, were compared with 35 children with fever due to infection and 48 healthy children. Levels of soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) in the healthy children were double those found in adults. All three soluble cell adhesion molecules and von Willebrand factor (vWF) were higher in the children with KD than in the healthy children, but only sE-selectin, a marker for activated endothelial cells, and sICAM-1 were higher than in the febrile children. The high levels of vWF in KD appear to reflect the prominent acute phase reaction. This information can help us to understand further the complex interactions between cytokines, circulating inflammatory cells and the vascular endothelium, and may lead to new therapeutic avenues in KD and other inflammatory diseases and vasculitides. PMID- 7542572 TI - Complement inhibition by human vitronectin involves non-heparin binding domains. AB - Vitronectin (complement S-protein), a multifunctional glycoprotein, inhibits complement-mediated cytolysis at two identified stages of terminal complement complex (TCC) formation: blocking of C5b-7 membrane binding, and prevention of C9 polymerization. However, the functional domain(s) of vitronectin involved in these reactions remains incompletely defined. In order to identify the complement inhibition site, a 12-kD heparin binding fragment and two other internal fragments (53 kD and 43 kD) of vitronectin were isolated after cyanogen bromide (CNBr) treatment of the native molecule. Potent inhibition of guinea pig erythrocyte (GPE) reactive lysis was demonstrated with native vitronectin, total CNBr digest and the 53-kD and 43-kD fragments, but only very poorly by the heparin binding 12-kD peptide. Similarly, the 43-kD fragment blocked the binding of C5b-7 to immobilized vitronectin, whereas the 12-kD fragment had no effect. These data localize the C5b-7 binding site to a 43-kD internal region. Further characterization of the fragments was carried out in an assay which detected C9 polymerization in the presence of C5b-8. Polymerized material was separated by PAGE, detected by autoradiography and quantified after excision from the gels. Results showed that polymerization did not occur in the presence of the 53-kD and 43-kD fragments. However, the 12-kD heparin binding fragment had no effect. It is proposed that prevention of C5b-8-induced C9 polymerization resides at a site in an internal region of the vitronectin molecule. PMID- 7542558 TI - Antiviral therapy for human immunodeficiency virus infections. AB - Depending on the stage of their intervention with the viral replicative cycle, human immunodeficiency virus inhibitors could be divided into the following groups: (i) adsorption inhibitors (i.e., CD4 constructs, polysulfates, polysulfonates, polycarboxylates, and polyoxometalates), (ii) fusion inhibitors (i.e., plant lectins, succinylated or aconitylated albumins, and betulinic acid derivatives), (iii) uncoating inhibitors (i.e., bicyclams), (iv) reverse transcription inhibitors acting either competitively with the substrate binding site (i.e., dideoxynucleoside analogs and acyclic nucleoside phosphonates) or allosterically with a nonsubstrate binding site (i.e., non-nucleoside reverse transcriptase inhibitors), (v) integration inhibitors, (vi) DNA replication inhibitors, (vii) transcription inhibitors (i.e., antisense oligodeoxynucleotides and Tat antagonists), (viii) translation inhibitors (i.e., antisense oligodeoxynucleotides and ribozymes), (ix) maturation inhibitors (i.e., protease inhibitors, myristoylation inhibitors, and glycosylation inhibitors), and finally, (x) budding (assembly/release) inhibitors. Current knowledge, including the therapeutic potential, of these various inhibitors is discussed. In view of their potential clinical the utility, the problem of virus-drug resistance and possible strategies to circumvent this problem are also addressed. PMID- 7542573 TI - Adhesion molecules intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and B7 are not expressed by epithelium in normal or inflamed colon. AB - Adhesion molecules are involved in facilitating cell-mediated immune events. Because lymphocyte-epithelial cell interaction has been implicated in the pathogenesis of colonic inflammation, we analysed expression of a range of adhesion molecules on colonic epithelium in vitro and in vivo using flow cytometry, immunohistochemistry and in situ hybridization. Expression of ICAM-1 by cell lines HT29 and int407 was increased by proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-1 but not by IL-6. Vascular cell adhesion molecule (VCAM) and E-selectin were not expressed. Immunohistochemistry using sections of inflamed colon from 16 patients with ulcerative colitis (UC), five patients with Crohn's disease (CD) and seven patients with normal colonoscopic biopsies, showed no expression of ICAM-1 on colonic epithelium. VCAM was seen in isolated lymphoid aggregates and E-selectin was expressed on endothelium. In situ hybridization showed no ICAM-1 or ICAM-3 mRNA in colonic epithelium. B7, the ligand for CD28, was not found on normal or inflamed colonic epithelium. The adhesion molecules ICAM-1, ICAM-3 and B7 are not involved in lymphocyte-epithelial cell interaction in the normal or inflamed colon. This may have implications for the development of T cell tolerance to intestinal luminal antigens. PMID- 7542574 TI - Allergen-stimulated T lymphocytes from allergic patients induce vascular cell adhesion molecule-1 (VCAM-1) expression and IL-6 production by endothelial cells. AB - Adhesion of inflammatory cells to endothelium is a critical step for their transvascular migration to inflammatory sites. To evaluate the relationship between T lymphocytes (TL) and vascular endothelium, supernatants from allergen stimulated TL obtained from patients sensitive to Dermatophagoides pteronyssinus (Dpt) versus healthy subjects were added to endothelial cell (EC) cultures. TL were stimulated by autologous-activated antigen-presenting cells (APC) previously fixed in paraformaldehyde to prevent monokine secretion. Two parameters were measured: the expression of adhesion molecule and the production of IL-6. Related allergen-stimulated TL supernatants from allergic patients induced an increase of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) expression when supernatants of the control groups (TL exposed to an unrelated allergen or not stimulated or TL obtained from healthy subjects) did not. E-selectin expression was not modulated whatever the supernatant added to EC culture. IL-6 production by EC was significantly enhanced after activation with related allergen stimulated TL supernatants from allergics compared with control supernatants. Induction of VCAM-1 expression was inhibited by adding neutralizing antibodies against IL-4, whereas IL-6 production and ICAM-1 expression were inhibited by anti-interferon-gamma (IFN-gamma) antibodies. Enhanced production of IL-4 and IFN gamma was detected in related allergen-stimulated TL supernatants from allergic subjects compared with the different supernatants. These data suggest that allergen-specific TL present in the peripheral blood of allergic patients are of Th1 and Th2 subtypes. Their stimulation in allergic patients may lead to the activation of endothelial cells and thereby participate in leucocyte recruitment towards the inflammatory site. PMID- 7542579 TI - Bone marrow and serum connective tissue polypeptides in idiopathic myelofibrosis. AB - The distribution of collagen type VI and tenascin has been determined in both normal and myelofibrotic bone marrow by immunohistological techniques. In normal sections positivity was demonstrated in the periosteum (collagen type VI and tenascin) and in the walls of small blood vessels (tenascin). In contrast, myelofibrotic bone marrow showed an increased deposition of both proteins, especially collagen type VI, although this increase was restricted to the later fibrotic stages of the disease. Serum concentrations of collagen type I (PICP), collagen type III (PIIIP) and laminin (laminin P1) related polypeptides were determined in a further 26 patients. PIIIP levels were significantly raised, in contrast to PICP and laminin P1 concentrations. All three markers, however, were significantly elevated in patients with active/transforming disease. Laminin P1 and PICP levels showed a strong correlation, indicating a relationship between basement membrane and interstitial collagen metabolism, although they do not appear to offer any advantage over PIIIP for the monitoring of disease activity. PMID- 7542577 TI - Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle. AB - In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111-242) containing the RNA recognition motif (RRM, aa 111-187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein. PMID- 7542580 TI - Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells. AB - We have previously reported that a peptide from chicken bursa of Fabricius, bursal antisteroidogenic peptide (BASP), inhibits luteinizing hormone-stimulated progesterone biosynthesis by chicken ovarian granulosa cells. The objective of this study was to determine the site(s) of BASP inhibition within the steroidogenic pathway of chicken granulosa cells. The effects of BASP on key steroidogenic enzymes, including adenylyl cyclase (AC), phosphodiesterase, the cholesterol side-chain cleavage enzyme complex and 3 beta-hydroxysteroid dehydrogenase were determined. Luteinizing hormone (10 ng/tube) stimulated a fivefold increase in granulosa cell progesterone production that was inhibited by BASP (0.06, 0.12 or 0.25 bursal equivalents) in a dose-dependent manner. Luteinizing hormone stimulated a sixfold increase in cyclic 3',5'-adenosine monophosphate (cAMP) formation, and this increase was potentiated by BASP in a dose-dependent manner. In addition, BASP stimulated cAMP formation in the absence of luteinizing hormone without affecting progesterone production. The AC activator forskolin (0.1 mM) stimulated a 4.5-fold increase in progesterone synthesis, which was inhibited by BASP. In the presence of forskolin. BASP increased cAMP formation in a dose-dependent manner. A fivefold increase in progesterone synthesis induced by the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (1.0 mM) was inhibited by BASP. In the presence of 3-isobutyl-1 methylxanthine, BASP increased cAMP formation in a dose-dependent manner. Finally, 22(R)-hydroxycholesterol (250, 500, 1,000, or 2,500 ng/tube) or pregnenolone (50, 100, 200, or 500 ng/tube) resulted in up to 15- or 10-fold increases in progesterone production, respectively. Increasing concentrations of BASP caused a dose-dependent suppression of the conversion of 22(R) hydroxycholesterol, but not pregnenolone, to progesterone. The inhibition of steroidogenesis by BASP is not associated with reduced cAMP levels, and BASP appears to strongly stimulate AC activity. In addition, these findings suggest that BASP may limit the availability of progesterone precursors by inhibiting the activity of the cholesterol side-chain cleavage enzyme complex. PMID- 7542576 TI - High numbers of autoantigen-reactive mononuclear cells expressing interferon gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) are present in cord blood. AB - Umbilical cord blood of neonates and peripheral blood of healthy adults were analysed by in situ hybridization for numbers of mononuclear cells (MNC) expressing the cytokines IFN-gamma, TGF-beta and IL-4 mRNA without culture and after culture in the presence of acetylcholine receptor (AChR), myelin basic protein (MBP) and peripheral myelin protein P2. These antigens were chosen since they represent autoantigens in putatively immune-mediated neurological diseases. The numbers of cells expressing cytokine mRNA after 72 h culture in the presence of AChR, MBP and P2 were higher in cord blood than in peripheral blood of healthy adults. IFN-gamma, TGF-beta and IL-4 were always elevated in parallel. In cord blood there was a pronounced reactivity to several of the tested antigens, while such broad reactivity was not found in peripheral blood of healthy adults. No differences in cytokine mRNA expression were found between cord blood and peripheral blood of adults when cells were analysed without culture. The results show a capacity of cord blood cells to react to several autoantigens by the up regulation of cytokine mRNA expression. PMID- 7542575 TI - Neutrophil adhesion molecules in term and premature infants: normal or enhanced leucocyte integrins but defective L-selectin expression and shedding. AB - The functional deficits of neonatal neutrophils are well documented and are thought to contribute to the increased susceptibility of newborn infants to infection. We measured the adhesion molecules L-selectin, CD11a/CD18 and CD11b/CD18 on neutrophils from the cord blood of term (n = 22) and premature (n = 32) infants using a whole blood method with flow cytometry and quantitative bead standards to enumerate cell surface receptors. We also assayed plasma for the shed form of L-selectin (sL-selectin). Our results suggested that L-selectin expression on term infant neutrophils is lower than that on adult neutrophils (unstimulated and stimulated, both P < 0.001), but that stimulated premature infant cell express higher L-selectin than term infants (P < 0.05); it is possible that this deficiency is caused by physiological changes occurring around the normal time of parturition. We observed reduced sL-selectin in term infants (P < 0.001) compared with adults, and even lower concentrations in premature infants (P < 0.001). The sL-selectin concentrations in plasma may be a reflection of granulopoiesis, which may be reduced in premature infants. Our results showed increased resting neonatal neutrophil expression of CD11b/CD18 compared with adults, and the absence of any neonatal deficit of the ability to up-regulate CD11b/CD18 expression on stimulation. These findings are contrary to previous reports. Further studies suggested that the isolation procedures used in previous reports reduces the capability of the cells to respond to a formyl methionine leucine phenylalanine (fMLP) stimulus. This effect is more marked in neonatal neutrophils, suggesting that the previously reported deficiency is in fact due to the isolation techniques used rather than the cells' innate ability to up regulate CD11b/CD18 expression. The results of our study lead us to propose that the adhesive function of neonatal neutrophils may be less defective than previously thought. PMID- 7542578 TI - Common variable immunodeficiency (CVID) and MxA-protein expression in blood leucocytes. AB - The underlying immunopathogenic mechanism of CVID has been suspected to involve a chronic viral infection or an autoimmune condition. However, formal proof of viral infection is lacking. Measurement of MxA-protein in leucocyte lysates is a sensitive test for evaluating the activation of the host's interferon system. Both viral infections and autoimmune diseases such as systemic lupus erythematosus (SLE) strongly induce MxA-protein in peripheral leucocytes. We therefore examined 15 patients with longlasting hypogammaglobulinaemia for MxA protein induction in vivo: 13 patients suffered from CVID, one from hyper-IgM syndrome, and one patient had chronic B lymphocytic leukaemia associated with immunoglobulin deficiency and chronic papilloma virus infection (condylomata accuminata). Only the latter patient exhibited a strong MxA-protein expression; two CVID patients were borderline positive, and the remaining 12 patients including the hyper-IgM syndrome were MxA-protein-negative. There was no relationship between MxA expression and low CD4/CD8 ratios or increased CD8/CD57+ T cell counts, although both conditions are often observed in CVID as well as in chronic viral infections. When exposed in vitro to interferon-alpha (IFN-alpha), peripheral blood leucocytes of four MxA-negative patients were capable of producing normal amounts of MxA-protein. Taken together, these results argue against a viral or autoimmune pathogenesis of CVID. PMID- 7542581 TI - Effects of somatotropin on the conceptus, uterus, and ovary during maternal recognition of pregnancy in cattle. AB - Effects of recombinant bovine somatotropin (rbST) on ovarian and uterine function and the production of components of the insulin-like growth factor (IGF) system were examined during the period of maternal recognition of pregnancy in cattle. Lactating dairy cows were treated with 25 mg/d rbST (n = 8) or saline (n = 8) for 16 d after estrus. Ovaries, uteri, and conceptuses were collected on Day 17 after estrus. The length (millimeters) of the conceptus was recorded. The concentration of IGF-I and the content of IGF-binding proteins (BP) in uterine flushings were determined. Corpora lutea (CL) were weighed, and the number of follicles (> or = 2 mm in diameter) were counted. Follicular fluid from the largest and second largest follicles was assayed for the concentration of IGF-I, IGFBP, progesterone, and estradiol. The length of the conceptus and the total amount of IGF-I in uterine fluid were similar for rbST and control. Recombinant bST increased 1) the weight of the CL, 2) the number of largest follicles (10 to 15 mm in diameter), 3) the concentration of IGF-I in the follicular fluid, 4) the follicular fluid content of IGFBP of the largest estrogenic follicle, and 5) the quantity of IGFBP in uterine flushings. The concentration of progesterone in the follicular fluid tended to be increased in rbST-treated cows, whereas the concentration of estradiol was similar to that of control cows. The concentration of progesterone in plasma was similar for rbST compared with control. In conclusion, the administration of rbST in lactating dairy cows for 16 d after estrus did not alter the growth of the conceptus collected on Day 17. The greatest responses to rbST were found within the ovary, where rbST increased the weight of the CL and altered the amount of IGF-I and IGFBP in the follicular fluid. PMID- 7542582 TI - Cytokeratin positivity in fine-needle aspirates of melanomas and sarcomas. AB - Cytokeratin (CK) positivity has been considered a specific marker for epithelial differentiation in cytologic specimens. After observing CK reactivity in fine needle aspirate (FNA) specimens of melanoma and sarcoma, a retrospective study of melanomas and sarcomas was undertaken to investigate the frequency of anomalous CK staining in these neoplasms. Cell block sections and/or restained smears from 36 melanomas and sarcomas were retrospectively stained for CK. An appropriate internal positive control (HMB-45), S100 protein, or vimentin) was also used to insure antigen preservation. Of the smears from 19 melanomas, five revealed focal strong CK positivity of neoplastic cells, two cases showed faint or equivocal staining, 11 cases were negative for CK, and one could not be interpreted due to inadequate controls. Of the smears from 14 sarcomas, two showed positivity for CK (one fibrosarcoma and one condrosarcoma), 11 were negative, and one had inadequate controls. The number of CK positive cells was less than that observed with the appropriate internal positive control antibodies. Destained Papanicolaou smears were superior to Diff-Quik smears for retrospective immunocytochemical stains. Cell block sections from four of the melanomas and one sarcoma demonstrated no aberrant staining. Since cytokeratin positivity occasionally is seen in nonepithelial neoplasms, its presence alone cannot be used to make a definitive diagnosis of carcinoma. Therefore, a panel of immunocytochemical stains should be utilized in diagnosis of FNA specimens. PMID- 7542583 TI - Cytoskeleton in myelin-basic-protein-deficient shiverer oligodendrocytes. AB - Production of CNS myelin by oligodendrocytes requires the regulated synthesis and assembly of cytoskeletal components. However, the molecular signals that mediate this process are not known. Here we use the shiverer mutant mouse, which is missing a large segment of the myelin basic protein (MBP) gene, to investigate the possible role in cytoskeletal assembly of an MBP gene product or of other myelin components whose expression may be linked to that of MBP. In axon-free cultures, we find that approximately half of shiverer oligodendrocytes have enlarged cell bodies, abnormal processes and fail to elaborate extensive membrane sheets. In those membrane sheets that are elaborated by shiverer oligodendrocytes, microtubular structures are abnormal in size and distribution. Additionally, 2',3'-cyclic nucleotide 3'-phosphohydrolase and microfilaments are not colocalized with microtubular structures as they would be in mature wild-type membrane sheets. These observations suggest that an MBP gene product has a direct or indirect role in regulating various aspects of cytoskeleton assembly in wild type oligodendrocytes. In the absence of this signal, oligodendrocytes apparently do not normally assemble cytoskeleton; this may be one important basis for the abnormal morphology of intact shiverer CNS. PMID- 7542584 TI - Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A. AB - Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542585 TI - Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP. AB - The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1 methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542589 TI - Relative bioavailability of nicotine from a nasal spray in infectious rhinitis and after use of a topical decongestant. AB - The relative bioavailability of nicotine from a nasal spray was assessed in 15 smokers suffering a common cold and rhinitis according to generally accepted criteria. The patients were given a single dose of 2 mg nicotine from the nasal spray with and without concurrent administration of a nasal vasoconstrictor decongestant, xylometazoline, in randomised order. Control session measurements were made in the disease-free state. Applying strict bioequivalence criteria, we found that common cold/rhinitis slightly reduced the bioavailability of nicotine, both in its rate and extent; the geometric mean of the ratio of Cmax, AUC and tmax were 0.81, 0.93 and 1.36, respectively. The nasal vasoconstrictor, xylometazoline, normalised the extent of the bioavailability of nicotine, but further prolonged the time for absorption to almost twice that measured in the disease-free state, increasing the tmax ratio to 1.72. The results suggest that a minor proportion of people stopping smoking with the help of a nicotine nasal spray may experience a minor reduction in the effect of the spray during common cold/rhinitis. However, the nicotine self-titration behaviour found with most smoking cessation products (except the nicotine patch) will automatically lead to an adjustment of the dosage to achieve the desired effect. PMID- 7542586 TI - Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. AB - A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps. PMID- 7542587 TI - Experimental evidence for RNA trans-splicing in mammalian cells. AB - We present evidence that mammalian cells have the ability to generate functional mRNA molecules by trans-splicing. Rat cells, transformed by an early SV40 DNA fragment (Bst/Bam) synthesize a truncated T antigen (T1 antigen), although the cells do not have a direct sequence homology for the T1 antigen at the DNA level. The Bst/Bam DNA fragment encodes exclusively for the second SV40 T antigen exon (aa 83-708) and contains the entire small t antigen intron. To synthesize the corresponding mRNA (T1 mRNA), the cells utilize a cryptic 5' splice site within the second exon (codons for aa 131/132) as donor site and the upstream small t antigen 3' splice site as the acceptor site. Since these sites are in an inverted order on the pre-mRNA, two Bst/Bam transcripts are required to generate one T1 mRNA molecule. HeLa cell nuclear extracts also performed the trans-splicing reaction in vitro. PMID- 7542588 TI - The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. AB - We have used either Escherichia coli or T7 RNA polymerase to transcribe in E. coli a series of lacZ genes that differ in the nature of their ribosome binding sites (RBS). Each T7 RNA polymerase transcript yields from 15- to 450-fold less beta-galactosidase than its E. coli polymerase counterpart, the ratio being larger when weaker RBS are used. The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E. coli RNA polymerase transcripts. T7 RNA polymerase transcribes the lacZ gene approximately 8-fold faster than the E. coli enzyme. We propose that this higher speed unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E. coli enzyme is used. This leads to degradation of the T7 transcript, unless the leading ribosome comes in time to shield the cleavage site: the weaker the RBS, the lower this probability and the more severe the inability of T7 RNA polymerase transcripts for beta-galactosidase synthesis. PMID- 7542591 TI - Distinct signaling properties identify functionally different CD4 epitopes. AB - The CD4 coreceptor interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF-AT transcription factor which is required for interleukin-2 gene expression. Whereas different anti-CD4 mAb or HIV-1 gp120 could all trigger activation of the protein tyrosine kinases p56lck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF-AT. Lack of full activation of NF-AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways. PMID- 7542592 TI - Characterization of critical residues in the cytoplasmic domain of the human interleukin-5 receptor alpha chain required for growth signal transduction. AB - Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, alpha and beta, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse beta chain (AIC2B) that were transfected with human IL-5 receptor (R)alpha cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL 3 induce tyrosine phosphorylation of beta chain and JAK 2 kinase. Phosphorylated beta receptor was co-precipitated with anti-JAK 2 antibodies, suggesting that both molecules were physically associated. IL-5 and IL-3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon-gamma responsive region of human Fc gamma 1 gene DNA element. A deletion mutant of hIL-5R alpha lacking the cytoplasmic part could bind hIL-5 normally in association with the beta chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane-proximal proline-rich element of the hIL-5R alpha cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL-5 inducible JAK 2 phosphorylation. PMID- 7542590 TI - The glycosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor zeta/ZAP-70-dependent and -independent signaling pathways in T cells. AB - The glycosylphosphatidylinositol (GPI)-anchored CD59 protein (human protectin) protects cells against complement-induced lysis, binds to CD2 and also transduces activation signals within T cells. We have further examined the biochemical signals transduced by CD59 and addressed its role in regard to the CD3-mediated signaling cascade. We show here that CD59 cross-linking induces a time-dependent activation of p56lck and of p70zap (ZAP-70) in CD3-positive Jurkat cells, leading to the stimulation of the T cell receptor zeta/ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. Cross-linking of CD59 on peripheral T cells and thymocytes induces tyrosine phosphorylations identical to those seen in Jurkat cells and this is followed by lymphokine production and proliferation. In contrast, only activation of CD59-associated p56lck occurs in CD3-negative Jurkat cells, while IL-2 production is impaired, consistent with the lack of ZAP-70 tyrosine phosphorylation observed in these cells. CD59 triggers activation events even in the absence of CD3/T cell receptor expression in Jurkat cells. CD59 cross linking synergizes with sub-optimal doses of phorbol ester for activation of the protein kinase C and of the p42mapk, as shown by in vitro phosphorylation of histone HIIIS and myelin basic protein, respectively, and leads to CD25 but not CD69 expression. In conclusion, at least two signaling pathways are triggered through CD59, the first one involving ZAP-70 activation and leading to IL-2 secretion and a second pathway observed in the absence of ZAP-70 activation leading to CD25 expression. These two pathways are likely to be involved in the modulation of T cell activation by CD59 protein. PMID- 7542594 TI - Hyaluronan binding by CD44 is regulated by a phosphorylation-independent mechanism. AB - CD44 is an adhesion receptor for which the major characterized ligand is the extracellular matrix glycosaminoglycan, hyaluronan. This interaction underlies CD44-mediated cell attachment, cell migration, and matrix remodelling during development and wound healing. Truncation of the CD44 cytoplasmic domain does not prevent cell surface expression of this hyaluronan receptor but it dramatically impairs ligand binding. In this study we have examined the role of phosphorylation in regulating this function by mutating the target serine residues to either neutral amino acids with the aim of creating a phosphorylation incompetent molecule, or to acidic residues to mimic a fully phosphorylated CD44. In transfected AKR1 cells the behavior of both the neutral and acidic mutants was indistinguishable from wild-type CD44, indicating that there is a phosphorylation independent mechanism involved in regulating hyaluronan binding. PMID- 7542593 TI - CD20 monoclonal antibodies decrease interleukin-4-stimulated expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) in human B cells by increasing the extent of its cleavage. AB - CD20 monoclonal antibody (mAb) B1 is known to inhibit B cell proliferation. We show that B1 reduced both anti-mu + interleukin-4 (IL-4)-induced DNA synthesis and the concomitant expression of CD23 at the surface of human tonsillar B cells. B1 mAb had no effect on CD23 mRNA levels. The disappearance of CD23 molecule from the surface correlates with an increase of soluble CD23 fragments in the culture medium, indicating that CD20 mAb B1 stimulated the cleavage of the molecule. B1 also inhibits IgE production by peripheral blood mononuclear cells cultured in the presence of IL-4. Suppression of IgE synthesis and enhancement of CD23 cleavage are concomitant but appear not to be functionally related. PMID- 7542595 TI - Comparative analysis of B7-1 and B7-2 expression in Langerhans cells: differential regulation by T helper type 1 and T helper type 2 cytokines. AB - Epidermal Langerhans cells (LC) are Ia-bearing potent antigen-presenting cells (APC) of dendritic cell lineage that play a crucial role in primary and secondary T cell-dependent immune responses. LC express several costimulatory molecules such as B7, which has been implicated as one of the important determinants of professional APC. Recently, B7 antigens have been shown to include three distinct molecules termed B7-1, B7-2, and B7-3, and the expression of B7-1 and B7-2 in LC has been already confirmed. However, little is known of the regulation of B7-1 and B7-2 expression in LC. We demonstrated that LC do not express B7-1 and B7-2 in situ; however, the expression of both molecules is rapidly induced during the first 3 days of culture, and high levels of expression are maintained at least until day 6. We show that the expression of B7-2 in LC is much higher than that of B7-1 in each experiment, and that B7-1 and B7-2 expression is reproducibly augmented by interleukin (IL)-4 in a dose-dependent manner; however, IL-2 affected expression very little. Finally, B7-1 expression is significantly and dose-dependently down-regulated by interferon (IFN)-gamma or IL-10, and B7-2 expression is consistently inhibited by IL-10, but not by IFN-gamma. The effects of these cytokines are active only in the induction phase (during first 3 days of culture) of B7 expression: the modulatory effects of cytokines are hardly detected in the plateau phase (days 4 to 6 of culture) of B7 expression in LC. These findings suggest that B7-1 and B7-2 expression are indeed selectively and differentially regulated by these T cell-derived cytokines, and that the cytokines may modulate the synthesis of B7 molecules rather than the degradation of already-expressed B7 molecules. PMID- 7542596 TI - The effects of natural altered peptide ligands on the whole blood cytotoxic T lymphocyte response to human immunodeficiency virus. AB - Cytotoxic T lymphocytes (CTL) directed against human immunodeficiency virus (HIV) 1 are detectable in the majority of infected individuals, and their early appearance as the initial viremia is suppressed is thought to represent a potent antiviral response. Variation which arises in CTL epitopes can affect recognition by CTL, and we have observed previously that variant epitopes in HIV-1 gag which arise in HIV-1-seropositive donors may act as T cell receptor (TCR) antagonists of their own CTL (Klenerman et al., Nature 1994, 369: 403). The most important question arising from these observations is the extent of these immune escape mechanisms in vivo. Here we show that fresh, uncultured lymphocytes taken directly from HIV-1-infected patients are susceptible to TCR antagonism by variants present within their own virus. In contrast to HLA Class II-restricted T cell responses, where anergy may be induced, we find that in vitro, natural variants may stimulate and sustain growth of CTL. These CTL lines retain lytic specificity exclusively for the original peptide. If this represents events in vivo, natural HIV altered peptide ligands (APL) have the capacity to inhibit the range of CTL directed against an epitope, not simply those clones selected in vitro. Partial activation of CTL by APL could also act to drive an ineffectual CTL response incapable of lysing infected cells bearing these natural antigenic variants. Distortion of lymphocyte populations and function by APL might represent a further mechanism of immune evasion by HIV. PMID- 7542597 TI - Prevention and treatment of Lewis rat experimental allergic encephalomyelitis with a monoclonal antibody to the T cell receptor V beta 8.2 segment. AB - The predominance of T cell receptor (TCR) V beta 8.2 utilization by encephalitogenic T cells induced in Lewis rats by immunization with myelin basic protein (MBP) is controversial. Thus, both an almost exclusive usage of V beta 8.2 [Burns, F. R., Li, X., Shen, N., Offner, H., Chou, Y. K., Vandenbark, A. A. and Heber-Katz, E., J. Exp. Med. 1989, 169: 27; Chluba, J., Steeg, C., Becker, A., Wekerle, H. and Epplen, J. T., Eur. J. Immunol. 1989. 19: 279] and a quite diverse V beta composition of CD4 T cells causing experimental autoimmune encephalomyelitis (EAE) [Sun, D., Gold, P. D., Smith, L., Brostoff, S. and Coleclough, C., Eur. J. Immunol, 1992. 22: 591; Sun, D., Le, J. and Coleclough, C., Eur. J. Immunol. 1993. 23: 494] have been reported. Using a recently developed monoclonal antibody (mAb) specific for TCR V beta 8.2, we show that postnatal treatment effectively eliminates V beta 8.2-bearing cells and prevents MBP-induced EAE in the majority of Lewis rats. Moreover, treatment of adult Lewis rats with V beta 8.2-specific mAb as late as on day 12 after MBP immunization suppressed the development of neurological symptoms. Thus, V beta 8.2-bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) V beta 8.2-expressing T cell subset provides an effective therapeutic strategy. PMID- 7542598 TI - IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules. AB - The expression and function of IgM and IgD antigen receptors were studied in a series of anti-hen egg lysozymes (HEL) immunoglobulin (Ig)-transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD+, IgM- B cells was twofold higher than on IgM+, IgD- B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down-regulation of IgD occurred following exposure to antigen in vitro. When regulation of CD80/CD86 co stimulatory molecules by surface Ig was examined, up-regulation of the former was initiated at lower antigen concentrations on IgM-, IgD+ compared to IgM+, IgD- B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells. PMID- 7542599 TI - Mechanism of enhanced antigen presentation by B cells activated with anti-mu plus interferon-gamma: role of B7-2 in the activation of naive and memory CD4+ T cells. AB - B cells activated with anti-mu antibody plus interferon (IFN)-gamma exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-mu, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-gamma further augmented the expression of B7-2 on anti-mu-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-mu plus IFN-gamma. These results suggest that IFN-gamma plays an important role in recruitment of naive T cells for an immune response. PMID- 7542602 TI - Restriction of self-antigen presentation to cytolytic T lymphocytes by mouse peptide pumps. AB - Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2Kd class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies. PMID- 7542600 TI - Cassette baculovirus vectors for the production of chimeric, humanized, or human antibodies in insect cells. AB - Plasmid cassette-transfer vectors pBHuC chi and pBHuC gamma l have been designed which enable the construction of recombinant baculoviruses directing the co expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti-human CD29 monoclonal antibody (Mu-K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch-K20-sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype. PMID- 7542601 TI - Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin. AB - The Gal beta(1-3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4+ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4+ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-gamma and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3- Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4- Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars. PMID- 7542606 TI - B7.2 provides co-stimulatory functions in vivo in response to staphylococcal enterotoxin B. AB - Excessive T cell activation induced by bacterial superantigens plays an important role in the pathology associated with Gram-positive bacteremia. To gain insight into the early phases of T cell activation by bacterial enterotoxins in vivo, we investigated the ability of antibodies to well-defined co-stimulatory molecules to inhibit T cell activation and the subsequent toxic shock syndrome induced in BALB/c mice following the injection of staphylococcal enterotoxin B (SEB). We demonstrate here that a single dose of anti-B7.2 antibodies, but not anti-B7.1 antibodies, significantly inhibits T cell activation, as judged by lower systemic IL-2 release, blastogenesis and IL-2 receptor expression, and reduces the lethal effect of SEB in D-galactosamine-sensitized mice. These results demonstrate that co-stimulation through the B7.2 molecule plays an important role in the activation of T cells in response to SEB in vivo and suggest alternative therapies for septic shock caused by bacterial enterotoxins based on blocking antibodies to co-stimulatory molecules. PMID- 7542608 TI - Transition and determinants of orthodontic root resorption-repair sequence. AB - When severe root resorption is observed during orthodontic treatment the clinician needs to know how the active process can be stopped. It is generally assumed that if active force is discontinued, the root resorption will end. The determinants of the resorption/repair sequence are, however, not well understood. The aim of the present study was to register and analyse determinants that may influence the extent of resorbed root surface, as well as the transition of a process of active root resorption into a process of tissue deposition/repair in the resorption lacunae. Using an experimental model that would simulate the first force-activation cycle, the upper first molars of rats were moved mesially by a fixed orthodontic appliance for periods between 2 and 21 days. The results revealed that (1) the extent of root resorption after 21 days corresponded with the maximal extent of the hyalinized zone; (2) a process of repair started from the periphery in the resorbed lacunae where the periodontal ligament (PDL) had been re-established, while ongoing active resorption was observed beneath the existing hyalinized tissue; and (3) root resorption continued in the area where hyalinized tissue persisted even after active force had terminated. It is hypothesized that determinants of continued resorption/repair generally seem to be associated with the persistence and removal of the necrotic tissue. PMID- 7542603 TI - Self and non-self peptides treat autoimmune encephalomyelitis: T cell anergy or competition for major histocompatibility complex class II binding? AB - In susceptible strains of mice, myelin basic protein (MBP) peptide Acl-ll induces experimental autoimmune encephalomyelitis (EAE) providing a useful model for human multiple sclerosis. Acl-11 binds major histocompatibility complex (MHC) class II molecules A alpha upsilon A beta upsilon. Here, we show that the Acl-11 peptide, when administered intraperitoneally in incomplete Freund's adjuvant (IFA) emulsion, can effectively treat Acl-11-induced EAE in mice. Treatment with Acl-11/IFA 9 days after initial immunization with Acl-11 in complete Freund's adjuvant (CFA) results in a loss of T cell proliferation to MBP Acl-11. This lack of T cell proliferation is not due to T cell anergy and is not specific. A similar lack of T cell proliferation and inhibition of EAE is observed when an ovalbumin peptide OVA323-339 or a sperm whale myoglobin peptide SWM110-121 are used to treat mice immunized with Acl-11. Interestingly, we show that previously unresponsive lymph node cells from treated mice respond normally if Acl-11 is presented by fresh antigen-presenting cells taken from normal mice. These results argue that the lack of T cell proliferation and inhibition of EAE is not due to specific T cell anergy as suggested by others. Instead this appears to be due to blocking of MHC class II molecules A alpha upsilon A beta upsilon by the treating peptides. PMID- 7542607 TI - Inhibition of constitutive nitric oxide synthase in the brain by pentamidine, a calmodulin antagonist. AB - Nitric oxide (NO) which is produced by activation of Ca2+/calmodulin-dependent NO synthase is known to induce neuronal damage. We examined the effects of 3'-azido 2',3'-dideoxythymidine (AZT, a reverse transcriptase inhibitor), pentamidine (a therapeutic drug for Pneumocystis carinii pneumonia) and calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W 7) on NO synthase activation. Although AZT had no effect on the activity of constitutive neuronal NO synthase, pentamidine inhibited the activation of neuronal NO synthase as did trifluoperazine and W-7. The inhibition by pentamidine was prevented by the addition of purified calmodulin. In addition, pentamidine inhibited calmodulin-dependent activation of neuronal NO synthase purified from rat cerebellum. From these results, it is suggested that pentamidine inhibits the neuronal NO synthase activation by probably acting as a calmodulin antagonist. PMID- 7542605 TI - Selective killing of T cells by immunotoxins directed at distinct V beta epitopes of the T cell receptor. AB - The potency and specificity of anti-T cell receptor (TcR)-directed immunotoxins were studied in two T cell leukemia lines, HPB-ALL and Jurkat, and in primary T cells. Immunoconjugates were synthesized using anti-CD3, or distinct anti-V beta antibodies cross-linked to CRM9, a binding site-mutant of diphtheria toxin. All TcR-expressing cells display the CD3 complex on the plasma membrane. HPB-ALL cells express the V beta 5 gene product in the beta subunit of the TcR, while Jurkat cells express V beta 8. V beta expression in primary T cells isolated from buffy coats is heterogeneous. Primary T cell populations expressing specific V beta epitopes in the TcR were generated by plating CD3+ T cells on V beta specific antibody-coated flasks or by positive immunomagnetic selection. Immunotoxins directed against the invariant CD3 epsilon epitope target and kill all T cells. Immunoconjugates targeted at distinct anti-V beta epitopes are specific for cells that express the corresponding gene product in the TcR. The results demonstrate the ability of anti-TcR-based immunotoxins selectively to kill T cells with defined V beta epitopes. These reagents may be clinically useful in disorders mediated by autoreactive T cell populations exhibiting V beta restriction and in the treatment of clonal TcR-expressing lymphomas. PMID- 7542604 TI - Activation of human peripheral blood dendritic cells induces the CD86 co stimulatory molecule. AB - Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA 4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC. PMID- 7542609 TI - Mechanical sensitivity of regenerating myelinated skin and muscle afferents in the cat. AB - These experiments describe the responses of myelinated skin and muscle afferent nerve fibres at a neuroma to stretch, local pressure and vibration in the anaesthetised cat. The sural nerve and the nerve supplying the medial gastrocnemius were studied. Neuroma formation was encouraged by placing the cut end of the nerve in a cuff made of synthetic material (Gore-tex). By 6 days after nerve section, the two nerves contained mechanically sensitive afferents. No motor fibres appeared to be mechanically sensitive. Mechanically sensitive sural afferents responded to ramp stretch of the nerve, applied at the cuff, with a single impulse or brief burst of impulses. The majority of gastrocnemius afferents responded to stretch with slowly adapting trains of impulses. Many muscle group II afferents exhibited a steady resting discharge, while group I afferents had an intermittent or bursting resting discharge or were silent. Those group I axons which showed resting activity had a low stretch threshold and were probably Ia fibres. Many of the silent units were also stretch sensitive. It is proposed that the spontaneously active units and silent units with low stretch thresholds were Ia fibres, while silent units with high stretch thresholds were Ib fibres. Both sural and gastrocnemius afferents responded to locally applied vibration. The mean peak response frequency for sural units was 170 Hz (+/- 70 Hz SD). For gastrocnemius units it was 325 Hz (+/- 86 Hz SD). Group I muscle afferents responded to higher frequencies of vibration than group II afferents. In four experiments the nerve was treated at a site a few millimetres proximal to the point of section with the axonal transport blocker colchicine. Twenty-five millimolar colchicine blocked impulse conduction at its point of application. Nevertheless, mechanically sensitive areas developed in the nerve just proximal to the treated region. Ten millimolar colchicine did not block impulse conduction, but led to dispersion of mechanosensitive areas to more proximal regions of the mechanosensitive areas to more proximal regions of the nerve. This result suggests that the disruption of orthograde axonal transport by colchicine leads to development of mechanically sensitive areas in axons further back from their cut ends. Local application of the drugs succinyl choline, tetra-ethyl ammonium and gadolinium had no effect on levels of resting activity or on mechanical sensitivity of afferents in the cuff. The potassium channel blocker 4 aminopyridine, on the other hand, produced an increase in the levels of resting activity and in the stretch responses of afferents.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542610 TI - [The effects of substance P and capsaicin on synaptic transmission in the peripheral reflex pathways of the rat caudal mesenteric ganglion]. AB - Peripheral nerve pathways in the caudal mesenteric ganglion of the rat were found. They preserve synaptic transmission after degeneration of preganglionic nerve fibres. To find the effects of substance P (SP) and capsaicin, the SP antagonist, on the synaptic transmission in peripheral reflex pathways of the caudal mesenteric ganglion rats were investigated using extracellular recording of action potentials from the ganglion nerves. Perfusion of the ganglion by solution containing SP (5 x 10(-7) and 1 x 10(-8) mol/l), facilitated the transmission, while capsaicin (5 x 10(-5) mol/l) inhibited it. The inhibitory effect grew at high-frequency stimulation of peripheral nerves. PMID- 7542611 TI - Exposure of adhesion molecules on activated platelets in patients with newly diagnosed IDDM is not normalized by near-normoglycemia. AB - It has been suggested that platelet hyperactivity contributes to the early evolution of diabetic vascular disease per se. This study directly evaluates the level of intravascular platelet activation in newly diagnosed IDDM patients before and after tight metabolic control. Platelet activation was determined by the Duesseldorf-III flow cytometry assay in 21 recent-onset hyperglycemic IDDM patients before insulin, after 3 days of treatment with intravenous insulin, and after 14 and 60 days of intensified conventional insulin therapy. The intravasal platelet activation status was quantified by the percentage of platelets exposing the activation-dependent molecules CD62 (P-selectin), thrombospondin (TSP), and CD63 (GP53) as well as the activated fibrinogen receptor (GPIIB/IIIA). Fifty matched normal subjects served as control subjects. Fourteen patients completed the 60-day study design. After initial recompensation, near-normoglycemic control was achieved after 14 days (fasting blood glucose, 117.0 +/- 19.0 mg/dl), and the HbA1 concentration was 7.6 +/- 1.2% after 60 days. CD62+ (4.0 +/- 4.5%), TSP+ (2.0 +/- 1.8%), CD63+ (11.0 +/- 7.0%), and activated-GPIIB/IIIA+ (7.6 +/- 7.7%) platelet levels were initially 5, 3.3, 5.7, and 2.8 times higher than the mean level of normal. There was no correlation with any of the nearly normalized metabolic parameters. Thus, more activated platelets circulate in newly diagnosed IDDM patients, which supports the assumption of a prethrombotic condition even in disease stages without apparent vascular damage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542613 TI - Keratins as markers of differentiated taste cells of the rat. AB - Cytokeratins in taste buds were immunocytochemically evaluated with monoclonal antibodies. In each of six different epithelial sites in the rat oral cavity, intragemmal cells of taste buds were immunoreactive for keratin polypeptides 8, 18, and 19, as well as for keratin 7, which has not been previously reported in taste buds. Keratin-18-like immunoreactivity was present in fewer than half of the intragemmal cells, whereas all intragemmal cells were immunopositive for keratins 7, 8, and 19. Apart from some salivary duct cells, no other cells in the tongue were immunoreactive for any of these four keratins. Morphological and immunocytochemical profiles indicate that taste buds are islets of simple epithelium embedded in an expanse of stratified squamous epithelium. These simple epithelial cells and their keratins are nerve-dependent, since denervation eliminated all four keratins and replaced elongated taste cells of the vallate papilla with stratified squamous epithelium. We conclude that antibodies against keratins 7, 8, or 19 are useful markers for intragemmal cells in studies of taste bud development, degeneration, regeneration, turnover and tissue culture. PMID- 7542612 TI - Sex-dependent expression of tenascin-C in the differentiating fetal rat testis and ovary. AB - The cellular mechanisms controlling sexual differentiation of fetal gonads are poorly understood. By examining the protein and mRNA expression of tenascin-C in correlation with the immunocytochemical detection of alpha-smooth muscle actin (alpha-SMA) and basement membrane heparan sulfate proteoglycan (HSPG) we demonstrate a clear-cut sex-and development-dependent expression pattern of tenascin-C in the rat testis, ovary and mesonephros. Immunocytochemistry and in situ hybridization of tenascin-C in 15-day-pc fetal testis and ovary showed protein and mRNA accumulation within the mesenchyme of the mesogonadal connection. In addition to the male and female mesonephros, some labeling could also be seen within the testicular tunica albuginea and intraovarian mesenchymal septa. In the 17-day-pc testis abundant accumulation of tenascin mRNA and protein appeared in the tunica and mediastinum testis, but not at all in the intratesticular mesenchyme. A similar pattern was still seen in the newborns where, however, a decrease in the anti-tenascin immunoreactivity of the tunica and mediastinum could be demonstrated. In contrast to the testis, expression of tenascin in 17-day-pc ovaries was widespread within the hilus and the entire intragonadal mesenchyme where it continued to accumulate also in newborns. Northern blot analysis of tenascin-C mRNAs showed one message of 8.0 kb in the 15 day-pc male and female gonads. An additional weak signal of 6.5 kb was seen in the female mesonephros. In the 18-day-pc testis, the 6.5-kb signal appeared stronger than the 8.0-kb signal. In contrast to the testis, the 6.5-kb message was weak in the developing ovary where the 8.0-kb signal had an intense peak on the day 18 pc. Further, in the ovary, mesenchymal accumulation of HSPG coincided with the spatial distribution of tenascin. In the testicular tunica and in the mesenchyme of the male and female genital ducts expression of tenascin was parallel with the differentiation of smooth muscle tissue, detected by labeling for alpha-SMA, which also indicated the tenascin-negative myoid cells of the testis. Our results indicate that tenascin expression in the fetal rat internal genitalia is involved in the differentiation of smooth muscle cells but not intratesticular myoid cells. In the ovarian mesenchyme, tenascin-C may have a specific function in the dynamic remodeling of the ovarian cords. PMID- 7542614 TI - Determination of the genomic structures of two nonallelic preproinsulin genes in Xenopus laevis using the polymerase chain reaction. AB - We have previously cloned cDNAs that correspond to two different nonallelic preproinsulin genes in the amphibian, Xenopus laevis (Shuldiner et al., 1989, J. Biol. Chem. 264, 9428-9432). The coding regions of the two genes are very similar (i.e., 93% amino acid identity). While both preproinsulin genes are expressed coordinately in the adult pancreas, they are differentially expressed in prepancreatic embryos during neurulation (Shuldiner et al., 1991, Proc. Natl. Acad. Sci. USA 88, 7679-7683). We now report the use of the polymerase chain reaction (PCR) to investigate the genomic structures of both Xenopus preproinsulin genes. First, the nucleotide sequence of a portion of the 5' untranslated region that was lacking in our cDNA clones was obtained using rapid amplification of cDNA ends (5' RACE). Then, oligonucleotide primers were designed that flanked putative exon-intron splice boundaries, and intronic sequences in each of the two nonallelic genes were amplified. We determined that both Xenopus preproinsulin genes contain two introns, one interrupting the 5' untranslated region, and the second interrupting the region encoding the C-peptide. The introns are similar in position, but of greater length than those reported in most other species. Interestingly, dideoxy sequence analysis of the PCR-amplified introns revealed that the exon-intron splice junctions are well conserved between the two nonallelic genes, but internal to these junctions, the respective introns diverge significantly from each other. Thus, ample time has elapsed since the duplication of these two genes for marked divergence to occur within the introns suggesting that these regions are not important for expression in the adult pancreas. From these studies, we predict that elucidation of the sequences of the 5' flanking regions of the two nonallelic preproinsulin genes will reveal conserved regions that will be important for coordinate expression, as well as less conserved regions that will either be unimportant for coordinate expression (i.e., pancreatic expression) or important for differential expression (i.e., prepancreatic expression). PMID- 7542615 TI - HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression. AB - Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. To test the hypothesis that intron-independent gene expression involves positive, cis-acting RNA sequence elements, we constructed chimeric genes in which various regions of the naturally intronless HSV-TK gene were inserted into an intronless variant of the highly intron-dependent human beta-globin gene. Using a transient transfection assay, we identified a 119-nucleotide sequence element contained within the transcribed region of the HSV-TK gene that enables efficient cytoplasmic accumulation of globin RNA in the absence of splicing. RNA UV-cross linking assays indicated that a 68-kD protein present in nuclear extracts of HeLa and COS cells specifically binds to this HSV-TK sequence element. This 68-kD protein was found to cross-react with an antiserum specific to hnRNP L. Recombinant hnRNP L was shown to bind with high sequence specificity to this RNA sequence element. Analysis of substitution mutants in this element indicated that binding of hnRNP L correlates with accumulation of the RNA in the cytoplasm. Thus, we conclude that (1) hnRNP L binds in a sequence-specific manner to this RNA sequence element that enables intron-independent gene expression, and (2) intron-independent pre-mRNA processing and transport involves sequence-specific RNA-protein interactions between cis-acting RNA sequence elements and proteins such as hnRNP L. This sequence element may be of general use for the efficient expression of cDNA versions of intron-dependent genes. PMID- 7542616 TI - GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3. AB - GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation. PMID- 7542617 TI - Receptor-mediated transfer of pSV2CAT DNA to a human hepatoblastoma cell line HepG2 using asialofetuin-labeled cationic liposomes. AB - Asialofetuin-labeled liposomes (AF-lps) were developed as a vector for gene transfer to hepatocytes. Plasmid pSV2CAT DNA which encodes bacterial chloramphenicol acetyltransferase (CAT) was associated with (meaning, in this report, the sum of 'to be adsorbed on the surface of' and 'to be encapsulated into the internal phase of') AF-lps (AF-lps-pSV2CAT) prepared by a tandem combination of the detergent removal and freeze-thaw methods. Ninety-six percent of input pSV2CAT was associated with AF-lps containing N-(alpha trimethylammonioacetyl)-didodecyl-D-glutamate chloride, and approx. two-thirds of the associated DNA was encapsulated into the internal phase. The uptake of AF-lps by the cultured human hepatoblastoma cell line HepG2, having asialoglycoprotein receptors (AGPR) on their plasma membrane, was decreased by the addition of free AF and cytochalasin B. AF-lps bound to HepG2 cells through specific interaction with AGPR, and were internalized into the cells by the receptor-mediated endocytotic pathway. HepG2 cells transfected by AF-lps-pSV2CAT showed a significantly higher CAT activity than those transfected by pSV2CAT associated with non-labeled control lps (N-lps-pSV2CAT) or a mixture of pSV2CAT and empty AF lps. Pretreatment with EDTA-encapsulated AF-lps increased the transfection efficiency of AF-lps-pSV2CAT. The CAT activity in A431 and Swiss/3T3 cells transfected with AF-lps-pSV2CAT was low and almost the same as those transfected with N-lps-pSV2CAT. Since DNA encapsulated in lps is likely to be protected against digestion by nucleases in the blood circulation, AF-lps could be used as a gene transfer vector targeting the hepatocytes in vivo. PMID- 7542620 TI - The influence of urinary incontinence on publicly financed home care services to low-income elderly people. AB - Urinary incontinence (UI) has been shown to be prevalent and a risk factor for permanent institutionalization; yet it is not routinely measured in research of home care utilization. A retrospective cohort design is used to directly estimate the effect of UI on the public costs of home care services to elderly individuals. Multivariate analyses controlling for other individual, household, and supply characteristics demonstrate that those with UI generate significantly greater public costs for home care services. Patterns of service use suggest palliative rather than rehabilitative service, raising questions regarding the effective use of resources. PMID- 7542619 TI - Evaluation of IBMX-enhanced ocular hypotension after adrenergic agonists in the rabbit eye. AB - BACKGROUND: 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, enhanced the reduction of intraocular pressure more after administration of norepinephrine and epinephrine than after isoproterenol. The question arises of whether or not the IBMX-induced enhancement of ocular hypotension is exclusively due to beta 2-adrenoceptor/cAMP stimulation. METHODS: In groups of eight rabbits the ocular hypotensive responses after selective adrenergic agonists were studied in the presence and absence of phosphodiesterase inhibition with IB-MX. RESULTS: Pretreatment with IBMX 1%, applied topically, did not enhance the ocular hypotensive responses after phenylephrine (alpha 1), B-HT920 (alpha 2) and dobutamine (beta 1). The ocular hypotensive responses induced by salbutamol (0.001-0.5%) and higher concentrations of terbutaline were significantly enhanced by IBMX. Combined treatments of terbutaline 0.01% and B-HT920 0.2%, dobutamine 3% and phenylephrine 2%, and dobutamine 3% and B-HT920 0.2% were not associated with enhanced ocular hypotensive responses in the presence of IBMX. The only combination that was associated with a significant enhancement of ocular hypotension when combined with 1% IBMX was phenylephrine 2% and terbutaline 0.01%. A subthreshold dose of phenylephrine 0.1% further increased the enhanced ocular hypotensive responses induced by salbutamol 0.025, 0.2 and 0.5% in combination with IBMX. CONCLUSIONS: Phosphodiesterase inhibition with IB-MX enhances the ocular hypotensive effect induced by catecholamines not only by beta 2-adrenoceptor/cAMP stimulation, but also by simultaneous alpha 1-adrenoceptor stimulation. PMID- 7542618 TI - Correlation of histological tumor vascularization and Doppler sonography in patients with malignant melanoma of the choroid. AB - BACKGROUND: In previous studies Doppler sonography was proven to the useful for diagnostics and follow-up of malignant melanomas of the choroid. METHODS: To evaluate the correlation between Doppler sonographical findings and the histological tumor vascularization, we examined five eyes of five patients with malignant melanoma of the choroid before enucleation with an ATL Ultramark-8 Duplex scanner and compared these results with computerized planimetry of histological tumor vascularization after immunohistochemical staining of vascular endothelium with anti-factor-VIII antibodies. RESULTS: We found no definite correlation of flow velocities with histological parameters of tumor vascularization. The tendency was for a decrease in maximum flow velocities as histological vascularization increased. CONCLUSIONS: Due to (1) the usual lack of sensitivity parameters of the used Duplex device for the clinical user and (2) the unpredictable branching pattern within a melanoma resulting in an unknown angle of incidence of the Doppler beam, we conclude that the quantitative results of Doppler sonography require cautious interpretation. Nevertheless, presence or absence of Doppler shifts is a valuable parameter for the follow-up of irradiated tumors. PMID- 7542621 TI - Serum levels of tumor necrosis factor (TNF), soluble receptors for TNF (55- and 75-kDa sTNFr), and soluble CD14 (sCD14) in epithelial ovarian cancer. AB - The preoperative serum levels of tumor necrosis factor (TNF), soluble receptors for TNF (55- and 75-kDa sTNFr), and soluble CD14 (sCD14) were retrospectively measured in 66 patients with epithelial ovarian cancer and in 59 patients with benign ovarian masses. The preoperative serum TNF and sCD14 levels were higher in patients with epithelial ovarian cancer than in those with benign ovarian disease (P = 0.001 and P < 0.0001, respectively). Among patients with advanced malignancy, preoperative serum TNF and sCD14 correlated with neither the common prognostic variables nor the clinical outcome of patients. The preoperative serum 55- and 75-kDa sTNFr levels were higher in patients with epithelial ovarian cancer than in those with benign ovarian disease (P < 0.0001 and P = 0.02, respectively). Among patients with advanced malignancy, preoperative serum 55- and 75-kDa sTNFr correlated with FIGO stage (IV vs III, P = 0.008 and P = 0.01, respectively) and with the clinical outcome of patients. Among patients followed after surgery and chemotherapy for advanced epithelial ovarian cancer, 55- and 75 kDa sTNFr levels were significantly higher in the samples drawn from patients with clinical evidence of disease when compared to those from patients without clinical evidence of disease; conversely, TNF and sCD14 levels were similar in the two groups. In conclusion, the preoperative serum levels of TNF, 55- and 75 kDa sTNFr, and sCD14 were significantly higher in patients with epithelial ovarian cancer than in those with benign ovarian disease. The measurement of serum TNF and sCD14 seemed to be of limited clinical value for the management of patients with advanced epithelial ovarian cancer. Conversely, the assay of serum 55- and 75-kDa sTNFr might have a potential clinical relevance, for both prognostic purposes and assessment of disease status. PMID- 7542622 TI - Expression and function of beta 1 and alpha v beta 3 integrins in ovarian cancer. AB - Ovarian cancer cells disseminate by implanting onto the peritoneal mesothelial cell surface of the abdominal cavity. A common feature of these peritoneal implants is the presence of tumor cell invasion into the submesothelial extracellular matrix (ECM). In view of the important role of integrins in ECM recognition and cell migration, we were interested in defining the pattern of integrin expression and function in ovarian cancer cell lines and primary tissue samples. The beta 1 integrin chain was expressed by CAOV-3, SKOV-3, OVCAR-3, and SW626 ovarian cancer cell lines, associated with expression of alpha 1, -2, -3, 5, and -6 chains. The alpha 4 chain was also expressed by two of the four lines. In addition to beta 1 integrins, the alpha v beta 3 integrin was also expressed, although there was no expression of beta 2, -4, and -7 chains. Immunoprecipitation of surface-labeled CAOV-3 cells with an anti-beta 1 antibody revealed a band at approximately 110-130 kDa consistent with the known molecular mass of the beta 1 chain, as well as several associated bands consistent with noncovalently linked integrin alpha chains. A similar pattern of beta 1 and alpha v beta 3 integrin expression was observed for primary ovarian cancer tissue samples. Ovarian cancer cell lines exhibited significant binding to collagen type I and laminin which was primarily mediated by beta 1 integrins. In contrast, ovarian cancer cell binding to fibronectin was mediated by both alpha 5 beta 1 and alpha v beta 3 integrins. Even though mesothelial cells were observed to express fibronectin mRNA and protein, binding of ovarian cancer cells to peritoneal mesothelium was not blocked by neutralizing antibodies to beta 1 or alpha v beta 3 integrins. These data suggest that functional integrins are commonly expressed by ovarian cancer cells, although they do not appear to mediate ovarian cancer cell implantation onto peritoneal mesothelium. The role that integrins play in the invasion of ovarian cancer cells into the submesothelial ECM deserves further investigation. PMID- 7542624 TI - Deconvolution of infrequently sampled data for the estimation of growth hormone secretion. AB - In this paper, the deconvolution of infrequently and nonuniformly sampled data is addressed. A nonparametric technique is worked out that provides a smooth estimate of the unknown input signal and takes into account nonnegativity constraints. In spite of the size of the problem, efficient algorithms for solving the constrained optimization problem and computing confidence intervals are proposed. The new technique is used to estimate growth hormone (GH) secretion after repeated GH-releasing hormone (GHRH) administration from samples of blood concentration. PMID- 7542623 TI - The types of neurons in the claustrum of bison bonasus: Nissl and Golgi study. AB - The Nissl staining and Golgi impregnation methods has been used to characterize the types of neurons in the claustrum of bison bonasus. Two fairly divergent subpopulations of cells can be distinguished by means of Golgi preparation technique. The first group--comprising the bulk of claustral neurons--is composed of only one type of large, efferent nerve cells with long axons and dendrites covered with spines. The somata and initial portion of dendritic processes are devoid of spines. The second group consists of large and medium-size interneurons with smooth infrequently branching dendrites. Amongst them two sub-populations can be observed: with dendritic swellings and with dendrites deveoid of them. In Nissl stained sections the claustral neuronal population consists of large multipolar cells among which fewer large or medium-size, spindle-shaped cells are dispersed. The multipolar cells and also spindle-shaped neurons contain widely distributed, deeply-stained, medium-sized pigment granules. No orderly alignment of cells can be seen. PMID- 7542625 TI - Prostaglandin E2 induces up-regulation of murine macrophage beta-endorphin receptors. AB - Cultured murine bone marrow macrophages specifically bound 125I-labeled beta endorphin. Binding was displaceable by 100 times molar excess of full-length beta endorphin but was insensitive to the opioid receptor antagonist, naloxone. Binding was inhibited by beta-endorphin's C-terminal tetrapeptide, lys-lys-gly glu, but not by the truncated N-terminal 27 amino acid fragment, indicating that binding of beta-endorphin to this receptor is dependent on its C-terminus. Macrophages incubated for 24 h with 10(-8)-10(-5) M prostaglandin E2 showed a dose-dependent increase in beta-endorphin binding, implying receptor up regulation. This was also observed in response to the phosphodiesterase inhibitor, isobutylmethylxanthine, indicating that regulation of these receptors may be mediated through a cAMP-dependent process. This is the first demonstration that beta-endorphin receptor expression can be positively regulated. PMID- 7542627 TI - IL-10 inhibits the adhesion of leukocytic cells to IL-1-activated human endothelial cells. AB - Cytokines play an important role in the recruitment of leukocytes from blood to sites of tissue injury and inflammation. Previous studies showed that the pro inflammatory cytokine, IL-1, induces the adhesion of leukocytes by up-regulating the expression of the adhesion molecules ICAM-1 and VCAM-1 on endothelial cells. IL-10, a pleiotropic mediator, inhibits the production of cytokines by macrophages and down-regulates the antigen-presenting function of macrophages, thereby acting as a suppressor of immune responses. This study was undertaken to evaluate the effect of IL-10 on the adhesion of leukocytic cells to human umbilical vein endothelial cells. IL-10 inhibited the adhesion of a human monocytic cell line (THP-1) and a human lymphoblastic T-cell line (MOLT-4) to IL 1-stimulated endothelial cells. IL-10 also down-regulated the expression of ICAM 1 and VCAM-1 on IL-1-activated endothelial cells. IL-10-treated THP-1 cells adhered less than untreated THP-1 cells to IL-1-activated endothelial cells, whereas direct treatment of MOLT-4 had minimal effect on its adhesion to cytokine activated endothelial cells. These results suggest that IL-10 counteracts the pro inflammatory effects of IL-1 and regulates the adhesion of leukocytic cells to endothelial cells. PMID- 7542626 TI - Molecular modeling of the steric structure of the envelope F1 antigen of Yersinia pestis. AB - Steric structure of the envelope F1 protein of Yersinia pestis was reconstructed by computer modeling taking into account structural similarities between F1 and interleukins (IL)-1 alpha, -beta, -ra and by using the known atomic coordinates for huIL-1 beta obtained by the X-ray crystallography. Of 18 hydrophobic positions forming a hydrophobic core in all the proteins studied with the IL-1 like conformation, 15 positions are occupied by hydrophobic residues in F1 protein as well. Of 8 homologous positions occupied by the amino acid residues of similar charge in all huIL-1 alpha, -beta, -ra, 5 positions are conserved in F1 antigen. The B-cell epitope accessible to antibodies in polymeric F1 is exposed as an hydrophilic loop at the surface opposite to the C-terminal sequence, forming a conserved binding site with periplasmic molecular chaperones. PMID- 7542628 TI - Anti-immunoglobulin and anti-CD40 stimulation induces CD25 expression by resting human tonsillar B lymphocytes. AB - In this report, the effect of ligation of a number of B-cell surface molecules upon expression of CD25, the 55-kDa inducible component of the IL-2 receptor complex found on T and B lymphocytes, is reported. IL-4 is the only cytokine apparently capable of promoting CD25 expression in human high-density quiescent tonsillar B cells; neither IL-10 nor IL-13 could induce CD25 expression. Cross linking of the antigen receptors or CD40 with antibody elicited CD25 expression in a dose-dependent manner. Stimulation with anti-CD40 promoted CD25 expression in approximately 25% of B cells, while anti-Ig caused 80% or more of cells to become CD25+. In experiments where the stimuli were used in combination, some additive effects upon CD25 expression were noted, but no obvious synergistic effects could be detected. PMID- 7542629 TI - Role of protein tyrosine kinases and phosphatases in isotype switching: crosslinking CD45 to CD40 inhibits IgE isotype switching in human B cells. AB - Protein tyrosine kinases and protein tyrosine phosphatases play an important role in the transduction of signals via antigen receptors in T and B cells, and in CD40-dependent B-cell activation. To examine the role of tyrosine kinases and phosphatases in B-cell isotype switching, we examined the effects of the engagement of the transmembrane phosphatase CD45 on the synthesis of IgE induced by IL-4 and anti-CD40 monoclonal antibody (mAb). Crosslinking CD45 to CD40 using biotinylated mAbs and avidin strongly inhibited CD40-mediated IgE synthesis in IL 4-treated human B cells. CD40/CD45 crosslinking did not affect epsilon germline transcription in B cells stimulated with IL-4, but strongly inhibited induction of S mu/S epsilon switch recombination as detected by a nested primer polymerase chain reaction assay. The B-cell src-type tyrosine kinase lyn, which is activated following CD40 engagement, is a potential target for the effects of CD45 observed in our experiments, because CD45/CD40 crosslinking resulted in the inhibition of CD40-mediated lyn phosphorylation and activation. These results suggest an important role for protein tyrosine kinases and phosphatases in CD40-mediated induction of isotype switching to IgE. PMID- 7542630 TI - Specificity and cross-reactivity of anti-galactocerebroside antibodies. AB - Anti-galactocerebroside (GalC) antibodies have been reported to inhibit myelin formation, cause demyelination, and block HIV-I infection of neural cells. We examined the binding of 3 monoclonal and polyclonal anti-GalC antibodies to a panel of purified glycolipids by ELISA and by an immunospot assay on nitrocellulose blots. All 3 antibodies bound strongly to GM1 ganglioside, monogalactosyl diglyceride, and asialo-GM1, and 2 of the antibodies bound to GD1b and psychosine. The anti-GalC antibodies also bound to 3 glycoprotein bands in human neuroblastoma cells on Western blot, and binding to the proteins was abolished by pre-treatment with pronase or with periodate which oxidizes the terminal carbohydrate residues. These results indicate that anti-GalC antibodies cross react with oligosaccharide determinants of other glycolipids and glycoproteins, and that these cross-reactivities may be responsible for some of the biological effects of the anti-GalC antibodies. PMID- 7542633 TI - Binding of native alpha 2-macroglobulin to human group G streptococci. AB - Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci. PMID- 7542631 TI - Comparative immunogenicity of conjugates composed of Escherichia coli O111 O specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes. AB - Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed. PMID- 7542634 TI - Humoral immunity to aerosolized staphylococcal enterotoxin B (SEB), a superantigen, in monkeys vaccinated with SEB toxoid-containing microspheres. AB - Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9 groups were vaccinated with the microspheres via combinations of mucosal and nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol. Monkeys primed with an intramuscular dose of the microspheres followed by an intratracheal booster all survived the SEB challenge. Overall, monkeys with an intratracheal booster generally had the highest antibody levels, which is consistent with their high survival rate and lower rate of illness. Protective immunity was correlated with antibody levels in both the circulation and the respiratory tract. The protection was not due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in cultures of circulating lymphocytes was not significantly reduced after the microsphere vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or hypersensitivity because the monkeys did not die immediately after SEB challenge and there were no significant differences in histamine levels between the vaccinated and control monkeys before and after SEB challenge. The antibodies seemed to neutralize the SEB that got into the airway and the circulation. PMID- 7542632 TI - Clearance of Pseudomonas aeruginosa from the murine gastrointestinal tract is effectively mediated by O-antigen-specific circulating antibodies. AB - The colonization of mucosal surfaces by Pseudomonas aeruginosa can lead to local or disseminated disease. Secretory immunoglobulin A (IgA) has been assumed to be responsible for preventing mucosal colonization by interfering with the binding of bacterial ligands to epithelial surface receptors. However, the efficacy of this mechanism of immunity derives little actual support from in vivo experiments. In an investigation of the role of local and systemic immunization strategies in reducing colonization of the gastrointestinal tract of mice by P. aeruginosa, the bacterial antigens that were potential targets for immune effectors promoting mucosal clearance were identified. Levels of gastrointestinal colonization were reduced when immunity to homologous O antigens, but not that to pili or flagella, was elicited. Oral vaccination with attenuated Salmonella typhimurium expressing P. aeruginosa serogroup O11 antigen elicited mucosal and serum IgA antibodies and serum IgG antibodies specific for the recombinant antigen. Oral challenge of immunized mice with P. aeruginosa serogroup O11 demonstrated protection against gastrointestinal colonization. Intraperitoneal immunization with a serogroup O11 high-molecular-weight O-polysaccharide antigen elicited only serum IgG and IgM antibodies yet was as effective as oral vaccination in protecting mice against gastrointestinal colonization. This finding was confirmed by the demonstration that intraperitoneal immunization with purified lipopolysaccharide was also protective against mucosal surface colonization. These results call into question the need for local immune effectors, particularly secretory IgA, directed at bacterial ligands for epithelial surface components, in protecting a mucosal surface from bacterial challenge. PMID- 7542635 TI - Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells. AB - Glycosphingolipids (GSL) isolated from the gram-negative lipopolysaccharide (LPS) free bacterium Sphingomonas paucimobilis have remarkable structural similarities with LPS and its hydrophobic part, termed lipid A. Like LPS, but in contrast to the structurally related ceramides and cerebrosides, GSL contain an alpha-linked, negatively charged pyranosidic glycosyl component adjacent to the lipid portion and are capable of forming membranes. Because of these similarities, it was of interest to investigate whether these GSL are also able to induce monokine production in human mononuclear cells (MNC). Our results show that a GSL containing four sugar residues (GSL-4A) induced the release of tumor necrosis factor, interleukin-6, and interleukin-1 in MNC, whereas GSL-1, containing only one glycosyl residue, was inactive. A minimal concentration of 1 microgram of GSL 4A per ml was necessary to induce monokine production in MNC, whereas LPS was as active at a 10,000-fold-lower concentration (0.1 ng/ml). Both GSL-4A-induced monokine production and LPS-induced monokine production were reduced by the bactericidal/permeability-increasing protein and GSL-1. In contrast to LPS, GSL 4A-induced monokine release could be inhibited neither by an anti-CD14 monoclonal antibody nor by lipid A partial structures. We therefore conclude that at the receptor level, different mechanisms are involved in the LPS- and GSL-4A-induced monokine release. PMID- 7542636 TI - A protein G-related cell surface protein in Streptococcus zooepidemicus. AB - This work describes the cloning and sequencing of a gene encoding a plasma protein receptor from Streptococcus zooepidemicus. This receptor, termed protein ZAG, is a 45-kDa protein that binds alpha 2-macroglobulin (alpha 2M), serum albumin, and immunoglobulin G (IgG). The IgG-binding activity is located in the C terminal part of the molecule and is mediated by two repeated domains highly homologous to each other as well as to the corresponding domains in streptococcal type III Fc receptors. The IgG-binding profile of protein ZAG is similar to that previously reported for S. zooepidemicus. Binding to serum albumin is mediated by a short amino acid sequence in the middle of the molecule. This domain shows homology to previously described albumin-binding proteins from streptococci, and the albumin-binding profile of protein ZAG is similar to that of streptococcal protein G. The N-terminal part of protein ZAG, which mediates binding to the plasma proteinase inhibitor alpha 2M, is composed of a unique stretch of amino acids. Protein ZAG competes for the same, or nearby, binding site(s) in alpha 2M as do two recently described Streptococcus dysgalactiae receptors, although the sequences of the alpha 2M-binding domains in these three receptors show only minor sequence similarities. PMID- 7542637 TI - Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes. AB - We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC). Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml [mean +/- standard error of six experiments; P < 0.001]). In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence. Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells. Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli. The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E. coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1 induced adherence. Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils. Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; [mean +/- standard error of four experiments; P < 0.001]). This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B. We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes. These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction. PMID- 7542640 TI - Restricted replication of Listeria monocytogenes in a gamma interferon-activated murine hepatocyte line. AB - The intracellular pathogen Listeria monocytogenes replicates mainly in resting macrophages and hepatocytes residing in infected tissues. Both innate and acquired resistance strongly depend on activation of listericidal capacities of macrophages by gamma interferon (IFN-gamma) produced by natural killer cells and T lymphocytes. In contrast to macrophages, hepatocytes have been considered to serve purely as a cellular habitat, prolonging survival of the pathogen in the host. By using an immortalized murine hepatocyte line, the relationship between L. monocytogenes and this cell type has been analyzed in more detail. Our data reveal that hepatocytes are able to eradicate listeriolysin-deficient (avirulent) L. monocytogenes but fail to control growth of listeriolysin-expressing (virulent) L. monocytogenes organisms. Following stimulation with IFN-gamma, hepatocytes gained the capacity to restrict growth of virulent L. monocytogenes, although less efficiently than the highly listericidal IFN-gamma-activated macrophages. Hepatocytes costimulated with a combination of IFN-gamma, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) expressed the highest antilisterial activities. Although IFN-gamma-stimulated hepatocytes produced demonstrable levels of reactive nitrogen intermediates (RNI), the results of inhibition studies do not support a major role for these molecules in antilisterial hepatocyte activities. In contrast, inhibition of RNI produced by macrophages neutralized their antilisterial effects. IFN-gamma-stimulated, L. monocytogenes-infected hepatocytes expressed TNF-alpha mRNA, suggesting that they are a source of this cytokine during listeriosis. These studies suggest a novel function for hepatocytes in listeriosis: first, IFN-gamma-stimulated hepatocytes could contribute to listerial growth restriction in the liver, and second, through secretion of proinflammatory cytokines, they could promote phagocyte influx to the site of listerial growth. PMID- 7542639 TI - Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne. AB - Although many cytokines have been implicated in the development and persistence of inflammatory immune responses, it is unknown if any of these are important in inflammatory acne. This study investigated the production of the proinflammatory cytokines interleukin-8 (IL-8), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) by human monocytic cell lines, ThP-1 and U937, and by freshly isolated peripheral blood mononuclear cells from acne patients. Both Propionibacterium acnes and supernatants obtained from 72-h P. acnes cultures could induce significant concentrations of IL-1 beta, TNF-alpha, and IL-8 by both cell lines and by peripheral blood mononuclear cells as determined by enzyme-linked immunosorbent assay. There was no significant difference between acne and non acne subjects. Endotoxin quantification and addition of polymyxin B to assays indicated no lipopolysaccharide (LPS) contamination. P. acnes supernatant was fractionated into components with molecular weights of < 3,000, < 10,000, and < 30,000 and assayed for the ability to induce IL-8 and TNF production in ThP-1 cells. Nearly 90% of the original activity was found in the < 30,000-molecular weight fraction, 50% was in the < 10,000-molecular-weight fraction, and only 15% remained in the < 3,000-molecular-weight fraction. The effluent from the < 3,000 molecular-weight fraction contained about 70% activity, indicating that the inducing factor was not retained in the membrane. Incubation of P. acnes supernatant with various concentrations of mutanolysin or lysozyme resulted in a loss of 60% of the original activity. The addition of jimson lectin, which binds peptidoglycan, resulted in a loss of 70% of the activity in a dose-response manner, whereas peanut lectin had little or no effect on the activity. Heating of the P. acnes supernatant to 65 degrees C also had no effect on the activity. Blocking of CD14, a receptor for both LPS and peptidoglycan, reduced cytokine production by > 50%, suggesting that the soluble stimulating factor may be a secreted form of peptidoglycan-polysaccharide. PMID- 7542641 TI - Expression of CD44 splice variants in human cutaneous melanoma and melanoma cell lines is related to tumor progression and metastatic potential. AB - Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastasis formation in a number of different animal and human cancers. Because human cutaneous melanoma is among the most aggressive human cancers, we explored expression of CD44 isoforms (CD44v) in lesions of melanocytic tumor progression. In addition, by RT-PCR and FACS analysis we assessed CD44v RNA species and cell surface expression of CD44v in cultured melanocytes isolated from human foreskin and in a panel of 2 non-, 2 sporadically and 2 highly metastatic human melanoma cell lines. We observed that all melanocytic lesions examined showed strong uniform expression of standard CD44 (CD44s) epitopes. We did not detect CD44v6 expression in the melanocytic lesions. However, CD44 isoforms containing v5 or v10 were differentially expressed. V5 was expressed in 16%, 0%, 20%, 67% and 58% of common nevi, atypical nevi, early primary melanomas (< or = 1.5 mm), advanced primary melanomas (> 1.5 mm) and metastases, respectively, and hence was related to tumor progression. In contrast, CD44v10 was expressed in all common nevi, whereas part of the atypical nevi and most primary melanomas and metastases lacked v10. CD44v RNA patterns were closely similar in cultured melanocytes and all melanoma cell lines. Melanocytes expressed high levels of CD44s but no CD44v, whereas all melanoma cell lines expressed CD44v at the surface. Interestingly, expression of v5 was strongly increased in the highly metastatic cell lines. Our results suggest a role for CD44 variant domains, particularly v5 and v10, in human melanocytic tumor progression. PMID- 7542638 TI - The inflammatory cytokine response to Chlamydia trachomatis infection is endotoxin mediated. AB - Chlamydia trachomatis is a major etiologic agent of sexually transmitted diseases. Although C. trachomatis is a gram-negative pathogen, chlamydial infections are not generally thought of as endotoxin-mediated diseases. A molecular characterization of the acute immune response to chlamydia, especially with regard to the role of its lipopolysaccharide (LPS), remains to be undertaken. We extracted 15 mg of LPS from 5 x 10(12) C. trachomatis elementary bodies (EB) for analysis of structure and biological activity. When methylated lipid A was subjected to high-pressure liquid chromatography followed by mass spectrometry, the majority of the lipid A was found to be pentaacyl. The endotoxin activities of whole C. trachomatis EB and purified LPS were characterized in comparison with whole Salmonella minnesota R595 and with S. minnesota R595 LPS and lipooligosaccharide from Neisseria gonorrhoeae. Both C. trachomatis LPS and whole EB induced the release of tumor necrosis factor alpha from whole blood ex vivo, and C. trachomatis LPS was capable of inducing the translocation of nuclear factor kappa B in a Chinese hamster ovary fibroblast cell line transfected with the LPS receptor CD14. In both assays, however, C. trachomatis was approximately 100-fold less potent than S. minnesota and N. gonorrhoeae. The observation that C. trachomatis is a weak inducer of the inflammatory cytokine response correlates with the clinical observation that, unlike N. gonorrhoeae infection, genital tract infection with C. trachomatis is often asymptomatic. The ability of specific LPS antagonists to completely inhibit the tumor necrosis factor alpha-inducing activity of whole C. trachomatis EB suggests that the inflammatory cytokine response to chlamydia infection may be mediated primarily through LPS. This implies that the role of other surface protein antigens, at least in terms of eliciting the proinflammatory cytokine response, is likely to be minor. PMID- 7542642 TI - Antipsychotic medication in the treatment of schizophrenia. AB - Antipsychotic medication remains the mainstay of both acute and long-term treatment for schizophrenia. Recent research has underscored the need for optimum dosing strategies. Relatively few patients benefit from high doses (e.g. greater than 15-20 mg per day of haloperidol or 500-800 mg/day of chlorpromazine). In poor or partial responders clozapine continues to be the treatment of choice. Risperidone is an effective antipsychotic with a good safety profile. Its potential advantages in terms of efficacy need to be further studied. An expanded data base from maintenance trials supports the use of continuing maintenance medication and provides guidelines for dosage requirements. PMID- 7542643 TI - CD14 and other recognition molecules for lipopolysaccharide: a review. AB - Lipopolysaccharide (LPS) or endotoxin elicits a broad, non-specific cascade of events in vivo, resulting in secretion of a variety of potent mediators and cytokines produced primarily by activated macrophages and monocytes. The overproduction of these effector molecules, such as interleukin-1 and tumor necrosis factor-alpha, contributes to the pathophysiology of endotoxic shock. Cellular recognition of LPS involves several different molecules, including cluster of differentiation antigen CD14. A thorough understanding of the interaction of LPS with cells of the immune system is necessary before effective preventative or therapeutic measures can be designed to limit the host response to endotoxin. This review discusses the role of CD14 and other LPS-recognition molecules in LPS-mediated macrophage activation. PMID- 7542644 TI - The presence and cytotoxicity of CD16+ CD2- subset from PBL and NK cells in long term IL-2 cultures enhanced by Prothymosin-alpha. AB - The aim of this study was to characterize the LAK cell subpopulation on which Prothymosin-alpha (ProT alpha) exerts its enhancing effect on cytotoxicity. We investigated the role of ProT alpha on LAK induction from peripheral blood lymphocytes (PBL) and NK-enriched cells, both cultured for 5 weeks with IL-2 and ProT alpha. The different cultures were separated into several subsets throughout the culture time using two color fluorescence activated cell sorting (FACS) and CD56, CD16, CD2 and CD8 monoclonal antibodies. Each cell subset was then tested for cytotoxicity against K562 and Daudi cells. The CD16+ CD2- subset from both, PBL and NK-enriched cells, was the only subset on which ProT alpha had an effect, significantly enhancing this subpopulation. Within the CD16 population, the cells CD16+ CD2+ were the most cytotoxic, although CD16+ CD2- cells were also cytotoxic. ProT alpha only potentiated the cytotoxicity of CD16+ CD2- subset significantly, with 29% and 41% for K562 and Daudi cells, respectively. PMID- 7542645 TI - Curative treatments of murine Colon26 solid tumors by immunochemotherapy with G CSF and OK-432. AB - In order to study the clinical usefulness of biological response modifiers (BRMs) in eliminating malignant solid tumors, we have investigated the effect of various combination therapies on the murine Colon26 solid tumor model. When the tumor bearing mice were treated with chemotherapeutics, G-CSF and OK-432 (streptococcal preparation), the tumors completely disappeared from all of the treated mice. When these survivors were rechallenged with Colon26 tumor cells on Day 120, all of them survived without showing any sign of recurrence or metastases. The results indicate that mice with malignant solid tumors, which can not be cured using chemotherapeutics alone, may be completely healed with a combination immuno chemotherapy. During the course of this combination therapy, study, it was found that there was a clear positive correlation between immunosuppressive acidic protein (IAP) levels and tumor sizes. Suppressor macrophages (sM phi) which produce IAP were found to be decreased in bone marrow and spleen of treated mice. This suggests that the combination therapy may make the mice recover from a suppressed immune state caused by sM phi. In conclusion, the combination therapy with chemotherapeutics and BRMs could cure the solid tumor-bearing mice very effectively through not only synergistic direct tumor cell destruction but also indirect immunomodulation of the host. PMID- 7542646 TI - Biliary secretion of antibody to dextran following oral immunization with dextran B512. AB - Anti-dextran in bile was induced to high levels by oral immunization with dextran B512. IgM anti-dextran were dominant in serum, whereas IgG anti-dextran was dominant in bile. The binding properties of these IgM and IgG antibodies were different, as determined by ELISA with several dextrans. Splenocytes produced equal amounts of IgG and IgM antidextran but cells from mesenteric lymph nodes (MLN) and Peyer's patches produced mainly IgG anti-dextran. Differences were observed among different strains of mice in their ability to produce anti-dextran in serum and bile upon immunization with dextran. BALB/c mice, which are intermediate responders in terms of their serum antibody levels, produced high levels of anti-dextran in bile. C3H/He and C57BL/6, which are high responders in terms of serum antibody levels, had intermediate responses in bile. DBA/2, which are low responders in terms of serum antibody levels, showed low responses in bile. The results provide further evidence of the existence of anti-dextran producing cells. These results indicate that B cells in systemic and mucosal associated lymphoid tissues from BALB/c, C3H/He, C57BL/6 and DBA/2 mice respond differently to oral immunization with dextran B512. PMID- 7542649 TI - The nitric oxide transduction pathway in Trypanosoma cruzi. AB - A nitric oxide synthase was partially purified from soluble extracts of Trypanosoma cruzi epimastigote forms. The conversion of L-arginine to citrulline by this enzyme activity required NADPH and was blocked by EGTA. The reaction was activated by Ca2+, calmodulin, tetrahydrobiopterin, and FAD, and inhibited by N omega-methyl-L-arginine. L-Glutamate and N-methyl-D-aspartate stimulated in vivo conversion of L-arginine to citrulline by epimastigote cells. These stimulations could be blocked by EGTA, MK-801, and ketamine and enhanced by glycine. A sodium nitroprusside-activated guanylyl cyclase activity was detected in cell-free, soluble preparations of T. cruzi epimastigotes. L-Glutamate, N-methyl-D aspartate, and sodium nitroprusside increased epimastigote cyclic GMP levels. MK 801 bound specifically to T. cruzi epimastigote cells. This binding was competed by ketamine and enhanced by glycine or L-serine. Evidence thus indicates that in T. cruzi epimastigotes, L-glutamate controls cyclic GMP levels through a pathway mediated by nitric oxide. PMID- 7542648 TI - The baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis. AB - The baculovirus p35 gene product inhibits virally induced apoptosis, developmental cell death in Caenorhabditis elegans and Drosophila, and neuronal cell death in mammalian systems. Therefore, p35 likely inhibits a component of the death machinery that is both ubiquitous and highly conserved in evolution. We now show for the first time that p35 also inhibits Fas- and tumor necrosis factor (TNF)-induced apoptosis. Additionally, p35 blocks TNF- and Fas-induced proteolytic cleavage of the death substrate poly(ADP-ribose) polymerase from its native 116-kDa form to the characteristic 85-kDa form. This cleavage is thought to be catalyzed by an aspartate-specific protease of the interleukin 1 beta converting enzyme family designated prICE (Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347). Our data suggest that p35 must directly or indirectly inhibit prICE. Given that p35 inhibits both TNF and Fas killing, along with previous reports of its ability to block developmental, viral, and x-irradiation-induced cell death, the present results indicate that TNF- and Fas-mediated apoptotic pathways must have components in common with these highly conserved death programs. PMID- 7542650 TI - A novel monovalent cation channel activated by inositol trisphosphate in the plasma membrane of rat megakaryocytes. AB - The activation of a monovalent cation current was studied in rat megakaryocytes using patch clamp techniques combined with photometric measurements of intracellular concentrations of Ca2+ ([Ca2+]i) and Na+. ADP evoked a release of [Ca2+]i and transiently activated a monovalent cation-selective channel, which, at negative potentials and under physiological conditions, would be expected to carry an inward Na+ current. The single channel conductance, estimated by noise analysis from whole cell currents at -50 to -60 mV was 9 picosiemens. Thapsigargin-induced [Ca2+]i increases failed to stimulate the monovalent cation current, suggesting that neither [Ca2+]i nor the depletion of internal Ca2+ stores were activators of this conductance. However, buffering of [Ca2+]i changes with 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showed that both activation and inactivation of the current were accelerated by a rise in [Ca2+]i. The monovalent cation conductance was activated by internal perfusion with inositol 1,4,5-trisphosphate, both in the presence and in the absence of a rise in [Ca2+]i. Internal perfusion with inositol 2,4,5-trisphosphate, the poorly metabolizable isomer of inositol trisphosphate, similarly activated the monovalent cation current, whereas 1,3,4,5-tetrakisphosphate neither activated a current nor modified the ADP-induced monovalent current. Heparin, added to the pipette, blocked activation of the channel by ADP. The intracellular concentration of Na+, monitored by sodium-binding benzofuran isopthalate, increased by 10-20 mM in response to ADP under pseudophysiological conditions. We conclude the existence of a novel nonselective cation channel in the plasma membrane of rat megakaryocytes, which is activated by IP3 and can lead to increases in cytosolic Na+ after stimulation by ADP. PMID- 7542647 TI - Cell adhesion molecule expression by vascular endothelial cells as an immune/inflammatory reaction in human colon carcinoma. AB - The cell adhesion of inflammatory cells to vascular endothelial cells is an important process in the recruitment of inflammatory cells to the site. In cancer tissue, infiltration of inflammatory cells has been suggested to be a mechanism of host resistance. To clarify this infiltration mechanism, we investigated cell adhesion molecule expression (E-selectin, P-selectin, and ICAM-1) in vascular endothelial cells by immunohistochemistry in colon carcinoma. Venules distributed along the invasive margin expressed E- and P-selectins and ICAM-1. These phenotypical features are identical to those of endothelial cells observed in active inflammatory lesions, and the vessels can, therefore, be designated as immunologically activated vessels. Nevertheless, the majority of blood vessels within the tumor lacked immunoreactivity for all these adhesion molecules and, therefore, could be designated as immunologically inactive vessels. Granulocytes, lymphocytes and macrophages, bearing the counter-receptors of these adhesion molecules, were more densely distributed along the invasive margin. In contrast, few inflammatory cells were present within the tumor. In conclusion, the present study has demonstrated the phenotypical heterogeneity of tumor vessels; those for inflammatory cell infiltration to the tumor and those for the nutrient supply to the tumor. PMID- 7542651 TI - The exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) binding sequences in a recombinant murine Fab fragment specific for the integrin alpha IIb beta 3 does not alter integrin recognition. AB - The murine monoclonal antibody OPG2 is an excellent paradigm of natural RGD ligands and binds specifically to alpha IIb beta 3 integrin. A reactive Arg103 Tyr104-Asp105 (RYD) tripeptide is located in an extended loop, the third complementarity-determining region of the heavy chain (H3). When compared to other RGD ligands, the RYD tripeptide of OPG2 is unique, in that the side chains are fixed in a stable orientation that we have defined by x-ray crystallography. In this study, we express OPG2 H chain segments (Fd) and kappa chains as components of active, Fab heterodimers by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing cDNA specific for each protein. Recombinant AP7 Fd segments are generated from the parent OPG2 Fd segments by replacement of Tyr104 with Gly, while recombinant AP7E Fd segments are produced from AP7 Fd segments, by exchange of Asp105 with Glu. Neither the free Fd segments nor the free kappa chains of OPG2 or AP7 can bind to alpha IIb beta 3. The AP7 Fab fragment, like the parent OPG2 Fab, binds strongly to purified alpha IIb beta 3 but weakly, if at all, to purified alpha V beta 3. The affinity of OPG2 and AP7 Fab fragments for gel-filtered platelets, whether nonstimulated or activated by 0.2 microM phorbol 12-myristate 13-acetate, is identical. As with other natural RGD ligands, the binding of recombinant OPG2 Fab or AP7 Fab fragments to purified alpha IIb beta 3 or to gel-filtered platelets is completely inhibited by the peptide RGDW or by addition of EDTA, AP7E Fab fragments do not bind at all to either purified alpha IIb beta 3 or platelets. Our results demonstrate, for the first time within a natural protein ligand, that the tripeptides RGD and RYD exhibit equivalent binding capacity and specificity for the integrin alpha IIb beta 3. PMID- 7542653 TI - Soluble ligands of the alpha v beta 3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells. AB - Binding of substrate-bound extracellular matrix proteins to cell surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the alpha v beta 3 integrin blocked the vitronectin or SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while antibodies against beta 1 integrins and the alpha v beta 5 integrin did not. Clustering of the alpha v beta 3 integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric Vn and SC5b-9, suggesting that aggregation of alpha v beta 3 was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125FAK. We conclude that ligation of the alpha v beta 3 integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9. PMID- 7542652 TI - Peptide-specific antibodies as probes of the topography of the voltage-gated channel in the mitochondrial outer membrane of Neurospora crassa. AB - The voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membranes is formed by a polypeptide (M(r) 31,000) coded by a nuclear gene whose cDNA sequence is known for several organisms. Antibodies have been raised against synthetic peptides corresponding to four different regions in the predicted sequence of the VDAC polypeptide of the fungus Neurospora crassa (residues 1-20, amino terminus; 195-210, 251-268, and 272-283, carboxyl terminus). Specificity of the antibodies has been characterized in terms of binding to peptides or fungal mitochondria on microtiter plates and binding to mitochondrial proteins of several species in Western blots. Reactivity of three of the four antibodies with fungal mitochondria in suspension increases with lysis of outer membranes, indicating that the respective epitopes (including those near the amino and carboxyl termini) are exposed on the surface of the outer membrane that faces inside the mitochondrion. Preincubation of mitochondria with a polyanion that modulates VDAC voltage dependence strongly inhibits binding of the antibody against residues 251-268, whose epitopes are on the outer mitochondrial surface. PMID- 7542654 TI - Transcriptional induction of endothelial nitric oxide synthase type III by lysophosphatidylcholine. AB - Endothelial synthesis of NO is catalyzed by constitutive NO synthase type III (NOS-III). NOS-III has been thought to be regulated mainly at the level of enzyme activity by intracellular calcium. We report that in human umbilical vein endothelial cells lysophosphatidylcholine (lyso-PC), a component of atherogenic lipoproteins and atherosclerotic lesions, increases NOS-III mRNA and protein levels. This leads to the augmentation of NOS-III activity and the enhancement of antiplatelet properties of endothelial cells. Importantly, nuclear run-off experiments demonstrate a transcriptional mechanism of regulation of NOS-III expression by lysophosphatidylcholine. As endothelium-derived NO appears to be an anti-atherogenic molecule, induction of NOS-III by lyso-PC may be a protective response that limits the progress of the atherosclerotic lesion and promotes its regression. PMID- 7542655 TI - Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells. AB - We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha 32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins. PMID- 7542656 TI - Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. PMID- 7542658 TI - Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM. AB - Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration. PMID- 7542657 TI - CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. AB - Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events. PMID- 7542660 TI - Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture. AB - We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three-dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with 3H-glucosamine and 3H-thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan-link protein complex. A low concentration of HC (10(-9) M) stimulated the incorporation of 3H-glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5-day culture in 10(-9) M HC. On the other hand, a high concentration of HC (10(-5) M) reduced both synthesis (-42%) and degradation (-46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5 day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10(-5) M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10(-5) M) concentration of HC was effective, inhibiting the incorporation of 3H-glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers. PMID- 7542661 TI - Magnesium-dependent stimulation of protein synthesis by the insulin mimic, pervanadate. AB - The insulin mimic, peroxide of vanadate (pervanadate), stimulated 35S-methionine incorporation into Xenopus oocyte protein in a Mg(2+)-dependent manner. Reducing the extracellular Mg2+ concentration from 1.0 to 0.1 mM decreased the pervanadate stimulated component of incorporation by 35%; with 0.01 mM Mg2+ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg2+ to 0.01 mM inhibited about 50% of the insulin-stimulated component of methionine incorporation. Mg2+ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn2+ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg2+. Reducing extracellular Ca2+ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 microliter droplet of buffer containing the Mg2+ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg2+ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg2+. In contrast, apparent extracellular Mg2+ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg2+, but not Ca2+, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg2+, and this change in membrane transport may be an early event in signalling the increase in translation. PMID- 7542659 TI - Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein. AB - Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity. PMID- 7542662 TI - Modulation of the adhesion of hemopoietic progenitor cells to the RGD site of fibronectin by interleukin 3. AB - The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions amd also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by alpha 5 beta 1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of alpha 5 and one antibody against beta 1 were used to study expression of the integrin. By flow cytometry the expression of alpha 5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation. PMID- 7542663 TI - Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis. AB - Transforming growth factor betas (TGF-beta s) are the defining members of a super family of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-beta s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-beta have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effect of IGF treatment on the expression and secretion of TGF-beta s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-beta 3 in a concentration-dependent manner at 24 and 48 hours; TGF-beta 1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type 1 IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-beta 1 and TGF-beta 3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-beta genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. PMID- 7542665 TI - Binding and endocytosis of 39 kDa protein by MDBK cells. AB - A 39 kDa protein copurifies with the low density lipoprotein receptor-related protein (LRP) and regulates ligand interactions with LRP. In our recent studies on the clearance of the 39 kDa protein in vivo, we demonstrated that once the liver LRP receptors were saturated, the kidney became the major organ responsible for the 39 kDa protein clearance (Warshawsky et al., 1993, J. Clin. Invest., 92:937-944). The current study was undertaken in order to investigate the potential binding and cellular processing of the 39 kDa protein by kidney-derived MDBK cells. Herein we demonstrate specific, high-affinity, saturable, and Ca(2+) dependent binding of the 125I-39 kDa protein to MDBK cells (Kd approximately 10 15 nM, 50-70,000 binding sites per cell). Cellular uptake and degradation of the 125I-39 kDa protein by MDBK cells was also demonstrated with kinetics typical of receptor-mediated endocytosis. Using chemical crosslinking we show that LRP in part mediates the binding of 125I-39 kDa protein to the MDBK cell surface. In addition, the presence of functional LRP on the MDBK cell surface was confirmed by the specific binding of activated alpha 2-macroglobulin, another ligand of LRP. Our data thus demonstrate the ability of kidney-derived MDBK cells to specifically bind, endocytose, and degrade the 39 kDa protein. PMID- 7542664 TI - Effects of dexamethasone, heat shock, and serum responses on the inhibition of Hsc70 synthesis by antisense RNA in NIH 3T3 cells. AB - A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5'-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA. PMID- 7542667 TI - A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines. AB - Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immunoelectron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluorescence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function. PMID- 7542666 TI - Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts. AB - The hyaluronan receptor CD44 is an abundant glycoprotein expressed on a variety of different cell types. In fibroblasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extraction. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton. In this study we examined the structural features of CD44 required for its Triton X-100 insolubility in murine fibroblasts. We expressed in L cells the wild-type hematopoietic form of CD44, a mutant CD44 lacking the cytoplasmic domain, and two mutant CD44 molecules with substituted transmembrane domains. Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined. No difference in detergent solubility was observed between wild-type and tailless mutant-transfected molecules. However, both CD44 mutants with a heterologous transmembrane domain, derived from either the CD3 zeta chain or CD45, were completely soluble in Triton X-100. These results demonstrate that the transmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells. No obvious colocalization of CD44 and actin stress fibers was observed before or after treatment with cytochalasin D, and no change in the detergent extraction profile of wild-type and mutant CD44 molecules was effected by cytochalasin D. In equilibrium density sucrose gradients the Triton-insoluble CD44 component was found in the low density fractions, indicating an association with Triton X-100 insoluble lipids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542668 TI - The state of actin assembly regulates actin and vinculin expression by a feedback loop. AB - Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament associated proteins which is linked to the state of actin polymerization in the cell. PMID- 7542669 TI - Alpha v beta 1 is a receptor for vitronectin and fibrinogen, and acts with alpha 5 beta 1 to mediate spreading on fibronectin. AB - We have shown previously that VUP was the only line out of ten human melanoma lines that failed to express the vitronectin receptor alpha v beta 3, but instead expressed alpha v beta 1. Levels of alpha v beta 1 expression were low on parental VUP cells so that iterative sorting by FACS, using an anti-alpha v antibody (13C2), was utilised to derive sublines with 8- to 10-fold higher amounts of cell surface alpha v beta 1. There was little difference between low (V-) and high (V+) alpha v beta 1-expressing sublines with regard to adherence to collagen type I, collagen type IV or laminin substrata. However, adherence to vitronectin and fibrinogen correlated closely with alpha v beta 1 expression (35 42% adhesion for V(+) lines versus 6-8% adhesion for V- lines on vitronectin, for example). Utilising a high alpha v beta 1-expressing subline (V + B2) we have shown that binding to vitronectin and fibrinogen was inhibited specifically by function-blocking antibodies to alpha v (17E6 and 14D9) and beta 1 (A11B2). V(+) sublines spread more compared with V(-) sublines on both vitronectin and fibronectin. However, neither alpha 5- nor alpha v-blocking antibodies had any effect on attachment or spreading of V + B2 on fibronectin whereas the combination of alpha 5 (PID6)- and alpha v(17E6)-blocking antibodies abrogated binding to fibronectin almost completely. This is the first report of an alpha v beta 1 integrin able to recognize vitronectin and fibrinogen, and also cooperate with alpha 5 beta 1 to mediate attachment to and spreading on fibronectin. PMID- 7542670 TI - Cerebral microenvironment influences expression of the vitronectin gene in astrocytic tumors. AB - Expression of the vitronectin gene was detected in advanced human astrocytoma by in situ hybridization, whereas vitronectin mRNA was undetectable in low grade tumors or in normal adult brain, indicating that vitronectin is a marker of malignant astrocytoma. We established a model of human astrocytoma by transplanting U-251MG human astrocytoma cells intracerebrally into acid mice (C.B.17 severe combined immunodeficient mice). In this model, tumors progressed rapidly and vitronectin mRNA was preferentially detected at the invading tumor margins, i.e. where tumor cells were adjacent to the normal brain tissue. Surprisingly, when U-251MG cells were injected subcutaneously into scid mice, vitronectin mRNA was undetectable throughout the tumor. Moreover, vitronectin mRNA or protein could not be detected among these cells in culture under a wide variety of growth conditions. These findings demonstrate that the cerebral microenvironment influences the expression of the vitronectin gene in malignant astrocytoma. Importantly, the vitronectin binding integrins alpha v beta 3 and alpha v beta 5 localized to distinct sites within these tumors, with beta 3 mRNA synthesized among invading cells, and alpha v and beta 5 mRNAs detected throughout the tumor. In vitro, both of these receptors were capable of promoting adhesion and invasion of astrocytoma cells on a vitronectin substratum. These findings implicate the expression of the vitronectin gene as a contributing factor to the biological behavior of astrocytomas within the cerebral microenvironment. PMID- 7542671 TI - Targeted expression of SV40 T antigen in the hair follicle of transgenic mice produces an aberrant hair phenotype. AB - Directed expression of SV40 large T antigen (TAg) in transgenic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultrastructure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormalities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy. PMID- 7542672 TI - Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. AB - Exogenous addition of neutralising antibody to transforming growth factor-beta 1,2 to cutaneous wounds in adult rodents reduces scarring. Three isoforms of transforming growth factor-beta (1, 2 and 3) have been identified in mammals. We investigated the isoform/isoforms of TGF-beta responsible for cutaneous scarring by: (i) reducing specific endogenous TGF-beta isoforms by exogenous injection of isoform specific neutralising antibodies; and (ii) increasing the level of specific TGF-beta isoforms by exogenous infiltration into the wound margins. Exogenous addition of neutralising antibody to TGF-beta 1 plus neutralising antibody to TGF-beta 2 reduced the monocyte and macrophage profile, neovascularisation, fibronectin, collagen III and collagen I deposition in the early stages of wound healing compared to control wounds. Treatment with neutralising antibodies to TGF-betas 1 and 2 markedly improved the architecture of the neodermis to resemble that of normal dermis and reduced scarring while the control wounds healed with scar formation. Exogenous addition of neutralising antibody to TGF-beta 1 alone also reduced the monocyte and macrophage profile, fibronectin, collagen III and collagen I deposition compared to control wounds. However, treatment with neutralising antibody to TGF-beta 1 alone only marginally reduced scarring. By contrast, wounds treated with neutralising antibody to TGF beta 2 alone did not differ from control wounds. Interestingly, exogenous addition of the TGF-beta 3 peptide also reduced the monocyte and macrophage profile, fibronectin, collagen I and collagen III deposition in the early stages of wound healing and markedly improved the architecture of the neodermis and reduced scarring. By contrast, wounds treated with either TGF-beta 1 or with TGF beta 2 had more extracellular matrix deposition in the early stages of wound healing but did not differ from control wounds in the final quality of scarring. This study clearly demonstrates isoform specific differences in the role of TGF betas in wound healing and cutaneous scarring. TGF-beta 1 and TGF-beta 2 are implicated in cutaneous scarring. This study also suggests a novel therapeutic use of exogenous recombinant, TGF-beta 3 as an anti-scarring agent. PMID- 7542673 TI - Clear cell papulosis: report of three cases of a newly recognized disease. AB - BACKGROUND: Clear cell papulosis is a newly described disease. Since the first report in 1987, no other cases have been reported. OBJECTIVE: Our purpose was to describe three more newly identified cases that further characterize this disease. METHODS: Formalin-fixed and paraffin-embedded biopsy specimens were used for histochemical and immunohistochemical studies. RESULTS: The three patients included two boys and, for the first time, a girl. All three had multiple white papules on the lower part of the abdomen, with or without scattered lesions along the milk lines bilaterally. The main histopathologic finding was the presence of clear cells scattered mainly among the basal cells of the acanthotic epidermis. The clear cells were variably stained by mucicarmine, colloidal iron, alcian blue (pH 2.5), periodic acid-Schiff, the anticytokeratin antibody AE1, carcinoembryonic antigen, epithelial membrane antigen, and gross cystic disease fluid protein-15. CONCLUSION: Clear cell papulosis is a unique clinicopathologic entity. The clear cells were confirmed to be sweat gland secretory cells by their positive immunostaining with gross cystic disease fluid protein-15. The latter was also present in eccrine sweat gland coil cells. However, whether the clear cells were eccrine or apocrine secretory cells could not be determined. PMID- 7542674 TI - Total serum protein and serum protein fractions in depression: relationships to depressive symptoms and glucocorticoid activity. AB - Recently, it has been reported that major depression is accompanied by changes in plasma protein concentrations indicative of an acute-phase protein (APP) response. The purpose of the present study was to examine total serum protein (TSP) and the electrophoretically separated major fractions of serum proteins (SP), i.e., albumin (Alb), alpha 1, alpha 2, beta and gamma globulin, in depression. Highly significant differences were found in TSP and the separated SP fractions between major depressed patients and normal controls and between melancholic and minor depressed patients. Major depressed subjects showed significantly lower TSP and Alb concentrations and a higher percentage of the alpha 1 globulin fraction than normal controls and minor depressed subjects. Major depressed subjects had significantly higher and lower percentages, respectively, of alpha 2 and gamma globulin fractions than normal controls. In depressed subjects, there were significant negative correlations between TSP or Alb concentrations and severity of illness. Psychomotor retardation and anorexia were psychopathological correlates of lower TSP and Alb concentrations while middle insomnia and psychomotor retardation were related to changes in the alpha 2 globulin fractions. Basal plasma cortisol values were significantly and positively related to serum alpha 2 globulin. The results support the view that major depression is accompanied by an APP response. PMID- 7542675 TI - Loratadine reduces allergen-induced mucosal output of alpha 2-macroglobulin and tryptase in allergic rhinitis. AB - BACKGROUND: Despite the wide use of antihistamines in the treatment of allergic rhinitis, little is known about effects of these drugs on airway mucosal indices, which specifically reflect either mast cell release activity (tryptase) or microvascular-epithelial exudation of bulk plasma (alpha 2-macroglobulin). OBJECTIVE: This study, involving subjects with seasonal allergic rhinitis, examines the effects of loratadine treatment on allergen-induced nasal mucosal output of tryptase and alpha 2-macroglobulin. Effects on nasal symptoms and eosinophils are also examined. METHODS: Placebo and loratadine (20 mg) were given orally once daily for 5 days at 6-week intervals. Nasal diluent and allergen challenges were carried out on day 5. The mucosa was lavaged with saline solution after each challenge, and nasal lavage fluid levels of tryptase and alpha 2 macroglobulin were determined. Nasal symptoms were scored, and nasal peak expiratory flow rates were measured. Superficial cells (eosinophils) were obtained with a brush device before and 24 hours after the allergen challenges. RESULTS: Allergen dose-dependently increased the nasal symptoms and the lavage fluid levels of alpha 2-macroglobulin and tryptase. Allergen also reduced the nasal peak expiratory flow rates. Loratadine inhibited the exudation of alpha 2 macroglobulin and reduced tryptase levels, nasal symptoms, and obstruction, but did not affect the number of eosinophils. CONCLUSION: The inhibitory effects of loratadine on nasal lavage fluid levels of alpha 2-macroglobulin suggest that histamine, through effects on microvascular H1-receptors, mediates allergen challenge-induced exudation of bulk plasma in acute allergic rhinitis. The reduced lavage fluid levels of tryptase suggest either that loratadine directly attenuates mast cell release activity or that loratadine, through inhibition of the exudation process, simply attenuates luminal entry of tissue solutes (in this case, tryptase). PMID- 7542677 TI - Prophylactic potential of selective reuptake inhibitors in suicidal patients. AB - Concentrations of cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) were similar in patients with major depressive disorder and in those with non depressive psychiatric problems, although concentrations were significantly lower in suicidal patients (particularly in those attempters who used violent methods) than in non-suicidal patients. In contrast, post mortem CSF 5-HIAA concentrations in suicide victims were significantly higher than in controls. In addition, there appear to be fewer imipramine binding sites in the left frontal cortex of the brain than the right in suicide victims, the converse being true for controls. The serotonergic dysfunction associated with suicidal behaviour is likely to be involved in other conditions such as depression, schizophrenia and anxiety disorder. Thus selective serotonin reuptake inhibitors may be useful in the treatment of various types of psychiatric disease. Fluvoxamine, in particular, is associated with a very low incidence of suicidality compared with other antidepressants. PMID- 7542676 TI - A pharmacological, pharmacokinetic and clinical overview of risperidone, a new antipsychotic that blocks serotonin 5-HT2 and dopamine D2 receptors. AB - Risperidone is a benzisoxazole derivative with antipsychotic activity that is chemically unrelated to other currently available antipsychotic agents. Its neuropharmacological properties, characterized by potent central antagonism of both serotonin 5-HT2 and dopamine D2 receptors, also differ from those of most other antipsychotic drugs. The pharmacokinetics of risperidone are well understood, having been studied in healthy subjects as well as in psychotic patients. The absolute oral bioavailability of risperidone is nearly 70%, and after oral administration, it is rapidly absorbed with the plasma level reaching a peak at about 1 h. 9-Hydroxyrisperidone, one of the metabolites of risperidone, is equally active with the parent compound and so the clinical activity of a dose of risperidone is due to the combined actions of both moieties. The plasma concentrations of risperidone and its active metabolite remain dose proportional even at doses exceeding the therapeutic range. In clinical trials with chronic schizophrenia patients, risperidone has an overall therapeutic activity comparable with that of haloperidol, but at doses that produce similar improvements in the positive symptoms of schizophrenia, risperidone has a greater effect on the negative symptoms and produces less extrapyramidal side effects than does haloperidol. However, additional controlled clinical studies are needed before the claims that risperidone is therapeutically superior to haloperidol can be considered to be established firmly. Although risperidone is effective in acute schizophrenia and in non-treatment-resistant schizophrenics, studies adequately comparing risperidone with clozapine in treatment-resistant schizophrenic patients remain to be published. In addition, risperidone has been reported to be of value in patients with schizodepressive disorders. The clinical success of risperidone suggests that the development of compounds with selective affinity for 5-HT2 or other serotonin receptors may result in even further improvements in the pharmacotherapy of psychiatric disorders. PMID- 7542679 TI - Differential distribution of single-stranded DNA, double-stranded DNA, and RNA in adenovirus-induced intranuclear regions of HeLa cells. AB - We investigated in great detail the fine spatial distribution of nucleic acids within adenovirus-infected HeLa cells by various immunogold labeling procedures. To detect DNA, we used the in situ terminal deoxynucleotidyl transferase immunogold technique. In addition to the expected evident label over the condensed host chromatin and the structures containing viral double- and single stranded DNA, label was consistently revealed over round fibrillar spots. By contrast, other virus-induced substructures, such as compact rings, crystalloids, clear amorphous inclusions, and electron-dense amorphous inclusions, displayed no significant label. Except for the viral single-stranded DNA accumulation sites, identical labeling pattern was obtained with the in situ nick-translation immunogold method. We further labeled the sections with anti-RNA antibodies. Label was present not only over the cytoplasm and the intranuclear fibrillogranular network but also quite obviously over the compact rings and interchromatin granule clusters. None was seen over the other nuclear structures of infected cells, notably over the fibrillar spots. We suggest that these fibrillar spots might be involved in the formation of the viral, non encapsidated, double-stranded DNA storage site. PMID- 7542678 TI - Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. AB - Technological developments have made possible extension of polymerase chain reaction (PCR) analysis to individual cells to localize DNA/RNA with non radioactive labels at the light microscopic level. This approach, in situ PCR, is particularly useful in resolving low-frequency message expression in mixed populations of cells and tissues. We have established a working protocol for direct in situ PCR and have utilized several controls to validate our results. In this report we outline the procedures for detecting either DNA or RNA in a rapid and reproducible manner. We evaluate the sequential steps required for this analysis, such as protease hydrolysis, DNAse digestion, "hot start" capabilities, and detection methods. We have applied these methods in several applications, including detection of the p53 gene in human tumor samples, localization of insulin-like growth factor-IA mRNA in cell lines with low levels of expression, and distribution of transferrin mRNA in lung cancer cell lines and tumors. We demonstrate from this study that the in situ PCR technique is an investigative approach capable of detecting specific DNA/RNA sequences at the cellular level and of identifying cells with low levels of mRNA expression. PMID- 7542680 TI - Myofibrillar ATPase activity of feline muscle fibers expressing slow and fast myosin heavy chains. AB - The interrelationships among myofibrillar ATPase activity (Quant-mATPase), qualitative myofibrillar ATPase staining after acid (Acid-mATPase) and alkaline (Alk-mATPase) preincubations, and myosin heavy chain (MHC) composition were determined in frozen sections of soleus (Sol) and medial gastrocnemius (MG) muscle fibers from adult control cats and cats 6 months after complete spinal cord transection (Sp). Fibers were categorized as either fast, slow, or fast and slow (Fast-Slow) based on monoclonal antibody labeling. Slow fibers had low Quant mATPase activity and stained lightly with Alk-mATPase and darkly with Acid mATPase. Fast fibers had high Quant-mATPase activity (approximately twice that of slow fibers) and stained darkly with Alk-mATPase and lightly with Acid-mATPase. Fast-Slow fibers had intermediate Quant-mATPase activity and stained intermediately for Acid-mATPase and darkly for Alk-mATPase. There was a positive linear relationship between Alk-mATPase and Quant-mATPase for all fibers of Sol and MG from control and Sp cats. There was a negative linear relationship between Acid-mATPase and Quant-mATPase for all fibers of Sol and MG. However, within the fast fiber population of the MG there was a positive relationship between these two measures of mATPase. In summary, quantitative and qualitative measures of mATPase are highly correlated with the types of MHC expressed by single fibers from control and Sp cat muscles. PMID- 7542681 TI - Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles. AB - We present here a new method for non-radioactive labeling of substance P (SP) to demonstrate the distribution of its binding sites in histological sections. The peptide was labeled at the primary amino group with a 1.4-nm gold particle. In Western blots of membrane fractions of rat spinal cord, specific binding occurred at 38 and 58 KD. This binding was competitively suppressed by adding the native peptide. In addition, the SP-gold conjugate was able to displace the corresponding 125I-labeled peptide from binding proteins. In histological sections, binding sites could be shown in various parts of rat brain and spinal cord. The distribution patterns were comparable to those found in studies using autoradiographic methods. Adding the native peptide or a neurokinin 1 receptor agonist markedly reduced labeling of the tissue, whereas only a slight reduction was obtained after adding neurokinin A. Therefore, SP could be specifically labeled with a 1.4-nm gold particle to demonstrate its binding sites. This new method combines the advantages of receptor-ligand affinity binding with a non radioactive detection system. It can be used for labeling peptides in general and therefore offers an alternative or addition to other methods used in study of the distribution of membrane receptors. PMID- 7542683 TI - Insulin-like growth factor binding proteins in arterial hypertension: relationship to left ventricular hypertrophy. AB - OBJECTIVE: It was reported previously that circulating insulin-like growth factor I levels are abnormally elevated in patients with essential hypertension and left ventricular hypertrophy. Tissue availability of the factor depends on the distribution of the circulating bound factor between its high- and low-molecular mass binding proteins, only the latter being able to cross the endothelium. The aim of this study was to investigate whether the presence of the different serum binding proteins is altered in patients with essential hypertension and left ventricular hypertrophy. DESIGN: The study was performed in 30 never-treated patients with essential hypertension and 30 age- and sex-matched normotensive subjects. Patients were separated into two groups according to the presence or the absence of echocardiographically determined left ventricular hypertrophy. METHODS: Plasma insulin-like growth factor I levels were determined by specific radioimmunoassay. The different molecular forms of its serum binding proteins were analysed by Western blotting using [125I]-labelled insulin-like growth factor I. A densitometric scanning of the blots was performed to analyse the quantitative relationships between the different forms of binding proteins. RESULTS: Insulin-like growth factor I levels were significantly higher in the hypertensive patients with than in the hypertensive patients without left ventricular hypertrophy or in the normotensive subjects. Compared with the normotensive subjects, both hypertensive patients subgroups exhibited increased high-molecular mass binding protein type 3 and decreased low-molecular mass binding proteins types 1 and 2. However, changes in the binding proteins were more marked in the hypertensive patients without than in the hypertensive patients with left ventricular hypertrophy. Accordingly, the ratio of low- to high-molecular mass binding proteins (an index of insulin-like growth factor I bioavailability) was higher in the hypertensive patients with than in the hypertensive patients without left ventricular hypertrophy. CONCLUSIONS: These results show that the distribution of the molecular forms of serum insulin-like growth factor binding proteins is altered in patients with essential hypertension, independently of insulin-like growth factor I levels. This suggests that regulation of the binding proteins is abnormal in essential hypertension. Whether the tissue availability of circulating insulin-like growth factor I is higher in hypertensive patients with than in hypertensive patients without left ventricular hypertrophy merits further investigation. PMID- 7542682 TI - Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart. AB - By using immunohistology combined with immunoblotting, cell culture, and RT-PCR, we show that the intermediate filament protein nestin is transiently expressed in the midembryonic mouse heart. Monoclonal antibody (MAb) Rat-401, known to react with nestin in neural and skeletal muscle cells, was also found to react with ventricular and atrial cells throughout the mouse heart from embryonic day 9 (E9) through E10.5. Both before (E8.5) and after (E11-adult) this brief period, staining with Rat-401 was absent from atrial and ventricular myocytes. To evaluate the specificity of staining with MAb Rat-401 in the heart, we used immunoblotting, cell culture, and RT-PCR to verify that the authentic nestin protein and mRNA were expressed in cardiomyocytes of the E10 mouse. Nestin expression is the first molecular marker for this distinct midembryonic period of heart development. PMID- 7542684 TI - Measurement of methemoglobin formation from oxyhemoglobin. A real-time, continuous assay of nitric oxide release by human polymorphonuclear leukocytes. AB - We have evaluated the spectrophotometric measurement (at 401 vs. 411 nm) of nitric oxide (NO)-dependent methemoglobin formation from oxyhemoglobin in order to assess NO release from human polymorphonuclear neutrophil leukocytes (PMN). S nitroso-D,L-acetyl-penicillamine (SNAP, 25-200 microM), a donor of NO, induced a dose-dependent methemoglobin formation. Furthermore, when PMN were activated with N-formyl-methionylleucyl-phenylalanine or phorbol myristate acetate in the presence of superoxide dismutase (SOD) and catalase, methemoglobin formation ensued. The amount of methemoglobin formed was dependent on the amounts of oxyhemoglobin and stimulus used, and the number of PMN in the assay. The NO synthase (NOS) inhibitors NG-monomethyl-L-arginine or nitro-L-arginine methyl ester did not affect methemoglobin generation from oxyhemoglobin induced by SNAP but inhibited that mediated by activated PMN with IC50 values of 250 microM and 340 microM, respectively. The substrate for NO formation from NOS, L-arginine in concentrations up to 1 mM did not significantly influence the methemoglobin formation either induced by SNAP or activated PMN. Exclusion of SOD did not affect SNAP-dependent oxidation of oxyhemoglobin. Exclusion of SOD from the cell containing system attenuated methemoglobin formation, and if catalase was also excluded the response was further reduced. Finally, PMN from a patient with X linked chronic granulomatous disease, unable to produce superoxide anions, showed a similar production of methemoglobin from HbO2 as did healthy PMN, activated with the respective agonists. We conclude that spectrophotometric measurement of methemoglobin formation from oxyhemoglobin in the presence of SOD and catalase is a suitable method for the measurement of NO release from PMN, with the benefits of a real-time, continuous assay. PMID- 7542685 TI - Serum IgG, IgM, and IgA reactivity to human papillomavirus types 11 and 6 virus like particles in different gynecologic patient groups. AB - Serum samples from several groups of patients attending a gynecology clinic were analyzed by ELISA for specific antibodies recognizing surface epitopes on intact human papillomavirus (HPV) types 6 and 11 L1 virus-like particles (VLPs) that were synthesized in vitro. In these samples, positive IgG and IgM reactivities to HPV-11 L1 VLPs were, respectively, 12% and 6% for 87 controls, 46% and 67% for 79 condyloma patients, 30% and 64% for 72 cervical intraepithelial neoplasia patients, 16% and 19% for 63 pregnant women at time of delivery, and 5% and 0 in their 63 newborns. IgA reactivities were low and not significantly different. The prevalence of IgG-positivity in HPV-6/11 DNA-positive patients increased from 46% with HPV-11 L1 VLPs to 76% when the sera were additionally screened with HPV-6 L1 VLPs. These data show that HPV-6 and -11 L1 VLPs are effective antigens for serologic studies and they detect type-specific antibodies. PMID- 7542686 TI - Growth of Staphylococcus aureus with nafcillin in vitro induces alpha-toxin production and increases the lethal activity of sterile broth filtrates in a murine model. AB - The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved. PMID- 7542687 TI - Decreased activation of the respiratory burst in neutrophils from AIDS patients with previous Pneumocystis carinii pneumonia. AB - Neutrophils from human immunodeficiency virus (HIV)-negative blood donors, asymptomatic HIV-positive patients, AIDS patients with previous Pneumocystis carinii pneumonia (PCP), and AIDS patients without previous PCP were compared for their ability to activate the respiratory burst, measured as luminol-amplified chemiluminescence. P. carinii, Staphylococcus aureus, phorbol-12-myristate-13 acetate, and FMLP were used to stimulate the neutrophils. When stimulated with P. carinii, neutrophils from PCP patients had a significantly lower response than the other groups, whereas no difference was found when S aureus was used. A somewhat but not significantly lower response to P. carinii was also seen in non PCP patients compared with HIV-negative donors. Priming of the neutrophils with recombinant granulocyte colony-stimulating factor (G-CSF) or recombinant human granulocyte-macrophage (GM)-CSF corrected this defect. A similar effect of these cytokines was seen on phagocytosis, whereas the chemiluminescence in unprimed cells did not correlate with phagocytosis. PMID- 7542689 TI - Polymerase chain reaction for assessing treatment response in patients with pulmonary tuberculosis. PMID- 7542688 TI - Genetic variation among Pneumocystis carinii hominis isolates in recurrent pneumocystosis. AB - Pneumocystis carinii hominis is a ubiquitous organism that causes pneumonia in immunocompromised persons. Paired P. carinii hominis isolates from human immunodeficiency virus-infected persons who had two episodes of pneumocystosis were examined for genetic heterogeneity. Genetic variation was detected by sequence comparison of a portion of the mitochondrial ribosomal RNA gene. In 5 of 10 patients experiencing two episodes of pneumocystosis, genetically distinct isolates were associated with each episode. These included 4 of 6 patients whose second episode of pneumocytosis occurred > 6 months after their initial bout. The genetic data support the hypothesis that some recurrent episodes of P. carinii hominis pneumonia are caused by reinfection rather than by reactivation of latent infection. PMID- 7542691 TI - Does acute ingestion of large amounts of alcohol cause pancreatic injury? A prospective study. AB - The contribution of ethanol to the pathogenesis of acute pancreatitis has been questioned for a long time. The authors asked whether acute ingestion of large amounts of alcohol may lead to pancreatic injury, as assessed by serum amylase levels, clinical picture, and abdominal ultrasound. Therefore, all patients (N = 122) admitted to our medical emergency ward with the diagnosis of alcohol intoxication were evaluated prospectively during a 12-mo period. Of these, 78 (56 M, 22 F; mean age 36 +/- 15) could be evaluated. The other 44 were excluded because of incomplete data (n = 18), mixed intoxications (n = 8), repeated admission (n = 9), incorrect diagnosis on admission (n = 7), and chronic pancreatitis (n = 2). Serum ethanol, amylase, and GOT were measured. Serum ethanol was 246 +/- 122 mg/dL (3-500 mg/dL), amylase 83 +/- 44 U/L (27-361 U/L), and GOT 25 +/- 37 U/L (5-271 U/L) without significant differences among the genders. No correlation between serum ethanol and serum amylase levels could be detected. PMID- 7542690 TI - Effects of tetraprenylacetone on pancreatic exocrine secretion and acute pancreatitis in two experimental models in rats. AB - The effects of tetraprenylacetone (TPN), an acyclic polyisoprenoid with antiulcer actions, on pancreatic exocrine secretion, and its preventive and therapeutic effects on acute pancreatitis in two experimental models were studied in rats. Intraduodenal administration of TPN (0, 100, 200, and 400 mg/kg/h) caused dose dependent increases in pancreatic juice and bicarbonate output without increasing protein output and plasma cholecystokinin (CCK) concentrations. TPN-stimulated pancreatic exocrine secretion was completely abolished by antisecretin serum but it was not by CCK receptor antagonist loxiglumide (50 mg/kg/h). In acute pancreatitis induced by four subcutaneous injections of 20 micrograms/kg cerulein at hourly intervals over, 3 h, TPN (400 mg/kg) given by an oral route either 1 h before the first cerulein injection or immediately after the last injection significantly reduced the increases in serum amylase and lipase activities and pancreatic wet wt. Pretreatment with TPN caused histologic improvements, whereas posttreatment failed to ameliorate histologic alterations. In severe type of acute pancreatitis induced by retrograde intraductal injection of 1.0 mL/kg of 4% sodium taurocholate, TPN exerted no apparent beneficial effects on biochemical and histologic alterations of acute pancreatitis. It is concluded that TPN given by an oral route stimulates pancreatic exocrine secretion through an increase in endogenous secretin release and causes beneficial effects on the experimental model of mild acute pancreatitis in rats. PMID- 7542692 TI - The effect of platelet activating factor antagonist (BN 52021) on acute experimental pancreatitis with reference to multiorgan oxidative stress. AB - Acute hemorrhagic pancreatitis was induced in Wistar rats using a retrograde intraductal injection of 5% Na-taurocholate. Rats were treated with platelet activating factor receptor (PAF) antagonist--BN 52021 (5 mg/kg) and sacrificed at 1 and 3 h after induction of acute pancreatitis. Malondialdehyde and sulfhydryl groups concentration were measured in pancreatic, lung, and liver tissue as a parameter of oxidant-antioxidant balance. We have shown that BN 52021 exerts only partial protecting effect against Na-TC-induced AP in rats. The positive effects of BN 52021 were expressed by: (1) Significant reduction of hyperamylasemia accompanied by lower malondialdehyde accumulation in pancreatic tissue; (2) Prevention of sulflhydryl groups depletion in lung tissue; (3) Diminution of necrotic and inflammatory changes in pancreatic tissue; and (4) Improvement of survival rate. We suggest that these effects may depend on the inhibition of PAF mediated activation and oxidant generation by phagocytes. PMID- 7542694 TI - Hepatitis C virus type I(1a) in northern China. AB - Twenty-four patients with hepatitis C virus (HCV) antibody from the Chinese North Western province of Jilin were further analysed by the immunoblot assay-2 (RIBA 2), reverse transcription-polymerase chain reaction (RT-PCR) for serum HCV RNA detection, and direct sequence-genotyping. Good concordance was found between the original second generation HCV antibody ELISA, RIBA-2, and serum HCV RNA. The occurrence of genotype I (1a), a genotype not previously reported in China, is described in 5(20.8%) of 24 cases, in association with genotypes II(1b) and III(2a) which were found in 16(66.7%) and 3(12.5%) of 24 cases, respectively. Imported blood products were unlikely to be the source of infection with genotype I (1a) but could not be definitively ruled out. PMID- 7542693 TI - Molecular epidemiology of hepatitis C virus infection amongst intravenous drug users in rural communities. AB - The prevalence of hepatitis C virus (HCV) infection amongst a group of intravenous drug users (IVDUs) resident in West Suffolk (East Anglia, England) was investigated and compared with the prevalence of infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In addition, both the level of HCV persistence, as defined by detection of viral RNA, and the HCV genotypes present in this population were determined. It was found that HCV antibodies were present in 59% of those tested; by comparison 22% had antibodies to HBV and 1% antibodies to HIV. HCV RNA was found in 44% of those with HCV antibody. HCV genotype 1 was the most prevalent within this population although both genotypes 2 and 3 were also represented. PMID- 7542695 TI - Inhibition of angiogenesis and growth of human non-malignant and malignant meningiomas by TNP-470. AB - Meningiomas are relatively common (22%) vascular brain tumors. 3-11% of meningiomas are malignant, and defy currently available therapy. Inhibition of neovascularization is one potential strategy for treating these hypervascular tumors. Inhibition of tumor-induced angiogenesis by TNP-470 (previously termed AGM-1470), a synthetic analogue of fumagillin, was tested on the growth of human non-malignant and malignant meningiomas in nude mice. TNP-470 significantly inhibited tumor neovascularization and tumor growth of both non-malignant and malignant meningiomas. TNP-470 is now in human trial and should be tested for efficacy in treating malignant or recurrent aggressive meningiomas. PMID- 7542697 TI - Na(+)-activated nonselective cation channels in primary olfactory neurons. AB - 1. Excised inside-out patch recordings were used to describe a novel cation channel from cultured lobster olfactory receptor neurons that is activated by [Na+]i. 2. The channel is reversibly activated by intracellular Na+ as low as 5 mM. The half-effect concentration of intracellular Na+ is approximately 60 mM at 60 mV. The dependence of the channel open probability on [Na+]i is sigmoidal with a Hill coefficient of 3.1, indicating that more than one Na+ must bind to activate the channel. 3. The channel is equally permeable to Na+, K+, and Li+. In symmetrical 210 mM Na+, the open channel current-voltage relationship shows slight inward rectification at positive potentials. The slope conductance of the channel is 107 pS between -90 and 0 mV. 4. Although the channel is not activated by voltage in the absence of intracellular Na+, the gating of the channel is dependent on voltage as well as [Na+]i and [Na+]o. 5. Both intracellular Ca2+ and Mg2+ reversibly affect channel activity in a concentration-dependent manner starting at 1 microM. Ca2+ decreases both the open probability and the single channel amplitude, whereas Mg2+ decreases the open probability but has no effect on the single channel amplitude. Ba2+ (5 mM), but not 20 mM Cs+ and 100 microM amiloride, reversibly block the channel. 6. We speculate that this novel cation channel regulates neuronal excitability by accentuating the rate and/or the magnitude of depolarization of the cell to odors. PMID- 7542696 TI - Analysis of c-erbB2 protein content of human glioma cells and tumor tissue. AB - This study was designed to determine whether or not overexpression of the c-erbB2 protein plays a role in the etiology of human gliomas. The c-erbB2 gene codes for a 185 kDa cell membrane glycoprotein (gp185c-erbB2), which is similar to the receptor for epidermal growth factor. In initial studies, four human glioma cell lines (A-172, U118MG, U138MG and SW608) were used to develop techniques for detecting and quantifying gp185c-erbB2, using immunofluorescence microscopy, immunoblot analysis and flow cytometry. A-172 cells were found to have the highest content of gp185c-erbB2. More detailed studies utilizing A-172 cells indicated that cellular gp185c-erbB2 content changed little in response to conditions affecting cellular proliferative status, including serum deprivation, growth in low glucose medium and treatment with dimethyl sulfoxide. Ten human glioma specimens were then analyzed for cellular gp185c-erbB2 fluorescence and DNA content, using A-172 cells as a biological standard. Results indicated that gp185c-erbB2 was expressed at levels comparable to that of A-172 cells in many specimens, and at a very high level in one specimen. These data reiterate the problem of the molecular heterogeneity of human gliomas and indicate that gp185c erbB2 may have a role in at least a subset of malignant glial tumors. PMID- 7542698 TI - Salamander olfactory bulb neuronal activity observed by video rate, voltage sensitive dye imaging. I. Characterization of the recording system. AB - 1. In this paper we describe properties of a video imaging system used to acquire voltage-sensitive dye fluorescence signals from the salamander olfactory bulb. Sources of noise in these signals were evaluated in preparations stained with the potentiometric probe RH-414. These were compared with noise levels in signals obtained from a light-emitting diode array designed to stimulate the experimental conditions with light levels similar to those seen in the salamander bulb recordings. These experiments define a number of determinants of video image quality to standardize optical voltage measurements in the salamander olfactory bulb. 2. Images were acquired at video rates using a Newvicon camera in a standard upright microscope and digitized with an eight-bit video frame grabber. 3. Sources of noise related to camera sensitivity, stability of illumination, and mechanical vibration were characterized. Camera dark noise was less than the pixel variability due to photon noise at the camera faceplate. This pixel noise was the limiting factor for discriminating the spatial and temporal properties of the optical responses. 4. No significant noise was found to be related to image digitization, transmission, or readout by the eight-bit frame grabber. Mechanical vibration, light stability, and other sources of noise could be controlled in vitro. In this condition, voltage-sensitive dye signal noise was similar to that in stimulated experiments using the light-emitting diode array. Higher levels of noise were found in vivo; some of this was reduced by sychronizing frame acquisition to the heartbeat. 5. On the basis of photodiode and video measurements, voltage-sensitive dye responses in the salamander olfactory bulb typically fell between 0.75% and 2.5% fractional change of background fluorescence. By appropriately adjusting the video signals before analog-to digital conversion, we could detect fractional changes of < 0.5%. 6. Both response averaging and low-bandpass spatial filtering improved the signal-to noise ratios of the images. For small numbers of averaged runs, the best improvement was obtained by low-bandpass spatial filtering. 7. Acquisition of high-spatial resolution video images permitted the use of low-bandpass spatial filters to suppress pixel noise. The degree of spatial enhancement depended on the relationship between the size of the structures of interest, pixel density, and the properties of the convolution filter kernel. This method avoided exposure of the preparation to prolonged illumination and the necessity of applying the large numbers of repeated stimuli required for averaging. PMID- 7542699 TI - Salamander olfactory bulb neuronal activity observed by video rate, voltage sensitive dye imaging. III. Spatial and temporal properties of responses evoked by odorant stimulation. AB - 1. Activity patterns across and within the laminae of the olfactory bulb were analyzed by imaging voltage-sensitive dye responses during odorant stimulation of all or part of the ventral olfactory mucosa. 2. The time course of the signals was generally characterized by a brief, small hyperpolarization, followed by a period of depolarization, and then a longer-lasting hyperpolarization similar to that seen with electric stimulation but with longer durations. 3. The activity was distributed nonhomogeneously across the bulbar laminae in the form of spatially segregated clusters having bandlike appearances. Clusters were observed with three monomolecular odorants, amyl acetate, ethyl-n-butyrate, and limonene, and with the complex odor of meal worms. Although response patterns to different odorants overlapped, they also showed differences in overall distribution. 4. Delivery of high odorant concentrations increased the size of the activated areas and accentuated the degree of response pattern overlap among different odorants. The general properties of the response patterns generated by each odorant were, however, similar at different odorant concentrations and in each of the animals tested. 5. The spatial and temporal distributions of the bulbar responses were somewhat similar regardless of whether the odorants were applied to local epithelial regions via punctate stimulation or to the entire mucosa. Certain regions did, however, have lower thresholds than others for eliciting bulbar activity in response to particular odorants. 6. Odorants applied to regions of the epithelium outside the areas of maximum sensitivity elicited odorant-related activity patterns with depolarizing and hyperpolarizing components similar to those seen with overall stimulation, but only if higher concentrations were used. Activation of distributed odorant sensitivities presumably gave rise to these patterns. 7. These data suggest that subsets of odorant receptor types are found in different areas of the olfactory epithelium, and demonstrate that there is widespread distribution across the epithelium of receptors sensitive to particular odorants. On the basis of the structure of these epithelial fields and the bulb response patterns that they relate to, these findings also provide evidence for complex spatial relationships between the olfactory epithelium and bulb. 8. The findings from this study suggest that representation of odor information in the salamander olfactory bulb does not occur by activation of a few selective bulbar regions, each related to a different odorant species. Instead, large regions of bulbar circuitry are involved in which molecular epitopes may be the unit of representation. Incorporation of new data presented here into a hypothesis of odor coding is discussed. PMID- 7542702 TI - The Relationship Between Undernutrition and Behavioral Development in Children. A report of the International Dietary Energy Consultative Group (IDECG) workshop on malnutrition and behavior. Davis, California, December 1993. PMID- 7542701 TI - Synergy between growth factors and transmitters required for catecholamine differentiation in brain neurons. AB - The phenotypically plastic neurons of the embryonic mouse striatum were used to explore mechanisms of catecholamine differentiation in culture. De novo transcription and translation of the CA biosynthetic enzyme, tyrosine hydroxylase (TH), was induced in striatal neurons exposed, simultaneously or sequentially, to the growth factor, acidic fibroblast growth factor (aFGF) and a catecholamine. Although dopamine was the most potent aFGF partner (ED50 = 4 microM), a number of substances, including dopamine (D1) receptor agonists, beta-adrenoceptor agonists, and dopamine uptake inhibitors also trigger TH induction when accompanied by aFGF. However, since none of the receptor antagonists nor transport blockers tested could inhibit dopamine's action, the mechanism remains obscure. Structure-activity analysis suggests that effective aFGF partners all contain an amine group separated from a catechol nucleus by two carbons. Thus, TH expression can be novelly induced by the synergistic interaction of aFGF, and to a lesser extent basic FGF, and a variety of CA-containing partner molecules. We speculate that a similar association between growth factor and transmitter may be required in development for the differentiation of a CA phenotype in brain neurons. PMID- 7542700 TI - Expression and in vitro function of beta 1-integrin laminin receptors in the developing avian ciliary ganglion. AB - In chick development, ciliary ganglion (CG) neurons go through a period of axon extension from approximately embryonic day (E)4 to E8, followed by a period of synaptogenesis and neuronal cell death. By examining the immunohistochemical localization of laminin, in conjunction with Dil labeling of the ciliary nerve projection, we have determined that the pathway taken by these neurons is rich in laminin expression. Therefore, laminins are good candidate molecules for mediating outgrowth of these neurons in vivo. In vitro, the ability of CG neurons to extend neurites on laminin-1 (EHS laminin, alpha 1 beta 1 gamma 1) is maximal up to E8, then declines dramatically. CG neuron outgrowth on laminin-1 requires the activity of beta 1-class integrins. We have used subunit-specific antibodies to determine which of the five beta 1-containing heterodimers known to be laminin receptors (alpha 1 beta 1, alpha 2 beta 1, alpha 6 beta 1, alpha 7 beta 1) are expressed, and which mediate neurite outgrowth. While we could not detect expression of alpha 2 or alpha 7, we have found that alpha 1, alpha 3 beta 1, and alpha 6 beta 1 are expressed on the surface of ciliary ganglion neuron cell bodies and axons, both in vitro and in vivo. Furthermore, antibodies against alpha 3 and alpha 6, but not alpha 1, interfered with CG neurite outgrowth on laminin-1 in vitro. Taken together, these data suggest that interactions of cell surface alpha 3 beta 1 and alpha 6 beta 1 integrins with laminin-1 are likely to mediate growth of CG neurons during pathfinding in vivo. PMID- 7542704 TI - Enduring cognitive effects of early malnutrition: a theoretical reappraisal. AB - This article presents a reappraisal of the literature on the enduring cognitive effects of early malnutrition. In addition to summarizing the existing empirical literature, we present a theoretical framework for determining whether the processes likely to be most vulnerable to early malnutrition were adequately assessed. The two types of information used to make this determination are clinical and experimental behavioral data as well as reported neural changes. One point of clear consensus is that animals exposed to early malnutrition exhibit lasting changes in the realm of emotionality, motivation, and/or anxiety. Because these alterations profoundly affect all aspects of behavioral functioning, including cognition, it is suggested that future research focus on these changes, rather than control for them as many past studies have done. The functional integrity of specific cognitive processes is less clear. The only cognitive processes for which enduring cognitive changes were demonstrated in rehabilitated animals--outside of effects mediated by these affective changes--are cognitive flexibility and, possibly, susceptibility to proactive interference. However, the inference that these are the only processes affected does not appear to be warranted on the basis of the evidence that several cognitive processes likely to be affected have not been fully assessed. Examples include executive functions linked to the prefrontal cortex (for example, attention), transfer of learning, procedural learning and long-term memory. Future research focusing on these specific cognitive functions as well as on these unequivocal affective changes should allow a more definitive conclusion regarding the enduring functional consequences of early malnutrition. PMID- 7542703 TI - Malnutrition and the brain: changing concepts, changing concerns. AB - Our conceptions of how malnutrition endured early in life affects brain development have evolved considerably since the mid-1960s. At that time, it was feared that malnutrition endured during certain sensitive periods in early development would produce irreversible brain damage possibly resulting in mental retardation and an impairment in brain function. We now know that most of the alterations in the growth of various brain structures eventually recover (to some extent), although permanent alterations in the hippocampus and cerebellum remain. However, recent neuropharmacological research has revealed long-lasting, if not permanent, changes in brain neural receptor function resulting from an early episode of malnutrition. These more recent findings indicate that the kinds of behaviors and cognitive functions impaired by malnutrition may be more related to emotional responses to stressful events than to cognitive deficits per se, the age range of vulnerability to these long-term effects of malnutrition may be much greater than we had suspected and the minimal amount of malnutrition (hunger) necessary to produce these long-term alterations is unknown. PMID- 7542705 TI - A review of studies of the effect of severe malnutrition on mental development. AB - This is a review of studies on the relationship between mental development and severe malnutrition. School-age children who suffered from early childhood malnutrition have generally been found to have poorer IQ levels, cognitive function, school achievement and greater behavioral problems than matched controls and, to a lesser extent, siblings. The disadvantages last at least until adolescence. There is no consistent evidence of a specific cognitive deficit. The evidence of a causal relationship is strong but not unequivocal because of difficulties in interpreting retrospective case control studies. Marked improvements in development can occur after adoption or intervention. Therefore, the outcome depends to a large extent on the quality of the subsequent environment. It is likely that extremely deprived environments would exacerbate the effects. There is limited evidence that other nutritional deficiencies may interact with previous malnutrition in affecting cognition. The mechanism linking malnutrition to poor development is still not established. PMID- 7542706 TI - Malnutrition and cognitive development: evidence from experimental/quasi experimental studies among the mild-to-moderately malnourished. AB - The purpose of this paper is to provide a review of the current literature on the relationship between malnutrition and cognitive development, with particular emphasis on data from experimental and quasi-experimental studies. The review provides a discussion of relevant issues of concern, including current knowledge regarding the specific outcomes affected by malnutrition, the significance of the timing of such insults, issues of differential responsivity, the role of specific nutrients, and the potential mechanisms. The implications of such findings for policy and directions for future research are discussed. PMID- 7542707 TI - Malnutrition and behavioral development: the nutrition variable. AB - During the last 50 years, the perception of nutrition variables that affect behavioral development has shifted, as have the scientific hypotheses that were addressed, the design of the studies that were conducted, the nature and composition of the dietary supplements that were given and compared and the interpretation of outcomes. Methods of diagnosing malnutrition and identifying the populations at risk of becoming malnourished are reviewed in relation to nutrition interventions. Even in dietary supplementation studies it can be difficult to isolate specific nutrient effects because of associations and interactions among dietary components. These and other problems associated with the study of possible effects of dietary energy, protein and micronutrients, and of breast vs. formula feeding on human development are examined. Where dietary intake data, biochemical indicators or clinical symptoms strongly suggest that presence of a single nutrient deficiency, the appropriate action may continue to be food supplementation or fortification, until the corresponding nutrient deficiency has been rectified in the habitual diet; where the nature of the deficiency is less clear, supplementation trials and programs aimed at improving dietary diversity and quality in general are more likely to show effects on indicators of behavioral development. PMID- 7542708 TI - Functional significance of the covariance between protein energy malnutrition and iron deficiency anemia. AB - Most of the correlational and experimental studies that have tested the hypothesis that mild-to-moderate protein-energy malnutrition (PEM) has an adverse effect on cognitive development disregarded the potential confounder effect of micronutrients. This omission may have been a critical flaw in study design because it is now recognized that iron deficiency increases the probability of deviations in the trajectory of children's motor and mental development from a normal developmental path. This paper discusses two frequently cited studies on the effects of PEM on neurointegrative and cognitive development and proposes that neither study can discard the hypothesis that effects attributed to protein and energy deficiency are, instead, determined by iron deficiency. PMID- 7542709 TI - Monoclonal antibodies raised against coccidia and malarial parasites recognize antigenic epitopes found in lankesterellid and adeleorin parasites. AB - Three murine monoclonal antibodies (MAbs) raised against poultry coccidia or murine malarial parasites were tested for cross-reactivity with 2 sporozoan parasites with different life histories and hosts: Lankesterella minima (Eimeriorina), an intraerythrocytic parasite of frogs that is transmitted by leeches; and Heptazoon catesbianae (Adeleinorina) that infects the red blood cells of frogs and is transmitted by mosquitoes. MAb 1209 recognized both refractile bodies of sporozoites of L. minima, using the indirect fluorescent antibody (IFA) technique and immunoelectron microscopy, and recognized antigens with relative rates of migration (M(r)) of 17, 23, 26, 43, and 48 kDa on a chemiluminescent western blot of L. minima sporozoite antigens. MAbs C(3)4F1 and E12 demonstrated spotty cytoplasmic staining and labeling of the anterior pellicle of L. minima sporozoites, respectively. Gamonts of H. catesbianae labeled with only MAb E12, using IFA. These gamonts exhibited staining similar to that observed with the L. minima sporozoites. The presence of the cross-reactive epitopes recognized by these MAbs in the same conserved locations suggests that these antigens are homologous. PMID- 7542712 TI - [Defensins, antimicrobial peptides, and their mechanism of actions]. PMID- 7542711 TI - Growth factors and the control of folliculogenesis. AB - There is increasing evidence that growth factors modulate folliculogenesis. However their precise role in the processes of follicular growth, differentiation and atresia is still unknown. Growth factors belong to complex systems, including all the factors structurally and functionally related, their receptor(s) and, in most cases, binding proteins or proteoglycans. In this review, the insulin-like growth factor (IGF) system is presented as a paradigm for the study of other growth factor systems in the ovary. IGFs are probably positive regulators of follicular development, as they enhance both proliferation and differentiation and amplify the action of gonadotrophins on follicular cells. In the ovaries of most mammals, there is little or no change in intrafollicular IGF concentrations during terminal follicular growth and atresia. By contrast, the concentrations of IGF-binding proteins < 40 kDa (mainly IGFBP-2, -4 and -5) decrease markedly during terminal follicular growth, but increase in atretic follicles. These variations could result from both changes in expression of these IGFBPs by follicular cells and in local degradation by gonadotrophin-induced specific protease(s). Experiments in vitro showed that these IGFBPs, present in a soluble form in follicular fluid, could play an important role by sequestrating the IGFs and decreasing their bioavailability. More generally, biological actions of growth factors critically depend on their bioavailability in the vicinity of the cell, resulting from the concentrations of the factors themselves, their binding proteins and the ability of specific proteases to degrade binding proteins or to cleave latent forms of growth factors. These mechanisms and their control by gonadotrophins throughout follicular development have now to be investigated for the different growth factor systems. PMID- 7542713 TI - Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse. AB - Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress. PMID- 7542710 TI - A monovalent cationic conductance that is blocked by extracellular divalent cations in Xenopus oocytes. AB - 1. Native Xenopus oocytes were voltage clamped and exposed to Ringer solutions containing low concentrations of divalent cations. Oocytes, held at -60 mV, developed a reversible non-inactivating smooth inward current (Ic) associated with an increase in membrane conductance. 2. Ic was selectively carried by cations (Na+, K+), indicating that the current was not the result of a non specific membrane breakdown, but was due instead to removal of a blocking effect of divalent cations on a specific population of endogenous ionic channels located in the oocyte membrane. 3. The blocking effects of Ca2+ and Mg2+ were voltage dependent, implying action at a binding site within the pore of the cationic channel. For example, the half-maximal inhibition (IC50) of Ic by Ca2+ was 61 microM in oocytes held at -60 mV and 212 microM in oocytes held at 0 mV. 4. The Ic channels could be unblocked by depolarization of the membrane even in the presence of physiological concentrations of Ca2+ or Mg2+. The unblocking of the channels was observed as a slowly developing outward current. 5. The novel cationic current was substantially reduced following in vitro maturation of oocytes by treatment with progesterone (10 microM, 4-5 h). 6. The physiological role of Ic channels remains to be elucidated. Nonetheless, their characteristics explain the ionic basis of the sensitivity of oocytes to reductions in extracellular divalent cations and raise the possibility that the channels play a role in calcium homeostasis. PMID- 7542714 TI - 5-Oxoprolinase deficiency associated with severe psychomotor developmental delay, failure to thrive, microcephaly and microcytic anaemia. PMID- 7542716 TI - [Heart failure as an initial manifestation of Whipple's disease]. PMID- 7542715 TI - C-fiber-evoked autonomic cardiovascular effects after injection of Piper betle inflorescence extracts. AB - Piper betle inflorescence extracts contain eugenol (6.2%) and safrole (78.9%). Intravenous injections of water extracts of P. betle inflorescence (PBE), eugenol, and safrole in rats induced hypotensive and bradycardiac effects, whereas both intraarterial and intrathecal injections of PBE, eugenol and safrole resulted in hypotensive and tachycardiac effects. Moreover, the effects of intravenous injections of PBE were reversed or inhibited by the pretreatment with bilateral vagotomy, atropine (1 mg/kg, i.p.) and capsaicin (100 mg/kg, s.c.). Effects of intraarterial injections of PBE on blood pressure were inhibited by the pretreatment with substance P (SP) antagonist (1 nmol, i.t.) and clonidine (2.5 micrograms, i.t.), while heart rate was only inhibited by the pretreatment with SP antagonist (1 nmol, i.t.). In addition, the tachycardia resulting from intrathecal injections of PBE was inhibited by pretreatment with propranolol (0.3 mg/kg, i.v.). Eugenol and safrole induced the same pattern on blood pressure and heart rate changes as PBE in rats after various treatments. This report suggests that acute administration of betel inflorescence extracts by different routes may activate C-fiber-evoked parasympathetic and sympathetic cardiovascular reflexes in rats. PMID- 7542717 TI - Metoprine, an inhibitor of histamine N-methyltransferase but not catechol-O methyltransferase, suppresses feeding in sated and in food deprived rats. AB - Metoprine is a histamine N-methyltransferase (HMT) inhibitor often used to elevate endogenous histamine (HA) levels when studying the role of brain HA. Since central histaminergic systems may be involved in the regulation of feeding, the effect of metoprine on food intake was studied in sated and in food deprived rats. The treatment caused a dose-dependent decrease in food intake in sated rats. It also suppressed deprivation-induced feeding. To clarify the specificity of the treatment, the effect of metoprine on another methylating enzyme, catechol O-methyltransferase (COMT), was examined indirectly by examining the ratio of the non-methylated dopamine metabolite, dihydroxyphenylacetic acid (DOPAC) to that of its methylated product homovanillic acid (HVA). The dopamine metabolites did not change in a manner consistent with COMT inhibition, but instead a transient decrease in DOPAC levels was observed. However, the suppression of feeding is considered to be related to the metoprine-induced inhibition of brain HA catabolism and not with the changes in dopaminergic systems. Metoprine had no effect on brain concentration of serotonin (5-HT) or its metabolite 5 hydroxyindoleacetic acid (5-HIAA). The results provide further support for the role of brain HA in the control of feeding behavior. PMID- 7542718 TI - Hepatitis C virus transmission by intravenous immunoglobulin. PMID- 7542719 TI - Hepatitis C virus transmission by intravenous immunoglobulin. PMID- 7542720 TI - Management of leucopenic sepsis. PMID- 7542721 TI - Angiogenesis and breast cancer metastasis. PMID- 7542722 TI - Treatment of acute myeloid leukaemia in pregnancy. PMID- 7542724 TI - Identification of an mRNA element promoting rate-limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme similar to RNase E. AB - We have identified an mRNA element that is involved in the initial cleavage of the pufBALMX mRNA species in Rhodobacter capsulatus. This endoribonuclease recognition site, the first to be identified in a bacterial species other than Escherichia coli, shows strong similarities to mRNA sequences cleaved by the endoribonuclease E in E. coli. The presence of an RNase E-like enzyme in R. capsulatus is further supported by in vitro cleavage of E. coli transcripts by R. capsulatus extracts at sites attributed to RNase E and by the cross-reaction of a polypeptide from R. capsulatus with antisera against E. coli RNase E. Our data provide evidence that mRNAs are degraded in different bacterial species by enzymes with similar recognition sequences and activities. We present a model that attributes the segmental differences in stability of the polycistronic puf transcript to a specific distribution of mRNA decay-promoting and mRNA decay impeding elements. PMID- 7542723 TI - Cultured peripheral blood mononuclear leukocytes from anorexia nervosa patients are refractory to visible light. AB - Cultured human peripheral blood mononuclear leukocytes [PBML] from patients with anorexia nervosa [AN] did not respond to light stimulation as PBML of normal controls [NC] did. During winter, visible light increased [3H]thymidine incorporation into DNA of NC-PBML stimulated with phytohemagglutinin [PHA]. This effect was enhanced by 10(-7) M melatonin. PHA-stimulated DNA synthesis of PBML from AN patients failed to respond to photic stimulation during winter, and their proliferative response to melatonin was significantly blunted. In vitro photic stimulation of NC-PBML reduced melatonin while increasing both serotonin and 5 hydroxyindole 3-acetic acid [HIAA] production in both basal and PHA-stimulated conditions. In contrast AN-PBML, that in darkness enhanced the oxidative deamination of serotonin into HIAA more than NC-PBML, did not switch their indole metabolism in response to light. Light did not inhibit the binding of both melatonin and serotonin to AN-PBML as occurred in NC-PBML. The present data suggest that AN-PBML do not respond to light in vitro, because of a failure in the regulation of serotonin and melatonin metabolism. PMID- 7542725 TI - Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1. AB - Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsular polysaccharide (CPS) and the O specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli. Independently of the O-type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage-encoded peptides were not successful. Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (LPS biosynthesis-interfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1. PMID- 7542727 TI - Getting the lead out.... PMID- 7542726 TI - Interpreting the literature on lead and child development: the neglected role of the "experimental system". AB - Controversy over lead's effect on children's cognition rests in part on the assumption that if such an effect exists it can be characterized by a single estimator (e.g., the same rate of decline in IQ with increasing exposure, the same neuropsychological presentation), which will be found by any study that is valid. Accordingly, efforts to resolve inconsistencies in study findings have focused almost exclusively on data analytic issues germane to bias, in particular confounding and its statistical control. Relatively little consideration has been given to the role of effect modification, i.e., the impact on effect estimation of differences in the "experimental systems" employed in human epidemiological studies. Lack of consistency in findings could be due to differences among study cohorts in exposure/toxicokinetic factors (e.g., dose, timing), differences in environmental characteristics (e.g., co-exposures, co-morbidity, developmental supports, assessment setting), or differences in the distribution of genetic characteristics that affect lead metabolism. Recent findings regarding lead's impact on the development of nervous system structure and function are consistent with the hypothesis that contextual factors affect the form in which lead toxicity is expressed and may contribute to the failure to date to identify a lead-associated "behavioral signature." Characterizing the neuropsychological effects of lead might be facilitated by greater use of a clinical "process" approach to assessment, which would permit the type of fine-grained analyses of lead-associated performance differences often employed in studies of behavioral toxicity in animal models. PMID- 7542728 TI - Lead research: where do we go from here? PMID- 7542729 TI - Bridging human and experimental animal studies of lead neurotoxicity: moving beyond IQ. PMID- 7542730 TI - A higher level of analysis: Bellinger's, interpreting the literature on lead and child development. PMID- 7542731 TI - Inconsistencies in the lead-effects literature exist and cannot be explained by "effect modification". PMID- 7542732 TI - Environmental lead and intellectual development: strengths and limitations of epidemiological research. PMID- 7542733 TI - Making models of real world events: the use and abuse of inference. PMID- 7542734 TI - Effects of early lead exposure: time to integrate and broaden our efforts. PMID- 7542735 TI - Lead and child development: uncertainties, possibilities, and explanations. PMID- 7542736 TI - Mechanisms in the induction of neuronal heterotopiae following prenatal cytotoxic brain damage. AB - Prenatal exposure to several neuroteratogens, such as ionizing radiation, ethanol, and cytotoxic drugs, induces the development of clusters of abnormally positioned neurons within the brain. These abnormalities have always been presumed to result from interference with normal neuronal migration, presumably via effects on radial glia. In our study, pregnant rats were injected with methylazoxymethanol acetate (MAM) on either E13, E14, or E15. Computerised reconstruction techniques, Golgi and immunocytochemical staining as well as electron microscopy were used to detect structural abnormalities of radial glia which might be responsible for the production of heterotopiae. Several structural abnormalities such as microcavitation, involvement of radial glial elements in rosettes, disturbance of the normal ventricular lining, and disruption of the attachment of radial glial endfeet to the pial surface were identified. We propose that periventricular heterotopiae result from disruption of the palisade arrangement of neuroepithelial cells in the ventricular zone and the involvement of radial glial elements in rosettes. Layer I heterotopiae may arise from abnormalities of the distal segments of radial glia and their attachment to the pia. No prenatal abnormalities in radial glia of the hippocampus were noted following MAM exposure at any of the 3 ages, consistent with the proposition that hippocampal heterotopiae arise by postnatal movements of pyramidal neurons without radial glial involvement. PMID- 7542737 TI - Implications of the use of neonatal birth weight, growth, viability, and survival data for predicting developmental neurotoxicity: a survey of the literature. AB - Current screening strategies for developmental neurotoxicants emphasize extensive behavioral and histological examination of the nervous system of maternally exposed offspring. In an ongoing effort to identify more rapid screening techniques which accurately predict developmental neurotoxicity, we conducted a literature review to investigate the suggestion that the Chernoff/Kavlock assay may adequately identify developmental neurotoxicants as well as developmental toxicants (58). We included information on a broad range of chemical classes including: pesticides, heavy metals, solvents, antiproliferative agents, and neuroactive drugs. For each chemical/agent, we recorded evidence of developmental neurotoxicity, teratological malformations of the nervous system, and associated information on the effects of that chemical on birth weight, growth, fetal viability, and/or neonatal survival (neonatal endpoints included in the Chernoff/Kavlock assay). Although complete Chernoff/Kavlock data were not always available, our results indicate that only 65% of developmental neurotoxicants affected at least one of the neonatal endpoints in the assay. Based on these results, we believe that reliance on the Chernoff/Kavlock assay as a primary developmental neurotoxicity screen could lead to a number of "false negatives" in hazard identification studies, and this assay should not be used to replace more comprehensive developmental neurotoxicity screening procedures. PMID- 7542738 TI - Inhibition of human immunodeficiency virus type 1 replication by 7-methyl-6,8 bis(methylthio)pyrrolo[1,2-a]pyrazine, an in vivo metabolite of oltipraz. AB - Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication in vitro (ED50 approximately 10 microM), undergoes extensive metabolism in vivo. Most of the orally administered drug undergoes opening of the dithiolethione ring, reduction, recyclization, and methylation to form 7-methyl-6,8 bis(methylthio)pyrrolo[1,2-a]pyrazine ("metabolite III"). We report here that metabolite III inhibits viral replication in vitro (ED50 approximately 25 microM) in acutely infected H9 and CEM T cell lymphoma cell lines. Although both metabolite III and oltipraz were able to inhibit phorbol-12-myristate-13-acetate stimulated viral replication in the chronically infected U1 promonocytic leukemia cell line, only metabolite III was able to inhibit phorbol-12-myristate-13 acetate-stimulated viral replication in chronically infected ACH-2 T cell lymphoma cells. The results with ACH-2 cells suggest that oltipraz inhibits an early stage of the viral life cycle, whereas metabolite III affects human immunodeficiency virus type 1 replication at a step distal to viral integration. This is consistent with the finding that oltipraz inhibits reverse transcriptase, whereas metabolite III does not. Although the mean ED50 for metabolite III in acutely infected peripheral blood mononuclear cells was 18 microM, the ED50 was below 5 microM in three of eight independent experiments. Studies of metabolite III in combination with oltipraz in acutely infected peripheral blood mononuclear cells demonstrated significant antiviral synergy. These results raise the possibility that the in vitro potency of oltipraz may underestimate its antiretroviral activity in vivo. Based on these results, the pharmacokinetics of oltipraz and metabolite III will be compared with the pharmacodynamic effects of orally administered oltipraz in a forthcoming phase I/II trial of oltipraz in patients with p24 antigenemia. PMID- 7542739 TI - WIN 17317-3: novel nonpeptide antagonist of voltage-activated K+ channels in human T lymphocytes. AB - We report the in vitro biological characterization of WIN 17317-3 (1-benzyl-7 chloro-4-n-propylimino-1,4-dihydroquinoline hydrochloride), a novel inhibitor of voltage-activated (n-type) K+ channels in human T lymphocytes. WIN 17317-3 inhibits 125I-charybdotoxin binding to n-type K+ channels with an IC50 value of 83 +/- 4 nM. WIN 17317-3 demonstrates competitive inhibition of 125I charybdotoxin binding by increasing its dissociation constant without changing the total number of channels bound and by having no effect on its dissociation rate constant. WIN 17317-3 inhibits whole-cell, n-type K+ currents with characteristics indicative of open channel block and has an IC50 value of 335 nM. The compound is 150-fold selective for n-type K+ channels, compared with Ca(2+) activated, charybdotoxin-sensitive K+ channels in smooth muscle. In purified CD4+ T lymphocytes activated with either anti-CD3 plus phorbol ester or anti-CD3 plus anti-CD28, WIN 17317-3 decreases interleukin-2 production with EC50 values of 0.8 microM and 1 microM, respectively. WIN 17317-3 is a novel, potent, and selective nonpeptide n-type K+ channel antagonist that inhibits interleukin-2 production in human T lymphocytes. PMID- 7542740 TI - A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes. AB - Microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of beta-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542742 TI - Association of insulin receptor substrate 1 with simian virus 40 large T antigen. AB - Mouse embryo cells expressing a wild-type number of insulin-like growth factor I receptors (IGF-IR) (W cells) can be transformed either by simian virus 40 large T antigen (SV40 T) or by overexpressed insulin receptor substrate 1 (IRS-1), singly transfected. Neither SV40 T antigen nor IRS-1, individually, can transform mouse embryo cells with a targeted disruption of the IGF-IR genes (R- cells). However, cotransfection of SV40 T antigen and IRS-1 does transform R- cells. In this study, using different antibodies and different cell lines, we found that SV40 T antigen and IRS-1 are coprecipitated from cell lysates in a specific fashion, regardless of whether the lysates are immunoprecipitated with an antibody to SV40 T antigen or an antibody to IRS-1. The same antibody to SV40 T antigen, however, fails to coprecipitate another substrate of IGF-IR, the transforming protein Shc, and two other signal-transducing molecules, Grb2 and Sos. Finally, an SV40 T antigen lacking the amino-terminal 250 amino acids fails to coprecipitate IRS-1 and also fails to transform R- cells overexpressing mouse IRS-1. These experiments indicate that IRS-1 associates with SV40 T antigen and that this association plays a critical role in the combined ability of these proteins to transform R- cells. This finding is discussed in light of the crucial role of the IGF-IR in the establishment and maintenance of the transformed phenotype. PMID- 7542741 TI - Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase. AB - Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca(2+)-mediated signaling in many cells. Yeast cells lacking functional calcineurin (cna1 cna2 or cnb1 mutants) display growth defects under specific environmental conditions, for example, in the presence of high concentrations of Na+, Li+, Mn2+, or OH- but are indistinguishable from wild-type cells under standard culture conditions. To characterize regulatory pathways that may overlap with calcineurin, we performed a synthetic lethal screen to identify mutants that require calcineurin on standard growth media. The characterization of one such mutant, cnd1-8, is presented. The CND1 gene was cloned, and sequence analysis predicts that it encodes a novel protein 1,876 amino acids in length with multiple membrane-spanning domains. CND1 is identical to the gene identified previously as FKS1, ETG1, and CWH53, cnd1 mutants are sensitive to FK506 and cyclosporin A and exhibit slow growth that is improved by the addition of osmotic stabilizing agents. This osmotic agent-remedial growth defect and microscopic evidence of spontaneous cell lysis in cnd1 cultures suggest that cell integrity is compromised in these mutants. Mutations in the genes for yeast protein kinase C (pkc1) and a MAP kinase (mpk1/slt2) disrupt a Ca(2+)-dependent signaling pathway required to maintain a normal cell wall and cell integrity. We show that pkc1 and mpk1/slt2 growth defects are more severe in the absence of calcineurin function and less severe in the presence of a constitutively active form of calcineurin. These observations suggest that calcineurin and protein kinase C perform independent but physiologically related functions in yeast cells. We show that several mutants that lack a functional vacuolar H(+)-ATPase (vma) require calcineurin for vegetative growth. We discuss possible roles for calcineurin in regulating intracellular ion homeostasis and in maintaining cell integrity. PMID- 7542744 TI - The phosphotyrosine interaction domain of Shc binds an LXNPXY motif on the epidermal growth factor receptor. AB - Shc is an SH2 domain protein that is tyrosine phosphorylated in cells stimulated with a variety of growth factors and cytokines. Once phosphorylated, Shc binds the Grb2-Sos complex, leading to Ras activation. Shc can interact with tyrosine phosphorylated proteins by binding to phosphotyrosine in the context of an NPXpY motif, where pY is a phosphotyrosine. This is an unusual binding site for an SH2 domain protein whose binding specificity is usually controlled by residues carboxy terminal, not amino terminal, to the phosphotyrosine. Recently we identified a second region in Shc, named the phosphotyrosine interaction (PI) domain, and we have found it to be present in a variety of other cellular proteins. In this study we used a dephosphorylation protection assay, competition analysis with phosphotyrosine-containing synthetic peptides, and epidermal growth factor receptor (EGFR) mutants to determine the binding sites of the PI domain of Shc on the EGFR. We demonstrate that the PI domain of Shc binds the LXNPXpY motif that encompasses Y-1148 of the activated EGFR. We conclude that the PI domain imparts to Shc its ability to bind the NPXpY motif. PMID- 7542743 TI - FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition. AB - The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action. PMID- 7542745 TI - The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase. AB - Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling. PMID- 7542747 TI - Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia. AB - BACKGROUND: In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS: We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS: Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild type and mutant receptors. CONCLUSIONS: Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia. PMID- 7542746 TI - Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions. AB - We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously. PMID- 7542748 TI - Granulocyte colony-stimulating factor, congenital neutropenia, and acute myeloid leukemia. PMID- 7542750 TI - The prevalence of hepatitis C in different countries of the ERA/EDTA area. PMID- 7542749 TI - Autoradiographic distribution of [3H]L-NG-nitro-arginine binding in rat brain. AB - The distribution of nitric oxide synthase (NOS), the enzyme which produces nitric oxide, has previously been studied in the rat central nervous system (CNS) using the NADPH-diaphorase technique and anti-NOS antibodies. However, the former method may not always be selective for NOS while the latter is not quantitative. Therefore a selective, quantifiable method would be desirable. L-NG-Nitro arginine, an inhibitor of NOS, is available in a tritiated form which we have shown to bind to NOS. We have now examined the regional distribution of [3H]L-NG nitro-arginine binding in the rat CNS using autoradiography. [3H]L-NG-nitro arginine specific binding was seen in a number of brain regions with the highest levels in the accessory olfactory bulb, the amygdaloid complex, the Islands of Calleja and the cerebellum. This regional distribution of [3H]L-NG-nitro-arginine binding sites in the rat CNS was, in general, similar to that seen with the NADPH diaphorase method and anti-NOS antibodies, consistent with the view that all three methods identify NOS in the CNS. Thus, [3H]L-NG-nitro-arginine appears to be a useful radioligand for studying the distribution of NOS in the CNS as its binding is quantifiable and apparently selective for NOS. PMID- 7542751 TI - Prevalence of anti-hepatitis C virus antibodies and hepatitis C virus viraemia in chronic haemodialysis patients in Libya. PMID- 7542752 TI - Hepatitis C virus infection among chronic dialysis patients in the southeast of France. Provence-Alpes-Cote d'Azur Nephrologists Group. PMID- 7542753 TI - Prevalence of anti-HCV among haemodialysis patients in Turkey: a multicentre study. PMID- 7542754 TI - Regulation of U-937 monocyte adhesion to cultured human mesangial cells by cytokines and vasoactive agents. AB - Leukocyte adhesion to kidney cells is an early event in renal inflammation, such as glomerulonephritis. We developed an experimental model of monocyte adhesion to cultured human mesangial cells. U-937 myelomonocytic leukaemia cells, similar to peripheral blood human monocytes, irreversibly bound to mesangial cell monolayers upon 30-180 min coincubations (to a max. of 13,600 +/- 1100/cm2 monolayer), as assessed by cell counting, U-937 labelling with 3H-thymidine, and colorimetry of nuclear staining with crystal violet. Adhesion was enhanced in mesangial cells proliferating in response to 17% fetal bovine serum, indicating expression of a proinflammatory phenotype. E. coli lipopolysaccharide (LPS), tumour necrosis factor-alpha (TNF-alpha) and protein kinase C activation with phorbol myristate acetate (PMA) potentiated monocyte binding during either coincubation or 24-h pretreatment (0.1 microM PMA, +200 +/- 21%). Binding was also promoted by pretreatment with vasoconstrictors, such as the thromboxane A2 mimetic, U-46619 (10 nM-1 microM, max. +35 +/- 3%), or 1 microM angiotensin II (+64 +/- 4%). To elucidate the mechanisms of monocyte adhesion, we analysed the adhesion molecules expressed by human mesangial cells, employing reverse transcription/polymerase chain reaction to detect ICAM-1, VCAM-1 and E-selectin gene expression. Proliferating cells express VCAM-1 and ICAM-1, confirmed by immunocytochemical staining and 79 +/- 3% inhibition of stimulated adhesion by pretreatment of mesangial cells with an anti-ICAM-1 monoclonal Ab. E-selectin transcription was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542755 TI - Changes in the kinetics and conductance of N-methyl-D-aspartate (NMDA)-receptor activated single channels with temperature. AB - Single channel currents activated by N-methyl-D-aspartate (NMDA) were recorded in inside-out membrane patches from hippocampal pyramidal cells. The effects of temperature on the conductance level and the kinetics of single channels were accurately determined by using a digital signal processing technique based on hidden Markov models. With increasing temperature, the probability of the channel being in the open state increases steeply, as does the amplitude of channel currents. The proportional change in channel conductance with increasing temperature is greater than the corresponding change in bulk conductivity of 150 mM electrolyte solutions. From the temperature dependence of channel conductance and kinetics, we derived several thermodynamical properties of the NMDA-receptor activated channel. Among these are the height of the energy barrier V presented to an ion traversing the pore, the activation energy Ea and the enthalpy delta H of the channel. PMID- 7542756 TI - Streptozotocin-induced diabetes decreases substance P levels in experimental arthritis in the rat knee. AB - Diabetic patients with sensory neuropathy are predisposed to disorders of the musculoskeletal system. It has been postulated that altered neurogenic inflammation, involving the neuropeptide substance P, may play a part in this phenomenon. We investigated the effect of streptozotocin-induced (STZ) diabetes on the development of an antigenic (mBSA) monoarthritis in the rat with particular reference to changes in substance P levels in dorsal root ganglia (DRG) and knee joint synovium. We found that STZ-induced diabetes of 24 weeks duration reduced the substance P content of L4/L5 DRG and knee joint synovial tissue. Induction of mBSA arthritis in diabetic rats resulted in diminished increases in synovial substance P and knee joint swelling compared to non diabetic arthritic controls. The results show that chronic STZ diabetes reduces neurogenic inflammatory responses in the rat knee which may render the joint more susceptible to arthritic attack. PMID- 7542757 TI - Localization of 12-lipoxygenase mRNA in cultured oligodendrocytes and astrocytes by in situ reverse transcriptase and polymerase chain reaction. AB - We studied the distribution of 12-lipoxygenase mRNA in glial cells. First, mRNA was detected from cellular extracts by soluble-phase reverse transcriptase polymerase chain reaction (RT-PCR). Taking into account that cell culture populations could not be 100% homogeneous, we then developed, for the first time, an in situ RT-PCR combined with immunocytochemistry with cell specific markers. Using this procedure we showed that 12-lipoxygenase mRNA was expressed both in mature oligodendrocytes and astrocytes. PMID- 7542758 TI - Enhancement of 7-nitro indazole-induced inhibition of brain nitric oxide synthase by norharmane. AB - 7-Nitro indazole (7-NI) has been used as a selective inhibitor of neuronal nitric oxide synthase (NOS) in vivo. This agent has a short duration of action which may be due to its metabolism. The structure of 7-NI resembles that of tryptophan which can be metabolized by the enzyme indolamine 2,3-dioxygenase (IDO). If 7-NI is also metabolized by this enzyme, then inhibition of IDO should augment the action of 7-NI on brain NOS activity. This possibility was examined by investigating the potential of norharmane, an IDO inhibitor, on the inhibitory effect of 7-NI on NOS catalytic activity (3, 4.5 and 7.5 h post-injection of 7 NI) in five brain regions. Norharmane, which alone did not alter NOS activity, enhanced the action of 7-NI on NOS activity in the cortex (4.5 and 7.5 h), hippocampus (3 h) and substantia nigra (3, 4.5 and 7.5 h) but not in the cerebellum or striatum. This suggests that IDO activity may, at least in part, be responsible for the relatively short duration of 7-NI action. PMID- 7542759 TI - Question of the month--January 1995. What is the optimal dose of 89Sr for bone pain palliation? PMID- 7542760 TI - Molecular and functional characterisation of E2F-5, a new member of the E2F family. AB - The transcription factor DRTF1/E2F is implicated in the control of cellular proliferation due to its interaction with key regulators of cell cycle progression, such as the retinoblastoma tumour suppressor gene product and related pocket proteins, cyclins and cyclin-dependent kinases. DRTF1/E2F DNA binding activity arises when a member of two distinct families of proteins, DP and E2F, interact as DP/E2F heterodimers. Here, we report the isolation and characterisation of a new member of the E2F family of proteins, called E2F-5. E2F 5 was isolated through a yeast two hybrid assay in which a 14.5 d.p.c. mouse embryo library was screened for molecules capable of binding to murine DP-1, but also interacts with all known members of the DP family of proteins. E2F-5 exists as a physiological heterodimer with DP-1 in the generic DRTF1/E2F DNA binding activity present in mammalian cell extracts, an interaction which results in co operative DNA binding activity and transcriptional activation through the E2F site. A potent transcriptional activation domain, which functions in both yeast and mammalian cells and resides in the C-terminal region of E2F-5, is specifically inactivated upon pocket protein binding. Comparison of the sequence with other members of the family indicates that E2F-5 shows a greater level of similarity with E2F-4 than to E2F-1, -2 and -3. The structural and functional similarity of E2F-5 and E2F-4 defines a subfamily of E2F proteins. PMID- 7542761 TI - Murine txk: a protein tyrosine kinase gene regulated by T cell activation. AB - To identify genes involved in signal transduction pathways that regulate T cell activation and development, murine fetal thymocytes were screened for expression of protein tyrosine kinase family members by the polymerase chain reaction. Using this approach, a non-receptor protein tyrosine kinase, txk, was identified and cloned. Tsk is expressed in thymocytes as early as fetal day 13.5 and its expression at the mRNA level continues throughout development. Txk transcripts are present in thymocytes, peripheral T cells and mast cell lines, but are not detectable in B cell macrophage/monocyte cell lines or in non-hematopoietic fetal or adult tissues. In both thymocytes and T cells, txk transcripts are down regulated after activation with PMA and ionomycin, concanavalin A or T cell receptor cross-linking. Sequence analysis indicates that txk contains SH2, SH3 and kinase catalytic domains and belongs to the tec family of cytoplasmic protein tyrosine kinases which includes tec, itk and btk. Its unique N-terminus contains a proline-rich region, but unlike the other tec family members, does not contain a pleckstrin homology domain. The restricted expression pattern of txk and its regulation by T cell activation make it an excellent candidate for involvement in signal transduction during thymocyte development. PMID- 7542762 TI - Direct and specific interaction of c-Src with Neu is involved in signaling by the epidermal growth factor receptor. AB - Transgenic mice expressing either the activated or wild type neu oncogene heritably develop metastatic mammary tumors. Tumor development in this transgenic mouse model correlates with activation of the Neu tyrosine kinase. Recently, we have shown that these Neu-induced mammary tumors possess elevated c-Src tyrosine kinase activity. Here, we demonstrate that c-Src requires tyrosine phosphorylated Neu for its ability to associate with Neu in vivo and this association is likely the result of a direct physical binding of c-Src SH2 domain to the tyrosine phosphorylated Neu. By contrast, the c-Src SH2 domain did not interact directly with tyrosine phosphorylated EGFR. Moreover, in established cell lines expressing elevated levels of EGFR, EGF stimulation results in transphosphorylation of Neu and formation of complexes between c-Src and tyrosine phosphorylated Neu. Taken together, these observations suggest that activation of c-Src by these two closely related EGFR family members results from a direct and specific interaction of c-Src with tyrosine phosphorylated Neu. PMID- 7542763 TI - A human homologue of the Drosophila tumour suppressor gene l(2)gl maps to 17p11.2 12 and codes for a cytoskeletal protein that associates with nonmuscle myosin II heavy chain. AB - Inactivation of the tumour suppressor gene lethal(2) giant larvae (D-lgl) of Drosophila leads to malignant transformation of the presumptive adult optic centers in the larval brain and tumours of the imaginal discs. These malignancies result from the disorganization of a cytoskeletal network in which the D-LGL protein participates. Here we describe the isolation of a cDNA encoding the human homologue to the D-lgl gene designated as hugl. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, we have prepared polyclonal rabbit antibodies against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues. PMID- 7542764 TI - Differential regulation of the activity of the 42 kD mitogen activated protein kinase (p42mapk) during enterocyte differentiation in vivo. AB - The family of mitogen activated protein (MAP) kinases appear to play a central role in relaying signals generated by receptor protein tyrosine kinases (RPTK) from the cell surface to the nucleus. We previously demonstrated that undifferentiated and mitotically active crypt cells have high levels of tyrosine phosphorylated proteins (DR Burgess, W Jiang, S Mamajiwalla and W Kinsey. 1989. J. Cell Biol., 109: 2139) possibly due to the activation of RPTKs and also have high pp60c-src protein tyrosine kinase activity (CA Cartwright, SN Mamajiwalla, SA Skolnik, W Eckhart and DR Burgess. 1993. Oncogene. 8: 1033) when compared to differentiated, non-mitotic villus cells. Since activation of RPTKs leading to cell proliferation or differentiation involves activation of the Ras-MAP kinase pathway, we chose to determine in this study if the activity of the MAP kinases were also regulated during differentiation of normal adult enterocytes. Our data show that although the 42 kD MAP kinase (p42mapk) was expressed in both crypt and villus cells, it was phosphorylated on tyrosine and active only in the crypt cells. Our data further suggest that p42mapk is inactivated during differentiation, possibly by a protein tyrosine phosphatase. Immunofluorescence studies revealed that p42mapk localized to the nuclei in both undifferentiated and differentiated enterocytes and colocalized with phosphotyrosine containing proteins at the region of the junctional complex. These results suggest that p42mapk and its regulators are tightly controlled during enterocyte differentiation in vivo and implicate p42mapk as a key regulatory molecule in the normal development of the adult intestinal epithelium. PMID- 7542765 TI - Strain variation limits protective activity of vaccines based on soluble Babesia canis antigens. AB - Groups of five dogs were immunized with vaccines containing soluble parasite antigens (SPA) derived from in vitro culture of Babesis canis parasites, either obtained commercially (Pirodog) or produced at laboratory scale. Both vaccines generated antibodies that reacted with parasitised erythrocytes (PE). Upon challenge infection with homologous parasites, protection was evident from less severe decreases of haematocrit values, and reduced morbidity. Vaccinated animals, however, were not protected against challenge infection with heterologous B. canis parasites. Recovery from challenge infection coincided with the production of antibodies against parasitized erythrocytes. The results suggest that SPA from B. canis carry strain-specific determinants that are crucial for inducing protection in dogs against challenge infection, and explain vaccination failures in the field. PMID- 7542766 TI - Update on talc, bleomycin, and the tetracyclines in the treatment of malignant pleural effusions. AB - Talc has been used to treat malignant pleural effusions (MPE) for over 30 years and is usually considered to be the most effective chemical agent for pleurodesis. Clinicians, aware of limited reports of serious adverse effects attributed to talc, have generally reserved it for selected patients who are refractory to first-line chemical sclerosants. Tetracycline and bleomycin have therefore been cited as the preferred agents for the treatment of MPE. Clinical studies reported over the last 5 years reflect a continuing interest in talc as a chemical sclerosant and provide evidence of its effectiveness with minimal side effects. Comparisons with other agents are necessary to evaluate further the application of talc insufflation in the thoracoscopy suite and talc slurry at the patient's bedside. As clinicians continue to debate the relative merits of various pleurodesis agents, talc appears to be a reasonable choice for the treatment of MPE. PMID- 7542767 TI - Effects of Isoprinosine on Echinococcus multilocularis and E. granulosus metacestodes. AB - An in vivo study of the effect of Isoprinosine on Echinococcus multilocularis and E. granulosus metacestodes (Cestoda) was performed. Short- and long-term treatments with different doses were tested in experimental hosts: jirds for E. multilocularis and mice for E. granulosus. Modifications in the weight of animals as well as macroscopical and ultrastructural aspects were registered for each animal group. The results obtained showed a considerable ultrastructural alteration in E. multilocularis metacestodes after short-term treatment with the highest dose, and the tissue transplants were negative. In E. granulosus cysts the ultrastructural damage was also very important and its severity increased as the dose was raised. For the two parasites, the macroscopical aspect and weight of the lesions were profoundly changed. PMID- 7542768 TI - Stimulatory effect of ursodeoxycholate on pancreatic exocrine secretion in an in vitro study. AB - Ursodeoxycholate (UDCA; 300 microM) significantly stimulates amylase secretion from isolated rat pancreatic acini but not Ca2+ mobilization. Removal of extracellular Ca2+ completely abolished the UDCA-stimulated secretory response. Ca ionophore (A23187) potentiated the response. However, staurosporine, a protein kinase C inhibitor, did not affect the UDCA-stimulated amylase secretion. These results indicated that UDCA directly acted on the pancreatic acini and stimulated amylase secretion by the mechanism of stimulation of influx of extracellular Ca2+, but not through inositol [1,4,5] triphosphate and diacylglycerol formation or Ca2+ mobilization. PMID- 7542769 TI - Epidermal growth factor receptor signaling in rat pancreatic acinar cells. AB - Epidermal growth factor (EGF) regulates pancreatic acinar enzyme secretion. The mechanism of action of EGF in pancreatic acinar cells is not clear. In the present study we investigated the role of heterotrimeric GTP-binding proteins (G proteins) in EGF receptor signal transduction. Pancreatic acini were isolated from rat pancreas by collagenase digestion and permeabilized by digitonin. Activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) was assessed using a radioreceptor assay specific for inositol 1,4,5 trisphosphate [IP3(1,4,5)]. For measurement of amylase secretion isolated pancreatic acini were incubated with secretagogues for 30 min at 37 degrees C. Amylase released into the medium was assessed by monitoring the hydrolysis rate of p-nitrophenyl-alpha,D-maltohepatoside. The weakly hydrolyzable GTP analogue guanosine 5'-[3-O-thio]triphosphate (GTP gamma S) and guanosine 5'-diphosphate (GDP) were used to activate and inhibit G protein-mediated signal transduction, respectively. EGF (90 nM) stimulated amylase release in isolated pancreatic acini. This effect was enhanced by guanosine 5'-[3-O-thio]triphosphate (0.1 mM), which stimulates G proteins. Guanosine 5'-diphosphate (1 mM), which inhibits the activity of heterotrimeric G proteins, had no effect on basal and EGF-induced amylase release. Lower EGF concentrations (20 nM) inhibited COOH-terminal cholecystokinin octapeptide (CCK8)-induced IP3(1,4,5) production and amylase release in pancreatic acini). However, in the presence of GDP, EGF had no significant effect on CCK8-stimulated amylase release. Furthermore, coincubation of the acini with CCK8, EGF, and GDP revealed that GDP reduces the inhibitory effect of EGF on CCK8-induced IP3(1,4,5) production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542770 TI - Effect of the cholecystokinin receptor antagonist loxiglumide on pancreatic exocrine function in rats after acute pancreatitis. AB - Recent studies have demonstrated that cholecystokinin (CCK) receptor antagonists not only reduce the severity of pancreatitis but also inhibit pancreatic regeneration after pancreatitis. This study was undertaken, therefore, to examine the effects of the CCK receptor antagonist loxiglumide on the exocrine pancreas when given after an episode of acute pancreatitis that was induced in rats by a 4 h subcutaneous infusion of 20 micrograms/kg body weight/h cerulein. Biochemical changes and secretory function in response to 100 ng/kg body weight cerulein were determined after a 6-day treatment with saline, loxiglumide (50 mg/kg body weight), or CCK-8 (2.5 micrograms/kg body weight), which was given three times a day starting 24 h after the induction of acute pancreatitis. In the saline treated rats, pancreatic enzyme contents and pancreatic juice and protein output were significantly low, whereas the pancreatic weight and protein and DNA contents were comparable to those of the controls without pancreatitis. Loxiglumide treatment, although significantly decreasing protein output, had no influence on pancreatic weight, protein and DNA contents, or pancreatic juice flow but increased the amylase and lipase contents compared to those of the saline-treated postpancreatitic rats. CCK-8 treatment also had no influence on pancreatic weight or protein and DNA contents but significantly increased the pancreatic enzyme contents and pancreatic juice and protein output compared to those of the saline-treated postpancreatitic rats. These results suggest that loxiglumide does not significantly inhibit the recovery of exocrine function but appears to accelerate the increase in pancreatic amylase and lipase contents even when given after an attack of acute pancreatitis. PMID- 7542771 TI - Effect of a novel cholecystokinin receptor antagonist, FK480, administered intraduodenally, on pancreatic secretion in rats. AB - The effect of a new cholecystokinin (CCK)-A receptor antagonist, FK480, developed in Japan, on pancreatic exocrine secretion stimulated by exogenous CCK and intraduodenal casein was investigated in vivo when administered to anesthetized rats intraduodenally, and its CCK antagonistic activity was compared with that of CR 1505. Intraduodenal administration of FK480 at graded doses of 0.0016-1.0 mg/kg-h produced dose-dependent inhibition of pancreatic juice volume and amylase output stimulated by intravenous infusion of CCK-8 at a dose of 0.06 micrograms/kg-h. The half-maximal inhibitory dose (ID50) of FK480 for CCK-8 stimulated amylase secretion was 0.025 mg/kg-h, whereas the ID50 of CR 1505 was 5.2 mg/kg-h, indicating that FK480 is 208 times more potent than CR 1505. Moreover, intraduodenal FK480 (0.2 mg/kg-h) significantly suppressed pancreatic juice volume and amylase output augmented by intraduodenal infusion of casein (400 mg/h). It is concluded that FK480 administered intraduodenally has a significant, potent inhibitory action on the exocrine pancreas stimulated by exogenous CCK and intraduodenal casein. PMID- 7542773 TI - Activation of trypsinogen in experimental models of acute pancreatitis in rats. AB - Trypsinogen activation peptide (TAP) concentration and alpha 2-macroglobulin trypsin complex (alpha 2M-T) activity were measured in two experimental models of acute pancreatitis in rats to evaluate the significance of activation of trypsinogen in acute pancreatitis. TAP concentration and alpha 2M-T activity in serum rose significantly in trypsin-taurocholate-induced hemorrhagic acute pancreatitis, while in cerulein-induced edematous acute pancreatitis they did not rise in spite of a similar increase in immunoreactive trypsin. When rats in trypsin-taurocholate-induced pancreatitis were treated by protease inhibitor (FUT 175; nafamostat mesilate; FUT group), alpha 2M-T activity in serum was significantly lower than that in nontreated controls (mean +/- SEM, 20.8 +/- 1.43 U/L in the FUT group vs 79.1 +/- 24.5 in controls; p < 0.01). The survival rate at 24 h was significantly improved in the FUT group compared with the controls (70 vs 43%; p < 0.05). The increase in TAP concentration in the FUT group was similar to that in controls. The TAP concentration in pancreatic tissue at 24 h was significantly (p < 0.01) lower in the survival group (7.8 +/- 0.8 ng/ml) than in the lethal group (25.9 +/- 3.7 ng/ml). Activation of trypsinogen and its subsequent enzyme activity play an important role in the evolution of severe acute pancreatitis. PMID- 7542772 TI - Effect of a new bombesin receptor antagonist, (E)-alkene bombesin isostere, on amylase release from rat pancreatic acini. AB - The short-chain pseudopeptide, [D-Phe6, Leu13 psi (CH2NH)Leu14]bombesin(6-14) (RDI), is reported to be a potent antagonist of bombesin, and development of this type of compound has greatly contributed to the investigation of biological actions of bombesin and its related peptides. We recently synthesized (E)-alkene bombesin isostere by replacing the peptide bond with an (E)-double bond: [D-Phe6, Leu13 psi [(E)CH = CH]Leu14] bombesin(6-14) (EABI). The present study examined the effect of EABI on amylase release from rat pancreatic acini. EABI showed no agonistic activity at concentrations up to 1 microM, and RBI showed slight agonistic activity at concentrations > 10 nM. EABI caused a dose-dependent inhibition of amylase release stimulated by 0.1 nM bombesin, with an IC50 of 6.7 +/- 1.7 nM, and induced almost-complete inhibition at 0.3 microM. RDI caused a dose-dependent inhibition of amylase release, with an IC50 of 68.7 +/- 16 nM. EABI caused a parallel and rightward shift of the entire dose-response curve of bombesin-stimulated amylase release, and the degree of the shift was dependent on the concentrations of EABI. EABI (100 nM) and RDI (100 nM) inhibited amylase releases stimulated by gastrin-releasing peptide (1 nM) and neuromedin-C (1 nM). In contrast, amylase release stimulated by cholecystokinin octapeptide (0.1 nM), carbachol (10 microM), vasoactive intestinal peptide (1 nM), and gastrin-17 (10 nM) was not inhibited by EABI and RDI. The results indicate that EABI is a potent and specific bombesin receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542774 TI - Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail. AB - Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1 15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183 501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment. PMID- 7542775 TI - A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. AB - Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta. PMID- 7542776 TI - Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display. AB - Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes. PMID- 7542778 TI - Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents. AB - Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR. PMID- 7542777 TI - Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression. AB - Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO. PMID- 7542779 TI - Identification of a retroviroid-like element from plants. AB - The biological nature of carnation small viroid-like RNA (CarSV RNA), a 275-nt circular molecule with self-cleaving hammerhead structures in its strands of both polarities, was investigated. The lack of infectivity observed in a series of transmission assays in carnation indicates that CarSV RNA, in spite of sharing structural similarities with viroid and viroid-like satellite RNAs from plants, does not belong to either of these two groups. Additional evidence in this direction comes from the observation that CarSV RNA also exists in carnation plants as DNA tandem repeats. In this respect, CarSV RNA is similar to a small transcript of a tandemly repeated DNA sequence of the newt genome. Moreover, CarSV and newt RNAs have similarities in their sequences as well as in some characteristics of their corresponding hammerhead structures. Further analyses have revealed that CarSV DNA is found directly fused to DNA sequences of carnation etched ring caulimovirus, a pararetrovirus, most likely in the form of an extrachromosomal element. The properties of the CarSV RNA/DNA system are those of a retroviroid-like element having some features in common with viroid and viroid-like satellite RNAs from plants and others with the newt transcript. PMID- 7542780 TI - The tRNA processing enzyme RNase T is essential for maturation of 5S RNA. AB - The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival. PMID- 7542781 TI - Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates. AB - Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained. PMID- 7542782 TI - Large secretory structures at the cell surface imaged with scanning force microscopy. AB - Scanning force microscopy was used to image rat basophilic leukemia (RBL-2H3) cell surfaces under different stimulation conditions that either permit or inhibit secretion. Cross-linking the surface IgE receptors with dinitrophenol conjugated bovine serum albumin initiates secretion in RBL cells with concomitant spreading of the cell body. Structures at the cell surface approximately 1.5 microns in diameter relate to secretion both spatially and temporally. The position of these surface pits and their sizes suggest that they may be related to the dense-core granules positioned along the cytoskeletal filaments in detergent-extracted, unactivated RBL cell processes. Topographic scanning force microscopy images of RBL cell surfaces at 2, 5, and 35 min after activation show that these structures persist and change in cross-sectional profile with time after activation. These structures may be related to the membrane retrieval mechanism of cells after intense stimulation. PMID- 7542784 TI - Binding studies of cationic thymidyl deoxyribonucleic guanidine to RNA homopolynucleotides. AB - Deoxyribonucleic guanidine is a potential antisense agent that is generated via the replacement of the negative phosphodiester linkages of DNA [--O--(PO2-)--O--] with positively-charged guanidinium (g) linkages [--NH--C(==NH2+)--NH--]. A pentameric thymidyl deoxyribonucleic guanidine molecule [d(Tg)4T-azido] has been shown to base pair specifically to poly(rA) with an unprecedented affinity. Both double and triple strands consisting of one and two equivalents of d(Tg)4T-azido paired with one equivalent of poly(rA) are indicated by thermal denaturation experiments. At an ionic strength of 0.22, the five bases of d(Tg)4T-azido are estimated to dissociate from a double helix with poly(rA) at > 100 degrees C! The effect of ionic strength on thermal denaturation is very pronounced, with stability greatest at low ionic strengths. The method of continuous variation indicates that there is an equilibrium complex with a molar ratio of d(Tg) to r(Ap) or d(Ap) of 2:1. Based on this evidence, models of the structures of d(Tg)9T-azido bound to r(Ap)9A are proposed. PMID- 7542783 TI - Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers. AB - c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors. PMID- 7542785 TI - The concentration of neurotransmitter metabolites in the cerebrospinal fluid of suicidal individuals: a meta-analysis. AB - A review of 27 research reports on the cerebrospinal fluid levels of neurotransmitter metabolites involving 1202 psychiatric patients found strong evidence for the involvement of the serotonin system in suicidal behavior. Attempted suicides, especially those using violent methods, had lower levels of CSF 5-HIAA as compared to psychiatric controls, and those making subsequent suicidal actions also had lower levels of CSF 5-HIAA. The meta-analysis permitted more reliable conclusions to be drawn than did each individual study alone. PMID- 7542787 TI - Mastering envy: from Freud's narcissistic wounds to Bettelheim's symbolic wounds to a vision of healing. PMID- 7542786 TI - Risperidone augmentation of clozapine. AB - While effective as a single agent in a significant proportion of treatment refractory patients, clozapine is often used in combination with other medications, including classical neuroleptics (1), mood stabilizers and antidepressants (2), benzodiazepines (3) and lithium (4). In Denmark, where clozapine has been in near continuous use since 1975, up to 60% of clozapine patients receive additional medication (4). Classical neuroleptics, often at antipsychotic levels, are the medications most frequently added, and are used in 30-35% of patients receiving clozapine (4,5). Clozapine, due to its low D2 blocking effect, may be therapeutically insufficient to contain symptoms and additional agents may be required (4). In controlled clinical trials risperidone has been shown to be superior to haloperidol (6), but has not yet been rigorously compared to clozapine. In light of its established efficacy and pharmacological profile (7) risperidone may be reasonably construed to be a medication with efficacy somewhere between clozapine and classical neuroleptics. As such, it may be especially well suited for use with clozapine as a part of an augmentation strategy. There are no reports in the literature describing the concurrent use of risperidone and clozapine. We present two cases demonstrating augmentation of clozapine's effects through combined use with clozapine. PMID- 7542788 TI - The value of religion in sustaining the self in extreme situations. PMID- 7542789 TI - Were the first organisms heat engines? A new model for biogenesis and the early evolution of biological energy conversion. PMID- 7542790 TI - Methimazole agranulocytosis treated with recombinant human granulocyte colony stimulating factor. PMID- 7542791 TI - Purification and immunohistochemical localization of a 17-kD porcine renal puromycin aminonucleoside binding protein. AB - We have purified a 17-kD puromycin aminonucleoside (PAN) binding protein from porcine kidney and identified it as the reported 17-kD protein kinase C inhibitor on the basis of its partial amino acid sequence. Of 54 determined amino acid sequences of the 17-kD porcine renal protein, 51 residues were identical to those of the 17-kD bovine brain protein kinase C inhibitor. However, our purified protein did not carry the inhibitory activity on protein kinase C. Immunohistochemical studies showed a unique intrarenal distribution of the 17-kD PAN-binding protein at the apical side of epithelial cells of Henle's loops in the inner medulla and of distal convoluted tubules. Immunoblot analysis revealed that the 17-kD PAN-binding protein was extractable by an isotonic buffer without sodium deoxycholate extraction. These results suggest that this protein binds loosely to the apical membranes of epithelial cells of Henle's loops and distal tubules and has specific functions related to tubular functions of these nephron segments at the apical side. Whether this protein is a real inhibitor of protein kinase C or not remains to be investigated. PMID- 7542792 TI - Ifosfamide and mesna: effects on the Na/H exchanger activity in renal epithelial cells in culture (LLC-PK1). AB - Ifosfamide (IF) is an alkylating cytostatic with urotoxic and tubulotoxic side effects which may result in the development of Fanconi syndrome in children. While the urotoxicity of IF is effectively prevented by the uroprotective thiol compound sodium-2-mercaptoethanesulfonate (Mesna), tubulo-toxicity of IF may occur even in the presence of Mesna and in the absence of any signs of urotoxicity. Using the renal tubular cell line LLC-PK1, we investigated whether there is a protective effect of Mesna or of its major dimeric metabolite Dimesna against metabolites of IF with respect to the Na/H exchanger activity. We tested the major IF metabolites 4-hydroperoxy-IF (4-OOH-IF), chloroacetaldehyde (CAA), and acrolein. All metabolites significantly inhibit the Na/H exchanger activity. Half-maximal inhibition of transport occurs at concentrations of 120 mumol/l (4 OOH-IF), 80 (CAA), and 60 mumol/l (acrolein) after 2 h of incubation. The onset of the inhibitory effect of all three metabolites is rapid. Complete inhibition of Na/H exchange by acrolein and CAA is present after a 6-hour exposure to 100 mumol/l of the respective metabolite, while 100 mumol/l 4-OOH-IF causes only 50% inhibition after 24 h of incubation. Dimesna, which the proximal tubular cell has to reduce to Mesna at the expense of intracellular glutathione before it exerts a uroprotective effect, has no protective effect in LLC-PK1 cells. Dimesna (0.3 mmol/l) displaces the dose-response curve for acrolein to the left, indicating an increased toxicity of the combination of acrolein plus Dimesna. Mesna (0.3 mmol/l) has a complete protective effect with respect to acrolein and CAA, while the protective effect versus 100 mumol/l of 4-OOH-IF is incomplete. We conclude that the function of the Na/H exchanger in LLC-PK1 cells is altered by metabolites of IF. The incomplete protection against the toxic effect of 4-OOH-IF by Mesna may explain the pathomechanism by which IF causes tubulotoxicity in the absence of urotoxicity. PMID- 7542793 TI - Nephrotoxicity of cyclosporin A and FK506: inhibition of calcineurin phosphatase. AB - Cyclosporin A (CsA; 50 mg/kg) and Fujimycine (FK506; 5 mg/kg), but not the related macrolide immunosuppressant rapamycin (5 mg/kg), caused a reduction of glomerular filtration rate, degenerative changes of proximal tubular epithelium, and hypertrophy of the juxtaglomerular apparatus in male Wistar rats when given for 10 days. The molecular mechanisms of CsA and FK506 toxicity were investigated. Cyclophilin A and FK506-binding protein, the main intracytoplasmic receptors for CsA and FK506, respectively, were each detected in renal tissue extract. In the kidney, high levels of immunoreactive and enzymatically active calcineurin were found which were inhibited by the immunosuppressants CsA and FK506, but not by rapamycin. Finally, specific immunophilin-drug-calcineurin complexes formed only in the presence of CsA and FK506, but not rapamycin. These results suggest that the nephrotoxic effects of CsA and FK506 is likely mediated through binding to renal immunophilin and inhibiting calcineurin phosphatase. PMID- 7542794 TI - Cross-talk between the Na(+)-K(+)-ATPase and the H(+)-ATPase in proximal tubules in suspension. AB - The cellular energy required for the activity of the Na(+)-K(+)-ATPase and of the H(+)-ATPase was estimated in intact proximal tubules in suspension. Both the fall in oxygen consumption (directly measured) and NADH oxidation (as estimated from exogenous substrate metabolism) were measured before and following application of ouabain (1 mM) to inhibit the sodium pump, following bafilomycin (0.1 mM) to inhibit the proton pump or following a combination of these inhibitors. The data demonstrate that the sodium pump utilizes 43% and the proton pump 19% of the phosphorylating NADH turnover of canine proximal tubules studied in vitro. However, a significant and stoichiometric stimulation of one pump was observed upon inhibition of the other. The NADH turnover related to the sodium pump increased from 308 to 402 (delta = 94) mumol.g-1 wet weight.h-1 following bafilomycin application and that of the proton pump from 136 to 230 (delta = 94) following ouabain application. This stimulation was largely abolished by inhibition of the Na+/H+ exchange occurring in either direction by amiloride or methylisobutylamiloride. It is concluded that a cross-talk occurs between the basolateral sodium pumps and the proton pumps located on the brush border membrane and/or on endosomes in proximal tubules. This cross-talk appears to be mediated by Na+/H+ exchange suggesting that both the proton pump and the Na+/H+ exchanger may contribute in a cooperative fashion to the proximal secretion of protons. PMID- 7542795 TI - Role of renal nerves in volume expansion in chronic hypoxic rats. AB - This work was designed to study the significance of the renal nerves in chronic hypoxic (high-altitude; HA) rats after saline loading. Female Wistar rats (200 290 g) under sodium pentobarbital (40 mg/kg i.p.) anesthesia were used in these experiments. Hypoxia was induced by placing the rats in an altitude chamber (5,500 m) for 4 weeks. Both the renal efferent nervous activity (RENA) and the renal afferent nervous activity (RANA) were recorded simultaneously throughout the experimental period. It was found that the responses of RENA and RANA to an intravenous saline infusion (10 ml, 10 min) were significantly different between the sea level (SL) control and HA rats. In SL rats, a depression of RENA was found; the depressed RENA had not recovered 80 min after cessation of the saline infusion. In HA rats, an initially depressed RENA was also found; however, it returned to the control level within 10 min following the cessation of saline infusion. RANA enhanced twice a few minutes after saline loading in SL rats; however, the changes of RANA in HA rats were not significant. In both groups of rats, whether renally denervated or intact, the amount of excretory urine and sodium after saline loading was unchanged. The renal norepinephrine levels were also measured by a high-pressure liquid chromatography system in both groups of rats, and it was found that the renal norepinephrine content of the HA rats was significantly higher than in the SL rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542796 TI - Indocyanine green videoangiography of idiopathic polypoidal choroidal vasculopathy. AB - PURPOSE: To identify the precise choroidal abnormalities associated with idiopathic polypoidal choroidal vasculopathy (IPCV), patients with IPCV were examined with indocyanine green (ICG) videoangiography. METHODS: Twelve patients with IPCV were examined using standard clinical, fluorescein, and ICG videoangiographic techniques. RESULTS: Indocyanine green videoangiography showed two basic choroidal vascular changes: a branching network of vessels in the inner choroid, and vascular dilations at the border of the network of vessels. The vascular dilations appeared to be associated with the exudative and hemorrhagic manifestations of IPCV. CONCLUSION: The choroidal vasculopathy seen in IPCV is distinct from the changes seen in other choroidal abnormalities. Recognition of these changes aids in diagnosis and patient management, since the clinical implications of IPCV differ from those of other similar entities. PMID- 7542797 TI - Simultaneous indocyanine green and fluorescein angiography. AB - PURPOSE: To facilitate the interpretation of the choroidal dye filling sequence, the use of simultaneous indocyanine green (ICG) and fluorescein angiography was evaluated. METHODS: A single-wavelength scanning laser ophthalmoscope (SLO) was modified to a two-wavelength system, permitting the simultaneous recording of ICG and fluorescein angiography. This method has been used in 340 cases. About two thirds of the patients had well-defined or occult choroidal neovascularization (CNV) in age-related macular degeneration (AMD). RESULTS: Simultaneous ICG and fluorescein angiography is feasible with a two-wavelength SLO and provides images of good quality. Two corresponding ICG and fluorescein angiography pictures can be presented as one combined red-green picture. CONCLUSION: This method has three advantages: (1) it allows a precise comparison of the transit of both dyes through both circulations, and there are no differences in the injected bolus nor in the actual blood pressure; (2) the important features of the ICG angiograms are fully aligned with the critical retinal vascular landmarks provided by the fluorescein images; and (3) it is very time efficient--with a single injection and one photographic session, immediate results are obtained. PMID- 7542798 TI - Individual lymphocyte subset composition determines cAMP response to isoproterenol in mononuclear cell preparations from peripheral blood. AB - The aim of the present study was to examine to what extent isoproterenol stimulated cAMP production in mononuclear cells, isolated from resting human subjects, was correlated with lymphocyte subset composition. Peripheral blood was collected from 14 healthy male subjects, who rested in the sitting position. Mononuclear cells were prepared by density centrifugation and subset composition was determined by two-colour flow cytometry. Cyclic AMP production was determined by a radioimmunoassay after inhibition of phosphodiesterase and stimulation with isoproterenol. NK-cells (CD3-CD56+) were positively correlated to total cAMP concentrations (basal cAMP plus increase in cAMP). Tcytotoxic cells (CD8+) were positively correlated to the relative increase in cAMP. Thelper cells (CD4+) were negatively correlated to total cAMP concentrations and B-lymphocytes (CD(19+20)+) were negatively correlated to the relative increase in cAMP. The results support the view that NK-cells and Tcytotoxic cells have higher beta 2-adrenoceptor densities and more receptors with high affinity than Thelper and B-cells. In conclusion, our study demonstrates that cAMP levels in blood mononuclear cells obtained from normal subjects show substantial variation. This variation is at least in part due to differences in lymphocyte subset composition in the same subjects. Therefore, it is important to know the lymphocyte subset composition when evaluating cAMP accumulation in mononuclear cell preparations from peripheral blood. PMID- 7542799 TI - One signal, two body axes. PMID- 7542800 TI - Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. AB - An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism. PMID- 7542801 TI - A role for CD5 in TCR-mediated signal transduction and thymocyte selection. AB - CD5 is a transmembrane protein that is expressed on the surface of T cells and a subset of B cells. The absence of CD5 rendered thymocytes hyperresponsive to stimulation through the T cell antigen receptor (TCR) in vitro. Selection of T cells expressing three distinct transgenic TCRs was also abnormal in CD5 deficient mice. These observations indicate that CD5 can influence the fate of developing thymocytes by acting as a negative regulator of TCR-mediated signal transduction. PMID- 7542802 TI - Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome. AB - The naturally transformable, Gram-negative bacterium Haemophilus influenzae Rd preferentially takes up DNA of its own species by recognizing a 9-base pair sequence, 5'-AAGTGCGGT, carried in multiple copies in its chromosome. With the availability of the complete genome sequence, 1465 copies of the 9-base pair uptake site have been identified. Alignment of these sites unexpectedly reveals an extended consensus region of 29 base pairs containing the core 9-base pair region and two downstream 6-base pair A/T-rich regions, each spaced about one helix turn apart. Seventeen percent of the sites are in inverted repeat pairs, many of which are located downstream to gene termini and are capable of forming stem-loop structures in messenger RNA that might function as signals for transcription termination. PMID- 7542803 TI - Molecular identification of an IgE-dependent histamine-releasing factor. AB - An immunoglobulin E (IgE)-dependent histamine-releasing factor (HRF) produced by lymphocytes of atopic children and present in biological fluids of allergic patients has been identified and purified. Amino-terminal sequencing revealed extensive homology to a mouse protein, p21, and its human homolog, p23. Both recombinant proteins caused histamine release from the human basophils of a subpopulation of donors, and this release was dependent on IgE. Polyclonal antibodies recognized and removed the biological activity of recombinant and native HRF. HRF identifies a heterogeneity of IgE and is believed to play a prominent role in chronic allergic disease processes. PMID- 7542804 TI - Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy. AB - Combinations of antiretroviral drugs that prevent or delay the appearance of drug resistant human immunodeficiency virus-type 1 (HIV-1) mutants are urgently required. Mutants resistant to 3'-azidothymidine (AZT, zidovudine) became phenotypically sensitive in vitro by mutation of residue 184 of viral reverse transcriptase to valine, which also induced resistance to (-)2'-deoxy-3' thiacytidine (3TC). Furthermore, AZT-3TC coresistance was not observed during extensive in vitro selection with both drugs. In vivo AZT-3TC combination therapy resulted in a markedly greater decreased in serum HIV-1 RNA concentrations than treatment with AZT alone, even though valine-184 mutants rapidly emerged. Most samples assessed from the combination group remained AZT sensitive at 24 weeks of therapy, consistent with in vitro mutation studies. PMID- 7542805 TI - Fluorescence and circular dichroism studies during the interactions of sulfated polysaccharides with antithrombin III. AB - The changes in relative fluorescence of antithrombin III (AT-III) during its interaction with sulfated xylans were compared with that of sulfated glycosaminoglycans by measuring the ratio of the increase in fluorescence of AT III in the presence of sulfated polysaccharide to the fluorescence of AT-III alone for various mass ratios. Interactions of corn cob xylan sulfate (CCXS) and sodium pentosan polysulfate (SP-54) with AT-III resulted in enhancements of relative fluorescence which were lower than commercial heparin. At mass ratios below 1, heparan sulfate and low molecular weight heparin (LMWH) gave increases in the relative fluorescence higher than that of commercial heparin, while highly sulfated semisynthetic chondroitin sulfates A and C gave much smaller increases. The relative fluorescence enhancements of AT-III by heparan sulfate, commercial heparin, LMWH and heparin derived pentasaccharide (HDP) increased with increasing mass ratios while the enhancements by CCXS, SP-54 and the highly sulfated chondroitin sulfates A and C were reversed at higher mass ratios. The estimated dissociation constants (kd) for the interaction of AT-III and the heparin-related compounds showed that heparin sulfate and LMWH gave the lowest kd values indicating a higher affinity for AT-III while commercial heparin and HDP gave higher kd values, indicating a lower affinity for AT-III. SP-54 gave a kd value lower than CCXS, indicating a greater affinity for AT-III. A comparison of the near ultraviolet (UV) circular dichroism (CD) spectrum of AT-III alone and during its interaction with oat spelts xylan sulfate (OSXS) showed enhancements of the two aromatic amino acid regions corresponding to phenylalanine and tryptophan. PMID- 7542806 TI - Toxicity of sulphur mustard in adult rat lung organ culture. AB - The toxicity of the chemical warfare agent sulphur mustard, (bis-(2 chloroethyl)sulphide, HD), was examined in adult rat lung organ cultures. Assessment of HD-induced damage by the MTT cytotoxicity assay indicated that the median lethal concentration (LC50) of HD in these cultures was reproducible, and in the microM range. Damage to the lung slices was expressed only after a latent period of 48 h and did not increase significantly with longer expression times. Histopathological examination of HD-treated lung cultures showed that the structural changes in the lung tissue paralleled the toxicity measured biochemically, and were also similar to the damage found in animals and man exposed to HD in vivo. This in vitro model offers a useful tool with which to study the toxicity and mechanism of action of sulphur mustard. PMID- 7542807 TI - Studies on reproductive toxicity of iloprost in rats, rabbits and monkeys. AB - A reproduction toxicological test program was performed with the carbaprostacyclin derivative iloprost, an analogue to the endogenous prostacyclin PGI2, in order to detect possible effects on fertility and reproductive performance, on preimplantational, embryonal and fetal development, on delivery as well as on lactation and postpartum development. While in humans iloprost is administered as an i.v. infusion for 6 h/day, it was administered i.v. to rats, rabbits and monkeys by continuous infusion with a subcutaneously implanted pump. No influence on mating or reproductive parameters was found after treatment of male or female rats during the premating phase up to day 7 post coitum (p.c.). Embryonal and fetal development were not remarkably impaired in rabbits or monkeys after treatment throughout the period of organogenesis. The only remarkable observations in the embryotoxicity and peri-/postnatal studies in the rat were defects on the digits (reductions of phalangeal structures) in single individuals. These malformations were interpreted as resulting from a compound related hypotonia with subsequent change in the regional blood flow and the consequence of temporary impairments of placental blood supply leading to hypoxia in the affected structures. PMID- 7542809 TI - Tacrolimus analysis: a comparison of different methods and matrices. AB - We determined the through blood and plasma concentrations of tacrolimus from the day of transplantation through 30 days posttransplantation in four liver and four kidney transplant patients by three different methods. The first method involved a solid phase extraction of the blood or plasma using Sep-Pak columns (SPs) followed by quantitation of tacrolimus using an enzyme-linked immunosorbent assay (ELISA); the second method involved a liquid-liquid extraction using methylene chloride (MC) followed by quantitation of tacrolimus using the ELISA, and the third method involved a high-performance liquid chromatography (HPLC) fractionation of the extract obtained from the solid-phase extraction and quantitation of tacrolimus in the fractions by ELISA. The trough plasma tacrolimus concentrations ranged from 0.1 to 5.2 ng/ml. While the trough plasma concentrations of tacrolimus were similar and independent of the method of analysis in kidney transplant patients and in liver transplant patients with normal biochemical profile, in patients with liver dysfunction, tacrolimus plasma concentrations were higher when measured by SP-ELISA and MC-ELISA methods as compared to the HPLC-ELISA method. In plasma samples obtained from liver transplant patients with liver dysfunction, the presence of some metabolites that cross-reacted with the antibody used in the ELISA could be documented in the HPLC fraction corresponding to the metabolites. This indicates that while tacrolimus metabolites that cross-react significantly with the antibody used in the ELISA do not accumulate in kidney transplant patients, they can appear in the plasma of patients, they can appear in the plasma of patients with liver dysfunction. The trough blood tacrolimus concentrations in patients were significantly higher than the corresponding plasma concentrations and ranged from 1.4 to 107 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542808 TI - Organophosphate detoxication potential of various rat tissues via A-esterase and aliesterase activities. AB - Paraoxon and chlorpyrifos-oxon, active metabolites of the organophosphorus insecticides parathion and chlorpyrifos, can be detoxified via A-esterases and aliesterases. These enzyme activities were measured in various tissues of Sprague Dawley rats. High A-esterase activities were detected in liver, serum and liver mitochondrial/microsomal fractions. Low or no A-esterase activities were detected in other tissues and tissue fractions. A-Esterase substrate:substrate activity ratios suggest that the substrates are probably not degraded by the same enzyme. Highest aliesterase activities were observed in the small intestine and liver with moderate activity in kidney, serum and lungs. Low activities were noted in brain, spleen and skeletal muscle. PMID- 7542810 TI - Stability of FK 506 in whole blood samples. AB - The effect of storage time and temperature on the stability of FK 506 in patient blood samples and quality control samples was assessed with the Abbott IMx microparticle enzyme immunoassay. Blood FK 506 concentrations measured at days 0, 2, 3, 4, 7, and 14 were stable at room (22 degrees C), refrigerator (4 degrees C), and freezer (-70 degrees C) temperatures for the 2-week study period. Patient samples stored at -70 degrees C for almost 1 year and then reanalyzed for FK 506 were also found to be stable. PMID- 7542811 TI - Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. AB - The use of allograft donor skin as a permanent skin transplant in full-thickness burns is limited by its immunogenic properties and by the inappropriateness of immunosuppression of a burn patient. Allograft skin will initially take on a full thickness wound, but it is ultimately rejected. This immunogenic response to allograft skin is directed primarily against the cells of the epidermis and the endothelial cells in the dermis. To date, it has not been possible to remove these immunogenic cells while maintaining the integrity of the nonimmunogenic components of allograft dermis. In this study, we have investigated a method of processing porcine skin to produce an acellular, structurally intact, dermal matrix. We have developed a process that de-epidermizes and decellularizes fresh porcine skin, while maintaining the basement membrane complex and the extracellular matrix structure of the dermis. Porcine dermis processed by this method was initially assessed for toxicity in a rat subcutaneous implant study. In vivo assessment confirmed the absence of local and systemic toxicity. Subsequently, we investigated the potential use of this matrix in combination with a meshed split-thickness autograft (STSG) as a permanent allograft in full thickness wounds in pigs. Histological analysis revealed that the dermal matrix supported fibroblast infiltration, neovascularization, and keratinocyte migration from an overlying STSG. There was no evidence of an inflammatory cell infiltrate or a cell-mediated immune response. This apparent lack of an immune response was also tested in vitro by assessing recipient lymphocyte proliferation in response to an extract of the dermal matrix. These results suggest that skin processed by this method has the potential to be used as a permanent dermal allograft to augment the performance of an STSG in the closure of full-thickness wounds. PMID- 7542813 TI - Influence of spontaneous portosystemic collateral pathways on portal hemodynamics in living-related liver transplantation in children. Doppler ultrasonographic study. AB - We investigated the influence of spontaneous portosystemic collateral pathways on the portal hemodynamics and examined the necessity for ligating these vessels in pediatric liver transplantation from living donors. We assessed portal blood flow before, during, and after surgery in 82 pediatric recipients (mean age, 4.2 years), using Doppler ultrasonography. When blood flow in the reconstructed portal vein was decreased (< 10 ml/min/kg body weight) and portosystemic collaterals persisted during surgery, those vessels were ligated and Doppler flowmetry was examined again. Spontaneous portosystemic collaterals were detected at one or more sites in 67 patients before transplantation. These collaterals had been ligated in 17 patients before intraoperative flowmetry. Among the remaining 50 patients, initial Doppler studies revealed a decrease in portal blood flow in 22 patients. Nine patients had hepatofugal splenic venous flow and 6 had no significant flow signals from the intrahepatic portal vein. Ligation of collaterals resulted in a remarkable increase in portal blood flow in 20 patients, all of whom are alive. The remaining 2 patients died of graft failure due in part to portal hypoperfusion. On the other hand, the collaterals were not ligated in 24 patients because adequate portal blood flow was confirmed by intraoperative flowmetry. Postoperatively, flow signals from the unligated collateral vessels gradually diminished, but they still persisted in 3 patients at 12 months after transplantation. Hepatofugal blood flow through the portosystemic collateral pathways may persist after implantation of a normal graft. If the patent collaterals significantly reduce the effective portal blood flow, these vessels should be ligated in order to avoid graft failure. PMID- 7542812 TI - Activation of the plasma contact system and hemodynamic changes after graft revascularization in liver transplantation. AB - In this study, the relation between activation of the plasma contact system and hemodynamic changes during orthotopic liver transplantation was evaluated. Nineteen consecutive courses of OLT in 17 adult patients were investigated. Veno venous bypass was used in all patients. Blood samples were drawn through all phases of the procedure, and analyzed for the following parameters using functional techniques (chromogenic peptide substrate assays): plasma kallikrein (KK), prekallikrein, functional plasma kallikrein inhibition, C1 inhibitor, and alpha 2-macroglobulin. Plasma high molecular weight kininogen (HK) degradation was evaluated using the immunoblotting technique. An abrupt rise in KK activities occurred within 1 min after portal reperfusion of the liver graft (7-16 U/L, P < 0.05). Simultaneously, proteolytic breakdown of HK was seen. The elevated KK activities were maintained the next 1 1/2 hr. Ten min after graft reperfusion, a significant increase in cardiac output compared with the anhepatic phase (7.2 12.4 L/min, P < 0.05) was found. At the same time, systemic vascular resistance fell significantly (817-408 dynes x sec/cm-5, P < 0.05). The increase in plasma KK activities accompanied by simultaneous degradation of HK seen immediately after reperfusion of the liver graft may be due to contact activation as recipient blood contacts with the underlying basement membrane of injured sinusoidal endothelium in the transplanted liver. We suggest that hemodynamic changes associated with the postreperfusion syndrome seen after revascularization of the liver in OLT could at least be caused in part by bradykinin release due to contact activation. PMID- 7542814 TI - Angiogenesis and vascularization of murine pancreatic islet isografts. AB - Although the microvascular endothelium has been identified as the primary target site for the initiation of pancreatic islet graft rejection, little is known of the microvascular and cellular mechanisms involved, partly due to the lack of adequate models. Herein, we present a model for the in vivo assessment of the microcirculation of pancreatic islet grafts in mice. Isolated islets of Langerhans from immunocompetent hairless mice and immunoincompetent athymic nude mice were transplanted syngeneically into a specially designed dorsal skinfold chamber mounted on nondiabetic recipients. The islets' microcirculation was visualized by means of intravital fluorescence microscopy, and microcirculatory parameters were quantitatively analysed over a period of 14 days in the awake animal. Between day 2 and 4 after transplantation 84% (31/37; hairless mice) and 69% (36/52; nude mice) of the islet grafts revealed capillary sprouts and formation of new microvessels. On day 6, these sprouts were found interconnected, and red blood cell movement within the newly formed microvascular network was observed. The process of angiogenesis and revascularization was completed within 10 days after transplantation yielding a glomerulus-like network of capillaries as known for pancreatic islets in situ. Functional capillary density of the islet grafts ranged between 650 and 700 cm-1 in both hairless and nude mice. Within the islets' microvessels neither accumulation of leukocytes nor leukocyte-endothelial cell interaction was observed, indicating the lack of rejection and inflammation in these syngeneic islet grafts. We propose that this model provides a wide spectrum of promising experimental approaches for the study of microcirculatory and cellular mechanisms in free pancreatic islet transplantation. PMID- 7542815 TI - The effect of FK506 on glycemic response as assessed by the hyperglycemic clamp technique. AB - We studied seven nondiabetic subjects with the autoimmune diseases psoriasis, multiple sclerosis, and primary biliary sclerosis who were to receive FK506 as experimental immunotherapy. All subjects underwent two standard oral glucose tolerance tests and two 180-min hyperglycemic clamps immediately before and 10 weeks after starting FK506. There was no significant difference in weight or HbA1c pre- vs. post-FK506 treatment. FK506 levels were therapeutic and non-toxic (0.1-1 ng/ml) for all subjects studied. Repeated measures analysis of variance for interaction between time and treatment was performed on insulin (after outlier removed) and glucose values from the OGTT. There was neither time-by treatment interaction, nor a treatment effect (P > 0.1). There were no significant differences in pre- vs. post-FK506 treatment values of plasma glucose during the hyperglycemic clamp mean acute insulin response to glucose (AIRG) 164 +/- 38 pmol/L vs. 148 +/- 46 pmol/L (P > 0.1); mean incremental area under the insulin curve (IAUC) during the first 10 min of the study, 473 +/- 109 pmol/L vs. 443 +/- 146 pmol/L (P > 0.1); total area under the insulin curve (TAUC) during the first 10 min of the study, 786 +/- 152 pmol/L vs. 781 +/- 18 pmol/L (P > 0.1); mean glucose infusion rate (GIR) 37.7 +/- 5.0 mumol/kg/min vs. 33.3 +/- 4.4 mumol/kg/min (P > 0.1); or mean insulin sensitivity index (ISI), 3.05 +/- 0.4 vs. 3.13 +/- 0.5 (P > 0.1). Mean steady-state insulin secretion (SSI) was significantly lower 244 +/- 43 pmol/L vs. 200 +/- 25.2 pmol/L (P = 0.03). Peak first-phase insulin secretion values of 321 +/- 62 pmol/L vs. 263 +/- 57 pmol/L approached significance (P = 0.07). No patient progressed to diabetes during the study. FK506 decreased steady-state insulin secretion during the last 60 min of the clamp, regardless of initial glucose tolerance. Insulin sensitivity and glucose infusion rate did not change in the group as a whole with FK506 treatment. PMID- 7542816 TI - Contribution of anti-Gal to primate and human IgG binding to porcine endothelial cells. PMID- 7542817 TI - Should Medicare provide reimbursement for prostate-specific antigen testing for early detection of prostate cancer? Part II: Early detection strategies. PMID- 7542818 TI - A prospective randomized comparison of transurethral resection to visual laser ablation of the prostate for the treatment of benign prostatic hyperplasia. AB - OBJECTIVES: Transurethral resection of the prostate (TURP) represents the accepted standard of surgical therapy for the management of symptomatic bladder outlet obstruction due to benign prostatic hyperplasia (BPH). However, this is a major operative procedure associated with significant perioperative morbidity. Visual laser ablation of the prostate (VLAP) utilizing a neodymium:yttrium aluminum-garnet laser represents a new technologic approach to the surgical management of BPH. We compared the relative safety and efficacy of these two surgical approaches in a prospective, randomized trial. METHODS: At 6 investigational sites in the United States, 115 men with symptomatic BPH more than 50 years of age and not in retention, were randomly assigned to undergo either TURP (59 patients) of VLAP (56 patients). VLAP patients received a mean of 10,200 J of energy delivered in a mean of 5.5 intraprostate laser applications. At preoperative baseline, 3 months, 6 months, and 1 year postoperatively, all patients underwent clinical evaluations, including ultrasonic prostatic volume determination, standardized American Urological Association (AUA)-6 symptom score, peak urine flow, postvoid residual urine volume, and quality-of-life assessment. RESULTS: Compared to TURP, the VLAP procedure required less time (23.4 versus 45.2 minutes; P < 0.01) and shorter hospitalization (1.8 versus 3.1 days, P < 0.01). VLAP was associated with a significantly lower rate of serious treatment-related complications compared to TURP (10.7% versus 35.6%; P < 0.01). Only One (2.2%) patient undergoing VLAP experienced a greater than 2.2 g/dL decrease in hemoglobin compared to 40% of TURP patients (P = 0.01). No patient in the VLAP group required blood transfusion compared with 3.4% of those undergoing TURP. Of the 115 patients, clinical outcomes measured at 1 year showed a mean improvement in AUA-6 symptom scores of -9.0 for VLAP compared with -13.3 for TURP (P < 0.04), mean increase in peak urinary flow rate of 5.3 cc/s for VLAP compared with 7.0 cc/s for TURP (P = 0.27), and mean decrease in postvoid residual urine volume of -55.4 cc for VLAP compared with -138.8 cc for TURP (P < 0.01). At 1 year, 78.2% of patients undergoing VLAP indicated that their quality of life was improved compared with 93.0% of patients undergoing TURP (P = 0.03). When compared with TURP, treatment of BPH with VLAP is associated with less hemoglobin decrease, a lower likelihood of serious complication, and requires less procedure time and a shorter hospital stay. Through a 1-year follow-up, VLAP produced significant improvement over baseline in objective and subjective outcome measures. However, for 1-year improvement in AUA-6 symptom score, postvoid residual urine volume, and quality of life, VLAP was less effective than TURP. CONCLUSIONS: In this initial study in the United States, with relatively low energy laser applications, VLAP did not result in as complete a removal of prostatic tissue as did TURP. Considering the lower morbidity, shorter procedure and hospitalization times, and the degree of effectiveness that was achieved even at the low-energy doses used in this study, VLAP appears to be a viable and safe alternative to standard TURP. PMID- 7542819 TI - Doxazosin in benign prostatic hyperplasia: effects on blood pressure and urinary flow in normotensive and hypertensive men. AB - OBJECTIVES: To assess the effects of doxazosin, a selective alpha 1 adrenoceptor inhibitor, on blood pressure and urinary flow in normotensive and hypertensive (sitting diastolic blood pressure more than 90 mm Hg) men with prostatic hyperplasia (BPH). METHODS: Patients (n = 232) with bladder outflow obstruction due to BPH, classified as normotensive or hypertensive, were enrolled into two, double-blind, placebo-controlled studies. After a washout period of at least 1 week, patients were randomized to doxazosin or placebo, and treatment was continued for 9 to 12 weeks. In addition to measures of standing and sitting blood pressures, the patients' response to treatment was also assessed with regard to urinary flow. Although the protocols differed, they were consistent enough to permit pooling of a number of variables. RESULTS: Results from the two studies demonstrated that doxazosin produced a clinically significant reduction in blood pressure only in hypertensive patients (systolic blood pressure/diastolic blood pressure: baseline 162/99 mm Hg, endpoint 143/89 mm Hg); little or no reduction was evident in normotensive patients (systolic blood pressure/diastolic blood pressure: baseline 139/82 mm Hg, endpoint 134/78 mm Hg). Similar effects in terms of uroflow were seen in hypertensive and normotensive patients. The maximum flow rate in hypertensive patients treated with doxazosin increased from 8.82 to 10.84 mL/s (+ 23%) and in normotensive patients treated with doxazosin from 8.52 to 10.90 mL/s (+ 28%). A greater than 30% improvement in maximum flow rate was achieved in 46 of 97 (47.4%) patients in the doxazosin group and 26 of 98 (26.5%) patients in the placebo group. Treatment with doxazosin was effective and generally well tolerated. The majority of side effects were mild or moderate, only slightly higher in the active treatment group compared with placebo, and similar in hypertensive and normotensive patients. CONCLUSIONS: Treatment with doxazosin is effective and well tolerated in the treatment of BPH. It appears to be a particularly appropriate therapy for men with both BPH and hypertension but can be safely administered to normotensive men without causing significant blood pressure reduction. The beneficial effects on urinary flow are similar, irrespective of blood pressure. PMID- 7542821 TI - Methodological and clinical comparison of the ACS prostate-specific antigen assay and the Tandem-E prostate-specific antigen assay in prostate cancer. AB - OBJECTIVES: The new ACS prostate-specific antigen (PSA) assay was methodologically and clinically compared with the established Tandem-E PSA assay. We intended to find a possible advantage in primary diagnosis and monitoring the recurrence of prostate cancer due to the additional better recognition of the free PSA form by the ACS PSA assay. METHODS: sera of 51 healthy men, 127 patients with hyperplasia, and 82 untreated patients with prostate cancer were analyzed by means of the Tandem-E PSA assay (Hybritech) and the ACS PSA assay (Ciba Corning). Follow-up was done on 12 cancer patients with recurrences. RESULTS: Both assays correlated very well (r = .98 for all studied men or hyperplasia patients or cancer patients). However, both assays did not yield comparable values: The ACS assay was characterized by nearly doubled values compared with the Tandem-E assay. At 95% specificity versus patients with benign hyperplasia, cutoff values were obtained as follows: 28.8 ng/mL for the ACS PSA assay and 15.2 ng/mL for the Tandem-E assay. At 95% specificity versus hyperplasia patients, we calculated sensitivities of 60% (ACS PSA) and 63% (Tandem-E PSA). Our longitudinal study revealed more prominent slopes for the ACS assay in patients with recurrent cancer disease. However, using either the ACS assay or the Tandem-E assay, the PSA increase started at the same time. In 1 patient, the increasing ACS PSA accelerated 3 months earlier than the increasing Tandem-E PSA did. CONCLUSIONS: The ACS assay has obviously higher PSA levels. The clinician is not familiar with such high PSA levels. The specificity-sensitivity profile nonetheless remains unchanged. If the PSA concentration is measured by the ACS assay, patients who relapse will reveal a more rapid PSA increase. Then, recurrent cancer disease may be detected earlier in some cases. PMID- 7542820 TI - Measurement of the proportion of free to total prostate-specific antigen improves diagnostic performance of prostate-specific antigen in the diagnostic gray zone of total prostate-specific antigen. AB - OBJECTIVES: This study examined the clinical significance of non-complexed (free) prostate-specific antigen (PSA) in the differential diagnosis of prostate cancer with an emphasis on patients with total PSA values between 4.0 and 10.0 ng/mL (the diagnostic gray zone). METHODS: Serum samples were obtained from three specimen banks. Patient samples consisted of 55 untreated histologically confirmed primary cancer, 62 men with untreated benign prostatic disease histologically confirmed by 6 negative sextant biopsies, and 64 asymptomatic healthy male controls with normal digital rectal examinations and PSA values less than 4.0 ng/mL. All patients were between the ages of 50 and 75 years. Total PSA levels were determined using the PA immunoassay performed on the TOSOH AIA-1200 automated immunoassay instrument. Free PSA levels were determined using a monoclonal-polyclonal antibody sandwich radioimmunoassay. The proportion of free to total PSA was calculated by dividing the patient's free PSA value by the total PSA value. RESULTS: When all subjects were included, both total PSA and the proportion of free to total PSA significantly differentiated between patients with prostate cancer and patients with benign histologic conditions (P < 0.0001). However, in men with total PSA values between 4.0 and 10.0 ng/mL, the proportion of free to total PSA significantly differentiated between patients with benign and malignant histologic conditions (P = 0.0004), whereas the total PSA did not (P = 0.13). Among this subgroup of patients, the analysis of sensitivity and specificity showed that the proportion of free to total PSA had a clearly higher specificity compared with that of the total PSA at the same level of sensitivity. CONCLUSIONS: Measurement of the free PSA level in a patient's serum and calculation of the proportion of free to total PSA enhances the ability to distinguish benign histologic conditions from cancer while retaining high sensitivity for detecting cancer in men who present with total PSA levels between 4.0 and 10.0 ng/mL. A large-scale population-based study is currently in progress to confirm this preliminary finding. PMID- 7542822 TI - Normal range prostate-specific antigen versus age-specific prostate-specific antigen in screening prostate adenocarcinoma. AB - OBJECTIVES: Prostate-specific antigen (PSA) has become the most useful serum tumor marker in the diagnosis and screening of prostate adenocarcinoma. The currently cited reference range of normal (0 to 4.0 ng/mL monoclonal) lacks both the sensitivity and specificity to be universally accepted as a screening test, and alternatives to serum PSA have been proposed, such as PSA density, PSA velocity, and age-adjusted PSA. Age-adjusted PSA takes into account the facts that as men grow older the prostate enlarges and that screening should have maximum sensitivity in younger men and maximum specificity in older men. METHODS: A population of 4,710 men with no known history of prostate adenocarcinoma underwent 5,629 examinations by transrectal ultrasound of the prostate (TRUS) from 1987 to 1994. This population consists of Mobile Urology Group, Mobile, Alabama, and Emory University, Atlanta, Georgia, patient databases. We have examined our data to determine the sensitivity, specificity, and positive predictive values for normal range PSA (0 to 4 ng/mL) versus age-specific PSA values. RESULTS: A total of 2040 patients had an abnormal digital rectal examination (DRE) and 3581 procedures were performed for an elevated PSA and a normal DRE. Biopsies were performed in 2,657 patients with 945 (35.6%) positive for cancer. Criteria for biopsy included elevated PSA (more than 4 mg/mL), PSA density more than 0.15 abnormal DRE, or suspicious TRUS. Patients were grouped according to decade: group 1 (ages 40 to 49 years, n = 183), group 2 (ages 50 to 59 years, n = 1018), group 3 (ages 60 to 69 years, n = 2358), and group 4 (ages 70 to 79 years, n = 1687). CONCLUSIONS: Use of the age-specific range for PSA increases the sensitivity in younger men more likely to benefit from treatment, and decreases the biopsy rate in older patients who may not be candidates for aggressive treatment. Age-adjusted PSA is the most valuable for patients over the age of 70 years of whom 22% would be spared TRUS with biopsy. PMID- 7542824 TI - Combined hormonal and radiation therapy for lymph node-positive prostate cancer. AB - OBJECTIVES: To evaluate the efficacy of combined radiation and hormonal therapy in patients with prostate cancer metastatic to the pelvic lymph nodes. METHODS: Fifty consecutive patients with node-positive prostate cancer were evaluated by the Departments of Urology and Radiation Oncology at the University of Pennsylvania and offered combined hormonal and radiation therapy. All patients received pelvic radiation to 45 Gy, with a boost dose to the prostate to 65 to 71 gy. Forty-five of the patients were treated with concurrent hormonal therapy consisting of diethylstilbestrol (2 patients), orchiectomy (18 patients), leuprolide (5 patients), or combined androgen blockade (20 patients); the other 5 patients declined hormonal therapy. Patients represented a group with locally advanced disease with a high incidence of T3 tumors (66%), high grade (74%; Gleason score more than 7), high prostate-specific antigen (PSA) (40%; more than 30.0 ng/mL), and a high incidence of gross (36%) or bilateral (30%) adenopathy and a high incidence of multiply involved lymph nodes (62%). RESULTS: Median follow-up of patients is 42 months (range, 10 to 102). All 5 patients declining hormonal therapy relapsed within 18 months and only 1 patient survived longer than 3 years. Among patients treated with combined hormonal and radiation therapy, the 6-year survival rate is 82%, the clinical disease-free survival at 6 years is 71%, and the probability of survival free of recurrence, with a PSA less than 0.2 ng/mL, is 62%. Only two PSA recurrences occurred, both in patients who elected to discontinue hormone therapy. There was no synergistic toxicity observed as a result of combined therapy. CONCLUSIONS: Combined hormonal and radiation therapy offers the potential for extended disease-free survival and may represent an effective treatment option for patients with locally advanced prostate cancer. PMID- 7542823 TI - The role of transrectal ultrasound-guided biopsy-based staging, preoperative serum prostate-specific antigen, and biopsy Gleason score in prediction of final pathologic diagnosis in prostate cancer. AB - OBJECTIVES: To evaluate the role of ultra sound-guided systematic and lesion directed biopsies, biopsy gleason score, preoperative serum prostate-specific antigen (PSA) as three objective and reproducible variables to provide a reliable combination in preoperative identification of risk of extraprostatic extension in patients with clinically localized prostate cancer. METHODS: The case records of 813 patients who underwent radical prostatectomy for clinically localized prostate cancer were analyzed. All had multiple systematic biopsies, two to three from each lobe, in addition to lesion-directed biopsies. Additionally, biopsies were done on seminal vesicles (SVs), if abnormal. Based on biopsy results, patients were classified as having stage B1 (T2a-T2b) or B2 (T2c) disease, depending on whether biopsies from one or both lobes were positive and stage C (T3) if there was evidence of SV involvement by biopsy of biopsies from areas of extracapsular extension as seen on transrectal ultrasound (TRUS) were positive. Logistic regression analyses with log likelihood chi-square test was used to define the correlation between individual as well as combination of preoperative variables and pathologic stage. RESULTS: On final pathologic examination, 473 (58%) patients had organ-confined disease, 188 (23%) had extracapsular extension (ECE), with or without positive surgical margins, and 72 (9%) had SV involvement. Eighty (10%) patients had pelvic lymph node metastases. Biopsy-based staging was superior to clinical staging in predicting final pathologic diagnosis. Logistic regression analyses revealed that the combination of biopsy-based stage, preoperative serum PSA, and biopsy Gleason score provided the best prediction of final pathologic stage. Probability plots constructed with these data can provide significant information on risk of extraprostatic extension in individual patients. CONCLUSIONS: This study demonstrates that TRUS-guided systematic biopsy in combination with preoperative serum PSA and biopsy Gleason score may provide a cost-effective approach for management decisions and prognostication in patients with prostate cancer. PMID- 7542825 TI - [The preoperative preparation of patients with thyrotoxicosis without the use of thyrostatics]. PMID- 7542827 TI - Response of foot-and-mouth disease virus C3 Resende to immunological pressure exerted in vitro by antiviral polyclonal sera. AB - The foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, FMDV has a high mutation rate and it has been proposed that selection exerted by antibodies of the host could play a major role in its evolution. In this work, antiserum-resistant variants of FMDV (Nr variants) were selected upon 25 serial passages of a cloned C3 Resende strain on secondary monolayers of fetal bovine kidney (FBK-2) cells in the presence of subneutralizing levels of antiviral polyclonal sera (APS). After serial passage under immune selective pressure, the five Nr variant populations selected from five independent serial passages--their controls remaining unmodified--acquired the following characteristics: (i) increased resistance to neutralization by APS; (ii) five different antigenic specificities detected by enzyme-linked and neutralization assays using monoclonal antibodies; (iii) the same modification (residue 146, S to L) at the major antigenic site of VP1 (G-H loop, the 135-160 region); and (iv) specific changes for each Nr population outside the major antigenic site of VP1 at residues 46, 48 and 49 of the 40-60 region of VP1 (B-C loop). These results extend our previous work on selection of Nr variants using polyclonal sera, and add new information with regard to antigenic variation, mainly concerning the involvement of the 40-60 region of VP1 in the process of immune selection. PMID- 7542826 TI - T-helper epitopes of the E7 transforming protein of cervical cancer associated human papillomavirus type 18 (HPV18). AB - The presence of T-helper epitopes within the E7 transforming protein of human papillomavirus type 18 (HPV18) was sought using a series of overlapping synthetic 15-20 mer peptides spanning the entire 105 amino acid sequence of this protein. Two H-2k restricted T-helper epitopes were defined, comprising 44VNHQHLPARRA55 and 81DDLRAFQQLF90 as the minimal T proliferative epitopes. Peptides containing these epitopes were able to provide cognate help to B epitopes from HPV18E7 protein for production of antibody to this protein in vivo in CBA/CaH mice. No H 2b or H-2d restricted epitopes were demonstrable, and in H-2d mice this was associated with poor antibody response to the E7 protein. There is no "promiscuous" T-helper epitope in HPV18 E7 comparable to the 49DRAHYNI55 sequence in HPV16 E7, and restricted T-helper epitope availability may be a determinant of poor immune responses to this protein after natural infection. PMID- 7542828 TI - Tumor necrosis factor alpha but not lymphotoxin is overproduced by blood mononuclear cells in multiple sclerosis. AB - The cytotoxic cytokines tumor necrosis factor alpha (TNF-alpha) and lymphotoxin (LT) possess toxic activity against myelin and/or oligodendrocytes in vitro. Multiple sclerosis (MS) plaques within the central nervous system (CNS) are infiltrated by peripheral blood mononuclear cells (PBMC). In this study the production of TNF-alpha and LT by PBMC in active MS were measured. PBMC were isolated from the blood of MS patients in relapse and also patients with other neurological diseases (OND) and healthy controls (HC). Isolated cells were cultured unstimulated or stimulated with phytohemagglutinin A (PHA), lipopolysaccharide (LPS) and myelin basic protein (MBP)--a hypothetical autoantigen for MS. Cytokine production was assessed using ELISA method. In the MS group, PBMC without stimulation as well as after stimulation with MBP displayed a significantly increased production of TNF-alpha. LT production was similar in MS and control groups. These results suggest that TNF-alpha but not LT is overproduced by PBMC during MS relapse. PMID- 7542829 TI - Risperidone versus zuclopenthixol in the treatment of acute schizophrenic episodes: a double-blind parallel-group trial. AB - A double-blind, randomized, multi-center, parallel-group study was conducted in Finland to compare the efficacy and safety of risperidone with zuclopenthixol in patients with acute exacerbations of schizophrenia or schizophreniform disorder. Ninety-eight patients were randomly assigned to treatment with risperidone (n = 48) or zuclopenthixol (n = 50), in variable doses, for 6 weeks. The mean daily doses of risperidone and zuclopenthixol at the end of the trial were 8 mg and 38 mg respectively. Efficacy was assessed throughout by the Positive and Negative Syndrome Scale for schizophrenia and Clinical Global Impression. Safety assessments included the Extrapyramidal Symptom Rating Scale, UKU Side-Effect Rating Scale, vital signs, body weight and laboratory screening. The results indicate that risperidone is at least as effective as zuclopenthixol for the treatment of acute schizophrenic episodes, with a trend towards greater improvement in the overall severity of symptoms. The onset of action was significantly shorter with risperidone than with zuclopenthixol. Although the general tolerability of the two drugs was comparable, fewer patients experienced extrapyramidal symptoms with risperidone, so that significantly fewer risperidone treated patients required antiparkinsonian medication. PMID- 7542830 TI - Sickle cell anaemia in Nigeria: a comparison between Benin and Lagos. AB - The clinical and haematological features of 77 patients of Bini and 107 patients of Yoruba origin with homozygous sickle cell (SS) disease have been compared. The Bini population were generally younger and had a slightly lower incidence of alpha thalassaemia but even after correction for age and alpha thalassaemia status, this group had significantly lower HbA2 and higher HbF and MCV values. Clinically the Bini group had significantly less dactylitis and more acute chest syndrome. The decreased frequency of dactylitis is consistent with the higher HbF level in the Bini population and the mechanism of the other effects are discussed. PMID- 7542831 TI - Vitamin A supplementation, morbidity, and serum acute-phase proteins in young Ghanaian children. AB - The association of vitamin A supplementation with concentrations of positive acute-phase proteins in the serum was investigated in the Child Health Study of the Ghana Vitamin A Supplementation Trials, a randomized, controlled trial of the effect of vitamin A on morbidity in children aged < 5 y. Mean serum concentrations of alpha 1-acid glycoprotein, serum amyloid A, and C-reactive protein did not differ overall between the vitamin A-supplemented and placebo treated groups. Treatment groups were then subdivided according to what symptoms children had experienced in the week before blood sampling. Acute-phase-protein responses to fever and cough were not affected by vitamin A supplementation. There was a tendency for vitamin A-supplemented children, but not placebo children, to have elevated acute-phase proteins in association with reported vomiting or severe diarrhea. The failure of unsupplemented children to mount an acute-phase response may have contributed to their increased morbidity from gastrointestinal symptoms. PMID- 7542832 TI - Malignant peritoneal mesothelioma after Thorotrast exposure. AB - A case of a malignant peritoneal mesothelioma in a 63-year-old male patient with a history of exposure to Thorotrast in 1945 is presented. There was no history of exposure to asbestos. The clinical manifestation was a serosal effusion, which required weekly ascites puncture until therapy with intraperitoneal bleomycin was initiated. The latter treatment led to a significant reduction of ascites without any influence on tumor progression. Unfortunately, intraperitoneal bleomycin was accompanied by pulmonary toxicity, but at a higher total dose than known for intravenous administration. Three years after diagnosis the patient is still alive, without relapse of ascites production after bleomycin had to be stopped. Considering the risk of pulmonary fibrosis with high-dose intraperitoneal bleomycin and the lack of efficacy on tumor reduction, bleomycin seems to offer no advantage with respect to cisplatin. PMID- 7542833 TI - The untreated patient with squamous carcinoma of the head and neck. AB - Of 3,482 patients with head and neck squamous carcinoma, 539 were unsuitable for radical treatment. Patients over 60 years of age were 1.6 times more likely not to receive radical treatment than younger patients. Patients who were unfit [Eastern Cooperative Oncology Group (ECOG) 1-4] were 1.4 times more likely not to receive radical treatment than those who were generally well. Patients with advanced primary tumours (T3, T4) were twice as likely to be considered unsuitable for radical treatment as patients with small tumours. Patients with neck node metastases were 2.1 times more likely to be considered unsuitable for radical treatment compared with those without nodes. In addition, if neck nodes were present their site influenced the decision to treat radically. Patients with disease low in the neck were 1.3 times more likely to be considered unsuitable for radical treatment than those with disease high in the neck. Survival for untreated patients was dismal. The median tumour specific survival was 5 months, and the 5-year survival was 4%. In contrast the 5-year survival for treated patients was 60%. Cox's regression on untreated patients showed that advanced age, poor general condition, and advanced nodal disease all adversely affected survival. PMID- 7542834 TI - Marker chromosome 21 identified by microdissection and FISH. AB - A child without Down syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with alpha satellite DNA probes for acrocentric chromosomes, and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. PMID- 7542835 TI - Familial inverted duplication 7p. AB - A 10-month-old female with developmental delay and failure to thrive was referred for genetic evaluation as part of an adoption assessment. Physical exam showed a mildly beaked nose and clinodactyly, but otherwise nothing remarkable. Chromosome analysis showed an inverted duplication of the p12.2-->p13 portion of chromosome 7[(46,XX, dup(7)(p13p12.2)]. The proposita's older brother, mother, and grandmother were cognitively delayed and had the same chromosome 7 duplication. A review of the literature showed no other cases involving this exact duplication. PMID- 7542836 TI - Risperidone for treatment-refractory schizophrenia. PMID- 7542837 TI - Two cases of risperidone-induced neuroleptic malignant syndrome. PMID- 7542838 TI - Two cases of risperidone-induced neuroleptic malignant syndrome. PMID- 7542839 TI - Use of risperidone with a patient sensitive to conventional neuroleptic drugs. PMID- 7542840 TI - Risperidone for treating dementia-associated aggression. PMID- 7542841 TI - Treating Alzheimer's disease. PMID- 7542842 TI - Molecular phylogeny of Guanarito virus, an emerging arenavirus affecting humans. AB - The nucleotide sequence of a portion of the nucleocapsid (N) gene of the Guanarito virus prototype strain (INH-95551) has been determined. It was obtained by direct RNA and polymerase chain reaction (PCR) fragment sequencing of the 3' end of the small (S) RNA fragment. A comparison of this 782-nucleotide segment was done with the known homologous gene sequences of five other arenaviruses: Junin, Machupo, Tacaribe, Pichinde, and lymphocytic choriomeningitis (LCM). Phylogenetic analysis of the N gene open reading frame showed that Guanarito virus is genetically distinct from other members of the Arenavirus family, with 32% nucleotide sequence divergence from Junin, 30% from Machupo, 32% from Tacaribe, 41% from Pichinde, and 45% from LCM. Comparison of amino acids encoded by this sequence region indicated a probable antigenic domain (amino acids 55-63) shared among all arenaviruses studied to date. Along with its host restriction and focal distribution, our data support the hypothesis that this virus has been evolving independently in its endemic focus, for some time. PMID- 7542844 TI - Mechanisms of cytoadhesion of flowing, parasitized red blood cells from Gambian children with falciparum malaria. AB - Adhesion of parasitized red blood cells to vascular endothelium is considered to be a major factor in the pathophysiology of falciparum malaria, and so the molecular mechanisms and rheologic characteristics of this interaction are of profound importance. We have investigated the adhesive behavior of wild-type parasite isolates cultured from the blood of Gambian children with falciparum malaria and allowed to flow over surfaces coated with formaldehyde-fixed human umbilical vein endothelial cells (HUVEC) or platelets. Parasitized cells were able to attach to HUVEC and/or to platelets, and studies with monoclonal antibodies showed that intercellular adhesion molecule-1 (ICAM-1) and CD36 antigen were the major mediators of adhesion for the two surfaces, respectively. The levels of adhesion to HUVEC and to platelets were highly variable but did not correlate with each other, so that different isolates express independently variable capacities to bind to the two receptors. Adhesion was stationary for platelets and generally at a higher level compared with binding to HUVEC, which was predominantly (about 60%) of a rolling type. The stationary component of adhesion to HUVEC represented a greater proportion of adhesion for the wild isolates than for laboratory-adapted strains, and this form of adhesion was relatively insensitive to antibody to ICAM-1. This suggests the existence of an additional endothelial cell-expressed receptor for the wild isolates. These studies show wide variation in the ability of wild isolates of Plasmodium falciparum to adhere to ICAM-1, CD36 antigen, and possibly other receptors in the presence of physiologically relevant flow.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542843 TI - Antibodies to a Plasmodium falciparum blood-stage antigen as a tool for predicting the protection levels of two malaria-exposed populations. AB - To evaluate the ability of antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) epitopes to discriminate between individuals well protected or poorly protected against malaria, a receiver operating characteristic analysis was performed in two populations living in Madagascar and Malawi. The definition of protection was based on longitudinal measurements of clinical malarial attacks during the season of high malaria transmission in the Madagascar study, and on a cross-sectional measurement of parasitemia in the Malawi study. Antibodies to peptides reproducing the 4-mer, 8 mer, and 11-mer of the Pf155/RESA were tested for their reactivities using the Falcon assay screening test-enzyme-linked immunosorbent assay. Maximal detection of poorly protected individuals (specificity = 100%) corresponded to high cutoff antibody titers (range = 1.65-3.0 optical density [OD] units in the Madagascar study and 0.67-1.42 OD units in the Malawi study) and a sensitivity less than 50%. For a given sensitivity of 50%, specificity ranged from 55% to 62% in the Madagascar study, and from 67% to 94% in the Malawi study. The antibody cutoff titers corresponding to minimal misclassification rates ranged from 0.24 to 1.73 OD units in the Madagascar study and from 0.15 to 0.55 OD units in the Malawi study. For each antibody, the highest detectability value as measured by the area under the curve was obtained for anti-R11 in the Malawi study (0.838). In demonstrating such qualities, antibodies to Pf155/RESA epitopes could be used for screening poorly protected populations in which malaria control programs have to be implemented. PMID- 7542845 TI - Characteristics of Shigella sonnei infection of volunteers: signs, symptoms, immune responses, changes in selected cytokines and acute-phase substances. AB - Shigella sonnei infection resulting from oral administration of 500 colony forming units was followed in 11 volunteers with the objective of studying the immune response and pathogenesis. Characterization of infection included recording of signs and symptoms, excretion of S. sonnei in stool, measurement of humoral tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma), C-reactive protein, IL-2 receptor, soluble CD8, antibody-antigen complexes, and endotoxin. Measurements were also made of the immune response including lymphocytes secreting antibody to S. sonnei O antigen and serum antibody to this antigen. Six of the volunteers developed typical shigellosis with excretion of bacteria in stool and systemic signs and symptoms, three excreted bacteria but did not show illness, and two showed no evidence of infection or illness. Shigellosis was characterized by excretion in stool of S. sonnei beginning on average 1.3 days after ingestion. Excretion of S. sonnei (mean of time of the first positive cultures) was followed in sequence by the onset of increases in TNF-alpha (10 hr), liquid stools (14 hr), fever and dysentery (18 hr), IFN-gamma (22 hr), and C-reactive protein (34 hr). A S. sonnei specific immune response was demonstrated somewhat later, between days 4 and 7 postinfection by antibody-secreting cells, and between days 7 and 14 postinfection by humoral antibody. Shigellosis was not associated with increased humoral IL-1 beta, endotoxin, or antigen-antibody complexes. PMID- 7542846 TI - Developmental perspective of sensory organization on postural control. AB - The development of sensory organization on postural control was studied using computerized dynamic posturography. Generalized postural stability increased with age but had not reached the adult level at the age of 15 years. The significance of each sensory component for postural control was analysed. Somatosensory function developed early and became comparable with the adult level at the age of 3-4 years. The visual function followed and reached the adult level at the age of 15 years. The vestibular function developed later, showing a considerably lower level even at the age of 15 years. Girls were superior to boys with respect to the vestibular function at the age of 7-8 years. The implication of this sexual difference for some developmental disorders is discussed. PMID- 7542847 TI - Ethanol-induced depletion of lymphocytes from the mesenteric lymph nodes of C57B1/6 mice is associated with RNA but not DNA degradation. AB - The effects of ethanol (EtOH) consumption by adult female C57B1/6 mice on lymphocyte populations of the mesenteric lymph nodes (MLNs) were determined by feeding mice with the Lieber-DeCarli liquid diet by a pair-feeding paradigm. Histological analysis of the MLNs of EtOH-fed mice showed a progressive loss of lymphocytes from the medullary regions at 3, 5, and 7 days after initiation of the EtOH diet. The stromal cells in the medullary region also demonstrated a progressive alteration in stellate morphological features at times corresponding to those of loss of lymphocytes from this region. Microscopic evaluation of the follicle regions of MLNs obtained from mice fed an EtOH-containing diet showed no appreciable alterations in morphological characteristics. The number of tingible body macrophages in the germinal centers of the follicles, however, was increased after 3 days of EtOH diet feeding and declined progressively after this time. Flow cytometric analysis of isolated lymphocytes showed a depletion of both T and B cell populations from the MLNs. In contrast to B cells, however, T cells were depleted through 7 days of EtOH diet feeding. Total RNA isolated from the MLNs of mice consuming the EtOH-containing diet was progressively degraded. No degradation of DNA was observed. These study results establish that continuous consumption of dietary EtOH adversely affects the cellularity of MLN, resulting in a progressive loss of lymphocytes that is associated with degradation of total RNA. PMID- 7542848 TI - Chronic alcohol administration stimulates nitric oxide formation in the rat liver with or without pretreatment by lipopolysaccharide. AB - This study examines the effect of chronic alcohol consumption on nitric oxide release from the liver of rats with or without lipopolysaccharide (LPS) (Escherichia coli) treatment. Reactive nitrogen intermediates (RNIs) in plasma were monitored with an NOx Analyzer, and nitric oxide (NO) production was measured as nitrite or nitrite + nitrate accumulation in perfusates of the perfused liver, and in supernatants of the freshly isolated hepatic cells after incubation for 3 hr in Hank's balanced salt solution buffer containing 1 mM L arginine. RNI concentration in plasma of control rats was 32.0 +/- 3.4 microM (mean +/- SE). Livers from diet-fed control rats produced RNIs at the barely detectable rate of 7.8 +/- 1.5 nmol/hr x g wet liver. Six hr after administration of LPS (1 mg/kg, i.v.), plasma RNI levels in diet-fed control rats increased to 426.9 +/- 29.4 microM, and RNI release from the perfused liver was also markedly elevated to 97.7 +/- 7.7 nmol/hr x wet g liver, indicating hepatic NO release as a potentially important source for the increased RNI in plasma. The presence of NG-monomethyl-L-arginine (0.5-1 mM) or the absence of L-arginine in the perfusate inhibited LPS-induced stimulation of RNI release. EGTA (1 mM) had little effect, indicating that the increased RNI release was likely to be due to inducible NO synthase activity. The release of RNIs by freshly isolated Kupffer cells increased 13-fold, and this small cell mass contributed almost half of the hepatic RNI production under these conditions. Plasma ALT concentration was elevated after LPS administration, indicating incipient liver damage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542849 TI - Ethanol suppresses Mycobacteria tuberculosis-induced mRNA for nitric oxide synthase in alveolar macrophages, in vivo. AB - Acute ingestion of alcohol [ethanol (ETOH)] adversely affects the immunocompetence of both naive individuals as well as chronic alcohol abusers. An increased incidence and severity of tuberculosis is found in chronic alcohol abusers. Nitric oxide (NO) produced by alveolar macrophages (AMs) may play a role in the in vitro killing of Mycobacterium avium and Mycobacterium tuberculosis (MTB). Moreover, tumor necrosis factor-alpha (TNF-alpha) is believed to be a primary cytokine mediator of NO production by AMs. Recent studies from our laboratory demonstrated that ETOH suppressed endotoxin-induced increases in both TNF-alpha and NO in AMs, in vivo. We tested the postulate that acute ingestion of ETOH can interfere with mycobacteria-induced upregulation of the NO system in AMs, in vivo. We show that heat-killed M. avium complex (MAC) and human virulent MTB instilled into rat lungs rapidly increased mRNA for inducible NO synthase II (iNOS) of AMs in fluid obtained by bronchoalveolar lavage (BAL fluid). This was associated with production of reactive nitrogen intermediates [(RNIs); NO2- and NO3-] in BAL fluid, lung homogenate, and AMs in the absence of a significant increase in BAL fluid TNF-alpha. A single dose of ETOH (5.5 g/kg, ip) administered 30 min before intratracheal administration of MAC or MTB attenuated both MAC and MTB-induced increases in RNI in BAL fluid, lung, and AMs, and the increase in mRNA for iNOS. Thus, mycobacteria upregulate iNOS mRNA and enhance RNI production by AMs without any increase in the production of TNF-alpha. Moreover, ETOH attenuates mycobacteria-induced upregulation of mRNA for iNOS and RNI production in the absence of ETOH-mediated suppression of TNF. Speculatively, ETOH-mediated inhibition of the AM NO system may offer an explanation for the increased severity of mycobacterial infections in alcoholics. PMID- 7542850 TI - Effect of ethanol on p36 protein kinase substrate and insulin receptor substrate 1 expression and tyrosyl phosphorylation in human hepatocellular carcinoma cells. AB - Ethanol inhibits insulin (IN) and epidermal growth factor (EGF)-induced hepatocyte DNA synthesis. Growth factor receptor kinases, such as IN and EGF, phosphorylate insulin receptor substrate (IRS-1) and p36 protein kinase substrate, respectively, on tyrosine residues. IRS-1 and p36 are thought to be important intracellular signal transduction molecules involved in the regulation of cell growth. These investigations explored the effect of ethanol additions on the expression and tyrosyl phosphorylation (TP) of p36 and IRS-1 in a human hepatocellular carcinoma cell line (FOCUS) in relationship to cell proliferation induced by IN and serum growth factor stimulation. It was found that p36 was constitutively and highly expressed in serum-starved cells and protein, and mRNA levels did not change with cell proliferation induced by growth factors. However, exposure of FOCUS cells to ethanol additions substantially inhibited TP of p36. The early TP of IRS-1 induced by IN stimulation was also reduced by ethanol additions. Finally, there was a parallel decrease of FOCUS cell proliferation in ethanol-exposed cultures. These studies suggest that one possible mechanism of ethanol inhibitory effect on cell proliferation is through reduced TP of putative intracellular signal transduction molecules, such as p36 and IRS-1. PMID- 7542851 TI - The segmental precision of the motor projection to the intercostal muscles in the developing chicken embryo. A differential labelling study using fluorescent tracers. AB - Each skeletal muscle in the vertebrate is innervated by a group of motoneurons called a motoneuron pool. Retrograde labelling of single motoneuron pools has suggested that the arrangement of motoneuron pools innervating different limb muscles does not change during the embryonic period when more than 50% of the motoneurons die. In this study we retrogradely labelled neighbouring intercostal motoneuron pools differentially with latex microspheres or dextran amines coupled to fluorescent dyes. We then mapped the positions of the differentially labelled motoneurons in whole-mount preparations using a computer-aided drawing system. While the intercostal motoneuron pools are clearly segregated even at early stages, there is some intermingling at the rostral and caudal ends. We used a logistic regression to determine the extent of segmental overlap, and to facilitate a quantitative comparison of the overlap at different stages. Statistical analysis shows that the overlap (expressed as the percentage of the length of the overlapping motoneuron pools) decreases modestly during the period of motoneuron death. Computer simulations suggest that this decrease does not result from random motoneuron death alone; one alternative possibility is selective death of motoneurons in the overlap zone. Occasional "rogue" motoneurons, that is, motoneurons of one pool that scatter into the neighbouring pool, are still present at the end of the period of cell death, representing a potential source of "noise" in the establishment of segmental patterns of connectivity. PMID- 7542852 TI - Innervation of the chicken parathyroid glands: immunohistochemical study with the TuJ1, galanin, VIP, substance P, CGRP and tyrosine hydroxylase antibodies. AB - The innervation of the chicken parathyroid glands was studied by immunohistochemistry using various antibodies. The parathyroid glands, as well as the carotid body and ultimobranchial gland, received branches originating from the vagus nerve. Numerous nerve fibers immunolabeled with the monoclonal antibody (TuJ1) against neuron-specific class III beta-tubulin isotype were found in the connective tissue capsule and septa penetrating into the parathyroid parenchyma. They were also prominent in the wall of blood vessels. Peptidergic nerve fibers immunoreactive for galanin, vasoactive intestinal polypeptide (VIP), substance P and calcitonin gene-related peptide (CGRP) were densely distributed in the capsule, septa and blood vessel walls of the parathyroid glands. In addition, some TuJ1-, substance P- and CGRP-immunoreactive fibers were detected in close association with the parenchymal cells of parathyroid glands. Tyrosine hydroxylase-immunoreactive fibers were concentrated around blood vessels and also in connective tissue stroma. PMID- 7542853 TI - Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. AB - OBJECTIVE: To compare two recently developed molecular techniques for quantitating the levels of hepatitis C virus (HCV) RNA in the serum of patients with a wide spectrum of chronic hepatitis C. DESIGN: Serum samples from 299 patients with HCV viremia, 101 control patients without HCV infection, and 19 consecutive patients receiving systemic interferon therapy were evaluated by a commercially available branched-chain DNA (bDNA) assay and a quantitative competitive polymerase chain reaction (PCR). SETTING: University-based hepatology clinics and reference virology laboratory. PATIENTS: Patients with HCV viremia as defined by results of qualitative RNA PCR, including 53 HCV-infected blood donors, 34 patients receiving renal dialysis, and 212 patients attending a hepatology clinic. RESULTS: Results of in vitro and in vivo experiments indicated that the sensitivity and dynamic range of the PCR assays were greater than those of the bDNA assay. Detection of HCV viremia by the bDNA assay was highly dependent on viral RNA titers, with a sensitivity of 5% at HCV RNA titers of 5.0 logs per mL or less and 94% at titers of 5.5 logs per mL or greater. The best correlation between assays was observed in specimens with HCV RNA titers between 6.0 and 7.5 logs per mL (r = 0.73). In patients with high-titer HCV viremia, including liver transplant recipients and patients with cirrhosis, quantitative PCR results were an average of 12-fold higher than bDNA assay results. Results of repetitive testing of discordant specimens showed that these discrepancies were caused by a high kit-to-kit coefficient of variation (112%) in the bDNA assay. Of 19 patients receiving interferon therapy, 9 (47%) became bDNA negative, but only 5 became quantitative PCR negative. The bDNA-negative, quantitative PCR-positive patients all had relapse when therapy was discontinued. CONCLUSIONS: The bDNA assay has a narrower linear range for quantitation of HCV viremia than quantitative PCR. Because persons with low HCV titers may respond well to therapy, seropositive persons with negative bDNA results should be retested with PCR-based assays. Similarly, the bDNA assay may underestimate the true degree of HCV viremia in persons with end-stage infection (> 10(7) RNA equivalents/mL of sera). Despite these limitations, the combination of bDNA- and PCR-based assays appears to be optimal for selecting and following patients during interferon therapy. PMID- 7542854 TI - Volunteer blood donors with antibody to hepatitis C virus: clinical, biochemical, virologic, and histologic features. The Hepatitis C Study Group. AB - OBJECTIVE: To assess the clinical significance of antibody to hepatitis C virus (anti-HCV) in volunteer blood donors. DESIGN: Prospective cohort study. SETTING: National Institutes of Health Clinical Center, a tertiary referral research hospital. PATIENTS: 60 anti-HCV-positive blood donors, divided into three groups of 20 persons each: Group I had normal alanine aminotransferase levels, group II had levels elevated to values less than twice the normal range, and group III had levels elevated to values greater than twice the normal range. MEASUREMENTS: Medical history, results of laboratory and virologic testing, and percutaneous liver biopsy findings. RESULTS: Participants with normal alanine aminotransferase levels were older and more often female than those with abnormal levels. The source of infection, duration of disease, symptom score, and amount of alcohol consumed were similar in the three groups. Hepatitis C virus RNA was detectable in 85% of participants, more commonly in the groups with elevated alanine aminotransferase levels (95%) than in the group with normal levels (65%); however, titers were similar in all groups. Examination of liver biopsy specimens showed chronic hepatitis in 54 participants (90%) and cirrhosis in 1 participant. The only normal liver biopsy specimens (n = 3) were those from participants who were HCV RNA negative and had normal alanine aminotransferase levels. CONCLUSIONS: Most blood donors with anti-HCV have chronic hepatitis C regardless of their serum alanine aminotransferase levels. Donors with normal alanine aminotransferase levels and no HCV RNA in their serum generally have normal liver histologic findings or minimal changes and have probably recovered from HCV infection. PMID- 7542855 TI - The hepatitis nucleocapsid as a vaccine carrier moiety. AB - The "carrier effect," defined as the provision of T cell recognition sites physically linked to B cell epitopes in order to provide Th cell function for antibody synthesis, is well known. Peptides, proteins, and more recently particulate protein antigens have been used for this purpose. The hepatitis B core antigen represents a highly immunogenic antigen in humans as well as in experimental animal models. Studies in mice have provided insight into this enhanced immunogenicity. For example, HBcAg directly activates B cells (i.e., T cell independence), HBcAg elicits strong T cell responses, and HBcAg is efficiently processed and presented by antigen presenting cells (APCs). These characteristics suggested that HBcAg may be an ideal carrier moiety for B cell epitopes requiring additional Th cell function. Therefore, a number of HBV and non-HBV B cell epitopes have been chemically linked or fused by recombinant methods to HBcAg as a method to increase immunogenicity with significant success. We have designed bacterial expression vectors that allow insertion of heterologous B cell epitopes at various positions within HBcAg particles and permit efficient purification of hybrid HBcAg particles. Studies of positional effects have demonstrated that an internal insertion into a dominant HBcAg specific B cell site represents a superior location for enhanced antibody production. Immunogenicity studies have been extended to protection against experimental challenge in several systems. For example, a malaria CS repeat sequence derived from P. berghei was inserted into HBcAg at the internal site, and purified hybrid HBcAg/CS particles were highly immunogenic and protected 100% of experimentally challenged BALB/c mice. This system has also been exploited for purposes of oral vaccination by expressing genes coding for hybrid HBcAg particles in live, avirulent vaccine strains of Salmonella species. PMID- 7542857 TI - In memoriam: Howard Temin, the fierce scholar. PMID- 7542858 TI - The nucleic acids. A backward glance. PMID- 7542856 TI - Genetics of retroviruses. PMID- 7542859 TI - Sex steroids and epidermal growth factor in benign prostatic hyperplasia (BPH). AB - Androgens provide the primary signal for the onset of DNA synthesis and cell division in normal prostate and benign prostatic hyperplasia (BPH). It is possible, however, that androgen mitogenic activity is in part indirect and mediated by peptide growth factors. In LNCaP cell lines, R1881 added to DCC-FCS medium increases DNA, epidermal growth factor (EGF) and EGF receptor (EGFR) levels: the antiandrogen hydroxy-flutamide prevents the increase of the growth factor and increases its receptor. In BPH tissue removed by transvesical prostatectomy, DHT, testosterone, 3 alpha-androstanediol and nuclear androgen receptors (AR) show a positive linear correlation with EGF: treatment with flutamide decreases significantly the EGF production. Androgens, therefore, represent important modulatory factors of prostatic EGF release. Moreover, androgens and EGF downregulate EGFR, which is probably internalized into the cell and degraded by lisosomes: in fact, a negative linear correlation between EGF, nuclear AR and the high- and low-affinity binding of EGFR is observed. These findings support the hypothesis that the growth-promoting effects of androgens in the prostate are in part mediated by peptide growth factors. The inhibitory effect of antiandrogens on prostatic cell proliferation may be the result of the decreased androgenic support and decreased EGF release and expression. PMID- 7542860 TI - Novel mutations in reverse transcriptase of human immunodeficiency virus type 1 reduce susceptibility to foscarnet in laboratory and clinical isolates. AB - Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 microM foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting > or = 8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (approximately 10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3' azido-3'-deoxythymidine (90-fold) and to the HIV-1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold). DNA sequence analysis of RT clones from resistant virus revealed the coexistence of two mutations in all clones: Gln 161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88-to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal structure of HIV-1 RT, the Gln-161 residue lies in the alpha E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the Beta5a strand of HIV-1 RT suggest that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs. PMID- 7542862 TI - Substance P stimulates murine epidermal keratinocyte proliferation and dermal mast cell degranulation in situ. PMID- 7542863 TI - Screening of potent inhibitors of collagen synthesis in cultured human skin fibroblasts. PMID- 7542861 TI - Topical application of calcitriol alters expression of filaggrin but not keratin K1 in mouse epidermis. AB - Calcitriol (1 alpha,25-dihydroxyvitamin D3) and its analogues are antiproliferative agents which promote epidermal differentiation in vitro, possibly reflecting their modes of action in the treatment of psoriasis. We examined the effect of calcitriol on early and late terminal differentiation in mouse epidermis in vivo using an immunofluorescence assay to detect keratin K1 and filaggrin expression. Pulse labelling with the tymidine analogue 5-bromo-2 deoxyuridine (BrdUrd) was performed by intraperitoneal injection of mice immediately or 16 h after a single topical application of 0.72 nmol calcitriol. The BrdUrd labelling index (LI) and keratin K1 or filaggrin expression of postmitotic cell cohorts were scored by paired immunofluorescence staining for up to 72 h after BrdUrd labelling. Calcitriol induced cell proliferation as shown by a 100% increase in the BrdUrd LI 17 h after application. The onset of keratin K1 expression in the postmitotic period was, however, unchanged in both series after calcitriol treatment. Filaggrin expression appeared earlier after calcitriol treatment than in control epidermis, probably reflecting altered cell kinetics with increased epidermal turnover. The results suggest that calcitriol only influences the later stages of the keratinocyte differentiation programme, possibly secondarily to its hyperproliferative effect. PMID- 7542864 TI - Autoregulation of histamine release via the histamine H3 receptor on mast cells in the rat skin. AB - The autoregulation of the histamine release via the histamine H3 receptor in the periphery was studied in vivo and in vitro. Antidromic electrical stimulation of the sciatic nerve caused a significant increase in histamine release in the subcutaneous perfusate in the rat hindpaw. (R)alpha-methylhistamine, a specific H3 receptor agonist, significantly and dose-dependently inhibited the increase in release of histamine by antidromic stimulation at intravenous doses of 0.25-2 mg/kg. Thioperamide (2 mg/kg, intraperitoneally), a specific H3 antagonist, prevented the inhibitory effect of (R)alpha-methylhistamine. Substance P perfusion (5-50 microM) also elicited a significant increase in histamine, and a significant inhibition by (R)alpha-methylhistamine and the antagonism of thioperamide were observed. (R)alpha-methylhistamine inhibited the histamine release by substance P from rat peritoneal mast cells in vitro, and thioperamide reduced the response to (R)alpha-methylhistamine. These data suggest that mast cells may have histamine H3 receptors, and that histamine probably modulates its own release through the H3 receptor in neurogenic inflammation. PMID- 7542865 TI - Salivary secretion and histopathological effects after single administration of the muscarinic agonist SNI-2011 in MRL/lpr mice. AB - The salivary secretion and the histopathological effects after administration of a single dose of cis-2-methylspiro (1,3-oxathiolane-5,3') quinuclidine hydrochloride hemihydrate (SNI-2011) were monitored in adult, male and female MRL/lpr mice, C57BL/6J mice and ICR mice. SNI-2011 (1-10 mg/kg, i.p.), dose dependently increased the secretion of saliva in MRL/lpr mice. The flow rate decreased gradually over the course of 60 min. The total volume of saliva, secreted in response to SNI-2011, was significantly higher in male than in female MRL/lpr mice, but there was no significant difference in this parameter between male and female C57BL/6J and ICR mice. Degeneration, apparent as atrophy and necrosis of serous cells in MRL/lpr mice, was reversed by treatment with SNI-2011 (3 and 6 mg/kg). These results suggest that SNI-2011 could be useful in the treatment of xerostomia in patients with Sjogren's syndrome. PMID- 7542866 TI - Rural general practitioners and palliative care in the north-west of New South Wales. AB - OBJECTIVES: To assess palliative care training, involvement and availability of services of 134 GPs in the inland North West of New South Wales. DESIGN: Questionnaire survey. Response rate 88.2%. RESULTS: Few had difficulties in managing terminally ill patients, but a significant minority found bereavement counselling very difficult. Over half expressed the view that the absence of debriefing opportunities was a problem. CONCLUSION: The findings of this survey confirm that rural general practitioners are heavily involved in looking after palliative care patients and require undergraduate, postgraduate and continuing education, both in community and hospital care. There appears to be a need for more nursing and medical palliative care consultant services to be available to rural general practitioners and for the appropriate associations to be more active in rural areas. PMID- 7542867 TI - On silence: a language for emotional experience. AB - Silence is an important aspect of human interaction, but is often experienced with discomfort and quickly filled with words. While quantitative parameters of silence such as timing and duration are easily recognised, qualitative experiential aspects are much more difficult to identify and describe. Emotions are experiential and complex, having antecedents in personal history, but words used to describe emotions are generally inadequate and simplistic. Silence is a useful experiential medium in which to identify and work with emotions. It is necessary to recognise what is being communicated by silence in each silence. This paper explores types of silences encountered in clinical work, and techniques to deal with them, avoiding symbolized language and technical terms of individual schools of psychotherapy. PMID- 7542868 TI - Increase of calcium levels in epithelial cells induces translocation of calcium binding proteins migration inhibitory factor-related protein 8 (MRP8) and MRP14 to keratin intermediate filaments. AB - Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca(2+)-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulphosuccinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca(2+)-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca(2+)-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca(2+)-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation. PMID- 7542869 TI - Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2. AB - The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase. PMID- 7542870 TI - Capture by chemical crosslinkers provides evidence that integrin alpha IIb beta 3 forms a complex with protein tyrosine kinases in intact platelets. AB - Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction. PMID- 7542872 TI - Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor. AB - Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules. PMID- 7542871 TI - Beta 1 integrins mediate adherent phenotype of human erythroblastic cell lines after phorbol 12-myristate 13-acetate induction. AB - We investigated the effects of phorbol ester (phorbol 12-myristate 13-acetate; PMA) treatment on the adhesive behaviour of three erythroleukaemia cell lines: HEL, LAMA-84 and AP217. In the three cell lines PMA induced an increase in expression of a megakaryocytic marker: alpha IIb beta 3 integrin, but did not promote activation of this receptor. Indeed, an antibody specific for the activated form of alpha IIb beta 3 failed to react with the three cell lines. PMA induction led to different adhesive phenotypes depending on the cell line; in fact LAMA-84 and HEL cells became adherent while AP217 cells remained non adherent. By studying cell surface receptors we found that the major difference between the adherent and the non-adherent cells was the expression of beta 1 integrins. After PMA induction, beta 1 integrin expression was totally abolished in AP217 cells and the amount of beta 1 mRNA was reduced preventing new synthesis of the subunit. In HEL and LAMA-84 cells, PMA treatment did not alter the overall level of beta 1 integrin but induced a new pattern of alpha-subunit expression: up-regulation of alpha 2 and alpha v subunits and down-regulation of alpha 4 and alpha 5 subunits. Function-perturbing antibodies against beta 1, alpha 4, alpha 5, alpha v and alpha 2 reduced adhesion of HEL cells to fibronectin or collagen, whereas antibodies against beta 3 or alpha v beta 3 did not. Our results favour the involvement of beta 1 integrins in PMA-induced adhesion of erythroleukaemia cells. PMID- 7542873 TI - Alterations of angiotensin II receptor contents in hypertrophied hearts. AB - Tsukuba hypertensive mice, which carry the human genes for renin and angiotensinogen, show cardiac hypertrophy as well as hypertension due to activation of the renin-angiotensin system (RAS). Here, we compared the cardiac angiotensin II (Ang II) receptor contents in these and normotensive control mice by means of ligand binding studies and competitive reverse transcription polymerase chain reaction analyses. The content of the Ang II receptor type 1 (AT1) was significantly higher at both the protein (2.5-fold; p < 0.01) and mRNA (1.4-fold; p < 0.05) levels in the hypertensive mice than that in control mice. Almost identical levels of the Ang II receptor type 2 (AT2) expression were identified at the mRNA levels in the two types of mice, although the levels were less than 20% of those of AT1 mRNA in control mice. These results suggest that AT1 in the heart is upregulated in response to Ang II-induced hypertrophic change and that, in particular, the upregulation of AT1 in particular contributes to the development and/or maintenance of cardiac hypertrophy in conjunction with the increase in Ang II production, because AT1 is responsible for cardiac hypertrophy related to the RAS. PMID- 7542874 TI - Elevated platelet calcium mobilization and nitric oxide synthase activity may reflect abnormalities in schizophrenic brain. AB - Schizophrenia has a diverse nature of clinical symptoms and a number of hypotheses have been suggested to explain its aetiological basis. In this study we have examined two aspects of membrane function, receptor-activated calcium mobilization and calcium activated nitric oxide synthase activity in schizophrenic subjects. Thrombin induces mobilization of calcium ions from intracellular stores. The platelet response of drug naive schizophrenics was found to be significantly increased over a range of thrombin concentrations (0.01 to 0.60 U/ml) compared to control subjects. Possible involvement of nitric oxide (NO) in the aetiology of schizophrenia was investigated. NO has been functionally linked to both dopaminergic and glutamatergic systems both of which are strongly implicated in the biochemical pathology of schizophrenia. Nitric oxide synthase (NOS) activity was determined in platelets of controls, schizophrenic and panic disorder subjects. Enzyme activity was found to be significantly higher in platelets of drug naive schizophrenic subjects compared to controls, drug treated schizophrenics and panic disorder subjects. It is suggested that there is an imbalance of the calcium-induced L-arginine- nitric oxide pathway in platelets of schizophrenic subjects which may be modified by neuroleptic treatment. This imbalance may be mirrored in the central nervous system in particular at the NMDA receptor. It is possible that such a disturbance in the L-arginine-nitric oxide pathway may have pathological implications in the aetiology of schizophrenia. PMID- 7542875 TI - Cytochrome P450 genes expressed in porcine ovaries: identification of novel forms, evidence for gene conversion, and evolutionary relationships. AB - In order to identify cytochrome P450s that are expressed in porcine ovaries, the RT/PCR methodology was used with a set of primers that corresponds to two conserved regions of the 2C gene subfamily of these enzymes. Five independent cDNA clones were isolated from preovulatory follicles (PF1, PF11, PF13, PF14, PF15) and six from the corpus luteum (CL1, CL6, CL7, CL8, CL12, CL13). The structural identities categorize these 11 P450s into two groups, group A consisting of PF1, CL7 and CL8 and group B consisting of the remaining clones. In addition, segments that are apparently interchanged between the clones were also revealed, implicating gene conversion events that have homogenized the sequence of these P450s. Furthermore, although the larger group of these porcine enzymes (group B forms) are structurally very similar to the known human P450s of the 2C gene subfamily, the group A forms are much more distantly related, implying that the 2C P450s might have evolved differently in the two species. PMID- 7542877 TI - Molecular genetic analysis of the duplication of human inducible nitric oxide synthase (NOS2) sequences. AB - In previous studies, we found multiple copies of inducible nitric oxide synthase gene (NOS2)-like sequences in the human genome and mapped them to the pericentric region of chromosome 17. Here, we describe the cloning and sequencing of exon22 regions from three of these NOS2-like sequences. We have also mapped another NOS2 like sequence to human chromosome 14. Since there are multiple NOS2-like sequences present in the human genome, we have also carried out Zoo Blot hybridisation analysis using a NOS2 cDNA probe. Our data suggest that duplication of NOS2-like sequences occurred very recently in primate evolution. PMID- 7542876 TI - Gramicidin D is a potent insulin secretagogue: dependence on extracellular calcium influx. AB - Gramicidin D, a sodium ionophore, was identified as a potent insulin secretagogue in the mouse beta-cell line, beta TC3. Gramicidin D stimulated insulin secretion by 3.2-fold relative to control cells incubated with vehicle alone. Using ion specific fluorescent probes, gramicidin D (1 microM) increased the intracellular concentrations of Na+ ([Na+]i) and Ca2+ ([Ca2+]i). By contrast, no changes in pHi were detected in cells exposed to ionophore. The increase in [Ca2+]i was biphasic and characterized by an initial peak at 1-2 minutes followed by a sustained second phase. The addition of EGTA (2 mM) to the extracellular medium abolished gramicidin D-induced increase in [Ca2+]i and insulin secretion. These parameters were also profoundly inhibited by the L-type Ca(2+)-channel inhibitor, verapamil (20 microM). These findings suggest that insulin secretion induced from beta TC3 cells by gramicidin D is mediated via the promotion of Ca(2+)-influx. PMID- 7542879 TI - Identification of a short form of the P2xR1-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea. AB - A truncated form of the P2xR1 purinoceptor subunit (which we designate P2xR1-2) was detected in rat pituitary gland and the secretory epithelial tissue (stria vascularis) of the cochlea using RT-PCR of solid-phase cDNA libraries. PCR products corresponding to the P2xR1 purinoceptor subunit (1) were obtained from vas deferens, brain and microdissected cochlear sensory epithelial tissues including organ of Corti, sacculus and crista ampullaris. Cloning and sequencing revealed that the P2xR1-2 product included an 85-bp insertion in a region corresponding to a novel C-terminal end of the second membrane spanning domain and continuing as the cytoplasmic domain. A stop codon sequence after the first 51 bp of the insert effectively truncates this subunit, reducing the final cytoplasmic domain by 90% compared with the previously published P2xR1(-1) sequence, thereby reducing the overall peptide by approximately 25%. The region of the receptor lost in the truncated version coded for a number of serine/proline rich regions which may act as potential intracellular regulatory sites. PMID- 7542878 TI - Immune response against the murine mdri protein induced by vaccination with synthetic lipopeptides in liposomes. AB - Intrinsically, or after exposure to chemotherapeutic drugs, many cancer cells overexpress a class of high molecular weight membrane glycoproteins associated with the multidrug resistance (mdr) of these cells. This report describes an immunization protocol eliciting autoantibodies specific to extracellular epitopes of the murine mdr 1 P-glycoprotein (Pgp). Synthetic peptides with the sequences of extracellular loops of murine Pgp were covalently coupled with four palmitic acid moieties per peptide molecule. These "lipopeptides" were reconstituted in the bilayer of liposomes containing lipid A and used to immunize mice. Antibodies against the lipopeptides corresponding to loop 2 and 4 were elicited in sera of immunized mice. They reacted specifically with extracellular epitopes of the naturally occurring murine Pgp. After interaction with resistant cancer cells, the antibodies induced an average 50% increase in cellular accumulation of doxorubicin and Bodipy-verapamil. In the presence of these antibodies the resistance of L1210 mdr cells was reduced from an LD50 of 4 x 10(-5) M to 5 x 10( 7) M doxorubicin. PMID- 7542881 TI - The effects of tyrphostins B42 and B46 on equine platelet function and protein tyrosine phosphorylation. AB - The effects of the protein tyrosine kinase inhibitors tyrphostins B42 and B46 on equine platelet function and protein tyrosine phosphorylation (PTP) were assessed. Tyrphostins B42 and B46 (both at 100 microM concentration) produced significant inhibition of thrombin-stimulated equine platelet aggregation. The effect of tyrphostin B46 was also time-dependent. The same concentration of these inhibitors produced very little or no inhibition of platelet-activating factor (PAF)-induced aggregation. The effects of tyrphostins B42 and B46 on thrombin- and PAF-stimulated PTP were generally similar, although some bands were more strongly inhibited when thrombin was the agonist. Therefore, although thrombin and PAF both act via G-protein coupled receptors, PAF may be capable of utilising an alternative signal transduction pathway to that used by thrombin. PMID- 7542880 TI - Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK. AB - Mechanical loading of cultured mesangial cells has been shown to induce both increased production of matrix proteins and cell proliferation. This in vitro phenomenon has been implicated in the progression of glomerular sclerosis observed in a variety of glomerular diseases. However, it is not yet known how cells sense mechanical stress and transfer this sense into biochemical and biological events. In this study, we show that mesangial stretching rapidly stimulates tyrosine phosphorylation of non-receptor tyrosine kinase pp125FAK. Since pp125FAK is a major constituent of focal adhesions, our findings suggest that the focal adhesion may be a site where mechanical forces are translated into biochemical events, and that pp125FAK may play an important role in this signaling cascade. PMID- 7542882 TI - A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release. AB - Previous studies have shown that 4-aminopyridine (4AP) induced Ca-influx effects the release of glutamate from nerve terminals (synaptosomes) isolated from rat cerebral cortex. We now show that the Ca-dependent component of this release is potentiated by preincubation of the synaptosomes with the immunosuppressant, FK506, an inhibitor of protein phosphatase-2B (calcineurin). FK506 did not inhibit the Ca-independent release of glutamate from a cytosolic pool. Examination of the effect of FK506 on the influx of Ca elicited by 4AP indicated that inhibition of calcineurin activity resulted in an increase of voltage dependent Ca-influx. Based on these results, we suggest that protein dephosphorylation effected by calcineurin may suppress voltage-dependent Ca channel activity and in so doing inhibits evoked glutamate release. Activation of calcineurin produced by initial Ca-entry may represent a negative feedback to limit the activity of Ca-channels coupled to the release of glutamate. PMID- 7542884 TI - Comparison of the enzymatic activities of human galactokinase GALK1 and a related human galactokinase protein GK2. AB - The GALK1 cDNA encoding human galactokinase was recently cloned and its cognate GALK1 gene shown to be involved in galactokinase deficient galactosemia. Previously, a separate human galactokinase cDNA, GK2, was cloned by complementation of a galactokinase deficient yeast mutant; however, the galactokinase activity of GK2 was not demonstrated in mammalian cells. To compare the relative galactokinase activity of GALK1 and GK2, their corresponding cDNAs were expressed in COS cells. Northern blot analysis indicated that both cDNAs were transcribed into mRNA transcripts of the expected size; however, only the GALK1 cDNA produced high levels of galactokinase activity. This result would suggest that GALK1 is the major enzyme for galactose metabolism while the role of GK2 remains uncertain. PMID- 7542883 TI - Inhibition of tumor necrosis factor alpha- and ceramide-induced internucleosomal DNA fragmentation by herbimycin A in U937 cells. AB - In many tumor cell lines, tumor necrosis factor alpha (TNF alpha) causes apoptosis with characteristic internucleosomal DNA fragmentation. However, the mechanism is largely unknown. Here we examined the involvement of protein tyrosine kinases by using their inhibitors. Among various tyrosine kinase inhibitors tested, only herbimycin A was found to inhibit internucleosomal DNA fragmentation but not apoptotic morphological changes and cell death induced by TNF alpha in U937 cells. Herbimycin A was able to block DNA fragmentation when it was added to the cell culture as late as 1.5 h after TNF alpha treatment. These results demonstrate that herbimycin A selectively inhibits a later event involved in the process of apoptois that results in internucleosomal DNA fragmentation. Sphingomyelinase and ceramide (Cer) induced internucleosomal DNA fragmentation was also inhibited by herbimycin A, supporting the hypothesis that Cer may be a novel second messenger mediating the cytotoxic effect of TNF alpha. PMID- 7542885 TI - Extracellular calcium causes the release of calcium from intracellular stores in chick osteocytes. AB - We have recently demonstrated that a rise in the extracellular divalent cation concentration induces a rapid elevation of cytosolic calcium in chick osteocytes. Here, we demonstrate that cytosolic calcium elevation that occurs in osteocytes on exposure to elevated extracellular calcium is independent of membrane voltage and is insensitive to modulation by organic calcium channel modulators, namely, BAY K 8644, nicardipine, and nifedipine. However, the calcium elevation was sensitive to modulation by an intracellular calcium antagonist, TMB-8, suggesting that the cytosolic calcium elevation was due to mobilization of this cation from an intracellular store. PMID- 7542886 TI - NF1 gene mutations in Japanese with neurofibromatosis 1 (NF1). AB - Neurofibromatosis 1 (NF1) is an autosomal dominant disease characterized by abnormalities in multiple tissues derived from the neural crest. We analysed 50 unrelated Japanese patients for NF1 mutations by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis for exons 28 to 36. Here, we demonstrate a single base pair (bp) insertional mutations in exon 31 in one patient (5843insA/5844insA/5845insA/5846insA) and a single adenine to guanine transitional mutation 4 bp upstream from the 3' end of intron 31 in two unrelated cases. The insertional mutation in exon 31 was novel and resulted in premature termination of the transcript. The other intron 31 mutations resulted in 4 bp insertions of cDNA between exon 31 and exon 32 with premature termination of the transcript, indicating that those transitions of intron 31 caused aberrant splice acceptor sites upstream from the 5' end of exon 32. However, as the same mutation of intron 31 has been reported previously in two cases of unrelated Caucasians, the splice junction mutation of intron 31 is thought to be common among different ethnic groups. PMID- 7542887 TI - Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers. AB - GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis. cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique. A number of regions were found to be strictly conserved between unrelated eukaryotes. These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme. PMID- 7542888 TI - Construction and characterization of adenoviral vector expressing biologically active brain-derived neurotrophic factor. AB - To deliver brain-derived neurotrophic factor (BDNF) to the central nervous system, we sought to attain adenovirus-mediated transfer and expression of the gene in both in vitro and in vivo experiments. For this purpose, we constructed AxCA-BDNF, a recombinant adenoviral vector containing the BDNF cDNA expression cassette. Reverse transcription polymerase chain reaction analyses of the infected HeLa cells and the transduced mouse brain revealed successful expression of the BDNF gene both in vitro and in vivo. The results of a survival assay of chick dorsal root ganglion cells showed that the produced BDNF was biologically active. We consider, therefore, this newly constructed recombinant adenovirus to be a useful tool to deliver BDNF to degenerating neurons and to be applicable to gene therapy of neurodegenerative diseases and nerve trauma. PMID- 7542889 TI - Nonionic contrast agents produce thrombotic effect by inducing adhesion of leukocytes on human endothelium. AB - The expression of P-selectin was more upregulated following the exposure to nonionic low osmolar contrast agents than to ionic contrast agents. Exposure to nonionic contrast agents led to a marked adhesion of leukocytes to endothelial cells. Thrombomodulin activity of endothelial cells was decreased, and plasminogen activator inhibitor-1 (PAI-1) and tumor necrosis factor (TNF) alpha in the supernatant were increased when leukocyte adhesion occurred after exposure to nonionic contrast agents. Results suggest that the adhesion of leukocytes to the endothelium increases procoagulant activity. PMID- 7542890 TI - Opioids inhibit the induction of nitric oxide synthase in J774 macrophages. AB - The effect of opioids on NO production by LPS-stimulated murine macrophages J774 has been investigated. Morphine (mu and k opioid receptor agonist), DAGO (selective mu receptor agonist) and U50-488H (selective k receptor agonist), added (10(-10)-10(-6) M) to the cells 0.5 h before activation with LPS, significantly inhibited NO production. This effect was reverted by naloxone (10( 12)-10(-8) M), an opioid specific antagonist. In contrast, DPDPE and deltorphin II respectively delta 1 and delta 2 receptor agonists (10(-10)-10(-6) M) did not affect NO generation. Morphine was not able to inhibit NO production when added after LPS challenge. The results of the present study indicate that opioids are able to inhibit NO formation in LPS-activated macrophages through the involvement of specific opioid receptors. Moreover, the ability of morphine to inhibit NO production only when given before LPS challenge suggests that the opiate inhibits the induction but not the activity of the inducible NO synthase. PMID- 7542891 TI - Fetuin is a substrate for Pasteurella haemolytica O-sialoglycoprotease. AB - A soluble bovine glycoprotein, fetuin, was used as an alternative substrate to identify O-sialoglycoprotease activity in culture supernatant protein fractions of Pasteurella haemolytica. An aliquot of a 24-hour incubation mixture containing fetuin and O-sialoglycoprotease was denatured and examined after gradient sodium dodecyl polyacrylamide gel electrophoresis. The Coomassie-Brilliant-Blue-stained gel was examined for the disappearance of the fetuin band at an apparent molecular mass of 64 KDa. Four major hydrolysis products were identified: an N terminal fragment of 45 kDa, a 20 kDa fragment at Val50, and two C-terminal fragments at Val273 and His287. PMID- 7542892 TI - Interaction of the cobalt complex of a bleomycin functional model with d(CGCAATTGCG)2: evidence of minor groove binding by 2D NMR methods. AB - The interaction between the cobalt complex of a bleomycin functional model and d(CGCAATTGCG)2 was determined by 2D NMR methods. The intermolecular NOE cross peaks between ligand protons and the DNA minor groove protons suggest that the cobalt complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. Also, the NOEs interactions of H8 pyridine proton and H2 imidazole proton in the metal-binding domain with H4' sugar proton of C9 and H4' sugar proton of A15 demonstrate that the sixth ligand site of cobalt complex, which is occupied by a solvent molecule, faces towards the minor groove of DNA. This binding model is in accord with the observed non-diffusion DNA cleavage by the metal-complexes of the bleomycin functional model compounds. PMID- 7542893 TI - Production and characterization of monoclonal antibodies against Panulirus interruptus hemocyanin. AB - Since the primary and higher-order structures of hemocyanin from the crustacean arthropod Panulirus interruptus have been elucidated completely, it should be possible to determine which regions of this immunogenic molecule are recognized most often by antibodies. Monoclonal antibodies were raised against subunits a and b of this hemocyanin, and fourteen of them were further characterized. The produced antibodies were of class IgG, subclasses 1 or 2a. Most of them had dissociation constants on the order of magnitude 10(-8)-10(-10), a few had lower affinities. Most clones showed no or negligible cross-reactivity with other crustacean hemocyanins. The reactivity of most other clones diminished with increasing sequence difference between the investigated hemocyanins. However, in a few instances a stronger reactivity with other hemocyanins was observed than with that from Panulirus interruptus. After complete denaturation of the hemocyanin there was no reaction with the monoclonal antibodies, indicating that the latter recognize conformational epitopes. Only one monoclonal antibody reacted with denatured hemocyanin. This antibody was also the only one which reacted with a CNBr digest, which means that it recognizes a sequential epitope. Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure. Limited proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment representing domains 2 and 3. It was determined on Western blots of the digest on which fragment epitopes for eleven of the monoclonal antibodies were located. PMID- 7542895 TI - Neutrophil response to endotoxin in the adult respiratory distress syndrome: role of CD14. AB - The role of both endotoxin and neutrophils in the development of acute lung injury continues to be debated. We hypothesized that early in the course of the development of the adult respiratory distress syndrome (ARDS) circulating neutrophils could be primed by endotoxin and that subsequent stimulated responses could be enhanced. Accordingly, neutrophils were isolated from patients at risk for and with ARDS. Unstimulated neutrophils from these patients neither produced nor were primed for superoxide production. Whereas phorbol myristate acetate stimulated superoxide production was preserved, indicating that the cells were capable of a response, patient neutrophils produced less superoxide than did cells from normal subjects when primed with endotoxin (lipopolysaccharide [LPS]) and stimulated with formyl-methionyl-leucine-phenylalanine (FMLP), suggesting that there was a defect in the signal transduction mechanism for LPS. This was confirmed by the finding that patient neutrophils also had both decreased baseline CD14 expression and less CD14 upregulation after LPS stimulation compared with neutrophils from normal subjects. The mechanisms that could account for the decreased CD14 expression were studied in vitro. Neutrophils from normal subjects both upregulate CD14 in response to LPS and shed CD14 over time, suggesting that in patients CD14 receptors could have been previously upregulated and shed. In addition, there is an association between CD14 expression and retention such that normal LPS-stimulated neutrophils which are not retained in a filtration system have decreased CD14 expression. Thus, in patients, those PMN most responsive to LPS could be preferentially sequestered and not available in the circulation for study. PMID- 7542894 TI - Inhibition of macrophages with gadolinium chloride abrogates ozone-induced pulmonary injury and inflammatory mediator production. AB - Acute inhalation of toxic doses of ozone (O3) induces macrophage accumulation in the lung and the release of cytotoxic and proinflammatory mediators. To evaluate the role of macrophages and their mediators in the pathophysiologic response of the lung to O3, we examined the effects of the macrophage inhibitor, gadolinium chloride (GdCl3), on O3-induced inflammation, mediator production, and lavage fluid protein levels. Rats were pretreated with GdCl3 (7 mg/kg, intravenously) or control 24 h prior to exposure to air or O3 (2 parts per million, 3 h). Animals were killed 48 h after exposure. GdCl3 pretreatment of rats was found to abrogate O3-induced increases in the number of cells, as well as the amount of protein recovered in bronchoalveolar lavage fluid. Following GdCl3 pretreatment of rats, lung lavage cells consisting of > 90% macrophages were found to produce significantly less nitric oxide and express less inducible nitric oxide synthase (iNOS) when compared to cells from rats exposed to O3. O3-induced alterations in superoxide anion production by alveolar macrophages, both in vitro and in situ, were also attenuated by GdCl3 pretreatment of rats. In addition, increases in tumor necrosis factor alpha (TNF-alpha) and fibronectin in lung tissue induced by O3 were reduced. Taken together, these data provide support for the hypothesis that macrophages contribute to the pathogenesis of O3-induced lung injury. PMID- 7542896 TI - Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. AB - Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability. PMID- 7542898 TI - [Changes in the use of percutaneous liver biopsy over time]. AB - AIM: To study, through the pathology diagnosis obtained, the current use of percutaneous liver biopsy and its change with time. MATERIALS AND METHODS: Pathology diagnosis of 759 liver biopsies performed during last 5 years are reviewed and compared with our previously published experience. RESULTS: Neoplasms have decreased from 16% of all pathologic diagnosis obtained in 1961-81 to less than 1% in the last 5 years. In 1985-87, liver cirrhosis was the final diagnosis in 60% of all biopsies, while currently it is only 18%, with a steady absolute number of cirrhosis each year. The most remarkable change is the number of biopsy diagnosis of chronic hepatitis, which has increased from 19% in 1985-87 to 51% in the last 5 years. Ten percent of all biopsies was performed in HIV positive patients; granulomatous hepatitis and tuberculosis were more prevalent in them. CONCLUSIONS: Percutaneous liver biopsy has been displaced for the diagnosis of liver cirrhosis and has been focussed on the diagnosis of chronic hepatitis. HIV-positive patients represent an important population for those who perform liver biopsy. PMID- 7542897 TI - Distribution of human i-NANC bronchodilator and nitric oxide-immunoreactive nerves. AB - We compared inhibitory nonadrenergic noncholinergic (i-NANC) neural relaxations, evoked by electrical field stimulation (EFS), at three levels (main [MA], proximal [PA], and distal [DA] airways) of isolated human airways and correlated these with nitric oxide synthase-immunoreactive (NOS-IR) nerves, using antiserum raised to rat cerebellar NOS. Maximal relaxations to papaverine (100 microM) were reduced in PA and DA (MA: 1,712 +/- 219 mg, n = 12; DA: 862 +/- 69 mg, n = 5, P < 0.05 versus MA); hence, subsequent relaxations were expressed as a percentage of the papaverine maximum. EFS elicited frequency-dependent relaxations that were largest in MA and reduced in PA and DA, especially at high stimulation frequencies (10 Hz EFS: MA: 51.6 +/- 3.7%, n = 12; PA: 30.5 +/- 6.0%, n = 6, P < 0.01 versus MA; DA: 17.8 +/- 3.6%, n = 5, P < 0.001 versus MA). The NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) (100 microM) and tetrodotoxin (3 microM) significantly inhibited i-NANC responses at all frequencies, leaving an L-NAME resistant non-neural relaxation at frequencies > 5 Hz which was reduced in PA and DA. Cumulative concentration-response studies to sodium nitroprusside (1 nM to 0.1 mM) and the NO donor 3-morpholinosydnonimine (1 nM to 1 mM) were not significantly different in PA and DA, suggesting impaired relaxation is not caused by impaired guanylyl cyclase activity. Total nerve density, shown by protein gene product 9.5 staining, was not significantly different in PA and DA; however, NOS-IR nerve density was reduced in PA and DA (NOS-IR [intercepts/mm2]: MA: 705 +/- 98, n = 6; DA: 284 +/- 32, n = 6, P < 0.01 versus MA). These studies demonstrate that i NANC neural relaxations are reduced in DA, apparently due to a decrease in the density of nitrergic innervation. PMID- 7542900 TI - Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia. AB - Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification. PMID- 7542899 TI - Systemic administration of rhIGF-I or rhIGF-I/IGFBP-3 increases cortical bone and lean body mass in ovariectomized rats. AB - The purpose of this study was to compare dose-related effects on cortical bone and lean body mass following subcutaneous administration of rhIGF-I alone, or bound to an equimolar amount of rhIGFBP-3 to adult Ovx rats. At the age of 16 weeks, rats were ovariectomized or sham-operated and were allowed 8 weeks to develop osteopenia. After being divided into control (saline treated) or treatment groups, rats were injected daily during an 8-week period with 0.9 and 2.6 mg/kg of rhIGF-I, or with 0.9, 2.6, and 7.5 mg/kg of rhIGF-I bound to rhIGFBP 3. Fluorescent bone markers were given 9 and 2 days prior to necropsy. Body weights and lean body mass were monitored throughout the experiment. Cortical bone histomorphometry was performed on tibial cross-sections at the tibiofibular junction, and endochondral bone growth was measured at the distal femoral metaphysis. All rats treated with rhIGF-I or the rhIGF-I/IGFBP-3 complex had increased body weights, corresponding to a dose-dependent increase in lean body mass. Endochondral growth was slightly increased in all experimental groups, but was not dose-dependent. A dramatic increase in periosteal, modeling-dependent formation, coupled with decreased or unchanged resorption on the endocortical envelope resulted in a dose-dependent increase in cortical thickness and cross sectional area in groups treated with the complex of rhIGF-I/IGFBP-3. This complex appeared to be more effective in promoting positive musculoskeletal changes than rhIGF-I alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542901 TI - Role of calcium channels in parathyroid hormone secretion. AB - Experimental evidence exists for the presence of parathyroid cell membrane calcium channels that respond to plasma calcium. In previous reports, the effects of various calcium channel agents on PTH secretion have revealed conflicting results. To resolve some of these inconsistencies, we have compared the pure calcium channel agonist, (+)202-791, and its antagonistic enantiomer (-)202-791 with other calcium channel agents--verapamil, nifedipine, and (+)Bay-K-8644. The agonist (+)202-791 enhanced 45Ca+2 uptake and decreased PTH secretion, while the antagonist (-)202-791 decreased 45Ca+2 uptake and increased PTH secretion. The calcium channel appears coupled to a G-protein as indicated by pertussis toxin treatment of the cells. The enantiomers (+/-)202-791 had little effect on intracellular cAMP production suggesting that the calcium channel may not be responsible for the previously observed calcium-mediated changes in cAMP. The antagonist (-)202-791 increased the phosphorylation of a 60-kd protein. The enantiomers (+/-)202-791 did not alter the effect of depolarizing concentrations of potassium on PTH secretion. Our results suggest that calcium channels provide a pathway for the movement of calcium across the plasma membrane and that this pool of calcium regulates, at least in part, PTH secretion. PMID- 7542902 TI - Potential activities of androgen metabolizing enzymes in human prostate. AB - The entire androgen metabolism of the human prostate is an integral part of the DHT mediated cellular processes, which eventually give rise to the androgen responsiveness of the prostate. Therefore, the potential activities of various androgen metabolizing enzymes were studied. Moreover, the impact of aging on the androgen metabolism and the inhibition of 5 alpha-reductase by finasteride were studied. In epithelium (E) and stroma (S) of normal (NPR) and hyperplastic human prostate (BPH), for each enzyme being involved in the conversion either of testosterone via DHT, 3 alpha- and 3 beta-diol to the C19O3-triols or from testosterone to androstenedione and vice versa, the amount (Vmax) and Michaelis constant (Km) were determined by Lineweaver-Burk plots. Furthermore, Vmax/Km quotients were calculated, which served as an index for the potential enzyme activity. 17 enzymes showed a mean Vmax/Km > or = 0.10. The top four were the 5 alpha-reductases in E and S of NPR and BPH. Among those, the highest activity was found in E of NPR (1.6 +/- 0.2). Moreover, in E a significant age-dependent decrease of 5 alpha-reductase activity occurred, whereas in stroma rather constant activities were found over the whole age range. Similar age-dependent alterations were found for the cellular DHT levels. Finally, the finasteride inhibition of 5 alpha-reductase (IC50;nM) was stronger in E (35 +/- 17) than in S (126 +/- 15). In conclusion, 5 alpha-reductase is: (a) the outstanding androgen metabolizing enzyme in NPR and BPH; (b) dictating the DHT enrichment in the prostate; (c) under the impact of aging; and (d) preferentially inhibited by finasteride in E. PMID- 7542903 TI - Hormonal regulation of peripheral-type benzodiazepine receptors. AB - Central benzodiazepine (BZ) receptors are located only in the central nervous system and mediate the clinical effects obtained by various BZs. In addition, there is another receptor that binds BZs with different drug specificities, which is located mainly on the outer mitochondrial membrane of various peripheral tissues. Peripheral BZ receptors (PBR) are composed of three subunits: an isoquinoline binding site, a voltage-dependent anion channel, and an adenine nucleotide carrier, with molecular weights of 18, 32, and 30 kDa, respectively. Complementary DNA of the isoquinoline binding subunit has been cloned in rat, calf, and human. The major role of PBR is in the regulation of steroid biosynthesis. Various PBR ligands stimulate the conversion of cholesterol into pregnenolone and the production of steroid hormones. The naturally occurring diazepam-binding inhibitor stimulates in vivo steroidogenesis via binding to PBR. In the female, PBR density is increased in rat and human ovary proportional with greater cell maturation and differentiation. In the male, testosterone modulates PBR density in the genital tract. These results show the strong relationship between PBR and the endocrine system. PMID- 7542905 TI - Correlation between the proportion of Philadelphia chromosome-positive metaphase cells and levels of BCR-ABL mRNA in chronic myeloid leukaemia. AB - We have sought to define the relationship between the proportion of marrow metaphases showing the Philadelphia chromosome (Ph) and levels of BCR-ABL mRNA assessed by quantitative polymerase chain reaction (PCR) in patients with chronic myeloid leukaemia (CML). From a total of 141 patients, 164 PCR assays were performed on peripheral blood samples taken within 2 weeks of a bone marrow specimen analysed by cytogenetics. BCR-ABL mRNA was quantified in all 106 PCR positive samples by competitive PCR; results ranged from < 10 to 3.4 x 10(6) transcripts/micrograms RNA. Twenty-one chronic-phase patients had a median of 5.0 x 10(5) BCR-ABL transcripts/micrograms RNA; no difference in levels of the fusion mRNA was found between 15 Ph-positive and six Ph-negative, BCR-ABL-positive patients. Ph-positive metaphases were not detected in any individual who was PCR negative (n = 58) and in only a single patient who was PCR positive with < 10(3) BCR-ABL transcripts/micrograms RNA (n = 44). Conversely, of 41 samples from patients in haematological remission who had > 10(3) BCR-ABL transcripts/micrograms RNA, 30 had at least one Ph-positive metaphase. The highest level of BCR-ABL transcripts at which Ph-positive metaphases were not detected was 1.5 x 10(4). For the 46 patients who had at least one Ph-positive metaphase, a good correlation (Spearman coefficient = 0.83, P < 0.0001) was found between the percentage of Ph-positive metaphases and BCR-ABL transcript levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542904 TI - Loss of neurofibromatosis type I (NF1) gene expression in pheochromocytomas from patients without NF1. AB - The neurofibromatosis type 1 (NF1) gene encodes a tumor-suppressor protein termed neurofibromin, which, in adults, is expressed predominantly in neurons, Schwann cells, and the adrenal medulla. Loss of NF1 gene expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 as well as in malignant melanomas and neuroblastomas from patients without NF1. Previously, we demonstrated the lack of neurofibromin expression in six pheochromocytomas from patients with NF1, supporting the idea that neurofibromin might be an essential regulator of cell growth in these cells. To determine whether NF1 gene expression is similarly altered in pheochromocytomas from patients without NF1, we examined 20 pheochromocytomas for the presence of NF1 RNA and neurofibromin by reverse transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Reduced or absent NF1 gene expression was documented in 7 of these 20 tumors (35%) including 1 of 4 sporadic tumors, 3 of 10 tumors from patients with multiple endocrine neoplasia (MEN) 2A, 2 of 4 tumors from patients with MEN2B, and 1 of 2 tumors from patients with von Hippel-Lindau syndrome. In addition, most of these tumors expressed predominantly the type 1 NF1 isoform (75% type 1 NF1 isoform expression) as opposed to other neural crest-derived tissues such as adrenal gland and Schwann cells, which express predominantly type 2 NF1. This type 1 isoform predominance was also observed in the rat pheochromocytoma PC12 cell line, suggesting that this change in isoform expression may be associated with the genesis of these tumors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542906 TI - Monochromosome transfers to Syrian hamster BHK cells via microcell fusion provide functional evidence for suppressor genes on human chromosome 9 both for anchorage independence and for tumorigenicity. AB - We previously identified an anchorage independence-suppressor gene, SAII, on rat chromosome (RNO) 5. RNO5 is homologous to human chromosomes (HSA) 1 and 9. In order to find the human homolog of the SAII gene, we transferred HSA1 and HSA9 to an anchorage-independent and tumorigenic Syrian hamster BHK 191-5C cell line by microcell fusion. For HSA9, we used a t(X;9)-derivative chromosome to force the retention of this chromosome in hybrids by hypoxanthine-aminopterin-thymidine (HAT) selection. To study the possible effect of the X portion of the der(9)t(X;9), we also transferred a normal X to 191-5C cells. For HSA1, a neo tagged chromosome was introduced. Following the transfer of der(9)t(X;9) to 191 5C cells, the hybrid cells became anchorage dependent and nontumorigenic, and, upon the loss of this chromosome, the cells regained their tumorigenic and anchorage-independent phenotypes. The transfer of HSAX or HSA1, on the other hand, affected neither of these phenotypes. These results provide functional proof of suppressor genes on HSA9 involving both anchorage independence and tumorigenicity. In addition, our data suggest the presence of another gene on HSA9 that causes a negative growth effect and whose phenotypic expression, contrary to the suppressor genes, is dosage dependent. PMID- 7542907 TI - Molecular analysis of a t(11;22) translocation junction in a case of Ewing's sarcoma. AB - Polymerase chain reaction (PCR)-directed sequence analysis was performed to characterize the genomic and cDNA breakpoint junctions of t(11;22) (q24;q12) translocation in a case of Ewing's sarcoma, in which the EWS gene located on chromosome 22 is rearranged with the FL11 gene located on chromosome 11. RNA-PCR revealed the novel chimeric product of EWS/FL11 gene on the derivative chromosome (der) 22, resulting from a probable fusion of EWS exon 7 to FL11 exon 9. Sequencing of the PCR-amplified genomic fragments of the fusion genes showed that the breakpoints on der(22) occurred in EWS intron 7 and, most probably, in FL11 intron 8. Those of the untranscribed counterpart on der(11) were located in the same FL11 intron and in EWS exon 11, with deletion of a considerable amount of sequences from both genes. These findings indicate asymmetric junction at the molecular level in the present t(11;22). None of the reported conserved sequences that mediate other cancer chromosome translocations was observed around the genomic junctions. Instead, a palindromic hexamer 5'-GCTAGC-3' was found to flank the breakpoints of both genes on der(22), which may have a functional significance in the genesis of the t(11;22). PMID- 7542908 TI - BCR/ABL fusion located on chromosome 9 in chronic myeloid leukemia with a masked Ph chromosome. AB - A reciprocal translocation, t(10;22)(q22;q11), resulting in a masked Ph chromosome was identified in a patient diagnosed with chronic myeloid leukemia (CML). Both homologs of chromosome 9 were of the normal pattern. Two signals for the ABL probe, both of them hybridized to chromosome 9, were demonstrated via fluorescence in situ hybridization (FISH). Furthermore, cohybridization with two differently labeled BCR/ABL translocation DNA probes indicated a BCR/ABL fusion apparently located on 9q34. Molecular studies revealed a rearrangement of the BCR region and expression of a chimeric BCR/ABL mRNA of CML configuration. These findings indicate that the BCR/ABL fusion resulted from an unusual relocation of the BCR gene from its normal position on 22q11 to 9q34 adjacent to the ABL gene. PMID- 7542910 TI - Molecular basis of 11q23 rearrangements in hematopoietic malignant proliferations. AB - Chromosomal abnormalities of the 11q23 band occur frequently in various hematopoietic malignant disorders. Because numerous partner chromosomes have been previously described, it is now important to determine the number of genes involved at 11q23 and to clarify the role of the partner genes. Recent efforts in several laboratories have identified a trithorax-related gene that is involved in most of the 11q23 abnormalities. The aim of this review is to summarize the recent data concerning these 11q23 rearrangements and the understanding of their consequences. PMID- 7542909 TI - Characterization of a t(10;11)(p13-14;q14-21) in the monoblastic cell line U937. AB - Previous analysis of the monoblastic cell line U937 has shown that several sublines contain a rearranged chromosome arm 11q. In order to determine the true nature of the rearrangement, fluorescence in situ hybridization (FISH) was carried out with various combinations of single copy anonymous markers, clones containing genes, a chromosome 10 paint, and an 11 centromere specific sequence. The rearrangement was deduced to be a reciprocal translocation between chromosomes 10 and 11 described as t(10;11)(p13-14;q14-21). The breakpoint on chromosome 11 is telomeric to the INT2 gene and the pHS11 probe at 11q13, and centromeric to the marker D11S36 localized to 11q14.3-q22.1 and the MLL gene at 11q23. Similar translocations have been reported in various acute leukemias, principally of the monocytic lineage, and also in T-cell precursor acute lymphocytic leukemias. Further characterization of the genetic rearrangements in U937 may lead to the isolation of genes important in leukemogenesis and provide an in vitro system for their study. PMID- 7542912 TI - Homozygous deletions and loss of expression of the CDKN2 gene occur frequently in head and neck squamous cell carcinoma cell lines but infrequently in primary tumors. AB - Deletion of 9p21-22 is a common genetic alteration in dysplastic, in situ, and invasive head and neck squamous cell carcinoma (HNSCC). However, a candidate tumor suppressor gene (TSG) at this site has thus far not been identified in HNSCC. We report homozygous deletion of the recently identified multiple tumor suppressor I (MTSI)/cyclin-dependent kinase-4-inhibitor (CDKN2) gene mapped to 9p21, which encodes the p16 protein, a regulator of cyclin-dependent kinase 4, in six of 16 HNSCC cell lines. We also show absence of the CDKN2 mRNA in all cell lines with CDKN2 deletion as well as in an additional two cell lines without deletion. Overall, we have identified 9p abnormalities in 12 of 16 (75%) cell lines, at least nine of which involved CDKN2. We further demonstrate that the CDKN2 deletion in HNSCC is located within a previously described region of allelic loss between D9S171 and IFNW, which spans a 4 cM region of 9p. However, examination of 36 primary tumors revealed genetic alterations in only seven of 36 (19%) tumors. These results suggest that genetic alterations at CDKN2 are frequent in HNSCC cell lines, but the role of this gene in primary tumors is less compelling. CDKN2 does not appear to be the only TSG on 9p21 in HNSCC, and our results suggest that another region of deletion exists proximal to the IFNW locus. PMID- 7542911 TI - Detection of multiple gains and losses of genetic material in ten glioma cell lines by comparative genomic hybridization. AB - A protocol for comparative genomic hybridization by use of nucleotides directly labeled with fluorochromes was used to map regions of deletion and amplification in ten glioma cell lines. The protocol greatly reduced experimental artifacts. We detected several genetic aberrations, including whole chromosome loss and gain, partial loss and gain, possible isochromosome, and higher level DNA amplification. The most frequent losses (in order of frequency) occurred on chromosomes 10, 18, 13, 11, 9, 14, 4, 6, 1, and X. The most common gain occurred on chromosome 7. Several sites of previously known and unknown amplifications were observed. PMID- 7542913 TI - Childhood acute lymphoblastic leukemia with equivocal chromosome markers of the t(1;19) translocation. AB - The t(1;19)(q23;p13) or its derivative encodes an E2A-PBXI fusion transcript and protein that has been shown to have important prognostic and therapeutic implications in patients with acute lymphoblastic leukemia (ALL). We describe two childhood cases in which a der(22)t(1;22)(q21-23;p13) cytogenetically mimicked a der(19)t(1;19)(q23;p13). In one case, which was phenotyped as early pre-B ALL with hyperdiploidy but lacked evidence of an E2A-PBX1 gene fusion by molecular study, the poor banding quality of chromosomes led to misinterpretation of the cytogenetic findings; a correct diagnosis was established only after analysis by the fluorescence in situ hybridization (FISH) method. The second case, which was classified as pseudodiploid pre-B ALL, had both a derivative 19 and a derivative 22 but lacked sufficient cells for evaluation of E2A-PBX1 gene fusion. This case was included in order to compare the der(19)t(1;19) and the der(22)t(1;22) and to pinpoint the difficulty in distinguishing these markers. FISH analysis can resolve diagnostic uncertainty in cases of ALL with equivocal chromosome 19 markers. PMID- 7542914 TI - Obstruction after self-expanding metallic stents in tuberculous bronchial stenosis. AB - Expandable metallic stents seemed to be a good method in tuberculous bronchial stenosis that does not respond to medical therapy. But there was no long-term follow-up study after stents insertion in tuberculous bronchial stenosis. We report a case of obstruction after successful Gianturco metallic stents insertion due to tuberculous bronchial stenosis. PMID- 7542915 TI - Foveal electroretinograms and choroidal perfusion characteristics in fellow eyes of patients with unilateral neovascular age-related macular degeneration. AB - AIMS/BACKGROUND: A prolonged choroidal filling phase on fluorescein angiography has been reported to be a common finding and associated with visual function abnormalities in patients with age-related macular degeneration (AMD). This investigation was carried out to determine whether this perfusion defect was related to the slowing of foveal cone electroretinogram (ERG) implicit time seen in patients with AMD. METHODS: Fluorescein angiograms and foveal cone ERGs were evaluated in the fellow eyes of 67 patients with unilateral neovascular AMD. RESULTS: Twenty eight (42%) of the eyes had a choroidal perfusion defect. ERG implicit times averaged 1 ms slower (p = 0.0167) and were more likely to be delayed (p = 0.0078) in eyes with abnormal choroidal perfusion than in eyes with normal choroidal filling; significant relations were found also after controlling for age. ERG implicit time was also inversely related to ETDRS visual acuity and positively related to the extent of macular drusen; and the latter showed a borderline significant tendency to be more prevalent in eyes with prolonged choroidal perfusion. However, an association of a delayed ERG implicit time with prolonged choroidal filling remained after controlling for age, acuity, and the extent of drusen. CONCLUSION: These findings further establish prolonged choroidal perfusion as a common finding in AMD and link it to retinal malfunction. PMID- 7542916 TI - Asymmetry of disciform scarring in bilateral disease when one eye is treated with radiotherapy. AB - AIMS/BACKGROUND: A previous study has shown that in age-related macular degeneration a high degree of concordance of disciform scar size occurs in the two eyes of any one patient. In a study of 35 patients with choroidal neovascular membrane who were treated with low dose ionising radiation to the macula of the affected eye, 11 were found to have bilateral disease. METHODS: The visual outcome and scar size and morphology in the two eyes of each of these patients were compared. RESULTS: In all radiotherapy treated eyes the appearance of the scar ranged from subtle subretinal pigmentary changes to dense subretinal fibrosis. By contrast all untreated fellow eyes showed marked subretinal scarring. Scars in radiotherapy treated eyes occupied an area that was approximately one third of that in untreated fellow eyes (3.8 mm2 v 11.7 mm2). Distance and near visual acuities in radiotherapy treated eyes were significantly better than that of untreated fellow eyes (p < 0.0033). Although untreated fellow eyes of necessity had longer follow up periods (64.5 months), the mean follow up time in treated eyes was 28 months by which time the disciform response is generally thought to have ceased evolving. CONCLUSION: This study has provided evidence in support of reduced scarring and maintenance of better central visual function in radiotherapy treated eyes when compared with untreated fellow eyes. PMID- 7542918 TI - Palmitoylation-induced conformational changes of specific side chains in the gramicidin transmembrane channel. AB - To gain insight into the structural consequences of acylation for membrane proteins, we have covalently attached palmitic acid to the ethanolamine end of gramicidin A (gA), which functions as a well-characterized cation-selective membrane channel. Next, we investigated by NMR methods the effect of acylation on the side chains of Trp9, Leu10, and Trp11, which are expected to be close to the acyl chain, and of Val7, which is expected to be far from the acyl chain. Two dimensional NMR spectroscopy in a sodium dodecyl sulfate (SDS) environment suggests that one of the beta-hydrogens of Leu10 of gA is severely shielded by a nearby aromatic ring. This shielding disappears upon acylation. Deuterium NMR spectra for labeled samples in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers show that, for the major gA conformation, the (deuterated) side chains of Trp9 and Leu10 are markedly influenced by acylation, whereas the side chains of Val7 and Trp11 are essentially unaffected. The NMR results in both environments suggest that the indole ring of Trp9 is situated near the side chain of Leu10 and moves away upon acylation. We propose that acylation provides a subtle mechanism to modulate protein and lipid interactions and to regulate the stability and function of proteins within membranes. PMID- 7542919 TI - The three-dimensional solution structure of RANTES. AB - The solution structure of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) has been determined using NMR spectroscopy. Backbone and side-chain 1H and 15N assignments have been obtained using a combination of two-dimensional homonuclear and three-dimensional heteronuclear spectra. Regular elements of secondary structure have been identified on the basis of a qualitative interpretation of NOE data, J(NH-H alpha) coupling constants, and amide exchange rates. Three-dimensional structures were calculated from a total of 2146 experimental restraints using a combination of distance geometry and simulated annealing protocols. For the 13 best structures the average backbone (N, C alpha, C) atomic rmsd from the mean coordinates for residues 5-65 is 0.64 A (+/- 0.14 A) for the dimer and 0.50 A (+/- 0.08 A) for the individual monomers. Each monomer consists of a three-stranded antiparallel beta-sheet (residues 26 30, 38-43, 48-51) in a Greek key motif with a C-terminal helix (56-65) packed across the sheet, an arrangement similar to the monomeric structure of other members of this chemokine family (IL-8, PF4, MGSA/Gro alpha, and MIP-1 beta). Overall, the RANTES dimer resembles that previously reported for MIP-1 beta. PMID- 7542917 TI - Myelinated axon number in the optic nerve is unaffected by Alzheimer's disease. AB - AIMS/BACKGROUND: Visual symptoms are a common but not invariable feature of Alzheimer's disease (AD) and such symptoms appear to become more pronounced as the severity of the dementia increases. Pathology in both the pregeniculate and cortical parts of the visual system has been suggested to underlie the visual deficits in AD. In order to investigate the former possibility, the effect of AD on the optic nerve was investigated. METHODS: Intraorbital segments of optic nerve were taken at autopsy from nine patients with AD and seven patients with no history of psychiatric or neurological disease and no abnormal neuropathology. All patients had functional vision before death and appeared free of retinal, optic nerve, or microvascular disease. The optic nerves were processed into resin, semi-thin sections cut perpendicular to the long axis of each optic nerve, and stained with paraphenylenediamine. The sections were then investigated using an image analysis system and standard morphometric techniques. RESULTS: There was no significant difference in the mean cross sectional neural area of AD compared with control optic nerves. Neither were there any significant differences between myelinated axon surface density, total axon number, or mean cross sectional axon area in AD compared with control optic nerves. CONCLUSION: These results indicate that optic nerve degeneration is not a feature of AD and suggest that the visual deficits in the disease result from cortical dysfunction. This view is supported by the fact that visuospatial dysfunction appears to be the most common visual problem in AD. PMID- 7542921 TI - Nuclear multicatalytic proteinase alpha subunit RRC3: differential size, tyrosine phosphorylation, and susceptibility to antisense oligonucleotide treatment. AB - Multicatalytic proteinases (MCPs) are macromolecular structures involved in intracellular degradation of many types of proteins. MCPs are composed of a 20S "core" which consists of both structural (alpha) and presumed catalytic (beta) subunits in association with complexes of accessory proteins. Immunohistochemical studies have shown MCP subunits to be largely cytoplasmic, although nuclear localization is also observed. Reverse transcription/polymerase chain reaction amplifications were performed with redundant primers to conserved regions within known subunits, in an attempt both to identify potential new subunits and to define the repertoire of subunits expressed in hepatocytes. No new subunits were identified, and we found that RRC3, an alpha subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant alpha subunit expressed in hepatocytes and hepatocyte-derived cell lines. Antibodies were developed against a unique C-terminal peptide region of RRC3. Immunohistochemical studies using affinity-purified antibodies showed that RRC3 has both cytoplasmic and nuclear localizations. Immunoprecipitation/immunoblot analyses showed that a significant proportion of nuclear RRC3 was associated with the nuclear scaffold (NS). NS RRC3 showed a significantly smaller M(r) (24,000) than the cytoplasmic form (M(r) 28,000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an M(r) of 24,000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of M(r) 28,000 was also observed in nuclei, and this M(r) 28,000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (M(r) 28,000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major M(r) 24,000 form associated with NS. Treatment with antisense oligonucleotides targeted to the initiation site of RRC3 reduced the growth of a hepatocyte-derived cell line by 95% and produced a marked morphological change (in the absence of overt toxicity). Under these treatment conditions, RRC3 mRNA was dramatically reduced. RRC3 protein was also dramatically reduced in the NS, but showed only a small reduction in cytosol, suggesting that the nuclear RRC3 may be important in cell growth and differentiation. PMID- 7542920 TI - Studies into the identity of the sites of insulin-stimulated insulin receptor serine phosphorylation. Characterization of synthetic peptide substrates for the insulin-stimulated insulin receptor serine kinase. AB - The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin receptor copurified with insulin-stimulated insulin receptor serine kinase activity. Of the 14 peptides studied, only 4 (1071--1080, 1290--1298, 1253 -1271, and 1313--1329) were phosphorylated on serine, with the serine phosphorylation stimulated 2--4-fold by insulin. Peptides 1071--1080 and 1290- 1298 were 3--7-fold better substrates for the serine phosphorylation than the other serine-phosphorylated peptides. Peptides 1071--1080 and 1313--1329 also exhibited insulin-stimulated phosphorylation on tyrosine. Two-dimensional thin layer tryptic mapping of the phosphorylated insulin receptor/insulin-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptides, called S1 and S2, that ran as a pair of closely migrating spots, and other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosposerine/phosphotyrosine ratios of approximately 6 and 0.96-1.50 for the in vivo and in vitro labeled receptor, respectively. S1 and S2 were not cleaved by V8. Of the serine-phosphorylated peptides, only 1290--1298 and 1071--1080 should be V8 resistant; 1290--1298 contains serine sites 1293/4 and migrated distinctly from S1 and S2 in tryptic maps. Peptide 1071--1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphopeptides, each containing phosphoserine and phosphotyrosine, which comigrated exactly with S1 and S2. Comigration was confirmed at a different pH and by mixing experiments. Radiosequenation showed that serine 1078 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it was no more than a minor site in vivo. It is concluded that serine 1078 of the insulin receptor is a major site of insulin stimulated phosphorylation in vivo and in vitro. The peptide sequences provide a range of substrates to facilitate the study, purification, and characterization of the insulin-stimulated insulin receptor serine kinase or kinases, and the identification of a major site of insulin-stimulated serine phosphorylation will help elucidate the function of the insulin receptor serine phosphorylation. PMID- 7542922 TI - High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus. AB - The reverse transcriptase (RT) of HIV-1 is a plausible target for therapeutic agents aimed at inhibiting propagation of the virus. We have used "irrational drug design", that is, combinatorial chemistry with oligonucleotide libraries, to identify high-affinity ligands aimed at HIV-1 RT. The methodology, termed SELEX (systematic evolution of ligands by exponential enrichment), was employed with a single-stranded DNA library. The selected ssDNA ligands bind HIV-1 RT with Kd values as low as 1 nM and inhibit the RNA-dependent DNA-polymerase activity of the enzyme with Ki values as low as 0.3 nM. We also demonstrate the high specificity of one ligand able to selectively discriminate between the reverse transcriptases of HIV-1, AMV, and MMLV. These ssDNA molecules may be useful as inhibitors or as models for the design of small molecule inhibitors of HIV-1 RT in vivo. PMID- 7542923 TI - Origins of high sequence selectivity: a stopped-flow kinetics study of DNA/RNA hybridization by duplex- and triplex-forming oligonucleotides. AB - Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affinity of circular triplex forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-nt target sequences which are fully complementary or which contain a single mismatch. Also studied for comparison are standard 12-nt Watson-Crick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermodynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide mismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported triplex formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA-RNA hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is reflected in large variations in dissociation, rather than association rates. Finally, (5) binding selectivity of circular ligands becomes significantly greater as pH is lowered; results indicate that the high sequence selectivity of the circular DNA ligand is due in large part to the special stability of the protonated C+G-C triad relative to unprotonated mismatched triads. The results are useful in the understanding of properties of nucleic acid complexes in general and give insight into optimum design for synthetic DNA binding ligands. PMID- 7542924 TI - Evidence for class-specific discrimination of a semiconserved base pair by tRNA synthetases. AB - Aminoacyl-tRNA synthetases have been divided into two classes based on the existence of two structurally distinct active sites. To date, few class-specific tRNA recognition features have been elucidated. High-resolution X-ray structures of representative class I and class II synthetases complexed to cognate tRNA substrates have been solved. In these structures, the class I enzyme approaches the end of the tRNA acceptor stem from the minor-groove side, while the class II synthetase approaches its cognate tRNA from the major-groove side. This distinction is reflected in the different initial sites (2'- or 3'-OH) of amino acid attachment. The role that the semiconserved G1.C72 terminal base pair plays in the aminoacylation of Escherichia coli tRNAs is probed in this in vitro study. We show here that class II alanyl-, prolyl-, and histidyl-tRNA synthetases are sensitive to changes at position 1 x 72. Previous work on class I synthetases and new data presented here with the valine-specific enzyme indicate that class I enzymes show little sensitivity to replacements of G1.C72. This work provides new evidence for class-specific differences in tRNA acceptor stem interactions that appear to be reflected not only in the initial site of aminoacylation but also in the mode of synthetase interaction with the semiconserved G1.C72 base pair proximal to the amino acid attachment site. PMID- 7542925 TI - SH2 domain structure and function. AB - An emerging theme in both the biology of signal transduction and the biochemistry of proteins has been the modular function of small protein domains. In some cases these can directly regulate catalytic activity. In others, they serve to interconnect important regulatory proteins. SH2 (src homology 2) domains represent some of the best studied models. Originally identified on the basis of homology in src and fps [1], SH2s are elements that ordinarily respond to tyrosine phosphorylation by binding the phosphorylated sequence. As such, they are key elements in tyrosine kinase regulation of cellular processes. Because SH2 interactions result from phosphorylation, such elements provide a regulatable circuitry along which signals can be transmitted in a timely manner. Because the regulation is based on a common mechanism, signal generators can target several different proteins coordinately. The PDGF receptor (PDGFr), for example, may interact with as many as ten different elements [2,3]. There are a number of excellent reviews on SH2 domains available [4-11]. This discussion will try to show how genetic, biochemical and biophysical results can be integrated in a satisfying way. PMID- 7542926 TI - Signal transduction by cell adhesion receptors. AB - Over the last few years, it has become clear that cell adhesion receptors function in signal transduction processes leading to the regulation of cell growth and differentiation. Signal transduction by both integrins and CAMs has been shown to involve activation of tyrosine kinases, while CAM signaling in neural cells involves G proteins as well. In the case of integrins, some of the downstream signaling events intersect with the Ras pathway, particularly the activation of MAP kinases. In fibroblasts, integrin mediated anchorage to the substratum regulates cell cycle traverse, while in epithelial cells, loss of anchorage can trigger programmed cell death. In many cell types, but particularly monocytic cells, integrin ligation has a profound impact on gene expression. Preliminary evidence also implicates CAMs and selectins in gene regulation. A consistent theme in signal transduction mediated by adhesion receptors concerns the role of the cytoskeleton. Integrin mediated signaling processes are interrupted by cytoskeletal disassembly. Identification of the APC and neurofibromatosis type 2 tumor suppressors suggest that cytoskeletal complexes also play a key role in signaling by cadherins and CD44, respectively. Thus, signaling by cell adhesion receptors may involve aspects that impinge on previously known signaling pathways including the RTK/Ras pathway and serpentine receptor/G protein pathways. However, novel aspects of signal transduction involving cytoskeletal assemblies may also be critical. PMID- 7542927 TI - Characterisation of the divalent cation channels of the hepatocyte plasma membrane receptor-activated Ca2+ inflow system using lanthanide ions. AB - The ability of Gd3+ to inhibit vasopressin-stimulated Ca2+ inflow to hepatocytes was compared with its effect on Mn2+ inflow. In the absence of Gd3+, the stimulation of Mn2+ inflow by vasopressin increased with increasing pH of the extracellular medium. Maximal inhibition of vasopressin-stimulated Ca2+ and Mn2+ inflow by saturating concentrations of Gd3+ was 70 and 30%, respectively. Gd3+ also inhibited thapsigargin-stimulated Ca2+ and Mn2+ inflow with maximal inhibition of 70 and 40%, respectively. It is concluded that vasopressin and thapsigargin each activate two types of Ca2+ inflow processes, one which is sensitive and one which is insensitive to lanthanides. The nature of the pore of the lanthanide-sensitive Ca2+ channel was investigated further using different lanthanides as inhibitors. Tm3+, Gd3+, Eu3+, Nd3+ and La3+ each inhibited vasopressin-stimulated Ca2+ and Mn2+ inflow but had no effect on Ca2+ inflow in the absence of an agonist, or on vasopressin-stimulated release of Ca2+ from intracellular stores. Maximal inhibition of vasopressin-stimulated Ca2+ inflow in the presence of a saturating concentration of each lanthanide ranged from 70-90%. An equation which describes a 1:1 interaction of the lanthanide with a putative binding site in the Ca2+ channel gave a good fit to dose-response curves for the inhibition of vasopressin-stimulated Ca2+ inflow by each lanthanide. Lanthanides in the middle of the series exhibited the lowest dissociation constant (Kd) values. The Kd for Gd3+ increased with increasing extracellular Ca2+ concentration, suggesting competitive inhibition of Ca2+ binding by Gd3+. In the absence of lanthanide, vasopressin-stimulated Mn2+ inflow was substantially reduced when the plasma membrane was depolarised by increasing the extracellular K+ concentration. Changing the membrane potential had little effect on the maximum inhibition by Gd3+ of vasopressin-stimulated Mn2+ inflow. The Kd for inhibition of vasopressin-stimulated Ca2+ inflow by Gd3+, measured at the lowest attainable membrane potential, was about 6-fold lower than the Kd measured at the highest attainable membrane potential. The idea that there is a site in the vasopressin-stimulated lanthanide-sensitive Ca2+ channel composed of carboxylic acid groups which bind Ca2+, Mn2+ or a lanthanide ion is consistent with the data obtained using the different lanthanides. PMID- 7542929 TI - Cervical cancer screening for hospital inpatients: report of an intervention study. AB - A pilot study of a cervical cancer screening service was carried out at a major teaching hospital in Perth. The service, for women inpatients aged 20 to 69 years, was staffed by a women's health nurse. The effect of providing the service (service) was compared with giving a leaflet on Pap smears to eligible women (education) and with no intervention (control). Of 517 women in the service group, 184 (36 per cent) needed a Pap smear and were well enough to be offered screening; only 29 of 184 (16 per cent) refused and 132 of 184 (72 per cent) were screened. Of those screened, 29 per cent had never had a Pap smear. Information on women in the education and control groups was obtained by mailed questionnaire. Of the eligible women in the service group, 72 per cent accepted screening in hospital, but only 24 per cent of eligible women in the education group and 20 per cent in the control group reported having a Pap smear in the four months since leaving hospital. The service group showed a very large effect relative to the control group (odds ratio (OR) 17.71, 95 per cent confidence interval (CI) 10.05 to 31.22), but there was no significant difference between the education and control groups. Other significant variables in the logistic regression model were age, marital status, and sex of the woman's general practitioner. The effect of offering the service was greater for women over 50 (OR 51.51, CI 19.01 to 139.60) A hospital-based cervical screening service provides an important opportunity for screening women who are not being reached by other services. PMID- 7542928 TI - The effect of an educational brochure on knowledge and early detection of melanoma. AB - Men over the age of 45 present with thicker, more advanced melanomas than younger people. A randomised trial was conducted in this group to evaluate whether an educational brochure would increase knowledge about melanoma and the ability to recognise and discriminate between pigmented skin lesions. Men in an industrial complex were allocated to an intervention group (n = 110) and two control groups (n = 96 and n = 108). The intervention group was given two educational brochures about melanoma. Their effect on knowledge and ability to detect pigmented lesions was assessed by a questionnaire and a self-examination body chart given before the brochure, and at four weeks and three months after return of the brochure. The control groups did not receive any educational material, but control group 2 received the questionnaire and chart. At the end of the study all participants were examined for pigmented lesions by doctors, whose counts were compared with those of the participants. There was a significant (19.8 per cent) increase in knowledge about melanoma in the intervention group (but not in the control groups), except for discrimination of photos of benign and malignant lesions. The educational material did not improve the ability of those in the intervention group to recognise and count their pigmented lesions nor to discriminate between benign and malignant pigmented lesions. The increased knowledge about melanoma was retained for at least three months. PMID- 7542930 TI - The endogenous scavengers in cerulein-induced acute pancreatitis. AB - Studies in animal models suggest that oxygen radicals are important in the pathogenesis of acute pancreatitis. Cerulein, a decapeptide isolated from the skin of the frog, Hyla caerula, is closely related to the C-terminus of cholecystokinin and it is a potent stimulant of pancreatic exocrine secretion. The aim of the present study was to measure the activity of endogenous scavengers, superoxide dismutase, catalase and glutathione levels in cerulein induced acute pancreatitis in rats. We found that the plasma amylase and ribonuclease levels in the pancreatitis group were both significantly high (p < 0.01, p < 0.05, respectively) when compared with the control group. Although superoxide dismutase and glutathione levels of pancreatic tissue were decreased significantly (p < 0.01, p < 0.01 respectively), we observed a significant increase (p < 0.01) in catalase activity in the cerulein treated group compared to the control group. Therefore, we concluded that the profound alteration of the activities of endogenous scavengers (superoxide dismutase, catalase) and glutathione depletion occurring after cerulein-induced pancreatitis seemed to be important in tissue injury and may provide the basis for successful therapy of the disease. PMID- 7542931 TI - In vitro effect of dipyrone on several peroxidase labelled immunoassays. AB - We studied the in vitro effect of dipyrone on the determination of free triiodothyronine (free T3), cortisol, progesterone, estradiol, carcinoembryonic antigen, human chorionic gonadotropin and alpha-fetoprotein measured with an immunoenzyme assay based on enhanced luminescence that uses peroxidase as label. We found significant interference from dipyrone (p < 0.01) in the determination of all the analytes mentioned: for progesterone and estradiol the interference was present at high doses of dipyrone; for free T3 and cortisol the minimum dipyrone concentration producing interference was 712 mumol/l and for carcinoembryonic antigen, human chorionic gonadotropin and alpha-fetoprotein 44 mumol/l. Dipyrone has an analytically and statistically significant interference effect on the determination of the mentioned analytes. PMID- 7542933 TI - Effect of acute glucose antagonism on hypophyseal hormones and concentrations of insulin-like growth factor (IGF)-I and IGF-binding proteins in serum, anterior pituitary, and hypothalamus of ewes. AB - The objectives of this study were to determine whether 2-deoxy-D-glucose (2DG), a metabolic inhibitor of glucose, 1) differentially affected release of LH, growth hormone (GH) and prolactin (PRL); 2) decreased anterior pituitary response to GnRH; and 3) altered concentrations of insulin-like growth factor I (IGF-I) and intensity of 125I-IGF-I binding to proteins (IGFBPs) in serum, anterior pituitary (AP), and hypothalamus (preoptic are [POA], medial basal hypothalamus [MBH], and stalk median eminence [SME]). In trial 1, mature, ovariectomized, crossbred ewes were treated with a single s.c. injection of either saline (n = 5) or 2DG (n = 5; 0.1 g/kg BW). Administration of 2DG suppressed (p < 0.05) concentrations of LH but did not affect (p > 0.05) serum concentrations of GH or PRL. In trial 2, ovariectomized ewes received injections of saline or 2DG, and each group was subdivided to receive either saline or GnRH analog (5 micrograms; Des-Gly10-[D Ala6]) 2 h later. Administration of 2DG suppressed (p < 0.05) serum concentrations of LH before administration of GnRH; however, release of LH in response to exogenous GnRH was greater (p < 0.05) in ewes that received 2DG. Treatment with 2DG did not affect (p > 0.05) concentrations of FSH before or after administration of GnRH. In trial 3, 10 ovariectomized ewes were slaughtered 2 h after treatment with saline (n = 5) or 2DG (n = 5). Serum and tissues collected at slaughter (AP, MBH, SME, and POA) were analyzed for concentrations of IGF-I by RIA and intensity of 125I-IGF-I binding to proteins by ligand blotting.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542932 TI - Efferent innervation of the rat testis. AB - Previous assessments of the autonomic nerve supply of the male genital tract concluded that the testis received sympathetic input primarily from paraaortic ganglia, particularly the spermatic ganglion. We challenged this consensus by using retrograde axonal tracing to examine the source and distribution of efferent fibers reaching the testis of adult rats. We also used immunohistochemical methods to assess putative neurotransmitters in testicular neurons of the abdominal and pelvic ganglia. The results indicate the majority of retrogradely labeled cell profiles were localized within the major pelvic (38%) and pelvic accessory ganglia (37%), and only a few labeled cell profiles were present in the paraaortic and spermatic ganglia. Injection of FluoroGold and Fast Blue dyes into the respective right and left testis demonstrated that 17% of the neurons in pelvic ganglia were labeled when tracers were microinjected beneath the capsule of the contralateral testis. About 8% of the neurons were labeled both with FluoroGold and Fast Blue, suggesting that certain neurons can provide simultaneous input to the left and right testicles. Immunohistochemical results showed that tyrosine hydroxylase, a marker for noradrenergic fibers, was present in over 33% of the cell profiles labeled with either FluoroGold or Fast Blue. Some 27% of the fluorescent-labeled cell profiles were positive for neuropeptide Y, while 22% were immunoreactive for vasoactive intestinal polypeptide. No evidence for vasoactive intestinal polypeptide immunoreactivity was detected within the testis, but neuropeptide Y-immunoreactive fibers were present in the tunica albuginea and testicular vasculature. Catecholamine fluorescent fibers were distributed sparsely throughout the periphery of the testis in association with the capsule, vasculature, and interstitium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542935 TI - Cyclic nucleotide-gated channels in visual and olfactory transduction. AB - Rod and cone photoreceptors are the light detectors in the visual system whereas olfactory receptor cells are the odorant detectors in the olfactory system. Despite the two very different types of stimuli, light in photoreceptors, and odorant molecules in olfactory receptor cells, the mechanisms of visual and olfactory transduction appear to have many homologies. Both stimuli trigger a chain of enzymatic events that terminates in a change in the concentration of a cyclic nucleotide: a decrease in the concentration of cGMP in photoreceptors, and an increase in the concentration of cAMP in olfactory receptor cells. These cyclic nucleotides directly gate cation channels and therefore a change in their concentration induced by the external stimulus is converted into an electrical signal. The analysis of the ionic selectivity properties of cyclic nucleotidegated channels in retinal rods, cones and in olfactory receptor cells shows that there are many similarities between these channels. They do not appreciably select between alkali monovalent cations and can be permeated and blocked by divalent cations. Their ionic permeation properties are consistent with the presence of a cation-binding site of high-field strength in the pore. PMID- 7542934 TI - [Evaluation of primary care treatment of oncology patients with opiates]. AB - OBJECTIVES: To evaluate how adequately pain in terminal cancer patients is managed by morphine taken orally. DESIGN: A descriptive, retrospective study, from January 1993 to March 1994. SETTING: Primary care. Four primary care teams of the 10th Health Area of Madrid. PATIENTS: 37 patients with terminal cancer who took morphine under the supervision of primary care doctors. MEASUREMENTS AND MAIN RESULTS: Average age was 64.6 +/- 14. The most common site of the primary tumour was lungs (n = 6), colon (n = 4) and stomach (n = 4). All the patients received conventional analgesics at first and 82% went on to weak opiates before progressing to morphine. Half the patients began with an oral solution of morphine and the other half with long-acting tablets. Increases in the doses of morphine were correct in 62% of cases. 41% of the patients took antiemetics and 55% laxatives. 82% of the bone and 100% of the brain metastases were correctly treated. CONCLUSIONS: In general, primary care doctors in our area correctly handle pain caused by terminal cancer. The most significant errors are to do with increases in doses of morphine and with the associated use of laxatives. These can easily be rectified so that our patients' quality of life can be improved. PMID- 7542936 TI - A genetic algorithm to search for optimal and suboptimal RNA secondary structures. AB - Genetic algorithms are a search method used in solving problems by selection, recombination and mutation of tentative solutions, until the better ones are achieved. They are very efficient when the 'building block' hypothesis is effective for the solutions, which means that a better solution can be obtained by assembling short 'motifs' or 'schemata' that can be retrieved in some other worse solutions. The additive nature of the secondary structure free energy rules suggests the validity of this hypothesis, and therefore the likely power of a genetic algorithm approach to search for RNA secondary structures. We describe in detail an original genetic algorithm specific for this problem. The sharing function used to obtain differentiated solutions is also described. It results in a greater effectiveness of the algorithm in retrieving a large number of suboptimal RNA foldings besides the optimal one. RNA sequences of different length are used to test the method. The PSTV viroid sequence has been studied. PMID- 7542938 TI - Immunocytochemistry, autoradiography, in situ hybridization, selective stains: complementary tools for ultrastructural study of structure-function relationships in the nucleus. Applications to adenovirus-infected cells. AB - A significant amount of new information on structure-function relationships in nuclei of adenovirus-infected cells has accumulated during the last decade as a result of the combined use of several new cytochemical techniques. Localization of viral DNA on ultrathin sections of infected cells has been investigated at the ultrastructural level by using specific DNA staining and immunocytochemistry with monoclonal anti-DNA antibodies. Both techniques, however, concomitantly visualize cellular and viral DNA. The specific stain for DNA reveals the configuration of the DNA molecules in the different nuclear substructures, whatever their synthetic activities. The immunodetection of DNA reveals that specific antibodies strongly bind to DNA of condensed host chromatin and to both encapsidated and nonencapsidated inactive viral genomes. However, the observation of an abnormally low level of labeling over the substructures in which synthetic activities of viral genomes are known to be intense demonstrates a serious limitation of this technique for the detection of active DNA. Postembedding in situ hybridization is the most useful method for identifying with certainty the structures containing defined nucleic acid sequences. By using a biotinylated viral DNA probe, in situ hybridization provides specific identification of structures containing either viral DNA or viral RNA molecules. In addition, with appropriate pretreatment of the sections, it is possible to reveal either all the viral DNA--that is, both double- and single-stranded DNA molecules (dsDNA, ssDNA)--or more specific species such as only ssDNA or only dsDNA molecules. The replicative and transcriptional activities of viral genomes are determined by high-resolution autoradiography. Autoradiography after a short pulse incorporation of appropriate radioactive precursors by infected cells reveals the sites of cellular and viral DNA replication or transcription. A short pulse followed by chase periods of different durations reveals the progressive migration of the cellular and viral synthesized products. The in situ distribution of the viral 72 kDa DNA-binding protein, a highly phosphorylated protein which protects the viral ssDNA, is revealed either by immunocytochemistry with specific antibodies or by the bismuth staining method which stains all highly phosphorylated proteins, including both cellular and viral proteins. The combined results of all these cytochemical procedures reveal the composition and functions of some of the structures induced by adenovirus infection. They demonstrate that viral genomes engaged in replication lead to the formation of the replicative foci in which two compartments rapidly develop, one of which results from the aggregation of single strands of viral DNA and their accompanying 72 kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542937 TI - Posterior extension of the chick nephric (Wolffian) duct: the role of fibronectin and NCAM polysialic acid. AB - The nephric duct of the chick embryo starts to form at about stage 10 of Hamburger and Hamilton ([1951] J. Morphol. 88:49-92) and extends posteriorly, fusing with the cloaca at about the end of the third day of incubation (HH stage 17). Evidence from the literature suggests that the extension involves active migration of the posterior tip. This investigation concerned some molecules that might control this migration: fibronectin, vitronectin, the beta 1 integrin receptor, and NCAM polysialic acid. The concentration of fibronectin in the extracellular matrix was found by immunocytochemistry to be negligible at the posterior end of the duct; treatment of the living embryo with GRGDS failed to halt further extension of the duct; SEM examination of embryos treated with the synthetic peptides of fibronectin GRGDS, GRDGS, SDGR, and GRGES, or with vitronectin, revealed negligible morphological effects on the duct. It is concluded that there is yet no evidence that fibronectin is an important factor in duct migration. NCAM polysialic acid had a similar distribution to fibronectin, but treatment of the living embryo with Endo-N caused cessation of extension of the duct. Endo-N is an enzyme that specifically degrades PSA without affecting the NCAM polypeptide itself. It is suggested therefore that PSA may play an important role in duct extension. The synthetic peptides of fibronectin each produced distinctive patterns of blebbing on the surfaces of cells in trunk mesoderm, but the duct cells were unaffected. GRGES and SDGR caused blebbing on cells in the somites and the anterior segmental plate, though not on cells in the posterior segmental plate. This suggests that integrin receptors change in the anterior segmental plate as the mesoderm forms somites from somitomeres. PMID- 7542939 TI - Nucleic acid compartmentalization within the cell nucleus by in situ transferase immunogold techniques. AB - In the present review, we report on recent results obtained by in situ transferase-immunogold techniques as to the ultrastructural distribution of DNA and RNA within the cell nucleus. Special emphasis is placed on the various nucleolar components and the various enigmatic structures of the extranucleolar region: interchromatin granules, coiled bodies, and simple nuclear bodies. These data are discussed in the light of our current understanding of the functional organization of the cell nucleus. PMID- 7542940 TI - Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. AB - Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons. PMID- 7542943 TI - [Peripheral blood stem cell transplantation: current status and perspectives]. AB - Peripheral blood stem cell (PBSC) transplantation is now widely used as technics of stem cell transplantation. If it is efficacious in terms of short-term engraftment, many questions remain unanswered: which is the best technic of mobilization, what role for growth factors after graft, which benefit to add bone marrow stem cells? So, comparative trials are necessary to compare bone marrow transplantation and peripheral stem cell transplantation in terms of long-term engraftment and anti-tumoral effect. However, the present main point is to better define the adequate estimation of circulating hematopoietic progenitors according to the technics of collection: evaluation of CFU-GM and CD34+ levels are inadequate for estimation of immature progenitor cells which vary a lot according to the types of chemotherapy or of growth factors used for mobilization. PMID- 7542944 TI - [Intensive chemotherapy with autologous bone marrow transplantation: assessment and limits. What is its future?]. PMID- 7542942 TI - The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs. AB - The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism. PMID- 7542941 TI - Functional analysis of selective interactions among rodent connexins. AB - One consequence of the diversity in gap junction structural proteins is that cells expressing different connexins may come into contact and form intercellular channels that are mixed in connexin content. We have systematically examined the ability of adjacent cells expressing different connexins to communicate, and found that all connexins exhibit specificity in their interactions. Two extreme examples of selectivity were observed. Connexin40 (Cx40) was highly restricted in its ability to make heterotypic channels, functionally interacting with Cx37, but failing to do so when paired with Cx26, Cx32, Cx43, Cx46, and Cx50. In contrast, Cx46 interacted well with all connexins tested except Cx40. To explore the molecular basis of connexin compatibility and voltage gating, we utilized a chimera consisting of Cx32 from the N-terminus to the second transmembrane domain, fused to Cx43 from the middle cytoplasmic loop to the C-terminus. The chimeric connexin behaved like Cx43 with regard to selectivity and like Cx32 with regard to voltage dependence. Taken together, these results demonstrate that the second but not the first extracellular domain affects compatibility, whereas voltage gating is strongly influenced by sequences between the N-terminus and the second transmembrane domain. PMID- 7542946 TI - [Immunotherapy of malignant diseases]. AB - Cancer immunotherapy has been carried out since the early fifties and first involved nonspecific system (BCG, Corynebacterium parvum, levamisole...). More recently, the production of cytokines as interferons or interleukin 2, the introduction of monoclonal antibodies have allowed a new development to cancer immunotherapy. Nevertheless, these new approaches have to be considered as a step in the biological therapy of cancer. PMID- 7542947 TI - [Cancer metastasis]. AB - Metastases result from the implantation of tumor cells into various organs, distant from the primary tumor. Among primary tumor cells, a small number undergo genetic events determining metastatic function acquisition. The metastatic function comprises the lack of homotypic intercellular connections, due to proteolytic enzyme effects and/or lack of adhesion molecule synthesis, stimulation of peri- and intratumoral angiogenesis, motility adhesion molecule synthesis, stimulation of peri- and intratumoral angiogenesis, motility and invasiveness, adhesiveness to acellular substrates or heterotypic cells, resistance to immune defenses, alteration of growth factor receptivity, and even growth autonomy. Formation of metastatic foci needs the establishment of an ecosystem born from the contribution of both host and tumor and in which respective cells interact. PMID- 7542945 TI - High-dose chemotherapy followed by hematological support: experience in the treatment of germ cell tumors. AB - Etoposide, bleomycin, and cisplatin based combination chemotherapy (BEP) and the surgical removal of residual disease is the standard treatment of metastatic germ cell tumors (GCT). Standard treatment including three cycles of BEP for good risk patients and four cycles of BEP for poor risk patients allows 95 and 65% cure rates in these groups respectively. However, about 10% of patients achieving CR after first line therapy eventually relapse. Conventional second line treatment (VeIP) includes a combination of vinblastin, ifosfamide and cisplatin or (VIP) with etoposide replacing vinblastin for patients pretreated by noncontaining etoposide chemotherapy regimens. These protocols induce a 60% CR rate and a 20 30% long term nonevolutive disease (NED) rate. Trials with high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) have been undertaken during the past decade. The main experiences were obtained with combinations of etoposide, platin derivatives, and either cyclophosphamide or ifosfamide. The majority of studies concerned heavily pretreated patients. Few studies have included HDCT and AHSCT as part of consolidation first line treatment for poor risk patients. A randomized multicenter French trial showed no significant benefit of this procedure (Droz, ASCO 1992). In refractory patients, HDCT and AHSCT induces a potential of 10% NED rate and this appears to be the maximum effect that can be obtained. An analysis of prognostic factors at first salvage chemotherapy revealed four adverse factors: high serum levels of HCG (> 10,000 mIU/ml) or AFP (> 1,000 g/ml); extragonadal origin; presence of lung metastases at salvage; and an incomplete response to first line treatment. Three prognostic groups have been defined according to a combination of these variables (Droz, Kramar, ASCO 1993). The use of HDCT followed by AHSCT as consolidation salvage treatment seems more promising since 25-45% of patients have long-term NED. It can be assumed that this procedure may increase the long term NED rate by 20% and produce a success rate up to 40%, but further studies are warranted in this setting. In order to evaluate HDCT with AHSCT rescue, an international randomized study has been undertaken in relapsed or first partial remission GCT patients. Randomization is stratified according to the three prognostic groups previously defined. Salvage regimens for eligible patients include two cycles of PEI or VeIP and in case of response, two more cycles of the same protocol (arm A) or one more cycle of PEI or VeIP followed by HDCT (CarboPEC: carboplatin, etoposide, and cyclophosphamide) (arm B).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7542949 TI - Inhibition by cromakalim, pinacidil, terbutaline, theophylline and verapamil of non-cholinergic nerve-mediated contractions of guinea-pig isolated bronchi. AB - Contractions induced by electrical field stimulation of sensory non-cholinergic excitatory nerves in guinea-pig isolated bronchi are due to the release of substance P (SP) and related tachykinins. Release of such neuropeptides are thought to play a pathophysiological role in asthma. Two K+ channel openers cromakalim (pD2 = 6.45; Emax = 95%) and pinacidil (pD2 = 6.06; Emax = 87%) were shown to concentration-dependently inhibit non-cholinergic nerve-mediated contractions in guinea-pig bronchi in vitro. Cromakalim (pD2 = 6.27; Emax = 25%) and pinacidil (pD2 = 6.03; Emax = 25%) each had a much lower inhibitory efficacy against contractions induced by exogenously applied SP but the same potency as found against contractile responses to non-cholinergic neurostimulation. Also the beta 2-adrenoceptor agonist terbutaline (pD2 = 8.29; Emax = 83%), the xanthine derivative theophylline (pD2 = 4.19; Emax = 100%) and the Ca2+ blocker verapamil (pD2 = 5.55; Emax = 100%) suppressed responses to non-cholinergic neurostimulation. Terbutaline (pD2 = 6.32; Emax = 74%), theophylline (pD2 = 3.25; Emax = 71%) and verapamil (pD2 = 4.01; Emax = 100%) had a 10-100-fold lower inhibitory potency against SP-induced contractions but each drug showed about the same efficacy as found against nerve-mediated contractions. Glibenclamide (1 microM) reversed the inhibitory effects of cromakalim and pinacidil on neurally mediated contractions but did not influence the effects of terbutaline, theophylline and verapamil. The results demonstrate that cromakalim, pinacidil, terbutaline, theophylline and verapamil inhibit non-cholinergic excitatory neurotransmission in guinea-pig bronchi and suggest that they act preferentially at a pre-junctional site. PMID- 7542951 TI - Professional presentations with posters. PMID- 7542950 TI - Inhibition of neutral endopeptidase potentiates compound 48/80-induced constriction of guinea-pig tracheal smooth muscle. AB - Our previous studies have shown that the inhibition of neutral endopeptidase, an enzyme which degrades tachykinins, increases anaphylactic construction of guinea pig tracheal smooth muscle. To investigate this observation further, we examined the effects of phosphoramidon, an inhibitor of a neutral endopeptidase, on constriction induced by the non-immunological mast cell degranulator-compound 48/80. Phosphoramidon produced significant leftward shift of the compound 48/80 concentration-response curve with corresponding decrease in the EC50 value from 51 (28-80) micrograms/ml to 42 (20-72) micrograms/ml. When added during the compound 48/80-induced constriction, phosphoramidon significantly increased the magnitude of this constriction by 69.7% after 30 min, and 78.9% after 45 min. Phosphoramidon was ineffective in tracheal rings from tachykinin-depleted guinea pigs. The incubation of tracheal rings with H1-histamine receptor antagonist (diphenhydramine HCl, 10 microM) and leukotriene receptor antagonist (ICI 198.615, 5 microM) significantly diminished the contractile response to compound 48/80 and prevented a phosphoramidon-dependent increase of this constriction. These results suggest that compound 48/80 induces the release of tachykinins by the stimulatory activity of histamine and leukotrienes. Anaphylactic release of tachykinins would therefore not depend directly on the antigen-antibody reaction. PMID- 7542952 TI - [Yersinia pestis superoxide dismutase]. AB - Superoxide dismutases with molecular weight of 65-67 and 200 kD have been identified in Yersinia pestis EV cells. These enzymes have some differences in antigenic specificity, resistance to heating and inhibitors (cyanides and hydrogen peroxide). The activity of Y. pestis superoxide dismutase is a thermoinducible sign unassociated with the presence of residual plasmids of the bacilli. Superoxide dismutase can be released from the cells into liquid culture medium. Patients inoculated with live plaque vaccine develop a humoral response to high molecular-weight superoxide dismutase. Immunization of guinea pigs with purified high molecular-weight superoxide dismutase ensures their specific protection after subcutaneous injection. PMID- 7542948 TI - Identification of three human sequences with viral superantigen-specific primers. AB - The open reading frame (ORF) in the long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) has recently been shown to encode multiple products including a negative acting factor (Naf) and a superantigen (Sag). Expression of superantigens from endogenous MMTV loci in the mouse results in the deletion of whole classes of T cells. In a PCR approach, with primers to the MMTV ORF and hybridization to MMTV specific probes, we have identified three human sequences. Direct sequencing of PCR products revealed that one of these products is related to a human autoantigen that is conserved among many species and is expressed in testes and sperm. The second sequence that we have identified is novel, and no evidence for expression of this sequence could be obtained. Finally, the third ORF-like sequence is a new member of a previously described family of human endogenous retroviruses (RTVL-I). This sequence is transcribed in several human cell lines, including B lymphoblastoid cells, and is thus the first demonstration that an RTVL-I-related sequence can be expressed. Taken together, these findings raise the intriguing possibility that the human genome contains superantigen-like sequences, some of which are also related to endogenous retroviruses, that may influence the T cell repertoire. PMID- 7542953 TI - Insulin-like growth factor binding protein production in human follicular thyroid carcinoma cells. AB - IGFs and IGF binding proteins (IGFBPs) appear to serve as regulators of non malignant thyroid cells from several species, but little is known about their role in thyroid malignancy. We have examined IGFBP production and hormonal regulation in two human thyroid follicular carcinoma cell lines; FTC-133 line derived from a local tumor recurrence and FTC-236 cells from a tumor metastasis. Under basal conditions these cell lines produced IGFBP-3, IGFBP-4 and IGFBP-2. In both cell lines, EGF or TPA stimulated IGFBP-3 production while TSH or forskolin inhibited IGFBP-3 production and reduced the stimulation of IGFBP-3 seen with EGF or TPA. IGFBP-4 production was increased in the presence of TSH, forskolin, and EGF and was reduced by TPA. mRNA assessment revealed that IGFBP-3 mRNA, more abundant in FTC-236 than FTC-133 cells, increased in the presence of EGF or TPA, while IGFBP-4 mRNA content was increased in the presence of TSH, EGF, and forskolin. These results indicate that IGFBP production in human thyroid follicular carcinoma clones is under specific hormonal regulation. IGFBP-3 production is increased by dedifferentiation factors such as EGF and TPA and inhibited by TSH and forskolin, which enhance differentiated function. The highly specific regulation of IGFBP-3 and IGFBP-4 suggests a potential role for these peptides in modulating malignant thyroid growth. PMID- 7542954 TI - Transcriptional regulation of insulin-like growth factor binding protein-1 expression by insulin and cyclic AMP. AB - In biological fluids, insulin-like growth factor binding proteins (IGFBPs) interact with the IGFs and modulate their effects. In this study, changes in IGFBP-1 expression were investigated under the influence of insulin and cAMP which may regulate expression of the IGFBP-1 gene in vivo during the perinatal period. Western ligand blot analysis of IGFBPs secreted by HepG2 human hepatoma cells showed that 24 h treatment with forskolin increased IGFBP-1 secretion by approximately 100%, whereas similar treatment with insulin resulted in a 50% reduction. After 24 h, the amounts of IGFBP-1 mRNA (measured by Northern blotting) were increased 2.5 times by forskolin and decreased by 65% by insulin. Transient transfection experiments showed that forskolin enhanced IGFBP-1 promoter activity by 70%, suggesting that stimulation of IGFBP-1 gene expression by cAMP is transcriptional, via a protein recognizing the cAMP responsive element (CRE) consensus sequence (nt -268 to -248). In contrast, modulation of gene expression by insulin is more complex, probably involving several levels of regulation. Complementary experiments (site-directed mutagenesis and/or use of a heterologous promoter) will be needed to confirm the functionality of the proteins interacting with the IRE (nt -285 and -276) and the CRE (between nt -268 and -248) described. PMID- 7542955 TI - Visuals that help, not hinder. PMID- 7542956 TI - If the light in your overhead projector suddenly dies in the middle of your presentation, what do you do? PMID- 7542957 TI - Evidence showing a different repertoire of antibodies against unfolded cobrotoxin in anticobrotoxin and anti-reduced and S-carboxymethylated cobrotoxin antisera. AB - Antibodies which specifically reacted with reduced and S-carboxy-methylated(RCM) cobrotoxin were purified from anticobrotoxin antisera and anti-RCM-cobrotoxin antisera, respectively. Results using a competitive immunoassay revealed that the antibodies from anti-RCM-cobrotoxin antisera had a greater affinity for RCM cobrotoxin than for cobrotoxin. Whereas the reactivity of the antibodies from the anticobrotoxin antisera toward cobrotoxin and RCM-cobrotoxin had a reversed order of binding. In contrast to observations made with S. aureus V8 protease-digest hydrolysates, the antigenic structures of RCM-cobrotoxin recognized by antibodies from anti-RCM-cobrotoxin antisera were notably affected following hydrolysis of RCM-cobrotoxin with chymotrypsin. Moreover, the chymotryptic hydrolysates showed a comparable reactivity as RCM-cobrotoxin toward the antibodies purified from anticobrotoxin antibodies, but a decrease in antigenicity with the V8 protease hydrolysates. These results reveal that the repertoire of antibodies against the unfolded cobrotoxin are not the same in anticobrotoxin and anti-RCM-cobrotoxin antisera. Moreover, it suggests that the repertoire of antibodies from the different sources against the same antigen can be differentiated by measurement of their reactivities with the proteolytic hydrolysates of the antigen. PMID- 7542958 TI - Calcium transport in human term placental mitochondria. AB - The kinetic characteristics of the transport of Ca2+ in the human term placental mitochondria previously depleted of their intramitochondrial calcium, was studied. Maximal Ca2+ uptake was observed in the presence of inorganic phosphate and ADP. Ca2+ transport showed a Michaelis-Menten behavior, with a Vmax of 61 nmol mg-1 min-1 and a Km of 118 microM. Ruthenium red inhibited mitochondrial Ca2+ uptake. Under optimal conditions of Ca2+ uptake, the cation inhibited progesterone synthesis in placental mitochondria. The kinetic parameters of Ca2+ transport agree with the physiological role of mitochondria in the human placenta. PMID- 7542959 TI - The chicken homeobox gene Hoxd-11 encodes two alternatively spliced RNA species. AB - Two forms of cDNA clones corresponding to Hoxd-11 were obtained by screening a chicken limb bud cDNA library. Comparison of the two cDNA sequences with the mouse cDNA and genomic sequence, shows that the shorter cDNA is missing 3 nucleotides, which encode an alanine residue, at the junction between exons 1 and 2. Analysis of the chicken genomic sequence shows two tandem CAG repeats at the intron 1-exon 2 junction, both of which could serve as splice acceptor sequences. Thus we hypothesize that the two spliced forms result from alternative use of two tandem splice acceptor sequences. Using reverse transcription polymerase chain reaction we examined expression of the two forms in 3 to 14-day chick embryos. All samples showed both species of RNA with approximately 2 times more of the larger from compared with the smaller form. PMID- 7542960 TI - Identification of L-methionine oxidation products in tripeptides, in Met enkephalin and in the bovine basic pancreatic trypsin inhibitor: 1H and 13C NMR study. AB - L-methionine (Met) oxidation products in tripeptides, in Met-enkephalin and in the bovine basic pancreatic trypsin inhibitor have been identified by 1H and 13C NMR spectroscopy. The oxidation of Met residues by stoichiometric amounts of chloramine-B, H2O2 and I2 yields a mixture of L-methionine sulfoxide (Met (O)) and dehydro-L-methionine (DH-Met), Met (O) and DH-Met, respectively, at pH 7.0 and 25.0 degrees C. The formation of DH-Met occurs only if the amino-group of Met is not derivatized. The analysis of 1H and 13C NMR spectra allows us to quantitate Met oxidation products, to ascertain the relative proportion of the R and S forms of Met (O) and DH-Met, and to reveal the presence of a Met residue at the N-terminal position in peptides and proteins. PMID- 7542961 TI - Free-beam and contact laser ablation of benign prostatic hyperplasia with the KTP/Nd:YAG laser: efficacy and versatility. AB - This report details the use of the free-beam Nd:YAG laser alone and in combination with contact vaporization for treatment of benign prostatic hyperplasia. Improvements in urinary flow rates and symptoms were noted along with a high degree of patient satisfaction. The portable KTP/Nd:YAG laser wavelengths and the variety of optical fiber configurations provide versatility to the surgeon. This study further supports the use of laser energy as a means to treat urinary outflow obstruction from prostatic enlargement in selected cases. PMID- 7542963 TI - Contact laser prostatectomy. AB - Laser prostatectomy is a recent technique that is evolving rapidly. As new techniques develop, it is important to discriminate between those that merely coagulate the prostate, those that vaporise the prostate and those that do a combination of both. Coagulation of the prostate alone results in prolonged obstruction and an increased incidence of urinary tract infection and post operative dysuria. Vaporisation alone can result in much more rapid dis obstruction but the procedure is relatively slow. For small prostates this is the treatment of choice. It has not been compared with transurethral incision of the prostate to date. This trial is indeed indicated. Hybrid procedures are increasing in popularity because they have the potential to combine the best aspects of both coagulation and vaporisation. There is relatively rapid coagulation of the bulk of the tissue but the vaporised channel allows more rapid dis-obstruction. This allows the patient to void more rapidly, thereby reducing the catheter time. The duration of dysuria appears to be reduced. These distinctions in the techniques of laser application and the treatment of prostatic outflow obstruction are important. PMID- 7542962 TI - Technique and results of interstitial laser coagulation. AB - By use of thermal "ablation" techniques to apply heat to the prostate from the urethra, the coagulation volume is limited by the limited penetration depth of suitable radiation sources, e.g., lasers, and by heat conduction. Secondarily, the coagulated tissue is removed by sloughing. Interstitial heat application was expected to overcome these problems. Our initial in vitro and animal studies using different light guides for interstitial Nd:YAG laser radiation showed that small, carbonized lesions were created by bare fibers, whereas large, homogeneous coagulation zones measuring up to 2 cm in diameter were produced by specially designed ITT (interstitial thermotherapy) fibers, which secondarily resulted in marked volume reduction by atrophy. Further experiments using such applicators resulted in an operation technique suitable for clinical routine in the treatment of symptomatic benign prostatic hyperplasia (BPH). These laser applicators are inserted into the prostate either transurethrally through a cystoscope under direct vision or percutaneously from the perineum under transrectal ultrasound guidance. The number of placements depends on the size and configuration of the gland. Radiation parameters were optimized for each system. To avoid charring, relatively low levels of laser power and long radiation periods (e.g., 7 W for 10 min) or power-formatting programs (e.g., stepwise reduction of power from 20 to 7 W for a total radiation time of 3 min) are applied. Beginning in July 1991, more than 350 unselected patients with BPH were treated with interstitial laser coagulation in our department, of whom 239 were followed for at least 1 year.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542964 TI - Transurethral laser ablation of the prostate--long-term results. AB - Continuing experience with the use of Nd:YAG laser-fibre prostatectomy over the past 4 years has produced encouraging results. Of the 198 patients we treated with the 60-W/60-s technique, 74 were available for evaluation after undergoing laser ablation of the prostate between October 1990 and July 1994. In all, 29/74 and 9/74 patients were available for 24 and 36 months of follow-up, respectively. The American Urological Association (AUA) symptom scores, peak uroflow (Qmax) rates and post-void residual urinary volumes show marked improvement. Following laser ablation of the prostate (LAP), complications have included 16 patients with post-procedural recurrent obstructive symptoms requiring revision. Three patients developed urinary tract infection, two developed bladder-neck stenosis, four fully anticoagulated patients with clot retention required blood transfusion and three patients had epididymo-orchitis. The results show a sustained reduction in AUA symptom scores and maintained improvement in peak flow rates and residual volumes, with stabilization of the above-mentioned parameters occurring within 12 36 months. The results demonstrate the continued durability of the outcome of LAP. PMID- 7542965 TI - Laser prostatectomy for patients with benign prostatic hyperplasia: a prospective randomized study comparing two different techniques using the Prolase-II fiber. AB - Laser prostatectomy for patients with complaints due to benign prostatic hyperplasia is a relatively new treatment option. The most effective procedure for coagulation and vaporization of the prostate is not yet known. In a prospective randomized study of 30 patients, 2 techniques for the delivery of laser energy were compared at 40 W for 90 s. The complications were minimal and antegrade ejaculation was preserved in 15 of 18 potent men. In 24 patients urodynamics evaluation was possible. In both groups a significant reduction in the symptom score was observed. The decrease in detrusor pressure at maximal flow and the increase in flow rate were, however, disappointing. No significant difference in the results was found between the two groups. The power setting needs to be changed in further studies. PMID- 7542967 TI - Evaluating the efficacy, safety, and cost of lasers for the treatment of benign prostatic hyperplasia. AB - This review highlights some of the caveats assessing the available clinical data base for determining the efficacy, safety, and cost of laser systems used to treat benign prostatic hyperplasia. Many new minimally invasive devices have recently been advanced in spite of the limited data base and the lack of properly designed clinical trials. There have been many claims regarding the effectiveness of laser prostatectomy based on the available clinical data. Some important general considerations for interpreting the clinical data base, critically analyzing the study design, and interpreting the data are discussed. Similarly, many claims have been made with respect to the cost of laser prostatectomy; however, insufficient objective data are available for clear determinations of cost. Some of the pertinent issues involved in the cost assessment of laser prostatectomy are also highlighted. It is hoped that this review will provide the reader with some additional insight into the critical assessment of the quality of studies that evaluate the safety, efficacy, and cost of new devices such as the laser. PMID- 7542968 TI - Urodynamics and laser prostatectomy. AB - A total of 81 patients with symptomatic bladder-outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH) underwent visual laser ablation of the prostate (VLAP) using a right-angled firing neodynium: YAG laser. The mean pre operative prostatic volume was 48.5 ml. All patients were discharged on the 1st post-operative day with an indwelling catheter. Two patients underwent a transurethral prostatectomy (TURP) after failing a trial without catheter on two occasions. Of the remaining 79 patients, 75 were evaluated 6 months post operatively. Mean symptom scores (I-PSS) decreased from 20.9 to 5.8, the mean maximal urinary flow rate increased from 7.9 to 16.4 ml/s and the mean residual volume decreased from 88.1 to 15.6 ml. Several different methods of evaluating BOO from pressure-flow measurements were used and all showed improvement. All the above-mentioned parameters showed a highly significant improvement (P < 0.01) at 6 months. PMID- 7542966 TI - A comparison of transurethral prostatectomy with visual laser ablation of the prostate using the Urolase right-angle fiber for the treatment of BPH. AB - The use of right-angle laser fibers for the treatment of benign prostatic hyperplasia (BPH) has gained widespread acceptance over the past several years. The number of right-angle fibers introduced into the marketplace has continued to grow, but most fibers have not been evaluated thoroughly with properly designed clinical trials. The Urolase fiber has undergone the most extensive clinical trials conducted to date. This report reviews the significant clinical trials with particular emphasis on the comparison of transurethral prostatectomy with visual laser ablation of the prostate. From these initial results, it would appear that laser ablation using the Urolase fiber is a safe and effective surgical therapy for men with symptomatic BPH. From our analysis, it would appear that transurethral prostatectomy provides a greater degree of symptomatic improvement as compared with transurethral resection of the prostate. It is important to consider that these are some of the earliest clinical trials assessing the efficacy of laser prostatectomy and that technological advancements are likely to improve the clinical outcome. PMID- 7542970 TI - Durability of laser fibers. AB - In 90 patients treated with laser prostatectomy using the Urolase (n = 50) or Ultraline (n = 40) laser fiber, the fiber-tip durability was investigated. In general the Urolase fiber tips were less damaged than the Ultraline fiber tips. At visual inspection, 62% of the Urolase fiber tips were graded as minimally damaged in comparison with 28% of the Ultraline group. The Urolase fiber tips are more susceptible than the Ultraline fiber tips to damage caused by tissue contact, whereas the latter seem more fragile. Transmission measurements were performed in a laboratory setting to estimate the loss of energy output at the fiber tip due to damage. These measurements showed a major loss in almost all fibers. None of the Ultraline fibers had less than 10% transmission loss, and 18% of the Urolaser fibers had a transmission value of more than 90%. Finally, there seemed to be a poor correlation between the visual aspects of the fibers used and the changes in transmission. PMID- 7542969 TI - The laser in the management of benign prostatic hyperplasia. PMID- 7542971 TI - Side-firing devices for laser prostatectomy. An overview. AB - Transurethral laser coagulation of the prostate has become an accepted treatment for benign prostatic hyperplasia (BPH). The most common method is the use of a sideward-firing fiber that, once inserted in the prostatic area, irradiates the abundant prostatic tissue with Nd:YAG laser light. In this study, eight different side-firing fibers that are commercially available were evaluated. The devices can be characterized by the way laser light is deflected sideward and by their thermal behavior. Most of the eight devices differ with regard to the angle at which the laser beam is deflected, the spot size on the irradiated tissue surface, and the heating of the device itself. Implementation of the optical and thermal characteristics of each device in the treatment protocol will contribute to the optimal use of laser energy for prostatectomy. PMID- 7542972 TI - TULIP: transurethral ultrasound-guided laser-induced prostatectomy. AB - Laser treatment has become increasingly attractive for treatment of bladder outlet obstruction due to benign prostatic hyperplasia (BPH). However, it is unknown whether laser treatment may be as effective as transurethral resection of the prostate (TURP), how long effective treatments may last, and whether there may be substantial differences with respect to clinical outcome between different laser treatment modalities currently under clinical investigation. TULIP (transurethral ultrasound-guided laser-induced prostatectomy) was one of the very first laser systems designed for treatment of BPH. The procedure is performed exclusively under transurethral ultrasound guidance, with which the surgeon has to become familiar. However, data reported from single-institution as well as multicenter studies demonstrate that the technique has only a small learning curve. The majority of patients treated by TULIP will experience substantial improvements in subjective and/or objective symptoms, which nevertheless do not appear to be as good as those seen after TURP. This is also shown by data from a prospective and randomized TULIP versus TURP study. However, it has to be emphasized that the clinical significance of such differences remains unknown. Advantageous for TULIP (which may be performed under analgosedation) are reduced blood loss, hospitalization, and rate of postoperative sexual dysfunction. Disadvantageous (as compared with TURP) are the delayed onset of success combined with the prolonged catheterization time and irritative symptoms in the early postoperative phase. In addition, as no tissue is obtained for histology examination, incidental prostate cancer may be missed. PMID- 7542974 TI - Leadership: taking a stand. PMID- 7542973 TI - Experience with the Ultraline and Urolase laser fibers: is there any difference? AB - Laser treatment of benign prostatic hyperplasia has enjoyed growing popularity among urologists over the last few years. Various applicators and techniques have been reported. Because this may result in a different overall performance, we performed a prospective randomized study comparing the results of treatment using the Ultraline fiber (n = 44) with that using the Urolase fiber (n = 49). Although different types of fibers and techniques were used, the results of this study were surprisingly similar for both fibers used. The uroflow for the Ultraline group increased from an average of 7.9 ml/s at baseline to 19.3 ml/s at 3 months and 16.9 ml/s at 6 months. In the patients treated with the Urolase fiber the uroflow improved from an average of 7.8 ml/s at baseline to 19.5 and 16.3 ml/s at 3 months and 6 months, respectively. The improvement in symptoms, reflected by changes in the I-PSS symptom scores, for the Ultraline group went from 21.0 at baseline to 7.9 at 3 months and 6.0 at 6 months. The Urolase patients improved from 21.0 at baseline to 8.2 and 5.6 at 3 and 6 month, respectively. The morbidity mainly consisted of a prolonged need for posttreatment catheterization and irritative symptoms lasting for about 2-4 weeks. From this study we conclude that the results achieved by laser treatment of the prostate using the Ultraline and Urolase fibers are both equivocal and excellent; however, the morbidity of these treatments remains considerable. PMID- 7542975 TI - A case of diabetic amyotrophy associated with 3243 mitochondrial tRNA(leu; UUR) mutation and successful therapy with coenzyme Q10. AB - We report the case of 71-year-old male who was once diagnosed as having diabetic amyotrophy, because of pronounced wasting in proximal muscles, massive weight loss, and development of paresthesia in his legs. Afterwards, ragged red fibers and mitochondrial tRNA mutation at position 3243 were documented in muscle biopsy. He had diabetes mellitus associated with 3243 mitochondrial DNA mutation, suggesting that clinically, diabetic amyotrophy may be overlapped with mitochondria-related disease entities in some parts. Coenzyme Q10 administration was effective in relieving the symptoms in his legs, fatigue, and residual urine in his bladder. These were confirmed with the improvement in neurological parameters. In conclusion, this case gives important help in understanding myopathy in diabetes. It would be important to check on the 3243 mitochondrial tRNA mutation in patients with diabetic amyotrophy and/or diabetic neuropathic symptoms. PMID- 7542976 TI - Nitric oxide is important for mouse beta-cell line killing by peritoneal exudate cells obtained from cyclophosphamide treated non-obese diabetic mice. AB - Macrophages from recent onset non-obese diabetic (NOD) mice showed cytotoxicity against the NOD mouse derived beta-cell line, MIN6N-9a. In this report, we examined whether nitric oxide is associated with beta-cell destruction. Peritoneal exudate cells (PEC), obtained from cyclophosphamide treated NOD mice showed higher cytotoxicity against MIN6N-9a compared to PECs from saline injected NOD mice (P < 0.01). This effect was suppressed in cells incubated with 0.5 mmol/l NG-methyl-L-arginine, a nitric oxide synthase inhibitor (P < 0.001). In addition, the nitrite concentration of the co-culture medium, as an index of nitric oxide production, increased in MIN6N-9a cells co-cultured with peritoneal exudate cells from cyclophosphamide injected NOD mice but not in co-culture with saline injected NOD mice (P < 0.05). Thus, nitric oxide plays an important role in beta-cell line destruction of macrophages obtained from NOD mice. PMID- 7542978 TI - BPH: The "forgotten" AHCPR clinical guideline. PMID- 7542979 TI - Glycoprotein hormones and pituitary adenomas. PMID- 7542977 TI - Presence of alternatively spliced-estrogen receptor mRNA variants in normal human uterine endometrium and endometrial cancer. AB - Presence of alternatively spliced-estrogen receptor (ER) mRNA variants has been revealed in the breast cancer tissues. The ER variants transcribed from these mRNA variants were supposed to cause changes in the estrogen responsiveness of breast cancer. Although uterine endometrial cancer also has an estrogen-dependent profile, these ER mRNA variants have not yet been reported in the tumor. In the present study, we attempted to detect the exon 7 deletion- (del.7-) and exon 5 deletion (del.5) ER mRNA variants in normal human uterine endometrium (hEM) and uterine endometrial cancer tissue (hEC) by the use of reverse transcription polymerase chain reaction-Southern blotting (RT-PCR-SB) with the PCR primers: hE4 (forward), hE6 (reverse), and hE8 (reverse), which were located in exons 4, 6, and 8, respectively. Two major products were generated from RNAs of both hEM and hEC with primers hE4 and hE8. The nucleotide sequence of the longer product was identical to exon 4-8 of human ER cDNA, whereas that of the shorter one completely deleted exon 7. Moreover, when the RT-PCR was done with the primers hE4 and hE6, the shorter product lacking exon 5 was detected with the longer one having the same sequence as exon 4-6 of human ER cDNA. Since the RT-PCR-SB with primers hE4 and hE8 produced a very low or undetectable level of the signals corresponding to del.5 ER mRNA variant, the level of del.7 ER mRNA variant seemed to be higher than that of del.5 ER mRNA variant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542980 TI - Glycoprotein hormone alpha-subunit production and plurihormonality in human corticotroph tumours--an in vitro and immunohistochemical study. AB - Glycoprotein hormone alpha-subunit (alpha SU) is a recognized product of clinically non-functioning, glycoprotein hormone-secreting and somatotroph adenomas but has not been studied systematically in corticotroph tumours. We have performed immunohistochemistry for alpha SU in a consecutive series of four corticotroph tumours causing Nelson's syndrome, three corticotroph macroadenomas, 12 corticotroph microadenomas and one adrenocorticotrophin-secreting bronchial carcinoid tumour. In addition we have assessed alpha SU secretion in vitro in corticotroph adenomas from two subjects with Cushing's disease and two subjects with Nelson's syndrome. Immunohistochemistry, performed after microwave treatment of sections to enhance antigen retrieval, demonstrated alpha SU positivity in 3/4 Nelson's tumours, 2/3 corticotroph macroadenomas, 7/12 microadenomas and one bronchial carcinoid. Eight of the 13 tumours positive for alpha SU were also immunostained after microwave pretreatment of sections for thyrotrophin (six positive), follicle-stimulating hormone (four positive), luteinizing hormone (three positive), beta-chorionic gonadotrophin (five positive), growth hormone (three positive) and prolactin (two positive) immunoreactivity. In vitro cell cultures of all four tumours studied secreted adrenocorticotrophin and three secreted alpha SU, with the variable presence of luteinizing hormone, follicle stimulating hormone, thyrotrophin, growth hormone and prolactin, in basal culture. The alpha SU secretion was augmented by phorbol ester (160 +/- 15%, SEM, n = 3 wells; p < 0.01) and 8-bromo-cAMP (138 +/- 8%; p < 0.05) in one tumour. These data indicate that plurihormonality and, in particular, alpha SU elaboration and secretion by corticotroph tumours is more common than hitherto recognized. Possible mechanisms include abnormal or deregulated gene expression, autocrine or paracrine effects or a stem cell origin of tumour.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542982 TI - Serum concentrations of insulin-like growth factors (IGFs), IGF binding proteins 1 and 3 and growth hormone binding protein in obese women and the effects of growth hormone administration: a double-blind, placebo-controlled study. AB - Obesity is associated with suppressed growth hormone (GH) concentrations but relatively little is known about insulin-like growth factors(IGFs) and binding proteins for GH and IGFs (GHBP and IGFBPs) and the modulatory effect of GH administration. In a double-blind, crossover design we studied the impact of 5 weeks of placebo or GH administration (0.03 mg.kg-1 body wt.day-1) in nine obese women (mean +/- SEM: age 30.4 +/- 2.4 years; body mass index 37.0 +/- 2.8 kg/m2) on IGF-I, IGF-II, IGFBP-1 and -3 and GHBP. Serum IGF-I (microgram/l) levels were subnormal and increased significantly following GH (117 +/- 16 (placebo) vs 434 +/- 33 (GH) vs 198 +/- 15 (control (p < 0.01)). By contrast, serum IGF-II (microgram/l) levels were in the normal range and remained unchanged (608 +/- 20 (placebo) vs 647 +/- 40 (GH) (NS)). Serum IGFBP-3 was in the normal range and increased significantly during GH treatment, although relatively less than IGF-I, such that the molar ratio between IGF-I and IGFBP-3 increased with GH treatment, whereas the ratio between IGF-I + IGF-II and IGFBP-3 remained unchanged. Serum IGFBP-1 was low in the placebo situation but became further and almost completely suppressed during GH treatment. During a 2-h hyperinsulinemic, euglycemic glucose clamp, IGFBP-1 decreased in the placebo study and remained suppressed during GH. Serum GHBP (nmol/l) levels were elevated substantially compared to non-obese controls (p < 0.001) and did not change during GH treatment (2.37 +/- 0.36 (placebo) vs 2.21 +/- 0.25 (GH) vs 0.80 +/- 0.19 (control)).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7542983 TI - [HLA-DR3 subtypes associated with immunization against HLA-1a antigen in families with alloimmune neonatal thrombopenia]. PMID- 7542981 TI - Serum levels of beta-subunit of chorionic gonadotropin in patients with pituitary tumors. AB - Many studies have shown that normal and tumoral pituitary is able to synthesize chorionic gonadotropin (CG). The aim of the present work was to investigate the circulating levels of free beta-subunit of CG (CG-beta) in a large number of patients with pituitary tumors in basal conditions and after thyrotropin releasing hormone (TRH) injection. The study includes 27 healthy subjects, 23 patients with prolactinoma, 20 with growth hormone-secreting adenoma and 77 with non-functioning pituitary adenoma (NFPA). The CG-beta was evaluated using a new one-step immunometric assay employing two monoclonal antibodies directed against epitopes present only on the free CG-beta and showing a detection limit of 0.04 U/l and a cross-reactivity with complete CG < 0.01%. In basal conditions, serum CG-beta was undetectable in healthy subjects and in the majority of patients, while in seven patients with NFPA and four with prolactinoma the CG-beta values ranged between 0.05 and 0.72 U/l. In these 11 patients serum levels of intact CG were found within the normal range (normal range < 5 U/l), while two patients with NFPA and one with prolactinoma had levels of free alpha-subunit inappropriately high with respect to gonadotropins and thyrotropin. Injection of TRH caused CG-beta to increase in two out of 16 patients with NFPA, whereas it was ineffective in 12 healthy subjects and 10 patients with prolactinoma. The present data indicate that detectable level of CG-beta not associated with hypersecretion of the intact CG molecule may be observed in about 10% of patients with NFPA or prolactinoma, while abnormal CG-beta responses to TRH are observed infrequently in individual patients with NFPA. PMID- 7542985 TI - The use of microcarrier beads in ion transport NMR studies of perfused cells. PMID- 7542984 TI - Determination of stereospecific assignments, torsion-angle constraints, and rotamer populations in proteins using the program AngleSearch. AB - A general program, AngleSearch, which calculates coupling constants and interproton distances for any molecular fragment and does a grid search to find torsion angles, rotamer populations, and stereospecific assignments which fit the measured data has been developed. The program takes full advantage of the fact that ratios of cross-peak intensities (measured in HNHB and HN(CO)HB experiments) can provide accurate ratios of coupling constants even for large molecules. AngleSearch is capable of: (a) analyzing any type of residue including protein, RNA, DNA, and ligand residues; (b) conformational grid searching in dihedral angle space using 6 degree steps; (c) averaging coupling constants and (l/r6) distances for rotamers undergoing fast exchange; (d) grid or Monte Carlo searching for populations of staggered rotamers; (e) using all available distance related data from ROESY and/or NOESY spectra; (f) using any available coupling constant data having known relationships to corresponding dihedral angles; and (g) directly using cross-peak intensities related to values of coupling constants. The program can also assist in the stereospecific assignment of the alpha-CH2 protons of glycine residues. The effects of the quality of the input data on the results of the AngleSearch calculations have been assessed. PMID- 7542986 TI - Recurrence of hepatitis C following liver transplantation: treatment with interferon. PMID- 7542987 TI - Biosynthesis of lipopolysaccharide O antigens. AB - Lipopolysaccharide O antigens are important virulence determinants for many bacteria. O-antigen synthesis is an interesting problem in cell-surface assembly. There are two known assembly pathways, which differ in the cellular location of their polymerization steps and in the direction of chain polymerization. Some reactions are shared with those for other surface polymers, such as capsular polysaccharides, and may be potential targets for therapeutic intervention. PMID- 7542989 TI - Klippel-Trenaunay syndrome: clinical features, complications and management in children. AB - Clinical features and management of 47 children with Klippel-Trenaunay syndrome treated since 1970 were reviewed. Haemangiomas and soft tissue and/or skeletal hypertrophy were present in all 47 patients; venous varicosities developed in 37 (79 per cent). There was no clinical evidence of macrofistulous arteriovenous communications in any patient. Thromboembolic episodes occurred in five children (11 per cent) and 25 (53 per cent) experienced thrombophlebitis. The Kasabach Merritt syndrome was observed in 21 (45 per cent) and six (13 per cent) presented with high-output cardiac failure. Other manifestations included haematuria in five (11 per cent), rectal or colonic haemorrhage in six (13 per cent), and vaginal, vulval or penile bleeding in six (13 per cent) children with visceral and pelvic haemangiomas. In 26 patients (55 per cent) symptomatic treatment only was required. Surgery was undertaken in selected cases for complications of the haemangioma, for cosmetic reasons and for chronic venous insufficiency. Only one of four children who underwent resection of varicose veins improved. There was no death, but significant morbidity was associated with the treatment of Kasabach Merritt syndrome and high-output cardiac failure. PMID- 7542988 TI - Rickettsia prowazekii, ribosomes and slow growth. AB - Some bacteria, such as Rickettsia prowazekii, grow slowly, not with anticipation of a future feast, but because it is evolutionarily advantageous to do so. This creates apparent paradoxes for understanding their physiology and biochemistry. These rickettsiae have a ribosome concentration higher than expected if these ribosomes support translation at rates comparable to those in Escherichia coli. PMID- 7542990 TI - SH3 domains. Minding your p's and q's. AB - SH3 domains mediate many important protein-protein interactions. The molecular basis of the binding of these domains to their ligands has been revealed, making it possible to identify SH3-binding sites in new proteins. PMID- 7542992 TI - [Efficacy and safety of loratadine vs terfenadine in the treatment of children with perennial allergic rhinitis. Capacity for inhibiting the positive cutaneous response after antihistaminic treatment]. AB - We studied 104 medical student from the UANL with symptoms of Perennial Allergic Rhinitis, including only 30 patients for this study; seven patients with Loratadine 10 mg, eight with Loratadine 20 mg, and 15 with Terfenadine 120 mg/day/21 days, 20 mg of Loratadine and 120 mg of Terfenadine demonstrated significant decrease of the symptoms (P > 0.005). Two patients had collateral effects with Terfenadine, but Terfenadine were more effective than the Loratadine in the reduction of the skin reactivity. PMID- 7542991 TI - A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. AB - BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together. PMID- 7542994 TI - Tetrahydro-9-aminoacridine presynaptically inhibits glutamatergic transmission in the rat amygdala. AB - The effect of the centrally active anticholinesterase inhibitor tetrahydro-9 aminoacridine (THA) on synaptic transmission was studied in rat amygdala neurons in the in vitro slice preparation. THA reversibly suppressed the excitatory postsynaptic potential (EPSP) in a concentration-dependent manner. Postsynaptic depolarization induced by alpha-amino-5-methyl-4-isoxazole propionate (AMPA) was not decreased by THA. These results demonstrate that THA has a presynaptic inhibitory action on the physiology of synaptic transmission in the amygdala. Pretreating the slices with atropine did not affect THA's effect, indicating that the presynaptic muscarinic receptors are not involved. PMID- 7542993 TI - Glutamic acid induces luteinizing hormone releasing hormone release via alpha receptors. AB - Glutamic acid (GA) and norepinephrine (NE) stimulate luteinizing hormone releasing hormone (LHRH) release via release of nitric oxide (NO) from NOergic neurons in the arcuate-median eminence region. To determine if GA releases LHRH via direct stimulation of NOergic neurons, or via stimulation of noradrenergic terminals, arcuate median eminence explants from male rats were incubated with various compounds, and the LHRH release into the medium was measured. GA-induced release of LHRH was completely blocked by phentolamine (1 microM), an alpha receptor blocker, which, by itself, had no effect on the release. Nitroprusside (NP), which spontaneously releases NO, more than doubled LHRH release. To determine if alpha receptors on the LHRH neuron are required for the action of NP, the tissue was incubated with phentolamine, plus NP. Phentolamine had no effect on the LHRH-releasing action of NP. The results are interpreted to mean that GA activates the release of NE from the noradrenergic terminals. This acts on alpha receptors on the NOergic neuron to produce the release of NO. This NO diffuses to the LHRH terminals and induces release of LHRH. PMID- 7542995 TI - Macroamylasemia associated with ulcerative colitis. AB - We describe a very rare case in which macroamylasemia was associated with ulcerative colitis of total colitis type. The patient's serum amylase isozyme pattern by electrophoresis showed a broad abnormal peak toward the side of the positive pole compared with regular salivary and pancreatic fractions. Sephadex G 200 column chromatography showed a sedimentation coefficient of 6.6 S. Amylase activity was bound to IgG. Double diffusion experiments demonstrated that amylase activity could be precipitated in gel by an antibody to the lambda chain. Although inflammatory bowel disease is occasionally associated with hyperamylasemia due to pancreatitis, we emphasize that, when hyperamylasemia is recognized in patients with inflammatory bowel disease, macroamylasemia also should be considered. PMID- 7542996 TI - Interferon-induced depression: just one of Bonhoeffer's exogene Reaktionstypen or a clue to understanding psychoimmunological aspects of depression? PMID- 7542998 TI - Iron overload augments the development of atherosclerotic lesions in rabbits. AB - Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n = 8), iron-overloaded (group B, n = 6), hypercholesterolemic (group C, n = 6), and untreated (group D, n = 6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P < .05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits. PMID- 7542997 TI - Enhanced revascularisation after angiogenic stimulation in a rabbit model of bilateral limb ischaemia. AB - OBJECTIVE: We previously demonstrated stimulation of collateral vessel formation in a rabbit model of unilateral limb ischaemia after administration of endothelial cell growth factor (ECGF). To distinguish clearly the effects of ischaemia alone from those of ischaemia combined with angiogenic stimulation in the same animal, a model of bilateral hindlimb ischaemia was used to evaluate further the angiogenic effect of ECGF. DESIGN: Ischaemia was produced in both hindlimbs of 11 rabbits by femoral artery excision. Beginning 10 days later, ECGF (8 mg in 3 ml of saline) was injected in one hindlimb while 3 ml of saline alone was injected in the other every other day for a total of five doses. OUTCOME MEASURES: Calf systolic blood pressure was measured in both limbs on postoperative days, 10, 30, and 50. On day 50, collateral formation was quantitated angiographically, and muscle samples were obtained for quantitation of capillary density and histologic studies. RESULTS: The mean calf systolic blood pressure in the both hindlimbs was similar on day 10 (36.9 +/- 2.3 versus 38.1 +/- 2.9 mmHg) but was significantly higher in the ECGF-treated limb on day 30 (68.9 +/- 3.1 versus 45.0 +/- 2.9 mmHg) and day 50 (83.0 +/- 3.0 versus 57.0 +/- 1.7; p < 0.0001 for both comparisons). On day 50, collateral vessels were significantly more numerous in the ECGF-treated limb (17.2 +/- 1.6 versus 11.0 +/ 0.8; p < 0.0006), as were capillaries (225.9 +/- 11.4 versus 159.6 +/- 12.9 per mm2; p < 0.002). CONCLUSION: Local administration of ECGF enhanced collateral development leading to significantly improved perfusion in the treated as compared with the untreated limb in the same animal. Exogenous administration of an angiogenic mitogen can upregulate the normal collateral response to ischaemia and may be useful in treating severe limb ischaemia. PMID- 7543000 TI - Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1. AB - The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGF beta 1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF beta 1 increased ecNOS mRNA in a dose-dependent manner. TGF beta 1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion were increased in TGF beta 1 treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGF beta 1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGF beta 1 response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGF beta 1. In conclusion, TGF beta 1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter. PMID- 7543001 TI - Acute-phase reactants as tumor markers. PMID- 7543002 TI - Snake-like chromatin in conjunctival cells of normal elderly persons and of patients with primary Sjogren's syndrome and other connective tissue diseases. AB - The aim of this study was to investigate the prevalence of snake-like chromatin in the nuclei of conjunctival cells of normal elderly persons, and to compare with findings in patients with connective tissue diseases other than primary Sjogren's syndrome and in age-matched patients with primary Sjogren's syndrome. Thirty-nine per cent of the eyes of normal controls contained snake-like chromatin. Snake-like chromatin has almost never been found in young normals, and the results of this study therefore raise the question of whether age may be a contributing factor. Snake-like chromatin was as frequent in patients with other connective tissue diseases as in patients with primary Sjogren's syndrome, and no correlation to the presence of keratoconjunctivitis sicca was found in this group. The presence of snake-like chromatin was significantly correlated to increased cell size (N/C ratio) and low goblet cell density. A marked correlation between snake-like chromatin and low values of break-up time was found in normals, whereas no correlation was found to the other standard tests for dry eye. PMID- 7543004 TI - Ocular hemodynamic changes in patients with high-grade carotid occlusive disease and development of chronic ocular ischaemia. II. Clinical findings. AB - Ocular changes due to chronic ischaemia are described in 45 patients with hemodynamically significant carotid occlusive disease. Regarding the ophthalmic artery flow before and after vasodilatory stimulus, the patients were divided into four different groups. Eyes with permanent anterograde ophthalmic artery flow had evidence of embolic features, but no signs of chronic hypoperfusion. In the group where anterograde ophthalmic artery flow changed to retrograde after 1 g intravenous acetazolamide, the patients had symptoms of critical ocular perfusion, but only subtle structural changes could be demonstrated. Chronic ocular ischaemic syndromes were found in the cases with retrograde ophthalmic artery flow, especially where bilateral severe carotid obstruction was present. PMID- 7543003 TI - In vitro studies of conjunctival cells from eyes with and without pseudoexfoliation. AB - The aim of the study was to examine for possible differences between cell cultures derived from eye with and without pseudoexfoliation. In both populations, scanning electron microscopy showed flattened epithelial cells and also spindleshaped fibroblast-like cells. The presence of these cell types was further confirmed by immunohistochemical demonstration of cytokeratin and vimentin in the cultured cells. The cells maintained in vitro showed a linear increase in uptake of leucine during a 12-h period. Within this period, the leucine recovered in the TCA precipitable fraction was considerably higher than the nonbound fraction. In cultures maintained in medium with and without L ascorbic acid, the presence of L-ascorbic acid significantly increase the uptake of leucine into TCA precipitable material, and to a similar extent in cultures from the two populations. In conclusion, cells derived from eyes with and without pseudoexfoliation material and maintained in vitro showed similar morphology, presence of intermediate filaments, as well as uptake of leucine under various culture conditions. PMID- 7542999 TI - Immunoselective targeting of an anti-thrombin agent to the surface of cytokine activated vascular endothelial cells. AB - An immunoconjugate was designed to target hirudin, a potent and specific inhibitor of thrombin, to the surface of activated endothelial cells. Hirudin was covalently cross-linked to the monoclonal antibody H18/7 that recognizes the extracellular domain of E-selectin (CD62E), an endothelium-leukocyte adhesion molecule that is expressed only on cytokine-activated endothelium. The hirudin H18/7 immunoconjugate selectively bound to interleukin-1-activated but not to unactivated cultured human umbilical vein endothelial cells with a temporal profile similar to that of inducible cell-surface procoagulant activity. When bound to activated endothelial cells, the hirudin-H18/7 immunoconjugate significantly inhibited endogenous thrombin activity generated from coincubated human plasma and fibrin clot formation on the monolayer surface. Cellular responses that are mediated via the thrombin receptor, such as increases in cytoskeletal F-actin content, also were significantly downregulated, and monolayers were protected from thrombin-induced disruption by this treatment. The ability to selectively antagonize thrombin-dependent processes at the endothelium blood interface may provide new insights into complex pathophysiological processes, such as thrombosis, inflammation, and atherogenesis. These studies also demonstrate the general feasibility of selective targeting of therapeutic agents to endothelial cells based on recognition of an activation-dependent surface phenotype. PMID- 7543005 TI - Action of quail and chicken interferons on a quail cell line, QT35. AB - Production of interferon (IFN) in quail cells (QT35) and the activity of quail IFN and heterologous avian IFN (chicken) on QT35 cells were examined. Quail cells produced IFN after induction by bluetongue virus serotype 10; chicken and quail IFN conferred antiviral resistance on the quail cells. Both chicken and quail IFN induced increased levels of 2',5'-oligoadenylate synthetase (2',5'-OAS) in QT35 cells and reduced levels of intracellular Salmonella typhimurium postchallenge. These results indicate that the quail cells produce IFN and respond to homologous and heterologous avian IFN as evidenced by enhanced (1) resistance to viral infection, (2) production of 2',5'-OAS, and (3) resistance to invasion by bacteria. QT35 quail cell monolayer cultures offer an alternative to primary chicken embryo fibroblasts cultures used for avian IFN studies. PMID- 7543008 TI - [Significance of increased alpha fetoprotein in gastric cancer]. PMID- 7543007 TI - [Nitric oxide: a nonadrenergic, noncholinergic mediator in the digestive system]. PMID- 7543006 TI - Drug sensitivity ("suicide") genes for selective cancer chemotherapy. PMID- 7543009 TI - Role of prostate-specific antigen and digital rectal examination in the detection of prostate cancer. AB - Prostate-specific antigen (PSA) is a kallikrein-like serine protease that is secreted exclusively by the epithelial cells of all types of prostatic tissue, benign and malignant. Its serum concentration is raised in men with prostatic disease including cancer. We have evaluated its usefulness in the diagnosis of prostate cancer by measuring serum PSA concentrations in 260 men aged 50 years or over. All had abnormalities at digital rectal examination (DRE) involving suspected cancer, signs and symptoms of benign prostatic hyperplasia and equivocal findings on DRE, and miscellaneous other conditions, including hematospermia, chronic prostatitis and microscopic hematuria. Transrectal prostatic needle biopsies were performed in the men with abnormal findings on DRE or elevated serum PSA (above 4 ng/ml). Serum PSA ranged from 4.0 to 9.9 ng/ml in 14 (5%) of the 260 men. Four of the men in this group (31%) who underwent prostatic biopsy had prostate cancer. Serum PSA levels greater than or equal to 10.0 ng/ml were found in 8 (3%) of the 260 men. 5 of these 8 (63%) who underwent prostatic biopsy had cancer. If DRE alone had been used to screen the men having biopsies, 4 of the 10 cancers (40%) would have been missed. If PSA alone had been used to screen these men, only 1 of the 10 cancers would have been missed. Serum PSA measurement was more reliable than DRE for detecting prostate cancer. Since these two methods do not always detect the same malignant tumor, the combined use of DRE and PSA testing affords a more complete evaluation of the prostate gland for malignant involvement. PMID- 7543010 TI - Schedule-intensified M-VAC chemotherapy for advanced urothelial cancer with recombinant human granulocyte colony stimulating factor (rhG-CSF). AB - M-VAC (Methotrexate, vinblastine, adriamycin and cisplatin) combination systemic chemotherapy is useful for treating invasive or metastatic transitional cell carcinoma. Granulocytopenia is the major dose-limiting factor of this chemotherapy and it takes 4 weeks or more to complete a single course of M-VAC. We have tried to shorten the period of M-VAC chemotherapy from 4 to 3 weeks by using rhG-CSF. With this modified M-VAC regimen, the number of days on which the absolute neutrophil count was less than 1000/mm3 was significantly reduced and the period to reach the neutropenia nadir was shortened. No severe side-effects were observed. In all patients treated with 2 courses of this modified M-VAC short regimen, the period of hospitalization could be reduced by 2 weeks. We emphasize the possibility of shortening the M-VAC regimen. PMID- 7543011 TI - Thermal damage to normal and hyperplastic prostates due to transurethral balloon laser therapy in dogs. AB - Fourteen dogs were studied to determine the level of thermal damage to the normal or benign hyperplastic prostate. Prostates were heated transurethrally by a balloon laser probe (Nd: YAG laser) at various temperatures for 20 min. Coagulative necrosis was observed in the prostate when it was heated above 45.4 degrees C for 20 min. Tissue damage in the bladder neck occurred above 49.4 degrees C, damage to the urethral mucosa occurred above 46.5 degrees C, and damage to the urethral sphincter occurred above 43.2 degrees C. It was calculated that there was a 50% probability of necrosis of the prostate gland occurring when prostatic tissue was treated at 45.0 degrees C for 20 min. Striated muscle cells in the urethral sphincter were more heat-sensitive than smooth muscle cells of the bladder neck or transitional cells of the urethral mucosa. This basic information is essential for the safe application of Nd: YAG laser hyperthermia to the prostate gland. PMID- 7543013 TI - Prostate-specific antigen as a tumor marker in prostate cancer. PMID- 7543012 TI - Clinical experience with yolk sac tumors and teratoma in children. AB - Between 1971 and 1993, 12 children with testicular germ cell tumors were treated at the Department of Urology, Faculty of Medicine, Kyoto University. Seven patients had yolk sac tumors and 5 had mature teratoma. Of the 7 patients with yolk sac tumors 6 had stage I and 1 had stage III tumors. Initial management of the stage I tumors consisted of high orchiectomy in 5 patients and high orchiectomy plus retroperitoneal lymph node dissection in 1 patient. Of these 6 patients, 4 were cured by surgery alone but lung metastases developed in the other 2 patients. One of them was salvaged with thoracotomy and chemotherapy but the other died of tumor. The patient with stage III tumor had bulky tumor spread to lung and retroperitoneum, but seems to have been cured by chemotherapy followed by resection of the residual mass although follow-up is still inadequate (14 months). Six of the 7 patients (85.7%) are alive 13 months to 21 years after diagnosis. Five patients with mature teratoma were treated by high orchiectomy or, more recently, enucleation and all are alive 4 months to 22 years after surgery. PMID- 7543014 TI - Aggressive pleomorphic CD2+, CD3-, CD56+ lymphoma with t(5;9)(q31;q34) abnormality. AB - CD56-positive lymphoma is a recently described entity which is characterized by predominantly extranodal involvement and an aggressive clinical course. We report one such case with involvement of the bone marrow and spinal cord at presentation, and associated with reactive hemophagocytic syndrome. The lymphoma cells had a highly pleomorphic appearance which is uncommon in CD56-positive lymphoma. Cytogenetic studies revealed a t(5;9)(q31;q34) abnormality. Analysis of more cases is required to determine if this is a recurring chromosomal translocation characteristic of the group of aggressive CD56-positive lymphoma. PMID- 7543015 TI - Human prostatic cancer cells are sensitive to programmed (apoptotic) death induced by the antiangiogenic agent linomide. AB - Human prostatic cancer cells have a remarkably low rate of proliferation even when they have metastasized to the bone and have become androgen independent (Berges et al., Clin. Cancer Res., 1:473-480, 1995). Due to this low proliferation, patients with such androgen-independent metastatic prostatic cancer cells are rarely treated successfully with the presently available chemotherapeutic agents. Therefore, new approaches are urgently needed which are not dependent on the rate of cancer cell proliferation for their effectiveness. One such approach is to inhibit the angiogenic response within localized and metastatic cancer deposits, since the resultant hypoxia-induced tumor cell death does not require cell proliferation. We have previously demonstrated that the quinoline-3-carboxamide, linomide, is an p.o. active agent which inhibits tumor angiogenesis and thus blood flow in a variety of rat prostatic cancers independent of their growth rate, androgen sensitivity, or metastatic ability. Because of its antiangiogenic effects, linomide treatment induces the hypoxic death of rat prostatic cancer cells, thus inhibiting their net growth and metastases. To determine whether human prostatic cancer cells are similarly sensitive to hypoxia-induced death caused by linomide inhibition of tumor angiogenesis, androgen-independent TSU and PC-3 human prostatic cancer cells were xenotransplanted into SCID mice that were either untreated or treated p.o. with linomide. These studies demonstrated that linomide treatment decreases microvessel density in both androgen-independent human prostatic cancers. Microvessel density was decreased from 1.8 +/- 0.4% of the total area in control tumors to 1.0 +/- 0.2% in linomide-treated TSU tumors [i.e., a 44% decrease in microvessel density (P < 0.05)]. Similarly, a 56% decrease (P < 0.05) was observed in the microvessel density of PC-3 tumors (i.e., 2.7 +/- 0.8% of the area in control tumor versus 1.2 +/- 0.2% in the linomide-treated tumors). This inhibition of angiogenesis increased cell death in both TSU and PC-3 cancer cells. This is reflected in both an increase in the area of necrosis and an increase in the apoptotic index in non-necrotic areas. In untreated TSU tumors, 40 +/- 2% of tumor volume was necrotic. Linomide treatment increased this necrotic percentage to 59 +/- 2% [i.e., 48% increase (P < 0.05)]. Linomide therapy also increased apoptotic cell death in non-necrotic tumor areas. In the untreated TSU tumors, 2.9 +/- 0.6% of tumor cells were apoptotic in the non necrotic areas, and in the linomide-treated TSU tumors this percentage increased to 3.6 +/- 0.4% [i.e., 24% increase (P < 0.05)].(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543016 TI - Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. AB - Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells. PMID- 7543018 TI - Alpha-fetoprotein (AFP) expression in clones of McA-RH 7777 rat hepatoma: correlation with the occurrence of homogeneously staining regions on chromosome 14. AB - alpha-Fetoprotein (AFP) is a well-established cell differentiation and tumor marker. We showed previously that McA-RH 7777 hepatoma cells are heterogeneous in terms of their AFP cellular expression. In the present study, we developed stable and unstable 7777 hepatoma clones in terms of their AFP phenotype: AFP-producing (AFP+) or AFP-nonproducing (AFP-) clones, and investigated in these clones (a) AFP phenotype related to protein and mRNA levels; (b) cellular morphology; and (c) expression of several liver-specific markers. Our results demonstrated that alpha-albumin expression paralleled that of AFP, from the absence of alpha albumin message in AFP- clones to high expression in AFP+ clones, suggesting that common mechanisms control the expression of both proteins in this hepatoma cell population. In addition, the karyotypes of the McA-RH 7777 hepatoma cell line and its 15 generated clones were analyzed and correlated to their AFP phenotypes. Only the stable AFP+ clones showed homogeneously staining regions on the chromosome carrying the AFP gene. These results strongly suggest that amplification of either structural or regulatory sequences of the AFP gene is involved in maintaining its high expression. PMID- 7543017 TI - Admixture of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor associated antigen gene results in enhanced specific T-cell responses and antitumor immunity. AB - At least two signals are required for the activation of naive T cells by antigen bearing target cells: an antigen-specific signal, delivered through the T-cell receptor, and a costimulatory signal delivered through the T-cell surface molecule CD28 by its natural ligand B7-1. The immunological benefit of coexpression of B7 with target antigen has been demonstrated with the use of several retroviral systems to transfect antigen-bearing cells. Although engineering recombinant constructs with genes for two or more antigens can mediate the dual expression of those antigens, disadvantages of this approach include the time for construction of each desirable combination and the inability to control differential expression levels of each gene product. An alternative approach would utilize separate constructs that could be admixed appropriately before administration. In this report we describe the functional consequences of the admixture of recombinant vaccinia murine B7-1 (rV-B7) to recombinant vaccinia expressing the human carcinoembryonic antigen gene (rV-CEA). Coinfection of cells resulted in high levels of cell surface expression of both the CEA and B7 molecules. Immunization of mice with various ratios (1:3, 1:1, 3:1) of rV-CEA and rV-B7 demonstrated that an admixture of rV-CEA and rV-B7 at a 3:1 ratio resulted in the generation of optimal CEA-specific T-cell responses. Next, we examined the efficacy of this admixture on antitumor activity. Typically, injection of murine carcinoma cells expressing CEA leads to the death of the host. One immunization of C57BL/6 mice with rV-CEA:rV-B7 (3:1) resulted in no tumor establishment. In contrast, administration of rV-CEA or rV-B7 alone had little or no antitumor effects. These studies demonstrate the advantages of the use of recombinant vaccinia viruses to deliver B7 molecules in combination with a tumor-associated antigen. The availability of the rV-B7 single construct and the ability to alter the B7 ratio could also have potential utility when coinfecting rV-B7 with recombinant vaccinia viruses containing genes for infectious agents or other tumor-associated antigen genes. PMID- 7543019 TI - Adhesion molecules on human myeloma cells: significant changes in expression related to malignancy, tumor spreading, and immortalization. AB - In order to evaluate putative changes of major adhesion molecule expression on plasma cells (PCs) associated with malignant transformation, tumor spreading, and immortalization, we have quantified and compared the expression of CD56, CD44, CD11a, CD49e, and CD45 RO/RA on normal PCs, malignant PCs from multiple myeloma patients in chronic phase, in accelerated phase with or without extramedullary progression, and from human myeloma cell lines. Plasma cell phenotype was defined with the use of two-color immunofluorescence in combination with B-B4 or anti CD38 antibodies. We found that all the adhesion antigens were expressed on normal PCs. Malignancy was characterized by an overexpression of CD56, whereas extramedullary spreading was associated with a dramatic down expression of CD56. Although CD44 remained unchanged, the subpopulation of PCs expressing CD11a, CD49e, and CD45RA/RO were significantly reduced during malignancy, and each of these negative subpopulations increased during disease acceleration. We demonstrated that CD11a and CD49e expression were correlated and defined the same subpopulation of PCs. The phenotype of HMCLs was similar to the expression profile of patients in accelerated phase with extramedullary spreading. In conclusion, we show that significant changes of PC phenotype were associated with malignancy, were correlated with the disease evolution, and could be of diagnostic and prognostic value in individuals with monoclonal gammopathy and patients with multiple myeloma. PMID- 7543020 TI - Ultraviolet B irradiation promotes tumorigenic and metastatic properties in primary cutaneous melanoma via induction of interleukin 8. AB - UV radiation has been shown to play a role in the initiation of human cutaneous melanoma, but its role in the development of malignant melanoma to the metastatic state is not very well defined. Although previous studies have concentrated on the effect of UV-B on the host immune response, the effect of UV-B on the tumor cells was not elucidated. Here we show that UV-B can induce interleukin 8 (IL-8) mRNA and protein secretion in human cutaneous melanoma with negligible expression of IL-8. UV-B-induced IL-8 was constitutively expressed 60 days after irradiation in tumors implanted in mice. Induction of IL-8 was UV-B dose dependent and blocked by cyclohexamide, indicating that de novo protein synthesis is required for its expression. The UV-irradiated cells demonstrated enhanced tumorigenicity and metastatic potential in nude mice. The increase in tumorigenicity and metastatic ability could be explained by the increase in Mr 72,000 type IV collagenase activity and angiogenesis attributed to the induction of IL-8 after irradiation. The acquisition of the metastatic phenotype induced by UV-B could not be attributed to abnormalities in the p53 or MTS-1 (p16INK4) genes. To the best of our knowledge, this is the first report to show that UV-B can increase the aggressiveness of human cutaneous melanoma for growth and metastasis. PMID- 7543021 TI - CD8 T cell activation after intravenous administration of CD3 x CD19 bispecific antibody in patients with non-Hodgkin lymphoma. AB - A bispecific antibody directed to T and B cells (CD3 x CD19 bsAb) was daily infused intravenously in escalating doses from 10 micrograms up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed a t1/2 of 10.5 h with peak levels of 200-300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor alpha was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon gamma. IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1 beta in serum). No gross changes in histology or number of IL-2R+, CD4+ or CD8+ cells were found in the lymph nodes after therapy, but one patient showed activated CD8+ T cells within the tumour nodules. In conclusion, after intravenously administered CD3 x CD19 bsAb only moderate toxicity was found, probably due to CD8+ T cell activation and cytokine release, without CD4+ T cell activation. PMID- 7543022 TI - Induction of tumor-specific T lymphocyte responses in vivo by prothymosin alpha. AB - We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy. PMID- 7543023 TI - Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. AB - CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating. PMID- 7543024 TI - Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. AB - Stat1 and Stat3 are latent transcriptional factors activated initially through phosphorylation on single tyrosine residues induced by cytokine and growth factor occupation of cell surface receptors. Here we show that phosphorylation on a single serine (residue 727) in each protein is also required for maximal transcriptional activity. Both cytokines and growth factors are capable of inducing the serine phosphorylation of Stat1 and Stat3. These experiments show that gene activation by Stat1 and Stat3, which obligatorily require tyrosine phosphorylation to become active, also depends for maximal activation on one or more of the many serine kinases. PMID- 7543027 TI - Cardiac arrest after intrauterine instillation of 32% dextran-70 during hysteroscopy. PMID- 7543026 TI - LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation. AB - The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD. PMID- 7543028 TI - Taxol encapsulation in poly(epsilon-caprolactone) microspheres. AB - Poly(epsilon-caprolactone) (PCL) microspheres containing taxol were prepared by the solvent evaporation method and tested for angiogenesis inhibition using the chick chorioallantoic membrane (CAM) model. Very high encapsulation efficiencies (95%) for taxol in PCL microspheres were obtained. In vitro release studies showed about 25% of the loaded drug was released in 6 weeks from microspheres containing 5% taxol. Studies with the CAM showed that taxol released from the microspheres induced vascular regression and inhibited angiogenesis. PMID- 7543025 TI - Histidine phosphorylation of P-selectin upon stimulation of human platelets: a novel pathway for activation-dependent signal transduction. AB - Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells. To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin. P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine. We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin. With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance of P-selectin are very rapid. Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine. These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling. PMID- 7543029 TI - Prolonged daily administration of oral etoposide in lymphoma following prior therapy with adriamycin, an ifosfamide-containing salvage combination, and intravenous etoposide. AB - Prolonged daily administration of oral etoposide has been reported to be active in refractory lymphoma. The purpose of this phase II trial was to confirm the activity of this schedule of etoposide in a selected group of heavily pretreated patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). A total of 26 patients (20 with NHL and 6 with HD) were entered in the trial; all had previously been treated with an Adriamycin-based chemotherapy, an ifosfamide containing salvage combination, and i.v. etoposide. Etoposide was given in a fixed oral daily dose of 100 mg over 3 weeks; the weekly dose (500-700 mg) was selected such that the average daily dose was approximately 50 mg/m2. Cycles were repeated on day 29. An objective response was seen in 16 patients (62%; 95% confidence interval, 42%-80%), with a complete response (CR) being observed in 3 cases (12%) and a partial response (PR), in 13 (50%). The median duration of PRs was 3 months. CRs lasted for 15 months in one patient and continue at 12+ and 20+ months in the remaining two patients. The overall actuarial survival for the entire group was 40% at 2 years; the median survival time was 12 months. The main toxicity was myelosuppression; WHO grade 3 or 4 leukopenia and thrombocytopenia developed in 31% and 12% of the patients, respectively. There was no drug-related death. We conclude that oral etoposide is an effective and tolerable palliative treatment for heavily pretreated lymphoma patients. PMID- 7543030 TI - Comparison of binding specificity and the function of two human IgM anti-lipid A monoclonal antibodies. AB - The interactions of two anti-lipid A monoclonal antibodies (mAb)--HA-1A and SdJ5 1.17.15--with their antigenic sites on lipid A, were compared using a dot-blot assay and lipid A structural analogues, as well as lipid A-high-density lipoprotein (HDL) complexes. The reactivities of both mAb were affected by the type of fatty acid side chains and by the phosphate group on the glucosamine residue II; however, the interaction of SdJ5-1.17.15 appeared to be more markedly affected by the fatty acid side chains. A determination of the biological significance of these antigenic differences was made. Human peripheral blood mononuclear cells (hPBMC) challenged with Escherichia coli 055:B5 lipopolysaccharide (LPS) pre-incubated with SdJ5-1.17.15 released significantly less tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), compared to hPBMC exposed to vehicle preincubated LPS. HA-1A did not attenuate the in vitro release of either cytokine. The ability of both mAb to neutralize the in vivo toxicity of LPS was also evaluated. Rats administered E. coli 055:B5 pre-incubated with SdJ5-1.17.15 had a significantly reduced 24-hr mortality rate compared to vehicle controls. HA-1A did not attenuate the in vivo mortality rate. Therefore, the reactivity of anti-lipid A mAb with the antigen is preferentially affected by different residues on the lipid A moiety. Thus, the differences in biological activity seen with SdJ5-1.17.15 and HA-1A may be due in part to differences in their recognition sites on lipid A. PMID- 7543031 TI - Hypertonic saline/dextran versus lactated Ringer's treatment for hemorrhage in dehydrated swine. AB - To determine the efficacy of low-volume resuscitation in dehydrated subjects, 7.5% hypertonic saline/6% dextran 70 (HSD) and lactated Ringer's (LR) treatments were compared in conscious pigs dehydrated for 48 hr prior to a 37% blood volume hemorrhage. Pigs randomized to treatment were resuscitated with equivalent sodium loads of either HSD (4 ml/kg) or LR (33.3 ml/kg) following the 60-min hemorrhage. Dehydration resulted in a 7-8% body weight loss. Mortality through 180 min of recovery was 1/13 (7.7%) for HSD, 1/11 (9.1%) for LR, and 4/8 (50%) for a group of dehydrated and untreated controls (DC). HSD and LR solutions elicited similar heart rate, cardiac output, arterial pressure, and oxygen transport responses in the recovery period following hemorrhage and resuscitation. HSD was as effective as LR in expanding plasma volume in dehydrated hemorrhaged pigs. Serum chemistries provided no evidence for a sustained systemic toxicity from HSD treatment. These findings support low-volume HSD resuscitation of hemorrhagic shock in moderately dehydrated subjects. PMID- 7543032 TI - Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification coupled with HPLC. AB - We describe a new strategy for quantifying mRNA by using polymerase chain reaction (PCR) coupled with HPLC. PCR-amplified products are directly analyzed with a specific HPLC column and are quantified by standardization against a housekeeping gene, beta-actin. To evaluate the experimental conditions, we examined the multidrug-resistance-associated mdr1 gene expression in two drug sensitive cell lines (RPMI-8226 and KB-3-1) and in their drug-resistant sublines. This revealed a significant overexpression of the mdr1 gene in chemoresistant sublines that paralleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods. Results were consistently reproducible, with imprecision (CV) < 25%. The reverse transcription option PCR/HPLC protocol described here is a sensitive, nonradioactive method for quantifying mdr1 mRNA in cell lines or clinical tumor samples, and can be applied to the simultaneous quantification of several mRNA species. PMID- 7543033 TI - Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum. AB - We developed a simple one-step dual-label immunoassay for simultaneous measurement of the free, noncomplexed form of prostate-specific antigen (PSA) and total PSA. The assay is based on time-resolved fluorescence and includes a stable fluorescent chelate of Eu to label a monoclonal antibody (mAb) that detects only free PSA, whereas a second mAb labeled with a fluorescent chelate of Tb provides equimolar detection of both free PSA and PSA complexed to alpha 1 antichymotrypsin. A third mAb on a solid phase captures the free and complexed forms of PSA in an equimolar fashion. The simultaneous measurement of the free-to total PSA ratio (F/T) with the one-step dual assay is not sensitive to variations in the sample volume. The discrimination between benign prostatic hyperplasia and prostate cancer patients, i.e., the area under the receiver-operating characteristic curve, increased from 0.64 (total PSA assay) to 0.78 and 0.81 when the F/T ratio was measured with single and dual assays, respectively. PMID- 7543036 TI - A case of Marden-Walker syndrome with Dandy-Walker malformation. AB - A 5-month-old girl with Marden-Walker syndrome is presented. This is a rare autosomal recessive syndrome. So far, approximately 20 cases have been described in the literature. The patient was hospitalized because of difficulty in feeding and slow spontaneous movements. Her parents were first cousins. She was diagnosed with clinical findings of growth and motor retardation, typical facial appearance, congenital heart disease, arachnodactyly, joint contractures, and a Dandy-Walker malformation on magnetic resonance imaging. PMID- 7543035 TI - Improving laboratory testing: can we get physicians to focus on outcome? AB - Managed care is changing the financing of healthcare and replacing open-ended reimbursement with fixed pricing schemes. Clinical laboratory tests will remain an important part of medical practice because laboratory information is essential for diagnosis and management of patients. The relative role of the hospital-based clinical laboratory, however, remains to be determined because healthcare organizations are reevaluating their services and attempting to drive down costs through reducing unit costs, decreasing utilization of services, and improving patient outcomes. The challenge for the clinical laboratory in managed care is to achieve appropriate utilization of laboratory tests so that clinical outcomes are optimized. The clinical laboratory at this medical center has used a number of approaches to improve utilization of thyroid function tests, isoenzyme tests for myocardial infarction, beta-human chorionic gonadotropin tests, and stat laboratory services for the emergency department. These experiences suggest that the laboratory can contribute to optimizing utilization of laboratory tests. This goal will require mutual cooperation of both the clinician and the clinical laboratory physician and (or) scientist. PMID- 7543034 TI - Amylase, lipase, pancreatic isoamylase, and phospholipase A in diagnosis of acute pancreatitis. AB - To determine the utility of serum amylase (AMY), lipase (Lp), pancreatic isoamylase (isoA), phospholipase A (PLA), and urine AMY in the diagnosis of acute pancreatitis, samples of serum and urine were obtained on admission and every day thereafter for 5 days from 384 patients with acute abdominal pain. Diagnostic accuracy, determined as the area under the receiver operating characteristic curve, was > 0.975 for serum AMY, Lp, isoA, and urine AMY. For each of these enzymes, a threshold value (twice to sixfold the upper limit of the reference values) offering diagnostic efficiency > 95% could be determined. In contrast, accuracy and efficiency of serum PLA were low. The profiles of these enzymes in acute pancreatitis decreased in a parallel fashion over 5 days except for PLA. We conclude that diagnostic utilities are similar for serum AMY, Lp, isoA, and urine AMY for acute pancreatitis, provided that an appropriate threshold is established. PMID- 7543037 TI - Assessing children's development using parents' reports. The Child Development Inventory. AB - The Child Development Inventory (CDI), completed by parents at home, assesses the development of social, self-help, motor, language, letter and number skills, and presence of symptoms and behavior problems of children between the ages of 15 months and 5 years. The results provide the pediatrician with a profile of the child's development, problems, and strengths, and are an aid to comprehensive assessment. CDI norms and validity were determined for a community sample of 568 children. The CDI developmental scales correlate closely with age (r = 0.84). CDI results identified all the normative group children who were enrolled in early childhood/special education (N = 26) and correlated with academic achievement for children in kindergarten (N = 132). CDI scales correlated with reading achievement in kindergarten as follows: general development 0.69, letters 0.56, language comprehensive 0.42, expressive language 0.36, and self-help 0.35. Thus, the CDI provides a useful measure of children's development and, because of its reliance on parental reports, offers an effective approach to developmental assessment in the busy pediatric practice. PMID- 7543038 TI - Intravenous morphine and nalbuphine increase histamine and catecholamine release without accompanying hemodynamic changes. AB - Patients receiving intravenous morphine at doses of 0.3 and 1.0 mg/kg for general anesthesia have been reported to show significant elevations in plasma histamine that are associated with hemodynamic changes. We undertook a prospective, randomized, double-blind trial in which 0.15 mg/kg morphine or 0.3 mg/kg nalbuphine was administered intravenously to normal volunteers. Thirteen of 15 subjects receiving morphine and 10 of 14 subjects receiving nalbuphine had elevations in plasma histamine levels and symptoms of histamine release within 5 minutes of drug administration. Six subjects in the morphine group and five in the nalbuphine group exhibited levels of plasma histamine > 2.0 ng/ml, but these levels were not associated with hemodynamic changes and occurred 10 to 15 minutes after drug administration. Our study suggests that the opiate-induced elevation of plasma histamine derives from cutaneous mast cells. PMID- 7543040 TI - Adequacy of mucosal biopsies for evaluation of intestinal cytokine-specific mRNA. Comparative study of RT-PCR in biopsies and isolated cells from normal and inflamed intestine. AB - Endoscopic biopsies are being increasingly utilized to investigate cytokine profiles in normal and diseased intestine. To evaluate the adequacy of mucosal biopsies as a source of cytokine-specific mRNA, we measured their content of interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2) mRNA by reverse transcription-polymerase chain reaction and compared it to that of autologous lamina propria mononuclear cells in control and inflammatory bowel disease involved specimens. High-quality total RNA was recovered more consistently from cell isolates than from biopsies. Interleukin-1 beta mRNA was reliably detected in both cell and biopsy samples, whereas IL-2 mRNA was measurable in all lamina propria cells but not always in biopsies. Compared to controls, levels of IL-1 beta were significantly elevated in Crohn's disease and ulcerative colitis cells and biopsies, and a weak but significant correlation existed between values derived from the two sources. In contrast, only ulcerative colitis cell isolates but not biopsies contained significantly reduced concentrations of IL-2 mRNA compared to those of control and Crohn's disease samples, and no correlation was found between cell and biopsy contents. Widely different levels of cytokine specific mRNA were present in closely adjacent biopsies, particularly for IL-2. These results suggest that mucosal biopsies are suitable to assess some but not all cytokine-specific mRNA due to variation in RNA recovery, intrinsic level of cytokine gene expression, and substantial variability of cytokine transcripts. PMID- 7543039 TI - A shared epitope found in the major paralysis inducing tick species of Africa. AB - Cross-reactivity between all the paralysis inducing tick species of veterinary relevance in Africa was demonstrated, by using a monoclonal antibody directed against the paralysis inducing toxin of Rhipicephalus evertsi evertsi. Western blot results, together with amino acid composition studies indicated that this monoclonal antibody recognizes protein bands of similar molecular mass and amino acid composition in R. evertsi evertsi and Ixodes rubicundus. This suggests that the Karoo paralysis toxin of I. rubicundus is possibly also a trimer with a high degree of homology to the spring lamb paralysis toxin of R. evertsi evertsi. The conclusive identity of these protein bands of I. rubicundus could not be shown. Bio-assay studies performed on 1-day-old chickens suggested that the anti-spring lamb paralysis toxin monoclonal antibody also recognizes the paralysis toxin present in Argas (Persicargas) walkerae, by rendering some degree of protection against the effect of this toxin. PMID- 7543042 TI - Prolactin replacement in adult dwarf mice does not reverse the deficit in tuberoinfundibular dopaminergic neuron number. AB - The lack of PRL synthesis in Ames dwarf mice coincides with a marked reduction in dopamine (DA) and in numbers of PRL-inhibiting tuberoinfundibular dopaminergic neurons in the hypothalamus (catecholaminergic area A12), as assessed by tyrosine hydroxylase (TH) immunoreactivity. This DA/TH deficit develops postnatally and can be prevented by PRL replacement initiated at 12 days of age. The present study tested whether a similar PRL treatment in adult dwarfs would reverse the A12 deficit, indicating that these neurons are quiescent due to absent PRL feedback stimulation, or would not reverse the deficit, suggesting that A12 neurons are either absent or refractory to PRL effect. At 60 days of age, Ames dwarf (df/df) mice received renal pituitary allografts from normal (DF/df) donors as a source of mouse PRL. Separate groups of dwarfs were treated sequentially with ovine PRL (50 micrograms/day, ip; 30 days) and vehicle (15 days) to assess whether the putative restorative effect of PRL regressed after hormone withdrawal. Brains were evaluated using DA histofluorescence and TH immunocytochemistry. Total numbers of TH-immunostained cells in A12 and medial zona incerta (area A13) regions were counted, and the intensity of TH immunostaining was assessed by computerized image analysis. The total A12 TH positive cell number was reduced (P < 0.01) in all PRL-treated dwarfs (1826 +/- 58) compared with that in normal mice (3340 +/- 180), and was not different from that in untreated dwarfs (1953 +/- 304) regardless of the PRL regimen. However, A12 perikarya in all PRL-treated dwarfs showed qualitatively increased histofluorescence and quantitatively increased TH immunostaining (P < 0.01) intensity compared with that in untreated dwarfs, an effect that regressed after ovine PRL withdrawal. Neither cell number nor staining intensity differed by gender. There were no significant differences in A13 cell numbers or staining intensity according to phenotype or PRL treatment. The present results indicate that the tuberoinfundibular dopaminergic neuronal population in adult Ames dwarf mice is permanently reduced, although extant A12 cells in dwarfs are responsive to either homologous or heterologous PRL feedback. Together with the previously reported effect of PRL treatment in neonatal dwarfs, the reduction appears to be the result of absent PRL stimulation during development. PMID- 7543043 TI - Suppression of development of experimental autoimmune thyroiditis by oral administration of thyroglobulin. AB - Experimental autoimmune thyroiditis (EAT), which to some extent represents an experimental model of human chronic lymphocytic thyroiditis, is an organ-specific autoimmune disease characterized by autoantibody production to thyroid antigens (Ag) and mononuclear infiltration of the thyroid gland. EAT induced by immunization with human thyroglobulin (hTG) with Freund's adjuvant in CBA/J (H 2K) mice is associated with prominent B and T cell responses. We report that oral administration of hTG effectively reduces the immune responses in EAT in mice in an Ag-specific manner. Both cellular and humoral immune responses are reduced in a dose-dependent manner. Histological evidence of disease is dramatically reduced. Suppression of the immune responses is seen 2 weeks after Ag challenge, with partial inhibition of proliferative and antibody responses. Six weeks after immunization, further inhibition is observed of both T and B cell responses. Hyporesponsiveness of T and B cell reactivity is seen only to hTG; T and B cell responses to other immunogens are not affected, including purified protein derivative and the nonrelated Ag BSA. This model may provide the basis for immunotherapy of autoimmune thyroid diseases in man. PMID- 7543041 TI - Intravenous contrast medium does not increase the severity of acute necrotizing pancreatitis in the opossum. AB - Contrast-enhanced computed tomography provides diagnostic and prognostic information in patients with acute pancreatitis. To evaluate whether contrast medium may worsen the severity of acute pancreatitis, we have used a model of necrotizing pancreatitis induced by ligating the common bile-pancreatic duct in opossums. Animals were infused with either saline or an ionic contrast agent 48 and 96 hr after induction of pancreatitis. Hyperamylasemia, pancreatic edema, acinar cell fragility, and macroscopic evidence of pancreatitis were comparable in both experimental groups. The microscopic extent of inflammation was similar in both groups, whereas acinar cell injury/necrosis was less in the contrast group. We conclude that administration of this ionic contrast agent during early stages of necrotizing pancreatitis in the opossum does not worsen the disease severity. The concept that administration of contrast medium during early stages of pancreatitis is dangerous should not be accepted until additional experimental and clinical studies support its validity. PMID- 7543044 TI - The role of the NPXY motif in the insulin receptor in tyrosine phosphorylation of insulin receptor substrate-1 and Shc. AB - The insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) and Shc on tyrosine residues, both of which associate with the protein-abundant Src homology/growth factor receptor-bound protein 2(ASH/GRB2) leading to p21ras activation. Juxtamembrane Tyr960 of the insulin receptor required for tyrosine phosphorylation of both IRS-1 and Shc is contained in the NPXY motif, which is also present in other tyrosine kinase receptors and oncogene products. In this study, the role of this motif in insulin's signaling was examined in Chinese hamster ovary cells expressing insulin receptors with mutations in this motif. All alterations in Tyr960 examined decreased tyrosine phosphorylation of both IRS 1 and Shc to a similar extent. The replacements of Asn957 and the deletion of NPE impaired tyrosine phosphorylation of Shc and IRS-1, although tyrosine phosphorylation of Shc was more severely affected than that of IRS-1. The amount of ASH/GRB2 bound to IRS-1 and Shc in vitro and in vivo was also decreased in these cells. These data suggest that the NPXY motif in the insulin receptor is important for tyrosine phosphorylation of both IRS-1 and Shc as well as subsequent signaling. PMID- 7543045 TI - Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation. AB - Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is uniquely regulated throughout MC3T3-E1 osteoblast differentiation: IGFBP-5 is first detectable in conditioned medium (CM) of replicating preosteoblasts (day 5); IGFBP-5 levels peak between culture days 8-12, then decline to almost undetectable levels in mature osteoblast cultures (> day 18) despite the persistence of IGFBP-5 messenger RNA. These observations suggest that IGFBP-5 concentrations may be regulated by posttranslational mechanisms. To determine whether proteolysis contributes to the disappearance of IGFBP-5 in CM of mature osteoblasts, serial samples of MC3T3-E1 cell CM obtained during a 30-day culture period were analyzed for IGFBP-5-degrading protease activity. Using [125I]recombinant human IGFBP-5 substrate zymography, we demonstrated that proteases with M(r) of 52-72 and 97 kilodaltons (kDa) were present in CM, and protease activity increased in concentration as cultures matured. The 52- to 72 kDa proteases were cation dependent and were inhibited by tissue inhibitor of metalloproteinase 1, a specific inhibitor of matrix metalloproteinases (MMPs), identifying them as MMPs. Furthermore, antisera to human MMP-1 and -2 immunoprecipitated IGFBP-5-degrading proteases with M(r) of 52 and 69/72 kDa, respectively, suggesting that homologous murine MMPs degrade IGFBP-5. Finally, MC3T3-E1 cell CM contained immunoreactive MMP-1 and -2, and MMP-2, in particular, increased significantly throughout differentiation. In contrast, the 97-kDa protease was neither inhibited by tissue inhibitor of metalloproteinase 1 nor immunoprecipitated by antisera to MMPs, suggesting that the 97-kDa protease is not a MMP. Together, these data suggest that MMPs along with an unidentified 97 kDa protease degrade IGFBP-5 in MC3T3-E1 cell cultures. Because truncated forms of IGFBP-5 have been shown to enhance the action of IGF in bone cells, IGFBP-5 proteases may be instrumental in IGF-mediated bone morphogenesis. PMID- 7543046 TI - Response of mouse lung to carbon deposition during injury and repair. AB - Increased respiratory disease and daily mortality rates are associated with higher levels of fine particulate air pollutants. We examined the possibility that deposition of even inert particles to previously injured lungs may accentuate pulmonary damage by investigating how the lung handles small carbon particles delivered during acute injury or during fibrotic repair. Mice received 2 mg carbon by intratracheal instillation into lungs already showing acute injury, 3 days after bleomycin (BL), or into lungs with fibrosis, 4 weeks after BL. At 3 days after BL, injury to the type I alveolar epithelium resulted in high protein levels in lavage fluid. Instilling carbon at this time induced a large increase in inflammatory cells, though many particles reached the interstitium, and a high proportion was retained up to 16 weeks later. However, fibrosis in these mice was equal to that found after BL alone. In the mice that received carbon 4 weeks after bleomycin, fibrotic repair had already occurred, and the epithelial surface was restored before particle instillation. After carbon, the subsequent inflammatory reaction cleared most particles, little reached the interstitium, and carbon retained at 16 weeks was not different from that in the carbon-only group. Instilling particles into fibrotic lung did not induce additional fibroblast growth or collagen production. The results indicate that instillation of fine particulates to the alveoli at a time of epithelial injury results in increased translocation to the interstitium. However, deposition of pure carbon into injured lungs does not further stimulate an ongoing fibrotic process, although it alters the patterns of particle deposition and retention in the lung. PMID- 7543048 TI - Distinct tissue distribution in pigs of tenascin-X and tenascin-C transcripts. AB - Tenascin-X and tenascin-C glycoproteins are phylogenetically conserved components of the extracellular matrix, although their specific roles remain to be determined. cDNA probes were produced from pig tenascin-X and tenascin-C genes and were used to examine the tissue distribution of the transcripts in 28 tissues from Large-White pigs, 4.5-42-months old (called adults) and 17 tissues from 87 day-old fetuses. The hybridization of Northern blots with tenascin-X probes revealed, in most tissues, a complex pattern of bands including a major band of about 13 kb, assumed to correspond to the main tenascin-X transcript. Hybridization with the tenascin-C probe showed two transcripts of 6.8 kb and 8.2 kb. The data from the ribonuclease-protection technique showed that both genes displayed large variations in the transcription levels among the tissues analysed. Overall, the tenascin-X gene was significantly expressed in two thirds of the tissues, and the tenascin-C gene in about 50% of them. The highest tenascin-X signals were observed in tendons, ligaments and, unexpectedly, in peripheral nerves. Other tissues, including colon, dermis, skin, heart, uterus, stomach, jejunum, placentae, aorta, lung, mammary and adrenal glands also exhibited significant signal intensities. In fetuses, mainly testes and skeletal muscle showed higher transcription levels than the adult counterparts. The tenascin-C gene was predominantly transcribed in the ligament, tendon, adrenal gland and colon, and more weakly in the stomach, jejunum, lung and spinal cord. In fetuses, the tenascin-C signal in the brain was higher than the signal in the brain of adult, whereas the reverse was true for the adrenal gland and the colon. Within a given tissue, the level of tenascin-X and tenascin-C transcripts varied greatly, indicating independent tenascin-X and tenascin-C transcription regulation mechanisms; this was particularly obvious in adult and fetal nerves but also in the dermis, skin, heart, uterus, placentae and aorta, where tenascin X RNA molecules were much more abundant than those of tenascin-C. In addition, similar differences were observed in the skeletal muscle and adrenal gland of fetuses. In contrast, the amount of tenascin-C transcripts in the fetal brain and adult spinal cord was higher than those for tenascin-X. Our results draw attention to a possible specific role of tenascin-X in the peripheral nerve physiology. PMID- 7543047 TI - The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. AB - ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers. PMID- 7543049 TI - Transient cognitive impairment due to glioblastoma. PMID- 7543050 TI - The effect of a 10-day space flight on the function, phenotype, and adhesion molecule expression of splenocytes and lymph node lymphocytes. AB - Previous studies have indicated that space flight affects the activation of lymphocytes from humans, monkeys, and rodents. In rats, where lymphocytes from blood, spleen, and lymph nodes have been tested, the accumulated data suggest that the effects of flight on various cells are lymphoid organ-specific. Thus, cells may be affected by variations in trafficking brought about by fluid shifts in microgravity (< 10(-3) g). In this study we examined lymphocyte activation (IL 2 production) as well as the expression of surface differentiation antigens and of adhesion molecules by splenocytes and lymph node lymphocytes (LNL) after a 10 day flight (Space Shuttle Mission STS-57). For splenocytes and LNL from flight (FLT) animals, IL-2 production decreased in response to the T cell receptor independent mitogen 12-O-tetradecanoylphorbol-13-acetate plus ionomycin, but was not affected by stimulation with the T cell receptor-dependent mitogens Concanavalin A or phytohemagglutinin. In addition, the percentage, as well as fluorescent intensity, of splenocytes which expressed CD8, CD4, or kappa increased after flight. The percentage of LNL expressing CD2 also increased but those expressing CD5 decreased. The percentage of cells expressing the integrins LFA-1 alpha and beta increased with splenocytes from FLT animals but decreased for LNL. In contrast, FLT animals showed a decrease in the percentage of selectin positive splenocytes. ICAM-1 expression did not change. In summary, these data are consistent with a model in which microgravity affects lymphocyte redistribution among organs, which in turn influences the activation potential of the cells. PMID- 7543051 TI - Increased LFA-1-mediated homotypic cell adhesion is associated with the G1 growth arrest induced by rapamycin in a T cell lymphoma. AB - The immunosuppressive macrolide, rapamycin, impedes the G1 to S cell cycle progression in cytokine-stimulated normal lymphocytes and in certain autonomously proliferating cell lines. Here, we found that the rapamycin-induced growth arrest augments homotypic aggregation in the YAC-1 T cell lymphoma. The growth arrest and increased aggregation were both blocked by the rapamycin antagonist, L 685,818, which interacts with the intracellular binding proteins mediating rapamycin's biochemical action. Moreover, rapamycin-induced aggregation was not seen in YAC-1 cells mutants selected for resistance to the drug's antiproliferative effect. Although the inhibition of G1/S progression induced by serum deprivation also resulted in increased cellular aggregation, cell cycle blockade in late G1 by mimosine, early S phase by hydroxyurea, or G2/M by nocodazole all failed to do so. Furthermore, the aggregation induced by rapamycin was blocked by antibodies to the alpha (CD11a) or beta (CD18) subunits of the integrin, LFA-1, or to its ligands, ICAM-1 and ICAM-2, and did not occur in LFA-1 deficient YAC mutants. However, the surface expression of LFA-1, ICAM-1, or ICAM 2 was not augmented in cells aggregated by rapamycin. Finally, the serine/threonine protein phosphatase inhibitor, okadaic acid, was found to abrogate rapamycin-induced aggregation. Therefore, rapamycin's impairment of YAC 1 cell growth in G1 is accompanied by enhanced LFA-1-mediated homotypic cell adhesion that may reflect an increase of the integrin's avidity for its ligands and may involve protein phosphorylation/dephosphorylation events. This suggests the existence of a link between cell cycle progression and "inside-out" LFA-1 signaling, possibly regulated by rapamycin's biochemical targets. PMID- 7543053 TI - Alterations of the cytoskeletal organization in tumor cell lines by a cardiotonic drug, vesnarinone, through protein tyrosine phosphorylation. AB - We describe nonspecific and moderate activation of cellular protein tyrosine phosphorylation by a chemical compound, vesnarinone, which results in enhanced synthesis and/or assembly of cytoskeletal proteins and morphological alterations in several transformed cells. In A431 cells, vesnarinone induced tyrosine phosphorylation of the overexpressed epidermal growth factor receptors (EGFR) as well as other unidentified proteins, increased the synthesis of cytokeratins, and caused amplification of the intermediate filament networks and cell flattening. The drug effects were abolished by tyrphostin, a protein tyrosine kinase inhibitor. Two other cell lines responded to the drug with increased synthesis of a cell type-specific cytoskeletal protein: vimentin in QG56 human lung carcinoma cells and alpha-tubulin in NIH3T3 cells transformed with v-src. In all cell lines tested, the drug-induced tyrosine phosphorylation was localized in cell-cell and cell-substrate contacts as detected by immunofluorescent staining. Responsive protein substrates and their sensitivity to the drug varied from one cell line to another as observed by immunoblot analysis. Vesnarinone exerted neither activating nor inhibitory effect on in vitro enzyme reactions including EGFR tyrosine kinase, v-src kinase, and protein tyrosine phosphatases. This suggests that vesnarinone indirectly activates tyrosine phosphorylation of membrane proteins related to cell adhesion, which influences a signaling pathway linked to the stress fiber assembly in certain cell lines. The possible mechanism by which vesnarinone induces the cellular responses is discussed. PMID- 7543052 TI - pp125FAK in human melanocytes and melanoma: expression and phosphorylation. AB - Focal adhesion kinase (pp125FAK) is a nonreceptor tyrosine kinase which colocalizes with integrins to focal contacts, sites where multiple proteins interact to regulate the assembly of the actin cytoskeleton. Autophosphorylation and activation of pp125FAK occur after integrin clustering or cell adhesion to ligands through cognate integrin receptors and are postulated to mediate integrin signaling events. In this report we examined pp125FAK expression and phosphorylation in normal human melanocytes, an adherent human metastatic melanoma cell line (SKMEL28), and a nonadherent human metastatic melanoma cell line (SKMEL1). We show that SKMEL28 cells express constitutively phosphorylated pp125FAK and that pp125FAK phosphorylation in melanocytes is induced by phorbol esters and growth factors present in melanocyte growth medium. Focal adhesion kinase phosphorylation could be enhanced by b1 integrin-activating antibodies in human melanocytes, but not in SKMEL28 cells. In contrast with SKMEL28 cells, constitutive phosphorylation of pp125FAK was not observed in SKMEL1 cells, and incubation with activating b1 integrin antibodies had no effect on pp125FAK phosphorylation. Absence of pp125FAK phosphorylation in SKMEL1 cells was not due to lack of expression of pp125FAK, as shown by immunoprecipitation of the pp125FAK protein from cell lysates. However, b1 integrin expression was significantly less in SKMEL1 cells than in human melanocytes and SKMEL28 cells. This study further supports the importance of integrins in pp125FAK-mediated signaling and indicates that transformation-related changes in pp125FAK phosphorylation exist in human melanocytes and melanoma cells. PMID- 7543054 TI - Tyrosine phosphorylation of p34cdc2 is regulated by protein phosphatase 2A in growing immature Xenopus oocytes. AB - Growing stage IV Xenopus oocytes are unresponsive to progesterone treatment. They contain a store of preMPF composed of tyrosine phosphorylated p34cdc2 and cyclin B2. The endogenous store of preMPF cannot be recruited by cdc25 protein phosphatase or cyclin protein microinjections. This is in contrast with full grown stage VI oocytes where microinjections of these proteins are known to activate the autoamplification of MPF. When cyclins are microinjected into stage IV oocytes, they associate with endogenous free p34cdc2 and the illegitimate complexes undergo phosphorylation on tyrosine 15. High doses of human cyclin A allow, however, part of the neoformed complexes to be activated as an histone-H1 kinase; this partial activation of p34cdc2 is sufficient to induce germinal vesicle breakdown in these small oocytes. Co-injections of cyclin A or cyclin B together with okadaic acid (10 microM in the microinjection solution), an inhibitor of protein phosphatase 2A (PP2A), lead to the full activation of neoformed p34cdc2/cyclin complexes. These results indicate that small oocytes possess an active tyrosine kinase that inactivates new p34cdc2/cyclin complexes. Inhibition of PP2A by okadaic acid prevents this inactivation reaction and conversely allows the illegitimate complex to be activated. Neither the activating phosphorylation on threonine 161 nor the inactivating phosphorylation on tyrosine 15 take place in stage IV enucleated oocytes. Altogether, our results show that the accumulation of inactive p34cdc2/cyclin B2 during the long-lasting prophase of the oocyte is positively controlled by PP2A through the tyrosine phosphorylation of p34cdc2. PMID- 7543056 TI - Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3. AB - The extracellular matrix proteins fibulin-1 (variants C and D) and fibulin-2 occur in basement membranes and in vessel walls and are thus potential candidates for cellular interactions. Recombinant forms of these proteins were obtained from stably transfected kidney cell clones and examined for cell-adhesion activity and binding to five different purified integrins. The two variants of mouse fibulin-1 were inactive in all these assays. Mouse fibulin-2, however, bound to alpha IIb beta 3 integrin almost as strongly as fibrinogen, while a lower activity was found for alpha V beta 3 and almost none for alpha 5 beta 1 integrin. Synthetic SVPRGDLDG peptide, corresponding to the single RGD site of mouse fibulin-2, was a strong antagonist of alpha IIb beta 3 integrin binding. Its affinity for alpha V beta 3 and alpha 5 beta 1 integrins was, however, 10- to 50-fold lower compared to GRGDS. Mouse fibulin-2 also promoted adhesion of thrombin-stimulated platelets and of some established cell lines which could be inhibited by RGD peptides. Human fibulin-2, in which the RGD sequence is changed to RSS, bound less strongly to alpha IIb beta 3 integrin and showed no cell-adhesion activity. Together these data suggest a potential role in hemostatic control for mouse fibulin-2 and possibly also for human fibulin-2. PMID- 7543055 TI - Amiloride suppresses erythropoietin-induced proliferation and MAP kinase, but potentiates differentiation of J2E cells. AB - The J2E erythroid cell line proliferates and differentiates in response to erythropoietin (epo). Here we demonstrate that the diuretic amiloride can suppress normal and hormone-induced cell division in a dose-dependent manner. In the presence of amiloride, cell numbers did not increase, [3H]thymidine incorporation decreased, and fewer cells were observed in the S, G2, and M phases of the cell cycle. In addition, the levels of proliferating cell nuclear antigen, a subunit of DNA polymerase delta, fell. In marked contrast, epo-initiated differentiation was potentiated when J2E cells were cultured with the drug: the number of benzidine-positive cells increased, hemoglobin content per cell rose, and more morphologically mature cells were produced. Immunoblotting with anti phosphotyrosine antibodies revealed that amiloride reduced the number of phosphorylated proteins in epo-stimulated cells. Moreover, the protein content of p42 and p44 MAP kinases was noticeably downregulated in amiloride-treated cultures. These data indicate that amiloride may interfere with epo-induced signaling cascades within J2E cells which result in restricted cell division and promotion of maturation. PMID- 7543057 TI - Association of Hb S/Hb lepore and delta beta-thalassemia/Hb lepore in Sicilian patients: review of the presence of Hb lepore in Sicily. AB - The hemoglobin (Hb) lepore-Boston is a beta-globin structural variant, produced in a reduced amount and formed from the fusion of N-terminus delta-(residues 1 87) and C-terminus beta-chains (residues 116-146). This type of fusion protein is quite common in Southern Italy (Campania, Calabria, and Sicily). We report here the hematological and hemoglobin data on 96 unrelated Sicilians with Hb lepore trait. Particularly interesting are the subjects where Hb lepore occurs with Hb S or Sicilian type delta beta-thalassemia. In these individuals, striking features are clinical variability and different hematological pictures. These observations underscore the importance of thalassemia screening in these geographic areas, such as Southern Italy, principally Sicily, where the mutations in globin gene clusters are especially prevalent. Moreover, as from the second half of the last century, owing to high migratory flux from Sicily to Northern Europe, North and South America, and Australia, the Hb lepore, as well as other hemoglobin variants, have become prevalent, making the identification of the heterozygotes a problem of general interest. PMID- 7543058 TI - The use of interferon-alpha and granulocyte colony-stimulating factor in hairy cell leukaemia with aplastic marrow. PMID- 7543059 TI - COP-BLAM regimen combined with granulocyte colony-stimulating factor and high grade non-Hodgkin's lymphoma. AB - The clinical efficacy of COP-BLAM chemotherapy combined with human recombinant granulocyte colony-stimulating factor (G-CSF) was evaluated in 104 previously untreated patients with non-Hodgkin's lymphoma (NHL). According to the method of Laurence et al., a modified COP-BLAM regimen was administered every 21 days. G CSF was added when the granulocyte count fell below 1000 x 10(9)/l. Ninety-eight of 104 (94.2%) patients achieved a complete remission; the 4-year survival rate was 82.4% with a median duration of observation of 26 months. Survival was significantly longer in patients with low serum LDH levels, B-cell type or low CRP or in earlier clinical stages, than in patients with high serum LDH levels, T cell type of higher CRP levels or in advanced clinical stages. The mean duration of administration of G-CSF was 5.4 days. Twelve patients developed infections during treatment. The adverse effects of G-CSF included interstitial pneumonia, bone pain and fever. Patients administered COP-BLAM combined with G-CSF achieved a high rate of remission and had a low incidence of infection. Nearly all the patients could be treated in 21-day cycles. The results suggest that G-CSF combined with COP-BLAM was effective in treating NHL, because the patients can tolerate a higher dose of the anticancer agents. PMID- 7543061 TI - The principal neutralizing determinant of HIV-1 located in V3 of gp120 forms a 12 residue loop by internal hydrophobic interactions. AB - The interactions of the peptide RP135a (RKSI-RIQRGPGRAFVT), corresponding to residues 311-326 of gp120 of HIV-1IIIB, with the anti-gp120 HIV-1IIIB neutralizing antibody 0.5 beta were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile-4, Ile-6 and Val-15 that create a 12-residue loop with these residues at the base and the conserved GPGR sequence at its top. PMID- 7543060 TI - Assessment of the value of treatment with granulocyte colony-stimulating factor in children with acute lymphoblastic leukemia: a randomized clinical trial. AB - The present trial was designed to test the effects of G-CSF on the duration of the second phase of induction chemotherapy in children with newly diagnosed acute lymphoblastic leukemia (ALL). A total of 32 patients were assigned randomly to a group that received (14 patients; group A) or a group that did not receive (18 patients; group B) G-CSF (10 g/kg/day subcutaneously and daily) throughout of the second phase of induction therapy. One of 14 (7.1%) patients in group A and 2 of 18 (11.1%) patients in group B completed the course of chemotherapy within the planned time. The median length of this phase was 37 days (range, 29 to 65; mean, 40; SD, 8.6) for patients in group A and 36 days (range, from 29 to 55; mean, 38; SD, 7.4) for those in group B, and the difference was not statistically significant. The number of days during which patients had granulocyte counts of less than 2 x 10(9)/l, the number of febrile episodes of unknown origin, the number of bacterial and fungal infections and the number of days of hospitalization did not differ in a statistically significant manner between the two groups. Our data suggest that G-CSF supportive therapy may be unnecessary in children with neutropenia of short duration, for whom the risk of infection is low. PMID- 7543062 TI - Lipopolysaccharide treatment in vivo induces tissue expression of GTP cyclohydrolase I mRNA. AB - A significant induction of GTP cyclohydrolase I (GTPCH) mRNA was observed in lung, heart and kidney of rats treated with lipopolysaccharide (LPS; 10 mg/kg i.v.). GTPCH mRNA levels in liver were high even in untreated rats, and remained elevated after LPS treatment. Parallel induction of nitric oxide synthase (NOS) mRNA was observed in these tissues of LPS-treated rats. Our results demonstrate induction of GTPCH mRNA after LPS treatment in vivo and provide molecular evidence for the increased GTPCH activity which may up-regulate NOS activity in vivo. PMID- 7543063 TI - Expression of transforming growth factor-beta mRNA in chicken ovarian follicular tissue. AB - RNA was isolated from chicken thecal tissue from the largest (F1), third largest (F3), and fifth largest (F5) preovulatory follicles, from small yolky follicles, and from granulosa tissue from F1 follicles. Transforming growth factor-beta (TGF beta) gene expression was measured using reverse transcription polymerase chain reaction. Thecal cells from all follicle sizes expressed all three isoforms of TGF-beta. TGF-beta 1 mRNA was detected in granulosa cells at levels comparable to those seen in thecal cells. However, TGF-beta 2 and TGF-beta 3 mRNA was expressed at significantly lower levels in granulosa than in thecal tissue. This is the first demonstration of TGF-beta gene expression in the ovary of a nonmammalian species. The similarities between mammalian and avian TGF-beta gene expression are remarkable, especially in light of the distinctive patterns of avian follicular development and the differing steroidogenic capacities of ovarian cell types of the two classes of vertebrates. PMID- 7543064 TI - Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2. AB - The Oct-2 transcription factor is expressed predominantly in B lymphocytes and has been shown previously to be important for the terminal phase of B-cell differentiation in mice. A number of genes specifically expressed in B cells contain Oct-2-binding sites in their regulatory regions. However, the analysis of expression levels of these genes in Oct-2-deficient B cells revealed that they were unaffected. Hence, there were no genes known that critically depend on Oct-2 for their expression. To understand the molecular basis for the Oct-2 effect on B cell development, we searched for Oct-2 target genes by subtractive cDNA cloning. We show here that expression of the murine CD36 gene in B cells and macrophages requires a functional Oct-2 protein. Nuclear run-on experiments demonstrate that this gene is regulated transcriptionally by Oct-2. Moreover, CD36 levels correlated with the levels of Oct-2 expression in several mouse B-cell and macrophage cell lines. Finally, compared to wild-type and heterozygous mice, CD36 mRNA levels were markedly reduced in spleens and B-cell-enriched splenocyte fractions from oct-2-/- mice. The data identify CD36 as the first target gene critically dependent on Oct-2 for its expression. Because CD36 expression is also dependent on Oct-2 in vivo, it is a candidate gene through which Oct-2 could affect B-cell differentiation. PMID- 7543065 TI - Regulation of angR, a gene with regulatory and biosynthetic functions in the pJM1 plasmid-mediated iron uptake system of Vibrio anguillarum. AB - AngR and the product(s) encoded in the trans-acting factor (TAF) region are necessary for the full expression of the pJM1 plasmid-mediated anguibactin iron uptake system in Vibrio anguillarum (Va). In this report, we analyzed the factors that affect the expression of the angR gene. Northern blot analysis showed that angR encodes a 3.1-kb transcript which is expressed only under iron-limiting conditions. Measurement of steady-state RNA levels show that, under iron-limiting conditions, angR is positively regulated at the transcriptional level by product(s) of the Va TAF region. However, this enhancement of angR expression by TAF does not occur at high levels of the AngR protein, as assessed by using an angR::lacZ fusion in the presence of a construct containing angR under the control of ptac. We also report that repression of angR by iron could possibly be mediated by an endogenous Va antisense RNA beta, which contains a stem-loop structure complementary to the stem-loop structure located at the 5' end of angR. PMID- 7543066 TI - Two domains with amino-acid sequence similarity are required for dihydroneopterin aldolase function in the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii. AB - The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) codes for a multifunctional enzyme (Fas) known to catalyse three consecutive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway. Previously, we predicted that a domain, designated FasB (amino acids (aa) 161 280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathway, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa identity with the DHNA of Streptococcus pneumoniae (Sp). We now extend these findings to show that a second domain, FasA (aa 39-160), whose function was previously unknown, shares 27% sequence identity with the adjacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were constructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect cells. No DHNA activity is associated with either fasA or fasB when produced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced FasAB polypeptide. The results of aa sequence alignments and expression studies suggest that FasA and FasB may be two subunits of the DHNA enzyme moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed. PMID- 7543067 TI - Glutathione-S-transferase activity and multidrug resistance phenotype in chronic lymphocytic leukemia: do they have any clinical relevance? AB - BACKGROUND: Lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL) have frequently been shown to be positive for the multidrug resistance (MDR) phenotype. However, this phenotype does not seem to be responsible for the resistance to alkylating agents usually employed in the management of CLL. METHODS: Lymphocytes from 42 patients were evaluated by flow cytometry for P-170 expression and by spectrophotometry for glutathione-S transferase (GST) activity. RESULTS: Our findings show that GST is not related to any clinical parameter but is increased in treated patients. Conversely, 85% of patients were positive for P-170 and this was related to the percentage of CD5/CD19-positive lymphocytes. CD5/CD19-negative patients were also negative for P-170. MDR was not related to any clinical parameter evaluated nor to GST activity in lymphocytes. CONCLUSIONS: MDR is constitutively expressed in B-cell chronic lymphocytic leukemia and seems to be related to a CD5/CD19 B-CLL phenotype. The increase of GST activity in treated patients is statistically significant (p < 0.005). PMID- 7543068 TI - Mobilization of circulating progenitor cells in multiple myeloma during VCAD therapy with or without rhG-CSF. AB - BACKGROUND: Circulating progenitor cells (CPC), when infused in large numbers, rapidly repopulate the marrow after myeloablation with high-dose therapy. In multiple myeloma (MM), as in other disorders, different chemotherapy regimens, including single-as well as multiple-agent chemotherapy, with or without hemopoietic growth factors, have been proposed to mobilize these progenitor cells into the blood. Here we report our experience with a drug combination called VCAD and compare the results to those obtained by adding rhG-CSF to the same combination. METHODS: Fourteen MM patients were given one course of VCAD, a chemotherapy association of vincristine 2 mg, cyclophosphamide 4 x 0.5 g/m2, adriamycin 2 x 50 mg/m2 and dexamethasone 4 x 40 mg, before undergoing apheresis to collect CPC for autografting. Seven also received rhG-CSF (filgrastim) 5 mcg/kg/day over the period of apheresis. These latter were allocated to rhG-CSF treatment sequentially from the time the drug became available for clinical use. RESULTS: Following VCAD-induced pancytopenia, CFU-GM peaked at a median of 853/mL (range 96-4352; 7.6 times basal level). RhG-CSF administration increased CFU-GM levels but not significantly. With rhG-CSF the CFU-GM peak was reached sooner, toxicity was reduced and granulocytopenia less protracted. Fewer aphereses were run in the rhG-CSF group, there were higher yields per single run, and patients began and completed their collection program more quickly. CONCLUSIONS: The VCAD association is able to mobilize CPC in patients with MM, and rhG-CSF is recommended as a fundamental part of the priming schedule. PMID- 7543069 TI - Granulocyte colony-stimulating factor (G-CSF) prevents dose-limiting neutropenia in lymphoma patients receiving standard dose chemotherapy. AB - In this study, nine patients with non-Hodgkin's lymphoma (n = 6) and Hodgkin's disease (n = 3) receiving different cytotoxic chemotherapy regimens were given granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg/day) from 48 hours after the end of chemotherapy to 48 hours before the next chemotherapy administration. The decrease in mean absolute neutrophil counts (ANC) and in mean platelet (Plt) counts was not significant when pre-therapy counts were compared with post-therapy ones (p < 0.375 and p > 0.4, respectively). The mean actual dose intensity was 92% (range 68-100%). G-CSF treatment after chemotherapy reduces neutropenia and permits administration of the full chemotherapy program. A wash-out period between G-CSF treatment and chemotherapy administration is needed to prevent the detrimental effect of chemotherapy on leukocyte and platelet recovery when repeated cycles of cytotoxic drugs and G-CSF are administered. PMID- 7543071 TI - Granulocyte colony-stimulating factor (G-CSF) administration increases PMN CD32 (FcRII) expression and FcR-related functions. AB - The phenotypical and functional properties of circulating neutrophils from ten patients suffering from intermediate- and high-grade non-Hodgkin lymphoma were investigated before and after rhG-CSF administration (5 micrograms/kg/day subcutaneously for 5 days). The following parameters were studied: flow cytometry evaluation of surface CD32, CD16, CD11b and CD18 by means of a whole blood method; whole blood phagocytosis by means of a flow cytometric assay; whole blood chemiluminescence using opsonized zymosan as a stimulus. A significant increase in the expression of surface CD32 was detected in all patients, while CD11b expression was found to be increased in only four of them. CD16 and CD18 expression did not change. A significant enhancement of phagocytosis and phagocytosis-associated chemiluminescence was also observed. These results show that rhG-CSF administration can increase both FcRII expression and FcR-related functions. PMID- 7543070 TI - Monoclonal expansion of large granular lymphocytes with a CD4+ CD8dim+/- phenotype associated with hairy cell leukemia. AB - Peripheral blood lymphoid cell expansions with an unusual CD3+, CD4+, CD8dim+/-, CD11b+, CD57+ immunophenotype have recently been reported. They frequently have the morphology of large granular lymphocytes (LGL) and can be either monoclonal or polyclonal. Their significance is still unclear and no association with hematological neoplasms has been described. We report the case of a patient with a monoclonal expansion of LGL associated with a B-cell-derived hairy cell leukemia. The two lymphoid clones were not physically associated since T-LGL were found in the peripheral blood and hairy cells were detected in the bone marrow and kidney. PMID- 7543072 TI - Secondary prophylaxis of venous thromboembolism: rational use of oral anticoagulants. PMID- 7543073 TI - [Receptive systems for drugs in salivary gland cells]. AB - Investigators have demonstrated many types of receptors or acceptors for endogenous substances in salivary glands. These suggest that salivary glands contain receptive systems for many drugs. These receptors can be classified into three types based on the property of saliva secretion: (1) receptors involved in fluid secretion, (2) receptors involved in exocytosis for the protein secretion, (3) receptors involved in both types of secretion. The receptors involved in fluid secretion include the group of alpha 1B, M3, NK-1 receptors coupled with IP3; the group of beta 2 and VIP receptors coupled with cAMP; and the group of P2Z and P2U receptors coupled with the ATP-gated calcium channel. Whereas alpha 1A, beta 1, VIP, M3, insulin and H2 receptors mediate exocytosis via the cAMP proteinkinase A pathway. Moreover, the another pathway of diacylglycerol proteinkinase C has also been demonstrated to be involved in the exocytosis occurring via NK-1 and M3 receptors. Thus, salivary gland cells have many kinds of drug-receptor system. These receptors are all positive to fluid and/or protein exocytosis. Recently our study on the inhibitory regulation of saliva secretion have suggested the existence of GABA and the GABA-synthetic/metabolic pathway, GABA(A) receptors and benzodiazepine (BDZ) receptors of both central and peripheral types, and furthermore, the coupling of GABA(A) and the central type of BDZ receptors. These receptors are involved in decreasing fluid secretion and amylase release elicited by secretagogues. In the future, mechanisms of the intracellular transduction elicited by BDZ or GABA must be clarified. PMID- 7543074 TI - [Bioassay for angiogenesis and analysis of its mechanism]. AB - To study the sites of action of cytokines and anti-angiogenic agents, bioassays have been developed to systematically investigate not only the entire process of angiogenesis in vivo but also its individual steps including capillary tube formation and proliferation of vascular endothelial cells (EC). Angiogenesis in vivo is quantitatively assayed by measuring the carmine dye content in Freund's complete adjuvant-induced mouse pouch granuloma after the intravenous administration of dye solution. Angiogenesis in vitro is quantified by counting the number of microvessels developed from blood vessels cultured in fibrin gel. Tube formation is quantitatively estimated by measuring the total length of capillary tubes from EC cultured in type I collagen gel. EC proliferation is assayed by measuring both the increase in cell number and 3H-thymidine incorporation into the cells. The competence and progression activities in the EC proliferation are analyzed by our convenient method. By these bioassays, the mechanism of an anti-differentiation agent can be easily clarified to develop new types of therapies for rheumatoid arthritis and diabetic retinopathy. PMID- 7543075 TI - Lipopolysaccharide priming of superoxide release by human neutrophils: role of membrane CD14 and serum LPS binding protein. AB - Previous studies have suggested that lipopolysaccharide (LPS) interactions with neutrophils and monocytes are mediated via the CD14 receptor, in the presence of serum factors such as LPS-binding protein (LBP) and septin. The present study was designed to test if CD14-mediated LPS priming of human neutrophils is dependent upon the presence of serum proteins and to evaluate the contribution of serum factors in LPS-neutrophil interactions. The results demonstrate that CD14 mediates the priming of neutrophil superoxide release by LPS both in the presence and in the absence of serum. However, priming by LPS is greatly enhanced in the presence of human serum, and the factor responsible for this phenomenon is LBP and not heat-sensitive proteins, such as septin. PMID- 7543077 TI - The prognostic influence of neuroendocrine cells in prostate cancer: results of a long-term follow-up study with patients treated by radical prostatectomy. AB - The distribution of immunohistochemically defined neuroendocrine (NE) cells in benign, pre-cancerous and neoplastic prostatic tissues and the prognostic value of these cells in prostate cancer were studied in the radical prostatectomy specimens of 90 patients from whom complete long-term follow-up data were available. The tissue blocks containing all the different Gleason patterns observed in a particular tumor were selected and immunostained. Since chromogranin B stained only a few cells compared to chromogranin A (CgA), NE cells were only defined by their reactivity with CgA. A semi-quantificative CgA score was assessed for all distinct pathological areas. Cox's regression model was used to analyze the influence of final TNM classification (TNM, 1992), Gleason sum score (GSS), age and CgA score on the probability of progression and tumor-specific death. NE cells were demonstrated in all normal prostatic tissues and in most hyperplastic and intra-epithelial neoplastic lesions. CgA staining was seen in 78% of the tumors. CgA scores were not related with Gleason growth patterns, GSS or TNM classification and had no prognostic value. The independent prognostic variables in Cox's regression model were: GSS and pT stage for progression and GSS for tumor-specific survival. Theoretically, NE cells could influence tumor behavior and this discrepancy suggests the need for experimental studies to investigate the role of NE cells in the normal and neoplastic prostate. PMID- 7543076 TI - Lipopolysaccharide-induced E-selectin expression requires continuous presence of LPS and is inhibited by bactericidal/permeability-increasing protein. AB - Endothelial cells stimulated by LPS express E-selectin, which plays an important role in mediating neutrophil adhesion during inflammation. E-selectin is induced within 1-2 h, peaks at 4-6 h, and gradually returns to basal level by 24 h. rBPI21, a recombinant N-terminal fragment of human bactericidal/permeability increasing protein (BPI), inhibited LPS-induced E-selectin expression when added at the same time as, and up to 6 h after, LPS. Delayed administration of rBPI21 also affected LPS-mediated activation of the nuclear factor, NF-kappa B. Two to 4 h following LPS addition to endothelial cells, when NF-kappa B was already activated, addition of rBPI21 resulted in marked reduction of NF-kappa B detectable at 4 or 6 h. These results indicate that endothelial activation requires continuous presence of LPS, and rBPI21 acts to reverse LPS-mediated endothelial activation by interrupting the on-going LPS signal. PMID- 7543079 TI - Insulin-like growth-factor-binding protein 3 is decreased in early-stage operable pre-menopausal breast cancer. AB - Insulin-like growth factor I (IGF-I) is a potent mitogen for human breast-cancer cells in vitro. In circulation, most of IGF-I is bound to IGF-binding protein 3 (IGFBP-3). This high-affinity binding is thought to have an important limiting effect on the availability of IGF-I for biological activity. To assess the availability of IGF-I for receptor binding, we determined serum levels of IGF-I and IGFBP-3 and IGF-I/IGFBP-3 ratios. In a case-control study, 150 women aged 38 to 75 years presenting with stage-I or -II breast cancer were investigated just prior to surgery (n = 76), or to irradiation one month after surgery (n = 74). The population-based control group consisted of 441 women of the same age having no breast cancer. Women reporting diabetes mellitus or other hormonal abnormalities were excluded. Premenopausal cases showed elevated IGF-I serum concentrations, decreased IGFBP-3 levels and increased IGF-I/IGFBP-3 ratios. The IGF-I/IGFBP-3 ratio was a significant breast-cancer risk factor, also after adjustment for age, family history, height, body-mass index, body-fat distribution, and serum levels of C-peptide. The relative risk was 7.34 for the highest compared with the lowest quintile of IGF-I/IGFBP-3. The presence or absence of tumor had no influence on these results. Increased levels of available IGF-I in the circulation of pre-menopausal women may contribute to the development of breast cancer. PMID- 7543078 TI - In situ expression of B7 and CD28 receptor families in human malignant melanoma: relevance for T-cell-mediated anti-tumor immunity. AB - Work in animal models has suggested that interactions of members of the B7 receptor family (e.g., B7-1, B7-2) on tumor cells with their ligands CD28 and CTLA-4 on cytotoxic T cells (CTL) are important for the induction of anti-tumor immunity against malignant melanoma (MM). To determine whether these molecules are of relevance for CTL responses against human MM, we studied the expression of B7-1, B7-2, CD28 and CTLA-4 in primary tumors of MM (PMM), MM metastases (MMM) and benign melanocytic nevi (BMN) by immunohistochemistry (IH) and by reverse transcription polymerase chain reaction (RT-PCR). By RT-PCR, B7-1 and B7-2 specific mRNAs were detected in most PMM, MMM and BMN. These PCR-signals were derived from CD45(+)-infiltrating leukocytes and not from tumor cells since (I) MMM depleted of CD45+ cells contained no B7-1 or B7-2 mRNA; and (2) by IH, B7-1 and B7-2 were found on infiltrating dendritic cells, macrophages and a variable proportion of tumor-infiltrating lymphocytes (TIL) but not on melanoma cells or nevus cells. The important exceptions were 5/5 spontaneously regressing PMM, in which B7-1 and B7-2 were expressed by melanoma cells, that were surrounded by TIL expressing CD28 but not CTLA-4. We conclude that, in PMM, MMM and BMN, the majority of TIL are CD28+ and that B7-1 and B7-2 are expressed by CD45(+) infiltrating antigen-presenting cells (APC) and TIL, but not by the tumor cells. However, in spontaneously regressing PMM, melanoma cells express B7-1, B7-2 and MHC class-I and -II antigens, particularly in areas with clinical and histological signs of an ongoing anti-tumor response. These data suggest that the absence of B7-1 and B7-2 favors the escape of MM from immunosurveillance, while B7-1, B7-2 expression enhances T-cell-mediated anti-tumor immunity. PMID- 7543082 TI - Therapy of human B-cell lymphoma bearing SCID mice is more effective with anti CD19- and anti-CD38-saporin immunotoxins used in combination than with either immunotoxin used alone. AB - The CD19+ CD38+ human Burkitt's lymphoma cell line Ramos grows aggressively when injected intravenously (i.v.) into severe combined immunodeficient (SCID) mice, killing 100% of animals within a 33-42 day period with widely disseminated disease. Treatment commencing 7 days after i.v. injection of Ramos cells, with 3 doses of an anti-CD19 immunotoxin (IT; BU12-SAPORIN) or an anti-CD38IT (OKT10 SAPORIN) led to a significant prolongation of survival compared with sham-treated controls; the anti-CD38 IT gave the greatest prolongation of survival, but all treated animals eventually succumbed to disease. When both ITs were used in combination at equivalent dose levels, the therapeutic outcome was significantly improved over that obtained for single IT therapy, with 20% of animals surviving disease-free to 300 days. When anti-CD38 IT was given in combination with anti CD19 antibody there was no therapeutic improvement over anti-CD38 IT used alone. However, when anti-CD19 IT was given in combination with CD38 antibody, a significant prolongation of survival ensued over that obtained with anti-CD19 IT alone, though this was not as significantly pronounced as that obtained when both ITs were used in combination and was only as good as the survival obtained with OKT10 antibody used alone. CD19 and CD38 are expressed on the surface of the vast majority of B-cell lymphoma and common acute lymphoblastic leukaemia cells, and our findings provide a sound rationale for a combination immunotoxin trial in these diseases directed against both these target molecules. PMID- 7543080 TI - Characterization of cell lines established from human hepatocellular carcinoma. AB - We characterized 8 human hepatocellular-carcinoma cell lines established from the primary tumors of Korean patients. All lines showed substrate adherence and one line from anaplastic tumor also grew as floating aggregates. Most cultured cells maintained many morphological characteristics of the original tumors from which they were derived. Doubling times varied from 34 to 72 hr. All lines showed relatively high viability and were not contaminated with Mycoplasma or bacteria. All lines showed aneuploidy and were proven to be unique by DNA fingerprinting analysis. Hepatitis-B-virus (HBV) DNA was integrated in the genomes of all lines. Two of the cell lines (SNU-354, SNU-368) showed expression of HBV and HBVx (HBx) transcripts. SNU-354 strongly expressed albumin, and SNU-368 expressed transferrin and insulin-like growth factor II. No lines produced alpha fetoprotein at the RNA and protein level. These cell lines represent useful tools for in vitro studies related to hepatocellular carcinoma. PMID- 7543081 TI - Retinoic acid and methotrexate specifically increase PHA-E-lectin binding to a 67 kDa glycoprotein in LA-N-1 human neuroblastoma cells. AB - Retinoic acid (RA) decreased growth and increased morphologic differentiation of human neuroblastoma LA-N-1 cells. These phenomena correlated with a specific enhancement of PHA-E lectin binding to a glycoprotein of MW 67 kDa (gp67). Gp67 was found susceptible to N-glycanase and displayed BSA binding by affinity chromatography analysis. The chemotherapeutic agent methotrexate (MTX) also reduced growth and induced differentiation of LA-N-1 cells. In addition, the cells responded to MTX as well as to doxorubicin by a marked increase in PHA-E binding to gp67. We conclude that reduced growth and induction of morphological differentiation of LA-N-1 cells correlates with increased binding of PHA-E to gp67. PMID- 7543084 TI - Bion's use of modern logic. AB - This paper introduces the reader to modern logic, more particularly the branch of logic called 'Model Theory', which is extensively used by Bion. Logic today is no longer described as the study of 'valid inference', but rather by the notion of consistency of beliefs. This central concern of the logician with consistency is also seen to be that of the psychoanalyst when he aims for eventual resolution of conflict between a patient's contradictory beliefs. Models are explained as the use of the already familiar to explain the unfamiliar, as when, for example, we invoke our familiar planetary system to understand the complex structure of the atom. In psychoanalysis we seek to find the old childhood models underpinning a patient's contemporary model of himself and others. Thus both Model Theory and psychoanalysis are interested in how we 'see the new in terms of the old'. Finally, the creation of models is assimilated to the integrative tendency of depressive activity and the dissolving or collapsing of models linked to paranoid schizoid activity. As these two processes in the individual are linked by Bion we can see human beings as continually oscillating between creating and destroying models of their inner and outer worlds. PMID- 7543083 TI - Effects of intravenous magnesium sulphate in suspected acute myocardial infarction on acute arrhythmias and long-term outcome. AB - A total of 252 patients with suspected acute myocardial infarction were included in a double blind study and randomised to 50 mmol magnesium sulfate infusion under 20 h or corresponding placebo. Acute myocardial infarction was verified in 117 patients and 59% of these had concomitant treatment with thrombolysis. One hundred ninety-four patients had Holter registrations during the first day in the coronary care unit. Intention-to-treat analysis showed an increase in long RR intervals (> 3 s) in the magnesium treated group (P = 0.006) and a tendency toward a reduction in episodes of ventricular premature complexes in triplets (P = 0.09). During hospital stay and a mean of 22 months follow-up, 23 fatal events occurred in the magnesium allocated group and 31 fatal events among the placebo allocated group (P = 0.1). Mortality rate from cardiac disease was reduced by 54% (95% C.I. 30-99%, P < 0.05). Subgroup analysis on acute myocardial infarction patients showed a 48% mortality risk reduction in the magnesium treated acute myocardial infarction group compared to the placebo treated acute myocardial infarction group (95% C.I. 23-104%, P = 0.06). There was no significant interaction between the effects of magnesium and thrombolytic treatment on total mortality or cardiac events. This study supports the results of other small double blind placebo controlled studies regarding effects of magnesium therapy on mortality in acute myocardial infarction, but are in discordance to the conclusion from the ISIS-4 study. The reasons for these discrepancies cannot be elucidated by our data. PMID- 7543085 TI - Serum hormones, follicular fluid steroids, insulin-like growth factors and their binding proteins, and ovarian IGF mRNA in sheep with different ovulation rates. AB - Ovulation rate, serum hormone concentrations, follicular fluid (FFL) concentrations of steroids and IGF, IGF binding protein (IGFBP) activity in FFL, and follicular IGF-I and -II mRNA were compared during the follicular phase among five genotypes of ewes: Finn (F), Composite III (C), 1/2 Booroola Merino (B) x 1/2 F (B x F), 1/2 F x 1/2 C (F x C), 1/2 B x 1/2 C (B x C). Composite III ewes were a Columbia x Suffolk x Hampshire crossbred. Ovulation rates for F (n = 7), C (n = 5), B x F (n = 6), F x C (n = 3), and B x C (n = 8) ewes were 3.1, 1.6, 3.8, 2.9, and 2.9 (Pooled SEM = .5), respectively. Concentrations of IGF-I in FFL were 53% greater (P < .05) in large (> or = 4.1 mm) than in small (< 4.1 mm) follicles but did not differ (P > .10) among genotypes. In contrast, FFL IGF-II concentrations were greater (P < .05) in B x C and B x F ewes than in C or F x C ewes but did not differ between small and large follicles. Ligand blotting revealed that IGFBP activity of three species (34, 27 to 29, and 24 kDa) were lower (P < .05) in FFL of large than in FFL of small follicles but did not differ (P < .10) among genotypes. Follicular wall IGF-I mRNA and IGF-II mRNA was detected in 5 and 32% of the samples from preovulatory follicles, respectively, using reverse transcriptase-PCR and ethidiumbromide staining. Ovarian IGF-I mRNA levels, assessed by Northern analysis, in B x F and B x C ewes were greater (P < .05) than those in C ewes; ovarian IGF-I mRNA levels in F and F x C ewes were intermediate and did not differ (P > .10) from those in C ewes. Small follicles from B x C and B x F ewes had severalfold greater (P < .05) estradiol concentrations than those from F or C ewes, whereas large follicles from B x F ewes had twice (P < .05) the estradiol concentrations of follicles from F or C ewes. Progesterone in FFL did not differ among genotypes. Serum LH, FSH, inhibin, IGF-I, and progesterone did not differ (P > .10) among genotypes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543086 TI - Hydrolysis of DNA and RNA through cooperation of two metal ions: a novel mimic of phosphoesterases. AB - The phosphodiester linkages in DNA and RNA are efficiently hydrolyzed through cooperation of the lanthanum(III) ion with the iron(III) or tin(IV) ion, although each of these metal ions exhibits poor activity. The conversion on the hydrolysis of adenylyl(3'-5')adenosine by the lanthanum(III)/iron(III) combination (10 mM each) at pH 7.0 and 50 degrees C for 5 min is 72 mol%, whereas the catalysis by the lanthanum or iron ion alone is only marginal. The activity of their combination in the hydrolysis of thymidyl(3'-5')thymidine is more than 15-fold greater than the sum of the activity of each of the two metal ions. PMID- 7543087 TI - P-glycoprotein and cell volume-activated chloride channels. AB - The multidrug resistance P-glycoprotein (P-gp) is an active drug transporter which can expel hydrophobic compounds from cells. Expression of P-gp has many effects on cells and tissues and the physiological function, or functions, of P gp are still unclear. Recently, expression of P-gp has been associated with altered activity of chloride channels which play a role in regulating cell volume of response to osmotic shock or nutrient uptake. The nature and physiological role of this association has been a subject of some debate. In this article, mechanisms by which P-gp might influence cell volume-activated chloride currents is discussed, and the potential physiological role of this regulation considered. PMID- 7543088 TI - Immunoelectron microscopic study of the distribution of porin on outer membranes of rat heart mitochondria. AB - The distribution of porin on the outer membranes of rat heart mitochondria has been studied by means of immunogold labelling with antibodies to the N-terminal part of the human protein. It was found that only a minority of isolated, unfixed mitochondria are labelled by these antibodies, with the gold particles frequently organized in threads or bands. Extensive immunogold labelling is frequently observed on regions of outer membranes stripped away from mitochondria and on regions separating two mitochondrial compartments whose cristae display different configurations (possibly representing two mitoplasts covered by a common outer membrane). Also, pairs of connected mitochondria are sometimes heavily labelled in the "neck" regions, which may represent the junctions involved in electrical communication between mitochondria in cardiac tissue. PMID- 7543089 TI - The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. AB - The particulate enzyme, endothelial nitric oxide synthase (eNOS), produces nitric oxide to maintain normal vasodilator tone in blood vessels. In this study, we demonstrate that eNOS is a Golgi-associated protein in cultured endothelial cells and intact blood vessels. Using a heterologous expression system in HEK 293 cells, we show that wild-type myristoylated and palmitoylated eNOS, but not mutant, non-acylated eNOS targets to the Golgi. More importantly, HEK 293 cells expressing wild-type eNOS release substantially more NO than cells expressing the mutant, non-acylated enzyme. Thus, eNOS is a novel Golgi-associated protein, and Golgi compartmentalization is necessary for the enzyme to respond to intracellular signals and produce NO. PMID- 7543090 TI - The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. AB - The cornified cell envelope (CE) is a 15-nm thick layer of insoluble protein deposited on the intracellular side of the cell membrane of terminally differentiated stratified squamous epithelia. The CE is thought to consist of a complex amalgam of proteins cross-linked by isodipeptide bonds formed by the action of transglutaminases, but little is known about how or in which order the several putative proteins are cross-linked together. In this paper, CEs purified from human foreskin epidermis were digested in two steps by proteinase K, which released as soluble peptides about 30% and then another 35% of CE protein mass, corresponding to approximately the outer third (cytoplasmic surface) and middle third, respectively. Following fractionation, 145 unique peptides containing two or more sequences cross-linked by isodipeptide bond(s) were sequenced. Based on these data, most (94% molar mass) of the outer third of CE structure consists of intra- and interchain cross-linked loricrin, admixed with SPR1 and SPR2 proteins as bridging cross-links between loricrin. Likewise, the middle third of CE structure consists largely of cross-linked loricrin and SPR proteins, but is mixed with the novel protein elafin which also forms cross-bridges between loricrin. In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. In addition, the data support a model for the intermediate to final stages of CE assembly: the proteins elafin, SPR1 and SPR2, and loricrin begin to be deposited on a preformed scaffold; later, elafin deposition decreases as loricrin and SPR accumulation continues to effect final assembly. The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events. PMID- 7543092 TI - Neuronal nitric oxide synthase. Expression in Escherichia coli, irreversible inhibition by phenyldiazene, and active site topology. AB - A gene coding for rat neuronal nitric oxide synthase (nNOS) has been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with a final yield of purified protein of approximately 1 mg/g of wet cells. The recombinant protein reconstituted with calmodulin and Ca2+ exhibits spectroscopic and catalytic properties identical to those reported in the literature for nNOS. Reaction of recombinant nNOS with phenyldiazene produces a phenyl-iron (Fe.Ph) complex with a maximum at 470 nm. Formation of this complex is paralleled by inactivation of the enzyme and is inhibited by arginine, the natural substrate of the enzyme. Phenyl-iron complex formation does not alter the rate of electron transfer from the flavin domain to cytochrome c. Addition of ferricyanide triggers migration of the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the nitrogens of pyrrole rings B, A, C, and D are formed in, respectively, approximately a 14:20:21:45 ratio. The regioisomer pattern indicates that the active site of NOS is open to some extent above all four pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus not only a new class of inhibitors of nNOS but provide useful information on the active site topology of the enzyme. PMID- 7543093 TI - Isolation of human aquaporin 3 gene. AB - Human aquaporin 3 (AQP3) gene was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and to comprise six exons distributing over 7 kilobases. The sizes of the exons are 171, 127, 138, 119, 218, and 1035 base pairs, and those of introns are approximately 3530, 300, 350, 330, and 90 base pairs, respectively. The initiation site of transcription was identified to locate 64 base pairs upstream of the first ATG codon by primer extension analysis and ribonuclease protection assay. The 5'-flanking region has a TATA box, two Sp1 sequences, and some consensus sequences including AP2 sites. With luciferase assay, the 5'-flanking region was demonstrated to have a promoter activity, which is up-regulated 4-fold by phorbol ester. These findings about the genomic clone of human AQP3 will contribute to elucidate the molecular mechanism of transcriptional regulation of AQP3. PMID- 7543094 TI - Lipopolysaccharide (LPS) neutralizing peptides reveal a lipid A binding site of LPS binding protein. AB - Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting. PMID- 7543091 TI - Activation of a cAMP-regulated Ca(2+)-signaling pathway in pancreatic beta-cells by the insulinotropic hormone glucagon-like peptide-1. AB - Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells. PMID- 7543095 TI - Transcription stimulation of the Fas-encoding gene by nuclear factor for interleukin-6 expression upon influenza virus infection. AB - We previously found that the level of Fas, a cell surface receptor for an apoptosis signal, increases at the mRNA level in influenza virus-infected HeLa cells prior to their death by apoptosis. Here we investigated the mechanism of activation of the Fas-encoding gene expression upon influenza virus infection. Nucleotide sequences for the binding of nuclear factor for interleukin-6 expression (NF-IL6), also known as CCAAT/enhancer-binding protein beta, were repeated 8 times in the 5'-end region of the human FAS gene, spanning from -1360 to +320. This region directed the expression of a downstream marker gene when introduced into HeLa cells and the activity of the FAS gene promoter was stimulated about 2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced in a DNA when human NF-IL6 was overproduced in a DNA co-transfection study. Moreover, the DNA binding activity of NF-IL6 increased after infection with the virus, whereas the amount of NF-IL6 seemed unchanged. The results suggest that NF-IL6 is activated upon influenza virus infection through post-translational modification and that the modified factor stimulates the transcription of the human FAS gene. PMID- 7543097 TI - The membrane-proximal scavenger receptor cysteine-rich domain of CD6 contains the activated leukocyte cell adhesion molecule binding site. AB - Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand. PMID- 7543096 TI - The activation state of the integrin alpha IIb beta 3 affects outside-in signals leading to cell spreading and focal adhesion kinase phosphorylation. AB - Integrins bind extracellular matrix and transduce signals mediating cell adhesion, spreading, and migration. It is unclear how these distinct responses follow from a common event: integrin clustering. We examined the relationship between integrin-mediated signals and the integrin's activation state using a cell line expressing alpha IIb beta 3 (Clone B) and a panel of monoclonal antibodies against this integrin. Non-activating antibodies used to cluster alpha IIb beta 3 stimulated focal adhesion kinase (FAK) phosphorylation, regardless of affinity, subunit specificity, or ligand-blocking phenotype. Coated on plastic, these antibodies supported cell adhesion, spreading, and FAK phosphorylation. In contrast, clustering of alpha IIb beta 3 induced with activating antibodies, or binding of soluble fibrinogen to antibody-activated alpha IIb beta 3, did not induce FAK phosphorylation. Thus, clustering of alpha IIb beta 3 on Clone B does not necessarily result in FAK phosphorylation. Coated on plastic, activating antibodies supported cell adhesion, but not spreading or FAK phosphorylation. Therefore, it appears the resting, not the active form of alpha IIb beta 3, induces cell spreading and FAK phosphorylation in Clone B. These data indicate that "inside-out" signals may alter not only the binding specificity of an integrin, but the "outside-in" biochemical signals that integrin initiates as well. This activation state-linked signaling represents a novel mechanism, which may explain how diverse cellular responses are induced by integrin-matrix interactions. PMID- 7543098 TI - Specificity of the PTB domain of Shc for beta turn-forming pentapeptide motifs amino-terminal to phosphotyrosine. AB - Shc phosphorylation in cells following growth factor, insulin, cytokine, and lymphocyte receptor activation leads to its association with Grb2 and activation of Ras. In addition to being a cytoplasmic substrate of tyrosine kinases, Shc contains an SH2 domain and a non-SH2 phosphotyrosine binding (PTB) domain. Here we show that the Shc PTB domain, but not the SH2 domain, binds with high affinity (ID50 approximately equal to 1 microM) to phosphopeptides corresponding to the sequence surrounding Tyr250 of the polyoma virus middle T (mT) antigen (LLSNPTpYSVMRSK). Truncation studies show that five residues amino-terminal to tyrosine are required for high affinity binding, whereas all residues carboxyl terminal to tyrosine can be deleted without loss of affinity. Substitution studies show that tyrosine phosphorylation is required and residues at -5, -3, 2, and -1 positions relative to pTyr are important for this interaction. 1H NMR studies demonstrate that the phosphorylated mT antigen-derived sequence forms a stable beta turn in solution, and correlations between structure and function indicate that the beta turn is important for PTB domain recognition. These results show that PTB domains are functionally distinct from SH2 domains. Whereas SH2 domain binding specificity derives from peptide sequences carboxyl-terminal to phosphotyrosine, the Shc PTB domain gains specificity by interacting with beta turn-forming sequences amino-terminal to phosphotyrosine. PMID- 7543099 TI - The chicken oocyte receptor for yolk precursors as a model for studying the action of receptor-associated protein and lactoferrin. AB - Receptor-associated protein (RAP) was originally described as a 39-kDa intracellular protein copurifying with mammalian low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR). RAP has a high affinity for LRP/alpha 2MR and interferes with the receptor's ability to bind a variety of ligands. The laying hen expresses, in a tissue-specific manner, at least four different proteins which belong to the same family of receptors as LRP/alpha 2MR. Here we show that the chicken also produces RAP, so far thought to be expressed only in mammals. Studies on the interaction of recombinant human RAP with the LDL receptor family in the chicken revealed that RAP binds with high affinity to the abundant oocyte receptor for yolk precursors (OVR) as well as to the somatic cell-specific LRP/alpha 2MR. Significantly, RAP interacts with a lower affinity with the LDL receptor, but does not bind to the oocyte-specific form of LRP. Binding of RAP to OVR inhibits the interaction of the receptor with all known physiological ligands, i.e. the yolk precursors very low density lipoprotein, vitellogenin, and alpha 2-macroglobulin. In COS cells transfected with OVR, RAP is internalized and degraded in a concentration-dependent and saturable manner. Lactoferrin, another protein with a high affinity for mammalian LRP/alpha 2MR, also binds to OVR and abolishes its interaction with yolk precursors. Cross-competition experiments show that RAP and lactoferrin recognize sites different from those involved in yolk precursor binding. The availability of pure OVR and LDLR enable us to determine kinetic parameters for the binding of RAP and lactoferrin to these receptors by surface plasmon resonance. Taken together, our results strongly suggest that chicken OVR, which is easily accessible and highly abundant in growing oocytes, represents a superior system for studying mechanistic and structural aspects of the interaction of ligands and modulating proteins with members of the LDL receptor gene family. PMID- 7543101 TI - Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli. AB - The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein. PMID- 7543100 TI - Sequence analysis of sarcosine oxidase and nearby genes reveals homologies with key enzymes of folate one-carbon metabolism. AB - Corynebacterial sarcosine oxidase, a heterotetrameric (alpha beta gamma delta) enzyme containing covalent and noncovalent FAD, catalyzes the oxidative demethylation of sarcosine to yield glycine, H2O2, and 5,10-CH2-tetrahydrofolate (H4folate) in a reaction requiring H4folate and O2. The sarcosine oxidase operon contains at least five closely packed genes encoding sarcosine oxidase subunits and serine hydroxymethyltransferase (glyA), arranged in the order glyAsoxBDAG. The operon status of a putative purU gene, found 340 nucleotides downstream from soxG, is not known. No homology with other proteins is observed for the smallest sarcosine oxidase subunits gamma and delta. The beta subunit (405 residues) contains an ADP-binding motif near its NH2 terminus, the covalent FAD attachment site (H175), and exhibits homology with the NH2-terminal half of dimethylglycine dehydrogenase (857 residues) and monomeric, bacterial sarcosine oxidases (approximately 388 residues), enzymes that contain a single covalent FAD. The alpha subunit (967 residues) contains a second ADP-binding motif within an approximately 280 residue region near the NH2 terminus that exhibits homology with subunit A from octopine and nopaline oxidases, heterodimeric enzymes that catalyze analogous oxidative cleavage reactions with N-substituted arginine derivatives. An approximately 380 residue region near the COOH terminus of alpha exhibits homology with T-protein and the COOH-terminal half of dimethylglycine dehydrogenase. These enzymes catalyze the formation of 5,10-CH2-H4folate, using different one-carbon donors. The results suggest that the alpha subunit and dimethylglycine dehydrogenase contain an NH2-terminal domain that binds noncovalent or covalent FAD, respectively, and a carboxyl-terminal H4folate binding domain. PMID- 7543102 TI - Signal transduction by a CD16/CD7/Jak2 fusion protein. AB - The addition of interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) to hormone-dependent cells induces tyrosine phosphorylation of Janus protein kinase 2 (Jak2) and activates its in vitro kinase activity. To explore the role of Jak2 in IL-3/GM-CSF-mediated signal transduction, we constructed a CD16/CD7/Jak2 (CD16/Jak2) fusion gene containing the external domain of CD16 and the entire Jak2 molecule and expressed this fusion protein using a recombinant vaccinia virus. The clustering of CD16/Jak2 fusion protein by cross-linking with an anti-CD16 antibody induced autophosphorylation of the fusion protein but did not induce the phosphorylation of either the endogenous Jak2 or the beta chain. Cross-linking of CD16/Jak2 stimulates the tyrosine phosphorylation of a large group of proteins that are also phosphorylated after the addition of IL-3 or GM-CSF and include proteins of 145, 97, 67, 52, and 42 kDa. Closer analysis demonstrated that the CD16/Jak2 phosphorylates Shc, a 52-kDa protein, and the 145-kDa protein associated tightly with Shc, as well as mitogen-associated protein kinase (pp42). Electrophoretic mobility shift assays demonstrate that CD16/Jak2 activates the ability of signal transduction and activation of transcription (STAT) proteins to bind to an interferon-gamma-activated sequence oligonucleotide in a manner similar to that seen after IL-3 treatment. Cross-linking of the CD16/Jak2 protein stimulated increases in c-fos and junB similar to IL-3 but did not cause major changes in the levels of the c-myc message, which normally increases after IL-3 treatment. Thus, a transmembrane CD16/Jak2 fusion is capable of activating protein phosphorylation and mRNA transcription in a manner similar but not identical to hematopoietic growth factors. PMID- 7543103 TI - Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries. AB - Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter. PMID- 7543104 TI - Cloning and characterization of multiple human genes and cDNAs encoding highly related type II keratin 6 isoforms. AB - The human type II keratin 6 (K6; 56 kDa) is expressed in a heterogeneous array of epithelial tissues under normal conditions, but is better known for its strong induction in stratified epithelia that feature an enhanced cell proliferation rate or abnormal differentiation. Previous work has established the existence of two functional genes encoding K6 protein isoforms in the human genome, although only a partial cDNA clone is available for K6a, the dominant human K6 isoform in skin epithelial tissues (Tyner, A., and Fuchs, E. (1986) J. Cell Biol. 103, 1945 1955). We screened human genomic and skin cDNA libraries with probes derived from the K6b gene, and isolated clones containing the full-length gene and cDNA predicted to encode K6a. A thorough characterization of a large number of genomic (57) as well as cDNA (64) clones further revealed the existence of as many as six different human K6 protein isoforms that are highly related at the gene structure, nucleotide sequence, and predicted amino acid sequence levels. Based on the information accumulated to date we propose an evolutionary model in which the multiplicity of human K6 genes is explained by successive gene duplication events. We further demonstrate that K6a is clearly the dominant K6 isoform in skin tissue samples and cultured epithelial cell lines and that the various isoforms are differentially regulated within and between epithelial tissue types. Our findings have direct implications for an understanding of the regulation and function of K6 during hyperproliferation in stratified epithelia and the search for disease-causing mutations in K6 sequences in the human population. PMID- 7543105 TI - Alpha v beta 5 integrin receptor-mediated endocytosis of vitronectin is protein kinase C-dependent. AB - Previous studies have demonstrated that the alpha v beta 5 integrin receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the beta 5 integrin or exogenous heparin, suggesting that both integrin receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha v beta 5 integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-alpha v beta 5 antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the alpha v beta 5 integrin following activation of protein kinase C. PMID- 7543106 TI - Effect of anthrax toxin's lethal factor on ion channels formed by the protective antigen. AB - Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment. PA63, a 63-kDa proteolytic fragment of PA, can be induced to form ionconductive channels in the plasma membrane of mammalian cells by acidification of the medium. These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA63 seen by electron microscopy of the purified protein. Here we report that the PA63-mediated efflux of 86Rb+ from preloaded CHO K1 cells under acidic conditions is strongly inhibited (> or = 70%) by LF or LFN, a PA-binding fragment of LF. Control proteins caused no inhibition. Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA63 channel. Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH approximately 5.3), suggesting that channel formation may also be retarded under these conditions. The relevance of these results to the location of the LF-binding site on PA63 and the mechanism of LF and EF translocation is discussed. PMID- 7543107 TI - Agonist-stimulated ligand binding by the platelet integrin alpha IIb beta 3 in a lymphocyte expression system. AB - The ligand binding activity of the platelet integrin alpha IIb beta 3 is initiated by agonist-generated intraplatelet signals. We studied this process in vitro by expressing recombinant alpha IIb beta 3 in Epstein-Barr virus immortalized B lymphocytes. We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing alpha IIb beta 3 to immobilized fibrinogen. Moreover, replacement of the transmembrane and cytoplasmic domains of the alpha and beta subunits of alpha IIb beta 3 with those of alpha L beta 2 significantly increased adherence, whereas replacement of only the cytoplasmic domains significantly decreased adherence. This suggests that transmembrane segments are involved in the agonist-induced modulation of alpha IIb beta 3 activity. Similar results were seen when the alpha IIb beta 3 activation-dependent monoclonal antibody PAC-1 was substituted for immobilized fibrinogen. We also found that the adherence of lymphocytes expressing beta 3 with either of the two alpha IIb/alpha L chimeras was similar to that of cells expressing alpha IIb beta 3, whereas the adherence of cells expressing alpha IIb with either of the two beta 3/beta 2 chimeras was substantially decreased, suggesting that the identity of the cytoplasmic domain of beta 3, but not of alpha IIb, is critical for alpha IIb beta 3 function. This report indicates that B lymphocytes contain signal transduction pathways involving protein kinase C that can increase the ligand binding activity of alpha IIb beta 3 and demonstrates the utility of these cells as an expression system for the study of agonist-stimulated alpha IIb beta 3 function. PMID- 7543108 TI - Biosynthesis of bikunin proteins in the human carcinoma cell line HepG2 and in primary human hepatocytes. Polypeptide assembly by glycosaminoglycan. AB - In this report we describe a series of experiments designed to probe the biosynthesis of the bikunin proteins. The bikunin proteins are serine proteinase inhibitors found in high concentrations in human plasma. The proteins are composed of two or three polypeptide chains assembled by a newly identified carbohydrate mediated covalent inter-chain "Protein-Glycosaminoglycan-Protein" (PGP) cross-link (Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751). In this study we show that transformed hepatocyte cell lines, exemplified by HepG2 cells, have lost the ability to produce these proteins. In contrast, primary human hepatocytes produce bikunin proteins identical to the proteins identified in human plasma. Pulse-chase analysis demonstrate that the PGP-mediated cross linking of the polypeptide chains occurs late in the secretary pathway. Moreover, the mechanism responsible for the formation of the PGP cross-link is divided in two steps involving a proteolytic cleavage followed by carbohydrate attachment. The results indicate that normal hepatocytes contain the biosynthetic machinery required for correct synthesis and processing. However, transformed cell lines are defective in several aspects of bikunin biosynthesis precluding such systems from being used as relevant in vitro models. PMID- 7543109 TI - IGF binding protein-3 protease regulation: how sweet it is! PMID- 7543110 TI - Insulin-like growth factor binding protein-3 proteolysis in children with insulin dependent diabetes mellitus: a possible role for insulin in the regulation of IGFBP-3 protease activity. AB - Limited proteolysis of serum insulin-like growth factor (IGF) binding protein (IGFBP)-3 has been described in various conditions and may increase the bioavailability of IGFs. The physiological regulators of serum IGFBP-3 protease activity are unknown. To characterize the relationship between insulin and IGFBP 3 protease activity, we have examined serum IGFBP-3 proteolysis in children with untreated insulin-dependent diabetes mellitus (IDDM) and have followed the effect of insulin therapy on serum IGFBP-3 proteolysis at 1 day, 1 week, and 1 month after the initiation of insulin therapy. Ligand blot analysis of sera from untreated children with IDDM showed that intact IGFBP-3 was 50 +/- 9% of the age matched control pool. After the initiation of insulin treatment, IGFBP-3 did not change significantly at 1 day after treatment but increased dramatically at 1 week (90 +/- 13%) and 1 month after treatment (102 +/- 13%). In contrast, when measured by immunoradiometric assay (which detects both intact and fragments of IGFBP-3), IGFBP-3 levels were 70% of the control pool before insulin therapy and did not increase significantly until 1 month after treatment. Immunoblot analysis demonstrated that intact IGFBP-3 doublet was diminished to 41 +/- 7% of controls, whereas the major IGFBP-3 fragment (30 kDa) was increased in IDDM sera before insulin therapy. After insulin, intact IGFBP-3 increased and the 30-kDa fragment decreased to values comparable to those observed in controls. In vivo IGFBP-3 proteolysis, which implies preassay exposure of serum IGFBP-3 to proteases, was estimated by immunoblot analysis. IGFBP-3 proteolysis was increased before insulin therapy (160 +/- 9%) and decreased to 81 +/- 9% at 1 week and to 71 +/- 11% at 1 month after insulin treatment. Residual serum IGFBP-3 protease activity was estimated by a 125I-IGFBP-3 degradation assay. Serum IGFBP-3 protease activity increased significantly in untreated diabetics, compared with activity in controls (128 +/- 5% vs. 99 +/- 11%). During insulin therapy, serum IGFBP-3 protease activity decreased gradually to 91 +/- 5% of control values at 1 month. Molecular sizes of the IGFBP-3 proteolytic fragments (30 kDa, 24 kDa, and 19 kDa) and inhibition profile of IGFBP-3 protease were similar in IDDM and pregnancy sera, indicating that similar proteases (cation-dependent serine proteases) were active in both conditions. These results suggest an important role of insulin in the regulation of IGFBP-3 protease activity. Increased IGFBP-3 proteolysis in the sera of children with IDDM may serve to counteract the catabolic state induced by insulin deficiency. PMID- 7543112 TI - Does thyroidectomy, radioactive iodine therapy, or antithyroid drug treatment alter reactivity of patients' T cells to epitopes of thyrotropin receptor in autoimmune thyroid diseases? AB - The effect of treatment on thyroid antibody production and T cell reactivity to thyroid antigens was studied in 15 patients with Graves' disease (GD) before and after thyroidectomy, 19 patients with GD before and after radioactive iodine (RAI) therapy, and 9 patients maintained euthyroid on antithyroid drugs (ATD). Twenty subjects matched for age and sex without known thyroid disease served as controls. In GD patients, the responses of peripheral blood mononuclear cells (PBMC) and TSH receptor (TSHR)-specific T cell lines to recombinant human TSHR extracellular domain, thyroglobulin, and TSHR peptides were examined on the day of surgery or RAI therapy (day 0) and also 6-8 weeks and 3-6 months thereafter. Reactivity to TSHR peptides before surgery was heterogeneous and spanned the entire extracellular domain. Six to 8 weeks after subtotal thyroidectomy, the number of patients' PBMC responding to any peptide and the average number of recognized peptides decreased. A further decrease in the T cell reactivity to TSHR peptides was observed 3-6 months after surgery. The responses of PBMC from Graves' patients before RAI therapy were less than those in the presurgical group. Six to 8 weeks after RAI therapy, the number of patients responding to any peptide and the average number of recognized peptides increased. Three to 6 months after RAI, T cell responses to TSHR peptides were less than those 6-8 weeks after RAI therapy, but still higher than the values on day 0. Responses of PBMC from patients with GD, maintained euthyroid on ATD, were lower than those before surgery or RAI therapy. The reactivity of T cell lines in different groups reflected a pattern similar to PBMC after treatment. TSHR antibody and microsomal antibody levels decreased after surgery, but increased after RAI therapy. The difference in the number of recognized peptides by patients' PBMC before RAI and surgery may reflect the effect of long term therapy with ATD in the patients before RAI vs. the shorter period in patients before surgery. The decreased T cell reactivity to thyroid antigens after thyroidectomy could be the result of removal of a major part of the thyroid gland or redistribution of suppressor inducer T cells. The increased T cell response after RAI therapy is probably epitope specific, rather than a response to the whole TSHR molecule. Synchronous recognition of peptides 158-176 and 248-263 is important for the development of GD, and the loss of recognition of one of these epitopes may be an early sign of immune remission and a predictor of euthyroidism. PMID- 7543111 TI - Greater secretion of growth hormone in black than in white men: possible factor in greater bone mineral density--a clinical research center study. AB - To determine why blacks have a higher bone mineral density (BMD) and lower incidence of osteoporosis and fractures than whites, we investigated whether the secretion of GH is higher in black than in white men. Measurements of GH were obtained at 20-min intervals over 24 h and analyzed by deconvolution. BMD was determined by dual energy x-ray absorptiometry in 16 normal black and 17 normal white men, aged 20-40 yr. The 24-h integrated GH concentration 942 +/- 174 vs. 602 +/- 104 micrograms/L; P = 0.0495) and GH secretory burst amplitude (0.499 +/- 0.163 vs. 0.169 +/- 0.027 micrograms/L.min; P = 0.0482) were higher in black than in white men. GH burst frequency, half-duration, mass, and half-life were not different in the 2 groups. The serum 17 beta-estradiol level (162 +/- 12 vs. 108 +/- 11 pmol/L; P = 0.0011) was higher, and the serum insulin-like growth factor binding protein 3 level (2.2 +/- 0.1 vs. 2.8 +/- 0.1 microgram/mL; P = 0.0001) was lower in black than in white men. BMD values for total body (1.22 +/- 0.02 vs. 1.14 +/- 0.02 g/cm2; P = 0.0041), forearm (0.69 +/- 0.01 vs. 0.66 +/- 0.01 g/cm2; P = 0.0211), trochanter (0.91 +/- 0.03 vs. 0.77 +/- 0.03 g/cm2; P = 0.0003), and femoral neck (1.08 +/- 0.03 vs. 0.93 +/- 0.03 g/cm2; P = 0.0007) were higher in black than in white men. Thus, serum 17 beta-estradiol level, GH secretion, and BMD values for the total body, forearm, trochanter, and femoral neck are greater in black than in white men. As estrogen is known to increase GH secretion and GH to increase bone mass, increases in circulating 17 beta estradiol may contribute to the higher GH secretion and bone mass in black men. PMID- 7543113 TI - Potential of testosterone buciclate for male contraception: endocrine differences between responders and nonresponders. AB - Suppression of serum LH and FSH, by testosterone (T) alone or in combination with other agents, has proved to be the most promising approach to male contraception. T enanthate, the only androgen preparation tested in male contraceptive efficacy trials so far, must be injected every week due to its short terminal elimination half-life of 4.5 days and leads to supraphysiological T serum levels. A new T ester synthesized under WHO and NIH auspices, testosterone buciclate (TB), showed a favorable pharmacokinetic profile, with a terminal half-life of 29.5 days when tested in hypogonadal men. Here we describe the results of the first clinical trial with TB for male contraception. After two control examinations, normal healthy male volunteers were given a single im injection of 600 mg TB (group I; n = 4) and 1200 mg TB (group II; n = 8) on day 0. Follow-up examinations were performed every 2 weeks up to week 32. In both groups mean serum T levels remained in the normal physiological range throughout the study course. Serum levels of dihydrotestosterone (DHT) showed a dose- and time-dependent increase, with serum levels slightly above the normal range in group II for several weeks and a maximal concentration of 3.8 +/- 0.5 nmol/L (mean +/- SE) in week 6. No suppression of spermatogenesis to oligozoospermia was observed in group I. However, in group II, spermatogenesis was suppressed to azoospermia in three of eight volunteers in week 10 that persisted up to weeks 14, 20, and 22, respectively. In these three men, LH and FSH were suppressed by TB injections to the respective assay detection limits, whereas in the other five subjects, mean serum levels were only decreased to values near the lower normal limit for LH and FSH, respectively. In addition, throughout the study course, a significant difference in serum sex hormone-binding globulin was detected between the responders (mean values, 21.2-26.4 nmol/L) and nonresponders (mean values, 36.2 46.3 nmol/L). Serum levels of LH as well as total and free T at baseline and after TB injection were lower in the responders than in the nonresponders. Both subgroups showed similar increases in serum LH and FSH after GnRH stimulation. In a newly introduced GnRH antagonist suppression test, serum LH and T were decreased to significantly lower levels in the responders. These results indicate a different hormonal equilibrium and probably different susceptibility to feedback regulation of the responders compared to the nonresponders.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543115 TI - GH and PRL gene expression by nonradioisotopic in situ hybridization in TSH secreting pituitary adenomas. AB - TSH-secreting pituitary adenomas are rare. The transcriptional expression (messenger ribonucleic acids: mRNAs) of TSH beta, GH, and PRL in five patients with TSH-secreting pituitary adenoma was studied by the in situ hybridization (ISH) method in order to elucidate their multiple hormone production. These patients showed inappropriately elevated serum TSH and alpha-subunit levels as well as pituitary mass lesions. The tissues from pituitary adenomas were obtained at the time of transsphenoidal surgery and revealed immunohistochemically the expression of alpha-subunit and TSH beta in all patients. Four adenomas were immunohistochemically associated with GH or PRL localization. The presence of pituitary-specific transcriptional factor Pit-1 was demonstrated in all adenomas in the nuclei of many cells. By ISH, signals for TSH beta mRNA were present in all five cases in many adenoma cells. Expression of GH mRNA and PRL mRNA were detected not only in four adenomas in which both hormonal products were immunolocalized but also in one adenoma that was immunohistochemically negative for GH and PRL. Combined staining by ISH and immunohistochemistry revealed the expression of GH mRNA and PRL mRNA in TSH beta-immunoreactive cells. Our findings indicate that TSH-secreting adenomas are multihormone-producing and could arise from precursor or stem cells rather than from differentiated TSH-secreting cells. It is suggested that ISH combined with immunohistochemistry may provide additional detailed information concerning the multidirectional histogenesis of this rare type of adenoma. PMID- 7543114 TI - Continuous infusion versus daily injections of growth hormone (GH) for 4 weeks in GH-deficient patients. AB - Endogenous GH secretion is pulsatile. Animal studies indicate that GH administered in a pulsatile manner induces growth and insulin-like growth factor I (IGF-I) generation more effectively than continuous administration. Short term human studies, however, have reported similar metabolic effects with constant and pulsatile GH delivery. This study was carried out to compare the metabolic effects of longer term continuous infusion vs. daily injections of GH. Thirteen GH-deficient patients were studied in a cross-over design. The patients were randomized to receive GH as a continuous sc infusion by means of a portable pump for 1 month and as daily sc injections (at 1900 h) for another month. An average daily GH dosage (+/- SEM) of 3.15 +/- 0.27 IU was administered during both periods. Steady state 24-h profiles of GH, IGF-I, IGF-binding proteins (IGFBPs), insulin, glucose, lipid intermediates, and other metabolites were monitored after each treatment period. At the end of each study period (at 0800 h), an oral glucose tolerance test was performed. The mean (+/- SEM) integrated levels of serum GH (micrograms per L) were higher after GH injection [2.51 +/- 0.54 (injection) vs. 1.77 +/- 0.35 (infusion); P < 0.02]. Continuous infusion induced higher nighttime than daytime GH levels (P = 0.01), indicating a diurnal variation in the absorption or clearance of GH. Serum IGF-I levels (micrograms per L) were slightly higher (P < 0.05, by analysis of variance) after continuous GH infusion [312.5 +/- 50.2 (injection) and 334.6 +/- 46.6 (infusion)]. Similarly, constant GH delivery induced higher IGFBP-3 levels (P < 0.05, by analysis of variance). Serum IGFBP-1 levels were similar on the two occasions. Daily GH injections increased daytime insulin levels (P < 0.05), whereas 24-h levels were similar (P = 0.14). The trend toward increased insulin levels after GH injections was also found during the oral glucose tolerance test (P = 0.07). Blood glucose levels were identical on the two occasions. Nocturnal levels of nonesterified fatty acids were higher (P < 0.05) after GH injection. We conclude that continuous sc infusion of GH induced serum IGF-I and IGFBP-3 levels more effectively than daily sc injections. The constant appearance of GH in the circulation did not impair glucose tolerance, but resulted in a less physiological diurnal pattern of nonesterified fatty acids. Our data do not support the concept that a pulsatile GH pattern is of critical physiological significance. PMID- 7543116 TI - Serum levels of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in healthy infants, children, and adolescents: the relation to IGF-I, IGF-II, IGFBP 1, IGFBP-2, age, sex, body mass index, and pubertal maturation. AB - Circulating IGF-I and -II are bound to specific insulin-like growth factor (IGF) binding proteins (IGFBPs), of which IGFBP-3 binds the majority of the IGFs. IGFBP 3 levels are regulated by GH and have been suggested to provide additional information on GH secretory capacity compared to IGF-I. However, the diagnostic value of IGFBP-3 is still controversial, perhaps because the quality of the available normative data for IGFBP-3 varies. It has recently been shown that a large number of individuals is required to establish reference ranges for IGF-I that take into account age, sex, body mass index (BMI), and pubertal stage. Therefore, we measured IGFBP-3, IGF-I, IGF-II, IGFBP-1, and IGFBP-2 levels by RIA in 907 healthy children to establish well characterized normative data on IGFBP-3 according to age, sex, and pubertal stage and to study the complex relationship between IGFs and their BPs in puberty. We found that IGFBP-3 levels increase with age in children, with maximal levels in puberty; girls experience peak values approximately 1 yr earlier than boys. Age, sex, height, BMI, and pubertal maturation were all important factors in determining the circulating levels of IGFBP-3, whereas IGF-I levels were unaffected by BMI. Comparison of IGFBP-3 with IGF-1 concentrations revealed that they did not exhibit the same developmental pattern in puberty. IGF-I levels increased to relatively higher levels than IGFBP 3, leading to an increasing molar ratio between IGF-I and IGFBP-3 in puberty, when growth velocity is high. Concomitantly, IGF-II and IGFBP-2 levels were unchanged throughout puberty, whereas IGFBP-1 levels declined with age in prepubertal children, with lowest values in puberty. There was a highly significant correlation between IGF-I and -II and IGFBP-3 on a molar basis (r = 0.84; P < 0.0001). Thus, we speculate that IGFBP-3 is pivotal for circulating IGF bioactivity and that the increase in the molar ratio between IGF-I and IGFBP-3 reflects an increase in free, biologically active IGF-I. In conclusion, we have provided normative data on a large group of healthy individuals and conclude that age, sex, height, BMI, and pubertal maturation have to be taken into account before a single IGFBP-3 value in a growth-retarded child can be evaluated properly. PMID- 7543117 TI - Identification of immunodominant epitopes in the core and non-structural region of hepatitis C virus by enzyme immunoassay using synthetic peptides. AB - Thirty-two synthetic peptides, components of the core and non-structural protein of Hepatitis C virus (HCV), were tested for their reactivities against antibodies in sera of healthy, HCV antibody positive of chronic liver disease patients. Among them, 8 of the core peptides, 4 of the NS4 peptides and 3 of the NS5 peptides reacted with the HCV infected sera. In particular, C22 (core peptide) and NS4-1924 (NS4 peptide) were most reactive with the serum samples giving a positive signal with commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our results indicate that the immunodominant regions of the HCV derived proteins are located at three regions in the core protein, three regions in the NS4 protein, and one region in the NS5 protein. These results indicate that the selected peptides are useful antigens in detecting antibodies in the sera from individuals infected with HCV. PMID- 7543118 TI - Immunohistochemical demonstration of different latent membrane protein-1 epitopes of Epstein-Barr virus in lymphoproliferative diseases. AB - AIM: To compare the immunoreactivity of monoclonal antibodies S12 and CS1-4, which recognise different epitopes of the Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1), in EBV associated benign and malignant lymphoproliferative disorders and control tissues processed using different methods. RESULTS: Both monoclonal antibodies gave comparable results on frozen tissue sections and formalin fixed, paraffin wax embedded samples from cases with Hodgkin's disease and infectious mononucleosis. In all cases S12 stained more cells than CS1-4. For EBV associated B and T non-Hodgkin's lymphomas, frozen tissue sections yielded better LMP-1 staining results than formalin fixed material. Again, in all these cases S12 stained more cells and gave stronger results than CS1-4. For EBV negative tissues, both monoclonal antibodies showed cross-reactivity with melanocytic-like cells in the basal cell layer of the skin, synaptophysin-like staining in layers three and four of the cortex of the brain, and myelin-like staining in peripheral nerves and peripheral ganglion cells. Staining with S12 was always much stronger. Moreover, in contrast to CS1-4, S12 stained pancreatic islands in formalin fixed material but not in frozen tissue sections and sporadically stained solitary epithelial cells in the large bowel especially in formalin fixed tissue sections. CS1-4 also cross-reacted with myoepithelial cells around hair follicles and other adnexa of the skin. CONCLUSION: The results indicate that for optimal detection of LMP-1, S12 yields better results than CS1-4 and that tissue processing is very important especially when B and T non-Hodgkin's lymphomas are examined. PMID- 7543119 TI - Selective media for isolation of Burkholderia (Pseudomonas) cepacia from the respiratory secretions of patients with cystic fibrosis. AB - One hundred and six specimens from 90 patients with cystic fibrosis were evaluated for the presence of Burkholderia cepacia using a current routine diagnostic protocol as well as a research protocol involving polymyxin B MacConkey agar without crystal violet, PC agar, OFPVL agar, and a selective brain heart infusion broth. Ten specimens from eight patients (8.9%) were positive by any method. The selective enrichment broth was the only medium that yielded B cepacia from all 10 positive samples, although the routine protocol was successful for eight of these. Transient carriage was identified in one patient. Epidemiological studies may be better served by the use of selective enrichment rather than selective solid media alone. Carrier status for B cepacia requires more strict definition if positive carrier status is to be accepted as having medical importance. PMID- 7543120 TI - Thalamocortical synapses between axons from the mediodorsal thalamic nucleus and pyramidal cells in the prelimbic cortex of the rat. AB - A combined anterograde axonal degeneration and Golgi electron microscopic (Golgi EM) study was undertaken to identify thalamocortical synaptic connections between axon terminals from the mediodorsal thalamic nucleus (MD) and pyramidal cells in layers III and V of the agranular prelimbic cortex in the rat. The morphological characteristics of thalamocortical synapses from MD were also examined by labeling axon terminals with anterograde transport of wheat germ agglutinin horseradish peroxidase (WGA-HRP). WGA-HRP labeled axon terminals from MD to the prelimbic cortex were small in size (0.5-1 microns in diameter), contained round synaptic vesicles, and formed axospinous synapses with asymmetrical membrane thickenings. With Golgi-EM methods, gold-toned apical dendrites in layer III were analyzed by reconstruction of serial ultrathin sections. Following lesions in the thalamus, degenerating thalamocortical axon terminals formed asymmetrical contacts exclusively on dendritic spines of the identified apical dendrites. More thalamocortical synapses were found on apical dendrites of layer V pyramidal cells than on apical dendrites of layer III pyramidal cells. In addition to thalamocortical synapses, a very few unlabeled symmetrical synapses were found on apical dendrites and somata of pyramidal cells, but they were not quantified and their sources are unknown. PMID- 7543121 TI - Zonal organization of the climbing fiber projection to the flocculus and nodulus of the rabbit: a combined axonal tracing and acetylcholinesterase histochemical study. AB - The localization and termination of olivocerebellar fibers in the flocculus and nodulus of the rabbit were studied with anterograde axonal transport methods [wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) and tritiated leucine] and correlated with the compartments in the white matter of these lobules delineated with acetylcholinesterase histochemistry (Tan et al. J. Comp. Neurol., 1995, this issue). Olivocerebellar fibers originating from the caudal dorsal cap travel through floccular compartments FC2 and FC4 to terminate as climbing fibers in floccular zones FZII and FZIV. Fibers from the rostral dorsal cap and the ventrolateral outgrowth traverse compartments FC1 and FC3, which are interleaved with compartments FC2 and FC4, and terminate in zones FZI and FZIII. Fibers from the rostral pole of the medial accessory olive traverse the C2 compartment and terminate in the C2 zone. FZI-III extend into the adjoining folium (folium p) of the ventral paraflocculus. The C2 zone continues across folium p into other folia of the ventral paraflocculus and into the dorsal paraflocculus. Four compartments and five zones were distinguished in the nodulus. Medial compartment XC1 contains olivocerebellar fibers from the caudal dorsal cap and subnucleus beta that terminate in the XZI zone. Olivocerebellar fibers from the rostral dorsal cap and the ventrolateral outgrowth occupy XC2 and terminate in XZII. The XC4 compartment contains fibers from both the caudal dorsal cap and from the rostral dorsal cap and the ventrolateral outgrowth. The latter terminate in a central portion of the XZIV zone. The dorsomedial cell column projects to the XZIII zone, which is present only in the dorsal part of the nodulus. The rostral medial accessory olive projects to the XZV zone, which occupies the lateral border of the nodulus. These results confirm and extend the conclusions of Katayama and Nisimaru ([1988] Neurosci. Res. 5:424-438) on the zonal pattern in the olivo-nodular projection in the rabbit. Additional observations were made on the presence of a lateral A zone (Buisseret-Delmas [1988] Neurosci. Res. 5:475-493) in the hemisphere of lobules VI and VII. Retrograde labeling of the nucleo-olivary tract of Legendre and Courville ([1987] Neuroscience 21:877-891) was observed after WGA-HRP injections into the inferior olive including the rostral dorsal cap and the ventrolateral outgrowth. The anatomical and functional implications of these observations are discussed. PMID- 7543122 TI - Saliva protein binding to layers of oral streptococci in vitro and in vivo. AB - This paper reports a system for measuring saliva protein binding to oral streptococci. Enamel chips with layers of Streptococcus gordonii Blackburn or Streptococcus oralis 10557 were incubated in vitro with whole saliva from eight persons. Blackburn bound significantly more amylase than 10557; no strain differences were seen for lysozyme or lactoferrin. There were significant correlations between saliva and bound amylase and lactoferrin. Blackburn and 10557 chips were then placed in ten subjects. Sites included the buccal left and right upper premolars and molars (UL, UR), labial upper central incisors (UC), and lingual lower central incisors (LL). That study was repeated three months later; chips with Streptococcus sanguis 13379 were also placed then. Blackburn bound significantly more amylase than the other strains. Blackburn and 10557 both bound the most amylase at UL and UR, and the least amylase at UC. However, strain 13379 bound less amylase at UL. That strain also bound significantly less sIgA at UL. All three strains bound the least sIgA at UC. Lysozyme and lactoferrin binding showed few differences among sites or strains. Bound protein concentrations were significantly correlated across sites and strains within subjects, but not correlated with whole saliva. Strain differences may reflect species differences in amylase binding, or differences in species-specific sIgA titers. Site differences may indicate local variation in protein availability. Differences between chip correlations with whole saliva in vitro and in vivo suggest that the salivary film may be modified as it flows over tooth surfaces. PMID- 7543123 TI - Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue. AB - Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors. PMID- 7543124 TI - Galanin infusion restores the blunted GH responses to GHRH administration during GH treatment in children with constitutional growth delay. AB - GH responses to GHRH (in basal conditions), GHRH (after six months of recombinant GH therapy) and GHRH plus galanin administration (after GH therapy) were determined in six children with constitutional growth delay (CGD). Before treatment, GHRH administration caused a clear rise of GH levels (mean GH peak: 38.0 +/- 7.2 ng/ml); mean GH net incremental area under the curve/120 min (GH nAUC) was 2631.0 +/- 519.8 ng/ml/120 min. During GH treatment, both height velocity and IGF-I levels significantly increased from 4.3 +/- 0.1 cm/yr to 9.4 +/- 0.9 cm/yr (p < 0.001) and from 218.7 +/- 12.3 ng/ml to 328.0 +/- 28.2 ng/ml (p < 0.001), respectively. After 6 months of GH therapy, mean GH peak (21.7 +/- 2.2 ng/ml) and mean GH nAUC (847.7 +/- 109.8 ng/ml/120 min) after GHRH administration were significantly lower (p < 0.05) than in basal conditions. Galanin infusion (10 micrograms/kg/bw) was able to completely restore the reduced GH responsiveness to GHRH administration. In fact, mean GH peak and mean GH nAUC after GHRH plus galanin were similar to those recorded after basal GHRH administration (GH peak: 37.5 +/- 4.0 ng/ml; GH nAUC: 2279.8 +/- 430.5 ng/ml/120 min). In conclusion, this study shows that the reduction of pituitary responsiveness to GHRH administration during GH treatment is not dependent on the depletion of the promptly GH releasable pool, since somatotroph refractoriness to GHRH can be overriden by the concomitant administration of galanin. PMID- 7543126 TI - Hydromorphone metabolite accumulation in renal failure. PMID- 7543127 TI - Hypercalcemia of malignancy in the palliative care patient: a treatment strategy. AB - Hypercalcemia of malignancy is most commonly due to the effects of parathyroid hormone-related peptide, which acts as a humoral factor to cause generalized osteoclast-mediated bone resorption and reabsorption of calcium by the kidney tubule, and may also act as a local resorptive factor adjacent to bone metastases. Local resorptive mechanisms are less common causes of malignant hypercalcemia than previously believed. Treatment begins with intravenous fluid rehydration, followed by a furosemide diuresis and the bisphosphonate pamidronate, 60-90 mg, intravenously. Gallium nitrate is an efficacious but inconvenient alternative to pamidronate. Calcitonin combined with pamidronate is a reasonable initial therapy for severe hypercalcemia to hasten normalization of the serum calcium. Steroids should be reserved for hypercalcemia due to tumor production of 1,25 dihydroxyvitamin D, or for steroid-responsive malignancies. Oral or parenteral bisphosphonates can be used to maintain normocalcemia. In addition to improving the morbidity of acute hypercalcemia, bisphosphonate therapy has been shown to reduce bone pain and pathological fractures in patients with bone metastases, and calcitonin also has a potent analgesic effect in these patients. Treatment for hypercalcemia should therefore be considered in the majority of patients in the palliative care setting. PMID- 7543128 TI - Opioid availability in Latin America: the declaration of Florianopolis. AB - Between March 27 and 29, 1994, a group of representatives of 32 palliative care programs from eight Latin American countries met under the auspices of the World Health Organization (WHO) Palliative Care Program for Latin America in Florianopolis, Brazil. The participants included physicians, nurses, psychologists, volunteers, drug regulators, hospital administrators, and representatives from the pharmaceutical industry. A comprehensive report by David Joranson (University of Wisconsin-Madison) was followed by a general discussion moderated by Jan Stjernsward, Chief of the Cancer Unit, World Health Organization, and by Eduardo Bruera, Coordinator of the WHO Cancer Pain and Palliative Care Program for Latin America. A number of issues related to opioid availability were identified and discussed. This declaration summarizes the main conclusions of the meeting. The attendants would like to encourage the widest possible distribution of this document. PMID- 7543129 TI - Cyfra 21-1 in bronchoalveolar lavage: preliminary results. PMID- 7543125 TI - Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro. AB - We investigated the effect of recombinant human IL-6 (rhIL-6) on cell proliferation using the MtT/E rat pituitary tumor cell line, which was recently established by Inoue et al. This cell line expresses the homeodomain protein Pit 1/GHF 1 and does not produce any significant amount of pituitary hormones, but retains its tumorigenicity by back-transplantation into rats, resulting in production of prolactin. MtT/E cells were seeded into Falcon 24-well plates at a density of 2 x 10(4) cells/well in a cultured medium, containing 10% horse serum and 2.5% fetal bovine, with test drug. After four-days (12 days for the time course study) incubations, the cells were counted using a hemocytometer. Incubation for 4 days with rhIL-6 caused concentration-dependent stimulation of MtT/E cell growth and [3H]-thymidine incorporation into MtT/E cells. Addition of 20 ng/ml rhIL-6 to the culture medium stimulated MtT/E cell growth in a time dependent manner, withdrawal of rhIL-6 from the culture medium reduced MtT/E cell growth, and re-addition of rhIL-6 to the culture medium again stimulated MtT/E cell growth. Among the cytokines tested, granulocyte colony-stimulating factor (rh G-CSF) also showed a slight but significant mitogenic activity on the MtT/E cells. Analysis of 125I-rhIL-6 binding to the MtT/E cells indicated a dissociation constant of 0.953 x 10(-9) mmol/l and the presence of 968 binding sites per cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543130 TI - Clinical significance of inverted PSA/PAP ratio in prostate cancer patients. PMID- 7543131 TI - Effects on glucose tolerance, insulin secretion, insulin-like growth factor 1 and its binding protein, IGFBP-1, in a randomized controlled diet and exercise study in healthy, middle-aged men. AB - OBJECTIVES: To study the effects of advice on diet, exercise and their combination on oral glucose tolerance (OGTT), insulin secretion, insulin-like growth factor-1 (IGF-1) and its binding protein, IGFBP-1. DESIGN: A 6-month, randomized, controlled intervention study. SETTING: Primary health care centres in Sollentuna, Stockholm and the Department of Medicine, Karolinska Hospital, Stockholm, Sweden. SUBJECTS: One hundred and fifty-seven normoglycaemic healthy men, mean age 46 years, range 35-60 years, with slightly to moderately raised cardiovascular risk factors. INTERVENTIONS: Advice on diet (D, n = 40), exercise (E, n = 39) a combination of both (DE, n = 39) and a control group (C, n = 39). MAIN OUTCOME MEASURES: An OGTT, insulin secretion, IGF-1 and its binding protein, IGFBP-1. RESULTS: The number of pathological OGTTs in the intervention groups decreased from 42/118 to 33/118 whilst the number in the control group did not change. Fasting insulin levels decreased in groups E and DE from 8.8-7.4 mU L-1 (P < 0.01) and from 8.3-6.7 mU L-1 (P < 0.01), respectively. Accordingly, the insulin area under the curve decreased from 5278 to 4828 (P < 0.05) in group E, and from 5482 to 4809 (P < 0.01) in group DE. IGF-1 only increased in group D. The most prominent changes were noted for IGFBP-1, which increased in all three intervention groups and to the highest degree in group DE (from 33.7-42.6 micrograms L-1, P < 0.001). CONCLUSIONS: A combination of increased exercise and improved diet, as well as increased exercise alone, favourably affect glucose and insulin homeostasis in middle-aged men with moderately elevated cardiovascular risk factors. The most marked changes were noted for IGFBP-1, possibly suggesting a decreased insulin secretion and an enhanced insulin sensitivity. PMID- 7543132 TI - Relationships among antigen presentation, cytokines, immune deviation, and autoimmune disease. PMID- 7543133 TI - Distinct roles for CD28 and cytotoxic T lymphocyte-associated molecule-4 receptors during T cell activation? PMID- 7543134 TI - Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium. AB - An important goal for the investigation of the proliferation of mammalian cells is to establish a fully defined condition for culturing them in vitro. Here, we report establishment of a fully defined culture condition that supports the primary culture of normal c-kit+IL-7 receptor (IL-7R)+ B precursor cells without the aid of stromal cell lines. This defined culture condition contains IL-7, the ligand for c-kit, transferrin, insulin, and bovine serum albumin as protein components. By using the cell lines derived from RAG2(-/-) mice, which do not differentiate into c-kit- stage, we have evaluated the role of each protein in the cell cycle progression of c-kit+IL-7R+ B precursor cells. Since B precursor cells can grow without insulin, c-kit remains a sole functional receptor tyrosine kinase for their growth. While both c-kit ligand (KL) and IL-7 are the requisite molecules for sustained proliferation of B precursor cells, each molecule plays distinct roles. IL-7 starvation results in prompt arrest of the cells at G1. An accumulation of the cells in the mitotic phase was also detected. Thus, the major role of IL-7 is to regulate the G1/S transition and the process of cytokinesis of B precursor cells. Although prolonged KL starvation over 48 h resulted in accumulation of G1 cells, its effect could not be detected within 24 h, which is long enough for all the cells to complete one cell cycle. This suggests that KL might be involved in the cell cycle progression of B precursor cells in a manner that its signal could still be effective in the one or two cell cycles that follow. Although molecular nature of the signals underlying the present observation awaits future investigation, the method described in this report would provide a useful model system for investigating the signaling pathways that are involved in the cell cycle progression of B precursor cells. PMID- 7543135 TI - Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells. AB - Previous experiments from this laboratory have shown that Lewis rats were protected from experimental allergic encephalomyelitis (EAE) induced by the injection of myelin basic protein (MBP) in Freund's complete adjuvant if they were treated with the encephalitogenic peptide of MBP covalently linked to mouse anti-rat immunoglobulin (Ig) D. It was suggested that this protection developed because the antibody-peptide conjugate targeted the peptide to B cells and that this mode of presentation induced a Th2-like T cell response that controlled the concomitant encephalitogenic Th1 reaction to the autoantigen. The current experiments were carried out to test this hypothesis and to examine the alternative explanation for the protective effect of the conjugate pretreatment, namely that it induced a state of nonresponsiveness in the autoantigenspecific T cells. It was shown that EAE induction was suppressed in Lewis rats when the antibody-peptide conjugate was injected intravenously 14 and 7 d before immunization with MBP in adjuvant, but that anti-MBP antibody titers were at least as high in these animals as in controls that were not pretreated with the conjugate before immunization. Lymph node cells from these pretreated animals, while proliferating in vitro to MBP as vigorously as those from controls, produced less interferon gamma and were very inferior in their ability to transfer disease after this in vitro activation. In contrast, these same lymph node cells from protected rats generated markedly increased levels of messenger RNA for interleukin (IL)-4 and IL-13. When these in vitro experiments were repeated using the encephalitogenic peptide rather than MBP as the stimulus, the proliferative response of lymph node cells from pretreated donors was less than that from controls but was still readily detectable in the majority of experiments. Furthermore, the cytokine expression induced by the peptide was similar to that elicited by whole MBP. While these results support the original hypothesis that the anti-IgD-peptide conjugate pretreatment protected rats from EAE by inducing a Th2-type cytokine response, a totally unexpected finding was that this pretreatment greatly reduced the level of leukocyte infiltration into the central nervous system. This result provides a direct explanation for the protective effect of the pretreatment, but it raises questions regarding migratory and homing patterns of leukocytes activated by different immunological stimuli. PMID- 7543136 TI - Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo. AB - Experimental autoimmune encephalomyelitis (EAE), an experimental model for the study of multiple sclerosis, is an autoimmune disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP). MBP-reactive CD4+ T cells, predominantly expressing the V beta 8.2 T cell receptor (TCR), migrate from the peripheral lymphoid organs and initiate the inflammatory response in the brain. We have previously shown that a single intrathymic injection of MBP or its major encephalitogenic peptide (p71 90), but not a nonencephalitogenic peptide (p21-40), induces antigen-specific systemic tolerance and inhibits the induction of EAE in Lewis rats. In this study, we investigated the mechanisms of induction and maintenance of acquired thymic tolerance in this model. First, we investigated which thymic cell is responsible for "induction" of systemic tolerance. Thymic dendritic-enriched cells, isolated by plastic adherence, when incubated in vitro with p71-90 and injected intravenously into Lewis rats, were capable of preventing the development of EAE, but his protection was lost in thymectomized recipients. In addition, intravenous injection of thymic dendritic cells isolated from animals that had been previously injected intrathymically with p71-90 but not p21-40 also prevented the development of EAE. Second, to determine the "effector" mechanisms involved in acquired thymic tolerance, we compared TCR expression in the brains of animals with actively induced EAE with TCR expression in animals that received intrathymic injection of p71-90 or p21-40. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique, we found increased expression of CD4 and V beta 8.2 message in brains of immunized animals compared with those of naive animals. In animals intrathymically injected with p71-90 but not p21-40, CD4 and V beta 8.2 transcript levels were significantly reduced compared with immunized controls. Immunohistologic studies of brain tissue and spleens with specific V beta 8.2 and control V beta 10 monoclonal antibodies confirmed these observations in vivo. These findings, taken together with recent data demonstrating that activated T cells circulate through the thymus, suggest that interaction of thymic dendritic cells with specific TCR of activated peripheral T cells can lead to inactivation of these antigen-specific cells and confirm the role of V beta 8.2-expressing T cells in EAE. PMID- 7543137 TI - Glycosylation of CD44 negatively regulates its recognition of hyaluronan. AB - Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44. PMID- 7543139 TI - CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. AB - The importance of the B7/CD28/CTLA-4 molecules has been established in studies of antigen-presenting cell-derived B7 and its interaction with the T cell costimulatory molecule CD28. CTLA-4, a T cell surface glycoprotein that is related to CD28, can also interact with B7-1 and B7-2. However, less is known about the function of CTLA-4, which is expressed at highest levels after activation. We have generated an antibody to CTLA-4 to investigate the consequences of engagement of this molecule in a carefully defined system using highly purified T cells. We show here that the presence of low levels of B7-2 on freshly explanted T cells can partially inhibit T cell proliferation, and this inhibition is mediated by interactions with CTLA-4. Cross-linking of CTLA-4 together with the TCR and CD28 strongly inhibits proliferation and IL-2 secretion by T cells. Finally, results show that CD28 and CTLA-4 deliver opposing signals that appear to be integrated by the T cell in determining the response to activation. These data strongly suggest that the outcome of T cell antigen receptor stimulation is regulated by CD28 costimulatory signals, as well as inhibitory signals derived from CTLA-4. PMID- 7543138 TI - Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. AB - CD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N-linked glycosylation. Furthermore, culture in tunicamycin for 2-3 d converted parental and inducible cell lines into cells showing constitutive CD44-mediated HA binding. Also, removal of cell surface glycosaminoglycan chains by culture of cells in p-nitrophenyl beta-D-xylopyranoside or treatment with chondroitinase ABC resulted in conversion of cells with an inactive CD44 receptor to an inducible state. These results indicate that carbohydrate side chains of CD44 and/or other molecules on the cell surface that interact with CD44 are potentially involved in regulating the HA-binding function of CD44 on the cell surface. PMID- 7543140 TI - Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1. AB - This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms. PMID- 7543142 TI - Mutational analysis of the membrane-proximal cleavage site of L-selectin: relaxed sequence specificity surrounding the cleavage site. AB - L-selectin expression is regulated in part by membrane-proximal cleavage from the cell surface of leukocytes and L-selectin-transfected cells. The downregulation of L-selectin from the surface of neutrophils is speculated to be a process involved in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. We previously reported that L-selectin is cleaved between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain. We demonstrate that replacing this cleavage domain of L selectin with the corresponding region of E-selectin prevents L-selectin shedding, as judged by inhibiting the generation of the 68-kD soluble and 6-kD transmembrane cleavage products of L-selectin. Unexpectedly, we found that point mutations of the cleavage site, as well as mutations of multiple conserved amino acids within the cleavage domain, do not significantly affect L-selectin shedding. However, short deletions of four or five amino acids in the L-selectin cleavage domain inhibit L-selectin downregulation. Mutations that appeared to inhibit L-selectin shedding resulted in higher levels of cell surface expression, consistent with a lack of apparent proteolysis from the cell membrane. One deletion mutant, I327 delta N332, retains the native cleavage site yet inhibits L selectin proteolysis as well. Restoring the amino acids deleted between I327 and N332 with five alanine residues restores L-selectin proteolysis. Thus, the proteolytic processing of L-selectin appears to have a relaxed sequence specificity at the cleavage site, and it may depend on the physical length or other secondary structural characteristics of the cleavage domain. PMID- 7543143 TI - Experimental autoimmune peripheral neuritis induced in BALB/c mice by myelin basic protein-specific T cell clones. AB - In vivo adoptive transfer of CD4+ T helper cell type 1 clones reactive with autologous myelin basic protein (MBP) may initiate an inflammatory demyelinating disease of the central nervous system called experimental autoimmune encephalomyelitis. Although MBP is also a component of peripheral nervous system (PNS) myelin, previous studies have failed to demonstrate inflammation in the PNS induced by MBP-reactive T cells. Here, we report on two MBP-specific T cell clones that preferentially initiate inflammatory and demyelinating peripheral neuritis when adoptively transferred to syngeneic recipients. The MBP epitope recognized by these clones spans the junction of exons 6 and 7 and, therefore, is present in the 21- and 18.5-kD but not the 14- and 17-kD MBP isoforms, in which exon 5 is spliced to exon 7. The data suggest that MBP may be processed and presented differently in the central nervous system and PNS, and they provide evidence for MBP as a potential target for autoimmune reactions in the PNS. PMID- 7543141 TI - Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. AB - L-selectin mediates leukocyte rolling on vascular endothelium at sites of inflammation and lymphocyte migration to peripheral lymph nodes. L-selectin is rapidly shed from the cell surface after leukocyte activation by a proteolytic mechanism that cleaves the receptor in a membrane proximal extracellular region. This process may allow rapid leukocyte detachment from the endothelial surface before entry into tissues. In this study, the structural requirements for regulation of human L-selectin endoproteolytic release were examined through analysis of chimeric selectin molecules and mutant L-selectin receptors. The use of chimeric selectins and a cytoplasmic tail truncation mutant demonstrated that the extracellular membrane-proximal 15-amino acid region of L-selectin is required for endoproteolytic release. The introduction of alanine-scanning mutations within this membrane-proximal region did not prevent endoproteolytic release, indicating that a specific amino acid motif was not an absolute requirement for cleavage. Furthermore, alterations within the putative primary cleavage site (K283-S284) resulted in either constitutive endoproteolytic release of the receptor or inhibition of cell activation-induced shedding to variable extents. The length of the membrane-proximal region was also critical since truncations of this region completely abolished endoproteolytic release. Thus, release of L-selectin is likely to be regulated by the generation of an appropriate tertiary conformation within the membrane-proximal region of the receptor which allows recognition by a membrane-bound endoprotease with relaxed sequence specificity that cleaves the receptor at a specific distance from the plasma membrane. These observations suggest a generalized protein-processing pathway involved in the endoproteolytic release of specific transmembrane proteins which harbor widely differing primary sequences at or neighboring their cleavage sites. PMID- 7543144 TI - Insulin-like growth factor induces phosphorylation of immunoreactive insulin receptor substrate and its association with phosphatidylinositol-3 kinase in human thymocytes. AB - Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1 transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160 180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells. PMID- 7543145 TI - Samarium-153-EDTMP for palliation of ankylosing spondylitis, Paget's disease and rheumatoid arthritis. AB - Samarium-153-EDTMP is an effective agent for palliation of widespread skeletal metastases because it concentrates in bone metastases which have an osteoblastic component. Similar concentration in areas of osteoblastic activity in ankylosing spondylitis, Paget's disease and rheumatoid arthritis suggests a possible new treatment approach. Three patients with ankylosing spondylitis, one patient with Paget's disease and one patient with rheumatoid arthritis were treated with 153Sm EDTMP. Objective and subjective improvement was noted, especially in ankylosing spondylitis patients. Samarium-153-EDTMP has disease-modifying potential in ankylosing spondylitis and Paget's disease and has palliative value in resistant rheumatoid arthritis. Further trials to determine optimal dose, treatment scheduling, long-term disease-modifying potential and toxicity are needed. PMID- 7543146 TI - Noninvasive measures of radiolabeled dextran transport in in situ rabbit lung. AB - Dextrans are nontoxic and can be obtained in a wide variety of molecular weights. The purpose of this study was to label 6-kDa and 40-kDa dextrans with gamma- (99mTc) and positron- (18F) emitting radioisotopes and monitor their transport across the pulmonary microvascular barrier. METHODS: External scan measurements for radiolabeled uncharged dextrans, albumin and red blood cells were obtained in eight blood-perfused in situ rabbit lung preparations. After 3 hr of external scanning, the lungs were removed for postmortem and extravascular distribution volume calculations. Extravascular distribution volumes were obtained in six additional rabbits following 4 hr of dextran perfusion to compare the effect of time. The normalized slope index (NSI), a measure of transvascular transport rate, was calculated for each diffusible tracer. RESULTS: The mean NSI for albumin (0.001676 +/- 0.000537 min-1) was significantly lower than NSI for the 40 kDa dextran (0.002303 +/- 0.0005426 min-1) as well as the 6-kDa dextran (0.004312 +/- 0.001134 min-1). The difference between the 6-kDa and the 40-kDa dextrans was also significant. After 4 hr of equilibration, distribution volumes were not significantly different than those obtained at 3 hr. CONCLUSION: Dextrans can be radiolabeled with gamma and positron emitters and small dextrans traverse the lung microvascular barrier more rapidly than albumin. Our results suggest that the use of small dextrans rather than albumin can reduce scan times in clinical applications and minimize motion artifact associated with the noninvasive gamma detection method. PMID- 7543148 TI - Use of bleomycin in radiochemotherapy. PMID- 7543147 TI - Measuring pulmonary vascular permeability. PMID- 7543149 TI - For whom the bell tolls--palliative care in the Third World. PMID- 7543150 TI - Dilemmas of life and death: Part one. PMID- 7543151 TI - Swallowing in motor neurone disease. PMID- 7543152 TI - Toward improved anti-HIV chemotherapy: therapeutic strategies for intervention with HIV infections. PMID- 7543153 TI - Analysis of a 2(9) full factorial chemical library. AB - Robotic synthesis is making possible the synthesis of large, systematically designed sets of compounds. We analyze a 512-compound set that is a 2(9) full factorial experimental design using a recursive partitioning algorithm, FIRM, and a high-dimension visualization tool, TempleMVV. These techniques are used to quickly and easily identify the main trends in the data set and also identify unusual observations. We show that analytical and visualization methods can be used synergistically to analyze a large, complex, high-dimensional data set. We also show that a fractional factorial design of 128 compounds would give essentially the same information. PMID- 7543154 TI - Localized prostate cancer. Diagnosis and treatment. PMID- 7543155 TI - Detection of antibodies against phenolic glycolipid-1 (PGL-1), 35-kDa and 30-40 kDa components of Mycobacterium leprae in the cerebrospinal fluid of of leprosy patients. AB - The involvement of the central nervous system (CNS) in lepromatous leprosy (LL) patients was investigated; 33 patients were examined clinically in detail and upper motor neuron involvement was observed in eight and lower motor neuron in three of these patients. Anti-Mycobacterium leprae antibodies could be detected in the CSF by PGL-1 enzyme-linked immunosorbent assay (ELISA) and monoclonal antibody (MAb) based competitive assays against defined epitopes on the 35-kDa protein and 30-40-kDa polysaccharide (lipoarabinomannan) antigens with MAbs MLO4 and ML34, respectively. Antibodies against PGL-1 and 35-kDa protein were observed in more subjects than antibodies against the 30-40-kDa antigen. Some correlation was observed between the upper motor neuron signs and antibody positivity for 35 kDa and PGL-1 antigens in the CSF of these patients. PMID- 7543157 TI - Biosynthesis and regulation of expression of the HNK-1 epitope on myelin associated glycoprotein in a transfected cell model system. AB - The HNK-1 antibody recognizes a carbohydrate epitope expressed by many cell adhesion molecules in the nervous system that has been proposed to be an important adhesive determinant. This epitope is particularly prominent on the myelin-associated glycoprotein (MAG) and is related to the antigenic target in an autoimmune mediated demyelinating neuropathy. Elucidation of the mechanisms underlying the biosynthesis and regulation of expression of the HNK-1 epitope is therefore likely to have important functional and clinical implications. In order to investigate its biosynthesis and the regulation of its expression, we have expressed both human and rat MAG in several different cell lines by retroviral infection. These studies indicate that the cellular milieu determines whether the HNK-1 epitope is expressed on the MAG polypeptide and provide an explanation for the significant variation in HNK-1 levels that has been noted in different species. Using a transfected human neuroblastoma line, we have determined that this epitope is present on the fourth and/or fifth immunoglobulin-like domain of rat MAG and that it is added intracellularly, probably in the trans Golgi. Finally we have found that expression of the HNK-1 epitope is increased by activation of different second messenger systems, providing direct evidence that its expression can be regulated independently from that of the MAG polypeptide. PMID- 7543156 TI - Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma. AB - BACKGROUND: Tumor angiogenesis, as assayed by microvessel density, has been proposed as an independent prognostic marker for clinical outcome in breast cancer patients. PURPOSE: The present study evaluated the microvessel density assay and assessed its utility, alone and together with the evaluation of other tumor characteristics, in predicting outcome in patients with invasive ductal carcinomas. METHODS: In a blinded design, cases of invasive ductal carcinoma were selected from a registry containing the records of and tumor specimens from 386 breast cancer patients treated at the Massachusetts General Hospital from 1977 through 1982. After the exclusion of ineligible patients and inadequate specimens, 220 patients were included in the study; their median time of follow up was 11.5 years. Half of these patients (n = 110) were positive for axillary lymph node metastases. Histologic sections of the tumors were stained immunocytochemically for factor VIII, a coagulation protein expressed by blood vessel endothelium, and for p53 protein. Independently, two analysts counted microvessels in three microscope fields selected from separate vascular regions of the tumor. Variability in microvessel scores between analysts and among different fields of the same tumor was summarized by the coefficient of variation. The kappa statistic tested for agreement between the analysts while correcting for chance agreement. The effects of tumor characteristics on metastasis-free survival and overall survival were tested univariately by the Harrington-Fleming rank test procedure. The effect of multiple factors on survival was tested under a Cox multivariate proportional hazards model. RESULTS: Microvessel count showed considerable variability between the two analysts and among regions within each tumor, with an overall concordance for tumor classification of 73%. Univariate analysis revealed no association between microvessel count and any other tumor or patient characteristic. Multivariate analysis indicated, for these patients, that nodal status and p53 staining predicted metastasis-free survival and that nodal status, estrogen receptor (ER) status and tumor grade predicted overall survival. CONCLUSIONS: Microvessel count showed much variation among different regions of each tumor. It did not predict metastasis-free survival or overall survival. Nodal status was the most powerful criterion to stratify these patients with invasive ductal carcinoma of the breast into different survival groups. Only ER status, tumor grade, and p53 staining had additional prognostic utility for these patients after they had been stratified by nodal status. PMID- 7543158 TI - Bovine opsin gene expression exhibits a late fetal to adult regulatory switch. AB - Rates of bovine photoreceptor gene transcription, as measured by nuclear run-on assays, exhibit gene-specific patterns of regulation. Here we investigate initiation and elongation in nuclear run-on assays with the use of sarkosyl to further understand the nature of these gene-specific elements. Opsin transcription, alone among several genes tested, proved sarkosyl-sensitive. This sensitivity is maximal in adult retinas, with inhibition first detected in mid third trimester fetal retinas. Therefore, opsin transcription appears to involve different regulatory elements in adult and fetal retinas, implying a fetal to adult switch in the control of opsin gene expression. Although this regulatory switch is initially activated at a time when the fetal outer nuclear layer of the retina first achieves adult-like morphology, further maturation of opsin regulation takes place postpartum since levels of sarkosyl sensitivity are almost 5-fold greater in adult retinas compared to the 7.5 month fetus. We also show that the sarkosyl-induced reduction of opsin transcription is not due to prevention of de novo RNA polymerase II initiation in the run-on reaction, suggesting the detergent alters a positive-acting, postinitiation component of the transcriptional apparatus. Since levels of opsin transcription with sarkosyl are similar to those of the other visual transduction genes with or without sarkosyl, this detergent-sensitive transcriptional component appears to account for the singularly high, gene-specific rate of opsin transcription in retinal photoreceptor cells. PMID- 7543159 TI - Effects of growth factors on phosphatidylinositol-3 kinase in astroglial cells. AB - Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of insulin like growth factor I (IGF1), insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factors (FGFs) on phosphatidylinositol-3 kinase (PI(3)-kinase). In astroglial cells cultured in serum-free medium, IGF1, PDGF, and EGF, which stimulate cell proliferation, increased PI(3)-kinase activity immunoprecipitated with anti-phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation, glutamine synthetase, and deiodinase activities and modify cell morphology, have no effect on PI(3)-kinase activity. Addition of 1 nM PDGF, 10 nM IGF1, or 100 nM EGF to the culture medium rapidly stimulated PI(3)-kinase activity which declined slowly after 2 min. The stimulation of PI(3)-kinase increased with growth factor concentration. The maximum increase in PI(3)-kinase activity occurred with 50 nM IGF1, 1 nM PDGF, or 100 nM EGF. Since insulin was active only at high concentration (1 microM), its effect was probably mediated through IGF1 receptors and not through insulin receptors. IGF1 and PDGF, to a lesser degree, also increased the PI(3)-kinase activity associated with pp60c-src protein. Immunoblots performed with an antibody directed against the p85-subunit of the PI(3)-kinase confirmed that IGF1 increased the number of PI(3)-kinase molecules associated with phosphotyrosine-containing proteins or with c-src protein. Each growth factor affects in a different manner the association of PI(3)-kinase with phosphotyrosine-containing proteins and with pp60c-src and thus probably modulates intracellular signals downstream of PI(3)-kinase in astroglial cells. PMID- 7543160 TI - Preparation of a monoclonal antibody to a glycidic epitope of the epidermal growth factor receptor that recognizes inhibitors of astrocyte proliferation and reactive microglia. AB - A mouse monoclonal antibody (5B9), directed against a carbohydrate epitope of human epidermal growth factor receptor (EGFR), recognized an 81-kDalton glycoprotein in buffer-soluble and detergent-solubilized rat brain extracts (BE). The glycoprotein was more abundant in extracts prepared from injured brain than in those from normal tissue. Removal from BE of the antigens recognized by 5B9 increased their astrocyte mitogenic activity. Sections of injured rat brain and cultures derived from damaged brain, enriched in microglia, showed 5B9 immunoreactivity in ED1-positive cells. The abundance of the glycoprotein recognized by 5B9 in injured, relative to normal, tissue, suggested that molecules with EGFR immunoreactivity may be expressed in reactive microglial cells and released after injury. PMID- 7543161 TI - [Recent progress and new directions in the study of molecular mechanisms of cancer metastasis]. AB - Metastatic cancers are major mortal diseases in Japan. To develop precise prognostic means and effective prevention therapies for malignant diseases, molecular mechanisms of cancer metastasis have to be elucidated. As it is necessary to coordinate metastasis research activities in various scientific fields, the Japanese Association for Metastasis Research has been established. The society plays an important role in the information exchange and the collaboration establishment among experimental and clinical investigators. Recent progress is observed in the development of experimental and spontaneous metastasis models including orthotopic implantation of human cancers and artificial bone metastasis models, using mice. A significant progress has been made in the study of angiogenesis. Angiogenesis inhibition is now believed to be the most-effective therapeutic approach for metastasis prevention. The investigation of metastasis-associated genes have also advanced. Most of such gene products appeared to be involved in the signal transduction and associated with the cytoskeletal network. Further investigations will lead us to the exploitation of therapeutic means against angiogenesis and metastasis, in the near future. PMID- 7543162 TI - [Inhibition of tumor cell metastasis in chick embryo by beta 1 integrin antisense oligonucleotide]. AB - The antimetastatic effect of integrin beta 1 subunit antisense oligonucleotide on human fibrosarcoma cell (HT-1080) in chick embryo was examined. Phosphorothioate modified antisense oligonucleotide (B1-1) was designed to hybridize specifically to integrin beta 1 subunit mRNA. Pretreatment of HT-1080 cells with B1-1 decreased the metastatic ability in chick embryonic liver and lung, while B1-1 showed no toxicity to HT-1080 nor chick embryo. Western blot analysis showed a reduction of the amount of cell surface integrin beta 1 subunit. B1-1 also inhibited HT-1080 attachment to laminin and fibronectin. These results indicate that the inhibition of tumor cell attachment by antisense oligonucleotide might be a good approach to the prevention of cancer metastasis. PMID- 7543163 TI - [Migration and adhesiveness of malignant glioma cells to fibronectin or vitronectin and their expression of integrin subunits]. AB - In order to better understand the cellular mechanism of glioma invasion, we investigated migratory responses and adhesiveness of human malignant glioma cells to fibronectin (FN) or vitronectin (VN). In addition, an expression of integrin subunits for FN and VN was analyzed by flow cytometry. All glioma cells tested migrated to both FN and VN to a various degree. Glioma cells which strongly migrated to FN or VN showed an intense expression of alpha 5 or alpha v, respectively, while there was no correlation between cell adhesiveness to FN or VN and intensity of the integrin expression. Studies using primary tumor cells from surgical specimen revealed that only an intensity of cell adhesiveness to FN was negatively correlated well with the degree of clinical invasion of gliomas. That is, the more glioma cells adhered to FN, the less the original tumor tissues showed tumor invasion. PMID- 7543164 TI - [Structure and function of cell adhesion molecules in selectin family and their roles in cancer metastasis]. AB - Cell adhesion molecules of selectin family are involved in the metastasis of cancer at the step of attachment of cancer cells to endothelial cells. P-selectin mediated adhesion of cancer cells with platelets, and L-selectin mediated adhesion of leukocytes to cancer cells would also be involved in the process of hematogenous metastasis through the microthrombus formation. Upon adhesion to cancer cells, platelets and leukocytes secrete cytokines that induce E-selectin expression of endothelial cells, and this further facilitates hematogenous metastasis. PMID- 7543165 TI - [Contribution of carbohydrate antigens to endothelial E-selectin binding in cancer cells]. AB - We first investigated the difference in carbohydrate antigens' expression between primary tumors and matched metastatic liver lesions of colorectal carcinomas, using immunohistochemical methods. Regarding the degree of expression, SLA, SLX and SPan-1 antigens were found to be expressed on a larger proportion of tumor cells in liver metastases than in primary tumors. Second, comparing the expression of carbohydrate antigens on highly metastatic colon cancer cell line to the parental line, the expression of SLA on the surface of highly metastatic variant was increased more intensively than the parental line. Finally, we investigated the contribution of carbohydrate antigens to endothelial E-selectin binding in colon and pancreatic cancer cells, and the results indicated that SLA might play a significant role in this binding. These findings suggest that the expression of carbohydrate antigens on the surface of cancer cells obviously contributed to E-selectin binding and also to metastatic potential. PMID- 7543166 TI - [E-selectin expression in serum and tissue correlates with distant metastasis of colorectal cancer]. AB - E-selectin is an adhesion molecule of endothelial cells that binds to cancer cells mediated by sialyl Lewis A (sLea) or sialyl Lewis X (sLex). It is suspected to be involved in hematogenous metastasis of tumors. Therefore, it is worth examining E-selectin expression in human colorectal cancer and its hepatic metastasis. In the present study, E-selectin was clearly revealed on the endothelial cells of small vessels adjacent to cancer nests in both primary and metastatic nests in immunohistochemistry. In these tissues, E-selectin was observed on the endothelial cells lining the lumen of small vessels. Its expression adjacent to cancer nests appears to be induced through some stimuli by cancer cells, since its degree of expression is inversely correlated to the distance of the blood vessels from the cancer nests (P < 0.001). Endothelial cells adjacent to the metastatic lesion expressed E-selectin more extensively than those adjacent to the primary foci. This is also in line with the finding on serum E-selectin levels which were significantly elevated in the metastatic group compared to the non-metastatic group. The serum E-selectin level may provide useful information in the diagnosis for hepatic metastasis of colorectal cancer, although the results are still very preliminary. PMID- 7543167 TI - [Clinical significance of circulating soluble E-selectin (ELAM-1) in patients with cancers]. AB - Cell adhesion mediated by E-selectin and its carbohydrate ligands, Le(a) and Le(x), plays an important role in cancer metastasis. We determined soluble E selectin concentration in the sera of patients with cancers using double determinant sandwich assay. The results indicated that the patients with metastatic colon cancers had significantly higher serum E-selectin concentration than the patients having no metastatic lesion. The highest frequency of metastasis was obtained in Le(a) HIGH E-selectinHIGH patients (80%), while the frequency was quite low in Le(a) LOW E-selectinLOW patients (17%). Expression of E-selectin, as well as its carbohydrate ligands, is suggested to be a risk factor for the metastasis of epithelial cancers. Soluble E-selectin-carbohydrate complex was also detected in the sera of patients, and the frequency was especially high in Le(a) HIGH E-selectinHIGH patients. PMID- 7543168 TI - Effects of cholecystokinin (CCK)-JMV-180 on the CCK receptors of rabbit pancreatic acini and gallbladder smooth muscle. AB - Effects of cholecystokinin (CCK)-JMV-180, a CCK analog, on the CCK receptor functions of isolated rabbit pancreatic acini and gallbladder smooth muscle were studied. When the pancreatic acini were incubated with increasing concentrations of CCK-8, stimulation of amylase release reached a maximum at 3 nM and then declined with the increasing concentration of CCK-8. CCK-JMV-180 also caused a dose-dependent amylase release stimulation, which plateaued and remained unchanged above 300 nM at about 50% of the maximal stimulation by CCK-8. CCK-JMV 180 above 100 nM caused a rightward shift of the downstroke of the dose-response curve for CCK-8 (pA2 = 7.5). In the gallbladder smooth muscle, CCK-8 caused a dose-dependent contraction, but CCK-JMV-180 totally lacked this property. Instead, CCK-JMV-180 caused a rightward shift of the dose-response curve for CCK 8 (pA2 = 7.9). These results suggest that CCK-JMV-180 distinguishes between the CCKA receptors associated with pancreatic exocrine secretion in the acini and those involved in contraction of the isolated gallbladder smooth muscle in rabbits. PMID- 7543169 TI - A self-paced case study approach to continuing education. PMID- 7543170 TI - Involvement of nitroxidergic and noradrenergic nerves in the relaxation of dog and monkey temporal veins. AB - We determined involvement of nitric oxide (NO) derived from perivascular nerve in venous relaxation. In helical strips of dog superficial temporal veins contracted with prostaglandin F2 alpha (PGF2 alpha) nicotine produced a contraction, which was reversed to a relaxation by prazosin. The relaxation was partially attenuated by timolol or metoprolol. The residual relaxation was not influenced by treatment with atropine or indomethacin and by endothelium denudation but was abolished by NG-nitro-L-arginine (L-NA), a NO synthase inhibitor, and hexamethonium. L- but not D-arginine reversed the inhibition induced by L-NA. Relaxations induced by NO were not influenced by L-NA. Similar results were also obtained in relaxations induced by transmural electrical stimulation that were sensitive to tetrodotoxin (TTX). In the monkey venous strips, relaxations induced by nicotine under treatment with prazosin were reduced by timolol. The relaxation observed with combined treatment with alpha- and beta-antagonists was abolished by L-NA, and L arginine restored the response. The presence of nerve fibers containing NO synthase immunoreactivity or NADPH diaphorase in the adventitia of dog and monkey veins was determined histologically. The findings so far obtained strongly suggest the presence of perivascular nerves that mediate venodilation via a release of NO. Contractions of the temporal vein appear to be mediated by norepinephrine (NE) released from adrenergic nerves that stimulates alpha 1 adrenoceptors, whereas relaxations are mediated by neurogenic NE, acting possibly on the beta 1-adrenoceptor subtype, in addition to NO derived from nerves. PMID- 7543171 TI - In vivo effects of pentaerythrityl-tetranitrate and isosorbide-5-mononitrate on the development of atherosclerosis and endothelial dysfunction in cholesterol-fed rabbits. AB - We wished to determine whether long-term treatment with organic nitrovasodilators has pharmacological effects on the development of atherosclerotic lesions and endothelial dysfunction in cholesterol-fed rabbits. For 15 weeks, six groups of 9 New Zealand White rabbits received a standard diet, which contained no admixture, pentaerythrityl-tetranitrate (PETN 6 mg/kg body weight/day), or isosorbide-5 mononitrate (ISMN 2 mg/kg body weight/day). In the other three groups, the same diets were further enriched with cholesterol (0,75%). Four rings of thoracic aorta were used for tension studies; these rings and the aortas from the aortic arch to bifurcation were then fixed in formol and stained with Sudan IV to determine the area of luminal atherosclerotic lesions by a computerized laser scanning approach. The cholesterol diet increased plasma levels of cholesterol from 69.8 +/- 10.4 to 907.1 +/- 85.5 mg/dl. A similar result was obtained in the group receiving PETN/cholesterol, but the group fed ISMN/cholesterol showed a significantly higher plasma level of cholesterol (1,165 +/- 81.4 mg/dl). Plasma levels of PETN metabolites were still detectable by gas chromatography/mass spectrometry after a 24-h in vivo washout period. The cholesterol diet induced a pronounced degree of atherosclerotic lesions in the aortic arch and the thoracic and abdominal aorta: 73.3 +/- 1.9, 46.3 +/- 2.5, and 49.6 +/- 3.6%, respectively. Additional treatment with PETN resulted in a reduction of this atherosclerotic area to 58.6 +/- 2.05% (p < 0.0001), 34.7 +/- 1.98% (p < 0.01), and 39.3 +/- 3.06% (p < 0.05). In contrast, ISMN had no significant effect on this parameter. The cholesterol diet also induced an endothelial dysfunction as indicated by the diminished vasorelaxation induced by acetylcholine (ACh). Treatment with PETN completely inhibited the development of endothelial dysfunction, whereas ISMN had no effect. In the three groups receiving a cholesterol diet, an increased extent of aortic lesions significantly correlated with increased endothelial dysfunction measured in the same preparations. The long-term treatment with PETN did not affect the vasorelaxing potency of PETN in aortic rings, and similar results were obtained in the case of ISMN. We conclude that long-term treatment with doses of PETN, which do not promote the development of in vitro vascular nitrate tolerance, may protect against atherosclerosis and endothelial dysfunction. This novel, yet unknown pharmacodynamic quality of nitrovasodilators like PETN may contribute to their long-term efficacy in coronary artery disease but may also imply new therapeutic indications in the future. PMID- 7543172 TI - Antiatherosclerotic effects of oral naftidrofuryl in cholesterol-fed rabbits involve inhibition of neutrophil function. AB - We investigated the action of oral naftidrofuryl, a serotonin (5-HT2)antagonist, on atheromatous plaque formation, endothelial function, and neutrophil activity in cholesterol-fed (1% for 12 weeks) rabbits. Cholesterol feeding caused almost complete (84 +/- 4%) coverage of the aortic surface with atheromas and a marked intimal thickening. The endothelium-dependent relaxation to acetylcholine (ACh 1 nM-10 microM) and substance P (30 nM) was considerably reduced, whereas the relaxing effect to the endothelium-independent nitric oxide donor linsidomine (SIN-1) (100 microM) was unchanged. Treatment of hypercholesterolemic rabbits with naftidrofuryl (50 mg/kg body weight) resulted in a marked (54 +/- 6%, p < 0.05) reduction in aortic plaque formation. Endothelium-dependent relaxation to ACh was significantly improved in rings of both thoracic aorta: 33 +/- 5 versus 14 +/- 5% (p < 0.05) and abdominal aorta 68 +/- 9 versus 37 +/- 10% (p < 0.05). Similar results were obtained with substance P, but the responses to SIN-1 were unchanged. Zymosan-induced, luminol-enhanced chemiluminescence of polymorphonuclear leukocytes (PMN) was markedly stimulated in cholesterol-fed rabbits. Naftidrofuryl reduced this hyperreactivity to that of control rabbits. There was no change by naftidrofuryl in any of these parameters in control rabbits, precluding a direct action of the compound in nonhypercholesterolemic conditions. These data demonstrate significant endothelium-protective actions of long-term oral naftidrofuryl in cholesterol-fed rabbits that involve inhibition of cholesterol-induced neutrophil activation. PMID- 7543173 TI - Effect of exogenous testosterone replacement on prostate-specific antigen and prostate-specific membrane antigen levels in hypogonadal men. AB - Previous studies have suggested that serum prostate-specific antigen (PSA) levels are under androgenic influence, especially in patients with adenocarcinoma of the prostate. PSMA (prostate-specific membrane antigen) is thought to reflect hormonal or clonal resistance or an independence with respect to testosterone regulation. The influence of testosterone on serum PSA expression in normal men is not clear. We studied the effect of exogenous testosterone administration on the serum levels of PSA and PSMA in hypogonadal men. Serial serum PSA, serum PSMA by Western blot, and serum total testosterone levels were obtained at intervals of every 2-4 weeks in 10 hypogonadal men undergoing treatment with exogenous testosterone, delivered as testosterone enanthate injection or by testosterone patch. Linear and quadratic orthogonal polynomial scores were calculated for PSMA, PSA, and testosterone. A 2-tailed, paired t-test failed to demonstrate a significant correlation between serum PSA (linear P = 0.432, quadratic P = 0.290) or PSMA (linear P = 0.162, quadratic P = 0.973) and serum testosterone levels. This study suggests that in hypogonadal men, neither PSMA nor PSA expression is testosterone-dependent. PMID- 7543176 TI - A novel monoclonal antibody, KOR-SA3544 which reacts to Philadelphia chromosome positive acute lymphoblastic leukemia cells with high sensitivity. AB - A monoclonal antibody which primarily reacted with Philadelphia chromosome (Ph1) positive ALL cells was produced. The reactivity of a monoclonal antibody, KOR SA3544 (IgG2a) was evaluated on normal hemopoietic cells, 68 leukemic cell lines and freshly obtained cells from 190 patients with leukemia and lymphoma. In cultured cells, KOR-SA3544 reacted with Ph1-positive ALL cell lines (5/5) and leukemic cell lines with 11q23 translocation (3/11). In lymphoid cells, KOR SA3544 was reactive with all of Ph1-positive ALL (26/26), a part of common ALL (5/38) and one case of early B precursor leukemia with 11q23 translocation, but not with peripheral lymphocytes. Normal mature granulocytes were also strongly stained. In myeloid leukemias, KOR-SA3544 was positive (16/56) only in patients with acute myeloid leukemia with FAB-M2 and overt leukemia following myelodysplastic syndrome, but neither with other types of myeloid leukemias nor with blast crisis in chronic myelogenous leukemia. KOR-SA3544 recognized a 90 KDa protein on the membrane of a leukemic cell line, KOPN-57bi. In normal bone marrow, CD19+/KOR-SA3544+ cells were not identified, while Ph1-positive ALL cells were strongly positive for both antibodies. KOR-SA3544 is useful not only for making the diagnosis of Ph1-positive ALL but for detection of the minimal residual disease during remission. PMID- 7543175 TI - Expression and function of the FAS antigen in B chronic lymphocytic leukemia and hairy cell leukemia. AB - The expression and function of the FAS antigen was analyzed in 21 patients with B chronic lymphocytic leukemia (CLL) and four with hairy cell leukemia (HCL) using a specific IgM monoclonal antibody and FACS analysis. The FAS antigen was expressed in a minority (5-41%, mean 15.6%) of the CLL cells in 10 of 21 CLL patients and this expression was not modified during spontaneous or hydrocortisone-induced apoptosis of CLL cells. In contrast, culture with gamma interferon (gamma-IFN) upregulated the expression of FAS in all CLL patients, with 65-100% (mean 84.8%) of the cells being positive after 2 days in vitro culture. Culture with alpha-IFN induced FAS expression in 15 of 19 CLL patients tested, with 15-74% (mean 34%) of the cells being FAS+ after 2 days culture. IL-4 and IL-10, lymphokines that inhibit and promote CLL apoptosis respectively, did not modify the expression of FAS. These results from FACS analysis were consistent with FAS mRNA analysis of fresh and cultured CLL cells, using a semi quantitative reverse transcriptase (RT)-PCR technique. Although IL-4 and IFNs prevent apoptotic cell death of CLL cells in vitro, the present results show that IFNs induce the expression of the apoptosis-inducing protein FAS. However, FAS+ CLL cells were not killed in the presence of anti-FAS monoclonal antibody (while the FAS+ Jurkat and four lymphoblastoid cell lines were killed). This resistance is not due to a mutated FAS protein, since only wild-type FAS cDNA was demonstrated in the leukemic cells of three CLL patients. In four HCL patients 34 53% (mean 44.5%) of the leukemic cells were FAS+ and they were also resistant to the anti-FAS mediated cytotoxicity. The combination of high bcl-2 protein levels and resistance to anti-FAS mediated cytotoxicity may contribute to the extended in vivo survival of CLL and HCL cells. PMID- 7543174 TI - Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia. AB - The expression of bcl-2 protein that is involved in preventing apoptosis in hemopoietic and other cells was evaluated by quantitative flow cytometry in various subpopulations in the normal fetal bone marrow (FBM) and in different types of acute myelogenous leukemias (AML). In the FBM the highest bcl-2 levels (mean antibody binding capacity 51 x 10(3) molecules/cell) were found in CD34+ intermediate sized blasts and myeloblasts, while a CD34+ subset of CD10+ lymphoblasts had low bcl-2 content (8-10 x 10(3) molecules/cell) and the CD34-, CD10+ lymphoblasts were, as expected from previous studies, bcl-2- (< 5 x 10(3) molecules/cell). Variable levels of bcl-2 (5.1-222 x 10(3)) were found in 43 tested cases of AML. The bcl-2 levels decrease with granulocytic and monocytic differentiation and, accordingly, cases of AML with M1 and M2 features showed significantly higher mean bcl-2 levels than the leukemias with promyelocytic (M3) or myelo-monocytic (M4/M5) features. Nevertheless, in seven cases of AML the bcl 2 levels were higher than seen in the normal FBM cells and none of these patients remain in remission after 2 years. Furthermore, in several AML cases intraclonal heterogeneity was observed. The undifferentiated smaller blasts with Class-IIdim display had higher bcl-2 content than the more differentiated larger blasts with more granular side scatter and Class-bright expression. In the same subsets of AML blasts the proliferative S-G2-M fractions showed a reciprocal correlation to bcl-2 content. Thus the higher bcl-2 levels may give a survival advantage and confer some degree of drug resistance to the least differentiated blast populations. The multi-parameter analysis described in this paper, including a combined bcl-2 and cytokinetic analysis of phenotypically defined subgroups of AML blasts, may detect early population shifts during relapse and also guide combination drug therapy. PMID- 7543177 TI - HIMeg-1, a cell line derived from a CML patient, is capable of monocytic and megakaryocytic differentiation. AB - We have characterized HIMeg-1, a subclone of the promegakaryoblastic cell line HIMeg, in terms of its capability of proliferation and differentiation when it is exposed to various agents. We observed that phorbol 12-myristate 13-acetate (PMA) arrested HIMeg-1 growth and induced expression of monocytic surface antigens CD11c and CD14, but not the megakaryocytic surface antigen CD14a. In addition, PMA treatment of HIMeg-1 led to rapid activation of mRNA expression of egr-1, a transcription factor involved in regulating differentiation of hematopoietic progenitor cells. On the other hand, treatment of HIMeg-1 with the activated peripheral blood lymphocyte-conditioned medium (PBL-CM) resulted in greatly enhanced incorporation of 3H-thymidine into newly synthesized DNA. This enhanced 3H-thymidine incorporation appears to be specific to HIMeg-1 since the same concentrations of PBL-CM had little effect on the growth of the megakaryoblastic leukemia cell line SAM-1. The PBL-CM-induced DNA synthesis in HIMeg-1 was associated with activation of CD41a and CD41b surface antigen expression and down regulation of expression of the erythroid marker glycophorin A and the early myeloid surface antigen CD33. HIMeg-1 capable of responding differentially to PMA and PBL-CM by changing its growth rate as well as its differentiation patterns will provide an ideal model to study the underlying mechanism regulating lineage restriction of hematopoietic progenitor cells. PMID- 7543178 TI - Hematopoietic growth factors in drug-induced agranulocytosis. PMID- 7543179 TI - Hematopoietic growth factors in drug-induced agranulocytosis. PMID- 7543180 TI - Avoidance of postoperative blockage of ventilation tubes. AB - While most of the complications of ventilation tubes are widely described in the literature, little is mentioned about postoperative blockage of these tubes. Generally, this blockage is caused by viscid secretion or a blood clot. This study was conducted to assess the effect of using a vasoconstrictor solution to cover the tympanic membrane after ventilation tube insertion to ensure hemostasis in the immediate postoperative period and to decongest the mucosa of the middle ear and the eustachian tube. Xylometazoline hydrochloride (Otrivin Nasal Drops 0.1% or Pediatric Nasal Drops 0.05%) was introduced into 60 ears in 32 patients undergoing myringotomy and ventilation tube insertion. The control group, consisting of 76 ears in 40 patients, underwent only myringotomy and ventilation tube insertion. In a follow-up period of 3 months, postoperative tube obstruction occurred 10.5% of the patients in the control group. No case of blocked tube occurred in the patients who received xylometazoline. PMID- 7543181 TI - Peripheral-blood stem-cell transplantation. PMID- 7543182 TI - Cloning and expression of cDNA encoding an antigenic Cryptosporidium parvum protein. PMID- 7543183 TI - Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19 deficient mice. AB - CD19 is the hallmark differentiation antigen of the B lineage. Its early expression has implicated a role for CD19 during the antigen-independent phases of B-cell development, whereas in mature B cells CD19 can act synergistically with surface immunoglobulin to induce activation. We have generated CD19 deficient mice and found that development of conventional B cells is unperturbed. However, mature CD19-/- B cells show a profound deficiency in responding to protein antigens that require T-cell help. This is accompanied by a lack of germinal centre formation and affinity maturation of serum antibodies. Thus CD19 is crucial for both initial B-cell activation by T-cell-dependent antigens and the maturation and/or selection of the activated cells into the memory compartment. An impairment in ligand-driven selection may also be responsible for the observation of a striking reduction in the B-1 (formerly Ly-1) B-cell subset, thought to develop under the control of self-antigens and bacterial antigens (reviewed in ref. 2). PMID- 7543184 TI - Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A system. AB - The effect of neuropeptide Y [NPY(1-36)] and related peptides on the voltage dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (IACh) of bovine adrenal chromaffin cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting IACh evoked by nicotine was NPY(1-36), NPY (16-36) > peptide YY(PYY) > [Leu31, Pro34]NPY. NPY(16-36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke IACh. NPY(16-36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in IACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3 isobutyl-1-methyl-xanthine (IBMX), decreased the inhibitory effect of NPY(16-36) on IACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14-24)amide] alone did not alter IACh, it potentiated the effect of NPY(16-36) in interaction experiments. While the NPY(16-36)-induced inhibition of IACh was reversed on washout of the peptide, the slightly shorter C terminal fragment NPY(18-36) caused a long-lasting depression of both IACh and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18-36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y3-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel. PMID- 7543185 TI - NMDA receptor heterogeneity in mammalian tissues: focus on two agonists, (2S,3R,4S) cyclopropylglutamate and the sulfate ester of 4-hydroxy-(S)-pipecolic acid. AB - Several potent and selective agonists of the glutamate (L-GLU) receptors of N methyl-D-aspartate (NMDA) type have been tested on the L-[3H]GLU binding to rat cortical membranes, on the depolarization of mouse cortical wedges and on the contraction of guinea pig longitudinal muscle myenteric plexus preparations with the aim of comparing the NMDA receptors present in the cortex and those present in the gut. When the depolarization of the cortical wedges was evaluated, the EC50 values of the agonists were (microM): (R,S)-(tetrazol-5-yl)-glycine (TG) 0.3; trans-4-hydroxy-(S)-pipecolic acid-4-sulfate (t-HPIS) 0.7; 1 aminocyclobutane-cis-1,3-dicarboxylic acid (ACBD) 0.8; NMDA 8; (2S,3R,4S) cyclopropylglutamate (L-CGA C) 12; quinolinic acid (QUIN) 400. When the contraction of the longitudinal muscle myenteric plexus was evaluated, the EC50 values were (microM): L-CGA C 1; TG 8; ACBD 50; t-HPIS 100; QUIN 500 and NMDA 680. When the displacement of NMDA specific L-[3H]GLU binding from rat cortical membranes was evaluated, the IC50 values were (microM): L-CGA C 0.003; TG 0.005; ACBD 0.044; t-HPIS 0.062; NMDA 0.31 and QUIN 15. No significant correlation was found when the EC50 values obtained in the ileum were plotted against the EC50 values obtained in the cortex (r = 0.47). In particular it was noted that L-CGA C was approximately three orders of magnitude more potent than NMDA when tested in the ileum but had a potency not significantly different from that of NMDA when tested in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543186 TI - Chronotropic and inotropic effects of histamine in developing chick heart: differential mechanisms before and after hatching. AB - Chronotropic and inotropic effects of histamine were examined in isolated atrial and ventricular preparations from embryonic and hatched chicken hearts. Histamine produced positive chronotropic and inotropic responses both in embryonic and hatched hearts. The responses to histamine in middle embryonic myocardia, which were observed in the micromolar range, were antagonized by H2 antagonists but not by H1, H3 antagonists and propranolol. Isobutyl-methylxantine, an inhibitor of phosphodiesterase, produced a leftward shift of the concentration-response curve for the chronotropic effect of histamine in the embryo. The responses to histamine in myocardia from hatched chicks, which were observed in the milimolar range, appeared concurrently with the responses to tyramine during development and were antagonized by beta adrenoceptor antagonists but not by any of the histamine antagonists. The positive inotropic response to histamine in hatched ventricular preparations were greatly attenuated by reserpine pretreatment or in the presence of desipramine. Thus, we demonstrated that exogenously applied histamine produces positive chronotropic and inotropic responses in developing chicken hearts and that the mechanisms are different between embryonic and hatched chicks: direct action on H2 receptors in the embryonic heart and release of norepinephrine from sympathetic nerve terminals in hatched hearts. PMID- 7543187 TI - Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs. AB - In guinea pig main pulmonary artery precontracted with noradrenaline, adenosine exerted an initial phasic contraction followed by a tonic contraction and a slow relaxation. After selective blockade by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX: 10 nM) of A1 receptors, adenosine only elicited a rapid relaxation. This initial response was characterized by use of adenosine (AR) and its analogues N6 cyclopentyl-adenosine (CPA), R-N6-phenylisopropyladenosine (R-PIA), 2 chloroadenosine (CADO), 5'-N-ethyl-carboxamidoadenosine(NECA), N6-2-(4 aminophenyl) ethyl adenosine (APNEA) and 2-p-((carboxyethyl)-phenethylamino)-5' carboxamidoadenosine (CGS 21 680). The order of potency of the adenosine analogues for purine-induced phasic contraction was CPA > R-PIA > NECA = APNEA > AR > CGS 21 680 suggesting the involvement of activation of A1 type adenosine receptors in the contraction phase. DPCPX antagonized the CPA-induced contraction with a pA2 = 9.27 +/- 0.26, but the Schild plot slope parameter was significantly lower than unity (0.58 +/- 0.09). In contrast, in electrically driven guinea pig atrial myocardium (a tissue reported to possess A1 receptors), the DPCPX-CPA antagonism was purely competitive (pA2 = 8.95 +/- 0.06; slope = 0.93 +/- 0.06). In the presence of 300 nM DPCPX, the rank order of potency for the purine-induced fast relaxation was NECA > CADO = AR > CGS 21 680 = R-PIA > CPA. The NECA- and adenosine-induced relaxation was influenced neither by 300 nM CP 66713 (an antagonist at A2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 microM NG-nitro-L-arginine: L-NOARG).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543190 TI - Evidence for 5-HT4 receptor subtype involvement in the enhancement of striatal dopamine release induced by serotonin: a microdialysis study in the halothane anesthetized rat. AB - The present study, using the in vivo intracerebral microdialysis method, investigated the role of different serotonin receptor subtypes in the control of dopamine (DA) release exerted by serotonin (5-HT) in the striatum of halothane anesthetized rats. Striatal dialysate DA content was reduced following the blockade of voltage-dependent Na+ channels by tetrodotoxin or by the removal of Ca2+ from the perfusion medium, and increased following depolarization with K+ ions. These findings demonstrate that under our experimental conditions, DA content reflects the neuronal origin of the neurotransmitter release. Drugs were locally applied by means of the microdialysis probe. One, 2.5 and 5 microM 5-HT significantly enhanced DA release in a concentration-dependent manner up to 157, 253 and 446% of basal values respectively. The effect induced by 1 microM 5-HT was not blocked by 10 microM (-)pindolol, a 5-HT1 receptor antagonist, 1 microM ketanserin or 10 microM cinanserin, both 5-HT2A antagonists. One or 10 microM ondansetron (GR 38032F), a selective 5-HT3 antagonist, were also ineffective. In contrast, 10 or 100 microM DAU 6285, a 5-HT3/4 antagonist, significantly reduced the effect of 5-HT on DA release (-20% and -60% respectively). Moreover, 100 microM BIMU 8, a selective 5-HT4 agonist, enhanced DA release (+85%) and this effect was reduced by 100 microM DAU 6285 (-40%). These results demonstrate that in vivo 5-HT exerts a facilitatory influence on striatal DA release and that the 5-HT4, but not the 5-HT1, 5-HT2 or 5-HT3, receptor subtype is implicated, at least partially, in this effect. PMID- 7543189 TI - Up-regulation of CYP2A5 expression by porphyrinogenic agents in mouse liver. AB - Coumarin 7-hydroxylase (COH) activity is catalyzed by the Cyp2a-5 gene product (CYP2A5 enzyme) in mice. Mouse hepatic CYP2A5 expression is often increased in conditions in which other P450 forms are repressed, e.g. after the administration of heavy metals and other toxic agents known to affect cellular heme balance. In this study, the effect of various porphyrinogenic chemicals on the expression CYP2A5 and the key enzymes in heme metabolism was studied. Administration of single doses of griseofulvin (1000 mg/kg), thioacetamide (10 mg/kg) and aminotriazole (1000 mg/kg) to DBA/2 and C57BL/6 mice produced up to 10-fold increases in hepatic COH catalytic activity. Dramatic, up to 130-fold increases in response to the inducers was observed in the amount of CYP2A5 steady-state mRNA. The mRNA contents of aminolevulinate synthase, ferrochelatase and heme oxygenase were also increased to a variable extent, possibly reflecting feed-back regulatory mechanisms. In D2 mice the CYP2A5 inducing effect of aminotriazole and thioacetamide, but not that of griseofulvin, pyrazole and phenobarbital, was abolished by exogenously administered heme arginate. In the B6 strain heme arginate treatment increased CYP2A5 expression but it did not affect the induction caused by porphyrinogenic agents. These results show that porphyrinogenic agents act as efficient inducers of CYP2A5, and suggest that regulation of the transcription of the Cyp2a-5 gene could in some instances involve heme-sensitive factors. PMID- 7543188 TI - Role of nitric oxide in penile erection and yawning induced by 5-HT1c receptor agonists in male rats. AB - The effect of the intracerebroventricular (i.c.v.) administration of NG-nitro-L arginine methyl ester and NG-monomethyl-L-arginine, two inhibitors of nitric oxide (NO) synthase, on penile erection and yawning induced by 1-(3-chlorophenyl) piperazine (m-CPP)- and N-(3-trifluoromethylphenyl)-piperazine (TFMPP), two selective 5HT1c receptor agonists, was studied in male rats. Both NO synthase inhibitors (50-500 micrograms i.c.v.) prevented dose-dependently the behavioural responses induced by m-CPP (0.5 mg/kg s.c.) or by TFMPP (1 mg/kg s.c.), but NG nitro-L-arginine methyl ester was about 4-5 times more potent than NG-monomethyl L-arginine. The D-isomer of NG-monomethyl-L-arginine, which does not inhibit nitric oxide synthase, was ineffective. The inhibitory effect of NG-nitro-L arginine methyl ester on m-CPP- and TFMPP-induced responses was prevented by the administration of L-arginine (1 mg i.c.v.). In contrast, NG-nitro-L-arginine methyl ester (20 micrograms) was ineffective when injected in the paraventricular nucleus of the hypothalamus, a brain area that plays a key role in the expression of these behavioural responses. m-CPP- and TFMPP-induced penile erection and yawning was prevented also by the i.c.v. administration of LY 83583 (50-200 micrograms) or methylene blue (50-400 micrograms), two inhibitors of guanylate cyclase but not by reduced hemoglobin (50-400 micrograms), a NO scavenger. The results suggest that central nitric oxide is involved in the expression of penile erection and yawning induced by 5-HT1c receptor agonists. PMID- 7543192 TI - Evidence for two types of non-NMDA receptors in rat cerebellar Purkinje cells maintained in slice cultures. AB - Pharmacological properties of non-NMDA receptors were investigated in Purkinje cells grown in rat cerebellar slice cultures and recorded in the whole-cell configuration of the patch-clamp technique. Dose-response curves for AMPA and domoate suggest that AMPA, in the concentration range tested, activated only AMPA receptors whereas, domoate activated two types of receptors, probably AMPA and kainate receptors, with EC50 values of 8 and 0.5 microM, respectively. The Scatchard analysis of the dose-response relationship for domoate also suggest that both kainate and AMPA receptors were activated by domoate with approximate affinities of 5 and 0.07 microM-1, respectively. The non-competitive non-NMDA receptors antagonist, GYKI 52466, reduced the amplitude of both AMPA- and domoate activated currents, with a greater potency in reducing currents evoked by AMPA (IC50 = 10 microM) than those induced by domoate (IC50 = 105 microM). These results suggest that, in addition to AMPA receptors, Purkinje cells express kainate receptors and that these two types of non-NMDA receptors can be distinguished from each other on the basis of several pharmacological properties, including affinity for AMPA, domoate and GYKI 52466. PMID- 7543191 TI - Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results. AB - The in vivo potencies of N omega-nitro-L-arginine (L-NA), N omega-monomethyl-L arginine (L-NMMA) and N omega-iminoethyl-L-ornithine (L-NIO) against brain nitric oxide synthase (NOS) were determined by assessing their ability to inhibit harmaline-induced increases in rat cerebellar cGMP. L-NA, L-NIO and L-NMMA were all able to completely prevent the harmaline-induced increase in cGMP with ID50s of 0.5, 30 and 55 mg/kg, respectively, and with the same order of potency as that seen for inhibition of cerebellar NOS in vitro. The inhibitory effects of low doses of L-NA on cerebellar cGMP were maintained for at least 8 hr. The ID50 of L NA for inhibition of cerebellar cGMP in vivo was similar to its ID50 for inhibition of cerebellar NOS ex vivo but only when NOS activity was assayed as an initial rate. However, doses of L-NMMA and L-NIO that inhibited harmaline-induced increases in cerebellar cGMP in vivo by 50% failed to inhibit NOS ex vivo. The methyl ester of L-NA, L-NAME, produced substantial inhibition of cerebellar NOS ex vivo when given either orally, intraperitoneally or intravenously but with a slower onset of action than L-NA. These results demonstrate that measurement of NOS activity ex vivo can accurately reflect the degree of inhibition of NOS in vivo with inhibitors that dissociate slowly from the enzyme such as L-NA, but only when the initial rate of NOS activity is measured. PMID- 7543193 TI - Panretinal cryopexy for the management of neovascularization of the iris. AB - Panretinal cryopexy was used for the treatment of 15 eyes with neovascularization of the anterior segment, treated with panretinal photocoagulation in the past. The eyes were classified preoperatively according to grade of neovascularization of the iris and anterior chamber angle using Weiss' and Gold's device system. Four eyes had rubeosis iridis with normal intraocular pressure and 11 had neovascular glaucoma. Rubeosis was secondary to proliferative diabetic retinopathy and/or central retinal vein occlusion. Nine eyes with grade 0, I and II neovascularization showed regression of neovascularization and controlled intraocular pressure. Six eyes with grade IV showed regression of neovascularization but uncontrolled intraocular pressure. All those eyes presented extensive peripheral anterior synechias. PMID- 7543194 TI - Reconstitution of signal transduction from the membrane to the nucleus in a baculovirus expression system: activation of Raf-1 leads to hypermodification of c-jun and c-fos via multiple pathways. AB - We have attempted to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus by reconstituting them in the baculovirus/Sf9 cell system. We have used this system to coexpress different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 and assayed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos. We observe that activation of ERK-1 via Raf-1 and p21ras dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. Our observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. Our data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors. PMID- 7543196 TI - UV activation of receptor tyrosine kinase activity. AB - The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumor promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as AP-1 and NF kappa B, as well as activation of the Src tyrosine kinase. This 'UV-response' has been well studied in mammalian cells and furthermore is conserved in yeast, however the most upstream components of this signal transduction pathway have remained elusive. Here we show that UV rapidly activates both the EGF receptor and insulin receptor, as shown by tyrosine phosphorylation of these receptors. We demonstrate that this activation is due to autophosphorylation as it only occurs in cells containing receptors with a functional kinase domain. We have further analysed the propagation of the UV-induced signal to downstream events such as, IRS-1 and Shc tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, leukotriene synthesis, MAP kinase activation and gene induction all of which are activated by UV irradiation. Importantly, we demonstrate that in cells expressing a 'kinase-dead' receptor mutant the UV-response is inhibited, blocking leukotriene synthesis, MAP kinase activation and transcriptional induction. Furthermore, prior-stimulation of cells with UV appears to reduce further responsiveness to addition of growth factor suggesting a common signaling pathway. These data demonstrate a critical role for receptor-mediated events in regulating the response mammalian cells to UV exposure. PMID- 7543195 TI - IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR. AB - Expression of the tumor suppressor IRF-1 results in the inhibition of cell growth and transcriptional activation of the IFN-beta gene. IFN production is not responsible for the IRF-1 mediated cell growth inhibition. It is shown here that activation of the IRF-1 causes induction of PKR expression. PKR is a serine/threonine kinase with tumor suppressor activity. IRF-1 mediated cell growth inhibition and IFN induction correlates with PKR expression. A catalytically inactive dominant negative PKR mutant abolishes both activities of IRF-1. These data demonstrate that the tumor suppressor activity of IRF-1 is mediated, at least in part, by PKR. PMID- 7543197 TI - X-ray crystal structure of a dimethylene sulfone-bridged ribonucleotide dimer in a single-stranded state. AB - A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide, r(Aso2U), where a bridging non-ionic dimethylene sulfone linker replaces the phosphodiester linking group found in natural RNA. Crystals of the single stranded state of r(Aso2U) were obtained from water at 50 degrees C. In these crystals, one hydrogen bond is formed between bases from different strands and base stacking occurs in intermolecular 'homo-A' and 'homo-U' stacks. Similar to typical oligoribonucleotides, the ribose rings adopt N-type conformations and dihedral angles chi are in the anti range. The all-trans rotamer of the CH2-SO2 CH2-CH2 bridge was found, which leads to a large adenine-uracil distance. Qualitative analysis of a NOESY spectrum of the Aso2U part in r(Uso2Cso2Aso2U) dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation observed in the crystal is also populated in solution. Comparison with the structure of r(Gso2C), which has been crystallized in the Watson-Crick paired state, shows that a rotation around zeta by +112 degrees leads from the observed, single-stranded state to a conformation that is compatible with formation of a duplex. A concerted trans/gauche flip of alpha and gamma then yields the standard conformer of A-type RNA helices. From the observed structure of r(Gso2C) and other oligonucleotides it is anticipated that this flip will also revert the ribose pucker from C2'-exo to C3'-endo. PMID- 7543198 TI - Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1. AB - Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex. PMID- 7543200 TI - Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein. AB - We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions. PMID- 7543199 TI - Purification and properties of human DNA helicase VI. AB - A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand. PMID- 7543201 TI - Perioperative management of thyroid disease. Prevention of complications related to hyperthyroidism and hypothyroidism. AB - Patients with underlying thyroid disease who are in need of surgery present a particular challenge to the surgeon responsible for their care and to the medical consultant who must offer clinical guidance. Often, underlying thyroid disease is difficult to detect clinically, because signs and symptoms of disease are varied or subtle. Furthermore, those with known disease who are receiving a seemingly stable medical regimen may still be at risk for associated complications. Only heightened clinical awareness, early and appropriate treatment, and delay of elective surgery results in an improved patient outcome. PMID- 7543202 TI - Enhancement of the vasoconstrictor response to KCL by nitric oxide synthesis inhibition: a comparison with noradrenaline. AB - The role of the vascular endothelium in the response to a vasoconstrictor agent acting through a non-receptorial mechanism, such as KCl, was tested in the isolated mesenteric vascular bed of the rat. It was confirmed that the vasoconstrictor response evoked by stimulation of sympathetic terminals was unaffected by 100 microM NG-nitro-D-arginine methyl ester (D-NAME), but was significantly potentiated by 100 microM NG-nitro-L-arginine methyl ester (L-NAME) and by removal of endothelium. Responses to exogenous noradrenaline (1-100 microM) were also enhanced by treatment with 100 microM NG-monomethyl-L-arginine (L-NMMA) and with L-NAME, but not with D-NAME. The potentiating effect of NO synthesis inhibitors was reversed by 1 mM L-arginine. Moreover, the noradrenaline induced vasoconstriction was significantly increased by endothelium-deprivation. Potassium chloride (80 mM) induced a vasoconstrictor response which was not modified by pretreatment with prazosin (0.1 microM) and yohimbine (0.1 microM). The response to KCl was unaffected by D-NAME (100 microM) but the L-stereoisomer induced a significant increase in the perfusion pressure. In endothelium-denuded preparations the vasoconstrictor response to KCl was greater than in control conditions and was quantitatively similar to that observed in L-NAME-treated preparations. The responses to electrical field stimulation, noradrenaline and KCl in endothelium-denuded preparations were not modified by L-NAME. The results suggest that an increase in vascular tone, per se, may represent a trigger for the release of endothelium-derived relaxing factor from endothelial cells. PMID- 7543203 TI - [Acute myeloid leukemias: role of growth factors to improve the efficacy of chemotherapy]. AB - Haematopoietic colony-stimulating factors are a group of molecules that can stimulate haemopoiesis. With the advent of recombinant DNA technology and the cloning of their genes, they can be utilized against malignant blood diseases. They might be useful in therapy of acute myelogenous leukaemia either by increasing the efficacy of antileukaemic drugs or by shortening the duration of granulocytopoenia following chemotherapy or bone marrow transplantation. Encouraging results have been obtained, but stimulation of leukaemic cells is not ruled out. Further clinical and biological investigations are required to provide insight into the role of cytokines in treatment of acute myelogenous leukaemia. PMID- 7543204 TI - Design of four-helix bundle protein as a candidate for HIV vaccine. AB - To be efficient, a synthetic vaccine should contain different T and B cell epitopes of human immunodeficiency virus (HIV) antigens, and the B epitope regions in the vaccine and in the HIV should be conformationally similar. We have suggested previously the construction of vaccines in the form of a protein with a predetermined tertiary structure, namely a four-alpha-helix bundle. Antigenic determinants of cellular and humoral immunity are blocks for the vaccine design. From experimentally studied HIV-1 T and B cell epitopes, we constructed a sequence of a four-helix protein TBI (T and B cell epitopes containing immunogen). The gene of the protein was synthesized and the protein was produced in C600 Escherichia coli cells under recA promoter from Proteus mirabelis. CD spectroscopy of the protein demonstrated that 30% of amino acid residues adopt an alpha-helical conformation. Mice immunized with TBI have shown both humoral and cellular immune responses to HIV-1. The obtained data show that the design of TBI was successful. The synthesized gene structure makes possible further reconstruction and improvement of the protein vaccine structure. PMID- 7543205 TI - Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment. AB - Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface. PMID- 7543206 TI - Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. AB - We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M 1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1). PMID- 7543208 TI - Ionizing radiation-induced expression of the genes associated with the acute response to injury in the rat. AB - Total-body irradiation of rats with doses ranging from an LD10/30 to an LD100/30 induced a dose-dependent increase in the concentration of serum protein associated with the acute response to the irradiation. However, this increase was reached at a later time and was not as pronounced as described previously during the typical acute phase of the response found experimentally (A. Koj, in Structure and Function of Plasma Proteins, Vol. 1, pp. 73-131, Plenum Press, London, 1974). The greatest increase in the serum concentrations of acute-phase proteins was found from the third to the seventh days postirradiation. At these times, the serum concentrations of alpha 2-macroglobulin, haptoglobin, fibrinogen and cysteine protease inhibitor were raised from two- to fivefold, whereas alpha 1-acid glycoprotein was increased sixfold. Incorporation of [35S]methionine into total serum and acute-phase proteins indicated that the increase in the concentration of the acute-phase proteins was preceded by their de novo synthesis in the liver. The results that were obtained by dot-blot analysis showed that the basic course of change in the relative mRNA concentrations in the liver for the acute-phase proteins examined correlated with the changes in their protein concentrations in the serum; only the relative increase in the concentration of alpha 1-acid glycoprotein mRNA was significantly lower than the increase in proteins in the serum, suggesting that a fraction of the serum alpha 1-acid glycoprotein had an extrahepatic origin. On the basis of these results we concluded that total-body irradiation increased the expression of acute-phase protein genes. PMID- 7543207 TI - Nucleotide sequence of a cDNA for a water channel protein (aquaporin) homolog from Atriplex canescens (Pursh.) Nutt. PMID- 7543209 TI - Treatment of local recurrences of rectal carcinoma. AB - From 1978-1992, 159 patients were treated for local recurrences of rectal carcinoma. They could be subdivided into three groups according to the type of primary treatment given; 82 patients underwent primary surgery without irradiation, 37 patients had preoperative and 40 patients postoperative radiotherapy. The localizations of the recurrences and the curative and palliative potentials of surgery and radiotherapy in the treatment of local recurrences were studied. There was no difference in the localisation of the recurrences in the three groups. Median time between initial surgery and recurrence was also almost the same in the three groups and 75% of the recurrences appeared within 2 years. Twenty percent of the patients in the primary surgery alone group, compared with 49% and 38% in the preoperative and postoperative irradiation groups, respectively, had distant metastases at the time of the diagnosis of local recurrence. The predominant symptom from the local recurrence was pain and, after treatment of the recurrence, pain relief was registered in 63%. In 66%, 16% and 22%, respectively, of the patients in the three groups, the intention of the treatment was curative, with either radiotherapy alone, radiotherapy combined with surgery or surgery alone. The 5 years-survival after recurrence was 6% in the primary surgery alone group and 0% in the other 2 groups. Of the 69 patients treated with a curative intention, 32% were locally symptom-free at death or the last follow-up. Our conclusion is that a local recurrence must be avoided due to the morbidity associated with local failure and the potentially low likelihood of curative treatment of a local recurrence. PMID- 7543210 TI - Lymphocytic choriomeningitis virus infection is associated with long-standing perturbation of LFA-1 expression on CD8+ T cells. AB - Flow cytometric analysis of splenocytes from mice infected with lymphocytic choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8+, but not on CD4+ T cells. Appearance of CD8+ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4+ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1hiMEL-14lo), but since about 80% of splenic CD8+ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8+LFA-1hi cells transiently express several markers of cellular activation, e.g. transferrin receptor, IL-2R alpha and beta. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8+LFA-1hi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up regulation of LFA-1 expression is a critical factor involved in directing activated CD8+ T cells to sites of viral infection. PMID- 7543211 TI - Influence of CD14, LBP and BPI in the monocyte response to LPS of different polysaccharide chain length. AB - In this study we examined the involvement of human serum, recombinant lipopolysaccharide binding protein (rLBP), recombinant (r)CD14, CD14 antibodies and recombinant bactericidal permeability-increasing factor (rBPI) in the induction of TNF by Salmonella minnesota LPS of different polysaccharide chain lengths. Soluble rCD14 and rLBP markedly enhanced LPS 6261 TNF production and to a lesser degree also enhanced TNF production from Re 595 LPS and lipid A DP. Addition of anti-CD14 antibodies resulted in nearly complete inhibition of LPS 6261-induced TNF production and partial inhibition of Re 595 LPS and lipid A DP induced TNF release. The ability of lipid A MP to induce TNF production increased with addition of rCD14. Addition of rLBP or anti-CD14 antibodies had no detectable effect on lipid A MP-induced TNF production. The effect of rBPI was also tested and the results showed that only the TNF-inducing ability from smooth LPS was completely inhibited by rBPI. Recombinant BPI was considerably less effective in inhibiting Re 595 LPS-induced TNF production, and lipid A DP was not affected by rBPI. Our data suggest that the ability of rLBP, rCD14, CD14 antibodies and rBPI to modulate LPS induced TNF production is strongly dependent on the LPS polysaccharide chain length. PMID- 7543213 TI - Characterization of chicken CD8-specific monoclonal antibodies recognizing novel epitopes. AB - CD8 is a heterodimeric cell surface glycoprotein expressed primarily on thymocytes and a subpopulation of mature T lymphocytes. It binds to the invariant part of the major histocompatibility complex class I molecule and participates in antigen recognition by the major histocompatibility complex class I restricted T cells. As in mammalian species, the majority of chicken thymocytes express both CD4 and CD8, whereas peripheral T cells are either CD4- or CD8-positive. We have created a panel of mouse monoclonal antibodies detecting different cell surface epitopes on chicken CD8. The antibodies precipitate a 32-34 kDa dimeric protein from surface labelled thymocytes under reducing conditions. The identical N deglycosylation pattern confirms that these MoAb precipitate the same heterodimeric molecule from chicken thymocyte lysates. Binding of 11-38 and 11-39 MoAb to peripheral blood T cells is totally inhibited by 11-39 and previously characterized CT8 and EP72 MoAb, further confirming their CD8 specificity. CD8 alpha-chain specificity of MoAb 11-39, 11-38, 11-30 and 11-13 is conclusively proven by staining COS-cells transfected with a plasmid containing CD8 alpha cDNA. However, MoAb 11-13, 11-30 and 11-38 do not compete with MoAb 11-39 in binding to CD8. These results demonstrate recognition of different epitopes by these MoAb. Monoclonal antibodies detecting novel epitopes on chicken CD8 provide a valuable tool for further studies on T cell development. PMID- 7543212 TI - Gliadin-specific T cell responses in peripheral blood of healthy individuals involve T cells restricted by the coeliac disease associated DQ2 heterodimer. AB - Coeliac disease (CD) is probably caused by an abnormal immune response towards wheat gliadin in the small intestine. We found that gliadin-specific T cells from the small intestinal mucosa of HLA-DQ2 positive CD patients were almost exclusively restricted by the disease-associated DQ2 molecule. In the peripheral blood of CD patients, a large proportion of gliadin-specific T cells were found to be restricted by DQ molecules, including DQ2, but many were instead restricted by DR or DP molecules of the patient. We have now investigated gliadin-specific T cell responses in peripheral blood from healthy individuals. Four of 20 persons tested had strong in vitro responses and were used as donors for gliadin-specific T cell clones. We found gliadin-specific T cells restricted by the CD-associated DQ2 molecule in peripheral blood for two of these four individuals. It is the presence of such T cells also in the small intestinal mucosa which seems typical of CD. PMID- 7543215 TI - T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors. AB - The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC-I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53 peptides as stimulator cells. T cell reactivity was observed towards both wild type and mutated p53 peptide epitopes with CTL precursor frequencies varying from 1:2 x 10(4) to 1:1.5 x 10(5). These results might suggest the presence of an ongoing immune response in normal individuals against cells expressing increased levels of p53 protein. PMID- 7543214 TI - Specific and long-lasting protection from collagen-induced arthritis and oil induced arthritis in DA rats by administration of immunogens. AB - DA rats develop transient arthritis after subcutaneous immunization with adjuvant oil, while chronic arthritis and collagen autoreactivity ensues when collagen is added to the oil. We show here that DA rats can be protected from oil-induced arthritis (OIA) and rat collagen-induced arthritis (rCIA) by addition of antigen to these arthritogenic inocula. We have investigated this remarkable phenomenon and demonstrate that both foreign and self antigens can be protective, apparently provided they are immunogenic; hence HSP-65kDa, ovalbumin, rat myelin basic protein, rat IgG and bovine albumin are effective while rat albumin is not. This protection is long-lasting and disease-specific because rats protected from rCIA resist a later attempt to induce arthritis, but not experimental autoimmune encephalomyelitis (EAE). Protection from rCIA depends neither on the blocking of humoral autoreactivity to collagen nor on a change in the isotype profile of anti collagen antibodies. We demonstrate that immunogens can also be protective when injected intraperitoneally only a few days before onset of arthritis. Our results indicate that protection is mediated through bystander immune reactions towards the co-immunized antigen and that the arthritogenicity of a given provocation, be it adjuvants, microbes or autoantigens, may be a complex net result of arthritogenic and contra-arthritogenic immune reactions. PMID- 7543217 TI - Anti CD5 sustains the proliferative response of IgM-activated human CD5+ B cells. AB - The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5+ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')2 fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5+ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25+ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5+ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5+ B cells favouring their proliferation and differentiation into autoantibody secreting cells. PMID- 7543216 TI - Augmented lung adenocarcinoma cytotoxicity by the combination of a genetically modified anti-Lewis Y antibody and antibodies to complement regulatory proteins. AB - Complement-dependent cytotoxicity (CDC) mediated by a chimeric anti-Lewis Y monoclonal antibody (cH18A; human IgG1) was investigated in this study. Human lung adenocarcinoma cell lines (PC7, PC9, and PC14) were used as the target cells. PC7 and PC9 cells, expressed Lewis Y antigen and were lysed by cH18A as effectively as by the parent mouse anti-Lewis Y antibodies (mH18A) in a concentration-dependent manner. PC14 cells did not express Lewis Y antigen and were not lysed by either cH18A or mH18A. cH18A mediated CDC activity against PC7 and PC9 cells was enhanced by the combined use of monoclonal antibodies directed against CD46(MCP), CD55(DAF), and CD59. These molecules are complement-regulatory proteins which protect host cells from CDC. PC7 and PC9 cells, showed high levels of surface expression of these proteins, PC7 cells were more susceptible to cH18A mediated CDC than PC9 cells. Use of multiple blocking antibodies to the complement-regulatory proteins produced more enhancement of cH18A-mediated CDC than a single antibody. Moreover, expression of CD55 and CD59 by PC7 and PC9 cells was decreased after treatment with PI-PLC, resulting in increased susceptibility to cH18A-mediated CDC. Although the reason is unknown, PC7 cells became more susceptible to CDC than PC9 cells after PI-PLC treatment even in the absence of cH18A. These data suggest that chimeric monoclonal antibodies can be used to induce CDC against lung adenocarcinoma, and that such CDC is potentiated by a variety of antibodies blocking compliment-regulatory proteins on the tumour cell surface. PMID- 7543218 TI - Clinical implications of clozapine discontinuation: report of an NIMH workshop. AB - In September 1994, the National Institute of Mental Health convened a group of scientists to discuss the clinical effects of rapid clozapine discontinuation, especially in light of the introduction of risperidone for the treatment of schizophrenia. Despite concern over recent reports of clinical deterioration (psychotic exacerbations, somatic withdrawal symptoms, and extrapyramidal side effects) in a few patients abruptly discontinued from clozapine, there is currently insufficient information to determine the magnitude of the problems associated with clozapine withdrawal. However, clinicians are reminded that the withdrawal schedule for clozapine indicates a gradual tapering schedule (unless the patient is experiencing severe side effects); that switching patients from clozapine to risperidone does not mean that such tapering is unnecessary; and that the use of risperidone may not produce all of the same effects as clozapine in some treatment-refractory patients. PMID- 7543219 TI - Congenital indifference to pain: long-term follow-up of two cases. AB - We are reporting the cases of two brothers with congenital indifference to pain, previously reported by one of us in 1965. This represents a nearly 30-year follow up. In this rare disorder of pain perception, all other sensory modes are intact. Management includes early recognition of the condition, prevention of injury, immobilization of injured extremities, and surgical correction of established deformities. PMID- 7543220 TI - Magnolol inhibits collagen-induced platelet serotonin release. PMID- 7543221 TI - DNA and protein adducts. AB - Application of methods for the measurement of DNA and protein adducts in environmental studies was surveyed. The methods included the 32P-postlabelling assay, immunoassay and synchronous fluorescence spectroscopy for DNA adducts. Additionally, methods for detecting excreted urinary RNA and DNA adducts were discussed. The protein adduct techniques included both immunological and chemical assays. The techniques have been applied in occupational and environmental studies, but usually one assay at a time. As specific DNA adducts can now be assayed for, it would be important to use these methods and specific protein adduct assays in the same studies. It is important to develop further specific adduct tests. This can be done with the help of standard compounds, which also allow quantitation in the assays. An international bank of standard compounds would be a major advancement to human biomonitoring. PMID- 7543222 TI - Circulating selectin- and immunoglobulin-type adhesion molecules in acute ischemic stroke. AB - BACKGROUND AND PURPOSE: Cellular adhesion molecules mediate adhesion between endothelial cells and leukocytes as a precondition for extravasation of leukocytes at sites of tissue injury. The pattern of release of circulating adhesion molecules has been characterized in patients with acute ischemic stroke. METHODS: Serum concentrations of soluble selectin-type adhesion molecules (solube endothelial leukocyte adhesion molecule-1 [sELAM-1], soluble lymph node homing receptor [sL-selectin]) and immunoglobulin-type adhesion molecules (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble intercellular adhesion molecule-1 [sICAM-1]) were serially determined (at hours 4, 8, and 10 and at days 1, 3, and 5) in 22 patients with acute ischemic stroke. As control subjects, age- and sex-matched individuals with (n = 40) and without (n = 22) vascular risk factors were studied. RESULTS: We observed increased concentrations of sICAM-1 and decreased levels of sL-selectin in patients with risk factors even in the absence of stroke. Patients with acute stroke had, in addition, an initial transient increase of sELAM-1 and a persistent increase of sVCAM-1. CONCLUSIONS: The results suggest a chronic alteration of expression of adhesion molecules sICAM-1 and sL-selectin in subjects with risk factors for atherosclerosis; they also indicate acute changes of levels of sELAM-1 and sVCAM-1 in response to acute ischemic stroke. Determination of soluble adhesion molecules could allow in vivo monitoring of the initial steps of leukocyte-mediated brain damage in acute ischemic stroke. PMID- 7543223 TI - Protection of human polymorphonuclear leukocyte function from the deleterious effects of isolation, irradiation, and storage by interferon-gamma and granulocyte-colony-stimulating factor. AB - BACKGROUND: Fungal infections represent a difficult challenge to clinicians caring for neutropenic patients with hematologic malignancies, as antifungal therapy often has limited success in that setting. One promising yet problematic alternative approach is leukocyte transfusion. The isolation of polymorphonuclear leukocytes (PMNs) induces apoptosis and functional deterioration, and irradiation to prevent transfusion-associated graft-versus-host disease causes further functional deterioration. STUDY DESIGN AND METHODS: The ability of interferon gamma and granulocyte-colony-stimulating factor (G-CSF), used both alone and in combination, to protect PMNs after 0 or 20 hours' storage in cell culture (as a model for function after transfusion) and irradiation with 0, 5, or 30 Gy was studied. RESULTS: Without cytokine treatment, 20-hour-old PMNs showed marked apoptosis, no appreciable chemotaxis, and no ability to kill Candida albicans. In contrast, cytokine treatment significantly reduced apoptosis and protected chemotaxis, C. albicans killing, and surface-receptor expression from both storage and irradiation. Although the majority of the benefit appeared to be due to G-CSF, consistent trends suggested better function of PMNs after combined treatment with interferon-gamma and G-CSF. CONCLUSION: Judicious use of cytokines may preserve PMN function. These findings have important implications for the transfusion of PMNs to cytopenic patients. PMID- 7543224 TI - Rho as a regulator of the cytoskeleton. AB - Many cell functions, including maintenance of morphology, aggregation, motility, membrane ruffling, smooth-muscle contraction, cytokinesis in mammals and bud formation in yeast, are regulated through the dynamic reorganization of actin filaments. Although it has long been known that Ca2+ is a key regulator of the cytoskeleton, evidence is now accumulating that Rho, a Ras-related small GTP binding protein, is another important regulator of these actin-dependent cell functions. PMID- 7543225 TI - The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA. AB - The RNP domain is found in a number of proteins involved in processing and transport of mRNA precursors. The crystal structure of a complex between the U1A spliceosomal protein and its cognate RNA hairpin at 1.92 A resolution reveals the molecular basis of sequence-specific RNA recognition by the RNP domain. PMID- 7543226 TI - [Ultrastructure of chloroplasts and activity of amylases in Chlorella cells during space flight]. AB - Ultrastructure of Chlorella cells grown in space flight was studied. Some differences in cell organells, chloroplasts in particular, were found in experimental cells in comparison to control. The considerable reduction in the number of reserve polysaccharides in chloroplasts and some decrease in stroma electron density were detected. Specific activity of amylases enhanced because of an increase in concentrations of mono- and disaccharides. The rearrangements of chloroplast ultrastructure, predominantly a decrease in reserve polysaccharides, are probably due to an increase of hydrolytic processes under microgravity. PMID- 7543227 TI - Expression of E-selectin in ischemic and reperfused human skeletal muscle. AB - This work was undertaken to assess the role of endothelial E-selectin in the development of neutrophil accumulation into the ischemic and reperfused human skeletal muscle and eventually in the genesis of ischemia-reperfusion syndrome. Twelve patients affected by abdominal aortic aneurysm who were undergoing reconstructive vascular surgery were studied. Muscle biopsies from the right femoral quadriceps were taken (1) immediately after anesthesia, as control samples, (2) before declamping the aorta, as ischemic samples, and (3) 30 minutes after reperfusion and then processed for immunohistochemical and ultrastructural analysis. Immunohistochemistry revealed a strong positive reaction for E-selectin on the venular endothelium during ischemia and reperfusion. Ultrastructural investigation showed that reactivity for E-selectin matched neutrophil accumulation of the skeletal muscle tissue. This phenomenon was dependent upon a complex series of events that included neutrophil adhesion to the inner surface of the postcapillary venules, passage through endothelial intercellular junctions, and migration distally into the interstitial spaces of the skeletal muscle tissue. Neutrophil tissue infiltration was also associated with ultrastructural signs of tissue damage at reperfusion. This is in agreement with accumulating evidence indicating a role for tissue infiltrating neutrophils in the genesis of toxic O2 free radicals. Our data suggest that E-selectin expression on the vascular endothelium of human skeletal muscle may represent a key regulatory point in the process of neutrophil tissue accumulation and indicate an active role for the venular endothelium in the development of human ischemia-reperfusion syndrome. PMID- 7543228 TI - [Morphine-induced hyperalgesia, allodynia and myoclonus--new side-effects of morphine?]. AB - During the last ten years hyperalgesia (H), allodynia (A) and myoclonia (M) has been reported at an increased frequency in human beings treated with morphine. The side effects are most common in cancer patients treated with high dose morphine, and has been reported for all routes of administration. The mechanisms are unknown, but human cases and experimental works have resulted in the following theories: 1) Morphine and morphine metabolites change the postsynaptic pain-transmission in dorsal horn neurones via non opioid-receptors (glycine and/or N-methyl-D-aspartate). 2) Morphine and morphine metabolites activate other opioid receptor populations. 3) Supplemental drugs in cancer management. 4) An abnormal metabolism of morphine or morphine metabolites. 5) A combination of one or more of the above-mentioned theories. The first mentioned theory is the most likely. The treatment of morphine induced H, A, and M seems to be to discontinue morphine administration and to initiate therapy with other opioids (fentanyl, sufentanyl, methadone or ketobemidone). PMID- 7543229 TI - Effects of chlorpyrifos on neuronal development in rat embryo midbrain micromass cultures. AB - Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide used to control mosquitos and household insects. In man it has toxic effects on the central nervous system, the cardiovascular system and the respiratory system. This study investigated the toxicity of CPF on nervous system development using the midbrain micromass culture system. Chlorpyrifos was tested as a marketed formulation and in 3 solvents. All demonstrated toxicity in midbrain micromass cells with IC50 values below 30 ug/mL, indicating a potent teratogen. PMID- 7543231 TI - Localization of a protective epitope on a Venezuelan equine encephalomyelitis (VEE) virus peptide that protects mice from both epizootic and enzootic VEE virus challenge and is immunogenic in horses. AB - In order to define more precisely the protective epitope encoded within the first 25 amino acids (aa) of the E2 glycoprotein of the Trinidad donkey strain of Venezuelan equine encephalomyelitis (VEE) virus, we examined the immunogenicity of smaller peptides within the first 19 aa. pep1-9 and pep3-10 elicited virus reactive antibody, but failed to protect mice from virus challenge. Additionally, pep3-10 was identified by a competitive binding assay using overlapping peptide octamers as the putative binding site of the antipeptide monoclonal antibody (mAb) 1A2B-10. Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus reactive antibody. PMID- 7543230 TI - Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella. AB - Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence. PMID- 7543232 TI - Platelet-derived growth factor receptor immunoreactivity in mesothelioma and nonneoplastic mesothelial cells in serous effusions. AB - Expression of the platelet-derived growth factor receptor (PDGFR) was detected by immunocytochemistry in normal and malignant mesothelial cells from 14 benign effusions and 22 mesotheliomas. Two well-characterized antireceptor monoclonal antibodies to the PDGFR alpha-subunit (PR292) and beta-subunit (PR7212) were used. PDGFR alpha-subunit outlined cell membranes intensely in nonneoplastic mesothelial cells, whereas it was focal in mesothelioma and limited to a few cases only. PDGFR beta-subunit was weakly expressed in the cytoplasm of normal mesothelium; in contrast, malignant mesothelial cells showed strong cytoplasmic staining. Our results show that normal mesothelium may be responsive to PDGF by the predominant expression of PDGFR-alpha and less by PDGFR-beta and indicate the presence of growth-stimulation loops in mesothelioma through PDGF/PDGFR-beta interaction. Also, scattered staining for the PDGFR alpha-subunit suggests that a PDGF-A chain/PDGFR-alpha interaction may also be active in mesothelioma cells growing in suspension. PMID- 7543233 TI - Usefulness of antral brushing samples stained with Diff-Quik in the cytologic diagnosis of Helicobacter pylori. A comparative methodologic study. AB - We studied the usefulness of antral cytologic specimens obtained by brushing and stained with Diff-Quik in the diagnosis of gastric colonization by Helicobacter pylori in 102 patients, comparing this method with other commonly employed techniques. We report on the sensitivity, specificity, predictive value for positive and negative cases of each of the techniques used, and diagnostic problems posed by each. In general, the Diff-Quik-stained samples proved to be the most useful. This technique is a simple, economical and practical method for the diagnosis of gastric colonization by H pylori. PMID- 7543234 TI - Intraoral and transoral fine needle aspiration. A review of 25 cases. AB - During a four year period, 25 intraoral and transoral fine needle aspirations were performed. The sites aspirated were the parapharyngeal space (7), palate (4), floor of mouth (3), tongue (3) and a variety of other intraoral sites (8). Squamous cell carcinoma, the most common malignancy encountered, was correctly diagnosed in six cases. Other malignancies correctly diagnosed were 2 malignant lymphomas, 1 mucoepidermoid carcinoma and 1 adenoid cystic carcinoma. One malignant case called "carcinoma, probably adenoid cystic carcinoma" proved to be a mucoepidermoid carcinoma. One case called "suspicious for squamous carcinoma" was found to be a branchial cleft cyst. In another case a diagnosis of carcinoma in situ of the tongue was suggested and confirmed by subsequent biopsy, but the underlying adenoid cystic carcinoma was missed. Of seven benign entities, five were correctly diagnosed by fine needle aspiration (FNA), and in three cases the FNA diagnosis was equivocal. Unsatisfactory aspirates were obtained in four cases. Despite some obvious problems, we consider FNA to be especially useful for the evaluation of parapharyngeal masses, which are usually not accessible to routine surgical biopsy, and for palatal masses, which are usually submucosal and therefore often difficult to biopsy. PMID- 7543235 TI - Cytologic diagnosis of intracranial germinomas in smear preparations. AB - Although cytologic smear preparations have been used in the intraoperative diagnosis of central nervous system tumors for a long time, detailed descriptions of the rarer tumors, including germinomas, have not been provided in the literature. We reviewed the cytologic features of five intracranial germinomas diagnosed with the smear technique. The smears showed sheets of large tumor cells intimately mixed with mature lymphocytes. The cells had delicate, vacuolated cytoplasm with distinct borders, while the nucleoli were prominent and elongated. There often was an amorphous, eosinophilic background of extracellular matrix. The main differential diagnoses were true pineal gland tumors, pituitary adenomas, craniopharyngiomas, pilocytic astrocytomas and parasellar meningiomas. PMID- 7543236 TI - Fine needle aspiration cytology of low grade endometrial stromal sarcomas. AB - The cytomorphology of endometrial stromal sarcoma (ESS) has been described. However, almost all of the reports are about the high grade type according to the newly revised histopathologic criteria; the cytomorphology of low grade ESS (endolymphatic stromal myosis) has yet to be characterized. This paper reports on two cases of recurrent low grade ESS diagnosed by fine needle aspiration and confirmed histopathologically. The aspirates consisted of extremely cohesive tissue fragments of monotonous, small (8-9 microns), oval cells tightly woven in a metachromatic, extracellular matrix on Diff-Quik-stained smears. In Papanicolaou-stained smears the transparency allowed visualization of bland, normochromatic, oval nuclei as well as small arterioles transecting the tissue fragment, whereas the extracellular matrix became invisible. The metachromatic, extracellular matrix in the smears correlated with the reticular fibrillar network wrapping around individual tumor cells on tissue sections, one of the histopathologic criteria that separates low grade from high grade ESS. PMID- 7543237 TI - Fine needle aspiration cytology of primary mediastinal germ cell tumors. AB - Fine needle aspiration cytology was performed on six patients with malignant mediastinal germ cell tumor: 1 pure seminoma, 1 pure embryonal carcinoma, 1 pure yolk sac tumor and 3 mixed germ cell tumors containing teratoma. Their cytologic features were compared with each other and with the cytologic features of thymoma, which arises commonly in the anterior mediastinum. A definitive cytologic diagnosis could be made only in the cases of seminoma because of its characteristic cytologic features. Cytochemical staining for germ cell alkaline phosphatase was helpful in diagnosing seminoma in the cytologic examination, while the presence of hyaline globule or alpha-fetoprotein immunostaining as the cytologic diagnostic feature of yolk sac tumor were not necessarily found in fine needle aspiration cytology. All epithelial, lymphocytic and mixed type thymomas were easily differentiated from the four types of germ cell tumor examined. PMID- 7543238 TI - Fine needle aspiration of prostatic rhabdomyosarcoma. A case report demonstrating the value of DNA ploidy. AB - A case of primary rhabdomyosarcoma of the prostate diagnosed by fine needle aspiration biopsy and confirmed with immunocytochemistry is described. Evaluation of DNA content of tumor cell nuclei by image analysis was performed to complement the diagnosis and clinical stage information and to further predict the tumor response to chemotherapy. Diploid tumors should prompt clinicians to use a more aggressive chemotherapeutic protocol to achieve as favorable a response to therapy as that seen in aneuploid tumors. The DNA content obtained with differently fixed and stained specimens yielded similar results, further confirming the wide applicability of image analysis as a diagnostic/prognostic tool in prospective and retrospective studies in pathology. PMID- 7543239 TI - Ziehl-Neelsen stain in the quest for elusive cestode larval hooklets. PMID- 7543240 TI - Charybdotoxin and its effects on potassium channels. AB - Over the last few years, a considerable amount of information has been obtained regarding K+ channels. Different areas of research have contributed to knowledge in this field. Charybdotoxin (ChTX), a 37-amino acid peptide isolated from venom of the scorpion Leiurus quinquestriatus var. hebraeus, represents a remarkable tool for studying K+ channels. With its use, it has been possible to purify the high-conductance Ca(2+)-activated K+ (maxi-K) channel to homogeneity and determine the subunit composition of this channel. This has led to the discovery of an auxiliary beta-subunit that, when coexpressed with the pore-forming subunit, mSlo, alters the biophysical and pharmacological properties of this latter subunit. With the feasibility of producing large amounts of ChTX by recombinant techniques and the knowledge of the three-dimensional structure of the peptide, it has been possible to carry out site-directed mutagenesis studies and obtain a picture of the interaction surface of the toxin with two channels, maxi-K and Shaker, and to derive a picture of the complementary surface of the receptor in these two channels. Finally, ChTX, and the more selective K+ channel toxins that were subsequently discovered, have provided us with unique tools not only to determine the functional role that K+ channels play in target tissues but also to develop the molecular pharmacology of these channels. PMID- 7543241 TI - Evidence for location of the CFTR in human placental apical membrane vesicles. AB - Ion transport (36Cl uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. 36Cl uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (Ko = Ki and Ko > Ki, where Ko is potassium concentration outside and Ki is potassium concentration inside the vesicles). The sensitivities of 36Cl uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of 36Cl uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the 36Cl uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3 thiotriphosphate) (0.8 mM) led to a decrease in total 36Cl uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of 36Cl uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of approximately 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles. PMID- 7543243 TI - CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells. AB - The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3 dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine. PMID- 7543242 TI - Ca(2+)-dependent Cl- current in canine tracheal smooth muscle cells. AB - Our goal was to investigate the role of Ca2+ entry in regulating Cl- current (ICl) in smooth muscle cells from canine trachealis. When studies were done using the perforated patch configuration, depolarization elicited a dihydropyridine sensitive Ca2+ current (ICa), followed in many cells by a sustained current. This sustained current reversed direction close to the Cl- equilibrium potential, consistent with its representing ICl. The ICl was also apparent as slowly deactivating tail currents seen upon repolarization to negative potentials. The Cl- channel blocker niflumic acid abolished both the sustained and tail currents, without affecting ICa. Several observations indicated that the ICl was dependent on Ca2+ entry. ICl was increased in magnitude when Ca2+ influx was augmented [by prolonging the depolarization or using BAY K 8644 or acetylcholine (ACh)] and decreased in magnitude when Ca2+ influx was reduced (using nifedipine). Based on these findings, we conclude that depolarization causes Ca2+ entry, with resultant elevation of cytosolic free Ca2+ concentration leading to activation of ICl (ICl(Ca)). We investigated whether Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was involved in activation of ICl(Ca), by depleting intracellular stores of Ca2+ using cyclopiazonic acid to block the sarcoplasmic Ca(2+)-adenosinetriphosphatase and repeated stimulation with ACh. In such Ca(2+) depleted cells, depolarization-mediated Ca2+ entry continued to activate ICl(Ca), suggesting that Ca(2+)-induced Ca2+ release was not required for its activation. We conclude that Ca2+ entry can activate Cl- channels in tracheal smooth muscle. This represents a positive-feedback system, which would promote excitation and contraction of airway muscle. PMID- 7543244 TI - Tyrosine kinase activation is necessary for inducible nitric oxide synthase expression by interleukin-1 beta. AB - The inflammatory cytokine interleukin-1 (IL-1) induces the inducible form of nitric oxide synthase (iNOS) with an increase in nitric oxide in rat mesangial cells. However, the cellular mechanisms that underlie the induction of iNOS by IL 1 beta in mesangial cells has not been clarified. Because we have shown that tyrosine kinase inhibitors attenuate IL-1 beta-induced cyclooxygenase expression and prostaglandin production, we investigated the effect of tyrosine kinase inhibitors on IL-1 beta-induced nitrite production and iNOS mRNA expression in rat mesangial cells. The tyrosine kinase inhibitors genistein and herbimycin A attenuated IL-1 beta-induced nitrite production in a dose-dependent manner. In addition, both of these inhibitors blocked IL-1 beta-induced iNOS mRNA expression. These data suggest that tyrosine kinase(s) plays a central role in IL 1 beta signaling to induce iNOS in rat mesangial cells. PMID- 7543245 TI - MCP-1-stimulated monocytes preferentially utilize beta 2-integrins to migrate on laminin and fibronectin. AB - Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins. PMID- 7543246 TI - Receptor-mediated clearance of G-CSF derivative nartograstim in bone marrow of rats. AB - To clarify the role of the granulocyte colony-stimulating factor (G-CSF) receptor in the nonlinear elimination of a recombinant human G-CSF derivative, nartograstim (NTG), the accompanying changes in the in vivo NTG total body clearance at steady state (CLss) or the early-phase tissue uptake clearance (CLuptake) in rats were compared with the change in the number of G-CSF receptors in bone marrow. The infusion rate-dependent decrease in CLss in control rats confirmed the existence of a saturable elimination mechanism for NTG. The Michaelis-Menten constant (Km) and maximal velocity for this saturable process were estimated to be 107 pM and 15.5 pmol.h-1.kg-1, respectively. The Km for this saturable process was comparable with the dissociation constant (Kd) for the specific binding of NTG to bone marrow cells. After administration of excess NTG, the CLuptake of tracer amounts of 1251-NTG by bone marrow and spleen, which corresponds to the receptor density in the tissues, was reduced at 2 h but gradually recovered. This change in CLuptake corresponds well to the change in the in vitro NTG-binding capacity in each isolated cell. This reduction in CLuptake might be due to the downregulation of G-CSF receptors on the cell surface. On the other hand, the saturable CLss in cyclophosphamide-treated rats was 17% of that in control rats, whereas the saturable CLss in rats given NTG repeatedly was twofold greater than in controls, which is associated with the upregulation of G-CSF receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543247 TI - Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin. AB - Lipopolysaccharide (LPS) produces a rapid and sustained reduction in the circulating concentration of insulin-like growth factor I (IGF-I), which may be responsible, in part, for the alterations in protein metabolism observed in these animals. The purpose of the present study was to determine whether this drop was due to a decreased hepatic production of IGF-I and/or an increased clearance of the peptide from the blood. Four hours after intravenous injection of LPS the plasma IGF-I concentration was decreased 50%. IGF-I release by in situ perfused livers from control rats was constant throughout the 60-min perfusion period and averaged 111 +/- 3 ng/min. In contrast, hepatic IGF-I output was decreased 46% by in vivo LPS. In contrast, livers from LPS-injected rats released more IGF binding proteins-1, -2 and -4 than did control livers. Hepatic cell isolation indicated that LPS decreased the IGF-I content in Kupffer and parenchymal cells, but not endothelial cells, by approximately 45%. Pharmacokinetic analysis of blood 125I IGF-I decay curves indicated that the half-life for whole body clearance of 125I IGF-I from the circulation was not altered by LPS. However, LPS increased 125I IGF-I uptake by spleen, liver, lung, and kidney while decreasing uptake by the pancreas and gastrointestinal tract. These results indicate that the LPS-induced decrease in blood IGF-I concentration is primarily due to a reduction in hepatic production, not a change in whole body peptide clearance, and that a decreased production by both parenchymal and Kupffer cells contributes to this alteration. PMID- 7543248 TI - Cation channel activated by muscarinic agonists on porcine adrenal chromaffin cells. AB - A large portion (70%) of the secretory response to muscarinic agonists in porcine adrenal chromaffin cells has previously been shown to be dependent on extracellular Ca2+ (Xu et al., J. Neurochem. 56: 1899-1896, 1991). Results presented here show that muscarinic agonists activate a cation-selective channel which is permeable to divalent cations. The muscarinic agonist, methacholine, was found to activate the uptake of Mn2+, which paralleled the ability of methacholine to activate 45Ca2+ uptake as shown previously. Secretion induced by methacholine was not affected by nifedipine, a compound that inhibits dihydropyridine-sensitive voltage-gated Ca2+ channels. In voltage-clamped cells, methacholine activated whole cell currents, which reversed at approximately -20 mV in standard salt solutions. However, with the standard whole cell configuration, the currents were slow to activate and were often erratic. In contrast, when the perforated-patch (nystatin) technique was used to measure whole cell currents, methacholine rapidly activated sustained inward currents. Ion-substitution experiments indicated that the inward currents were carried by Na+, Ba2+, or Ca2+ but not by Cl-. Single-channel currents activated by methacholine were observed in outside-out vesicles, which were electrically accessed using the perforated-patch technique. These channels reversed at -15 mV, had a slope conductance of 20 pS, and were 14-fold more likely to be open in the presence of methacholine. These channels are probably responsible for the extracellular Ca(2+)-dependent secretory response to muscarinic receptor stimulation in porcine adrenal chromaffin cells. PMID- 7543250 TI - Leukotoxin, 9,10-epoxy-12-octadecenoate causes edematous lung injury via activation of vascular nitric oxide synthase. AB - We examined the mechanism of leukotoxin, 9,10-epoxy-12-octadecenoate (Lx)-induced lung injury in blood-free, physiological salt solution-perfused rat lungs under constant flow conditions. Mean pulmonary arterial (Ppa) and pulmonary capillary pressure (Pcap, estimated by the double-occlusion method), wet lung weight (WLW), pulmonary capillary filtration coefficient (Kfc), lung perfusate lactate dehydrogenase (LDH) activity, and nitrite levels were assessed. Bolus injection of Lx (200 microM) caused insidious and significant lung weight gain, which was not associated with remarkable elevation of Ppa or Pcap but was associated with an increase of perfusate LDH activity and nitrite levels. Lx (20 microM) elevated Kfc, indicating that Lx had affected pulmonary vascular permeability. Because Lx causes endothelium dependent pulmonary vasodilation, we studied the effect of NG monomethyl-L-arginine (L-NMMA), NG-monomethyl-D-arginine (D-NMMA), superoxide dismutase (SOD), human oxyhemoglobin (oxyHb), and methylene blue (MB) on Lx induced lung injury. L-NMMA, SOD, and oxyHb, but not MB or D-NMMA, protected the lungs against Lx (200 microM)-induced injury. Lx increased pulmonary vascular permeability and caused lung injury. Because both nitric oxide synthase inhibitors and SOD inhibited the Lx-induced lung injury, it is possible that peroxynitrite is involved in the mechanism whereby Lx causes lung injury. PMID- 7543249 TI - Substance P induces ion secretion in mouse small intestine through effects on enteric nerves and mast cells. AB - We used genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice and congenic WBB6F1 +/+ normal (+/+) mice to examine the role of mast cells in substance P induced intestinal ion secretion. Isolated sheets prepared from segments of the midportion of the small intestine were studied in Ussing chambers. Substance P caused a dose-dependent increase in short-circuit current (Isc) that was approximately 50% less in intestine from W/Wv than from +/+ mice. Similar results were obtained for substance P-(4-11) (the COOH terminus) and substance P methyl ester [a selective neurokinin (NK)-1 agonist]. Histamine H1 or H2 antagonists reduced the Isc responses to substance P in intestine from +/+ mice but had no effect in intestine from W/Wv mice. In addition, reconstitution of intestinal mast cells in W/Wv mice by intravenous injection of +/+ bone marrow cells normalized the tissues' secretory responses to substance P or substance P methyl ester. However, in W/Wv and +/+ mice, the selective NK1 antagonist CP-96345 virtually abolished intestinal responses to substance P, and the responses were also markedly inhibited by neural blockade with tetrodotoxin. In contrast, in tetrodotoxin-pretreated intestine, histamine antagonism caused a further reduction in the responses to substance P only in +/+ mouse tissues. Taken together, our results suggest that the effects of substance P on intestinal Isc KN1 receptors but that the neuropeptide acts via effects on enteric nerves and mast cells. The data thus support the concept that mast cells and enteric nerves participate in the regulation of substance P-induced intestinal ion secretion. PMID- 7543251 TI - Selective neuronal nitric oxide synthase inhibition blocks furosemide-stimulated renin secretion in vivo. AB - The macula densa is a regulatory site for renin. It contains exclusively the neuronal isoform of nitric oxide synthase (NOS), suggesting NO could stimulate renin secretion through the macula densa pathway. To test whether neuronal NOS mediates renin secretion, renin was stimulated by either the renal baroreceptor or the diuretic furosemide (acting through the macula densa pathway). Renin secretion rate (RSR) was measured in 12 Inactin-anesthetized rats at normal (104 +/- 3 mmHg) and reduced renal perfusion pressure (65 +/- 1 mmHg), before and after selective blockade of the neuronal NOS with 7-nitroindazole (7-NI, 50 mg/kg ip). 7-NI had no effect on basal blood pressure (102 +/- 2 mmHg) or renal blood flow (RBF). Decreasing renal perfusion pressure doubled RSR from 11.8 +/- 3.3 to 22.9 +/- 5.7 ng ANG I.h-1.min-1 (P < 0.01) (ANG I is angiotensin I). Similarly, in 7-NI-treated rats, reduced perfusion doubled RSR from 8.5 +/- 1.8 to 20.5 +/- 6.2 ng ANG I.h-1.min-1 (P < 0.01). Renal hemodynamics and RSR were measured in response to 5 mg/kg iv furosemide in 12 control rats and 11 rats treated with 7 NI. Blocking neuronal NOS did not alter blood pressure (102 +/- 2 mmHg), RBF (5.8 +/- 0.4 ml.min-1.g kidney wt-1), or renal vascular resistance (18.7 +/- 1.4 mmHg.ml-1.min.g kidney wt).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543252 TI - Oxytocin as an antidiuretic hormone. I. Concentration dependence of action. AB - Circulating concentrations of oxytocin increase to 10-40 pM in rats in response to osmotic stimuli, suggesting that oxytocin could play a role in regulation of water balance. The present studies tested whether oxytocin at such concentrations increases osmotic water permeability (Pf) in isolated perfused terminal inner medullary collecting ducts (IMCD). In IMCD segments from Sprague-Dawley rats, 20 pM oxytocin added to the peritubular bath caused a two- to threefold increase in Pf, whereas 200 pM oxytocin increased Pf by five- to sixfold (n = 8, P < 0.01). IMCD from Brattleboro rats, which manifest central diabetes insipidus, exhibited a 2.8-fold increase in Pf in response to 20 pM oxytocin and a 4.7-fold increase in response to 200 pM oxytocin. However, in Brattleboro rats, the response to 20 pM oxytocin was dependent on prior water restriction of the rats. Immunoblotting showed no change in the expression of the aquaporin-CD water channel in Brattleboro rats in response to water restriction. Nevertheless, immunofluorescence studies of inner medullary tissue from Brattleboro rats revealed a marked redistribution of the aquaporin-CD water channels to a predominantly apical and subapical localization in IMCD cells in response to water restriction, similar to the redistribution seen in response to vasopressin. Mathematical modeling studies revealed that the measured increase in Pf in response to oxytocin is sufficient to generate a concentrated urine. We conclude that oxytocin can function physiologically as an antidiuretic hormone, mimicking the short-term action of vasopressin on water permeability, albeit with somewhat lower potency. PMID- 7543253 TI - Modification of cerebral laser-Doppler flow oscillations by halothane, PCO2, and nitric oxide synthase blockade. AB - We investigated whether nitric oxide (NO) played a role in the generation of cerebrocortical flow oscillations and their modification by hypocapnia, hypercapnia, and halothane administration. Parietal cortical laser-Doppler flow (LDF) was monitored transcranially in anesthetized (barbiturate + 0-1.0% halothane), artificially ventilated, adult male Sprague-Dawley rats. Thirty minutes after infusion of N omega-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg i.v.) mean arterial pressure (MAP) increased from 105 +/- 10 to 132 +/- 15 mmHg (P < 0.02), while mean LDF decreased from 159 +/- 36 to 135 +/- 30 perfusion units (PU, P < 0.05). Oscillations in LDF at a frequency of 6.3-7.8 cycles/min and amplitude of 10% were induced or augmented by L-NAME but not by D-NAME or indomethacin (2 mg/kg i.p.). L-arginine (200 mg/kg) abolished the oscillations post-L-NAME at constant MAP. Sodium nitroprusside infusion (10(-5) M, 5-50 microliters/min) reversed the L-NAME-induced increase in MAP and decrease in mean LDF but did not attenuate the flow oscillations. Hypocapnia post-L-NAME decreased LDF to 110 +/- 20 PU (P < 0.001) and augmented the flow oscillations (amplitude: 11-31%). Hypercapnia (5% CO2) or halothane (0.4-1.0%) suspended the oscillations in the presence of L-NAME. The results suggest that NO synthase activity inhibits cerebrocortical flow oscillations, and NO is not an obligatory mediator of the effects of halothane, hypocapnia, and hypercapnia on oscillatory activity. PMID- 7543254 TI - Baseline arterial pressure affects sympathoexcitatory responses to ventricular premature beats. AB - The seconds to minutes before sudden cardiac death are characterized by fluctuations of arterial pressure, cardiac rhythm, and probably sympathetic nerve activity. We explored the interrelations among these factors in seven patients undergoing clinical electrophysiological testing. We measured muscle sympathetic nerve activity (SNA) and arterial pressure responses to ventricular premature beats induced throughout the cardiac cycle under three conditions: 1) lowered arterial pressure and elevated SNA produced by intravenous nitroprusside, 2) baseline arterial pressure and SNA during saline infusion, and 3) elevated arterial pressure and decreased SNA activity produced by intravenous phenylephrine. Sympathetic responses to premature beats were inversely related to diastolic pressure. The magnitude of the sympathetic response was directly related to the prevailing arterial pressure and inversely related to baseline SNA. These data demonstrate that sympathoexcitation evoked by ventricular dysrhythmias is determined importantly by the prevailing arterial pressure and possibly by the background R-R interval and level of sympathetic activity. This effect may influence hemodynamic and electrophysiological stability during dysrhythmias. PMID- 7543255 TI - Nitric oxide and prostaglandins interact to mediate arteriolar dilation during cortical spreading depression. AB - We examined whether blockade of prostaglandin synthesis by indomethacin could attenuate the effect of nitric oxide synthase (NOS) inhibition on cerebral arteriolar dilation during cortical spreading depression (CSD). CSD was induced by microinjection of 5% (670 mM) KCl onto the cerebral cortex of anesthetized adult rabbits. A closed cranial window and intravital microscopy were used to measure pial arteriolar diameter, and NOS activity was determined by the conversion assay of [14C]arginine to [14C]citrulline. CSD dilated pial arterioles by 47 +/- 3% (baseline = 80-88 microns) (n = 21, P < 0.05), and inhibition of NOS by NG-nitro-L-arginine (L-NNA) (15 mg/kg iv) reduced dilation during CSD by over one-half (n = 8, P < 0.05) without altering the onset latency to CSD. After indomethacin administration (15 mg/kg iv), CSD dilated arterioles from 73 +/- 2 to 152 +/- 6 microns (n = 4, P < 0.05). However, after administration of both indomethacin and L-NNA (n = 5), CSD-induced arteriolar dilation was not different from the situation where indomethacin alone was given. Thus indomethacin completely abolished the inhibitory effect of L-NNA on CSD-induced dilation. Administration of L-NNA inhibited NOS activity in brain cortex almost completely (n = 8, P < 0.05), whereas indomethacin itself had no effect (n = 8). In addition, L-NNA inhibited topical acetylcholine (10(-5) M)-induced arteriolar dilation (n = 3, P < 0.05), and this effect was not altered by indomethacin (n = 4). In summary, L-NNA reduced arteriolar dilation during CSD. However, after administration of indomethacin, L-NNA does not reduce CSD-induced arteriolar dilation. PMID- 7543256 TI - Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. AB - Caldesmon phosphorylation has been proposed to be involved in regulation of smooth muscle contraction. Mitogen-activated protein (MAP) kinase has been suggested to be the caldesmon kinase; stimulation-induced MAP kinase activation in intact vascular smooth muscle, however, has not been demonstrated. We measured temporal profiles of MAP kinase activation in response to histamine stimulation and membrane depolarization in intact swine carotid artery. Phosphotyrosine levels of 42- and 44-kDa MAP kinases were elevated during contraction in response to histamine or KCl. The temporal profile of MAP kinase activation/inactivation was similar to that for contraction/relaxation of the vascular tissue in response to KCl or histamine stimulation. MAP kinase activated during contractile stimulation phosphorylates caldesmon with a specific activity significantly greater than that for myelin basic protein-(95-98). We propose that MAP kinase is activated in response to all forms of contractile stimulation. We also suggest that activated MAP kinase phosphorylates and disinhibits the effects of caldesmon on actin-myosin interactions. This disinhibition allows an inherent level of myosin ATPase activity to be expressed. PMID- 7543257 TI - Modulation of resistance artery force generation by extracellular Ca2+. AB - We tested the hypothesis that increasing extracellular Ca2+ (Cao) over a physiological concentration range depresses vascular smooth muscle force generation by altering the intracellular Ca2+ (Cai)-force relationship. Mesenteric resistance arteries were isolated from Wistar rats; Cai and isometric force were measured using a fura-based method and wire myography. Vessels were depleted of releasable Cai by repeated contraction with norepinephrine; Cao was then cumulatively added back from 0.025-2.5 mM in the presence of an agonist. With norepinephrine, serotonin, prostaglandin F2 alpha, and K+, Cao from 0.025 to 0.8 mM induced a graded increase in Cai and active stress. With the receptor agonists but not K+ raising Cao from 0.8 to 1.6 mM and from 1.6 to 2.5 mM decreased active stress to 82 +/- 6 and 54 +/- 6% of maximum, respectively, P < 0.05. Although there was a transient decrease in Cai in response to both 1.6 and 2.5 mM Cao, steady-state Cai only decreased significantly in response to 2.5 mM Cao (85 +/- 3% of maximum). Inhibition of the sarcoplasmic reticulum Ca(2+) adenosinetriphosphatase with 1 microM thapsigargin had no effect on the decrease in force induced by high Ca2+. The decrease in active stress induced by 1.6 and 2.5 mM Cao was inhibited by Ca2+ channel antagonists and by blockade of Ca(2+) activated K+ channels with charybdotoxin (with 1.6 mM Cao, control tension = 67 +/- 10% of maximum vs. charybdotoxin = 99.2 +/- 1%, P < 0.05; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543258 TI - Effect of nitric oxide synthase inhibition on postischemic cerebral hyperemia. AB - We tested the hypothesis that inhibition of nitric oxide synthase (NOS) activity in brain before ischemia decreases postischemic hyperemia. Pentobarbital anesthetized piglets underwent 15 min of complete global cerebral ischemia induced by elevation of intracranial pressure followed by 20 min of reperfusion. Before ischemia the animals were randomly assigned to receive either intravenous N omega-nitro-L-arginine methyl ester (L-NAME 10 mg/kg, n = 6, or 50 mg/kg, n = 6) or an equal volume of saline (10 ml, n = 8). Serial cerebral blood flow (radiolabeled microspheres) was measured at baseline and during ischemia and reperfusion. Forebrain postischemic hyperemia was documented after administration of saline (42 +/- 4 to 88 +/- 10 ml.min-1.100 g-1) and 10 mg/kg L-NAME (36 +/- 4 to 59 +/- 9 ml.min-1.100 g-1) but not after 50 mg/kg L-NAME (29 +/- 3 to 34 +/- 7 ml.min-1.100 g-1). However, the percent reduction in cerebral vascular resistance (CVR) fell during reperfusion to a similar extent in all three groups because of differences between groups in cerebral perfusion pressure changes during the protocol. CVR fell to the lowest level at 8 min of reperfusion in the saline treated animals (2.0 +/- 0.16 to 0.68 +/- 0.05 mmHg.ml-1.min.100 g) compared with the L-NAME-treated animals (50 mg/kg: 4.0 +/- 0.3 to 1.8 +/- 0.2 mmHg.ml 1.min.100 g).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543259 TI - Salt discriminability is related to number of regenerated taste buds after chorda tympani nerve section in rats. AB - Transection of the chorda tympani nerve (CTX) impairs taste-guided discrimination of NaCl from KCl in rats. We wanted to determine whether this discrimination recovers after chorda tympani regeneration. Experiment 1 showed that few taste buds regenerated 14 days after CTX, whereas substantial regeneration occurred 42 days after surgery. Experiment 2 demonstrated that rats trained before CTX could clearly discriminate the two salts when tested starting 49 days after surgery, whereas rats tested starting 8 days after surgery were severely impaired in this task. Rats tested starting 28 days after CTX were unimpaired, moderately impaired, or severely impaired on the discrimination task. Overall, discrimination performance was significantly related to the number of regenerated taste buds. Unilaterally transected rats tested shortly after surgery were nearly as competent as controls. These results indicate that rats can recover the ability to discriminate NaCl from KCl after regeneration of anterior tongue taste buds. PMID- 7543260 TI - Acidic fibroblast growth factor (FGF) but not basic FGF induces sleep and fever in rabbits. AB - Acidic fibroblast growth factor (FGF) and basic FGF belong to a growth factor family. Interleukin-1, another member of that family, is involved in sleep regulation. FGFs and interleukin-1 share structural and functional features. We therefore determined whether acidic FGF and basic FGF were somnogenic. Male New Zealand White rabbits were provided with electroencephalographic (EEG) electrodes, a brain thermistor, and a lateral intracerebroventricular (icv) cannula. The animals were injected icv with isotonic NaCl (control) and on separate days with one of three doses of acidic or basic FGF (0.01, 0.1, or 1.0 micrograms) or with heat-treated acidic FGF (1.0 micrograms). The EEG, brain temperature, and motor activity were recorded for 23 h. The biological activity of basic FGF was determined in vitro by its ability to induce DNA synthesis in rat aortic smooth muscle cells. Acidic FGF induced prolonged dose-related increases in non-rapid eye movement sleep beginning in the 1st postinjection h and continuing for 12-23 h after the treatment. Acidic FGF also induced fevers of approximately 1 degree C after the 1.0 micrograms dose. Both activities of acidic FGF were lost after heat treatment. In contrast, basic FGF lacked somnogenic and pyrogenic activity, although it did induce DNA synthesis. Current results suggest that acidic FGF is part of the complex cytokine network in brain involved in sleep regulation. PMID- 7543261 TI - In vivo platelet deposition on polytetrafluoroethylene coated with fibrin glue containing fibroblast growth factor 1 and heparin in a canine model. AB - BACKGROUND: We previously reported that the coating of expanded polytetrafluoroethylene (ePTFE) with fibrin glue containing fibroblast growth factor 1 (FGF-1) and heparin accelerates endothelial coverage of grafts implanted into animals. We report here the effect of this surface modification on early platelet deposition. MATERIALS AND METHODS: Nine dogs received 7-cm ePTFE grafts, 60-microns internodal distance, 4-mm internal diameter, as bilateral aortoiliac implants, one coated (luminal cross section and abluminal surface) with fibrin glue (fibrinogen 32.1 mg/mliters, thrombin 0.32 U/mliters) containing FGF-1 (11 ng/mliters and heparin (250 U/mliters), the other uncoated. After 5, 30, or 120 minutes of circulation with blood containing autologous platelets radiolabelled with indium 111, gamma emissions were quantitated on explants and correlated to surface areas measured by computerized planimetry. RESULTS: Both global and segmental comparisons showed significantly (P < 0.05, Student's t-test) less platelet deposition on coated than on uncoated grafts after 120 minutes of circulation, but no difference at 5 and 30 minutes. CONCLUSIONS: In this model, ePTFE coating with fibrin glue containing FGF-1 and heparin shows no adverse effect on early platelet deposition. PMID- 7543263 TI - Amylase measurement--which method? PMID- 7543264 TI - Cutaneous bacillary angiomatosis in a patient with chronic lymphocytic leukemia. AB - BACKGROUND: Bacillary angiomatosis is a recently described vascular disorder that is associated with infection by Bartonella henselae (formerly known as Rochalimaea henselae) and Bartonella quintana (formerly known as Rochalimaea quintana); this disorder usually occurs in patients with human immunodeficiency virus infection. We report a case of cutaneous bacillary angiomatosis that occurred in a patient with chronic lymphocytic leukemia. OBSERVATIONS: A 55-year old man with chronic lymphocytic B-cell leukemia, Rai stage IV, presented with multiple angiomatous papules that clinically resembled pyogenic granulomas. Histopathologic examination revealed circumscribed lobules of small vessels with plump endothelial cells, numerous neutrophils, and abundant nuclear dust; these features were diagnostic for bacillary angiomatosis. The diagnosis was confirmed by the Grocott-Gomori methenamine-silver nitrate stain that revealed argyrophilic bacteria and by ultrastructural demonstration of bacillary structures with trilaminar walls. Treatment with clarithromycin led to complete resolution of the lesions within 4 weeks. CONCLUSIONS: This case emphasizes that (1) bacillary angiomatosis must be considered in the differential diagnosis of vascular lesions in immunocompromised patients without human immunodeficiency virus infection, (2) Grocott-Gomori methenamine-silver nitrate stain is a simple and satisfactory alternative to the Warthin-Starry stain for the demonstration of bacilli in this condition, and (3) clarithromycin is an effective oral antibiotic for the treatment of this disease. PMID- 7543262 TI - Shear stress-mediated changes in the expression of leukocyte adhesion receptors on human umbilical vein endothelial cells in vitro. AB - Extensive monocyte recruitment is an early phenomenon associated with the development of atherosclerotic lesions, suggesting an active role for the involvement of adhesion receptors expressed by endothelial cells. In this study we describe the contribution of hemodynamic shear forces in regulating the expression of a few of the monocyte adhesion receptors, including intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E selectin on endothelial cells. A parallel plate flow chamber and recirculating flow loop device was used to expose human umbilical vein endothelial cells (HUVECs) to different levels of shear (2-25 dyn/cm2). Subsequently the cells were analyzed either for shear induced changes in the mRNA levels of adhesion receptors by Northern blot analyses or for changes in the surface expression of ICAM-1 using flow cytometry. Results from the fluorescence analysis showed a transient increase in the surface expression of ICAM-1, 12 hr after exposure to 25 dyn/cm2 shear, returning to basal levels within 24 hr. This was quite different from the time dependent response of ICAM-1 to lipopolysaccharide (LPS), where ICAM-1 expression was maximally induced 18-24 hr post-stimulus. ICAM-1 mRNA level appeared slightly elevated after exposure to shear for 1 hr, compared to basal values, but dropped below basal levels within 6 hr. This biphasic response was seen irrespective of the magnitude of applied shear stress. VCAM-1 mRNA expression, in contrast, decreased below the baseline expression within an hour after onset of flow, and appeared to be considerably down-regulated within 6 hr. After exposure to shear for 24 hr, no increase in mRNA levels could be detected for either molecule, at any shear magnitude. E-selectin mRNA was less responsive to shear stress, especially at the lower magnitudes of shear. After an hour of exposure to flow E-selectin mRNA level appeared slightly reduced compared with control levels, but it remained at this level even after 6 hr of flow. These results indicate that the expression of adhesion receptors is sensitive to local shear stresses in a manner that is molecule specific in the short term even though prolonged exposure to flow results in similar down-regulation for both ICAM-1 and VCAM-1. PMID- 7543265 TI - Behavioral vs biochemical prediction of clinical stability following haloperidol withdrawal in schizophrenia. AB - BACKGROUND: We sought to identify haloperidol-treated subjects who relapsed within 6 weeks of placebo replacement and those who did not, using multivariate analysis. METHODS: In the week prior to discontinuation of haloperidol treatment, global behavioral ratings and a lumbar puncture for cerebrospinal fluid monoamine metabolities were obtained in 88 patients with chronic schizophrenia. Logistic regression analyses were used to evaluate two competing models of relapse prediction. The models were then compared using receiver operating characteristic analysis and a final combined model was derived. RESULTS: The behavioral model was less variable in its prediction than the cerebrospinal fluid monoamine model. The final model consisted of increased psychosis, decreased anxiety, higher cerebrospinal fluid homovanillic acid levels, and lower cerebrospinal fluid 5 hydroxyindoleacetic acid levels. CONCLUSIONS: Several monoamine systems are involved in psychotic relapse within 6 weeks of haloperidol withdrawal. Future studies of relapse prediction should include both clinical and biological measures to fully assess relapse risk. PMID- 7543266 TI - Metabolism and biological activities of inositol pentakisphosphate and inositol hexakisphosphate. PMID- 7543267 TI - Cyclic guanosine 3',5'-monophosphate mediates 3-morpholinosydnonimine-induced inhibition of human natural killer cells. AB - Nitric oxide (NO) donors were used to investigate the effect of NO on and the role of cyclic GMP in the regulation of human natural killer (NK) cell function. NO-producing drugs, molsidomine and its metabolite 3-morpholinesydnonimine (SIN 1), inhibited NK cell-mediated cytotoxicity significantly at 0.04-5 mM. At 1 mM, SIN-1 completely inhibited NK cell activity while molsidomine decreased NK cell mediated cytolysis by 35% of the control value. These data suggest that NO from exogenous NO-donors may down-regulate NK cell cytotoxic function. The stimulatory effect of interferon-gamma (IFN-gamma) on human NK cell-mediated killing could not overtake the NK cell inhibition induced by the NO releasing drugs, indicating different modes of action for IFN-gamma and SIN-1. The results in the present study also showed that SIN-1 (1 mM) stimulated cyclic GMP production 37-fold in NK cells. In the presence of 0.5 mM IBMX, a phosphodiesterase inhibitor, the increase in cyclic GMP was even more pronounced, demonstrating a relation between cyclic GMP stimulation and NK cell inhibition by SIN-1. Further evidence for mediation via cyclic GMP was provided by the finding that methylene blue (20 microM), an inhibitor of soluble guanylate cyclase, decreased both the inhibition of SIN-1-induced NK cell cytotoxicity as well as cyclic GMP formation. Moreover, membrane-penetrating cyclic GMP and its analogues inhibited NK cell-mediated cytolysis significantly. Molsidomine was without effect on cyclic GMP levels. Our data indicate that cyclic GMP may play a role in human NK cell regulation and suggest that the inhibitory effect of cGMP may be elicited by NO. PMID- 7543269 TI - Skin microdialysis: detection of in vivo histamine release in cutaneous allergic reactions. AB - In vivo histamine release in cutaneous allergic reactions was chronologically examined using the skin microdialysis technique. Antigen challenge was made in ovalbumin-sensitized guinea pigs 1 h after the implantation of the microdialysis probe. A marked histamine release after intradermal injection of ovalbumin solution was observed in the early phase (up to 40 min). No isolated delayed histamine release was observed thereafter over 8 h. Also, in an atopic volunteer, a marked release of histamine after intradermal injection of mite extract was observed only in the early phase, although erythema and induration at the site of injection were continuously observed in the late phase (up to 4 h). These findings suggest that the skin microdialysis technique is a useful, simple technique for chronological determination of chemical mediators released in the allergic skin in vivo. PMID- 7543268 TI - Relative susceptibility of cytoskeleton-associated and soluble neurofilament subunits to aluminum exposure in intact cells. A possible mechanism for reduction of neurofilament axonal transport during aluminum neurotoxicity. AB - Previous studies have demonstrated the appearance of phosphorylated neurofilament (NF) subunits within perikaryal cytoskeletons following aluminum exposure. In order to examine the mechanisms leading to this altered distribution of NF subunits, we carried out biochemical analyses of NF subunits in Triton-insoluble and -soluble fractions derived from aluminum-treated NB2a/d1 cells. In addition to increases in the Triton-insoluble cytoskeleton, increases in all three NF subunits were also detected within the Triton-soluble fraction of aluminum treated cells. To address the nature of this increase in Triton-soluble subunits, aluminum-treated and untreated cultures were harvested in the absence of Triton and fractionated by established procedures to yield fractions greatly enriched for perikarya and neurites, respectively. Each of these subcellular fractions was then subjected to further homogenization in the presence of 1% Triton and centrifugation to yield Triton-insoluble cytoskeletons and Triton-soluble material derived from perikarya and axonal neurites, respectively. Resulting Triton-soluble fractions were "clarified" by high-speed centrifugation to eliminate oligomeric assemblies or soluble neurofilaments. Immunoblot analysis demonstrated quantitative recovery of the aluminum-induced increase in Triton soluble NF subunits in the perikaryal fraction. Additional aluminum-treated and untreated cultures were pulse-chase radiolabeled with [35S]methionine and fractionated into Triton-insoluble and soluble fractions from isolated perikarya and axonal neurites. Autoradiographic analysis of immunoprecipitated NF subunits revealed that aluminum treatment delayed the translocation of newly synthesized subunits into neurites and resulted in the accumulation of radiolabeled subunits within the Triton-soluble fraction of perikarya. These findings suggest that aluminum may exert a relatively greater effect on NF subunits that have not yet undergone axonal transport and/or incorporation into Triton-insoluble structures vs those that have already deposited into axons. This possibility was supported by the observation that a higher concentration of aluminum was required to alter the electrophoretic migration of in vitro reassembled neurofilaments vs that required for unassembled NF subunits. These findings provide possible mechanisms for the accumulation of NF subunits in perikarya during aluminum intoxication. PMID- 7543270 TI - Antiviral action of oligodeoxyguanylic acids against human immunodeficiency virus type 1. AB - Deoxyguanylic acids, but not other deoxynucleotides, as short as 3- to 4-mer, were effective in preventing HIV-1-induced cytopathicity. In addition, they prevented giant cell formation of infected Sup-T1 cells, and p24 production in HIV-1 infected H9 cells. Phosphorylation at either the 5'- or 3'-end enhanced these activities. Furthermore, 5'-phosphorylated phosphorothioate tetradeoxyguanylic acid was effective in reducing HIV production in chronically infected cells (H9/IIIB). The search for the target steps of this compound revealed that it inhibits at least 3 steps in the life cycle of HIV: interaction with CD4 (measured by inhibitory effect on the syncytia formation between Sup-T1 and H9/IIIB cells), reverse transcriptase, and step(s) after integration. These results suggest that phosphorylated phosphorothioate tetradeoxyguanylic acid may be a novel candidate for a therapeutic agent of AIDS. PMID- 7543271 TI - Two new human monoclonal antibodies against HIV type 1 glycoprotein 120: characterization and neutralizing activities against HIV type 1 strains. AB - Two human IgGk monoclonal antibodies (HuMAbs), termed 48-16 and 50-61A, were derived by Epstein-Barr virus transformation of B cells from two HIV-1-infected donors. These HuMAbs recognized discrete, nonoverlapping, and conformational or discontinuous epitopes on the gp120 envelope protein of HIV-1. The binding affinities of 48-16 and 50-61A for recombinant gp120 from HIV-1LAI strain, reflected by their dissociation constants, were estimated to be 2-5 x 10(-9) and 2.4 x 10(-10) M, respectively. 48-16 was shown to react with a conserved determinant present on a variety of divergent laboratory isolates, residing outside the CD4-binding site and the V3 region, which remains to be determined. 48-16 did not display, however, any detectable functional activity. 50-61A exhibited a more restricted recognition pattern, but was able to completely inhibit the 2 HIV-1 laboratory strains LAI and SF2 in a concentration range of 0.5-10 micrograms/ml, as measured by an antigen capture assay. The ability of 50 61A to block the interaction between recombinant gp120LAI and recombinant as well as cellular CD4 indicated that 50-61A epitope was localized near or within the CD4-binding side. We also demonstrated that 50-61A- and 48-16-defined epitopes (or closely related epitopes) were immunogenic in infected humans, since serum samples from 45 seropositive subjects were able to inhibit both gp120LAI-HuMAb recognitions. However, the presence of "50-61A-like" antibodies in these sera could not be associated with their neutralizing activities of HIV-1LAI infection. Interest in producing such antibodies for characterization of the human B cell repertoire to HIV-1 and their potential use in passive immunotherapy or vaccination strategy against AIDS are discussed. PMID- 7543272 TI - Prospective trials for the radiotherapeutic treatment of bone metastases. AB - Radiation therapy is a proven technique to relieve the pain of bone metastases. Focal painful metastases can be palliated in up to 80 percent of patients. The technique to use for the treatment varies depending on the extent of disease, the prognosis of the patient and physician preference. Most studies indicate that the local treatment of bone metastases with single large treatments is as efficacious as extended courses of treatment. For patients with wide-spread disease, two forms of systemic radiotherapy are available: hemibody irradiation and intravenous injection of radionuclides. Studies have shown the combination of either focal irradiation and hemibody irradiation or focal irradiation and the injection of strontium (89) prolongs the pain-free duration of the patients. All palliative patients with symptomatic bone metastases should be evaluated for radiotherapy. PMID- 7543273 TI - Hospice and palliative care: attitudes and practices of the physician faculty of an academic hospital. AB - It has been suggested that physicians, particularly in academic hospitals, are resistant to the hospice approach to palliative care for terminally ill patients. It is of interest, therefore, to assess the attitudes and practices of the physician faculty of an academic hospital where a hospice program has been in existence for more than 10 years. This was assessed with two faculty surveys. All 966 physician faculty that were on staff at Northwestern Memorial Hospital in the fall of 1993 were sent a survey about their opinion of hospice care (Survey A). Then, all physicians who had referred patients to the hospice program between September 1993 and September 1994 at Northwestern Memorial Hospital received a survey letter after the death of their patient (Survey B). Seventy-seven percent of faculty physician respondents to Survey A had either referred patients, or knew of colleagues who had referred patients to a hospice program. Ninety-four percent of those who answered "yes" to the question about referrals reported satisfaction with their care. Ninety-four percent would refer patients in the future and 96 percent thought the hospice program was a valuable resource to the medical center. Of the respondents to Survey B, nearly 100 percent thought the referral had been handled in an "excellent" or "good" fashion, that communication with hospice staff was "excellent" or "good," that symptom control was "excellent" or "good," that their patients and families had received "excellent" or "good" psychosocial support, and that their patients and families were satisfied with the hospice care they received.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543274 TI - Renal allograft rejection in children and young adults: the Banff classification. AB - In the Banff classification, arteritis and tubulitis are regarded as the principal histological lesions indicating acute renal allograft rejection. To test this claim, we examined 51 biopsies obtained from 21 children and young adults with transplant rejection. Two reviewers, blind to the clinical course, graded the biopsies according to the Banff scheme. In patients without significant tubulitis (borderline changes), rejection tended to be reversed easily (88%), often with methylprednisolone pulse (52%). In patients with arteritis or significant tubulitis (Banff I-III), rejection was reversed in only 23% (P < 0.001), in 9% with steroids, and in 14% with OKT3. Salvage of the graft was achieved in 26 of 35 (74%) with a score < 5 but in only 1 of 12 (8%) with a score > or = 5 (P < 0.001). All 6 patients with vasculitis lost their grafts despite methylprednisolone pulse and OKT3. We conclude that the Banff classification predicts accurately the outcome of renal allograft rejection in children and may aid in choosing appropriate therapy. PMID- 7543275 TI - Endothelial cell control of vasomotor tone. PMID- 7543276 TI - Production of interferons and lymphokines in leukocyte cultures of patients with schizophrenia. AB - Recently, several lines of evidence have suggested the possible of immunological dysfunction in the pathogenesis of schizophrenia. We therefore investigated the ability to produce interferons and lymphokines in response to mitogenic or viral stimulation in a whole blood assay of 37 schizophrenic patients (DSM-III-R) and of 42 healthy blood donors. Phytohaemagglutinin (PHA) was used for the induction of interleukin-2 (IL-2), interferon gamma (INF gamma), interleukin-6 (IL-6) and the soluble interleukin-2 receptor (sIL-2R) and Newcastle Disease Virus (NDV) for the induction of interferon alpha 2 (INF alpha 2). All lymphokines and, in addition, the sIL-2R in the sera were determined by ELISA technique. The psychopathological status of the patients was assessed by psychiatrists according to internationally accepted standards. The patient group showed a trend to lower levels of the interferons alpha 2 and gamma and a significant decrease of IL-2 production. The sIL-2R levels were significantly increased in the sera of schizophrenic patients. The latter increase was associated with a poor assessment of prognosis (Strauss and Carpenter). This association appears to be of interest. However, its significance is not understood, since longitudinal studies could not be performed. PMID- 7543277 TI - Identification of residues involved in ligand binding to the neurokinin-2 receptor. AB - Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and location of these receptor residues suggest that the distance between bound peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thus receptor subtype selectivity. While many single residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists. PMID- 7543278 TI - Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB. AB - SecB is a chaperone dedicated to protein translocation in Escherichia coli. SecB binds to a subset of precursor proteins, and targets them in a translocation competent state to the SecA subunit of the translocase. The nature and kinetics of the interaction of SecB with polypeptides were studied by spectroscopic techniques using the reduced form of bovine pancreatic trypsin inhibitor (BPTI) as a model substrate. Binding of SecB to BPTI resulted in an increase in the fluorescence of the surface-exposed tryptophan residue 36 of SecB. SecB reversibly binds BPTI in stoichiometric amounts. Labeling of BPTI with the fluorophore acrylodan allowed the analysis of the binding reaction at nanomolar concentrations. High-affinity binding (KD of 5.4 nM) of labeled BPTI to SecB resulted in a blue shift of the acrylodan emission maximum and an increase in the fluorescence quantum yield, suggesting that BPTI binds in an apolar environment. Stopped-flow acquisition of rate constants of complex formation between SecB and BPTI yielded a second-order binding rate constant of 5 x 10(9) M-1 s-1, and a dissociation rate constant of 48 s-1. These data demonstrate that in vitro, the association of SecB with polypeptide substrates is limited by the rate of collision. In vivo, SecB binding is selective, and predominantly occurs with nascent polypeptides. Since these chains are not expected to fold into stable structures, SecB association may be governed by "more or less" specific interactions and be limited by the rate of chain elongation rather than the rate of folding. PMID- 7543279 TI - Redesigning the topology of a four-helix-bundle protein: monomeric Rop. AB - The topology of alpha-helices and beta-sheets in folded proteins is largely specified by the connectivities of the loops and turns which join them. We have used the protein Rop to test the feasibility of using short glycine-rich linkers to reconnect the alpha-helices within a four-helix-bundle protein. In wild-type Rop the four-helix-bundle structure is formed by the association of two identical helix-turn-helix monomers. Our redesigns encode Rop as a single chain to create a monomeric, rather than a dimeric, form of the protein. Characterization of a series of such variants demonstrates that new connections of this type can be used to generate stable, native-like proteins. The length of the connections is of key importance; if the loops are too short, correct association of the helices is prevented, and misfolded, higher order oligomers occur. Designs with sufficiently long loop connections, however, generate exclusively the desired monomeric form of the protein. Moreover, the successful monomeric designs bind Rop's RNA substrate with affinities that are equal to that of the wild-type protein. This result provides strong confirmation that the positioning of the helices in the monomeric variants is closely similar to that in wild-type Rop. PMID- 7543282 TI - Interaction of partially structured states of acidic fibroblast growth factor with phospholipid membranes. AB - Although acidic fibroblast growth factor (aFGF) lacks a conventional signal sequence, it is often found complexed to sulfated proteoglycans on the external surface of cells. The protein also forms a "molten globule"-like state at neutral pH and physiological temperatures as well as at acidic pH in the presence of physiological ionic strength or moderate quantities of polyanions. These states display a marked tendency to aggregate. Such observations suggest that related partially structured states might be involved in the membrane translocation of aFGF. To explore this hypothesis, we examined the interaction of this growth factor with lipid vesicles as well as the effect of such surfaces on the structure of the protein. We find that these states interact with negatively charged but not neutral phospholipid unilammelar vesicles at acidic pH, inducing bilayer disruption. The rate of leakage of a liposome-entrapped fluorescent probe is proportional to the logarithm of the aFGF concentration, suggesting competition between protein self-association and membrane binding. Liposome leakage can be also induced at neutral pH by partial unfolding of aFGF at or above physiological temperature in contrast to most control proteins. The importance of partially folded hydrophobic surfaces in aFGF self-association and membrane binding is further suggested by the fact that thermally unfolded aFGF does not aggregate, in contrast to states observed at intermediate temperatures or transiently during unfolding at high temperatures. In contrast to heparin, a polyanion which stabilizes the native structure of aFGF, negatively charged phospholipid membranes appear to enhance the disruption of aFGF tertiary structure at submicellar concentrations of sodium dodecyl sulfate but stabilize the remaining secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543281 TI - Analysis of gp39/CD40 interactions using molecular models and site-directed mutagenesis. AB - The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF beta/TNFR as a guide, we previously reported a site-directed mutagenesis study in which we identified residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of these models has allowed us to extend our mutagenesis analysis of gp39/CD40 interactions. These experiments have led to the identification of additional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that contribute to the gp39/CD40 interaction. We also further explored the importance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The results of these studies have enabled us to approximately outline the binding sites in gp39 and CD40. It appears that the gp39/CD40 interaction is centered on at least two clusters of residues and involves residues of two adjacent gp39 monomers. The molecular regions involved in the gp39/CD40 interaction essentially correspond to those in the homologous TNF-beta/TNFR system. PMID- 7543284 TI - Hyperbaric oxygenation reduces the cytostatic activity and transcription of nitric oxide synthetase gene of mouse peritoneal macrophages. AB - The effects of hyperbaric oxygenation (HBO) and hyperbaric air (HBA) on the cytostatic activity, peroxynitrite synthesis and transcription of the inducible nitric oxide synthetase (iNOS) gene of murine peritoneal macrophages were studied in vitro. Exposure of mice to HBO or HBA significantly reduced the cytostatic activity, peroxynitrite synthesis and transcription of iNOS mRNA of their macrophages stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma). These results indicate that HBO and HBA treatments of mice reduce the cytostatic activity of peritoneal macrophages by reducing iNOS mRNA synthesis. PMID- 7543280 TI - Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes. AB - Vesicular stomatitis virus buds from domains of the plasma membrane that have a unique protein and lipid composition. Fluorescence digital imaging microscopy and resonance energy transfer were used to determine how the two viral envelope associated proteins, the G and the M proteins, could alter the lateral distribution of lipids in large unilamellar vesicles and form domains. The G protein formed large domains in vesicles containing phosphatidic acid but not with phosphatidylserine, while the M protein formed domains enriched in both acidic phospholipids. Domains enriched in sphingomyelin were observed only when both the G protein and the M protein were present in vesicles containing phosphatidic acid. Phosphatidylcholine and gramicidin (chosen to represent a host membrane protein) were excluded from the domains. Cholesterol was induced to partition into the domains only in vesicles containing phosphatidic acid and sphingomyelin along with both of the proteins. Phosphatidylethanolamine was not enriched or depleted in the domains. Domains of similar composition were formed using vesicles made from dioleoylphospholipids and the lipids extracted from BHK 21 plasma membranes, indicating that the fatty acid composition was not as important as the polar head groups of the phospholipids. The phospholipid and cholesterol compositions of the domains formed by the G and the M proteins in vesicles were very similar to the composition of the viral envelope, suggesting that the domains represent the areas in the plasma membrane where the virus buds. This study provides a model for selective lipid and protein sorting that occurs in biological membranes. PMID- 7543285 TI - Medical marijuana: to prescribe or not to prescribe, that is the question. PMID- 7543286 TI - Marijuana as medicine: making a silk purse out of a sow's ear. PMID- 7543283 TI - Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. AB - The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication. PMID- 7543287 TI - The medical use of marijuana: the case for clinical trials. PMID- 7543288 TI - Samarium-153-EDTMP in bone metastases. AB - Thirty-five patients with painful bone metastases arising from a variety of tumor types underwent a clinical trial in which 153Sm-EDTMP was injected as a single intravenous dose. The injection ranged in amount from 330 MBq to 1110 MBq of 153Sm-EDTMP. Pain relief usually occurred within one week after administration. The duration of pain relief lasted from 2 to 17 weeks. A detectable degree of pain palliation was experienced by 80% of the treated patients; 54% reported substantial or complete pain relief. Due to the small number of patients, no clear-cut dose-related response was detectable. Moderate myelosuppression was observed in one patient (WHO grade III). The metastatic lesion-to-normal bone ratios remained constant (varying from 1.5 to 4.8) for at least 5 days post injection. 153Sm cleared very rapidly from the blood. Less than 1% of the injected dose remained in circulation at 4 hours post-injection. No local accumulation of the tracer could be detected outside the skeleton. Urinary excretion was quite complete at 6 hours post-injection. The biodistributions of 153Sm-EDTMP and 99mTc-DPD are very similar in metastatic and normal bone; thus, bone scanning can be used for patient selection and followup. According to our results, it seems that higher doses of 153Sm-EDTMP can be given safely and without any irreversible myelosuppression. PMID- 7543289 TI - A low pH 111In-bleomycin complex: a tracer for radiochemotherapy of head and neck cancer. AB - Bleomycin (BLM) is a well known natural antibiotic. It is toxic to dividing cells and has been used for the treatment of several forms of cancer. BLM has been labeled with various cations, but most of them have turned out be unstable in in vivo experiments. In-BLM demonstrated high bone marrow uptake, but using 111In bleomycin complex (BLMC) formed at low pH, the low in vivo stability and high bone marrow seeking behavior of the molecule could be avoided. The idea of using BLMC in combined radiotherapy and chemotherapy is intriguing. In this study we examined the effects of 111In-A'2a-c-BLMC in the treatment of 31 head and neck cancer patients. Findings were compared with those of surgery, and pre-operative radiology. The injected activity was 85-110 MBq, and the specific activity was approximately 100 MBq/mg. The half-life of 111In activity in serum varied from 1.5 to 3.1 hours. Maximum activity in the urine was achieved in all patients within 3 hours, and the average half-life in urine was 2 hours. In most patients 50% was excreted within 3 hours, in some 70%; in all patients > 95% of the activity was excreted within 22 hours. In surgical samples from 24 patients the best tumor-to-tissue ratios were: fat 60:1, bone 17:1, muscle 12:1, blood 3.6:1. All patients were examined on the injection day with ultrasonography of the neck. Using 111In-BLMC we missed a few small lymph nodes in 2 patients, but there were no false positive findings.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543290 TI - Patterns of care in advanced HIV disease in a tertiary treatment centre. AB - A retrospective chart review of all in-patient deaths in 1992 was undertaken to examine patterns of care in advanced HIV disease at St Paul's Hospital, Vancouver, Canada. St Paul's Hospital cares for approximately 75% of the Province of British Columbia's AIDS caseload. This represents about 18% of Canada's caseload. Data were collected on demographic characteristics, the utilization of home care and community services, income and social support, symptom presentation at terminal admission and the utilization of acute hospital care and hospital based palliative care. A total of 126 deaths were reviewed. All but two subjects were homosexual/bisexual men. The median age at death was 39 years (range 24-67). Four patterns of care at death were identified: (1) aggressive therapy with resuscitation 24 (19%), (resuscitation was initiated in 58%); (2) aggressive therapy with a no resuscitation order 49 (39%), in which the palliative period was a median of three days; (3) death on the palliative care unit 33 (29%), with a median survival once palliative of 20 days; and (4) death on the palliative care unit following respite admissions 16 (13%), with a median survival once palliative of 64 days. Despite a well known and respected Palliative Care Unit and community palliative care programme, there is a marked trend towards death occurring during aggressive therapy with a 200% increase in the initiation of resuscitation compared to the previous three years. No-one has been discharged alive from hospital following the initiation of resuscitation since 1988. This study illustrates the need for providers and persons infected with HIV to reconsider expectations about treatment outcomes in advanced HIV disease. PMID- 7543293 TI - Multicentre evaluation of the Serono SR1 assay for prostate specific antigen. AB - This report describes the findings of an evaluation of the validity of the new, highly automated Serono SR1 enzyme immunoassay for prostate specific antigen performed at three European sites. Within-run precision was below 5.5% at the clinically important decision point of 4 micrograms/l, less than 10% below 3 micrograms/l and less than 5% above 4 micrograms/l. Between-run precision was below 12% at each site and between-site precision was less than 14%. Dilution series were shown to be parallel to the standard curve on 12 samples with neat prostate specific antigen concentrations ranging from 20 to 88 micrograms/l (linear regression slopes and correlation coefficients of 1.00 to 1.06 and 0.972 to 1.000 respectively). Comparisons with Hybritech Tandem-E and -R and Roche Cobas Core prostate specific antigen methods used routinely at the three sites were good (linear regression slopes 1.04 to 1.13, correlation coefficients 0.96 to 0.99), but results were significantly lower than the Ciba Corning ACS180 (slope 0.66, correlation coefficient 0.99). The assay presents a precise, robust and valid diagnostic tool offering additional advantages of non-isotope automation. PMID- 7543292 TI - In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene. AB - The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5 aza-2'-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q + chromosome in the AraC-resistant cell line and a (Xq:11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the rat dck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of the dck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence of dck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in the dck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functional dck deficiency that may be the consequence of mutations occurring in the dck gene. PMID- 7543294 TI - Distribution of Tenr, an RNA-binding protein, in a lattice-like network within the spermatid nucleus in the mouse. AB - In a molecular screen for cDNAs that encode protamine RNA-binding proteins, we obtained seven independent clones that encode Tenr, a testis nuclear RNA-binding protein. Tenr is a 72-kDa protein that has one copy of a previously described RNA binding domain. Northwestern blotting experiments showed that a maltose-binding protein-Tenr fusion binds to a variety of RNAs in vitro and that it does not bind to single-stranded or double-stranded DNA. The Tenr gene is transcribed exclusively in the testis, and its mRNA is restricted to cells from the pachytene spermatocyte stage through the round spermatid stage. Immunolocalization of the Tenr protein within the testis showed that it is first detected postmeiotically, demonstrating that the Tenr mRNA is under translational control. The Tenr protein is localized to round and early elongating spermatid cells, and confocal microscopy revealed a lattice-like nuclear distribution suggesting association with the nuclear scaffold. We suggest that the Tenr protein may be involved in testis-specific nuclear posttranscriptional processes such as heterogeneous nuclear RNA (hnRNA) packaging, alternative splicing, or nuclear/cytoplasmic transport of mRNAs. PMID- 7543297 TI - Human fallopian tube as an extraovarian source of relaxin: messenger ribonucleic acid expression and cellular localization of immunoreactive protein and 125I relaxin binding sites. AB - By means of specific human relaxin primers that originated from relaxin A and B chains, a monoclonal antibody, and 125I-relaxin, the expression of mRNA and immunoreactive protein and the presence of binding sites for relaxin were investigated in human fallopian tubes. Reverse transcription polymerase chain reaction (RT-PCR) analysis of total RNA isolated from tubal tissues revealed the predicted 434-bp fragments originating from both the H1 and H2 relaxin genes. Restriction enzyme digestion of the RT-PCR products with Msp I (Hpa II), present only in the relaxin H1 sequence, resulted in the anticipated 175- and 259-bp fragments, whereas digestion with Hpa I, which is present in the relaxin H2 sequence and should have resulted in 188- and 246-bp fragments, induced a limited and partial digestion of the product. Codigestion of the RT-PCR product with Msp I+Hpa I also resulted in 175- and 259-bp fragments. The immunoreactive relaxin protein was present primarily in the tubal epithelial cells of the ampullary and isthmus regions, and with weaker intensity in tubal smooth muscle cells. Immunoreactive relaxin either was barely present or was absent in other tubal cell types. The intensity of immunostaining for relaxin in the epithelial cells appeared not to be cycle-dependent; however, these cells showed a lower immunostaining at the late secretory phase of the menstrual cycle than at other reproductive stages and an absence of immunostaining during the postmenopausal period.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543291 TI - Molecular characterization of CD34+ human hematopoietic progenitor cells. PMID- 7543295 TI - Partial complementary deoxyribonucleic acid cloning of equine relaxin messenger ribonucleic acid, and its localization within the equine placenta. AB - To determine the site of relaxin gene expression in equine placentae, a set of degenerate oligonucleotide primers was made according to the published amino acid sequence of the A- and B-chain of equine relaxin (eRXN). Total cellular RNA (tcRNA) from equine placentae at about 120 and 300 days of pregnancy was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with use of these primers. A single amplification product of approximately 430 bp was detected in each case by agarose gel electrophoresis. The PCR product was ligated into Bluescript plasmid and sequenced to confirm the identity of the clone as an eRXN cDNA fragment. Nucleic acid sequence analysis revealed a 428-bp eRXN cDNA fragment encoding for parts of the A- and B-chain and the connecting peptide (109 residues). Northern analysis of tcRNA from placentae of 120 and 300 days of pregnancy was carried out with use of antisense digoxigenin-labeled cRNA generated from the cDNA clone, and a single transcript of approximately 1 kb was detected. In situ hybridization on placental tissue at 120 and uteroplacental tissue at 300 days of pregnancy indicated that only the fetal trophoblastic cells expressed eRXN mRNA transcripts. The identity of these cells was confirmed by their positive staining with an antibody specific for equine trophoblast (cell surface) protein. Relaxin peptide was also detected immunohistochemically in samples of the same placental tissues. This is the first report of the nucleic acid sequence of eRXN. The study identified fetal trophoblast cells as the site of eRXN mRNA expression and protein secretion in the equine placenta. PMID- 7543296 TI - Neutralization of tumor necrosis factor alpha (TNF alpha) action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF alpha p60 receptor. AB - Antisense oligodeoxyribonucleotide inhibition of gene expression was used to test whether the p60 form of the tumor necrosis factor alpha (TNF alpha) receptor (Rp60) is responsible for mediating the negative effect of TNF alpha on the development of rat blastocysts in vitro. The antisense oligonucleotide was designed to overlap the translation initiation codon of the TNF alpha Rp60 mRNA. Preliminary experiments showed that concentrations of oligonucleotides above 10 microM in the culture medium were embryotoxic over 24 h. When used at nontoxic concentrations (8 microM), antisense oligonucleotides specifically decreased the abundance of intact TNF alpha Rp60 transcripts by 80% within 3 h of exposure. In contrast to results with control embryos, mRNA for the second form of TNF alpha receptor, TNF alpha Rp80, was detected in blastocysts exposed to antisense oligonucleotides to TNF alpha Rp60. Antisense oligonucleotides to TNF alpha Rp60 blocked the 25-30% decrease in cell proliferation induced by 50 ng/ml TNF alpha added to a standard culture medium and by 5 ng/ml TNF alpha added to a medium that had been conditioned by rat uterine cells. Sense oligonucleotides had no such protective effect. Because uterine cells from diabetic rats secrete higher levels of TNF alpha than those from control rats, antisense oligonucleotides were also tested in a medium that had been conditioned by diabetic uterine cells (cell secreted TNF alpha concentration was 50 pg/ml in this medium, and no exogenous TNF alpha was added). Addition of antisense oligonucleotides to TNF alpha Rp60 improved the quality of this medium with respect to cell proliferation but failed to correct the high frequency of dead cells observed in the blastocysts. PMID- 7543298 TI - Carbohydrates mediate the adherence of hamster sperm to oviductal epithelium. AB - The lower isthmus of the mammalian oviduct appears to serve as a reservoir for sperm that are retained by adherence to the epithelium. By inhibiting sperm binding within excised hamster oviducts and making use of carbohydrate probes, we have characterized the adherence of sperm in the reservoir and established a potential biochemical mechanism for the adherence and release of sperm. Fetuin and its terminal sugar, N-acetylneuraminic acid, interfered with the adherence of sperm to the oviductal epithelium. Labeled fetuin bound to the acrosomal region of fresh epididymal sperm, but not hyperactivated sperm, which have previously been reported to release from epithelial binding. Western blots labeled with fetuin and sialic acid-recognizing lectins identified proteins at several molecular masses that are candidates for the sperm surface component involved in adherence. Labeling of some of these candidates was reduced in samples from hyperactivated sperm. These results indicate that a sialylated oligosaccharide similar to that found on fetuin may be recognized by a sperm surface component and mediate binding to the oviductal epithelium. Release may be accomplished by loss or modification of the component during capacitation or hyperactivation. PMID- 7543299 TI - Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicle rupture. AB - Ovulation is accompanied by a large increase in the permeability of the capillaries surrounding the follicle, beginning a few hours after the ovulatory stimulus. The resulting edema may play a role in ovulation as well as in the formation and vascularization of the CL. Vascular endothelial growth/permeability factor (VEG/PF) is both a specific mitogen for endothelial cells and a potent stimulator of vascular permeability. The purpose of this study was to determine whether or not the ovulatory stimulus induces an increase in the expression of VEG/PF in the ovary. Female rats were primed with 8 IU of eCG at 27 days of age. Follicular maturation and ovulation were induced with an injection of hCG (5 IU) in the early afternoon on the second day after priming. Ovaries were removed 0, 1, 2, 4, 8, 10, and 18 h later. VEG/PF mRNA levels were compared through use of quantitative reverse transcription-polymerase chain reaction (RT-PCR). There was a marked increase (approximately 8-fold) in steady state levels of the transcripts for VEG/PF120 and VEG/PF164 between 1 and 4 h after hCG in whole ovaries. Increases were detectable both in granulosa cells and in thecal/stromal tissue. The high level of expression was maintained at 10 and 18 h (Day 1 CL). Thus, the preovulatory increase in follicular vascular permeability is closely associated with a marked, sustained increase in VEG/PF expression. VEG/PF, therefore, may play an important role in that increase and, consequently, in the process of ovulation as well as the subsequent vascularization of the CL. PMID- 7543300 TI - Long distance pathways of diffusion for dextran along fibre bundles in brain. Relevance for volume transmission. AB - Texas Red-labelled dextran with a mol. wt of 3000 g mol-1, a marker for the extracellular space, was injected unilaterally into the neostriatum of adult rats (0.3-30 micrograms microliter-1) and its distribution evaluated 1 min to 5 h later. Diffusion in the neuropil was observed with clearance starting after 30 min. After 10-15 min strong labelling along the myelinated fibre bundles was observed in the entire neostriatum. After about 20 min the labelling along the fibres reached into the corpus callosum and the overlaying deep layers of the cerebral cortex. A marked cellular uptake and accumulation of labelled dextran was found in putative perivascular pericytes. Thus, in the living brain preferential extracellular fluid pathways for diffusion exist, especially along fibre bundles, which allow the exchange of chemical signals between two distant regions. These may represent extracellular fluid pathways for volume transmission. PMID- 7543301 TI - Developmental change of the potentiation of NMDA response by spermine. AB - Developmental change in the potentiation of N-methyl-D-aspartate (NMDA) responses by spermine was investigated on the ventromedial hypothalamic neurones acutely dissociated from the rats aged between 5 and 21 days, using a nystatin perforated patch clamp recording in a whole cell mode. Spermine potentiated the NMDA response in a concentration dependent manner between 10(-5) M and 10(-5) M at all ages examined. This potentiation decreased significantly with age. On the other hand, spermine did not affect the kainate and AMPA responses at any age. This developmental change of the modulation of NMDA responses might influence to or be influenced by the behavioural and neuronal changes related to the VMH in the early postnatal life. PMID- 7543302 TI - Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions. AB - In higher plants anion channels have recently been suggested to play key roles in controlling cellular functions, including turgor- and osmoregulation, stomatal movements, anion transport, signal transduction and possibly also signal propagation. In guard cells and roots, physiological functions of anion channels have been proposed which will be discussed here. In initial investigations it was proposed that anion channels in the plasma membrane of guard cells provide a prominent control mechanism for stomatal closing. The proposed model suggests that anion channel activation and the resulting anion efflux from guard cells cause membrane depolarization, thereby driving K+ efflux through outward rectifying K+ channels required for stomatal closing. This article provides a brief review of new and recent insights into the molecular properties and cell biological functions of anion channels in guard cells. Furthermore, recently implicated putative functions of anion channels in roots during salt stress, xylem loading and Al3+ tolerance are addressed. PMID- 7543304 TI - Enhancement of murine bone marrow macrophage differentiation by beta-endorphin. AB - The present study was performed to investigate the effect of beta-endorphin on macrophage colony-stimulating factor (M-CSF)-induced differentiation of macrophages from bone marrow cells in a semisolid culture system. beta-endorphin increased the number of macrophage colonies when bone marrow cells were cultured in the presence of M-CSF plus lipopolysaccharide (LPS). This was not the case with LPS-unresponsive C3H/HeJ mouse bone marrow cells. alpha-endorphin and gamma endorphin were as effective as beta-endorphin in enhancing the colony formation. Exogenous interleukin-1 (IL-1), but neither IL-6 nor tumor necrosis factor (TNF), collaborated with beta-endorphin even in the absence of LPS, suggesting that IL-1 is a primary mediator of the effect of LPS. Indeed, anti-IL-1 antibody abolished the collaborative effect of beta-endorphin with LPS. Moreover, IL-1 was effective even for C3H/HeJ mouse bone marrow cells. Naloxone, an antagonist of endorphins for opioid-receptors, completely abrogated the effect of beta-endorphin. In a single-cell culture system, the collaboration between beta-endorphin and IL-1 was revealed by the increase in number and size of macrophage colonies, but collaboration between beta-endorphin and LPS did not occur. These results indicate that, in mixed cell culture, beta-endorphin acts in concert with paracrinal IL-1 induced by LPS to enhance M-CSF-dependent macrophage differentiation from immature precursor cells. PMID- 7543303 TI - Granulocyte colony-stimulating factor versus placebo in addition to penicillin G in a randomized blinded study of gram-negative pneumonia sepsis: analysis of survival and multisystem organ failure. AB - Sepsis is a common cause of morbidity and mortality. Neutrophils are the major defense against bacterial invasion, and granulocyte colony-stimulating factor (G CSF) augments both neutrophil number and function. In our study, 160 rabbits were inoculated transtracheally with 0.5 mL of a solution containing 10(4) colony forming units per milliliter of Pasteurella multocida. Twenty-four hours later, chest x-rays and quantitative blood cultures demonstrated pneumonia and bacteremia. Therapy was then begun with penicillin G and either recombinant human G-CSF (rG-CSF; 5 to 8 micrograms/kg subcutaneously) or placebo every day for 5 days. Arterial blood gases and 23 other parameters of organ function were performed before inoculation and serially thereafter. All rabbits underwent histologic examination of organs at the time of septic death or when sacrificed on day 6. A total of 149 rabbits survived long enough to initiate therapy. A significant increase in leukocytes by day 4 was found in the rG-CSF-treated group. There was a trend towards improved survival in the rG-CSF group (77% v 67%; P = .13, n = 149). Analysis of pretreatment variables revealed sepsis induced leukopenia (< or = 2,800/microL) as the only predictor of significantly improved survival with rG-CSF treatment (57% v 39%; P = .04, n = 73). The majority of the survival benefit occurred within the first 24 hours of treatment. This was before the time that a significant difference in mean white blood cell (WBC) count was observed between the study groups, making intravascular leukocytosis an unlikely explanation for the survival advantage in the rG-CSF group. No significant difference in laboratory variables reflecting organ function was demonstrated between the groups. Histologic grading of inflammation (0, normal, to 6, necrosis) in seven organs revealed that the surviving rabbits had mild but statistically significant increased inflammation in the liver, spleen, and noninoculated lung in the rG-CSF versus placebo groups (liver: 2.6 v 1.5, P < or = .0001; spleen: 3.2 v 2.3, P < or = .0001; and noninoculated lung: 2.9 v 2.5, P = .04). Administration of rG-CSF, in addition to penicillin G, in immune competent rabbits with gram-negative sepsis complicated by leukopenia significantly improved survival over antibiotics alone. The administration of rG CSF in early sepsis for a short therapeutic duration was not associated with any clinically evident toxicity. Clinical trials using rG-CSF in septic patients with leukopenia are indicated. PMID- 7543305 TI - Protective effect of granulocyte colony-stimulating factor against T-cell meditated lethal shock triggered by superantigens. AB - The bacterial superantigens (SAg), toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB), are powerful T-cell stimulators, triggering systemic release of lymphokines causing lethal shock in D-galactosamine (D-Gal) sensitized mice. We show that pretreatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) protects mice against T-cell-mediated SAg shock. In mice challenged with D-Gal/TSST-1, lethal shock was caused within 30 hours. In contrast, animals pretreated with two consecutive subcutaneous injections of 2 micrograms rhG-CSF with a 12-hour time interval showed only marginal signs of illness and no lethality after challenge with D-Gal/TSST-1. Mice treated with 5 micrograms rhG-CSF either 12 or 6 hours in advance also survived otherwise lethal doses of D-Gal/TSST-1. The protective effects of rhG CSF pretreatment was also evident against lethal doses of D-Gal/SEB challenge and this protection was accompanied by suppression of systemic interleukin-2. However, rhG-CSF affected neither the proliferative responses of SAg-reactive T cells in vivo or in vitro nor their interleukin-2 production in vitro, implying that rhG-CSF may indirectly interfere with cytokine synthesis in T cells but not with T-cell-SAg binding itself. These results represent another beneficial effect of rhG-CSF as an anti-inflammatory agent against T-cell-mediated toxicity triggered by SAg. PMID- 7543306 TI - Induction of apoptosis in human eosinophils by anti-Fas antibody treatment in vitro. AB - Fas antigen (CD95) can induce apoptosis of cells such as lymphocytes and neutrophils. To determine whether Fas antigen is involved in eosinophil apoptosis, we examined its expression and function on eosinophils in vitro. Purified human eosinophils expressed low but consistently detectable levels of Fas antigen. Culture of eosinophils in up to 10 ng/mL interleukin-5 (IL-5) prolonged eosinophil survival; incorporation of 1 to 1,000 ng/mL Fas antibody led to significant reductions in IL-5-induced eosinophil viability after 48 to 72 hours of culture. Reductions in survival could not be overcome by IL-5 and also occurred in the absence of exogenous IL-5. Preactivation of eosinophils with platelet-activating factor (PAF) significantly reduced eosinophil viability without altering the survival-reducing effects of Fas antibody treatment. In contrast, RANTES did not affect eosinophil viability or Fas antibody-induced reductions in eosinophil survival. After treatment with Fas antibody, electron microscopy of eosinophils and gel electrophoresis of DNA extracted from eosinophils demonstrated changes consistent with apoptosis. These data demonstrate that Fas antigen can modify eosinophil survival by inducing apoptosis through a pathway that is, at least in part, independent of the survival promoting effects of IL-5. PMID- 7543309 TI - Noncycling state of peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor and other cytokines. AB - Incubation with high doses of tritiated thymidine in vitro was used to determine the percent of progenitor cells in the S phase of the cell cycle. Peripheral blood (PB), bone marrow (BM), and spleen populations from mice injected with granulocyte colony-stimulating factor (G-CSF) at 5 micrograms/day for 5 days and BM cells from uninjected littermates were assayed. Although the percentage of progenitor cells in S phase in the marrow (47% +/- 5%) and spleen (52% +/- 9%) was increased significantly in G-CSF-treated mice, only a small proportion of PB progenitor cells (PBPC) were in S phase (7% +/- 4%). In normal human subjects injected with G-CSF at 5 or 10 micrograms/kg/d, the proportions of PB myeloid (-1 +/- 4%) and erythroid (0% +/- 8%) progenitor cells in S phase were very low compared with the proportion of myeloid progenitor cells in S phase in normal BM (34% +/- 10%). Similarly, the large majority of steady-state PBPC and PBPC mobilized by interleukin-3 in combination with either granulocyte-macrophage colony-stimulating factor or G-CSF were also found not to be in S phase. Experiments indicated that the low percentages of PBPC in S phase were not ascribable either to inhibitory elements in the blood or to reduced responsiveness to growth factors. PMID- 7543308 TI - Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. AB - The hematologic disorder paroxysmal nocturnal hemoglobinuria (PNH) occurs following an acquired somatic mutation in the Piga gene within a bone marrow stem cell. The progeny of this mutated cell cannot synthesize glycosylphosphatidylinositol (GPI) anchors, with a resultant deficiency in surface expression of all GPI-linked proteins. The protean clinical manifestations of PNH presumably result from the deficiency of these GPI-linked surface proteins. To explain the observation that neutrophils are affected at a significantly higher percentage than circulating erythrocytes and to analyze the proliferative rates of erythroid production in PNH, we studied 25 patients using flow cytometry. The fluorescent dye thiazole orange was used to detect reticulocytes, and CD59 monoclonal antibody was used to identify GPI-deficient cells. In contrast to the mature circulating erythrocytes, the percentage of abnormal reticulocytes was similar to the percentage of affected neutrophils. However, the vast majority of reticulocytes was completely GPI-deficient, ie, were type III cells, even in patients with only modest numbers of circulating type III erythrocytes. In addition, greater than 5% type II reticulocytes were identified in only 3 patients, although greater than 5% type II mature erythrocytes were identified in 10 of 25 patients. The results show that the erythroid and neutrophil bone marrow precursors have an equivalent proliferative advantage in PNH. The data also have important implications for the origin of type-II erythrocytes in PNH. PMID- 7543307 TI - Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells. AB - The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL-7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c-kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H-thymidine incorporation. Low level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance. PMID- 7543310 TI - Endoscopic laser ablation of the prostate. A new technique for treating prostate problems. AB - Bladder outflow obstruction due to benign prostatic enlargement affects up to a third of men over the age of 50. To date, the only reliable form of treatment is surgical, mainly by transurethral resection of the prostate (TURP). An alternative technique, endoscopic laser ablation of the prostate (ELAP) has recently been introduced to overcome some of the drawbacks of TURP. ELAP involves directing laser energy via a specially designed side-firing fibre at the enlarged portion of the prostate. The fibre, passed into the prostate through a cystoscope, carries the energy from the laser to the prostate causing heating of the prostate and subsequent shrinkage of the gland. The operation is performed under general or spinal anaesthetic and is quicker to perform than a TURP. Because the laser causes virtually no bleeding, patients can be treated as day cases or go home the day after surgery. A catheter is left in the bladder and is removed as an out-patient a few days later. The early results of ELAP are comparable to TURP, but complications and length of hospital stay are less. PMID- 7543312 TI - ABC of medical computing. Illustrating and presenting your data. PMID- 7543311 TI - Potentiation of warfarin by interferon. PMID- 7543313 TI - Guidelines to reduce x ray examinations in accident departments. Implementing guidelines may not improve outcome for patients. PMID- 7543315 TI - Mechanism of the myelin basic protein-induced insulin and glucagon release from isolated rat pancreatic islets. AB - The central nervous system myelin basic protein (MBP) stimulates the release of several peptide hormones including insulin and glucagon. This could be associated with the development of hyperglycaemia in neurological disorders such as stroke, in which MBP is known to leak into blood circulation. In the present study the mechanism of insulin and glucagon release was investigated by using short-term incubation of isolated rat pancreatic islets. Incubation with MBP in the absence of Ca2+ resulted in approx. 11-fold stimulation of insulin and glucagon release. The stimulation dwindled with increasing Ca2+ concentration and was 6.5-fold at 0.5 mM and 2-fold at 2.5 mM Ca2+. When MBP and glucose at various concentrations were simultaneously present in the incubation mixture, stimulation of insulin release was the sum of the stimulation induced by these two agents separately both at the 0.5 and 2.5 mM Ca2+ concentrations. Glucose at concentrations of 10 or 15 mM did not suppress MBP-stimulated glucagon release. Caffeine-evoked increase in intracellular Ca2+ was without effect on MBP-stimulated insulin or glucagon release but enhanced glucose-induced insulin release. The Ca2+ channel blocker diltiazem had no effect on MBP-stimulated insulin release at concentrations where glucose-stimulated release was inhibited. Ruthenium red inhibited both MBP- and glucose-stimulated insulin release as well as MBP-induced glucagon release. Staurosporine (inhibitor of protein kinase C) had no effect on MBP-induced insulin release, although it partially inhibited glucose-stimulated release. Maleylation of MBP abolished its insulin- and glucagon-releasing activity by approx. 90%. These results suggest that MBP exerts its insulin releasing effect by mechanisms different from those of glucose-stimulated insulin release and does not require Ca2+ channels or protein kinase C. The relation of MBP-induced insulin and glucagon release to Ca2+ concentration is probably explained by enhanced self-aggregation of MBP or by increased ability of MBP to interact with islet cell membranes in the absence of Ca2+, or both. It is concluded that MBP-induced hormone release appears to be mediated by membrane fusion and oligomerization of MBP. The mechanism thus resembles that of various toxins and other cytotoxic agents. PMID- 7543314 TI - An immunohistochemical study of odontogenic mixed tumours. AB - Five cases of odontogenic mixed tumour comprising of an ameloblastic fibroma, an adenomatoid odontogenic tumour, an odonto-ameloblastoma and two ameloblastic fibro-odontomas were immunohistochemically investigated. Odontogenic epithelial cells were fully positive for cytokeratin detected by antibody KL-1, although there were some differences in its intensity. In contrast, for tenascin, only immature dental papilla-like mesenchymal tissue, especially around the dental lamina-like odontogenic epithelium, was positive, while the myxomatous area and connective tissue were negative. Positive vimentin staining was observed in some areas of immature dental papilla-like cells as well as the basement membrane of odontogenic epithelium in the ameloblastic fibroma, suggesting that this tumour had developed at the early stage of tooth formation. Proliferating nuclear cell antigen-positive cells were generally rarely seen, but were frequently observed in epithelial cells of the ameloblastic fibroma and odonto-ameloblastoma. These observations suggest that tumour cells in each odontogenic mixed tumour possess characteristic proteins associated with proliferation potential and that ameloblastic fibroma and odonto-ameloblastoma have higher proliferation potential among the tumours examined. PMID- 7543317 TI - Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis. AB - In order to identify a possible hereditary predisposition to the development of obstructive pulmonary disease of unknown origin, we have looked for the presence of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations in unrelated patients with no signs of Cystic Fibrosis (CF). We screened for 70 common mutations, and also for rare mutations by denaturing gradient gel electrophoresis analysis. In this search, different CFTR gene mutations (R75Q, delta F508, R1066C, M1137V and 3667ins4) were found in five out of 16 adult Italian patients with disseminated bronchiectasis, a significant increase over the expected frequency of carriers. Moreover, three rare CFTR gene DNA polymorphisms (G576A, R668C, and 2736 A-->G), not deemed to be the cause of CF, were found in two patients, one of which was a compound heterozygote with R1066C. These results indicate that CFTR gene mutations, and perhaps also DNA polymorphisms, may be involved in the etiopathogenesis of at least some cases of bronchiectasis. PMID- 7543316 TI - Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation. AB - The trinucleotide expansion mutation causing myotonic dystrophy is in the 3' untranslated region of a protein kinase gene. The molecular mechanisms by which the expanded repeat causes the clinically variable and multisystemic disease, myotonic dystrophy, are not understood. It has been particularly difficult to rationalize the dominant inheritance with the fact that the expansion mutation lies outside of the protein-encoding gene elements, and should not be translated into protein. Here we use muscle biopsies from classical adult-onset myotonic dystrophy patients to study the accumulation of transcripts from both the normal and expanded DM kinase genes in patient muscle, and compare the results to normal and myopathic controls. We found relatively small decreases of DM kinase RNA in the total RNA pool from muscle; however, these reductions were not disease specific. Analysis of poly(A)+ RNA showed dramatic decreases of both the mutant and normal DM kinase RNAs, and these changes were disease-specific. Our findings are consistent with a novel molecular pathogenetic mechanism for myotonic dystrophy: both the normal and expanded DM kinase genes are transcribed in patient muscle, but the abnormal expansion-containing RNA has a dominant effect on RNA metabolism by preventing the accumulation of poly(A)+ RNA. The ability of the expansion mutation to alter accumulation of poly(A)+ RNA in trans suggests that myotonic dystrophy may be the first example of a dominant-negative mutation manifested at the RNA level. PMID- 7543318 TI - The lactase persistence/non-persistence polymorphism is controlled by a cis acting element. AB - Lactase activity is present at high levels in the small intestine of some human adults and not others. This is due to a genetically determined polymorphism which affects the developmental regulation of the expression of the lactase gene. This polymorphism is of considerable interest in relation to cultural differences in nutrition but despite exhaustive studies, the molecular basis has not yet been found. It has not even been shown whether the sequence differences reside within or adjacent to the lactase gene itself or in a trans-acting factor. We have therefore exploited known DNA 'marker' polymorphisms within the exons of the lactase gene to examine the expression of the individual lactase mRNA transcripts from persistent and non-persistent individuals in order to determine whether the regulation is in cis or trans. Our results show that in certain lactase persistent individuals one allele of the lactase gene is expressed at much lower levels than the other and these individuals tend to have intermediate lactase activities. It is proposed that these people are heterozygous for the lactase persistence and non-persistence alleles and that this means that the nucleotide substitutions responsible for the lactase persistence/non-persistence polymorphism are cis-acting. This narrows down considerably the area of the genome that needs to be searched for the relevant sequence differences. PMID- 7543319 TI - Expression patterns of two human genes coding for different rab GDP-dissociation inhibitors (GDIs), extremely conserved proteins involved in cellular transport. AB - We have analysed the expression patterns of two human genes coding for two different rab GDIs, rab GDI alpha/XAP-4 and rab GDI beta, proteins involved in the regulation of vesicle-mediated cellular transport. The gene sequences are extremely conserved in evolution, with substantial homology preserved across three eukaryotic kingdoms. Although the sequence homology between the two human rab GDIs studied is very high, their expression patterns are completely different. The Northern blot analysis and in situ hybridization to sections of mouse embryos and postnatal tissues have revealed that the rab GDI alpha/XAP-4 is expressed predominantly in neural and sensory tissues and may thus serve a specific function in neural signal transmission. In contrast to rab GDI alpha/XAP 4, the human rab GDI beta is expressed ubiquitously. PMID- 7543320 TI - A point mutation in exon 2 of the CD40 ligand gene causes the simultaneous expression of two defective mRNA species in X-linked hyperimmunoglobulinemia M. PMID- 7543322 TI - Use of bare fiber with siderfiring Albarran bridge in laser therapy of the prostate. AB - Laser coagulation of the prostate has been performed using a bare fiber passed through a sidefiring Albarran bridge containing a distal gold-plated reflector with a deflecting mechanism. The system and the fiber can be used for several dozen treatments. Transurethral laser coagulation with the new Albarran bridge and the noncontact Nd:YAG laser was performed on 45 patients for obstructive symptoms caused by benign prostatic hyperplasia. The dosimetry was 1000 J per 1 cc of prostatic tissue at 60 W for 60 seconds. Successful results were obtained in 38 patients (85%). A significant reduction in obstructive symptoms from a mean AUA-6 Symptom Score of 21.2 preoperatively to 9.1 at 3 months and 7.6 at 6 months was associated with an increase in the peak urine flow rate from 6.1 mL/sec preoperatively to 13.1 mL/sec at 3 months and 15.7 mL/sec at 6 months. The residual urine volume averaged 190 mL preoperatively and 35 mL at 6 months. Transurethral laser coagulation of the prostate represents a useful alternative to transurethral resection, especially in the high-risk patient with an enlarged median lobe or a small prostate. The treatment is bloodless and, with the aid of the modified Albarran bridge, can be performed with a standard urologic Nd:YAG laser. The new Albarran bridge also can reduce the cost of laser treatment. PMID- 7543321 TI - Contact laser vaporization techniques for benign prostatic hyperplasia. AB - Contact laser applications for the relief of bladder outlet obstruction caused by an enlarged prostate are different from the noncontact Nd:YAG laser methods. The noncontact techniques rely on coagulation necrosis or high power-density vaporization. The pure contact Nd:YAG laser allows cutting, coagulation, and vaporization of tissue with minimal penetration beyond the contact surface. In the contact laser prostatectomy technique, the laser probe directly touches and vaporizes the prostatic tissue. This results in immediate removal of the obstructing tissue, in a manner similar to the standard electrosurgical transurethral resection (TURP), and offers the patient the potential for decreased catheter time and a more rapid resolution of symptoms. Our initial experience suggests that the contact technique (contact laser ablation of the prostate or CLAP) may be better suited for the smaller prostate gland (i.e., less than 20-30 g). For prostates larger than 30 g, a newly described procedure known as coagulation and hemostatic resection of the prostate (CHRP) can be used. This method combines initial noncontact coagulation of the prostate with vaporization of a channel. The goal of CHRP is to allow more rapid removal of the catheter with a continued improvement in urine flow secondary to the coagulation effects. The contact laser is specifically designed to vaporize tissue such as the prostate and allows immediate observation of a TUR defect. Improvements in the delivery system and in the size of the contact laser probes have made CLAP a useful modality for the treatment of symptomatic benign prostatic hyperplasia. PMID- 7543324 TI - Transurethral ultrasound-guided laser-induced prostatectomy (TULIP): past, present, and future. AB - The TULIP system was the first apparatus designed specifically for laser ablation of the prostate. Laser energy is delivered under ultrasound guidance with the probe moved distally from the bladder neck at the rate of 0.5 to 1 mm/sec. The aim is to produce a deep level of coagulation necrosis. Postoperatively, patients have a suprapubic catheter until they can void comfortably. As of 1994, 2-year results showed a 69% reduction in the mean Boyarsky symptom score (N = 57) v 63% at 6 months (N = 212) and a mean increase in the peak urine flow rate of 68% (N = 46) v 59% at 6 months (N = 195). At present, it is not clear whether TULIP will become commercially available in the United States. PMID- 7543323 TI - Interstitial laser therapy outcomes in benign prostatic hyperplasia. AB - Two hundred thirty-nine patients with benign prostatic hyperplasia (BPH) were treated using interstitial laser coagulation (ILC or LITT; laser-induced interstitial thermotherapy), and the long-term results were determined. In this minimally invasive procedure, special laser light guides were inserted into the adenoma tissue in order to achieve primary large-volume coagulation necrosis and secondary shrinking, leaving the urethra intact. The AUA Symptom Scores improved markedly, from an average of 25 pretreatment to an average of 6 at 1 year. Peak urine flow rates improved commensurably, from 8 mL/sec pretreatment to 18 mL/sec at 1 year. Postoperative irritative symptoms occurred occasionally, affecting only 12% of patients. Significant complications were rare, with 4% of patients suffering strictures and 7% retrograde ejaculation. No patients suffered impotence or incontinence. Only 9.6% of patients required retreatment for BPH during 12 months of follow-up. Twelve patients were treated with subsequent transurethral laser incision of the bladder neck in order to shorten the time required for improvement of voiding function. PMID- 7543327 TI - Ultrasound monitoring during laser-assisted transurethral resection of the prostate. AB - Initial studies utilizing the original visual laser ablation prostatectomy (VLAP) technique of coagulation and a pilot study applying laser energy to prostate cancer led to the realization that these procedures could be monitored effectively in real time by ultrasound. Physical and chemical changes occur in prostate tissue with heating by laser energy, and these changes can be detected, not only as cavitation when the prostate tissue is vaporized, but also as a hyperechoic alteration that presumably is cell death leading to necrosis. Utilizing real-time monitoring helps assure the efficacy of the procedure and predict greater cavitation from slough of dead tissue. Monitoring by ultrasound scanning also allows following of the change of directions of the laser beam in tissue, which could be dangerous to the patient. With ultrasonography, one can make sure that the neurovascular bundle is not compromised and that the energy is not allowed to proceed past the posterior capsule of the prostate into the rectal wall. This is particularly helpful in patients with a high bladder neck but with minimal prostatic tissue posteriorly at the base. Also, the amount of tissue and the length from the verumontanum to the external sphincter can be accurately assessed and correlated with the lesion created at that level to avoid damage to the external sphincter. PMID- 7543329 TI - Prostatic aperture resulting from visual laser ablation: classification system based on follow-up endoscopy. AB - To study the evolving prostatic aperture created by visual laser ablation (VLAP), we performed 38 video-endoscopies in 24 men with prostatism at various intervals 2 weeks to 1 year after treatment. Complete healing was generally observed within 3 to 4 months, never before 6 weeks; and in some patients, tissue sloughing was still apparent beyond 6 months. By review of the cystoscopic findings and video hard copies, three independent observers classified the healed prostatic apertures with great uniformity into one of four categories: (I) minimal change (lateral lobes still meet in midline throughout gland length) (N = 3); (II) minor aperture (opening less than 50% of cystoscopic field over less than 50% of gland length) (N = 5); (III) major aperture (opening more than 50% of cystoscopic field over more than 50% of gland length) (N = 11); and (IV) full ablation (nearly complete replacement of lobar configuration with a general concavity) (N = 5). Clinical outcomes (symptom scores, uroflow rates) matched with follow-up cystoscopic categories but not with any other readily identifiable measures. The four-category system proved to be simple, reproducible, and clinically relevant. If a standardized tissue aperture is the ultimate aim of new methods to ablate the prostate, the proposed system for classifying the aperture could have a considerable future application. PMID- 7543326 TI - Local anesthesia for laser prostatectomy. AB - The technique of visual laser-assisted prostatectomy (VLAP) with a noncontact right-angle delivery system (Urolase) under local anesthesia is described. The advantages of local anesthesia include the facilitation of early patient discharge (2-3 hours after the procedure), avoidance of the risks of spinal and general anesthesia in high-risk patients, and, potentially, the enabling of VLAP as an outpatient office procedure. This technique was employed in 52 men with symptomatic benign prostatic hyperplasia (BPH) as an outpatient procedure without significant morbidity. The mean AUA Symptom Scores and uroflow measures all improved significantly with 1-year follow-up. Outpatient VLAP under local anesthesia is a promising treatment alternative for men with BPH. PMID- 7543330 TI - Complications of laser prostatectomy. AB - The appeal of laser therapy is rooted in its absence of complications relative to the gold standard of transurethral electroresection. As in any evaluation of a new medical intervention, efficacy must be weighed against the degree of complications that accompany it. Although there has been a relative paucity of literature specifically addressing the safety of this new modality, several studies are presented testifying to the clinical efficacy and relative absence of complications of laser prostatectomy. PMID- 7543328 TI - MRI evaluation of cavitation induced by laser prostatectomy. AB - Magnetic resonance images obtained by endorectal surface coils have shown histopathological changes in the prostate. In patients who underwent laser prostatectomy, MR imaging demonstrated how deeply the laser energy penetrated and how different changes were induced by the coagulation and vaporization techniques. Cavitation effects were quantitatively analyzed, and the processes of reepithelization after sloughing of necrotic tissues were observed in gadolinium enhanced T1-weighted images. PMID- 7543325 TI - Transurethral balloon laser thermotherapy for symptomatic benign prostatic hyperplasia: preliminary clinical results. AB - Between April 1993 and May 1994, 66 patients were treated with transurethral balloon laser thermotherapy (TUBAL-T) for the relief of bladder outlet obstruction secondary to benign prostatic hyperplasia. TUBAL-T, with a urethral cooling system, employs a balloon catheter and irradiating laser through 360 degrees to produce deep coagulation and necrosis of the prostatic tissue while preserving the urethral mucosa. The procedure was implemented under local topical anesthesia. Baseline AUA Symptom Scores, peak uroflow rates, postvoiding residual urine volumes (PVR), and prostatic volumes were measured before and at 1, 3, 6, and 12 months after treatment. The mean symptom score decreased from 18.8 preoperatively to 9.8, 6.9, 7.4, and 4.8 at 1, 3, 6, and 12 months, respectively. The mean peak uroflow rate increased from 6.4 mL/sec to 9.1, 11.2, 10.1, and 10.4 mL/sec at 1, 3, 6, and 12 months, respectively. As for the mean PVR, statistically significant reductions were clearly observed at 3 and 6 months after treatment. However, at 1 and 12 months, the difference was not statistically significant. In follow-up for as long as 12 months after the procedure, 23 of 26 patients (88%) showed an improvement of 50% or more in the AUA Symptom Scores. Of 20 available patients, 12 (60%) showed an improvement of 50% or higher in the peak uroflow rates, and 10 (50%) showed an improvement of 50% or higher in PVR. The mean prostatic volume reductions at 3, 6, and 9 months were 12%, 16%, and 14%, respectively. The serum prostate specific antigen concentration increased to four times the baseline concentration on the 7th day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543332 TI - Laser coagulation prostatectomy: evolution of clinical practice and treatment parameters. AB - Laser coagulation prostatectomy has evolved into an accepted option for the management of bladder outlet obstruction secondary to benign prostatic hyperplasia. Clinical results document the safety and efficacy of this operation. Rapidly expanding clinical experience and basic research efforts in recent years have provided a scientific foundation and an improved qualitative understanding of the laser-tissue interactions during Nd:YAG laser coagulation of the prostate. Today, truly quantitative laser dosimetry is becoming available to guide the operative approaches to laser prostatectomy with the increasing variety of Nd:YAG delivery systems. The development of laser coagulation prostatectomy is traced to the present, and accumulated clinical experience and laboratory data are incorporated into a rational approach to operative and perioperative patient management utilizing this technology. PMID- 7543331 TI - Evaluating the cost of lasers for the treatment of benign prostatic hyperplasia. AB - Cost is an extremely complex equation with many variables, but itemized hospital bills are useful in analysis because they reflect costs as defined by an institution. Using this data source, we found that the total cost of an outpatient visual laser-assisted prostatectomy ($6,872.42) is almost the same as that of transurethral resection with a 3-day hospital stay ($6,925.00). Use of local anesthesia, improvement in the efficiency of tissue vaporization, and increases in fiber durability to permit multiple use would reduce laser prostatectomy costs. The true economic impact of laser prostatectomy awaits a long-term analysis that takes into account the costs of late complications and of reoperation. PMID- 7543333 TI - [Gamma/delta T lymphocytes]. PMID- 7543334 TI - Early neurodevelopmental growth in children with vertically transmitted human immunodeficiency virus infection. AB - OBJECTIVE: To examine mental and motor development in children with vertically transmitted human immunodeficiency virus (HIV) infection in the first 30 months of life. DESIGN: Prospective longitudinal study comparing two groups: children with HIV infection and HIV-exposed but uninfected children. SETTING: Pediatric Immunodeficiency Clinic at Boston (Mass) City Hospital, Boston University Medical Center. STUDY PARTICIPANTS: Twenty-four children with vertically transmitted HIV infection and 27 children who were born to HIV-infected mothers and became HIV negative served as controls. Socioeconomic status, gestational age, and prenatal drug exposure were comparable in the two groups. MEASUREMENTS/RESULTS: Using the Bayley Scales of Infant Development, all children were assessed at least once between 4 and 16 months and again between 17 and 30 months of age. Individual mean mental and motor scores were calculated for the early and later age span. Motor development in the infected group was delayed in comparison to the seroreverter group in both age spans and remained stable in both groups over time. Mental development was comparable in the two groups at 4 to 17 months, but HIV infection was associated with delay in mental development at 17 to 30 months of age. CONCLUSION: Early and persistent delay in motor development and deceleration in mental development in late infancy distinguishes many children who are HIV infected from exposed but uninfected children, but there is significant variability in early neurodevelopmental outcome among children with HIV infection. PMID- 7543335 TI - Ferrous ions detected in iron-overloaded cord blood plasma from preterm and term babies: implications for oxidative stress. AB - Redox active iron chelatable to bleomycin is often present in the plasma of cord blood samples taken from preterm and term babies. The low caeruloplasmin and high ascorbate levels in plasma at birth may allow this iron to exist in the reduced ferrous state. In support of this postulate thirteen cord blood samples showing the presence of low molecular mass iron were able to degrade DNA in the presence of bleomycin and plasma. PMID- 7543336 TI - Endoscopic mucosal resection for superficial carcinoma and high-grade dysplasia of the esophagus. AB - Endoscopic mucosal resection (EMR) is a recently introduced therapeutic method based on the principles of the strip biopsy for resection of flat lesions of the gastrointestinal tract. Eleven asymptomatic patients (nine men, two women) with superficial carcinoma or high-grade dysplasia of the intrathoracic esophagus were treated by EMR during a 6-month period. The patients' mean age was 64 years (range, 49-78). The site of the lesions was the middle third of the esophagus in eight, upper third in two, and lower third in one patient. All patients in this series had a flat (II) type of superficial cancer. The procedure was carried out in all 11 patients without complication. Histopathological examination of the specimens revealed squamous cell carcinoma in nine patients. The remaining two patients were confirmed to have dysplasia only. Free margins measured greater than 5 mm in all cases. No recurrence was detected in a mean follow-up of 8 (5 10) months. For superficial flat lesions, EMR proved to be a safe and curative procedure that provided good quality of life following resection. However, larger trials are needed to confirm these results. Applying EMR to esophageal dysplasia could decrease the incidence of esophageal cancer. PMID- 7543338 TI - Immunophenotypic features of uterine stromal cells. CD34 expression in endocervical stroma. AB - CD34 is a myeloid progenitor cell antigen present in endothelial cells and some other mesenchymal cells, including perivascular and periadnexal dermal fibroblasts. It was evaluated immunohistochemically in uterine stromal tissue and in 4 aggressive angiomyxomas and 6 endometrial stromal sarcomas with potentially related and similar stromal tissues. The stromal cells in normal endocervix and endocervical polyps were strongly CD34 positive irrespective of the cycle phase, and negative for muscle actins. Ectocervical stroma was variably but generally less CD34 reactive. In the endometrium, the CD34 reactivity was limited to the stromal cells of the basal endometrium and was found only in 4 of 20 from proliferative endometria and 1 of 8 from secretory endometria. The uterine cervical and myometrial smooth muscle tissues showed CD34 positive cells only between the muscle bundles and around the vessels. In pelvic aggressive angiomyxomas and endometrial stromal sarcomas the tumour cells were CD34 negative and only the vascular endothelial cells were positive. Endothelial cell-specific antigen, CD31, was identified only in endothelial cells and was not present in the endocervical stroma. These results illustrate the particular immunohistochemical profile of endocervical stromal tissue, namely the strong CD34 expression. The CD34 reactivity of the endocervical tissues should be noted and not confused with neoplasms known to be strongly CD34 positive, such as angiosarcomas, Kaposi's sarcomas and some other spindle cell sarcomas. PMID- 7543337 TI - The significance of atypical adenomatous hyperplasia and prostatic intraepithelial neoplasia for the development of prostate carcinoma. An update. AB - The term prostatic intraepithelial neoplasia (PIN) is an accepted diagnosis in pathology of the prostate. The diagnostic difference between atypical adenomatous hyperplasia (AAH) and adenosis is still under debate. A number of questions remain about the significance of grading of AAH and PIN, the biology of AAH and PIN as precursors of carcinoma, the possibility of treatment of AAH and PIN and whether AAH- and PIN-associated cancers differ from non-associated carcinoma. This paper reviews the results and discussions at the First International Consultation Meeting on Atypical Adenomatous Hyperplasia and Prostatic Intraepithelial Neoplasia and the Origins of the Prostatic Carcinomas. AAH is an architectural atypia of the prostate. The histological and cytological features of AAH are intermediate between BPH and low-grade carcinoma of the prostate. Cell kinetic findings show no distinct neoplastic pattern. AAH may be a precursor of transition zone carcinoma but the findings to date are inconclusive. Follow up studies should address whether the association of AAH and carcinoma is incidental or whether transition occurs between AAH and carcinoma. In contrast, PIN is an accepted preneoplastic lesion and the most likely precursor of the dorso peripheral zone carcinoma. The diagnosis of high-grade PIN is clinically important, because high-grade PIN is associated with carcinoma in a high percentage of patients (38-100%). AAH- and PIN-associated cancers may not differ from other prostatic cancers. At present treatment for AAH and PIN without carcinoma is not indicated, but high-grade PIN warrants surveillance and follow up of the patient to identify a possible coexisting cancer. It must be stressed that AAH and PIN are multifocal lesions and both are age-associated. PMID- 7543340 TI - URO-9 monoclonal antibody is not a marker for normal human bladder epithelial cells. PMID- 7543339 TI - Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis. AB - To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P < 0.01) and in vitro angiogenesis (2.4 times, P < 0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner. PMID- 7543342 TI - An improved method for the isolation of type II and Clara cells from mice. AB - Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens. PMID- 7543341 TI - Quinine sensitive changes in cellular Na+ and K+ homeostasis of COS-7 cells caused by a lipophilic phenol red impurity. AB - An impurity of phenol red (PRI) has been shown to markedly alter the intracellular Na+ and K+ homeostasis of several cell types. The effect of PRI seems to involve intracellular Ca(++)-dependent mechanisms. Using COS-7 cells as a model, we further characterized the mechanism of action of PRI by measuring cellular Na+/K+ contents and 86Rb+ efflux. Similar to human skin fibroblasts, in COS-7 cells calmodulin inhibition moderated the cationic transport effects of PRI. A TMB-8 dependent intracellular Ca++ pool does not seem to be involved in these transport events. We found no evidence for participation of the transcriptional-translational machinery in the effect of PRI. Both quinine and quinidine are able to prevent nearly all changes caused by PRI in the cellular Na+/K+ contents and 86Rb+ efflux. Although phenol red contained multiple impurities by high performance liquid chromatography (HPLC), phenolphthalein, a structurally close relative of phenol red, was free of any detectable contamination. Phenolphthalein elicited qualitatively similar transport changes to those observed during exposure to PRI. Regardless of the exact mechanism of action, we propose that the as yet unidentified substance is not a cellular toxin, rather it is a cationic transport modulator. Directly or indirectly, it may interact with the cellular Ca++/calmodulin system and activate some quinine/quinidine sensitive transport processes. This transport process is likely to be a Ca(++)-sensitive K+ channel but, due to the lack of specificity of quinine and quinidine, other transport mechanisms must be also considered. The chemical nature of PRI may be similar to phenolphthalein. PMID- 7543344 TI - Repeated inhalation challenge with exogenous and endogenous histamine released by acetaldehyde inhalation in asthmatic patients. AB - We previously reported that inhaled acetaldehyde, a metabolite of ethanol and a main factor in alcohol-induced asthma, causes bronchoconstriction indirectly through endogenously released histamine in asthmatic subjects. No study has examined the difference between tachyphylaxis in response to endogenous as opposed to exogenous histamine. Therefore, we examined tachyphylaxis occurring in response to repeated inhalation of histamine or acetaldehyde in nine asthmatic subjects. The mean acetaldehyde concentration causing a 20% decrease in FEV1 increased significantly from 18.4 (geometric standard error of the mean (GSEM = 0.14) to 45.2 (GSEM = 0.14) mg/ml over a period of 1 h (p < 0.002), whereas the mean histamine concentrations causing a 20% decrease in FEV1 were identical. No correlations were observed between the change in bronchial responsiveness to each solution and the change in baseline FEV1. These results suggest that tachyphylaxis in response to histamine is observed only when the latter is released endogenously. We believe that this is the first report suggesting tachyphylaxis caused by endogenous histamine. PMID- 7543343 TI - Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver. AB - Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (> 97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a "spindle cell," consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture. PMID- 7543345 TI - Acute and chronic effects of cigarette smoking on exhaled nitric oxide. AB - Cigarette smoking is associated with an increased risk of respiratory tract infections, chronic airway disease, and cardiovascular diseases, all of which may be modulated by endogenous nitric oxide (NO). We have investigated whether cigarette smoking reduces the production of endogenous NO. We compared exhalations of 41 current cigarette smokers with normal lung function and 73 age matched non-smoking controls. Peak exhaled NO levels were measured by a modified chemiluminescence analyzer. The effects of inhaling a single cigarette in smokers were also measured. In control subjects we also measured the effects of inhalation of NO itself and carbon monoxide, both constituents of tobacco smoke. Peak exhaled NO concentrations were significantly reduced in smokers (42 +/- 3.9 compared with 88 +/- 2.7 parts per billion in nonsmokers, p < 0.01), with a significant relation between the exhaled NO and cigarette consumption (r = 0.77, p < 0.001). Smoking a single cigarette also significantly (p < 0.02), but transiently, reduced exhaled NO. Inhalation of carbon monoxide and NO had no effect on exhaled NO in normal subjects. Cigarette smoking decreased exhaled NO, suggesting that it may inhibit the enzyme NO synthase. Since endogenous NO is important in defending the respiratory tract against infection, in counteracting bronchoconstriction and vasoconstriction, and in inhibiting platelet aggregation, this effect may contribute to the increased risks of chronic respiratory and cardiovascular disease in cigarette smokers. PMID- 7543346 TI - A dominant human leucocyte antigen DR4-Dw15 restricted mycobacterial 65 kDa antigen-specific T-cell immunity in Chinese patients with rheumatoid arthritis. AB - The human leucocyte antigen DR4-associated immune responses to Mycobacterium tuberculosis 65 kDa heat shock protein were considered to be relevant to the pathogenesis of rheumatoid arthritis (RA). In the Chinese population, DR4-Dw15 was found to be the predominant DR4 subtype in RA. To further define the immune responses associated with DR4-Dw15 molecules, the proliferative responses of peripheral (PBMC) and synovial mononuclear cells (SFMC) to mycobacterial 65 kDa antigen were evaluated. The SFMC of all of our RA patients responded significantly to 65 kDa mycobacterial antigen. The responses of PBMC to this antigen in RA were much lower than those of SFMC. Our results further indicated that relatively low numbers of peripheral antigen-specific T-cells, but not incompetence of peripheral antigen presenting cells, might be related to the observed low responsiveness to 65 kDa antigen in PBMC of RA patients. Of utmost importance, DR4-Dw15 was proved to be one of the major restrictive molecules in mycobacterial 65 kDa antigen-specific immune responses in Chinese RA patients. PMID- 7543347 TI - Determination of the apparent synovial permeability in the knee joint of patients suffering from osteoarthritis and rheumatoid arthritis. AB - The concentrations of alpha 1-acid glycoprotein (alpha 1-antitrypsin (alpha 1 AT), ceruloplasmin (Cp) and alpha 2-macroglobulin (alpha 2-MG) in serum and in knee joint synovial fluid of patients suffering from rheumatoid arthritis (RA) and osteoarthritis (OA) were determined and apparent synovial permeability (SP) to each protein calculated. The results showed that the rheumatoid synovia were significantly more permeable (P < 0.001) than the osteoarthritic synovia. Cp and alpha 2-MG showed the greatest average increase in apparent SP, about five times the values for OA joints. Apparent SP reflected disease activity rather well, since the patients with the more active disease had the highest values, six times that of the OA values. Although the values for the small proteins alpha 1-AGP and alpha 1-AT were greater in RA joints, more intense inflammation resulted in a greater increase in apparent SP to larger proteins, so that the apparent SP for alpha 2-MG and Cp are more reliable for evaluating disease activity. Apparent SP as determined by this and previous studies appeared to be a much more accurate and sensitive measure than the synovial fluid/plasma protein concentration ratio. Knowledge of the apparent SP could be a useful parameter in evaluating synovitis since the exudative flare-ups usually parallel the intensity of the inflammation. PMID- 7543348 TI - A comparative study of tenidap, a cytokine-modulating anti-rheumatic drug, and diclofenac in rheumatoid arthritis: a 24-week analysis of a 1-year clinical trial. AB - Tenidap is a novel anti-rheumatic drug that combines cytokine modulation with cyclo-oxygenase inhibition. This 24-week, multicentre, double-blind, randomized study compared the clinical efficacy, biochemical effects and safety of tenidap 120 mg/day (once daily) with diclofenac 150 mg/day (50 mg t.i.d) in the treatment of 384 patients with active rheumatoid arthritis. After 24 weeks, improvement with tenidap was significantly greater than with diclofenac for all five primary efficacy parameters, two of the four secondary efficacy parameters and 11 of the 13 Arthritis Impact Measurement Scales assessments. The superior efficacy of tenidap was apparent after 4 weeks of treatment with further improvements observed by 24 weeks. The probability of discontinuation due to lack of efficacy was significantly greater in the diclofenac group. Tenidap but not diclofenac was associated with significant, rapid and sustained reductions in C-reactive protein and serum amyloid A levels and with a significant reduction in plasma interleukin 6. The nature and frequency of side-effects were similar in the two groups as was the discontinuation rate for treatment-related safety reasons. Tenidap was associated with an equal incidence of elevated transaminases, but a higher incidence of mild (> or = 500 mg/24 h < 1500 mg/24 h) non-progressive, proteinuria of proximal tubular origin compared with diclofenac. PMID- 7543349 TI - CD5 antigen expression in B cell chronic lymphocytic leukaemia. PMID- 7543351 TI - Development of an efficient serum-free semisolid culture system for the evaluation of hematopoietic progenitors. AB - Three different combinations of serum-free (SF) media proposed by Drouet et al., Ieki et al., and Wu et al. were tested to assess their ability to replace fetal calf serum (FCS) in a human hematopoietic progenitor semisolid culture system using human bone marrow and peripheral blood stem cells. This study confirmed that two (Drouet and Wu) of the three SF media tested were able to induce progenitor growth in the presence of either 5637-conditioned medium or a defined combination of growth factors: stem cell factor (SCF), interleukin-1 (IL-1), interleukin-3 (IL-3), and erythropoietin (EPO). The best results were obtained using the SF media described by Wu. The number of 5637-stimulated CFU-GM obtained with the Wu SF media was not different from that obtained with FCS-supplemented media when cultures were assessed on day 14 (79 +/- 14 versus 104 +/- 17), day 18 (64 +/- 11 versus 79 +/- 12), or day 21 (58 +/- 13 versus 62 +/- 12). Similar findings were obtained when the previously defined combination of growth factors was used to stimulate progenitor growth. Because the Wu medium was the most efficient SF medium to promote hematopoietic progenitor growth, we attempted to improve its efficacy by modifying the concentrations of the various components. A reduction in the concentration of bovine serum albumin (BSA) dramatically reduced the total number of clonogenic elements. Soybean lectin was not essential for colony proliferation; however, its presence had an favorable effect on the overall appearance of the colonies (greater number of cells per colony).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543350 TI - Photodynamic activity on human larynx epidermoid carcinoma cell line, HEp-2. PMID- 7543352 TI - Comparison of subcutaneous and intravenous administration of recombinant human granulocyte-macrophage colony-stimulating factor for peripheral blood stem cell mobilization. AB - In an effort to determine whether subcutaneous or continuous intravenous infusion administration of rhGM-CSF results in better hematopoietic progenitor mobilization, the findings of two sequential clinical trials were reviewed. Patients who had received prior chemotherapy for leukemia, lymphoma, multiple myeloma, breast cancer, or other solid tumors and were candidates for high-dose therapy received rhGM-CSF, 250 micrograms/m2/day, either as a continuous intravenous infusion (trial 1) or subcutaneously (trial 2) for stem cell mobilization. At least five apheresis collection procedures were performed to collect a target number of 6.5 x 10(8) mononuclear cells (MNC)/kg. For the 37 patients in trial 1, the collections contained a median of 7.99 x 10(8) MNC/L (range 6.42-21.36) and a median of 5.27 x 10(4) CFU-GM/kg (range 0.28-19.35). In trial 1, 25 patients were autografted with their cells and recovered 0.5 x 10(9) granulocytes/L at a median of 12 days (range 6-16). For the 33 patients in trial 2, the autograft product contained a median of 7.63 x 10(8) MNC/kg (range 6.51 22.66) and 6.31 x 10(4) CFU-GM/kg (range 0.06-60.4). In trial 2, 25 patients were autografted. The median time to reach 0.5 x 10(9) granulocytes/L was 11 days (range 9-26). All patients received rhGM-CSF after peripheral stem cell transplant. No significant differences in the collected products or the time to hematopoietic recovery was found between the two trials (p > 0.05). The mobilization effects of subcutaneous rhGM-CSF in these pretreated patients were similar to those of intravenous rhGM-CSF. PMID- 7543353 TI - Long-term developmental outcomes of low birth weight infants. AB - Advances in neonatal medicine have resulted in the increased survival of infants at lower and lower birth weight. While these medical success stories highlight the power of medical technology to save many of the tiniest infants at birth, serious questions remain about how these infants will develop and whether they will have normal, productive lives. Low birth weight children can be born at term or before term and have varying degrees of social and medical risk. Because low birth weight children are not a homogeneous group, they have a broad spectrum of growth, health, and developmental outcomes. While the vast majority of low birth weight children have normal outcomes, as a group they generally have higher rates of subnormal growth, illnesses, and neurodevelopmental problems. These problems increase as the child's birth weight decreases. With the exception of a small minority of low birth weight children with mental retardation and/or cerebral palsy, the developmental sequelae for most low birth weight infants include mild problems in cognition, attention, and neuromotor functioning. Long-term follow-up studies conducted on children born in the 1960s indicated that the adverse consequences of being born low birth weight were still apparent in adolescence. Adverse sociodemographic factors negatively affect developmental outcomes across the continuum of low birth weight and appear to have far greater effects on long term cognitive outcomes than most of the biological risk factors. In addition, the cognitive defects associated with social or environmental risks become more pronounced as the child ages. Enrichment programs for low birth weight children seem to be most effective for the moderately low birth weight child who comes from a lower socioeconomic group. Continued research and attempts to decrease the rate of low birth weight and associated perinatal medical sequelae are of primary importance. Ongoing documentation of the long-term outcome of low birth weight children needs to be mandated, as does the implementation of environmental enrichment programs to help ameliorate the long-term consequences for infants who are born low birth weight. PMID- 7543354 TI - Effects of nitric oxide (NO) synthesis inhibition on the development of supersensitivity to stereotypy and locomotion stimulating effects of methamphetamine. AB - The present study examined the effects of nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (LNAME; 30 and 60 mg/kg, i.p.) on the development of supersensitivity to stereotypy as well as locomotion stimulating effects of methamphetamine (MA) (3.22 and 0.805 mg free base/kg, s.c., respectively). Rats treated with MA for 10 days showed enhancement in MA-induced stereotypy and locomotor activity. Rats pretreated with LNAME prior to MA also showed enhancement in the two types of behavior, also they showed significantly reduced stereotypy scores compared to those treated with MA alone. The results suggest that NO synthesis is not critically involved in the development of behavioral supersensitivity to stereotypy stimulating as well as locomotion stimulating effect of MA. However, No synthesis may have a modulatory role in behavioral sensitization in stereotypy. PMID- 7543355 TI - Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction. AB - Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts, L-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL42.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543356 TI - Brain-derived neurotrophic factor (BDNF) prevents lesion-induced axonal die-back in young rat optic nerve. AB - Lesions of the optic nerve in young animals lead to rapid retrograde degeneration of the axon stumps and to death of retinal ganglion cells. We injected different neurotrophic factors into the eye at the time of an intracranial freeze-crush lesion of the optic nerve in 8 day old rats. Optic nerve axons were visualized by anterograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and by electron microscopy. The lesion induced a rapid die-back of the axons, which could be prevented by BDNF and to a lesser extent by neurotrophin-3 (NT-3) or ciliary neurotrophic factor (CNTF). No effect was seen in animals injected with nerve growth factor (NGF) or a mixture of acidic and basic fibroblast growth factor (FGF). In contrast to this effect on the axons, none of these factors was able to counteract the rapidly progressing degeneration of the retinal ganglion cells. These results suggest a selective influence of BDNF on the mechanisms responsible for the maintenance of optic nerve axons. PMID- 7543357 TI - Ionophoretically applied substance P activates hamster suprachiasmatic nucleus neurons. AB - ionophoretic ejection of substance P (SP) activated 31% and suppressed 9% of hamster suprachiasmatic nucleus (SCN) cells in vitro. Hamster SCN cells did not demonstrate variation in sensitivity to SP across the circadian phases tested. SP modulated the response of 47% of hamster SCN cells to the excitatory amino acid (EAA) agonists glutamate and N-methyl-D-aspartate (NMDA). The results indicate that SP can alter both the spontaneous and EAA-evoked firing rate characteristics of hamster SCN neurons. PMID- 7543358 TI - Urethral bleeding as a presenting sign of benign prostatic hyperplasia in the dog: a retrospective study (1979-1993). AB - Nineteen dogs were presented to the Murdoch University Veterinary Hospital (MUVH) because of blood loss from the urethra, unassociated with other clinical signs of prostatic disease. Blood loss was intermittent in all cases, but was severe enough to require blood transfusion in one case. Prostatic enlargement was detected on rectal palpation in all cases, and benign prostatic hyperplasia was diagnosed after elimination of other prostatic diseases (i.e., infection, cysts, neoplasia) using diagnostic imaging and clinical laboratory tests. Castration resulted in cessation of blood loss; whereas antiandrogen therapy, an alternative treatment to castration, was less effective. PMID- 7543360 TI - A common RNA loop motif as a docking module and its function in the hammerhead ribozyme. AB - Here I present a three-dimensional model of a novel element of RNA tertiary structure. A common loop motif composed of adjacent, sheared G.A and A.N non canonical base pairs is proposed to form long-range tertiary interactions with other RNA residues. The widespread distribution of this G.A/A.N docking module suggests that the putative long-range docking interaction plays an important role in specifying the tertiary structure of large RNAs, and perhaps the quaternary structure of some intermolecular RNA-RNA interactions. Application of this docking module hypothesis to the hammerhead ribozyme provides crucial constraints for the calculation of three-dimensional models of its self-cleaving conformation. PMID- 7543359 TI - Real and artificial histories. PMID- 7543361 TI - The fine art of pore formation. PMID- 7543362 TI - The domain structure of the ion channel-forming protein colicin Ia. AB - Colicin Ia undergoes a transition from a soluble to a transmembrane state, forming an ion channel to effect its bactericidal activity. The X-ray crystal structure of soluble colicin Ia at an effective resolution of 4 A reveals that the molecule is highly alpha-helical and has an unusually elongated 'Y'-shape. The stalk and two arms of the 'Y' form three discrete structural domains which most likely correspond to the three functional regions identified for the channel forming colicins. The channel-forming region of colicin Ia can be located to the larger of the two arms, the insertion domain, by its structural similarity to the ten alpha-helix motif found for the ion channel-forming fragments of colicins A and E1. The domain arrangement found in this structure provides novel insights into the mechanism of membrane insertion of colicin Ia. PMID- 7543363 TI - Enhanced formation of secondary dentin in the absence of nerve supply to feline teeth. AB - This investigation was designed to study the formation of secondary dentin in permanent teeth of young cats after denervation. In eight animals, cervical dentin was exposed bilaterally in the mandibular canines, 7-10 d after unilateral resection of the inferior alveolar nerve. The observation intervals were 30 d, 90 d and 180 d, after which histological examination of dentin was performed. In order to verify the loss and regeneration of pulpal innervation and an intact blood supply, blood flow responses to electrical stimulation of the tooth and to i.v. injections of substance P (SP) were recorded by laser Doppler flowmetry before and at 7-10 d after denervation, at the end of the experiments, and at predetermined intermediate intervals. SP-induced vasodilation was significantly enhanced at 1 wk and 30 d postoperatively and was normalized to control values at 90 and 180 d. Vasodilation in response to electrical tooth stimulation, which was absent after denervation, reappeared after 90 d in two of four cats. There was no irregular dentin formation under the exposed dentin at any time in denervated or control teeth. Formation of regular secondary dentin appeared to be enhanced on the denervated side at 30 d and 90 d postoperatively, whereas at 180 d there was no difference between sides. The results indicate that intradental nerves influence secondary dentin formation in feline permanent teeth. PMID- 7543366 TI - Anti-angiogenic activity of triterpene acids. AB - Ursolic acid (UA) and oleanolic acid (OA) were examined for anti-angiogenic activities by using the chick embryo chorioallantoic membrane (CAM) assay. The presence of UA or OA inhibited angiogenesis in a dose-dependent manner; the doses required for half-maximal inhibition (ID50) were 5 micrograms and 40 micrograms per CAM, respectively. UA was a more potent angiogenic inhibitor than OA. We also tested for inhibitory effect on the proliferation of bovine aortic endothelial cell. They effectively inhibited the proliferation of bovine aortic endothelial cell in a concentration-dependent manner. The IC50 values of anti-proliferative effects were determined to be 5 microM for UA and 20 microM for OA. Based on these results, we speculated that the inhibitory effects on bovine aortic endothelial cell proliferation of UA and OA might be important for anti angiogenesis. PMID- 7543364 TI - Bisbenzimide: a fluorescent counterstain for tissue autoradiography. AB - Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus, bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies. PMID- 7543365 TI - Projections of neurochemically specified neurons in the porcine colon. AB - The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5 HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics. PMID- 7543367 TI - Presence of iron catalytic for free radical reactions in patients undergoing chemotherapy: implications for therapeutic management. AB - We investigated the kinetics of generation of iron 'catalytic' for free radical reactions in children with diagnosed acute lymphoblastic leukaemia (ALL) who received high-dose methotrexate infusions. In 76% of the chemotherapy courses studied, 'catalytic' iron appeared in plasma in the concentration range from 0.1 to 3 mumol/l. Positive correlations between maximum levels of 'catalytic' iron and plasma hepatic enzymes could be established in the majority of cases and in one subset of patients (low and medium risk ALL) mean 'catalytic' iron levels correlated well to clinically observable toxicities. The damaging potential of 'catalytic' iron was also demonstrated experimentally: oxidative damage to proteins was significantly (P < 0.05) higher in plasma samples showing the presence of 'catalytic' iron and in addition a strong correlation (r = 0.95, P < 0.02) was seen between plasma concentration of 'catalytic' iron and the ability of the plasma to stimulate lipid peroxidation. Our data show that chemotherapy releases 'catalytic' iron which may relate to toxic side effects. Hence binding this 'catalytic' iron by judicious co-administration of iron chelating agents could be beneficial in minimizing the iatrogenic adverse effects of chemotherapy of acute leukaemia. PMID- 7543368 TI - The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. AB - HIV-1 Rev protein directs nuclear export of pre-mRNAs and mRNAs containing its binding site, the Rev response element (RRE). To define how Rev acts, we used conjugates between bovine serum albumin (BSA) and peptides comprising the Rev activation domain (BSA-R). BSA-R inhibited Rev-mediated nuclear RNA export, whereas a mutant activation domain peptide conjugate did not. BSA-R did not affect the export of mRNA, tRNA, or ribosomal subunits, but did inhibit export of 5S rRNA and spliceosomal U snRNAs. BSA-R was itself exported from the nucleus in an active, saturable manner. Thus, the Rev activation domain constitutes a nuclear export signal that redirects RRE-containing viral RNAs to a non-mRNA export pathway. PMID- 7543370 TI - Tiazofurin induces a down-modulation of ICAM-1 expression on K562 target cells impairing NK adhesion and killing. AB - Tiazofurin treatment of K562 leukemia cells in vitro depletes the metabolites of the guanylate biosynthetic pathway, inducing erythroid differentiation, that, in turn, alters the phenotypic profile. As a consequence, K562 cells possibly modify their interaction with immune cells. Here we describe the binding and killing activity of peripheral blood NK cells against differentiating K562 cells and the correlation between their altered binding capacity and ICAM-1 expression levels in differentiating K562 cells. We found that decreased percentages of NK (and T) cells were bound to differentiating K562 cells generating a decreased cytotoxic activity. This corresponded to decreased expression of ICAM-1, as detected by FACS analysis and Western blot. Erythroid differentiation, binding and killing reduction, and ICAM-1 down-modulation were completely abrogated by guanosine treatment. Tiazofurin causes a decrease in lymphocyte recognition and binding to K562 target cells. This can be ascribed to the down-modulation of ICAM-1 expression on target cells, which, therefore, can escape killing, acquiring a selective survival advantage. PMID- 7543372 TI - In vitro effects of cocaine on cytokine secretion induced in murine splenic CD4+ T cells by antigen-specific stimulation. AB - The in vitro effects of cocaine on antigen-specific-induced cytokine production by murine splenocytes was evaluated both by quantitation by ELISA of the cytokines in culture supernatants and by flow cytometric analysis of the frequency of the cytokine-producing CD4+ T cells. Spleen cells from mice immunized with ovalbumin (OVA) were restimulated with OVA in the presence or absence of cocaine for different periods of time and then evaluated for production of cytokines. Exposure to cocaine was found to reduce the levels in culture supernatants of IL2 and IFN-gamma, whereas IL4 and IL5 levels were not changed. Flow cytometric analysis showed that cocaine increased the frequency of IL2- but not of IL4-producing CD4+ T cells. Kinetics studies indicated that the in vitro antigen-specific-induced production of IL2 is faster than that of IL4 and that cocaine did not affect the production kinetics of either cytokine. Collectively, the results suggest that in vitro cocaine acts by interfering with the secretion rather than with the synthesis of cytokines and that the drug exerts different effects on cytokines with different production kinetics. PMID- 7543371 TI - Effects of cyclosporin A, rapamycin, and FK520 on peripheral T-cell deletion and anergy. AB - In this study we compare the effects of cyclosporin A (CsA), FK520 (an agent similar to FK506), and rapamycin (RAPA) on peripheral T-cell deletion induced by either superantigens or anti-TCR alpha beta mAb, and on anergy induced by superantigens in mice. CsA enhanced T-cell deletion and blocked anergy induction (in residual T cells), while FK520 and RAPA had no effects on these processes. CsA also enhanced apoptosis of stimulated T cells in vitro, where cell death occurred without prior proliferation and in the absence of phagocytes. Our data suggest that CsA exerts these effects through a calcineurin-independent pathway, and this may be relevant to the development of tolerance in some models. PMID- 7543369 TI - X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. AB - The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12 FK506. PMID- 7543373 TI - Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity. AB - We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages. PMID- 7543374 TI - Minor histocompatibility antigen-dependent rejection of Pam 212 epidermoid carcinoma by DBA/2 mice. AB - Since Pam 212 cells express low levels of class I major histocompatibility (MHC) antigens, we tested their ability to present alloantigens or minor histocompatibility (mH)/minor lymphocyte stimulatory (mls) antigens in disparate hosts. After subcutaneous injection, Pam 212 cells grew progressive tumors in normal BALB/c mice but were rejected rapidly by naive C3H mice (3 weeks) and slowly by DBA/2 mice (8 weeks). Pam 212 cells (high or low class I MHC expression) induced a strong primary MLR in DBA/2 T cells, but a weak BALB/c T cell response. In contrast, splenic APC (BALB/c) did not induce an MLR, suggesting that Pam 212 cells represented mH antigens to naive DBA/2 T cells. This MLR was blocked by anti-TCR alpha/beta, anti-class II, and anti-CD4 monoclonal antibodies, but was independent of ICAM-1 and B7. Repeated immunization using IFN-gamma-treated Pam 212 cells induced anti-Pam 212 CTL in DBA/2 mice but not in BALB/c mice. DBA/2 T-cell responses did not appear to be mls (MMTV superantigen)-specific, because Pam 212 cells did not express MMTV mRNA detectable by RT-PCR. Pam 212 cells presented non-lymphoid-associated mH antigens that served as potent stimuli for tumor rejection in mH/mls-disparate hosts, which is similar to tumor rejection mediated by MHC alloantigens. PMID- 7543376 TI - Classification of mutations at the human hprt-locus in T-lymphocytes of bus maintenance workers by multiplex-PCR and reverse transcriptase-PCR analysis. AB - Polymerase chain reaction (PCR)-based screening methods were used to classify mutations arising in vivo at the hypoxanthine guanine phosphoribosyl-transferase (hprt) locus in small samples of human T-lymphocyte clones (< 5 x 10(4) cells) from 29 bus maintenance workers exposed to diesel exhaust, and 14 control individuals. All subjects were healthy, non-smoking males. Among 462 T-cell mutants studied by multiplex-PCR of genomic DNA, only 12 (2.6%) deletions were found: three total deletions, five partial exon deletions and four mutants with one or two exons deleted. Point mutations were classified in 323 mutants using reverse transcriptase-PCR amplification: 74 (22.9%) of these had splice site mutations and 241 (74.6%) had coding errors. Splice mutation was more frequent among the garage workers (24.8%) as compared to the controls (19.5%), possibly reflecting a polycyclic aromatic hydrocarbon-specific mutation induction in these workers. Our results also show that both gene deletion and splice mutation at the hprt-locus in T-cells of healthy non-smokers could be less frequent than previously reported. PMID- 7543377 TI - Relationship between hprt mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers. AB - We have studied the mutant frequency in the human gene for hypoxanthine-guanine phosphoribosyl transferase (hprt) using the T-cell cloning assay, the aromatic DNA adduct level using the 32P-postlabelling assay, and related the levels of these biomarkers to the genotypes for glutathione transferase (GST mu) and N acetyltransferase (NAT2) in non-smoking bus maintenance workers exposed to diesel exhaust. No difference in mutant frequency was observed between the 47 exposed (8.6 x 10(-6), age range 27-65) and the 22 control individuals (8.4 x 10(-6), age range 23-61), while the difference in adduct level (3.2 versus 2.3 x 10(-8)) was highly significant (P = 0.0009). Both mutant frequency and adduct level were highest in the 16 most heavily exposed workers. Overall, a significant increase of mutant frequency was observed with adduct level (P = 0.008) as well as with age (P < 0.0001). The age dependence was higher in the GSTM1-negative slow acetylators (3.1%/year) as compared to the three other genotype combinations (2.4 2.5%/year). There was no significant difference in mutant frequency or in adduct level between the GSTM1-negative (49.3% of the population) and positive individuals, or between the slow (60.9% of the population) and rapid acetylators. Among the slow acetylators, however, a significantly higher adduct level (P = 0.03) was obtained for the GSTM1-negative individuals as compared to the GSTM1 positive individuals. These results suggest a possible role of both GST mu and NAT2 for individual susceptibility to carcinogen exposure. PMID- 7543375 TI - The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism. AB - The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis. PMID- 7543379 TI - Vascular cell adhesion molecule-1 is induced on vascular endothelia and medial smooth muscle cells in experimental cardiac allograft vasculopathy. AB - BACKGROUND: Cardiac allograft vasculopathy (CAV) is the major cause of late death among heart transplant recipients. The pathogenesis of CAV is poorly understood. METHODS AND RESULTS: To better characterize CAV, we performed immunohistochemical analysis of vascular lesions in a previously described murine model of CAV. The B10.A strain hearts were transplanted heterotopically into B10.BR strain recipients. The cardiac allografts were harvested from 1 to 2 months after implantation. The majority of epicardial and intramyocardial coronary arteries in explanted hearts had developed intimal thickening. The cellular infiltrate of the intimal thickening, major histocompatibility (MHC) antigens, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression were studied with the use of immunohistochemistry. In experimental CAV in mice, the cellular infiltrate of expanded intima consisted of macrophages, T lymphocytes, and smooth muscle cells. A substantial number of macrophages and T lymphocytes within the expanded intima expressed MHC class II antigen, a marker of cellular activation. The vessel wall cells also appeared to be activated due to their expression of endothelium-leukocyte adhesion molecules. The vascular endothelium of cardiac allografts displayed ICAM-1, VCAM-1, and unmatched MHC antigen (MHC class I in this model) upregulation. The medial smooth muscle cells also expressed VCAM-1 and unmatched MHC antigen. CONCLUSIONS: These findings suggest that (1) the cellular infiltrate of the expanded intima in experimental CAV is similar to that of human CAV, (2) experimental CAV is a local immune mediated process requiring active participation of donor vessel wall cells and recipient mononuclear cells, and (3) coexpression of adhesion molecules and unmatched MHC antigen identifies endothelial cells as immune targets for activated host mononuclear cells. Furthermore, the presence of both VCAM-1 and unmatched MHC antigen supports a central role for medial smooth muscle cells as allogeneic immune stimulator. PMID- 7543378 TI - Synergistic increase in chromosomal breakage within the euchromatin induced by an interaction of the benzene metabolites phenol and hydroquinone in mice. AB - The hematopoietic and carcinogenic effects of benzene may result from an interaction of various benzene metabolites. Following the co-administration of phenol and hydroquinone, a synergistic increase in myelotoxicity and genotoxicity has been observed in the bone marrow of mice. To understand the mechanisms underlying these synergistic genotoxic effects we have studied the origin of micronuclei (MN) formed in bone marrow erythrocytes following the co administration of these two metabolites. Phenol and hydroquinone were administered to male CD-1 mice by i.p. injection three times at 24 h intervals. The frequency of MN was evaluated in bone marrow cells harvested 24 h following the final dose. A marked increase in MN was observed in mice co-administered phenol and hydroquinone, which was significantly greater than that observed with the individual metabolites. Labeling with the CREST antibody and multicolor fluorescence in situ hybridization with the mouse major and minor satellite probes indicated that both chromosomal loss and breakage and occurred. The major increase in MN induced by the phenol and hydroquinone combination originated from breakage in the euchromatic region of the mouse chromosomes. The origin of MN in mice co-administered phenol and hydroquinone differed substantially from that induced by hydroquinone alone, but was almost identical to that seen in MN from benzene-treated mice. These results strongly support the hypothesis that interactive effects among benzene metabolites play an important role in the genotoxic and carcinogenic effects of benzene. PMID- 7543380 TI - Upregulation and modulation of inducible nitric oxide synthase in rat cardiac allografts with chronic rejection and transplant arteriosclerosis. AB - BACKGROUND: The Lewis-F344 rat cardiac transplantation model produces cardiac allografts with chronic rejection characterized by arteriosclerotic lesions composed of macrophages and smooth muscle cells. Modulation of the inflammatory response with a diet deficient in essential fatty acids protects against the development of intimal thickening. Little is known about the components of the inflammatory response mediating this process. The cytokine-inducible isoform of nitric oxide synthase (iNOS) regulates the high-output nitric oxide pathway that confers activation properties to macrophages and regulates vasomotion, monocyte adherence, and smooth muscle cell proliferation in the vasculature. The purpose of the present study was to determine whether the iNOS pathway was upregulated during the course of chronic cardiac rejection. METHODS AND RESULTS: We studied iNOS mRNA and protein expression patterns in a series of Lewis-F344 cardiac allografts with early and late chronic rejection and after modulation of the inflammatory response (in an effort to attenuate arteriosclerosis). Relative gene transcript levels were measured with a 32P-dCTP reverse-transcriptase polymerase chain reaction assay designed to amplify iNOS mRNA. The distribution of the iNOS gene product was examined by immunocytochemistry with a polyclonal antibody against iNOS. NOS transcript levels increased significantly in cardiac allografts (days 7, 14, 28, and 75) compared with paired host hearts (exposed to the same circulation) and syngrafts (P < .003). Immunostaining localized the iNOS antigen within subpopulations of mononuclear inflammatory cells in cardiac allografts- presumably, activated macrophages. The number of iNOS-positive mononuclear cells was 25-fold higher in cardiac allografts compared with paired host hearts and syngrafts (P < .009). In cardiac allografts of 75 days or older, there also was striking iNOS staining within some medial and intimal smooth muscle cells in various vessels. Modulation of the inflammatory response (with a diet deficient in essential fatty acids) produced significant decreases in the intimal thickening score and in the percentage of diseased vessels in 28-day cardiac allografts compared with allografts from rats fed a control diet. There was a correlate decrease in iNOS transcript levels and in the number of iNOS-positive mononuclear cells in the 28-day cardiac allografts from rats fed the essential fatty acid-deficient diet. CONCLUSIONS: The early and persistent upregulation of iNOS in chronic cardiac rejection and the coincident reduction in arteriosclerosis and downregulation of iNOS suggest that this inducible regulator may contribute to the inflammatory response mediating transplant arteriosclerosis. PMID- 7543381 TI - Adjunctive selectin blockade successfully reduces infarct size beyond thrombolysis in the electrolytic canine coronary artery model. AB - BACKGROUND: An adjunctive pharmacological strategy to thrombolytic therapy that is tailored to limit reperfusion injury after thrombolysis could further maximize the unquestioned benefit of restoring flow to ischemic myocardium. Ischemia reperfusion injury exhibits features characteristic of an acute inflammatory response, including the rapid activation and infiltration of neutrophils. The initial process of neutrophil migration from the circulation to injured tissue is modulated by a group of adhesion molecules called selectins. The purpose of the present study was to assess the efficacy of a selectin blocker (CY 1503) given as an adjunct to thrombolytic therapy to interfere with the inflammatory response after ischemia-reperfusion and subsequently reduce myocardial infarct size in the electrolytic canine model. METHODS AND RESULTS: A fully occlusive thrombus was formed in the left circumflex coronary artery by electrolytic injury in 20 anesthetized open-chest dogs. After occlusion, an infusion of 1 mg/kg recombinant tissue-type plasminogen activator (rTPA) was administered over 20 minutes with either a bolus of placebo or the selectin blocker CY 1503 (40 mg/kg). At the onset of reperfusion, 20 micrograms/kg per minute rTPA was administered for 1 hour to prevent reocclusion. After 1 hour of reperfusion, infarct size, myocardial myeloperoxidase activity, and reperfusion arrhythmias were measured. In CY 1503-treated dogs, there was a significant 69% reduction in infarct size when expressed as a percentage of the area at risk (6.7 +/- 8.4% versus 21.8 +/- 13.6%; P = .008) and a marked reduction in myeloperoxidase activity (0.014 +/- 0.009 versus 0.0370 +/- 0.025 U/min per gram; P = .02) compared with the placebo group. There was no difference between the groups in the occurrence of reperfusion arrhythmias. CONCLUSIONS: Selectin blockade as an adjunct to rTPA mediated thrombolysis significantly reduces infarct size and myocardial neutrophil infiltration well beyond thrombolysis alone in the electrolytic canine model. These data suggest that selectin blockade is extremely effective at reducing ischemia-reperfusion injury and myocardial infarct size in this model and that the neutrophil is a potent mediator of ischemia-reperfusion injury. PMID- 7543382 TI - Longitudinal gradients for endothelium-dependent and -independent vascular responses in the coronary microcirculation. AB - BACKGROUND: Coronary microvessels (< 300 microns in diameter) have been demonstrated to be important in the regulation of local resistance and flow. Recent studies also suggest that these microvessels are more responsive to physiological and pharmacological stimuli than conduit vessels. However, little is known regarding the relative sensitivity of different microvascular segments in response to flow (shear stress) and agonists. The goal of this study was to test the hypothesis that a longitudinal gradient for shear stress- and agonist induced dilation exists in the coronary microcirculation. METHODS AND RESULTS: Experiments were performed in four different sizes of porcine subepicardial coronary arterial microvessels: small arterioles (40 +/- 1-micron ID with resting tone); intermediate arterioles (60 +/- 1 micron); large arterioles (106 +/- 4 micron); and small arteries (179 +/- 9 microns). Vessels were isolated and cannulated to allow luminal pressure and flow to be independently controlled. All vessels developed active tone (to approximately 65% to 75% of maximum diameter) at their control luminal pressures and showed graded dilations to stepwise increases in shear stress (0 to 10 dynes/cm2). For arterioles, the magnitude of the dilations increased as vessel size increased. The highest shear stress produced 21 +/- 3%, 32 +/- 2%, and 52 +/- 5% increases in diameter in small, intermediate, and large arterioles, respectively. Small arteries dilated only 22 +/- 6%. The endothelium-dependent vasodilator substance P (SP) produced dose dependent dilation of all vessels with a threshold at 10(-16) mol/L. Arterioles were maximally dilated at 10(-9) mol/L SP. However, this dose produced only 80% dilation in small arteries. The ED50 for SP was shifted to the right by two orders of magnitude in small arteries compared with the arterioles. Adenosine preferentially dilated small arterioles, and the dose-response curves shifted to the right for larger vessels. The thresholds for adenosine-induced dilation were 10(-12), 10(-11), and 10(-9) mol/L for small, intermediate, and large arterioles, respectively. The endothelium-independent vasodilator nitroprusside produced identical dose-dependent dilations in all vessel segments. CONCLUSIONS: The results indicate that the pig coronary circulation exhibits a heterogeneity in physiological and pharmacological responses along the microvascular network. Small arterioles are more sensitive to adenosine, but large arterioles are more responsive to shear-stress stimulation. We speculate that site-specific preferential responses may play a crucial role in coordinating overall vascular function in the coronary microvascular network. PMID- 7543383 TI - Preferential salivary-type hypoamylasemia in obesity. PMID- 7543384 TI - Alpha-fetoprotein and the acute phase response. A study using acute pelvic inflammatory disease as a model system. AB - Alpha-fetoprotein (AFP) is used as a tumor marker for hepatomas and germ cell tumors. In healthy non-pregnant adults the serum concentration (S-AFP) is very low and we examined whether it was affected when the acute phase response was activated, using patients with acute pelvic inflammatory disease (PID) as a model system. In 70 PID patients the median S-AFP was 1.2 kIU/l (range: 0.5-5.9 kIU/l), within the normal range. S-AFP did not correlate with the clinical grade of disease nor with the serum concentrations of acute phase reactants and albumin. Thus, S-AFP is not significantly affected by activation of the acute phase response and presence of infection should not per se interfere with the use of S AFP as a tumor marker. However, a negative correlation was found with the serum concentration of alpha-2-macroglobulin (P = 0.05), but whether this has any biological significance remains to be clarified. PMID- 7543385 TI - Skipping of exon 12 as a consequence of a point mutation (1898 + 5G-->T) in the cystic fibrosis transmembrane conductance regulator gene found in a consanguineous Chinese family. AB - A point mutation (1898 + 5G-->T) located five base pairs downstream from the donor splice site in intron 12 of the CFTR gene has been identified in a consanguineous CF patient of Chinese origin. To determine if this nucleotide substitution could affect mRNA splicing, PCR analysis was performed with RNA isolated from the lymphoblastoid cell line of the mother of the deceased patient. While exon 12-minus transcript was detected in this sample, it was also found in individuals without 1898 + 5G-->T, albeit in a smaller proportion. Using a sequence polymorphism associated with each of the two alleles in the mother, however, we showed that mutant transcript was almost exclusively produced by the 1898 + 5G-->T allele. Skipping of exon 12 would result in the deletion of 29 amino acids from the first nucleotide binding domain of CFTR, rendering the protein non-functional. The possibility of a low level (< or = 2.5%) of normal transcript from the mutant allele cannot be excluded and it may explain the pancreatic sufficient phenotype of the patient. The 1898 + 5G-->T mutation was found in two other CF patients of Chinese origin, but it was not detected in 192 CF chromosomes of Caucasian origin and 30 other chromosomes from Chinese individuals without a family history of CF. PMID- 7543387 TI - Detection of circulating adhesion molecules ICAM-1, VCAM-1 and E-selectin in Wegener's granulomatosis, systemic lupus erythematosus and chronic renal failure. AB - The adhesion molecules ICAM-1, VCAM-1 and E-Selectin are regulated by proinflammatory cytokines and play an important role in the binding and activation of leukocytes in inflammatory diseases. This study measured the serum concentrations of circulating adhesion molecules (cICAM-1, cVCAM-1, cE-Selectin) by sandwich ELISA in systemic vasculitis with renal involvement (Wegener's granulomatosis WG, n = 25; systemic lupus erythematosus SLE, n = 50) in comparison to chronic glomerulonephritis (n = 10), stable renal allograft function and end-stage renal disease (CAPD/hemodialysis, each n = 10). Both cICAM 1 and cVCAM-1 levels, but not cE-Selectin, were significantly increased (p < 0.001) in active WG compared to healthy controls. cICAM-1, not cVCAM-1 differed significantly between active and inactive WG. Only cVCAM-1 was significantly elevated in active/inactive SLE (p < 0.01). WG with rapidly progressive glomerulonephritis had significantly raised levels of cICAM-1 and cVCAM-1, but not cE-Selectin, compared to controls (p < 0.005). In lupus nephritis only cVCAM 1 was significantly elevated (p < 0.01). cICAM-1 and cVCAM-1 levels were significantly raised in WG patients, that were hemodialysed and in patients with hemodialysis because of different reasons when compared to controls. However, cVCAM-1 but not cICAM-1 was significantly higher in WG with HD than without HD. Patients with chronic glomerulonephritis, renal allografts or CAPD also had significantly raised cVCAM-1 concentrations. These data suggest, that levels of circulating adhesion molecules might reflect different pathophysiologic processes in systemic vasculitides and endothelial/immune activation in non-inflammatory renal diseases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543386 TI - Dinucleotide repeat polymorphism at the human CD59 locus. PMID- 7543390 TI - Radiotherapy in the management of bone pain. AB - Radiotherapy may be indicated for the management of bone metastases because of associated pain, fracture, or neurologic complications. For metastatic bone pain, simple low-dose radiation treatment is usually effective for local problems. When there are multiple sites of bone pain, external beam irradiation using wide-field hemibody techniques is highly effective. An alternative to this approach is the administration of radioisotopes that may localize to the sites of bone metastases, either because they are tumor specific (radioiodine for thyroid cancer) or bone seeking (radioactive phosphorus [32P] and strontium [89Sr]). The primary treatment of pathologic fracture is surgery where possible, but radiotherapy has a major role in postoperative treatment and in treatment of fractures that are inoperable either because of their site, such as a rib or pelvis, or because of the general poor condition of the patient. For neurologic complications such as spinal cord compression or nerve root compression, radiotherapy appears to be as beneficial as decompressive surgery in most situations, except where there is bony instability. The role of radiotherapy in the prophylactic setting is discussed. Prevention of pathologic fracture and spinal cord compression may be possible in high-risk patients. PMID- 7543389 TI - Cancer metastasis and adhesion molecules. AB - Malignant tumor cells express various cell adhesion molecules. Experimental evidence indicates that dysregulation of adhesion mechanisms plays an important role in tumor metastasis. The results of clinical studies corroborate this notion, and altered expression of cell adhesion molecules has been related to the prognosis or biologic behavior of human primary tumors and their metastases. The molecular events underlying the spread of malignant tumor cells to distant sites from the view point of cell adhesion are reviewed in this article. PMID- 7543391 TI - Management of pain in skeletal metastases. AB - Bone metastases can cause pain in several ways, including bone destruction, compression of nerve roots and spinal cord, and reactive muscle spasm. Pain management is correspondingly diverse, including primary and secondary analgesics, physical and psychologic nondrug methods, and modification of daily activities. Careful evaluation of the neuropathologic mechanisms underlying the patient's pain is the first step, followed by an explanation to the patient. Palliative radiation therapy is generally the preferred treatment, in addition to drug therapy with a combination of a nonsteroidal antiinflammatory drug and an opioid used in accordance with the World Health Organization Method for Relief of Cancer Pain. Alternative strategies are needed for neuropathic and functional muscle pains that are opioid resistant. Pain management is only 1 part of palliative care that also addresses psychologic, social, and spiritual aspects of suffering. PMID- 7543388 TI - Mesangiolysis associated with severe glomerular endocapillary proliferation of CD57 large granular lymphocytes. AB - A 54-year-old man developed renal failure, with renal biopsy findings of diffuse mesangiolysis with severe endocapillary proliferation. Immunohistochemical studies revealed that CD3-CD56-CD57+ large granular lymphocytes were present predominantly within glomerular tufts. Intercellular adhesion molecule-1 was more preferentially expressed in the glomerular endothelial cells with severe endocapillary proliferation as compared to those without endocapillary proliferation. These findings suggest that CD57+ large granular lymphocytes caused glomerular endothelial injury by a cell-mediated cytolytic mechanism, resulting in the development of mesangiolysis and microaneurysm formation. PMID- 7543393 TI - The systemic treatment of bone metastases. AB - Bone metastases are a major source of morbidity in patients with metastatic cancer. There are effective systemic treatments available for patients with breast and prostate cancers, who are the majority of patients with bone metastases. Unfortunately, < 50% of patients benefit from currently available endocrine or cytotoxic chemotherapy, although problems of response assessment often lead to under reporting of true response rates. A better understanding of the mechanisms underlying the development of osteolytic bone metastases and the crucial role of increased osteoclastic activation provided the rationale for the use of the bisphosphonates. They reduce hypercalcemic episodes, pain, and pathologic fractures, and can induce radiologic healing of skeletal metastases. The development of potent, easily tolerated oral preparations will lead to their increased use by patients with osteolytic bone metastases. Radioisotopes used either alone or with external beam radiotherapy are effective in palliating the pain of skeletal metastases in patients with breast and prostate cancer and result in little associated subjective toxicity. Most studies have lacked placebo treated controls, and the results of multicenter studies evaluating the benefits of radioisotopes with respect to pain relief and quality of life are awaited. These treatments combined with local treatments such as radiotherapy, surgery, and analgesia can provide effective palliation of symptoms for the majority of patients. PMID- 7543394 TI - Differential nitric oxide synthase activity in human platelets during normal pregnancy and pre-eclampsia. AB - 1. Nitric oxide released from endothelial cells is a potent vasodilator that might play an important role in cardiovascular regulation during pregnancy. Platelets, like endothelial cells, contain a constitutive form of nitric oxide synthase. 2. The present study aimed to measure the activity of this nitric oxide forming enzyme in normotensive pregnant and non-pregnant women, as well as in women who had developed pre-eclampsia. Nitric oxide synthase activity was measured in the platelets of 21 normotensive pregnant women, 16 non-pregnant women and seven pregnant women who had developed pre-eclampsia. 3. The nitric oxide synthase activity was significantly higher in normotensive pregnant women [36.8 +/- 2.7 pmol h-1 mg-1 of protein (mean +/- SEM), P < 0.001] than in non pregnant control subjects (16.8 +/- 1.4 pmol h-1 mg-1 of protein) and in women with pre-eclampsia (24.5 +/- 2.1 pmol h-1 mg-1 of protein, P < 0.01). 4. These data suggest that nitric oxide synthesis is increased during normal pregnancy, possibly contributing to the vasodilatation associated with this condition. Nitric oxide generation, however, may be inappropriately low in pregnant women developing pre-eclampsia, thus leading to an enhanced vasoconstriction. PMID- 7543395 TI - Endothelium-dependent relaxation of small arteries from essential hypertensive patients: mechanisms and comparison with normotensive subjects and with responses of vessels from spontaneously hypertensive rats. AB - 1. Impaired endothelium-dependent relaxation has been previously demonstrated in blood vessels of hypertensive rats and in humans with essential hypertension. Arteries from spontaneously hypertensive rats have been shown to produce, in response to high concentrations of acetylcholine, a vasoconstrictor substance called endothelium-derived contracting factor, the production of which can be inhibited by indomethacin or other cyclo-oxygenase inhibitors, suggesting that it is a prostanoid. The mechanisms involved in endothelium-dependent relaxation of human arteries are unclear, and the potential generation of endothelium-derived contracting factor by endothelium in human hypertension has not been established. 2. We investigated the effects of acetylcholine on precontracted small arteries dissected from gluteal subcutaneous fat biopsies from normotensive subjects and subjects with borderline and mild essential hypertension. Vessels from normotensive subjects and those from borderline hypertensive patients, precontracted by noradrenaline, were relaxed completely by acetylcholine, whereas those from patients with mild essential hypertension relaxed slightly but significantly less, indicating that generation of endothelium-derived relaxing factor (endothelium-derived nitric oxide) was only minimally reduced or that production of minor amounts of endothelium-derived contracting factor occurred in small arteries from these hypertensive subjects. This impairment of endothelium dependent relaxation was not corrected by indomethacin, which indicated that the contribution of endothelium-derived contracting factor, if any, was minimal in this subset of essential hypertensive patients. In contrast, mesenteric small arteries of adult spontaneously hypertensive rats presented strong contractions in response to the higher concentrations of acetylcholine, which were abolished by exposure to indomethacin. 3. The relaxation induced by acetylcholine in arteries from both hypertensive and normotensive humans was partially blunted (by 30%) by pretreatment with 0.1 mmol/l NG-nitro-L-arginine methyl ester or NG-nitro monomethyl-L-arginine (inhibitors of nitric oxide synthase) and by 10 mumol/l Methylene Blue (a blocker of soluble guanylate cyclase), indicating the role of endothelium-derived nitric oxide and the generation of its intracellular second messenger cyclic guanosine monophosphate in acetylcholine-induced relaxation. The remaining relaxation elicited by acetylcholine could be blocked with 30 mmol/l KCl or with 10 mumol/l ouabain (inhibitor of Na+, K(+)-ATPase), and, when combined with NG-nitro-L-arginine methyl ester, these interventions abolished acetylcholine-induced relaxation. Tolbutamide at 2 mmol/l or 10 mumol/l glyburide (blockers of ATP-sensitive potassium channels) partially inhibited NG-nitro-L arginine methyl ester-resistant endothelium-dependent relaxation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543396 TI - Effect of blockade of nitric oxide synthesis on the renin secretory response to frusemide in conscious rabbits. AB - 1. The enzyme nitric oxide synthase is present in the macula densa and may participate in the control of renin secretion by the adjacent juxtaglomerular cells. In the present study, we investigated the effect of inhibiting nitric oxide synthase on the renin secretory response to frusemide, which stimulates renin secretion by blocking Na(+)-K(+)-2Cl- co-transport in the macula densa. 2. Injection of frusemide in 12 conscious rabbits elicited a transient increase in mean arterial pressure from 84 +/- 2 to 92 +/- 3 mmHg at 5 min (P < 0.01) and a sustained increase in heart rate from 246 +/- 6 to 281 +/- 10 beats/min at 45 min (P < 0.01). Plasma renin activity increased from 8.0 +/- 1.2 to 14.3 +/- 1.8, 12.4 +/- 1.6 and 11.6 +/- 1.5 pmol 2 h-1 ml-1 at 15, 30 and 45 min respectively (P < 0.01). There were no changes in plasma sodium and potassium concentrations or osmolality. 3. Inhibition of nitric oxide synthase with NG-nitro-L-arginine methyl ester increased mean arterial pressure by 9 mmHg, decreased heart rate and plasma renin activity, and markedly suppressed the renin response to frusemide (from 4.6 +/- 0.7 to 7.6 +/- 1.7, 4.7 +/- 1.0 and 4.6 +/- 0.7 pmol 2 h-1 ml-1 at 15, 30 and 45 min respectively). By contrast, infusion of an equipressor dose of phenylephrine did not suppress the renin response to frusemide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543397 TI - Inducible nitric oxide synthase: regulation subserves function. PMID- 7543399 TI - The role of nitric oxide in the pathogenesis of virus-induced encephalopathies. PMID- 7543392 TI - Nitric oxide and its role in orthopaedic disease. AB - Nitric oxide (.NO) is synthesized by the enzyme nitric oxide synthase (NOS). There are 2 constitutive forms of NOS (cNOS) and 1 inducible form (iNOS). Cells containing cNOS rapidly and transiently produce small amounts of NO in response to agonists that raise cytosolic levels of free Ca2+, whereas cells expressing inducible iNOS produce large amounts of .NO for extended periods after a lag of several hours during which time the enzyme is induced. Until recently, the 2 constitutive isoforms of NOS were thought to be confined to endothelial cells (eNOS) and brain (bNOS or nNOS). However, eNOS and bNOS have been identified in an increasing variety of additional cells. Many, if not most, types of cells are capable of expressing iNOS in response to cytokines, endotoxin, and phagocytosis. Regulation of iNOS occurs at transcriptional, translational, and posttranslational levels. Because .NO is rapidly diffusible and soluble in hydrophobic and aqueous environments, it is well suited to its role as an intercellular messenger with the unique ability to penetrate solid tissue. However, it is rapidly inactivated by hemoglobin. The biochemistry of .NO is dominated by its rapid reaction with oxygen and transitional metals, notably iron. The former reaction may be protective, as when neutralizing superoxide (.O2 ), or harmful in forming additional highly damaging radicals such as peroxynitrite. Interaction of .NO with iron-containing proteins has a number of sequelae, including the activation of guanylate cyclase, inhibition of mitochondrial respiration, and inhibition of cell division. Nitric oxide has been implicated in a number of conditions of orthopaedic interest, including inflammation, arthritis, osteoporosis, sepsis, ligament healing, and aseptic loosening of joint prostheses. PMID- 7543400 TI - Nitric oxide formation in the vascular wall: regulation and functional implications. PMID- 7543401 TI - Regulation and dysregulation of constitutive nitric oxide synthases types I and III. PMID- 7543398 TI - A new nitric oxide scavenger, imidazolineoxyl N-oxide derivative, and its effects in pathophysiology and microbiology. PMID- 7543402 TI - T cell apoptosis triggered via the CD3/T cell receptor complex and alternative activation pathways. PMID- 7543403 TI - Rate and treatment of pelvic recurrence after abdominoperineal resection and low anterior resection for rectal cancer. AB - PURPOSE: This study was designed to analyze factors of importance for local recurrence after radical surgery for rectal cancer and to analyze course and outcome of treatment of pelvic recurrence. METHODS: One hundred seventy-five patients treated for rectal cancer with low anterior resection (LAR) or abdominoperineal resection (APR) were studied, retrospectively. Seventy-four patients had LAR and 101 had APR. RESULTS: The two groups were comparable with respect to Dukes classification, histologic differentiation, and male to female ratio. The rate of pelvic recurrence was 18 percent for LAR and 24 percent for APR (not significant). Recurrence rates were 27 percent after stapled anastomoses and 10 percent after handsewn anastomoses respectively (P = 0.09). Twenty five had pelvic recurrence diagnosed without signs of distant metastatic disease. They were treated with radiotherapy, palliative operations, or analgesics. The group receiving radiotherapy had a significantly longer survival (15.9 months) compared with other groups (2.4 months; P < 0.001). CONCLUSIONS: There is no difference in local recurrence rate after LAR and APR. Radiotherapy seems to increase survival in patients with an unresectable recurrence and should be offered irrespective of pain. PMID- 7543404 TI - Immunohistochemical localization of carcinoembryonic antigen as a predictor of lymph node status in submucosa-invasive colorectal carcinoma. AB - PURPOSE: Submucosa-invasive colorectal carcinoma is a colorectal carcinoma extending only into the submucosal layer. To clarify the metastatic potential of submucosa-invasive colorectal carcinoma, we studied the relationship between the immunohistochemical staining pattern of carcinoembryonic antigen (CEA) and that of lymphatic invasion/lymph node metastasis. METHODS: We investigated 49 submucosa-invasive colorectal carcinomas resected surgically or endoscopically. CEA distribution patterns of the neoplastic tissues were divided into three patterns: Pattern 1 = luminal type; Pattern 2 = apical cytoplasmic type; and Pattern 3 = diffuse cytoplasmic type. We also observed the submucosal stromal staining of CEA. RESULTS: Lymphatic invasion and lymph node metastasis were found in 48.8 percent (21/43) and 11.6 percent (5/43) of the Pattern 2/Pattern 3 cases, whereas these were seen in none (0/6) of Pattern 1 cases. Lymphatic invasion and lymph node metastasis were found in 63.3 percent (19/30) (chi-squared = 21.94; P < 0.001) and 16.7 percent (5/30) of the positive stromal CEA cases, whereas these were seen in 10.5 percent (2/19) and none (0/14) of the negative stromal CEA cases, respectively. CONCLUSION: Pattern 2/Pattern 3 and stromal CEA can be predictors of the lymph node metastasis with 11.6 percent and 16.7 percent risks. PMID- 7543405 TI - Neurological examination of the preterm and full-term infant at risk for developmental disabilities using the Dubowitz Neurological Assessment. AB - The Dubowitz Neurological Assessment of the Preterm and Full-term Infant was used to evaluate infants at risk for developmental disabilities. Criteria for categorising the results of the Dubowitz Neurological Assessment were developed by documenting the findings on a group of relatively low risk infants managed in a NICU. The criteria, expressed as a number of deviant responses on items in the assessment, were used to test the concurrent and predictive validity. The Dubowitz Neurological Assessment was found to correlate with a perinatal risk rating and the findings on brain ultrasonography, indicating concurrent validity. Predictive validity was demonstrated by correlation with neurodevelopmental outcome and neurological status at 1 year corrected age. The sensitivity and specificity of the assessment were also used to develop a management protocol for graduates of the NICU. PMID- 7543406 TI - Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein. AB - The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and 31P, 13C, and 1H nuclear magnetic resonance spectroscopy (NMR), and electron magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5-, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and 13C- and 1H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% alpha-helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of alpha-helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. PMID- 7543407 TI - Neuropharmacology of progressive myoclonus epilepsy: response to 5-hydroxy-L tryptophan. AB - Low concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5 HIAA) in cerebrospinal fluid (CSF) of patients with progressive myoclonus epilepsy (PME) suggest hypofunctional serotonergic neurotransmission. To study this hypothesis, we enrolled 6 patients with PME [Unverricht-Lundborg disease (U L), mitochondrial encephalomyopathy, or Lafora disease] in a controlled, double blinded, dose-ranging, cross-over add-on pilot clinical trial of 5-hydroxy-L tryptophan (L-5-HTP) plus carbidopa after 2 other patients had received open label L-5-HTP for compassionate use. Prestudy CSF 5-HIAA concentrations were low (< 20 ng/ml) in 6 patients regardless of the etiology of PME. One patient with U L disease showed clinical improvement and a fivefold increase in CSF 5-HIAA, and 1 with Lafora disease showed a twofold increase in CSF 5-HIAA without improvement. A patient with Lafora disease reported enough improvement in myoclonus-evoked convulsions to continue chronic use of the drug. One patient with mitochondrial encephalomyopathy developed status epilepticus during treatment with L-5-HTP. As a group, patients had no statistically significant changes in myoclonus evaluation scale scores, subjective and objective measures of ataxia, seizure frequency, antiepileptic drug (AED) levels, or routine blood tests. These data suggest a serotonergic abnormality regardless of the underlying etiology of PME, but one that seldom responds to acute treatment with L-5-HTP. PMID- 7543409 TI - Characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) that interacts with RNA. AB - The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 degrees C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA-free ferredoxin. The RNA-bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe-4S](2+;1+) cluster with an EPR spectrum (g = 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of -360 mV was determined for the RNA free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as delta S degree = -230 J.K-1.mol-1. No electrochemical response was obtained with the RNA-bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster. PMID- 7543408 TI - Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein. AB - The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sep1. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein. PMID- 7543410 TI - The species-specific differences in the cAMP regulation of the tissue-type plasminogen activator gene between rat, mouse and human is caused by a one nucleotide substitution in the cAMP-responsive element of the promoters. AB - In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein. PMID- 7543411 TI - Epitope mapping of monoclonal antibodies to keratin 19 using keratin fragments, synthetic peptides and phage peptide libraries. AB - To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding. PMID- 7543413 TI - Decreased spontaneous motor activity and startle response in nitric oxide synthase inhibitor-treated rats. AB - In the central nervous system, nitric oxide has been proposed to be a retrograde messenger mediating learning and synaptic plasticity. Since only pretraining injections of nitric oxide synthesis inhibitors were shown to impair learning, we examined the possibility that systemic administration of these inhibitors might influence some non-specific aspects related to the organism's general psychophysiological status. Intraperitoneal administration of NG-nitro-L-arginine methyl ester (30 or 100 mg/kg) 60 min pre-test to adult rats resulted in: (i) altered exploratory pattern and reduced locomotion in a novel environment; (ii) reduced startle response to either acoustic or electric stimuli; and (iii) cardiovascular alterations. In addition, intracerebroventricular administration of N-nitro-L-arginine (10 microliters of a 10 mM solution) diminished the acoustic startle response. Specificity of these effects through nitric oxide was supported by the ability of the nitric oxide precursor, L-arginine, to prevent the inhibitors actions. These findings indicate that nitric oxide inhibitors interfere with the general psychophysiological status of the organism. PMID- 7543414 TI - Enrichment of bone marrow and blood progenitor (CD34+) cells by density gradients with sufficient yields for transplantation. AB - We have evaluated the use of iso-osmolar Percoll density gradients to enrich CD34+ hematopoietic progenitor cells and to reduce T cells for purposes of bone marrow or mobilized peripheral blood cell transplantation (BMT or PBCT). Samples from 12 normal BM donors and 11 patients undergoing mobilization of PB cells using chemotherapy and G-CSF were placed over a five-step density gradient from 40 to 50% Percoll. In BM, low-density fractions 1 to 3 (40 to 45% Percoll) accounted for 3% of starting nucleated cells with a 10- to 20-fold enrichment of hematopoietic progenitors (CD34+ cells) and a 20-fold reduction of CD4+ and CD8+ T cells. In PB, fractions 1 to 3 accounted for 20 to 30% of the starting nucleated cells with a five-fold enrichment of hematopoietic progenitors. Based on these values, such populations have been used for clinical transplantation using a single apheresis. The reduced cell numbers in the low-density fractions can facilitate tumor purging, and the reduced T cell numbers present in the marrow may ameliorate graft-vs.-host disease. PMID- 7543412 TI - ATP-sensitive K+ channels mediate regulation of substance P release via the prejunctional histamine H3 receptor. AB - Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the subcutaneous perfusate in the rat hindpaw. The active L-enantiomer of cromakalim, lemakalim (50 micrograms/kg, i.v.), a selective K+ channel activator, significantly inhibited the immunoreactive substance P release. Glibenclamide (10 mg/kg, i.v.), an ATP sensitive K+ channel blocker, abolished the response to lemakalim on the release of immunoreactive substance P. R(-)-alpha-methylhistamine (1 mg/kg, i.v.), a specific histamine H3 receptor agonist, significantly inhibited the release of immunoreactive substance P. Glibenclamide (10 mg/kg, i.v.) antagonized the inhibitory effect of R(-)-alpha-methylhistamine. Tetraethylammonium (10 mg/kg, i.p.), a K+ channel blocker, also reduced the inhibitory effect significantly. These results suggest that the inhibition of substance P release from sensory nerve endings via prejunctional histamine H3 receptors may be achieved by activating the ATP-sensitive K+ channel coupled to the histamine H3 receptor in the rat skin. PMID- 7543415 TI - The effect of basic and acidic fibroblast growth factors (bFGF and aFGF) on the growth of leukemic blast progenitors in acute myelogenous leukemia. AB - The effect of basic and acidic fibroblast growth factors on leukemic blast progenitors was studied in 14 patients with acute myelogenous leukemia and in one patient with chronic myelocytic leukemia in myeloid crisis. bFGF and aFGF stimulated blast-colony formation by leukemic blast progenitors cultured in methylcellulose in two patients. In the other 13 patients, no significant effect of either FGF on blast-colony formation was noted. The combination of bFGF or a FGF and G-CSF, GM-CSF, interleukin-3, or stem cell factor (SCF) had a synergistic effect on blast-colony formation in three patients. In the other patients, however, synergism between FGF and CSF was not detected. In fact, bFGF was found to suppress the stimulation of blast-colony formation due to GM-CSF in one of 10 patients and that due to SCF in four of eight patients. aFGF suppressed the stimulation of blast-colony formation due to GM-CSF in two of 11 patients and that due to SCF in four of eight patients. The results show that bFGF and aFGF do not directly play a major role in leukemic hematopoiesis but that they may modulate the cytokine network affecting leukemic cell growth. PMID- 7543417 TI - Nonspecific binding of anti-CD34 antibody QBend10 to nonviable cells. PMID- 7543416 TI - The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases. AB - Thrombopoietin (TPO) is a growth and differentiation factor for megakaryocyte lineage cells. The receptor for TPO, c-MPL, is a member of the hematopoietic cytokine receptor family and has previously been shown to rapidly activate one or more cytoplasmic tyrosine kinases after ligand binding. In this study, we found that activation of the TPO receptor rapidly induced tyrosine phosphorylation of two members of the Jak tyrosine kinase family, JAK2 and TYK2, but not JAK1 or JAK3, in two different factor-dependent hematopoietic cell lines. The activation of both JAK2 and TYK2 was dose- and time-dependent and was associated with rapid tyrosine phosphorylation of a series of STAT proteins including STAT1, STAT3, and STAT5. Gel-shift assays indicated that one or more of these STATs is likely to participate in the formation of specific DNA-binding complexes. The activation of tyrosine kinases and signal propagation through tyrosine phosphorylation are likely to represent important initial steps in mediating the activities of TPO in myeloid cells. PMID- 7543418 TI - Differential effects of TGF-beta 1 on normal and leukemic human hematopoietic cell proliferation. AB - We evaluated the effects of transforming growth factor-beta 1 (TGF-beta 1) on the growth of hematopoietic progenitors in normal donors and in patients with hematologic malignancies now designed as clonal disorders of multipotential stem cells. TGF-beta 1 at 80 pM exhibited differential effects on the normal hematopoietic progenitors when cells were stimulated with different growth factors, such as G-CSF, GM-CSF, interleukin-3 (IL-3), or stem cell factor (SCF). The suppressive effect by TGF-beta 1 was increased for growth with GM-CSF, IL-3, and SCF, and growth with G-CSF was unaffected in hematologic malignancies, TGF beta 1 suppression for growth with G-CSF was increased for essential thrombocythemia (ET) and polycythemia vera; chronic myelogenous leukemia (CML) in chronic phase; CML in accelerated phase; CML in myeloid crisis; myelodysplastic syndrome (MDS) in refractory anemia; MDS in refractory anemia with an excess of blasts; and acute myeloblastic leukemia (AML). In CML-myeloid crisis and AML, TGF beta 1 almost completely abolished the growth, with some patient-to-patient variation. The mean ED50s for the growth of leukemic blast progenitors were 1.6, 1.2, 0.7, and 0.2 pM in the presence of G-CSF, GM-CSF, IL-3, and SCF, respectively, c-myc and c-myb antisense oligonucleotides significantly suppressed the growth of leukemic blast progenitors, but not that of clonogenic cells from normal donors and patients with ET. We also demonstrated that TGF-beta 1 inhibits mRNA expression by AML blasts for c-myc and/or c-myb. When the data are taken together, growth suppression by TGF-beta 1 appears to increase with the progression of clonal evolution in hematologic malignancies. PMID- 7543421 TI - Formation and properties of C1-inhibitor polymers. AB - Heating of the serpin C1-inhibitor above 55 degrees C induced the formation of inactive polymers. Western blotting of non-denaturing gels showed that the polymers bound to the conformation specific monoclonal antibody 4C3, suggesting that a similar conformational change to that occurring in complexed or cleaved inhibitor had taken place. N-Terminal analysis of tryptic peptides which bound to 4C3 showed that the epitope resides within residues 288-444, a region which includes parts of beta-sheets A and C. alpha 1-Antichymotrypsin, alpha 2 antiplasmin, angiotensinogen and thyroxine binding globulin also polymerised on heating, indicating that this is a property of many serpins. PMID- 7543419 TI - Improved specificity of ribozyme-mediated cleavage of bcr-abl mRNA. AB - Ribozyme-mediated cleavage of bcr-abl mRNA has been shown to be associated with the concomitant cleavage of bcr mRNA. The possible role of this ribozyme as a clinical therapeutic constrained us to improve its target specificity. Consequently, three modified ribozymes (Rz7-9) were created, each carrying a single base mismatch in the sequence immediately 5' of the cleavage site, while a fourth ribozyme (Rz10) was targeted to a nearby site. Each was compared with the parent ribozyme for its ability to cleave synthetic bcr-abl and bcr substrates, and it was shown that alteration of the second base 5' of the cleavage site created a ribozyme with significantly improved specificity for its substrate. Rz10 was shown to exclusively act on bcr-abl, but the efficiency of cleavage was reduced compared to that shown by ribozymes 6, 8, and 9. PMID- 7543420 TI - Direct synergistic effects of IL-4 and IL-11 on proliferation of primitive hematopoietic progenitor cells. AB - The present studies have investigated, for the first time, the synergistic effects of interleukin-4 (IL-4) and IL-11 on the growth of single murine bone marrow progenitor cells. These studies suggest that IL-4 and IL-11 are synergistic hematopoietic growth factors, enhancing colony formation of bone marrow progenitors from normal mice in the presence of colony-stimulating factors or stem cell factor, whereas neither IL-4 nor IL-11, alone or in combination, resulted in colony formation. However, in the presence of a neutralizing anti-TGF beta antibody, IL-11 plus IL-4 induced clonal growth of primitive Lin-Sca1+ progenitors. Furthermore, here we report several observations extending the knowledge about IL-4 and IL-11 as synergistic factors. In addition to the established ability of IL-11 to enhance IL-3- and GM-CSF-induced colony formation, IL-11 also enhanced the number of G-CSF- and CSF-1-stimulated colonies of mature (Lin-) and primitive (Lin-Sca-1+) hematopoietic progenitors cultured at the single-cell level. In contrast, IL-4 bifunctionally regulated the growth of Lin- progenitors, whereas the growth of single Lin-Sca=1+ progenitors was unaffected or enhanced in the presence of IL-4. Finally, IL-4 and IL-11, in combination, potently synergized to enhance the high-proliferative-potential colony-forming cell colony formation of Lin-Sca-1+ progenitors in response to all four CSFs and to SCF. PMID- 7543422 TI - Improved detection of nitric oxide radical (NO.) production in an activated macrophage culture with a radical scavenger, carboxy PTIO and Griess reagent. AB - An improved method for the detection of nitric oxide radicals (NO. in cultures of activated macrophages was developed, involving a nitric oxide radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-3-oxide-1-oxyl (carboxy PTIO) and Griess reagent. A murine macrophage-like cell line, J774.1, was activated with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), which induced the production and secretion of NO2- into the culture supernatant. Addition of carboxy PTIO to the activated macrophages increased the amount of NO2 to 4- to 5-fold without cell damages, probably because carboxy PTIO rapidly reacted with NO. to form NO2-, which was finally assayed by the Griess reaction. PMID- 7543424 TI - Use of dextran-primed extracorporeal circuit in the canine model for venous return: with special references to the effects of two dihydropyridine-type Ca2+ channel blockers. AB - 1. A successful canine "venous return (VR)" model is described, in which all hemodynamic parameters were stable for about an hour, by minimizing extracorporeal circuit primed with dextran (DX) so that the dilution of the blood was kept about 33%. 2. Increases in VR by norepinephrine (1 micrograms/kg) and dose-dependent decreases in VR by glyceryl trinitrate (1-30 micrograms/kg) were clearly observed. 3. While 1 microgram/kg of nifedipine produced a small, but significant decrease in VR, only a tendency for decrease was observed with higher doses which produced a definite fall in blood pressure. 4. Nisoldipine (1 microgram/kg) caused a small decrease in VR. However, higher doses that produced a large and persistent hypotension produced an increase in VR due probably to intense reflex translocation of the blood. The increase in VR was reversed with carvedilol, a beta-blocker with alpha-blocking activity. PMID- 7543423 TI - APO-1(CD95)-mediated apoptosis in Jurkat cells does not involve src kinases or CD45. AB - Tyrosine phosphorylation has been reported to be an early event required for APO 1/Fas(CD95) signalling in lymphocytes [Eischen, C.M., Dick, C.J. and Leibson, P.J. (1994) J. Immunol. 153, 1947-1954]. We have compared two mutant Jurkat cells, one largely deficient in expression of CD-45 (J45.01) and a second one deficient in expression of p56lck (JCaM1.6) with wild type Jurkat cells for their ability to undergo APO-1-induced apoptosis. No significant difference was observed among the three cell lines. In the mutant Jurkat cells APO-1 triggering did not result in increased tyrosine phosphorylation of cytosolic proteins. Furthermore, herbimycin A did not inhibit but rather augmented apoptosis at concentrations which effectively degraded the src related kinases lck and fyn. The data suggest that APO-1-mediated signalling is independent from src kinases and CD45. PMID- 7543425 TI - Insulin-activated amiloride-blockable nonselective cation and Na+ channels in the fetal distal lung epithelium. AB - 1. The apical membrane of fetal distal lung epithelium had two types of amiloride blockable Na(+)-permeant cation channels; (1) nonselective cation (NSC) channel with a single channel conductance of 27 pS and (2) Na+ channel with a single channel conductance of 12 pS around resting membrane potential. 2. The IC50 of amiloride to the Na+ channel was 1-2 microM, while the IC50 of amiloride to the NSC channel was less 1 microM. The open probability of the Na+ channel was about 10-fold larger than that of the NSC channel. 3. Insulin (100 nM) increased the open probability of both channels. PMID- 7543426 TI - Suppressing effects of neuroactive peptides on the inward current caused by achatin-I, an Achatina endogenous peptide. AB - 1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and [SER2]-Mytilus inhibitory peptide ([SER2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. FMRFamide, perfused at 3 microM, suppressed markedly the Iin elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3 microM also suppressed the Iin of v-RCDN, but did not affect that of PON. Oxytocin at 1 microM suppressed the Iin of PON, but did not affect that of v RCDN. [Ser2]-MIP at 3 microM did not affect the Iin of v-RCDN. 4. The dose response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the Iin of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm2, 10(-3) M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543427 TI - The effects of pregnancy and parturition on the levels of substance P-like immunoreactivity in different areas of the hypothalamus. AB - 1. The concentrations of substance P-like immunoreactivity (SPLI) have been measured in the rostral and caudal areas of the hypothalamus of male rats and of virgin, pregnant and puerperal female rats. 2. The rostral:caudal ratio of SPLI is similar in males and virgin females, but diminishes in pregnancy and decreases further during the puerperium. In the pre-optic area, the SPLI concentration fell significantly during parturition, from 241.8 to 177.2 pmols/g wet weight (P < 0.05), and in the medio-basal hypothalamus, the concentration rose during pregnancy and parturition, from 87.4 to 145.5 pmols/g wet weight (P < 0.001). 3. The results are discussed in relation to the endocrine and nociceptive aspects of pregnancy and parturition. PMID- 7543428 TI - Pharmacological evidence that nitric oxide can act as an endogenous antipyretic factor in endotoxin-induced fever in rabbits. AB - 1. This study investigates the effects of the nitric oxide donors on lipopolysaccharide-induced fever in rabbits, and the effect of brain nitric oxide synthase inhibition on the febrile response in pyrogen tolerant animals. 2. The febrile response was reduced by intravenous injections of the nitric oxide donors molsidomine (1.0 mg/kg) and isosorbide dinitrate (0.5 mg/kg) 60 min after intravenous treatment with lipopolysaccharide. 3. The magnitude of fever was also attenuated by intracerebroventricular administration of molsidomine (75 micrograms). 4. Intracerebroventricular pretreatment with the nitric oxide synthase inhibitor, NG-nitro-L-arginine (100 micrograms) 10 min before the injection of lipopolysaccharide significantly enhanced the febrile response in pyrogen tolerant animals. 5. The results suggest that nitric oxide is involved in the central mechanisms of thermoregulation during fever as one of the effective endogenous antipyretics. PMID- 7543429 TI - The human umbilical vein in normal, hypertensive and diabetic pregnancies: immunomorphological and ultrastructural evidence. AB - Mother-fetus exchanges at the placental level are found to be altered in women affected by hypertensive or diabetic pregnancies following the onset of microenvironmental, circulatory, trophic or tissue disorders. Our aim was therefore to assess the alterations occurring within the umbilical cord, particularly its venous endothelial component and underlying smooth muscle layer, using transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemical analyses. Immunohistochemical data appear to support the ultrastructural evidence for an activated state of these vascular structures, in both conditions (hypertension and diabetes). Furthermore, mainly during diabetic pregnancies, extracellular matrix molecules such as tenascin and fibronectin also quantitatively increase at the vein wall level. The umbilical cord seems to be a structure capable of responding actively to abnormal microenvironmental conditions which seriously threaten the health of the fetus and also the mother herself. PMID- 7543430 TI - [The value of self-assessment in management of patients with benign prostatic hyperplasia]. AB - Since benign prostatic hyperplasia (BPH) is a very common and slowly progressive disease which requires surgical treatment in only one-third of those affected, surveillance of the condition on the basis of subjective symptoms is of considerable importance. For this reason, the WHO developed a prostate symptom score (WHO-PSS or I-PSS), which includes both irritative and obstructive symptoms, and which has become accepted all over the world. This score can be used both in investigative studies as a means of monitoring the efficacy of therapy, and as a very good instrument for patient management in the doctor's office. PMID- 7543431 TI - Selected metabolic alterations in the ischemic heart and their contributions to arrhythmogenesis. AB - Myocardial ischemia in vivo is associated with dramatic electrophysiologic alterations which occur within minutes of cessation of coronary flow and are rapidly reversible with reperfusion. This suggests that subtle and reversible biochemical and/or ionic alterations within or near the sarcolemma may contribute to the electrophysiologic derangements. Our studies have concentrated on 2 amphipathic metabolites, long-chain acylcarnitines and lysophosphatidylcholine (LPC) which have been shown to increase rapidly in ischemic tissue in vivo and to elicit electrophysiologic derangements in normoxic tissue in vitro. Incorporation of these amphiphiles into the sarcolemma at concentrations of 1 to 2 mol%, elicits profound electrophysiologic derangements analogous to those observed in ischemic myocardium in vivo. LPC is produced in endothelial cells and myocytes in response to thrombin. Thus, activation of the coagulation system during ischemia may result in extracellular production and accumulation of LPC. The pathophysiological effects of the accumulation of both amphiphiles are thought to be mediated by alterations in the biophysical properties of the sarcolemmal membrane, although there is a possibility of a direct effect on ion channels. Inhibition of carnitine acyltransferase I in the ischemic cat heart was found to prevent the increase in both long-chain acylcarnitines and LPC and to significantly reduce the incidence of malignant arrhythmias including ventricular tachycardia and fibrillation. This review focuses on the influence of these amphiphiles on cardiac ionic currents observed during early ischemia and presents data supporting the concept that accumulation of these amphiphiles within the sarcolemma contributes to changes in ionic conductances leading to electrophysiological derangements. The contribution and the accumulation of these amphiphiles to alterations in intracellular Ca2+ as related to changes in Na/K ATPase activity and intracellular Na+ are examined. Other alterations occur during early myocardial ischemia in addition to the events reviewed here; however, the results of multiple studies over the past 2 decades indicate that accumulation of these amphiphiles contributes importantly to arrhythmogenesis and that development of specific inhibitors of carnitine acyltransferase I or phospholipase A2 may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease in man. PMID- 7543432 TI - The significance of immunoglobulin M antibody response to hepatitis C virus core protein in patients with chronic hepatitis C. AB - The significance of immunoglobulin (Ig) M antibody to hepatitis C virus core protein (IgM anti-HCV core) was studied in 41 patients with chronic hepatitis C virus (HCV) infection diagnosed by the polymerase chain reaction (PCR). IgM anti HCVcore was tested with a solid-phase enzyme-linked immunoassay. The results were correlated with clinical features, liver histology findings evaluated by the histological activity index, and virological features such as genotypes and viremic levels assessed by a branched DNA assay. IgM anti-HCVcore was found in 29 (71%) patients, and its occurrence was only related to viremic levels. A significant relationship was observed between viremic levels and IgM anti-HCVcore cut-off index (rs = .42, P < .01). Of the eight low viremic (branched DNA negative) patients, only two (25%) tested positive for IgM anti-HCVcore with a low cut-off index of < 3, whereas 27 (82%) of the 33 highly viremic (branched DNA positive) patients had IgM anti-HCVcore (P < .01). After a 28-week interferon alpha course (IFN-alpha), sustained aminotransferase normalization after therapy withdrawal was achieved by only two (13%) of the 16 patients with IgM anti HCVcore cut-off index > 3 compared with 11 (44%) of the 25 patients with that < 3 (P < .05). IgM anti-HCVcore cut-off index decreased after therapy in patients who cleared the virus in sera but increased again after reappearance of viremia. These findings suggest that IgM anti-HCVcore may serve as a simple serological indicator of active virus replication and have relevance to the outcome of antiviral therapy. PMID- 7543433 TI - The virological and histological states of anti-hepatitis C virus-positive subjects with normal liver biochemical values. AB - We investigated anti-hepatitis C virus (HCV) titers, HCV RNA levels in liver and serum, genetic variability in the hypervariable region of the genome, the form of the virus in the circulation, and liver histology in 21 anti-HCV-positive subjects with sustained normal liver biochemical values. Titer of anti-HCV was determined by second generation anti-HCV-passive hemagglutination assay, and HCV RNA levels were semiquantitated by reverse transcriptase polymerase chain reaction (PCR). In 19 (90%) of the 21 subjects who had a higher titer of anti-HCV (> or = 2(14)), HCV RNA was detected in both serum and liver, and histological examination showed minimal or mild chronic hepatitis in all. In the remaining 2 patients who had a lower titer of anti-HCV, HCV RNA was not detected in serum and liver, and liver histology was normal. Anti-HCV titers and HCV RNA levels in serum and liver in the 19 HCV RNA-positive subjects were compared with those levels in the 41 patients with biopsy-proven chronic hepatitis C and elevated serum aminotransferase levels as a control group. There were no significant differences in viral levels in serum and liver between the two groups. To further investigate virological differences between the two groups with regard to degree of genetic variability and the form in the circulation, we performed the PCR single strand conformation polymorphism (PCR-SSCP) of the hypervariable region 1 and the immunoprecipitation analyses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543434 TI - Screening for hepatocellular carcinoma in chronic carriers of hepatitis B virus: incidence and prevalence of hepatocellular carcinoma in a North American urban population. AB - OBJECTIVE: To prospectively determine the prevalence and annual incidence of hepatocellular carcinoma in hepatitis B carriers in a heterogeneous urban North American population and to assess the diagnostic accuracy of tests used for screening for this cancer. DESIGN: Prospective cohort study of 1,069 chronic carriers of hepatitis B virus using screening with alpha-fetoprotein alone or in combination with ultrasonography every 6 months. RESULTS: The mean age of the cohort was 39 +/- 12 years (+/- SD), 65% were men, 71% were Asians. At the first screening visit, serum alpha-fetoprotein was > or = 20 micrograms/L in 4%. In those subjects who were also screened by ultrasonography during the first visit, 9% were found to have focal lesions. Only 3 subjects were found to have hepatocellular carcinoma at the first screening, giving a prevalence of 281/100,000 chronic carriers of hepatitis B virus. The cohort was followed for 2,340 person-years (mean, 26 months follow-up, with a range from 6 to 60 months). During this period, 11 more subjects, 10 men and 1 woman, were diagnosed to have hepatocellular carcinoma (annual incidence, 470/100,000). In men only, the annual incidence was 657/100,000. During the study, 5 subjects died from hepatocellular carcinoma (annual mortality rate, 214/100,000). Sensitivity and specificity of serum alpha-fetoprotein > 20 micrograms/L were 64.3% and 91.4%, respectively. For ultrasonography, sensitivity was 78.8% and specificity 93.8%. CONCLUSIONS: These data suggest that the incidence and prevalence of hepatocellular carcinoma in hepatitis B carriers in our area, an urban North American setting, are as high as in countries where hepatitis B is endemic. Current screening tests have significant false-positive and false-negative rates raising questions about the cost-benefit of screening for hepatocellular carcinoma in our study population. PMID- 7543435 TI - Autoantibodies to BCOADC-E2 in patients with primary biliary cirrhosis recognize a conformational epitope. AB - Primary biliary cirrhosis (PBC) is an autoimmune disease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoantigens recognized by autoantibodies in PBC are members of the 2-oxo-acid dehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate dehydrogenase complex (PDC-E2) and the E2 component of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2). The autoantibody response to PDC-E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational components. The presence of a single immunodominant epitope in PDC-E2 is unusual when contrasted to the immune response to autoantigens in other human autoimmune diseases. We have mapped the epitope recognized by antimitochondrial autoantibodies (AMA) specific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 complementary DNA (cDNA) and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, enzyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the absence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid. PMID- 7543436 TI - Serum concentration of intercellular adhesion molecule-1 in patients with hepatocellular carcinoma is a marker of the disease progression and prognosis. AB - Serum levels of soluble forms of intracellular adhesion molecule-1 (sICAM-1) and lymphocyte function-associated antigen-3 (sLFA-3) in 122 patients with chronic liver disease including hepatocellular carcinoma (HCC) were measured by enzyme linked immunosorbent assays. Serum levels of sICAM-1 in patients with HCC were significantly higher than those of chronic hepatitis (CH) and cirrhosis. On the other hand, serum levels of sLFA-3 in patients with HCC were almost the same as those of cirrhosis. Western blot analyses showed that molecular sizes of sICAM-1 and sLFA-3 detected in the sera were 90 kd and 50 kd, respectively, indicating that both molecules include whole extracellular domains. In patients with HCC, circulating sICAM-1 levels were significantly (P < .001) correlated with tumor volume (r = .50), total bilirubin (r = .38), serum aspartate aminotransferase levels (r = .51), and gamma-globulin (r = .63). Furthermore, serum sICAM-1 levels were significantly elevated in patients with multiple HCC (tumor number > 3) or HCC with tumor embolus in the first branch or trunk of portal vein. Survival periods were analyzed in relation to serum sICAM-1 levels in patients with HCC who had been treated by transcatheter arterial chemoembolization. The HCC patients with < 1,000 ng/mL of serum ICAM-1 showed significantly (P = .0005) longer survival than those with higher levels of the molecule. The same results were obtained when only patients with moderately differentiated HCC were analyzed (P = .02). Analyses by Cox's proportional hazard model showed that sICAM-1 is a significant (P = .032) prognostic factor for patients with HCC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543437 TI - Enhanced nitric oxide synthase activity in portal hypertensive rabbits. AB - Portal hypertension (PHT) is characterized by splanchnic hyperemia caused by a reduction in mesenteric vascular resistance. Mediators of this hyperemia include nitric oxide (NO). This is based on several reports indicating a marked splanchnic hyporesponsiveness in PHT to vaso-constrictor stimuli, both in vitro and in vivo, and a subsequent reversal using specific inhibitors of NO synthase (NOS). The objective of this study was to determine directly if the generation of NO is altered in PHT vasculature. Thus, we compared NOS activity in the hyperemic vasculature of normal rabbits and rabbits with PHT (after undergoing partial portal vein ligation). Nicotinamide adenine dinucleotide phosphate diaphorase staining indicated the presence of NOS within the vascular endothelium. Ca(2+) dependent NOS activity was significantly increased (P < .05) in PHT particulate fractions from the superior mesenteric artery and thoracic aorta, but not from the portal vein. There was no change in NOS activity within the cytosolic fractions. Arterial wall cyclic guanosine monophosphate (cGMP) levels and plasma nitrite levels were both significantly increased in PHT. These results show enhanced NOS activity in PHT hyperemic vessels concurrent with increased tissue cGMP levels. We conclude that enhanced NO synthesis contributes to the hyperdynamic circulation of PHT. PMID- 7543438 TI - Liver transplantation for hepatitis C: the promise and the challenge. PMID- 7543439 TI - Differentiation and proliferative activity in benign and malignant cartilage tumors of bone. AB - To assess the histological grade in benign and malignant cartilage tumors of bone by more objective methods, we examined the differentiation and proliferative activity of tumor cells in six enchondromas, five chondroblastomas, and 13 chondrosarcomas immunohistochemically. A variable number of cells in all tumors showed S-100 protein and vimentin immunoreactivity. In fully differentiated cartilage of enchondromas and low grade chondrosarcomas, tenascin, which is an extracellular matrix glycoprotein, was present in small amounts or absent but was increased at the periphery of tumor lobules and even in the matrix throughout the high grade chondrosarcomas. Higher rate and intensity of proliferating cell nuclear antigen (PCNA) reactivity were found in chondrosarcomas, especially in spindle-shaped cells of high grade tumors, than in enchondromas. The distribution of PCNA-positive cells almost corresponded to the regions with tenascin reactivity. One tumor of high grade chondrosarcoma showed p53 protein immunoreactivity. Aberrant expression of cytokeratin was observed in four chondroblastomas. The expression of desmin was identified in relatively large proportions of enchondromas and chondrosarcomas, regardless of their benign or malignant nature and histological grade. Smooth muscle or muscle-specific actins also were present in a smaller number of tumors. Based on these findings, it is concluded that unusual staining characteristics were present, in addition to those of a chondroblastic nature, in the cartilage tumors of bone. Tenascin and PCNA positivity of various degrees in all chondroblastomas may suggest that they are chondrogenic tumors having a relatively high proliferative activity, albeit their benign clinical course. Proliferative activity of tumor cells in enchondromas and chondrosarcomas correlated well with their histological grade. Tenascin may play a role in promoting tumor cell proliferation of cartilagenous neoplasms and, on the other hand, the alterations of extracellular matrix involving tenascin synthesis seem to be a result of tumor development. PMID- 7543440 TI - Insulin-like growth factor 2 and insulin-like growth factor binding protein 2 expression in hepatoblastoma. AB - The expression of insulin-like growth factor 2 (IGF2) and insulin-like growth factor binding protein 2 (IGFBP2) in 11 cases of hepatoblastoma was studied by means of in situ mRNA hybridization using digoxigenin (DIG)-labeled riboprobes. The results showed that both IGF2 and IGFBP2 transcripts are present in hepatoblastoma and that their expression is inversely correlated with the degree of tumor cell differentiation. The data suggested that IGF2 and IGFBP2 gene expression could be regarded as a marker for assessment of the degree of differentiation in hepatoblastoma. PMID- 7543441 TI - Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. AB - The most common carcinomas metastatic to the ovary that mimic ovarian primaries are colonic adenocarcinomas and endometrial carcinomas. Conventional histochemical staining procedures, even in combination with additional immunohistochemical assays, are of limited value in distinguishing between these metastases and primary ovarian carcinomas. In this study we investigated whether the application of monoclonal antibodies against keratins 7, 8, and 20 could help in differentiating between these categories. The reactivity patterns of 40 carcinomas metastatic to the ovary were compared with those of their primary carcinomas on the one hand and with various primary ovarian carcinomas and mesotheliomas on the other. Colon cancer metastatic to the ovary was keratin 7 negative and keratin 20 positive in 94% of the cases; in contrast, all primary ovarian carcinomas were keratin 7 positive and keratin 20 negative, with the exception of two cases of mucinous cystadenocarcinoma. Ovarian metastases of gastric cancer usually contained keratins 7 and 20. Metastases of endometrial cancer to the ovary and primary ovarian carcinomas usually showed similar keratin expression. We propose that keratin 7 and 20 antibodies may be of help to distinguish between primary ovarian carcinomas and carcinoma metastases in the ovary. PMID- 7543442 TI - Menstrual cycle dependent expression of CD44 in normal human endometrium. AB - CD44 is a cell surface glycoprotein known to be a hyaluronate receptor, which functions in lymphocyte homing and cancer metastasis. We have previously shown that CD44 molecules are expressed in normal endometrial tissue. In this study we immunohistochemically examined expression of CD44 glycoproteins in normal human endometrial tissue to gain insight into roles of CD44 molecules. No expression of the CD44 standard form was observed with 11 endometria in the proliferative phase. On the contrary, 13 of 19 secretory phase endometria were stained intensely by anti-CD44 standard form anti-body; all CD44-positive cases were in the mid and late secretory phases. Among the 13 CD44-positive endometria, four also were positive for the CD44 variant with exon v6. CD44 molecules were present at all but the luminal cell surfaces. These results indicate that expression of CD44 depends on menstrual cycle, suggesting that CD44 molecules might be involved in implantation of fertilized ovum in endometrium. PMID- 7543443 TI - Mucin expression in atypical adenomatous hyperplasia of the prostate. AB - Prostatic atypical adenomatous hyperplasia (AAH) is a small glandular proliferation that has histological similarities with well-differentiated adenocarcinoma. To determine the histochemical profile of AAH, we assessed the production of total neutral mucin, total acidic mucin, and sulfated acidic mucin in 24 cases of AAH, five cases of Gleason primary pattern 1 and 2 adenocarcinoma, and 29 cases of adjacent benign and hyperplastic prostatic tissue. All specimens were formalin-fixed transurethral resections, and the diagnosis in each was confirmed by evaluation of the keratin 34B-E12 immunoreactive basal cell layer (intact in benign and hyperplastic epithelium, fragmented in AAH, and absent in cancer). The extent of mucin staining was measured semiquantitatively in 10% increments according to the number of stained glands. Neutral mucin was found in all but two cases, and there was no apparent difference in the amount of staining in benign glands, AAH, and cancer (mean number of stained glands, 43%). Total acidic mucin was more common in AAH (63% of cases; mean, 11% of glands) and adenocarcinoma (60% of cases; mean, 30% of glands) than in benign glands (0% of cases). Similarly, nonsulfated acidic mucin was more common in AAH (63% of cases; mean, 12% glands) and adenocarcinoma (60% of cases; mean, 8% of glands) than in benign glands (0% of cases); the pattern and intensity of staining for nonsulfated acidic mucin appeared to be similar to that for total acidic mucin in AAH and cancer. These findings indicate that there is a close relationship in mucin expression between AAH and well-differentiated adenocarcinoma. Identification of acidic mucin should be used cautiously as an adjunct in the diagnosis of adenocarcinoma but is useful in separating some cases of AAH and adenocarcinoma from benign prostatic epithelium. PMID- 7543444 TI - Opposing effects of interferon-gamma on iNOS and interleukin-10 expression in lipopolysaccharide- and mycobacterial lipoarabinomannan-stimulated macrophages. AB - Previous studies have demonstrated that, like bacterial lipopolysaccharide (LPS), arabinofuranosyl-terminated lipoarabinomannan (AraLAM) from an attenuated strain of Mycobacterium induces potent early gene (c-fos, KC, JE and TNF-alpha) responses in murine macrophages, whereas extensively alpha-Manp capped LAM (ManLAM) from virulent M. tuberculosis do not. In this study we have extended analysis of the influence of mycobacterial LAM on macrophage function by demonstrating that AraLAM (but not ManLAM), like bacterial LPS, is a potent stimulator of inducible nitric oxide synthase (iNOS) expression independent of the autocrine activity of co-stimulated tumour necrosis factor-alpha (TNF-alpha) release. The inability of ManLAM to induce iNOS expression was not due to induction of the 'deactivating' cytokine interleukin-10 (IL-10). Indeed, like LPS, AraLAM was also a potent inducer of IL-10 expression. However, analysis of AraLAM- or LPS-induced responses in the presence of interferon-gamma (IFN-gamma) showed that, whereas IFN-gamma acts as a potent co-stimulus for iNOS, it completely inhibits the IL-10 response. Hence, the presence of IFN-gamma early in infection will have an important immunomodulatory role in determining the macrophage response. These results have important implications for the pathogenesis of virulent and avirulent mycobacteria in vivo. PMID- 7543445 TI - Immunological and biochemical characterization of biglycan-like haemopoietic factor. AB - Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L 929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells. PMID- 7543446 TI - Porcine E-selectin: cloning and functional characterization. AB - E-selectin, a member of the selectin family, is believed to play an important role in mediating the initial adhesive events between leucocytes and the endothelium during inflammation. A monoclonal antibody against human E-selectin was found to cross-react with the porcine equivalent, a glycoprotein of 92,000 MW. Isolation of the cDNA for porcine E-selectin showed that it was 71% homologous with human E-selectin but had two less short consensus repeats. The porcine adhesion molecule could also support the adhesion of both porcine and human neutrophils. Expression of E-selectin on interleukin-1 alpha (IL-1 alpha)- or tumour necrosis factor-alpha (TNF-alpha)-activated porcine aortic endothelial cells in culture was prolonged, persisting for up to 48 hr. Binding studies using a chimeric molecule consisting of the lectin domain of porcine E-selectin and the epidermal growth factor (EGF) domain of human E-selectin fused to the human IgG constant region, further characterized porcine E-selectin as recognizing mainly granulocytic leucocytes and a subpopulation of lymphocytes. PMID- 7543447 TI - Regulation of CD59 expression on K562 cells: effects of phorbol myristate acetate, cross-linking antibody and non-lethal complement attack. AB - CD59 is the major membrane attack complex of complement (MAC) inhibiting protein on human cells. Its regulation is therefore an important factor in determining the fate of cells at sites of complement activation. We have chosen the K562 erythroleukaemia cell line as a model for studies of the regulation of CD59 expression, because it has previously been reported that phorbol 12-myristate 13 acetate (PMA) caused a 15-fold up-regulation of CD59 mRNA in these cells, implying a substantial capacity for CD59 synthesis. However, no assessment of CD59 protein expression was made in these studies. We show here that surface expression of CD59, as assessed by flow cytometry, was increased four-fold over a 16-hr incubation with PMA, whereas surface expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) was not altered. The newly expressed CD59 was functionally active and anchored through glycosyl phosphatidylinositol (GPI). Increased expression was dependent upon de novo protein synthesis. CD59 released into cell supernatant was also increased seven fold by PMA, this 'secreted' CD59 retained its GPI anchor. Non-lethal complement attack did not alter CD59 expression but antibody cross-linking of CD59 caused a rapid loss of the CD59-antibody complexes. However, CD59 was quickly restored to pre-attack levels. This rapid restoration was not dependent upon protein synthesis, suggesting release from preformed stores. PMID- 7543449 TI - A mutation in the alpha 3 domain of Db that abrogates CD8 binding does not affect presentation of an immunodominant H-Y peptide. AB - The peptidic nature of the male (H-Y) antigen, a model minor histocompatibility antigen in H-2b mice, has recently been demonstrated. In this study we show that the H-Y peptide, which is recognized by PM-1, a Db-restricted cytotoxic T lymphocyte (CTL) clone, is absent in male H-2d spleen cells but present in male H 2d spleen cells that also express a transgenic Db molecule under its endogenous promoter. This result indicates that both the H-Y and the Db gene products are essential and sufficient for production of the Db-restricted H-Y peptide. By comparing the ability of the PM-1 clone and bulk CTL generated in a secondary mixed lymphocyte culture to recognize H-Y peptidic material eluted from affinity purified Db molecules and separated by reversed-phase high-performance liquid chromatography (HPLC), we provide evidence that there is an immunodominant H-Y epitope that is presented by the Db molecule. Furthermore, the presentation of this epitope is not affected by a mutation in the alpha 3 domain of Db (asp227 to lys227), which abrogates CD8 binding, since similar amounts of H-Y peptide were eluted from affinity-purified wild-type or mutant Db molecules. However, the generation of the H-Y epitope is dependent on the presence of beta 2 microglobulin, since it is absent in male H-2b mice that lack a functional beta 2 microglobulin gene. The implications of these findings on T-cell development are discussed. PMID- 7543448 TI - Human CD4-CD8- alpha beta + T-cell receptor T cells recognize different mycobacteria strains in the context of CD1b. AB - Double-negative alpha beta+ T-cell receptor (TCR) human T cells have been reported to recognize antigen in the context of the HLA class I-like (Ib) CD1 complex. In particular, the CD1b molecule has been shown to act as the element of genetic restriction for antigens derived from Mycobacterium tuberculosis. The stenotopic nature of these major histocompatibility complex (MHC) class Ib molecules raised the question of whether the antigenic moiety recognized by CD4 CD8- alpha beta+ TCR T cells was shared by different mycobacteria. We demonstrate here that a CD4-CD8- alpha beta+ TCR T-cell line and three clones raised against M. tuberculosis proliferated following stimulation with soluble extracts from organisms of the M. tuberculosis complex, M. leprae and 10 out of 16 tested isolates of M. avium complex; however, four species of weakly or non-pathogenic mycobacteria were not stimulatory. Furthermore, the M. tuberculosis soluble extract (MTSE)-derived, recognized antigenic moiety proved to be proteinase K resistant and to have a molecular weight greater than 5000 MW, thus it differed from the reported antigenic moiety, recognized by CD4-CD8- gamma delta+ TCR cells. Our results suggest that a common antigenic moiety, presented by CD1b molecules to CD4-CD8- alpha beta+ TCR T cells, is shared by many mycobacterial species. Therefore they raise interest in the question of whether CD4-CD8- alpha beta+ TCR T cells, elicited by M. tuberculosis, may play a role in the natural history of other mycobacterial infections. PMID- 7543451 TI - Plasma from women with preeclampsia increases endothelial cell nitric oxide production. AB - In preeclampsia, a factor in the maternal circulation alters endothelial function via a reduction in nitric oxide synthesis. We measured the in vitro effects of 2% plasma from women with preeclampsia, compared with 2% plasma from normotensive pregnant women, on cultured endothelial cell nitrite production and nitric oxide synthase activity. On finding differential effects, we measured the effects on cellular viability (assessed by lactate dehydrogenase levels) and performed a time course study. Endothelial cell nitrite production was found to be higher after exposure to plasma from the preeclamptic group than the normotensive pregnant group. The effects of long-term exposure (120 hours) were similar to those of short-term exposure (24 hours). In addition, nitric oxide synthase activity was significantly greater after exposure to preeclamptic plasma than after exposure to normotensive pregnant plasma. No differential effect on cellular viability was found. Contrary to our hypothesis, exposure of endothelial cells to preeclamptic plasma resulted in increased nitric oxide production and nitric oxide synthase activity. PMID- 7543450 TI - A peptide of Chlamydia trachomatis shown to be a primary T-cell epitope in vitro induces cell-mediated immunity in vivo. AB - Chlamydiae are a major cause of infertility and preventable blindness and there is currently no effective vaccine in humans or rodents against these organisms. We have previously shown that a peptide of 12 amino acids (termed TINKP) from a conserved region of the major outer membrane protein (MOMP) of Chlamydia trachomatis (C. trachomatis) is a primary T-cell epitope in humans. Here we showed that when dendritic cells (DC) from C3H or BALB/c mice were pulsed in vitro with the peptide they stimulated proliferation of syngeneic T cells in vitro indicating that the peptide is also a primary T-cell epitope in mice. Since the skin is a rich source of DC, we immunized mice from each strain with an intradermal injection of the peptide. Humoral and cell-mediated immunity to peptide, MOMP or whole elementary bodies (EB) of C. trachomatis (F/NI1/GU) were assessed. No antibody response to TINKP was observed. However, immunized mice showed recall responses to all three chlamydial antigens. T-cell-mediated immunity in the absence of antibody was induced by a single injection of the peptide intradermally. C. trachomatis isolated from the human genital tract causes salpingitis in mice. Preliminary studies in susceptible C3H mice indicated that intradermal injection of peptide conferred some protection against the development of salpingitis. Thus, a primary T-cell epitope identified by in vitro stimulation using DC can also initiate cell-mediated immunity in vivo and this approach may be useful in the development of vaccines. PMID- 7543452 TI - Nitric oxide synthase inhibition in spontaneously hypertensive rats. Systemic, renal, and glomerular hemodynamics. AB - To investigate the prolonged effects of nitric oxide inhibition on systemic, renal, and glomerular hemodynamics, the effects of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on cardiac index, renal micropuncture results, urinary excretion, and histology were obtained in 20-week old male spontaneously hypertensive rats (SHR) that were divided into two groups: untreated and L-NAME-treated (50 mg/L), each followed for 3 weeks. Cardiac index and effective renal plasma flow decreased (P < .01) in L-NAME-treated SHR, exhibiting a positive correlation (r = .816; P < .0001). Single-nephron plasma flow (123 +/- 8 versus 80 +/- 12 nL/min per gram; P < .01) and ultrafiltration coefficient (P < .05) were also reduced in L-NAME-treated SHR versus controls. Most notably, the L-NAME-treated SHR had increased afferent (4.4 +/- 0.3 versus 9.5 +/- 1.3 U; P < .01) and efferent (1.4 +/- 0.1 versus 2.7 +/- 0.3 U; P < .01) glomerular arteriolar resistances versus controls. These functional changes were associated with significantly altered afferent arteriolar (P < .001) and glomerular (P < .005) histological injury scores accompanied by marked proteinuria (P < .001). Because of the intense afferent glomerular artery constriction and lesser increase in efferent glomerular arteriolar resistance associated with reduced single-nephron plasma flow, glomerular capillary pressure did not increase in the L-NAME-treated SHR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543453 TI - Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats. AB - The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide. PMID- 7543455 TI - Blood-sparing drugs: aprotinin, tranexamic acid, and epsilon-aminocaproic acid. PMID- 7543454 TI - Structural versus functional modulation of the arterial baroreflex. AB - Structural changes in large arteries are often considered the predominant mechanism responsible for decreased baroreflex sensitivity and baroreceptor resetting in hypertension, atherosclerosis, and aging. Recent work has demonstrated that "functional" mechanisms, both at the level of the peripheral sensory endings and within the central nervous system, contribute significantly to altered baroreflex responses. We have conducted both reductive studies of mechanoelectrical transduction in cultured baroreceptor neurons and integrative studies with in vivo recordings of the activity of baroreceptor afferent fibers and efferent sympathetic nerves. Results suggest that the primary mechanism of mechanical activation of baroreceptor neurons involves opening of stretch activated ion channels susceptible to blockade by gadolinium. Baroreceptor nerve activity is modulated by the activity of potassium channels and the sodium potassium pump and by paracrine factors, including prostacyclin, oxygen free radicals, and factors released from aggregating platelets. Endothelial dysfunction and altered release of these paracrine factors contribute significantly to the decreased baroreceptor sensitivity in hypertension and atherosclerosis. The central mediation of the baroreflex depends on the pulse phasic pattern of afferent baroreceptor discharge. Baroreflex-mediated inhibition of sympathetic nerve activity is well maintained during pulse phasic afferent activity. Continuous, nonphasic baroreceptor discharge or a rapid (> 1.5 Hz) pulse phasic discharge results in disinhibition of sympathetic activity. This disinhibition during continuous baroreceptor input is exaggerated with aging. Thus, a defect in central mediation of the baroreflex may be a major cause of the impaired baroreflex and sympathoexcitation in the elderly. In summary, functional neural mechanisms, in addition to structural vascular changes, contribute importantly to altered baroreflex responses in normal and pathophysiological states.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543456 TI - Insulin resistance, insulin-like growth factor I and breast cancer: a hypothesis. AB - Consideration of existing data regarding clinical and biochemical risk factors for the development of breast cancer leads to the hypothesis that enhanced insulin-like growth factor I (IGF-I) activity plays a significant role in the development of this disease. Abnormal IGF-I activity may be related to events occurring prenatally, during puberty, or during adult life. Insulin resistance, a common feature in populations characterized by high caloric intake, may result in the amplification of IGF-I action at the tissue level by altering serum concentrations of IGF-I binding proteins. Several approaches toward testing the hypothesis are proposed, and potential opportunities for clinical application are described. PMID- 7543457 TI - Studies of in vivo recruitment and activation of cytotoxic lymphocytes using a gelatin-sponge model of concomitant tumor immunity. AB - We have earlier described a sponge model of concomitant tumor immunity that permits the capture and isolation of effector T lymphocytes that mediate the rejection of a secondary EMT6 tumor implant. In this study, we have employed the sponge model to study lymphocyte homing and in situ activation during the development of concomitant tumor immunity. Our results demonstrate that EMT6 specific CTL in animals bearing primary EMT6 tumors home preferentially to sponges implanted with EMT6 tumor cells, as compared with contralateral sponges lacking tumor cells or sponges injected with antigenically distinct 168 tumor cells. We further show that recruitment is selective and is not in response to a foreign-body reaction to the sponge. In addition, we show that EMT6-specific CTL were recovered from sponges injected either with intact EMT6 tumor cells or with a mixture of EMT6-derived membranes and supernatant. In contrast, cells accumulating in sponges injected with membranes or supernatant alone were not cytolytic. Thus, maximal recruitment, retention, and activation of CTL precursors require putative chemo-attractive factors secreted by tumor cells as well as interaction with tumor antigen. PMID- 7543458 TI - Patterns of splice variant CD44 expression by normal human urothelium in situ and in vitro and by bladder-carcinoma cell lines. AB - CD44 core and splice variant exon expression was investigated in the stratified transitional epithelium of the urinary tract and in normal and malignant bladder epithelial cell lines in vitro. Antibodies against core CD44 epitopes and splice exon variants v3, v4/5, v5 and v6 showed an intense reaction in the basal and lower intermediate urothelial cell layers, which was consistently lost from the upper intermediate and superficial cell layers. Seven independent cell lines established from normal human urothelium expressed complex multiple-spliced CD44 mRNA transcripts when tested by RT-PCR and were positive with antibodies against CD44 core epitopes and splice variants v3, v4/5, v5 and v6. Of the 13 bladder carcinoma cell lines, all were positive for CD44 core. The more differentiated cell lines had retained some splice-variant antibody reactivity and showed multiple but less complex CD44 mRNA transcript patterns, compared with normal cells. Anaplastic cell lines did not react with variant antibodies and did not contain multiple-spliced CD44 transcripts. These data suggest that loss of alternatively-spliced CD44 may reflect a selection pressure in the evolution of anaplastic bladder cancers. PMID- 7543459 TI - Induction of protective immunity to schistosomiasis with immunologically cross reactive Lumbricus molecules. AB - Immunologically cross-reactive molecules of Schistosoma japonicum and Lumbricus terrestris were identified by antibodies derived from human and rodent sera. Pooled IgG from schistosomiasis patients but not uninfected individuals bound multiple antigens of identical molecular weight in both soluble S. japonicum worm antigen preparations (SWAP) and soluble earthworm preparations (SEWP). These antigens had molecular weights corresponding to 18, 40, 62, 64, 74, 97, and > 110 kDa. Three of these antigens of 74, 97 and > 110 kDa were immuno-affinity purified using antibodies derived from schistosomiasis patients' sera. Vaccination of mice with SEWP produced murine antibodies which bound parasite molecules of 40, 74, 97, and > 110 kDa and induced 36% protection from S. japonicum infection (P < 0.05). Antibody production to S. japonicum paramyosin, a molecule previously shown to induce protection from schistosome infection, was prominently expressed in the protected murine immune sera. The study shows that Lumbricus sp. represent a potential source for paramyosin and other candidate vaccine molecules for schistosomiasis. PMID- 7543460 TI - Ultrastructural and cytochemical aspects of the cuticle of adult Wuchereria bancrofti (Nematoda: Filarioidea). AB - Because of the practical limitations of obtaining viable adult forms of the Wuchereria bancrofti, the major species responsible for human lymphatic filariasis, only few ultrastructural studies were carried out. Adult worms present the cuticle as the interface structure between host and parasite. Cuticle structure and the demonstration of the presence of basic proteins, lipids, small amounts of terminal carbohydrate residues, phospholipids and collagen in the cuticle was undertaken on thin sections of embedded parasites. Using immunocytochemical methods, antigenic epitopes similar to those found in the extra cellular matrix of vertebrates were localized on thin sections of the Lowicryl embedded adult filariae. PMID- 7543461 TI - Skin microvascular architecture and perfusion studied in human postmastectomy oedema by intravital video-capillaroscopy. AB - Breast cancer treatment often causes chronic arm oedema. Oedema depends on the balance between microvascular filtration and lymph drainage, but little is known about the microvessels in postmastectomy oedema (PMO). We investigated cutaneous capillary density in PMO, since capillary density is one of the factors influencing fluid load on the lymphatic system. Video-capillaroscopy allows the skin microcirculation to be examined in vivo and recorded for later analysis. Patient arm volumes, measured optoelectronically, increased by 30 +/- 13%. The forearm skin of each arm was examined in 7 normal subjects (mean age 22 years) and 15 PMO patients (mean age 61 years). Native capillaroscopy was found to underestimate capillary density. Venous congestion increased the number of capillaries detected by native capillaroscopy by 26-28%. Fluorescein detected 19 27% more capillaries than native capillaroscopy. Using fluorescein, no significant difference in mean capillary density was found between the right (50 mm-2) and left (48 mm-2) arms of normal subjects (p = 0.53). Surprisingly, cutaneous capillary density in the swollen arm (33 mm-2) was not significantly less than in nonswollen arms (35 mm-2) of patients, despite a mean 14% increase in skin area. Capillary density decreased significantly with age. Relative to the volume and area changes, the density results indicated that cutaneous neovascularization had occurred in the swollen arm. PMID- 7543463 TI - Alterations of ocular nitric oxide synthase in human glaucoma. AB - PURPOSE: The authors recently reported that sites of outflow resistance and regulation in the human eye are highly enriched in the endothelial isoform of nitric oxide (NO) synthase (ecNOS). In vasculature, ecNOS activation is associated with altered vascular resistance and, in the stomach, defects in NO are associated with pathologic gastric hypertension. Because human glaucoma sometimes is associated with an increase in intraocular pressure and resistance changes in the aqueous outflow pathway (OP), the authors have investigated the possibility that alterations in NO or defects in NO-synthesizing tissues might exist in glaucomatous eyes. METHODS: Occurrence, distribution, and extent of sites of ocular NO production in the anterior segments of 16 normal eyes (10 patients) and 17 eyes (12 patients) with a history of primary open-angle glaucoma (POAG) were determined using the NO-indicator marker, NADPH-diaphorase (NADPH-d), which is known to colocalize with ecNOS immunoreactivity. Analysis of NADPH-d reactivity in tissues was combined with examination of overall cell distribution and use of neuron-specific markers. RESULTS: The ciliary muscle (CM) and OP of glaucomatous eyes showed marked differences in the amount and distribution of NADPH-d and alterations in gross structure. NADPH-d reactivity was decreased in trabecular meshwork (TM) and Schlemm's canal, and there was a marked reduction of anterior longitudinal CM fibers that insert near (and may normally regulate resistance in) the TM. CONCLUSION: Abnormalities in NO or NO-containing cells occur in POAG. These abnormalities may be causally related to glaucoma or may be a manifestation of the disease or its treatment. In either case, such alterations, together with recent pharmacologic studies showing that NO-mimicking nitrovasodilators alter IOP, indicate that NO has relevance to the course, treatment, or both, or some forms of this disease. PMID- 7543462 TI - Identification of an extensive system of nitric oxide-producing cells in the ciliary muscle and outflow pathway of the human eye. AB - PURPOSE: Nitric oxide (NO) is an important intracellular and intercellular regulator in nerve tissue as well as in vascular endothelium, smooth muscle, and certain other cell types. In the ocular anterior segment, studies in rat have revealed a comparatively restricted distribution of the NO synthetic enzyme, NO synthase (NOS-1). Recent physiological studies, however, have shown that NO mimicking nitrovasodilators can alter intraocular pressure in monkeys through an action (at least in part) on outflow resistance. The current studies, which determine the sites of NO synthesis in the human outflow pathway, were performed to provide an anatomic and biochemical explanation for these observations. METHODS: The occurrence and distribution of sites of ocular NO production in postmortem human eyes were determined using the isozyme-independent NO-indicator marker, NADPH-diaphorase (NADPH-d), together with direct biochemical assay and immunocytochemical localization of specific NO synthase (NOS) isoforms. RESULTS: The ciliary muscle (CM) and outflow pathway of normal human eyes were found to be substantially enriched in NADPH-d, the majority of which, by immunological analysis, consisted not of NOS-1 (brain or bNOS) but rather of NOS type 3 (endothelial cell or ecNOS). Biochemical analysis confirmed the NADPH-dependent production of NO and, unlike the primarily soluble distribution of bNOS, activity was found in both particulate and soluble fractions. NO reactivity was enriched in major sites of outflow resistance (trabecular meshwork and Schlemm's canal) as well as in collecting channels and was particularly prominent in the CM, especially in the anatomically distinct longitudinal subgroup of CM fibers that insert near (and may normally play a role in regulating resistance in) the trabecular meshwork. CONCLUSION: The human outflow pathway and CM are enriched sites of NO synthesis. These sites are anatomically distributed in such a manner as to suggest that one possible role for NO in the anterior segment may be to modulate outflow resistance either directly at the level of the trabecular meshwork, Schlemm's canal and collecting channels, or indirectly through alteration in the tone of the longitudinal CM. PMID- 7543464 TI - Integrin-dependent tyrosine phosphorylation in corneal fibroblasts. AB - PURPOSE: A major pathway for intracellular signaling from cell surface receptors, such as integrins, involves intracellular phosphorylation. In corneal fibroblasts, the authors have investigated the role of tyrosine phosphorylation in integrin-dependent cell adhesion to extracellular matrix. METHODS: Antibodies were used to detect phosphotyrosine-containing proteins, including focal adhesion kinase in lysates and immunoprecipitates of corneal fibroblasts. The authors used anti-phosphotyrosine antibodies to localize phosphotyrosines in fixed cultured corneal fibroblasts. Similarly, immunocytochemical detection of vinculin was used to identify focal adhesions, the subcellular structures in which integrins organize attachment to matrix extracellularly and to cytoskeletal components intracellularly. RESULTS: Suspension of corneal fibroblasts produced a dramatic decrease in detectable phosphotyrosines. During integrin-dependent fibroblast attachment to exogenously supplied fibronectin, the cytoplasmic phosphotyrosine kinase, focal adhesion kinase (FAK), pp125FAK, became tyrosine phosphorylated. However, FAK was not phosphorylated during fibroblast attachment to vitronectin or polylysine or when cells were kept in suspension. In addition, the treatment of suspended cells with antibody to the extracellular domain of fibronectin receptor caused FAK phosphorylation. Phosphotyrosine was colocalized with vinculin in newly formed focal adhesions. Focal adhesion formation was prevented by herbimycin A, an inhibitor of tyrosine kinases. CONCLUSIONS: In corneal fibroblasts, fibronectin receptor-specific signal transduction from extracellular matrix during the formation of focal adhesions requires tyrosine kinase activation, including phosphorylation of FAK. This underscores a role for the fibronectin receptor in signaling from the extracellular matrix in corneal fibroblasts. PMID- 7543466 TI - Incidental prostate cancer detected by transurethral resection. PMID- 7543465 TI - Human scleral permeability. Effects of age, cryotherapy, transscleral diode laser, and surgical thinning. AB - PURPOSE: To determine the in vitro permeability of human sclera to compounds varying in molecular weight. To evaluate the effects of age, cryotherapy, transscleral diode laser, and surgical thinning on scleral permeability. METHODS: Scleral tissue from 97 human eye bank eyes was tested individually in a two chamber Ussing apparatus with the following hydrophilic radiolabeled compounds on one side of the chamber: 5-fluorouracil, sucrose, dexamethasone, methotrexate, inulin, and three separate dextran polymers (MWt = 10,000, 40,000, and 70,000). Scleral hydration levels were obtained on 20 more scleral specimens. Additional groups of scleral specimens were treated with either a cryotherapy probe, a transscleral diode laser retinopexy probe, or partial thickness lamellar dissection, and specimens were mounted in the Ussing chambers for testing. Scleral tissue was digested to measure the amount of radioactivity present. Scleral sections were examined with electron microscopy. RESULTS: Scleral hydration was maintained during the perfusion. The mean scleral permeability (cm/second x 10(-6) +/- SD) was established for each of the above compounds. Age, cryotherapy, or diode laser treatment did not alter permeability or ultrastructure of the sclera. Surgical thinning significantly increased the scleral permeability to dexamethasone (P = 0.011) and methotrexate (P = 0.037). CONCLUSION: This study establishes baseline human scleral permeability to a series of hydrophilic compounds with various molecular weights. Age, cryotherapy, and diode laser treatment do not alter the permeability or ultrastructure of the sclera, whereas surgical thinning significantly increases permeability. PMID- 7543467 TI - Long-term follow-up and outcome of infants with non-organic failure to thrive. AB - Sixty-one children diagnosed as having non-organic failure to thrive during infancy were reviewed at an average of 5 years after their initial presentation, and were compared to a control group matched for age, sex, social class, and ethnic affiliation. Children with a previous history of failure to thrive were found to be shorter and, in particular, to gain less weight. Birthweight percentile and maternal height of children with failure to thrive were lower than those of matched children from the control group. They also had more learning difficulties and evidenced developmental delay. The degree of growth retardation and the duration of follow-up had no significant effect on the outcome. In contrast, birthweight, maternal height, and social status and, to a lesser extent, paternal parameters were good predictors of catching-up capabilities of these infants in terms of weight and height. Children who caught up faster had better school performances and came from families of higher socioeconomic status. Failure to thrive is a multifactorial process involving biological, nutritional, and environmental factors. All these components should be considered in long-term follow-up and management. PMID- 7543468 TI - Characterization of late gene promoters of Chlamydia trachomatis. AB - Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies. Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins. The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes. Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C. trachomatis genomic libraries. Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced. The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known. The 5' ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone like genes (hctA and hctB) were mapped, and a consensus -10 promoter region of TATAAT was derived from their upstream DNA sequences. In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial sigma 66, while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added sigma 66. These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on sigma 66, which is homologous to the major sigma factors of other eubacteria. PMID- 7543470 TI - Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP). AB - trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan. Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon. Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not. these studies also show that this regulation is mediated by the trp RNA-binding attenuation protein. Deletion and point mutations indicated that regulation is dependent on a series of G/UAG trinucleotide repeats surrounding the putative ribosome-binding site for trpG. Our results are consistent with a model in which the tryptophan-activated trp RNA-binding attenuation protein and ribosomes compete for binding to trpG mRNA. PMID- 7543469 TI - Conformational analysis of the Campylobacter jejuni porin. AB - The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins. PMID- 7543471 TI - Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection. AB - F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection. PMID- 7543473 TI - Substrate requirements for ErmC' methyltransferase activity. AB - ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented. PMID- 7543472 TI - Organization and expression of the Rhodobacter sphaeroides cycFG operon. AB - The Rhodobacter sphaeroides cycFG operon has been cloned, sequenced, and mapped to approximately coordinate 2500 of chromosome I. The cycF gene encodes cytochrome c554, a member of the class II family of soluble cytochrome c proteins. The cycF open reading frame includes a 20-amino acid extension at its N terminus which has not been detected in cytochrome c554. Antiserum against cytochrome c554 shows that this protein is localized to the periplasm of wild type cells, which suggests that this N-terminal extension functions as a signal peptide. The predicted cycG gene product is a diheme cytochrome c with a subunit molecular mass of approximately 32 kDa. While a cytochrome with the properties predicted for CycG has not been reported for R. sphaeroides, we have tentatively identified this protein as a heme-staining polypeptide that is associated with membranes. CycG could have an overall structure similar to that of several other electron carriers, since the similarity between the predicted amino acid sequence of CycG and other multiheme cytochrome c proteins extends throughout the polypeptide. The cycFG transcript is approximately 1,500 nucleotides long and has a single 5' end 26 nucleotides upstream of the start of cycF translation. Expression of cycFG is regulated at the level of mRNA accumulation, since approximately fivefold-higher levels of both cycF-specific transcript and cytochrome c554 protein are detected in cell extracts from aerobic cultures in comparison with those from anaerobically grown cells. Although cytochrome c554 was detected under all growth conditions tested, the highest levels of this protein were found when cells generate energy via aerobic respiration. PMID- 7543474 TI - Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon. AB - The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited. PMID- 7543475 TI - A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions. AB - Caulobacter crescentus differentiates prior to each cell division to form two different daughter cells: a monoflagellated swarmer cell and a nonmotile stalked cell. Thus, one might expect that developmentally expressed genes would be regulated by mechanisms different from those used to regulate the expression of the biosynthetic genes. To determine a consensus promoter sequence for genes involved in biosynthetic or housekeeping functions, DNA fragments containing the regulatory regions of the ilvD, ilvR, cysC, pleC, and fdxA genes were cloned. S1 nuclease protection mapping and primer extension techniques were used to identify the transcription initiation sites. Comparison of the regulatory regions of these genes with those of the published sequences of the ilvBN, rrnA, trpFBA, dnaA, dnaK, hemE, and rsaA genes has resulted in the identification of a putative promoter consensus sequence. The -35 region contains the sequence TTGACGS, which is similar to the Escherichia coli -35 region, while the -10 region, GCTANAWC, has a more balanced GC content than the corresponding region in E. coli. Oligonucleotide-directed site-specific mutagenesis of both the ilvBN and pleC promoters indicates that mutations that make a promoter more like the consensus result in increased promoter activity, while mutations decreasing similarity to the consensus result in decreased promoter activity. PMID- 7543477 TI - Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus. AB - Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5 bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70 dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences. PMID- 7543476 TI - CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258. AB - The CadC protein from the cadA cadmium resistance operon of Staphylococcus aureus plasmid pI258 regulates transcription of this system in vitro. The CadC protein was overproduced in Escherichia coli cells and partially purified. Gel shift assays of the proposed cadA operator/promoter region DNA showed specific association with the CadC protein. Control arsenic resistance operator/promoter DNA from the same plasmid was not shifted by the CadC protein. Cd2+, Bi3+, and Pb2+ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions -7 to +14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro. PMID- 7543478 TI - Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli. AB - The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB. PMID- 7543479 TI - Transcriptional analysis of rolling circle replicating plasmid pVT736-1: evidence for replication control by antisense RNA. AB - Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus. Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined. This plasmid possesses all the features necessary for rolling circle replication. The present study involved a transcriptional analysis of pVT736-1. Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter. Expression of these promoters varied with growth phase. In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein. To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis. PMID- 7543480 TI - Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12. AB - We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8 phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3 deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture. PMID- 7543483 TI - Monoclonal antibodies against native ovarian tumor cells: specificity and characterization of the antigen. A preliminary report. AB - Monoclonal antibodies were prepared against native human ovarian carcinoma cells derived from the ascitic fluid of a patient (BH). One antibody, HC7R7, was selected on the basis of its binding to tumor BH cells, to other ovarian tumor cell lines (CAOV-3 and GZL-8), but not to the patient's fibroblasts. One hundred cell smears from ascitic and pleural effusions of tumor-suspected patients were immunostained with HC7R7. All serous ovarian carcinomas and half of the breast carcinomas stained positive with HC7R7; cells from noncancer ovarian aspirates were negative. Mesothelial cells were also stained with HC7R7. A correlation was noted between HC7R7 and OC-125 staining of ovarian tumor cells but not between HC7R7 and c-neu staining of breast tumor paraffin sections. The location of HC7R7 positive material in ovarian tumor cell lines (CAOV-3 and GZL-8) differed from that in the breast tumor cell line (MCF-7). CAOV-3 and GZL-8 showed membrane binding while, in MCF-7, not fully identified intracellular organelles were stained. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots from membrane and cytosol fractions of GZL-8 and MCF-7 showed HC7R7 binding to three protein bands in the membrane fraction and to three other bands in the cytosol, all in the 29- to 68-kDa range. Two of the bands were glycoproteins. The only band that was different in the GZL-8 and MCF-7 cells was a 43-kDa glycoprotein, which was more pronounced in the MCF-7 cells. The possible significance of the new HC7R7 antibodies for detection and survey of ovarian malignancies is discussed. PMID- 7543482 TI - Effects of spo0 mutations on spo0A promoter switching at the initiation of sporulation in Bacillus subtilis. AB - Transcriptional analyses of the Bacillus subtilis sporulation initiator gene spo0A revealed that promoter switching from the vegetative (Pv) to the sporulation-specific (Ps) promoter did not occur in the spo0A, spo0B, spo0E, spo0F, and spo0H mutants. The sof-1 mutation in spo0A restored the promoter switching in the spo0F mutant. These results strongly suggest that Spo0A plays a central role in the regulation of its own promoter switching. PMID- 7543481 TI - Growth properties associated with A-U replacement of specific G-C base pairs in 16S rRNA from Escherichia coli. AB - Mutations that disrupt each of seven specific G-C base pairs in 16S rRNA from Escherichia coli confer loss of expression of a plasmid-encoded 16S rRNA selectable marker (spectinomycin resistance). However, A-U replacement of G-C base pairs at nucleotides 359/52 or 1292/1245 in 16S rRNA permits normal expression of the marker. By contrast, A-U replacements at 146/176, 153/168, 350/339, or 1293/1244 are associated with loss of expression of the marker. These genetic studies are designed to determine the importance of specific base pairs by assessment of the structural and functional impairments of 16S rRNA molecules resulting from expression of base pair substitutions at these positions. PMID- 7543484 TI - New knowledge in primary biliary cirrhosis. AB - The autoantibodies characteristic of the disease react against mitochondrial enzymes essential to the function of all body cells, yet the disease is highly focused on only one tissue: the epithelial lining of intrahepatic bile ductules. The explanation may lie in tissue-specific cell-surface expression of a triggering antigen. PMID- 7543485 TI - An approach to antiretroviral treatment of HIV disease. New approaches. AB - Viral resistance limits the value of drugs that act through competitive inhibition of HIV reverse transcriptase (RT). Thus, the emphasis of current research is on agents with other mechanisms. The possibilities include noncompetitive RT inhibitors, drugs against an entirely different enzyme--HIV proteinase, and measures to enhance immunity, either globally or against HIV specifically. PMID- 7543486 TI - Rapid genotyping of hepatitis C virus isolates by dideoxy fingerprinting. AB - A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a 33P-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6% acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a & 1b); a ddC reaction confirmed 1a and 1b typing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543487 TI - Attachment of an oligopeptide epitope to the C-terminus of recombinant SIV gp160 facilitates the construction of SMAA complexes while preserving CD4 binding. AB - A small 14 amino acid oligopeptide tag (termed SV5-Pk) was fused onto the carboxy terminus of simian immunodeficiency virus gp160 expressed from a recombinant baculovirus. The presence of the Pk tag had no obvious effect on the expression and glycosylation of gp160 and did not interfere either with CD4 binding or with cleavage at its maturation site by the protease furin. The presence of the Pk tag did, however, facilitate the simplified purification of full-length gp160 and its incorporation into immunogenic solid matrix-antibody-antigen (SMAA) complexes. PMID- 7543489 TI - Detection of California serogroup viruses using universal primers and reverse transcription-polymerase chain reaction. AB - Universal primers have been identified and a protocol developed that are suitable for rapid detection of California encephalitis (CE) complex viruses in a reverse transcription-polymerase chain reaction (RT-PCR) assay. These primers correspond to sequences in the coding regions of the G2 glycoprotein of the middle-size RNA segment. The identities of the amplified products were confirmed by sequencing on the clones or PCR products. The technique is capable of detecting 40 plaque forming units (PFU) directly on an ethidium bromide-stained agarose gel and the sensitivity increases to 0.4-1 PFU when a radiolabeled probe is used as the detector. PMID- 7543488 TI - Application of the polymerase chain reaction to the detection of African horse sickness viruses. AB - The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA. PMID- 7543492 TI - FK506 augments activation-induced programmed cell death of T lymphocytes in vivo. AB - FK506 is an immunosuppressive drug that inhibits T cell receptor-mediated signal transduction. This drug can induce immunological tolerance in allograft recipients. In this study, we investigated the in vivo effects of FK506 on T cell receptor-mediated apoptosis induction. Injection of anti-CD3 antibody (Ab) in mice resulted in the elimination of CD4+ CD8+ thymocytes by DNA fragmentation. FK506 treatment significantly augmented thymic apoptosis induced by in vivo anti CD3 Ab administration. Increased thymic apoptosis resulted in the disappearance of CD4+ CD8+ thymocytes after anti-CD3 Ab/FK506 treatment. DNA fragmentation triggered by FK506 was induced exclusively in antigen-stimulated T cells, since enhanced DNA fragmentation induced by in vivo staphylococcal enterotoxin B (SEB) injection was confirmed in SEB-reactive V beta 8+ thymocytes but not in SEB nonreactive V beta 6+ thymocytes. In addition to thymocytes, mature peripheral T cells also die by activation-induced programmed cell death. A similar effect of FK506 on activation-induced programmed cell death was observed in SEB-activated peripheral spleen T cells. In contrast, cyclosporin A treatment did not enhance activation-induced programmed cell death of thymocytes and peripheral T cells. Apoptosis is required for the generation and maintenance of self-tolerance in the immune system. Our findings suggest that FK506-triggered apoptosis after elimination of antigen-activated T cells may represent a potential mechanism of the immunological tolerance achieved by FK506 treatment. PMID- 7543490 TI - Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells. AB - 8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA). We observed that RI alpha decreased within 24 h of 8-Cl cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer. PMID- 7543493 TI - Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. AB - Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia. PMID- 7543491 TI - Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha. AB - IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression. PMID- 7543494 TI - Effects of rhIGF-I administration on bone turnover during short-term fasting. AB - Insulin-like growth factor-I (IGF-I) is a nutritionally dependent bone trophic hormone which stimulates osteoblast function and collagen synthesis in vivo and in vitro. We hypothesized that in the fasting state, IGF-I levels would decline significantly and would establish a model in which we could investigate the effects of IGF-I administration on bone turnover. We therefore studied 14 normal women ages 19-33 (mean, 24 +/- 4 [SD] years) during a complete 10-d fast. After 4 d of fasting, subjects were randomized to receive rhIGF-I or placebo subcutaneously twice a day for 6 d. Bone turnover was assessed using specific markers of formation (osteocalcin and type I procollagen carboxyl-terminal propeptide [PICP]) and resorption (pyridinoline, deoxypyridinoline, type I collagen crosslinked N-telopeptide [N-telopeptide] and hydroxyproline). Serum levels of PICP and osteocalcin decreased from 143 +/- 52 to 60 +/- 28 ng/ml (P = 0.001) and from 7.6 +/- 5.4 to 4.2 +/- 3.1 ng/ml (P = 0.001) respectively with 4 d of fasting. Urinary excretion of pyridinoline and deoxypyridinoline decreased from 96 +/- 63 to 47 +/- 38 nmol/mmol creatinine (P < 0.05) and from 28 +/- 17 to 14 +/- 11 nmol/mmol creatinine (P < 0.05) respectively. Mean IGF-I levels decreased from 310 +/- 81 to 186 +/- 78 ng/ml (P = 0.001). In the second part of the experimental protocol, serum osteocalcin and PICP levels increased 5- and 3 fold, respectively with rhIGF-I administration and were significantly elevated compared with the placebo group at the end of treatment (20.9 +/- 17.3 vs. 5.9 +/ 6.4 ng/ml for osteocalcin [P < 0.05] and 188 +/- 45 vs. 110 +/- 37 ng/ml for PICP [P < 0.05]). In contrast, all four markers of bone resorption, including urinary pyridinoline, deoxypyridinoline, N-telopeptide and hydroxyproline were unchanged with rhIGF-I administration. This report is the first to demonstrate that bone turnover falls rapidly with acute caloric deprivation in normal women. RhIGF-I administration uncouples bone formation in this setting by significantly increasing bone formation, but not resorption. These data suggest a novel use of rhIGF-I to selectively stimulate bone formation in states of undernutrition and low bone turnover. PMID- 7543495 TI - Treatment of marrow stroma with interferon-alpha restores normal beta 1 integrin dependent adhesion of chronic myelogenous leukemia hematopoietic progenitors. Role of MIP-1 alpha. AB - The mechanisms by which interferon-alpha (IFN-alpha) restores normal hematopoiesis in chronic myelogenous leukemia (CML) are not well understood. We have recently demonstrated that IFN-alpha acts directly on CML hematopoietic progenitors to restore their adhesion to marrow stroma by modulating beta 1 integrin receptor function. In the present study we examined the effect of IFN alpha treatment of marrow stroma on subsequent adhesion of CML progenitors. Stromal layers were preincubated with IFN-alpha (10,000 microns/ml) for 48 h. Subsequent coincubation with CML progenitors for 2 h resulted in significantly increased adhesion of CML progenitors. We demonstrated that alpha 4 beta 1 and alpha 5 beta 1 integrin receptors were involved in the enhanced adhesion of CML progenitors, suggesting that IFN-alpha-treated stroma can upregulate CML integrin function. This effect is due, at least in part, to IFN-alpha-induced increased stromal production of the chemokine macrophage inflammatory protein-1 alpha (MIP 1 alpha), which upregulates beta 1 integrin-dependent adhesion of CML progenitors to stroma. Thus, IFN-alpha treatment of marrow stroma restores beta 1 integrin dependent adhesion of CML progenitors, at least in part through induction of MIP 1 alpha production. These observations provide further insights into mechanisms by which IFN-alpha may restore normal hematopoiesis in CML. PMID- 7543501 TI - Postnatal development and synaptic connections of hilar mossy cells in the hippocampal dentate gyrus of rhesus monkeys. AB - Mossy cells of the hippocampal dentate gyrus were analyzed through postnatal development. At birth, a few thorny excrescences were found on the proximal dendrites of mossy cells, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number and complexity until the third month. After that age, the complexity of thorny excrescences is so great that an increase in spine density can be seen only in electron microscopic preparations. An increase in the number of pedunculate spines per unit length of distal dendrite was detected via light microscopy during the first 9 postnatal months. The somata and dendrites of mossy cells displayed adult-like characteristics after the ninth postnatal month. Mossy fiber terminals at birth frequently displayed immature ultrastructural characteristics and formed synapses with dendritic shafts and spines. At later postnatal ages and in adults, axospinous synapses were found almost exclusively. This is consistent with the postnatal development of the complex spines of the mossy cells. Axons of mossy cells were generally confined to the hilus in our 150-microns-thick sections, where they gave rise to several collaterals. The axon terminals from these collaterals formed asymmetric synapses with dendrites and dendritic spines in the hilar region of the dentate gyrus. These data provide the first anatomical evidence that hilar mossy cells of the primate dentate gyrus have excitatory projections similar to their equivalent cell type in subprimates. The present study indicates that mossy cells of the dentate gyrus are in a more advanced stage of development at birth and mature faster than similar neurons of the human hippocampus. This may represent a faster maturation of hippocampal circuitry in nonhuman primates compared to that in the human. PMID- 7543499 TI - Unexpected dystonia while changing from clozapine to risperidone. PMID- 7543500 TI - Critique of the Canadian Multicenter Placebo-Controlled Study of Risperidone and Haloperidol. PMID- 7543502 TI - Allergen-specific IgE production of committed B cells from allergic patients in vitro. AB - The allergen-specific in vitro IgE synthesis in blood leukocytes from patients with allergy was monitored outside the pollen season with recombinant and natural pollen allergens and was compared with the total IgE production. The addition of interleukin-4 (IL-4) and antibody to CD40 increased the amount of total IgE by up to 20-fold in the culture supernatants of peripheral blood leukocytes from patients with allergy that could be antagonized by a neutralizing anti-IL-4 antibody in a dose-dependent manner. In contrast to total IgE, the amount of allergen-specific IgE was not affected by IL-4, and anti-CD40 or anti-IL-4, treatment. With oligonucleotides specific for IgE, complementary DNA from the amino terminal of the IgE heavy chain could be reversely transcribed and amplified by polymerase chain reaction from RNA of patients' unstimulated blood leukocytes, indicating that the IgE secretion in the cultures is due to a de novo IgE synthesis. It is concluded that the peripheral blood of patients with allergy contains long-lived allergen-specific B cells, which are not responsive to IL-4 mediated signals. These results may have implications for attempts to modulate specific IgE production in allergic patients with cytokines or cytokine antagonists. PMID- 7543503 TI - Allergen challenge-induced entry of alpha 2-macroglobulin and tryptase into human nasal and bronchial airways. AB - BACKGROUND: Microvascular-epithelial exudation of bulk plasma may characterize inflammatory airway diseases. This study compares the acute allergen challenge induced mast cell and exudative responses in nasal and bronchial airways. The focus is on alpha 2-macroglobulin as an index of luminal entry of plasma exudates. METHODS: Separate nasal and bronchial allergen challenges were carried out outside the pollen season in eight patients with pollen-induced seasonal allergic rhinitis. The levels of different-sized plasma proteins (albumin molecular weight, 66,000 d and alpha 2-macroglobulin molecular weight, 725,000 d) and tryptase were determined in pre- and postchallenge nasal lavage and bronchoalveolar lavage (BAL) fluids. Diluent and increasing doses of allergen were sprayed into the right nasal cavity, and each challenge was followed by a nasal lavage (volume, 15 ml) with a "nasal pool" device (recovery, > 80%). Endobronchial allergen challenge (individual doses) and BAL (volume, 2 x 25 ml) were performed in a lobe bronchus through a fiberoptic bronchoscope (recovery, 30%). Saline challenge and BAL were carried out in the contralateral lung as control. RESULTS: The levels of albumin, alpha 2-macroglobulin, and tryptase increased dose-dependently in postchallenge nasal lavage fluids (p < 0.05) and correlated to nasal symptoms. In particular, albumin and alpha 2-macroglobulin correlated (r = 0.98, p < 0.001). Both alpha 2-macroglobulin and tryptase, but not albumin, were increased in BAL fluids from the allergen-challenged side (p < 0.05). CONCLUSION: Local allergen challenge causes luminal entry of tryptase and alpha 2-macroglobulin in the nose and bronchi of patients with allergy. We suggest that mast cell and plasma exudation responses may be similar in human nasal and bronchial airways and that albumin levels (in BAL fluids) may not well reflect the exudation process in bronchial airways. PMID- 7543497 TI - Collagen-induced arthritis in the BB rat. Prevention of disease by treatment with CTLA-4-Ig. AB - Antigen-specific T cell activation requires two independent signalling events, one mediated through T cell receptor engagement by the antigen-presenting cell expressed peptide/class II major histocompatibility complex, and the second through the cognate interactions of costimulatory molecules expressed on the T cell and antigen-presenting cell. There is evidence from in vitro and in vivo experimental systems suggesting that the CD28/B7 costimulatory pathway is crucial for induction of maximal T cell proliferation and T helper-B cell collaboration for IgG production. This pathway can be blocked by CTLA-4-Ig, a soluble form of CTLA-4 which binds with high avidity to the CD28 ligands, B7-1 and B7-2. Here, we show that CTLA-4-Ig treatment prevents clinical and histological manifestations of disease in a collagen-induced arthritis model of rheumatoid arthritis in the diabetes resistant BB/Wor rat, when therapy is initiated before immunization with bovine type II collagen (BIIC). Anti-BIIC antibody titers are reduced in CTLA-4 Ig-treated rats compared to diseased control animals. Histologically, joints from CTLA-4-Ig-treated animals show no histological abnormalities, in contrast to control antibody-treated animals, which show complete erosion of the articular cartilage and bone. Despite the efficacy of CTLA-4-Ig in preventing clinical and histological signs of arthritis and reducing antibody responses to BIIC, delayed type hypersensitivity responses to collagen 18 d or more after CTLA-4-Ig treatment ends are similar in CTLA-4-Ig-treated and untreated rats, suggesting that the prolonged disease suppression observed does not result from induction of T cell anergy. PMID- 7543504 TI - The production of medical audiovisual teaching materials--Chinese style. AB - The production of medical audiovisual material for the advancement of medical education in China is a problem requiring an urgent solution. To achieve faster, better and more economical results, the production of audiovisual material must be undertaken with careful planning and efficient management. A systematic approach is essential to control production. The procedures described are: leadership and management; choice of subject and medium; production; distribution and evaluation. PMID- 7543498 TI - Colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils. AB - Since colchicine-sensitive microtubules regulate the expression and topography of surface glycoproteins on a variety of cells, we sought evidence that colchicine interferes with neutrophil-endothelial interactions by altering the number and/or distribution of selectins on endothelial cells and neutrophils. Extremely low, prophylactic, concentrations of colchicine (IC50 = 3 nM) eliminated the E selectin-mediated increment in endothelial adhesiveness for neutrophils in response to IL-1 (P < 0.001) or TNF alpha (P < 0.001) by changing the distribution, but not the number, of E-selectin molecules on the surface of the endothelial cells. Colchicine inhibited stimulated endothelial adhesiveness via its effects on microtubules since vinblastine, an agent which perturbs microtubule function by other mechanisms, diminished adhesiveness whereas the photoinactivated colchicine derivative gamma-lumicolchicine was inactive. Colchicine had no effect on cell viability. At higher, therapeutic, concentrations colchicine (IC50 = 300 nM, P < 0.001) also diminished the expression of L-selectin on the surface of neutrophils (but not lymphocytes) without affecting expression of the beta 2-integrin CD11b/CD18. In confirmation, L-selectin expression was strikingly reduced (relative to CD11b/CD18 expression) on neutrophils from two individuals who had ingested therapeutic doses of colchicine. These results suggest that colchicine may exert its prophylactic effects on cytokine-provoked inflammation by diminishing the qualitative expression of E-selectin on endothelium, and its therapeutic effects by diminishing the quantitative expression of L-selectin on neutrophils. PMID- 7543496 TI - Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis. AB - Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS. PMID- 7543507 TI - Ethnic-based learner response to child accident prevention illustrations. AB - This article presents findings of a field study during development of audiovisual learning materials for child accident prevention in South Africa. The results indicate some common features, on the one hand, as well as specific differences, on the other hand, in the appropriateness of instructional illustrations for children across certain ethnic and language groups. PMID- 7543506 TI - An 'interactive' poster display. AB - Posters are a commonly used method of communication at scientific meetings. How can a significant amount of information be provided on a limited surface area, in a manner that will interest the viewer? This article describes a simple and inexpensive answer. By being interactive, a poster display overcomes the problems normally associated with posters. PMID- 7543505 TI - Colour teaching aids in oral disease for hospital ward staff. AB - Provision of adequate care for the mouths of patients in hospitals, particularly those such as the elderly in long-stay wards, is frequently overlooked. An important contributing factor is the lack of dental training of the carers. As part of a wider initiative to address this problem a set of encapsulated, full colour illustrated sheets has been designed by a team of dentists for use by medical, nursing and allied staff at ward level. The 20 sheets illustrate features of a healthy mouth together with 16 common oral disorders. Each set is held in a loose-leaf ring binder. Over 100 sets are now in use within the West of Scotland, with an encouraging response to the circulated appraisal forms. PMID- 7543508 TI - Trichrome staining of Gyrodactylus sclerites and soft tissues following fixation in ammonium picrate-glycerin, including an improved rendition of the haptoral bars of G. turnbulli. AB - A simple technique using modified Mallory stain in the transferral of Gyrodactylus specimens from ammonium picrate-glycerin to a permanent mountant is described. Hamuli, their connecting bars and the penis sclerites are well defined by the technique as are muscles and tendons, cell nuclei, tegument and gland cells. As well as being useful in the study of general anatomy, the technique enhances the observation of the taxonomically important ventral and dorsal bars. In order to show this, improved illustrations of the dorsal and ventral bars of G. turnbulli are given along with explicit demonstrations of differences in morphology of the ventral bars of G. bullatarudis and G. rasini-two easily confused species. PMID- 7543509 TI - Relative perforin- and Fas-mediated lysis in T1 and T2 CD8 effector populations. AB - IFN-gamma-secreting T1 and IL-5-secreting T2 subsets of CD8 effectors were generated in vitro using freshly isolated cells from wild-type and perforin knockout mice stimulated with allogeneic Ag-presenting cells. Both T1 and T2 effectors from wild-type mice exhibited perforin-mediated cytolysis. T1, but not T2, populations from perforin knockout mice displayed significant lysis by the Fas-mediated pathway. Th1 cells have recently been shown to be regulated by Fas and we speculate that Fas-mediated mechanisms are involved in the regulation of both Th1 and T1 populations of T cells. PMID- 7543510 TI - Antigen-dependent clonal expansion of a trace population of antigen-specific CD4+ T cells in vivo is dependent on CD28 costimulation and inhibited by CTLA-4. AB - The importance of CD28 costimulation to a primary T cell response in vivo was assessed in an adoptive transfer system where a small population of peptide specific CD4+ TCR transgenic T cells can be physically tracked. Ag-dependent clonal expansion of the transgenic T cells in draining lymph nodes was blocked by cyclosporin A and required a CD28 signal that was completely inhibited by CTLA-4 Ig or a combination of anti-B7-1 and anti-B7-2 mAbs, but not by either Ab alone. In vivo treatment with the combination of anti-B7-1 and anti-B7-2 mAbs also blocked conversion of the Ag-specific T cells to the activated phenotype. In contrast, anti-CTLA-4 Fab greatly enhanced the in vivo clonal expansion of the Ag specific T cells. These results suggest that Ag-driven proliferation and phenotype conversion of naive CD4+ T cells is dependent on CD28-derived signals and is inhibited by CTLA-4. PMID- 7543513 TI - Stimulation of germinal center B lymphocyte proliferation by an FDC-like cell line, HK. AB - To identify the signals given to the germinal center (GC) B cells by FDC, we have established an FDC-like cell line, HK. HK cells have the important functions of FDC, which are the preferential binding and stimulation of GC B cells. Cell-cell interaction between IgD- B cells and HK cells resulted in the rescue of these cells from apoptosis, whereas the majority of unbound B cells or B cells cultured in the absence of HK cells underwent apoptosis. We investigated the effects of HK cells on the subpopulations of tonsillar B lymphocytes that are at different stages of maturation and differentiation. The subpopulations of tonsillar B cells were purified by panning and/or MACS according to the surface expression of IgD, CD38, and CD44. Although HK cells alone did not stimulate B cell proliferation, HK cells in the presence of either anti-mu or anti-CD40 significantly enhanced the proliferation of B cells. The data show that HK cells preferentially bind, rescue, and stimulate GC B cells (IgD-CD38+) generating CD38-CD44+ memory B cells. The costimulation activity of HK cells is also provided in a form of soluble factor(s). Hence, the phenotypic and functional analysis of HK cells suggests that these cells may be derived from FDC. PMID- 7543512 TI - IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes. AB - IL-10 affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells IL-10 stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, STAT1 alpha and STAT3, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel IL-10-stimulated STAT protein with an M(r) of 70 kDa that is recognized by the anti-STAT3 Ab but is not observed in T cells. IL-10 treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of tyk2 and Jak1, but not Jak2 or Jak3. Selective modulation of immune responsiveness by IL-10 in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs. PMID- 7543511 TI - Human anti-mouse xenorecognition. Provision of noncognate interactions reveals plasticity of T cell repertoire. AB - DAP.3 transfectants expressing native H-2E molecules with or without human LFA-3 and ICAM-1 (intercellular adhesion molecule-1) failed to induce proliferation by human peripheral blood T cells. Introduction of sequence from the DR beta 2 domain into the H-2E molecule led to the induction of detectable proliferation, which was substantially augmented by co-expression of human LFA-3 and ICAM-1 to levels comparable to those induced by DAP.3 cells co-expressing wild-type DR alloantigens with human LFA-3/ICAM-1. In marked contrast, cells expressing native H-2A molecules together with human accessory molecules provoked strong primary proliferative responses. The results of Ab inhibition experiments confirmed that this was caused by direct xenorecognition. In limiting dilution assays the frequency of anti-H-2A, IL-2-secreting, CD4+ human T cells was only fivefold lower than that measured against a DR alloantigen expressed on the same background. No measurable frequency was recorded against H-2E-expressing cells. Evidence to suggest that this difference was a result of isotype-specific differences in the interaction with CD4 was provided using transfectants expressing DR alloantigens with either the H-2E or H-2A beta 2 domain. DR molecules with the H-2A beta 2 domain stimulated a substantially stronger response than those with the H-2E beta 2 domain. These results challenge the view that xenogeneic T cell responses between evolutionarily distant species are weak; further emphasize the influence of the interaction between the T cell co-receptor molecule CD4, with its MHC class II molecular ligand on the strength of primary xenoresponses; and suggest that MHC class II isotypes may differ substantially in their interaction with CD4. PMID- 7543514 TI - Interactions between IL-4, anti-CD40, and anti-immunoglobulin as activators of locomotion of human B cells. AB - The locomotor properties of small, surface IgM+ and surface IgD+ B cells from the human tonsil were studied using polarization assays and collagen gel invasion assays. These cells gave poor locomotor responses when freshly isolated from the tonsil; but 30 to 40% of the cells polarized and invaded collagen gels after overnight culture in IL-4 or anti-CD40. IL-13 had a similar but weaker effect. Culture with anti-CD40 and IL-4 together gave a higher proportion of polarized cells than either alone, and culture in anti-CD40, IL-4, and anti-IgM gave a still higher proportion (> 60% of B cells polarized). Polarization increased gradually, during hours of culture, unlike the typical rapid response to chemoattractants. We also studied the immediate (< 30 min) effects of chemoattractants on B cell polarization. B cells cultured overnight then washed, but not B cells fresh from the tonsil, polarized immediately in response to anti CD40. Similar responses to anti-IgM and anti-IgD, both pre- and postculture were also observed, but the response of cultured cells was stronger. IL-4-cultured B cells invaded collagen gels incorporating anti-IgM, anti-IgD, or anti-CD40 in higher numbers than control gels. Most of the invading cells were surface IgM+. These results suggest that locomotor activation in B cells requires two steps. The capacity for locomotion is growth-related and is first activated by IL-4 or by anti-CD40, enhanced by the presence of anti-IgM. Following activation, the cells respond rapidly to chemoattractants such as anti-Ig or anti-CD40. PMID- 7543515 TI - Costimulation of human CD4+ T cells with LFA-3 and B7 induce distinct effects on AP-1 and NF-kappa B transcription factors. AB - We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either B7 or LFA-3 resulted in distinct cytokine profiles. We now demonstrate that B7, but not LFA 3, strongly costimulated IL-2 transcription and mRNA expression in CD4+ T cells. Maximal increase in IL-2 transcription was recorded with CHO-DR/B7/LFA-3, suggesting a cooperative effect of B7 and LFA-3 at the transcriptional level. Gel shift analysis demonstrated that stimulation of CD4+ T cells with CHO-DR and staphylococcal enterotoxin A was sufficient to induce significant amounts of NF kappa B binding proteins, whereas induction of AP-1 binding proteins required costimulation. LFA-3 induced moderate levels of AP-1, but did not influence the levels of NF-kappa B, while B7 costimulation strongly induced both AP-1 and substantially enhanced NF-kappa B binding proteins. The CHO-DR/B7/LFA-3 triple transfectant induced a further increase in AP-1 and NF-kappa B binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the NF-kappa B complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The AP-1 binding proteins contained c-Jun, Jun-D, and Fra-1, but marginal amounts of Jun-B and c-Fos. Our results indicate distinct effects of B7 and LFA-3 costimulation on the activity of AP-1 and NF-kappa B. These may partly account for the differential effects of B7 and LFA-3 costimulation on IL-2 expression. PMID- 7543516 TI - Soluble antigen can impede affinity maturation and the germinal center reaction but enhance extrafollicular immunoglobulin production. AB - The primary immune response to T cell-dependent Ags develops in two pathways. These comprise the extrafollicular pathway, in which foci of Ab-secreting cells develop, and the intrafollicular pathway that gives rise to germinal centers and affinity maturation. We have previously shown that de-aggregated (4-hydroxy-3 nitrophenyl) acetyl (NP) coupled to the protein carrier human serum albumin (HSA), (NP-HSA), injected 6 days after challenge immunization with aggregated NP HSA, resulted in impaired development of NP-specific, higher-affinity cells. Studies presented here describe the cellular basis underlying this impairment of affinity maturation. Using multiparameter flow cytometry, we show that mice injected with soluble NP-HSA ("tolerant" mice) develop significantly fewer NP binding IgG1+ B220+ cells of germinal center origin than do the control ("immune") mice. In addition, using immunohistology, we noted that the spleens of tolerant mice had a marked reduction in the number of germinal centers that contained lambda-bearing cells, these being characteristic of the NP response in C57BL/6 mice. Curiously, germinal centers in the spleens of tolerant mice had more than twice the volumes of those in the immune spleens. In contrast to its effect on the germinal center pathway, soluble Ag enhanced the extrafollicular pathway, reflected by the increased numbers of B cells secreting IgM and IgG1 Abs specific for NP, HSA, and undefined Ags. Thus, soluble NP-HSA given after challenge immunization can impede affinity maturation of NP-specific cells, but enhance the extrafollicular pathway. These results are discussed in the context of the known capacity of some persisting Ags, e.g., malaria parasites, to frustrate affinity maturation and memory cell generation. PMID- 7543518 TI - Human anti-Gal heavy chain genes. Preferential use of VH3 and the presence of somatic mutations. AB - Anti-Gal is the most abundant natural Ab known in humans. It interacts specifically with the carbohydrate structure Gal alpha 1-3 Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope), constitutes approximately 1% of circulating Ig, and is found in all three isotypes in the serum. Anti-Gal is produced in Old World monkeys, apes, and humans, and in no other mammalian species. The objective of this study was to determine the VH genes involved in the synthesis of anti-Gal. B lymphocytes from various individuals were transformed by EBV, the clones producing anti-Gal were isolated, the specificity and affinity of the Abs were determined, and the VH genes were sequenced. The affinity of anti-Gal clones for the free radiolabeled alpha-galactosyl epitope ranged between 1.1 x 10(6) M-1 and 5 x 10(8) M-1. Eight of the nine clones studied used VH3 family genes and one clone used a VH1 family gene. Four of the five VH3 genes used were found to form a cluster of related sequences, suggesting that functional constraints may lead to the use of VH3 genes with structural motifs that are suited for specific interactions with the alpha-galactosyl epitope. Comparison with known germline sequences for all of the clones studied and analysis of autologous germ-line genes in two of the clones indicate that anti-Gal VH genes undergo somatic mutations. These somatic mutations may provide a pool of variants that are available for affinity maturation. PMID- 7543517 TI - Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. AB - To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance. PMID- 7543519 TI - Identification of recombinant filarial proteins capable of inducing polyclonal and antigen-specific IgE and IgG4 antibodies. AB - Filarial infection is characterized by an immune response associated with the production of Ag-specific IgG4 and IgE and IL-4 and IL-5. To identify filarial Ags capable of inducing such responses and to analyze the role Ags themselves play in sustaining it, 24 recombinant filarial parasite proteins were screened for their ability to be recognized by sera from 67 individuals with tissue invasive filarial infections. Among the recombinant proteins that were recognized by IgG4 or IgE Abs in 25% of the sera or more, two were selected on the basis of their ability to elicit polyclonal and Ag-specific IgE/IgG4 Abs in vitro. Ov27 (analogous to Ov7/cystatin, a cysteine protease inhibitor) and OvD5B (analogous to Ov33, an aspartyl protease inhibitor) induced both a polyclonal and Ag specific IgE/IgG4 response that was blocked by neutralizing Abs to IL-4 and to IL 13 or by soluble IL-4 receptors. Recombinant human IFN-gamma and IL-12 also led to a decrease in the production of polyclonal and Ag-specific IgE/IgG4 Abs. In addition, these two recombinant proteins preferentially stimulated the secretion of IL-4, IL-5, and IL-10 (in contrast to IFN-gamma). The data suggest that certain epitopes on filarial Ags preferentially elicit a Th2-type response and provide an in vitro model for dissecting the mechanisms underlying this preferential response. PMID- 7543520 TI - Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor infiltrating lymphocytes. AB - A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA-A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti-peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24+ B cell lines pulsed with the peptide and HLA-A24+ tyrosinase+ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24+ melanoma patients. PMID- 7543522 TI - Variation of LPS-binding capacity, epitope expression, and shedding of membrane bound CD14 during differentiation of human monocytes. AB - The myeloid differentiation Ag CD14 is considered to play a critical role in the binding of LPS to monocytes. To determine if differences in LPS-binding capacities of cells could reflect a variability of CD14 molecules, we analyzed the interactions of various reagents with these molecules in human blood monocytes and in promyelocytic (HL60) and monocytic (THP-1) cell lines. The expression of CD14 epitopes was analyzed with the fluorescent anti-CD14 mAbs My4 and LeuM3. Expression of LPS-binding sites (LPS+ molecules) was detected with LPS FITC. THP-1 cells stimulated with calcitriol (VitD3), as well as the majority of blood monocytes (50-90%) were My4+/LPS+. However, untreated THP-1 cells, and a substantial population (10-50%) of human monocytes from healthy donors, were My4+/LPS-, thus suggesting the existence of CD14 isoforms with different LPS binding capacities. In line with this assumption, monocytes stimulated with PMA selectively shed LeuM3+ molecules, but almost no My4+ and LPS+ constituents. Analysis of monocytes after treatment with phosphatidylinositol-specific phospholipase C indicated that among CD14 molecules with LPS-binding capacity, some are susceptible and others are resistant to the enzyme, each type being mainly expressed by a different monocyte subset. Studies of uninduced and chemically induced THP-1 cells showed that wheat-germ agglutinin blocked the binding of My4 to constitutive, but not to chemically inducible CD14. The overall results suggest the existence of at least three different forms of CD14, which may reflect different stages of cell maturation. PMID- 7543521 TI - Enhancement by the complement membrane attack complex of tumor necrosis factor alpha-induced endothelial cell expression of E-selectin and ICAM-1. AB - Although TNF-alpha and several products of the activated complement system (e.g., C3b, iC3b, and C5a) are known to modulate endothelial cell function in vitro, relatively little is known about the potential modulatory role of the membrane attack complex (MAC) in endothelial cell activation. Using an in vitro neutrophil endothelial adhesion assay and a quantitative whole cell ELISA to measure endothelial E-selectin and intracellular adhesion molecule-1 (ICAM-1) expression, we examined the modulatory role of the MAC in TNF-alpha-induced neutrophil endothelial cell adhesive interactions. Activation of quiescent human umbilical vein endothelial cells (HUVECs) with TNF-alpha results in a concentration dependent increase in neutrophil adhesion measured at 4 h. Assembly of sublytic concentrations of the MAC on endothelial cells did not result in changes in neutrophil-HUVEC adhesion measured at 4 h. Activation of HUVECs with TNF-alpha followed by assembly of the MAC resulted in a marked increase in neutrophil binding as compared with that observed in cells treated with TNF-alpha alone. Blocking studies of mAb revealed that in either TNF-alpha-stimulated or TNF-alpha and MAC-activated endothelial cells enhanced neutrophil binding was nearly entirely attributable to E-selectin and ICAM-1. This conclusion was further supported by a whole-cell ELISA, which provided evidence that the MAC augments TNF-alpha-induced up-regulation of both E-selectin and ICAM-1. This study provides data that support the conclusion that the distal complement system (MAC) can enhance TNF-alpha-induced proinflammatory endothelial cell functions. PMID- 7543523 TI - Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein. AB - The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33 kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased. PMID- 7543528 TI - Epitope specificity of monoclonal anti-beta 2-glycoprotein I antibodies derived from patients with the antiphospholipid syndrome. AB - beta 2-Glycoprotein I (beta 2GPI) has been identified as a cofactor in the recognition of the phospholipid Ag cardiolipin (CL) by anticardiolipin Ab (aCL) purified from patients with autoimmune diseases. However, there is considerable controversy as to the exact nature of the epitopes to which these Abs are directed. mAb derived from patients with the antiphospholipid syndrome bound to CL only in the presence of beta 2GPI. Synthetic peptides that span the fifth C terminal domain of beta 2GPI supported the binding of one of the mAbs to CL in a beta 2GPI-free system. These peptides possessed the phospholipid binding sequence Cys281-Lys-Asn-Lys-Glu-Lys-Lys-Cys288. Three of the mAbs bound to beta 2GPI that had been adsorbed on gamma-irradiated microtiter plates. Binding to beta 2GPI was inhibited in a dose-dependent manner by the peptides from the carboxyl-terminal end of beta 2GPI and soluble beta 2GPI, indicating that the mAb bound to peptides and beta 2GPI in free solution. Thus, mAbs derived from patients with the antiphospholipid syndrome have specificity for epitopes on the fifth domain of beta 2GPI. Our results support the idea that beta 2GPI acts as a primary Ag for these Abs. PMID- 7543527 TI - Self-thyroid epithelial cell (TEC)-reactive CD8+ T cell lines/clones derived from autoimmune thyroiditis lesions. They recognize self-thyroid antigens directly on TEC to exhibit T helper cell 1-type lymphokine production and cytotoxicity against TEC. AB - Self-thyroid epithelial cell (TEC)-reactive CD8+ and CD4+ T cell lines were established by culturing T cells that infiltrate in autoimmune thyroiditis lesions. We investigated the properties of CD8+ T cell lines and clones in comparison with previously characterized CD4+ T cell lines/clones. Although the recognition of self-Ag by anti-TEC CD4+ T cell lines/clones required the cooperation of syngeneic spleen cells as APC, a representative CD8+ line (N4C) was stimulated with syngeneic TEC in the absence of APC. Precise analysis of MHC restriction using N4C-derived clones revealed that CD8+ clones recognize self-Ag on TEC in the context of class I MHC molecules. Most CD8+ clones were also found to express TCR with V beta specificities that were different from those observed for anti-TEC CD4+ clones. N4C cells produced IL-2, IFN-gamma, and TNF-alpha beta, but not IL-4 and IL-5 after stimulation with TEC, thus exhibiting the profile of lymphokine production similar to that expressed by CD4+ Th1 on one hand, but on the other, they showed the functional property that has not been observed for anti-TEC CD4+ clones. Namely, they elicited appreciable levels of cytolytic effects on syngeneic TEC in a short-term (4-h) 51Cr release assay. Thus, these results indicate that self-TEC-reactive CD8+ T cell lines/clones recognize Ag directly on TEC in a class I MHC-restricted way so as to exhibit various functions including the Th1-like profile of lymphokine production and anti-TEC cytolysis. The results are also discussed in terms of the nature of self-Ag presented with class I MHC molecules on TEC, as well as the potential roles of anti-TEC CD8+ T cells in the pathogenesis of thyroiditis. PMID- 7543524 TI - L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin. AB - Emigration of leukocytes at sites of inflammation is initiated by the selectin family of carbohydrate-binding adhesion molecules. Molecular crossbridges initiate rolling of cells along the vascular endothelium where chemokines such as IL-8 and platelet activating factor (PAF) may be presented to their receptors on the leukocyte surface resulting in cell stimulation. Integrin activation appears to be a requirement for subsequent cell localization and diapedesis into the tissue. Several recent reports have demonstrated that ligation and cross-linking of neutrophil L-selectin results in neutrophil activation, including intracellular calcium release, superoxide production, and induction of mRNA for production of IL-8 and TNF-alpha. The purpose of this study was to examine whether ligation and cross-linking of L-selectin would specifically result in activation of beta 2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-microns diameter) were adsorbed with albumin or fibrinogen and added in excess to human neutrophils in a shear-stirred suspension. Following stimulation the kinetics of bead capture by neutrophils was continuously measured in real time on the flow cytometer. The onset of bead binding was detected in the presence of extremely low concentrations of PAF (10 pM) or formyl peptide (0.2 nM) stimulation. Ligation of L-selectin with whole IgG DREG200 or DREG56 Ab, but not controls (anti-CD44, -CD45, -CD11a), resulted in a significant potentiation of bead binding. Cross-linking F(ab')2 fragments of DREG200 with a goat anti-mouse F(ab')2 secondary Ab also stimulated beta 2 integrin-dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing gamma 4 or gamma 1 isotypes of human Fc domain also stimulated cell adhesion when cross-linked. Surface expression of CD18 and an activation dependent epitope, as detected with mAb24, also increased in response to L selectin cross-linking. Cross-linking L-selectin induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells. We propose that cross-linking of L-selectin results in a cell signal that directly stimulates beta 2-integrin adhesive responses. PMID- 7543525 TI - The antirheumatic drug disodium aurothiomalate inhibits CD4+ T cell recognition of peptides containing two or more cysteine residues. AB - The mechanism of action of antirheumatic gold drugs, such as disodium aurothiomalate (Au(I)TM), has not been clearly identified. Gold drugs inhibit T cell activation induced by mitogen and anti-CD3 mAb in vitro at relatively high concentrations. However, since gold drugs fail to induce immunosuppression in vivo, the pharmacologic relevance of this finding is doubtful. In this study, we asked whether Au(I)TM interferes with processing and presentation of defined Ags to T cells. Using a panel of murine CD4+ T cell hybridomas, we found that low concentrations of Au(I)TM (< or = 10 microM) led to a markedly reduced IL-2 release of T cell hybridoma clones that recognized peptides containing two or more cysteine (Cys) residues, such as bovine insulin A1-14. Since disodium thiomalate alone had no effect, the inhibition was due to Au(I). IL-2 production induced by anti-CD3 mAb stimulation was not affected by the low concentration of Au(I)TM used. Au(I)TM had no effect on the presentation of peptides containing no or only one Cys residue(s). In contrast to the unmodified insulin peptide A1-14, Au(I) could not inhibit recognition of an insulin peptide in which Cys residues in positions 6 and 11 were replaced by serine. Most likely, the observed inhibition is mediated by formation of chelate complexes between Au(I) and two Cys thiol groups of the affected antigenic peptides. The peptide-specific inhibitory effect of Au(I) on Ag presentation described here might contribute to the therapeutic effect of Au(I) compounds in rheumatoid arthritis. PMID- 7543529 TI - TABS, a T cell activation antigen that induces LFA-1-dependent aggregation. AB - We describe here a mAb, DATK44, which induces homotypic aggregation of TK1 cells (a CD8 lymphoma). The glycoprotein recognized by DATK44 is of approximate m.w. 50 kDa and is expressed by monocytes, neutrophils, and subsets of lymphocytes, as well as on the high endothelial venule in peripheral and mesenteric lymph nodes. We named this Ag TABS (T cell activation B cell subset Ag), as TABS appears on T lymphocyte activation and is expressed at low and high levels by B cells. TABS is differentially regulated during T lymphocyte development, CD4+veCD8+ve thymocytes being TABShigh, while single positive CD4+ve and CD8+ve thymocytes are TABSdull CD4-veCD8-ve thymocytes are clearly split into dull and bright populations by the mAb. On exit from the thymus, T lymphocytes cease to express TABS, but T lymphocyte activation results in re-expression of TABS. TABS also shows tight coregulation with heat stable Ag on resting lymphocytes, but coexpression of these two molecules is lost upon lymphocyte activation. DATK44-induced aggregation of TK1 cells is temperature sensitive and blocked by pretreatment of the cells with metabolic inhibitors, genestein, dibutyl cAMP or cytochalasin B, while colchicine, staurosporin, sphingosine, okadaic acid, and W7 are without effect. DATK44-induced TK1 cell aggregation appears to be mediated by the LFA-1 pathway, as aggregation is blocked by anti-LFA-1 and anti-ICAM-1 mAbs but not by Abs capable of blocking CD44 and alpha 4 beta 7-mediated adhesion. Thus, TABS appears to be an adhesion inducer that selectively activates LFA-1-mediated lymphocyte aggregation. PMID- 7543530 TI - Cross-linking cell surface class II molecules stimulates Ig-mediated B cell antigen processing. AB - Th cells bind to peptide-class II complexes presented on B cell surfaces. Recent evidence indicates that upon cross-linking, class II molecules transduce signals that modulate a variety of B cell functions. One possible function of class II signaling is to regulate the assembly of processed Ag-class II complexes. Here we show that cross-linking B cell surface class II molecules augments the processing and presentation of an Ek-restricted Ag to a specific T cell hybrid. Significantly, class II cross-linking only affects processing initiated by Ag binding to the surface Ig. The processing of Ag taken up by fluid phase pinocytosis is not affected by class II cross-linking, nor is the presentation of an antigenic peptide that does not require processing. Augmentation of Ag processing is enhanced by treatment of B cells with dibutyryl cAMP, a second messenger in the class II signaling pathway. The cross-linking of class II molecules does not alter the rate or number of Ig molecules internalized or the biosynthesis or expression of Ek molecules. Moreover, changes in the expression of the B7 family of costimulatory molecules or the adhesion molecule LFA-1 (CD11a/CD18) induced by class II cross-linking do not appear to account for the augmentation of processing observed here. Thus, the cross-linking of class II molecules on B cell surfaces selectively stimulates Ig-mediated Ag processing, indicating that a step in this pathway is a target of class II-mediated signaling events. PMID- 7543532 TI - Activated T cells can induce high levels of CTLA-4 expression on B cells. AB - Engagement of the TCR/CD3 complex together with ligation of CD28 by its counterstructures B7-1 (CD80) and B7-2 (CD86) on APC are required for mitogenic T cell activation. After activation, T cells not only express B7-1 and B7-2 molecules, but a second receptor for the B7 ligands, CTLA-4, can be found on their surfaces. We here show that B cells can be induced to express CTLA-4 on the plasma membrane. Similar to what has been reported for T cells, CTLA-4 expression on B cells was transient. Purified B cells did not express CTLA-4 when mitogenically activated with alpha IgM and CD40 Ab, but did express the molecule when cultured in the presence of membranes from activated T cells, which suggests that induction of CTLA-4 expression on B cells was dependent on direct cell-cell contact of B lymphocytes and activated T cells. CTLA-4 molecules isolated from either T or B cells were biochemically indistinguishable. Moreover, because the ability of chimeric B7-1/Ig proteins to bind to activated B cells was correlated with CTLA-4 expression levels on these cells, we conclude that B cell-expressed CTLA-4 has ligand binding capacity. These data suggest that costimulatory receptors and their specific ligands not only play a role in T cell stimulation, but contribute in a direct fashion to the regulation of B cell responses. PMID- 7543531 TI - Neuropeptides modulate immune deviation induced via the anterior chamber of the eye. AB - Ag presentation via the anterior chamber of the eye results in a form of immune deviation termed anterior chamber-associated immune deviation (ACAID). The hallmarks of this response are the suppression of delayed-type hypersensitivity with simultaneous induction of Ab production. In this study, we examined the role of the neuropeptides vasoactive intestinal peptide (VIP) and substance P (SP) and found that the levels of these two peptides are controlled by neurogenic stimulation of the eye by light, and that these molecules determine the outcome of Ag presentation in the eye. Mice reared under diurnal conditions had VIP in the iris and ciliary body (not free in aqueous humor) and low levels of SP. Mice that were reared in the dark (or dark-adapted) did not contain detectable levels of VIP but had high levels of SP. The adaptation of diurnal mice to the dark eliminated VIP and increased SP, while adapting dark-reared mice to the diurnal cycle increased VIP and reduced SP. We then tested the hypothesis that immune reactions resulting from Ag presentation in the eye were linked to SP and VIP. We found that a VIP receptor antagonist, when injected into the eye with Ag, reversed ACAID in diurnal mice, while a SP receptor antagonist restored ACAID to dark-adapted mice. We further determined that injection of Ag or TNF-alpha induced VIP release, while SP was liberated into the aqueous humor following reexposure of dark-reared mice to light. Our results demonstrate a close linkage of intraocular immune reactions to neuropeptide levels in the eye. PMID- 7543526 TI - Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis. AB - Experimental autoimmune encephalomyelitis (EAE), induced in Lewis rats by injection of myelin basic protein (MBP) and adjuvant, is a T cell-mediated autoimmune disease. Earlier studies from our laboratory have shown that oral administration of guinea pig MBP before encephalitogenic challenge induces T cell anergy and results in the suppression of clinical signs and CNS histopathologic changes of EAE. In contrast, oral administration of rat MBP did not confer a similar degree of protection. This study was undertaken to determine the tolerogenicity of the synthetic peptide 68-88 derived from guinea pig (GP) MBP and rat MBP. These peptides differ by a single amino acid at position 80. Lewis rats fed GP 68-88 were protected from EAE induced with GP 68-88 or rat 68-88. In contrast, feeding rats 68-88 did not protect the animals from challenge with either peptide. Measurement of the frequency of peptide-reactive Th1 cells showed results consistent with the clinical picture. The in vitro proliferative response was significantly suppressed following oral administration of either whole GP MBP, the GP peptide, or the rat peptide, irrespective of clinical status. These results extend our earlier observation at the whole molecule level that GP but not rat MBP confers oral tolerance. These findings suggest that small structural differences at the amino acid level can produce dramatic differences in clinical outcome, with important implications for the design of multiple sclerosis clinical trials. PMID- 7543534 TI - CD28-mediated signaling in vivo prevents activation-induced apoptosis in the thymus and alters peripheral lymphocyte homeostasis. AB - Activation of T lymphocytes can result in a functional immune response, anergy or apoptosis. Functional T cell activation requires the interaction of the TCR with Ag presented by MHC molecules on APC concurrent with appropriate interactions between cell surface accessory molecules. Interestingly, the level of CD28 expression is regulated during T cell development as well as during T cell activation and proliferation, suggesting that CD28 could play a role in determining the outcome of activation of TCR during T cell ontogeny. We identify, herein, a novel function of murine CD28 in the regulation of activation-induced apoptosis in thymocytes. In vivo, or combined in vivo and in vitro treatment with mAbs to CD28 prevents apoptosis of CD4+CD8+ thymocytes induced by Abs to the TCR complex. Prolonged administration of anti-CD28 Abs increased the number of both CD4+CD8- and CD4-CD8+ T cells in the thymus, while the number of CD4+CD8+ T cells is relatively unchanged. Furthermore, this treatment leads to a dramatic enlargement of peripheral lymphoid organs characterized primarily by the expansion of B cells. The number of CD4+CD8- T cells in the spleen of anti-CD28 treated mice is also moderately increased, while the number of CD4-CD8+ cells is relatively unchanged. PMID- 7543533 TI - IgE-dependent expression of mRNA for IL-4 and IL-5 in human lung mast cells. AB - By using the reverse transcriptase (RT)-PCR and in situ hybridization we have studied the expression of mRNA for IL-5 and IL-4 in human lung mast cells induced by cross-linkage of high affinity Fc epsilon Rs. Lung mast cells were purified using affinity magnetic selection with mAb YB5.B8 against c-kit to achieve a final mast cell purity > 93%. Purified mast cells were precultured with stem cell factor (SCF) (10 ng/ml) and myeloma IgE (3 micrograms/ml) for 16 h before challenge with anti-IgE (1 or 10 micrograms/ml). IgE-dependent activation of lung mast cells caused expression of IL-5 mRNA, which was evident by 2 h and persisted for up to 48-72 h in all of 12 experiments, whereas IL-4 mRNA expression was of a shorter duration and was demonstrable in 6 of 13 experiments. We confirmed that mast cells, and not T cells, were the source of these cytokine messages by using reverse transcriptase-PCR in cell preparations containing known numbers of mast cells and T cells, in situ hybridization in enriched mast cell preparations, and double in situ hybridization-immunocytochemical staining. IL-5 mRNA expression did not require the pretreatment of cells with SCF, whereas expression of IL-4 mRNA seemed to require both anti-IgE and SCF. The strength of IL-5 mRNA signal was related to anti-IgE concentration. Immunoreactive IL-5 was detectable 8 h after anti-IgE challenge, and 10(6) mast cells generated a mean of 731 +/- 400 pg of IL-5 into the supernatant during 48-h culture, but no IL-4 product was detectable. These findings demonstrate the capacity of human lung mast cells to transcribe IL-4 and IL-5 after IgE-dependent activation and to synthesize and release immunoreactive IL-5. PMID- 7543535 TI - Characterization of distinct stages during the differentiation of human CD69+CD3+ thymocytes and identification of thymic emigrants. AB - During thymocyte development, CD69 expression is induced at an early stage of positive selection. To examine the differentiation of human CD69+CD3+ thymocytes, discrete phenotypes were defined by the relative expression of CD69, CD27, CD1, and CD45RA/RO: CD3+CD69- thymocytes were CD27-CD1+CD45RA-/RO+ (phenotype I), whereas the CD3+ CD69+ population could be subdivided into CD27-CD1+CD45RA-/RA+ (phenotype II), CD27+CD1+CD45RA-/RO+ (phenotype III), CD27+CD1-CD45RA-/dull/RO+ (phenotype IV), and CD27+CD1-CD45RA+/ROdull (phenotype V) thymocytes. Phenotype I thymocytes were CD4+CD8 alpha beta + double positive (DP). Phenotype II thymocytes contained DP and CD4+CD8 alpha dullCD8 beta- cells, whereas phenotype III thymocytes were DP and mostly CD4dullCD8 alpha beta+, indicating that CD27 on DP cells may be a marker for CD8-committed cells. Results obtained with SCID-hu mice, transplanted with human fetal thymus and liver, showed that immature human CD69-CD3+ thymocytes were corticosteroid-sensitive, whereas essentially all CD69+CD3+ cells were resistant. During differentiation of one cohort of corticosteroid-resistant CD69+CD3+ thymocytes, phenotype II and III thymocytes, disappeared within a week, whereas the percentage of phenotype IV and especially V thymocytes increased, suggesting that the latter represent the end stages in differentiation. Recent thymic emigrants in SCID-hu mice were identified as CD69 CD27+CD1-CD45RA-/dull/RO+ or CD45RA+/ROdull cells. Because phenotype IV and phenotype V thymocytes rapidly lose CD69 expression in cell culture, these thymocytes are probably the cells that are exported from the thymus. In conclusion, after the acquisition of CD69, thymocyte differentiation appears to continue in an ordered pattern, and cells that eventually leave the thymus are CD69-CD1-CD45RA+ or CD45RA-/dull. PMID- 7543536 TI - Ly-6C is a marker of memory CD8+ T cells. AB - This study examined long-term phenotypic and functional effects of TCR ligation in vivo. Flow cytometric analysis of T cells from mice treated with anti-CD3 revealed an increase in CD44 expression in both the CD4+ and CD8+ populations. The phenotypic changes were a result of TCR engagement, because treatment with staphylococcal enterotoxin B (SEB) resulted in a preferential increase in CD44 expression on the SEB-reactive V beta 8 T cells. In addition, the percentage of cells expressing Ly-6C increased among the CD8+ subset after anti-CD3 treatment and in the V beta 8+ CD8+ subset after treatment with SEB. Finally, the TCR transgenic (Tg) mouse strain 2C was used to confirm that the phenotypic changes can be induced by exposure to a physiologic ligand (H-2Ld). Before treatment, nearly all of the Tg+CD8+ cells were CD44low/Ly-6C-. Tg+ peritoneal exudate T cells isolated from mice challenged with P815 cells (H-2Ld) up-regulated Ly-6C and secreted higher levels of IFN-gamma on a per Tg+ CD8+ T cell basis after treatment. Taken together, these data indicate that in vivo TCR/CD3 engagement results in phenotypic and functional changes in T cells. Furthermore, Ly-6C expression correlates with an increase in IFN-gamma production after antigenic stimulation of CD8+ T cells, suggesting that it is a "memory" marker that correlates with Ag-specific functional changes in CD8+ T cells. PMID- 7543537 TI - Dissection of strain difference in acquired protective immunity against Mycobacterium bovis Calmette-Guerin bacillus (BCG). Macrophages regulate the susceptibility through cytokine network and the induction of nitric oxide synthase. AB - Protection against infection with intracellular pathogens operates in two stages, early innate resistance and late acquired protective immunity (API), in inbred mouse strains. Although both C57BL/10 (B10) and BALB/c mice bear the susceptible phenotype of innate resistance, Calmette-Guerin bacillus (BCG) vaccination generated efficient API in B10 but not in BALB/c mice. Employing a specific nitric oxide (NO) synthase inhibitor, we revealed that NO production plays a pivotal role in the API of B10 mice. Consistent with this, expressions of the inducible isoform of NO synthase (iNOS) protein and mRNA were significantly higher in the spleen of B10 mice than in that of BALB/c mice. Furthermore, IFN gamma, a potent inducer of iNOS, and mRNAs for IL-12 (p40); an inducer of IFN gamma and IL-2 were also vigorously expressed in the spleen of B10 mice compared with that of BALB/c mice. In an attempt to clarify the mechanism by which the different capacities for API are generated, we analyzed the cytokine network between T cells and macrophages in both B10 and BALB/c mice. We found that multiple functions of macrophages, which include capacities to express IL-12 (p40) mRNA in response to BCG and to express mRNAs for iNOS and IL-12 (p40) in response to IFN-gamma, were impaired in BALB/c mice as compared with B10 mice. However, T cells appeared to express comparable level of IFN-gamma mRNA in both strains when stimulated with IL-12. Taken together, these results indicate that the macrophage functions play a pivotal role in both the induction and effector phases of API to determine the susceptibility of mice to BCG infection. PMID- 7543543 TI - The spectrum of hepatitis C antibody positive disease in a teaching hospital. AB - OBJECTIVE: To survey clinical associations of a positive hepatitis C virus antibody (HCV-antibody) test. DESIGN: Retrospective analysis of all positive HCV antibody tests reported by a Virology Department. SETTING: A complex secondary and tertiary care teaching hospital. PATIENTS: 144 confirmed cases with HCV antibody in serum, identified from approximately 2,500 clinical specimens tested. RESULTS: Over 99% of positive tests were in the following groups: liver dysfunction; multiply transfused individuals; intravenous drug abusers; patients with bleeding disorders; and renal replacement therapy. Amongst the patients with liver disease--the only group in which a known epidemiological association was not the indication for testing--over 25% of patients had no risk factor identifiable, representing 7.5% of the total group of HCV-antibody positive patients. CONCLUSION: Selective HCV-antibody testing in a U.K. hospital population reveals a substantial population of positive patients, and risk factors for hepatitis C are not always present. PMID- 7543540 TI - Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation. AB - Adhesion to the adhesion protein, VCAM-1, on vascular endothelium is proposed to be an important factor in the selective accumulation of eosinophils at sites of allergic inflammation. To determine whether eosinophil adhesion to VCAM-1 is also associated with an alteration of eosinophil function, human peripheral blood eosinophils were isolated from allergic donors and incubated in VCAM-1-coated wells. Spontaneous adherence of isolated eosinophils to VCAM-1-coated wells was greater than cells incubated in FCS-treated control wells (38.0 +/- 1.6% vs 17.1 +/- 1.9%, n = 16, p < 0.0001). In addition, eosinophils incubated in VCAM-1 coated wells spontaneously generated modest but significant amounts of superoxide anion (O2-; 2.0 +/- 1.3 vs 00.5 +/- 0.5 nmol/5 x 10(5) cells, n = 9, p = 0.029). Moreover, when 100 nM FMLP was added to eosinophils in the presence of VCAM-1, significantly greater O2- generation occurred (7.2 +/- 0.9 vs 5.4 +/- 1.0 (FCS control) nmol/5 x 10(5) cells, n = 9, p = 0.009). Adhesion, as well as the spontaneous and enhanced O2- generation to FMLP activation, was blocked by the monoclonal anti-alpha 4 integrin Ab, HP 1/2, implying involvement of an alpha 4 integrin-VCAM-1 interaction. In contrast, the anti-CD18 mAb, L130, inhibited the spontaneous and enhanced O2- generation to FMLP without affecting adhesion, suggesting an involvement of CD18 molecule(s) only in VCAM-1-enhanced respiratory burst. Finally, 1 microM genistein, a tyrosine kinase inhibitor suppressed the VCAM-1-enhancing effect on eosinophil O2- generation and VCAM-1-induced tyrosine phosphorylation, suggesting a role for tyrosine phosphorylation in this eosinophil functional up-regulation. Our observations suggest that eosinophil adhesion to VCAM-1 may be an important step in determining the eventual functional activity of these cells as they migrate from the circulation to the airways and contribute to the allergic inflammatory process. PMID- 7543539 TI - P-selectin regulates platelet-activating factor synthesis and phagocytosis by monocytes. AB - Adhesion molecules on endothelial cells or platelets may regulate localization and activation of leukocytes at sites of tissue injury, infection, or thrombosis. In these studies, we found that human peripheral blood monocytes adhered specifically to immobilized P-selectin (CD62P), Chinese hamster ovary cells transfected with a cDNA for P-selectin, or endothelial cells stimulated to express P-selectin on the cell surface. P-selectin did not directly stimulate synthesis of the lipid autoacoid platelet-activating factor (PAF); however, incubation on immobilized P-selectin primed monocytes for increased synthesis of PAF in response to opsonized zymosan particles. P-selectin did not stimulate increased surface expression of integrin CD11b/CD18 and did not enhance binding of iC3b-coated erythrocytes, a CD11b/CD18-mediated functional response. P selectin increased PAF production by monocytes incubated with unopsonized zymosan particles that stimulate this response by interaction with the beta-glucan receptor. Further, phagocytosis of unopsonized zymosan particles, another response triggered by the beta-glucan receptor, was increased following the adherence of monocytes to P-selectin. These data suggested that P-selectin primed monocytes for increased PAF synthesis through regulation of the beta-glucan receptor or regulation of signal transduction mechanisms that are linked to the receptor. P-selectin expressed on endothelial cells or platelets may serve both to localize monocytes at sites of vascular inflammation or thrombosis and to prime the cells for subsequent responses that augment inflammation. PMID- 7543538 TI - Endotoxin receptors (CD14) are found with CD16 (Fc gamma RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase. AB - CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein on the surfaces of monocytes and polymorphonuclear leukocytes (PMN) that binds and initiates cellular responses to bacterial LPS. PMN also contain an intracellular pool of CD14 that can be deployed rapidly to the cell surface in response to stimulation with a variety of agonists. To determine which of the well-characterized subcellular compartments of PMN contains CD14, cells were cavitated and fractionated on Percoll gradients. The gradient fractions were assayed for CD14 by ELISA and Western blot and for the marker proteins beta-glucuronidase (azurophil granules), vitamin B12 binding protein (specific granules), alkaline phosphatase (secretory vesicles and plasma membrane), and HLA (plasma membrane). Approximately one-half of the CD14 ran with plasma membrane fractions and one half with intracellular membranes of light density. Both intracellular and cell surface CD14 were associated tightly with membrane, and both forms showed identical electrophoretic mobility. The intracellular CD14 was clearly not present in azurophil granules or specific granules, but ran precisely with alkaline phosphatase, a marker for secretory vesicles. Parallel studies showed that an additional GPI-linked protein, Fc gamma RIII (CD16), also fractionated precisely with CD14 and alkaline phosphatase. Association of CD14 with secretory vesicles were confirmed by studies on cells stimulated with the formyl peptide fNLLP for 20 min at 37 degrees C before fractionation. This treatment caused translocation of CD14 from intracellular fractions to plasma membrane fractions. No release of the specific granule marker vitamin B12 binding protein was observed under these conditions, whereas two other GPI-anchored proteins, alkaline phosphatase and CD16, moved coincidentally with CD14 to comigrate with the plasma membrane. Time course studies of CD14 and CD16 surface expression confirmed the rapid and coordinate up-regulation of these proteins. Thus, the intracellular compartment containing CD14 and CD16 had the properties of secretory vesicles. These vesicles may represent a specialized membrane domain of PMN enriched in GPI-anchored proteins. PMID- 7543541 TI - IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor potentiate basophil mediator release stimulated by eosinophil granule major basic protein. AB - We have examined the potential of IL-3, IL-5, and granulocyte-macrophage (GM)-CSF to enhance basophil activation by eosinophil granule major basic protein (MBP). Preincubating basophil-containing mononuclear cells with 0.01 to 10 ng/ml IL-3 or IL-5 for 15 min at 37 degrees C caused a concentration-dependent enhancement of histamine release stimulated by 1.5 microM MBP. Statistically significant enhancement was evident at 1 ng/ml and was maximal at 10 ng/ml. Preincubation with GM-CSF similarly enhanced MBP-induced histamine release. A 10- to 15-min preincubation with IL-5 maximally increased the level of MBP-stimulated histamine release. Preincubation of cells with 10 ng/ml IL-3 or IL-5 reduced the MBP concentrations required for histamine release by three- to fourfold and enhanced the rate of MBP-induced histamine release. MBP-stimulated histamine release before or after cytokine priming was independent of cytotoxicity as measured by 51Cr release. Consistent with a direct action of the cytokines on basophils, flow cytometric analysis demonstrated the presence of IL-3 and GM-CSF receptors on basophils. MBP also stimulated low levels of leukotriene C4 (LTC4) release from basophils of 84 to 99% purity, and experiments using enriched (18-63%) basophil preparations demonstrated that preincubation with IL-3, IL-5, and GM-CSF also potentiated MBP-stimulated leukotriene C4 release up to threefold in parallel with histamine release. These results indicate that IL-3, IL-5, and GM-CSF may contribute to the pathogenesis of allergic and other disorders characterized by eosinophilia in part through potentiation of basophil mediator release stimulated by MBP. PMID- 7543542 TI - Characterization of HLA-A 0201-restricted cytotoxic T cell epitopes in conserved regions of the HIV type 1 gp160 protein. AB - CTL activity is a major component of the host immune response associated with control of HIV replication in the course of infection. Emerging populations of HIV overcome the protective effector mechanisms with variant sequences unrecognized by CTL. Therefore, a critical element for containment of virus spread might be the establishment of an immune response against highly conserved epitopes. In this study, we selected a panel of nonamer or decamer peptides, with demonstrated binding affinity for HLA-A 0201, to define novel highly conserved envelope-derived epitopes of HIV-1. CTL activities were characterized from PBMC of five HLA-A2+, HIV-1-infected individuals given recombinant gp160. CTL activity derived from patient PBMC stimulated in vitro with peptide was demonstrated against at least two novel minimal env-encoded conserved epitopes. One epitope, KLTPLCVTL (aa 120-128), is highly conserved among HIV-1 strains of the B subtype. Analysis of a CTL clone reactivity to a distinct epitope (aa 814-823) demonstrated fluctuations in the recognition of peptides corresponding to natural virus variants found in vivo. PMID- 7543544 TI - Diagnostic and therapeutic problems due to cat scratch disease. PMID- 7543545 TI - Pemphigus sera recognize conformationally sensitive epitopes in the amino terminal region of desmoglein-1. AB - To identify regions of the Desmoglein-1 (Dsg1) extracellular domain that are targeted by pemphigus antisera, cDNA sequences encoding various regions of the Dsg1 extracellular domain were ligated to sequences encoding the E-cadherin extracellular anchor and transmembrane and cytoplasmic domains. These constructs were then expressed in mammalian cells, and pemphigus sera were tested for the ability to recognize the Dsg1 extracellular domains. When analyzed by immunoblot, very few pemphigus foliaceus sera recognized the Dsg1 domains. To determine whether pemphigus sera recognize non-denatured Dsg1 domains, constructs were expressed in cultured cells and tested for reactivity with pemphigus sera using live-cell immunofluorescence. The pemphigus foliaceus sera reacted strongly with the Dsg1 extracellular domain by live-cell immunofluorescence and recognized predominantly the amino-terminal region of the Dsg1 extracellular domain. In addition, sera from patients with pemphigus vulgaris also demonstrated strong reactivity with the Dsg1 extracellular domain when tested using live-cell immunofluorescence. In contrast, sera from normal human controls and sera from bullous pemphigoid patients did not react with the Dsg1 extracellular domain. These data indicate that both pemphigus foliaceus and pemphigus vulgaris sera react with conformationally sensitive epitopes in the amino-terminal region of Dsg1. PMID- 7543546 TI - Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues. AB - We performed a systematic study on deiminated proteins present in rat epidermis. Proteins extracted from various epidermal samples were resolved by either one- or two-dimensional gel electrophoresis and Western blotted to nitrocellulose membranes. Deiminated proteins were detected by modification of citrulline residues followed by probing with an anti-modified citrulline monospecific antibody. The cornified layer of adult plantar skin gave multiple series of isoelectric variants, most of which were found to be differentially deiminated type II keratins (60 kDa, and 67 kDa or above). The whole epidermis of 5-day-old rat back skin showed isoelectric variants of 60-kDa keratin as major deiminated components, and deiminated 55-kDa keratin and deiminated filaggrin as minor spots. In addition, we found highly deiminated proteins (200-220 kDa) thought to be derived from trichohyalin. The immunoreactivity of deiminated proteins was mainly localized in the granular and cornified layers of epidermis. Co localization of deiminated filaggrin and keratins in the granular layer suggests the possible role of protein deimination during the terminal stage of epidermal differentiation. PMID- 7543547 TI - Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration. AB - Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration. PMID- 7543548 TI - Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++) induced keratinocyte differentiation. AB - Proliferation and differentiation in many cells are linked to specific changes in transmembrane ion fluxes. Previously, we have identified a nonspecific cation channel in keratinocytes, which is permeable to and activated by Ca++. To test whether this cation channel might serve as a pathway for Ca++ entry, we examined the effect of blocking this channel on membrane currents, markers of differentiation, and intracellular Ca++. In patch clamp studies, 10(-8) to 10(-6) M amiloride decreased the single-channel open probability. The same concentrations of amiloride inhibited the calcium-induced formation of cornified envelopes and activity of transglutaminase in a dose-dependent fashion. Amiloride inhibited the long-term rise of intracellular Ca++ induced by raised extracellular Ca++, without blocking the initial increase of intracellular Ca++. Amiloride at concentrations of 10(-7) to 10(-3) M did not change the resting intracellular pH of keratinocytes, although concentrations of 10(-6) M or greater inhibited the recovery from NH4(+)-induced acidification. To test whether the effect of amiloride was toxic, we measured DNA synthesis in the presence or absence of amiloride. DNA synthesis was unchanged, suggesting that amiloride's actions were not due to toxic effects. Although the exact mechanisms of amiloride's action remains to be determined, these experiments suggest that this compound may inhibit keratinocyte differentiation by blocking the nonspecific cation channel. PMID- 7543550 TI - Reduced type I and type III procollagens in photodamaged adult human skin. AB - We have quantitatively assessed the relation between type I and type III procollagen precursor levels and the severity of clinical photodamage in human skin. Levels of procollagen, pN collagen (collagen without the carbroxypropeptide), and/or pC collagen (collagen without the aminopropeptide) were determined by radioimmunoassay, Western blot, and immunohistology in punch biopsy specimens from mildly and severely photodamaged forearm skin and from sunprotected underarm and buttock skin of the same subjects. Collagen precursor levels in forearm and underarm skin were expressed relative to buttock levels for comparison. In the mildly photodamaged group, collagen precursors in the forearm did not differ from those in the underarm by any measurement, except for type I collagen precursors measured by radioimmunoassay, which were reduced 16%. In severely photodamaged forearm skin, both type I and type III collagen precursor levels, measured by radioimmunoassay, were significantly reduced (approximately 40%). Western analysis revealed similar significant reductions in type I and type III collagen precursor levels in severely photodamaged forearm skin compared with the sun-protected underarm. Immunohistology localized both type I and III pN collagens predominantly to the extracellular papillary dermis. Relative staining intensities of type I and type III pN collagen were also significantly reduced in severely photodamaged forearm skin. Multiple linear regression modeling of all data demonstrated that reductions in collagen precursor levels were significantly correlated (p < 0.03) with the severity of photodamage, but not with chronologic age. These data demonstrate, by three independent methods, coordinate reductions of both type I and type III collagen precursors in photodamaged human skin, and the degree of reduction correlated with the degree of photodamage. It is likely that such changes in collagen precursors lead to reduced levels and/or altered organization of fibrillar collagen, and thus may contribute to the wrinkled appearance of photodamaged human skin. PMID- 7543549 TI - Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent. AB - The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the anti-genicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculo-proteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P 40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens. PMID- 7543552 TI - When families request that 'everything possible' be done. AB - The paper explores the ethical and psychological issues that arise when family members request that "everything possible" be done for a particular patient. The paper first illustrates this phenomenon by reviewing the well known case of Helga Wanglie. We proceed to argue that in Wanglie and similar cases family members may request futile treatments as a means of conveying that (1) the loss of the patient is tantamount to losing a part of themselves; (2) the patient should not be abandoned or disvalued in any way; or (3) the patient is owed special obligations by virtue of the special relationship in which the family and the patient stand. We maintain that families can best express these important messages by caring for patients, rather than by making requests for futile interventions. Likewise, when life-sustaining measures are futile, health providers can best fulfill their professional obligations by assuring patients' dignity and comfort, rather than by applying futile interventions. PMID- 7543553 TI - Effects of undernutrition and diabetes on serum and liver mRNA expression of IGFs and their binding proteins during rat development. AB - The purpose of the present study was to investigate the influence of nutrients and insulin on IGFs and their binding proteins (IGFBPs) during the fetal and neonatal periods of three rat populations: (a) rats undernourished by a 35% reduction in the diet from day 16 of gestation, (b) streptozotocin-induced diabetic rats from the same day, or 4 days after birth, and (c) control rats. Fetuses from the diabetic population showed a decrease in insulinemia at 19 and 21 days, along with an increase in glycemia at all stages. Neither glycemia nor insulinemia changed in the fetuses of undernourished mothers, but body weight was decreased at birth. Serum IGF-II decreased at 18 and 19 days of gestation in fetuses from undernourished mother, and increased at 18, 19 and 21 days in fetuses from diabetic mothers. Serum IGFBPs of low molecular weight (IGFBP-1 and IGFBP-2) increased in the three fetal populations studied, although no changes in serum IGFBPs were found from the effect of undernutrition or diabetes, but fetal liver IGFBP-1 mRNA expression was found to decreased in undernourished and diabetic animals as compared with controls. In neonatal rats, body weight, insulinemia and serum GH decreased in both undernourished and diabetic rats vs controls, while glycemia decreased in the undernourished and increased in the diabetic group. Serum IGF-II decreased only in diabetic rats and serum IGF-I decreased in both groups. The neonatal serum 30 kDa complex (IGFBP-1 and -2) also increased in undernutrition and diabetes parallel to the expression of mRNA. But, taken together, the changes in IGFBP peptide levels and liver mRNA expression strongly suggest that the 30 kDa complex seems to be composed mostly of IGFBP-1 in the diabetic group and of both IGFBP-1 and -2 in the undernourished animals. The studies of liver mRNA expression of IGFs and IGFBPs confirm the different metabolic control mechanism for the availability of IGFs by the IGFBPs, depending on the animal's maturity. The different adaptation shown by the diabetic neonatal population was confirmed by correlation studies between body weight, glycemia, insulinemia, IGF-I and IGFBPs. The different mechanism of adaptation in diabetic vs undernourished rats seems to be probably due to the decisive role played by hyperglycemia in the diabetic population, and also shows the crucial influence of nutritional status on IGFs and IGFBPs. PMID- 7543554 TI - Circulating insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and tissue mRNA levels of IGFBP-2 and IGFBP-4 in the ovine fetus. AB - The IGF-binding proteins (IGFBPs) are a family of at least six structurally related proteins, which bind the IGFs and modulate their actions, including the regulation of pre- and postnatal growth. In this study we have examined the relationship between circulating and tissue mRNA levels of IGFBPs and related this to circulating IGFs in the fetal sheep over the gestational period when rapid growth and development occurs. Circulating IGFBP-2, as measured by Western ligand blot (WLB), increases between early and mid gestation, remains high, then declines throughout late gestation (P = 0.0002). Circulating IGFBP-3 increases throughout gestation, as measured by WLB or RIA (P = 0.04 and P = 0.0001 respectively), as does circulating IGFBP-4 (P = 0.004). These ontogenic changes in circulating IGFBPs-2 and -4 are paralleled by changes in liver mRNA for these proteins and, for IGFBP-2, by those in kidney IGFBP-2 mRNA also. This suggests that liver and kidney may be the primary contributors to circulating IGFBP-2 and the liver to circulating IGFBP-4, IGFBP-2 mRNA is present in the heart and lung in early gestation but barely detectable in these tissues after approximately 60 days gestation. IGFBP-4 mRNA is also present in the heart in early but not late gestation, but is abundant in the lung throughout gestation. These results demonstrate tissue specific and developmental regulation of IGFBPs-2 and -4 at the mRNA level. To assess any role the circulating IGFs may play in mediating these changes in IGFBPs, or vice versa, both plasma IGF-I and IGF-II were measured by RIA. Circulating IGF-I increases as gestation progresses (P = 0.0001), while circulating IGF-II increases between early and mid gestation, remains high (P = 0.01), then declines. Circulating IGF-I is positively correlated with fetal weight (r = 0.66, P = 0.03), circulating IGFBP-3 (r = 0.54, P = 0.01) and IGFBP-4 (r = 0.52, P = 0.01). Circulating IGF-II positively correlates with circulating IGFBP-2 (r = 0.48, P = 0.02) throughout gestation and at 1 day postnatally. These relationships are consistent with circulating IGF-I influencing IGFBPs-3 and -4, and similarly, IGF-II determining IGFBP-2, or vice versa. Alternatively, these correlations may reflect coordinate regulation of IGF and IGFBP by a common factor. PMID- 7543555 TI - Investigation into the potential physiological sources of rat milk IGF-I and IGF binding proteins. AB - We have previously reported the presence of IGF-I and IGF-binding proteins (IGFBP 2, -3 and -4) in rat milk. Herein, the potential sources of rat milk IGF-I and IGFBPs were investigated. Lactating dams (day 14 postpartum) were separated from their pups and injected intraperitoneally with 0.45 microCi 125I-IGF-I or 125I IGFBP-3. After 3 h, serum and milk of rats receiving 125I-IGF-I contained 7642 +/ 3121 and 14,455 +/- 7837 c.p.m./ml respectively. Serum and milk of rats given 125I-IGFBP-3 contained 7232 +/- 1366 and 10,371 +/- 4091 c.p.m./ml respectively. Sephacryl S-200 gel filtration chromatography demonstrated that the 125I-IGF-I in both serum and milk was primarily in the 150 kDa IGF-binding complex, whereas the distribution of 125I-IGFBP-3 differed between serum and milk. In serum, most of the 125I-IGFBP-3 was in the 150 kDa fraction, while most 125I-IGFBP-3 in milk was in the 40 kDa fraction. Northern analysis of liver showed IGFBP-1 and -3 mRNA expression, with variable expression of IGFBP-2 and -4 mRNA. In contrast, mammary tissue expressed only IGFBP-2 and -4 mRNA, suggesting that these IGFBPs in milk may arise from de novo synthesis within the mammary gland. The lack of detectable IGFBP-3 mRNA in mammary tissue and the translocation of 125I-IGFBP-3 from the serum suggest that milk IGFBP-3 arises from the maternal circulation. PMID- 7543551 TI - Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine. AB - The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects. PMID- 7543556 TI - Prostate-specific antigen in a community screening program. AB - BACKGROUND: This study was designed to determine who participates in community based prostate-specific antigen (PSA) screening programs and what serum PSA levels can be expected. METHODS: A descriptive analysis of men who participated in an annual community health screening program was used to compare men who chose PSA screening with those who did not. The relationship of demographic variables to PSA level was evaluated by multivariate regression analysis. Data were available on 5548 men, 6% of whom were black. RESULTS: The population of PSA screening participants included proportionately more middle-aged white men with higher median income, as compared with men who did not participate. Those who did not participate in the screening were more likely to be either very old or very young. PSA levels increased with age, and the percentage of men with elevated PSA levels increased with age. One tenth (9.6%) of all participants had PSAs between 4 ng/mL and 10 ng/mL, and 1.9% had levels greater than 10 ng/mL. Within 1 year of the screening, 1.7% of the screened participants had a diagnosis of prostate cancer. The mean PSA in this group was 15.9 ng/mL. CONCLUSIONS: These data confirm the need for age-specific PSA reference ranges. It is likely that the same reference range can be used for all racial ethnic populations. PMID- 7543557 TI - Prostate cancer screening. PMID- 7543558 TI - Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production. AB - The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA. PMID- 7543559 TI - Paclitaxel and carboplatin in combination in the treatment of advanced non-small cell lung cancer: a phase II toxicity, response, and survival analysis. AB - PURPOSE: To determine the activity and toxicity of combination paclitaxel (24 hours) and carboplatin in advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Eligibility required measurable disease (stage IV or stage IIIB with malignant pleural effusion), Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1, absolute neutrophil count > or = 2,000/microL, platelet count > or = 100,000/microL serum creatinine concentration < or = 1.5 mg/dL, and bilirubin level < or = 2 mg/dL. Paclitaxel was initially administered at a dose of 135 mg/m2/d, followed by carboplatin on day 2 at a targeted area under the concentration-time curve (AUC) of 7.5 using the Calvert formula. Granulocyte colony-stimulating factor (G-CSF) 5 micrograms/kg subcutaneously (SC) on days 3 to 17 was introduced during the second and subsequent cycles. In patients who sustained less than grade 4 myelosuppression, the paclitaxel dose was sequentially escalated 40 mg/m2 per cycle to a maximum of 215 mg/m2. Treatment was repeated at 3-week intervals for six cycles. RESULTS: From June 1993 through February 1994, 54 patients were enrolled; 53 are assessable for toxicity and response. The median age was 62 years (range, 34 to 84). Sixty-nine percent were male, 65% had adenocarcinoma, and 93% had stage IV disease. Two hundred sixty-eight cycles were administered; 32 patients (59%) completed all six cycles. Twenty-five unanticipated hospitalizations occurred during treatment (9.3% of cycles) in 20 patients (37%). Myelosuppression was the principal toxicity; grade 3 or 4 granulocytopenia occurred in 57% of patients after the first cycle, but decreased to 35% during the second cycle after introduction of G CSF and consistently remained < or = 22% during subsequent cycles. Seven episodes of neutropenic fever occurred, all during the first cycle. Grade 3 or 4 thrombocytopenia and anemia occurred in 47% and 33% of patients, respectively. Eight patients (15%) required platelet transfusions and 16 (30%) required packed RBC support. Neuropathy, myalgias/arthralgias, and thrombocytopenia, although generally mild, were cumulative. The paclitaxel dose was boosted to 215 mg/m2 in > or = 70% of patients who received three or more cycles. At an AUC of 7.5, the median first-cycle carboplatin dose was 424 mg/m2 (range, 273 to 709 mg/m2). The objective response rate was 62%, with five (9%) complete responses and 28 (53%) partial responses. The median progression-free survival time was 28 weeks and the median survival time 53 weeks. The 1-year survival rate is 54%. CONCLUSION: The paclitaxel-carboplatin combination is active in advanced NSCLC and may enhance survival; it merits further investigation in phase III trials. PMID- 7543560 TI - CD44 variant expression is a common feature of epithelial ovarian cancer: lack of association with standard prognostic factors. AB - PURPOSE: CD44 is a hyaluronic acid receptor that exists as a standard 90-kd form (CD44S) as well as several CD44 variant isoforms produced through alternative splicing. Expression of CD44 variants is associated with clinically aggressive behavior in some human tumors. The purpose of the present study is to define the expression of CD44 variant isoforms in ovarian cancer and to investigate whether the expression of these molecules is associated with adverse prognosis. MATERIALS AND METHODS: Six specimens of normal ovarian surface epithelium (NOSE) and 31 separate cases of newly diagnosed ovarian cancer were studied by a combination of reverse-transcription polymerase chain reaction (RT-PCR) and immunoperoxidase staining. Clinical correlation was made between CD44 variant expression and stage (I/II v III/IV), residual disease (< or = 2.0- v > 2.0-cm mass), age (< or = 65 v > 65 years), histology (papillary serous v other), grade, and survival. RESULTS: RT-PCR analysis revealed that NOSE predominantly expressed transcripts for CD44S, as well as a restricted pattern of transcripts characteristic of CD44 splice variants. CD44S and CD44 variant exon nine sequences (CD44-9v) were focally expressed in one of two NOSE specimens examined by immunoperoxidase staining. In comparison, the majority (71%) of ovarian cancer specimens expressed a complex pattern of CD44 splice variants by RT-PCR analysis. Immunoperoxidase studies revealed that the majority of ovarian cancer specimens expressed both CD44S and CD44-9v, whereas expression of sequences from variant exons 3, 4, and 6 was uncommon. There was no association between CD44 variant expression (transcript or protein) and stage, residual disease, age, histology, grade, or survival. CONCLUSION: Expression of CD44S and CD44-9v is a common feature of epithelial ovarian cancer cells. The lack of a significant association between CD44 variant expression and prognosis suggests that other factors may be more important in determining the clinical behavior of this disease. PMID- 7543561 TI - Subsets of CD34+ cells and rapid hematopoietic recovery after peripheral-blood stem-cell transplantation. AB - PURPOSE: To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets. PATIENTS AND METHODS: Based on data from 59 patients, we calculated for each day after PBSC transplantation the dose of CD34+ cells that resulted in rapid recovery of either neutrophils or platelets in the majority (> 70%) of patients. Using dual-color flow cytometry, subsets of peripheral-blood CD34+ cells were quantified and the numbers of CD34+ cells belonging to each of the reinfused subsets correlated with hematopoietic recovery following high-dose chemotherapy. RESULTS: The calculated threshold values with a high probability of engraftment showed a steep dose-effect relationship between CD34+ cell dose and time to recovery of both neutrophils or platelets. Predominantly CD34+ cells with the phenotype of myeloid precursors were mobilized. A minority of CD34+ cells expressed the erythroid and megakaryocytic lineage-associated antigens and a low but distinct population of CD34+ cells expressed antigens associated with multipotent stem cells. Analysis showed that the number of CD34+CD33- cells (r = .74, P < .05), as well as the number of CD34+CD41+ cells (r = -.81, P < .005), correlated significantly better with time to neutrophil and platelet recovery, respectively, than with the total number of CD34+ cells (r = -.55 and r = -.56, respectively). CONCLUSION: The numbers of CD34+CD33- cells and CD34+CD41+ cells may help to predict short-term repopulation capacity of PBSCs, especially when relatively low numbers of CD34+ cells per kilogram are reinfused. PMID- 7543563 TI - Effect of granulocyte colony-stimulating factor on the rate of neutrophil engraftment following peripheral-blood stem-cell transplantation. PMID- 7543562 TI - Paclitaxel activity in heavily pretreated breast cancer: a National Cancer Institute Treatment Referral Center trial. AB - PURPOSE: To provide paclitaxel, an investigational drug at the inception of this study, to women with chemotherapy-refractory metastatic breast cancer and to evaluate response and toxicity in these patients. PATIENTS AND METHODS: Two hundred sixty-seven patients with progressive disease (PD) following at least two chemotherapy regimens for metastatic breast cancer and a contraindication to further doxorubicin treatment received paclitaxel either at 175 mg/m2 intravenously (IV) over 24 hours or at 135 mg/m2 if they had prior irradiation to 30% of marrow-bearing bone or a cumulative dose of mitomycin > or = 20 mg/m2. RESULTS: In a subgroup of patients (n = 172) with measurable disease, four complete responses (CRs) and 36 partial responses (PRs) occurred, for an overall response rate of 23% (95% confidence interval [CI], 17% to 30%). No differences in response rates were noted according either to the number of prior chemotherapy regimens received or to whether patients were considered refractory to doxorubicin. The dose and schedule used in this trial resulted in febrile neutropenia in 45% of patients and a hospitalization rate of 49%. CONCLUSION: Paclitaxel's activity in this multiinstitutional trial in heavily pretreated patients confirms the encouraging results attained in single-institution trials. Although at this dose and schedule paclitaxel may be considered too myelosuppressive for palliative care, supportive measures such as colony stimulating factors and antibiotics were not used prophylactically. Current research efforts are focusing on whether paclitaxel's activity against breast cancer is dose- and/or schedule-dependent, and on what role it has in patients with less advanced disease. PMID- 7543564 TI - ICAM-1 and VCAM-1 expression in accelerated cardiac allograft arteriopathy and myocardial rejection are influenced differently by cyclosporine A and tumour necrosis factor-alpha blockade. AB - Expression of the vascular cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) occurs in allograft myocardium and in coronary arteries, promoting adhesion and transendothelial migration of inflammatory cells. We therefore investigated, in cholesterol-fed rabbits 9-10 days following heterotopic cardiac transplantation, whether the reduction of both myocardial rejection and graft arteriopathy with cyclosporine A (CsA) or graft arteriopathy alone with tumour necrosis factor alpha soluble receptor (TNFsr) was associated with suppression of ICAM-1 and VCAM 1 expression. Host hearts showed negative immunostaining for these adhesion molecules, whereas donor specimens from untreated (control) rabbits showed moderate immunostaining for ICAM-1 and weaker immunostaining for VCAM-1 in the coronary arteries, myocardium (cardiac myocytes), and perivenular regions. The selective reduction of the coronary arteriopathy with TNFsr was associated with somewhat reduced expression of these adhesion molecules in the arteries, whereas CsA also suppressed myocardial rejection and markedly decreased both vascular and myocyte expression of ICAM-1 and VCAM-1. PMID- 7543565 TI - A murine osteoblast cell line (MC3T3) produces a soluble elastogenic compound. AB - Elastofibromas are localized proliferations of mesenchymal cells that produce an exuberant amount of elastin-rich extracellular matrix. Recently periosteal fibroblasts have been proposed to be the proliferating cell. The hypothesis has been tested that osteocytes or osteoblasts contribute to the formation of elastofibromas by secreting a compound(s) that enhances elastin production. Media conditioned by murine calvarial osteoblasts (MC3T3) increased tropoelastin synthesis in bovine ligamentum nuchae fibroblasts. Addition of MC3T3-conditioned medium to bovine ligamentum nuchae fibroblast cultures produced a two-fold increase in tropoelastin RNA. The maximal increase in tropoelastin RNA was between 16 and 24 h; tropoelastin mRNA had returned to control values by 40 h. A similar increase in tropoelastin protein production was detected. The soluble elastogenic compound was neither interleukin-1 (IL-1) nor IL-6. These results support the hypothesis that an interaction between bone and perosteum may be involved in the formation of elastofibromas. PMID- 7543566 TI - Diagnostic yield of the neurologic assessment of the developmentally delayed child. AB - OBJECTIVE: The aim of this study was to determine the etiologic yield of the neurologic assessment of a consecutive cohort of developmentally delayed children. STUDY DESIGN: A retrospective chart review was carried out on all patients referred to a single university-based pediatric neurologist for evaluation of global developmental delay from July 1991 to December 1993. Patients referred because of isolated speech or motor delay or autism or those who had been previously evaluated by another neurologist were excluded. RESULTS: A total of 77 patients were identified; 47 were male, and 62 were referred by a pediatrician. Neurologic evaluation did not confirm global delay in 10, and 8 did not complete diagnostic evaluation; one child was included in both groups. Of the remaining 60, an etiologic diagnosis was suspected by the referring physician at the time of referral in 13. Although parents suspected a delay at a mean age of 0.66 (+/- 0.69) year, children were examined by the neurologist at a mean age of 3.58 (+/- 2.42) years. Twenty-five were mildly delayed, 23 were moderately delayed, and 12 were severely delayed. Diagnostic studies (history, physical examination, and selected investigations, including screens for metabolic disease, karyotype, fragile X testing, electroencephalography, and neuroimaging) yielded an etiologic diagnosis in 38 (63.3%) of the 60 patients. Etiologic categories included cerebral dysgenesis (16.7%), hypoxic-ischemic encephalopathy (10.0%), chromosomal abnormalities (10%), toxins (8.3%), metabolic disorders (5.0%), and neurocutaneous (3.3%), neuromuscular (3.3%), genetic/dysmorphic (3.3%), and epileptic (3.3%) syndromes. Etiologic yield was equivalent across categories and degree of developmental delay. CONCLUSION: Referral to a pediatric neurologist and application of a selected battery of investigations yield etiologic findings with important implications with respect to management, prognosis, and recurrence risk estimate in a significant portion of globally delayed children. PMID- 7543567 TI - A specific cystic fibrosis mutation (T3381) associated with the phenotype of isolated hypotonic dehydration. AB - We carried out molecular screening for mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in eight children of Sardinian descent seen because of hypotonic dehydration associated with hyponatremia, hypochloremia, hypokalemia, and metabolic alkalosis; none had pulmonary or pancreatic involvement. All the patients had the T3381 mutation either in homozygosity or compound heterozygosity with another CF mutation. The T3381 mutation was not detected in patients with CF who had classic symptoms or in healthy persons of the same descent. These data suggest that the T3381 mutation is associated with a specific mild CF phenotype. PMID- 7543569 TI - The in vitro immunosuppressive activity of the C15-demethylated metabolite of FK 506 is governed by ring- and open-chain tautomerism effects. AB - The FK-506 C15-demethylated metabolite, isolated from human liver microsomes and identified by fast atom bombardment mass spectrometry and NMR spectroscopy, is under the influence of ring- and open-chain tautomerism effects as demonstrated by fast atom bombardment mass spectrometry. Ring- and open-chain tautomerism effects are strongly influencing the in vitro immunosuppressive potency expressed by the measure of the inhibition of the mixed lymphocyte reaction, as demonstrated by the clear differences observed in immunosuppressive activity between the FK-506 C15-demethylated ring- and open-chain tautomeric conformations and depending on the tautomer-receptor specific interactions modulating the immunosuppressive activity of the FK-506 C15-demethylated metabolite. PMID- 7543568 TI - Endogenous nitric oxide modulates acetylcholine-induced edema and vasoconstriction in isolated perfused rabbit lungs. AB - The modulatory role of endogenous nitric oxide (NO) on pulmonary edema induced by acetylcholine (ACh), capsaicin, substance P (SP) and 5-hydroxytryptamine (5-HT) was investigated by using an inhibitor of NO synthase, N-omega-nitro-L-arginine (L-NNA). The effects of endogenous NO on the hemodynamic response to ACh, 5-HT and SP were also investigated. The capillary filtration coefficient (Kf,c), the total pressure gradient (delta Pt) and its four components [arterial (delta Pa), pre- (delta Pa') and post-capillary (delta Pv'), and venous gradient (delta Pv)] were evaluated on isolated, ventilated, perfused rabbit lungs. ACh (10(-8) to 10( 4) M) and SP (10(-10) to 10(-6) M) induced a concentration-dependent increase in the Kf,c. Capsaicin (10(-4) M) and 5-HT (10(-4) M) also increased this parameter. L-NNA (10(-4) M) completely inhibited the effects of ACh and capsaicin on the Kf,c, without preventing the effects of SP and 5-HT. ACh induced a concentration dependent vasoconstriction in the precapillary segment. Pretreatment with L-NNA enhanced this increase in delta Pa' but also increased delta Pv' and delta Pv. 5 HT increased delta Pt and delta Pa proportionally to the concentration. This effect was enhanced by L-NNA, which also increased delta Pa'. SP had no significant hemodynamic effect. Pretreatment with L-NNA did not modify the response to SP. Sodium nitroprusside (10(-5) M) induced a left shift of the concentration-response curve to ACh on the Kf,c, although it did not change the response to SP. Sodium nitroprusside also inhibited the hemodynamic effect of ACh. It was concluded that endogenous NO is involved in ACh-and capsaicin-induced edema via a prejunctional stimulatory effect on the C-fibers. Endogenous NO can also modulate ACh- and 5-HT-induced vasoconstriction by exerting a vasodilator action on the whole pulmonary vascular bed. PMID- 7543570 TI - Inhibition of L-type calcium currents in guinea pig ventricular myocytes by the kappa-opioid agonist U50488H does not involve binding to opiate receptors. AB - In guinea pig ventricular myocytes the kappa-opioid agonist trans(+/-)-3,3 dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate salt (U50488H) inhibited L-type calcium currents (ICa) as measured by the patch clamp technique in a dose-dependent fashion. The basal, as well as the isoprenaline and 8-Br-cAMP-stimulated, ICa were found to be reduced by U50488H. The inhibition was reversible and was not prevented on preincubation with pertussis toxin or the receptor antagonists naloxone and naltrexone. Naltrexone alone also caused an inhibition of ICa. Leucine enkephaline, a peptide opiate agonist, had no effect on ICa. U50488H did not alter the current/voltage relationship of the calcium current. The inhibition was independent of the cytosolic calcium concentration because it was also observed in the presence of 10 mM BAPTA in the pipette. If the compound was applied intracellularly via a perfused patch pipette there was no inhibition of ICa. The calcium current stimulated by the dihydropyridine calcium agonist Bay K 8644 was also reduced by U50488H. Conversely the inhibition of ICa by U50488H could be antagonized by Bay K 8644. In conclusion, these results demonstrate that binding to specific membrane receptors is not involved in the inhibition of L-type calcium current by U50488H and other nonpeptide opioid agonists. A direct interaction with the channel molecule at the exterior of the cell is probably involved. PMID- 7543572 TI - Neuropeptide metabolism on intact, regional brain slices: effect of dopaminergic agents on substance P, cholecystokinin and Met-enkephalin degradation. AB - Neuroleptic drugs have been shown to affect the level and messenger ribonucleic acid of specific neuropeptides. The effect of subchronically administered neuroleptics on neuropeptide metabolism, however, has not been systematically characterized. In the present study, the effect of neuroleptics and other dopaminergic compounds on substance P (SP), cholecystokinin and met-enkephalin degradation was determined on intact, regional, rat brain slices. After 7-day administration of haloperidol (1 mg/kg) or chlorpromazine (20 mg/kg), SP degradation was decreased in caudate-putamen and nucleus accumbens. After administration of the dopaminergic agonist apomorphine (5 mg/kg, b.i.d.), SP degradation was increased in the nucleus accumbens. The dopamine D2-receptor antagonist sulpiride (100 mg/kg, b.i.d.) produced no effect on SP degradation. Met-enkephalin degradation was decreased after haloperidol administration in both frontal cortex and caudate-putamen and unaffected by apomorphine administration. The metabolism of cholecystokinin was not affected by neuroleptic treatment. Studies performed with specific peptidase inhibitors suggested that neutral endopeptidase 24.11, metalloendopeptidase 24.15 and aminopeptidases degrade SP on caudate-putamen and nucleus accumbens slices. Therefore, alterations in these peptidases may be responsible for the change noted in SP degradation after dopaminergic compound administration. These metabolic changes noted after neuroleptic administration may therefore contribute to neuroleptic-induced alterations in regional peptide levels. PMID- 7543571 TI - Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. III. Agonist actions of the novel alkaloid epibatidine and analysis of type II current. AB - The nicotinic-agonist properties of epibatidine, an alkaloid originally isolated from the Ecuadorian frog Epipedobates tricolor, was investigated in rat hippocampal neurons grown in culture. The ability of epibatidine enantiomers to evoke nicotinic whole-cell currents of type IA, sensitive to blockade by alpha bungaro-toxin and methyllycaconitine and presumably subserved by an alpha-7-based nAChR, and of type II, sensitive to blockade by dihydro-beta-erythroidine and presumably subserved by an alpha 4 beta 2-based nAChR, was studied and compared with that of other nicotinic agonists. Epibatidine elicited type IA currents with EC50 values of 2.9 and 4.3 microM for the (-)- and (+)- enantiomers, respectively. The potency of the agonists in evoking type IA currents was as follows: (-)-epibatidine > (+)- anatoxin-a > (+)-epibatidine > (-)-nicotine > DMPP > cytisine > acetylcholine. The EC50 values of (-)- and (+)- epibatidine in eliciting type II currents were 19 and 15 nM, respectively. The order of potency of the agonists in evoking type II currents was: (+)-epibatidine > (-) epibatidine > cytisine > (+)-anatoxin-a > (-)-nicotine > acetylcholine > DMPP. Although these agonists produced nearly identical maximal type IA responses, the size of the maximal type II responses varied with the agonist. Cytisine and (+) anatoxin-a were the least efficacious agonists in inducing type II currents. Enantiomers of epibatidine showed the least stereoselectivity, those of nicotine showed a moderate stereoselectivity, and those of anatoxin-a exhibited the highest stereoselectivity in inducing either type of currents. In summary, these results suggest that epibatidine can be used as a novel nicotinic agonist for the study of type II currents, and that the nicotinic acetylcholine receptor related to type II currents may be one of the key target sites through which agonists such as epibatidine and nicotine elicit their behavioral actions in vivo. PMID- 7543573 TI - Irsogladine activates gap-junctional intercellular communication through M1 muscarinic acetylcholine receptor. AB - Irsogladine, an agent that protects gastric mucosa against various ulcerogenic stimuli through increasing cyclic AMP in surface mucous cells, has been reported to dose-dependently (10(-7) to 10(-5) M) facilitate gap-junctional intercellular communication (GJIC) in gastric epithelial cells. The beta adrenergic agonist, isoproterenol, stimulates GJIC in resting cells and inhibits GJIC in cells activated by 3-isobutyl-1-methylxanthine. In this study, we investigated whether irsogladine acts on GJIC in a manner similar to that shown by isoproterenol. Irsogladine, which bound to M1 muscarinic acetylcholine receptors (mAChR), did not inhibit, but failed to further facilitate the 3-isobutyl-1-methylxanthine enhanced GJIC, measured by Lucifer yellow transfer. The enhancement of GJIC by irsogladine was inhibited by the M1 mAChR antagonist, pirenzepine. A selective M1 mAChR agonist, McN-A-343, enhanced GJIC. Isoproterenol (10(-8) to 10(-6) M), which alone did not affect GJIC, inhibited the GJIC enhanced by 10(-5) M irsogladine. Conversely, 10(-10) to 10(-6) M irsogladine, which alone did not affect GJIC, inhibited the GJIC enhanced by 10(-5) M isoproterenol. McN-A-343 also converted the action of 10(-5) M isoproterenol from facilitation to inhibition of GJIC. These results indicate that GJIC is heterologously down regulated by cross-talk between M1 mAChR and beta adrenergic receptors. In addition, the effects of irsogladine and isoproterenol at low concentrations suggest the involvement of another mechanism for down-regulating GJIC. PMID- 7543574 TI - Modification of the behavioral effects of (+/-)BAY k 8644, cocaine and d amphetamine by L-type calcium channel blockers in squirrel monkeys. AB - Behavioral effects of the 1,4-dihydropyridine L-type calcium channel activator (+/-)BAY k 8644 [(+/-)methyl-1,4-dihydro-2,6-dimethyl-3- nitro-4-(2 trifluoromethylphenyl)-pyridine-5-carboxylate] the monoamine reuptake inhibitor cocaine and the monoamine releaser d-amphetamine were determined alone and after pretreatment with the structurally distinct L-type calcium channel blockers nimodipine (1,4-dihydropyridine), verapamil (phenylakylamine), diltiazem (benzothiazepine) and flunarizine (diphenylalkylamine) in squirrel monkeys responding under a 10-response fixed-ratio schedule of stimulus-shock termination. When administered alone, (+/-)BAY k 8644 (0.1-0.56 mg/kg) produced dose-dependent decreases in rates of responding. Pretreatment with nimodipine (3.0-10 mg/kg) or verapamil (1.0-3.0 mg/kg) produced dose-dependent rightward shifts of the (+/-)BAY k 8644 dose-response curve. In contrast, pretreatment with flunarizine (3.0 mg/kg) produced a leftward and downward shift of the (+/-)BAY k 8644 dose-response curve. Pretreatment with diltiazem (10-17.8 mg/kg) did not modify the (+/-)BAY k 8644 dose-effect curve. In addition, stereoselectivity was evident in the behavioral effects of (+/-)BAY k 8644 with the S(-)-enantiomer being approximately 3-fold more potent than the racemate. When administered alone, cocaine (0.1-5.6 mg/kg) and d-amphetamine (0.1-3.0 mg/kg) produced dose dependent decreases in rates of responding. Pretreatment with flunarizine (3.0 mg/kg) produced rightward shifts of the cocaine and d-amphetamine dose-response curves. Pretreatment with nimodipine (10 mg/kg), verapamil (3 mg/kg) or diltiazem (17.8 mg/kg) did not modify the effects of cocaine or d-amphetamine. The results of the present study suggest that the behavioral effects of (+/-)BAY k 8644 are differentially modified by L-type calcium channel blockers interacting with different sites on the channel and also suggest that the calcium channel blockers can be distinguished based on their differing interactions with (+/-)BAY k 8644, cocaine and d-amphetamine. PMID- 7543575 TI - Neuropeptide Y (18-36) modulates chromaffin cell catecholamine secretion by blocking the nicotinic receptor ion channel. AB - Neuropeptide Y (NPY) is a widely distributed peptide with varied activities including inhibition of [3H]NE secretion from chromaffin cells. In the present study, we investigated the mechanism through which NPY and NPY fragments inhibit nicotinic receptor induced influx of 22Na+ and 45Ca++ into bovine chromaffin cells. Fragments of NPY, including NPY13-36, NPY18-36 and NPY26-36, are more potent inhibitors of 45Ca++ and 22Na+ influx than NPY. High [K+]- and BAY K 8644 induced 45Ca++ influx and veratridine-induced 22Na+ influx are not inhibited by either NPY or NPY fragments. Thus, the site of NPY or NPY fragment action is not voltage-gated Ca++ or Na+ channels. A significant amount of acetylcholine-induced 45Ca++ influx still occurs in the presence of the voltage-gated Ca++ channel blockers: nifedipine (L-type), omega-conotoxin-GVIA (N-type) and omega-agatoxin IVA (P-type). NPY18-36, in the presence of these channel blockers, inhibited the residual nicotinic receptor-induced Ca++ influx. The response to NPY 18-36 is not pertussis toxin sensitive. The rank orders of potency for inhibition of 45Ca++ and 22Na+ are the same: NPY18-36 > or = NPY26-36 > NPY13-36 > NPY13-36 > NPY > or = NPYfree acid. Moreover, the IC50 values for NPY18-36 inhibition of 45Ca++ influx and 22Na+ influx are similar, 0.9 x 10(-6) M and 2.03 x 10(-6) M, respectively. Regression analysis for inhibition of these two phenomena produced a correlation coefficient of .9697 (P < .0003).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543576 TI - Shared care between general practitioners and urologists in the management of benign prostatic hyperplasia: a survey of attitudes among clinicians. AB - Recent community-based population surveys have revealed a much greater prevalence of benign prostatic hyperplasia than previously suspected. From these data it has been projected that there may be more than 2 million men in the UK whose quality of life is to some extent impaired by this disorder. Since there are only 330 fully trained urologists in this country it will not be feasible for every individual presenting with prostatism to be assessed by a specialist. In an attempt to provide a more rational basis from which family practitioners can decide whether or not to refer a patient for a specialist opinion a 'shared care' flow diagram was developed and assumptions contained within field tested by means of a postal questionnaire which was sent to 2020 urologists, family practitioners and other interested clinicians. There was general agreement with most of the precepts set out in the flow diagram, the main exception was a rejection of the suggestion that every patient with prostatism should have a prostate-specific antigen level determined before referral. We conclude that there seems a consensus among respondents that a shared care approach to the management of BPH may both improve the standard of care provided in this area by family practitioners and allow hard pressed urologists to focus greater attention on those patients whose conditions require surgical expertise to resolve. PMID- 7543577 TI - Syntheses, calcium channel agonist-antagonist modulation activities, and voltage clamp studies of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-pyridinylpyridine-5 carboxylate racemates and enantiomers. AB - A novel group of racemic isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 pyridinylpyridine-5-carboxylate isomers [(+/-)-12-14] were prepared using a modified Hantzsch reaction that involved the condensation of nitroacetone with isopropyl 3-aminocrotonate and 2-, 3-, or 4-pyridinecarboxaldehyde. Determination of their in vitro calcium channel-modulating activities using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays showed that the 2-pyridinyl isomer (+/-)-12 acted as a dual cardioselective calcium channel agonist (GPLA)/smooth muscle selective calcium channel antagonist (GPILSM). In contrast, the 3-pyridinyl [(+/-)-13] and 4-pyridinyl [(+/-)-14] isomers acted as calcium channel agonists on both GPLA and GPILSM. The agonist effect exhibited by (+/-)-12 on GPLA was inhibited by nifedipine and partially reversed by addition of extracellular Ca2+. In anesthetized rabbits, the 4 pyridinyl isomer (+/-)-14 exhibited a hypertensive effect that was qualitatively similar to that exhibited by the nonselective agonist Bay K 8644 and the 3 pyridinyl isomer (+/)-13, whereas the 2-pyridinyl isomer (+/-)-12 induced a hypotensive effect similar to that of the calcium channel antagonist nifedipine. Similar results were obtained in a spontaneously hypertensive rat model. In vitro studies showed that the (+)-2-pyridinyl enantiomer (+)-12A exhibited agonist activity on both GPILSM and GPLA, but that the (-)-2-pyridinyl enantiomer (-)-12B exhibited agonist activity on GPLA and antagonist activity on GPILSM. Whole-cell voltage-clamp studies using isolated guinea pig ventricular myocytes indicated that (-)-12B inhibited the calcium current (ICa), that (+)-12A increased slightly ICa, and that (+/-)-12 inhibited ICa but the latter inhibition was less than that for (-)-12B. (-)-12B effectively inhibited ICa at all membrane potentials examined (-40-50 mV), whereas (+)-12A exhibited a weak agonist effect near the peak of the I-V curve. The 2-pyridinyl isomers (enantiomers) 12 represent a novel type of 1,4-dihydropyridine calcium channel modulator that could provide a potentially new approach to drug discovery targeted toward the treatment of congestive heart failure and probes to study the structure-function relationships of calcium channels. PMID- 7543578 TI - Anti-AIDS (acquired immune deficiency syndrome) agents. 17. New brominated hexahydroxybiphenyl derivatives as potent anti-HIV agents. AB - Sixteen biphenyl derivatives were synthesized and evaluated for their inhibitory activity against HIV-1 replication in acutely infected H9 cells. 3-Bromo- (4) and 3,3'-dibromo-4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2,2'- bis(methoxycarbonyl)biphenyl (5) demonstrated potent anti-HIV activity with EC50 values of 0.52 and 0.23 micrograms/mL and therapeutic index values of > 190 and > 480, respectively. A comparison of the anti-HIV activity of these biphenyl derivatives suggested that the types of substituents on the phenolic hydroxy groups rather than the number of bromine(s) on the aromatic rings are important to the enhanced anti-HIV activity. Compounds 4 and 5 also showed potent inhibitory activity against HIV-1 reverse transcriptase in a template-primer dependent manner. The site of inhibition of HIV could be related to inhibition of this enzyme. Compounds 4 and 5 did not induce virus expression from the chronic HIV-1-infected cell lines ACH-2 and U1. Furthermore, these two agents did not inhibit an increase in virus production from the chronic HIV-1-infected cell lines when the phorbol ester PMA was present. PMID- 7543579 TI - Novel, potent, and orally active substance P antagonists: synthesis and antagonist activity of N-benzylcarboxamide derivatives of pyrido[3,4-b]pyridine. AB - A series of 4-phenylisoquinolone derivatives were synthesized and evaluated for NK1 (substance P) antagonist activity. Highly potent antagonists, 4-phenyl-3 isoquinolone-N-benzylcarboxamides (11), were discovered from the structure activity relationship studies on the isoquinolone-urea lead 1a. Optimization of the activity in this series resulted in the development of 5-phenyl-6-pyrido[3,4 b]pyridine-N-benzylcarboxamides (30) which are highly potent orally active NK1 antagonists. Among the compounds synthesized, N-[3,5-bis(trifluoromethyl)benzyl] 7,8-dihydro-N,7-dimethyl-8-oxo-5- (substituted phenyl)-6-pyrido[3,4 b]pyridinecarboxamides (30a,f,g) showed excellent antagonist activities with IC50 values (in vitro inhibition of [125I]-BH-SP binding in human IM-9 cells) of 0.21 0.34 nM and ED50 values (in vivo inhibition of capsaicin-induced plasma extravasation in guinea-pig trachea, iv) of 0.017-0.030 mg/kg. These compounds exhibited significantly potent activity upon oral administration with ED50 values of 0.068-0.17 mg/kg. Conformational studies on 30g indicated that the two stable conformers of 30g are quite similar to those of CP-99,994. PMID- 7543580 TI - The impact of psychosocial stressors on African-American and Latino preschoolers. AB - Particularly for children of color, one's productivity and prosperity as an adult depend to a significant degree on one's education as a child. Education is both the attainment of skills and the development of character. In the National Academy of Science's 1993 publication, Losing Generations: Adolescents in High Risk Settings, it is clear that the single common pathway to high-risk adolescent behavior is early academic failure. Coupled with the dire effects of poverty, institutional racism, and the disintegration of public school education, the fate of many African-American and Latino preschoolers may become one of functional illiteracy or worse. Project CHILD (Community Health Initiatives Against Learning Difficulties) was a pilot community program that aimed both to identify youngsters at risk of early academic failure and to obtain a profile of learning and developmental difficulties for each child. Psychoeducational and medical screenings were the primary tools used. Parental empowerment was critical to effect beneficial changes for the children within the schools. The program succeeded in its goals. However, it also was apparent that the preschoolers were adversely affected by many stressors both within and without the families. Primary child health care must become more community-based and comprehensive to offset the effects of poverty. PMID- 7543581 TI - Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries. AB - Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure. PMID- 7543582 TI - Host cell requirements for efficient infection of quiescent primary B lymphocytes by Epstein-Barr virus. AB - Quiescent primary B lymphocytes are efficiently immortalized by Epstein-Barr virus (EBV). This process requires both the delivery and expression of the viral genome and results in activation of the cell division cycle. Infection of B lymphocytes depends on a direct interaction between the viral glycoprotein gp340/220 and CD21, the C3dg complement receptor. This interaction is required for the adsorption of EBV. In addition, several lines of evidence suggest that the interaction of EBV with CD21 modulates the phenotype of cells. CD21 forms part of a multimeric signal transduction complex with CD19, TAPA-1, and Leu-13. In normal B lymphocytes, CD19 becomes tyrosine phosphorylated following stimulation of the antigen receptor and recruits the signal-transducing enzyme phosphatidylinositol 3-kinase kinase. Here, we investigated the involvement of signal transduction pathways in efficient infection. Protein synthesis is not required for events leading to the transcription of the viral genome, suggesting that the early stages of infection do not depend on the expression of novel cell genes and consistent with the Wp promoter being the first viral promoter used upon infection. Since the stimulation of cells with gp340/220 leads to an increase in the level of CD19 tyrosine phosphorylation, we investigated the potential contribution of both tyrosine and phosphatidylinositol 3-kinase kinases to efficient infection. Both kinases contribute to the posttranscriptional control of viral gene expression following infection, but neither is required for the entry or initial transcription of the virus. Thus, it appears that EBV exploits a host signal transduction pathway to efficiently infect primary cells. PMID- 7543583 TI - Characterization of the echovirus 7 receptor: domains of CD55 critical for virus binding. AB - CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates complement activity by accelerating the decay of C3/C5 convertases. Recently, we and others have established that this molecule acts as a cellular receptor for echovirus 7 and related viruses. DAF consists of five domains: four short consensus repeats (SCRs) and a serine/threonine-rich region, attached to the cell surface by a glycosylphosphatidyl inositol anchor. Chinese hamster ovary cells stably transfected with deletion mutants of DAF or DAF-membrane cofactor protein recombinants were analyzed for virus binding. The results indicate that the binding of echovirus 7 to DAF specifically requires SCR2, SCR3, and SCR4. There is also a nonspecific requirement for the S/T-rich region which probably functions to project the binding region away from the cell membrane. The three nonpeptide modifications of DAF, N-linked glycosylation, O-linked glycosylation, and the glycosylphosphatidyl inositol anchor, are not required for virus binding. The SCRs of membrane cofactor protein, the closest known relative of DAF, cannot substitute for those of DAF with retention of virus binding activity. The monoclonal antibody used to identify DAF as an echovirus receptor, and which inhibits binding of the virus (monoclonal antibody 854), binds to SCR3. PMID- 7543584 TI - Biological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformation. AB - The glycoprotein (G) of rabies virus can assume at least three different conformations: the native (N) state detected at the viral surface above pH 7; the activated (A) hydrophobic state, which is probably involved in the first steps of the fusion process; and the fusion-inactive (I) conformation. There is a pH dependent equilibrium between these states, the equilibrium being shifted towards the I state at low pH. It has been supposed that the transition from the N to the I state mediates membrane fusion. By using a lipid-mixing assay, we studied the kinetics of fusion and fusion inactivation for two rabies virus strains, PV and CVS. In addition, by using electron microscopy and a trypsin sensitivity assay, we analyzed the kinetics of the conformational change towards the I state for both strains. Although the PV strain fuses faster, inactivation and the conformational change of PV G occur more slowly than for the CVS strain. This suggests that the structural transition towards the I state is irrelevant to the fusion process. Immunofluorescence and immunoprecipitation experiments performed with infected cells and two different monoclonal antibodies, one specific for the N form of G and one which recognizes both the N and the I states, suggest that G is transported in an I state-like conformation through the Golgi apparatus and acquires its N structure only near or at the cell surface. We propose that the role of the I state is to avoid unspecific fusion during transport of G in the acidic Golgi vesicles. PMID- 7543585 TI - Detection of an RNase H activity associated with hepadnaviruses. AB - Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex. PMID- 7543587 TI - Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell. AB - Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID- 7543586 TI - Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding. AB - The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies. PMID- 7543589 TI - Cyclophilin binding to the human immunodeficiency virus type 1 Gag polyprotein is mimicked by an anti-cyclosporine antibody. AB - Human immunodeficiency virus type 1 is unique among retroviruses in that infectivity requires specific incorporation into virions of the cellular protein cyclophilin A through interactions with the Gag polyprotein. Here we show that monoclonal antibody B11 1.4, which recognizes a cyclophilin-binding epitope on cyclosporine, detects denatured or native human immunodeficiency virus type 1 capsid. B11 1.4 does not recognize the capsids of other retroviruses, and binding is inhibited by cyclosporine or by cyclophilin A. PMID- 7543591 TI - Characterization of diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V beta bias. AB - The glycoprotein B (gB) from herpes simplex virus type I is a major target of cytotoxic T lymphocytes (CTL) in C57BL/6 mice. The majority of these T cells are directed to a single Kb-restricted determinant, gB498-505. We have analyzed the T cell receptor (TCR) usage in gB-specific CTL lines derived shortly after virus infection. The CTL populations preferentially used two V beta regions, a dominant V beta 10 element and a subdominant V beta 8 element. Detailed sequence analysis revealed considerable TCR beta-chain heterogeneity despite a striking level of predicted amino acid conservation at the V beta-D beta junction. This junction forms part of the third hypervariable loop of the TCR thought to directly contact the major histocompatibility complex-bound antigenic peptide. The results reveal considerable diversity within the primary T cells responding to a single viral determinant while still maintaining a high degree of TCR V beta bias and sequence conservation at the V-D-J junction. PMID- 7543590 TI - Induction of macrophage procoagulant activity by murine hepatitis virus strain 3: role of tyrosine phosphorylation. AB - The induction of a unique macrophage procoagulant molecule by murine hepatitis virus strain 3 correlates with the severity of viral hepatitis. The role of tyrosine phosphorylation in the signalling pathway leading to procoagulant expression was studied. Murine hepatitis virus strain 3 initiated a rapid increase in phosphotyrosine accumulation. Tyrosine kinase inhibition precluded this increase and abrogated expression of the virus-induced procoagulant mouse fibrinogen-like protein (musfiblp) gene. These findings suggest that manipulation of this signalling pathway in vivo might represent a novel approach to treating this disease. PMID- 7543588 TI - The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding. AB - The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components. PMID- 7543594 TI - Isolation of novel human endogenous retrovirus-like elements with foamy virus related pol sequence. AB - A new class of reverse transcriptase coding sequences was detected in reverse transcribed RNAs from human placenta by polymerase chain amplification with primers in highly conserved regions of the pol gene of mammalian retroviruses and retrotransposons. Using one of these novel sequences as a probe to screen a human genomic library, we isolated retrovirus-like elements bordered by long terminal repeats and having a potential leucine tRNA primer-binding site. Determination of the complete nucleotide sequence (6,591 bp) of one of these elements, termed HERV L (for human endogenous retrovirus with leucine tRNA primer), revealed domains of amino acid similarities to retroviral reverse transcriptase and integrase proteins. In addition, a region with homologies to dUTPase proteins was found unexpectedly downstream from the integrase domain. Amino acid sequence and phylogenetic analysis indicate that the HERV-L pol gene is related to that of foamy retroviruses. HERV-L-related sequences are detected in several mammalian species and have expanded in primate and mouse genomes up to 100 to 200 copies. PMID- 7543592 TI - Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells. AB - The bovine papillomavirus E5 protein is a 44-amino-acid membrane-associated protein that forms a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor in rodent and bovine fibroblasts, resulting in sustained receptor activation and cell transformation. We report here that high level expression of the E5 protein caused a reduction in the level of the mature form of the PDGF beta receptor in acutely and stably transformed mouse C127 cells. To explore in more detail the interaction of the E5 protein and the PDGF beta receptor, we tested the abilities of various E5 point mutants to bind the PDGF receptor, to induce PDGF receptor down-regulation and tyrosine phosphorylation, and to transform cells. A transformation-competent mutant, like the wild-type E5 protein, bound the receptor and induced receptor tyrosine phosphorylation and down-regulation. Transformation-defective E5 proteins either failed to interact with the endogenous PDGF beta receptor in mouse fibroblasts or underwent an aberrant interaction with the receptor. Mutation of glutamine at position 17, aspartic acid at position 33, or both carboxyl-terminal cysteine residues required for E5 homodimerization interfered with stable complex formation with the PDGF receptor, tyrosine phosphorylation and down-regulation of the receptor, and cell transformation. Point mutations at several other carboxyl terminal positions generated transformation-defective E5 proteins that formed a complex with the PDGF receptor and induced receptor tyrosine phosphorylation but did not induce PDGF receptor down-regulation. Either PDGF receptor activation is not sufficient for transformation of C127 cells or the receptors that are tyrosine phosphorylated in response to these mutant E5 proteins are not fully activated and therefore are not able to deliver a mitogenic signal. PMID- 7543596 TI - Autoantibodies to prostate specific antigen in patients with benign prostatic hyperplasia. AB - PURPOSE: We tested for a possible autoimmune process in benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Titers of IgG antibodies to prostate specific antigen (PSA) were measured in the sera of 85 BPH patients, 20 controls and 17 chronic prostatitis patients by enzyme-linked immunosorbent assay. RESULTS: The mean anti-PSA titers in the BPH group were significantly higher than in the controls and prostatitis group (p < 0.0005). Accordingly, 59% of BPH patients could be defined as responders to PSA compared to none among the controls (p < 0.0005). CONCLUSIONS: Circulating autoantibodies to PSA were shown to exist in the sera of BPH patients. This observation suggests that autoimmune processes may have a role in BPH. PMID- 7543593 TI - Modification of retroviral RNA by double-stranded RNA adenosine deaminase. AB - In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions. PMID- 7543595 TI - Full-length proviruses of baboon endogenous virus (BaEV) and dispersed BaEV reverse transcriptase retroelements in the genome of baboon species. AB - Baboon endogenous virus (BaEV) is present in multiple copies in many Old World monkey species. BaEV proviruses may contain open reading frames for all major genes, as is indicated by the rescue of infectious virus particles from baboon and gelada tissues after cocultivation with permissive cells. We have analyzed full-length BaEV proviral structures in a baboon (Papio cynocephalus) genomic library and found no evidence for the rearrangements or large deletions commonly observed in endogenous virus genomes from other mammalian species. The two proviruses studied were integrated next to or nearby long interspersed repeat sequence (LINE) transposable elements. Additionally, isolated dispersed fragments with 100% and approximately 77% homology, respectively, to part of the BaEV reverse transcriptase gene were detected. These presumed retroelements were present in an approximately 10-fold excess compared with the full-length proviral genomes. PCR amplification and sequencing of BaEV reverse transcriptase and env fragments from the lambda clones and from the genomic DNA of other baboon species showed that there is little sequence variation present in BaEV DNA in the baboon genome. PMID- 7543597 TI - The American Urological Association symptom index: does mode of administration affect its psychometric properties? AB - PURPOSE: We determine whether self-administration and interviewer administration of the American Urological Association (AUA) symptom index are equivalent. MATERIALS AND METHODS: The AUA symptom index was administered twice to 41 visually impaired or illiterate men. Men who were able were randomized to self administration first and then interviewer administration (43) or the reverse (40). RESULTS: The scores in each group were reliable. However, because we could not reject a differential carryover effect, the nonsignificant test of mode effect in the crossover design may have been invalid. At time 1 there was no significant difference between group mean scores for randomized patients (p = 0.13). CONCLUSIONS: Although the AUA symptom index should be self-administered when possible, interviewer administration appears to be acceptable. PMID- 7543599 TI - Placement of the urethral stent made of shape memory alloy in management of benign prostatic hypertrophy for debilitated patients. AB - PURPOSE: A new urethral stent made of shape memory alloy was placed to relieve prostatic obstruction in 17 patients in whom other approaches were contraindicated. MATERIALS AND METHODS: All patients were unable to tolerate intervention with sedation and positioning. Placement of the shape memory alloy stent mounted on a 16F Foley catheter is similar to insertion of a urethral catheter except for the heat-sensitive expansion. RESULTS: Each device was easily implanted with the patient on a flat examination table. There was no migration or incrustation of the shape memory alloy stent during the indwelling period. CONCLUSIONS: Clinical results demonstrate that the shape memory alloy urethral stent might be the only choice for management of prostatic obstruction in debilitated patients. PMID- 7543600 TI - Benign prostatic hyperplasia. PMID- 7543598 TI - The effect of zanoterone, a steroidal androgen receptor antagonist, in men with benign prostatic hyperplasia. The Zanoterone Study Group. AB - PURPOSE: Zanoterone (100 to 800 mg.) versus placebo was studied in 463 patients with benign prostatic hyperplasia. MATERIALS AND METHODS: Study end points were maximum urinary flow rate, American Urological Association symptom index, prostate volume, prostate specific antigen and sex steroid concentrations after 6 months of treatment. RESULTS: Mean increases in maximum urinary flow rate were 2 to 3-fold over placebo, although only the 200 mg. group had significant results (1.7 ml. per second, p = 0.026). There were no statistically significant differences between the zanoterone and placebo groups in symptom index or prostate volume. Estradiol and testosterone concentrations, and the incidence of breast pain and gynecomastia increased significantly with zanoterone compared with placebo. Prostate specific antigen levels decreased significantly. CONCLUSION: Zanoterone did not demonstrate a favorable risk-to-benefit profile for the treatment of benign prostatic hyperplasia. PMID- 7543603 TI - Prostate cancer. PMID- 7543602 TI - Failure of focal prostate cancer on biopsy to predict focal prostate cancer: the importance of prevalence. AB - PURPOSE: We investigated whether the reported correlation between focal prostate cancer on biopsy and the presence of clinically insignificant cancer applies to men with an elevated serum prostate specific antigen level or a positive biopsy from an area of palpable abnormality. MATERIALS AND METHODS: A total of 33 men with these findings and focal cancer on biopsy underwent radical perineal prostatectomy, with step-section pathological analysis of all specimens and volume determination of all stage pT2a tumors. RESULTS: Pathological examination revealed that 17 tumors (52%) were stage T3, 10 (30%) stage T2b to c and 6 (18%) stage T2a, with 4 (12%) significant (volumes 0.8 to 1.2 ml.) and 2 (6%) insignificant (volumes 0.1 and 0.3 ml.) neoplasms. Thus, focal prostate cancer on biopsy correlated with significant cancer in 94% of the cases. CONCLUSIONS: In patients with another variable controlling for a high prevalence of significant prostate cancer, the focal nature of a positive biopsy probably reflects only sampling limitations and should not influence treatment. To restrict prostate biopsy to men with a high prevalence of significant cancer, targeted area biopsy alone must replace sextant biopsy in men with a palpable prostatic abnormality but a normal prostate specific antigen level. PMID- 7543601 TI - The lack of predictive value of prostate specific antigen density in the detection of prostate cancer in patients with normal rectal examinations and intermediate prostate specific antigen levels. AB - PURPOSE: The management of patients with a normal digital rectal examination and a prostate specific antigen (PSA) level of 4.0 to 10.0 ng./ml. remains controversial. To improve the specificity of cancer detection in this group, PSA density has been recommended with biopsies based on a PSA density of 0.15 or more. To evaluate PSA density as a discriminator of prostate cancer we enrolled patients in a prospective study. MATERIALS AND METHODS: A prospective evaluation was done of 44 consecutive patients with a palpably normal digital rectal examination and a serum PSA level of 4.0 to 10.0 ng./ml. enrolled during a 13 month period. All patients underwent transrectal ultrasound with sextant biopsies regardless of calculated PSA density. RESULTS: Overall, 8 of 44 men (18%) had prostate cancer. There was no significant difference in the mean PSA density between the patients with positive and negative biopsies (mean 0.12 and 0.15, respectively, p = 0.258). Also, there was no significant association between PSA or PSA density and a positive biopsy in multivariate analysis (p = 0.863). Receiver operating characteristic curves for PSA and PSA density failed to demonstrate any superior benefit for PSA density in this patient population. A PSA density of 0.15 was an unreliable indicator of cancer (sensitivity 12.5%, specificity 61.1% and positive predictive value 6.7%). CONCLUSIONS: In our study, PSA density did not discriminate between patients with positive and negative biopsies, and in fact most cancers would not have been detected if a PSA density of 0.15 or more had been used as the sole indication for biopsy. Therefore, we recommend systematic biopsies in these patients independent of calculated PSA density. PMID- 7543604 TI - Diagnostic significance of prostate specific antigen and the development of a mass screening system for prostate cancer. AB - PURPOSE: The significance of prostate specific antigen (PSA) in the mass screening for prostate cancer was investigated. MATERIALS AND METHODS: A total of 4,375 PSA, digital rectal and transrectal ultrasonographic examinations was performed on 3,526 subjects. RESULTS: Among the 3 modalities, PSA had the highest sensitivity (80.4%) and positive predictive value (45.1%). We established age specific reference PSA ranges for normal Japanese men, and demonstrated the significance of PSA density and PSA velocity. CONCLUSIONS: The significance of PSA as a prostate cancer screening modality should be evaluated across multiple age ranges and in combination with previous PSA data and/or prostate volume. PMID- 7543606 TI - Prostate specific antigen based disease control following ultrasound guided 125iodine implantation for stage T1/T2 prostatic carcinoma. AB - PURPOSE: We report the prostate specific antigen (PSA) based recurrence-free survival rate after 125iodine (125I) implantation for early stage prostatic carcinoma. MATERIALS AND METHODS: A total of 197 patients with clinical stage T1 and T2 prostatic carcinoma underwent outpatient 125I seed implantation. Followup was 1 to 7 years (median 3). Pretreatment serum PSA levels were elevated (greater than 4.0 ng./ml.) in 138 patients (70%). There were 105 well differentiated (Gleason score 2 to 4), 87 moderately differentiated (Gleason score 5 to 6) and 5 indeterminate tumors. The prescribed minimum prostatic dose was 160 Gy. The total dosage of 125I implanted ranged from 15 to 62 mCi. (median 37). Staging lymph node dissection and seminal vesicle biopsies were not routinely performed. RESULTS: Among 138 patients with an elevated PSA level before implantation and no prior hormonal treatment, the PSA value returned to normal in 98% and decreased to less than 1.0 ng./ml. in 82% within 24 months of treatment. In 97% of those 138 patients the PSA level decreased to less than 1.0 ng./ml. at 48 months after implantation. Of 8 patients with an increasing PSA value 5 also had clinically evident failure. The actuarial rate of chemical (increasing PSA) or clinical failure at 5 years following implantation was 7%, with 15 patients still at risk at 5 years. There was a trend for higher failure rates among patients with higher pretreatment PSA levels (p = 0.57), Gleason scores 5 and 6 versus 2 to 4 (p = 0.51) or higher stage of disease (p = 0.17). CONCLUSIONS: There is a high rate of clinical and chemical freedom from progression following 125I implantation for select patients with early stage prostatic carcinoma. PMID- 7543605 TI - Free, complexed and total serum prostate specific antigen: the establishment of appropriate reference ranges for their concentrations and ratios. AB - PURPOSE: Prostate specific antigen (PSA) exists in the serum in several molecular forms that can be measured by immunodetectable assays: free PSA, PSA complexed to alpha 1-antichymotrypsin (complexed PSA) and total PSA, which represents the sum of the free and complexed forms. We determined the normal distribution of values and established the appropriate reference ranges for these 3 molecular forms of PSA and their ratios (free-to-total, complexed-to-total and free-to-complexed PSA). Knowing the amount and ratio of these molecular forms appears to be useful in enhancing the ability of PSA to distinguish potentially curable prostate cancer from benign prostatic hyperplasia and in decreasing the number of unnecessary prostate biopsies. MATERIALS AND METHODS: A total of 422 healthy men 40 to 79 years old was randomly chosen from the male population of Olmsted County, Minnesota and underwent a detailed clinical examination that included digital rectal examination, serum PSA determination and transrectal ultrasound to exclude the presence of prostate cancer. Using newly developed, monoclonal monoclonal immunofluorometric assays for each molecular form, the free, complexed and total PSA, and the ratios of these 3 forms were determined for each study participant. RESULTS: All 3 molecular forms correlated directly with patient age (r = 0.45, r = 0.43 and r = 0.45, respectively). Using the 95th percentile, the recommended age-specific reference ranges for the free, complexed and total PSA forms, respectively, are 0.5, 1.0 and 2.0 ng./ml. for men 40 to 49 years old; 0.7, 1.5 and 3.0 ng./ml. for men 50 to 59 years old; 1.0, 2.0 and 4.0 ng./ml. for men 60 to 69 years old, and 1.2, 3.0 and 5.5 ng./ml. for men 70 to 79 years old. With regard to each of the ratios (free-to-total, complexed-to-total and free-to complexed PSA) none correlated with patient age. As a result, the appropriate upper limit of normal (95th percentile) for all 3 ratios is constant for men of all ages. These reference ranges are greater than 0.15 for free-to-total PSA ratio, less than 0.70 for complexed-to-total PSA ratio and greater than 0.25 for free-to-complexed PSA ratio. The free-to-total PSA ratio will have its greatest value for men with a serum PSA value between 2 and 10 ng./ml. CONCLUSIONS: The establishment of appropriate reference ranges for free, complexed and total PSA as well as the ratios will allow the practicing urologist to incorporate these new parameters into the diagnostic evaluation of men at risk for early, potentially curable prostate cancer. PMID- 7543607 TI - Prostate cancer. PMID- 7543609 TI - Hyperplasia of prostatic mesonephric remnants: a potential pitfall in the evaluation of prostate gland biopsy. PMID- 7543610 TI - Re: Selection of optimal prostate specific antigen cutoffs for early detection of prostate cancer: receiver operating characteristic curves. PMID- 7543608 TI - Critical evaluation of salvage surgery for radio-recurrent/resistant prostate cancer. AB - PURPOSE: A positive post-irradiation prostatic biopsy associated with an increasing prostate specific antigen level but no palpable evidence of local progression may identify a subgroup of patients who could be cured by salvage surgical therapy. MATERIALS AND METHODS: Between 1967 and 1992, 132 patients underwent salvage surgery, including radical retropubic prostatectomy in 79, anterior exenteration in 38, total exenteration in 5 and bilateral pelvic lymphadenectomy only in 10. RESULTS: The 10-year cancer-specific survival rate in the prostatectomy group was 72%. Local control was equivalent among the surgical groups. Radical retropubic prostatectomy patients with negative surgical margins and nonaneuploid tumors demonstrated a significant survival advantage. Adjuvant hormonal therapy improved the disease-free survival rate in patients with nonaneuploid tumors. CONCLUSIONS: Radical retropubic prostatectomy can achieve excellent survival with low morbidity in select patients. Patients with clinical stage T2 or less disease and with prostate specific antigen detected cancers (52% and 75%, respectively) had pathological stage T2 disease. Thus, by using modern diagnostic techniques patients can be identified who may be cured with salvage surgery. PMID- 7543611 TI - An increase of neuropeptide Y but not nitric oxide synthase-immunoreactive nerves in the bladder neck from male patients with bladder neck dyssynergia. AB - PURPOSE: To determine the distribution of neuropeptides in male patients with bladder neck dyssynergia and benign prostatic hyperplasia. MATERIALS AND METHODS: Bladder neck tissue, obtained from male patients with bladder neck dyssynergia (BND) and control patients with benign prostatic hyperplasia (BPH), was studied immunohistochemically for protein gene product 9.5 (a general neuronal marker), vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, substance P, growth associated protein 43 and nitric oxide synthase. RESULTS: In the bladder neck from control patients, the greatest density of nerves contained protein gene product 9.5, followed in decreasing order by neuropeptide Y; vasoactive intestinal polypeptide; calcitonin gene-related peptide; nitric oxide synthase; substance P and serotonin. The neuropeptides were found in the smooth muscle and were also associated with blood vessels. In patients with BND there was a statistically significant increase (P < 0.05) in the density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves in the smooth muscle and the base of the mucosa but not in blood vessels in the bladder neck, while the density of the other neuropeptides studied, nitric oxide synthase and serotonin did not significantly change from that of control tissue. Growth associated protein 43-immunoreactive nerves were absent from the bladder neck from both groups of patients. CONCLUSION: It is suggested that the increase in density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves, part of the sympathetic contractile system of the bladder neck, may exacerbate bladder outlet obstruction and thus play a role in the pathogenesis of BND. PMID- 7543612 TI - alpha-blockade in the treatment of symptomatic benign prostatic hyperplasia. AB - PURPOSE: The clinical and urodynamic effects of oral alpha 1-selective adrenoceptor blockers in the treatment of symptomatic benign prostatic hyperplasia were quantified, and side effects and patient tolerance were assessed. MATERIALS AND METHODS: A total of 29 original reports of placebo controlled clinical trials of alpha-blockers in which results were adequately presented was identified and reviewed, along with additional pertinent literature. We assumed that the efficacy of the different alpha-blockers was basically the same and the weighted average treatment effect was calculated in comparison with placebo. RESULTS: The average improvement in maximum urine flow rate was 1.5 ml. per second but this rate would probably approach 1.8 to 1.9 ml. per second if all dosages had been titrated up to the highest level tolerated. Overall symptom score decreased by 14% and residual urine volume decreased by 29%. A slight decrease in detrusor pressure during voiding was suggested. CONCLUSIONS: alpha-Blockers were beneficial in the treatment of benign prostatic hyperplasia. Tolerance to treatment appeared to develop in a large proportion of patients after 6 months of therapy. However, for patients who benefit from long term use of alpha-blockers effective treatment might be maintained for years. PMID- 7543613 TI - Shock wave lithotripsy with the Dornier MFL 5000 lithotriptor using an external fixed rate signal. AB - PURPOSE: We examine the effects of fixed rate shock wave administration on the cardiac rhythm and treatment efficacy of a tubless lithotriptor (Dornier MFL 5000*). A secondary goal was to examine the treatment efficacy of fixed shock wave administration compared to R wave triggered lithotripsy. MATERIALS AND METHODS: In this prospective study Holter monitoring was used before, during and after nonR wave triggered shock wave lithotripsy. RESULTS: An increase in premature ventricular contractions was noted during shock wave lithotripsy. However, there were no episodes of significant ventricular ectopia, ventricular tachycardia, asystole or heart block as a result of nonR wave triggered shock wave administration. NonR wave gated shock wave lithotripsy expedited patient treatment and (mean treatment time 46 +/- 21 minutes)., minimized the use of sedation during treatment and produced results similar to R wave gated shock wave lithotripsy with the MFL 5000 lithotriptor. CONCLUSIONS: With adequate precautions, fixed rate shock wave administration would appear to be a reasonable option to treat urolithiasis with the MFL 5000 lithotriptor as with other newer lithotriptors. PMID- 7543614 TI - The utility of prostate-specific antigen for detecting prostate cancer. PMID- 7543615 TI - The utility of prostate-specific antigen for detecting prostate cancer. PMID- 7543616 TI - The utility of prostate-specific antigen for detecting prostate cancer. PMID- 7543617 TI - The utility of prostate-specific antigen for detecting prostate cancer. PMID- 7543618 TI - The utility of prostate-specific antigen for detecting prostate cancer. PMID- 7543619 TI - [Pain management in advanced pediatric cancer patients--a proposal of the two step analgesic ladder]. AB - Pain treatment for 17 pediatric cancer patients in our institution was evaluated and disirable cancer pain management for children was discussed. Most of the patients (aged 1-17 years) suffering severe pain for about one month were in the advanced stage of the malignant diseases (e.g. leukemia). The pain etiology was mostly tumor-associated while therapy-related pain accounted for 23.5%. These pains were treated with NSAIDs or pentazocine before the consultation with inadequate relief. Oral morphine sulfate or continuous intravenous morphine chloride was administered to 16 patients with successful pain relief and side effects such as nausea (52.9%) and drowsiness (41.2%). It took 5.5 days on average until adequate pain control methods were determined. It is known that most NSAIDs frequently used for pediatric pain possibly cause adverse effects such as platelet dysfunction or mucous membrane injury with a suppository, which could lead to a fatal disorder in clinically ill pediatric cancer patients. Moreover sufficient doses for the pain relief are not necessarily given to the pediatric patients because of a limit to the dosage of NSAIDs. The period of pediatric cancer pain in which the patient require a methodical treatment and receive benefit from pain relief is relatively short in the advanced stage, not to mention the early stage in which chemotherapy is efficacious against cancer disease itself. Therefore, to obtain effective pain control within a short time, the authors propose the pain management for advanced pediatric cancer patients by the two-step analgesic ladder prescribing weak or strong opioid analgesics first, adapted from the three-step ladder of the WHO Cancer Pain Relief, 1986. PMID- 7543620 TI - [Influence of the hypotensive anesthesia using halothane or trinitroglycerin on coronary blood flow--comparison of normal group and normovolemic hemodilution group]. AB - We divided adult mongrel dogs under halothane anesthesia (1 MAC: 0.86%) into the normal group (N-group: no-hemodilution, with Hct 38 +/- 7%) and the hemodilutional group (H-group: normovolemic hemodilution, with Hct 38 +/- 4%-->21 +/- 4%). For dilution of blood, Dextran 70 was used. In both groups, control values of respiratory and circulatory parameters and coronary blood flow (CBF) were measured. We repeated these measurements after animals had been anesthetized by two different methods:halothane hypotensive anesthesia and trinitroglycerine hypotensive anesthesia. Under hypotensive anesthesia with halothane, CI, LV dp/dt max, and LVSWI decreased significantly as compared to control values in both group. CBF also decreased significantly as compared to the control values in both groups. Under trinitroglycerine hypotensive anesthesia, CI, LV dp/dt max and LVSWI decreased significantly from the control values in both groups as observed under halothane hypotensive anesthesia. Differing from these findings, under trinitroglycerine hypotensive anesthesia no difference from the control group was observed in CBF in N-group, while in H-group CBF decreased significantly from the control value. It was indicated from our results that the increase in coronary blood flow induced by trinitroglycerine becomes obscure under hemodilutional state. PMID- 7543621 TI - [Effects of sairei-to (TJ-114) on the expression of adhesion molecule in anti-GBM nephritic rats]. AB - In order to clarify the antinephritic mechanisms of Sairei-to (TJ-114), the effects of TJ-114 on the expression of adhesion molecules were evaluated in rats with anti-GBM nephritis. TJ-114 was administered orally once a day from the day (1st day) after anti-GBM serum injection throughout the experiment. TJ-114 inhibited crescent formation in glomeruli at the 10th day compared to control rats with anti-GBM nephritis. The ICAM-1- or VCAM-1-positive area in the glomeruli was increased in the nephritic control rats at the 10th day. In contrast, TJ-114 prevented increase in the ICAM-1- or VCAM-1-positive area in the glomeruli of nephritic rats. TJ-114 inhibited increase in the number of LFA-1- or VLA-4-positive cells in the glomeruli. One of the constituent components of TJ 114, Syo-saiko-to (TJ-9) at the dose of 750 mg/kg, also significantly inhibited increase in the ICAM-1- or VCAM-1-positive area in the glomeruli. Gorei-san (TJ 17), another component of TJ-114, inhibited increase in the ELAM-1-positive area and increase in the number of VLA-4-positive cells in the glomeruli. Prednisolone markedly inhibited increase in the positive area of those adhesion molecules and increase in the number of ligand-molecule-positive cells in the glomeruli. These results indicate that the antinephritic action of TJ-114 may be partially due to inhibition of adhesion molecule expression in the glomeruli. PMID- 7543622 TI - Transcellular biosynthesis of lipoxin A4 during adhesion of platelets and neutrophils in experimental immune complex glomerulonephritis. AB - Polymorphonuclear neutrophils are important effectors of injury in host defense and inflammation. Many inflammatory diseases are self-limiting, raising the possibility that compounds are generated in vivo during the course of inflammation that inhibit neutrophil recruitment and tissue destruction. Lipoxins, a more recent addition to the families of bioactive eicosanoids, are potential candidates in this regard. Lipoxins are generated via pathways that initially involve the dual lipoxygenation of arachidonic acid and are potent inhibitors of several neutrophil trafficking events in vitro. Here, we present evidence that lipoxin A4 is generated in rat kidneys during experimental immune complex-mediated glomerulonephritis in vivo. Renal lipoxin A4 levels were markedly reduced by prior depletion of animals of either neutrophils or platelets, suggesting that most lipoxin A4 generated in vivo was derived from transcellular biosynthetic pathways during platelet-neutrophil interactions. Electron microscopic examination of glomerulonephritic kidneys revealed areas of intimate contact between neutrophils and platelets within the lumen of glomerular capillaries. P-selectin on platelets is an important mediator of platelet neutrophil adhesion in vitro and in vivo. Prior treatment of animals with a blocking monoclonal antibody (mAb) against P-selectin (mAb CY1747), but not an isotype-matched non-blocking control mAb (mAb PNB1.6), caused striking inhibition of lipoxin A4 generation without attenuating neutrophil recruitment. Anti-P selectin mAb also blunted transcellular lipoxin A4 generation during coincubations of activated neutrophils and platelets in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543624 TI - Urinary excretion of protectin (CD59), complement SC5b-9 and cytokines in membranous glomerulonephritis. AB - Protectin (CD59) is a low molecular weight glycophosphoinositol-anchored inhibitor of the membrane attack complex of complement (MAC) that is present, for example, on the membranes of endothelial cells and on epithelial cells of glomeruli and distal tubuli. To examine for the possibility that CD59 becomes detached from cell surfaces following cell injury, this study evaluated renal excretion of CD59 in patients with idiopathic membranous glomerulonephritis (MGN; N = 21), diabetic nephropathy (DNP; N = 15) and in healthy control subjects (N = 13). CD59 in human urine was quantitated by a competitive solid-phase radioimmunoassay having approximately 13 kDa soluble urinary CD59 as a standard. Immunofluorescence microscopy demonstrated a decreased expression of CD59 in the glomeruli of MGN patients. Using a Triton X-114 phase separation method 91 to 97% of urinary CD59 was found to be in a soluble form without anchor-associated phospholipid. The mean (+/- SEM) level of urinary CD59 was 5.6 +/- 0.2 micrograms/ml in MGN patients, 3.7 +/- 0.4 micrograms/ml in healthy controls (P < 0.001) and 2.6 +/- 0.1 in DNP patients (P < 0.001). When related to urinary creatinine (UCr) the corresponding values were 11.9 +/- 5.6, 4.8 +/- 0.3 (P = 0.021) and 4.4 +/- 0.2 (P < 0.002), respectively. The amount of CD59 in urine correlated with the urinary excretion of soluble terminal complement complexes, SC5b-9 (r = 0.594, P < 0.006) in MGN patients. The excretion of CD59 also correlated with the excretion of the inflammatory mediator IL-1 beta (r = 0.671, P = 0.001) but not with TNF-alpha (r = 0.314, P = 0.178). No correlation of CD59 excretion was observed with duration of the disease level of proteinuria, serum albumin concentration or serum creatinine level. Based on these findings we speculate that the increased excretion of CD59 into urine in MGN patients is due to complement activation and inflammation induced shedding of CD59 from glomerular cells. PMID- 7543623 TI - ICAM-1 and VCAM-1 in human renal allograft rejection. AB - Light microscopy studies have demonstrated heightened ICAM-1 and VCAM-1 expression in renal allograft rejection in experimental animals and in humans, and administration of ICAM-1 blocking antibodies has been shown to prolong graft survival in nonhuman primates. We used a precise ultrastructural immunogold localization technique to identify the exact sites of expression of ICAM-1 and VCAM-1 in both normal human kidney and in renal allograft rejection. In the normal kidney ICAM-1 is moderately strongly expressed in glomeruli, on the endothelium and parietal epithelium and in the interstitium, on the endothelium of peritubular capillaries, arterioles and small arteries, on fibroblast-like interstitial cells and on the brush border of proximal tubules. In contrast, in normal kidney, VCAM-1 expression is restricted to the parietal epithelium and the basolateral surfaces of a few proximal tubule cells. In allograft rejection, although ICAM-1 expression appears to be increased, its pattern of distribution is similar to that seen in the normal kidney. However, VCAM-I in allograft rejection is widely expressed on the endothelium of peritubular capillaries and arterioles in association with adhesion of mononuclear leukocytes within these vessels. The tubular expression of VCAM-1, although still focal in nature, is increased on the basolateral surfaces in association with lymphocytic infiltration of tubules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543627 TI - [The correction of the hormonal disorders in the combined treatment of Sudeck's syndrome]. AB - On the basis of the results of complex examination of the patients with the Sudeck syndrome, the disorders in reproductive hormones in individuals of both sexes were revealed. The character and degree of pronouncement of these changes were defined, their correction was performed. The result obtained is positive. PMID- 7543628 TI - Extracellular matrix proteins in colorectal carcinomas. Expression of tenascin and fibronectin isoforms. AB - BACKGROUND: Interactions of tumor cells and extracellular matrix (ECM) components are crucial determinants of tumor cell spreading and metastatic activity. Particularly tenascin (TN) as a member of the adhesion modulating family of ECM and its alternatively spliced isoforms became the matter of interest in ECM changes associated with malignancy. EXPERIMENTAL DESIGN: We analyzed the composition of the stromal- and basement membrane-associated ECM of colorectal adenomas and carcinomas using indirect immunofluorescence. Tenascin was investigated by immunoblot of snap frozen tumor specimens. RESULTS: Fibronectin (FN), TN, and chondroitin sulfate proteoglycan were the major components of the tumor stroma. Normal basement membrane components like laminin (LM), collagen type IV, and heparan sulfate proteoglycan were down-regulated. In the center of the tumor, tumor glands were surrounded by discontinuous basement membranes. At the tumor-host interface and in solid, poorly differentiated tumors, no immunoreactivity with normal basement membrane components was found. However, in cases with pericellular anti-LM staining, LM immunoreactivity was also found at the tumor-host interface. An alternatively spliced isoform of TN with a molecular weight of 330 kDa was found in seven of 15 carcinomas. In four of these cases, an alternatively spliced isoform of FN containing the ED-B segment was present. CONCLUSIONS: The coexpression of alternative splicing of FN and TN suggests that there may be common regulation mechanisms. The matrix composition found in the present study resembles that of healing wounds and probably favors the invasive spread of tumor cells. PMID- 7543626 TI - Partial cloning and characterization of an arginine decarboxylase in the kidney. AB - Using homology-based polymerase chain reaction (PCR) amplification, we demonstrate the presence of arginine decarboxylase mRNA in tissues involved in arginine metabolism (brain, kidney, gut, adrenal gland, and liver of the rat) but not in organs (lung, heart, and spleen) in which arginine metabolism is low or absent. The polymerase chain reaction product from the kidney had a nucleotide sequence 61% identical to that of the E. coli biosynthetic arginine decarboxylase. On a whole tissue basis, kidney homogenates were three times more active than brain homogenates at decarboxylating [1-14C]arginine. Subcellular fractionation localized the arginine decarboxylase activity of the kidney to the mitochondria fraction. Agmatine, one of the products of arginine decarboxylation, was found to inhibit nitric oxide formation by post-mitochondrial supernatants of the brain or kidney. We propose that arginine is metabolized to two structurally different signaling molecules, nitric oxide and agmatine. Furthermore, agmatine can influence the nitric oxide synthase pathway. PMID- 7543625 TI - Urinary endothelin and renal vasoconstriction with cyclosporine or FK506 after liver transplantation. AB - Transplant immunosuppression using either cyclosporine (CsA) or FK506 leads to renal vasoconstriction. To examine the role of endothelin (ET) in this process, we measured plasma and urinary ET before and at intervals for two years after liver transplantation. Urinary prostacyclin (as 6-keto-PG-F1 alpha), thromboxane, glomerular filtration rate and renal plasma flow were also measured. Forty-four patients were treated with CsA-based regimens and 31 patients with FK506-based regimens. Prednisone doses after one year were lower with FK506 (5.5 +/- 0.5 vs. 10.5 +/- 0.5 mg/day) by study design. Circulating plasma ET remained above normal, but not different from pre-transplant levels. Urinary ET was elevated before transplant (24.6 +/- 3.4 ng/day vs. normal 16 +/- 1.5 ng/day, P < 0.05) and rose further after transplantation (48.5 +/- 13 ng/day, P < 0.05), remaining elevated for two years. 6-keto-PG-F1 alpha fell from 2567 +/- 338 ng/day to subnormal levels and remained suppressed (1158 +/- 128 ng/day, P < 0.01). Over the same period GFR fell (84 +/- 3 ml/min to 60 +/- 3 ml/min, P < 0.01) and renal vascular resistance index rose (11,119 +/- 561 to 23,279 +/- 1692 d.s.cm-5.m-2, P < 0.01). Similar changes were observed both with CsA and FK506-based immunosuppression. No changes in ET were attributable to dihydropyridine calcium channel blockers. These results demonstrate that urinary ET changes independently from plasma ET after transplantation. Elevated ET and suppression of endothelium derived prostacyclin persist with intense renal vasoconstriction for at least two years after transplant. PMID- 7543629 TI - Virus-induced increases in bronchiolar mast cells in Brown Norway rats are associated with both local mast cell proliferation and increases in blood mast cell precursors. AB - BACKGROUND: Parainfluenza type 1 (Sendai) virus-induced bronchiolitis during early life in rats induces increases in bronchiolar mast cells that persist for months after infection and are associated with airway obstruction and airway hyperresponsiveness. Brown Norway (BN) rats are highly susceptible, and Fischer 344 (F344) rats are relatively resistant to, Sendai virus-induced increases in airway responsiveness and mast cell density. EXPERIMENTAL DESIGN: To identify mechanisms responsible for the virus-induced mast cell increases, BN rats were studied using in vivo bromodeoxyuridine labeling, in vitro culture of bone marrow, blood, and lung mast cell progenitors (colony-forming unit-mast cell (CFU MC)), and in vivo treatment with the rodent mast cell stabilizers disodium cromoglycate and nedocromil sodium. Bone marrow, blood, and lung CFU-MC were also quantitated in F344 rats. RESULTS: At 10 days after inoculation, there was a fivefold increase (p = 0.001) in the bromodeoxyuridine labeling index of bronchiolar mast cells in virus-inoculated BN rats. Viral inoculation increased CFU-MC/ml blood greater than fivefold (p < 0.05) in BN rats at 10 days after inoculation, and BN rats had greater numbers of both blood and lung CFU-MC than did F344 rats. Treatment with inhibitors of mast cell degranulation had no effect on Sendai virus-induced bronchiolar mast cell increases in BN rats. CONCLUSIONS: Virus-induced increases in bronchiolar mast cells result from proliferation of preexisting mast cells and may be augmented by recruitment of circulating mast cell precursors. PMID- 7543631 TI - Effect of growth factors on cell proliferation and epithelialization in human skin. AB - The failure of chronic wounds to heal remains a major medical problem. Recent studies have suggested an important role for growth factors in promoting wound healing. We investigated the mitogenic effect of basic fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF), comparing their effects with those of media alone (MEM) in a human skin explant model. A stable organ culture system for maintaining the histologic structure of human epidermis for 10 days in vitro was developed. DNA synthesis was measured on Days 1, 3, and 7 of organ culture using [3H]thymidine ([3H]thy) uptake and expressed as cpm/mg dry weight (mean +/- SEM). FGF, IGF-1, and EGF were each capable of stimulating [3H]thy uptake on Day 1 of culture (2372 +/- 335 FGF, 2226 +/- 193 IGF-1, 4037 +/- 679 EGF vs 1108 +/- 70 MEM, P < 0.05). IGF-1 and EGF also stimulated [3H]thy uptake on Days 3 and 7 of culture. The organ culture system was further employed to observe epidermal outgrowth. Longest keratinocyte outgrowth from the explant periphery (simulating epithelial regeneration from the wound edge) was observed on Day 7. EGF resulted in maximum stimulation of epithelial outgrowth (440 +/- 80 microns), followed by FGF (330 +/- 56 microns), IGF-1 (294 +/- 48 microns), and MEM (189 +/- 50 microns). We postulate, therefore, that FGF, IGF-1, and EGF are important mitogens for wound healing and that EGF in particular is capable of stimulating epithelialization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543632 TI - Autolymphocyte therapy. III. Effective adjuvant adoptive cellular therapy with in vivo anti-tumor specificity against murine melanoma and carcinoma using ex-vivo activated memory T-lymphocytes. AB - Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease based upon ex vivo activation of lymphocytes by either the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC) or using low doses of the mitogenic monoclonal antibody OKT3 and a mixture of previously prepared cytokines (T3CS). We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts (TBH) using an MLC supernatant or T3CS without the use of tumor antigen results in the expansion of the CD44+ (memory) T-cell subset. These CD44+ T-cells are the principal mediators of anti-tumor specificity in the ALT-cell population in advanced metastatic murine tumors and are able to protect against tumor challenge in healthy syngeneic mice (HSM). To determine if ALT is effective in an adjuvant setting, C57BL/6J splenocytes from HSM and TBH with B16 melanoma or Lewis lung (3LL) carcinoma were activated ex vivo using T3CS. Mice were implanted with either B16 melanoma or 3LL carcinoma and then underwent surgical excision of tumor. Tumor excised mice (TEM) then received small numbers (10(6)) of ALT-cells derived from 3LL-TBH or B16-TBH splenocytes, HSM-derived ALT-cells, fresh splenocytes derived from 3LL-TBH or B16-TBH, or CD44-depleted ALT-cells. Significant anti-tumor activity as shown by prolonged survival (Day 100), cure of disease, and rejection of a local and systemic tumor rechallenge was demonstrated in 3LL-TEM that received B16-derived ALT-cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543630 TI - Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan. AB - BACKGROUND: A delayed revascularisation model has been used to assess the angiogenic activity of hyaluronan fragments on impaired wound healing. EXPERIMENTAL DESIGN: Revascularisation of single, full thickness skin autografts in rats was delayed by subjecting isolated grafts to a sublethal cryoinjury (freeze injury) before implantation. Hyaluronan fragments were delivered to the grafts using slow release pellets (ethylene vinyl acetate copolymer). Rates of release were measured in vitro by ELISA. The angiogenic response to the application of 100 micrograms of low (1 to 4 kDa) molecular weight hyaluronan and 100 micrograms of medium (33 kDa) molecular weight hyaluronan was tested in separate groups of 15 rats and was compared with unstimulated cryoinjured controls (n = 40). The effect of low (1 to 4 kDa) molecular weight hyaluronan on uninjured grafts was also investigated. Return of graft blood flow was measured on anesthetised animals over a 10-day period using laser Doppler flowmetry and 133Xe clearance. Quantitative histologic assessment of the graft vasculature was performed on Days 3, 7, and 10 after implantation. RESULTS: The 1- to 4-kDa hyaluronan fragments increased blood flow (p < 0.001), as measured by both flow measuring techniques, and increased graft vessel growth, as assessed histologically at each of the three time points (p < 0.05). By contrast, the 33 kDa fragments had no such effect on graft blood flow or vessel growth. Low molecular weight hyaluronan had no effect on either graft blood flow or on vessel growth in uninjured skin grafts. CONCLUSIONS: The hypothesis that there may be physiologic regulation of angiogenesis by hyaluronan and its metabolites is supported by the results of these studies. The data provide further evidence of the utility of the cryoinjured graft model for the study of in vivo angiogenesis. PMID- 7543633 TI - Hepatic function is preserved following liver-directed, adenovirus-mediated gene transfer. AB - Inherited and acquired disorders of the liver are attractive targets for gene therapy. Hepatic cells are susceptible targets for shuttle vectors because of a diversity of protein and viral receptors and accessibility of a selective afferent blood supply. Preservation of existing hepatic cell integrity and metabolic function is of paramount importance for successful whole animal gene therapy trials. In this report, we examine hepatic cell function and integrity following adenovirus-mediated reporter gene transfer to the liver in vivo. E1 deleted, replication-defective adenovectors encoding the LacZ gene driven by the human CMV promoter were delivered to the liver by isolated portal perfusion. The gene transfer rate, as determined by specific histochemical staining, approached 30% with recombinant protein detectable by Western blot throughout the course of study. Hepatic cell integrity as assessed by histology and hepatic enzyme profile (serum aspartate aminotransferase, gamma-glutamyl transpeptidase) demonstrated normal cellular architecture and no significant difference between transfected liver and controls. Hepatic synthetic and metabolic function, as determined by albumin levels, prothrombin time, and bilirubin, were similar between the two study groups. This study demonstrates that efficient adenovirus-mediated gene transfer and expression in the rat liver do not compromise hepatic cell metabolism and integrity. PMID- 7543635 TI - Late endothelial function of free and pedicled internal mammary artery grafts. AB - The internal mammary artery has greater long-term patency than the saphenous vein when used for coronary bypass grafting. Therefore, bilateral use of the internal mammary artery for grafting with the right internal mammary artery used as a "free" graft may result in improved graft survival. The study objectives were to compare the endothelial-dependent and -independent vasodilatory response in free and pedicled internal mammary artery grafts in patients who had previously undergone coronary surgery. Free (group 1, n = 8) and pedicled (group 2, n = 7) internal mammary artery grafts were studied by comparing the response to selective infusion of the endothelial-dependent vasodilator substance P (1.4 up to 22.4 pmol/min in doubling dose increments) followed by isosorbide dinitrate (2 mg over 2 minutes), in patients undergoing coronary angiography, 1 month to 6 years after coronary surgery. Maximal dilatory response to substance P was 8.7% +/- 1.8% in pedicled grafts compared with 8.8% +/- 2.3% in free grafts (p = not significant), with the dose response for both groups being similar. Infusion of isosorbide dinitrate produced only minimal further dilatation in both groups. No significant difference was found in endothelium-dependent and -independent vasodilatory response between free and pedicled internal mammary artery grafts, suggesting that the use of the free right internal mammary artery and other arterial grafts may enhance graft survival. PMID- 7543636 TI - Topical use of aprotinin in cardiac surgery. PMID- 7543638 TI - The effect of a shared decisionmaking program on rates of surgery for benign prostatic hyperplasia. Pilot results. PMID- 7543634 TI - Supplemental L-arginine during cardioplegic arrest and reperfusion avoids regional postischemic injury. AB - Unenhanced hypothermic cardioplegia does not prevent postischemic endothelial and contractile dysfunction in hearts subjected to antecedent regional or global ischemia. This study tested the hypothesis that supplementing blood cardioplegic solution and reperfusion with the nitric oxide precursor L-arginine would preserve endothelial function, reduce infarct size, and reverse postcardioplegia regional contractile dysfunction by the L-arginine-nitric oxide pathway. In 23 anesthetized dogs, the left anterior descending coronary artery was ligated for 90 minutes, after which total bypass was established for surgical "revascularization." In 10 dogs, unsupplemented multidose hypothermic blood cardioplegic solution was administered for a total of 60 minutes of cardioplegic arrest. In eight dogs, L-arginine was given intravenously (4 mg/kg per minute) and in blood cardioplegic solution (10 mmol) during arrest. In five dogs, the nitric oxide synthesis blocker N omega-nitro-L-arginine (1 mmol) was used to block the L-arginine-nitric oxide pathway during cardioplegia and reperfusion. Infarct size (triphenyltetrazolium chloride) as percent of the area at risk was significantly reduced by L-arginine compared with blood cardioplegic solution (28.2% +/- 4.1% versus 40.5% +/- 3.5%) and was reversed by N omega-nitro-L arginine to 68.9% +/- 3.0% (p < 0.05). Postischemic regional segmental work in millimeters of mercury per millimeter (sonomicrometry) was significantly better with L-arginine (92 +/- 15) versus blood cardioplegic solution (28 +/- 3) and N omega-nitro-L-arginine (26 +/- 6). Segmental diastolic stiffness was significantly lower with L-arginine (0.46 +/- 0.06) compared with blood cardioplegic solution (1.10 +/- 0.11) and was significantly greater with N omega nitro-L-arginine (2.70 +/- 0.43). In ischemic-reperfused left anterior descending coronary arterial vascular rings, maximum relaxation responses to acetylcholine, the stimulator of endothelial nitric oxide, was depressed in the blood cardioplegic solution group (77% +/- 4%) and was significantly reversed by L arginine (92% +/- 3%). Smooth muscle function was unaffected in all groups. We conclude that cardioplegic solution supplemented with L-arginine reduces infarct size, preserves postischemic systolic and diastolic regional function, and prevents arterial endothelial dysfunction via the L-arginine-nitric oxide pathway. PMID- 7543637 TI - A human CD4- CD8- T-cell receptor alpha beta + T leukemic cell line undergoing phytohemagglutinin-induced apoptosis. AB - A human CD4- CD8- alpha beta T-cell receptor (TCR alpha beta)+ T leukemic cell line, L-KAW, was established from the peripheral blood of a 6-year-old male patient with T-cell acute lymphoblastic leukemia. Its phenotype was found to be CD7+CD2+CD3+CD4-CD8- TCR alpha beta +. The cell line proved susceptible to killing by exposure to a mitogenic concentration of phytohemagglutinin (PHA) within several hours, with biochemical analyses demonstrating extensive degradation of DNA to oligonucleosomal bands characteristic of apoptosis. Chromatin condensation and cell shrinkage features of apoptotic cells could also be clearly identified under the electron microscope. The apoptosis-related Fas antigen was expressed on L-KAW cells and DNA fragmentation was induced by incubation with anti-Fas monoclonal antibody. The c-myc mRNA levels declined within 15 min to 3 h after the addition of PHA. These results suggest that L-KAW cells are specifically sensitive to induction of apoptosis in response to PHA without preactivation. They may, therefore, be considered an analog of activated T-cells and prove useful as a model system for characterizing biochemical and molecular mechanisms underlying the process of this type of cell death. PMID- 7543639 TI - Patient reactions to a program designed to facilitate patient participation in treatment decisions for benign prostatic hyperplasia. AB - Patients often want considerable information about their conditions, and enhanced patient participation might reduce unwanted practice variation and improve medical decisions. The authors assessed how men with benign prostatic hyperplasia reacted to an education program designed to facilitate participation in decisionmaking, and how strongly ratings of their symptom state and the prospect of complications predicted their treatment choice. A prospective cohort study was conducted in three hospital-based urology practices: two in prepaid group practices, and one Veterans Administration clinic. Four hundred twenty-one men with symptomatic benign prostatic hyperplasia without prior prostatectomy or benign prostatic hyperplasia complications were enrolled, and 373 provided usable ratings. Subjects participated in an interactive videodisc-based shared decisionmaking program about benign prostatic hyperplasia and its treatment options, prostatectomy, and "watchful waiting." They rated the length, clarity, balance, and value of the program and were followed for 3 months to determine if they underwent surgery. Patients rated the program as generally clear, informative, and balanced. Across all three sites, 77% of patients were very positive and 16% were generally positive about the program's usefulness in making a treatment decision. Logistic models predicting choice of surgical treatment documented the independent importance of negative ratings of the current symptom state (odds ratio 7.0, 95% confidence interval 2.9-16.6), as well as the prospect of postoperative sexual dysfunction (odds ratio 0.20, 95% confidence interval 0.08-0.48) in decisionmaking. Patients rated the Shared Decisionmaking Program very positively and made decisions consistent with their assessed preferences. These results suggest that patients can be helped to participate in treatment decisions, and support a randomized trial of the Shared Decisionmaking Program. PMID- 7543640 TI - [Chronic myeloid leukemia (CML): a therapeutic dilemma]. PMID- 7543642 TI - Colonic strictures in children with cystic fibrosis on low-strength pancreatic enzymes. PMID- 7543644 TI - Palliative care for patients with terminal renal failure. PMID- 7543645 TI - Tacrolimus and cardiotoxicity in adult liver transplant recipients. PMID- 7543646 TI - Effects of nitric oxide synthesis inhibition with or without nitric oxide inhalation on responses to systemic cocaine administration in rats. AB - The effects of N omega-nitro-L-arginine methyl ester (L-NAME) i.v. and nitric oxide (NO) inhalation on integrated systemic responses to cocaine were studied in lightly anesthetized, paralyzed, and mechanically ventilated rats. Cocaine (4 mg/kg/min i.v.) produced seizures then isoelectric electrocephalographic (isoEEG) activity as well as an initial increase in systolic blood pressure and heart rate, then progressive cardiovascular system depression culminating in asystole. Pretreatment with L-NAME (2 mg/kg/min i.v.) for 30 min significantly reduced the incidence of seizure as compared to saline treated animals (saline 7/8; L-NAME 3/8). Doses of cocaine that produced arrhythmias, isoEEG and asystole were significantly lower in the L-NAME treated animals as compared to the saline group. L-NAME did not affect peak systolic blood pressure and heart rate responses to cocaine. No inhalation (80 ppm) did not affect CNS and cardiovascular responses to cocaine in control animals but enhanced the effects of L-NAME on cocaine toxicity. The results show that pretreatment with L-NAME reduces the central nervous system stimulatory effect of cocaine (reduced seizure incidence) and enhances its depressant effect on both the central nervous system (lower does for isoEEG) and the cardiovascular system (lower dose for arrhythmias and asystole), but does not affect the cardiovascular stimulatory action of cocaine. NO inhalation does not protect against any of the systemic effects of cocaine in animals with normal or suppressed NO production. PMID- 7543643 TI - CD44 isoforms in prognosis of breast cancer. PMID- 7543641 TI - Colonic strictures in children with cystic fibrosis on low-strength pancreatic enzymes. PMID- 7543647 TI - Acute and chronic effects of fluoxetine and haloperidol on mouse brain serotonin and norepinephrine turnover. AB - Evidence from clinical studies suggests that the noradrenergic system may play a role in the pathophysiology of antidepressant- and neuroleptic-induced akathisia. In limited previous neurochemical research, acute treatment with selective serotonin reuptake inhibitors (SSRIs) has been reported to increase rat brain norepinephrine (NE) turnover or release. We have examined the neurochemical effects of 2 hr, 4 day, and 20 day treatment with the SSRI fluoxetine, and the neuroleptic haloperidol, on regional brain monoamine metabolism. Short and long term fluoxetine treatment produced substantial decreases (to 65-79% of control) in serotonin (5HT) turnover. However no effects of fluoxetine on mouse brain NE turnover--as assessed by forebrain, hypothalamus, and hindbrain 3-methoxy-4 hydroxyphenylglycol (MHPG) levels or MHPG/NE ratios--were observed. Acute (2 hr) fluoxetine also did not alter regional NE turnover in rat brain. In contrast, haloperidol tended to increase MHPG levels and MHPG/NE ratios in the mouse brain regions studied. The persistence of decreased 5HT turnover during fluoxetine treatment, the lack of an effect of fluoxetine on NE turnover, and the increased NE turnover seen after haloperidol may have important implications regarding drug responsivity and the mechanism of akathisia induction. PMID- 7543649 TI - Effect of hexachlorocyclohexane isomers on calmodulin mRNA expression in the central nervous system. AB - Three different calmodulin genes that encode the same protein have been found in the brain of all mammalian species so far examined. Little is known about the factors involved in regulating the expression of this gene family in the central nervous system. We have investigated the possibility of differential expression of two calmodulin genes, CaM I and CaM II, which are expressed strongly in neuronal cells in the adult rat brain, after treatment with the gamma (lindane) and the delta isomers of the hexachlorocyclohexane (HCH). In this study a decrease of CaM I mRNA (mainly in the 4.0 kb transcript) was found in the cortex of the rats after 24 h of isomer administration. CaM I expression seemed to be more sensitive to delta isomer action, whereas the gamma isomer acted mainly at CaM II level. The levels of mRNA of calmodulin CaM II gene were also found to decrease after lindane administration; delta-HCH produced an increase of this transcript. These results were obtained by Northern blot analysis and confirmed by means of in situ hybridization. Our results suggest that levels of neuronal calmodulin mRNA species are modified in response to changes in neuronal activity. PMID- 7543648 TI - Double in situ hybridization study on coexistence of mu-, delta- and kappa-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia. AB - Coexistence of the mRNA for each subtype of opioid receptor (OPR) with the mRNA for preprotachykinin A (PPTA), a precursor protein of substance P (SP), in the rat dorsal root ganglia was examined by double in situ hybridization technique. About 90% and 30% of PPTA mRNA-positive neurons expressed mu- and kappa-OPR mRNAs at high level, respectively. However, only about 3% of PPTA mRNA-positive neurons expressed delta-OPR mRNA at high level. These results suggest that mu- and kappa OPRs exist on most of and a part of the primary afferent terminals containing SP, respectively. On the other hand, among the neurons which highly expressed mu-, delta- or kappa-OPR mRNA, PPTA mRNA was not expressed in about 58%, 95% or 24% of those neurons, respectively. These findings suggest the possibility that OPRs co exist with other neurotransmitters and/or neuromodulators than SP in the primary afferent neurons. PMID- 7543650 TI - Modified trichrome staining technique of the nerve to determine proximal nerve viability. AB - Normal myelinated fibers are stained "red" by modified trichrome staining of the frozen section of the nerve. We have used this technique for rapid assessment of the anatomical integrity of the proximal nerve stump by identifying well preserved myelinated fibers. This technique can also identify degenerating and degenerated myelinated fibers, fibrosis, and inflammatory cells. We report three cases in which the practical usefulness of this technique is demonstrated. PMID- 7543652 TI - Insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the kidney of the chronically hypoinsulinemic rat and hyperinsulinemic rat. AB - Acute streptozotocin (STZ)-induced diabetes in rats causes a transient increase in insulin-like growth factor-I (IGF-I) in the kidney, followed by a rapid renal hypertrophy and constant renal hyperperfusion. However, renal IGF-I levels return to normal within 4 days. Thus, hyperperfusion, which is independent of renal hypertrophy of the chronically diabetic kidney, is not explained by increased renal IGF-I. We studied IGF-I and IGF-I receptor gene expression in the kidney of rats with long-standing STZ-induced diabetes. IGF-I mRNA level in the chronically diabetic kidney was approximately 50% of that in control rats, whereas IGF-I receptor mRNA was increased approximately threefold. Ten days' treatment with insulin 65 days after induction of diabetes resulted in a glucose-dependent decrease in IGF-I receptor mRNA. Chronic hyperinsulinemia with near normoglycemia did not change gene expression of either IGF-I or IGF-I receptor. The studies suggest that glucose levels per se, independent of insulin levels, play an important role in the regulation of IGF-I receptor gene expression in the chronically diabetic kidney. Furthermore, kidney hyperperfusion in chronic diabetes is coupled with the increase in IGF-I receptor mRNA, despite normal kidney IGF-I levels. PMID- 7543651 TI - Role of galanin in the regulation of somatotrope and gonadotrope function in young ovulatory women. AB - The aim of the study was to elucidate the role of the neuropeptide galanin in the regulation of somatotropic and gonadotropic function in normal women. Thirteen normally ovulating (aged 28 to 40 years), non-obese (body mass index, 18.4 to 27.1 kg/m2) women with infertility due to a tubal or male factor were studied. Each woman underwent three tests: (1) bolus intravenous (IV) injection of growth hormone (GH)-releasing hormone (GHRH) (1-29)NH2 1 microgram/kg plus gonadotropin releasing hormone (GnRH) 100 micrograms at time 0; (2) IV infusion of porcine galanin 500 micrograms in 100 mL saline from -10 minutes; and (3) bolus IV injection of GHRH(1-29)NH2 1 microgram/kg plus GnRH 100 micrograms at time 0 plus IV infusion of porcine galanin 500 micrograms in 100 mL saline from -10 to +30 minutes. All results are expressed as the mean +/- SEM. GH peak after GHRH was 14 +/- 5 micrograms/L; porcine galanin significantly increased serum GH (GH peak, 7.3 +/- 1.2) with respect to baseline levels. No significant differences were observed between either GH peak or GH absolute values after galanin as compared with GHRH alone. Porcine galanin significantly enhanced GH response to GHRH (peak, 31.4 +/- 4.4 micrograms/L) with respect to either GHRH or galanin alone. Luteinizing hormone (LH)/follicle-stimulating hormone (FSH) peaks after GnRH were 16.5 +/- 5.3 and 17.4 +/- 4 IU/L, respectively. Porcine galanin did not cause significant increases in serum LH and FSH levels with respect to baseline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543653 TI - Leukocyte adhesion. Missing link in angiogenesis. PMID- 7543654 TI - Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. AB - Endothelial adhesion molecules facilitate the entry of leukocytes into inflamed tissues. This in turn promotes neovascularization, a process central to the progression of rheumatoid arthritis, tumor growth and wound repair. Here we test the hypothesis that soluble endothelial adhesion molecules promote angiogenesis. Human recombinant soluble E-selectin and soluble vascular cell adhesion molecule 1 induced chemotaxis of human endothelial cells in vitro and were angiogenic in rat cornea. Soluble E-selectin acted on endothelial cells in part through a sialyl Lewis-X-dependent mechanism, while soluble vascular cell adhesion molecule 1 acted on endothelial cells in part through a very late antigen (VLA)-4 dependent mechanism. The chemotactic activity of rheumatoid synovial fluid for endothelial cells, and also its angiogenic activity, were blocked by antibodies to either soluble E-selectin or soluble vascular cell adhesion molecule-1. These results suggest a novel function for soluble endothelial adhesion molecules as mediators of angiogenesis. PMID- 7543655 TI - Origin of life. RNA seeks its maker. PMID- 7543657 TI - Expression of the alpha 2-macroglobulin receptor on human neoplastic fibroblastoid cells. AB - The alpha 2-macroglobulin membrane-associated receptor (alpha 2MR) has been previously detected on hepatocytes, fibroblasts, macrophages, syncytiotrophoblasts and recently on human malignant blood cells of myelomonocytic leukemia. In cells growing in vitro from human germ cell tumors alpha 2MR mRNA was detected by Northern blotting. Endocytosis of alpha 2M from culture medium was detected in these cells by indirect immunofluorescence. In cell extracts alpha 2M and its degradation products were detected by immunoblotting. The cells expressing alpha 2MR and internalizing alpha 2M were identified as fibroblasts both by their morphology and expression of vimentin intermediate filaments. The role and function of alpha 2MR receptor in the analyzed neoplastic cells of teratomatous origin is discussed. PMID- 7543658 TI - [Anticonvulsive drug therapy. Historical and current aspects]. AB - The development of new antiepileptic drugs in recent years has enlarged the number of anticonvulsant compounds for the treatment of intractable focal epilepsies. The anticonvulsant potency of these drugs is usually compared by the number of patients who achieve a reduction in seizure frequency of more than 50%. Such an effect can be observed in approximately 20-30% of patients with pharmacoresistant focal epilepsies and is about the same with all the new compounds. In addition to the influence on focal seizures some of the novel anticonvulsant drugs exhibit efficacy in generalized seizures or in Lennox Gastaut syndrome. In general there are fewer side effects in newly developed drugs than in standard anticonvulsants. However, in some cases characteristic side effects may occur: weight gain, depression or psychosis from vigabatrin; lamotrigine may provoke allergic rashes and felbamate may cause gastrointestinal side effects and sleeplessness. Apart from felbamate, there are no interactions with an antiepileptic comedication or they are of little importance. The development of the new anticonvulsants follows a rational design based on pathophysiological aspects: the main aim is to influence synaptic transmission, resulting in an increase in inhibitory and a decrease in excitatory transmitters. Thus, vigabatrin and tiagabine enhance the endogenous GABA amount, whereas felbamate and remacemide interact with the NMDA-receptor complex. Because it is not possible to draw sufficient conclusions from add-on studies in clinical testing it is necessary to establish new forms of trial design. Monotherapy designs are favored because they lack possible interactions with comedication and make the anticonvulsant efficacy of the compound better comparable to those of established anticonvulsants.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543659 TI - [Craniectomy in space-occupying middle cerebral artery infarcts]. AB - Space occupying supratentorial ischemic stroke has a high mortality. The benefit of decompressive surgery in these patients is still matter of debate. In a prospective study we performed craniectomy in 37 patients with acute middle cerebral artery infarction and progressive deterioration under conservative antiedematous therapy. Twenty-one patients treated conservatively during the same period served as control group. All survivors were reexamined between one to two years after surgical decompression. In addition, neuropsychological tests were performed, including an Aachener Aphasie Test (AAT) in those patients with infarction of speech-dominant hemisphere. Clinical evaluation was graded using the Barthel index (BI). Mortality rate in the operated group was 37%. Twenty three patients survived acute stroke and were reexamined. Despite complete hemispheric infarction, no patient suffered from complete hemiplegia or was permanently wheel chair bound. In speech dominant hemispheric infarction (n = 8) only mild to moderate aphasia could be detected. Mean BI was 64. Mortality rate in the conservatively treated group was 76%. The clinical outcome following craniectomy for the treatment of severe ischemic hemispheric infarction is unexpectedly good. Therefore, decompressive surgery should be considered in cases of space-occupying hemispheric infarctions and conservatively uncontrollable intracranial pressure. PMID- 7543656 TI - Template switching between PNA and RNA oligonucleotides. AB - The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information. PMID- 7543660 TI - [Copolymer 1 in therapy of multiple sclerosis]. PMID- 7543661 TI - Inhibition of axoplasmic transport in the rat vagus nerve alters the numbers of neuropeptide and tyrosine hydroxylase messenger RNA-containing and immunoreactive visceral afferent neurons of the nodose ganglion. AB - Previous work showed that axotomy-induced deafferentation of the placode-derived visceral afferent neurons of the nodose ganglion altered their expression of some neuropeptides and tyrosine hydroxylase. The present studies were designed to selectively evaluate the loss of axonal transport on the numbers of vasoactive intestinal polypeptide, tyrosine hydroxylase, and calcitonin gene-related peptide mRNA-containing and immunoreactive neurons in the nodose ganglion of the adult rat. Vinblastine (0.15 mM) application to the cervical vagus nerve was used to block axonal transport between ganglionic perikarya and peripheral targets. In situ hybridization histochemistry with 35S-labeled oligonucleotide probes was used to both quantify the number of mRNA-containing neurons and to assess the density of mRNA expression per neuron, and immunocytochemistry was used to visualize the number of immunoreactive neurons. The efficacy of vinblastine to inhibit axonal transport was verified by evaluating the build-up of calcitonin gene-related peptide immunoreactive in the vagus nerve immediately rostral to the site of drug application. The absence of vinblastine-induced neuronal damage was verified by the relative absence of degenerating nerves in the vagus nerve caudal to the site of drug application. Vinblastine treatment of the vagus nerve increased the numbers of vasoactive intestinal peptide mRNA-containing neurons and vasoactive intestinal peptide-immunoreactive neurons in the nodose ganglion at three, seven and 14 days, and increased the numbers of calcitonin gene-related peptide mRNA-containing and calcitonin gene-related peptide-immunoreactive neurons in the nodose ganglion at one, three and seven days. The average labeling density of vasoactive intestinal peptide mRNA-containing neurons was also increased following vinblastine treatment. Vinblastine treatment of the cervical vagus nerve, however, led to the appearance of low-labeling density calcitonin gene-related peptide mRNA-neurons and resulted in reduction of the average labeling density for calcitonin gene-related peptide mRNA-containing neurons. In contrast, application of vinblastine to the cervical vagus nerve, decreased the number of tyrosine hydroxylase mRNA-containing and tyrosine hydroxylase immunoreactive neurons in the nodose ganglion. In summary, inhibition of the axoplasmic transport between the periphery and the visceral sensory perikarya appeared to alter vasoactive intestinal peptide, calcitonin gene-related peptide, and tyrosine hydroxylase expression and content in visceral sensory neurons of the nodose ganglion. These data suggest the presence of an axonally transported influence on the regulation of neuropeptide and neurotransmitter enzyme synthesis in mature placode-derived visceral sensory neurons. PMID- 7543664 TI - Voltage-gated ionic currents in mature oligodendrocytes isolated from rat cerebellum. AB - Mature rat cerebellar oligodendrocytes were isolated in culture using a serum free medium and identified using anti-galactocerebroside (GalC) and OL-1 antibodies. The morphology of such oligodendrocytes changes with time in culture from a multipolar shape at about 4 days in vitro (DIV) to a monopolar shape at about 8 DIV, a transition that has been previously described only in situ. Voltage-gated ionic currents were characterized at both oligodendrocyte developmental stages using the whole cell configuration of the patch-clamp technique. No differences between these two stages were detected, both types expressed a large K+ inward rectifying current similar to that described for oligodendrocytes from other vertebrate neuronal structures. This current could play an important role in the control of oligodendrocyte resting membrane potential. Our culture system provides a valuable model, close to the situation encountered in situ, not only to study the process of oligodendrocyte maturation, but also to identify the possible interactions between oligodendrocytes and cerebellar neurons such as granular and Purkinje cells during development. PMID- 7543663 TI - Long-term enhancement of dopamine release by high frequency tetanic stimulation via a N-methyl-D-aspartate-receptor-mediated pathway in rat striatum. AB - We studied the effects of high frequency tetanic stimulation of the striatum on the KCl (20 mM)-evoked dopamine release in rat striatal slices. The KCl-evoked dopamine release was potentiated by high frequency tetanic stimulation (10-20 Hz) of the striatum including the corticostriatal fibers, and this potentiation was observed until 3 h after high frequency tetanic stimulation. Potentiation of dopamine release after high frequency tetanic stimulation was induced not only by KCl but also by glutamate in Mg(2+)-free medium, N-methyl-D-aspartate in Mg(2+) free medium, and by DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. 2-amino-5-phosphovalerate, 3-[(+/-)-2-carboxypiperazine-4-yl]-propyl-1 phosphonate or dibenzocycloheptaneimine, N-methyl-D-aspartate receptor inhibitors, abolished enhancement by tetanus, whereas, 6,7-dinitroquinoxaline-2,3 dione, an antagonist of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ionotropic receptors, or L-2-amino-4-phosphonobutyrate, an antagonist of glutamate metabotropic receptors, showed no effect. Moreover, pretreatment with glutamate or N-methyl-D-aspartate in the absence of Mg2+ also facilitated dopamine release evoked by KCl concentrations. When extracellular Ca2+ was removed from the medium during pretreatment, potentiation by glutamate disappeared. We conclude that activation of N-methyl-D-aspartate receptors on dopaminergic nerve terminals in the striatum produces the long-term changes in efficacy of the response to KCl or glutamatergic agents. That is, plastical phenomena could exist at presynaptic levels between glutamatergic neurons and dopaminergic neurons in striatum. PMID- 7543662 TI - NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons. AB - Nitric oxide, a simple gas which serves as a neurotransmitter in the CNS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfaction has been questioned by experiments in which nitric oxide synthase, the enzyme that generates nitric oxide, could not be localized to the olfactory epithelium. We have localized nitric oxide synthase to the olfactory neurons in adult rat and catfish olfactory epithelia using a modified nicotinamide adenine dinucleotide phosphate diaphorase technique. In the rat, staining was also found in cells with morphology reminiscent of microvillar olfactory cells. In contrast, the respiratory epithelium and the sustentacular cells in the olfactory epithelium displayed no staining. The nicotinamide adenine dinucleotide phosphate diaphorase reaction, which has been shown to co-localize with immunohistochemical staining for nitric oxide synthase in the brain, was stimulated by addition of the nitric oxide synthase substrate L-arginine, and was inhibited by the nitric oxide synthase inhibitor L-NG-nitro arginine, indicating that staining was specific for nitric oxide synthase. Unilateral bulbectomy, which causes degeneration of mature olfactory neurons on the bulbectomized size, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were substantiated by biochemical assays for nitric oxide synthase by monitoring the production of [3H]-L-citrulline from [3H]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of mature olfactory neurons in rat and catfish olfactory epithelial suggesting the presence of nitric oxide synthase in these cells. Our histological and biochemical findings, in conjunction with data from other research, are supportive of a role for nitric oxide synthase in olfactory function. PMID- 7543665 TI - Guidelines for the use of G-CSF. PMID- 7543666 TI - Changing perspectives in benign prostatic hyperplasia. PMID- 7543667 TI - Prognostic value of CD44 splice variant expression in ovarian cancer. AB - In 44 ovarian cancers, CD44 variant (CD44v) expression was investigated immunohistochemically using a variant-specific polyclonal antibody. Patients with CD44v-positive carcinomas had a significantly shorter disease-free survival than patients with CD44v-negative tumors. Overall survival was also significantly reduced for stages III and IV of the International Federation of Gynecology and Obstetrics. Furthermore, a highly significant inverse correlation was observed between CD44v expression and preoperative platelet count. Urinary neopterin concentration, a marker of cell-mediated immunostimulation, did not differ between CD44v-positive and -negative ovarian cancer patients. Moreover, in seven ovarian carcinoma cell lines, modulation of CD44v expression was analyzed by living cell radioimmunoassay. Interferon-alpha, interferon-gamma, tumor necrosis factor, transforming growth factor-beta, all-trans retinoic acid and cisplatin did not affect CD44v expression. PMID- 7543668 TI - Absence of cumulative bone marrow suppression in heavily pretreated ovarian cancer patients undergoing salvage chemotherapy with paclitaxel. AB - This retrospective study was undertaken to investigate whether paclitaxel was associated with cumulative bone marrow toxicity in patients undergoing salvage chemotherapy for refractory ovarian cancer. Seventy-seven patients were treated with paclitaxel 135 mg/m2 every 21 days, with granulocyte-colony-stimulating factor (G-CSF) support as necessary according to standard criteria. The mean white blood cell nadir was significantly higher and the incidence of severe leukopenia (Gynecologic Oncology Group grade 3-4) significantly lower after ten cycles than after the first cycle for the entire study population (3.4 vs. 1.6 x 10(3)/mm3 and 29 vs. 77%, respectively) and the patients who received G-CSF (3.5 vs. 1.4 x 10(3)/mm3 and 33 vs. 89%, respectively), but did not differ significantly for the patients who did not require G-CSF (2.9 vs. 2.5 x 10(3)/mm3 and 40 vs. 59%, respectively). The mean hematocrit and platelet nadirs, as well as the incidence of severe anemia and thrombocytopenia, did not differ significantly after ten cycles from those after the first cycle for the entire study population and both subgroups. Thirty-two (42%) patients received G-CSF, each initiated within four cycles. The indications for initiating G-CSF support were febrile leukopenia (53%) and treatment delay (47%). The average duration of G-CSF support was 4.6 days, and did not increase significantly as the number of paclitaxel cycles increased. We conclude that paclitaxel was not associated with cumulative bone marrow toxicity in patients undergoing salvage chemotherapy for refractory ovarian cancer. PMID- 7543670 TI - Time-course changes in pancreatic laboratory and morphologic parameters in two different acute pancreatitis models in rats. AB - The aim of this work was to study in rats the temporal course of laboratory parameters and morphologic features in acute pancreatitis induced by cholecystokinin octapeptide (CCK-8) or by a closed duodenal loop. Pancreatitis was induced either with an overdose of CCK-8 (3 x 75 micrograms/kg at 1 h intervals) or by ligation of the duodenum on both sides of the bilio-pancreatic duct. The animals were examined at 0, 2, 4, 8, 16 and 24 h after AP induction. In CCK-8-induced acute pancreatitis, the pancreatic weight/body weight ratio (8.2 +/ 1.1 mg/g) and the amylase level (44.8 +/- 7.5 x 10(3) U/ml) were significantly increased vs. the controls (4.5 +/- 0.8 mg/g and 3.3 +/- 0.2 x 10(3) U/ml, respectively) 2 h after the intervention. The plasma CCK was significantly increased at 4 h (4.55 +/- 1.7 pM) and remained elevated thereafter. The tissue malonyldialdehyde concentration was significantly elevated at 8 h (0.28 +/- 0.07 mumol/mg pancreas) vs. the controls (0.20 +/- 0.02 mumol/mg pancreas). In closed duodenal loop-induced acute pancreatitis, the ratio pancreatic weight/body weight steadily increased during the study; it reached its maximum level at 24 h (7.1 +/ 0.5 mg/g) vs. the sham-operated control (4.8 +/- 0.9 mg/g). The serum amylase level was significantly elevated at 2 h (47.1 +/- 9.3 x 10(3) U/ml), and then decreased steadily. Plasma CCK values were significantly higher than the controls throughout the study. A significant increase in the tissue malonyldialdehyde concentration (0.94 +/- 0.15 mumol/mg vs. 0.20 +/- 0.01 mumol/mg pancreas) appeared at 4 h. Our data indicate that in CCK-8-induced acute pancreatitis the laboratory signs of pancreatitis are most expressed at 4 h, whereas the morphologic changes culminate 8 h, following the last CCK injection. In closed duodenal loop-induced acute pancreatitis, the histologic findings showed a progressive deterioration. Endogenous CCK and oxygen-derived free radicals seem to play a role in the pathogenesis of both types of acute pancreatitis. PMID- 7543671 TI - PSA assay of dried blood samples from the ear lobe on a filter paper with special reference to prostatic mass screening. AB - To eliminate complicated procedures for taking blood samples in the field work for prostatic mass screening, we developed a method for PSA testing using blood samples taken from the ear lobe and dried on a filter paper. The stability, the diagnostic efficacy, and the cost-benefit effect of the method were confirmed in hospitalized patients, as well as in subjects from our mass screening program. PMID- 7543669 TI - [Results of studies of prostate-specific antigen and prostate-specific antigen density in patients with prostatic hypertrophy and prostatic cancer]. AB - The authors investigated the PSA concentration and the preoperative prostate volume of 113 histologically proved BPH and 31 prostate cancer patients. There was no correlation between the age of the patients and the volume of the prostate. Whereas, correlation was proved between the marker concentration and the prostate volume (p < 0.0001). The prostate volume and the PSA concentration ratio correlated between the BPH and tumorous patients (PSA density). The difference was highly significant (p < 0.001). The use of PSAD improves further the diagnostical value of PSA. PMID- 7543672 TI - [Interdependence of the immune system and structure of extracellular matrix proteins]. AB - The major proteins from plasma and extracellular matrices (ECM) are described. The interaction of lymphoid cells with their microenvironment is critical for their growth and function. The interaction with ECM may play a significant role in defining the biological properties of many cells. ECM form a network of bioactive proteins, that is being linked with an increasing number of processes, including antigen-independent activation, proliferation, homing and cell migration. Some aspects of the participation of the extracellular matrix in the language of intracellular communication is discussed. PMID- 7543673 TI - Beta 1 integrins in third trimester human placentae: no differential expression in pathological pregnancy. AB - Integrins are a group of cell surface receptors that play important roles in cell cell and cell-extracellular matrix interactions. The expression of trophoblast cell surface integrin subunits changes during placental development in normal pregnancy but the functional significance is unknown. The aim of this study was to investigate the expression of beta 1 integrins and their extracellular matrix ligands in human placenta and membranes in normal and pathological pregnancy using an avidin-biotin-peroxidase technique. Expression of the beta 1 integrins was similar in all study groups. Whilst there was some heterogeneity of expression of specific integrin alpha chains this was not characteristic of defined subject groups, variations occurring within all groups. Two distinct trophoblast subpopulations were demonstrated in the chorion laeve according to differential expression of beta 1 integrins. Trophoblast immediately adjacent to maternal decidua, which expressed alpha 1 rather than alpha 2, also comprised the majority of trophoblast in the basal plate; possession of the alpha 1, alpha 3, alpha 5, alpha 6 rather than alpha 2, alpha 3, alpha 5, alpha 6 phenotype may be important in the invasive potential of trophoblast populations. The results obtained in the present study indicate that the integrin phenotypes of third trimester uteroplacental tissues are similar in normal and pathological pregnancy, including pre-eclampsia, before and after labour. PMID- 7543674 TI - Angiogenesis and the placental environment. AB - Rapid growth and vascularization of the human placenta are characteristic of early pregnancy and are accomplished in an unusually hypoxic environment. Stimulation of placental growth through hypoxia-induced angiogenesis may therefore be of particular importance. We have previously found that several varieties of vascular endothelial growth factor (VEGF) mRNA, including VEGF165, are present in cultured placental fibroblasts. We hypothesized that hypoxia would increase the transcription and translation of VEGF by these cells and provide one mechanism linking placental development with its environment. Placental fibroblasts were grown in aerobic or anaerobic atmospheric conditions for 72 h. By 24 h the oxygen tension of the anaerobic culture media was significantly less than that of the aerobic cultures. RNA was extracted from the cells at 24, 48 and 72 h. Following reverse transcription polymerase chain reaction (RT-PCR) stronger signals for VEGF were always found in the anaerobic cultures and this was confirmed by competitive PCR. mRNA for VEGF165 was represented most strongly but the anaerobic cultures also showed clearly mRNA for VEGF121, VEGF189 and VEGF206. The VEGF protein was also measured in the aerobic and anaerobic culture medium. By 72 h the average concentration of VEGF was significantly higher (P = 0.01) in the anaerobic culture medium. VEGF production is one mechanism through which oxygen supply may influence placental development. Examples of this may include the compensatory placental hypertrophy associated with maternal anaemia and with reproduction at high altitude. PMID- 7543676 TI - Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity. AB - To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL 2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding. PMID- 7543675 TI - Kinking of DNA and RNA by base bulges. PMID- 7543677 TI - Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells. AB - The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1 transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways. PMID- 7543678 TI - Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site. AB - Microbial community structure in natural environments has remained largely unexplored yet is generally considered to be complex. It is shown here that in a Mid-Atlantic Ridge hydrothermal vent habitat, where food webs depend on prokaryotic primary production, the surface microbial community consists largely of only one bacterial phylogenetic type (phylotype) as indicated by the dominance of a single 16S rRNA sequence. The main part of its population occurs as an ectosymbiont on the dominant animals, the shrimp Rimicaris exoculata, where it grows as a monoculture within the carapace and on the extremities. However, the same bacteria are also the major microbial component of the free-living substrate community. Phylogenetically, this type forms a distinct branch within the epsilon Proteobacteria. This is different from all previously studied chemoautotrophic endo- and ectosymbioses from hydrothermal vents and other sulfidic habitats in which all the bacterial members cluster within the gamma-Proteobacteria. PMID- 7543679 TI - Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor. AB - A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser desorption-ionization mass spectrometry based on the molecular mass and on post source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala 268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis. PMID- 7543681 TI - Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach. AB - Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs- a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology. PMID- 7543680 TI - Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. AB - E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation-sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism of transcriptional inactivation of some genes. We found that E-cadherin expression negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property. PMID- 7543682 TI - Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. AB - P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin. PMID- 7543683 TI - An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor. AB - We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay. Neither the nonphosphorylated tyrosyl peptide nor an unrelated phosphotyrosyl peptide previously shown to inhibit the signal transducers and activators of transcription factor (STAT) blocked binding of the hER to the ERE. The hER phosphotyrosyl peptide was shown by molecular sizing chromatography to dissociate the hER dimer into monomers. The hER specifically bound the 32P-labeled phosphotyrosyl peptide, indicating that the inhibition of ERE binding was caused by the phosphotyrosyl peptide binding directly to the hER and blocking dimerization. These data suggest that the phosphorylation of tyrosine-537 is a necessary step for the formation of the hER dimer. In addition, we propose that the dimerization of the hER occurs by a previously unrecognized Src homology 2 domain (SH2)-like phosphotyrosyl coupling mechanism. Consequently, the phosphotyrosyl peptide represents a class of antagonists that inhibits estrogen action by a mechanism other than interacting with the receptor's hormone binding site. PMID- 7543684 TI - Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. PMID- 7543685 TI - Somatostatin in a water-restricted environment: fluorescence and circular dichroism study in AOT reverse micelles. AB - Steady-state and time-resolved fluorescence emission from the single tryptophan residue of somatostatin, and the kinetics of quenching of this emission, were studied in aqueous solution and in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT)/water/isooctane, a system that mimics the water-membrane interface well. Incorporation into micelles caused blue shifts and reduced band widths of the emission peaks and altered the quantum yields with respect to emission from bulk water. Steady-state anisotropy values also increased considerably on micellization. These observations point to reduced polarity of the environment around the Trp residue of the peptide, as well as restricted freedom of its rotational motions, due to transfer from the aqueous to the micellar phase. Fluorescence emission kinetics of the Trp moiety followed biexponential decay laws in both aqueous and micellar media. Static and dynamic quenching constants were measured for acrylamide and CCI4 quenchers localized in the micellar and organic pseudophases, respectively, using both steady-state and time-resolved experiments. The efficiency of dynamic quenching by acrylamide became vanishingly small in going from water to reverse micelles, in sharp contrast to the comparable quenching efficiencies exhibited by CCI4 in micelles and acrylamide in water. The circular dichroic (CD) spectrum of the native peptide in water indicated the possibility of some amount of beta-type secondary structure being present. Conformational analysis of CD spectra in micelles showed that the relative amount of this structural feature was enhanced for the micellized peptide but was insensitive to the water content of micelles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543686 TI - Anticoagulants and hematomas in free flap surgery. AB - A review of systemic anticoagulant use in 517 free flap procedures was performed to determine the associated risk of hematoma formation. Patients were divided retrospectively (not randomly) into five groups: no anticoagulation (227 flaps, 5.3 percent hematomas), low-dose heparin bolus of 2000 to 3000 units and postoperative infusion at a rate of 100 to 400 units/hr for 5 to 7 days (192 flaps, 6.7 percent hematomas), intraoperative bolus of 5000 units of heparin without postoperative anticoagulation (46 flaps, 6.5 percent hematomas), high dose heparin infusion at a rate of 500 to 1200 units/hr (30 flaps, 20 percent hematomas), and dextran 40 infusion at a rate of 25 ml/hr (22 flaps, 9.1 percent hematomas). Intraoperative blood loss was similar for all groups. The flap loss rate was lower in the bolus (1.0 percent) and low-dose (1.0 percent) heparin groups than in the no-anticoagulation group (4.4 percent), but this difference was not statistically significant. The pedicle thrombosis rate also was lower in the bolus (2.2 percent) and low-dose (2.1 percent) heparin groups than in the no anticoagulation group (6.2 percent). A cause-and-effect relationship between the use of anticoagulants and flap loss or prevention of thrombosis could not be established. We can conclude, however, that the use of low-dose heparin does not increase significantly the risk of hematoma or intraoperative bleeding. PMID- 7543688 TI - [The present status of radiotherapy in the treatment of non-small cell bronchial cancer]. AB - GOAL. To re-define the type and place of radiotherapy in non-small cell bronchial carcinoma, where the disease is localised, either operable or non-operable, and in metastatic disease. METHOD. A review of the literature, review of the principle randomised trials and non-randomised trials describing innovative procedures, and a commentary and debate. For operable disease the indications for post-operative radiotherapy are redefined and the treatments envisaged such as those proposed using new adjuvents which may or may not include radiotherapy are discussed. For non-operable disease, different possibilities have been explored, including increased doses of treatment, modifications of the fraction regime, the association of radio-chemotherapy either concomitantly or successively. In metastatic disease the place of mediastinal radiotherapy is discussed and the possibility of radiation of the metastatic sites is recalled. PMID- 7543687 TI - In-vitro exposure of guinea pig main bronchi to 2.5 ppm of nitrogen dioxide does not alter airway smooth muscle response. AB - In order to investigate whether the oxidant airborne pollutant nitrogen dioxide (NO2) affects airway smooth muscle responsiveness, the contractile response of guinea pig main bronchi after in vitro exposure to 2.5 ppm of nitrogen dioxide was studied. Main bronchi were cannulated and exposed for 2 or 4 h to a constant flow of either NO2 or air. After exposure, bronchial rings were obtained and placed in a 37 degrees C jacketed organ bath filled with Krebs-Henseleit solution. Concentration-response curves were performed for acetylcholine (10(-9) 10(-3) M), substance P (10(-9)-10(-4) M), and neurokinin A (10(-10)-10(-5) M), and voltage-response curves (12-28 V) were performed for electrical field stimulation. There was no significant difference in either the smooth muscle maximal contractile response, or sensitivity between the bronchi exposed to NO2 and those exposed to air. We conclude that in vitro exposure to 2.5 ppm of NO2 does not alter airway smooth muscle responsiveness in guinea pigs. PMID- 7543689 TI - [Open thoracic surgery of giant bullous pulmonary emphysema in adults]. AB - Ninety-two adult patients underwent open surgery for unilateral (60) or bilateral (32) emphysematous bullae filling at least one third of an hemithorax: 56 were apical, 34 were basal and 2 were apical on one side and basal on the other side. A first group of 18 patients were admitted in critical care medicine, after an acute complication, in a state of respiratory distress which was not controlled by oxygen therapy and/or drainage, 17 of them being mechanically ventilated at time of operation. In this group 9 patients died, 8 of them with bilateral bullae and 5 with cor pulmonale. A second group of 74 patients underwent surgery after a delay during which symptoms appeared or worsened, complications were observed or a radiological opacity was discovered. In this group 5 patients died. During a mean 4.8 year follow-up, 55 patients among the 71 survivors with no associated cancer were in a better functional state and remained clinically stable. The patients history and the results of surgery suggest that: 1) giant bullae should be resected when they increase in volume, when they are bilateral and when symptoms progress; 2) criteria for surgery are not limited to patients having compression of lower lobes from apical bullae; 3) it is necessary to operate on both sides for bilateral disease in elective surgery; 4) when there is a state of respiratory distress after a complication of bilateral bullae, the mortality is prohibitive; 5) cor pulmonale is of poor prognosis; 6) diffuse lung disease and associated diseases add to mortality and morbidity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543690 TI - Molecular implications of the antiandrogen withdrawal syndrome. PMID- 7543693 TI - [Lipodystrophy of the mesentery or mesenteric panniculitis]. PMID- 7543692 TI - [Mesenteric lipodystrophy]. AB - This case report describes the clinic and pathologic findings of an inflammatory pseudotumor of the mesentery in a 43 years old male. Previous to the hospital admission he had several episodes of intestinal occlusion and an abdominal mass. Computed tomography and mesenteric angiography were very helpful. Laboratory tests were unspecific, and the final diagnosis was obtained after surgery and histologic study of the lesion. We review published literature on inflammatory pseudotumors. PMID- 7543691 TI - [Massive paracentesis and administration of dextran 70 vs albumin in cirrhotic patients with tense ascites]. AB - Some studies have demonstrated that paracentesis for large-volume extraction of ascites produces renal failure and hyponatremia, and intravenous infusion of plasma expanders can overcome this complications. We performed a survey where we compared effectiveness of dextran 70 vs albumin on prevention of adverse effects and cost differences. Two random groups were formed, 8 cirrhotic patients with tense ascites in each group. Paracentesis with extraction of more than 5 liters was performed. The group A received human albumin and group B dextran 70, both received 6 g per liter of extracted liquid. 24 hours before and 48 hours after of ascites extraction, we performed hepatic function test, blood chemistry with renin and aldosterone. Clinical results and biochemistry test were similar in both groups without statistical significance (p > 0.05). Amount of plasma expander was almost the same, but the cost in group A was $266 USD and in group B $20.8 USD. Azotemia was present in 12.5% in group A and hyponatremia in 12.5% in both groups, without symptoms. The results show that dextran 70 produces the same effect like albumin in the treatment of ascites after large-volume paracentesis with lower cost. PMID- 7543696 TI - Estrogen: key player in heart disease among women. PMID- 7543694 TI - [The endoscopic treatment of carcinoma of Vater's ampulla]. AB - The ampullary carcinoma is a rare tumor. Its early and accurate diagnosis will lead to early treatment and subsequent better prognosis. Endoscopic retrograde cholangiopancreatography (ERCP) has been shown to be one of the best diagnostic tools. Furthermore an endoscopic biliary drainage procedure: endoscopic sphincterotomy or endoprosthesis placement, can be performed immediately following the diagnostic procedure. PATIENTS AND METHODS: Endoscopic drainage was attempted in 20 patients with a success rate of 90 percent. There were 8 females and 12 males, with a mean age of 71 years (range 43-92), and were admitted to the hospital with obstructive jaundice. Endoscopic insertion of a biliary endoprosthesis (9 cms long 3.2 mm [10 Fr] diameter), was successful in 18 patients whom made uneventful recovery and their jaundice resolved completely. RESULTS: In 18 of them an endoscopic sphincterotomy was carried out. Two patients developed cholangitis, the endoprosthesis were removed and a new one inserted. The survival rate of these patients was 3 and 18 months respectively. Failure of endoscopic sphincterotomy or endoprosthesis insertion in 2 remaining patients, was ascribed to an inability to cannulate the papilla due to infiltrating tumor. The median survival time in 15 remaining patients was 4 months (1-18 months) and died by metastatic disease. Five patients underwent Whipple's procedure, and all survived the operation. Two died, by metastatic disease, with a median survival time of 16 months and 3 still alive at 46, 25 and 18 months post-operatively. Carcinoma of the ampulla of Vater is not resectable in 25-50% of the patients because of metastatic disease, deep extension of the tumor or general contraindications for major surgery. CONCLUSIONS: Endoscopic sphincterotomy or endoprosthesis insertion as a definitive treatment modality should be reserved for poor surgical candidates and those patients with limited life expectancy due to metastatic disease. PMID- 7543695 TI - [The surgical possibilities in patients with adenocarcinoma of the pancreas]. AB - OBJECTIVE: To analyze the types of surgical procedures which can be performed in patients with pancreatic adenocarcinoma and its results. SUMMARY BACKGROUND DATA: Until the 80's the incidence of resection for pancreatic adenocarcinoma was low with a high morbidity and mortality rates, and the 5-year survival rate below 8 per cent. During the last decade many reports in the international literature are showing very low morbidity and mortality rates and much better long-term survival rates (35-40 per cent). The palliative procedures had not change. METHODS: The retrospective analysis of charts of patients with any surgical procedures and diagnosis of pancreatic adenocarcinoma, between 1962 and 1991, was performed, such analysis included demographic data, surgical procedures, operative morbidity and mortality, and long-term results. RESULTS: There were 410 patients, 52 per cent were males and 48 per cent were females. Tumor resection was possible in only 20 per cent of the patients (pancreatoduodenectomy 69 per cent, distal pancreatectomy 17 per cent and total pancreatectomy 14 per cent). The remaining 80 per cent were candidates for palliative procedures (73 per cent) and diagnostic procedures (27 per cent). The resected group showed a high operative morbidity and mortality rates in particular total pancreatectomy, and the 5-year survival rate was 8 per cent. This figure was 4 per cent with the Whipple's procedure. When palliative or diagnostic procedures were performed, 97 per cent were dead during the first 12 months, and there were no survivors after 20 months. CONCLUSIONS: Our results are equal to those reported in the world literature until 1985, after that date better results are increasingly reported, but we shall wait at least the next decade in order to know if there are some changes in our results. There has been no changes with palliative procedures. PMID- 7543697 TI - Regulation of ion channels by ABC transporters that secrete ATP. PMID- 7543698 TI - CFTR as a cAMP-dependent regulator of sodium channels. AB - Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP dependent regulator likely accounts for abnormal sodium transport. PMID- 7543699 TI - High-dose paclitaxel with granulocyte colony-stimulating factor in patients with advanced breast cancer refractory to anthracycline therapy: a European Cancer Center trial. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) is a novel cytostatic agent that has shown interesting antitumor activity in patients with advanced breast cancer. Depending on variable patient characteristics and amount and type of prior therapy, as well as the applied dose and schedule of paclitaxel, response rates have varied from 13% to 62%. However, optimal dose and schedule are still unknown. We studied a high-dose (250 to 300 mg/m2) 3-hour paclitaxel infusion schedule in a poor prognostic group of breast cancer patients who progressed or relapsed while taking anthracyclines. This regimen was given every 3 weeks. Twenty-one of the 36 patients studied had increased liver enzymes and 18 had documented liver metastases. The objective response rate was only 6%, but response rate by disease site indicated that soft tissue lesions responded in 30% of cases. For a better comparison with other reported data a uniform definition of "anthracycline refractory" is needed. Neuropathy, which was found to be dose limiting, and arthralgia/myalgia syndrome were the most frequently occurring toxicities. Both severe myelosuppression (and infections) and severe diarrhea and mucositis were reported more frequently in patients with liver dysfunction. As higher peak levels, increased areas under the concentration time curves, and longer times during which plasma concentrations were above the threshold level of 0.1 mumol/L were found in patients with elevated liver enzymes, a correlation with the observed toxicities is assumed. Further pharmacodynamic studies in such patients receiving a 3-hour infusion seem warranted. PMID- 7543700 TI - Treatment of metastatic breast cancer with combination paclitaxel/cyclophosphamide. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) has demonstrated substantial single-agent activity against both minimally pretreated and resistant metastatic breast cancer. Evaluation of paclitaxel-based combinations has demonstrated appreciable activity, but also toxicity, for the paclitaxel/doxorubicin combination. The paclitaxel/cyclophosphamide combination is potentially attractive because of the significant single-agent activity of both drugs against metastatic breast cancer and the paucity of shared nonhematologic toxicities. This combination is being evaluated in women with doxorubicin-refractory metastatic breast cancer. Dose escalation to paclitaxel 200 mg/m2 over 24 hours and cyclophosphamide 2,000 mg/m2 administered every 21 days with granulocyte colony-stimulating factor support has been performed. The final dose level was complicated by dose-limiting toxicity. The maximum tolerated dose for this combination is currently being defined. There is preliminary evidence of sequence-dependent toxicity. Grade 4 neutropenia and neutropenic fevers are more frequent in cycles in which paclitaxel is administered first. PMID- 7543702 TI - Update: the M.D. Anderson Cancer Center experience with paclitaxel in the management of breast carcinoma. AB - The first of three trials at M.D. Anderson Cancer Center investigating paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in metastatic breast cancer was a phase II study involving 25 patients (297 courses) previously treated with only one chemotherapy regimen; the patients received paclitaxel 250 mg/m2 infused over 24 hours without granulocyte colony-stimulating factor (G-CSF). Complete (12%) and partial responses (44%) led to median durations of response and survival of 9 and 21 months, respectively. The median paclitaxel dose was 200 mg/m2. Despite profound neutropenia (median granulocyte count of 0.3 cells x 10(9)/L for the first three courses), infection occurred in 42% of patients but only 6% of courses. In a phase I trial of paclitaxel 125 mg/m2 over 24 hours followed by doxorubicin 60 mg/m2 using G-CSF at 5 micrograms/kg days 5 through 19, dose-limiting mucositis with neutropenic fever occurred at the starting dose, so the maximum tolerated dose was one dose lower: paclitaxel 125 mg/m2 (over 24 hours) followed by doxorubicin 48 mg/m2 over 48 hours. Among 10 patients, there was one complete response and seven partial responses (overall response, 80%). Suspecting a schedule-dependent interaction between drugs, a phase I trial of the reverse sequence yielded a maximum tolerated dose, defined by neutropenic fever without mucositis, of doxorubicin 60 mg/m2 (over 48 hours) followed by paclitaxel 150 mg/m2 (over 24 hours) in 21 patients. A pharmacokinetic study in which the sequence of administration of paclitaxel over 24 hours and doxorubicin over 48 hours was alternated in courses 1 and 2 indicated that when paclitaxel by 24-hour infusion is given first, doxorubicin plasma levels at the end of infusion were an average 70% higher and doxorubicin clearance was reduced approximately 30% compared with the reverse sequence. Similarly, the incidence of grade 2 or 3 mucositis was 70% with the paclitaxel/doxorubicin sequence versus only 10% with the reverse sequence. We concluded that paclitaxel slows doxorubicin metabolism and that when used together in this schedule, doxorubicin should precede paclitaxel. In the third trial paclitaxel without G-CSF was administered to two groups of heavily pretreated patients: (1) those with only two prior chemotherapy regimens (inclusive of adjuvant therapy) received paclitaxel 175 mg/m2 over 24 hours and (2) those with three or more prior regimens received paclitaxel 150 mg/m2 over 24 hours. Response rates in both regimens were approximately 20%. We conclude that paclitaxel has significant antitumor activity in metastatic breast cancer, especially in patients with limited prior therapy, without need for G CSF.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543701 TI - Memorial Sloan-Kettering Cancer Center experience with paclitaxel in the treatment of breast cancer: from advanced disease to adjuvant therapy. AB - The novel chemotherapeutic agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) has received more attention than perhaps any other drug in the past two decades in its development for the treatment of breast cancer. Its unique mechanism of action, demonstrated safety profile, and significant antitumor activity in thousands of patients treated in clinical trials thus far have led to recent US Food and Drug Administration approval of its use against chemotherapy-refractory metastatic breast cancer. This report will summarize the series of clinical investigations with paclitaxel in patients with metastatic breast cancer that have been completed over the past 3 years at Memorial Sloan Kettering Cancer Center and address present and future avenues of research. PMID- 7543706 TI - CD14-lipopolysaccharide receptor activity in hepatic macrophages after cholestatic liver injury. AB - BACKGROUND: Increased septic complications and altered cytokine responses to bacterial endotoxins are lethal consequences of chronic liver disease. Kupffer cells (HM phi) control the clearance of lipopolysaccharide (LPS) and subsequent cytokine responses. The purpose of this study is to determine the phenotype and function of HM phi after cholestatic liver injury. METHODS: Male Sprague-Dawley rats underwent 4 days of common bile duct division and ligation or sham laparotomy. HM phi were isolated by collagenase-pronase perfusion and purified by centrifugal elutriation. RESULTS: Indirect immunofluorescence and Western blot analysis with monoclonal antibodies OX42 and ED9 showed that HM phi from cholestatic rats expressed MAC-1 (CD11b) and the LPS receptor CD14, respectively. HM phi from sham animals were negative for CD14 and CD11b by use of immunofluorescence; however, Western blot detected low levels of CD14 in controls. Functional analysis for tumor necrosis factor-alpha release after LPS stimulation showed that HM phi isolated from cholestatic livers exhibited serum (normal rat serum) dependent stimulation (1 ng/ml LPS + 1% normal rat serum; n = 12; p < 0.05 at 8 hours, t test) consistent with the presence of CD14 on their cell surface. CONCLUSIONS: This study shows that HM phi isolated after common bile duct division and ligation are phenotypically and functionally different from normal HM phi. The appearance of serum-dependent LPS responses within the injured liver may play an important role in the immunologic alterations associated with hepatic disease. PMID- 7543703 TI - Intestinal reperfusion up-regulates inducible nitric oxide synthase activity within the lung. AB - BACKGROUND: This study examines the hypothesis that pulmonary inducible nitric oxide synthase (iNOS) activity is up-regulated during intestinal reperfusion and that inhibition of NO generation exacerbates pulmonary microvascular dysfunction. METHODS: Sprague-Dawley rats underwent intestinal ischemia and reperfusion (IIR) or sham operation (SHAM). Pulmonary iNOS activity was measured by quantitating the conversion of L-arginine (L-Arg) to L-citrulline. Another set of animals undergoing IIR or SHAM received an inhibitor of NOS (NG-nitro-L-arginine methylester; L-NAME; 20 mg/kg intravenously), substrate for NO generation (L-Arg; 300 mg/kg intravenously), or vehicle (normal saline solution; 3 ml). Pulmonary microvascular dysfunction was then quantitated by measuring the extravasation of Evans blue dye (EBD) into the lung. RESULTS: Inducible NOS activity was six times greater in the lungs of animals sustaining IIR when compared with SHAM (p = 0.0005). The concentration of EBD within the lungs of animals sustaining IIR was 30% greater than SHAM (p < 0.05). Inhibiting NOS with L-NAME significantly increased pulmonary EBD concentration of both IIR and SHAM groups when compared with normal saline solution-treated animals (p < 0.0001). Treatment with L-Arg prevented this IIR-induced increase in pulmonary dye extravasation. CONCLUSIONS: These data suggest that pulmonary iNOS activity is up-regulated in animals sustaining IIR and that this may serve as a compensatory protective response to remote organ injury. PMID- 7543705 TI - Prolongation of islet allograft survival with an antibody to vascular cell adhesion molecule 1. AB - BACKGROUND: The purpose of this study was to determine whether an antibody to vascular cell adhesion molecule 1 (VCAM1) prolongs the survival of neovascularized pancreatic islet allografts. METHODS: We treated CBA (H-2k) recipients of BALB/c (H-2d) islet allografts with anti-VCAM1 antibody (400 micrograms/day for 20 days). Sensitized recipients of islet grafts also were treated with anti-VCAM1. To study mechanism we performed mixed lymphocyte reactions (MLRs) with anti-VCAM1 and studied the graft infiltrate in treated recipients. RESULTS: Anti-VCAM1-treated CBA recipients showed prolonged graft survival with indefinite survival in five of nine cases. Anti-VCAM1 prevented proliferation in an MLR but not when added 36 hours after the beginning of the MLR. Anti-VCAM1 did not prolong allograft survival in sensitized recipients and did not prevent lymphocytic infiltration of the graft at 7 days. CONCLUSIONS: Anti-VCAM1 prolongs allograft survival in neovascularized islets in which the donor vascular endothelium plays little or no role in immunogenicity. VCAM1 appears to be important in the afferent phase (lymphocyte activation) of the allograft response. Once activated, either late in an MLR or in sensitized recipients, lymphocytes are not dependent on VCAM1 for function. Finally, anti VCAM1 does not appear to affect the homing of lymphocytes to the allograft. PMID- 7543704 TI - Cytochrome P450IIIA activity and cytokine-mediated synthesis of nitric oxide. AB - BACKGROUND: Although nitric oxide synthase (NOS) is a cytochrome P450-like hemoprotein with additional sequence homology to cytochrome P450 reductase, the role of the cytochrome P450 system in cytokine-mediated NO synthesis is unknown. METHODS: To characterize the role of the P450 system in the synthesis of NO, NO production, NOS enzyme activity, and steady state NOS mRNA and protein expression were characterized in the setting of P450 isoform activity inhibition by using a model of isolated rat hepatocytes in primary culture. Cimetidine (0 to 10 mmol/L) was chosen as a specific inhibitor of P450IIIA activity. NO production was induced by interleukin-1 (50 ng/ml) and tumor necrosis factor (500 units/ml) and quantified by measurement of its metabolite, nitrite, in the culture medium. Steady state NOS mRNA and protein expression were determined by reverse transcriptase polymerase chain reaction and immunoblot analysis, respectively. NOS enzyme activity was measured by the conversion of tritiated-L-arginine to tritiated-L-citrulline. RESULTS: Inhibition of P450IIIA activity was associated with a concentration-dependent decrease in cytokine-mediated NO production. Levels of NOS mRNA and protein were not altered. The NOS enzyme assay was notable for stable concentrations of intermediate, N-OH-L-arginine, and decreased production of the final end product, L-citrulline. Dixon plot kinetic analysis of cimetidine-mediated inhibition of NOS yielded an inhibition constant Ki = 1.76 mmol/L. CONCLUSIONS: These results indicate that cytochrome P450IIIA isoform may play a posttranslational role in cytokine-mediated NO synthesis in this model of isolated rat hepatocytes in primary culture. PMID- 7543711 TI - [Use of a 7.5% NaCl/6% Dextran 70 solution in the prevention of hemodynamic disorders during surgery of abdominal aortic aneurysms]. AB - The experience in the application of infusion solutions Ringer lactate (RL) and 6% of Dextran 70 is shown in order to replenish intravascular volume in 19 patients with the abdominal aorta aneurysm after the removal of the aortal clamp and hypertonic-hyper oncotic 7.5% NaCl/6% Dextran 70 solution in 4 to 5 ml/kg dose in 9 patients. The application 7.5% NaCl/6%Dextran 70 at the optimum time, 15-20 min before the removal of aortal clamp, in a dose of 4-5 ml/kg allowed the support of normal hemodynamic function and decreased the need for some solutions or blood, as well as the incidence of intra- and postoperative complications. PMID- 7543708 TI - Detection of an antigenic marker expressed by peripheral blood monocytes and platelets by a new monoclonal antibody, UN8. PMID- 7543709 TI - Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis. AB - A haemolytic toxin was purified by ion-exchange chromatography and FPLC gel filtration from the nematocysts of the jellyfish Carybdea marsupialis. Sheep red cells, but not human or rabbit red cells, were susceptible to lysis by the toxin. The toxin is a protein with an apparent molecular mass of about 102-107 kDa, is heat labile, highly unstable in polar media, inactivated by reducing agents, and devoid of phospholipase activity. The experimental data speak in favour of a pore forming mechanism of toxin action. PMID- 7543710 TI - RNA fingerprinting and differential display using arbitrarily primed PCR. AB - RNA fingerprinting by arbitrarily primed PCR can be used to detect and clone transcripts that are differentially expressed between cells that have been subject to different environments or developmental programs. The method also allows an estimate of the number of genes that are differentially expressed under various circumstances. When many experimental treatments are compared in parallel, intersecting regulatory pathways are reflected in genes that are perturbed by more than one treatment. PMID- 7543712 TI - [Biochemistry, molecular mechanism of action and biological effects of endotoxin]. AB - This review is a brief attempt at providing an overview of a subject of enormous complexity-endotoxins and the mediators associated with its biological effects. More specifically it deals with biochemistry and biology of endotoxin, detection of endotoxin with the Limulus amebocyte lysate test, the molecular mechanisms and biological effects, and in the last part with future aspects of therapeutical strategies. It seems certain that the subject will become even more complex and possibly controversial as scientific knowledge further involves. However, because of the high mortality rate of patients suffering from gram-negative sepsis all efforts have to be made to find effective therapeutical strategies. PMID- 7543707 TI - [A world without poliomyelitis?]. PMID- 7543713 TI - Effect of low-dose subcutaneous heparin on postoperative wound hematomas: randomized clinical trial on hospitalized inguinal hernia patients in Germany. AB - A randomized prospective study of low-dose heparin was performed in 89 surgical patients during and after Bassini-Lotheissen repair. The patients were divided into two groups: (1) Low-dose heparin: 5000 units 2 hours before operation and 5000 units every 8 hours for 5 days after the operation. (2) Dextran 40 (Rheomacrodex); administered during the operation, with an additional infusion of dextran (500 ml/day) for the next 3 days. The frequency of wound complications in 45 the heparin-treated patients was 22.0% (comprising 10 hematomas), compared to only 4.5% in the dextran-treated patients (n = 44 patients). These findings are significant (p < 0.05). Low-dose heparin should therefore be administered routinely only to patients with increased risk of thromboembolism. PMID- 7543714 TI - Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide. AB - Vaccine development against Group B Neisseria meningitidis is complicated by the nature of the capsular polysaccharide, which is alpha 2-8-linked poly-sialic acid, identical in structure to the poly-sialic acid found in many mammalian tissues during development. To test the feasibility of a vaccine based on this polysaccharide, we synthesized several conjugates of meningococcal B polysaccharide linked to a carrier protein (tetanus toxoid or diphtheria CRM197), via an adipic acid dihydrazide (ADH) spacer. All conjugates induced a strong immune response. However, most of the antibodies were not directed against the Meningococcus B polysaccharide and could not be inhibited by the purified polysaccharide alone. Further investigations showed that the antibodies recognized an epitope composed by the junction between the spacer and the polysaccharide and protein, that is not present in the native polysaccharide and is generated during the coupling reaction. This epitope becomes immunodominant with respect to the poorly immunogenic polysaccharide. While the majority of the immune response is directed against the above epitope, the conjugates induced also an immune response against the Meningococcus B polysaccharide. The anti Meningococcus B antibodies elicited are of the IgM and IgG class and are inhibitable by the polysaccharide. Moreover, they are bactericidal, thus suggesting that they would induce protection against disease. PMID- 7543716 TI - Isolation and characterization of a highly attenuated respiratory syncytial virus (RSV) vaccine candidate by mutagenesis of the incompletely attenuated RSV A2 ts-1 NG-1 mutant virus. AB - Ts-1, a temperature sensitive (ts) mutant of RSV, was previously derived from RSV A2 virus by mutagenesis with 5-fluorouracil (5-FU). Ts-1 was attenuated for adult volunteers and seropositive children but retained a low level of virulence in seronegative infant vaccinees as indicated by the occurrence of upper respiratory tract disease. Ts-1 NG-1, a more defective derivative of ts-1, was produced by mutagenesis of ts-1 with nitrosoguanidine. However, ts-1 NG-1 still retained a low level of virulence for the upper respiratory tract and showed some genetic instability in chimpanzees. With renewed interest in the goal of developing a live, attenuated RSV vaccine, we have now attempted to further attenuate ts-1 NG 1 by mutagenesis with 5-FU and 5-azacytidine. Four mutants that are phenotypically different from the ts-1 NG-1 parental virus were identified. Each of the four mutants was more restricted in replication in BALB/c mice compared with the ts-1 NG-1 parental virus. One of the ts-1 NG-1 derivatives, termed A-20 4, which showed the lowest (35 degrees C) in vitro shutoff temperature and which was also completely restricted in replication in BALB/c mice, was selected for further evaluation in seronegative chimpanzees. A-20-4 did not cause rhinorrhea in chimpanzees but induced detectable titers of serum RSV neutralizing antibodies in 2 of 4 chimpanzees. Apparent complete protection to subsequent challenge with wild-type RSV was observed in each of the four chimpanzees previously immunized with A-20-4.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543717 TI - [Ischemic type lesions of the bile ducts after liver transplantation: 2 years results]. AB - Ischemic type lesions (ITL) after orthotopic liver transplantation are characterized by bile duct necroses leading to alterations of the ductal lumen, biliary leakage, cast formation and, thereby, to cholestasis. After exclusion of causative factors, such as arterial thrombosis, ABO incompatibility and chronic rejection, ITL occurred in 21 of 165 patients. The rate of ITL after UW preservation was higher (25%) in grafts preserved for > 10 hours in comparison to < 10 hours (7%; p < 0.05). Treatment consisted of endoscopic and percutaneous intervention, surgical revision and retransplantation. Retransplantation is indicated in many patients with lesions of extra- and intrahepatic bile ducts (type A). Other methods are of palliative character and may only be successful in type B (extrahepatic) lesions. Morbidity in patients with ITL is considerable. In comparison to patients without ITL, mortality, however, is not increased. PMID- 7543715 TI - Recombinant BCG strains expressing the SIVmac251nef gene induce proliferative and CTL responses against nef synthetic peptides in mice. AB - CTL responses are known to be important for the control of HIV and SIV infections. Such responses are targeted against various components of these viruses including regulatory proteins like Nef. The SIVmac251nef gene was cloned in Mycobacterium bovis BCG under the control of P(AN), a promoter from Mycobacterium paratuberculosis. Nef was expressed as a fused polypeptide with ORF2, an open reading frame adjacent to P(AN). Mice inoculated with rBCG(SIVmac251nef) exhibited proliferative and CD8+ cytotoxic T-cell (CTL) responses against several Nef synthetic peptides. A mapping of the epitopes recognized by CTLs revealed that the central region of Nef is mainly involved in responses. This region had already been demonstrated to induce CTLs in experimentally SIV-infected macaques as well as in HIV-infected individuals. These results demonstrate the feasibility of constructing BCG vaccine strains expressing nef for eliciting cytotoxic responses. PMID- 7543718 TI - Early trilineage recovery by granulocyte colony-stimulating factor in a patient with aplastic anemia. PMID- 7543719 TI - The pharmacological manipulation of fetal haemoglobin: trials using hydroxyurea and recombinant human erythropoietin. AB - Hydroxyurea (HU) and recombinant human erythropoietin (rHuEpo) have been used in several studies to elevate Hb F level in sickle cell disease (SCD) patients and hence to ameliorate the clinical presentations of the disease. The treatment protocol and doses have varied in the different studies. We studied the effects of HU+rHuEpo combination therapy in sickle cell anaemia (SCA patients) to investigate the Hb F manipulation and hence treatment of SCA. Six patients with severe SCA were selected for treatment with HU (20-25 mg/kg body weight) and rHuEpo (400-800 U/kg body weight) combination therapy for 4 weeks followed by HU (20-25 mg/kg body weight) maintenance therapy for 6 months to 1 year. Iron and folic acid were administered during HU+rHuEpo treatment. Signs, symptoms and complications were recorded to obtain the severity index. Only patients with a severity index > or = 6 were included in the study. Haematological and biochemical parameter values, Hb A2, Hb F, Hb F distribution, Hb F cells, bilirubin level and reticulocyte count were assessed at least on 2-3 occasions prior to initiation of the therapy protocol to establish baseline values. During the treatment period, the clinical presentations were monitored and the estimation of the laboratory parameters was carried out every 4-8 weeks. The results of these parameters during HU and rHuEpo combination therapy and HU maintenance therapy were compared with baseline values using paired t test. The elevation in the level of Hb F, Hb F cells, total haemoglobin, red cell count and MCV were significant (p < 0.005), while reticulocyte count and total bilirubin were significantly decreased (p < 0.05). Each patient showed an individual pattern of Hb F elevation. The increase in Hb F level was correlated with the haematological and biochemical parameters using the General Linear Model Programme of Statistical Analysis System. In general, the clinical presentation improved as Hb F level increased in each patient. In addition, the positive correlation with the haematological parameters and negative correlation with reticulocytes and total bilirubin confirmed the beneficial effect of elevated Hb F level on reducing red cell haemolysis. No correlation could be demonstrated between the pretreatment Hb F level and the increase in Hb F during the treatment period. Daily doses of HU with a single intravenous rHuEpo and iron supplementation elevate Hb F and Hb F cells in SCA patients. The Hb F level can be maintained high on HU therapy alone.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543720 TI - Methylprednisolone in advanced chronic lymphocytic leukaemia: rationale for, and effectiveness of treatment suggested by DiSC assay. AB - The effect of methylprednisolone on fresh cells from patients with chronic lymphocytic leukaemia (CLL) has been studied using the differential staining cytotoxicity (DiSC) assay resulting in LC90s of < or = 0.2 to 2,000 micrograms/ml. Cells from previously treated patients were, on average, significantly more sensitive to methylprednisolone than those from untreated patients (mean LC90 = 5.7 micrograms/ml, n = 61 vs 31.0 micrograms/ml, n = 17, respectively; p < 0.05). Twelve patients with advanced disease were given high dose methylprednisolone (1 g/m2/day i.v. x 5 days). In 7 cases, > or = 3 courses were given; 3 patients did not respond (2 achieved palliation) and 4 (57%) achieved a good partial response. These latter 4 patients were all clinically resistant to chlorambucil and anthracyclines and 2 were resistant to fludarabine. In 5 cases, 1 or 2 courses were given but no patients responded. The 8 nonresponders survived a median of 3.5 months whilst the responders have survived a median of 28.5+ months (3 of 4 still alive). This work suggests a rationale for why CLL patients resistant to standard chemotherapy may benefit from high-dose methylprednisolone therapy. Due to cost and toxicity associated with therapy, the decision to treat would be best made on the basis of a DiSC assay result. This pilot study requires confirmation with a well-designed controlled clinical trial. PMID- 7543722 TI - Decay-accelerating-factor-deficient erythrocytes during the long-term clinical course of patients with paroxysmal nocturnal hemoglobinuria. AB - We performed long-term follow-up studies of 5 patients with paroxysmal nocturnal hemoglobinuria (PNH). The percentages of decay-accelerating-factor (DAF) deficient erythrocytes were almost stable or slowly increased during the 2-4 years' observation periods when the effects of treatment with drugs or blood transfusion and pregnancy were excluded. However, a tendency of increase in the percentages of DAF-deficient erythrocytes was observed along with the duration of the disease. Our findings suggest that the growth advantage of PNH clones beyond the normal hematopoiesis may be different from the other clonal stem cell disorders. PMID- 7543721 TI - CD34+ human hemopoietic progenitor cells of the bone marrow differ from those of the peripheral blood: an immunocytochemical and morphometric study. AB - CD34+ progenitor cells were harvested from bone marrow and peripheral blood from 10 healthy donors by immunomagnetic isolation and enrichment procedures. The CD34+ cell population was investigated using a battery of enzyme reactions and monoclonal antibodies on cytospin preparations. Additionally, morphometric measurements were carried out and also liquid suspension culture studies were performed to ascertain vitality and stem cell character. More than 95% of the total yield of medullary CD34 progenitors expressed CD45 (LCA), CD43 (MT1) and beta-glucuronidase. Reactivity with CD33 (My9), CD15 (LeuM1), CD38 (Leu17), CD20 (L26) and Ret40f (glycophorin C) was assumed to be in keeping with a transition into more differentiated elements of the various hemopoietic lineages. Morphometric analysis revealed conspicuous heterogeneity of the CD34+ cell population considering size measurements. This finding was in line with the diversities of antigen expression, indicating the more committed nature of CD34+ stem cells derived from the bone marrow in comparison with those progenitors isolated from the peripheral blood. Moreover, proliferation marker staining by PCNA disclosed a positivity in a considerable number of progenitors in contrast to the findings in CD34+ cells that are found in the peripheral blood. PMID- 7543723 TI - [Single channel analysis of aconitine blockade of calcium channels in rat myocardiocytes]. AB - Ventricular myocardiocytes from neonatal Wistar rats were isolated and cultured. Aconitine, Ca2+ channel blocker verapamil or Ca2+ channel activator BAY K8644 were added to the bath solution separately. Using the cell-attached configuration of the patch clamp technique, the single channel activities of L type Ca2+ channel were recorded before and after addition of all three drugs. The results showed the blocking effect of aconitine (50 micrograms.ml-1) on L type Ca2+ channels. Its mechanism may be relevant to the decrease in both open state probability and the mean open time of Ca2+ channel. The difference was statistically significant compared with control group (P < 0.01). The amplitude of Ba2+ currents, which flow through open L type Ca2+ channel was unchanged. PMID- 7543724 TI - [Influence of calcium concentration on 4-aminopyridine induced histamine release from isolated peritoneal mast cells]. AB - 4-Aminopyridine (4-AP) has been shown to induce histamine release from isolated peritoneal mast cells (PMC) in mice and rats. In the presence of extracellular calcium at normal level (0.9 mmol.L-1) histamine release induced by 4-AP (13.6 mmol.L-1) from mice PMC was 33.0% +/- 4.6%, while at low calcium concentration (0.5 mmol.L-1) and in calcium-free medium this parameter decreased to 25.5% +/- 4.2% and 16.3% +/- 3.7% respectively. Histamine release in response to 4-AP (10 mmol.L-1) from rat PMC was 39.1% +/- 6.7% (0.9 mmol.L-1 calcium), while at low calcium concentration (0.5 mmol.L-1) and in calcium-free medium this parameter decreased to 29.3% +/- 4.7% and 20.2% +/- 2.9% respectively. Results of statistical analysis indicate that 4-AP induced histamine release is related to Ca2+ concentration. When rat PMC were preincubated in calcium-free medium with EDTA 0.1 mmol.L-1 for 180 min 4-AP induced histamine release was 13.8% +/- 1.6%. This shows that 4-AP also elicited mobilization of endogenous calcium stores in mast cells. The mechanism of 4-AP induced histamine release was discussed. PMID- 7543726 TI - Reversibility of white matter changes and dementia after treatment of dural fistulas. AB - We describe two patients with dural fistulas who presented with dementia and diffuse white matter signal changes on MR that significantly improved after surgery. One patient had preoperative embolization. PMID- 7543725 TI - Rapamycin: a bone sparing immunosuppressant? AB - Immunosuppressant therpay is associated with osteoporosis both clinically, post transplantation, and experimentally. In rats, cyclosporin A (CsA) and FK506 induce a state of high turnover rapid bone loss. After 14 days of administration in immunosuppressive doses, the more recently discovered immunosuppressant, rapamycin, resulted in no change of cancellous bone volume. A longer study over 28 days has now been carried out; contrasting the new drug with CsA and FK506. Sixty, 10-week-old Sprague-Dawley rats were randomly divided into five groups of 12 rats each. The first group served as an aging control. The remaining four groups received, by daily gavage, a combined vehicle placebo, CsA 15 mg/kg, FK506 5 mg/kg, and rapamycin 2.5 mg/kg, respectively. CsA- and FK506-treated rats, but not those treated with rapamycin, demonstrated high turnover osteoporosis with raised serum 1,25(OH)2D (p < 0.05) and elevated serum osteocalcin (p < 0.05). The trabecular bone area was decreased by 66% (p < 0.01) in the CsA group and 56% (p < 0.05) in the FK506-treated group compared with the control animals. The CsA- and the rapamycin-treated groups failed to gain weight and developed severe hyperglycemia (> 20 mmol/l, p < 0.001) by day 14 but which largely resolved by day 28. Unlike the groups treated with CsA and FK506, rapamycin-treated rats had no loss of trabecular bone volume but there was increased modeling and remodeling and a decreased longitudinal growth rate. Rapamycin may thus confer a distinct advantage over the established immunosuppressants in not reducing bone volume in the short term.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543727 TI - Stenosis severity and the occurrence of ventricular ectopic activity during acute coronary occlusion during balloon angioplasty. AB - To elucidate the incidence and determinants of early ventricular arrhythmias (VA) during acute coronary occlusion, continuous electrocardiographic, heart rate, and blood pressure recordings were performed in 152 patients during standardized balloon occlusions of significant (50% to 95%) coronary artery stenoses. A control group of 13 patients with chronic total occlusion of a coronary artery was also studied. None of them developed VA during balloon inflation in the preexisting total occlusion of the artery. Balloon occlusion of a coronary artery was associated with occurrence of ventricular ectopy in 18 patients (VA group, 12%). The VA group had milder stenosis severity (72% vs 81%, p < 0.001) than the rest of the patients, and none of them had visible collaterals to the occluded vessel. The VA group also had ST-segment deviations more often (p < 0.05) during occlusion than patients with no VA. Occlusion of the left anterior descending artery caused VA more often (p < 0.05) than occlusion of the left circumflex or right coronary artery. No clinical or hemodynamic variable or medication was associated with the occurrence of VA. In stepwise logistic regression analysis, the only significant predictors of ventricular ectopic activity were the stenosis severity and the anterior site of coronary occlusion. Even a nonstenotic plaque can be so fragile that it is prone to rupture. The present findings suggest that such an occlusion may result in electrical instability more easily than occlusion of a more advanced coronary lesion. PMID- 7543729 TI - Use of prostate specific antigen assays. PMID- 7543728 TI - CD4 predicts nonlymphocytic lineage in acute leukemia. Insights from analysis of 125 cases using two-color flow cytometry. AB - The classification of acute leukemia into lymphoid or nonlymphoid is of critical therapeutic importance. Two-color flow cytometric analysis has emerged as a valuable addition to morphology and cytochemistry for the distinction of acute lymphocytic leukemia (ALL) and acute nonlymphocytic leukemia (ANLL). By careful selection of monoclonal antibody (mAb) combinations, diagnostic accuracy, and cost effectiveness may be enhanced compared to flow cytometry using one-color analysis. The sensitivity and specificity of a mAb panel were assessed in the determination of nonlymphocytic lineage in acute leukemia. One hundred twenty five consecutive cases of acute leukemia were analyzed in which Wright's-stained smears, cytochemical stains, and immunophenotyping studies had been performed. The antibody panel included the nonlymphoid markers CD13, CD33, CD14, and CD4 in combination with CD2, as well as a broad panel of lymphoid and nonlineage specific markers. Of the 125 cases of acute leukemia studied, 85 cases (68%) were nonlymphocytic and 32 cases (26%) were lymphocytic (28 cases B cell, 4 cases T cell). CD13 and CD33 were very sensitive in the detection of ANLL, being expressed on 94% and 93% of ANLL cases, respectively. Sixty-five percent of cases of ANLL were CD4+ (CD2-). However, CD4+ (CD2-) had a much higher specificity (91%) for ANLL than CD13 (75%) or CD33 (84%), which were expressed in a significant number of ALL. When leukemic cells were positive for CD4 (CD2-) and either CD13 or CD33, specificity and positive predictive value (PPV) for ANLL rose to 96% and 98%, respectively. The combination of CD4 positivity with either CD13 or CD33 has higher specificity and PPV than the traditional positivity for both CD13 and CD33 (specificity 89%, PPV 96%). Careful analysis of the sensitivity, specificity, and predictive values of mAbs using this method has also allowed us to establish a more cost-effective and diagnostically relevant mAb panel. Our studies show that CD4 is underappreciated as a very specific and moderately sensitive marker for ANLL. PMID- 7543731 TI - Interstitial pneumonia induced by combination therapy with low-dose cytarabine and granulocyte colony-stimulating factor. PMID- 7543730 TI - Fetal hemoglobin expression in compound heterozygotes for -117 (G-->A)A gamma HPFH and beta zero 39 nonsense thalassemia. AB - The -117(G-->A)A gamma hereditary persistence of fetal hemoglobin (Greek HPFH) and beta zero 39-thal mutations are rather frequent in Sardinia so that their interaction is to be expected. Characterization of eight compound heterozygotes for these defects indicated that HPFH was linked to haplotype VII and beta zero 39-thal to haplotype II. Haplotype II beta zero 39-thal chromosome carries the A gamma T gene which is a useful marker of gamma-gene expression. Since the Hb F level in these compound heterozygotes was significantly higher than in 46 -117 HPFH carriers, the A gamma I, A gamma T, and G gamma globin level was determined. A gamma T was underexpressed while G gamma was significantly increased, which suggest that in -117 A gamma HPFH/beta zero 39-thal healthy subjects the increase in Hb F production is determined only by the -117 mutated A gamma gene and the adjacent G gamma gene. PMID- 7543732 TI - Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression. AB - Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. PMID- 7543733 TI - Ovarian angiogenesis. Phenotypic characterization of endothelial cells in a physiological model of blood vessel growth and regression. AB - Angiogenesis occurs during embryogenesis and is a down-regulated process in the healthy adult that is almost exclusively linked to pathological conditions such as tumor growth, wound healing, and inflammation. Physiological angiogenic processes in the adult are restricted to the female reproductive system where they occur cyclically during the ovarian and uterine cycle as well as during pregnancy. By systematically analyzing the phenotypic changes of endothelial cells during bovine corpus luteum (CL) formation and regression, we have established a physiological model of blood vessel growth and regression. Quantitation of vessel density, percentage of vessels with lumen, and ratio of Bandeiraea simplicifolia-I to von Willebrand Factor-positive endothelial cells were established as parameters of angiogenesis. Sprouting endothelial cells invade the growing CL and continue to grow throughout the first third of the ovarian cycle. Thereafter the mature CL is characterized by a dense network of vessels with gradually decreasing vessel density. During luteolysis and for several weeks thereafter (regressing and residual CL) all newly formed vessels regress, which is accompanied by gradual foreshortening and rounding of endothelial cells and subsequent detachment. Based on histochemical detection of nucleosomal fragmentation products physiological blood vessel regression in the cyclic CL does not appear to involve endothelial cell apoptosis. Lectin histochemical analysis revealed a distinct alteration of endothelial cell glycoconjugate expression during ovarian angiogenesis comparable with the distinct pattern of hyperglycosylation of cultured migrating endothelial cells (up-regulation of binding sites for Lycopersicon esculentum lectin, wheat germ agglutinin, neuraminidase-treated peanut agglutinin, and Ricinus communis agglutinin-I on sprouting ECs). Northern blot analysis of glycosyltransferases during the different stages of angiogenesis revealed an up-regulation of beta galactoside alpha 2,6-sialyltransferase and alpha 1,3-galactosyltransferase mRNA expression during the angiogenic stages of CL formation. These data establish the ovarian angiogenesis model as a suitable experimental system to study the functional and phenotypic properties of endothelial cells in sprouting and regressing blood vessels and provide additional evidence for the importance of endothelial cell surface glycoconjugates during angiogenesis. PMID- 7543734 TI - Cellular localization of transforming growth factor-beta expression in bleomycin induced pulmonary fibrosis. AB - Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF beta 1 expression in this model of pulmonary fibrosis. PMID- 7543735 TI - L-selectin and alpha 4 beta 7 integrin homing receptor pathways mediate peripheral lymphocyte traffic to AKR mouse hyperplastic thymus. AB - Before the development of thymic lymphoma, AKR mice undergo a striking lymphoid hyperplasia of the thymic medulla. We have previously shown that there is a marked increase in traffic of B and T lymphocytes from the periphery into the preneoplastic, hyperplastic thymuses of these mice, in contrast to the scant traffic of such cells to normal thymuses. The traffic of lymphocytes to lymph nodes and Peyer's patches is controlled in part by the interaction of lymphocyte adhesion molecules called homing receptors with their tissue-selective endothelial ligands known as vascular addressins. We have investigated the roles of homing receptors and vascular addressins in the traffic of lymphocytes to the AKR hyperplastic thymus. We demonstrate that development of hyperplasia is accompanied by an increase in the number of thymic medullary blood vessels with high endothelial venule morphology and expression of the peripheral node addressin (PNAd) and the mucosal addressin (MAdCAM-1). In vitro and in vivo functional assays show that the addressin/homing receptor pairs PNAd/L-selectin and MAdCAM-1/alpha 4 beta 7 are involved in lymphocyte traffic to the hyperplastic thymus. These results indicate that molecular adhesion mechanisms involved in tissue-selective migration of lymphocytes to peripheral lymph node and to mucosal lymphoid tissues play a role in the recruitment of B and T lymphocytes to the AKR thymus and thus in the pathogenesis of thymic hyperplasia. PMID- 7543738 TI - Bacillary angiomatosis. AB - The acquired immunodeficiency syndrome and other causes of immunosuppression have ushered in a variety of opportunistic infections. One of these is bacillary angiomatosis, a vasoproliferative lesion whose principal causative agent is Rochalimaea henselae. Bacillary angiomatosis, while preponderantly a cutaneous affliction, can be systemic, including involvement of the head and neck mucous membranes. Molecular technology and epidemiologic studies used to identify the bacterial agent of bacillary angiomatosis have also uncovered R henselae as the organism responsible for most cases of cat-scratch disease. Why the same organism promotes two different histopathologic lesions, as seen in bacillary angiomatosis and cat-scratch disease, is unknown. PMID- 7543736 TI - Qualitative alterations in laminin expression in experimental lupus nephritis. AB - Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis. PMID- 7543737 TI - Repetitive mechanical strain suppresses macrophage uptake of immunoglobulin G complexes and enhances cyclic adenosine monophosphate synthesis. AB - Uptake of immunoglobulin G (IgG) complexes by macrophages (M phi) may play an important role in disease states characterized by increased levels of circulating immune complexes. In sites such as the glomerular mesangium M phi may be subjected to repetitive mechanical strain, although in vitro studies of M phi endocytosis are typically carried out with cells grown on rigid surfaces. We undertook the present study to determine whether repetitive mechanical strain could modulate M phi endocytosis of IgG complexes. IgG complex uptake was significantly diminished in M phi that were subjected to repetitive mechanical strain using parameters corresponding to peak and minimal intraglomerular pressures compared with control, and uptake varied according to the amount of mechanical strain applied. There was no significant difference in surface binding of IgG between M phi subjected to strain and those not. Mechanical strain did not significantly influence the rate of IgG complex degradation. Inhibition of nitric oxide synthase and guanylate cyclase activity did not alter the effect of mechanical strain, although this effect was potentiated by 3-isobutyl-1 methylxanthine (IBMX). Angiotensin II, which has been shown to reduce adenosine 3',5'-cyclic monophosphate (cAMP) production in M phi, significantly attenuated the suppressive effect of mechanical strain on IgG complex uptake as well as another inhibitor of cAMP generation, indomethacin. Enzyme immunoassay demonstrated significantly enhanced levels of cAMP in M phi that were subjected to mechanical strain compared with control, an effect that was potentiated by IBMX and attenuated by angiotensin II and indomethacin. These results demonstrate that repetitive mechanical strain significantly reduces IgG complex uptake by M phi, most likely by enhancing cAMP synthesis. Such an effect might play a significant role in macromolecule handling by M phi in sites in which they are subjected to repetitive mechanical deformation such as the glomerular mesangium. PMID- 7543739 TI - [Magnetic resonance spectroscopy. A new technic for the study and the diagnosis of prostatic disease]. PMID- 7543741 TI - Postburn pancreatitis. AB - OBJECTIVE: The authors examined the prevalence and complications of pancreatitis in severely burned patients. Factors predictive for the development of pancreatitis after burns are considered. SUMMARY BACKGROUND DATA: Pancreatitis has been documented at necropsy after burns; however, it is not clinically recognized as a common complication of burn injury. Recent improvements in survival rates could yield previously unrecognized complications, such as pancreatitis, particularly in those patients who previously would have not survived. The hypothesis is that pancreatitis is a frequent complication after major burn injury and causes significant morbidity for patients with large burns. METHODS: This retrospective review of adult patients with large burns examines postburn pancreatitis using stepwise logistic regression analysis. RESULTS: Forty nine of 121 (40%) patients developed hyperamylasemia or hyperlipasemia well after the admission period (23 +/- 3 days), and all enzyme abnormalities were temporally associated with emerging infections. Most of these patients (40/49, 82%) had symptoms of pancreatitis. Three patients (6%) had pancreatic pseudocysts or abscesses. Inhalation injury (p = 0.0001), associated trauma (p = 0.0311), and escharotomy (p = 0.0415) were risk factors for pancreatitis. Using Fischer's exact test, patients with pancreatitis had increased mortality and length of stay. Patients with high enzyme elevations and > or = 50% body surface area burned were at severe risk of pancreatic pseudocyst or abscess development (43%; 90% confidence interval of 23-77%). CONCLUSIONS: Pancreatitis is a frequent complication after large burn injuries. Patients at high risk for pancreatitis complications should receive surveillance examinations during their acute hospitalization. PMID- 7543740 TI - Elevated plasma transforming growth factor-beta 1 levels in breast cancer patients decrease after surgical removal of the tumor. AB - OBJECTIVE: The authors determined whether untreated breast cancer patients have elevated plasma levels of transforming growth factor-beta 1 (TGF-beta 1). SUMMARY BACKGROUND DATA: Increased plasma TGF-beta 1 levels recently were found after chemotherapy in patients with advanced breast cancer. However, it currently is unknown whether this elevation in plasma TGF-beta 1 is caused by chemotherapy induced normal tissue damage or whether it results from the presence of the tumor. METHODS: An enzyme-linked immunosorbent assay was used to measure plasma TGF-beta 1 levels in 26 newly diagnosed breast cancer patients before and after definitive surgery. Patients were grouped by postoperative tumor status: 1) negative lymph nodes (group 1); 2) positive lymph nodes (group 2); and 3) overt residual disease (group 3). The site of TGF-beta 1 production in the tumors was localized by immunohistochemistry and in situ hybridization. RESULTS: Plasma TGF beta 1 levels were elevated preoperatively in 81% of the patients; TGF-beta 2 and TGF-beta 3 were undetectable. The preoperative TGF-beta 1 levels in the three patient groups were similar; however, the postoperative plasma TGF-beta 1 levels differed by disease status. The mean plasma TGF-beta 1 level in group 1 (n = 12) normalized after surgery (19.3 +/- 3.2 vs. 5.5 +/- 1.0 ng/mL, p < 0.001). In contrast, the mean plasma TGF-beta 1 levels remained above normal in both group 2 (n = 9) and group 3 (n = 5) after surgery. Transforming growth factor-beta 1 expression was found to be preferentially increased in the tumor stroma. CONCLUSIONS: Breast tumors result in increased plasma TGF-beta 1 levels in 81% of patients. After surgical removal of the primary tumor, the plasma TGF-beta 1 level normalizes in the majority of patients; persistently elevated levels correlate with the presence of lymph node metastases or overt residual tumor. These findings suggest that the usefulness of TGF-beta 1 as a potential plasma marker for breast tumors deserves further study. PMID- 7543744 TI - [Fulguration of extrasystolic ventricular focus]. AB - A case is presented of symptomatic premature ventricular contractions refractory to drug therapy with right bundle branch block QRS morphology and left axis deviation in a 68-year-old female without structural heart disease. Endocardial mapping localized the extrasystolic focus at meso-inferoapical region of the left ventricular septum suggesting an origin from the Purkinje network of the left posterior fascicle. Catheter ablation with direct-current energy abolished extrasystolic complexes, without complications. The patient remained asymptomatic over a follow-up of 3 months. PMID- 7543742 TI - Resection margin for squamous cell carcinoma of the esophagus. AB - OBJECTIVE: The safe resection margin in esophagectomy for esophageal squamous cell carcinoma (SCC) was determined based on the extent of epithelial and subepithelial accessory lesions from the main lesions of esophageal SCC. BACKGROUND: There have been many reports on the high incidence of a positive resection margin for esophageal cancer. Although there were some studies on the relationships of the proximal clearance to postoperative local recurrence, no pathologic study on the resection margin has been reported. METHODS: Four hundred twenty specimens of a whole resected esophagus were examined histopathologically and the longitudinal length from the main lesion to the five types of accessory lesions was measured on microscopic slides. RESULTS: Contiguous intraepithelial carcinoma existed in 69 (46%) of 150 sites of main lesions restricted to the mucosa or submucosa and subepithelial lesions existed in 131 (54%) of 245 sites and 82 (55%) of 150 sites of main lesions invading an adventitia and into neighboring structures, respectively. The risk of a positive resection margin due to subepithelial lesions was below 5% at 10 mm in the main lesion, restricted to the submucosa or the muscularis propria, and at 30 mm in the main lesion, invading the adventitia in the potentially curative operation cases. CONCLUSION: These clearances of the resection margin, in which the risk of a positive resection margin is below 5%, are acceptable, although these clearances should only be accepted after the extent of epithelial accessory lesions is accurately determined by the Lugol's stain method. PMID- 7543743 TI - [Interatrial aneurysm as a cause of supraventricular arrhythmia in a newborn infant]. AB - Atrial septal aneurysm is a rare malformation that may occur as an isolated abnormality or in association with various cardiac defects or with connective tissue disease. The reported incidence in adults is 0.2% and in children 4.9% which suggests spontaneous regression with advancing age. Atrial septal aneurysm may be complicated by thromboembolism, valvular obstruction or arrhythmias. Supraventricular arrhythmias have been reported in as much as 16% of the cases, most commonly paroxysmal supraventricular tachycardia, supraventricular extrasystoles and atrial flutter. The relationship between the arrhythmias and the atrial septal aneurysm is still controversial. Two cases of atrial septal aneurysm in neonates are described, who presented with arrhythmias: supraventricular extrasystoles with aberrant conduction and atrial flutter with variable conduction, respectively. One of them required treatment with various anti-arrhythmia agents. During follow-up the arrhythmias remitted with spontaneous involution of the aneurysm. In the presence of the supraventricular arrhythmias in neonates, atrial septal aneurysm needs to be ruled out as a potential cause. PMID- 7543748 TI - Amylase secretion by cultured porcine parotid cells. AB - The similarity of porcine and human physiology and the availability of slaughterhouse tissues suggests the use of porcine parotid cells as a model for amylase secretion. A procedure is described for the isolation of porcine parotid cells by collagenase-P/dispase digestion of the tissue. The preparation consisted of individual cells and small aggregates that were maintained in primary culture, during which the cells formed aggregates that firmly attached to the plastic substrate. The amylase content of the cultured cells remained adequate for assay of secretory activity during culture for one week after isolation. Depending upon variations in experimental treatments, the cultured cells secreted approx. 35-65% of cellular amylase in response to a carbachol challenge. The cells were slightly responsive to long exposures to isoproterenol, and were unresponsive to nicotine, elevated extracellular K+ or substance P. Secretion induced by carbachol required extracellular Ca2+, was inhibited by atropine and occurred with a nearly linear response over a 30-min period. The Ca2+ ionophore A23187 was also a potent secretagogue for amylase secretion, producing levels of secretion equal to that induced by carbachol. The ease of preparation and the retention of amylase during primary culture suggests that the preparation will be useful in studies on muscarinic receptor-mediated control of amylase secretion. PMID- 7543745 TI - Characterization of the neurogenic plasma extravasation in the airways. AB - Release of neuropeptides from sensory nerves causes an increase in vascular permeability, plasma extravasation and edema. The sensory nerves in the airways can be activated by electrical stimulation of the vagus nerve or by application of chemical and mechanical irritants, such as capsaicin, hypertonic saline, isocapnic hyperpnea and cigarette smoke. In rodent airways, the neurogenic plasma extravasation is mediated by tachykinins released from the capsaicin-afferent nerve fibres, and involves activation of neurokinin-1 tachykinin receptors. In peripheral guinea-pig airways, neurokinin-2 tachykinin receptors have also been implicated in the neurogenic plasma exudation. The tachykinins can increase vascular permeability by both a direct effect on venular endothelium, and indirect mechanisms involving mast cell activation and serotonin release. Tachykinins and their receptors are present in the human airways. Release of tachykinins, following antigen challenge, has been demonstrated in the nose and lower airways. In humans, tachykinins have been shown to increase plasma exudation in the nasal mucosa, but whether neurogenic inflammation also occurs in the lower airways still remains to be proven. PMID- 7543746 TI - Tachykinin antagonists and the airways. AB - There is now convincing evidence for the presence of substance P (SP) and neurokinin A (NKA) in human airway nerves. Studies on autopsy tissue, on bronchoalveolar lavage fluid and on sputum suggest that SP may be present in increased amounts in the asthmatic airway. Substance P and NKA are potent bronchoconstrictors of human airways, asthmatics being more sensitive than normal persons. The major enzyme responsible for the degradation of the tachykinins, the neutral endopeptidase, is present in the airways and is involved in the breakdown of exogenously administered SP and NKA, both in normal and asthmatic persons. Other, less well documented airway effects of SP and NKA include mucus secretion, vasodilation and plasma extravasation, as well as the chemoattraction and stimulation of various cells presumed to be involved in asthmatic airway inflammation. NK2 receptors and, to a lesser extent, NK1 receptors have been shown to be involved in bronchoconstriction, whereas NK1 receptors were found to be involved in mucus secretion, microvascular leakage and vasodilatation, and in most of the effects on inflammatory cells. The first clinical trial with FK224, a peptide NK1 and NK2 receptor antagonist, and CP99994, a nonpeptide NK1 receptor antagonist, are negative. However, FK224 failed to block the bronchoconstrictor effect of NKA in asthmatics and the dose of CP99994, needed to antagonize tachykinin effects in man, remains to be determined. PMID- 7543751 TI - The chloride effect in the human embryonic haemoglobins. AB - The interactions of the three human embryonic haemoglobins with chloride ions have been investigated. Each of the three embryonic haemoglobins exhibits a unique pattern of oxygen-affinity-dependence on chloride ion concentration. Human embryonic haemoglobin Portland (zeta 2 gamma 2) is found to be completely insensitive to chloride ion concentration. Haemoglobin Gower I (zeta 2 gamma 2) shows a small concentration dependence, whilst haemoglobin Gower II (alpha 2 epsilon 2) exhibits a dependence approaching that of the adult protein. The degree of co-operativity for each protein is essentially chloride concentration independent. The chloride-dependent and -independent components of the alkaline Bohr effects have been measured for each of the embryonic haemoglobins and compared with that of the adult protein. Both the chloride-binding data and the Bohr effect have been analysed in terms of the recently developed allosteric model proposed by Perutz [Perutz, Fermi, Poyart, Pagnier and Kister (1993) J. Mol. Biol. 233, 536-545]. PMID- 7543749 TI - Effects of chronic hypoxia on the secretory responses of rat salivary glands. AB - Female Wistar rats were placed for 3 weeks in a simulated chamber evacuated by a vacuum pump and maintained at 40.5 kPa (7100 m). Dose-response curves were obtained through the sequential injection, via the femoral vein, of increasing doses of methacholine, methoxamine, isoprenaline and substance P. The secretory activity in the parotid gland after exposure to chronic hypoxia was significantly decreased for all agonists studied, and the submaxillary gland showed the same behaviour except in relation to isoprenaline, which did not show a significant difference compared to controls. These data suggest that changes in the number or sensitivity of autonomic receptors and/or alterations in the intracellular signals caused by hypoxia may be involved in the reduction in salivary secretory responses. PMID- 7543757 TI - Single-strand-specific nucleases. AB - Single-strand-specific nucleases, which act on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids, are multifunctional enzymes and are ubiquitous in distribution. They find wide application as analytical tools in molecular biology research, although enzymes such as P1 nuclease are also used for production of flavor enhancers such as 5' IMP and 5' GMP. Because these enzymes are mainly used as analytical tools, very little attention was paid to aspects relating to their structure-function relationships. However, during the last few years considerable developments have taken place in this area. Single-strand-specific nucleases, their purification, characteristics, biological role, and applications have been reviewed. PMID- 7543758 TI - Impaired antigen presentation by splenocytes of ethanol-consuming C57BL/6 mice. AB - Excessive alcohol consumption impairs T-cell-dependent immune function. Whether this impairment results from the direct inhibition of helper T (Th) cells or from inhibition of the cells that process and present antigen to Th cells is unclear. The present study examines the effect of dietary alcohol on the ability of spleen cells from C57BL/6 mice to present antigen to T-cell hybridomas. We find that ethanol consumption impairs the ability of spleen cells to present hen egg lysozyme (HEL) in vitro. This impairment was seen for native HEL protein, a hapten-modified HEL, and a peptide bearing a minimal T-cell epitope (HEL 51-60) that requires no additional enzymatic processing. These results suggest that deficiencies in immune responsiveness in alcohol-consuming individuals may include antigen presentation. PMID- 7543752 TI - Nitric oxide production by Sertoli cells in response to cytokines and lipopolysaccharide. AB - Nitric oxide (NO) is formed from L-arginine residues by nitric oxide synthase (NO Synthase) in many types of cells and acts as an intercellular messenger in several physiological systems. In the present study, we demonstrate that a combination (CL) of interleukin-1 alpha, interferon gamma, tumor necrosis factor alpha and lipopolysaccharide induces nitrite (NO2) production in cultured rat Sertoli cells. This biosynthesis of NO2- requires a lag time period of 18 hr and then increases for at least 96 hr; it is prevented by two NO Synthase inhibitors, NG-monomethyl-L-arginine and aminoguanidine. Northern blot analysis shows the induction of a macrophage-like NO Synthase mRNA synthesis in Sertoli cells cultured for a minimum of 6 hr in the presence of CL, with maximal levels after 12 to 30 hr of incubation. These results indicate for the first time that cultured rat Sertoli cells express an inducible NO Synthase isoform in response to a combination of cytokines and lipopolysaccharide. PMID- 7543753 TI - Functional link between phosphorylation state of membrane proteins and morphological changes of human erythrocytes. AB - The Tyr-phosphorylation of the cytoplasmic domain of the major membrane-spanning band 3, rather than the Ser/Thr-phosphorylation of the membrane proteins (spectrin and band 3 itself), might be functionally related to certain morphological changes of human erythrocytes. This view is supported by the following lines of evidence: a) vanadate or its derivative pervanadate (vanadyl hydroperoxide), which markedly increase the Tyr-phosphorylation of band 3 (without practically affecting the Ser/Thr-phosphorylation of spectrin) promotes a crenation of human erythrocytes; b) okadaic acid, which selectively increases the Ser/Thr-phosphorylation of spectrin and other membrane proteins, does not promote any shape change, at least at a level detectable with scanning electron microscopy. PMID- 7543755 TI - Ion channels and calcium signalling in plants: multiple pathways and cross-talk. PMID- 7543756 TI - PSA screening: a public health dilemma. AB - Screening for prostate cancer with serum prostate specific antigen (PSA) is one of the most controversial practices in health care today and guidelines from professional and policy organizations are contradictory. Prostate cancer is unique because of a wide discrepancy between prevalent asymptomatic cancer and clinical disease and an uncertain natural history. Proponents and critics of mass screening agree that PSA can detect early cancer and that definitive data that PSA reduces prostat cancer mortality are not available. Differences in screening recommendations reflect distinct viewpoints regarding what constitutes screening benefit and what level of evidence is needed to endorse a screening practice. This review describes what is known about the areas most relevant to the conflicting perspectives: the epidemiology and natural history of prostate cancer, the ability of PSA to detect disease and operational characteristics of PSA as a screening test, and the efficacy of treatment of early disease. PMID- 7543750 TI - Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4. AB - Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation. PMID- 7543761 TI - Prolonged alteration of vasoconstrictor and vasodilator responses in rat knee joints by adjuvant monoarthritis. AB - Both neurogenic influences and the regulatory neuropeptide substance P (SP) have been implicated in the development of joint inflammation. Using the laser Doppler perfusion imaging technique to quantify relative changes in joint blood flow, the effects of nerve stimulation and topical SP application were examined in normal and chronically inflamed rat knee joints. Synovial inflammation was induced by unilateral intra-articular injection of Freund's complete adjuvant and experiments were carried out 1 week and 3 weeks later. Normal knees showed a frequency-dependent vasoconstriction in response to saphenous nerve stimulation over the range of 5-30 Hz and a dose-dependent vasodilation in response to SP administration. These vasoactive responses were completely abolished in the chronically inflamed knee joint, the abolition persisting throughout the investigation. Since articular cartilage is critically dependent on synovial fluid formation for its nutrition, loss of neurovascular control of the synovial microcirculation could contribute to the degenerative changes that commonly accompany chronic inflammatory joint diseases. PMID- 7543760 TI - Neuropeptidergic and autonomic control of the vasculature of the rat knee joint revealed by laser Doppler perfusion imaging. AB - An imaging technique (laser Doppler perfusion imaging, LDI), based on measurement of backscattered Doppler-broadened laser radiation, was used to provide two dimensional images of perfusion in the exposed rat knee joint capsule. In control animals, frequency-dependent constriction of knee joint blood vessels occurred during electrical stimulation of sympathetic nerve fibres whilst dose-dependent vasodilatation of these vessels was elicited by direct application of the sensory neuropeptide substance P (SP) to the capsule. Intra-articular injection of the neurotoxic agent capsaicin did not affect vasoconstrictor responses when tested 5 7 days later, but substantially reduced dilator responses to SP. These findings indicate that capsaicin is selectively neurotoxic for sensory unmyelinated fibres but not sympathetic postganglionic fibres and is also capable of modifying receptor-mediated effects of SP. LDI is a useful method for mapping tissue perfusion, particularly in structures such as joints where the spatial distribution of blood flow is heterogeneous. PMID- 7543759 TI - Evidence of feline immunodeficiency virus replication in cultured Kupffer cells. AB - OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection. PMID- 7543762 TI - Chloride current activated by hypotonicity in cultured human astrocytoma cells. AB - Cultured astrocytoma cells were voltage clamped with pipettes where [Ca2+] in the pipette was buffered to 10(-7) M with 10 mM 1,2-bis(O-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) and membrane currents recorded. In isotonic solution predominantly K+ currents were evoked by depolarizing or hyperpolarizing commands and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 1 mM) had little effect on inward and outward currents evoked by voltage steps to negative and positive potentials. If the osmolarity of the bathing solution was reduced from 280 to 200 mosmol l-1, a large current developed, which rectified outwardly and reversed close to the equilibrium potential for chloride ions, ECl. Upon stepping to potentials positive to about +30 mV in hypotonic solution, this outward current inactivated over 2-3 s; this decline was greater with N-methyl-D glucamine chloride (NMDG-Cl) in the pipette than when KCl or sodium glutamate was present. Holding at various positive potentials inactivated the current, with half-inactivation occurring around +50 mV. The current reactivated over 2-3 s at potentials negative to about +30 mV. The current evoked by hypotonic solution was inhibited by various putative chloride channel blockers with the order of potency DIDS > SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) approximately NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) > niflumic acid > 9-AC (anthracene 9-carboxylic acid). The currents inhibited by these compounds reversed close to the calculated ECl. It is concluded that hypotonic solution evokes a large anionic current in these cultured astrocytoma cells largely carried by chloride ions. This current is absent in isotonic solution, when currents are carried mainly by potassium ions. It is likely that the current elicited by hypotonic solution is part of the mechanisms regulating astrocyte volume in vivo. PMID- 7543747 TI - The nitrergic transmitter of the anococcygeus--NO or not? AB - Nonadrenergic noncholinergic (NANC) relaxations of the anococcygeus muscle are reduced by inhibitors of nitric oxide synthase (NOS). Since NOS can be detected within 6-hydroxydodpamine-resistant nerve tracts running through the muscle, it seems clear that these NANC relaxations result from activation of the L arginine/NO pathway within the prejunctional nerve terminal, an example of so called "nitrergic" transmission. However, a number of substances (hydroquinone, superoxide anions, hydroxocobalamin) profoundly reduce relaxations to exogenous NO but do not affect those to nitrergic field stimulation; such observations have raised questions over the nature of the substance actually released from the nitrergic nerves. Several possible explanations are discussed: (1) NO is released attached to a carrier molecule, perhaps in the form of a nitrosothiol; (2) NO is released in a modified redox form; (3) NO is released as a free radical, but is protected within the neuroeffector junction by other substances which preferentially interact with scavenger molecules; and (4) NO is released as a free radical and, because of a rapid and unhindered rate of diffusion over short distances (100-200 microM), it is less susceptible than exogenous NO to scavenger molecules. As yet, there is insufficient experimental evidence to decide which, if any, of these explanations is correct. PMID- 7543754 TI - Suppression of experimental antigen-induced arthritis in transgenic mice producing human alpha-fetoprotein. AB - Experimental arthritis was induced in the knee joint of transgenic mice expressing human alpha-fetoprotein by immunization with methylated bovine serum albumin in Freund's complete adjuvant. In the control experiment with normal C57BL/6 mice, definite arthritis was observed in 55.6% (5/9) of the mice, but in only 21.1% (4/19) of the transgenic mice. This result suggested that human alpha fetoprotein functioned as an immunosuppressant to ameliorate the development of the immune disease. PMID- 7543765 TI - Biological response modifiers. PMID- 7543767 TI - Breast cancer angiogenesis: therapy target and prognostic factor. PMID- 7543764 TI - Kinetics of prostate-specific antigen after manipulation of the prostate. AB - Kinetics of prostate-specific antigen (PSA) were investigated after manipulation of the prostate in two groups of patients: those treated with digital rectal examination (DRE), and those with needle biopsy. 8 patients had serial PSA measurements to study the effect of DRE (group 1). 7 of 8 patients had PSA baseline values < 10 ng/ml. Blood samples were taken at 1 min, 30 min, 1, 3, 6, 12 and 24 h after DRE. Some patients were further monitored for 5 days with one blood sample taken at the same time each day. Statistically significant increased PSA levels were found after DRE (P < 0.001). Maximal increase was 70%. In most patients, peak levels were found between 30 and 60 min after DRE. Based on the results, it is concluded that after DRE it is prudent to wait 3 days before PSA is determined. 7 patients had serial PSA measurements after transrectal prostate needle biopsy (group 2). PSA sampling was similar as in the previous group. All patients had increased PSA levels after biopsy (range 1.3-9.5-fold). After 5 days, only 2 of 7 patients had returned to baseline levels. We conclude that biopsies of the prostate induce an important and long-lasting PSA elevation. PMID- 7543766 TI - Medical management of non-small cell lung cancer. PMID- 7543763 TI - Activity-induced facilitation of L-type Ca2+ current in cardiomyocytes isolated from guinea-pig ventricles. AB - The facilitation of L-type Ca2+ current (ICa), which sometimes occurs with an increase in stimulation frequency, was investigated in single guinea-pig ventricular cardiomyocytes using whole-cell recording and K(+)-free solutions. With a holding potential of -80 mV, an increase in frequency from 0.5 to 1, 2, 3 or 4 Hz caused either a small or large initial reduction, or a transient enhancement (facilitation) of peak ICa, which developed rapidly and was followed by a reduction of ICa. Reducing the frequency to 0.1 or 0.2 Hz caused a depression of ICa on the first pulse that was followed by a slower increment. Transient facilitation and depression were entirely absent when either Ba2+ or Na+ was used as the charge carrier in Ca(2+)-free solutions. High concentrations of isoprenaline (1-3 microM), forskolin (1-3 microM), or 8-(4-chlorophenylthio) cAMP (150 microM) suppressed but did not abolish the incidence and size of facilitation; employing a holding potential of -40 mV also suppressed the incidence of ICa facilitation. Lower isoprenaline concentrations (0.1 and 0.3 microM) greatly enhanced the incidence and magnitude of the transient facilitation occurring with an increase in stimulation rate, but did not diminish the magnitude of the ensuing reduction. When facilitation occurred with 0.1 mM EGTA in the dialysate, or the usual 5 mM EGTA with 0.01 microM extracellular isoprenaline, it developed more slowly after an increase in frequency. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, an increase in stimulation rate from 0.5 to 1, 2 or 3 Hz sometimes caused a large and sustained facilitation of ICa, which developed over tens of seconds, declined slowly with continued stimulation and was maintained after returning to 0.5 Hz. It is concluded that the levels of intracellular cAMP and Ca2+ mediate the initial sensitivity of ICa to changes in stimulation rate, to the extent that they determine whether or not transient facilitation will occur. Because the reduction of ICa was relatively constant, facilitation dictates the level of steady-state ICa that will be reached at the higher rate. Taken together with the fact that facilitation can be modulated, the results argue for separate mechanisms of facilitation and reduction for ICa. It is suggested that the mechanism of facilitation is partly enzymatic, insofar as sustained facilitation could be a manifestation of a stimulatory Ca(2+)-dependent process, which is normally counteracted by the action of phosphodiesterases.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543769 TI - Does solute stereochemistry influence percutaneous penetration? AB - The stratum corneum, the rate-limiting barrier to percutaneous penetration, is made up of several components, principally keratin and ceramides. These are potential sources of chiral discrimination that could result in differential diffusion rates, dependent upon the stereochemistry of the solute. Although binding to keratin can occur it is not a stereoselective process [percent binding to solubilised epidermal keratin: (R)-propranolol 7.9 +/- 1.7, (S)-propranolol 8.3 +/- 2.0]. On the other hand, studies with ceramide monolayers produced qualitative evidence of dose-dependent stereoselective interaction when the pure diastereomers of ephedrine were present in the aqueous subphase which suggested that differences in diffusion rates might occur in skin. However, the differences in permeation rates in vitro for these diastereomers through human skin were not statistically different [(+)-(1S,2R)-ephedrine 119.1 +/- 2.6 micrograms/cm2, (-) (1R,2S)-ephedrine 107.0 +/- 3.9 micrograms/cm2, 12 h]. Time averaging, involving contributions from binding to all lipid headgroups present in the intercellular channels, may obscure specific differential interactions. Further, any stereospecific interaction may be subtle and readily overwhelmed if diffusant concentration is greater than the capacity of the skin to differentiate between stereoisomers. Evidence for intrinsic stereoselectivity in skin permeation has therefore yet to be obtained. PMID- 7543771 TI - Tumour angiogenesis and tumour cell proliferation as prognostic indicators in gastric carcinoma. AB - Tumour growth depends on neovascularisation and tumour cell proliferation. Factor VIII-related antigen (F-VIII RA) localises to vascular endothelium. Expression of proliferating cell nuclear antigen (PCNA) is correlated with cell proliferation. We investigated the correlation between the expression of these antigens and prognosis in gastric carcinoma. A total of 108 specimens resected from patients with gastric carcinoma were investigated by staining with monoclonal antibodies against F-VIII RA and PCNA. Microvessel count (MVC; the mean number of microvessels in the five areas of highest vascular density at 200 x magnification) and PCNA labelling index (PCNA LI; percentage of positive cells in more than 500 tumour cells) were determined. The results showed that prognosis was significantly worse in patients who had a tumour with a high MVC (16 or greater) or a high PCNA LI (42% or greater) than in those patients who had a tumour with a low MVC (less than 16) or a low PCNA LI (less than 42%). Furthermore, MVC was significantly associated with the risk of hepatic recurrence. In conclusion, both MVC and PCNA LI may be good prognostic indicators in patients with gastric carcinoma. PMID- 7543774 TI - Clinical evaluation of serum alpha-fetoprotein and circulating immune complexes as tumour markers of hepatocellular carcinoma. AB - To evaluate the diagnostic application of serum alpha-fetoprotein (AFP) and circulating immune complexes (CICs), AFP, 3% polyethylene glycol (PEG)-CICs, 4% PEG-CICs, and C1q-CICs were determined in 101 patients with cirrhosis alone, 101 sex-matched and age-matched cirrhotic patients with hepatocellular carcinoma (HCC) and 54 healthy controls. Multivariate analysis indicated that AFP (odds ratio 1.014; 95% confidence interval 1.004-1.024) and 3% PEG-CICs (odds ratio 1.011; 95% confidence interval 1.005-1.017) are associated, in a dose-related fashion, with an increased risk for HCC. A receiver operative characteristic (ROC) curve was used to determine the optimal cut-off values of AFP (120 ng ml-1) and 3% PEG-CICs (310 micrograms aggregated IgG equivalent ml-1). The area under ROC curve was 0.875 for AFP and 0.812 for 3% PEG-CIC. Both AFP and 3% PEG-CICs show a high specificity (100%) and positive likelihood ratio. The sensitivity was 65.3% for 3% PEG-CICs and 67.3% for AFP. Determination of both markers in parallel significantly increase the diagnostic accuracy (92.1%) and sensitivity (84%), with a high specificity (100%) and positive likelihood ratio (> 84). In conclusion, both 3% PEG-CICs and AFP are independent risk factors of HCC, and may be used as complementary tumour markers to discriminate HCC from cirrhosis. Determination of 3% PEG-CICs should be performed in cirrhotics negative for AFP to improve detection of HCC. PMID- 7543772 TI - Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. AB - The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp. PMID- 7543775 TI - Synergistic inhibition of HIV-1 reverse transcriptase DNA polymerase activity and virus replication in vitro by combinations of carboxanilide nonnucleoside compounds. AB - The carboxanilides UC84 and UC38 are nonnucleoside inhibitors of both the RNA dependent and DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase (RT). We have previously shown that UC84 and UC38 bind to the same site as nevirapine but interact with different RT mechanistic forms, with UC84 preferentially binding to the RT-primer/template complex and UC38 binding only to the RT-primer/template-dNTP ternary complex [Fletcher, R. S., et al. (1995) Biochemistry 34, 4346-4353]. Here we demonstrate that combinations of UC84 and UC38 inhibit RT DNA polymerase activity in vitro in a synergistic manner. This synergy was noted primarily in reactions containing high concentrations of primer/template and Km levels of dNTP substrate and was independent of both primer/template identity and the molar ratio of UC84:UC38. Combination indices were in the range of 0.4-0.6, indicating substantial synergy in the inhibition of RT activity. More importantly, combinations of UC84 and UC38 also showed a high degree of synergy in inhibiting HIV-1 replication in both MT-4 and cord blood mononuclear cells. We believe this to be the first example of synergistic inhibition of HIV-1 RT by combinations of structurally related nonnucleoside inhibitors. PMID- 7543777 TI - Molecular cloning and expression of DNA encoding ovine interleukin 2. AB - We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL 2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep. PMID- 7543776 TI - Identification of the peptide binding motif for HLA-B44, one of the most common HLA-B alleles in the Caucasian population. AB - Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9. PMID- 7543773 TI - Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta. AB - The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL 64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors. PMID- 7543768 TI - Prostate cancer: a continuum of controversy. PMID- 7543778 TI - Soluble T-cell products antigenically related to T-cell receptors. AB - Human peripheral blood mononuclear cells (PBMC) cultured with or without concanavalin A (Con A) secrete soluble TCR alpha and TCR beta chains. Soluble TCR like products of three different relative molecular masses--185,000 (Mr 185 kDa), 152 and 42 kDa--which are antigenically similar to those on T lymphocytes have been found in sera from healthy adult donors. These findings suggest that soluble TCRs, antigenically similar to cell surface TCRs, are secreted from T lymphocytes. PMID- 7543770 TI - Tumour vasculature--a potential therapeutic target. AB - The tumour vasculature is vital for the establishment, growth and metastasis of solid tumours. Its physiological properties limit the effectiveness of conventional anti-cancer strategies. Therapeutic approaches directed at the tumour vasculature are reviewed, suggesting the potential of anti-angiogenesis and the targeting of vascular proliferation antigens as cancer treatments. PMID- 7543779 TI - IL-8 and angiogenesis: evidence that human endothelial cells lack receptors and do not respond to IL-8 in vitro. AB - It has been established that IL-8 triggers angiogenesis in vivo, but this effect may be mediated either by IL-8-recruited leukocytes or by direct actions of IL-8 upon endothelial cells (EC). We have approached this question by examining interactions of recombinant human IL-8 with cultured large vessel and microvascular human EC. We are unable to detect specific IL-8 binding to cultured human umbilical vein endothelial cells (HUVEC) or leukocyte-like IL-8 receptor mRNA expression by either cultured HUVEC or human dermal microvascular endothelial cells (DMEC). We find no alteration of cytoplasmic calcium concentration ([Ca2+]i) in either cell type in response to IL-8 treatment. Finally, we find no IL-8-induced change in EC proliferative rates in the presence or absence of endothelial cell growth factor. Our data favour an indirect action for IL-8 as an angiogenic factor. PMID- 7543783 TI - Comparison of two anti-hepatitis C virus enzyme-linked immunosorbent assays: Wellcozyme VK45 and Ortho 2.0. AB - BACKGROUND: The aim of the present study was to compare 2 anti-HCV ELISA tests with respect to sensitivity and specificity in detecting Hepatitis C antigen. MATERIALS AND METHODS: A 3rd-generation anti-HCV ELISA (Wellcozyme anti-HCV VK45) was compared with a 2nd-generation anti-HCV ELISA (Ortho HCV 2.0) in various serum panels: A) anti-HCV ELISA-positive samples of blood donations (n = 536), B) non-A, non-B hepatitis patients (n = 188), C) multi-transfused patients (n = 79), D) hemodialysis patients (n = 473), and E) random blood donors (n = 1,080). RESULTS: Of 248 cDNA polymerase chain reaction (cDNA-PCR) positive samples in panels A, B, and C, ELISA-VK45 detected 247 (99.6%) and Ortho-2 248 (100%). The cDNA-PCR-positive sample missed by ELISA-VK45 showed isolated anti-C33c reactivity in a 2nd-generation recombinant immunoblot (RIBA-2). Of 281 RIBA-2 positive samples, ELISA-VK45 detected 274 (97.5%) and Ortho-2 279 (99.3%). ELISA VK45-negative, RIBA-2-positive samples showed combined anti-C100/5-1-1 reactivity in RIBA-2 in 6/7 cases and anti-C22 and C33c reactivity in one. Ortho-2-negative, RIBA-2-positive samples showed combined anti-C100/5-1-1 reactivity in RIBA-2 in 2/2 cases. The specificities of ELISA-VK45 and Ortho-2 were not significantly different in 1,080 blood donors. CONCLUSION: It is concluded that the ELISA-VK45 lacks sensitivity because a cDNA-PCR-confirmed positive sample was missed in the assay. The specificity of both ELISAs was comparable. PMID- 7543782 TI - Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells. AB - BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension. PMID- 7543780 TI - Differential effect of rapamycin and cyclosporin A in proliferation in a murine T cell line expressing either intermediate or high affinity receptor for IL-2. AB - It has been reported that rapamycin (rap), cyclosporin A (CsA) and FK506 have immunosuppressive effect during the activation process of murine T cells. These drugs were investigated for their suppressive effect on a murine T cell line expressing intermediate (TS1 beta) or high (TS1 alpha beta) affinity IL-2R. Rap and CsA strongly inhibit the IL-2-dependent proliferation of TS1 alpha beta cells while they minimally affect the IL-2-mediated proliferation of TS1 beta cells. FK506 does not have any effect on the IL-2-driven proliferation of either TS1 beta or TS1 alpha beta cells. Simultaneous addition of Rap and CsA or Rap and FK506 inhibit the IL-2-mediated proliferation of TS1 beta and TS1 alpha beta cells and therefore FK506 does not revert the inhibition mediated by Rap in TS1 alpha beta cells. Neither Rap nor CsA affect IL-2R expression and internalization in TS1 alpha beta cells. CsA and Rap strongly inhibit the appearance of DNA binding activity of NF-AT and to a lesser extent NF-kappa B. Rap inhibits IL-2 stimulated phosphatidylinositol 3 (PI3) kinase activity in TS1 alpha beta cells. In TS1 beta cells, Rap activates PI3 kinase on its own, inhibiting the IL-2 stimulated PI3 kinase to a lesser extent. These results suggest that PI3 kinase is a target for Rap action. Our results strongly suggest that we have Rap and CsA sensitive and resistant activation pathways operating in TS1 beta and TS1 alpha beta cells. PMID- 7543781 TI - Effect of granulocyte colony-stimulating factor (G-CSF) on chemotherapy-induced oral mucositis. AB - In this study, the ability of granulocyte colony-stimulating factor (G-CSF) to treat or prevent chemotherapy-induced oral mucositis in patients with advanced breast cancer was evaluated. A total of 14 patients who received intraarterial (i.a.) adriamycin (ADM) preoperatively were divided into two groups according to whether or not G-CSF was given. Thus, group A (n = 7) was given G-CSF and group B (n = 7) was not. G-CSF therapy reduced both the incidence and duration of ADM induced oral mucositis, and a positive correlation was also seen between the incidence of mucositis and ADM-induced leukopenia (< 2,000/mm3). Group A was further divided into two subgroups according to whether G-CSF was given after or before the leukopenia had dropped below 2,000/mm3: group A-1 (n = 3) and group A 2 (n = 4), respectively. ADM-induced mucositis was observed in two of the three patients in group A-1, but in none of the four patients in group A-2. These results strongly support the idea that G-CSF can effectively treat and prevent ADM-induced oral mucositis. PMID- 7543784 TI - [Model calculations for HIV screening of blood and plasma donors with a combination of 2 screening tests: test strategies, validity, costs and effectiveness]. AB - OBJECTIVE: The strategies for combining two screening tests for HIV infections in blood or plasma donors are formulated in biometric terms and analyzed with respect to their value, i.e. their validity, cost and effectiveness. DESIGN: Biometrical modeling using assumptions on the validity of the single tests, the conditional correlations between them, as well as on the cost of testing and the consequences of false-negative or false-positive test results. RESULTS: If the test combination is defined as positive whenever at least one of the single tests is positive, then this rule (the 'believe the positive' rule, BTP), due to its lower specificity, has extremely low positive predictive values. In case of high prevalence rates of the infection (e.g. 1:1,000), the BTP rule leads to lower total cost than single testing, unless the latter has very high sensitivity (e.g. 99%). For smaller prevalence rates (< 1:50,000), which are more typical of the selected group of blood or plasma donors, combination testing is of little value because the extra cost of detecting one additional infection (compared with single testing) may reach several 100 million DM. CONCLUSION: The cost for detecting additional cases of HIV infection by using combination instead of single testing in HIV screening is so high that this decision requires a public consensus. PMID- 7543786 TI - Failure of normalisation of alpha fetoprotein concentration after successful treatment of teratoma. PMID- 7543787 TI - Fibroblast growth factors: at the heart of angiogenesis. AB - Acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF 2) are ubiquitous cytokines found in many tissues. They have effects on multiple cell types derived from mesoderm and neuroectoderm, including endothelial cells. In this review the structure and function of the fibroblast growth factor family and its receptors are described. The evidence implicating both FGF-1 and FGF-2 in the control of blood vessel formation is presented and their involvement in normal and pathological angiogenesis during adult life is then described in more detail. PMID- 7543788 TI - CD4+ cytolytic T cells can destroy autologous and MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG. AB - Mycobacterium bovis-BCG infected macrophages were exposed in vitro to PPD stimulated T lymphocytes from tuberculin responsive donors or to a panel of mycobacterial-antigen specific CD4+ T cell clones. Both polyclonal and clonal T cells caused considerable antigen-specific lysis of autologous or MHC class II matched macrophages. However, lysis of infected macrophages did not significantly affect the number of viable mycobacteria which were released into the culture media from lysed macrophages. In tuberculosis, CD4+ cytolytic T cells may be primarily involved in tissue destruction and lack a significant role in acquired cellular immunity. PMID- 7543785 TI - Palliative therapy of melanoma patients with fotemustine. Inverse relationship between tumour load and treatment effectiveness. A multicentre phase II trial of the EORTC-Melanoma Cooperative Group (MCG). AB - Fotemustine (FM) is a new chloronitrosurea (CNU), chemically characterized by the graft of an aminophosphonic acid on the CNU radical, which makes it highly lipophilic. Following single-institution phase I and II studies with remarkably high response rates of some 40%, including brain metastases of 25% and more, the EORTC-MCG started a multicentre phase II trial to confirm these results according to EORTC guidelines. Treatment consisted of an induction cycle of FM (100 mg/m2 on days 1 + 8 + 15), followed by maintenance courses (q3w). Fifty-four patients were entered by 11 institutions. General interest in this promising new agent, however, led clinicians of six additional institutions to join the EORTC trial and 90 more patients were included in only 4 months. This rapidly rising accrual rate became inversely related to the physicians' adherence to the eligibility criteria: palliation of symptoms rather than clinical research was the dominant reason to start treatment. Clinical characteristics and results in the eligible vs non-eligible patient group (in parentheses) were as follows: male/female 29/26 (68/65), mean age 54 years (53), ECOG-PS 0-1 (0-4), CR 2 (0), PR 10 (2), NC 17 (5) and for brain metastases: PR 4 (1), NC 2 (1), for an ORR of 12% (5%). Median duration of response was 6 months (range 4-16). The clinically relevant toxicity was limited to the haematopoiesis with delayed platelet nadirs (30% grade III+IV), granulocyte (25% grade III + IV) and the gastrointestinal tract: nausea and vomiting (26% grade II, 18% III, 1% IV). This study confirms that FM is active in melanoma including brain metastases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543789 TI - Summit of the Americas and its follow-up: PAHO's Role. Pan American Health Organization. PMID- 7543790 TI - Inter-American conference on society, violence, and health. PMID- 7543791 TI - Parents and families of children with communication disorders. AB - The role of parents in the development of the communicative competence of their children has been of growing interest to researchers and practitioners in the field of speech and language therapy. Researchers have attempted to define and test specific hypotheses regarding the nature and degree of the influence of parental interaction styles upon the developing child's pragmatic skills. The literature includes studies regarding normal children and those with speech and language impairments. Practitioners have endeavoured to move towards a firmer framework of partnership with parents which will ultimately be of benefit to children with speech and language impairments and their families. PMID- 7543793 TI - Dynamics of fibrovascular tissue ingrowth in hydrogel foams. AB - We have investigated and quantified the degree of fibrovascular tissue ingrowth in cylindrical poly(vinyl alcohol) (PVA) foams of 12.5 mm diameter, 5 mm thickness, and 71% porosity implanted in the mesentery of rats over a period of 25 days. Fibrovascular tissue penetrated the center of PVA foams 5 days postimplantation yet the void fraction available for cell seeding was 55% and the volume average pore diameter was 190 (+/- 39) microns. By 10 days postimplantation the void fraction had decreased to 32% and the volume average pore diameter was 121 (+/- 20) microns. As time elapsed fibrovascular tissue continued to expand and fill the remaining pore space. At 15 days postimplantation the void space was impractical for cell seeding and continued to decrease through the remainder of the study. Our data suggest that hydrogel foams with a polydispersed pore morphology can be prevascularized with adequate space for cell seeding as the volume of tissue penetrating the foam is limited by the smaller pores in the foam structure; however, available void space for cell seeding decreases with time. PMID- 7543792 TI - [The child at high risk of imminent death: integrated management]. AB - The authors report on their experience of the integrated medical and psychological care in a pediatric intensive care unit. They describe an individualized psychological management of the child at high risk of imminent death. The helpful attitudes and people are identified such as a loving human presence, a climate of trust, life openness and tolerance to child's reactions and receptiveness to the still present life. With the provided support, the child possibly goes through his difficult experience in a more serene way, at his own rythm, with the respect and the acceptance for his individual expression. PMID- 7543794 TI - Detection of autoantibodies against bullous pemphigoid and pemphigus antigens by an enzyme-linked immunosorbent assay using the bacterial recombinant proteins. AB - To develop a new method to evaluate autoantibodies in various autoimmune bullous skin diseases, we examined reactivity of bullous pemphigoid (BP) and pemphigus vulgaris (PV) patients' sera with partial bacterial fusion proteins of the 230 kD BP antigen (BPAG1) and PV antigen, respectively, by an enzyme-linked immunosorbent assay (ELISA), and compared the results with those of immunoblotting. We used two fusion proteins derived from the mouse BPAG1 and a fusion protein derived from the amino-terminus (EC1-2) of PV antigen. For both BP and PV sera, the ELISA scores were well correlated with the reactivities on immunoblot assays. The present study indicates that ELISA using recombinant antigen proteins in various autoimmune bullous skin diseases will be a new useful technique to detect the autoantibodies. However, development of recombinant proteins with the entire molecule and correct conformation will be necessary to establish a perfect ELISA system in the future. PMID- 7543795 TI - Histaminergic nerves demonstrated in the skin. A new direct mode of neurogenic inflammation? AB - An intradermal administration of histamine into human skin results in a local erythema, edema and often also the sensations of itch and/or pain. These effects have classically been attributed to the presence of histamine-containing mast cells. However, in the present investigation, we report the observation of histamine-immunoreactive nerves in the skin of Sprague-Dawley rats using a new and highly sensitive immunohistochemical approach. These data suggest a more direct route of cutaneous histamine effects, mediated exclusively by the peripheral nervous system. The findings could also give a new basis for explaining histamine-related issues, such as itch. PMID- 7543796 TI - [Digestive cryptosporidiosis in young children in an urban area in Gabon]. AB - A 27 month epidemiological survey of cryptosporidiosis in stools was conducted in 288 Gabonese children aged between 0 and 2 years (mean 10.2 months). By at least one of two staining techniques, acid-fast and direct immunofluorescence, the rate of infection was determined to be 24%. The rate was 28% in cases of acute diarrhea, in striking contrast with the low prevalence of 14.8% in uninjured gastroenteritus subjects (p < 0.05). The notion of asymptomatic carriers was clearly demonstrated. The maximum of 34.4% was observed for infants aged between 6 and 12 months (p < 0.02). In cases of malnutrition, this rate increased to 31.8%, whereas it was 16.8% in eutrophic children (p < 0.01). For the rainy and dry seasons, it was 31.7% and 19.4%, respectively (p < 0.02). Water is emerging as an important vehicle for the transmission and spread of Cryptosporidium sp. in tropical areas. PMID- 7543798 TI - Cutaneous ciliated cyst: a case report. AB - We report a case of a cutaneous ciliated cyst in the buttock of a 16-year-old girl. We describe the pathological and immunohistochemical data on this very uncommon skin lesion, whose histogenesis remains a matter of controversy. PMID- 7543801 TI - Ultrasonography of the fetal neck in the first and early second trimesters. Part 1. Normal appearance. AB - The soft tissues of the neck of 196 normal fetuses were examined with ultrasonography during the first and early second trimesters, and the criteria for normality are described here. The dorsal pseudomembrane is the most important feature of this area in a normal fetus. This feature appears as a single echogenic line lying parallel to the occiput and the upper cervical spine but separated from them by an anechoic area, which should be less than 2.8 mm deep before 18 weeks gestational age. The membrane is best observed when the fetus is in the "neck-up" position with the neck flexed. Although the superior-inferior extent of the membrane is variable, the occurrence of lateral extension, septations or hydrops or a bulbous appearance indicates abnormality. The membrane should move freely with the motion of the neck, which allows it to be differentiated from the normal amnion, amniotic bands or synechiae. PMID- 7543799 TI - Action of 5-hydroxytryptamine, substance P, thyrotropin releasing hormone and clonidine on spinal neuron excitability. AB - We investigated the influence of four substances on the excitability of lumbar motoneurons. These substances, three of which coexist in the same bulbospinal descending pathways that end, for the most part, around motoneurons (MNS), are: 5 hydroxytryptamine (5-HT), substance P (SP) and thyrotropin-releasing hormone (TRH). We also studied the effects of clonidine, an alpha 2 noradrenergic (NA) agonist. This study was carried out in rats spinalized at T5 and treated three weeks earlier with 5-7 dihydroxytryptamine (5-7 DHT). Under these conditions, the following responses were observed: 5-HTP (5-HT precursor) intraperitoneally (I.P.), 5-HT intrathecally (I.T.), TRH (I.P. or I.T.) and substance P (I.T.) all elicited strong excitation of MNS as measured by integrated EMG of the hindlimb muscles; substance P reduced by almost half the response to 5-HTP given one hour and 24 hours later; TRH given acutely did not modify the response to 5-HTP, but given chronically for 21 days markedly increased the response to this substance. Clonidine by itself decreased the excitability of MNS and antagonized the excitatory effects of 5-HTP and TRH. In two separate pilot trials, cyproheptadine, a 5-HTP antagonist, decreased the manifestations of spasticity in a patient with a partial spinal lesion. It would appear that clonidine may have potential use in the management of spasticity. PMID- 7543797 TI - Extracellular matrix proteins and VLA integrins expression in the microenvironment of human lung carcinoma. AB - In order to gain an insight into interactions between human cancer and the surrounding host tissues, surgical samples of lung carcinoma of distinct histological types were examined for the expression of extracellular matrix (ECM) proteins and very late antigen (VLA) integrins, by means of a panel of monoclonal antibodies and immunohistochemistry of frozen tissue sections. It has been found that fibronectin (FN), tenascin (TN) and to a lesser degree collagen IV were abundant in the immediate vicinity of the tumor, but only TN penetrated tumor mass. FN isoforms were scarce or undetectable within the tumor area. The walls of blood vessels in the vicinity of the tumor showed increased an expression of collagen IV and laminin. The latter was occasionally absent within the basal membrane of cancer cells. The expression of EMC proteins was inversely proportional to the intensity of mononuclear tumor infiltrating cells (TIC). VLA integrins were present on both types of the cells: TIC and tumor cells. Percentage of positive TIC varied from 20% to 70%, depending on VLA integrin tested. VLA-3 was demonstrated on most of the cells of squamous carcinoma, but was almost absent on those of anaplastic small cell carcinoma one. In metastatic lymph node, VLA-4 was strongly expressed on tumor cells comparing to lymphoid ones. These data show that VLA integrins and their EMC ligands play apparently an important, but still obscure role in the interactions between lung carcinoma and its host. PMID- 7543800 TI - Breast cancer angiogenesis: a quantitative morphologic and Doppler imaging study. AB - BACKGROUND: Tumor growth and metastases require the development of new vessels (angiogenesis). Angiogenesis, assessed by microvessel count using immunocytochemical stain of endothelial cells, has been shown to predict metastases and correlate with early death. Recently developed color Doppler mapping can detect the "tumor flow signals" in breast cancer and help to distinguish it from benign lesions. The question is, does this tumor vascularization assessed by color Doppler mapping correlate with the angiogenesis assessed by immunocytochemistry? METHODS: Eighty-four patients admitted for breast surgery were studied. The final diagnosis was made by pathology for 52 malignancies and 32 benign lesions. The color Doppler mapping of the breast lesion was made preoperatively. The following parameters were assessed: (a) vessel location (peripheral or central); (b) density of color Doppler signals; and (c) maximum systolic velocity. Tumor angiogenesis was assessed by microvessel count under light microscopy using the platelet/endothelial cell adhesion molecule antibodies (CD31) method. The correlation between maximum velocity and microvessel count of breast cancer was examined. The clinical significance of maximum flow velocity of breast cancer with various clinicopathologic factors was assessed. RESULTS: Color signals were detected in 48 cases of 52 malignancies (92%). All tumors demonstrated signals at the periphery of the lesion but in only 13 (27%) were the signals detected within the tumor. Color signals were scored as + + or + + + in 44 (92%) patients. Pulsed wave blood flow was shown in all these 48 tumors, with maximum velocities varying from 4 to 36 cm/s. Among the 32 benign lesions, color signals were detected in 10 (31%) and all were peripheral and scored subjectively as +. Evaluation of these color Doppler mapping parameters shows no significant correlation with microvessel counts using CD31 monoclonal antibodies. However, there was a positive association (p < 0.05) between nodal metastases and higher tumor flow velocity in T1 (< 2 cm) breast tumors but not in larger tumors. CONCLUSION: Although the color Doppler mapping has been shown to be useful in distinguishing benign from malignant breast lesions, the intensity of signal and velocity of flow had no correlation with the extent of angiogenesis of breast cancer. The presence of high-flow tumor signal in early breast carcinoma is significantly associated with the presence of axillary lymph node metastases. PMID- 7543802 TI - Cryptogenic organizing pneumonia in the immunocompromised patient: radiologic findings and follow-up in 12 patients. AB - OBJECTIVE: To assess the radiographic, computed tomography (CT) and pathologic findings and response to therapy of cryptogenic organizing pneumonia in immunocompromised patients. PATIENTS AND METHODS: The study group consisted of 12 consecutive immunocompromised patients (seven men and five women), examined between July 1988 and July 1993, who had pathologically proven cryptogenic organizing pneumonia and for whom chest radiographs and CT scans had been obtained within 9 days of diagnosis. The 12 patients were treated with corticosteroids and underwent follow-up radiography. RESULTS: The findings of chest radiography included consolidation (in 10 patients), reticulation (in 3) and small nodules (in 2). None of the radiographic abnormalities were localized to particular zones of the lung. The CT findings included ground-glass attenuation (in nine patients), consolidation (in six), small nodules (in five) and reticulation (in two). In 8 of the 12 patients (67%) these abnormalities were most marked in the subpleural and peribronchovascular regions. The follow-up radiographs showed improvement in 11 (92%) of the patients. CONCLUSIONS: The most common radiographic abnormality associated with cryptogenic organizing pneumonia in these immunocompromised patients was consolidation, and the most common CT finding was ground-glass attenuation with or without associated consolidation or nodules. Most of the patients responded to corticosteroid therapy, irrespective of the initial radiologic pattern. PMID- 7543805 TI - Duplex ultrasonography criteria for internal carotid stenosis of more than 70% diameter: angiographic correlation and receiver operating characteristic curve analysis. AB - OBJECTIVE: To determine duplex ultrasonography criteria for detecting stenosis of 70% or more in the internal carotid artery, to be used as a means of selecting candidates suitable for endarterectomy (a procedure suggested by the North American Symptomatic Carotid Endarterectomy Trial as appropriate for symptomatic patients with 70% to 99% stenosis). PATIENTS AND METHODS: Between Jan. 1, 1991, and Apr. 30, 1993, 120 patients underwent internal carotid angiography at a tertiary care hospital for transient ischemic attacks, asymptomatic bruits or preoperative assessment. Of these, 83 also underwent duplex ultrasonography within 31 days of angiography. The angiographic and sonographic studies for 145 vessels in 75 of the patients (50 men and 25 women) were suitable for further study. The sonographic criteria were selected on the basis of a receiver operating characteristic curve relating peak systolic velocity of the internal carotid artery to the degree of stenosis determined angiographically. RESULTS: Angiography indicated that 33 of the vessels had stenosis of 70% or more and that 12 additional vessels were completely occluded. The combined sonographic criteria of peak systolic velocity of less than 40 cm/s or 175 cm/s or more yielded 91% sensitivity and 60% specificity for angiographically determined stenosis of 70% or more. CONCLUSION: In this patient population the combined criteria of peak systolic velocity of less than 40 cm/s or 175 cm/s or more were highly sensitive and reasonably specific for internal carotid artery stenosis of 70% or more. PMID- 7543806 TI - Mammographic features of pure mucinous carcinoma of the breast with pathological correlation. AB - OBJECTIVE: To determine the mammographic features of pure mucinous carcinoma of the breast and correlate them with the pathologic features. PATIENTS AND METHODS: The authors analysed 23 tumours identified as pure mucinous carcinoma of the breast in 22 patients, aged 40 to 92 (mean 64.9) years at the time of mammography (which took place between November 1975 and July 1992). The review covered mammographic features, clinical findings, pathological features and nodal status. RESULTS: Twenty (87%) of the lesions were palpable, and 4 (17%) were mammographically occult. At mammography, eight cases (35%) presented as ill defined masses without calcifications, five (22%) as well-defined masses without calcifications, four (17%) as ill-defined masses with calcifications, one (4%) as a focal asymmetric opacity and one (4%) as pleomorphic calcifications only. Eleven (65%) of the 17 mammographically identified masses were irregular in shape, 4 (24%) were oval, and 2 (12%) were lobular. In 11 (61%) of the 18 cases involving masses or an opacity, the lesions were denser than normal breast parenchyma; in the others the lesions were isodense. All of the hyperdense lesions were greater than 1.9 cm in greatest diameter, as determined mammographically, whereas only one of the isodense lesions was greater than 1.9 cm in greatest diameter. In 17 (77%) of the 22 cases in which the border of the lesions could be evaluated histologically, the lesions had well-defined, pushing borders; the others had ill-defined, irregularly outlined borders. Calcifications were seen histologically in seven cases (30%) and were correlated with mammographically suspicious pleomorphic calcifications in three. Fourteen (82%) of the 17 patients who underwent axillary node dissection were free of nodal disease. CONCLUSIONS: The most common mammographic appearance of pure mucinous carcinoma was an ill-defined mass and the second most common was a well-defined mass. No spiculated lesions were observed in this patient group. Microcalcifications were seen in some cases but were less common than in breast cancers in general. Lesions more than 1.9 cm in greatest diameter tended to be hyperdense. A substantial proportion of pure mucinous carcinomas may be mammographically occult. PMID- 7543804 TI - Ultrasonography of jejunal intussusception in children. AB - OBJECTIVE: To describe the ultrasonographic findings in different forms of jejunal intussusception in three children. PATIENTS AND METHOD: Two children with hamartomatous polyps of the jejunum acting as lead points for antegrade jejunoileocolic intussusception and retrograde jejunoduodenogastric intussusception respectively and one child with idiopathic postoperative intussusception were examined by ultrasonography. RESULTS: The findings of the US studies were abnormal and different in each case, depending on the underlying condition and the direction (antegrade or retrograde) of the intussusception. The hamartomatous polyps were seen as hyperechoic solid masses but could not be diagnosed more specifically with US. A target lesion was found in the case of idiopathic postoperative intussusception. The US results prompted the next imaging procedure, air enema in the one patient in whom the intussusception had reached the colon and preoperative barium meal in all of the patients. Surgery was performed without the delay that usually occurs with jejunal intussusception. CONCLUSION: In the appropriate clinical setting, US should be used to look for jejunal intussusception, so that suitable diagnostic gastrointestinal studies can be performed and delay in diagnosis avoided. PMID- 7543803 TI - Osteonecrosis associated with HIV infection. AB - OBJECTIVE: To report cases of osteonecrosis in patients who were HIV positive but had no apparent risk factors for osteonecrosis. PATIENTS AND METHODS: A computer search of records from two university-affiliated teaching hospitals yielded eight cases of osteonecrosis in HIV-positive patients and patients with AIDS. The patients' charts and radiographic records were reviewed to determine possible risk factors for osteonecrosis. RESULTS: In four of the eight patients no definite risk factors for osteonecrosis could be found. CONCLUSION: Osteonecrosis may be a musculoskeletal complication of HIV infection. PMID- 7543807 TI - Malpositioning of intravascular stent during transfemoral repair of transjugular intrahepatic portosystemic shunt: fixation with another intravascular stent. AB - The author describes a 43-year-old man in whom an intravascular stent was dislodged into the inferior vena cava during repair of a transjugular intrahepatic portosystemic shunt. The malpositioned stent was fixed in position by another stent to prevent central embolization. PMID- 7543808 TI - Soft-tissue recurrence of osteosarcoma: ultrasound findings. AB - The authors describe a 26-year-old woman with soft-tissue recurrence of osteosarcoma, which presented as focal soft-tissue swelling after limb salvage surgery and insertion of a metallic endoprosthesis. Because of the clinical presentation, ultrasonography was performed. The characteristic ultrasonographic pattern of soft-tissue tumours is summarized here, and the role and limitations of ultrasonography in this situation are discussed. PMID- 7543809 TI - Expansile subchondral degenerative bone cyst secondary to osteoarthritis. AB - A 53-year-old man known to have osteoarthritis of the left knee was examined because of a soft-tissue swelling overlying the pes anserinus tendon. Plain radiography and computed tomography revealed an expansile subchondral cyst of the proximal tibia. The cyst was multiloculated, had a sclerotic margin and an extraosseous soft-tissue component, and interrupted the cortex. Proof of a degenerative cyst was obtained by open biopsy. Such presentation in the knee is rare. Awareness of the possible aggressive behaviour of such degenerative cysts should help avoid confusion with neoplasm. PMID- 7543811 TI - Residents' corner. Answer to case of the month #32. Metastatic calcification of the lungs. PMID- 7543812 TI - Concomitant use of thioridazine with risperidone. PMID- 7543813 TI - Apoptosis as a mechanism of skin renewal: Le(y)-antigen expression is involved in an early event of a cell's commitment to apoptosis. AB - Skin renewal is a typical example of the active participation of a cell in its own death process. Cells arising from mitotic activity in the stratum germinativum of the epidermis continuously migrate upwards to the stratum corneum, where dead cells are eventually desquamated. Recent studies have suggested that apoptosis is involved in the dynamic process of skin renewal. However, this still remains to be further elucidated. In this paper, we investigated the involvement of apoptosis in the skin renewal process. Changes in the morphology of cells in different epidermal layers were compared with histochemical analyses of the extent of DNA fragmentation, as determined by nick end-labelling, and of the reactivities to a monoclonal antibody directed to Le(y) antigen, difucosylated type 2 chain determinant, which has a close association with apoptosis, and to a monoclonal antibody directed to the proliferating cell nuclear antigen. The results show that apoptosis proceeds concomitantly with cell movement in the epidermis. It seems likely that commitment of a cell to death by apoptosis occurs in the epidermal tissue immediately after completion of cell proliferation, and that Le(y)-antigen expression may be involved in the entire apoptotic process including this early event. PMID- 7543814 TI - Cytokeratin expression in bovine corpora lutea. AB - Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corpora lutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed "zonation" such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts. PMID- 7543810 TI - Lipoma arborescens of the shoulder: magnetic resonance imaging findings. AB - Lipoma arborescens is a rare intra-articular lesion that typically affects the knee. This condition represents villous lipomatous proliferation of the synovial membrane. The magnetic resonance imaging findings are diagnostic and include a frond-like, fatty synovial mass and associated joint effusion. The authors describe a 90-year-old woman with lipoma arborescens of the shoulder. To the authors' knowledge, lipoma arborescens has not previously been reported in this joint. PMID- 7543816 TI - Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents. AB - The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations. PMID- 7543817 TI - Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas. AB - Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas. PMID- 7543818 TI - Murine epidermal growth factor (EGF) fragment (33-42) inhibits both EGF- and laminin-dependent endothelial cell motility and angiogenesis. AB - Laminin, murine epidermal growth factor (mEGF), and the synthetic laminin peptide Lam.B1(925-933) (a linear peptide from the B1 chain of murine laminin, CDPGY1GSR amide) all stimulate endothelial cell motility above basal rates, whereas a synthetic mEGF fragment, mEGF33-42 (a linear peptide from the C-loop of mEGF, acetyl-C-[S-Acm]-VIGYSGDR-C-[S-Acm]-amide), inhibits motility. In both human SK HEP-1 and embryonic chick endothelial cells, mEGF33-42 blocks both EGF- and laminin-stimulated locomotion of endothelial cells. In vivo, mEGF33-42 also blocks both laminin- and mEGF-induced angiogenesis in the chick. In the human cell line. Lam.B1(925-933) has an additive effect in coincubation with either laminin or mEGF, but it blocks their effects in the chick cells. Lam.B1(925-933) alone stimulates angiogenesis in the chick but blocks laminin-induced angiogenesis. Thus, mEGF33-42 acts as a general laminin antagonist, whereas Lam.B1(925-933) acts as a laminin agonist in human cells, but in chick cells it acts as a partial antagonist. We propose that the presence of an anionic group at the eighth residue of mEGF33-42 may be the source of the antagonistic effects seen with this peptide as compared with the laminin fragment. These findings have important implications in the design of human antiangiogenic agents, and also in the use of chick models in the study of human disease. PMID- 7543819 TI - Suppressed transformation and induced differentiation of HER-2/neu-overexpressing breast cancer cells by emodin. AB - The amplification and overexpression of the HER-2/neu proto-oncogene, which encodes the tyrosine kinase receptor p185neu, have been observed frequently in tumors from human breast cancer patients and are correlated with poor prognosis. To explore the potential of chemotherapy directed at the tyrosine kinase of p185neu, we have found that emodin (3-methyl-1,6,8-trihydroxyanthraquinone), a tyrosine kinase inhibitor, suppresses autophosphorylation and transphosphorylation activities of HER-2/neu tyrosine kinase, resulting in tyrosine hypophosphorylation of p185neu in HER-2/neu-overexpressing breast cancer cells. Emodin, at a 40-microM concentration, which repressed tyrosine kinase of p185neu, efficiently inhibited both anchorage-dependent and anchorage-independent growth of HER-2/neu-overexpressing breast cancer cells. However, the inhibition was much less effective for those cells expressing basal levels of p185neu under the same conditions. Emodin also induced differentiation of HER-2/neu overexpressing breast cancer cells by exhibiting a morphological maturation property of large lacy nuclei surrounded by sizable flat cytoplasm and by showing a measurable production of large lipid droplets, which is a marker of mature breast cells. Therefore, our results indicate that emodin inhibits HER-2/neu tyrosine kinase activity and preferentially suppresses growth and induces differentiation of HER-2/neu-overexpressing cancer cells. These results may have chemotherapeutic implications for using emodin to target HER-2/neu-overexpressing cancer cells. PMID- 7543815 TI - Visualization of silver-enhanced reaction products from protein-and immuno colloidal gold probes by laser scanning confocal microscopy in reflection mode. AB - We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold labelled antibody to mouse immunoglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markers will probably be of considerable advantage in cytochemical analysis. PMID- 7543820 TI - Functional hyaluronan receptors are expressed on a squamous cell lung carcinoma cell line but not on other lung carcinoma cell lines. AB - We investigated the production of hyaluronan and the presence of hyaluronan receptors in a panel of human lung carcinoma cell lines, consisting of small cell carcinomas (SCLC) and non-small cell carcinomas (non-SCLC). These transformed cell lines produced only minute amounts of hyaluronan, whereas normal lung fibroblasts synthesized high amounts. CD44 molecules (an integral membrane glycoprotein suggested previously to function as a hyaluronan receptor) were differentially expressed on non-SCLC cell lines but essentially not on the SCLC cell lines. In contrast, RHAMM molecules (receptor for hyaluronan-mediated motility) were preferentially expressed on SCLC cells. Although all the lung tumor cell lines expressed various amounts of CD44 and RHAMM, only the SCLC line U-1752 could bind [3H]hyaluronan. The binding sites were saturated with about 19,700 hyaluronan molecules (Mr 1.4 x 10(6)) bound per cell with a Kd of 0.16 x 10(-9) M. CD44 molecules were responsible for the binding activity since Hermes-1 antibodies that block the binding of hyaluronan to CD44 blocked the binding of [3H]hyaluronan to U-1752 cells. 4-Phorbol 12-myristate 13-acetate (PMA) treatment of U-1752 cells both increased the hyaluronan-binding activity in U-1752 cells as well as induced abrogation of cell-cell and cell-matrix interactions. Addition of hyaluronan inhibited the PMA-induced disassembly of the cells. The fact that CD44 molecules are able to bind [3H]hyaluronan only on the SCLC line U-1752 but not on other lung carcinoma cell lines may be of value as a marker for squamous cell carcinoma differentiation. Furthermore, the inhibitory effect of hyaluronan on the PMA-promoted cell disassembly suggest that hyaluronan surrounding squamous cell carcinoma cells may affect their migration and invasiveness. PMID- 7543821 TI - Interferons up-regulate with different potency HLA class I antigen expression in M14 human melanoma cell line. Possible interaction with glucocorticoid hormones. AB - The relative potency of interferon alpha (IFN alpha), interferon beta (IFN beta), and interferon gamma (IFN gamma) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFN gamma > IFN beta > IFN alpha) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expression, (c) glucocorticoid hormones, when associated with IFN alpha or IFN gamma, inhibit the HLA class I enhancement induced by IFN alone, and, finally, (c) the association between 1 microM prednisone or 1 microM deflazacort and IFN beta seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma. PMID- 7543824 TI - Studies on antiulcer agents. II. Antiulcer properties of N-(1H-tetrazol-5-yl)-2 anilino-5-pyrimidinecarboxamides inhibiting release of histamine from passively sensitized rat peritoneal mast cells. AB - With the aim of applying mast cell-stabilizing agents as antiulcer agents, N-(1H tetrazol-5-yl)-2-anilino-5-pyrimidinecarboxamides were synthesized, and initially evaluated pharmacologically for activity in the rat passive cutaneous anaphylaxis test by oral administration. The most active compound 6 was proved to inhibit potently the release of histamine from passively sensitized rat peritoneal mast cells in vitro. When compared with other mast cell-stabilizing agents and an antiulcer agent, compound 6 was found to show excellent gastric mucosal protection and gastric antisecretion activities. Furthermore, compound 6 revealed good activity against acidified aspirin ulcer in rats and water-immersion stress ulcer in rats. PMID- 7543822 TI - In vitro and in vivo antitumor activity of a chimeric anti-CD19 antibody. AB - Mouse monoclonal antibodies to CD19 detect an antigenic determinant expressed exclusively on the surface of B lymphocytes, and have previously been shown to be potentially useful therapeutic reagents for human B cell lymphoma. We report the production and characterization of a mouse/human chimeric antibody, cCD19, with potent in vivo antitumour activity. The genes encoding the variable domains for heavy (VH) and light (VL) chains were subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and kappa), and co transfected into non-secreting Sp2/0 mouse myeloma cells. Intraperitoneal administration of cCD19 produced inhibition of growth of subcutaneous CD19+ Sultan human B lymphoma tumours in scid/scid mice. When the antibody was administered 18 and 20 days after subcutaneous tumour inoculation, an approximately 30% reduction in tumour size was noted by day 29. cCD19 faithfully mimicked the in vitro binding characteristics of mCD19 as (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed CD19, (b) cCD19 was able to inhibit the binding of mCD19 on CD19+ cells completely and (c) the affinity of binding of the two antibodies was not significantly different [Ka = (2.03 +/- 1.5) x 10(8)]. In bio-distribution studies, up to 14.8% of the total injected antibody dose per gram of tissue was localized in CD19+ Sultan tumours at 24 h approximately, 14.4% was present in the tumors at 48 h, and about 13.7% at 72 h. These levels were comparable to mCD19 administered in the same fashion. cCD19 conjugated to idarubicin was specifically and strongly cytotoxic to CD19+ cells cultured in vitro, and demonstrated an IC50 of 0.17 microM, similar to that of mCD19 (0.32 microM) and approximately 14-fold greater than the IC50 of free idarubicin. The specific cytotoxic capacity of cCD19 and its likely reduced immunogenicity suggest that it may potentially be of use in the treatment of refractory B cell lymphoma in humans. PMID- 7543827 TI - Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. AB - To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543825 TI - Contractile responsiveness of ventricular myocytes to isoproterenol is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture. AB - Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543826 TI - Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to beta adrenergic agonists. AB - Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10(-7) mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543823 TI - Inhibitory effects of HgCl2 on excitation-secretion coupling at the motor nerve terminal and excitation-contraction coupling in the muscle cell. AB - 1. Indirect and direct twitch (0.1-Hz) stimulation of the rat phrenic nerve diaphragm disclosed that the inhibitory effect of HgCl2, 3.7 x 10(-5) M, on the neuromuscular transmission and in the muscle cell, was accelerated by 10-sec periods of 50-Hz tetanic stimulation every 10 min. This activity-dependent enhancement suggested an inhibitory mechanism of HgCl2 related to the development of fatigue, like membrane depolarization or decreased excitability, decreased availability of transmitter, or interference with the factors controlling excitation-secretion coupling of the nerve terminal, i.e. (Ca2+)0 or (Ca2+)i, and excitation-contraction coupling in the muscle cell, i.e., (Ca2+)i. 2. During both indirect and direct stimulation, HgCl2-induced inhibition was enhanced markedly by pretreatment with caffeine, which releases Ca2+ from endoplasmic and sarcoplasmic reticulum in the nerve terminal and muscle cell, respectively. This caffeine-induced enhancement was completely antagonized by dantrolene, which inhibits the caffeine-induced release. However, dantrolene alone did not antagonize the HgCl2-induced inhibition. 3. Since caffeine depletes the intracellular Ca2+ stores of the smooth endoplasmic reticulum, HgCl2 probably inhibits by binding to SH groups of transport proteins conveying the messenger function of (Ca2+)i. In the muscle cell this leads to inhibition of contraction. In the nerve terminal, an additional enhancement of the HgCl2-induced inhibition, by inhibiting reuptake of choline by TEA and tetanic stimulation, suggested that HgCl2 inhibited a (Ca2+)i signal necessary for this limiting factor in resynthesis of acetylcholine. 4. The (Ca2+)0 signal necessary for stimulus induced release of acetylcholine was not affected by HgCl2. Hyperpolarization in K(+)-free solution antagonized the inhibitory effect of HgCl2 at indirect stimulation, and Ca(2+)-free solution enhanced the inhibitory effect at direct stimulation. K+ depolarization, membrane electric field increase with high Ca2+, membrane stabilization with lidocaine, and half-threshold stimulation, did not change the inhibitory effect of HgCl CH3HgCl. 1.85 x 10(-5) M, disclosed a synergistic interaction with caffeine during direct, but not during indirect, stimulation. PMID- 7543828 TI - Evidence that expression of inducible nitric oxide synthase in response to endotoxin is augmented in atherosclerotic rabbits. AB - Atherosclerotic lesions contain monocytes/macrophages and vascular smooth muscle cells and thus may have an increased capacity for generation of nitric oxide by inducible nitric oxide synthase (NOS). We used three approaches (contractile responses, generation of L-citrulline from L-arginine, and staining with NADPH diaphorase) to test the hypothesis that after administration of lipopolysaccharide (LPS) in vivo, generation of nitric oxide by inducible NOS is augmented in atherosclerotic arteries. New Zealand White (normal, n = 18) and Watanabe heritable hyperlipidemic (atherosclerotic, n = 21) rabbits were anesthetized and injected intravenously with vehicle or LPS. Contractile responsiveness of aortic segments was examined in vitro 4 hours after injection of LPS in vivo. There was a substantial (approximately fivefold) decrease in contractile sensitivity of aortas from LPS-treated atherosclerotic rabbits and a small (approximately twofold) decrease in normal rabbits. Incubation of aortic segments with aminoguanidine, which inhibits inducible NOS, restored contractile responsiveness after LPS treatment. In vitro assay of conversion of [14C]L arginine to [14C]L-citrulline by aortic segments demonstrated marked (approximately fivefold) increase in calcium-independent conversion of [14C]L arginine by LPS-treated atherosclerotic, but not normal, aortas. NADPH-diaphorase staining demonstrated positive cells only in the endothelium of normal rabbits and in the lesions and media of the atherosclerotic aortas in both vehicle- and LPS-treated rabbits. The general distribution of these NADPH-diaphorase-positive cells resembled that of smooth muscle cells and not macrophages. Thus, impairment of contractile responses, generation of L-citrulline, and staining with NADPH diaphorase suggest that atherosclerotic arteries have increased capacity for generation of nitric oxide by inducible NOS. PMID- 7543829 TI - Nonreentrant mechanisms underlying spontaneous ventricular arrhythmias in a model of nonischemic heart failure in rabbits. AB - BACKGROUND: The goal of this study was to define the mechanisms of spontaneously occurring ventricular arrhythmias in the setting of nonischemic heart failure. METHODS AND RESULTS: Three-dimensional cardiac mapping from 232 intramural sites was performed in four rabbits with heart failure induced by combined aortic regurgitation and aortic stenosis and in four control rabbits. During the development of heart failure, serial echocardiographic examination demonstrated a progressive increase in left ventricular (LV) chamber dimensions and a decrease in LV systolic function over 19 +/- 2 months. Serial Holter monitoring demonstrated spontaneously occurring premature ventricular complexes (PVCs) (up to 13,000 per day) and couplets in all four rabbits with heart failure, and runs of nonsustained ventricular tachycardia (VT) up to 26 beats long in three. Mapping of spontaneous rhythm was performed for up to 60 minutes. None of the control rabbits demonstrated spontaneous arrhythmias during mapping. Three rabbits with heart failure demonstrated isolated PVCs, and two demonstrated couplets and runs of nonsustained VT up to 4 beats long. The three-dimensional activation sequence of 50 sinus beats (42 from rabbits with heart failure; 8 from control rabbits), 19 PVCs, and 37 beats of couplets and nonsustained VT was determined and the mechanism of arrhythmia defined for all ventricular ectopic beats analyzed. Normal sinus beats from the failing rabbits activated rapidly, with a total activation time of 28 +/- 1 ms (P = .18 versus sinus beats from control hearts, 26 +/- 1 ms). Sinus beats preceding PVCs in the rabbits with heart failure activated in a similar fashion, with a total activation time of 26 +/- 1 ms. In each case, these PVCs initiated in the subendocardium by a nonreentrant mechanism based on the absence of intervening electrical activity between the termination of the preceding beat and the initiation of the next (225 +/- 7 ms), despite the presence of multiple intervening electrode recording sites. Couplets and monomorphic and polymorphic VTs were due to repetitive nonreentrant activation at the same or different subendocardial sites. Total activation time of beats of VT averaged 44 +/- 1 ms and did not differ from that of isolated PVCs (43 +/- 2 ms, P = .65). Pathological analysis of tissue demonstrated myocardial fiber hypertrophy, degenerative changes, and interstitial fibrosis throughout the failing hearts. CONCLUSIONS: Spontaneously occurring PVCs, couplets, and VT in a model of nonischemic heart failure are due to nonreentrant mechanisms such as triggered activity or abnormal automaticity. Approaches to the treatment of spontaneously occurring ventricular arrhythmias in patients with nonischemic heart failure should be directed at nonreentrant mechanisms. PMID- 7543830 TI - Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase. PMID- 7543831 TI - Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. AB - BACKGROUND: In animal models of myocardial infarction (MI), inflammatory responses compromise microcirculation during reperfusion and restrict functional recovery. To investigate cardiac inflammatory responses in patients with acute MI, we examined the cardiac release of cytokines, the expression on neutrophils of the beta 2-integrin Mac-1 (CD11b/CD18) and L-selectin (CD62L), and the cardiac release of thrombomodulin as a marker of endothelial injury. METHODS AND RESULTS: In 12 patients with acute anterior MI, blood samples were obtained from the coronary sinus and from the aorta immediately before and after recanalization of the coronary occlusion by balloon angioplasty. Twelve patients undergoing elective balloon angioplasty served as control subjects. Plasma concentrations of interleukin (IL)-1 beta, IL-6, IL-8, tumor necrosis factor-alpha, and thrombomodulin were determined by immunoassay, and surface expression of CD11b and CD62L was assessed by flow cytometry. Differences in coronary sinus and arterial blood were found in IL-6 before (median, 6.3 ng/L, P = .01) and after (13.4 ng/L, P = .002) recanalization and in IL-8 after recanalization (10.7 ng/L, P = .02). The cardiac release of both cytokines significantly (P < or = .03) increased with reperfusion. Cytokine release after reperfusion was associated with significant transcardiac gradients in surface expression on neutrophils of CD11b (10.1 mean channel of fluorescence intensity [mean fl], P = .01) and CD62L (-87 mean fl, P = .007) and with a thrombomodulin release (4.5 micrograms/L, P = .004). Transcardiac gradients in IL-1 beta and tumor necrosis factor-alpha were not found. None of the changes found in MI were detectable in the control group. CONCLUSIONS: As evidence of cardiac inflammatory responses in reperfused acute MI, the study demonstrates cardiac neutrophil activation with signs of endothelial injury and a release of the proinflammatory cytokines IL-8 and IL-6. These findings may assist in the design of pharmacological interventions aimed at reducing microvascular reperfusion injury. PMID- 7543832 TI - Is IGF binding protein-3 assessment helpful for the diagnosis of GH deficiency? AB - OBJECTIVE: The measurement of serum immunoreactive IGFBP-3 levels has been proposed as a screening test to identify children with growth hormone deficiency (GHD). We tested the sensitivity and specificity of the IGFBP-3 assessment in comparison with the measurement of IGF-I. DESIGN: We assessed the IGFBP-3 and IGF I circulating levels in normal subjects and patients with GHD or idiopathic short stature (ISS). PATIENTS: Eighty-two normal subjects, 16 GHD, and 10 children with ISS were studied. Controls were divided into three age groups: group A, 1-4 years (n = 16); group B, 5-9 years (n = 35), and group C, 10-14 years (n = 31). MEASUREMENTS: All subjects underwent standard anthropometry. In short patients, GH secretory status was assessed by clonidine and arginine stimulation tests. IGFBP-3 and IGF-I circulating levels were measured by radioimmunoassay. RESULTS: IGFBP-3 and IGF-I levels were closely related (r = 0.51, P < 0.0001) and IGFBP-3 was less age dependent than IGF-I (r = 0.57, P < 0.02 vs r = 0.64, P = 0.0001). Sensitivity (true positive ratio) and specificity (true negative ratio) of IGFBP 3 measurement were 50 and 92% respectively, whereas sensitivity and specificity of IGF-I assessment were 75 and 90% respectively. Below the age of 5 years, sensitivity was 20% for IGFBP-3 and 40% for IGF-I; specificity was 94% for IGFBP 3 and 88% for IGF-I. CONCLUSIONS: IGFBP-3 measurement had poor sensitivity in detecting growth hormone deficient patients, offering no diagnostic advantage over IGF-I, even in the first years of life, although, due to the high specificity, the finding of subnormal levels of IGFBP-3 was strongly suggestive of growth hormone deficiency. The presence of low IGFBP-3 and IGF-I levels in a short child with normal GH response to provocative tests should prompt further investigations, such as the determination of spontaneous GH secretion or assessment of the GH binding proteins together with an IGF-I and/or IGFBP-3 generation test, in order to identify neurosecretory dysfunction or GH receptor deficiency. Finally, we believe that there is no definitive test for diagnosing or excluding growth hormone deficiency and detailed analysis of the results of endocrine tests, clinical findings and other laboratory and radiological information is necessary to maximize diagnostic accuracy. PMID- 7543833 TI - Characterization of human larynx carcinoma cell lines HLaC'79 and HLaC'82: a common origin but diverged malignancies. AB - As part of a study on the relationship of tumour phenotype and behaviour, we have characterized two head and neck squamous cell carcinoma cell lines, derived from human laryngeal carcinomas and designated HLaC'79 and HLaC'82. Cytogenetic analysis revealed that HLaC'79 and HLaC'82 shared 10 major chromosome rearrangements indicating that the cell lines had a common origin. In the extremely complex chromosomal patterns, abnormalities were found in chromosomes 1, 3 (surplus 3q) and 5 (i(5p) x 2). Both cell lines displayed constitutive expression of vimentin and were capable of anchorage-independent growth in agarose gels. However, in spite of their common origin specific differences were found. Cells of HLaC'79 were spindle shaped and formed tumours in athymic mice. In contrast, cells of HLaC'82 had a compact morphology, contained less vimentin, were more contact inhibited and were not tumorigenic. These results indicate that malignant transformation in HLaC'82 was partially reversed. PMID- 7543834 TI - Cloning and identification of genes differentially expressed in metastatic and non-metastatic rat rhabdomyosarcoma cell lines. AB - The objective of this study was to identify genes involved in invasion and metastasis using a rat rhabdomyosarcoma model (SMF-A and RMS-B cell lines). The SMF-A cell line was established from a metastatic nodule of an induced rhabdomyosarcoma in syngeneic F344 rats. Two cell lines with defined metastatic potentials, SMF-Ai and SMF-Da, were cloned from the SMF-A line. The cell line SMF Ai is tumorigenic, highly invasive and highly metastatic. On the other hand, the revertant line SMF-Da is less tumorigenic, non-invasive and non-metastatic. We have isolated from a SMF-Ai cDNA library eight cDNA clones which are differentially expressed by the metastatic SMF-Ai and the non-metastatic SMF-Da cell line using Northern blot analysis. Five of these clones, smf-4, smf-6, smf 41, smf-42 and smf-44, are overexpressed in the SMF-Da cell line and have homology with beta-2-microglobulin, lactate dehydrogenase, ribosomal protein L38, ribosomal protein S4 and acidic ribosomal phosphoprotein P1, respectively. The three other clones, smf-7, smf-40 and smf-61, are overexpressed in SMF-Ai. Clones smf-40 and smf-61 show significant homology with the human TB3-1 gene and the human fus gene respectively. The clone smf-7 has no significant homology with known sequences. We also analyzed the expression of these clones in other rat rhabdomyosarcoma cell lines (RMS-B and their clones) and in tumors obtained by injection of these cell lines into rats or nude mice. Smf-61 and smf-7 were the only clones with a differential expression pattern associated with the invasive or metastatic potential of all cell lines examined. A preliminary study of the expression of smf-7 and smf-61 in other cancer cell lines also showed mRNA expression in two human rhabdomyosarcomas and a human epidermoid carcinoma suggesting the existence of genes homologous to smf-7 and smf-61 clones in human cancers. Our findings suggest an association between the expression of smf-7 and smf-61 and invasive or metastatic potential of rhabdomyosarcoma cells. PMID- 7543835 TI - Retroviral expression in a patient who has paraarticular ossification. AB - Reverse transcriptase activity is reported in the mononuclear blood cells isolated from a patient in whom paraarticular ossification developed after surgery for an aneurysmal bone cyst. The enzyme was purified to apparent homogeneity by chromatography before being characterized biochemically for its template specificity and ionic requirement. The enzyme was able to transcribe poly(rA).(dT)12-18 very efficiently in the presence of Mn++ ions. Viral particles were observed in the HuT-78 cell line, cocultured with the mononuclear cells of the patient. No viral particles were observed in HuT-78 cells before the coculture. The patient was found seronegative for HIV-1, HIV-2, and HTLV-1. These results suggest that a new retrovirus infecting mononuclear blood cells may be involved in the development of ectopic ossification. This hypothesis is strengthened by the previous finding of a retrovirus in the mononuclear blood cells of a patient with benign osteopetrosis, and by the fact that HTLV-1 infected T-lymphocytes acquire the ability to secrete factors responsible for the lytic bone lesions observed in the patients. A family of human bone diseases that reflect T-cell dysfunction(s) and are caused by lymphotropic viruses may exist. PMID- 7543836 TI - Pericyte differentiation. AB - Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo. PMID- 7543837 TI - Adhesion molecule expression on peripheral blood mononuclear cells in rheumatoid arthritis: positive correlation between the proportion of L-selectin and disease activity. AB - The expression of a series of adhesion molecules (the integrin family, the immunoglobulin superfamily, CD44 and the selectin family) on peripheral blood mononuclear cells (PBMC) from RA patients was investigated by flow-cytometry. L Selectin (CD62L) in the selectin family was more significantly expressed on PBMC from RA patients as compared to those from normal controls. Further, RA patients exhibited a slight but significant increase of VLA-alpha 2 (CDw49b), p150,95 (CD11c), and VNR-beta (CD61) in the integrin family. The enhanced expression of L selectin was positively correlated with disease activity. An increase in the proportion of L-selectin+CD4+ cells in PBMC from patients with RA and its clinical significance is reported. PMID- 7543839 TI - [Therapy of peripheral facial nerve paralysis and hearing loss]. PMID- 7543838 TI - [Venous angioplasty and Wallstent implantation in emergency treatment of superior vena cava syndrome caused by tumor]. AB - HISTORY AND CLINICAL FINDINGS: A 58-year-old man was hospitalized because of threatened asphyxia resulting from massive obstruction to neck vein flow and increased venous markings over the ventral and dorsal thorax. The previous year he had received radiotherapy (total of 55.8 Gy) for squamous cell carcinoma in the right upper lobe of the lung (primary stage T2 N2 M0). The signs of venous congestion had developed over several months. TESTS: Emergency phlebography demonstrated obstruction to superior vena cava (SVC) flow by tumour compression. TREATMENT AND COURSE: It was possible to pass the SVC obstruction with a hydrophilic guide-wire and then perform a balloon angioplasty, followed later by implantation of two intraluminal expandable stents ("Wallstent"). The dyspnoea quickly improved after the successful recanalization. The day after the intervention palliative radiotherapy of the SVC obstruction was begun. But increased venous markings over the thorax recurred, but without dyspnoea, after 4 1/2 months free of signs of venous congestion. Repeat phlebography again demonstrated complete obstruction of the SVC by the tumour. Recanalization by balloon angioplasty was again achieved. CONCLUSIONS: This case underlines the value of percutaneous balloon angioplasty with stent implantation as supplementary treatment in the late stages of SVC obstruction by tumour. PMID- 7543841 TI - Aprotinin. A review of its pharmacology and therapeutic efficacy in reducing blood loss associated with cardiac surgery. AB - Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) experience transient haemostatic defects as a result of adverse changes to their blood components, blood cells and specific coagulation proteins. Aprotinin is a naturally occurring serine protease inhibitor isolated from bovine lung tissue which inhibits kallikrein and plasmin. A high dose aprotinin regimen (aprotinin 280mg loading dose over 20 to 30 minutes after anaesthesia induction followed by 70 mg/h for the duration of the operation and 280mg added to the priming fluid of the CPB circuit) has been used during CPB in order to reduce perioperative bleeding. Recent clinical trials confirm the efficacy of high dose aprotinin in reducing blood loss and transfusion requirements associated with primary cardiac procedures such as coronary artery bypass graft (CABG) or heart valve replacement surgery. High dose aprotinin is also effective in procedures known to possess a high risk for excessive blood loss, such as repeat CABG or heart valve replacement surgery, cardiac surgery in patients with infective endocarditis, or in patients receiving aspirin (acetylsalicylic acid) before surgery. Studies indicate that low dose aprotinin (280mg added to CPB pump prime fluid) is effective in reducing blood loss and transfusion requirements in patients undergoing primary CABG surgery. Additionally, low dose aprotinin regimens (both 280mg added to CPB pump prime fluid and 50% of the high dose regimen) have shown some benefit in repeat CABG surgery; however, more studies are needed to confirm these results. Data from clinical trials indicate that aprotinin is well tolerated. The types and incidences of adverse events reported with aprotinin therapy are generally consistent with those associated with major cardiac surgery, and are not significantly different from those observed in control groups. A trend towards lower graft patency rates, detected by ultrafast computerised tomography (CT), has been observed in aprotinin recipients in 2 US trials. These differences did not reach statistical significance and should be interpreted with caution since the ability of ultrafast CT to determine graft patency has not been validated. Mildly elevated plasma creatinine levels are more commonly observed in aprotinin-treated patients; these changes are transient in the majority of patients. Both high dose and low dose aprotinin regimens (280mg added to CPB pump prime fluid or 50% of the high dose regimen) have reduced blood loss and transfusion requirements in patients undergoing primary and repeat cardiac surgery. The role of aprotinin in paediatric cardiac surgery needs further clarification, while well-designed studies comparing aprotinin with other agents which inhibit fibrinolysis are also awaited with interest.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543840 TI - The optimal management of hairy cell leukaemia. AB - Hairy cell leukaemia is an uncommon B cell chronic lymphoproliferative disorder characterised by circulating lymphocytes displaying prominent cytoplasmic projections. Therapy is initiated for severe cytopenias or recurrent infections. Splenectomy, the first standard treatment, is now less commonly used as primary treatment. Interferon-alpha (IFN alpha) induces partial responses in most patients but complete responses in only a few. Adverse effects from IFN alpha are common but not life-threatening. The ability of two newer purine analogues, pentostatin (2'-deoxycoformycin) and cladribine (2-chlorodeoxyadenosine), to induce long-lasting complete remissions in the majority of patients has revolutionised the treatment of this disease. Cladribine is emerging as the treatment of choice because of its favourable toxicity profile, brief duration of treatment, high percentage of unmaintained complete remissions and low incidence of relapse. PMID- 7543843 TI - The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity. AB - The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF-4B. We have added six histidine residues to the C-terminus of Tif3 protein (Tif3-His6p) and purified the tagged protein by affinity chromatography. Tif3-His6p stimulates translation and mRNA binding to ribosomes in a Tif3-dependent in vitro system. Furthermore, it binds to single stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro. In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF-4B. A role for Tif3/eIF-4B and RNA annealing activity in the scanning process is proposed. PMID- 7543845 TI - Effect of an equal volume replacement with 500 mL 6% hydroxyethyl starch on the blood and plasma volume of healthy volunteers. AB - The effect of an equal volume replacement with 6% low molecular weight (200,000) hydroxyethyl starch (substitution ratio 0.60-0.66) (Elohaes) on the blood volume and plasma volume of eight healthy volunteers was investigated with special attention to volume overshooting. After replacement of 500 mL whole blood, blood volume was measured repeatedly for 8 1/2 h using chromium-51 labelled erythrocytes. Red cell volume and plasma volume were calculated from standard formulae. After replacement, blood and plasma volumes were unchanged from control (4754 +/- 630 mL and 2900 +/- 450 mL, respectively). The haematocrit decreased from 43.7 +/- 2.5% to 39.0 +/- 1.4% (1 h after starch infusion) and 41.1 +/- 1.9% (7.5 h after starch infusion). There were no significant changes in haemodynamic variables (systolic and diastolic blood pressure; heart rate), liver function (bilirubin; transaminases), renal function (serum creatinine; serum sodium) or coagulation (activated partial thromboplastin time). Alpha-amylase was significantly increased, which is a well known effect of starches. The replacement of 500 mL whole blood by hydroxyethyl starch maintains isovolaemia for at least 8 h in normal volunteers without any significant volume overshooting; this is not what was seen in earlier studies. This should increase safety in patients with cardiac disease. PMID- 7543846 TI - The acrosome reaction in digitonin-permeabilized sea urchin sperm in the absence of the natural inducer. AB - In many species, the acrosome reaction of sperm is an obligatory step in fertilization. Increases in [Ca2+]i and pHi, activation of adenylyl cyclase and inositol trisphosphate generation accompany the egg jelly-induced acrosome reaction of sea urchin sperm. The signaling mechanisms involved are unknown. We used digitonin, a cholesterol-complexing compound, to selectively permeabilize the plasma membrane of sea urchin sperm suspended in a medium that mimics the cytosolic ion composition. Within 6 to 8 min, 30 to 50 microM digitonin allowed incorporation of the membrane-impermeant dye Hoechst 33258 into the sperm, staining exclusively the nucleus. No alterations in sperm morphology were caused by digitonin at the concentrations used, however, it irreversibly permeabilized the plasma membrane. Permeabilized sperm retained lactate dehydrogenase and actin. When incubated in Ca(2+)-containing permeabilization buffer (pH 7.8), sperm were capable of undergoing spontaneously the acrosome reaction; this reaction was pH dependent and displayed an absolute Ca2+ requirement. Electron microscopy indicates that the acrosome reaction undergone by permeabilized sperm resembled that induced by egg jelly. Additionally, rhodaminyl-phalloidin staining of sperm reacted under permeabilizing conditions revealed a fluorescent filament in the acrosomal tubule region, demonstrating the occurrence of actin polymerization. Thus, in permeabilized sperm the machinery necessary to perform a [Ca2+]i- and pHi-sensitive acrosome reaction is functionally preserved. Permeabilized sperm offer new avenues to study the molecular bases of the sea urchin sperm acrosome reaction. PMID- 7543848 TI - Overexpression of insulin-like growth factor binding protein-3 by senescent human fibroblasts: attenuation of the mitogenic response to IGF-I. AB - We have previously demonstrated that senescent human diploid fibroblasts (HDF) produce large amounts of IGF-binding protein-3 (IGFBP-3) in comparison to early passage vigorously proliferative HDF. In order to determine whether this excess IGFBP-3 accumulation plays a role in the observed attenuation of DNA synthesis in senescent HDF, we examined the response of these cells to IGF-I and two IGF-I functional analogs, [QAYL]IGF-I and insulin, both of which have extremely low binding affinity for IGFBP-3 but which exert their mitogenic effect via the IGF-I plasma membrane receptor. Senescent HDF showed an increased sensitivity of DNA synthetic response to [QAYL]IGF-I and insulin compared to IGF-I. IGF binding activity was significantly higher in conditioned medium of senescent HDF than the medium of young HDF, and virtually all of this enhanced binding capacity could be accounted for by IGFBP-3. Addition of recombinant IGFBP-3 to young cells at a constant molar ratio of 1:1 with respect to IGF-I significantly attenuated the response to IGF-I and abolished the response at a 2:1 molar ratio. These data indicate that IGFBP-3 accumulated in medium of senescent HDF can bind and sequester IGFs when present in molar excess and thereby account for a significant part of the attenuated response of senescent HDF to IGF-I. PMID- 7543844 TI - Use of multicolour chromosome painting to identify chromosomal rearrangements in human lymphocytes exposed to bleomycin: a comparison with conventional cytogenetic analysis of Giemsa-stained chromosomes. AB - Exchange aberrations induced by bleomycin were identified by multicolour fluorescence in situ hybridisation (FISH) with probes for chromosomes 1, 2, and 3. The frequency and distribution of aberration types were compared to conventional metaphase analysis of Giemsa-stained chromosomes from the same human lymphocyte cultures. The total percentage of exchanges detectable by painting three pairs of chromosomes with separate colours was calculated as 40%. Giemsa staining revealed predominantly asymmetric chromosome exchanges, which are expected to comprise 50% of the total induced exchanges. Genomic exchange frequencies were, therefore, determined by multiplying the observed frequencies from FISH analysis by 2.5 and the number of asymmetric exchanges identified in Giemsa-stained slides by 2.0. By these calculations, the genomic exchange frequency calculated from chromosome painting exceeded that estimated by Giemsa staining. This difference was due to the identification by chromosome painting of a unique class of cells in which chromosomes had undergone complex exchanges (nonreciprocal exchanges involving multiple mutual sites). The percentage of cells exhibiting exchanges was similar for both methods. PMID- 7543847 TI - Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells. AB - Microtubules are critically involved in membrane traffic and maintenance of epithelial cell polarity. In this study we examined the effect of microtubule disruption by colchicine on 1) the subcellular organization of the apical endocytic apparatus, 2) apical endocytosis, and 3) subcellular distribution of gp330 and Aquaporin-1 water channels in renal proximal tubule cells. Rats were treated in vivo with colchicine for 5 h before fixation of the kidneys, and sections of proximal tubules were studied using electron microscopy, morphometry and immunocytochemistry. In proximal tubule cells from colchicine-treated animals, virtually no endocytic invaginations are present, indicating that colchicine blocks the formation of endocytic invaginations. Furthermore, no large endocytic vacuoles are present, also consistent with a block of endocytosis. This was confirmed by functional studies revealing a colchicine-induced arrest in apical endocytosis of peroxidase. There is a marked reduction in dense apical tubules (the exocytic vehicle for membrane recycling) but an extensive accumulation of small vesicles, suggesting a disruption in membrane recycling. This disruption may be primary or secondary to the block of endocytosis. Immunofluorescence and immunoelectron microscopy reveal extensive colchicine induced changes in the subcellular distribution of gp330 and Aquaporin-1. The localization of gp330 is normally restricted to the apical endocytic apparatus. However, after colchicine treatment gp330 is localized to numerous vesicles distributed throughout the entire cytoplasm, as previously shown (Gutmann et al., Am. J. Physiol. 257, C397-C407 (1989). Also Aquaporin-1 water channels, which are normally almost exclusively present in the plasma membranes, are redistributed to small vesicles in addition to the plasma membrane localization after colchicine treatment. In summary, the present study suggests that cytoplasmic microtubules are critically involved in 1) formation and stabilization of endocytic invaginations, 2) formation of large endocytic vacuoles, 3) apical endocytosis, 4) maintaining the polarized distribution of vesicles in the apical part of the cell, and 5) maintaining the polarized organization of gp330 in the apical endocytic apparatus, and maintaining Aquaporin-1 water channels in the plasma membranes. PMID- 7543842 TI - Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. AB - Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions. PMID- 7543849 TI - Beta 1 integrins do not have a major role in keratinocyte intercellular adhesion. AB - Integrins of the epidermis have been implicated both in intercellular adhesion and in cell-substratum adhesion. In the present study the role of alpha 2 beta 1 and alpha 3 beta 1 integrins has been evaluated further using human keratinocyte culture. alpha 3 beta 1 but not alpha 2 beta 1 strongly colocalizes with talin in adhesion plaques, consistent with a role for the former in adhesion to endogenous matrix. Upon elevation of the extracellular Ca2+ concentration from 30 microM to 1.0 mM, which is known to induce the organization of intercellular junctions, all three integrin subunits redistribute to concentrate along the cell-cell borders, but alpha 3 redistributes more slowly. Blocking antibody to E-cadherin, which has previously been shown to delay the establishment of cell-cell adhesion upon Ca2+ elevation, delays the redistribution of alpha 2 beta 1 and alpha 3 beta 1 integrins. Elevation of the Ca2+ concentration also induces a rapid morphological change in the keratinocytes and organization of the culture into colonies with tight cell-cell connections. Blocking antibodies to beta 1 or to alpha 3, but not to alpha 2, delays this morphological change and the organization into colonies; however, the effect is much more pronounced in subconfluent cultures. These data are consistent with the hypothesis that anti-beta 1 or anti-alpha 3 antibodies affect cell-cell interactions primarily through their previously described inhibition of motility. Stratification of the culture, which follows the formation of intercellular interactions, is normal in the presence of blocking antibody to beta 1 integrin. In summary, these data suggest that integrins do not play a major role in intercellular keratinocyte adhesion, but may appear to do so under certain conditions because of their involvement in motility. PMID- 7543850 TI - Heat stress or rotavirus infection of human epithelial cells generates a distinct hyperphosphorylated form of keratin 8. AB - The two major intermediate filament (IF) phosphoglycoproteins of human simple epithelia are keratins 8 and 18 (K8/18). Previous studies showed that heat stress and, in some cases, viral infection of cultured cells can be associated with alteration in IF organization. Here we show that heat stress of the human colonic cell line HT29 increased K8 and K18 phosphorylation and glycosylation and generated a distinct hyperphosphorylated form of K8 (HK8) that was previously noted upon G2/M arrest of epithelial cells. In contrast, rotavirus infection of HT29 cells resulted in phosphorylation changes similar to those induced by heat stress but did not alter K8/18 glycosylation. The identity of HK8 was determined using phosphatase treatment, tryptic phosphopeptide mapping, and K8-specific antibodies. A time course of heat stress showed that the increased phosphorylation and glycosylation of K8/18 occurred several hours after induction of heat shock protein 70 expression. Rotavirus altered the organization of the K8/18 network in most infected cells, whereas the effect of heat stress on K8/18 organization was less prominent. However, in vitro filament assembly of purified K8/18 was not significantly altered after isolation from heat-stressed or rotavirus-infected cells, despite having increased solubility and hyperphosphorylation. Our results indicate that increased glycosylation and/or phosphorylation of K8 and K18, in association with heat stress or rotavirus infection, does not significantly alter in vitro keratin filament assembly, whereas the association of these modifications with filament reorganization in cells is more dramatic. Generation of HK8 appears to be a late event and to occur in common with several forms of stress including heat, viral infection, and arrest of cells in G2/M. PMID- 7543853 TI - Characterization of the 2H12 antigen as a nonshuttling human isoelectric variant of the nucleolar protein B23. AB - It has become obvious that a better understanding of the nucleolar compartment should encompass the elucidation of structural and functional relationships between its molecular constituents. Using a mouse monoclonal antibody referred to as 2H12, we have identified a human epitope that appears to be implicated in the regulatory events governing the elaboration and stabilization of the nucleolar architecture. By immunofluorescence and immunoblotting, the 2H12 monoclonal was shown to be directed against a nucleolar protein with a relative mobility of 38 40 kDa and an isoelectric point of 5.1 that is present in human cells, regardless of their proliferation state. No reactivity was detected in cells from other species, implying that the targeted epitope could be unique to humans. Investigation of the fate of the epitope throughout the cell cycle led to evidence that its immunoreactivity was phosphodependent and suggested that the disassembly and reassembly of the nucleolar apparatus during cell division is accompanied by dephosphorylation/phosphorylation modifications at this site. In a series of double immunofluorescence experiments and two-dimensional immunoblotting analyses, it was demonstrated that the 2H12 antigen corresponds to an isoelectric variant of the human nucleolar protein B23 that is most prominent during interphase. Tightly associated with the nuclear matrix, this human B23 isoelectric variant did not shuttle between the nucleus and the cytoplasm but remained sequestered within the human nucleolus during mobility assays in human murine heterokaryons. PMID- 7543852 TI - Different response to retinoic acid of two teratocarcinoma cell lines. AB - Retinoic acid (RA), a well-known inducer of differentiation, has been shown to regulate its own receptor gene expression in F9 teratocarcinoma cells. The homologous regulation of receptors by RA might be critical for RA-induced F9 cell differentiation. F9 cell lines from two different laboratories, named F9-1 and F9 2, were compared for retinoic acid receptor (RAR) and retinoid x receptor (RXR) gene expression in response to RA. The data show that both F9-1 and F9-2 cell lines are embryonal carcinoma cells, but of different phenotypes and different sensitivity to RA. In F9-1 cells, RA regulates all three RARs (alpha, beta, and gamma), two RXRs (alpha and gamma), two activin receptors (ActR II and IIB), and tissue-specific plasminogen activator (t-PA) gene expression. In F9-2 cells RA regulates only the RAR beta, RXR alpha, and t-PA genes. The induction of mRNA levels was much higher in F9-1 than in F9-2 cells. Different basal RAR gamma and RXR gamma mRNA levels were also noted. In these two cell lines F9-2 cells expressed greater amounts of RAR gamma 1, gamma 2, and gamma 3 mRNA isoforms, but lacked RXR gamma mRNA compared with F9-1 cells. Since RAR gamma 1 has been shown to exert an antagonistic effect on other types of RA receptors, the decreased sensitivity of F9-2 cells to RA might be due to its high level of RAR gamma 1 and/or low level of RXR gamma. This notion was in part supported by gel shift assay which demonstrated constitutive binding of RAR gamma to a RA responsive element (RAR beta E) in F9-2 cells. Further, the binding of nuclear protein to RAR beta E was increased upon RA treatment in F9-1 cells, but not in F9-2 cells. These differences in the regulation of RA receptors might determine the sensitivity of the two substrains of F9 cells to RA. PMID- 7543854 TI - MG160, a membrane protein of the Golgi apparatus which is homologous to a fibroblast growth factor receptor and to a ligand for E-selectin, is found only in the Golgi apparatus and appears early in chicken embryo development. AB - While over 20 intrinsic proteins of the Golgi apparatus have been identified and sequenced, there is no information on their developmental history, i.e., whether all Golgi proteins are expressed simultaneously or whether there is a hierarchical or stage-specific order of their expression during embryonic development. In this study we have examined the emergence and distribution of MG160 during the development of chicken embryos. MG160 is a conserved membrane sialoglycoprotein of the Golgi apparatus of most cells displaying over 90% amino acid sequence identities with two apparently unrelated molecules, namely CFR, a chicken fibroblast growth factor receptor, and ESL-1, a ligand for E-selectin (Gonatas et al., J. Biol. Chem. 1989, 264, 646-653; Burrus and Olwin, J. Biol. Chem. 1989, 264, 18647-18653; Burrus et al., Mol. Cell Biol. 1992, 12, 5600-5609; Gonatas et al., J. Cell Sci. 108, 457-467; Steegmaier et al., Nature 1995, 373, 615-620). This study was carried out by in situ hybridization, using a 56-mer antisense probe for the chicken homologue of MG160 which differs only by four bases from the corresponding segment of the rat cDNA and by immunocytochemistry and Western blotting using a polyclonal antiserum against MG160. The protein was ubiquitously and exclusively localized in the Golgi apparatus and appeared early in development within the ectoblast and primitive endoblast prior to the formation of the primitive streak. At 2 to 3 days, MG160 was particularly prominent in the notochord, neural tube, somites, and cartilage cells. In organs with central lumens, such as the neural tube, the Golgi apparatus, visualized by immunostaining for MG160, was elongated and it was located at the apical pole of cells. In 6-day-old embryos, the ongoing physiologic degeneration of the notochord was accompanied by fragmentation of the immunostained Golgi apparatus and decreased labeling of the mRNA for MG160. In order to gain information on possible interactions between MG160 and basic fibroblast growth factor (bFGF), the localization of both molecules was studied by immunocytochemistry in 3-day old chicken embryos. While MG160 was ubiquitous in the Golgi apparatus of all cells and tissues, endogenous bFGF was no detected, while exogenous bFGF bound only to basement membranes. These results indicate that MG160 is a primordial protein of the Golgi apparatus and are consistent with the hypothesis that the binding of MG160 to fibroblast growth factors and E-selectin is not related to the still unknown principal function of MG160 in the Golgi apparatus. PMID- 7543851 TI - Structural determinants of calcium signaling by RGD peptides in rat osteoclasts: integrin-dependent and -independent actions. AB - Extensive characterization of the vitronectin receptor (VNR), a member of the integrin group of cell adhesion molecules, which is abundantly expressed in osteoclasts, has revealed a role for this receptor in osteoclast adhesion as well as bone resorption. Earlier evidence from our laboratory suggests that VNR is also capable of transducing intracellular signals following receptor ligand interaction, although this function is poorly understood. Thus, addition of peptides containing the minimal tripeptide Arg-Gly-Asp (RGD) integrin recognition sequence elicits transient increases in intracellular free calcium ions, with maximal responses seen with a bone sialoprotein peptide, BSP-IIA. In the present study we have attempted to determine some of the structural requirements for calcium signaling in osteoclasts using derivatives of the peptide PRGDN/T sequence found in bone sialoprotein. While some peptides, such as the parent sequence PRGDN, can induce both signaling and retractile events, it was found that minor structural modifications yielded peptides such as PRADN which elicited a transient increase in intracellular free calcium ions without promoting a reduction in osteoclast spread area (retraction). Conversely, certain other modifications resulted in peptides, such as PrGDN and benzoyl-RGDN, which effect osteoclast retraction, while having minimal Ca2+ signaling capabilities. Osteoclast adhesion, and hence retraction, are known to be RGD-dependent and integrin-dependent events. However, intracellular Ca2+ signaling is RGD independent and, based on lack of inhibition by an anti-beta 3 integrin antibody F11 and echistatin, very likely integrin-independent. These data suggest that signaling is not always via VNR and as yet unknown receptors on the osteoclast membrane play a role in osteoclast signaling and hence function. PMID- 7543856 TI - High endocytotic activity occurs periodically in the endplate region of denervated mouse striated muscle fibers. AB - High endocytotic activity after denervation of skeletal muscle occurs in a proportion of muscle fibers (both slow and fast fiber types) in the endplate region. The present study was performed in order to examine if a periodicity in the endocytotic activity could explain why the process is not observed in all fibers at a given time. Three markers, horseradish peroxidase (HRP), rhodamine B isothiocyanate-labeled dextran, and fluorescein isothiocyanate-labeled dextran were used to demonstrate endocytotic activity of muscle fibers of the denervated mouse hemidiaphragm in vivo. Acetylcholine esterase staining was used in conjunction with HRP uptake to determine the proportion of denervated muscle fibers with endocytotic activity in the endplate region at any one time. The results show that 25-50% of the muscle fibers display high endocytotic activity in the endplate region at a given time 10 days after denervation. The existence of a periodicity in this endocytotic activity is suggested by results obtained using two different endocytotic markers administered at time intervals of 0-7 days. We conclude that loss of contact with the innervating motorneuron induces a high endocytotic activity which occurs periodically in the perisynaptic region of skeletal muscle fibers. PMID- 7543855 TI - The role of DNA ploidy in the differentiation of WEHI-3B D- leukemia cells transfected with the granulocyte colony-stimulating factor receptor gene. AB - Granulocyte colony-stimulating factor (G-CSF) exerts various biological effects through occupancy of its receptor (G-CSFR). WEHI-3B D- myelomonocytic leukemia cells do not express the G-CSFR, do not respond to G-CSF or to retinoic acid by the induction of granulocytic maturation, contain a near tetraploid content of DNA, and form tightly aggregated colonies. However, they still maintain the capacity to differentiate since they respond to vitamin D3 by the formation of mature cells. Transfection of the G-CSFR gene into WEHI-3B D- cells resulted in three major changes. G-CSFR-expressing clones (a) acquired the capacity to respond to the differentiation-inducing properties of G-CSF and retinoic acid, (b) formed colonies which exhibited a dispersed phenotype, and (c) exhibited near diploid DNA ploidy. In contrast, WEHI-3B D- cells transfected with vector alone behaved like parental WEHI-3B D- cells. The findings imply that the near diploid phenotype is required for WEHI-3B D- leukemia cells to respond to certain inducers of differentiation. PMID- 7543857 TI - Ilimaquinone inhibits the cytotoxicities of ricin, diphtheria toxin, and other protein toxins in Vero cells. AB - Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum [P. A. Takizawa, J. K. Yucel, B. Viet, D. J. Faulkner, T. Deerinck, G. Soto, M. Ellismann, and V. Malhotra, Cell (1993) 73, 1079-1090]. We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner. The inhibition was reversed upon the removal of IQ. Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ. However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin. Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin. The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity. In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells. Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ induced breakdown product of the Golgi membranes. Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga like toxin in Vero cells. Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT). Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells. PMID- 7543858 TI - Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes. AB - In the present study we investigated the Fas-mediated cellular events using the human leukemic T cell line, JURKAT. Ligation of the Fas receptor with a monoclonal antibody (IgM) resulted in the rapid (within 3 h) induction of apoptosis and was characterized by a sequence of distinct morphological and biochemical events. Thus, plasma membrane blebbing, condensation of the chromatin, and formation of high-molecular-weight (HMW) DNA fragments were the earliest events observed (by 45 min). They were followed by cleavage of DNA into oligonucleosomal-length fragments (laddering pattern) and the formation of apoptotic bodies, and finally, rounding of the apoptotic cells and complete cleavage of DNA into oligonucleosomal-length fragments occurred. The mitochondria remained structurally intact up to the stage of oligonucleosomal-length DNA cleavage, and the ability of the cells to exclude trypan blue was not compromised throughout the time course of the experiments. In contrast to many other model systems, apoptosis in JURKAT cells after anti-Fas treatment did not require the presence of extracellular Ca2+ or Mg2+ and was only partially inhibited by Zn2+. In addition, Fas-mediated apoptosis was unaffected by the presence of free radical scavengers or inhibitors of protein phosphatases, protein kinases, and nitric oxide synthesis. However, the serine protease inhibitors, N-tosyl-L phenylalanine chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI) prevented anti-Fas-induced apoptosis in JURKAT cells. Low concentrations of these inhibitors blocked oligonucleosomal-length, but not HMW, DNA fragmentation. The latter required a higher concentration of TPCK or DCI to block. In addition, low concentrations of DCI also prevented Fas-mediated plasma membrane blebbing. In summary, our results suggest that proteolysis plays a central role in Fas mediated apoptosis and that distinct proteolytic enzymes are involved in HMW DNA fragmentation, and oligonucleosomal-length DNA fragmentation, as well as in plasma membrane blebbing. PMID- 7543859 TI - RAPD analysis of isolates of Burkholderia pseudomallei from patients with recurrent melioidosis. AB - Twenty-seven isolates of Burholderia pseudomallei (formerly Pseudomonas pseudomallei) from ten patients with recurrent melioidosis were analysed by RAPD. In two cases RAPD patterns in recurrent isolates differed from the original isolates; one was considered a likely reinfection while the other may represent relapse from one of two strains initially infecting the patient. In two cases where a change in antibiotic resistance had occurred between original and relapse isolates, slight changes in RAPD patterns were found with one of the four primers used. In the other six cases the relapse was clearly due to the original strain re-emerging unchanged, with identical RAPD patterns with all four primers. PMID- 7543860 TI - Antigenic analysis of SAT 2 serotype foot-and-mouth disease virus isolates from Zimbabwe using monoclonal antibodies. AB - This paper compares strains of foot-and-mouth disease (FMD) serotype SAT (South African Territories) 2 viruses isolated from Zimbabwe and other African countries using monoclonal antibodies (MAb). A sandwich-ELISA was used to examine the relative binding of anti-SAT 2 MAb to the various viruses. The MAb-binding profiles of viruses isolated from field samples were compared using hierarchical cluster analysis. Viruses were obtained from game animals, mainly African buffalo (Syncerus caffer) which is the natural host and reservoir for SAT serotypes in Africa, and from cattle showing clinical signs of FMD, as well as from animals suspected of carrying the virus subclinically. Some isolates have been adapted for use as vaccine strains. The results showed that most of the Zimbabwe isolates collected between 1989 and 1992 were an antigenically closely-related group. Although differences were observed between Zimbabwe isolates collected between 1989 and 1992 and those collected in 1987, there was no correlation with the different MAb binding patterns within the 1987 group and the epidemiological information received from the field. Similar profiles were observed for many SAT 2 viruses, including viruses isolated over a 50-year period and from geographically distant areas. This indicates an inherent stability in antigenic profiles of SAT 2 viruses. The MAb panel was capable of assessing antigenic variation, since very different profiles were obtained for some isolates. The work also allowed comparison and characterization of anti-type SAT 2 MAb from different laboratories. The findings are discussed with reference to selection of vaccine strains. PMID- 7543862 TI - Subretinal neovascularization. PMID- 7543861 TI - Molecular characterization of a virulence-associated epitope on the lipopolysaccharide of Legionella pneumophila serogroup 1. AB - For identification of lipopolysaccharide (LPS)-associated epitopes of Legionella pneumophila serogroup 1, LPS of strain Philadelphia 1 was investigated using monoclonal antibodies (MAbs). The O-specific chain of LPS is a homopolymer of 5 acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-D-glycero- L-galacto- nonulosonic acid. At least four immunoaccessible epitopes were recognized by different MAbs on the intact LPS. After O-deacetylation of LPS, the reactivity of one of the MAbs (MAb 3/1) was lost, indicating thus that the corresponding epitope is associated with the 8-O-acetyl group. Since the reactivity pattern of the MAb 3/1 is identical with those of the MAb 2 which was considered as a virulence marker for serogroup 1, this epitope may be involved in mediating virulence in L. pneumophila. Four MAbs specific to strains of serogroup 1 other than the monoclonal subtype Philadelphia recognized epitopes on the O deacetylated LPS of strain Philadelphia 1 and, therefore, the virulence associated epitope blocks recognition of the immunodeterminants that are accessible on the intact LPS of the strains lacking this epitope. PMID- 7543863 TI - Nucleocytoplasmic transport: factors and mechanisms. AB - In the past two years, our knowledge concerning the mechanisms of nucleocytoplasmic transport through the nuclear pore complex (NPC) has considerably expanded. The application of in vitro systems that reconstitute nuclear protein import has allowed the identification of cytosolic factors that are required for the import process. Microinjection into Xenopus oocytes and yeast genetic systems have provided interesting candidates for RNA export mediators. Functional and structural analysis of nucleoporins has demonstrated the involvement of NPC components in the transport process. Finally, new concepts have emerged such as the integration of the mechanisms of the nuclear protein import and RNA export reactions and the assembly of the transport machinery at specialised domains of the NPC. PMID- 7543865 TI - High HbF in pregnancy is associated with the Xmn I polymorphism at the -158bp of the G gamma-globin gene. AB - HbF was measured in the peripheral blood of 354 women during the first trimester of pregnancy. In those (n = 6) with HbF > 3% and in 30 randomly selected pregnant women with normal HbF values, analysis of the G gamma content and the Xmn I polymorphism was performed. The frequency of the Xmn I polymorphism was significantly higher (P < 0.001) in the group with high HbF (0.67) compared to the control group (0.10). The G gamma chain content evaluation revealed a newborn ratio in all 6 high HbF subjects. Six months after delivery, HbF was measured in all women with high HbF during pregnancy, revealing normal values. These results suggest that the presence of the Xmn I polymorphism is a strong inducer for the elevation of HbF during pregnancy. PMID- 7543864 TI - Effect of aging on the female reproductive system: evidence for a role of uterine senescence in the decline in female fecundity. AB - OBJECTIVE: To determine the effects of age on reproductive performance of women using oocyte donation as an in vivo model. SETTING: Oocyte donation and IVF programs at the Instituto Valenciano de Infertilidad. PATIENTS: Seventy-six women undergoing 90 cycles of ovum donation, who were recipients of 36 donors undergoing IVF, and 9 fertile women. DESIGN: Prospective longitudinal study: [1] recipients underwent an artificial cycle to demonstrate adequate response of the endometrium to exogenous steroids; [2] oocytes from the same cohort of follicles were distributed randomly into recipients younger and older than 40 years; and [3] pregnancies were followed during the first trimester. MAIN OUTCOME MEASURES: Endometrial histology, fertilization, embryo quality, pregnancy, implantation, and abortion rates in both groups of recipients. Serum E2, P and beta-hCG levels during initial pregnancy. RESULTS: Similar implantation rates but significantly higher abortion rates were detected in women > 40 years despite an appropriate action of P on the endometrium and the transfer of embryos in similar number and quality. The secretion of E2 and P by the placenta started earlier in pregnancies included in the group < 40 years. CONCLUSIONS: Age increases pregnancy losses in ovum donation patients after implantation is completed. This is accompanied by a retardation of steroid synthesis and suggests that the mechanism(s) responsible for placenta formation and functioning in the uterus is affected by age. Thus, uterine aging also is a factor influencing the poor reproductive performance of women with advancing age. PMID- 7543866 TI - An extra gonadal non-gestational choriocarcinoma in a woman treated for an endometrial carcinoma. AB - A patient was presented with a non-gestational non-gonadal choriocarcinoma and hyperthyroidism. Five years earlier, at the age of 36, she underwent an abdominal hysterectomy with bilateral adnexectomy for endometrial carcinoma. Despite intensive treatment with multiple chemotherapy the patient died. We concluded that this non-gestational, non-gonadal choriocarcinoma was a recurrence of her previous endometrial carcinoma or a new extra (non) gonadal germcell tumor with choriocarcinoma. PMID- 7543868 TI - A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes. AB - We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits. PMID- 7543867 TI - [Pseudo AV dissociation caused by interpolated junctional extrasystoles in the presence of a first-degree AV block]. AB - This presentation reports an electrocardiogram showing first degree A-V block with a very prolonged P-R interval of 0.80 sec. On several occasions an arrhythmia occurred, characterized by what looked like an A-V junctional escape rhythm with A-V dissociation. This was suggested by a variable and, at first glance, haphazard relationship between QRS complexes and P waves. Analysis of the tracing suggested that the pattern was due to an interpolated A-V junctional extrasystole, followed by a sinus beat with an inordinately long P-R interval, whose duration was 1.18 sec. This very prolonged A-V conduction time made it difficult to recognize the relationship between P waves and QRS complexes, so that the pattern appeared as an A-V dissociation. PMID- 7543869 TI - Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs. AB - Reduced-stringency PCR was used to isolate a cDNA encoding a novel human FK506 binding protein (FKBP) homolog. The encoded 38-kDa protein (FKBPr38) contains at its N-terminus a domain that is 33% identical to FKBP12. FKBPr38 is a member of a subclass of immunophilins, whose other members include FKBP52 and CyP40 (cyclophilin 40), that contain a three-unit tetratricopeptide repeat (TPR). In addition, FKBPr38 contains a consensus leucine-zipper repeat. The presence of the TPR domain and leucine zipper suggest that FKBPr38 may form homo-multimers or interact with other, as yet unidentified, proteins. PMID- 7543870 TI - Presence of an additional PstI fragment in intron 1 of the chicken growth hormone encoding gene. AB - A previously unreported 196-bp PstI fragment was found in intron 1 of the gene encoding chicken growth hormone (cGH) when a PCR assay for an MspI restriction fragment length polymorphism was established. A pair of PCR primers was designed according to the published cGH sequence and used to amplify a fragment which contained two MspI sites, one polymorphic and another non-polymorphic. However, amplification of genomic DNA from two strains of meat-type chickens and three strains of White Leghorn chickens yielded a PCR product which was about 200 bp larger than expected. The fragment from one of the meat-type chickens was subcloned into the vector pCR-Script SK+, and sequenced. It revealed the presence of an extra fragment of 196 bp which was flanked by the PstI sites and occurred at nt +308 of the previously reported cGH sequence. PMID- 7543871 TI - Activity and stability of Bacillus cereus penicillinase entrapped in aerosol OT reverse micelles. AB - The kinetics of enzyme catalyzed hydrolysis of penicillin G and stability of the enzyme alpha-penicillinase, entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, such as, water pool size (related to Wo), pH and temperature, were optimized for maximum activity of penicillinase in water/aerosol OT/isooctane reverse micelles. The enzyme showed maximum activity of Wo - 14 and pH, 7.0. At any temperature the enzyme was to be more active in reverse micelles than in aqueous solution. At optimum conditions of Wo, pH and temperature the enzyme was 100% more active in reverse micelles than its maximum activity in aqueous solution. In both the systems, the activity starts falling at and above 25 degrees C. CD Spectral studies showed that the enzyme in reverse micelles possesses more helical structure than it has in aqueous solution and at the optimum conditions in which it showed maximum activity, the alpha-helicity was also maximum. The enzyme was very stable in reverse micelles at and above room temperature compared to the same in aqueous solution. PMID- 7543872 TI - Induction of feline immunodeficiency virus-specific cell-mediated and humoral immune responses following immunization with a multiple antigenic peptide from the envelope V3 domain. AB - Cytotoxic T-cell determinants should be an important component of a vaccine against feline immunodeficiency virus (FIV). Epitope mapping studies have revealed an immunodominant neutralization epitope within the third variable (V3) domain of the viral envelope glycoprotein comprizing 17 amino acids (residues 390 406: RAISSWKQRNRWEWRPD). We have investigated the induction of FIV-specific cytotoxicity and anti-peptide antibody in cats immunized with a multiple antigenic peptide (MAP) containing this epitope. Virus-specific lymphocytotoxicity was determined using autologous or allogeneic skin fibroblasts as target cells labelled with chromium-51 and pulsed with overlapping 10 amino acid peptides. Cytotoxic effector cells derived from fresh peripheral blood were detected in five out of 10 immunized cats. The cell-mediated immune response appeared to be directed to envelope peptide 1 (RAISSWKQRN) and peptide 2 (SWKQRNRWEW), with recognition of peptide 3 (QRNRWEWRPD) in only one cat. An antibody response to the 17 amino acid peptide immunogen was detected in seven immunized cats, which was directed to envelope peptides 2 and 3. These results suggest that different epitopes may be recognized by the cell-mediated and humoral immune responses. None of the cats was protected from challenge with the Glasgow8 isolate of FIV (FIV/GL-8). This study has implications for vaccine strategies using synthetic peptides to induce virus-specific cell-mediated immune responses. PMID- 7543873 TI - Lymphocyte recognition elements on the VP1 protein of Theiler's virus. AB - Theiler's virus is a murine picornavirus that persists in the central nervous system in susceptible mouse strains, and gives rise to immune mediated demyelinating disease. Antiviral CD4 T cells are necessary to protect from overwhelming virus replication in the acute phase of the disease, and are thought to act by stimulating the antibody response. The present study used overlapping synthetic peptides to map the location of epitopes recognized by CD4 T cells. One T-cell epitope was identified between amino acids 33-47 of VP1, which was recognized by virus-reactive T cells. 'Cryptic' epitopes were also present within VP1 at positions 153-167, 166-180, 225-239 and 233-247. A linear B-cell epitope was identified in the C-terminal region 225-276. Immunization of CBA mice with inactivated virus, but not peptides containing VP1 B- or T-cell epitopes, reduced the virus titre in the CNS in the acute phase of the disease. PMID- 7543875 TI - Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum. AB - The adhesion of erythrocytes infected with Plasmodium falciparum (P. falciparum) is one of the major pathological features of severe malaria. Several potential receptors to endothelium for falciparum-infected erythrocyte on endothelium have been described. Recently, the malaria binding site on ICAM-1(CD54) has been mapped to a site distinct but overlapping with the LFA-1 (CD11a/CD18) site. We detected by flow cytometry, confocal laser microscopy and immunoprecipitation, a molecule expressed at the surface of erythrocytes infected with mature stages of the M96 strain of P. falciparum that was recognized by a monoclonal antibody (mAb) (TS1/22) directed against an LFA-1 epitope. However, this molecule was not recognized by mAbs directed against other epitopes of LFA-1 or against other integrins. Furthermore, the mAb TS1/22 partially inhibited cytoadherence of parasitized red blood cells to human-brain microvascular endothelial cells. The expression of a molecule sharing an epitope with human LFA-1 integrin on the parasitized erythrocyte surface could be involved in the sequestration of these cells and thus in the pathogenesis of severe disease. PMID- 7543876 TI - Identification of MIC 11 antigen as an epitope of the CD59 molecule. AB - The MIC 11 antigen is expressed on human cells and is characterized by reaction with a monoclonal antibody (mAb), 16.3A5. The gene controlling MIC 11 was recently mapped to the p13 region of chromosome 11 within 500 kb of the gene encoding CD59, a complement regulatory protein. The present report investigates the antigenic relationship between these cell-membrane determinants and sets out evidence that MIC 11 and CD59 are encoded by the same gene. Western blotting of human erythrocyte membrane proteins and purified membrane CD59 showed that 16.3A5 anti-MIC 11 antibody bound to a 19-24,000 MW band with the characteristic appearance of CD59 protein, and gave staining patterns identical to those obtained with the CD59 antibody, BRIC 229. The binding of 16.3A5 monoclonal IgG to purified urine-derived CD59 in enzyme-linked immunosorbent assay (ELISA) was inhibited by YTH 53.1 rat CD59 antibody, indicating that the MIC 11 epitope is the same as, or close to, that recognized by CD59 antibodies such as YTH 53.1, BRIC 229 and 2/24. Prior exposure of erythrocytes to 16.3A5 anti-MIC 11 also reduced the ability of the CD59 antibodies, BRIC 229 and YTH 53.1, to block the complement-inhibiting function of membrane CD59. Anti-MIC 11 antibody alone, however, had no inhibitory effect on CD59 function. This may be due to its relatively low binding affinity or to some slight difference in epitope specificity. Further studies using immunofluorescence showed that the MIC 11 epitope, like CD59, is absent from EBV-B cells lacking GPI-anchored proteins and from a B-cell line specifically deficient in CD59 protein. Overall, the results provide strong evidence that MIC 11 is a determinant on the CD59 molecule. PMID- 7543877 TI - Comparable profiles of the immunoglobulin heavy chain complementarity determining region (CDR)-3 in CD5+ and CD5- human cord blood B lymphocytes. AB - It has been suggested that CD5+ B cells are closely related to the pathogenesis of autoimmune disease or chronic lymphocytic leukaemia, whereas the origin and physiological role of CD5+ B cells remain controversial. To study the molecular differences between CD5+ and CD5- B cells in terms of immunoglobulin gene structure, we sorted both subsets from new-born cord blood and analysed the complementarity determining region (CDR)-3 profiles of the immunoglobulin heavy chain (IgH) gene. The CDR-3 sequences from both CD5+ and CD5- B cells represented the same incidence and length of N-region, and the same usage of D and JH segments. When translated into amino acids, 24 of 32 clones (75%) and 25 of 37 clones (65.8%) from their respective subset were productive, and the composition of the deduced amino acids were similar between both subsets. These data suggest that CD5+ B cells are not a distinct lineage presenting a biased immunoglobulin repertoire in B cells. PMID- 7543878 TI - Cytokines as adjuvants for vaccines: antigen-specific responses differ from polyclonal responses. AB - The use of cytokines in the administration of vaccines has a unique value in obtaining the appropriate immune response and in ensuring a protective outcome. Earlier studies indicating that cytokines can influence the generation of a particular antibody isotype may represent an oversimplification of a more complex problem. Several studies discussed in this review show that the effect of a given cytokine on the immune response depends on whether one examines the antigen specific response or the polyclonal response (i.e., total serum immunoglobulins). Further, a balanced regulation of immune responsiveness is important in maintaining homeostasis of the immune system. Consequently, for any vaccine that uses cytokines to boost the response, due consideration must be given to these important variables. PMID- 7543879 TI - Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium. AB - The mechanisms by which Aspergillus fumigatus colonizes the respiratory mucosa are unknown. Culture filtrates of eight of nine clinical isolates of A. fumigatus slowed ciliary beat frequency and damaged human respiratory epithelium in vitro. These changes appeared to occur concurrently. Culture filtrates of two clinical isolates of Candida albicans had no effect on ciliated epithelium. We have purified and characterized cilioinhibitory factors of a clinical isolate of A. fumigatus. The cilioinhibitory activity was heat labile, reduced by dialysis, and partially extractable into chloroform. The activity was associated with both high and low-molecular-weight factors, as determined by gel filtration on Sephadex G 50. A low-molecular-weight cilioinhibitory factor was further purified by reverse phase high-performance liquid chromatography and shown by mass spectrometry to be gliotoxin, a known metabolite of A. fumigatus. Gliotoxin significantly slowed ciliary beat frequency in association with epithelial damage at concentrations above 0.2 microgram/ml; other Aspergillus toxins, i.e., fumagillin and helvolic acid, were also cilioinhibitory but at much higher concentrations. High-molecular weight (> or = 35,000 and 25,000) cilioinhibitory materials had neither elastolytic nor proteolytic activity and remain to be identified. Thus, A. fumigatus produces a number of biologically active substances which slow ciliary beating and damage epithelium and which may influence colonization of the airways. PMID- 7543874 TI - Evidence of post-transcriptional regulation of L-selectin gene expression in rat lymphoid cells. AB - Early investigations of lymphocyte migration in the rat operationally identified a lymphocyte membrane protein, designated 'A.11', which mediates lymphocyte adherence to lymph node (LN) high endothelial venules (HEV). To determine the primary structure of A.11 and examine its expression in lymphoid cells, we constructed an expression phage cDNA library of rat thoracic duct lymphocytes (TDL) and performed screening by immunoselection (utilizing an anti-A.11 polyclonal antiserum) as well as by hybridization selection. We have isolated a approximately 1.6 kb clone, RS-2, and sequencing revealed that it encodes rat L selectin. The clone contains the complete coding sequence, a 105-bp 5' untranslated region and a 359-bp 3' untranslated region. Transfection of RS-2 cDNA into 70Z/3 cells conferred binding to HEV concomitant with expression of A.11, providing direct evidence that A.11 is rat L-selectin. Metabolic radiolabelling studies revealed that thymocytes synthesize markedly less L selectin than do TDL or LN lymphocytes. However, Northern blot studies using RS-2 as a probe indicate that thymocytes possess more L-selectin RNA than does TDL. Together, these data provide evidence that post-transcriptional events contribute to regulation of L-selectin expression in thymocytes. PMID- 7543882 TI - Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. AB - A serologically typical strain of Burkholderia pseudomallei (strain 304b) was found to produce two S-type lipopolysaccharides (LPS) differing in the chemical structures of their O-polysaccharide (O-PS) components. Structural analysis revealed that one O-antigenic polysaccharide (O-PS I) is an unbranched high molecular-weight polymer of 1,3-linked 2-O-acetyl-6-deoxy-beta-D-manno heptopyranose residues. The other LPS O antigen (O-PS II) is an unbranched polymer of repeating disaccharide units having the structure -3)-beta-D glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which ca. 33% of the L 6dTalp residues bear 2-O-methyl and 4-O-acetyl substituents while the other L 6dTalp residues carry only 2-O-acetyl substituents. Analysis of a serologically atypical strain of B. pseudomallei (strain 824a) produced a single LPS O-PS which was chemically identical to the 6-deoxy-D-manno-hepan O-PS I. The production of two distinct LPS raises the interesting question of their relative immunogenicities and consequently their relative importance for diagnostic serology and for the possible development of conjugate vaccines. PMID- 7543880 TI - Signal transduction responses following adhesion of verocytotoxin-producing Escherichia coli. AB - Attaching and effacing adhesion to epithelial cells is a pathognomonic feature of infection by both enteropathogenic Escherichia coli (EPEC) and certain strains of verocytotoxin-producing E. coli (VTEC). EPEC adhesion to tissue culture epithelial cells results in activation of the phosphatidylinositol pathway, with elevated levels of inositol 1,4,5-triphosphate and cytosolic free calcium. In this report, we show that VTEC also activate this signal transduction pathway in infected epithelial cells. Specifically, increased levels of inositol 1,4,5 triphosphate and intracellular free calcium were observed in HEp-2 cells infected with VTEC of serotype O157:H7. VTEC of serotypes O157:H7 and O113:H21 also induced increases in intracellular calcium levels in the human intestinal crypt like cell line T84, even with minimal or no attaching and effacing activity as monitored by transmission electron microscopy. In contrast to EPEC, VTEC failed to induce tyrosine phosphorylation of epithelial cell proteins in HEp-2 and T84 cells, as determined by indirect immunofluorescence microscopy. These findings suggest that signal transduction responses to VTEC, including elevated levels of inositol triphosphates and intracellular free calcium, are independent of formation of the attaching and effacing lesion. Our findings also show that VTEC pathogenesis may involve signal transduction pathways that are distinct from those induced by EPEC infection. PMID- 7543888 TI - Pseudomonas pseudomallei-insulin interaction. PMID- 7543884 TI - Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes. AB - To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface specific immunoprecipitation of [35S]methionine-labeled proteins (200, 28, and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of 200 and 220 kDa were isolated from a MAb 44.18 affinity matrix. Calf serum antibodies to these isolated antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf serum antibodies identified major labeled proteins with sizes of 200 and 72 kDa by surface-specific immunoprecipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationale for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines. PMID- 7543885 TI - Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes. AB - Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined. PMID- 7543883 TI - Conjugates of synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria meningitidis. AB - Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease. PMID- 7543886 TI - Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides. AB - The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied. Substance P was attacked primarily at the Phe-8-Gly-9 bond by a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-acting prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopeptidase (FALGPA-peptidase). Bradykinin was cleaved at Phe 5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase. Angiotensin I was rapidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPase. All these enzymes were assumed to be cell associated and were easily extracted with a mild (0.05 to 0.1%) Triton X-100 treatment. Because it was conceivable that the hydrolysis of substance P at the Phe-8-Gly-9 bond was catalyzed by a CTLP described earlier (V.-J. Uitto, D. Grenier, E. C. S. Chan, and B. C. McBride, Infect. Immun. 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventional fast protein liquid chromatography procedures. For kinetic studies, Phe-8(4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, epsilon = 545 M-1 cm-1) was synthesized to replace substance P as a substrate in kinetic studies. In reversed-phase chromatography, both NSP and substance P gave identical results with both whole cells and the purified enzyme. The CTLP has a mass of 95 kDa, and its activity is suggested to be based on an active seryl residue, on an active imidazole group, and on an active carboxyl group but not on metal cations. The enzyme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p nitroaniline (SAAPFNA, a typical chymotrypsin substrate) at a high rate and several proteins, such as calf thymus histone, human plasma fibrinogen, milk caseins, and gelatin. Among the substrates tested, substance P showed the highest affinity (Km = 0.22 mM) for the purified enzyme. Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/liter, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAPFNA by whole cells and the purified CTLP. The hydrolysis of NSP by whole cells and purified CTLP was slightly inhibited by chlorhexidine. The results demonstrated the versatility and the effectiveness of the outer membrane of T. denticola in occasioning a rapid breakdown and inactivation of human bioactive peptides and other peptidolytic catalyses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543887 TI - The tetrasaccharide L-alpha-D-heptose1-->2-L-alpha-D-heptose1--> 3-L-alpha-D heptose1-->(3-deoxy-D-manno-octulosonic acid) and phosphate in lipid A define the conserved epitope in Haemophilus lipopolysaccharides recognized by a monoclonal antibody. AB - A murine monoclonal antibody, MAHI 3 (immunoglobulin G2b), that is broadly reactive with Haemophilus influenzae lipopolysaccharides (LPSs) but nonreactive with all enterobacterial LPSs tested was generated by fusing mouse myeloma cells with spleen cells of BALB/c mice immunized with azide-killed H. influenzae RM.7004. MAHI 3 bound to all H. influenzae, all other human Haemophilus spp., all Bordetella pertussis and Bordetella parapertussis, and all Aeromonas spp. tested but not to any Neisseria or Moraxella catarrhalis strains, as determined by enzyme immunoassay, colony dot immunoblotting, and immunoblotting. In an inhibition enzyme immunoassay, MAHI 3 reacted with all 45 H. influenzae LPSs tested but not with the LPS from the rough mutant I69 Rd-/b+, which has only 3 deoxy-D-manno-octulosonic acid (P) [Kdo(P)] and lipid A. The antibody was not inhibited by H. influenzae lipid A or lipid-free polysaccharide isolated after mild acid hydrolysis. Only native LPSs show positive inhibitory activity, indicating that part of lipid A is involved in the binding of MAHI 3. From the results, it is indicated that the structural element recognized by MAHI 3 is Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1-->Kdo together with part of lipid A, including the phosphate. PMID- 7543881 TI - Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552. AB - Three mutants of Mycoplasma hominis PG21 were isolated and shown to contain alterations in the size of a repeat-containing gene encoding a surface-localized 135-kDa antigen designated Lmp1. The mutants were isolated by cultivating M. hominis for a 3-month period in the presence of Lmp1-specific monoclonal antibody (MAb) 552. The epitope for MAb 552 was localized at the repeated part of the protein. The gene encoding Lmp1 is part of a transcriptional complex that contains 9.5 direct repeats of 471 bp each. Pure cultures of mutant strains were obtained by subcloning, and three mutants were characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells in culture medium was, however, increased, indicating that the repeated elements may be of importance for repulsion of the cells. PMID- 7543889 TI - Laser photocoagulation of choroidal neovascularization in pathologic myopia: long term results. AB - Direct laser photocoagulation of macular choroidal neovascularization (CNV) in pathologic myopia is still controversial. However, previous studies have reported encouraging results. One-hundred and thirty-three consecutively treated eyes were followed, in a prospective manner, for at least 12 months (108 of these were followed for 24 months, 84 for 36 months, 63 for 48 months, and 47 for 60 months) to evaluate the potential benefits and complications of laser treatment of extra foveal macular CNV in pathologically myopic eyes. Pre-treatment mean visual acuity was 0.36. Post-treatment mean visual acuity was 0.33 at 1-year examination, 0.31 at 2 years, 0.32 at 3 years, 0.34 at 4 years, and 0.33 at 5 years. During the total follow-up period mean visual acuity did not change. Neovascular recurrences occurred in 58% of treated eyes by the end of the five year follow-up. At final examination, complete destruction of CNV was achieved in 100 eyes (75%). These findings suggest that laser photocoagulation of macular choroidal neovascularization in pathologic myopia may be effective in preventing or limiting severe and irreversible loss of vision. PMID- 7543890 TI - Prostate-specific antigen concentration: influence of age and ethnicity. AB - This pilot study evaluated the influence of age and ethnicity on serum prostate specific antigen (PSA) concentration in Asian and white men without a clinical diagnosis of prostate cancer. Between October and December 1993, 1260 patients who underwent serum PSA determination (Hybritech Tandem-R assay, San Diego, California) at Straub Clinic & Hospital were retrospectively analyzed. Of these, 885 (70%) men aged 40 to 79 years were either Asian (Chinese, Filipino, Japanese, and Korean) or white and had a serum PSA less than 10.0 ng/ml. The PSA for the entire group was 2.1 +/- 2.0 ng/ml (mean +/- SD). PSA correlated with age (r = 0.31, p = 0.0001) and age accounted for 10% of the variance in serum PSA. Using the regression formula, serum PSA increased 2.5% (0.06 ng/ml) per year of age. The entire study group was about equally divided between whites (49%) and Asians (51%). Nearly three-fourths of the Asian men were Japanese. The mean PSA was very close in the Asian and white groups. There was no direct correlation between serum PSA and ethnicity (r = 0.03; p = 0.3201). Ethnicity contributed 0.1% of the variance in PSA. In conclusion, this preliminary study suggests serum PSA increases with age in Asian and white men without a clinical diagnosis of prostate cancer. No difference was found in PSA between men of Asian and white ethnicity. PMID- 7543891 TI - Ultrasound-guided high dose rate conformal brachytherapy boost in prostate cancer: treatment description and preliminary results of a phase I/II clinical trial. AB - PURPOSE: To improve results for locally advanced prostate cancer, a prospective clinical trial of concurrent external beam irradiation and fractionated iridium 192 (Ir-192) high dose rate (HDR) conformal boost brachytherapy was initiated. METHODS AND MATERIALS: Between November 1991 and February 1994, 99 implants were performed on 33 patients with prostatic adenocarcinoma at William Beaumont Hospital. Using AJCC staging criteria, 9 patients had T2b tumors, 17 patients had T2c tumors, and 7 patients had T3 disease. Patients were treated with (a) 45.6 Gy whole pelvis external irradiation and (b) three HDR fractions of 5.5 Gy each (18 patients) or 6 Gy each (15 patients) to the prostate. Transperineal needle implants using real-time ultrasound guidance with interactive on-line isodose distributions were performed on an outpatient basis during weeks 1, 2, and 3 of external irradiation. Acute toxicity was scored using the Radiation Therapy Oncology Group (RTOG) morbidity grading system. RESULTS: This technique of concurrent external pelvic irradiation and conformal HDR brachytherapy was well tolerated. No significant intraoperative or perioperative complications occurred. Three patients (9%) experienced Grade 3 acute toxicity (two dysuria and one diarrhea). All toxicities were otherwise Grades 1 or 2 and were primarily as expected from pelvic external irradiation. Persistent implant-related toxicities included Grades 1-2 perineal pain (12%) and hematospermia (15%). Median follow-up time was 13 months. Serum prostatic-specific antigen (PSA) levels normalized in 91% of patients (29 out of 32) within 1-14 months (median 2.8 months) after irradiation. PSA levels were progressively decreasing in the other three patients at last measurement. Prospectively planned prostatic rebiopsies done at 18 months in the first 10 patients were negative in 9 out of 10 (90%). CONCLUSIONS: Acute toxicity has been acceptable with this unique approach using conformal high dose rate Ir-192 boost brachytherapy with concurrent external irradiation. The initial tumor response as assessed by serial PSA measurement and rebiopsy is extremely encouraging. Dose escalation will proceed in accordance with the protocol guidelines. Further patient accrual and longer follow-up will allow comparison to other techniques. PMID- 7543892 TI - Prostate cancer and radiation therapy--the message conveyed by serum prostate specific antigen. AB - PURPOSE: Prostate-specific antigen (PSA) is a powerful pretreatment prognosticator and a sensitive post-treatment outcome measure for clinically localized prostate cancer treated with radiation therapy. Today, the pretreatment serum PSA level appears to supersede both grade and T-stage as a determinant of outcome. This study was undertaken to attempt a reconciliation between the old (pre-PSA) and the new (PSA) data-in particular to address the question of why stage and grade apparently play so little role in this PSA era. METHODS AND MATERIALS: We analyzed the outcome of two cohorts of men with T1-T4, NO, or NX, MO prostate cancer, one group (648 patients) treated and followed in the pre-PSA era (1966-1988), another group (707 patients) treated and followed in the PSA era (1987-1993)--who received definitive radiation as their only initial treatment. The patterns of relapse and prognostic factors for these groups were compared and contrasted using univariate and multivariate techniques. RESULTS: At a median follow-up of 6.5 years, the relapse patterns in the pre-PSA series were: local in 109 (17%), nodal in 17 (3%), and distant metastatic in 186 (29%). Actuarial local and metastatic rates at 5 years were 13 and 26%, respectively. Local recurrence was only weakly predictable, Gleason grade being the only significant, albeit weak, covariate. Metastatic failure, however, was highly significantly and meaningfully correlated with Gleason grade and T-stage. Because metastasis was the most common adverse end point in this series, overall freedom from progression also correlated with grade and stage. At a median follow-up of 31 months, the patterns of failure in the PSA series were: local in 77 (11%), nodal in 3 (< 1%), and distant metastatic in 24 (3%). Actuarial local and metastatic rates at 5 years were 30 and 6%, respectively. Local recurrence was highly and meaningfully correlated with pretreatment PSA level, which was the only significant determinant of this end point. Metastatic failure was highly correlated with Gleason grade and T-stage, with PSA playing a much lesser, though significant role. The inversion of failure patterns (local vs. distant) between the two series was striking. The high incidence of local failure in the PSA series was almost entirely related to positive prostatic biopsies pursuant to the investigation of the postradiation rising PSA profile. Of the 77 local recurrences, 69 (90%) were identified in this way. Among 99 men with rising PSA values who underwent investigation (CT scans, bone scans, and biopsies), disease was found in 86, and the patterns of disease in these 86 were: local only in 62 (72%), local and metastatic in 7, and metastatic in 17 (20%). The most common event in the PSA series was the rising PSA profile, and this, too, strongly correlated with the pretreatment PSA level. CONCLUSION: Based on our earlier finding that the major source of pretreatment serum PSA in patients with clinically localized disease is the primary tumour itself and on the findings in the present report, we conclude that the new major message conveyed by serum PSA relates to the primary tumor and its likely outcome. Gleason grade and T-stage remain major determinants of metastatic relapse. The total and permanent eradication of prostate cancer from the prostate with conventional doses of external beam radiation therapy is harder to achieve than generally appreciated. PMID- 7543893 TI - Adjuvant radiotherapy for pathologic stage T3/4 adenocarcinoma of the prostate: ten-year update. AB - PURPOSE: To determine the role of adjuvant postoperative radiotherapy (RT) following radical prostatectomy (RP) in a group of patients with pathologic Stage T3/4 adenocarcinoma of the prostate followed for a median of 10 years after treatment. METHODS AND MATERIALS: Between 1970 and 1983, 159 patients underwent RP for newly diagnosed adenocarcinoma of the prostate and were found to have pathologic Stage T3/4. Forty-six received adjuvant RT and 113 did not. Radiotherapy usually consisted of 45-50 Gy to the whole pelvis followed by a boost to the prostate bed of 10-15 Gy, to a total dose of 55-65 Gy. Patients were analyzed with respect to survival, disease-free survival, local control, and freedom from distant metastases. A rising prostate-specific antigen in the absence of other evidence of relapse was scored as a separate category of recurrence. RESULTS: Both groups of patients have been followed for a median of 10 years. The actuarial survival at 10 and 15 years was 62% and 62% for the RT group compared to 52% and 37%, respectively, for the RP group (p = 0.18). The disease-free survival for the Rt group was 55% and 48% at 10 and 15 years, respectively, compared to 37% and 33% for the RP group (p = 0.16). Similarly, there was no difference in the rate of distant metastases between the two groups. In contrast, the local relapse rate was significantly reduced by the addition of postoperative radiotherapy. The actuarial local control rate at 10 and 15 years was 92% and 82%, respectively, for the RT group vs 60% and 53% for the RP group (p = 0.002). CONCLUSIONS: While postoperative pelvic RT significantly improves local control compared to RP alone for pathologic Stage T3/4 prostate cancer, it has no impact on distant metastases and consequently does not improve survival. These data are consistent with the conclusion that many patients with pathologic Stage T3/4 prostate cancer have occult metastases at presentation and will not be cured by local therapies alone. The optimal treatment for this patient population remains to be established. PMID- 7543896 TI - CrmA-inhibitable cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein during Fas- and tumor necrosis factor-induced apoptosis. AB - Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that, when stimulated with ligand or cross-linking antibody, trigger apoptotic cell death by a mechanism that has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor than can inhibit interleukin-1 beta converting enzyme (ICE) and ICE-like proteases. We showed previously that expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways (Tewari, M., and Dixit, V.M. (1995) J. Biol. Chem. 270, 3255-3260). Here we report that the 70 kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage was inhibited by expression of CrmA, but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway. PMID- 7543897 TI - Characterization of the antigenic specificity of four different anti-(alpha 2-->8 linked polysialic acid) antibodies using lipid-conjugated oligo/polysialic acids. AB - A rapid, sensitive, and facile method for screening and characterizing anti polysialic acid (polySia) antibodies using lipid-conjugated oligo/polysialic acids (oligo/polySia) was developed, which is based on an enzyme-linked immunosorbent assay. Homooligo/polymers of alpha 2-->8-linked N-acetylneuraminic acid (Neu5Ac), N-glycoly-neuraminic acid, and 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE). Using this method, the anti-polySia equine antibody, H.46, bound to (-->8Neu5Ac alpha 2-->)n-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as 25 pg/well of oligo/polyNeu5Ac-PE, while 0.4 ng/well of oligo/polyNeu5Ac-PE to be detected. mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of polymerization (DP), the minimum chain length for immunoreactivity of the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP 3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3 and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding that is of practical value for identifying shorter oligoSia chains in glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate between short and long chains of polySia when both are expressed on the same molecule. PMID- 7543899 TI - Epitopic regions for antibodies against tumor necrosis factor alpha. Analysis by synthetic peptide mapping. AB - The location of biologically relevant epitopes on human tumor necrosis factor alpha (hTNF-alpha) was evaluated by testing the immunoreactivity of anti-TNF alpha antibodies against 149 sequential, overlapping octamer peptides. A goat polyclonal antibody raised against recombinant hTNF-alpha, which neutralizes hTNF alpha biological activities, reacted with oligopeptides corresponding to hTNF alpha residues 7-11, 17-23, 30-39, 42-49, 106-112, and 135-142. A possible assembled epitopic region (residues 25, 27, and 144) for neutralizing murine monoclonal antibodies designated 11D7G4 and 9C4G5 was deduced from the fact that they bound to tripeptides, mimicking a discontinuous epitope. These antigenic regions wer found to include of adjoin poorly conserved amino acids and they were located in the turns between beta-sheets on the surface of the molecule. Three of the sequential epitopic regions and an assembled region were closely related to the receptor binding sites proposed in several other studies. These antibodies appear to neutralize TNF-alpha activities by directly masking receptor binding sites. PMID- 7543900 TI - Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain. AB - We have analyzed two human immunodeficiency virus (HIV-1) reverse transcriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-primer. These enzymes, G262A and W266A, were previously shown to have greatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3' azidodeoxythymidine 5'-triphosphate. Here we describe their processivity and error specificity. The results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence-dependent termination of processive synthesis and in error specificity, and (iii) the magnitude of the mutator effect relative to wild-type enzyme for deletions in homopolymeric sequences decreases as the length of the run increases. Thus amino acid substitutions in a subdomain thought to interact with the duplex template-primer confer a strand slippage mutator phenotype to a replicative DNA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity. These effects, obtained in aqueous solution during catalytic cycling, are consistent with and support the existing crystallographic structural model. PMID- 7543898 TI - Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. AB - The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3 kinase (Pandey, A., Lazar, D.F., Saltiel, A. R., and Dixit, V.M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule. PMID- 7543895 TI - The replication of an IncL/M plasmid is subject to antisense control. AB - A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnal gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed. PMID- 7543894 TI - Localization of ornithine decarboxylase (ODC) in the cochlea of the immature rat. AB - Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents. PMID- 7543901 TI - Non-opioid-based approaches to burn pain. AB - Burn pain is almost always acute, and treatment strategies are often on the opposite end of the spectrum from chronic pain. However, many of the techniques developed for chronic pain can be useful for burn pain, particularly when the problem involves characteristics of both. The cognitive styles that patients bring to burn care and the manner in which they interpret nociception provide a rich source of intervention strategies. Learning theory can be extremely useful in pain control, particularly if such principles are structured into the overall manner in which care is provided (e.g., medication schedules and therapy approaches). Because patients in the burn unit often undergo significant distress, less conventional approaches such as hypnosis may assume a legitimate role. Finally, the needs of children have been neglected in the burn pain literature, and efforts on adjunctive techniques should also consider this population. PMID- 7543903 TI - Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: implications for potentiation of growth factor action by heparin. AB - Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1987): J Cell Physiol 131:123-130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factors for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates from the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interaction of FGF with FGFR, possibly by the formation of a ternary complex. PMID- 7543904 TI - Progesterone receptor does not form oligomeric (8S), non-DNA-binding complex in intact cell nuclei. AB - We raised a polyclonal antibody, alpha D, against a synthetic peptide (amino acids 522-535) of chicken progesterone receptor (PR). The sequence is located between the DNA-binding domain and the hormone-binding domain in the region within the sequences required for stability of the oligomeric form of PR. In the immunoblot, alpha D reacted with both A and B forms of PR. In the sucrose gradient and dot-blot the antibody did not recognize the so-called 8S form of PR, which is an oligomeric complex of PR and other proteins. When the oligomeric complex was dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 4S form. To study whether a similar complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, freeze-drying-vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear staining was detected independent of the fixation technique. When receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (8S). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an increased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These results suggest that the oligomeric complex (8S), present in tissue extracts, does not exist in intact cell nuclei. They also call into question the proposed role of hsp90 in regulating progesterone receptor function. PMID- 7543905 TI - Effect of substance P on uterine mast cell cytokine release during the reproductive cycle. AB - There is increasing evidence that neuropeptides, steroid hormones and inflammatory cytokines influence the immune response during the reproductive cycle. In the present study, we focus on the effects of neuropeptide Substance P (SP) during the pre-implantation stage of embryo development (day 4 of pregnancy), at pro-estrus and di-estrus (two phases with different hormonal states). We found heterogeneous responses to SP and anti-IgE by the rat uterine mast cells (MCs), as detected by ELISA. In fact, MCs purified from uteri on day 4 of pregnancy released histamine, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in response to anti-IgE, but not to SP. When pre-incubated with SP, the release to anti-IgE was significantly enhanced compared to anti-IgE alone. Exposure of SP to antibodies to SP, prior to pre-incubation with MCs, negated the SP effect on IgE-mediated release. At the pro-estrus phase SP showed similar behavior as on day 4 of pregnancy, whereas at the di-estrus phase SP alone was capable of inducing release of histamine and cytokines from purified uterine MCs. Moreover, non-quantitative RT-PCR analysis of the TNF-alpha mRNA level suggested an SP stimulation at the di-estrus phase, but neither on day 4 of pregnancy nor at the pro-estrus phase. Taken together, these data strongly suggest that SP can modulate IgE-mediated uterine MC release of histamine and inflammatory cytokines in different ways, depending on the phase of the reproductive cycle. PMID- 7543906 TI - Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S nitrosocysteine. AB - Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 x 10(-7) M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS. PMID- 7543907 TI - Subclass of IgG antibody to GM1 epitope-bearing lipopolysaccharide of Campylobacter jejuni in patients with Guillain-Barre syndrome. AB - Sera of patients who develop Guillain-Barre syndrome (GBS) subsequent to Campylobacter jejuni enteritis frequently have IgG anti-GM1 antibody. Lipopolysaccharide (LPS) of C. jejuni isolated from a GBS patient has a GM1 ganglioside-like structure. IgG subclass distribution of the anti-GM1 antibody in GBS patients is mainly restricted to IgG1 and IgG3. Since IgG antibodies to bacterial polysaccharide generally are restricted to IgG2 subclass, some investigators have assumed that either the general rules for immune response to LPS are broken in the patients or an alternative antigen has yet to be identified. To clarify whether the LPS participates in the production of the anti GM1 antibody, we investigated the subclass of IgG antibody to the LPS that bears GM1-like structure. The subclasses of IgG antibody to the LPS were restricted predominantly to IgG1 and IgG3. The GM1 epitope-bearing LPS may function in the production of the anti-GM1 antibody in patients with GBS subsequent to C. jejuni infection. PMID- 7543902 TI - The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo. AB - The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM-1 (or of its Fab fragment) with arterial SM actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not exerted on skeletal muscle actin. After microinjection of the alpha-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM actin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha skeletal [SK] actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an actin isoform plays a role in the regulation of polymerization in vitro and in vivo. PMID- 7543908 TI - Differential recognition of peptide analogs by naive verses activated PLP 139-151 specific CD4+ T cells. AB - CD4+ T cells specific for PLP 139-151 induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease multiple sclerosis (MS) in both clinical course and histopathology. Conservative and nonconservative amino acid substitutions were introduced at three TcR or MHC contact residues within PLP 139-151 to identify fine specificity requirements, at the polyclonal level, for stimulating naive encephalitogenic T cells and for reactivating pre-primed autoreactive T cells as measured by T cell proliferation, cytokine induction, and functional encephalitogenic potential. The results indicate that peptides with substitutions at position 145 exhibited a significantly diminished ability to induce active disease, but these substitutions had little or no effect on the ability to activate PLP 139-151-primed T cells for proliferation or disease transfer. A conservative or a nonconservative substitution at position 144 ablated both encephalitogenic potential in active and adoptive EAE models and the ability to induce proliferative responses in T cells primed to the native peptide. A nonconservative lysine for glycine, but not a conservative serine substitution, at position 146 had similar effects. In contrast to their inability to induce active EAE and stimulate in vitro proliferation of PLP 139-151-primed T cells, the Y144 and the 146 analog peptides were able to suboptimally reactivate these cells for transfer of adoptive EAE. Furthermore, the nonencephalitogenic K146 peptide was found to exacerbate in vivo induction of EAE induced by priming with a suboptimal dose of PLP 139-151. These data support the hypothesis that naive neuroantigen-specific CD4+ T cells have more stringent activation requirements than do PLP 139-151-specific T cells which have previously encountered antigen. The finding that the analog peptides induced differential patterns of cytokine production, with LT/TNF-alpha production but not IFN-gamma production correlating with full encephalitogenic potential, suggests different functional outcomes may result from differential levels of signal transduction triggered by the substituted peptides. The significance of these results to the potential development of autoimmune disease via molecular mimicry and for the development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed. PMID- 7543909 TI - Developmental changes in the distribution of NADPH-diaphorase-containing neurons in telencephalic nuclei of the zebra finch song system. AB - Extensive recent research has focused on the potential role of nitric oxide (NO) in synaptic plasticity. Could the capacity to synthesize NO be associated with neural and behavioral plasticity in the song system? The timing of song learning and of major developmental changes in song system anatomy are known. We searched for an association between NO and these developmental events by observing the distribution of neurons staining for NADPH-diaphorase, an enzyme used in the synthesis of NO, in the brains of zebra finches. Both male and female brains were taken at different developmental ages from day 21 to adulthood. We found that the incidence of stained neurons in the song system nuclei is lower than in surrounding areas. The incidence of staining decreases with development, with most of the decrease occurring prior to the auditory learning phase of song learning. The developmental changes were quantified for area X and found to be highly significant, with a 56% decrease in staining frequency from day 21 to adulthood in males and a 23% decrease in females for the equivalent region. We also found a sexual dimorphism in the song system of adult birds, consisting of a reduced incidence of stained neurons in song system nuclei area X, high vocal center (HVC), and nucleus robustus (RA) archistrialis in males compared with females. These findings suggest that NO is less involved in the plasticity underlying song acquisition than in the earlier formation of the song system. PMID- 7543910 TI - Morphological classification of retinal ganglion cells in mice. AB - Mice have been used for extensive studies on optic nerves and retinal ganglion cells, but mouse retinal ganglion cells have not been classified morphologically. In the present study, normally placed retinal ganglion cells and displaced retinal ganglion cells in pigmented and albino mice were classified morphologically using horseradish peroxidase. These cells were classified into three types according to the sizes of the soma and the dendritic field: type I cells, large soma and large dendritic field; type II cells, small-to-medium soma and small dendritic field; and type III cells, small-to-medium soma and large dendritic field. Some ganglion cells had both symmetric and asymmetric cells. Each type was further subdivided according to the termination level of dendrites in the inner plexiform layer and the dendritic branching pattern. Except for type III displaced ganglion cells, dendrites of the normally placed ganglion cells and the displaced ganglion cells ramify in the outer two-fifths of the inner plexiform layer (sublamina a) or the inner three-fifths of the inner plexiform layer (sublamina b). Type III displaced ganglion cells ramify only in sublamina a. Dendrites of some normally placed type I ganglion cells ramify in both sublaminae. Displaced biplexiform cells were observed, the dendrites of which ramify in both the inner and the outer plexiform layers. All cell types were found in both mouse strains. PMID- 7543913 TI - Recruitment of immunocompetent cells after dentinal injuries in innervated and denervated young rat molars: an immunohistochemical study. AB - The dental pulp represents a peripheral end-organ deprived of a collateral nerve supply. After inferior alveolar nerve (IAN) axotomy, rat molar pulp is denervated over a period of at least 6 days. Therefore, rat molar pulp was used as an experimental model to study the effect of sensory nerve fibers on influx of immunocompetent cells after dentinal injury. In the present study we performed a quantitative analysis of CD43+, CD4+, CD11b+, and I-A antigen-expressing cells subjacent to dentinal cavities in denervated and innervated first mandibular molars. For visualization of nerve fibers, antibodies to protein gene product (PGP) 9.5, the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the sympathetic neuropeptide Y (NPY) were used. Immunohistochemistry was performed by the avidin-biotin-peroxidase method. In the innervated teeth, a correlation between increased sensory nerve density and influx of immunocompetent cells was found. Compared to the contralateral innervated molars, a significant reduction in recruitment of immunocompetent cells was found in the denervated pulp tissue subjacent to the dentinal cavities. The rat molar represents a unique model to illustrate the influence of sensory nerves and neuropeptides on inflammation and recruitment of immunocompetent cells. PMID- 7543912 TI - Effects of fixation and paraffin embedding on the immunohistological detection of cell-associated HIV-1 by different monoclonal antibodies. AB - This study evaluates a panel of monoclonal antibodies (MAbs) to HIV-1 antigens (DuPont anti-gp120, gp41, p24; Olympus anti-gp120/160, gp41, p24A, p24B, p55, p18A, p18B, reverse transcriptase) for their ability to detect the virus in tissues after exposure to various fixatives (100% acetone, 10% formaldehyde, 2.5% glutaraldehyde, 4% paraformaldehyde/1% glutaraldehyde, Bouin's fluid) and after paraffin embedding. Acetone, 10% formaldehyde, and Bouin's fluid all preserved a wide range of viral epitopes compared with other fixatives. The most robust MAbs were DuPont p24 and Olympus p55, which produced excellent staining regardless of the fixative used. Embedding in paraffin variability influenced the capacity of MAbs to detect HIV-1 epitopes on fixed cells. Certain antibodies (e.g., DuPont gp24, Olympus p24B) produced good staining, whereas other epitopes (e.g., DuPont gp120, formaldehyde) were destroyed. In some cases, paraffin embedding revealed antigenic sites that had been formerly masked (e.g., Olympus gp120 and p24A; formaldehyde and glutaraldehyde fixation). These results indicate that HIV-1 antigens can be detected by immunohistology on cells exposed to most common fixatives. Therefore, retrospective analysis of pathological material is possible, provided that the antibodies are matched to the fixative used to preserve the tissue. PMID- 7543914 TI - Fluorescent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells. AB - The fungal metabolite brefeldin A (BFA) interferes with vesicular trafficking in most animal cells. To gain insight into the mechanism of BFA action, we esterified it to the fluorophore, boron dipyromethene difluoride (BODIPY). BODIPY BEA localized predominantly in the endoplasmic reticulum (ER) and Golgi complex of viable cells and was extracted by detergent treatment, suggesting it interacts primarily with lipid bilayers. The localization of the conjugate is conferred by BFA, since free BODIPY or BODIPY esterified to cyclopentanol did not specifically localize to internal membranes. BODIPY-BFA exhibited a similar biological activity to BFA, but only when used at higher concentrations and after a delay. HPLC analysis revealed that over this period, cells converted BODIPY-BFA to species co-eluting with free BODIPY and BFA. Therefore, BODIPY-BFA is probably inactive until BFA is released by cellular esterases. The specific localization of BODIPY-BFA to the ER and Golgi complex suggests that BFA might exert its effects on vesicular trafficking by perturbing the lipid bilayer of its target organelles. Because BODIPY-BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions, it should be useful for visualizing the ER in living cells. PMID- 7543911 TI - Platelet receptor desensitization induced by elevated prostacyclin levels causes platelet-endothelial cell adhesion. AB - OBJECTIVES: The purpose of this study was to investigate the role of platelet prostacyclin receptor desensitization in platelet-endothelial cell adhesion. BACKGROUND: Platelet-endothelial cell adhesion is regulated by endothelial cell derived mediators, such as prostacyclin and endothelium-derived relaxing factor. Prostacyclin activates platelet adenylate cyclase and augments cyclic adenosine monophosphate formation by way of specific membrane receptors. Platelet exposure to prostacyclin or chemically stable analogs results in a time- and dose dependent prostacyclin receptor desensitization as it occurs during infusion therapy with prostacyclin analogs or in pathophysiologic situations such as acute myocardial infarction. METHODS: Adhesion of washed and radiolabeled human platelets stimulated with thrombin to cultured umbilical vein endothelial cells was measured under control conditions and under conditions of platelet prostacyclin receptor desensitization induced by incubation with the prostacyclin analog iloprost (10 to 100 nmol/liter) for 3 h. RESULTS: Thrombin (0.08 to 0.2 U/ml) increased platelet adhesion in a dose-dependent manner from 2.7 +/- 0.3% to 6.4 +/- 0.6% (mean value +/- SEM). Preincubation of platelets resulted in a dose dependent down-regulation of 3H-iloprost binding up to 58.8 +/- 6.7% of control platelets with 100 nmol/liter of iloprost. Co-incubation of prostacyclin receptor desensitized platelets with endothelial cells resulted in a marked augmentation of thrombin-induced adhesion up to 28.6 +/- 4.5%. Approximately the same increase in platelet adhesion was seen after complete abrogation of endothelial cell prostacyclin synthesis by pretreatment with aspirin. Comparison of iloprost induced receptor desensitization and increased platelet-endothelial cell adhesion indicated a positive correlation. CONCLUSIONS: Platelet prostacyclin receptor desensitization was observed in humans in vivo during acute myocardial infarction or during therapeutic administration of prostacyclin analogs. In vitro platelet prostacyclin receptor desensitization caused a marked augmentation of platelet endothelial cell adhesion. This increase in adhesion might result in an enhanced tendency toward thrombus formation in humans. PMID- 7543915 TI - Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain III. AB - Perlecan is the best-characterized basement membrane heparan sulfate proteoglycan. It has a large (approximately 400 KD) core protein consisting of five distinct domains. Domain III, a centrally located domain, contains three globular domains separated by cysteine-rich epidermal growth factor (EGF)-like repeats. Domain III has overall homology with the N-terminus of the laminin alpha 1-chain. The aim of this study was to map a library of nine rat monoclonal antibodies (MAbs) against murine perlecan core protein, using recombinant whole Domain III and defined subdomains of Domain III. ELISA and Western blotting showed that six of the nine MAbs recognized Domain III of perlecan, three of them mapping to globular Subdomain IIIc, and the other three recognized epitopes within the cysteine-rich regions. All six MAbs stained every basement membrane of several mouse organs as well as some connective tissues, including cartilage. Therefore, several distinct epitopes of perlecan Domain III are present in most if not all basement membranes and are not obscured by intermolecular interactions. These precisely mapped antibodies may therefore be useful in understanding the function of perlecan and its core protein. PMID- 7543918 TI - Mast cells in aural polyps: a preliminary report. AB - Mast cells are a rich source of potent biologically active mediators and are found in connective tissue, associated with blood vessels in many varied inflammatory conditions. Mast cells have been described in nasal polyps and turbinates and in adenoidal tissue in the upper aerodigestive tract. As the middle ear lining is contiguous with the nose and the nasopharynx, the presence of mast cells in aural polyps is interesting. This preliminary study investigated the presence of mast cells in inflammatory aural polyps using light microscopy. All patients presenting to the department in one year were included. Patients with previous ear disease or surgery and in whom cholesteatoma was suspected were excluded. Except for one patient mast cells were seen in all aural polyps. The implications of these findings is discussed. Further work is needed using electron microscopy. PMID- 7543917 TI - Effect of protease inhibitors on peptide-stimulated amylase secretion from dispersed pancreatic acini. AB - Cholecystokinin (CCK) and bombesin stimulate dose-dependent amylase secretion from dispersed pancreatic acini. To establish whether cellular proteases can reduce secretion by degrading these regulatory peptides, the effect of protease inhibition on CCK and bombesin stimulated amylase secretion was investigated. A spectrum of protease inhibitors, including bacitracin, phenylmethylsulfonylfluoride, captopril, bestatin, phosphoramidon, and 1,10 phenanthroline, were investigated. Bacitracin (0.35 mM) increased the acinar amylase secretory response to CCK and bombesin substantially, suggesting that these two peptides are degraded by an endopeptidase from pancreatic acinar cells. In contrast, PMSF (1 mM) inhibited CCK and bombesin stimulated amylase release, suggesting a covalent interaction with this inhibitor and CCK or bombesin receptors. Other protease inhibitors either had minimal or no effects on acinar cell secretion. These results suggest bacitracin is a valuable enzyme inhibitor that can potentiate the effect of CCK and bombesin on acinar cells. In contrast, PMSF should be avoided when using these secretagogs to study pancreatic function. PMID- 7543916 TI - Contrast staining with reduced osmium complexes. PMID- 7543919 TI - Graded changes in the response of individual human basophils to stimulation: distributional behavior of events temporally coincident with degranulation. AB - Human basophils, stimulated with either anti-IgE antibody or formyl-methionine leucine-phenylalanine, were examined by two measures of the cell response that may reflect degranulation. Flow cytometric measurement of either of these two measures, changes in forward scatter intensity (an indirect measure of the basophil size) or changes in the intensity of acridine orange-loaded cells (which labels basophil granules), allowed an assessment of the distribution of single cell responses. With regard to the latter technique, structures that appeared to be basophil granules were shown to metachromatically label with low concentrations of acridine orange, which has little or no effect on histamine release. During stimulation these labeled granules were lost, leading to decreased fluorescence. Changes in either the forward scatter parameter or acridine orange labeling occurred on the same time scale as histamine release, differentiating these measures of the basophil response from early signal transduction events. Challenging basophils with a combination of phorbol myristate acetate and ionomycin caused 100% histamine release and allowed a measurement of the maximum change in forward scatter intensity or loss of acridine orange labeling. The flow cytometric distributions after this treatment were then compared with the distributions obtained by challenging cells with several concentrations of anti-IgE antibody or formyl-methionine-leucine phenylalanine, which induced various submaximal responses. These flow cytometric distributions demonstrated that single cells could be found in intermediate states of activation, i.e., the response of all cells was graded according to the strength of the stimulus. These studies lead to the general conclusion that all aspects of the basophil response, including those late events in the basophil response we have studied here, as well as early events that we have studied previously, are graded in a continuous manner, according to the magnitude of the stimulus. PMID- 7543920 TI - Monomeric bovine IgG2 is a potent stimulus for bovine neutrophils. AB - Bovine plasma contains factors that can stimulate bovine neutrophils. Bovine plasma at 1:1,000,000-1:1,000 dilution induced a dose-dependent superoxide production in bovine neutrophils. When bovine plasma was fractionated with a protein G column, only the IgG fraction contained induction activity. At similar concentrations purified IgG induced a much stronger response than that of plasma IgG. Purified monomeric bovine IgG induced a dose-dependent increase in superoxide production. The maximum induction can be achieved at 100 micrograms/ml of bovine IgG. When subclasses of bovine IgG were examined, monomeric bovine IgG2 potently stimulated bovine polymorphonuclear leukocytes. In contrast, bovine IgG 1 failed to induce a response at similar concentrations, and neither bovine IgG F(ab')2 and Fc were effective. Both recombinant bovine granulocyte-macrophage colony-stimulating factor (r-BoGM-CSF) and recombinant bovine granulocyte colony stimulating factor (r-BoG-CSF) primed bovine neutrophils for superoxide production induced by bovine IgG. The above results suggest that: (1) bovine plasma contain factors that can activate bovine neutrophils; (2) bovine plasma IgG is the major component that is responsible for bovine neutrophil activation; (3) bovine plasma contains factors that can inhibit the effect of bovine IgG; (4) monomeric bovine IgG2, but not IgG1, can activate bovine neutrophils directly without Fc receptor cross-linkage; (5) the integrity of bovine IgG is important in bovine polymorphonuclear leukocyte activation; and (6) bovine neutrophil activation induced by bovine IgG can be primed by r-BoGM-CSF or r-BoG-CSF. PMID- 7543924 TI - Hematoxylin-lac-curcuma polychrome stain for mucin. AB - A polychrome method for detection of mucin substance in paraffin section is produced by sequential stepwise staining of hematoxylin, crude lac extract (Laccifer lacca), and crude curcuma extract (khamin shan-Curcuma longa). The name LacCur stain is proposed. After a tissue section is deparaffinized and rehydrated, it is stained with Weigert's hematoxylin for 7 minutes. After a quick wash, it is stained for at least 3 hours with lac dye mordanted with aluminum chloride. Washed again and premordanted with ferric chloride for 1 minute, in the last step, it is counterstained with curcuma dye for 5 minutes. With this staining method, the nuclei are stained black, mucin deep red, and organelles and ground substances brownish yellow. The method and outcome colors are comparable to the widely used Mayer's mucicarmine staining method. It costs less than the Mayer's mucicarmine staining method and the procedure is not complicated. PMID- 7543923 TI - Regulation of NK cells through the 80-kDa TNFR (CD120b). AB - By using monoclonal antibody specific for tumor necrosis factor receptor80 (TNFR80) (CD120b) and TNFR60 (CD120a), we determined which receptor transduces the signals involved in activating natural killer (NK) cells. Purified CD56+CD3- large lymphocytes express TNFR80 but not TNFR60 and interleukin-2 (IL-2) up regulates TNFR80 expression, consistent with NK cells being activated in vivo. Treatment of NK cells with anti-TNFR80 for 18 h enhanced the NK activity detected on K562 target cells mimicking the effect of TNF. In combination with IL-2, TNF enhanced the development of lymphokine-activated killing. However, only anti TNFR80 abrogated IL-2 induction of lymphokine-activated killer cell activity. The activity of TNF or anti-TNFR80 was selective for NK cytotoxic function because they did not directly mimic IL-2 activation or induce significant proliferation, expression of cell surface activation antigens (CD25 or HLA-DR), or interferon gamma secretion. These results indicate that TNFR80 is an important signal transducing receptor for the differentiation of NK cells induced by TNF and IL-2. PMID- 7543922 TI - Effect of PGE2 and of agents that raise cAMP levels on macrophage activation induced by IFN-gamma and TNF-alpha. AB - The effect of prostaglandin (PG) E2 on macrophage activation by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) was evaluated. Murine macrophages infected with Leishmania enriettii or Leishmania major were activated by exposure to IFN-gamma (10-50 U/ml) and TNF-alpha (30-3000 U/ml), leading to intracellular parasite destruction within 24-48 h. Leishmanicidal activity was markedly increased when activation was performed in the presence of PGE2 (10(-9) 10(-7) M) or arachidonate (10(-5) M, a PG precursor), concomitant with enhanced nitrite release and glucose oxidation through the hexose monophosphate shunt pathway. Conversely, activation was reduced by indomethacin and hydrocortisone, two inhibitors of PG synthesis. Parasite killing and nitrite production were fully restored by exogenous PGE2, indicating that inhibition by these drugs was related to their ability to block PG production. PG can stimulate adenylate cyclase, thus raising intracellular cAMP levels. Accordingly, dibutyryl-cAMP, theophylline (which prevents cAMP breakdown), and forskolin (an activator of adenylate cyclase) all stimulated macrophage activation. Finally, PGE2 and cAMP enhanced expression of inducible nitric oxide synthase mRNA in response to IFN gamma and TNF-alpha, and this effect was inhibited by the cAMP antagonist 2'-O methyl adenosine. These findings are consistent with the hypothesis that PGE2 acts as a positive agonist in macrophage activation by IFN-gamma and TNF-alpha via its capacity to modulate intracellular cAMP levels. PMID- 7543921 TI - Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up regulation and IL-6 production and proliferation. AB - CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation. PMID- 7543925 TI - Comparison of human polymorphonuclear neutrophil elastase, polymorphonuclear neutrophil cathepsin-G, and alpha 2-macroglobulin levels in healthy and inflamed dental pulps. AB - Polymorphonuclear neutrophils (PMNs) are found in dental pulp secondary to carious exposures, periodontal disease, or trauma. Lysosomal degranulation of these cells liberates cellular proteases, including elastase (PMN-E) and cathepsin-G (PMN-CG), which produce connective tissue degradation. However, nonspecific pulpal tissue destruction can be modified by a naturally occurring serum protease inhibitor alpha 2-macroglobulin (A2-M). This study relates the concentrations of human PMN-E, PMN-CG, and A2-M in healthy and inflamed pulpal samples. Evaluation of 21 specimens yielded statistically significant differences between healthy and moderate to severely inflamed pulps for all groups (p < 0.05). No significant correlation was detected among human PMN-E, PMN-CG, and A2 M in the healthy tissues (P > 0.05). However, in the moderate to severely inflamed pulps, there was a significant correlation between PMN-CG and A2-M (p < 0.05). PMID- 7543926 TI - Time-resolved signaling pathways of nerve growth factor diverge downstream of the p140trk receptor activation between chick sympathetic and dorsal root ganglion sensory neurons. AB - We have recently shown that the small GTP binding protein p21ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observed within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543927 TI - Melatonin biosynthesis in cultured chick retinal photoreceptor cells: calcium and cyclic AMP protect serotonin N-acetyltransferase from inactivation in cycloheximide-treated cells. AB - The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N-acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 microM forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of approximately 30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K+, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1 methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K+ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca2+ influx through dihydropyridine-sensitive channels. MDL-12330A, and inhibitor of K(+)-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K+. This result suggests that the effect of Ca2+ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization evoked Ca2+ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules. PMID- 7543928 TI - Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies. AB - Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4 propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro. PMID- 7543929 TI - d-fenfluramine increases striatal extracellular dopamine in vivo independently of serotonergic terminals or dopamine uptake sites. AB - The effect of various doses of the serotonin (5-HT) release-inducing agent d fenfluramine (d-fenf) on extracellular dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) was studied in vivo in the striatum of halothane-anesthetized rats, following systemic and local administration. At 5 and 10 but not 2.5 mg/kg, d-fenf administered intraperitoneally significantly increased DA extracellular concentration and reduced DOPAC outflow. A concentration-dependent enhancement of DA dialysate content was also found following intrastriatal application (5, 10, 25, and 50 microM). The bilateral administration of 5,7-dihydroxytryptamine into the dorsal raphe nucleus, which markedly depleted 5-HT in the striatum, did not modify the effect on extracellular DA concentration of 25 microM d-fenf locally applied into the striatum. The enhancement of extracellular DA level induced by 25 microM d fenf was slightly but significantly reduced by the local application of 25 microM citalopram. The blockade of DA uptake sites by nomifensine (0.1, 0.3, and 1 microM) did not modify significantly the effect of d-fenf. The rise of DA outflow induced by 25 microM d-fenf was strongly reduced in the presence of 1 microM tetrodotoxin (TTX) or by the removal of Ca2+ from the perfusion medium. The results obtained show that d-fenf increases the striatal extracellular DA concentration by a Ca(2+)-dependent and TTX-sensitive mechanism that is independent of striatal 5-HT itself or DA uptake sites. PMID- 7543931 TI - In vivo electrochemical monitoring of serotonin in spinal dorsal horn with Nafion coated multi-carbon fiber electrodes. AB - Biosensors sensitive for in vivo monitoring of serotonin (5-HT) in the CNS by differential normal pulse voltammetry were constructed by coating treated multicarbon fiber electrodes (mCFEs) with Nafion (N-mCFE). In vitro sensitivities of mCFE and N-mCFE were compared in solutions ranging from 5 nM to 20 microM of uric acid (UA), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. The mCFEs were three to seven times less sensitive for 5-HIAA or UA than for 5-HT. Nafion treatment dramatically decreased sensitivity for 5-HIAA and UA of N-mCFEs (approximately 10(3) times), whereas it remained in the nanomolar range for 5-HT. In vivo, in the dorsal horn of the lumbar spinal cord of anesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 +/- 3% at 180 min) of peak 3 of oxidation current (characteristic of 5 hydroxyindoles) monitored with mCFEs, but with N-mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 +/- 5% at 180 min. The 5-HT release inducer p-chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 +/- 3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with N-mCFEs PCA induced a rapid increase of peak 3N (137 +/- 6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 +/- 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N-mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 +/- 6% at 90 min) in peak 3N.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543932 TI - Characterization of the binding of [3H]NS 257, a novel competitive AMPA receptor antagonist, to rat brain membranes and brain sections. AB - The binding of [3H]NS 257 (1,2,3,6,7,8-hexahydro-3-(hydroxyimino)-N,N [3H]dimethyl-7-methyl- 2- oxobenzo[2,1-b:3,4-c']dipyrrole-5-sulfonamide) to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [3H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 mM. In the presence of thiocyanate [3H]NS 257 bound to a single population of binding sites with an affinity of 225 +/- 8 nM and a binding site density of 0.61 +/- 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [3H]NS 257 for both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and L-glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline 2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > L-glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [3H]NS 257 binding sites in rat brain was similar to that of [3H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate. PMID- 7543930 TI - Multiple levels for regulation of TrkA in PC12 cells by nerve growth factor. AB - TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140trkA was rapidly followed by down-regulation of cell surface and total cellular gp140trkA. At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression. PMID- 7543934 TI - Expression of inducible nitric oxide synthase in cerebral endothelial cells is regulated by cytokine-activated astrocytes. AB - A nitric oxide (NO) synthase (NOS) can be induced in both astrocytes and cerebral endothelial cells with a combination of interleukin-1 beta/interferon-gamma or lipopolysaccharide/interferon-gamma, respectively. Exogenous NO, either from the chemical donor spermine NONOate or from activated astrocytes, affected the expression of inducible NOS in cerebral endothelial cells. In cerebral endothelial cells pretreated with spermine NONOate the induction of NOS was reduced, as revealed by mRNA expression and nitrite accumulation. Cytokine treated astrocytes generating NO and placed in close proximity to endothelial cells decreased the expression of NOS induced by cytokines in endothelial cells. In addition, it was apparent that cytokine-activated astrocytes released a factor(s) that initiated transcriptional induction of NOS in cerebral endothelium. This suggests that astrocytes activated by cytokines in vivo could influence expression of inducible NOS in cells of the adjacent microvasculature. PMID- 7543933 TI - A charybdotoxin-sensitive, Ca(2+)-activated K+ channel with inward rectifying properties in brain microvascular endothelial cells: properties and activation by endothelins. AB - A charybdotoxin-sensitive, Ca(2+)-activated K+ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and 86(Rb+)-influx and efflux experiments. Channel activity was dependent on the presence of Ca2+ on the cytosolic face of the membrane with a threshold concentration of 100 nM. It was inhibited by charybdotoxin (IC50 30 nM) and quinine (IC50 0.1 mM) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC50 0.7 nM) and endothelin-3 (EC50 7-10 nM). The actions of endothelins were prevented by BQ-123 (Ki = 8 nM) in a competitive fashion, hence suggesting the involvement of an ETA-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca(2+)-activated K+ channels for mediating K+ movements across the blood-brain barrier is discussed. PMID- 7543936 TI - Analysis of connections between nitric oxide synthase neurons in the myenteric plexus of the guinea-pig small intestine. AB - In the myenteric plexus of the guinea-pig ileum, a sub-population of descending interneurons contains nitric oxide synthase. Final neurons in descending motility pathways, inhibitory circular muscle motor neurons, also contain nitric oxide synthase. In this study we used ultrastructural immunocytochemistry to determine whether nitric oxide synthase descending interneurons provide inputs to all nitric oxide synthase neurons. The presence of nitric oxide synthase inputs to 35 nitric oxide synthase nerve cells from three animals was examined. Nine nerve cells from one ganglion were studied in serial section. Every nerve cell received inputs (close contacts and synapses) from nitric oxide synthase terminals. The number of inputs to the nine serially sectioned neurons ranged from 13 to 45. The inputs were found in about equal numbers on the cell bodies and the dendrites. There was no significant correlation between the size of nitric oxide synthase neurons and the number of nitric oxide synthase inputs they received. There was also no correlation between the number of nitric oxide synthase inputs and the number of 5-hydroxytryptamine inputs (determined in a previous study) received by nitric oxide synthase neurons. Random sections through an additional 26 nitric oxide synthase neurons (seven in the same ganglion and 19 from another two myenteric ganglia from different animals) were examined and nitric oxide synthase synapses and close contacts were observed on each neuron. Nitric oxide synthase interneurons and motor neurons are morphologically indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543935 TI - Induction of heme oxygenase-1 mRNA and protein in neocortex and cerebral vessels in Alzheimer's disease. AB - Previous studies demonstrated the specific association of heme oxygenase (HO)-1 protein to the neurofibrillary pathology of Alzheimer's disease (AD). In this study, we used reverse transcription-polymerase chain reaction methods to show the increased expression of HO-1 but not HO-2 mRNA transcripts in cerebral cortex and cerebral vessels from subjects with AD compared with age-matched non-AD controls. Neither the HO-1 nor the HO-2 mRNA levels was altered in the cerebellum, a brain region usually spared from the pathological alterations of AD. There was no clear evidence that the expression of HO-1 in these tissues was related to postmortem interval, cause of death, or the age of the subjects studied. Using immunoblotting methods, we further showed that HO-1 protein content was increased in neocortical and vascular samples from AD subjects compared with controls. Our findings suggest the specific induction of HO-1 mRNA and protein in the cerebral cortex and cerebral vessels but not HO-2 mRNA or protein in association with the pathological lesions of the disease. PMID- 7543937 TI - Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location. AB - Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used for in situ and in vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable by in situ immunocytochemistry. However, B-type cells could be seen to express all three subunits in vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543938 TI - Unilateral basal ganglia involvement in primary progressive aphasia. PMID- 7543940 TI - Spinal cord compression in prostate cancer. AB - Approximately 200,000 men will be diagnosed with prostate cancer in 1994. While localized disease is potentially curable with surgery or radiation therapy, metastatic disease is incurable. The most frequent site of metastasis is bone. Spinal cord compression occurs in approximately 7% of men with prostate cancer. Back pain often heralds the diagnosis of spinal cord compression. In prostate cancer patients with back pain or signs of myelopathy or radiculopathy, plain radiographs of the spine and magnetic resonance imaging should be performed. Early diagnosis is of utmost importance. The neurologic status prior to treatment is the major determinant influencing outcome. Following diagnosis, corticosteroid therapy should begin immediately. Hormonal therapy should be instituted in those patients who have not previously undergone hormonal manipulation. The standard approach to definitive therapy is radiation. Surgical decompression plays a role in patients with severe myelopathy, spinal instability, and in those patients whose neurologic status deteriorates during or after radiation therapy. PMID- 7543942 TI - Motoneuron properties after operantly conditioned increase in primate H-reflex. AB - 1. Monkeys can increase (HRup conditioning mode) or decrease (HRdown conditioning mode) the triceps surae (TS) H-reflex in response to an operant conditioning task. This conditioning modifies the spinal cord. To define this spinal cord plasticity and its role in the behavioral change (H-reflex increase or decrease), we have recorded intracellularly from TS motoneurons in conditioned animals. The present report describes data from HRup animals and compares them with data from previously studied naive (NV; i.e., unconditioned) animals. 2. Thirteen monkeys (Macaca nemestrina, male, 3.8-7.1 kg) were exposed to the HRup conditioning mode, in which reward occurred when H-reflex size in one leg (i.e., the trained leg) was above a criterion value. Conditioning was successful (i.e., increase of > or = 20%) in 12 of the 13 animals. At the end of conditioning, H-reflex size in the trained leg averaged 188% of its initial value, whereas size in the control leg averaged 134% of its initial value. 3. Intracellular recordings were obtained from 136 TS motoneurons on trained (UT + motoneurons) and control (UC + motoneurons) sides of the successful animals. Measurements included axonal conduction velocity, input resistance, time constant, electrotonic length, rheobase, firing threshold to current injection, afterhyperpolarization duration and amplitude, and composite homonymous and heteronymous excitatory postsynaptic potential (EPSP) size and shape. Results were compared with intracellular data from NV animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543939 TI - The pathophysiology and management of spine metastasis from lung cancer. PMID- 7543941 TI - The role of endothelial cells in tumor invasion and metastasis. AB - Metastasis is one of the most devastating aspects of cancer. It is a complex multistep processes that results in spread of tumorigenic cells to secondary sites in various organs. The actual events that are involved in metastasis are the subject of several recent reviews [1-3]. Upon growth of neoplastic cells beyond a certain mass (2 mm in diameter) an extensive vascularization through angiogenesis occurs. The new capillary network provides a supply of nutrients and gas exchange that allows further growth and development of the tumor mass. The network of the blood vessels also provides an entry site into the circulation for the neoplastic cells that detach from the tumor mass. Only a small percentage of circulating tumor cells (< 0.01%) survive travel in the circulation and arrest in the capillary beds of distant organs, extravasate and proliferate within the organ parenchyma producing a successful metastasis [1]. Vasculature plays an important role in several steps of the metastatic process; 1) at the site of metastasis, vessels capture the cancer cell and provide the entry route into the secondary organ, and 2) through angiogenesis, vascular endothelial cells provide the supply of nutrients for the growth of the primary tumor mass and the route of intravasation. The lining of all blood vessels are covered with endothelial cells which play an active role in both processes. The metastatic properties of cancer cells have been extensively studied. Here, we will discuss the role of endothelial cells in the metastatic process with focus on their interaction with cancer cells at the site of extravasation. PMID- 7543943 TI - Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons. AB - 1. We determined the effect of the neuropeptide galanin on barium currents (IBa) flowing through voltage-gated calcium channels. We voltage clamped parasympathetic neurons dissociated from mudpuppy cardiac ganglia using both the standard whole cell and the perforated-patch variations of the patch-clamp technique. 2. Galanin produced a concentration-dependent inhibition of IBa. The maximal inhibition was 50-60% and the concentration that produced half-maximal inhibition (IC50) was 0.42 nM. In mud-puppy parasympathetic neurons, omega conotoxin-GVIA (CTX)-sensitive channels are the predominant type of calcium channels, and only a small portion of IBa is contributed by dihydropyridine sensitive channels. Galanin preferentially inhibited a portion of the CTX sensitive current. 3. In currents recorded with the standard whole cell technique, activation of IBa was slowed in the presence of galanin. In contrast, in the majority of neurons studied with the perforated-patch technique, galanin decreased IBa without altering the kinetics of current activation. With both recording methods, the decrease in IBa was greatest with voltage steps to 0 mV and persisted with steps to +50 mV. For control currents, large depolarizing voltage steps (+70 to +120 mV) did not markedly facilitate IBa when either recording technique was used. However, the degree of facilitation in galanin was significantly greater with the standard whole cell recording technique. 4. IBa exhibited inactivation under the conditions of these experiments. Inactivation of IBa recorded during a 900-ms depolarizing voltage step was fitted to a double exponential. Galanin decreased the amplitude of IBa but did not alter the time constants of inactivation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543944 TI - Inwardly rectifying currents of saccular hair cells from the leopard frog. AB - 1. Inwardly rectifying currents were characterized in sensory hair cells isolated from the saccules of leopard frogs, using the whole cell configuration of the patch-clamp technique in voltage-clamp mode. 2. Two types of inwardly rectifying currents were distinguishable based on their ionic selectivity, activation and deactivation kinetics, voltage dependence, dependence on external K+ and sensitivity to divalent cations. 3. One inwardly rectifying current displayed K+ selectivity, rapid monoexponential activation (tau approximately 1 ms at -120 mV), steep voltage dependence, dependence of the activation voltage range on external K+ and block by external Ba2+. We refer to this current as IK1, consistent with the terminology used for a similar current in cardiac cells. In 5 mM external K+, IK1 activated negative to -60 mV, was half-activated at -86 mV and fully activated by -110 mV. 4. The other inwardly rectifying current was a mixed K+/Na+ current with slow sigmoidal activation (slow tau approximately 100 ms at -120 mV) and deactivation, shallow voltage dependence and no dependence of the activation curve on external K+ and which was blocked by external Cd2+. This current was called Ih because of its similarities to Ih of photoreceptors. Ih activated negative to -50 mV, was half-activated at -90 mV and was fully activated at -130 mV. 5. A correlation between cell shape and the type of inwardly rectifying current was noted; the more spherical cells had Ih alone and the more cylindrically shaped cells had Ih and IK1. 6. The mean resting potential of 115 cells with IK1 and Ih was -68 +/- 0.5 mV (mean +/- SE) and that of 53 cells with Ih alone was -50 +/- 0.5 mV. This suggests that IK1 contributes to the more negative resting potential of the cylindrical cells. 7. In current-clamp mode, the voltage responses to current steps of the two cell populations differed. Small negative current steps evoked faster, smaller responses in cells with IK1 and Ih than in cells with Ih alone. In cells with Ih alone, long (> 100 ms) negative current steps evoked a hyperpolarization that partly repolarized as Ih activated. Cells with Ih alone showed electrical resonance at rest whereas cells with IK1 resonated only in response to positive current steps. 8. A model developed to explain electrical resonance in bullfrog saccular hair cells was adapted to include Ih or IK1 and Ih.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543945 TI - Excitatory postsynaptic currents and glutamate receptors in neonatal rat sympathetic preganglionic neurons in vitro. AB - 1. We obtained whole cell patch-clamp recordings from visually identified sympathetic preganglionic neurons (SPNs) in thin (200-300 microns) transverse spinal cord slices of neonatal rats (1-14 days postnatal). Exogenous application of glutamate (100 microM), N-methyl-D-aspartate (NMDA; 100 microM), kainate (100 microM), quisqualate (1 microM), and alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA; 50 microM) induced inward currents at a holding potential of -30 mV. 2. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation either in the dorsal horn or the lateral funiculus. They reversed at 1.2 +/- 4.6 (SD) mV and could in most cases (49 of 51) be separated into two components. 3. In the presence of DL-2-amino-5-phosphonovalerate (10-40 microM) the current-voltage (I-V) relationship of the remaining EPSC was linear. When stimulated in the lateral funiculus, its rise time (10-90%) and the time constant of the monoexponential decay were 1.6 +/- 1.0 and 5.5 +/- 2.7 ms, respectively. By contrast, when stimulated in the dorsal horn, this component had a rise time (10-90%) of 3.0 +/- 0.8 ms and a decay time constant of 13.7 +/- 7.6 ms. 4. We studied the NMDA receptor-mediated component of the EPSCs after superfusion of 6 cyano-7-nitroquinoxaline-2,3-dione (5 microM). The I-V relationship of this component had a region of negative slope conductance between -30 and -80 mV, which was abolished in Mg(2+)-free saline. The rise time (10-90%) ranged from 3.3 to 9.5 ms and the decay was biexponential. Both decay time constants increased with depolarization. Mg(2+)-free saline reduced this voltage sensitivity. 5. At a membrane potential of -80 mV and in 1 mM extracellular Mg2+, the NMDA receptor mediated component represented 74.8 +/- 11.2% of the total charge carried by the EPSCs evoked by stimulation in the dorsal horn. In contrast, when stimulated from the lateral funiculus, 28.9 +/- 18.9% of the total charge carried during the EPSC was mediated by the NMDA receptor-mediated component. The contribution of the NMDA receptor-mediated component increased in both cases with depolarization. In addition, in 2 of 18 SPNs the EPSC evoked in the dorsal horn was exclusively carried by NMDA receptors. 6. We conclude that L-glutamate or a related substance mediates the fast excitatory input onto SPNs. Viscerosomatic and supraspinal inputs form synapses with different topographical locations on the SPN.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7543946 TI - Adhesive properties of proteolipid protein are responsible for the compaction of CNS myelin sheaths. AB - We have studied the molecular function of proteolipid protein (PLP), the main integral membrane protein of CNS myelin, by generating mice lacking PLP expression. Here, we demonstrate that these PLP-minus mice show no pleiotropism as mice carrying point mutations within the PLP gene. The expression of other myelin genes (myelin basic protein, MBP; myelin associated glycoprotein, MAG; UDP galactose-ceramide galactosyl transferase, CGT) is unimpaired on the RNA level. Protein level immunofluorescence analysis by confocal microscopy reveals that in PLP-minus mice there is a complete absence of PLP, a scattered appearance of MBP, and MAG expressed more widely in regions lacking MBP staining, which may be a compensatory mechanism. In electron microscopy the myelin lamellae of the ensheathed CNS axons are loosely wrapped with wide extracellular spaces between turning loops. Intraperiod dense lines are missing. The lateral loops of the paranode form regular axoglial junctions. In PLP-minus mice axons form regular axoglial junctions. In PLP-minus mice axons with large diameters are loosely myelinated, whereas small axons remain unmyelinated. Functionally, the mutant mice show deficits in their locomotor activity. We propose that adhesion properties of the extracellular domains of PLP are responsible for the tight apposition of the plasma membrane processes of oligodendrocytes wrapping axons to form the compact myelin sheath. PMID- 7543947 TI - Alterations in cerebral cortical galanin concentrations following neurotransmitter-specific subcortical lesions in the rat. AB - Galanin is associated with multiple projection neurons, and its immunoreactivity in the cerebral cortex may be derived from diverse sources. We investigated the effects of subcortical lesions on cerebral cortical galanin concentrations. Lesions of the anterior noradrenergic bundle (ANB) comparably reduced cerebral cortical galanin and norepinephrine (NE) concentrations. The effects of the ANB lesions on galanin were immediate and became most pronounced 1 week later. Extensive unilateral lesions of the nucleus basalis of Meynert (NBM) decreased galanin concentrations, although not as markedly as after ANB lesions. The NBM lesions had no additional effect in the presence of an ANB lesion. Decreases in cerebral cortical galanin concentrations depended upon the extent and the duration of the NBM lesion and were not as pronounced as the decreases in markers of cholinergic activity. Acute treatments with physostigmine, which inhibit cerebral cortical AChE, had no effect on galanin concentrations. The depletion of galanin following an NBM lesion was most pronounced within hours of the insult, while the depletion of ChAT following the same lesions required several days to develop. Cortical concentrations of galanin and 5-HT increased 1 hr after dorsal raphe nucleus (DRN) lesions and then decreased 7 d later. Six weeks later, galanin concentrations recovered in the cerebral cortex despite the continued depletion of 5-HT. These studies suggest that a substantial portion of cerebral cortical galanin may derive from noradrenergic neurons and may be modulated by cortically-projecting ACh and 5-HT neurons. PMID- 7543948 TI - Deprivation of dietary nucleotides results in a transient decrease in acid soluble nucleotides and RNA concentration in rat liver. AB - This study examines the contribution of dietary nucleotides to liver nucleotide pools in rats. Liver acid-soluble nucleotides, DNA and RNA concentrations were monitored in two groups of rats fed either a diet supplemented with nucleotides or a diet free of nucleotides for 3 wk. Significantly lower concentrations of ATP, ADP, GTP and CDP as well as of RNA were found after 1 wk in the rats fed a nucleotide-free diet compared with those fed the nucleotide-supplemented diet; concentrations remained lower after 2 wk except for ATP and ADP. No changes over time were observed in the rats fed the nucleotide-supplemented diet. Between wk 2 and 3 an increase in both acid-soluble nucleotides and RNA was observed in the rats fed the nucleotide-free diet, reaching the values found in the rats fed the nucleotide-supplemented diet. These findings, which indicate that dietary nucleotides are utilized at least in part by the liver to maintain the cell nucleotide pools and that diets devoid of nucleotides affect hepatic nucleotide metabolism and RNA, support the hypothesis that liver nucleotide metabolism is modulated by the availability of dietary nucleotides. PMID- 7543952 TI - [Targeted toxin therapy in the treatment of leukemia]. AB - A chimeric toxin in which the cell surface binding domain of Pseudomonas exotoxin was replaced with mature human granulocyte colony-stimulating factor (G-CSF) was produced in Escherichia coli, partially purified and tested for its biological activity on a G-CSF-dependent murine myeloid leukemia cell line, NFS60. This fusion protein, termed as G-CSF-PE40, can displace [125I] G-CSF binding to its receptor. After 48 hrs incubation in the presence of G-CSF, G-CSF-PE40 inhibited protein synthesis and revealed cytotoxicity in NFS60 cells in a concentration dependent manner. The half maximal dose of G-CSF-PE40 for protein synthesis inhibition (ID50) in NFS60 cells was estimated at around 100pM. Additionally, relatively low concentration of G-CSF-PE40 stimulated DNA synthesis in NFS60 cells after 16 hrs' incubation in the absence of G-CSF, suggesting that G-CSF PE40 also can transduce a transient mitogenic signal. Thus, G-CSF-PE40 may be useful in the selective elimination of myeloid cells expressing G-CSF receptors, especially in combination with chemotherapeutic agents like cytosine arabinoside. PMID- 7543951 TI - Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular phylogenies. AB - Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results. PMID- 7543954 TI - [The effects of recombinant human granulocyte colony-stimulating factor on protracted neutropenia in patients with acute myelogenous leukemia. (rG.CSF Clinical Study Group)]. AB - A clinical study to investigate the efficacy and safety of recombinant human G CSF (rG.CSF) was performed in patients with acute myelogenous leukemia who had had protracted neutropenia. The drug was administered d.i.v. at a dose of 5 micrograms/kg. Sixty-four patients entered the study, of whom 61 patients were evaluable for safety and 58 patients evaluable for efficacy. The treatment produced an early recovery in neutrophil count in the patients who had had protracted neutropenia (< 1,000/microliters) of over 10 days. Among relapsed cases and cases showing > 20% blasts in the bone marrow, many showed blast stimulation in response to rG.CSF, suggesting difficulty in attaining complete remission by subsequent chemotherapy in such cases. The present data indicates that it is desirable to use the drug in lower-blast-count states in order to attain safe and sufficient therapeutic effects in patients with acute myelogenous leukemia who have had protracted neutropenia. PMID- 7543953 TI - [Treatment of acute promyelocytic leukemia--combined use of all-trans retinoic acid and granulocyte colony-stimulating factor]. AB - We evaluated the effect of treatment by all-trans retinoic acid (ATRA) in 9 patients with acute promyelocytic leukemia (APL). Of 6 patients who had circulating leukemic blasts before treatment, 3 initially received ATRA alone but died of respiratory failure due to retinoic acid syndrome (RAS). High dose steroid therapy did not rescue RAS in these patients. Another 3 who were given intensive chemotherapy followed by ATRA and/or granulocyte colony-stimulating factor (G-CSF) achieved complete remission (CR). Of 3 patients without peripheral leukemic blasts before treatment, 1 received intensive chemotherapy followed by G CSF and reached CR, 1 who had been previously given ATRA did not respond to ATRA, and 1 did not initially respond sufficiently to ATRA alone but responded dramatically to ATRA plus G-CSF. In the treatment of APL, appropriate combination of ATRA, G-CSF and chemotherapy should always be taken into consideration. In addition, RAS have to be carefully avoided when applying ATRA therapy in patients who have circulating leukemic blasts before treatment. PMID- 7543955 TI - [A randomized controlled study of rG.CSF in patients with neutropenia after induction therapy for acute myelogenous leukemia. (rG.CSF Clinical Study Group)]. AB - A randomized treated/non-treated study of rG.CSF (5 micrograms/kg/d, d.i.v.) in patients with acute myelogenous leukemia was conducted to assess its efficacy on fever (> or = 38 degrees C) or documented infection after induction therapy. Of 95 patients enrolled, 46 patients were evaluable for safety and 43 for efficacy in the treated group of 47 patients while 37 of 48 patients were eligible for data analysis in the untreated group. Mare patients showed a recovery in the blood neutrophil count (to > 1,000/microliters) during rG.CSF treatment (14 days) than in the non-treated group (p = 0.039) while the number of febrile patients and duration of fever did not significantly differ between the two groups. The treatment with rG.CSF enabled an early recovery in neutrophil count in the patients with neutropenia and overt signs of infection after induction therapy, but there was no hastened allevistion of symptoms of infection in these patients. PMID- 7543959 TI - A possible role for nitric oxide in modulating the functional cyclosporine toxicity by arginine. AB - The renal damage consequent to cyclosporine A (CsA) administration ranges from hemodynamic alterations to irreversible chronic lesions. The initial vasoconstriction depends upon the imbalance between the various modulators of the renal vascular tone, among which the most powerful are endothelins and nitric oxide (NO). CsA could play a crucial role by inhibiting the Ca++/calmodulin mediated activation of the constitutive NO synthase (NOS) isoform, which converts L-arginine (L-Arg) into NO and citrulline, with a 1:1 stoichiometry. To investigate the possibility of modulating CsA nephrotoxicity with L-Arg we studied six groups (G) of Lewis rats treated with daily gavage up to eight weeks: G1, CsA 40 mg/kg; G2, G1 plus L-Arg 300 mg/kg; G3, G2 plus the competitive inhibitor of NOS, NG-nitro-L-Arg (L-NNA); G4, L-Arg alone; G5, L-NNA alone; and G6, controls receiving vehicle alone. After eight weeks L-Arg treated rats were protected against the toxic effects of CsA [creatinine (Cr) values, G2, 0.62 +/- 0.05 mg/dl vs. G1, 0.99 +/- 0.16 mg/dl, P < 0.001; proteinuria (P), G2, 7.2 +/- 1.02 mg/day vs. G1, 15.1 +/- 1.9 mg/day, P < 0.01]. The administration of L-NNA abolished the protective effect of L-Arg (G3, Cr 1.23 +/- 0.16 mg/dl; P 16.9 = 2.3; P < 0.02 and P < 0.005, respectively vs. G2). The levels of Cr in G2 rats were superimposable to control groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543957 TI - [A case of tuberculous pleurisy aggravated by interferon treatment for chronic hepatitis C]. PMID- 7543962 TI - Rapid DNA fragmentation from hypoxia along the thick ascending limb of rat kidneys. AB - Extensive DNA fragmentation, a marker for programmed cell death, was selectively and rapidly induced by hypoxia in the thick ascending limbs of rat kidneys. In isolated perfused kidneys, DNA breaks were present in medullary tubules as early as after 10 minutes of local hypoxia and were prevented by reduction of metabolic work. In a model of radiocontrast-induced acute renal failure, DNA breaks were detected selectively along thick ascending limbs as early as 15 minutes following insult, preceding overt morphological damage. Hypoxia induces rapid DNA fragmentation along thick ascending limbs, where programmed cell death could play an important role in nephron injury and kidney failure. PMID- 7543960 TI - Renal growth hormone--insulin-like growth factor-I system in acute renal failure. AB - The renal growth hormone--insulin-like growth factor-I system in acute ischemic renal failure. Recovery from acute tubular necrosis (ATN) is accelerated by IGF-I therapy. Furthermore, the local renal growth hormone-IGF-I system may participate in the natural repair. We examined the IGF-I system in rat kidneys subjected to 60 minute ischemia compared to sham operated controls. Two days after injury, growth hormone receptor mRNA and IGF-I mRNA levels fell approximately 9 to 33% of control values. This was associated with a reduction in kidney immunoreactive IGF I levels. In contrast, IGF-I receptor mRNA abundance was unchanged. However, plasma membrane IGF-I receptor binding on day 2 and day 7 was near double the control values (P < 0.01). Scatchard analysis revealed a near twofold increase in receptor number. Since receptor mRNA levels were unchanged, this implies receptor protein up-regulation. In contrast to unchanged IGF-I receptor mRNA levels, the abundance of mRNA levels of insulin-like growth factor binding proteins (IGFBP) 2, -3, -4 and -5 fell approximately 14 to 62% of control levels day 2 after injury (P < 0.05), suggesting reduced IGFBP production. Thus, the renal response to ischemic ATN, namely, low IGFBP mRNA levels and high IGF-I receptor number, may function to increase IGF-I bioavailability and thereby enhance the reparative actions of local and circulating IGF-I in ischemic ATN. PMID- 7543961 TI - Progression of overt nephropathy in non-insulin-dependent diabetes. AB - The detection of overt albuminuria (> 300 mg/g creatinine) in the absence of azotemia was used to diagnose early nephropathy in 34 Pima Indians with NIDDM of 16 +/- 1 years duration. Differential solute clearances were performed serially to define the course of the glomerular injury over 48 months. At baseline, the GFR (107 +/- 5 ml/min), filtration fraction and sieving coefficients of relatively permeant dextrans (< 52 A) were all depressed below corresponding values in 20 normoalbuminuric Pima Indians with a similar duration of NIDDM. Over the ensuing 48 months the GFR (-34%) and filtration fraction (-13%) in the nephropathic patients declined further. The sieving coefficients of large, nearly impermeant dextrans (> 56 A radius) increased selectively and fractional clearances of albumin and IgG increased correspondingly by > 10-fold. Analysis of the findings with pore theory revealed: (1) a progressive decline in pore density and the ultrafiltration coefficient (Kf); and (2) broadening of glomerular pore size distribution that resulted in greater prominence of large pores (> 70 A radius). We conclude that increasing loss of intrinsic ultrafiltration capacity is the predominant cause of the early and progressive decline in GFR that follows the development of nephropathy in NIDDM. We speculate that progressive impairment of barrier size-selectivity contributes to but does not fully account for the increasingly heavy proteinuria that is observed early in the course of this disorder. PMID- 7543949 TI - Nucleic acid-binding metabolic enzymes: living fossils of stereochemical interactions? AB - Recently, a series of intriguing observations expanded the list of a number of metabolic enzymes known to be associated with various forms of nucleic acids, including single- and double-stranded DNA, cognate and noncognate RNAs, and specific tRNAs. There is no clear reason why such a phenomenon should take place in contemporary cell physiology, or, further, why such a property has evolved at all. Sixteen known cases are presented in an attempt to delineate any common features of these enzymes. Apart from their ancient nature, as judged by their wide distribution and their participation in fundamental biochemical pathways, it appears that these enzymes do not share any structural or functional characteristics. Given that most of these proteins require nucleotide-based cofactors for their activity, it is proposed that they may represent genuine molecular fossils of the transition from an RNA to a protein world. Their nucleic acid-binding properties are in keeping with previously proposed hypotheses regarding the origins and evolution of nucleotide-based cofactors. The mode of interaction between these proteins and their nucleic acid substrates remains unclear, but it may represent an extended form of stereochemical interactions that have been proposed for the origins of the genetic code. PMID- 7543958 TI - Suppression of corneal graft rejection by subconjunctival injection of FK-506 in a rat model of penetrating keratoplasty. AB - The usefulness of the subconjunctival injection of FK-506 for suppression of allograft rejection was investigated in a rat model of orthotopic penetrating keratoplasty. Fischer rats were used as donors and Dark Agouti rats, as recipients. FK-506 was administered subconjunctivally in a dosage of 0.3 mg/kg/day for 15 consecutive days after penetrating keratoplasty was performed. Allograft rejection occurred within 8 to 10 days after keratoplasty in all untreated rats (n = 6). None of the FK-506-treated rats (n = 6) exhibited graft rejection during the 3-week observation period. Histologic examination showed marked infiltration of mononuclear cells in the stroma of corneal grafts from untreated rats 3 weeks after grafting. Inflammatory cells were only occasionally observed in grafts from FK-506-treated rats. Donor-specific cytotoxic T lymphocyte activity was completely suppressed in FK-506-treated rats 3 weeks after grafting. Our results indicated that subconjunctival injection of FK-506 effectively prevented corneal allograft rejection in a rat model of penetrating keratoplasty. PMID- 7543956 TI - [A randomized double-blind controlled study of recombinant human granulocyte colony-stimulating factor in patients with neutropenia induced by consolidation chemotherapy for acute myeloid leukemia. (rG.CSF clinical study group)]. AB - A multicenter, randomized, double-blind controlled study was performed to evaluate the efficacy and safety of recombinant human granulocyte colony stimulating factor (rG.CSF) in reducing infectious morbidity and neutropenia induced by consolidation chemotherapy for acute myeloid leukemia (AML). One hundred and twenty-four eligible patients were randomized to receive either rG.CSF (5 micrograms/kg/d d.i.v.; 59 patients) or placebo (65 patients) for 14 days from the day after chemotherapy. All of them were included in the safety analysis, while 57 patients receiving rG.CSF and 64 patients receiving placebo were included in the efficacy analysis. The duration of neutropenia as well as the incidence of fever and febrile neutropenia, and frequency of antibiotic therapy required, were all significantly reduced in the rG.CSF group. No serious adverse reactions were encountered; there was no significant difference between the two groups in terms of incidence of adverse events. These results demonstrate that rG.CSF is beneficial to alleviate neutropenic episodes induced by consolidation chemotherapy in patients with AML. PMID- 7543963 TI - [Practical teaching of nursing students. II. Organizational standard for groups of students at the surgical intensive care unit. Standards of the Wurzburg University Surgery Department]. PMID- 7543964 TI - Trisomy 4 in 'stem cell-like' leukemic cells of a patient with AML. AB - CD34 positive progenitor cells were analyzed in the bone marrow aspirate from a patient with newly diagnosed AML FAB M4. The patient had trisomy 4 as sole cytogenetic abnormality and a dominant population of CD34 negative leukemic blasts. Karyotyping of the FACS isolated, minor subpopulation of CD34+/CD38-, 'stem cell'-like cells (incidence 0.29%) revealed trisomy 4 in 11/13 metaphases. No metaphases were obtained in the CD34 negative subpopulation. The experiments point to the existence of leukemic stem cells in the CD34+/CD38- compartment in AML patients with trisomy 4. PMID- 7543966 TI - RNA for reverse PCR analysis can be obtained from bone marrow cells prepared for cytogenetic analysis and stored at -20 degrees C for several years. PMID- 7543968 TI - Dielectrophoretic separation and enrichment of CD34+ cell subpopulation from bone marrow and peripheral blood stem cells. AB - Dielectrophoresis has been used to enrich selected cell subpopulations in a mixed cell population by exploiting differential dielectric properties. Six-fold enrichment of stem cells expressing the CD34+ antigen has been achieved for bone marrow samples and peripheral blood, without the requirement for initial chemical treatment associated with immunoadsorption techniques. PMID- 7543967 TI - Primary chemotherapy for clinical stage II nonseminomatous germ cell testicular tumors: selection criteria and long-term results. AB - OBJECTIVE: To determine the treatment option for patients with low-volume stage II nonseminomatous germ cell testicular tumors (NSGCTT) that yields the best survival, is associated with the least morbidity, and avoids "double therapy"- that is, chemotherapy and retroperitoneal lymph node dissection (RPLND). DESIGN: We reviewed our institutional experience with 28 patients with stage II NSGCTT who received primary chemotherapy between August 1983 and October 1992. MATERIAL AND METHODS: The 28 study patients (mean age, 28 years; range, 20 to 52) with low volume stage II NSGCTT were treated with bleomycin, etoposide, and cisplatin. The correlation of response rates with volume of disease and predominant histologic cell type was determined. The duration of survival was measured from the initiation of chemotherapy to the appearance of progressive disease or death or the date of last follow-up visit. RESULTS: Of the 28 patients treated, 27 (96%) achieved a complete response--20 (71%) with only chemotherapy and an additional 7 (25%) with chemotherapy plus surgical treatment. Twenty-seven patients (96%) remained free of disease after a median follow-up of 72 months. The most frequent complication was cisplatin-associated paresthesias or tinnitus which was noted in 13 patients (46%). In 11 of 15 patients (73%), attempts to have children have been successful. CONCLUSION: Excellent long-term survival rates in patients with stage II NSGCTT can be achieved with primary chemotherapy. In this series, 71% of patients were spared RPLND. The need for postchemotherapy RPLND seemed to be related to the initial metastatic tumor volume and possibly the histologic features of the primary tumor. Continued refinement in surgical techniques and chemotherapeutic regimens will necessitate the comparison of these two treatment approaches in a randomized prospective trial. PMID- 7543950 TI - Investigating hypothetical products from noncoding frames (HyPNoFs). AB - Hypothetical Products from Noncoding Frames (i.e., HyPNoFs) are hypothetical, not coded proteins, translated from alternate reading frames (i.e., coding + 1 and coding + 2) of cDNAs. HyPNoFs of CD4, PKC, oncostatin, bcl-2 proto-oncogene, tumor suppressor p53, cystic fibrosis transmembrane regulator (CFTR), and tumor necrosis factors alpha and beta were searched as query sequences vs the SWISS PROT data bank. Homology searchers carried out revealed that hypothetical products (i.e., HyPNoFs) may share high similarity with real protein products actually coded. Sequence similarity of hypothetical products to real proteins is sometimes very high, suggesting common conformational features, according to the Sander and Schneider cutoff value. This finding supports the hypothesis that eukaryotic DNA, currently considered to be monocistronic, might occasionally have polycistronic regions, carrying different protein messages on overlapping frames. As yet, polycistronic genes have been observed in viral genomes only. The presence of polycistronic regions in eukaryotic genes is likely reminiscent of an ancient strategy, rather than a present feature of the genome in eukaryotes. These data suggest that thorough investigation of HyPNoFs is likely to improve our ability to trace genes' evolution and to investigate structure-function relationships of protein and DNA sequences. PMID- 7543965 TI - Induction of CD80 expression in low-grade B cell lymphoma--a potential immunotherapeutic target. AB - The CD80 antigen (B7) is expressed on activated B lymphocytes. It is thought to be important in eliciting a T cell response via its ligands CD28 and CTLA-4 when antigen is presented in the presence of the MHC-1 peptide. Low-grade B cell lymphomas analysed by flow cytometry express CD80 very poorly. However, when grown in vitro using the IL-4/anti-CD40 stromal cell culture system, following depletion of T and IgD-bearing cells, a monoclonal B cell expansion occurs. Cells harvested at days 10-13 express the antigen strongly, regardless of the histological subtype of lymphoma. Further investigation of CD80-mediated immune functions may be possible using this system as a basis for testing immunotherapy. PMID- 7543969 TI - The D2 dopamine receptor occupancy of risperidone and its relationship to extrapyramidal symptoms: a PET study. AB - Risperidone is a recently introduced neuroleptic distinguished by a decreased incidence of extrapyramidal side effects (EPS). The mechanism of its low EPS is unclear. Since it has been shown that EPS is related to the level of D2 receptor occupancy, we studied nine patients receiving 2-6 mg/day of risperidone using [11C]-raclopride PET scans in order to determine the in vivo D2 receptor binding characteristics of risperidone. The mean level of receptor occupancy was 66% at 2 mg; 73% at 4 mg; and 79% at 6 mg. Three patients, those with the highest receptor occupancies, exhibited mild EPS, though none required anitparkinsonian medications. Our results suggest that at doses of 4-6 mg the in vivo D2 receptor occupancy of risperidone is similar to that of typical neuroleptics and higher than that of clozapine. This would suggest that the EPS benefits of risperidone cannot be explained by a low D2 binding but may be related to its high 5-HT2 affinity. However, the emergence of EPS at higher levels of D2 receptor occupancy, in this study and in previous clinical trials, would suggest that risperidone's high 5-HT2 affinity provides only a relative protection from EPS. And once the D2 occupancy exceeds a certain threshold this 'relative' 5-HT2 mediated protection from EPS may be lost. PMID- 7543972 TI - Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture. AB - Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell membrane glycoprotein that has been implicated in leukocyte/endothelial cell interactions in inflammation. In this study, we report the expression of VCAM-1 in primary cultures of human brain microvessel endothelial cells (HBMEC) under standard conditions and following bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma) treatment. Surface expression was detected and quantitated by light and immunogold electron microscopy and ELISA. Unstimulated cerebral endothelial cells (EC) constitutively expressed low levels of VCAM-1. LPS, TNF-alpha, or IL-1 beta increased the overall intensity of surface staining and the percentage of cells expressing VCAM-1 in a time- and concentration-dependent manner. LPS had the most potent effect, followed by TNF-alpha and then IL-1 beta. IFN-gamma did not upregulate VCAM-1. The level of VCAM-1 expression increased by 12-24 hr and returned to unstimulated levels by 48 hr. Immunoelectron microscopy demonstrated that VCAM-1 was preferentially localized on the apical as compared to the basal surface in both unstimulated and cytokine-treated cells. In addition, the intensity of immunostaining was significantly greater in stimulated versus unstimulated EC. The polarization and significant upregulation of VCAM-1 after cytokine treatment suggest a possible role of this adhesion molecule in inflammatory and autoimmune processes within the central nervous system. PMID- 7543971 TI - Subcutaneous implanted chambers in different mouse strains as an animal model to study genetic stability during infection with Lyme disease Borrelia. AB - Tissue metal net cages were implanted subcutaneously in BALB/cJ and C3H/Tif mice as an experimental model of Borrelia burgdorferi infection. B. burgdorferi sensu stricto strain Sh2-82 could be isolated up to 14 weeks after the inoculation. However, a significant difference in infectivity between the two mice strains was observed. C3H/Tif mice were more susceptible to developing chronic B. burgdorferi s.s. infections than BALB/cJ mice. Although a B. burgdorferi infection was established, no rearrangements in the ospA and ospB genes were observed in any of the infected mice. PMID- 7543973 TI - [Hydrocephalus in the pediatric age group: considerations on a series of 245 shunted patients]. AB - A series of 245 hydrocephalic patients in the pediatric age group who underwent 544 surgical shunting procedures is reviewed in this paper. The subsequent average of 1.5 operations per patient appears therefore significantly low, when compared with the Literature data. The authors emphasize that this result may be due mainly to the fact that all the surgical procedures were performed by the same surgeons, even if two other concepts appear extremely important in this regard: the great care in prevention of shunt infections and the use of one-piece shunting devices. A pattern for the "clinical surveillance" of hydrocephalic children is proposed and the neuropsychological follow-up of the series is described in the paper. Authors conclude that serious damage to cerebral parenchyma due to intracranial hypertension related to the hydrocephalic condition may be avoided by an accurate selection of patients to be operated on, a correct surgical timing and a careful postoperative control. PMID- 7543975 TI - Increased laminin A expression in regenerating myofibers in neuromuscular disorders. AB - Laminin is a basement membrane (BM) glycoprotein composed of three of five subunits, the A, M, B1, B2, and the S chain. Four forms of laminin, A-B1-B2, A-S B2, M-B1-B2, and M-S-B2, have been identified. Laminin is implicated in various biological processes such as cell adhesion and differentiation. We studied immunohistochemically the expression of the four laminin subunits A, M, B1, B2 as well as of neural cell adhesion molecule (N-CAM, CD56), a marker of regenerating myofibers, in various neuromuscular disorders. In normal muscle, the predominant subunits of myofiber laminin were M, B1, and B2. The A chain was only faintly expressed in myofiber BM. In inflammatory myopathies and dystrophinopathies myofiber laminin A expression was greatly increased. An average of 80% and 63% of laminin A-positive myofibers in inflammatory myopathies and dystrophinopathies, respectively, were additionally CD56 positive. Laminin A and CD56 expression in denervating diseases and mitochondrial myopathies were negligible. Expression of M, B1, and B2 subunits did not seem to be altered in the diseased conditions examined above. The data suggest that laminin A is upregulated in inflammatory myopathies and dystrophinopathies and, most markedly in regenerating myofibers. PMID- 7543974 TI - Repertoire cloning of a human IgG inhibitor of alpha IIB beta 3 function. The OG idiotype. AB - A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit beta 3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (alpha IIb beta 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti-beta 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified alpha IIb beta 3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to alpha IIb beta 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed with one of three V lambda genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline V lambda gene segment. Fab using H23 bind to alpha IIb beta 3, while those using H21 do not. Based on sequence homology, both H21 and H23 use VH gene segments belonging to the VH4 gene family. Thus, the idiotype OG is restricted to the VH4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of alpha IIb beta 3. PMID- 7543970 TI - [Observations of the dynamics of the symbol formation process in Jung]. PMID- 7543980 TI - Vasoconstrictor mechanism of neuropeptides augmented after endothelial removal in isolated, perfused canine basilar arteries. AB - In order to investigate the functional role of endothelium and vasoeffector mechanism in cerebrovascular responses to neuropeptides, the stainless steel cannula inserting method was applied to examine the responses to intraluminally applied bradykinin, substance P and vasopressin in isolated and perfused canine basilar arteries. In control vessels with intact endothelium, each neuropeptide induced a monophasic dilation at lower doses, and a biphasic response, i.e., an initial dilation followed by a secondary constriction, at higher doses. The dilation was significantly reduced and the constriction was significantly enhanced, while the dilation to papaverine was not modified by endothelial removal with intraluminal saponin. The same tendency was observed in the responses after extraluminal treatment with oxyhaemoglobin. The monophasic constrictions to prostaglandin F2 alpha and potassium chloride were significantly potentiated by the endothelial removal. The augmented constrictions to the neuropeptides were significantly diminished by indomethacin (a cyclooxygenase inhibitor), OKY-046 (a thromboxane synthetase inhibitor) and nimodipine (a dihydropyridine calcium antagonist), but not by AA-861 (a lipoxygenase inhibitor). These results suggest that the neuropeptide causes an endothelium dependent dilation and a constriction of smooth muscles, and that the enhanced constriction might be relevant in part with thromboxane A2, linked with calcium influx into smooth muscle cells in cerebral arteries. PMID- 7543977 TI - In vivo evidence for the reversible action of the monoamine oxidase inhibitor brofaromine on 5-hydroxytryptamine release in rat brain. AB - We have used intracerebral microdialysis to examine the reversibility of the action of brofaromine, a selective inhibitor of monoamine oxidase-A (MAO, E.C. 1.4.3.4.), on 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) output in rat frontal cortex. Brofaromine significantly increased the 5-HT output to about 200% of basal values 4 h after the s.c. administration of 10 and 30 mg/kg (but not 3 mg/kg) and reduced the concentration of 5-HIAA in the dialysate dose-dependently (61%, 53% and 41% of basal value with doses of 3, 10 and 30 mg/kg, respectively). At this time, cortical 5-HT concentration was increased and cortical 5-HIAA concentration was decreased in a dose-dependent manner. Treatment of rats with 10 mg/kg brofaromine plus 2.5 mg/kg of the irreversible MAO-B inhibitor L-deprenyl increased the concentration of 5-HT in the dialysate more than did brofaromine alone (503% vs 206% of the basal value, 4h after administration). Similarly, clorgyline (5 mg/kg) plus L-deprenyl (2.5 mg/kg) increased the concentration of 5-HT in the dialysate to 461% of the control value. This indicates that the concurrent inhibition of both types of MAO increases 5-HT output more than the selective blockade of either enzyme subtype. We have used this characteristic to examine, in vivo, the reversibility of the interaction of brofaromine with MAO-A. The output of 5-HT and 5-HIAA was examined 19-21 h after treatment with L-deprenyl plus clorgyline or L-deprenyl plus brofaromine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543978 TI - Involvement of the Na,K-ATPase in the induction of ion channels by palytoxin. AB - The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin. PMID- 7543979 TI - Selective neurotoxicity of ruthenium red in primary cultures. AB - The inorganic dye ruthenium red (RuR) has been shown to be neurotoxic in vivo when injected intracerebrally. In this work the toxicity of RuR was compared in primary cultures of rat cortical neurons, cerebellar granule neurons and cerebellar astroglia. Microscopic examination of the cultures revealed that RuR penetrates the somata of both types of neurons used and produces vacuolization and loss and fragmentation of neurites. In contrast, no RuR was seen inside cultured astrocytes and no morphological signs of damage were observed in these cells. RuR toxicity was also assessed by immunocytochemistry of alpha-tubulin and by biochemical measurement of the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by the cultured cells. The morphological alterations in the neurons were closely correlated with loss of tubulin immunoreactivity and particularly with a notable decrement in the ability to reduce MTT. Using the latter parameter, it was found that neuronal damage was independent of the age of the cultures, augmented progressively with time of incubation with RuR, from 8 to 24 h, and showed a clear dose-response curve from 20 to 100 microM RuR. Astrocytes showed only a slight decrease in MTT reduction after 24 h of incubation with 100 microM RuR. It is concluded that RuR seems to be toxic for neurons but not for astroglia, and that this selectivity is probably related to the ability of the neurons to internalize the dye. The possible mechanisms of RuR penetration and neuronal damage are discussed. PMID- 7543976 TI - In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide. AB - (6R)-5,6,7,8-Tetrahydro-L-biopterin(H4biopterin) is well known as a cofactor of enzymes that hydroxylate aromatic amino acids. More recent work has revealed an essential role of H4biopterin in the biosynthesis of nitric oxide (NO), an intercellular messenger molecule synthesized from L-arginine by different NO synthase isozymes in many species and tissues. While the function of H4biopterin in aromatic amino acid hydroxylation is well established, the role of this pteridine in NO synthesis is, as yet, elusive. Current experimental evidence hints at a dual mode of action of H4biopterin, involving both an allosteric effect on the NO synthase protein and participation as a reactant in L-arginine oxidation. As discussed in detail in the present article, the latter effect of this pteridine may be related to the protection of NO synthase from feedback inhibition by NO. PMID- 7543981 TI - Primary craniofacial chordoma: case report. AB - A 37-year-old man presented with right facial pain and a nonpalpable mass over the malar eminence. An incisional biopsy via the intraoral route was performed and interpreted as a vascular malformation with degenerative changes. His symptoms persisted, and a repeat biopsy was suggestive of an epithelioid nerve sheath tumor. Total resection of the tumor was planned to include the infraorbital and malar regions, the infratemporal fossa, and the pterygopalatine fossa. At surgery, the tumor was removed with tumor-free margins obtained along the course of the maxillary nerve just before its entrance into the cavernous sinus. The pathological findings and the immunohistochemistry demonstrated a typical chordoma with no chondroid or sarcomatous dedifferentiation. We think that with greater use of immunohistochemical markers and electron microscopy, patients with chordoma in this location may be diagnosed promptly and accurately. PMID- 7543983 TI - Distribution and morphological characterization of viscerofugal projections from the large intestine to the inferior mesenteric and pelvic ganglia of the male rat. AB - Viscerofugal neurons are enteric neurons in the myenteric plexus of the stomach and intestine that project to the prevertebral ganglia as the afferent limb of intestino-intestinal reflexes. This study characterizes viscerofugal projections to the inferior mesenteric ganglion and investigates the possibility of similar projections to the major pelvic ganglia in the male rat. The colon and rectum were examined for retrogradely labelled neurons following the injection of retrograde tracer into the inferior mesenteric or major pelvic ganglia, or following the application of tracer to the caudal end of the cut intermesenteric nerves, or either end of the cut hypogastric nerves. All labelled viscerofugal neurons were found in the myenteric plexus and were often grouped near the mesenteric attachment. The number of viscerofugal neurons projecting to the inferior mesenteric ganglion via the lumbar colonic nerves increases along the length of the large intestine with the maximum number of viscerofugal neurons found in the rectum. Some viscerofugal neurons from the distal colon and rectum reach the inferior mesenteric ganglion via the hypogastric nerves. A similar number and distribution of viscerofugal neurons project via the inferior mesenteric ganglion into the intermesenteric nerves as terminate in the inferior mesenteric ganglion. Very few viscerofugal neurons project to the neurons of the major pelvic ganglia via the rectal nerves, and no viscerofugal neurons project caudally in the hypogastric nerves to these ganglia. The majority of labelled neurons resembled Dogiel type I morphology. Thus the inferior mesenteric ganglion receives a substantial innervation from viscerofugal neurons of the large intestine, with the greatest supply from the distal colon and rectum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7543982 TI - The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors. AB - In order to provide further information about the types of spinal neuron which possess neurokinin-1 receptors, we have carried out pre-embedding immunocytochemistry on sections of rat lumbar spinal cord with an antiserum raised against a synthetic peptide corresponding to part of the sequence of the receptor, and combined this with post-embedding immunocytochemistry to detect GABA and glycine. Numerous neuronal cell bodies showing neurokinin-1 receptor immunoreactivity were seen in lamina I, laminae III-VI, the lateral spinal nucleus and the area around the central canal. Most of the cells observed in lamina III were small and had relatively restricted dendritic trees which could often not be followed into lamina II, however some larger cells in laminae III and IV had dendrites which extended through lamina II and into lamina I. Cells of the latter type are likely to represent a major target of substance P released from small-diameter primary afferents in the superficial dorsal horn. The great majority (255 out of 283) of spinal neurons which possessed neurokinin-1 receptor immunoreactivity, including all of those in lamina I, were not GABA- or glycine immunoreactive, however a few cells in the deep part of the dorsal horn and the lateral spinal nucleus and several cells near the central canal were GABA immunoreactive, and some of these were also glycine-immunoreactive. These results suggest that substance P acts through neurokinin-1 receptors mainly on excitatory neurons within the spinal cord. PMID- 7543984 TI - Distribution of nitric oxide synthase in the central nervous system of Macaca fuscata: subcortical regions. AB - The distribution of nitric oxide synthase-immunoreactive neurons was studied in the Macaca fuscata by immunohistochemistry using antiserum against nitric oxide synthase. In the macaque lower brainstem, many nitric oxide synthase-containing cell bodies were found in the gigantocellular and parvocellular reticular nuclei, the nucleus of the spinal tract of trigeminal nerve, the cochlear nucleus, the prepositus hypoglossi and the nucleus of the solitary tract. Many nitric oxide synthase-immunoreactive perikarya were observed in the laterodorsal pedunculopontine tegmental nucleus complex of the macaque pontine and midbrain tegmentum. In addition, there were many highly immunoreactive cell bodies in the superficial layers of the inferior and superior colliculi. In the forebrain, numerous cell bodies were observed in the caudate nucleus, putamen, nucleus accumbens, nucleus of the diagonal band, anterior perforated substance and amygdaloid complex. Whereas most of these neurons were labeled highly intense for nitric oxide synthase, there were also many lightly labeled nitric oxide synthase immunoreactive neurons in the substantia innominata, globus pallidus, ansa peduncularis and lateral hypothalamic nucleus. The present observation indicated some species difference in the distribution of central nitric oxide synthase containing neurons. Furthermore, the present neuroanatomical evidence that nitric oxide synthase is distributed in a variety of specific neuronal systems, with some differences in the patterns of cytoplasmic localization, further indicates the neural messenger role of nitric oxide in the central nervous system. PMID- 7543985 TI - Internalization of IgG in motoneurons of patients with ALS: selective or nonselective? AB - The hypothesis that abnormal antibodies may be involved in the pathogenesis of ALS has been supported in part by IgG's being present within motoneurons of ALS patients more frequently than in motoneurons of controls. IgG, as well as other serum proteins, is also present in motoneurons of normal human and animal spinal cords. We attempted to determine whether the IgG found in motoneurons of ALS patients was localized by an immune-specific or nonspecific process. To address this question, we used immunocytochemistry to evaluate the presence and relative density of different serum proteins in spinal cords from nine patients with ALS. Both IgG and alpha 2-macroglobulin (alpha 2Mac) were present in motoneurons in all nine cases. More important, there was a close concordance between the IgG and alpha 2Mac immunolabeling of motoneurons. The presence of a nonimmune plasma protein--alpha 2Mac--in a similar distribution to IgG and with a similar intensity implies that the internalization of these proteins in motoneurons of patients with ALS is best explained by a nonselective mechanism of endocytosis of extracellular fluid. PMID- 7543986 TI - Canine distemper virus-specific antibodies in multiple sclerosis. AB - Current postulates support the idea that MS is triggered by an infectious agent or agents through an autoimmune reaction directed against brain antigens in genetically susceptible individuals. Evidence for an infectious etiology of MS is indirect. We have proposed that MS may, in some instances, be due to a zoonotic infection and that canine distemper virus, a measles-like virus in dogs, is a likely candidate in the causation of this disorder. The high homology between canine distemper and measles virus proteins has made it extremely difficult to distinguish distemper from measles antibodies serologically. We now provide evidence that humans can be infected with this neurotropic dog virus. Furthermore, a high titer of canine distemper virus antibodies is significantly associated with MS. Identification of the etiologic agent in MS may lead to the elucidation of disease pathogenesis and to disease prevention through appropriate public health and vaccination programs. PMID- 7543988 TI - Anti-MAG antibody-associated polyneuropathies: improvement following immunotherapy with monthly plasma exchange and IV cyclophosphamide. AB - Treatment outcomes of sensory-motor polyneuropathies associated with anti-myelin associated glycoprotein (MAG) antibodies have varied even with relatively intensive immunosuppression. We used plasma exchange and cyclophosphamide to treat four patients with anti-MAG antibody-associated polyneuropathies whose symptoms had progressed in the preceding year. Treatment courses consisted of five to seven monthly regimens of plasma exchange on 2 consecutive days followed by intravenous cyclophosphamide (1 g/m2). Effects of treatment were quantitatively measured with hand-held dynamometry. All four patients showed improvement in both strength and sensation in the 5 to 24 months after treatment. We conclude that selected patients with sensory-motor polyneuropathies associated with high-titer serum IgM autoantibodies against MAG may have quantitative and useful functional improvement after immunotherapy. The improvement continues after completion of treatment and may persist for 1 to 2 years or longer. PMID- 7543987 TI - Altered expression of transforming growth factor-beta in Alzheimer's disease. AB - We compared immunohistochemical expression of the transforming growth factor betas (TGF-beta 1, TGF-beta 2, and TGF-beta 3) using brain tissue from patients with nondominantly inherited Alzheimer's disease (NDAD) (n = 9), autosomal dominantly inherited Alzheimer's disease with linkage to 14q24.3 (FAD-14) (n = 4), and cognitively normal controls (n = 10) to determine whether their pathologic changes are associated with an altered distribution of the TGF-betas. We found increased expression of TGF-beta 2 in large, tangle-bearing neurons with widespread staining of glia in NDAD and FAD-14 patients compared with control cases. This result was confirmed with sandwich ELISA assays of brain tissue, which showed TGF-beta 2 levels in AD and NDAD to average 3.2 times the average level of control cases. Despite proximity of TGF-beta 1 and TGF-beta 3 to the sites of susceptibility loci on chromosomes 19 and 14, we did not find that TGF beta 1 and TGF-beta 3 were selectively altered in any AD subtypes. However, selective induction of TGF-beta 2 may occur in NDAD and FAD-14. PMID- 7543989 TI - Analysis of spinocerebellar ataxia type 1 (SCA1)-related CAG trinucleotide expansion in Japan. AB - Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat. We analyzed CAG repeat expansion in 25 families in the northeast of Japan with hereditary ataxia of Menzel type. Twenty of 38 patients in 12 families had expanded allele for SCA1. The number of CAG repeats correlated with the age at onset. Although the relationship between anticipation and the number of CAG repeats in successive generations was not ascertainable, there was a tendency to paternal bias for the accelerated age at onset. Study of the number of CAG repeats in various tissues showed no differences in the repeat length in lymphocytes, muscle, or brain; sperm, however, showed an obvious expansion. This may be a clue to a possible mechanism for the molecular basis of paternal anticipation of the disease. The SCA1 gene was transcribed from both wild and mutated alleles in muscles of affected individuals, but the repeat length was the same for both the muscle cDNA and the lymphocyte genomic DNA. These results suggest that, in the area of Japan where SCA1 is prevalent, 48% of families with spinocerebellar degeneration have SCA1 mutation. PMID- 7543991 TI - Sendai virosomal infusion of an adeno-associated virus-derived construct containing neuropeptide Y into primary rat brain cultures. AB - A novel neuronal gene-delivery system was investigated in primary neuron-enriched cultures with respect to driving the expression of neuropeptide Y (NPY). This delivery system consists of an adeno-associated virus-derived (AAV) plasmid, pJDT95npy, encapsulated in reconstituted Sendai virosomes. pJDT95npy contains full length rat NPY cDNA inserted downstream from the P40 promoter in a cap-gene deleted AAV-derived construct. The rep-sequences under control of the P5 and P19 promoters are intact. Virosomally encapsulated pJDT95npy drove the expression of NPY mRNAs, predominantly by P40. Total cellular NPY immunoreactivity and release in the presence of depolarization increased following pJDT95npy-transfection. Neither empty virosomes nor virosomes containing pJDT95 affected NPY mRNA expression or immunoreactivity. This study demonstrates that an AAV-derived plasmid can drive exogenous gene expression in intact neurons after infusion by Sendai virosomes. PMID- 7543993 TI - Fibroblast growth factors alter light responses and dark voltage in retinal rods of the frog (Rana temporaria). AB - Fibroblast growth factors (FGF-1 and FGF-2) were applied intracellularly via whole-cell patch-clamp electrodes while the membrane voltage was recorded simultaneously. During recording the exchange of substances by diffusion between cytosol and pipette medium affects the cell's function. Under control conditions, the loss of nucleotides is reflected by a slow hyperpolarization of the dark voltage and prolongated light responses. Addition of FGF-1 and FGF-2 to the pipette medium accelerated the time course of the hyperpolarization and intensified the prolongation of the light responses. The depolarization of photoreceptor cells after intracellular application of the nitric oxide (NO) synthase cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and the stabilization of light response recovery by L-arginine is abolished by FGF-2. FGF 2 was ineffective when it was applied together with the calcium chelator ethylene glycol-bis(2-aminoethylether)tetraacetate (EGTA). The results indicate a possible role of FGF in the regulation of NO and calcium in photoreceptor cells and may explain protective effects of FGF in degenerative processes of photoreceptor cells. PMID- 7543990 TI - The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat. AB - The intra-adrenal distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibres was studied in rats subjected to various denervations. Splanchnic nerve section eliminated the NOS-immunoreactive nerve fibres which innervate adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels and zona glomerulosa, which instead were affected by adrenal demedullation. Guanethidine, 6-hydroxydopamine (6-OHDA) and capsaicin treatments, however, did not produce any change. These results suggest that nitrergic nerves which innervate adrenal medullary cells are extrinsic (largely preganglionic sympathetic), whilst those innervating the zona glomerulosa and the majority of adrenal vessels are intrinsic, and that they do not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and 6-OHDA, or the sensory neurotoxin, capsaicin. PMID- 7543994 TI - Ribotyping on small-sized spirochetes isolated from subgingival plaque. AB - In the present study DNA restriction patterns and corresponding ribotypes of 17 subgingival small-sized spirochetes (1:2:1 and 2:4:2 isolates), 2 Treponema socranskii strains and two Treponema denticola strains were examined. Purified chromosomal DNA was digested by BamHI, HindIII, PstI and ClaI. The DNA fragments were separated in a horizontal slab of 0.7% agarose containing ethidium bromide and transferred by nylon membranes. Hybridization was carried out with digoxigenin-labelled copy DNA of 16S and 23S ribosomal RNA from Escherichia coli. Depending on the restriction endonuclease used, up to 4 distinct bands were observed for the 2:4:2 isolates and the T. denticola strains. For each of the endonucleases used, identical band patterns were always observed for this group of isolates, and these patterns differed persistently from the T. denticola strains. For the 1:2:1 strains, up to 11 distinct bands were observed after digestion with HindIII, whereas a maximum of 6 bands were observed when PstI or ClaI was used. By using ClaI, the examined 1:2:1 isolates were separated into 8 groups, whereas PstI and HindIII separated these isolates into 5 groups. The ribotyping showed that the tested 1:2:1 spirochetes were more heterogeneous than the 2:4:2 spirochetes examined. PMID- 7543992 TI - Infrequent co-existence of nitric oxide synthase and parvalbumin, calbindin and calretinin immunoreactivity in rat pontine neurons. AB - Neurons in the laterodorsal tegmental nucleus (LDTg), ventrolateral dorsal tegmental nucleus (LDTgV), pedunculopontine tegmental nucleus (PPTg), lateral and medial parabrachial nuclei (LPB and MPB) were immunoreactive to brain nitric oxide synthase (NOS) or isoform I. Double-labeling experiments showed that very few NOS-containing neurons in the pons were immunoreactive to any of the three calcium-binding proteins: calbindin-D 28K (CB-IR), parvalbumin (PV-IR) and calretinin (CR-IR). These findings extend our previous observation in the neocortex and suggest that a population of central NOS-containing neurons can be neurochemically characterized as CB/CR/PV deficient. PMID- 7543995 TI - Poster sessions: planning through presentation. PMID- 7543996 TI - Association of endogenous avian viral and endogenous viral genes with feed conversion and six-week body weight in broilers. AB - The consistency of the effect of selection on the frequencies of endogenous avian viral (eav) and endogenous viral (ev) specific restriction fragment length polymorphism (RFLP) bands was studied in two broiler lines selected from a single base population and in an F2 population derived from a reciprocal cross of both lines. One broiler line (FC line) was selected for low feed conversion ratio and the other line (GL line) was selected for high 6-wk body weight. In the F2 population, the band frequencies were determined in groups representing separate tails of the distribution of two production traits, namely, low feed conversion ratio between 29 and 42 d of age and body weight at 42 d of age. The F2 population consisted of 288 females belonging to 24 full-sib families. To rule out family effects, the tails for these production traits were composed by either the best or by the worst female performer for each trait in each full-sib family. In total, 29 HindIII-eav, 34 MspI-eav, and 21 BamHI-ev bands could be distinguished by RFLP analysis. This report describes the influence of selection on 11 potentially interesting bands. Two bands, the 9.5-kb HindIII-eav and the 15 kb MspI-eav band, which were found both in higher frequencies in the parental FC line, were also found in higher (P < or = .05) frequencies in the F2 tail with a favorable feed conversion ratio. A third band, the 6.5-kb HindIII-eav band, present in lower frequencies in the parental GL line, was also present in lower (P < or = .05) frequencies in the F2 tail of birds with heavy body weight. PMID- 7543998 TI - Amniotic alpha-fetoprotein in the prenatal diagnosis of congenital nephrotic syndrome of the Finnish type. AB - Congenital nephrosis of the Finnish type (CNF) is rare outside Finland, where the incidence may be as high as 1 in 2000 live births. Neonates with the disorder develop renal failure within the first months of life and without renal transplantation, the prognosis is extremely poor. This case report describes a woman's three pregnancies, two of which were affected with CNF. PMID- 7543997 TI - Attitudes to prenatal screening, diagnosis and research among pregnant women who accept or decline an alpha-fetoprotein test. AB - The aim of the study was to describe the opinion of pregnant women who had accepted or declined an alpha-fetoprotein (AFP) test, not only on AFP screening in general, but also on whether every pregnant woman should be offered amniocentesis (AC)/chorionic villus sampling (CVS) and an ultrasound scan for fetal malformations. An additional aim was to describe pregnant women's attitudes concerning continued research in the prenatal field. The study was performed as a questionnaire study in two regions over a 1-year period from 1 October 1988 to 30 September 1989. Results are based on answers from 3331 women who had taken an AFP test and 336 women who had declined the offer of a test. A total of 79 per cent of the women thought that an AFP test, 70 per cent that an ultrasound scan for fetal malformations, and 26 per cent that AC or CVS should be offered to all pregnant women. Fifty-nine per cent of the women were positive towards continued research in the prenatal field. Women who had had an AFP test were generally much more positive towards screening and research than women who had declined, who were generally against. Women who had left school without a high school degree were on average more positive towards the screening issues that women who had this degree. In conclusion, the results obtained in this study strongly suggest that women's attitudes are very dependent on how the prenatal screening programme is already organized in their local area. PMID- 7544000 TI - Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation. AB - The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL 4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3. PMID- 7543999 TI - Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization. AB - Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation). PMID- 7544001 TI - Interleukin 2 signaling involves the phosphorylation of Stat proteins. AB - One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. PMID- 7544004 TI - Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. AB - Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation. PMID- 7544002 TI - Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor. AB - She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide. PMID- 7544003 TI - The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen. AB - Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages. PMID- 7544006 TI - Induction of mammary epithelial hyperplasias and mammary tumors in transgenic mice expressing a murine mammary tumor virus/activated c-src fusion gene. AB - Activation of the c-Src tyrosine kinase has been implicated as an important step in the induction of mammary tumors in both mice and humans. To directly assess the effect of mammary gland-specific expression of activated c-Src, we established transgenic mice that carry a constitutively activated form of c-src under transcriptional control of the murine mammary tumor virus long terminal repeat. Female mice derived from several independent transgenic lines lactate poorly as a consequence of an impairment in normal mammary epithelial development. In addition to this lactation defect, female mice frequently develop mammary epithelial hyperplasias, which occasionally progress to frank neoplasias. Taken together, these observations suggest that expression of activated c-Src in the mammary epithelium of transgenic mice is not sufficient for induction of mammary tumors. PMID- 7544005 TI - Isolation of a primate embryonic stem cell line. AB - Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species. PMID- 7544007 TI - Olfactory transduction is intrinsically noisy. AB - The sources of noise that limit olfactory signal detection were investigated in dissociated rat olfactory receptor cells. Near-threshold odorant-evoked currents exhibited large random fluctuation. However, similar fluctuations were observed in the absence of applied odorants when currents were induced by elevating the intracellular cyclic AMP concentration. This suggests that the fluctuations reflect noise intrinsic to the transduction mechanism, rather than the quantal nature of an odorant stimulus. For many odorants, this intrinsic noise may preclude the reliable detection of single odorant molecules. PMID- 7544008 TI - Interleukin 1 beta up-regulates the expression of sulfoglucuronosyl paragloboside, a ligand for L-selectin, in brain microvascular endothelial cells. AB - Treatment of cultured bovine brain microvascular endothelial cells (BMECs) with interleukin 1 beta (IL-1 beta), an inflammatory cytokine, was shown to induce the accumulation of sulfoglucuronosyl paragloboside (SGPG), a glycolipid bearing the HNK-1 epitope. This resulted in the attachment of a greater number of human lymphocytes to the treated than to the untreated BMEC monolayers. Attachment of human lymphocytes to the IL-1 beta-activated BMEC cells could be blocked either by incubation of the human lymphocytes with an anti-L-selectin antibody or by application of an anti-SGPG antibody to the BMECs. These results suggest that SGPG may act as an important ligand for L-selectin for the regulation of the attachment of activated lymphocytes and their subsequent invasion into the nervous system parenchyma in inflammatory disorders of the central and peripheral nervous systems. PMID- 7544010 TI - The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase. AB - Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS. PMID- 7544013 TI - Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors. AB - A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer). PMID- 7544015 TI - The protective effect of thromboxane synthetase inhibitor UK 38485 against bile duct ligation induced liver injury. AB - In order to elucidate the relation between tissue eicosanoids and liver injury due to bile duct obstruction, we have examined the effects of iloprost, a stable analogue of prostaglandin I2 (PGI2), and UK 38485 (UK), an inhibitor of thromboxane synthetase, on prostaglandin E2 (PGE2) and leukotriene C4(LTC4) in guinea pig liver. 56 male guinea pigs were divided into the following groups: (i) sham operations (SHAM), (ii) bile duct ligated (BDL) group, (iii) guinea pigs given UK (5 micrograms/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation), and (iv) guinea pigs treated with iloprost (ILO) (2 micrograms/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation). Liver damage was assessed by blind quantitation of liver cell necrosis. Bile duct ligation caused an increase in tissue PGE2-like activity and a decrease LTC4-like activity. But the most pronounced elevation of PGE2-like activity was observed in ILO treated group. The LTC4-like activity level improved significantly in the UK-treated BDL group compared with the BDL only and ILO treated animals. Also, UK was found to be beneficial in preventing the liver cell necrosis due to cholestasis. It is concluded that the ratio of PGE2/LTC4 in liver is a valuable marker for cholestatic injury. PMID- 7544011 TI - Interleukin 4 signals through two related pathways. AB - The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors. PMID- 7544014 TI - Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80). AB - To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy. PMID- 7544012 TI - Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha melanocyte-stimulating hormone. AB - alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines. PMID- 7544009 TI - TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides. AB - The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA. PMID- 7544016 TI - Mechanisms of viral inhibition by interferons. AB - Interferons (IFNs) are a family of related proteins grouped in four species (alpha, beta, gamma and omega) according to their cellular origin, inducing agents and antigenic and functional properties. Their binding to specific receptors leads to the activation of signal transduction pathways that stimulate a defined set of genes, whose products are eventually responsible for the IFN antiviral effects. Their action against viruses is a complex phenomenon. It has been reported that IFNs restrict virus growth at the levels of penetration, uncoating, synthesis of mRNA, protein synthesis and assembly. This review will attempt to evaluate evidence of the involvement of the IFN-inducible proteins in the expression of the antiviral state against RNA or DNA viruses. PMID- 7544017 TI - Association of Chairmen of Departments of Physiology 1994 survey results. PMID- 7544018 TI - Developing visual aids for presentations. PMID- 7544019 TI - Reference values for fetal hemoglobin concentration in blood of steel-mill workers. AB - The concentration of fetal hemoglobin (HbF) in blood of 195 healthy men and 39 women was measured by alkali denaturation method of Singer et al. Arithmetic mean in the group of men was 0.325% whereas in women it was 0.399%. The difference between the means was statistically significant. Frequency histograms showed two unlike subpopulations with wider span of values in women, different standardised skewness and kurtosis. Kolmogorov-Smirnoff test revealed a significant abnormality of both distributions. The data presented may serve for the comparison of HbF values obtained in persons living and working in similar microclimatic conditions. PMID- 7544020 TI - The galanin-induced feeding stimulation is mediated via alpha 2-adrenergic receptors in goldfish. AB - In the present study we examined the effects of intracerebroventricular (ICV) or intraperitoneal (IP) galanin administration on food intake in satiated goldfish, at different time intervals after injections, 0-2, 2-8 and 0-8 h. We found that food intake was increased by ICV administered galanin (1 and 3.33 micrograms) at 2 and 8 h postinjection, while no modifications on feeding were observed after intraperitoneal injection in any of the studied time intervals. The galanin receptor antagonist, galantide, blocked the galanin-induced feeding increase. Pretreatment with yohimbine (alpha 2-adrenergic receptor antagonist), but not with prazosin (alpha 1-adrenergic receptor antagonist) antagonized the stimulatory effect of galanin on ingestive behavior. These results suggest that galanin is involved in the central regulation of feeding in goldfish, being the food intake stimulatory effect mediated by the alpha 2-adrenergic system. PMID- 7544021 TI - Tests for detection and identification of mycobacteria. How should they be used? PMID- 7544022 TI - [Development of an exposure index of air pollution caused by motor vehicles for use in epidemiological studies]. AB - Air pollution due to motor vehicles is a worrying issue, in particular in the urban environment, all the more as a number of pollutants such as benzene and PAH are known to have carcinogenic effects. Epidemiological studies seem to be required, so a cumulated score of exposure must be developed. A correlation with pathology should be investigated. The calculation of an index of exposure to motor vehicle-related pollution requires a good assessment of pollutant emissions and a precise knowledge of pollutant transfer mechanisms by advection and spreading in built-up areas. In this study, it is proposed to use current theoretical and experimental knowledge to develop a calculation algorithm for this index. The following issues will be addressed in the presentation: an analysis of epidemiological requirements and of constraints of data acquisition using questionnaire surveys; a model for assessing, for each residential area, the average annual concentration of gaseous pollutants from motor vehicles. It will consider the environment geometry, traffic emissions and wind distributions. The model CALINE3 is used for open areas near roads and highways and the Danish model OSPM is introduced for street canyon environments. PMID- 7544024 TI - Road traffic nuisance in residential and commercial areas. AB - Air pollution from traffic is one of the main factors considered in the environmental appraisal of road schemes. Currently this appraisal concentrates on the emission and roadside concentration of those regulated pollutants which are potentially harmful to the health or well-being of human, animal or plant life, or to ecological systems. However, vehicle emissions, especially those from diesel vehicles, may also cause a number of aesthetic and nuisance problems, such a visibility reduction, urban soiling and physical irritation. A methodology to investigated the subjective nuisance effects of air pollution from road traffic on the public has been developed and tested. The data indicates that vehicle derived air pollution was an issue of high relative importance to the public when compared to other major social issues. On a local level, the physical presence of road traffic and its associated pollution appeared to be the largest contributors to outdoor public environmental nuisance. Indoors, the public appeared to experience only minor disturbances from vehicle-derived pollution, with the average respondent being not very bothered by vehicle-derived smoke, fumes and odour, dust and dirt and general air pollution. Noise appeared to cause the greatest indoor traffic-related nuisance, although many respondents complained about soiling from dust/dirt. The surveys suggest that outdoor disturbance from vehicle-derived air pollution was fairly high, with smoke, fumes and odour causing the greatest annoyance. The main reason given for disturbance from outdoor smoke, fumes and odour and dust/dirt was concern that they would harm the public's health. Other important specific reasons included soiling and the smell of the fumes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544023 TI - Perceived traffic air pollution, associated behavior and health in an alpine area. AB - The relationship of traffic air pollution, perception of exhaust fumes/soot and behavioral impact or symptoms/illnesses was investigated in two surveys (adults: aged 25-65, N = 1989, 62% participation; children: aged 8-12, N = 796, 85% participation) in 13 small alpine communities (Tyrol/Austria) by means of questionnaire responses and air pollution measurements. Although pollutant levels complied with current WHO guidelines, adult respondents felt annoyed by odourous traffic fumes (39.7%) or visible dust/soot (26.9%). Logistic regression analysis revealed that accompanying noise annoyance, rated impairment of life quality, protesting behaviour, noise- and odour-sensitivity was directly associated with perceived air quality, while age above 45 years, smoking, and social support was inversely associated with perceived air quality. Among the symptoms, feelings of fatigue/exhaustion/low mood/nervousness and irritation of the eyes and stomach aches showed a significant association with rated air quality. Children in the traffic exposed areas spend less time outdoors and reported perception of car fumes was significantly associated with recurrent colds, chronic bronchitis and an index of hyperreactive airways. Measured indices of pollution (traffic counts, NO2) were not associated with any of the children's reported illnesses. PMID- 7544026 TI - Paclitaxel and ifosfamide: a multicenter phase I study in advanced non-small cell lung cancer. AB - Both paclitaxel and ifosfamide have significant single-agent activity in non small cell lung cancer. We designed a phase I study combining escalating doses of paclitaxel, administered by 24-hour infusion, with ifosfamide given at a dose of 1.6 g/m2 daily x 3. Paclitaxel dose levels were 135, 170, 200, 250, and 300 mg/m2. The goal of the study was to determine the maximum tolerated dose and dose limiting toxicities of paclitaxel when used in this combination. Dose escalation was possible because of the use of filgrastim, a granulocyte colony-stimulating factor. Twenty-five patients at three institutions were treated. The dose limiting toxicity of the combination was granulocytopenia, with other toxicities being generally mild to moderate. The maximum tolerated dose of paclitaxel was 300 mg/m2, and the recommended phase II dose is 250 mg/m2. There was a suggestion of a dose-response curve with paclitaxel as all three partial responses were seen at the 250 mg/m2 dose level. An additional II patients had objective regression or stable disease lasting for 9 to 30 weeks. A phase II study of this combination is currently being planned by the Cancer and Leukemia Group B. PMID- 7544025 TI - Paclitaxel by 24- or 1-hour infusion n combination with carboplatin in advanced non-small cell lung cancer: the Fox Chase Cancer Center experience. AB - A phase II trial of combination paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) and carboplatin included 54 chemotherapy-naive patients with advanced non-small cell lung cancer. Eligibility mandated Eastern Cooperative Oncology Group performance status of 0 or 1 and adequate hematologic, renal, and hepatic function. Paclitaxel 135 mg/m2 over 24 hours preceded carboplatin dosed to an area under the concentration-time curve of 7.5. Six planned courses were given every 3 weeks. Granulocyte colony-stimulating factor was introduced during the second and subsequent cycles, and paclitaxel increased 40 mg/m2/cycle (maximum, 215 mg/m2) in patients with absolute neutrophil and platelet nadirs exceeding 500/microL and 50,000/microL, respectively. Grade 3 or 4 neutropenia, observed in 54% of patients during cycle 1, declined to 35% during cycle 2 and to 22% or less during cycles 3 through 6. Neuropathy, myalgias/arthralgias, and thrombocytopenia were mild but cumulative. In 53 evaluable patients, the objective response rate was 62%, with 9% complete remissions and a median response duration of 6 months (range, 1 to 19+ months). At median potential follow-up of 16 months, 9% of patients remain progression free (52+ to 80+ weeks). Median survival is 12.5 months; 1-year survival is 54%. Paclitaxel/carboplatin is highly active in advanced non-small cell lung cancer; granulocyte colony-stimulating factor abrogates neutropenia as the dose-limiting toxicity, but has no effect on the cumulative incidence of thrombocytopenia or treatment delays. One-hour paclitaxel infusion is minimally myelosuppressive, logistically easier, and less costly. A follow-up study combined paclitaxel (175 mg/m2) over 1 hour followed by carboplatin (area under the concentration-time curve, 7.5). In the absence of grade 4 myelosuppression, paclitaxel was increased 35 mg/m2/cycle (maximum, 280 mg/m2). Granulocyte colony-stimulating factor was implemented only after neutropenic fever or grade 4 neutropenia. Of 17 patients entered, 13 are evaluable for toxicity and seven for response. Four patients have sustained a partial response, two a minor response, and one stable disease. The incidence of grade 3 or 4 neutropenia, thrombocytopenia, and anemia in cycle 1 was 38%, 16%, and 0%, respectively, and 72%, 28%, and 28%, respectively, during cycle 2. Major nonhematologic toxicities include myalgias and arthralgias (54%) and fatigue and neuropathy (78%), the latter cumulative and progressive over successive cycles. Preliminary data suggest comparable activity for the 1- and 24 hour paclitaxel infusions in combination with carboplatin. The more severe neuropathy of the 1-hour paclitaxel/carboplatin combination may be related to the paclitaxel dosing schema (175 mg/m2 to as high as 280 mg/m2).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7544027 TI - Four-day paclitaxel infusion with cisplatin for patients with lung cancer. AB - Lung cancer cell lines are between seven and 1,000 times more sensitive to paclitaxel (Taxol; Bristol-Myers Squibb Co, Princeton, NJ) when exposed for 120 hours (5 days) compared with 3-hour exposure. A phase I study of 4-day infusion of paclitaxel plus bolus cisplatin for patients with lung cancer has defined the recommended phase II dose. In this study, paclitaxel infused at 30 mg/m2/d for 4 days followed by a cisplatin bolus of 80 mg/m2 after infusion completion was associated with acceptable hematologic toxicity. Nine of the 16 patients with non small cell lung cancer treated with at least two cycles of this regimen attained an objective tumor response (one complete response and eight partial responses; overall response rate, 56%). The recommended phase II dose of a 4-day infusion of paclitaxel plus bolus cisplatin followed by the administration of granulocyte colony-stimulating factor has not yet been determined. PMID- 7544028 TI - Paclitaxel and carboplatin with and without filgrastim support in patients with metastatic non-small cell lung cancer. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Oncology, Princeton, NJ) and carboplatin have each shown activity against non-small cell lung cancer and they are synergistic in vitro. We thus designed a phase I study to define the maximum tolerated dose and dose-limiting toxicity of the combination with and without filgrastim support. With an initial fixed dose of paclitaxel 135 mg/m2 given as a 24-hour infusion, carboplatin was administered in escalating doses in cohorts of three patients, based on a target area under the concentration-time curve (AUC) of 5, 7, 9, or 11 using Calvert's formula: dose (mg) = target AUC x (glomerular filtration rate + 25). Dose escalations were based on course I toxicities. Filgrastim 5 micrograms/kg was administered with the first cycle only after grade 4 neutropenia occurred in two of three patients at the prior dose level. One hundred five courses of paclitaxel and carboplatin have been administered in 26 patients. Dose-limiting toxicity (grade 4 neutropenia) occurred in two patients at level 2 (cycle I). Filgrastim was instituted thereafter with cycle I for the next four levels. Grade 4 thrombocytopenia was seen at level 4; thus, the carboplatin dose was de-escalated in the next level, but the paclitaxel dose was escalated. The regimen has been well tolerated. One patient had a complete response and 12 had partial responses, for an overall response rate of 50%. There is a suggestion of a dose-response effect with both paclitaxel and carboplatin. The combination of paclitaxel and carboplatin is active in non-small cell lung cancer, and the recommended phase II doses for the combination without filgrastim support are paclitaxel 175 mg/m2 as a 24-hour infusion with the carboplatin dose based on a target AUC of 7. The phase II dose with filgrastim support will be defined as dose escalation of paclitaxel continues. PMID- 7544030 TI - Dose-finding and sequencing study of paclitaxel and carboplatin in non-small cell lung cancer. AB - A dose-finding study was set up to identify the optimal dose of the combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) and carboplatin for phase II studies in patients with advanced chemotherapy-naive non small cell lung cancer (NSCLC). The influence of drug sequence on the toxicity and pharmacokinetics of both agents was also assessed. To develop an ambulatory regimen for palliation of advanced NSCLC, paclitaxel was infused over 3 hours with standard premedication and carboplatin over 30 minutes. Cycles were repeated every 4 weeks. At each dose level, at least six patients were randomized to receive either paclitaxel followed by carboplatin or the reverse sequence. In the second and following cycles the alternate sequence was administered. The pharmacokinetics of both paclitaxel and carboplatin were compared in the first two cycles in at least two patients per dose level. Sixty-two patients have been entered in this study. Paclitaxel was increased from 100 mg/m2 in 25 mg/m2 increments up to a maximum of 225 mg/m2 combined with a fixed carboplatin dose (300 mg/m2). Thereafter, the drug doses were increased to a maximum of 400 mg/m2 carboplatin and 250 mg/m2 paclitaxel. In 243 cycles, the most frequent side effects were neutropenia, alopecia, and mild emesis. Only one patient developed a major hypersensitivity reaction to paclitaxel. Bone pain, myalgia, and peripheral neurotoxicity occurred more frequently at paclitaxel doses above 200 mg/m2. No significant differences in toxicity or in the pharmacokinetics of either drug were observed between the two drug sequences. The pharmacokinetics of paclitaxel were nonlinear and consistent with saturation. At the highest paclitaxel dose (250 mg/m2 with carboplatin 350 mg/m2) a toxic death due to severe leukopenia, thrombocytopenia, and hemorrhage occurred. Safe doses for phase II trials in untreated NSCLC are 200 mg/m2 paclitaxel with 300 mg/m2 carboplatin. Of 50 evaluable patients, five of the six major responses were observed at paclitaxel doses of 175 mg/m2 and above, which suggests a dose-response relationship for paclitaxel in NSCLC. PMID- 7544029 TI - A phase I study of ifosfamide/carboplatin/etoposide/paclitaxel in advanced lung cancer. AB - A phase I study was conducted to define the maximally tolerated dose and toxicity profile of the ifosfamide/carboplatin/etoposide/paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) (ICE-T) regimen in advanced lung cancer. This chemotherapy program uses paclitaxel given as a 24-hour continuous infusion in conjunction with full-dose ICE chemotherapy with growth factor support. The dosage of paclitaxel was escalated from 75 to 225 mg/m2. Thirty-four patients have been accrued to date onto this study. Because hematologic dose-limiting toxicity was defined in terms of neutropenia and/or thrombocytopenia exceeding 7 days' duration, no patient demonstrated what was defined by the protocol as dose limiting toxicity. Nonetheless, substantial hematologic toxicity was observed. Overall, 26% had fever and neutropenia, 56% had grade 4 neutropenia, and 26% had grade 4 thrombocytopenia. In all cases, hematologic toxicity was short term and reversible. While grade 3 and 4 myelosuppression was frequently observed, it was not dose related (in terms of paclitaxel dosage). Nonhematologic toxicity also was not dose related and, with only a few exceptions, was not clinically significant. Among 27 patients evaluable for response, 41% achieved an objective response, including 15% with a complete response. All of five patients with small cell lung cancer responded (including two with a complete response). Among 22 patients with non-small cell lung cancer, 27% achieved an objective response (also including two with a complete response). The results of this study suggest that with growth factor support, it is possible to safely administer full-dose, single-agent paclitaxel in conjunction with full-dose ICE chemotherapy. We will soon be initiating a phase II study of the ICE-T regimen using paclitaxel at 225 mg/m2 as a 24-hour continuous infusion in advanced lung cancer. We will also conduct a phase I study of ICE-T, with paclitaxel administered as a 3-hour continuous infusion. PMID- 7544032 TI - [Multiple sclerosis]. AB - Multiple sclerosis is a chronic inflammatory disease of the central nervous system which affects young and middle-aged adults. The clinical symptoms and course vary considerably; most patients experience a relapsing-remittent course which may become secondary progressive, a benign course with a favourable prognosis is seen in 30-40%. 10-15% experience a primary progressive course, with a less favourable prognosis. Magnetic resonance imaging, MRI, has become the most important paraclinic investigation and MRI with paramagnetic contrast enhancement reveals "subclinical" disease activity. Demyelination is caused by phagocytosis of microglial cells which appear to be activated by cytokines produced by T helper cells. A delayed type autoimmune response to myelin antigens is probably involved, initiated by a viral infection. Immunosuppressive treatment, corticosteroids and interferons have been shown to affect the MRI behaviour, and reduce the frequency of relapse. The effect on the natural course of the disease has been less convincing and no treatment will reverse long standing neurological dysfunction. PMID- 7544031 TI - Osteogenesis imperfecta complicated by osteosarcoma. PMID- 7544033 TI - Beta-adrenergic control of c-fos expression in fetal and neonatal rat tissues: relationship to cell differentiation and teratogenesis. AB - beta-Adrenergic receptors appear in noradrenergic target tissues well before the arrival of nerve terminals, and are thought to play a role in the control of cell differentiation. We examined the ability of beta-agonists to stimulate expression of the nuclear transcription factor, c-fos, in developing rat liver and heart. This factor has been shown to associated with trophic activation of genes involved in both cell differentiation and cell growth. In response to terbutaline, a beta 2-selective agonist, marked stimulation of c-fos was demonstrated in the liver, which contains beta 2-receptors, on Gestational Day 20, as well as on Postnatal Days 1 and 8. In the heart, which contains predominantly beta 1-receptors, isoproterenol (a non-subtype-selective beta agonist) was more effective that terbutaline, indicating that either receptor subtype can elicit stimulation of c-fos. In both tissues, the response magnitude increased with age, rather than following changes in receptor number, which increase in the heart but decrease in the liver; the same pattern has been seen for the ability of beta-agonists to promote cell differentiation at the expense of replication; the implication is that ontogenetic changes in post-receptor coupling are much more important than is the number of receptors in determining neurotrophic influences on gene expression and cell development. In keeping with the view that fetal/neonatal beta-adrenergic stimulation of c-fos is related to cell differentiation rather than simply to growth, repeated administration of isoproterenol to neonatal rats did not elicit cardiac hypertrophy, whereas the same treatment did produce hypertrophy in adult rats. The intracellular signaling cascade from beta-receptor to c-fos expression may thus provide one of the basic cellular mechanisms for trophic control of differentiation by biogenic amines, and for the teratologies associated with beta-adrenergic agonist therapy. PMID- 7544035 TI - A proposal of FK506 optimal dosing in living related liver transplantations. AB - We analyzed the relation between FK506 trough levels (ELISA: patients 1-41, IMx: patients 42-70) and rejection and/or viral infection episodes, retrospectively, in the first 70 consecutive cases of living related liver transplantation. Twenty patients (28.6%) had rejection episodes. Of the 13 patients who had evidence of rejection during the first 3 months, 6 patients without infection and 7 patients with viral infection showed low concentrations of FK506 (< 5 ng/ml). Twelve patients were treated and improved with high dose steroid administration and an increase in the FK506 dosage. One patient died of refractory rejection. Nine patients had evidence of rejection after the first 3 months. In 3 patients, weaning from FK506 initiated the rejection episodes. Five patients repeated rejection and 4 patients required a third immunosuppressant (azathioprine). Viral infection included CMV (11 cases), EBV (13 cases), HZV (3 cases), and HSV (1 case). Excess immunosuppression might have been the cause, but no clear correlation was found. We propose that the optimal dosage of FK506 obtained by monitoring the trough levels using the IMx method should maintain a 10-20 ng/ml level during the first month, and a 5-10 ng/ml level at the second and third months. PMID- 7544034 TI - Fas-mediated cytotoxicity. An immunoeffector or immunoregulatory pathway in T cell-mediated immune responses? AB - Fas/Fas ligand interactions serve as a signaling pathway for apoptosis (1-3), an important regulatory mechanism in the development and function of the immune system (4-9). Recent evidence that Fas-dependent apoptosis is also an important mode of T cell cytotoxicity (10-13) suggested that Fas might play a critical role in the effector phase of T-dependent immune responses, such as allograft rejection. We observed that Fas transcripts are constitutively expressed in syngeneic and allogeneic murine cardiac transplants, while Fas ligand (FasL) is up-regulated only in rejecting allografts. Surprisingly, the absence of an intact Fas/FasL pathway did not alter the tempo of allograft rejection, even CD4 dependent rejection. These results indicate that Fas/FasL interactions are not essential mediators of T cell-induced allograft damage. Rather, as suggested in other studies, the Fas pathway may be principally involved in the regulation of clonal expansion and subsequent contraction of T cell populations during immune responses. PMID- 7544036 TI - Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells. AB - Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations. PMID- 7544038 TI - Antithrombotic effect of FK506 versus prothrombotic effect of cyclosporine in vivo. PMID- 7544037 TI - The direct effect of injectable cyclosporine and its vehicle, cremophor, on endothelial vascular cell adhesion molecule-1 expression. Ricinoleic acid inhibits coronary artery endothelial activation. AB - As in humans, rabbit coronary artery endothelium basally expresses vascular cell adhesion molecule-1 (VCAM-1). Treatment with parenteral cyclosporine (CsA) to prevent graft rejection in rabbits receiving heterotopic heart transplantation reduced VCAM-1 expression in coronary arteries not only in transplanted, but also in native rabbit hearts. To explore the mechanism of this effect, we co-incubated cultured human saphenous vein endothelial cells for 24 hr with CsA or its vehicle (containing polyoxyethylated castor oil, or Cremophor, and ethanol), at concentrations compatible with those achievable in plasma during administration of parenteral preparations of CsA. Cells were then stimulated with TNF alpha or IL-4 to induce VCAM-1 expression, assessed by a cell-surface enzyme immunoassay. Both CsA and vehicle inhibited IL-4-stimulated VCAM-1 expression in a dose dependent manner (from [OD mU, mean +/- SEM] 230 +/- 5 to 165 +/- 3 for CsA 50 ng/ml, and to 181 +/- 6 for the corresponding vehicle concentration; P < 0.05 for both comparisons). To investigate whether this vehicle effect also occurs in vivo, we treated 9 New Zealand White rabbits with saline (n = 3), CsA (10 mg/kg/day, n = 3), or vehicle at corresponding doses (n = 3) for 6 weeks. Profiles of coronary arteries (> or = 48 for each group) were semiquantitatively scored (0-5) for VCAM-1 in immunostained heart cross-sections. Administration of both CsA and vehicle significantly reduced VCAM-1 expression compared with saline. Two vehicle components, ethanol and ricinoleic acid, were further evaluated directly on endothelial cells in vitro. While ethanol was ineffective, the monounsaturated fatty acid ricinoleic acid inhibited IL-4-stimulated VCAM-1 expression in a dose-dependent manner (IC50 between 10 and 100 microM). Thus, a fatty acid component of CsA vehicle exerts direct endothelial effects, potentially limiting arterial leukocyte recruitment during parenteral CsA treatment. This observation reveals a novel mechanism for CsA as an inhibitor of leukocyte-endothelial interactions, and furnishes a new potential rationale for the therapeutic action of unsaturated fatty acids in graft coronary disease. PMID- 7544042 TI - An unusual late relapse in pathologic stage I non-seminomatous germ cell tumor: case report and review of the literature. AB - Late relapses are exceedingly uncommon in patients with pathologic stage (PS) I non-seminomatous germ cell tumors (NSGCT) of the testis treated with inguinal orchiectomy and retroperitoneal lymph node dissection (RPLND). We describe an unusual patient with PSI NSGCT who relapsed 40 months after surgery. The only evidence of recurrent disease was an elevated serum alpha-fetoprotein level, and complete remission was achieved following the prompt institution of combination chemotherapy. To our knowledge, this is the first report of a late relapse in PSI NSGCT of the testis manifested only as an elevated tumor marker. PMID- 7544040 TI - Esophageal prosthesis in palliation of malignant esophageal obstruction. AB - Esophageal intubation with a plastic prosthesis is a well established palliative treatment for esophageal carcinoma. The technique is safer with endoscopy than previous surgical techniques. Advantages of stent include rapid and long lasting relief of dysphagia in most patients with carcinoma esophagus. Repeated procedures are not required. Placement of prosthesis is the treatment of choice in BEF. Cost is less compared to other palliative modalities such as laser. SEMS have distinct advantages over conventional prosthesis as they may be inserted with less trauma and fewer complications. Diet needs occasionally to be limited to soft or blenderized foods to prevent occlusion. A disadvantage of uncovered SEMS is short duration of palliation due to tumor ingrowth which can be overcome with availability of covered SEMS. Starvation is the most common cause of death in patients with esophageal malignancy. Prosthesis combats deterioration and leads to rapid weight gain. Overall, single time procedure without general anaesthesia, short hospital stay and immediate improvement in dysphagia are considerable gains. PMID- 7544039 TI - Experience with intraluminal radiotherapy in advanced oesophageal cancer. AB - Despite improvement in the diagnostic modalities, surgical technique, chemotherapy and radiotherapy, mortality and morbidity due to carcinoma esophagus continues to be dismal. Combination of external and intraluminal radio therapy (ILRT) has emerged as a powerful and promising palliative therapy in this disease. Thirty four patients with inoperable cancer esophagus treated with ILRT during June 1991 to December 1993 were evaluated to assess its palliative effects. Seventeen of these patients had received additional chemotherapy and external radiotherapy. Thirteen patients received only radiotherapy (both external radiotherapy + ILRT) and the remaining 4 received only ILRT. They were followed up for a mean period of 8.3 months (range 2 to 28 months) during which one patient was lost to follow up. Eight had a follow up of less than 6 months. Eight (33%) amongst the remaining 25 patients were considered disease free, 15 had recurrent and progressive disease, one developed metastasis and one patient died. In 21 (66%) dysphagia markedly improved. Nine (26%) patients survived beyond 1 year and the median survival for all patients was 8 months. Associated radiation morbidity was documented in 13 (38%) patients. We conclude that combination of external radiotherapy and ILRT is an effective and safe therapy for inoperable esophageal malignancies. PMID- 7544043 TI - Apparent lack of response of salmon affected by pancreas disease to pancreatic enzyme replacement therapy. AB - A trial was conducted to establish whether there would be any benefit in feeding mammalian pancreatic enzymes to farmed salmon clinically affected by pancreas disease. Pancreatic enzymes were incorporated at a rate of 4 kg/tonne of feed and fed at 0.6 to 0.9 per cent bodyweight/day for 41 days to one cage of salmon. The weights, lengths and condition factors were established for the fish before and after the feeding trial and compared with those for an adjacent cage of untreated fish. Histopathology and blood amylase and lipase activities were monitored weekly. No significant increases in weight or condition factor, and no reduction in mortalities were recorded. Histopathology confirmed the presence of pancreas disease throughout the trial and there were concurrent skeletal and cardiac myopathies. Blood amylase activities showed no significant change and were considered to be within normal ranges; blood lipase activity remained undetectable. PMID- 7544041 TI - Comparative evaluation of commonly used catheters through histopathological changes induced in bladder urothelium. AB - There is growing concern about the toxic potential of chemicals coating various catheter materials, especially due to lack of international standards for toxicity testing. The present study evaluates the toxicity potential in terms of pathological changes induced by the catheters commonly used in practice, i.e. latex-, silicone- and Teflon-coated catheters. Teflon-coated catheters appear to be superior to others in the present study. PMID- 7544044 TI - Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. AB - We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription. PMID- 7544045 TI - Vaccination of cattle with the N-terminus of L2 is necessary and sufficient for preventing infection by bovine papillomavirus-4. AB - We have previously shown that cattle vaccinated with L2, the minor structural protein of bovine papillomavirus-4 (BPV-4), do not develop alimentary papillomas upon challenge with BPV-4. Analysis of the B and T cell response in L2-vaccinated animals showed that the majority of the response was directed against the N terminus and C-terminus of L2 with little response against the middle portion. Cattle were vaccinated with the N-terminus or the C-terminus of L2. The animals vaccinated with the N-terminus were completely protected from viral challenge, whereas the animals vaccinated with the C-terminus were not. Further analysis with synthetic overlapping peptides spanning the entire N-terminus mapped a B cell immunodominant epitope at amino acid 101-120. This epitope was recognised by all vaccinated animals. PMID- 7544047 TI - Nucleotide sequence analysis of the M genomic segment of El Moro Canyon hantavirus: antigenic distinction from four corners hantavirus. AB - El Moro Canyon hantavirus (ELMC, previously known as HMV-1) is associated with the western harvest mouse Reithrodontomys megalotis. The interpretation that ELMC is a novel hantavirus was based upon comparisons of the nucleotide sequence of the S genomic segment with those of other hantaviruses. We now show that the ELMC M genome, like the S genome, is genetically similar to but distinct from that of Four Corners hantavirus (FC). The ELMC M genome is 3801 nt in length and encodes a glycoprotein precursor of 1139 amino acids. The G1 and G2 genes are 71 and 73.1% identical to those of FC and the corresponding glycoproteins are 73.5 and 82.3% identical. A portion of the G1 glycoprotein of ELMC that is homologous to an important linear epitope of FC differs from the FC epitope by 10 of 31 residues. That domain and flanking sequences were expressed in Escherichia coli. G1 antibodies from 3 of 19 FC-infected patients showed cross-reactivity with ELMC by Western blot assay. The portion of the ELMC G1 antigen recognized by those antibodies is the region of homology to the FC epitope. These data support our previous interpretation that ELMC is distinct from FC and other hantaviruses. PMID- 7544046 TI - Analysis of human immunodeficiency virus type 1 integrase mutants. AB - The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication. PMID- 7544048 TI - Specificity and neutralizing capacity of three monoclonal antibodies produced against the envelope glycoprotein of simian immunodeficiency virus isolate 251. AB - Three mouse monoclonal antibodies (mAb) were produced against soluble recombinant vaccinia virus gp140 from SIV-mac251. Two mAbs (1B9 and 6C11) were mapped at the aa 411-430 sequence within the V4 domain, and the third mAb (3C8) recognizes a conformation-dependent epitope on the external envelope glycoprotein. This was shown by its loss of reactivity in Western blot and ELISA with dithiothreitol reduced gp140. mAb 3C8, but not 1B9 and 6C11, cross-reacts well with gp140 and gp125 from HIV-2ROD, indicating that this discontinuous epitope includes conserved regions localized within the external envelope glycoprotein. Analysis of the neutralizing activities of the mAbs showed that only mAb 1B9 is able to inhibit both syncytium formation and SIVmac251 infection of human peripheral blood lymphocytes. PMID- 7544049 TI - Most of the VP1 unique region of B19 parvovirus is on the capsid surface. AB - B19 parvovirus is pathogenic in man and a vaccine is desirable. In convalescence after acute infection, the dominant humoral immune response is directed to the minor capsid protein called VP1, which differs from the major capsid protein by an additional NH2-terminal 227 amino acids. We have previously shown that this unique region contains multiple linear neutralizing epitopes. We produced seven recombinant B19 capsids that contained progressively truncated VP1 unique region sequences, each fused to a Flag peptide (AspTyrLysAspAspAspAspLys) at the NH2 terminus. Capsids containing normal VP2 and truncated Flag-VP1 proteins and, in some cases, only truncated Flag-VP1 chimeric proteins, were analyzed by ELISA, affinity chromatography, and electron microscopy using anti-Flag monoclonal antibody. All regions examined showed binding to anti-Flag antibody in multiple assays, indicating that most of the VP1 unique region is external to the capsid and accessible to antibody binding. These results have implications for the design of a B19 parvovirus vaccine and the use of empty capsids for presentation of heterologous protein antigens. PMID- 7544050 TI - Polymerase substrate depletion: a novel strategy for inhibiting the replication of the human immunodeficiency virus. AB - Mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase, shows strong anti-HIV activity in vitro in both human peripheral blood CD4+ lymphocytes and macrophages, as well as established human cell lines. MPA shows its greatest antiviral effects during the early stages of HIV infection. By limiting the rate of de novo synthesis of guanosine nucleotides, this drug apparently blocks the activity of reverse transcriptase, which is required for the formation of the HIV DNA provirus. MPA provides a novel strategy for inhibiting the replication of HIV and should be considered in clinical trials of antiviral therapies. PMID- 7544051 TI - Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex dependent epitope on gp 120. AB - The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization. PMID- 7544053 TI - [Inhibitors of NO-synthase and guanylate cyclase block the modulation of cholinoreceptor plasticity in snail neurons by 15-hydroxyeicosatetraenoic acid]. AB - Probable participation of two second messengers, NO and cGMP, in short- and long latent effects of 15(S)-hydroxy-(5Z,8Z,11Z, 13E)-eicosatetraenoic acid (15-HETE, acyclic eicosanoid) on plasticity of somatic acetylcholine receptors on identified neurons of Helix lucorum was studied using two-electrode voltage clamp technique. It was shown that inhibitor of NO synthase N omega-Methyl-L-Arginine and inhibitors of soluble guanylate cyclase LY-83,583 and methylene blue disturbed short- and long-latent modulatory effects of 15-HETE on depression depth of the inward current evoked by rhythmic acetylcholine application on the neuron soma. We suppose that NO and cGMP participate in short- and long-latent effects of 15-HETE on plasticity of acetylcholine receptors. PMID- 7544055 TI - [Vaginal mixed tumor or spindle cell epithelioma--a case report]. AB - A case of mixed tumor of the vagina or spindle cell epithelioma is presented and the literature on this rare type of tumor is reviewed. Immunohistochemical findings suggest an epithelial origin. Follow-up studies indicate benign behavior. However, recurrent tumors were reported suggesting careful follow-up observation after excision of extended primary tumors. PMID- 7544056 TI - [The day care unit--a patient and personnel oriented and cost effective patient management?]. AB - Based on the increasing number of chronically ill patients with restricted life expectancy it was necessary to reorganize the health care system. Aim of the study was to evaluate the benefits for patients and health care workers and the costs of day hospital care. In March 1991 a day clinic with 20 beds for HIV antibody negative and HIV-infected patients was established. The number of patients treated increased from 122 patients in April 1991 to 487 patients in March 1993. During a 6 1/2-month period in 1994 2745 patients--33% were HIV infected - were treated and diagnosed on this ward. The mean duration of hospital stay was 1.8 days. From January 1994 till 19th July 1994 200 diagnostic invasive procedures were performed, 150/200 were bronchoscopies. During the same period 158 non invasive diagnostic procedures were done. 436 cycles of chemotherapy were applicated and 277 patients were treated with blood products. 55/2745 patients (2%) had to be admitted to the ward because of worsening of the clinical condition. Based on the increasing number of patients who preferred the day clinic to the ward, it was an optimal sort of taking care for patients. Based on the results of questionnaires answered by nurses, working at the day clinic is beneficial for health care workers. Since the mean duration of hospital stay is reduced to 1.8 days the day clinic is cost-effective. PMID- 7544054 TI - [Persistent granulocytosis and hyperuricemia after a single G-CSF injection (Filgrastim) in treatment of chemotherapy-induced myelosuppression in metastatic breast carcinoma]. AB - We report about a patient with metastatic breast cancer treated with chemotherapy regime (CMF), who developed a febril leukopenia as well as a hyperuricemia. A single subcutaneous application of 300 micrograms Filgrastim led to a severe overstimulation with leukocytosis of max. 50,000 for about 8 days and hyperuricemia. PMID- 7544058 TI - Disappearance of neutrophil fluctuations in a child with cyclic neutropenia by combination therapy of granulocyte colony-stimulating factor and high-dose immunoglobulin. AB - We report on a patient with cyclic neutropenia who was treated by granulocyte colony-stimulating factor (G-CSF) and high-dose immunoglobulin. The serial examination revealed cyclic fluctuations in the numbers of neutrophils, monocytes, platelets in peripheral blood, and in the serum G-CSF concentration. Bone marrow examination confirmed a cyclic fluctuation of both progenitor cells (CFU-GM) and CD10-positive B cells. The therapy of G-CSF followed by high-dose immunoglobulin achieved a disappearance of neutrophil oscillations. It suggested that the combination therapy of G-CSF with high-dose immunoglobulin might be effective for cyclic neutropenia. PMID- 7544057 TI - Increasing bone density in myeloma patients after the administration of clodronate. AB - The use of bisphosphonates in hypercalcemia is fully accepted, but the long term therapy with bisphosphonates is still controversial. The aim of our study was to evaluate the influence of clodronate on the bone density of myeloma patients. 20 patients were included in the study. A total dose of 3000 mg clodronate was administered in 4 to 6 hourly infusions of 600 mg a day, once in 3 months. The effect of clodronate on bone density was evaluated by CT-densitometry over a period of 6 months. At the beginning of May 1994, 15 patients had completed at least 2 estimations of bone density. The amount of hydroxyapatite had increased in 9 patients, remained unchanged in 1 of them, and decreased in 4 of them during the 6 months. The mean bone density before the administration of clodronate was 2.6 SD (standard deviation of European standard bone density for the respective age and sex). After 6 months of therapy, bone density had increased to -2.3 SD. The mean amount of hydroxyapatite in spongiosa rose from the mean value of 32.71 mg/ml before clodronate administration to 38.91 mg/ml after the 6-month treatment period. The mean increase in calciumhydroxyapatite in trabecular bone mass was 6.2 mg. Clodronate contributed to alleviating bone pain in the majority of patients, but this effect is difficult to evaluate because of other treatment modalities administered concomitantly. The tolerance of clodronate was very good. No impairments of renal function, nor other adverse effects were observed. Only in 2 patients the decrease in calcium concentration caused slight tetania.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544059 TI - [Obsessive-compulsive disorder and depression]. AB - Obsessive-compulsive disorders (OCD) are unique among the anxiety disorders. They have many characteristics in common with depressive disorders, both from clinical as well from psychopathological and neurochemical perspectives. The clinical response to serotonergic antidepressants is also similar. But, in spite of the common aspects, there are at every level important peculiarities which definitively differentiate OCD from depressive disorders. We present data on neuroendocrinological challenges and on brain regional blood flow measured with the SPECT. OCD patients not fulfilling diagnostic criteria for depression have an exaggerated response to a serotonergic challenge, measured by the increase in Prl, Cor and GH plasma concentrations when compared to controls. The differences are higher when controlling for the presence of depressive (subsyndromal) symptoms. Patients with OCD and comorbid depression have a blunted response to the CMI challenge. SPECT shows an increased blood flow in basal ganglia and hippocampus on the left side. In the presence of a comorbid depression, the regional brain blood flow shows a marked decrease in wide range of brain regions. Therefore, the presence of depressive symptoms and even more of diagnostic criteria for major depression significantly changes neuroendocrinological challenge tests and regional blood flow in OCD patients, making them look like depressive patients. Although depressive symptoms are present in many OCD patients and that there is a significant comorbidity between OCD and depressive disorders, we believe that they can be differentiated from the biological point of view. Furthermore, the association of depressive symptoms and of depression diagnostic criteria to OCD masks the biological finding, neurochemical and of brin regional blood flow, of OCD. This is an important factor to be considered when carrying out research in this field. PMID- 7544061 TI - The burden of prostatic hyperplasia. PMID- 7544062 TI - [Effect of growth hormone and thyroxine on renal development in Snell pituitary dwarf mice]. AB - To elucidate the effects of growth hormone (GH) and thyroxine (T4) on renal development, we biochemically and morphologically examined the renal tissue of Snell pituitary dwarf mice (dw/dw) at different developmental stages. Differences in DNA, RNA and insulin-like growth factor-1 (IGF-1) content between the dwarf and normal control mice first became apparent on the 10th day of age, and from this time that of dwarf mice showed no further increase in DNA and RNA content and decrease in IGF-1 content, whereas that of the normal control mice continued to increase. These findings indicated that GH and T4 may have effects on the renal development, and deficiency of these hormones to affect renal development from the 10th day of age. PMID- 7544064 TI - [A case of tacrolimus-induced glucose intolerance following renal allografting]. AB - Living renal transplantation (Tx) was carried out on a 41-year-old male undergoing hemodialysis for a six-month period because of end-stage renal failure due to chronic glomerulonephritis. Tacrolimus (FK 506) was used as one of immunosuppressants. The graft worked immediately after Tx. However, his blood sugar level rose extremely high and use of insulin (IS) was required. At the second postoperative day, 0.3 mg/kg/day of FK506 was administered and the trough level (TL) was as high as 65 ng/ml. The serum IS level decreased from the pre-Tx value of 22 microU/ml to 12 microU/ml. With decrease in the dose of FK506, the TL was normalized, and the dose of IS could be decreased. FK506 has been reported to inhibit IS secretion. Therefore, we must be careful to evaluate the blood glucose level in the use of FK506 for patients with poor glucose tolerance. PMID- 7544052 TI - Congruences in Chinese and Western medicine from 1830-1911: smallpox, plague and cholera. AB - A close examination of three examples, smallpox, plague and cholera, suggest that for acute infectious diseases the Chinese viewed the symptomatologies, the causes, and the rational treatments of these illnesses in many ways similar to that of their contemporary Western counterparts. Rather than holding an opposing, clashing or incongruent system of medical thoughts for these common, well recognized infectious diseases, the Chinese were prepared, by a long tradition of ontological thinking, to be receptive to the adoption, incorporation or modification of Western medical ideas in the late nineteenth century. PMID- 7544060 TI - Human cervical cancer. Retinoids, interferon and human papillomavirus. AB - Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16 immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways). PMID- 7544063 TI - [Value of serum PSA and PAP measurement with newly developed latex turbidimetric immunoassay]. AB - Serum prostate specific antigen (PSA) and prostatic acid-phosphatase (PAP) levels in normal controls, and patients with prostate cancer, benign prostate hypertrophy (BPH) and other urological diseases were examined with a newly developed latex turbidimetric immunoassay (LPIA ACE PSA, LPIA ACE PAP, IATRON LABORATORIES, INC., Tokyo, Japan). The advantageous characteristics of this method are small amount (10 microliters) of serum required and short time (about 20 min.) for performing this assay. There was a high linear correlation between LPIA ACE PSA and MARKIT-F PA (r2 = 0.953), between LPIA ACE PSA and TANDEM-E PSA (r2 = 0.881) and between LPIA ACE PAP and ABBOTT-PAP EIA (r2 = 0.946). When the BPH patients (n = 110) were used as negative controls, the cut-off value of PSA was determined to be 4.3 ng/ml. Using this level as the cut-off value, the sensitivity was 78% (42 positive/54 untreated prostate cancer patients), specificity (negative rate in BPH patients) was 95% and efficiency was 89%. In a follow-up study of prostate cancer, the PSA value was elevated above the cut-off value in 68% at the time of clinical progression. These findings suggest that LPIA ACE PSA is a useful tool for serum PSA measurement. The cut-off value of PAP measured with LPIA ACE PAP was 9.0 ng/ml, which was determined by the same method as PSA. The sensitivity, specificity and efficiency ware 39%, 96% and 77%, respectively. These findings indicate that PAP is less useful than PSA in the diagnosis of prostate cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544065 TI - Anti-neutrophil cytoplasm antibodies and anti-glomerular basement membrane antibodies: two coexisting distinct autoreactivities detectable in patients with rapidly progressive glomerulonephritis. AB - Circulating autoantibodies against the Goodpasture antigen (alpha 3 chain of type IV collagen) in the glomerular basement membrane (anti-GBM) and anti-neutrophil cytoplasm antibodies (ANCA) are each associated clinically with the development of a rapidly progressive glomerulonephritis. Antibodies with both these specificities coexist in a subset of patients, raising the possibility that they might be a result of cross-reactivity. In this study we have shown that 21% of patients with anti-GBM antibodies also had ANCA, and by using cross-inhibition assays, antigen-specific enzyme-linked immunosorbent assays, and Western blot analysis, these were shown to be two distinct populations of autoantibodies. In patients with both specificities, a greater proportion of the ANCA had specificity for myeloperoxidase (73.5%) than in patients with ANCA alone (36.6%). The presence of ANCA should be ascertained in all patients with anti-GBM disease as the prognosis for these double-positive patients may be dependent on both populations of antibodies. PMID- 7544067 TI - Elevated maternal serum alpha-fetoprotein levels: what is the risk of fetal aneuploidy? AB - OBJECTIVE: Our purpose was to present the findings of a project to determine the efficacy of including routine fetal karyotyping in the investigation of an elevated maternal serum alpha-fetoprotein concentration. STUDY DESIGN: Targeted ultrasonographic examinations were performed in 658 patients with elevated maternal serum alpha-fetoprotein levels. The scans were normal in 557 women, of whom 427 consented to amniocentesis; 435 fetuses were karyotyped. In the 101 patients with abnormal ultrasonographic examinations 75 had fetal karyotyping. RESULTS: In the 435 fetuses with normal scans, two had karyotypic anomalies, a 47,XYY and an inherited balanced translocation. Three fetuses with normal karyotypes and high amniotic fluid alpha-fetoprotein levels had congenital nephrosis. In the 101 patients with abnormal scans, 75 fetuses were karyotyped. There were four aneuploidies. Among the 26 patients with abnormal scans who declined amniocentesis one fetus with multiple anomalies was karyotyped after delivery and triploidy was discovered. CONCLUSIONS: These results provide little justification for including fetal karyotyping in the investigation of elevated maternal serum alpha-fetoprotein when the targeted ultrasonographic examination is normal. When it is abnormal, selective rather than routine karyotyping is more appropriate. PMID- 7544066 TI - Lead-induced nephropathy: relationship between various biological exposure indices and early markers of nephrotoxicity. AB - Lead nephropathy in adults is silent and insidious, characterized by the absence of proteinuria in its early phase. Of the early markers of nephrotoxicity, urinary N-acetyl-beta-D-glucosaminidase (NAG) appears to be the only one that is elevated in early lead nephropathy. However, the elevation in urinary NAG activity may be a response to a sharp increase in renal burden of lead. Its usefulness as a marker of chronic lead nephropathy is thus in doubt. There is a need, then, to identify a reliable early biological indicator of lead-induced kidney damage. Furthermore, there is also a need to identify suitable markers of chronic exposure to describe meaningful dose-response and dose-effect relationships. Traditionally, blood lead (PbB) was used, but the current blood lead level (PbBrec) is more an indicator of recent exposure. Time-integrated blood lead indices (PbBint) derived from repeated serial PbB measurements can be used as indices of chronic exposure. In 128 lead-exposed workers, the PbBint was the most important exposure variable in describing the variability in urinary alpha 1-microglobulin (U alpha 1 m), urinary beta 2-microglobulin (U beta 2m), and urinary retinol binding protein (URBP). U alpha 1m was the only marker that was significantly higher in the exposed group, with a good dose-response and dose effect relationship with PbBint. The lack of dose-response and dose-effect relationships in other studies may be due to inappropriate exposure markers as well as less sensitive response markers. PbBint has a better correlation than PbBrec. Furthermore, U alpha 1m may be the most sensitive of the markers because of its higher molecular weight. PMID- 7544069 TI - The interaction of nitric oxide and superoxide in the human fetal-placental vasculature. AB - OBJECTIVE: Our purpose was to study the interaction of nitric oxide and superoxide anion on the vasculature of the isolated perfused human placental cotyledon. STUDY DESIGN: Isolated placental cotyledons were preconstricted with the thromboxane mimetic U46619, and fetal perfusion pressure was used as an index of response. The interaction of nitric oxide and O2- was studied by three protocols: (1) with endogenous nitric oxide, (2) with addition of exogenous nitric oxide, and (3) with inhibition of nitric oxide synthesis by the nitric oxide synthase inhibitor N-nitro-L-arginine. O2- was generated by infusion of purine-xanthine oxidase. Statistical significance of response to treatment was determined by paired t test. RESULTS: Infusion of xanthine oxidase with purine in the presence of endogenous nitric oxide resulted in vasodilation (p < 0.05). Vasodilation was more pronounced in the presence of exogenous nitric oxide (p < 0.008). Coinfusion of xanthine oxidase with purine in the presence of N-nitro-L arginine to inhibit nitric oxide synthesis resulted in vasoconstriction. CONCLUSION: We conclude that, rather than superoxide inactivating nitric oxide, interaction of the two radicals generates in the placental vasculature a vasodilator, which may be peroxynitrite. PMID- 7544071 TI - An increase in expression of the lipocalin 24p3 is found in mouse uterus coincident with birth. AB - OBJECTIVE: To provide novel insights into the molecular events associated with parturition, a differential screen was made of a mouse uterine complementary deoxyribonucleic acid library to identify selectively expressed genes in late pregnancy. STUDY DESIGN: A differential hybridization was used to screen a mouse complementary deoxyribonucleic acid library prepared from late pregnancy uterus. A 1 kb clone was isolated that was subsequently identified as 24p3, a member of the lipocalin family. By use of radiolabeled complementary deoxyribonucleic acid probes prepared from this clone Northern hybridizations were conducted against total ribonucleic acid purified from mouse uterus collected during late pregnancy and the first week after birth and a variety of other mouse tissues to determine whether this gene is selectively expressed in the uterus coincident with parturition. RESULTS: Low levels of message for the 24p3 gene could be detected in uterine ribonucleic acid, but there was a massive increase in the level of message for this gene on the days surrounding birth. Northern hybridizations conducted against additional tissues collected from both pregnant and nonpregnant mice did not detect message to a similar degree as found in the uterus. CONCLUSIONS: The uterus constitutes a major site of expression of this gene, particularly near birth. The expression of this gene coincident with birth suggests a potential physiologic role of the neutrophil with the induction of labor. PMID- 7544068 TI - Prenatal diagnosis of diverse chromosome abnormalities in a population of patients identified by triple-marker testing as screen positive for Down syndrome. AB - OBJECTIVE: Our purpose was to determine the incidence of all types of chromosome abnormalities (i.e., trisomy 21 and other abnormalities) in women receiving prenatal chromosome analysis after a Down syndrome screen-positive result by maternal serum triple-marker testing (alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol analyses). STUDY DESIGN: A total of 11,434 patients between 15.0 and 21.9 weeks' gestation received second-trimester Down syndrome risk evaluation by triple-marker testing. By use of a 1:270 midtrimester Down syndrome risk cutoff value, and after ultrasonographic confirmation of gestational age, 677 patients were screen positive for Down syndrome (corrected screen-positive rate 5.92%). Karyotypes were reviewed for 468 (69%) of these patients who received prenatal chromosome analysis. RESULTS: In addition to 12 cases of Down syndrome, 12 other fetal chromosome abnormalities were found (i.e., 5.13% had a chromosome abnormality of some type). Expressed as a proportion of all patients with a corrected Down syndrome screen-positive result, at least 3.69% had a chromosome abnormality. The overall spectrum of abnormal karyotypes (approximately 50% autosomal trisomy, 25% structural and 25% sex chromosome abnormality) appears to be comparable to that seen in patients undergoing amniocentesis because of advanced maternal age. CONCLUSIONS: As is the case for women of advanced maternal age, preamniocentesis counseling for patients with positive triple-marker testing results should reflect the relatively high probability that an abnormality other than Down syndrome may be identified. PMID- 7544072 TI - Cytokeratin expression in the developing vagina of the postnatal gerbil (Meriones unguiculatus). AB - During postnatal development the vaginal epithelium of the Mongolian gerbil is transformed from two to three layers into a stratified, first mucified subsequently keratinized squamous epithelium. Changes in the expression of cytokeratins were studied and the immunohistochemical results compared with the ultrastructural findings at the corresponding stage. The first 10 postnatal days (days pn) were characterized by a moderate, positive immunoreaction for pancytokeratin in all vaginal cell layers. A faint reaction was caused by mAB CK 18.01 against CK 1, 5, 6 and 8. The appearance of mucous granules in the luminal cells after 15 pn seemed to coincide with an increase in cytokeratins. The immunoresponse for pancytokeratin in these cells was very intense compared with the reaction in the basal cell layers. Mucocytes during development and at proestrus were the only cells which reacted faintly positive with mAB against CK 18 alone. The keratinizing epithelium, which differentiates after day 40 pn, reacted strongly positive for pancytokeratin in the keratinizing layers, desquamating, fully keratinized cells, however, showed a negative reaction. The data indicate that mucocytes are not transformed squamous keratinized cells, but represent a cell category with its individual differentiation potential. Vimentin was not expressed. Neither the epithelium of the sinus vagina nor of the Mullerian vagina displayed any response. PMID- 7544076 TI - T-cell receptor use in multiple sclerosis. PMID- 7544074 TI - [Effects of high doses of aprotinin on intra- and postoperative blood loss in surgery with artificial circulation]. AB - A total of 100 patients subjected to multiple aortocoronary shunting under artificial circulation were examined. All of them were administered aprotinin or placebo in standard doses in a double blind test. The drug was infused over the entire course of the operation, total dose being 6 million KIU. The groups of patients did not differ by duration of artificial circulation and aorta clamping, or by the number of shunts used. The data permit a conclusion that prolonged intraoperative infusion of aprotinin in a dose of 6 million KIU resulted in a twofold reduction of the blood loss during and after the operation, thus permitting less frequent and lower in volume transfusions of donor blood during the operation and in the early postoperative period. PMID- 7544073 TI - Macromolecular permeability of chick wing microvessels: an intravital confocal study. AB - The development of the vertebrate limb requires the formation of a normal vasculature to nurture the soft and hard tissue phenotypes. The pattern of embryonic limb bud vessels has been extensively studied, but little is known about the permeability characteristics of the developing circulation. In the present study, the microvascular endothelial cell phenotype was examined by in vivo confocal microscopy following the systemic injection of a graded series of fluorescent dextrans (40,000, 70,000, 150,000 molecular weight) into chick embryos at stages 21-23 in order to determine how selective is the endothelial lining of microvessels as a partition between the blood vessels and the interstitium. Videodensitometry, over a gray scale range of 0-255, was used to quantitate the amount of tracer found within the interstitial compartment of the limb. The tracers of larger molecular weight (70,000, 150,000) were confined exclusively to the vascular lumina, whereas that of smaller molecular weight (40,000) was found to cause perivascular brightening due to extravasation into the surrounding interstitium. The reported differences in permeability were not dependent upon the stage of the embryo used in this study, but were due to the size of the tracer. These data indicate that embryonic wing microvessels demonstrate permselectivity to macromolecular efflux across the endothelium. The present results provide a basis for additional studies concerned with the dynamic characteristics of the limb microvasculature and challenge our concepts about the role of diffusible morphogens in vertebrate limb development. PMID- 7544075 TI - A review of T-cell receptors in multiple sclerosis: clonal expansion and persistence of human T-cells specific for an immunodominant myelin basic protein peptide. AB - Understanding the immune response to myelin antigens in regard to the peptide/MHC/TCR complex is important in defining pathogenesis of demyelinating autoimmune diseases and in developing antigen-specific therapies. We previously reported that individual multiple sclerosis patients may use certain dominant TCR V beta chains to recognize immunodominant MBP peptides. In examining the TCR beta chain usage, we observed repeated TCR VDJ sequences among different T-cell lines isolated from the same patient. This suggested that a few expanded T-cell clones may dominate the immune response to immunodominant MBP peptides. Here, we report experiments where TCR rearrangements were used as a probe for the clonal origin of MBP specific T-cells cultured from blood lymphocytes of MS patients and normal subjects. In two patients with the DR2 haplotype that were analyzed in detail, the T-cell response to MBP was focused on the MBP (84-102) peptide and in vivo expanded population(s) dominated the response to the MBP (84-102) peptide. Two MBP (84-102) specific T-cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha and beta chain sequences as well as identical sequences of a TCR gamma chain or of a second TCR alpha chain rearrangement. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time. PMID- 7544070 TI - Epithelial cells in peritoneal fluid--of endometrial origin? AB - OBJECTIVE: Our purpose was to examine the immunohistochemical properties of epithelial cells in peritoneal fluid and to compare the staining characteristics with cells of endometrium, menstrual effluent, peritoneum, and endometriotic lesions. STUDY DESIGN: Samples of menstrual effluent, endometrium, and peritoneal fluid and biopsy specimens of endometriotic lesions and peritoneum from 16 patients were examined. Monoclonal antibodies against vimentin, cytokeratin 18 and 19, and the monoclonal antibody BW495/36, staining an epithelial marker present in endometrium and absent in peritoneal epithelium, were used. RESULTS: All but one sample of menstrual effluent and peritoneal fluid cells stained positively with antibodies against vimentin and cytokeratin 18 and 19. BW495/36 stained 14 of 16 menstrual effluent samples and nine of 16 peritoneal fluid cell samples. Endometriotic specimens showed staining with all markers. No major differences in staining properties were observed in menstrual effluent, endometrium, and peritoneal fluid cells between patients with or without endometriosis. CONCLUSION: These results support the contention of transport of menstrual detritus to the peritoneal cavity in women with patent fallopian tubes. PMID- 7544078 TI - The relationship between human multiple sclerosis and rodent experimental allergic encephalomyelitis. PMID- 7544077 TI - Unique T-cell receptor junctional sequences found in multiple sclerosis and T cells mediating experimental allergic encephalomyelitis. AB - We have used two approaches to isolate TCR sequences that are unique to patients with multiple sclerosis. One strategy was to sequence TCR gene rearrangements directly from MS lesions. The second strategy utilized T-cell clones with a selectable mutation that are found only in MS patients. The selection of T-cell clones with mutations in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene was used to isolate T-cells reactive to myelin basic protein (MBP) in patients with multiple sclerosis (MS). These T-cell clones are activated in vivo, and are not found in healthy individuals. The third complementarity determining regions (CDR3) of the T-cell receptor (TCR) alpha and beta chains are the putative contact sites for peptide fragments of MBP bound in the groove of the HLA molecule. The TCR V gene usage and CDR3s of these MBP-reactive hprt- T-cell clones are homologous to TCRs from other T-cells relevant to MS, including T cells causing experimental allergic encephalomyelitis (EAE) and T-cells found in brain lesions and in the cerebrospinal fluid (CSF) of MS patients. In vivo activated MBP-reactive T-cells in MS patients may be critical in the pathogenesis of MS. PMID- 7544080 TI - T-cell receptor alpha and beta CDR3 expression by myelin basic protein-specific human T-cell clones. PMID- 7544082 TI - Development of a transfection system for myelin basic protein-specific human alpha beta T-cell receptors. PMID- 7544079 TI - Limited restriction in alpha beta T-cell receptor usage of T-cell clones specific for myelin basic protein (a.a. 84-102) and hsp65 (a.a. 3-13) peptides within major histocompatibility complex-identical individuals. PMID- 7544081 TI - Myelin basic protein-reactive human T-cell clones: stimulation by diverse microbial antigens. PMID- 7544083 TI - Clonal depletion of human myelin basic protein-reactive T-cells by T-cell vaccination. PMID- 7544084 TI - T-cell receptor V beta spectrotypes of central nervous system T-cells during acute relapsing experimental autoimmune disease. PMID- 7544085 TI - Dominant usage of T-cell receptor alpha gene segments from one parent in experimental autoimmune encephalomyelitis induced in F1 mice. PMID- 7544086 TI - [Embolization by direct puncture of hypervascularized ORL tumors]. AB - Direct intratumoral embolization was used to treat five patients with vascularized tumors of the head and neck: 2 nasopharyngeal angiofibroma, 2 glomus jugulare tumors and 1 hemangiopericytoma. Embolization was performed by direct puncture and intratumoral injection of a plastic mixture under angiographic control. In 4 patients, embolization was performed preoperatively. Devascularization was induced in at least 90% of the volume of these tumors, thus facilitating subsequent surgical removal of the tumor. In 1 patient, embolization was performed with palliative intent. Good regression of symptoms was obtained. PMID- 7544090 TI - Inhibition of angiogenesis and metastases of the Lewis-lung cell carcinoma by the quinoline-3-carboxamide, Linomide. AB - Linomide has antitumor effects when administered in vivo but not in vitro. Recent data indicate that at least part of this effect can be attributed to anti angiogenic properties. The aim of the present investigation was to study the anti angiogenic effects of Linomide on early tumor-induced angiogenesis in vivo, using a newly developed skinfold chamber technique in the mouse, and to relate this to the effect of Linomide on the number of metastases that develop from a s.c. implanted tumor. Tumor spheroids of Lewis lung cell carcinoma (LLC) with a diameter of about 800 microns were implanted in dorsal skinfold chambers inserted on CB6/Fl mice. Tumor cells were pre-labelled with a fluorescent vital dye (CMTMR), which allowed the estimation of the growth of the implanted tumor spheroids. Linomide, given orally from day 7 to day 11, reduced the incidence of lung metastasis arising from LLC tumors at day 21 in a dose-dependent manner, a 55% reduction being found at a dose of 50 mg/kg/day (N = l9 for the controls and N = 10 for treatment groups). In the dorsal skinfold chamber, the vascular network in Linomide treated animals (100 mg/kg/day, N = 22) was more heterogenous, large areas within the tumor being completely avascular; in addition, capillary density at the tumor site was reduced by 34% 6 days after implantation and by 39% after 14 days. In the control group (N = 16), tumor volume doubling time was not significantly different in the early avascular part of the observation period as compared to the later vascular phase; this indicates that the growth of these micro-tumors in the early avascular phase is not angiogenesis dependent. However, in the Linomide treated animals, tumor volume doubling time was significantly prolonged by 42% during the later part of the observation period. Taken together, the data indicate that the prolongation of the tumor doubling time is due to the anti-angiogenic activity of Linomide. PMID- 7544089 TI - Expression of a reverse transcriptase activity in a cell line established from peritoneal macrophages of mice treated with N-methyl-N-nitrosourea. AB - We have established a cell line from peritoneal macrophages of mice treated intraperitoneally with N-methyl-N-nitrosourea. The present communication describes the identification of an RNA-dependent DNA-polymerase activity in a particulate fraction from supernatants of the cell culture. This activity is similar to retroviral Reverse Transcriptase (RT) based on its template specificity and ionic preference. The proof of the retrovirus-like nature of RT was obtained by ultracentrifugation of the pelleted proteins secreted in the medium on a sucrose gradient. The main RT activity was obtained in fractions of 1.14-1.16 g/ml densities, which are comparable to those of type C retroviruses. The presented data support constitutive expression of the retrovirus gene in a chemically transformed cell line. PMID- 7544087 TI - High-dose methotrexate, vincristine and cisplatin as salvage treatment for relapsed non-seminomatous germ-cell cancer. AB - Eight patients with non-seminomatous testicular cancer relapsing after primary chemotherapy were treated with salvage chemotherapy consisting of high-dose methotrexate (12 g/m2), vincristine (1.2 mg/m2) weekly for four weeks, followed after an interval of four weeks by 3 times 100 mg/m2 cisplatin (50 mg/m2 on day 1 and 2) every 10 days. This regimen resulted in 2 partial (PR) and 2 complete responses (CR). The two patients achieving CR remain disease-free for 43+ and 53+ months. Toxicity was mainly methotrexate-related and could be ameliorated to a large extent by leucovorin rescue. This small study shows that methotrexate, vincristine, followed by cisplatin is effective in the treatment of relapsed non seminomatous testicular cancer at the cost of manageable toxicity. PMID- 7544091 TI - Production of acidic and basic fibroblast growth factor by the hormone independent breast cancer cell line MDA-MB-231. AB - Normal, as well as the majority of malignant, mammary epithelial cells will proliferate upon stimulation by FGFs. However estrogen-independent MDA-MB-231 cells (which are able to grow in vitro in serum-free medium) are not significantly stimulated by exogenous FGFs even though they possess high and low affinity receptors for these growth factors. Biological assays, measuring CCL39 fibroblast proliferation or PC12 pheochromocytoma cell neurite outgrowth, demonstrated the presence of FGF activity in MDA-MB-231 cell extracts and also in the culture medium conditioned by these cells. This biological activity decreased in the presence of neutralizing anti-FGF antibodies. Using immunohistochemical methods FGF1 and FGF2 immunoreactivity was detected in MDA-MB-231 cells. After purification by heparin-Sepharose chromatography and Western blot analysis, M(r) 18000 molecules showed the same physicochemical characteristics as FGFI and FGF2. These results demonstrate the production and release of FGF related molecules by MDA-MB-231 cells and suggest an autocrine stimulation of these cells. PMID- 7544088 TI - Immunohistochemical analysis of cytokeratin #8 as a prognostic factor in invasive breast carcinoma. AB - Cytokeratin #8 (CK8) was analysed by immunohistochemistry using a monoclonal antibody in 101 operable invasive breast carcinomas (IBCs). The immunohistochemical staining pattern, and its correlations with patient characteristics and prognosis were studied. Sixty-five (65%) out of 101 patients had CK8 positive tumors. The incidence of CK8 positivity in patients with estrogen receptor (ER) positive tumors was significantly higher than that in patients with ER negative tumors. The same tendency was seen between the CK8 positivity and progesterone receptor (PR) status; however, no statistical significance was found. Furthermore, the incidence of CK8 positivity in premenopausal patients was lower than that in postmenopausal patients with no statistical significance. Relapse-free survival (RFS) for patients with CK8 positive tumors was significantly better than that for patients with CK8 negative tumors. The multivariate analysis of factors related to RFS showed that CK8 staining positivity was an independent prognostic indicator of IBCs. These results suggest that CK8 expression may be one of the prognostic factors of IBCs. PMID- 7544092 TI - Nitric oxide, peroxynitrite and nitroso-compounds formation by ultraviolet A (UVA) irradiated human squamous cell carcinoma: potential role of nitric oxide in cancer prognosis. AB - Ultraviolet A (UVA) irradiated human squamous cell carcinoma (SCC-13) releases nitrogen oxides, i.e. nitric oxide (NO), peroxynitrite (ONOO-), nitrosocompounds, ammonia (NH3) and hydroxylamine (H2NOH) formed from L-arginine. Formation and/or release of these nitrogen oxides was time and concentration-dependently stimulated by UVA and decreased by N-monomethyl-L-arginine (L-NMMA), a compound that inhibits NO synthase activity. UVA irradiation of SCC-13 cells resulted in concomitant increase in soluble guanylate cyclase (sGC) which was inhibited by L NMMA. The increased NO and ONOO- production evoked by dibutyryl cGMP and 3 isobutyl-l-methyl-xanthine (IBMX) represents an additional positive feedback mechanism that could serve to maintain NO and ONOO- release for extended periods following UVA radiation. Using an in vitro chemical model system, it was demonstrated that oxidation of NH3 to NO by hydroxyl radical (.OH) at physiological pH is chemically feasible. UVA irradiated SCC-13 cells induced a luminol-enhanced chemiluminescence signal that reaches a peak within 1 min. The modulation of this signal by ebselen is consistent with a rate-determining step corresponding to the disproportionation of a luminol-superoxide (O2-) complex. UVA irradiated SCC-13 cells promptly increased malondialdehyde (MDA) production with subsequent decrease of plasma membrane fluidity. Desferrioxamine tested in UVA irradiated SCC-13 cells showed a concentration dependent decrease in MDA production with subsequent restoration of the membrane fluidity to the normal level. Furthermore, it was shown that squamous cell carcinoma possesses higher NO synthase and sGC activity as compared to normal keratinocytes. Such an increase in NO production may be directly related to the poor prognosis of squamous cell carcinoma. PMID- 7544097 TI - The nuclear estrogen receptor R-II of the goat uterus: distinct possibility that the R-II is the deglycosylated form of the nonactivated estrogen receptor (naER). AB - Structural and functional characteristics of the goat uterine nuclear estrogen receptor R-II have been subjected to comparison with those of the nonactivated estrogen receptor (naER), purified from the cytosol. The two proteins have the same molecular mass, 66 kDa; they display identical peptide maps and are both recognized by anti-estrogen receptor (R-I) IgG. Both are tyrosine kinases and bind with equal affinity to a column of anti-phosphotyrosine IgG-Sepharose. On the other hand, while naER is a glycoprotein, the R-II does not show any sign of glycosylation. Unlike the naER, the R-II is incapable of dimerization with estrogen receptor activation factor (E-RAF) and, as a consequence, bind to the DNA. R-II has a higher estradiol binding capacity and the resultant reduction in its affinity for the hormone in comparison with the naER. Further, the sedimentation behavior and the Stokes radius of the naER indicate a globular nature in the shape of the protein. The corresponding data for the R-II reveal that the protein has a distinct nonglobular shape. Deglycosylation of the naER using a glycopeptidase resulted in the total conversion of the distinct physical features of the naER to the R-II category. This treatment resulted, without effecting any reduction in its molecular mass, in the loss of the E-RAF dimerization capacity of the naER. The Stokes radius and the sedimentation coefficient of the protein underwent drastic changes and became closely similar to those of the R-II. In addition, the deglycosylation introduced a several-fold enhancement in the capacity of the naER to bind estradiol with a concomitant decrease in its affinity, similar to the corresponding properties of the R-II. The R-II is shown to have a conformational structure different from that of the naER, to interact with the nuclear RNA polymerase II. It is also shown here that the R-II phosphorylates two subunits (molecular mass 91 and 20 kDa) in the RNA polymerase II, in addition to the 40-kDa subunit phosphorylated by the naER. The results clearly indicate the possibility that the nuclear R-II estrogen receptor is the deglycosylated naER. PMID- 7544098 TI - Peroxisomal membrane protein PMP68 of mouse liver: cloning of a cDNA encompassing the nucleotide binding fold and epitope mapping of monoclonal antibodies to the expressed protein. AB - We have isolated and sequenced a cDNA which encodes 376 amino acids toward the carboxy-terminus, and encompassing the putative nucleotide binding fold, of PMP68 (mouse liver peroxisomal integral membrane protein of 68 kDa) the major integral membrane protein of mouse liver peroxisomes. The protein sequence predicted from this cDNA shows 97.9% amino acid identity to this same region of rat liver PMP70, a member of the ATP-binding cassette protein superfamily (K. Kamijo, S. Taketani, S. Yokota, T. Osumi, and T. Hashimoto, 1990, J. Biol. Chem. 265, 4534-4540). The section of the cDNA encoding the hydrophilic and putative cytoplasmic domain of PMP68 was expressed as a recombinant fusion protein in bacteria. Two monoclonal antibodies raised against this protein have been epitope-mapped to peptides generated by cyanogen bromide cleavage of the fusion protein. Antibody 1A4 recognizes a peptide whose sequence contains the first motif of the putative nucleotide binding fold of PMP68, and antibody 8F11 recognizes a carboxy-terminal peptide which includes the second motif of this nucleotide binding fold. These antibodies are expected to be useful in the elucidation of the biological function of this putative membrane transporter. PMID- 7544094 TI - Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. AB - The bacterial community structures of phosphate- and non-phosphate-removing activated sludges were compared. Sludge samples were obtained from two sequencing batch reactors (SBRs), and 16S rDNA clone libraries of the bacterial sludge populations were established. Community structures were determined by phylogenetic analyses of 97 and 92 partial clone sequences from SBR1 (phosphate removing sludge) and SBR2 (non-phosphate-removing sludge), respectively. For both sludges, the predominant bacterial group with which clones were affiliated was the beta subclass of the proteobacteria. Other major groups represented were the alpha proteobacterial subclass, planctomycete group, and Flexibacter-Cytophaga Bacteroides group. In addition, several clone groups unaffiliated with known bacterial assemblages were identified in the clone libraries. Acinetobacter spp., thought to be important in phosphate removal in activated sludge, were poorly represented by clone sequences in both libraries. Differences in community structure were observed between the phosphate- and non-phosphate-removing sludges; in particular, the Rhodocyclus group within the beta subclass was represented to a greater extent in the phosphate-removing community. Such differences may account for the differing phosphate-removing capabilities of the two activated sludge communities. PMID- 7544093 TI - Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana. AB - Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria. PMID- 7544096 TI - Free radicals induce reversible membrane-cytoplasm translocation of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes. AB - We investigated the role of oxygen free radicals in the modulation of glyceraldehyde-3-phosphate dehydrogenase binding to the erythrocyte membrane. Previous studies have demonstrated that in vitro tyrosine phosphorylation of Band 3 prevents the binding of various glycolytic enzymes to its cytoplasmic domain. Since these enzymes are inhibited in their bound state, the functional consequence of Band 3 tyrosine phosphorylation in red blood cells should be to increase glycolysis. To generate free radicals, we used an azo-compound, the hydrophilic 2,2'-azobis(2-amidinopropane) hydrochloride, which, at 37 degrees C and in the presence of oxygen, decomposes and produces peroxyl radicals at a constant rate. The reaction of peroxyl radicals with intact red cells induced a time-dependent loss of the membrane-bound glycolytic enzyme, glyceraldehyde-3 phosphate dehydrogenase, associated with a concomitant decrease in enzyme activity. At the same time, Band 3 was phosphorylated in tyrosine. These results were completely reversible in plasma after removal of the oxidative stress. The peroxyl radicals also enhanced the production of lactate in intact cells. Our data reveal a powerful mechanism of erythrocyte metabolic regulation that can boost or reduce energy production in times of special need such as during a free radical attack. PMID- 7544095 TI - Aromatic-degrading Sphingomonas isolates from the deep subsurface. AB - An obligately aerobic chemoheterotrophic bacterium (strain F199) previously isolated from Southeast Coastal Plain subsurface sediments and shown to degrade toluene, naphthalene, and other aromatic compounds (J. K. Fredrickson, F. J. Brockman, D. J. Workman, S. W. Li, and T. O. Stevens, Appl. Environ. Microbiol. 57:796-803, 1991) was characterized by analysis of its 16S rRNA nucleotide base sequence and cellular lipid composition. Strain F199 contained 2-OH14:0 and 18:1 omega 7c as the predominant cellular fatty acids and sphingolipids that are characteristic of the genus Sphingomonas. Phylogenetic analysis of its 16S rRNA sequence indicated that F199 was most closely related to Sphingomonas capsulata among the bacteria currently in the Ribosomal Database. Five additional isolates from deep Southeast Coastal Plain sediments were determined by 16S rRNA sequence analysis to be closely related to F199. These strains also contained characteristic sphingolipids. Four of these five strains could also grow on a broad range of aromatic compounds and could mineralize [14C]toluene and [14C]naphthalene. S. capsulata (ATCC 14666), Sphingomonas paucimobilis (ATCC 29837), and one of the subsurface isolates were unable to grow on any of the aromatic compounds or mineralize toluene or naphthalene. These results indicate that bacteria within the genus Sphingomonas are present in Southeast Coastal Plain subsurface sediments and that the capacity for degrading a broad range of substituted aromatic compounds appears to be common among Sphingomonas species from this environment. PMID- 7544099 TI - Buthionine sulfoximine pretreatment potentiates the effect of isolated lung perfusion with doxorubicin. AB - BACKGROUND: Although surgical resection remains the mainstay of treatment for metastatic pulmonary sarcoma, 5-year survival approaches only 25%. Chemotherapy has been limited by tumor resistance and systemic toxicity. We assessed the efficacy of L-buthionine-SR-sulfoximine, an inhibitor of glutathione synthesis, as a sensitizer for isolated lung perfusion. METHODS: In experiment 1, sarcoma bearing rats (n = 20) received either buthionine sulfoximine via intraperitoneal injection or Hespan. After the last injection, tumor glutathione levels were measured. In experiment 2, rats (n = 60) were injected with sarcoma intravenously. On day 6, animals were pretreated with either buthionine sulfoximine or Hespan intraperitoneally. On day 7, rats underwent isolated lung perfusion (Hespan or doxorubicin) or intravenous therapy (Hespan or doxorubicin). On day 14, tumor nodules were counted. RESULTS: Buthionine sulfoximine effectively depleted tumor glutathione. Animals treated with intravenous therapy had no response to therapy, whereas those animals treated with doxorubicin isolated lung perfusion alone had a limited response. Buthionine-sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion led to a 13 fold reduction in tumor nodules and 5 complete responses. CONCLUSIONS: Buthionine sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion is superior to intravenous doxorubicin and doxorubicin isolated lung perfusion alone for the treatment of metastatic pulmonary sarcoma. PMID- 7544100 TI - Does aprotinin increase the myocardial damage in the setting of ischemia and preconditioning? AB - BACKGROUND: Aprotinin reduces postoperative bleeding in cardiac operations, but its association with perioperative myocardial infarction remains controversial. Ischemic preconditioning is a novel method of myocardial protection. METHODS: To answer whether aprotinin increases postischemic myocardial damage and also to characterize the effect of aprotinin on ischemic preconditioning, four groups of sheep were fully heparinized to keep activated clotting time readings greater than 750 seconds and subjected to 60 minutes of normothermic regional ischemia (diagonal artery occlusion) with 3 hours of reperfusion. Group I was the control with no treatment, group II received aprotinin (1 million KIU load followed by 250,000 KIU/h), group III underwent ischemic preconditioning (three 5-minute intervals of ischemia and reperfusion) before prolonged 1-hour ischemia, and group IV underwent similar ischemic preconditioning and received aprotinin. Area at risk was delineated by monastryl blue pigment, and infarction size by tetrazolium staining. RESULTS: The ratios of weight of area at risk to left ventricular weight and left ventricular weight to body weight were constant between groups. Infarction size to area at risk ratio data demonstrated that aprotinin increases infarction size by 60% (infarction size to area at risk ratio from 52% +/- 10% to 84% +/- 10% for I versus II; p < 0.001). Aprotinin also attenuates the protective effect of ischemic preconditioning (infarction size to area at risk ratio from 25% +/- 4% to 41% +/- 6%; p < 0.001). CONCLUSIONS: In the setting of ischemia, aprotinin increases myocardial damage. If, however, the heart is provided with protective preconditioning, then the deleterious effect of aprotinin may be neutralized. From these data we suggest that aprotinin should not be used routinely in cardiac operations unless extensive blood loss is anticipated, such as in redo open heart operations. PMID- 7544101 TI - Myocardial mitochondrial calcium accumulation modulates nuclear calcium accumulation and DNA fragmentation. AB - BACKGROUND: Previously, we have shown that normothermic global ischemia increases cytosolic calcium accumulation in both the mature and aged heart. Increased nuclear and mitochondrial calcium accumulation was shown to occur in the aged but not the mature heart, and these age-related differences were associated with increased DNA fragmentation and decreased cellular viability only in the aged heart. METHODS: To investigate the relationship between increased mitochondrial and nuclear calcium and DNA fragmentation, mature and aged rabbit hearts were subjected to normothermic global ischemia with and without the addition of ruthenium red to block mitochondrial calcium influx. Cytosolic calcium accumulation was measured in a parallel experiment using fura-2. RESULTS: Ruthenium red ameliorated mitochondrial calcium accumulation and was associated with both decreased DNA fragmentation and decreased nuclear calcium accumulation. CONCLUSIONS: Nuclear calcium accumulation was correlated with increased mitochondrial calcium accumulation but not increased cytosolic calcium accumulation in the aged heart. Modulation of mitochondrion "futile calcium cycling" may be of significance in the modulation of ischemic myocardial injury. PMID- 7544102 TI - Neonatal pulmonary vascular disease in hypoplastic left heart syndrome. AB - A term neonate with hypoplastic left heart syndrome underwent surgical palliation using the Norwood procedure on day 2 of life. The postoperative course was complicated by recurrent systemic desaturation leading to death. Postmortem histologic examination of lung tissue confirmed the clinical impression of fixed pulmonary vascular disease. Some neonates with a restrictive interatrial communication may have pulmonary vascular disease at birth, which will negatively influence survival irrespective of the type of palliation undertaken. PMID- 7544103 TI - The gvpA/C cluster of Anabaena flos-aquae has multiple copies of a gene encoding GvpA. AB - Southern analysis of genomic DNA from Anabaena flos-aquae revealed that the genes encoding the two authenticated protein components of cyanobacterial gas vesicles, GvpA and GvpC, were carried on the same 4.9-kb HindIII restriction fragment. By comparing the hybridization intensities observed when either gvpA- or gvpC specific oligonucleotides are bound to this HindIII fragment, we calculated that the A. flos-aquae genome contains seven copies of gvpA and a single copy of gvpC. The nucleotide sequence of the longest cloned section of the gvpA/C cluster of A. flos-aquae DNA revealed the presence of four complete copies of gvpA and part of a fifth copy located upstream from a single copy of gvpC; no clones carrying the entire gvpA/C-bearing HindIII fragment were identified. The distribution of Sau3A restriction sites throughout the gvpA/C-bearing genomic HindIII fragment resembled that seen in the cloned portion of the gvpA/C cluster and is consistent with that expected for a cluster containing seven copies of gvpA and one copy of gvpC. The length of transcripts that hybridize to both gvpA and gvpC on Northern blots was consistent with a 7gvpA + 1gvpC transcriptional unit. PMID- 7544104 TI - Extramedullary hematopoiesis during therapy with granulocyte colony-stimulating factor. PMID- 7544105 TI - Artifactual hypoglycemia associated with hematopoietic cytokines. AB - We observed apparent hypoglycemia in seven patients manifesting granulocytosis associated with hematopoietic cytokine treatment or with a leukemoid reaction. All seven patients had confounding preanalytic conditions of specimen transport delay and lack of antiglycolytic agents. Cytokine-stimulated leukocytes may cause artifactual hypoglycemia by consuming glucose in vitro, possibly leading to unnecessary diagnostic evaluation. The glucose depletion was faster in blood drawn from patients receiving granulocyte colony-stimulating factor (0.29 mmol/L/h) than in blood from a control group (0.17 mmol/L/h) or from a group with leukemia (0.23 mmol/L/h). Stabilization with sodium fluoride (60 mmol/L) slowed the glucose depletion in both the cytokine group (0.13 mmol/L/h) and the leukemic group (0.09 mmol/L/h), which were then statistically indistinguishable from the control rate (0.10 mmol/L/h). In blood obtained from patients being treated with hematopoietic cytokines or who have leukemoid reactions, an antiglycolytic agent should be used whenever separation of plasma might be delayed more than 1 hour. PMID- 7544107 TI - 'Minimal deviation' endometrioid carcinoma with oncocytic change of the endometrium. AB - We report an unusual adenocarcinoma of the endometrium in an 80-year-old woman. The tumor diffusely involved the entire thickness of the myometrium. The cervix, ovaries, and fallopian tubes were unremarkable. Microscopic examination revealed an extremely well-differentiated endometrioid adenocarcinoma with mild cytologic atypia and slightly distorted glands infiltrating almost the entire thickness of the myometrium. In addition, the neoplastic endometrioid cells showed extensive oncocytic change. Endometrial curettings had been interpreted as simple endometrial hyperplasia with eosinophilic metaplasia. The significance of this lesion is discussed in terms of possible confusion with benign endometrial lesions as well as benign cervical lesions. PMID- 7544106 TI - Elastin fibers resembling Sporothrix schenkii in the skin of a patient with acquired immunodeficiency syndrome. AB - We describe a patient with the acquired immunodeficiency syndrome who presented with an erythematous skin rash on his trunk and extremities. Initial histologic examination of a skin biopsy revealed silver-staining elements resembling Sporothrix schenkii. Additional histochemical and ultrastructural studies revealed that these elements were elastin fibers rather than fungi. The literature describing pseudofungal infections is reviewed. PMID- 7544109 TI - The replication of a necrogenic cucumber mosaic virus satellite is temperature sensitive in tomato. AB - Lethal necrosis development in tomato plants infected with cucumber mosaic virus (CMV) strain D containing the necrogenic satellite D-CARNA 5 and held at 32 degrees C is shown to be impaired. CARNA 5 accumulation in tomato at 32 degrees C is reduced about 100-fold compared to accumulation in plants held at 24 degrees C, while viral RNA accumulation is reduced about 5-fold. CMV-infected tomato held for 3 days at 24 degrees C prior to shift to 32 degrees C do not develop lethal necrosis. Longer incubations at 24 degrees C prior to shift to 32 degrees C allow necrosis to develop. CMV-infected plants held for up to 4 weeks at 32 degrees C required an additional 8-10 days at 24 degrees C to develop necrosis. Necrogenic CMV-infected plants held at 24 degrees C and analyzed 3 days p.i. contained detectable amounts of ss- and ds-CARNA 5; upon shift to 32 degrees C, such CARNA 5 declined to undetectable levels and lethal necrosis did not occur. There appear to be temperature-sensitive factors that are required for efficient satellite replication which are not required for efficient viral RNA replication. Whether these factor(s) are of host or satellite origin is uncertain. PMID- 7544110 TI - Effect of temperature on cucumber mosaic virus satellite-induced lethal tomato necrosis is helper virus strain dependent. AB - The effect of temperature on the response of tomato (Lycopersicon esculentum Mill. cv. Rutgers) to infections with the necrogenic cucumber mosaic virus (CMV) satellite D-CARNA 5 was investigated with each of four CMV strains D, 1, Y and S functioning as helper virus. At 24 degrees C lethal necrosis was observed in all infections. However, at 32 degrees C the response varied from total absence or reduction of necrosis with some strains to accelerated lethal necrosis with others. The total lack of necrotic response with CMV-S and the aggravated necrosis with CMV-Y at the higher temperature both turned out to be independent of the coinfecting satellite, and rather to correlate with the changing rate of viral RNA accumulation in tomato, which probably was responsible for the changes in pathogenic response. However, when CMV-D was helper virus, satellite accumulation decreased, while with CMV-1 it increased, respectively, while viral RNA accumulations were not seriously affected. Although these profound effects of temperature seem to link the necrotic response of tomato to the competitive replication dynamics of the infecting virus/satellite combination in the case of CMV-D/D-CARNA 5, temperature effects at other levels of disease induction probably play an important role as well. PMID- 7544108 TI - Ovine aortic smooth muscle cells allow the replication of visna-maedi virus in vitro. AB - Visna-maedi virus induces in sheep an interstitial lung disease characterised by an accumulation of smooth muscle cells (SMC) or myomatosis. Infection by HIV-1 has been recently associated with disorders of the vessel-derived cells: primary pulmonary hypertension, coronary artery disease and smooth muscle tumors in humans. We hypothesized that, besides their regular targets (i.e. macrophages and lymphocytes), lentiviruses could infect smooth muscle cells. Smooth muscle cell cultures derived from ovine aorta were infected with visna-maedi virus strain K1514. The cultured cells were smooth muscle cells as demonstrated by their antigenic expression of alpha-actin and vimentin. The lentiviral infection of the smooth muscle cells was demonstrated by a typical cytopathic effect (syncytia), the expression of virus specific antigens, and the presence of genomic RNA detected by Northern blot analysis and RT PCR. The detection of a reverse transcriptase activity, the presence of viral RNA in supernatants of infected smooth muscle cells detected by RT PCR and their ability to infect ovine permissive fibroblasts demonstrated a productive infection. The ability of smooth muscle cells to be infected by lentiviruses may participate in the pathogenesis of the tissue damage associated with the lentiviruses such as myomatosis in sheep and vascular disease in humans. PMID- 7544111 TI - Biological and molecular studies on Syrian hamster intracisternal R-type particles. AB - Retrovirus-like intracisternal R-type particles (IRP) are structures present in Syrian hamster (Mesocricetus auratus) cells cultured in vitro where they appear either spontaneously or under chemical induction conditions. We have tested several chemical inducers and ten different cell lines, looking for the best IRP induction conditions. BHK21 cl. 13 showed the highest inducibility one day after a 24 h treatment with 1 microgram/ml of 5-aza-2'-deoxycytidine. Using detergent treatments and sucrose gradients, we obtained semi-purified IRP cores. A 7.2 kb RNA associated with the core fraction was revealed by hybridization with total Syrian hamster genomic DNA, but not with Syrian hamster intracisternal A particle (IAP) specific probes. This suggests that the IRP genes are distinct from IAP ones. PMID- 7544112 TI - Inhibitory effect of tyrphostin on the replication of herpes simplex virus type 1. AB - Tyrphostins 9 and 47, inhibitors of protein-tyrosine kinase, inhibited the replication of herpes simplex virus type 1 (HSV-1), whereas tyrophostin 1, which does not inhibit protein-tyrosine kinase, did not affect the replication of HSV 1. The inhibitory effect of tyrphostin 9 was more potent than that of tyrphostin 47, and the IC50 of tyrphostin 9 was 40 nM. Sodium orthovanadate, an inhibitor of protein phosphotyrosine phosphatase, increased HSV-1 plaque formation and its effect was partly reversed by tyrphostin 9. The phosphorylation of viral phosphoproteins was decreased by tyrphostin 9 in a dose-dependent manner, but the tyrphostin 9-induced reduction of protein synthesis was not dose-dependent. At the late stage of infection, tyrosine phosphorylation was demonstrated in HSV-1 phosphoproteins. These results indicate that protein-tyrosine kinase is involved in the replication of HSV-1 and that tyrphostin can inhibit the synthesis and post-translational phosphorylation of the viral proteins. PMID- 7544114 TI - Characterisation of Pasteurella multocida isolated from fowl cholera outbreaks on turkey farms. AB - Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate Pasteurella multocida isolates from outbreaks of fowl cholera on 7 turkey farms in New South Wales. While only a single isolate was available from 5 of the farms, multiple isolates, 4 and 12 respectively, were available from the other 2 farms. The available field evidence suggested that 8 outbreaks had occurred with one farm suffering 2 outbreaks. The isolates obtained were all confirmed as Pasteurella multocida. Biochemical profiles allocated the isolates to 4 groups, 3 being variants of P multocida subsp multocida and the fourth being P multocida subsp septica. REA performed with HpaII established 7 groups. Ribotyping using the HpaII digests probed with the 16S rRNA operon of Haemophilus paragallinarum recognised the same 7 groups as REA. Unlike the biochemical profiles, both REA and ribotyping provided a fine subdivision that identified outbreaks as either related or unrelated. The REA and ribotyping patterns as well as biochemical profiles were stable for all isolates from the outbreaks in which multiple isolates were obtained from either the same bird or from different birds. REA and ribotyping were found to be superior to biotyping methods for the investigation of fowl cholera outbreaks. PMID- 7544113 TI - Detection of group A rotavirus by reverse transcriptase and polymerase chain reaction in feces from children with acute gastroenteritis. AB - RT-PCR was employed to detect rotavirus infection in 450 fecal samples from children with acute diarrhoea. It was compared with enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE) for rotavirus detection. A total of 67 samples were found positive by at least one of the three techniques. Of these 67 samples, 51 were positive by all three methods, 6 were positive by ELISA and RT-PCR but negative by PAGE, 3 were positive by PAGE and RT PCR and negative by ELISA, 3 and 4 samples were exclusively positive by RT-PCR and ELISA respectively. These results indicate that RT-PCR is a sensitive and specific assay for detection of group A rotaviruses in stool samples from cases of acute diarrhoea. PMID- 7544116 TI - Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets. AB - This study explores the role of cyclic nucleotides (i.e. cyclic AMP and cyclic GMP) in store-regulated external Ca2+ entry in human platelets. To stimulate store-regulated external Ca2+ entry, thapsigargin was used to deplete Ca2+ from the dense tubules, and sodium nitroprusside and iloprost respectively were used to stimulate endogenous cyclic GMP and cyclic AMP formation. Pretreatment with sodium nitroprusside and iloprost (a) attenuated the thapsigargin-evoked external Ca2+ entry and (b) reduced the rate of Ca2+ release from the dense tubules. The effects on external Ca2+ entry and Ca2+ release from the dense tubules were exerted independently and were apparently mediated through activation of the respective cyclic nucleotide-dependent protein kinases. Both sodium nitroprusside and iloprost reduced tyrosine kinase phosphorylation of a number of proteins, particularly a 72 kDa protein band. Both agents also attenuated the thapsigargin evoked tyrosine kinase phosphorylation of the 72 kDa band. Intracellular Ca2+ depletion resulted in a reduction in tyrosine kinase-mediated phosphorylation of a number of protein bands, including the 72 kDa band and the further attenuation of thapsigargin-mediated tyrosine phosphorylation of this band. The effects of the cyclic nucleotides on cellular Ca2+ homoeostasis in thapsigargin-treated platelets were not exerted via acceleration of Ca2+ extrusion or Ca2+ sequestration into the mitochondria. We conclude that cyclic nucleotides participate in store-regulated control of external Ca2+ entry by slowing down the rate of external Ca2+ entry and Ca2+ release from intracellular Ca2+ stores. These effects are apparently mediated via cyclic nucleotide-dependent protein kinases and the attenuation of protein phosphorylation by tyrosine kinases. PMID- 7544117 TI - Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. AB - We have developed a monoclonal antibody AF-28 that specifically recognizes a neo epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...). PMID- 7544115 TI - Control of coronary blood flow by autacoids. AB - Coronary flow and thus myocardial perfusion is regulated by myogenic, metabolic, humoral and neuro-hormonal factors which closely interact with local autacoids released from the endothelial lining of the coronary bed. In a number of disease states an impaired synthesis and release of autacoids decisively limit the overall capacity of coronary regulation and adaptation of myocardial perfusion to increased metabolic demands. The important factors for these control mechanisms are analyzed and reviewed in this article. PMID- 7544120 TI - Calcium dependent K-channels in guinea pig and human urinary bladder. AB - This study provides evidence for the presence of large conductance Ca(2+) dependent K-channels in guinea pig and human urinary bladder smooth muscle. A23187, a Ca(2+)-ionophore, increased charybdotoxin and iberiatoxin sensitive 42K efflux in human urinary bladder smooth muscle cells, suggesting that large conductance Ca(2+)-dependent K-channels are present in these cells. NS004, a large conductance Ca(2+)-dependent K-channel opener, relaxed guinea pig bladder strips precontracted with 15 mM KCl which is inhibited by iberiatoxin. In addition, NS004 also evoked an iberiatoxin sensitive increase in 86Rb/42K efflux in guinea pig and human urinary bladder smooth muscle cells, demonstrating that NS004 activates large conductance Ca(2+)-dependent K-channels to achieve its relaxation effect in the bladder. PMID- 7544121 TI - Vitamin-D-dependent transcriptional regulation of the intestinal plasma membrane calcium pump. AB - The vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was shown to increase intestinal plasma membrane calcium pump (PMCA) gene expression. The present study was done to determine whether gene transcription is involved in this process. Nuclei were isolated from duodena of vitamin-D-deficient chicks given 1,25(OH)2D3 intracardially at various times before experiment. The abundance of PMCA RNA in the nuclear and total cellular fractions, measured by a ribonuclease protection assay, was significantly increased above control values at 1.5 hr. and maximized at 3 hr. post-dose. As shown, cross-contamination of nuclear PMCA RNA by cytosolic RNA cannot account for these results. These studies are the first to show that 1,25(OH)2D3 regulates expression of a plasma membrane calcium pump gene by increasing the rate of transcription. PMID- 7544119 TI - Differential display identifies developmentally regulated genes during somatic embryogenesis in eggplant (Solanum melongena L.). AB - Changes in gene expression patterns during early somatic embryogenesis of eggplant (Solanum melongena L.) were characterized by means of differential display. Eight products of polymerase chain reaction (PCR) derived from newly expressed mRNAs after four days of culture were identified and cloned. One clone, pTM002, was found to contain a 416 bp insert and identical to the proximal arbitrary primer at both ends. Based on the terminal sequences, reverse transcription PCR (RT-PCR) was carried out to monitor the expression level of pTM002. The results indicated that the message of pTM002 was increased to correlate with the intensities obtained in the differential display. The significance of this method in investigation of differentially expressed genes is also discussed. PMID- 7544122 TI - Immunosuppression by FK506 markedly prolongs expression of adenovirus-delivered transgene in skeletal muscles of adult dystrophic [mdx] mice. AB - Adenovirus-mediated gene transfer into skeletal muscles of adult immune competent animals has been limited by the fact that a cellular immune attack of the host against transduced muscle fibers prevented long-term transgene expression. In this study we treated adult dystrophic [mdx] mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 10 and 30 days after adenovirus-mediated reporter gene transfer and compared the transduction level to saline-injected controls. After 30 days, transgene expression was no longer demonstrable in the control group, whereas it remained at about 70% of the 10-day transduction value in the FK506 treated group. In addition, we demonstrated a reduction in the number of CD3 and CD8 positive T-lymphocytes in the muscles of the immunosuppressed group compared to controls. PMID- 7544123 TI - Transferrin induces nitric oxide synthase mRNA in rat cultured aortic smooth muscle cells. AB - Incubation with holo-transferrin, but not apo-transferrin, induced the nitrite accumulation in the culture medium of rat aortic smooth muscle cells concentration- and time-dependently, which occurred after a lag period of 6 hours. The accumulation of nitrite was inhibited by N omega-nitro-L-arginine and cycloheximide, but not by calmidazolium. Ferric or ferrous iron had no effect on the accumulation. In reverse transcription-coupled polymerase chain reaction analysis, one amplified fragment (580 bp) corresponding to the inducible form of nitric oxide synthase (iNOS) mRNA was expressed after incubation with holo transferrin for 10 hours. These results suggest that holo-transferrin activates expression of iNOS mRNA in rat aortic smooth muscle cells accompanied by nitric oxide accumulation via a pathway dependent on L-arginine in the culture medium. PMID- 7544118 TI - Effect of transforming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican. AB - We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF. PMID- 7544126 TI - Functional modulation of human "ganglionic-like" neuronal nicotinic acetylcholine receptors (nAChRs) by L-type calcium channel antagonists. AB - Recent studies suggest that the neuronal nicotinic acetylcholine receptors present on chromaffin cells contain a 1,4-dihydropyridine-sensitive site whose occupation blocks membrane depolarization (1). In the present study, several L type Ca2+ channel blockers inhibited the activation of the nAChRs present in the human neuroblastoma cell line, IMR 32, in a dose-dependent manner with IC50 values ranging from 0.8-3 microM. In contrast, omega-Conotoxin GVIA and omega Agatoxin IVA had no effect up to 100 microM. Furthermore, the inorganic channel blocker, cadmium, had no effect either alone or on the modulatory role of the L type antagonists, suggesting that the effects of these agents on nAChRs are not mediated via an interaction with calcium channels but possibly via a direct interaction with the nAChR ionophore. PMID- 7544124 TI - Models of in vitro angiogenesis: endothelial cell differentiation on fibrin but not matrigel is transcriptionally dependent. AB - Endothelial cells can be induced to form a branching network of tubular structures using a variety of cell culture conditions. We have examined this differentiation process using several sets of conditions: plating human umbilical vein endothelial cells (HUVEC) on Matrigel, adding collagen to the apical surface of HUVEC grown on fibronectin, and plating HUVEC on fibrin in the presence of FGF 1. We determined that although the first two conditions produce dramatic morphologic changes in the HUVEC population, gene transcription and translation are not required for the regulation of the process. Rather, post-translational events are involved since the Matrigel-dependent process could be inhibited by the addition of nocodazole, suramin or H7, a protein kinase inhibitor. In contrast, the fibrin matrix-dependent differentiation pathway involved transcriptional and translational events since the addition of either actinomycin D or cycloheximide inhibited this pathway. A modified differential display of RNA extracted from HUVEC after 0, 2, 5, and 24 hours on fibrin revealed expression of a novel cDNA. PMID- 7544125 TI - Exon skipping caused by a base substitution at a splice site in the GTP cyclohydrolase I gene in a Japanese family with hereditary progressive dystonia dopa responsive dystonia. AB - We report a novel mutation at a splice site in the GTP cyclohydrolase I gene in a Japanese family with hereditary progressive dystonia with marked diurnal fluctuation (HPD)/dopa responsive dystonia (DRD). Reverse transcriptase-initiated PCR (RT-PCR) of lymphocyte mRNA showed both normal and small size fragments in the HPD patient and his asymptomatic mother. Sequence analysis revealed that skip splicing of exon 1 to exon 3 occurred in the small fragment. The patient and his mother were heterozygous for G --> C substitution at conserved consensus sequence GT at 5' end of the intron 2. Quantitative RT-PCR showed that the expression of normal GTP cyclohydrolase I mRNA decreased in their lymphocytes, while the HPD patient had more expression of mutant GTP cyclohydrolase I mRNA than his asymptomatic mother. PMID- 7544127 TI - Role of Sp1 in transcriptional activation of human nitric oxide synthase type III gene. AB - Endothelial nitric oxide synthase (eNOS or NOS-III) is constitutively expressed. To elucidate the mechanism by which the basal expression of NOS-III gene is activated, we constructed in a luciferase vector, pXP1, serial 5'-deletion mutants of a 1.3-kb 5'-flanking fragment and transiently expressed them in cultured human endothelial cells. The promotor activity was detected in the 198/+22 region which contains several putative Sp1 binding sites. DNase I footprinting assays coupled with gel shift assays revealed the GC box(-104/-90) to be the Sp1 binding site. Site-directed mutation of 4 crucial bases in this site reduced the promotor activity by > 90%. These findings provide strong evidence that binding of Sp1 or closely related protein to this site is required for the activation of basal NOS-III transcription. PMID- 7544129 TI - Modulation of extraluminally induced vasoconstrictions by endothelium-derived nitric oxide in the canine basilar artery. AB - The present study was undertaken to investigate the role of endothelium in extraluminally induced vasospasm of the cerebral artery using isolated perfused canine basilar arteries. The extraluminal applications of high K+ and prostaglandin F2 alpha (PGF2 alpha) induced concentration-dependent vasoconstriction. Both constrictive responses were significantly enhanced by denuding endothelium. Additionally, the responses in the endothelium-intact arteries were markedly augmented by intraluminal perfusion with NG-monomethyl-L arginine (L-NMMA). These results suggest that the inhibition of nitric oxide (NO) synthase in endothelium enhances increase in transmembrane Ca(2+)-influx which is a common constrictive mechanism to the vasoconstrictors. The augmentative action induced by L-NMMA was inhibited by intraluminal perfusion of L-arginine, but not by D-arginine. Furthermore, the augmentation was not observed in the arteries without endothelium. These results suggest that the endothelium may have a great significance on responsiveness to extraluminal vasoactive substances and that endothelium-derived NO may modulate the extraluminally induced vasoconstriction which is responsible for cerebral vasospasm after subarachnoid hemorrhage. PMID- 7544128 TI - Expression and distribution of cholesterol 7 alpha-hydroxylase in rat liver. AB - The hydroxylation of cholesterol by cholesterol 7 alpha-hydroxylase (CYP7) to 7 alpha-hydroxycholesterol is the rate-limiting step in the production of bile acids. An anti-peptide antibody targeted to the C-terminus of CYP7 was produced by immunising rabbits with the synthetic peptide Tyr-Lys-Leu-Lys-His. The antibody bound to a single band of 54 kDa from rat hepatic microsomal fractions. The intensity of the band was subject to a diurnal variation and showed a significant increase (P < 0.01) in apoprotein at night. Treatment of rats with cholestyramine increased CYP7 apoprotein in the morning (P < 0.005) and at night (P < 0.005), but diurnal variation was maintained. CYP7 catalytic activity, measured using a specific gas chromatography/mass spectrometry assay, showed similar changes in the pattern of diurnal variation and induction. The distribution of CYP7 in rat liver tissue sections was investigated by immunocytochemistry. In sections from rats treated with cholestyramine, there was an even distribution of immunoreactivity, except in the proximal perivenous hepatocytes where immunoreactivity was slightly more intense. A similar distribution was found in sections from untreated rat liver, except immunoreactivity was overall slightly less intense. This study shows that the C terminus of CYP7 is a useful epitope for the targeting of anti-peptide antibodies. PMID- 7544130 TI - Inhibition of pituitary adenylate cyclase activating polypeptide induced relaxation of guinea-pig tracheal smooth muscle by charybdotoxin. AB - The aim of the present study was to investigate whether or not charybdotoxin (CAS 95751-30-7, ChTX), a selective and potent Ca(2+)-dependent K+ channel blocker, inhibits the relaxation of guinea-pig tracheal smooth muscle induced by pituitary adenylate cyclase activating polypeptides with 27 residues (PACAP27) and with 38 residues (PACAP38). Two forms of PACAP were discovered in hypothalamic tissues, and are known to increase the tissues cyclic AMP levels and to be independent of beta-adrenoceptors. The relaxant effects of these polypeptides were evaluated by measuring the isometric tension of tracheal smooth muscle of guinea-pig in vitro. Both forms of PACAP showed dose-dependent relaxant effects. The pD2 of PACAP27 was 7.01 +/- 0.04 and that of PACAP38 was 6.43 +/- 0.05. ChTX (10(-12)-3 x 10(-9) mol/l) did not affect the resting tension of the guinea-pig tracheal smooth muscle. ChTX (10(-8) mol/l) slightly increased the tension, in some experiments being considered as a phasic tension change. ChTX (10(-8) mol/l) caused a small but significant rightward shift in the concentration-response curves of PACAP27 and PACAP38. ChTX decreased the pD2 of PACAP27 to 6.74 +/- 0.03 and that of PACAP38 to 6.25 +/- 0.04. These results suggest that cyclic AMP-mediated activation of Ca(2+)-dependent K+ channels may play an important role in the relaxation of the guinea-pig tracheal smooth muscle induced by both forms of PACAP as well as beta-agonist. PMID- 7544133 TI - Use of reverse ligation-PCR to identify transcriptional start sites in GC-rich TATA-less genes: application to the rat IGFBP-2 gene. AB - TATA-less genes are often GC-rich in the region of transcriptional initiation and the corresponding mRNAs are prone to the formation of secondary structure. These properties have made it difficult to determine unambigously the start sites of transcription for some of these genes by conventional assays such as primer extension and nuclease protection. Using the TATA-less rat IGFBP-2 gene, we demonstrate that tobacco acid pyrophosphatase-reverse ligation polymerase chain reaction (TAP-RLPCR), a novel and sensitive assay, can be used to map the start sites of these genes. First, the validity of TAP-RLPCR was demonstrated by mapping the transcription start site of the rat insulin-like growth factor 1 (IGFBP-1) gene to the correct position (nucleotides -173 relative to ATG, +1). Using total RNA obtained from the rat liver cell line BRL-3A, the transcription start sites of the rat IGFBP-2 gene were mapped to a narrow cluster extending from nucleotides -86 to -90 (ATG, +1), 39 bp downstream of three adjacent GC boxes that are essential to the transcriptional activity of the gene. The assay was also used to map the start sites of a luciferase reporter gene driven by the fragment -1,295 to -32 of the rat IGFBP-2 promoter after transfection in the human embryonic kidney cell line 293. The hybrid gene utilized the same transcription start sites as the rat IGFBP-2 gene, indicating that the elements required for positioning of the transcription initiation complex are contained within the 3' end terminating at nucleotide -32.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544132 TI - Isolation of decidual lymphocytes from chorionic villus samples: phenotypic analysis and growth in vitro. AB - PROBLEM: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CONCLUSION: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells. PMID- 7544131 TI - Expression of complement regulatory proteins on human eggs and preimplantation embryos. AB - PROBLEM: To investigate the relation between the complement system and reproduction, expression of complement regulatory proteins (C3b receptors and inhibitor of the membrane attack complex) were screened on unfixed human eggs and preimplantation embryos. METHODS: Unfixed unfertilized oocytes and preimplantation embryos obtained from an in vitro fertilization program were stained by indirect immunofluorescence using monoclonal antibodies raised against membrane cofactor protein, (MCP or CD46), decay accelerating factor (DAF or CD55), protectin (CD59), human C3b/C4b receptor (CR1 or CD35), and major histocompatibility complex class I antigen (MHC class I). RESULTS: CD55 and CD59 were both expressed by the plasma membrane of unfertilized oocytes and pre implantation embryos. CD46 was not expressed by unfertilized oocytes but appeared at the 6-to-8 cell stage embryo when human gene expression first occurs. CD35 and MHC class I antigens were not expressed at all on oocytes and preimplantation embryos. CONCLUSIONS: Selective expression of complement regulatory proteins (DAF and protectin) associated with the lack of MHC class I antigens may represent an immune protective mechanism by which human oocytes and preimplantation embryos escape complement-mediated damage during their travel through the female genital tract. Furthermore, participation of these complement regulatory proteins including MCP in cell to cell interaction during fertilization and/or implantation cannot be excluded. PMID- 7544136 TI - Posttransplantation lymphoproliferative disorder in patients under primary tacrolimus (FK 506) immunosuppression. AB - Posttransplantation lymphoproliferative disorder (PTLD) is a well-described complication of the systemic immunosuppression required for successful organ transplantation. Lesions of PTLD often occur in the region of the head and neck and require otolaryngologic evaluations. Although the majority of reported cases of PTLD are associated with cyclosporine immunosuppression, recently, PTLD has been described in patients treated solely with the newer systemic immunosuppressive agent tacrolimus (FK 506). As an introduction to tacrolimus and to PTLD as one of its complications, a case of PTLD presenting as airway obstruction in a child treated solely with tacrolimus immunosuppression is described. In addition, a review of tacrolimus and PTLD in patients under tacrolimus immunosuppression is presented to familiarize the otolaryngologist with this important new immunosuppressive agent and a potential complication of its use. PMID- 7544135 TI - Inhibition of the angiotensin converting enzyme by perindoprilat and release of nitric oxide. AB - Experiments were designed to investigate the mechanism underlying the endothelium dependent relaxations to perindoprilat, a converting enzyme inhibitor, in canine coronary arteries previously exposed to bradykinin. Rings suspended in organ chambers were exposed to bradykinin for 3 min and washed extensively for 150 min. In rings previously exposed to the peptide, bradykinin induced relaxations which were augmented in the presence of perindoprilat; this response was not affected by indomethacin, but nitro-L-arginine induced a rightward shift of the relaxation to the peptide without affecting its maximal effect. In canine coronary arteries previously exposed to the peptide, perindoprilat caused endothelium-dependent relaxations (IC50 = 7.83), which had been observed previously at concentrations where the converting enzyme inhibitor did not augment the response to bradykinin. Carboxypeptidase B, but not aprotinin, impaired the relaxation to perindoprilat, suggesting a contribution of bradykinin. The relaxation to perindoprilat was not affected by the B1 antagonist Leu8-des-Arg9-bradykinin. However, the bradykinin B2 antagonist HOE-140 displayed a noncompetitive antagonism against the response to perindoprilat. The response to the converting enzyme inhibitor was not affected by indomethacin but was impaired significantly by nitro-L-arginine. The present findings suggest that in canine coronary arteries previously exposed to bradykinin, the relaxation to perindoprilat is mediated mainly by endothelium derived nitric oxide. In addition, the response to perindoprilat may be due to factors other than just protection of bound bradykinin from degradation. PMID- 7544137 TI - Immunohistochemistry: a new outlook in histopaleopathology. AB - To date, rehydration techniques on mummified tissues as well as the methods for histological specimen preparation with standard basic stains and with histochemical techniques are commonly known and well established. The application of immunohistochemical techniques is a further development in the study of mummified tissues. Similarly to what is done on fresh and fixed tissues, reaction sensitivity and specificity must be guaranteed. While reaction sensitivity depends on serum quality and the detection method employed, specificity is guaranteed by serum quality but also by method standardisation. Method standardisation is the most serious problem in the study of mummified tissues. Mummified tissues have different antigen preservation characteristics, varying from subject to subject, and even within the same subject, depending on the topographic region and the site. These changes are due to different mummification methods and to subsequent environmental conditions in which the mummy was preserved. The Streptavidin-Biotin-Peroxidase Complex (SABC) technique guarantees excellent detection sensitivity. However, the method accuracy and reliability can be ensured only through a rigorous case by case examination with internal controls on structures in which the antigens we are looking for are surely expressed. Today, with these artifices (technique optimisation), cell differentiation phenotype markers can be detected which are useful in paleopathological studies and in the diagnosis of morphologically doubtful lesions. PMID- 7544138 TI - Heart rate decrease after inhibition of nitric oxide release in the anaesthetized dog. AB - In 5 anaesthetized vagotomized dogs the administration of an inhibitor of the synthesis of nitric oxide (NO) was seen to reduce the heart rate, although the arterial pressure was prevented from increasing. Such a finding suggests that the inhibition of nitric oxide release can produce bradycardia without the involvement of baroreceptor reflexes. Since NO inhibition is known to increase the concentration of adenosine in the myocardium, in 3 additional dogs, dipyridamole, which also potentiates the effect of adenosine, was infused: a reduction in heart rate was observed also in these animals. In both groups of animals, before and after the administration of the relevant compound, the increase in ABP obtained with aortic constriction caused the same reduction in heart rate, which which was attributed to a reduction of the basal sympathetic discharge, which was shown not to be affected by NO inhibition. It is concluded that NO inhibition can cause bradycardia without the intervention of any nervous mechanism but with the possible intervention of an increase in myocardial adenosine concentration. PMID- 7544140 TI - Three-dimensional location of the main immunogenic region of the acetylcholine receptor. AB - About two-thirds of the antibodies to the nicotinic acetylcholine (ACh) receptor in patients with the autoimmune disease myasthenia gravis bind to the main immunogenic region (MIR). This is small, well-defined region on each of the two alpha subunits, containing residues 67-76 (alpha 67-76). By determining the structure of the ACh receptor complexed with two different fragments of an MIR directed antibody, we have determined the three-dimensional location of the MIR (and therefore residues alpha 67-76) to be at the extreme synaptic end of each alpha subunit. The antibody fragments extend from the binding site away from the receptor axis and into the synaptic cleft, minimizing any steric interference neighboring ACh receptors might have with their binding. PMID- 7544139 TI - Patch sensor detection of glutamate release evoked by a single electrical shock. AB - We tried to detect minimal stimulation-induced glutamate overflow from the surface of a hippocampal slice using an outside-out patch electrode excised from pyramidal cell membranes. The amplitude of the stimulation-induced patch current was dependent on the distance between the slice surface and the tip of patch sensor. The current-voltage relations of the stimulation-induced patch current were similar to those of the current evoked puff by application of L-glutamate to the patch. This indicates that the stimulation-induced patch current was produced by glutamate released from presynaptic terminals, and thus this technique may be useful in the study of transmitter release evoked by minimal electrical stimulation in brain slices. PMID- 7544142 TI - Neuroantibodies: ectopic expression of a recombinant anti-substance P antibody in the central nervous system of transgenic mice. AB - Recombinant antibodies are efficiently secreted by cells of the nervous system. Thus, their local expression in the CNS of transgenic mice could be used to perturb the function of the corresponding antigen. As a first application of this approach, we have generated transgenic mice that express antibodies against the neuropeptide substance P, under the transcriptional control of the promoter of the neuronal gene vgf. The transgenic antibodies are expressed in a tissue specific and developmentally regulated manner and are effective in competing with the endogenous substance P, as demonstrated by a marked inhibition of neurogenic inflammation and by motor deficits. This phenotypic knockout approach may provide a complementary alternative to gene knockout by homologous recombination. PMID- 7544141 TI - Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth. AB - Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo. PMID- 7544144 TI - Pancreatic cancer: a plea for more trials. PMID- 7544147 TI - Cholera in the Americas. PMID- 7544145 TI - Problem areas in pain and symptom management in advanced cancer patients. PMID- 7544148 TI - AIDS surveillance in the Americas. PMID- 7544146 TI - Epidermal growth factor-induced protection of tumour cell susceptibility to cytolysis. AB - Using radiobinding, transfection and colorimetric assays, the biological significance of epidermal growth factor (EGF) and its receptor on established human tumour cell lines was investigated. The intensity of class I major histocompatibility antigen (MHC) and EGF receptor (EGFR) expression on 20 tumour cell lines was investigated and showed no direct correlation (coefficient of correlation r = 0.43 and P = 0.06). furthermore, transfection of the beta 2 microglobulin gene into a class I negative bladder tumour cell line, resulting in the re-expression of fully assembled cell surface class I antigens, did not result in alteration of EGFR expression. However, there was an inverse correlation between the intensity of EGFR expression and the stimulatory response of cells to exogenously added EGF. The per cent inhibitions of cell proliferation by EGF at 100 ng/ml for A431 (highest EGFR expressor) and Scaber (lowest EGFR expressor) were 37 and -7%, respectively. The results also showed that cell lines isolated from testis tumours positive for epithelial markers (using pan keratin antibody LP34 as an epithelial marker), expressed significantly lower EGFR levels than cell lines from bladder tumours. The expression of EGFR receptor was not modulated by interferons (IFN-alpha and -gamma and only a minor effect with IFN beta) or active supernatant containing a mixture of cytokines. Whilst the pretreatment of tumour cells with IFNs resulted in a significant increase in the susceptibility of tumour cells to interleukin-2-activated peripheral blood mononuclear cells, EGF treatment resulted in their protection. Thus, the per cent killing at an effector:target ratio of 20:1 for untreated cells and EGF (100 ng/ml), IFN-alpha (1000 U/ml), -beta (2000 U/ml) and -gamma (100 U/ml) were 53%, 33% (P = 0.004), 64% (P = 0.004), 69% (P = 0.001) and 66% (P = 0.001), respectively. These results indicate the complex interactions between EGF and EGFR and their relevance in modifying tumour cell behaviour. The hypothesis that the resistance to cytolysis of tumour cells induced by EGF stimulation may be a factor in the accelerated tumour growth seen in patients after traumatic tissue damage is discussed. PMID- 7544143 TI - Independent mechanisms for long-term depression of AMPA and NMDA responses. AB - While the mechanisms responsible for LTP and LTD of excitatory synaptic responses mediated by AMPA receptors (AMPARs) have been extensively characterized, much less is known about the regulation of NMDA receptors (NMDARs) by synaptic activity. In hippocampal CA1 cells, prolonged low frequency afferent stimulation depresses synaptic responses mediated by either NMDARs or AMPARs. However, this apparently similar LTD is accompanied by a change in the coefficient of variation (CV) of only the AMPAR-mediated synaptic responses; the CV of the NMDAR-mediated synaptic responses is unaffected. Moreover, by varying the pattern of synaptic stimulation, the responses mediated by one receptor subtype can be modified without affecting the responses mediated by the other. These results indicate that the mechanisms underlying activity-dependent plasticity of NMDAR-mediated synaptic responses are different from those responsible for plasticity of AMPAR mediated synaptic responses. PMID- 7544149 TI - Health conditions in the Americas, 1994. PMID- 7544151 TI - Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide. AB - In 1990 we reported that GP Ib-IX complexes accumulated within the surface connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide. PMID- 7544150 TI - Cholera in 1994. Part I. AB - In 1994, Vibrio cholerae O1 biotype El Tor, the agent responsible for the seventh cholera pandemic which began in 1961, continued to spread in all regions of the world (Map 1). In all, 384,403 cholera cases were officially reported to WHO in 1994 (a 2% increase over 1993), reversing the downward trend which started in 1992. A total of 10,692 deaths were reported in 1994, increasing the reported global case-fatality ratio (CFR) to 2.8% from 1.8% in 1993. Cholera cases were notified from 94 countries/areas, the highest number ever reporting in one year. (Table 1 and Fig. 1). The year was marked by the dramatic cholera outbreak that devastated the Rwandan refugee camps in Goma, Zaire in July. Major outbreaks also affected Afghanistan, Brazil, Guinea, Guinea-Bissau and Somalia. Africa reported a rise in the number of cholera cases, and was the continent accounting for the largest proportion of all reported cases. The incidence of cholera cases in the Americas continued to fall and reported CFR was the lowest recorded since the disease reached Latin America in 1991. Asia reported a 17% increase in cholera cases compared with 1993. Europe, which usually reports only imported cases, registered a significant increase in the number of indigenous cholera cases. Oceania reported 6 cases, 5 of which were imported, and no deaths (Figs. 2 and 3). The new V. cholerae strain O139 (Bengal) has affected 10 countries in Asia since it was first detected in India at PMID- 7544152 TI - Flow cytometric analysis of decay-accelerating factor (CD55) on neutrophils from aplastic anaemia patients. AB - Using a flow cytometric analysis, CD55 (decay-accelerating factor), CD59 and CD58 have been measured on neutrophils from 12 aplastic anaemia (AA) patients who were long-term survivors after immunosuppressive therapy (IS), 17 healthy individuals, four patients with PNH, and six patients with other haematological disorders. The neutrophils from normal control patients and the six patients with other haematological disorders showed 98 +/- 2% (mean +/- SD) positive granulocytes for CD55. Corresponding values were low (12%, 26%, 51% and 58%) on the primarily PNH patients. Among the 12 AA patients examined, seven had normal and five low values (59% in two, 70%, 71% and 82%). Among the five AA patients who showed CD55 neutrophil deficiency, four had showed an incomplete response after the initial IS treatment and the other relapsed following an initial haematological complete response; three cases had a positive Ham's test and two were negative. Our data suggest that the development of PNH clones is a frequent finding in AA long-term survivors, mainly in those who had shown an incomplete response following IS. Neutrophil CD55 expression analysis by flow cytometry could be useful to detect clonal evolution in these patients. PMID- 7544153 TI - Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development. AB - INT3 is interrupted by retroviral DNA insertion in approximately 18% of primary Czech mouse mammary tumors induced by mouse mammary tumor virus. One consequence of these insertions is the production of a 2.4-kilobase, tumor-specific RNA transcript encoding the entire intracellular domain of the Int3 protein which is initiated from the 3' long terminal repeat promoter of the inserted viral genome. Female mice (FVB-3) transgenic for a genomic fragment comprised of this truncated region of INT3 express the 2.4-kilobase truncated INT3 transcript and exhibit focal mammary tumors at 100% penetrance. INT3 is a member of a family of genes, highly conserved through evolution and characterized by Drosophila melanogaster Notch and Caenorhabditis elegans lin-12, the function of which relates to cell fate determination. Upon transfection into the appropriate hosts, expression vectors of truncated Notch and lin-12, representing their respective cytoplasmic domains, have been demonstrated to effect their complete gene function with respect to cell fate determination. This suggests that the extracellular portion of these proteins function only to regulate activity. Reciprocal transplantation of transgenic FVB-3 and normal mammary tissue to the epithelium-divested fat pads of the respective donor females demonstrates that FVB-3 mammary epithelium is unable to grow and/or to functionally differentiate. However, normal epithelium grows and fully differentiates in transgenic FVB-3 fat pads, indicating that the dysfunction of FVB-3 mammary glands is due to a deficiency inherent in their epithelium. Electron microscopy reveals that transgenic INT3 epithelial cells do not form intercellular junctional complexes in the developing subadult mammary gland. The hormonal stimulation of pregnancy overcomes the deficiency for ductal growth so apparent in the virgin gland such that pregnant FVB-3 glands produce complete ductal systems. Nevertheless, during pregnancy, FVB-3 mammary cells fail to form secretory lobules and to produce milk. Examination of INT3 expression by immunocytochemistry and reverse transcriptase-PCR show that INT3 is expressed constitutively in mammary stroma and epithelia at all stages of postpubertal mammary evolution. These results indicate that deregulated expression of a truncated Int3 in mammary epithelial cells limits their capacity to perform the cell fate decisions required for morphogenesis and functional differentiation. PMID- 7544154 TI - 12-O-tetradecanoylphorbol-13-acetate promotion of transgenic mice expressing epidermal-targeted v-fos induces rasHA-activated papillomas and carcinomas without p53 mutation: association of v-fos expression with promotion and tumor autonomy. AB - Transgenic mice that expressed v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos) developed preneoplastic epidermal hyperplasia and hyperkeratosis after long latency and an associated wound promotion stimulus. To assess the requirements for papilloma formation and malignant conversion and determine the sensitivity to a chemical promotion stimulus, HK1.fos mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). HK1.fos mice were sensitive to TPA promotion but developed papillomas only after long latency (20-30 weeks of promotion) and in relatively few numbers per animal, suggesting the necessity of an additional genetic event prior to overt lesion formation. Consistent with this idea, at 60 weeks, on cessation of TPA promotion, these HK1.fos TPA-papillomas were found to be autonomous, TPA independent tumors which persisted, grew larger, and converted to malignancy. Analysis of HK1.fos tumor RNA and DNA identified endogenous c-rasHa mutations at codons 12 and 61 in papillomas and carcinomas; however, no p53 tumor suppressor gene mutations were detected. These data indicate that epidermal expression of v fos induces sensitivity to TPA promotion, but since additional genetic events, such as endogenous c-rasHa activation, appear to be required in tumorigenesis, v fos may predominantly play a role in the mechanism of promotion to achieve papilloma autonomy and TPA independence. Furthermore, spontaneous malignant conversion in this model does not appear to involve mutations in the p53 tumor suppressor gene. PMID- 7544156 TI - Studies of cloning, chromosomal mapping, and embryonic expression of the mouse Rab geranylgeranyl transferase beta subunit. AB - The mouse Rab geranylgeranyl transferase beta subunit has been cloned from a mouse E8.5 embryonic cDNA library. Sequence comparison reveals 97.4% sequence identity at the amino acid level to the rat clone isolated from an adult rat brain cDNA library. This gene, given a gene symbol of Rabggtb, is mapped in the distal region of mouse chromosome 3. It is ubiquitously expressed in adult animals but displays an interesting pattern of expression during a specific time of embryonic development. The expression of this gene can be detected in the whole embryos during early embryonic stages and is specifically concentrated in the developing brain, heart, and liver between gestation stages of E11.5 and E13.5. In addition, the expression of this gene is induced by retinoic acid in a mouse embryonal carcinoma cell line, P19. PMID- 7544155 TI - Expression of the proto-oncogene rhombotin-2 is identical to the acute phase response protein metallothionein, suggesting multiple functions. AB - Rhombotin-2 (RBTN-2) is a LIM domain protein that, with the exception of thymocytes, is widely expressed during fetal development. Although RBTN-2 is crucial for normal erythropoiesis, the ectopic expression of RBTN-2 in T lymphocytes results in T-cell proliferation and leukemogenesis. Thus, while a proliferative function for RBTN-2 has been established in T-cells, neither its role in erythropoiesis nor its function(s) in other tissues are known. We have examined the expression and location of RBTN-2 in normal and malignant cells. Similar to fetal development, RBTN-2 RNA was detected in all normal adult tissues tested with the exception of colon and thymocytes. RBTN-2 RNA was not detected in all primary tumors and tumor cell lines, indicating RBTN-2 expression is not ubiquitous in proliferating cells. Using polyclonal antisera, RBTN-2 was detected predominantly in the nucleus of human hematopoietic cells. Significantly, human leukemic T cells with disruption of the RBTN-2 locus and thymocytes from transgenic mice with enforced expression of RBTN-2 showed similar nuclear location of RBTN-2 protein, consistent with the notion that RBTN-2 acts as a transcriptional regulator in T-cell proliferation. Surprisingly, in normal tissues, RBTN-2 showed a strikingly similar distribution to that of metallothionein-1, having both nuclear and cytoplasmic localization that suggested that RBTN-2 may be involved in the acute phase response. Indeed, similar to metallothionein-1, RBTN-2 mRNA was induced in thymocytes of mice exposed to zinc and in human thymocytes treated with the phorbol ester 12-O tetradecanoylphorbol-13-acetate. Since the LIM domain permits binding of multiple protein partners, the specific function of RBTN-2 may depend upon subcellular sequestration through interaction with different cofactors. Thus, in addition to its roles in erythropoiesis and T-cell leukemia, RBTN-2 may also be involved in the acute phase response. PMID- 7544157 TI - Elevated levels of insulin-like growth factor binding protein-1 in fetal distress. AB - OBJECTIVE: To investigate the association between fetal distress (abnormal cardiotocograph tracing and/or a low fetal pH) and the levels of fetal IGFBP-1. DESIGN: Prospective comparative study. SUBJECTS AND METHODS: Twenty-two women in labour with evidence of fetal distress defined by FIGO criteria and 19 women in uncomplicated labour. The gestation range was 37 to 42 weeks and birthweight range was 2500 to 4240 g. IGFBP-1 was determined by radioimmunoassay. RESULTS: The umbilical levels of IGFBP-1 were significantly higher in the study group compared with the control group (median 282.5 micrograms/l versus 128 micrograms/l, P = 0.0046; Mann-Whitney U test). There was a significant inverse correlation between fetal IGFBP-1 and cord pH (r = 0.58, P < or = 0.0001). There was no difference between the maternal serum levels of IGFBP-1 in the two groups. CONCLUSION: Umbilical IGFBP-1 is elevated in association with fetal distress. PMID- 7544159 TI - Macrocyclic lactone synthesis by lipases in water-in-oil microemulsions. AB - Five microbial lipases from Chromobacterium viscosum, Candida cylindracea, Pseudomonas (source Fluka), Pseudomonas (source Genzyme) and lipoprotein lipase ex Microbial (Genzyme) have been screened for lactonisation activity towards 16 hydroxyhexadecanoic acid (HHA) in a variety of different w/o microemulsion systems. With the exception of Candida cylindracea (CC), all the lipases exhibited lactonisation activity although they were inherently more active in microemulsion systems based on the anionic surfactant sodium bis(2 ethylhexyl)sulphosuccinate (AOT) than in those based on the cationic surfactant cetyltrimethylammonium bromide (CTAB). Lactone yields are typically 50-60% and are markedly better than those reported previously using microemulsions in combination with chemical catalysts. Lipase stability is superior in the CTAB microemulsion systems, while lipase stability in the low water content AOT microemulsion systems was still good with the exception of CC lipase, which is rapidly inactivated. Buffering the water pools of AOT microemulsions using diglycine buffer at pH 8.0 improved biocatalyst stability. The lactonisation activity of lipases in CTAB w/o microemulsion systems compares favourably with that obtained using the same preparations as a solid suspension in the corresponding water-saturated organic solvent. In addition, the unusual solubility properties of microemulsions allowed the use of considerably higher concentrations of substrate in the microemulsion systems as compared to water saturated organic solvents such as n-heptane. Lactone yields obtained at equivalent concentrations in the corresponding organic solvents containing conventional condensation catalysts were consistently measured at approx. 10%. PMID- 7544160 TI - Zonal distribution of receptor binding of trypsin-activated alpha 2 macroglobulin, alpha 2-macroglobulin receptor-associated protein, lactoferrin and transferrin on rat liver parenchymal cells. AB - Periportal and perivenous rat liver parenchymal cells were isolated according to the digitonin-collagenase perfusion method. Affinities and maximal specific binding of a conjugate of glutathione S-transferase and the alpha 2-macroglobulin receptor-associated protein (GST-39kDaP), of lactoferrin and of transferrin to freshly isolated periportal parenchymal cells in vitro were not significantly different from values obtained with perivenous cells. It is concluded that the receptors for these three ligands show a zonally homogeneous expression in rat liver. The zonal homogeneity in binding observed for GST-39kDaP is at variance with the 1.5-fold higher periportal over perivenous binding of trypsin-activated alpha 2-macroglobulin. Since GST-39kDaP as well as trypsin-activated alpha 2 macroglobulin are ligands for the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein, it is suggested that GST-39kDaP can bind to (an) additional receptor(s) with a higher perivenous expression. The zonal homogeneity observed with lactoferrin, an inhibitor of ligand binding to the lipoprotein remnant receptor, may indicate zonal homogeneity of the lipoprotein remnant receptor. The observed zonal homogeneity of the transferrin receptor suggests an equal and essential need for iron by parenchymal cells across the rat liver acinus in vivo. PMID- 7544158 TI - Low brain serotonin turnover rate (low CSF 5-HIAA) and impulsive violence. AB - The findings of a series of studies by the authors support the idea that most impulsive offenders who have a tendency to behave aggressively while intoxicated have a low brain serotonin turnover rate. The impulsive violent offenders with the lowest CSF 5-HIAA concentrations have diurnal activity rhythm disturbances, and are also prone to hypoglycemia after an oral glucose challenge. Low CSF 5 HIAA combined with hyoglycemic tendency also predicts future violence under the influence of alcohol. Sons of alcoholic fathers, who have committed violent crimes, have very low CSF 5-HIAA concentrations. Vagal tone does not correlate significantly with CSF 5-HIAA but correlates with enhanced insulin secretion, which is most prominent in subjects with intermittent explosive disorder. A polymorphism of tryptophan hydroxylase (TPH) gene is associated with low CSF 5 HIAA and a history of suicide attempts. PMID- 7544161 TI - Effect of retinoids on fetal lung development in the rat. AB - We investigated the effect of retinoids on fetal lung development in the rat. The concentration of retinyl palmitate increased rapidly to a peak on day 17 of gestation and decreased to a minimum on day 21 of gestation; there was a slight increase after birth. Retinoid acid receptor (RAR)-alpha and -beta mRNA were detected in all samples obtained from perinatal and adult rat lung, and only a trace of RAR-gamma mRNA was detected in the fetuses on days 15, 17 and 19 of gestation and in the adults by reverse transcriptase-polymerase chain reaction. After a maternal retinol deficiency of 28 days' duration, fetal body and lung weights were significantly lower than those of controls; the concentrations of retinyl palmitate and phosphatidylcholine (PC) in the lung after a maternal retinol deficiency of 14, 21, or 28 days were significantly lower than those of controls. Expression of RAR-beta mRNA in the group with 28-day retinol deficiency was lower than in controls, that of RAR-alpha mRNA was increased and that of RAR gamma mRNA was not influenced by retinol deficiency. The rate of choline incorporation into PC in fetal lung explants was significantly higher in the group treated with retinoic acid (RA) than in controls. RA enhanced the effect of epidermal growth factor on choline incorporation and prevented that of dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544162 TI - [Cough induced by nasal vasoconstrictors]. PMID- 7544163 TI - Cellular interactions in tissue factor expression by blood monocytes. AB - Tissue factor (TF) is the cellular receptor for factor VIIa. In complex with TF, factor VIIa initiates coagulation by activation of factor IX and factor X. TF is normally not in contact with blood, but is expressed on extravascular cells which forms a haemostatic envelope ready to activate coagulation when vascular integrity is disrupted. However, under pathological conditions monocytes, but probably not endothelial cells, are stimulated to express TF activity on the surface of the cells and may thereby trigger activation of blood coagulation. For several years we have observed some individuals (high responders) with a very high response to lipopolysaccharide (LPS) as judged by induction of TF activity in their monocytes in whole blood. This phenomenon has been shown to be mediated by a P-selectin dependent interaction between granulocytes, platelets and monocytes where the release of platelet factor 4 (PF4) plays an important role. It is concluded that cellular interactions play a central role in the expression of TF activity in circulating monocytes. PMID- 7544164 TI - Gramicidin tryptophans mediate formamidinium-induced channel stabilization. AB - Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl. PMID- 7544168 TI - Effect of streptozotocin diabetes on polysomal aggregation and protein synthesis rate in the liver of pregnant rats and their offspring. AB - To study the effect of diabetes on hepatic protein synthesis and polysomal aggregation in pregnant rats, female rats were treated with streptozotocin prior to conception. Some animals were mated, and studied at day 20 of pregnancy, whereas, others were studied in parallel under non pregnant conditions. The protein synthesis rate measured with an "in vitro" cell-free system was higher in pregnant than in virgin control rats. It decreased with diabetes in both groups, although values remained higher in diabetic pregnant rats than in the virgin animals. The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they showed a higher protein synthesis rate than either their respective mothers or virgin rats. Liver RNA concentration was higher in control and diabetic, pregnant rats than in virgin rats, and the effect of diabetes decreasing this parameter was only significant for pregnant rats. Liver RNA concentration in fetuses was lower than in their mothers, and did not differ between control and diabetic animals. The decreased protein synthesis found in diabetic animals was accompanied by disaggregation of heavy polysomes into lighter species, indicating an impairment in peptide-chain initiation. PMID- 7544165 TI - Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells. AB - A clone of human HeLa cells stably transfected with mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ("induced cell pair") to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j (residual state)), and a closed state (gamma j(closed state)). The gamma j(main state) was 198 pS, and gamma j(residual state) was 36 pS (temperature, 36-37 degrees C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, Vj. The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, Po, decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: Vj,o = -44 mV; z = 6). The temperature coefficient, Q10, for gamma j(main state) and gamma j(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40 degrees C). This difference suggests interactions between ions and channel structure in case of gamma j(residual state). In cell pairs with many channels, the gap junction conductance at steady state, gj, exhibited a bell-shaped dependency from Vj (Boltzmann fit, negative Vj, Vj,o = -45 mV, gj(min) = 0.24; positive Vj, Vj,o = 49 mV, gj(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for Vj gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state. PMID- 7544169 TI - Mitoxantrone plus vinorelbine with granulocyte-colony stimulating factor (G-CSF) support in advanced breast cancer patients. A dose and schedule finding study. AB - Mitoxantrone (MTZ) and vinorelbine (VNR) have shown a good efficacy in advanced breast cancer. We conducted a phase I-II trial to determine the MTDs and best schedule of these drugs, in advanced breast cancer patients, when granulocyte colony stimulating factor (G-CSF) support was given. The starting dose-intensity level was MTZ 3 mg/m2/week + VNR 15 mg/m2/week; dose was escalated at each step by 1 mg/m2/week for MTZ and 5 mg/m2/week for VNR, until dose limiting toxicity (DLT) developed in 33% or more of the patients at the first course. G-CSF 5 micrograms/kg/day d3-13 was administered at each cycle from dose level 2 on. For each dose step we planned 3 different schedules (a = total dose of MTZ on day 1; b = total dose d1 and 8; c = weekly schedule). At the time of this analysis (December 1993) 43 patients with locoregionally advanced or metastatic breast cancer have entered this study, 23 of whom had received prior chemotherapy other than adjuvant. Toxicity has been primarily hematologic. Non hematologic toxicity never caused interruption of dose escalation. Overall 8 patients developed DLT at the first course. Dose escalation was stopped at level 3 in patients receiving schedules a or b, and in those receiving schedule c the dose was escalated until level 5. The MTD was MTZ 6 mg/m2 and VNR 30 mg/m2 weekly. Age, dose level, and PS were found to be correlated with neutrophil and platelet nadirs, but dose level was the only independent variable predictive of myelotoxicity at multiple regression analysis. Forty-one patients were evaluable for response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544167 TI - Flexibility of myosin attachment to surfaces influences F-actin motion. AB - We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. PMID- 7544166 TI - Multiple conductance channel activity of wild-type and voltage-dependent anion selective channel (VDAC)-less yeast mitochondria. AB - Yeast mitoplasts (mitochondria with the outer membrane stripped away) exhibit multiple conductance channel activity (MCC) in patch-clamp experiments that is very similar to the activity previously described in mammalian mitoplasts. The possible involvement of the voltage-dependent anion-selective channel (VDAC) of the outer membrane in MCC activity was explored by comparing the channel activity in wild-type yeast mitoplasts with that of a VDAC-deletion mutant. The channel activity recorded from the mutant is essentially the same as that of the wild type in the voltage range of -40 to 30 mV. These observations indicate that VDAC is not required for MCC activity. Interestingly, the channel activity of the VDAC less yeast mitoplasts exhibits altered gating properties at transmembrane potentials above and below this range. We conclude that the deletion of VDAC somehow results in a modification of MCC's voltage dependence. In fact, the voltage profile recorded from the VDAC-less mutant resembles that of VDAC. PMID- 7544172 TI - Reliability of the Assessment of Communicative Effectiveness in Severe Aphasia. AB - The Assessment of Communicative Effectiveness in Severe Aphasia (ACESA) was developed to measure the communicative effectiveness of people with severe aphasia following a stroke. The reliability was evaluated. Ten patients with severe communication difficulties (particularly limited expression) were assessed and videoed. The videos were rated by three trained raters. Reliability was calculated to demonstrate intrarater and interrater reliability, and test-re-test reliability. Reliability was good but needed to be improved across raters. It was concluded that the ACESA has the potential to be a useful clinical and research tool. PMID- 7544171 TI - Distribution of vitronectin mRNA during murine development. AB - Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS-specific vascular marker. PMID- 7544170 TI - Steel and c-kit in the development of avian melanocytes: a study of normally pigmented birds and of the hyperpigmented mutant silky fowl. AB - We describe here the expression of c-kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso-laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c-kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c-kit-positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c-kit positive cells are present in the Steel factor-producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111-2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c kit expressing melanoblasts migrate initially in the dorso-lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant. PMID- 7544173 TI - Distinct effect of G-CSF on the growth and differentiation of myeloid progenitor cells from chronic idiopathic neutropenia. AB - Chronic idiopathic neutropenia (CIN) is a disorder characterized by severe neutropenia and a maturational arrest of the neutrophil precursors in the bone marrow. We examined the effect of recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the growth and maturation of the myeloid progenitor cells from a patient with CIN. The patient's marrow cells showed poor colony forming activity, but a normal differentiating capacity to the stimulation with rhG-CSF, although they displayed a normal colony forming capacity in the presence of GM-CSF. Our observation indicates the distinct effect of rhG-CSF on the growth and maturation of the myeloid progenitors from a CIN patient. PMID- 7544175 TI - Small intestinal malabsorption and colonic fermentation of resistant starch and resistant peptides to short-chain fatty acids. AB - Some starch and protein, as well as fiber, remains unabsorbed in the small intestine and is degraded by anaerobic bacteria to short-chain fatty acids, hydrogen, methane, and carbon dioxide in the large intestine. The production of butyrate from starch has received the most attention, because butyrate seems to possess several important functions in the large bowel, including antineoplastic properties. In 16.6% fecal homogenates, starch polysaccharides, whether digestible or resistant to in vitro hydrolysis by amylase, pectin, and glucose, were all completely degraded to equal amounts of short-chain fatty acids (mean 60 wt/wt%; range 49-67 wt/wt%). However, starch that was resistant to hydrolysis by amylase was much more slowly fermented with the production of proportionally less butyrate and propionate than digestible starch (butyrate, 15 and 33%, respectively; propionate, 3 and 20%, respectively). The daily intake of 35 g resistant starch (100 g amylomaize starch) by 7 ileostomy subjects increased ileal dry-matter effluent by 38 +/- 2 g/day, due exclusively to increased excretion of carbohydrates of nonfiber origin (starch-polysaccharides and oligo- and monosaccharides) from 14 +/- 1 to 51 +/- 2 g/day, with no change in excreted nonstarch polysaccharides, nitrogen, and ileal volume. The ileal excreted resistant starch increased the formation of total short-chain fatty acids by 50% in fecal homogenates incubated with ileal dry matter from the amylomaize starch period, with comparatively little effect on the ratio of produced butyrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544177 TI - [Clinical study of TCM-WM on aplastic anemia complicated with hepatitis C]. AB - The testing kit of second generation for serum anti-HCV was used in 82 cases of aplastic anemia (AA). The results showed that positive rate was 69.4% (43/62) in the patients of AA with transfusion, this was significantly higher than that in the patients of AA without transfusion. There was no difference of anti-HCV antibody positive rate between chronic AA and acute AA (P > 0.05), incidence rate of post-transfusion hepatitis C (PTHC) in AA was 33.9% (21/62), among which the incidence rate in acute and chronic AA were 68.8% (11/16) and 21.7% (10/46) respectively (P < 0.01). The anti-HCV positive patients were divided into two groups: PTHC and non-PTHC, there was no statistical difference of their transfusion volume, hemoglobin, white blood cell between these groups. Response rate of AA was lower in anti-HCV positive patients than that in negative patients (P < 0.05). Acute, icteric PTHC was predominant in patients with AA. The patients with AA complicated with PTHC was liable to bleed and be infected. PTHC has been an important complication in patients with AA. The better response was obtained by TCM-WM therapy in the patients. PMID- 7544176 TI - Male golden hamster in male reproductive toxicology testing: assessment of protective activity of selenium in acute cadmium intoxication. PMID- 7544178 TI - Toxic hematoma: an unusual and previously undescribed type of thyrotoxicosis. AB - The present case describes a new and unusual variant of thyrotoxicosis: transient hyperthyroxinemia following acute and massive hemorrhage into a previously normal thyroid gland. A 74-year-old woman presented with a large painful neck mass, palpitations, rapid atrial fibrillation, and hyperthyroxinemia following a fall and neck trauma while treated with excessive oral anticoagulants. Ultrasound and computerized tomography of the neck were consistent with a massive intrathyroidal hematoma. Elevation in serum T4 and T3 levels with suppressed TSH normalized over 3-4 weeks in parallel with hematoma shrinkage. Her tests were consistent with extensive bleeding into thyroidal tissue producing release of stored hormone. Her clinical course was compatible with the known disappearance rate of thyroxine and she returned to her euthyroid status without antithyroid therapy. PMID- 7544174 TI - Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review. AB - During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from placebo-controlled double-blind clinical trials is reviewed. The effect of alpha 1-blockers on symptoms and objective parameters for bladder outlet obstruction is well documented. However, alpha 1-blockers carry a small but significant incidence of adverse effects. Parameters for identification of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit or unwilling to undergo surgical resection of the prostate will benefit from such therapy. PMID- 7544179 TI - A polymorphism in the extracellular domain of the thyrotropin receptor is highly associated with autoimmune thyroid disease in females. AB - We and others have described previously a polymorphism at the first position of codon 52 (C52 --> A52) of the human thyrotropin receptor (hTSHr) gene. To determine its potential significance, we studied female (n = 100) and male (n = 25) patients with autoimmune thyroid disease (Graves' disease, n = 91; Hashimoto's thyroiditis, n = 34) and normal individuals [n = 121, female (n = 69), male (n = 52)]. Screening was performed using AciI restriction enzyme digestions of PCR-amplified genomic DNA. All codon 52 polymorphisms were verified by direct DNA sequencing. Data were analyzed using Chi-square or Fisher exact tests and p-values were corrected for multiple comparisons. Our studies demonstrated that this polymorphism is highly associated with autoimmune thyroid disease in the female population (corrected p = 0.008). We found no such association in the male population. Within females, there was a greater association between Graves' disease and the polymorphism (corrected p = 0.017) than between Hashimoto's thyroiditis and the polymorphism (corrected p = 0.090). The polymorphism was present in a higher proportion of Graves' disease patients with Graves' ophthalmopathy and pretibial dermopathy (40%) or Graves' ophthalmopathy, pretibial dermopathy, and acropachy (60%), than in patients with Graves' disease alone (15%), or Graves' disease and Graves' ophthalmopathy alone (17%). In conclusion, a polymorphism (C52 --> A52) of the hTSHr is associated with autoimmune thyroid disease in females. PMID- 7544180 TI - Characterization of the allergen group VI in timothy grass pollen (Phl p 6). I. Immunological and biochemical studies. AB - For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pI 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phl p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients' serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes. PMID- 7544181 TI - Studies of the specificities of IgE antibodies found in sera from subjects with allergic reactions to penicillins. AB - Penicillins are immunogenic when administered to humans and in some instances they can also be allergenic, inducing specific IgE antibodies. Whilst the major haptenic group, the penicilloyl, is well characterised, less is known about the relative importance of the different parts of the structure for antibody binding and how this can influence the specificity of patients response. In order to investigate this further, sera from subjects who had suffered an IgE-mediated reaction to penicillins were studied using the radioallergosorbent test (RAST) and RAST inhibition. The assays employed reagents related to the penicillins causing the reaction. Using 173 sera, positive RAST results were only found with reagents based on benzyl penicillin (BP) and amoxicillin (AX). Fifty-three positive sera were selected for further studies and categorized into three groups: (A) sera only RAST positive to AX, (B) sera only positive to BP and (C) sera positive to both penicillins. RAST inhibition studies were then carried out using monomeric penicilloyl conjugates and compounds representing parts of the penicilloyl structures of BP and AX. For all three groups, monomeric penicilloyl conjugates were the most efficient inhibitors but there were differences for the other compounds. Group A sera were also inhibited by the side chain amoxicillin, whereas group B sera were poorly inhibited by all other inhibitors. Group C sera showed two patterns of inhibition, both consistent with their more cross-reactive profile. PMID- 7544183 TI - AMPA elicits long-lasting, partly hypothermia-sensitive calcium responses in acutely dissociated or cultured embryonic brainstem cells. AB - This study aimed at testing if, and under which conditions, long-lasting cytosolic calcium responses can be induced in dissociated embryonic brain cells exposed to alpha-amino-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor agonists. Rat brainstem cells (gestation days 13-14; mean crown-rump lengths 8-11 mm) were mechanically dissociated and loaded with the fluorescent calcium marker Fluo-3 after in vitro delays ranging from 20 min to 6 days. The cells were exposed to various concentrations of AMPA, domoic acid or kainic acid. The evoked fluorescence changes, indicating variations of cytosolic calcium, were recorded and analysed either with a video-microscope or a laser cytometer. Even at the earliest stages, non-desensitizing (or partly desensitizing) calcium responses to AMPA were found. In addition, sequential exposure to AMPA followed either by domoic acid, or by AMPA in the presence of aniracetam, revealed the existence of cells bearing predominantly desensitizing receptors. The non desensitizing as well as desensitizing response components were blocked by 6,7 dinitroquinoxaline-2,3-dione (DNQX). When the experiments were conducted at 24 degrees C, the cytosolic calcium levels generally returned close to pre-stimulus baseline levels after washout. In contrast, when the working temperature was slightly raised (to 27 degrees C), complex secondary calcium rises were observed not only during prolonged stimulation, but also after short agonist application. The calcium modulation might be correlated with some form of cellular "learning" in the embryonic brain. Under particular conditions, where the regulation processes are either switched off by cell programmes or simply overloaded, the cascade of events comprising secondary calcium rises may lead to cell death. PMID- 7544182 TI - An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins. AB - A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex. PMID- 7544185 TI - [Value of a specific bronchial provocation test in comparison with other methods in diagnosis of atopic bronchial asthma]. AB - The aim of this study was to determine the relation between specific Bronchial Provocation Test (sBPT), the serum specific IgE level (sIgE) and specific basophil histamine release (Dpt HR) in asthma patients, sensitive to Dermatophagoides pteronyssinus (Dpt). The studies were carried out on 30 mild asthma patients with positive case history and strongly positive skin tests with Dpt. In all the patients were performed following tests: BPT with histamine, serum specific IgE level against Dpt (sIgE Dpt), spontaneous basophil HR (spHR), anti-IgE depended basophil HR (aIgE HR), Dpt depended basophil HR (Dpt HR), sBPT with Dpt (sBPT Dpt). The positive results of sBPT Dpt were observed in 27 patients. Twenty six of them had the early? asthmatic response (EAR) at least, 19 -dual asthmatic response (EAR and LAR) and isolated late asthmatic response (LAR) demonstrated only one patient. The correlation coefficient (r) between sIgE and Dpt HR was 0.4 p < 0.04, between sIgE and sBPT Dpt r = -0.4, p < 0.03, and between sBPT Dpt and Dpt HR r = -0.37, p < 0.05. The results showed a significant correlation between sIgE and Dpt HR, sIgE and sBPT Dpt, sBPT Dpt and Dpt HR and these tests appear to be of value in casual diagnosis of allergic bronchial asthma, but only sBPT makes possible to observe LAR. PMID- 7544186 TI - Plasma galanin concentrations in obese, normal weight and anorectic women. AB - OBJECTIVE: Galanin is believed to play a role in the control of eating behavior. No information is available on its concentrations in the biological fluids in human obesity, and this study aimed to clarify this. MEASUREMENTS: We measured plasma galanin and serum insulin levels in 30 obese, 35 normal weight and 11 anorectic women. RESULTS: Mean galanin values were quite similar in obese and control subjects (76.8 +/- 3.20 vs 76.1 +/- 2.33 pg/ml) and only slightly reduced in anorectic patients (67.9 +/- 2.30 pg/ml). Insulin levels were significantly increased and decreased in obese and anorectic patients, respectively, compared to controls. Insulin correlated positively with BMI in the whole group of subjects studied (r = 0.72, P < 0.0001) and in the obese subgroup (r = 0.56, P < 0.02). No correlations could be detected between WH ratio, insulin and galanin concentrations and between galanin and BMI. CONCLUSIONS: In conclusion, plasma galanin concentrations appear to be comparable in obese, normal weight and anorectic subjects. This does not exclude a role of galanin in the regulation of eating behavior since variations of the peptide in discrete brain areas may not be detectable in general circulation and peripheral sources of the peptide may contribute to its plasma levels. Also, our data suggest that galanin does not play a major role in the regulation of insulin secretion in humans. PMID- 7544188 TI - "Rebound" phenomenon of hepatitis C viremia after interferon therapy in "relapsed" patients with chronic hepatitis C. AB - The correlation between serum hepatitis C virus (HCV)-RNA and aminotransferase levels following completion of interferon therapy was evaluated in eight relapsed patients with chronic hepatitis C. Both HCV-RNA and aminotransferase levels were significantly increased in the relapsed patients 1 month after completion of therapy, compared to pretreatment values, despite aminotransferase levels being normal and HCV-RNA being undetectable by reverse transcription polymerase chain reaction assay at the end of therapy. The serum levels of HCV-RNA and aminotransferase were significantly elevated 1 and 2 months post-treatment. They then decreased to pretreatment values 3-5 months after the completion of therapy. Thus, in relapsed patients after the completion of therapy, the changes in HCV RNA levels preceded the elevation in aminotransferase levels. These findings suggest a correlation between serum HCV-RNA levels and aminotransferase levels in relapsed patients with chronic hepatitis C after the completion of interferon therapy. PMID- 7544187 TI - Methylphenidate treatment of poststroke depression in a patient with global aphasia. AB - A 76-year-old male with poststroke depression, seizure disorder, and global aphasia improved markedly when treated with methylphenidate. The use of psychostimulants in this population as well as their effect on seizure threshold is discussed. PMID- 7544189 TI - Hepatocyte regeneration in chronic hepatitis C and interferon treatment: analysis of immunohistological identification of proliferating cell nuclear antigen (PCNA). AB - To evaluate the usefulness of proliferating cell nuclear antigen (PCNA) immunostaining in the assessment of the efficacy of interferon (IFN) therapy in chronic hepatitis C, we investigated the proliferative activity of hepatocytes in 67 patients with chronic hepatitis C, using this immunostaining method. The percentage of PCNA-positive hepatocytes was 2.4% in patients with chronic persistent hepatitis, 2.5% in those with chronic aggressive hepatitis 2A, and 3.9% in those with chronic aggressive hepatitis 2B. The PCNA count increased with the progression of the liver disease. Patients were classified as complete, partial, and non-responders to IFN; the percentage of PCNA-positive hepatocytes before IFN therapy was 1.6% in the complete responders, 3.9% in the partial responders, and 4.9% in the non-responders. There was a significant negative correlation between the percentage of PCNA-positive hepatocytes and the response to IFN treatment. Thirty-two of 53 cases (60.4%) in which the PCNA labeling index (LI) was less than 5.0 were complete responders compared with 13 of 14 cases (92.9%) in which the PCNA LI was higher than 5.0, representing partial responders or non-responders (P < 0.001). Most complete responders had a low PCNA LI, irrespective of HCV genotype. Our findings indicate that PCNA immunostaining is a simple and reliable index of cell proliferation in liver regeneration, and may be a useful predictor of the response to IFN treatment in chronic hepatitis C. PMID- 7544184 TI - [Spontaneous histamine release by basophils and bronchial hyperreactivity in patients with hay fever]. AB - Basophil spontaneous histamine release test was performed in seventeen patients suffered from seasonal allergic rhinitis before, 2 weeks after immunotherapy and during pollen season. During pollen season at the same day non-specific bronchial hyperreactivity to histamine was estimated. Values of spontaneous basophil histamine release before immunotherapy were not different from control group but significantly increased after immunotherapy and during pollen season. There was no correlation between degree of nonspecific bronchial hyperreactivity and values of spontaneous histamine release from basophils. PMID- 7544190 TI - alpha-Phenyl N-tert-butyl nitrone (PBN) increases the cortical cerebral blood flow by inhibiting the breakdown of nitric oxide in anesthetized rats. AB - The effects of intravenous administration of alpha-phenyl N-tert-butyl nitrone (PBN) on cortical cerebral blood flow (CBF) were examined in Wistar rats under pentobarbital anesthesia and artificial ventilation. The cortical CBF in parietal cortex was measured by laser Doppler flowmetry. Intravenous administrations of 2 mg/kg and 20 mg/kg of PBN dose-dependently produced significant increases in cortical CBF and decreases in systemic blood pressure (BP). To examine whether these increased responses in cortical CBF produced by PBN were associated with the vasodilatation system of nitric oxide (NO), the NO synthase inhibitor L-NG nitroarginine (L-NOArg), which is an analog of L-arginine, was used to inhibit the NO-related-vasodilatative system. Since the PBN-induced responses in the cortical CBF were much attenuated in L-NOArg-treated rats (30 mg/kg, i.v.), it was inferred that NO-related vasodilatation was strongly associated with the PBN induced increase in cortical CBF. PMID- 7544191 TI - Preparation of cDNA-silica using reverse transcriptase and its DNA sequence determination. AB - A new method for producing macroporous silica (suitable for high-performance liquid chromatography) with covalently attached DNA is presented. The method uses (dT)18-silica as a primer, annealed to a poly(A)-RNA template, which is then transcribed using reverse transcriptase. The RNA template is eluted and single stranded cDNA-silica is recovered. The cDNA-silica can be sequenced using the dideoxy method. These methods provide a facile method for producing cDNA-silica of demonstrable authenticity and provide a unique approach to DNA and RNA sequencing. PMID- 7544192 TI - All the colors of the mango. PMID- 7544194 TI - A plethora of cardiac chloride conductances: molecular diversity or a related gene family. AB - Recent electrophysiologic studies have provided evidence suggesting that as many as six different Cl- conductances can be identified in the sarcolemma of cardiac myocytes isolated from various animal species and areas of the heart. These include Cl- conductances activated by stimulation of protein kinase A, protein kinase C, extracellular ATP, intracellular Ca2+, membrane stretch, and a basally active Cl- conductance. Many basic biophysical and pharmacological properties of these channels are presently unknown, and the only molecular information presently available suggests that the cAMP-activated Cl- conductance is due to cardiac expression of an isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel normally found in epithelial cells. We used the polymerase chain reaction (PCR) to amplify four distinct regions corresponding to the cardiac CFTR gene product from several cardiac tissues to determine if the molecular distribution of CFTR matches the distribution of cAMP-dependent Cl- channels in native myocytes. Amplification of regions corresponding to the first nucleotide binding domain (NBD1), transmembrane segments (TS) VII-XII, and the regulatory (R) domain showed a precise correlation to tissues that electrophysiologically exhibit sarcolemmal cAMP-dependent Cl- channels, whereas region TS I-VI exhibited a distribution independent of the presence of cAMP dependent Cl- channels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544193 TI - Reentry: insights from theoretical simulations in a fixed pathway. AB - This review article summarizes theoretical insights into the principles and mechanisms associated with reentrant activity in cardiac tissue. A mathematical ring model is used in computer simulations to investigate, at the cellular level, mechanistic aspects of initiation, perpetuation, and termination of reentry. Taking advantage of the ability to compute membrane processes in this model, we relate dynamic properties of the reentrant action potential (e.g., beat-to-beat alternans) to the underlying kinetics of membrane ionic channels. Effects on reentry of inhomogeneities in refractoriness, excitability, cellular coupling at gap junctions, and fiber cross-section are also studied. PMID- 7544195 TI - Involvement of tachykinins in plasma extravasation induced by bradykinin and low pH medium in the guinea-pig conjunctiva. AB - 1. The effect of bradykinin, capsaicin, substance P and low pH medium on plasma extravasation in the guinea-pig conjunctiva has been studied. Evans blue dye was measured in the conjunctiva after local instillation of the agents into the conjunctival sac. 2. Bradykinin (2-50 nmol), capsaicin (20-50 nmol) and substance P (0.5-5 nmol) caused a dose-dependent increase in plasma extravasation with the following order of potency: substance P > bradykinin = capsaicin. The effect of capsaicin (50 nmol) and substance P (5 nmol) was abolished by the tachykinin NK1 receptor antagonist, CP-99,994 (8 mumol kg-1, i.v.) (P < 0.01), whereas CP 100,263 (8 mumol kg-1, i.v.) the inactive enantiomer of CP-99,994 was without effect. CP-99,994 inhibited by 70% (P < 0.01) the effect of bradykinin. 3. The kinin B2 receptor antagonist, Hoe 140 (icatibant, 10 nmol kg-1, i.v.) abolished the response to bradykinin (50 nmol) (P < 0.01), but did not affect the responses to capsaicin (50 nmol) or substance P (5 nmol). Plasma extravasation induced by low pH medium (pH 1) was abolished by CP-99,994 (P < 0.01) and by Hoe 140 (P < 0.01). 4. The present findings suggest that: endogenous or exogenous tachykinins increase plasma extravasation in the guinea-pig conjunctiva by activation of NK1 receptors; bradykinin-induced plasma extravasation is mediated by tachykinin release from sensory nerve endings; low pH media cause plasma extravasation via release of kinins that by activation of B2 receptors release tachykinins from sensory nerve endings. PMID- 7544196 TI - Inhibition of rat colon contractility by prostacyclin (IP-) receptor agonists: involvement of NANC neurotransmission. AB - 1. The possibility that prostacyclin (IP-) receptor agonists inhibit spontaneous contractions of the rat isolated colon by activating enteric neurones has been investigated. Cicaprost was used as the test agonist because of its high stability, selectivity and potency (IC50 = 3.8 nM). 2. The Na+ channel blockers saxitoxin (STX, 1 nM) and tetrodotoxin (TTX, 1 microM), whilst having little effect on resting spontaneous activity, virtually abolished the inhibitory actions of cicaprost (10 nM) and nicotine (3 microM); inhibitory responses to isoprenaline (20 nM) were not affected. Phentolamine (1 microM), propranolol (1 microM) and atropine (1 microM) had no effect on cicaprost inhibition. These data are compatible with release of inhibitory NANC transmitter(s) by cicaprost. 3. A transmitter role for nitric oxide was investigated. The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM) inhibited the actions of both cicaprost (10 nM) and nicotine (3 microM) by 50 60%, but did not affect responses to isoprenaline (20 nM) or sodium nitroprusside (1-5 microM). The enantiomeric D-NAME (100 microM), which has negligible NOS inhibitory activity, had no effect on the action of cicaprost. 4. The involvement of purinergic transmitters was also investigated. Desensitization to the inhibitory action of ATP did not affect cicaprost responses. The P2x/P2y-receptor antagonist, suramin, at 300 microM blocked ATP responses, but not those due to adenosine; it did not affect cicaprost inhibition. The selective adenosine A1 receptor antagonist, DPCPX, used at a sufficiently high concentration (5 microM) to block adenosine A2-receptors, did not affect cicaprost inhibition. Apamin (25 nM), a blocker of calcium activated K+ channels on smooth muscle, abolished or markedly reduced the inhibitory actions of ATP and adenosine, and partially inhibited cicaprost and nicotine responses. The combination of L-NAME(100 microM) and apamin (25 nM) abolished cicaprost and nicotine responses.5. Investigation of vasoactive intestinal peptide (VIP) as a potential transmitter showed that its inhibitory action on the colon (IC50 = 50 nM) was partially inhibited by TTX (1 microM). alpha-Chymotrypsin abolished the effect of VIP but had no effect on cicaprost inhibition. Attempts to inhibit VIP responses using peptide antagonists and by agonist desensitization were unsuccessful.6. KCI (40 mM) contracted the colon and abolished spontaneous activity. Under these conditions,isoprenaline, sodium nitroprusside and ATP induced relaxation, whereas cicaprost (10-3 10 nM) had no effect. Cicaprost inhibited both the tone and the spontaneous activity induced by the EP1/EP3-receptor agonist, sulprostone (8.6 nM) but not when either TTX (1 microM) or KC1 (40 mM) was also present. On KCl-treated preparations, the prostacyclin analogue, iloprost (10-500 nM), induced contraction,presumably due to activation of EP-receptors.7. It is concluded that IP-receptor agonists inhibit the contractility of rat colon by stimulating the release of at least two transmitters from NANC enteric neurones. Nitric oxide appears to be one of the transmitters. The second transmitter mechanism is apamin-sensitive; the experimental results do not support ATP, adenosine or VIP as transmitter candidates. However, further studies using more potent and selective receptor antagonists are required. PMID- 7544197 TI - Subtypes of tachykinin receptors on tonic and phasic neurones in coeliac ganglion of the guinea-pig. AB - 1. Intracellular recording techniques were used to investigate the characteristics of tachykinin receptors and their subtypes in tonic and phasic neurones, which constituted two major neuronal populations in the coeliac ganglion of the guinea-pig. 2. In 95% of phasic neurones a long-lasting after hyperpolarization (LAH), 5-8 s in duration and 10-20 mV in amplitude, was observed following action potentials evoked by passing a train of depolarizing current pulses into the neurones. In contrast, LAH was observed in only 4% of tonic neurones. 3. In most tonic neurones, substance P (SP), neurokinin A (NKA) and senktide induced depolarizations, whereas in phasic neurones they usually inhibited LAH but rarely induced depolarization. 4. Tonic and phasic neurones were further classified into three groups based on their responses (depolarization for tonic neurones and LAH inhibition for phasic neurones) to these tachykinin receptor agonists: (1) neurones responsive to SP, NKA and senktide (71-78%); (2) those responsive to senktide but not to SP and NKA (12 23%) and (3) those not responsive to any of the three agonists (7-11%). 5. GR71251 (5 microM), an NK1-selective tachykinin receptor antagonist, depressed the depolarization in tonic neurones and the LAH inhibition in phasic neurones induced by SP and NKA, but not those induced by senktide. 6. Selective NK2 receptor agonists, [Nle10]NKA4-10, [beta-Ala8]NKA4-10 and GR64349, were without effect in both tonic and phasic neurones. Furthermore, an NK2 receptor antagonist, L659,877, did not inhibit the depolarization induced by NKA, SP or senktide in tonic neurones. 7. It is suggested that NK1 and NK3 receptors are present on a large proportion of coeliac ganglion neurones. In tonic neurones both subtypes of tachykinin receptors are coupled to membrane depolarization,whereas in phasic neurones activation of these receptors leads to inhibition of LAH. The present study also suggests that NKA evokes the depolarization in tonic neurones and the LAH inhibition in phasic neurones via NK1, but not NK2 receptors. PMID- 7544199 TI - Ion selectivity of the channels formed by pardaxin, an ionophore, in bilayer membranes. AB - Using the method of bilayer membranes at the tip of a patch pipette, the properties of the ionic channels produced by the ionophore toxin pardaxin were investigated. At low toxin concentrations, voltage-dependent, single-channel events were measured. The current-voltage curves were non-linear when determined in Tris-Cl solution, but were linear in K(+)-HEPES solution. Using asymmetric ion solutions, the ionic selectivity of pardaxin channels was estimated from the reversal potentials obtained. The sequence of the relative permeabilities for monovalent cations was Tl+ > Rb+ > Cs+ > K+,NH4+ > methylamine+ > Li+ > dimethylamine+ > Na+. Except for Li+, the selectivity sequence fitted the cations relative hydrated size. For bivalent ions the permeability of Ba2+, Sr2+, and Mn2+ relative to Mg2+ changed according to the relative hydrated size. For anions the selectivity sequence was I- > NO3- > Br- > Cl- > ClO4- > SCN- > BF- > HCOO- > F- > CH3COO-. The selectivity sequence for the small anions (I-, NO3-, Br-, Cl-) was different from their hydrated size. Pardaxin channel showed a modest ion selectivity between small anions and cations (PK:PCl:PNa = 1.28:1.00:0.56). Pardaxin is proposed as a biophysical model to study ionic channel selectivity. PMID- 7544198 TI - The anti-emetic effects of CP-99,994 in the ferret and the dog: role of the NK1 receptor. AB - 1. The selective NK1 receptor antagonist, CP-99,994, produced dose-related (0.1 1.0 mg kg-1, s.c.) inhibition of vomiting and retching in ferrets challenged with central (loperamide and apomorphine), peripheral (CuSO4) and mixed central and peripheral (ipecac, cisplatin) emetic stimuli. 2. Parallel studies with the enantiomer, CP-100,263 (1 mg kg-1, s.c.), which is > 1,000 fold less potent as a NK1 antagonist, indicated that it was without significant effect against CuSO4, loperamide, cisplatin and apomorphine-induced emesis. Against ipecac, it inhibited both retching and vomiting, expressing approximately 1/10th the potency of CP-99,994. 3. The 5-HT3 receptor antagonist, tropisetron (1 mg kg-1, s.c.) inhibited retching and vomiting to cisplatin and ipecac, but not CuSO4 or loperamide. 4. CP-99,994 (1 mg kg-1, i.v.) blocked retching induced by electrical stimulation of the ventral abdominal vagus without affecting the cardiovascular response, the apnoeic response to central vagal stimulation or the guarding and hypertensive response to stimulation of the greater splanchnic nerves. CP-99,994 (1 mg kg-1, i.v.) did not alter baseline cardiovascular and respiratory parameters and it failed to block the characteristic heart rate, blood pressure and respiratory rate/depth changes in response to i.v. 2-methyl-5-HT challenge (von Bezold-Jarisch reflex). 5. Using in vitro autoradiography, [3H]-substance P was shown to bind to several regions of the ferret brainstem with the density of binding in the nucleus tractus solitarius being much greater than in the area postrema. This binding was displaced by CP-99,994 in a concentration-related manner. 6. In dogs, CP-99,994 (40 micrograms kg-1 bolus and 300 micrograms kg-1 h 1, i.v.) produced statistically significant reductions in vomiting to CuSO4 and apomorphine as well as retching to CuSO4. 7. Together, these studies support the hypothesis that the NK1 receptor antagonist properties of CP-99,994 are responsible for its broad spectrum anti-emetic effects. They also suggest that CP 99,994 acts within the brainstem, most probably within the nucleus tractus solitarius although the involvement of the area postrema could not be excluded. PMID- 7544200 TI - Retrons: retroelements of no known function. PMID- 7544201 TI - Palliation of obstructive nephropathy due to malignancy. AB - OBJECTIVE: To conduct a prospective study of patients with malignant obstructive uropathy treated actively by percutaneous nephrostomy and J-J ureteric stents. PATIENTS AND METHODS: Forty-two patients (27 men, 15 women, median age 62 years, range 29-83) with obstructive nephropathy secondary to malignancy underwent urinary diversion followed, where appropriate, by active treatment of the underlying cancer. RESULTS: The median survival of all patients was 133 (range 7 712) days. Seventeen patients (40%) survived for > 6 months and five (12%) for < 1 month. Patients who had received no prior therapy and for whom further therapeutic options were available were more likely to benefit from urinary diversion. In nine patients (21%) nephrostomy insertion failed to relieve renal failure. In 20 patients (48%) obstructive nephropathy recurred. The procedure was complicated by urinary tract or nephrostomy site infection in 16 patients, by septicaemia in six patients, by percutaneous urine leak in 13 patients and by pelvi-calyceal perforation in two patients, but not by haemorrhage or death. The median percentage of the patients' remaining life which was spent in hospital was 23.6% (range 2.2-100). CONCLUSIONS: Patients likely to benefit from nephrostomy were those for whom there were therapeutic options available for the treatment of their malignancy. Prolonged survival is possible in obstructive nephropathy secondary to malignancy, which should no longer be cited as an absolute contra indication to urinary diversion. Patients unlikely to benefit from urinary diversion can also be identified and they should not routinely undergo this intervention. PMID- 7544202 TI - Prognostic value of prostate-specific antigen minimum after orchidectomy in patients with stage C and D prostatic carcinoma. AB - OBJECTIVE: To determine the significance of the minima of prostate-specific antigen (PSA) levels after orchidectomy in patients with advanced carcinoma of the prostate (stage C and D disease), to thereby discriminate patients who have a statistically significant difference in prognosis and thus individualize their adjuvant treatment. PATIENTS AND METHODS: A retrospective analysis of 50 patients (mean age 75.8 years) with extracapsular prostate cancer (58% stage C, 42% stage D disease) was performed. PSA measurement and clinical assessment were continued at 3-monthly intervals until there was evidence of clinical progression. The minimum values of PSA were determined, the patients grouped at intervals of 1 ng/mL and the groups compared statistically on the basis of disease progression. RESULTS: Serum PSA levels decreased in all patients to a minimum after 3-6 months. There was a statistically significant difference in the probability of, and time to, disease progression for PSA minima at 1 ng/mL and 10 ng/mL. Of the 13 patients in group 1 (with PSA minima < 1 ng/mL), 11 stayed in remission during a mean follow-up duration of 45.9 months. Of 25 patients in group 2 (with PSA minima between 1 and 10 ng/mL) 19 developed progression after a mean remission period of 16.7 months, while all 12 patients in group 3 (with PSA minima > 10 ng/mL) progressed after a mean remission period of 12.5 months. CONCLUSION: Post orchidectomy PSA minima are an excellent prognostic factor with significant predictive value. While patients in group 1 tend to have a favourable prognosis after orchidectomy alone, group 2 and 3 patients need adjuvant treatment as early as possible. Group 3 patients, with their high probability of early progression, also form a uniform cohort for studies concerning new therapeutic modalities. If the PSA minimum was included in further examinations, the results of treatment should improve. PMID- 7544203 TI - Evaluation of patients with diseases of the prostate using prostate-specific antigen density. AB - OBJECTIVE: To compare the efficacy of two tests, prostatic-specific antigen (PSA) and the PSA/prostate volume ratio (PSAD), as diagnostic and staging markers to discriminate patients with benign prostatic hyperplasia (BPH) from patients with cancer of the prostate (CaP). PATIENTS AND METHODS: Prostate gland volumes were estimated in 60 patients with BPH and 88 patients with clinically organ-confined CaP by performing transrectal ultrasonography (TRUS) and using the prolate ellipse formula. Serum PSA concentration was determined using an enzyme immunoassay. In patients with BPH, the prostates were removed either by transurethral resection or retropubic prostatectomy. Patients with CaP underwent laparoscopic pelvic lymphadenectomy followed by radical perineal prostatectomy. PSAD was calculated by relating the serum PSA level to the TRUS-estimated prostate volume. RESULTS: The median PSA level was 4.4 ng/mL in patients with BPH, 9.3 ng/mL in patients with CaP-NO disease and 24 ng/mL in those with CaP-N+ disease. However, imposing a PSA limit of 4 ng/mL for the diagnosis of CaP gave a positive predictive value of only 64.8%, whereas a limit of 10 ng/mL gave a positive predictive value of 71.4%. In contrast, the median PSAD was 0.086 ng/mL/cm3 in patients with BPH, but was 0.295 ng/mL/cm3 in patients with NO disease and 0.775 ng/mL/cm3 in those with N(+)-disease. With a limit of 0.15 ng/mL/cm3 the positive predictive value of PSAD was 81%. Furthermore, a limit of 0.6 ng/mL/cm3 revealed a positive predictive value of 81% for the diagnosis of metastatic lymph node involvement. CONCLUSIONS: There was a considerable overlap of PSA concentrations in patients with BPH and CaP, and PSA was not sufficiently accurate to distinguish between them. In contrast, PSAD enhanced the discrimination between BPH and CaP and may provide additional information about the status of the lymph nodes in patients with CaP. PMID- 7544204 TI - Prostate-specific antigen density: correlation with histological diagnosis of prostate cancer, benign prostatic hyperplasia and prostatitis. AB - OBJECTIVE: To assess the additional value of prostate-specific antigen density in the diagnosis of prostate cancer in patients who undergo prostate biopsies. PATIENTS AND METHODS: The study comprised 376 patients with symptoms of prostatism who were undergoing prostate biopsy. Digital rectal examination (DRE) and transrectal ultrasonography (TRUS) were performed and the prostate specific antigen level (PSA) and density (PSAD) were determined for each patient. RESULTS: Both PSA and PSAD significantly differentiated (P < 0.001) between benign and malignant histology. Of the 376 patients, 91 (24%) had a PSA level in the intermediate range (4.0-10.0 ng/mL). In these patients PSAD was significantly better than PSA in differentiating between benign and malignant histology (P = 0.027 vs 0.316). With a PSAD limit of 0.15 ng/mL/cm3 in these patients, the sensitivity was 92% and the specificity was 54% for the diagnosis of prostate cancer. No patient with a positive biopsy had a PSAD < 0.11 ng/mL/cm3. No limiting value could be found for PSAD that combined both an acceptable sensitivity and specificity. Of the patients with a malignancy detected by the biopsy, 92% also had a suspect DRE. CONCLUSION: In patients with intermediate PSA levels, PSAD is of limited additional value when compared to DRE in correctly diagnosing prostate cancer. Acute prostatitis is also a possible cause of elevated PSA. Both PSA and PSAD had no additional value in differentiating between benign prostatic hyperplasia (BPH) and histologically proven extensive prostatitis. PMID- 7544205 TI - The diagnostic accuracy of digital rectal examination, transrectal ultrasonography, prostate-specific antigen (PSA) and PSA density in prostate carcinoma. AB - OBJECTIVE: To evaluate the efficacy of digital rectal examination (DRE), transrectal ultrasonography (TRUS), prostate-specific antigen (PSA) and PSA density (PSAD) in the diagnosis of prostate cancer (CaP). PATIENTS AND METHODS: Retrospective data were analysed from a selected population of 159 patients (mean age 66.7 years, range 50-83), 56 with histologically diagnosed CaP and 103 with benign prostatic hyperplasia (BPH). RESULTS: Among the four methods, DRE was found to have accuracy, sensitivity and specificity rates of 79.9, 91 and 73.8% respectively. The most common clinical practice, the combination of PSA (> 4 ng/mL), DRE and TRUS, showed a higher accuracy, a similar specificity rate, but a lower sensitivity (84.2, 91.2 and 71.4%, respectively). Rates from the combination of PSAD (> 0.15 ng/mL/cm3) with DRE and TRUS were not significantly different from those obtained using the combination of PSA, DRE and TRUS. CONCLUSION: PSAD alone or in combination did not improve the diagnostic value of PSA. We cannot claim DRE was the best method for the diagnosis of CaP, because this study group did not represent a true screening population. However, this study revealed that DRE should not be omitted from the physical examination of patients and, despite technological developments, it remains a major tool in the diagnosis of CaP. PMID- 7544207 TI - Microwave hyperthermia in benign prostatic hypertrophy: a controlled clinical trial. AB - OBJECTIVE: To compare the effects of microwave hyperthermia in benign prostatic hypertrophy (BPH) to sham treatment. PATIENTS AND METHODS: The trial included 96 patients with proven symptomatic bladder outflow obstruction (BOO) caused by BPH. Patients underwent a full subjective and objective assessment (including urodynamics) before inclusion and again 3 and 6 months later. They were randomly assigned to receive 1 h of microwave hyperthermia or a sham treatment. Of the 96 patients, 93 were assessed at 3 months and 62 at 6 months after treatment. RESULTS: There was no statistically significant difference in the objective measures of BOO between the treated and control groups of patients. There was an improvement of approximately 40% in all the subjective measures, but there was no significant difference between the treated and control groups. CONCLUSION: Microwave hyperthermia, within the parameters defined in this trial, resulted in no significant difference from sham treatment in subjective or objective outcome. PMID- 7544206 TI - Acid mucin and high molecular weight cytokeratin in prostatic lesions: evaluation of a combined histochemical and immunohistochemical stain. AB - OBJECTIVE: To evaluate the presence of acid mucins and high molecular weight cytokeratin (KER) in prostatic lesions using a combined histochemical and immunohistochemical stain consisting of Alcian blue at pH 2.5(AB) with a strept avidin-biotin complex (SAB) staining for KER (SAB-KER). MATERIALS AND METHODS: Sections were obtained from archival paraffin blocks which included 20 cases of prostatic carcinoma, 30 cases of benign hyperplasia, and five cases of basal cell hyperplasia. Sections were stained for mucosubstances using the AB stain, for KER using SAB-KER and by both AB and SAB-KER, the combined stain (CS). RESULTS: With the CS stain KER, which is present in the prostatic basal cells, was not detected in malignant glands and in 60% of these cases intraluminal blue-stained acidic mucin was seen. On the other hand, all benign hyperplastic prostatic glands were devoid of intraluminal acidic mucin and showed staining for KER of their basal cells. Areas of basal cell hyperplasia were strongly positive for KER and intraluminal acidic mucin was seen in one case. Each of the stains separately gave similar results to the CS method but the contrast between the areas of carcinoma and hyperplasia was accentuated by the CS, and small foci of carcinoma were easily detected. CONCLUSION: The combined AB+SAB-KER stain is quicker to perform and allows the simultaneous appraisal of acid mucin and KER. PMID- 7544209 TI - Epidemiology of benign prostatic hyperplasia. PMID- 7544212 TI - Evaluating symptoms and functional status in benign prostate hyperplasia. PMID- 7544211 TI - Diagnosing benign prostatic hyperplasia versus prostate cancer. PMID- 7544213 TI - Benign prostatic hyperplasia: treatment guidelines and patient classification. PMID- 7544214 TI - Alpha-adrenergic blocking drugs in bladder outflow obstruction: what potential has alpha 1-adrenoceptor selectivity? PMID- 7544208 TI - Shared care for benign prostatic hyperplasia: a feasibility study. AB - OBJECTIVE: To determine the proportion of patients with symptoms of urinary outflow obstruction secondary to benign prostatic hyperplasia (BPH) who could be managed in the community after assessment in a shared-care clinic (managed by nursing staff, supervised by a consultant) to which they had direct access. PATIENTS AND METHODS: A total of 127 men were referred to one consultant urologist in a 9-month period for assessment of possible urinary outflow obstruction. All were investigated using urine analysis, serum prostate-specific antigen level, urea and electrolytes, plain abdominal X-ray, renal ultrasonography and urinary flow rate. Additional investigations were undertaken as required. The proportion of men who could have been investigated in a shared care clinic and then managed in the community was determined. RESULTS: Of 127 men, 88 (69%) were found to have uncomplicated outflow obstruction secondary to BPH; of these 49 (38%) could have been managed in the community after assessment in the shared-care clinic and a further 27 (21%) could have been managed in the community after additional investigation by a specialist. Twelve men (9%) were found to have uncomplicated outflow obstruction and chose to undergo transurethral resection of the prostate. CONCLUSION: Many men with uncomplicated outflow obstruction could be assessed in a shared-care clinic and then managed in the community. A shared-care protocol for the management of these men has now been introduced in this unit. PMID- 7544210 TI - Objective evaluation of bladder outlet obstruction. PMID- 7544215 TI - Prostatic growth: new insights into hormonal regulation. PMID- 7544216 TI - Alpha 1-adrenoceptor selectivity: clinical or theoretical benefit? PMID- 7544217 TI - Efficacy and safety of tamsulosin in the treatment of benign prostatic hyperplasia. PMID- 7544218 TI - Clinical research criteria: response criteria for evaluating efficacy of treatments for benign prostatic hypertrophy. PMID- 7544221 TI - Segment III cholangiojejunostomy for palliation of malignant hilar obstruction. PMID- 7544220 TI - Expression of tenascin in gastric carcinoma. AB - Tenascin expression was determined by an immunohistochemical technique in 120 surgical specimens of gastric carcinoma to investigate its relationship with clinicopathological factors. Tenascin expression was more prominent in the neoplastic area than in the adjacent non-neoplastic mucosa. Tenascin was frequently observed in gastric mucosa with diffuse chronic gastritis, glandular atrophy and intestinal metaplasia. In the neoplastic area, tenascin expression was positive in 72 cases (60 per cent). Tumours with a high frequency of tenascin expression included: Borrmann type II (19 of 20), well or moderately differentiated tumours (52 of 63), tumours with expansive growth and with an intermediate growth pattern (40 of 42), and those with a medullary or intermediate-type stroma (55 of 73). There was no significant relationship between tenascin expression and age, sex, depth of tumour invasion, lymph node metastasis, invasion to lymphatic vessel, venous invasion and the 4-year survival rate. PMID- 7544219 TI - Effect of postoperative insulin-like growth factor I supplementation on protein metabolism in humans. AB - Insulin-like growth factor I (IGF-I) has been shown experimentally to exert a nitrogen-sparing effect in both animals and humans. Its effects on protein metabolism during the first 5 days after radical large bowel resection were investigated. Nineteen patients were randomly allocated to receive either human recombinant IGF-I (2 x 80 micrograms/kg body weight subcutaneously, n = 10) or placebo (n = 9) starting on the morning of the first day after operation. All patients received parenteral nutrition (glucose 3 g and nitrogen 0.1 g/kg/day). The mean(s.e.m.) urinary nitrogen:creatinine ratio was significantly reduced in patients who received IGF-I compared with those given placebo (275.0(17.1) compared with 386.3(23.6), P < 0.01). The mean urinary 3 methylhistidine:creatinine ratio and nitrogen balance were lower in the IGF-I group, but not significantly so. Plasma IGF-I concentrations were four times higher in the treated group compared with those in the placebo group. Insulin like growth factor binding protein 3 (IGFBP-3) increased significantly after the start of IGF-I treatment, compared with mean postoperative levels. The mean blood glucose concentration was significantly lower in the IGF-I group than the placebo group (P < 0.05) but no patient had a hypoglycaemic attack. The authors conclude that IGF-I does not significantly influence the nitrogen balance, but that results indicate a possible nitrogen-sparing effect in patients after major abdominal operations. IGF-I therapy is safe, and may be of value in catabolic patients after serious injury and major operations. PMID- 7544222 TI - Excitatory amino acid receptor ligands: asymmetric synthesis, absolute stereochemistry and pharmacology of (R)- and (S)-homoibotenic acid. AB - The (R)- and (S)-forms of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) were synthesized, using (S)-BOC-phenylalanine as a chiral auxiliary and their absolute stereochemistry correlated with that of (R) Br-HIBO. The enantiomeric excesses for (R)-HIBO (1) (> 99.5%) and (S)-HIBO (2) (99.5%) were determined using chiral HPLC. Whereas compounds 1 and 2 were equipotent inhibitors of the binding of [3H]glutamic acid in the presence of calcium chloride, 2 showed AMPA agonist activity and 1 very weak NMDA agonist activity. PMID- 7544223 TI - Inhibition of nitric oxide synthase dramatically potentiates seizures induced by kainic acid and pilocarpine in rats. AB - We investigated whether the severity of convulsions evoked by kainic acid and pilocarpine is modified in nitric oxide synthase inhibitor-treated rats. We found that chronic treatment (4 days) with NW-nitro-L-arginine greatly potentiates seizures induced by both convulsants suggesting a potential role for nitric oxide in mechanisms regulating seizure induction and propagation. PMID- 7544225 TI - Humoral responses to linear epitopes on the HIV-1 envelope in seropositive volunteers after vaccine therapy with rgp160. AB - Humoral responses to the HIV-1 envelope were investigated in 30 human volunteers enrolled in a phase I vaccine therapy trial of rgp160 (LAI/LAV) using two techniques that emphasize detection of antibody response against linear (continuous) epitopes: immunoblotting and PEPSCAN. Seven fusion proteins containing large portions from constant regions 1, 2, 3, and 5, and variable region 3 of gp120 and two regions in the transmembrane protein, gp41, were employed in immunoblots to quantitatively measure immune response as a function of immunization. In addition, the entire gp160 (LAI/LAV) envelope protein was constructed in duplicate sets of 211 overlapping 12-mer peptides to fine-map the changes. Immunoblotting defined significant changes in reactivity to epitopes in constant regions; of 28 volunteers completing the trial, the percentage with reactivity against C1 changed from 62 to 100%; for C2, from 0 to 46%; for C3, from 0 to 82%; and for a constant region in gp41, from 25 to 68%. PEPSCAN on a subset (n = 8) of these volunteers identified new reactivity to epitopes throughout the envelope, concentrated in V1, C3, and C5 in gp120 and several peptides in gp41. Completely immunized patients responded to double the number of linear epitopes compared with two patients receiving alum alone. The results verify that the response to rgp160 is significantly broadened after immunization, providing additional evidence that HIV-1-infected volunteers can expand their antibody repertoire against a protein from a pathogen during chronic infection with that same pathogen. These results expand those previously obtained in this patient cohort, by defining explicitly the immunogenic regions recognized postvaccination and by providing methodology for quantitating those changes. PMID- 7544227 TI - Standardization of allergenic extracts by basophil histamine release. AB - BACKGROUND: Allergenic extracts are standardized by using the ID50EAL (Intradermal Dilution for 50 mm sum of Erythema Determines the Allergy Unit) skin test technique to assign allergy units to reference preparations. When new batches of extracts are manufactured, they are compared with the reference by RAST inhibition or other in vitro techniques. This study was designed to determine whether basophil histamine release might be an additional useful in vitro test for the standardization of allergenic extracts. METHODS: Basophil histamine release, skin tests, and RAST were compared using several different allergenic extracts at many different strengths. Allergy units were calculated using skin test results and basophil histamine release. RESULTS: Basophil histamine release, skin tests, and RAST correlated well (P < .01). Basophil histamine release-derived allergy units and skin test-derived allergy units were highly correlated (P < .01). CONCLUSION: Basophil histamine release appears to be a useful tool for the standardization of allergenic extracts. PMID- 7544224 TI - Effects of excitatory amino acid lesions upon neurokinin B and acetylcholine neurons in the nucleus basalis of the rat. AB - The nucleus basalis magnocellularis (NBM) contains cholinergic neurons that project to the neocortex and is densely innervated by excitatory amino acid containing terminals. A dysfunction in the balance of excitatory inputs or an alteration in the sensitivity of NBM cells to glutamate may underlie the selective vulnerability to aging. Some large NBM neurons contain neurokinin B (NKB) mRNA. The present study investigated whether alpha-2-amino-3-hydroxy-5 methyl-isoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) differentially destroy NKB-containing, NKB-receptive, or cholinergic NBM cells, and whether this vulnerability is altered by aging. Injections of AMPA or NMDA significantly decreased neocortical ChAT activity, as compared to control levels, across all three age groups, with no interaction between lesion and age group. The results of in situ hybridization histochemistry and NKB receptor studies suggest that NKB-containing neurons in the NBM, and the neurons they innervate, are not vulnerable to NMDA or AMPA in either young or old rats. While NKB mRNA positive cells were diffusely distributed throughout the basal forebrain, only a small proportion of the large NBM cells contained NKB mRNA. The results suggest that NKB does not extensively colocalize with acetylcholine within the basal forebrain of rats and that NBM NKB neurons do not directly innervate cholinergic cells. PMID- 7544226 TI - Anti-beta 2-glycoprotein I antibodies. PMID- 7544230 TI - Human type I interferon receptor, IFNAR, is a heavily glycosylated 120-130 kD membrane protein. AB - Type I interferons (IFNs) bind and signal through cell surface receptors that share at least one common component. One candidate for such a component is the interferon-alpha receptor (IFNAR). Genetic studies have shown that the IFNAR gene product is required for response to many type I interferons. However, these studies also suggest that the IFNAR protein interacts with an additional receptor component(s) to form functionally complete type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors, little biochemical characterization of IFNAR and its function has been reported. To facilitate biochemical studies of the IFNAR gene product, a monoclonal antibody, GB8, recognizing the extracellular domain of IFNAR was prepared. The epitope for GB8 maps to the second extracellular domain of IFNAR between amino acids 278 and 293. GB8 identifies IFNAR in western blots of cell membranes as a broad band with molecular mass ranging from 100 to 150 kD in membranes from CHO cells overexpressing the human IFNAR gene to 136-150 kD in Daudi cell membranes. Such variations in the mean value and the range of molecular mass between IFNAR in different cell lines suggest differences in glycosylation. The majority of glycosylation is N-linked, although there may also be a small amount O-linked oligosaccharide. Deglycosylation of IFNAR in Daudi cell membranes results in a 70 kD IFNAR species, indicating that nearly half of the apparent molecular mass of Daudi cell IFNAR is contributed by carbohydrate moieties. PMID- 7544229 TI - Up-regulation of MHC class I by flavivirus-induced peptide translocation into the endoplasmic reticulum. AB - Flavivirus infection of mammalian cells increases the cell surface expression of major histocompatibility complex (MHC) class I molecules, the recognition elements for cytotoxic T cells. Here, we show that the mechanism for flavivirus induced up-regulation of class I MHC involves an increase in peptide supply to the endoplasmic reticulum. Flavivirus-mediated peptide supply for MHC class I assembly is independent of the peptide transporters for class I antigen presentation, since infection of class I MHC peptide transport-deficient cell lines with flaviviruses results in the cell surface expression of biologically functional class I MHC peptide complexes. The flavivirus-induced supply of antigenic peptides to the endoplasmic reticulum is not restricted to flavivirus encoded peptides and independent of interferon. The data imply that peptide availability regulates surface expression of class I MHC restriction elements and suggests a mechanism for flavivirus-induced immunopathology. PMID- 7544228 TI - Effect of procaterol, a beta 2-adrenoceptor agonist, on skin whealing response caused by inflammatory mediators in asthmatic children. AB - BACKGROUND: Beta 2-adrenoceptor agonists have been shown to reduce allergen induced skin whealing responses via inhibition of mediator release. OBJECTIVE: To study whether beta 2-adrenoceptor agonists have a direct action against inflammatory mediator-induced skin whealing responses. METHODS: We examined the effect of procaterol, a beta 2-adrenoceptor agonist, on skin whealing responses to histamine, platelet-activating factor (PAF), substance P, or bradykinin in eight asthmatic children in a double-blind, randomized, cross-over study. Two hours after taking procaterol (50 micrograms) or placebo orally, the subjects were given these mediators intradermally at a concentration of 10(-5) M. RESULTS: Procaterol has a small but significant inhibitory effect on wheal formation following the intradermal injection of histamine and PAF by an average of 15% (P < .05) and 18% (P < .05) respectively but not against substance P or bradykinin. CONCLUSIONS: Our results suggest that although beta 2-adrenoceptor agonists may have an inhibitory effect against plasma exudation from microvasculature in the human skin, bronchodilatory effects are more prominent. PMID- 7544231 TI - Hemin inhibits the interferon-beta-induced antiviral state in established cell lines. AB - Hemin and other metalloporphyrins are known as very versatile compounds in nature, because they are able to carry out numerous functions in a free state or in association with specific proteins. When Friend murine erythroleukemia cells are treated with IFN-beta plus 100 microM hemin, the antiviral state is not observed, whereas the antiviral effect of IFN-gamma is unaffected by hemin treatment. This inhibitory effect of hemin is not restricted to erythroid cells. In fact, it is also observed in murine L929 and in human cell lines treated with IFN-beta. Neither trivalent iron in other forms nor hemin analogs (such as protoporphyrin IX or Sn(2+)-protoporphyrine IX) mimic this effect. Conversely, Co(3+)-protoporphyrin IX was as effective as hemin. At the transcriptional level, results obtained by run-on assays on nuclei from IFN-treated cells indicate that hemin does not completely inhibit IFN-beta induction of 2-5A synthetase gene(s) at 6 h of treatment but abolishes it at 24 h. In addition, hemin is able to inhibit the accumulation of IFN-induced 2-5A synthetase mRNAs. Experiments carried out to investigate the hemin effect on the early steps of the IFN signaling pathway indicate that hemin interferes with the ability of type I IFN to bind to its receptor, probably by a direct action on the IFN molecule. PMID- 7544232 TI - Combined transarterial targeting locoregional immunotherapy-chemotherapy for patients with unresectable hepatocellular carcinoma: a new alternative for an old problem. AB - The prognosis for patients with advanced (stage III and IV) hepatocellular carcinoma (HCC) remains poor. Liver resection and liver transplantation have limited effects on overall survival. Our study was carried out to assess a novel therapeutic approach, which includes transarterial locoregional chemotherapy and in vivo locoregional dual immunostimulation, in patients with unresectable HCC. A group of 20 patients with stage III and IV hepatocellular carcinoma had 10 courses (once per day) of transarterial targeted locoregional immunotherapy with interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), emulsified in a Lipiodol Urografin mixture. The target organs were the spleen and the liver tumor itself. One course of intrahepatic locoregional targeting transarterial chemotherapy was given 10 days after completion of immunotherapy (mitomycin C, carboplatin, Farmorubicin, Leucovorin, 5-fluorouracil, and IFN-gamma). This was followed after 2 months by another course of transarterial targeted locoregional immunotherapy chemotherapy. All patients survived the operation and had a mean survival time of 18 months (4-22 months). There was a decrease in the tumor size of 14 of the 20 patients. Serum alpha-fetoprotein (AFP) levels declined in 14 patients, reaching normal levels in 12 patients. These preliminary results indicate that combined locoregional immunotherapy-chemotherapy is a promising therapeutic approach in patients suffering from advanced nonresectable HCC and merits further evaluation. PMID- 7544234 TI - Effect of rhinovirus 39 (RV-39) infection on immune and inflammatory parameters in allergic and non-allergic subjects. AB - The economic impact and medical complication rate of the common cold are well documented, but many of the physiological, inflammatory, and immune responses to common cold viruses have only recently been investigated. The purpose of this study was to compare selected systemic immune and inflammatory responses to experimental rhinovirus (RV)-39 challenge in seronegative allergic rhinitis and non-allergic rhinitis subjects. Peripheral blood was obtained before (baseline), during (acute), and 23 days after (convalescent) RV-39 intranasal challenge and assayed for leucocyte histamine release, serum immunoglobulins, allergen-specific IgE antibodies, plasma histamine, and platelet aggregation. All subjects were infected, as manifested by viral shedding in nasal secretions or seroconversion. RV-39 infection induced significant acute increases in serum IgE, leucocyte histamine release, and platelet aggregation, but caused no changes in serum IgG, serum IgA, serum IgM, and plasma histamine. The first change was confined to the allergic rhinitis subjects. There was no evidence that the acute rise in total serum IgE was due to an elevation of a pre-existing, pollen-specific serum IgE antibody. The results show that intranasal challenge with RV-39 induced changes in systemic immune and inflammatory parameters with a unique response pattern in allergic rhinitis subjects. PMID- 7544235 TI - Nizatidine inhibits reactions in skin-prick tests. PMID- 7544233 TI - Characterization of IgE-binding epitopes on Candida albicans enolase. AB - Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-I-360), E (16.2 kDa; F-171-D-317) and F (13.0 kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein. PMID- 7544236 TI - Endothelin-1 expression in blood vessels of DOCA-salt hypertensive rats treated with the combined ETA/ETB endothelin receptor antagonist bosentan. AB - In previous studies it has been shown that blood vessels of deoxycorticosterone acetate (DOCA) salt hypertensive rats present significantly higher immunoreactive ET-1 (ir-ET-1) content and increased ET-1 gene expression. DOCA-salt hypertensive rats respond to treatment with the combined ETA/ETB endothelin receptor antagonist bosentan with lowering of blood pressure. In the present study, we investigated the ir-ET-1 levels and the expression of the ET-1 gene in blood vessels of DOCA-salt hypertensive rats treated or not treated with bosentan. Blood pressure was significantly lower in bosentan-treated rats (185 +/- 6 mmHg; 1 mmHg = 133.3 Pa) compared with DOCA-salt hypertensive rats (203 +/- 4 mmHg; p < 0.01). Plasma ir-ET-1 concentration was slightly but significantly elevated (p < 0.01) in DOCA-salt hypertensive rats compared with uninephrectomized control rats, and was further increased (p < 0.01) in bosentan-treated rats. The tissue wet weight and ir-ET-1 content of segments of thoracic aorta were significantly increased (p < 0.01) in DOCA-salt hypertensive rats in comparison with control rats, but were similar in bosentan-treated DOCA-salt rats. The abundance of ET-1 mRNA measured by Northern blot analysis in thoracic aorta and the ir-ET-1 content were attenuated by bosentan treatment. Tissue wet weight and ir-ET-1 content in the mesenteric vascular bed were similar in bosentan-treated and -untreated DOCA salt rats, and were significantly higher in both groups than in control rats (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544237 TI - The complete 1H NMR assignments of aminoglycoside antibiotics and conformational studies of butirosin A through the use of 2D NMR spectroscopy. AB - The complete proton assignments of the aminoglycoside antibiotics, butirosin A, kanamycin A and kanamycin B, at pH 6.5 have been made through the use of various homonuclear and heteronuclear 2D NMR methods. Butirosin A NOESY experiments suggest a stacking arrangement between the xylose and 2,6-diamino-2,6 dideoxyglucose rings, while the 2-deoxystreptamine ring and its substituent, the (S)-4-amino-2-hydroxybutyryl group, extend away from the stacked rings. Informative long-range NOEs were observed for butirosin A but not with kanamycin A or kanamycin B. Many intra-ring NOEs were observed with all three aminoglycosides that confirm the proton assignments made in this study. PMID- 7544240 TI - Charge dissociation and the rising phase of gating currents. AB - Gating currents from voltage-sensitive channels are generally attributed to the translocation or redistribution of ionic charge associated with the channel molecule. Such charge moves in the direction of the applied field to produce a decreasing current in the external circuit. An early rising phase for the gating current is observed for a number of channel systems and might be either some special kinetic redistribution of charge or an experimental artifact. A model that produces net charge in the channel through sequential molecular dissociation of a charged channel segment gives a rising phase for the gating current. Translocation of the charged segment produces the decay phase for a biphasic gating current. This kinetic molecular explanation constitutes a physical explanation for the biphasic gating currents that is consistent with present views of channel structure. PMID- 7544238 TI - NMR reinvestigation of two N-acetylneuraminic acid-containing O-specific polysaccharides (O56 and O24) of Escherichia coli. AB - Structures for the N-acetylneuraminic acid (Neu5Ac)-containing O56 and O24 polysaccharides of Escherichia coli have been reported previously. During these studies unusual chemical shifts had been observed for the NMR signals for H-3eq and C-3 of the Neu5Ac residues of both polysaccharides. In further pursuing this phenomenon, we have reinvestigated the O56 and O24 polysaccharides as well as derived oligosaccharides by one- and two-dimensional NMR spectroscopy. The results showed that structures of both polysaccharides (PSs) had to be modified and formulated as [formula: see text] 2D ROESY spectra revealed a strong NOE between H-3eq of Neu5Ac and the protons of the side-chain sugar (H-3 and H-5 of alpha-D-Gal p in the O56 PS and H-3 of alpha-D-Glc p in the O24 PS) and also between H-3ax of Neu5Ac and H-3 of beta-D-Glc p in the main chain. This indicated a close spatial association of the seven-linked alpha-Neu5Ac and the side-chain residues alpha-D-Gal p (O56 PS) and alpha-D-Glc p (O25 PS), respectively. The strong long-range spatial contacts caused the unusual chemical shifts of H-3eq and C-3 of Neu5Ac. PMID- 7544239 TI - TIPS for portal decompression to allow palliative treatment of adenocarcinoma of the esophagus. AB - We describe a case in which a transjugular intrahepatic portosystemic shunt (TIPS) was inserted to enable palliative treatment of an adenocarcinoma of the esophagus. The cirrhotic patient presented with esophageal varices in contact with a tumor of the esophagus. By relieving portal pressure, TIPS reduced the risk of hemorrhage during laser resection and prevented recurrence of esophageal varices. PMID- 7544242 TI - Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina. AB - Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed. PMID- 7544241 TI - Origins of nerve fibers containing nitric oxide synthase in the rat celiac superior mesenteric ganglion. AB - The origin of nitric oxide synthase-containing nerve fibers in rat celiac superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the celiac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons. PMID- 7544244 TI - Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response. AB - TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production. PMID- 7544243 TI - [Preliminary observation on the destructive effect of chemical disinfectants in serum markers of hepatitis B and C]. AB - Chloroform, 40% formaldehyde, 3% hydrogen peroxide solution and 75% alcohol were used to act on hepatitis B surface antigen (HBeAg), hepatitis B e antigen (HBaAg) and anti-hepatitis C virus antibody (Anti-HCV) positive serum for 5, 15, 30 and over 60 minutes. They were examined by enzyme-linked immunosorbent assay and observed for the effect. The destructive effect of chloroform on HBsAg and anti HCV positive derum was marked. It was discovered that 40% formaldehyde solution destroyed HBsAg, HBeAg, and anti-HCV positive serm, while 75% alcohol also showed destructive effect on HBsAg, HBeAg and anti-HCV positive serum. PMID- 7544245 TI - Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor V beta 2 gene usage in microscopic polyarteritis. AB - Antigen recognition by T lymphocytes is mediated by cell surface receptors T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the alpha and beta polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the beta chain (V beta) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR V beta genes. To determine if the dominant usage of a V beta gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific V beta gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (D beta) J beta region. A significant increase in the mean percentage expression of the V beta 2.1 gene was seen in vasculitis patients (11.4 + 1.0% (mean + s.e.m.)) compared with controls (6.6 + 0.6%; P < 0.003). The most marked increase was seen in microscopic polyarteritis (13.9 + 1.7%; P < 0.0001). There were also increases in the expression of V beta 3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of V beta 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR V beta 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR V beta 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding V beta 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis. PMID- 7544246 TI - Autoantibodies to malondialdehyde-modified epitope in connective tissue diseases and vasculitides. AB - Malondialdehyde (MDA), a peroxidative end-product released during polyunsaturated fatty acid degradation, reacts strongly with lysine residues of cellular proteins. MDA-modified proteins become immunogenic and may elicit specific autoantibody formation. We hypothesized that systemic diseases in which inflammatory events occur, could be an interesting model for studying oxidative stress. A few studies have suggested that MDA-modified proteins may exist in systemic diseases, and that autoantibodies to MDA-modified structures might reflect this oxidative process. Autoantibodies to MDA-modified epitope(s) were therefore assayed in sera of patients with systemic lupus erythematosus (SLE, n = 29), scleroderma (SCL, n = 11), giant cell arteritis (GCA, n = 11), periarteritis nodosa (PAN, n = 10), rheumatoid arthritis (RA, n = 9), and healthy subjects (HS, n = 32). Significantly increased anti-MDA-modified epitope(s) autoantibodies were found in patients with SLE and also in other systemic diseases such as PAN and SCL. Autoantibodies to MDA-modified epitope(s) were predominantly of IgM isotype, with low levels of IgG and no IgA activity. In SLE, anti-MDA-modified epitope(s) autoantibody titres correlated strongly with systemic lupus activity measure (SLAM, r = 0.702, P = 0.0001), anti-nuclear antigen autoantibodies (ANA, r = 0.4, P = 0.029), IgG anti-cardiolipin (r = 0.558, P = 0.03) and the steroid drug regimen (r = 0.52, P = 0.004). Autoantibodies to MDA-modified epitope(s) may reflect oxidative modifications occurring in systemic diseases, and might be useful as clinical markers of SLE activity if further investigated. PMID- 7544247 TI - IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp). AB - CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant. PMID- 7544248 TI - A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil based adjuvants. AB - TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2 type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL 2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer. PMID- 7544249 TI - Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C. AB - The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays. PMID- 7544250 TI - Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1. AB - Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1. PMID- 7544251 TI - Cell-mediated immune responses of lambs to challenge with bovine respiratory syncytial virus. AB - The lamb is a good model to study the pathogenesis and immune responses to infections with respiratory syncytial virus (RSV) as lambs experimentally infected with bovine or human RSV may develop overt clinical disease. In the present study the development of cellular cytotoxic responses was studied in splenic, pulmonary and peripheral blood mononuclear cells obtained from lambs after primary and secondary infection with bovine RSV. Infection with bovine RSV was followed by the appearance of cytotoxic cells in the peripheral blood, the spleen and lung lavage fluids. These effector cells lysed virus-infected targets in a self-restricted manner. Depletion techniques revealed that cytotoxic activity was largely due to OvCD8+ cells. When effector cells obtained from primed lambs were stimulated with inactivated bovine RSV or with virus-infected cells in vitro, virus-specific cytotoxicity was significantly increased. PMID- 7544254 TI - Monoclonal antibodies specific to the glutamic acid decarboxylase 65 kDa isoform derived from a non-obese diabetic (NOD) mouse. AB - Two monoclonal antibodies specifically recognizing the 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD) were generated by fusion of spleen cells of a non-obese diabetic (NOD) mouse which had received a single intraperitoneal injection of 0.2 ml complete Freund's adjuvant followed three days later by one administration of a subdiabetogenic dose of streptozotocin (80 mg/kg body weight) three days before the fusion experiment was performed. Both monoclonals belong to the IgG1 isotype and were screened with an enzyme-linked immunosorbent assay using rat brain extract as a natural source of GAD and additionally with a capture assay by means of immunoglobulins of a patient with Stiff-man syndrome. The specific binding to the 65 kDa isoform of the enzyme was detected by a radioligand and an enzyme-linked immunosorbent assay using recombinant human glutamic acid decarboxylase specific for both the 67 and 65 kDa isoforms. Both monoclonal antibodies recognize the same antigenic epitope, which is located in the N-terminal region of the first 17 amino acids detected by fragments of human pancreatic 65 kDa GAD. Three out of 30 sera from Type 1 diabetic patients specifically displaced the binding of the monoclonals from 125I-labelled GAD65 measured by radio-immunoassay. A striking binding of both monoclonals M61/8F9 and M61/7E11 to the islets of cryosections of human, monkey, pig and rat pancreas but not to mouse pancreas was detectable. The antibodies failed to bind on the cell surface of viable rat islet cells. It is concluded that also in the diabetes prone NOD mice GAD65 autoantibodies occur although GAD65 was not detectable in the mouse islets. PMID- 7544252 TI - CD5 expression on B cells may be an activation marker for secretion of anti myelin antibodies in patients with polyneuropathy associated with monoclonal gammopathy. AB - B cells expressing the CD5 marker belong to a subpopulation with potential autoreactive properties. Increased proportions of CD5+ B cells have been reported in autoimmune diseases. In patients with monoclonal gammopathy and demyelinating polyneuropathy, the M-component often consists of autoantibodies reacting with myelin components. We therefore investigated if CD5+ B cells were involved in the production of anti-myelin antibodies. There was no difference of mean value of CD5+ B cells between patients and controls. However, the proportion of CD5+ B cells was significantly correlated with the amount of anti-myelin antibodies. In seven patients, CD5+ B cells were enriched using an immunomagnetic technique. The number of CD5+ and CD5- B cells secreting anti-myelin antibodies was determined by ELISPOT. In two patients with high levels of antibodies, antibody-secreting cells were mainly, but not exclusively, CD5+ B cells. In five patients with low levels of antibodies, most cells secreting anti-myelin antibodies were CD5-. We conclude that CD5 expressed on B cells may be an activation marker, reflecting B cells producing high amounts of anti-myelin antibodies in patients with polyneuropathy associated with monoclonal gammopathy. PMID- 7544256 TI - [Response of ovarian endodermal sinus tumor to combination chemotherapy with cis platinum, vincristine and bleomycin or vincristine, actinomycin and cyclophosphamide]. AB - The treatment results of 63 cases of ovarian endodermal sinus tumor admitted to Peking Union Medical College Hospital from August 1977 through April 1992 by combination chemotherapy were reported. Patients were divided into three groups. The first group received full courses of vincristine, actinomycin and cyclophosphamide (VAC) or cis-platinum, vincristine and bleomycin (PVB) chemotherapy in adequate dosages (37 cases). The second group also received VAC or PVB chemotherapy but in inadequate dosages (17 cases). The third group took no VAC or PVB chemotherapy but some other chemotherapeutic drugs (9 cases). 28 of the 63 patients died. The 35 surviving patients have been followed for 1 to 15 years. The sustained remission rates were 81.1%, 23.5% and 11.1% for the first, second and third group respectively. As the ovarian endodermal sinus tumor is very sensitive to VAC or PVB chemotherapy an extensive or aggressive cytoreductive surgery and retroperitoneal lymphadenectomy seemed to be not necessary. Such a point of view is demonstrated by our preliminary study. The treatment results for the primary cases are significantly better than that of the recurrent cases. The sustained remission rate is 94.7% for the former while that for the latter is 66.7%. PMID- 7544255 TI - Treatment of invasive squamous cell carcinoma complicating anal Buschke Lowenstein tumor: a case history. AB - We present here the case of a patient with Buschke-Lowenstein tumor of the anus complicated by invasive squamous cell carcinoma metastatic to the inguinal and periaortic pelvic lymph nodes. Successful local palliation was achieved with 5 fluorouracil and mitomycin-C-based chemotherapy combined with radiation therapy, but systemic progression was unaffected. PMID- 7544257 TI - [Histogenesis of pleomorphic adenoma of the salivary gland]. AB - Six parotid pleomorphic adenomas were studied histochemically by electron microscopy after staining with ruthenium red. It was found that the myxoid region was filled with ruthenium red granules. Similar positive ruthenium red-stained intracellular granules were observed within the Golgi-derived vacuoles of the neoplastic myoepithelial cells in the myxoid region. Some cells located at the periphery of the double-layer ductal structure had the features of myoepithelial cell, they also showed similar positive ruthenium red-stained granules in the intracellular vacuoles and intercellular space. No ruthenium red-stained granules was obtained in neoplastic ductal epithelium and the mucus in ductal cavities were negative for ruthenium red stain. The observations indicate that the neoplastic myoepithelial cells in the myxoid region probably derive from the periphery of the double-layer ductal structure, they secrete the proteoglycans forming the myxoid region and play an important role in the histogenesis of pleomorphic adenoma. PMID- 7544258 TI - [An approach to determining the effect on salivary amylase by green tea extract]. AB - The inhibition effect of three Chinese tea and a kind of acid tea without fluoride to human salivary amylase were studied. The inhibited ability of polyphenols and fluoride were further studied, simultaneously, the tea: saliva ratio were also tested in vitro. The results indicated that the inhibition effect was in this order: acid tea, Ulong tea, longjing, huacha. The polyphenols play important role in the inhibition of salivary amylase, the optimal ratio between tea and saliva was 0.6:1. PMID- 7544260 TI - The toxicity of zinc to terrestrial isopods in a "standard" laboratory test. AB - A method is described for assessing the effects of metals on the food consumption rate of isopods from measurements of fecal production. The effects of zinc in the diets of two isopod species, Porcellio scaber and Oniscus asellus, were tested. The metal was fed to the isopods on leaves of field maple (Acer campestre) contaminated with concentrations ranging between 1000 and 10,000 micrograms Zn g 1 leaf dry wt. Significant reductions in feeding rates were observed of the highest concentrations of zinc. The test employed in this study is quick, cheap, and relevant for estimating sublethal effects of metals on isopods. PMID- 7544253 TI - Production of immunoglobulins by human sIgD+ and sIgD- human blood B lymphocytes in response to stimulation with activated T cells and agonistic antibodies; effect of IL-10, IL-2 and mode of activation of T cells. AB - Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed. PMID- 7544259 TI - Fractionated irradiation alters enteric neuroendocrine products. AB - Radiation profoundly alters the contractile activity of the small intestine and colon. We hypothesized that some motor changes of the gut might be secondary to impaired neural input to smooth muscle or abnormal release of gut endocrine peptides. The density of products within peptidergic and cholinergic nerves and gut endocrine cells was estimated in six normal controls and six dogs who had received 1500 cGy in six equal fractions of 250 cGy. Choline acetyltransferase, acetylcholinesterase, vasoactive intestinal peptide (VIP), substance P, peptide YY (PYY), and motilin were measured in tissue specimens divided into mucosal submucosal (MS) and muscularis externa (ME) layers. Tissue samples were obtained from the duodenum, jejunum, ileum, and proximal and distal colon. In addition, serum levels of motilin and PYY were determined before and during the administration of 1500 cGy in four separate dogs instrumented to record upper gut contractile activity. Intrinsic cholinergic activity as estimated by choline acetyltransferase activity was unchanged, while acetylcholinesterase activity increased in the MS layers of distal small bowel and colon. VIP was increased in the MS layers of jejunum and proximal colon as well as in the ME layers the jejunum and ileum. By contrast, substance P increased in the jejunal and proximal colonic MS layers and in the ME layers of the jejunum and ileum. Duodenal and jejunal motilin levels markedly decreased after radiation exposure, while serum motilin levels continued to cycle at a decreased peak level with migrating motor complexes. Colonic PYY remained unchanged but serum PYY levels decreased after irradiation. Increased neuronal synthesis and inhibition of neurotransmitter release are potential explanations for elevated tissue concentrations of VIP, substance P, and acetylcholinesterase. There appeared to be differences in the sensitivity of gut endocrine cells to irradiation. Changes in gut regulatory peptides and cholinergic enzyme activity occur with fractionated doses of abdominal irradiation, while the same schedule of irradiation produces striking changes in the canine small intestinal and colonic motor activity. It is therefore likely that alterations of contractile events may be produced by changes in gut neuroendocrine products. PMID- 7544261 TI - The number of components in a mixture determines whether synergistic and antagonistic or additive toxicity predominate: the funnel hypothesis. AB - A new hypothesis, the funnel hypothesis, was derived to explain the variation in toxicity of equitoxic multicomponent mixtures of nonspecific toxicants (narcotics). The variation is explained in terms of the volumes associated with solute-solute and solute-solvent interactions. The hypothesis predicts that as the number of components in a mixture increases, the range of deviation from toxic additivity decreases. It also predicts that the toxicity of mixtures measured using biological endpoints that require high toxicant concentrations will deviate more from toxic additivity than endpoints that require low concentrations. Aquatic toxicity data for equitoxic mixtures of narcotic toxicants to a range of aquatic organisms compiled from the literature support the predictions of the funnel hypothesis. PMID- 7544262 TI - The effects of cadmium on the successional stages of a freshwater protozoa community. AB - Microcosms containing protozoan were used to observe the differential effects of cadmium on the successional stages of a community. After 7, 14, and 21 days, 500 micrograms/liter cadmium was added. Some environmental parameters such as pH, conductivity, and nutrient levels affect metal toxicity. The effect of cadmium on diversity, density, and biomass was evident 48 hr after the addition of the metal. The pollutant caused a transition period in each treatment, of a different duration, followed by a recovery of the community. In both the control and the treatments, trophic structure was defined by bacterivore-detritivore, nonselective, and photoautotroph organisms. PMID- 7544263 TI - Applicability of CoMFA in ecotoxicology: a critical study on chlorophenols. AB - Comparative molecular field analysis (CoMFA) is used to relate ecotoxicological data with steric and electrostatic fields of chemicals forming an intentionally selected homogeneous set, the chlorophenols, for which reliable data are abundant in literature. Among these data, those concerning 16 different biological systems were selected, leading to predictive CoMFA QSAR in 14 of the cases. This is attested by cross-validation and bootstrapping, which also authorize the prediction of the chlorophenols toxicity values, when they are missing. The quality of obtained CoMFA models and the applicability of the method are discussed. The results are very promising, and they encourage further investigation into CoMFA in ecotoxicology. PMID- 7544264 TI - Biochemical responses and environmental contaminants in breams (Abramis brama L.) caught in the river Elbe. AB - Adult breams (Abrama brama L.) were caught in October 1992 at seven stations in the river Elbe and at one nonpolluted reference site, the Belauer See. The locations of the sampling stations extended from the city of Steti (Tschechien Republic) to the city of Hamburg. Indices of biochemical effects in microsomal and cytosolic fractions of livers were studied by measuring cytochrome P450 dependent monooxygenases and glutathione-S-transferase (GST) activities. In addition, levels of mercury and 35 polychlorinated biphenyl (PCB) congeners were analyzed in livers of breams. Fish caught in the River Elbe exhibited a significant increase of cytochrome P450-mediated monooxygenase activities and the detoxifications enzyme GST compared to the reference site. At two stations of the river Elbe (Steti and Dresden) elevated activities of ethoxyresorufin-O deethylase (EROD) were analyzed. These effects were discussed as effects from the pulp mill industries at station Steti and high concentrations of PCBs in the livers of breams at station Dresden. A significant reduction of GST activities was observed at station Dresden compared to those at Steti. These findings were probably a synergistic effect of high mercury concentrations at Dresden. The results presented in this study suggest that breams can be successfully employed for monitoring biological effects in the river Elbe. PMID- 7544265 TI - Preliminary investigations into the bioaccumulation of mercury by the liverwort Scapania undulata L. (Dum). AB - Studies were made of the aquatic liverwort Scapania undulata (L.) Dum from streams in the Polish Sudeten Mountains. It was found that this species growing in water with mercury concentrations below the detection limit (10(-3) micrograms/liter) contains from 0.025 to 4.7 mg Hg/kg dry wt. According to the bioassay the lethal concentration of Hg2+ (24-hr LC100) for S. undulata appeared to be 500 or 1000 micrograms/liter, depending on the population the liverworts were chosen from. Exposure to 16 micrograms Hg2+/liter for 14 days may be recognized as harmless for S. undulata. After completion of the experiment S. undulata, treated with 64-512 micrograms Hg2+/liter, accumulated respectively 46 2800 times more mercury than the blank. PMID- 7544267 TI - Effect of cadmium chloride on physiological color changes of the fiddler crab, Uca pugilator. AB - The effects of cadmium exposure on the color change system of the fiddler crab, Uca pugilator, were determined. Crabs were either immersed in 10 ppm cadmium chloride solutions or injected with 8.5 micrograms cadmium/g body weight. The crabs exposed to cadmium, either by injection or immersion, were less able to bring about dispersion of the pigment in their integumentary melanophores than were the unexposed crabs. Cadmium did not affect the melanophores directly. This decreased black pigment dispersion was apparently due to effects of cadmium on the neuroendocrine processes that control the melanophores. The neuroendocrine complex in the eyestalk is the source of a black pigment-dispersing hormone (BPDH). Eyestalks of crabs kept in clean water contained 3.27 times more BPDH than did the eyestalks of the cadmium-exposed crabs. Histological studies revealed that cadmium exposure results in depletion of the neurosecretory material in the eyestalks and brain. It is hypothesized that cadmium inhibits BPDH synthesis in neurosecretory (neuroendocrine) cells, resulting in decreased ability of exposed crabs to disperse their black pigment. PMID- 7544266 TI - Histopathological changes induced by chronic nonlethal levels of elsan, mercury, and ammonia in the small intestine of Channa punctatus (Bloch). AB - Histopathological changes in the intestine of Channa punctatus induced by chronic nonlethal levels of Elsan (211 ppb), mercury (16.7 ppb), and ammonia (15.64 ppm) were studied at 7-day intervals for 90 days and the data were presented only for days (7, 28, 63, and 90) when the most conspicuous changes were noted after treatment. In the earlier phases of Elsan treatment (7 and 28 days) overall destruction of the structure of villus and other layers was prominent. Histopathology of the intestine of C. punctatus, after 63-day Elsan exposure, could be described as collapsed villi with the tips merged with each other to give a flattened appearance. Ninety-day Elsan exposure demonstrated severe damage in the longitudinal muscle layer. After 7-day mercury treatment a high degree of necrosis was indicated by submucosal area, whereas 28-day mercury treatment revealed collapsed villi due to necrotic mucosal cells and goblet cells. Mercury treatment for 63 days caused disarray of all the layers, but some improvement of villus organization was noted in fish treated with mercury for 90 days. Seven-day exposure to ammonia deteriorated the normal structure of the villus, whereas in 28-day ammonia exposure, lesions were predominant in the submucosal layers. Sixty three-day ammonia treatment demonstrated an effect similar to that produced by Elsan, associated with a more complete destruction of all the layers. Fishes under ammonia treatment for 90 days demonstrated extensive damage to the mucosal folds. The major changes in thickness of different layers of the intestine were also evaluated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544269 TI - Naphthalene-induced atresia in the ovary of the crayfish, Procambarus clarkii. AB - Female crayfish, Procambarus clarkii, were exposed to 10 ppm naphthalene to determine its effect on ovarian development. Ovaries were removed for examination after 1, 2, 4, 6, 8, 10, 12, and 15 days of exposure to the naphthalene. Crayfish that had been exposed to naphthalene for 15 days were then returned to clean freshwater for 1, 2, 4, 5, 7, 10, and 15 days to determine whether the ovaries would recover from the naphthalene exposure. The ovarian index, which reflects the weight of the ovary, decreased gradually over the 15-day naphthalene exposure period. The previtellogenic and vitellogenic oocytes degenerated. Intense atresia along with yolk resorption led to this decrease in the ovarian index. In contrast, the ovaries of the control crayfish developed into ripe gonads. However, immature oocytes reappeared in the ovaries of the crayfish subjected to depuration, which suggests that the ovary of P. clarkii can recover from naphthalene-induced atresia. PMID- 7544271 TI - Insulin-like growth factor II (IGF-II) is more potent than IGF-I in stimulating cortisol secretion from cultured bovine adrenocortical cells: interaction with the IGF-I receptor and IGF-binding proteins. AB - Although the stimulating effect of insulin-like growth factor I (IGF-I) on adrenal steroidogenesis has been well established, the role of IGF-II in the adult adrenal gland remains unknown. We, therefore, investigated the effect of recombinant human IGF-II on cortisol and cAMP synthesis from adult bovine adrenocortical cells. IGF-II, time and dose dependently, stimulated basal cortisol secretion maximally 3-fold. In combination with ACTH, IGF-II (13 nM) synergistically increased cortisol secretion from 1-fold (10(-8) M ACTH) to 28 fold of untreated control levels. In contrast, IGF-I at equimolar concentrations did not show an effect on basal cortisol secretion, and in combination with ACTH elicited a significant weaker stimulatory effect than IGF-II (22-fold increase). The synergistic effect of IGF-II on ACTH-promoted cortisol secretion was paralleled by accumulation of cAMP in the culture medium. Although both IGF receptors are present in adult bovine adrenocortical cells, the effect of IGF-II seems to be mediated through interaction with the IGF-I receptor, as [Arg54,55]IGF-II, which only binds to the IGF-I receptor, was equipotent to native IGF-II, whereas [Leu27]IGF-II, which preferentially binds to the type II IGF receptor, did not show any effect. By Western ligand blotting, four different molecular forms of IGF-binding proteins (IGFBPs) were identified in conditioned medium of bovine adrenocortical cells with apparent molecular masses of 39-44, 34, 29, and 24 kilodaltons. ACTH treatment increased the abundance of all binding proteins, on the average, 2.3-fold, except for the 29-kDa band, which was predominantly induced 6.8-fold. Additionally, [des1-3]IGF-I, a truncated IGF variant that exhibits only minimal binding to IGFBPs, was significant more potent than IGF-I and elicited the same maximum stimulatory effect on cortisol secretion as IGF-II and [des1-6]IGF-II. In conclusion, these results demonstrate that 1) IGF-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I; 2) this effect is mediated through interaction of IGF-II with the IGF-I receptor; 3) bovine adrenocortical cells synthesize various IGFBPs that are induced differentially by ACTH; and 4) IGFBPs apparently play a modulatory role in IGF-induced stimulation of adrenal steroidogenesis. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the interactions among locally produced IGFs, IGFBPs, and the IGF-I receptor can be evaluated. PMID- 7544270 TI - Relationship of aquatic natural organic material characteristics to the toxicity of selected insecticides. AB - Toxicities of the commercial insecticide formulations of azinphos methyl, chlorpyrifos, fenvalerate, and methyl parathion were evaluated in streamwater samples containing natural organic material (NOM) using a modification of a bacterial bioluminescence assay. Toxicity reduction of azinphos methyl was significantly (P < 0.05) correlated with water sample nonvolatile total solids (NVTS) concentration. Toxicity reductions of fenvalerate and of methyl parathion were significantly correlated to E4/E6 absorbance ratio of NOM, and to the proportion of NOM having molecular weight greater than 3700 Da. Toxicity reductions of fenvalerate and methyl parathion had significant correlations to the concentration of NVTS in water samples. Linear regressions of insecticide EC50 values to these NOM characteristics were used to mathematically model the effect of NOM on insecticide toxicity. Insecticide logKOW values were significantly correlated to the slopes of the EC50-total organic carbon (TOC) concentration regression lines. This confirms that the level of association of some nonionic organic contaminants with NOM is a function of the contaminant's water solubility. No single NOM characteristic was the best predictor of toxicity for all insecticides examined. However, these findings suggest that the influence of NOM on toxicity results primarily from differences in TOC content and abundance of larger molecular size NOM. PMID- 7544268 TI - The effects of a secondary-treated bleached kraft mill effluent on aquatic organisms as assessed by short-term and long-term laboratory tests. AB - The chronic effects of secondary-treated effluent from a bleached kraft mill were assessed by means of long-term and short-term laboratory tests. In the long-term test, the effects of the effluent on the life cycle of fathead minnows (Pimephales promelas) were studied. In this experiment, which began with the egg stage and continued through to sexual maturity and reproduction, the fish were exposed in the laboratory to well water (control) and five concentrations (viz., 1.25, 2.5, 5, 10, or 20%) of effluent for 275 days. The effluent concentrations did not significantly affect the hatching of the eggs, the mortality of the hatched fish, the incidence of visible morphological abnormalities, the mortality and the hatchability of the first generation eggs and larvae, and the weights of minnows at various stages of development. Based on a conservative evaluation of the data, a significant finding of this work was that effluent concentrations > or = 2.5% caused lower egg production as well as changes in the gender balance (i.e., increased numbers of individuals with male secondary sexual characteristics) of the fish. Further work is required to understand the causes and ecological significance of these findings. Two short-term tests, each lasting 7 days, were also run. In one, even 100% effluent did not reduce the survival or growth of minnow larvae, correctly predicting the lack of effluent effects on similar endpoints in the long-term test. In the other short-term test, while the survival of Ceriodaphnia was also unaffected by 100% effluent, their reproductive capacity was reduced, but only at effluent concentrations an order of magnitude greater than those affecting the reproduction of minnows in the long-term test. PMID- 7544273 TI - Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. AB - Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity. PMID- 7544275 TI - Interleukin-1 induces insulin-like growth factor binding protein-3 gene expression and protein production by Leydig cells. AB - Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4 , and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis. PMID- 7544274 TI - Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice. AB - Transgenic mice that constitutively overexpressed rat insulin-like growth factor binding protein-1 (IGFBP-1) were generated to determine the effects of overexpression of IGFBP-1 on growth and development. In offspring of three of the founders that showed high levels of expression, birth weight was significantly reduced to approximately 83-92% of the weight of their nontransgenic littermates. The transgenic mice gained less weight and were approximately 3.5-8 g lighter than nontransgenic littermates at 40 days of age. The difference in body weight between transgenic and wild-type mice was most apparent around the time of weaning when transgenic mice showed a more marked growth deceleration than wild type mice. No significant catch-up growth was apparent over the first 3 months of life. In addition, offspring of all three high-expressing founders demonstrated fasting hyperglycemia. The transgene was highly expressed in the brain, uterus, lung, kidney, and heart, but little expression was detected in the liver. The weight of the brain relative to body weight was significantly reduced in transgenic mice compared with wild-type mice, whereas the relative weight of most other organs was similar to wild-type mice. These data demonstrate that IGFBP-1 may function to inhibit IGF action in vivo and that this inhibition selectively impairs development of organs such as the brain. PMID- 7544276 TI - Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis. AB - We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis. PMID- 7544272 TI - Cell-specific expression of activin and its two binding proteins in the rat decidua: role of alpha 2-macroglobulin and follistatin. AB - Rat decidual tissue is formed by two distinct decidual cell populations located either antimesometrially or mesometrially in the uterus. They differ in morphology, the genes they express, the proteins they secrete, and the role they play during pregnancy. Recently, we have shown that rat decidua expresses follistatin and alpha 2-macroglobulin (alpha 2-MG), two binding proteins to activin. In the present study, we determined whether the decidua of pregnant and pseudopregnant rats also expresses activin, whether activin messenger RNA (mRNA) is confined to a particular cell population, and whether it is regulated by its binding proteins. Decidual and placental tissues were collected at different stages of pseudopregnancy or pregnancy. mRNA expression was examined by in situ hybridization, reverse transcription-polymerase chain reaction, and Northern analysis. Developmental studies revealed that activin A became highly expressed in the antimesometrial decidua only from day 11 at a time when this tissue was undergoing extensive degeneration. Very little activin A mRNA could be detected in the mesometrial decidua. However, late in pregnancy, significant expression of activin A mRNA was detected in the mesometrial decidua undergoing extensive cell death at this stage. Developmental study revealed that activin A mRNA became expressed in the antimesometrial decidua only when follistatin mRNA disappeared from this tissue. Furthermore, mesometrial decidua expressing the most alpha 2-MG mRNA had reduced levels of activin A mRNA. These data suggest that follistatin and alpha 2-MG may, by binding to activin, prevent activin A from stimulating the expression of its own gene. To examine this possibility we first established that the rat decidua expresses activin receptor II at a constant level between days 11 15. Then we examined whether follistatin and alpha 2-MG down-regulate activin expression in a simian virus 40-transformed decidual cell line (GG-AD). These cells express activin A mRNA in abundance, very little follistatin, and no alpha 2-MG. Follistatin and alpha 2-MG caused a dose-related decrease in activin A mRNA levels in these cells. The same inhibitory effect was observed with activin A blocking antibody. In summary, the results of this investigation demonstrate that rat decidual tissue expresses mRNAs for activin A and its two binding proteins; follistatin and alpha 2-MG. The expression of each mRNA is cell specific and developmentally regulated. The finding that both activin-binding proteins and antibody inhibit the expression of activin A in cultured decidual cells suggests that activin regulates its own gene expression in the decidua.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7544282 TI - Conformation of a tetradecapeptide epitope of myelin basic protein. AB - The peptide AcAla-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr, which comprises the first 14 residues of the acetylated N-terminus of myelin basic protein, is an epitopic site for two monoclonal antibodies to the human protein. The conformations of the tetradecapeptide in aqueous solutions were investigated employing high-resolution 1H- and 13C-NMR spectroscopy. Two-dimensional techniques were used to assign the spectra observed from both nuclei. Nuclear Overhauser-effect data, amide proton temperature coefficients, 13C spin-lattice relaxation times, distance geometry calculations and dynamic simulated annealing provided evidence that the solution conformations of the tetradecapeptide included a nascent alpha-helix in the N-terminal segment, and a loop extending from Ser7 to Ser12 that bring His10 and Tyr14 into close proximity. PMID- 7544280 TI - Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate. AB - Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding. PMID- 7544278 TI - CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. AB - We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies. PMID- 7544283 TI - A novel approach to introduce site-directed specific cross-links within RNA protein complexes. Application to the Escherichia coli threonyl-tRNA synthetase/translational operator complex. AB - We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The first step involves site directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3' or 5' phosphate, a 4-[-N-(2 chloroethyl)-N-methylamino]benzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4-dinitro-5-fluorophenylazide) is condensed to the modified nucleotide. This method was used to induce specific cross-links between Escherichia coli threonyl-tRNA synthetase and the leader region of threonyl-tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl-tRNA synthetase can be cross linked to the enzyme, depending on the nucleotide modified. PMID- 7544279 TI - Prevalence of epilepsy and epileptic seizures in 10-year-old children: results from the Metropolitan Atlanta Developmental Disabilities Study. AB - With reported prevalence rates of 4-9 cases per 1,000 children, childhood epilepsy is a major public health concern. Reported prevalence rates vary, mainly because researchers often use different epilepsy definitions. In addition, total prevalence may be underestimated if incomplete case-ascertainment methods are used. We used a multiple-source case-ascertainment method that included obtaining information from electroencephalogram laboratories to estimate the prevalence of epilepsy and to classify seizure types among 10-year-old children. In the metropolitan Atlanta (GA, U.S.A.) area, we found a lifetime prevalence of childhood epilepsy of 6 per 1,000 (95% confidence interval, 5.5-6.5) 10-year-old children. However, using capture-recapture analysis, this prevalence may be as high as 7.7 per 1,000. Proportionately more boys than girls had epilepsy. The prevalence did not vary appreciably by race. Partial seizures, including secondarily generalized seizures, were the most common seizure type (58%). Of the children with epilepsy, 35% had another developmental disability (mental retardation, cerebral palsy, visual impairment, or hearing impairment). An accurate estimate of the public health burden of childhood epilepsy and determination of possible risk factors for idiopathic epilepsy both depend on conducting complete community-based case ascertainment and obtaining detailed clinical data. PMID- 7544281 TI - Chemical structures of the core region of Campylobacter jejuni O:3 lipopolysaccharide and an associated polysaccharide. AB - The complete structure for the core oligosaccharide region of the water-insoluble low-M(r) lipopolysaccharide of Campylobacter jejuni serotype O:3 from phenol/water extraction of bacterial cells was assigned through studies on derivatives of the liberated oligosaccharide. Structure determinations were performed using 1H-NMR and 31P-NMR spectroscopies, methylation analysis supported by fast-atom-bombardment mass spectrometry, and Smith degradation experiments. It was concluded that the complete chains in the core oligosaccharide had the following structure in which a proportion of the terminal residues were phosphorylated: [formula: see text] From a similar series of experiments, it was concluded that an associated polysaccharide, which was isolated from the water phase of the phenol/water extracts, had the following repeating unit in which a proportion of the previously unknown L-glycero-D-ido-heptose (L-alpha-D-ido-Hep) residues were present as 3-hydroxypropanoyl esters, and were not covalently linked to the lipopolysaccharide: [formula see: text] PMID- 7544277 TI - Somatostatin may participate in the antiinflammatory actions of glucocorticoids. AB - Glucocorticoids are potent antiinflammatory agents. They inhibit leukocyte chemotaxis and vascular permeability and generally suppress the expression of many inflammatory mediators. Recent reports suggested that somatostatin (Sms) had significant immunomodulatory properties in vitro and in vivo. In this study we examined the effects of glucocorticoids on immunoreactive somatostatin expression in aseptic inflammatory sites of Sprague-Dawley rats given carrageenin sc. The progress of the inflammatory reaction was studied over a 7-h period with respect to the volume and cellularity of the exudate and the levels of the inflammatory mediators expressed in the inflammatory site, including immunoreactive substance P (sP), corticotropin-releasing hormone (CRH), and tumor necrosis factor-alpha (TNF alpha). Dexamethasone significantly reduced the volume and cellularity of the inflammatory exudates; in parallel, the levels of immunoreactive sP, CRH, and TNF alpha were significantly suppressed by this glucocorticoid. In contrast, immunoreactive Sms was stimulated by dexamethasone in a time-dependent fashion. These findings suggest another mechanism for suppression of the inflammatory reaction by glucocorticoids via stimulation of local Sms expression, which occurs in parallel to the inhibition of the local inflammatory mediators sP, CRH, and TNF alpha. PMID- 7544285 TI - 1H and 15N assignment of NMR spectrum, secondary structure and global folding of the immunophilin-like domain of the 59-kDa FK506-binding protein. AB - FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence specific assignment of the 1H and 15N resonances was achieved using two dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100 K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure. PMID- 7544287 TI - Structural elucidation of the O-antigen lipopolysaccharide from two strains of Plesiomonas shigelloides that share a type-specific antigen with Shigella flexneri 6, and the common group 1 antigen with Shigella flexneri spp and Shigella dysenteriae 1. AB - Sugar and methylation analyses of native polysaccharides together with one dimensional 1H- and 13C-NMR spectroscopy revealed that the two polysaccharides from strains 22074 and 12254 of Plesiomonas shigelloides are identical. The structure of the polysaccharide from strain 22074 was deduced from a uronic acid degradation and by NMR spectroscopy where heteronuclear multiple bond connectivity and two-dimensional nuclear Overhauser effect spectroscopy experiments established the pentasaccharide repeating unit as-->4)-alpha-D-GalpA (1-->3)-alpha-D-GlcpNAc-(1-->3)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha L-Rhap-(1-->. PMID- 7544284 TI - Site-specific and complete enzymic deglycosylation of the native human chorionic gonadotropin alpha-subunit. AB - Numerous studies have shown that glycosylation of the alpha-subunit of human chorionic gonadotropin (alpha hCG) is essential for the biological activity of this hormone. To obtain detailed insight into the function of N-glycosylation, the availability of site-specifically and fully deglycosylated alpha-subunits obtained under non-denaturing conditions is a prerequisite. NMR spectroscopy in combination with FAB-mapping demonstrates that only Asn52 of the alpha-subunit is accessible to digestion by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F under native conditions. Treatment of native alpha hCG with endo-beta-N acetylglucosaminidase B results in full deglycosylation yielding alpha hCG with one GlcNAc residue at both Asn52 and Asn78. PMID- 7544286 TI - Structures of the two polymers present in the lipopolysaccharide of Burkholderia (Pseudomonas) cepacia serogroup O4. AB - Like several other strains of Burkholderia (Pseudomonas) cepacia, the reference strain for serogroup O4 in the French typing scheme [Heidt, A., Monteil, H. & Richard, C. (1983) J. Clin. Microbiol. 18, 738-740] produces a lipopolysaccharide containing two distinct polymers. Attempts to separate the polymers chromatographically were unsuccessful, but the periodate-resistant major polymer could be isolated by application of the Smith degradation technique to the mixture. By means of chemical and NMR spectroscopic analysis, the following structure could be assigned to the repeating unit of the major polymer: -->3) alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->. The following structure of the repeating unit of the minor polymer was established from similar studies of its degradation product, resulting from the oxidation of L-rhamnose (Rha), and of the original mixture: -->3)-alpha-D-GalpNAc-(1-->3)-beta-D-GalpNAc (1-->4)-alpha-L-Rhap- (1-->. Individually, the polymers have recently been found in related strains of B. cepacia. The minor polymer was identified as the O antigen in serotype A of a Canadian typing scheme [Beynon, L. M. & Perry, M. B. (1993) Biochem. Cell Biol. 71, 417-420], and the major polymer in serotype C of a Japanese typing scheme [Paramonov, N. A., Shashkov, A. S., Knirel, Y. A., Soldatkina, M. A. & Zakharova, I. Y. (1994) Bioorg. Khim. 20, 984-993]. In the case of the O4 strain studied here, both polymers were produced under a variety of growth conditions. PMID- 7544288 TI - Immunohistochemical staining for endotoxin using horseshoe crab factor C in fecal peritonitis. AB - The object of the present study was to apply a new immunohistochemical staining method to the in vivo determination of endotoxin localization. The immunohistochemical staining method requires factor C (an initiation factor in the Limulus clotting system which is mediated by endotoxin) as a specific ligand of endotoxin, and a newly developed murine monoclonal antibody to factor C. The blood endotoxin level and endotoxin localization in the rat were determined before and at 6, 12 and 24 h after intraperitoneal injection of 0.25 g/kg of fresh rat feces. The greatest blood endotoxin level was achieved at 12 h after the injection, and uptake of endotoxin was evident in Kupffer cells in the liver at 24 h after the injection. There has been no report on determining endotoxin localization in cases of endotoxemia attributed to fecal peritonitis. This new immunohistochemical staining method for determining endotoxin localization will contribute to the histopathological diagnosis of endotoxemia in humans. PMID- 7544289 TI - Expression of glutamic acid decarboxylase mRNA in striatum and pallidum in an animal model of tardive dyskinesia. AB - Long-term administration of neuroleptics can induce tardive dyskinesia in humans. Oral movements with the same distinctive form observed in humans with tardive dyskinesia are observed in rats treated with haloperidol for 8 and 12 months but not 28 days. We have examined the effects of these long-term haloperidol treatments on the levels of mRNA encoding glutamic acid decarboxylase (GAD, M(r) 67,000), the rate-limiting enzyme of GABA synthesis, in the striatum and pallidum of adult rats. Despite the differences in behavior, GAD67 mRNA was increased in the striatum and entopeduncular nucleus (internal pallidum) after both 28 days and 8 months of haloperidol administration. In contrast, only long-term haloperidol treatments (8 and 12 months) decreased GAD67 mRNA in globus pallidus (external pallidum). This effect contrasted with the increased level of GAD67 mRNA we have previously observed in the globus pallidus after short-term haloperidol treatment (3-14 days), a regimen that induces catalepsy. Together with data indicating a loss of GAD activity in target areas of the globus pallidus in humans with tardive dyskinesia, the results suggest that decreased GABAergic transmission in the projection neurons of the external pallidum may play a critical role in the motor side effects associated with long-term neuroleptic therapy. PMID- 7544295 TI - Degradation of vitronectin by matrix metalloproteinases-1, -2, -3, -7 and -9. AB - The susceptibility of vitronectin (Vn) purified from human plasma to digestion by matrix metalloproteinases (MMPs) was examined. MMP-2, -3, -7 and -9 except for MMP-1 degraded Vn into multiple fragments. MMP-7 showed the highest activity to the substrate among these MMPs, digesting 8-, 30- and 44-fold more preferentially than MMP-2, -3, and -9, respectively. These data suggest that MMP-2, -3, -7 and 9 may be responsible for the pathological degradation and/or normal turnover of Vn. PMID- 7544291 TI - Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA. AB - A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and S641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution S641A loses RNA polymerase activity but is able to synthesize DNA. PMID- 7544296 TI - The modulation and characterisation of the Ca(2+)-induced Ca2+ release mechanism in cultured human myometrial smooth muscle cells. AB - The process of Ca(2+)-induced Ca2+ release (CICR) was studied in saponin permeabilised human myometrial smooth muscle cells in which the sarcoplasmic reticulum (SR) was pre-loaded with 45Ca2+. A rise in the free Ca2+ concentration of the bathing solution from 100 nM to 10 microM increased the rate of 45Ca2+ loss, while a reduction to 10 nM decreased the rate of 45Ca2+ loss. Ruthenium red (20 microM) lowered the basal rate of 45Ca2+ loss and reduced CICR. Caffeine did not activate 45Ca2+ release although ryanodine induced 45Ca2+ release and CICR was augmented in the presence of caffeine. These data suggest the operation of a Ca(2+)-activated 45Ca2+ release process which is similar in many of its properties to the CICR process described in many cells. The basic properties of the CICR mechanism in myometrium differs in many respects from the CICR process recently described in human vascular smooth muscle. PMID- 7544294 TI - Selective inhibition of gastrulation in the starfish embryo by albuside B, an inosine analogue. AB - External application of 0.2-100 micrograms/ml albuside B inhibits gastrulation of the starfish (Asterina pectinifera) embryo. Treated embryos retain the late blastula morphology with the vegetal plate. However, the vegetal plate is unreactive to soybean agglutinin, a probe for observing the progenitor cells of the archenteron (mesendoderm) in a normal embryo. The effective period of the treatment is limited from 4 to 6 h after fertilization, a period immediately before the onset of blastulation. RNA synthesis is unaffected during the period of sensitivity. The selectivity of the inhibition shows that albuside B may be a useful tool for studying the mechanisms of mesendoderm differentiation. PMID- 7544297 TI - The first untranslated exon of the human tenascin-C gene plays a regulatory role in gene transcription. AB - The transcription of the human tenascin-C (TN-C) gene is directed by a single promoter. Here we demonstrate, in transiently transfected cells, that two distinct regions of the untranslated 179 bp-long exon 1 play antagonistic roles in transcriptional regulation: bases from 1 to 20 strongly increase the transcription of the reporter gene CAT directed by the human TN-C gene promoter, while bases from 79 to 179 significantly reduce this activation. PMID- 7544293 TI - Interaction of rat liver lysosomes with basic polypeptides. AB - In order to gain knowledge on the interaction of lysosomes with proteins, we have assessed the equilibrium densities of the lysosomal membrane and matrix markers after in vitro incubation of rat liver lysosomes with various polypeptides. The addition of basic polypeptides, polylysine or protamine, to the suspension of lysosomes brought about a profound alteration of lysosomal membrane, causing extensive leakage of lysosomal matrix enzymes. Electron microscopic observation revealed a remarkable aggregation of lysosomes by the basic polypeptides. Polyglutamic acid, an acidic polypeptide, did not produce such effect. ATP was found to stabilize lysosomes during incubation, particularly with basic polypeptides. PMID- 7544290 TI - Evidence for apoptotic cell death in Alzheimer's disease. AB - We provide evidence for apoptosis in Alzheimer's disease using the in situ labeling technique TUNEL (terminal transferase-mediated dUTP-biotin nick end labeling). The technique specifically detects apoptotic cells by utilizing terminal transferase to incorporate biotinylated nucleotides into the fragmented DNA of apoptotic cells. The labeled cells are visualized by reaction with avidin peroxidase and a suitable substrate. Sections from the hippocampus of Alzheimer diseased (AD) brains and non-AD brains were examined for apoptosis. While considerable variation in the quantity of apoptotic cells was observed among individual samples, the incidence of apoptosis in AD brains was elevated in comparison to age-matched, non-AD brains in specific regions of the hippocampal formation. Immunostaining indicated that both neurons and astrocytes were undergoing apoptosis, although the majority of the TUNEL-positive cells appeared to be glial, based on the location of the stained cells. These data suggest that apoptosis may be involved in both the primary neuronal cell loss and in the glial response that is a component of AD. PMID- 7544292 TI - Cannabinoids enhance human B-cell growth at low nanomolar concentrations. AB - This study examined the effect of cannabinoid ligands on human tonsillar B-cells activated either through cross-linking of surface immunoglobulins or ligation of the CD40 antigen. The two synthetic cannabinoids, CP55,940 and WIN55212-2, as well as delta 9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, caused a dose-dependent increase of B-cell proliferation and displayed EC50 at low nanomolar concentrations. This cannabinoid-induced enhancing activity was inhibited by pertussis toxin which suggested a G-protein-coupled receptor process. In addition, the absence of antagonistic effect of SR141716A, a specific CB1 receptor antagonist, together with the demonstration that human B-cells displayed large amount of CB2 receptor mRNAs, led us to assume that the growth enhancing activity observed on B-cells at very low concentrations of cannabinoids could be mediated through the CB2 receptor. PMID- 7544299 TI - Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. AB - We studied the effects of protein tyrosine kinase inhibitors (genistein and tyrphostin) on receptor-activated increases in cellular Ca2+ ([Ca2+]i), and protein tyrosine phosphorylation in cultured canine femoral arterial smooth muscle cells. Fura-2 imaging analysis showed that each agonist evoked a transient increase in ([Ca2+]i) followed by a sustained plateau phase. Experiments in Ca(2+)-free medium showed that 70-80% of the transient increase in [Ca2+]i evoked by either agonist is due to influx of extracellular Ca2+ whereas the plateau phase is only due to Ca2+ entry. Pre-incubation with genistein or tyrphosin markedly inhibited the transient rise in [Ca2+]i evoked by serotonin or phenylephrine. Immunoblot analysis of cell extracts with antiphosphotyrosine antibodies revealed that serotonin and phenylephrine also evoked an increase in tyrosine phosphorylation of several substrates. These increases were abolished by tyrosine kinase inhibitors. One of the major substrates was recognized by an an antibody for rasGAP. These data suggest that receptor-activated increases in [Ca2+]i in vascular smooth muscle cells may be coupled to receptor-activated increases in protein tyrosine phosphorylation. PMID- 7544298 TI - A novel beta 1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle. AB - The mRNA's of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human beta 1 subunit by reverse transcriptase polymerase chain reaction. This fourth beta 1 variant, named beta 1D, is specific for skeletal and cardiac muscle. The determined genomic organization of the 3'-region of the human beta 1 gene reveals that beta 1D is produced by alternative splicing of mRNA. In addition, we show that the expression of beta 1D is developmentally regulated during murine myoblast differentiation, suggesting a role for beta 1D in myogenesis. PMID- 7544301 TI - Voltage-dependent calcium channel beta-subunits in combination with alpha 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G alpha o in rat frontal cortex. AB - The dihydropyridine-sensitive calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel beta-subunit antiserum abolished the ( )-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the beta subunit binding domain of the calcium channel complex, also attenuated (-)-BayK 8644 activation of GTPase. This study suggests that the calcium channel beta subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal. PMID- 7544300 TI - Effect of glucagon on insulin receptor substrate-1 (IRS-1) phosphorylation and association with phosphatidylinositol 3-kinase (PI 3-kinase). AB - In the present study we have examined the levels and phosphorylation state of the insulin receptor and insulin receptor substrate 1 (IRS-1) as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of rats treated with glucagon. There was a decrease in the insulin-stimulated receptor and IRS-1 phosphorylation levels which was paralleled by a reduced association between IRS-1 and PI 3-kinase in vivo in the liver and muscle of glucagon-treated rats. These observations suggest that glucagon, probably acting through cAMP, may impair insulin signaling in the three early steps in insulin action after binding. PMID- 7544305 TI - Novel expression of luteinizing hormone/chorionic gonadotropin receptor gene in rat prostates. AB - The reverse transcription-polymerase chain reaction (RT-PCR) amplified an expected 255 bp luteinizing hormone/chorionic gonadotropin (LH/CG) receptor sequence from rat prostates. Northern blotting demonstrated that prostates contain 4.3, 3.3, 2.6, 1.8, 0.8 and 0.2 kb LH/CG receptor transcripts. Western immunoblotting and ligand blotting demonstrated that prostates also contain an 80 kDa receptor protein which can bind 125I-labeled hCG and this binding was inhibited by excess unlabeled hCG. In situ hybridization and immunocytochemistry revealed that while the receptors are most abundant in epithelial cells, they are scarcely found in the stroma. The ventral lobe contained more receptors than the lateral lobe and the receptors in peripheral acini of the ventral lobe are higher which progressively decreased towards central acini. In summary, prostate glands express LH/CG receptor gene. The cellular, topographical and lobular distribution of receptors suggest that LH may directly regulate prostate functions. PMID- 7544302 TI - Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s. AB - The replacement of either Tyr-181 or Tyr-188 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) by the corresponding HIV-2 RT amino acids Ile-181 or Leu-188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L-697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U-90152S, a highly specific inhibitor (IC50, 0.29 +/- 0.01 microM) of HIV-1 RT, inhibited the recombinant Y181I and Y188L HIV-1 RT mutants with IC50 values of 3.6 +/- 0.15 microM and 0.71 +/- 0.02 microM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV-1 RT showed > 150-fold resistance to U-90152S. An HIV-2 RT mutant containing amino acids 176-190 from HIV-1 RT acquired full sensitivity to U-90152S (IC50, 0.26 +/- 0.01 microM). It is concluded that simultaneous mutations at Tyr-181 and Tyr-188 of HIV-1 RT promotes resistance to U-90152S. PMID- 7544304 TI - Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene. AB - Previous studies have indicated that human chorionic gonadotropin (hCG) has immunoregulatory properties and alters the functions of human lymphocytes. However, it has not been determined whether the gene encoding the receptors for hCG/luteinizing hormone (LH) is expressed in human lymphocytes. Total peripheral mononuclear lymphocytes isolated from blood samples of pregnant women by Ficoll Paque gradient centrifugation contained mRNA transcripts encoding the hCG/LH receptors and a 50 kDa receptor protein which can bind 125I-hCG. T-Lymphocytes isolated from total mononuclear cell fractions also contained these receptor mRNA transcripts as well as the receptor protein. The levels of receptor transcripts and receptor protein are lower in lymphocytes than in ovarian tissue. These findings suggest that the immunoregulatory actions of hCG are probably mediated by specific receptors in T-lymphocytes from pregnant women. PMID- 7544303 TI - Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3. AB - The growth and differentiation of megakaryocytes are regulated by thrombopoietin (TPO), a recently characterized cytokine which exerts its effects via a member of the hematopoietin receptor superfamily, c-Mpl. Since many cytokines which bind hematopoietin receptors activate the STAT family of transcription factors, we investigated whether STAT proteins were activated by TPO. TPO induced the formation of a DNA-binding complex recognizing a known STAT-binding sequence. STAT5 was a major component of this DNA-binding complex, and STAT5 was tyrosine phosphorylated in response to TPO. Additionally, TPO-induced the tyrosine phosphorylation and DNA-binding activity of STAT3. Together with the recent demonstration of JAK2 activation in response to TPO, the data presented here define a rapid signaling pathway likely to be important in TPO-induced gene regulation. PMID- 7544308 TI - The extracellular matrix in higher plants. 4. Developmentally regulated proteoglycans and glycoproteins of the plant cell surface. AB - The review focuses on recent evidence that two classes of cell-surface protein, one consisting largely of proteoglycans and the other of glycoproteins, may function during plant development. One class, the arabinogalactan proteins (AGPs), includes some of the extracellular proteoglycans in plant secretions, as well as related molecules that are found at the outer face of the plasma membrane where they present an array of complex carbohydrate structures to the extracellular matrix (cell wall). Recent evidence implicates cell-surface AGPs in the control of cell proliferation and morphogenesis. For example, immunodetection methods have shown that the developmentally regulated appearance of carbohydrate epitopes in these proteoglycans correlates with the formation of anatomical patterns. Likewise, the members of a second class, the hydroxyproline-rich glycoproteins (HRGPs, or extensins) of the cell wall, are developmentally regulated and their occurrence also correlates with changes in anatomy. Recent observations suggest that HRGP-like domains are present in plasma membrane proteins that bridge the wall and cytoskeleton. The roles of oxidative cross linking and wall protein insolubilization during development and defense responses are also discussed, with particular reference to the roles of hydrogen peroxide, oxidases, and HRGPs in the processes. The survey emphasizes the value of monoclonal antibodies for revealing molecular and developmental changes in AGPs and HRGPs at the plant cell surface. PMID- 7544309 TI - RNA structure at high resolution. AB - Studies of RNA structural motifs at high resolution by NMR and X-ray crystallographic methods have provided many insights into the fundamental forces that give rise to the unique structural characteristics of RNA. Non-Watson-Crick purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine base pairing, as well as base-phosphate and base-ribose hydrogen bonding, are important forces for folding and stabilizing RNA structures. Base stacking is as important in determining RNA conformations as hydrogen bonding interactions. With the noncanonical interactions, many single-stranded loop regions such as hairpin loops, bulge loops, and internal loops fold into well-defined secondary structures. Loop-loop and loop-helix interactions can produce tertiary structures such as pseudoknots. Also, single strands adjacent to helical regions can form tertiary contacts with base-paired nucleotides of the helices. As we learn more about the structures of the important motifs we can ask more specific questions about the mechanisms of RNA-mediated functions. Conformational flexibility rather than a specific shape of the RNA may be important for some biological reactions. However, knowledge of the structures and the ease of conformational change of the molecules involved in any process are essential for understanding and eventually controlling the process. PMID- 7544306 TI - Proliferation of the murine corticotropic tumour cell line AtT20 is affected by hypophysiotrophic hormones, growth factors and glucocorticoids. AB - In pituitary-dependent hyperadrenocorticism (Cushing's disease), the disturbed regulation of ACTH secretion is associated with neoplastic transformation of corticotropic cells. As these two phenomena are almost indissolubly connected, it is of prime importance to elucidate the factor(s) that induce corticotropic cell proliferation. Here we report on the effects of hypophysiotrophic hormones and intrapituitary growth factors on the proliferation and hormone secretion of the murine corticotropic tumour cell line AtT20/D16v, as measured by DNA content, and ACTH concentration in culture media. In addition, sensitivity to the inhibitory effect of cortisol was assessed under various conditions. Corticotropin releasing hormone (CRH) and vasopressin (AVP) induced proliferation of AtT20-cells. In contrast to that caused by AVP, the CRH-induced proliferation was associated with increased ACTH secretion, which could be inhibited by cortisol. Insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) also stimulated the proliferation of AtT20-cells. The proliferation of AtT20-cells was significantly inhibited by cortisol in all tests. The IGF-I-induced proliferation was the least sensitive to inhibition by cortisol. The growth factors did not stimulate ACTH secretion but IGF-I differed in that it prevented the inhibition of basal ACTH secretion by cortisol. Additional experiments (Western ligand blot analysis) concerning the relative insensitivity of IGF-I induced proliferation to inhibition by cortisol revealed that IGF-I increased the concentration of a 29 kDa IGF binding protein (IGFBP) in the culture medium. The concentration of the 29 kDa IGFBP was slightly decreased by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544311 TI - Structural and serological studies of lipopolysaccharides from proposed new serotypes (O25 and O26) of Serratia marcescens. AB - The surface polysaccharides of the two most recently proposed O-serotype strains of Serratia marcescens, O25 and O26, were characterised in terms of their chemical structure and immunological reactions. No polymer was isolated from O25, which was shown to lack both capsular K-antigen and smooth, O-antigenic lipopolysaccharide. A neutral polysaccharide was isolated from O26 and shown to be a polymer of rhamnose and N-acetylgalactosamine of the type previously found in the O9 and O15 reference strains. Serological cross-reactions among all three strains were demonstrated by using both whole-cell enzyme-linked immunosorbent assay and immunoblotting of lipopolysaccharide resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. No acidic polysaccharide was found in O26 and this was consistent with the absence of an immunogenic capsule. Thus, neither strain qualifies for inclusion as a new serotype in either an O-typing or a K-typing scheme. PMID- 7544310 TI - A novel beta 1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration. AB - It is generally accepted that the beta 2-integrin is restricted to mononuclear leukocytes. The objective of this study was to determine whether neutrophils can also express beta 1-integrin (specifically alpha 4 beta 1) and whether this can support neutrophil adhesion to endothelial cells and to extracellular matrix. We stimulated neutrophils with dihydrocytochalasin B (DHCB) and various chemotactic stimuli and observed that chemotactic stimuli induced neutrophil adhesion via beta 2-integrin (CD18), whereas DHCB and either fMLP, PAF, or IL-8 induced adhesion to endothelium or protein-coated plastic that was not inhibitable by anti-CD18 antibody. beta 2-integrin-deficient cells, which did not respond to chemotactic stimuli alone, also adhered avidly in the presence of chemotactic stimuli and DHCB. The induced neutrophil adhesion was inhibited by antibody to beta 1- or alpha 4-integrin chains, but only if an anti-beta 2-integrin antibody was also present. Flow cytometry revealed increased expression of both beta 1 and alpha 4 in the presence of fMLP plus DHCB. Transendothelial migration of neutrophils induced by chemotactic stimuli alone also increased expression of beta 1 and alpha 4. Transmigration across deendothelialized membranes induced a similar beta 1 expression on neutrophils suggesting that events other than an endothelial signal elicited beta 1-integrin expression. Transmigration-induced beta 1-dependent expression translated into only modest adhesion to protein coated plastic. These data suggest that both a pharmacological (DHCB) and a physiological (transmigration) stimulus can invoke expression of alpha 4 and beta 1 on human neutrophils to mediate adhesion. PMID- 7544312 TI - Deletion of beta 1 integrins in mice results in inner cell mass failure and peri implantation lethality. AB - Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass. PMID- 7544307 TI - A maternal RNA localized in the yellow crescent is segregated to the larval muscle cells during ascidian development. AB - A cDNA library prepared from one-cell zygotes of the ascidian Styela clava was screened with probes from isolated cellular fractions to identify clones encoding RNAs localized in the yellow crescent or myoplasm, a cytoskeletal domain with multiple developmental roles. The differential screen yielded five overlapping cDNA (Styela clava yellow crescent or ScYC) clones encoding a 1.2-kb polyadenylated RNA (yellow crescent or YC RNA) which is present throughout embryonic development. In situ hybridization confirmed that YC RNA is localized in the yellow crescent. Antisense probes containing the 3' region of YC RNA hybridize with multiple maternal and zygotic RNAs, suggesting sequence homologies with other transcripts. YC RNA was first detected during oogenesis when transcripts accumulate in the perinuclear region of vitellogenic oocytes and are gradually translocated to the cortex. The YC transcripts are localized in the cortex of unfertilized eggs but after fertilization segregate with the myoplasm to the yellow crescent. During cleavage most YC transcripts enter the primary muscle cell lineage. YC RNA is also present in the secondary muscle cells. The YC transcripts are retained in the myoplasm of oocytes and eggs extracted with the non-ionic detergent Triton X-100, suggesting that they are associated with the cytoskeleton. The nucleotide sequence of the longest ScYC clone contains a short open reading frame (ORF). The YC ORF would encode a putative polypeptide of 49 amino acids, which shows no significant homology to known proteins. Several features of the YC RNA, however, suggest that it functions as an RNA rather than as a protein coding molecule. We conclude that the myoplasm contains a novel maternal RNA which is associated with the cytoskeleton and segregated to the muscle cells during ascidian embryogenesis. The YC RNA may be a new member of a growing family of noncoding RNAs that play important roles in growth and development. PMID- 7544316 TI - Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium. AB - There exists a plethora of tyrosine kinases that play essential roles in regulation of eukaryotic proteins. Several dual specificity kinases that phosphorylate proteins on threonine, serine, and tyrosine residues also play critical roles in eukaryotic phosphorylation cascades. In contrast, very few prokaryotic proteins have been shown to be phosphorylated on tyrosine residues, and the functions of the rare examples remain obscure. Furthermore, no dual specificity kinases have been described in prokaryotes. Our results indicate that PutA protein from the bacterium Salmonella typhimurium autophosphorylates on several threonine, serine, and tyrosine residues. PutA protein both represses the proline utilization (put) operon and degrades proline to glutamate. These two opposing functions are regulated by the availability of proline and the membrane sites needed for the proline dehydrogenase activity of PutA protein. In addition, these functions are modulated by phosphorylation of PutA protein. The rate of dephosphorylation of PutA protein is determined by the availability of proline and membranes. Dephosphorylated PutA protein has a higher DNA binding affinity than the phosphorylated protein and thus may prevent toxic overexpression of PutA protein in the absence of available membrane sites. PMID- 7544317 TI - Prooxidant and antioxidant hepatic factors in rats chronically fed an ethanol regimen and treated with an acute dose of lindane. AB - While acute lindane treatment and chronic ethanol feeding to rats have been associated with hepatic oxidative stress, the possible roles of these stresses in the pathogenesis of hepatic lesions reported in acute lindane intoxication and in those observed in some models of chronic alcoholism have not been established. Our previous studies in rats chronically fed ethanol regimens and then treated with a single intraperitoneal (i.p.) dose of lindane (20 mg/kg) showed that while lindane per se was invariably associated with hepatic oxidative stress, chronic ethanol feeding only produced this stress when the dietary level of vitamin E was relatively low. Chronic ethanol pretreatment did not significantly affect the lindane-associated oxidative stress, and neither chronic ethanol feeding nor acute lindane, single or in combination, produced any histologic and biochemical evidence of liver damage. In the present experiment, the acute dose of lindane was increased to 40 mg/kg, and we have studied a larger number of prooxidant and antioxidant hepatic factors. Male Wistar rats (115.5 +/- 5.4 g) were fed ad lib for 11 weeks a calorically well-balanced and nutritionally adequate basal diet, or the same basal diet plus a 32% ethanol/25% sucrose solution, also ad lib, and were then injected i.p. with a single dose of lindane or with equivalent amounts of corn oil. The results indicated that acute lindane treatment to naive rats increased practically all the prooxidant hepatic factors examined (cytochromes P450 and b5, NADPH cytochrome c reductase, NADPH oxidase), as well as the generation of microsomal superoxide radical and thiobarbituric acid reactive substances of liver homogenates, but did not modify any of the antioxidant hepatic factors studied. Conversely, the chronic administration of ethanol alone did not significantly affect the prooxidant hepatic factors but reduced some of the antioxidants (i.e., the activities of GSH-Px and the contents of alpha tocopherol and ubiquinols 9 and 10). Although chronic ethanol pretreatment further increased the superoxide generation induced by lindane per se, it did not increase but generally reduced the effects of lindane per se on the other prooxidant factors studied. Furthermore, although acute lindane administration to ethanol-pretreated rats was associated with decreases in GSH and catalase (not affected by ethanol or lindane treatment alone), it did not substantially modify the reducing effects of ethanol feeding per se on GSH-Px, alpha-tocopherol, and ubiquinols. Once again, neither chronic ethanol feeding nor lindane treatment, single or in combination, was associated with any evidence of liver damage. PMID- 7544314 TI - Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice. AB - Signaling by tyrosine kinases is required for the induction of synaptic plasticity in the central nervous system. Comparison of fyn, src, yes, and abl nonreceptor tyrosine kinase mutant mice shows a specific requirement for Fyn in the induction of long-term potentiation at CA1 synapses in the hippocampus. To identify components of a Fyn-dependent pathway that may be involved with hippocampus function we examined tyrosine-phosphorylated proteins in kinase mutant mice. We found that nine proteins were hypophosphorylated specifically in fyn mutants. One of the hypophosphorylated proteins was focal adhesion tyrosine kinase (FAK). FAK also showed reduced activity in immunocomplex kinase assays only in fyn mutants. FAK is expressed at very high levels in the brain but in contrast to non-neural cells, FAK was not restricted to focal adhesion contacts. FAK was found in axons, dendrites, and the intermediate filament cytoskeleton of astrocytes. Brain extracts from the mutants also show specific patterns of compensatory changes in the activity of the remaining Src family kinases. Tyrosine phosphorylation is a critical regulator of FAK, and impairments in FAK signal transduction in fyn mutants may contribute to the mutant neural phenotype. PMID- 7544315 TI - Targeted disruption of the pituitary glycoprotein hormone alpha-subunit produces hypogonadal and hypothyroid mice. AB - Pituitary thyrotropin (TSH) and gonadotropins (LH and FSH) are thought to be critical for thyroid and gonadal development and function. Each of these pituitary hormones is a heterodimer composed of a common alpha-subunit and unique beta-subunit, and heterodimerization is required for function. No mutations in the alpha-subunit or any of the beta-subunit genes have been reported in mice. To assess directly the functional role of TSH, LH, and FSH in thyroid and gonadal development, we created a disruption of the alpha-subunit gene by homologous recombination. The homozygous mutant animals were hypogonadal and exhibited profound hypothyroidism resulting in dwarfism. Thyroid development was arrested in late gestation, but GnRH neuron migration, development of secondary sex organs, and fetal and neonatal gonadal development were normal. This establishes the importance of thyrotropin in ontogeny and reveals that fetal pituitary gonadotropins are not required for sexual differentiation or genital development in male or female fetuses. The pituitary cells that produce TSH beta-subunit exhibited dramatic hypertrophy and hyperplasia as a result of the lack of thyroid function. This proliferation response occurred at the expense of somatotrope and lactotrope cells, consistent with a derivation of these three cell types from a common precursor. PMID- 7544313 TI - Consequences of lack of beta 1 integrin gene expression in mice. AB - beta 1 integrins are cell-surface receptors that mediate cell-cell and cell matrix interactions. We have generated a null mutation in the gene for the beta 1 integrin subunit in mice and embryonic stem (ES) cells. Heterozygous mice are indistinguishable from normal littermates. Homozygous null embryos develop normally to the blastocyst stage, implant, and invade the uterine basement membrane but die shortly thereafter. Using beta 1 integrin-deficient ES cells we have established chimeric embryos and adult mice. Analysis of the chimeric embryos demonstrated the presence of beta 1 integrin-deficient cells in all germ layers indicating that beta 1-null cells can differentiate and migrate in a context of normal tissue. When evaluated at embryonic day 9.5 (E9.5), embryos with a beta 1-null cell contribution below 25% were developing normally, whereas embryos with a contribution above this threshold were distorted and showed abnormal morphogenesis. In adult chimeric mice beta 1 integrin-deficient cells failed to colonize liver and spleen but were found in all other tissues analyzed at levels from 2%-25%. Immunostaining of chimeric mice showed that in cardiac muscle, there were small, scattered patches of myocytes that were beta 1-null. In contrast, many myotubes showed some beta 1-null contribution as a result of fusion between wild-type and mutant myoblasts to form mixed myotubes. The adult chimeric brain contained beta 1-null cells in all regions analyzed. Also, tissues derived from the neural crest contained beta 1 integrin-deficient cells indicating that migration of neuronal cells as well as neural crest cells can occur in the absence of beta 1 integrins. PMID- 7544318 TI - An aid to empowerment: a caregiver well-being scale. AB - To aid medical social workers in the promotion and support of well-being among older family caregivers, a simple, valid, and reliable scale was developed. This scale has two subscales, one that assesses the extent to which a caregiver's basic human needs are being met and one that assesses the extent to which a caregiver can satisfactorily meet his or her predictable activities of daily living. Developed from a model that focuses on a person's strengths, this tool was created to help social work practitioners enable caregivers to become more in touch with their own abilities. Both subscales had high internal reliability and construct validity. This scale may have value in other areas of caregiving. Implications for using this scale in practice are presented. PMID- 7544321 TI - Tumor-derived factor synergizes with IFN-gamma and LPS, IL-2 or TNF-alpha to promote macrophage synthesis of TNF-alpha and TNF receptors for autocrine induction of nitric oxide synthase and enhanced nitric oxide-mediated tumor cytotoxicity. AB - Evidence has previously been presented for an immunomodulatory role of a soluble activity, designated as tumor-derived recognition factor (TDRF), which was produced constitutively by P815 mastocytoma, L 1210 leukemia and other murine tumor targets. TDRF synergized with IFN-gamma and IL-2 to promote TNF-alpha and mRNA synthesis and release by murine macrophages for increased autocrine induction of nitric oxide (NO)-mediated tumor cytotoxicity. We have now further assessed the modulatory role of TDRF on TNF-alpha, TNF receptors (TNF-R) and NOS mRNA synthesis. Macrophages activated by INF-gamma priming and triggering by rTNF alpha bacterial lipopolysaccharide (LPS) of IL-2 evoked greater NO generation in the presence than in the absence of L1210 targets. TDRF-containing culture fluid from L1210 targets was subsequently confirmed to synergize with IFN-gamma and rTNF-alpha, LPS or IL-2 triggering agents to promote increased TNF-alpha mRNA for autocrine induction of NOS mRNA synthesis with resultant augmentation of NO generation. IFN-gamma selectively upregulated TNF-R1 mRNA expression, whereas either IL-2 or LPS upregulated only TNF-R2 mRNA expression. TDRF combined with IFN-gamma to further upregulate TNF-R1 mRNA and with either IL-2 or LPS to further upregulate TNF-R2, mRNA expression. These findings indicate that TDRF activity synergizes with either IL-2 or LPS triggering agents for enhanced activation of IFN-gamma-primed macrophages by promotion of TNF-alpha and TNF-R mRNA synthesis for autocrine induction of NOS with resultant increased NO mediated tumor cytotoxicity. PMID- 7544320 TI - Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation. AB - To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (delta F 508, G542X, G551D, 621 + 1 G-->T, N1303K, W1282X) using allele specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the delta F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A-->G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles. PMID- 7544319 TI - Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations. AB - A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency. PMID- 7544322 TI - Cardiovascular responses to phenylephrine during acute experimental anaemia in anaesthetized cats. AB - Experiments were performed on anaesthetized artificially ventilated cats to study the effects of phenylephrine (PE) on cardiovascular responsiveness, before and after induction of experimental anaemia. Acute anaemia was induced by replacement of blood by dextran in three steps of 20% each of total estimated blood volume. The effect of PE (20 micrograms/kg) was investigated at four stages: control and after 1st, 2nd and 3rd exchanges of blood. Induction of anaemia produced a significant increase in heart rate (HR) and cardiac output (CO) and a decrease in right atrial pressure (RAP). No significant change in mean arterial pressure (MAP), LV dP/dt max and blood gas tension was observed. Administration of bolus dose of PE produced a rapid rise in MAP, LVdP/dt max, and a decrease in HR without a change in the RAP. The pattern of response to PE was similar after induction of acute anaemia, however the magnitude of the response was significantly reduced. The attenuation in the response to PE was related to the fall in the haematocrit (HCT) level. This shows that induction of experimental anaemia, produced an increase in CO due to an increase in HR and SV and the effect of PE on cardiovascular responsiveness was significantly attenuated. The reduced sensitivity to PE during acute anaemia could be due to many factors such as inadequate O2 supply, effect of local vasodilating agents or some other cardiotonic agents which are known to contribute to vascular responsiveness. PMID- 7544323 TI - GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i. AB - It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca2+]i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium-sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca2+]i within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1 aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca2+]i. The increases in [Ca2+]i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca2+]i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca2+]i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca2+]i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [Ca2+]i and are able to respond to neuronally released neurotransmitters. PMID- 7544326 TI - Changes of acute phase proteins glycosylation profile as a possible prognostic marker in myocardial infarction. AB - In 24 consecutive patients with myocardial infarction, the concentrations of C reactive protein, alpha 1-acid glycoprotein and alpha 1-antichymotrypsin, as well as acid alpha 1-glycoprotein and alpha 1-antichymotrypsin glycosylation profiles were estimated. Blood samples were taken at admission, after 4, 8, 12 and 24 h, on 2, 3, 6, 9, and 12 days of hospitalization. All studied patients were divided into 2 groups: 12 patients without clinical or radiological symptoms of acute heart failure and 12 patients with acute heart failure. The results of all investigations were tested statistically to appraise significance of differences between the two investigated groups. At admission, as well as after 6 and 12 h, C reactive protein concentration was significantly higher in patients who developed heart failure. Similarly, the glycosylation profile of alpha 1-antichymotrypsin, reported as reactivity coefficient, was of good prognostic value from the first time-point on. Development of acute cardiac failure seemed to correlate more with the magnitude of inflammatory reaction (measured by changes in acute phase proteins) than with enzymatically estimated infarct size. PMID- 7544324 TI - Elevated intracellular levels of cAMP induce olfactory ensheathing cells to express GAL-C and GFAP but not MBP. AB - The primary olfactory pathway contains non-myelinating glial cells, called ensheathing cells, that exhibit a variety of phenotypes depending on their immediate environment. In vivo, these cells normally possess a mixture of astrocyte- and Schwann cell-specific phenotypic features. When co-cultured with dorsal root ganglion neurons, their phenotype can become more like that of a myelinating Schwann cell. The objective of this study was to determine whether ensheathing cells would express a myelinating phenotype in culture in the absence of neurons but in the presence of cAMP analogues that are known to induce the expression of myelin associated molecules in Schwann cell cultures. The ensheathing cell cultures were initiated using the nerve fiber layers of Theiler stage 23 rat olfactory bulb primordia and were fed for 1 day to 3 weeks with serum containing (1% or 10% FBS) or serum-free media to which was added different concentrations of dBcAMP (0.1 to 1 mM) or forskolin (10 microM). These cultures were double-labelled with a rabbit polyclonal antibody to S100 in combination with mouse anti-GAL-C (O1 and BRD1 hybridomas) or anti-MBP monoclonal antibodies. The remaining cultures were double-labeled with a rabbit polyclonal antibody to GFAP in combination with the BRD1 antibody. Treatment with dBcAMP or forskolin failed to induce ensheathing cells to express MBP regardless of the concentration. On the other hand, the treatment induced approximately one tenth of the cells to express GAL-C, and virtually all of the cells to express GFAP. These results indicate that although ensheathing cells can synthesize myelin associated molecules, the cAMP second messenger system appears to play a lesser role in controlling the expression of a myelinating phenotype in ensheathing cells than it does in Schwann cells. PMID- 7544327 TI - Risk, autonomy, and responsibility. Informed consent for prenatal testing. PMID- 7544325 TI - Change of phosphotyrosine immunoreactivity on microglia in the rat substantia nigra following striatal ischemic injury. AB - Using immunohistochemistry, we investigated changes in phosphotyrosine (P-Tyr) immunoreactivity on the microglia of the rat substantia nigra (SN) following striatal ischemic injury produced by transient middle cerebral artery (MCA) occlusion. Anterograde axonal degeneration in the SN due to striatal ischemic injury was detected by depletion of calcineurin immunoreactivity in that region from 1 day after operation. From 3 days to 1 month (the longest period examined in this study) after MCA occlusion, there was a significant increase in P-Tyr immunoreactivity in the SN ipsilateral to the MCA occlusion. Also, light microscopic observation showed that the microglia exhibited an increased immunoreactivity for P-Tyr and characteristic morphological changes in the ipsilateral SN. The present results indicate that a signal transducing cascade(s) associated with tyrosine phosphorylation may be involved in the activation of the microglia in the SN responding to anterograde degeneration of the striatonigral pathway. PMID- 7544328 TI - In Britain: fewer conflicts of conscience. PMID- 7544329 TI - Euthanasia in Britain. PMID- 7544331 TI - Lectin-binding sites and silver affinity of the macula densa basement membranes in the rabbit kidney. AB - Fluorochrome-labelled lectins and the Jones method of silver impregnation preceded by different oxidation and enzymatic digestion procedures were used to study the patterns of glycosylation and silver affinity of the macula densa (MD) and thick ascending limb (TAL) basement membranes of the rabbit kidney. The goal of this study was to analyse the morphological basis of MD basement membrane permeability and its possible role in modulation of the signal involved in tubuloglomerular feedback control of the juxtaglomerular apparatus. The lectin binding pattern and silver affinity of basement membrane differed clearly from those of the TAL basement membrane. The former had greater WGA and Con A affinity than the latter. Furthermore, the MD basement membrane lost argyrophilia in permanganate oxidized sections whereas that of the TAL did not. The cell coat of MD cells differed from that of the TAL cells in that it had N-acetyl neuraminic acid and Con A binding sites. Our results suggest that the MD basement membrane has a distinctive macromolecular composition which may be related to its permeability to high molecular weight molecules. PMID- 7544330 TI - NADPH diaphorase and nitric oxide synthase are expressed by the majority of intramural neurons in the neonatal guinea pig urinary bladder. AB - The distribution of neurons expressing NADPH diaphorase activity was examined histochemically in whole mount preparations of the neonatal guinea pig urinary bladder. NADPH diaphorase positive neurons were abundant in the intramural ganglia in both the detrusor and trigone regions of the bladder. Labelled nerve fibres were found in the ganglion interconnectives and in smooth muscle bundles. Mucosal epithelial cells and endothelial cells lining the blood vessels supplying the bladder were also found to express NADPH diaphorase activity. In order to verify that NADPH diaphorase activity represented the presence of nitric oxide synthase in bladder neurons, a well characterised tissue culture preparation was employed. This also provided an opportunity to estimate the proportion of the total population of bladder neurons which expressed NADPH diaphorase activity. Using a combination of histochemical and immunohistochemical techniques, NADPH diaphorase positive neurons were found to constitute approximately 90% of the total neuronal population, which was identified by labelling with an antiserum to the general neuronal marker protein gene product 9.5. Almost all neurons (99%) which expressed NADPH diaphorase activity in culture were also found to be immunoreactive for nitric oxide synthase. These findings indicate that nitric oxide may play a role in the neural control of bladder function, and this possibility is discussed. PMID- 7544332 TI - Isolation and characterisation of a hyaluronan binding protein, hyaluronectin, from human placenta and its colocalisation with hyaluronan. AB - Hyaluronan (HA) is a major component of the extracellular matrix and is known to influence cell behaviour and to play a role in angiogenesis, morphogenesis and tissue remodelling, although little is known concerning the regulation of these effects. Until now its detection in the placenta has been by indirect methods, which has led to conflicting conclusions as to its distribution and hence its role. Hyaluronectin (HN) is one of a group of proteins with HA binding ability which may regulate the effects of HA. Although nervous tissue HN has been partly characterised with regard to its distribution, structure and biochemistry, little is known about the mesenchymal isoform and its distribution in placenta has not previously been reported. Using specific probes we have characterised the distribution of HA and HN in human placental tissue. At all stages of development studied (8, 10, 12, 30 and 38 wk gestation) HA and HN were unequivocally colocalised, being distributed in the extracellular matrix of stromal tissue of placental villi, chorioallantoic membranes and umbilical cord. Particularly strong immunoreactivity was observed in the villous stroma immediately adjacent to fibrinoid depositions at sites of denudation of the trophoblast layer. Extraction and characterisation of the HN from placental villi have revealed 4 major glycoproteins of 47, 52, 57 and 67 kDa, this being a different pattern and smaller molecular range than observed for the nervous tissue form. This is the first direct demonstration of the presence of HA and HN in the placenta and identifies an abundant new source of mesenchymal HN. The functions of mesenchymal HN are unknown but may include ion exchange, immunosuppression and regulation of the effects of HA in such roles as maintenance of tissue architecture, cell migration and angiogenesis. PMID- 7544333 TI - Ultrastructural localisation of substance P, vasoactive intestinal peptide and somatostatin immunoreactivities in the submucous plexus of guinea pig ileum. AB - The submucous neurons, especially those related to the lymphatic vessels, together with their associated synapses, were studied ultrastructurally with respect to their immunoreactivities for 3 types of neuropeptides, namely substance P (SP), vasoactive intestinal peptide (VIP) and somatostatin (SOM). With the antibodies directed against the 3 types of neuropeptides, a variable number of submucous neurons including those contacting the lymphatic vessels were immunostained. Based on the immunoreactivities and synaptic relations with the submucous neurons contacting the lymphatic vessels, at least 4 types of synaptic relations with the submucous neurons contacting the lymphatic vessels, at least 4 types of synaptic configurations were observed: immunopositive terminals with positive neurons, immunopositive terminals with negative neurons, immunonegative terminals with positive neurons and immunonegative terminals with negative neurons. All 4 types of synaptic configurations were observed in SP and VIP immunostained specimens, with the exception of type 3 which was not encountered in samples immunoreacted for SOM. When the proportions of all 4 types of peptidergic immunopositive terminals contacting the lymphatic vessel-associated neurons were totalled, the value exceeded 100%, suggesting the coexistence of 2 or more neuropeptides in the same terminals. Furthermore, some immunoreactive axon terminals made direct synaptic contacts with positive neurons suggesting the formation of the so-called 'peptide neuron chain'. It is speculated from this study that the submucous neurons receive multiple peptidergic inputs. The various synaptic contacts would imply a complicated reflex pathway in the submucous plexus. PMID- 7544334 TI - A comparative study by retrograde neuronal tracing and substance P immunohistochemistry of sympathetic preganglionic neurons in spontaneously hypertensive rats and Wistar-Kyoto rats. AB - A comparative morphological study of the sympathetic preganglionic neurons that innervate the superior cervical ganglion (SPN-scg) was made in spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats. The cytoarchitectonics and dendroarchitectonics of the SPN-scg were studied following retrograde transport of choleragen subunit B horseradish peroxidase conjugate (CB HRP) and Fluorogold. Significant differences were observed in the maximum and minimum diameters of neurons of the nucleus intermediolateralis pars principalis (ILp) and in the minimum diameter of neurons in the nucleus intermediolateralis pars funicularis (ILf) between SHR and WKY rats (P < 0.01). These diameters were decreased in neurons of SHR. The distribution patterns of dendrites of SPN-scg also showed differences between SHR and WKY rats. The dendritic distribution patterns showed the following changes in SH rats: (1) the mediolaterally oriented dendrites were reduced in number, (2) the ladder-like configuration of the medially oriented grey-matter dendrites was less prominent, (3) the medially oriented dendrites formed a triangular or dome-like configuration, and (4) the white matter dendritic plexuses and subependymal plexuses were reduced. Similar differences between SHR and WKY rats were also observed in our immunohistochemical study of substance P-like fibres. In addition, the SP study has also shown a close association of SP fibres with the central canal both in SHR and WKY rats; some of the SP fibres penetrated the ependymal lining to run longitudinally up or down the central canal. This finding suggests the presence of substance P-positive neurons contacting the cerebrospinal fluid. PMID- 7544338 TI - Effect of hypoxia on reflex responses of tracheal submucosal glands. AB - The effects of moderate sustained normocapnic hypoxia on tracheal submucosal gland reflex responses were studied. Experiments were performed in anesthetized, paralyzed, and mechanically ventilated dogs. The changes in the number of secreting glands and volume of secreted fluid in the subsequent period of time were recorded after 15-30 min of controlled ventilation with room air [arterial PO2 (PaO2) 86 +/- 3 Torr], hypoxic gas mixture (PaO2 49 +/- 4 Torr), or 100% O2 (PaO2 339 +/- 39 Torr), under isocapnic and isohydric conditions. The hillocks method was used to quantify the changes in submucosal gland secretion. The changes in secretion 30 s after stimulation of pulmonary C-fiber receptors by right atrial injection of capsaicin (10 micrograms/kg; n = 10) were markedly lower during moderate hypoxia than in normoxia or hyperoxia. Differences in the number of liquid droplets and the volume of secreted fluid were statistically significant (P < 0.05 and P < 0.001, respectively). Stimulation of airway rapidly adapting receptors by lung deflation increased airway secretion; the number of "hillocks" and the volume of secreted fluid were lower in hypoxic than in hyperoxic state. Differences between response curves for the number of glands activated and secreted volume were statistically significant (P < 0.05 and P < 0.001). The number of glands activated by substance P given locally by arterial infusion was not affected by the state of oxygenation, but the calculated volume of secreted fluid was lower during the hypoxic state than under hyperoxic condition (P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544337 TI - A new assay method for immunosuppressants with a tacrolimus (FK506)-like mode of action. PMID- 7544340 TI - Leukocyte adhesion induced by inhibition of nitric oxide production in skeletal muscle. AB - Superfusion of rat cremaster muscles with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) elicited significant leukocyte adhesion to postcapillary venules (20- to 30-microns diameter), an effect that was attenuated by pretreatment with L-arginine (an NO precursor) or sodium nitroprusside (SNP) (an exogenous source of NO). In contrast to the effects of pretreatment, addition of SNP or L-arginine to the superfusate 30 min after the initiation of NO synthase inhibition failed to reverse the L-NAME induced leukocyte adherence. However, this effect was reversed by administration of an anti-CD18 monoclonal antibody or 8-bromoguanosine 3',5'-cyclic monophosphate 30 min after L-NAME superfusion was initiated. These findings indicate that L-NAME promotes leukocyte adhesion to venular endothelium by a CD18 dependent mechanism in skeletal muscle and suggest that the failure of L-arginine or SNP to reverse L-NAME-induced leukocyte adherence is not due to a defect in signaling events that occur subsequent to activation of guanylate cyclase by NO derived from these agents. Because the simultaneous administration of superoxide dismutase (scavenges superoxide radicals) and SNP or L-arginine, but not superoxide dismutase alone, decreased L-NAME-induced leukocyte adherence, our results suggest that leukocyte adhesion caused by NO synthase inhibition may result in the generation of superoxide. PMID- 7544335 TI - Identification of heterogeneous cell populations in normal human intervertebral disc. AB - There is clear evidence that tissues related to the intervertebral disc, such as articular cartilage, contain several phenotypically different chondrocytic cell populations. Histological data for the disc suggest the same may be true for the annulus fibrosus and nucleus pulposus, but this has not been shown directly. For the first time, cells from adult human nondegenerative nucleus pulposus and annulus fibrosus were recovered after enzymatic digestion and maintained in an alginate bead culture system for up to 6 wk. The cells remained viable and produced matrix, but did not divide. Cultured cells were stained simultaneously for the presence of chondroitin sulphate and keratan sulphate, or types I and II collagen. The majority of the cells from both the annulus fibrosus and the nucleus pulposus produced both keratan sulphate and chondroitin sulphate (> 60%), a few only detectable levels of one or the other, but a significant population produced neither. This is an indication of a population of cells with a nonchondrocytic phenotype. In nondegenerative discs, the majority of the annulus fibrosus cells produced both types I and II collagen but the majority of nucleus pulposus cells produced only type II collagen. These observations are consistent with the presence of at least 2 phenotypically stable populations of cells in the adult human intervertebral disc and with the view that the phenotype of the major population of the annulus is different from that of the nucleus. PMID- 7544342 TI - Clinical experience with risperidone. PMID- 7544339 TI - Sensory nerve- and neuropeptide-mediated relaxation responses in airways of Sprague-Dawley rats. AB - We examined the role of sensory nerves in mediating nonadrenergic inhibitory responses in airway segments isolated from male Sprague-Dawley rats. In the presence of adrenergic blockade, capsaicin (Cap; 1 microM) elicited marked relaxation responses in isolated bronchi precontracted with bethanechol (Beth). Cap-induced inhibitory responses were unaffected by tetrodotoxin (TTX), were attenuated by incubation of the airway with indomethacin (Indo), phosphoramidon, or RP-67580, but were abolished by previous exposure of the airway to Cap and by denuding the epithelium. Substance P (SP; 1 microM), neurokinins A and B (1 microM), and calcitonin gene-related peptide (0.1 microM) relaxed Beth-contracted airway segments to a similar extent. The SP-induced responses were unaffected by adrenergic blockade or by pretreatment with either TTX, phosphoramidon, or Cap, but were attenuated by RP-67580 and abolished by Indo and by denuding the epithelium. In anesthetized mechanically ventilated rats, Cap (50 and 100 micrograms/kg i.v.) elicited a dose-dependent reversal of the increase in lung resistance induced by an infusion of Beth. The Cap-induced bronchodilation was unaffected by pretreatment with propranolol alone or in combination with hexamethonium. SP (44 nmol/kg iv) also evoked bronchodilatory responses in intact animals, which were unaffected by propranolol and hexamethonium but were abolished by treatment of the animals with Indo. Electrical-field stimulation (EFS) evoked nonadrenergic noncholinergic relaxation responses in contracted airway segments. These EFS-induced inhibitory responses were markedly attenuated by treatment of the airway segment with TTX, Cap, or RP-67580. We conclude that neuropeptides released from Cap-sensitive sensory nerves have potent inhibitory effects in rat airways that are mediated, in part, by activation of neurokonin NK1 receptors on epithelium and subsequent release of an inhibitory prostaglandin(s). PMID- 7544336 TI - TAN-1511 A, B and C, microbial lipopeptides with G-CSF and GM-CSF inducing activity. AB - The microbial lipopeptides, TAN-1511 A, B and C, were isolated from the culture broth of Streptosporangium amethystogenes subsp. fukuiense AL-23456. Their structures were elucidated on the basis of their reactions and spectroscopic analyses. These lipopeptides were mixtures of molecules having different lengths of fatty acids. The metabolites stimulated the proliferation of bone marrow cells from BALB/c female mice at very low concentrations (concentration giving 30% increase: A and B, 0.313 ng/ml; C, 1.25 ng/ml). We confirmed that chemically synthesized TAN-1511 A analogue [(2R,6R)-2-tetradecanoylamino-6,7- bis(hexadecanoyloxy)-4-thiaheptanoyl-Gly-Gly-Gly-Glu-Thr-Thr -OH] stimulated the proliferation of bone marrow cells in a manner similar to that of natural TAN 1511 A. This analogue induced the secretion of both granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), and potentiated the generation of Gr-1 positive cells in the bone marrow cell culture. Moreover, it effected the G-CSF mediated restoration of granulocytopoiesis in a murine leukopenia model. PMID- 7544343 TI - Gene regulation by antisense DNA produced in vivo. AB - Antisense technology has been widely used for regulating gene expression. Single stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein. PMID- 7544341 TI - Role of C fibers in physiological responses to ozone in rats. AB - The purpose of this study was to evaluate the role of C fibers in airway responsiveness after exposure to ozone (O3) in rats. The role of C fibers in the decreases in heart rate (HR) and core body temperature (Tc) that occur after inhalation of O3 was also examined. Neonatal rats were treated with capsaicin (Cap) or the vehicle used to dissolve capsaicin (Veh). Cap has been shown to cause permanent destruction of C fibers. When they reached adulthood, conscious minimally restrained rats were exposed to 2 ppm O3 or to air for 3 h. Two hours after the cessation of exposure, rats were anesthetized and instrumented for the measurement of pulmonary mechanics and airway responsiveness to inhaled aerosolized methacholine. O3 had no effect on baseline pulmonary conductance (GL) in either Veh or Cap rats but did cause a decrease in dynamic compliance (Cdyn) in Cap rats (P < 0.05). In Cap rats, O3 exposure caused a marked increase in airway responsiveness; the doses of inhaled aerosolized methacholine required to decrease GL and Cdyn by 50% were 6.5-fold and 9.8-fold lower in O3-compared with air-exposed rats (P < 0.005). In contrast, in Veh rats, O3 did not alter responsiveness. During O3 exposure, there was a profound, almost 50%, decrease in HR as measured with implanted electrodes. A decrease in Tc (measured with a rectal probe) of approximately 2.5 degrees C also occurred during O3 exposure. There was no significant effect of Cap pretreatment on the magnitude of these O3 induced changes in HR and Tc. Our results are consistent with the hypothesis that C fibers act to inhibit the development of hyperresponsiveness elicited by O3 inhalation but do not contribute to O3-induced changes in HR or Tc. PMID- 7544344 TI - Identity of the segment of human complement C8 recognized by complement regulatory protein CD59. AB - CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma. PMID- 7544345 TI - Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity. AB - In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT. PMID- 7544347 TI - Targeted inactivation of the mouse alpha 2-macroglobulin gene. AB - The mouse alpha 2-macroglobulin gene was inactivated in embryonic stem cells by homologous recombination. Liver alpha 2-macroglobulin mRNA and plasma protein was absent in homozygotes and reduced to 50% in heterozygotes. alpha 2-Macroglobulin deficient mice were viable and produced normally sized litters with normal sex ratio over 3 generations. Characterization of adult homozygotes included diets with different fat content, treatments with endotoxin, bleomycin, carbon tetrachloride, and ethionine to test for immune system, lung, liver, and pancreas toxicity, respectively. Knock-out mice were more resistant to endotoxin but more sensitive to a choline-free diet supplemented with ethionine. Regulation of murinoglobulin mRNA expression during pregnancy was analyzed as a possible back up mechanism for the deficiency in alpha 2-macroglobulin. In addition, expression of mRNA was studied, coding for alpha 2-macroglobulin receptor/lipoprotein receptor-related protein, low density lipoprotein receptor, and very low density lipoprotein receptor and for some common ligands, i.e. apolipoprotein E, lipoprotein lipase, and the 44-kDa heparin binding protein. Their differential regulation in the knock-out mice relative to C57B1 mice was evident and is discussed. The impressive 15-fold increase in maternal liver murinoglobulin mRNA at partum in the knock-out mice indicated increased consumption, compared to only 4-fold in normal mice. Thus, murinoglobulin appears as the major proteinase inhibitor around partum, obviously solicited to a much greater extend in alpha 2 macroglobulin-deficient mice. PMID- 7544349 TI - Interaction of single-chain urokinase and plasminogen activator inhibitor type 1. AB - Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. 125I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of 125I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. 125I-scu-PA.PAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type plasminogen activator in lacking a complete zymogen conformation. PMID- 7544350 TI - Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits. AB - Protein-tyrosine phosphorylation has recently been suggested to play an important role in synaptic transmission at the neuromuscular junction. The role of tyrosine phosphorylation in the modulation of synaptic function in the central nervous system, however, is not clear. In this study, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrates that there are high levels of phosphotyrosine, which co-localizes with glutamate receptors at excitatory synapses on cultured hippocampal neurons. In addition, the tyrosine phosphorylation of various subtypes of glutamate receptors were examined using subunit-specific antibodies. Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and are classified into three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based on their electrophysiological and pharmacological properties. NMDA receptors play a central role in synaptic plasticity, synaptogenesis, and excitotoxicity and are thought to be heteromeric complexes of the two types of subunits: NR1 and NR2(A D) subunits. Immunoaffinity chromatography of detergent extracts of rat synaptic plasma membranes on anti-phosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immunoprecipitation of the NR1, NR2A, and NR2B subunits with subunit specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies confirmed that the NR2A and NR2B subunits but not the NR1 subunit were phosphorylated on tyrosine residues. No tyrosine phosphorylation of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunits was detected. It was estimated that 2.1 +/- 1.3% of the NR2A subunits and 3.6 +/- 2.4% of the NR2B subunits were tyrosine-phosphorylated in vivo. In addition, endogenous protein tyrosine kinases in synaptic plasma membranes phosphorylated the NR2A subunit in vitro, increasing its phosphorylation 6-8-fold but did not phosphorylate NR1 or NR2B. These studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of the NR2 subunits may be important for regulating NMDA receptor function. PMID- 7544346 TI - Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. AB - Vascular endothelial growth factor (VEGF) is an endothelial specific angiogenic mitogen secreted from various cell types including tumor cells. Increasing evidence suggests that VEGF is a major regulator of physiological and pathological angiogenesis, and the VEGF/VEGF receptor system has been shown to be necessary for glioma angiogenesis. Hypoxia seems to play a critical role in the induction of VEGF expression during glioma progression. C6 glioma cells provide an in vivo glioma model for the study of tumor angiogenesis, and the expression of VEGF in C6 cells has been shown to be up-regulated by hypoxia in vitro. However, little is known about the molecular mechanism of hypoxic induction of VEGF. Here, we demonstrate that hypoxic induction of VEGF in C6 cells is due to both transcriptional activation and increased stability of mRNA. Nuclear run-on assays revealed a fast and lasting transcriptional activation, whereas the determination of mRNA half-life showed a slower increase of mRNA stability during hypoxia. Reporter gene studies revealed that hypoxia responsive transcription activating elements were present in the 5'-flanking region of the VEGF gene. These results suggested that several distinct molecular mechanisms were involved in hypoxia-induced gene expression and were activated in a biphasic manner. PMID- 7544348 TI - Identification of nitric oxide synthase as a thiolate-ligated heme protein using magnetic circular dichroism spectroscopy. Comparison with cytochrome P-450-CAM and chloroperoxidase. AB - Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size. PMID- 7544351 TI - Biochemical characterization of a human band 4.1-related protein-tyrosine phosphatase, PTPH1. AB - PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M(r) of 120,000 on SDS gels. The native enzyme dephosphorylated both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP. The Km values for the two substrates were similar (1.45 microM for MBP and 1.6 microM for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in Km but had no effect on Vmax. Removal of the NH2 terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited its activity toward MBP. The dephosphorylation of RCML by full-length PTPH1 was enhanced up to 6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing the isolated catalytic domain were unaffected. These results suggest that in addition to a potential role in controlling subcellular localization, the NH2-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function. PMID- 7544353 TI - T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38. AB - Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells. PMID- 7544354 TI - Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation-defective ras-transfected keratinocyte line. AB - The role of ras on protein kinase C (PKC) signaling was examined in two keratinocyte cell lines. Increasing the level of extracellular calcium from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c-H-ras-transfected subline (ras HaCaT). Raising extracellular calcium also induced a transient increase in membrane-associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras-HaCaT cells. Partial purification of PKC from the membrane/particulate fraction revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti-PKC alpha antibody. In ras-HaCaT, the major expressed isoform is a 130 KD species recognized by the PKC beta antibody. The kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras HaCaT membrane/particulate cell fraction by immunoblotting using affinity purified antibodies against PKC alpha, beta, delta, epsilon and zeta revealed a 130 KD band reacting with the PKC delta antibody. Increased expression of this high molecular weight protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ras-HaCaT using the PKC delta antibody also revealed a 130 KD species. Analysis of the PKC delta immunoprecipitate demonstrated a phospholipid, but not calcium-dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium-independent) PKC (nPKC) isoform and increased expression in the ras-transfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras associated inhibition of differentiation in HaCaT. PMID- 7544352 TI - Generation of a monoclonal antibody that recognizes the amino-terminal decapeptide of the B-subunit of Escherichia coli heat-labile enterotoxin. A new probe for studying toxin assembly intermediates. AB - Cholera toxin and the related Escherichia coli heat-labile enterotoxin are hexameric proteins comprising one A-subunit and five B-subunits. In this paper we report the generation and characterization of a monoclonal antibody, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B subunit (EtxB) of E. coli heat-labile enterotoxin. The monoclonal antibody is unable to precipitate native B-subunit pentamers, thus making LDS47 a useful probe for studying the early stages of enterotoxin biogenesis. The use of LDS47 to monitor the in vivo turnover of newly synthesized B-subunits in the periplasm of E. coli demonstrated that (i) the turnover of unassembled B-subunits followed an apparent first order process and (ii) it occurred concomitantly with the assembly of native B-pentamers (k = 0.317 +/- 0.170 min-1; t1/2 = 2.2 min). No other proteins were co-precipitated with the newly synthesized B-subunits; a finding that implies that unassembled B-subunits do not stably associate with other periplasmic proteins prior to their assembly into a macromolecular complex. The use of overlapping synthetic peptides corresponding to the entire EtxB polypeptide demonstrated that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of the B-subunit. From the x-ray structural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., Dauter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mol. Biol. 230, 890-918), this region appears to resemble a curved finger that clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become partially buried upon assembly and thus inaccessible to recognition by the monoclonal antibody. PMID- 7544356 TI - Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice. AB - We have investigated the phenotypic and functional characteristics of murine pre B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/lpr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1-, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12-14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 x 10(6) pre-B cells had readily detectable serum levels of IgM (54 +/- 26 micrograms/ml) and IgG (60 +/- 95 micrograms/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (> 80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. PMID- 7544355 TI - PC12-E2 cells: a stable variant with altered responses to growth factor stimulation. AB - A variant cell line, designated E2, characterized by more rapid responses to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) and markedly more robust responses to interleukin-6 and 8-Br-cAMP, has been subcloned from the rat PC12 cell line. The enhanced responsiveness to NGF in E2 cells is not due to receptor overexpression as judged by TrkA protein levels and tyrosine kinase activity, but may be associated with the increased and prolonged tyrosine phosphorylation of ERK1 (extracellular signal regulated kinase 1) and ERK2. The rapid morphological differentiation induced by different growth factors in E2 cells is mediated in a transcription-independent manner suggesting that E2 cells may constitutively express some differentiation-associated molecules that allow direct entry into the neuronal program. PMID- 7544357 TI - Interaction of CD44 variant isoforms with hyaluronic acid and the cytoskeleton in human prostate cancer cells. AB - CD44 is a glycosylated adhesion molecule which may undergo alternative splicing of 10 possible exons to generate variant isoforms. A number of CD44 variant isoforms expressed by tumor cells have been correlated with metastatic and proliferative behavior. In this study, we have characterized CD44 isoform expression on three prostate cancer cell lines: ALVA-31, PPC-1, and LNCaP. Using reverse transcriptase-polymerase chain reaction, we have found that ALVA-31 and PPC-1 cells express multiple CD44 isoforms, including CD44s (standard form), CD44E (epithelial form), and an exon 14-containing form. In addition, two smaller forms have been detected: one using an alternative donor splice site within exon 5, and a novel form omitting exon 5 entirely. The CD44 isoforms expressed by ALVA 31 and PPC-1 cells appear to be preferentially located on the cell surface. By contrast, LNCaP cells do not express any of the CD44 forms at the RNA or protein level. Both PPC-1 and ALVA-31 cells display tumorigenesis and invasiveness in nude mice, whereas LNCap cells exhibit a less malignant phenotype, suggesting a correlation between CD44 variant (CD44v) expression and aggressive prostate tumor behavior. Functional characterization reveals that CD44 mediates prostate cell adhesion to extracellular hyaluronic acid (HA). In addition, the CD44 cytoplasmic domain binds specifically to ankyrin, a membrane cytoskeletal protein. Double immunofluorescence labeling and confocal microscopic analyses indicate that HA binding induces the HA receptor (i.e., CD44) to form capped structures. Importantly, intracellular ankyrin is preferentially accumulated underneath HA receptor-capped structures. These results suggest that cytoskeletal proteins such as ankyrin are closely associated with CD44-mediated signaling events induced by HA. Finally, HA-mediated transmembrane interactions between CD44 isoforms and cytoskeletal proteins (i.e. ankyrin) may play a pivotal role in regulating tumor cell behavior during human prostate cancer development. PMID- 7544358 TI - Evidence for the presence of a Hg-inhibitable water-permeability pathway and aquaporin 1 in A5 salivary epithelial cells. AB - The molecular basis of water-permeability in salivary and other exocrine glands is not understood. We have examined two well-studied salivary epithelial cell lines for evidence of a Hg-inhibitable water-permeability pathway. A5 and HSG cells are derived from rat and human submandibular glands, respectively. Only A5 cells demonstrated such a pathway. The rate of A5 cell osmotic shrinkage was inhibited about fivefold in the presence of 300 microM HgCl2. To determine if this activity was associated with the expression of the prototypical water channel (aquaporin, AQP) AQP1, we used three separate experimental approaches; Northern analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis of isolated mRNA, and Western analysis of cell membranes. All three methods yielded positive results with A5 cells and negative results with HSG cells. The approximately 800 bp product of RT-PCR was analyzed further by sequencing and restriction enzyme digestion. The results were consistent with the previously reported coding region sequence for rat kidney AQP1. The aggregate data demonstrate that marked differences in water-permeability and water channel expression exist in these two salivary epithelial cell lines. PMID- 7544359 TI - Synergistic interaction of interleukin-1 beta and growth factors in primary cultures of rat aortic smooth muscle cells. AB - Activated macrophages release cytokines and growth factors that may contribute to the growth of vascular smooth muscle cells in injured blood vessels. In the present study, we investigated the interactions between interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (FGF-2) in primary rat aortic smooth muscle cells, relative to their effects on DNA synthesis and cell proliferation. We report that femtomolar levels of IL-1 beta, which alone were non-mitogenic or weakly mitogenic, synergistically increased FGF-2-induced [3H]thymidine incorporation and cell proliferation. The potentiating effect of IL-1 beta extended to PDGF-AB and EGF, but not to IGF-1-induced thymidine incorporation. An antagonist of the IL-1 receptor, IL-1ra, blocked the co-mitogenic effect of IL-1 beta. Stimulation of cells with FGF-2 and IL-1 beta increased both DNA content and proliferation, an observation that was consistent with the thymidine incorporation experiments. An inhibitor of NO synthase, N5-iminoethyl L-ornithine (L-NIO), did not block the co-mitogenic effect of IL-1 beta, despite effective inhibition of NO synthase activity, suggesting that the synergistic interaction between IL-1 beta and FGF-2 was independent of the NO/cGMP pathway. The mechanism of co-mitogenesis appeared to be independent of the intermediacy of PDGF-AA, IL 6, and prostanoids, and was not associated with increased levels of c-fos mRNA, FGF receptor-1 protein, or FGF-2-induced early and delayed tyrosine phosphorylation events. We conclude that IL-1 beta interacts with FGF-2 to amplify the proliferation of primary rat aortic smooth muscle cells, an effect that may be important in vascular smooth muscle cell proliferation following vascular injury. PMID- 7544360 TI - Induction of angiogenesis by smooth muscle cell-derived factor: possible role in neovascularization in atherosclerotic plaque. AB - The development of atherosclerotic plaque is associated with neovascularization in the thickened intima and media of vascular walls. Neovascularization may have a role in the progression of atherosclerotic plaque as well as in the development of intraplaque hemorrhage. However, the mechanism and stimulus for neovascularization in atherosclerotic plaque are unknown. We postulated that smooth muscle cells (SMCs), a major cellular component in the vascular wall, might contribute to the induction of neovascularization in atherosclerotic plaque through the secretion of an angiogenic factor. We observed that endothelial cells (ECs) cultured on collagen gel with SMC-conditioned medium became spindle shaped, invaded the underlying collagen gel, and organized a capillary-like branching cord structure in the collagen gel. The conditioned medium also stimulated EC proliferation and increased the EC-associated plasminogen activator activity. The angiogenic factor in SMC-conditioned medium was retained in a heparin-Sepharose column and eluted with 0.9 M NaCl. Neutralizing anti-vascular endothelial growth factor (VEGF) antibody attenuated the angiogenic activity in the conditioned medium, including the induction of morphologic changes in ECs, mitogenic activity, and increased plasminogen activator activity associated with ECs. Immunoblotting analysis confirmed the secretion of VEGF from SMCs. These observations indicate that SMC may be responsible for the neovascularization in atherosclerotic plaque through the secretion of VEGF. PMID- 7544361 TI - Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos. AB - Human embryos have been biopsied at either the cleavage or the blastocyst stage of development. One to two blastomeres were removed from cleavage-stage embryos and 2-6 cells from blastocysts. The biopsy specimens were subjected to gene amplification by the polymerase chain reaction (PCR) and a comparison made of amplification efficiencies of two unique target sequences, one located within the beta-globin gene and containing the sickle-cell locus and the other a polymorphic dinucleotide repeat. When the cleavage-stage biopsy sample consisted of an intact blastomere with a clearly discernible nucleus, an amplification efficiency of 89% was achieved for each target locus. This was similar to that achieved with cleavage-stage biopsy samples consisting of two blastomeres or with blastocyst biopsy samples consisting of 2-3 trophectoderm cells. When biopsy samples consisted of four or more trophectoderm cells, both target loci were amplified in all samples tested. When the biopsy sample was heterozygous at the dinucleotide repeat locus and the biopsy consisted of one or more intact cells with a clearly discernible nucleus, both alleles were amplified in > 80% of biopsy samples. When four or more trophectoderm cells were used for the PCR, both alleles were amplified in all heterozygous samples. Target sequences were never amplified from biopsy samples which lysed prior to transfer into the reaction tube. Analysis of DNA fragments amplified from the dinucleotide repeat locus indicated that in most cases faithful amplification of biopsy DNA template had taken place.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544362 TI - The effect of high-dose medium- and long- term progestogen exposure on endometrial vessels. AB - A total of 19 paraffin-embedded endometrial tissue blocks were obtained from high dose progestogen-exposed patients. A labelled streptavidin-biotin-alkaline phosphatase method was used with antibodies against von Willebrand factor (vWF) and CD34. The density of CD34 and vWF positive (CD34+ and vWF+) vessels in progestogen-exposed endometria (103 +/- 9.6/mm2 and 106 +/- 8.7/mm2) was significantly lower than in endometria from women with normal cycles (169 +/- 9.3/mm2 and 136 +/- 8.0/mm2) (P < 0.05). In women with normal menstrual cycles the concentration of CD34+ vessels was significantly higher than the number of vWF+ vessels (P = 0.0001). By comparison, the concentration of CD34+ vessels was similar to the concentration of vWF+ vessels in progestogen-exposed endometria. The ratios of vascular density as determined by vWF+ and CD34+ staining the control and progestogen groups were 0.81 and 1.05 respectively (P = 0.0001). Dilated venules were seen in the progestogen group. This study has demonstrated firstly that CD34 antibody detected the endothelial cells in a higher proportion of small endometrial vessels than vWF, and secondly that high-dose progestogen exposure significantly decreased the density of microvessels and increased the number of dilated venules in endometrium. PMID- 7544363 TI - Fluorescence of experimental endometriosis in rabbits, using tamoxifen-eosin association. AB - A major problem with endometriosis is the detection of microscopic and atypical lesions. An incomplete surgical eradication may lead to recurrence of the disease. This study aimed to investigate the diagnostic improvement of endometriosis by the use of tamoxifen-eosin induced fluorescence. The experimental study was performed on surgically induced endometriosis in the rabbit. Endometriosis was induced by grafting endometrium onto the broad ligament in 10 rabbits. After 5 weeks, the fluorescence of excised endometriosis was studied after systemic injection of tamoxifen and local application of eosin. Healthy peritoneal samples served as controls. The fluorescence of endometriotic foci was also compared with (n = 5) or without (n = 5) tamoxifen. Fluorescence excitation was carried out using a 150 W filtered lamp connected to an optical fibre. Fluorescence emission was measured using an optical fibre connected to a spectrofluorometer. Spectral analysis showed a specific fluorescence of endometriosis 72 h after systemic injection of tamoxifen and eosin application. This result is explained by binding to oestrogen receptors of tamoxifen which was protonized to form an ionic pair with eosin. Histological study of samples from the graft of endometrial tissue showed that experimental endometriosis had developed in eight out of the 10 rabbits. However, the fluorescence was not significantly different among the 10 rabbits. This observation was in accordance with previous studies in which endometriosis was confirmed by routine histological techniques or electron microscopy in 70-80% of cases. Consequently, the fluorescence of the two samples which did not present histological evidence of endometriosis indicates the presence of microscopic endometriotic foci.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544364 TI - Genetic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis revealed by restriction fragment length polymorphism of the rRNA gene region. AB - The restriction fragment length polymorphism patterns of rDNAs from Pseudomonas aeruginosa strains isolated from the respiratory tracts of patients suffering from cystic fibrosis were obtained to evaluate the genetic polymorphism of this population of strains. Eighty-seven P. aeruginosa strains isolated from 87 patients from diverse areas of France and the ATCC 10145 strain were examined. Four restriction enzymes were used: BamHI, ClaI, EcoRI, and PstI. Forty-nine strains (56%) were in the three most frequent ribotypes (ribotypes R1 to R3). In addition, hierarchical clustering analysis of the data showed that 71 of the 88 strains (81%) clustered at a distance of less than one-third of the greatest distance observed in the total population. This indicates that clinical isolates implicated in the pathology of cystic fibrosis present a low degree of heterogeneity of rRNA operons, in contrast to the heterogeneity of strains of P. aeruginosa isolated from patients with various other pathologies. This relative homogeneity of rRNA genes was observed independently of the clinical status of the patient and the colony morphology. PMID- 7544365 TI - Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens. AB - In a prospective comparative study, 2,696 consecutive fresh stool specimens over the course of 1 year were examined for Giardia lamblia and Cryptosporidium parvum by using a direct immunofluorescent-monoclonal antibody stain (for unspun specimens) and conventional staining methods (chlorazol black E for Giardia cysts and modified Kinyoun acid-fast for Cryptosporidium oocysts). The direct immunofluorescent-monoclonal antibody method resulted in a significantly increased detection rate for both giardia (118 versus 79 specimens, 49.4%; P = 0.006) and cryptosporidia (39 versus 23 specimens, 69.6%; P = 0.055). PMID- 7544366 TI - Intraerythrocytic presence of Bartonella henselae. AB - Recent reports in the medical literature emphasize the risk of zoonotic disease and the high degree of prevalence of asymptomatic feline infection with Bartonella (Rochalimaea) henselae. While investigating Bartonella bacteremia in cats, we used transmission electron microscopy to demonstrate B. henselae in the erythrocytes of persistently bacteremic cats. PMID- 7544369 TI - Immunogenicity of biliary epithelial cells: study of the expression of B7 molecules. AB - Efficient antigen presentation requires the provision of a co-stimulatory signal, the best characterized of which is provided by the B7 molecules. It is unclear whether biliary epithelial cells expressing Class II major histocompatibility complex molecules can function as antigen presenting cells, although this has been suggested as an important mechanism in the initiation and/or perpetuation of some immune-mediated liver diseases, including primary biliary cirrhosis and liver allograft rejection. We have found that human intrahepatic biliary epithelial cells do not express B7-1 (CD80) or B7-2 in vitro, even after activation with high doses of interferon-gamma, tumour necrosis factor-alpha or phorbol myristate acetate, or in vivo. However, they express similar levels of class II major histocompatibility complex antigens to those expressed by professional antigen presenting cells (Epstein-Barr virus transformed B cells). It is therefore unlikely that biliary epithelial cells stimulate efficient primary T cell activation. It may be possible that these non co-stimulatory class II major histocompatibility complex positive cells play a role in modulating immune responses in the liver. PMID- 7544368 TI - In patients with orthotopic liver transplantation, serum markers of cholestasis are unreliable indicators of biliary secretion. AB - BACKGROUND/AIMS: In patients after orthotopic liver transplantation, treatment with the novel immunosuppressant FK 506 may lead to elevated levels of alkaline phosphatase, gamma glutamyl transferase and bilirubin. Up to now it was unclear whether the excretory capacity of the liver in such patients is impaired. METHODS: We measured quantitatively the secretion of bile acids, phospholipids and cholesterol using the duodenal perfusion method, which allows assessment of biliary secretion without interruption of the enterohepatic circulation. Six healthy volunteers served as controls. RESULTS: All patients studied after orthotopic liver transplantation had elevated concentrations of serum alkaline phosphatase and gamma glutamyl transferase, whereas only half of them had slightly abnormal serum bilirubin levels. On average, the FK 506-treated patients excreted 1.23 +/- 0.27 mmol/h bile acids, 0.23 +/- 0.04 mmol/h phospholipids and 0.11 +/- 0.02 mmol/h cholesterol, which was not significantly different from the healthy controls. CONCLUSIONS: The normal secretion rates of biliary bile acids and lipids in FK 506-treated patients with elevated serum alkaline phosphatase and gamma glutamyl transferase indicate that the excretory capacity of the transplanted liver has completely recovered 2-3 months after surgery. In addition, in the majority of these patients elevated serum levels of alkaline phosphatase, gamma glutamyl transferase and bilirubin do not reflect impaired biliary secretion. PMID- 7544367 TI - Combined inhibition effects of tacrolimus and methylprednisolone on in vitro human lymphocyte proliferation. AB - Tacrolimus (FK 506) has synergistic immunosuppressive effects in combination with corticosteroids. A steroid dose-lowering effect can be explained partly by the inhibition by FK 506 of cytochrome P-450 IIIA, which metabolizes corticosteroids. We investigated the influence of combinations of FK 506 and methylprednisolone (MPL) on the suppression of in vitro human lymphocyte proliferation. To quantify the type of drug interaction (synergistic, additive, and antagonistic), three methods (Drewinko's statistical analysis, median-effect principle, and normal distribution model) were used. The interaction appeared synergistic in most combination ratios, but the extent of synergism varied depending on the quantitative method. There may be optimal combination ratios of FK 506 and MPL which achieve more effective immunosuppressive effects. PMID- 7544370 TI - Probable G-CSF-induced hepatitis and pancreatitis in an HIV-seropositive patient. PMID- 7544371 TI - IL-4 plus CD40 monoclonal antibody induces human B cells gamma subclass-specific isotype switch: switching to gamma 1, gamma 3, and gamma 4, but not gamma 2. AB - Stimulation with IL-4 plus CD40 mAb is known to induce production of IgE and IgG4. In this study, we determined the IgG subclass specificity of IL-4 plus CD40 mAb stimulation for human purified B cells. We determined true in vitro switching by the generation of switch circular DNA (S gamma/S mu) representing primary S mu/S gamma events and production of gamma subclass-specific germ-line transcripts by a combination of reverse transcription-PCR and restriction endonuclease digestion. We simultaneously measured changes in the levels of IgG subclass proteins produced. Forty-two clones of circular switch DNA were identified and sequenced. The IgG subclass of S gamma fragment in the S gamma/S mu chimeric PCR products was determined by analyzing key S gamma nucleotides. The switch-deleted clones were found to consist of S gamma 1/S mu, S gamma 3/S mu, and S gamma 4/S mu chimeric switch sequences, showing that such switching had occurred. No S gamma 2/S mu chimeric switch sequences were found. While a consensus sequence was not identified at the S gamma/S mu breakpoints, four contiguous guanines (GGGG) were noticeably present in the S gamma region near the breakpoint. The induction of gamma 1, gamma 3, and gamma 4 switch circles in human purified B cells was accompanied by enhanced production of IgG1, IgG3, and IgG4 but not IgG2. Similarly, stimulation with IL-4 alone induced gamma 1, gamma 3, and gamma 4 but not gamma 2 germ-line transcripts. These results demonstrate that IL-4 plus CD40 mAb induces Ig isotype switch from mu to gamma 1, gamma 3, and gamma 4 but not to gamma 2. PMID- 7544372 TI - Stimulation of human bone marrow stromal cell tyrosine kinases and IL-6 production by contact with B lymphocytes. AB - The interaction of normal human B cell precursors with the bone marrow (BM) stromal cells that support their growth and maturation is mediated by adhesion of B cell CD49d/CD29 (alpha 4 beta 1 integrin or VLA-4) to stromal cell signal transduction, we generated CD49d-deficient variants of the RAMOS early b Burkitt's lymphoma cell line. RAMOS was chosen due to its lack of expression of CD49e (alpha 5 integrin) and CD44, which could potentially interact with stromal cells or their extracellular matrix. In contrast to the parental RAMOS cell line, CD49d-deficient RAMOS cell lines did not adhere to primary human BM stromal cells. Tyrosine phosphorylation of several stromal cell proteins was stimulated within 5 to 10 min of contact with either CD49d-expressing or CD49d-deficient RAMOS. These proteins included a 110-kDa substrate tentatively identified as pp125FAK. In contrast, mAb cross-linking of stromal cell CD106 had no significant effect on protein tyrosine phosphorylation. Il-6 production by stromal cells was enhanced up to fourfold over 3 days when stromal cells were cultured in contact with RAMOS cells, regardless of CD49d expression. Stromal cell IL-6 production was not significantly enhanced by RAMOS cells cultured above the stromal cells in Transwell inserts. Thus, we have demonstrated through the use of paired CD49d expressing and CD49d-deficient RAMOS cell lines that protein tyrosine kinase activity and IL-6 production by primary human BM stromal cells can be enhanced in a manner that is dependent on B cell contact but independent of CD49d-mediated adhesion. These results indicate the potential for B cells to interact with cells in their microenvironment and activate feedback mechanisms mediated by stromal cells, which may regulate lymphopoiesis. PMID- 7544373 TI - Initiation of the phosphocholine-specific response to Proteus morganii. B cell transfectants expressing unmutated VH/VL can respond to stimulation by P. morganii antigen. AB - We wished to resolve a paradox of how the response to the phosphocholine (PC) determinant of Proteus morganii could be initiated from a precursor B cell whose receptor, in unmutated form as Ab, appears to be unable to bind Ag. Unmutated VH and unmutated VL constructs were co-transfected into the B cell lymphoma M12.4 to study stimulation via membrane lg (mlg). The same VH construct was expressed in an L+, H- hybridoma line to characterize Ab binding. The unmutated Ab showed no detectable binding in ELISA to the PC-containing Ag from P. morganii PC(PM). By contrast, the unmutated mlg mediated mobilization of calcium in response to the PC(PM) Ag. Single-positive B cell lines of mlgM, mlgD double-positive lines were all capable of responding. The degree of signaling depended greatly on high receptor number, and only a fraction of cells in the population responded. Inhibition of the PC(PM)-induced calcium response by free PC indicated that the response was Ag-specific. A transfectant B cell line expressing moderate levels of a high affinity, mutated mlgM readily responded to PC(PM). These observations indicate that the unmutated lg as a receptor is capable of interacting with PC(PM) and suggest that the immune response to PC(PM) could originate from the precursor B cell expressing the unmutated mlg. The role of mlgD vs mlgM is discussed in terms of the requirement for high receptor number in the signaling process. PMID- 7544375 TI - Alloreactive 2C T cells recognize a self peptide in the context of the mutant Kbm3 molecule. AB - Previous studies hav shown that 2C T cells have dual specificity for Ld and Kbm3 alloantigens and are positively selected in the context of Kb. Analysis of Kbm3 eluted peptides reveals a single HPLC fraction that is a central component of the Kbm3 allodeterminant. Peptides with biologic and biochemical properties indistinguishable from those of the Kbm3 allopeptide were also isolated from Kb and Kbm8, despite the fact that cells bearing these class I molecules are not efficient targets for 2C T cells. The allopeptide was isolated and compared with p2Ca, the Ld allopeptide recognized by 2C T cells in the context of Ld. The Kbm3 allopeptide was found to be biochemically and functionally different from p2Ca. Therefore, the 2C TCR has dual specificity for two different peptides presented in the context of two structurally distinct class I molecules. The possibility of a common peptide being bound by Kb, Kbm3, and Kbm8 suggests that the peptide defined as an allodeterminant in the context of Kbm3 may also function in the positive selection of 2C T cells when presented in the context of Kb and Kbm8 during thymic development. In support of this view, the allopeptide was also found in Kb and Kbm8 thymic peptide eluates. PMID- 7544376 TI - RANTES-activated human T lymphocytes. A role for phosphoinositide 3-kinase. AB - RANTES (regulated on activation, normal T cell expressed and secreted), a member of the chemokine family, is a potent chemoattractant for CD4+/CD45RO human T lymphocytes, but the signal-transduction mechanisms utilized by RANTES are poorly defined. In freshly isolated human T lymphocytes loaded with fura-2 acetoxymethyl, the CD3 mAb, UCHT-1, but not RANTES, elicited elevation of intracellular calcium levels. However, RANTES produced a bell-shaped chemotactic response and an increase in polarization of the T lymphocytes. Immunoprecipitates of phosphoinositide (PI) 3-kinase, derived from T lymphocytes stimulated with RANTES, contained increased in vitro PI 3-kinase activity compared with that present in immunoprecipitates derived from vehicle-treated cells. RANTES induction of PI 3-kinase activity was maximal at 10 to 100 ng/ml. Furthermore, the fungal metabolite, wortmannin, which is a potent PI 3-kinase inhibitor, inhibited RANTES-induced T lymphocyte migration, polarization, and increased PI 3 kinase activity. Our results show that RANTES activation of T lymphocytes seems to be independent of detectable elevation of cytosolic-free calcium, but the functional effects of chemotaxis and polarization, induced by RANTES, seem to involve the putative PI 3-kinase signal-transduction pathway. PMID- 7544378 TI - A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2. AB - The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses. PMID- 7544374 TI - Characterization of human CD7 transgenic mice. AB - CD7 is a 40-kDa transmembrane glycoprotein member of the lg gene superfamily expressed on most peripheral blood T lymphocytes and NK cells. CD7 is also expressed on myeloid, NK, B, and T cell precursors during adult hematopoiesis. Because Thy-1 is absent in human thymocytes and peripheral blood T cells and shows structural similarities to the human CD7 gene, we have suggested that human CD7 may be a functional homologue in humans of mouse Thy-1. To study the tissue specific expression of the CD7 gene utilizing its own promoter, we constructed transgenic mice that contained both the coding and flanking regions of the human CD7 gene. We found that human CD7 was expressed in transgenic mice in T, B, NK, and myeloid lineages and was induced with T cell activation. Unlike the expression of CD7 in humans, the CD7 transgene was present on mature B lymphocytes and macrophages. Like mouse Thy-1, transgenic human CD7 was expressed in immature and mature T cells and in Sca-1+ bone marrow mononuclear cells. Unlike mouse Thy-1, the human CD7 transgene was not expressed in mouse brain or fibroblasts. The human CD7 transgene was expressed during fetal development before mouse Thy-1 in fetal liver mononuclear cells. Expression of the human CD7 transgene did not alter mouse thymopiesis or Thy-1 expression. Taken together, these data demonstrated that the CD7 transgene contained sufficient regulatory regions to direct hematopoietic expression and mitogenic induction. The pattern of CD7 transgene expression more closely resembled that of CD7 in humans than that of mouse Thy-1. PMID- 7544377 TI - Activation of nuclear factor-kappa B and gene expression in human endothelial cells by the common haptens nickel and cobalt. AB - Nickel chloride (NiCl2) and cobalt chloride (CoCl2), two haptens frequently leading to contact hypersensitivity in industrialized countries, induce gene transcription of adhesion molecules ICAM-1, VCAM-1, and E-selectin in endothelial cells. In search of transcriptional mechanisms underlying their gene-inductive effects, we studied the capacity of both haptens to activate nuclear factor (NF) kappa B, a transcription factor involved in inducible expression of adhesion molecules. Using electrophoretic mobility shift assays, a strong increase of NF kappa B DNA binding was detected after stimulation of HUVEC with NiCl2 or CoCl2. Supershift analysis using antisera against p50 and p65 confirmed the authenticity of the induced NF-kappa B complex. Neutralizing Abs against TNF-alpha and IL-1 did not inhibit metal hapten-induced activation of NF-kappa B, thus ruling out action via an indirect autocrine pathway. In addition, NiCl2-induced activation of NF-kappa B and adhesion molecule expression was inhibited by the antioxidant pyrrolidine dithiocarbamate, indicating the involvement of redox-dependent mechanisms. Furthermore, NiCl2 was found to induce dose-dependency mRNA production and protein secretion of the NF-kappa B-controlled proinflammatory cytokine IL-6. Our data suggest that distinct allergens represent a new class of so far unknown agents that induce NH-kappa B binding activity that subsequently modulates transcription of cytokine and adhesion molecule genes. Thus, pathomechanisms leading to contact hypersensitivity to NiCl2 and CoCl2 appear to involve not only Ag-specific Langerhans- and T cell-dependent events but also include direct effects on other immunocompetent cells such as the endothelium. PMID- 7544380 TI - IL-3-induced histamine release from human basophils. Dissociation from cationic dye binding and down-regulation by Na+ and K+. AB - The effect of recombinant human IL-3 on human basophils from normal subjects was evaluated by the decrease of toluidine blue-positive basophils (TB+) and by histamine release. IL-3 (0.001 to 100 ng) caused the decrease of TB+ without concomitant histamine release, whereas anti-IgE-induced TB+ decrease was accompanied by histamine release. IL-3 enhanced both anti-IgE-induced TB+ decrease and histamine release. IL-3-induced TB+ decrease was dose- and Ca2+ dependent and related to the time of incubation (detectable after an incubation of 5 min and maximal after incubation of 2 h). When extracellular Na+ was replaced isosmotically with choline, N-methyl-D-glucamine, or glucose, histamine release also was observed after direct stimulation with IL-3. The effect was dose dependent, related to the time of incubation (detectable after 30 min and maximal after 60 min), and required extracellular Ca2+. The increase in extracellular Na+ or K+ concentrations was accompanied by a reduction of histamine release, with a more marked effect on IL-3- than on anti-IgE-induced histamine release. In line with these results, the Na+ ionophore gramicidin D, which increases Na+ influx, and the K+ channel blocker 4-aminopyridine, which decreases K+ efflux, dose dependently inhibited anti-IgE- and IL-3-induced histamine release. The inhibitory effects on Na+ and K+ were slightly additive, suggesting an action via the same pathway, which most probably is membrane potential. These results indicate that: 1) IL-3 can induce TB+ decrease without concomitant histamine release, 2) basophils from normal subjects can release histamine on challenge with IL-3 once the inhibitory effect of Na+ is removed, and 3) Na+ and K+ down regulate IL-3 as well as anti-IgE-induced histamine release. PMID- 7544379 TI - A crucial role for beta 2 integrin in the activation of eosinophils stimulated by IgG. AB - An IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for eosinophil activation. Recent evidence suggests that cellular adhesion, especially that through the beta 2 integrin, is an important step in cellular activation and accumulation. Therefore, we investigated the role of adhesion molecules in IgG-stimulated eosinophil functions. Cross-linking of eosinophil cytophilic IgG by anti-IgG immobilized to tissue culture plates induced degranulation, whereas soluble anti-IgG did not. Similarly, eosinophils exposed to human IgG immobilized to plates adhered and degranulated; in addition, adherence and subsequent degranulation were inhibited by mAbs to CD18 and CD11b, but not by mAb to CD29, suggesting an important role of beta 2 integrin for these responses. Eosinophil degranulation induced by IgG covalently coupled to Sepharose 4B beads was also inhibited by mAb to CD18. Furthermore, fibrinogen, a ligand for CD11b/18, showed synergistic enhancement of IgG-induced degranulation when it was co-immobilized with IgG to plates. A morphologic study showed that eosinophils, stimulated by immobilized IgG, protrude numerous pseudopods; this morphologic change was inhibited by mAb to CD18. This cellular adhesion seems to affect the early signaling events in eosinophils because the production of inositol phosphates was abolished by mAb to CD18. Interestingly, although superoxide production by eosinophils triggered by immobilized IgG was inhibited by mAb to CD18, superoxide production and morphologic change of neutrophils were not. These results suggest that cell adhesion through CD11b/18 is a crucial step for the activation, signaling, and effector function of eosinophils stimulated by IgG. PMID- 7544381 TI - A proteoglycan (aggrecan)-specific T cell hybridoma induces arthritis in BALB/c mice. AB - Aggrecan, the high buoyant density cartilage proteoglycan (PG), has been shown to induce progressive polyarthritis and ankylosing spondylitis in genetically susceptible BALB/c mice. To further characterize the nature of the autopathogenic effector T cells operating in these mice and to determine the region(s) of the PG molecule recognized by these T cells, we generated PG-specific T cell hybridomas from arthritic mice. One of the PG-specific T cell hybridomas (5/4E8), when injected into naive irradiated BALB/c mice, was capable of inducing clinical and histopathologic signs of arthritis. Massive swelling and redness of the paws dominated the clinical picture. A reactive synovial cell proliferation, the accumulation of hybridoma and inflammatory cells in the enlarged joint space, the loss of PG from the superficial layer of the articular cartilage, and the erosion of articular surface were identical histopathologic signs to those found either in primary or adoptive transfer of PG-induced arthritis. The PG-specific and arthritogenic T cell hybridoma (5/4E8) expressed TCR-alpha beta + (V beta 4), CD4+, and CD8- phenotypes and belonged to the Th1 subset, as the cells secreted IL-2 and IFN-gamma, but not IL-4 upon PG stimulation, and the response was MHC class II (I-Ad)-restricted. These observations provide direct evidence that PG specific Th cells play crucial roles in autoimmune arthritic processes. PMID- 7544383 TI - Reversal of acute experimental autoimmune encephalomyelitis and prevention of relapses by treatment with a myelin basic protein peptide analogue modified to form long-lived peptide-MHC complexes. AB - Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease induced by immunization with myelin basic protein (MBP), proteolipid protein, or encephalitogenic peptides from these myelin components. EAE resembles basic protein multiple sclerosis in some of its clinical and histologic features, and serves as an experimental model for this and other autoimmune diseases. In this study, we examine i.v. peptide therapy of EAE in detail, and show that repeated i.v. injections of MBP peptides effectively treat EAE in (PLJxSJL)F1 mice. In this study, administration of the immunodominant epitope (MBP Ac1-11) prevents MBP-induced disease, whereas the subdominant epitope MBP 31-47 is neither required nor sufficient. Intravenous administration of substituted MBP peptide analogues is also effective in treating EAE, provided the peptide side chains presumed to be involved in TCR contact and MHC binding are preserved. A substituted MBP peptide analogue that forms long-lived peptide-MHC complexes in vivo is more effective than the unmodified MBP peptide. Lower doses of the substituted peptide analogue are effective, and the effect is longer lasting than treatment with the unmodified peptide. Clinical signs of EAE are reversed by injection of the substituted peptide during the acute phase of disease. Moreover, treatment of mice in the remission phase of EAE results in a dramatically reduced incidence of relapse. In summary, we have shown that EAE can be reversed after onset and treated during remission with an MBP peptide analogue that has been modified for improved therapeutic potency. PMID- 7544382 TI - Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses. AB - Cytotoxic T lymphocyte responses in HIV infection can be impaired through variation in the epitope regions of viral proteins such as a gag. We report here an analysis of variant epitope peptides in three gag epitopes presented by HLA B8. Fifteen variant peptides were examined for their binding to HLA-B8; all but one bound at concentrations comparable to known epitopes. All except two of those that bound could be recognized by CTL from an HLA-B8 positive HIV-1-infected patient and were therefore immunogenic. However, in a hemophiliac patient studied in detail, there was a failure to respond to two immunogenic peptide epitopes representing virus present as provirus in the patient's peripheral blood. In one case, the patient's CTL had previously responded to the peptide; in the other case, there was a good response to a peptide of closely related sequence. Thus there was a selective failure of the CTL response to some proviral epitopes. This impaired reaction to new variants could contribute to the loss of immune control of the infection. PMID- 7544385 TI - A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats. AB - We compared the T cell responses of Lewis (LEW) and DA rats to guinea pig myelin basic protein (MBP), the synthetic peptides corresponding to the epitopes that are encephalitogenic in the LEW strain (MBP73-86, MBP68-86, and MBP87-99), and bovine proteolipid protein (PLP). DA and LEW rats were susceptible to experimental autoimmune encephalomyelitis (EAE) induced with MBP or MBP68-86, but the peptide was less active in DA rats than in intact MBP molecule. MBP73-86 and MBP87-99 induced EAE in LEW rats but not in the DA strain. MBP89-169 was also encephalitogenic in DA rats. Encephalitogenic CDa+ T cell lines and clones derived from MBP-sensitized DA rats secreted IFN-gamma and TNF-alpha and proliferated to MBP and MBP89-169, but not to MBP68-88. However, T cells from MBP68-86-sensitized DA or LEW rats proliferated specifically in an I-A-restricted response to MBP68-86. T cells from MBP87-99-immunized LEW rats responded to MBP87 99 in the context of I-E, whereas the peptide-specific response of MBP87-99 immunized DA rats was I-A-restricted, although FACScan analysis indicated that DA rats express both I-A and I-E. DA rats were also highly susceptible to EAE induced with PLP; 0.6 nmol was Encephalitogenic for DA rats, but did not induce clinical EAE in LEW rats. Although both DA and LEW rats are highly susceptible to EAE, we demonstrate marked differences in the array of myelin epitopes capable of inducing the disease, as well as the MHC restriction of these epitopes, between the two rat strains. PMID- 7544386 TI - Animal models of scleroderma: contrasts and comparisons. PMID- 7544387 TI - Adhesion molecules in scleroderma: collagen binding integrins. AB - In summary, adhesion molecules are likely to play a prominent role in scleroderma pathogenesis and evolution. Endothelial adhesion molecules required for leukocyte extravasation are upregulated in affected tissue, though the mechanism is unclear. Certainly, endothelial adhesion molecule expression is seen in the context of other diseases not characterized by fibrosis. Adhesion molecules on the fibroblast, particularly those that play a role in fibroblast collagen interactions, may be very important. The ability of fibroblasts to organize collagen fibrils, and to exert forces across collagenous tissue, is likely to involve a prominent role of alpha 2 beta 1 integrin. Enhanced organization and contraction of newly formed collagen, as well as unregulated procollagen production, may be intimately linked in this disease process. At least two factors that strongly enhance fibroblast force generation could potentially influence other aspects of scleroderma. TGF beta is a potent stimulus for collagen production and has been found to be elevated in lesional scleroderma. Endothelin 1 is also a potent vasoconstrictor and is elevated in scleroderma patient serum as well [60,62-65]. Its apparent role in other fibrocontractive diseases suggests that its potential role in the pathogenesis of scleroderma deserves additional attention. PMID- 7544384 TI - The IFN pregnancy recognition hormone IFN-tau blocks both development and superantigen reactivation of experimental allergic encephalomyelitis without associated toxicity. AB - Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease if the central nervous system (CNS). Recently, the type I IFN, IFN-beta-1b was demonstrated to be a useful immunotherapy for MS. During treatment with IFN-beta 1b, toxicity at higher doses has been observed. IFN-tau, discovered for its role in the reproductive cycle, possesses all of the functions normally ascribed to the type I IFNs but lacks the toxicity normally associate with IFN treatment in vitro. We have examined the effects of IFN-tau treatment on experimental allergic encephalomyelitis (EAE), an animal model useful for the study of MS. EAE is a model of Ag-induced autoimmunity that can be modulated by bacterial superantigen to resemble the relapsing-remitting pattern of autoimmune disease observed in MS. IFN-tau was able to prevent development of EAE as effectively as IFN-beta but without associated toxicity such as lymphocyte suppression and weight loss. In addition, IFN-tau was able to prevent superantigen reactivation of EAE akin to the reduction in disease exacerbations observed in IFN-beta-1b treated MS patients. Mechanisms by which IFN-tau may prevent EAE include reduced proliferation in response to the autoantigen myelin basic protein and reduced TNF alpha production. Thus, IFN-tau may prove to be a promising new IFN therapy for MS in light of its ability to prevent EAE and the lack of toxicity exhibited by this novel IFN. PMID- 7544388 TI - Mast cells: accessory cells which potentiate fibrosis. PMID- 7544390 TI - Light and electron microscopic localization of lipids in Blastocystis hominis. AB - Localization of the lipid of Blastocystis hominis was histochemically demonstrated. B. hominis was fixed with glutaraldehyde, treated with detergents, and stained with Sudan black B or Nile blue. The central vacuole of B. hominis stained with Sudan black B showed many black droplets with great variation in staining intensity. In Nile blue staining, the central vacuole showed many pinkish granules or homogeneous blue reactions. These results indicated the presence of neutral lipids and/or acidic lipids in the central vacuole. At the ultrastructural level, the central vacuole and some cytoplasmic vesicles showed enhanced electron density after post-fixation with imidazole-buffered osmium tetroxide solution. Great variation in the density and distribution of the electron-dense materials was observed in the central vacuole. Since the electron dense vesicles were frequently observed in the cytoplasm, B. hominis may accumulate the lipid in the central vacuole, the function of which is not well known. PMID- 7544389 TI - Early results with a new procedure for hypoplastic left heart syndrome and aortic atresia. AB - One patient with hypoplastic left heart syndrome and another with aortic atresia and a complex form of functional single ventricle, who were candidates for the Norwood operation, received a new palliative procedure. This involved anastomosis of the dome of the pulmonary artery to the undersurface of the transverse arch of the aorta with placement of a fenestrated patch in the main pulmonary artery to divide the systemic and pulmonary circulations. Both patients have survived this initial procedure and will receive second stage palliation at 6 months of age. Shorter circulatory arrest and cardiopulmonary bypass times are required, and this procedure may be a safer alternative than the Norwood for neonates with hypoplastic left heart syndrome and aortic atresia. PMID- 7544391 TI - Ultrastructural localization of penicillin-binding sites in Staphylococcus aureus using penicillin G-BSA-gold. AB - Penicillin G (PcG) conjugated to bovine serum albumin (BSA) was found to retain antibacterial activity against Staphylococcus aureus as judged by growth inhibition and by ultrastructural analyses of the PcG-BSA-treated bacterium. Using the PcG-BSA conjugated to colloidal gold, binding sites of PcG were localized in S. aureus with electron microscopy using a postembedding labeling technique. To our surprise, labeling was observed both on the cell wall and in the cytoplasm of S. aureus. A part of the labeling observed may be attributed to penicillin-binding proteins (PBPs) or their precursors. Also there may be other molecules which bind with PcG in the cell wall and cytoplasm of S. aureus. PMID- 7544392 TI - Mortality from benign prostatic hyperplasia: worldwide trends 1950-92. AB - STUDY OBJECTIVE: To provide a systematic overview of worldwide trends in mortality from benign prostatic hyperplasia (BPH) over the past four decades. DESIGN: This was a descriptive analysis based on age adjusted mortality rates for BPH between 1950 and 1992 for 41 countries from five continents. SETTING: Official death certifications from the World Health Organization database. MAIN RESULTS: In the 1950s, the highest age adjusted (on the world standard population) mortality rates for BPH in Europe were in Denmark (22.8/100,000) and Germany (18.1), followed by Scandinavian countries, the UK, and Switzerland. Italy had rates around 10/100,000, and rates were lower in eastern and southern Europe (5-8/100,000). Between 1950 and 1990, a fall of over 90%, or even 95%, was observed in most western European countries. Thus, in the early 1980s, overall mortality from BPH ranged between 0.5 and 1.5/100,000 in most western European countries. In proportional terms, similar reductions were registered in other developed countries of North America, Asia (that is, Japan or Singapore), and Oceania. A fall in rates was also observed in eastern Europe and in Latin America, particularly from the late 1970s onwards, although these reductions were generally much smaller. Thus, in the early 1990s, most countries in these areas had BPH rates between 1 and 5/100,000. The pattern of trends was, at least qualitatively, similar at younger ages, although most falls were proportionally greater. CONCLUSIONS: The most probable interpretation of these trends is that therapeutic improvements--including more widespread and timely surgery, introduction of less invasive techniques, such as transurethral prostatectomy, and possibly the development of medical treatments--have had a favourable and substantial impact on BPH mortality. There are, however, areas of the world, including several countries of western Europe and South America, where rates are still very high. PMID- 7544393 TI - Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4. AB - The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4. PMID- 7544395 TI - A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. AB - Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2 F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes. PMID- 7544396 TI - CD2 regulates responsiveness of activated T cells to interleukin 12. AB - Interleukin (IL) 12 is a 70-kD heterodimeric cytokine produced by antigen presenting cells (APCs) such as macrophages in response to infectious pathogens and interferon (IFN) gamma. The varied immunomodulatory effects of IL-12 include the stimulation of proliferation and IFN-gamma production by T cells, and it also has a central role in the development of the T helper cell type 1 immune phenotype. We undertook the production of antibodies capable of modulating the response of T cells to IL-12, and in the process we discovered two antibodies that inhibited the ability of IL-12 to stimulate T cell proliferation. In this report, we demonstrate that these anti-bodies recognize CD2, and we show how antibodies directed toward either the adhesion domain of CD2 or its ligand, CD58, specifically inhibit IL-12 induced proliferation and IFN-gamma production by phytohemagglutinin-activated T cells, leaving the response to IL-12 unaffected. A three-to fourfold reduction in proliferation and IFN-gamma production was observed at IL-12 concentrations as high as 1 nM, with complete inhibition occurring at < or = 1 pM. This novel effect is not directly mediated at the level of the IL-12 receptor, as shown by the inability of these antibodies to block IL 12 binding to activated T cells. Furthermore, by using activating pairs of CD2 antibodies, we show that CD2 stimulation strongly synergizes with IL-12, even at 0.1 pM, in inducing both T cell proliferation and IFN-gamma production. Cytolytic T lymphocyte-associated antigen 4-immunoglobulin-mediated inhibition of the B7/CD28 interaction did not affect the T cell response to either IL-12 or IL-2, but the removal of APCs selectively diminished the proliferative response to IL 12. Based on this data, we hypothesize that CD2 has a central role in an IL 12/IFN-gamma positive feedback loop between T cell and APC, providing the key functional link via a CD2/CD58 interaction that controls T cell responsiveness to IL-12. This model provides a basis for future investigations aimed at defining the signaling mechanisms that mediate this cytokine-specific regulatory effect of CD2, and it offers insight into how a cytokine receptor and distinct adhesion molecule can interact to modulate responsiveness to that cytokine. In addition, it underscores the possibility that the clinical potential of an immunomodulatory drug like IL-12 may be governed by the presence or absence of specific costimulation. PMID- 7544398 TI - A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities. AB - It is generally accepted that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus-specific CD8+ CTLs of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships between the serotype specificities of T cells and the amino acid sequence of the recognized peptides, we examined the ability of this viral peptide D4.NS3.500-508 (TPEGIIPTL) to stimulate T lymphocytes of an HLA-B35 positive, dengue virus type 4-immune donor. Peptide stimulation of the PBMC generated dengue virus-specific, HLA-B-35-restricted CD8+ CTL clones. These clones lysed dengue virus-infected autologous cells, as well as autologous target cells pulsed with this peptide. Four patterns of dengue virus serotype specificities were demonstrated on target cells infected with dengue-vaccinia recombinant viruses or pulsed with synthetic peptides corresponding to amino acid sequences of four dengue virus serotypes. Two serotype-specific clones recognized only D4V. Three dengue virus subcomplex-specific clones recognized D1V, D3V, and D4V, and one subcomplex-specific clone recognized D2V and D4V. Three dengue virus serotype-cross-reactive clones recognized D1V-D4V. Thus, a single nine-amino acid peptide induces proliferation of a heterogeneous panel of dengue virus-specific CD8+ CTL clones that are all restricted by HLA-B35 but have a variety of serotype specificities. Peptides that contain a single amino acid substitution at each position of D4.NS3.500-508 were recognized differently by the T cell clones. These results indicate that a single epitope can be recognized by multiple CD8+ CTLs that have a variety of serotype specificities, but the manner of recognition by these multiple CTLs is heterogeneous. PMID- 7544394 TI - Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor. AB - Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines. PMID- 7544397 TI - Monoclonal immunoglobulin A antibody directed against serotype-specific epitope of Shigella flexneri lipopolysaccharide protects against murine experimental shigellosis. AB - To determine the role of humoral mucosal immune response in protection against shigellosis, we have obtained a monoclonal dimeric immunoglobulin A (IgA) antibody specific for Shigella flexneri serotype 5a lipopolysaccharide (mIgA) and used a murine pulmonary infection model that mimics the lesions occurring in natural intestinal infection. Adult BALB/c mice challenged with 10(7) S. flexneri organisms developed a rapid inflammatory response characterized by polymorphonuclear cell infiltration around and within the bronchi and strong systemic interleukin 6 response. Implantation of hybridoma cells in the back of mice, resulting in the development of a myeloma tumor producing mIgA in the serum and subsequently secretory mIgA in local secretions, or direct intranasal administration of these antibodies, protected the animals against subsequent intranasal challenge with S. flexneri serotype 5a. Absence of histopathological lesion and significant decrease in bacterial load of the lungs and of systemic interleukin 6 response were the three major criteria of protection. This protection was shown to be serotype-specific and dependent on local concentration of mIgA. These data demonstrate that mucosal antibodies directed against a single polysaccharidic surface epitope of Shigella can protect against the disease. PMID- 7544400 TI - Prostate-specific antigen testing to screen for prostate cancer. AB - Prostate cancer is a common cause of cancer-related morbidity and mortality in men. Prostate-specific antigen (PSA) measurement to screen for prostate cancer has been promoted as a way to reduce morbidity and mortality from prostate cancer. This paper examines the usefulness of PSA screening for asymptomatic prostate cancer, focusing on outcomes for all patients screened. The sensitivity and specificity of PSA testing for prostate cancer are low and have not been studied properly in asymptomatic men being screened for prostate cancer. PSA screening detects localized prostate cancer undetected by digital rectal examination in fewer than 1% of men screened. The effectiveness of early treatment of prostate cancer, compared with deferral of treatment until symptoms develop, is unproven, and good survival rates have been reported among patients who defer aggressive treatment. Complications of treating prostate cancer with radical prostatectomy or radiation treatment include death, impotence, urethral stricture, incontinence, and rectal injury. At the present time, there is insufficient evidence to support a policy of PSA screening, and its use should be discouraged until randomized controlled trials demonstrate benefit from PSA screening. PMID- 7544403 TI - Dopaminergic and serotonergic neurotransmitters in bone marrow transplant patients. AB - Depigmentation of neurons in the substantia nigra (SN) is found in patients dying after bone marrow transplantation (BMT). This study examined neurochemical striatal changes related to BMT. Caudate nucleus and putamen of 6 BMT subjects and 10 age-matched controls were analyzed for levels of dopamine (DA), homovanillic acid (HVA), 5-hydroxyindoleamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA), by high pressure liquid chromatography-electron capture detection (HPLC-ECD). In addition, assays of the enzymatic activities of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) were performed. Cholinergic markers, ChAT and AChE, were reduced in BMT caudate (p < 0.05) but not in the putamen. A recovery toward normal cholinergic enzymatic activity was identified with increased post transplant survival time. The level of DA was reduced 50% in BMT caudate and putamen while HVA was increased 30%, however, neither reduction achieved statistical significance. Increasing post-transplant survival time correlated with decreased levels of DA in caudate nucleus and putamen in the early post transplant period, while HVA was increased over the same interval but tended to return to normal levels with increasing survival time. Two-fold increases of BMT caudate 5-HT (p < 0.003) and 5-HIAA were found; similar changes were noted in putamen 5-HT and 5-HIAA (p < 0.0008). Significant increases in MAO-A and B were found in BMT caudate (p < 0.0001 and p < 0.06, respectively) and putamen (p < 0.0005 and p < 0.006, respectively). No statistically significant changes were noted in the 5-HT, 5-HIAA, or MAO A or MAO B with increasing post-transplant survival. Whether these changes are the result of physiologic or toxic effects is unknown. PMID- 7544399 TI - Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas mediated apoptosis. AB - We have identified a serine/threonine kinase that is rapidly activated during Fas mediated apoptosis. Fas-activated serine/threonine kinase (FAST) is phosphorylated on serine and threonine residues in Jurkat cells. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1, a nuclear RNA-binding protein that has been implicated as an effector of apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program. Our results introduce Fast and TIA-1 as components of a molecular cascade involved in signaling Fas-mediated apoptosis. PMID- 7544404 TI - Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembly. AB - Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs. PMID- 7544405 TI - Coccidioidal antigen reactive CD4+ T-lymphocytes in the cerebrospinal fluid in coccidioides immitis meningitis. AB - CSF lymphocytes from patients with Coccidioides immitis meningitis exhibited a significant antigen-specific response to in vitro stimulation with C. immitis antigens. In some patients, lesser responses to control antigens (Candida and PPD) were also detected. Antigen-specific responses by CSF lymphocytes were seen early in the course of this disease as well as several years after patients had entered remission. When compared to CSF cells, the response of autologous peripheral blood mononuclear cells was similar but of a much smaller magnitude and at times undetectable. Fluorescence activated cell sorting revealed an increased percentage of CD3+ (T-cells), CD4+ (helper/inducer) and CD3+/HLA-DR+ (activated T-cell) cells in the CSF of C. immitis meningitis patients compared to their blood. Most of the antigen-specific proliferative response resided in the CD4+ lymphocyte subset. CSF T-cell proliferation assays may have a role in the diagnosis of C. immitis meningitis. PMID- 7544402 TI - Ganglioside-like epitopes of lipopolysaccharides from Campylobacter jejuni (PEN 19) in three isolates from patients with Guillain-Barre syndrome. AB - Sera from patients with Guillain-Barre syndrome (GBS) frequently have anti-GM1 antibody. We earlier showed that an lipopolysaccharides (LPS) from Campylobacter jejuni (PEN 19) isolated from a GBS patient has a GM1 ganglioside-like structure. Aspinall et al. (Biochemistry, 61 (1994) 335-337) reported that OH 4382 has an LPS that bears a CD3 ganglioside-like structure and that OH 4384 has an LPS that bears a GT1a-like structure; both strains were isolated from patients with GBS. They also suggested a GM1-like structure is present in the LPSs from OH 4384, but failed to show the presence in the LPSs from OH 4382. To clarify the pathogenesis of GBS after infection by C. jejuni (PEN 19), we investigated the carbohydrate structures of the three strains by thin-layer chromatography immunostaining with cholera toxin and monoclonal anti-ganglioside antibodies. We found that both OH 4382 and OH 4384 have an LPS with the GM1 epitope as well as one with the GT1a or GD3 epitope. PMID- 7544401 TI - Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis. AB - In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis. PMID- 7544408 TI - Prefabricated vascularized periosteal grafts using fascial flap transfers. AB - The purpose of this study was to demonstrate the feasibility of prefabricated vascularized periosteal grafts using fascial flap transfers. Fifty rats were divided into two (Group A, underwent periosteal wrapping, and Group B underwent fascial wrapping). Fascial flap from the medical leg, which were nourished by the saphenous artery and vein, were used for all grafts. In Group A, the fascia was transferred onto the proximal tibial periosteum. Two weeks postoperatively, the elevated fascioperiosteal flap was wrapped around a 4-mm hydroxapatite cube. In Group B, the fascial flap was immediately wrapped around the hydroxyapatite cube. In both groups, the hydroxapatite-wrapped cube was transferred to the inguinal region. Three days postoperatively, five rats from group A were sacrificed. On histologic examination, these five showed no necrosis of the fascioperiosteal flap and no bone formation. The remaining 45 rats were sacrificed 5 weeks postoperatively. Microangiography was performed on five rats in Group A, revealing neovascularization of the hydroxyapatite. On histologic examination, new bone formation was confirmed in 15 of the remaining 20 rats in Group A, and in three of the 20 rats in Group B. These experiments demonstrated that the prefabricated vascularized periosteal graft, which has good osteogenic capacity, could be carried out using a fascial flap transfer. PMID- 7544410 TI - First-trimester manifestation of intrahepatic cholestasis of pregnancy and high fetoplacental hormone production in a triploid fetus. A case report. PMID- 7544409 TI - Transvaginal probe ultrasonography. Diagnostic or outcome advantages in women with molar pregnancies. AB - This study attempted to determine whether patients with molar pregnancy initially evaluated by transvaginal ultrasound had earlier diagnoses and superior outcomes than did similar patients initially evaluated by only transabdominal scans. The medical records of 71 patients with molar pregnancy evacuated at the University of California at Los Angeles and affiliate hospital Olive View Medical Center between 1975 and 1988 were reviewed. Eight patients did not have ultrasound imaging prior to evacuation, and three had missing films. The remaining 60 patients were divided into two cohorts: 19 had transvaginal scans, while 41 had transabdominal scans only. The groups were compared retrospectively. Transvaginal pelvic ultrasound did not appear superior to the less expensive and less invasive transabdominal approach in identifying patients with molar pregnancy. These patients generally present with uteri over 10 weeks in size and often much larger; the gestation can be well visualized abdominally through the bladder window. Our study demonstrated no differences in outcome between patients from the two imaging groups. PMID- 7544406 TI - Glycine induces two distinct membrane currents in neonatal rat sympathetic preganglionic neurones in vitro. AB - 1. The effects of glycine (Gly) on neonatal (12- to 16-day-old) rat sympathetic preganglionic neurones (SPNs) in transverse (500 microns) thoracolumbar spinal cord slices were studied by whole-cell patch-clamp techniques. 2. Gly elicited three types of membrane currents when applied to SPNs by pressure ejection (100 mM; 20-180 ms pulse duration): (1) an outward current (20/94 cells); (2) an inward current (30/94); and (3) a biphasic response (44/94) consisting of an outward followed by an inward current. 3. The Gly-induced outward current (IGly,(out)) had a mean reversal potential of -67 mV, was reversed in a low (5.7 mM) chloride Krebs solution, and was reversibly eliminated by strychnine (0.1-1 microM). 4. The Gly-induced inward current (IGly,(in)) had a mean reversal potential of -41 mV, was reduced in a Na(+)-free and increased in a high (15 mM) K+ solution; strychnine at the high concentration of 1 microM reduced the response by an average of 53%. 5. The electrophysiological and pharmacological characteristics of the biphasic response suggest that it was a combined response of outward and inward currents. 6. The results show that Gly elicits two distinct membrane currents, a Cl(-)-dependent outward current and a cationic inward current, which are mediated by strychnine-sensitive and strychnine-resistant Gly receptors. Functionally, activation of both types of Gly receptors reduces neuronal excitability and attenuates synaptic transmission. PMID- 7544407 TI - Electrophysiological and metabolic effects of a convulsant barbiturate on dissociated mouse primary sensory neurons. AB - 1. The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarizes dorsal root ganglion (DRG) neurons. We have applied microfluorimetric and whole-cell patch clamp techniques to investigate the mechanisms underlying this response in freshly dissociated mouse DRG cells. 2. Application of CHEB (2-200 microM) raised cytosolic calcium concentration ([Ca2+]i) rapidly and reversibly in 55% of eighty-three neurons tested. This population did not correlate with other classifications of sensory neurons based on either cell size or the expression of membrane currents. 3. The response was dependent on external calcium and was reduced by 81 +/- 22% by Ruthenium Red. A rise in [Ca2+]i was still seen with the membrane potential clamped at -70 mV, excluding membrane depolarization and activation of voltage-dependent Ca2+ channels as the principal mechanism for the response. 4. The rise in [Ca2+]i was associated with an increase in membrane conductance and a current, ICHEB, which was inward at -70 mV. Both the rise in [Ca2+]i and the current showed 'run-down' under whole-cell recording conditions. When K+ conductances were blocked, the reversal potential of ICHEB was close to 0 mV. This was independent of the Cl- reversal potential, suggesting that ICHEB is carried as a non-specific cation current. 5. In contrast to the change in [Ca2+]i, ICHEB was not dependent on external Ca2+ and the current was still seen when [Ca2+]i as strongly buffered by the pipette filling solution. These data suggest that CHEB opens a non-selective cation channel permeant to Ca2+, raising [Ca2+]i and further depolarizing the cell membrane potential. The exact nature of this conductance remains unknown. These actions could readily account for the convulsant actions of the drug, depolarizing neurons and increasing transmitter release. 6. It was also noted that CHEB increases autofluorescence derived from mitochondrial NAD(P)H. Further examination of this phenomenon using the dye rhodamine 123 to follow changes in mitochondrial potential (psi m) suggested that CHEB is a potent inhibitor of mitochondrial respiration, probably acting at complex I. These effects appeared to be quite distinct from the action of CHEB at the level of the plasma membrane. PMID- 7544411 TI - Midtrimester Rh sensitization associated with circulating anticardiolipin antibodies and elevated maternal serum alpha-fetoprotein. A case report. AB - The incidence of antepartum Rh isoimmunization has been limited by third trimester Rh immune globulin (RhIg) administration. Prophylactic failures are uncommon but can occur if sensitization takes place prior to the 28th week of gestation. We report a case of midtrimester Rh sensitization in an anticardiolipin antibody-positive primipara coincident with the discovery of an elevated maternal serum alpha-fetoprotein value, oligohydramnios and fetal growth retardation. This case suggests that fetal-maternal hemorrhage and subsequent sensitization may be facilitated by anticardiolipin antibody-induced placental damage. Prophylactic midtrimester RhIg administration might avoid sensitization in similar cases. PMID- 7544412 TI - Evidence of common and genus-specific epitopes on Ornithodoros spp. Tick (Acari: Argasidae) salivary proteins. AB - New Zealand White rabbits were repeatedly infested with Ornithodoros turicata (Duges), Ornithodoros talaje (Guerin-Meneville), and Ornithodoros coriaceus (Koch) at 2-wk intervals. Blood samples were taken from each animal 10 d after each infestation and the titer of anti-tick antibody was determined by enzyme linked immunosorbent assay. Subsequent cross-reactivity studies demonstrated that the antitick antisera nonspecifically bound to salivary gland extract proteins prepared from several other tick genera and species' Amblyomma maculatum (Koch), Dermacentor andersoni (Stiles), Dermacentor variabilis (Say), and Ornithodoros moubata (Murray). Absorption of the antisera against an immobilized extract of A. maculatum substantially increased specificity at the genus level. Western blots of electrophoretically separated Ornithodoros salivary gland extract samples were used to further compare the specificity of absorbed and nonabsorbed antitick antisera. The blots demonstrated that many of the Ornithodoros salivary gland extract proteins bear genus specific epitopes. Some differences were noted among the Ornithodoros species examined with respect to the degree of antigenic relatedness with the ixodid ticks. PMID- 7544413 TI - Use of flow cytometry to identify a Caulobacter 4.5 S RNA temperature-sensitive mutant defective in the cell cycle. AB - Flow cytometry was used to screen a collection of temperature-sensitive mutants for those blocked at discrete points in the cell cycle with respect to the replicative status of the chromosome. At the non-permissive temperature, one such mutant, LS439, could not initiate new rounds of DNA replication and arrested primarily as cells with two completed chromosomes Extended incubation at the restrictive temperature resulted in filament formation. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. A 0.2 kb fragment of DNA located immediately upstream of the Caulobacter homolog of the Escherichia coli dnaX gene was able to completely rescue the temperature sensitive phenotype of LS439. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype. PMID- 7544414 TI - The mitochondrial LSU rDNA of the brown alga Pylaiella littoralis reveals alpha proteobacterial features and is split by four group IIB introns with an atypical phylogeny. AB - The mitochondrial DNA region coding for the large ribosomal RNA subunit from the brown alga Pylaiella littoralis (L.) Kjellm was sequenced. The LSU rRNA was folded into a secondary structure and aligned with homologous, mitochondrial and eubacterial sequences. Taking into account the primary and secondary structure levels, the mitochondrial LSU rRNA of P. littoralis shares more structural features with alpha-proteobacterial genes than do those of the green alga Prototheca wickerhamii and land plants. In phylogenetic trees, branches leading to brown algae, red algae, the protozoan Acanthamoeba castellanii and land plants, respectively, emerge approximately at the same time, as they do in nuclear-gene based phylogenies. This suggests that there is only one origin for the mitochondrial rRNA genes found in these lineages. The LSU rDNA is split by four group IIB introns. The first two introns each contain one open reading frame which encodes a reverse transcriptase-like protein. Comparison of their amino acid sequences with those of other reverse transcriptase-like genes contained in group II introns shows that these genes are more closely related to plastid and cyanobacterial homologous genes than to any known mitochondrial intronic reverse transcriptase. PMID- 7544415 TI - Structure of the osmo-regulated H2O-channel, AQP-CHIP, in projection at 3.5 A resolution. AB - An osmo-regulated H2O-channel, aquaporin-CHIP, from bovine red blood cell membranes was purified and reconstituted with lipids, forming two-dimensional crystalline patches that diffract to about 3.0 A resolution. Electron diffraction patterns and high-resolution images of the crystalline patches embedded in glucose were recorded and used to calculate the projection map at 3.5 A resolution. The map confirms that the osmo-regulated H2O-channel basic packing unit is a tetramer and begins to reveal it's structural design. The basic architecture of the H2O-channel protein consists of a trapezoid-like envelope and a substructure located within the trapezoid that could play a crucial role in the channel structure itself; near this substructure there is a region of very low density, which is the probable site of the channel. The trapezoid-like envelope is composed of high density regions many of which can be interpreted as projections of alpha-helices along their axes. PMID- 7544417 TI - Cell dissociation with papain reduces the density of cGMP-activated channels of the retinal rod. AB - A proteolytic enzyme, papain, is the enzyme commonly used for dissociation of retinal neurons. To ask whether papain modifies the activity of ion channels, cGMP-activated channels were recorded in inside-out membrane patches excised from the bullfrog rod outer segment, and the activity was compared between cells dissociated using papain and cells dissociated mechanically. While the affinity to cGMP, I-V relation, channel kinetics, and single-channel current amplitude were almost the same in both papain-treated and the control preparations, the total current flowing through the patch membrane of the papain-treated cells was reduced to about 20% of the control, suggesting that papain reduced the density of cGMP-activated channels. PMID- 7544416 TI - Expression of immunoreactive nitric oxide synthase isoforms in rat kidney. Effects of dietary salt and losartan. AB - Immunohistochemical studies have shown expression of two different isoforms of NOS in the juxtaglomerular apparatus (JGA). Antibodies to a Ca(++)-calmodulin dependent isoform purified from rat brain (B-NOS) label the macula densa cells whereas antibodies to an isoform purified from rat aortic smooth muscle cells in culture (VSM-NOS) induced with lipopolysaccharide and interferon gamma label the afferent arteriole. Since dietary salt intake and angiotensin II (Ang II) are determinants of renal NO generation, we have tested the hypothesis that salt intake can regulate the immunohistochemical expression of these NOS isoforms through an effect of Ang II. In 4 of 5 paired studies, the immunostaining for both B-NOS and VSM-NOS was more intense in rats that had received a low salt (LS), compared to a high salt (HS), diet. Infusion of the Ang II type 1 (AT1) receptor antagonist, losartan, enhanced the intensity of immunoreactive staining for both isoforms. In conclusion, the immunohistochemical expression of NOS isoforms in the JGA is increased by dietary salt restriction; this effect cannot be ascribed to Ang II acting on type 1 receptors. PMID- 7544418 TI - Suppressive effects of tranilast on pulmonary fibrosis and activation of alveolar macrophages in mice treated with bleomycin: role of alveolar macrophages in the fibrosis. AB - We have reported that tranilast, an anti-allergic drug that inhibits chemical mediator release from mast cells, suppresses bleomycin (BLM)-induced pulmonary fibrosis in mice through mechanisms other than inhibiting chemical mediator release from mast cells. The purpose of this paper is to examine the effect of tranilast on alveolar macrophage (AM) activation and on the development of fibrosis in ICR mice instilled with BLM intratracheally. Twenty eight days after the BLM instillation (0.01 mg/mouse), AM often migrated into alveolar spaces surrounding the fibrotic areas. Flow cytometry analysis for the size and density of AM (MAC-1 positive cells) suggested that AM were activated not only in the earlier acute inflammatory phase, but also in the later chronic phase. The p.o. administration of tranilast suppressed an increase of AM activity to produce reactive oxygen species in BLM-instilled mice, and it inhibited the subsequent development of pulmonary fibrosis. In vitro treatment with tranilast suppressed the reactive oxygen species production from murine peritoneal macrophages. However, several different anti-oxidants failed to inhibit the development of fibrosis. These results suggest that the activation of AM plays an important role in the development of fibrosis, and it is likely that tranilast suppresses fibrosis by inhibiting AM activation but not by scavenging reactive oxygen species. PMID- 7544420 TI - Desensitization of capsaicin-sensitive sensory neurons in rat stomachs on chronic treatment with sodium taurocholate. AB - We examined the effects of chronic treatment with 10 mM sodium taurocholate (TC) on gastric functions, capsaicin-sensitive afferent neurons and the gastric mucosa in male rats. Stomachs were mounted in Lucite chambers, and then the transmucosal potential difference (PD), luminal pH and gastric mucosal blood flow (GMBF) in response to TC or capsaicin was determined. In normal animals, 10 mM TC caused a reduction in PD, and increases in luminal pH and GMBF. Capsaicin (1 mg/ml) produced an apparent increase in GMBF without any change in PD or luminal pH. After 4- or 12-week treatment with TC, the basal PD was significantly reduced, and the luminal pH tended to increase. The increase in GMBF in response to TC or capsaicin was profoundly suppressed in TC-pretreated animals. The calcitonin gene related peptide release in response to capsaicin was significantly reduced after 4 weeks treatment with TC. There were no microscopical changes in the oxyntic mucosa until 4 weeks after TC treatment except for exfoliation of surface cells. However, an increase in inflammatory cell infiltration was observed 12 weeks later. We conclude that chronic treatment with TC causes desensitization of capsaicin-sensitive afferent neurons and reduces GMBF, which may result in the production of gastritis. PMID- 7544419 TI - Effect of vitamin E on keratinocyte-modulation induced by lauroylsarcosine. AB - The effect of vitamin E on the modulation of keratinocytes was studied in rats. A 1% lauroylsarcosine (LS) ointment caused skin erythema with keratinocyte-damage. A 30% vitamin E ointment markedly alleviated this erythema and protected keratinocytes from cell damage. Vitamin E (100 micrograms/ml) was also effective on LS (7.5 micrograms/ml)-induced proliferative reduction of cultured keratinocytes. On the other hand, ointment containing superoxide dismutase (SOD) (99,000 U/g) decreased the LS-induced erythema, suggesting that superoxide anion (O2-) produced from keratinocytes play an important role in the skin irritation. Indeed, LS induced O2- production from cultured keratinocytes. The O2- was significantly reduced by vitamin E and SOD, although vitamin E had no effects on O2- production in a xanthine-xanthine oxidase system, unlike the effect observed with SOD. These results indicate that vitamin E is an inhibitor of keratinocyte modulation. PMID- 7544422 TI - Euthanasia. PMID- 7544421 TI - Calcitonin gene-related peptide mediated neurogenic vasorelaxation in the isolated canine lingual artery. AB - The nature of neurogenic relaxation was investigated in ring preparations of canine lingual artery. In all experiments, the preparations were previously treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. In the presence of norepinephrine (10(-5) M) to induce tone, electrical stimulation (10 V, 4 to 16 Hz, for 45 sec) produced relaxation of the rings in an endothelium-independent fashion. The relaxant response in endothelium denuded rings was not changed by propranolol (10(-5) M), and atropine (10(-5) M) did not affect the relaxation elicited by electrical stimulation in endothelium intact rings. NG-monomethyl-L-arginine (10(-4) M) or NG-nitro-L-arginine methyl ester (10(-4) M), a nitric oxide (NO) synthase inhibitor, had no effect on the electrical stimulation-induced relaxation of endothelium-denuded rings. Human calcitonin gene-related peptide (CGRP)-(8 - 37) (2 x 10(-8) M), a CGRP1-receptor antagonist, inhibited neurogenic relaxation of endothelium-denuded rings; substance P (10(-6) M) failed to mimic the observed effect of electrical stimulation. The demonstrated effect of electrical stimulation was inhibited by glibenclamide (10(-5) M), but not tetraethylammonium (2 x 10(-4) M); glibenclamide abolished the relaxation in response to exogenous CGRP or the ATP sensitive K+ channel opener cromakalim (10(-6) M) in endothelium-denuded rings. Moreover, tetrodotoxin (3.13 x 10(-6) M) inhibited the relaxation of endothelium denuded rings induced by electrical stimulation. The relaxation was selectively inhibited when endogenous CGRP had been depleted from perivascular nerves by capsaicin (10(-6) M). These results suggest that CGRP, but not NO, released from non-adrenergic non-cholinergic nerves by electrical stimulation produces relaxation of canine lingual artery that is mediated by activation of CGRP1 receptors. PMID- 7544424 TI - Biosynthesis of glycosylphosphatidylinositol anchors. PMID- 7544425 TI - Role of membrane permeability in determining antibiotic resistance in Pseudomonas aeruginosa. PMID- 7544423 TI - Bacterial expression and purification of human protein prenyltransferases using epitope-tagged, translationally coupled systems. PMID- 7544426 TI - Chromatographic study of spirosin by use of monoclonal antibodies to spirosin of Yersinia enterocolitica. AB - Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies were designated MAbs-S44 and S50. They were IgG2b and IgG2a, respectively, both with kappa light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli. PMID- 7544427 TI - Extracellular antigens of Actinobacillus actinomycetemcomitans Y4 in severe alveolar bone loss patients studied by two-dimensional electrophoresis and western blots. AB - The human immune response to extracellular substances (ES) from Actinobacillus actinomycetemcomitans Y4 were analyzed. Twenty-nine periodontal patients with generalized severe alveolar bone loss and 13 healthy volunteers were examined for serum IgG antibody titers against ES. Among the patients, 17 had higher antibody titers than healthy individuals (high-titer patients) but the remainder (low titer patients) did not. Two-dimensional electrophoresis (2DE) and Western blots demonstrated that two proteins (40 and 37 kDa) and three smeared substances reacted with IgGs from high-titer patients, but not with IgGs from healthy volunteers. The low-molecular-mass smear at the acid side reacted with over 90% of all patients. This smear reacted with anti-A. actinomycetemcomitans lipopolysaccharide (LPS) monoclonal antibody which recognized LPS from each A. actinomycetemcomitans serotype. The high molecular mass smear at the acid side might be serotype-specific O-antigens of LPS. Another high molecular mass smear which located from the alkaline to the neutral side reacted with anti-serotype-b specific capsular polysaccharide monoclonal antibody. Moreover, the 40- and 37 kDa proteins reacted with this anti-capsular polysaccharide antibody. This results suggested that 40- and 37-kDa proteins which reacted with 100% or 88% of high-titer patients might be glycoproteins linked with capsular polysaccharide. PMID- 7544429 TI - Cytotoxic chemotherapy for adrenocortical carcinoma. AB - The efficacy of mitotane in providing objective tumour responses in patients with adrenocortical carcinoma (ACC), has been recently questioned. Experience with non specific chemotherapy is limited. Tumour responses have been reported with cisplatin administered as a single agent or in combination. Other reports however failed to show benefit from cytotoxic chemotherapy. The very low number of patients included in each study, mostly of them previously treated with mitotane, may account for these controversial results. The finding that multidrug resistance mediated by MDR-1/P-glycoprotein can be reverted by mitotane provides a rational basis for exploring the use of mitotane in combination with chemotherapeutic agents. In a multicenter cooperative (SWOG) phase II study, a combination of mitotane+cisplatin appeared active in advanced ACC, with 30% response rate in 37 eligible patients. These results prompted us to evaluate the activity of a combination chemotherapy of Eto-poside, Adriamycin and Cisplatin (EAP) in association with mitotane (4 g daily per os). Up to now we treated 6 patients, obtaining 3 partial responses. Recently, new drugs as suramin and gossypol have been show to have some activity in patients with surgically unresectable ACC, suggesting the need for further investigation. In conclusion, cytotoxic drugs+mitotane and new adrenocorticolytic/cytotoxic agents, should be explored as first line treatments in patients with advanced ACC. However, due to the extreme rarity of the disease, coordinated multicenter investigations should be highly encouraged. PMID- 7544428 TI - Frequent detection of hepatitis C virus subtype 3a (HCV-3a) isolates in Thailand by PCR using subtype-specific primers. AB - By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or -2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and -1b) and 2 (HCV-2a and -2b). PMID- 7544430 TI - Metastatic tumors of the adrenals. AB - Adrenals are a common site of metastasis for many solid tumors. Adrenal metastases, and related symptoms of adrenal failure, are usually overlooked in clinical practice. This is probably due to the functional compensation of the adrenal glands and to the fact that signs and symptoms of adrenal insufficiency are aspecific, and often masked by symptoms of the neoplastic disease. In some tumors in which adrenal involvement is particularly frequent, adrenal evaluation should be an essential part of the preoperative diagnostic work-up. In case of demonstration of metastatic involvement the patient could be spared a useless resection of the primary tumor. However, in selected patients, even after the demonstration of an adrenal metastasis, radical surgery could still be considered for tumors with favorable biological behaviour. In patients with widespread disease, if clinical indicators of possible adrenal involvement are present an adequate palliative therapy should be started, thus ameliorating the quality of life of the patients. PMID- 7544431 TI - Mutation of a highly conserved base in the yeast mitochondrial 21S rRNA restricts ribosomal frameshifting. AB - A mutation shown to cause resistance to chloramphenicol in Saccharomyces cerevisiae was mapped to the central loop in domain V of the yeast mitochondrial 21S rRNA. The mutant 21S rRNA has a base pair exchange from U2677 (corresponding to U2504 in Escherichia coli) to C2677, which significantly reduces rightward frameshifting at a UU UUU UCC A site in a +1 U mutant. There is evidence to suggest that this reduction also applies to leftward frameshifting at the same site in a -1 U mutant. The mutation did not increase the rate of misreading of a number of mitochondrial missense, nonsense or frameshift (of both signs) mutations, and did not adversely affect the synthesis of wild-type mitochondrial gene products. It is suggested here that ribosomes bearing either the C2677 mutation or its wild-type allele may behave identically during normal decoding and only differ at sites where a ribosomal stall, by permitting non-standard decoding, differentially affects the normal interaction of tRNAs with the chloramphenicol resistant domain V. Chloramphenicol-resistant mutations mapping at two other sites in domain V are described. These mutations had no effect on frameshifting. PMID- 7544432 TI - Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). AB - cDNAs encoding P2x purinoceptors from human bladder smooth muscle and from rat PC 12 cells were expressed in oocytes and human embryonic kidney 293 cells. Agonist potencies of 2-methylthio-ATP = 2-chloro-ATP = ATP > = 2'- and 3'-O-(4 benzoylbenzoyl)-ATP > or = adenosine-5'-O-(3-thio)-triphosphate > or = P1,P5 di(adenosine-5') pentaphosphate >> ADP prevailed for both P2x purinoceptors. There were two main differences in agonist sensitivity between the two receptors. First, ATP was 10 times more potent at the receptor from bladder (EC50, 0.8 microM) than at the receptor from PC-12 cells (EC50, 8.2 microM). Second, alpha,beta-methylene-ATP and L- and D-beta,gamma-methylene-ATP were agonists in cells expressing the bladder smooth muscle receptor (EC50, 1-3 microM) but were ineffective in cells expressing the PC-12 receptor. The P2 purinoceptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, and pyridoxal-5-phosphate acted similarly at both receptor forms, producing noncompetitive inhibition, with IC50 values of 1-5 microM for suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and 10-20 microM for pyridoxal-5-phosphate. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid distinguished receptor subtypes, producing potent inhibition of the bladder smooth muscle P2x-mediated response, with an IC50 value of 3 microM; it inhibited the PC-12 form by < 40% at 100 or 300 microM. This study thus defines the pharmacological properties of homo-oligomeric forms of these two types of cloned P2x receptor channels. PMID- 7544433 TI - Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. AB - In brain and other tissues, nitric oxide (NO) operates as a diffusible second messenger that stimulates the soluble form of the guanylyl cylase enzyme and so elicits an accumulation of cGMP in target cells. Inhibitors of NO synthesis have been used to implicate NO in a wide spectrum of physiological and pathophysiological mechanisms in the nervous system and elsewhere. The function of cGMP in most tissues, however, has remained obscure. We have now identified a compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), that potently and selectively inhibits NO-stimulated guanylyl cyclase activity. In incubated slices of cerebellum, ODQ reversibly inhibited the NO-dependent cGMP response to glutamate receptor agonists (IC50 approximately nM) but did not affect NO synthase activity. The compound did not affect synaptic glutamate receptor function, as assessed in hippocampal slices, nor did it chemically inactivate NO. ODQ did, however, potentially inhibit cGMP generation in response to NO-donating compounds. An action on NO-stimulated soluble guanylyl cyclase was confirmed in studies with the purified enzyme. ODQ failed to inhibit NO-mediated macrophage toxicity, a phenomenon that is unrelated to cGMP, nor did it affect the activity of particulate guanylyl cyclase or adenylyl cyclase. ODQ is the first inhibitor that acts selectively at the level of a physiological NO "receptor" and, as such, it is likely to prove useful for investigating the function of the cGMP pathway in NO signal transduction. PMID- 7544434 TI - RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth. AB - RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3. PMID- 7544436 TI - Individual mouse alpha-fetoprotein enhancer elements exhibit different patterns of tissue-specific and hepatic position-dependent activities. AB - Transcription of the mouse alpha-fetoprotein (AFP) gene, which is expressed at high levels in the visceral endoderm of the yolk sac and fetal liver and at low levels in the fetal gut, is regulated by three distinct upstream enhancer regions. To investigate the activities of these regions, each enhancer was individually linked to a heterologous human beta-globin promoter fused to the mouse class I H-2Dd structural gene. When tested in transgenic mice, the beta globin promoter alone has minimal activity. We find that all three enhancers activate the beta-globin promoter in an AFP-like pattern; i.e., activity is detected in the yolk sac, fetal liver, and fetal gut. The enhancers remain active in the livers and guts of adult mice, consistent with previous studies showing that postnatal AFP repression is due not to the loss of enhancer activity but to a dominant repressor region. Enhancer III also functions in the brain. In addition, these studies reveal that the three enhancers exhibit different position-dependent activities in the adult liver. Enhancers I and II are most active in hepatocytes surrounding the central vein, with a gradual decrease in activity along the hepatic plates toward the portal triad. Enhancer III is active exclusively in hepatocytes surrounding the central vein. These data represent the first examples of individual control elements exhibiting positionally regulated activity in adult liver. PMID- 7544437 TI - Functional regions of the mouse interleukin-10 receptor cytoplasmic domain. AB - The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested. PMID- 7544435 TI - The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase activating protein-associated p62 protein in cells expressing v-Src or activated c-Src. AB - CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation. PMID- 7544438 TI - Teaching tape for the motor section of the unified Parkinson's disease rating scale. AB - We developed a teaching tape of the motor section of the Unified Parkinson's Disease Rating Scale (UPDRS) to provide investigators with a visual document of three raters' interpretations of the scoring system for each item except rigidity. The rate of agreement for the selected samples was always significant, with Kendall's coefficient of concordance W ranging between 0.97 and 0.62. We also provided full UPDRS ratings on sample patients that may be used for training and for multicenter studies to assure uniformity of rating. The study identified several items of the UPDRS motor examination for which written instructions were vague, including speech, action tremor, finger taps, rapid alternating movements, and postural stability. Future versions of the scale should address these problems and correct ambiguities. This project offers the first attempt to provide a visual analog for the UPDRS. PMID- 7544440 TI - Treatment of unresectable hepatocellular carcinoma. PMID- 7544441 TI - Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin. AB - The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth. PMID- 7544439 TI - A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. AB - BACKGROUND: Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. METHODS: An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (PDs) to assess abnormalities of ion transport specific to cystic fibrosis. The safety of this treatment was monitored by nasal lavage and biopsy to assess inflammation and vector replication. RESULTS: The adenoviral vector was detected in nasal-lavage fluid by culture, the polymerase chain reaction (PCR), or both in a dose-dependent fashion for up to eight days after vector administration. There was molecular evidence of gene transfer by reverse transcriptase PCR assays or in situ hybridization in five of six patients treated at the two highest doses. However, the percentage of epithelial cells transfected by the vector was very low (< 1 percent), and measurement of PD across the epithelium revealed no significant restoration of chloride transport or normalization of sodium transport. At the lower doses of vector, there were no toxic effects. However, at the highest dose there was mucosal inflammation in two of three patients. CONCLUSIONS: In patients with cystic fibrosis, adenoviral vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer. PMID- 7544442 TI - Identification of a mouse male-specific transplantation antigen, H-Y. AB - The male-specific transplantation antigen, H-Y, causes rejection of male tissue grafts by genotypically identical female mice and contributes to the rejection of human leukocyte antigen-matched male organ grafts by human females. Although first recognized 40 years ago, the identity of H-Y has remained elusive. T cells detect several distinct H-Y epitopes, and these are probably peptides, derived from intracellular proteins, that are presented at the cell surface with major histocompatibility complex (MHC) molecules. In the mouse, the gene(s) controlling H-Y expression (Hya) are located on the short arm of the Y chromosome between the zinc-finger genes Zfy-1 and Zfy-2. We have recently identified Smcy, a ubiquitously expressed gene, in this region and its X-chromosome homologue, Smcx. Here we report that Smcy encodes an H-YKk epitope that is defined by the octamer peptide TENSGKDI: no similar peptide is found in Smcx. These findings provide a genetic basis for the antigenic difference between males and females that contributes towards a tissue transplant rejection response. PMID- 7544444 TI - Effects of deprenyl on monoamine oxidase and neurotransmitters in the brains of MPTP-treated aging mice. AB - Deprenyl is a selective monoamine oxidase B (MAO-B) inhibitor and has been used in the treatment of Parkinson's disease. However, it is not known whether deprenyl effects are symptomatic or pharmacological. Aging mice were partially lesioned with MPTP. Control and MPTP-treated mice were given deprenyl in drinking water for 14 days. Brain tissue (including the striatum, olfactory tubercle and cerebral cortex) was assayed for MAO-B and neurotransmitter levels. The results show that deprenyl treatment, given alone or after MPTP, reduced MAO-B activity in all the three regions. No change was seen in dopamine (DA), 3,4 dihydroxyphenyl acetic acid (DOPAC), and homovanillic acid (HVA) content in any of the three areas. Cortical norepinephrine (NE) levels were also unaltered. However, striatal serotonin (5-HT) levels were decreased while its metabolite, 5 HIAA levels were significantly increased in the olfactory tubercle in animals receiving deprenyl alone. These data suggest that deprenyl treatment reduces MAO B activity in regions in addition to the striatum without affecting norepinephrine, dopamine (DA) and its metabolites. PMID- 7544443 TI - Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. AB - The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may provide a mechanism for a variety of short- and long-term calcium dependent signalling events in the nervous system. PMID- 7544445 TI - Effect of domoic acid on metabolism of 5-hydroxytryptamine in rat brain. AB - Domoic acid (Dom) is a neurotoxic secondary amino acid that interacts with the glutamate receptors, producing neurological problems. In the present work, we study the effects of Dom on the levels of serotonin (5-HT) and 5 hydroxyindoleacetic acid (5-HIAA) in discrete rat brain regions. The effects of Dom on the brain metabolism of serotonin are also discussed in this paper. Dom stimulates the rat brain serotoninergic system, increasing differentially the synthesis and the catabolism of 5-HT and the elimination of 5-HIAA. PMID- 7544446 TI - Nitroarginine, an inhibitor of nitric oxide synthetase, attenuates ammonia toxicity and ammonia-induced alterations in brain metabolism. AB - We have proposed that acute ammonia toxicity is mediated by activation of the N methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroarginine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (approximately 50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D aspartate receptors. PMID- 7544449 TI - Effect of thiocyanate on AMPA receptor mediated responses in excised patches and hippocampal slices. AB - The binding affinity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for [3H]AMPA is increased 10-30-fold by the chaotropic anion thiocyanate. The present experiments tested if thiocyanate alters AMPA receptor mediated current fluxes and if any such effects are reflected in the waveform of synaptic responses. Currents were measured after a step application of glutamate or AMPA to patches excised from pyramidal cells of hippocampal slice cultures. Application of 1 mM AMPA produced responses with an average peak amplitude of 86 pA at -50 mV and a 10-90% rise time of 1.7 +/- 0.1 ms; the responses desensitized to a steady-state level below 10% of the peak current with a time constant of 11.1 +/- 0.7 ms. Glutamate in presence of D-amino-phosphonopentanoate produced similar responses which were inhibited by 6-cyano-7-nitro-quinoxaline-dione and enhanced by aniracetam or cyclothiazide and thus are characteristic for AMPA receptors. Thiocyanate accelerated the decay of AMPA responses two-fold and reduced the peak current by 30-50% with an EC50 of 3.2 mM which is comparable to its EC50 for enhancing binding. Effects on the desensitization of glutamate induced responses were much smaller and only evident at the highest thiocyanate concentration; no effect was seen on response amplitude. Binding and physiological effects can be adequately explained by assuming that thiocyanate enhances conversion from the sensitive to the desensitized state of the receptor and reduces ligand dissociation from the desensitized state. Synaptic responses were measured in disinhibited hippocampal slices. Perfusion with 20 mM sodium thiocyanate increased the slope of the field excitatory postsynaptic potential by 44.9 +/- 4.2% and reduced its decay time by 10.4 +/- 4.3%. The former effect appears to result at least in part from an increase in transmitter release since it was accompanied by a decrease in paired-pulse facilitation and was reduced in magnitude after enhancing transmitter release. The decrease in the decay time constant points to an effect of thiocyanate on AMPA receptors in situ which is similar to that seen in excised patches. These results demonstrate that an increase in binding affinity may be indicative of reduced rather than enhanced current flow through AMPA receptors. In addition, the results provide further evidence that the kinetics of the AMPA receptor channel contribute significantly to at least the decay phase of fast excitatory synaptic responses. PMID- 7544450 TI - Maternal and umbilical serum concentrations of granulocyte colony-stimulating factor and its messenger RNA during clinical chorioamnionitis. AB - OBJECTIVE: To determine whether women with chorioamnionitis developed elevated serum granulocyte colony-stimulating factor (G-CSF) concentrations and, if so, whether the G-CSF crossed to the fetus. METHODS: We quantified G-CSF before, during, and after delivery and in the cord blood of 12 women with clinical chorioamnionitis and 12 matched controls. We also measured G-CSF messenger RNA (mRNA) transcripts in mononuclear cells isolated from maternal and cord blood at delivery. RESULTS: At study entry, G-CSF concentrations were much higher in women with chorioamnionitis (1397 +/- 950 pg/mL, mean +/- standard error of mean) than in controls (17 +/- 5 pg/mL, P < .05). At delivery, concentrations had increased in both groups (2752 +/- 1100 pg/mL in the chorioamnionitis group, 165 +/- 61 pg/mL in controls; P < .05). After delivery, G-CSF concentrations had diminished to 839 +/- 594 pg/mL in women with chorioamnionitis and to 83 +/- 16 pg/mL in controls (P < .05). Concentrations in the cord blood were 2729 +/- 974 pg/mL for the chorioamnionitis group and 51 +/- 17 pg/mL for controls (P < .05). Granulocyte colony-stimulating factor mRNA transcripts were more abundant in women with chorioamnionitis than in controls but were scarce in all matched cord blood samples tested. CONCLUSION: Serum G-CSF concentrations were elevated during clinical chorioamnionitis, and similar levels were found in maternal and cord serum. Because G-CSF mRNA levels were very low in cord mononuclear cells, the G CSF in cord serum might be maternal in origin. PMID- 7544447 TI - Nutritional insult and recovery in the neonatal rat cerebellum: insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). AB - Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p < 0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158 +/- 18 to 210 +/- 18 ng/ml in C but remained low in D (47 +/- 6 ng/ml and 25 +/- 3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49 +/- 5 to 110 +/- 7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p < 0.05 and p < 0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84 +/- 0.83 vs 8.56 +/- 0.92 ng/g, p < 0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p < 0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32-34 K IGFBPs were increased 3-4 fold in D vs C, reflecting high levels of IGFBP-1 and/or -2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and -4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system. PMID- 7544448 TI - Regulation of neuronal nitric oxide synthase by histone, protamine, and myelin basic protein. AB - We examined the effects of endogenous basic proteins rich in the amino acid L arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively. PMID- 7544451 TI - [Palliative endoscopic treatment of proximal malignant biliary stenosis]. AB - 24 patients (median age 71.5 years) with inoperable proximal malignant biliary obstruction were treated by insertion of endoscopic endoprostheses from January 1991 to August 1994. 10 patients had gallbladder cancer, 6 cancer of body or tail of pancreas, 5 cholangiocarcinoma and 3 other metastatic malignancy, respectively. 13 patients had type I, 10 had type II and 1 had type III proximal biliary stenosis (Bismuth classification). Stent occlusion or dislocation required a secondary stent insertion in 9 patients. In all cases there was adequate biliary drainage after stent insertion. Complications were: early cholangitis developed in 2 patients, late cholangitis in 5, stent dislocation in 3. One patient underwent an operation because of necrotising cholecystitis and subhepatic abscess. There was no bleeding, retroperitoneal perforation or pancreatitis. 18 patients died (median survival time 28 weeks) and 6 have been alive at the time of review for 15 weeks in average. Endoscopic stent insertion can be applied effectively also in the palliative treatment of proximal malignant biliary obstruction. PMID- 7544452 TI - Calcium induces tyrosine phosphorylation of a novel p120GAP-associated protein of 65 kDa. AB - Several tyrosine-phosphorylated proteins have been identified that associate with p120GAP, the GTPase activating protein of p21ras. In keratinocytes, calcium induced the tyrosine phosphorylation of a 65 kDa p120GAP-associated protein (p65Ca). This protein did not comigrate with two previously reported p120GAP associated proteins, i.e. a 68 kDa protein from src-transformed cells (p68) and an insulin-induced protein of 60 kDa (p60(2C4)). P65Ca was neither recognized by poly(U)-sepharose, which efficiently precipitates p68, nor did it crossreact with antibodies against p68. In addition, a monoclonal antibody directed to p60(2C4) did not recognize p65Ca. From these results we conclude that p65Ca is different from p68 and p60(2C4) and thus, a novel p120GAP-associated protein. Since calcium has an important, tyrosine kinase dependent, role in the differentiation of keratinocytes, phosphorylation of p65Ca may be important for this differentiation process. However, surprisingly, calcium induced the phosphorylation of a similar sized p120GAP-associated 65 kDa protein in fibroblast cell lines. PMID- 7544453 TI - Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines. AB - We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation. PMID- 7544454 TI - Media calcium attenuates mitochondrial 1,25(OH)2D production in phosphorus or vitamin D-deprived rats. AB - Although PTH and hypophosphatemia are the best known stimulators of 25 hydroxyvitamin D-1 alpha-hydroxylase, 1,25(OH)2D3 production in rats and humans can be modulated by circulating calcium, independent of PTH. To test whether calcium modulates this function directly in mitochondria, we examined effects of calcium on 1 alpha-hydroxylase in isolated mitochondrial preparations under basal and stimulated conditions. Rats were fed a low phosphorus (or matched control) diet for 4 or 7 d or a vitamin D-deficient (or matched control) diet for 2, 4, or 7 wk. Renal mitochondria were isolated and assayed for 1 alpha-hydroxylase activity in the presence or absence of added calcium. Calcium did not alter 1 alpha-hydroxylase in rats on control diets. After 4 d of low phosphorus diet, 1 alpha-hydroxylase was increased 2-fold over basal activity; media calcium prevented this stimulatory response. By 7 d the calcium effect was not evident. After 4 wk of vitamin D deprivation, activity was approximately 30-fold greater than controls; calcium reduced this response significantly (15-fold). A significant, but less marked inhibition of activity by calcium was present in rats subjected to 7 wk of vitamin D deprivation. Extramitochondrial calcium can directly modulate 1,25(OH)2D3 production, but this effect appears to be secondary to the primary physiologic regulators of this function. The calcium effect can be overcome after longer term exposure to phosphorus deprivation, but is sustained in the presence of long term vitamin D deprivation. PMID- 7544457 TI - Silent victims revisited: the special case of domestic violence. PMID- 7544456 TI - Outcome of infants weighing less than 800 grams at birth: 15 years' experience. AB - OBJECTIVE: Mortality and neurodevelopmental morbidity among infants weighing less than 800 g at birth are compared in three separate studies from the same intensive care nursery during an almost 15-year period. METHODS: The survival and neurodevelopmental outcome of 210 infants with birth weights less than 800 g admitted to the University of Washington neonatal intensive care unit between 1986 and 1990 are compared with those of two previous cohorts (1977 through 1980 and 1983 through 1985) of extremely low birth weight (ELBW) infants from the same nursery. RESULTS: Annual admissions of these ELBW infants nearly doubled from 1977 to 1990, whereas nursery survival rose from 20% between 1977 and 1980, to 36% between 1983 and 1985, to 49% in this current study of births between 1986 and 1990. The greatest increase in survival among the three studies occurred among infants with birth weights less than 700 g. Female survival was 20% higher than male survival in each of the time periods. The prevalence of major neurosensory impairments did not differ significantly among the three study groups (19%, 21%, and 22% respectively); male survivors were more commonly affected across time periods. There were no differences in mean cognitive test scores between the current 1986 through 1990 birth cohort (94) and the two previous cohorts (1977 through 1980, 98; 1983 through 1985, 89). CONCLUSIONS: The experience of our center with these ELBW infants over time seems reassuring to the extent that progressive increases in nursery survival have not resulted in increased neurodevelopmental morbidity. PMID- 7544455 TI - Neutrophil pool sizes and granulocyte colony-stimulating factor production in human mid-trimester fetuses. AB - We quantified neutrophils and neutrophil progenitors, and assessed granulocyte colony-stimulating factor (G-CSF) production in the liver and bone marrow of 20 human abortuses after elective pregnancy termination between 14 and 24 wk of gestation. Mature neutrophils were not observed in any of the liver specimens, but were present in the bone marrow as early as 14 wk. The concentrations of neutrophils in the fetal marrow were extremely low, by comparison with term infants and adults, with less than 5% of the nucleated cells being segmented neutrophils, band neutrophils, or metamyelocytes compared with 31-69% in term infants. Despite the low neutrophil populations, progenitors which had the capacity for clonal maturation into neutrophils in vitro were abundant in the fetal liver and fetal bone marrow. In addition, such progenitors had a dose response relationship to recombinant G-CSF similar to that of progenitors from the bone marrow of healthy adults. At each gestational age tested, stimulation of mononuclear cells from fetal liver with IL-1 alpha generated less G-CSF protein and fewer G-CSF mRNA transcripts than did stimulation of mononuclear cells from fetal bone marrow. Mononuclear cells from the fetal bone marrow produced less G CSF protein and mRNA than did mononuclear cells from the blood of adults. Thus, the liver of the mid-trimester human fetus is almost devoid of neutrophils, and the bone marrow contains a significantly lower proportion of neutrophils than does the marrow of term neonates or adults. These findings correlate with IL-1 alpha-induced production of G-CSF in these organs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544459 TI - A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein. AB - We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. PMID- 7544458 TI - Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction. AB - G-->A hypermutation is a remarkable phenomenon resulting from retroviral reverse transcription in the presence of highly biased dNTP concentrations. Of the three reverse transcriptases (RTases) available, those of human immunodeficiency virus type 1 (HIV-1), avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MoMLV), the HIV-1 enzyme showed the greatest sensitivity to biased [dCTP]/[dTTP] ratios. The HIV-1 RTase was able to discriminate between dUTP, dITP and the four DNA precursors and was insensitive to pH. There was little preference for nucleotide contexts. A few exceptionally modified sequences were found presumably resulting from G-->A hypermutation and multiple strand transfer. This particular predilection of the HIV-1 and, by extrapolation, the lentiviral RTases towards G-->A hypermutation suggests that the phenomenon may have contributed to the remarkably elevated A content of these retroviral genomes. PMID- 7544460 TI - Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus. AB - Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. PMID- 7544461 TI - Solid phase synthesis of 5'-diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp-oligoribonucleotides for use as primers for influenza A virus RNA polymerase in vitro. AB - We have synthesized four different 5'-diphosphorylated oligoribonucleotides, varying in length from 11 to 13 nucleotides by a new solid phase method. After deprotection and partial purification the 5'-diphosphorylated oligoribonucleotides could be converted to capped (m7Gppp) oligoribonucleotides using guanylyl transferase. Radiolabelled capped oligoribonucleotides acted as primers for the influenza A virus RNA polymerase in vitro. The solid phase method described here should also allow the addition of 5'-diphosphates to synthetic oligodeoxyribonucleotides and be capable automation. PMID- 7544463 TI - "Well-determined" regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA. AB - Recent structural analyses of genomic RNAs from RNA coliphages suggest that both well-determined base paired helices and well-determined structural domains that are identified by "energy dot plot" analysis using the RNA folding package mfold, are likely to be predicted correctly. To test these observations with another group of large RNAs, we have analyzed 15 ribosomal RNAs. Published secondary structure models that were derived by comparative sequence analysis were used to evaluate the predicted structures. Both the optimal predicted fold and the predicted "energy dot plot" of each sequence were examined. Each prediction was obtained from a single computer run on an entire ribosomal RNA sequence. All predicted base pairs in optimal foldings were examined for agreement with proven base pairs in the comparative models. Our analyses show that the overall correspondence between the predicted and comparative models varied for different RNAs and ranges from a low of 27% to high of 70%, with a mean value of 49%. The correspondence improves to a mean value of 81% when the analysis is limited to well-determined helices. In addition to well-determined helices, large well determined structural domains can be observed in "energy dot plots" of some 16S ribosomal RNAs. The predicted domains correspond closely with structural domains that are found by the comparative method in the same RNAs. Our analyses also show that measuring the agreement between predicted and comparative secondary structure models underestimates the reliability of structural prediction by mfold. PMID- 7544464 TI - Caudal raphe-dorsal vagal complex peptidergic projections: role in gastric vagal control. AB - Subpopulations of raphe pallidus (Rpa) and raphe obscurus (Rob) neurons containing TRH, serotonin (5-HT), and substance P contribute projections to the dorsal vagal complex (DVC). Activation of Rpa and Rob neurons induces a vagal cholinergic-dependent stimulation of gastric secretory and motor function and modulates resistance of the gastric mucosa to gastric injury in rats and cats. The caudal raphe nuclei-DVC pathways containing TRH/5-HT are involved in mediating cold-induced vagal stimulation of gastric function and erosion formation. These results suggest that Rpa/Rob-DVC projections containing TRH/5-HT may be an important pathways in the medullary regulation of vagal activity to the viscera. PMID- 7544465 TI - Subcutaneous injections of the tachykinin senktide reduce alcohol intake in alcohol-preferring rats. AB - The present study evaluated the effect of SC injections of the selective NK3 tachykinin agonist, Suc-[Asp6,MePhe8]substance P(6-11), also referred to as senktide (SENK), on 8% alcohol intake in genetically selected alcohol-preferring rats. Animals were offered access to 8% ethanol for 2 h/day (between 1800 and 2000 h) and to tap water for 4 h/day (between 1800 and 2200 h); SENK was injected 10 min before access to fluids. The peptide significantly reduced alcohol intake at doses of 125 and 250 micrograms/kg, but not at 62.5 micrograms/kg. The reduction in alcohol intake was accompanied by a sharp increase in water intake, so that total fluid intake was never significantly modified. The same SC doses of SENK did not modify water intake in rats with access to water, as the only fluid, for 4 h/day. In food-deprived rats food intake was not altered by 125 micrograms/kg, whereas 250 micrograms/kg produced a reduction in food intake that was smaller in intensity and shorter lasting than the reduction in alcohol intake. The same doses of SENK did not modify 0.1% saccharin intake, nor did they elicit major competing behaviors. The results of the present study are in keeping with those obtained following central injection of NK3 agonists, and show that a behaviorally selective reduction of alcohol intake can be evoked also by peripheral administration of SENK. PMID- 7544466 TI - Effect of ribonuclease A and deoxyribonuclease I on immunostaining of Ki-67 in fixed-embedded sections. AB - Immunostaining of the cell cycle-associated Ki-67 antigen was studied on routinely formalin-fixed and paraffin-embedded tissue sections, using the Ki-67 specific monoclonal antibody MIB-1. Immunomorphological analysis of the Ki-67 immunostaining pattern was carried out following tissue pre-treatments including combinations of microwave heating and trypsinization, as well as of ribonuclease and deoxyribonuclease pre-digestion of the sections. The nucleolar Ki-67 immunostaining after slide pre-treatment by microwave heating followed by trypsinization was reduced only by ribonuclease pre-digestion, if this latter was used before heating. Ki-67 immunostaining was not significantly reduced by deoxyribonuclease treatment. We conclude that Ki-67, located in the nucleoli, may be associated there with nucleolar RNA. PMID- 7544467 TI - [The hemolysis and solubilization behavior of nonionic polymer surface-active agents classes]. AB - The solubilization of poorly water soluble drugs in surfactant solutions is still a problem in pharmaceutical technology. There doesn't exist any surfactant which can be applied unrestricted in parenteral dosage forms. For this reason, alkylpolyglycosides and fatty esters of dextran were investigated. Their solubilization behaviour and hemolytic activity was tested and compared to established surfactants. For solubilization, only water soluble surfactants can be used. That means, that only alkylpolyglycosides with two or more glucose units and dextran esters with a degree of substitution of 0.1 or less may be applied. The determination of the CMC of alkylpolyglycosides by solubilization of fluorescent substances shows a plot, typical for monomeric surfactants. The dextran esters show no CMC. They actually are polymeric surfactants and form monomolecular micelles. The differences in hemolytic activity of the alkylpolyglycosides run up to the factor 100. The hemolytic activity increase in this class of surfactants with the elongation of the alkyl chain and decreases with increasing degree of glycosylation. It doesn't exist any correlation between the CMC and the hemolytic activity. For diazepam the differences in solubilization behaviour are rather low. The solubilization behaviour is more effective than that of Solutol HS 15. The hemolytic activity of the dextran fatty esters is low, compared with the alkylpolyglycosides. PMID- 7544462 TI - Synthesis, deprotection, analysis and purification of RNA and ribozymes. AB - Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity. PMID- 7544469 TI - Spontaneous feeding-related monoamine changes in rostromedial hypothalamus of the obese Zucker rat: a microdialysis study. AB - Using microdialysis in freely moving rats, we have been able to observe changes in monoamines from the ventromedial and paraventricular hypothalamic nuclei before, during, and after spontaneous feeding. Because the genetically obese Zucker rat shows abnormalities related both to feeding and monoamines, it was of interest to investigate possible particularities in the monoaminergic variations around spontaneous feeding. The profile of changes in Zucker rats was grossly similar to that of Wistar rats: increases in 5-hydroxy-tryptamine (5-HT), 5 hydroxyindolacetic acid (5-HIAA), and dopamine (DA), and decrease in dihydroxyphenylacetic acid (DOPAC). However, the release in monoamines (5-HT and DA) was more dramatic and longer-lasting than in Wistar rats, while the changes in the metabolites were proportionnally less pronounced. This suggests that high concentrations of these feeding-related amines are released and remain in the synaptic cleft of the obese rat, possibly because they are required in larger amounts to bring about satiety. The hyperphagia of the obese Zucker rat may therefore be the result of a resistance to these prandially released satiety promoting neurosubstances. PMID- 7544468 TI - Stress-induced changes in regional monoamine metabolism and behavior in rats. AB - Groups of adult, male, Sprague-Dawley rats were subjected to either restraint at room temperature or cold restraint at 4 +/- 1 degree C for 2 h. Spontaneous motility, measured immediately after the stress, was decreased and defecation scores were increased in the cold restraint group but not in the restraint group. The rats were then sacrificed and prefrontal cortex, nucleus accumbens, and striatum dissected out. The levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were measured in these areas by high performance liquid chromatography (HPLC) with electrochemical detection. DA levels were decreased in the prefrontal cortex (-68%), nucleus accumbens (-40%), and striatum (-76%) of the cold restraint group when compared to the controls. DA levels were decreased only in the prefrontal cortex of the restraint rats (-31%) when compared to the controls. The cold restraint group also had increased levels of DOPAC in the prefrontal cortex (+66%) and nucleus accumbens (+65%) and increased levels of HVA in the prefrontal cortex (+43%) when compared to the control group. Our results therefore indicate that cold restraint activates DA metabolism in the prefrontal cortex, nucleus accumbens, and striatum while restraint activates DA metabolism in the prefrontal cortex alone. Thus, cold restraint may be a more potent stressor than restraint alone. PMID- 7544470 TI - Roles of peptides in enteric neuromuscular transmission. PMID- 7544471 TI - Inhibitory effect of ODN, a naturally occurring processing product of diazepam binding inhibitor, on secretagogues-induced insulin secretion. AB - Diazepam binding inhibitor (DBI1-86) is a peptide that is present in large amounts in the intestine and pancreas and which inhibits glucose-stimulated insulin release from both perfused pancreas and isolated islets in low nanomolar concentrations. Here, DBI33-50 (also known as ODN, octadecaneuropeptide), one of the naturally occurring processing products of DBI1-86, and certain synthetic modified derivatives, have been shown to inhibit glucose and glibenclamide stimulated insulin secretion from isolated rat islets and glibenclamide stimulated insulin secretion from hamster-insulinoma (HIT-T15) beta-cell line. DBI17-50 (TTN; triakontatetraneuropeptide), another prominent processing product of DBI, had no effect. The 50% inhibitory concentration (IC50) for the effect of ODN on insulin secretion induced by 8.3 of 16.7 mM glucose was approximately the same: 5 to 6 nM. Moreover, ODN inhibited insulin release induced by 0.01 or 1 microM glibenclamide with a similar IC50 (8 to 10 nM) in both isolated pancreatic islets and in HIT-T15 beta-cells. At concentration up to 1 microM, ODN had no effect on insulin secretion induced by PACAP (pituitary adenylate cyclase polypeptide), BAYK 8644 (methyl-(1,4-dihydro-2,6-dimethyl-3-nitro-4,2 trifluoromethylphenyl) pyridine-5-carboxylate), and only marginally it affected IBMX-(isobutylmethylxanthine) induced insulin secretion. This indicates that ODN does not act directly on ATP-regulated K+ channels, voltage dependent Ca2+ channels or cAMP production. In contrast, ODN inhibited insulin secretion induced by sodium nitroprussiate in a manner that is independent from the presence of extracellular Ca2+. These results suggest that ODN or ODN-like peptide fragments of DBI, may inhibit glucose or glibenclamide-induced insulin secretion via a signaling pathway that regulate the cytoplasmic free Ca2+ concentration. PMID- 7544472 TI - [From antinuclear antibodies to corresponding antigens. Physiopathological value]. PMID- 7544474 TI - [Funeral rites, a symbolic act]. PMID- 7544473 TI - [Hectic fever and granulocytosis]. PMID- 7544475 TI - [Information about GCSF by a nurse]. PMID- 7544476 TI - New developments in the clinical application of auditory evoked potentials with children with multiple handicaps. PMID- 7544477 TI - History and state-of-the-art in behavioural methods for hearing assessment in low functioning children. PMID- 7544479 TI - New diagnostic applications and physiological functions of prostate specific antigen. AB - Prostate specific antigen was, until recently, thought to be a highly specific biochemical marker of prostatic epithelial cells. Using highly sensitive techniques, we have recently found that PSA is present in 30-40% of breast tumors and at a lower percentage in other tumors including lung, colon, ovary, liver, kidney, adrenal and parotid tumors. Others have found PSA in skin and salivary gland tumors and in normal endometrium. We found PSA in normal breast, milk of lactating women and in amniotic fluid. The physiological role of PSA in these tissues and tumors is currently unknown. It appears that PSA is either a growth factor or a growth factor regulator and that it may play a role in fetal development. A substrate for PSA in these tissues has not as yet been identified. PMID- 7544480 TI - TPS--a cytokeratin marker for therapy control in breast cancer. PMID- 7544478 TI - Regulation of the enzymatic activity of prostate-specific antigen and its reactions with extracellular protease inhibitors in prostate cancer. AB - Prostate-specific antigen (PSA) is a tissue-specific serine protease similar in structure to the trypsin-like glandular kallikreins but which is unique inasmuch as the enzyme activity is similar to that of chymotrypsin. The active enzyme is a single chain glycoprotein of 237 amino acids. The major form of PSA in serum is complexed to alpha 1-antichymotrypsin (ACT). A small amount is free, non complexed despite a large excess of ACT. This suggests that the form in serum lacks enzyme activity. Although serum PSA concentrations are regularly abnormally high (above 4 micrograms/L) in prostate cancer (CAP), the utility of PSA measurements in the early detection of CAP is limited, as many tumors are undetected at a cut-off of 4 micrograms/L. Also, 25% of all men with benign prostate hyperplasia (BPH) have serum PSA levels above 4 micrograms/L. Using assays specially developed to measure free and complexed forms of PSA in serum, we found the proportion of PSA-ACT complexes to be higher in CAP than in BPH, but the ratio of free-to-total PSA in serum to be lower. Using an abnormally low ratio of free-to-total PSA to detect CAP increases diagnostic specificity by 15 to 20%, compared to using a high serum PSA concentration. This suggests that the ratios of free-to-total PSA significantly increase the ability to distinguish BPH from localized CAP. The molecular basis is unclear, but may be related to the high incidence of prostate tumor cells producing both PSA and ACT. This is in contrast to the lack of ACT production in BPH epithelium. Possibly owing to lack of ACT production in BPH areas, conditions are not optimal for complex formation, whereas tumors producing both ACT and PSA may promote the formation of PSA-ACT complexes in CAP. PMID- 7544481 TI - Biochemistry of prostate specific antigen, PSA. AB - Human prostate specific antigen, PSA, is a product of the human glandular kallikrein gene locus on chromosome 19 that is almost selectively expressed by prostate tissue. PSA is one of the dominating prostate derived proteins in seminal fluid. The mature form of PSA, a single chain glycoprotein of 237 amino acids, is a serine protease manifesting restricted chymotrypsin-like activity. PSA is mainly responsible for gel dissolution in freshly ejaculated semen by proteolysis of the major gel forming proteins, semenogelin I and II, and fibronectin. In semen approximately two thirds of PSA is enzymatically active. The remaining 30-40% is inactive due to internal cleavage(s). A few per cent of PSA in semen is complexed to the protein C inhibitor. PSA complexed to alpha 1 antichymotrypsin (ACT) constitutes the predominant molecular form of serum PSA, although complex formation is slow between the purified proteins in vitro. PSA also forms stable complexes with alpha 2-macroglobulin in vitro but as this results in encapsulation of PSA and complete loss of the PSA-epitopes, the in vivo significance of this complex formation is presently unclear. A free, non complexed form of PSA constitutes a minor fraction of the serum PSA despite the large molar excess of antiproteasees such as ACT. In patients with carcinoma of the prostate the serum PSA level increases. Analysis of the serum level of PSA is used both for diagnosing and monitoring patients with carcinoma of the prostate (CAP).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544483 TI - Prostate-specific antigen: its clinical use and application in screening for prostate cancer. AB - Prostate cancer in most European countries is the second most frequent cancer in males and the second most frequent cause of cancer death. Prostate specific antigen (PSA) is an important tumour marker, which relates to many aspects of this disease. It has been shown that PSA is helpful in the early diagnosis of prostate cancer and in this respect is superior to other available tests like rectal examination and transrectal ultrasonography. PSA is also helpful in staging of locally confined disease. It can be used to identify or exclude local extension of disease, if combined with T category and grade of differentiation determined on biopsy. The same parameters also give an indication of the presence of lymph node metastases, which may prevent unnecessary and invasive staging procedures in certain groups of patients with favourable prognostic factors and a low PSA value. PSA is less suitable as a marker for metastatic disease. Progression of untreated prostate cancer in various stages can be monitored by PSA. The true value of the marker in this respect is still underexplored. It may be possible that PSA will be shown to differentiate effectively between aggressive and non-progressive disease. In this respect, it could become an essential tool to identify those patients that may not require treatment at all. PSA is also a useful marker for therapy response. An elevation of PSA after radical prostatectomy indicates local or metastatic progression, which will occur within 1-2 years. PSA is an androgen dependent enzyme and decreases under endocrine treatment. It is unexplained why in spite of its endocrine dependent character, PSA rises with endocrine independent progression of prostate cancer. PMID- 7544482 TI - Clinical usefulness of free and complexed PSA. AB - We selected serums from 51 fully characterized prostate cancer patients and 48 biopsy-proven BPH patients in order to test the ability of the ratio of the free/total PSA in distinguishing between CaP and BPH patients in the best case scenario. The 51 cancer patients had cancer volumes ranging from 2.0-17.8 mL and had a median % free PSA of 8.9%. The 48 BPH patients, which had prostate volumens ranging from 36.9-313.8 mL, had a median % free PSA of 16.5%, almost twice that of the CaP patients. We also examined the physiological variation of serums drawn on the same patient over a reasonably short time (mean of 22 days). The variation between consecutive redraws on the same patient was measured to be 30% (95% confidence interval) in the PSA range of 4-10 micrograms/L, measured on the Hybritech Tandem-R PSA Assay. PMID- 7544484 TI - Standardization of PSA determinations. AB - Standardization of determinations for prostate specific antigen (PSA) has become an important issue due to the widespread use of these determinations for prostate cancer screening. Standardization of this assay is complicated due to the occurrence of two major forms of PSA in serum, the free antigen and a complex between PSA and alpha 1-antichymotrypsin (PSA-ACT). These two forms of PSA are recognized differently by different antibodies, but by careful selection of antibodies, it is possible to design methods that measure each form equally. It is suggested, that standards for PSA and PSA-ACT should be prepared and established as international standards. Furthermore, reference methods should be established on the basis that these standards and carefully selected reference antibodies. PMID- 7544485 TI - A new mathematical procedure for the assessment of test performance of PSA assays. AB - A new procedure has been worked out and adapted on microsoft Excel that allows to calculate from one set of e.g. 100 triplicate measurements the critical limit, the limit of detection and the power of definition. The limit of detection is defined as the analyte concentration which can be differentiated from the blank with a confidence of > or = 95%. The power of definition defines the number of values that can be differentiated in a certain interval with a predefined probability (e.g. > or = 95%). The lower limit of quantification is obtained graphically from the precision profile as the intersection of a line, representing the maximum acceptable CV, with the upper edge of the confidence interval. PMID- 7544486 TI - A universal calibrator for prostate specific antigen (PSA). AB - We describe for the first time a protocol to purify an in vitro made PSA-ACT complex to apparent homogeneity by using a combination of gel filtration and ion exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. Purification of the PSA-ACT complex was highly reproducible. An extinction coefficient of 1.0 and 280 nm (L x g-1 x cm-1) was assigned to the PSA-ACT complex based on amino acid analysis. Molecular weight was assigned by taking cDNA of ACT (plus 26% carbohydrate) and the molecular weight of PSA (28,430) which totals 89,280. Two common calibrators were made consisting of 100% PSA-ACT complex and/or 90% PSA-ACT complex plus 10% free PSA (90:10 calibrator). The 90:10 calibrator is recommended as a universal calibrator for the international standardization of PSA immunoassays. PMID- 7544488 TI - CYFRA 21-1--clinical applications and analytical requirements. AB - CYFRA 21-1 has proved to be a useful marker for non-small-cell lung cancer (NSCLC), which is the major form of lung cancer. Its most effective application is in monitoring. CYFRA 21-1 provides diagnostic information about the success of primary surgery, the response to chemotherapy and the detection of relapse. It is also an independent prognostic factor. The diagnostic potential may not be fully used because decision-making is currently based on group reference ranges. It seems useful to carry out systematic studies to investigate if the application can be improved and extended by using individual reference ranges for decision making. In contrast to most of the other tumour markers the comparability of the commercial CYFRA 21-1- assays currently available on the market is good. The high degree of comparability should be maintained by international standardization. The analytical performance of the currently available commercial CYFRA 21-1 tests meets requirements derived from its current clinical applications. However, there are no data available about the analytical performance under field conditions. CYFRA 21-1, an established tumour marker for lung cancer, should be included in external quality assurance schemes. PMID- 7544487 TI - The biochemistry of CYFRA 21-1 and other cytokeratin-tests. AB - Soluble forms of keratins in human sera seem to be useful analytes for the monitoring of cancer patients. CYFRA 21-1 is a new test measuring keratin 19 in human blood. The test was developed as sandwich ELISA based on two monoclonal antibodies, both reacting with keratin 19. The epitopes recognised by the antibodies are located on the rod region of the molecule. In sera from malignant patients CYFRA 21-1 detects immunoreactive compounds which appear to be larger than keratin 19 itself, indicating the presence of oligomers in these sera. In a comparison study the reactivity of other keratin tests (TPA, TPS, TPAcyk) with the keratins 8, 18 and 19 were measured both in solution and on immunoblots. The reactivity pattern was found to be different what may explain the different diagnostic properties of the individual tests. PMID- 7544489 TI - [Galanin research progress]. PMID- 7544490 TI - [Abnormality in nitric oxide and nitric oxide synthase potential relevance to disease]. PMID- 7544491 TI - The RNA component of human telomerase. AB - Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells. PMID- 7544492 TI - Functional characterization and developmental regulation of mouse telomerase RNA. AB - Telomerase synthesizes telomeric DNA repeats onto chromosome ends de novo. The mouse telomerase RNA component was cloned and contained only 65 percent sequence identity with the human telomerase RNA. Alteration of the template region in vivo generated altered telomerase products. The shorter template regions of the mouse and other rodent telomerase RNAs could account for the shorter distribution of products (processivity) generated by the mouse enzyme relative to the human telomerase. Amounts of telomerase RNA increased in immortal cells derived from primary mouse fibroblasts. RNA was detected in all newborn mouse tissues tested but was decreased during postnatal development. PMID- 7544493 TI - Conformation and function of the N-linked glycan in the adhesion domain of human CD2. AB - The adhesion domain of human CD2 bears a single N-linked carbohydrate. The solution structure of a fragment of CD2 containing the covalently bound high mannose N-glycan [-(N-acetylglucosamine)2-(mannose)5-8] was solved by nuclear magnetic resonance. The stem and two of three branches of the carbohydrate structure are well defined and the mobility of proximal glycan residues is restricted. Mutagenesis of all residues in the vicinity of the glycan suggests that the glycan is not a component of the CD2-CD58 interface; rather, the carbohydrate stabilizes the protein fold by counterbalancing an unfavorable clustering of five positive charges centered about lysine-61 of CD2. PMID- 7544494 TI - [Single channel properties of NMDA receptors in cortical neurons]. AB - The cell-attached and inside-out configurations of the patch-clamp techniques were used to investigate single channel properties of NMDA receptors in cultured intact neurons mechanically isolated from rat cereberal cortex. Recordings were made in Mg(2+)-free solutions. A channel, with a conductance of 35 pS was studied in detail with either NMDA or L-aspartate in the patch pipette. NMDA channels were permeable to Na+ K+, but not to Cl-, the mean open times and open probabilities of these channels were decreased with increasing hyperpolarization. Distributions for the open times, closed times and burst durations required two component fits. Channel openings were suppressed by APV. When Mg2+ was included in the pipette, the mean open times were significantly diminished in a concentration- and voltage-dependent manner. Decreasing the bath temperature prolonged channel open times and decreased current amplitudes. The results indicate that there is an intrinsic voltage dependence of NMDA channel kinetics in the intact neurons, suggesting that the normal function of the NMDA channel may be dependent on some intracellular biochemical processes. PMID- 7544495 TI - Neurosyphilis, or chronic heavy metal poisoning: Karen Blixen's lifelong disease. AB - BACKGROUND: Since the 1490s, the treatment of syphilis has consisted of heavy metals--first mercurial and later arsenic and bismuth preparations. Tabes dorsalis, as described by Duchenne in the 1850s, is made up of various characteristic neurologic symptoms. "Gastric crises," sudden stabbing pains followed by vomiting and diarrhea, was originally included by Duchenne, but later, syphilologists disputed its relevance to syphilis. Poisoning by heavy metals, including mercury, may produce similar pain reactions and tabes-like neurologic symptoms. METHODS: According to an earlier published pathography, the Danish author Karen Blixen (1885-1962), also known under the pseudonym Isak Dinesen, suffered from a lifelong disease described as tabes dorsalis. She got syphilis in 1914 and took mercury pills for a year, after which she experienced a severe mercurial intoxication. The Wassermann reaction (WR) in peripheral blood was positive only once, in 1915, before treatment with arsphenamine (Salvarsan), which she received during hospitalization in Copenhagen in 1915 to 1916. Her spinal fluid was examined several times from 1915 to 1956. Apart from an increased number of cells in 1915, the fluid remained unremarkable and the WR was always negative. RESULTS AND CONCLUSIONS: It was postulated that her illness, ending with a cachectic state, was the result of heavy metal poisoning from the various treatments and not a monosymptomatic tabes dorsalis with negative serology. PMID- 7544496 TI - [Good pain relief or euthanasia?]. PMID- 7544497 TI - [Infusion of barbiturates as a compenent in the treatment of refractory pain in patients with terminal cancer]. AB - Four patients with advanced cancer were referred to the Pain Team because of refractory pain symptoms. They received intravenous barbiturates prior to death. All attempts at palliative treatment had been exhausted. The symptoms were described by patients and family as unendurable. After adding intravenous thiopental, relief and tranquility were achieved. All four patients died peacefully. The need to sedate to a state of unconsciousness or semi consciousness to relieve symptoms in dying cancer patients is extremely rare. After all viable alternatives have been explored, titrated barbiturate infusion as an adjuvant may fill a void in the care of especially challenging patients as they approach death from terminal cancer. PMID- 7544500 TI - Aprotinin in orthotopic liver transplantation. PMID- 7544501 TI - Compassionate use of FK 506 in liver transplantation. PMID- 7544499 TI - Hepatitis C virus markers and de novo liver disease after transplantation: a prospective study. PMID- 7544498 TI - Organophosphate pesticides--neurological and respiratory toxicity. PMID- 7544502 TI - Bone marrow transplantation without protective environment: evaluation of infectious complications. PMID- 7544504 TI - Multilineage amplification of graft-vs-host disease-resistant chimerism following rat vascularized bone marrow allotransplantation. PMID- 7544503 TI - Peripheral blood stem cell mobilization following salvage chemotherapy (IAPVP-16) plus granulocyte colony-stimulating factor and autografting for non-Hodgkin's lymphoma. PMID- 7544505 TI - Porcine epitopes recognized by mouse anti-human monoclonal antibodies. PMID- 7544507 TI - Effect of different fluid-loading solutions on renal tubular cells in brain-dead dogs. PMID- 7544508 TI - Effects of early interferon alfa therapy for hepatitis C virus infection recurrence after liver transplantation. PMID- 7544509 TI - [The effect of the components of the extracellular matrix on the spreading of rat keratinocytes in the substrate during cultivation in a low-calcium medium]. AB - Two-day-old newborn rat keratinocytes were plated in serum-free low-Ca medium on different substrates. The culture conditions prevented from keratinocyte aggregation, and therefore allowed to observe the interaction between a single cell and the substrate without interference of cell-cell contacts. It is shown that separate cells spread on type I collagen, whereas cell colonies developed on fibronectin and non-treated glasses. Cellular actin distribution, revealed by rhodamine-phalloidin staining, suggested that on type I collagen cell-substrate interactions differed from those on other substrates. While cells on type I collagen had large radial phillopodia with rich actin staining, they had marginal actin rings and small phillopodia plated on fibronectin or glass. Taking together differences in cytoskeletal actin distribution and shapes of colonies type I collagen is supposed to be more powerful than fibronectin and glass in binding the corresponding cell receptors, thus preventing from colony formation. PMID- 7544506 TI - Hepatitis C following renal transplantation: histopathological correlations. PMID- 7544511 TI - [Continuous subcutaneous infusion of opioids in cancer patients]. AB - This review article describes pharmacokinetics, pharmaco-dynamics, side effects and the practical use of continuous subcutaneous infusion of opioids in cancer patients with pain. Clinical studies have shown that the analgesic effects of continuous subcutaneous infusion of morphine are comparable to continuous intravenous morphine, and that the treatment modality is associated with a low frequency of side-effects and complications. Continuous subcutaneous infusions of morphine are therefore recommended as the treatment of choice for cancer patients with pain, when oral analgesic treatment is no longer possible. PMID- 7544512 TI - [Perinaud's oculoglandular syndrome as a manifestation of cat-scratch disease]. AB - The first Danish case of Parinaud's oculoglandular syndrome, a manifestation of cat-scratch disease, is reported in a 35-year-old man presenting with an enlarged preauricular lymph node and an ipsilateral conjunctival granuloma. Surgical removal of the granulomatous lesion was followed by rapid healing. The diagnosis was verified by demonstrating a high antibody tire against Rochalimaea (Bartonella) henselae. On subsequent questioning the man gave a history of acquiring a kitten six weeks before his illness. The importance of eye examination in patients presenting with preauricular lymphadenopathy is emphasized. PMID- 7544510 TI - [The amplification and overexpression of mdr-family genes in ethidium bromide resistant Chinese hamster CHO-K1 cells and in the hybrids of sensitive and resistant cells]. AB - Stable mutant cells Cebr-1 and Cebr-2, resistant to ethidium bromide (EB) in concentration of 2 micrograms/ml, have been isolated by a multistep selection in Chinese hamster ovary cells. It was shown that Cebr-1 and Cebr-2 cells acquired a cross-resistance to unrelated drugs. Stable changes in the structure of chromosomes 1, 2, 5 and 8 were revealed by karyological analysis. Overexpression and amplification of mdr genes were detected in Cebr-2 cells using Northern RNA and Southern DNA blot hybridization. Two independent hybrids Hybr-1 and Hybr-2 were obtained by fusion of Cebr-2 cells with Chinese hamster lung V-79 RJK cells, sensitive to EB. Hybr-2 cells were characterized by the same level of EB resistance as Cebr-2 cells. Hybr-1 cells have a lower level of EB-resistance than Cebr-2 cells. Hybr-2 cells have demonstrated amplification and overexpression of mdr gene, the same as in Cebr-2 cells, whereas in Hybr-1 cells no mdr gene amplification was observed, but the level of mdr gene expression was higher than in sensitive cells. The data suggest that resistance of Chinese hamster cells to EB is mediated by amplification and overexpression of mdr genes. PMID- 7544513 TI - Economic issues and the management of benign prostatic hyperplasia. AB - Enormous financial resources are expended worldwide on the treatment of the urologic complications and symptoms induced by benign prostatic hyperplasia (BPH). Even for its surgical management, where the best data exist, current international accounting of these expenditures remains very poorly documented. On February 8, 1994, the Department of Health and Human Services of the US government released clinical guidelines for the diagnosis and management of BPH. Imaging of the upper urinary tract as a routine diagnostic procedure is not recommended in these guidelines unless a comorbidity indicating its need exists. Diagnostic cystoscopy to assist in the decision of the need to treat is not recommended. Adherence to these two principles along with adherence to the strategies of management presented in the guidelines and discussed herein has the potential of achieving profound financial savings without impairing quality of care worldwide. PMID- 7544515 TI - Natural history of benign prostatic hyperplasia: appropriate case definition and estimation of its prevalence in the community. AB - There is no consensus about a case definition of benign prostatic hyperplasia (BPH). In the present study, BPH prevalence rates were determined using various case definitions based on a combination of clinical parameters used to describe the properties of BPH: symptoms of prostatism, prostate volume increase, and bladder outflow obstruction. The aim of this study--in a community-based population of 502 men (55-74 years of age) without prostate cancer--was to determine the relative impact on prevalence rates of the inclusion of these different parameters (and of different cutoff values for these parameters) in a case definition of BPH. There is agreement that age is the dominant determinant of BPH. However, of 28 different case definitions that were formulated, only eight gave a statistically significant increase in the prevalence of BPH with age. The highest overall prevalence of 19% (95% confidence interval [CI], 15-23%) occurred using the definition that combines a prostate volume > 30 cm3 and an International Prostate Symptom Score (IPSS) > 7. The lowest prevalence rate of 4.3% (95% CI, 2-6%) occurred using the definition that combines a prostate volume > 30 cm3, an IPSS > 7, a maximum flow rate < 10 mL/s, and the presence of a postvoid residual urine volume > 50 mL. Thus, prevalence rates depend very much on the parameters used in a case definition. Follow-up will establish which men will eventually request a workup and treatment for BPH and will help determine the best clinical definition of BPH. PMID- 7544514 TI - Using prostate-specific antigen to eliminate unnecessary diagnostic tests: significant worldwide economic implications. AB - As the 21st century approaches, it is necessary for urologists worldwide to diagnose and stage malignancies in a cost-effective manner. With the recent advances in the use of prostate-specific antigen (PSA), it is now possible to avoid many time-consuming, expensive, and invasive procedures that have been commonplace in the past. PSA has replaced transrectal ultrasonography (TRUS) as a first-line diagnostic test for evaluating men for early, curable prostate cancer. In older men, the use of age-specific reference ranges for serum PSA can significantly decrease the number of prostate biopsies that are routinely performed. With regard to staging procedures, PSA can now be used successfully to eliminate the bilateral pelvic lymphadenectomy in 30% of men with clinically localized disease and the radionuclide bone scan in 40% of patients with newly diagnosed prostate cancer. In the discussion that follows, the use of serum PSA in the diagnosis and staging of prostate cancer will be discussed. In addition, the economic benefit achieved through proper use of this tumor marker will be reviewed. PMID- 7544516 TI - Worldwide patterns of prevalence and mortality from benign prostatic hyperplasia. AB - Benign prostatic hyperplasia (BPH) can be found in 88% of autopsies in men > or = 80 years, with compatible symptomatology reported in nearly 50% of men aged > or = 50 years in the general population. Despite such a common occurrence, little is known with any certainty about the epidemiology of BPH (for which "prostatism" is a commonly, and wrongly, used synonym). Knowledge of risk factors is sparse: analytic epidemiologic studies of BPH are difficult to conduct. It is essential to establish an epidemiologic definition of BPH for these reasons. Both BPH and prostatism are the problems that seem set to increase in absolute terms. They are clearly identified as priority areas for research into their causes and treatment. However, it is clear that there is a great need for more epidemiologic information, particularly regarding prostatism, whose occurrence is unknown in many parts of the world. PMID- 7544517 TI - Characteristics of prostate cancer found with early detection regimens. AB - This article reviews the clinical and pathologic characteristics of nonpalpable (Stage T1c) prostate cancer reported in five studies published to date. Stage T1c tumors more closely resemble palpable prostate cancers (Stage T2) than they do incidental prostate cancers detected in cystoprostatectomy specimens. The mean tumor volume of Stage T1c tumors is about 50 times greater (2.0 mL vs 0.04 mL) than incidental cystoprostate cancers. Only about 20-25% of T1c tumors have a tumor volume < 0.5 mL; tumors > 0.5 mL are considered to have significant biologic potential. Futhermore, 30-50% of T1c tumors have penetrated the prostatic capsule, 20-30% demonstrate positive surgical margins, and 5-10% demonstrate seminal vesicle invasion and/or positive pelvic lymph nodes. Possible reasons for nonpalpability of T1c tumors include tumor location in the central and/or anterior zones of the prostate and increased gland volume due to benign prostatic hyperplasia, making T1c tumors more difficult to palpate. A combination of serum prostate-specific antigen (PSA) density < 0.1 and favorable needle biopsy pathology (no Gleason 4 or 5 pattern in any core, < 3 cores positive for cancer, and < 50% involvement of any single core) will identify about 75% of men with tumors < 0.5 mL who might be followed without immediate treatment. Overall, however, these data indicate that most T1c tumors are significant cancers > 0.5 mL that warrant aggressive treatment in accordance with age and other health considerations. PMID- 7544518 TI - Comparative ribotyping of Staphylococcus intermedius from dogs, pigeons, horses and mink. AB - Strains of Staphylococcus (S.) intermedius from dogs, pigeons, horses and mink were typed by comparison of rRNA gene restriction fragment length polymorphisms (ribotyping) and the resulting ribotypes examined by cluster-analysis. Digestion of whole-cell DNA with HindIII resulted in 9 ribotypes with 3 to 4 bands. Separation of isolates from different host animal species was not possible. EcoRI yielded 11 different patterns with 4 to 9 fragments. The EcoRI-ribotypes of all canine strains grouped in one cluster encompassing four closely related ribotypes. Isolates were indistinguishable with respect to their origin from cases of canine pyoderma or from healthy carriers. In contrast, pigeon and equine strains expressed variety of ribotypes including some strains with patterns similar to canine isolates. However, for canine versus pigeon and equine strains the difference in distribution among ribotypes was significant (P < 0.001). The data might suggest that pigeons and horses carry S. intermedius transiently and might be able to spread these bacteria to other animal species. PMID- 7544519 TI - Immunohistochemical detection of p53 protein expression in HPV-induced condyloma acuminatum. AB - Immunohistochemical peroxidase staining for p53 protein was performed on 22 condyloma acuminatum tissue samples from patients infected with human papillomavirus (HPV). The purpose of our study was to understand the benign character of this syndrome. The patients studied were infected by HPV type 6 and 11. Two monoclonal antibodies, PAbs DO-1 and 240, were used to detect the p53 protein. Overexpression of wild-type p53 was found in the nuclei of the basal cell layers. In healthy tissues and non-infected patients no p53 protein expression was detected. We would like to speculate that infection with HPVs and their viral protein E7, which is implicated in disruption of normal growth, may regulate the induction of wild-type p53 over-expression, as is known for DNA damaging agents such as UV- or X-radiation. PMID- 7544520 TI - Expression of transglutaminase I in human anagen hair follicles. AB - The expression of the transglutaminase I in human anagen hair follicles was studied by an immunohistochemical staining. In the bulbar and suprabulbar portions of anagen hair follicles, transglutaminase I was detected on the hair cuticle and the three layers of the inner root sheath. Subsequently, the positive staining became translocated to the inner site of the outer root sheath in the middle part of the hair follicle. In the upper portion of the hair follicle transglutaminase I was detected in the internal part of the outer root sheath and the surface epidermis. Therefore, it was suggested that the expression of transglutaminase I might be closely associated with the terminal keratinization in the anagen hair follicles. PMID- 7544521 TI - Ultrastructure of murine epidermis treated with the vitamin D3 analogue KH-1060. AB - A new highly potent analogue (KH-1060) of vitamin D3 has been recently shown to stimulate the growth and differentiation of keratinocytes. This study was intended to determine the effects of this new analogue on epidermis at the ultrastructural level. KH-1060 was applied topically on the backs of hairless mice for 4 weeks; the skin was then studied by routine electron microscopy. The effects were compared with those of betamethasone-17-valerate and with concomitant treatment of KH-1060 following betamethasone. KH-1060 stimulated normal function of keratinocytes and formed a thick epidermis. The ultrastructure of the thick epidermis represents an enhanced normal process of keratinization and proliferation. Moreover, KH-1060 diminished the atrophogenic effects of betamethasone. PMID- 7544525 TI - bcr-abl hybrid messenger RNA in a patient with Philadelphia-negative essential thrombocythemia. AB - We describe a case of Philadelphia-negative essential thrombocythemia in whom bcr abl hybrid messenger RNA was detected. The patient suffered from frequent splenic infarctions and myelofibrosis. Interestingly, a transformation to acute leukemia which was commonly seen in patients with bcr-abl-positive chronic myelogenous leukemia did not occur until he died from heart failure due to severe anemia 8 years after the diagnosis. The heterogeneity of bcr-abl-positive thrombocythemia is emphasized. PMID- 7544524 TI - Primary T cell CD30-positive anaplastic large-cell lymphoma associated with adult onset celiac disease and presenting with skin lesions. AB - We report the case of a 52-year-old woman with primary CD30+ anaplastic large cell lymphoma of T cell phenotype with skin involvement, stage IVB, fulfilling almost all the clinical, histopathologic and immunophenotypic criteria for this disease, associated with adult-onset celiac disease. The diagnoses of malignancy and celiac disease were made during the same clinical episode. The clinical course of the patient has been extremely favorable and she is in complete remission, 15 months after finishing consolidation therapy. PMID- 7544523 TI - Complete remission in a patient with hypoplastic acute lymphoblastic leukemia induced by granulocyte-colony-stimulating factor. AB - We report here a case of hypoplastic leukemia with T cell markers in whom complete remission was obtained with granulocyte-colony-stimulating factor (G CSF) alone. A 23-year-old male was diagnosed with hypoplastic leukemia: Hb 2.6 g/dl, platelet count 29.0 x 10(9)/l after transfusion, WBC 2.9 x 10(9)/l, hypocellular bone marrow with 70.7% blasts. He was given G-CSF 300 micrograms/day by intravenous drip infusion without antileukemic agents for severe pneumonia. After the administration of G-CSF for 15 days, hematological examination and bone marrow findings had improved to normal, and complete remission was obtained. However, the patient relapsed 45 days after discontinuation of G-CSF. The characteristics of the relapsed leukemia cells were similar to those on admission: negative for myeloperoxidase and positive for T cell markers (CD2 and CD7). The possibilities for the differentiation of leukemic cells and the recovery of normal hematopoiesis with G-CSF are discussed. PMID- 7544522 TI - Production and expression of granulocyte- and macrophage-colony-stimulating factors in newborns: their roles in leukocytosis at birth. AB - We analyzed the production and expression of three colony-stimulating factors (CSFs) in neonates to clarify the mechanism of leukocytosis at birth. Serial blood samples (n = 23) were collected from mothers, cord blood, and from newborn infants on days 1, 5, and 30 after birth. The serum levels of granulocyte-CSF (G CSF), granulocyte/macrophage/CSF (GM-CSF) and macrophage-CSF (M-CSF) were measured by ELISA. The G-CSF levels on day 1 after birth were significantly higher than those thereafter, and they were also higher in the mothers than those on days 5 and 30 after birth. The GM-CSF levels did not change significantly during the neonatal period. The serum M-CSF levels were higher on postnatal day 1 than at other times, and gradually decreased thereafter. To confirm the production sites of G-CSF and M-CSF, the mRNA for these CSFs in peripheral mononuclear cells (MNCs) from healthy adults, mothers, and cord blood were analyzed by PCR. The expression of G-CSF and GM-CSF mRNA was undetectable in MNCs from adults, mothers, and cord blood, while these cells expressed low levels of M CSF mRNA. After stimulation with lipopolysaccharide or phorbol myristate acetate, the MNCs expressed high levels of G-CSF and GM-CSF mRNA. The levels of G-CSF PCR products in cord MNCs were lower than those in adult and maternal MNCs. The expression of M-CSF mRNA was virtually unchanged by stimulation. To detect the localization of G-CSF and M-CSF in the placenta and umbilical cord, these tissues were immunocytochemically stained with anti-G-CSF and anti-M-CSF antibodies, G CSF and M-CSF were expressed in trophoblasts and decidual stromal cells, whereas the umbilical cord did not express these CSFs. Moreover, large amounts of G-CSF and M-CSF were detected in the supernatant of cultured trophoblasts and decidual stromal cells. The expression of G-CSF and M-CSF in these cells was confirmed by PCR. These findings suggested that G-CSF and M-CSF produced in the placenta (trophoblasts and decidual stromal cells) are the major factors that induce leukocytosis in newborn infants at birth. PMID- 7544526 TI - [Cytokines in the treatment of blood diseases]. AB - Cytokines are a class of signal peptides which represent a major communication network in living organism. Over the last decade, the discovery, cloning and purification of hematopoietic cytokines (interleukins, hematopoietic growth factors) has increased our understanding of the regulation, proliferation, differentiation and function of hematopoietic cells. More recently, the large scale production of the recombinant forms of these molecules has enabled to treat the patients with pharmacologic doses of cytokines. The therapeutic activity of interferon-alpha (IFN-alpha) has been demonstrated in patients with chronic myeloid leukaemia and other chronic myeloproliferative syndromes. IFN-gamma is useful in the prevention of infections in patients with chronic granulomatous disease. Erythropoietin (EPO) was the first hematopoietic growth factor available for clinical use, initially to treat anaemia in renal failure patients. The next cytokines introduced into the clinic were granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF). They are used successfully in haematological malignant disorders to stimulate granulopoiesis after chemotherapy or bone marrow transplantation and to help mobilise marrow stem cells for peripheral blood stem cell transplantation. Interleukin (IL)-1, -2, -3, -4, -6 and -11 have been tested in clinical trials. However, the value of these agents remains to be established. PMID- 7544527 TI - NG-methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin. AB - Sensorineural hearing loss is a major sequela of the bacterial meningitis associated in particular with Streptococcus pneumoniae. Recent studies have shown pneumolysin, a toxin elaborated by S. pneumoniae, to be cytotoxic to the guinea pig cochlea. The mechanisms of this cytotoxicity are, however, not fully understood. In the present study this deleterious action of pneumolysin has been shown to be blocked by pretreating the cochlea with NG-methyl-L-arginine, a known inhibitor of nitric oxide synthesis. Furthermore, pretreatment of the cochlea with MK-801, an NMDA receptor antagonist, was also found to confer marked protection from the action of pneumolysin. This latter finding is consistent with previous reports that excess stimulation of NMDA receptors within the cochlea, an event known to lead to excess nitric oxide release, have similar effects on the cochlea as pneumolysin perfusion. It would therefore appear that nitric oxide may represent a significant link in the chain of events leading to the deafness of bacterial meningitis. PMID- 7544531 TI - G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells. AB - Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl ) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells. PMID- 7544529 TI - Ca2+ release and activation of K+ and Cl- currents by extracellular ATP in distal nephron epithelial cells. AB - We have measured ionic currents and changes in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular ATP in single epithelial cells of the distal nephron from toad (A6 cells). ATP increased [Ca2+]i and concomitantly activated ionic currents. The ATP concentration for half-maximal increase in [Ca2+]i was approximately 10 microM. Current activation and elevation of [Ca2+]i also occurred in Ca(2+)-free bath solutions but were abolished by loading the cells via the patch pipette with 10 mM 1,2-bis(2-aminophenoxy)ethane N,N,N',N' tetraacetic acid (BAPTA) or by preincubating the cells with 10 microM BAPTA acetoxymethyl ester for 120 min. ATP-activated currents reversed at -53.9 +/- 1.9 mV (n = 22). Tetraethylammonium (TEA, 25 mM), a K+ channel blocker, partially blocked this current but did not affect the Ca2+ transients. The TEA-insensitive component of the current reversed close to Cl- equilibrium potential. 5-Nitro-2 (3-phenylpropylamino) benzoic acid, a putative Cl- channel blocker (100 microM), abolished nearly completely the ATP-activated current. Suramin (100 microM), a P2 purinergic receptor antagonist, strongly attenuated both Ca2+ transients and currents. In cell-attached patches, single channel currents activated by ATP could be observed, i.e., an inwardly rectifying K+ channel with a slope conductance for inward currents of approximately 32 pS and an ohmic Cl- channel with a conductance of 34 pS. It is concluded that ATP activates both Cl- and K+ channels in distal nephron epithelial cells by a Ca(2+)-dependent mechanism. PMID- 7544533 TI - Insulin and counterregulatory hormones influence acute-phase protein production in human hepatocytes. AB - After trauma or sepsis, the liver undergoes a reprioritization of export protein synthesis with elevated production of some acute-phase reactants and reduced production of others. We have examined the effects of combinations of insulin and the counterregulatory hormones (dexamethasone, glucagon, and epinephrine), in the presence or absence of interleukin (IL)-6, on the production by isolated hepatocytes of the positive acute-phase proteins C-reactive protein, alpha 1 antichymotrypsin, alpha 1-acid glycoprotein, and haptoglobin, and the negative acute-phase proteins prealbumin and transferrin. The effect of IL-6 on the production of the above proteins was influenced significantly by insulin and all of the counterregulatory hormones. Significant three-way interactions as well as higher order interactions between the stress hormones and insulin were seen in the case of C-reactive protein. The results indicate that both positive and negative acute-phase proteins respond differently to insulin and the counterregulatory hormones and that the potential exists for the regulation of synthesis of individual acute-phase reactants by interaction between the cytokine network and the classical endocrine hormones. PMID- 7544528 TI - Combined laser surgery and adjuvant intralesional interferon injection in patients with laryngotracheal papillomatosis. PMID- 7544534 TI - Differential projection of cholinergic and nitroxidergic neurons in the myenteric plexus of guinea pig stomach. AB - The aim of this study was to investigate the organization of myenteric circuits in the guinea pig stomach. Intracellular neurobiotin injections followed by choline acetyltransferase (ChAT) immunohistochemistry and NADPH-diaphorase reaction were used to identify projections of cholinergic and nitroxidergic neurons. Neurons were classified as motor neurons based on varicose endings in the muscle or the occurrence of retraction bulbs, as nonmotor neurons if varicose endings terminated onto other ganglion cells, or as multitargeted neurons. ChAT positive cells are composed of 64% motor, 27% nonmotor, and 9% multitargeted neurons. The percentages for NADPH-reactive motor, nonmotor, and multitargeted neurons were 57, 39, and 4%, respectively. The majority of ChAT-positive motor (81%) and nonmotor neurons (85%) had ascending projections. In contrast, the majority of NADPH-reactive motor (86%) and nonmotor neurons (86%) had descending projections. Cell bodies of ascending neurons were smaller in size than the descending neurons. The results indicate that ChAT- and NADPH-neurons in the stomach have preferred projections, the former being primarily ascending, the latter mainly descending neurons. This suggests the existence of a basic circuit for polarized reflexes in the myenteric plexus of the stomach, which might mediate descending relaxation and ascending excitation. PMID- 7544530 TI - Drugs activating G proteins disturb cycling of ADH-dependent water channels in toad urinary bladder. AB - In the toad urinary bladder, antidiuretic hormone (ADH)-mediated changes in water permeability depend on exocytic insertion and endocytic retrieval of water channels into and from the apical membrane, respectively. Because GTP-binding proteins (G proteins) are well-recognized regulators of vesicular trafficking throughout the cell, we tested the hypothesis that drugs interfering with G protein would modify the hydrosmotic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluorescein isothiocyanate (FITC)-dextran, 70 kDa]. Mastoparan (4 microM) and compound 48/80 (poly-p-methoxyphenylethylmethylamine; 50 micrograms/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added to the serosal side (400 microM), inhibited ADH- and 8-bromoadenosine 3',5'-cyclic monophosphate-induced transepithelial water flow by > 50% and simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC dextran uptake observed using fluorescence microscopy both in scraped cells and in the intact bladder was granular, suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are both probes of G proteins, increased FITC-dextran uptake only in the presence of ADH and a transepithelial osmotic gradient, i.e., under conditions where water channel-carrying endosomes presumably cycle. Therefore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical membrane and/or the water channel-carrying vesicles. PMID- 7544532 TI - Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. AB - Although initially described over 30 years ago, the blood-follicle barrier has remained a biological enigma. In this study, we characterize the blood-follicle barrier with respect to its regulation of intrafollicular inter-alpha-trypsin inhibitor protein (I alpha I) influx after an ovulatory stimulus. We have found that I alpha I is localized within the ovarian vasculature but is excluded from the follicular compartment until an ovulatory stimulus is given. Within minutes after an ovulatory dose of human chorionic gonadotropin, I alpha I is localized within the follicular fluid of responding follicles where this protein becomes associated with and stabilizes the newly synthesized hyaluronic acid-rich cumulus extracellular matrix. Analysis of this process has shown that intravenous injection of sodium nitroprusside or excess substrate for nitric oxide synthase, L-arginine, mimics the effect of gonadotropic hormones on the influx of I alpha I into the follicular compartment of preovulatory follicles. Moreover, intravenous injection of specific nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine, inhibits gonadotropin-mediated intrafollicular influx of I alpha I and also inhibits ovulation in the mouse. PMID- 7544535 TI - Leukotriene D4 facilitates airway smooth muscle cell proliferation via modulation of the IGF axis. AB - The insulin-like growth factor (IGF) axis is involved in regulating proliferation in a variety of cell types, including airway smooth muscle. Because airway hyperplasia is a characteristic feature of asthma and other lung diseases, we examined the interaction of the potent proinflammatory eicosanoid leukotriene D4 (LTD4) with the IGF axis in regulating airway smooth muscle cell mitogenesis. In cultured rabbit airway smooth muscle cells, IGF-I but not LTD4 was mitogenic at submaximal concentrations. The combination of the two agents exerted a significant synergistic effect on airway smooth muscle cell mitogenesis. Analysis of airway smooth muscle cell conditioned medium by Western ligand blotting demonstrated a marked LTD4-induced reduction in the levels of the predominant IGF binding protein IGFBP-2, which is elaborated into the conditioned medium. The latter effect on IGFBP-2 release was not associated with a reduction in IGFBP-2 mRNA levels; however, LTD4-treated airway smooth muscle conditioned medium demonstrated the presence of a lower molecular weight form of IGFBP-2 by cross linking to IGFs and specific proteolysis of radiolabeled IGFBP-2. IGFBP-2 was also noted to be associated with airway smooth muscle cell membranes, where it was protected from LTD4-induced proteolysis. Finally, exogenous administration of IGFBP-2 was found to inhibit the promitogenic effect of IGF-I in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544536 TI - Reactive species in ischemic rat lung injury: contribution of peroxynitrite. AB - Lung ischemia-reperfusion represents a potentially important mechanism for diverse forms of tissue injury associated with decreased pulmonary flow. Previous studies demonstrated oxidative injury in ischemic-reperfused lungs. The present study was designed to evaluate the contribution of nitric oxide and peroxynitrite in tissue injury. The levels of the stable decomposition products of nitric oxide and peroxynitrite, nitrite plus nitrate, were twofold greater than control during reperfusion after 60 min of ischemia. Inhibition of nitric oxide synthesis by endotracheal insufflation of 5 mM NG-nitro-L-arginine methyl ester, 30 min before the induction of ischemia, decreased the production of lung thiobarbituric acid reactive substances (TBARS) by 67% (P < 0.05, n = 5), TBARS released into the lung perfusate by 55% (P < 0.05, n = 5), lung-conjugated dienes by 61% (P < 0.05, n = 5), and dinitrophenylhydrazine-reactive protein carbonyl levels by 86% (P < 0.05, n = 5). Amino acid analysis of tissue homogenates from lungs exposed to 60 min of ischemia and 60 min of reperfusion revealed a 1.8-fold (P < 0.05, n = 5) increase in nitrotyrosine concentration compared with 2 h continuously perfused lungs. Inhibition of nitric oxide synthesis abolished the increase in nitrotyrosine levels. Furthermore, lungs exposed to 60 min of reperfusion after 60 min of ischemia showed specific binding of an anti-nitrotyrosine antibody. In reperfused tissues, antibody binding was observed throughout the lung. The binding was blocked with excess of nitrotyrosine, and minimal binding was observed in nonperfused blood-free control lungs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544537 TI - Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5 beta 1, or TNF-alpha exposure. AB - Endothelial permeability can be altered by tumor necrosis factor-alpha (TNF alpha), a cytokine released in association with inflammation-induced tissue injury. In the subendothelial matrix, fibronectin (Fn) influences endothelial cell adhesion by the interaction of integrins with RGD and non-RGD attachment sites in Fn. We compared the effect of TNF-alpha, RGD-containing peptides (GRGDSP), or antibody to alpha 5 beta 1-integrins on the protein permeability of bovine lung endothelial monolayers as assessed by transendothelial 125I-labeled albumin clearance. We also examined the influence of purified human plasma fibronectin (hFn) on this permeability response. TNF-alpha, RGD peptides, and antibodies to alpha 5 beta 1-integrins elicited a dose- and time-dependent increase in protein permeability as well as a reorganization and/or disruption of the endogenous Fn matrix. A control RGE peptide (GRGESP) as well as immunoglobulin G purified from nonimmune rabbit serum did not increase endothelial protein permeability or disrupt the endogenous fibrillar Fn pattern in the matrix. Likewise, a LDV peptide derived from the alternatively spliced type III connecting segment (IIICS) within bovine Fn (bFn) was unable to increase permeability of the bovine endothelial monolayer. Co-incubation of purified soluble hFn (300 or 600 micrograms/ml) with either TNF-alpha, the RGD peptide, or the antibody to alpha 5 beta 1-integrins prevented the increase in endothelial permeability. This protective effect was also observed when the purified hFn (600 micrograms/ml) was added after the TNF-alpha-induced increase in endothelial permeability had taken place. Immunofluorescent analysis confirmed the incorporation of the hFn into the subendothelial matrix and its co-localization with the endogenous bFn. The similar alteration of the subendothelial matrix after exposure to RGD peptides, anti-alpha 5 beta 1-antibodies, or TNF-alpha, coupled with the ability for hFn to attenuate the permeability increase typically elicited by all three agents, suggests that disruption of cell-matrix interactions may be the mechanism by which TNF-alpha alters endothelial permeability. PMID- 7544538 TI - Developmental regulation of ACE gene expression by endogenous kinins and angiotensin II. AB - Angiotensin converting enzyme (ACE, i.e., kininase II), a key regulator of kinins and angiotensin II (ANG II) generation, is developmentally regulated and its expression is induced at a specific time point (day 15) of postnatal kidney development. The present study tested the hypothesis that endogenous kinins and ANG II regulate the developmental expression of the renal ACE gene. In the first protocol, newborn rats received the kallikrein inhibitor, aprotinin (100,000 KIU.kg-1.day-1 sc), or the kinin B2 receptor antagonist, HOE-140 (600 micrograms.kg-1.day-1 sc), or 0.9% saline, from birth until postnatal days 5, 15, or 20. Aprotinin prevented the postnatal rise in renal kallikrein activity without affecting blood pressure in either developing or adult rats. Chronic kallikrein blockade significantly attenuated the postnatal induction of both serum ACE activity (-11% vs. controls) and kidney ACE activity and mRNA (-50% vs. controls). In addition, aprotinin attenuated the postnatal rise of ACE activity in the developing lungs. Kidney renin mRNA and ANG II contents were not altered by aprotinin. HOE-140 also attenuated the postnatal rise in kidney ACE mRNA ( 25%) and activity (-40%) without affecting blood pressure. Infusion of aprotinin or HOE-140 via osmotic minipumps for 7 days in adult rats was not associated with any changes in renal or pulmonary ACE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544539 TI - TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells. AB - Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC. PMID- 7544540 TI - Molecular analysis of spontaneous glomerulosclerosis in Os/+ mice, a model with reduced nephron mass. AB - Oligosyndactyly mice (ROP Os/+) are a radiation-induced mutant strain with reduced glomerular number and increased glomerular size. We found that they develop glomerulosclerosis. At 3 mo, ROP Os/+ mice had diffuse mesangial expansion by light microscopy, whereas their +/+ littermates did not. Electron microscopic morphometry revealed a twofold increase in mesangial areas but no changes in the thickness of glomerular basal laminae. Mean glomerular volume was increased 1.8-fold. Cell number and thymidine labeling index were increased 1.3- and 2.4-fold, respectively. The amount of glomerular type IV collagen and tenascin but not laminin was increased by immunofluorescence microscopy. mRNA levels in microdissected glomeruli were measured by competetive reverse transcription-polymerase chain reaction and corrected for cell number. alpha 1 Chain type IV collagen and tenascin mRNAs were increased 3.2-fold and 1.8-fold, whereas laminin B1 mRNA levels were not. The levels of 72-kDa collagenase mRNA were increased 1.6-fold. Transforming growth factor-beta 1 mRNA levels were elevated 1.8-fold, but platelet-derived growth factor-B mRNA levels remained normal. This is the first analysis of glomerular molecular and cellular changes in a model of congenital nephron reduction. PMID- 7544545 TI - Cortex, XI. Amyloid plaques. PMID- 7544541 TI - Myocardial and endothelial protection by TMS in ischemia-reperfusion injury. AB - N,N,N-trimethylsphingosine (TMS), a stable synthetic sphingosine derivative, was investigated in a feline model of myocardial ischemia (90 min) and reperfusion (270 min) injury. TMS (60 micrograms/kg), administered intravenously 10 min before reperfusion, significantly attenuated myocardial necrosis (15 +/- 3 vs. 31 +/- 4% necrosis of area at risk, P < 0.01) and cardiac myeloperoxidase activities, a marker of neutrophil accumulation, compared with vehicle-treated cats. Endothelium-dependent relaxation to acetylcholine in ischemic-reperfused coronary artery rings treated with TMS was also significantly preserved compared with vehicle (73 +/- 4 vs. 34 +/- 4% vasorelaxation, P < 0.01). Polymorphonuclear neutrophil (PMN) adherence to coronary endothelium 270 min after reperfusion was markedly attenuated in the TMS group compared with vehicle-treated cats (37 +/- 5 vs. 76 +/- 5 PMN/mm2, P < 0.01). TMS also attenuated upregulation of P-selectin on coronary venular endothelium by immunohistochemistry. This was consistent with in vitro findings that TMS attenuates PMN adherence to thrombin-stimulated coronary endothelium and P-selectin upregulation on thrombin-stimulated cat platelets. A sphingolipid derivative, TMS at physiological concentrations exerts cardioprotective actions and preserves coronary endothelial function following myocardial ischemia and reperfusion in vivo. The effects appear to be mediated by the inhibition of PMN-endothelial interaction and subsequent accumulation into the ischemic myocardium. Thus TMS may be a useful agent in attenuating myocardial reperfusion injury. PMID- 7544544 TI - Ventricular sensory neurons in canine dorsal root ganglia: effects of adenosine and substance P. AB - Effects elicited by adenosine and substance P on ventricular sensory endings of 14 dorsal root ganglion afferent neurons were studied in situ in anesthetized dogs. Sensory-field application of adenosine (1 microM) increased the activity of these neurons by 179%. Application of a nonspecific adenosine antagonist to epicardial sensory fields suppressed ongoing activity in all 14 neurons by 39%. Application of an A1- or A2-adenosine-receptor antagonist suppressed activity generated by 10 of these neurons by 44 and 59%, respectively. Adenosine applied after A1- or A2-receptor blockade increased activity in 10 neurons by 131 and 145%, respectively, indicating that A1- and A2-receptor effects were not additive. Application of substance P (1 microM) to identified sensory fields increased activity in 12 of these neurons by 169%, whereas application of a substance P-receptor antagonist reduced activity generated by these neurons by 75%. Myocardial ischemia increased activity of nine neurons associated with left ventricular sensory fields by 320%, an effect that was counteracted by the nonspecific adenosine-receptor antagonist. It is concluded that A1- and A2 adenosine receptors, as well as substance P receptors, are present on ventricular epicardial sensory nerve endings of dorsal root ganglion neurons that are tonically active during normal states, becoming further activated during ischemia. PMID- 7544542 TI - Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. AB - The role of chronic fluid shear stress on endothelial constitutive nitric oxide synthase (cNOS) levels may have an important role in vessel diameter control. We subjected primary human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC, passages 2-14) to steady laminar shear stress. In both cell types, the intracellular level of cNOS was elevated within 3 h of flow exposure at 25 dyn/cm2 and remained elevated at 6 and 12 h of flow exposure, compared with stationary controls, as indicated by digital immunofluorescence microscopy. Shear stress exposure for 6 h caused a 2.2 +/- 0.3- and 2.8 +/- 0.3 fold elevation of cNOS protein levels in BAEC (n = 3, P < 0.01) and HUVEC (n = 3, P < 0.01), respectively, in the presence or absence of 1 microM dexamethasone. Dexamethasone suppresses induction of the inducible NOS gene, indicating that cNOS was elevated by fluid shear stress. Flow exposure at 4 dyn/cm2 caused no enhancement of cNOS levels in either cell type. The flow induction of the cNOS protein levels was not blocked by preincubation of BAEC with 100-400 microM of NG nitro-L-arginine methyl ester, indicating that flow-induced NO (or guanosine 3',5'-cyclic monophosphate) was not involved in the elevation of cNOS levels. Protein kinase C inhibitor H-7 (10 microM) had no effect on induction of NOS protein in BAEC exposed to 25 dyn/cm2. The cNOS mRNA levels were found to be elevated by two- to threefold in BAEC after 6 or 12 h of flow exposure at either 4 or 25 dyn/cm2, and this induction of NOS mRNA occurred in the presence of dexamethasone. The elevation of cNOS levels by chronic flow exposure may provide a mechanism for chronic regulation of vessel diameter by endothelial response to prevailing blood flow. PMID- 7544546 TI - Is risperidone a substitute for clozapine for patients who do not respond to neuroleptics? PMID- 7544543 TI - Cytokine signaling during myocardial infarction: sequential appearance of IL-1 beta and IL-6. AB - The purpose of this study was to investigate the significance of the sequential changes in proinflammatory cytokines observed in the plasma of patients early after myocardial infarct: a rise in interleukin (IL)-1 beta (308 +/- 126 vs. 141 +/- 78 pg/ml, P < 0.01) between 0 and 2 h followed by an IL-6 peak (49 +/- 24 vs. 14.5 +/- 13 pg/ml, P < 0.01) 4-9 h later. No significant changes in tumor necrosis factor-alpha (TNF-alpha) were observed at this early stage. The linkage between IL-1 beta and IL-6 secretions is supported by 1) the ability of patient's plasma drawn early after myocardial infarction to induce IL-6 mRNA and protein synthesis in cells that may be potential targets in vivo (fibroblasts and endothelial cells), 2) suppression of this activity by antibodies against IL-1 beta, and 3) a delay between IL-1 beta and IL-6 peaks in vivo (4-9 h), which is similar to the time required for maximal IL-6 production in IL-1 beta stimulated target cells in vitro (6 h). This sequential signaling might serve as the basis for an amplification mechanism of proinflammatory cytokines. In fact, a much greater synthesis of C-reactive protein was observed in hepatocytes when stimulated with conditioned medium of fibroblasts or endothelial cells that had previously been incubated with plasma of patients. The results of our work strongly suggest that, by inducing IL-6 in potential target cells, IL-1 beta could act as the primary, but indirect, signal that stimulates acute-phase protein synthesis after myocardial injury. PMID- 7544547 TI - Pharmacokinetic interaction between risperidone and clozapine. PMID- 7544548 TI - Role of interleukin-4 and interleukin-13 in synthesis of IgE and expression of CD23 by human B cells. PMID- 7544549 TI - Cytokine modulation of basophil histamine release in wasp-venom allergy. AB - We report the effect of interleukin-3 (IL-3) and of other cytokines on antigen induced basophil histamine release in wasp-venom-allergic subjects. Leukocytes from 12 patients with documented anaphylactic sensitivity to wasp venom were preincubated in the presence or absence of IL-3, granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-5, IL-8, or stem cell factor (SCF). Washed cells were then exposed to venom and to other secretagogues, and histamine release in the supernatant was measured fluorometrically. Preincubation of leukocytes with IL-3, GM-CSF, or IL-5 (0.02-2 ng/ml), but not with IL-8 and SCF, caused a dose dependent enhancement of antigen-induced basophilic histamine release in all subjects tested. Mean maximum increase was about 100% for IL-3, IL-5, and GM-CSF. The priming effect of IL-3 was rapid, persisted up to 12 h, and was not accompanied by a change in cellular histamine. IL-3 had a comparable enhancing effect when basophils were triggered with anti-IgE or N formylmethionylphenylalanine (FMP). By contrast, IL-3 had no effect on substance P-induced histamine release. The significant enhancement of basophil releasability to antigen in wasp-venom allergy by very low concentrations of IL 3, GM-CSF, and IL-5 suggests that cytokines in the basophil (mast-cell?) microenvironment could be critical factors in determining the variability of sting reactions in Hymenoptera-venom-allergic subjects. PMID- 7544550 TI - Influence of hyposensitization on soluble interleukin-2 receptor, eosinophil cationic protein, in vitro lymphocyte proliferation, in vitro lymphocyte adhesion, and lymphocyte membrane markers in childhood asthma. AB - Soluble interleukin-2 receptor (sIL-2R), eosinophil cationic protein (ECP), the lymphoproliferative response to house-dust mite (HDM), adhesion to human umbilical vein endothelial cells (HUVEC), and lymphocyte membrane markers were studied in three groups of children: healthy children, asthmatic children without hyposensitization (HS), and asthmatic children with HS. HS was associated with significantly lower numbers of peripheral blood eosinophils (PBE) and lower sIL 2R serum levels and with a tendency to lower ECP serum levels and lymphoproliferative response to HDM. There were no changes in the T-lymphocyte phenotypic markers CD4 and CD8 among the three groups. The interleukin-2 receptor (IL-2R, CD25) on HDM-stimulated T lymphocytes increased over unstimulated T lymphocytes in the three groups. The CD25 expression was higher on HDM-stimulated lymphocytes in both asthmatic groups than in healthy children. Adhesion of lymphocytes on HUVEC increased significantly after HDM stimulation in asthmatic children without HS, whereas no change was observed in the two other groups. However, there was no change in the expression of adhesion molecules CD29 and CD11a on lymphocytes in either of the groups. This study provides further evidence that HS can modify lymphocyte and eosinophil functions. PMID- 7544551 TI - Aprotonin and transurethral resection of the prostate. PMID- 7544553 TI - Bleomycin and hyperoxia exposure in the operating room. PMID- 7544552 TI - The influence of preoperative concentrations of beta-endorphin and met-enkephalin on the duration of analgesia after transurethral resection of prostate. AB - beta-Endorphin (beta-EP) and methionine-enkephalin (M-EK) are endogenous peptides that play a role in the modification of pain perception and analgesia threshold. In order to understand more about pathophysiology of pain in association with neuroaxial blocks, we evaluated cerebrospinal fluid (CSF) concentrations of beta EP and M-EK prior to spinal anesthesia (SA) in patients undergoing transurethral resection of prostate (TURP) to determine the correlation between preanesthesia concentrations and the duration of postoperative analgesia and opioid requirements. Twenty-five healthy patients undergoing TURP under SA were enrolled. beta-EP and M-EK were measured with a competitive radioimmunoassay. Mean preoperative beta-EP and M-EK concentrations were 153 +/- 44 and 38 +/- 5 pg/mL, respectively. Those with beta-EP concentrations > 153 pg/mL had significantly longer analgesia (P < 0.01), and lower utilization of morphine in the first postoperative day (P < 0.01). Moreover, patients with milder postoperative pain (visual analog scale score < 4/10) had significantly higher beta-EP concentrations (P < 0.01). A similar correlation was not found with M-EK values. These data suggest that preoperative CSF beta-EP, but not M-EK, concentrations correlate with the duration and quality of postoperative analgesia, as well as opioid requirements after spinal anesthesia. PMID- 7544555 TI - Development of monoclonal antibodies and capture immunoassays for feline immunodeficiency virus. AB - We generated monoclonal antibodies (MAB) against feline immunodeficiency virus (FIV) and characterized these MAB by single competition enzyme immunoassays (EIA), immunoblot analysis, and radioimmunoprecipitation. Four MAB identified 3 distinct epitopes of the FIV p24/26 gag major core protein. One MAB recognized the p16/17 gag protein; none recognized envelope proteins. We developed an FIV p26 antigen capture EIA that proved more sensitive (0.5 ng of p26/ml), less expensive, and less time-consuming than reverse transcriptase assay. The same MAB were used to develop an antibody EIA specific for FIV p26. The MAB and capture assays reported should prove useful in FIV diagnosis and research. PMID- 7544556 TI - Evaluation of marking materials for cutaneous surgical margins. AB - Microscopic evaluation of the margins of excised cutaneous neoplasms is of paramount importance for determining that neoplastic tissue does not extend to the excision borders. Dyes or pigments that indelibly mark the tissue should be more reliable than sectioning techniques or suture markers for identifying the surgical margins before and after tissue processing. We evaluated 5 pigments to select a material that could be rapidly applied by surgeons, and readily identified on histologic section by the pathologists. Twenty normal canine skin specimens were assigned to each of 5 groups. Each group was treated with artists' pigments in acetone, India ink in acetone, alcian blue, typists' correction fluid, or a commercially available marking kit. Ten specimens within each group were marked before formalin fixation, and 10 were marked after fixation. Application properties, fixation and processing properties, and microscopic characteristics were evaluated for each material. Application properties were acceptable for all marking materials on unfixed specimens, and for alcian blue, India ink in acetone, and correction fluid on fixed specimens. Fixation and processing properties were acceptable for all materials except correction fluid. All marking materials survived fixation and processing, and colors were readily visualized under the microscope. Microscopic characteristics were acceptable for alcian blue, India ink in acetone, and the commercial kit. Overall, alcian blue was the best marking material, with India ink in acetone and the commercial kit also acceptable. Correction fluid and artist' pigments in acetone were not acceptable because pigment fragmentation and incomplete tissue coverage hindered microscopic evaluation of resection margins. PMID- 7544557 TI - Interview with Steven A. Krilis. What is going to happen tomorrow for beta 2 glycoprotein I? PMID- 7544554 TI - [Use of serotonin adipinate in acute oral poisoning]. AB - Ninety-five patients with acute poisonings with psychotropic agents, barbiturates, amitriptyline, leponex, cholinolytics, and organophosphorus insecticides were treated by gastro-enteroadsorption making use of activated coal. In order to enhance propulsive function of the gastrointestinal tract, serotonin adipinate was repeatedly administered in a dose of 5 to 20 mg. Evacuation of the enteroadsorbent in the rectum was enhanced fivefold in comparison with a control group of similar patients administered traditional drugs (proserine, pituitrin, cerucal). As a result of such therapy, the toxicogenic phase of poisoning was shortened and mortality in the observed group reduced twofold in patients poisoned with psychotropic agents, 5.4 times in those with barbiturate poisoning, and 3.5 times in those with organophosphorus poisoning. PMID- 7544558 TI - The use of metallic stents in the treatment of malignant superior vena caval obstruction. AB - Superior vena caval obstruction due to malignancy is conventionally treated by radiotherapy and/or chemotherapy. In patients with unresolved or recurrent obstruction after treatment, expandable metallic stents can be percutaneously placed within the vena cava for relief of symptoms. In this series, metallic stents were successfully deployed in 11 consecutive patients with bronchial carcinoma. Gianturco Z stents were used in 10 patients and Strecker stents in one. There were 2 minor procedural complications of no sequelae. All patients had partial or full relief of symptoms after the procedure. On follow-up (mean 3.9 months), 9 patients had no recurrent symptoms up till the time of death or the present time. Two patients had recurrent obstruction, both within a week of the procedure. Based on our experience, percutaneous stenting was an effective means of palliation in this group of patients when other treatment modalities failed. PMID- 7544559 TI - Surgical management of the locally advanced pelvic malignancy--the colorectal surgeon's view. AB - About 10% of patients with newly diagnosed colorectal cancer will have locally advanced disease without gross evidence of distant or discontiguous intraabdominal metastases. Long-term cure or disease-free survival frequently follows extended resection for a locally advanced primary colorectal carcinoma. The use of adjuvant combined chemo-radiation therapy is still being evaluated. While complete resection of recurrent disease sometimes provides the best long term survival when compared with other treatment modalities, few patients are suitable candidates for curative resection. In our experience, major extended resection for recurrent pelvic disease is largely palliative and is rarely associated with long-term survival. PMID- 7544560 TI - [Cancer of the rectum in young adults. Apropos of 38 cases]. AB - This retrospective study is based upon 38 cases of rectal carcinomas occurring in young individuals aged under 25, collected during the 15 year period from January 1978 to December 1993. The mean age of the patients was 23 (range: 14 to 25), with a gender ratio of 2.1. Histology revealed a well differentiated adenocarcinoma in 12 cases, a moderately or slightly differentiated and undifferentiated carcinoma in 14 cases, a colloid carcinoma in 10 cases and a villous tumor with malignant change in 2 cases. Curative surgical excision was possible in only 11 patients. Sixteen patients underwent a palliative procedure, while 12 refused surgery. No treatment was proposed to 3 patients with visceral metastases. Only 11 patients could be followed up, with a mean 5-year survival rate of 10%. The prognosis of rectal carcinoma in young individuals is poor because of late diagnosis at an advanced stage and the aggressive histologic forms encountered. PMID- 7544561 TI - Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. AB - The present study compared two microwave based antigen-retrieval solutions in their ability to unmask antigenic determinants in formalin-fixed and paraffin embedded tissues for Immunostaining. In this regard, two widely used antigen retrieval solutions, namely 0.05 M glycine-HCl buffer, pH 3.6, containing 0.01% (w/v) (EDTA) and 0.1 M sodium citrate buffer, pH 6.0, were evaluated for (1) their effectiveness in unmasking a wide range of antigenic determinants (2) their ability to yield reproducible results (3) the lack of deleterious effects in any antibody antigen systems of interest. Both of these antigen-retrieval solutions resulted in greatly improved immunostaining following microwave-heating of dewaxed tissue sections for 2 x 5 min. Glycine-HCl buffer solution resulted in stronger immunostaining with antibodies to nuclear antigens [androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR), p53, proliferating cell nuclear antigen (PCNA), Ki-67 and MIB-1], cytoplasmic antigens (actin and factor VIII) and cell-surface antigens [Cu-18, epithelial membrane antigen (EMA) and MT 1 (CD43)], whereas sodium citrate buffer yielded superior immunostaining with antibodies to vimentin, and some cell-surface antigens [common leukocyte antigen (CLA) (CD45) and UCHL-1 (CD45RO)]. The effect of unmasking the epitopes recognized by antibody to PCNA was equally effective with either of the antigen retrieval solutions. Antibodies to pan-keratin, prostatic acid phosphatase (PAP), B lymphocyte antigen (BLA.36, CD20CY) and L26 (CD20) exhibited no enhancement in the intensity of staining with either of the antigen-retrieval solutions. PMID- 7544562 TI - Expression of CD44 in prostate cancer cells: association with cell proliferation and invasive potential. AB - High level expression of the cell surface adhesion molecule CD44 standard form and its splice variants, have been causally linked to tumor metastasis. In this study, we investigated the significance of CD44 expression in human prostatic carcinoma cells. Immunocytochemistry showed high level expression of CD44 in cells from a high grade prostate tumor, and two androgen-independent, invasive prostatic carcinoma lines, PC-3 and TSU-Pr1. Normal prostatic epithelial cells and LNCaP, a low metastatic, androgen sensitive cell line, expressed none to a very low level of CD44, although mRNA transcripts were detected in all cell lines. Immunoprecipitation detected two proteins of M(r) approximately 140 kDa and 210 kDa in PC-3, and predominantly the M(r) approximately 95 kDa protein in TSU Pr1, but none in LNCaP. Most importantly, a neutralizing antibody to CD44 inhibited cell proliferation and basement membrane invasive activity, suggesting a definitive role of CD44 in prostate tumor growth and metastasis. PMID- 7544563 TI - CD44 participates in tumor cell adhesion to endothelial cells in the experimental metastatic process in B16BL6 melanoma cells. AB - There are several pieces of evidence suggesting a relationship between CD44 expression and tumor metastasis, but the role of CD44 in the metastatic processes is unclear. We analysed the role of CD44 in the experimental metastatic processes of B16BL6 melanoma cells using anti-CD44 monoclonal antibody (clone IM7). B16BL6 melanoma cells expressed CD44 and these cells treated with anti-CD44 monoclonal antibody increased in the experimental metastatic ability, indicating that CD44 participates in metastatic processes in B16BL6 cells. Furthermore, the adhesiveness of B16BL6 cells to endothelial cells and the retention of these cells in the lung increased by treatment with anti-CD44 monoclonal antibody. These results suggested that the CD44-mediated adhesion of tumor cells to endothelial cells was involved in the experimental metastatic process of B 16BL6 cells. PMID- 7544565 TI - Enhancement effect of an anti-angiogenic agent, TNP-470, on hyperthermia-induced growth suppression of human esophageal and gastric cancers transplantable to nude mice. AB - The effect of a combination of TNP-470 (AGM-1470, an anti-angiogenic agent) and hyperthermia on tumor growth was examined using human esophageal (ESO-2) and gastric (NSC-8) cancers transplantable to nude mice. TNP-470 alone at a dose of 30 mg/kg three times a week for 2 weeks was sufficient to obtain an antitumor effect. A combination of this dose of TNP-470 and 43 degrees C hyperthermia for 30 min inhibited tumor growth markedly in comparison with either treatment alone. It was considered that angiogenesis after hyperthermia was inhibited by TNP-470, and then regrowth of the tumor cells was potently suppressed by reduction of O2 pressure, pH and nutrient supply in the tumor tissue. PMID- 7544564 TI - Macrophage colony stimulating factor inhibits experimental liver metastases from colon cancer. AB - This study investigated the inhibitory effect of human macrophage colony stimulating factor (hM-CSF) on experimental liver metastases. Murine colon cancer cell lines C-26 and C-36 were each injected into the spleen of BALB/c mice and the number of subsequent liver metastases was counted. An optimal dose (3,000U/body/day) of hM-CSF to inhibit liver metastases was identified. Additionally, hM-CSF (3,000U/body/day) administration for 7 days before and after the injection of the same cancer cell lines was investigated. The mean number of liver metastases decreased significantly in the hM-CSF group compared with the saline group. These results suggest potential therapeutic benefit for the use of hM-CSF in the adjuvant treatment of liver metastases. PMID- 7544568 TI - Immunohistochemical localization of CD44 variants 5 and 6 in human gastric mucosa and gastric cancer. AB - The aim of this study was to compare the distribution of CD44 variants 5 and 6 in normal mucosa and gastric cancer and to determine their relationship with histoclinical Parameters of the disease. Experimental material included 112 paraffin blocks of various human gastric carcinomas. Both variants of CD44 were detected immunohistochemically with primary antibodies deriving from Bender Med. Systems. In normal gastric mucosa positive reactions with both antibodies were observed in surface epithelium, parietal cells, myocytes and vascular endothelia. They were also found in intestinal enterocytes, esophageal epithelium and myoepithelial. Additional reactivity with antibodies against variant 6 was observed in Paneth cells. In gastric cancer, variants 5 and 6 were demonstrated in 91% and 64% of cases respectively, without significant correlation with the tumor type. The occurrence of variant 6 correlated positively with tumor size (p = 0.081) and negatively with histological grading (p = 0.093). The relationship with metastases was insignificant. PMID- 7544566 TI - Cell proliferation and tumor angiogenesis in oral squamous cell carcinoma. AB - Cell proliferation and vascularization play an important role in the metabolic functions of normal and malignant tissues. Tumor cell proliferation and density of microvessels (DM) in 33 specimens of oral squamous cell carcinoma (SCC) were assessed by immunohistochemical staining of proliferating nuclear cell antigen (PCNA) and JC/70A, respectively. Their staining patterns were compared to the clinicopathologic findings. The PCNA labeling index (LI) showed significantly higher values in advanced carcinomas (T3 and T4) (mean, 20.19 +/- 12.79) than in early carcinomas (T1 and T2) (mean, 9.58 +/- 4.01; P < 0.01). The DM (mean, 105.92 +/- 22.65) and PCNA LI (mean, 25.69 +/- 13.32) of tumors with lymph node metastasis were significantly higher than in tumors without nodal involvement (DM, mean, 72.6 +/- 24.30; P < 0.01) (PCNA LI, mean, 10.25 +/- 3.99; P < 0.001). Marginal DM values were significantly correlated and directly proportional to the mode of tumor invasion. Evaluation of PCNA and DM at the periphery of the tumor may help to identify oral SCC that have higher malignant potential. PMID- 7544571 TI - Correlation of cell surface fluorescence with conventional PAP analysis of cells of cytological interest obtained from cervical scrapes. AB - Archival PAP stained cervical smears were destained and treated with a fluorescent probe for a cell surface enzyme (GB). Cells which exhibited cell surface fluorescence were demonstrated to be cells of cytological interest in the analysis of cervical smears. These cells could be directly related to PAP and reclassified by subsequent restaining with PAP. Fluorescent cell surface technology was shown to be compatible with conventional PAP staining. PMID- 7544572 TI - Prognostic value of tumour microvessel density in cancer of the uterine cervix stage IB to IIB. AB - High intensity of tumour angiogenesis has been correlated with an increased potential of metastasis and poor prognosis in human malignancies. We investigated 43 patients with cervical cancer stages IB (n = 13), IIA (n = 8) and IIB (n = 22). All patients were treated by radical hysterectomy and lymphadenectomy. In the tumour specimen blood vessels were highlighted by staining endothelial cells for factor VIII. Microvessels were counted on a 200x field (0.74 mm2) in the most active areas of neovascularisation. The mean microvessel counts per field for stage IB, IIA and IIB tumours were 59.6 +/- 28.1, 56.3 +/- 24.3 and 55.7 +/- 55.6, respectively (p-value = n.s.). We found no significant correlation of microvessel density and established prognostic factors like pelvic lymph node involvement, vascular space invasion and stromal reaction. Patients with tumours showing low microvessel density (< 40 microvessels per field) had a significantly poorer recurrence-free interval (log-rank test: p-value = 0.01). PMID- 7544570 TI - Biochemical characteristics and clinical applications of alpha-fetoprotein isoforms. AB - Alphafetoprotein (AFP) is an oncofetal glycoprotein that frequently reappears in sera of patients affected by hepatocellular carcinoma (HCC) and yolk-sac tumours (YST). In these cases AFP determination represents a useful marker for monitoring the therapeutic response and the clinical evolution of the neoplastic disease. AFP is a heterogeneous molecule with respect to the carbohydrate moiety and the different AFP glycoforms can be separated and characterized by their affinity for lectins, such as Concanavalin A (con A), and Lens culinaris agglutinin (LCA). Increased production of LCA-reactive AFP has been proposed as a specific and early marker for HCC, while Con A non-reactive AFP could be a marker for YST and gastrointestinal malignancies. In this review the molecular basis of AFP microheterogeneity and the clinical application of AFP isoforms will be discussed. PMID- 7544567 TI - Nitric oxide and peroxynitrite production by human erythrocytes: a causative factor of toxic anemia in breast cancer patients. AB - In the present study we demonstrated that human erythrocytes possess a NO synthase (NOS) that can be activated by oxidative stress and Ca2+ accumulation to produce nitric oxide (NO), and that this activation could be involved in the pathogenesis of toxic anaemia in breast cancer patients. By causing oxidative stress in human erythrocytes with hydrogen peroxide (H2O2) (100 microM), or by increasing the intracellular calcium concentration using various doses (up to 100 microM) of the calcium ionophore A23187, a gradual increase in both NO and peroxynitrite (ONOO-) release that was inhibited by N-monomethyl-L-arginine (L NMMA) (1mM) was observed. Time-dependent experiments using hemolysates showed a linear rise of NO production which was elevated by 60% in the presence of superoxide dismutase (SOD) (100 U). NOS isolated from hemolysates was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins. In reconstitution experiments, when purified NOS, isolated from erythrocytes, was added to purified soluble guanylate cyclase (sGC), isolated from endothelial cells, in the presence of the appropriate cofactors and substrates, a linear increase in cGMP production at various concentrations (up to 50 microM) of H2O2 was observed. Furthermore, it was shown that erythrocytes from breast cancer patients were subjected to higher oxidative stress by ONOO- (100 microM), with a consequential increase of membrane rigidity, than erythrocytes from healthy individuals. Such mechanic changes may result in shortening of the lifespan of erythrocytes, a feature of toxic anemia in cancer patients. PMID- 7544573 TI - Circulating adhesion molecules and tumor necrosis factor receptor in multiple sclerosis: correlation with magnetic resonance imaging. AB - Adhesion molecules are important in T-cell trafficking to sites of inflammation. We determined levels of circulating vascular cell adhesion molecule-1 (VCAM-1), L selectin, and E-selectin in the serum of 147 patients with definite multiple sclerosis of the remitting-relapsing or secondary progressive type. Soluble VCAM 1 and L-selectin concentrations were increased compared to levels in a large group of control subjects. Levels were highest in patients with gadolinium enhancing lesions on magnetic resonance imaging (VCAM-1: 1,011 +/- 276 vs 626 +/- 87 ng/ml; L-selectin: 1,130 +/- 272 vs 793 +/- 207 ng/ml [mean +/- standard deviation]; p < 0.0001 vs patients without enhancing lesions). Serum levels of soluble tumor necrosis factor receptor (60 kd) were also raised (2.64 +/- 1.23 vs 2.17 +/- 0.69 ng/ml in subjects with other neurological diseases and 2.1 +/- 0.77 ng/ml in healthy control subjects; p < 0.05). Soluble VCAM-1 and L-selectin levels were correlated to concentrations of soluble tumor necrosis factor receptor. In 13 patients with viral encephalitis, similar observations were made. Raised levels of soluble VCAM-1 and L-selectin probably reflect cytokine-induced endothelial cell and T-lymphocyte/monocyte activation occurring in the process of T-cell migration into the central nervous system. Tumor necrosis factor-alpha may be critically involved. PMID- 7544574 TI - Ligand-dependent and -independent activation of the transcription factor gamma RF 1 in a cell-free system. AB - gamma RF-1 is a recently identified transcription factor induced by interferon gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity. PMID- 7544569 TI - Comparison of prostate specific membrane antigen, and prostate specific antigen levels in prostatic cancer patients. AB - PSA (prostate specific antigen) and PSMA (prostate specific membrane antigen) serum levels were determined in over 235 prostate cancer patients from 8 different United States clinical urological cancer centers. The clinical data were not known until after the serum assay results were shared with all participants to attempt to eliminate possible clinical bias. PSA values are useful in the clinical diagnosis and staging of prostate cancer patients, and generally fall to low values in response to effective treatment, e.g., surgery, hormones, radiation, chemotherapy. PSMA values are not related to clinical stage but if elevated can fall in response to effective treatments. In contrast, PSMA values can be elevated post-treatment in the presence of very low PSA levels (0.01 to 0.00). The elevated PSMA levels predicted a state of clinical progression or clinical resistance in most cases (> 70%). PSMA levels in this study were of better prognostic value than PSA. PMID- 7544575 TI - Nitric oxide-mediated inactivation of mammalian ferrochelatase in vivo and in vitro: possible involvement of the iron-sulphur cluster of the enzyme. AB - To investigate the role of the iron-sulphur cluster in mammalian ferrochelatases, the terminal enzyme of the haem biosynthetic pathway, we examined the interaction of nitric oxide (NO) and ferrochelatase. When macrophage cell line RAW 264.7 cells were treated with interferon-gamma and lipopolysaccharide NO synthesis in the cells was stimulated, and a decrease in ferrochelatase activity was observed, with no change in the amount of ferrochelatase. The addition of NG-monomethyl-L arginine, a selective inhibitor of NO synthesis, reduced the effect of interferon gamma and lipopolysaccharide, while the effect of NG-monomethyl-L-arginine was suppressed by the addition of L-arginine, a substrate of NO synthase. When purified recombinant human ferrochelatase was treated with 3 morpholinosydnonimine, a NO-generating compound, ferrochelatase activity decreased with disappearance of characteristic absorbance spectra of the iron sulphur cluster. S-Nitroso-N-acetylpenicillamine also reduced the activity, in a dose-dependent manner. These results indicate that ferrochelatase activity can be modulated by NO synthesis probably through disruption of the iron-sulphur cluster. We propose that inactivation of ferrochelatase mediated by NO (or NO derived species) may play a role in the regulation of haem metabolism. PMID- 7544576 TI - Structural and functional analysis of the canine histamine H2 receptor by site directed mutagenesis: N-glycosylation is not vital for its action. AB - G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s). PMID- 7544578 TI - CD95 (Fas/APO-1) antibody-mediated apoptosis of human retinal pigment epithelial cells. AB - CD95 (Fas/APO-1) is a cytokine receptor protein that signals apoptosis. Here we report that human retinal pigment epithelial cells express CD95 but are rather resistant to agonistic CD95 antibodies. Resistance to CD95 antibodies is overcome by preexposure to the cytokines, tumor necrosis factor-alpha or interferon-gamma, or by coexposure to CD95 antibodies and inhibitors of RNA or protein synthesis. The cells are resistant to tumor necrosis factor-alpha even in the presence of cycloheximide and only moderately sensitized to tumor necrosis factor-alpha mediated toxicity by coexposure to actinomycin D. CD95-mediated apoptosis is inhibited by dexamethasone both after cytokine sensitization and upon coexposure to CD95 antibodies and actinomycin D or cycloheximide. Induction of apoptosis via CD95 may be involved in the regulation of retinal pigment epithelial proliferation at the vitreoretinal interface. PMID- 7544580 TI - Calcium dependent activation of skeletal muscle Ca2+ release channel (ryanodine receptor) by calmodulin. AB - In this study terminal cisternae vesicles from rabbit skeletal muscle were fused into planar bilayers and the effect of calmodulin on single Ca2+ release channel currents was investigated. In the presence of 10(-7) and 10(-9) M free [Ca2+], nanomolar concentrations of calmodulin activated the channel by increasing the open probability of single-channel events in a dose dependent manner. The activatory effect of calmodulin was reversed by 10 microM ruthenium red. At 10( 5) M free [Ca2+], calmodulin (0.1-1 microM) inhibited channel activity. Calmodulin overlays were carried out using concentrations of Ca2+ similar to those used for the planar lipid bilayer assays. In the presence of 10(-7) M [Ca2+], calmodulin bound to the ryanodine receptor, to a region defined by residues 2937-3225 and 3546-3655. These results suggest that calmodulin may activate the Ca(2+)-release channel (ryanodine-receptor) by interacting with binding sites localized in the central portion of the RYR protomer. PMID- 7544581 TI - Production of recombinant PSA and HK2 and analysis of their immunologic cross reactivity. AB - Measurements of prostate-specific antigen (PSA) in serum are widely used to monitor patients with prostate cancer, but the attenuation of the assay response by PSA complexed to protease inhibitors has been shown to affect the results in certain assay designs. Moreover, the human glandular kallikrein-2 (hK2), a kallikrein-like serine protease that is 80% similar to PSA, might interfere with the specific detection of PSA by immunological cross-reactivity. We have expressed hK2 and PSA in eucaryotic cells using the Semliki Forest Virus expression system and studied the reactivity of 18 monoclonal anti-PSA IgGs. Five of them cross-reacted with identical affinities to recombinant hK2 whereas 13 recognized PSA alone. The antibodies that recognized both PSA and hK2 bind to a region of the protein that is exposed when PSA is complexed to alpha-1 antichymotrypsin. PMID- 7544579 TI - Identification of an EWS-pseudogene using translocation detection by RT-PCR in Ewing's sarcoma. AB - The presence of a t(11;22)(q24;q12) translocation is one of the characteristic features of the Ewing family of tumors. The detection of the fusion gene product by RT-PCR using primers at both sides of the breakpoints has been advocated as a diagnostic tool. By applying this technique appropriate internal controls are required. We found that the use of normal non-rearranged EWS mRNA as an internal control for RNA quality may lead to conflicting data. We obtained PCR products of the expected size for the normal EWS mRNA in both RNA and DNA samples, suggesting, the existence of one or more EWS pseudogenes. A 109 bp sequence at the 5' end of this PCR-product contained a correctly spliced exon junction and was 97% homologous to the EWS cDNA sequence. Similarly two such junctions were found in a 346 bp sequence of the 3' end, which was 89% homologous. Hence EWS should not be used as an internal control for the RNA quality in a RT-PCR based test for the presence of the translocation. PMID- 7544582 TI - Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver. AB - In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in non-synaptosomal rat brain mitochondria and rat liver mitochondria, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that nitric oxide synthase located in mitochondria is involved in the regulation of mitochondrial oxidative phosphorylation is discussed. PMID- 7544577 TI - Platelet phospholipase D is activated by protein kinase C via an integrin alpha IIb beta 3-independent mechanism. AB - Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation. PMID- 7544583 TI - Requirement of p56lck in T-cell receptor/CD3-mediated apoptosis and Fas-ligand induction in Jurkat cells. AB - Interactions of Fas (CD95) and its ligand (Fas-L) has been shown to play a pivotal role in T cells receptor (TCR)/CD3 activation-induced cell death via apoptosis. Although several lines of evidence suggest involvement of protein tyrosine kinase (PTK) activity in this process, the role of src family PTK p561ck (lck) is not known. We report here that, contrary to wild type Jurkat, the lck deficient mutant JCaM is resistant to anti-CD3-induced apoptosis and fails to express Fas-L mRNA upon anti-CD3 treatment. However, both Jurkat and JCaM were found to constitutively express Fas and were equally sensitive to anti-Fas mediated apoptosis. If stimulated with PMA plus ionomycin, JCaM expressed Fas-L mRNA and underwent apoptosis. These findings indicate that p56lck is required for TCR/CD3-mediated Fas-L induction but not for the transduction of Fas receptor mediated death signal. PMID- 7544584 TI - p-chlorophenylalanine-induced serotonin depletion: reduction in exploratory locomotion but no obvious sensory-motor deficits. AB - Para-chlorophenylalanine (PCPA) depletes central serotonin (5-hydroxytryptamine, 5-HT) by inhibiting tryptophan hydroxylase, an enzyme necessary for the synthesis of 5-HT. The effects of a wide range of PCPA doses (150-1000 mg/kg) on spontaneous exploratory locomotor activity in a novel environment, activity in running wheels and a number of sensory-motor capacities were examined. Administration of 1000 mg/kg PCPA reduced whole brain levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid to 9.4 and 8.2% of control levels, respectively. Treatment with PCPA produced a dose-dependent decrease in exploratory locomotion in an unfamiliar automated open field relative to vehicle treated animals. Further, all measures of general, horizontal and vertical activity were suppressed by PCPA treatment. In contrast to previous work, hyperactivity of rats chronically housed in cages with running wheel access was not observed. In their home cages, some PCPA-treated rats exhibited hyper reactivity to cutaneous stimulation. No other sensory-motor deficits were apparent. Previous theories of 5-HT function state that its action may be to inhibit motor activity or promote sleep. The present results challenge this view and suggest that 5-HT, at least in certain environments, may stimulate locomotor activity without directly controlling various sensory-motor capacities in rats. PMID- 7544585 TI - Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli. AB - A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process. PMID- 7544588 TI - The role of growth factors in the suppression of active cell death in the prostate: an hypothesis. AB - Regression of the rat ventral prostate occurs when the level of 5 alpha dihydrotestosterone, the trophic hormone, drops below the threshold required to suppress apoptosis. The induction of apoptosis in the ventral prostate is accompanied by the increase in the steady-state level of a number of mRNAs coding for proteins that are involved in the latter stages of apoptosis and thus represent secondary thanatogens. These include proteases (cathepsins, plasminogen activators, and collagenase), clusterin, poly(ADP)ribose polymerase, tenascin, and several unidentified genes, as well as several RNases and the classical Ca2+,Mg(2+)-dependent endonuclease. In addition, insulin-like growth-factor binding protein 5 (IGFBP-5) is induced de novo. We propose that IGFBP-5 may serve to trigger the apoptotic process through the attenuation of the insulin-like growth factor signalling system (which is necessary for cell survival), and as such, represents a primary thanatogen in the prostate. PMID- 7544586 TI - The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation. AB - Recent evidence suggests that tumor necrosis factor alpha, Fas, and ionizing radiation employ the sphingomyelin pathway to trigger apoptosis. The sphingomyelin pathway is initiated by hydrolysis of plasma membrane sphingomyelin to generate ceramide via a sphingomyelinase. Ceramide serves as a second messenger stimulating a cascade of kinases and transcription factors that activate a final common pathway of programmed cell death. The extent to which this signaling system is used in apoptosis induced by other toxic modalities is not known, but accumulating evidence suggests that it is a commonly employed pathway that could be exploited therapeutically. PMID- 7544589 TI - Fluorescence correlations, single molecule detection and large number screening. Applications in biotechnology. AB - Fluorescence correlation spectroscopy (FCS), when carried out under conditions with low background as obtained in very small volume elements, is a powerful tool for examining molecular interactions as well as their time dependence. Interactions of biological importance which can be analyzed are hybridization between nucleic acid primers and DNA or RNA targets, between peptide ligands and isolated as well as cell-bound receptors, between antigen and antibodies. Since the interaction can be analyzed rapidly in small volumes without the need for separating unbound from bound ligand, an important application of FCS is envisaged in large-scale drug screening. The sensitivity has been advanced to the point that detection of single dye molecules is possible in the submillisecond range. This opens up the possibility for detecting rare events such as the appearance of pathogens in the early phase of infection or mutants exhibiting unusual properties when screening combinatorial libraries. PMID- 7544591 TI - How to search for RNA structures. Theoretical concepts in evolutionary biotechnology. AB - The relation between RNA sequences and minimum free energy secondary structures is viewed as a mapping from sequence space into shape space. The properties of such mappings depend strongly on the ratios of the numbers of sequences and structures and, hence, substantial differences are observed between samples of structures derived from AUGC, pure AU or pure GC sequences. Statistical analysis of large samples is used to demonstrate that structures from AUGC sequences are much less sensitive to point mutations than those from sequences containing exclusively AU or GC. The frequency with which a structure is realized in sequence space is inversely proportional to some power c > 1 of the structure's frequency rank, thus following a (generalized) Zipf law. For long sequences the exponent approaches c = 1. An inverse folding algorithm is used to compute samples of sequences folding into the same secondary structure. These sequences are distributed randomly in sequence space. Common structures form extended neutral networks along which populations can migrate through the entire sequence space without changing structure. In this migration, moves of Hamming distance d = 1 and d = 2 are accepted in order to allow for base and base pair exchanges, respectively. Around any arbitrarily chosen sequence a ball that contains sequences folding into all common structures can be drawn. This ball has a diameter that is much smaller than the diameter of sequence space. Hence, only a small fraction of sequence space needs to be searched in order to find a given structure. The results derived from the mapping of sequences into structures are used to suggest a rationale for evolutionary searches on RNA structures: selection cycles with high and low mutation rates applied in alternation. Generalizations of the results to RNA 3-D structures and protein structures are discussed. PMID- 7544590 TI - Chemistry and applications of oligonucleotide analogues. AB - This review is aimed at biochemists and molecular biologists, and covers the chemistry and key features involved in the solid-phase synthesis of a variety of the better known DNA and RNA analogues by the phosphoramidite and H-phosphonate methods. A wide spectrum of biological applications such as inhibition of gene expression, translation arrest, RNA processing, affinity purification of RNA protein complexes, in situ hybridization, and synthetic ribozymes are then discussed in some detail, enabling the molecular biologist to get an idea of what is possible using the current technology. PMID- 7544592 TI - Immunogenicity expressed in patients with bacteraemia of an epitope shared by enterobacterial and neisserial porin proteins. AB - A monoclonal antibody (MAb) against an epitope (Po I) on an Escherichia coli O55 porin protein has shown broad cross-reactivity with other Enterobacteriaceae and with both pathogenic and non-pathogenic Neisseriaceae. In this study, we have measured antibody levels against the Po I site in patients with bacteraemia in order to examine the immunogenicity of the Po I domain in humans. A MAb-based competition ELISA (cELISA) was used. Only 20% of healthy controls had detectable levels of anti-Po I antibodies in serum. Of patients bacteraemic with enterobacteria (n = 45), 11% and 58% showed elevated antibody levels compared to healthy controls with the first and second serum specimens, respectively, and 73% of these patients showed > or = 10% increase in the antibody levels. Of patients bacteraemic with N. meningitidis (n = 20), only 30% showed > or = 10% increase in the antibody levels when paired serum specimens were tested. Levels of competing antibodies were similar in the cELISA with N. meningitidis (B: 15: P1, 7, 16) OM coat or E. coli O55 OM coat. The results demonstrated that the highly conserved porin protein domain Po I expressed immunogenicity in humans when present in bacteria which caused bacteraemia. This finding represents a challenge in further investigations on the immunobiological role of the cross-reacting antibodies. PMID- 7544593 TI - Endocytosis in mouse blastocysts: characterization and quantification of the fluid phase component. AB - Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4 degrees C. Insulin stimulated uptake of 3H-dextran by 30% (P < 0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin treated blastocysts contained 38% more 3H-dextran (38%; P < 0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased 3H dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 +/ 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/l and 10 g BSA/l, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544595 TI - Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse. AB - The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/ )(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544596 TI - Delivery of purified, functional CFTR to epithelial cells in vitro using influenza hemagglutinin. AB - To assess the feasibility of protein replacement as a potential therapy for cystic fibrosis, we have evaluated the ability of influenza hemagglutinin (HA) to mediate the delivery of purified cystic fibrosis transmembrane conductance regulator (CFTR) to recipient cells in vitro. CFTR was purified from both CHO cells and Sf9 cells and reconstituted into two different types of vesicular delivery vehicles. In one, CFTR and HA were co-reconstituted into the same lipid vesicle. After binding to the cell surface, delivery of CFTR to the recipient cell was achieved by a transient, low-pH activation of the fusion activity of HA. A second delivery strategy used HA virosomes together with purified CFTR that had been reconstituted into vesicles containing gangliosides, a receptor for HA. After binding of the HA virosomes and CFTR-containing vesicles to the recipient cells, delivery to the plasma membrane again was achieved by a transient pH drop. Delivery of functional CFTR was assessed using the SPQ fluorescence assay. Functional CFTR was detected in a fraction (> 20%) of the recipient cells using this assay. Quantitative binding and fusion assays using radiolabeled virosomes and lipid vesicles showed that on the order of 1,000 of the added CFTR-containing vesicles bound to each C127 cell under the conditions of our delivery protocols. However, only a fraction of these vesicles fused and delivered CFTR to the cell plasma membrane. The two delivery strategies were found to be approximately equivalent in their ability to deliver active CFTR, and there were no significant differences between deliveries using purified CFTR from either cell source. These feasibility studies suggest that purified CFTR can be delivered to a recipient cell in a functional form and therefore represent a significant step in establishing the concept of protein replacement as a therapy for cystic fibrosis. PMID- 7544587 TI - Expression of tenascin during carcinogenesis and involution of hormone-dependent tissues. AB - Cytotactin/tenascin/hexabrachion, now referred to as tenascin-C (TN-C), is a hexameric glycoprotein of the extracellular matrix of mesenchymal tissue constituents. A high expression was found in embryonic development and during carcinogenesis of almost all organs. TN-C expression by the mesenchyme thereby appears to be induced by paracrine-acting, epithelial-derived (growth) factors. In normal adult organs there is little, if any, TN-C expression. In the human endometrium for instance, tenascin expression is low in the normal proliferative endometrium and undetectable in the normal secretory endometrium. In this paper, the appearance and expression of TN-C in hormone-dependent tissues, regressing hormone-dependent tissues, and tumors of the endometrium, breast and prostate is reviewed. Further, the regulation of TN-C expression is summarized and possible functions of TN-C during regression and carcinogenesis of hormone-dependent tissues are discussed. PMID- 7544594 TI - Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. AB - Interleukin-10 (IL-10) is a potent regulatory cytokine that decreases inflammatory responses and T-cell stimulation. We have found that respiratory epithelial lining fluid (ELF) from patients with cystic fibrosis (CF) contains significantly less soluble IL-10 than ELF of healthy control subjects. Although macrophages from the chronically infected lungs of CF patients appear to be one source of IL-10, little or no intracellular IL-10 was found in bronchoalveolar lavage macrophages from healthy control subjects, suggesting that there must be another source of this cytokine in healthy lungs. We found that bronchial epithelial cells from healthy control subjects constitutively produce IL-10, which appears to be downregulated in CF patients. It is thus likely that the bronchial epithelium plays an important role in regulating the local immune response, producing IL-10 to decrease inflammation in the healthy lung. Conversely, downregulation of epithelial IL-10 production in CF airways may contribute to enhancing local inflammation and tissue damage. PMID- 7544597 TI - Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells. AB - The role of sulfhydryl groups (SH) and disulfide bonds as well as disulfide oxidoreductases in regulation of the catalytic activity of the membrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatment of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N ethylmaleimide (NEM) (10 to 50 microM) or with the intramolecular disulfide forming agent diamide (20 to 100 microM) resulted in the reduction of NO synthase activity in a dose-dependent fashion. Similar loss of enzyme activity was observed when purified NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity. In contrast, when intact PAEC or isolated total membranes derived from PAEC were treated with increasing concentrations (1 to 5 mM) of the disulfide reducing agent dithiothreitol (DTT), but not oxidized DTT, NO synthase activity was increased by 20 to 85%. DTT reduction of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/thioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide treated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fold more efficient than the DTT-induced reduction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544598 TI - [Calcium channels in bullfrog saccular hair cells]. AB - In order to understand the cochlear physiology at a cellular and molecular level, Ca2+ channels were studied in cell-attached recordings from the basolateral membrane of the bullforg saccular hair cells with the EPC-9 patch-clamp system. Pipettes contained 110 mM Ba2+ and the membrane potential was zeroed with isotonic potassium aspartate. Data acquisition and analysis were performed using E9SCREEN and M2LAB software. L-type channel was distinguished by a single-channel conductance of 26 pS, activation range between -10 and +40 mV and intense activity even at a holding potential of -40 mV. The L-type channel showed characteristic bursts of brief openings (mode 1) interrupted occasionally by longer openings (mode 2). Bay K 8644 promoted the mode 2 activity and nifedipine inhibited L-type channel activity. Another type of calcium channels, 20 pS channel, was detected by -50 to +10 mV depolarizing steps from a holding potential of -40 mV or -80 mV. This channel was insensitive to dihydropyridines and resembled the N-type channel. PMID- 7544599 TI - [Morphologic characteristic of substance P immunoreactivity in facial nerve of normal cats]. AB - This paper presents the morphologic charactistic of substance P immunoreactivity (SP-IR) in the facial nerve and geniculate ganglion of normal cats by using immunohistochemical technique and immunoelectronic microscopy. SP-IR positive cells are more in geniculate ganglion than the other parts of the facial nerve. These cells are black. The sizes of their somata are different, and the shapes are round, elliptic or irregular. The somata are connected with each other by their central and peripheral synapses in the shapes of spider's web or root of a tree. Among the cells the bipolar and pseudounipolar cells are also found. SP-IR positive fibers curve and some of them have obvious expansive bodies, which are beaded, different in diameter. Under electronic microscope SP-IR positive cells show thicker electronic density than the others, and SP-IR positive products with thick electronic density are found in the nuclei, nucleomembranes, outer membranes of mitochndria and plasma. The nerve fibers are myelinated and the myelin sheath is concentric, and in the axoplasms there are nervous microfilament and a bit of mitochndria. PMID- 7544600 TI - Clinico-pathological criteria with prognostic relevance in hepatoblastoma. AB - We investigated clinical data and histological specimens of 46 patients with a hepatoblastoma (HB) for prognostic criteria. Disease-free survival (DFS) of 23 patients treated in the German Cooperative Study HB-89 (1988-1990) was 83%, in contrast to 40% in 10 children with other chemotherapy regimes (1977-1987) and 38% in 13 with only a tumour resection (P = 0.005). Tumour residence after resection (R category) correlated significantly with probability of DFS (P = 0.0001). This was also the case for pT status, according to the pTNM classification for liver carcinoma (P = 0.0007), involvement of one or both liver lobes (P = 0.004), multiplicity of tumour nodes (P = 0.001), vascular invasion (P = 0.0006) and expression of nucleolar organiser regions as an indicator for proliferation activity of tumour cells (P = 0.05). Patients' age and histopathological subtypes could only indicate outcome, while tumour size and serum alpha-fetoprotein values were not significantly related to prognosis. In multivariate analysis, pT status and R categories remained significant. These should be applied in all cooperative trials on HB. PMID- 7544604 TI - Demonstration of the high-affinity IgE receptor on human Langerhans cells in normal and diseased skin. AB - Epidermal dendritic cells of normal adult foreskin, and of lesional skin from patients with atopic eczema, stasis eczema and urticaria pigmentosa are shown to be highly reactive with two different monoclonal antibodies (29C6 and 6F7) specific for extracellular domains of the alpha-chain of the high-affinity IgE receptor. By their distribution pattern, the reactive cells are Langerhans cells. This is confirmed by immunoelectron microscopic demonstration of Birbeck granules in the labelled epidermal cells. Very weak staining is observed on the same cells with an antibody (Tu1) against the low-affinity IgE receptor. Pre-incubation of the sections with IgE partially blocks binding of 6F7 antibody. Langerhans cells, together with dermal mast cells, can therefore bind IgE with high efficiency, and may in this way participate in IgE-mediated cutaneous diseases. PMID- 7544602 TI - The effect of systemically administered rhIGF-I/IGFBP-3 complex on cortical bone strength and structure in ovariectomized rats. AB - The action of systematically administered recombinant human insulinlike growth factor-I (rhIGF-I) complexed to its natural binding protein-3 (rhIGFBP-3) on cortical bone dynamic, structural, and mechanical properties was tested in previously ovariectomized (Ovx) rats. Bilateral ovariectomy or sham surgery was performed on 16-week-old female Sprague-Dawley rats. Eight weeks after surgery basal Sham and Ovx rats were killed to establish baseline cortical bone values before the initiation of treatment with rhIGF-I/IGFBP-3 complex. At that time, Ovx rats had increased body weight and body fat mass with reduced femoral BMC and BMD relative to basal Shams. Bone formation rates in Ovx rats were increased on both cortical envelopes relative to time-matched controls. The thickness of the inner lamellar bone layer and average cortical width were reduced due to increased endocortical erosion. A similar ratio between Sham and Ovx rats in body mass and composition and femoral BMC and BMD continued throughout the experiment. Sixteen weeks after surgery bone formation rates at both cortical envelopes in Ovx rats were reduced relative to Shams, but endocortical erosion remained high causing a further decrease in thickness of the inner lamellar layer. As a result of periosteal bone modeling. Ovx rats exhibited a larger femoral cross-sectional area and periosteal perimeter, as well as a thicker outer lamellar layer. Newly deposited periosteal bone increased ultimate torque values in the Ovx rats relative to Shams at 16 weeks. Treatment of Ovx rats with the rhIGF-I/IGFBP-3 complex increased body weight, lean body mass, and femoral BMC and BMD.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544607 TI - Nitric oxide synthesis in retinal photoreceptor cells. AB - Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 microM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 microM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544608 TI - Expression of glycine receptor subunits and gephyrin in single bipolar cells of the rat retina. AB - We studied the expression of glycine receptor (GlyR) subunits and gephyrin in the adult rat retina. Reverse transcribed RNA was amplified by polymerase chain reaction (RT-PCR) with primers designed to recognize GlyR alpha 1, alpha 2, alpha 3, beta subunits, and gephyrin. Using RNA isolated from the whole retina, signals for all four GlyR subunits and gephyrin could be observed. In rod bipolar cells, in contrast, we detected a subset of GlyR subunits, alpha 1 and beta, and no gephyrin. Patch-clamp recording employing two subtype-specific blockers of the GlyR, picrotoxinin and cyanotriphenylborate (CTB), indicated that the GlyR in rod bipolar cells is a heteromeric protein composed of the alpha 1 and beta subunit. Moreover, the absence of detectable amounts of gephyrin mRNA suggests that the anchor protein is not required for the function of GlyRs in rod bipolar cells. PMID- 7544601 TI - Response of femoral neck to estrogen depletion and parathyroid hormone in aged rats. AB - Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated at 3 months of age and maintained untreated for 1 year after surgery. Baseline control and OVX rats were killed at the beginning of treatment when the rats were 15 months of age and 1 year postovariectomy. The remaining rats were treated with hPTH 1-34 (80 micrograms/kg BW, 5 days/week) or vehicle for 10 weeks. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal femur from each rat. Baseline OVX rats exhibited cancellous and cortical osteopenia at the femoral neck as their mean cancellous bone volume and cortical width were significantly decreased compared to the means for baseline control rats. In addition, baseline OVX rats had increased osteoblast and osteoclast surfaces and a greater cancellous bone formation rate than baseline control rats. OVX rats remained osteopenic with no further bone loss from the femoral neck after 10 weeks of vehicle treatment. In contrast, cancellous bone volume and cortical width in OVX rats treated with PTH were increased to the level of vehicle-treated control rats. The hormone restored lost bone in the femoral neck of OVX rats by markedly stimulating both cancellous and cortical bone formation. These histomorphometric findings in concert with recent biomechanical studies of bone strength indicate that the femoral neck of aged OVX rats is a promising sample site for studies of the prevention and treatment of bone loss induced by estrogen depletion. PMID- 7544603 TI - Monospecific monoclonal antibodies to keratin 1 carboxy terminal (synthetic peptide) and to keratin 10 as markers of epidermal differentiation. AB - Monospecific antibodies to individual keratin polypeptides can be used to examine the tissue and cellular coexpression of members of keratin pairs. Monospecific monoclonal and polyclonal antibodies have been raised to keratins 1 and 10 using both crude cytoskeletal extracts and synthetic peptides. The tissue distribution of these keratins has been determined against a panel of freshly frozen normal tissues from humans, rodents and pigs. Epidermal expression has been examined in psoriatic plaques, and healing wounds, as examples of epidermal hyperproliferation. Cultured keratinocytes in monolayer (low calcium), stratified (high calcium), and complex cultures, transformed keratinocytes, and tumour cell lines, have been examined for the in vitro expression of these keratins. The sensitivity and precise localization of reactivity with these monospecific antibodies gives a highly accurate picture of individual cell expression. There is confirmation of coexpression of keratins 1 and 10 in epidermal and mucosal sites, and with keratin 16 in hyperproliferative states. These monospecific antibodies provide an important means of examining keratin expression in epidermal tumours and keratinizing disorders, and of seeking keratin mutations in cell lines and in skin diseases. PMID- 7544605 TI - Malignant carcinoid syndrome associated with cutaneous scleroderma. PMID- 7544606 TI - GABA immunoreactivity in the nucleus isthmo-opticus of the centrifugal visual system in the pigeon: a light and electron microscopic study. AB - The present study examined GABA immunoreactivity within the retinopetal nucleus isthmo-opticus (NIO) of the pigeon centrifugal visual system (CVS) using light- (immunohistofluorescence, peroxidase anti-peroxidase: PAP) and electron- (postembedding GABA immunogold) microscopic techniques. In some double-labeling experiments, the retrograde transport of the fluorescent dye rhodamine beta isothiocyanate (RITC) after its intraocular injection was combined with GABA immunohistofluorescence. GABA-immunoreactive (-ir) somata were demonstrated within the neuropilar zone of the NIO adjacent to the centrifugal cell laminae whereas the centrifugal neurons were always immunonegative. A quantitative ultrastructural analysis was performed which distinguished five categories of axon terminal profiles (P1-5) on the basis of various cytological criteria: type of synaptic contact (symmetrical or asymmetrical); shape, size, and density of synaptic vesicles as well as the immunolabeling (positive or negative), size of profile and appearance of hyaloplasm. Numerous GABA-ir afferents to centrifugal neurons via axon terminal types P2a, P2c, and P3 were observed which comprised 47.1% of the total input. Moreover, the data suggest that some of the P2a terminals, which make up 26.4% of the input, stem from the intrinsic GABA-ir interneurons, whereas the latter receive P1, P3, but also P2 terminal input, indicating that interneurons may contact other interneurons via type P2a axon terminals. The results also suggest that the GABA-ir P3 or the immunonegative P1b and P5 axon terminals are of extrinsic origin arising from cells in the optic tectum whereas the P2c and P4 axon terminals are associated with extra-tectal input to the NIO. The GABAergic innervation of centrifugal neurons within the NIO may be the basis for the demonstrated facilitatory effect of the centrifugal output upon ganglion cell responses. This is relevant to hypotheses regarding CVS involvement in attentional mechanisms through selective enhancement of retinal sensitivity depending on the location of meaningful or novel stimuli. PMID- 7544609 TI - Excitatory amino acid receptors modulate habituation of the response to visual stimulation in the cat superior colliculus. AB - In visual neurones of the superficial layers of the superior colliculus (SSC), repetitive stimulation causes a progressive decline in the size of the response to the stimulus, usually known as response habituation or response adaptation. A mechanism has been proposed in which habituation results from coactivation of excitatory and inhibitory neurones, and the responses of the inhibitory neurones block the response to subsequent stimulus presentations. Excitatory amino acid (EAA) neurotransmitters mediate visual responses via NMDA and non-NMDA receptors in cat SSC. We have investigated the role of these receptors in the generation of response habituation. Following the iontophoretic application of the EAA antagonists CNQX, AP5 or CPP, repetitive visual stimulation paradigms which normally produce response habituation no longer do so. Indeed the response to each presentation of the stimulus is similar. Intravenous administration of the dissociative anesthetic ketamine (2-10 mg/kg) had similar actions to iontophoretically applied NMDA antagonists. The data imply that intracollicular mechanisms activated by NMDA and non-NMDA receptors contribute to the generation of the inhibitory responses in SCC which lead to response habituation. Furthermore, the effects seen with ketamine anesthesia suggest that the use of ketamine in studies of sensory systems may result in the lack of habituation. PMID- 7544611 TI - Maternal serum screening for Down's syndrome in the first trimester of pregnancy. PMID- 7544612 TI - Molecular modeling based mutagenesis defines ligand binding and specificity determining regions of fibroblast growth factor receptors. AB - The fibroblast growth factor receptor 2 (FGFR2) and the keratinocyte growth factor receptor (KGFR) have different ligand binding specificities despite differing only in the second half of their immunoglobulin-like (Ig-like) domain III. Three-dimensional model structures were generated for domain III on the basis of variable (V) Ig domains. The region that differs between the two receptors is predicted to include two loops: one connects beta-strands F-G and is analogous to the complementarity determining region 3 (CDR3) of immunoglobulins; the other connects beta-strands D-E. These regions were targeted for mutagenesis. Single mutations in the F-G loop were found to only slightly alter ligand binding, whereas a double mutant, KGFR Y345-->S,Q348-->I, acquired significant affinity for bFGF. Notably, the affinity of this double mutant KGFR for KGF and aFGF was essentially unaltered. A mutant FGFR2, in which the D-E beta-hairpin (T319TDKEI) is replaced with the KGFR D-E beta-hairpin (S319SNA), has 9-fold reduced affinity for bFGF. These results demonstrate that the F-G or CDR3 analogous loop in FGFRs plays a key role in determining ligand binding and specificity. In addition, however, the protein loop connecting beta-strands D and E may also be involved in ligand binding. Several point mutations in FGFR2, shown recently to give rise to multiple inherited skeletal defects, are localized according to our models to the F-G or D-E loops of domain III. Our results strongly suggest that these naturally occurring mutations specifically alter ligand binding by FGFR2. PMID- 7544613 TI - Agouti antagonism of melanocortin binding and action in the B16F10 murine melanoma cell line. AB - Several dominant mutations at the murine agouti locus result in the expression of a number of phenotypic changes, including a predominantly yellow coat color, obesity, and hyperinsulinemia. The mutants exhibit ectopic overexpression of normal agouti protein, suggesting that agouti regulates coat coloration by direct antagonism of the alpha-melanocyte-stimulating hormone receptor. We have tested this hypothesis by examining agouti inhibition of both melanocortin-stimulated cyclic adenosine monophosphate production and the binding of a radioactive melanocortin analog in the murine B16F10 melanoma cell line. Inhibition of melanocortin-induced cyclic nucleotide accumulation did not require preincubation of the cells with agouti and was independent of the agonist used. Furthermore, inhibition of both agonist binding to and activation of melanocortin receptor could be described by a simple competitive model with similar inhibition constants of 1.9 and 0.9 nM, respectively. The mutually exclusive binding of agouti and melanocortin was verified by cross-linking experiments using a radiolabeled alpha-melanocyte-stimulating hormone analog. Competitive inhibition of alpha-melanocyte-stimulating hormone binding can account for the effects of agouti on coat coloration and suggests the possibility that the other phenotypic changes observed on agouti overexpression may be due to direct action of agouti at a novel melanocortin receptor(s). PMID- 7544614 TI - Association of rat C-reactive protein and other pentraxins with rat lipoproteins containing apolipoproteins E and A1. AB - C-Reactive protein (CRP) is a member of the pentraxin family of proteins, ubiquitous components of animal serum. This study suggests that, in serum, rat CRP is complexed with lipoprotein and may interact directly with apolipoprotein E. When mixed with diluted rat serum, radiolabeled rat CRP showed a slightly higher sedimentation coefficient (about 15%) than that of the free protein. Elimination of calcium or addition of O-phosphorylethanolamine (O-PE), a low molecular weight compound that binds tightly to rat CRP in a calcium-dependent manner, abolished this difference. Adsorption of rat serum on a rat CRP affinity gel and elution with PE resulted in the isolation of material containing high levels of apolipoproteins E and A1. The affinity-purified preparation interacted with rat CRP and altered the sedimentation coefficient of the latter to the value observed in whole serum. Conversely, rat CRP increased the sedimentation coefficient of the major component of the affinity-purified material or to diluted rat serum, human serum amyloid P (SAP) and hamster female protein (FP), two other members of the pentraxin protein family, also had slightly higher sedimentation coefficients. In contrast, human CRP showed no evidence of an interaction in rat serum or with the affinity-purified proteins. This selectivity coincided with the ability of these pentraxins to bind to O-PE with high affinity. The sedimentation properties of serum lipoproteins, radiolabeled with [3H]cholesterol, also suggested an interaction with rat CRP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544610 TI - Interlaminar connections of the superior colliculus in the tree shrew. II: Projections from the superficial gray to the optic layer. AB - This study of the tree shrew, Tupaia belangeri, provides evidence for an intracollicular pathway that arises in the superficial gray layer and terminates in the optic layer. As a first step, Nissl, myelin, and cytochrome oxidase stains were used to identify the layers of the superior colliculus in the tree shrew. Second, anterograde and retrograde axonal transport methods were used to determine relationships between laminar borders and patterns of connections. Intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase showed that the border between the superficial gray and optic layers in the tree shrew is marked by a sharp decrease in the density of retinotectal projections. The optic layer also could be distinguished from the subjacent intermediate gray layer by differences in connections. Of the two layers, only the intermediate gray layer received projections following injections of wheat germ agglutinin conjugated to horseradish peroxidase within substantia nigra pars reticulata. Similarly, following injections of horseradish peroxidase or biocytin in the paramedian pons, the intermediate gray but not the optic layer contained labeled cells of origin for the main premotor pathway from the tectum, the predorsal bundle. Next, cells in the superficial gray layer were intracellularly injected with biocytin in living brain slices. Axons were traced from narrow and wide field vertical cells in the deep part of the superficial gray layer to the gray matter surrounding the fiber fascicles of the optic layer. Small extracellular injections of biocytin in brain slices showed that the optic layer gray matter contains a population of stellate cells that are in position to receive the input from the superficial layer. Finally, small extracellular injections of biocytin in the intermediate gray layer filled cells that sent prominent apical dendrites into the optic layer, where they may be directly contacted by the superficial gray layer cells. Taken together, the results support the hypothesis that the optic layer is functionally distinct from its adjacent layers, and may provide a link in the transfer of information from the superficial, retinal recipient, to the intermediate, premotor, layer of the superior colliculus. PMID- 7544615 TI - Structure of the carboxy-terminal region of thyroid hormone nuclear receptors and its possible role in hormone-dependent intermolecular interactions. AB - The thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors. To understand the molecular basis of ligand-dependent transactivation, we studied the structure of their carboxy-terminal activation domain. We analyzed the structures of the peptides derived from the C-terminal sequences of human TR subtypes beta 1 (h-TR beta 1) and alpha 1 (h-TR alpha 1) and a human TR mutant, PV, by circular dichroism (CD). Mutant PV has a C-terminal frameshift mutation and does not bind to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3). Analyses of the secondary structures of the peptides by CD indicate that five amino acids, EVFED, are part of an amphipathic alpha-helix which is required to maintain the structural integrity of the hormone binding domain. A monoclonal antibody, C4 (mAb C4), which recognizes both h-TR beta 1 and h-TR alpha 1 was developed. Using a series of truncated mutants and synthetic peptides, we mapped the epitope of mAb C4 to the conserved C-terminal amino acids, EVFED. Analysis of the binding data indicates that binding of T3 to either h-TR beta 1 or h-TR alpha 1 was competitively inhibited by mAb C4. Deletion of C-terminal amino acids including EVFED led to a total loss of T3 binding activity. Thus, part of the T3 binding site is located in this five amino acid segment. T3 may transduce its hormonal signal to the transcriptional machinery via interaction with EVFED at the C-terminus of TRs. PMID- 7544616 TI - Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA. AB - An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly. PMID- 7544618 TI - Functional and histopathologic studies of primate pulmonary allografts preserved for 24 hours with a form of modified extracellular solution. AB - BACKGROUND: The purpose of this investigation was to evaluate the efficacy of a solution (Ep4) for long-term hypothermic pulmonary allograft preservation in a primate model using both functional and histopathologic criteria. METHODS: Twenty seven Japanese monkeys were divided into donor group and three study groups. The animals in group I underwent acute left lung transplantation (n = 5). Group II consisted of animals which received left pulmonary allografts preserved for 24 hours by simple hypothermic immersion in Ep4 solution (n = 6). The temporary contralateral (right) pulmonary artery occlusion test was performed immediately after transplantation and on postoperative day 7 to assess lung function in the allografts. This test was also performed in the control group (group III, n = 5). The recipient animals in groups I and II were also subjected to serial open lung biopsies and bronchoscopic assessments after transplantation. RESULTS: Temporary right pulmonary artery occlusion did not show any significant differences in gas exchange capacity and pulmonary hemodynamics between groups I and II. Histopathologic examination did not show significant differences in the pulmonary allografts between groups I and II for a period of 4 weeks after transplantation. Serial bronchoscopic and histologic examinations also showed no significant differences in bronchial healing between these two groups. CONCLUSIONS: Long-term hypothermic pulmonary allograft preservation with Ep4 solution does not impair pulmonary function immediately after transplantation. The results of this study indicate that extracellular electrolyte composition solutions such as Ep4 should be subjected to clinical trials. PMID- 7544617 TI - Anisotropy decay measurement of segmental dynamics of the anion binding domain in erythrocyte band 3. AB - Time-resolved anisotropy was utilized to detect nanosecond segmental motions of the band 3 intramembrane domain. Band 3 at lysine 430 was fluorescently labeled in ghost membranes by fluorescein or eosin maleimide treatment of intact human erythrocytes followed by hypotonic lysis. Single lifetimes for fluorescein (3.8 4.1 ns) and eosin (3.2-3.4 ns) were observed. Phase-modulation measurement of anisotropy decay indicated a segmental motion model, r(t) = exp(-t/tau 1c)[r infinity + (ro-r infinity) exp(-t/tau 2c)], defined by rotational correlation times corresponding to band 3 segmental motion (tau 1c, 30-70 ns) and rapid fluorescein motion in its binding pocket (tau 2c, 200-400 ps), and a residual anisotropy (r infinity, 0.23-0.28) describing hindered fluorescein motion. In PBS at pH 7.4, tau 1c, tau 2c, and r infinity were 44 ns, 307 ps, and 0.24, respectively, predicting a steady-state anisotropy of 0.24, in agreement with the measured value of 0.23. Factors that might influence band 3 structure/dynamics were examined. Whereas pH (range 5-10) had little effect on r(t), [NaCl] addition (0-150 mM) remarkably decreased tau 1c from 68 to 44 ns. The decrease in tau 1c correlated with solution ionic strength, and did not depend on osmolality (studied by mannitol addition), or specific anion interactions (comparing Cl, Br, F, SO4, citrate). The ionic strength effect was not observed in fluorescein labeled carbonic anhydrase and trypsin-cleaved band 3, suggesting a specific effect on intact band 3. Anisotropy decay was relatively insensitive to external lectin or internal 2,3-DPG binding, but was sensitive to temperature, membrane fluidity, urea denaturation, fluid-phase viscosity, and aldehyde fixation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544619 TI - Histamine release from pulmonary mast cells after lung transplantation in rats. AB - BACKGROUND AND METHODS: On the assumption that lung mast cells might respond and release histamine after lung transplantation, the number of mast cells and tissue histamine content were investigated for 2 weeks after rat orthotopic left lung isotransplantation (Lewis rat to Lewis rat) and allotransplantation (Lewis rat to Brown Norway rat). The allografts were rejected histologically by day 7 (grade A4). RESULTS: In the isografts, both the numbers of mast cells and histamine content were lower (p < 0.05) on day 4 but higher (p < 0.001) on day 14 compared with the nontransplanted donor left lungs (control). In the allografts, the numbers of mast cells on day 7 and histamine content on day 1 were lower (p < 0.05) than controls and both continued to be low thereafter. On day 7, the histamine content of the allografts was low (p < 0.05) compared with the level of the isografts. Conversely, no significant change was found in histamine content of the untransplanted right native lungs except for on day 14 after isotransplantation, when the content was higher than the controls, suggesting the possibility of a systemic signal for proliferation of mast cells. CONCLUSIONS: Our data indicated that degranulation of the pulmonary mast cells and consequent histamine release localized in the grafts occurred under the microenvironment of reimplantation and rejection after rat lung transplantation. PMID- 7544620 TI - Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and interferon-gamma in human pulmonary allografts. AB - BACKGROUND: Cytokines participate in host responses to allografts, largely through recruiting and activating various regulatory and effector cells. We performed this study to determine the feasibility of using polymerase chain reaction methodology to define the expression of three important cytokines (tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) in human pulmonary allografts. METHODS: Twenty-six graft-derived samples (11 transbronchial biopsy and 8 macrophage and 7 lymphocyte cell pellets isolated from bronchoalveolar lavage) were obtained from 13 lung transplant recipients and treated as follows: extraction of RNA; reverse transcription of RNA to complementary DNA; polymerase chain reaction amplification of cDNA with oligonucleotide primers specific for the three cytokines; gel electrophoresis of the polymerase chain reaction products; and verification of correct cytokine message by Dot blot technique (with specific 32P-labeled oligonucleotide probes). RESULTS: Concomitant pathologic evaluation of biopsy specimens from these 13 recipients showed five diagnostic groups: "normal" (no rejection/infection), n = 2; acute rejection, n = 4; nonspecific inflammation, n = 3; infection, n = 3; and obliterative bronchiolitis, n = 1. Interleukin-2 was expressed predominantly in acute rejection and infection (seven of ten and five of six samples positive, respectively), whereas tumor necrosis factor-alpha was expressed mainly in nonspecific inflammation (four of five samples) and somewhat less in rejection (six of ten). Interferon-gamma was expressed less frequently (in two of six samples with infection, but in none of ten with rejection and none of five with nonspecific inflammation). Serial data from one patient (6 months apart) showed considerable increase in interleukin-2 and interferon-gamma expression as she progressed from normal histologic status to obliterative bronchiolitis. CONCLUSIONS: Cytokine gene transcripts can be determined from minute samples derived directly from pulmonary allografts. Although our data are insufficient to make definitive conclusions, the suggestion of trends of cytokine expression in different posttransplantation pathologic conditions may indicate a useful role for this approach in the clinical evaluation of the lung transplant recipient. PMID- 7544622 TI - The determination of binding constants of micellar-packaged gramicidin A by 13C and 23Na-NMR. AB - Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30 degrees C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+, and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same ffmperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts. PMID- 7544621 TI - Evidence that donor pretreatment with FK506 has a synergistic effect on graft prolongation in hamster-to-rat heart xenotransplantation. AB - BACKGROUND: Concordant xenografts are rejected in a different fashion than are discordant xenografts or allografts. We tested the effect of donor pretreatment with the use of FK506 combined with posttransplantation FK506 treatment and splenectomy and analyzed the mechanism of rejection in hamster-to-rat heart xenotransplantation. METHODS: Heart xenotransplantation from hamster to rat was carried out under immunosuppression with the use of donor pretreatment with FK506, posttransplantation FK506 treatment and splenectomy, followed by complement-dependent cytotoxicity assay, delayed type hypersensitivity test, and histologic analysis. RESULTS: The cardiac graft survived 3 days without donor pretreatment, whereas it survived 4.8 days with donor pretreatment with FK506 (5 mg/kg for 3 days). The graft survival was synergistically enhanced by donor pretreatment, posttransplantation FK506 therapy, and splenectomy, and the longest survival was 43.6 +/- 7.7 days in the group with donor pretreatment, posttransplantation FK506 therapy for 4 weeks, and splenectomy. Delayed-type hypersensitivity response was suppressed significantly in the donor pretreatment group. The antibody typed by immunoglobulin M was mainly detected in the rat serum with the rejected grafts by complement-dependent cytotoxicity assay. Correlating with this complement-dependent cytotoxicity titer, neutrophil infiltration and vasculitis of the coronary vessels were recognized in the rejected grafts, and the marginal zone of the white pulp expanded in the spleen. CONCLUSIONS: Donor pretreatment with FK506 combined with posttransplantation FK506 therapy and splenectomy suppresses the outlet antigen, immunoglobulin M production, and lymphocyte activation, thereby prolonging the graft survival in hamster-to-rat heart xenotransplantation. PMID- 7544623 TI - Ion pair binding of Ca2+ and Cl- ions in micellar-packaged gramicidin A. AB - The two independent NMR experiments were performed to investigate the interaction between CaCl2 and the gramicidin A (GA) ion transport channel, using 13C-enriched GA and GA molecules incorporated into dodecylphosphocholine (DPC) micelles. The chemical shifts of C-13 labeled carbonyl carbons vs. CaCl2 concentration demonstrate that Ca2+ and Cl- ions interact as an ion pair within the GA structure with the Cl- ion located near the position of the carbonyl group of the Trp11 residue some 5.5 A from the mouth of the GA helix, and the Ca2+ ion bound at the position of the carbonyl group of the Trp15 residue some 2.5 A from the entrance to the helical pore. The measurements of the 35Cl line-widths and transverse relaxation times illustrate that the interaction occurs between Cl- ions and GA in DPC when in CaCl2 solution, that no interaction is detected between Cl- ions and GA in DPC when in NaCl solution, and that the interaction between Cl- ions and GA in DPC when in MgCl2 solution is much weaker than in CaCl2 solution. In short, a Cl- ion can enter the GA when it is paired with a divalent Ca2+ ion; and Ca2+ and Cl- ions as a pair exchange rapidly with sites of the GA dimer. PMID- 7544624 TI - Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage gated ion channels when incorporated into planar lipid bilayers. AB - Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl. The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented. PMID- 7544625 TI - Purification of an angiogenesis inhibitor from culture medium conditioned by a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. AB - We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three step procedure, gel filtration chromatography, ion-exchange chromtography, and reverse-phase HPLC, 200 micrograms of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass (M(r)) of 24,000 (24K) under reducing conditions and M(r) 22K under nonreducing conditions on SDS-PAGE. On reverse zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP, hCHIAMP at a dose of as little as 0.45 microgram significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mumol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors. PMID- 7544626 TI - Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis. AB - Cultured chondrocytes respond to insulin-like growth factors (IGFs) by increasing the production of proteoglycans and insulin-like growth factor binding proteins (IGF-BPs). To investigate the biological effects of IGFs and IGF-BPs, isolated bovine articular and ovine growth-plate chondrocytes were cultured at high density in the presence of IGF-1, and its truncated form, des (1-3) IGF-I. Both growth factors stimulated the production of IGF-BPs in articular and growth-plate chondrocyte monolayers. Western ligand blots showed that bovine articular chondrocytes released two forms of IGF-BPs into conditioned medium with molecular weights of 29 and 31 kDa. Ovine growth-plate chondrocytes released four different forms of IGF-BPs of approx. 22, 24; 29-30 and 34 kDa. IGF-I and des (1-3) IGF-I stimulated total proteoglycan synthesis by articular chondrocytes up to 1.5-fold. The truncated analogue was more potent at lower concentrations, particularly in stimulating incorporation of newly synthesized proteoglycans into the cell-layer. The maximal stimulation of proteoglycan synthesis in ovine growth-plate chondrocyte culture was 3-fold with des (1-3) IGF-I, while IGF-I enhanced proteoglycan production by only 2-fold over the concentrations used. Our results suggest that endogenous IGF-BPs in chondrocyte cultures act as a part of a feed back mechanism which diminishes the bioactivity of IGF-I. PMID- 7544630 TI - [Recombinant reverse transcriptase from Rous sarcoma virus. Kinetics and inhibition of DNA polymerase activity]. AB - Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E. coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer and dTTP as nucleotide substrate. Kinetic constants for steady state conditions were determined. The substrate initial velocity patterns point to an ordered mechanism which results in the formation of a ternary complex, in which the template-primer is the first to bind to the enzyme. Inhibition of the DNA-polymerase activity of the enzyme by various inhibitors was studied. Analysis of final products of the DNA-polymerase reaction revealed the presence of distribution syntheses of the DNA chain by the alpha alpha-subunit form of the enzyme. PMID- 7544628 TI - Optimal timing of granulocyte colony-stimulating factor (G-CSF) administration after bone marrow transplantation. A prospective randomized study. AB - The positive role of G-CSF in hastening the myeloid recovery of patients undergoing allogeneic bone marrow transplantation (ALLO-BMT) or autologous bone marrow transplantation (ABMT) has recently been established. Considerable knowledge about adequate doses and route of administration has been accumulated in the past few years. Nonetheless, the optimal time to start growth-factor administration remains undetermined. We have performed a stratified study according to the source of hematopoietic progenitors (ALLO-BMT or ABMT), underlying disease and its stage, and acute graft-versus-host disease (GVHD) prophylaxis regimen and randomized patients in two arms: group A, which started G CSF on day 0 (36 patients), and group B, which started on day +7 post-BMT (39 patients). The same dose (5 micrograms/kg/day) and route of administration were employed in both groups. We found no significant differences in the time to reach an absolute neutrophil count (ANC) of 0.1, 0.5, and 1 x 10(9)/l and 50 x 10(9) platelets/l (medians: 10 and 11, 14.5 and 14, 17 and 16, 23 and 24 days, respectively, in groups A and B). We did not find differences in the days of fever or days on antibiotic treatment with less than 1 x 10(9)/l ANC, rate of bacteriemia, or days of hospitalization in both groups. In contrast, a considerable saving of G-CSF in B group was found (mean days of infusion in group A, 18, versus 11 in group B) (p < 0.0001). This is equivalent to a saving of 1120 $US per patient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544627 TI - Prostate cancer in the era of prostate-specific antigen. AB - The number of cases of prostate cancer diagnosed increased by 15% in the past year. This trend is likely to continue as prostate-specific antigen testing was approved for screening during the past year. The result is that mroe patients are being diagnosed at an earlier and presumably curable stage, although the long term impact on survival has not been proven. Two large national randomized comparisons were initiated to assess the value of early detection (Prostate, Lung, Colorectal, and Ovarian cancer screening trial) and of early treatment (Prostate Cancer Intervention Versus Observation Trial). Evolving data from early detection studies suggest that the majority of cancers diagnosed are clinically significant. Further, criteria for "deferred treatment" approaches are likewise beginning to evolve. The more widespread use of nomograms based on digital rectal examination findings, Gleason grade, and baseline prostate-specific antigen has led to more refined treatment selection. Patients deemed incurable by local means are being referred for combined modality approaches. For more advanced disease, several new hormonal approaches are evolving, and for those with relapsing tumors, responses to antiandrogen withdrawal were extended to Casodex, (Zeneca Pharmaceuticals, Macclesfield, UK) and a number of nonhormonal treatments such as combinations of estramustine and etoposide estramustine and vinblastine, and doxorubicin and ketoconazole are showing clinical benefit. PMID- 7544634 TI - [Fast methods of bacterial and fungal identification in sepsis from blood culture flasks]. PMID- 7544629 TI - Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients. AB - The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted from delta TCS-1-positive T lymphocytes. At concentrations of 1.25-10 microM, the three antisense ODN did not inhibit any erythrocyte or granulocyte monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, of delta TCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies. PMID- 7544633 TI - Effects of FK506 on an experimental model of colitis in rats. AB - AIM: To assess the effects of FK506, a newly developed immunosuppressant, on experimental colitis in rats. METHODS: Experimental colitis was induced by a single colonic instillation of hapten 2,4,6-trinitrobenzene sulphonic acid (TNB) in anaesthetized rats. Rats received 30 mg TNB dissolved in 0.25 mL of 50% ethanol, and were sacrificed on day 5 following 4 days dosing with FK506 (0.25, 0.5, 1.0, 2.0 mg/kg, s.c.) or vehicle. Mucosal prostanoid concentrations were determined using high performance liquid chromotography. Tissue myeloperoxidase activities were measured. The effects of FK506 on superoxide radical formation by neutrophils in both rats and humans were also estimated in vitro. RESULTS: Administration of FK506 significantly reduced the colonic damage in a dose dependent manner. Activities of myeloperoxidase and concentrations of 6-keto prostaglandin F1 alpha (6-keto-PGF1 alpha), PGF2 alpha and PGE2 in colonic tissue increased significantly following induction of experimental colitis, however, FK506 did not affect these changes. FK506 reduced stimulant-induced superoxide radical formation by neutrophils in rats and humans. CONCLUSION: FK506 decreased superoxide radical generation by neutrophils, which might contribute to the lessening of colonic damage in this model. PMID- 7544632 TI - Intraluminal pressure modulates the magnitude and the frequency of induced vasomotion in rat arteries. AB - Arterial vasomotion and its relation to intraluminal pressure were investigated in vitro in isolated rat arteries. Femoral arteries (mean diameter = 768.2 +/- 25 microns, n = 5) and mesenteric arteries (mean diameter = 393.4 +/- 32 microns, n = 5) were used in this study. Arterial segments were excised, mounted on microcannulas and perfused with Tyrode's solution at a constant flow (100 microliters/min). After equilibration, intraluminal pressure was stepwise changed from 0 to 120 mm Hg. The changes in the outer diameter of the vessels were measured continuously over a period of 4 h after the equilibration. Vasomotion was induced by constrictor agonists (norepinephrine 10(-6) M for mesenteric arteries and norepinephrine 10(-6) M + Bay K8644 10(-7) M for femoral arteries) and was maintained only in the presence of the above-mentioned drugs. Both vasomotion magnitude and frequency are modulated by pressure. Vasomotion frequency increases with pressure increase. When intraluminal pressure varied between 0 and 120 mm Hg, vasomotion frequency varied between 0.19 and 0.49 Hz for mesenteric arteries and between 0.04 and 0.23 Hz for femoral arteries. Thus, vasomotion frequency differed clearly between the two vessel types. Vasomotion amplitude shows a biphasic relationship with a maximum occurring at about 40 mm Hg for mesenteric arteries and 50 mm Hg for femoral arteries. Based on these findings, it is hypothesized that vasomotion amplitude relates to the active mechanical properties of the artery and, in particular, to its contractile capacity. PMID- 7544631 TI - Utilization of activated U937 monocytic cells as a model to evaluate biocompatibility and biodegradation of synthetic calcium phosphate. AB - The use of calcium phosphate biomaterials as a bone substitute necessitates the use of normative biocompatibility and biodegradation techniques which must be fast, simple and reproducible. In the present study, we have developed an in vitro model to study and to compare different calcium phosphate ceramics. After activation with 1,25-dihydroxy-vitamin D3 and phorbol 12,13-dibutyrate, the monoblastic U937 cells became multinucleated, expressed tartrate-resistant acid phosphatase and several markers of monocyte/macrophage differentiation. Activated U937 cells did not express the vitronectin receptor (VNR) (as revealed using monoclonal antibodies 23C6 or 13C2) but around 25% of the cells were strongly reactive with 211D, a novel monoclonal antibody that recognizes an osteoclast specific membrane antigenic determinant. These cells remain active/viable with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP) ceramics. In conclusion, activated U937 cells are good candidates to use in a normative in vitro method to evaluate new biomaterials. PMID- 7544635 TI - [Therapeutic approach to prostatic adenoma in primary care]. PMID- 7544636 TI - A rapid latex immunoassay for the detection of plasmin-alpha 2-plasmin inhibitor complex. Utilization of two monoclonal antibodies differentially recognizing respective components of the complex. AB - Among six monoclonal antibodies raised against the human plasmin-alpha 2-plasmin inhibitor complex (PPI), three antibodies were found to recognize the plasmin part (group 1) and another three the alpha 2-plasmin inhibitor (alpha 2-PI) part (group 2) of the complex. One of the group-1 monoclonal antibodies, designated JIPPI-3, specifically reacted with a segment of plasmin containing kringles 2 and 3. Although all three group 2 antibodies reacted with both alpha 2-PI and PPI on immunoblotting and ELISA, one of them, JIPPI-50, was unable to react with alpha 2 PI, when the antibody had been covalently conjugated to Sepharose 4B gels and tested for reactivity against the antigens in solution. The results indicated that the epitope for this antibody had been buried in nascent alpha 2-PI, but had been exposed by complex formation with plasmin or by possible conformational changes induced in the alpha 2-PI molecule on insolubilization to nitrocellulose membranes or immunoplates. By utilizing a set of JIPPI-3 and JIPPI-50, individually coated onto latex beads, PPI could be measured in plasma in the range of 0.8-100 micrograms/ml without interference by coexisting plasminogen (120-200 micrograms/ml) or alpha 2-PI (70 micrograms/ml). This measurable range seems to cover the level of PPI clinically observed under hyperfibrinolytic states. PMID- 7544637 TI - A monoclonal antibody with high affinity for a neo-antigenic site in fibrinogen degraded by polymorphonuclear leukocyte-derived elastase. AB - Elastase, released by stimulated polymorphonuclear leukocytes (PMN), is thought to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD) especially pulmonary emphysema. A test that can detect release of elastase activity from PMN would be valuable to monitor therapy or to identify patients at risk. The authors aimed to isolate and characterize monoclonal antibodies (mAb) with a high affinity for a neo-antigenic determinant in a high molecular weight degradation product of fibrinogen (Fbg) generated by PMN-derived elastase. Using synthetic peptides, they mimicked the new amino or carboxy terminal sequences of the A alpha-, B beta- and gamma-chains of Fbg that are generated by elastase. These synthetic peptides (A alpha 22-36, A alpha 350-360, B beta 44-55, and gamma 295-305), uni-directionally coupled to a carrier protein, were used to generate mAb specific for elastase-degraded fibrinogen (EDF). mAb that appeared to be specific for a neo-antigenic determinant (neotope) consisting of the new amino terminal amino acid(s) of the Fbg A alpha-chain that is generated by elastase activity were isolated only with the A alpha 22-36 synthetic peptide. One mAb, designated as EF1-4, was further characterized and had an approximately 75-fold higher affinity for EDF, as compared with Fbg, in solution. Using the other peptides, no mAb specific for elastase generated fibrinogen degradation products were obtained.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544639 TI - Early CD34high cells can be separated into KIThigh cells in which transforming growth factor-beta (TGF-beta) downmodulates c-kit and KITlow cells in which anti TGF-beta upmodulates c-kit. AB - We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c-kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF-beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF-beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors. PMID- 7544638 TI - Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors. AB - We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL 3)/granulomonocyte-CSF (GM-CSF)/erythropoietin (Epo), FL potentiates colony forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, whereas M-CSF alone stimulates prevalent monocytic differentiation but little cell proliferation: combined M-CSF+FL treatment causes both proliferation and almost exclusive monocytic differentiation (97% monocytes in fetal calf serum-rich (FCS+) culture conditions, mean value). At primitive HPC level, FL potentiates the clonogenetic capacity of colony-forming units-blast (CFU-B) and high proliferative potential colony-forming cells (HPP-CFC) in primary and secondary culture; KL exerts a similar action, and additive effects are induced by FL combined with KL. More important, addition of FL alone causes a significant amplification of the number of long-term culture-initiating cells (LTC-ICs), ie, putative repopulating HSCs, whereas this effect is not induced by KL. The FL effects correlate with flt3 mRNA expression in HPCs differentiating throught the erythroid or GM pathway in liquid suspension culture: (1) flt3 mRNA is expressed in freshly purified, resting HPCs; after growth factor stimulus the message (2) is abruptly down-modulated in HPC erythroid differentiation, but (3) is sustainedly expressed through HPC GM differentiation and abolished in GM precursor maturation. This pattern contrasts with the gradual downmodulation of c-kit through both erythroid and GM HPC differentiation. The results indicate that FL exerts a stimulatory action on primitive HPCs, including a unique expanding effect on putative stem cells, whereas its distal proliferative/differentiative action is largely restricted to CFU-GM and monocytic precursors. The latter effect is potentiated by KL and M CSF, thus suggesting that the structural similarities of FL, KL, M-CSF, and their tyrosine kinase receptors may mediate positive interactions of these growth factors son monocytic differentiation. PMID- 7544640 TI - CD34+ progenitor cells from asymptomatic patients are not a major reservoir for human immunodeficiency virus-1. AB - Controversy exists as to whether hematopoietic progenitor cells are infected by human immunodeficiency virus-1 (HIV-1) in vivo. Most studies have focused on patients with acquired immunodeficiency syndrome (AIDS)/AIDS-related complex, and little data are available on asymptomatic patients with well preserved CD4+ T cell counts. To determine if CD34+ hematopoietic progenitor cells are infected early in the course of HIV-1 disease, we evaluated 10 asymptomatic HIV-1 seropositive (HIV-1+) patients. The CD34+ cell fraction was purified by a two step procedure consisting of both affinity chromatography and fluorescence activated cell sorting that resulted in a median purity of over 99%. Using conventional and nested polymerase chain reaction (PCR) assays, we evaluated the presence and frequency of HIV-1 proviral DNA. Both bone marrow mononuclear cells and CD34- cells from all 10 patients were strongly positive for the HIV-1 pol and/or gag gene sequences. In contrast, sorted CD34+ cells from only two of 10 patients were positive, and the number of copies of proviral DNA in these samples was estimated to be from 2 to 5 per 250,000 cells. To test the in vitro functional capacity of CD34+ progenitors, these cells were assayed in both methylcellulose and long-term stromal culture. We found no significant reduction in the number of colony-forming unit-erythroid (CFU-E), burst-forming unit erythroid (BFU-E), or colony-forming unit-granulocyte macrophage (CFU-GM) colonies, or in the frequency of cobblestone area forming cells from limit dilution analysis in HIV-1+ asymptomatic patients. Pooled methylcellulose colonies generated from CD34+ cells were HIV-1- in nine of 10 samples. All progeny from long-term cultures of CD34+ cells were HIV-1-. We conclude that the CD34+ hematopoietic progenitor compartment is not infected in the majority of asymptomatic HIV-1+ patients, and that these cells may represent a suitable target for strategies designed to protect developing CD4+ T cells from infection. PMID- 7544641 TI - Steel factor (c-kit ligand) promotes the survival of hematopoietic stem/progenitor cells in the absence of cell division. AB - It is known that the majority of primitive hematopoietic progenitors are in a noncycling quiescent state. In addition, normal hematopoietic progenitors and progenitor cell lines show an absolute dependence on growth factors for their survival in vitro, yet the effect of growth factors on progenitor cell survival has not been separated from effects on both proliferation and differentiation. Using an in vitro assay system, we examined whether growth factors could promote the survival of stem cells in culture in the absence of cell division. These studies show that steel factor (SLF) and, to a lesser extent, interleukin-3 (IL 3) directly promoted the survival of elutriated bone marrow progenitor cells (countercurrent centrifugal elutriation [CCE]-27) that are enriched for primitive hematopoietic progenitors that respond to the combination of SLF plus IL-3. Furthermore, SLF promoted the survival of short-term reconstituting cells (STRC), and long-term reconstituting cells (LTRC) with trilineage reconstitution potential in vivo. In comparison, granulocyte colony-stimulating factor (G-CSF), IL-6, leukemia inhibitory factor, IL-11, IL-1, granulocyte macrophage CSF (GM CSF), and macrophage CSF (M-CSF) had no effect on the survival of these cells. In the presence of mitotic inhibitors (nocodazole or aphidicolin), SLF promoted the survival of CCE-27 progenitor cells that respond to the combination of SLF plus IL-3 in vitro and STRCs and LTRCs that are detected in vivo. Taken together, these data show that SLF can directly promote the survival of hematopoietic progenitor cells in the absence of cell division. PMID- 7544642 TI - Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects. AB - Despite considerable effort, the expansion of long-term culture-initiating cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medium exchange. The use of frequent medium exchange was, therefore, examined in cultures of CD34-enriched cells. In stromal-free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no feeding for 14 days gave similar results. Six different growth factor combinations, reported by other groups to give optimal expansion of CD34 enriched cells, were tested in comparison with the control combination of IL-3/GM CSF/Epo/SCF. None of the combinations resulted in improved colony-forming unit granulocyte macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enriched cell cultures, the effect of preformed stroma was next examined. Preformed stroma increased cell (3-fold), CFU-GM (5-fold), and LTC IC (3-fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near input level. The lack of LTC-IC expansion in CD34-enriched cell cultures prompted experiments to examine the effect of cell purification. Parallel cultures were performed at CD34+lin- cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34+lin- cells in addition to the CD34- accessory cells required to give the desired percentage. Also, MNC from the same source (approximately 2% CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss of culture performance with purification was likely due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were found to decline significantly with increasing CD34+lin- cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU GM output from cultured CD34-enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula resulting in a growing stromal layer during the culture led to LTC-IC expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7544643 TI - Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. AB - The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS-binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies. PMID- 7544644 TI - Both tumor necrosis factor receptors, TNFR60 and TNFR80, are involved in signaling endothelial tissue factor expression by juxtacrine tumor necrosis factor alpha. AB - We have investigated the role of the two distinct tumor necrosis factor (TNF) receptors (TNFR60 and TNFR80) in endothelial cell activation employing an in vitro model of tumor necrosis factor alpha (TNF-alpha)-dependent tissue factor production of human umbilical vein endothelial cells (HUVECs). In this model, tissue factor is produced either on addition of exogeneous TNF-alpha, or by induction of endogenous TNF-alpha via adhesion molecule-linked signal pathways. Under both conditions, tissue factor expression could be partially blocked by antagonistic antibodies against either TNFR60 or TNFR80 and was fully inhibited by simultaneous application of both antibodies. Selective inhibitors of either TNFR60 or TNFR80-induced signal pathways inhibited tissue factor expression, and selective triggering of either of the two TNF receptors by agonistic antibodies induced this response in HUVECs. Furthermore, a coculture system of HUVECs and Chinese hamster ovary transfectants expressing a noncleavable, exclusively membrane-bound form of TNF-alpha resulted in a potent activation of HUVECs with synergistic action of both TNF receptors. Together, these data underline the importance of juxtacrine pathways in endothelial cell activation of procoagulant functions and show that membrane TNF-alpha and both TNFR types play a critical role. PMID- 7544645 TI - B-cell homotypic adhesion through exon-A restricted epitopes of CD45 involves LFA 1/ICAM-1, ICAM-3 interactions, and induces coclustering of CD45 and LFA-1. AB - Lymphocyte interactions with other leukocytes and other cell types, as well as with components of the extracellular matrix, are one of the key steps in the immune response. Three novel monoclonal antibodies (MoAbs) have been produced and selected for their ability to induce intercellular adhesion in B cells. These three MoAbs immunoprecipitated a polypeptide of 220 kD, displaying specific phosphotyrosine phosphatase activity that has been identified as CD45. These MoAbs recognize epitopes located on the alternative spliced exon-A-encoded region of CD45. These epitopes are of polypeptidic nature, but they can be masked by addition of carbohydrate during CD45 biosynthesis. Interestingly enough, CD45 epitopes recognized by these MoAbs appeared to be selectively expressed on both peripheral blood and tonsillar B lymphocytes as well as on peripheral blood natural killer (NK) cells. CD45-mediated intercellular adhesion was abrogated upon incubation with anti-leukocyte function-associated antigen 1 (anti-LFA-1), intercellular cell adhesion molecule 1 (ICAM-1), and ICAM-3 MoAbs, thus indicating that this phenomenon involved both LFA-1/ICAM-1 and LFA-1/ICAM-3 cell adhesion pathways. Moreover, CD45-mediated cell aggregation was also inhibited by preincubation with some conventional anti-CD45 MoAbs. Interestingly, the triggering of cell aggregation through CD45 induced membrane surface relocation of CD45 and LFA-1 molecules, with both of them colocalizing at cell-cell contact areas of B-cell aggregates. Studies with inhibitors of both phosphotyrosine phosphatase and tyrosine kinase activities suggest that CD45 phosphotyrosine phosphatase activity could be involved in CD45-mediated cell aggregation. Taken together, these results support the notion that CD45 is a key molecule in the regulation of LFA-1-mediated cell-cell interactions. PMID- 7544646 TI - The receptor tyrosine kinase p185HER2 is expressed on a subset of B-lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia. AB - The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B-lymphoblastic leukemias. PMID- 7544647 TI - Refined mapping of genomic rearrangements involving the short arm of chromosome 9 in acute lymphoblastic leukemias and other hematologic malignancies. AB - Deletions of chromosomal band 9p21 have been detected in various tumor types as well as in more than 20% of acute lymphoblastic leukemia (ALL). These deletions frequently include the entire interferon (IFN) gene cluster as well as the methylthioadenosine phosphorylase (MTAP) gene. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) was proposed as a candidate tumor-suppressor gene on 9p21 because it is frequently deleted in cell lines derived from multiple tumor types. To determine if CDKN2 or another closely related gene on 9p is the target of 9p deletions in ALL and other hematologic malignancies, we analyzed 20 primary patient samples (13 ALL, 2 acute myeloid leukemias [AML], and 5 non-Hodgkin's lymphomas [NHL]) with 9p rearrangements using Southern blot analysis, fluorescence in situ hybridization (FISH), and single-strand conformation polymorphism (SSCP) for alterations of CDKN2. Homozygous deletions of the CDKN2/CDKN2B (p15) region were detected in 10 cases (50%; 6 ALL, 2 AML, and 2 NHL). In 1 additional case, the intensity of the Southern blot band was significantly reduced, suggesting a CDKN2 deletion in a subpopulation of the malignant cells. No CDKN2 or CDKN2B rearrangements were seen. The IFN gene cluster was homozygously deleted in 2 of 15 (13%) analyzed cases, whereas the MTAP gene was deleted in 6 of 15 cases (40%). In addition, hemizygous deletions of the CDKN2 region were identified in 6 ALL cases using interphase FISH. No point mutation of the coding region of CDKN2 was detected by SSCP in these cases. We conclude that CDKN2 is the most frequently homozygously deleted marker on 9p. The absence of point mutations in the coding region of CDKN2 in cases with hemizygous 9p deletions and the frequent codeletion of MTAP, CDKN2B, and other yet unidentified neighboring genes suggest that the simultaneous deletion of these genes may be necessary for the selective growth advantage of malignant cells. PMID- 7544648 TI - Expression of Fas/Apo-1 (CD95) and apoptosis in tumor cells from patients with plasma cell disorders. AB - Fas/Apo-1 antigen (CD95) is a cell surface molecule that directly mediates apoptosis. Fas expression was studied in five plasma cell lines, 11 multiple myeloma cases, and three plasma cell leukemia (PCL) cases. Induction of apoptosis by anti-Fas antibody was studied in five plasma cell lines and fresh plasma cells from eight patients. Apoptosis was confirmed by morphologic analysis alone or in combination with DNA electrophoresis analysis. Four of the five cell lines showed Fas expression, three of which showed induction of apoptosis by anti-Fas antibody. One cell line, RPMI 8226, showed the highest sensitivity for Fas mediated apoptosis. High bcl-2 expression was found in KMS12PE, which showed resistance to Fas-mediated apoptosis despite its Fas expression. Plasma cells from seven fresh cases, including all five cases with high serum lactate dehydrogenase (LDH), showed expression of Fas antigen. Fas-induced apoptosis was found in five cases at various levels, although significant induction of apoptosis was found in only one case. Interestingly, Fas-independent apoptosis was induced during culture without anti-Fas antibody in cases with high serum LDH. These results indicate that plasma cells from aggressive myeloma with high LDH express Fas antigen and undergo apoptosis through either Fas-mediated or Fas independent pathways. An understanding of the mechanism of apoptosis in malignant plasma cells should contribute to investigations of the pathophysiology of and therapy for myeloma/PCL. PMID- 7544649 TI - Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in severe combined immune deficient mice given human peripheral blood lymphocytes. AB - CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV-transformation, whereas anti-CD20 antibodies had no effect. Thus, anti-CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome. PMID- 7544650 TI - Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N brasiliensis infection. AB - We assessed the effects of the c-kit ligand, stem cell factor (SCF), in the jejunal mucosal mast cell hyperplasia that occurs during infection with the intestinal nematodes, Nippostrongylus brasiliensis or Trichinella spiralis in rats. Compared with vehicle-treated rats, rats treated with SCF (25 micrograms/kg/d, intravenous [i.v.] for 14 days) during N brasiliensis infection exhibited significantly higher levels of the rat mucosal mast cell (MMC) associated protease, rat mast cell protease II (RMCP II) in the jejunum and serum on day 8 of infection, but not on days 10 or 15 of infection. By contrast, in comparison to rats treated with normal sheep IgG, rats treated with a polyclonal sheep antirat SCF antibody exhibited markedly decreased numbers of jejunal MMCs, levels of jejunal RMCP II, and serum concentrations of RMCP II during infection with either nematode, particularly at the earlier intervals of infection (< or = day 10). Taken together, these findings indicate that SCF importantly contributes to MMC hyperplasia and/or survival during N brasiliensis or T spiralis infection in rats, but that levels of endogenous SCF are adequate to sustain near maximal MMC hyperplasia during infection with these nematodes. Notably, treatment of rats with SCF somewhat increased, and treatment with anti-SCF significantly decreased, parasite egg production during N brasiliensis infection. This finding raises the interesting possibility that certain activities of intestinal MMCs may contribute to parasite fecundity during infection with this nematode. PMID- 7544651 TI - Rapid and sustained allogeneic transplantation using immunoselected CD34(+) selected peripheral blood progenitor cells mobilized by recombinant granulocyte- and granulocyte-macrophage colony-stimulating factors. PMID- 7544652 TI - Biochemical characterization, developmental expression, and induction of the immune protein scolexin from Manduca sexta. AB - The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis. PMID- 7544653 TI - Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs. AB - The mechanisms by which the early limb cell condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin-C is associated with articular cartilage and joint tissue development; others have shown that tenascin-C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin-C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin C and syndecan-3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan-3 rich perichondrium while tenascin-C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo-distally and joint formation proceeded with age, we observed that syndecan-3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin-C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone-derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well-defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin-C gene expression in articular cap, fragmentation of tenascin-C gene expression along the periosteum, reinitiation of syndecan-3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly, tenascin-C and syndecan-3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues. PMID- 7544656 TI - The sulphated carbohydrate-protein linkage region isolated from chondroitin 4 sulphate chains of inter-alpha-trypsin inhibitor in human plasma. AB - Inter-alpha-trypsin inhibitor (ITI) in human plasma has a unique structural architecture composed of three polypeptide chains (H1, H2 and L chains), which are linked to each other through a chondroitin 4-sulphate chain. The structure of the carbohydrate-protein linkage region of the chondroitin 4-sulphate chain attached to the L chain was investigated. The peptide-chondroitin sulphate fraction was isolated by anion-exchange chromatography after exhaustive digestion with lysyl endopeptidase and then V8 protease. The chondroitin 4-sulphate chain was released from the peptides by beta-elimination using NaB3H4 and then digested with chondroitinase ABC. These treatments resulted in a single 3H-labelled hexasaccharide alditol fraction derived from the linkage region which had been associated with the L chain. Chemical and enzymatic analyses as well as fast-atom bombardment-mass spectrometry (FAB-MS) analysis revealed that the 3H-labelled hexasaccharide alditol had the following structure: delta HexA-alpha 1-3GalNAc(4 sulphate)beta 1-4GlcA beta 1-3Gal(4-sulphate)beta 1-3Gal beta 1-4Xyl-ol (where delta HexA is 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and Xyl-ol is xylitol). The structure contained the novel 4-sulphated Gal residue, which was previously demonstrated in a linkage hexasaccharide isolated from chondroitin 4 sulphate of rat chondrosarcoma (Sugahara et al., J. Biol. Chem., 263, 10168 10174, 1988) and of whale cartilage (Sugahara et al., Eur. J. Biochem., 202, 805 811, 1991). The above disulphated hexasaccharide alditol was the only component detected in the linkage region fraction of the chondroitin 4-sulphate chain of ITI, which implies some biological significance of this novel structure. PMID- 7544655 TI - Surveillance of colony-stimulating factor use in US academic health centers. AB - OBJECTIVE: To characterize and evaluate hematopoietic colony-stimulating factor (CSF) use, including cost implications, in US academic health centers. DESIGN: An observational study of patients who received granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) from September 1 to October 15, 1993. SETTING: Thirty academic health centers in the US. PARTICIPANTS: Five hundred sixty-five patients were evaluated. MAIN OUTCOME MEASURES: The appropriateness of CSF use was assessed, based on consensus-derived indication guidelines and the Food and Drug Administration-approved product labeling. Indication, type of CSF, and dosage were considered in determining the appropriateness of CSF therapy. RESULTS: Based on indication evaluation criteria, 71% of CSF use was appropriate, 7% was inappropriate, and 22% was unproven, although the majority of unproven use was deemed promising by the expert panel. Based on dosage evaluation criteria, 51% of CSF use was appropriate, 27% was inappropriate, and 22% was for promising and other unproven indications. More than 90% of the patients studied received G-CSF. Approximately 3.4% of patients who received G-CSF had an adverse event, compared with 22% of those who received GM-CSF. Approximately $791,000 was spent on CSF therapy in the 565 patients: $401,000 (51%) on appropriate indications and doses, $160,000 (20%) on inappropriate doses for appropriate indications, $124,000 (16%) on promising indications, and $106,000 (13%) on unproven or inappropriate indications. CONCLUSIONS: Substantial costs are incurred currently for CSF therapy without adequate literature support. Further studies are warranted to justify promising but unproven uses of CSFs, as well as to clarify proper dosing, monitoring, and relative safety of CSFs. PMID- 7544654 TI - Quantitative immunocytochemical study of secretory protein expression in parotid glands of rats chronically treated with isoproterenol. AB - Chronic treatment of mice and rats with isoproterenol (IPR) causes marked hypertrophy and hyperplasia of the salivary glands, and alters the expression of several secretory proteins. We used quantitative postembedding immunogold labeling to study the cellular responses in the rat parotid gland during daily (up to 10 days) injections of IPR and during recovery (up to 14 days) after cessation of IPR treatment. Labeling densities of acinar cell secretory granules with antibodies to amylase and protein SMG-B1 (cross-reactive with the rat homologue of Parotid Secretory Protein, PSP) fell to 10% of control levels after 8-10 IPR injections, then increased during recovery, paralleling previous biochemical determinations of changes in protein and mRNA levels. With antibodies to proline-rich proteins (PRP), labeling densities initially fell, then subsequently showed considerable variability, but never exceeded control levels. These results contrast with biochemical determinations showing a marked induction of PRP synthesis, and may have both immunological and structural explanations. Occasional intercalated duct cells located close to the acini underwent differentiation toward an acinar-like phenotype as a result of IPR treatment. After 1-2 IPR injections, the secretory granules of these cells labeled with antibodies to amylase and PRP. Subsequently, the granules appeared electron lucent and were increased in size and number. These observations support earlier work, suggesting that intercalated duct cells may differentiate into other gland cell types. PMID- 7544657 TI - Hydrophobic interactions and the design of receptor mimetic peptides. AB - The possibility that hydrophobic interactions may be used as a basis for the design of receptor mimetic peptides for small peptide hormones that lack the potential to adopt amphiphilic secondary structures was tested by designing and characterizing receptor mimetic peptides for gamma-endorphin. The receptor mimetic peptides were designed to exhibit a pattern of hydrophobic surfaces in an antiparallel orientation matching that of the peptide hormone in an extended conformation. An ELISA-based assay was used to determine the relative binding affinities of receptor mimetic peptides, control peptides and antisense peptides to gamma-endorphin immobilized on a surface. The inhibition constant for the best gamma-endorphin receptor mimetic peptide was 1.6 microM. No binding was detected for scrambled control peptides or the antisense-derived peptide mimetic to the limit of their respective solubilities. Sera from rabbits immunized with a gamma endorphin receptor mimetic peptide were used to immunopurify the ligand-binding domain of the human opiate receptor and were cross-reactive with purified bovine opiate receptor. These results suggest that patterns of hydrophobicity can provide a rational basis for designing receptor mimetic peptides and may provide an explanation for the ability of some antisense peptides to bind to their cognate hormones and to elicit antibodies cross-reactive with hormone receptors. PMID- 7544659 TI - Taking control of control charts. PMID- 7544660 TI - Enhancement of norepinephrine and angiotensin II-induced renal effects by NG nitro-L-arginine, a nitric oxide synthase inhibitor. AB - We investigated whether endogenous nitric oxide (NO) has a role as an inhibitory modulator of norepinephrine (NE)- and angiotensin II (Ang II)-induced renal effects in anesthetized dogs. Intrarenal arterial infusion of NE (100 ng/kg/min) or Ang II (10 ng/kg/min) decreased renal blood flow (RBF), glomerular filtration rate (GFR) and urine formation. The NE- or Ang II-induced renal effects were augmented by the intrarenal administration of a NO synthase inhibitor, NG-nitro-L arginine (NOARG), at doses (10 and 40 micrograms/kg/min) which did not affect the mean arterial blood pressure and heart rate. The stimulating activity of NOARG on NE- or Ang II-induced renal effects were abolished by the simultaneous administration of L-arginine, a NO precursor. These findings suggest that endogenous NO, which is probably generated within the kidney, functions as an inhibitory modulator in NE- or Ang II-induced renal vasoconstriction and antidiuresis. PMID- 7544663 TI - Localization of a locus for the striated form of palmoplantar keratoderma to chromosome 18q near the desmosomal cadherin gene cluster. AB - Palmoplantar keratoderma is a frequent hereditary disorder of keratinization in humans. Various clinically, histopathologically and genetically distinct phenotypes can be diagnosed. Recently, mutations in the keratin genes have been identified in palmoplantar keratoderma: mutations in the keratin 9 gene causing the epidermolytic form, and mutations in the keratin 1 gene in a non epidermolytic form. We have now investigated a family with the striated form of palmoplantar keratoderma (type Brunauer-Fuhs-Siemens) for linkage to either the type II keratin gene cluster on chromosome 12q or the type I keratin gene cluster on chromosome 17q. After excluding both type I and type II keratin genes we have mapped a locus for this form of palmoplantar keratoderma to chromosome 18q12 with a maximum two-point lod score of 3.3 at theta = 0.00 at D18S536. A cluster of desmosomal cadherin genes has been mapped to this region making them good candidates for this form of PPK. These findings indicate that hyperkeratosis of palms and soles is clinically as well as genetically heterogeneous. PMID- 7544661 TI - Non-intercalative and sequence-selective interaction of nitropyrene/acridine skeleton with nucleotides: application of the dextran-coupling method. AB - Water-insoluble typical intercalators, acridine and pyrene, were converted to water-soluble species, Dex-Pyr and Dex-Acr, respectively, by coupling with dextran polymer. By the use of these dextran-coupled intercalators, interactions of acridine- and pyrene-skeletones with various nucleotides in aqueous solutions were analyzed. PMID- 7544664 TI - Genetic linkage studies in non-epidermolytic palmoplantar keratoderma: evidence for heterogeneity. AB - The palmoplantar keratodermas (PPK) are a group of skin diseases characterized by thickening of the skin of the palms and soles due to abnormal keratinization. We have performed linkage analysis on families affected with three distinct forms of non-epidermolytic PPK (NEPPK): focal, diffuse and punctate. Genetic heterogeneity was demonstrated, with focal NEPPK linked to the region on chromosome 17 harbouring the type I keratin cluster, diffuse NEPPK linked to the region on chromosome 12 containing the type II keratin cluster, and in the punctate NEPPK pedigrees, linkage was excluded to both of these keratin clusters. This study provides evidence for genetic differences between these forms of NEPPK and also between NEPPK and epidermolytic PPK (EPPK) in which mutations in keratin 9 have been demonstrated. PMID- 7544658 TI - Zinc-containing innervation of the subicular region in the rat. AB - The subiculum is densely innervated by zinc-containing axonal terminals, but the cells of origin of those zinc-containing afferents have not previously been identified. In the present work the zinc-specific retrograde tracing method was employed to locate the zinc-containing neurons afferent to the subicular complex. Following microinfusions into the subicular region, the somata of zinc-containing neurons were found in the hippocampus, the pre- and para subiculum, retrosplenial, cingulate, and perirhinal cortices, and in the anterodorsal nucleus of the thalamus. The results show another component of the zinc containing associational network that interconnects the cerebral cortex and amygdalohippocampal systems of the brain. PMID- 7544662 TI - Effect of lipophilicity of trichosporin-Bs on ion-channel formation and catecholamine-releasing activity. AB - A series of peptaibols, trichosporin (TS) -Bs, induced voltage-activated conductance in lipid bilayer membranes and catecholamine secretion from bovine adrenal chromaffin cells. The order of activities is the same as that of the lipophilicity rank of TS-Bs derived from the reversed-phase HPLC capacity factors. PMID- 7544665 TI - Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B. AB - It has previously been suggested that keratinocytes might provide a suitable target cell for delivery of factor IX to the systemic circulation for gene therapy of haemophilia B. Here, an investigation of the use of cellular gene promoters specific for keratinocytes was undertaken to examine whether factor IX could be passed from the epidermis to the systemic circulation. Utilizing two bovine cytokeratin gene promoters, BKIII and BKVI, three lines of transgenic mice were generated with targeted expression of human factor IX in the epidermis. All three transgenic mouse lines secreted epidermally derived human factor IX into the blood system. Most effective factor IX expression (46 ng/ml steady-state levels of circulating human factor IX) was obtained utilizing the BKVI gene promoter, the human homologue of K10, which is expressed exclusively in differentiated keratinocytes, localized distal to the basement membrane. This report demonstrates, for the first time, that human factor IX can be efficiently synthesized and secreted from keratinocytes in situ, and can cross the epidermal basement membrane to reach the systemic circulation. The transgenic mouse model will provide a good in vivo system with which to optimize the efficiency of different keratin gene promoter constructs for delivery of therapeutic gene products to the serum, especially for those promoters, such as K10, which are not effectively expressed in vitro. PMID- 7544666 TI - Characterization of a gene transcribed in the L2 and L3 layers of the tobacco shoot apical meristem. AB - Recent genetic and molecular studies have been initiated to uncover the fundamental cellular processes unique to the shoot apical meristem. Our previous work resulted in the isolation of a cDNA clone derived from tobacco apex RNA, A3, that appeared to be transcriptionally restricted to the shoot apex regardless of developmental stage. Here the DNA sequence and in situ RNA analysis of A3 is presented. The A3 gene potentially encodes a small hydrophilic polypeptide, the sequence of which is unique to current data bases. It has been found that transcripts of the A3 gene are confined to the subepidermal and internal cell layers of the tobacco shoot apical meristem throughout development, become localized to undifferentiated floral organ primordia, and diminish as the developmental potential of the meristematic tissue becomes restricted. PMID- 7544668 TI - Evaluation of high-performance liquid chromatography for the separation and determination of arsenic species by on-line high-performance liquid chromatographic-hydride generation-atomic absorption spectrometry. AB - An on-line high-performance liquid chromatographic-microwave assisted oxidation hydride generation-atomic absorption spectrometric (HG-AAS) system (using columns of different kinds) has been developed for the determination of arsenite, arsenate, dimethylarsinate (DMA), monomethylarsonate (MMA), arsenobetaine (AsB) and arsenocholine (AsC) in environmental samples. Ion-pair reversed-phase chromatography using tetrabutylammonium phosphate as the ion-pair reagent and anion-exchange chromatography were evaluated and the analytical performances of each are reported. The detection limits were 97-143 and 10-30 micrograms l-1 for ion-pair reversed-phase and anion-exchange chromatography, respectively. The Hamilton PRP-X 100 anionic column was proposed for the determination of the six species; AsB can be quantitated independently of AsC by taking the difference between readings at pH 6 and pH 10.7. The proposed methods were applied to water samples and sediments and their potential for future application was demonstrated. PMID- 7544667 TI - [Preoperative prediction of the presence of lymph node metastasis of prostatic carcinoma: reliability and clinical significance]. AB - A total of 28 patients with clinically localized prostate cancer have undergone laparoscopic pelvic staging lymphadenectomy. In 21% of the patients pelvic lymph node metastases were diagnosed. If the Gleason score on needle biopsy was less than 6, the likelihood of lymph node metastases was 15%, whereas 50% of patients with a Gleason score of 6 or more had lymph node spreading; Whatever cutoff was used, the preoperative PSA value unreliable to predict the regional nodal status. PMID- 7544669 TI - Influence of salts on protein interactions at interfaces of amphiphilic polymers and adsorbents. AB - The protein-binding capacity of two different amphiphilic adsorbents was investigated to determine the effect of solvent additives on the binding of proteins in hydrophobic-interaction chromatography. There was no simple correlation between binding capacity and the lyotropic series such as those suggested by the two different theories proposed by Arakawa and Narhi and Melander and Horvath. Proteins are known to be dynamic flexible objects which continuously undergo changes in conformation and which may well be influenced by chaotropic salts. Are conformational changes of proteins at interfaces an important parameter involved in protein interactions with amphiphilic polymers and adsorbents? In an attempt to answer this question, the reactivity of the thiol group in human serum albumin (HSA) toward N-ethyl-3-(2 pyridyldisulfanyl)propionamide dextran was used as a model system to evaluate its correlation with the lyotropic series. The results indicate that the thiol disulfide exchange reaction at interfaces of amphiphilic polymers is influenced by the type of salt used. PMID- 7544670 TI - Octreotide, but not bromocriptine, increases circulating insulin-like growth factor binding protein 1 levels in acromegaly. AB - Twenty-three patients with active acromegaly underwent serum sampling for growth hormone (GH), insulin and insulin-like growth factor binding protein 1 (IGFBP-1) after placebo or single doses of octreotide or bromocriptine. Integrated 24-h serum GH levels decreased by 90% after octreotide and 49% after bromocriptine. A statistically significant correlation between the course of GH levels after octreotide and bromocriptine was observed (p < 0.001). Octreotide, but not bromocriptine, induced a significant increase in integrated 24-h serum IGFBP-1 levels to 37.4 times the baseline values. Bromocriptine caused a non-significant increase in integrated 24-h serum IGFBP-1 levels, which argues against a direct regulatory effect of GH on IGFBP-1 production in acromegaly. In conclusion, octreotide induces in acromegaly the production of IGFBP-1, which occurs independently of the number of somatostatin receptors on the GH-secreting pituitary adenoma. The supposed inhibitory effect of IGFBP-1 on the biological effect of IGF-1 might result in an additional clinical benefit in acromegalic patients as compared to treatment directed at the pituitary level. PMID- 7544671 TI - [The isolation and characteristics of cloned strains of Pseudomonas pseudomallei phages]. AB - On the basis of museum strains of the melioidosis agent Pseudomonas pseudomallei, 14 pure lines of bacteriophages were isolated belonging to morphological groups IV and V according to the classification by A. S. Tikhonenko. These phages show a broad bacteriolytic range as far as museum P. pseudomallei cultures are concerned. It was determined that some phages isolated from the same melioidosis culture and belonging to the same morphological type display different bacteriolytic activity. It is demonstrated that some strains of melioidosis agent are characterized by polylysogeny. PMID- 7544673 TI - Opioid availability in Latin America: the Declaration of Florianopolis. AB - This declaration was formulated by consensus of the various government agencies and other organizations involved. It is hoped that it will be widely circulated. PMID- 7544672 TI - Intestinal neuronal dysplasia. Why does it only occur in parts of Europe? PMID- 7544674 TI - Bowel obstruction in home-care cancer patients: 4 years experience. AB - Conservative management of bowel obstruction in advanced cancer patients has been recognized as efficacious in controlling distressing symptoms such as nausea, vomiting and pain. A retrospective analysis of prevalence, treatment and outcome was performed. A group of 25 patients with signs of bowel obstruction were studied from 1001 consecutive patients receiving palliative care at home. A score for total distress was calculated to assess the gastrointestinal symptoms at diagnosis, after 1 week of treatment and 1 day before death. The mean survival was 19 days. Six patients were admitted to hospital, 3 of whom underwent surgery and died in the immediate postoperative period. The patients diagnosed in hospital and discharged received a nasogastric tube (7 cases) and a central venous line. The nasogastric tube was removed after gastrointestinal secretions had been controlled by a combination of drugs, and parenteral nutrition or hydration, mainly started in hospital, was discontinued only a few days before death (13 cases, mean duration 19 days). The distress symptom score was statistically reduced after 1 week of treatment and on the day before death when compared to the referral time. The most frequent combination of drugs was octreotide, haloperidol and low doses of morphine. A subcutaneous route was preferred for administering the drugs. Utilization of a wide range of drugs permits good symptom control for bowel obstruction in patients followed at home. PMID- 7544675 TI - Spindle cell lipomas. A report of two cases: one with multiple lesions. AB - BACKGROUND: Spindle cell lipoma is an unusual and histologically distinctive form of lipoma occurring primarily in older individuals. OBJECTIVE: The dermatologic surgeon and pathologist should be familiar with this variant of lipoma. METHODS: We are reporting two cases of spindle cell lipomas with the results of histologic examination with special stains and immunohistochemical studies. RESULTS: The neoplasms consisted of lobulated masses of mature adipose tissue with areas of spindle cell proliferation. There was strong positive staining with antibodies for vimentin within the areas of spindle cells proliferation. One of the cases is unusual in that multiple lesions are occurring in a young patient. CONCLUSION: Recognition of the histologic pattern is important in distinguishing the benign spindle cell lipoma from the malignant liposarcoma. PMID- 7544676 TI - Mechanism of dextran transport across rabbit intestinal tissue and a human colon cell-line (CACO-2). AB - The in vitro permeabilities of 14C labeled dextrans (10, 40, and 70 kD) were calculated from mass transport across Peyer's patches and non-patch tissues derived from rabbit jejunum, and a human colon cell line (Caco-2) grown as a monolayer on polycarbonate filters. Size distribution of dextrans did not change upon transport as judged from size exclusion chromatography. Permeabilities decreased in a size-dependent manner. Ranking of permeabilities for dextran 10 and 40 kD were: Caco-2 > non-patch tissue > Peyer's patches; while dextran 70 kD demonstrated no difference among the barriers. Tissue resistance, expressed as 1/(permeability.tissue thickness) was virtually the same in Peyer's patches and non-patch tissue, suggesting that tissue thickness and not interaction determines the difference in permeability. ATP depletion with ouabain, Na(+)-azide and 2 deoxy-D-glucose, and low temperature (4 degrees C) did not result in reduced permeabilities suggesting passive transport. The results suggest that the investigated intestinal barriers transport dextrans in a similar fashion independent of their source. However, comparison of the ratios dextran 10 kD/mannitol and PEG 900/mannitol between rabbit tissue and Caco-2 monolayers suggests Caco-2 monolayers may serve as a model to study absorption potential of potentially harmful compounds in coeliac disease, gastroenteritis, and colon carcinoma. PMID- 7544677 TI - Retroviral-mediated transduction of the fanconi anemia C complementing (FACC) gene in two murine transplantation models. AB - Fanconi anemia (FA) is a well-known genetic syndrome manifested by bone marrow failure, variable physical anomalies, and cancer susceptibility. This disorder is marked by genotypic and phenotypic heterogeneity and consists of four distinct complementation groups A, B, C, and D. The defective gene responsible for the C group of FA, FACC, was identified by cDNA complementation cloning, and we have recently proposed a trial of gene therapy for group C FA. No animal model yet exists for FA. Consequently, we have studied the effects of constitutive expression of human FACC in two murine transplantation models. In the first model, we demonstrated transduction of FACC to reconstituting stem cells of mutant W/WV mice. In the second model, we demonstrated transduction of FACC to hematopoietic cells transplanted to the bone marrows and spleens of non myeloablated BALB/c mice. Our data suggest that retroviral-mediated transfer of the normal human FACC cDNA to hematopoietic progenitor and stem cells of mice is feasible and not associated with direct harmful effects to the hematopoietic organ. PMID- 7544681 TI - Picture story. From protoxin to pore. PMID- 7544682 TI - A sweet success. PMID- 7544680 TI - Translocation of an RNA duplex on a ribozyme. AB - RNA cleavage by the Tetrahymena ribozyme requires recognition of the reaction site helix by the catalytic apparatus. This binding can occur in several registers, each of which results in reaction at a different nucleotide in the helix. We now identify commensurate sets of 2'-hydroxyl interactions on both strands of the reaction-site helix that account for its translocation into alternative binding registers. These results indicate that the ribozyme has a relatively rigid substrate-binding pocket into which the helix can bind in different alignments. A similar mechanism of reaction site recognition is proposed to occur during intron circularization and ribozyme polymerase activity. Translocation of the reaction site duplex provides an example of structural heterogeneity in packing of helices during the tertiary folding of RNA. PMID- 7544679 TI - Study of the activation mechanism of human GRF(1-29)NH2 on rat mast cell histamine release. AB - Human growth releasing factor (GRF) (1-29)NH2 releases histamine from pleural and peritoneal rat mast cells by a non cytotoxic and non immunological mechanism. Pretreatment of cells with pertussis toxin markedly inhibits the secretion, suggesting a possible function of a Gi-protein in the activation pathway. In order to determine the role of cAMP on GRF mediated secretion, mast cells were preincubated with isobutylmethylxanthine (IBMX) or cholera toxin, since both drugs greatly and enhance cAMP levels. IBMX inhibits mediator secretion while, in contrast, cholera toxin is ineffective to modify histamine release. The PKC activator TPA amplifies the response of mast cells to human GRF, shifting the dose-response curve to the left. The pretreatment of mast cells with the phosphatase inhibitor okadaic acid exerts no effect on the dose-response function curve to GRF. The response to human GRF does not depend on extracellular calcium, but there is a good correlation between the percent of histamine released and 45calcium uptake. The kinetic of calcium uptake is fast, maximum uptake being reached in 30 seconds. PMID- 7544678 TI - The alpha isoform of protein kinase C inhibits histamine-stimulated adenylate cyclase activity in a particulate fraction of the human gastric cancer cell line HGT-1. AB - The isoform of protein kinase C responsible for the inhibition of histamine stimulated adenylate cyclase by the phorbol ester, 12-O-tetradecanoylphorbol 13 acetate (TPA), has been investigated in a particulate fraction prepared from the human gastric cancer cell line HGT-1. The alpha and epsilon isoforms of protein kinase C were detected in HGT-1 cells and in a 40,000 x g particulate fraction by immunoblotting procedures. The inhibitory effect of TPA on histamine-stimulated adenylate cyclase was enhanced by the presence of Ca2+, but decreased in a concentration-dependent manner by anti-peptide antibody to protein kinase C alpha, but not to protein kinase C epsilon. Addition of Ca2+ and TPA to the 40,000 x g particulate fraction stimulated the phosphorylation of the protein kinase C substrate myelin basic peptide 4-14. Protein kinase C alpha is probably the isoform responsible for inhibition of histamine-stimulated adenylate cyclase in HGT-1 cells. PMID- 7544683 TI - Proteinase inhibitor homologues as potassium channel blockers. AB - We report here the NMR structure of dendrotoxin I, a powerful potassium channel blocker from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), calculated from an experimentally-derived set of 719 geometric restraints. The backbone of the toxin superimposes on bovine pancreatic trypsin inhibitor (BPTI) with a root-mean-square deviation of < 1.7 A. The surface electrostatic potential calculated for dendrotoxin I and BPTI, reveal an important difference which might account for the differences in function of the two proteins. These proteins may provide examples of adaptation for specific and diverse biological functions while at the same time maintaining the overall three dimensional structure of a common ancestor. PMID- 7544684 TI - Effects of hypertonic saline dextran on the postoperative evolution of Jehovah's Witness patients submitted to cardiac surgery with cardiopulmonary bypass. AB - Hypertonic saline-dextran (HSD) solutions have been used for hemorrhagic shock, aortic aneurysm, and cardiopulmonary bypass surgery (CPB). Jehovah's Witness patients refuse blood and derivatives even under life-threatening conditions. A negative fluid balance for Jehovah's Witnesses would avoid further hemodilution. In this study we compared clinical, hemodynamic, laboratory evolution, and fluid balance of 20 Jehovah's Witnesses over the first 72 h following CPB. Ten received HSD immediately prior to CPB. All patients survived and were maintained in stable hemodynamic and metabolic condition throughout the study period. HSD induced high cardiac output, low vascular resistance immediately after administration. Vascular resistance remained low until the end of CPB. HSD patients ran a slightly negative fluid balance, while control patients ran a large positive fluid balance. HSD pretreatment is now used routinely for Jehovah's Witnesses undergoing CPB in our facility. PMID- 7544685 TI - Sterility of repackaged filgrastim and sargramostim. PMID- 7544687 TI - In vitro evaluation of Pyrularia thionin-anti-CD5 immunotoxin. AB - The membrane-active peptide, Pyrularia thionin, purified from Pyrularia pubera, was covalently conjugated to an anti-CD5 monoclonal antibody. The membrane-active properties of thionin were not affected by the conjugation. The immunotoxin killed CD5+ lymphocytes in vitro at a concentration of 0.1 nmol/10(7) cells after 2 h of incubation. The immunotoxin also inhibited the proliferation of T cells in vitro, stimulated either by mitogens or in the mixed lymphocyte reaction. It was shown by electron paramagnetic resonance of spin probes and differential scanning calorimetry that the ability of the immunotoxin to perturb the lipid phase of membranes is close to that of unconjugated thionin. The results obtained suggest that Pyrularia-thionin-anti-CD5 conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5+ leukemia and lymphomas. PMID- 7544686 TI - Triiodothyronine decreases the accumulation of 1,2-diacylglycerol in rat hearts. AB - 1,2-Diacylglycerol (DAG) is an intracellular signal mediator that may initiate protein synthesis and cardiac hypertrophy via activation of protein kinase C. The amounts of 1,2-DAG and its fatty acid components in the myocardium was assessed and compared with the concentrations of RNA and DNA in cardiac hypertrophy induced by administering triiodothyronine (T3) with and without cycloheximide, an inhibitor of protein synthesis. After the first injection of T3 no cardiac hypertrophy was observed, and there were no differences in myocardial 1,2-DAG content or in RNA and DNA concentrations. Cardiac hypertrophy was present on days 3 and 7 after a daily injection of T3. Myocardial RNA concentration was increased by 26% on day 3 and by 34% on day 7, whereas the myocardial 1,2-DAG content was decreased by 8% on day 3 and by 24% on day 7. Pretreatment with cycloheximide of T3-treated rats prevented the development of cardiac hypertrophy, but elevated the RNA concentration and lowered the 1,2-DAG content compared with the findings in T3-treated rats. Analysis of the fatty acid components of 1,2-DAG revealed that the amounts of 16:0, 18:1 and 18:2 were decreased, accompanied by an elevation of RNA concentration and a decrease in 1,2-DAG content. It seems that RNA synthesis preceded the alteration in 1,2-DAG. These findings suggest that 1,2 DAG is not involved in the initiation of cardiac hypertrophy induced by T3, but is affected by the enhanced concentration of RNA resulting from the introduction of thyroid hormones into the myocardium. PMID- 7544688 TI - Neutrophil activation and adhesion molecule expression in a canine model of open heart surgery with cardiopulmonary bypass. AB - OBJECTIVE: The aim was to determine whether, in a canine model, changes in surface expression of the neutrophil adhesion molecules CD11b/CD18 and L-selectin during and after open heart surgery with cardiopulmonary bypass can be used to identify subjects at risk for postoperative pulmonary dysfunction. METHODS: Adult mixed breed dogs underwent cardiopulmonary bypass and were compared to "sham bypass" controls. Flow cytometry was performed on blood from the two groups of dogs and changes in CD11b/CD18 adhesion molecules and L-selectin were investigated. RESULTS: Flow cytometry on blood from bypass dogs showed increased CD18 expression during and after cardiopulmonary bypass and a reciprocal decrease in L-selectin expression. Sham animals showed no significant change. In the bypass animals, changes in adhesion molecule expression were not evenly distributed across the population of circulating neutrophils; however, they were indicative of a percentage of activated cells. There was a significant negative linear relationship between the percentage of activated cells and arterial oxygenation 3 h after bypass (r = -0.80, P < 0.001). From this analysis, 11 animals were identified as "high" responders and seven as "low" responders, with different patterns of cellular activation and oxygenation during and after bypass. High responders had an average of 40(SEM 5)% activated cells during bypass with a persistently raised percentage of activated cells [38(3)%] 3 h later, whereas low responders had only 22(6)% activated cells during bypass and 11(2)% activated cells 3 h after bypass. High responder animals had a marked and continued deterioration in PO2 after bypass [to 25(6)% of baseline 3 h after bypass] whereas low responder animals showed recovery of oxygenation after the first hour postbypass and improved to 80(8)% of baseline at 3 h. CONCLUSIONS: Changes in adhesion molecule expression serve as a marker of neutrophil activation during cardiopulmonary bypass. The percentage of activated neutrophils in the circulation within 3 h after cardiopulmonary bypass may be predictive of an ongoing inflammatory process that is linked to pulmonary dysfunction. PMID- 7544690 TI - Effects of nitric oxide synthase inhibition on myocardial capillary permeability and reactive hyperaemic response. AB - OBJECTIVE: The aim was to examine the role of nitric oxide (NO) in the myocardial microcirculation. METHODS: Open chest anaesthetised dogs received intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA; 0.5 mg.kg-1 over 30 min). Myocardial microvascular extraction fraction of a small hydrophilic solute (technetium 99m labelled diethylenetriaminepenta-acetate [99mTc-DTPA]), and the regional myocardial plasma flow rate, were determined by the single injection, residue detection method, and the capillary permeability-surface area product was calculated. The tone in intramyocardial resistance vessels was assessed by the local 133Xe washout method during baseline conditions, in response to intracoronary acetylcholine, and during peak reactive hyperaemia after 10 s or 30 s of myocardial ischaemia, respectively. RESULTS: In eight open chest dogs, L NMMA attenuated the increase in myocardial plasma flow rate in response to intracoronary acetylcholine by 32(SEM 7)%, but failed to alter baseline myocardial plasma flow rate significantly, as determined by the local 133Xe washout method. L-NMMA did not influence myocardial microvascular permeability to 99mTc-DTPA. However, intracoronary L-NMMA decreased the peak reactive hyperaemic myocardial plasma flow rate after 10 s, but not 30 s, of coronary occlusion. CONCLUSIONS: In open chest dogs, microvascular NO synthesis is not a major determinant of baseline myocardial plasma flow rate, and does not appear to influence myocardial microvascular permeability significantly. In this model, NO intervenes in the regulation of the peak reactive hyperaemic plasma flow rate following brief, but not more prolonged, periods of coronary occlusion. PMID- 7544693 TI - Antibodies against leukocyte function-associated antigen-1 and against intercellular adhesion molecule-1 together suppress the progression of experimental allergic encephalomyelitis. AB - We obtained the evidence that coadministration in vivo of mAbs against leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM 1) suppressed the progression of experimental allergic encephalomyelitis (EAE) in rats. The suppressive effect in vivo of coadministration of the mAbs during the induction phase was not prominent, but the administration of these mAbs during the effector phase markedly suppressed the progression of clinical illness and prevented the infiltration of encephalitogenic cells into the central nervous system. However, administration of the mAb to LFA-1 alone or ICAM-1 alone did not show such suppressive effects. These findings suggest that LFA-1 and ICAM-1 are critically involved in the development of EAE and that the administration together of mAbs against adhesion molecules including LFA-1 and ICAM-1 might provide a new immunotherapeutic approach for the treatment of multiple sclerosis. PMID- 7544691 TI - A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1. AB - Lipopolysaccharide (LPS) triggers a wide range of cellular responses in mammalian cells. Several proteins, including CD14, have been reported to possess LPS binding capacity. However, the signal transduction molecule(s) and pathway(s) through which LPS induces cellular responses have not been identified. A rabbit antiserum (5299), which had been made against the N-terminal 20-amino-acid sequence of the cytokine, soluble immune response suppressor, unexpectedly activated the human monocyte cell line THP-1 as assessed by tumor necrosis factor (TNF alpha) production. Normal rabbit serum or IgG caused no TNF production, while antiserum 5299 induced TNF at a 1:10(4) dilution. The activity of antiserum 5299 was not due to endotoxin contamination, since antiserum 5299 contained less endotoxin than required for activation of THP-1 and the TNF-inducing activity disappeared when the antiserum was boiled. Moreover, the activity was recovered in purified immunoglobulin G fraction from the antiserum (5299 IgG). These results suggest that antiserum 5299 contains an antibody which recognizes a signal-transducing molecule. THP-1 desensitized by incubation with LPS for 24 hr could not respond to LPS or antiserum 5299 while responsiveness to phorbol ester remained, suggesting that the signals induced by the antiserum 5299 and LPS are transduced through at least a partially common pathway. Western blotting with antiserum 5299 showed a 69-kDa protein which rapidly appeared in Triton X-100 insoluble fraction after stimulation with LPS or 5299 IgG, suggesting an association of 69-kDa protein with the cytoskeleton. Furthermore, two mutant cell lines were isolated (T-15 and T-25) from gamma-irradiated THP-1 which lack responsiveness to LPS. In these mutant cell lines, the 69-kDa protein was not observed in Triton X-100-insoluble fraction after stimulation with LPS. It is proposed that this protein may play an important role in LPS-stimulated signal transduction. PMID- 7544694 TI - Loss of L-selectin (CD62L) on human neutrophils following exudation in vivo. AB - L-selectin, an adhesion molecule expressed on the surface of peripheral blood neutrophils, mediates the rolling of neutrophils along vascular endothelium. Stimulation of neutrophils in vitro causes decreased expression of L-selectin on the surface of neutrophils due to shedding. In this study, we have demonstrated that human exudative neutrophils isolated from both skin lesions and from pus exhibit little expression of L-selectin. Although a dramatic accumulation of exudative neutrophils is observed in the skin lesions within 24 hr, there is no accompanying increase in soluble L-selectin. In addition, the levels of soluble L selectin in the extracellular tissue fluid (97.2 +/- 12.7 micrograms/ml, n = 8) are only 20% of that observed in peripheral plasma (482.4 +/- 35.9 micrograms/ml). These data suggest that shedding of L-selectin from the surface of neutrophils occurs in the peripheral circulation as a prelude to diapedesis of neutrophils into peripheral tissues. PMID- 7544689 TI - Nitric oxide synthesis in cardiac myocytes and fibroblasts by inflammatory cytokines. AB - OBJECTIVE: The aim was to investigate nitric oxide (NO) synthase activity in cultured neonatal rat cardiac myocytes and fibroblasts upon treatment with inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), IL-2, IL-6, IL-8, transforming growth factor beta (TGF-beta) and gram negative bacterial lipopolysaccharide (LPS). METHODS: NO and guanosine 3',5'-cyclic monophosphate (cGMP) synthesis was measured in cultured neonatal rat cardiac myocytes and fibroblasts, using Griess reagent and an enzyme immunoassay kit, respectively. The expression of inducible NO synthase (iNOS) mRNA and protein was assayed by northern and western blotting, respectively. RESULTS: Incubation of cardiac myocytes for 24 h with IL-1 beta (10 ng.ml-1) or LPS (1 microgram.ml-1) caused significant increases in NO and cGMP production. TNF alpha, IL-2, IL-6, IL-8, and TGF-beta showed no significant effect on their production. IL-1 beta induced NO and cGMP production in a time and dose dependent manner. IL-1 beta also increased iNOS mRNA and protein accumulation in cardiac myocytes. Simultaneous incubation of IL-1 beta with NG-monomethyl-L-arginine, genistein, calphostin C, cycloheximide, or actinomycin D completely inhibited the IL-1 beta induced NO production by cardiac myocytes. TGF-beta, dexamethasone, or cyclosporin A also dose dependently inhibited the IL-1 beta induced NO production. Exposure to IL-1 beta for 12-24 h decreased the beating rate of cardiac myocytes, but addition of dexamethasone completely overcame this inhibition. In contrast to cardiac myocytes, incubation of cardiac fibroblasts for 24 h with IL-1 beta or LPS showed no significant effect on NO or cGMP production. CONCLUSIONS: These observations suggest that IL-1 beta/LPS responsive iNOS, which is an important regulator of contractile function of the heart, is present in cardiac myocytes but not in cardiac fibroblasts. PMID- 7544692 TI - Mechanisms of enhancement of natural killer activity by an antibody to CD44: increase in conjugate formation and release of tumor necrosis factor alpha. AB - In this report, we found that an anti-CD44 mAb, S5, augmented conjugated formation between the effector cells (canine PBMC) and an NK-sensitive target cell line [canine thyroid adenoma carcinoma cell line (CTAC)], as determined by two different methods: conjugate enumeration using fluorescence microscopy and two-color flow cytometric analysis. However, no increase in conjugate formation was seen when an NK-resistant target was used. This enhancement of initial conjugate formation could not be blocked using an antibody to CD18 (60.3), indicating that S5 possibly acted through another adhesive molecule(s). But the NK killing activity can be partially blocked by 60.3, indicating that this molecule may be important for later adhesive events. We also found that S5 stimulated the release of a heat labile cytotoxic factor from canine PBMC. This factor was found to lyse only TNF alpha-sensitive targets (CTAC and L929 cell lines) and caused apoptosis in the target cells. Its bioactivity was neutralized by polyclonal antibody to TNF alpha. All of these observations were consistent with the fact that the factor was TNF alpha. Our data suggested that the two mechanisms responsible for the enhancement in canine NK activity by an mAb to CD44 were an increase in conjugate formation and the release of TNF alpha. PMID- 7544696 TI - [Isolation, purification and characterization of human proteinase inhibitor, alpha 2-macroglobulin]. AB - A study of the isolation, purification and characterization of human proteinase inhibitor, alpha 2-macroglobulin (alpha 2-M) is presented. The alpha 2-M, with a purity of more than 90% and subunit molecular weight of 185kDa, was isolated by ammonium sulfate fractionated precipitation from human blood plasma and purified by DEAE cellulose DE52 column chromatography. It was found to be the same as the standard counterpart provided by sigma company. This purification procedure of alpha 2-M can be done with low cost, high efficiency and in large scale preparation. PMID- 7544697 TI - [Establishment and biological characteristics of the nude mice xenograft model from human hepatocellular carcinoma]. AB - The human hepatocellular carcinoma was successfully heterotrasplanted into nude mice and 10 passages were made seriously during 11 months. Initial take rate was 14.3% and then the take rate increased to 100% after the 3rd passage. Histological, morphological, ultrastructural and biological investigation and chromosome analysis showed that the characteristic of transplanted tumor was identical to that of characteristics of human donor tumor. Through serial passages no metastatic signs were found grossly and under the microscope in the lymph nodes, liver and lung of the nude mice. Result also showed that the model retained some functions of the original tumor. The transplanted tumor could secret a-fetoprotein (AFP), r-glufamyl transpeptidase (r-GT). Acidic isoferritin (AIF) also could be detected. The tumor tissue of xenograft model was cultured in vitro in order to develop a permanent cell line. We found that the tumor specimen of the later passages grew more rapidly than the earlier ones. Up to now 3 cell passages were made. In this paper, a reliable animal model of human hepatocellular carcinoma is provided for studies of clinical diagnosis, treatment, antitumor drug screen test, etc. PMID- 7544695 TI - [Expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 in kidney of patients with lupus nephritis and membranoproliferative glomerulonephritis]. AB - Immunohistochemical and computer-imaging analysis techniques were used to detect the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in the kidney of 25 patients with lupus nephritis (LN) type IV and in 15 patients with membranoproliferative glomerulonephritis (MPGN) type I. Results showed that ICAM-1 and VCAM-1 expressions were increased significantly in the kidneys of patients with MPGN and LN and that ICAM-1 in the glomeruli of LN patients correlated well with glomerular endothelial cell proliferation. ICAM-1 and VCAM-1 may well take a role in the pathogenesis of LN and MPGN. PMID- 7544699 TI - Keratan sulphate is a marker of differentiation of ramified microglia. AB - Recently we reported that the keratan sulphate epitope recognised by the monoclonal antibody 5D4 is expressed by a population of ramified microglia in adult rats. As ramified microglia is believed to differentiate from ameboid microglia during postnatal development, we studied the rat brain from birth to 90 postnatal days of life with the monoclonal antibody 5D4. Contrary to all the other microglia markers until now described, keratan sulphate is not expressed by ameboid microglia and by macrophages but appears on the surface of microglia only when the cells are differentiated and show ramified processes. The keratan sulphate positive cells become evident at different times in different central nervous system areas; the first were localised in the pyriform cortex and brainstem from the end of the second postnatal week. These observations suggest that keratan sulphate expression on microglia cells is induced by differentiation and by a resting functional state. Moreover the 5D4 monoclonal antibody showed a strong diffuse positive staining of some cortical, thalamic and white matter areas during the first two postnatal weeks. This staining was transient and it does not seem biologically correlated with the expression of the keratan sulphate on differentiated microglia. PMID- 7544698 TI - Migration of dopaminergic neurons in the embryonic mesencephalon of mice. AB - Migration of dopamine (DA)-containing neurons and its guiding cues were histologically examined in the embryonic mesencephalon of normal mice. Cells immunoreactive (ir) for tyrosine hydroxylase (TH), a DA-synthesizing enzyme, were first detected on embryonic day 10 (E10) in the medio-basal part of the mesencephalon and were distributed throughout the entire length of the ventral mesencephalic wall at E12. By E14, TH-ir cells were located laterally along the ventral pial surface to form the primordia of the substantia nigra. Experiments with a single injection of bromodeoxyuridine, a thymidine analog, demonstrated that cells generated in the ventricular surface of the ventral mesencephalon at E11 migrated ventrally and then moved laterally to form the substantia nigra and the ventral tegmental area. Electron microscopic examination of the ventral mesencephalon of E12 mice disclosed that in the dorsal part ventrally migrating immature neurons made close contacts with the processes of radial glial cells. The expression of tenascin was transiently seen on radial glial processes between E10 and E13 coincident with the period of the ventral migration of mesencephalic DA neurons. By double immunostaining of E13 mesencephalon, ventrally migrating TH ir cells were seen to be apposed to tenascin-bearing radial glial processes. On the other hand, laterally migrating neurons in the basal part of the mesencephalon were observed by electron microscopy to contact with tangentially arranged nerve fibers which were immunopositive for the 160 kDa neurofilament polypeptide at the light microscopic level from E10. Double immunostaining of E13 mesencephalon demonstrated that laterally migrating TH-ir cells were intermingled among neurofilament-ir fiber bundles. The cells of origin of the tangential nerve fibers were detected in the lateral part of the mesencephalon, when a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) was injected into the basal part of the mesencephalon of fixed E12 mice. The present results suggest that guiding cues of the radial migration of mesencephalic DA neurons represent processes of radial glial cells which express tenascin. On the other hand, tangentially arranged nerve fibers originating from the lateral part of the mesencephalon may provide a scaffolding along which the mesencephalic DA neurons subsequently migrate laterally to form the ventral tegmental area and the substantia nigra. PMID- 7544700 TI - Expression of acidic FGF mRNA in rat auditory brainstem during postnatal maturation. AB - In situ hybridization was used to investigate the mRNA distribution of acidic and basic fibroblast growth factor (aFGF and bFGF) in the auditory brainstem of neonatal and adult rats. bFGF mRNA was not detected at any age. In adult rats, aFGF mRNA was strongly expressed in the principal neurons of the anteroventral and posteroventral cochlear nuclei, but not in the octopus cells. In the dorsal cochlear nucleus, aFGF mRNA was seen only in scattered smaller vertical cells. aFGF was strongly expressed in the nucleus of the trapezoid body and in all periolivary cell groups, but not in the medial and lateral olivary nuclei. No expression was observed in the lemniscal nuclei or in the central nucleus of the inferior colliculus, but large neurons in the external zone of the colliculus were labeled. Developmentally, low levels of aFGF expression appeared in the cochlear nuclei and olivary nuclei between P0 and P6. This expression increased rapidly during the onset of hearing, between P10 and P14, and reached adult level by P14-P17. Labeling in collicular neurons appeared slightly later. The results suggest that the appearance of strong aFGF mRNA expression is related to the onset of function. PMID- 7544701 TI - The expression, origin and function of tenascin during peripheral nerve formation in the chick. AB - During development of the peripheral nervous system, the extracellular matrix molecule tenascin has been found to be closely associated with growing axons. However, its origin and function in peripheral nerve formation are far from clear. In this study, we examined the expression of tenascin during outgrowth of sensory, motor and sympathetic preganglionic axons, and assessed its origin and function in peripheral nerve formation. During outgrowth of sensory and motor axons, a high concentration of tenascin and its mRNA was found to surround sensory and motor axons in the newly formed spinal nerves. The source of this tenascin was examined through a series of surgical manipulations. Neural crest removals did not alter the distribution of tenascin protein or its mRNA surrounding the spinal nerves. Transplantation of quail somites into chick embryos showed that, similar to the distribution of tenascin, there is a high concentration of somitic cells surrounding the spinal nerves. Moreover, somite removals resulted in a reduction of the tenascin and tenascin mRNA surrounding the spinal nerves. Taken together, these results suggest that the majority of the tenascin surrounding the spinal nerves is of somitic origin. Possible functions of tenascin associated with peripheral nerve formation were examined through injections of tenascin or its antiserum into individual somites prior to or during axon outgrowth. Injections of tenascin or its antiserum did not alter the trajectory of peripheral axons in the anterior half of the somite, nor produce gross abnormalities in the morphology of peripheral nerves, suggesting that tenascin does not play a crucial role in the early formation of peripheral nerves. PMID- 7544703 TI - Development of a crossed corticotectal pathway following cerebral hemispherectomy in cats: a quantitative study of the projecting neurons. AB - A hypothetical mechanism for the partial sparing of visual function in the contralateral visual field following cerebral hemispherectomy early in life is the formation of a new corticotectal pathway arising from the remaining primary visual cortex (areas 17 and 18) that projects to the contralateral superior colliculus. To test this hypothesis, the left superior colliculus of intact adult and neonatal (5-15 days old) cats and of adult cats with a left cerebral hemispherectomy sustained neonatally (7-9 days old) or in adulthood, was injected with WGA-HRP and the brains were processed for combined TMB/DAB histochemistry. The primary visual cortex was examined, labelled neurons were counted and the cross sectional area of their somata was measured. The left primary visual cortex of intact adult animals exhibited a mean of 959.68 labelled cells +/- 406.5 (S.E.), with a mean soma size of 366.7 microns2 +/- 131.2. For the neonatal intact cats, there was a mean of 75.31 +/- 21.08 cells within the left primary visual cortex which exhibited a mean soma size of 249.56 microns2 +/- 68.18. The peak cell size distribution for both intact groups was similar at 300 microns2. Virtually no labelled neurons were detected in the right primary visual cortex of intact animals (neonatal or adult). For neonatal-hemispherectomized cats, the remaining right primary visual cortex exhibited a mean cell count of 351.09 +/- 126.3 cells, with a mean soma size of 436.1 microns2 +/- 131.5, and a peak cell size distribution of 400 microns2. Finally, for adult-hemispherectomized animals, the contralateral primary visual cortex exhibited 68.27 +/- 20.13 neurons having a mean soma size of 486.6 microns2 +/- 143.2 with a peak cell size distribution of 500 microns2. These results indicate that reorganization of the corticotectal pathway occurs in both adult- and neonatal-hemispherectomized cats but is more pronounced in neonatal-lesioned animals. In addition, the cells of origin of this reorganized pathway tended to be larger, perhaps in response to a greater axonal arborization. PMID- 7544704 TI - Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays. AB - We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1 antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays. PMID- 7544707 TI - Stability of FK506 (tacrolimus) in whole-blood specimens. PMID- 7544708 TI - Novel nonseparation sandwich-type electrochemical enzyme immunoassay system for detecting marker proteins in undiluted blood. AB - A novel nonseparation electrochemical enzyme immunoassay (NEEIA) for detecting marker proteins in undiluted blood is described. The approach is based on preferential electrochemical measurement of surface-bound enzyme-labeled reporter antibody (E-Ab), relative to an excess of this reagent in the sample solution. NEEIAs are carried out on microporous membranes coated with a thin, circular area of gold. The gold serves simultaneously as a working electrode and solid phase for immobilized capture anti-protein antibodies. In the assay, analyte protein is incubated concurrently with the Ab-coated gold surface and excess E-Ab conjugate. Detection of bound E-Ab is achieved by introducing the substrate for the enzyme through the back side of the membrane. The product of bound E-Ab is detected immediately by oxidation or reduction at the gold electrode, and the resulting current is proportional to the concentration of protein in the sample. The feasibility of the NEEIA approach is demonstrated via the detection of prostate specific antigen in undiluted plasma samples (n = 64), with alkaline phosphatase as the label. Use of multiple gold films deposited on the same porous membrane to perform simultaneous NEEIAs is also described. PMID- 7544705 TI - HPLC-microparticle enzyme immunoassay specific for tacrolimus in whole blood of hepatic and renal transplant patients. AB - Tacrolimus is a relatively new immunosuppressant used in organ transplantation to prevent graft rejection. However, its use is not devoid of side effects, making it important to maintain blood concentrations within therapeutic ranges. Several analytical methods are currently available for routine drug monitoring. However, these methods are based on use of the same monoclonal antibody, which also cross reacts with some metabolites, resulting in overestimation of some blood concentrations. Even though this antibody appears appropriate for therapeutic drug monitoring, no reference method measures only the parent drug, mainly because of the poor absorptivity of tacrolimus in ultraviolet light. We have developed a method displaying an increased specificity towards the unchanged drug, using conventional equipment available in most clinical laboratories. After chromatographic separation of the blood extract, the tacrolimus fraction is analyzed by an automated microparticle enzyme immunoassay (MEIA) performed on the IMx analyzer (Abbott Labs.). This method is linear from 0 to 40 micrograms/L, yields CVs from 8.5% to 18.2%, and has a detection limit of 5 micrograms/L. Tacrolimus concentrations obtained by HPLC-MEIA in hepatic and renal transplant patients are from 47.5% to 18.8% lower than those obtained by MEIA, according to liver function tests and metabolite accumulation, even though no significant differences were observed between the methods for drug-free blood samples supplemented with known amounts of tacrolimus. PMID- 7544710 TI - Relationship between anti-phospholipid and anti-endothelial cell antibodies III: beta 2 glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules. AB - OBJECTIVE: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome. METHODS: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied. RESULTS: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1). CONCLUSIONS: These findings indicate that the close association between anti-endothelial and anti phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation. PMID- 7544709 TI - Distribution of neuropeptide-containing nerve fibers in the synovium and adjacent bone of the rat knee joint. AB - OBJECTIVE: The innervation of normal rat synovium and adjacent bone, especially the communicatory nerve fibers between them, were investigated. METHODS: Nerve fibers were visualized using immunohistochemistry. Antisera to substance P(SP) and antisera to calcitonin gene-related peptide (CGRP) were employed to identify sensory nerves, while antisera to neuropeptide Y (NPY) were used to identify sympathetic nerves. RESULTS: In the synovium, SP-positive nerve fibers were found in the lining cell layer, with some fibers branching toward the joint space. In the sublining layer of the synovium, SP-positive and CGRP-positive fibers were observed mainly near the blood vessels. NPY-positive fibers were predominantly seen as a network around the blood vessels in the sublining layer. A large number of SP-positive and CGRP-positive fibers were seen near the attachment of the meniscus to the synovium. Toluidine blue positive mast cells were detected in close proximity to all three types of fibers, especially near the blood vessels. In bones forming the knee joint, nerve fibers immunoreactive to SP, CGRP and NPY were present in the bone marrow and some of these fibers communicated with the synovium through the synovium-bone junction located in the bare area. CONCLUSION: The role of nerves, especially the role of the communicatory nerves between the synovium and the bone marrow, should be considered in the pathophysiolosy of erosive arthritis. PMID- 7544712 TI - The effect of storage temperature on the stability of frozen erythrocytes. AB - A systematic study on the stability of frozen erythrocytes was performed. Washed and concentrated erythrocytes were mixed with an equal volume of cryoprotective solution containing 24% (w/w) hydroxyethyl starch (HES) and 60 mmol/liter NaCl according to an established protocol. Volumes of 250 microliters of this mixture were filled into polypropylene tubes and cooled to -196 degrees C with a rate of 293 degrees C/min by immersion in liquid nitrogen. The storage temperature was then varied from -10 to -75 degrees C and could be identified as the predominant factor influencing hemolysis kinetics. The effect of storage temperature on the frozen erythrocytes after thawing was evaluated by measuring the hemolysis in a dilute, isotonic NaCl solution (saline stability). A strong time dependence was found within the temperature range studied and could be described by an exponential kinetic law. A stability prediction was made for storage temperatures lower than those examined. Temperature ranges of qualitatively different hemolysis kinetics were identified and compared to devitrification behavior of intra-and extracellular solutions. The intracellular solution was simulated by a concentrated mixture of dried erythrocytes and water. The devitrification behavior was studied using DSC techniques. A rapidly frozen mixture was annealed at selected temperatures which fall into the range of storage temperatures for frozen erythrocytes. This paper tentatively interprets the devitrification data with respect to the means for cell damage during storage. The results are reviewed with respect to the design of a safe storage procedure. PMID- 7544702 TI - Sustained inhibition of acetylcholinesterase activity does not disrupt early geniculocortical ingrowth to developing rat visual cortex. AB - Esterase activity of endogenous transiently expressed acetylcholinesterase was locally suppressed in visual cortex of infant rats for 2-5 days by the irreversible inhibitor phospholine iodide, delivered from Elvax implants. Tissue processed for anterograde movement of the carbocyanine dye DiI or anterograde transneuronal transport of wheat germ agglutinin-horseradish peroxidase revealed normal geniculocortical growth into layer IV of visual cortex. These results suggest that the catalytic activity of transiently expressed acetylcholinesterase may play little, if any, role in early development of thalamocortical systems. PMID- 7544706 TI - Interferences by cephalosporin antibiotics in urinary tests for metabolic disorders. PMID- 7544714 TI - Treatment of IPF. What does the future hold? PMID- 7544711 TI - Hepatitis C virus antibodies and systemic lupus erythematosus. PMID- 7544713 TI - The triphasic volume response of human red cells in low ionic strength media: demonstration of a special bicarbonate transport. AB - When human red cells are first placed in isotonic media of low ionic strength there is a triphasic volume change. The initial shrinkage (phase A) starting immediately is followed by a rapid reflation (phase B). The final shrinkage (phase C) is slower and has more variable properties. The first two phases need Cl- and HCO3- and they have been shown to involve both the band 3 anion exchanger and the red cell carbonic anhydrase. Phase C is only seen when phase B is present. Both phase B and phase C were accelerated by the lower monohydric alcohols. With methanol the acceleration was maximal near 2M, thereafter inhibition developed. The hypothesis is advanced that phase C involves a permeability for bicarbonate which is independent of the band 3 anion exchanger, and which may be a dimer of carbonic anhydrase. The unique kinetics, the physiological significance, and implications of the special bicarbonate transport system are discussed. PMID- 7544715 TI - High amylase levels in pleural effusion. PMID- 7544716 TI - [Transrectal hyperthermia in the treatment of benign prostatic hyperplasia]. AB - A group of 53 patients with benign prostatic hyperplasia underwent transrectal hyperthermia using Prostathermer (Biodan) from September 1992 to March 1993. 41 of the 53 patients were followed up for 6 months. The subjective symptoms improved in 75.6% (31/41) of the treated patients. The mean maximum urine flow rate increased by 3.1 +/- 2.6 ml/s. The mean residual urine reduced 38.4 +/- 25.2 ml. The mean prostate volume remained almost unchanged. The results showed that transrectal hyperthermia of the prostate gland is as effective as transurethral approach for the treatment of benign prostatic hyperplasia. PMID- 7544717 TI - Leu-7 immunoreactivity in cytologic specimens of thyroid lesions, with emphasis on follicular neoplasms. AB - Anti-Leu-7 (HNK-1, CD 57) antibody, a marker for natural killer lymphocytes, was employed by Ghali et al. (Hum Pathol 1992;23: 21-25) to study surgically resected formalin-fixed, paraffin-embedded thyroid lesions. They demonstrated strong immunoreactivity of this antibody with thyroid carcinomas, both follicular and papillary, and only occasional weak immunoreactivity with colloid goiters and follicular adenomas. We studied cytologic specimens (primarily fine-needle aspiration biopsy specimens) from 44 thyroid lesions, including 10 follicular carcinomas, 14 follicular adenomas, seven adenomatous nodules, six papillary carcinomas, and seven cases of Hashimoto's thyroiditis. All follicular carcinomas exhibited immunoreactivity to anti-Leu-7 antibody, usually of a moderate to strong degree (9/10); however, six of 14 follicular adenomas yielded similar results. The patterns of immunoreactivity in the other lesions were similar to those previously described (Ghali et al., Hum Pathol 1992;23:21-25). It does not appear that anti-Leu-7 antibody can be used as a specific marker of malignancy in the cytologic assessment of follicular neoplasms of the thyroid. PMID- 7544718 TI - Serous effusion cytology in gynecologic malignant mixed mullerian tumors. AB - We reviewed 51 serous effusions (50 peritoneal/one pleural) from 38 patients with uterine (30 cases) and ovarian (eight cases) malignant mixed Mullerian tumors (MMMT). There were 16 patients (42%) with positive effusion cytology specimens; 13 cases (81%) were diagnosed as adenocarcinoma with three cases (19%) interpreted as having a sarcomatous component. Eight of 16 positive effusion specimens had cell block material available for immunoperoxidase (IP) study that included cytokeratin (AE1/3), vimentin, muscle specific actin (HHF) and S-100 protein to determine if unsuspected mesenchymal components were present in the cases originally diagnosed as carcinoma (six cases), or sarcomas (two cases). In the six cases originally interpreted as carcinoma, three were diagnosed as adenocarcinoma and three as poorly differentiated carcinoma. All three of the cases considered adenocarcinoma and two of those diagnosed as poorly differentiated carcinoma reacted only with AE1/3 and vimentin. The remaining case, considered a poorly differentiated carcinoma, stained only with vimentin. In the two cases having cell blocks interpreted as having a sarcomatous component, only vimentin was positive in one while AE1/3, vimentin, HHF, and S 100 were positive in the other. The case where all immunohistochemical stains were reactive contained both carcinomatous and sarcomatous components. In the three cases considered sarcomatous, the cytomorphologic features helpful in the recognition of a mesenchymal component included a dissociated smear pattern of pleomorphic round to oval cells and/or spindle cells. In retrospect, the IP stains did not alter any of the original diagnoses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544721 TI - Molluscum contagiosum presenting as cellulitis in an AIDS patient: cytologic and ultrastructural features. AB - This report describes the cytologic features of molluscum contagiosum presenting clinically as a left thigh abscess in a 29-yr-old male with AIDS. The fine-needle aspiration findings are correlated with the histologic and ultrastructural findings. Molluscum contagiosum's clinical presentation, natural course, and induction of host immune responses in immunocompetent and immunosuppressed individuals as identified by light microscopy, immunocytochemically and ultrastructurally are reviewed. Molluscum contagiosum presenting clinically and cytologically as an abscess is unusual. In this report, we emphasize the inclusion of molluscum contagiosum in a differential diagnosis of an abscess, particularly in immunosuppressed patients, and discuss the cytologic differential diagnoses. PMID- 7544720 TI - Fine-needle aspiration cytology of seminoma: a review of 16 cases. AB - We report the cytomorphologic features of 16 fine-needle aspiration (FNA) biopsies of seminoma obtained from 16 male patients. The aspirates included two primary gonadal tumors (one occurring in a cryptorchid testis), two primary mediastinal tumors, and 12 metastases (two with unknown primaries). Analysis of the aspirates revealed a primarily dispersed cell population of large cells with scant to moderately abundant cytoplasm. The nuclei were round to slightly irregular, had finely granular chromatin, and had either one central prominent nucleolus or two to three smaller nucleoli. Variable numbers of lymphocytes and plasma cells were intermingled with the tumor cells. Only a few cases had epithelioid histiocytes or the characteristic "tigroid" background. The cytologic features of the metastases were distinctive and were considered diagnostic for therapeutic management. In six cases, an initial diagnosis of seminoma by FNA biopsy identified the neoplasm as germ cell in origin rather than other neoplasms in the differential diagnosis, thereby expediting therapeutic management. PMID- 7544722 TI - Fine-needle aspiration cytology of carcinosarcoma of the parotid gland: cytohistological and immunohistochemical findings. AB - Carcinosarcoma or true malignant mixed tumor of salivary glands is a very rare neoplasm which shows malignant features of the epithelial and mesenchymal components. We hereby present cytological, pathological, and immunohistochemical findings of one such case, first examined by fine-needle aspiration cytology. The aspirate was cell-rich and consisted of large cell clusters which at low magnification showed an arrangement reminiscent of a jigsaw puzzle. An amorphous substance was observed in the middle of some of these clusters. There were also numerous dissociated cells which often displayed marked atypia. Histology showed a tumor with malignant epithelial and mesenchymal components. The carcinomatous areas consisted of cells arranged in solid nests or glandular structures. The sarcomatous areas showed osteosarcoma, low-grade chondrosarcoma, and undifferentiated components. On immunohistochemistry, the tumor coexpressed epithelial and mesenchymal markers. Cytokeratin, keratin, and epithelial membrane antigen were mainly localized to the carcinomatous portion, whereas vimentin and neuron-specific enolase were restricted to the sarcomatous areas. S-100 protein was positive in both. Our findings support previous views that this tumor may be related to pleomorphic adenoma. PMID- 7544719 TI - Cytologic diagnosis of papillary cystic neoplasm of the pancreas. AB - The diagnosis of papillary cystic neoplasm of the pancreas (PCNP) on cytologic material allows important therapeutic decisions to be made before surgery. We report two cases in which PCNP was diagnosed preoperatively by fine-needle aspiration biopsy, and review the cytologic features of PCNP previously reported in the literature. The distinctive cytologic features of this tumor included papillary structures composed of tumor cells surrounding myxoid material with delicate central vascular cores, and globoid structures composed of amorphous myxoid material. The cytologic characteristics of PCNP are reviewed here. Definitive diagnosis of PCNP can be reliably achieved by fine-needle aspiration cytology. PMID- 7544724 TI - Differential diagnostic dilemmas in malignant fine-needle aspirates of liver: a practical approach to final diagnosis. AB - We retrospectively reviewed two hundred malignant fine-needle aspirates of liver. Of these 49.5% were metastatic neoplasms, 32% were hepatocellular carcinomas, and 18.5% presented with diagnostic dilemmas. In less than half of these diagnostic challenges, differential diagnosis was between hepatocellular carcinoma and metastatic adenocarcinoma. The remainder of cases involved a variety of metastatic neoplasms. Cytomorphology in association with immunocytochemistry resolved the diagnostic problems in about half of the problematic cases. Three cases were undifferentiated and remained unclassifiable. We conclude that approximately 80% of malignant lesions of liver can be correctly diagnosed by thorough cytomorphologic analysis and good clinical correlation, and 20% pose differential diagnostic dilemmas. PMID- 7544725 TI - [Pathological study of hepatoid gastric adenocarcinomas]. AB - Routine examination of pathologic sections in 500 cases of primary gastric adenocarcinomas was carried out by light microscopy. Regions of hepatocellular carcinomatoid differentiation were discovered in 12 of the 500 cases. In 11 of them, histopathologic features were described in great detail and the hepatocellular carcinomatoid regions were studied by four kinds of immunohistochemistrical methods. The results indicate that positive staining for AFP was seen in 9 cases but negative in 9 controls, weakly positive for CEA in 10 cases, but strongly positive in 8 controls. Staining for alpha 1-AT and alpha 1 ACT showed no differences as compared to controls. The clinical pathological features and its relationship with AFP are discussed. It is suggested that hepatoid gastric adenocarcinoma is a subtype of gastric adenocarcinoma. PMID- 7544726 TI - [The effect of diethyl-dithiocarbamate on the distribution of pingyangmycin in vivo]. AB - Diethyl-dithiocarbamate (DDC) is a compound with wide spectrum of pharmacologic activities. It has been shown to enhance the tumor cytotoxic effect of Pingyangmycin (PYM). In this paper, the effect of DDC on the distribution of 99mTc-labeled PYM in mice bearing subcutaneous transplant of Ehrlich carcinoma is reported. The results indicate that DDC did not affect the absolute concentration of PYM in the tumor but it accelerated its excretion in the urine and decreased the concentration of PYM in the non-tumorous tissues. Therefore, DDC could help alleviate the damaging effect of PYM on normal tissues without affecting its tumor cytotoxic activity. PMID- 7544727 TI - Starving tumors. PMID- 7544723 TI - Alteration of Wilms' tumor staging by cytologic detection of malignant cells in chylous ascites. AB - Accurate staging as per National Wilms' Tumor Study (NWTS) criteria is essential for therapy of Wilms' tumor. We report a unique case of a 9-mo-old white male in whom the diagnosis based upon clinical, radiological, surgical, and initial histopathologic findings was Stage I Wilms' tumor without regard to a positive cytologic examination of a small amount (5 ml) of chylous ascitic fluid obtained intraoperatively. This problem was clarified later at the NWTS panel review when the deeper recuts of a renal hilar lymph node revealed a single cluster of Wilms' tumor cells in capsular sinus, resulting in upstaging of tumor to Stage III. The significance of these findings in relation to staging will be discussed. PMID- 7544729 TI - Role of specific successive contacts between extracellular matrix proteins and epidermal Langerhans cells in the control of their directed migration. AB - To migrate from epidermis to regional lymph nodes, antigen-bearing epidermal Langerhans cells (LC) must move through extracellular matrix (ECM) of various composition. The present study was designed to contrast the ability of basement membrane (BM)- and dermis-ECM components to successively stimulate the adhesion of normal human epidermal LC, in vitro. For this purpose, we used highly enriched LC suspensions (70%-80%), allowed them to attach to one ECM substrate, and then studied the readhesion properties of these recovered ECM-attached LC to the same and different ECM substrates. Each of four ECM molecules (laminin (LM), fibronectin (FN), type I and type IV collagen) was tested in pairs. Readhesion of recovered LM and type IV collagen-attached cells did not affect readhesion to FN and type I collagen, indicating that the interaction of LC with the BM components can be normally followed by interaction with the dermis-ECM molecules. In contrast, readhesion of recovered FN-, type I collagen-, and type IV collagen attached cells to LM was significantly reduced. The findings indicate that following contact to BM components, epidermal LC are able to attach to ECM proteins present in the dermis, whereas once they have made contact with ECM present in the dermis, they reduce their binding capacity to the BM laminin, suggesting the contact with the dermal components could prevent LC from reentering the epidermal compartment. Binding to LM and FN was also shown to induce a decline in the expression of CD1a, known as a specific marker restricted to epidermal LC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544730 TI - Peptide binding to MHC class I molecules analyzed by confocal microscopy. PMID- 7544731 TI - Five-year follow-up of patients with benign prostatic hyperplasia treated with finasteride. AB - In 18 of 55 original patients who completed 5 years of treatment with finasteride, significant reductions in prostate size were noted at 1 year and sustained thereafter. Symptom scores in these same patients were significantly improved or stable over the 5 years while maximal urinary flow rates were unchanged. Data from 15 of 18 other patients who dropped out of the study before 5 years showed changes in prostate size, symptom score and flow rates that were similar to those noted in patients treated for 5 years. No side effects were noted in this study except for sexual dysfunction, which occurred in less than 5% of the patients. With few exceptions, finasteride appears to arrest the process of BPH over a 5 year period as indicated by sustained reductions in prostate size accompanied by either symptomatic improvement or stability in all other patients. PMID- 7544732 TI - Effects of the nonsteroidal antiandrogen Casodex on lipoproteins, fibrinogen and plasminogen activator inhibitor in patients with benign prostatic hyperplasia. AB - The effect of the nonsteroidal antiandrogen Casodex (176334) on a number of risk factors for cardiovascular diseases was investigated in a double-blind, randomized, placebo-controlled study comprising 27 evaluable patients with benign prostatic hyperplasia who received either placebo or Casodex at a dosage of 50 mg daily for 24 weeks. We hypothesized that the 30-35% increase in serum levels of testosterone and estradiol seen during treatment with Casodex might induce changes in various risk factors. We found no statistically significant change in total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides. Apolipoproteins A1 and B also remained unchanged, along with plasma concentrations of fibrinogen and plasminogen activator inhibitor 1. Casodex has the potential to become an important drug for treatment of prostatic diseases. Our study does not suggest that the drug is associated with an increased cardiovascular risk. PMID- 7544728 TI - Delivery to lysosomes in the human carcinoma cell line HEp-2 involves an actin filament-facilitated fusion between mature endosomes and preexisting lysosomes. AB - We have addressed the following question: what is the mechanism behind the delivery of internalized molecules from mature endosomes to lysosomes in HEp-2 cells, and which role does the cytoskeleton play in this process? Quantitative electron microscopy and immunogold labeling revealed that whereas the cytoskeleton was not of importance for endosome maturation, actin filaments facilitated fusion of mature endosomes with preexisting lysosomes. Delivery to lysosomal degradation was not dependent on protein synthesis as determined biochemically, but was reduced by cytochalasin D. Observations made by electron microscopy as well as by video microscopy of living cells showed that the concerted action of actin filaments and microtubules was responsible for the random distribution and movement of endocytic organelles throughout the cell. Actin microfilaments, however, seem to facilitate perinuclear clustering and frequent fusion of mature endosomes and lysosomes, while microtubules play a role in preventing formation of large lysosome aggregates by separating endosomes and lysosomes and moving them toward the cell periphery. Taken together, our data suggest that delivery of internalized molecules to lysosomal proteolysis takes place by fusion of mature endosomes with preexisting lysosomes and that actin microfilaments somehow facilitate this step. PMID- 7544734 TI - Effect of ejaculation on prostate-specific antigen levels in normal men. AB - While several manipulations such as digital rectal examination and cystoscopy have been shown to increase prostate-specific antigen (PSA) levels, lack of ejaculation for a period of time has been implicated as a cause of decreased serum PSA concentrations. However, the effect of ejaculation on the serum PSA concentration is not clear. This work determined the effect of ejaculation on serum PSA levels in normal men. Blood samples of 19 healthy subjects were taken before and 5 consecutive days after ejaculation. One subject was withdrawn from the study because of ejaculation during sleep, since another ejaculation was prohibited during the study. The mean PSA levels of the men before and 5 consecutive days after ejaculation were 0.79, 0.72, 0.63, 1.01, 0.71, and 0.74 ng/ml--not statistically significantly different. We conclude that ejaculation does not seem to affect the serum PSA concentration. PMID- 7544733 TI - Prostate-specific antigen density and age-specific prostate-specific antigen values: the solution of prostate cancer screening? AB - The results of 225 systematic prostate biopsies from 1992 to 1993 were evaluated retrospectively. The parameters prostate-specific antigen (PSA) density and age specific PSA values were compared with digital rectal examination, transrectal ultrasound, and PSA as single parameters and possible combinations. The PSA density proved to have the highest specificity of all single parameters, but the sensitivity was low. Age-specific PSA values are offering a good compromise of sensitivity and specificity as compared with fixed cutoff levels. Since there is no sufficient screening parameter up to now, a combination of all parameters is recommended for screening of early prostate cancer. PMID- 7544736 TI - Late effects of intravesical bacillus Calmette-Guerin immunotherapy on bladder mucosa infiltrating lymphocytes: an immunohistochemical study. AB - During intravesical bacillus Calmette-Guerin (BCG) treatment for the prophylaxis of recurrent superficial bladder carcinoma, patients typically show a local inflammatory response involving mainly T lymphocytes, most of which have the helper-induced phenotype (CD4+) (CD4+/CD8+ ratio > 1). To evaluate whether this immunophenotypic profile of the lymphocytes persists also after the completion of this immunotherapy, we examined bladder biopsy specimens during the posttreatment follow-up period of 24 patients, previously submitted to a 2-year BCG administration. The intensity of inflammatory response differed among the patients and in 10 of them even between the scar and the normal mucosa of the bladder. A reversal to the pretreatment CD4+/CD8+ ratio < 1 occurred in the majority of subjects, including the 3 patients with histologically confirmed tumour recurrence. In addition, 11 tumour-free patients showed prevailing CD4+ cells in the scar mucosa and prevailing CD8+ in the normal mucosa of their bladder or vice versa. From these findings it appears that the long-term host response to BCG does not depend exclusively on an intense, long-lasting local mononuclear immune reaction. PMID- 7544735 TI - Prostate-specific antigen as a unique routine test in monitoring therapy for inoperable prostate cancer: comparison with radionuclide bone scan and prostatic acid phosphatase. AB - The aim of the present investigation was the evaluation of cost-effectiveness of variables used in monitoring patients with inoperable prostate cancer. Prostate specific antigen (PSA), prostatic acid phosphatase (PAP), and radionuclide bone scan were considered. The tumor marker positivity was assessed according to dynamic criteria (> 50% increase between consecutive samples). 108 patients entered the study; 72 patients treated with a luteinizing hormone-releasing hormone analogue were followed up for periods ranging from 12 to 64 months. PSA and PAP levels were measured using immunometric assays. Both cutoff-based and dynamic, serial sample-based decision criteria were employed. With respect to a positive bone scan, PSA showed negative predictive values of 82 and 77%, respectively, using 4 and 10 ng/ml as cutoff points. Progression of the disease to the bone occurred in 20 patients: in 17 PSA was the first indicator of progression, in the other 3 PAP anticipated PSA for a very short time (3-4 months), which was not of relevance to clinical decisions. PAP is less specific and sensitive than PSA; PAP may eventually provide information on disease status in a limited percentage of patients with advanced prostate cancer treated with androgen ablation, being differently regulated with respect to PSA. No increasing PSA profile was detected in patients who responded to the therapy. From the results of the present investigation, we draw the following conclusions: (1) PSA can be used reliably as a unique tool in the follow-up of patients for the early detection of progressive disease, and (2) dynamic criteria of evaluation of serial PSA determinations probably provide more effective and earlier clinical information. PMID- 7544737 TI - Detection of tumor cells in the bone marrow, peripheral blood, and apheresis products of breast cancer patients using flow cytometry. AB - One of the possible drawbacks to autologous bone marrow (BM) and peripheral blood progenitor cell (PBPC) transplantation in breast cancer patients is the potential for tumor cell contamination in the transplanted product. To assess the presence of breast cancer cells, we have developed a flow-cytometric method using cytokeratin-FITC and CD45-phycoerythrin (PE) to detect very low levels of cytokeratin-positive (CK+) tumor cells in mononuclear cell (MNC) preparations. In a model system using PBMNC and the breast cancer cell line CAMA, the sensitivity of detection of this flow-cytometric method was one tumor cell in 200,000 MNC. This method was used to evaluate BM, PB, and apheresis products (AP) from 44 patients with metastatic breast cancer. When possible, stained cytologic examination was performed on smears of the unprocessed specimens and on flow cytometry-sorted cells. Results indicated that CK+ tumor cells could be detected by flow cytometry in all three specimen types. When present, however, the tumor content (per MNC) tended to be higher in BM than in PB or AP. Samples from a given patient taken serially over the course of chemotherapy revealed variable results, suggesting that the presence of tumor contamination may be sporadic and requires evaluation of each stem cell product. Of 75 samples tested with both flow cytometry and cytology, the results were concordant in 54 cases (72%). In the remaining samples, flow cytometry only was positive in 15 cases (20%), and cytology only was positive in six cases (8%). This flow-cytometric technique is useful in the evaluation of transplant products for CK+ tumor cell contamination. PMID- 7544738 TI - Role of suramin as an IL-1 inhibitor in suppression of acute myelogenous leukemia progenitor proliferation. AB - Interleukin-1 (IL-1) modulates both autocrine and paracrine growth-stimulatory mechanisms of acute myelogenous leukemia (AML) cell proliferation. Recent studies show that blocking the interaction between IL-1 and its receptor may suppress this proliferation. Because suramin, a polysulfonated naphthylurea originally described as an antitrypanosomal agent, was found to inhibit the binding of several growth factors to their receptor, we tested its effect on AML progenitor proliferation. We first examined the effect of suramin on murine EL-4.6.1 cells that express type I IL-1 receptors and found that suramin inhibited the binding of IL-1 to its receptor. We then tested the effect of suramin on AML progenitors using bone marrow samples from 17 patients with AML. In all experiments, suramin inhibited AML blast proliferation in a dose-dependent fashion at concentrations ranging from 30 to 240 microM. IL-1 beta (100 U/mL) partially reversed this inhibitory effect. Suramin also inhibited normal early and mature hematopoietic progenitors, as assessed by both the delta assay and the mixed colony culture assay; however, at the same concentration, suramin suppressed the colony growth of colony-forming units granulocyte-macrophage (CFU-GM) by only 45% compared with an 89% suppression of AML progenitors. IL-1 beta did not negate this inhibitory effect, which suggests that another growth inhibitory mechanism might be operative. Our data suggest that suramin may inhibit AML progenitor proliferation by blocking the interaction of IL-1 and its receptor; therefore, further studies are warranted to evaluate suramin's therapeutic potential in patients with AML. PMID- 7544740 TI - Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells. AB - The present studies evaluated effects of recombinant human (rhu) and murine (rmu) flt3 ligand (flt3-L) on colony formation by subsets of myeloid stem and progenitor cells present in low-density (LD) and cell-sorted CD34 hu cord blood (CB) and bone marrow (BM) cells and unseparated mu BM cells. By itself, flt3-L had weak colony-stimulating activity. It stimulated small dispersed CFU-GM-type colonies, but not BFU-E, CFU-GEMM, or HPP-CFC colonies, from LD and CD34 huCB and BM. However, flt3-L had additive to greater-than-additive effects on colony number and size by CFU-GM stimulated with GM-CSF or IL-3, with or without Steel factor (SLF); by CFU-G stimulated by G-CSF with or without SLF; by CFU-M stimulated by CSF-1; and by BFU-E, CFU-GEMM, and HPP-CFC stimulated by Epo with or without IL-3 or SLF. Flt3-L enhanced the effects of SLF, alone and in combination with other CSFs. Similar effects were apparent on LD and sorted CD34 cells and also at the level of single sorted and isolated CD34 cells/well. Flt3-L enhanced expansion of immature subsets of huCD34(+)-column separated CB CFU-GM stimulated by the potent combination of SLF and PIXY321 (a GM-CSF/IL-3 fusion protein). While flt3-L did not enhance the replating capacity of CFU-GEMM plated in the presence of Epo and SLF, it enhanced numbers of these CFU-GEMM colonies with the capacity to be replated. Flt3-L effects were not species-specific; rhu and rmu forms were active on huCB/BM and muBM. These results demonstrate the potent direct-acting stimulating/costimulating activities of flt3-L in vitro on myeloid stem/progenitor cells. PMID- 7544741 TI - Pulmonary toxoplasmosis in HIV-infected patients: usefulness of polymerase chain reaction and cell culture. AB - Toxoplasmosis is a serious opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The lung is a major site of infection after the central nervous system. The aim of the study was to assess the polymerase chain reaction (PCR) and cell culture for the detection of Toxoplasma gondii. One hundred and thirty two human immunodeficiency virus (HIV)-infected patients with respiratory manifestations, who underwent fibreoptic bronchoalveolar lavage, were investigated. Detection of Toxoplasma gondii was compared using three techniques: Giemsa staining; polymerase chain reaction with specific primers derived from the P30 gene; and culture on the MRC5 cell line. Toxoplasma gondii was detected in the same four samples by all three techniques. We conclude that PCR adds little to conventional (and cheaper) tools already used to diagnose pulmonary toxoplasmosis. PMID- 7544742 TI - Multimedia presentation of lung sounds as a learning aid for medical students. AB - New educational technologies might help to compensate for the decrease in time and emphasis dedicated to physical examination in medical training. This may, in particular, be applicable for improving the skills in auscultation of the chest. We investigated whether a multimedia presentation of acoustic and graphic characteristics of lung sounds could improve the learning of pulmonary auscultation by medical students, in comparison with conventional teaching methods. We studied 48 medical students without clinical experience, who had received conventional formal teaching on chest examination. Chest auscultation skills were evaluated using an inaccuracy score for the student's auscultation report on three patients, selected according to a standardized procedure. After a baseline evaluation, 27 students in groups of 5-10, participated in a multimedia seminar on lung sounds during which digitized lung sounds were played and the corresponding time-expanded waveform and frequency spectrum were commented on and displayed on a computer. The remaining 21 students received conventional bedside training, acting as control group. The following week, all the students underwent a second evaluation of chest auscultation skills. No differences in the inaccuracy score were observed between the two groups in the preliminary test. However, in the second postintervention assessment, the inaccuracy score of the students who had followed the seminar (11.2 +/- 1.3 points) was significantly lower than that of the controls (16.6 +/- 1.6 points). The answers to a feedback questionnaire confirmed that the great majority of the students found the association of the acoustic signals with their visual image to be useful for learning and understanding lung sounds.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544739 TI - Stem cell factor production by human marrow stromal fibroblasts. AB - To characterize the production of stem cell factor (SCF, the ligand for the c-kit receptor protein) and its regulation by inflammatory cytokines and glucocorticoids, primary marrow stromal fibroblasts were isolated from normal individuals and two patients with Diamond-Blackfan anemia. Unstimulated normal marrow stromal fibroblasts constitutively expressed a low level of SCF mRNA (9 +/ 2 copies/cell [mean +/- SEM]), continually secreted soluble SCF into the supernatant of 1- to 5-day-old cultures (0.16 +/- 0.02 to 0.73 +/- 0.04 ng/mL per 10(6) cells, respectively), and expressed membrane-bound SCF. Stimulation with interleukin-1 beta (IL-1 beta) only modestly increased SCF mRNA levels, soluble SCF production at 24 hours, and membrane-bound SCF. In comparison, hydrocortisone or tumor necrosis factor alpha (TNF-alpha) exposure increased SCF mRNA levels 3.5 to four-fold above controls, but with different kinetics. The peak TNF-alpha effect was at 6 hours, with return to near control levels at 24 hours, whereas hydrocortisone induced maximal mRNA increases at 12 to 18 hours, and the levels remained high at 24 hours. Similarly, a sustained increase in soluble SCF production was detected during 1 to 5 days of hydrocortisone exposure (0.27 +/- 0.03 to 1.10 +/- 0.08 ng/mL per 10(6) cells), while TNF-alpha stimulation modestly increased the production of soluble SCF in 24-hour cultures only. Unstimulated normal marrow fibroblasts expressed predominantly the long species of alternatively spliced SCF mRNA, and the relative amounts of long and short mRNAs did not change after stimulation with IL-1 beta, hydrocortisone, or TNF alpha. SCF production by marrow stromal fibroblasts from a symptomatic patient with Diamond-Blackfan anemia was equivalent to simultaneously studied normal marrow fibroblasts. In contrast, marrow fibroblasts from a Diamond-Blackfan anemia patient in untreated hematologic remission constitutively expressed high levels of SCF mRNA (21 +/- 4 copies/cell) and soluble protein (0.40 ng/mL per 10(6) cells at 24 hours). Together, these observations suggest that SCF is constitutively produced by fibroblasts in the human marrow microenvironment and that hydrocortisone induces a modest but sustained increase in SCF gene expression and protein production, compared to only a transient increase induced by TNF-alpha. In addition, these findings support the hypothesis that endogenous or corticosteroid-induced increases in the production of SCF could play a physiologic role in the clinical improvement of congenital anemia. PMID- 7544744 TI - The transmembrane signalling. PMID- 7544743 TI - Nitric oxide is involved in heat-induced HSP70 accumulation. AB - Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock-induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO-synthase inhibitor N omega-nitro-L-arginine (L-NNA). L-NNA also markedly attenuated the heat shock-induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock-induced activation of HSP70 synthesis. PMID- 7544745 TI - An artificial intelligent diagnostic system with neural networks to determine genetical disorders and fetal health by using maternal serum markers. AB - OBJECTIVE: To develop an artificial intelligent diagnostic system with neural networks to determine genetical disorders and fetal health problems by using maternal serum markers ('Triple Test') and maternal age. STUDY DESIGN: A total of 112 pregnant women were referred to Fetal Medicine Unit of Hacettepe University Hospital for fetal ultrasonography and chromosome analysis with different indications. All patients underwent genetic amniocentesis or fetal blood sampling under ultrasound guidance. Gross malformations and hydrops fetalis were detected in 15 and 5 fetuses, respectively. We have found chromosomal abnormality in 7 cases. 'Triple Test' is offered to all patients and serum levels of alpha fetoprotein, human chorionic gonadotropin and unconjugated estriol were analyzed by radioimmunoassay. In this study, we have used supervised artificial neural network structure to develop a diagnostic system. Our system's input parameters are maternal age, gestational age and 'Triple Test' results. Our system consists of two different artificial neural network modules whose decision-making logics are different. One of them is designed to search genetical disorders while the other one is for the assessment of fetal well-being. Confusion matrix is used for statistical evaluation. RESULTS: The discriminatory power of the artificial neural network to search genetical disorders and fetal well-being is found to be highly significant (z = 10.583 and z = 10.424, respectively). CONCLUSION: This system brings objectively to the evaluation of 'Triple Test' results and can be used both for the detection of genetical disorders and fetal well-being. Nevertheless, the analysis program's performance is limited to input information and knowledge and medical expert expert can not get more than he or she has donated the system. PMID- 7544747 TI - Late histopathologic changes and healing in an improved rodent model of acute necrotizing pancreatitis. AB - Studies of experimental pancreatitis have generally focussed on early time points rather than the stages of healing and resolution or scarring. We recently characterized a new pancreatitis model of moderate severity by combining intraductal infusion of very low concentrations of glycodeoxycholic acid with intravenous caerulein. This study evaluates late histopathologic changes and gross complications in this pancreatitis model compared to the traditionally used high-dose bile salt model in rats. After 14 days, histopathologic features of caerulein pancreatitis were not different from saline controls. High-dose intraductal bile salt infusion resulted in widespread chronic inflammation, acinar dilation and atrophy, marked reactive stromal proliferation, and ductular budding with periductal fibrosis. In contrast, animals receiving low-dose intraductal bile salt infusion combined with intravenous caerulein demonstrated a moderate degree of chronic inflammation and acinar atrophy along with an intermediate degree of periductal fibrosis and stromal reaction. We conclude that due to its moderate degree of injury, this model may be useful for the study of tissue injury and repair following acute pancreatitis. PMID- 7544746 TI - Superovulation, IGFBP-1 and birth weight. AB - In this study, the effect of superovulation on the circulating levels of insulin like growth factor binding protein-1 (IGFBP-1) has been investigated. IGFBP-1 levels were measured in singleton pregnancies achieved either naturally (n = 203) or following superovulation, in-vitro fertilisation and embryo transfer (IVF-ET) with either pituitary desensitisation with buserelin and superovulation with human menopausal gonadotrophin (b/hMG) followed by IVF-ET (n = 15) or with clomiphene citrate and hMG (CC/hMG) followed by IVF-ET (n = 15, 1st trimester only). The circulating levels of IGFBP-1 were similar in all three groups during the first trimester, and in both normal and b/hMG pregnancies in the second, but were significantly higher during the third trimester in b/hMG pregnancies than in normal pregnancies (P = 0.0002). The birth weights were significantly lower in the b/hMG group (P = 0.04), but not in the CC/hMG group compared with natural conceptions. Gestational age at delivery was similar in control and b/hMG pregnancies, but significantly reduced in CC/hMG pregnancies (P = 0.04). These data suggest that pregnancies achieved following superovulation with b/hMG are associated with elevated levels of IGFBP-1 during the third trimester of pregnancy and reduced birth weight. PMID- 7544749 TI - Biochemical and genetic screening of colorectal cancer. PMID- 7544748 TI - Activated immunocompetent cells in rat colitis mucosa induced by dextran sulfate sodium and not complete but partial suppression of colitis by FK506. AB - Administration of dextran sulfate sodium (DSS) solutions to rats induced colitis which resembled mucosal lesions of human ulcerative colitis. Recent reports have shown that some cytokines are related to the pathogenesis of ulcerative colitis. In the present report, we describe the production of two cytokines in colitis mucosa in this DSS model. Using a cytotoxicity assay and a radioimmunoassay, we observed significant increases in levels of tumor necrosis factor-alpha (TNF alpha) in the colitis mucosa and detected interleukin-1 alpha in the mucosa of 3 of 5 DSS rats and an increase in TNF-alpha had a tendency to be inhibited by treatment with FK506. Immunohistochemical investigation of DSS mucosa showed that the number of activated T cells increased at the earlier phase of inflammation. Luminol-dependent chemiluminescence values and myeloperoxidase activities were increased in the late phase of colitis and were suppressed by the FK506 treatment. These findings may support the role of TNF-alpha and T-cell activation in the pathogenesis of DSS colitis. PMID- 7544750 TI - Calcium currents in human and canine jejunal circular smooth muscle cells. AB - BACKGROUND & AIMS: Although calcium plays an essential role in intestinal smooth muscle contractile activity, calcium entry pathways in canine and human small intestine are largely unknown. The goal of this study was to characterize calcium channels, a potential entry pathway for calcium, in isolated circular smooth muscle cells of canine and human jejunum. METHODS: Single freshly dissociated human and canine jejunal circular smooth muscle cells were studied using single channel and perforated whole-cell patch clamp recordings as well as fluorescence dual wavelength ratio imaging. RESULTS: An inward whole-cell current was identified that was carried by a 17 pS (80 mmol/L Ba2+) dihydropyridine sensitive, barium-permeable channel. The current was potentiated by BayK 8644 (1 mumol/L; n = 3; 82% +/- 34%), acetylcholine (1 mumol/L; n = 8; 42% +/- 5%), and erythromycin (1 mumol/L; n = 9; 70% +/- 11%) and was completely blocked by nifedipine (1 mumol/L; n = 6) or diltiazem (200 mumol/L; n = 4). Application of BayK 8644 (1 mumol/L), acetylcholine (1 mumol/L), or erythromycin (1 mumol/L) to Fura-2-loaded smooth muscle cells bathed in Krebs' solution containing 2.54 mmol/L calcium increased intracellular calcium levels. CONCLUSIONS: A calcium entry pathway was identified in canine and human jejunal circular smooth muscle cells. The pathway was mediated by a dihydropyridine-sensitive calcium channel. The channel allowed the entry of significant amounts of calcium at physiological extracellular calcium concentration. PMID- 7544751 TI - Detection of decay-accelerating factor in stool specimens of patients with colorectal cancer. AB - BACKGROUND & AIMS: Colorectal cancers have an increased expression of decay accelerating factor (DAF). The aim of this study was to determine whether stool specimens of patients with colorectal cancer contain increased amounts of DAF. METHODS: DAF was measured using an immunoassay in the stool specimens of 40 persons with colorectal cancer, 18 with colorectal adenomatous polyps, 13 with upper gastrointestinal cancer, and 41 without gastrointestinal disease. RESULTS: Stool DAF concentrations in patients with colorectal cancer (0-9.8 ng/g stool; median, 1.6 ng/g) were significantly higher than those in patients with adenoma (0-6.4 ng/g; median, 0 ng/g) (P < 0.05), patients with upper gastrointestinal cancer (0-3.1 ng/g; median, 0 ng/g) (P < 0.05), and subjects without gastrointestinal disease (0-3.4 ng/g; median, 0 ng/g) (P < 0.01). Resection of colorectal cancers caused a marked decrease in stool DAF concentrations. The stool DAF test was positive in a substantial portion of patients with colorectal cancer whose tumors were small ( < 2 cm), at an early TNM stage, or unassociated with fecal occult blood positivity. The sensitivity of the test for colorectal cancer was 55%, and the specificity was 85%. CONCLUSIONS: The measurement of stood DAF deserves evaluation as a test for detection of colorectal cancer. PMID- 7544752 TI - The role of endogenous glucocorticoids in rat experimental models of acute pancreatitis. AB - BACKGROUND & AIMS: Cytokines activate the hypothalamic-pituitary-adrenal axis and suppress inflammation by stimulating glucocorticoid secretion. The state of adrenocortical function during acute pancreatitis and its role in this disease were determined. METHODS: Cerulein-induced pancreatitis or closed duodenal loop pancreatitis was produced in rats that had undergone adrenalectomy or sham adrenalectomy, and the serum corticosterone and interleukin 8 levels and the intensity of the pancreatitis were examined. RESULTS: Serum corticosterone levels were significantly higher than basal levels in both models of experimental pancreatitis. In both models, adrenalectomy increased serum amylase and pancreatic edema and produced more severe inflammation. Adrenalectomy significantly increased mortality in animals with closed duodenal loop pancreatitis. Exogenous hydrocortisone administered to adrenalectomized animals suppressed the elevation of serum interleukin 8 levels and decreased both the severity of pancreatitis and mortality. CONCLUSIONS: These results suggest that the adrenocortical function is stimulated during acute pancreatitis and that the secretion of endogenous glucocorticoids may play an important role in mitigating the progress of this disease, probably by inhibiting cytokine production. PMID- 7544753 TI - Analysis of insulin-like growth factor binding proteins in the tammar wallaby, Macropus eugenii. AB - The insulin-like growth factors (IGFs) are important regulators of growth and development in eutherian mammals. In this study we have analyzed circulating insulin-like growth factor binding proteins (IGFBPs) by Western ligand blotting (WLB) and neutral gel filtration, and hepatic IGFBP mRNA transcripts in the wallaby, Macropus eugenii, a marsupial in which the fetus is born at an immature stage compared to eutherian mammals. Plasma from male and female adults, lactating mothers, and pouch young contains an IGF binding species consisting of a 42- to 50-kDa doublet, 30, 28, 24, and approximately 200 kDa, as shown by WLB. This pattern of IGFBPs is very similar to that observed in human and sheep. Neutral gel filtration revealed IGF binding activity of molecular size 70-160 kDa. When electrophoresed under nonreducing, denaturing conditions, the 70- to 160-kDa IGF binding activity appeared as a 42- to 50-kDa doublet, indicating the presence of a high-molecular-weight circulating IGF binding complex. These properties are again analogous to those observed for eutherian IGFBPs. Northern analysis of total wallaby liver RNA detected transcripts of 1.8 kb for IGFBP-1 and 1.6 kb for IGFBP-2, the same as those observed in the sheep and rat. Transcripts of 3.2 kb were detected for IGFBP-4, larger than the major band of 2.6 kb observed for the sheep and rat. Transcripts representing wallaby IGFBP-3 mRNA were not detected using a rat IGFBP-3 cDNA probe. Circulating IGFBPs were analyzed in plasma from wallaby mothers and their pouch young. There were no changes in circulating IGFBPs in the wallaby mothers throughout lactation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544754 TI - An enhancer deletion affects both H19 and Igf2 expression. AB - The distal end of mouse Chromosome 7 contains four tightly linked genes whose expression is dependent on their parental inheritance. Mash-2 and H19 are expressed exclusively from the maternal chromosome, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf2) are paternally expressed. The identical expression during development of the 3'-most genes in the cluster, Igf2 and H19, led to the proposal that their imprinting was mechanistically linked through a common set of transcriptional regulatory elements. To test this hypothesis, a targeted deletion of two endoderm-specific enhancers that lie 3' of H19 was generated by homologous recombination in embryonic stem cells. Inheritance of the enhancer deletion through the maternal lineage led to a loss of H19 gene expression in cells of endodermal origin, including cells in the liver, gut, kidney, and lung. Paternal inheritance led to a very similar loss in the expression of Igf2 RNA in the same tissues. These results establish that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes. Mice inheriting the enhancer deletion from fathers were 80% of normal size, reflecting a partial loss-of-function of Igf2. The reduction was uniformly observed in a number of internal organs, indicating that insulin-like growth factor II (IGFII), the product of Igf2, acts systemically in mice to affect prenatal growth. A modest decline in Ins-2 RNA was observed in the yolk sac. In contrast Mash-2, which is expressed in spongiotrophoblast cells of the placenta, was unaffected by the enhancer deletion. PMID- 7544756 TI - A clinical study of lectin-reactive alpha-fetoprotein as an early indicator of hepatocellular carcinoma in the follow-up of cirrhotic patients. AB - Levels of two types of lectin-reactive alpha-fetoprotein (AFP), designated AFP-L3 and AFP-P4+P5, were analyzed with Lens culinaris agglutinin A and AFP-P4+P5 with erythroagglutinating phytohemagglutinin, respectively, in an attempt to determine the utility and significance of these macromolecules as early indicators of hepatocellular carcinoma during the periodic follow-up of cirrhotic patients. The subjects were 51 of 190 consecutive cirrhotic patients in whom hepatocellular carcinoma developed during a 6-year follow-up period and 21 cirrhotic patients without hepatocellular carcinoma. Serum AFP levels were of limited value to diagnose and predict hepatocellular carcinoma. The relative levels of AFP-L3 and AFP-P4+P5 in patients with hepatocellular carcinoma at the time of tumor detection were significantly higher than those in patients with cirrhosis. The sensitivity was 61%, and the specificity was 90%. Fourteen patients (48%) of 29 patients with small hepatocellular carcinomas less than 2 cm in diameter showed elevated percentage of lectin-reactive AFP. Retrospective examination of 21 patients who were positive for lectin-reactive AFP at diagnosis of hepatocellular carcinoma showed that 41% of them had already expressed lectin-reactive AFP 12 months before the direct detection of hepatocellular carcinoma by diagnostic imaging. These results lead us to conclude that the level of lectin-reactive AFP is a suitable predictive marker for the early recognition of hepatocellular carcinoma in the follow-up of patients with cirrhosis, and that measurements of the level of lectin-reactive AFP should be added to the screening methods that are now in use. PMID- 7544755 TI - In vitro activation of woodchuck lymphocytes measured by radiopurine incorporation and interleukin-2 production: implications for modeling immunity and therapy in hepatitis B virus infection. AB - Cellular immune responses to hepatitis B virus (HBV) play an important role in the resolution of acute infection. They also influence the course of chronic infection and disease but are inadequate to completely clear the infection. Woodchuck hepatitis virus (WHV) infection of the woodchuck can provide a model to study these processes. Lymphocyte responses of woodchucks were assessed by in vitro proliferation and/or interleukin (IL)-2 assays using mitogen (Concanavalin A [ConA]), cytokine (IL-2), superantigen (Staphylococcus aureus enterotoxin B [SEB]), major histocompatibility complex (MHC) allo-antigen (mixed lymphocyte reaction [MLR]), and viral antigens (woodchuck hepatitis virus core antigen [WHcAg] and woodchuck hepatitis virus surface antigen [WHsAg]). ConA-stimulated woodchuck lymphocytes underwent cell division based on cell counting experiments and produced IL-2 as detected using an IL-2-dependent murine cell line but failed to incorporate sufficient tritiated thymidine; however, they did incorporate sufficient tritiated adenosine and deoxyadenosine to permit development of a meaningful proliferation assay. The IL-2 assay was sensitive and specific for detection of woodchuck IL-2 induced by mitogen, superantigen, and MLR, as shown by quantitative titration analysis and anti-body neutralization of ConA supernatant activity. Cyclosporin A and FK506 specifically inhibited ConA- and SEB-induced IL-2 production by woodchuck lymphocytes. Positive two-way MLRs were detected by IL-2 production and proliferation assay between woodchucks from different geographic regions, thus indicating divergence among MHC molecules; however, occasional negative MLR reactions among indigenous pairs of woodchucks indicated that some woodchucks were mutually immunocompatible to some degree. The radioadenosine proliferation assay was sensitive for detecting peripheral blood lymphocyte responses to WHcAg and WHsAg in adult woodchucks with recently resolved acute infections. The above systems should facilitate the design of adoptive therapy and liver transplantation experiments in the woodchuck, and also enable modeling of immune responses that promote and maintain chronic hepadnavirus infection. PMID- 7544757 TI - Downregulation of phorbol 12-myristate 13-acetate-induced tumor necrosis factor alpha and interleukin-1 beta production and gene expression in human monocytic cells by human alpha-fetoprotein. AB - We previously identified a specific receptor of alpha-fetoprotein (AFP) on human monocytes. Although AFP alters many immune cell functions, the effect of AFP on monocyte cytokine production is unknown. Because tumor necrosis factor--alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are important cytokines in immunoregulation, we investigated whether AFP could modulate TNF-alpha and IL-1 beta production in U937, a human monocytic cell line. Our results showed that U937 cells secreted TNF-alpha and IL-1 beta in response to either phorbyl 12 myristate 13-acetate (PMA) or IFN-gamma + LPS. In contrast, AFP significantly suppressed PMA-induced TNF-alpha and IL-1 beta production by U937 cells in a time and dose dependent fashion. Pretreatment of U937 cells with AFP resulted in maximal inhibition of PMA-stimulated TNF-alpha and IL-1 beta production by 58% and 67% respectively. AFP also inhibited interferon-gamma plus lipopolysaccharide (IFN-gamma + LPS)-induced TNF-alpha and IL-1 beta production. Furthermore, Northern blot analysis showed that AFP suppressed PMA-mediated TNF-alpha and IL-1 beta messenger RNA (mRNA) expression. PMA-induced prostaglandin E2 (PGE2) production by U937 cells was enhanced by AFP. Pretreatment with indomethacin, a cyclooxygenase inhibitor, reversed AFP-inhibited TNF-alpha production by 78%. Thus, we conclude that AFP downregulates TNF-alpha and IL-1 beta production via a PGE2-dependent mechanism. PMID- 7544759 TI - Immunosuppression: now we have choices. PMID- 7544758 TI - Expression of glycoconjugates during intrahepatic bile duct development in the rat: an immunohistochemical and lectin-histochemical study. AB - We investigated the expression of carbohydrate residues on the developing intrahepatic bile ducts of rats. At 17 days of gestation, immature biliary cells around the portal vein close to the hepatic hilum assumed one of the following forms: slitlike lumen, incomplete, or complete bile ductule-like structures. These immature biliary elements then rapidly spread throughout the liver along with development. At birth, a few mature interlobular bile ducts became visible in the portal tracts. The cytoplasm of immature biliary cells stained weakly for concanavalin A, Erythrina crista galli agglutinin, and Limax flavus agglutinin, whereas the luminal surface of immature biliary cells at 17 days of gestation was positive for lectins, similar to those that are expressed on the luminal surface of the mature bile ducts, including concanavalin A, succinyl wheat germ agglutinin, Vicia villosa agglutinin, soybean agglutinin, peanut agglutinin, Erythrina crista galli agglutinin, and Limax flavus agglutinin. As development progressed, the number of lectins binding to the cytoplasm of biliary cells gradually increased, and lectin bindings to the luminal surface of biliary cells gradually became intense. Immature biliary epithelial cells of three structures expressed similar carbohydrate residues in their cytoplasm and luminal surfaces. This study suggested that the profile of carbohydrate residues on the biliary epithelium changes with development. Therefore, this profile could be a useful tool with which to evaluate the development of the biliary tree as well as associated disorders. PMID- 7544760 TI - Pulmonary B-cell non-Hodgkin's lymphomas. The value of immunohistochemistry and gene analysis in diagnosis. AB - We reviewed 45 pulmonary B-cell non-Hodgkin's lymphomas to determine whether their morphology and immunohistochemical features were those of lymphomas arising from mucosa-associated lymphoid tissue (MALT), as described in other sites. The polymerase chain reaction was used to provide further information on clonality. We found that these lymphomas infiltrate the pulmonary interstitium along bronchovascular bundles and interlobular septa, subsequently spilling out into airspaces and finally destroying the alveolar architecture of the lung. Central hyaline sclerosis and vascular infiltration were common features. All lymphomas stained CD20 positive and were accompanied by variable numbers of reactive CD3 positive T-cells. Cytokeratin staining with CAM 5.2 was useful in identifying lymphoepithelial lesions. CD21 staining of follicular dendritic cells revealed germinal centres where they were not recognizable on H & E staining. The polymerase chain reaction was performed on paraffin tissue from 28 patients. Twenty were low grade, of which 12 showed a clonal band and eight showed a polyclonal smear pattern. Eight were high grade, of which one revealed a clonal band. Three produced polyclonal smear patterns and four cases were inadequate samples. In one patient who had lymphoma at a second extranodal site, identical bands were identified, evidence for 'homing' of lymphoid cells towards mucosal epithelium. PMID- 7544761 TI - Reactive pulmonary lymphoid disorders. AB - The two main reactive pulmonary lymphoid disorders are lymphoid interstitial pneumonia and follicular bronchitis/bronchiolitis, both pathological entities with a variety of aetiologies. We reviewed the morphological and immunohistochemical features of 26 cases with one or other of these two diagnoses, to explore the possibility that they represented overlapping patterns of hyperplasia of the bronchopulmonary immune system. The polymerase chain reaction was used to determine the clonality of the infiltrates. Histologically, there was a spectrum of changes with two main components. An interstitial infiltrate of mainly T lymphocytes, plasma cells and histiocytes predominated in lymphoid interstitial pneumonia, whilst lymphoid follicles predominated around airways in follicular bronchitis/bronchiolitis. Classification of the disorder rested on which component the pathologists believed to be dominant. In two cases, histology and immunohistochemistry suggested lymphoma, and in one of these cases this diagnosis was confirmed by the polymerase chain reaction. One case of lymphoid interstitial pneumonia produced three bands. The remainder produced polyclonal patterns when samples were adequate. Clinically, there was no clear difference between patients with the two disorders, or patients with pathological features of both. PMID- 7544762 TI - Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-beta in human post-burn hypertrophic and mature scars. AB - The distributions of the small proteoglycans, decorin and biglycan and the large proteoglycan, versican, in normal skin and post-burn hypertrophic and mature scars, were compared using monoclonal and polyclonal antibodies to the core proteins. Biglycan and versican were virtually undetectable in normal dermis but readily seen in hypertrophic scars. Staining for decorin was strong throughout the dermis in normal skin but restricted to the deep dermis and a narrow zone under the epidermis in hypertrophic scar--areas which did not stain for versican. Decorin was absent or reduced in the nodules in these specimens. In mature post burn scars, staining for all three proteoglycans demonstrated an intensity that was intermediate between that in normal dermis and that in the nodules of the hypertrophic scars. Transforming growth factor-beta was present in the nodules of hypertrophic scars but the deep dermis of these specimens stained most intensely for this cytokine which was also found in the dermis of mature scars but was not detectable in normal dermis. The apparent co-distribution of decorin and transforming growth factor-beta suggests that this proteoglycan may play an active role in the resolution of the scars. Changes in proteoglycan type and distribution could possibly account, at least in part, for the derangement of collagen and the altered physical properties of hypertrophic scar tissue. PMID- 7544763 TI - Immunoreactivity of sinusoids in hepatoblastoma: an immunohistochemical study using lectin UEA-1 and antibodies against endothelium-associated antigens, including CD34. AB - Sinusoids are found not only in the normal liver but also in certain liver tumours, including hepatoblastoma, the most common malignant liver tumour in childhood. In this study, sinusoids in 12 hepatoblastomas, of various subtypes, and in normal liver were investigated with UEA-1 and antibodies against von Willebrand's factor, CD31 and CD34 to detect differences of possible diagnostic significance. In the normal liver, staining of sinusoids was seen with all these markers, but it was focal and confined to a few sinusoids near the portal tracts. In hepatoblastoma, the endothelial markers reacted with the sinusoids to varying extents. UEA-1 and anti-CD34 usually stained the majority of these vessels, anti CD34 staining greater numbers of sinusoids and with greater intensity. Immunostaining revealed that both number and spatial organization of sinusoids in hepatoblastoma are dependent on the subtype. In addition to staining of endothelium, one of the two small cell hepatoblastomas exhibited strong immunoreactivity of the tumour cells for CD34. These findings show that the marked difference in sinusoidal immunoreactivity for CD34 between normal liver and hepatoblastoma could be useful for discriminating between non-neoplastic liver tissue and highly differentiated fetal hepatoblastoma. Our findings also show that small cell hepatoblastoma, in addition to acute leukaemia, should be considered when immunoreactivity for CD34 is found in small round and blue cell tumours in childhood. PMID- 7544764 TI - Pseudoangiomatous hyperplasia of male breast. AB - Ninety-three male breast specimens have been examined for the presence of pseudoangiomatous hyperplasia of the mammary stroma which has hitherto been described almost exclusively in females. Forty-four cases (47.4%) showed some degree of hyperplasia, varying from small microscopic foci to extensive change involving 90% of the mammary tissue. All but one were found in association with gynaecomastia, early and intermediate stage. The association between pseudoangiomatous hyperplasia and benign proliferative lesions mirrors that reported in the female breast, and our findings suggest that the change may represent a stage in the maturation of newly formed mammary stroma. PMID- 7544768 TI - TNF alpha blockade in rheumatoid arthritis: rationale, clinical outcomes and mechanisms of action. AB - Tumor necrosis factor alpha (TNF alpha) is a cytokine with many biological functions of relevance to inflammatory disease. Although only one of several inflammatory mediators produced in abundance in rheumatoid arthritis (RA), experimental data suggest that it is in a dominant position within a cytokine hierarchy and is therefore a prime target for directed immunotherapy in this disease. We have targeted TNF alpha in vivo using a chimerised monoclonal anti TNF alpha antibody and have now demonstrated beneficial responses to treatment in three different clinical trials. The results confirm that TNF alpha is of central importance in the inflammatory process in RA and define a new treatment strategy in this disease. PMID- 7544765 TI - Cutaneous ciliated cyst of the lower limb in a male. AB - Cutaneous ciliated cysts of the lower limb are rare. Almost exclusively occurring in women they have been widely regarded as Mullerian heterotopias. A second case in a man is reported, the literature reviewed and the origin of this lesion briefly discussed. PMID- 7544770 TI - Perianal basal cell carcinoma. PMID- 7544766 TI - Comparison of liver histology in chronic active hepatitis C and chronic active hepatitis B. AB - OBJECTIVE: To study the histological features of chronic active hepatitis C (CAH C) and to compare these with those of chronic active hepatitis B (CAH-B). METHODS: Thirty-two liver biopsy specimens from patients with chronic active hepatitis and presence of antibodies to hepatitis C on second generation enzyme immunoassay were studied and compared with those in 34 patients with CAH-B. Seventeen of the 32 CAH-C patients had fully developed or developing cirrhosis of liver whereas the remainder had only chronic active hepatitis. RESULTS: Among 32 patients with CAH-C, fatty change (20), Kupffer cell hyperplasia (30), sinusoidal lymphocytosis (27) lymphoid follicles aggregates in portal tracts (26) and bridging necrosis (16) were regular features. Focal necrosis, bile duct necrosis, cholestasis and ground glass cells were however seen much less often. On the other hand, in patients with CAH-B, fatty change (no patient), sinusoidal lymphocytosis (one patient) and lymphoid follicles/aggregates in portal tracts (one patient) were rare. Also, Kupffer cell hyperplasia (22 patients) was seen less commonly in patients with CAH-B as compared to CAH-C. Focal necrosis (34 patients), bile ductular proliferation (9 patients), cholestasis (17 patients) and ground glass cells (15 patients) were more prominent in CAH-B. CONCLUSION: Presence of certain histological features can help in distinguishing between CAH C and CAH-B. PMID- 7544767 TI - Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide. AB - Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed. PMID- 7544769 TI - Adjuvants, endocrines and conserved epitopes; factors to consider when designing "therapeutic vaccines". AB - Research into immunity to complex intracellular parasites has recently placed emphasis on the identification of peptide sequences recognised by T-cells, often with the dual objectives of finding species-specific protective epitopes, and of understanding selection of Th1 versus Th2 response patterns. In this review it is suggested that although such work is interesting, it will not achieve these objectives, which must, however, be addressed before we can design the new generation of therapeutic vaccines which may eventually replace antimicrobial drugs in the treatment of infection. First, we suggest that the balance of Th1 to Th2 lymphocyte activity is not determined by epitopes, but rather by adjuvant effects of microbial components which we have barely begun to define, and local endocrine effects mediated by conversion of prohormones into active metabolites by enzymes in lymph node macrophages. Cytokines play a role as mediators within these pathways. In chronic disease states there is a tendency for T-cell function to shift towards Th2. We describe immunopathological consequences of this tendency, including a putative role for agalactosyl IgG, and review evidence for involvement of changes in the endocrine system, brought about not only by the cytokine-hypothalamus-pituitary-adrenal axis, but also by direct actions on peripheral endocrine organs of excess levels of cytokines such as TNF alpha, TGF beta and IL-6. We summarise evidence that the epitopes that are targets for protective cell-mediated responses to complex organisms are usually not species specific. In tuberculosis, cellular responses to species-specific components appear to be associated with immunopathology rather than protection. Finally, we discuss how application of these principles has led to remarkable results in the immunotherapy of tuberculosis, including multidrug-resistant disease. PMID- 7544771 TI - The sensitization of mucosal mast cells during infections with Trichostrongylus colubriformis or Haemonchus contortus in sheep. AB - Responses of isolated mucosal mast cells (MMC) during infections with either Trichostrongylus colubriformis or Haemonchus contortus were examined by measuring the release of sheep mast cell protease (SMCP) in a degranulation assay. MMC from sheep immune to T. colubriformis released maximal amounts of SMCP and histamine within 0.5h of incubation with larval antigen whereas maximum secretion of leukotrienes occurred 3h after addition of antigen. It was only after 8 weeks of a primary T. colubriformis infection, that MMC released significantly elevated levels of SMCP (23%); this occurred when the worm burden was being rejected. In contrast, the SMCP release from MMC of immune sheep was significantly higher at 40%, and occurred within 1-4 days after challenge (DAC). The SMCP release peaked at 6-8 DAC at 51%, and declined after 56 DAC to < 25%. MMC isolated from the duodenum and mid-small intestine of immune sheep released 2-3 times higher proportion of SMCP than did cells recovered from the terminal ileum. Mast cell numbers were similar in the 3 regions but the quantity of globule leucocytes (GL) was 2.5 times higher in the duodenum. During infections with H. contortus in the abomasum, MMC isolated from the small intestine released greater levels of SMCP when incubated with larval antigens than did abomasal MMC. There was no increased release during the first 12 weeks of a primary infection although the SMCP release (23%) from immune MMC at 7-10 DAC was significantly enhanced. Once again the release from MMC isolated from the three intestinal regions of sheep immune to H. contortus was lowest in the terminal ileum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544772 TI - Acute suppurative parotitis and parotid abscess in children. AB - Acute suppurative parotitis and parotid gland abscess are infrequently encountered among children. Fourteen children were diagnosed in the Sheba Medical Center as suffering from these infections and their clinical features are described. Discussion reveals neither significant differences nor predominating factors to differentiate between these two entities. PMID- 7544775 TI - Vascular permeability factor, tumor angiogenesis and stroma generation. AB - Vascular permeability factor (VPF/VEGF) is a highly conserved multifunctional cytokine that acts directly on endothelial cells (ECs) to activate phospholipase C and induce [CA2+]i transients. Two high-affinity receptors, both tyrosine kinases, have been described. VPF/VEGF has at least two important roles in tumor biology: (1) it potently increases microvascular permeability to plasma proteins, thereby modifying the tumor extracellular matrix to promote the ingrowth of fibroblasts and new blood vessels, and (2) it is a selective EC mitogen. VPF/VEGF is also involved in several other nonmalignant processes with a pathogenesis analogous to that of tumor stroma generation, including wound healing and rheumatoid arthritis. PMID- 7544774 TI - Expression of integrin and CD44 adhesion molecules on neuroblastoma: the relation to tumor aggressiveness and embryonic neural-crest differentiation. AB - The immunohistological expression of integrins and CD44 cell adhesion molecule was analyzed on neuroblastoma (NB) specimens to study the potential role of these molecules in normal differentiation and in the transformation of neural crest derivatives. None of the specimens expressed the alpha 5 beta 1 integrin heterodimer; the expression of alpha 3 beta 1 heterodimer was maintained during all stages of differentiation; alpha 1 beta 1 heterodimer was expressed on undifferentiated neuroblasts and on Schwann cells, but was lost on ganglion cells. In contrast alpha 2 beta 1, alpha 6 beta 1, alpha 6 beta 4 and alpha V beta 1 expression was usually restricted to cells differentiated in the Schwann cell lineage. Alpha V beta 3 was expressed on tumors developed in the mediastinum. CD44 was strongly detected on differentiated ganglioneuroblastomas, stage 1 and 2 ganglioneuromas, as well as low-grade stage 4S NB and normal neuroblasts migrating in the fetal adrenal gland. CD44 expression was observed on Schwann cells and ganglion cells; in contrast, it was expressed on only 50% stage 3 and 4 undifferentiated NB. None of these specimens expressed exons V5, V7 or V6. In a few specimens, an intracellular expression of exons V8-V10 was observed in ganglion cells. The expression of CD44 on NB may reflect its pattern of expression on sympatho-adrenal precursors and arrest differentiation at these stages. Conversely, CD44 expression may be silenced during malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544773 TI - Expression of beta 1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity. AB - Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 microgram/ml) of anti-beta 1 integrin antibody. The amounts of total cellular beta 1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of beta 1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more beta 1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of beta 1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells. PMID- 7544777 TI - CD44 isoforms in metastatic cancer. AB - CD44 designates a large family of proteins generated from one gene by alternative splicing. Variants of CD44 (CD44v) differ from the standard form (CD44s) by usage of ten variant exons in various combinations. Some variants have been causally related to the metastatic spread of rat tumor cells. In human mammary carcinomas and colorectal carcinomas, the expression of CD44v has also been correlated with more progressed tumor stages. Moreover, the expression of CD44v on mammary and colorectal carcinomas correlates with a bad prognosis for patient survival. The biochemical features of these CD44 isoforms that may account for both their normal functions and their roles in tumor progression are discussed. PMID- 7544778 TI - Fibronectin and its alpha 5 beta 1 integrin receptor in malignancy. AB - Fibronectin and the alpha 5 beta 1 integrin play complex roles in malignancy. While normal cells usually deposit a fibronectin matrix around themselves, malignant cell lines often fail to do so. Decreased expression of the alpha 5 beta 1 integrin, which directs fibronectin matrix deposition, is one of the reasons for deficient matrix deposition. The defect can be corrected by expression of additional alpha 5 beta 1 from transfected cDNAs and exacerbated by selecting cells for loss of all alpha 5 beta 1. Tumorigenicity is suppressed in the alpha 5 beta 1 overexpressors and enhanced in the alpha 5 beta 1-negative cells. Synthetic peptides that bind to the alpha 5 beta 1 and other integrins can modulate their functions and suppress malignancy and metastasis in vivo. The signaling mechanisms that underlie these tumor-suppressive effects of the fibronectin matrix and the integrins are an important target for ongoing study. PMID- 7544779 TI - Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11 cis retinol dehydrogenase. AB - PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11 cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde. PMID- 7544776 TI - Mast cell accumulation at sites of murine tumor implantation: implications for angiogenesis and tumor metastasis. AB - To investigate the role of mast cells during the process of tumor angiogenesis, we compared the rates of tumor vascularization, growth and metastasis in control WBB6F1(-)+/+ mice and in their mast-cell-deficient WBB6F1-W/Wv littermates injected with MB49 murine bladder carcinoma cells. Our results demonstrate that in mast-cell-deficient mice injected with tumor cells, there is a decreased number of capillaries at the tumor periphery, reduced tumor size relative to control mice, and an absence of metastases. In contrast, when tumor cells were inoculated intravenously, both strains of mice showed high numbers of lung metastases. These results suggested that the reduction of blood vessels at the tumor periphery may lead to a reduction in the number of metastatic cells shed into the circulation in mast-cell-deficient mice. PMID- 7544780 TI - Differential gene expression in healing rat corneal epithelium. AB - PURPOSE: The authors used and validated a recently developed method, mRNA differential display, to detect and clone genes that are differentially expressed in healing compared to stationary corneal epithelium. METHODS: RNAs from unwounded and 18-hour postwound corneal epithelia were isolated and subjected to mRNA differential display analysis. The generated cDNAs were used as probes in Northern blot analysis and in situ hybridization to confirm their differential expression and to clone longer or full-length cDNAs from a healing corneal epithelial cDNA library. RESULTS: Changes in the pattern of gene expression in healing epithelium, compared with that in stationary cells, were noted. To date, 15 combinations of 5'- and 3'- primers were used with approximately 1500 mRNA species screened. Differential expression of nine mRNA species were observed. These included four known proteins. They are nonmuscle tropomyosin TM-1, cytokeratin K14, small GTP binding protein rab 11, and amyloid beta-A4 precursor like protein-2. One is a sequence with homology to type II cytokeratin, and four represent genes with sequences that are unreported. The differential expression of five of these genes was confirmed by Northern blot analysis, in situ hybridization, or both. CONCLUSION: mRNA differential display provides a unique and powerful experimental system to study differential gene expression in wound healing and cell migration. Using this system, differential expression of nine genes was observed. Detection of genes differentially expressed in healing epithelium may prompt studies that will define the specific role of each of the proteins in wound healing. PMID- 7544781 TI - Fetal topography of bovine rhodopsin mRNA suggests retinotopographically determined gene expression. AB - PURPOSE: To understand the developmental processes in the differentiating bovine retina, topographic accumulation of rhodopsin mRNA in staged fetal and adult retinas was analyzed. METHODS: Isolated retinas were spread on a nylon membrane with the photoreceptor cells facing the membrane and dissected into 25-mm square tissue segments, sometimes with as many as 150 segments/eye. Subsequent to disruption of the tissue in each segment, rhodopsin and beta-actin mRNA levels were quantitated with a solution hybridization assay. Slight variations in RNA extraction efficiency and retinal segment size were corrected using beta-actin mRNA as an internal standard. RESULTS: Analysis of multiple fetal and adult bovine retinas revealed a relatively static central-to-peripheral gradient of rhodopsin mRNA level that appears at the time of transcriptional induction (6 to 6.5 months of gestation) and persists into adulthood. After induction of rhodopsin mRNA expression, increase of rhodopsin mRNA levels was detected simultaneously in all retinal segments. Furthermore, the rate of increase in rhodopsin mRNA levels in peripheral and central regions was identical. CONCLUSIONS: Fetal induction of rhodopsin mRNA expression occurs simultaneously in all photoreceptor cells across the retina, but the levels are set according to a topographically predetermined pattern. This suggests that regulation of accumulation of rhodopsin mRNA during development is determined according to spatial coordinates before gene induction, most likely in a nonphotoreceptor retinal cell type. PMID- 7544783 TI - Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. AB - PURPOSE: The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. METHODS: A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration. RESULTS: Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta 1 and anti-alpha 5 antibodies each inhibited haptotactic cell migration to fibronectin and GRGDSP peptide. CONCLUSIONS: Epidermal growth factor provides an important stimulus of haptotactic cell migration of human corneal epithelial cells. Stimulation of cell migration by EGF was maximal in the range of 5 to 10 ng/ml; this response was completely blocked by incubation with an anti-EGF receptor antibody. Function-blocking integrin antibodies, specifically anti-beta 1 and anti-alpha 5, inhibited integrin-mediated cell migration to fibronectin and GRGDSP peptide. These data suggest that EGF represents an essential initial stimulus for haptotactic cell migration of human corneal epithelial cells; furthermore, integrins are important in mediating cell migration to fibronectin and GRGDSP: PMID- 7544784 TI - Role of transcription, translation, and protein turnover in controlling the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the lens. AB - PURPOSE: To determine the principle site (epithelium or superficial cortex) of gene transcription and mRNA translation for the regulatory enzyme of lens cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). To evaluate the contribution of waning enzyme synthesis versus enzyme turnover by proteolysis in accounting for the disappearance of HMGR protein from elongated fiber cells. METHODS: Young rats were treated with lovastatin, a drug that increases transcripts of the HMGR gene and translation of HMGR mRNA in lens secondary to inhibiting cholesterol biosynthesis. The relative concentration of HMGR mRNA in lens epithelium and superficial cortex was estimated by a competitive reverse transcriptase-polymerase chain reaction system. Relative HMGR protein levels were estimated by Western blot analysis. Because lovastatin is cleared rapidly from the lens, the half-life of HMGR protein in epithelium and cortex was estimated by following the disappearance of the increased pool of enzyme protein from each compartment with time after halting drug treatment. RESULTS: Between 75% and 90% of the total content of HMGR mRNA and protein in the epithelium and the superficial cortex of control rat lens was located in the cortex. Treatment with lovastatin increased the content of the mRNA in epithelium and cortex by approximately 0.4-fold and HMGR protein content approximately 5 fold. Although the concentration of HMGR mRNA and protein was similarly increased in epithelium and superficial cortex, approximately 85% to 90% of the total increase in mRNA and protein content was located in the cortex because of that area's greater mass. The half-life for the disappearance of the increased pool of HMGR protein from epithelium and cortex was similar at approximately 14 to 17 hours. CONCLUSIONS: The bulk of HMGR gene transcription and mRNA translation apparently is confined to elongating fiber cells. The 10-fold greater increase in enzyme protein than mRNA levels after lovastatin treatment indicates that enzyme concentration in lens is controlled mainly by effects on HMGR mRNA translation or rates of HMGR proteolysis. The observed rapid turnover of enzyme protein in the epithelium and the superficial cortex, if applicable to the deeper cortex and the homeostatic state (absence of drug exposure), suggests that the gradual disappearance of HMGR protein from the lens could be caused by waning of enzyme synthesis rather than to proteolysis in the absence of continuing enzyme synthesis. PMID- 7544785 TI - Substance P immunoreactivity in rat von Ebner's gland. AB - The present immunohistochemical study revealed substance P-immunoreactive neuronal elements in the von Ebner's gland of rats. Immunoreactive ganglion cells were observed as single cells or groups of several immunoreactive ganglion cells among intra-lingual muscles, at the base of the vallate papillae and near the von Ebner's gland. Very numerous substance P-immunoreactive varicose nerve fibres ran closely associated with the serous cells and excretory duct cells, and were seen to run along blood vessels in the gland. Since substance P-immunoreactive ganglion cells were present near the glands, the immunoreactive varicose nerve fibres in the von Ebner's gland may be partly derived from the intra-lingual ganglion cells. These substance P-immunoreactive varicose nerve fibres may have an effect on the secretory activity of the serous cells and duct cells, and on the vasodilation of blood vessels of the von Ebner's gland. Actin immunoreactivity was seen in numerous myoepithelial cells embracing serous cells and duct cells, and in the smooth muscle cells of blood vessels of the gland. By using a double immunolabelling technique with anti-substance P and anti-actin sera, substance P-immunoreactive varicose nerve fibres were found to be in close contact with myoepithelial cells. PMID- 7544782 TI - A major human lacrimal gland mRNA encodes a new proline-rich protein family member. AB - PURPOSE: To examine the existence of novel protein products of the human lacrimal gland. METHODS: cDNA clones corresponding to a highly abundant human lacrimal gland mRNA were isolated and sequenced. Tissue distribution of expression was studied by Northern blot analysis, RNase protection analysis, and in situ hybridization. RESULTS: A highly abundant 600-base mRNA was identified, and corresponding cDNA clones were isolated. The mRNA has a 134-residue open reading frame encoding a secreted protein of 13458 Da. This protein shows 45.5% similarity to human salivary acidic proline-rich protein PRP 1 and has a similar domain structure, but, unlike other members of the proline-rich protein family, it lacks a conserved repetitive domain. The lacrimal proline-rich protein is encoded by a single gene, designated as LPRP. Expression of LPRP was also detected in the human submandibular, von Ebners, sublingual, and parotid glands. LPRP was expressed in the acinar cells of the lacrimal gland, and in the submandibular gland expression of the LPRP and PRP 1 genes was localized to the serous acini and demilunes. CONCLUSIONS: The human lacrimal gland produces a previously unknown member of the proline-rich protein family. By analogy with other proline-rich proteins, this LPRP most likely mediates protective functions in the eye, such as modulation of the microflora. In contrast to other proline rich protein genes, LPRP is expressed in the lacrimal acinar cells, and other anterior exocrine glands. LPRP should be a useful marker for human lacrimal gland acinar cell function in vitro. PMID- 7544788 TI - Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels. AB - A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis. PMID- 7544786 TI - Kinetics of tyrosine phosphorylation when IgE dimers bind to FC epsilon receptors on rat basophilic leukemia cells. AB - Previously, we demonstrated that aggregates of the high affinity receptor for IgE (Fc epsilon RI), formed by the binding of chemically cross-linked oligomers of IgE, continue to signal early and late cellular responses long after the formation of new aggregates is blocked. In the present work, we explore quantitatively the relationship between aggregation of the receptors and one of the earliest biochemical changes this initiates. We compare the time course of aggregate formation, inferred from studies of the binding of dimers of IgE, and the time course of phosphorylation of tyrosines on receptor subunits when the receptors are aggregated. A simple model does not fit the data. It appears that aggregates formed late in the response are less effective signaling units than those formed initially. We propose new explanations for the persistence of the response and the unusual kinetics. PMID- 7544790 TI - Osmotic loading of neutralizing antibodies demonstrates a role for protein tyrosine phosphatase 1B in negative regulation of the insulin action pathway. AB - Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus. PMID- 7544791 TI - Differential modulation of the RNA-binding proteins IRP-1 and IRP-2 in response to iron. IRP-2 inactivation requires translation of another protein. AB - Iron regulatory proteins (IRPs)-1 and -2 bind specific mRNA hairpin structures known as iron-responsive elements and thereby post-transcriptionally regulate proteins involved in iron uptake, storage, and utilization. In this study, we compared modulation of the RNA-binding activities of IRP-1 and IRP-2. We show that in vitro RNA-binding can be inhibited for each IRP by the alkylation of free sulfhydryl groups with N-ethylmaleimide, or by oxidation with diamide. The in vivo iron regulation of IRP-1 and IRP-2 appeared to involve different pathways. Both proteins are activated in Ltk- cells following iron chelation. This induction, however, was distinguishable by the addition of translation inhibitors, which temporarily delayed activation of IRP-1 by up to 8 h, but fully blocked IRP-2 induction for up to 20 h. The activation of IRP-2 was also prevented by transcription inhibition with actinomycin D. Further analysis revealed that, while both IRPs are rapidly inactivated following iron treatment of iron-depleted cells, the repression of IRP-2 was again completely translation dependent. Immunoblot analysis suggests that iron modulation of IRP-1 activity is predominantly a posttranslational process. This contrasts with IRP-2, whose activation reflected the accumulation of stable IRP-2 protein by de novo synthesis. IRP-2 inactivation/degradation occurred upon readdition of iron, but it required translation of another protein. The existence of an independent regulator of IRP-2 may help explain the differential regulation and expression of the two IRP proteins in different tissues and cell lines. PMID- 7544795 TI - Biogenesis of synaptic vesicles in vitro. AB - Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved. PMID- 7544789 TI - Interleukin-9 induces tyrosine phosphorylation of insulin receptor substrate-1 via JAK tyrosine kinases. AB - Interleukin (IL)-9 stimulates the proliferation of a variety of hematopoietic lineages through its interaction with a receptor of the cytokine receptor superfamily. In the studies presented here, we have begun to characterize the downstream signaling pathways activated by IL-9. In addition to the activation of JAK1 and JAK3 tyrosine kinases, IL-9, unlike most hematopoietic cytokines but similar to IL-4, induces the tyrosine phosphorylation of a 170-kDa protein that is related to the insulin receptor substrate-1 (IRS-1). We further demonstrate for the first time that IRS-1 is not only associated with JAK1 but also tyrosine phosphorylated and functionally involved in IL-9 signaling in TS1 lymphocytes transfected with the murine IRS-1 cDNA. Cotransfection studies and in vitro experiments directly demonstrate that JAK1, JAK2, or JAK3 is capable of tyrosine phosphorylating IRS-1, suggesting a functional role for these kinases in vivo. Lastly, we demonstrate that IL-9 induces the tyrosine phosphorylation of Stat3 and in this regard differs from IL-4, which triggers tyrosine phosphorylation of Stat6. Taken together, these results strongly suggest that IL-9 and IL-4 utilize common and unique signaling pathways via inducing the similar and distinct tyrosine-phosphorylated proteins. PMID- 7544794 TI - Shc isoform-specific tyrosine phosphorylation by the insulin and epidermal growth factor receptors. AB - Insulin stimulation of Chinese hamster ovary cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IR/ER) resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform with a relatively low extent of 46-kDa Shc tyrosine phosphorylation. In contrast, EGF stimulation resulted in the tyrosine phosphorylation of both the 52- and 46-kDa Shc isoforms. Consistent with these differences, Grb2 predominantly bound to the 52-kDa Shc isoform following insulin stimulation, whereas Grb2 associated with both the 52- and 46-kDa Shc isoforms after EGF stimulation. Further, in vitro kinetic analysis demonstrated that the insulin receptor has a 4-fold greater Vmax with no significant difference in the Km for the purified 52-kDa Shc isoform compared with the 46-kDa Shc isoform. However, the EGF receptor displayed the identical Vmax and Km for tyrosine phosphorylation of both of these species. In direct contrast to the EGF receptor, we also observed significant differences in binding interactions between the insulin receptor with the 52- and 46-kDa Shc isoforms in vitro. These data demonstrate that the predominant insulin-dependent Shc signaling pathway occurs via the 52-kDa Shc isoform, whereas the EGF receptor can effectively use both the 52- and 46-kDa Shc species. PMID- 7544787 TI - Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells. AB - The growth potentiating effects of the insulin-like growth factor (IGF)-I and IGF II are modulated by a family of six insulin-like growth factor binding proteins (IGFBPs). Despite the similarity in amino acid sequences of the IGFBPs, their effects on the growth of bone cells differ. Studies on the molecular mechanisms for IGFBP-4 actions revealed that coincubation of bone cells with IGFBP-4 and 125I-IGF-I or 125I-IGF-II decreased the binding of both of these ligands in a dose-dependent manner. In addition, IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF receptor. These data in conjunction with data showing that IGFBP-4 had no effect on cell proliferation induced by analogs of IGF-I or IGF II, which exhibited > 100-fold reduced affinity for binding to IGFBP-4 suggest that IGFBP-4 may inhibit IGF action by preventing the binding of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5 treatment increased the binding of IGF tracer to bone cells but did not increase the binding of 125I-IGF I to type I IGF receptor. Studies on the mechanism by which IGFBP-5 increased the binding of 125I-IGF tracer to bone cells suggest that IGFBP-5 could facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface binding sites independent of IGF receptors. These data in conjunction with the findings that IGFBP-5 potentiated cell proliferation even in the presence of those same IGF analogs that exhibited > 200-fold reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in part stimulate bone cell proliferation by an IGF independent mechanism involving IGFBP-5-specific cell surface binding sites. PMID- 7544793 TI - Identification of Lck-binding elements in tip of herpesvirus saimiri. AB - A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri. PMID- 7544799 TI - CD44 is the major peanut lectin-binding glycoprotein of human epidermal keratinocytes and plays a role in intercellular adhesion. AB - Although binding of peanut agglutinin (PNA) to keratinocytes is often used as a marker of terminal differentiation, the identity of the PNA-binding glycoproteins has been unclear. We now show that an antiserum raised against the glycoproteins recognises isoforms of CD44, the most abundant of which could be labelled with [35S]sulphate, indicating the presence of glycosaminoglycan side chains. RT-PCR analysis showed that keratinocytes expressed at least 5 forms of CD44 containing different numbers of exons from the variable region of the extracellular domain and also expressed the standard 'haemopoietic' form of CD44 which lacks the variable exons. Standard and variant isoforms of CD44 were expressed both by proliferating keratinocytes and cells undergoing terminal differentiation, although the level of CD44 mRNAs decreased when keratinocytes were placed in suspension to induce differentiation. The role of CD44 in intercellular adhesion was investigated by plating keratinocytes onto a rat pancreatic carcinoma line transfected with different CD44 isoforms. Keratinocyte adhesion to transfectants expressing variant exons 4-7 was greater than to cells expressing standard CD44 and could be inhibited with hyaluronan or digestion with hyaluronidase. These observations confirm earlier predictions that the PNA-binding glycoproteins of keratinocytes play a role in intercellular adhesion. PMID- 7544796 TI - Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes. AB - Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2 terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes. PMID- 7544797 TI - Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders. AB - Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and classical rhizomelic chondrodysplasia punctata are lethal genetic disorders caused by defects in peroxisome biogenesis. We report here a characterization of the peroxisomal matrix protein import capabilities of fibroblasts from 62 of these peroxisome biogenesis disorder patients representing all ten known complementation groups. Using an immunofluorescence microscopy assay, we identified three distinct peroxisomal protein import defects among these patients. Type-1 cells have a specific inability to import proteins containing the PTS1 peroxisomal targeting signal, type-2 cells have a specific defect in import of proteins containing the PTS2 signal, and type-3 cells exhibit a loss of, or reduction in, the import of both PTS1 and PTS2 proteins. Considering that the common cellular phenotype of Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum's disease has been proposed to be a complete defect in peroxisomal matrix protein import, the observation that 85% (40/47) of the type-3 cell lines imported a low but detectable amount of both PTS1 and PTS2 proteins was surprising. Furthermore, different cell lines with the type-3 defect exhibited a broad spectrum of different phenotypes; some showed a complete absence of matrix protein import while others contained 50-100 matrix protein-containing peroxisomes per cell. We also noted certain relationships between the import phenotypes and clinical diagnoses: both type-1 cell lines were from neonatal adrenoleukodystrophy patients, all 13 type-2 cell lines were from classical rhizomelic chondrodysplasia punctata patients, and the type-3 import defect was found in the vast majority of Zellweger syndrome (22/22), neonatal adrenoleukodytrophy (17/19), and infantile Refsum's disease (7/7) patients. Our finding that all type-1 cell lines were from the second complementation group (CG2), all 13 type-2 cell lines were from CG11, and that cells from the eight remaining complementation groups only exhibit the type-3 defect indicates that mutations in particular genes give rise to the different types of peroxisomal protein import defects. This hypothesis is further supported by correlations between certain complementation groups and particular type-3 subphenotypes: all patient cell lines belonging to CG3 and CG10 showed a complete absence of peroxisomal matrix protein import while those from CG6, CG7, and CG8 imported some peroxisomal matrix proteins. However, the fact that cell lines from within particular complementation groups (CG1, CG4) could have different matrix protein import characteristics suggests that allelic heterogeneity also plays an important role in generating different import phenotypes in certain patients.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7544798 TI - Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma. AB - We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell extra-cellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express alpha 1, alpha 2, alpha 3, alpha 6, beta 1 and beta 4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or downregulation of these subunits. Function-blocking experiments using inhibitory anti-integrin subunit antibodies showed a > 5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-beta 1 or anti-alpha 3 antibodies, whereas anti-alpha 2 or -alpha 6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-beta 1 and -alpha 2 antibodies but not by anti-alpha 3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or capacity to form colonies. Thus under our culture conditions breast acinar formation is at least a two-step process involving beta 1-integrin dependent cellular growth followed by polarization of the cells into organized structures. The regulation of this pathway appears to be impaired or lost in the tumor cells, suggesting that tumor colony formation occurs by independent mechanisms and that loss of proper integrin-mediated cell-ECM interaction may be critical to breast tumor formation. PMID- 7544792 TI - Post-transcriptional regulation of Na+/glucose cotransporter (SGTL1) gene expression in LLC-PK1 cells. Increased message stability after cyclic AMP elevation or differentiation inducer treatment. AB - We have further investigated the molecular basis of increased differentiation regulated expression of SGTL1, a Na+/glucose cotransporter, in the renal epithelial cell line LLC-PK1. Treatment of confluent monolayers either with the differentiation inducer hexamethylene bisacetamide (HMBA) or with cyclic AMP elevating agents promoted increased levels of the SGLT1 mRNA, the immunodetectable 75-kDa cotransporter subunit, and the transport activity. Two molecular species of SGLT1 mRNA (2.2 and 3.9 kilobases (kb)) are transcribed from the same gene in LLC-PK1 cells and differ only in the length of the 3' untranslated region. The larger transcript is less stable (t1/2 = 2 h) than the smaller one (t1/2 = 10 h) in control, confluent monolayers. The 3.9-kb species was stabilized from degradation after either cyclic AMP elevation (t1/2 = 14 h) or HMBA addition (t1/2 = 8 h), with negligible effects on the stability of the 2.2-kb species (t1/2 = 11 h). Inhibition of translation by cycloheximide resulted in a 10-fold increase in the t1/2 of the 3.9-kb transcript and a 2-fold increase in that of the 2.2-kb species in control monolayers. Our results demonstrate that post-transcriptional regulation of message stability plays a major role in differentiation-dependent SGTL1 expression promoted by either HMBA or cyclic AMP. PMID- 7544800 TI - Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. AB - The redox status of human spermatozoa was found to have a profound influence on the fertilizing potential of these cells in association with qualitative and quantitative changes in the patterns of tyrosine phosphorylation. In general, oxidizing conditions enhanced tyrosine phosphorylation and stimulated sperm function, whereas reducing conditions had the opposite effect. Unstimulated human spermatozoa exhibited low levels of spontaneous acrosomal exocytosis and sperm oocyte fusion and minimal reactive oxygen species generation, while phosphotyrosine expression was largely confined to a single protein of 116 kDa. However, if the spermatozoa were exposed to oxidizing conditions through the addition of exogenous H2O2, or the stimulation of endogenous NADPH-dependent reactive oxygen species generation, then a dramatic increase in tyrosine phosphorylation was observed (major phosphotyrosyl bands at 222 kDa, 200 kDa, 159 kDa, 133 kDa, 116 kDa and 82 kDa) in concert with the functional activation of the spermatozoa. A causal association between reactive oxygen species generation, tyrosine phosphorylation and sperm function was indicated by studies with the ionophore, A23187, which induced high rates of spermoocyte fusion together with enhanced rates of reactive oxygen species production and the increased expression of phosphotyrosyl proteins. This functional response to A23187 could be abrogated, without any concomitant change in sperm motility or viability, by using membrane permeant thiols or catalase to suppress the reactive oxygen species-induced increase in phosphotyrosine expression. The fact that the biological responses of human spermatozoa to biological agonists (recombinant human ZP3 and progesterone) could also be inhibited by catalase indicated the general relevance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544801 TI - The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus. AB - FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos 7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59 HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i.e. in microtubule depolymerizing conditions). Exposure of cells to 1 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7544802 TI - Heart CD36 expression is increased in murine models of diabetes and in mice fed a high fat diet. AB - High levels of CD36 expression are found in triglyceride storing and secreting cells such as differentiated adipocytes and mammary secretory epithelial cells and in some capillary endothelial cells. We have found high levels of CD36 in the capillary endothelium of murine adipose tissue and in cardiac and skeletal muscles. Muscle cells themselves were CD36 negative. No CD36 was found in brain endothelium. Cardiac and skeletal muscle tissues are highly oxidative and catabolize long-chain fatty acids as a source of energy while brain tissue does not use long-chain fatty acids for energy production. Since capillary endothelial cell CD36 expression appeared to correlate with parenchymal cell fatty acid utilization and since CD26 has been identified recently as a long-chain fatty acid-binding protein, we examined heart tissue CD36 expression in murine models of insulin-dependent (nonobese diabetic, NOD) and non-insulin-dependent diabetes mellitus (KKAY). Diabetic NOD and KKAY mice had serum triglyceride levels 2.6- and 4.2-fold higher, respectively, than normal mice and exhibited 7- and 3.5-fold higher levels of heart microsomal CD36, respectively, than control mice. Mice fed a 40% fat diet expressed heart tissue CD36 at a level 3.5-fold higher than those fed a 9% fat diet. These data suggest that endothelial cell CD36 expression is related to parenchymal cell lipid metabolism. PMID- 7544803 TI - Advanced glycation endproducts interacting with their endothelial receptor induce expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. AB - Vascular cell adhesion molecule-1 (VCAM-1), an inducible cell-cell recognition protein on the endothelial cell surface (EC), has been associated with early stages of atherosclerosis. In view of the accelerated vascular disease observed in patients with diabetes, and the enhanced expression of VCAM-1 in diabetic rabbits, we examined whether irreversible advanced glycation endproducts (AGEs), could mediate VCAM-1 expression by interacting with their endothelial cell receptor (receptor for AGE, RAGE). Exposure of cultured human ECs to AGEs induced expression of VCAM-1, increased adhesivity of the monolayer for Molt-4 cells, and was associated with increased levels of VCAM-1 transcripts. The inhibitory effect of anti-RAGE IgG, a truncated form of the receptor (soluble RAGE) or N acetylcysteine on VCAM-1 expression indicated that AGE-RAGE-induced oxidant stress was central to VCAM-1 induction. Electrophoretic mobility shift assays on nuclear extracts from AGE-treated ECs showed induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter, which was blocked by anti-RAGE IgG or N-acetylcysteine. Soluble VCAM-1 antigen was elevated in human diabetic plasma. These data are consistent with the hypothesis that AGE-RAGE interaction induces expression of VCAM-1 which can prime diabetic vasculature for enhanced interaction with circulating monocytes. PMID- 7544806 TI - Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. AB - In vivo, endothelial cells (EC) are subjected to hemodynamic forces which may influence the production of nitric oxide. This study was designed to examine the effect of cyclic strain on the expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic EC. EC were grown on flexible membranes which were subjected to deformation at 60 cycles/min with -5 or -20 kPa of vacuum. This results in an average strain of 6 and 10%, respectively, which is transmitted to the attached cells. Northern blot analysis of total cytosolic RNA demonstrated an increase in eNOS gene expression with both strain regimens but the increase with 10% average strain was greater than that at 6%. Nuclear runoff transcription assays confirmed the induction of eNOS transcripts. Western blot analysis showed an increase in eNOS level after 24 h of cyclic 10% average strain compared with controls or 6% average strain. Immunohistochemical staining of EC for eNOS was increased in the high strain periphery (7-24% strain) of membranes deformed with 20 kPa vacuum. These results demonstrate that cyclic strain upregulates the expression of eNOS transcripts and protein levels in bovine aortic EC thus emphasizing the importance of hemodynamic forces in the regulation of eNOS in vivo. PMID- 7544808 TI - Antibody-dependent complement-mediated cytotoxicity in sera from patients with HIV-1 infection is controlled by CD55 and CD59. AB - Various immune mechanisms have been reported to contribute to the progressive destruction of Th cells in HIV-1-infected patients. Among these, complement mediated lysis of infected cells has been suggested. An increased sensitivity of lymphocytes from HIV-1-infected patients to lysis by monoclonal antibodies directed to MHC class I antigen and complement has been directly correlated with a decreased expression of the decay accelerating factor (CD55). It also has been reported that the expression of the membrane inhibitor of reactive lysis (CD59) is decreased during HIV-1 infection. We examined the effect of antibodies in the serum of HIV-1-positive individuals and normal human serum (NHS) as source of complement on several HIV-1-infected cell lines differing in their expression of CD55 and CD59. When HIV-1-infected target cells without membrane expression of CD55 and CD59 were used, a highly significant cytotoxic effect was observed in the presence of heat inactivated anti-HIV-1-positive sera and NHS, while heat inactivated anti-HIV-1-negative sera and NHS were unable to induce cytolysis. Similar results were obtained using purified IgG isolated from HIV-1-positive sera and either NHS or guinea pig serum as source of complement. Lysis of HIV-1 infected cells correlated with expression of viral antigens on the cell surface. HIV-1-infected CD55 and CD59 positive target cells showed specific lysis, when the function of these molecules was abrogated by blocking antibodies to CD55 and CD59. The finding of anti-HIV-1-specific cytotoxic antibodies in sera from HIV-1 infected patients should be considered in the pathogenesis of the HIV-1 infection. PMID- 7544805 TI - Regulation of arachidonic acid, eicosanoid, and phospholipase A2 levels in murine mast cells by recombinant stem cell factor. AB - The current study evaluates the capacity of recombinant rat stem cell factor (rrSCF) to regulate enzymes that control AA release and eicosanoid generation in mouse bone marrow-derived mast cells (BMMCs). Initial studies indicated that rrSCF provided for 24 h inhibited the release of AA into supernatant fluids of antigen- and ionophore A23187-stimulated BMMCs. Agonist-induced increases in cellular levels of AA were also inhibited, albeit to a lesser degree by rrSCF. To determine the inhibitory mechanism, several steps (e.g., mobilization of cytosolic calcium, release of BMMC granules, and regulation of phospholipase A2 [PLA2] activity) that could influence AA release were measured in rrSCF-treated cells. rrSCF did not alter the capacity of BMMCs to mobilize cytosolic calcium or release histamine in response to antigen and ionophore. BMMCs released large amounts of PLA2 with characteristics of the group II family in response to antigen and ionophore A23187. rrSCF treatment of BMMCs reduced the secretion of this PLA2 activity by BMMCs. Partial purification of acid-extractable PLA2 from rrSCF-treated and untreated BMMCs suggested that rrSCF decreased the quantity of acid-stable PLA2 within the cells. In contrast to group II PLA2, the quantity of cPLA2 (as determined by Western blot analysis) increased in response to rrSCF. To assess the ramifications of rrSCF-induced reductions in AA and group II PLA2, eicosanoid formation was measured in antigen- and ionophore-stimulated BMMCs, rrSCF-inhibited (100 ng/ml, 24 h) prostaglandin D2 (PGD2), thromboxane B2, and leukotriene B4 by 48.4 +/- 7.7%, 61.1 +/- 10.0% AND 38.1 +/- 3.6%, respectively, in antigen-stimulated cells. Similar patterns of inhibition were observed in ionophore-stimulated BMMCs. The addition of a group I PLA2 or exogenous AA to BMMCs reversed the inhibition of eicosanoid generation induced by rrSCF. Together, these data indicate that rrSCF differentially regulates group II and cytosolic PLA2 activities in BMMCs. The resultant reductions in eicosanoid generation suggest that group II PLA2 provides a portion of AA that is used for eicosanoid biosynthesis by BMMCs. PMID- 7544804 TI - Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs. AB - Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2 macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity. PMID- 7544809 TI - Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection. AB - It has been suggested that immune selection pressure exerted by the cytotoxic T lymphocyte (CTL) response could be responsible for viral persistence during chronic hepatitis B virus infection. To address this question, in the current study we compared the DNA and amino acid sequences of, and the CTL responses to, multiple HLA-A2-restricted CTL epitopes in the hepatitis B virus in several HLA A2-positive patients with acute and chronic hepatitis. Our results indicate that the CTL response to these epitopes is barely detectable in the majority of patients with chronic hepatitis. Further, we show that the weak CTL response is not secondary in infection by mutant viruses lacking these epitopes, and we show that the CTL response did not select for escape mutants in any of these patients. We conclude that an ineffective hepatitis B virus specific CTL response is the primary determinant of viral persistence in chronic hepatitis and that immune selection of viral variants is not a common event in the majority of patients. PMID- 7544807 TI - Dexamethasone enhances insulin-like growth factor-I effects on skeletal muscle cell proliferation. Role of specific intracellular signaling pathways. AB - IGF-I stimulation of cell proliferation and c-Fos expression in skeletal muscle cells is markedly enhanced by dexamethasone. The effect of dexamethasone is not mediated by changes in IGF-binding proteins, as evidenced by similar effects of dexamethasone on the actions of insulin, PDGF-BB, and the IGF-I analogue long R3IGF-I. Dexamethasone also does not alter autocrine IGF-II secretion by muscle cells. To investigate the mechanism of the augmentation of IGF-I action, the effects of dexamethasone on intracellular IGF-I signaling pathways were determined. In dexamethasone-treated cells, the levels of IGF-I receptor tyrosine phosphorylation and receptor-associated phosphatidylinositol 3-kinase activity were increased. Dexamethasone-treated cells also showed increased and prolonged tyrosine phosphorylation of the Shc proteins. In contrast, dexamethasone decreased both tyrosine phosphorylation and expression of insulin receptor substrate 1 (IRS-1) and IRS-1-associated phosphatidylinositol 3-kinase activity. Thus, distinct signaling pathways activated by the IGF-I receptor in skeletal muscle cells are differentially regulated by dexamethasone. Potentiation of IGF-I action correlates with increased IGF-I receptor-associated phosphatidylinositol 3 kinase activity and tyrosine phosphorylation of Shc, but appears to be independent of activation of the IRS-1/phosphatidylinositol 3-kinase signaling pathway. PMID- 7544810 TI - CFTR and differentiation markers expression in non-CF and delta F 508 homozygous CF nasal epithelium. AB - Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations. Our results show that the steady-state levels of CFTR, CK13, CK18, CK18, CK14, or DP 1 mRNA transcripts in delta F 508 CF nasal polyps were not significantly different from those of non-CF tissues. A variability in the CFTR mRNA transcript level and in the pattern of CFTR immunolabeling has been observed between the different tissue samples. However, no relationship was found between the level of CFTR mRNA transcripts and the CFTR protein expression and distribution, either in the non-CF or in the CF group. The histological observations of non-CF and CF nasal polyp tissue indicated that the huge variations in the expression and distribution of the CFTR protein were associated with the variations in the degree of surface epithelium remodeling and inflammation in the lamina propria. A surface epithelium, showing a slight basal cell hyperplasia phenotype associated with diffuse inflammation, was mainly characterized by a CFTR protein distribution at the apex of ciliated cells in both non-CF and CF specimens. In contrast, in a remodeled surface epithelium associated with severe inflammation, CFTR protein presented either a diffuse distribution in the cytoplasm of ciliated cells, or was absent. These results suggest that abnormal expression and distribution of the CFTR protein of CF airways is not only caused by CFTR mutations. Airway surface epithelium remodeling and inflammation could play a critical role in the posttranscriptional and/or the posttranslational regulation of the CFTR protein expression in non-CF and CF airways. PMID- 7544812 TI - Cutaneous reactions to recombinant cytokine therapy. AB - Cytokines are critical to several fundamental homeostatic mechanisms such as fever, acute phase reactions, wound healing, hematopoiesis, inflammation, cellular and humoral immune responses, and tumor regression. As a result of advances in recombinant DNA technology, recombinant cytokines are available as therapeutic agents. They have been used for metastatic cancers and immunodeficiencies, as a therapy for naturally occurring or drug-induced anemias or leukopenias, and they have also been applied to some cutaneous disorders. Cytokine therapy can result in toxic reactions that affect many organ systems, especially the skin. These reactions are common and diverse, ranging from minor injection site reactions, pruritus, and flushing to life-threatening autoimmune disorders, severe erythroderma, or bullous skin reactions. This review focuses on the major cytokines that are in current clinical use or under investigation and describes the cutaneous complications of these agents. PMID- 7544813 TI - Surfactant-induced dermatitis: comparison of corneosurfametry with predictive testing on human and reconstructed skin. AB - BACKGROUND: Surfactants elicit alterations in the stratum corneum. Predictive tests that avoid animal experimentation are needed. OBJECTIVE: This study compares three methods of rating and predicting shampoo-induced irritation. METHODS: Corneosurfametry entails collection of stratum corneum followed by brief contact with diluted surfactants and measurement of variations in staining of samples. RESULTS: Corneosurfametry appears to correlate well with in vivo testing in volunteers with sensitive skin. However, corneosurfametry presents less interindividual variability than in vivo testing and allows better discrimination among mild products. Morphologic information about surfactant-induced loosening of corneocytes may be increased by testing surfactants on human skin equivalent. Results are similar to those provided by specimens used for corneosurfametry. CONCLUSION: The corneosurfametric prediction of surfactant irritancy correlates with in vivo testing and with in vitro evaluation on human skin equivalent. PMID- 7544811 TI - Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse. AB - Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs. PMID- 7544814 TI - Chlorpyrifos formulation effect on airborne residues following broadcast application for cat flea (Siphonaptera: Pulicidae) control. AB - Airborne residues of 3 chlorpyrifos formulations were measured up to 50 h after broadcast treatment for flea control in residences. Insecticide formulation, time after treatment, ventilation regime, and height above floor affected airborne residues. Before spraying, chlorpyrifos residues were low or undetected. In nonventilated residences, chlorpyrifos residues peaked in 0-6 h after treatment at 38 ng/liter for emulsifiable, remained low at < 12 ng/liter for the microencapsulated, and sharply peaked in 1-2 h after treatment at 52 ng/liter for the aerosol formulation. Residues for all formulations then slowly declined through 50 h. In ventilated residences, chlorpyrifos residues peaked from 2 to 10 h after treatment at 25-27 ng/liter for emulsifiable, remained low at < 10 ng/liter, and sharply peaked in 0-2 h after treatment at 21 ng/liter for the aerosol formulation. Maximum airborne concentrations were 74 ng/liter for emulsifiable, 17 ng/liter for microencapsulated, and 61 ng/liter for aerosol chlorpyrifos. PMID- 7544815 TI - Prostate conditions, treatment decisions, and patient preferences. PMID- 7544816 TI - Implications of the most bothersome prostatism symptom for clinical care and outcomes research. AB - OBJECTIVES: Because treatment of benign prostatic hyperplasia (BPH) is based largely on patients' symptoms, understanding and measuring the impact of these symptoms from the patient's perspective is critically important for clinical care. Such knowledge also is crucial for comparing patient-weighted outcomes because the increasing array of medical and surgical BPH treatments differ in their impact on specific symptoms. Our purpose was to determine the most bothersome symptom in older men seeking evaluation for symptomatic BPH and to examine whether age, comorbidity, or urodynamic evidence of prostatic obstruction were important covariates. DESIGN: Prospective evaluation of a consecutive series. SETTING: Veterans Affairs urology clinic. PARTICIPANTS: 115 men (age 69 +/- 6 years) presenting for initial evaluation of prostatism. MEASUREMENTS: Scores on standard symptom index and patients' reports of the most bothersome symptom. Bladder outlet obstruction was assessed by multichannel videourodynamic evaluation. RESULTS: An "irritative" symptom (frequency, urgency, or nocturia) was cited as most bothersome significantly more often than an "obstructive" symptom (weak stream, hesitancy, etc.) (53 vs. 35%, P < .05); older men were significantly more likely to name an irritative symptom as most bothersome (chi 2 for trend = 6.63, P < .025). Even among men with prostate obstruction, most cited an irritative symptom as the most bothersome, regardless of the severity of obstruction. These associations were not confounded by comorbid conditions or medications that independently may cause symptoms. CONCLUSIONS: Because irritative symptoms are most bothersome, have a diverse differential diagnosis, and do not respond as well to BPH treatment, neither providers nor researchers should rely solely on global assessments of symptom severity and bother in assessing men with voiding symptoms. Additional focus on individual symptom impact and etiology is needed, especially in older men. PMID- 7544817 TI - Chemotaxis of T lymphocytes on extracellular matrix proteins. Analysis of the in vitro method to quantitate chemotaxis of human T cells. AB - The present report compares a variety of T cell purification protocols and chemotaxis procedures in assessing chemokine-induced T cell migration using a microchemotaxis assay. Rapidly purified T cells are capable of directly responding to the beta chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, and RANTES in the absence of alpha CD3 stimulation as previously described (Taub, D.D. and Oppenheim, J.J. (1993) Cytokine 5, 175). However, T cell purification schemes involving prolonged 37 degrees C incubations generally produce non-motile T lymphocytes that require stimulation with alpha CD3 antibody for 6-12 h in culture to recover chemotactic mobility. This loss of chemotactic potential appears to be due to prolonged 37 degrees C incubations as rapidly purified T cells lose migratory activity upon incubation at 37 degrees C. Radiolabeled binding analysis revealed that beta chemokine binding sites are downregulated as short as 2 h after incubation at 37 degrees C. T cells require the presence of extracellular matrix molecules to facilitate T cell migration. While many of these proteins permit chemotactic activity, human plasma and foreskin fibronectin were found to be the most effective matrix molecule for T cell migration. Kinetic analysis of T cell activation revealed that 6-12 h of anti-CD3 stimulation was optimal to restore the ability of purified T cells to migrate in response to the chemokines MIP-1 alpha, MIP-1 beta, RANTES, and IL-8. However, rapidly dividing T cells (> or = 48 h post alpha CD3 mAb stimulation) fail to migrate in response to any chemotactic stimulus. Together, these results suggest that the measurement of T cell migration, using microchemotaxis chambers, is a multifactorial process with strict environmental and activation requirements. PMID- 7544818 TI - Sandwich immunoassay specific for the N-terminal sequence of osteocalcin. AB - A monoclonal antibody (10B) against the N-terminal sequence of human osteocalcin was selected to characterize its epitope and species specificity. The cross reactivity of 10B with human and rat osteocalcin demonstrated that the reactivities of 10B with both human and rat osteocalcins were very similar. The pin technology method was used to determine the epitope and clearly demonstrated that the epitope recognized by 10B was localized to residues 12-16, the sequence of which is identical in rat and human osteocalcin molecules. This monoclonal antibody was found to be useful for designing region-specific sandwich immunoassay systems for human and rat N-terminal osteocalcin (N-OC) molecules using rabbit anti-hN20 and anti-rN20 polyclonal antibodies, respectively. The osteocalcin levels in serum determined by this N-OC method were stable during prolonged storage at 25 degrees C and the method could be usefully applied in the development of immunoassay systems for many osteocalcin molecules from many other species. PMID- 7544819 TI - Effect of mosquito age and reproductive status on melanization of sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae. AB - Malaria-refractory and -susceptible strains of the mosquito vector, Anopheles gambiae, differ in their response to negatively-charged Sephadex CM-25 beads. CM 25 beads elicit a much stronger melanization reaction in refractory mosquitoes than in susceptible mosquitoes. Light microscopic and scanning electron microscopic studies documented a progression from early stages with small spots of melanin adhering to CM-25 beads to late stages where spots had grown and coalesced to form a dark dense capsule. This reaction occurred maximally during the first 18 hr after inoculation; female mosquitoes aged 3-5 days showed 48% of beads heavily melanized by 18 hr postinoculation in the refractory strain and 0 10% in the susceptible strain. Female mosquito age and reproductive status strongly affected the ability to melanize beads. All of the beads were completely melanized in the refractory strain on the day immediately following eclosion (Day 0); thereafter these levels decreased steadily until the last time point on Day 7, when only 23% of beads were melanized in this strain. In the susceptible strain, 53% of beads were heavily melanized on Day 0 and 0-10% were melanized at all other times. At Day 1 and 2 after blood feeding, 85 and 88% of beads, respectively, were heavily melanized in refractory females in comparison with control mosquitoes of the same age which heavily melanize 23-58% of the beads. Blood feeding had little effect on the ability to melanize beads in the susceptible strain. PMID- 7544820 TI - Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo. AB - The integrin CD11b/CD18 promotes leukocyte extravasation during inflammation. Filamentous hemagglutinin (FHA) of Bordetella pertussis binds to CD11b/CD18, raising the possibility that peptides derived from FHA might inhibit leukocyte migration. The Arg-Gly-Asp (RGD) sequence of FHA has been suggested to modulate binding of ligands to CD11b/CD18. Peptides derived from this region inhibited adherence and transendothelial migration of neutrophils in vitro and prevented recruitment of leukocytes into the cerebrospinal fluid in an experimental model of meningitis in rabbits. The mechanism of the antiinflammatory effect may involve modulation of the activity of CD11b/CD18 through peptide interaction with the leukocyte response integrin/integrin-associated protein complex. PMID- 7544821 TI - Phytanic acid oxidation: topographical localization of phytanoyl-CoA ligase and transport of phytanic acid into human peroxisomes. AB - To understand the possible role of phytanoyl-CoA ligase, present in the membrane, in the oxidation of phytanic acid in the matrix of peroxisomes (Pahan, K. and I. Singh. 1993. FEBS Lett. 333: 154-158) we examined the transport of phytanic acid/phytanoyl-CoA into peroxisomes and the topology of the active site of phytanoyl-CoA ligase in the peroxisomal membrane. The increase in lignoceroyl-CoA ligase as compared to no change in the activities of palmitoyl-CoA and phytanoyl CoA ligases when peroxisomes were disrupted with detergent or sonication and inhibition of the activities of both palmitoyl-CoA and phytanoyl-CoA ligase by impermeable inhibitor of acyl-CoA ligases (mercury-dextran) and trypsin treatment in the intact peroxisomes. On the other hand, the lignoceroyl-CoA ligase activity was inhibited by mercury-dextran and trypsin only in the disrupted peroxisomes. Taken together, these studies support the conclusion that the enzymatic site of phytanoyl-CoA ligase is on the cytoplasmic surface of peroxisomal membrane. This implies that phytanoyl-CoA is synthesized on the cytoplasmic surface of peroxisomal membrane and is translocated through the membrane for its alpha oxidation to pristanic acid in the matrix of peroxisomes. To delineate the transport for phytanic acid through the peroxisomal membrane, we examined cofactors and energy requirements for its transport into peroxisomes. The similar rates of transport of phytanoyl-CoA and phytanic acid under conditions favorable for fatty acid activation (presence of ATP, CoASH, and MgCl2) and the lack of transport of phytanic acid when ATP and/or CoASH were removed or replaced with their inactive analogues (ATP and/or CoASH) from assay medium clearly demonstrates that the transport of phytanic acid requires prior synthesis of phytanoyl-CoA by phytanoyl-CoA ligase. The prerequisite activation of phytanic acid to phytanoyl-CoA for its alpha-oxidation only in intact peroxisomes, and oxidation of free phytanic acid in digitonin-permealized peroxisomes or isolated matrix, suggests that phytanoyl-CoA ligase (in peroxisomal membrane) regulates the oxidation of phytanic acid in peroxisomes by providing phytanoyl-CoA for its transport into peroxisomes. PMID- 7544822 TI - Chylous ascites in children demonstrated by Tc-99m dextran lymphoscintigraphy. PMID- 7544823 TI - Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state. AB - Swelling-induced loss of organic osmolytes from cells is mediated by an outwardly rectified, volume-sensitive anion channel termed VSOAC (Volume-Sensitive Organic osmolyte/Anion Channel). Similar swelling-activated anion channels have been described in numerous cell types. The unitary conductance and gating kinetics of VSOAC have been uncertain, however. Stationary noise analysis and single-channel measurements have produced estimates for the unitary conductance of swelling activated, outwardly rectified anion channels that vary by > 15-fold. We used a combination of stationary and nonstationary noise analyses and single-channel measurements to estimate the unitary properties of VSOAC. Current noise was analyzed initially by assuming that graded changes in macroscopic current were due to graded changes in channel open probability. Stationary noise analysis predicts that the unitary conductance of VSOAC is approximately 1 pS at 0 mV. In sharp contrast, nonstationary noise analysis demonstrates that VSOAC is a 40-50 pS channel at +120 mV (approximately 15 pS at 0 mV). Measurement of single channel events in whole-cell currents and outside-out membrane patches confirmed the nonstationary noise analysis results. The discrepancy between stationary and nonstationary noise analyses and single-channel measurements indicates that swelling-induced current activation is not mediated by a graded increase in channel open probability as assumed initially. Instead, activation of VSOAC appears to involve an abrupt switching of single channels from an OFF state, where channel open probability is zero, to an ON state, where open probability is near unity. PMID- 7544826 TI - Neurodevelopmental status at age five years of neonates treated with extracorporeal membrane oxygenation. AB - OBJECTIVE: To determine the neurodevelopmental status at age 5 years among children who received extracorporeal membrane oxygenation (ECMO) in the newborn period as a treatment for severe cardiorespiratory failure. METHODS: We conducted a prospective cohort study of 103 five-year-old ECMO-treated children born between June 1984 and July 1988, and treated at our institution. Thirty-seven healthy control children were recruited locally. The assessment protocol included a complete neuropsychologic assessment, psychosocial assessment with parent questionnaires, a standard neurologic evaluation, assessment of gross motor and fine motor function, a medical history, and physical examination. RESULTS: Major disability was present in 17 of the ECMO cohort. Eleven ECMO-treated children (11%) were mentally retarded, one of whom was profoundly impaired. Two additional children had severe learning disabilities. Cerebral palsy was diagnosed in 5 (5%) ECMO-treated children, but all cases were mild in nature and the patients were walking unaided. One child has paraplegia. The mean Full Scale, Verbal, and Performance IQs of the EMCO-treated children were within the normal range, but as a group were significantly lower than in control children (96 vs 115, p < 0.001). Children treated with ECMO had increased risk relative to the control children for academic difficulties at school age (49% VS 22%, P < 0.01) and a higher rate of behavioral problems reported by parents (42% vs 16%, p = 0.01). CONCLUSIONS: The rate of major disability was comparable to that in other high-risk populations. The high rate of behavioral problems and increased risk of subsequent school failure among nonretarded ECMO-treated children supports the need for close follow-up of these children after hospital discharge. PMID- 7544824 TI - Characterization of the voltage-dependent properties of a volume-sensitive anion conductance. AB - Outwardly rectified, swelling-activated anion conductances have been described in numerous cell types. The major functional variable observed amongst these conductances is the extent and rate of depolarization-induced inactivation. In general, the conductances can be divided into two broad classes, those that show rapid inactivation in response to strong depolarization and those that show little or no voltage dependence. The swelling-activated anion conductance in rat C6 glioma cells is inactivated nearly completely by membrane depolarization above +90 mV and reactivated by membrane hyperpolarization. The kinetics of inactivation and reactivation are fit by single and double exponentials, respectively. Voltage-dependent behavior is well described by a simple linear kinetic model in which the channel exists in an open or one of three inactivated states. pH-induced changes in voltage-dependent gating suggest that the voltage sensor contains critical basic amino acid residues. Extracellular ATP blocks the channel in a voltage-dependent manner. The block is sensitive to the direction of net Cl- movement and increases open channel noise indicating that ATP interacts with the channel pore. Blockage of the channel with ATP dramatically slows depolarization-induced inactivation. PMID- 7544828 TI - Failure of granulocyte colony-stimulating factor in alloimmune neonatal neutropenia. PMID- 7544829 TI - Ultrastructure of Trichophyton mentagrophytes stained with neutral red. AB - The ultrastructure of Trichophyton mentagrophytes cells stained with neutral red was investigated using electron microscopy and X-ray microanalysis. Fixatives containing molybdenum and chromium were used to prevent the outflow of neutral red. Electron-dense particles composed of metals and dye were observed exclusively in the vacuoles, which were increased in number and size, but not near the cell wall. Results indicate that neutral red passes directly through the fungal cell wall and is incorporated into the vacuoles. PMID- 7544830 TI - Induction of cytochrome P-450 by 5-methoxypsoralen in the yeast Saccharomyces cerevisiae. AB - Yeast cells (D7 strain) incubated in the presence of 5-methoxypsoralen (5-MOP) increase the activity of the monooxygenase system cytochrome P-450 dependent (cytochrome P-450 level and 7-ethoxycoumarin-O-diethylase activity). Northern analysis of cytochrome P-450 specific RNA shows that 5-MOP treatments induce an increase in mRNA. The induction of cytochrome P-450 appears to occur at the transcriptional level. The capacity of 5-MOP to induce the cytochrome P-450 system in eukaryotic cells, in which it is known to be involved in the metabolism of the psoralen, may decrease the availability of the compound for photo-induced genotoxic reactions, which may explain the good tolerance in patients. PMID- 7544827 TI - Effect of recombinant human erythropoietin on the switchover from fetal to adult hemoglobin synthesis in preterm infants. AB - To determine whether recombinant erythropoietin (r-HuEpo) administered to very low birth weight infants could increase hemoglobin F synthesis, or delay its developmentally programmed decline, we determined serially the synthesis of hemoglobins A and F in 15 preterm infants receiving either a placebo or r-HuEpo. There was no difference between the two groups in the proportion of hemoglobin F being synthesized in relation to postconceptional age. PMID- 7544825 TI - Retrograde but not anterograde bead movement in intact axons requires dynein. AB - Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. PMID- 7544831 TI - Functional reconstitution of ion channels from Paramecium cortex into artificial liposomes. AB - Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless di- or trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca2+ release, and the prospects of their purification are discussed. PMID- 7544832 TI - Combining prostate markers may yield useful screening tool. PMID- 7544833 TI - Tumor antigen-specific immunization of bone marrow transplantation donors as adoptive therapy against established tumor. AB - BACKGROUND: Persistence of the underlying malignancy remains the main obstacle to the successful treatment of human malignancies with high-dose chemoradiotherapy and bone marrow transplantation. PURPOSE: The aim of this study was to determine whether antigen-specific antitumor immune responses, elicited in normal donor mice by immunization with the soluble form of a surrogate tumor antigen (i.e., ovalbumin [OVA]), can be transferred via bone marrow transplantation into lethally irradiated, syngeneic recipient mice. An additional goal was to evaluate the ability of these adoptively transferred bone marrow cells to eradicate established recombinant OVA-expressing lymphomas that recurred after lethal-dose total-body irradiation (TBI). METHODS: Female C57BL/6 donor mice were immunized twice with OVA emulsified in a muramyl-dipeptide-containing adjuvant. Syngeneic mice bearing a day-10 or day-11, approximately 1-cm subcutaneous E.G7-OVA tumor (E.G7-OVA tumor cells were derived from transfection of EL-4 thymoma tumor cells using the coding sequence of chicken OVA gene complementary DNA) were treated with TBI and reconstituted with bone marrow from nonimmune or OVA-immunized mice. In subsequent experiments, tumor-bearing mice, treated with TBI and OVA-immune bone marrow, were given additional therapy either with a single OVA immunization or by the adoptive transfer of 1 x 10(7) in vitro activated spleen cells derived from OVA-immune donor mice and cultured 5 days with irradiated E.G7-OVA cells before transfer. RESULTS: E.G7-OVA tumor-bearing mice given TBI and OVA-immune bone marrow showed a significantly increased cure rate when compared with that among controls reconstituted with nonimmune bone marrow after TBI (logrank, P < .01). The antitumor effect of immune bone marrow was abrogated by T-cell depletion of the marrow graft (P < .016). The antitumor effect of immune marrow was enhanced by the addition of OVA immunization of tumor-bearing recipients (P < .015). OVA-specific cytotoxic T-lymphocyte (CTL) activity was recovered from tumor-bearing recipients of immune marrow 14 days after bone marrow transplantation. The antitumor effect observed following the adoptive transfer of immune marrow was further augmented by the addition of 1 x 10(7) splenic E.G7-OVA specific in vitro activated CTLs derived from OVA-immune mice (P < .03). CONCLUSION: These studies establish the principle that antigen-specific T-cell immunity against a defined tumor-specific antigen can be transferred with bone marrow from an immune donor. IMPLICATIONS: Active immunization of normal human bone marrow or T-cell donors with a refined, safe tumor antigen and transfer of immunity to the patient may represent a novel strategy for circumventing the obstacle of host immune suppression associated with the tumor-bearing state. PMID- 7544834 TI - Quality of life in phase II trials: a study of methodology and predictive value in patients with advanced breast cancer treated with paclitaxel plus granulocyte colony-stimulating factor. AB - BACKGROUND: Despite the clinical benefit that may be associated with reduction of tumor volume, chemotherapy may produce physical or psychological distress that could compromise a patient's quality of life. Although palliation may be as relevant as tumor response in patients with metastatic breast cancer, quality of life is not commonly evaluated in phase II clinical trials of new therapeutic agents. PURPOSE: We evaluated the utility of quality-of-life assessment in two phase II clinical trials of patients receiving paclitaxel (Taxol) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) as salvage therapy for metastatic breast cancer. METHODS: A battery of instruments (i.e., Memorial Symptom Assessment Scale [MSAS], Functional Living Index-Cancer [FLIC], Rand Mental Health Inventory [MHI], Brief Pain Inventory [BPI], and Memorial Pain Assessment Card [MPAC]) designed to capture information about social, psychological, and functional aspects of quality of life, as well as symptom prevalence and distress, was completed prior to treatment; serial assessments were obtained at regular intervals during the treatment period. Univariate and multivariate analyses were performed evaluating base-line quality-of-life parameters and standard prognostic factors in relation to outcome measures of survival, tumor response, and toxicity. For 30 consecutive patients with extensive prior chemotherapy for metastatic disease, longitudinal data were analyzed associating tumor response to changes in quality-of-life scores throughout the course of treatment with paclitaxel. RESULTS: Base-line scores of two validated quality-of-life instruments, the MSAS and the FLIC, independently predicted the overall survival (P < .01 for each). In this model, however, neither standard prognostic factors nor quality of life instruments predicted the likelihood of tumor response or the probability of encountering grade 3 or grade 4 nonhematologic toxicity. With serial assessments of quality of life, the majority of patients who achieved partial tumor response or stable disease reported improved or unchanged quality-of-life scores, while those patients with progressive disease experienced rapid deterioration in quality of life. CONCLUSIONS: Base-line quality-of-life assessment may provide prognostic information distinct from that obtained through standard prognostic indicators alone. The combination of two factors--extent of disease and a base-line quality of-life assessment--predicted survival more accurately than either used separately. Evaluation of quality-of-life outcomes in relation to tumor response may illuminate previously unmeasured palliative effects of chemotherapy, such as pain relief, as well as the burdens it imposes. IMPLICATIONS: Information obtained from quality-of-life assessment in conjunction with phase II testing of new chemotherapeutic agents for metastatic breast cancer can guide quality-of life evaluation planned in large, randomized future studies. PMID- 7544838 TI - Hyperamylasemia after post-chemotherapy retroperitoneal lymph node dissection for testis cancer. AB - PURPOSE: Postoperative hyperamylasemia was evaluated in patients undergoing post chemotherapy retroperitoneal lymph node dissection for testis cancer. MATERIALS AND METHODS: Serum levels of amylase, lipase and bilirubin were evaluated prospectively in the immediate postoperative period in 39 consecutive patients who underwent post-chemotherapy retroperitoneal lymph node dissection. RESULTS: Hyperamylasemia was found in 16 patients (41%), hyperlipasemia in 17 (43%) and hyperbilirubinemia in 9 (23%). Peak elevations were observed at 24 hours postoperatively and most returned to normal at 1 week. Elevation of these parameters was significantly associated with length and difficulty of the procedure. No patient demonstrated clinical acute pancreatitis. CONCLUSIONS: Extended retraction of the pancreas during post-chemotherapy retroperitoneal lymph node dissection may cause a minor reversible injury to the pancreas expressed as hyperamylasemia, hyperlipasemia and, rarely, jaundice. These findings are important to consider in the differential diagnosis of hyperamylasemia following post-chemotherapy retroperitoneal lymph node dissection. PMID- 7544837 TI - "Wildcatting" for breakthroughs in urothelial cancer detection and management--a frustrating business. PMID- 7544839 TI - Changes in pressure-flow parameters in patients treated with transurethral microwave thermotherapy. AB - PURPOSE: We document changes in pressure-flow study parameters in patients treated by transurethral microwave thermotherapy. MATERIALS AND METHODS: Pressure flow study tracings before and after therapy from 75 patients with benign prostatic hyperplasia were analyzed. Patients were stratified according to the predominant type of obstruction at screening (constrictive or obstructive). RESULTS: An improvement in Madsen score and flow rates was noted at 6 months in both groups. In contrast to compressive obstruction patients, however, those with constrictive obstruction also showed significant changes in pressure-flow study parameters, including detrusor pressure at maximum flow, maximum flow rate and urethral resistance factor. CONCLUSIONS: Patients with predominantly constrictive obstruction are the best candidates for microwave thermotherapy. PMID- 7544836 TI - Histopathological features and p53 nuclear protein staining as predictors of survival and tumor recurrence in patients with transitional cell carcinoma of the renal pelvis. AB - PURPOSE: We determine which histopathological features are predictive of recurrence and cause-specific survival in renal pelvic transitional cell carcinoma. MATERIALS AND METHODS: Univariant and multivariant analysis was done on material from 67 patients. RESULTS: Univariate and multivariate analysis identified lamina propria invasion, grade 3 tumors and capillary-lymphatic invasion as predictors of disease-specific survival. Multicentric disease was the sole independent predictor of recurrence. Multicentric disease and lamina propria invasion were independent predictors of recurrence-free survival. Also, p53 over expression was not statistically associated with any of the studied prognostic factors. CONCLUSIONS: Histopathological features remain the cornerstone of prognostic assessment for renal pelvic transitional cell carcinoma. PMID- 7544835 TI - Prostatic intraepithelial neoplasia is a risk factor for adenocarcinoma: predictive accuracy in needle biopsies. AB - PURPOSE: High grade prostatic intraepithelial neoplasia is considered the most likely precursor of prostatic adenocarcinoma. However, the natural history and predictive value of prostatic intraepithelial neoplasia for cancer are unknown. MATERIALS AND METHODS: To examine the predictive value of high grade prostatic intraepithelial neoplasia, we conducted a retrospective clinic based comparative study of 100 patients with high grade prostatic intraepithelial neoplasia and 112 without prostatic intraepithelial neoplasia on needle biopsies matched for digital rectal examination findings, patient age and serum prostate specific antigen level. Only patients who had 1 or more followup biopsies were included. RESULTS: Adenocarcinoma was identified in 35% of subsequent biopsies from patients with prostatic intraepithelial neoplasia, compared with 13% of control biopsies. The likelihood of finding cancer increased as the interval from initial biopsy increased. High grade prostatic intraepithelial neoplasia, patient age and serum prostate specific antigen concentration were jointly highly significant predictors of cancer, with prostatic intraepithelial neoplasia providing the highest risk ratio of 14.93 (95% confidence intervals 5.6 to 39.8). No other candidate predictor was significant, including digital rectal examination findings, transrectal ultrasound results, amount of prostatic intraepithelial neoplasia on biopsy and architectural pattern of prostatic intraepithelial neoplasia. CONCLUSIONS: These results indicate that the presence of high grade prostatic intraepithelial neoplasia on needle biopsy is strongly predictive of carcinoma. Prostatic intraepithelial neoplasia should be reported in needle biopsies and biopsy repeated. These finding support the hypothesis that prostatic intraepithelial neoplasia is a precursor of prostate cancer. PMID- 7544840 TI - The value of prostate specific antigen in early diagnosis of prostate cancer: the study of men born in 1913. AB - PURPOSE: We attempt to elucidate the performance of serum prostate specific antigen (PSA) as a screening test for prostate cancer. MATERIALS AND METHODS: We analyzed sera stored since 1980 in a nested case-control study. RESULTS: The 36 patients with prostate cancer had a markedly greater mean serum PSA level than did 68 subjects without cancer (22.71 versus 2.67, respectively, p < 0.0001). Among the controls 24% had a serum PSA of greater than 4.0 micrograms/l. A serum PSA value exceeding 4.0 micrograms/l. in 1980 was associated with a 20-fold excess risk of cancer between 1981 and 1986, and an 8-fold risk between 1987 and 1991. CONCLUSIONS: Diagnosis of clinically significant prostate cancer can be advanced substantially by serum PSA testing. However, the large number of high serum PSA values in men who remained free of clinical disease emphasizes the need for a more specific screening test. PMID- 7544841 TI - Serial prostate specific antigen measurements and progression in untreated confined (stages T0 to 3NxM0, grades 1 to 3) carcinoma of the prostate. AB - PURPOSE: The contribution of serial prostate specific antigen (PSA) determinations was studied to obtain better understanding of the natural history of clinically confined prostate carcinoma. MATERIALS AND METHODS: Serial PSA determinations in 29 patients with untreated confined prostate carcinoma were correlated to the clinical course for a mean of 39 months. RESULTS: Disease progressed locally in 13 patients after a mean of 31 months. Metastatic progression was not observed. Neither grade, stage, PSA changes nor initial PSA showed significant differences with respect to interval of progression between patients with and without progression. CONCLUSIONS: PSA does not parallel clinical progression in patients selected for watchful waiting. PMID- 7544842 TI - Seminal vesicle biopsy in prostate cancer staging. AB - PURPOSE: Prostate cancer patients were differentiated using seminal vesicle biopsies related to clinical staging, prostate specific antigen (PSA), Gleason scores and PSA density values. MATERIALS AND METHODS: A total of 75 patients underwent seminal vesicle biopsies. Patients with no seminal vesicle invasion and negative lymphadenectomy results underwent radical prostatectomy. RESULTS: Efficacy of seminal vesicle biopsy was 90.9%. Infiltration rates were 69% for disease stage T2b or greater, 68% for PSA greater than 10 ng./ml., 78% for PSA greater than 20 ng./ml. and 73% for type 4 and/or 5 Gleason scores. For the prognosis of seminal vesicle invasion, 0.40 was the best PSA density cutoff point. CONCLUSIONS: We recommend seminal vesicle biopsy in patients with stage T2b or greater disease, and with a lower clinical stage when the PSA level is 20 ng./ml. or greater and/or the Gleason score is 7 or greater. PMID- 7544844 TI - The central role of prostate specific antigen in diagnosis and progression of prostate cancer. PMID- 7544843 TI - Prostate specific antigen after radiotherapy for prostate cancer: a reevaluation of long-term biochemical control and the kinetics of recurrence in patients treated at Stanford University. AB - PURPOSE: We evaluated prostate specific antigen (PSA) evidence for control of prostatic cancer after irradiation. MATERIALS AND METHODS: We studied 110 patients for whom more than 2 PSA measurements were obtained to establish trends and the initial measurement was done between April 1985 and January 1988. RESULTS: A total of 42 patients (38%) had stable, normal PSA levels with followup averaging 12.4 years (range 4.4 to 24.8). Increasing clinical stage or Gleason score correlated significantly with risk for PSA relapse, as did pretreatment PSA level. Short PSA doubling times were associated with distant metastasis rather than with local recurrence. CONCLUSIONS: We found that irradiation durably controlled 38% of prostatic cancers of various stages and grades and is unlikely to accelerate tumor growth. PMID- 7544845 TI - Long-term effects of finasteride on invasive urodynamics and symptoms in the treatment of patients with bladder outflow obstruction due to benign prostatic hyperplasia. AB - PURPOSE: We assess the long-term effects of finasteride on bladder outlet obstruction and symptoms in the treatment of patients with benign prostatic hyperplasia. MATERIALS AND METHODS: Of the original 36 patients assigned to treatment with 5 mg. finasteride daily (group 1) or placebo (group 2) for 6 months 27 completed an open extension study of 5 mg. finasteride for 4 more years. The possible relief of bladder outlet obstruction was monitored with repeated pressure-flow studies at baseline, 6 months and 4.5 years. RESULTS: The treatment resulted in a further slight decrease in detrusor pressure at maximum flow rate in group 1 and a significant decrease in group 2 during the 4-year period, whereas improvement in maximum flow rate did not achieve statistical significance. Concomitantly, there was a significant improvement in obstructive and irritative symptoms. CONCLUSIONS: Finasteride decreases bladder outlet obstruction moderately and only occasionally relieves it completely. However, the decrease in obstruction achieved in many patients is sufficient to improve the symptoms significantly. The beneficial effect is long-lasting. PMID- 7544846 TI - The generation of nitric oxide and its roles in neurotransmission and neurotoxicity. AB - The N-methyl-D-aspartate (NMDA) receptor plays a key role in synaptic plasticity and is thought to underlie memory, learning and development of the nervous system. The NMDA receptor is a ligand-gated ion channel complex that contains distinct recognition sites for endogenous and exogenous ligands, including glutamate, glycine, Mg2+, Zn2+ and noncompetitive blockers such as MK-801. In the central nervous system, nitric oxide (NO) is produced in some neurons following activation of excitatory amino acids receptors, particularly those of the NMDA receptor. Nitric oxide is synthesized from a L-arginine by the cytoplasmic enzyme nitric oxide synthase (NOS) which is a calcium dependent enzyme, and this pathway is inhibited by the analogues of L-arginine such as NG-monomethyl-L-arginine (L NMMA) and is augmented by NMDA receptor activation. Activation of the NMDA receptor results in the elevation of intracellular calcium ([Ca2+]i) which in turn activates NOS via the calcium-calmodulin complex. Nitric oxide is not a classical neurotransmitter in the central nervous system since it is not released by exocytosis and does not interact with a receptor protein but rather diffuses rapidly across the membrane and binds with the iron in heme-containing proteins. Nitric oxide can serve as both an oxidizing and reducing agent. It has strong affinity for heme proteins such as guanylyl cyclase, but there is evident that NO may have a regulatory role by oxidizing sulfhydryl groups of non-heme proteins such as those on the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544847 TI - [Use of intra-tissue electrophoresis and electrophonophoresis in the multimodal treatment of edematous forms of pancreatitis]. AB - The experience of treatment of 305 patients with the edematous++ forms of pancreatitis is assigned. The application efficacy of intra-tissue (pancreatic) electrophoresis and electrophonophoresis using inhibitors, cytostatics, antibacterial and the other medicinal agents was studied. Decrease of frequency of the destructive pancreatitis appearance and the treatment results improvement were promoted by application of the intra-tissue electrophonophoresis with the chemotherapeutic agents in the complex treatment of an acute or exacerbated pancreatitis. PMID- 7544848 TI - Functional characteristics of mature granulocytes in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid. AB - All-trans retinoic acid (ATRA) is a differentiating agent that has been successfully used in the treatment of patients with acute promyelocytic leukemia (APL). Functional properties of peripheral blood neutrophils from a patient with APL during treatment with ATRA have been studied. Wright stain of patient neutrophils showed hypogranulation and loose nuclear chromatin when compared with normal neutrophils. These cells were of lower density than normal neutrophils and separated on density gradient centrifugation with mononuclear cells. Surface antigen expression by FACS distinguished these cells from lymphocytes. The histograms showed a population of larger cells expressing CD18 and CD11b, distinct from the smaller cells which did not express CD11b. fMLP caused an increase in intracellular calcium (measured spectrophotometrically) that was inhibited by the calcium chelator BAPTA. Actin polymerization following cell activation was measured using NBD-phallacidin staining and FACS. Both IL-8 and fMLP caused rapid increases using F-actin content (2.5-3.0 fold), which were of greater magnitude than generally seen with normal neutrophils. Treatment with BAPTA before activation with fMLP did not blunt the actin responses, despite complete inhibition of an intracellular calcium increase. In summary, neutrophils derives from differentiated APL cells express CD18/CD11b, and exhibit a similar degree of actin polymerization in response to fMLP and IL-8, independent of an increase in intracellular calcium. Although the actin responses are greater than normal neutrophils, most properties are similar, supporting the contention that these cells can protect the host. The exaggerated actin response to inflammatory mediators, however, may play a role in the 'retinoic acid syndrome'. PMID- 7544851 TI - Long-term follow-up of a randomized post-induction therapy trial in acute myelogenous leukemia (a Southeastern Cancer Study Group trial). AB - A phase III clinical trial was designed to determine if more intensive induction and consolidation therapy for acute myeloblastic leukemia increases the remission rate and prolongs survival. A minor objective was to determine if the use of non cross resistant drugs was more effective than the same drugs used for induction. Patients with untreated leukemia between the ages of 15 and 50 were given daunorubicin 45 mg/m2 for the first 3 days of a 10-day continuous infusion of cytosine arabinoside, initially at a dose of 2000 mg/m2 but reduced to 100 mg/m2 because of toxicity. Those under 36 achieving a complete remission and with an histocompatible donor were assigned to a transplant arm. The rest were randomized to receive one of three consolidation arms: A, cytosine arabinoside, 200 mg/m2 daily for 7 days and daunorubicin 45 mg/m2 daily for 3 days for three courses; B, one course as in Arm A followed by amsacrine, 120 mg/m2 daily for 5 days followed by a 5-day continuous infusion of azacytidine, 150 mg/m2/day; C, thioguanine and cytosine arabinoside, 100 mg/m2 every 12 h and daunorubicin 10 mg/m2 daily for 5 days for three courses followed by four maintenance courses of cytosine arabinoside, 100 mg/m2 daily for 5 days and daunorubicin, 45 mg/m2 for 2 days every 13 weeks. From 1981 to 1986, 398 eligible patients were enrolled and 219 achieved a complete remission. The initial induction dose of cytosine arabinoside was reduced after five of 29 patients exhibited fatal gastrointestinal toxicity. Only 11 patients were assigned to the transplant arm. There were no significant differences in the consolidation arms. The 5 year disease-free survivals were 38, 31 and 27% in arms A, B, and C respectively. Intensive consolidation therapy with the same or different drugs used in induction was as effective as lower dose consolidation followed by maintenance therapy. PMID- 7544854 TI - Synergistic effects of basic fibroblast growth factor and hematopoietic growth factors on colony formation of K562 human leukemic cells. AB - The effect of basic fibroblast growth factor (bFGF) alone and in combination with other hematopoietic growth factors on the colony formation of K562 human leukemic cells was studied using soft agar colony assay. bFGF was found to have a weak colony-stimulating activity on K562 cells derived from the blastic crisis cells of human chronic myelogenous leukemia and to potentiate the K562 cell colony stimulating activity of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) at a low concentration of below 1 ng/ml. These findings suggested that bFGF stimulates the growth of human leukemic cells directly in vivo alone and in synergy with other hematopoietic growth factors. PMID- 7544852 TI - Antibodies are capable of directing superantigen-mediated T cell killing of chronic B lymphocytic leukemia cells. AB - The bacterial superantigen staphylococcal enterotoxin A (SEA) is a highly potent activator of cytotoxic T cells when presented on MHC class II molecules of target cells. Our earlier studies showed that such SEA-directed T cells efficiently killed chronic B lymphocytic leukemia (B-CLL) cells. With the ultimate goal to replace the natural specificity of SEA for MHC class II molecules with the specificity of a monoclonal antibody (mAb), we initially made a mutated protein A SEA (PA-SEAm) fusion protein with > 100-fold reduced binding affinity for MHC class II compared to native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage specific (CD19, CD20) or associated (CD37, CD40) mAbs. The PA-SEAm protein was 10-100-fold more potent against mAb coated compared to uncoated HLA class II+ B-CLL cells. No correlation was seen between the amount of mAb bound to the cell surface and sensitivity to lysis. Preactivation of B-CLL cells by phorbol ester increased their sensitivity, and lysis was dependent on ICAM-1 molecules. However, no preactivation of the target cells was needed when a cocktail of two or four mAbs was used. Circulating leukemia and spleen cells were equally well killed. We conclude that the natural target specificity of SEA, MHC class II, can be reduced by mutagenesis and novel binding specificity can be introduced by linkage to tumor reactive mAbs. Our findings encourage the construction of recombinant SEA mutant fusion proteins for specific T cell therapy of hematopoietic tumors such as B-CLL. PMID- 7544850 TI - Responsiveness to stem cell factor (SCF) of peripheral blood colony-forming cells from patients with myelodysplastic syndromes (MDS). AB - We have previously reported that serum stem factor (SCF) levels are significantly lower in patients with myelodysplastic syndrome (MDS) than normal controls. We have now studied the effects of adding SCF to cultures of blood mononuclear cells from patients with MDS and normal subjects. Three of 17 patients with MDS showed marked increases in erythroid colony numbers with SCF + erythropoietin compared to interleukin-3 + erythropoietin. In two cases (1RA and 1ISA) the erythroid colony numbers became normal. The same RA patient also showed a marked increase in myeloid colony numbers, which were undetectable with granulocyte-macrophage colony-stimulating factor alone, but within the normal range when SCF was added. A fourth patient (ISA) showed a 4.7-fold increase in myeloid colonies with SCF, but no erythroid response. The normal subjects showed a trend towards increased numbers of myeloid colonies with SCF; erythroid colonies did not increase. A correlation was found between the MDS patients' haemoglobin levels and erythroid colony numbers with and without SCF, but there was no correlation between erythroid or myeloid colonies in the presence of SCF and their serum SCF level. PMID- 7544853 TI - Production of hematopoietic regulatory factors in cultures of adult and fetal mouse organs: measurement by specific bioassays. AB - Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues. PMID- 7544855 TI - Chronic myelogenous leukemia (CML) associated with granulocyte colony-stimulating factor (G-CSF)-producing mesothelioma. PMID- 7544849 TI - Molecular study of the interferon genes in chronic myeloid leukemia. AB - The interferons alpha, beta, and w (IFNA, IFNB, IFNW), are a family of genes that have been mapped on the short arm of chromosome 9 (9p21-22). Deletions of genetic material on 9p are frequently observed in hematological diseases, particularly in lymphoid neoplasias. In this paper we have performed the molecular studies of IFNA and IFNB genes in chronic myeloid leukemia (CML) in order to determine if the deletions of these genes are prevalent in this pathology. Forty CML patients, Philadelphia positive or with BCR/ABL rearrangement, were studied at diagnosis. The analysis of IFNA and IFNB genes was performed by Southern and dot blot techniques. Homozygous or hemizygous deletions of IFNA and IFNB genes could not be detected, indicating that deletions of these genes would not be present or would be a very infrequent event in the chronic phase of the CML patients. PMID- 7544856 TI - From neuropathology to neurodevelopment. PMID- 7544859 TI - Arterial embolisation to treat uterine myomata. AB - Haemorrhage, probably related to hypervascularisation, is the commonest complication of uterine myomata and is difficult to treat. 16 patients, aged 34 48 years, with symptomatic uterine myomata, for which a major surgical procedure was planned after failure of medical treatment, were treated by selective free flow arterial embolisation of the myomata with Ivalon particles. With a mean follow-up of 20 months (range 11-48) in the responders, symptoms resolved in 11 patients; menstrual cycles returned to normal in ten of these. Three patients had partial improvement. Two failures required surgery. In 14 cases embolisation caused pelvic pain, which required analgesia in all. PMID- 7544857 TI - Chemotherapy for elderly patients with lung cancer. PMID- 7544858 TI - Adverse cutaneous reactions to thiacetazone for tuberculosis treatment in Tanzania. AB - Because thiacetazone has been linked with serious adverse cutaneous reactions, we undertook 1 year of systematic surveillance for cutaneous thiacetazone-associated adverse reactions within the national tuberculosis programme of Tanzania. For individual cases, we collected information on age, sex, interval between commencing thiacetazone-containing treatment and occurrence of adverse reaction, most severe clinical presentation (toxic epidermal necrolysis, rash without necrolysis, itching without rash), and outcome (dead or alive) within 2 weeks of onset. Univariate and multivariate analyses were done of variables relevant to outcome. 1273 patients with adverse reactions were reported. The frequency of fatal outcome from any cutaneous reaction was 3.1 per 1000 among all tuberculosis patients, and 19.1% among patients with toxic epidermal necrolysis. About 60% of all adverse reactions and deaths occurred within 20 days of starting thiacetazone. Case fatality from adverse cutaneous reactions was considerably less frequent than reported previously, suggesting that improved management might allow retention of thiacetazone in the armamentarium of national tuberculosis programmes even where infection with HIV is prevalent. PMID- 7544860 TI - Neuroleptic sensitivity to risperidone in Lewy body dementia. PMID- 7544861 TI - Severe rhabdomyolysis associated with tacrolimus. PMID- 7544862 TI - Possible involvement of tyrosine kinase in the LPS-promoted initiation of L arginine-induced relaxation of rat aorta mediated by induction of no synthase. AB - Tyrosine kinase inhibitors herbimycin A, genistein and erbstatin analog prevented endotoxin (LPS)-promoted initiation of L-arginine (Arg)-induced relaxations and cGMP formation in rat thoracic aorta, which appear to be mediated by nitric oxide synthase expressed by LPS in the vascular smooth muscle. Similarly, interleukin-1 beta (IL-1 beta) triggered initiation of Arg-induced relaxation of the arteries. In addition, in the aortic smooth muscle cells cultured in the presence of Arg, LPS- or IL-1 beta-triggered accumulation of nitrite was suppressed by the tyrosine kinase inhibitors. These results suggest that tyrosine kinase is involved in the LPS- and IL-1 beta-promoted induction of nitric oxide synthase in the vascular smooth muscle, which in turn mediates production of NO from added Arg, thus stimulating formation of cGMP and causing relaxation. Alternatively, it is possible that LPS acts indirectly through cytokines such as IL-1 beta. PMID- 7544864 TI - Phylogenetic analyses of 55 retroelements on the basis of the nucleotide and product amino acid sequences of the pol gene. AB - Comparisons of pol gene nucleotide and reverse transcriptase (RT) amino acid sequences of 47 retroviruses, 3 caulimoviruses, and 5 hepadnaviruses showed that approximately one-third of the gene at the 5' end is much more conserved than other pol regions. The most conserved regions on both the nucleotide and amino acid sequences were chosen for construction of phylogenetic trees. The maximum parsimony and distance-matrix methods were used for analyses of aligned amino acid sequences; these two methods, and the compatibility method, were used to analyze the aligned nucleotide sequences. Essentially identical majority-rule consensus trees were produced by these different methods from both the pol gene nucleotide and RT amino acid sequences, which divided the 55 retroelements into six major groups. The reliability of the phylogenetic trees was probed with the bootstrapping of 100 replicates of the original sequence alignments. The grouping results were shown to be statistically significant by multiple comparisons with the least-significant-difference procedure. PMID- 7544863 TI - 7-Nitro indazole derivatives are potent inhibitors of brain, endothelium and inducible isoforms of nitric oxide synthase. AB - The effect of 7-nitro indazole (7-NI) and a range of substituted indazole derivatives on nitric oxide synthase (NOS) enzyme activity in homogenates of rat cerebellum, bovine endothelial cells and lung from endotoxin-pretreated rats was investigated. 3-bromo 7-nitro indazole was either equipotent (IC50, 0.86 +/- 0.05 microM c.f. 0.78 +/- 0.2 microM, n = 6, P > 0.05) or approximately 4x (IC50, 0.17 +/- 0.01 microM c.f. 0.71 +/- 0.01 microM, n = 6, P < 0.05) or 20x (IC50, 0.29 +/ 0.01 microM c.f. 5.8 +/- 0.4 microM, n = 6, P < 0.05) more potent than 7-NI as an inhibitor of bovine endothelial, rat cerebellar and rat lung NOS enzyme activity respectively. 2,7-dinitro indazole also inhibited NOS in all three tissue sources with a potency similar to that of 7-NI. These results suggest that 3-bromo 7-NI and 2,7-dinitro indazole may prove to be useful additional tools with which to examine the biological properties of nitric oxide (NO). PMID- 7544865 TI - Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae. AB - DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA. PMID- 7544866 TI - PCR-based analysis of voltage-gated K+ channels in vascular smooth muscle. AB - Irregularities in K+ currents form the basis of several cardiovascular dysfunctions, among which are arrhythmias and vasospasms. The developmental regulation of voltage-gated K+ channels, however, has been difficult to study. A novel approach was therefore employed to examine these channels in muscle tissue. Primers for a PCR-based analysis were designed using published nucleic acid sequences for voltage-gated K+ channels. Final selection of the primer pairs was based on the homology present in the S4 and H5 transmembrane domains. A specific band was amplified with these primers using RNA isolated from both rat A10 vascular smooth muscle cells and rat heart tissue. PMID- 7544867 TI - The proteoglycan perlecan is expressed in the erythroleukemia cell line K562 and is upregulated by sodium butyrate and phorbol ester. AB - Perlecan is a modular heparan sulfate proteoglycan that harbors five domains with homology to the low density lipoprotein receptor, epidermal growth factor, laminin and neural cell adhesion molecule. Using a monoclonal antibody directed against the laminin-like domain of perlecan, we have recently shown that perlecan is widely expressed in all lymphoreticular systems. To investigate further this observation we have studied the expression of perlecan in two human leukemic cell lines. Using reverse transcriptase-PCR, ribonuclease protection assay, and metabolic labeling we detected significant perlecan expression in the multipotential cell line K562, originally derived from a patient with chronic myelogenous leukemia. In contrast, the promyelocytic cell line HL-60 expressed perlecan at barely detectable levels. These results were intriguing because the K562 cells do not assemble or produce a classical basement membrane. Following induction with either sodium butyrate or the phorbol diester 12-0 tetradecanoylphorbol-13-acetate (TPA), K562 and HL-60 differentiate into early progenitor cells with erythroid or megakaryocytic properties, respectively. Following treatment of K562 and HL-60 cells with either of these agents, perlecan expression was markedly increased in K562 cells. In contrast, we could detect perlecan protein synthesis in HL-60 cells only at very low levels, even after induction with TPA or sodium butyrate. Collectively, these results indicate that perlecan is actively synthesized by bone marrow derived cells and suggest that this proteoglycan may play a role in hematopoietic cell differentiation. PMID- 7544868 TI - Alterations of a dominant epitope of lymphocytic choriomeningitis virus which affect class I binding and cytotoxic T cell recognition. AB - We have investigated mutation of a dominant cytotoxic T cell (CTL) epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Five NP peptide analogs with single substitutions at the predicted anchor residues (designated by the wild type amino acid, the position number and the new amino acid: P2A, P2R, M9L and M9K) and at a non-anchor position (S5N) were examined for binding to class I, H-2 Ld molecules. Each of the substitutions decreased or abolished the capacity of the NP peptide to increase cell surface Ld expression and to induce Ld stabilization in the cell lysates, indicating that these substitutions significantly affected peptide binding to Ld. We tested the peptide analogs for recognition by bulk primary CTL specific for LCMV, and for their ability to stimulate in vitro the CTL originally induced by wild type LCMV. Except for the M9L change, all mutations reduced CTL recognition by at least 100 fold, and the analogs failed to stimulate the CTL in vitro. The M9L peptide was recognized by the CTL and stimulated CTL in vitro almost as well as wild type; however, this peptide induced Ld stabilization in the cell lysates to a much lesser extent than wild type. Overall, this study demonstrates that mutations in the NP epitope affected peptide binding to the Ld molecule and CTL recognition. PMID- 7544869 TI - Aberrant cytokine regulation in macrophages from young autoimmune-prone mice: evidence that the intrinsic defect in MRL macrophage IL-1 expression is transcriptionally controlled. AB - We have reported that, when compared to macrophages from normal strains, macrophages from the autoimmune-prone MRL and NZB mouse strains demonstrate dramatically reduced IL-1 expression in response to LPS. In MRL mice, this is an intrinsic defect which is unmodified by age, the progression of disease, or the presence of the Ipr gene. Here we report that the key events leading to aberrant IL-1 expression appear to be transcriptional, based on the following three sets of findings. (1) Nuclear run-on analysis demonstrates that the patterns of IL-1 transcription in MRL/+ and BALB/c macrophages are distinct, as the former is clearly more transient. The reduction in MRL/+ IL-1 transcription coincides with a reduction in the levels of nuclear NF-KB and precedes a drop in IL-1 mRNA steady-state levels. (2) Reduced levels of IL-1 transcripts are found in both nuclear and cytosolic fractions of MRL/+ macrophages, arguing against faculty IL 1 mRNA transport into the cytosol as a contributing factor in the establishment of this defect. (3) In the presence of actinomycin D, the rate of RNA degradation is similar in MRL/+ and BALB/c macrophages. Moreover, in vitro RNA decay assays demonstrate that even in the absence of metabolic inhibitors, there is no evidence for an accelerated decay of IL-1 mRNA during exposure to lysates isolated from MRL/+ vs BALB/c macrophages. Taken together, these findings argue that transcription is the predominant level at which this striking example of cytokine dysregulation is controlled. PMID- 7544871 TI - Myoblast allotransplantation in primates. PMID- 7544870 TI - Comparative molecular modelling of the Fas-ligand and other members of the TNF family. AB - A number of proteins with significant similarity to the tumour necrosis factor (TNF) have been identified over the last years. Upon interaction with their cognate receptor (members of the TNF-receptor family), all members of this protein family induce either cell death or proliferation/differentiation of the receptor-bearing cells. One of the last identified members of the TNF family is the apoptosis-inducing ligand of the Fas-receptor, termed Fas-ligand (FasL). Here we report the cloning and sequencing of the mouse cDNA for the FasL. Using knowledge-based protein modelling, we demonstrate that all members of the TNF family form trimeric complexes, and define the residues located at the subunit interfaces. The resulting structurally corrected multiple sequence alignment allows the identification of residues potentially involved in receptor recognition, and should help design mutagenesis experiments for structure function relationship studies. PMID- 7544872 TI - Production of gliotoxin during the pathogenic state in turkey poults by Aspergillus fumigatus Fresenius. AB - Turkey poults were given either of two different dosages of two different gliotoxin-producing strains of Aspergillus fumigatus. Infected lung tissue was examined postmortem for the presence of gliotoxin. Gliotoxin was found in lung tissue of ten poults infected with one strain and in seven of ten poults infected with the other strain. Concentrations of gliotoxin in the tissue exceeded 6 ppm in some of the infected tissues. The concentration of gliotoxin found in infected tissue did not appear to be correlated with the dosage of organism given. Considering the pathologic changes observed in turkey poults with aspergillosis and the production of gliotoxin during the pathogenic state in turkey poults, gliotoxin is considered likely to be involved in avian aspergillosis. PMID- 7544873 TI - An outbreak of dermatophytosis in pigs caused by Microsporum canis. AB - An outbreak of dermatophytosis caused by Microsporum canis in a porcine farm is described. The morbidity was 100% among sows, 95% among new-borns and 75% among feedlot animals. Microsporum canis was also isolated from walls and environmental air. PMID- 7544875 TI - Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes. AB - Control of actin filament length and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends. Tropomodulin is a capping protein for the pointed end of the actin filament; it is associated with the free, pointed ends of the thin filaments in striated muscle, where it is thought to bind to both tropomyosin and actin. In embryonic chick cardiac myocytes, tropomodulin assembles after the thin, as well as the thick, filaments have become organized into periodic I and A bands, suggesting that tropomodulin might be involved in maintaining actin filament length. Here we show that microinjection of an antibody that inhibits tropomodulin's pointed-end-capping activity in vitro results in a marked elongation of actin filaments from their pointed ends and a > 80% reduction in the percentage of beating cells. This demonstrates that pointed-end capping by tropomodulin is required to maintain actin filament length in vivo and that this is essential for contractile function in embryonic chick cardiac myocytes. PMID- 7544876 TI - Inward membrane currents and electrophysiological responses to GnRH in ovine gonadotropes. AB - We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10 nM), identifying it as a Na+ current. Slowly inactivating inward current, activated at potentials more positive than -30 mV, remained in Na(+)-free medium or in the presence of 1 microM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedipine about 70% of this high voltage activated Ca2+ current was abolished, leaving a slowly inactivating component. No transient Ca2+ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 microM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K+ currents sensitive to membrane voltage and intracellular Ca2+ concentration ([Ca2+]i) were present. The resting membrane potential lay between -20 and -75 mV (mean = -43 +/- 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at -20 to -35 mV) for up to 1 h. Apamin, the SK-type Ca(2+)-dependent K+ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca2+ channel blockade did not. Microfluorimetric detection of [Ca2+]i showed that 10 nM GnRH induced [Ca2+]i oscillations. We conclude that Ca2+ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca2+]i fluctuations may activate the apamin-sensitive, Ca(2+)-dependent SK-type K+ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na+ current spikes may be crucial for activation of the nifedipine- and CGTX sensitive high-voltage-activated Ca2+ channels. PMID- 7544877 TI - Hormonal regulation of human follicle-stimulating hormone-beta subunit gene expression: GnRH stimulation and GnRH-independent androgen inhibition. AB - Previous studies from our laboratory demonstrated that chronic testosterone administration to castrated transgenic mice suppressed human follicle-stimulating hormone-beta (FSH beta) mRNA levels transcribed from a human transgene to approximately 20% of control values. In the present study we used primary pituitary cultures prepared from the transgenic mice and in vivo experiments in hypogonadal (hpg) mice carrying the human transgene to assess the role of hypothalamic gonadotropin-releasing hormone (GnRH) in this inhibitory action. The levels of human FSH beta mRNA in monolayer cultures of pituitary cells were decreased by 24-hour treatments with 10 nM testosterone propionate or 5 alpha dihydrotestosterone to 13 and 26% of control values, respectively, in the absence of GnRH. For the in vivo experiments we introduced the 10-kb human FSH beta transgene into the hpg genetic background by selective crossbreeding. Daily injections of 1 microgram GnRH for 14 days induced expression of the human FSH beta gene in male and female mice. Maximal effects were obtained by GnRH treatment of gonadectomized, hpg transgenic mice. Human FSH beta mRNA levels rose to approximately 4- or 10-fold that of control males and females, respectively. The stimulation was blocked completely by simultaneous administration of testosterone propionate in males and partially by estradiol in females. Pituitary content of immunoreactive FSH paralleled the mRNA changes. These data suggest that testosterone feedback inhibits the human FSH beta subunit gene directly at the pituitary gland in addition to the indirect mechanism of GnRH suppression. Furthermore, the in vitro data indicate that the suppression of human FSH beta gene expression is at least partly a direct androgen effect that does not require aromatization of testosterone to estradiol. PMID- 7544874 TI - Ca(2+)- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. AB - Chemoattractants stimulate neutrophil migration by activating signalling pathways including repeated transient increases in intracellular free calcium, [Ca2+]i. A motile neutrophil sends out many pseudopods, some of which adhere to the substrate; to continue moving forward the cell must release these attachments. Adhesion can be actively regulated, and neutrophils in which [Ca2+]i transients are inhibited become stuck on fibronectin or vitronectin, extracellular matrix proteins that neutrophils encounter in vivo. Function-blocking antibodies to beta 3 integrins or the alpha v beta 3 heterodimer restore motility on vitronectin to [Ca2+]i-buffered cells (B. Hendey, M.A.L., E. Marcantonio and F.R.M., manuscript submitted), indicating that an alpha v beta 3-like integrin is responsible for the [Ca2+]i-sensitive adhesion. We show that the density of alpha v beta 3 integrins in the adherent membrane of neutrophils migrating on vitronectin is much higher at the leading edge than at the rear, but [Ca2+]i buffering or inhibition of Ca(2+)-calmodulin-activated protein phosphatase 2B (calcineurin) leads to accumulation of alpha v beta 3 on the adherent surface at the rear of the cell. We show that the polarized distribution of alpha v beta 3 integrins in migrating neutrophils is maintained by [Ca2+]i-dependent release of adhesion followed by endocytosis of these integrins and recycling to the leading edge. PMID- 7544879 TI - Activation of central GABAA-but not of GABAB-receptors rapidly reduces pituitary LH release and GnRH gene expression in the preoptic/anterior hypothalamic area of ovariectomized rats. AB - gamma-Aminobutyric acid (GABA) exerts an inhibitory action on gonadotropin releasing hormone (GnRH) release from the hypothalamus. In vivo, this inhibitory action appears to be mediated via the GABAA receptor since in ovariectomized (ovx) rats and sheep direct application of muscimol (MUS), a GABAA agonist, into the preoptic area (POA), the site were the GnRH cell bodies are located, caused an immediate reduction of LH release. This effect may be the result of an inhibition of GnRH release but also GnRH biosynthesis may be affected. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) we now addressed the question, whether an acute inhibition of the GnRH pulse generator in ovx rats by GABA involves reduction of GnRH biosynthesis as determined by GnRH mRNA levels in micropunches of the POA. To activate either the GABAA or GABAB receptor, we injected intraventricularly (icv) MUS or baclofen (BAC). Intracerebroventricular injection of 10 nmol MUS caused a rapid and lasting inhibition of LH release from about 7.5 ng/ml (pretreatment value) to approximately 1.5 ng/ml. Neither application of BAC or saline (control injections) affected LH secretion. Two hours after icv injections, rats were decapitated and GnRH mRNA levels were determined. MUS induced a pronounced decrease of GnRH levels in the POA (control rats: 2.26 pg GnRH mRNA; MUS-treated rats: 0.85 pg, n = 10/group). BAC was without any effect on GnRH mRNA levels. Thus, we confirm the inhibitory action of GABA on LH release in vivo which is exerted via the A-subtype of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544881 TI - Rescue of injury-induced neurofilament loss by BDNF gene transfection in primary septo-hippocampal cell cultures. AB - We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls. PMID- 7544880 TI - Effects of progesterone on prolactin, hypothalamic beta-endorphin, hypothalamic substance P, and midbrain serotonin in guinea pigs. AB - Unlike rats, but similar to primates, guinea pigs exhibit prolonged function of the corpus luteum and elevated progesterone secretion after ovulation. The gonadotropins, estrogen (E) and progesterone (P) have been examined throughout the guinea pig estrous cycle. However, neither prolactin secretion nor its regulation by steroid hormones has been characterized, perhaps due to the lack of a specific radioimmunoassay. beta-Endorphin (BE), substance P (SP), and serotonin (5-HT) increase prolactin secretion in rats and monkeys. BE and SP neurons in guinea pigs and 5-HT neurons in monkeys contain progestin receptors which could mediate neuroendocrine effects of steroid hormones. Therefore, the effects of E and P on prolactin, BE, SP, and 5-HT and its metabolite 5-HIAA were examined in guinea pigs which were ovariectomized, E treated (28 days), and E+P treated (14 days E+14 days E+P). The rat NB2 lymphoma cell line was used as a bioassay for serum prolactin. BE and SP levels were measured by radioimmunoassay in four hypothalamic areas: the preoptic region (POA), the mediobasal hypothalamus (MBH), the dorsomedial hypothalamus (DMH), and the mamillary bodies (MB). 5-HT and 5 HIAA were measured in the midbrain raphe area by high-pressure liquid chromatography. E alone had little effect on serum prolactin levels, but E+P significantly increased prolactin as compared with ovariectomized controls. The BE levels increased with E treatment and remained elevated with E+P treatment in MBH and POA. The BE content was stimulated in DMH and MB by E+P treatment and not with E alone. The SP content in MBH, DMH, and MB increased in E-treated guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544882 TI - Distribution of secretoneurin-like immunoreactivity in comparison with that of substance P in the human spinal cord. AB - Secretoneurin (SN), a neuropeptide of 33 amino acids, was determined in comparison with substance P (SP) by immunocytochemistry in normal human spinal cord. The density of secretoneurin-like immunoreactivity (SN-IR) was high in the superficial dorsal horn and in the lateral column of autonomic arcs. The ventral horn displayed low to moderate density of SN-IR and prominently outlined motoneurons. The congruent distribution of SN and SP to the termination of primary afferents may indicate that SN is involved in modulation of pain. PMID- 7544878 TI - Galanin-binding sites in the female rat brain are regulated across puberty yet similar to the male pattern in adulthood. AB - The neuropeptide galanin (GAL) has been implicated in a variety of neuroendocrine functions and has been shown to be regulated by gonadal hormones in several brain regions. We have used slice binding and quantitative autoradiography techniques to determine whether the activation of GAL pathways across puberty in female rats is associated with changes in the density of GAL binding in telencephalic and diencephalic regions as we previously observed in male rats. We have also asked whether sex differences in GAL immunoreactivity and GAL gene expression detected in some brain regions would be paralleled by sex differences in 125I-GAL-binding density in adult male and female rat brains. To control for intrinsic differences in the level of endogenous GAL synthesis and release, brain slices from prepubertal female and adult male and female rats were treated with guanosine 5' triphosphate (GTP) to induce dissociation of endogenous GAL from its binding sites prior to incubation with radiolabeled ligand. 125I-GAL binding was significantly reduced in seven brain regions of adult compared with prepubertal female rats. These regions included the islands of Calleja (p < or = 0.03), the medial amygdaloid nucleus, posterodorsal division (p < or = 0.05), median eminence (p < or = 0.02), medial habenular nucleus (p < or = 0.05), rhomboid thalamic nucleus (p < or = 0.05), and paraventricular (p < or = 0.05) and intermediodorsal (p < or = 0.02) thalamic nuclei. Only one region, the lateral preoptic area, exhibited significantly enhanced 125I-GAL binding in adult female (p < or = 0.04) compared with prepubertal animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544885 TI - Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template. AB - Avian myeloblastosis virus and Maloney murine leukemia virus RNase H-reverse transcriptases pause when they encounter a 2'-5' linkage or a 2'-thiophosphate in their template RNAs, but eventually read through these backbone modifications. Both reverse transcriptases pause after the 2'-5' linkage but before the 2' thiophosphate. These results suggest that in the absence of precise information concerning the behavior of a given reverse transcriptase with respect to a particular lesion or modification, caution should be exercised in the interpretation of primer extension data that is being used to determine the existence of, or map the position of, a crosslink, site of chemical modification or non-standard linkage in an RNA template. PMID- 7544884 TI - Altered pharmacokinetic and tumour localization properties of Fab' fragments of a murine monoclonal anti-CEA antibody by covalent modification with low molecular weight dextran. AB - This paper describes the modification of Fab' fragments of ZCE025, a murine monoclonal antibody recognizing carcinoembryonic antigen (CEA), with oxidized dextran of low molecular weight. This modification altered the in vitro and in vivo characteristics of the Fab'. The dextran conjugated fragments exhibited markedly reduced renal uptake and excretion and the prolonged residence time of the Fab' in the vascular compartment. The result was enhanced tumour localization of the derivative compounds compared with underivatized Fab', even though the process of chemical coordination reduced the immunoreactivity of the molecule. The isoelectric point of the molecule was much lower than the unaltered Fab' and previous research has shown this to reduce markedly its potential for inducing a human anti-mouse antibody (HAMA) response. Thus, the conjugation of Fab' fragments with low molecular weight oxidized dextrans can increase the bioavailability of these fragments for tumour localization, while simultaneously reducing their immunogenic potential and renal accumulation problems. PMID- 7544886 TI - Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5' triphosphate as substrate. AB - L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. PMID- 7544887 TI - Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA. AB - The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1 expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV 1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect. PMID- 7544889 TI - Targeting pyrimidine single strands by triplex formation: structural optimization of binding. AB - Recent reports describe a new strategy for the binding of single-stranded pyrimidine sequences by triple helix formation. In this approach, a double-length purine-rich oligonucleotide binds a target strand, folding back to form an antiparallel pur.pur.pyr triple helix. We now describe a series of studies in which sequence and structural variations are made in such purine-rich ligands, in an effort to optimize binding properties. Comparison is made between the use of two separate strands and the use of single two-domain ligands; the latter are found to bind more tightly and to aggregate less in media containing Na+ or K+. Placement of mismatched bases in the target shows that sequence selectivity of binding is as high as that for Watson-Crick duplex formation. Variation of the lengths and sequences of loops bridging the binding domains demonstrates that dinucleotide loops composed of pyrimidines give the highest stability. Oligoethylene glycol-derived loop replacements are shown to give good binding affinity as well. The binding of an RNA target is shown to occur with the same affinity as the binding of DNA. In general, it is found that circular variants bind more tightly than do either separate strands or singly-linked ligands and unlike linear oligomers, the circular compounds do not aggregate to a large extent even in buffers containing 100 mM K+. Such structurally optimized ligands are useful in expanding the number of possible naturally-occurring sequences which can be targeted by triplex formation. PMID- 7544883 TI - Laser ablation of prostatic hypertrophy. AB - Laser prostatectomy offers the urologist a new tool for the management of symptomatic benign prostatic hypertrophy. Future studies will determine the long term benefits of this new procedure in a properly selected group of patients as a possible alternative to transurethral prostatectomy. PMID- 7544890 TI - Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications. AB - RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments. PMID- 7544888 TI - A novel IL-4 responsive element of the E alpha MHC class II promoter that binds to an inducible factor. AB - Interleukin-4 (IL-4) is a lymphokine with important role in the growth and differentiation of T and B lymphocytes. In the latter, IL-4 induces transcriptionally MHC class II gene expression. Using the M12 mouse lymphoid cell line, we have determined an IL-4 response sequence (ILRS) in the proximal promoter region of the E alpha class II gene. The ILRS extends from -80 to -111 and includes the MHC class II X motif and 19 bp of additional 5' sequence. In mouse lymphoid cells, IL-4 activates a complex (Nuclear Factor-IL-4, NFIL-4), that binds to a novel element within the ILRS. Similar IL-4 inducible complexes bind to the interferon-gamma response element of the Fc gamma receptor (GRR), the acute phase response element (APRE) of the alpha 2 macroglobulin promoters and an INF beta promoter site, overlapping the PRDII/NF kappa B element. The factor contacts all these elements through their common GGAA motif. NFIL-4 is immunologically unrelated to NF kappa B or STAT 1 proteins that also recognize the above elements. Activation of NFIL-4 requires tyrosine phosphorylation, occurs within 2 min and persists as long as IL-4 is present. NFIL-4 has an apparent molecular weight of 75 kDa as determined by sedimentation through glycerol gradients. PMID- 7544891 TI - An efficient strategy for the synthesis of circular RNA molecules. PMID- 7544892 TI - A rapid RT-PCR method for detection of intact RNA in formalin-fixed paraffin embedded tissues. PMID- 7544893 TI - CHILD syndrome: analysis of abnormal keratinization and ultrastructure. AB - A new patient with CHILD syndrome (congenital hemidysplasia, ichthyosiform erythroderma, and limb defects), the thirtieth in the literature, was observed for over three years. Initially, the right-sided lesion spared the breast area. At 10 months of age the trunk lesion extended to cover the entire area of the right chest. At age 20 months the patient developed linear, bandlike, keratotic, brown-black lesions on her left thigh that subsided within six weeks, leaving a slight hyperpigmentation. This patient was studied by routine histologic methods as well as with markers of keratinization and electron microscopy. In hematoxylin and eosinstained sections, parakeratosis and orthokeratosis alternated. In some parakeratotic areas, large granular cells, and in others, ghost granular cells, were present. The latter showed basophilic cytoplasm, and palestaining or vacuolated nucleus and were seen either above the normal granular layer or without it. Although regional variations existed, basal cell-type keratins as recognized by AE1 continued to be expressed in suprabasal layers. Filaggrin- and involucrin-positive layers were expanded, particularly the latter, down to the lower prickle cell layer. Ultrastructurally, numerous lamellar or membranous structures were found in upper layers of the epidermis, both intracellulary and intercellularly. Normal cementsomes coexisted with these abnormal lamellar structures, and it was thought that the latter represent modified cementsomes because the discharge of those from the cell periphery was often detected. PMID- 7544896 TI - Potter's syndrome in the second trimester--prenatal screening and pathological findings in 60 cases of oligohydramnios sequence. AB - Fifty-two second-trimester and eight third-trimester (> 28/40) autopsies with clinical or pathological evidence of oligohydramnios sequence ('Potter's syndrome') were reviewed. Twenty-eight cases had renal anomalies (71 per cent in terminations following prenatal ultrasound), 27 had no renal malformation (35 per cent with chorioamnionitis), and five had external assessments only. In 15 cases, the renal lesion was part of a multiple malformation syndrome. Seven cases had a lesion which either recurred in a sibling in the same family or was a recognized autosomal recessive syndrome. Three cases had an abnormal karyotype, two of which had renal anomalies. Maternal serum alpha-fetoprotein (AFP) did not discriminate between cases with renal malformations and those without. Pulmonary hypoplasia was commoner in third-trimester than in second-trimester cases. External appearance and absent umbilical artery were not reliable predictors of underlying internal anomalies. These findings reflect the shift from postnatal to prenatal diagnosis in modern practice. In this series, mainly second-trimester cases, 50 per cent of cases had no malformations, in a condition which is traditionally associated with renal disease. The high incidence of chorioamnionitis suggests that the mechanism of oligohydramnios is occult amniotic fluid leakage. Prenatal diagnosis of oligohydramnios in the second trimester is dependent on ultrasound scanning and a full post-mortem examination is necessary to identify any underlying fetal cause. PMID- 7544895 TI - Turtle lung cells produce a melanization-stimulating activity that promotes melanocytic differentiation of avian neural crest cells. AB - We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC-2, was found to produce a putative melanization-stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC-2-derived MSA was different from that of basic fibroblast growth factor (bFGF), alpha-melanocyte stimulating hormone (alpha-MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation. PMID- 7544894 TI - Bilateral pseudocyst of the auricle in an infant girl. AB - A 6-week-old girl developed bilateral auricular swelling in the absence of trauma. Clinical and histologic findings were consistent with pseudocyst of the auricle. This case is unusual given the age and sex of the patient and the bilateral presentation of the pseudocysts. PMID- 7544897 TI - Chorionic villus sampling by biopsy forceps. Results of 1580 procedures from a single centre. AB - The results of a prospective series of 1580 chorionic villus sampling (CVS) procedures using biopsy forceps are presented. Most of the procedures (1442), including 11 sets of twins, were performed by the transcervical approach (TC CVS), using a curved-shank thin forceps, and 138 by the transabdominal approach (TA-CVS), using a trocar-guided straight thin forceps. The mean gestational age for TC-CVS was 10.9 weeks, and in 233 cases (16 per cent) the procedure was carried out between the 12th and 14th weeks. The mean gestational age for TA-CVS was 16.7 weeks. The major indication for CVS was advanced maternal age (92.7 per cent in the TC and 91.8 per cent in the TA approach), and indications for abnormal ultrasound findings were more common in the TA approach (4.5 per cent) than in TC-CVS (0.07 per cent). Although sampling was apparently accomplished in all the procedures, in 3.1 per cent of the TC-CVS and 2.2 per cent of TA-CVS procedures, the samples were less than 1 mg after dissection. A cytogenic report was obtained in 96.1 per cent of the TC-CVS and 90.6 per cent of the TA-CVS. Maternal serum alpha-fetoprotein (MSAFP) was measured before and after TC-CVS and the post-CVS MSAFP was positively correlated with the sample weight. Second trimester amniocentesis following CVS was required in 5.2 per cent (TC-CVS) and 6.5 per cent (TA-CVS), due to the failure to obtain a cytogenetic report or diagnostic confirmation. The follow-up to the 20th week was 100 per cent by ultrasound scan, and 88.6 per cent from the 21st week to 1 week after delivery. Fetal loss rates within 2 weeks of the procedure were 1.7 per cent (TC-CVS) and 0.8 per cent (TA-CVS) and total fetal loss accumulated to 1 week after delivery was 4.6 per cent (TC-CVS) and 5.9 per cent (TA-CVS). Factors found to increase significantly fetal loss in the TC-CVS series were maternal age and the collection of very small samples, but not the number of forceps insertions. PMID- 7544899 TI - Growth, hormonal status and protein turnover in rats fed on a diet containing peas (Pisum sativum L.) as the source of protein. AB - The inclusion of peas (Pisum sativum L.) as the source of protein in the diet of growing rats brings about a reduction in growth rate as well as the impairment in the liver, muscle and spleen weights as compared with casein fed controls. Also, a fall in plasma glucose, triglycerides and protein was observed in the legume fed animals, while no changes in cholesterol levels were found. Furthermore, the rats fed on the diet containing peas showed lower levels of plasma insulin, corticosterone, IGF-I and T4 as compared with casein controls. Liver and muscle total protein (mg) and total DNA (mg) were markedly decreased in the legume fed animals, but DNA/g, protein/DNA and RNA/protein ratios were similar in both dietary groups. Likewise, liver and muscle fractional synthesis rates were similar in the casein and legume groups, while the whole body protein synthesis is assumed to be lower in the legume fed animals due to differences in body weights. It is concluded that animals fed on a diet containing peas (Pisum sativum L.) as the only source of protein showed less adverse effects than those found with other legumes such as Vicia faba L. or Phaseolus vulgaris L., in which protein quality, antinutritional factors and nutrient availability could be involved. PMID- 7544898 TI - Analysis of maternal serum alpha-fetoprotein and free beta human chorionic gonadotrophin in the first trimester: implications for Down's syndrome screening. AB - The aim of this study was to determine the maternal population, pregnancy, serum alpha-fetoprotein (AFP) and free beta subunit of human chorionic gonadotrophin (F beta hCG) parameters in a large series of women attending prenatal clinics before 15 weeks' gestation and to assess the practical problems of population screening for Down's syndrome in the first trimester using these markers. Serum samples were collected from 8600 women attending prenatal clinic booking visits. Maternal serum AFP and F beta hCG medians were calculated for each day of gestation (49 104 days), using both dates and ultrasound estimates of gestation. The effects of maternal weight, twin pregnancies, and threatened abortion on AFP and F beta hCG levels were analysed. The median age of the population was 27.1 years and the median weight 62.1 kg. Twenty-six per cent of samples were collected before 70 days and 50 per cent before 78 days' gestation. Eighty-nine per cent of all samples had gestational estimates by dates, 60 per cent by ultrasound and 52 per cent by both dates and ultrasound. The AFP median was 5 kU/l at 49 days, 5.9 kU/l at 70 days, and 17.9 kU/l at 100 days. The peak median F beta hCG level was 66.4 ng/ml at 64 days, falling to 20.6 ng/ml at 100 days' gestation. Both AFP and F beta hCG levels showed log Gaussian distributions but the standard deviation for AFP was 20 per cent greater than that found in the second trimester. AFP and F beta hCG levels showed an inverse relationship with maternal weight and were increased in twin pregnancies (1.68 and 1.97 multiples of the median, respectively). AFP and F beta hCG can be readily measured in a large screening population in the first trimester. Down's syndrome screening protocols based on these markers could be refined by the use of gestations in individual days but AFP is likely to be a less effective marker and detection rates are likely to be lower than in the second trimester. To realize the potential of first-trimester screening, more women should be encouraged to attend the prenatal clinic in early pregnancy and ultrasound dating should be carried out for all pregnancies at this stage. PMID- 7544900 TI - Olanzapine, an atypical antipsychotic, increases rates of punished responding in pigeons. AB - The effects of olanzapine [LY 170053; 2-methyl-4-(4-methyl-1-piperazinyl)-10H thieno[2, 3b] [1,5]benzodiazepine), a potential atypical antipsychotic, were determined in pigeons whose keypeck responding was punished. These effects were compared to the anxiolytic agents chlordiazepoxide and pentobarbital, and to other antipsychotic agents. Keypeck behavior was maintained under a multiple FR30 FR30 schedule, signalled by white and red stimulus lights, respectively. Each component of the schedule alternated every 3 min with a 30-s timeout. During the white keylight component, responding was maintained by food presentation. During the red keylight component, responding was maintained by food and simultaneously suppressed by electric shock presentation, with response rates being only about 5% of those during the white stimulus light. Olanzapine (0.01-1.0 mg/kg) increased punished responding at doses below those which had an effect on unpunished responding. Clozapine (0.01-1.0 mg/kg), ritanserin (0.1-3.0 mg/kg), and, to a lesser extent, risperidone (0.1-1.0 mg/kg) were also effective at increasing punished responding. Generally, the maximum effect seen with olanzapine was equal to that seen with ritanserin, and it exceeded that seen with clozapine. However, these effects were generally less than those seen with chlordiazepoxide and pentobarbital. Haloperidol (0.01-0.1 mg/kg) was completely without effect on punished responding, while it caused decreases in unpunished behavior. These results provide further evidence that olanzapine has a profile in behavioral tests unlike the typical antipsychotic haloperidol. Moreover, this profile is similar to clozapine, a clinically effective antipsychotic with an atypical profile. PMID- 7544901 TI - Replicable RNA vectors: prospects for cell-free gene amplification, expression, and cloning. PMID- 7544902 TI - Tachykinin receptors mediating contraction of guinea pig lung strips. AB - The aim of this study was to determine which tachykinin receptors mediate contraction in guinea pig lung parenchymal strips in vitro. Contraction caused by selective neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 (NK-3) receptor agonists and the natural agonists substance P (SP) and neurokinin A (NKA) was measured in the absence or presence of the NK-1 antagonist CP-96,345 and/or the NK-2 antagonist SR 48968. The NK-1 agonist [Sar9, Met(O2)11]-substance P and the NK-2 agonist [Nle10]-neurokinin A 4-10 caused similar concentration dependent contractions that were inhibited by CP-96,345 and SR 48968, respectively. The NK-3 agonist [MePhe7]-neurokinin B also caused contraction, albeit at 10-fold higher concentrations, and this contraction was unaffected by either the NK-1 or NK-2 antagonist or the combination of both antagonists. Either CP-96,345 or SR 48968 alone had little effect on NKA-mediated contraction but administration of both antagonists virtually eliminated force generation. SP induced tension was partially inhibited by SR 48968 and unaffected by CP-96,345. A second NK-1 receptor antagonist CP-99,994 also had no effect on SP-mediated tension. Therefore, NK-1, NK-2, and NK-3 agonists can all cause contraction in guinea pig lung strips, NKA-induced tension is mediated by both NK-1 and NK-2 receptors, and SP-induced contraction is mediated in part by NK-2 receptors. Both the SP and the [MePhe7]-neurokinin B data suggest that activation of a third neurokinin receptor subtype that is unaffected by an NK-1 receptor antagonist or an NK-2 receptor antagonist can cause contraction in guinea pig lung strips. PMID- 7544904 TI - [Usefulness of Doppler color in the endometrial assessment of post-menopausal women]. AB - We performed this study for investigate the uterine flow velocity waveforms and color flow imaging in women with postmenopausal bleeding. Our results suggest that the color doppler is an important method for the evaluation of women with abnormalities at the ultrasound examination. PMID- 7544905 TI - Interferon induction in swine lymphocyte antigen-defined miniature pigs. AB - Interferon was induced in two groups of swine lymphocyte antigen (SLA)-defined miniature pigs with polyinosinic: polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose. The group 1 pigs were low antibody-response phenotypes (SLAa/a, SLAa/c, SLAc/c), and the group 2 pigs were high antibody response phenotypes (SLAd/d, SLAd/g, SLAg/g). Six hours after induction the antiviral tires were not influenced by the SLA group, but higher titres were observed in females. Higher antiviral titres were found in group 2 pigs before treatment and 24 hours after treatment, and higher titres were found in female pigs. The antiviral titres before and after treatment were also influenced by the sire. Group 2 pigs had a lower total leucocyte counts before treatment, and there was a significant reduction in leucocyte numbers in both groups six hours after induction, due mainly to a large reduction in lymphocyte counts. PMID- 7544903 TI - Inhibition of gastrin-stimulated enterochromaffin-like cell proliferation and mucosal histamine production in the rat stomach by the somatostatin analogue octreotide. AB - The effect of octreotide, a potent and long-acting analogue of somatostatin, on gastrin-stimulated proliferation and function of enterochromaffin-like (ECL) cells were examined in rats. Animals were divided into four groups and each group was continuously infused with saline, octreotide alone (40 micrograms/kg per day), gastrin alone (60 nmol/kg per day), or octreotide (40 micrograms/kg per day) plus gastrin (60 nmol/kg per day) respectively for 9 days via osmotic minipumps. Gastrin induced the increase of the bromodeoxyuridine labeling index and density of oxyntic mucosal ECL cells as well as oxyntic mucosal histidine decarboxylase activity. Octreotide completely abolished the gastrin-induced increases in the labeling index and density of ECL cells and oxyntic mucosal histidine decarboxylase activity. These results indicate that octreotide inhibits gastrin-stimulated proliferation of ECL cells and histamine production by these cells. PMID- 7544907 TI - Apoptosis in the central nervous system of developing mouse fetuses from 5 azacytidine-administered dams. AB - To determine the effect of 5-azacytidine (5 Az), a cytidine analogue, on the developing fetus, different strains of mice were injected with various doses of the agent, ip, at 11 days of gestation. Light microscopically, severely pyknotic cell death profiles were observed in different layers of the developing brain and spinal cord (central nervous system) on the day after the injection. Electron microscopy of the pyknotic cells revealed the shrinkage of the cell body, to various degrees margination of the nuclear chromatin, condensation of nuclear materials, fragmentation of the affected cells, and, finally, neat "bite-size" bodies engulfed by surrounding normal neuroepithelial cells and macrophage-like cells. DNA fragmentation was detected in the nuclei of the pyknotic cells by an in situ detection method for fragmented DNA ends. These changes were consistent with the criteria of apoptosis. PMID- 7544914 TI - Risk factor exposure among hepatitis C virus RNA positive Swedish blood donors- the role of parenteral and sexual transmission. AB - The potential modes of transmission for hepatitis C virus (HCV) infections were studied using a multivariate analysis of risk factor exposure among 51 2nd generation anti-HCV and HCV-RNA positive and matched anti-HCV negative blood donors. The following variables were found to be independently associated with anti-HCV and HCV-RNA positivity: intravenous drug use (IVDU) (p < 0.001), blood transfusion (p < 0.01), tattoos (p < 0.001), previous hospitalization (p < 0.05), history of sexually transmitted disease (STD) (p < 0.001) and lack of travels outside of Europe (p < 0.05). Among the 23 HCV-RNA positive donors without a history of IVDU or blood transfusion, an increased frequency of hospitalization (p = 0.017) and history of STD (p = 0.023) were found. Five of 22 sexual partners of the 51 index blood donors were HCV-RNA positive and in one of these couples sexual transmission was suspected. Anti-HCV and HCV-RNA positive donors were more often seropositive for herpes simplex virus type 2 (HSV-2) antibodies than were HCV-negative controls (p = 0.015). Sexual transmission of HCV may occur, but the possible role of HSV-2 requires further investigation. PMID- 7544908 TI - Chemical denaturation of ovalbumin abrogates the induction of oral tolerance of specific IgG antibody and DTH responses in mice. AB - We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA, for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction. PMID- 7544906 TI - Indirect immunohistochemistry and immunoelectron microscopy distribution of eight epidermal-dermal junction epitopes in the pig and in isolated perfused skin treated with bis (2-chloroethyl) sulfide. AB - Sulfur mustard (bis [2-chloroethyl] sulfide, HD) is a potent cutaneous vesicant that causes gross blisters by separation of the epidermal-dermal junction (EDJ). The EDJ of the skin is a highly specialized and complex structure composed of several components and plays a major role in the integrity of the skin. The isolated perfused porcine skin flap (IPPSF) was dosed with 0.2 mg/ml (n = 4), 5.0 mg/ml (n = 4), and 10.0 mg/ml (n = 5) HD or ethanol (n = 4) for 8 hr (dose response study) and 10.0 mg/ml HD or ethanol for 1, 3, 5, and 8 hr (n = 4/treatment) (time-response study). Successful EDJ mapping was carried out in normal pig skin (NPS), ethanol-treated IPPSFs, and HD-treated IPPSFs using the following antibodies: laminin, type IV collagen, fibronectin, GB3 (Nicein), bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). Two mouse anti-human monoclonal antibodies, L3d and 19-DEJ-1 (Uncein), did not cross-react with the EDJ of the pig. Antibody staining in NPS, ranging from very intense for laminin and type IV collagen to weak for fibronectin, was generally more discrete than in the IPPSF. No differences in staining were noted between the ethanol and nonblistered areas of the HD-treated IPPSFs. In HD-blistered areas, BP stained only the epidermal hemidesmosomes, and laminin, fibronectin, and GB3 stained primarily the dermis with fragments attached to the basal pole of the stratum base cells, while type IV collagen and EBA stained only the dermis. Mapping of these epitopes determined that the precise plane of EDJ separation in the HD treated skin occurred beneath the hemidesmosomes within the upper portion of the lamina lucida. The conservation of human epitopes in the EDJ of the pig further emphasizes the similarities between human skin and pig skin. Therefore, pig skin and the IPPSF may be used to study HD-induced vesication and blistering diseases. PMID- 7544913 TI - Proliferation of IL-2 activated lymphocytes preferably occurs in aggregates by cells expressing the CD57 antigen. AB - Aggregates of lymphocytes were investigated in long-term cultures of IL-2 stimulated peripheral blood mononuclear cells. Cells in clusters formed broad contact areas where coated pits and vesicles were often detectable. Proliferating cells were preferably found in aggregates indicating that the cell clusters represent proliferation centres in which cell division may be promoted by accessory signal transduction mediated by the close cell-to-cell contact. In bulk cultures CD57+ cells yielded increased proliferation capacity compared to CD57- cells. In addition, CD57+ cells were preferably localized in aggregates where they occupied the same position at the periphery of the clusters than the proliferating cells, suggesting that both cell types may be identical. It is discussed that the CD57 antigen represents a differentiation marker which is upregulated when CD57- cells start to proliferate assisted in cell aggregates. PMID- 7544911 TI - Administration of antibodies to hyaluronanreceptor (CD44) delays the start and ameliorates the severity of collagen II arthritis. AB - Hyaluronanreceptor (CD44) has been shown to be involved in lymphocyte homing during normal leucocyte circulation and during leucocyte extravasation into sites of tissue inflammation. In addition, interaction with CD44 molecule induces T cell activation and production of cytokines, such as interferon-gamma. In this study we have examined what influence interaction with the CD44 receptor would have on collagen II-induced arthritis in mice. Mice were immunized with rat collagen II and administered with injections of a monoclonal anti-CD44 antibody. Seventeen days after the outbreak of the disease, all of the anti-CD44 treated animals remained clinically health, whereas 37% of the controls displayed arthritis (P < 0.001). Ten days later the prevalence of arthritis was 26% and 65% (P < 0.05), respectively. Furthermore, the severity of the arthritis was significantly ameliorated by the anti-CD44 treatment. Serum levels of interferon gamma were significantly higher in collagen II immunized animals having been treated with anti-CD44, compared to the controls. Delayed-type hypersensitivity (DTH) response was significantly decreased in the anti-CD44 treated animals, indicating a functional suppression of T cells. In contrast, T cell independent experimental inflammation was not affected by the administration of CD44 antibodies. Our results suggest that interaction with CD44 down-regulates T lymphocyte/monocyte mediated inflammatory reaction, possibly by triggering of interferon-gamma release. PMID- 7544910 TI - Stimulation of the same B-cell population by thymus-independent dextran and by thymus-dependent oligosaccharide-carrier. AB - The immune response of adult BALB/c mice against the bacterial antigen dextran B1355S (Dex) is well characterized as thymus independent (TI type 2) and Igha linked. The antisera consist of mainly IgM/lambda antibodies directed against the alpha(1-->3) glucosidic linkage. This study describes the immune response against the alpha(1-->3) linkage in the thymus dependent form (TD), i.e. tetra- or heptasaccharides (N4 or N7) of glucose as hapten coupled to chicken serum albumin (CSA) as carrier. Whereas athymic BALB/c-nu/nu mice did not respond to the TD antigens N4-CSA and N7-CSA, euthymic BALB/c showed high anti-Dex antibody titres of IgM and, after 2 degrees immunization, a class switch to IgG (mainly IgG1) isotypes with lambda light chains. The hapten N4 inhibited Dex-binding of M104E or of antisera from Dex or N4-CSA or N7-CSA immunized mice at 1.7-10 x 10(-4)M. The idiotype composition of these antibodies resembled those after Dex immunization. We conclude that the same Dex-specific precursor B cells have been stimulated by either form of antigen. The ontogenic development of a Dex-specific response could not be accelerated by the aid of T cells, even of adult origin. It seems, therefore, that the maturation of antigen specific B cells is the limiting step in ontogeny. PMID- 7544912 TI - Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction. AB - We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-alpha 2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with alpha 2M. PMID- 7544909 TI - Macrophage response to microbial pathogens: modulation of the expression of adhesion, CD14, and MHC class II molecules by viruses, bacteria, protozoa and fungi. AB - The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA-1), and CD54 (ICAM-1) molecules in unprimed bone marrow-derived mononuclear phagocytes (BMM phi) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down-regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon gamma was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell-mediated macrophage activities while viruses, protozoa and fungi are poor in this respect. PMID- 7544915 TI - TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. AB - TNF receptor-associated factor (TRAF) proteins are candidate signal transducers that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. The role of TRAFs in the TNF-R2 and CD40 signal transduction pathways, which result in the activation of transcription factor NF-kappa B, was investigated. Overexpression of TRAF2, but not TRAF1 or TRAF3, was sufficient to induce NF-kappa B activation. A truncated derivative of TRAF2 lacking an amino-terminal RING finger domain was a dominant-negative inhibitor of NF-kappa B activation mediated by TNF-R2 and CD40. Thus, TRAF2 is a common mediator of TNF-R2 and CD40 signaling. PMID- 7544916 TI - Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression. AB - Platelet activation plays an important role in the pathomechanisms of arterial vascular disorders including stroke, peripheral arterial disease (PAD), and myocardial infarction. Circulating activated platelets may be useful markers of local thrombotic events occurring in these diseases. Using flow cytometry circulating activated platelets can be detected by determining: 1. the platelets' shape change on the basis of the different light scatter properties of discocytes and spherocytes, 2. the expression of platelet bound fibrinogen or conformation specific neoantigens on fibrinogen and on its platelet receptor, and 3. the exposure of granule membrane proteins such as P-selectin as a result of platelet secretion. The concentration-effect relationships were determined for the ADP and U46619 induced shape change, fibrinogen binding, and expression of P-selectin. The EC50 for the shape change was 4 times lower than the EC50 for the fibrinogen binding and 29 times lower than the EC50 for the expression of P-selectin. These data clearly demonstrate that the shape change is a more sensitive indicator of platelet activation in vitro than fibrinogen binding or P-selectin expression. Both the shape change and fibrinogen binding were reversible, whereas the expression of P-selectin was irreversible upon stimulation. Reversibility of the shape change may be responsible for the fact that in patients with stroke or PAD the fraction of discocytes did not differ from controls, whereas more than 80% of them revealed a significantly higher fraction of P-selectin positive platelets. Thus the determination of the P-selectin expression reveals a higher diagnostic sensitivity for detecting a platelet activation in vivo than the determination of the shape change. PMID- 7544920 TI - Endoscopic palliation for unresectable pancreatic carcinoma. AB - Endoscopic stenting has revolutionized the palliation of malignant biliary obstruction. Individuals with biliary obstruction due to pancreatic malignancy are best managed by a team approach comprising individuals with expertise in oncology, surgery, endoscopy, and radiology. The clinical value of alleviating jaundice and associated symptoms of anorexia, pruritus, and malaise cannot be overestimated. These quality-of-life factors deserve more attention in future studies to define subgroups of patients most likely to derive benefit from the array of treatment options. Further technical improvements are required to solve the most important clinical problem of late stent occlusion. New developments such as expandable metallic stents and refinements in existing equipment and techniques have already resulted in measurable gains and hold great promise for future expansion of their use in malignant gastric outlet obstruction secondary to tumor infiltration of the duodenum. Corresponding advances are being made in the fields of percutaneous transhepatic intervention and hepatobiliary surgery such that further prospective randomized trials are necessary to define optimal therapy. PMID- 7544919 TI - Surgical palliation of unresectable pancreatic carcinoma. AB - The decision to perform surgical versus nonoperative palliation for unresectable pancreatic cancer is influenced by a number of factors. In most cases, patient symptoms clearly dictate the management. In patients with symptoms of duodenal obstruction at the time of presentation, surgery is the only option. In patients with obstructive jaundice alone, the options for management must be weighed against factors such as overall health status, projected survival, and procedure related morbidity and mortality. A prospective multicenter trial recently analyzed factors influencing perioperative morbidity and mortality following both curative and palliative surgery for pancreatic cancer. This analysis demonstrated that preoperative diabetes, low Kanofsky's index, and liver metastases are significant risk factors in predicting perioperative morbidity and mortality in patients undergoing palliative procedures for pancreatic cancer. Another analysis focusing on tumor characteristics suggested that for patients with Stage I and Stage II disease (i.e., with no evidence of systemic metastases), survival and the potential for late duodenal obstruction favor surgical management. In summary, although patient management must be individualized, most patients with pancreatic cancer in good medical health and with no evidence of systemic disease are most appropriately managed with surgical palliation. This option affords patients the best chance of avoiding the late complications of recurrent jaundice, duodenal obstruction, and disabling pain. Surgical palliation can generally be completed with an acceptable perioperative morbidity and mortality and a hospital stay of approximately 2 weeks. Finally, only surgical exploration can offer full opportunity for resection for cure. PMID- 7544917 TI - Renal insufficiency and hypertension as long-term complications in liver transplantation. PMID- 7544918 TI - Iliac vein compression syndrome from urinary bladder distension due to prostatism. AB - Urinary bladder distention is a rarely reported cause of iliac vein compression. We report a case of bilateral lower extremity and scrotal edema resulting from marked bladder distention. The bladder was readily palpable and a 4,100 cm3 postvoid residual urine volume was found. No other cause of lower extremity and scrotal edema was found. The patient improved after bladder drainage and transurethral prostatectomy (TURP) for moderate benign prostatic hypertrophy. PMID- 7544922 TI - Infrared angiography of the anterior ocular segment. AB - We performed angiography with indocyanine green (ICG) and fluorescein using a scanning laser ophthalmoscope (SLO) in the anterior segments of seven normal and 35 diseased eyes. ICG angiography revealed the radical stromal vessels and minor arterial circle in brown irides, which were not detected by fluorescein angiography. High penetration of infrared fluorescence through the pigmented tissue and absence of extravasation of ICG facilitated demonstration of the fine structure of iris rubeosis and its origins from stromal vessels. In 11 diabetic eyes, the iris rubeosis showed three basic patterns of location: along the pupillary margin; originating from the iris root; and arising from stromal radial vessels near the collarette. ICG gonioangiography with SLO showed fine structure of angle rubeosis because of its high resolution and greater depth in focus. Rubeotic vessels in the chamber angle were perfused by neovascular trunks which arose from the iris root in all 12 rubeotic eyes. Rubeosis in the iris and the angle consistently showed no extravasation of the ICG dye, while fluorescein quickly leaked out. ICG angiography with SLO in the anterior ocular segment proved to be a useful means to study the structure and the hemodynamics of normal and newly formed vessels. PMID- 7544921 TI - Percutaneous palliation of unresectable pancreatic cancer. AB - Percutaneous biliary drainage is one of several methods of palliating patients with unresectable carcinoma of the pancreas. In most cases, patients with unresectable carcinoma of the pancreas causing biliary obstruction are optimally managed by endoscopic stent placement. Percutaneous biliary drainage may be performed when endoscopic biliary drainage is unsuccessful or with obstruction at or above the hepatic duct bifurcation. Patients with common bile duct obstruction today are optimally managed by placement of expandable metal endoprostheses. In such patients with short life expectancies, symptoms of jaundice can be effectively palliated with a low rate of recurrence and with a complication rate no higher than that associated with percutaneous biliary drainage. Metal endoprostheses are associated with less pain during placement than are plastic endoprostheses and may have a lower rate of recurrence of jaundice than plastic endoprostheses in patients with common bile duct obstruction due to carcinoma of the pancreas. Patients with hilar biliary obstruction are better treated with internal-external biliary drainage catheters. PMID- 7544927 TI - Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia. AB - BACKGROUND AND PURPOSE: A process resembling programmed cell death appears to contribute to postischemic neuronal loss in several models of stroke. Because the expression of the bcl-2 gene has been shown to rescue neurons from programmed cell death due to other causes, we determined whether it would be similarly neuroprotective in stroke. METHODS: Replication of defective herpes viral vectors that transduce bcl-2 (HSVbcl2) or Escherichia coli lacZ (HSVlac) were injected into two sites in the rat cerebral cortex 24 hours before induction of neocortical focal ischemia by tandem permanent occlusion of the right middle cerebral artery and ipsilateral common carotid artery. Local ischemic damage was determined 24 hours after occlusion by staining with 2% 2,3,5 triphenyltetrazolium chloride. RESULTS: Expression of bcl-2 in cerebral cortex was confirmed by immunohistochemistry in animals injected with the HSVbcl2 expression vector. Viable tissue was significantly increased at the injection sites in HSVbcl2- but not HSVlac-injected animals. The protection observed in the HSVbcl2 animals was localized to the injection sites. CONCLUSIONS: These data indicate that bcl-2 expression protects neurons in vivo from ischemic injury and suggest the feasibility of gene therapy for stroke and perhaps other neurological diseases in which programmed cell death is involved. PMID- 7544923 TI - Neurogenic inflammation in human airways: is it important? PMID- 7544928 TI - PPBP [4-phenyl-1-(4-phenylbutyl) piperidine], a potent sigma-receptor ligand, decreases brain injury after transient focal ischemia in cats. AB - BACKGROUND AND PURPOSE: We tested the hypothesis that administration of 4-phenyl 1-(4-phenylbutyl) piperidine (PPBP), a potent sigma-receptor ligand, during transient focal ischemia would affect early postischemic brain injury. METHODS: Halothane-anesthetized cats underwent left middle cerebral artery occlusion for 90 minutes followed by 4 hours of reperfusion. Control cats received saline (n = 10). Experimental cats (2 groups, n = 10 per group) were treated with PPBP at a rate of 0.1 mumol/kg per hour (PPBP-0.1) or administered 1 mumol/kg per hour (PPBP-1) intravenously from 75 minutes after initiation of ischemia and continuing during the 4 hours of reperfusion. RESULTS: As measured by the microsphere method, blood flow to the ipsilateral caudate nucleus was decreased similarly in all groups during ischemia. Blood flow to the ipsilateral inferior temporal cortex was decreased during ischemia in all groups but was higher in cats subsequently treated with PPBP at the highest dose, even before drug administration. There was no difference in blood flow to the ipsilateral caudate nucleus or inferior temporal cortex (area of greatest cortical injury) during reperfusion. Triphenyltetrazolium-determined injury volume of the ipsilateral cerebral hemisphere (control, 29 +/- 5%; PPBP-0.1, 17 +/- 3%; PPBP-1, 6 +/- 1% of ipsilateral hemisphere; mean +/- SEM) and caudate nucleus (control, 49 +/- 5%; PPBP-0.1, 39 +/- 6%; PPBP-1, 25 +/- 5% of ipsilateral caudate nucleus) was less in cats treated with 1 mumol/kg per hour of PPBP compared with cats treated with saline. Cats treated with 0.1 mumol/kg per hour had a 45% smaller hemispheric injury volume than the control group without differences in intraischemic blood flow. Recovery of somatosensory evoked potential amplitude was greater in cats treated with PPBP-1 compared with control (control, 18 +/- 11%; PPBP-0.1, 30 +/- 14%; PPBP-1, 54 +/- 14% of baseline). CONCLUSIONS: These data indicate that sigma receptors may play an important role in the mechanism of acute injury in both the cortex and the caudate nucleus after 90 minutes of transient focal ischemia in the cat. Because PPBP afforded protection when administered at the end of ischemia and during reperfusion, sigma-receptors may contribute to the progression of injury in ischemic border regions. PMID- 7544926 TI - Demonstration of increased endothelial-leukocyte adhesion molecule-1 mRNA expression in rat ischemic cortex. AB - BACKGROUND AND PURPOSE: Leukocyte infiltration from circulating blood into ischemic brain tissue contributes significantly to ischemic injury. The role of adhesion molecules in leukocyte attachment and infiltration in ischemic tissue has been emphasized. The aim of the present study was to evaluate whether endothelial-leukocyte adhesion molecule-1 (ELAM-1 or E-selectin) mRNA expression is upregulated in focal brain ischemia. METHODS: Northern blot analysis with the use of poly(A) RNA isolated from the ischemic and nonischemic rat cortex at 2 and 12 hours after permanent occlusion of the middle cerebral artery (PMCAO) was used to examine ELAM-1 mRNA expression. The temporal expression profile of ELAM-1 mRNA in the ischemic cortex was further evaluated with the use of a quantitative reverse transcription and polymerase chain reaction technique. RESULTS: A very low level of ELAM-1 mRNA was detected in the sham-operated cortex or in the nonischemic cortex. The expression of ELAM-1 mRNA in the focal ischemic cortex was significantly induced by PMCAO, reaching a peak level at 12 hours (6.9-fold increase compared with sham surgery cortical samples, P < .01) and remained elevated for up to 2 days (3.3-fold increase, P < .01) after PMCAO. CONCLUSIONS: The demonstration of upregulated ELAM-1 mRNA expression after focal stroke suggests that ELAM-1 may play an important role in leukocyte infiltration into the ischemic brain and that ELAM-1 may provide a potential therapeutic target in ischemic stroke. However, the demonstration of translated ELAM-1 and its cellular localization in the ischemic tissue is required when specific antibodies become available. PMID- 7544925 TI - Adenosine modulates N-methyl-D-aspartate-stimulated hippocampal nitric oxide production in vivo. AB - BACKGROUND AND PURPOSE: Adenosine acts presynaptically to inhibit release of excitatory amino acids (EAAs) and is thus considered to be neuroprotective. Because EAA-stimulated synthesis of nitric oxide (NO) may play an important role in long-term potentiation and excitotoxic-mediated injury, we tested the hypotheses that adenosine agonists attenuate basal and EAA-induced NO production in the hippocampus in vivo and that adenosine A1 receptors mediate this response. METHODS: Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of adult Sprague-Dawley rats under pentobarbital anesthesia. Probes were perfused for 5 hours with artificial cerebrospinal fluid containing 3 mumol/L [14C]L-arginine. Recovery of [14C]L-citrulline in the effluent was used as a marker of NO production. In 10 groups of rats, time-dependent increases in [14C]L-citrulline recovery were compared between right- and left-sided probes perfused with various combinations of N-methyl-D-aspartate (NMDA), adenosine agonists, adenosine antagonists, and the NO synthase inhibitor N omega-nitro-L arginine methyl ester (L-NAME). RESULTS: Recovery of [14C]L-citrulline during perfusion with artificial cerebrospinal fluid progressively increased to 141 +/- 27 fmol/min (+/- SEM) over 5 hours. Contralateral perfusion with 1 mmol/L NMDA augmented [14C]L-citrulline recovery to 317 +/- 62 fmol/min. Perfusion of 1 mmol/L L-NAME with NMDA inhibited [14C]L-citrulline recovery compared with NMDA alone. Perfusion with 0.1 mmol/L 2-chloroadenosine attenuated basal as well as NMDA-enhanced [14C]L-citrulline recovery. This action of 2-chloroadenosine was reversed by infusion of 0.1 mmol/L 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist. Infusion of 0.1 mmol/L (2S)-N6-[2-endo norboryl]adenosine, a specific A1 receptor agonist, also attenuated the 0.1 mmol/L and 1 mmol/L NMDA-enhanced [14C]L-citrulline recovery. CONCLUSIONS: Using an indirect method of assessing NO production in vivo, these data are consistent with in vitro results showing that NMDA receptor stimulation enhances NO production. Furthermore, we conclude that stimulation of A1 receptors can attenuate the basal as well as NMDA-induced production of NO. Because NMDA receptor stimulation amplifies glutamate release, our data are consistent with presynaptic A1 receptor-mediated inhibition of EAA release and consequent downregulation of NO production. PMID- 7544924 TI - Direct inhibition of protein Ca by site directed thrombin inhibitors: implications in anticoagulant and thrombolytic therapy. PMID- 7544929 TI - Cytokines and acute-phase response in acute stroke. PMID- 7544934 TI - Influence of DNA ploidy and adjuvant treatment on progression and survival in patients with pathologic stage T3 (PT3) prostate cancer after radical retropubic prostatectomy. AB - OBJECTIVES: To determine whether adjuvant treatment (AT: hormonal or radiation) affects outcome in pathologic Stage T3 (pT3) prostate cancer when analyzed according to DNA ploidy. METHODS: The predictive value of nuclear DNA ploidy and AT on clinical and prostate-specific antigen (PSA) progression and on overall and cause-specific survival after radical retropubic prostatectomy was assessed in 894 patients with pT3 prostate cancer. RESULTS: Mean follow-up was 6.7 years (range, 0.3 to 20). Mean age was 66 years (range, 39 to 79). Six hundred sixty patients (74%) had no immediate AT, 131 (15%) had early adjuvant radiotherapy (ART), and 103 (12%) had early adjuvant orchiectomy (AHT). DNA diploid tumors were found in 445 patients (52%), tetraploid tumors in 346 (41%), and aneuploid tumors in 59 (7%). DNA ploidy was a significant (P < 0.05) prognostic indicator for clinical systemic progression-free survival. With PSA progression (more than 0.2 ng/mL) as an endpoint, ploidy was an even more powerful predictor for outcome (P = 0.004). Use of early AHT or ART was associated with decreased overall clinical progression for diploid and nondiploid tumors (P < 0.001 and P < 0.001, respectively). With respect to PSA progression, ART and AHT were equally effective and superior to no AT only in patients with diploid tumors. However, in patients with nondiploid tumors, only AHT appeared to have improved PSA progression-free survival (P < 0.001) over ART or no AT, which are similar in outcome. CONCLUSIONS: In the present nonrandomized study, AHT was as effective as ART for all endpoints except for PSA more than 0.2 ng/mL progression, for which it appeared to be superior to ART for patients with nondiploid tumors. PMID- 7544930 TI - A rapid staining and clearing technique for detecting filarial larvae in alcohol preserved vectors. PMID- 7544933 TI - Open pelvic lymph node dissection for prostate cancer: a reassessment. AB - OBJECTIVES: To provide a risk-to-benefit analysis of open staging pelvic lymph node dissection (PLND) for prostate cancer. METHODS: The medical records of all patients presenting with prostate cancer from July 1989 to April 1994 were reviewed. A total of 245 patients with clinically localized disease were selected to undergo radical retropubic prostatectomy (RRP) preceded by open PLND. Univariate and multivariate analyses were performed to evaluate the predictive value of the preoperative serum prostate-specific antigen (PSA) concentration, clinical stage, and Gleason score with regard to final nodal status. The cost and morbidity associated with PLND in the setting of RRP was also defined. RESULTS: Overall, only 16 patients (6.5%) had lymph node metastases. Lymph node involvement correlated significantly with elevated serum PSA values (P = 0.0001), high Gleason score (P = 0.0022), and advanced clinical stage (P = 0.0001). Lymph node metastases were particularly uncommon in patients with nonpalpable tumors (1 of 67 [1.5%]), PSA values less than 10 (2 of 154 [1.3%]), and Gleason score less than 6 (1 of 26 [3.8%]). Overall, 179 patients (73.1%) presented with at least one or more of these favorable characteristics, and only 4 (2.2%) had lymph node involvement. Complications related to the lymphadenectomy occurred in 10 patients (4.1%). The cost per metastasis diagnosed in patients with low-risk characteristics was approximatley $43,600. CONCLUSIONS: An open staging PLND may no longer be justified on a routine basis in patients undergoing radical retropubic prostatectomy. PMID- 7544932 TI - A multicenter, randomized, prospective study of endoscopic laser ablation versus transurethral resection of the prostate. AB - OBJECTIVES: To assess the safety and efficacy of endoscopic laser ablation of the prostate (ELAP), performed with the Urolase fiber and the neodymium:yttrium aluminum-garnet laser, compared to transurethral resection of the prostate (TURP) in patients with bladder outflow obstruction secondary to benign prostatic hyperplasia (BPH). METHODS: In this multicenter, prospective, randomized study, a total of 151 patients were treated (ELAP, 76; TURP, 75) of whom 137 completed 1 year follow-up (ELAP, 67; TURP, 70). Safety parameters included measurement of preoperative and 24-hour postoperative sodium, hematocrit, and hemoglobin values and careful monitoring of adverse events. Efficacy was assessed with the American Urological Association symptom score (6), urinary flow rates, and residual urinary volume measurements. RESULTS: There was 1 death in each group during the study period unrelated to the treatment procedure. There was a clinically significant improvement in all efficacy parameters in both groups. Between group comparisons favored TURP in maximum flow rate, residual urinary volume, and symptom score. ELAP had a better safety profile than TURP in the defined safety parameters of drop in hemoglobin and hematocrit values. In 16% of patients, a blood transfusion was required after TURP compared with no transfusions in the ELAP group. Urinary tract infections and dysuria were more frequent in the ELAP group. CONCLUSIONS: ELAP performed with the Urolase fiber is a useful alternative therapy to TURP in patients presenting with bladder outflow obstruction secondary to BPH. PMID- 7544935 TI - Prostatic carcinoma presenting with painless parotid mass. AB - Metastasis of prostate cancer to the parotid gland is exceedingly rare, with only 4 cases reported in the literature. We present the case of an 83-year-old white man who had a painless parotid mass that was diagnosed as metastatic prostate cancer. Immunohistochemical staining with prostate-specific antigen was positive. This is the first case of prostate cancer presenting with a painless parotid mass. Prostate cancer can be metastatic to the head and neck region, and this should be kept in mind when treating patients with this disease. PMID- 7544931 TI - Should Medicare provide reimbursement for prostate-specific antigen testing for early detection of prostate cancer? Part III: Management strategies and outcomes. PMID- 7544937 TI - Product lookback for anti-HCV-positive blood donors. PMID- 7544936 TI - Prevalence of antibodies to Rochalimaea species (cat-scratch disease agent) in cats. PMID- 7544939 TI - [Small bowel transplantation]. PMID- 7544940 TI - Distribution of silver stained proteins (SSPs) in ring-shaped nucleoli of leukemic lymphocytes. AB - The distribution of silver stained proteins (SSPs) was investigated in ring shaped nucleoli of lymphocytes of the peripheral blood of patients suffering from chronic lymphocytic leukemia (B type). As expected, SSPs were present in fibrillar centers and adjacent nucleolar regions which appeared as bud and protrusion-like structures. The inhibition of the nucleolar RNA transcription by cultivation of leukemic lymphocytes at low temperature (4 degrees C) resulted in a significant reduction of these structures at fibrillar centers of ring-shaped nucleoli and decreased the number of cells which contained a large number of such structures at fibrillar centers of their ring shaped nucleoli. There was no substantial difference in the visualization and distribution of SSPs after fixation with phosphate-buffered glutaraldehyde or formaldehyde in distilled water and postfixation in ethanol or methanol-glacial acetic acid mixture. PMID- 7544938 TI - Oral desensitization in the treatment of human immune diseases. AB - Oral desensitization or oral tolerance is induced by giving antigenic peptides by the mucosal route. In man only the oral route has been used up to now. Experiments in animal models of human autoimmune diseases, have shown that it is not necessary to use the primary antigen responsible for disease induction. Antigens implicated in secondary immune phenomenon can act similarly by means of the so-called "bystander suppression". Thus for diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) candidate antigens for desensitization are available. Many patients with MS have immunity to myelin basic protein (MBP). A recent controlled trial giving MBP to patients with MS is discussed (Weiner et al., Science 259, p. 1321, 1993). No clear-cut effect was obtained. Collagen II is used to induce experimental arthritis in rats; signs of immunity against it can be found in patients with RA. Collagen-induced arthritis has been successfully modified in animals by feeding of collagen II. In man one open uncontrolled trial and one other placebo controlled blind trial have been reported, and these are discussed (Trentham et al., Science 261, 1727, 1993). These trials suggest that oral desensitization might be useful and devoid of side effects. Subreum is a peptic E. coli extract containing heat shock protein 60. Its efficacy as a disease-controlling agent in RA has been documented (Clin. Exp. Rheum. 11, p. 121, 1993). It is given orally. Data suggesting that Subreum acts by oral desensitization are discussed. Considering the low incidence of side effects observed with oral desensitization, this therapeutic approach should also be tested in other forms of arthritis and other inflammatory diseases. PMID- 7544941 TI - The damaging effect of UV rays below 320 nm on the rabbit anterior eye segment. II. Enzyme histochemical changes and plasmin activity after prolonged irradiation. AB - Prolonged irradiation of the rabbit eyes with UVB rays (312 nm) caused serious enzymatic disturbances in the cornea and lens and the development of an inflammatory reaction in the whole anterior eye segment, particularly in the cornea. In the corneal stroma many inflammatory cells with high activities of acid glycosidases and lysosomal proteases were present. This was accompanied with significantly elevated plasmin activity in the tear fluid (1.6 IU/ml). Plasmin appeared also in the aqueous humour (0.8 IU/ml). For the treatment of these changes catalase (1 mg/1 ml saline), aprotinin (100 micrograms/1 ml saline) and catalase-aprotinin mixture (1:1) were applied on the eye surface during irradiation. The catalase-aprotinin mixture was most efficient and decreased plasmin activity in the tear fluid and diminished disturbances of the anterior eye segment. Obviously both, active oxygen species and elevated plasmin activity in the tear fluid contribute to the damage of the anterior eye segment and development of intracorneal inflammation after irradiation of the eye with UVB rays. PMID- 7544942 TI - Differential expression of cytokeratin mRNA in hamster squamous epithelia as observed by in situ hybridization using human cytokeratin cRNA probes. AB - In this hybridohistochemical study, we investigated the expression of cytokeratin (CK) mRNA in the keratinizing squamous epithelium of hamster cheek pouch and esophagus, using eight different digoxigenin-labelled RNA probes complementary to human CK mRNAs. CK 4, CK 6, CK 8, CK 14, and CK 15 RNA probe obviously hybridized with hamster counterpart mRNA(s) in the cheek pouch as well as in the esophageal epithelium. However, using the CK 10-specific RNA probe, only the cheek pouch epithelium exhibited a positive reaction. We were not able to detect any positive signal for the CK 18 or for the CK 19 RNA probe. We observed three different CK mRNA distribution patterns in the cheek pouch epithelium, and four in the esophageal epithelium. The differences in expression and distribution pattern of CK mRNAs between the two types of epithelia suggest that the hamster CK polypeptide family comprises at least six different species. We also conclude that human cRNA probes for CK mRNAs may provide a way to detect changes in CK expression and distribution during induced non-neoplastic and neoplastic changes in the hamster cheek pouch model. This may also help to elucidate the molecular pathogenesis of squamous-cell carcinoma. PMID- 7544943 TI - Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8. AB - In order to explore the origin and significance of Merkel cells in the hairy skin of mammals, the development of Merkel cells and nerve endings in the dorsolateral skin of C57BL mouse embryos was studied in serial cryostat sections. At 13 and 14 days of gestation, application of a monoclonal antibody to mouse keratin 8 (mK8) resulted in specific immunofluorescence of all cells in the epidermis and periderm. The periderm retained specific staining until it was shed, around 18 days. At 15 days, mK8-specific staining elsewhere was restricted to scattered immature Merkel cells in the developing tylotrich follicles and the adjacent epidermis. Between 16 and 17 days, these cells assembled within the basal epidermal layer, caudal to each tylotrich follicle, to form a disc-shaped rudiment of a 'haarscheibe' or touch dome. No Merkel cells were found in association with the later developing awl and zigzag follicles. In mice homozygous or hemizygous for the Tabby mutation, in which tylotrich follicles never form, no Merkel cells were found in any part of the dorsolateral skin. In mice homozygous for the recessive downy mutation, in which all three types of hair are present but reduced in size, Merkel cell development was the same as in wild-type mice. Nerve endings were located in the upper dermal mesenchyme by a monoclonal antibody to neural cell adhesion molecule. This antibody also stained plasma membranes in specific parts of the hair follicles during their development. From 14 to 19 days, none of the nerve endings were seen in contact with the epidermis or the follicle epithelium, even in areas where Merkel cells were located. These findings support the view that both location and early differentiation of Merkel cells in the dorsolateral epidermis are independent of neural influences but linked to the development of tylotrich follicles. PMID- 7544944 TI - [Effect of tremulacin on actions of SRS-A and histamine]. AB - The effect of tremulacin (TRC) extracted from Mao Bai Yang (Folia Populus tomentosa Carr) on actions of SRS-A and histamine were investigated by using isolated guinea pig ileum and spectrofluorometric assay. TRC was found to inhibit the contraction of isolated guinea pig ileum induced by histamine and SRS-A, in a dose-dependent manner with IC50 of 1.78 x 10(-4) mol.L-1 and 2.51 x 10(-4) mol.L 1, respectively. TRC at the dose of 10(-4) mol.L-1 inhibited SRS-A release from sensitized isolated guinea pig lung. While at the dose of 10(-5) mol.L-1 inhibited histamine release from the peritoneal mast cells in sensitized rats. These results indicate that inhibition of the release of histamine and SRS-A may play an important role in the mechanism of antiinflammatory actions of TRC. PMID- 7544945 TI - [Studies on gelatin microspheres-in-oil emulsion of pingyangmycin]. AB - As a drug for lymphosarcoma, pingyangmycin (A5) is not widely used because of its toxicities, short half-life and low affinity to lymph. For the purpose of delivering A5 to the lymph system to strengthen and sustain its effects and to lower its toxicities, its gelatin microspheres-in-oil emulsion (S/O) was studied in vitro and in vivo. By ultrasonication, gelatin microspheres with diameters of 1.67 +/- 0.69 microns were homogeneously dispersed in oil to form the S/O, which was a pseudoplastic flow and stable under 0 degrees C for at least 1 month. With the content of 14.03 +/- 0.15 mg.ml-1, A5 released from the emulsion in a zero order rate with t0.5 of 12.0 h. In vivo experiments showed that the S/O emulsion exhibited potent lymphotropicity, prolonged plasma concentration and a probably lower pulmonary toxicity. PMID- 7544946 TI - The effect of beta-aminopropionitrile on bleomycin-induced lung injury in rats. AB - The effect of beta-aminopropionitrile on collagen cross-links, lysyl oxidase and prolyl hydroxylase and particular collagen type content in rat lungs after bleomycin treatment was investigated. It was stated, that beta-aminopropionitrile significantly diminishes elevated dihydroxylysinonorleucine to hydroxylysinonorleucine ratio, prevented increase of lysyl oxidase activity and increase in type I collagen content in the lungs. It is suggested, that beta aminopropionitrile may be useful in the treatment of lung fibrosis. PMID- 7544951 TI - Serum guanase activity in hepatitis C virus infection. PMID- 7544950 TI - Properties of mitochondrial DNA metabolising enzymes; implications for chemotherapy. PMID- 7544947 TI - Dual response of cerebrocortical blood flow and arterial blood pressure to transient CO2 stimulus after inhibition of nitric oxide synthesis in rats. AB - Inhibition of nitric oxide synthase (NOS) by Nitro-L-arginine-methyl-ester (L NAME 15 mg and 70 mg/kg i.v.) in 16 male Wistar rats anaesthetized with urethane, paralysed and artificially ventilated, increased significantly local peripheral vascular resistance in the parietal cortex (CVR) along with augmentation of the mean arterial blood pressure (MAP) and no change of the local cerebrocortical blood flow (CBF) recorded with a Laser-Doppler-Flowmeter. In 11 rats L-NAME reversed a pressor effect of brief hypercapnia induced by 10% CO2/air mixture (PaCO2 84.1 +/- 5 mm Hg) into a depressor response, reduced CBF response proportionally to the reduction of MAP and did not influence CVR response to CO2. In 5 rats L-NAME did not abolish the central pressor effect of a CO2-stimulus and significantly augmented CO2-induced vasodilatatory response in the cortex (43.4 +/- 24% before L-NAME and 137.8 +/- 38.8% after L-NAME) by a larger reduction of CVR (-11 +/- 8% before L-NAME and -47.1 +/- 7.6% after L-NAME). It is concluded that NO does not mediate the vasodilatatory effect of brief hypercapnia in the cortex. NO appears critical for the central pressor effect of CO2. In those rats in which the central pressor effect of a CO2-stimulus was not abolished by an NOS blocker, an increased CBF and augmented decrease in CVR was observed during brief hypercapnia. Possible mechanisms of this dual responsiveness of cortical blood flow and arterial blood pressure to CO2, induced by inhibition of NOS, are discussed. PMID- 7544949 TI - Normal uric acid concentrations in a purine nucleoside phosphorylase (PNP) deficient child presenting with severe chicken pox, possible immunodeficiency and developmental delay. PMID- 7544948 TI - Modulation of arabinosylcytosine metabolism during leukemia therapy. PMID- 7544952 TI - Stimulation by 6-azauridine of de novo pyrimidine biosynthesis in BHK 165-23 cells. PMID- 7544953 TI - [A long-term course of experimentally produced choroidal neovascularization in the rat]. AB - We studied morphologically a long-term course of experimental choroidal neovascularization (ChNV) induced by krypton laser photocoagulation in the rat retina. Fifty-two weeks after photocoagulation, ChNV was enveloped completely by the retinal pigment epithelium. Vascular endothelial cells of ChNV were thin, with many fenestrations and wide lumen. The ChNV maintained the morphological characteristics of mature leaky capillaries similar to choriocapillaris. The lumen of the neovascularizations tended to be compressed by massive collagen fibers produced by the retinal pigmented epithelium. We found that experimental ChNV in the rat retina retains the characteristics of leaky capillaries for a long time unlike that in the monkey ChNV. PMID- 7544954 TI - Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas. AB - This study examined the presence of substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities in various milks and infant formulas. Rat milk was obtained from lactating dams between parturition and weaning (0, 2, 5, 10, 15, and 20 d postpartum). Samples of human milk were obtained from seven multiparous, nonsmoking white women, and newborn infant formulas were purchased from local stores. Substance P and CGRP were measured by competitive enzyme immunoassay using acetylcholinesterase-peptide conjugates as tracers. In rats, substance P and CGRP were below detectable concentrations in amniotic fluid from the last day of gestation. In contrast, in milk the concentrations of substance P and CGRP-like immunoreactivities were high on the first day of lactation (3.1 +/- 0.2 and 23.1 +/- 1.5 micrograms/L, respectively), then dropped after day 2 (1.6 +/- 0.7 and 7.5 +/- 0.4 microgram/L, respectively) and remained fairly constant until weaning. Significant concentrations of substance P and CGRP were found in human milk (129.2 +/- 27 ng/L and 4.5 +/- 0.7 microgram/L, respectively, at 15 wk), but substance P or CGRP could not be detected in any of the formulas tested. These data show that milk contains high concentrations of immunoreactive substance P and CGRP. In rats the absence of peptides in amniotic fluid suggests that there is a flood of peptides into the gastrointestinal tract of neonates when suckling is initiated. Significant concentrations of substance P and CGRP in human milk but not in infant formulas may therefore have physiologic implications for neonatal nutrition. PMID- 7544955 TI - Spontaneous regression of hepatocellular carcinoma. AB - Spontaneous regression of cancer is a rare phenomenon seldom described in patients with hepatocellular carcinoma. A case of spontaneous regression of hepatocellular carcinoma is reported and compared with the reports published in the English literature. A 52-yr-old man presented with biopsy-proven hepatocellular carcinoma, which was considered to be unresectable at initial laparotomy. The tumor subsequently regressed without specific treatment, as assessed radiologically and by normalization of a previously elevated alpha fetoprotein level. At repeat laparotomy 14 months after initial diagnosis, intraoperative ultrasound failed to disclose a hepatic mass, and multiple biopsies showed no evidence of malignancy. To date, only nine case reports of apparently spontaneous regression of hepatocellular carcinoma have been published in the English literature. Clinical characteristics discriminating these patients from less fortunate patients with hepatocellular carcinoma could not be identified. The mechanisms underlying this intriguing phenomenon remain unknown. PMID- 7544956 TI - Early dysphagia after placement of a self-expanding esophageal stent: severe esophagitis mimicking luminal occlusion. PMID- 7544957 TI - Diabetic iris neovascularization. AB - PURPOSE: To compare the validity of careful slit-lamp biomicroscopic examination of the pupillary margin to screening gonioscopic examination in the early detection and prevention of neovascular glaucoma in diabetic patients. METHODS: We examined two patients with histories of diabetes mellitus. RESULTS: Angle neovascularization developed before iris neovascularization in both patients. CONCLUSION: We believe screening gonioscopic examination is valuable in patients with diabetes. PMID- 7544958 TI - Postatrophic hyperplasia of the prostate. A histologic mimic of prostatic adenocarcinoma. AB - Clusters of atrophic prostatic acini that display proliferative epithelial changes are referred to as postatrophic hyperplasia (PAH). PAH is histologically similar to adenocarcinoma and may cause diagnostic confusion. Despite the importance of distinguishing PAH from carcinoma, the last systematic study of this lesion was reported > 40 years ago, and many contemporary pathologists are unfamiliar with this lesion. We reviewed 100 consecutive whole-mount radical prostatectomy specimens removed for carcinoma to determine the incidence of PAH. In addition, 11 prostatic needle biopsy specimens with PAH were evaluated to further characterize the lesion in limited specimens. PAH was identified in 18 radical prostatectomies (18%), including 10 unicentric and eight multicentric cases. It was found exclusively in the peripheral zone in all but two cases, which had additional involvement of the transition zone. PAH consisted of a microscopic lobular cluster of small acini with irregular atrophic-appearing contours lined by cuboidal cells with mild nucleomegaly and micronucleoli; mildly enlarged nucleoli were focally present in 39% of cases. Within the small acinar cluster, a larger dilated acinus was usually present centrally, which was lined by flattened to cuboidal epithelial cells. The basal cell layer at the periphery of each acinus was invariably present but often inconspicuous. Immunohistochemical staining for high-molecular-weight keratin (antibody 34 beta E12) showed the presence of an intact basal cell layer in seven of 10 cases and a focally fragmented basal cell layer in three other cases. PAH was associated with patchy chronic inflammation in 16 of 18 prostatectomy cases; stromal changes were present in all cases, ranging from smooth atrophy to dense sclerosis with compression of acini. No intraluminal basophilic mucin was identified, but two needle biopsies showed PAH with focal mucinous metaplasia. Crystalloids were not seen in any case. Focal partial acinar involvement by high-grade prostatic intraepithelial neoplasia was present in adjacent acini in two cases. Adjacent acini also invariably showed typical changes of atrophy. In the needle biopsy specimens, PAH showed the same features as those in prostatectomies, but often only a portion of the lesion was sampled. PAH is distinguished from carcinoma by its characteristic architecture, intact or fragmented basal cell layer, inconspicuous or mildly enlarged nucleoli, and adjacent acinar atrophy with stromal fibrosis or smooth muscle atrophy. Distinguishing PAH from carcinoma is most difficult in needle biopsy specimens in which only a portion of the lesion is sampled, and awareness of this entity assists in this distinction. PMID- 7544959 TI - CD34 and thyroid fibrous tumor. PMID- 7544960 TI - Inhalation anesthetics inhibit the release of endothelium-derived hyperpolarizing factor in the rabbit carotid artery. AB - BACKGROUND: Inhalation anesthetics may interfere with the synthesis or action of endothelium-derived vasoactive factors. We investigated the effects of desflurane, enflurane, halothane, isoflurane, and sevoflurane on the release of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the isolated endothelium-intact carotid artery of the rabbit. METHODS: Isolated segments of the carotid artery were suspended in Krebs-Henseleit solution (37 degrees C) and preconstricted with phenylephrine (1 microM). Relaxations caused by acetylcholine (ACh) (0.03-10 microM) or sodium nitroprusside (0.01-10 microM) were compared in the presence or absence of the nitric oxide synthase inhibitor NG-nitro-L-arginine (0.1 mM) in segments exposed to desflurane (8%), enflurane (2 4%), halothane (2-3.5%), isoflurane (2-4%), or sevoflurane (2%) as well as in NG nitro-L- arginine-treated segments exposed to enflurane (2%) in combination with the KCa(+)-channel blocker tetrabutylammonium (0.3 mM) or the cytochrome P450 inhibitor clotrimazole (3 microM). RESULTS: Desflurane, enflurane, and sevoflurane selectively inhibited the ACh-induced release of EDHF. Halothane and isoflurane also weakly affected the nitric oxide-mediated relaxant response to ACh. The inhibitory effect of these two anesthetics on EDHF release was concentration-dependent. Relaxations induced by sodium nitroprusside were not inhibited by any of the anesthetics tested. Three structurally unrelated cytochrome P450 inhibitors clotrimazole (0.1 mM), metyrapone (1 mM), and SKF525a (proadifen, 0.1 mM) abolished the EDHF-mediated relaxation elicited by ACh. The pharmacologic profile of the inhibitory effect of enflurane on the release of EDHF closely resembled that of clotrimazole but not that of tetrabutylammonium. Moreover, all anesthetics inhibited the cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin by rabbit liver microsomes in a concentration-dependent manner. CONCLUSIONS: Inhalation anesthetics significantly attenuate the EDHF mediated relaxant response to ACh in the rabbit carotid artery. This effect appears to be attributable to inhibition of the cytochrome P450-dependent synthesis of EDHF by the endothelium. PMID- 7544961 TI - A MspI polymorphism at the transferrin (TF) locus in cattle. PMID- 7544962 TI - Angiogenesis as a prognostic marker in early head and neck cancer. AB - Experimental evidence suggests that tumor growth beyond the earliest stages is dependent on angiogenesis, or neovascularization, and that angiogenesis may also promote metastasis. Recent clinical studies demonstrate that angiogenesis is a prognostic marker in breast, lung, and prostate cancer. To investigate whether tumor angiogenesis also correlates with metastasis and survival in early head and neck carcinoma, we quantified the microvascularity of 106 primary carcinomas prior to treatment and correlated the counts with eventual outcome after 3 to 15 years of follow-up. Microvessels were stained immunocytochemically for von Willebrand factor and then counted by light microscopy. Microvessels were counted per 200x and 400x fields, and their density was graded from 1 to 4, in the area of most intense neovascularization. We found that neither microvessel counts nor density grades correlated with metastatic disease, local recurrence, or survival in early head and neck carcinoma. These results are in contradistinction to those recently reported for other tumor sites. PMID- 7544963 TI - [Limitation of clinical usefulness of tumor marker]. AB - The clinical features and limitations of the usefulness of 10 kinds of main tumor markers such as AFP, CEA, CA 19-9 etc. are described in detail in this paper. This review is concerned with the limitation of the clinical usefulness of tumor marker. Evaluation of tumor marker is directed toward usefulness for diagnosis and the monitoring of cancer. The main weak points in the diagnosis of cancer by tumor marker have been insufficient sensitivity for detection of cancer and specificity to cancer. The sensitivity for detection of early cancer by tumor marker is especially low, and many tumor markers have ordinarily high false positive incidence for benign liver disease. On the other hand, limitation of specificity to organ is found in most tumor markers. Most tumor markers generally considered to be organ-specific are in fact, non-organ-specific, such as CA 19-9, which is thought to be specific to pancreatic cancer. On the other hand, there are few strictly organ-specific tumor markers such as prostate-specific antigen (PSA) and PIVKA-II. The usefulness of tumor marker for monitoring cancer is considered far better than that for diagnosis of cancer. However, a tumor marker in cases with less than the cut-off value is not available for monitoring of cancer. PMID- 7544964 TI - [Intrapleural bleomycin for management of malignant pleural effusions]. AB - We studied the efficacy of intrapleural administration of bleomycin for the management of malignant pleural effusions of non-small cell lung cancer in 24 cases. Bleomycin 60 mg was administered into the pleural space after tube drainage. If the effusion continued, one additional dose was given. The efficacy was seen in 18 cases (75%). The main adverse drug reaction was transient fever among others. There was little toxicity and no cases of pulmonary fibrosis. Intrapleural administration of bleomycin is useful in management of malignant pleural effusions. PMID- 7544965 TI - [Pharmacokinetic investigation of 5-day multiple dose of tropisetron capsule in patients who had received cisplatin and the usefulness of tropisetron capsule in the treatment of nausea and vomiting]. AB - We have investigated the pharmacokinetics of a 5-day multiple oral dose of 5 mg tropisetron capsule in patients with malignant tumour who had received cisplatin single administration. Its anti-emetic effects on acute and delayed emesis and vomiting were also investigated. During the 5 days after this administration, changes in the release and metabolism of serotonin were investigated. The results may be summarized as follows: 1) The pharmacokinetic parameters of tropisetron revealed no significant change in the data between day 1 and day 5. Also, the parameters were almost similar to those observed in healthy adult males (clinical phase I study); Cmax. T1/2 and AUC 0-24 hrs on day 1 were 9.1 +/- 2.1 ng/ml, 12.5 +/- 4.2 hrs, 85.5 +/- 22.7 ng.hr/ml, respectively. The urinary excretion of the parent drug up until 24 hours after administration on day 1 was 3.8% of the dose administered and 2.9% of the total dose after the last administration; no difference in the urinary excretion rate was observed in healthy subjects. It was thus suggested that hydration accompanied by cisplatin administration did not affect the pharmacokinetics of tropisetron. 2) The changes in the amount of urinary excretion of 5-hydroxy-indoleacetic acid (5-HIAA) during 5 days after cisplatin administration were observed; urinary excretion of 5-HIAA increased 2.3 times (1.3-5.4 times) the baseline on the average during 6 to 12 hours on day 1. In 6 out of 10 patients, the increases in urinary excretion of 5-HIAA showed double or more the baseline during 2 to 5 days after cisplatin administration. Serotonin was thus deemed to be related to the development of delayed emesis. 3) The anti-emetic effects of tropisetron on acute and delayed emesis and vomiting were rated as "markedly effective" in 6 out of 11 patients (54.6%) and "effective" in 1 out of 11 patients (9.1%) on day 1; vomiting did not occur in any of these 7 patients. Tropisetron also controlled emesis and vomiting during days 2-5, and was rated as "almost favorable" in 6 out of 11 patients (54.5%). Further, in 4 out of 6 patients, in whom the urinary excretion of 5-HIAA was increased on day 2 onwards, vomiting did not occur during the time when the urinary excretion of 5-HIAA was increasing. On the basis of the above results, tropisetron is deemed to have certain antiemetic effects on delayed vomiting as well. Its 5-HT3 receptor mediated mechanism was similarly seen to inhibit acute nausea and vomiting. PMID- 7544966 TI - Multimodality treatment in advanced primary liver cancer. AB - This paper reports the long-term results of multimodality treatment in 1639 patients with pathologically proven primary liver cancer (PLC) over the past three decades. In this series, patients in subclinical stage constituted 23.9% (391/1639), moderate stage 63.0% (1032/1639), and late stage 13.2% (216/1639). There were 381 patients (23.2%) with small PLC (< = 5 cm). The PLC was coexistent with liver cirrhosis in 86.4% (1416/1639). The 5-year survival after resection of PLC was 45.5% for the whole series (n = 896), and 62.7% for patients with a tumor < = 5 cm (n = 345). The 5-year survival after hepatic artery ligation (HAL) and hepatic artery infusion (HAI) (n = 124) was 18.1%, while the 5-year survival after palliative resection (n = 175) was 12.5%. The 5-year survival of 40 patients receiving second-look resection after cytoreduction therapy was 68.4%. Cryosurgery was performed on 107 PLC patients, the 5-year survival being 22.0% for the whole series, and 48.8% for the 32 patients with small PLC. The 5-year recurrence rate after resection was 55.3%. The 5-year survival after reoperation for recurring tumor (n = 90) was 40.8%. It is suggested that surgery remains the modality of choice, and it plays a more important role in the treatment of PLC. The results of palliative surgery (HAL + HAI) seem better than those of palliative resection; combined multimodality treatment and second-look resection, and new surgical techniques might offer hope for unresectable advanced PLC. PMID- 7544969 TI - [Esterase and amylase: two biochemical markers in the polytypic Idotea chelipes (crustacea, isopoda valvifera)]. AB - In polytypic Idotea chelipes species, the slow esterase constitutes a subspecific biochemical marker which discriminates, without ambiguity, each of three subspecies. Amylase 2 is a diagnostic locus which separates I. chelipes bocqueti from the two other subspecies where each is characterized by particular frequencies of the most frequent allele. The analysed electrophoretic zymogram and the intersubspecific cross-breeding results attribute a monomeric structure for this enzyme which depends on diallelic gene. PMID- 7544968 TI - [Yield of ultrasonography-guided transrectal biopsy in the diagnosis of carcinoma of the prostate]. AB - OBJECTIVES: The present study analyzes the combination of digital rectal examination (DRE), prostate specific antigen (PSA), transrectal ultrasound (TRUS) and ultrasound-guided transrectal biopsy in the diagnosis of prostate cancer. METHODS: 115 of 224 patients (51.3%) were biopsied due to a suspicious DRE and/or PSA > 4 ng/ml, and/or DPSA > 0.15, and/or the presence of a hypoechoic lesion on TRUS. RESULTS: Adenocarcinoma of the prostate was diagnosed in 28 (24.3%) of these 115 patients. The positive predictive value (PPV) for carcinoma of the prostate was 4.76% if PSA was < 4 ng/ml, 5.12% if PSA was 4.1 to 10 ng/ml, and 48% if PSA was > 10 ng/ml. If DPSA is considered, the PPV is 7.4% if PSA is 4.1 to 10 ng/ml and 51.02% if PSA is > 10 ng/ml. If DRE is suspicious, the PPV is 43.6%, with a sensitivity of 85.7% and a specificity of 63.9%. The PPV for carcinoma of the prostate was 34% for a hypoechoic lesion on TRUS, 50% if DRE is suspicious, 53.5% is PSA is > 10 ng/ml and 65% if DRE is suspicious and PSA > 10 ng/ml. CONCLUSIONS: The detection rate of carcinoma of the prostate is increased when DRE, PSA, TRUS and US-guided transrectal biopsy are used in combination. PMID- 7544967 TI - Two concentrations of topical tretinoin (retinoic acid) cause similar improvement of photoaging but different degrees of irritation. A double-blind, vehicle controlled comparison of 0.1% and 0.025% tretinoin creams. AB - BACKGROUND AND DESIGN: The efficacy of topical tretinoin (all-trans-retinoic acid) in treating photoaging is well established. Questions that remain are (1) whether irritation causes all or part of the improvement; (2) the concentration of tretinoin that maximizes clinical response with minimal side effects; and (3) the effects of long-term treatment on components of the cutaneous immune system. To address these issues, 99 photoaged patients completed a 48-week study using 0.1% tretinoin cream (n = 32), 0.025% tretinoin (n = 35), or vehicle (n = 32) once daily in a double-blind manner. Before and after treatment, we assessed histologic features, keratinocyte expression of HLA-DR and intercellular adhesion molecule-1, numbers of epidermal Langerhans' cells and epidermal and dermal T lymphocytes, and vascularity as measured by dermal endothelial cell area. RESULTS: Both 0.1% and 0.025% tretinoin produced statistically significant overall improvement in photoaging of the face compared with vehicle; there were no clinically or statistically significant differences in efficacy between the two concentrations of tretinoin. After 48 weeks, 0.1% and 0.025% tretinoin produced similar statistically significant epidermal thickening (by 30% and 28%, respectively) compared with vehicle (11% decrease) and increased vascularity (by 100% and 89%, respectively) compared with vehicle (9% decrease). By various analyses, irritant side effects (erythema and scaling) were statistically significantly greater with 0.1% tretinoin than with 0.025% tretinoin. No significant changes occurred in any immunologic markers when tretinoin and vehicle treatments were compared. CONCLUSIONS: Tretinoin 0.1% and 0.025% produce similar clinical and histologic changes in patients with photoaging, despite significantly greater incidence of irritation with the higher concentration. The separation between clinical improvement and irritation suggests that mechanisms other than irritation dominate tretinoin-induced repair of photoaging in humans. PMID- 7544971 TI - Presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle. AB - Antibodies reacting with porcine circovirus (PCV) were found in sera of humans, mice, and cattle by means of an indirect immunofluorescence assay (IFA) and an ELISA. In man, the highest seroprevalence (23.9% in IFA and 30.2% in ELISA) was found among hospitalized patients with fever of partially unclear etiology. Non hospitalized "healthy" persons of the former German Democratic Republic showed a significantly higher number of positive sera (IFA = 20%) than blood donors from Berlin-West (IFA = 8.6%). Murine sera reacted positive with PCV in IFA between 12 to 69% in different breeding groups and about 35% of cattle sera were found reactive with PCV in IFA. Double-staining IFAs, immuno-electron microscopy and immunoblotting showed that non-porcine antibodies reacted with PCV structural antigen. Mathematical analysis revealed that in ELISA, non-porcine antibodies reacted specifically with PCV. Loss of binding specificity of non-porcine antibodies in ELISA after storage of sera and lower maximal optical densities obtained at equal titers in ELISA with non-porcine than with porcine sera suggest that antibodies in man, mice and cattle are caused by related species specific viruses sharing antigenic epitopes with PCV. PMID- 7544972 TI - Satellite RNA associated with bamboo mosaic potexvirus shares similarity with satellites associated with sobemoviruses. AB - A putative nonstructural protein encoded by a satellite RNA associated with bamboo mosaic potexvirus shares 46% identity with the capsid protein of satellite virus of panicum mosaic sobemovirus. The sequence similarity among satellite plant viruses which have no apparent relationship implies a common origin. PMID- 7544970 TI - Relative reactivity of the V3 loop PND of HIV-1 subtypes A, B, C, D, and F with sera from selected Ugandan localities. AB - Synthetic peptides comprising the predicted principal neutralizing determinant (PND) in new African and North American HIV-1 clones were tested in ELISA for reactivity with ninety six serum samples from asymptomatic donors in six selected localities in Uganda. Irrespective of the geographical origin of the samples, the majority of the test sera cross-reacted at high intensities with the peptides derived from the North American clone, BRT3.6 (Group B), the Ugandan clone, CUG045, (Group C), and the Romanian clone, FRMA (Group F). The frequency of reactivity of the peptides from BRT3.6, CUG045, and FRMA were within the ranges of 57-100%, 50-100%, and 57-100%, respectively, for the sera collected from these disparate localities. In contrast to these findings, the V3 peptides derived from the other Ugandan isolates showed a more restricted pattern of reactivity with the same serum samples: AUG06c (1-63%), DUG23c (2%), and DUG044 (38-87%). The results from ELISA inhibition assay indicated that the V3 peptide from BRT 3.6, CUG045, and FRMA express closely related antigenic specificities quite distinct from those in AUG06c and DUG044. The residues comprising the PND in BRT 3.6, CUG045, and FRMA appear to be well conserved in the HIV-1 subtypes prevalent in the selected Ugandan locales. PMID- 7544973 TI - [Synthesis of a highly active recombinant Thermus aquaticus His6-DNA-polymerase in Escherichia coli cells and a rapid method of purifying it]. PMID- 7544974 TI - [Angiotensin-converting enzyme in an OAT-octane reversed micelle system: interaction with the matrix]. AB - Regulation of catalytic activity and intramolecular structure of the bovine lung angiotensin-converting enzyme was studied using reversed micelles in a sodium docusate-water-octane system, which model the enzyme's environment in vivo. The catalytic parameters of monomeric and dimeric forms of the enzyme in the reversed micellar system were evaluated. The catalytic activity of the angiotensin converting enzyme extracted from bovine lung with Triton X-100 did not depend on the detergent concentration at a constant level of hydration. An artificially hydrophobized form of the angiotensin-converting enzyme was obtained by modifying the enzyme with stearic acid chloride. The modification leads to the dependence of the catalytic activity on the surfactant concentration, which provides evidence that the enzyme interacts with the micellar matrix. The modified enzyme showed a significant increase in catalytic activity in the reversed micellar system. PMID- 7544976 TI - T-cell autoimmunity in multiple sclerosis. PMID- 7544975 TI - Complement regulatory molecules: application to therapy and transplantation. PMID- 7544977 TI - Could HLA-DRB1 be the protective locus in rheumatoid arthritis? AB - Extensive studies in different ethnic groups have associated the susceptibility to development of rheumatoid arthritis (RA) with the third hypervariable region of the major histocompatibility complex (MHC) HLA-DR beta 1 molecule. On the basis of recent findings in the experimental mouse model of collagen-induced arthritis, Eric Zanelli, Miguel Gonzalez-Gay and Chella David propose that the HLA-DRB1 locus is associated with protection to RA and that the actual arthritogenic peptide-presenting molecule is HLA-DQ. Thus, the development of RA would depend upon the expression of the susceptible DQ allele and the nonprotective DRB1 alleles, along with environmental factors that trigger the autoimmune process. PMID- 7544979 TI - GPI-linked molecules on lymphoid cells of allogeneic BMT patients. PMID- 7544980 TI - The chemokines and the histamine-releasing factors: modulation of function of basophils, mast cells and eosinophils. PMID- 7544978 TI - Does endotoxin stimulate cells by mimicking ceramide? AB - Recent studies indicate that tumor necrosis factor (TNF) and interleukin 1 (IL-1) stimulate cells via the intracellular messenger ceramide. Bacterial endotoxin (lipopolysaccharide; LPS) shows strong structural and functional resemblance to ceramide. Here, Samuel Wright and Richard Kolesnick review data suggesting that LPS may provoke cellular responses by mimicking the action of ceramide. PMID- 7544981 TI - Signal transduction and cytokine production by human basophils. PMID- 7544982 TI - Improvement of hammerhead ribozymes cleaving mdr-1 mRNA. AB - Overexpression of the mdr-1 gene is one of the mechanisms involved in therapy induced drug resistance. Gene-specific reduction of mdr-1 overexpression in human cancer using antisense technology may be an efficient tool for the reduction of multiple drug resistance (MDR). The application of catalyticly active RNA species -the so-called ribozymes--represents a possible improvement of this molecular strategy using oligonucleotides due to the catalytic potential of ribozymes. In the present paper we investigated the catalytic activity of ribozymes directed against three different cleavage sites on the mdr-1 mRNA. We designed ribozymes against cleavage sites at position 2408 (GUC, ribozyme III), 2429 (CUC, ribozyme I), and 2440 (GUC, ribozyme II). At all these cleavage sites we investigated ribozymes containing a 14 nucleotide complementary sequence to the target RNA (ribozymes I14-III14); at two sites (I and II) additional ribozymes with a 24 nucleotide hybridizing sequence have been tested. Catalytic activity was dependent on ribozyme to target ratio, pH, MgCl2 concentration, and incubation time. The highest cleavage activity was found with ribozyme II14, which cleaved 91% of an 292 nucleotides long in vitro transcript of the mdr-1 mRNA within 15 h in the presence of 10 mM MgCl2. Ribozymes I14 and II24 exhibited very similar but 6-fold reduced activity compared to ribozyme II14. Ribozyme III14 showed little and ribozyme I24 no cleavage activity. PMID- 7544983 TI - Loss of nocturnal blood pressure fall after liver transplantation during immunosuppressive therapy. AB - Hypertension, which develops after organ transplantation during immunosuppression with cyclosporine (CSA), is often associated with a loss of nocturnal decrease in blood pressure. Few data correlate circadian blood pressure patterns before transplant with those observed at fixed time points after transplant, or address the role of alternate immunosuppressive agents such as FK506. FK506 is unrelated structurally to CSA and less often leads to hypertension early after transplant. The present study compared nocturnal blood pressure patterns in patients with end stage liver disease (ESLD) before transplant to those of transplant recipients receiving either FK506 (0.15 mg/kg/day) plus prednisone or CSA (2 to 3 mg/kg/day) plus prednisone and azathioprine after orthotopic liver transplantation. Overnight ambulatory blood pressure profiles were studied in 13 pretransplant ESLD patients and in 34 patients (FK506: n = 13; CSA: n = 21) treated with different steroid doses (24 +/- 11 mg/day FK506; 34 +/- 3 mg/day CSA), according to protocol, 4 weeks (range, 2 to 7 weeks) after liver transplant. Mean blood pressure and heart rate values from awake and nocturnal 5-h time blocks were compared to 13 normotensive control subjects. Patients with ESLD were normotensive and maintained a normal nocturnal blood pressure fall (125 +/- 3/74 +/- 2 mm Hg awake; 109 +/- 3/60 +/- 2 mm Hg nocturnal). Awake ambulatory blood pressures were higher in CSA patients than in FK506 patients (148 +/- 3/95 +/- 2 v 128 +/- 3/78 +/- 2 mm Hg, respectively; P < .01), despite reduced glomerular filtration rates in both transplant groups. Both immunosuppressive regimens led to a loss of nocturnal blood pressure fall, as compared to ESLD patients or normotensive controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544984 TI - Controlled, targeted, intracellular expression of ribozymes: progress and problems. AB - Significant progress has been made in the development of ribozymes for a wide variety of intracellular applications, including human gene therapy. Further advances are likely to come from innovative strategies to improve the delivery, expression, co-localization, targeting specificity and substrate turnover of ribozymes. In order to stimulate problem solving in these areas, this article discusses examples of recent successes in intracellular ribozyme applications, and identifies some of the obstacles that remain. In addition, some testable, but as yet untried, ideas for overcoming several of these obstacles are presented. PMID- 7544985 TI - Application of a fluorescent redox dye for enumeration of metabolically active bacteria on albumin-coated titanium surfaces. AB - A bacterial staining method using fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is described for quantifying actively respiring bacteria that adhere to commercially pure titanium surfaces coated with cross linked albumin. This has not been possible to date using ordinary DNA stains such as propidium iodide (PI) or Hoechst, both of which produce a very bright background. With this technique, it was demonstrated that the cross-linked albumin inhibited the adherence of Staphylococcus aureus and Staph. epidermidis to the titanium surface. PMID- 7544988 TI - Surgical strategies in managing organ malperfusion as a complication of aortic dissection. AB - Between December 1978 and March 1994, 48 of 312 patients who underwent surgery for aortic dissection were diagnosed with major vascular complications. There were 18 patients with type A dissection and 30 patients with type B. In 23 patients with acute dissection, the site of vascular obstruction was the abdominal aorta in 12 patients, brachiocephalic artery in 7, iliac artery in 4, left common carotid artery in 3 and thoracic aorta in 2. In 26 patients with chronic dissection, the site of vascular obstruction was the abdominal aorta in 13 patients, brachiocephalic artery in 10, renal artery in 5, iliac artery in 4, superior mesenteric artery in 2, left common carotid artery in 2 and celiac artery in 1. Fifteen patients underwent proximal repair of the aorta during the acute stage, including the ascending aorta in 6 patients, from ascending aorta to arch in 7, arch to descending aorta in 1, thoracoabdominal aorta in 1, and entry closure in 1. In the acute stage, eight patients had palliative surgery, including aortic fenestration in four patients, axillo-femoral bypass in two, cross-over bypass to the iliac or femoral artery in one, bypass to superior mesenteric artery in one, bypass to the renal artery in one, and ileum resection in one. During the chronic phase, seven patients with type B dissection, who had malperfused unilateral renal artery, underwent proximal aortic repair.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544987 TI - Interleukin-6 and acute-phase proteins in head and neck cancer. AB - The acute-phase response is the answer of the organism to a disturbance of its homeostasis and is characterized by dramatic changes in the concentration of some plasma proteins defined as acute-phase proteins. In recent years several data have shown that interleukin-6 (IL-6) is the major inducer of acute-phase protein synthesis in human hepatocytes. Recently, we demonstrated higher IL-6 serum levels in head and neck cancer (HNC) patients than in healthy subjects. In the present study we examined the relationship between levels of IL-6 and of several acute-phase proteins, including C-reactive protein (CRP), alpha 1-antitrypsin (ATT), alpha 1-acid glycoprotein (AAG), haptoglobin (HPT) and fibrinogen. Eighteen patients were studied and had squamous cell carcinoma of the larynx (n = 9), oral cavity (n = 4), oropharynx (n = 3) and hypopharynx (n = 2). Proteins were measured at three time points before and three time points after surgery. Significant (P < 0.0001) relationships were found between IL-6 and CRP (r = 0.69), and fibrinogen (r = 0.51), whereas no correlation was found with AAT (r = 0.13, P = 0.56), AAG (r = 0.38; P = 0.07) and HPT (r = 0.16; P = 0.46). These data strongly suggest that IL-6 may play a key role in acute-phase protein synthesis in HNC and in regulation of the complex host response to malignancies. PMID- 7544986 TI - A rapid protocol for isolation of RNA from mycobacteria. AB - Isolation of total cellular RNA from members of mycobacteria has been a labour intensive task involving large volumes of cells, multiple extractions of cell lysates with phenol-chloroform followed by caesium chloride centrifugation. A simple and rapid procedure is reported for isolation of RNA from mycobacteria using as few as 1 x 10(7) cells. The RNA thus isolated when analysed on ethidium bromide gels contained 16S and 23S RNA as major species. Further, the RNA was used for amplification of an internal segment of hsp65 gene by reverse transcription followed by PCR. PMID- 7544989 TI - [Successful treatment of subacute sclerosing panencephalitis with long-term intrathecal large dose of alpha-interferon--a case report]. AB - Recent effective treatments for subacute sclerosing panencephalitis (SSPE) are oral inosiplex and intrathecal interferon therapy. The former seems to be effective for life expectancy but not for neurological disability, and the latter is a debating antiviral treatment although favorable reports are increasing. A 12 year-10 month-old boy with SSPE was presented as a successful case of prolonged effectiveness in both neurological symptoms and life expectancy. There are no significant side effects by the 200 weeks treatment of intrathecal alpha interferon of large dose (6 million unit/dose/week) and ordinary dose of inosiplex (100 mg/kg/day). Neurological disability index (Dyken) improved from 70% to 10%; speech and higher cerebral functions improved from aphasia and almost vegetative states to verbal communicable level; motor dysfunction from bedridden to wheel chair level; and myoclonic and other seizures were controlled. Laboratory data also improved; periodic synchronized discharge (PSD) and other paroxysmal discharges disappeared and alpha activity that had once disappeared in the background activity of active stages reappeared in the EEGs. Measles antibodies in serum and CSF improved, and oligoclonal bands disappeared. However diffuse brain atrophy remained on neuroimaging. Long-term intrathecal large dose treatment with alpha-interferon is effective and safe. It should be started as soon as the diagnosis is made, and a trial of large-dose and long-term therapy may be worthy even for more advanced cases irrespective of progress of neuroimages. PMID- 7544991 TI - Interleukin-11 induces phosphatidic acid formation and activates MAP kinase in mouse 3T3-L1 cells. AB - Interleukin-11 (IL-11) stimulated [3H]phosphatidic acid (PA) formation in [3H]arachidonic acid (AA) prelabelled quiescent mouse 3T3-L1 cells. When IL-11 stimulated 3T3-L1 cells were incubated with NaF, a phosphatidic acid phosphohydrolase (PAP) inhibitor, increased PA formation was observed. In the presence of ethanol, phosphatidylethanol accumulated at the expense of PA. These results indicated that the formation of PA upon IL-11 stimulation was a result of phospholipase D (PLD) activation. Endogenous accumulation of PA by NaF treatment or exogenously added PA enhanced tyrosine phosphorylation of two proteins of 44 KDa (p44) and 47 KDa (p47) whereas tyrosine phosphorylation of other proteins was not affected. Among various PA species, dipalmitoyl PA was found to be most effective in enhancing tyrosine phosphorylation of these proteins. p44 and p47 cross reacted with anti-MAP kinase monoclonal antibody (MoAb) in both immunoprecipitation and western blot analysis. Lysates from IL-11-induced or PA induced cells stimulated phosphorylation of a synthetic peptide substrate for MAP kinase, indicating the activation of MAP kinase in the induced cells. These studies suggest that one of the cellular signalling mechanisms of IL-11 in 3T3-L1 cells involves the activation of phospholipase D to produce the second messenger PA. The increased level of PA enhances tyrosine phosphorylation of p44 and p47 which belong to the members of MAP kinase family and thus transduces some of the mitogenic signals of IL-11 in this cell line. PMID- 7544990 TI - The effects of N-ras oncogene expression on PDGF-BB stimulated responses in cultured mouse myoblasts. AB - The role of the ras oncogene in the signalling pathway triggered by platelet derived growth factor BB (PDGF-BB) has been investigated in a cell line which normally differentiates into myotubes. Following the activation of the N-ras oncogene, however, the cells proliferate and form foci. PDGF-BB stimulated the phosphorylation of tyrosine in several cellular proteins of molecular weight 185, 160, 94, 54, 44, 42 kDa and furthermore Ca2+ was released from internal stores. Activation of the N-ras gene by treatment of cells with dexamethasone (DEX) inhibited these responses to PDGF-BB. On the other hand, both ras-induced and non induced cells responded to bradykinin (BK), foetal calf serum (FCS) and ionomycin (ION) by releasing Ca2+ from intracellular stores. The inhibition of the response to PDGF-BB in ras-activated cells has been further investigated. The binding of [125I]-PDGF-BB to its receptors was low and western blotting showed a low level of PDGF-BB receptor protein. This was in marked contrast to the receptor number seen in cells grown in growth medium or fusion promoting medium. These results indicate that cells transformed with the N-ras oncogene fail to respond to platelet-derived growth factor and exhibit a very low level of PDGF receptors. This suggests a role for the ras oncogene in the earliest steps of the signalling pathway. PMID- 7544992 TI - A novel neuropeptide cellular mechanism in amphibian interrenal steroidogenesis. AB - Interrenals of female Rana esculenta were incubated with gonadotropin-releasing hormone (GnRH), 9-ketoreductase inhibitor (palmitic acid), acetyl salicyclic acid, prostaglandin F2 alpha (PGF2 alpha), forskolin, isobutylmethyl xanthine (IBMX), dibutyril cyclic adenosine monophosphate (dbcAMP). Prostaglandin E2 (PGE2), PGF2 alpha, testosterone and 17 beta-estradiol were assessed on the incubation media. In addition, in the same interrenals, 9-ketoreductase and aromatase activities were evaluated. GnRH increased PGF2 alpha, 17 beta estradiol, 9-ketoreductase and aromatase, and decreased PGE2 and testosterone. PGF2 alpha increased 17 beta-estradiol and aromatase, and decreased testosterone. Palmitic acid counteracted GnRH effects, while forskolin, IBMX and dbcAMP showed the same PGF2 alpha effects. These results suggest that GnRH stimulates 9 ketoreductase enhancing PGF2 alpha which in turn activates aromatase through cAMP mediation in the interrenal of Rana esculenta. PMID- 7544993 TI - Intracellular calcium as a second messenger following growth stimulation of human keratinocytes. AB - The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing. PMID- 7544994 TI - Immunohistology of skin pathergy reaction in Behcet's disease. AB - The immunophenotypic characteristics of the skin pathergy reaction (SPR) at 48 h in Behcet's disease (BD) were investigated in 12 patients with BD and in five controls. The findings in 11 positive and one negative SPR lesions of patients with BD were evaluated in comparison with those of normal adjacent skin and with the negative pathergy biopsies from the controls. Positive SPR biopsies showed variable epidermal thickening and cell vacuolization, as well as subcorneal pustule formation. In the SPR dermis, a variable dense focal mononuclear cell (MNC) infiltrate was seen around vessels and skin appendages, extending into the deep dermis. The MNC infiltrate was predominantly composed of T lymphocytes and monocytes/macrophages. The majority of the T lymphocytes were CD4+, and almost all expressed CD45RO. Approximately half of the infiltrating cells strongly expressed HLA-DR. Neutrophils constituted less than 5% of the infiltrating cells, but were present as clusters of elastase-positive cells at the needle-prick sites. Vessels within the lesion showed marked congestion and endothelial swelling. The endothelial cells expressed ICAM-1 strongly, and E-selectin moderately. VCAM-1 was not expressed on endothelial cells. The basal and mid epidermal layers of keratinocytes expressed HLA-DR and ICAM-1 strongly, particularly so in areas close to the dermal MNC infiltrates. In negative pathergy biopsies, there were increased numbers of neutrophils and a few small clusters of macrophages and T lymphocytes only at the needle-prick site, and the endothelial cells of vessels close to these areas expressed E-selectin weakly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7544995 TI - Human piebaldism: relationship between phenotype and site of kit gene mutation. AB - Human piebaldism is a rare autosomal dominant disorder characterized by congenital depigmented patches of skin and hair. Piebaldism results from mutations of the kit proto-oncogene, which encodes a cell-surface receptor, tyrosine kinase, whose ligand is the stem/mast cell growth factor. We report four unrelated patients with piebaldism and consider the variations in phenotype in relation to the site of the kit gene mutation. PMID- 7544996 TI - Response of subcutaneous and cutaneous metastases of malignant melanoma to combined cytostatic plus interferon therapy. AB - A chemotherapy regimen consisting of dacarbazine (DTIC), vincristine, bleomycin and lomustine (CCNU) was combined with natural leucocyte interferon (IFN) in the treatment of 37 patients with cutaneous and subcutaneous metastases of malignant melanoma. Twenty-five also had concomitant lymph node, visceral, bone or brain metastases. Fifteen patients (41%) experienced complete response (CR) and 10 (27%) partial response (PR) of the superficial lesions. In addition, three patients were rendered surgically tumour-free after PR or disease stabilization during drug therapy. The median overall duration of response was 10.2 months (range 1-53 months). In disseminated disease, the combined therapy produced favourable results, particularly with regard to superficial lesions. It is also possible that this therapy may retard the growth of aggressive subcutaneous metastases in patients who have previously had multiple surgical procedures in an attempt to stabilize their disease. In a small proportion of patients, the long term complete remission with the drug therapy alone, or in combination with surgery, suggests that this regimen might have been curative. PMID- 7544997 TI - Sudden death following thyroxine administration. PMID- 7544998 TI - Growth hormone and thyroid function: is secondary thyroid failure underdiagnosed in growth hormone deficient patients? AB - Thyroid hormones and the GH/IGF-1 system show considerable mutual interference which may have physiological, pathophysiological and clinical importance. GH therapy of children and adults may induce a fall in serum T4, which seems to be due to an effect on the deiodination of T4 to T3. Animal studies suggest that the alterations in thyroid hormones in tissue may be much more prominent than the changes observed in serum. It is possible that the GH deficiency seen in the majority of patients with pituitary/hypothalamic disorders may mask secondary hypothyroidism in some patients by giving a relatively high serum T4. GH therapy may then unmask the hypothyroidism. In accordance with such a mechanism GH deficient children evaluated thoroughly to exclude secondary thyroid failure before GH administration do not develop thyroid insufficiency during GH substitution therapy. It is suggested that thyroid insufficiency should be considered in GH deficient patients with low normal serum T4. PMID- 7544999 TI - Interactions between atrial natriuretic factor (ANF) and thyrotropin or somatostatin in their effects on thyroid growth processes; studies in vitro and ex vivo in vitro. AB - The goal of our present study has been to examine the effects of the atrial natriuretic factor (ANF) on the growth processes in rat thyroid lobes. In the initial in vitro experiment, thyroid lobes were preincubated with rat ANF (Sigma) for 30 min in RPMI 1640 medium with 3H-thymidine (2 microCi/ml), and later on 15% fetal calf serum (FCS), Hepes buffer and the remaining tested substances [TSH 20 mIU/ml, somatostatin (SS) 10(-7)M] were added. Preincubations with ANF were not conducted in the controls and in the group exposed to TSH alone. Incubations of all the examined groups (controls, TSH alone, ANF alone, ANF together with TSH or ANF together with SS) with 3H-thymidine were carried out for 4 hours. We obtained the following results: at none of the examined concentrations (10(-5)M, 10(-7)M, 10(-9)M), did ANF significantly affect the rate of 3H-thymidine incorporation in vitro. Neither did TSH alone nor ANF with TSH jointly significantly influence the process in question. However, we observed increased rates of the 3H-thymidine uptake, following the joint exposure of thyroid lobes to ANF (10(-7)M or 10(-9)M) and SS (10(-7)M), when compared to ANF alone. In the ex vivo in vitro experiment, direct intrathyroidal microinjections of ANF alone or jointly with TSH or SS, were carried out. Twenty four (24) hours after the microinjections, all the animals were sacrificed by decapitation, the thyroid lobes being collected and incubated for 4 hours with 3H-thymidine (2 microCi/ml).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545000 TI - Therapeutic 131I dose in hyperthyroidism: role of pretreatment with thionamide. AB - Radioiodine therapy has become a cornerstone of treatment of hyperthyroidism. However, the timing of its administration varies between 1) the time of initial diagnosis with concurrent therapy with beta adrenergic blocking drugs or 2) following induction of euthyroidism with thioamide, Propylthiouracil or Methimazole. This study assessed 24-HR 131I uptake values and the thyroid scan in 24 subjects with hyperthyroidism at the time of diagnosis and again after attaining the euthyroid state with Propylthiouracil or Methimazole. Propylthiouracil of Methimazole was withdrawn seven days prior to the second 24 HR 131I uptake and scan. In all subjects, as a group, 24-HR 131I uptake increased following antithyroid therapy as compared to the time of initial of diagnosis [76 + 5% Vs. 54 + 4%; p < 0.01]. The thyroid gland size decreased in nine of twenty four subjects, but remained unchanged in the remaining subjects. Since 24-HR 131I uptake and the gland size are the major factors influencing the therapeutic radioiodine dosage, it is possible that initial therapy with thioamide drugs may reduce the therapeutic dose of 131I in subjects with hyperthyroidism belonging to both groups, i.e., Graves' disease and Multinodular toxic goiter by inducing a rise in 24-HR 131I uptake. Furthermore, the shrinkage of thyroid glands may further decrease the radioiodine dosage in patients with Graves' disease. PMID- 7545001 TI - Serum concentrations of apolipoproteins in patients with thyroid dysfunction. AB - We measured serum concentrations of apolipoproteins (apo A-I, A-II, B, C-II, C III and E) in patients with thyroid disease (hyperthyroidism; n = 44, hypothyroidism; n = 15) and in normal subjects (n = 89). We found that apoA-II, B and C-III concentrations revealed significant difference among three groups of the normal (apoA-II; 31.4 +/- 4.9 mg/dl, apoB; 85.8 +/- 16.3 mg/dl, apoCIII; 7.45 +/- 2.99 mg/dl), hyperthyroidism (apoA-II; 29.8 +/- 5.4, apoB; 63.4 +/- 18.9, apoC-III; 6.28 +/- 2.45) and hypothyroidism (apoA-II; 27.5 +/- 5.3, apoB; 108.0 +/- 30.9, apoC-III; 9.43 +/- 2.74). Thyroid hormones showed clear negative correlation to apoB (r = 0.70, p < 0.001). Furthermore, apoC-III was also found to be negatively correlated with thyroid hormone concentrations (r = 0.47, p < 0.001). PMID- 7545002 TI - Percutaneous ethanol injection therapy of autonomous nodule and amiodarone induced thyrotoxicosis. AB - A patient with amiodarone-induced thyrotoxicosis and autonomous nodule was treated with percutaneous ethanol injection (PEI) in 8 sessions. Preinjection thyroid hormone levels showed a marked elevation, peaking before the third session. The thyroid hormone increments following each procedure never exceeded 20% of the preinjection levels. FT4 plasma levels thereafter declined to within the normal range by the sixth session (day 21), while FT3 levels, though markedly reduced, were still slightly elevated; also, the thyroid hormone increments following ethanol injection were not observed after the fifth session. These findings suggest that a significant, but not sustained, increase in thyroid hormone levels is induced by PEI and may account for the lack of acute deterioration of clinical status, which remained under control with medical treatment alone. Normal serum thyroid hormone levels were observed at the 3 and 12 month follow-up. The use of percutaneous ethanol injection therapy for amiodarone-induced hyperthyroidism should be restricted to patients with preexisting thyroid hyperfunctioning nodule, and it may be a practical alternative to surgery in addition to medical treatment. Special caution should be exercised with patients with severe underlying heart disorders, since their clinical status might seriously worsen in case of acute elevations of serum thyroid hormones following ethanol injection. To this purpose, a close monitoring of serum thyroid hormones is recommended in order to institute a prompt adjustment in their medical therapy and/or in their PEI protocol. PMID- 7545003 TI - Adsorption and covalent immobilization of human serum albumin (HSA) and gamma globulins (gamma G) onto poly(styrene/acrolein) latexes with pyrene, dansyl, and 2,4-dinitrophenyl labels. AB - The poly(styrene/acrolein) latexes (P(SA)1 and P(SA)2), differing in poly(acrolein) content, were synthesized by the emulsifier-less emulsion precipitation polymerization of styrene and acrolein. The fraction of poly(acrolein) in the surface layer was 0.35 and 0.50, for the P(SA)1 and P(SA)2 latex, respectively. Latexes were labelled with 2,4-dinitrophenylhydrazine (DNPH), dansylhdrazine (DAH), and 1-aminopyrene (APY). Surface concentration of labels varied from 4.20.10(-7) mol m-2 (for APY label on P(SA)1 latex) to 1.54.10(-6) mol m-2 (for DNPH label on P(SA)2 latex) reflecting the fraction of polyacrolein in the surface layer and bulkiness of the label. The differences between adsorption and covalent immobilization of human serum albumin and gamma globulins onto the P(SA)2 latex and onto its derivatives labelled with the 2,4 dinitrophenyl (DNP), dansyl (DA), and pyrene (PY) groups were small. The observation conforms to the hypothesis that polyacrolein forms domains on the surface of the P(SA) latexes and that after labelling some aldehyde groups are still available for the covalent immobilization of proteins. Labelled and parent latexes were used in the model slide and turbidimetric aggregation tests for the goat anti-HSA. The fluorescent latexes, labelled with APY and DAH, and latexes labelled and with DNPH were found to be suitable for the model tests, similarly as the nonlabelled ones, however, some differences in the sensitivity, depending on the presence and the nature of labels, were noticed. The standard goat anti HSA serum (Sigma) was detected at maximum dilution equal to 2000 in the slide test, and in the dilution region from 1.8.10(3) to 4.7.10(6) times in the turbidimetric test. PMID- 7545006 TI - Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites. AB - A solvent-exposed loop, comprising residues 98-119 of S. typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme.PRPP.orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744 10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H. Scapin, G., Sacchettini, J. C., & Grubmeyer, C. (1995) Biochemistry 34, 10755 10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545005 TI - Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase. AB - Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5 phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10 fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2 hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12 fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545008 TI - Interfacial binding of human gastric lipase to lipid monolayers, measured with an ELISA. AB - Two sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin- peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN.m-1 were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mumol/(min.mg of enzyme)]. At a given lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545007 TI - Cross-linking of charybdotoxin to high-conductance calcium-activated potassium channels: identification of the covalently modified toxin residue. AB - High-conductance calcium-activated potassium (maxi-K) channels are composed of two subunits, alpha and beta. The pore-forming alpha subunit is a member of the mSlo family of K+ channels, whereas the beta subunit is a novel protein that modulates the biophysical and pharmacological properties of the channel complex. In the presence of a bifunctional cross-linking reagent, monoiodotyrosine charybdotoxin ([125I]ChTX) is covalently incorporated specifically into Lys69 of the beta subunit, which is located in a large extracellular loop of this protein. Using variants of ChTX which retain their channel-blocking activity and in which individual Lys residues have been mutated, we have identified the corresponding amino acid in ChTX that is involved in the cross-linking reaction. All of the ChTX mutants investigated bind to the maxi-K channel and display the same pharmacological profile as native ChTX in competition binding experiments. Whereas substitution of amino acids at positions 11 and 31 of ChTX yields wild type cross-linking patterns, the peptide without a Lys at position 32 fails to incorporate into the beta subunit of the maxi-K channel. Given the model for the interaction between ChTX and the outer vestibule of the maxi-K channel that has been proposed (Stampe et al., 1994), our data constrain the maximum distance between the pore of this channel and the region in the extracellular loop of the beta subunit where the cross-linking reaction takes place to 11 A. This topological limit helps define structural features of the maxi-K channel that may aide in probing the functional interaction between alpha and beta subunits of the channel complex. PMID- 7545004 TI - The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate. AB - The three-dimensional structure of Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5--phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base orotic acid has been solved and refined with X-ray diffraction data extending to 2.3 A resolution to a crystallographic R-factor of 18.7%. The complex was generated by carrying out catalysis in the crystal. Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287 1294] revealed that the enzyme backbone undergoes only small movements. The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5' phosphate binding loop, and a long, solvent-exposed loop at the dimer interface. The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP.orotate complexes, with its anomeric carbon moving approximately 7 A across the binding cavity. In the PRPP.orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate with the substrate in the OMP complex. PMID- 7545010 TI - [Modes of functioning of an ensemble of discrete active elements in a membrane]. AB - The behaviour of multichannel system as system of cellular automata was investigated. The action of the every channel is a stationary markov process. Computer simulation of the multichannel system demonstrate that according the values of parameters, system demonstrate the various types of behaviour: the weak oscillations about some stationary value, synchronous and chaotic oscillations and beatings. PMID- 7545009 TI - Study of LDL and acetylated LDL endocytosis by mononuclear cells in HIV infection. AB - Activated lymphocytes have a high level of low density lipoprotein (LDL) uptake as compared to resting lymphocytes, whereas scavenger receptors for acetylated LDL (Ac-LDL) are expressed on limited number of immune cells, i.e., monocytes/macrophages. The endocytosis of LDL and Ac-LDL by mononuclear cells was studied during in vitro and in vivo HIV infection, in order to use LDL and Ac-LDL as carriers of antiviral and/or immunomodulatory drugs towards lymphocytes and monocytes. The uptake of LDL and Ac-LDL was analyzed by cytofluorimetry. LDL endocytosis in PHA/IL2-activated lymphocytes was higher than in resting lymphocytes. In vitro HIV infection of PHA/IL2-activated lymphocytes did not alter the high LDL endocytosis in lymphocytes. CD4+ and CD8+ cells. In a group of 12 symptomatic patients there was no alteration of LDL endocytosis in lymphocytes, CD4 and CD8 lymphocytes. In another group of 23 individuals, the Ac LDL endocytosis mediated by CD14+ monocytes was unaltered in asymptomatic patients (n = 6) and in some symptomatic patients (n = 6, CD14+ cells > 100/mm3). On the contrary, in other symptomatic patients (n = 11, CD14+ cells < 100/mm3), the number of Ac-LDL+ CD14+ cells decreased, whereas their efficiency of Ac-LDL endocytosis increased as compared to those of other HIV+ patients. In conclusion, the use of lipoproteins as carriers to increase the drug delivery to CD4+ lymphocytes and to CD14+ monocytes can be envisaged, since: (i) the LDL endocytosis was not impaired in CD4 lymphocytes of HIV+ patients, and (ii) the Ac LDL uptake by monocytes was altered only in some patients of stage IV. PMID- 7545011 TI - Synchrony measures for biological neural networks. AB - Synchronous firing of a population of neurons has been observed in many experimental preparations; in addition, various mathematical neural network models have been shown, analytically or numerically, to contain stable synchronous solutions. In order to assess the level of synchrony of a particular network over some time interval, quantitative measures of synchrony are needed. We develop here various synchrony measures which utilize only the spike times of the neurons; these measures are applicable in both experimental situations and in computer models. Using a mathematical model of the CA3 region of the hippocampus, we evaluate these synchrony measures and compare them with pictorial representations of network activity. We illustrate how synchrony is lost and synchrony measures change as heterogeneity amongst cells increases. Theoretical expected values of the synchrony measures for different categories of network solutions are derived and compared with results of simulations. PMID- 7545013 TI - [An endogenous inhibitor of monoamine oxidase A (tribulin A) from brain: purification and structure identification]. AB - The endogenous monoamine oxidase inhibitor, tribulin, contains several components which selectively (or nonselectively) inhibit monoamine oxidases A and B. The pig brain tribulin component selectively inhibiting monoamine oxidase A was purified and identified as 4-hydroxyphenylethanol using gas chromatography-mass spectrometry. This compound was also found in the rabbit brain tribulin fraction which selectively inhibits monoamine oxidase A but has no influence on monoamine oxidase B. 4-Hydroxyphenylethanol inhibits monoamine oxidase A in an incompetitive manner with respect to the substrate, serotonin (Ki = 1.4 mM). Possible pathways of 4-hydroxyphenylethanol synthesis and its biological importance as the monoamine oxidase A inhibiting component of tribulin are discussed. PMID- 7545012 TI - On the relationship between the number of negative slope regions in the voltage current curve of the Hodgkin-Huxley model and its parameter values. AB - We consider the problem of the existence of a negative slope region (NSR) in the voltage-current curve of the neuronal membrane and the relationship between this phenomenon and the membrane parameters. For the Hodgkin-Huxley model it is proposed to determine the dependence of the number of NSR on the values of the maximal sodium (gNa) and potassium (gk) conductances. The method is suggested for constructing the boundaries on the (gNa, gk) plane, in passing through which the number of NSR changes to 1. Using the method we partition the (gNa, gk) plane into the regions corresponding to the curves with the different number of NSR. This number can be changed from 0 to 2 in changing the values of gNa and gk over the physiologically possible range. PMID- 7545014 TI - [Penicillin acylase from Escherichia coli: catalytically active subunits]. AB - Gel filtration under denaturing conditions was used to isolate the alpha- and beta-subunits of penicillin acylase (PA). Refolded subunits were obtained through removing urea by dialysis. Both renatured subunits were catalytically active during hydrolysis of phenylacetic acid p-nitroanilide; this activity decreased after addition of a serine-specific inhibitor--phenylmethanesulfonyl fluoride. The subunits were also active in reversed micelles of Aerosol OT (AOT) in octane, the optimum hydration degree being 11.9 and 17.5 for the light (alpha) and heavy (beta) subunits, respectively. The positions of the maxima were consistent with both theoretically calculated optimum hydration degrees and the earlier reported profile of enzymatic activity for native PA in reversed micelles. PMID- 7545015 TI - Effects of Yersinia enterocolitica infection on growth of the body and internal organs in newborn colostrum-deprived piglets. AB - Yersinia enterocolitica enteritis in newborn, colostrum-deprived piglets fed a human milk formula caused a reduced milk intake and decreased gain in weight but not length of the body. In infected piglets, the weight of the liver was less than in controls but other abdominal organs were unaffected. The weight of the caecum and small intestinal muscle were greater, but the length and weight of the small intestine and colon were not different. In infected piglets, the RNA concentration of the small intestinal mucosa was elevated whereas the concentrations of DNA and protein were unchanged, and total lactase activity was reduced. After antibiotic therapy, the liver weight was greater and the body weight increased at the same rate as the controls but was still lower at 14 days. The body weight of the control piglets increased linearly over the 14 days after birth. The stomach, pancreas and spleen grew more rapidly, but the liver more slowly, than the body as a whole but the kidneys had a minimum relative weight at 5 days. The total lactase activity and protein and RNA concentrations of the small intestinal mucosa decreased with age, the RNA to a greater extent than the protein. PMID- 7545017 TI - Regional distribution and characterization of nitric oxide synthase activity in the brain of the common marmoset. AB - The distribution of nitric oxide synthase (NOS) within the brain of the common marmoset, a non-human primate species, was investigated using the [3H]L citrulline formation assay and Western blot analysis. No hemispheric asymmetry of specific NOS activity was shown. The highest levels of NOS were found in the putamen and caudate nucleus--more than twice those in the cortex and the cerebellum, the brain regions with the lowest activities. The regional distribution pattern was similar to that in the ferret brain and contrasted to that in the mouse and bovine brain. Analysis of NOS catalytic activities in subcellular fractions revealed marked differences in the subcellular localization. Neuronal NOS accounted mainly for the measured catalytic activity in the brain. Differences in the regional distribution pattern of brain NOS activity among species may be indicative of diversities in the functional role of nitric oxide and NOS in mammals. PMID- 7545016 TI - Ca2+ permeable AMPA/kainate channels permit rapid injurious Ca2+ entry. AB - Small subsets of central neurons possessing Ca2+ permeable AMPA/kainate channels can be identified by a histochemical stain based on kainate-stimulated Co2+ uptake (Co2+(+)neurons) and are unusually vulnerable to AMPA/kainate receptor mediated injury. Using brief kainate exposures (which selectively destroy Co2+(+) neurons) along with kainate triggered 45Ca2+ influx measurements, we estimate kainate to cause an unusually high rate of Ca2+ influx into Co2+(+) neurons. Also, while fura-2 Ca2+ imaging revealed low (10 microM) kainate exposures to preferentially induce intracellular free Ca2+ ([Ca2+]i) elevations in Co2+(+) neurons, intense (100 microM) kainate exposures used in the 45Ca2+ influx studies triggered comparable [Ca2+]i rises in all neurons. These findings suggest that the exceptional vulnerability of Co2+(+) neurons to AMPA/kainate receptor mediated injury reflects a high rate of agonist triggered Ca2+ influx, and that [Ca2+]i rises may only poorly reflect influx rate. PMID- 7545018 TI - Nitric oxide synthase inhibited by audouine in the rat brain. AB - Nitric oxide synthase (NOS) synthesizes nitric oxide (NO) from L-arginine (Arg) which has a guanidino group in its molecule. Audouine, a derivative of Arg, is the diguanidino compound. In this study, the effects of audouine on rat brain NOS activity were investigated by measuring nitrite and nitrate formation. Audouine inhibited NOS activity in a competitive (Ki = 2.10 microM) and partially uncompetitive (Ki = 49.7 microM) manner. Audouine is not substituted at the guanidino nitrogen, in contrast to most previously reported NOS inhibitors which were synthesized by substituting the guanidino nitrogen of Arg. Audouine is a novel inhibitor of NOS and should be useful for investigating the chemical nature of NOS and the roles of NO in the central nervous system. PMID- 7545019 TI - The neuritogenic effect of myenteric plexus on striatal neurones in co-culture involves nitric oxide. AB - We reported previously that myenteric plexus explants promoted striatal neurite elongation in co-culture and that this effect was abolished by tetrodotoxin (TTX). Here we demonstrate that the nitric oxide synthase blocker N-nitro-L arginine methyl ester significantly reduced the neuritogenic effect of the myenteric plexus whereas the nitric oxide donor, sodium nitroprusside (SNP), partially reversed the blocking effect of TTX. 2-Chloroadenosine (2-CA), a stable analogue of adenosine, which is produced following release of ATP from enteric neurones, further enhanced the effect of SNP. Basic fibroblast growth factor or neurotrophin-3 in combination with 2-CA and SNP were only marginally neuritogenic in striatal cultures alone. These results suggest that NO is involved in the trophic effects of myenteric plexus explants on striatal neurones. PMID- 7545020 TI - Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-gamma 1 pathways. AB - Receptor tyrosine kinases are known to be important in growth and differentiation. We have recently found that c-kit, the tyrosine kinase receptor for steel factor, also regulates cell-matrix adhesion. Because Steel factor helps regulate cell migration and localization, this may be an important biologic function. Integrin adhesiveness is regulated within minutes by c-kit. The signaling pathways for tyrosine kinase stimulation of integrin adhesiveness and their relation to pathways that regulate growth and differentiation over much longer time periods remain uncharacterized. We have studied the effector pathways by which receptor tyrosine kinases regulate cell-matrix adhesion using wild-type and mutant forms of the platelet-derived growth factor (PDGF) receptor, which is closely related to c-kit. The PDGF receptor expressed in mast cells is as potent as c-kit in stimulating adhesion to fibronectin. We show that induction of adhesion is regulated through two independent pathways of phosphatidylinositol 3 kinase (PI3K) and phospholipase C-gamma 1 (PLC gamma)-protein kinase C by elimination of autophosphorylation sites required for activation of PI3K and PLC gamma or in combination with downregulation of protein kinase C or wortmannin. By contrast, a receptor mutated in both the PI3K and PLC gamma association sites can still stimulate mast cell growth, indicating a crucial role of these effector molecules in regulating adhesion rather than cell growth. PMID- 7545021 TI - Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells. AB - To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop. PMID- 7545022 TI - Granulocyte colony-stimulating factor downregulates allogeneic immune responses by posttranscriptional inhibition of tumor necrosis factor-alpha production. AB - We report downregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic immune responses in vitro. G-CSF did not affect the proliferative response of peripheral blood mononuclear cells (PBMC) against allogeneic Daudi cells but did inhibit tumor necrosis factor (TNF)-alpha secretion. In contrast with G-CSF, granulocyte-macrophage (GM)-CSF and interleukin (IL)-3 enhanced alloactivation-induced TNF-alpha production. G-CSF-mediated suppression of TNF alpha production was not affected by fixation of stimulators. G-CSF did not inhibit TNF-alpha mRNA expression or accelerate mRNA degradation, whereas pentoxifylline inhibited the expression of TNF-alpha mRNA. These results indicate that G-CSF acts directly on responder cells and modulates TNF-alpha production at posttranscriptional levels. Suppression of TNF-alpha secretion was accompanied by an increase of intracellular cyclic adenosine monophosphate (cAMP) concentration in alloactivated PBMC. The cell-permeable cAMP analogue, dibutyryl cAMP, suppressed TNF-alpha secretion without affecting TNF-alpha mRNA expression. G-CSF showed an inhibitory effect on the development of cytotoxic effector cells against allogeneic Daudi cells. Anti-TNF-alpha monoclonal antibody (MoAb) also inhibited the induction of cytolytic activity, and the inhibitory effects of G CSF and anti-TNF-alpha MoAb on killer activity generation were overcome by adding exogenous TNF-alpha. Hence, impaired generation of cytolytic effector cells by G CSF is believed to be the result of reduced TNF-alpha production. Collectively, the results described above suggest that G-CSF downregulates allogeneic immune responses by posttranscriptionally inhibiting TNF-alpha production. PMID- 7545023 TI - Regulatory role of CD43 leukosialin on integrin-mediated T-cell adhesion to endothelial and extracellular matrix ligands and its polar redistribution to a cellular uropod. AB - CD43 is a cell surface-associated mucin that is abundantly expressed by most leukocytes, and that appears to function as a negative regulator of cell surface interactions, providing a repulsive barrier around cells. We have analyzed herein the ability of anti-CD43 monoclonal antibody (MoAb) to upregulate both beta 1 and beta 2 integrin-mediated cell adhesion and to promote redistribution of the CD43 molecule into a cellular uropod. Engagement of CD43 with specific antibodies enhanced the cell adhesion to both 80- and 38-kD fibronectin fragments as well as to the endothelial cell ligands vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, an effect that was mediated through the alpha 5 beta 1, alpha 4 beta 1, and alpha L beta 2 integrins, respectively. This effect on cell adhesion was achieved in Jurkat leukemic T cells by anti-CD43 MoAb alone; however, in T lymphoblasts, the activation of cell adhesion required the concomitant ligation of CD3 with suboptimal doses of anti-CD3 MoAb. Immunofluorescence analysis showed that the engagement of CD43 was accompanied by a differential redistribution of CD43 into a well-defined cytoplasmic projection or uropod, whereas the beta 1 or beta 2 integrins remained uniformly located on the contact area with substrata. This change in the localization of CD43 did not require costimulation and was induced directly by engagement of CD43 in T lymphoblasts. Other stimuli of cell adhesion in the form of cross-linked anti-CD3 MoAb or phorbol esters did not induce uropod formation or CD43 redistribution. In addition, we observed that prolonged co-culture of resting peripheral blood T lymphocytes with endothelial cells, in the absence of anti-CD43 MoAb, induced uropod formation and redistribution of CD43 in T cells. Interestingly, the myosin disrupting drug butanedione monoxime inhibited the redistribution of CD43 induced by the specific MoAb, whereas the stimulation of cell adhesion induced by engagement of CD43 was preserved in the presence of this drug. These observations indicate that the signaling inducing integrin-mediated cell adhesion by CD43 takes place independently from the receptor redistribution. Altogether, these results indicate that CD43 has a regulatory role on both integrin-mediated T-cell adhesion and cellular morphology. PMID- 7545027 TI - Expression of HLA-DR (major histocompatibility complex class II) on neutrophils from patients treated with granulocyte-macrophage colony-stimulating factor for mobilization of stem cells. PMID- 7545026 TI - Do steady-state peripheral blood progenitor cell (PBPC) counts predict the yield of PBPC mobilized by filgrastim alone? PMID- 7545028 TI - Negative inotropic effect of carbachol on rat atria mediated by nitric oxide. AB - In this paper we show that isolated rat atria synthetized nitric oxide (NO) and its acts as intracellular messenger, increasing cGMP production that in turn modulates the muscarinic cholinergic dependent inhibition of contractility. Carbachol activating M2 muscarinic acetylcholine receptors (M2 mAchR) activated phosphoinositide turnover, stimulated nitric oxide synthase and increased production of NO. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase activities, shifted to the right the dose-response curve of carbachol upon contractility. Moreover, sodium nitroprusside and 8-bromo cGMP, induced negative inotropic effect. These results suggest that carbachol activating M2 mAchR exerts inotropic negative effect associated to an increase production of NO. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via phospholipase C activation. This in turn, triggers cascade reactions leading to the production of NO, that contribute to the inotropic negative action of low concentrations of carbachol. PMID- 7545025 TI - Correlation of multidrug resistance (MDR1) protein expression with functional dye/drug efflux in acute myeloid leukemia by multiparameter flow cytometry: identification of discordant MDR-/efflux+ and MDR1+/efflux- cases. AB - Resistance to chemotherapy is a major factor limiting successful treatment of acute myeloid leukemia (AML); one of the best characterized drug resistance mechanisms is extrusion of drugs by the energy-dependent multidrug resistance (MDR1) transport protein. Expression of MDR1 is common in AML and has been linked to lower remission induction rates and decreased remission durations. Because MDR1 efflux function may be modified by drugs such as cyclosporin A, accurate identification of MDR1+/efflux+ AML cases will be critical to identify patients who may benefit from therapies that contain such MDR1 modulators. We have optimized single and multiparameter flow cytometric assays to detect efflux of drugs or fluorescent dyes by previously cryopreserved AML blasts. These assays allowed precise identification of efflux by leukemic blasts, and correlation with CD34 and MDR1 expression. We subsequently studied a series of 60 previously untreated AML cases. Functional efflux was identified in 39 cases and was significantly correlated with MDR1 expression (P = .0002). However, discrepant cases were identified; 10 cases were efflux+ without significant MDR1 expression, whereas 6 MDR1+ cases were efflux-. There was also a highly significant correlation of efflux with CD34; 31 (79%) of the 39 efflux+ cases were CD34+ in comparison with only 5 (24%) of the 21 efflux- cases (P < .0001). Multivariate analysis showed that efflux was significantly associated with independent effects of both CD34 (P = .0011) and MDR1 expression (P = .034); the majority of efflux+ cases were CD34+, whereas 5 of the 6 MDR1+ efflux- cases lacked CD34 expression. Cyclosporin A blocked efflux in all but 2 cases regardless of MDR1 expression. Functional efflux in AML is frequently detected without the classic MDR1+ phenotype indicating that alternate non-MDR1-mediated efflux mechanisms may be important. Efflux assays may better identify patients who would benefit from therapies that include efflux modulators. PMID- 7545024 TI - Evidence for antigenic selection of large granular lymphocytes in a patient with Wiskott-Aldrich syndrome. AB - It is now recognized that CD3+ large granular lymphocyte (LGL) proliferations may be clonally derived from their normal CD3+LGL+ counterpart, but the nature of the pressure responsible for the proliferation of these cells remains unclear. We approached this problem by analyzing the diversity of the T-cell receptor repertoire of LGL developed in different clinical settings. Two of our patients had typical lymphoproliferative disorders. The third case was much more unusual, as the LGL proliferation was associated with a Wiskott-Aldrich syndrome. Our data relative to the patients with the lymphoproliferative disorders only suggest that these LGL were clonally expanded. The data relative to the patient with Wiskott Aldrich syndrome were more unexpected, as the T-cell repertoire of the LGL appeared to have common features with that of the other T-cell populations analyzed. These LGL were characterized by the clonal expansion of a few TCRBV segments that shared common amino acid motifs in the junctional region of the T cell receptor. This common pattern of junctional diversity associated with different TCRBV segments is, therefore, consistent with a strong on-going antigenic selection process, possibly related to the pathogenesis of Wiskott Aldrich syndrome. Furthermore, the finding that the same TCRBV segments were also highly expanded among other T-cell subpopulations questions the malignant nature of this LGL proliferation. PMID- 7545029 TI - A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present. PMID- 7545030 TI - Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor. AB - Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface. PMID- 7545032 TI - Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells. AB - By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access. PMID- 7545031 TI - Loss of cell surface syndecan-1 causes epithelia to transform into anchorage independent mesenchyme-like cells. AB - Simple epithelial cells are polygonal in shape, polarized in an apical-basal orientation, and organized into closely adherent sheets, characteristics that result from a variety of cellular specializations and adhesive proteins. These characteristics are lost when the epithelia transform during embryogenesis into mesenchymal cells or after neoplasia into invasive carcinoma cells. Of the syndecan family of transmembrane heparan sulfate proteoglycans, simple epithelia produce predominantly syndecan-1, which is found at basolateral surfaces and within adhesive junctions. To elucidate the function of this syndecan-1, normal murine mammary gland epithelia were made deficient in syndecan-1 by transfection with an expression vector containing the syndecan-1 cDNA in the antisense configuration. Several independently derived clones of stable transfectants contained the antisense cDNA in their genome and expressed the antisense transcript. These grew either as epithelial islands of closely adherent polygonal cells, identical to both the parental cells and the vector-only control transfectants, or as individual elongated fusiform cells that invaded and migrated within collagen gels, like mesenchymal cells, but were anchorage independent for growth. The clones that retained epithelial characteristics were moderately deficient in cell surface syndecan-1 (greater than 48% of control levels) but did not differ from control cells in expression of beta 1-integrins and E-cadherin, or in F-actin organization. However, the clones of fusiform cells were severely deficient in cell surface syndecan-1 (less than 12% of control levels) and showed rearranged beta 1-integrins, markedly reduced E-cadherin expression, and disorganized F-actin filaments, but retained mammary epithelial markers. Therefore, depleting epithelia of cell surface syndecan-1 alters cell morphology and organization, the arrangement and expression of adhesion molecules, and anchorage-dependent growth controls. Thus, cell surface syndecan-1 is required to maintain the normal phenotype of simple epithelia. PMID- 7545033 TI - The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm. AB - In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100 fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed. PMID- 7545034 TI - Risperidone: review and assessment of its role in the treatment of schizophrenia. AB - OBJECTIVE: To review pharmacologic, pharmacokinetic, therapeutic, and safety information for the antipsychotic agent risperidone and to evaluate its place in the treatment of schizophrenia. DATA SOURCES: MEDLINE and Exerpta Medica databases; Janssen Pharmaceuticals; Food and Drug Administration Psychopharmacology Advisory Committee; PJB Publications; published articles and abstracts; unpublished research reports and abstracts. STUDY SELECTION: In vivo animal studies (pharmacology); volunteer studies (pharmacokinetics); clinical case reports, open clinical studies, and controlled clinical studies (clinical efficacy and adverse effects; long-term studies; studies in special populations. DATA EXTRACTION: Relevant data were extracted from published and unpublished source documents, evaluated, and summarized in tables for comparative review. Abstracts were used in 6 of 8 pharmacokinetic studies, 4 of 8 open clinical studies, and 2 of 9 controlled clinical studies. Use of abstracts limits the extent of data extracted. DATA SYNTHESIS: Risperidone is an effective treatment for positive symptoms of schizophrenia and may be effective for negative symptoms. Efficacy in treatment-resistant patients is inconclusive. In patients in whom low-to-moderate dosage is effective, there should be fewer and less severe adverse effects with risperidone than with haloperidol. Optimal dosage for schizophrenia appears to be 6 mg/d. Although data are limited, risperidone may be toxic on overdose. CONCLUSIONS: Risperidone is a useful addition to the antipsychotic drug armamentarium, but it should be viewed as an atypical antipsychotic agent. It is reasonable to consider a trial of risperidone in treatment-resistant schizophrenic patients prior to the use of clozapine and as a first-line treatment for newly diagnosed patients with schizophrenia. PMID- 7545036 TI - Comparative uptake of a pyrethroid and organophosphate insecticide by selected aquatic insects. PMID- 7545037 TI - Lindane and DDT-induced changes in rat harderian N-acetyltransferase activity, melatonin levels, and porphyrin concentration. PMID- 7545038 TI - Determination of organochlorine pesticide residues in human adipose tissue: 1992 study in Mexico. PMID- 7545040 TI - Exceptionally stable nucleic acid hairpins. AB - Hairpins represent the dominant secondary structure element in RNA. Certain sequences are found with exceptional frequency in many RNAs and are characterized by exceptionally high thermodynamic stability. Stable RNA hairpins define nucleation sites for folding, determine tertiary interactions in RNA enzymes, protect mRNAs from degradation, and are recognized by RNA-binding proteins. The structures of several stable DNA and RNA hairpins have revealed networks of stabilizing interactions within the hairpin loop: non-Watson-Crick base pairs and base-phosphate and base-sugar contacts. The unusual stability of these structural elements can be used to stabilize RNA and DNA structures and to protect antisense oligonucleotides and mRNAs against exonucleolytic degradation. PMID- 7545039 TI - Electron microscopic study of guinea pig skin exposed to sulphur mustard. PMID- 7545035 TI - Effect of human immunodeficiency virus infection on haematopoiesis. AB - The pathogenesis of peripheral blood cytopenias in AIDS patients is clearly multifactorial. Among the various contributing mechanisms, those involving a direct role of HIV-1 have been actively investigated in the past few years. It has now been convincingly demonstrated that HIV can impair the survival/proliferative capacity of purified haematopoietic progenitor cells. Although a subset of haematopoietic progenitor cells are perhaps susceptible to HIV-1 infection, both in vitro and in vivo, the suppressive effect does not require either active or latent infection and is probably mediated by the interaction of viral or virus-associated proteins with the cell membrane of haematopoietic progenitor cells. Both the viral load and the biological characteristics of the virus play an important role in suppression, since different isolates displayed different inhibitory activity. Haematosuppression is not a specific property of monocytotropic versus lymphocytotropic or low replicating versus high-replicating isolates, and it will be important to exactly establish which viral component is crucial to suppression of haematopoietic progenitor cells. Since the haematopoietic stem cell is the common progenitor to both the myeloid and lymphoid lineages, the capacity of HIV to impair the growth of early haematopoietic progenitor cells could contribute not only to the frequent occurrence of anaemia, granulocytopenia and thrombocytopenia in AIDS patients, but also to the inability of the bone marrow to reconstitute a functional pool of mature CD4+ T-cells. It is also possible that haematopoietic progenitor cells committed to the T-lymphoid lineage are impaired by HIV in their differential pathway within the thymus (Bonyhadi et al, 1993). Infection of megakaryocytes could result in underproduction of platelets and possibly represents a major pathogenetic mechanism of HIV-related thrombocytopenia. Infection of monocytes and T-lymphocytes leads in vitro and probably also in vivo to deranged cytokine production. In the first stages of the disease, increased cytokine production, consequent to a chronic immune activation, is probably responsible for the myelodysplastic/hyperplastic alterations observed at the bone marrow level. In more advanced stages of the disease, the general decline in immune function, the consequent imbalance in cytokine production, and the increase in viral burden, may contribute to dysregulated haematopoiesis and peripheral blood cytopenias. PMID- 7545041 TI - Structure-function of the channel-forming colicins. AB - The channel-forming colicins are plasmid-encoded bacteriocins that kill E. coli and related cells and whose mode of action is of interest in related problems of protein import and toxicology. Colicins parasitize metabolite receptors in the outer membrane and translocate across the periplasm with the aid of the Tol or Ton protein systems. X-ray structure data for the channel domain and colicin are available. Residues have been identified that affect the channel ion selectivity and particular helices implicated in channel structure and in conformational changes required for binding or insertion of the channel into the membrane. Unique aspects of the colicin channel system are the involvement of protein import in the gating process, the existence of multiple open and closed states, and the existence and action of an immunity protein that involves specific intramembrane helix-helix interactions with transmembrane helices of the colicin channel-forming domains. PMID- 7545042 TI - Dynamic modelling of a helical peptide in solution using NMR data: multiple conformations and multi-spin effects. AB - Nuclear Overhauser effect (NOE) measurements on molecules in solution provide information about only the ensemble-averaged properties of these molecules. An algorithm is presented that uses a list of NOEs to produce an ensemble of molecules that on average agrees with these NOEs, taking into account the effect of surrounding spins on the buildup of each NOE ('spin diffusion'). A simplified molecular dynamics simulation on several copies of the molecule in parallel is restrained by forces that are derived directly from differences between calculated and measured NOEs. The algorithm is tested on experimental NOE data of a helical peptide derived from bovine pancreatic trypsin inhibitor. PMID- 7545043 TI - Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis (delta F508) AB - OBJECTIVE: To evaluate direct polymerase chain reaction amplification of mutation on single embryo cells for the routine preimplantation diagnosis of cystic fibrosis. DESIGN: Direct polymerase chain reaction amplification of mutation was performed to identify the cystic fibrosis delta F508 mutation in human blood DNA, single lymphocytes, embryos, and embryo cells obtained by biopsy. Preimplantation diagnosis was performed for a couple who were heterozygous carriers of the delta F508 mutation. SETTING: Laboratory for preimplantation diagnosis in a reproductive medicine unit. MAIN OUTCOME MEASURE: Correct diagnosis of homozygous normal, heterozygous, and homozygous abnormal DNA of the cystic fibrosis delta F508 mutation. RESULTS: 45 blood samples (18 homozygous normal, 17 heterozygous, and 10 homozygous abnormal) and 204 single lymphocytes from known sources showed 100% amplification and were diagnosed correctly. 17 human embryos and 52 normal nucleated embryo cells obtained by single cell embryo biopsy also showed 100% amplification. After a miscarriage of the initial pregnancy (diagnosed at preimplantation to be homozygous normal) in the heterozygous carrier couple, fetal tissue was confirmed to be homozygous normal. CONCLUSION: Direct polymerase chain reaction amplification of mutation is a simple, fast, reliable test for the common cystic fibrosis mutation (delta F508) in blood DNA and single cells and should be applicable to routine programmes of general screening, maternal blood examination, and preimplantation diagnosis. PMID- 7545044 TI - Commentary: Preimplantation diagnosis raises a philosophical dilemma. PMID- 7545045 TI - Systematic review of clinical efficacy of topical treatments for head lice. AB - OBJECTIVES: To collect and evaluate all trials on clinical efficacy of topical treatments for head lice. DESIGN: Systematic review of randomised trials identified from following data sources: Medline, International Pharmaceutical Abstracts, Science Citation Index, letters to key authors and companies, and hand search of journals. SETTING: Trials in schools or communities. SUBJECTS: Patients infested with lice. MAIN OUTCOME MEASURE: Cure rate (absence of live lice and viable nits) on day 14 after treatment. RESULTS: Total of 28 trials were identified and evaluated according to eight general and 18 lice specific criteria. Of the 14 trials rated as having low to moderate risk of bias, seven were selected as they used the main outcome measure. These seven trials described 21 evaluations of eight different compounds and placebo (all but two evaluations were of single applications). Only permethrin 1% creme rinse showed efficacy in more than two studies with the lower 95% confidence limit of cure rate above 90%. CONCLUSIONS: Only for permethrin has sufficient evidence been published to show efficacy. Less expensive treatments such as malathion and carbaryl need more evidence of efficacy. Lindane and the natural pyrethrines are not sufficiently effective to justify their use. PMID- 7545047 TI - Treatment of choroidal neovascularization in age-related macular degeneration with interferon alpha-2a: a short term, nonrandomized pilot study. AB - A total of 11 patients exhibiting in 11 eyes a subfoveal or juxtafoveal choroidal neovascular membrane due to age-related macular degeneration (AMD) were treated with interferon alpha-2a (Roferon) according to a protocol that was designed for an independent, randomized, open-label pilot study to evaluate the efficacy and safety of this therapy. However, none of the patients agreed to be randomized and all elected to be treated. The average follow-up period after treatment was 8.2 months. In 6 of 7 eyes with classic choroidal neovascularization the membrane increased in size during and after treatment. Of these 6 eyes, 5 lost more than 6 lines of visual acuity. In 2 of 4 eyes with occult neovascularization the lesion enlarged. In the other 2 eyes the lesion became atrophic in 1 case and remained stable in size in the other. Visual acuity decreased by 3-9 lines in 3 eyes with occult neovascularization. Of the 11 patients, 2 discontinued interferon treatment after 8 weeks because of severe adverse reactions. From the data collected thus far in this study and in the majority of published series, we conclude that interferon alpha-2a in the dose range of 3-6 million IU given three times weekly over 8-12 weeks is neither effective nor safe for the treatment of subfoveal neovascularization in AMD. PMID- 7545046 TI - Surface antigen analysis of group B Neisseria meningitis outer membrane by monoclonal antibodies: identification of bactericidal antibodies to class 5 protein. AB - Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes. PMID- 7545051 TI - A new method of human cardiac troponin I and troponin T purification. AB - New rapid and effective procedure for simultaneous purification of human cardiac troponin I and troponin T has been developed. Affinity chromatography on immobilized monoclonal antitroponin I antibody C5 was used for purification of the whole troponin complex with a yield of 120 mg from 100 g of tissue. Isolated troponin I and troponin T (about 20 and 35 mg from 100 g tissue) were obtained by conventionally used ion-exchange chromatography. Antibody C5 recognizes conservative epitope of troponin I, therefore the method is applicable for purification of skeletal and cardiac troponin from a number of different animal species. PMID- 7545049 TI - Telling a story. PMID- 7545050 TI - Cystic fibrosis and pancreatic cancer cells synthesize and secrete MUC1 type mucin gene product. AB - The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by cystic fibrosis cells (CFPAC-1), a pancreatic cancer cell line derived from a patient with cystic fibrosis, and pancreatic cancer (SW-1990) cell lines. High molecular weight glycoproteins (HMG) were quantified by [3H]-glucosamine labeling and chromatography on sepharose CL 4B. Mucin gene expression was determined by using cDNA probes for 2 distinct intestinal mucins (MUC2 and MUC3) and one stomach mucin (MUC1). The specific mucin core epitopes were confirmed by immunoblots using antibodies that recognize T, Tn, sialosyl Tn, MUC1, MUC2, and MUC3. The results of these experiments demonstrate that CFPAC-1 cells contained 1.25 fold and 1.4 fold more HMG in the membrane and cytosolic fractions, however, secreted 4-fold more HMG into the medium compared to SW-1990 cells. The HMG of SW-1990 was found to be mucinous in nature and not proteoglycans, as it was not susceptible to hyalurinidase, heparinase and chondroitinase ABC. The HMG of CFPAC-1 was also predominantly (80%) mucinous but with small amounts of proteoglycans. mRNA and immunoblot analysis suggest that these CFPAC-1 and SW-1990 cells predominantly express MUC1 apomucin, small amounts of MUC2 apomucin, and no MUC3. Pulse chase labeling and immunoprecipitation of MUC1 type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin gene product were present in both cell lines, corresponding to the known length polymorphism of this mucin. Both T and Tn antigens were significantly higher in CFPAC-1 and SW-1990 cells as compared to sialosyl Tn antigen. These findings were associated with the increased activities of polypeptidyl N-acetylgalactosaminyl transferase and b1,3 galactosyltransferase. These investigations demonstrate for the first time that cystic fibrosis cells (CFPAC-1) secrete and synthesize high amounts of mucin which is associated with high levels of MUC1 mRNA, low levels of MUC2 mRNA and non detectable MUC3 mRNA. PMID- 7545048 TI - Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding. AB - Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix. Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT. PMID- 7545052 TI - A method for quantification and correction of proteins after transfer to immobilization membranes. AB - A method is described for quantification of specific proteins after electrophoresis and transfer to immobilization membranes for Western blots. This method is analogous to methods used to correct the amounts of specific transcripts detected on Northern blots. Ponceau S staining of proteins bound to immobilization membranes is efficient and accurate compared to antibody binding in terms of time, effort and cost. Comparison of Ponceau S to other detection and staining methods for quantifying proteins and the basic chemistry of Ponceau S are summarized. PMID- 7545053 TI - Potato mitochondrial manganese superoxide dismutase is an RNA-binding protein. AB - An RNA-binding protein present in potato mitochondrial lysates was purified and identified as manganese-containing superoxide dismutase (MnSOD). Using a gel mobility shift assay we found that proteins from mitochondrial lysates bind with high affinity to in vitro transcripts of mitochondrial orf206, encoding a subunit of the ABC-type heme transporter. By ammonium sulfate fractionation and two subsequent chromatographic steps on MonoQ columns we purified a 28 kDa protein to apparent homogeneity. Protein sequencing identified the purified polypeptide as manganese-containing superoxide dismutase, which is a specific enzymatic scavenger of superoxides in mitochondria. Using gel mobility shift and competition assays, we show that RNA-binding of MnSOD of potato is not influenced by 400 mM KCl or heparin and is specific to heteropolymeric RNAs. The labeled mitochondrial transcript could be competed with low amounts of unlabeled transcript while binding was stable to competition with large amounts of tRNA or high concentrations of NADH and NADPH. The purified MnSOD of potato mitochondria was UV-cross-linked to the mitochondrial transcript. The Mn- and Fe-containing SODs from Escherichia coli showed no binding to the RNA by either gel mobility shift or UV-cross-linking. Enzyme activity assays revealed that binding of RNA to the mitochondrial MnSOD does not significantly influence enzyme activity. This indicates that the RNA-binding feature of MnSOD of potato mitochondria is probably not involved in modulating SOD enzyme activity and suggests a function different from superoxide degradation as ist biological role. PMID- 7545055 TI - Biochemical and functional markers of lung damage after bleomycin treatment. AB - Lung function tests, including spirometry, and pulmonary transfer factor for carbon monoxide (TLCO), serum angiotensin-converting enzyme (sACE), serum copper (sCU++) and serum procollagen III peptide (sPIIIP) were measured in 20 patients with metastatic testicular carcinoma, before and after treatment with cisplatin, bleomycin, vinblastine or etoposide. A significant decrease of TLCO, total lung capacity and vital capacity was observed at the end of the treatment. No recovery of pulmonary function had taken place 2 yrs after the last dose of bleomycin. This result suggests that bleomycin-induced lung damage is not completely reversible. Serum angiotensin-converting enzyme, remained unmodified, whilst sPIIIP was found to be significantly increased at the end of chemotherapy, but normal 2 yrs after the completion of chemotherapy. Serum Cu++ showed a tendency to decline. No significant correlation was found between sACE, sCu++ and sPIIIP and lung function tests in the follow-up period. These results question the possibility that these markers could be useful indicators of bleomycin-induced lung damage. PMID- 7545054 TI - Genetic functions of isolated maize mitochondria under model changes of redox conditions. AB - The template functions of maize mitochondrial genome have been studied under model conditions of redox potential created by the addition of potassium ferricyanide as an oxidising agent and sodium dithionite as a reducing agent. The addition of both potassium ferricyanide and sodium dithionite to the isolated mitochondria causes the changes in the activity of the mitochondria RNA and DNA synthesis. The data obtained indicate the possibility of redox regulation of genetic functions in isolated mitochondria. PMID- 7545056 TI - Cytokine gene expression in epidermis with biological effects following injection of naked DNA. AB - The epidermis is readily accessible for genetic manipulation and is easily monitored. Using pig skin because it is very similar to human skin morphologically, we have developed a method to transiently express biologically active factors in epidermis. Following direct injection of naked plasmid DNA into skin, DNA is taken up and transiently expressed at high levels by epidermal keratinocytes. Injection of interleukin-8 plasmid DNA into skin results in the appropriate biological response of neutrophil recruitment, demonstrating functional utility. In addition to this model's therapeutic uses, the biological effects of structural gene products on the epidermis could also be studied in vivo. PMID- 7545060 TI - Risperidone in the treatment of patients with chronic schizophrenia: a multi national, multi-centre, double-blind, parallel-group study versus haloperidol. Risperidone Study Group. AB - BACKGROUND: This study was performed in order to evaluate the short-term efficacy and safety of fixed risperidone doses compared to haloperidol. METHOD: In a multi national, parallel-group, double-blind study, patients with chronic schizophrenia (DSM-III-R) were randomly assigned to risperidone 1, 4, 8, 12 or 16 mg or haloperidol 10 mg daily for 8 weeks. Efficacy was assessed by the Positive and Negative Syndrome Scale for schizophrenia (PANSS) and clinical global impression (CGI), and safety primarily by the Extrapyramidal Symptom Rating Scale (ESRS). RESULTS: One thousand three hundred and sixty-two patients were evaluated. The optimum risperidone doses were 4 mg and 8 mg, with response rates of 63.4% (56.8%; 69.7%) and 65.8% (59.2%; 71.9%) respectively. Response rate in haloperidol-treated patients was 58.7% (52.0%; 65.3%); the 95% confidence intervals (CI) of the differences between risperidone 4 mg or 8 mg and haloperidol were (- 4.3%; 13.7%) and (- 1.9%; 16.0%) respectively. There were no significant differences in CGI scores at endpoint between risperidone 4 mg, 8 mg, 12 mg and 16 mg and haloperidol (3.0, 3.0, 3.2, 3.1 and 3.1 respectively); the 95% CI of the differences between risperidone 4 mg or 8 mg and haloperidol were ( - 0.4; 0.1) and ( - 0.3; 0.2) respectively. Mean shifts to the maximum total ESRS scores versus baseline (mean (confidence interval)) were significantly greater in haloperidol-treated patients (5.1 (4.0; 6.2)) than in the risperidone 1, 4, 8 and 12 mg groups (1.1 (0.3; 1.9); 1.8 (0.9; 2.7); 2.7 (1.8; 3.6) and 3.2 (2.3; 4.1) respectively (P < 0.05)). CONCLUSION: Risperidone is an effective antipsychotic for the treatment of chronic schizophrenia; doses of 4 and 8 mg seem to be optimal and have a lower incidence of side-effects than haloperidol. PMID- 7545058 TI - Expression and distribution of cell-membrane complement regulatory glycoproteins along the human respiratory tract. AB - Complement in the human respiratory tract protects the host from invading microorganisms and from other inhaled insults. However, complement may also lyse the host's respiratory tract cells, leading to tissue injury. In many extrapulmonic tissues, cells express cell-membrane complement regulatory glycoproteins that protect the cells from complement-induced lysis. To determine whether these glycoproteins are expressed in human respiratory tract tissue, we studied tissue biopsies of healthy and diseased human respiratory tract from nose to alveoli for the presence of four cell-membrane complement regulatory glycoproteins (membrane cofactor protein [MCP], decay-accelerating factor [DAF], CD59, and complement receptor type 1 [CR1]) using an immunoperoxidase technique. In addition, to establish a model for in vitro studies of these glycoproteins in respiratory cells, we studied whether they are expressed in cultured nasal epithelial cells, using the same technique. Altogether, 26 tissue specimens from 22 patients were studied. We found that normal human respiratory tract from nose to alveoli express MCP, DAF, and CD59, but not CR1, and that this expression increases in inflammation and in lung cancer. In addition, expression in nasal epithelial cells is retained under cell culture conditions. These findings suggest that human respiratory tract tissue may regulate complement activation on its surface in order to avoid self-injury. We propose that imbalances in the mechanism that regulates cell-membrane complement may predispose the respiratory tract to tissue injury and disease, and that iatrogenic modulation of such imbalances may help to prevent these adverse consequences. PMID- 7545059 TI - Secretion of chemokines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. AB - We have demonstrated the detection of proallergic cytokines in the nasal secretions after antigen challenges. Our aim was to determine the secretion kinetics of chemokines (interleukin [IL]-8, macrophage inflammatory protein-1 alpha [MIP-1 alpha], and RANTES) and other cytokines (IL-1 beta and granulocyte/macrophage colony-stimulating factor [GM-CSF] after allergen challenges and their inhibition by steroid therapy. Ten allergic patients were given either beclomethasone dipropionate (BDP) or placebo in a double-blind, randomized, crossover manner. Allergen challenges were performed after 1 wk of treatment. Nasal secretions were collected serially for 11 h after allergen challenge by a matrix method. Subjects maintained symptom scores at each time point of nasal secretion recovery. Cytokines were measured by specific enzyme linked immunosorbent assays. The mean peak values for each cytokine and total symptom scores during the early (ER) and/or late-phase reactions (LPR) were significantly reduced during the BDP treatment period (p < 0.05). The levels of cytokine correlated (p < 0.05) with corresponding total symptom scores during ER (IL-1 beta and MIP-1 alpha) and LPR (all cytokines). Our findings document local elevations of IL-1 beta, GM-CSF, and chemokines in the nasal secretions after allergen challenges and their inhibition by steroids. We speculate that the inhibition of cytokine production and secretion in the nasal mucosa may contribute to the clinical efficacy of topical steroids. PMID- 7545061 TI - Functional and neurochemical profile of place learning after L-nitro-arginine in the rat. AB - Rats in which near-total inhibition of the nitric oxide synthesizing enzyme (NOS) had been obtained by a 5-day pretreatment during which two daily injections of L nitro-arginine (50 mg/kg per injection) had been administered were subjected to five sessions of training on a place learning task (one session per day). NOS inhibition was associated with significantly impaired task acquisition but on the fifth training session normal task proficiency was achieved. Subsequent pharmacological and behavioral challenges established that dissimilar neural substrates mediated the task in NOS-inhibited and normal animals. In NOS inhibited rats the neural substrate of task mediation depended less than normally on cholinergic mechanisms while potentially relying more on catecholaminergic mediation. In a separate experiment rats that had acquired the place learning task to asymptotic quality of performance were subjected to a L-nitro-arginine treatment similar to the pretreatment of the first experiment. Such a NOS inhibition did not impair subsequent task performance. It is concluded that nitric oxide participates in processes subserving acquisition rather than performance of at least some forms of place learning. PMID- 7545062 TI - A urodynamic study of laser ablation of the prostate and a comparison of techniques. AB - OBJECTIVE: To evaluate the effect of laser ablation of the prostate on symptomatic and urodynamic parameters and to compare laser delivery systems. PATIENTS AND METHODS: The study comprised 81 patients of whom 79 were waiting for transurethral resection of the prostate (TURP) and two who presented in acute urinary retention. The 79 patients (median age 65 years, range 45-82) underwent pre-operative urodynamics and all patients completed American Urological Association (AUA) symptom score questionnaires before surgery. Visually-guided laser ablation of the prostate (VLAP) was performed and the urodynamics and symptom scores were repeated 3 months later. RESULTS: The improvements in symptom scores and flow rates were statistically significant and comparable with other published data. There were also significant improvements in voiding pressure. There were no significant differences between the various laser fibres used. There were few complications. CONCLUSION: The effectiveness of VLAP in improving symptoms and flow rates in patients with benign prostatic hyperplasia (BPH) is confirmed. The procedure also reduced voiding pressure, confirming the relief of bladder outflow tract obstruction. VLAP is confirmed as a safe and effective treatment for BPH. Continued follow-up is needed to determine the long-term effects. PMID- 7545057 TI - Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia. AB - Pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB), is a rare inherited disorder characterized by pyloric stenosis and blistering of the skin as primary manifestations. We demonstrate that in one PA-JEB patient the disease resulted from two distinct mutations in the beta 4 integrin gene alleles. The paternal mutation consists of a one base pair deletion causing a shift in the open reading frame, and a downstream premature termination codon. The maternal mutation occurs in a donor splice site, and results in in-frame exon skipping involving the cytoplasmic domain of the polypeptide. Our results implicate mutations in the beta 4 integrin gene in some forms of PA-JEB. PMID- 7545063 TI - Transrectal ultrasonography versus digitally guided prostate biopsies in patients with palpable lesions on digital rectal examination. AB - OBJECTIVE: To compare the efficacy of digital and ultrasonographic guidance of biopsies when there is a palpable lesion on the prostate. PATIENTS AND METHODS: Forty patients with a palpable nodule confined to a single lobe of the prostate underwent digitally guided biopsies of this lesion or biopsies directed by transrectal ultrasonography (TRUS) and/or systematic biopsies. RESULTS: According to the histopathological examination, 21 patients were found to have prostate carcinoma (PCa) using TRUS-guided biopsies, whereas digitally guided biopsies revealed PCa in 18 patients but did not detect three cases. In the study group, the prostate of 10 patients had a normal ultrasonographic appearance and two patients had PCa revealed by systematic biopsies. CONCLUSION: We suggest that in the presence of a palpable lesion in the prostate, regardless of TRUS findings, systematic biopsies are mandatory. Present data indicates that digitally guided biopsies are unnecessary as they do not provide additional information over systematic or TRUS-guided biopsies. PMID- 7545064 TI - Diabetes mellitus increases nitric oxide synthase in penises but not in major pelvic ganglia of rats. AB - OBJECTIVE: To determine the effect of diabetes mellitus (DM) on erectile function and evaluate the levels of nitric oxide synthetase (NOS) activity in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Rats were studied at 9 weeks and 14 weeks after the induction of DM by streptozotocin and compared with untreated control rats. Erectile potency was assessed physiologically by testing and recording mating behaviour. NOS activity was assayed in penile tissues and major pelvic ganglia (MPG) by conversion of [3H] L-arginine to [3H] citrulline. Histological, ultrastructural and immunohistochemical studies of penile tissues were performed in similar groups of rats. RESULTS: Diabetes mellitus adversely and significantly degraded all parameters of mating behaviour, thus indicating defective erectile potency. However, NOS activities in penile tissues from both groups of diabetic rats were significantly higher than those in controls (P < 0.01). In MPG, NOS activities were not significantly different between diabetic and control rats (P > 0.05). Histological, ultrastructural and immunohistochemical studies of penile tissues revealed no significant differences between control and diabetic rats, indicating an intact effector organ (smooth muscles) in rats with up to 14 weeks of DM. CONCLUSION: The impotence frequently observed in diabetic subjects would suggest that despite the increase in NOS activity in the penis, the pharmacological action of nitric oxide is impaired. PMID- 7545065 TI - Primary seminoma of the prostate. PMID- 7545069 TI - Challenging the predictors. PMID- 7545066 TI - A new flavor in phosphotyrosine recognition. AB - The phosphotyrosine-binding (PTB) domain, recently identified in a number of proteins, specifically recognizes tyrosine-phosphorylated sequences in other proteins. Although similar in function to the well-studied SH2 domain, the PTB domain appears to be structurally unrelated. PMID- 7545067 TI - Brain-derived neurotrophic factor works coordinately with partner molecules to initiate tyrosine hydroxylase expression in striatal neurons. AB - Previous studies demonstrated that the cooperative interaction of acidic fibroblast growth factor (aFGF) and a partner molecule could induce the novel expression of the catecholamine (CA) biosynthetic enzyme, tyrosine hydroxylase (TH) in striatal neurons [Du and Iacovitti, J. Neurosci., in press; Du et al., J. Neurosci., 14 (1994) 7688-7694; Iacovitti et al., submitted]. The present study demonstrates that in addition to aFGF, brain-derived neurotrophic factor (BDNF) is also capable of moderate levels of TH induction (30% TH+ striatal neurons) when administered at high concentrations (100 ng/ml). As with aFGF, BDNF's activity depended on its coupling to an appropriate partner molecule; the most potent of which were 10 microM dopamine (DA) and 50 microM mazindol. BDNF + DA induced TH expression was first evident after at 12 h; peaked by 18 h and declined by 4 days in culture. Cyclohexamide eliminated nearly all and alpha amanitin reduced by half the TH induction elicited by DA and BDNF; indicating that both de novo transcription and translation were required for increased expression. In contrast with aFGF and BDNF, other putative dopamine differentiation factors, such as glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), were able to elicit barely detectable (10%) levels of TH induction, regardless of the partner molecule used. These studies suggest that aFGF and/or BDNF may work coordinately with partner molecules to initiate TH expression; while a number of factors including, CNTF and GDNF, may be involved in its subsequent modulation. PMID- 7545070 TI - A slippery grip. PMID- 7545071 TI - RNA origami. PMID- 7545073 TI - An RNA pocket for an aliphatic hydrophobe. AB - We have isolated an RNA with specific affinity for the L-valine side chain, using selection-amplification. The active RNA secondary structure, identified by repeated selection, is a highly conserved asymmetric (4:10) internal loop adjacent to required G-U pairs. The binding free-energy per methylene is up to 1.5 kcal mol-1, and very dependent on group position. Amino acid binding is L stereoselective and distinguishes aliphatic sidechains by size and, given the same total size, by configuration. Though aliphatic-RNA interactions have frequently been neglected, their avidity and specificity seem sufficient for a biological role. PMID- 7545072 TI - Representation of the secondary and tertiary structure of group I introns. AB - Group I introns, which are widespread in nature, carry out RNA self-splicing. The secondary structure common to these introns was for the most part established a decade ago. Information about their higher order structure has been derived from a range of experimental approaches, comparative sequence analysis, and molecular modelling. This information now provides the basis for a new two-dimensional structural diagram that more accurately represents the domain organization and orientation of helices within the intron, the coaxial stacking of certain helices, and the proximity of key nucleotides in three-dimensional space. It is hoped that this format will facilitate the detailed comparison of group I intron structures. PMID- 7545068 TI - Role of nitric oxide in prevention of ethanol-induced gastric damage by CuNSN a copper-chelating compound. AB - CuNSN a bis (2-benzimidazolyl)thiother complex with copper, has been shown to prevent the formation of acute gastric mucosal lesions induced by acetylsalicylic acid and ethanol. In the present study we have investigated the role of NO in CuNSN protection from ethanol-induced gastric damage. For this purpose we have used the inhibitor of NO biosynthesis, NG-nitro-L-arginine (L-NNA) as well as L- or D-arginine. Gastric mucosal damage caused by ethanol was dose-dependently increased by i.v. administration of graded dose of L-NNA. The effect of L-NNA was completely antagonized by the administration of L-arginine while D-arginine did not cause a reduction in the damage. Treatment with CuNSN has shown a significant protection against the damage produced by ethanol. This protection was not reversed by L-NNA and was significant as compared to the corresponding control group. The combination of L-NNA plus L-arginine potentiates this protection. These results suggest that NO synthesis is not involved in the protection afforded by CuNSN. PMID- 7545074 TI - Structure-based drug design ten years on. PMID- 7545075 TI - Signalling an interest. PMID- 7545076 TI - Lessons from molecular matchmakers. PMID- 7545077 TI - Structure of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse nonnucleoside inhibitors. AB - We report the structure of HIV-1 reverse transcriptase (RT) complexed with the nonnucleoside inhibitor TIBO R 86183 at 3.0 A resolution. Comparing this structure with those of complexes of HIV-1 RT/alpha-APA R 95845 and HIV-1 RT/nevirapine provides a basis for understanding the nature of nonnucleoside inhibitor binding, the structure of the binding site and the interactions between the bound inhibitors and surrounding amino acid residues as well as for understanding mechanisms of inhibition by and resistance to nonnucleoside inhibitors. All three inhibitors considered assume a similar butterfly-like shape and bind to HIV-1 RT in a very similar way. Important differences occur in the conformation of amino acid residues that form the binding pocket. PMID- 7545078 TI - Human antibody reactivity against the lower matrix protein (pp65) produced by cytomegalovirus. AB - The lower matrix protein (pp65) is a major product of many laboratory strains of cytomegalovirus (CMV). It is thus an integral part of many CMV serological assays based on native antigen. Recombinant fragments of pp65 have previously been investigated for their usefulness in more-defined assays. The latter antigens have, however, failed to develop a positive response with serum samples derived from a substantial number of infected individuals. Here we show that the human humoral immune response to CMV pp65 is highly diverse and recognizes at least seven distinct but in some cases partly overlapping epitopes. Most of these epitopes could not be mimicked by any of the investigated recombinant or synthetic antigens. Furthermore, when we investigated the ability of human CMV seropositive serum samples to block the reactivity of pp65-specific antibodies recognizing five different epitopes within pp65, it was evident that several sera did not contain significant levels of antibodies against any of these or overlapping structures. It was thus concluded that the antibody response against CMV pp65 is weak in some CMV-infected individuals, making this antigen unsuitable for use alone in serological screening systems for CMV infection. PMID- 7545079 TI - Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies. AB - We compared flow cytometric immunophenotyping results obtained by using the lysed whole blood method of sample preparation with those obtained by using Ficoll Hypaque-separated cells on 44 consecutive specimens from patients with various hematologic malignancies. When the samples were analyzed as a group, seven antigens (CD2, CD3, CD5, CD11c, CD20, CD22, and CD34) demonstrated significantly different percentages of positively staining cells. When the samples were grouped by disease, results for patients with acute lymphocytic leukemia were discordant for CD22 and HLA-DR and results for patients with hairy cell leukemia were discordant for CD34. Most of the differences, however, were not with antigens critical to the evaluation of the malignancy. Additionally, the most frequent reason for differences in the percentage of positive cells was due to isotype control-based placement of the quadrant markers and not an actual discrepancy in staining. However, analysis of the CD34 antigen yielded eight instances in which staining of Ficoll-Hypaque-separated cells was essentially negative, but a clearly positive population was evident with the lysed preparation. This finding has important implications because of the prognostic significance of this antigen. Further studies are needed to determine the cause of this phenomenon. PMID- 7545080 TI - Infection with human T-lymphotropic virus types I and II results in alterations of cellular receptors, including the up-modulation of T-cell counterreceptors CD40, CD54, and CD80 (B7-1). AB - To examine the phenotypic alterations associated with human T-lymphotropic virus types I and II (HTLV-I and -II) infection, long-term cell lines (n = 12 HTLV-I cell lines; n = 11 HTLV-II cell lines; n = 6 virus-negative cell lines) were analyzed for the cell surface expression of various lineage markers (i.e., myeloid, progenitor, and leukocyte), integrin receptors, and receptor counterreceptor (R-CR) pairs responsible for cellular activation. As expected, all cell lines expressed the markers characterizing the leukocyte lineage (CD43, CD44, and CD53). Of the progenitor-myeloid markers examined (CD9, CD13, CD33, CD34, and CD63), only the percent expression of CD9 was significantly increased on HTLV-I and -II-infected cell lines as compared with that on virus-negative cell lines. Analysis of the beta 1 integrin subfamily (CD29, CD49b, CD49d, CD49e, and CD49f) showed no significant change, except that CD49e was significantly decreased on the HTLV-infected cell lines. For the beta 2 integrin subfamily, the cell surface density was increased for CD18 and CD11a, while the CD11c molecule was expressed exclusively on the HTLV-I- and HTLV-II-infected cell lines. Analysis of several R-CR pairs (CD2-CD58, CD45RO-CD22, CD5-CD72, CD11a-CD54, gp39 CD40, and CD28-CD80) demonstrated that comparable levels of expression of the Rs (CD2, CD45RO, CD5, and CD28) and of some of the CRs (CD58, CD22, and CD72) were in all cell lines; however, CD54, CD40, and CD80 were expressed constitutively on the HTLV-I- and HTLV-II-infected cell lines. Functionally, the expression of these R-CR pairs did not appear to affect the autologous proliferation since monoclonal antibodies to these R-CR pairs were not able to inhibit proliferation of the infected cell lines. Taken together, our results indicate that HTLV-I and II can modulate the expression of several T-cell activation molecules and CRs normally expressed on alternate cell types. PMID- 7545082 TI - Pain management now part of standards for care in cancer centres. PMID- 7545083 TI - Palliation of bone pain in patients with metastatic cancer using strontium-89 (Metastron). AB - Bony metastasis is the most common cause of cancer pain. Strontium-89 (Sr-89), or Metastron, therapy has been shown to be effective for the palliation of pain due to skeletal metastases. By reducing opioid analgesics intake and restoring mobility, Sr-89 improves the patient's quality of life. Sr-89 is given conveniently as an outpatient procedure, and when necessary it can be repeated at 3-month intervals. Sr-89 is useful as an adjunct to local external beam radiation (EBR) because Sr-89 will target all skeletal metastases, including those not included in the EBR field. Because Sr-89 is a beta-emitting radionuclide with a long physical half-life (50.5 days), precautions should be taken by the caretaker(s) against Sr-89 contamination from the patient's blood or excretions, particularly if the patient is incontinent. PMID- 7545081 TI - Characterization of the recognition site and diagnostic potential of an enterovirus group-reactive monoclonal antibody. AB - The epitope for a monoclonal antibody reactive against different enteroviruses was characterized. The epitope could be located to residues 40 to 48 of VP1, and the most-essential residues for monoclonal antibody binding coincided with those conserved among the majority of known enteroviruses, indicating a high diagnostic potential. PMID- 7545084 TI - Monoclonal antibody BW835 defines a site-specific Thomsen-Friedenreich disaccharide linked to threonine within the VTSA motif of MUC1 tandem repeats. AB - mAb BW835 (IgG1) has been generated to breast cancer cell lines by alternating injections of MCF-7 or SW-613 cells and has been demonstrated to be of value in the serodiagnosis of mammary carcinoma. BW835 defines a carbohydrate epitope on integrated or secreted MUC1 glycoforms from carcinoma cells and human milk. To identify BW835-reactive glycopeptides on MUC1, proteolytic fragments of the mucin obtained by digestion with the Gly-C-specific endopeptidase IV from papaya corresponding to low molecular mass fragments (< 10 kilodaltons) of the tandem repeat domain were screened. A glycosylated fragment (glycopeptide 17) containing the mAb HMFG-2-defined epitope was highly reactive to BW835 antibody, while nonglycosylated tandem repeat peptide TAP25 or its in vitro-glycosylated N acetylgalactosamine (GalNAc) derivatives were unreactive. Glycopeptide 17 bound to peanut agglutinin and to a Thomsen-Friedenreich antigen (TF alpha)-specific mAb (A78-G/A7). Binding of BW835 to glycopeptide 17 or to MUC1 was competitively inhibited by peanut agglutinin and by the synthetic glycopeptides TF alpha Ser or TF alpha Thr but not by their beta-anomers. Evidence for site specificity of binding by BW835 to glycopeptide 17 was revealed by demonstrating nonreactivity of the antibody to other TF alpha-expressing glycoproteins with peptide moieties lacking MUC1-specific motifs at putative glycosylation sites. The epitope of BW835 was localized to threonine within the VTSA-peptide motif by site-specific enzymatic beta-galactosylation of the synthetic tandem repeat peptide TAP25 GalNAc1 TAPPAHGVT(-O-alpha GalNAc)SAPDTRPAPGSTAPPA. This is the first report on a TF alpha-specific mAb that shows a strict peptide sequence dependency of binding. PMID- 7545086 TI - Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. AB - CD3+ peripheral blood T lymphocytes were evaluated for expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen and potent angiogenic factor. VEGF mRNA expression was confirmed in CD3+ cells and Jurkat cells, a human T-cell line, by reverse transcription-PCR and in CD4+ and CD8+ T cell subtypes by Northern blot hybridization. Steady-state levels of VEGF mRNA were inducible in CD3+ T cells by hypoxia, a known inducer of VEGF mRNA accumulation. Secreted VEGF was detected in CD4+ and CD8+ T cell- and Jurkat cell conditioned medium, indicating that T lymphocytes are capable of exporting bioactive concentrations of VEGF into the extracellular space. Human prostate and bladder cancers (prostatic adenocarcinoma and transitional cell carcinomas) were evaluated for VEGF mRNA expression by in situ hybridization. Tumor-infiltrating lymphocytes (TIL), identifiable immunocytochemically as T cells, along with tumor cells in these cancers, expressed VEGF mRNA. TIL in bladder cancers could be labeled with a specific anti-VEGF mAb, indicating that TIL are likely to be able to secrete VEGF protein in situ at bioactive concentrations. The finding that peripheral T cells and TIL in human tumors synthesize a factor known to be a specific mediator of neovascularization suggests a role for T lymphocytes as cellular effectors of angiogenesis. PMID- 7545088 TI - Effects of anti-nuclear factor kappa B reagents in blocking adhesion of human cancer cells to vascular endothelial cells. AB - Transcription factor nuclear factor kappa B (NF kappa B) controls gene expression of a number of genes including cell adhesion molecules such as E-selectin, intercellular adhesion molecule 1, and vascular adhesion molecule 1. These cell adhesion molecules are known to play important roles in a critical step of tumor metastasis, arrest of tumor cells onto the venous or capillary bed of the target organ. NF kappa B is activated by extracellular signals such as those elicited by proinflammatory cytokines, tumor necrosis factor and interleukin 1 (IL-1). Here we demonstrate that IL-1 beta induces nuclear translocation of NF kappa B in human umbilical vein endothelial cells, followed by induction of cell surface expression of E-selectin, intercellular adhesion molecule-1, and vascular adhesion molecule 1, and subsequently augments adhesion of those cancer cells expressing sialyl Lewis X antigen, a ligand to E-selectin. We have also demonstrated that the adhesion of tumor cells to IL-1 beta-treated human umbilical vein endothelial cells can be inhibited by anti-NF kappa B reagents such as N-acetyl L-cysteine, aspirin, or pentoxifylline. These observations indicate the involvement of NF kappa B in cancer metastasis and the feasibility of using anti-NF kappa B reagents in preventing metastasis. PMID- 7545091 TI - [Experience in establishing special multiple-function demonstration rooms]. PMID- 7545087 TI - CD30 ligand signal transduction involves activation of a tyrosine kinase and of mitogen-activated protein kinase in a Hodgkin's lymphoma cell line. AB - CD30 is a transmembrane receptor of the nerve growth factor/tumor necrosis factor receptor superfamily. Its expression associated with Hodgkin's lymphoma and a subset of non-Hodgkin's lymphoma. Recently, its ligand (CD30L) has been cloned. CD30L enhances the proliferation of peripheral T cells and the Hodgkin's cell line HDLM-2 but seems to exert antiproliferative effects on large cell anaplastic lymphoma cell lines. Since tyrosine kinases are critical regulators of cell growth, we investigated whether CD30L induced changes in cellular tyrosine phosphorylation in CD30-positive lymphoma cell lines. Stimulation with CD30L or with an agonistic mAb against CD30, M44, induced a rapid, transient, and concentration-dependent tyrosine phosphorylation of a cytosolic protein of M(r) 42,000 (p42) in the Hodgkin's lymphomas cell line HDLM-2 but not in other CD30 positive lymphomas. In HDLM-2 cells, the phrobol ester phorbol 12-myristate 13 acetate also stimulated tyrosine phosphorylation of p42, and this effect was enhanced by M44. In marked contrast, agents stimulating the protein kinase A pathway, like forskolin or dibutyryl cAMP, did not affect tyrosine phosphorylation of P42. By immunoprecipitation with mAbs against mitogen activated protein kinase (MAPK; p42ERKII), a M(r) 42,000 protein was identified which comigrated with p42 on SDS gels and which was phosphorylated on tyrosine residues in response to stimulation of CD30. Immune complex kinase assays showed that M44 mAb induced the activation of MAPK (p42ERKII) and the phosphorylation of a MAPK substrate, myelin basic protein. Taken together, the results suggest that CD30L induces the tyrosine phosphorylation and activation of the MAPK p42ERKII isoform in HDLM-2 cells. These findings may have implications for the understanding of the pathogenesis of Hodgkin's disease. PMID- 7545089 TI - Alterations of cell surface carbohydrates and inhibition of metastatic property of murine melanomas by alpha 1,3 galactosyltransferase gene transfection. AB - Three melanomas of C57BL/6 mice (BL6, JB/MS, and JB/RH) share several phenotypic properties. All these cells contain melanoma-specific ecotropic C-type retrovirus that encodes melanoma-associated antigen recognizable by MM2-9B6 mAb. They do not express H-2Kb molecules, and the alpha-galactosyl epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) they fail to react with soybean agglutinin (SBA), peanut agglutinin (PNA), and vicia villosa (VV) lectins. Previously, we found that failure of BL6 melanoma cells to express alpha-galactosyl epitopes is due to down-regulation of alpha 1,3 galactosyltransferase (alpha 1,3GT) gene expression. To evaluate the possible role of alpha-galactosyl cell membrane carbohydrates in regulation of metastatic properties, individual clones isolated from BL6, JB/MS, and JB/RH melanomas were transfected with alpha 1,3GT cDNA. This resulted in appearance of alpha-galactosyl epitopes, as well as of carbohydrates reacting with SBA, PNA, or VV lectins, but did not affect expression of H-2 class I molecules or melanoma associated antigen. Appearance of SBA, PNA, and VV lectin binding carbohydrates in the alpha 1,3GT gene-transfected melanoma cells is a result of reduction of cell membrane sialylation and unmasking of these carbohydrates. Reduction in cell membrane sialylation in the alpha 1,3GT gene-transfected melanoma cells is probably due to the competition between alpha 1,3GT with alpha 2,3 sialyltransferase or alha 2,6 sialyltransferase for the common acceptor N acetyllactosamine in the Golgi apparatus. As a result of this competition, cell membranes of alpha 1,3GT gene-transfected melanoma cells became galactosylated and less sialylated. In parallel with alteration of cell membrane carbohydrates, transfection of the alpha 1,3GT gene leads to the loss of metastatic properties of the transfected melanoma cells in the immunocompetent and immunosuppressed C57BL/6 mice. Thus, the use of specific glycosyltransferase cDNA transfection presents direct experimental confirmation of the importance of cell membrane carbohydrates in the regulation of metastatic properties of tumor cells. PMID- 7545085 TI - Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through immunoconjugate formation. AB - Bis-indolyl-(seco)-1,2,9a-tetrahydrocyclopropa[c]benz[e]indol-4-on e compounds are synthetic analogues of CC-1065 that are highly cytotoxic toward a broad spectrum of tumor cell lines. One of these compounds, called DC1, was conjugated to antibodies via novel cleavable disulfide linkers. Conjugates of DC1 with murine mAbs anti-B4 and N901 directed against tumor-associated antigens CD19 and CD56, respectively, proved to be extremely potent and antigen selective in killing target cells in culture. DC1 conjugates with humanized versions of anti B4 and N901 antibodies were also constructed and demonstrated to be as cytotoxic and selective as the respective murine antibody conjugates. The anti-B4-DC1 conjugate showed antitumor efficacy in an aggressive metastatic human B-cell lymphoma survival model in SCID mice and completely cured animals hearing large tumors. Anti-B4-DC1 was considerably more effective in this tumor model than doxorubicin, cyclophosphamide, etoposide, or vincristine at their maximum tolerated doses. PMID- 7545092 TI - Clinical evaluation of a monoclonal antibody to serum KM01 for the diagnosis of hepatocellular carcinoma. AB - In order to evaluate a monoclonal antibody KM01 which was developed in mice immunized against a human colon carcinoma cell line, serum levels of KM01 and other tumor markers were studied in patients with both hepatocellular carcinoma and liver cirrhosis and in patients with liver cirrhosis alone. The KM01 levels in the sera of 50 patients with hepatocellular carcinoma plus liver cirrhosis and 50 patients with liver cirrhosis were measured using an enzyme immunoassay method and compared with various tumor markers including alpha-fetoprotein (AFP), DUPAN 2, and protein induced vitamin K absence or antagonist-II (PIVKA-II). The mean serum level (+/- S.D.) and sensitivity of KM01 in patients with hepatocellular carcinoma plus liver cirrhosis were 734 (+/- 716) units/ml and 64%, respectively, and they were significantly higher than those of liver cirrhosis patients (P < 0.001). Three out of 9 cases showing negative serum AFP levels had positive serum KM01 levels. Although the sensitivity of serum KM01 level for hepatocellular carcinoma was inferior to serum AFP and plasma PIVKA-II values, the sensitivity of a combination assay of serum KM01 or AFP was increased to 88%. Clinical data of the patients with markedly elevated serum KM01 levels (more than 1000 units/ml) were compared with patients with moderately elevated levels (530-1000 units/ml); serum bilirubin and alkaline-phosphatase were statistically higher in the former group (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545090 TI - Possible regulation of capacitative Ca2+ entry into colonic epithelial cells by NO and cGMP. AB - A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry. PMID- 7545095 TI - Elevation of serum soluble vascular cell adhesion molecule-1 (sVCAM-1) levels in bronchial asthma. AB - We have previously shown the elevation of serum soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) in patients with bronchial asthma during asthma attacks. In the present study, we extended our earlier study by measuring serum sVCAM-1 levels by ELISA in 45 patients with bronchial asthma (23 atopic and 22 non-atopic) during asthma attacks and in stable conditions in order to assess further the state of adhesion molecules in allergic inflammation of bronchial asthma. The levels of sVCAM-1 in sera obtained during bronchial asthma attacks were higher than those in sera obtained in stable conditions. These findings were observed regardless of atopic status. To examine the regulatory mechanism in the elevation of serum sVCAM-1 levels, serum tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA. TNF-alpha levels in sera obtained during bronchial asthma attacks were higher than those in sera obtained in stable conditions. The nature of change in serum TNF-alpha levels correlated with the nature of change in serum sVCAM-1 levels, but serum TNF-alpha levels did not correlate with serum sVCAM-1 levels. These results suggest that higher levels of sVCAM-1 during asthma attacks may reflect the up-regulation of VCAM-1 expression in allergic inflammation, and that a soluble form of VCAM-1 molecules may be useful markers for the presence of allergic inflammation. TNF alpha is shown to enhance the expression and release of VCAM-1 in vitro, however; the regulatory mechanism in the elevation of serum sVCAM-1 levels remains to be clarified. PMID- 7545094 TI - Reduced antigen-presenting function of human Epstein-Barr virus (EBV)-B cells and monocytes after UVB radiation is accompanied by decreased expression of B7, intercellular adhesion molecule-1 (ICAM-1) and LFA-3. AB - In this study, the effect of ultraviolet-B (UVB) radiation on antigen-presenting function was studied, to investigate whether antigen-presenting cells (APC) are inhibited by UVB through a common mechanism. Two types of human APC were used: EBV-B cells and monocytes, and these were irradiated in vitro with single low doses of UVB (range 0-200 J/m2). Irradiation of EBV-B cells or monocytes resulted in similar dose-dependent reduction in APC function, when determined by the allogeneic mixed leucocyte reaction (MLR) or Candida albicans- or tetanus toxoid specific T cell response. Our study shows that the reduced APC function was not likely to be caused by alterations in antigen processing or cytokine production. However, UVB-irradiated APC displayed marked changes in adhesion molecule expression. Irradiated EBV-B cells showed reduced expression of ICAM-1 (30%), LFA 3 (25%) and B7-1 (35%), while expression of HLA-DR, CD19 and LFA-1 was not affected. UVB irradiation of monocytes did result in reduction in the expression of HLA-DR (30%), LFA-3 (40%), ICAM-1 (65%) AND B7-1 and B7-3 (90%), but had no effect on CD14, LFA-1 and ICAM-3 expression. Addition of non-irradiated cells (but not the supernatant of these cells) or CD28 antibodies partly restored T cell activation, indicating that UVB-induced reduction in APC function is at least partly mediated via impairment of co-stimulatory molecule expression. PMID- 7545093 TI - Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and macrophage inflammatory protein (MIP)-1 beta production by synovial T cells. AB - Earlier studies from this laboratory provided evidence for restricted cytokine expression in the T cell population in RA tissues. Specifically, IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) gene expression levels were low. The selective chemoattractant and activation effects of chemokines on leucocytes identify them as potentially ideal candidates in mediating selective inflammatory processes in RA. Accordingly, we undertook studies to examine constitutive chemokine gene expression in RA tissues. RANTES, monocyte chemotactic protein-1 (MCP-1) and MIP 1 beta gene expression was examined in both the T and non-T cell populations in RA peripheral blood (PB), synovial fluid (SF) and synovial tissues (ST). Our results identified elevated levels of both RANTES and MIP-1 beta gene expression in circulating RA PB and SF T cells. By contrast, MCP-1 expression was virtually absent in RA PB, yet elevated MCP-1 mRNA levels were detected primarily in the non-T cell populations of the SF and ST samples. Histological examination of affected rheumatoid joints revealed extensive RANTES and MIP-1 beta expression in sites of lymphocyte infiltration and cell proliferation, namely the synovial lining and sublining layers. Fractionation or RA ST patient samples revealed that RANTES expression was restricted to the T cells, whereas MIP-1 beta expression was detected in both T and non-T fractions. These data suggest that MCP-1, MIP-1 beta and RANTES may have a central role in the trafficking of reactive molecules involved in immunoregulation and in the inflammatory processes in RA. PMID- 7545096 TI - Depression in caregivers of demented patients is associated with altered immunity: impaired proliferative capacity, increased CD8+, and a decline in lymphocytes with surface signal transduction molecules (CD38+) and a cytotoxicity marker (CD56+ CD8+). AB - Changes in relevant immune parameters, including function, were found to be associated with depression in elderly caregiver wives of demented patients. We studied the relationship between immune cell phenotype and T cell proliferative capacity of such caregivers to levels of stress and depression over the course of a support group intervention. The data indicate the strongest association between depression (of all stress parameters) and impaired T cell proliferative capacity. Depression was also most strongly (of stress parameters) associated with a shift in T cell populations with an increase in CD8+ T cells, and a reduced percentage of CD38+ cells in both CD8+ and CD4+ T cell populations. Since CD38 is a signal transduction factor, it was interesting that a decreased percentage of CD38+ cells correlated with impaired T cell function (proliferation). Another significant difference was the reduction in natural killer (NK) cells as well as the percentage of the CD56+ component of the CD8+ population. This latter subset is important in MHC-unrestricted cytotoxicity, and has been found expanded in healthy centenarians. This study shows that both chronic stress, and depression in particular, and age have deleterious effects on T cells, and together could significantly contribute to the higher risk of disease and mortality associated with being a caregiver of a demented individual. PMID- 7545099 TI - Insulin-like growth factor (IGF)-binding proteins inhibit the smooth muscle cell migration responses to IGF-I and IGF-II. AB - Smooth muscle cells have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the role of IGF-binding proteins (IGFBPs) in this process has not been determined. As IGFBPs are synthesized and secreted by smooth muscle cells and bind to IGFs with high affinity, this study was undertaken to determine the ability of IGFBP-1 and -2 to modulate cellular migration in response to IGF-I and -II. Confluent monolayers of porcine vascular smooth muscle cells were wounded with a razor blade. After wounding, IGF-I and -II induced increases of 159 +/- 49% (mean +/- SD) and 108 +/- 33%, respectively, above control values in the number of cells migrating over a fixed distance in 4 days. The addition of IGFBP-1 caused a 19-21% inhibition of IGF-I- or IGF-II-stimulated migration, whereas the addition of IGFBP-2 inhibited the effect of both by greater than 60%. The addition of IGFBP-2 alone had no effect, whereas IGFBP-1 addition was associated with a 42 +/- 12% increase. In contrast, [Trp221]IGFBP-1 in which the RGD sequence was changed to WGD, thus eliminating its capacity to bind to the alpha 5 beta 1 integrin, inhibited IGF-I-stimulated migration by 67 +/- 17%. An IGF analog that has a reduced affinity for IGFBP-2-stimulated migration equally well as IGF-I alone even in the presence of IGFBP-2. Likewise, the addition of insulin, which cannot bind to IGFBPs, at supraphysiological concentrations that are adequate to activate the IGF-I receptor resulted in a similar increase in migration. In summary, IGF-I and -II stimulate smooth muscle cell migration after wounding. This migratory response is modulated by IGFBPs. Both IGFBP-1 and IGFBP-2 appear to neutralize the effects of the IGFs by inhibiting their interaction with IGF receptors, but IGFBP-1 also has a direct stimulatory effect that requires an intact RGD integrin recognition sequence. PMID- 7545097 TI - Defective functional response to membrane stimuli in lymphocytes from patients with benign prostatic hyperplasia. AB - Benign prostatic hyperplasia (BPH) is a local disturbance in the prostate that may involve an inflammatory infiltrate predominantly composed of activated lymphocytes and macrophages. The activation and proliferative response of T lymphocytes to different mitogenic signals has been analysed in peripheral blood mononuclear cells (PBMC) from 45 patients with BPH and 55 healthy controls. The PBMC obtained from the patients showed a significant specific impairment in proliferation, CD25 expression and IL-2 production in response to stimulation with lectins (phytohaemagglutinin (PHA), concanavalin A (Con A)), that was not corrected by the addition of IL-2 or of phorbol esters (phorbol myristate acetate (PMA)). Also, the CD28 response was defective in patient PBMC. Activation with anti-CD3 or anti-CD2 MoAbs was normal, but the addition of PMA to these stimuli provoked a significant defective response. Only the use of transmembrane stimuli (PMA and ionomycin) elicited responses similar to those found in the control group. The results indicate that peripheral T lymphocytes from BPH patients show a functional impairment that is mainly explained by an alteration of membrane signals (PHA, CD28) and is distal to protein kinase C (PKC) activation. PMID- 7545098 TI - A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA. AB - Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL 60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment. PMID- 7545100 TI - Hypothalamic nitric oxide synthase gene expression is regulated by thyroid hormones. AB - We investigated the effects of thyroid status on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). Propylthiouracil (PTU)-induced hypothyroidism in male rats produced a highly significant reduction in NOS gene transcripts in the PVN and SON, as assessed by quantitative in situ hybridization histochemistry with a specific oligodeoxynucleotide probe. The addition of T3 (40 micrograms/kg) to the PTU containing diet completely prevented the reduction in NOS transcripts. Hyperthyroidism, induced by adding 160 micrograms/kg T3 to the food, more than doubled the prevalence of NOS transcripts in the PVN and SON after a similar time. Up-regulation of NOS gene transcripts induced by the osmotic stimulus of chronic salt loading was markedly attenuated by PTU-induced hypothyroidism. These results demonstrate a major effect of thyroid status on regulation of NOS gene expression in the hypothalamus. PMID- 7545101 TI - Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells. AB - GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP 2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545102 TI - Regulation of inducible nitric oxide production by cytokines in human thyrocytes in culture. AB - We now report on the induction and modulation of NO production by cytokines in primary cultures of human thyrocytes and the effect of NO on iodine organification by human open thyroid follicles in the process of thyroid hormone biosynthesis. Although unstimulated thyrocytes produced little NO (measured as nitrite), interleukin-1 alpha or beta (IL-1 alpha/beta) substantially increased NO formation. Interferon-gamma (IFN-gamma) by itself failed to stimulate NO formation but markedly increased the IL-1-stimulated NO production. Tumor necrosis factor-alpha alone did not induce NO production but did so slightly in the presence of IFN-gamma. Induction of NO formation by thyrocytes upon stimulation with IL-1 alpha + IFN-gamma was accompanied by the synthesis of tetrahydrobiopterin (BH4), an obligatory cofactor of NOS. Coinduction of NO and BH4 synthesis in thyrocytes was preceded by coexpression of messenger RNAs for NOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme for de novo synthesis of BH4. NO synthesis was prevented by an inhibitor of GTPCH, 2,4 diamino-6-hydroxypyrimidine, and this inhibition was completely reversed by administration of sepiapterin, a substrate for BH4 synthesis via pterin salvage pathway. In contrast to IFN-gamma, some cytokines such as interferon-alpha, IL-4, and transforming growth factor-beta 1 inhibited the IL-1-induced NO production. Finally, a possible role of NO on thyroid hormone synthesis was investigated. Iodine organification by human open thyroid follicles was inhibited by two kinds of NO donor but not by cell permeable cyclicGMP analog. Thus, cytokines such as IL-1, IL-1/IFN-gamma, and tumor necrosis factor-alpha/IFN-gamma stimulate human thyrocytes to produce NO; this process can be modulated by other cytokines and coregulated with a cofactor BH4 biosynthesis, and resulting NO may affect cell function including thyroid hormone synthesis. PMID- 7545104 TI - Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining. AB - The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1 ATPase found in hypothyroid newborns. All of these changes were reversed by the administration of thyroid hormones. ATP in vitro promotes the conversion of HFP into LFP and increases the respiratory control ratio in hypothyroid newborns, although this effect was not observed after thyroid hormone treatment. The effect of thyroid hormones on both the postnatal changes in mitochondrial populations and in F0,F1-ATPase activity was prevented by cycloheximide, but not by streptomycin. Thus, the observed effects of thyroid hormones on neonatal mitochondria must be accomplished by the induction of the synthesis of some nuclei-coded protein, possibly involved in F0,F1-ATPase assembly. PMID- 7545103 TI - Expression of c-kit ligand messenger ribonucleic acids in human ovaries and regulation of their steady state levels by gonadotropins in cultured granulosa luteal cells. AB - The c-kit ligand (KL), a ligand for the c-kit protooncogene receptor tyrosine kinase, is an important regulator of germ cell development in rodent gonads. However, no information about the role of KL in the ovaries of women or higher primates has been available. We studied the expression of KL messenger RNA (mRNA) in human ovaries and the effect of purified hCG and recombinant human FSH (rhFSH) on KL mRNA steady state levels in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. KL complementary DNA was generated by reverse transcription-polymerase chain reaction from human ovarian tissue RNA. Two alternatively spliced KL transcripts encoding 248-amino acid (aa) and 220-aa membrane-associated KL proteins were observed in GL cells and ovarian tissue. In Northern blot analysis of human ovarian and GL cell RNA, a major transcript of approximately 6.0 kilobases was detected. Specific mRNA transcripts for KL were detected in dot blot filter hybridization analyses, and the steady state levels of these mRNAs were lowered in cultured GL cells by both gonadotropins in a distinct time- and concentration-dependent manner. The KL mRNA levels of untreated and hCG- or rhFSH-stimulated GL cells were determined at 2- to 3-day intervals between days 2-10 of culture. An 8-h treatment with hCG was shown to decrease KL mRNA levels on days 2, 3, 5, and 7 of culture, whereas rhFSH decreased KL mRNA levels on days 5 and 7 of culture. Time-course and concentration-dependence studies were performed on days 2-7 of culture. Both gonadotropins decreased KL mRNA levels as early as 2 h after treatment. The maximal response to hCG and rhFSH treatment was observed at 7-24 h. Concentration dependence studies performed 8 or 24 h after treatment indicated that the maximal inhibition occurred with 10-100 ng/ml hCG and 100-300 ng/ml rhFSH. We conclude that 1) the KL transcripts encoding 248- and 220-aa transmembrane proteins are expressed in vivo in the human ovary and in cultured human GL cells; and 2) KL transcript levels are rapidly decreased by gonadotropins in a time- and concentration-dependent manner in cultured GL cells. Thus, KL expression is hormonally regulated in human granulosa cells, and this growth factor may control the function of the ovarian follicle during the human menstrual cycle. PMID- 7545107 TI - Use of an expandable Ultraflex stent in the treatment of malignant rectal stricture. AB - Obstructive colorectal cancer is usually an indication for surgical intervention. For patients with metastatic disease, medical contraindications, or those who refuse surgery, palliation by nonoperative means is necessary. The advent of expandable metallic stents may allow for speedy palliation. We have treated one patient with unresectable colon cancer with a new selfexpanding endoprosthesis made of nitinol (Ultraflex, Microvasive, Watertown, MA, U.S.A.). PMID- 7545105 TI - Isolation of a preadipocyte cell line from rat bone marrow and differentiation to adipocytes. AB - A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3 adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and glycerol release in differentiated adipocytes, but not preadipocytes. In addition, differentiated adipocytes contain messenger RNA encoding the brown adipose-specific protein, thermogenin. Thus, this rat preadipocyte cell line can be differentiated into adipocytes that histologically and functionally resemble brown adipose tissue. PMID- 7545111 TI - Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen, APO 1)-mediated apoptosis. AB - CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class I mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one call line but was not promoted by the treatment with anti HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95 mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells. PMID- 7545106 TI - Contribution of L- and non-L-type calcium channels to voltage-gated calcium current and glucose-dependent insulin secretion in HIT-T15 cells. AB - The pharmacological properties of voltage-gated Ca current and glucose-dependent insulin secretion were determined using the HIT insulinoma line to understand the role of Ca channels in stimulus-secretion coupling. The L-type Ca channel antagonist nimodipine inhibited a maximum of 50-55% of the peak Ca current, suggesting that L- and non-L-type channels contribute to Ca current. The L agonist BAY K 8644 increased Ca current by 155%, whereas the N-channel blocker omega-conotoxin MVIIA reversibly blocked 35% of the peak Ca current. Total block with nimodipine and MVIIA was additive. Conotoxin MVIIC did not affect HIT Ca current. Prolonged depolarizations elicited rapidly and slowly inactivating Ca currents. Nimodipine partially inhibited transient current, but fully inhibited slowly inactivating current, suggesting that the former is mediated by L- and N channels, and the latter is mediated by L-channels. Like slowly inactivating Ca current, glucose-dependent insulin secretion was fully inhibited by nimodipine and insensitive to MVIIA. BAY K potentiated secretion and antagonized nimodipine block. These results suggest that persistent Ca current is mediated by L-channels and is strongly coupled to insulin secretion, whereas transient Ca current is mediated by L- and N-channels and is weakly coupled. Sustained Ca influx may be preferentially coupled because glucose persistently depolarizes HIT cells and inactivates more transient Ca channel pathways. PMID- 7545112 TI - Spreading of the immune response to different myelin basic protein peptides in chronic experimental autoimmune encephalomyelitis in B10.RIII mice. AB - B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89-101 (VHFFKNIVTPRTP). To investigate the basis for the chronicity of the disease, the subsequent development of an immune responses to other parts of the MBP protein were investigated. Onset of disease occurs 9-25 days after immunization with MBP89-101. T cell responses towards a series of MBP peptides were assessed in an enzyme-linked immunospot assay detecting single cells secreting IFN-gamma. There were responses not only to MBP89-101, but also towards peptides derived from sequences outside of MBP89-101. These peptides were of two kinds: those with sequences completely outside the 89-101 stretch of MBP; and those sharing a short sequence with MBP89-101 depending on alternative splicing of MBP mRNA. Immunization with these peptides also produced chronic EAE and a spreading of the immune response to other MBP peptides. Immunization with stepped peptides around the relevant region (MBP87-110) showed that peptides sharing a 6 amino-acid motif induced EAE after immunization. After MBP89-101 peptide immunization, T cells isolated from lymph nodes did not cross-react in vitro to the other peptides sharing this motif. We suggest that one mechanism for the development of relapses during the disease course is the recruitment of new T cells with specificity for MBP peptides not derived from the peptide used for immunization. This is the first time such a mechanism has been demonstrated in a chronic autoimmune disease model. PMID- 7545108 TI - Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites. AB - The NagC repressor controls the expression of the divergently transcribed nagE nagBACD operons involved in the uptake and degradation of the amino sugars, N acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS). PMID- 7545110 TI - The peripheral T cell repertoire: independent homeostatic regulation of virgin and activated CD8+ T cell pools. AB - Mature T cells may be produced in the thymus, or by expansion in the periphery. While thymus output of virgin cells ensures repertoire diversity, peripheral expansion increases the size of rare clones, and thus the efficiency of immune responses. We studied the role of both phenomena in the generation of the CD8+ T cell pool using RAG-/- female mice expressing a transgenic T cell receptor specific for the male antigen; nude mice injected with peripheral T cells; and euthymic irradiated chimeras injected with bone marrow and mature T cells. Our results show that the total number of virgin and activated T cells, each constituting about half of the peripheral T cell pool, was regulated independently, revealing an efficient mechanism to maintain repertoire diversity while optimizing the immune response. PMID- 7545113 TI - Characterization of mouse CD53: epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection. AB - The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4-8-thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4-8-thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25- subset, but down-regulated on the later CD44high25+, CD44low25+ and CD44low25- stages. Also, the subsequent transient TcR-/low CD4-8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the re-expression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2b/influenza nucleoprotein-peptide specific TcR, back-crossed onto an H-2q or H-2b background of RAG-2-deficient mice. CD4+8+ thymocytes from non-selecting H-2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H-2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histocompatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation. PMID- 7545109 TI - Growth regulation by cell shape alteration and organization of the cytoskeleton. AB - The correlation between cellular growth and microfilament-dependent morphology was investigated. It was found that the proliferative growth of various nontransformed cell lines does not only depend on cell adhesion to a suitable substratum and cell flattening but also on intact microfilaments. Disintegration of microfilaments by cytochalasin D (CD) as well as lactrunculin (LAT)-A and LAT B is correlated with a strong decline of the number of DNA-synthesizing cells during a period of 8 to 12 h after application of the drug. RNA and protein synthesis are reduced already after a preincubation time of 2 h. Although microinjected rhodamine-phalloin is colocalized with microfilaments there is no stabilizing effect against CD even at high phaloidin concentrations. Microinjection of DNAse I results in a strong alteration of the microfilament system. The disorganization of microfilaments was correlated with a moderate decrease of protein synthesis 2 h after microinjection, whereas RNA synthesis remained unchanged, if RNase-free DNase I was used. The number of DNA synthesizing cells was somewhat diminished 24 h after microinjection. Furthermore, the microfilament system is disorganized by microinjection of gelsolin and gelsolin segment 1 + 2, respectively. The severing the microfilaments by gelsolin is correlated with a significant restriction of RNA and protein synthesis during a period of 2 to 4 h after injection, but the labeling index remaining unchanged. Therefore, we assume that inhibition of the G0-G1-S transition is only caused by a disintegration of microfilaments lasting longer than 4 h. The significance of microfilament organization for growth regulation is discussed. PMID- 7545114 TI - Macrophages, but not dendritic cells, present collagen to T cells. AB - Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen alpha chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen. PMID- 7545115 TI - Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo. AB - Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')2 fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F.R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364:806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment. PMID- 7545117 TI - Schistosoma-specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2. AB - Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty-eight CD3+, CD4+, CD8- parasite-specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3+, CD4+, CD8- clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite-specific clones produced interleukin (IL) 4 and interferon (IFN)-gamma in large amounts. However, they generally produced more IL-4 than IFN-gamma; variations in IL-4/IFN-gamma ratios were accounted for by differences in IFN-gamma production since IL-4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL-4 and more IFN-gamma than parasite-specific T cell clones. Most clones produced IL-2, and IL-2 production did not differ between the two types of clones. Parasite-specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN-gamma and the lowest IL-4 producers, compared to those of resistant subjects. Thus, parasite specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL-4 than IFN-gamma (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN-gamma than IL-4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN-gamma is required for full protection. PMID- 7545116 TI - Characteristics of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells. AB - Dendritic cells (DC) are the main antigen-presenting cells for the initiation of primary T cell-mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen-independent manner. We studied the adhesion characteristics of human CD4+ T cells to DC generated from CD34+ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha. A majority of these cells had the morphology, phenotype and functions of DC. CD4+ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA-1/ICAM, ICAM/LFA-1, CD2/LFA-3 and CD28/CD80, were involved in the transient adhesion of DC to CD4+ T cells in antigen-independent and specific alloantigen-dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4+ T cells to antigen-presenting DC was stronger than that of resting CD4+ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA-1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4+ T cells to specific DC differed in part, since ICAM-3 on resting T cells and ICAM-1 on alloreactive T lymphocytes preferentially bound LFA-1. Studies of interactions between DC and phorbol ester activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4-mediated regulation of CD4+ T/DC adhesion was suggested by the observation that preincubation of CD4+ T cells and DC individually with anti-CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4+ T cells. PMID- 7545118 TI - Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases. AB - APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca2(+)-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40 kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a molecular mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn2(+)-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO 1L. PMID- 7545119 TI - Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines. AB - Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in single-gene-transfected tumors. However, T cells in B7 and IL 7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28+ and CD25-, while in IL 7 transfected tumors, T cells were mainly CD28- and CD25+. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28+ and CD25+. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity. PMID- 7545120 TI - The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the V chi Ox1 gene. AB - Priming mice with a chicken gamma globulin (CGG) carrier protein significantly accelerated the onset of somatic mutation in the V chi Ox1 gene when the mice were subsequently immunized with 2-phenyl-5-oxazolone (phOx) coupled to CGG. The first mutations were already detected 7 days after immunization, while in the true primary response, they are not apparent until day 10. It was also found that comparing the mutation pattern of V chi Ox1 genes from hybridomas derived after immunization with phOx coupled to different carriers revealed quite distinct patterns of somatic mutation. Analysis of hybridoma sequences from the primary immune response to phOx-ovalbumin showed that the codons for Ser29, Ser31 and Lys45 were hot-spots for somatic mutation. Thus, the frequency and pattern of somatic mutations in the V chi Ox1 gene depends on the available T cell help as well as on the complex structure of the immunizing antigen. PMID- 7545121 TI - Soluble interleukin-6 receptor strongly increases the production of acute-phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes. AB - Interleukin-6 (IL-6) interacts with a system of receptors, which include a 80-kDa IL-6-binding subunit (IL-6R) and a transducing element (gp130). The soluble form of IL-6R (sIL-6R) can bind its ligand and induce cellular responses by association with gp130, thus acting as an IL-6 agonist. We and others have previously shown that the responsiveness to IL-6 is different in hepatoma and human primary hepatocytes. We therefore compared the effects of sIL-6R on the two types of cells, and on the B9 hybridoma, another IL-6-sensitive cell line. Human primary hepatocytes, hepatoma cells PLC/PRF/5, and B9 cells were incubated with different concentrations of IL-6, sIL-6-R, or both. The hepatocyte culture supernatants were tested for their content of acute-phase proteins (APP). The proliferation of B9 cells was assessed by a colorimetric method. Results showed that sIL-6R alone markedly increased the production of APP by hepatoma cells in a dose-dependent manner, but affects only minimally primary hepatocytes and the proliferation of B9 cells. The combinations of IL-6R and its ligand enhanced the effects of Il-6 alone in both PLC/PRF/5 and B9 cells, but had no effect on primary hepatocytes. An immunohistochemical study indicated that the cell-surface expression of IL-6R was dramatically lower in hepatoma cells than in primary hepatocytes. In conclusion, our results show that the expression of IL-6R is low in the hepatoma cell PLC/PRF/5 when compared with primary hepatocytes and that this difference can, at least partly, explain their deficient responsiveness to IL-6. On the other hand, it appears that IL-6R expression by primary hepatocytes is sufficient and that circulating sIL-6R is unlikely to play a significant role in the modulation of IL6 effects. PMID- 7545122 TI - Activity of SR 142801 at peripheral tachykinin receptors. AB - The pharmacological profile of the novel tachykinin NK3 receptor antagonist SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl) 4-phenylpiperidin-4-yl)-N-methylacetamide), was studied at tachykinin NK1, NK2 and NK3 receptors, in several in vitro bioassays. In the guinea-pig isolated ileum longitudinal muscle preparation, SR 142801 (10 nM-1 microM) caused an insurmountable antagonism of tachykinin NK3 receptor-mediated contractions produced by senktide (apparent pKB = 9.27). The blockade induced by SR 142801 was essentially irreversible, since it was not removed by washout (up to 2 h) and was increased by prolonging the incubation from 15 to 120 min. SR 142801 showed similar antagonist potency at rat tachykinin NK3 receptors (portal vein) and rabbit tachykinin NK2 receptors (pulmonary artery) (pKB = 7.49 and 7.66, respectively), whereas it was distinctly less potent at hamster tachykinin NK2 receptors (trachea; pKB = 6.84) and inactive at guinea-pig tachykinin NK1 receptors (ileum, longitudinal muscle). In the guinea-pig whole ileum SR 142801 (100 nM) did not affect the contraction produced by capsaicin (1 microM). The combined SR 142801 pretreatment and tachyphylaxis of neuronal CGRP (calcitonin gene-related peptide) receptors produced a slight (about 25%), but significant reduction of the response to capsaicin, suggesting that tachykinin NK3 receptors play a minor role in capsaicin-induced neuronal excitation of afferent nerves in the guinea-pig ileum. PMID- 7545126 TI - Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation. AB - The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells. PMID- 7545128 TI - Polyomavirus middle T selective action on cytokeratin 14 gene expression in liver nonparenchymal epithelial cells. AB - We reported recently that liver nonparenchymal epithelial cells (LECs) constitute a small population of cells scattered throughout biliary structures and the Glisson's capsule, containing the unusual cytokeratin (CK) pair CK8/CK14 (Blouin et al., Differentiation, 1992, 52, 45). The transfection of polyomavirus middle T oncogene (MT) into the LEC line T51B leads to the loss of their CKs, due to a down-regulation of CK14 gene expression (Royal et al., Cell Growth Differ., 1992, 3, 589). In the present work, we examined CK gene expression at both mRNA and protein levels following polyomavirus small T oncogene (ST), MT, or large T oncogene (LT) transfection of T51B cells, MT transfection of rat hepatic cell lines containing different subsets of CKs, and MT transfection of rat keratinocytes. Immunofluorescence staining revealed that MT indeed induced an inhibition of CK14 gene expression and a loss of CK8/CK14 intermediate filaments (IFs) in liver cells, whereas ST and LT had no effect. Moreover, CK14 was the only CK gene whose expression was inhibited in MT-containing hepatic cells, in the sense that the expression of the CK7, CK8, CK18, and CK19 genes was not affected. Two-dimensional SDS-PAGE of the Triton-resistant cytoskeletal proteins and Northern blotting of the CK mRNA content confirmed these findings. The transfer of the MT oncogene into the keratinocytes did not result in the loss of CK5/CK14 IFs nor the inhibition of CK14 gene expression. These results show that the polyomavirus oncogene action on CK gene expression is restricted to an MT effect on CK14 in rat LECs. PMID- 7545127 TI - Cell-to-cell contact modulates the expression of the beta 1 family of integrins in primary cultures of thyroid cells. AB - The expression of the beta 1 family of integrins was studied in normal thyroid tissue cultures and monolayer cell cultures. The expression of the various subunits was measured by flow cytofluorometry with specific monoclonal antibodies and by Northern analysis. In monolayer cell cultures but not in tissue cultures, the expression of the alpha 3 subunit on the cell membrane progressively increased soon after plating, reaching a 30-fold higher intensity. The alpha 2 subunit, not detectable in native follicular cells, was expressed de novo and reached a remarkable high level. Up-regulation of alpha 2 and alpha 3 in monolayer cell cultures was serum-independent and preceded the expression of proliferating cell nuclear antigen, [3H]thymidine incorporation, and cell replication. Northern analysis demonstrated an increased level of beta 1 integrin mRNA. The increase of alpha 2 and alpha 3 was readily reversible since the expression of these molecules returned to a lower level when cultures reached a high cell density. Down-regulation did not occur until cell cultures were confluent. When cells from high cell density and low integrin expression were harvested and sparsely seeded in culture, up-regulation of integrins was observed again, while rapid reaggregation of isolated cells inhibited this phenomenon. Altogether these data suggest that cell-to-cell contact may regulate the expression of beta 1 integrins in thyroid primary cultures. PMID- 7545125 TI - The role of inducible nitric oxide synthase in vascular hyporeactivity of endotoxin-treated and portal hypertensive rats. AB - The involvement of the inducible nitric oxide (NO) synthase in the vascular hyporeactivity in portal vein-ligated rats was assessed in isolated perfused mesenteric arterial beds. Aminoguanidine, a selective inhibitor of the inducible NO synthase, restored the pressor responses to methoxamine in arteries of endotoxin-treated rats, but was ineffective in hyporeactive portal vein-ligated vessels. NG-Nitro-L-arginine methyl ester enhanced the responsiveness both in portal vein-ligated and sham-operated rats, without changing the difference between the two groups. These results not only indicate that the inducible NO synthase is not involved in the hyporeactivity to methoxamine in mesenteric arteries of portal hypertensive rats, but also suggest a role for factors other than NO. PMID- 7545124 TI - Role of thromboxane A2 in bradykinin-induced human isolated small bronchi contraction. AB - We previously demonstrated that the bradykinin-induced contraction of human isolated small bronchi is inhibited by indomethacin, capsaicin (N-methyl-N-6 nonenamide) and ruthenium red but not by tachykinin receptor antagonists. The thromboxane A2 receptor (TP receptor) antagonist GR32191 ((1R-(1 alpha(Z),2 beta,3 beta,5 alpha))-(+)-7-(5-(((1,1'-biphenyl)-4-yl)- methoxy)-3-hydroxy-2-(1 piperidinyl)cyclopentyl)-4-heptenoic acid, hydrochloride) (10(-10) to 10(-8) M) dose dependently inhibited the effect of bradykinin, suggesting the mediation of the TP receptor in the action of bradykinin. With higher concentrations of GR32191 (10(-7) and 10(-6) M) bradykinin induced a relaxation which was inhibited by indomethacin and by the bradykinin B2 receptor antagonist Hoe 140 (D Arg0[Hyp3,Thi-5,D-Tic7,Oic8]bradykinin). The thromboxane A2 synthase inhibitor dazoxiben (4-(-2-(1H-imidazol-1-yl)ethoxy) benzoic acid hydrochloride) 10(-6) M inhibited the bradykinin-induced contraction, suggesting that thromboxane A2 was involved in TP receptor stimulation. The thromboxane A2 mimetic U-46619 (9,11 dideoxy-11 alpha,9 alpha-epoxy-methano-prostaglandin F2 alpha)-induced contraction of human distal bronchi was not inhibited by capsaicin and ruthenium red. Our data suggest that bradykinin contracts human isolated small bronchi through thromboxane A2 release. The inhibitory effect of ruthenium red and capsaicin on the bradykinin response may be due to inhibition of thromboxane A2 release or arachidonic mobilisation. PMID- 7545130 TI - Induction of beta 1 integrin synthesis by recombinant platelet-derived growth factor (PDGF-AB) correlates with an enhanced migratory response of human dermal fibroblasts to various extracellular matrix proteins. AB - Cell migration plays a major role during wound healing and is tightly controlled by a variety of growth factors and extracellular matrix proteins. The experiments reported here have been designed to study whether defined beta 1 integrins are involved in the platelet-derived growth-factor-AB (PDGF-AB)-modulated migratory response to collagen type I and to fibronectin. Preincubation of fibroblasts with PDGF-AB resulted in an up to 2.5-fold increase in the migratory response to collagen type I as well as fibronectin and to enhanced synthesis and cell surface expression of the alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits. Function-blocking monoclonal antibodies against the common beta 1 integrin subunit dose-dependently inhibited the PDGF-AB-augmented migration of fibroblasts to collagen type I and fibronectin. The PDGF-AB-induced migration to collagen type I was also inhibited by antibodies against the alpha 2 integrin subunit, whereas the corresponding migration to fibronectin was almost completely blocked by the combined application of antibodies against the alpha 3 and the alpha 5 integrin subunits. Taken together, up-regulation of integrin synthesis and expression by human recombinant PDGF-AB correlate with an increase in the migratory response of dermal human fibroblasts to various extracellular matrix proteins and thus may contribute to an efficient regulation of cell migration during wound healing and tissue repair. PMID- 7545129 TI - Cell surface proteolysis by serine proteinases enhances RGD-sensitive melanoma cell adhesion on fibrinogen and vitronectin. AB - Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-medicated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 microM of trypsin, chymotrypsin, or plasmin for 30 min at 37 degrees C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate alpha v beta 3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against alpha v beta 3 integrin subunits by 70%, but not that of monoclonal antibody against alpha 2, alpha 3, or alpha 6 subunit, indicating that cell surface proteolysis exposed more alpha v beta 3 integrin on the cell surface. These results suggest that exposure of alpha v beta 3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin. PMID- 7545123 TI - Effects of nitric oxide availability on responses of spinal wide dynamic range neurons to excitatory amino acids. AB - The role of nitric oxide (NO) in responses of spinal dorsal horn neurons to excitatory amino acids and to cutaneous mechanical stimuli was examined. Extracellular recordings were made from wide dynamic range neurons excited with iontophoretically applied excitatory amino acid agonists, N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainic acid. Nitric oxide availability was decreased by iontrophoretic application of NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L NAME) or L-N5-(1-iminoethyl)ornithine (L-NIO), or elevated by the NO donating compound, S-nitroso-N-penicillamine (SNAP). When cells were excited with successive application of NMDA and non-NMDA excitatory amino acid receptor agonists, application of NO synthase inhibitors led to a decrease in responses to NMDA in 60% of neurons. In more than a third of the cells tested, inhibition of NO synthase caused reciprocal changes in responses to glutamate receptor agonists: NMDA-evoked responses were significantly decreased whereas responses to the non-NMDA receptor agonists (AMPA or kainic acid) were increased. Application of the NO donating compound, S-nitroso-N-penicillamine, revealed an opposite tendency, increasing responses to NMDA in more than half of the neurons tested. In approximately 40% of the cells, reciprocal changes in responses to excitatory amino acid receptor agonists of NMDA versus non-NMDA types were observed after application of S-nitroso-N-penicillamine, such that the increase in NMDA responses was accompanied by decreases in the responses to kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545132 TI - Nitric oxide and hypoxic pulmonary hypertension. PMID- 7545133 TI - Inhibitory effect of terfenadine, a selective H1 histamine antagonist, on alcoholic beverage-induced bronchoconstriction in asthmatic patients. AB - We wanted to evaluate the effect of terfenadine, a selective H1-receptor antagonist, on alcoholic beverage-induced bronchoconstriction. Eight patients with alcohol-induced asthma received terfenadine (60 mg, twice on the test day) or placebo, with the last dosing 2 h before the test in a double-blind, randomized, cross-over manner. On two separate study days, each subject drank the same brand and volume of alcoholic beverage (beer or Japanese sake), and bronchoconstriction was assessed as change in peak expiratory flow (PEF) over 120 min postchallenge. Inhalation challenges were performed with the same alcoholic beverage with which they had been orally challenged. The mean (SEM) percentage fall in PEF 15, 30, 45, 60, 90 and 120 min after the oral alcohol challenge was significantly reduced from 12.0 (3.1), 17.0 (1.7), 15.8 (2.3), 15.2 (3.4), 16.6 (4.8) and 14.7 (5.2)%, to 2.6 (1.8), 2.1 (1.6), 3.9 (1.2), 5.7 (2.2), 6.5 (2.6), 5.1 (1.6)%, respectively, by terfenadine. No significant bronchoconstriction was observed after the inhalation challenge. We conclude that the release of histamine makes a major contribution to alcoholic beverage-induced bronchoconstriction in Japanese asthmatic patients, and that histamine H1 antagonists may be effective in preventing alcoholic beverage-induced bronchoconstriction. PMID- 7545134 TI - The features of the spatial structure of the gramicidine A-cesium complex. AB - Earlier obtained two-dimensional 1H-NMR spectroscopy data were used to analyze the spatial structure and conformational mobility of the double right increases decreases pi pi 7.2 LD helix of the complex formed by gramicidine A and Cs+ ions in an organic solvent (a chloroform-methanol mixture). Analysis of the experimental data permitted the determination of a set of conformations for each of the high-mobility residue side chains in the solution. The energy refinement of the most probable conformation of the double right increases decreases pi pi 7.2 LD helix was made and conformational rearrangements of the tryptophan residue side chain were studied in detail. PMID- 7545131 TI - A chimeric proinsulin-CD5 protein expressed in AtT-20 cells is directed to the cell surface via the constitutive pathway. AB - A chimeric gene encoding mouse proinsulin fused to the transmembrane and the cytoplasmic domains of the CD5 antigen of human T lymphocytes was expressed in AtT-20 cells to assess the relative strength of signals that influence the sorting of secretory proteins to the regulated or constitutive pathway in endocrine cells. Transfected cells expressing the antigen at the surface were purified by fluorescence-activated cell sorting and analyzed by Northern and Western blots. They contained a mRNA of 1.4 kb hybridizing with an insulin cDNA probe and two immunoreactive insulin forms of 21 and 24 kDa, recognizable by antibodies against both insulin and C peptide. The surface density of these antigens was not increased following KCl stimulation of the cells, suggesting that they were not stored within the cells in significant amounts. This was confirmed by immunoelectron microscopy which showed the antigen attached to membranes, in the Golgi, in endosomes, and at the cell surface, but not in secretory granules. These results indicate that the proinsulin-CD5 fusion protein was transported to the cell surface via the constitutive pathway and partly recycled by endocytosis. They also suggest that the signals that direct proinsulin into storage granules may no longer be dominant when fused to transmembrane and cytosolic sequences derived from a constitutively secreted molecule. PMID- 7545135 TI - Self-association of plasma membrane Ca(2+)-ATPase by volume exclusion. AB - At enzyme concentrations above 40 nM the configuration of the purified plasma membrane Ca(2+)-ATPase is that of calmodulin-insensitive dimers. Dilution of the enzyme generates progressively higher proportions of calmodulin-sensitive monomers with lower Vmax and Ca2+ sensitivity than the dimeric enzyme. Dimerization from monomeric state had not been documented before. We investigated whether concentration by volume exclusion, obtained by addition of a large molecular weight dextran to a monomeric Ca(2+)-ATPase would elicit dimer-like behavior. Dextran induced self-association of monomers, as monitored by fluorescence energy transfer, but the Ca2+ sensitivity of the re-associated monomers was lower than that of the native dimers. These results suggest that the self-association reaction is structurally but not functionally reversible, and also document the existence of a hitherto unknown kinetic state of the oligomerized Ca(2+)-ATPase, with high Vmax but low Ca(2+)-sensitivity. PMID- 7545137 TI - Open trials in colorectal cancer. PMID- 7545138 TI - Update on urology--prostatic cancer. 2--Diagnosis and assessment of prostatic cancer. PMID- 7545141 TI - Basophilic degeneration of the myocardium: histological, immunohistochemical and immuno-electronmicroscopic studies. AB - We studied basophilic degeneration of heart muscle cells in 100 consecutive autopsies (74 males, 26 females). Except for two infants, the age of individuals whose tissue was studied ranged from 10 to 90 years (mean age, 68.3). Using histochemical and immunohistochemical methods, we found the frequency and extent of basophilic degeneration and the materials in type IV glycogenosis (Andersen's disease) were reactive to monoclonal antibodies raised against polyglucosan extracted from the myocardium of a patient with Lafora's disease. Basophilic degeneration was found in 98% of the entire study population and in 100% of those aged over 60. The reactivity for polyglucosan became more intense after diastase digestion. Immuno-electronmicroscopy using an immunogold method showed that the fibril-like structures of basophilic degeneration were specifically labelled by the monoclonal antibodies to polyglucosan. PMID- 7545142 TI - Atrophic variants of dermatofibroma and dermatofibrosarcoma protuberans. AB - Dermal atrophy of more than 50% of the locoregional dermis may be the predominant histopathological feature in dermatofibroma and dermatofibrosarcoma protuberans. This may cause diagnostic difficulties. In the present study 26 cases of atrophic dermatofibroma were compared with three cases of atrophic dermatofibrosarcoma protuberans. Clinically, both conditions mostly occurred on the (upper) trunk of females. While atrophic dermatofibroma usually presented as a reddish, umbilicated lesion (0.5-1cm), often suspected to be a basal cell carcinoma, atrophic dermatofibrosarcoma protuberans showed irregularly arranged tan-brown plaques (3-6 cm). Histologically, atrophic dermatofibroma showed a regular silhouette with a smooth nodular (9/26) or scalloped lower margin with an intervening lace-like pattern of superficial fatty tissue infiltration (17/26) and variable sclerosis: atrophic dermatofibrosarcoma protuberans showed a deep, irregular infiltration of fatty tissue in a lacelike/honeycomb and/ or multilayered pattern, but no sclerosis. Immunohistochemically, atrophic dermatofibroma was mostly negative with QBEnd 10 (CD34;24/26), variably positive for factor XIIIa (20/26) and metallothionein (11/26). Labelling for factor XIIIa and metallothionein was usually seen in 'early' (metabolically active) lesions, while 'late' sclerotic ones were negative. In contrast to atrophic dermatofibroma all three atrophic dermatofibrosarcoma protuberans showed a consistently uniform profile: CD34 positive, factor XIIIa and metallothionein negative. Our study delineates atrophic dermatofibroma and atrophic dermatofibrosarcoma protuberans as distinct entities clearly distinguishable from each other by clinicopathologic criteria. PMID- 7545140 TI - [Obstetric prognostic factors of newborn infants with very low birth weight (< or = 1,500 gram) with reference to survival rate and early childhood development]. AB - Prognostic factors influencing survival in 235 very low birthweight prematures (< or = 1500 g) born between 1986 and 15.11. 1993 at the Department of Obstetrics and Gynaecology, University Hospital of Cologne, were retrospectively evaluated. Chromosomal anomalies and severe congenital malformations were excluded. Of 180 singletons 84 were classified as appropriate-for-gestational-age (AGA) and 96 as small-for-gestational-age (SGA). By interrogating the attending paediatricians data regarding the early development of 62/65 surviving singletons born between 1986 to 1990 were recorded (follow-up rate 95%). Survival was significantly correlated to singleton pregnancy (p < 0.05), female sex (p = 0.001) and in the AGA-prematures to prenatal corticoid prophylaxis. With similar mean birthweight SGA-singletons showed a three weeks higher mean gestational age; the mortality showed an inverse correlation to birthweight and gestational age being 11% higher in the AGA-group compared with the SGA-group (32% versus 21%). At the age of between 11 months and 6 years severe handicaps and developmental retardations were found more often in previous AGA-prematures (6/26) than in previous SGA prematures (4/36); type and degree of later handicap were not correlated to birthweight. According to our results survival rates of very low birthweight prematures are strongly influenced by singleton pregnancy, by fetal sex, by gestational age and in the AGA-group by prenatal corticoid prophylaxis; mortality shows an inverted correlation to birthweight and gestational age, whereas the later prognosis of survivors does not seem to be influenced by birthweight or gestational age. PMID- 7545136 TI - Divergence of insulin-like growth factors I and II in the elasmobranch, Squalus acanthias. AB - Recent studies have shown that vertebrates, including teleostean fishes, amphibians, birds and mammals, contain two distinct insulin-like growth factor (IGF) genes. In contrast agnathans, represented by hagfish, apparently have only one IGF that has features characteristic of both IGF-I and IGF-II. Between these groups the elasmobranchs occupy a critical position in terms of the phylogeny of IGFs. We sought to determine if gene duplication and divergence of IGF-I and IGF II occurred before or after divergence of elasmobranchs from other vertebrates by cloning IGF-like molecules from Squalus acanthias. Our analysis shows that Squalus liver produces two distinct IGF-like molecules. One has greater sequence identity to, and conserved features characteristic of, known IGF-I molecules, while the other is more IGF-II like. These results suggest that the prototypical IGF molecule duplicated and diverged in an ancestor of the extant gnathostomes. PMID- 7545143 TI - Tumor-infiltrating T lymphocytes from renal-cell carcinoma express B7-1 (CD80): T cell expansion by T-T cell co-stimulation. AB - B7-1 (CD80) provides co-stimulation for T-cell activation by interacting with CD28 or CTLA4. Here we demonstrate the expression of B7-1 in freshly isolated and cultured lymphocytes from renal-cell carcinoma. In fresh preparations of lymphocytes infiltrating renal-cell-carcinoma tissue, B7-1 mRNA could readily be detected by reverse transcription PCR, and 2-color flow-cytometry analysis revealed substantial B7-1 expression on T cells from these isolates. As expected, tumor-derived T cells also expressed CD28, the B7 receptor. While B7-1 expression of tumor-derived T cells was maintained during culture in interleukin-2 supplemented medium, CD28 expression was further enhanced. We also show that B7-1 is functionally involved in T-cell expansion: anti-B7-1 MAb inhibited the PHA induced proliferation of tumor-derived B7-1+ T cells (35%) in the absence of exogenous antigen-presenting cells, indicating that B7-1 mediates T-T cell co stimulation (self-co-stimulation). Our data demonstrate that T cells infiltrating renal-cell carcinoma express B7-1, and that mutual co-stimulation via the B7 1/CD28 pathway contributes to the interleukin-2-driven expansion of tumor-derived T cells in vitro. The frequency of B7-1+ T cells in tumor lesions and the level of B7-1 on these cells may determine the time course of T-cell expansion in vivo. Self-co-stimulation, however, might also represent one mechanism leading to the state of suppression or anergy characteristic of tumor-infiltrating lymphocytes. PMID- 7545139 TI - An in vitro model for arsine toxicity using isolated red blood cells. AB - A novel test system using isolated red blood cells (RBCs) and arsine (AsH3) in aqueous solution was developed to allow quantitation of AsH3 exposure and to study the toxicity of AsH3 in vitro. In this system AsH3 gas was generated and dissolved in aqueous solution, the concentration was measured, and the standardized solution was mixed with rat or dog red blood cells (RBCs). AsH3 was found to be stable in solution at neutral pH for several hours, but was lost quickly from solution as the acidity was increased to pH 2. Approximately 74% of the initial 0.56 mM AsH3, measured as total arsenic, was found to be taken up by, or strongly associated with, dog RBCs within 5 min of incubation and 82% of the initial 0.49 mM AsH3 was found in rat RBCs after 10 min incubation. Following hypotonic lysis of rat RBCs, 55% of the cell-associated arsenic was found in the membrane fraction with the balance found in the cytosolic fraction. The in vitro technique was used to examine factors influencing AsH3 toxicity using hemolysis as the end point. Hemolysis levels in dog and rat RBC incubations were found to increase with time after exhibiting a lag phase of about 30 min. At the AsH3 concentrations used, maximum levels of hemolysis were observed by 2 hr; maximum hemolysis at room temperature for dog RBCs was 20% and for rat RBCs was 22%. Increasing the temperature from room temperature to 37 degrees C resulted in increased hemolysis in dog RBCs (36%) and rat RBCs (90%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545144 TI - Significance of tenascin serum level as tumor marker in primary colorectal carcinoma. AB - Tenascin serum levels were evaluated in 118 patients with primary colorectal carcinoma and in a control group of 51 healthy persons in a double-sided sandwich ELISA. The data were correlated with post-operative TNM-staging. Patients with colorectal carcinomas had significantly higher serum levels of tenascin than the control group. At the 95% level of specificity, sensitivity was 25%. Tumor grading obviously had no influence on the level of tenascin in serum. With increasing pT-category, tenascin levels increased as well. In patients with distant metastatic disease, serum tenascin levels were significantly higher than in patients without distant metastases. These data suggest that, in colorectal carcinoma, the preoperative level of serum tenasin reflects the total tumor burden and correlates with metastatic disease. Our observation warrants a prospective study of the relevance of tenascin serum levels with regard to prognosis and as an indicator of relapse. PMID- 7545146 TI - Growth patterns in selected organs of the miniature swine as determined by gross macromolecular composition. AB - As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, the brain, kidney, liver, pancreas, and spleen from male animals were examined for changes in RNA, DNA, and protein contents from 1 to 196 d after birth. Distinct patterns were found for changes with age in macromolecular levels. Protein levels increased from d 1 to 56 in all organs except spleen, in which little change was noted. Gel electrophoresis showed little qualitative change in the liver protein profile during this period. A fat-free, non-nucleic acid, protein-containing fraction, insoluble in hot alkali, appeared in the brain after approximately 1 wk following birth. DNA concentrations decreased markedly from d 1 to d 196 for brain, kidney, and spleen but decreased more gradually for liver and pancreas. RNA levels declined slightly or remained the same in all organs except pancreas, where a large increase occurred from d 1 to weaning (56 d). Growth proceeded in all organs by increases in cell number (hyperplasia), as evidenced by increases in total (level or concentration x organ weight) DNA, or by hypertrophy, as evidenced by increases in the ratio of protein to DNA or by a combination of both processes. Hypertrophic growth was attained by d 56 and continued to sexual maturity in all organs except spleen. Hyperplastic growth continued to sexual maturity in all organs except brain. PMID- 7545145 TI - Administration of porcine somatotropin by sustained-release implant: growth factor and metabolic responses in crossbred white and genetically lean and obese boars and gilts. AB - Differences in endocrine and metabolic responses to porcine somatotropin administered by daily injection or sustained-release implant (pST-SR) have been previously observed in genetically lean and obese, gilts and barrows. The current study extended those findings by examining responses to pST-SR in gilts and boars of a contemporary crossbred line, as well as lean and obese lines. Pigs were treated with 0, 1, or 2 pST-SR implants inserted subcutaneously behind the ear. The osmotically driven pST-SR implants delivered 2 mg of recombinant pST/d. Pigs were bled on d 0, 7, 14, 28, and 42 after implantation. Sera were assayed for pST, insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein-2 (IGFBP-2), insulin, glucose, and blood urea nitrogen (BUN). Circulating pST concentrations were increased in a dose-dependent manner (P < .001) in the pST-SR treated pigs, but remained elevated (P < .05) only in the 4 mg of pST-SR/d group on d 42. Significant effects of line, dose, time, line x dose, and time x dose were noted for IGF-I. Serum IGF-I was elevated in a dose-dependent manner over the 42-d period in all pST-treated swine. Examination of the line x dose x time interaction indicated that the IGF-I response to pST-SR was greatest in the obese line compared with the lean and crossbred lines. Conversely, serum IGF-II responded to pST-SR to the least extent in the obese pigs. Circulating IGFBP-2 concentrations were reduced by pST-SR, but were not affected by line. The BUN concentrations were reduced by pST-SR. An interaction of line x dose x time (P < .001) indicated that the response was greater in the obese line. Line x dose x time interactions were also noted for insulin and glucose concentrations, which were elevated by pST-SR in a dose-response manner in all lines, but to a much greater extent in the obese pigs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545147 TI - Physiological and cognitive correlates of child abuse. AB - OBJECTIVE: To compare the physiological responses of abused children to different stimuli with responses of children in a reference group and to correlate the physiological responses with intellectual and personality functioning. METHOD: Abused children were compared with a reference group on two batteries of tests that were administered on separate days. In one session, children were shown slides with emotional or cognitive content while heart rate, pulse height, skin conductance, electromyography, and skin temperature were measured. In the other session, intellectual and personality functioning was measured using the WISC-R, Quick Neurological Screening Test, and the Junior Eysenck personality inventory. RESULTS: Abused children had smaller changes in pulse height in the first two stimulus conditions presented ("No Signal" and "Math"), but their electrodermal responses were lower throughout all stimulus conditions. Abused children also had higher introversion and lower Verbal and Full Scale IQ scores. Verbal and Full Scale IQ scores were inversely related to the severity of abuse that had been experienced. When these variables were used in a discriminant function analysis, children were assigned to the correct group 86% of the time. CONCLUSION: These findings support a model that describes the effects of abuse as delaying cognitive development and inhibiting physiological responsiveness to the environment. PMID- 7545148 TI - Case study: corticosteroid treatment of language regression in pervasive developmental disorder. AB - The authors describe a child whose language and behavior regressed at 22 months and in whom pervasive developmental disorder was later diagnosed. At 6 years, he displayed a profound receptive-expressive aphasia accompanied by behavioral disturbances characterized by hyperactivity, impaired social interactions, tantrums, gestural stereotypies, and echolalia. A single-photon emission computed tomography scan and steady-state auditory evoked potentials suggested bitemporal and left frontal pathophysiology. The overall profile resembled Landau-Kleffner syndrome, but no electroencephalographic disturbance was evident. Corticosteroid treatment resulted in amelioration of language abilities and behavior. These findings suggest that the factors underlying language regression in pervasive developmental disorder can, in special circumstances, be amenable to pharmacological treatment. PMID- 7545151 TI - Isolation and characterization of ropA homologous genes from Rhizobium leguminosarum biovars viciae and trifolii. AB - ropA encodes a 36-kDa outer membrane protein of Rhizobium leguminosarum bv. viciae strain 248 which constitutes the low-M(r) part of antigen group III (R.A. de Maagd, I.H.M. Mulders, H.C.J. Canter Cremers, B.J.J. Lugtenberg, J. Bacteriol. 174:214-221, 1992). We observed that genes homologous to ropA are present in strain 248 as well as in other R. leguminosarum strains, and we describe the cloning and characterization of two of these genes. Sequencing of a 2.2-kb Bg/II fragment from R. leguminosarum bv. viciae strain 248 that hybridizes with ropA revealed one large open reading frame of 1,074 bp encoding a mature protein of 38.096 kDa. Homology between this gene and ropA is 91.8% on the DNA level. Homology on the amino acid level is only 69.9% as a result of a frameshift. On the basis of homology and immunochemical characteristics, we conclude that this gene encodes the high-M(r) part of the outer membrane protein antigen group III that is repressed during symbiosis. We named this gene ropA2. The second gene that we cloned was the ropA homologous gene of R. leguminosarum bv. trifolii strain LPR5020. Except for amino acid 43, the N-terminal part of the corresponding protein appeared to be identical to the first 51 amino acids of RopA of strain 248. The transcription start sites of both genes were determined, and the promoter regions were compared with that of ropA of strain 248. No clear consensus sequence could be deduced. The relationship of ropA and ropA2 of R. leguminosarum bv. viciae strain 248 with two similar genes from Brucella abortus is discussed. PMID- 7545149 TI - Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP. AB - The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer. PMID- 7545150 TI - Quantification of group A colicin import sites. AB - Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duche, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively. PMID- 7545152 TI - Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. AB - We previously reported on the occurrence, partial purification, and preliminary characterization of the first reported bacterial nitric oxide synthase. The soluble Nocardia enzyme, designated NOSNoc, has now been purified 1,353-fold by a combination of 2',5'-ADP-agarose affinity chromatography and hydroxylapatite chromatography. NOSNoc runs as a band of M(r) 51,900 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass was estimated to be 110.6 +/- 0.5 kDa by gel filtration, indicating that the native enzyme exists as a homodimer in solution. An N-terminal 15-amino-acid sequence was determined for NOSNoc, showing it to be different from known mammalian NOSs. NG-Hydroxy-L arginine was confirmed to be an intermediate in the enzymatic reaction by stoichiometric determinations of oxygen uptake, NADPH oxidation, NO formation as measured by nitrite determinations, citrulline formation, and kinetic studies. NOSNoc was competitively inhibited by NG-methyl- and NG-nitro-L-arginine with either L-arginine or NG-hydroxyl-L-arginine as the substrate. Furthermore, the stability and pH and temperature optima of NOSNoc have been established. PMID- 7545153 TI - Fis activates the RpoS-dependent stationary-phase expression of proP in Escherichia coli. AB - Fis is a general nucleoid-associated protein in Escherichia coli whose expression is highly regulated with respect to growth conditions. A random collection of transposon-induced lac fusions was screened for those which give increased expression in the presence of Fis in order to isolate a ProP-LacZ protein fusion. We find that proP, which encodes a low-affinity transporter of the important osmoprotectants proline and glycine betaine, is transcribed from two promoters. proP1 is transiently induced upon subculture and is upregulated by increases in medium osmolarity. As cells enter stationary phase, a second promoter, proP2, is strongly induced. This promoter can also be induced by high medium osmolarity in exponential phase. The activity of proP2 depends on Fis and the stationary-phase sigma factor sigmas. In the presence of Fis, proP2 expression is increased over 50-fold, as judged by the LacZ activity of cells carrying the proP-lacZ fusion as well as by direct RNA analysis, making this the most strongly activated promoter by Fis that has been described. Two Fis binding sites centered at positions -41 (site I) and -81 (site II) with respect to the transcription initiation site of P2 have been defined by DNase I footprinting. Mutations in site I largely abolish stationary-phase activation, while mutations at site II have a minor effect, suggesting that direct binding of Fis to site I is important for Fis-mediated activation of this promoter. In addition to Fis and sigmas, sequences located over 108 bp upstream of the proP2 transcription initiation site are required for efficient expression. PMID- 7545156 TI - Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes. AB - The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did. PMID- 7545157 TI - Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation. AB - A gene was cloned from Streptomyces coelicolor A3(2). It encodes a protein of 368 amino acid residues with a high degree of similarity to prokaryotic release factor 2. However, it has neither an internal stop codon nor the Shine-Dalgarno like sequence immediately upstream of the assumed frameshift position. The gene is expressed and functional in Escherichia coli as peptide chain release factor 2. The transcription start site is at or adjacent to the translational start site. The size of the mRNA detected by hybridization suggests that the gene (prfB) is monocistronic in S. coelicolor A3(2). However, about 80 bp upstream of the gene there is an operon which is composed of two genes encoding eukaryotic type serine/threonine kinases. PMID- 7545155 TI - Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content. AB - Molecular typing allowed the separation of the species Bacteroides fragilis into two genotypically distinct groups. A unique set of 50 strains of B. fragilis carrying the chromosomal metallo-beta-lactamase gene cfiA was subjected to a comparative analysis with respect to sets of up to 250 randomly collected strains devoid of this gene. The two groups were found to be distinct on the basis of the following results: (i) ribotyping, after DNA digestion with AvaI, revealed a practically homogeneous DNA fragment pattern for the cfiA-positive strains and distinct multiple patterns for the cfiA-negative strains; (ii) PCR, arbitrarily primed with an experimentally selected decamer, generated fragment patterns typical for the strains of each group; (iii) the three insertion sequences described to date in the species B. fragilis, i.e., IS4351, IS942, and IS1186, were all but confined to the cfiA-positive group, in which they were capable of providing promoter sequences for the transcription of cfiA; and (iv) the cepA gene, encoding the so-called endogenous cephalosporinase of B. fragilis, was found exclusively in the cfiA-negative group, in which it was present in ca. 70% of the strains. The cfiA-, cepA-negative fraction was not characterized further. In a natural population of 500 randomly selected strains of B. fragilis, the cfiA positive and cfiA-negative groups represented ca. 3 and 97% of the strains, respectively. Analysis of 82 metabolic traits revealed no difference between the two groups. PMID- 7545154 TI - Lateral transfer of rfb genes: a mobilizable ColE1-type plasmid carries the rfbO:54 (O:54 antigen biosynthesis) gene cluster from Salmonella enterica serovar Borreze. AB - Plasmid pWQ799 is a 6.9-kb plasmid isolated from Salmonella enterica serovar Borreze. Our previous studies have shown that the plasmid contains a functional biosynthetic gene cluster for the expression of the O:54 lipopolysaccharide O antigen of this serovar. The minimal replicon functions of pWQ799 have been defined, and a comparison with nucleotide and protein databases revealed this replicon to be virtually identical to ColE1. This is the first report of involvement of ColE1-related plasmids in O-antigen expression. The replicon of pWQ799 is predicted to encode two RNA molecules, typical of other ColE1-type plasmids. RNAII, the putative replication primer from pWQ799, shares regions of homology with RNAII from ColE1. RNA1 is an antisense regulator of DNA replication in ColE1-related plasmids. The coding region for RNAI from pWQ799 shares no homology with any other known RNAI sequence but is predicted to adopt a secondary structure characteristic of RNAI molecules. pWQ799 may therefore represent a new incompatibility group within this family. pWQ799 also possesses cer, rom, and mob determinants, and these differ minimally from those of ColE1. The plasmid is mobilizable in the presence of either the broad-host-range helper plasmid pRK2013 or the IncI1 plasmid R64drd86. Mobilization and transfer of pWQ799 to other organisms provides the first defined mechanism for lateral transfer of O-antigen biosynthesis genes in S. enterica and explains both the distribution of related plasmids and coexpression of the O:54 factor with other O-factors in different Salmonella serovars. The base composition of the pWQ799 replicon sequences gives an average percent G+C value typical of Salmonella spp. In contrast, the percent G+C value is dramatically lower with rfb0:54, consistent with the possibility that the cluster was acquired from an organism with much lower G+C composition. PMID- 7545159 TI - Risperidone in the treatment of affective illness and obsessive-compulsive disorder. AB - BACKGROUND: Risperidone is a new-generation atypical antipsychotic agent with potent dopaminergic and serotonergic antagonist activity. Compared with traditional dopamine-blocking neuroleptics, risperidone is more effective in treating negative symptoms of schizophrenia and may be less likely to cause extrapyramidal symptoms or tardive dyskinesia. Although risperidone is marketed for the treatment of schizophrenia, its novel psychopharmacologic effects and potentially mild side effect profile suggest the possibility of other therapeutic applications. An open prospective study was undertaken to determine whether risperidone might diminish psychosis, severe agitation, or rapid cycling in patients having acute and chronic primary affective illnesses (bipolar and major depressive disorder) and to document response characteristics and side effects. Additionally, a small number of patients with refractory obsessive-compulsive disorder (OCD) without comorbid tic or delusional disorders were given open trials of risperidone added to their medication. METHOD: Outpatients who fulfilled DSM-IV criteria for bipolar I, bipolar II, or major depressive disorder and suffered from psychosis or agitation associated with their illness (N = 20) and those who had treatment-refractory DSM-IV OCD (N = 5) were started on open trials of risperidone at daily doses of 1 to 1.5 mg. Doses were adjusted upwards to a maximum of 6 mg depending on clinical response. RESULTS: Seventeen (85%) of 20 patients (13 bipolar, 4 major depressive disorder) showed complete or partial improvement after treatment with risperidone doses ranging from 1 to 6 mg/day (mean = 3.5 mg). Beneficial effects included decreases in agitation, psychosis, sleep disturbance, and rapid cycling. Four patients (20%) discontinued risperidone because of intolerable side effects. Five patients with refractory OCD also showed significant symptomatic improvement after the addition of risperidone. CONCLUSION: The findings suggest that (1) risperidone may be useful in the acute/p.r.n. and chronic treatment of psychosis, agitation, and cycling accompanying affective illness, and (2) risperidone may be useful in augmenting pharmacologic response in OCD. PMID- 7545161 TI - Oocyte Gal alpha 1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. AB - The Gal alpha 1-->3Gal structure is displayed on the zona pellucida glycoprotein ZP3 on murine oocytes. This trisaccharide has been implicated in sperm-zona pellucida adhesive events thought to be essential to fertilization in the mouse. To determine directly if this molecule is required for fertilization, we have generated mice that are deficient in a gene (alpha 1,3GT) encoding the UDP Gal:beta-D-Gal-alpha 1-->3Gal-galactosyltransferase enzyme responsible for Gal alpha 1-->3Gal synthesis and expression. These mice develop normally and exhibit no gross phenotypic abnormalities. The Gal alpha 1-->3Gal epitope is absent from the vascular endothelium and other tissues in alpha 1,3GT (-/-) adult mice. By contrast, alpha 1,3GT (-/-) mice, like humans, develop naturally occurring anti alpha-galactoside antibodies normally absent in wild type mice. Female alpha 1,3GT (-/-) mice yield oocytes that are devoid of the Gal alpha 1-->3Gal epitope; however, these mice are fully fertile. These observations indicate that the Gal alpha 1-->3Gal moiety is not essential to sperm-oocyte interactions leading to fertilization or to essentially normal development. They further suggest that alpha 1,3GT (-/-) mice will find utility for exploring approaches to diminish anti-Gal-dependent hyperacute xenograft rejection, which presents a major barrier to the use of porcine and other non-primate organs for xenotransplantation in humans. PMID- 7545158 TI - Cytochemical localization of glycogen in Chlamydia trachomatis inclusions. AB - The origin and distribution of glycogen in inclusions of Chlamydia trachomatis were demonstrated with silver proteinate stain for electron microscopy. Glycogen particles were detected in all developmental stages of C. trachomatis, as well as free in the inclusions. Intrachlamydial glycogen was most common in elementary bodies but was also detected in intermediate forms and reticulate bodies (RBs). Abnormal divisions and breakdown of cytoplasmic membranes were common in RBs. Cytoplasmic contents, including glycogen particles, were released into the inclusions after rupture of the outer membranes of abnormal RBs and intermediate forms. From these observations, we conclude that glycogen in inclusions of C. trachomatis originates in the organisms themselves. PMID- 7545160 TI - Risperidone treatment of refractory acute mania. PMID- 7545162 TI - Staphylococcus aureus expresses a major histocompatibility complex class II analog. AB - Staphylococcus aureus expresses various surface proteins which specifically recognize and bind to different host molecules. We have previously identified a bacterial protein that exhibits a broad specificity and binds to several mammalian extracellular proteins. The gene encoding this bacterial component has now been cloned and sequenced. The deduced protein consists predominantly of six repeated domains of 110 residues. Each of the repeated domains contain a subdomain of 31 residues that share striking sequence homology with a segment in the peptide binding groove of the beta chain of the major histocompatibility complex (MHC) class II proteins from different mammalian species. The purified recombinant bacterial protein bound several mammalian proteins, including recombinant osteopontin, suggesting a protein-protein interaction and also specifically recognized a 15-amino acid residue synthetic peptide. Taken together, these results suggest that the bacterial protein resembles mammalian MHC class II molecules with respect to both sequence similarities and peptide binding capabilities. PMID- 7545164 TI - cAMP activation of CAAT enhancer-binding protein-beta gene expression and promoter I of acetyl-CoA carboxylase. AB - The acetyl-CoA carboxylase (ACC) gene contains two distinct promoters, denoted PI and PII. PI is responsible for the generation of class I ACC mRNAs which are induced in a tissue-specific manner under lipogenic conditions. PII generates class II ACC mRNAs which are expressed constitutively. During 30A5 preadipocyte differentiation, both promoters are activated; the preadipocytes must be pretreated with cAMP for this activation to occur. In this report, we present evidence that CAAT enhancer-binding protein-beta (C/EBP-beta) is induced and involved in the PI activation by cAMP. Expression of the reporter gene under the control of the PI promoter is activated within 3 h after treatment of 30A5 cells with a cyclic AMP analogue, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, and 3-isobutyl-1-methylxanthine, in association with the accumulation of C/EBP-beta mRNA and protein. These accumulations were inhibited in the presence of H8, a protein kinase inhibitor; H8 also inhibited activation of PI by cAMP. However, the induction of reporter gene expression and the increase of C/EBP-beta mRNA by cAMP were not affected by treatment with tumor necrosis factor alpha, which completely inhibited the accumulation of C/EBP-alpha mRNA. Overexpression of C/EBP-beta by transfection with the C/EBP-beta gene led to increased binding of C/EBP-beta to DNA and partial PI activation. cAMP did not affect the amount of C/EBP-beta binding to the DNA but did promote phosphorylation of C/EBP-beta and PI activation. As in the case of C/EBP-alpha, C/EBP-beta bound to the CCAAT box of the PI promoter. These results indicate that cAMP not only induces, but also activates, bound C/EBP-beta through phosphorylation for PI activation. Our studies also indicate that cAMP induces C/EBP-alpha. C/EBP-beta induction, however, precedes that of C/EBP-alpha. PMID- 7545166 TI - Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. AB - We investigated the influence of the activation state of integrin alpha 5 beta 1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked alpha v beta 3 expression and adhered to fibronectin through alpha 5 beta 1. Mel57 cells adhered through alpha v beta 3 and alpha 5 beta 1. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6-10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of alpha v beta 3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory beta 1-integrin antibodies or Mn2+ induced alpha 5 beta 1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn2+, alpha 5 beta 1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn2+ or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn2+, and TS2/16, alpha 5 beta 1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for alpha 5 beta 1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin. PMID- 7545165 TI - Activation of protein kinase C alpha inhibits signaling by members of the insulin receptor family. AB - Stimulation of the activity of protein kinase C by pretreatment of cells with phorbol esters was tested for its ability to inhibit signaling by four members of the insulin receptor family, including the human insulin and insulin-like growth factor-I receptors, the human insulin receptor-related receptor, and the Drosophila insulin receptor. Activation of overexpressed protein kinase C alpha resulted in a subsequent inhibition of the ligand-stimulated increase in antiphosphotyrosine-precipitable phosphatidylinositol 3-kinase mediated by the kinase domains of all four receptors. This inhibition varied from 97% for the insulin receptor-related receptor to 65% for the Drosophila insulin receptor. In addition, the activation of protein kinase C alpha inhibited the in situ ligand stimulated increase in tyrosine phosphorylation of the GTPase-activating protein associated p60 protein as well as Shc mediated by these receptors. The mechanism for this inhibition was further studied in the case of the insulin-like growth factor-I receptor. Although the in situ phosphorylation of insulin-receptor substrate-1 and p60 by this receptor was inhibited by prior stimulation of protein kinase C alpha, the in vitro tyrosine phosphorylation of these two substrates by this receptor was not decreased by prior stimulation of the protein kinase C alpha in the cells that served as a source of the substrates. Finally, the insulin-like growth factor-I-stimulated increase in cell proliferation was found to be inhibited by prior activation of protein kinase C alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545163 TI - Crkl is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia. AB - The deregulated tyrosine kinase activity of the Bcr/Abl protein has been causally linked to the development of Philadelphia (Ph) chromosome-positive leukemia in mice and man. Abnormally tyrosine-phosphorylated substrates of the Bcr/Abl kinase in Ph-positive cells are likely to contribute to leukemogenesis by interfering with normal signal transduction pathways. We have previously shown that the adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase, and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Ph-positive patient material, containing an active Bcr/Abl protein. We demonstrate here that this protein is Cbl, originally discovered as an oncogene which induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl. PMID- 7545169 TI - P-glycoprotein. Associations between domains and between domains and molecular chaperones. AB - P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain. PMID- 7545168 TI - Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor. AB - Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine kinase activity, GH has recently been shown to promote the association of its receptor with JAK2 tyrosine kinase, to activate JAK2, and to promote the tyrosyl phosphorylation of both GH receptor (GHR) and JAK2. In this work, we examined whether tyrosines 333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH. Tyrosines 333 and 338 in rat full-length (GHR1-638) and truncated (GHR1-454) receptor were replaced with phenylalanines and the mutated GHRs expressed in Chinese hamster ovary cells. These substitutions caused a loss of GH-dependent tyrosyl phosphorylation of truncated receptor and a reduction of GH-dependent phosphorylation of the full length receptor. Consistent with Tyr333 and/or Tyr338 serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GH.GHR.JAK2 complexes. The Tyr to Phe substitutions did not substantially alter GH-dependent JAK2 association with GHR or tyrosyl phosphorylation of JAK2. These results suggest that Tyr333 and/or Tyr338 in GHR are phosphorylated in response to GH and may therefore serve as binding sites for SH2 domain-containing proteins in GH signal transduction pathways. PMID- 7545170 TI - Aggregation of IgE receptors in rat basophilic leukemia 2H3 cells induces tyrosine phosphorylation of the cytosolic protein-tyrosine phosphatase HePTP. AB - The cDNA encoding the rat equivalent of the human hematopoietic tyrosine phosphatase, also known as leukocyte phosphatase, was isolated from a rat basophilic leukemia mast cell cDNA library. By two-dimensional electrophoresis, the protein expressed in the mast cells was of a size (40 kDa) and pI (6.9) predicted from the deduced amino acid sequence. Thus, although previously shown to be preferentially expressed in T cells and B cells, the phosphatase is also found in mast cells. By immunofluorescence microscopy, rat hematopoietic tyrosine phosphatase localized to discrete, globular compartments within the cytoplasm and was not found either in the nucleus or associated with the cell surface membrane. Aggregation of high affinity IgE receptors in the mast cells induced tyrosine phosphorylation of the phosphatase. The tyrosine phosphorylation was mimicked by stimulation with calcium ionophore A23187 but not by direct activation of protein kinase C. Since phosphorylation of the phosphatase was dramatically reduced when the cells were activated in Ca(2+)-free media, it is dependent on a rise in intracellular Ca2+. These data strongly suggest that hematopoietic tyrosine phosphatase may be involved in the IgE receptor-mediated signaling cascade. PMID- 7545167 TI - Evolutionary conservation of the sulfated oligosaccharides on vertebrate glycoprotein hormones that control circulatory half-life. AB - The circulatory half-life of the mammalian glycoprotein hormone lutropin is controlled by its unique Asn-linked oligosaccharides, which terminate with the sequence SO4-4-GalNAc beta 1,4GlcNAc. A cluster of basic amino acids essential for recognition of the alpha subunit by the glycoprotein hormone:N acetylgalactosaminyltransferase is located within two turns of an alpha helix (Mengeling, B.J., Manzella, S.M., and Baenziger, J.U. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 502-506). The amino acids within this region are virtually invariant in the alpha subunits of all vertebrates, indicating that the recognition determinant utilized by the N-acetylgalactosaminyltransferase has been conserved in species ranging from teleost fish to mammals. We demonstrate that the glycoprotein hormone:N-acetylgalactosaminyltransferase and the N acetylgalactosamine-4-sulfotransferase responsible for the synthesis of these unique sulfated oligosaccharides are expressed in the pituitaries of vertebrates ranging from teleost fish to mammals. Furthermore, we show that Asn-linked oligosaccharides terminating with SO4-4-GalNAc beta 1,4GlcNAc are present on the alpha and beta subunits of the salmon glycoprotein hormone GTH II. Asn-linked oligosaccharides terminating with SO4-4-GalNAc beta 1,4GlcNAc are unique structural features of the glycoprotein hormones that have been conserved during vertebrate evolution, suggesting they are critical for the expression of hormone biologic activity. PMID- 7545173 TI - P-selectin glycoprotein ligand-1 is the major counter-receptor for P-selectin on stimulated T cells and is widely distributed in non-functional form on many lymphocytic cells. AB - P-selectin glycoprotein ligand-1 (PSGL-1) is the high affinity counter-receptor for P-selectin on myeloid cells (Sako, D., Chang, X.J., Barone, K.M., Vachino, G., White, H.M., Shaw, G., Veldman, G.M., Bean, K.M., Ahern, T.J., Furie, B., Cumming, D. A., and Larsen, G. R. (1993) Cell 75, 1179-1186). Here we demonstrate that PSGL-1 is also widely distributed on T- and B-lymphocytic tumor cell lines, resting peripheral blood T and B cells, and on stimulated peripheral blood T cell and intestinal intraepithelial lymphocyte (IEL) lines. However, the majority of PSGL-1-positive resting peripheral blood lymphocytic cells and lymphoid tumor cell lines do not display significant P-selectin binding. In contrast, in vitro stimulated peripheral blood T cell and IEL lines avidly bind P-selectin, and PSGL 1 is the sole high affinity counter-receptor mediating this binding. During the course of in vitro stimulation, cell surface expression levels of PSGL-1 do not change as P-selectin binding increases. Rather, the activities of two glycosyltransferases reportedly involved in the production of functional PSGL-1 in myeloid cells are substantially higher in the stimulated T-lymphocytic lines than in resting T lymphocytes, consistent with the hypothesis that activation dependent post-translational events contribute to the expression of functional PSGL-1 on lymphocytes. PMID- 7545171 TI - Alpha 2-macroglobulin functions as a cytokine carrier to induce nitric oxide synthesis and cause nitric oxide-dependent cytotoxicity in the RAW 264.7 macrophage cell line. AB - Nitric oxide (NO) is an important mediator of macrophage activities. We studied the regulation of macrophage NO synthesis by alpha 2-macroglobulin (alpha 2M), a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor-beta (TGF-beta). Native alpha 2M and the alpha 2M receptor-recognized derivative, alpha 2M-methylamine (alpha 2M-MA), increased nitrite generation by the RAW 264.7 murine macrophage cell line. The level of nitrite accumulation, which is an index of NO synthesis, was comparable to that observed with interferon-gamma. Native alpha 2M and alpha 2M-MA also increased inducible nitric oxide synthase (iNOS) mRNA levels and substantially reduced the number of viable cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium/succiny l dehydrogenase assay or trypan blue exclusion. At slightly higher alpha 2M concentrations, [3H]thymidine incorporation was inhibited. All of these activities were counteracted nearly completely when the iNOS competitive inhibitor NG-monomethyl-L-arginine was included. By in situ nick translation, native alpha 2M and alpha 2M-MA increased the percentage of cells with detectable single strand chromatin nicks from 4 to 12 and 17%, respectively. This change suggested apoptosis; however, electron microscopy studies demonstrated variability in the morphology of injured cells. To determine the mechanism by which alpha 2M increases macrophage NO synthesis, we studied proteolytic alpha 2M derivatives that retain partial activity. A 600-kDa derivative that retains growth factor binding activity increased RAW 264.7 cell NO synthesis and iNOS mRNA levels comparable to native alpha 2M and alpha 2M-MA. The purified 18-kDa alpha 2M receptor-binding fragment had no effect on NO synthesis or iNOS expression. Thus, the growth factor-carrier activity of alpha 2M and not its receptor-binding activity is essential for NO synthesis regulation. A TGF-beta-neutralizing antibody mimicked the activity of alpha 2M, increasing RAW 264.7 cell NO synthesis and decreasing cellular viability. These studies demonstrate that alpha 2M can regulate macrophage NO synthesis and profoundly affect cellular function without gaining entry into the cell and without binding specific plasma membrane receptors. PMID- 7545174 TI - Interferon-inducible protein 10 and macrophage inflammatory protein-1 alpha inhibit growth factor stimulation of Raf-1 kinase activity and protein synthesis in a human growth factor-dependent hematopoietic cell line. AB - Stimulatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), act in a synergistic manner to stimulate the growth of hematopoietic progenitor cells, an effect also demonstrated for the growth factor-dependent human hematopoietic cell line MO7e. While little is known about the mechanisms responsible for mediating synergistic interactions of cytokines, Raf-1, a component of the MAP kinase signaling pathway, is thought to play a role in the stimulatory response evoked by several cytokines, including SLF and GM-CSF. Interferon-inducible protein-10 (IP-10) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are members of the chemokine family of suppressive cytokines. Prior exposure of hematopoietic cells to chemokines, including IP-10 and MIP-1 alpha, inhibits the synergistic action of growth factors on stimulating cell proliferation. We report that treatment of MO7e cells with the combination of GM-CSF and SLF directly stimulates statistically significant synergistic increases in the phosphorylation and activation of Raf-1 kinase, and in cellular protein synthesis levels. Pretreatment of MO7e cells with IP-10 or MIP-1 alpha blocked synergistic growth factor action, resulting in statistically significant suppression of cell proliferation, protein synthesis, and Raf-1 phosphorylation and activation. IP-10 and MIP-1 alpha treatment also evoked significant increases in intracellular cAMP levels. Pretreatment of cells with agents which serve to raise intracellular cAMP levels, or with cAMP analogs inhibited the synergistic actions of GM-CSF and SLF in a manner similar to IP-10 and MIP-1 alpha. In addition, treatment of cells with a potent inhibitor of cAMP dependent protein kinase A blocked the suppressive action of MIP-1 alpha and IP 10 on Raf-1 kinase activity and on MO7e cell proliferation. The ability of IP-10 and MIP-1 alpha to antagonize the synergistic action of GM-CSF and SLF appears to involve inactivation of Raf-1 and the down-regulation of protein synthesis. Our findings suggest that both MIP-1 alpha and IP-10 mediate their suppressive effects in MO7e cells by stimulating increases in cellular cAMP levels and activating protein kinase A, a mechanism we believe to be unique to these chemokines and not one applied to all growth suppressive members of the chemokine superfamily (for example, interleukin 8 and platelet factor 4). PMID- 7545172 TI - Differential binding of the NFE3 and CP1/NFY transcription factors to the human gamma- and epsilon-globin CCAAT boxes. AB - Naturally occurring nondeletional mutations affecting the distal CCAAT box of the human gamma-globin gene promoter result in hereditary persistence of fetal hemoglobin in adult life. Although the distal CCAAT box is the target of several factors, including CP1/NFY, CDP, GATA-1 and NFE3, only NFE3 binding activity is consistently sensitive to well characterized mutations in this region such as G 117-->A, C-114-->T, and delta 13 hereditary persistence of fetal hemoglobin. We extensively characterized the binding specificities of NFE3 and demonstrated that NFE3 has unique properties with respect to other CCAAT box-binding proteins. Affinity-purified NFE3 from erythroid K562 cells binds the distal but not the proximal human gamma-globin CCAAT box, the single CCAAT box of the human epsilon globin promoter, and the proximal CCAAT box of the evolutionarily related Galago crassicaudatus gamma-globin gene. Within the epsilon-globin CCAAT box, NFE3 represents the major and almost exclusive binding activity. Disruption of such a binding site essentially inactivates the epsilon-globin promoter, suggesting that NFE3 plays an important role in the embryonic expression of this gene. PMID- 7545175 TI - Identification and initial characterization of a specific proteasome (prosome) associated RNase activity. AB - We have identified and characterized a specific nuclease activity to be tightly associated with proteasomes. Using tobacco mosaic virus RNA (TMV-RNA) as substrate to analyze and quantify the cleavage reaction, we supply several lines of evidence that this nuclease activity is an integral part of proteasomes. Thus, RNase activity was coincident with the elution profiles of proteasomes at each stage of purification. Proteasomal nuclease activity was resistant to strong dissociation conditions using 480 mM KCl, 0.5% sodium lauroylsarcosinate, and 6 M urea. This nuclease activity remained associated with an urea-resistant subcomplex of the proteasome comprising a specific set of proteins. Finally the digestion of TMV-RNA led to a well defined pattern of RNA fragments while 5 S ribosomal RNA and globin mRNA were not degraded. These results provide further evidence that proteasomes are able to discriminate between different RNAs, and the possible involvement of proteasomes in translation control is discussed. PMID- 7545176 TI - Subdivision of Burkholderia pseudomallei ribotypes into multiple types by random amplified polymorphic DNA analysis provides new insights into epidemiology. AB - Ribotyping has previously been used for epidemiological studies of Burkholderia pseudomallei (previously Pseudomonas pseudomallei). We show here that random amplified polymorphic DNA (RAPD) analysis allows subdivision of strains of the same ribotype. With five different primers, no two epidemiologically unrelated isolates of any single ribotype in this study of 102 isolates from humans, goats, cats, and soil had identical RAPD patterns. Conversely, RAPD analysis showed clonality for isolates from each of two animal outbreaks of melioidosis and from a nontropical focus of animal and human melioidosis spanning 25 years. Some soil isolates were identical to epidemiologically related animal and human isolates as determined by RAPD typing. There was no evidence that the clinical outcome of melioidosis was related to RAPD patterns. PMID- 7545177 TI - Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes. AB - The genus Arcobacter encompasses gram-negative, aerotolerant, spiral-shaped bacteria formerly designated Campylobacter cryaerophila. Two genus-specific 16S rRNA-based oligonucleotide DNA probes (23-mer and 27-mer) were developed. The probes hybridized with strains of Arcobacter butzleri (n = 58), Arcobacter cryaerophilus (n = 19), and Arcobacter skirrowii (n = 17). The probes did not cross-react with any of the reference strains of Campylobacter, Helicobacter, including "Flexispira rappini," or Wolinella. The 27-mer hybridized with 61 Arcobacter spp. field isolates originating from late-term aborted porcine (n = 54) and equine (n = 2) fetuses and humans with enteritis (n = 5). The species of Arcobacter isolates (n = 56) recovered from aborted livestock fetuses were determined by ribotyping and were as follows: A. cryaerophilus group 1A (11 of 56; 20%), A. cryaerophilus group 1B (37 of 56; 66%), A. butzleri (5 of 56; 9%), and unknown (3 of 56; 5%). The five human field strains were identified as A. butzleri. A species-specific DNA probe (24-mer) for A. butzleri was also developed since there is evidence that this organism may be a human pathogen. This probe hybridized with previously characterized strains of A. butzleri (n = 58), with 10 field strains identified as A. butzleri by ribotyping and with 2 strains having an indeterminate ribotype. The A. butzleri-specific probe did not cross-react with strains of A. skirrowii (n = 17) and A. cryaerophilus (n = 19). PMID- 7545179 TI - Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods. AB - Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping. PMID- 7545180 TI - Significance of Salmonella typhi bacteriuria. AB - Bacteriuria due to Salmonella typhi usually occurs following recent typhoid fever or in chronic carrier states. Data from 18 patients with S. typhi bacteriuria, seen during 5 years, were analyzed. Fourteen patients had localized urinary tract infection due to S. typhi. Four others had bacteriuria, probably associated with typhoid fever. Localized abnormalities of the urinary tract and kidneys and also systemic diseases were found to predispose patients to S. typhi bacteriuria. Local abnormalities encountered included urolithiasis (n = 3), prostatic hypertrophy (n = 1), and tuberculosis (n = 1). One renal transplant recipient and another with lupus nephritis had S. typhi bacteriuria. One had associated strongyloidosis, and another was pregnant. PMID- 7545178 TI - Phenotypic characterization of epidemic versus sporadic strains of methicillin resistant Staphylococcus aureus. AB - Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%. PMID- 7545181 TI - Differentiation of Bartonella-like isolates at the species level by PCR restriction fragment length polymorphism in the citrate synthase gene. AB - The citrate synthase gene (gltA) of Bartonella henselae was cloned and sequenced to compare genetic divergence among alpha and gamma branches of the class Proteobacteria and to develop enhanced genotypic reagents for B. henselae identification. B. henselae gltA is 1,293 nucleotides in length and 63 to 66% homologous with corresponding gene sequences of Rickettsia prowazekii, Escherichia coli, and Coxiella burnetii. The observed genetic variability suggests that gltA sequences can provide a useful means for studying moderate divergence among related bacteria. Oligonucleotides specific for B. henselae gltA were evaluated for the ability to prime PCR amplification within the alpha and gamma branches of the proteobacteria. Under the conditions used, only B. henselae, Bartonella quintana, and R. prowazekii template DNAs yielded amplification products (approximately 380 bp). DNAs from 28 Bartonella-like isolates of feline origin were amplified by B. henselae primers and analyzed for restriction fragment length polymorphism. The resulting patterns for all 28 isolates were similar or identical to that of the recognized B. henselae strain. Current studies are aimed at optimization of PCR conditions for specificity and sensitivity of amplification of Bartonella sequences from clinical isolates. PMID- 7545182 TI - Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate. AB - A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory. PMID- 7545183 TI - Identification of Bartonella (Rochalimaea) species among fastidious gram-negative bacteria on the basis of the partial sequence of the citrate-synthase gene. AB - The bacterial genus Bartonella (Rochalimaea) includes emerging human pathogens with five recognized species. These are fastidious gram-negative bacteria, exhibiting few phenotypic characteristics and whose identification relies upon serotyping, cellular fatty acid analysis, and molecular typing. Most of the isolates have been recovered from the blood of patients, and three of the four pathogenic Bartonella species are associated with infectious endocarditis. We performed PCR-restriction fragment length polymorphism (RFLP) analysis of the blood culture bottle supernatant for the routine identification of Bartonella species among fastidious gram-negative bacteria. The amplification of the citrate synthase gene with primers previously reported (R. L. Regnery, C. L. Spruill, and B. D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) yielded a 379-bp product from Bartonella species and a 382-bp product for Capnocytophaga ochracea but no product from any of the other 15 genotypically or phenotypically related species tested. We determined the sequences of the citrate-synthase gene-amplified products for Bartonella species and C. ochracea in order to predict the optimal restriction enzyme to be used in RFLP analysis. TaqI and AciI allowed identification of Bartonella species and C. ochracea. We propose that acridine orange and Gram staining, followed by PCR-RFLP analysis of the blood bottle supernatant, be included in the examination of blood samples from patients with suspected infectious endocarditis. PMID- 7545185 TI - Interference by hydroxyethyl starch used for vascular filling in latex agglutination test for cryptococcal antigen. AB - The glucuronoxylomannan component of the Cryptococcus neoformans capsular polysaccharide confers serotype specificity, and its detection in cerebrospinal fluid or serum by the latex agglutination test is used for diagnosis. Low molecular-weight hydroxyethyl starches can be used as an alternative to albumin for vascular filling. This study reports the occurrence of a false-positive result with the Pastorex Cryptococcus test (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) for a patient receiving hydroxyethyl starch characterized by a substitution ratio of 0.6 (Elohes, Biosedra, Sevres, France). PMID- 7545188 TI - Postnatal changes in the uncrossed retinal projection of pigmented and albino Syrian hamsters and the effects of monocular enucleation. AB - Anterograde and retrograde tracing techniques have been used to study the uncrossed retinal projection in neonatal pigmented and albino Syrian hamsters. The total number of retinal ganglion cells projecting ipsilaterally peaks at postnatal days 2-4 (P2-P4) and declines to adult values by P12. The change in cell numbers has a similar time course in albino and pigmented animals. Although the population of uncrossed cells in the temporal retina of albino hamsters is always less than that in pigmented hamsters, no difference between the colour phases was found for the population of uncrossed cells in nasal retina. Differential cell death also contributes to the adult albino decussation pattern in hamsters: The relative loss of cells from temporal retina in albinos (72%) is greater than that in pigmented animals (56%). The additional loss in albinos does not appear to depend on binocular interactions: The same proportion (30%) of uncrossed cells is "rescued" from death by neonatal monocular enucleation in both colour phases. Flat-mount preparations showing the distribution of uncrossed fibres reveal that a distinct focus of terminals emerges in rostral superior colliculus, which is topographically appropriate for a binocular mapping, at the peak of uncrossed ganglion cell numbers (P4). Comparison of uncrossed terminal distributions and ganglion cell death reveals considerable refinement of the terminals prior to the main phase of cell death. Monocular enucleations performed some time after birth have a greater effect on uncrossed terminal distributions than on cell death. These observations suggest that independent mechanisms may be involved in the regulation of terminal distributions and of cell numbers in the developing uncrossed retinal pathways. PMID- 7545184 TI - Concentration of sputum by cytocentrifugation for preparation of smears for detection of acid-fast bacilli does not increase sensitivity of the fluorochrome stain. AB - To evaluate the usefulness of cytocentrifugation for detection of acid-fast bacilli (AFB) in sputum specimens, we compared this method to a traditional concentration method for the preparation of smears. A total of 844 sputum specimens (from 579 patients) of adequate volume that were submitted for detection of mycobacteria were evaluated. A portion of each specimen was used for cytocentrifugation; the remainder was processed by our decontamination concentration protocol (2% sodium hydroxide-N-acetyl-L-cysteine; centrifugation at 3,600 x g for 15 min) for preparation of smears and culture. All smears were stained with auramine O. Ninety-four cultures from 46 patients gave positive results, and AFB were seen in one or both smears from 53 specimens; 3 of the latter specimens (positive by both smear methods) were culture negative. Of the 50 AFB smear-positive and culture-positive specimens, 46 were smear positive by traditional concentration, and 47 were positive by the cytocentrifugation smear (P, not significant). Cultures of all specimens that were smear positive by only one method grew nontuberculous mycobacteria. The routine use of cytocentrifugation for concentrating sputum specimens increases the cost of smear preparation but does not increase detection of AFB by auramine O staining; however, it would be useful in handling emergent requests for AFB smears. PMID- 7545187 TI - CD19 and immunophenotype of bone marrow plasma cells in monoclonal gammopathy of undetermined significance. AB - AIMS: To determine whether a particular phenotype or antigen is preferentially related to monoclonal gammopathies of undetermined significance (MGUS). METHODS: Bone marrow specimens from 56 patients with MGUS were stained immunocytochemically (ABC peroxidase) for CD38, CD56, CD9, CD10, CD19, CD20, CD22, and MB2. Specimens from patients recently diagnosed with multiple myeloma and reactive bone marrow samples were studied in parallel. RESULTS: CD38 was expressed on all plasma cells from all MGUS samples tested, while 36% were positive for CD56, CD9 and MB2 were both expressed strongly; CD20 was moderately expressed, and staining for CD10 and CD22 was uncommon. For these five B cell antigens there was no clear difference between their expression in MGUS and in multiple myeloma. A great difference was found for CD19: in MGUS this antigen was expressed on 2-91% of plasma cells (mean 35%) and 77% patients had > 10% positive plasma cells; in multiple myeloma its expression was low and only 12% patients had > 10% positive plasma cells. When these results were converted to numbers of CD19 positive plasma cells per 100 nucleated bone marrow cells, reactive bone marrow and MGUS specimens had a similar number of positive plasma cells. There was no correlation between expression of any of the antigens tested. CONCLUSIONS: Many of the so-called pre-B, B or activation antigens are present on plasma cells from MGUS specimens, and expression of CD9, CD10, CD20, CD22, MB2, and CD38 in MGUS was very similar to that in multiple myeloma. CD56 was frequently expressed in MGUS. In this series CD19 was highly expressed in MGUS but not in multiple myeloma. Plasma cells bearing this antigen could represent the non-neoplastic process and determination of its expression could be useful for the diagnosis of MGUS. PMID- 7545186 TI - Activated phenotype in neutrophils and monocytes from patients with primary proliferative polycythaemia. AB - AIM: To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines. METHODS: Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry. Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli. Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-gamma (IFN-gamma) by enzyme linked immunosorbent assay (ELISA). RESULTS: Patients with PPP (n = 18), when compared with normal subjects (n = 10), had increased percentages of CD64+ monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%). Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36). These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia. The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n = 5). G-CSF, GM-CSF and IFN-gamma concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples. There was no correlation between cytokine concentrations and the expression of CD11b, CD14, CD18, and CD64 on patients' phagocytes. CONCLUSIONS: Increased expression of CD11b and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these cells are activated in vivo in patients with PPP. The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone. PMID- 7545189 TI - Lateral superior olive projections to the inferior colliculus in normal and unilaterally deafened ferrets. AB - We have examined the projection from the lateral superior olive (LSO) to the inferior colliculus (IC) in the ferret, with particular interest in the laterality of the projection and in the effects of unilateral cochlear removal in infancy. Large or small deposits of the retrograde tracer wheat germ agglutinin horseradish peroxidase (WGA-HRP) were made in the IC of anesthetized adult ferrets that either were normally hearing or had been unilaterally deafened in infancy (P5 or P25). After 2 days, the ferrets were perfused, and frontal sections of the brainstem were treated with tetramethyl benzidine. For large deposits of WGA-HRP, equal numbers of labelled neurons were found evenly spread through both LSOs. Smaller deposits of WGA-HRP produced four results that contrasted with previous reports on the cat. First, many more labelled neurons were found in the contralateral than in the ipsilateral LSO. Second, the relative number of labelled neurons in each LSO was independent of whether the deposits were in the ventral or in the dorsal IC. Third, the total number of labelled LSO neurons was independent of whether the deposits were in the ventral or in the dorsal IC. Fourth, the proportion of ipsilateral to contralateral labelled neurons was slightly higher in the medial LSO than in the lateral LSO. Ventral IC deposits resulted in more labelled neurons in the medial LSO, and dorsal IC deposits resulted in more labelled neurons in the lateral LSO, as expected. Neonatal cochlear removal did not change any of these results. We conclude that, in the ferret, the organization of the crossed and uncrossed projections from the LSO to the IC differs from that of the cat, and any similarity with the optic chiasm is not obvious. PMID- 7545190 TI - Direct projections from the dorsolateral pontine tegmentum to pudendal motoneurons innervating the external urethral sphincter muscle in the rat. AB - Direct projections from the dorsolateral pontine tegmentum to pudendal motoneurons innervating the external urethral sphincter and the external and sphincter muscles were examined in the rat by the tract-tracing methods utilizing retrograde transport of cholera toxin B subunit and anterograde transport of biotinylated dextran amine. The dorsolateral pontine tegmental region, corresponding to the micturition reflex center of Barrington, was confirmed to send bilaterally, with an ipsilateral dominance, projection fibers to the spinal parasympathetic nucleus (inferior intermediolateral nucleus). The micturition reflex center of Barrington, however, did not seem to send many projection fibers to the ventral horn of the lumbosacral cord segments, whereas the region immediately ventral to the micturition reflex center of Barrington was found to send bilaterally, with a contralateral dominance, projection fibers to the dorsolateral group of pudendal motoneurons in both the male and female rats. In the female rat, the dorsolateral group of pudendal motoneurons are comprised primarily of motoneurons that innervate the external urethral sphincter muscle. The dorsomedial group of pudendal motoneurons, which contain motoneurons that innervate the external and sphincter and the bulbocavernosus muscles, did not seem to receive major projections from the dorsolateral pontine tegmental regions. It was also observed that the locus coeruleus sent some projection fibers bilaterally to the spinal parasympathetic nucleus but only a few to the ventral horn of the lumbosacral cord segments. Thus, the present results indicate that the dorsolateral group of pudendal motoneurons containing those innervating the external urethral sphincter muscle receive pontospinal projection fibers mainly from the dorsolateral pontine tegmental region immediately ventral to the micturition reflex center of Barrington. PMID- 7545192 TI - Telecommunications and nursing education. AB - Telecommunications are becoming increasingly important to nursing educators. At the University of Nebraska Medical Center College of Nursing, communication by two-way television, computers, facsimile machines, and telephone conferences is essential to the administration and operation of a school with four divisions located across 500 miles. Two-way television is available through one system that uses satellite and fiberoptic technology and another that uses telephone lines. The four campuses of the college share classes, administrative meetings, and conferences through television. Faculty members teaching via TV are oriented to designing instructional material for transmission and to the minor quirks of the technology. Students in TV classes must be aware of their responsibility for active involvement in learning. Studies have found no significant differences in the grades of students in "live" classrooms and those in TV classrooms, but both faculty and students prefer the face-to-face situation. The College of Nursing uses computers extensively on an internal network linking the four campuses for E mail, file transfer, computer-assisted instruction, and administrative information sharing. Another computer network, Synapse Health Resources Online, links the college and the Medical Center with health professionals in rural areas throughout the state. PMID- 7545191 TI - The impact of a family systems nursing approach: nurses' perceptions. AB - Relationships between nurses and families form the foundation of effective nursing practice. This article describes a study of medical-surgical nurses' perceptions of the impact of a Family Systems Nursing Continuing Education Project (FSNP). Four major themes emerged from the study: 1) reduction in family crises during acute hospitalizations; 2) increased efficiency and productivity of discharge planning; 3) increased family involvement in multidisciplinary conferences with nurse-family interactions described as collaborative; and 4) improvement in nursing competence and confidence while caring for families. Implications for clinical practice, continuing education, and research are presented. PMID- 7545195 TI - Regulation of Ito cell proliferation by soluble factors. PMID- 7545193 TI - Celiac sprue and macroamylasemia: potential clinical and pathophysiological implications. Case study. AB - In a patient with celiac sprue, abdominal complaints were ascribed to acute pancreatitis in the setting of hyperamylasemia and choledocholithiasis. The problem turned out to be macroamylasemia associated with elevated serum immunoglobulin A. The patient remained stable for 4 years with no specific intervention except gluten restriction. An association between celiac sprue or other malabsorptive states and macroamylasemia requires more study. PMID- 7545194 TI - Expression of cell-matrix adhesion molecules in the liver and their modulation during fibrosis. PMID- 7545196 TI - Serum assays for liver fibrosis. PMID- 7545197 TI - Soluble E-selectin and vascular cell adhesion molecule-1 (VCAM-1) in primary biliary cirrhosis. AB - BACKGROUND/AIMS: E-selectin and vascular cell adhesion molecule-1, important in leucocyte adhesion, have recently been detected in soluble form in the circulation. However, their clinical significance remains unclear. Our aims were to determine whether the levels of these molecules are increased in primary biliary cirrhosis, and to relate these to histological disease stage, biochemical measures of liver damage and to lymphocyte activation. METHODS: We studied 42 patients with primary biliary cirrhosis, nine with primary sclerosing cholangitis, 14 with alcoholic liver disease and 17 healthy subjects. Circulating E-selectin and vascular cell adhesion molecule-1 levels were measured by enzyme linked immunosorbent assay. In subgroups of patients with primary biliary cirrhosis, hepatic bile acid uptake and excretory rates and T-cell activation were also determined. RESULTS: Soluble E-selectin and vascular cell adhesion molecule-1 levels were significantly elevated in primary biliary cirrhosis compared to healthy controls. However, there was no difference between primary biliary cirrhosis and other liver disease groups. In primary biliary cirrhosis, both adhesion molecules correlated with disease stage, but differed in their relationships with specific liver function tests. They did not correlate with either hepatic bile acid uptake or excretion, or lymphocyte activation. CONCLUSIONS: We conclude that soluble E-selectin and vascular cell adhesion molecule-1 are elevated in chronic liver diseases. In primary biliary cirrhosis, they reflect the stage of disease and may reflect the degree of leucocyte adhesion and migration. PMID- 7545198 TI - T cell autoreactivity against a 28 kD biliary protein (B1-p28) in primary biliary cirrhosis. AB - BACKGROUND/AIMS: Cellular immune responses against biliary epithelial cells are important in understanding the pathogenesis of primary biliary cirrhosis. We previously reported a biliary antigen B1 which stimulated peripheral T lymphocytes of primary biliary cirrhosis as a possible, non-mitochondrial target of biliary epithelial cells. Further characterization of B1 was performed. METHODS: To confirm localization of B1 in biliary epithelial cells, a mouse monoclonal antibody was raised against B1. As B1 could be separated into two main components by SDS-PAGE under reducing condition, these components were individually cut out from Western blots and converted into antigen-bearing particles for a proliferation assay to detect the antigenic component responsible for stimulating lymphocytes. The relation of this proliferation to HLA DR antigens was also analyzed. RESULTS: Immunohistochemically, B1 was shown to be specifically expressed on biliary epithelial cells of human liver tissue. Of two components from B1 under reducing condition, prominent proliferation against B1 p28, a 28 kD component of B1 was detected in primary biliary cirrhosis. The reactivities were significantly higher than chronic liver diseases (p < 0.001) or normal controls (p < 0.001). Furthermore, high responders to B1-p28 were observed frequently in primary biliary cirrhosis patients with HLA DR8 (p < 0.02), which was susceptible to the development of primary biliary cirrhosis. CONCLUSIONS: These data suggest B1-p28 expressed on biliary epithelial cells is a candidate for the target antigen of primary biliary cirrhosis in addition to mitochondrial autoantigens. Further characterization of B1-p28 may provide new insight into the autoimmune mechanisms of primary biliary cirrhosis. PMID- 7545200 TI - Flow cytometric analysis of protein-tyrosine phosphorylation in peripheral T cell subsets. Application to healthy and HIV-seropositive subjects. AB - This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine phosphorylation levels in T cells subsets following stimulations which mimic physiological activation. Overall data obtained in healthy subjects, using two- or three-color immunofluorescence, indicated that: (1) most CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinking. Nevertheless, the two subsets follow similar kinetic patterns and tend to retain a homogeneous profile. Processing of samples from HIV-seropositive patients showed heterogeneous phosphorylation levels in both subsets, when compared to normal donors. This assay should, in the future, lead to easy and rapid exploration of the signal transduction pathway in different subsets of T cells under normal and pathological conditions. PMID- 7545201 TI - Effects of monoclonal anti-c-kit antibody (ACK2) on melanocytes in newborn mice. AB - Previous studies indicate that c-Kit is required for postnatal melanocyte development. To understand the precise mechanisms of c-Kit dependence, we studied melanocyte development in newborn C57BL/6 mice by means of peritoneal injection of a monoclonal anti-c-Kit antibody (ACK2), which blocks c-Kit functions. The mice were injected once or more with ACK2 at various intervals after birth. In experiment 1, skin samples were examined on day 10 post-partum and in experiment 2 they were examined daily until day 10 post-partum. We studied melanocytes in the hair follicles, epidermis, and dermis by light and electron microscopy with dopa reactions and immunohistochemistry. Epidermal melanocytes in untreated mice were dopa negative and c-Kit positive on day 0 post-partum but became dopa positive soon thereafter. In ACK2-treated mice, the earlier the mice received ACK2 injections after birth, the fewer melanocytes they had, not only in the epidermis, but also in follicles. In these mice, melanocytes that had undergone apoptosis in the dermis and the follicles were detected ultrastructurally. Some appeared to have produced tyrosinase, because they had dopa-positive melanosomes. These results suggest that melanocytes in newborn mice are c-Kit dependent and undergo apoptosis when c-Kit receptors are blocked by ACK2 in the early days after birth. During this c-Kit-dependent period, melanocytes differentiate from dopa negative to positive and migrate from the epidermis to hair follicles. PMID- 7545199 TI - Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis. AB - BACKGROUND/AIMS: Little is known about the pathogenesis of liver damage related to hepatitis C virus. The presence of steatosis or increased ferritin levels, and preliminary data on the relevance of iron as a prognostic factor prompted us to ascertain whether hepatitis C virus-related liver damage might be mediated by iron accumulation. METHODS: We evaluated the degree of hepatic inflammation and steatosis, serum ferritin, transferrin saturation and iron levels, tissue iron concentrations and iron index, liver glutathione and malondialdehyde in 33 males and 20 females with chronic hepatitis C virus- or hepatitis B virus-related hepatitis (42 + 11). We also considered six patients with both alcohol abuse and hepatitis C virus, four males with chronic alcoholic liver disease and four males with genetic hemochromatosis, giving a total of 67. All diagnoses were histologically confirmed. Patients with cirrhosis were excluded. RESULTS: Our data show that: 1. Steatosis is more frequent in hepatitis C virus and hepatitis C virus+alcohol abuse patients; 2. In males, serum ferritin and tissue iron are significantly higher in hepatitis C virus- than in hepatitis B virus-positive patients (p < 0.01 and 0.05); transferrin saturation is higher (p < 0.05) in hepatitis C virus-positive than in hepatitis B virus-positive patients only when males and females are considered together; 3. Serum ferritin and transferrin saturation only correlate with liver iron (r = 0.833 and r = 0.695, respectively, p = 0.00001); tissue iron is significantly higher in hepatitis C virus- than in hepatitis B virus-positive patients (p < 0.05); 4. In patients with chronic hepatitis, serum ferritin is a better marker of liver iron storage than transferrin saturation, both in males and in females; 5. Hepatitis C virus positive patients have higher malondialdehyde levels and activation of turnover of glutathione, probably in response to free-radical-mediated liver damage. Females have lower liver iron levels but similar trends. CONCLUSIONS: These findings suggest that hepatitis C virus-related liver damage is characterized by increased iron storage (possibly induced by the virus) which elicits a free radical-mediated peroxidation, with consequent steatosis and activation of glutathione turnover. PMID- 7545202 TI - Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors. AB - In vitro angiogenesis models suggest that new blood vessel formation requires the induction and secretion by endothelial cells of matrix metalloproteinases. These enzymes assist in the controlled proteolytic degradation of the surrounding extracellular matrix during blood vessel formation. The results of in vitro studies cannot be extrapolated directly to the process of in vivo angiogenesis because the type of matrix employed and the repertoire of enzymes secreted by cells in vivo differ dramatically from in vivo conditions. To investigate the in vivo role of matrix metalloproteinases in blood vessel development, we looked for the presence of these proteinases in endothelial cells involved in fetal angiogenesis and in neovascularization of certain invasive skin tumors using immunofluorescent staining. In fetal tissue, interstitial collagenase was present in both early microvessels developing from undifferentiated mesoderm and in microvessels involved in elongation and sprout formation from preexisting blood vessels. In aggressive skin tumors, i.e., morpheaform and recurrent basal cell carcinomas and squamous cell carcinomas, there was a marked increase in the number of collagenase-containing blood vessels, often extending into the tumor nests. Immunofluorescent staining failed to detect stromelysin, matrilysin, or gelatinase A and B (72- and 92-kDa type IV collagenases, respectively) in fetal or tumor blood vessels. These findings are consistent with the hypothesis that proteolytic degradation of the extracellular matrix is required for the formation of new blood vessels. Interstitial collagenase appears to play an important role in this process. PMID- 7545205 TI - Contamination of mercurochrome and chlorhexidine-cetrimide in a teaching hospital. AB - A study on the contamination of antiseptics was done in a teaching hospital, during March and April 1989. Ninety-five samples of 9 antiseptics from 23 wards were cultured. Results showed that 2 of 12 and 3 of 5 samples of mercurochrome and chlorhexidine-cetrimide compound were contaminated. This led to a wider scale study on the two products. It was found that over one-half of the samples of mercurochrome and 6 of 54 samples of chlorhexidine-etrimide compound were contaminated. Mercurochrome was contaminated by gram-positive and chlorhexidine cetrimide compound by gram-negative bacteria. Contamination of mercurochrome was found in samples taken from the pharmacy and wards, and of chlorhexidine cetrimide compound from wards only. Mercurochrome was removed from the hospital formulary and the use of chlorhexidine-cetrimide compound was more restricted. PMID- 7545206 TI - The use of dolls to interview young children: issues of symbolic representation. AB - A very common practice in the investigation of suspected sexual abuse is to use anatomically detailed dolls to interview children. The use of such dolls is particularly advocated for very young children. For a doll to be useful, however, children must accept and use it as a representation of themselves. Our previous research on 2- and 3-year-old children's understanding of symbolic objects led us to hypothesize that such very young children might have difficulty understanding and using a doll as a self-representation. In the study reported here, 2-1/2-, 3 , and 4-year-old children played some games with an experimenter, and they were interviewed immediately afterward. The children did, as expected, have difficulty using the doll as a self-representation and mapping from themselves to the doll. As a consequence, they provided more correct information in their direct (verbal and nonverbal) responses to the interviewer's questions than they demonstrated on the doll. Implications and limitations of this research are discussed with respect to interviewing young children. PMID- 7545203 TI - Cognate interaction between human lymphocytes and eosinophils is mediated by beta 2-integrins and very late antigen-4. AB - Here the cognate interactions between human eosinophils and lymphocytes are studied. These interactions were measured in a double-colored FACS analysis by applying fluorescent red eosinophils (stained with hydroethidine, 40 mumol/L) and fluorescent green lymphocytes (stained with sulfidofluorescein diacetate, 100 mumol/L) in a ratio of 1:3. When normal eosinophils were mixed with a total lymphocyte preparation in stirred suspensions (37 degrees C), no physical interaction was present between both cell types. However, the addition of phytohemagglutinin and PMA resulted in a clear aggregation response between both cell types (up to 30% of the eosinophils interacted with lymphocytes after 15 minutes). CD8(+)- and CD4(+)-positive T cells contributed equally to the heterotypic aggregation response. It is interesting that when lymphocytes were pretreated with phytohemagglutinin or eosinophils with phorbol myristate acetate and subsequently washed, the cells still interacted with unstimulated counterparts. The heterotypic interaction between lymphocytes and eosinophils is blocked by monoclonal antibodies directed against the beta-chain of the beta 2 integrins (CLB LFA1/1; CD18) and the alpha-chain of very late antigen-4 (VLA-4) (HP2/1; CD49d), indicating that both the beta 2-integrins and VLA-4 contributed to this heterotypic interaction. When eosinophils bound to PHA-treated lymphocytes, the cells exhibited an activated phenotype that was characterized by an enhanced expression of CD66b and CD11b. The cells interacted with each other provided that an intact cellular metabolism was present--that is, no interaction was seen after treatment with the glycolysis inhibitor sodium mono iodoacetate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545204 TI - Occurrence of multiple aberrantly spliced mRNAs of the LDL-receptor gene upon a donor splice site mutation that causes familial hypercholesterolemia (FHBenevento). AB - A novel point mutation of the LDL-receptor gene was found in an Italian patient with homozygous familial hypercholesterolemia. The SSCP analysis of the promoter and of 16 out of the 18 exons of the LDL-receptor gene was negative, suggesting that the mutation might be located in the region of the gene encompassing exons 14 and 15, a region that had not been amenable to polymerase chain reaction (PCR) amplification from genomic DNA. This region was amplified from cDNA by reverse transcription PCR (RT-PCR). RT-PCR of proband cDNA generated three fragments of 800, 600, and 550 bp, respectively, as opposed to a single 720 bp fragment obtained from control cDNA. The sequence of these fragments showed that: i) in the 800-bp fragment exon 14 continued with the 5' end of intron 15 (90 nucleotides), which in turn was followed by exon 16; ii) in the 600-bp fragment exon 14 was followed by the 5' end of exon 15 (50 nucleotides), which continued with exon 16; iii) in the 550-bp fragment exon 14 joined directly to exon 16. These abnormally spliced mRNAs resulted from a G-->A transition at the +1 nucleotide of intron 15, which changed the invariant GT dinucleotide of the 5' donor splice site. That was associated with the activation of two cryptic donor splice sites in intron 15 and exon 15, respectively, and the use of an alternative splicing leading to the skipping of exon 15. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was one-fourth that found in control cells. These abnormally spliced mRNAs are predicted to encode three abnormal receptor proteins: the first would contain an insertion of 30 novel amino acids; the second would be a truncated protein of 709 amino acids; the third would be devoid of the 57 amino acids of the O-linked sugar domain. Ligand blot experiments indicated that the amount of LDL-receptor present in proband's fibroblasts was approximately one-tenth that found in control cells. PMID- 7545207 TI - Epidemiologic survey and genetic analysis of endemic hepatitis C virus infection in a Japanese town with a high prevalence of hepatitis B virus carriers. AB - Mass screening for hepatitis C virus antibody was carried out in 875 inhabitants (313 men and 562 women) of a town in Japan with a high rate of hepatitis B virus infection. The overall rate of positivity for anti-HCV was 8.8% (6.4% in men and 10.1% in women). The rate of positivity for hepatitis B virus surface antigen was 11.2%. Five subjects (0.6%) were positive for both markers. HCV-RNA was detected in 65 (88.4%) of 77 individuals who were positive for anti-HCV and in 1 (1.5%) of 60 individuals negative for anti-HCV. The genotype of the HCV genome was determined by PCR analysis using type-specific primers in 60 individuals. HCV type 1b was detected in 51 subjects (85%), type 2a in 3 subjects (5%), and type 2b in 6 subjects (10%). None of the individuals was infected with more than one genotype. The nucleotide sequences of the partial nonstructural 5 region of HCV type 1b genotype obtained from 6 individuals showed at least 92.0% homology in the nucleotide sequence, and 94.8% homology in the amino acid sequence. Homology among these clones was greater than their homology with previously described type 1b sequences. The findings suggest that there was a specific local origin of HCV infection, although it was not possible to identify any single source of HCV infection. The results also indicate that presence of asymptomatic HCV carriers. PMID- 7545208 TI - Intrafamilial transmission of hepatitis C virus in hemodialysis patients. AB - The prevalence of hepatitis C virus (HCV) infection in chronic hemodialysis patients ranges from 20 to 50% and these patients may serve as a reservoir of infection for their household contacts. The aim of this study was to investigate the prevalence of anti-HCV in hemodialysis patients and their families, and to evaluate possible routes of infection. One hundred eighty-six family members of 84 hemodialysis patients and 529 healthy adults were enrolled. The family members consisted of 50 spouses, 96 children, 11 parents, 29 siblings, and other relatives living together with the patients. Serum samples were collected for testing anti-HCV. Exposure to risk factors was obtained by a questionnaire and an interview. The results showed that prevalence of anti-HCV in hemodialysis patients was 44%, whereas in family members it was 5.4%, not significantly different from that of age-matched healthy adults (standardized morbidity rate = 1.51, P = 0.390). The anti-HCV rate in family members tended to increase with age, and a spouse of an infected hemodialysis patient had a higher risk of HCV infection than other family members (15% vs. 2.6%, odds ratio 6.6, P = 0.058). Except for the age factor, no difference was found between seropositive and seronegative family members with respect to risk factors such as blood transfusion, surgery, frequent injections, dental procedures, or acupuncture. It was concluded that, although the anti-HCV positivity of hemodialysis patients is high, the risk of HCV infection for their family members is not higher than that of the general population. Among family members, spouses of seropositive hemodialysis patients have the highest risk of HCV infection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545210 TI - Hepatitis C virus genotypes in French blood donors. AB - The prevalence of different hepatitis C virus (HCV) genotypes in HCV infected individuals and the relation between the HCV genotypes and the source of the infection are controversial. The aim of this study was to determine the HCV genotypes in French blood donors. Fifty-one anti-HCV positive blood donors were studied with detectable serum HCV RNA by nested polymerase chain reaction (PCR) using primers derived from the 5' non-coding region. For genotyping HCV, we used a method based on analysis of the restriction fragment length polymorphisms (RFLP) after amplification by PCR of the HCV non-structural 5 (NS5) genome domain. Using this technique, the genotypes of 39 of the 51 blood donors (76%) were determined. Three previously described genotypes were found: 19 blood donors were infected by HCV genotype I (37%), 14 were infected by HCV genotype II (27%), 3 were infected by HCV genotype III (6%), and 3 were coinfected by two genotypes (6%). All three blood donors infected with two different genotypes were intravenous drug abusers. A past history of intravenous drug abuse was more frequent in blood donors with HCV genotype I. However, there was no difference in alanine transaminase (ALT) levels, histological lesions, and RIBA-2 patterns in blood donors infected with either HCV genotype I or HCV genotype II. These findings indicate that most HCV genotypes isolated from French blood donors belong to HCV genotype I and HCV genotype II, and that risk factors for HCV infection may differ for different genotypes of HCV. PMID- 7545211 TI - Detection of antibodies to L1, L2, and E4 proteins of human papillomavirus types 6, 11, and 16 by ELISA using synthetic peptides. AB - Antibodies against eight synthetic peptides spanning different epitopes located on L1, L2, and E4 proteins of human papillomavirus (HPV) types 16, 6, and 11 were examined in sera from 73 women infected by HPV and from 139 healthy controls. Only three of these peptides were reactive. Two located on proteins L2 and E4 of HPV 16 seem type specific since antibodies to these peptides were detected, respectively, in 21% and 15% of the HPV 16 infected patients and in 2.5% and none of women infected by other HPVs. The third peptide located on the L1 protein of HPV 6 bears a common epitope since antibodies to this peptide were detected not only in 85% of women infected by HPV 6 or 11, but also in 82% of women infected by other HPVs, and in 74% and 71% of the control groups (10-12-year-old children and adults, respectively). In conclusion, none of the peptides investigated seems useful to develop ELISAs for serological diagnosis of HPV infection. PMID- 7545212 TI - Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia. AB - The second envelope protein (E2) of the hepatitis C virus (HCV) was cloned and expressed in Chinese hamster ovary (CHO) cells. This E2 glycoprotein was purified using ion exchange and lectin chromatography and used to construct an enzyme immunoassay for HCV E2 antibodies. The assay was shown to have good specificity, and detection of E2 antibodies was positively correlated (97.3%) to the presence of HCV RNA in serum and plasma. A high concordance between HCV 2.0 and E2 EIA reactivities was also observed. E2 antibody was the first serological marker to appear in 3/5 HCV seroconversion panels. This work demonstrated that 42.4% of core and 15.4% of NS3 indeterminate specimens also contained antibodies to E2, suggesting that HCV infection had occurred in these individuals. The E2 antibody assay was used to evaluate HCV 2.0 EIA-positive, HCV 3.0 EIA-negative plasma donors with indeterminate reactivity on RIBA HCV 2.0 or MATRIX HCV 1.0. Several HCV 3.0-negative specimens were shown to contain E2 antibodies in addition to an original indeterminate serological marker, primarily core. It is concluded that anti-E2 is a useful marker for determining HCV infection, and that the presence of antibodies to two nonoverlapping viral gene products suggests true HCV exposure. New HCV 3.0 blood screening tests should detect HCV 2.0-positive donors who present with an indeterminate pattern by RIBA or MATRIX and who also carry E2 antibodies. PMID- 7545213 TI - Hepatitis C virus infection in Schistosomiasis mansoni in Brazil. AB - The involvement of the hepatitis C virus (HCV) in the severity of liver disease in chronic schistosomiasis was investigated in 215 Brazilian patients with S. mansoni infections, but without evidence of hepatitis B surface antigen (HBsAg). Forty-three had hepatointestinal (HIS) and 172 had hepatosplenic schistosomiasis (HSS), and 135 had compensated (HSSC), and 37 had decompensated (HSSD) liver disease. Fifty-two (24%) were found to have evidence of HCV infection (seropositive for anti-HCV antibodies and/or HCV-RNA). These comprised 35 (95%) of the 37 with HSSD, 16 (12%) of the 135 with HSSC, and 1 (2.4%) of the 43 with HIS, compared with only 1 (2%) of 50 control patients without S. mansoni. Testing of matched liver tissue and peripheral blood mononuclear cells (PBMCs) from 25 patients (6 HSSC and 19 HSSD) with HCV infections showed that 17 (68%) had "active" viral infections, in that negative strand HCV-RNA (the presumed replicative intermediate of the virus) could be detected in liver and/or PBMCs. Among these 25, negative strand HCV-RNA was found in 16 (84%) of the 19 with chronic active hepatitis, but in only 1 (17%) of the 6 with mild or inactive disease (P < 0.01). HCV-RNA was detected in matched spleen specimens from 9 of 10 patients (all of whom were also positive in PBMCs), suggesting that the spleen is an important extrahepatic reservoir of the virus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545209 TI - A helper T-cell antigen enhances generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro. AB - A T-cell helper for generation of hepatitis C virus-specific cytotoxic T lymphocytes was studied in three patients with chronic hepatitis C. In all three, human leukocyte antigen B44-restricted cytotoxic T lymphocytes recognizing an epitope in hepatitis C virus nucleocapsid protein residues 81-100 were generated from the peripheral blood lymphocytes by repeated stimulation with a synthetic hepatitis C virus nucleocapsid peptide. The proliferative response of peripheral blood lymphocytes to hepatitis C virus nucleocapsid protein residues 1-120 was observed in one patient, and was ascribed to CD4+ T cells. The helper T cells recognized a major epitope in residues 21-40 and a minor epitope(s) in residues 81-110. They produced interferon gamma, but interleukin 4 was not detectable in the T-helper cell culture supernatants. The hepatitis C virus nucleocapsid protein residues 1-120 and the major helper T-cell epitope enhanced generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro, although the protein alone did not generate them. In the other two patients, the protein did not enhance generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro. The results suggest that a hepatitis C virus-specific helper T-cell epitope is helpful for inducing a strong specific cytotoxic T-lymphocyte response. PMID- 7545214 TI - Treatment of chronic hepatitis C with cirrhosis with recombinant human granulocyte colony-stimulating factor plus recombinant interferon-alpha. AB - Interferon therapy in cirrhotic patients with hepatitis C virus infection is not efficient. In an attempt to improve the response rate, a pilot study using recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone, and in combination with recombinant interferon-alpha (rIFN), was carried out. Fifteen cirrhotic patients with hepatitis C virus infection were randomly allocated into 3 groups to receive treatment: 0.5, 1, or 1.5 micrograms/kg of rhG-CSF daily for 4 weeks, followed by a 4 week resting period, and by the same dose of rhG-CSF daily, plus 6 MU of rIFN 3 times weekly for 4 weeks. They then continued to receive 6 MU of rIFN alone for 4 weeks, followed by 3 MU of rIFN for 16 weeks. After the 4 weeks of treatment with rhG-CSF alone, no changes in alanine aminotransferase (ALT) levels were observed. No changes occurred during the resting period. Three of 10 patients who ended the rhG-CSF plus rIFN treatment period had normal ALT values. During the treatment with rIFN alone, two responders suffered a relapse. The other responder had normal ALT until the first month of follow-up. In summary, the combination of rhG-CSF and rIFN seems promising for the treatment of patients with chronic hepatitis C and liver cirrhosis. PMID- 7545215 TI - Mapping of a human T-lymphotropic virus type I gag protein epitope that cross reacts with anti-Plasmodium falciparum antibodies. AB - Studies in Palawan, Philippines, and Irian Jaya, Indonesia, showed that indeterminate human T-lymphotropic virus type I (HTLV-I) Western blot immunoreactivity is due to cross-reacting anti-Plasmodium falciparum antibodies. To further define this immunoreactivity, mapping studies were conducted using the HTLV-I p19 protein to identify the precise epitope that reacts with these antibodies. Anti-P. falciparum antibody-positive sera from Palawan, Philippines, and Irian Jaya, Indonesia, were studied using overlapping synthetic peptides. Immunoreactivity was localized to residues 108-120 of p19. Further analysis of the sera with 5 biotinylated synthetic peptides showed that the cross-reactive epitope consists of the sequence PDSDPQI (amino acid residues 110-116), which was shown to be homologous to a 7 amino acid sequence on the Exp-1 protein of the P. falciparum blood stage parasite. This is the first study that identifies a specific HTLV-I protein epitope that cross-reacts with malaria antibodies. PMID- 7545216 TI - Peroxisomal disorders: a review. AB - The peroxisomal disorders represent a group of inherited diseases in man in which there is an impairment in one or more peroxisomal functions. The disorders known up to now are usually subdivided into three groups depending upon whether there is a more generalized, multiple or single loss of peroxisomal functions. In this paper we will briefly describe the peroxisomal disorders known thus far with the biochemical abnormalities identified. Furthermore, we will describe a straightforward approach for the postnatal identification of patients suspected to suffer from a peroxisomal disorder which is of great importance since reliable prenatal diagnostic methods have become available for each of these disorders. PMID- 7545217 TI - Pain relief into practice: rhetoric without reform. PMID- 7545218 TI - Prospective evaluation of hydrocortisone and suramin in patients with androgen independent prostate cancer. AB - PURPOSE: To assess efficacy of intermittent infusion of suramin in patients with androgen-independent prostate cancer who have had disease progression on hydrocortisone. PATIENTS AND METHODS: Chemotherapy-naive patients with progressive androgen-independent prostate cancer were given hydrocortisone 40 mg/d and monitored for treatment effect. At the time of disease progression, suramin was administered on a pharmacokinetically derived, 2-week dosing schedule. RESULTS: Thirty patients with a median Karnofsky performance status (KPS) of 90% were treated with hydrocortisone. No responses were seen in 12 patients with measurable disease or 29 patients with abnormal bone scans. Thirty patients had an increasing prostate-specific antigen (PSA) level before treatment and six (20%) had a more than 50% decline in PSA from the baseline value for a median of 16 weeks (range, 12 to 52+). Twenty-eight patients had disease progression after a median of 7 weeks (range, 3 to 23), and two patients have continued to receive hydrocortisone for 44 and 52 weeks. Twenty-eight patients received hydrocortisone and suramin, with median suramin concentrations of 97 to 170 micrograms/mL for 4 weeks. No responses in measurable disease and no improvements in bone scans were seen. Five patients (18%) showed a more than 50% decline in PSA levels from baseline, of whom three had previously responded to hydrocortisone. Only two of 24 patients who did not show a posttherapy decline in PSA levels after hydrocortisone had a reduction in PSA levels with the addition of suramin. Toxicity profiles were acceptable with each agent, although a higher proportion of subjects showed hematologic, cardiac, and neurologic events when suramin was added. CONCLUSION: Suramin has limited efficacy in patients with androgen-independent prostate cancer who have had disease progression after hydrocortisone. PMID- 7545219 TI - Phase I study of paclitaxel and topotecan in patients with advanced tumors: a cancer and leukemia group B study. AB - PURPOSE: To define the dose-limiting toxicities (DLTs) and the recommended phase II doses of paclitaxel combined with topotecan, without and with filgrastim support. PATIENTS AND METHODS: Patients with advanced solid tumors and a maximum of one prior chemotherapy regimen for metastatic disease were eligible if they had a performance status of 0 to 1 and normal renal, hepatic, and bone marrow function. Prior treatment with taxanes or comptothecin analogs, and prior pelvic irradiation were not allowed. Patients with a history of cardiac disease or on medications known to affect cardiac conduction were excluded. The dose of topotecan was fixed at 1.0 mg/m2/d for 5 days. The dose of paclitaxel was escalated until the maximum-tolerated dose (MTD), without and with filgrastim 5 micrograms/kg subcutaneously (SC) on days 6 to 14, was reached. Paclitaxel was administered over 3 hours on day 1 before topotecan. Treatment cycles were repeated every 21 days. RESULTS: Of 46 patients entered, 45 were assessable for toxicity and 34 for response. The principal toxicity was neutropenia. Without filgrastim, the MTD of paclitaxel was 80 mg/m2 on day 1 in combination with topotecan 1.0 mg/m2/d for 5 days. With filgrastim, the dose of paclitaxel was escalated to 230 mg/m2 in combination with the same dose of topotecan. At this dose level, one patient had hematologic DLT and a second patient developed neuromuscular DLT. Three patients had a partial response (PR): one with head and neck cancer, a second with non-small-cell lung cancer, and the third with colon cancer. CONCLUSION: We conclude that paclitaxel can be given at clinically relevant doses in combination with topotecan and filgrastim. The recommended dose for phase II studies is paclitaxel 230 mg/m2 on day 1 and topotecan 1.0 mg/m2/day for 5 days with filgrastim 5 micrograms/kg on days 6 to 14. PMID- 7545220 TI - Evaluation of reproductive capacity in germ cell tumor patients following treatment with cisplatin, etoposide, and bleomycin. AB - PURPOSE: To evaluate the infertility rate in patients with germ cell tumors receiving chemotherapy with cisplatin, etoposide (VP-16), and bleomycin (PVP16B). PATIENTS AND METHODS: Thirty patients were evaluated. All patients had undergone chemotherapy with two to four cycles of PVP16B. A single semen analysis was performed 24 to 78 months following initiation of chemotherapy. All 30 patients were continuously disease-free. Eight of these patients had also undergone nerve sparing retroperitoneal lymph node dissection (RPLND). RESULTS: The median sperm concentration was 33.9 x 10(6), with a median volume of 3.2 mL. The median total sperm count was 86.4 x 10(6). Oligospermia (< 40 x 10(6) total sperm count) was found in 13 patients (43%), including six (20%) who were azoospermic. There was a high incidence of morphologically abnormal sperm, with only one patient having more than 50% normal spermatozoa. Only 13 patients (43%) had sperm motility greater than 50%. Five patients had positive semen antisperm immunoglobulin G (IgG). Eight patients fathered children, including three with document oligospermia. CONCLUSION: Patients with germ cell tumors successfully treated with PVP16B chemotherapy are at substantial risk for persistent semen abnormalities. However, some patients with oligospermia will slowly recover and others are still capable of reproductive capacity despite continued oligospermia. PMID- 7545221 TI - Phase Ia/Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-2/neu. AB - PURPOSE: MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS: Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS: Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION: MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2. PMID- 7545223 TI - Effect of precultivation conditions on colicin susceptibility in Escherichia coli. AB - The effect of 20 colicins and cloacin was studied after various precultivations. Nutrient agar supplemented with subinhibitory concentration of EDTA used for precultivation or elevating the growth-temperature of the inoculum from 37 degrees C to 42 degrees C increased the susceptibility of wild-type (smooth) Escherichia coli strains to the inhibitory action of some colicins. There were great differences among the colicins in respect to these effects. In case of rough mutants, their sensitivities did not change or eventually decrease after EDTA or heat pretreatment. The LPS pattern in SDS-PAGE of smooth cells grown in EDTA-containing nutrient medium changed in some degree towards the rough character. In case of precultivation at 42 degrees C this change was less considerable. It is supposed that both factors applied during precultivation have influence on colicin sensitivity by means of the change of receptor activity caused by LPS modification. PMID- 7545222 TI - Free-riding and the prisoner's dilemma: problems in funding economic analyses of phase III cancer clinical trials. AB - PURPOSE: Both economic and clinical data on new agents are important to policy makers who approve pharmaceuticals for widespread use. Randomized clinical trials have been used to evaluate both clinical results and total medical costs associated with new agents. With new expensive pharmaceutical agents, early assessments of economic benefit have taken on greater importance to physicians and patients. Who should provide financial support to these integrated economic and clinical analyses in clinical trials? Here we describe issues that hinder funding of economic analyses and propose potential support mechanisms. RESULTS: The Cancer and Leukemia Group B (CALGB), a large, national cooperative group of academic and community hospitals in the United States, designed a non-small-cell lung cancer (NSCLC) treatment trial to compare two widely used supportive care regimens that varied 20-fold in cost. One important objective of this trial was to compare the cost-effectiveness of the two regimens. While funding for the clinical trial was supported by grants from the National Cancer Institute and the pharmaceutical companies involved in the trial, no specific funding agency was willing and/or able to provide financial support for the economic analyses. After 2 years of planning, the clinical trial was retracted when the funding for the economic analyses could not be secured. The prisoner's dilemma, individual reluctance to support a common social good, explains the lack of funding. CONCLUSION: Economic theory predicts difficulties in evaluating cost effectiveness of new pharmaceuticals and reluctance to support economic analyses of clinical trials. Economic analyses will require new sources of funds that will not take scarce resources from clinical trials groups. Options for funding include a new federal agency, coordinated work by existing agencies, or academic centers for economic analysis. PMID- 7545224 TI - A phase II trial of mitoxantrone plus cyclophosphamide and 5-fluorouracil in modulation with levo-folinate for advanced breast cancer patients. AB - Advanced breast cancer remains a major clinical problem. Current chemotherapy regimens are able to induce a clinical response in many patients but do not appear to influence significantly patients' survival. The use of new drugs such as mitoxantrone with a predicted lower toxicity and biochemical modulation of 5 fluorouracil with levo-folinate are extensively studied research areas that could combine good therapeutic efficacy with the maintenance of an acceptable quality of life. 34 patients with advanced breast carcinoma were included in the study. Only 4 women had received prior chemotherapy for advanced disease. Treatment plan was: 5-fluorouracil 400 mg/m2 + l-leucovorin 100 mg/m2 days 1-3, cyclophosphamide 600 mg/m2 and mitoxantrone 12-14 mg/m2 on day 3, q28. G-CSF (5 micrograms/kg/d days 7-14) was routinely delivered to the patients with the aim of maintaining dose intensity. 15 patients obtained a response for an overall response rate of 44%. Mean duration was 10.2+ and 11+ months for complete and partial responses respectively. Mean overall survival was 14.4+ months. A high complete response rate was seen in liver metastasis (44%), while lung lesions had a lower probability of response (25%). Toxicity was globally mild with 23% of grade 3 vomiting and 15% of grade 3-4 leukopenia. Two cases of cardiotoxicity were reported. No difference in response rate or toxicity was identified between patients receiving two different mitoxantrone doses (12 or 14 mg/m2). The schedule employed appears to be well tolerated and active in the treatment of advanced breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545225 TI - Inactivating and noninactivating Ca(2+)- and voltage-dependent K+ current in rat adrenal chromaffin cells. AB - The properties of Ca(2+)- and voltage-dependent K+ currents and their role in defining membrane potential were studied in cultured rat chromaffin cells. Two variants of large-conductance, Ca2+ and voltage-dependent BK channels, one noninactivating and one inactivating, were largely segregated among patches. Whole-cell noninactivating and inactivating currents resulting from each of these channels were segregated among different chromaffin cells. Cell-to-cell variation in the rate and extent of whole-cell current decay was not explained by differences in cytosolic [Ca2+] regulation among cells; rather, variation was due to differences in the intrinsic properties of the underlying BK channels. About 75% of rat chromaffin cells and patches express inactivating BK current (termed BKi) while the remainder express noninactivating BK current (termed BKs). The activation time course of both currents is similar, as is the dependence of activation on [Ca2+] and membrane potential. However, deactivation of BKi channels is slower than that of BKs channels. The functional role of these BK channel variants was studied in current-clamp recordings. Although both BKi and BKs currents contribute to action potential repolarization, cells expressing BKi current are better able to fire repetitively in response to constant current injection. Blockade of BKi current by charybdotoxin abolishes this behavior, showing that afterhyperpolarizations mediated by BKi current are permissive for repetitive firing. Thus, important properties of chromaffin cell membrane excitability are determined by the type of BK current expressed. PMID- 7545226 TI - Altered reactivity of human cerebral arteries after subarachnoid hemorrhage. AB - To investigate the effects of subarachnoid hemorrhage (SAH) on the responsiveness of human cerebral arteries to vasoactive substances, the authors measured the isometric tension generated in helical strips of basilar and middle cerebral arteries isolated from human cadavers. Contractions caused by KCl, prostaglandin F2 alpha, noradrenaline, and serotonin were reduced in arteries obtained from cadavers with aneurysmal SAH damage and compared to those obtained from cadavers with no indication of intracranial diseases. Endothelium-dependent relaxation elicited by substance P and bradykinin, and endothelium-independent relaxation induced by prostaglandin I2 and nitroglycerin were also markedly decreased in arteries affected by SAH. However, the reduction in relaxation response to prostaglandin I2 was significantly less than that to the other vasodilator agents. These results indicate that human cerebral artery functions are severely impaired after SAH and that poor responses to vasoactive agents may result primarily from dysfunction of smooth-muscle cells. PMID- 7545227 TI - Congenital astroblastoma: an immunohistochemical study. Case report. AB - Astroblastoma is a rare type of glial tumor, usually occurring in older children and young adults. It has a distinctive histological appearance that is characterized by a radiating arrangement of tumor cells that form perivascular pseudorosettes. The authors report only the second case of astroblastoma presenting in congenital form. Following subtotal tumor resection, the infant received 10 courses of chemotherapy consisting of vincristine, etoposide, and carboplatinum. Evidence is presented for a tumor response to chemotherapy, a previously unreported observation. The child is alive 2.5 years after diagnosis with satisfactory functional status. Immunohistological and ultrastructural features of this tumor are presented. The discussion focuses on the biology, natural history, and management of this unusual neoplasm. PMID- 7545228 TI - Complete resection before development of drug resistance is essential for survival from advanced hepatoblastoma--a report from the German Cooperative Pediatric Liver Tumor Study HB-89. AB - Clinical data and tumor histology of 37 patients with advanced and/or metastatic hepatoblastoma (32 stage III and 5 stage IV) treated according to the protocol of the German Cooperative Pediatric Liver Tumor Study HB-89 from 1988 to 1992 were studied for prognostic factors. Twenty-three patients (73%) were free of tumor 9 months to 5 years (median, 36 months) after treatment, whereas 4 experienced progressive disease, 7 had local relapse, and 3 had recurrent metastases. None of 2 patients with primary lymph node involvement or 5 with primary metastases remained disease-free. Chemotherapy with ifosfamide, cisplatin, and adriamycin was effective in reduction of tumor to resectability in 33 (89%) patients. Drug resistance developed in 6 of 11 patients treated with four or more courses of chemotherapy as could be shown by monitoring of serum-alpha-fetoprotein (AFP) and serial investigations of tumor expansion with sonography and computed tomographic (CT) scan. Only 1 of these patients survived after a liver transplantation. Completeness of tumor resection at second- or third-look laparotomy was significantly related to disease-free survival (P < .0001). Patients with initial serum-AFP values < 100 ng/mL or > 1,000,000 ng/mL had a worse outcome than those with immediate levels (P = .044). The rate of decrease of serum-AFP during chemotherapy was significantly related to prognosis (P = .003). Growth pattern of tumor within the liver (ie, defined nodes versus diffusely disseminated) (P = .011) and vascular tumor invasion (P = .026) were valuable prognostic factors, whereas tumor volume, local infiltration of surrounding tissue, histological subtypes, and epithelial differentiation were not significantly related to the outcome.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545229 TI - Calcium entry through a subpopulation of AMPA receptors desensitized neighbouring NMDA receptors in rat dorsal horn neurons. AB - 1. A Ca(2+)-dependent interaction between non-NMDA and NMDA receptors was studied in embryonic rat dorsal horn neurons grown in tissue culture using perforated patch recording. Specifically, non-NMDA receptors were found to induce reversible inhibition of NMDA receptors in a manner dependent on the presence of extracellular Ca2+. 2. Non-NMDA receptor-induced inhibition of NMDA receptors was mediated by the elevation of intracellular Ca2+ concentration produced by Ca2+ entry through a subpopulation of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionate (AMPA) non-NMDA receptors. Furthermore, Ca2+ entry through the AMPA channels alone is sufficient for desensitization of NMDA channels to occur. 3. Imaging of neuritic sites of Ca2+ revealed that Ca(2+)-permeable AMPA channels are often co-localized with NMDA channels on dorsal horn neurons, indicating that the Ca(2+)-mediated interaction between receptors may occur within small dendritic domains. 4. The ability of Ca(2+)-permeable AMPA channels to inhibit adjacent NMDA channels may contribute to the postsynaptic integration of excitatory input. PMID- 7545230 TI - Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus. AB - 1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by alpha-amino-3- hydroxy-5-methyl-4 isoxazolepropionate receptor (AMPAR)/kainate receptor and N-methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/- 0.1 ms (mean +/- S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/- 0.2ms. 5. The peak current-voltage (I-V) relation of AMPAR-mediated currents in Na(+)-rich extracellular solution showed a reversal potential of -4.0 +/- 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/- 1.6 pS using non-stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in hippocampal basket cells were studied in isolation in the presence of CNQX. Deactivation of NMDARs activated by glutamate pulses occurred bi-exponentially with mean tau values of 266 +/- 23 ms (76%) and 2620 +/ 383 ms (24%). 7. The peak I-V relation of the NMDAR-mediated component in Na(+) rich extracellular solution showed a reversal potential of 1.5 +/- 0.6 mV and a region of negative slope at negative membrane potentials in the presence of external Mg2+, due to voltage-dependent block by these ions. The conductance of single NMDAR channels in the main open state was 50.2 +/- 1.8 pS.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545231 TI - Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum. AB - 1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR-95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR-95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545232 TI - Pacemaker current i(f) in adult canine cardiac ventricular myocytes. AB - 1. Single cells enzymatically isolated from canine ventricle and canine Purkinje fibres were studied with the whole-cell patch clamp technique, and the properties of the pacemaker current i(f) compared. 2. Steady-state i(f) activation occurred in canine ventricular myocytes at more negative potentials (-120 to -170 mV) than in canine Purkinje cells (-80 to -130 mV). 3. Reversal potentials were obtained in various extracellular Na+ (140, 79 or 37 mM) and K+ concentrations (25, 9 or 5.4 mM) to determine the ionic selectivity of i(f) in the ventricle. The results suggest that this current was carried by both sodium and potassium ions. 4. The plots of the time constants of i(f) activation against voltage were 'bell shaped' in both canine ventricular and Purkinje myocytes. The curve for the ventricular myocytes was shifted about 30 mV in the negative direction. In both ventricular and Purkinje myocytes, the fully activated I-V relationship exhibited outward rectification in 5.4 mM extracellular K+. 5. Calyculin A (0.5 microM) increased i(f) by shifting its activation to more positive potentials in ventricular myocytes. Protein kinase inhibition by H-7 (200 microM) or H-8 (100 microM) reversed the positive voltage shift of i(f) activation. This effect of calyculin A also occurred when the permeabilized patch was used for whole-cell recording. 6. These results indicate i(f) is present in ventricular myocytes. If shifted to more positive potentials i(f) could play a role in ischaemia-induced ventricular arrhythmias. The negative shift of i(f) in the ventricle might play a role in differentiating non-pacing regions of the heart from those regions that pace. PMID- 7545233 TI - Tachykinins as mediators of slow EPSPs in guinea-pig gall-bladder ganglia: involvement of neurokinin-3 receptors. AB - 1. The effects of endogenous tachykinins and related peptides on intact guinea pig gall-bladder neurones were investigated with single-electrode voltage- and current-clamp recording techniques. 2. Pressure ejection of substance P (100 microM) caused a long lasting membrane depolarization that was associated with a decrease in input resistance. In cells that were voltage-clamped to their resting membrane potential, substance P activated an inward current. 3. The reversal potentials of the substance P-induced depolarization and inward current were congruent to 0 mV. In a low-Na+ solution, the substance P-induced depolarization and inward current were reduced in amplitude. 4. Substance P increased the excitability of neurones, as evidenced by a greater anodal break activity and an increase in the number of action potentials generated during a depolarizing current pulse. 5. Substance P, neurokinin A (NKA) and neurokinin B (NKB) were applied by superfusion to determine the relative potencies of these tachykinins. NKB was the most potent, with an EC50 of 24 nM. The EC50 values for NKA and substance P were 47.8 and 281 nM, respectively. 6. The neurokinin-3 (NK-3) receptor agonist senktide depolarized neurones with an EC50 of 6.3 nM. Neither the NK-1 receptor agonist [Sar9,Met(O2)11]-substance P nor the NK-2 receptor agonist [beta-Ala8]-NKA(4-10) caused a measurable depolarization. 7. The NK-3 antagonist [Trp7,beta-Ala8]-NKA (4-10) inhibited the responsiveness of gall bladder neurones to substance P with a KB (dissociation constant of receptor antagonist) of 49 nM, and depressed both capsaicin-induced depolarizations and stimulus-evoked slow EPSPs. 8. These data indicate that tachykinins mediate slow EPSPs in guinea-pig gall-bladder ganglia by activating NK-3 receptors on gall bladder neurones. It is proposed that in response to inflammation or high intraluminal pressure, tachykinins may be released within ganglia by sensory fibres and act directly on intrinsic neurones to facilitate ganglionic transmission. PMID- 7545234 TI - Hypoxia-induced brain angiogenesis in the adult rat. AB - 1. Prolonged hypoxia increases the brain vascularity. Here we studied the protein and deoxyribonucleic acid (DNA) content of isolated cerebral microvessels in hypoxic and control rats. 2. Adult male Wistar rats that were subjected to hypobaric hypoxia at 50.5 kPa (0.5 atm) for periods of 1, 2, or 3 weeks and normoxic littermate controls were used. Cerebral microvessels were harvested by bulk isolation from the pooled cortical mantles of three to six rats in each group. The isolated microvessels were assayed for their protein and DNA content. 3. Microvessel protein yield increased from 0.31 to 0.45 mg of microvessel protein (g wet wt)-1 of cerebral cortex after 1 week of hypoxia, but did not increase further during up to 3 weeks of hypoxia. In contrast, microvessel DNA yield did not increase during the first week of hypoxia, but increased significantly after 2 weeks of hypoxia and continued to increase up to 56 micrograms of microvessel DNA (g cerebral cortex)-1 at 3 weeks of hypoxia (normoxic mean was 32 micrograms DNA g-1). 4. The cell size index (mg protein:mg DNA) of isolated cerebral microvessels increased after 1 week of hypoxia, suggesting microvascular hypertrophy, but returned to control by the second week of hypoxia and decreased to below control levels by the third week of hypoxia, suggesting microvascular hyperplasia. These results indicate that the increased vascularity of the brain in hypobaric hypoxia progresses from an early phase of microvascular hypertrophy to later microvascular hyperplasia. PMID- 7545238 TI - Top cancer-related news stories focus on fraud, breast cancer, and the hope of early detection. PMID- 7545237 TI - Good growth response to growth hormone treatment in the ring chromosome 15 syndrome. AB - Ring chromosome 15 syndrome is a rare condition in which severe growth retardation is a major finding. We report a 4 year old boy with the karyotype 46,XY, r(15)(p11.2q26.2) whom we have treated with recombinant human growth hormone (GH) for two years. During the first year of treatment, the insulin-like growth factor I increased from subnormal 4.2 nmol/l to normal 13.8 nmol/l and the insulin-like growth factor binding protein 3 levels increased from 2.6 to 3.8 mg/l, whereas high binding protein 1 concentrations normalised from 52.0 to 16.7 micrograms/l. During the two years of treatment his relative height improved from -6.2 SD to -4.4 SD and the predicted adult height from 159.6 cm to 163.5 cm. Owing to the good growth response, we have decided to continue GH treatment. PMID- 7545236 TI - Biological function of GABAA/benzodiazepine receptor heterogeneity. AB - gamma-Aminobutyric acid (GABA) is the most prominent of the inhibiting neurotransmitters in the brain. It exerts its main action through GABAA receptors. The receptors respond to the presence of GABA by the opening of an intrinsic anion channel. Hence, they belong to the molecular superfamily of ligand-gated ion channels. There exist in the brain multiple GABAA receptors that show differential distribution and developmental patterns. The receptors presumably form by the assembly of five proteins from at least three different subunits (alpha 1-6, beta 1-3 and gamma 1-3). The regulation of functional properties by benzodiazepine (BZ) receptor ligands, neurosteroids, GABA and its analogs differs dramatically with the alpha variant present in the complex. Additional variation of the GABAA receptors comes with the exchange of the gamma subunits. No clear picture exists for the role of the beta subunits, though they may play an important part in the sensitivity of the channel-receptor complex. The effects of BZ receptor ligands on animal behavior range from agonist effects, e.g. anxiolysis, sedation, and hypnosis, to inverse agonist effects, e.g. anxiety, alertness, and convulsions. The diversity of effects reflects the ubiquity of the GABAA/BZ receptors in the brain. Recent data provide some insight into the mechanism of action of BZ ligands, but no clear delineation can be drawn from a single ligand to a single behavioral effect. This may be due to the fact that intrinsic efficacies of the ligands differ between receptor subtypes, so that the diversity of native receptors is further complicated by the diversity of the mode the ligands act on GABAA receptor subtypes. The behavioral actions of alcohol (ethanol) are similar to those produced by GABAA receptor agonists. In agreement, alcohol-induced potentiation of GABAergic responses has often been observed at behavioral, electrophysiological and biochemical levels. Thus, there is clearly a GABAA-dependent component in the actions of alcohol. However, the site and mode of action of ethanol on GABAA/BZ receptors remain controversial. PMID- 7545239 TI - Expression of alpha v beta 5 integrin is necessary for efficient adenovirus mediated gene transfer in the human airway. AB - Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways. PMID- 7545235 TI - Gradients of CSF monoamine metabolites: a comparison between male and female volunteers. AB - Lumbar puncture at the L4-5 level was performed on 12 healthy male and 12 healthy female volunteers. Confirming previous results, we found pronounced gradients in cerebrospinal fluid (CSF) 5-HIAA and HVA (5-hydroxyindoleacetic acid and homovanillic acid). We also found a gradient in 4-hydroxy-3-methoxyphenylglycol (HMPG), but only in the male volunteers. We also found that tapping-time was significantly longer in females than in males. One reason for this discrepancy may be that an estimate of the spinal distance was greater in males, which might indicate that a hydrodynamic factor plays a role. On taking tapping-time into consideration, the 5-HIAA and HMPG concentrations were significantly higher in males than in females. PMID- 7545240 TI - Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys). AB - The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at a site in the viral RNA genome which is designated the primer-binding site (PBS). The HIV-1 PBS is an 18-nucleotide sequence that is complementary to the 3'-terminal 18 nucleotides of tRNA(3Lys), which is used as the primer for reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA(3Lys), suggesting that other cellular tRNAs might not function as primers for reverse transcription. To investigate this possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3'-terminal nucleotides of tRNA(1,2Lys), tRNA(Ile), and tRNA(His), which previous studies have identified to be packaged into HIV-1 virions along with tRNA(3Lys). We demonstrate that infectious viruses which utilized tRNA(1,2Lys), tRNA(Ile), and tRNA(His) in reverse transcription can be recovered. However, the appearances of viruses with PBSs complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type PBS complementary to tRNA3(Lys). Furthermore, only the first 9 nucleotides of the 18 nucleotide PBSs were sufficient for HIV-1 to utilize the alternate tRNA primers in reverse transcription, demonstrating that HIV-1 does not require the complete 18-nucleotide PBS to utilize these tRNA primers for reverse transcription. These results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription. PMID- 7545242 TI - Vaccinia virus gene A18R encodes an essential DNA helicase. AB - The vaccinia virus A18R protein is a DNA-dependent ATPase that contains the canonical sequence motifs associated with the DEXH group of DNA and RNA helicases. Investigation of A18R protein function during infection indicated it functions in the early and late phases of vaccinia virus transcription. The A18R protein shares sequence similarity with the mammalian DNA helicase ERCC3. The ERCC3 protein has a dual function: it is a component of the transcription factor TFIIH and is an essential participant in the cellular nucleotide excision repair pathway. Here we present evidence that the A18R protein is a DNA helicase that unwinds duplex DNA in a 3'-to-5' direction. The A18R helicase was inactive on RNA DNA and RNA-RNA hybrids. The A18R unwinding activity was most efficient on DNA substrates with lengths of 20 nucleotides or less, and its unwinding activity was not stimulated by the addition of Escherichia coli single-strand-binding protein (SSB), the bacteriophage T4 gene 32 SSB, or the vaccinia virus I3L protein, a putative SSB. We have used an electrophoretic gel mobility shift assay to show that the A18R protein forms a stable complex with single-stranded DNA, and to a lesser extent RNA, in a reaction that does not require ATP. PMID- 7545241 TI - Human T-lymphotropic virus type 1 peptides in chimeric and multivalent constructs with promiscuous T-cell epitopes enhance immunogenicity and overcome genetic restriction. AB - Conventional strategies of viral peptide immunizations often elicit low-affinity antibody responses and have limited ability to elicit immune responses in outbred animals of diverse major histocompatibility (MHC) haplotypes. This genetically restricted T-cell-stimulatory activity of peptides is a serious obstacle to vaccine design. However, the use of promiscuous T-cell epitopes may circumvent this problem. Promiscuous T-cell epitopes from tetanus toxin (amino acids [aa] 580 to 599) and the measles virus F protein (aa 288 to 302) that bind to several isotypic and allotypic forms of human MHC class II molecules have been identified and have been used in highly immunogenic constructs to overcome haplotype restricted immune responses. Chimeric and beta-template peptide constructs incorporating known human T-lymphotropic virus type 1 (HTLV-1) B- and T-cell epitopes from the surface envelope protein gp46 (SP2 [aa 86 to 107] and SP4a [aa 190 to 209]) and promiscuous T-cell peptides were synthesized, and their immunogenicities were evaluated in both rabbits and mouse strains of divergent haplotypes (C3H/HeJ [H-2k], C57BL/6 [H-2b], and BALB/c [H-2d]). In addition, peptide preparations were structurally characterized by analytical high performance liquid chromatography, mass spectrometry, and circular dichroism. In contrast to their linear forms, the chimeric constructs of both the SP2 and SP4a epitopes displayed alpha-helical secondary structures. Immunogenicity of the peptide constructs was evaluated by direct and competitive enzyme-linked immunosorbant assay (ELISA), as well as by radioimmunoprecipitation, syncytium inhibition, and antigen-induced lymphocyte proliferation assays. Immunization with the SP4a peptide without conjugation to a carrier protein produced antibodies specific for SP4a in two mouse strains (C3H/HeJ and C57BL/6). However, BALB/c mice failed to respond to the peptide, indicating that the T-cell epitope of the SP4a sequence is MHC restricted. In contrast, the chimeric constructs MVF SP2 and SP4a-measles virus F protein were highly immunogenic, producing elevated ELISA titers after only two immunizations. Elicited antibodies recognized native forms of gp46 in ELISAs and radioimmunoprecipitation assays, as well as inhibited HTLV-1-mediated syncytium formation. In addition, chimeric constructs were effective at induction of lymphocyte proliferation to the T-cell epitope, SP4a, in each strain of immunized mice. Our data demonstrate that the antibody response to retroviral peptides is enhanced by promiscuous peptide constructs, in part because of the ability of such constructs to promote appropriate secondary structural forms of viral epitopes. In addition, these constructs promote virus specific helper T-cell responses, thereby overcoming genetically restricted immune responses to the synthetic peptides. PMID- 7545243 TI - Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules. AB - The entry of human immunodeficiency virus type 1 into cells proceeds via a fusion mechanism that is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. Species- and tissue-specific restrictions to viral entry suggested the participation of additional membrane components in the postbinding fusion events. In a previous study (H. Golding, J. Manischewitz, L. Vujcic, R. Blumenthal, and D. Dimitrov, J. Virol. 68:1962-1968, 1994), it was found that phorbol myristate acetate (PMA) inhibits human immunodeficiency virus type 1 envelope-mediated cell fusion by inducing down modulation of an accessory component(s) in the CD4-expressing cells. The fusion inhibition was seen in a variety of cells, including T-cell transfectants expressing engineered CD4 receptors (CD4.401 and CD4.CD8) which are not susceptible to down modulation by PMA treatment. In the current study, it was found that preincubation of A2.01.CD4.401 cells with soluble monomeric gp120 for 1 h at 37 degrees C primed them for PMA-induced down modulation (up to 70%) of the tailless CD4 receptors. The gp120-priming effect was temperature dependent, and the down modulation may have occurred via clathrin-coated pits. Importantly, nonhuman cell lines expressing tailless CD4 molecules did not down modulate their CD4 receptors under the same conditions. The gp120-dependent PMA-induced down modulation of tailless CD4 receptors could be efficiently blocked by the human monoclonal antibodies 48D and 17B, which bind with increased avidity to gp120 that was previously bound to CD4 (M. Thali, J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski, J. Virol. 67:3978-3988, 1993). These findings suggest that gp120 binding to cellular CD4 receptors induces conformational changes leading to association of the gp120-CD4 complexes with accessory transmembrane molecules that are susceptible to PMA-induced down modulation and can target the virions to clathrin-coated pits. PMID- 7545245 TI - Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers. AB - We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs. PMID- 7545244 TI - Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding. AB - We investigated the binding of conformation-dependent anti-V2, anti-V3, and anti CD4-binding site monoclonal antibodies to monomeric and virion-associated gp120 from human immunodeficiency virus type 1 isolates displaying marked differences in cell tropism. For all viruses examined, we found that the half-maximal binding values of the anti-V2 and anti-CD4-binding site antibodies with virion-associated gp120 were higher than those with monomeric gp120, but the maximum amount of antibodies bound was diminished only for one of the anti-V2 antibodies tested. These observations suggest that upon gp120 oligomerization, the V2 loop and CD4 binding site undergo conformational changes and that particular epitopes within these domains are occluded in the oligomeric gp120. In contrast, although the overall binding patterns and half-maximal binding values of the anti-V3 loop antibodies tested were similar with monomeric and oligomeric gp120, all the V3 loop epitopes examined were less accessible to antibody binding on the virion surface. This masking of the V3 loop is more pronounced for the primary-like macrophage-tropic isolates examined. Lastly, we observe that upon soluble receptor-virion binding, specific V3 loop epitopes that differ for viruses displaying different tropisms are exposed. PMID- 7545246 TI - Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits. AB - The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli. The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51. The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase. When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases. The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51. On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis. These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M. Hottiger, V.N. Podust, R.L. Thimmig, C.S. McHenry, and U. Hubscher. J. Biol. Chem. 269:986-991, 1994). Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA. PMID- 7545249 TI - A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse transcriptase: coexpression of HIV reverse transcriptase and herpes simplex virus thymidine kinase. AB - Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase. PMID- 7545248 TI - Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity. AB - Vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS) when intranasally applied. We have examined cellular inflammatory changes in the CNS following VSV infection. As early as 1 day postinfection (p.i.), astrocytes were activated in the olfactory bulb (OB). This was followed by activation of microglia, first observed in the OB at day 3 p.i. Expression of inducible nitric oxide synthase was observed in activated microglia in the OB at day 3 p.i., and increased inducible nitric oxide synthase expression coincided with decreased virus titers in tissue homogenates. Expression of major histocompatibility complex (MHC) class I molecules on astrocytes and microglial, endothelial, and ependymal cells was also rapidly induced and followed by induced expression of MHC class II molecules on astrocytes and microglial and endothelial cells. Consistent with the pattern of viral dissemination, MHC molecules were expressed temporally from the rostral-to-caudal direction. Infiltration of CD8+ cells was observed as early as 1 day p.i. in the OB. CD4+ cells were detected in the OB at day 4 p.i. Increasing T-cell infiltration coincided with decreased virus titers. In contrast, B-cell infiltration of the CNS was not detected until day 14 p.i., after the virus was cleared and mice were showing behavioral signs of recovery. Breakdown of the blood-brain barrier was detected beginning at day 6 p.i., was most severe at day 8 p.i., and was followed by full recovery. Collectively, these data show that both innate immunity (production of nitric oxide) and acquired immunity (expression of MHC molecules and T-cell infiltration) are activated following VSV infection in the CNS. PMID- 7545251 TI - Chlormadinone acetate withdrawal syndrome under combined androgen blockade for advanced prostate cancer. AB - Between July 1991 and December 1994 at Tsukuba Gakuen Hospital, we treated 19 consecutive men with advanced adenocarcinoma of the prostate (five at stage C, four at stage D1 and 10 at stage D2). Of these, 14 patients underwent castration (two patients) or received LH-RH analogue (12 patients) plus chlormadinone acetate for combined androgen blockade. We report three representative cases of sequential prostate specific antigen (PSA) elevation following initial response to this combined androgen blockade. Discontinuation of chlormadinone acetate resulted in decline of the serum PSA level. This suggests that trial chlormadinone acetate withdrawal in patients showing increasing levels of PSA during combined androgen blockade should be considered before initiation of alternative treatment. PMID- 7545250 TI - Preoperative endocrine therapy in patients with locally advanced prostate cancer. AB - Recently, there has been increased interest in the application of preoperative endocrine therapy prior to radical prostatectomy for locally advanced cancer in order to enhance surgical curability and increase survival. Between 1986 and 1993, 40 patients with prostate cancer were given endocrine therapy before radical prostatectomy. Fifteen patients had stage B2 disease and 25 stage C. The median duration of preoperative endocrine therapy was 3.8 months, and all the patients subsequently underwent radical prostatectomy, pelvic lymphadenectomy and castration. There was an average 25.5% (0-71.8%) decreases in the maximal cross sectional area of the prostate gland as determined by transrectal ultrasonography. Twenty-four of 25 patients with elevated levels of serum prostate-specific antigen (PSA) showed normal values after preoperative endocrine therapy. Evaluation of treatment-related histological effects, divided into three grades, revealed that 17 patients had pronounced, 11 moderate and 12 poor or no regression. Thirteen of the 40 patients (33%) demonstrated pathological downstaging of disease status from the diagnosis made at the initial clinical examination. After a median follow-up period of 36 months (3-100 months), 36 of the 40 patients are disease-free; two died of cancer 43 and 50 months after surgery, respectively. These results suggest that preoperative endocrine therapy may play an important role in the management of locally advanced prostatic cancer. PMID- 7545253 TI - Streptococcal pneumoniae polysaccharide increases IgA-class antibody activity under the immunological memory of a protein antigen: two signals on experimental IgA nephropathy. AB - We designed the following experiment in order to clarify the factors that induce a hyper-immune state of IgA. Six-week-old Balb/c mice were immunized with bovine gammaglobulin (BGG) at 0 and 2 weeks, followed by the administration of phosphorylcholine-BGG (PC-BGG) at 3 and 5 weeks to obtain an immunological memory. At 6 weeks, we divided the mice into three groups: one was a saline group used as a control, another was a PC-BGG group used to investigate T-cell dependent antigen, and the last was a streptococcal pneumoniae polysaccharide (R36A) group used to investigate T-cell independent antigen. We compared the antibody activity in response to BGG, and glomerular immune deposition among the groups. In the control group, antibody activities did not change, and all stainings on glomerular immune deposits were negative. In the PC-BGG group, IgG class antibody activity was significantly suppressed (p < 0.05), but IgA- and IgM class antibodies were not affected. The intensity of glomerular deposition of IgM was level one positivity (TFS: 116.7 +/- 20.2 (mean +/- SD)). In the R36A group, polysaccharide produced significant increases (almost four times) in IgA-class and IgM-class antibody activity under the condition of immunological memory (IgA: p < 0.05, IgM: p < 0.005). The intensity of IgA was between weak and level one positivity (TFS: 60.8 +/- 6.3), but the intensity of IgM was weak positive (TFS: 36.7 +/- 10.4). This became a predominant glomerular deposition of IgA in the R36A group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545255 TI - Adenoma-carcinoma sequence: a reappraisal with immunohistochemical expression of ferritin. AB - To examine the biological significance of ferritin (FRN) expression, a retrospective immunohistochemical study was performed in normal colonic mucosae (n = 8), adenomas (n = 88), and colorectal carcinomas (n = 104). FRN was present in some epithelia in the crypt base of normal colonic mucosae. Significant cytoplasmic staining for FRN was revealed in 26 (29.5%) cases of adenoma and 54 cases (51.9%) of adenocarcinoma. The cancer cells had a higher proportion of FRN expression than those of adenomas or non-neoplastic mucosae (P < 0.001). Expression of FRN showed a positive association with the degree of dysplasia (P = 0.039) and the distal location of adenoma (P = 0.013). FRN expression tended to be associated with the tumor size (P = 0.083), but no substantial difference was observed among the histologic types of adenoma (P = 0.754). The results suggest that cytoplasmic FRN expression is associated with cellular proliferation. The proliferative index shows a significant difference through the adenoma-carcinoma sequence. Further investigation is necessary to clarify the clinical implication of FRN expression in tumor cells and normal-appearing mucosae. PMID- 7545254 TI - Intraoperative peritoneal washing cytology with the rapid immunoperoxidase method using microwave irradiation. AB - The rapid immunocytochemical staining of gastrointestinal cancer-associated antigens, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), for intraoperative peritoneal washing cytology of pancreatic or biliary carcinoma can be completed in only 25 minutes by using microwave irradiation to accelerate incubation of the primary antibody. Only 3-second irradiation at 500 W for fresh smears produced specific antigen staining of malignant cells similar to that obtained with longer incubation, and the sensitivity of intraoperative cytology was greater than by the conventional staining method. The rapid immunocytochemical staining method of peritoneal washings at laparotomy can provide sensitive information for determining more aggressive therapy for intraperitoneal spread of malignant cells before closing the abdominal wall. PMID- 7545252 TI - Leukemoid reaction resulting from multiple cytokine production in metastatic mucoepidermoid carcinoma with central necrosis. AB - We report a male patient with metastatic high-grade mucoepidermoid carcinoma and associated leukemoid reaction. The patient was transferred to our hospital due to persistent spiking fever, marked granulocytosis, and suspected liver abscess. After thorough bacteriological studies, including cultures of blood and material aspirated from the "liver abscess", no evidence of infection was documented. The patient suffered from persistent spiking fever for more than 4 weeks in spite of empirical antibiotic treatment, and repeated aspiration of the presupposed liver abscess. He underwent exploratory laparotomy for intended surgical evacuation of the liver abscess and bacteriological diagnosis. The operative findings were compatible with metastatic carcinoma with multiple liver and retroperitoneal lymph node involvement and tumor necrosis. The pathology report indicated high grade mucoepidermoid carcinoma. Immunohistochemistry showed positive staining for interleukin-1 alpha (IL-1 alpha) and IL-6. Elevation of cytokine levels in the necrotic tumor fluid, including IL-1 alpha, IL-6, granulocyte-macrophage colony stimulating factor and granulocyte colony-stimulating factor was confirmed by ELISA test. This case shows that multiple cytokine production from a metastatic tumor and its central necrotic area in the liver can produce a febrile leukemoid reaction mimicking a pyogenic liver abscess. PMID- 7545257 TI - Prospective randomized study of hyperthermia combined with chemoradiotherapy for esophageal carcinoma. AB - From January 1988 to June 1992, 66 patients with resectable squamous cell carcinoma of the thoracic esophagus underwent preoperative adjuvant therapy. These patients were prospectively divided into two treatment groups; 32 were treated with radiofrequency wave local hyperthermia combined with chemoradiotherapy (hyperthermochemoradiotherapy; HCR), while the remaining 34 patients were treated with chemoradiotherapy alone (CR). There were no procedural complications in either group and the postoperative mortality was zero. In the HCR group, no viable cancer cells were found within the entire 5 mm-width slices of the resected specimen in eight patients (25%), while only two (5.9%) in the CR group (P < 0.05) demonstrate no viable cancer cells. The cumulative 3-year survival rate was 50.4% in the HCR group and 24.2% in the CR group. The present prospective trial demonstrated that the addition of hyperthermia to chemoradiotherapy resulted in a better local control and an improved long-term survival when treating patients with advanced esophageal carcinoma. PMID- 7545256 TI - Overexpression of P-glycoprotein in untreated AFP-producing gastric carcinoma. AB - P-glycoprotein (P-gly), which is responsible for the phenotypic expression of multidrug resistance in cancerous tissue was stained immunohistochemically in previously untreated alpha-fetoprotein (AFP)-producing (n = 20) and nonproducing gastric cancers (n = 20). P-gly, AFP, and carcinoembryonic antigen(CEA) were stained in formalin-fixed paraffin-embedded tissue sections immunohistochemically using the monoclonal antibody JSB-1, anti-AFP, and anti-CEA, respectively. DNA ploidy pattern was determined by Fluorescence Activated Cell Sorter (FACS) analyzer. P-gly was significantly overexpressed in AFP producing gastric cancers (60%) than in AFP nonproducing ones (20%) (P < 0.01). When the result of P-gly staining was analyzed among the AFP-positive cases, P-gly positivity did not emerge either as a significant prognostic factor or as a predictor of the metastatic potentiality of the tumor. The intrinsic overexpression of P-gly in AFP producing gastric cancers proves its biological and morphological similarities to hepatocellular carcinoma. The significantly (P < 0.05) higher incidence of P-gly in diploid tumors indicate that expression of this phenotype might be related to the differentiation of the tumor. P-gly was overexpressed in AFP producing gastric carcinoma and the existing drug resistance, frequent recurrence, and poor prognosis might be explained by presence of P-gly in this carcinoma. PMID- 7545247 TI - Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences. AB - The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins. PMID- 7545258 TI - An APL-programmed genetic algorithm for the prediction of RNA secondary structure. AB - The possibilities of using a genetic algorithm for the prediction of RNA secondary structure were investigated. The algorithm, using the procedure of stepwise selection of the most fit structures (similarly to natural evolution), allows different models of fitness or driving forces determining RNA structure to be easily introduced. This can be used for simulation of the RNA folding process and for the investigation of possible folding pathways. Such an algorithm needs several modifications before it can predict RNA secondary structures. After modification, a fair number of correct stems are predicted, even when using computationally quick, but very crude, fitness criteria such as stem length and stacking energy, including elements of tertiary structure (pseudoknots). The fact that genetic algorithm simulation includes both stem formations and stem disruption allows one to observe intermediate structures that may be used in combination with phylogenetic or experimental research. PMID- 7545259 TI - Protocol recommended by the CSGMT/JEMS.MMS for the short-term mouse peripheral blood micronucleus test. The Collaborative Study Group for the Micronucleus Test (CSGMT) (CSGMT/JEMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan). AB - Although the target cells for the bone marrow (BM) and peripheral blood (PB) micronucleus tests are the same, erythroblasts, the PB method offers important advantages over the BM method. We propose a protocol for the short-term peripheral blood micronucleus test. This assay is intended primarily for the identification of a wide variety of chemical clastogens and spindle poisons, and secondarily for risk assessment. The recommended experiment size, seemingly small, has adequate detection power. Experimental results obtained from Collaborative Study Group for the Micronucleus Test (CSGMT) studies and data collected from a survey of the literature provided the basis of the proposed protocol. Our protocol, designed for mice, includes the following features. (i) The maximum tolerated dose (MTD) is determined experimentally with a small number of animals treated i.p. or per os (or by other routes, if called for) in a dose finding test, which can be conducted simultaneously with tests for finding both number of treatments and optimal sampling time. (ii) At least three groups of five mice (at least four effective animals per group), males or females, are given i.p. or per os doses of, for example, the MTD, 1/2 MTD and 1/4 MTD once, twice or more, 24 h apart. (iii) Peripheral blood samples are taken before treatment (the 0 time control) and twice at 48 and 72 h for a single treatment, once between 24 and 36 h after the second treatment for double treatments, or once 24 h after the final treatment for multiple dosing. Or, if an optimal sample time is established in a preliminary test, samples are taken at that time, (iv) Samples are stained with acridine orange, and 2000 immature erythrocytes per animal are examined. (v) The combined data of 0 time samples are the negative control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545260 TI - Collaborative study of interlaboratory variability in Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101. Association of Microbial Mutation Testing Laboratory C.O. AB - A collaborative study of interlaboratory variability in bacterial mutagenicity induced by mitomycin C (MMC) and bleomycin (BLM) was performed using the four strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101. Thirteen laboratories participated in this study. The four strains and two chemicals were sent from a central source to each laboratory. Each strain was cultured in nutrient broth containing antibiotics and laboratories were requested to ensure that the spontaneous counts for marker check fell within a specified range in the preparation of the original stock culture. Concerning the response to the chemicals, most interlaboratory variability was within a 4-fold range for tests with TA102, TA2638 and WP2/pKM101. From the results of the statistical analysis using the linear regression test, positive results in TA102, TA2638 and WP2/pKM101 and negative results in WP2 uvrA/pKM101 were obtained by MMC testing in all of the laboratories. On the other hand, with BLM testing, considerable interlaboratory variability was observed between the strains, largely because of the variation in results of the repeat experiments. Overall mean spontaneous revertant counts in all laboratories were within acceptable ranges for all four bacterial strains. In conclusion, it is judged that among Japanese laboratories, there is excellent agreement in the tested response of these bacterial strains to a strong mutagen such as MMC, as well as uniformity in the spontaneous reversion rates. However, for chemicals such as BLM that induce a weak response, it may be necessary to repeat experiments several times to obtain clear results. In this study, TA102 was shown to be a useful strain for routine mutagenicity testing. This necessitated control over culture maintenance by the addition of tetracycline and strict selection in the preparation of the original stock cultures. PMID- 7545261 TI - Analysis of chemically induced spindle aberrations in male mouse germ cells: comparison of differential and immunofluorescent staining procedures. AB - Differential staining of spindle and chromatin was adapted to meiotic cells of male mice. Immunostaining with antitubulin antibodies was developed. Both methods were used to test acrylamide, diazepam and vinblastine for their potential to cause spindle disturbances in male mouse germ cells. The analyses were performed after in vivo treatment, comparing the classical safranin/brilliant blue spindle staining to an immunofluorescent assay. The two staining methods complemented each other. Differential staining seems to be more sensitive to the detection of misplaced chromatin whereas the immunostaining is superior when scoring for spindle structure aberrations. All three chemicals showed spindle activity. Acrylamide mainly caused multipolar spindles which possibly develop from a separation of mother and daughter centrioles during an unspecific meiotic block. Diazepam elevated the level of monopolar spindles and induced the loss of single chromosomes, suggesting an effect on motor proteins. Vinblastine was the strongest spindle poison by far. It produced high numbers of shortened and monopolar spindles as well as multiple chromosome loss. PMID- 7545264 TI - Short note: are electron-transport and electron-transfer involved in intracellular signaling? AB - In this paper, the possibility of electron-transfer and subsequent electron transport across membrane proteins (ion channels) is studied with respect to the ion channel function. The electronic properties of the ionic channel protein interface with the electrolytes, and the properties of the same ion channel protein as a solid-state biopolymer are used as the physical basis to explain these elementary charge transport phenomena. It is proposed that, by means of the occurrence of those two processes, there can be electrons participating in the activation of the G-proteins and subsequent intracellular signaling. As another example, the same analysis is used to propose the involvement of electrons in the neuromodulation of the ionic channels. PMID- 7545262 TI - Modulation of skeletal muscle protein synthesis by amino acids and insulin during sepsis. AB - Effects of different concentrations of insulin and amino acids on protein synthesis in skeletal muscle of young, fed septic rats were determined in the perfused rat hindlimb. Rates of protein synthesis in gastrocnemius were measured by incorporation of [3H]-phenylalanine into protein. Perfusion of hindlimb muscles from young, fed control rats with medium containing either insulin and a complete mixture of amino acids at plasma concentration (1x) or a mixture of amino acids at 10-fold (10x) plasma concentration resulted in an approximately twofold stimulation of the rate of protein synthesis. The effect of amino acids on protein synthesis was partly accounted for by elevated concentrations of branched-chain amino acids ([BCAA] leucine, isoleucine, and valine). In young, fed septic rats, the rate of protein synthesis in muscle perfused with buffer containing the normal concentration of amino acids was reduced 40% as compared with control levels (P < .05). In contrast to controls, addition of insulin (1,000 microU/mL) did not augment protein synthesis in muscle from young, fed septic rats perfused with the complete mixture of amino acids. Addition of insulin 10,000 microU/mL stimulated protein synthesis approximately 80% in gastrocnemius of septic rats (P < .05). However, the rate of protein synthesis remained less than that observed in young, fed control rats at similar insulin concentrations. Perfusion with medium containing 10x plasma amino acids stimulated protein synthesis approximately fourfold in young, fed septic rats as compared with control animals. In contrast to controls, BCAA at 10x plasma concentration did not augment protein synthesis in young, fed septic rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545265 TI - Heterogeneity in chemical mutagen-induced chromosome damage after G2 phase exposure to bleomycin, ara-C and gentian violet in cultured lymphocytes of beta thalassaemia traits. AB - Chemical mutagen-induced chromosome damage was analysed in cultured peripheral blood lymphocytes from beta-thalassaemia traits and healthy individuals. This was promoted by the fact that beta-thalassaemia trait is present in 1-17% of different population groups in India. To study mutagen-induced chromosome instability, G2 lymphocytes were exposed to bleomycin, ara-C or gentian violet in 48-h cultures. Spontaneous chromosome aberration frequencies in lymphocytes from beta-thalassaemia traits were found to be in the normal range. In all three clastogen-treated lymphocytes from beta-thalassaemia traits, there is a degree of hypersensitivity, when the results are averaged over a number of individuals, but some individuals overlap within the normal range. The heterogeneity in chemical mutagen sensitivity observed in beta-thalassaemia traits is discussed in terms of the oxidative damage consequent on the genetic and biochemical features peculiar to the beta-thalassaemia trait cell. PMID- 7545263 TI - Plasma levels of insulin-like growth factor binding protein-3 in acute trauma patients. AB - Insulin-like growth factors (IGFs) are a family of polypeptides that regulate cell growth. Their action and bioavailability are modified by binding proteins such as IGF binding protein-3 (IGFBP-3). Plasma IGFBP-3 level was found to be growth hormone (GH)-dependent, which makes detection of IGFBP-3 useful in the evaluation of GH secretion. In the early catabolic flow phase of severe injury, when plasma levels of GH and IGF-1 are low versus uninjured levels, the role of IGFBP-3 has not been investigated. We have measured basal levels of these polypeptide hormones in 16 adult (13 men and three women aged 47 +/- 7 years) severely injured (Injury Severity Score, 32 +/- 2), hypermetabolic resting energy expenditure [REE] to basal energy expenditure [BEE] ratio, 1.30 +/- 0.05), ventilator-dependent, multiple-trauma patients within 48 to 60 hours of injury when the patients were receiving maintenance fluids without calories or nitrogen. These basal values were compared with those of 16 age-matched postabsorptive normals. In the catabolic flow phase of injury, plasma levels of GH, IGF-1, and IGFBP-3 were significantly reduced by 50%, 46%, and 45%, respectively. There was a significant linear inverse relationship between IGFBP-3 and age and also a positive correlation between IGFBP-3 and IGF-1 in both control and injured subjects. The ratio of IGFBP-3 to IGF-1 was not changed in trauma victims. Measurement of plasma IGFBP-3 levels has potential as a marker for monitoring GH therapeutic efficacy. PMID- 7545267 TI - Hodgkin's disease--new insights from immunoglobulin genetics. PMID- 7545266 TI - Hodgkin's disease with monoclonal and polyclonal populations of Reed-Sternberg cells. AB - BACKGROUND: There is strong evidence that Reed-Sternberg cells have a lymphoid phenotype, but clonally rearranged genes for B-cell and T-cell antigen receptors have not been demonstrable in tumor tissue from most patients with Hodgkin's disease. To elucidate this issue, we assayed single Reed-Sternberg cells from 12 patients with classic Hodgkin's disease of a B-cell immunophenotype to detect rearranged immunoglobulin variable-region heavy-chain (VH) genes. METHODS: We isolated single Reed-Sternberg cells from frozen sections that had been immunostained for CD30. The rearranged VH genes of these cells were amplified by the polymerase chain reaction and analyzed by gel electrophoresis and nucleotide sequencing. RESULTS: In all 12 patients, the Reed-Sternberg cells studied contained rearranged VH genes. Three patterns were observed: in three patients the rearrangements in each patient were identical, in six patients all the rearrangements were unrelated and unique, and in three patients both identical and unrelated rearrangements were detected. Apparently somatic mutations of VH genes were present in some Reed-Sternberg cells but absent in others. CONCLUSIONS: Reed-Sternberg cells with B-cell phenotypes have rearranged VH genes; therefore, these cells arise from B cells. The pattern of VH gene mutations suggests that Reed-Sternberg cells can correspond to either immunologically naive or memory B cells. In half our patients the population of Reed-Sternberg cells was polyclonal; in the other half, monoclonal or mixed cell populations were found. Correlation with the clinical stage suggests that polyclonal Hodgkin's disease can present as a widespread lymphoma. PMID- 7545268 TI - Effects of acute systemic treatment with the 5 HT-uptake blocker alaproclate on tissue levels and release of substance P in rat periaqueductal grey. AB - The effects of acute systemic treatment with alaproclate, a serotonin uptake blocker on regional brain tissue levels of substance P, neurokinin A and cholecystokinin were studied in the rat. The peptide levels of all three peptides were increased (23-35%) in the rat periaqueductal grey 60 min after treatment with alaproclate (20 mumol/kg peroral, p.o.), compared to controls. In the cingulate cortex, the tissue levels of substance P and cholecystokinin were increased (19-32%) after subcutaneous (s.c.) treatment with alaproclate, compared to controls. Higher tissue levels of all three peptides (20-38%) in the periaqueductal grey, and lower levels of substance P and cholecystokinin in the cingulate cortex were found following saline s.c. compared to saline p.o., probably due to different degrees of stress. In microdialysis experiments, a s.c. injection of either saline (2 ml/kg), alaproclate (20 mumol/kg) or morphine (3 mg/kg) was found to slowly increase the substance P release in the periaqueductal grey. Experiments with the selective 5-HT neurotoxin, 5,7-dihydroxytryptamine indicated no neuronal co-existence of substance P and serotonin in the periaqueductal grey and cingulate cortex. In conclusion, acute treatment with the serotonin uptake blocker alaproclate increases both the tissue level and the release of substance P in the periaqueductal grey. PMID- 7545270 TI - Managed care for children with neurodevelopmental disabilities. PMID- 7545271 TI - The role of the pediatrician in caring for children with developmental disabilities: overview. AB - Pediatricians can help children with developmental disabilities to reach their potential. In addition to the issues relating to direct care outlined above and in the other articles in this issue of Pediatric Annals, clinicians should advocate on a community-wide level. This includes working with neighborhood schools, community health nurses, disease-oriented volunteer groups, and neighborhood agencies to provide access to services, support, and education for children and their families. It also includes championing the cause of children with disabilities with local and state governments. Because of the respect that pediatricians command in the community, they can play a major role in the lives of these children and their families. PMID- 7545272 TI - Common behavioral and emotional problems in children with developmental disabilities. PMID- 7545269 TI - Differential effects of 3-isobutyl-1-methylxanthine injected intrathecally or intracerebroventricularly on antinociception induced by opioids administered intracerebroventricularly in the mouse. AB - Various doses of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. IBMX (0.01 to 1 ng) pretreatment i.t. for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by i.c.v. administered morphine (2 micrograms), beta-endorphin (1 microgram), and U50, 488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide), 60 micrograms. However, pretreatment with IBMX i.c.v. did not affect the inhibition of the tail-flick response induced by morphine, beta-endorphin, and U50, 488H administered i.c.v. Neither i.c.v. nor i.t. pretreatment with IBMX attenuated the inhibition of the tail-flick response induced by D-Pen2-D-Pen5-enkephalin (DPDPE; 10 micrograms) administered i.c.v. Our results suggest that spinal but not supraspinal cAMP phosphodiesterases are involved in mediating antinociception induced by morphine, beta-endorphin and U50, 488H administered supraspinally. However, neither spinal nor supraspinal cAMP phosphodiesterase is involved in mediating antinociception induced by DPDPE administered supraspinally. PMID- 7545275 TI - Helping families with children with developmental disabilities. AB - The physician can help the child with developmental disabilities maximize his or her potential and minimize the adverse effects on the family by understanding the emotional and other impacts of a developmental disability; providing information about diagnosis, prognosis, and associated problems; providing nonjudgmental listening; providing information about community resources, including parent support groups; ensuring adequate communication with everyone involved in the care of the child; and referring the family to appropriate agencies and providers. PMID- 7545274 TI - Feeding and nutrition in children with developmental disabilities. AB - Feeding is a complex physiologic process that is further complicated by social and cultural influences. Feeding development, although dependent on structural integrity and neurologic maturation, is a learned progression of behaviors influenced by sensory motor development and experience. Children learn how to eat. Children with developmental disabilities frequently develop problems with feeding that can lead to malnutrition and respiratory symptoms. Feeding disorders are often the result of multiple interacting variables that have disrupted feeding development and the feeding relationship. The pediatrician who has a solid understanding of these variables can understand the problems, sort out the contributing causes, and intervene effectively. PMID- 7545276 TI - Transition into adulthood. PMID- 7545273 TI - MD's DD BASICS: identifying common problems and preventing secondary disabilities. PMID- 7545277 TI - Pulmonary manifestations of cat-scratch disease; a case report and review of the literature. PMID- 7545279 TI - Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice. AB - Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5 +/- 2.8 pA/pF (n = 29) in wild-type cells, and about half that value, i.e. 12.4 +/- 1.6 pA/pF (n = 20) in heterozygotes (P = 0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527 +/- 162 pA/pF (n = 24) from wild-type, 316 +/- 35 pA/pF (n = 21) from heterozygote and 419 +/- 83 pA/pF (n = 7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells. PMID- 7545280 TI - Multiple ion binding sites in Ih channels of rod photoreceptors from tiger salamanders. AB - The mechanism of ion permeation in K+/Na(+)-permeable Ih channels of tiger salamander rod photoreceptors was investigated using the whole-cell voltage-clamp technique. Ih channels showed features indicative of pores with multiple ion binding sites: in mixtures of K+ and thallium (T1+), the amplitude of the time dependent current showed an anomalous mole fraction dependence, and K+ permeation was blocked by other permeant ions (with K0.5 values: T1+, 44 microM; Rb+, 220 microM and NH4+, 1100 microM) as well as by essentially impermeant ions (Cs+, 22 microM Ba2+, 9200 microM) which apparently block Ih by binding in the pore. In contrast, Na+ had little blocking action on K+ permeation. The block by all of these ions was sensitive to external K+ with the block by Cs+ being the least sensitive. Na+ was more effective than K+ in reducing the block by T1+, Rb+ and NH4+, but was less effective for the block by Cs+ and Ba2+. The blocking action of Cs+ and Ba2+ was non-competitive, suggesting that they block Ih channels at independent sites. Based on the efficacy of block by the different ions, the degree to which K+ and Na+ antagonize this block and the noncompetitive blocking action of Cs+ and Ba2+, the permeation pathway of Ih channels appears to contain at least three ion binding sites with at least two sites having a higher affinity for K+ over Na+ and another site with a higher affinity for Na+ over K+. PMID- 7545278 TI - Stimulation of Na+/H+ exchange activity by endothelin in opossum kidney cells. AB - Endothelin-1 (ET-1) controls multiple aspects of kidney function. In this study we have analysed the effects of ET-1 on apical Na+/H+ exchange activity in opossum kidney (OK) cells. ET-1 (at 10(-10) M and 10(-8) M) activated Na+/H+ exchange activity within 5 min of exposure. ET-1 (10(-8) M) prevented PTH-induced (parathyroid hormone; 10(-8) M) inhibition of Na+/H+ exchange activity; it also abolished transport inhibition in response to 10(-3) M IBMX (isobutyl methylxanthine) and 3 x 10(-7) M TPA (phorbol 12-myristate 13-acetate), but had no effect on the 8-bromo-cAMP-induced (10(-4) M) decrease of transport rate. Basal cAMP content, IBMX- and PTH-stimulated cAMP production were unaffected by ET-1 (10(-8) M). The stimulatory action of ET-1 (10(-8) M) on Na+/H+ exchange activity was prevented by calphostin C (10(-8) M). These data document that OK cells might serve as a useful in vitro model for analysis of cellular mechanisms involved in endothelin action; proteine kinase C activation seems to participate in the observed endothelin effects. PMID- 7545282 TI - The effect of a chemical phosphatase on single calcium channels and the inactivation of whole-cell calcium current from isolated guinea-pig ventricular myocytes. AB - A chemical phosphatase, butanedione monoxime (BDM, at 12-20 mM), reduced open probability (P0) of single cardiac L-type Ca2+ channels in cell-attached patches from guinea-pig ventricular myocytes, without effect on the amplitude of single channel current, the mean open time or the mean shorter closed time, but it increased mean longer closed time and caused a fall in channel availability. A decrease in the mean time between first channel opening and last closing within a trace was principally due to an inhibition of the longer periods of activity. As a result, the time course of the mean currents, which resolved into an exponentially declining and a sustained component, was changed by an increase in the rate of the exponential phase and a profound reduction of the sustained current. Essentially similar results were obtained when studying whole-cell Ba2+ currents. The inactivation of the whole-cell Ca2+ currents was composed of two exponentially declining components with the slower showing a significantly greater sensitivity to BDM, an effect that was much more pronounced in myocytes exposed to isoprenaline with adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) in the pipette solution. The actions of BDM, which are the opposite of those produced by isoprenaline, suggest that the level of phosphorylation affects processes involved in the slow regulation of channel activity under basal conditions and that several sites (and probably several kinases) are involved. Channels with an inherently slow inactivation would seem to be converted into channels with a rapid inactivation by a dephosphorylation process. PMID- 7545283 TI - RNA ligands to human nerve growth factor. AB - High affinity RNA ligands to human nerve growth factor (NGF) were selected from pools of random RNA using SELEX [Tuerk, C. and Gold, L. (1990) Science, 249, 505 510]. Nerve growth factor, which is a protein required for the development of neurons, is not known to bind nucleic acids as part of its natural function. We describe two of the selected RNA molecules in detail. One of them is highly structured, folding into a pseudoknot with an additional hairpin-loop; this structure provides salt-resistant binding to NGF. The other is unstructured and elevated salt concentrations inhibit its binding. These molecules compete with each other for NGF binding. Our RNAs may furnish useful diagnostic tools for the study of an important neurotrophic protein; additionally, they illustrate another example of the potential for nucleic acids to take part in novel binding interactions. PMID- 7545284 TI - Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system. AB - Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3' phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks. PMID- 7545281 TI - Multiple calcium channel subtypes in isolated rat chromaffin cells. AB - By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of --80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (IBa) showed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 microM and 10 microM respectively, during short (15-50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 microM) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of IBa was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin omega-conotoxin GVIA (GVIA; 1 microM) blocked 31% of IBa; blockade was irreversible upon removal of the toxin from the extracellular medium. omega-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of IBa. omega-Conotoxin MVIIC (MVIIC; 5 microM) produced a 36% blockade of IBa; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 microM), GVIA (1 microM), IVA (100 nM) and MVIIC (3 microM) produced partial inhibition of IBa, which were additive. Our data suggest that the whole cell IBa in rat chromaffin cells exhibits at least four components.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545287 TI - Rapid preparation of plasmid templates suitable for a DNA sequencer without RNase treatment. PMID- 7545285 TI - Oxidative base damage in RNA detected by reverse transcriptase. AB - Oxidative base damage in DNA and metabolic defects in the recognition and removal of such damage play important roles in mutagenesis and human disease. The extent to which cellular RNA is a substrate for oxidative damage and the possible biological consequences of RNA base oxidation, however, remain largely unexplored. Since oxidatively modified RNA may contribute to the high mutability of retroviral genomic DNA, we have been interested in developing methods for the sequence specific detection of such damage. We show here that a primer extension assay using AMV reverse transcriptase (RT) can be used to reveal oxidatively damaged sites in RNA. This finding extends the currently known range of RNA modifications detectable with AMV reverse transcriptase. Analogous assays using DNA polymerases to detect base damage in DNA substrates appear to be restricted to lesions at thymine. Oxidative base damage in the absence of any detectable chain breaks was produced by dye photosensitization of RNA. Six out of 20 dyes examined were capable of producing RT detectable lesions. RT stops were seen predominantly at purines, although many pyrimidine sites were also detected. Dye specific photofootprints revealed by RT analysis suggests differential dye binding to the RNA substrate. Some of the photoreactive dyes described here may have potential utility in RNA structural analysis, particularly in the identification of stem-loop regions in complex RNAs. PMID- 7545286 TI - Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution. AB - The number and location of all of the pseudouridine (phi) residues in Saccharomyces cerevisiae small subunit (SSU) ribosomal RNA have been determined by a reverse transcriptase sequencing method [Bakin, A. and Ofengand, J., 1993, Biochemistry, 32, 9754-9762]. Thirteen residues were found in addition to the previously described m1acp3 phi 1189. The residues were scattered throughout the molecule with three being in expansion segments. No phi was found in the three highly conserved single-stranded sequence elements common to all SSU RNAs. Specifically, phi 563, the analog of phi 516 (Escherichia coli) and phi 517 (Bacillus subtilis) were not found. Eight of the phi were located identically to those in mammalian SSU RNA and three were near to mammalian phi residues in the secondary structure. There was no discernible correlation between the sites for phi and the known locations of the methylated nucleosides as exists in large subunit (LSU) RNAs. Comparison of the structural context in which phi was found in SSU RNA with that in LSU RNA showed a differential bias suggestive of possible different roles for phi in the two rRNAs. This work also identified the locations of three putative new modified bases in SSU rRNA, and revealed 15 sequence differences between the yeast strain used here and the reported sequence. PMID- 7545288 TI - A simple procedure for gel electrophoresis and northern blotting of RNA. PMID- 7545289 TI - High throughput RNase protection assay. PMID- 7545290 TI - Severe heart failure: a focus on the quality of care. AB - Heart failure usually originates from conditions such as coronary heart disease, valvular disease, hypertension or cardiomyopathy. Heart failure may develop after several heart attacks as a result of extensive myocardial damage. Despite many advances in treatment for heart disease, chronic heart failure is a terminal condition with a death rate as high as that for many malignant tumours. PMID- 7545291 TI - [Preoperative evaluation of cancers of the esophagus. Contribution of in vivo staining]. AB - OBJECTIVES: We assessed the effectiveness of a dye-endoscopy to detect inapparent multicentric carcinomas of the oesophagus. METHODS: The study included 95 patients scheduled for oesophagectomy. A lugol stain was applied to the oesophageal mucosa and any non-stained area was biopsied. The proximal oesophagus was reexamined on the pathology specimen obtained after oesophagectomy. RESULTS: Intramucosal epithelioma of the proximal oesophagus was found in 7 patients (7.4%) and severe dysplasia was seen in 3 (3.1%). Abnormal stains were seen in 33 patients including 9 with diffuse parakeratosis. There were 24 focal anomalies including 6 cases of intramucosal carcinoma, 3 severe dysplasias, 3 gastric metaplasias and 12 parakeratoses. One intramucosal epithelioma observed on the surgical specimen had not been detected by the staining technique. Overall sensitivity was 85.7% and specificity was 71.7%. Including focal anomalies only, specificity was 79.5%. CONCLUSION: Vital staining is a useful tool for pre operative assessment of cancer of the oesophagus, in particular for superficial forms. PMID- 7545292 TI - Immunosuppressive agents induce the reduction of local pancreatic blood flow. PMID- 7545294 TI - Microsurgical evaluation of experimental pancreatitis after allopurinol: a secretory serological and morphological study. AB - The purpose of the present work was to evaluate the efficacy of allopurinol in rat cerulein-induced acute pancreatitis, by studying amylase and lipase concentrations in bile-pancreatic juice and serum, blood leukocytes, and pancreatic histology. Supramaximal doses of cerulein, injected intraperitoneally twice with a 1-h interval (50 micrograms/kg), caused a reduction in juice amylase and lipase, high levels of the two enzymes in serum, leukocytosis, and severe histological damage to the pancreas, including the presence of diffuse cellular necrosis. These responses were not substantially altered by cotreatment with allopurinol (40 mg/kg i.p. twice with a 1-h interval) or by posttreatment with allopurinol (60 mg/kg i.p. 3 h after cerulein). In contrast, pretreatment with allopurinol (40 mg/kg allopurinol, 1 h, 20 mg/kg allopurinol + 50 micrograms/kg cerulein, 30 min, 40 mg/kg allopurinol, 30 min, 50 micrograms/kg cerulein) had a clear protective effect on pancreatic morphology and function, as shown by higher juice amylase and lipase values, lower serum amylase and lipase concentrations, and an almost normal histological picture of the pancreas. We concluded that allopurinol is effective in preventing the acute pancreatitis caused by administration of supramaximal doses of cerulein, by blocking the free radical generation in pancreatic tissue. PMID- 7545293 TI - On the importance of cholecystokinin in neonatal pancreatic growth and secretory development in guinea pigs. AB - The role of cholecystokinin (CCK) in pancreatic growth and secretory development in fetal and neonatal guinea pigs was investigated by CCK receptor blockade. For the last 20 days of gestation, sows received the CCKA receptor antagonist, MK329 (25 nmol/kg/h) by continuous subcutaneous infusion. Alternatively, neonates from untreated females received an MK329 infusion for the first 4 or 15 days following birth. Pancreatic weight, DNA, RNA, protein, and amylase content per 100 g body weight and secretory responses to CCK, carbamylcholine, and phorbol ester were determined at birth and 4 days in animals receiving MK329 in utero and were measured at 4 and 15 days in neonatally infused animals. No significant changes in pancreatic growth parameters were seen in MK329-treated animals compared to controls, except for a small (14%; p < 0.02) decrease in weight after 15 days of neonatal exposure. Enhanced amylase secretion in response to CCK and carbamylcholine was seen in all groups receiving MK329 (all p values < 0.00001). Pancreatic growth and secretion were not inhibited by CCKA receptor blockade, which suggests that the effects of CCK mediated by the CCKA receptor are not essential for growth or development of the pancreatic amylase secretory response in the neonatal guinea pig. PMID- 7545295 TI - Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity. AB - Initial studies suggested that major histocompatibility complex class I restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development. PMID- 7545298 TI - Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries. AB - When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells. PMID- 7545297 TI - Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors. AB - The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3' deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy. PMID- 7545299 TI - The interferon-inducible double-stranded RNA-activated protein kinase self associates in vitro and in vivo. AB - The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo. PMID- 7545300 TI - Toward the therapeutic editing of mutated RNA sequences. AB - If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing. PMID- 7545296 TI - Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity. AB - In the tumor-bearing host, T cells invariably fail to induce a clinically significant antitumor immune response. Although model systems support the existence of tumor peptide antigens, the molecular interactions critical for antigen presentation by the tumor cell remain unresolved. Here, we demonstrate that human follicular lymphoma cells are highly inefficient at presenting alloantigen despite their strong expression of major histocompatibility complex and low-to-intermediate expression of some adhesion and B7 costimulatory molecules. Activation of follicular lymphoma cells via CD40 induces or up regulates both adhesion and B7 costimulatory molecules essential to repair this defect. More importantly, once primed, alloreactive T cells efficiently recognize unstimulated follicular lymphoma cells. Thus, correction of defective tumor immunity requires not only expression of major histocompatibility complex but also sufficient expression of multiple adhesion and costimulatory molecules. PMID- 7545302 TI - High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli. AB - The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development. PMID- 7545301 TI - Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E selectin and intercellular and vascular cell adhesion molecules 1. AB - To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q. PMID- 7545305 TI - Effects of isradipine and darodipine on serotonergic system of the rat brain. AB - Isradipine and darodipine are dihydropyridine calcium antagonists that easily pass into the brain, showing high affinity for cerebral L-type voltage-sensitive calcium channel (VSCC). These drugs were IP administered to rats to study their effects on serotonergic systems of discrete brain areas. Isradipine (0.05-5.0 mg/kg) and darodipine (0.3-20 mg/kg) increased the 5-HIAA/5-HT ratio, mostly enhancing the metabolite (5-HIAA) content in various brain areas, suggesting that serotonin (5-HT) turnover was increased. This increase appeared to depend on facilitation of serotonergic neurotransmission, because low doses of isradipine (< 0.075 mg/kg) or darodipine (< 0.6 mg/kg) enhanced the number of head twitches induced by L-5-hydroxytryptophan (L-5-HTP). However, higher doses of isradipine (1.5 mg/kg) or darodipine (5 mg/kg) also appeared to stimulate a negative feedback mechanism, which predominated over the facilitation when the serotonergic neurotransmission was strongly activated. Thus, higher drug doses decreased both the serotonin turnover and the number of head twitches on rats treated with L-5-HTP. It was speculated that the observed effects were due to brain VSCC blockade, although the studied compounds showed a peculiar profile of properties when compared to other previously studied calcium antagonists. Moreover, it was concluded that darodipine appeared to be more effective and selective than isradipine regarding the effects on brain serotonergic systems. PMID- 7545304 TI - Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells. AB - We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5 10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation. PMID- 7545303 TI - Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. AB - Triggering of the Fas/APO-1 cell-surface receptor induces apoptosis through an uncharacterized chain of events. Exposure of Fas-sensitive cells to an agonist monoclonal antibody induced cell death and a 200-300% elevation in endogenous levels of the sphingolipid ceramide, a proposed intracellular mediator of apoptosis. In contrast, similar treatment of Fas-resistant cells caused insignificant changes in ceramide levels. Because resistant cell lines expressed the Fas antigen, these results indicate that these cells have a defect in the proximal signaling events leading to ceramide generation. Exposure of the resistant cell lines to a synthetic analog of ceramide induced apoptosis, thus bypassing Fas resistance and indicating that the signaling pathways downstream of ceramide were intact. Furthermore, activation of protein kinase C with the diacylglycerol analog phorbol 12-myristate 13-acetate significantly reduced Fas induced cytotoxicity, suggesting opposing roles for ceramide and protein kinase C in regulation of apoptosis. These results provide evidence for ceramide as a necessary and sufficient lipid mediator of Fas-mediated apoptosis and suggest this process may be modulated via activation of additional signal-transduction pathways. PMID- 7545310 TI - Telomerase and DNA end replication: no longer a lagging strand problem? PMID- 7545309 TI - Call to desegregate microbial databases. PMID- 7545308 TI - A novel IRMA and ELISA for quantifying cytokeratin 8 and 18 fragments in the sera of healthy individuals and cancer patients. AB - Cytokeratins 8 and 18 are most frequently co-expressed in simple epithelia and carcinomas. Fragments of these cytokeratins are released into the systemic circulation where they can be quantified and utilized as tumour markers. The present paper reports on a solid-phase sandwich monoclonal immunoassay, considered "epithelial tissue-specific", which recognizes fragments of human cytokeratins 8 and 18 of different sizes (10-50 kD). The evaluated ELISA and IRMA assays exhibited a detection limit of 0.1 microgram 1-1 and characteristically demonstrated a within-assay coefficient of variation (CV) of 1-4% and a between assay CV of 3-5%. The long-term (300 days) repeatability of lyophilized samples tested in the assay was estimated to have a less than 10% CV. Of apparently healthy individuals, 95% were found to have serum concentrations of less than 0.95 micrograms 1-1. A highly significant difference was found between apparently healthy individuals and pancreatic cancer patients. Patients with metastatic disease showed a sensitivity of 93% and those with local disease 83%, at 95% specificity. This TPAcyk assay revealed good correlation (0.98) with the TPS assay detecting the specific M3 epitope of the tissue polypeptide antigen. The correlation with the long established polyclonal assay of tissue polypeptide antigen (TPA) was estimated to be 0.9. PMID- 7545306 TI - Calcium-and G-protein-related spasmolytic effects of nonsteroidal anti inflammatory drugs on rat uterus contractions in vitro. AB - The effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) acetylsalicylic acid, metamizole, phenylbutazone, indometacin, piroxicam, naproxen, tolmetin, diclofenac, and mefenamic acid on methacholine (10 mumol/l), prostaglandin F2 alpha (1 mumol/l), and KCl (60 mmol/l) induced contractions of isolated rat uterus were assayed. All of these cause a concentration-dependent inhibition of methacholine and prostaglandin F2 alpha-induced contractions with the exception of acetylsalicylic acid, metamizole, and naproxen. All except acetylsalicylic acid and metamizole relaxed in a concentration-dependent manner the tonic contractions induced by KCl. CaCl2 (0.1-10 mmol/l) totally counteracted the relaxant effects of naproxen and tolmetin, but not those of the other NSAIDs. Bay K8644 did not revert the effect of the NSAIDs. Pertussis toxin (50 micrograms/l) did not modify the effect of indometacin, mefenamic acid, and tolmetin, but partially antagonized the effects of diclofenac and naproxen and increased the effect of phenylbutazone and piroxicam. These results suggest that some of the NSAIDs assayed induce smooth muscle relaxation by mechanisms independent of prostaglandin synthesis inhibition, but related to the inhibition of extracellular calcium influx through mechanisms related or unrelated to pertussis toxin sensible G proteins. PMID- 7545307 TI - [Aprotinin: its biological effect and clinical usefulness]. PMID- 7545312 TI - Inhibition of cell cycle progression by the alternatively spliced integrin beta 1C. AB - Integrins regulate cell growth, differentiation, and behavior in many systems. Integrin beta 1C (beta 1S) is an alternatively spliced variant of integrin beta 1 with a specific cytoplasmic domain and is expressed in several human tissues. Human beta 1c transiently expressed in mouse 10T1/2 fibroblasts showed a diffuse pattern of cell surface staining, whereas beta1 localized to focal adhesions. Moderate concentrations of beta 1C had no effect on actin stress fibers or focal adhesions, but markedly inhibited DNA synthesis. Inhibition by beta 1C mapped to the late G1 phase of the cell cycle, near the G1-S boundary. Thus, alternative splicing of beta1 results in transmission of distinct signals that may regulate growth in vivo. PMID- 7545313 TI - A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. AB - Endogenously synthesized antigenic determinants are generally presented on major histocompatibility complex (MHC) class I molecules, whereas exogenous determinants are presented by MHC class II molecules. Here, it is shown that exogenous antigens chaperoned by a heat shock protein can be channeled into the endogenous pathway, presented by MHC class I molecules, and recognized by CD8+ T lymphocytes. This pathway is functional only in a subset of macrophages among the cell types tested. These observations provide a basis for the tumor-specific and virus-specific immunogenicity of cognate heat shock protein preparations and offer a mechanism for the classical phenomenon of cross-priming. PMID- 7545311 TI - Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division. AB - The protein tyrosine kinase c-Src is transiently activated at the transition from the G2 phase to mitosis in the cell cycle of mammalian fibroblasts. Fyn and Yes, the other members of the Src family present in fibroblasts, were also found to be activated at mitosis. In cells microinjected with a neutralizing antibody specific for Src, Fyn, and Yes (anti-cst.1) during G2, cell division was inhibited by 75 percent. The block occurred before nuclear envelope breakdown. Antibodies specific for phosphatidylinositol-3 kinase alpha and phospholipase C gamma 1 had no effect. Microinjection of the Src homology 2 (SH2) domain of Fyn was also inhibitory. Functional redundancy between members of the Src family was observed; a Src-specific antibody had no effect in NIH 3T3 cells but inhibited cell division in fibroblasts in which the only functional Src family kinase was Src itself. Thus, Src family kinases and proteins associating with their SH2 domains are required for entry into mitosis. PMID- 7545314 TI - 'Doing the right thing': the symbolic meanings and experiences of having an HIV antibody test. AB - Very few studies have been undertaken to explore in depth the reasons why individuals seek HIV antibody testing. This paper discusses the socio-cultural meanings surrounding the HIV antibody test, using the findings from a qualitative study directed at understanding why 'low risk' individuals make the decision to have an HIV test, their experiences of testing and their use of the result, especially in negotiating sexual relationships. It is concluded that the less obvious reasons for taking the test include pressure from parents or lovers, as a symbolic closure or commencement of a sexual relationship, the discourse of mutuality, the privileging of 'knowledge' and notions of responsibility and purity. Implications for AIDS education programs and policy are addressed. PMID- 7545316 TI - [The pathogenetic therapy of acute respiratory diseases by aprotinin inhalations]. AB - Inhalations of natural protease inhibitor aprotinin in the form of finely divided aerosol against acute respiratory infections caused by influenza virus, parainfluenza viruses, adenoviruses, and mixed infections produced subjective effect as early as the treatment day 1. Objectively, aprotinin therapy was associated with a 1.5-2-fold reduction in the duration of systemic and respiratory symptoms compared to placebo. As a rule, the inhalations were well tolerated and caused neither local irritation nor allergy. No hepatic, hematopoietic toxicity has been documented. Aprotinin inhalations are thought promising against influenza and acute respiratory infections. PMID- 7545315 TI - Comparative results in detection of HCV antibodies by using a rapid HCV test, ELISA and immunoblot. AB - Viral hepatitis C is a major problem of post-transfusion hepatitis. The best measure for preventing hepatitis C infection in transfusion is blood donor screening. There are many methods for detecting hepatitis C virus antibodies. ELISA is one of the sensitive screening methods. However it needs equipment, technical skills and it is time consuming. The Genelabs Diagnostics HCV-SPOT is a simple, rapid test for the detection of anti-HCV. This paper reports comparative results in detection of HCV antibodies by using GLD HCV-SPOT, ELISA and GLD/DBL HCV BLOT. One hundred and ninety-two specimens from blood donors patients with chronic hepatitis, cirrhosis, hepatoma and miscellaneous chronic liver diseases were tested by HCV-spot assay and HCV ELISA. The positive specimens were confirmed by the immunoblot assay. Only one of 140 samples negative by HCV-SPOT was positive by ELISA. The sensitivity and specificity of HCV-SPOT assays were 97.6% and 92.6% respectively. The positive predictive value was 78.8%, negative predictive value was 99.3%, accuracy was 93.6%. The rapid HCV-SPOT assay can be used in the management of transplantation graft procedures or emergency blood screening for transfusion. In addition, it can be used in small, local hospitals without any expensive equipment. PMID- 7545317 TI - One month follow-up of haemostatic variables in patients undergoing aortocoronary bypass surgery. Effect of aprotinin. AB - It is already known that activation of the coagulation and fibrinolytic system occurs in patients undergoing cardiopulmonary bypass (CPB). We have thus studied twenty patients (10 treated with aprotinin during CPB and 10 untreated) both during the intraoperative period and during thirty days follow up. In untreated patients D-dimer levels increased 4-fold during CPB and the levels were above baseline for the whole follow up (p < 0.0001). D-dimer levels were reduced in aprotinin treated patients in comparison to untreated patients (p = 0.0172); levels then gradually increased to the values of the untreated patients over the following 24 h later and remained higher during the thirty day follow up. The behavior of haemostatic variables in the 24 h after CPB did not vary between untreated and aprotinin treated patients. In particular, five minutes after protamine sulphate administration, levels of F1 + 2 and TAT rose significantly (p = 0.0054, p = 0.0022 respectively), whereas fibrinogen significantly decreased (p < 0.0001) and PAI-1 antigen levels were reduced. Two days after CPB the concentrations of F1 + 2 and TAT lowered, whereas fibrinogen and PAI-1 antigen levels increased. On the 5th, 8th and 30th days after CPB, F1 + 2 and TAT levels remained higher than those reported at baseline in both groups of patients, whereas fibrinogen levels increased over basal levels in aprotinin treated patients only. Thus, in addition to the activation of the coagulation and fibrinolytic system occurring during the intraoperative period, in patients undergoing CPB, there are alterations of haemostatic variables up to thirty days from surgery. PMID- 7545318 TI - Inhibition of thrombin by antithrombin III and heparin cofactor II in vivo. AB - The critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate. PMID- 7545319 TI - Protein tyrosine kinase activation is required for LPS and PMA induction of tissue factor mRNA in human blood monocytes. AB - Tissue factor (TF) is a transmembrane glycoprotein which assembles with factor VIIa on cell surfaces to form a proteolytically active cofactor-enzyme complex; the TF/VIIa complex initiates the coagulation protease cascade. In response to bacterial lipopolysaccharide (LPS) and phorbol-12 myristate 13-acetate (PMA), monocytes synthesize and express TF on their surface. However, the mechanisms by which LPS and PMA activate TF synthesis by human blood monocytes are not fully understood. As it has been established that LPS and PMA activate protein tyrosine kinase (PTK) in monocytes, we studied the role of PTK in LPS and PMA induction of TF by human blood monocytes. Both LPS- and PMA-induced TF activity was inhibited in a concentration-dependent manner by the protein tyrosine kinase-specific inhibitors herbimycin A and genistein. TF antigen determination confirmed that LPS- and PMA-induced cell surface TF protein levels decreased in parallel to TF functional activity under herbimycin A and genistein treatment. Northern blot analysis of total RNA from LPS- and PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to herbimycin A and genistein. The rate of decay of LPS-induced TF mRNA, evaluated after the arrest of transcription by actinomycin D was not affected by genistein and herbimycin A, suggesting that the inhibitory effects occur at least partly at the transcriptional level. We conclude that LPS- and PMA-induced TF production by human monocytes is dependent on tyrosine kinase activation. PMID- 7545321 TI - Vampire bat salivary plasminogen activator evokes minimal bleeding relative to tissue-type plasminogen activator as assessed by a rabbit cuticle bleeding time model. AB - Cuticle bleeding time (CBT) measurements in anesthetized rabbits were performed to assess the potential bleeding risks which may accompany the administration of tissue-type plasminogen activator (tPA) or vampire bat salivary plasminogen activator (BatPA). The dose of BatPA or tPA used in this study, 42 nmol/kg, was previously shown to be efficacious using a rabbit femoral artery thrombosis model (Gardell et al, Circulation 84:244, 1991). CBT was determined by severing the apex of the nail cuticle and monitoring the time to cessation of blood flow. CBT was minimally elevated (1.6-fold, p = NS) following bolus intravenous administration of BatPA; in contrast, bolus intravenous administration of tPA dramatically elevated CBT (6.2-fold, p < 0.05). Rabbits treated with tPA, but not BatPA, displayed profound activation of systemic plasminogen and consequent degradation of Factor VIII and fibrinogen. Elevations in CBT after the administration of tPA were reversed by the replenishment of plasma Factor VIII activity to 40% of control, but were unaffected by complete replenishment of plasma fibrinogen. The results of this study suggest that the administration of BatPA, at a dose that promotes thrombolysis, may evoke a minimal bleeding risk, relative to an equi-efficacious dose of tPA. In addition, the tPA-provoked proteolytic consumption of Factor VIII may be a key contributor to the heightened bleeding risk. PMID- 7545320 TI - Lymphocyte adhesion to human endothelial cells induces tissue factor expression via a juxtacrine pathway. AB - To study the effect of lymphocyte adhesion on the procoagulant activity of endothelial cells, we have stimulated HUVECs with interferon-gamma to upregulate adhesion molecules. Subsequent addition of lymphocytes induced the expression of tissue factor (TF) by HUVECs. Both CD4+ and CD8+ T-cells promoted this TF synthesis via distinct adhesion molecules (CD4+ T-cells: E-selectin and ICAM-1; CD8+ T-cells: MHC-I molecules). In addition, tumor necrosis factor-alpha and beta (TNF alpha, TNF beta) and platelet-activating factor (PAF) were involved in lymphocyte-mediated TF expression on HUVECs. We demonstrate that PAF plays a pivotal role in this process. Adhesion of lymphocytes to endothelial cell surface molecules induced the release of PAF. PAF, in turn, caused the production of TNF alpha and TNF beta, both of which are potent stimulators of TF expression. PMID- 7545323 TI - Targeting platelets containing electro-encapsulated iloprost to balloon injured aorta in rats. AB - Drugs can be electro-encapsulated within platelets and targeted to damaged blood vessels by exploiting the platelet's natural haemostatic properties to adhere to collagen and other vessel wall constituents revealed by injury. A rat aorta balloon angioplasty model has been used to study the effect on platelet deposition of giving iloprost loaded platelets i.v. during the balloon injury. After labelling the circulating platelets with 111-Indium before balloon injury, time course studies showed maximum platelet deposition on the injured aorta occurred at about 1 h post-injury and the deposition remained stable over the next 2-3 h. When iloprost-loaded platelets were given i.v. during injury and the circulating platelet pool labelled with 111-Indium 30 min later, platelet deposition, measured at 2 h postinjury, was substantially and significantly reduced compared with control platelet treatment. Some anti-proliferative effects of iloprost-loaded platelets given i.v. during injury have also been observed. Whereas the incorporation of [3H]-thymidine into aorta intima-media DNA at 3 days post injury was 62-fold higher in balloon injured rats than in control sham operated rats, thymidine incorporation into intima/media of rats which had received iloprost loaded platelets during injury was reduced as compared with rats subjected only to the injury procedure. The reduction was only of near significance, however, but at 14 days after injury the total DNA content of the aorta intima/media of rats given iloprost loaded platelets during injury was significantly reduced. Although iloprost loaded platelets can clearly inhibit excessive platelet deposition, other encapsulated agents may have greater anti proliferative effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545325 TI - Composite graft (Medtronic-Hall) replacement of the ascending aorta and aortic valve in aortic aneurysms: what is adequate follow-up? AB - Between 1987 and 1994, 39 patients underwent 41 replacements (2 reoperations) of the aortic valve and ascending aorta by composite grafts with mechanical valves. One patient had annuloaortic ectasia, one had a sinus of valsalva aneurysm, 13 patients had a Debakey type I acute dissection, 10 had a Debakey type II acute dissection including two Marfan patients, and 14 had atherosclerotic aneurysms. 6 patients (15%) died within a postoperative period of 30 days. The mean follow-up time was 40 months (1-82 months). Twenty-six patients were restudied by clinical examinations and computed tomography of the chest (CT). Two patients required emergency reoperation due to disruption of the proximal aortic anastomosis and right coronary anastomosis. Both patients had been diagnosed to have Marfan disease. Anastomotic dehiscence of composite grafts has a potentially high risk of lethal complications. In follow-up examination computed tomography was performed as a simple and accurate method to detect complications such as pseudoaneurysms, but up to now did not give the indications for reoperation. We suggest that complications may occur not only in the early postoperative period so that regular CT-scan control studies (every 6 to 12 months) should be performed in all patients who undergo composite graft replacement. PMID- 7545326 TI - Early clinical results with VDD pacing in patients with atrioventricular block. AB - In patients with intact sinus node function and atrioventricular block III(o) VDD pacing with a single pass lead may have advantages compared to conventional DDD systems. The purpose of this study was to evaluate the reliability of a new VDD pacemaker with regard to problems encountered with the implantation and the stability of the atrial sensing potential in the postoperative course. 24 patients (10 male, 14 female; age 61 +/- 17 years) underwent implantation of the Intermedics UNITY 292+07 VDD pacemaker. Patients were analyzed postoperatively with respect to stimulation parameters applied. The mean follow-up was 10+/- 3 months. While early on 23 of 24 patients were paced in a VDD mode, one patient was programmed to the vvi mode due to atrial flutter. One patient died early after aortic valve replacement, while another lost his atrial sensing one month postoperatively. Two patients were reprogrammed to the vvi mode because of atrial arrhythmias. The other 20 patients demonstrated stable atrial sensing potential aside from unchanged ventricular stimulation parameters. No infectious or unchanged ventricular stimulation parameters. No infectious or technical problems were observed. From these results it is concluded that VDD pacing may represent an excellent alternative in patients with intact sinus node function and AV block III(o). The atrial sensing was found to be reliable with the additional technical advantage that the single pass lead is less prone to dislocation than the atrial leads in DDD pacing. PMID- 7545322 TI - Anticoagulant-induced pseudothrombocytopenia and pseudoleucocytosis. AB - Pseudothrombocytopenia (PTP) is the phenomenon of falsely low platelet counts due to in vitro platelet clumping in the presence of platelet autoantibodies and anticoagulants. We assessed anticoagulant-dependence, time course of platelet counts and impact of different counter devices on the phenomenon. Blood of 10 persons with previously recognized pronounced EDTA-dependent PTP was collected into 7 different anticoagulants and counted after different intervals in parallel in a Coulter T540 and a Coulter STKS counter and by phase contrast microscopy. With the Coulter T540 model PTP was most pronounced in blood samples anticoagulated with EDTA, Na-oxalate or Na-citrate. In the STKS counter EDTA, heparin and oxalate presented as the worst anticoagulants. The time course of platelet counts was significantly different between the two counters. Our results demonstrate that PTP is not restricted to EDTA, but is also present with other anticoagulants. In contrast, pseudoleucocytosis was observed only in EDTA anticoagulated blood in the Coulter T540 device. We investigated the expression of platelet integrins and activation antigens on platelets of persons with anticoagulant-dependent PTP and in healthy controls without PTP. In the presence of EDTA the expression of GpIIb/IIIa was significantly reduced in the PTP subjects compared to control. Activation antigens CD62, CD63 and thrombospondin antigen were upregulated in the presence of EDTA. These alterations in the expression of platelet antigens could also be induced on platelets of normal donors by incubation with sera of PTP subjects and EDTA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545327 TI - The thrombosed popliteal aneurysm with distal arterial occlusion--successful therapy by interdisciplinary management. AB - Aneurysms of the popliteal artery are rare. Their rate of incidence is reported from 0.1% up to 2.8%. Whereas surgical treatment in an asymptomatic stage bears no problems, the symptomatic stage in a high percentage of patients leads to extremity loss due to thrombosis or embolism. In these cases, amputation rates are reported from 16 to 69%. In the period 1981 - 1994 we saw 39 patients suffering from 58 popliteal aneurysms: 53.4% of these aneurysms were symptomatic. 24.1% of the popliteal aneurysms angiographically showed an occlusion of the popliteal and peripheral outflow tract with concomitant critical limb ischemia. By applying a preoperative local catheter fibrinolysis the outflow tract could be reopened in 13 ischemic extremities and a receiving segment could be recanalized for reconstruction. Following this procedure, in spite of the high number symptomatic cases we gained very good postoperative results in 70.7% of the treated extremities after a maximum follow-up time of 62 months. An aneurysm is commonly seen as a contraindication for the application of lytic therapy. But in the presence of critical extremity ischemia due to a thrombosed or embolizing popliteal aneurysm, preoperative catheter fibrinolysis can often help to save the extremity. PMID- 7545324 TI - A structural/functional domain on human CD36 is involved in the binding of anti Nak(a) antibodies. AB - The human CD36 antigen is an integral membrane glycoprotein expressed by platelets, monocytes, endothelial cells and various tumor cell lines. CD36 acts as a receptor for thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein. Individuals possessing the Nak(a)-negative phenotype do not express CD36 and risk developing anti-CD36 isoantibodies upon blood transfusion or during pregnancy. In the present study, we have examined the interaction of an anti-Nak(a) serum with recombinantly expressed CD36. Results obtained show that five functional CD36 monoclonal antibodies (OKM5, FA6-152, L103, ESIV-C7 and 10/5) prevent the binding of the anti-Nak(a) serum whereas a single monoclonal antibody (13/10) has no effect. Consistent with this result, an epitope map of CD36 generated using cross blocking experiments, indicates that the inhibitory monoclonal antibodies recognize closely-related epitopes whereas 13/10 reacts with a distinct CD36 determinant. Furthermore, we have demonstrated, in a recent study, that OKM5, FA6 152, L103 and 10/5 bind to the same CD36 domain defined by amino acids 155 to 183. Taken together, our results indicate that the 155-183 sequence is important for the binding of the anti-Nak(a) serum to CD36 and may represent a surface exposed, immunogenic and presumably functional region on human CD36. PMID- 7545330 TI - The failure of retrograde continuous warm-blood cardioplegia to resuscitate cardiac function in experimental acute coronary artery occlusion and reperfusion. AB - The effects of retrograde continuous warm-blood cardioplegia (RCWBC) on myocardial preservation during surgical revascularization for acute coronary artery occlusion were investigated using an isolated in-situ dog heart model. The left anterior descending artery (LAD) was occluded for 60 minutes followed by 60 minutes of cardioplegic arrest and reperfusion after release of the coronary artery occlusion. Thirty one animals were divided into 3 groups according to the manner of cardioplegic arrest. The first group of animals (n=10) received multiple doses of cold St. Thomas' Hospital solution delivered antegradely through the aortic root. The second group of animals (n=11) received the same dose of the crystalloid solution delivered retrogradely through the coronary sinus. The third group of animals (n=10) received RCWBC through the coronary sinus. In the animals which were capable of supporting the working mode after reperfusion (8 hearts in each group), regional myocardial function in the occluded LAD distribution measured by sonomicrometer as well as global myocardial function evaluated by left-ventricular stroke-work index were not significantly improved during reperfusion by RCWBC. Corresponding to the functional data, myocardial pH in the occluded LAD distribution was not significantly increased by RCWBC. Although RCWBC maintained myocardial pH in the circumflex artery distribution at a significantly higher level than the other two groups of hearts undergoing cold crystalloid cardioplegia, RCWBC resulted in a substantial decline of myocardial pH in the right-ventricular free wall. These results suggest that RCWBC after 60 minutes of LAD occlusion may not provide a significant benefit in myocardial preservation compared to cold crystalloid cardioplegia delivered through either an antegrade or retrograde manner. PMID- 7545328 TI - Continuous retrograde cerebral perfusion aids repair of aortic laceration during sternal re-entry. AB - We successfully repaired an anticipated large laceration of the ascending aorta occurring during sternal re-entry in a 68-year old woman undergoing replacement of a prosthetic aortic valve. Cerebral protection was ensured by using continuous retrograde cerebral perfusion via femoro-femoral bypass with deep hypothermia. The cooling time was 20 minutes. The minimum nasopharyngeal temperature was 15 degrees C and the duration of the retrograde perfusion was 11 minutes at a rate of 600 ml per minute. The postoperative course was uneventful and no neurological deficits occurred. PMID- 7545331 TI - Reduction of reperfusion injury in rat skeletal muscle following administration of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor. AB - We used cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 (TXA2) receptor, and superoxide dismutase (SOD) with catalase to examine their protective effect against reperfusion injury in rat skeletal muscle. In 5 groups of 6 wistar rats three hours of ischaemia were induced in one hind limb by application of a tourniquet to the proximal thigh; the contralateral limb served as an internal, nonischaemic control. The first group did not receive any drug nor was it reperfused. In the other four groups, normal saline (reperfusion control), dimethylsulphoxide (DMSO), cinnamophilin, or SOD with catalase was given before removal of the tourniquet and one hour of reperfusion followed. Skeletal muscle injury was measured by a quantitative spectrophotometric assay of triphenyltetrazolium chloride (TTC) reduction and by muscle weight gain. One hour of reperfusion significantly (p<0.05) lowered TTC reduction in ischaemic limbs in the reperfusion control group in comparison with the rats in 3h ischaemia alone. Among the four reperfusion groups, only the cinnamophilin group had significantly lower decrease of TTC reduction and significantly lower muscle weight gain. These results demonstrate the protective effect of cinnamophilin against reperfusion injury of the ischaemic skeletal muscle in rats. PMID- 7545333 TI - Early and late prognosis following valve replacement for bacterial endocarditis of the native valve. AB - Between 1983 and 1922 70 patients of the author's institution underwent valve replacement for bacterial endocarditis of native valves. In 22 cases the source of infection could be identified. Among them the most frequent source of infection was dental manipulation (7 patients). The predominating causative microorganism was Streptococcus viridans followed by Staphylococcus aureus. Preoperative complications, the intraoperative finding of extensive destructions, and the histological finding of acute changes influenced significantly the occurrence of perioperative complications. 20 patients died (28.6% overall mortality), there were 9 perioperative deaths (perioperative survival 87.1%). The one-year survival was 73.8% the five-year survival 61.5%. Significant risk factors for the prognosis were preoperative complications, positive bacteriological result of the analyzed resected valves, perioperative complications, duration of the postoperative antibiotic treatment, and the postoperative NYHA functional class. Therefore one should intervene surgically before preoperative complications appear. Perioperative complications must by treated immediately, and an adequate postoperative antibiotic therapy must be guaranteed. PMID- 7545332 TI - Preoperative risk indicators of death at an early and late stage after coronary artery bypass grafting. AB - The aim of the study was to describe mortality during a period of two years after coronary artery bypass surgery (CABG) in relation to preoperative risk factors. Included were all the patients in western Sweden in whom CABG was performed between June 1988 and June 1991, without concomitant procedures or re-operations. The study was prospective in design. In all, 2000 patients with a median age of 64 years were operated upon. Early (within 30 days) mortality was 3.0% and late (30-day-2-year) mortality was 4.2%. Total two-year mortality was 7.1%. For patients undergoing coronary artery bypass grafting, the factors found to be independently predictive of early mortality were female sex, renal dysfunction (creatine clearance < 60ml/min), left main stenosis, number of diseased vessels, previous myocardial infarction, and functional class. We found that a history of congestive heart failure, a history of cerebrovascular disease, diabetes mellitus, renal dysfunction and intermittent claudication were independent risk factors for late mortality. In conclusion, with the exception of renal dysfunction, preoperative risk factors for death within 30 days after CABG differ from risk factors for death between 30 days and two years after CABG. PMID- 7545334 TI - Combined coronary bypass grafting and mitral valve surgery: Early and late results. AB - It is reported that the mortality rate for combined coronary bypass grafting and mitral valve replacement is greater than for either isolated operation. To evaluate the effects of various predicting factors we analyzed the results of 67 consecutive patients undergoing combined mvr and CABG. The mean age was 61.3 +/- 7 years. There were 55 males and 12 females and the mean follow-up was 59.4 +/- 7 months. The hospital mortality rate was 13.4% (9/67). Preoperative NYHA functional class (p<0.05), left-ventricular motion score (increased scores indicating impaired function, (p<0.05), and aortic cross-clamp time (p<0.05) were associated with hospital mortality. There was no significant relationship of age (>60), cause of mitral valve disease, severity of mitral regurgitation, number of grafts, or previous myocardial infarction with hospital mortality. There were 7 late deaths, and survival at five years was 76.1%. Although there was a trend for preoperative NYHA class and aortic cross-clamp time to be associated with late survival, the only factor significantly related to late survival was global wall motion score (p<0.05). Severity of mitral regurgitation and cause of mitral valve disease have been reported as being related to late survival, but we have found no such relationship. Our results indicate that both hospital and late mortality are strongly correlated with preoperative left ventricular function. PMID- 7545335 TI - Changes of white blood cells, immunosuppressive acidic protein, and interleukin-2 receptor after open heart surgery. AB - There are a lot of immunological changes after major surgery, especially in open heart surgery when blood flows through a cardiopulmonary bypass system. We monitored leukocyte count, lymphocyte subpopulation count, soluble IL- 2 receptor (sIL-2r) and serum IL-2 levels, and an immunosuppressive acidic protein (IAP) level before and on the 1st, 7th, and 14th day after open heart surgery. It was noted that there were significant increases of total leukocyte and granulocyte counts for more than two weeks. But total lymphocyte, T-lymphocyte, b-lymphocyte, natural killer cell, suppressor T-cell, and helper T-cell all showed significant decreases in absolute numbers for more than one week as compared to the preoperative values. Serum IAP level was elevated for more than two weeks after operation. Serum IL-2 decreased but serum sIL-2R increased in the postoperative period. It is concluded that there is a period of immunosuppression after open heart surgery which lasts for at least one week and perhaps more than two weeks. PMID- 7545336 TI - Individual anatomy demands various techniques in correction of an anomalous origin of the left coronary artery in the pulmonary artery. AB - Anomalous origin of the left coronary artery (ALCA) from the pulmonary artery is a rare cardiac anomaly. It can result in decreased myocardial perfusion and impaired left-ventricular function, which can lead to congestive heart failure or even myocardial infarction. Reconstructive surgery of the left coronary artery is the desirable measure in the management of this anomaly. Since July 1992, five patients with ALCA from the pulmonary artery were treated surgically. Age at operation ranged from 3 months to 11 years. Three patients had congestive heart failure and one was in cardiogenic shock. Operative techniques included "tunnel type" surgery in three cases and aortic reimplantation in two. Two operative variations performed successfully in this series are described. There were no postoperative deaths. At the latest follow-up (mean 14 months), all anastomoses were patent and showed antegrade flow. For those patients with ALCA from the pulmonary artery, direct reimplantation of the ALCA to the aorta is the most physiologically appropriate reconstructive solution, and offers good early and late results. Intrapulmonary tunnel from aortopulmonary window to coronary artery is recommended for children in whom aortic reimplantation is not anatomically feasible. PMID- 7545337 TI - The PTB domain: a new protein module implicated in signal transduction. AB - Src homology 2 (SH2) domains have been identified in a large number of proteins involved in signal transduction downstream of receptor tyrosine kinases. They allow cytoplasmic signalling proteins to bind specifically to other polypeptides that are phosphorylated on tyrosine in response to growth factor stimulation. A novel phosphotyrosine-binding (PTB) domain has been identified recently in the amino terminus of Shc, an adaptor molecule that appears to be involved in Ras activation PTB domains are longer than SH2 domains, and recognize phosphotyrosine in the context of amino-terminal residues, in contrast to SH2 domains, which recognize them in the context of carboxy-terminal residues. PMID- 7545338 TI - Pre-rRNA processing and the path from the RNA world. PMID- 7545339 TI - Prevalence of antibodies to hepatitis C virus and other markers in Jamaica. AB - Three population groups, 1500 blood donors, 513 antenatal women representing a normal population group and 250 sicklers representing a multiply transfused group were studied to determine the prevalence of hepatitis C viral (HCV) infection in Jamaica. The relationship to liver enzyme levels, hepatitis B infection, syphilis and HIV infection was also investigated. Sera were screened by enzyme-linked immunoassay (EIA) for anti-HCV C100-3 and subsequently tested by a supplementary second generation recombinant immunoblot assay (RIBA). In the blood donors, the prevalence of anti-HCV was low, 0.3%-0.4%, the same level as that reported by several European countries. In the multiply transfused sicklers, the prevalence was more than seven times higher. No HCV infection was detected in the antenatal group. There was little correlation between HCV infection and surrogate markers alanine aminotransferase (ALT) and antibody to hepatitis B core antigen (anti HBc) and no correlation with sexually transmitted diseases. PMID- 7545340 TI - Prostate cancer screening--is the controversy resolved? PMID- 7545329 TI - Demonstration of vascular ring anatomy with ultrafast computed tomography. AB - An infant with double aortic arch is reported in whom ultrafast computed tomography with three-dimensional volume reconstruction provided great anatomic detail of the vascular anomaly. This technique and magnetic resonance imaging may obviate the need for invasive angiography. PMID- 7545341 TI - [The high risk patient with symptomatic prostatic hyperplasia--a therapeutic dilemma?]. AB - Since a few year, a lot of new methods in therapy of symptomatic benign prostate hyperplasia are available. Until now, none of these methods could reach the outcome of surgical therapy. Therefore, surgery is remaining the golden standard in the therapy of BPH. Alternative methods are of great importance in the treatment of "high risk patients". It may be possible to improve the quality of life. Laser therapy and intraurethral catheters are superseding transurethral and suprapubic catheters. The indication for the several methods has to been proven critically. PMID- 7545343 TI - Biodegradable microspheres. XVIII: The adjuvant effect of polyacryl starch microparticles with conjugated human serum albumin. AB - The adjuvant effect of polyacryl starch microparticles was examined in mice using covalently coupled human serum albumin as a model antigen. Mice were immunized using different administration routes and the humoral and cellular immune responses were followed. The intraperitoneal immunizations gave the strongest humoral immune response, almost as high as with Freund's Complete Adjuvant (FCA), and the response after intravenous and intramuscular immunizations were somewhat lower. The delayed type hypersensitivity response was, after intramuscular immunization, about the same as after intraperitoneal immunizations with FCA. The results shows that polyacryl starch microparticles is a promising candidate for a new adjuvant for human use. PMID- 7545344 TI - Potentiality of gelatin microsphere as immunological adjuvant. AB - This paper describes a new attempt to enhance the production of antibody by delivery of an antigen to phagocytic antigen-presenting cells (e.g. macrophages) using gelatin microspheres. A model protein antigen, human gamma globulin (HGG), was incorporated into microspheres composed of gelatin which have an opsonic ability for macrophage phagocytosis. Subcutaneous injection of the microspheres induced the production of HGG-specific IgG antibody in the mouse serum to a great extent compared with that of HGG in soluble form or in Freund's incomplete adjuvant (FIA) form. There was an optimal concentration of cross-linking agent (glutaraldehyde) for the highest production of antibody. When gelatin microspheres were cross-linked at lower concentrations of glutaraldehyde, they were more extensively swollen in an aqueous solution, leading to an increase in the size of hydrated microspheres because of their lower cross-linking densities. The increased size of microspheres caused a decrease in their macrophage phagocytosis, whereas the release rate of HGG from the microspheres increased as the concentration of cross-linking agent became low. The balance of the two factors, the microsphere susceptibility to macrophage phagocytosis and the rate of HGG release, seemed to affect the efficacy of gelatin microspheres to enhance the antibody production. In addition, incorporation of HGG into gelatin microspheres enhanced the delayed-type hypersensitivity reaction. Moreover, the microspheres developed a strong secondary response in comparison with FIA. The gelatin microspheres induced a minimal inflammatory response around the injection site in contrast to FIA. These findings demonstrate that the gelatin microsphere is promising as an adjuvant to enhance both humoral and cellular immune responses to antigen. PMID- 7545345 TI - The journey. PMID- 7545342 TI - [Masked course of Whipple disease with uveitis, infection, endocardial involvement and abdominal lymphomas--case report and review of the literature]. AB - Whipple's disease is a systemic disease which may virtually affect any organ system, but in many cases it involves the small intestine causing gastrointestinal symptoms. The differential diagnosis is difficult since symptoms may be nonspecific. We report the case of a 44-year old white male patient with a history of migrating arthralgia and chronic fatigue. The patient newly developed an uveitis and underwent a vitrectomy; the further clinical work-up including gastroscopy with intestinal biopsy revealed no sufficient diagnosis. Subsequently, the patient's condition deteriorated with marked weight loss, fever and progressive weakness. An anaerobic sepsis with a corynebacterium was confirmed and with i.v.-antibiotics the patients's condition improved markedly. The further examinations disclosed enlarged mesenteric lymph nodes and the involvement of other organs (endocard, liver). CT-guided biopsy only showed fatty degeneration, but operative adenectomy confirmed Whipple's disease. The patient remained without relapse on long-term antibiotic treatment with doxycycline until today. Obviously, in our case the intestinal biopsies failed to detect Whipple's disease after the successful initiation of antibiotic treatment. In the absence of gastrointestinal findings and with concomitant secondary diseases the definitive diagnosis can be difficult. In addition, the previous uveitis and the endocardial involvement are most interesting. PMID- 7545348 TI - Quantification of macrophage-derived substance P receptor mRNA using competitive polymerase chain reaction. PMID- 7545346 TI - Detection of kappa-opioid receptor mRNA in immature T cells. AB - The R1.1 cell line has been shown to express kappa-opioid receptors on the cell surface. Our analysis shows that the R1.1 cell line exhibits a CD4NEG CD8NEG CD3LOW CD25LOW cell surface phenotype, characteristic of thymocytes in one of the early stages of differentiation. We have developed reverse transcriptase polymerase chain reaction (RT-PCR) conditions that permit the detection of mRNA coding for the kappa-receptor. Using cell fractionation techniques we have isolated CD4NEG CD8NEG thymocytes, and analysis by RT-PCR shows that these primary immature thymocytes also express the kappa-opioid receptor. We hypothesize that the expression of kappa-opioid receptor may be a marker which is characteristic of immature T development. PMID- 7545350 TI - [Significance of prostate fluid carcinoembryonic antigen in the diagnosis of prostate cancer]. AB - Clinical studies of carcinoembryonic antigen (CEA) in prostate fluid were performed on 121 men; 29 patients with prostate cancer, 65 with benign prostatic hypertrophy, 10 with prostatitis and 17 without any prostatic diseases. The CEA level in prostate fluid in the prostate cancer group was significantly higher than that in any other group. However, it could not demonstrate any particular advantage when compared with the prostate specific antigen (PSA) in serum. Additional research was done comparing the sensitivity and specificity of CEA level in prostate fluid in the population with slightly elevated PSA level (3.0 14.9 ng/ml). The sensitivity and the specificity were 82% and 83%, respectively. These findings suggest the usefulness of the measurement of prostate fluid CEA as an adjunctive tool in the diagnosis of prostate cancer in the population with a slightly elevated PSA level. PMID- 7545347 TI - Morphine alteration of histamine release in vivo. AB - Studies were performed to evaluate the effects of either acute or chronic morphine exposure on histamine release in vivo and supporting studies in vitro. In order to effectively assess histamine release in swine, studies were undertaken to evaluate the effectiveness of compound 48/80 as an intradermal skin test and determine its ability to release histamine in swine cells. Compound 48/80 skin testing was found to be a useful measure of histamine release in swine as evidenced by dose dependent wheal and flare reaction in vivo and histamine release from swine cells in vitro. Acute effects of morphine were determined on swine administered a single injection of morphine alkaloid. Skin tests using intradermal compound 48/80 and histamine, were performed using compound 48/80 both prior to, and 24 h following initiation of morphine treatment. Morphine tolerant swine were subjected to in vivo skin tests and the resulting wheal and flare responses measured. In select swine skin samples from the test sites were measured for mast cell numbers. Swine dermal mast cells were found to release histamine in a dose dependent manner upon compound 48/80 exposure. Both acute and chronic morphine-treated swine had significantly depressed responses to compound 48/80, however this difference was not due alteration in mast cell number or morphology, and skin responsiveness to histamine remained intact. PMID- 7545349 TI - Expression of cannabinoid receptor mRNA in murine and human leukocytes. AB - delta 9-tetrahydrocannabinol, the major psychoactive cannabinoid of marijuana modulates immune cells in vivo and in vitro. It is possible that the drug exerts it's effect either by inserting into and disrupting the cell membrane (nonreceptor mechanism) or by binding to a cannabinoid receptor moiety and thus altering cell function through some form of signal transduction. In the present study, we confirm and extend the findings that mouse and human immune cells express specific cannabinoid binding sites and cannabinoid receptor mRNA. Reverse transcription-polymerase chain reaction analysis showed the presence of receptor mRNA not only in the neuroblastoma cell line (N18TG-2), but also in mouse splenocytes and in cell lines such as NKB61A2 (a mouse natural killer-like), CTLL2 (a mouse IL2-dependent T cell), THP-1 (a human monocytic cell) and Raji (a human B cell) but not in Jurkat (a human T cell). Furthermore, the receptor mRNA was expressed in purified populations of resting splenic T and B lymphocytes but not in resting populations of enriched splenic macrophages. Finally, LPS stimulated Raji and PMA-stimulated THP-1 human cell lines showed increased levels of the cannabinoid receptor mRNA. These results suggest cannabinoid receptors have biological relevance in lymphoid cells because: receptor mRNA is detected in some resting immune cells but not others and the mRNA increases during cell activation. The major psychoactive component of marijuana, delta9 tetrahydrocannabinol (THC), has been shown to modulate human and mouse immune responses both in vitro and in vivo (1,2).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545354 TI - Activity staining of mammalian ribonuclease inhibitors after electrophoresis in sealed vertical slab polyacrylamide gels. AB - A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating at 37 degrees C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex formation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples. PMID- 7545351 TI - [Post-TUR-P stress urinary incontinence successfully treated by periurethral injection of autologous fat to the prostate: a case report]. AB - A 77-year-old man had been suffering from stress urinary incontinence for 2 years since he had received transurethral resection of prostate (TUR-P) for benign prostatic hypertrophy. A 60-min pad test yielded 3 g of urine. Prostatic urethra was widely open and the external sphincteric injury was suggested because of the short membraneous urethra on the urethrogram. Urethral pressure profile indicated his maximal urethral closing pressure (MUCP) of 24 cmH2O and functional urethral length of 1.6 cm and cystometry demonstrated an underactive bladder, indicating that his incontinence was caused by sphincteric injury. Autologous fat injection therapy was performed in the lithotomy position under spinal anesthesia. Fifteen ml of subcutaneous fat was obtained from his lower abdomen by liposuction through a 15G needle, and 10 ml was injected submucosally from the perineum at 6 o'clock area of the prostatic apex under the guidance of transrectal echography using a 15G needle. The patient became completely dry after the procedure. MUCP and FUL increased to 35 cmH2O and 1.9 cm, respectively, although longer follow up is necessary. The advantage of autologous fat injection to the prostate for post-TUR P SUI patients is briefly discussed. PMID- 7545353 TI - Bone marrow of patients with active multiple myeloma: angiogenesis and plasma cell adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. AB - Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination. PMID- 7545352 TI - Clinical and histological features retain their prognostic impact under interferon therapy of CML: a pilot study. AB - In 55 patients with Ph1+ CML under interferon (IFN) monotherapy, an immunohistochemical and morphometric study on pretreatment bone marrow biopsies was performed to evaluate the prognostic impact of clinical as well as histological disease features. For identification of megakaryocytes we used the PAS stain and CD61 to calculate the subfraction of precursors (pro- and megakaryoblasts). Demonstration of macrophages and their different subsets was carried out by PG-M1 (CD68) and the GSA-1 lectin. The erythroid precursors were stained by Ret40f (anti-glycophorin C). Density of argyrophilic (reticulin plus collagen) fibers was determined by applying Gomori's silver impregnation method. Clinical variables like state of hematological response to IFN administration, age, spleen and liver size, myeloblasts plus promyelocytes, basophils as well as basophils and eosinophils exerted a predictive capacity by univariate statistical analysis. However, when entering these factors into previously published risk models, i.e., the so-called Sokal score and its modifications, to assess subgroups with different survival patterns or relative risk groups, a clear-cut discrimination was not feasible. Bone marrow features of prognostic value consisted of megakaryocytes and their precursors, fibers, and pro- and erythroblasts. Only when including histological variables into a formerly reported Cox model, could a significant separation of patients into the different categories or relative risk groups be computated. In conclusion, the present data emphasize the prognostic impact of histological parameters to be considered in all clinical trials on CML. PMID- 7545355 TI - Monoclonal antibody-based immunoassay for evaluation of lipoprotein oxidation. AB - Numerous reports indicate that the oxidation of low-density lipoprotein (LDL) can significantly change its metabolic and physiological properties. Most methods for evaluation of LDL oxidation require isolation of lipoprotein, making the procedure laborious and increasing the probability of artifactual modification of LDL. In this paper we describe an immunochemical approach which can be used to measure the oxidation of isolated LDL and apoprotein B in unfractionated serum and to evaluate the effects of antioxidants on these processes. The procedure is based on differential recognition by monoclonal antibodies of native and oxidized lipoproteins. The results obtained with our assay indicate a strong correlation between the changes of apo B epitope expression during oxidation and the formation of conjugated dienes, changes in lipoprotein electrophoretic mobility, and interaction with fibroblast and macrophage receptors. The sensitivity of apo B to oxidation varies greatly among serum samples obtained from individual donors. These differences do not correlate with the differences in sensitivity to oxidation of LDL isolated from the blood samples of the same donors. It is also shown that apo B oxidation in serum can be progressively inhibited in the presence of increasing amounts of various antioxidants. PMID- 7545358 TI - Role of prophylactic gastroenterostomy for unresectable pancreatic carcinoma. AB - Methods of palliation and the use of prophylactic gastroenterostomy in the treatment of unresectable pancreatic carcinoma remain controversial. Gastroenterostomy has been linked with various complications. We conducted a 10 year (1982-1992) retrospective review of patients who had unresectable pancreatic carcinoma and underwent biliary decompression without prophylactic gastroenterostomy. 50 patients were studied. Only four patients (8%) developed duodenal obstruction and required reoperation for therapeutic gastroenterostomy. The mean time to obstruction was 15.75 months, whereas the mean overall survival was 12.99 months. The mean survival of patients who underwent therapeutic gastroenterostomy was 32.25 months, with an average palliation of 16.5 months after the second operation. We conclude that pancreatic carcinoma has a rapid natural progression, and most patients do not survive long enough to obstruct. The ones who do obstruct are unique in that they survive for a long period of time. We recommend that routine prophylactic gastroenterostomy is unnecessary, and selective use of gastroenterostomy should be exercised in case of present or impending duodenal obstruction. PMID- 7545356 TI - Detection of adenylate cyclase-coupled receptors in Xenopus oocytes by coexpression with cystic fibrosis transmembrane conductance regulator. AB - To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists. PMID- 7545357 TI - Appearance and distribution of laminin A chain isoforms and integrin alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits in the developing human small intestinal mucosa. AB - BACKGROUND: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. METHODS: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4, four potential receptors, at the epithelial mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. RESULTS: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins alpha 6 beta 1 and alpha 6 beta 4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the alpha 2 beta 1 and alpha 3 beta 1 integrins was found time- and site-restricted. The alpha 2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the alpha 3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19-20-week-old crypts. CONCLUSIONS: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. PMID- 7545360 TI - Clinical and laboratory diagnosis of acute pancreatitis. AB - Determination of serum pancreatic enzymes remains the gold standard for the diagnosis of acute pancreatitis. Clinical symptoms and signs are of major importance in suspecting the disease, but they are not accurate enough to confirm the diagnosis. Among pancreatic enzymes, total amylase, pancreatic isoamylase and lipase are preferred, since simple, rapid and unexpensive enzymatic methods are commercially available. More expensive and cumbersome methods (e. g. ELISA for pancreatic elastase) are required if a significant delay to hospital admission occurs. In that case, other serum enzymes are usually normal or only lightly increased. To early define the etiology of acute pancreatic serum pancreatic enzymes lack of value. With this purpose, determination of AST, bilirubin and alkaline phosphatase may allow to distinguish between biliary and non-biliary origin of the disease. PMID- 7545359 TI - [Surgery of loco-regional recurrences in colorectal cancer]. AB - By the term of local recurrence (LR), after apparently curative resection of a colorectal cancer, we mean the recurrence of the disease at the site of the original tumor, in correspondence to the regional lymph nodes or to intestinal anastomosis. The frequency of LR, which differs widely from one case to another because of the unhomogenity of the samples under examination, is on average around 16%, although this figure is to be considered an underestimation since it is not supported by routine autopsies. The authors analyse, together with the data in the literature, the results of their personal experience relating to 1164 patients undergoing surgery for colorectal cancer in the space of 18 years, in order to evaluate the true effectiveness of surgery on patients affected by LR. Surgery on the original tumor had a curative aim in 791 (68%) of these cases, 33 patients died during the postoperative period and 37 were lost to follow-up. Among the remaining 721 patients, recurrence was observed in 218 cases (30.2%): in 81 of these was confirmed a LR, with simultaneous distant metastasis in 45. During the same time span, 15 patients were admitted who were affected by LR from colorectal cancers treated surgically in other hospitals. Of these, only 2 had concomitant distant metastasis. 58 of these 96 patients (60.4%) underwent surgery, which had a curative aim in 22 cases (37.9%)/ Up to present, 12 patients undergoing curative surgery are still alive, 4 (33.3%) being affected by further relapse. The data reported demonstrate that in patients with proven LR surgical intervention is widely indicated, being the only therapeutic approach able to provide tangible results. However, the choice of exeresis must be carefully weighed: surgery of LR can be said to be truly curative in few cases, and thus the operative risks may not be acceptable in situations where limited results, in terms of duration and quality, are foreseen. PMID- 7545363 TI - Function of hematopoietin receptor subunits in hepatic cells and fibroblasts. PMID- 7545364 TI - Membrane-bound and soluble interleukin-6 receptor: studies on structure, regulation of expression, and signal transduction. PMID- 7545362 TI - Transcription factors NF-IL6 and APRF involved in gp130-mediated signaling pathway. PMID- 7545361 TI - Gabexate mesilate in the treatment of acute pancreatitis. AB - Despite controversial experimental and clinical findings, the use of Aprotinin (A) in the treatment of acute pancreatitis (AP) is still widespread in our country. Compared to A, Gabexate Mesilate (FOY) should have the following advantages: lack of antigenicity, low molecular weight with better cellular penetrance, wide inhibitory spectrum (against phospholipase A2 too) and good tolerability. In order to verify the efficacy of FOY versus, we decided to carry out a prospective multicenter (34 centers) randomized double blind trial in moderate-severe AP with FOY 3g/day versus A 1,500,000 U.I.K./day infused for 7 days with central venous line. Based on standard clinical, biochemical and radiological data, 199 patients suffering from AP were enrolled to the study from January 1989 to December 1990. The main admission criteria were: onset of the disease from no more than 72 hours, at least 2 Ranson positive criteria and C.T. evidence of acute pancreatic damage. Seventeen no protocol patients were excluded. The Etiology in the 182 analyzed patients (106 male, 76 female; mean age 57.6 years, range 21-91) were: biliary 102 (56%), alcohol 47 (25.8%), post operative 13 (7.1%), unknown causes 13 (7.1%) and others 7 (3.8%). Median Ranson's Score was 3. CT examination showed 66 AP with edema, while 116 cases (63.7%) were recognized as severe forms with pancreatic necrotic involvement of 30% of the gland in 71 (61.2%), 50% in 31 (26.7%), and more than 50% in 14 (12.1%). Out of 182 patients, 91 were treated with A and 91 with FOY. The two groups were comparable regarding the main prognostic factors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545365 TI - Isolation of two interleukin-6 response element binding proteins from acute phase rat livers. AB - Proteins binding at the IL-6 response element of the rat alpha 2 macroglobulin gene were purified by a combination of conventional chromatographic procedures and binding-site specific DNA affinity chromatography. The proteins were purified from the nuclei of rat livers, excised at the peak of an experimentally induced acute phase response. By this procedure three polypeptides of 92, 91 and 86 kD were enriched more than 6,000-fold. Partial proteolysis with lysyl endopeptidase and aminoacid sequence analysis of proteolytic peptides identified the 86 and 91 kD species as the transcription factor Stat3 and the 92 kD species as a Stat factor distinct from Stats 1 and 3. cDNA clones for Stats 1, 3 and this 92 kD factor were isolated from a cDNA library prepared with mRNA from acute phase rat livers. Parts of their DNA sequences were determined and the sequences of the purified peptides were found in these cDNA sequences. Thus, the identity of the factors as Stat3 and a Stat factor different from Stats 1 and 3 was confirmed. These results suggest, that APRF/Stat3 and p91/Stat1 are not the only factors mediating the effects of IL-6 on class 2 acute phase genes. The 92 kD Stat factor binding at the IL-6 RE probably also functions as a transcription factor in the cytokine-induced activation of the alpha 2M gene. PMID- 7545366 TI - Functional analysis of IL-6 and IL-6DBP/C/EBP beta by gene targeting. PMID- 7545367 TI - In lethally irradiated mice interleukin-12 protects bone marrow but sensitizes intestinal tract to damage from ionizing radiation. AB - Administration of IL-12 prior to lethal irradiation, protected a significant fraction of mice from 60Co-gamma radiation-induced lethal hematopoietic syndrome. Radioprotection was associated with an increase in the number of c-kit+ bone marrow cells (BMC) in IL-12 treated mice compared to saline-treated mice. Even after supralethal doses of radiation (1200 cGy), IL-12-treated mice had twofold greater numbers of c-kit+ BMC than controls. However the mice receiving IL-12 and 1200 cGy died of the gastrointestinal (GI) syndrome, evident by gross necroscopy and histological evaluation, within 4 to 6 days after irradiation. Induction of the GI syndrome in mice not treated with IL-12 required radiation doses of 1600 cGy. Thus, at doses of radiation at which IL-12 still protects c-kit+ hematopoietic cells, it sensitizes the intestinal tract to damage. Radioprotection with IL-12 was abrogated by anti-IL-1R or anti-SCF antibody, but not anti-IFN gamma antibody. In contrast, anti-IFN gamma antibody abrogated sensitization of the intestinal tract by IL-12. PMID- 7545368 TI - Pleiotropic defects of IL-6-deficient mice including early hematopoiesis, T and B cell function, and acute phase responses. PMID- 7545370 TI - Interleukin-6-type cytokine-induced changes in acute phase protein glycosylation. AB - The plasma levels and the glycosylation of acute-phase proteins (APP) are subject to marked changes during acute and chronic inflammation. The pathophysiological variations in different glycoforms of APP in serum most likely result from changes in the glycosylation process during their biosynthesis in the parenchymal cells of the liver. This is suggested from in vitro studies with isolated hepatocytes and hepatoma cell lines. Inflammatory cytokines appear to regulate the changes in glycosylation independent from the rate of synthesis of the APP. In addition, other humoral factors like corticosteroids and growth factors are involved. The interplay of these factors is determined by the stage of the disease (as in rheumatoid arthritis) or the physiological situation (as in pregnancy). The changes in glycosylation of specific APP might affect the operation of the immune system. PMID- 7545369 TI - Interleukin (IL)-11--mediated signal transduction. AB - IL-11 is a multifunctional cytokine biologically related to IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF). It has been shown that these cytokines can utilize common signal transducer, gp130. We have demonstrated that Jak tyrosine kinases, MAP kinases and pp90rsk are highly activated by IL-11 and related cytokines. In addition, we have identified pp90rsk as one of the H7 sensitive protein kinases critical for primary response gene expression induced by IL-11. Furthermore, activation of 3CH134 (a MAP kinase phosphatase) gene by IL-11 suggested that a MAP kinase phosphatase may be involved in IL-11-mediated signal transduction. Our data also suggested that tyrosine phosphorylation of Stat91 and related transcriptional factors is involved in IL-11 signaling but is not sufficient for the activation of primary response genes such as JunB, tis11, tis8 and MAP kinase phosphatase in mouse preadipocytes. The understanding of signal transduction pathways mediated by IL-11 and related cytokines may help to define the common and unique biological properties of these growth factors. PMID- 7545373 TI - Interleukin-6-type cytokines affect glycosylation of acute phase proteins in vitro. PMID- 7545371 TI - Interleukin-6: effects on tumor models in mice and on the cellular regulation of transcription factor IRF-1. PMID- 7545372 TI - Selectin-P (PADGEM, GMP-140)-mediated adhesion of human platelets to neutrophils in vitro and immune complex-induced peritonitis in rats is influenced by interleukin-8. PMID- 7545374 TI - Effects of HGF and RA on the class 1 and class 2 rat acute phase proteins. PMID- 7545377 TI - Plasma acute phase proteins and metalloproteins in children with neuroblastoma at diagnosis. PMID- 7545376 TI - The LIF-response element confers LIF-induced transcriptional control in P19 embryonal carcinoma cells. PMID- 7545379 TI - Reliability and sensitivity of intelligibility judgments by severely and profoundly hearing-impaired children. PMID- 7545375 TI - In vivo properties of oncostatin M. PMID- 7545380 TI - The effect of 6% hydroxyethyl starch and desmopressin infusion on von Willebrand factor: ristocetin cofactor activity. AB - Hydroxyethyl starch is commonly used as a plasma volume expander in the surgical patient. Although it is generally considered a safe plasma substitution, some reports of an acquired von Willebrand's disease-like syndrome have been documented. To examine this further, von Willebrand factor: ristocetin cofactor activity (RCoF) was measured in two groups of patients perioperatively, following hydroxyethyl starch infusion and at 30, 60, and 240 minutes following either deamino-8-D-arginine vasopressin (DDAVP) (group I, n = 12) or saline (group II, n = 11). Following hydroxyethyl starch infusion, ristocetin cofactor activity decreased to 58 percent (group I) and 55 percent (group II) of their respective baseline values. After infusion of DDAVP, mean ristocetin cofactor activity in group I increased significantly to 95 percent at 30 minutes and 100 percent of baseline at 60 minutes. Mean ristocetin cofactor activity levels in group II, however, remained decreased, 69 percent and 57 percent of baseline at the same time points. There was no statistical difference between groups before or immediately after hydroxyethyl starch administration or at 240 minutes post-DDAVP or saline infusion. It is our conclusion that DDAVP is a safe therapy for the mild coagulapathy infrequently associated with hydroxyethyl starch administration. PMID- 7545381 TI - Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes. AB - Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl) adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D. PMID- 7545378 TI - Signal transduction through IL-6 receptor: involvement of multiple protein kinases, stat factors, and a novel H7-sensitive pathway. PMID- 7545382 TI - Expression of CD44 on rheumatoid synovial fluid lymphocytes. AB - OBJECTIVES: To investigate the involvement of the adhesion molecule CD44 in the homing of lymphocytes to synovial tissue, by examining the density of expression and molecular mass of CD44 on rheumatoid synovial fluid lymphocytes. METHODS: Twenty patients with rheumatoid arthritis were studied. Peripheral blood and synovial fluid lymphocytes were isolated by Ficoll-Hypaque sedimentation. CD44 expression was analysed by two colour flow cytometry of CD3 positive T lymphocytes with calculation of mean fluorescence intensity. Expression of activation markers M21C5, M2B3, interleukin (IL)-2 receptor and transferrin receptor was quantitated. In addition, CD44 molecular mass was examined by Western blot in six patients. RESULTS: CD44 expression was markedly increased on synovial fluid T lymphocytes of rheumatoid patients relative to peripheral blood lymphocytes from the same individuals. CD44 molecular mass on peripheral blood mononuclear cells was 88 kDa, but that on synovial fluid lymphocytes was only 83 kDa. CD44 expression correlated significantly with expression of activation markers M21C5, M2B3, and the IL-2 receptor. CONCLUSIONS: Alterations in density of expression or of the molecular mass of CD44 could contribute to local tissue injury, either directly by facilitating adhesion, or indirectly through effects on other adhesion molecules. PMID- 7545383 TI - Thermococcus peptonophilus sp. nov., a fast-growing, extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents. AB - Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7-2 microm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85-90 degrees C (range 60-100 degrees C), pH 6 (range pH 4-8), a NaCl concentration of 3% (range 1-5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005-5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. PMID- 7545384 TI - Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. AB - Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents. PMID- 7545385 TI - Involvement of transglutaminase in the formation of covalent cross-links in the cell wall of Candida albicans. AB - Activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N epsilon-(gamma glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms of Candida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall. PMID- 7545386 TI - Mycobacterial pseudotumors of lymph node. A report of two cases diagnosed at the time of intraoperative consultation using touch imprint preparations. AB - OBJECTIVE: The purpose of this report is to emphasize the diagnostic utility of intraoperative touch imprint preparations in the evaluation of Mycobacterium avium-intracellulare-infected lymph node biopsy specimens obtained from human immunodeficiency virus-positive patients. PATIENTS: Two human immunodeficiency virus-positive men, 41 and 29 years of age, with marked lymphadenopathy clinically suspicious for malignant neoplasm. RESULTS: Intraoperative touch imprints of two lymph node biopsy specimens rapidly demonstrated the presence of numerous intracellular organisms in Mycobacterium avium-intracellulare-infected lymph nodes, including one case of mycobacterial spindle cell pseudotumor. CONCLUSIONS: Intraoperative touch imprint preparations are helpful in the evaluation of lymph node biopsy specimens obtained from human immunodeficiency virus-positive patients and allow rapid diagnosis of Mycobacterium avium intracellulare infection. PMID- 7545387 TI - Amyloid in adrenal gland pheochromocytomas. AB - OBJECTIVE: To assess the frequency of amyloid deposits in adrenal gland pheochromocytomas. DESIGN: We evaluated 22 pheochromocytomas using routine hematoxylin-eosin and Congo red stains, polarization microscopy, and electron microscopy. Cases with amyloid were further characterized immunohistochemically. RESULTS: Congo red and polarization microscopy revealed characteristic apple green birefringence, not abolished by potassium permanganate incubation, in only one neoplasm. In a subset of the remaining tumors, yellow or yellow-green birefringence not characteristic of amyloid was found. We performed electron microscopy on the tumor with characteristic apple-green birefringence and six other neoplasms with abundant yellow or yellow-green birefringence. Amyloid was identified in two tumors, including the Congo red-positive tumor, while the remaining five neoplasms contained collagen. Immunohistochemically, the amyloid deposits were reactive with component P and immunoglobulin kappa- and lambda light chains in both tumors. Adsorption studies using human urine with monoclonal immunoglobulin kappa- or lambda-light chains abolished reactivity, consistent with specific immunostaining for both light chains. Transthyretin was present in one tumor. The neoplastic cells expressed chromogranin, neuron-specific enolase, and [Leu5]-enkephalin, although the amyloid was not reactive. CONCLUSIONS: We conclude that amyloid is infrequent in pheochromocytomas. Electron microscopic examination is more sensitive than Congo red stain and, in our opinion, is the best technique for the demonstration of amyloid deposits. PMID- 7545390 TI - Binding of cell-surface expressed CD44 to hyaluronate is dependent on splicing and cell type. AB - CD44 is a major cell-surface receptor for hyaluronate (HA). By alternative RNA splicing a large number of CD44 variants are generated. To explore the role of CD44 splicing in the regulation of cell binding to HA, three different isoforms of CD44 were transfected in the CD44 negative B-cell lymphoma line Namalwa and in the fibroblastoid cell line COS7. We observed that whereas the standard form of CD44 (CD44s) mediated adhesion of Namalwa to HA, Namalwa transfected with CD44v3 10 or CD44v8-10 was unable to bind to either immobilized or soluble HA. After stimulation of CD44 with an activating anti-CD44 mAb or with phorbol ester, the binding of CD44s to HA was 5- to 10-fold higher than that of the other two isoforms. By contrast, COS7 cells transfected with CD44s, CD44v8-v10, or CD44v3 v10 bound equally effectively to HA. Hence, in addition to alternative splicing, cell type determines CD44 binding to HA. PMID- 7545389 TI - Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF-alpha in lipopolysaccharide-treated RAW 264.7 murine macrophages. AB - Extracellular ATP potentiates, by activation of P2y-type purinergic receptors, the production of NO induced by low doses of lipopolysaccharide (LPS) in the murine macrophagic cell line RAW 264.7 (Tonetti et al. (1994) Biochem. Biophys. Res. Commun. 203, 430-434). Release of TNF-alpha, known to be an autocrine factor for iNOS expression, was enhanced, too, following exposure of either LPS-induced or uninduced cells to externally added micromolar ATP. Reverse transcription-PCR experiments showed that extracellular ATP increases mRNA levels of both inducible NO synthase (iNOS) and of TNF-alpha to extents comparable to those of enzymatic and biological activities, respectively. These data demonstrate that activation of purinergic receptors by extracellular ATP results in an enhanced expression of the iNOS and TNF-alpha genes. PMID- 7545388 TI - Contributions of NO synthase and heme oxygenase to cGMP formation by cytokine and hemin treated brain capillary endothelial cells. AB - Two mechanisms contribute to cGMP formation by soluble guanylyl cyclase (i) NO production by NO synthase and (ii) CO production by heme oxygenase. We analyze here the contributions of these two pathways to IL1, TNF, lipopolysaccharide and hemin treated brain capillary endothelial cells. Cytokines and LPS induced cGMP formation in manners that were completely prevented by LY 83,583, methylene blue and by cyclosporin A. They were partially inhibited by inhibitor of NO synthase. Cyclosporin A acts by a posttranscriptional mechanism. Cells constitutively expressed mRNAs for heme oxygenase-1. Expression was enhanced by hemin but not by IL1 or lipopolysaccharide. Induction of heme oxygenase-1 and its inhibition by Sn protoporphyrin IX had no effect on cGMP levels. PMID- 7545391 TI - The interferon-inducible autoantigen, IFI 16: localization to the nucleolus and identification of a DNA-binding domain. AB - The human IFI 16 gene encodes a nuclear protein whose expression is associated with activation and differentiation of cells of the myeloid lineage, and which has recently been postulated to be a frequent target of human autoantibodies. We have used a unique monoclonal antibody in immunofluorescence studies to define the intranuclear distribution of IFI 16 antigen in primary human leukocyte sub populations. We demonstrate that IFI 16 is expressed both within the nucleoli and nucleoplasm of mononuclear cells, but is not present in granulocytes. IFI 16 expression correlated with that of the known nucleolar antigen B23, suggesting that the function of IFI 16 is restricted to cells that have nucleoli and therefore are not terminally differentiated. These findings were supported by immunofluorescence studies in IFN-gamma-stimulated HL-60 cells and by biochemical fractionation of Daudi nuclear extract. IFI 16 was extracted from whole nuclei at approximately 200-300 mM NaCl, within the range described for known transcription factors. Together with our previous findings, these results support a possible role of IFI 16 in binding nucleic acid in vivo. Furthermore, a DNA-binding site was localized within a fusion protein containing the amino-terminal 159 amino acids. As the nucleic acid-binding domains of several proteins are targeted by autoantibodies in SLE, this region may be of significance in the pathogenesis of autoimmune disease. PMID- 7545392 TI - LFA-3 delta D2: a novel in vivo isoform of lymphocyte function-associated antigen 3. AB - Lymphocyte function-associated antigen 3 (LFA-3) has previously been described as occurring as two isomers in vivo, a transmembrane (TM) form and a glycosyl phosphatidylinositol (GPI)-linked form, differing only in their membrane anchoring mechanism. A third isoform, LFA-3 delta D2, which has the cytoplasmic tail of TM LFA-3 but a truncated extracellular domain, has been identified in vitro. We report that the LFA-3 delta D2 isoform, identified by RT-PCR analysis and DNA sequencing, is also present in vivo and appears to share a signal sequence with the TM and GPI isoforms. Expression of LFA-3 delta D2 was observed in both normal and diseased human buccal mucosa and gingiva. Thus, while specific functional differences between isoforms remain to be established, our results show that LFA-3 delta D2 is constitutively expressed in vivo, along with the other, previously described, isoforms of LFA-3. PMID- 7545393 TI - Inhibition of NF-kappa B by pyrrolidine dithiocarbamate blocks endothelial cell activation. AB - Endothelial cell activation is achieved by the rapid, protein synthesis independent induction of a characteristic set of genes. Because of the abundance of binding sites for the transcription factor NF-kappa B in the regulatory region of the aforementioned genes, we hypothesized that this factor might play a key role. Reactive oxygen intermediates act as second messengers in the activation of NF-kappa B. We have used the antioxidant pyrrolidine dithiocarbamate to analyze the effect of NF-kappa B inhibition on TNF alpha-induced EC activation in vitro. We show that pyrrolidine dithiocarbamate strongly reduces the TNF alpha-mediated induction of E-selectin, VCAM-1, ICAM-1, PAI-1, tissue factor, IL-8 and I kappa B alpha. We present evidence identifying NF-kappa B as a central of EC activation. Therefore, this factor may represent a prime target for therapeutic intervention in pathologic conditions associated with EC activation such as allo- and xenograft rejection, atherosclerosis, ischemic reperfusion injury and vasculitis. PMID- 7545395 TI - cDNA cloning and gene expression of lebocin, a novel member of antibacterial peptides from the silkworm, Bombyx mori. AB - A cDNA encoding lebocin, a novel member of insect antibacterial peptides, was isolated from the fat body cDNA library of Bombyx mori larvae immunized with Escherichia coli. The cDNA was 844 bp long and had an open reading frame (ORF) containing a probable signal peptide (16 amino acids), a putative prosegment (104 amino acids) and a mature peptide (32 amino acids) followed by 27 additional amino acids at its carboxyl-terminus. Northern blot analysis showed that lebocin gene expression was inducible by bacterial injection, occurred tissue specifically in the fat bodies and continued at least for 48 h post-infection. These results suggest that lebocin has a unique precursor structure and shows typical gene expression pattern as insect antibacterial peptide. PMID- 7545396 TI - Novel isoform of beta 1 integrin expressed in skeletal and cardiac muscle. AB - We describe a novel isoform of the beta 1 integrin subunit called beta 1D, which contains a unique cytoplasmic domain, and is expressed specifically in skeletal and cardiac muscle. The beta 1D isoform arises from splicing into the final transcript of an additional exon located between exons 6 and 7. The nucleotide sequence of beta 1D predicts a cytoplasmic domain of 50 amino acids in which the last 21 amino acids of the beta 1A integrin isoform are replaced by a related sequence of 24 amino acids. A beta 1D-specific anti-peptide polyclonal antibody was prepared and immunoprecipitation of tissue extracts with subsequent immunoblotting showed expression of beta 1D isoform only in striated muscle cells. PMID- 7545394 TI - Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR. AB - In a search for new alpha-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known alpha-subunits. RT-PCR based on two degenerate primers revealed the expected band of 495 base pairs and an additional band of 540 base pairs reflecting the alternative splice product of Gs alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits Gi alpha-2 and Gs alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as Gz alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified Gs alpha-L (differing from Gs alpha-S by an insertion of 45 base pairs), G16 alpha, (a member of the pertussis toxin insensitive Gq-family), and two new variants of both Gs alpha-S and Gs alpha-L each containing a C-A-G triplet. With G16 we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of Gs alpha lead to an insertion of a Ser-residue, which results in the consensus sequence of a phosphorylation site for protein kinase C (Ser-X-Lys), making these variants candidates for protein kinase C-sensitive cyclic AMP formation. PMID- 7545397 TI - Inhibition of tumor necrosis factor induced human aortic endothelial cell adhesion molecule gene expression by an alkoxybenzo[b]thiophene-2-carboxamide. AB - The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation. PMID- 7545398 TI - Interleukin-1 beta induces expression of adhesion molecules in human vascular smooth muscle cells and enhances adhesion of leukocytes to smooth muscle cells. AB - Increased expression of cell adhesion molecules is an important pathological event during the development of atherosclerosis. The smooth muscle cell (SMC) is one of the cell types present in the atherosclerotic lesion. To evaluate the regulation of adhesion molecules in human vascular SMCs and its possible role, we studied the expression of adhesion molecules in SMCs stimulated with interleukin 1-beta (IL-1 beta), a pleiotropic cytokine that is involved in the pathological development of vascular diseases including atherosclerosis and restenosis. Our data demonstrated that IL-1 beta markedly induced the adhesiveness of human vascular SMCs for monocytes and neutrophils in a concentration (10 pM - 10 nM)- and time (0.5-24 h)-dependent manner. The maximal induced adhesion by IL-1 beta (1 nM) was reached at 4 h, with 4.6-fold and 3.3-fold for monocytes and neutrophils, respectively. This induction was dose-dependently inhibited by the IL-1 receptor antagonist (IL-1 ra). The IL-1 beta-induced expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin 1 (ELAM-1) on SMCs was examined by reverse transcription/polymerase chain reaction (RT/PCR). Unstimulated, serum-deprived SMCs expressed a low or undetectable level of mRNA for these adhesion molecules. The expression of ICAM-1 and VCAM-1 but not ELAM-1 mRNA was significantly induced with IL-1 beta in a concentration (1 fM - 1 nM)- and time (0.5 - 24 h)-dependent manner. The maximal increase in ICAM-1 and VCAM-1 mRNAs was reached at 4 h after IL-1 beta stimulation. The IL-1 beta-induced adhesion of SMCs for monocytes was partially inhibited by monoclonal anti-human ICAM-1 and anti-human VCAM-1 antibody, but not by anti-human ELAM-1 antibody. Pretreatment of monocytes with anti-human integrin beta 2 antibody significantly reduced the adhesion of monocytes to IL-1 beta-stimulated SMCs. These results suggest that IL-1 beta is a potent inducer for ICAM-1 and VCAM-1 expression in human vascular SMC, and could play a role in the pathogenesis of atherosclerosis by recruitment and retention of inflammatory cells such as monocytes and neutrophils in the lesions. PMID- 7545400 TI - [Nd:YAG laser surgery in treatment of allergic rhinitis]. AB - The purpose of our study was to evaluate the effects of laser turbinectomy on local allergic inflammation by measuring the secretion of mediators (histamine, bradykinin, and TAME-esterase activity) in nasal lavage fluid after nasal provocation with different allergen concentrations. Our study included fifteen patients, aged fifteen to 35, who displayed perennial house-dust-mite rhinitis (positive prick test, RAST class > 2 and positive nasal provocation with Dermatophagoides pteronyssinus [D.p.] extract) and hypertrophic inferior turbinates. Rhinomanometry (Rhino-Test 441, Allergopharma, Reinbeck, Germany) and nasal provocation with D.p. extract (Allergopharma, Germany) followed by lavage were performed in all patients. The procedure was repeated three and twelve months after neodymium:YAG laser turbinectomy. Three and twelve months after laser turbinectomy, we found a significant improvement of nasal flow (p < 0.01, p < 0.05) and resistance (p < 0.01, p < 0.01) but no change of mediator levels in nasal lavage after allergen provocation, suggesting that laser turbinectomy has no effects on local allergic inflammation. PMID- 7545401 TI - Consequences of antisense human chorionic gonadotrophin-alpha subunit cDNA expression in human choriocarcinoma JAR cells. AB - The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-alpha cDNA expression vector. The vector directs the synthesis of antisense hCG-alpha subunit mRNA which would then bind to sense hCG-alpha subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-alpha cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-alpha and -beta subunit mRNA levels. The decrease of hCG-beta subunit transcripts was unexpected and it was not due to contamination of antisense hCG-alpha cDNA construct with hCG-beta sequence. The transcription of hCG-alpha and -beta subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steady-state hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector. PMID- 7545402 TI - IGF, type I IGF receptor and IGF-binding protein mRNA expression in the developing mouse lung. AB - The IGFs are important mitogens involved in lung growth and development. The regulation of IGF action depends not only on the expression of IGFs and IGF receptors, but also on the modulation of IGF activity by IGF-binding proteins (IGFBPs). In this study, we describe the mRNA expression of IGF-I, IGF-II, type I IGF receptor, IGFBP-2, IGFBP-4 and IGFBP-5 during mouse lung development as studied by in situ hybridization techniques. The IGF, type I IGF receptor and IGFBP-2, -4 and -5 genes were expressed in developing lung as early as embryonal day 12.5. Expression of IGFBPs-1, -3 and -6 was below detection. IGF and IGFBP-2 mRNAs were expressed both in mesenchymal and epithelial cells. Type I IGF receptor transcripts were also observed throughout the developing lung, with the exception of the epithelial cells of the bronchi after embryonal day 15. Furthermore, mRNA expression of IGFBPs-4 and -5 was noted in neighbouring cell types, and after embryonal day 15, co-expression of the type I IGF receptor and IGFBP-4 transcripts was detected. The observed expression patterns imply that the IGFBP-2, -4 and -5 genes are differentially regulated during embryonic development and suggest that each may have a discrete function. A possible role for IGFBPs-2, -4 and -5 is to participate in the regulation of cell-specific IGF responses during mouse lung development. PMID- 7545399 TI - [Imaging time-dependent changes in the nasal mucosa using the most modern morphing software]. AB - Changes in the nasal mucous membrane lasting longer than 1-2 minutes have, until now, escaped dynamic observation. Even the congestion and decongestion of the turbinates during the nasal cycle and after application of vasoconstrictive nose drops have been documented only by instant stills. For this reason, we developed a method which permits documenting time-dependent endonasal mucosal changes with their natural dynamics on time-lapse video film. Instead of primarily recording a continuous video film, we processed instant stills. Computer interpolation of intermediate steps gives the viewer a dynamic impression. As continuous video endoscopy is not feasible, we produced the continuous film by recording video sequences of approximately 30 seconds length in defined time intervals endoscopically, selecting single stills (so-called original photographs) from each sequence, and splicing them, producing the missing intermediate photographs by a special computer technique (morphing), similar to mathematical interpolation. In this way we could demonstrate the changes of the right and left inferior turbinate during a nasal cycle of lasting five hours and the effect of xylometazolin nose drops on the nasal mucosa. The practical value of this technique, lies in the comprehensive realistic observation of dynamic processes of the nasal mucosa with respect to physiology and pathophysiology. PMID- 7545403 TI - Characterization of rat neuronal nitric oxide synthase expressed in Saccharomyces cerevisiae. AB - A cDNA encoding rat neuronal nitric oxide synthase (nNOS) was cloned into the yeast expression vector pMA56 to generate pA379. Transformation of Saccharomyces cerevisiae strain BJ2168 with this plasmid resulted in the synthesis of nNOS at levels of 0.5-1.0 mg/liter. The protein is localized in the cytosol and is catalytically active as determined by the conversion of [3H]-L-arginine to [3H]-L citrulline and NO. The enzyme was purified by calmodulin-Sepharose affinity chromatography and its catalytic activity was found to be both calcium and calmodulin dependent. Overexpression of nNOS in S. cerevisiae and purification of the recombinant protein will facilitate detailed characterization of this important enzyme. PMID- 7545404 TI - Methylation status of CpG sites in the mouse and human CFTR promoters. AB - To determine whether a relationship exists between DNA methylation and CFTR gene expression, we investigated the methylation status of CpG sites in the mouse and human CFTR promoters. Tissues and previously characterized cell lines that vary with respect to CFTR expression were selected for analysis using the methylation sensitive restriction endonuclease Hha I. We find that CpG sites are not methylated in high and low CFTR-expressing cell lines, whereas in the very low or non-CFTR-expressing cell lines, the CpG sites are partially or completely methylated. However, none of these sites were methylated in any of the tissues examined irrespective of the state of CFTR expression. Therefore, we conclude that the CFTR promoter belongs to the class of CpG-rich promoters in which the associated CpG sites are not methylated in tissues and that an inverse correlation between methylation and CFTR expression can only be found in cell lines. PMID- 7545408 TI - Rapid PCR for RNA differential display in a conventional heat block thermal cycler. PMID- 7545405 TI - A risk-benefit assessment of tacrolimus in transplantation. AB - Tacrolimus is a new macrolide immunosuppressant that was isolated from Streptomyces tsukubaensis in 1984. In vitro, on a molecular basis, tacrolimus is 50 to 100 times more potent that cyclosporin. Since 1989, numerous clinical trials have been completed comparing the usefulness of tacrolimus with that of cyclosporin for baseline immunosuppression in solid organ transplantation. Almost all of these studies demonstrated the superior immunosuppressive potency of tacrolimus. However, in most of these trials, tacrolimus-based immunosuppression was associated with a higher overall frequency of drug-related adverse effects compared with cyclosporin. Based on the efficacy and tolerability profile of tacrolimus that has been demonstrated in the various clinical trials so far, tacrolimus is a relevant alternative to cyclosporin for baseline immunosuppression in liver transplantation. In addition to primary immunosuppression, conversion from cyclosporin to tacrolimus should be performed during episodes of severe graft rejection (e.g. resistant to steroid bolus therapy). The future role of tacrolimus for baseline immunosuppression in kidney and thoracic organ transplantation has yet to be defined by ongoing studies. PMID- 7545406 TI - Acetylcholinesterase histochemistry in the macaque thalamus reveals territories selectively connected to frontal, parietal and temporal association cortices. AB - The patterns of histochemical staining for acetylcholinesterase (AChE) activity in the macaque thalamus were analyzed and compared with the distribution of cells and terminals labeled from injections of axonal tracers in the dorsolateral and orbital prefrontal cortex, in area 7a of the posterior parietal cortex and in the polysensory cortex of the superior temporal sulcus. AChE histochemistry is very useful in delineating the thalamic nuclei connected with the association cortex and in uncovering thalamic subdivisions that are barely evident on cytoarchitectonic grounds. Moreover, AChE activity reveals previously unrecognized heterogeneities within several thalamic nuclei, like the ventral anterior (VA), where a new ventromedial subdivision (VAvm) is described, the medial pulvinar (PulM) or the mediodorsal nucleus (MD). In this nucleus three distinct chemical domains are present: the medial, ventral and lateral sectors characterized by low, moderate and high AChE activities, respectively. The staining pattern of the lateral sector is markedly heterogeneous with patches of intense AChE activity surrounded by a moderately stained matrix. The MD medial sector is connected with the orbitofrontal cortex, whereas the AChE-rich patches in the lateral sector are selectively connected with the dorsolateral prefrontal, parietal and temporal association cortices. In the PulM, a dorsomedial AChE-rich patch is selectively connected with the orbitofrontal cortex, whereas the surrounding territory, which shows moderate AChE activity, is preferentially connected with the parietal and temporal cortices. Chemically specific domains in the anterior, ventral anterior, midline, and intralaminar thalamic nuclei are also connected with the examined association cortices. These findings indicate that the topographic patterns of the thalamo-cortical connections of primate association areas conform to the chemical architecture of the thalamus. This implies that because each cortical area is connected to a particular set of thalamic regions, the influence of the thalamus on cortical function is exclusive for each area, highly diverse among the various association areas, and subject to a wide range of modulation at the thalamic level. PMID- 7545407 TI - Biotinylated hyaluronan as a probe for detection of binding proteins in cells and tissues. AB - A convenient and reliable method for preparing and using biotinylated hyaluronan for detection of hyaluronan-binding proteins is described. The biotinylated hyaluronan can be used to detect binding proteins in transblots after electrophoresis or as a histological probe for localization of binding proteins in cultured cells and tissue sections. PMID- 7545411 TI - Neurodevelopmental outcome in very low birthweight infants with necrotizing enterocolitis requiring surgery. AB - OBJECTIVE: To assess the effect of necrotizing enterocolitis (NEC) on neurodevelopmental outcome. METHODOLOGY: Neurodevelopmental outcome of 20 very low birthweight (VLBW) infants who developed NEC requiring surgery was compared with 40 matched infants controlled for gestation, birthweight, and year of admission. Twenty-nine VLBW infants who developed NEC and did not require surgery were also compared. RESULTS: Infants with NEC needing surgery were of 26 +/- 2 weeks gestation and weighted 892 +/- 192 g at birth. Infants with NEC managed medically were of higher gestation (27 +/- 2 weeks) but similar birthweights. More infants with NEC requiring surgery required inotropic support. At follow up, NEC surgery infants had a significantly higher incidence of developmental morbidity. 11 of 20 compared with 11 of 40 matched controls (Fisher's exact test P = 0.0493), and six of 29 infants with NEC managed medically (Fisher's exact test P = 0.0174). CONCLUSIONS: These findings stress the importance for close follow up for neurodevelopmental sequelae in VLBW infants who have had NEC requiring surgery. PMID- 7545410 TI - Engineering a peptide epitope display system on filamentous bacteriophage. AB - The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein-protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines. PMID- 7545409 TI - Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR. AB - The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest. PMID- 7545413 TI - AMPA-, but not NMDA-, receptor blocker, NBQX, prevents seizure induction in magnesium-deficient rats. PMID- 7545412 TI - Brain catecholamines, serotonin and their metabolites in mice selected for low (MGL) and high (MGH) blood magnesium levels. AB - Brain noradrenaline, dopamine, DOPAC (3-4 dihydroxyphenylacetic acid), HVA (homovanillic acid), serotonin and 5-HIAA (5-hydroxyindolacetic acid) were determined by high performance liquid chromatography with amperimetric detection in adult male mice from three different strains : mice with genetically low (MGL) or high (MGH) blood magnesium levels, obtained by selective breeding and outbred Swiss albino mice. Noradrenaline levels were significantly higher (P < or = 0.001) in MGL than in MGH and Swiss mice : DOPAC levels were lower (P < or = 0.001) in MGL than in MGH and Swiss. Little or no differences were found for these variables between MGH and Swiss mice. MGL and MGH animals had similar brain dopamine, HVA and serotonin contents. These results suggest that the mere selection for genetic traits inducing low blood magnesium levels increases the synthesis of noradrenaline or decreases its catabolism. The above data together with the higher urinary noradrenalin excretion previously observed in the MGL line might account for the higher sensitivity and/or reactivity of MGL animals to stress. Swiss mice had significantly lower (P < or = 0.001) brain dopamine and serotonin contents than both MGL and MGH lines; indeed Swiss mice and MGL/MGH mice were issued from very different populations and had vastly different stocks of genes. Brain 5-HIAA content was also found higher (P < or = 0.01) in MGH than in MGL and Swiss mice; this latter result needs to be confirmed by further research. PMID- 7545414 TI - Relationships between cellular condensation, preosteoblast formation and epithelial-mesenchymal interactions in initiation of osteogenesis. AB - Initiation of osteogenesis or bone formation is dependent on cell and tissue interactions. We investigated the events between 4 and 7 days of incubation that translate epithelial-mesenchymal signalling into overt differentiation of osteoblasts and deposition of bone in the mandibles of chick embryos. Condensation of mandibular mesenchyme (the membranous skeleton), visualized with PNA-lectin, occurred at H.H. mid-26 (5.75 days), lasted 12 h and preceded osteoblast differentiation by 1.5 days. As determined from 3D-reconstruction all mandibular membrane bones arose from a single condensation closely associated with the stomodeal epithelium. The finding that the osteogenic condensation in the mandibular arch is a major branch of a common condensation that provides osteogenic mesenchyme to both maxillary and mandibular arches establishes a closer link between mechanisms controlling development of the skeleton in these two arches than previously suspected. Preosteoblasts (alkaline phosphatase positive cells) form in the mandible at H.H. early 25, which is before condensation but after the epithelial-mesenchymal interaction upon which preosteoblast formation and condensation depend--neither form in isolated mesenchyme, whereas both form after recombination of mesenchyme and epithelium. Tenascin was present in the mandibular epithelium only at H.H. stage 19 but not in the mesenchyme at any age. Therefore, the epithelial-mesenchymal interaction controls initiation of osteogenesis at the preosteoblast stage. Preosteoblasts then condense, transform into osteoblasts and deposit bone matrix. Differentiation of preosteoblasts precedes condensation which amplifies their number. This is in contrast with chondrogenesis where condensation triggers prechondroblast differentiation. PMID- 7545415 TI - Intra-arterial administration of the angiogenesis inhibitor TNP-470 blocks liver metastasis in a rabbit model. AB - We evaluated the best route of administration of TNP-470, an angiogenesis inhibitor, by comparing the anti-tumour effects and toxicity following injection via the hepatic artery, the portal vein, or the jugular vein in a rabbit model of liver metastases. Following the injections of 1 x 10(6) VX2 carcinoma cells into the portal vein of rabbits, 50 mg of TNP-470 was injected continuously into the hepatic artery, portal vein, or jugular vein for 7 days. The number of tumours on the surface of the liver was counted 14 days following the start of the infusion, and the serum glutamic-oxaloacetic transamine (GOT), glutamic-pyruvic transaminase (GPT) and total bilirubin concentrations were examined. In addition, a coloured silicon rubber was injected into the vessels of the liver to visualise the capillary networks around the tumours and assess the degree of suppression of angiogenesis by TNP-470. The mean number of tumours following intra-arterial injection (17.5 +/- 2.9) was significantly less than the control (237.0 +/- 34.0) (P < 0.05). The mean numbers of the tumours following intraportal (89.1 +/- 16.0) and intravenous (140.6 +/- 31.2) injection were both less than the controls (215.3 +/- 45.5, 284.8 +/- 55.4 respectively), but the differences were not significant. We conclude that intra-arterial injection of TNP-470 is the most effective method for preventing liver metastases in this model. PMID- 7545417 TI - Prostate-specific antigen in serum of women with breast cancer. AB - Prostate-specific antigen (PSA) was recently found in 30% of female breast tumours. In this study we have examined if PSA circulates in the blood of breast cancer patients and if serum PSA has any clinical application. We have compared serum PSA levels between women with and without breast cancer, between women with PSA-positive and PSA-negative breast cancer and between women with breast cancer before and after surgical removal of the tumour. We found that for women > or = 50 years, there is no difference in serum PSA between normal or breast cancer patients. We also could not find any difference in presurgical or post-surgical serum PSA between women who have PSA-positive or PSA-negative breast cancer. We found no correlation between PSA concentrations in matched presurgical and post surgical sera, between presurgical sera and tumour cytosols and between post surgical sera and tumour cytosols. High-performance liquid chromatography has shown that PSA in normal male serum consists mostly of PSA bound to alpha 1 antichymotrypsin (molecular weight approximately 100,000), and PSA in breast tumours and presurgical and post-surgical serum consists mostly of free PSA (molecular weight approximately 33,000). These data suggest that female serum PSA is not associated with tumour PSA levels. We speculate that most of the circulating PSA in women originates from the normal breast. It appears that serum PSA in women does not have potential for breast cancer diagnosis or monitoring, but our previous data are consistent with the view that tumour PSA concentration is a favourable prognostic indicator in women with breast cancer. PMID- 7545419 TI - Dermo-epidermal separation is associated with induced tenascin expression in human skin. AB - Tenascin, a large glycoprotein of the extracellular matrix, shows a site restricted distribution during embryogenesis, and can be found in adults in a variety of pathological conditions. In normal skin, tenascin is expressed at low levels, but it is upregulated in skin tumours, in a number of skin diseases with epidermal hyperproliferation and during wound healing. Several tenascin variants have been described, and these arise by alternative splicing. Using a monoclonal antibody recognizing all tenascin variants, and polyclonal antibodies specific for the large tenascin variants, we have investigated tenascin expression in bullous diseases such as epidermolysis bullosa, pemphigus, bullous pemphigoid and pemphigoid gestationis. By immunohistochemistry, we have found increased tenascin staining in all patient skin samples, with a more pronounced tenascin expression in samples of autoimmune bullous diseases. The large tenascin variants seem to be major forms of tenascin occurring in healthy skin. In patients with blistering diseases, however, these large variants appear to represent a subpopulation of the induced tenascin accumulation. These findings suggest different functions for the tenascin variants in normal and diseased skin. PMID- 7545416 TI - Immunofluorometric analysis of p53 protein and prostate-specific antigen in breast tumours and their association with other prognostic indicators. AB - Mutation and overexpression of p53 occurs in 20-40% of breast cancers and has been shown to be an independent prognostic indicator. Recently we have demonstrated prostate-specific antigen (PSA) expression in breast tumours to be suggestive of favourable prognosis, but quantitative relationships between PSA and p53, and between these and other prognostic factors in breast cancer, have not been investigated. Time-resolved immunofluorometric procedures were used to quantify both p53 protein and PSA in 200 breast tumour extracts, which were also assayed for oestrogen (ER) and progesterone receptors (PGR), epidermal growth factor receptors (EGFR), cathepsin D and HER-2/neu, and characterised for S-phase fraction and DNA ploidy. Weak Spearman correlations were found between p53 and ER (r = - 0.18, P = 0.010), PGR (r = - 0.15, P = 0.0385) and S-phase fraction (r = 0.17, P = 0.016), while PSA was correlated only with PGR (r = 0.16, P = 0.025). Wilcoxon rank sum analysis revealed that levels of ER (P = 0.0001), PGR (P = 0.0001), S-phase fraction (P = 0.0001) and EGFR (P = 0.0014) differed significantly between the two groups categorised as p53 negative or p53 positive. Tumours classified as PSA negative or PSA positive were found to differ with respect to PGR (P = 0.0091) and S-phase fraction (P = 0.011) in a similar analysis. Contingency tables indicated significant negative associations between the status of p53 and that of ER (P = 0.003) and PGR (P = 0.001) and between PSA and S-phase fraction (P = 0.012), and positive associations between p53 and EGFR (P = 0.017), HER-2/neu (P = 0.008), S-phase fraction (P = 0.001) and aneuploidy (P = 0.007), and between PSA and both ER (P = 0.061) and PGR (P = 0.010). No significant associations were found between p53 and PSA. Our results demonstrate that the presence of p53 in breast tumours relates to several other variables which are suspected to predict aggressive tumour phenotypes and that the presence of PSA relates to variables associated with good prognosis. PMID- 7545420 TI - Subsets of human dermal dendrocytes. PMID- 7545418 TI - Are fine-needle breast aspirates representative of the underlying solid tumour? A comparison of receptor levels, ploidy and the influence of cytokeratin gates. AB - Fifty-three solid and 33 fine-needle aspirate (FNA) samples (20 paired) of human breast carcinomas were examined by flow cytometry. Experiments were conducted to assess whether FNA samples were phenotypically representative of the solid tumour. Quantification of oestrogen receptor (ER), epidermal growth factor receptor (EGFR), c-erbB-2 receptor levels and ploidy were examined on the total and cytokeratin-positive cell populations. The absolute number of molecules of cytokeratin per cell expressed on the FNA (n = 33) and solid tumour (n = 53) samples showed no significant difference, but, on a proportional basis, there was a significant difference between the two samples (P = 0.004), with lower expression exhibited by the FNAs. Examination of paired data showed no significant difference in the percentage of cytokeratin-positive cells (P = 0.51) or in the number of cytokeratin molecules expressed (P = 0.25). While the correlation for ER expression between paired tumour and FNA samples in the absence of cytokeratin gating was P = 0.06, r2 = 0.18, clear correlation was shown when a cytokeratin gate was used (P = 0.005, r2 = 0.4). Repeating this experiment for EGFR, it was found that no correlation was seen between FNA and solid tumour (P = 0.2, r2 = 0.14) in ungated populations, but use of the cytokeratin gate improved the correlation (P = 0.05, r2 = 0.3). A similar finding was seen with c-erbB-2 expression (P = 0.2, r2 = 0.1) without cytokeratin gating and when it was employed (P = 0.05, r2 = 0.4). Ploidy data showed concordance in 18/20 cases. Three cases of aneuploidy were missed by FNA, and this was because of an insufficient number of cells for analysis. The presented data suggest that FNAs are representative of solid tumours and may be useful for measuring receptor levels on clinical material when cytokeratin gating is used. However, observation by light microscopy is still necessary to confirm the presence of tumour cells in FNAs subjected to flow cytometry. PMID- 7545421 TI - Beta 2-integrins in different forms of urticaria. AB - As urticarial lesions involve tissue invasion by inflammatory cells, and as beta 2-integrins play a central part in adhesion of leucocytes to endothelia, allowing their migration into the tissues, we have explored the distribution and sequential expression of these molecules in tissue sections from different forms of urticaria. Prick test weals (of 10 min duration) to common inhalant allergens showed only a minor increase of CD18, whereas in a case of cold urticaria CD11b and CD18 molecules were increasingly upregulated within the first 30 min after elicitation of the lesions. Skin test sites in delayed pressure urticaria, and urticarial lesions (> 6 h duration) of acute and chronic recurrent urticaria also showed marked upregulation of CD11b and CD18, and to a lesser extent of CD11a, but this did not strongly correlate with the intensity of the mixed cellular infiltrate. Non-lesional skin showed expression of beta 2-integrins in chronic urticaria, delayed pressure urticaria, and less so in acute urticaria, suggesting generalized leucocyte activation. This analysis of integrins thus suggests an early and extensive involvement of these molecules in the pathological events associated with the evolution of urticarial lesions. PMID- 7545422 TI - The effect of haemopoietic growth factors on the cell cycle of AML progenitors and their sensitivity to cytosine arabinoside in vitro. AB - This study examines the effect of pretreatment in liquid culture of acute myeloid leukaemic (AML) progenitors with recombinant human IL-3 or G and GM-CSF. Prior to and following cytokine priming, the sensitivity of cells to cytosine arabinoside (Ara-C) at concentrations ranging from 10(-12) to 10(-4) M was assessed in clonogenic culture. In addition, the initial percentage of AML cells in S phase was assessed and their subsequent kinetic response to cytokine treatment evaluated by FACScan analysis. Light-density marrow cells (LDMCs) from 19 AML patients were initially T-cell and monocyte depleted in order to remove potential sources of endogenous cytokine production prior to in vitro investigation. LDMCs were incubated in liquid phase for 7 d in a chemically defined complete medium with or without cytokines. Clonogenic data from fresh AML LDMCs not pretreated with growth factors demonstrated a heterogenous response to Ara-C. In only 4/15 marrows tested clonogenically was there any improvement in sensitivity to Ara-C following cytokine priming. S-phase data on all 19 marrows were similarly variable either before or after cytokine preincubation. There was no discernible correlation between clonogenic and kinetic data, nor could any relationship be established between in vitro findings and the FAB subtypes of patients or clinical outcomes. In summary, it would appear that the cell-cycle status of AML cells is likely to be only one of many contributory factors governing the sensitivity of AML progenitors to Ara-C. The clinical response of AML patients to cytokine therapy in association with cell-cycle-specific cytotoxic agents may therefore be variable and unpredictable. PMID- 7545425 TI - A specific chromosome abnormality of t(4;12)(q11-12;p13) in CD7+ acute leukaemia. AB - Three cases of acute leukaemia with t(4;12) (q11-12;p13) karyotypic abnormalities were analysed. They had the following common clinical and biological characteristics: (1) dysplasia of three haemopoietic lineages: (2) absent or low myeloperoxidase activity: and (3) retention of platelets in the peripheral blood and megakaryocytes in the bone marrow. There were increased numbers of basophils in the bone marrow and peripheral blood in two of the cases. In all, the blast cells displayed the unique immunophenotype CD7+CD13+CD34+HLA-DR+. The blasts analysed in one case expressed c-kit on the membrane surface. These findings suggest that the t(4;12) (q11-12;p13) abnormality is associated with a particular type of acute leukaemia, one in which the morphology and immunophenotype suggest that the translocation may have occurred at an early stage of haemopoiesis. PMID- 7545424 TI - Transplanted sickle-cell disease patients with autologous bone marrow recovery after graft failure develop increased levels of fetal haemoglobin which corrects disease severity. AB - Bone marrow transplantation (BMT) is the only curative therapy for sickle-cell disease (SCD), but is not devoid of failure risk. Nine patients with severe SCD were grafted in our institution between 1988 and 1993. Six patients successfully engrafted, but three failed to engraft and had delayed autologous recovery. All patients had, prior to BMT, low levels of fetal haemoglobin (HbF < or = 3.5%). No change in HbF occurred in successfully grafted patients. In the three patients with graft failure HbF increased and remained persistently present at a high level (> or = 22%) 14 months, 16 months and 39 months post BMT, although two of the three patients were homozygous for either the Benin or the Central African Republic haplotype, a characteristic associated with low HbF level. Of interest, these three previously severely affected patients remain free of vaso-occlusive events. The mechanism responsible for the expression of high levels of HbF in our three patients with graft failure is not understood, but it protects them from the recurrence of severe vaso-occlusive crises. PMID- 7545423 TI - In vivo effects of interleukin-11 and stem cell factor in combination with erythropoietin in the regulation of erythropoiesis. AB - In this study we evaluated the in vivo effects of interleukin-11 (IL-11) and stem cell factor (SCF), in combination with erythropoietin (EPO) on murine erythropoiesis. Mice were treated for 7 d with IL-11. SCF and EPO, each at three dose levels. In total, 27 different dose combinations were tested. IL-11 as well as SCF could only marginally stimulate erythroid progenitor cell numbers, but IL 11 in combination with SCF was able to increase BFU-E and CFU-E numbers 4-fold, in the absence of exogenous EPO. This resulted in an increased reticulocyte count. In contrast with the stimulatory effect on immature erythroid cell stages, IL-11 treatment induced a mild anaemia, which probably resulted from a plasma volume expansion. The additional treatment with EPO resulted in strong synergistic effects on CFU-E numbers. The combination of high-dose IL-11 and high dose SCF was able to increase the overall efficiency of EPO-induced erythroid amplification, which was reflected by a left-shift of the in vivo EPO dose response curve. The stimulating effects of IL-11 and SCF were further demonstrated when the effects on the reticulocyte count of a single high-dose EPO injection were assessed in normal and SCF+IL-11 treated mice. Whereas a single EPO dose increased the reticulocyte count by a factor of 3, IL-11 + SCF pretreatment increased this to a factor of 7. This study shows that in vivo SCF and IL-11 are important modulators of red blood cell production. First, these factors probably increase the input from the stem cell compartment into the erythroid lineage, where subsequently EPO is required for further amplification. Additionally, however, IL-11 and SCF increase the overall efficiency of EPO induced amplification, probably due to a stimulatory effect on late-stage erythroid cells and to a redistribution of cells from marrow to spleen. PMID- 7545426 TI - The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors. AB - We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 +/- 2.5 CFU-GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 +/- 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MIP-1 alpha, and the addition of IL-12 did not further enhance expansion of progenitors. These results indicate that accessory cells, lost in CD34+ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34+ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential. PMID- 7545427 TI - Mobilization of CD34-positive tumour cells in a patient with testicular mixed germ cell tumour. AB - Tumour cells from a patient with recurrent testicular germ cell cancer and bone marrow infiltration were found to express CD33 and CD34 in the absence of other haemopoiesis-associated antigens. After myelosuppression and treatment with G-CSF for stem cell mobilization, CD34-positive tumour cells were detected in the peripheral blood in addition to normal haemopoietic progenitor cells. The tumour cells were decreased in the leukapheresis product. Retrospectively, the appearance of tumour cells in the peripheral blood after stem cell mobilization was the first indication of impending relapse. PMID- 7545429 TI - Serum cytokine levels correlate with clinical parameters in Hodgkin's disease. AB - BACKGROUND: It has been suggested that cytokines are involved in the pathogenesis of Hodgkin's disease. Enhanced expression of various cytokines has been demonstrated in cell lines and biopsy specimens from patients with Hodgkin's disease (HD). PATIENTS AND METHODS: In this investigation 14 cytokines were analysed by ELISA in sera of a large panel of patients with HD and compared with clinical and serological parameters. RESULTS: Increased levels of soluble IL-2 receptors (sIL-2R), IL-6, IL-7, IL-8 and G-CSF, were found in many patients with HD as opposed to healthy individuals. In contrast, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, TNF alpha, TNF beta and GM-CSF were rarely detectable. Serum concentrations of sIL-2R, IL-6 and IL-7 were significantly correlated with advanced stage of HD and, together with G-CSF levels, with the presence of B symptoms. In addition, elevated cytokines correlated with several laboratory parameters. In the majority of patients the serum levels of cytokines decreased after therapy. However, elevated cytokine levels persisted in some patients in complete remission. Patients with normal IL-6 levels had better event-free survivals than patients with elevated IL-6 levels but this difference has not reached significance. CONCLUSION: Our results indicate that enhanced levels of sIL-2R, IL-6, IL-7, IL-8 and G-CSF, are correlated with disease activity and clinical symptoms in HD. PMID- 7545428 TI - Short-term weekly chemotherapy followed by high-dose therapy with autologous bone marrow transplantation for lymphoblastic and Burkitt's lymphomas in adult patients. AB - BACKGROUND: Type and duration of treatment for highly aggressive non-Hodgkin's lymphoma has been a matter of debate over the past decade. To determine the therapeutic efficacy of an abbreviated treatment regimen, 26 patients with newly diagnosed highly aggressive lymphomas, 17 of them belonging to the International Working Formulation (IWF) group I and 9 with Burkitt's lymphoma (IWF J), were entered in a study using short-term weekly chemotherapy followed by high-dose therapy and autologous bone marrow transplantation. PATIENTS AND METHODS: Besides histology, requirements for entry into to the study were age between 16 and 60 years, stage 1 bulky disease and elevated LDH or stage II to IV disease with or without bulk or elevated LDH, and an absence of HIV infection or CNS involvement at diagnosis. The treatment plan was 12 weeks of MACOP-B or VACOP-B chemotherapy followed by high dose therapy and autologous bone marrow transplantation in first complete remission. RESULTS: Twenty patients (76%), 16 (62%) of those on MACOP-B or VACOP-B, 1 who had received 2 cycles of ProMACE-CytaBOM prior to MACOP-B and 3 after a first salvage regimen, achieved complete remissions. Seventeen patients (65%) were transplanted in first remission, and 15 (58%) after induction treatment with only MACOP-B or VACOP-B. Reasons for not being given high dose therapy and autologous bone marrow transplantation (ABMT) were failure to achieve complete remission in 6 patients, early relapse in 2 and severe pulmonary toxicity associated with chemotherapy in 1. The median time of follow-up was 45 months. At 3 years, the estimated event-free survival was 31% (CI 14%-50%) and the overall survival 48% (CI 25%-67%). There were no deaths from toxic effects of treatment. Pretreatment factors associated with relapse were stage III or IV disease, age over 30 years and bone marrow involvement. Logrank analysis showed that age was the only factor significantly associated with poor event-free survival. CONCLUSION: Short-term weekly chemotherapy followed by high-dose therapy with the CBV regimen in first remission is not a higly effective treatment for advanced lymphoblastic and Burkitt's lymphomas. The 30% rate of failure to achieve partial remission after 6 weeks and/or complete response after 12 weeks of MACOP-B or VACOP-B treatment, as well as the 42% failure rate to undergo ABMT in first remission, suggest that more aggressive chemotherapy should be used in the beginning. PMID- 7545431 TI - Lung function impairment in long-term survivors of Hodgkin's disease. AB - BACKGROUND: Treatment of Hodgkin's disease (HD) involves radiation and chemotherapy, modalities known to cause lung injury. PATIENTS AND METHODS: In Norway, between 1980 and 1988, 129 patients aged less than 50 years at the time of diagnosis, had curative treatment with thoracic radiation alone or combined modality therapy for supradiaphragmatic HD. We have examined 116 (90%) of these patients by interview, chest X-ray and lung function tests, 5-13 years after treatment. RESULTS: Nearly 30% of the patients had dyspnoea on exertion and associated reductions in total lung capacity (TLC), forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1) and gas transfer (TLCO) (p < 0.05). Radiographic evidence of no, slight or moderate fibrosis occurred in 32%, 54% and 14% of the patients, respectively. Moderate fibrosis was associated with reductions in FVC, FEV1 and TLCO (p < 0.05). Radiation plus chemotherapy containing bleomycin-anthracyclines (median cumulative bleomycin dose 120 mg) was associated with decreases in FVC and TLCO (p < 0.05). In the multivariate analysis, chemotherapy with bleomycin-anthracyclines was the only significant predictor for lung function impairment. CONCLUSION: More than five years after therapy, respiratory symptoms and reduction in lung function were diagnosed in nearly one-third of otherwise healthy HD survivors. PMID- 7545430 TI - Outcome analysis after post-chemotherapy surgery in patients with non seminomatous germ cell tumours. AB - BACKGROUND: The goal of the study was to analyse long-term outcome after post chemotherapy surgery in male patients with non-seminomatous germ cell tumours (NSGCT). PATIENTS AND METHODS: We reviewed the charts of 111 patients with metastatic NSGCT treated at a single institution from 1979 to 1993 who underwent post-chemotherapy resection of residual masses after normalisation of tumour markers. The prognostic relevance of the extent of tumour residuals, the comprehensiveness of surgery and the histology at resection was analysed. RESULTS: Thirteen patients (12%) harboured viable cancer, and 46 patients (41%) mature teratoma at post-chemotherapy surgery. Only seven (54%) of the 13 patients with viable cancer remained continuously disease-free. Incomplete surgery predicted an impaired outcome (62% vs. 86% survival), although only one recurrence was observed at a site of an incompletely resected mass. Moreover, a progression-free interval < or = 3 months after post-chemotherapy surgery was correlated with worse subsequent survival (p = 0.0001). CONCLUSIONS: Post chemotherapy surgery remains an essential part of the treatment for metastatic NSGCT. Resection of viable cancer and mature teratoma is of therapeutic benefit. Viable cancer at surgery defines the need for further chemotherapy. PMID- 7545434 TI - Subunit dissociation and unfolding of macrophage NO synthase: relationship between enzyme structure, prosthetic group binding, and catalytic function. AB - Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545432 TI - Uncoupling of the proliferation and differentiation signals mediated by the murine macrophage colony-stimulating factor receptor expressed in myeloid FDC-P1 cells. AB - The macrophage colony-stimulating factor (M-CSF) regulates proliferation and differentiation of cells belonging to the monocytic lineage. We have investigated the nature and origin of the proliferation and differentiation signals derived from the M-CSF receptor (Fms) by mutating Fms at the four tyrosine autophosphorylation sites and examining their biological effects in an FDC-P1 clone. Wild-type Fms stimulated both growth and differentiation of FDC-P1 cells in response to M-CSF stimulation. In contrast, both proliferation and differentiation were differentially disrupted by mutations affecting the four tyrosine autophosphorylation sites. These analyses revealed that: (a) none of the four autophosphorylation sites studied (Y697, Y706, Y721, and Y807) were essential for M-CSF-dependent proliferation of the FDC-P1 clone; (b) Y697, Y706, and Y721 sites, located in the kinase insert region of Fms, were not necessary for differentiation, but their presence augmented this process; (c) mutation of the Y807 site totally abrogated the differentiation of the FDC-P1 clone and simultaneously increased the rate of M-CSF-dependent proliferation; and (d) conversely, increasing the intracellular cAMP level blocked the growth signal in the FDC-P1 clone but had no effect on differentiation. These results suggest that autophosphorylation of Fms at the Y807 site controls the balance between signals for growth and differentiation. PMID- 7545435 TI - Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase. AB - Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication. PMID- 7545433 TI - Coexpression of the c-kit and stem cell factor genes in breast carcinomas. AB - Expression of the c-kit tyrosine kinase growth factor receptor has been reported in some breast tumors; however, no data exist concerning expression of its ligand, stem cell factor. The aim of this study was to determine how frequently the c-kit and stem cell factor genes were coexpressed in breast tumors and tumor derived cell lines and to determine whether coexpression of c-kit and stem cell factor could result in growth stimulation of breast tumor cells. Expression of the c-kit and stem cell factor genes in tissue specimens and cell lines was determined using an RNase protection assay, with confirmation of c-kit protein expression by immunohistochemistry and Western blotting in tumor tissue and cell lines, respectively. Of 11 tumor specimens studied, 9 expressed variable but detectable quantities of c-kit; 7 of 13 tumor-derived cell lines also expressed c kit. All tumor specimens and cell lines expressed detectable stem cell factor mRNA, suggesting that an autocrine growth loop could exist in the majority of breast carcinomas. To determine the biological effects of coexpression of c-kit and stem cell factor, the MCF-7 cell line, which expresses only stem cell factor, was transfected with a c-kit expression vector. Coexpression of c-kit and stem cell factor in MCF-7 cells resulted in an enhanced growth rate and cloning efficiency but not a loss of the dependence of this cell line upon estrogen. Analysis of subclones expressing different amounts of c-kit protein revealed that, although they all showed enhanced growth relative to control transfectants in serum-free medium containing IGF-1, only the highest c-kit expressor responded with additional growth to exogenous soluble stem cell factor. However, all c-kit expressing clones, but not control clones, showed growth inhibition when exposed to a blocking anti-c-kit antibody. This blocking antibody also significantly inhibited the growth of the established ZR75-1 cell line in serum-free medium containing IGF-1. Taken together, these data suggest that coexpression of stem cell factor and c-kit could be responsible for growth deregulation in a significant number of breast carcinomas. PMID- 7545436 TI - Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. AB - The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blocker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors. PMID- 7545439 TI - Fibronectin receptors in preimplantation development: cloning, expression, and localization of the alpha 5 and beta 1 integrin subunits in bovine trophoblast. AB - The fibronectin receptor, alpha 5 beta 1, may be involved in many aspects of early development, including migration of endodermal and mesodermal cells during formation of the placenta, trophoblastic outgrowth in culture, and development of an invasive phenotype by fetal cytotrophoblasts. In contrast to the human blastocyst, the bovine blastocyst elongates in the uterine lumen for several days until it begins attachment, and the fetal trophoblast limits its invasion to the maternal epithelium. Fibronectin receptor expression was characterized in bovine embryos before and after their attachment to the uterus. Initially, the polymerase chain reaction (PCR) was conducted with degenerate oligonucleotide primers to isolate bovine cDNAs for the alpha 5 and beta 1 subunits. Bovine specific primers were then constructed to assay for alpha 5 and beta 1 mRNA expression in embryo RNA during the morula through the attachment stages using reverse-transcriptase PCR. Northern blot analysis was used to quantify mRNA levels from Days 15 to 21. Integrin and fibronectin protein expression was assessed by immunohistochemical examination of embryo sections. Both alpha 5 and beta 1 subunit mRNAs were expressed throughout the stages examined. Expression of both subunit proteins was found in the endoderm at Day 14 but not at Day 18 or later. Fibronectin reactivity was not present at any of the stages examined. Between Days 18 and 21, beta 1-reactivity appeared on the lateral surfaces of the trophoblast cells. Day 24 trophoblast binucleate cells showed intense staining with the beta 1 antibody, suggesting that a beta 1-integrin is involved in binucleate cell migration. PMID- 7545437 TI - Regulation of gonadotropin subunit messenger ribonucleic acid expression in gonadotropin-releasing hormone (GnRH)-deficient female rats: effects of GnRH, galanin, GnRH-associated peptide, neuropeptide-Y, and thyrotropin-releasing hormone. AB - Gonadotropin subunit mRNA expression is differentially regulated during the 4-day estrous cycle in rats, with LH-beta and FSH-beta mRNA expression rapidly increasing on proestrus. Studies in an ovariectomized (OVX) GnRH-deficient female rat model have shown that GnRH pulses can increase alpha and FSH-beta mRNA concentrations, but LH-beta mRNA is unchanged. Thus, the factors required for physiologic regulation of the LH-beta gene are not fully understood. To determine whether or not the proestrous ovarian hormone environment is required to allow increased expression of the LH-beta gene, GnRH pulses were administered to GnRH deficient (phenoxybenzamine-treated) intact female rats on proestrus. Both LH and FSH secretion and alpha and FSH-beta mRNA concentrations were increased, but LH beta mRNA expression was unaltered. The effect of co-administration of GnRH and specific neurohormones (GnRH-associated peptide [GAP], galanin, neuropeptide-Y [NPY], and thyrotropin-releasing hormone [TRH] was also examined in OVX rats receiving estradiol (E2) and progesterone (P) replacement. Alpha and FSH-beta mRNA concentrations increased 2-fold in response to pulsatile GnRH, and no further increase was seen after the addition of GAP, galanin, or TRH. It was of interest that NPY blocked the GnRH-induced rise in alpha and FSH-beta mRNA. LH beta mRNA expression was not increased by GnRH pulses alone or by addition of any of the neuropeptides. Further studies determined that continuous GnRH was no more effective than pulsatile GnRH in stimulating a rise in LH-beta mRNA. The results indicate that GnRH pulses are not sufficient to enhance LH-beta mRNA expression in the GnRH-deficient female rat.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545440 TI - Estradiol and progesterone influence the synthesis of gonadotropins in the absence of gonadotropin-releasing hormone in the ewe. AB - Acute actions of estradiol and progesterone on synthesis and secretion of gonadotropins without the confounding effects of endogenous steroids and GnRH were examined by using the ovariectomized (OVX), hypothalamic-pituitary disconnected (HPD) ewe. Thirty-two OVX ewes were subjected to HPD and randomly assigned to eight groups (4 ewes/group). An additional four OVX ewes served as controls. Each of 12 OVX-HPD ewes received 4 s.c. implants of estradiol 24 h after HPD (implantation was designated as Time 0 h). Sixteen OVX-HPD ewes received no treatment and served as contemporary controls. The remaining four OVX HPD ewes received implants of estradiol for 48 h and were administered 16 mg progesterone i.m. twice daily in the last 24 h of estradiol treatment. Blood samples and pituitary glands were collected from OVX-HPD control and steroid treated ewes at 0, 12, 24, and 48 h. Amounts of gonadotropin subunit mRNAs and serum concentrations of gonadotropins were decreased at 24 h after HPD and remained unchanged thereafter in OVX-HPD controls. Although treatment with estradiol decreased pituitary content of LH and steady-state levels of mRNA for LH beta-subunit (p < 0.05) compared to contemporary controls, serum concentrations of LH were not affected. Serum concentrations and pituitary content of FSH decreased (p < 0.05) 24 h after initiating treatment with estradiol, but steady-state levels of FSH beta-subunit mRNA were unchanged. There was a transient decrease in pituitary content of FSH at 24 h of estradiol treatment. Steady-state levels of mRNA for alpha-subunit were unaffected by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545438 TI - Growth hormone suppression of apoptosis in preovulatory rat follicles and partial neutralization by insulin-like growth factor binding protein. AB - A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545441 TI - Hormonal regulation of urokinase- and tissue- type plasminogen activator in rat Sertoli cells. AB - Tissue type (t) and urokinase type (u) plasminogen activators (PA) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon hormonal stimulation or the presence of adjacent spermatogenic cells. Furthermore, in cultured Sertoli cells, tPA has been shown to respond to FSH induction whereas uPA appears to be nonresponsive to gonadotropins. In the present study we analyzed the production of PA by Sertoli cells and regulation of this production by FSH during puberty. Cultured Sertoli cells under basal conditions secreted predominantly uPA. This production was high in 10-day-old animals and gradually decreased in older animals. The treatment of cultures with FSH or dibutyl cAMP induced production of tPA by Sertoli cells but at the same time produced a decrease in uPA activity. Northern blot analysis revealed that the control of PA synthesis is at the steady-state level of their mRNAs. Moreover, the use of cycloheximide, a protein synthesis inhibitor, showed that while tPA stimulation did not require intermediary protein synthesis, the decrease in uPA production was dependent upon protein synthesis. The differences in PA production by Sertoli cells obtained after FSH stimulation could explain the cyclic production of the two enzymes during the spermatogenic cycle and reinforce the hypothesis that different roles are played by the two enzymes in the several events occurring in testis development. PMID- 7545446 TI - Symmetry and structure of RNA and DNA triple helices. AB - Despite wide interest in nucleic acid triple helices, there has been no stereochemically satisfactory structure of an RNA triple helix in atomic detail. AN RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244, pp. 99-101; S. Arnott, P. J. Bond, E. Selsing, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3, pp. 2459-2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new model for an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3-11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from the introduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455-462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu, H. T. Miles, K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1, pp. 11-12]. The above triple helices have Watson-Crick-Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16, pp. 907-916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson-Crick-reverse Hoogsteen, Donohue [J. Donohue (1953) Proceedings of the National Academy of Science USA, Vol. 39, pp. 470-475] (reverse Watson-Crick)-Hoogsteen, and Donohue-reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain. PMID- 7545442 TI - Acute-phase proteins and immunoglobulin G against Porphyromonas gingivalis in peri-implant crevicular fluid: a comparison with gingival crevicular fluid. AB - This investigation had 2 aims: 1) to determine the levels of acute-phase proteins and immunoglobulin G (IgG) against Porphyromonas gingivalis in peri-implant crevicular fluid (PICF) and their association with the clinical condition of the peri-implant mucosa; and 2) to compare the inflammatory and immunological responses at implants and teeth as reflected by the gingival crevicular fluid (GCF) and PICF levels of acute-phase proteins and immunoglobulins. Thirty-one partially edentulous subjects were recruited for this study. PICF was sampled from 1 healthy and 1 inflamed site from each patient; GCF was sampled from an additional 21 healthy and 27 inflamed tooth sites of the same patients. GCF and PICF were collected with paper strips (for 30 s) and analysed using enzyme-linked immunosorbent assays for alpha 2-macroglobulin, alpha 1-antitrypsin, transferrin, lactoferrin and IgG against P. gingivalis. This investigation demonstrated that the absolute amounts of the acute-phase proteins and IgG against P. gingivalis are higher in GCF and PICF from inflamed than healthy sites. No significant differences were observed between PICF and GCF components at either healthy or inflamed sites, suggesting that inflammatory and immune events are similar in the peri-implant mucosa and gingiva in humans and that PICF and GCF production is governed by similar mechanisms. PMID- 7545445 TI - Comparison of base inclination of ribo-GC and deoxyribo-GC polymers, and synthesis of poly(rGrC)-poly(rGrC). AB - The inclination angle between the base normal and the helix axis, and the axes around which the bases incline, are measured for ribo-GC polymers in buffer by using flow linear dichroism (LD), and compared to measurements for deoxyribo-GC polymers in buffer and under dehydrating conditions. A new method is designed to synthesize poly(rGrC)-poly(rGrC), which is not available commercially, in large quantities. The LD of this RNA reveals inclination angles that are similar to the B-form DNA in buffer, although the axes are different. The CD of poly(dGdC) poly(dGdC) under the dehydrating conditions is similar to poly(rGrC)-poly(rGrC), indicating it is in the A form, and the LD gives larger inclination angles than either the B form or the corresponding RNA. Poly(dG)-poly(dC) is in the A form in buffer. Comparison among poly(rG)-poly(rC) in buffer, and poly(dG)-poly(dC) in buffer under dehydrating conditions, reveals similar inclination angles and axes, although the LD shows that the DNA has the largest inclination angles. Except for poly(rGrC)-poly(rGrC), which has a unique reduced dichroism, all the axes for G are similar, as are the axes for C. PMID- 7545447 TI - Carcinogenic effects of cytostatic protocols in CBA/Ca mice. AB - An in vivo mouse model was developed in order to study the characteristics of secondary tumor induction by cytostatic drug combinations used in human anticancer treatment. In this model we have proved the carcino-leukemogenic effects of widely used chemotherapeutical drug combinations (CHOP, COPP, COPBLAM, VAM). The carcinogenic hazards of cyclophosphamide and other alkylating drugs could also be demonstrated in our model. PMID- 7545444 TI - Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH dependent current fluctuations. AB - To probe protonation dynamics inside the fully open alpha-toxin ion channel, we measured the pH-dependent fluctuations in its current. In the presence of 1 M NaCl dissolved in H2O and positive applied potentials (from the side of protein addition), the low frequency noise exhibited a single well defined peak between pH 4.5 and 7.5. A simple model in which the current is assumed to change by equal amounts upon the reversible protonation of each of N identical ionizable residues inside the channel describes the data well. These results, and the frequency dependence of the spectral density at higher frequencies, allow us to evaluate the effective pK = 5.5, as well as the rate constants for the reversible protonation reactions: kon = 8 x 10(9) M-1 s-1 and koff = 2.5 x 10(4) s-1. The estimate of kon is only slightly less than the diffusion-limited values measured by others for protonation reactions for free carboxyl or imidazole residues. Substitution of H2O by D2O caused a 3.8-fold decrease in the dissociation rate constant and shifted the pK to 6.0. The decrease in the ionization rate constants caused by H2O/D2O substitution permitted the reliable measurement of the characteristic relaxation time over a wide range of D+ concentrations and voltages. The dependence of the relaxation time on D+ concentration strongly supports the first order reaction model. The voltage dependence of the low frequency spectral density suggests that the protonation dynamics are virtually insensitive to the applied potential while the rate-limiting barriers for NaCl transport are voltage dependent. The number of ionizable residues deduced from experiments in H2O (N = 4.2) and D2O (N = 4.1) is in good agreement. PMID- 7545443 TI - Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors. AB - We studied the ionic permeability of cGMP-dependent currents in membrane patches detached from the outer segment of retinal cone and rod photoreceptors. Reversal potentials measured in membranes exposed to symmetric Na+ but with varying cytoplasmic Ca2+ concentrations reveal that the permeability ratio, PCa/PNa, is higher in the cGMP-gated channels of cones (7.6 +/- 0.8) than in those of rods (3.1 +/- 1.0). Ca2+ blocks both channels in a voltage-dependent manner. At any Ca2+ concentration, the channel block is maximal near the ionic reversal potential. The maximal block is essentially identical in channels of cones and rods with respect to its extent and voltage and Ca2+ dependence. The Ca2+ block is relieved by voltage, but the features of this relief differ markedly between rods and cones. Whereas the Boltzmann distribution function describes the relief of block by hyperpolarizing voltages, any given voltage is more effective in relieving the Ca2+ block in cones than in rods. Similarly, depolarizing voltages more effectively relieve Ca2+ block in cones than in rods. Our results suggest that channels contain two binding sites for Ca2+, one of which is similar in the two receptor types. The second site either interacts more strongly with Ca2+ than the first one or it is located differently in the membrane, so as to be less sensitive to membrane voltage. The channels in rods and cones differ in the features of this second site. The difference in Ca2+ permeability between the channels is likely to result in light-dependent changes in cytoplasmic Ca2+ concentration that are larger and faster in cones than in rods. The functional differences between channels, therefore, may be critically important in explaining the differences in the phototransduction signal of the two photoreceptor types. PMID- 7545448 TI - A comparison of the effects of tranexamic acid and low-dose aprotinin on blood loss and homologous blood usage in patients undergoing cardiac surgery. AB - OBJECTIVES: To assess the relative efficacy of a "low-dose" aprotinin regimen and tranexamic acid on blood loss and homologous blood usage in patients undergoing primary cardiac surgery. DESIGN: The trial was prospective, randomized, and controlled. SETTING: A single center study in a regional cardiothoracic unit in the UK. PARTICIPANTS: 75 Patients, age 18 years or over, who were scheduled for routine primary cardiac surgery. INTERVENTIONS: The patients were randomly allocated to receive neither drug nor placebo, a total of 5 g of tranexamic acid, or a total of 2 x 10(6) kallikrein inhibitory units of aprotinin in the perioperative period. MEASUREMENTS AND MAIN RESULTS: The volume of blood loss and blood replacement were measured in the operative and postoperative periods. Hemoglobin concentration, platelet count, and white cell counts were determined preoperatively and at 24 hours postoperatively. Patients receiving tranexamic acid or aprotinin showed a significant reduction in postoperative blood loss (median[interquartile range] 375 mL [252 to 542] and 230 mL [137 to 547]), respectively, compared with the control group (615 mL [430 to 861]). Blood usage was also reduced in patients in both the tranexamic acid group (600 mL [415 to 800]) and the aprotinin-treated group (420 mL [350 to 887]) compared with the control group (1,050 mL [0 to 1,337]). There was no significant difference in blood loss or homologous blood use between patients treated with tranexamic acid or aprotinin. CONCLUSIONS: Tranexamic acid is as effective as low-dose aprotinin in the reduction of postoperative blood loss and homologous blood transfusion in patients undergoing primary cardiac surgery. PMID- 7545449 TI - Effects of aprotinin on blood loss, heparin monitoring tests, and heparin doses in patients undergoing coronary artery bypass surgery. AB - OBJECTIVES: Evaluation of the effect of aprotinin on heparin monitoring tests, on heparin doses, and on perioperative blood loss in patients undergoing coronary artery bypass grafting. DESIGN: Randomized, prospective open study. SETTING: The study was performed in the departments of Anaesthesiology and Haematology of the Erasme University Hospital, Brussels. PARTICIPANTS: Twenty-six patients undergoing primary coronary artery bypass grafting. INTERVENTIONS: Twelve patients received aprotinin, and 14 received no treatment. MEASUREMENT AND MAIN RESULTS: Aprotinin significantly reduced blood loss during coronary artery bypass surgery and was associated with a slight reduction in the amount of heparin administered. Activated coagulation time and activated partial thromboplastin time were prolonged by the addition of aprotinin. Activated coagulation time and activated partial thromboplastin time were poorly correlated with heparin assays. On the other hand, two other clotting tests designed to monitor heparin therapy, namely Titrarine (Stago, Asniere, France) and Heptest (Haemachem, St. Louis, MO), gave very good correlation with amidolytic heparin assays and can be used during extracorporeal circulation. Thrombin time showed a good correlation with amidolytic heparin assays after protamine administration and can be useful to detect residual heparin after heparin neutralization by protamine. PMID- 7545451 TI - Pseudomyxoma of the right atrium in a patient with heparin-associated thrombocytopenia and antiphospholipid antibodies. PMID- 7545450 TI - Warfarin pretreatment does not lead to increased bleeding tendency during cardiac surgery. AB - OBJECTIVE: To study the influence of preoperative warfarin anticoagulation on postoperative blood loss and allogeneic blood requirement. DESIGN: Retrospective study with a matched control group. SETTING: University and community hospital, single institutional. PARTICIPANTS: 240 patients scheduled for cardiac surgery. INTERVENTIONS: Warfarin sodium was administered to 125 patients preoperatively to prevent thromboembolic complications of their underlying heart disease, 115 served as control. Approximately half of the patients in each group received aprotinin treatment during operation. MEASUREMENTS AND MAIN RESULTS: The international normalized ratio (INR) of the prothrombin time was preoperatively 2.4 (95% confidence interval [CI 0.95]: 2.3 to 2.6) in the warfarin group and 1.1 (CI 0.95: 1.0 to 1.1) in the control group (p < 0.05), and postoperatively 1.9 (CI 0.95: 1.8 to 2.1) (warfarin) and 1.5 (CI 0.95: 1.4 to 1.5) (control) (p < 0.05). The postoperative blood loss after 6 and 12 hours was 381 (CI 0.95: 329 to 434)/505 (CI 0.95: 439 to 571) mL (warfarin) compared with 472 (CI 0.95: 403 to 541)/612 (CI 0.95: 527 to 697) mL (control) (p < 0.05). This difference was blunted when the proteinase inhibitor aprotinin was used. Neither preoperative nor postoperative prothrombin time correlated with blood loss. Despite lower heparin consumption the activated coagulation time (ACT) during CPB was significantly prolonged in the warfarin group. CONCLUSIONS: Warfarin pretreatment does not lead to increased bleeding but may even have a beneficial anticoagulant effect that may lead to better preserved postoperative hemostasis and reduced blood loss. PMID- 7545453 TI - Involvement of substance P in pial arteriolar vasodilatation during pneumococcal meningitis in the rat. AB - The present study investigated whether the endogenous vasodilating neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) contribute to pial arteriolar vasodilatation in the early phase of pneumococcal meningitis in the rat. Using a closed cranial window preparation we measured the diameters of pial arterioles during 4 h after pneumococcal infection. Arteriolar vasodilatation in infected rats was significantly attenuated by pretreatment with the SP-antagonist spantide up to 3 h after infection. Neither post-treatment with spantide (administered 2 h after infection) nor pretreatment with the CGRP antagonist CGRP(8-37) affected the pneumococci-induced pial arteriolar dilatation. These data suggest that SP is involved as a mediator in early pneumococci-induced dilatation of pial arterioles. PMID- 7545455 TI - Tumor necrosis factor alpha-induced up-regulation of interleukin-3 receptor mRNA expression in a CD34-positive hematopoietic cell line. AB - Regulation of interleukin-3 receptor (IL-3R) gene expression by tumor necrosis factor alpha (TNF alpha) was investigated using an IL-3-dependent CD34-positive hematopoietic cell line (KMT2) and a human megakaryocytic cell line (CMK). KMT2 expressed IL-3R alpha-subunit mRNA, whereas the level of expression of IL-3R beta subunit mRNA was low. CMK expressed IL-3R beta-subunit mRNA more strongly. The expression of IL-3R mRNA varied in the progenitor cells of different lineages. TNF alpha markedly enhanced expression of IL-3R beta-subunit mRNA in KMT2, whereas it only slightly augmented IL-3R alpha-subunit mRNA level. TNF alpha weakly augmented IL-3R mRNAs in CMK. However, the enhancement of IL-3R beta subunit mRNA in CMK was hardly detectable. The effects of TNF alpha on IL-3R mRNA expression were completely different in a primitive and in a more committed hematopoietic cell line. Addition of TNF alpha to KMT2 resulted in increased numbers of IL-3R on the cell surface without increased IL-3R affinity. The combination of IL-3 with TNF alpha abolished TNF alpha-induced inhibition of proliferation of KMT2. These results indicate that TNF alpha modulates the IL-3 responsiveness of primitive hematopoietic cells through up-regulation of the expression of IL-3R mRNAs, especially that of IL-3R beta-subunit mRNA. Phorbol ester (TPA) enhanced the IL-3R mRNA expression in KMT2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545452 TI - Projection sites of superficial or deep dorsal horn in the dorsal reticular nucleus. AB - The termination sites in the medullary dorsal reticular nucleus (DRt) of axons arising from superficial or deep dorsal horn cells were analysed by anterograde tracing with biotinylated dextran (BD). Following BD injections in spinal laminae I-III or in these laminae plus the dorsal funiculus, labelled fibres and boutons were located in the dorsal most ipsilateral portion of the DRt. Injections covering laminae III-V or laminae I-V resulted in bilateral labelling of the ventral DRt and ipsilateral labelling of the dorsal DRt. These findings, together with previous data concerning the origin of DRt projections to the superficial or deep dorsal horn, suggest the occurrence of reciprocal anatomical loops connecting the dorsal or the ventral areas of both regions. PMID- 7545457 TI - Angiogenic factors stimulate mast-cell migration. AB - Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell-shaped dose-response curve. Another potent angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase-inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions. PMID- 7545456 TI - T-lymphoblastic lymphoma/leukemia cells negative for CD52 and CD59. PMID- 7545458 TI - Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors. AB - We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst-promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony-stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO. PMID- 7545454 TI - Effect of growth factors on substance P mRNA expression in axotomized dorsal root ganglia. AB - The effects of NGF, BDNF, NT-3, BDNF plus NT-3 and LIF on substance P (SP) mRNA levels were analysed in axotomized dorsal root ganglia (DRGs) in vivo by quantitative in situ hybridization. The growth factors were applied on to the transected sciatic nerve. SP mRNA levels were decreased significantly 3 days after axotomy. NGF (1 microgram) fully counteracted the down-regulation of SP mRNA. Neither BDNF (10 micrograms) nor NT-3 (10 micrograms) alone had any effect. However, co-administration of BDNF together with NT-3 (10 micrograms) distinctly reversed the decrease in SP mRNA. A similar effect was seen with a high (1.5 micrograms) but not a low dose of LIF (0.15 microgram). Our data suggest that the present method of delivering growth factors to the transected sciatic nerve is a valid way to study in vivo effects of growth factors on peptide expression in DRGs. Moreover, SP expression is regulated by several growth factors in vivo such as NGF, BDNF plus NT-3 as well as the neuroimmune factor LIF. PMID- 7545459 TI - Combined transplantation of allogeneic bone marrow and CD34+ blood cells. AB - Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA-identical and MLC- sibling donors were mobilized with granulocyte colony stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti-CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively. Group I patients were given platelet transfusions within 18 days and red blood cells within 10 days, whereas for group II patients, these time points were 26 and 17 days, respectively. These same transfusions could be ceased within 38 and 24 days, respectively, in control patients. The addition of about 2% more peripheral blood CD3+ cells (group I patients) did not result in higher grades of acute GVHD (median grade II) as compared with the controls (median grade II). Four of five group II patients showed no signs of acute GVHD. These data suggest that the addition of immunoselected allogeneic CD34+ progenitor cells to BM cells may accelerate hematopoietic recovery. PMID- 7545460 TI - The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. AB - Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin-6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545461 TI - Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro. AB - Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages. PMID- 7545462 TI - Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3. AB - Fibrin clot retraction may be important in resolution of thrombi and, in platelets, is mediated by integrin alpha IIb beta 3 (GPIIb-IIIa). Nucleated cells that lack alpha IIb beta 3 can retract fibrin clots, and we now report that integrin alpha v beta 3 can support this process. In addition, we compared the capacities of recombinant beta 3 integrins to mediate clot retraction in Chinese hamster ovary and M21 melanoma cells. We found that alpha v beta 3, but not alpha IIb beta 3, could spontaneously support retraction. Transferring the cytoplasmic domain of alpha v to alpha IIb enabled the resulting chimeric alpha IIb beta 3 to support clot retraction. The capacity of the alpha v cytoplasmic domain to support clot retraction was not caused by activation of the ligand binding function of alpha IIb beta 3 or by enhancement of alpha IIb beta 3's capacity to stimulate the formation of focal adhesions or the tyrosine phosphorylation of pp125FAK. These experiments define requirements for alpha IIb beta 3-mediating clot retraction, establish the capacity of alpha v beta 3 to mediate this process, and suggest differing functional roles of the alpha v and alpha IIb cytoplasmic domains. PMID- 7545463 TI - Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy. AB - The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII. PMID- 7545464 TI - Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow. AB - In vitro studies have defined an essential role for stromal cells in supporting B cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed "reticular stromal" cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed. PMID- 7545465 TI - CD44 (Pgp-1) inhibits CD3 and dexamethasone-induced apoptosis. AB - Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones (GCH) induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, indicating that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether signals activated by adhesion receptors have a similar activity, we analyzed the effect of CD44 (Pgp 1) adhesion molecule receptor stimulation on T-cell apoptosis induced by three stimuli (anti-CD3 MoAbs, dexamethasone [DEX] treatment, and exposure to ultraviolet irradiation [UV]) on a 3DO T-cell line. The results show that CD44 engagement, either by hyaluronic acid (HA) or anti-CD44 MoAbs, inhibits DNA fragmentation and apoptosis induced by DEX and anti-CD3 MoAbs, whereas that induced by UV, a p53-dependent phenomenon, was not inhibited. Furthermore, the antiapoptotic effect exerted through CD44 activation does not seem related to overexpression of bcl-2 or to have appreciable effects on cell proliferation. Our results indicate that adhesion molecules modulate T-cell survival by counteracting apoptosis induced by DEX or anti-CD3 MoAbs. PMID- 7545467 TI - Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT. AB - Nuclear factor of activated T cells (NFAT) was originally described as a T-cell specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM 1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF) alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent. PMID- 7545466 TI - Evidence for a signaling role for the alpha chains of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors: divergent signaling pathways between GM-CSF/IL-3 and IL-5. AB - In the present study, we have used a human erythroleukemia cell line, TF-1, that proliferates in response to granulocyte macrophage colony stimulating factor (GM CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) to investigate the role of receptors for these cytokines in signal transduction mechanisms involved in proliferative responses. The receptors for GM-CSF, IL-3, and IL-5 each possess a cytokine specific alpha subunit, but all three share a common beta chain. Using an immunoblotting system designed to detect phosphotyrosine containing proteins and a permeabilized cell system to detect rapid changes in phosphate turnover on proteins, we show that while GM-CSF and IL-3 use tyrosine phosphorylation to mediate mitogenic signal transduction, IL-5 uses tyrosine dephosphorylation in its signaling pathway. The use of different signaling pathways by these cytokines can be confirmed in a biologic system whereby the proliferation induced in culture by GM-CSF and IL-3 is inhibited by tyrosine kinase inhibitors, but that induced by IL-5 is enhanced. Conversely, GM-CSF- and IL-3-induced proliferation is stimulated by a tyrosine phosphatase inhibitor, yet IL-5-induced proliferation is inhibited. Inhibitors of protein kinase C inhibit IL-3- and GM-CSF-, but not IL-5-induced proliferation. We suggest that, because all these cytokines share the identical beta chain of their receptors, the cytokine specific alpha chain mediates the linkage of each receptor to the individual biochemical signal transduction pathways responsible for the different biologic activities of these cytokines. PMID- 7545468 TI - Delineation of the dendritic cell lineage by generating large numbers of Birbeck granule-positive Langerhans cells from human peripheral blood progenitor cells in vitro. AB - It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14- progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens. PMID- 7545469 TI - Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules. AB - Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P-selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P-selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules. PMID- 7545470 TI - Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte: endothelial cell interactions. AB - The extravasation of leukocytes from the lumen of the vessel to a site of inflammation requires specific binding events. The interaction of leukocytes with endothelium, via specific receptors, may provide intracellular signals that activate extravasating cells. In the present study, we have investigated the production of chemokines, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) during monocyte: endothelial cell interactions. Both unstimulated and interferon-gamma (IFN-gamma)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of IL-8 and MCP-1. The addition of enriched monocytes with unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with IFN-gamma preactivated HUVECs demonstrated little additional increase in IL-8 and MCP-1 production in coculture assays compared with unstimulated HUVEC. Northern blot analysis paralleled the protein data, demonstrating upregulated expression of IL 8 and MCP-1 mRNA in stimulated and unstimulated coculture assays. Culture of enriched monocytes and endothelial cells in transwells demonstrated no increases in IL-8 or MCP-1, indicating the necessity for cellular contact for chemokine production. In previous investigations, we have demonstrated that increased monocyte-derived MIP-1 alpha production was induced by intracellular adhesion molecule-1 (ICAM-1) interactions on activated HUVECs. In contrast, addition of anti-ICAM-1 monoclonal antibodies (MoAbs) did not diminish the production of IL-8 and MCP-1 in the present study. Furthermore, neither antibodies to IL-1 nor tumor necrosis factor (TNF) diminished the production of either IL-8 or MCP-1. However, when soluble matrix proteins were added to the coculture to block cellular interactions, the chemokine protein and mRNA levels were significantly decreased. IL-8 production was decreased by both soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggesting differential activation pathways. These results indicate that IL-8 and MCP-1 production are increased during monocyte and endothelial cell interactions in part due to matrix protein binding mechanisms. This mechanism may serve a role in cell activation, production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses. PMID- 7545472 TI - Epstein-Barr virus modulates de novo protein synthesis in human neutrophils. AB - Neutrophils and macrophages represent the first line of defense against microbial invaders. However, the role of phagocytes in host response to viral infection is poorly understood. We have previously shown that Epstein-Barr virus (EBV) interacts with human monocytes and modulates cytokine production in this cell type, but its effects on neutrophils are still unknown. In the present study, we investigated the presence of EBV receptor (CR2 or CD21) on neutrophils by cytofluorometry using five different anti-CD21 monoclonal antibodies (MoAbs), as well as fluoroscein isothiocyanate-EBV (FITC-EBV). Whereas no significant amount of neutrophils reacted with anti-CD21 MoAbs, studies with FITC-EBV indicated that viral particles bind to 30% of cells (in some individuals, EBV binds to more than 50% of neutrophils). This interaction is specific as it was completely inhibited by nonconjugated virus or with labeled virus preincubated with neutralizing MoAbs. After EBV treatment, cellular aggregation was observed in neutrophil cultures, an indication that neutrophils were activated. Although EBV did not induce respiratory burst activity in neutrophils, pretreatment with infectious particles enhanced (priming effect) the fMLP-induced O2- release in neutrophils. Instead of restricting our analysis to specific cytokine genes, we investigated the effects of EBV on neutrophil transcriptional events in general. The effect of this virus on de novo synthesis of total cellular RNA was first investigated by measuring the incorporation of [5-3H] uridine into total RNA. The results showed that RNA synthesis in neutrophils was significantly increased (2.3- to 21.3-fold) by EBV compared with the unstimulated controls. Live and UV-inactivated virus markedly induced RNA synthesis, whereas heat-inactivated virus lost this ability. Induction of RNA transcription was EBV specific, as an EBV-neutralizing antiserum abolished this effect. Induction of protein synthesis was also studied by measuring the incorporation of [35S] methionine and [35S] cysteine into secreted and intracellular proteins in neutrophils incubated with EBV. The synthesis of both secreted and cytoplasmic proteins was induced by EBV. One- and two dimensional gel electrophoresis analysis showed that EBV modulates protein synthesis, because activation of the synthesis of certain proteins was accompanied by the inhibition of others. Interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) synthesis was found to be induced by EBV. Therefore, modulation of host-response proteins such as IL-1Ra could be one of the many mechanisms by which this virus avoids rejection.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545471 TI - Blockage of the early events of mitogenic signaling by interferon-gamma in macrophages in response to colony-stimulating factor-1. AB - Inhibition of cell proliferation is an important biologic function of interferons (IFNs), which has been exploited in therapeutic treatment of certain hematologic malignancies. However, the molecular mechanism was not clear. We have recently shown that IFNs (alpha/beta and gamma) inhibit protein kinase C (PKC)-dependent (such as PDGF and phorbol ester) but not PKC-independent (such as epidermal growth factor) activation of Raf-1 and mitogen-activated protein kinases (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, 1994), suggesting a novel mechanism by which IFNs execute their antiproliferative function. Monocytes/macrophages are primary targets in vivo for IFN-gamma, the major activity of macrophage-activating factor. In the present study, mechanism of IFN gamma-induced antiproliferative action in macrophages in response to colony stimulating factor-1 (CSF-1) has been investigated. Our results show that antiproliferative effect of IFN-gamma overrode mitogenic effect of CSF-1 and phorbol ester, as measured by early gene expression, DNA synthesis and cell proliferation. Although activation, phosphorylation, and turnover of the CSF-1 receptor and CSF-1-induced increase in diacylglycerol production remained normal, IFN-gamma blocked CSF-1-stimulated activation of mitogen-activated protein kinases, Raf-1 kinase, increase in GTP-bound Ras and tyrosine phosphorylation, and activation of protein kinase C delta (PKC-delta). PKC-delta was required for CSF-1-induced mitogenic signaling and a primary target for IFN-gamma-induced inhibition. Interestingly, although phorbol myristate acetate stimulated Ras activation, PKC-delta did not appear to be an upstream activator of Ras. These studies clearly indicated that IFN-gamma specifically inhibits PKC-delta activation, resulting in blockage of the early events of mitogenesis in macrophages in response to CSF-1. PMID- 7545473 TI - Calcium-loaded erythrocytes have a defect in complement regulation distinct from that resulting from exposure to 2-aminoethylisothiouronium bromide. AB - Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs. PMID- 7545475 TI - Reduced expression of vascular cell adhesion molecule-1 on bone marrow stromal cells isolated from marrow transplant recipients correlates with a reduced capacity to support human B lymphopoiesis in vitro. AB - A common sequela to allogeneic or autologous bone marrow transplantation (BMT) is a delay in the reconstitution of a functional B-cell immune response. Therefore, we examined whether the posttransplant BM microenvironment is deficient in supporting the proliferation and/or differentiation of B-cell precursors. BM stromal cell cultures were established from patients who received allogeneic or autologous BMT for acute lymphoblastic leukemia, Hodgkin's disease, or non Hodgkin's lymphoma. These cultures were then compared with normal donor BM stromal cell cultures for expression of adhesion molecules and the capacity to support the adhesion and interleukin-7 (IL-7)-dependent growth of normal B-cell precursors. Analysis of BM stromal cell cultures established from 28 BMT recipients showed a significantly reduced expression of cell surface vascular cell adhesion molecule-1 (VCAM-1/CD106), compared with normal donor BM stromal cells. Transplant BM stromal cell CD106 expression was responsive to regulatory cytokines in a manner qualitatively comparable with normal donor BM stromal cells. The level of B-cell precursor adhesion to transplant BM stromal cells correlated with the level of CD106 expression. Of 19 evaluable transplant BM stromal cell cultures, eight exhibited a reduced capacity to support the growth of CD19+/light chain- normal B-cell precursors. The capacity of transplant BM stromal cells to support B-cell precursor growth correlated with the level of CD106 expression, and the level of B-cell precursor adhesion. Our collective results may provide new mechanistic insight into why B-cell recovery is delayed post-BMT and underscore the importance of VCAM-1/CD106 in regulating B lymphopoiesis. PMID- 7545476 TI - Allogeneic blood stem cell transplantation: peripheralization and yield of donor derived primitive hematopoietic progenitor cells (CD34+ Thy-1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease. AB - Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy 1dim and CD34+ Thy-1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7-fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4 fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3 ). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight. There was no significant correlation between the total number of CD34+ cells transfused and the time to reach PMNC >0.5 x 10(9)/L or platelets > 50 x 10(9)/L posttransplant, nor was there a correlation found between the total number of CD3+, CD4+, and CD8+ cells transfused and the development of acute graft-versus host disease (GVHD). The actuarial probability of developing acute GVHD in 38 evaluable patients was 48%. In 13 patients followed longer than 100 days posttransplant, the actuarial probability of developing chronic GVHD was 66% (median follow-up, 264 days). PMID- 7545477 TI - Monocytosis and high serum macrophage colony-stimulating factor in Waldenstrom's macroglobulinemia. PMID- 7545474 TI - Detection of maternal cells in human umbilical cord blood using fluorescence in situ hybridization. AB - Cord blood is a potential source of hematopoietic stem cells for transplantation and is being used on a growing number of patients. However, there are concerns that cord blood might be contaminated with maternal cells that could lead to graft-versus-host disease. To ascertain the extent to which maternal cell contamination of cord blood occurs, we examined 49 cord blood samples from male babies for maternal cells by fluorescence in situ hybridization using probes to the X and Y chromosomes. A minimum of 1,000 nuclei were scored from each sample, and maternal cells were found in 7 of the 49 cord bloods, at levels ranging from 0.04% to 1.0%. In addition, in 39 and 27 of the cord blood samples, respectively, we examined the CD8+ and CD34+ cell populations for maternal cells. Maternal cells were found in 5 of the 39 CD8 fractions and in 1 of the 27 CD34 fractions, at levels similar to that found in the unfractionated cord blood. In sum, maternal cells were found in either the unseparated mononuclear fraction or the CD8 or CD34 fractions in 10 of the 49 cord blood samples (20%). These results show that maternal cells are present in a substantial number of cord bloods, and that some of these maternal cells are T cells. PMID- 7545479 TI - The detection of specific acute phase serum protein complexes and immune complexes by congo red binding. AB - The study concerns the problem of selective binding of Congo Red dye to serum proteins. The dye-binding structural motifs were assumed to appear in proteins as the result of local disruptions of tertiary interaction associated with biological activities. Functional structures of serum proteinase inhibitors and haptoglobins induced by interaction of these proteins with their natural substrates were found to bind Congo Red. Soluble immune complexes formed by bivalent hapten-bridged anti-TNP rabbit antibodies also appeared able to interact with the dye. The possible use of these effects for diagnostic analysis was suggested. PMID- 7545480 TI - Therapeutic effects of the angiogenesis inhibitor TNP-470 against carcinomatous peritonitis in mice. AB - The therapeutic effects of the new anti-angiogenesis factor TNP-470 were examined against carcinomatous peritonitis in mice. In the first experiment using carcinomatous peritonitis caused by i.p. inoculation of 10(6) M5076 tumor cells, TNP-470 solution was injected i.p. in a bolus of 50 mg/kg body weight into two groups of 10 mice either 1 or 8 days after the i.p. inoculation. The administration of TNP-470 on day 1 extended the survival time of the mice compared with 10 control mice receiving no treatment, whereas TNP-470 given on day 8 did not affect the survival time. In the next experiment on the M5076 tumor, TNP-470 solutions at 100 or 300 mg/kg were injected i.p. in a bolus into two groups of 20 mice 1 day after the inoculation 10(6) tumor cells, respectively. The administration of TNP-470 at 100 mg/kg also had an inhibitory effect. However, TNP-470 at 300 mg/kg caused toxic death in half of the mice. Next, we examined the effects of TNP-470 on another type of carcinomatous peritonitis model, which was caused by i.p. inoculation of 10(6) B16 melanoma cells. In this experiment, TNP-470 solutions in a bolus of 150 mg/kg were injected i.p. into six groups of 10 mice each on day 1 only (group 1), on days 1 and 4 (group 2), on days 1, 4 and 7 (group 3), on day 8 only (group 4), on days 8 and 11 (group 5), or on days 8, 11 and 14 (group 6), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545478 TI - The effect of caerulein-induced acute pancreatitis on the levels of serotonin (5 HT) and 5-hydroxyindoleacetic acid (5-HIAA) in various rat tissues. AB - The effects of caerulein-induced acute pancreatitis (AP) on the levels of 5-HT and 5-HIAA in pancreas, liver, brain, kidney and different parts of the digestive tract in rats have been studied. The acute pancreatitis was induced by the continuous intravenous infusion of caerulein in the doses of 5 x 10(-6) g/kg/h and 7.5 x 10(-6) g/kg/h for 12 hours, and the 5-HT and 5-HIAA concentrations were determined by the method of Curzon and Green. The changes evoked by caerulein varied qualitatively and quantitatively between organs. The significant decrease of both 5-HT and 5-HIAA was observed in pancreas, liver, brain and kidney while in the stomach there was a significant fall of 5-HT level accompanied by the relevant increase of 5-HIAA level. In the duodenum, an opposite effect was noticed, the level of 5-HT was higher than normal whereas the 5-HIAA level was reduced. The significant decrease of the 5-HT and 5-HIAA levels in pancreas observed in the present study suggests the importance of 5-HT in the development of acute pancreatitis. PMID- 7545482 TI - Evidence that the expression of the gene for NADPH-cytochrome P-450 reductase is n-alkane-inducible in Candida maltosa. AB - A gene coding for NADPH-cytochrome P-450 reductase of an n-alkane-assimilating yeast, Candida maltosa, was isolated and sequenced. Northern analysis and assay of the expression of the reporter gene under the control of the promoter of this gene showed that the transcriptional level was induced 4 to 8-fold in cells grown on n-alkane relative to cells grown on glucose. PMID- 7545481 TI - Controlled release of poly-D,L-lactic acid containing bleomycin. AB - By use of four types of in vivo degradable polylactic acid (PLA), i.e. PLA with an average molecular weight of 1500 (1500DL), 2200 (2200DL), 2800 (2800DL) and 3500 (3500DL), preparations of bleomycin (BLM)-containing solid forms (polymers) were tested. The in vitro release of BLM from the polymers was also examined in an immersion system. By the melt-pressing technique, five types of BLM (2.5 mg) containing solid forms, i.e. 1500DL polymer, 2200DL polymer, 2800DL polymer, 3500DL polymer and 1500DL + 3500DL (a mixture of 1500DL and 3500DL) polymer were prepared. In all five types of polymers, cumulative BLM release was controlled to less than 5% by the third day and no initial burst of the release was observed. BLM release from the polymer continued for 3 weeks at the shortest and 6 weeks at the longest. Various polymers containing BLM could be useful for the site of drug administration or anti-cancer release pattern. PMID- 7545483 TI - Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen. AB - PURPOSE: Although the significance of leukocytospermia in semen remains controversial, evidence exists that white blood cells (WBCs) may adversely affect sperm function and act as a potential cofactor in male infertility. Nevertheless, the mechanisms by which leukocytes may alter sperm function in vitro is unknown. Recent investigations suggest that reactive oxygen species (ROS) generated by the polymorphonuclear (PMN) granulocytes could adversely affect sperm function. The objective of this study was to investigate the relationship between the presence of leukocytospermia as determined by the Endtz test and the excessive formation of ROS. METHODS: The WBC concentration and ROS formation in human semen, obtained from men consulting for infertility, were assessed and compared to that of normal donors. ROS was measured by a chemiluminescence assay using luminol and a Berthold luminometer. The WBC concentration was determined with a myeloperoxidase staining technique (Endtz test). Specimens were obtained from 94 subjects (20 donors, 74 patients). RESULTS: Of the 20 donors, 2 were Endtz positive and ROS positive; 18 were Endtz negative with 2 (11%) ROS positive. In the patient group (n = 74), 26 (35%) were ROS positive, and 17 were Endtz positive and found to be ROS positive. Of the 57 Endtz-negative patients, 9 (15%) were ROS positive. The positive Endtz test results correlated strongly with positive ROS formation in patients and donors (P < 0.001). CONCLUSIONS: Our results indicate that the simple, cost-efficient Endtz test could be used as an indicator of excessive ROS formation in semen. PMID- 7545484 TI - Production of insulin-like growth factor binding proteins (IGFBPs) by human endometrial stromal cell is stimulated by the presence of embryos. AB - PURPOSE: To identify IGFBPs among endometrial secretory products and study their role in implantation and early embryo development. METHODS: Two-cell CB6F1 mouse embryos were cultured alone or with human endometrial stromal cells in RPMI 1640 plus 10% fetal calf serum (FCS) with or without addition of IGF-I (20 micrograms/ml), IGF receptor antibody (0.1 microgram/ml), progesterone (P) (20 ng/ml) and relaxin (R) (20 micrograms/ml). On the designated day, the medium was changed to protein-free RPMI and incubated for 16 h. Both conditioned medium and conditioned protein-free medium were then collected for protein analysis and immunoradiometric assay. Cells were fixed with 4% paraformaldehyde for immunohistochemical staining. RESULTS: IGFBP1 (31 kDa), IGFBP2 (36 kDa), IGFBP3 (45 kDa and 50 kDa) and an unknown IGFBP (25 kDa) were identified in conditioned medium of human endometrial stromal cells cultured alone or cocultured with mouse embryos. Secretion of IGFBPs by endometrial stromal cells was stimulated in the presence of mouse embryos as well as by P and R. IGFBP3 appears to be more responsive to embryonic signals. On the other hand, the secretion of IGFBP1 was greatly stimulated by P and R. Immunolocalization revealed that all three BPs were present in both embryonic and endometrial cells and their immunological staining was heavily increased by P and R. CONCLUSIONS: Endometrial stromal cells were able to synthesize and secrete IGFBPs to modify IGF action on embryo development. Secretion of IGFBPs was stimulated by embryonic signals and was hormonally dependent. The fact that IGFBP3 was more responsive to embryonic signals suggests that it may be important in early implantation. On the other hand, IGFBP1 production was highly responsive to both P and R, suggesting that it may be important throughout pregnancy. In addition, the fact that IGFBPs were located in endometrial and embryonic cells may suggest that these secretory products have autocrine and/or paracrine effects on both types of cells. PMID- 7545488 TI - Cytokine production in long-term marrow cultures after autologous bone marrow transplantation. AB - We compared the release of granulocyte-macrophage colony-stimulating factor (GM CSF), granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor alpha (TNF alpha) in the supernatant of long-term bone marrow cultures (LTBMC) derived from 10 control patients and from 14 patients before and 3 months after autologous bone marrow transplantation (BMT). The three cytokines were spontaneously present in the supernatant of cultures established from patients before and after autologous BMT, while GM-CSF remained undetectable in the supernatants of control patients. The maximal levels of cytokines were produced after the first week and were not statistically different between control, patients before and after grafts although the granulocyte-macrophage colony forming unit (CFU-GM) production in long-term culture (LTC) was lower in patients after graft compared with control patients (median values at LTC initiation: 32 and 158, respectively, P < 0.001 and median values of the total production: 510 and 12406, respectively, P < 0.002). However, GM-CSF was more frequently detected in patients after graft than in control patients. This study demonstrated that the production of GM-CSF, G-CSF and TNF alpha is not impaired in patients after graft (medians 0, 870.5, 173.5 pg/ml and ranges 0-31.2, 0-10 000 and 0-1426, respectively) compared with control patients (medians 0, 69, 66 pg/ml and ranges 0, 0-13 280 and 0-1318, respectively), although patients after graft were shown to have lower marrow CFU-GM counts. These results suggest that the ability of the accessory cells to produce these cytokines was not reduced after autologous BMT. PMID- 7545490 TI - Mobilization of peripheral blood progenitor cells by high-dose Ara C, VP-16 and recombinant human granulocyte colony-stimulating factor: factors affecting progenitor cell yields. AB - Forty seven patients with hematological malignancies were treated with high doses of cytosine arabinoside (Ara C; 12 g/m2) and etoposide (VP-16), followed by recombinant human granulocyte colony-stimulating factor (rhG-CSF; 50 micrograms/m2). Peripheral blood progenitor cells (PBPC) were collected during rapid leukocyte recovery using a CS-3000 blood cell separator. A blood volume of 9 liters was processed in each apheresis, with 162 apheresis procedures performed. The mean numbers of mononuclear cells (MNC) and colony-forming unit granulocyte-macrophage (CFU-GM) harvested per apheresis were 4.4 x 10(8)/kg and 142.5 x 10(4)/kg, respectively. A sufficient number of CFU-GM for engraftment (> 30 x 10(4)/kg) could be harvested by a single apheresis in 35 of 47 patients (74%). Various factors that influence the collection of progenitor cells were analyzed by univariate and multivariate analyses. The number and duration of previous chemotherapy cycles were the most significant factors affecting CFU-GM yield. In patients with malignant lymphoma, age also had an influence in addition to these two factors. MNC harvested had an impact on CFU-GM yields by univariate analysis. These observations suggest that high-dose Ara C plus VP-16 followed by G-CSF is an effective regimen for harvesting PBPC. PBPC should be collected in the early stage of first-line chemotherapy. PMID- 7545485 TI - Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA. AB - 6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and nuclease protection assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by nuclease protection assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor. PMID- 7545491 TI - Single dose of filgrastim (rhG-CSF) to predict mobilisation of haemopoietic progenitors in patients with haematological malignancies. PMID- 7545486 TI - Oligoclonal beta-galactoside-specific gamma immunoglobulins allow the immunocytochemical detection of cellular antigenic determinants expressed in mouse brain after surgical injury. AB - Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells. PMID- 7545487 TI - Differential effects of the immunosuppressants cyclosporin A, FK506 and KM2210 on cytokine gene expression. AB - Cyclosporin A (CsA) inhibited interleukin 2 (IL-2), IL-3, interferon gamma (IFN gamma), GM-CSF and tumor necrosis factor alpha (TNF alpha) mRNA expression in spleen cells stimulated with concavalin A (Con A) when determined by the semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. FK506 which has a similar immunosuppressive mechanism to that of CsA also showed the same inhibitory effects except for decreased IL-5 and IL-6 mRNA expression. In contrast, both CsA and FK506 enhanced transforming growth factor beta (TGF beta) and IL-1 beta mRNA expression. Another immunosuppressant KM2210 did not show any inhibitory effects on cytokine gene expression but rather enhanced IL 10, IL-6, TGF beta and IL-1 mRNA expression, thus suggesting that KM2210 has a completely different immunosuppressive mechanism from that of CsA and FK506. Anti TFG beta 1 antibody abrogated the suppression by KM2210 of BALB/c anti-3H/He mixed lymphocyte reaction (MLR) whereas this antibody did not abrogate the suppression by CsA and FK506 of BALB/c anti-C3H/He MLR. These results indicate that TGF beta is one of the major cytokines in KM2210 immunosuppression, in addition to IL-10, but not in immunosuppression by CsA and FK506. PMID- 7545492 TI - Identification of murine loci associated with susceptibility to chronic experimental autoimmune encephalomyelitis. AB - B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89-101. The disease is associated with the major histocompatibility complex (MHC) (eae1). We have now investigated the importance of non-MHC regions for the EAE susceptibility in a cross between RIIIS/J and B10.RIII mice which share the MHC region but differ in disease susceptibility. Linkage analysis using microsatellite markers spanning the genome identified a region (eae2) on chromosome 15 which showed linkage to disease (P = 0.0002). Our data also suggest linkage to a second region (eae3) on chromosome 3 (P = 0.0024), and provide evidence for locus interactions between eae2 and eae3. These results provide clues to the genetic basis of multiple sclerosis. PMID- 7545489 TI - Role of granulocyte colony-stimulating factor in preventing ceftazidime-induced myelosuppression in vitro. AB - Ceftazidime has been reported to cause myelosuppression both in vitro and in vivo. Granulocyte colony-stimulating factor (G-CSF) is known to enhance the proliferation and differentiation of myeloid cells. The present study was carried out to define the role of G-CSF in preventing the ceftazidime-induced suppression of BM progenitor cells in vitro, and to define the mechanisms involved in ceftazidime-induced myelosuppression. Our results show that G-CSF was able to maintain the proliferative activity of BM cells in the presence of ceftazidime if it was added to the culture medium during the early phase of exposure of BM to ceftazidime. Monoclonal antibody to TNF completely inhibited the ceftazidime induced myelosuppression. The suppressive effect on BM was mediated via CD3+ T cells whereas macrophages conferred protection against this suppression. TNF induced suppression of BM was inhibited by G-CSF. These data suggest that G-CSF prevents the ceftazidime-induced myelosuppression by antagonizing the suppressive effect of TNF and by enhancing the proliferative activity of BM. PMID- 7545494 TI - Generation and characterization of a delta F508 cystic fibrosis mouse model. AB - We have generated mice carrying the most common mutation in cystic fibrosis (CF), delta F508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. delta F508-/- mice die from peritonitis and show deficiencies in cAMP-activated electrogenic Cl- transport. These mice produce delta F508 transcripts and show the temperature-dependent trafficking defect first described for the human delta F508 CFTR protein. A functional CFTR Cl- channel not demonstrated by null CF mice or present at 37 degrees C was detected following incubation of epithelial cells at 27 degrees C. Thus, these mice are an accurate delta F508 model and will be valuable for testing drugs aimed at overcoming the delta F508 trafficking defect. PMID- 7545493 TI - Mutation of a type II keratin gene (K6a) in pachyonychia congenita. AB - Pachyonychia congenita (PC) is a rare autosomal dominant condition characterized by multiple ectodermal abnormalities. Patients with Jadassohn-Lewandowsky Syndrome (MIM #167200; PC-1) have nail defects (onchyogryposis), palmoplantar hyperkeratosis, follicular hyperkeratosis and oral leukokeratosis. Those with the rarer Jackson-Lawler Syndrome (MIM #167210; PC-2) lack oral involvement but have natal teeth and cutaneous cysts. Ultra-structural studies have identified abnormal keratin tonofilaments and linkage to the keratin gene cluster on chromosome 17 has been found in PC families. Keratins are the major structural proteins of the epidermis and associated appendages and the nail, hair follicle, palm, sole and tongue are the main sites of constitutive K6, K16 and K17 expression. Furthermore, mutations in K16 and K17 have recently been identified in some PC patients. Although we did not detect K16 or K17 mutations in PC families from Slovenia, we have found a heterozygous deletion in a K6 isoform (K6a) in the affected members of one family. This 3 bp deletion (AAC) in exon 1 of K6a removes a highly conserved asparagine residue (delta N170) from position 8 of the 1A helical domain (delta N8). This is the first K6a mutation to be described and this heterozygous K6a deletion is sufficient to explain the pathology observed in this PC-1 family. PMID- 7545496 TI - [A clinical and endoscopic study of the treatment of benign prostatic hypertrophy using focused transrectal ultrasound]. AB - OBJECTIVE: To evaluate the clinical and endoscopic effects of transrectal focal ultrasound (Sonablate) on benign prostatic hypertrophy. METHOD: This was a phase II study, performed in 13 patients with benign prostatic hypertrophy. Patients were evaluated before treatment, and 45 days and 90 days after treatment by means of a symptom score (1-PSS), uroflowmetry, estimation of prostatic volume by transrectal ultrasonography, and urethroprostatic endoscopy. A transducer with a focal length of 3.5 cm was used in 12 patients. One patient was treated with a transducer with a focal length of 3 cm. We used an acoustic energy level at the focal point of 1,680 W/cm2 for the last two patients. RESULTS: With a follow-up of 3 months, the symptom score was only moderately improved (decreasing from 20 +/- 7 to 14 +/- 9, p < 0.03), with no improvement of uroflowmetry and tissue destruction was not demonstrated on endoscopy in any of the patients. CONCLUSION: The therapeutic applications of focal ultrasound thermotherapy are potentially very vast, but, up until now, have only rarely exceeded the experimental stage. Our data emphasize that future developments are essential before this technology can be included in therapeutic alternatives for benign prostatic hypertrophy. PMID- 7545495 TI - Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I. AB - Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2 (2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer. PMID- 7545497 TI - [Adenocarcinoma of the rete testis. Apropos of a case]. AB - In the light of a case report, the authors recall the clinical signs of adenocarcinoma of the rete testis and the precise histological and immunohistochemical features allowing confirmation of this very rare diagnosis. They emphasize the persistent controversies concerning the treatment of this testicular tumour. PMID- 7545499 TI - Antigen-specific immunodeficiency and its relation to the spectrum of American cutaneous leishmaniasis. AB - Delayed-type hypersensitivity (DTH) and antibodies against Leishmania have been studied in 207 Venezuelan patients with localized, muco-cutaneous and diffuse forms of American cutaneous leishmaniasis, representing the clinical spectrum of this disease. Muco-cutaneous disease appears to be related to inadequate immunomodulation or defective killing mechanisms; about 40% of these patients show exaggerated DTH, which is inversely related to antibody levels and is more pronounced in less extensive lesions. Patients with diffuse disease present severe antigen-specific immunodeficiency, apparently limited to T cell-mediated protection, DTH and its in vitro correlates. Treatment of patients with diffuse cutaneous leishmaniasis using a combination of chemotherapy and combined vaccine immunotherapy (heat-killed promastigotes plus BCG) has induced clinical inactivity and positive DTH in about one third of these patients, accompanied by marked lowering of antibody levels. These results are discussed in terms of Type 1 T cell responses, protective in cell-mediated immune reactions, and Type 2 T cell responses, non-protective in cell-mediated reactions, in the spectrum of leishmaniasis. Factors related to the induction of favorable Type 1 responses to intracellular pathogens are discussed in terms of a possible mechanism of the combined vaccine efficacy and priorities in vaccine development. PMID- 7545498 TI - [Carcinoma of Bellini's tubules]. AB - Carcinoma of the papillary ducts of Bellini is a rare form of renal cancer. Patients are usually asymptomatic middle-aged men who frequently have metastatic disease at the time of diagnosis. The diagnosis is based on histological examination of the nephrectomy specimen with a precise immunohistochemical examination. Radical nephrectomy is the first-line treatment and adjuvant chemotherapy, recommended by certain authors, does not appear to improve the usually poor prognosis of these tumours. PMID- 7545502 TI - Molecular evidence for the importation of Old World Leishmania into the Americas. AB - A brief review of work carried out by the laboratory on the systematics of trypanosomatids during the last three years is given. The principal line of research has been on the taxonomy of New World Leishmania and one of the topics studied has been the determination of the autochthonous origin of certain Leishmania species found in the New World. Controversy exists as to whether the etiological agent of American Visceral Leishmaniasis is indigenous. Here, we present evidence from enzyme electrophoresis and schizodeme analysis indicating that L. chagasi has a recent origin and that it is similar to L. infantum. We also describe L. major-like isolates which have been found in the New World and present evidence suggesting that some of these populations may have been imported into the Americas. Reference strains from the subgenus Viannia are examined and compared with other Old World and New World species by enzyme electrophoresis. The results are analyzed numerically and we show that the Viannia species are a group of parasites indigenous to the New World that cluster separately from other Leishmania species. The numerical analyses also indicate that the subgenus forms a monophyletic group in contrast to the subgenus Leishmania which appears to be polyphyletic. PMID- 7545500 TI - Calcium homeostasis in Trypanosoma cruzi. AB - By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545501 TI - The cutaneous lesion in American leishmaniasis: leukocyte subsets, cellular interaction and cytokine production. AB - Interactions between immunocompetent cells require the participation of T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). These interactions are mediated by interlinking cytokines, which are important in determining the type of immune response. In the present study, we have shown that in American cutaneous leishmaniasis (ACL) lesions, most infiltrating T cells expressed the alpha beta TCR including those selectively migrating to the epidermis. In contrast, gamma delta T cells were abundant in localized (LCL) and scarce in muco-cutaneous (MCL) and diffuse (DCL) cutaneous leishmaniasis, suggesting a role in effective granulomas. There were differences in the expression of LFA-1 alpha and beta subunits, with most cells expressing LFA-1 beta. The ratio LFA-1 beta/LFA-1 alpha was higher in LCL (11.8:1) than in MCL (3.3:1) and DCL (2.4:1). Similar results were observed in Leishmania mexicana infected C57BL/6 mice. DCL lesions showed a higher proportion of LFA-1 alpha+ cells than MCL and LCL lesions. A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of the cytokine profiles showed that most T cells present in the MCL and DCL lesions secrete a mixture of Type 1 and Type 2 cytokine patterns, but in DCL granulomas predominate the Type 2 cytokines. In LCL the cytokine patterns show a preponderance of INF gamma over IL-4, and low levels of IL-5 and IL-10, suggesting a Type 1 cytokine profile. PMID- 7545503 TI - The chronic presence of the parasite, and anti-P autoimmunity in Chagas disease: the Trypanosoma cruzi ribosomal P proteins, and their recognition by the host immune system. AB - Molecular expression cloning techniques revealed that patients with the severest clinical form of Chagas disease, chronic Chagas heart disease, presented a strong humoral response against the cloned C-terminal portion of a Trypanosoma cruzi ribosomal P protein. Parasite P antigens identification led to characterize the ribosomal P protein system in T. cruzi. Their exposed location on the ribosome, and the "amplification" of their parasite specific, serine free C-terminal domain, generate a strong parasite specific anti-P response, that in certain cases may induce anti-P autoimmunity. PMID- 7545505 TI - Serum concentrations of granulocyte colony-stimulating factor (G-CSF) determined by a highly-sensitive chemiluminescent immunoassay during the clinical course of subacute thyroiditis. AB - Granulocyte colony-stimulating factor (G-CSF) concentrations in serum were determined for the first time by a newly developed and highly sensitive chemiluminescent immunoassay (the limitation of detection, 0.5 pg/ml) in ten patients with subacute thyroiditis, during treatment with glucocorticoid or indomethacin. Before therapy, circulating neurophil counts significantly increased to 5.15 +/- 2.07 x 10(3)/microliters compared with the convalescent phase (2.94 +/- 1.07 x 10(3)/microliters), and the data were correlated with individual serum G-CSF levels (r = 0.854, P < 0.01). Serum concentrations of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM CSF) were less than the detectable threshold of ELISA. During two weeks of glucocorticoid therapy, although the circulating neutrophil counts increased from 5.15 +/- 2.46 x 10(3)/microliters to 7.73 +/- 1.64 x 10(3)/microliters (P < 0.01), serum G-CSF levels were depressed from 25.1 +/- 15.3 pg/ml to 13.8 +/- 13.9 pg/ml (P < 0.01). These data indicate that G-CSF is one of the mediators of the increase of neutrophils in subacute thyroiditis, while it does not contribute to steroid-induced neutrophilia. PMID- 7545504 TI - Trypanosoma and Leishmania have clonal population structures of epidemiological significance. AB - This paper presents three results concerning the population structure of Trypanosoma cruzi, the agent of Chagas disease: (1) The mode of propagation of T. cruzi in nature is clonal; sexual reproduction is either totally absent or so rare that it leaves no traces in the population structure of the parasite. (2) The genetic diversity of the clonal lineages is large: extant T. cruzi represent lineages of descent that have evolved independently for long time spans (up to 40 million years). (3) Some genetically identical clonal lineages ("clonets") are geographically widespread ("ubiquitous"). However, most clonets are endemic, restricted in geographic distribution. These results have each in turn consequences of epidemiological significance: (1) In a sexually-reproducing organism the individual genotype is ephemeral; the entity that persists and evolves is the species ("gene pool"), and a few individuals contain most of the genetic variability of the species. In a clonally-propagating organism, the entity that persists and evolves is the clonal lineage; the genetic diversity of the species can only be captured by extensive sampling of distinct lineages. (2) The extensive genetic divergence among clonal lineages implies proportionally diverse biological characteristics, which are likely to include pathological effects, host propensity, vulnerability to drugs and vaccines, and other medically significant attributes. The extant T. cruzi lineages diverged much before human origins; hence, specific adaptation to human hosts, to whichever extent it exists, has evolved independently in separate lineages, and may not have evolved at all in some T. cruzi. (3) Epidemiological surveys and medical characterization, including search for specific vaccines and drugs, should not proceed randomly; rather, preliminary surveys must identify those clonets that are ubiquitous and target them for investigation. Review of published literature shows that Leishmania (and other parasitic protozoa) also has a clonal population structure. We advance a taxonomic and nomenclatural proposal that is appropriate for clonal organisms, yet simple. PMID- 7545507 TI - Nitric oxide plays no role in ACTH release induced by interleukin-1 beta, corticotropin-releasing hormone, arginine vasopressin and phorbol myristate acetate in rat pituitary cell cultures. AB - To investigate the role of nitric oxide (NO) in pituitary ACTH secretion, the effect of a nitric oxide synthase (NOS) inhibitor, N omega-Nitro-L-arginine (Nitro-Arg), on ACTH secretion induced by interleukin (IL)-1 beta, corticotropin releasing hormone (CRH), arginine vasopressin (AVP) and phorbol myristate acetate (PMA) was examined in rat anterior pituitary cell cultures. Nitro-Arg did not affect IL-1 beta-induced ACTH release in pituitary cell cultures incubated with Dulbecco modified Eagle's medium, Basal Medium Eagle or Krebs Ringer bicarbonate glucose buffer. It did not affect CRH-, AVP- or PMA-induced ACTH release either. These results suggest that NO does not play an important role in ACTH release at the pituitary level. PMID- 7545508 TI - Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains. AB - The major outer surface proteins of Lyme disease spirochaetes are differentially expressed in different isolates. Borrelia afzelii strain F1 expresses none, or very low amounts, of the OspA and OspB proteins. To elucidate the mechanisms that control the expression of these abundant surface proteins the ospAB operon of B. afzelii F1 was cloned, sequenced and compared to the previously sequenced ospAB operon of B. afzelii ACAI and Borrelia burgdorferi B31. The two B. afzelii strains showed almost 100% identity at the DNA level, although Coomassie-stained gels and Western blot analyses showed significant variation in the Osp protein content. Transcriptional analysis revealed that the amount of ospAB mRNA produced in B. afzelii F1 varies more than the amount of protein, suggesting that the expression of OspA and OspB proteins is regulated at both the transcriptional and the translational level. Furthermore, the inverse relationship between the transcription of ospC and the ospAB operon could indicate coregulation of these separately encoded operons. PMID- 7545506 TI - Changes in serum levels of IGF-I and IGFBP-3 in children with chronic renal failure. AB - The significance of serum insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) levels in uremia is still controversial. In this study we measured serum IGF-I by immunoradiometric assay (IRMA) and IGFBP-3 levels by specific radioimmunoassay (RIA) in 28 children (aged 3-16 years) with end-stage (n = 14, on hemodialysis) and pre-terminal renal failure (n = 14) and in 15 age-matched healthy children. Thyroid function of the patients was also investigated and GH-stimulation tests with L-Dopa and insulin were performed. Neither IGF-I nor IGFBP-3 levels significantly correlated with mean height SDS for bone age or for chronological age in either non dialysis patients or pubertal (n = 10) or prepubertal patients (n = 18). These data was consistent with the concept that growth in CRF was not related to abnormalities in serum IGF-I or IGFBP-3 levels. PMID- 7545509 TI - A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases. AB - A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designed hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron restriction. The results indicate that the availability of iron modulates the expression of the hly gene. PMID- 7545511 TI - Four glucosyltransferases, GtfJ, GtfK, GtfL and GtfM, from Streptococcus salivarius ATCC 25975. AB - The four recombinant glucosyltransferases (GTFs), GtfJ, GtfK, GtfL and GtfM, that had previously been cloned from Streptococcus salivarius ATCC 25975, were individually expressed in Escherichia coli and their glucan products and kinetic properties were analysed. GtfJ was a primer-dependent GTF which synthesized an insoluble glucan composed mainly of alpha-(1-->3)-linked glucosyl residues in the presence of dextran T-10. GtfK was primer-stimulated, and produced a linear soluble dextran without any detectable branch points both in the absence and in the presence of dextran T-10. GtfL was primer-independent and produced a mixed linkage insoluble glucan composed of approximately equal proportions of alpha-(1- >3)- and alpha-(1-->6)-linked glucosyl residues. GtfL was inhibited by dextran T 10. GtfM was primer-independent and produced a soluble dextran with approximately 5% alpha-(1-->3)-linked glucosyl residues. GtfM was essentially unaffected by the presence of dextran T-10. The results confirmed that each enzyme represented one of the four possible combinations of primer-dependency and product solubility and that each possessed unique biosynthetic properties. The soluble dextrans formed by GtfK and GtfM, as well as the mixed-linkage insoluble glucan formed by GtfL, were also capable of acting as primers for the primer-dependent GtfJ and the primer-stimulated GtfK. Unexpectedly, the linear dextran produced by GtfK was by far the least effective either at priming itself or at activating and priming the primer-dependent GtfJ. PMID- 7545510 TI - A Bacillus subtilis spore coat polypeptide gene, cotS. AB - A gene, cotS, encoding a spore coat polypeptide of Bacillus subtilis, was isolated from an EcoRI fragment (5.4 kb) of the chromosome by using synthetic oligonucleotide probes corresponding to the NH2-terminal amino acid sequence of Cot40-2 previously purified from the spore coat of B. subtilis. The nucleotide sequence (2603 bp) was determined and sequence analysis suggested the presence of two contiguous ORFs, ORF X and cotS, followed by the 5'-region of an additional ORF, ORF Y, downstream of cotS. The cotS gene is 1053 nucleotides long and encodes a polypeptide of 351 amino acids with a predicted molecular mass of 41083 Da. The predicted amino acid sequence was in complete agreement with the NH2 terminal amino acid sequence of Cot40-2. The orfX gene is 1131 nucleotides long and encodes a polypeptide of 377 amino acids with a predicted molecular mass of 42911 Da. The gene product of cotS was confirmed to be identical to Cot40-2 by SDS-PAGE and immunoblotting from Escherichia coli transformed with a plasmid containing the cotS region. Northern hybridization analysis indicated that a transcript of cotS and orfX appeared at about 5 h after the onset of sporulation. The transcriptional start point determined by primer extension analysis suggested that -10 and -35 regions are present upstream of orfX and are very similar to the consensus sequence for the sigma k-dependent promoter. Terminator-like sequences were not found in the DNA fragment (2603 bp) sequenced in this paper, which suggested that the cotS locus may be part of a multicistronic operon. The cotS gene is located between dnaB and degQ at about 270-275 degrees on the genetic map. Insertional mutagenesis of the cotS gene by introducing an integrative plasmid resulted in no alteration of growth or sporulation, and had no effect on germination or resistance to chloroform. PMID- 7545512 TI - Heterogeneity among 16S-23S rRNA intergenic spacers of species within the 'Streptococcus milleri group'. AB - The 16S-23S rRNA intergenic spacer has been suggested as a suitable region of the bacterial genome from which to derive useful taxonomic information, particularly with regard to identification at the species level. To investigate this approach as an aid to the identification of the three species comprising the 'Streptococcus milleri group' (SMG), the spacers of isolates of Streptococcus intermedius, Streptococcus anginosus and Streptococcus constellatus were amplified by PCR and length polymorphisms determined by agarose gel electrophoresis. Phenotypically atypical isolates which had been identified presumptively as belonging to these three species were also included. Spacers from two representatives of each spacer length found within the three SMG species were sequenced. 16S-23S rRNA intergenic spacer length polymorphisms allowed discrimination between S. anginosus (350 bp or 450 bp amplification product) and S. constellatus (380 bp amplification product), species that are difficult to differentiate phenotypically. S. intermedius (330 bp or 450 bp amplification product) and S. anginosus (350 bp or 450 bp amplification product) were not reliably distinguished by this method but are phenotypically distinct. Sequencing data demonstrated that the spacers had a central region of highly variable length flanked by conserved regions which included a single tRNA(Ala) gene. Polymorphism in the length of the 16S-23S spacer determined by PCR provides a rapid and useful adjunct to strain identification for S. anginosus and S. constellatus, which are not readily differentiated phenotypically. PMID- 7545514 TI - Acute pancreatitis following liver transplantation. AB - BACKGROUND: Acute pancreatitis (AP) has not been recognized as a frequent complication following orthotopic liver transplantation (OLT). Reports have suggested that this condition is associated with high morbidity and mortality rates. STUDY DESIGN: A retrospective review of 354 patients who underwent OLT at the University of Toronto, with at least one-year follow-up, was done to identify the incidence, etiology, risk factors, and severity of pancreatitis following OLT, as well as to discuss its management. A possible correlation between AP and hepatitis B was also investigated. Prognostic score systems, including Ranson, Imrie, and APACHE II, were also evaluated. RESULTS: Acute pancreatitis occurred in 5.9 percent of the OLT recipients. Based on clinical presentation and diagnostic tests, AP could be subdivided into three distinct categories: biochemical AP, significant increase of serum amylase or lipase levels in absence of objective radiologic or surgical evidence of the disease; clinical AP, pancreatitis with radiologic or surgical evidence of the disease; and AP in the critically ill patient. Biochemical AP occurred in the early postoperative period and was consistently a benign condition. Clinical AP occurred after a delay and was associated with a 40 percent mortality rate. Acute pancreatitis in the critically ill patient was only one component of multiorgan failure, which was lethal in each case. About one-half of the patients with biochemical and clinical AP were hepatitis surface antigen positive prior to OLT. The incidence of AP was significantly higher in patients with hepatitis B (17 percent) than in patients without hepatitis B (3 percent) (p < 0.01). APACHE II was the best prognostic score system in the clinical group with a positive and negative predictive value for a score equal to or greater than 10 points of 67 and 100 percent, respectively. CONCLUSIONS: Acute pancreatitis is not an uncommon complication of OLT. Clinical presentation and diagnostic tests have important prognostic values. In some patients, hepatitis B virus may play an important role. APACHE II scores appear to be useful in the evaluation of clinical AP following OLT. Treatment of AP should be similar in both the transplant and nontransplant setting. PMID- 7545513 TI - Paracoccus thiocyanatus sp. nov., a new species of thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb. nov. with emendation of the genus. AB - A facultatively chemolithotrophic thiocyanate-degrading bacterium, strain THI 011T, which was previously isolated from activated sludge and tentatively named Thiobacillus sp., was studied taxonomically and phylogenetically. This bacterium utilizes thiocyanate as sole energy source and the specific growth rate for chemolithoautotrophic growth with thiocyanate was 0.059 h-1. Molecular phylogenetic relationships of strain THI 011T to Thiobacillus versutus and members of the genus Paracoccus were elucidated by comparing 16S rRNA gene sequences. Binary sequence comparisons showed that strain THI 011T was most related to Paracoccus aminophilus, at a similarity level of 97.0%, and T. versutus was most similar to Paracoccus denitrificans, at a level of 99.1%. A neighbour-joining phylogenetic tree showed that strain THI 011T formed a cluster together with T. versutus and known species of the genus Paracoccus within the alpha-3 subclass of the Proteobacteria. DNA-DNA hybridization assays and phenotypic studies indicated that strain THI 011T differed from T. versutus and known species of the genus Paracoccus. On the basis of these results, we propose to classify strain THI 011T into a new species of the genus Paracoccus with the name Paracoccus thiocyanatus sp. nov. We also propose to transfer T. versutus to the genus Paracoccus and present an emended description of the genus. PMID- 7545515 TI - Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium. AB - Rabbit hearts were preconditioned with four 5 min coronary artery occlusions 24 h before 30 min coronary occlusion with 120 min reperfusion. Preconditioning significantly reduced the percentage of myocardium infarcting within the risk zone from 49.1 +/- 4.3% to 31.8 +/- 3.5% (P < 0.05). When the protein kinase C (PKC) inhibitor, chelerythrine, was administered just before preconditioning, the delayed protection against infarction 24 h later was abolished. We conclude that the delayed cytoprotective response associated with ischaemic preconditioning of myocardium is likely to involve the early activation of one or more PKC subtypes. PMID- 7545516 TI - Effect of a new non-steroidal anti-inflammatory drug, nitroflurbiprofen, on the expression of inducible nitric oxide synthase in rat neutrophils. AB - The effects of a non-steroidal anti-inflammatory drug, flurbiprofen, and its nitro-derivative, nitroflurbiprofen, on inducible nitric oxide synthase in rat neutrophils were examined. Nitroflurbiprofen was shown to inhibit nitric oxide synthase induction caused by lipopolysaccharide administration, while flurbiprofen had no effect on nitric oxide synthase induction. This inhibitory action may be ascribed to nitric oxide released from nitroflurbiprofen. PMID- 7545518 TI - Group B Streptococcus and E. coli LPS-induced NO-dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets. AB - 1. The effects of endotoxin (E. coli lipopolysaccharide, LPS) and heat inactivated group B Streptococcus (GBS) were studied on the contractile responses to noradrenaline (NA) in isolated pulmonary arteries and on the activity of the constitutive and inducible nitric oxide synthase (NOS) in lung fragments of neonatal piglets. 2. Short-term (< or = 5 h) incubation with LPS (1 micrograms ml 1) or GBS (3 x 10(7) colonies forming units ml-1) did not modify the vascular responsiveness to NA (10(-8) M-10(-4) M) in isolated intrapulmonary arteries. However, long-term incubation (20 h) with LPS or GBS produced a significant reduction in the maximal contractile responses and shifted the concentration response curve for NA downwards. 3. Endothelium removal or the cyclo-oxygenase inhibitor meclofenamate (10(-5) M) did not affect the GBS- and LPS-induced hyporesponsiveness to NA. 4. The presence of the nitric oxide (NO) precursor, L arginine (10(-5) M), 30 min prior to the contractility challenge increased the LPS- and GBS-induced pulmonary vascular hyporesponsiveness to NA. In contrast, the addition, prior to the challenge with NA, of the NOS inhibitor NG-nitro-L arginine methyl ester (L-NAME, 10(-4) M) or coincubation with dexamethasone (3 x 10(-6) M), a potent inhibitor of the induction of NOS, or with the protein synthesis inhibitor cycloheximide (10(-5) M) completely restored the reactivity to NA in LPS- and GBS-treated pulmonary arteries. 5. The incubation for 20 h of lung fragments with LPS and GBS produced a significant increase in the Ca2+ independent (inducible) NOS activity determined by the conversion of radiolabelled L-arginine to citrulline, but did not modify the constitutive NOS activity. This NOS induction was abolished by coincubation with dexamethasone (3 X 10-6 M).6. These results demonstrated that prolonged incubation with GBS and LPS causes an induction of NOS activity which results in a reduced vascular responsiveness to NA in pulmonary arteries of neonatal piglets. Thus, induction of NOS seems to be responsible for the delayed pulmonary vascular hyporesponsiveness induced by GBS (a Gram-positive) and E. coli (a Gram negative), the most common causal agents of neonatal sepsis. PMID- 7545517 TI - Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum. AB - 1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2 thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization. After addition of apamin and L-NOARG, the ij.p. was blocked, and EFS produced a pure NANC e.j.p.; ADPPS produced depolarization, action potentials and contraction.8. Suramin (100 microM) blocked the depolarization, action potentials and contractions produced by ADP beta S in the presence of apamin and L-NOARG, without affecting the responses produced by the NK1receptor agonist, [Sar9}SP sulphone.9. We conclude that NK1 and NK2 receptors cooperate in producing NANC excitation and contraction of the circular muscle in the guinea-pig proximal duodenum. Activation of either TK receptor produces membrane depolarization and both receptors contribute to generate action potentials which are essential for producing muscle contraction, via nifedipine-sensitive calcium channels. It appears that endogenous ATP chiefly acts as an inhibitory transmitter but, after blockade of NANC inhibitory mechanism(s),ATP may act as a fast signalling excitatory transmitter. PMID- 7545520 TI - Effect of nitric oxide on integrity, blood flow and cyclic GMP levels in the rat gastric mucosa: modulation by sialoadenectomy. AB - 1. The effects of the nitrosothiol, S-nitroso N-acetylpenicillamine (SNAP) which liberates nitric oxide (NO), on ethanol-mediated gastric damage, blood flow and cyclic GMP levels in siaoloadenectomized (SALX) rats have been investigated. 2. Intraluminal instillation of ethanol (5-50% w/v) dose-dependently induced haemorrhagic damage and decreased NO synthase activity in the gastric mucosa. Both the extent of mucosal damage and inhibition of NO synthase activity were exacerbated in SALX rats. 3. Epidermal growth factor administration (5 and 10 micrograms kg-1, s.c.) reduced mucosal damage but did not restore NO synthase activity in ethanol-treated SALX rats. 4. SNAP infusion (0.01-1.0 micrograms kg-1 min-1, i.v.) attenuated haemorrhagic damage in ethanol-treated rats. The reduction in mucosal damage was significantly greater in SALX rats. 5. SNAP administration also caused an increase in gastric mucosal blood flow and cyclic GMP levels in control rats and both responses were augmented in SALX animals. 6. These data suggest that SALX is associated with increases in mucosal susceptibility to ethanol-mediated damage and reduces mucosal NO synthase activity. Epidermal growth factor does not appear to influence mucosal NO synthase in ethanol-treated rats. Furthermore, SALX augments the responsiveness of the gastric mucosa to NO administration. Therefore, factors from the salivary glands influence gastric NO formation and mucosal responsiveness to a NO donor. PMID- 7545519 TI - Subcellular localization and molecular pharmacology of distinct populations of [3H]-AMPA binding sites in rat hippocampus. AB - 1. The subcellular distributions of [3H]-alpha-amino-3-hydroxy-5- methylisoxazolepropionate ([3H]-AMPA) and [3H]-kainate binding sites in rat hippocampus were investigated by cell fractionation techniques. 2. Two major populations of [3H]-AMPA sites were detected with the majority of binding located intracellularly in the microsomal (P3) fraction. Most of the remaining sites were in the synaptosomal membrane fraction but some were also present in the nuclear fraction. In contrast, essentially all of the [3H]-kainate binding sites were in the synaptosomal membrane fraction. 3. Saturation binding analysis yielded KD and Bmax values for [3H]-AMPA of 147 nM and 2.6 pmol mg-1 protein respectively for the synaptosomal membrane-associated sites and 129 nM and 5.3 pmol mg-1 protein respectively for the microsomal sites. 4. Both main populations of [3H]-AMPA sites displayed the same rank order of inhibition by competitive ligands, the apparent Mr values of GluR1 subunits were equivalent, suggesting the same degree of post-translational modification and the hydrodynamic properties of the receptor complexes were identical. 5. These data are consistent with the hypothesis that the movement of AMPA receptors between cellular compartments in the postsynaptic neurone could constitute one mechanism underlying long-term potentiation in the hippocampus. PMID- 7545521 TI - Kallikrein rK10-induced kinin-independent, direct activation of NO-formation and relaxation of rat isolated aortic rings. AB - 1. rK10, a weak T-kininogenase isolated from the rat submandibular gland, is a protein belonging to the rat kallikrein family. In the present work, we have studied the biological effects of rK10 with respect to its ability to alter vascular resistance, either directly like rK9, i.e. another kallikrein-like protein, trypsin and thrombin, or through the release of kinins like tissue kallikrein (rK1). The direct effect was studied by its vasomotor activity on rat isolated aortic rings since this preparation was insensitive to the action of kinins. Its ability to induce altered vascular resistance through kinin generation was investigated by blood pressure studies in whole animals. The studies were performed in comparison to rK1. 2. Unlike rK1, which induces hypotension when administered intravenously to rats (delta BP = -56 +/- 5 mmHg, 5 micrograms kg-1), rK10 did not have any effect on systemic blood pressure (delta BP = -3 +/- 1, 5 micrograms kg-1, i.v.). 3. rK10 was without effect on uncontracted aortic rings, but showed a concentration-dependent (10(-8)-10(-6) M) relaxant effect on tissue precontracted with phenylephrine (10(-6) M). After removal of endothelial cells, no relaxation was observed. The relaxant response to rK10 was transient. rK1 (with and without endothelium), bradykinin and T-kinin (with endothelium) had no effect on contracted or uncontracted aortic rings. 4. The relaxant effect of rK10 was dependent on its enzymatic activity since preincubation with aprotinin (1.02 mM) significantly reduced vasorelaxation from 74 +/- 4% to 24 +/- 3%. 5. The relaxant effect was not inhibited by the kinin antagonist Hoe 140 (10-7 M; 34 +/- 4% without,versus 30 +/- 2% with Hoe 140), but was totally inhibited by the NO-synthase inhibitor N omega.nitro-L-arginine methyl ester (L-NAME) (2.5 x 10-4 M; 27 +/- 3% without and 2 +/- 1% with L NAME).6. These results show that rKlO has the ability to induce vascular relaxation by a specific, direct effect on endothelial cell NO-synthesis, dependent on rK1O proteolytic activity, but independent of its ability to generate kinin. This effect, or its T-kininogenase activity in blood, was not sufficient for rK1O to have an effect on peripheral vascular resistance since intravenous injections of rK1O, unlike rKl, did not induce hypotension. Thus, rKlO does not seem to play a role in blood pressure homeostasis but may have a local effect on vascular resistance. PMID- 7545522 TI - Hypercholesterolemia and endothelial dysfunction. AB - Endothelial cells respond to hemodynamic forces with the expression of different phenotypes with disparate functional properties. At arterial bends and flow dividers, cells are relatively deprived of fluid-shear-stress-induced cell differentiation and exhibit phenotypes with increased mitotic rate, decreased intercellular contact, increased permeability for macromolecules, and the expression of molecules favoring constriction, adhesion, and thrombosis. Arterial sites covered by such cells are vulnerable to the atherogenic effects of hypercholesterolemia. A hallmark of endothelial cells exposed to hypercholesterolemia is a reduced capacity to release endothelium-derived relaxing factor. Pharmacological interventions that suppress or stimulate nitric oxide production appear to enhance or reduce the atherogenic effects of hypercholesterolemia. PMID- 7545523 TI - [Concentration of plasmin complexes with alpha-2-macroglobulin and alpha-2 antiplasmin in blood plasma and serum]. AB - A methodological approach has been developed for measuring the concentrations of proteinase complexes with their inhibitors in various biological fluids. This approach is based on the use of polyclonal antibodies to the complex complements and their detection by enzyme immunoassay. Appreciable individual differences in the levels of alpha 2-macroglobulin and plasmin in donor blood plasma samples were revealed. Complexes of this inhibitor with plasmin are responsible for about 3% of the total alpha 2-macroglobulin pool in blood plasma and sera. PMID- 7545524 TI - Use of bioassays in assessing health hazards from complex mixtures: a rash analysis. AB - The Finney harmonic mean model for joint toxicity of ingredients in mixtures can be used to estimate the toxicity of the neat compound if each component can be substituted in potency-adjusted-doses for any of the other components. Chemical analysis of constituent substances and their associated concentrations and relative toxicological potency values (computed according to the RApid Screening of Hazard (RASH) method) were used to compare the toxicities as predicted from ingredients of cigarette smoke, PAHs in diesel exhaust, asphalt, coal tar, pitch, and creosote with the measured toxicities of the corresponding neat mixtures. Accuracy for cigarette smoke condensate, coal tar, pitch, and creosote were within a factor of three based on the PAH fraction; asphalt was within a factor of 18; but the PAH fraction of diesel particulate was again found to be inadequate to describe the composite toxicity of diesel emissions. PMID- 7545525 TI - Influence of nitric oxide synthase inhibition on the development of rapid tolerance to ethanol. AB - We recently reported that the nitric oxide (NO) synthase inhibitor L nitroarginine (L-NA) blocks the development of rapid tolerance to the motor incoordinating effect of ethanol in the tilt-plane test. To clarify the mechanism of L-NA blockade of tolerance, four additional experiments were carried out using the same test. The first demonstrated that L-NA prevented the development of rapid tolerance to ethanol when injected prior to ethanol either on both Days 1 and 2 or only on Day 1. In the second experiment, tolerance was blocked only when L-NA was injected before but not after behavioral testing on Day 1. In the third, L-NA blocked the enhancement of rapid tolerance to ethanol induced by D cycloserine (CS), an agonist at the N-methyl-D-aspartate (NMDA) receptor. In the last experiment, L-NA pretreatment did not influence blood ethanol disappearance curves on Day 1, or ethanol concentrations in brain, tail blood or decapitated trunk blood on Day 2. These data argue against state-dependent learning as the basis of the L-NA effect, and confirm and extent our previous observation that NO plays a role in the development of rapid tolerance to ethanol. PMID- 7545528 TI - [Acceleration of the regeneration of skeletal muscles in adult rats by dextran derivatives]. AB - Dextran derivatives were obtained by controlled successive substitutions of carboxymethyl, carbomethyl-benzylamide and carboxymethyl-benzylamide sulfonate groups on glucose residues. Among these derivatives, RGT11 was selected since it mimicked some properties of heparin or heparan sulfate to stabilise and protect heparin binding growth factors such as FGFs and TGF-beta. Furthermore RGT11 inhibited plasmine and leukocyte elastase. In previous works, we have explained the healing effects of RGT obtained in skin or bone repair models by these protecting and inhibiting properties. We now present the results obtained after a single injection of RGT11 in a regenerating crushed extensor digitorum longus (EDL) muscle. After 8 days, RGT11 injected muscles contained 10 times the number of fibers than controlled injected muscles. Fibers were organized in bundles of normal size. Histological analysis indicated that regeneration was comparable to that observed after 3 weeks without RGT. Hence in vivo, RGT11 could act by protecting the heparin binding growth factors involved in the natural process of muscle regeneration and therefore favour their actions. This family of polymers should offer a new pharmaceutical potential for treating muscle atrophy and destruction and is worth studying further. PMID- 7545526 TI - Selection of more pathogenic hepatitis C virus genotype II during long-term follow-up of interferon-treated patients. AB - The behavior of hepatitis C virus (HCV) infection with regards to type and number of HCV genotypes (tested with genotype-specific nested polymerase chain reaction) was evaluated in 60 patients with anti-HCV-positive chronic active hepatitis without cirrhosis [17 untreated and 43 subjects undergoing single or repeat courses of interferon (IFN) therapy] during a mean follow-up period of 76 +/- 18 months. In untreated patients (2 genotype I, 6 genotype II, 9 mixed infections) 4 out of 9 mixed infections selected for genotype II at the end of follow-up. Of the 43 treated patients 10 were long-term responders with histological remission, 6 were short-term responders, and 22 did not respond. Fifteen of the latter patients received another course of IFN therapy, and only 3 patients responded. Eight of the 10 responders had infection with a single genotype (4 gt I, 3 gt II, 3 gt III). After IFN therapy, all but 2 patients cleared the HCV infection. The responders to the second IFN course (1 gt I, 1 gt II, 1 gt III) remained viremic. Of the short-term responders, 2/6 patients had genotype II and 4 had a mixed infection (3 gt II +/- I and 1 gt II +/- III); gt III became prevalent in the latter in all but one patient. Of the nonresponders 18/24 had more than one genotype, 5 were genotype II at baseline and one had genotype I. At the end of the follow-up period 15/18 with mixed infection had selected for gt II (P < 0.01 vs. untreated patient).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545527 TI - Post-capillary venules in the "milky spots" of the greater omentum are the major site of plasma protein and leukocyte extravasation in rodent models of peritonitis. AB - Intraperitoneal injection of inflammatory agents in the mouse and rat causes plasma protein and leukocyte extravasation into the peritoneal cavity. Following an intraperitoneal injection of zymosan A, the milky spots of the omentum were the only abdominal sites detected where intravenously administered Monastral Blue labeled interendothelial cell gaps responsible for plasma extravasation. In addition, when colored microspheres were intraventricularly administered to quantify blood flow, the omentum was the only abdominal organ which showed an increase in blood flow during zymosan A peritonitis. A combination of light and electron microscopy, plus measurement of myeloperoxidase activity (a marker of neutrophil accumulation) demonstrated that the omental milky spots are the major route through which leukocytes migrate into the peritoneal cavity. Identical structures in the pleura likewise are the sites of protein leakage into the pleural cavity. In contrast, selective sites of protein and cellular extravasation could not be detected in the synovial lining of the inflamed knee joint. PMID- 7545535 TI - Ultrastructural and immunohistochemical observations on intralobular fibroblasts of human breast, with observations on the CD34 antigen. AB - Understanding the diverse pathological processes which are initiated in the ultimate organizational units of the mammary parenchyma--the lobules and ductules -will to a large extent be based on understanding the normal breast. During the routine diagnostic evaluation of 11 surgical specimens for a variety of benign and malignant breast lesions, grossly normal mammary parenchyma, subsequently confirmed as histologically normal by light microscopy, was sampled for this purpose. Tissue was studied by histology, light microscope immunohistochemistry, transmission electron microscopy, scanning electron microscopy, and ultrastructural immunohistochemistry. A number of features hitherto disregarded in the literature are described in detail, or are elaborated with respect to earlier descriptions. These include: the presence of solitary cilia and lipid rich residual bodies in intralobular fibroblasts, and the frequent association of inflammatory cells (lymphocytes, plasma cells, macrophages, mast cells) with the cell bodies and processes of fibroblasts. For the first time also, CD34, best known as an endothelial cell marker, has been demonstrated by ultrastructural immunohistochemistry on the cell surface of fibroblasts. Finally, scanning electron microscopy has demonstrated new features of the intercellular matrix in which the fibroblasts and inflammatory cells are located. The significance of these findings is discussed in terms of a postulated immune surveillance role of the mononuclear cells in collaboration with the fibroblasts. PMID- 7545530 TI - Alterations of substance P metabolism and neuropeptidases in Alzheimer's disease. AB - The levels of specific neuropeptides and the activity of neuropeptidases known to degrade neuropeptides have been shown to be altered in senile dementia of the Alzheimer's type (SDAT) postmortem brain. We examined the metabolism of exogenous substance P, a neuropeptide with a putative role in SDAT pathology, in human postmortem temporal cortex, hippocampus, and caudate membrane homogenates from SDAT and sex- and age-matched control brain. The activity of two neuropeptidases known to metabolize substance P, neutral endopeptidase E.C.3.4.24.11 and metalloendopeptidase E.C.3.4.24.15, was also determined in the same postmortem, human tissue samples. The metabolic half-life of substance P was significantly increased in SDAT, postmortem, temporal cortex. The increased half-life of substance P was correlated with a decreased activity of the metabolic enzyme metalloendopeptidase 24.15 in temporal cortex. Substance P metabolism was not significantly altered in hippocampus or caudate tissues from SDAT brain compared to controls. The alteration of neuropeptidases demonstrated in this study and as shown by altered substance P metabolism may have a significant effect on the level of several biologically important neuropeptides in SDAT brain. PMID- 7545532 TI - Signalling by the p60c-src family of protein-tyrosine kinases. AB - The c-src gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c src, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-src PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalling proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-src PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-src family in transgenic mice has confirmed important roles for p56lck and p59fym(T) in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60c-src in bone maintenance and for p59fym(B) in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-src PTKs operate and about the other signalling proteins with which they interact. The c-src PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-src PTKs also associated with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences. PMID- 7545529 TI - Hyperadrenocorticism and food restriction-induced life extension in the rat: evidence for divergent regulation of pituitary proopiomelanocortin RNA and adrenocorticotropic hormone biosynthesis. AB - The increased diurnal elevation of plasma corticosterone (B) induced by food restriction (FR) may play a role in the life span extension of FR. We investigated whether FR alters adrenocorticotropic hormone (ACTH) and proopiomelanocortin (POMC) mRNA levels in plasma and anterior pituitary (AP), since these molecules both regulate and can be suppressed by B. Measurements were made in 3-month-old male Fischer 344 rats that had been ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Plasma B was 2-fold higher in FR rats in the PM samples, but did not differ in AM samples. By contrast, plasma ACTH did not differ in the PM samples of FR and AL rats and was 20% lower in AM samples (p < .05) of FR rats. AP content of ACTH was 50% lower in FR rats in both AM and PM samples (p < .01). In contrast, AP contents of POMC and mRNA, primary transcript, and processing intermediate were not reduced in FR rats, and PM content of POMC primary transcript was elevated in FR rats (p < .05). The reduced pituitary and plasma ACTH of FR rats may be the consequence of their elevated plasma B levels. This study also suggests that factors other than elevated ACTH account for FR induced hyperadrenocorticism. These results also indicate that POMC mRNA and ACTH biosyntheses are differentially regulated in FR rats. PMID- 7545531 TI - Substance-P-mediated intestinal inflammation: inhibitory effects of CP 96,345 and SMS 201-995. AB - The proinflammatory peptide substance P (SP) has been shown to be intimately involved in the local inflammatory processes of Trichinella-spiralis-induced murine intestinal inflammation. Significant increases in SP, increased myeloperoxidase levels coupled with local morphological deterioration of the jejunum and impaired lymphocyte responses to exogenous SP in vitro have been associated with the model. We have recently determined that the elimination of increased levels of SP via anti-SP antibody therapy can spare the murine gastrointestinal tract much of the pathologies associated with the parasitic infection. Here we further demonstrate that the somatostatin analogue SMS 201-995 as well as the SP receptor antagonist CP 96,345 can effectively decrease the inflammation and lost lymphocyte function seen in the jejunum of T. spiralis infected mice. Again, both intestinal morphology and myeloperoxidase levels were shown to return to normal values upon treatment. The above results suggest that SP is an important modulator of gastrointestinal inflammation. PMID- 7545534 TI - Proliferation and progesterone production of ovine luteal cells from several stages of the estrous cycle: effects of fibroblast growth factors and luteinizing hormone. AB - This study was conducted to evaluate the effects of fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), or luteinizing hormone (LH) on proliferation and progesterone secretion of ovine luteal cells from days 5, 10, or 15 after estrus (estrus = day 0; n = 4 or 5 ewes/day). After enzymatic dispersion, luteal cells were incubated in the presence or absence of various doses of FGF-1, FGF-2, LH, or fetal bovine serum (FBS) (positive control) in serum-free media for 7 days in 24-well plates. Cells were counted on day 7 of culture and media analyzed for progesterone concentration. For all treatments, maximal effects (Emax) and dissociation constants (KD) were calculated. In addition, luteal cells were cultured in eight-chamber slides and treated as above, but on day 7 of culture cells were fixed and stained for the presence of 3beta-hydroxy-delta 5-steroid dehydrogenase (3beta HSD). The number of steroidogenic (3beta HSD positive) cells per unit area was counted for control cultures (no treatment) and cultures treated with the most effective doses of FGF 1, FGF-2, LH, OR FBS in proliferation and (or) progesterone assays. FGF-1, FGF-2, AND FBS stimulated (p < 0.05) proliferation of luteal cells from all stages of luteal development in a dose-dependent manner. In addition, LH increased (p < 0.01) the number of 3beta HSD-positive cells across all stages of luteal development. Moreover, LH and FBS increased (p < 0.05) progesterone secretion by luteal cells from all stages in a dose-responsive manner, but the effects of FGF 1 and FGF-2 were variable. For proliferation, the Emax of all factors was greatest (p < 0.01) on day 5, whereas the KD values were similar across days of the estrous cycle. For progesterone production, the Emax and KD of LH and FBS were similar and did not differ across the estrous cycle. These data demonstrate the luteal cells from the early luteal phase of the estrous cycle exhibit the greatest ability to proliferate and (or) increase their progesterone secretion in response to FGF-1, FGF-2, LH, or FBS. In addition, although LH does not affect the total number of luteal cells in culture, it does increase the number of steroidogenic cells. These data indicate that in addition to LH, fibroblast growth factors may be involved in regulation of luteal growth and differentiation in ewes. PMID- 7545536 TI - Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze substitution. AB - CUT-1 and CUT-2 are two distinct protein components of cuticlin, the insoluble residue of the cuticles of nematodes. In previous experiments of gold-immuno labelling on sections of chemically fixed Caenorhabditis elegans, CUT-1 and CUT-2 epitopes were specifically lost. Cryo-immobilization of C. elegans under high pressure followed by freeze-substitution, however, resulted in a good preservation of these antigenic sites and of the ultrastructure of the worms. The entomopathogenic nematode Heterorhabditis sp. processed by the same cryopreparation protocol has shown a strong reactivity with anti-sera raised against CUT-1, CUT-2 and against the whole cuticlin residue of C. elegans. The localization of these epitopes was conserved across the two species. PMID- 7545533 TI - Treatment of ovariectomized rats with the complex of rhIGF-I/IGFBP-3 increases cortical and cancellous bone mass and improves structure in the femoral neck. AB - Sixteen-week-old Sprague-Dawley rats were ovariectomized (Ovx) or sham-operated and housed for 8 weeks to develop osteopenia prior to systemic administration of rhIGF-I (0.9 and 2.6 mg/kg) alone or the rhIGF-I/IGFBP-3 (0.9, 2.6 and 7.5 mg/kg) complex. After 8 weeks of treatment, proximal femurs were fixed, embedded, and cut through the midneck region. Structural and dynamic histomorphometric analyses were performed using standard techniques. Ovx increased endocortical resorption and modeling-dependent periosteal formation which resulted in decreased cortical bone area. Despite increased bone formation, trabecular number, thickness, and area were all reduced due to increased resorption. Structural changes following Ovx included fewer struts and nodes, a higher percentage of the simpler strut forms, and reduced endocortico-trabecular connectivity. Eight weeks of treatment with rhIGF-I or rhIGF-I/IGFBP-3 promoted periosteal and endocortical bone formation and reduced the endocortical resorption induced by Ovx. Both rhIGF-I formulations stimulated bone formation on existing trabecular surfaces which increased trabecular thickness and area but not trabecular number. These treatments prevented further deterioration of the trabecular network caused by Ovx and preserved endocortico-trabecular connectivity. In summary, changes in the femoral neck following Ovx appear to be similar in rats and humans. The highest dose of rhIGF-I/IGFBP-3 used in this study showed the best results in promoting cortical and cancellous bone formation, and appears to be promising therapy for human osteopenias. PMID- 7545537 TI - A strategy to discover circulating angiogenesis inhibitors generated by human tumors. AB - The phenomenon of inhibition of tumor growth by tumor mass has been studied in many experimental animal systems and has been observed in several clinical scenarios. Not until the recent discovery of angiostatin, a circulating angiogenesis inhibitor generated in the presence of a murine Lewis lung tumor, has a satisfactory mechanism been proposed to explain this phenomenon. Thus far, no other animal or human tumors are known to generate angiostatin. In this study, we utilized a mouse corneal neovascularization model to detect circulating inhibitors of angiogenesis generated by three human tumors grown in immunodeficient mice: (a) the PC-3 human prostate carcinoma; (b) the CCL188 human colon carcinoma; and (c) the UBC urinary bladder carcinoma. Mice bearing these three primary tumors demonstrated significant inhibition of angiogenesis in the cornea induced by a pellet containing basic fibroblast growth factor. Corneas of mice bearing s.c. prostate and colon carcinomas showed significant inhibition of vessel length, clock-hours of neovascularization, and vessel density. However, corneas of mice bearing s.c. bladder carcinomas demonstrated significant inhibition of vessel density only. Three colon carcinomas (clone A, CX-1, and MIP101), the MDA-MB-435S breast carcinoma, the MM-AN melanoma, and the JE-3 choriocarcinoma did not significantly inhibit corneal neovascularization. PMID- 7545538 TI - Abnormal retention of intron 9 in CD44 gene transcripts in human gastrointestinal tumors. AB - We have recently identified a new exon of the CD44 gene and demonstrated abnormal retention of a noncoding section, intron 9, in mRNA from bladder carcinomas. To analyze this further, the present study examined CD44 gene expression in cell lines from 14 esophageal, 3 colonic, and 4 breast carcinomas and in fresh samples from 20 colorectal carcinomas and corresponding normal colonic mucosa, using reverse transcriptase followed by the polymerase chain reaction (RT-PCR). This confirmed that there was abnormal assembly of several exons of the gene in cell lines and in tumor tissues from these organs. However, the most striking new finding was that intron 9 was present in RNA from 11 esophageal, 3 colon, and 1 breast carcinoma cell line, respectively. This was confirmed by RNase and DNase digestion analysis. Moreover, it was detected both in nuclear and cytoplasmic mRNA fractions, indicating that abnormal splicing of pre-mRNA occurs in cancer cells. The abnormal retention of intron 9 in CD44 gene transcripts was also demonstrated in tumor tissues from 16 (80%) of 20 patients with colon carcinoma, but there was no correlation with Dukes' stage. The biological significance of these observations is not yet understood. However, it is clear that, as with the abnormal expression pattern of CD44 variant exons, intron 9 retention is a good candidate molecular diagnostic tool for colorectal carcinomas. PMID- 7545539 TI - Nitric oxide production in relation to spontaneous B-cell lymphoma and myositis in SJL mice. AB - SJL mice spontaneously develop B cell lymphomas (historically described as reticulum cell sarcomas) by 12 months of age and inflammatory muscle disease (myositis) by 6 months of age. Tumors originate in mesenteric lymph nodes and in Peyer's patches and resemble human germinal center lymphomas. The growth of reticulum cell sarcomas is completely dependent on cytokine production by normal T cells. The spontaneous myositis, which resembles human idiopathic myositis, is characterized by various abnormalities in skeletal muscle, including infiltration with inflammatory cells consisting primarily of macrophages. The participation of different cytokines in the pathogenesis of the lymphoma and the massive invasion of macrophages into muscle tissues led us to investigate the possible involvement of nitric oxide (NO.), which is known to be synthesized by activated macrophages under inflammatory conditions. Elevated NO. production, measured by urinary nitrate excretion, by SJL mice in comparison with BALB/c control mice was observed as early as 7 weeks of age. Both aging and degree of spontaneous myositis correlated with increased nitric oxide production. Oral administration of N-monomethyl-L-arginine, an inhibitor of nitric oxide synthase (NOS), reduced urinary nitrate excretion and also the severity of myositis. Immunohistochemical analysis revealed the presence of inducible NOS (iNOS) in cells in the spleen, lymph nodes, and skeletal muscle. The iNOS is primarily responsible for the enhanced nitric oxide production. Morphology of cells that stained positive for iNOS was similar to that of macrophages infiltrating into the affected tissues. Chronic production of elevated amounts of nitric oxide by the SJL mice, therefore, provides a useful in vivo model for future studies of cellular damage resulting from endogenously produced NO.in combination with oxygen radicals. PMID- 7545542 TI - Targets for specific therapies in leukemia. PMID- 7545540 TI - Bispecific antibody-mediated target cell-specific costimulation of resting T cells via CD5 and CD28. AB - Induction of T-cell activation requires multiple signals provided by cell surface receptor interactions and/or cytokines. T-cell stimulation via the T-cell receptor/CD3 complex provides an important initial activation event which, when combined with the proper costimulatory signals, results in an activated effector T cell. In this report, we have investigated the effectiveness of epithelial glycoprotein-2- (EGP-2) positive tumor target cells to induce specific T-cell stimulation via CD3, CD5, and CD28 using various combinations of bispecific monoclonal antibodies (BsMab) directed against CD3, CD5, or CD28 on the one hand and the pancarcinoma-associated antigen EGP-2 on the other. Induction of T-cell activation was investigated by assessment of CD69 expression, induction of proliferation, and acquirement of cytolytic potential. EGP-2-specific induction of T-cell activation was observed using combinations of BsMab which simultaneous ligated CD3/CD5, CD3/CD28, or CD3/CD5/CD28 with EGP-2. Activation with CD3-, CD5 , or CD28-based BsMab alone did not result in significant induction of T-cell activation in the presence or absence of EGP-2-positive target cells. Simultaneous ligation via CD5/CD28 resulted in partial T-cell activation, including CD69 up-regulation and increased cytolytic activity. Stimulation via CD3 and CD5 or CD28 could be further increased by the addition of exogenously added recombinant Interleukin 2. In contrast, T-cell activation by simultaneous ligation of CD3/CD5/CD28 could not be further augmented by addition of exogenous interleukin 2, indicating that T-cell activation via the combination of CD3, CD5, and CD28 results in complete T-cell activation. Our results show that rapid and target cell-specific induction of T cells is possible using combinations of BsMab directed against different costimulatory molecules. Simultaneous costimulation via CD3/CD5/CD28 results in the most complete activation of T cells. PMID- 7545543 TI - c-fos is required for malignant progression of skin tumors. AB - The proto-oncogene c-fos is a major nuclear target for signal transduction pathways involved in the regulation of cell growth, differentiation, and transformation. Using the multistep skin carcinogenesis model, we have directly tested the ability of c-fos-deficient mice to develop cancer. Upon treatment with a tumor promoter, c-fos knockout mice carrying a v-H-ras transgene were able to develop benign tumors with similar kinetics and relative incidence as wild-type animals. However, c-fos-deficient papillomas quickly became very dry and hyperkeratinized, taking on an elongated, horny appearance. While wild-type papillomas eventually progressed into malignant tumors, c-fos-deficient tumors failed to undergo malignant conversion. Experiments in which v-H-ras-expressing keratinocytes were grafted onto nude mice suggest that c-fos-deficient cells have an intrinsic defect that hinders tumorigenesis. These results demonstrate that a member of the AP-1 family of transcription factors is required for the development of a malignant tumor. PMID- 7545541 TI - Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin type glycoproteins. AB - E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2 3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression. PMID- 7545546 TI - Contribution of B cell subsets to delayed development of MAIDS in xid mice. AB - C57BL/6 (B6) mice develop a syndrome of progressive lymphoproliferation and immunodeficiency, murine AIDS (MAIDS), when infected with an etiologic replication-defective virus termed BM5def. Induction of MAIDS requires the presence of CD4+ T cells and B cells. B6 mice with altered conventional B cell function and a deficit in CD5+ B cells due to the xid mutation develop disease with a greatly prolonged latency. The association of this mutation with resistance to MAIDS was confirmed in studies of P.xid mice. To test the hypothesis that conventional B cells are required for rapid induction of disease, B6.xid mice were injected with spleen cells from nude mice or were given bone marrow from aged donors. Both sets of recipients developed advanced disease by 10 weeks post infection, suggesting that resistance to MAIDS in xid mutants may be due to effects of B cells other than the CD5+ subset. PMID- 7545545 TI - TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1. AB - Patients with the genetic disorder ataxia telangiectasia (AT) have mutations in the AT mutated (ATM) gene, which is homologous to TEL1 and the checkpoint gene MEC1. A tel1 deletion mutant, unlike a mec1 deletion, is viable and does not exhibit increased sensitivity to DNA-damaging agents. However, increased dosage of TEL1 rescues sensitivity of a mec1 mutant, mec1-1, to DNA-damaging agents and rescues viability of a mec1 disruption. mec1-1 tel1 delta 1 double mutants are synergistically sensitive to DNA-damaging agents, including radiomimetic drugs. These data indicate that TEL1 and MEC1 are functionally related and that functions of the ATM gene are apparently divided between at least two S. cerevisiae homologs. PMID- 7545544 TI - Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. AB - Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD. PMID- 7545547 TI - Production and characterization of the extracellular domain of human CD7 antigen: further evidence that CD7 has a role in T cell signaling. AB - CD7 is a T cell-associated antigen which appears early in ontogeny and persists on circulating T cells. It appears to have a significant role in T cell development and function. The precise mechanism by which this molecule mediates its effect is not known. In this paper, we expressed the extracellular domain of CD7 in the baculovirus system and used this product to study the function CD7 might have in T cell activation. The recombinant protein was found to be structurally similar to the native CD7 and recognized by monoclonal and polyclonal antibodies to CD7. This protein inhibited T cell proliferation induced by anti-CD3/anti-CD7 costimulation. It also inhibited the augmentation effect of anti-CD7 on suboptimal PHA stimulation. However, it did not block T cell proliferation induced by optimal doses of PHA, staphylococcal entertoxin A or B. Interestingly, the recombinant protein inhibited antigenic- and alloantigenic induced T cell proliferation. The latter finding strongly suggests that a ligand for CD7 exists and crosslinking CD7 by this ligand may be responsible for the costimulatory role it plays in T cell activation. PMID- 7545548 TI - A novel T-cell differentiation antigen expressed in immature human thymocytes and neuroglial cells. AB - We report a novel human thymocyte differentiation antigen ICT-1 with a molecular weight of 49 kDa that is noncovalently associated with another 12-kDa protein. The ICT-1 antigen is expressed in 50-70% of total thymocytes, but not in resting or PHA-activated peripheral blood T-cells and bone marrow cells. The thymocytes expressing ICT-1 antigen appear after the 18th week of gestation during fetal development. Since the distribution pattern of the ICT-1 antigen within thymus partly overlaps with that of the CD1 antigens, we investigated whether ICT-1 was one of the CD1 antigen family. However, the failure of anti-ICT-1 antibody to react with mouse L cells transfected with cDNA of CD1a, -b, and -c and the different histologic distribution patterns from that of CD1d strongly suggest that the anti-ICT-1 antibody recognizes an antigen distinct from CD1. Furthermore, ICT-1 is also expressed in human neuroglial cells such as oligodendroglioma, glioblastoma multiforme, Ewing's sarcoma, and cerebellar astrocyte. Hence we believe that the ICT-1 antigen may be a novel thymus-leukemia (TL) antigen or a nonclassical MHC class I antigen. PMID- 7545549 TI - Differences in the recognition of CTL epitopes during primary and secondary responses to herpes simplex virus infection in vivo. AB - The immune response to HSV infection in C57BL/6 mice includes a CTL population that recognizes the virion envelope glycoprotein gB. However, previous studies showed that CTL specific for other viral determinants were also responding to HSV infection. These studies demonstrate that an additional determinant is the HSV immediate-early protein ICP27. During the primary response, both gB- and ICP27 specific CTL were detected in the draining lymph node. In response to reinfection, ICP27-specific CTL were present early in the lymph node, while the appearance of gB-specific CTL activity was delayed. Analysis of the primary amino acid sequence of ICP27 predicted two potential Kb-binding epitopes, one of which sensitized uninfected cells for lysis by HSV-specific CTL. In addition, ICP27 epitope-specific CTL activity was detected in the splenic memory CTL pool. These results show that CTL which recognize different antigens may also exhibit differences in how they respond to HSV reinfection in vivo. PMID- 7545553 TI - Soluble P-selectin is released into the coronary circulation after coronary spasm. AB - BACKGROUND: The glycoprotein P-selectin is an adhesion molecule involved in the property change of leukocytes at the initiation of the inflammatory process. The purpose of the present study was to determine whether acute myocardial ischemia induced by coronary spasm causes an acute inflammatory response in the coronary circulation. METHODS AND RESULTS: We examined plasma soluble P-selectin levels in the coronary sinus and the aortic root simultaneously in 16 patients with coronary spastic angina before and after left coronary artery spasm induced by intracoronary injection of acetylcholine and in 15 patients with stable exertional angina before and after acute myocardial ischemia induced by rapid atrial pacing. Ten control patients with chest pain but normal coronary arteries and no coronary spasm also received intracoronary acetylcholine. Plasma soluble P selectin levels were increased significantly in the coronary sinus (32.8 +/- 3.6 to 52.8 +/- 5.9 ng/mL, P < .001) and in the aortic root (34.6 +/- 3.7 to 41.9 +/- 4.4 ng/mL, P < .05) after the attacks in the coronary spastic angina group but remained unchanged in the stable exertional angina group after the attacks and in the control group after the administration of acetylcholine. Furthermore, the coronary sinus-arterial difference of soluble P-selectin increased significantly after the attacks in the coronary spastic angina group (-1.8 +/- 2.2 to 10.9 +/- 2.7 ng/mL, P < .001). CONCLUSIONS: Our data indicate that soluble P-selectin is released into the coronary circulation after coronary artery spasm. We conclude that coronary artery spasm may induce the leukocyte adhesion in the coronary circulation and may lead to myocardial damage. PMID- 7545551 TI - Products from alkylation of DNA in cells by melphalan: human soft tissue sarcoma cell line RD and Escherichia coli WP2. AB - Alkylation of DNA was studied after treatment with [3H]melphalan (phenylalanine mustard; 1-2 microM) using a human tumour cell line, RD, in culture, or Escherichia coli (WP2 or WP2-uvrA strains) in growth medium. After 6 h at 37 degrees C, treated cells were isolated and re-suspended in fresh growth media. Samples were taken at times up to 48 h for isolation of DNA, and in some cases also RNA and protein (which were found to be alkylated to about the same extent as DNA). Alkylated DNA was analysed as previously described (M.R. Osborne and P.D. Lawley, Chem.-Biol. Interact 89 (1993) 49-60). The four principal products, mono-7-alkylguanine (G-M-OH); mono-3-alkyladenine (A-M-OH); and the cross-linked products G-M-G and A-M-G, were identified in DNA from melphalan treated cells, and quantitatively determined. In each case, alkylation of cellular macromolecules was maximal after about 6 h. In DNA of the human tumour cell line, the relative amounts of adenine products decreased with time, most markedly with A-M-OH to 42% of the 2-h value after 48 h. In DNA of both bacterial strains, A-M OH was virtually undetectable even at early times. Comparisons between the time course of relative decreases in amounts of these alkylpurine products and the corresponding values for alkylated DNA in vitro suggest that the adenine products are subject to removal by repair enzyme action in E. coli of either strain. PMID- 7545550 TI - Effects of cyclosporin A and FK506 on nitric oxide and tetrahydrobiopterin synthesis in bacterial lipopolysaccharide-treated J774 macrophages. AB - We evaluated the effects of the immunosuppressants cyclosporin A (CsA) and FK506 on the synthesis of nitric oxide (NO) induced by bacterial lipopolysaccharide (LPS) in J774 macrophages. CsA and FK506 each inhibited NO production by LPS in a concentration-dependent fashion, but the cytotoxicity was also evident at higher concentrations (100 microM). Neither CsA nor FK506 had any effect on the activity of NO synthase (NOS) that had already been induced. Findings indicated that CsA and FK506 inhibit the induction of NOS, rather than its catalytic activity. CsA and, to a lesser extent, FK506 increased the synthesis of tetrahydrobiopterin (BH4), an essential cofactor of NOS. Thus, inhibition of NO formation by CsA or FK506 is unlikely to associated with a change in BH4 synthesis caused by these agents in LPS-treated J774 macrophages. PMID- 7545552 TI - Increased soluble form of P-selectin in patients with unstable angina. AB - BACKGROUND: P-selectin in platelets and endothelial cells mediates adhesive interaction with leukocytes to form thrombi. The purpose of the present study was to investigate the plasma levels of P-selectin in patients with unstable angina and in those with stable effort angina of different pathophysiologies. METHODS AND RESULTS: Plasma P-selectin levels were determined by a monoclonal antibody based enzyme immunoassay on plasma samples taken from 12 patients with unstable angina, 11 patients with stable effort angina, and 15 healthy volunteers. Patients with unstable angina had angina at rest associated with ECG changes. In patients with unstable angina, plasma P-selectin levels within 1 hour (361 +/- 90 ng/mL) and at 3 hours (282 +/- 56 ng/mL) after angina were significantly (P < .05) higher than those in volunteers (177 +/- 31 ng/mL). Plasma P-selectin levels at 5 hours after attack (242 +/- 46 ng/mL) did not differ from those in volunteers. Although patients with stable effort angina developed angina with ST segment depressions by treadmill exercise, their plasma P-selectin levels did not change (before, 178 +/- 45; immediately after, 186 +/- 36; and 1 hour after the exercise, 179 +/- 34 ng/mL). CONCLUSIONS: Plasma P-selectin levels after angina increased significantly in patients with unstable angina but did not in patients with stable effort angina. These findings may contribute to understanding of the pathophysiology of the acute coronary syndrome of unstable angina. PMID- 7545554 TI - Increased central nervous system monoamine neurotransmitter turnover and its association with sympathetic nervous activity in treated heart failure patients. AB - BACKGROUND: Congestive heart failure is a debilitating disease characterized by impaired cardiac function with accompanying activation of a variety of neural and hormonal counter-regulatory systems. Abnormal activity of the sympathetic nervous system and renin-angiotensin-aldosterone axis and a predisposition to the generation of fatal ventricular arrhythmias are often associated with the development of the disease. Although the underlying cause of sudden death in these patients remains to be unequivocally elucidated, abnormally increased cardiac sympathetic nervous activity may be involved. METHODS AND RESULTS: Twenty two patients with severe congestive heart failure (New York Heart Association functional class III or IV with left ventricular ejection fraction of 18 +/- 1%) and 29 healthy male volunteers participated in this study. By combining direct sampling of internal jugular venous blood via a percutaneously placed catheter with a norepinephrine and epinephrine isotope dilution method for examining neuronal transmitter release, we were able to quantify the release of central nervous system monoamine and indoleamine neurotransmitters and investigate their association with the increased efferent sympathetic outflow that is variably present in treated patients with this condition. Mean cardiac norepinephrine spillover was 145% higher in treated heart failure patients than in healthy subjects (P < .05), with norepinephrine release from the heart in 6 of 22 patients being more than the highest control value. Raised internal jugular venous spillover of epinephrine (26 +/- 12 versus 2 +/- 4 pmol/min, P < .05) and of norepinephrine and its metabolites (2740 +/- 480 versus 875 +/- 338 pmol/min, P < .05), indicative of increased central nervous system turnover of both catecholamines, occurred in cardiac failure and was quantitatively linked to the degree of activation of the cardiac sympathetic nervous outflow, as was the jugular overflow of the principal serotonin metabolite, 5-hydroxyindoleacetic acid. CONCLUSIONS: An association between the degree of activation of central monoaminergic neurons and the level of sympathetic nervous tone in the heart was identified in treated patients with heart failure. Epinephrine neurons in the brain may contribute to the sympathoexcitation that is seen in this condition, with the activation of sympathoexcitatory noradrenergic neurons, most likely those of the forebrain, playing an accessory role. PMID- 7545555 TI - Protection against ischemic injury by nonvasoactive concentrations of nitric oxide synthase inhibitors in the perfused rabbit heart. AB - BACKGROUND: The functional and metabolic effects of inhibitors of nitric oxide (NO) synthase on ischemic hearts have not been investigated. This work was designed to perform such a study in isolated perfused rabbit hearts submitted to low-flow ischemia. METHODS AND RESULTS: After a 30-minute equilibration period, the hearts were submitted to low-flow ischemia for 60 minutes followed by reperfusion for 30 minutes. Functional and metabolic parameters were followed in hearts perfused with or without inhibitors of NO synthase or NO precursors, which were added 15 minutes before ischemia but were absent during reperfusion. Ischemic contracture was delayed and reduced in hearts perfused with 1 mumol/L L N-monomethylarginine (L-NMMA) or 1 mumol/L L-N-arginine methylester, two inhibitors of NO synthase, but not with D-N-monomethylarginine, the inactive enantiomer of L-NMMA. The protection was suppressed by addition to the perfusate containing L-NMMA of 1 mmol/L L-arginine or 0.1 mmol/L sodium nitroprusside but not by addition of 10 mumol/L 8-bromo cGMP, a cGMP analogue. The functional protection by 1 mumol/L L-NMMA was related to a stimulation of glycolysis from exogenous glucose and a preservation of the glycogen stores. This resulted in a better maintenance of high-energy phosphates and a lower acidosis as measured by 31P nuclear magnetic resonance spectroscopy. During reperfusion, functional recovery was more than doubled, and enzyme release was halved in L-NMMA-treated hearts compared with controls. The functional and metabolic protection was maximal at 1 nmol/L to 1 mumol/L L-NMMA, ie, below the vasoactive concentrations of the inhibitor. CONCLUSIONS: Nonvasoactive concentrations of NO synthase inhibitors protect the heart against ischemic damage; this relates to a stimulation of glycolysis from exogenous glucose. PMID- 7545558 TI - Vascular proliferation and angiogenic factors in psoriasis. AB - Psoriasis is a common, chronic skin disorder characterized by hyperproliferation of the epidermis, inflammatory cell accumulation and increased tortuosity and dilatation of dermal papillary blood vessels. Research into the pathogenesis of psoriasis has concentrated mainly on the interplay between inflammatory cells and epidermal proliferation. Central to the proposed pathogenetic pathway are cytokines produced by activated keratinocytes, which are thought to induce both keratinocyte proliferation and lymphocyte migration. Cytokines also mediate upregulation of adhesion molecules on vascular endothelium which in turn permits lymphocyte recruitment. The close spatial relationship between altered microvasculature and epidermis is clearly important in psoriasis. Consequently, understanding the mechanisms underlying vascular changes is fundamental to an elucidation of pathogenetic mechanisms in psoriasis. PMID- 7545557 TI - Effects of premature beats on repolarization of postextrasystolic beats. AB - BACKGROUND: A short-long-short sequence of cycle lengths predisposes to reentrant tachyarrhythmias. There is limited information about the effects of premature ventricular contractions (PVCs) on repolarization of postextrasystolic depolarizations (PEDs). Such information would contribute to understanding the mechanism for facilitating reentry with short-long-short cycle lengths. METHODS AND RESULTS: We introduced PVCs, over a range of coupling intervals and during a range of basic drive cycle lengths (BCLs), and determined PED repolarization. Our results from whole-animal experiments, isolated cell studies, and computer simulations are reported. In the whole-animal experiments, PED refractory periods (RPs) were longer than RPBCL. The greatest difference between RPPED and RPBCL (delta RPmax) occurred after short coupling interval PVCs and was 4.3 +/- 0.8, 4.2 +/- 0.8, and 2.1 +/- 0.5 ms (mean +/- SEM) during drives with short, intermediate, and long BCLs, respectively. The diastolic interval preceding the PED (DIPED) was inversely related to the coupling interval between the basic drive beat and the PVC and directly related to RPPED. PED action potential durations (APDs) of isolated canine myocytes were 9.8 +/- 4.9 ms (mean +/- SEM) longer than APD BCL (n = 19). The DiFrancesco-Noble membrane equations were used in simulations of action potential propagation in a one-dimensional cable, with stimulation protocols duplicating those in the animal experiments. PVCs prolonged APDPED, and APDPED was prolonged more during short than during long BCL drives. There was a direct relation between DIPED and APDPED. Analysis of the membrane currents over the time course of the PVCs and PEDs suggested that the ionic basis for PED repolarization prolongation was the interaction of Ito and Ik. Hyperpolarizing constant-current injections introduced immediately after the spike of isolated myocyte action potentials caused APD prolongation. This observation is consistent with the Ito and Ik interaction causing PED repolarization prolongation. CONCLUSIONS: PED repolarization prolongation could provide sites for unidirectional block to propagation of PVCs after PEDs and could facilitate initiation of reentrant tachyarrhythmias after short-long-short sequences of cycle lengths. PMID- 7545556 TI - Trandolapril decreases prevalence of ventricular ectopic activity in middle-aged SHR. AB - BACKGROUND: Although severe arrhythmias are still a major problem in patients with left ventricular hypertrophy (LVH), the relationship between ventricular remodeling and its regression or prevention, and the prevalence of ventricular premature beats (VPB) or more sustained arrhythmias are still poorly explored in hypertensive heart disease. METHODS AND RESULTS: Holter monitoring was used to quantify supraventricular premature beats and VPB and heart rate (HR) in middle aged spontaneously hypertensive rats (SHR) and Wistar rats treated for 3 months with trandolapril (ACE inhibitor, 0.3 mg/kg per day). Hypertrophy and fibrosis were morphometrically determined. Statistical analysis was performed with the use of simple regression and multivariate data analysis (cluster and correspondence analysis). SHR have higher cardiac mass and fibrosis, more VPB, and a decreased HR. Cluster analysis demonstrated that trandolapril was only effective in SHR. Trandolapril significantly reduced cardiac hypertrophy, fibrosis, and VPB incidence and increased the HR. Simple regression analysis showed that VPB incidence correlated to both hypertrophy and fibrosis. Correspondence analysis evidenced a strong correlation between hypertrophy, fibrosis, and VPB, but only for severe hypertrophy, and the correlation disappeared for moderate hypertrophy. CONCLUSIONS: After trandolapril treatment, the regression of VPB incidence not only is linked to hypertrophy and fibrosis, but additional causal factors also are involved including the myocardial phenotype and new calcium metabolism. Our model of Holter monitoring in conscious middle-aged SHR and multivariate data analysis might be useful in correlating myocardial structural modifications and ectopic activity. PMID- 7545559 TI - Altered lipid peroxidation/glutathione ratio in experimental extrahepatic cholestasis. AB - 1. Lipid peroxidation can occur in the presence of a cellular antioxidant-oxidant imbalance, but the role of lipid peroxides in cholestasis is not well understood. 2. This study was undertaken in order to: (i) evaluate the behaviour of a product of lipid peroxidation (thiobarbituric acid-reactive species), and of an important antioxidant tripeptide, reduced glutathione, in the course of experimental extrahepatic cholestasis; and (ii) ascertain whether there was a link between this aspect and the alterations in liver morphology. 3. Forty-five male Sprague Dawley rats (250-300 g) were double bile duct ligated and followed from 1 to 28 days. At the end of each experimental period, blood and liver samples were collected for thiobarbituric acid-reactive species and glutathione determinations. 4. Bile duct ligated rats showed a marked increase in liver weight which was related to cholestasis duration and to some anatomical alterations such as bile duct proliferation and dilation and liver fibrosis (periportal, perivenular, perineoductular and parenchymal). 5. An increase in serum lipid peroxidation was also observed but this was not linked to hepatic thiobarbituric acid-reactive species. Erythrocyte and hepatic glutathione decreased in relation to cholestasis duration. Serum lipid peroxides and erythrocyte glutathione were correlated with liver cell necrosis. 6. In conclusion, experimental extrahepatic cholestasis determines bile duct proliferation and fibrosis, the degree of which is directly related to the duration of cholestasis itself and to liver cell necrotic phenomena.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545560 TI - Neuropharmacology of nicotine: effects on the autoregulation of acetylcholine release by substance P and methionine enkephalin in rodent cerebral slices and toxicological implications. AB - 1. The neuronal release of acetylcholine (ACh) and its autoregulation by neuromodulators, substance P (SP) and methionine enkephalin (MEK), have been studied using superfused rodent cerebral slices. Nicotine exerts significant effects on autoregulation of ACh release, which may have toxicological implications. 2. Positive and negative feedback systems have been postulated for autoregulation of ACh release. The components of the positive feedback system include a muscarinic (Ms) receptor, SP, and activation of Ca2+ influx. Low levels of ACh in the biophase of the cholinergic synaptic gap may trigger the positive feedback system, and high levels of ACh may trigger the negative feedback system. 3. There are also neuronal pathways for direct reciprocal regulations of SP and MEK. 4. Low concentrations of nicotine triggers the release of ACh followed by MEK and SP. Release of SP causes neurogenic inflammation. 5. Nicotine and its metabolite, cotinine, activate platelet activating factor (PAF)-hydrolase and thereby enhance the turnover rate of PAF. This effect may contribute to tobacco induced arterial thrombosis in peripheral and central nervous systems. PMID- 7545563 TI - Longitudinal studies of platelet cyclic AMP during healthy pregnancy and pregnancies at risk of pre-eclampsia. AB - 1. Platelet behaviour in vitro in relation to cyclic AMP was studied longitudinally during pregnancy and in the same women when they were not pregnant. Subjects comprised a group of healthy primigravidae and a group of women deemed at risk of pre-eclampsia, on the basis of a previous history of the condition. 2. There was a progressive decline during pregnancy in sensitivity of platelets to inhibition of the arachidonic acid-induced release reaction by agents which act via cyclic AMP. This effect was maximum at 36 weeks' gestation. 3. Basal platelet cyclic AMP levels, and those in the presence of a phosphodiesterase inhibitor, did not change throughout the period of the study. 4. By contrast, platelet cyclic AMP accumulation in response to a variety of adenylate cyclase stimulators was reduced from early pregnancy, throughout the gestational period, compared with post-natally. This effect was noted when platelets were incubated with prostaglandins acting via different surface receptors or with forskolin and was most marked on co-incubation with a phosphodiesterase inhibitor. 5. Compared with healthy women, platelets from women with a previous history of pre-eclampsia tended to accumulate less cyclic AMP in response to adenylate cyclase stimulators. This was the case both during pregnancy and post-natally. Further investigation of adenylate cyclase activity in platelets in relation to pre-eclampsia is required. PMID- 7545562 TI - Lipopolysaccharide induces upregulation of neutral endopeptidase 24.11 on human neutrophils: involvement of the CD14 receptor. AB - 1. As lipopolysaccharide is a major stimulator of neutrophil responses during Gram-negative bacterial infections, we studied its effect on the membrane expression of neutral endopeptidase 24.11/CD10 on neutrophils in a model of endotoxaemia in vitro. Lipopolysaccharide added to human whole-blood induced a marked and sustained CD10/neutral endopeptidase upregulation that was already detectable at 0.1 ng/ml and was maximal at a lipopolysaccharide concentration of 10 ng/ml. 2. We observed that neither tumour necrosis factor-alpha nor any newly synthesized protein was involved in the upregulation observed after 1 h incubation with 10 ng/ml lipopolysaccharide. 3. We further studied whether the lipopolysaccharide-induced CD10/neutral endopeptidase upregulation was mediated by lipopolysaccharide binding to the neutrophil CD14 receptor. Incubation of whole blood with an anti-CD14 monoclonal antibody before the addition of 0.1 ng/ml or 0.5 ng/ml lipopolysaccharide resulted in complete inhibition of CD10/neutral endopeptidase upregulation. In contrast, at a lipopolysaccharide concentration of 10 ng/ml, the anti-CD14 monoclonal antibody had an incomplete blocking effect. 4. The differential requirement for the CD14 receptor, depending on the lipopolysaccharide dose, was confirmed by the study of a patient suffering from paroxysmal nocturnal haemoglobinuria (in whom a complete defect in neutrophil CD14 expression was previously documented). 5. We finally confirmed these results using purified neutrophils, demonstrating that lipopolysaccharide induced CD10/neutral endopeptidase upregulation depends on direct interaction with neutrophil CD14. PMID- 7545564 TI - Use of neodymium yttrium aluminum garnet laser in long-term palliation of airway obstruction. AB - Palliation of acute airway obstruction using the neodymium yttrium aluminum garnet (Nd-YAG) laser was studied in 54 patients who presented over a 42-month period to the Yale cardiothoracic surgery service. Thirty-seven patients had bronchogenic carcinoma; 27 had stage IIIB or IV disease. Nine patients had endobronchial metastases from a primary nonbronchogenic carcinoma. Eight patients had benign disease. A total of 109 Nd-YAG laser tumor ablations were performed. In addition, 32 patients underwent postoperative brachytherapy. Median survival for all patients was 12 months. Patients with bronchogenic carcinoma had a median survival of five months. Fifteen of 20 patients (75%) alive at the time of follow up reported continued palliation as shown by an improved postoperative Karnofsky score. There was no survival benefit from Nd-YAG laser ablation of endobronchial bronchogenic carcinoma; however, the Nd-YAG laser provided good to excellent palliation in the majority of patients on long-term follow-up. PMID- 7545561 TI - Nitric oxide: role in neurotoxicity. AB - 1. Nitric oxide (NO) is a novel neuronal messenger molecule which interacts with surrounding neurones, not by synaptic transmission but by diffusion between cells. 2. NO is produced following stimulation of the enzyme, NO synthase (NOS). After synthesis, NO exerts its biological actions by diffusion to the site of action. Therefore, the way to regulate the physiological actions of NO is to regulate NOS. 3. NOS is activated by the influx of calcium from glutamate activated N-methyl-D-aspartate receptors. Overactivation of these receptors leads to overproduction of NO and neuronal cell death. 4. NOS can be regulated at the catalytic site, at the flavo-proteins, at the calmodulin site and by phosphorylation. 5. In excess, NO is toxic to neurones. This toxicity is mediated largely by an interaction with the superoxide anion, presumably through the generation of the oxidant, peroxynitrite. 6. NO or peroxynitrite-mediated neuronal injury involves the activation of the nuclear protein, poly(ADP ribose)synthetase. PMID- 7545566 TI - Interaction between rose bengal and different protein components. AB - Bindings of rose bengal to several proteins were determined by Sephadex G-75 chromatography. Their respective blocking effect against dye uptake was demonstrated in an assay using a rabbit corneal epithelial cell layer. The total binding capacity of nonmucin proteins was measured using fluorometry and Scatchard analysis. The results showed that albumin, lactoferrin, transferrin, and lysozyme could--but serum prealbumin, IgA, carboxymethyl cellulose (CMC), and Sepharose 4B-purified porcine stomach mucin (PSM) could not--bind rose bengal. Lysozyme formed precipitates with rose bengal. Sufficient concentrations of albumin, lactoferrin, transferrin, or lysozyme premixed with rose bengal could block dye uptake by cells, but IgA and serum prealbumin could not. Premixed PSM was not as effective as precoated PSM in blocking dye uptake. The dissociation constant (Kd) was 1.2 x 10(-7) M, 3.6 x 10(-7) M, 3.9 x 10(-7) M, and 1.6 x 10( 6) M for albumin, transferrin, lactoferrin, and lysozyme, respectively. Based on these values, the total maximal binding capacity of nonmucin proteins in normal 7 microliters tears was extrapolated to be 0.249 micrograms rose bengal, which is too small to explain the negative staining of rose bengal on the normal ocular surface. Rose bengal, but not fluorescein, could interact with carbohydrate containing Sephadex, CMC, and PSM to slow down its elution via Sephadex column chromatography. Therefore, the normal negative staining to rose bengal might be caused by the blocking effect of preocular mucus tear layer, which serves as a diffusion barrier. Rose bengal remains a unique dye for detecting the protective function of the preocular mucus tear. PMID- 7545565 TI - Expression of genes coding growth factors and growth factor receptors in differentiated and dedifferentiated human corneal endothelial cells. AB - Growth and differentiation of human corneal endothelial cells are shown to be influenced by growth factors and by the extracellular matrix. Under standard culture conditions, these cells tend to dedifferentiate to a fibroblast-like phenotype. The aim of this study was to examine the expression of genes encoding growth factors and growth factor receptors in differentiated and dedifferentiated human corneal endothelial cells and in keratocytes (stromal fibroblasts) by means of Northern blot analysis. Expression of genes coding basic fibroblast growth factor (FGF), FGF receptor-1 (flg-type), epidermal growth factor receptor, and transforming growth factor-beta 1 in keratocytes and human corneal endothelial cells could be confirmed. In contrast, significant expression of the gene encoding acidic FGF was not detected. However, expression of the genes encoding vascular endothelial growth factor (VEGF) and the high-affinity tyrosine kinase receptor for VEGF (flt-1) in human corneal endothelial cells could be demonstrated for the first time. Moreover, exogenous basic FGF seemed to have a positive influence on the VEGF gene expression level. The flt-1 gene was expressed in dedifferentiated, but not in differentiated human corneal endothelial cells. The results indicate a possible role of VEGF in the differentiation of human corneal endothelial cells. PMID- 7545568 TI - Palliative surgery for thoracic malignancies. PMID- 7545567 TI - SV40-immortalized and primary cultured human retinal pigment epithelial cells share similar patterns of cytokine-receptor expression and cytokine responsiveness. AB - Retinal pigment epithelial (RPE) cells produce and respond to a variety of cytokines; however, molecular and biochemical studies are restricted by the limited access to large numbers of pure cells and the variability associated with different donor sources. Despite success in establishing primary human RPE (HRPE) cell cultures, the inability to sustain consistent proliferation rates and morphology over several passages remains a concern. This problem was approached by using an immortalized line of simian virus (SV)40 transformed fetal HRPE cells (SVRPE). Cytokine production, receptor expression and responsiveness in the SVRPE cell line was analyzed to determine the usefulness of this model for studying HRPE-cytokine interactions. Using reverse transcriptase polymerase chain reaction (RT-PCR), HRPE and SVRPE cells demonstrated an identical pattern of interleukin-1 receptor (IL-1R), IL-2R (alpha sub-unit), IL-6R, interferon (IFN)-gamma R and tumor necrosis factor-alpha (TNF)R p55 expression. No amplification products for TNFR p75 or granulocyte/macrophage colony stimulating factor (GM-CSF)R were demonstrated in either population. IFN-gamma stimulation induced surface human leukocyte antigen (HLA)-DR in both SVRPE and HRPE, while TNF treatment induced surface expression of intercellular adhesion molecule (ICAM)-1 on SVRPE and upregulated ICAM from basal levels on HRPE. Both cell types showed amplification products for interleukin (IL)-1 beta, IL-6 and transforming growth factor (TGF) beta 1 using RT-PCR. The bioassays demonstrated that both populations of unstimulated cells constitutively secrete very low levels of TGF-beta and no IL 6.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545569 TI - Surgical palliation of head and neck cancer. AB - Palliative care in head and neck cancer has not been studied systematically. Patients with incurable head and neck tumors may live months and even years. Ideal palliation should enable them to engage in a normal life before death ensues. It is likely that our improving ability to treat these tumors without achieving cures will cause people to live longer with their cancer. Hence, the need for palliation will probably increase. Also, treatments that cure patients produce conditions that require palliation. Achievement of the best possible function is the major consideration in dealing with head and neck tumors. Difficulty with speech, swallowing, oral hygiene, and laodorous tumors are all common. Depression too should be addressed in a comprehensive fashion by the "head and neck team". The surgeon, radiotherapist, and medical oncologist will need help from dentists, prosthodontists, dental hygienists, psychiatrists, physiatrists, occupational and physical therapists, visiting nurses, nutritionists, and social workers. Palliative care in the hospital is the least desirable, although often unavoidable. Proper hospice support will benefit patients and their families. PMID- 7545570 TI - Management of extremity metastatic bone cancer. AB - It is important to have an aggressive and appropriate treatment program for patients with metastatic cancer to bone. Metastasis to bone should no longer be considered the beginning of the end. It should be an invitation for appropriate orthopedic oncologic involvement and management. Impending pathologic fractures of the long bones can be quickly and relatively easily managed with intramedullary rod fixation. Patients with adenocarcinoma should be monitored periodically to identify the focus of metastatic lesions early. Patients with large lesions previously considered disastrous can be managed successfully using modular oncology systems. Patients with metastatic cancer to bone deserve evaluation and management by orthopaedic oncologists familiar with their treatment. PMID- 7545571 TI - [Differentiated surgical therapy of esophageal carcinoma]. AB - Between September 1985 and December 1993 280 patients suffering from esophageal cancer underwent treatment at the department of general and abdominal surgery at the University of Mainz. Of the 167 patients operated, 152 patients had an esophageal resection performed in 113 cases as abdominothoracic resection and in 39 cases via the transmediastinal approach. 104 patients were curative resected (R0). Recurrence-free and total survival were correlated to the extent of the mediastinal lymph node dissection supposing comparable operative stress. The mean recurrence-free and the mean total survival after abdominothoracic resection with 2-field lymph node dissection were 42.4 months resp. 47.3 vs. 18.9 months resp. 25.2 months after transmediastinal resection (p = 0.015 and p = 0.035). We suggest a differentiated surgical approach concerning abdominothoracic resection with 2-field lymph node dissection for limited tumor size (pT1-3 pN0-1 M0), if the operative risk is tolerable. The transmediastinal resection appears to be only enough radical in cases with early tumor stages (UICC 0-I) and also is advantageous for risk patients with simultaneously more advanced tumor (palliative resection), because of the lower operative stress. PMID- 7545572 TI - [Janeway laparoscopic gastrostomy]. AB - The percutaneous endoscopic gastrostomy is the procedure of first choice for the creation of a gastrostomy. If that is not possible a laparoscopic access should be preferred to a laparotomy. In tumor patients there seem to be some advantages of the laparoscopic Janeway gastrostomy compared to Kader-Stamm fistulas. We describe the technique and discuss the results of 5 patients without complications referring to current publications. In conclusion the Janeway gastrostomy is a good palliation because it is an effective, safe and simple method with easy handling, and little requirement for nursing care. PMID- 7545573 TI - [Sudden graying or head hair becoming white]. PMID- 7545574 TI - A role for mesenchyme-derived tachykinins in tooth and mammary gland morphogenesis. AB - Tachykinin peptides such as substance P (SP) function as neurotransmitters and neuromodulators in the mammalian central and peripheral nervous systems. Here, we provide evidence that they may also play an important role in the morphogenesis of some nonneural organs where epithelial-mesenchymal interactions are involved. We show the following. (1) mRNA encoding tachykinin precursor proteins is expressed transiently in condensing mesenchyme during the development of mouse tooth germ, mammary gland, limb bud, external auditory meatus and genital tubercle. (2) In developing tooth germ and mammary gland; mRNA encoding the neutral endopeptidase (NEP) that degrades secreted tachykinins is spatially and temporally co-expressed with tachykinin precursor mRNA. (3) SP and the mRNA encoding SP receptors are also expressed in the developing tooth germ. (4) Tooth development in explant cultures is blocked both by tachykinin-precursor-specific antisense oligonucleotide and by an SP receptor antagonist: in both cases the block is relieved by exogenous SP. Together, these findings suggest a surprising new role for tachykinins in tooth and mammary gland morphogenesis, and possibly also in limb, ear and external genitalia morphogenesis. PMID- 7545575 TI - The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes. AB - Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5 trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 microM ryanodine and 4-8 microM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 microM. Prior injection of either 50 microM ruthenium red, 50 microM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10-20 mM caffeine or 200 microM ryanodine, but prior injection of 50 microM ruthenium red or procaine completely inhibited the effect of 10-20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 microM concentration of ruthenium red or procaine only partially blocked the effect of 200 microM ryanodine, but 200 microM ruthenium red or procaine completely blocked the effect of 200 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545576 TI - Reduced contractile effect of endothelin-1 and noradrenalin in human umbilical artery from pregnancies with abnormal umbilical artery flow velocity waveforms. AB - This study on the human umbilical artery was undertaken in order to elucidate possible correlations between changes in response to vasoactive substances in vitro and abnormal umbilical artery flow velocity waveforms in vivo associated with preeclampsia and intrauterine growth retardation. The vascular reactivity to endothelin-1, noradrenalin, serotonin, the thromboxane A2 analogue U46619, substance P and prostacyclin was determined in umbilical artery segments from 13 normal pregnancies and 29 pregnancies complicated with preeclampsia and/or intrauterine growth retardation with normal or abnormal umbilical flow velocity waveforms. The contractile effect in vitro of endothelin-1 and noradrenalin was reduced in segments from pregnancies complicated by abnormal umbilical flow velocity waveforms in vivo. No differences were detected in the contractile effect of serotonin and U46619, or in the relaxatory effect of substance P and prostacyclin. In conclusion, endothelin-1- and noradrenalin-related mechanisms could be involved in the abnormal umbilical flow velocity waveforms associated with preeclampsia and intrauterine growth retardation. PMID- 7545577 TI - Treatment of patients with Ullrich-Turner syndrome with conventional doses of growth hormone and the combination with testosterone or oxandrolone: effect on growth, IGF-I and IGFBP-3 concentrations. AB - Thirty-nine girls with Ullrich-Turner syndrome (UTS) (median age 9.5 years) were treated with growth hormone (GH) with either 12 or 18 IU/m2 per week for 12 months followed by combination therapy with either oxandrolone (Ox) (0.0625 mg/kg/day po) or low-dose testosterone (T) (5 mg in every 2 weeks). Growth velocity improved significantly after 12 IU/m2 per week (6.4 +/- 1.7 cm/year vs 4.0 +/- 1.3 cm/year, x +/- SD, P < 0.001) and 18 IU/m2 per week of GH (6.5 +/- 1.3 cm/year vs 4.5 +/- 1.4 cm/year, P < 0.001). Ox, but not T was effective in maintaining growth velocity during the 2nd year of therapy (6.9 +/- 1.3 vs 5.3 +/ 1.5 cm/year). Basal insulin-like growth factor-I (IGF-I) concentrations were in the lower normal range and increased significantly in patients treated with 18 IU/m2 per week (357 +/- 180 ng/ml vs 160 +/- 84 ng/ml) and 12 IU/m2 per week (273 +/- 121 ng/ml vs 140 +/- 77 ng/ml). IGF-I concentrations increased further after addition of Ox (533 +/- 124 ng/ml, P < 0.001) or T (458 +/- 158, P < 0.05). IGFBP 3 concentrations were in the upper normal range before therapy and increased only moderately in both GH dosage groups. However, IGF binding protein-3 (IGFBP-3) concentrations were not affected by additional Ox or T treatment. CONCLUSIONS: 1. Conventional GH doses are effective in increasing growth velocity in UTS, especially, when combined with Ox.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545580 TI - Functional study on vasodilator effects of prostaglandin E2 in the newborn pig cerebral circulation. AB - Cerebral vascular reactivity to prostaglandin E2 was investigated in newborn pigs using closed cranial windows. Exogenous prostaglandin E2 is a dilator of pial arterioles that elevates cyclic AMP in cortical cerebrospinal fluid. Pial arterioles are less sensitive to prostaglandin E2 than to the prostacyclin receptor agonist iloprost, but their maximal responses to the dilator prostanoids are similar. The cerebrovascular effects of prostaglandin E2 and iloprost are not additive. Pretreatment with either iloprost or prostaglandin E2 decreases pial arteriolar responsiveness to iloprost without affecting responses to isoproterenol. The homologous desensitization of pial arterioles suggests that auto- and cross-tachyphylaxis in vascular effects of iloprost and prostaglandin E2 occur at the receptor level. Indomethacin, which selectively inhibits prostacyclin receptor-mediated responses in cerebral vascular smooth muscle, greatly reduces the vascular responses to prostaglandin E2. These results suggest that vasodilator effects of prostaglandin E2 in the newborn cerebral circulation are mediated via prostacyclin receptors coupled to adenylyl cyclase. PMID- 7545581 TI - Functional aspects of developmental toxicity of polyhalogenated aromatic hydrocarbons in experimental animals and human infants. AB - A scientific evaluation was made of functional aspects of developmental toxicity of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in experimental animals and in human infants. Persistent neurobehavioral, reproductive and endocrine alterations were observed in experimental animals, following in utero and lactational exposure to PCBs, PCDDs and PCDFs. The lowest observable adverse effect levels (LOAELs) for developmental neurobehavioral and reproduction endpoints, based on body burden of TCDD-toxic equivalents (TEQs) in animals, are within the range of current background human body burdens. Relatively subtle adverse effects on neurobehavioral development and thyroid hormone alterations have also been observed in infants and children exposed to background levels. Exclusive use of the toxic equivalency factor (TEF) approach may underestimate the risk of neurodevelopmental effects, because both Ah receptor dependent and independent mechanisms may be involved in these effects. The use of marker congeners and/or bioassays based on Ah receptor mediated mechanisms are rapid, low cost pre screening alternatives for expensive and time consuming gas chromatographic-mass spectrometric analysis. PMID- 7545579 TI - Bone mineralisation in type 1 glycogen storage disease. AB - Radial bone mineral content (BMC) was measured using single photon absorptiometry in 11 prepubertal children, aged 3.4-12.6 years, with glycogen storage disease type 1 (GSD-1), 2 of whom were receiving granulocyte colony stimulating factor (G CSF) therapy for chronic neutropenia. Patients were short (median height SD score -1.35, range -3.74 to -0.27), and had reduced BMC Z scores (median 1.79, range 6.35 to +0.27) and radial bone width Z scores (median -0.72, range -2.00 to +0.68). Those receiving G-CSF did not differ significantly from the rest of the group. Generally dietary calcium intake was low and urinary calcium excretion increased. Urinary lactate excretion was high but did not correlate with BMC Z scores. Factors regulating bone metabolism (parathyroid hormone and 25-hydroxy vitamin D concentrations) and markers of bone formation (osteocalcin and skeletal alkaline phosphatase) were not increased implying that there was no compensation for increased bone resorption. CONCLUSION: Patients with GSD-1 may be at increased risk of fracture in later life and require close attention to metabolic control and calcium balance. PMID- 7545582 TI - A NIH/3T3 cell line stably expressing human cytochrome P450-3A4 used in combination with a lacZ' shuttle vector to study mutagenicity. AB - An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ' gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B1 were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B1 increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B1. The system described in this paper opens the possibility to study the capacity of single human cytochrome P450s to activate xenobiotics into mutagenic metabolites. Since activation, phase II metabolism, DNA repair and an endpoint for mutations are all present in one cell, this system will be useful in screening as well as in mechanistic studies. PMID- 7545578 TI - Phenotype of the Williams-Beuren syndrome associated with hemizygosity at the elastin locus. AB - To correlate presence or absence of a 7q11 microdeletion with the clinical picture of the Williams-Beuren syndrome (WBS), we investigated 29 patients with a clinical diagnosis of WBS or WBS-like features, aged 1-30 years, using molecular analysis and/or fluorescent in situ hybridization (FISH). Deletions at 7q11 were found in 75% of the patients (22 out of 29). Nine deletions occurred on a paternal, and ten on a maternal chromosome; three deletions were demonstrated by FISH only, and parental origin could thus not be determined. All deletion patients aged between 2 years and puberty displayed a distinct pattern of facial features (including periorbital fullness, short nose with flat bridge, wide mouth, and full lips and cheeks), the characteristic outgoing social behaviour, as well as moderate growth and mental retardation. Two-thirds (15 out of 22) had a cardiovascular malformation, but only one third (7 of 22) had supravalvular aortic stenosis (SVAS). A stellate iris pattern was also present in one-third of the patients only. In the four adult patients with 7q11 deletions, there was prominence of the lower lip whereas fullness of cheeks and periorbital tissue was not seen. CONCLUSION: This study confirms that WBS has a unique clinical picture which can be diagnosed clinically, but also shows that the relative frequency of individual features may have been overemphasized in the past, and that a minority of patients may exist who are clinically indistinguishable from WBS but who appear to have no deletion at 7q11. PMID- 7545583 TI - Calcitonin gene-related peptide, substance P and nitric oxide are involved in cutaneous inflammation following ultraviolet irradiation. AB - Evidence from our previous work suggests that neurogenic mediators contribute to the inflammation following ultraviolet (UV) irradiation of the skin. We have investigated whether calcitonin gene-related peptide (CGRP), substance P and nitric oxide (NO) participate in the cutaneous inflammatory reaction of the rat hind paw and ear to UV irradiation. Skin blood flow was measured by laser Doppler technique. Oedema was quantified using a spring loaded micrometer to measure ear thickness. UV irradiation of the rat skin lead to a long lasting increase in skin blood flow. This increase was dose dependently attenuated by the CGRP receptor antagonist CGRP-(8-37) (0.15 nmol in 25 microliters to 6.0 nmol in 25 microliters, s.c.) up to 51% with a maximum of effectiveness at 24 h post irradiation. The inhibitor of NO synthase NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 nmol in 25 microliters, s.c.) attenuated skin blood flow by 38%. Concurrent injections s.c. of CGRP-(8-37) (1.5 nmol in 12.5 microliters) and L-NAME (25 nmol in 12.5 microliters) demonstrated an augmentive effect in attenuating skin blood flow. The tachykinin NK1 receptor antagonist CP 96,345 (6.0 nmol in 25 microliters, s.c.) attenuated skin blood flow by 27%. NG Nitro-D-arginine methyl ester hydrochloride (D-NAME) and CP-96.344 showed no effects on skin blood flow after UV irradiation. CGRP-(8-37) (0.6 nmol in 10 microliters) i.d. and L-NAME (10 nmol in 10 microliters) i.d. had no effect of oedema formation after UV irradiation. Furthermore, post UV irradiation enhanced CGRP- and NO synthase-immunoreactivity in nerve fibres in the exposed skin area were visible. Taken these findings together we suggest the involvement of the neuropeptides CGRP and substance P and of neuronal NO on the vasodilatory component of the UV-induced inflammatory reaction of the rat skin. CGRP contributing to UV-induced vasodilation acts in an endothelial NO-independent manner. PMID- 7545584 TI - Neurochemical heterogeneity of the primate nucleus accumbens. AB - In order to further investigate the neurochemical anatomy of the primate nucleus accumbens (NAC), the distributions of the neuropeptides leucine-enkephalin (Leu ENK), neurotensin (NT), and substance P (SP) and of haloperidol-induced c-fos expression were investigated in the macaque monkey using immunohistochemical methods. To define the boundaries of the NAC, dopamine (DA) and tyrosine hydroxylase (TH) immunohistochemistry was performed. In addition, to formulate the distinction between subdivisions of the nucleus accumbens, immunohistochemistry for calbindin-D28 (CBD) and SP was employed. In general, the medial part of NAC, which consisted of small to medium-sized cells, was low for CBD immunoreactivity and moderate to high for SP immunoreactivities, while the dorsolateral part, which was composed of small cells, showed the opposite pattern of immunostaining for CBD and SP. Many Leu-ENK-immunoreactive perikarya were observed in the dorsal NAC at its middle and caudal levels. There were moderate densities of Leu-ENK-positive fibers throughout the medial part of the NAC. At the dorsolateral margin of the NAC, Leu-ENK-positive fibers formed patches. Most NT-positive perikarya were found in the dorsolateral subdivision. SP-positive perikarya were scarce in the NAC. Dense distribution of NT- and SP-containing fibers or puncta were observed in the mediodorsal part (medial subdivision), where a dense field of DA-immunoreactive fibers was observed. The ventral part (ventral subdivision) contained moderate numbers of NT- and SP-immunoreactive fibers. Haloperidol-induced c-fos expression was very extensive in the medial half of NAC, particularly in the mediodorsal region, which overlapped with the DA and peptide-rich region. The present study indicates that the NAC of the primate can be subdivided into at least three subterritories, the dorsolateral, medial and ventral subdivision, by neuropeptide histochemistry as well as by the response of its constituent neurons to haloperidol. PMID- 7545587 TI - B-50 (GAP-43) immunoreactivity is rarely detected within intact catecholaminergic and serotonergic axons innervating the brain and spinal cord of the adult rat, but is associated with these axons following lesion. AB - The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B 50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts. PMID- 7545586 TI - Transection of rat fimbria-fornix induces lasting expression of c-Jun protein in axotomized septal neurons immunonegative for choline acetyltransferase and nitric oxide synthase. AB - The fimbria-fornix (FF) fiber tract was unilaterally transected in adult rats by a stereotaxic knife cut. In the axotomized neurons of the medial septal nucleus (MS) and ventral diagonal band of Broca (VDB), the expression of Jun, Fos, Krox, CREB transcription factors, choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) were studied by immunocytochemistry. In addition, NADPH-diaphorase (NDP) and acetylcholine esterase (AChE) were visualized by activity assays. For retrograde tracing of axotomized neurons, either HRP-coupled gold was injected in the entorhinal cortex prior to axotomy, or Fast Blue was injected into the transection site subsequently to FF transection. Following FF transection c-Jun and in a less extend JunD were expressed in axotomized MS and VDB neurons. Expression levels rose at 24 h, but not at 18 h, postaxotomy, reached their maximal levels between 5 and 7 days, and then gradually declined. Up to 100 days, c-Jun was still present in a substantial number of septal neurons. JunB, Krox-20, Krox-24, c-Fos, and pan-Fos immunoreactivities (IR) were not detectable in axotomized septal neurons and CREB-IR did not change compared to the intact contralateral side. ChAT-IR dramatically declined over 36 h, and furthermore AChE reactivity had substantially fallen after 5 days. The number and intensity of cytoplasmic neuronal NOS-IR and NDP which generated congruent temporospatial patterns gradually fell between 3 and 5 days postaxotomy. The surviving neurons labeled by NOS and NDP showed a high coexpression of c-Jun, whereas c-Jun was almost completely absent in neurons stained for ChAT and AChE. Finally, ChAT-IR and NDP reaction labeled different subpopulations. Our findings demonstrate a lasting expression of the c-Jun transcription factor in axotomized MS and VDB neurons that might indicate the regenerative propensity of damaged neurons. The decrease of NOS and NDP in MS and VDB neurons demonstrates that neuronal populations respond to axotomy with an individual regulation of NOS expression. PMID- 7545585 TI - Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas. AB - The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas. NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed. Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity. PMID- 7545588 TI - Isolation of Coxiella burnetii and of an unknown rickettsial organism from Ixodes ricinus ticks collected in Austria. AB - Two strains of Coxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time from Ixodes ricinus ticks collected in the Alpine region of Tirol, Austria. The C. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes with C. burnetii and R. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms. PMID- 7545591 TI - Possibility of identification of hematopoietic stem cells using a conventional blood cell counter. PMID- 7545590 TI - Constitutive and selective expression of basic fibroblast growth factor in human leukaemia cell lines. AB - We have previously shown that basic fibroblast growth factor (bFGF) stimulates megakaryocytopoiesis and granulopoiesis in vitro and that normal haematopoietic cells and several leukaemic cell lines express FGF receptors. In this paper, we demonstrate by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) that bFGF mRNA is expressed in two leukaemia cell lines with megakaryocytic features (Meg-01 and K562), in two lymphocytic cell lines (Hut 78 and CA) and in normal human peripheral blood mononuclear cells. In addition, the conditioned media of Meg-01, but not Dami, contained a potent fibroblast-stimulating activity which could be neutralized by bFGF antibodies. Furthermore, bFGF antibody significantly inhibited the autocrine growth of Meg-01 cells in vitro. However, we could not detect cell-associated 18 kDa bFGF or HMW bFGF by immunofluorescence, immunoprecipitation or Western blotting. These data indicate that bFGF is expressed by certain haematopoietic cells and support further a role of this FGF prototype in haematopoiesis. PMID- 7545592 TI - A severe case of ovarian hyperstimulation syndrome despite the prophylactic administration of intravenous albumin. AB - OBJECTIVE: To evaluate the role of the recently suggested IV administration of human albumin solution as an effective preventive measure of severe ovarian hyperstimulation in high-risk patients. CASE: Presented here is, to the best of our knowledge, the first case of early severe ovarian hyperstimulation necessitating early transabdominal aspiration of ascites and intensive fluid and colloid (dextran and albumin) replacement, which developed despite administration of IV human albumin solution in an attempt to prevent severe ovarian hyperstimulation. CONCLUSIONS: Because IV albumin does not prevent severe ovarian hyperstimulation in absolute terms, its use in high-risk patients should not lead one astray. Further research should be directed at investigating the fundamental cause of ovarian hyperstimulation syndrome and developing a reliable predictive test for this complication. PMID- 7545593 TI - Appearance of lymphocytes positive for E-selectin ligands during inflammatory reactions in vivo. PMID- 7545589 TI - Mixed haematopoietic colony formation via immature blast cell clusters on foetal mesenchymal cell layers distinguishes stem cells from peripheral blood, cord blood, bone marrow and blood stem cells mobilized by granulocyte-macrophage colony-stimulating factor. AB - Multilineage colony formation was evaluated from healthy donors' bone marrow (BM), peripheral blood (PB) and cord blood (CB) and compared with blood stem cell (BSC) harvests of sarcoma patients mobilized with granulocyte-macrophage colony stimulating factor (GM-CSF). The test was a modified CFU-blast assay performed with and without an irradiated foetal mesenchymal cell layer (HFFF). These non transformed mesenchymal cells served as a good source of haematopoietically active stroma cells in that cytokine expression patterns (interleukin (IL)-6, granulocyte (G)-CSF, GM-CSF) and adhesion molecules on HFFF cells were qualitatively identical to BM-derived fibroblasts, but the expression density of adhesion receptors was significantly higher. This HFFF layer stimulated blood stem cells of GM-CSF-treated patients significantly more than a cocktail of exogenous growth factors with IL-1, IL-6, and stem cell factor (SCF). The reverse was true for multilineage colonies from healthy donors' PB, BM, and CB. According to these results, stem cells of GM-CSF-treated patients are functionally distinct due to their dependence on stroma-derived factors and/or matrix-adhesion interactions and can be reproducibly evaluated on these mesenchymal cells. PMID- 7545596 TI - The kinetics of E-selectin expression and leukocyte traffic in BCG/PPD delayed hypersensitivity in the pig. PMID- 7545597 TI - Adhesion of eosinophils to E- & P-selectin. PMID- 7545595 TI - Endothelial selectin expression and leukocyte trafficking in cutaneous DNFB induced delayed hypersensitivity. PMID- 7545598 TI - Cyclic AMP modulates adipogenesis in 3T3-F442A cells. PMID- 7545599 TI - Critical role for the phosphorylation of tyrosines 674 and 675 in signalling by the Trk tyrosine kinase. PMID- 7545594 TI - The L-selectin counter-receptor in porcine lymph nodes. PMID- 7545600 TI - Cytokine-induced E-selectin expression by human umbilical vein endothelial cells (HUVEC). A role for protein tyrosine kinases? PMID- 7545601 TI - Construction and expression of a functional chimeric receptor from PDGFr-beta and c-erbB-2. PMID- 7545602 TI - Stimulation of platelets with platelet-activating factor induces changes in the subcellular distribution and activity of the tyrosine kinase pp60c-src. PMID- 7545603 TI - Thudichum Medal Lecture. The acetylcholine receptor: a model for allosteric membrane proteins. PMID- 7545604 TI - Differential effects of wortmannin on growth hormone dependent signalling pathways in 3T3-F442A cells. PMID- 7545605 TI - Immunoglobulins and alpha-1-proteinase inhibitor in human intervertebral disc material. PMID- 7545607 TI - Effect of superoxide dismutase on nitric oxide production by RAW264 macrophages. PMID- 7545608 TI - Interactions of diesel engine emissions with extracellular biological fluids. PMID- 7545606 TI - The mitogen-activated protein kinase cascade in rat islets of Langerhans. PMID- 7545609 TI - Damage to DNA and RNA by tumour promoter-derived alkoxyl radicals: an EPR spin trapping study. PMID- 7545613 TI - The effect of cytokines on tissue factor expression in HL-60 and U937 cell lines. PMID- 7545610 TI - Exogenous CD59 incorporated into U937 cells through its glycosyl phosphatidylinositol anchor becomes associated with signalling molecules in a time dependent manner. PMID- 7545612 TI - A role for phosphoinositide 3--kinase in RANTES induced chemotaxis of T lymphocytes. PMID- 7545614 TI - Endotoxin causes reciprocal changes in hepatic nitric oxide synthesis and gluconeogenesis. PMID- 7545611 TI - The phosphoinositide 3-kinase inhibitor wortmannin inhibits CD28-mediated T cell co-stimulation. PMID- 7545615 TI - Inhibition of nitric oxide synthase by L-arginine metabolites. PMID- 7545616 TI - Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of pp42 mitogen-activated protein kinase by protein kinase C and protein kinase A in EAhy926 cells. PMID- 7545617 TI - The cloning and expression of the alpha subunit of equine glycoprotein hormones. PMID- 7545618 TI - Regulation of Ca2+ release from the ryanodine receptor of sarcoplasmic reticulum. PMID- 7545619 TI - Detection of human 11 beta-hydroxysteroid dehydrogenase isoforms using reverse transcriptase-polymerase chain reaction and localization of the type 2 isoform to renal collecting ducts. AB - 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), responsible for the interconversion of hormonally active cortisol to inactive cortisone, dictates specificity for the mineralocorticoid receptor (MR) in the distal nephron and colon. Two isoforms of human 11 beta-HSD have been cloned, an NADP(H)-dependent (type 1) dehydrogenase/oxo-reductase enzyme, and a high-affinity NAD-dependent (type 2) unidirectional dehydrogenase. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of RNA extracted from human adult tissues, type 1 11 beta-HSD mRNA was found in decidua, placenta, liver, lung, spleen, kidney medulla, cerebellum and pituitary, but was absent in kidney cortex, sigmoid and rectal colon, salivary gland and thyroid. In contrast, type 2 11 beta HSD mRNA was found only in placenta and in the classical mineralocorticoid target tissues, kidney cortex, kidney medulla, sigmoid and rectal colon, salivary gland, and colonic epithelial cell lines (AAC1 and RGC28). In situ hybridization studies of renal cortex, cortico-medullary junction and medulla using a 35S-labeled antisense cRNA probe for type 2 human 11 beta-HSD, revealed specific localization of type 2 11 beta-HSD mRNA expression exclusively to renal cortical and medullary collecting ducts. Type 1 and type 2 isoforms of human 11 beta-HSD are expressed in a distinct tissue-specific fashion, in keeping with the proposed differences in their physiological roles. Type 2 11 beta-HSD is found predominantly in mineralocorticoid target tissues where it serves to protect the MR in an autocrine fashion. PMID- 7545620 TI - Gonadotropin-releasing hormone overcomes follicle-stimulating hormone's inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells. AB - The effect of a gonadotropin-releasing hormone-agonist (GnRH-a) on the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5), a physiological marker for atresia, was investigated. Granulosa cells obtained from diethylstilbestrol (DES)-treated immature female rats were cultured in serum-free medium for 72 h with GnRH-a and the conditioned media were subjected to immunoblot analysis using rat IGFBP-5 specific antibody. GnRH-a caused a dose-dependent (ED50 = 8.6 x 10( 11) M) accumulation of IGFBP-5, which migrated as 35 (non-glycosylated) and 36 kDa (glycosylated) bands under reducing conditions. A maximally effective dose of GnRH-a (10(-9) M) caused a 4-fold increase in IGFBP-5 accumulation. In contrast, increasing doses of porcine follicle-stimulating hormone (pFSH) caused a biphasic effect on IGFBP-5 accumulation. A low dose of pFSH (0.25 ng/ml) increased and higher doses of pFSH (22.5 ng/ml) decreased the 35 and 36 kDa IGFBP-5 bands. In the presence of high doses of pFSH (20.75 ng/ml), a 22 kDa band corresponding to a cleaved IGFBP-5 fragment appeared in the media. When the granulosa cells were cultured with a saturating dose of pFSH, co-addition of GnRH-a dose dependently inhibited the FSH effects (ED50 = (2.3-3.7) x 10(-10) M). The GnRH-a effects were completely blocked by co-incubation with GnRH-antagonist. IGFBP-5 mRNA accumulation levels were increased by GnRH-a in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545621 TI - A quantitative assay for IGF-I and IGF binding protein mRNAs: expression in malignant melanoma cells. AB - The quantification of messenger RNA is central in studies of gene expression. We describe a quantitative assay for specific mRNAs (QASM) that measures mRNAs for insulin-like growth factor-I, IGF binding proteins (IGFBPs) -2, -3, -4, and -5, and beta-actin. The assay utilizes reverse transcription and polymerase chain reaction, followed by an ELISA based DNA assay technique. The use of internal (competitive) quantification standards gave poorly linear results, while external standards gave linear and reproducible results. QASM results correlated with IGFBP protein concentrations in conditioned medium and with mRNA levels determined by Northern blotting. QASM was used to study IGFBP expression in human malignant melanoma cells. Messenger RNA for IGFBP-2, -3, and -5 were present, while IGF-I and IGFBP-4 mRNAs were not detected. IGFBP-2 and -3 expression was increased in a dose dependent manner by treatment with IGF-I. Protein concentrations in conditioned media paralleled mRNA levels. QASM is a sensitive, specific, and reproducible approach to determining mRNA levels. PMID- 7545622 TI - Expression and regulation of aromatase cytochrome P450 in THP 1 human myeloid leukaemia cells. AB - Aromatase cytochrome P450 mRNA and activity was strongly expressed in THP 1 myeloid leukaemia cells after treatment with phorbol-myristate-acetate (PMA) and dexamethasone, low level expression was caused by calcitriol. mRNA species of 4.0, 3.0, 2.4 and 1.1 kb size were differentially stimulated. After calcitriol mediated differentiation (72 h, measured by CD 14 expression) mRNA expression was further enhanced by PMA (45-fold), dexamethasone (15-fold), oestradiol (3.7 fold), testosterone (2.5-fold) and androstenedione (3.5-fold). Forskolin, cAMP and follicle stimulating hormone had no stimulatory effect. Oestradiol formation from testosterone (oestradiol radioimmunoassay in culture supernatants) increased to > 2000 pg/ml/10(6) cells/24 h after PMA-stimulation, mirrored mRNA expression and was suppressed below 10% of original values in the presence of 4-OH androstenedione. Exons I.2 and I.4 were expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. A new splicing variant was expressed after calcitriol-stimulation, which did not hybridize to an exon II derived oligonucleotide but to an exon III-derived one. Local aromatisation of androgens into oestradiol may be important in the concerted crosstalk of cells of the monocyte/macrophage lineage with their respective tissues in inflammation and bone metabolism. PMID- 7545623 TI - Expression of the relaxin gene in rat tissues. AB - The peptide hormone relaxin is produced at high levels in the corpus luteum of the rat ovary during pregnancy. The biological effects of the hormone include remodelling of collagen in target tissues such as the cervix, and inhibition of uterine contractility. In addition, many paracrine actions for the hormone have been proposed, however sites of relaxin production outside the ovary have not been well characterized. We have investigated relaxin gene expression in a range of rat tissues using the techniques of reverse transcription/polymerase chain reaction (RT/PCR), RNase protection and immunohistochemistry. Relaxin mRNA was detected by RT/PCR in brain, uterus, prostate gland, pancreas and kidney, with other tissues giving weak signals. Relaxin gene expression in brain was detected by RNase protection, and relaxin-like immunoreactivity was observed in the arcuate nucleus of the hypothalamus of rat brain. This characterization of sites of relaxin gene expression provides further evidence for proposed paracrine actions of relaxin which may be important in non-pregnant and male rats in addition to the pregnant female. PMID- 7545624 TI - KAL, a gene mutated in Kallmann's syndrome, is expressed in the first trimester of human development. AB - Kallmann's syndrome (KS) is characterised by the association of anosmia and isolated hypogonadotrophic hypogonadism (IHH). Mutations of the KAL gene which is located at Xp22.3 cause X-linked KS (XKS). In this study we used the reverse transcriptase polymerase chain reaction and in situ hybridisation to examine the developmental expression of KAL in the first trimester of pregnancy, the earliest stage of human gestation examined thus far. At 45 days after fertilisation KAL mRNA was detected in the spinal cord, the mesonephros and metanephros but not in the brain. Later in gestation, at 11 weeks, the gene was expressed in the developing olfactory bulb, retina and kidney. This expression pattern correlates with the clinical findings in XKS since olfactory bulb dysgenesis with subsequent defective neural migration causes anosmia and IHH. Additionally, renal agenesis occurs in 40% of patients. Therefore this study provides strong evidence that KAL expression is required for the normal development of the olfactory bulb and kidney in the first trimester of human pregnancy. PMID- 7545626 TI - Treatment of nondilatable malignant pharyngoesophageal strictures by Montgomery salivary bypass tube: a new approach. PMID- 7545625 TI - Inhibition of serine/threonine protein phosphatases enhances agonist-stimulated cAMP accumulation in UMR 106 osteoblast-like cells. AB - Protein phosphatases regulate the activity of signal transduction mechanisms by dephosphorylating activated components. By utilizing selective inhibitors of these phosphatases, we investigated their role in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line. PTHrP, PTH and PGE2 stimulated cAMP accumulation up to 100-fold. Calyculin A, a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A), did not affect basal levels of cAMP, but concentrations of 10(-11) M to 10(-8) M increased PTHrP-, PTH-, and PGE2 stimulated cAMP accumulation up to 1.7-fold, and this increase was concentration dependent. Similar results were obtained with tautomycin, another potent inhibitor of PP1 and PP2A. In contrast, okadaic acid, a potent inhibitor of PP2A which inhibited PP1 less potently, did not enhance PTHrP-, PTH-, or PGE2 stimulated cAMP accumulation. The effect of calyculin A on agonist-stimulated cAMP accumulation persisted in cells treated with isobutyl methylxanthine, a phosphodiesterase inhibitor. When the effect of calyculin A was compared with that of 4 beta-phorbol 12-myristate 13-acetate (PMA), it was found that while PMA enhanced both the receptor and forskolin-stimulated cAMP accumulation, calyculin A had no effect on the forskolin-stimulated cAMP accumulation. The effect of calyculin A on PTHrP- and PTH-stimulated cAMP accumulation persisted in cells treated with PMA. These results suggest that protein phosphatases play an important role in agonist-stimulated cAMP accumulation in osteoblast-like cells, and that PP1 but not PP2A may be the major phosphatase involved. In contrast to activation by protein kinase C, the site of action for the phosphatase appears to be predominantly at a step prior to the activation of adenylyl cyclase in the cAMP signal transduction pathway. PMID- 7545628 TI - Dying patients deserve a 'true continuum of care'. PMID- 7545627 TI - Short-patch reverse transcription in Escherichia coli. AB - Chimeras of RNA and DNA have distinctive physical and biological properties. Chimeric oligonucleotides that contained one, two or three ribonucleotides whose phosphodiester backbone was covalently continuous with DNA were synthesized. Site directed mutagenesis was used to assess genetic information transfer from the ribonucleotide positions. Transfer was scored by the formation or reversion of an ochre site that also corresponded to a restriction cleavage site. This allowed physical as well as genetic assay of mutational events. Bases attached to the ribonucleotides were able to accurately direct the synthesis of progeny DNA. The results suggest that in vivo DNA polymerases utilize a "running start" on a DNA backbone to continue across a covalent backbone junction into a region of ribonucleotides and then back again onto a normal DNA backbone. The phenomenon is designated short-patch reverse transcription (SPRT) by analogy to short-patch mismatch correction and reverse transcription as the term is generally used. The possibility is considered that SPRT contributes to an unrecognized pathway of mutagenesis. PMID- 7545632 TI - Expression of a novel beta 1 integrin in the dysplastic progression of the cervical epithelium. AB - Epithelial cell interactions with matrices and basal membranes are central for tissue organization, and integrins are a family of adhesion molecules that play a major role in these interactions. We have analyzed the expression of a novel integrin alpha chain, alpha 10.1.2, in the squamous epithelium of the portio vaginalis uteri from patients with cervical intraepithelial neoplasia (CIN) and from control samples without apparent cervical abnormalities. The localization of beta 1 and alpha 6 chains was also investigated, together with the distribution of intraepithelial HLA-DR and CD1c-positive Langerhans cells. In the normal cervical epithelium, all of the integrin chains were detected in the basal cell layer, albeit with a different localization in the apical, lateral, and basal cell surfaces. Langerhans cells were evenly distributed in the deep 2/3 of the squamous epithelium. Expression of alpha 10.1.2 was reduced or absent in all of the CIN 1 specimens in which the other integrin chains were either normal or slightly reduced. CIN 2 was characterized by overexpression of integrins, namely of beta 1 and of alpha 10.1.2 chains which were consistently detected also in suprabasal cell layers. None of the integrin chains was found in CIN3 samples in which Langerhans cells were also absent. Thus, modulation and redistribution of integrins occur in the progression of cervical dysplasia, and lack of integrin expression characterizes high-grade lesions. As in other dysplasias and cancers of squamous epithelia, the localization of alpha 10.1.2 chains provides reliable diagnostic and possibly prognostic criteria. PMID- 7545631 TI - Abnormal expression of the retinoblastoma gene in ovarian neoplasms and correlation to p53 and K-ras mutations. AB - We analyzed the expression of the retinoblastoma (Rb) gene in a group of ovarian neoplasms previously characterized for mutations in the p53 suppressor gene and the Ki-ras oncogene. Using immunohistochemical techniques, a total of 59 ovarian neoplasms spanning the histiologic spectrum from benign to malignant were examined for the expression of the Rb protein. All benign cystic adenomas and low malignant potential tumors exhibited normal expression of the Rb protein. Abnormalities in Rb protein staining were noted in 3 of 22 (14%) ovarian carcinomas. The staining patterns included tumors that were totally or focally negative for Rb protein. One tumor focally expressed Rb. This tumor demonstrated a direct juxtaposition of sections of Rb expressing and nonexpressing malignant epithelial cells. Two of the three tumors with abnormal Rb expression also had p53 mutations and staining on serial sections demonstrated that selected ovarian cancer cells possessed mutations in both oncogenes. These data suggest that the loss of Rb gene expression may play a role in the pathogenesis of a small number of invasive ovarian malignancies, but not in noninvasive ovarian neoplasms. PMID- 7545630 TI - High prevalence of hepatitis C in Egyptian patients with chronic liver disease. AB - The highest prevalence rates of hepatitis C virus infection in the world have been recently reported among Egyptian blood donors and frequent recipients of transfusions and other blood products. This is the first report, however, demonstrating hepatitis C as the most frequent association with chronic liver disease in Egypt. Of 1023 patients referred to the Liver Institute in Menoufia governorate for evaluation of chronic liver disease, 752 (73.5%) had antibodies to hepatitis C compared with 168 (16.4%) with hepatitis B surface antigen. Hepatitis C antibody was more common in patients with active schistosomiasis and patients without hepatitis B surface antigenaemia. Of 100 patients having liver biopsies, histological findings consistent with chronic viral hepatitis or its complications were found in 89 and antibody to hepatitis C was present in 75 (84.3%) of these patients with chronic hepatitis, active cirrhosis or hepatocellular carcinoma. These data pointing to the importance of hepatitis C as a cause of chronic liver disease in Egypt emphasise the necessity of studies delineating its routes of transmission in this country. PMID- 7545629 TI - Angiogenesis in early choroidal neovascularization secondary to age-related macular degeneration. AB - BACKGROUND: The morphological features of angiogenesis in early choroidal neovascularization secondary to age-related macular degeneration are yet to be fully described. METHODS: Six eyes from five patients which on clinical and histological examination showed advanced age-related macular degeneration and early choroidal neovascularization have been studied by transmission electron microscopy. RESULTS: Pre-existing choroidal capillaries and venules showed changes which included endothelial cell budding, pericyte enlargement, endothelial cell sprout formation and the development of intrachoroidal new vessels. In one case, an endothelial cell sprout continuous with an intrachoroidal vessel penetrated Bruch's membrane. Examination of early subretinal pigment epithelial new vessels showed them to spread between the inner layers of Bruch's membrane within the space usually occupied by the basal linear deposit and drusen. New vessel formation took place in blind pouches at the margins of new vessel networks, either in the absence of pericytes or in the presence of mainly myofibroblast-like pericytes. CONCLUSION: This ultrastructural study describes two phases of new vessel growth associated with the onset of choroidal neovascularization secondary to age-related macular degeneration. The initial intrachoroidal phase appears to be a "low-turnover" form of neovascularization which may lead to new vessels penetrating Bruch's membrane. Extensive subretinal pigment epithelial neovascularization, on the other hand, results from a "high-turnover" phase of neovascularization characterized by extensive endothelial cell proliferation and migration. Pericyte phenotypic changes associated with these different phases of neovascularization appear to relate to the dynamics of angiogenesis taking place in each process. PMID- 7545634 TI - Recombinant human G-CSF: how wide is the field of clinical applicability? PMID- 7545636 TI - Human granulocyte colony-stimulating factor (rHuG-CSF) for treatment of neutropenia in Shwachman syndrome. AB - We report a boy affected by Shwachman syndrome (SS) who presented severe neutropenia and frequent suppurative infections, which we treated successfully with granulocyte colony-stimulating factor (rHuG-CSF). Daily injections of 7.5 micrograms/kg/day significantly increased the absolute neutrophil count and he was free from infections. In order to avoid the risk of side effects and to improve the child's quality of life, we intermittently administered lower doses of rHuG-CSF. A weekly dose of 2 micrograms/kg/day was able to maintain the absolute neutrophil count high enough (0.58-1.2 x 10(9)/L) to prevent suppurative infections. During the follow-up period (2.5 years) we tried suspending rHuG-CSF twice, but the absolute neutrophil count (0.18-0.31 x 10(9)/L) fell significantly and suppurative infections reoccurred (otitis, perianal abscess). rHuG-CSF may be a useful therapeutic agent in patients with symptomatic neutropenia in SS. PMID- 7545633 TI - Evidence for a protein synthesis-dependent and -independent TNF alpha cytolytic mechanism. AB - The analysis of the effect of the protein synthesis inhibitors emetine (EM) or actinomycin D (ACT-D) on the TNF alpha-mediated cytolysis of L929 target cells demonstrates a biphasic, concentration (10(-12)-10(-4) M)-dependent curve indicative of two cytolytic mechanisms operative in L929 cells. One TNF alpha cytolytic mechanism is dependent on protein synthesis in the target cells, while the other cytolytic mechanism is protein synthesis independent. Both TNF alpha cytolytic mechanisms cause apoptosis (fragmentation of DNA) as shown by the TNF alpha-mediated release of tritiated thymidine, Apoptag, and DAPI staining, in the presence or absence of EM or ACT-D. The two cytolytic mechanisms are also similar in their requirement for lipoxygenase enzymes as shown by the ability of nordihydroguaiaretic acid (10(-6)-10(-5) M) and ketoconazole (4 x 10(-6)-2 x 10( 5) M) to block TNF alpha-mediated lysis of the target cells. However, the two cytolytic mechanisms differ in their requirement for the production of oxygen free radicals. The oxygen free radical scavengers, dimethylsulphoxide (0.2-0.4 M) and glutathione (2 x 10(-6)-10(-5) M) block the TNF alpha-mediated cytolysis of target cells in the absence of protein synthesis inhibitors, but not in the presence of EM or ACT-D. PMID- 7545635 TI - Treatment of normal donors with rhG-CSF 16 micrograms/kg for mobilization of peripheral blood stem cells and their apheretic collection for allogeneic transplantation. AB - BACKGROUND: Utilization of peripheral blood stem cells (PBSC) in allogeneic transplantation requires a method for their mobilization and collection that is not inconvenient for the donor. METHODS: We administered rhG-CSF (filgrastim) 16 micrograms/kg subcutaneously for 4 days in five normal subjects (age 18-31, M = 3, F = 2), previously selected as HLA-identical donors of siblings with leukemia. All the donors gave written informed consent. On days 4 and 5 (in one donor on day 6 too), 10:l leukapheretic collection was performed with a CS-3000 (Baxter) or an AS-104 (Fresenius) cell separator through the antecubital vein. RESULTS: The WBC count reached a median peak of 57.0 x 10(9)/L on day 5. The peripheral blood CFU-GM peaked to a median level of 8908/mL on day 5 with a median increase over baseline values of 39.1 times. The CD34+ cells peaked to (median) 147.0 x 10(6)/L on day 4 with a median increase of 65.3 times. A lesser enrichment was recorded for BFU-E (median increase 12.7 times) and CFU-GEMM (median increase 15.2 times). Even CD3+ and CD56+CD3- cells increased (median 1.7 and 1.5 times, respectively). A median of 771 x 10(8) MNC (range 672-1378), 116.4 x 10(6) CFU-GM (range 47.7-145.1) and 754 x 10(6) CD34+ cells (range 477-2599) were apheretically collected. Concerning side effects, mild to moderate back pain and general minor discomfort were reported by all donors. The platelet level regularly but transiently decreased after completion of the apheretic procedures with a median nadir of 69 x 10(9)/L (range 43-126) on (median) day 7, but in no case did thrombocytopenia cause bleeding. The thrombocytopenia was more pronounced with the CS-3000 than the AS-104 apparatus. CONCLUSIONS: rhG-CSF 16 micrograms/kg x 4 days is an efficient schedule for PBSC mobilization in healthy donors, but lower doses and even a single apheresis procedure might prove similarly adequate. PMID- 7545637 TI - Trilineage response to granulocyte colony-stimulating factor administration in a patient with myelodysplastic syndrome. AB - We report a 72-year-old man with refractory anemia with excess of blasts who presented severe pancytopenia and pneumonia and received granulocyte colony stimulating factor (G-CSF) treatment over a 6-week period. In addition to a dramatic increase in mature neutrophils, platelet count and hemoglobin level, the patient achieved a hematological remission which continued for more than 5 months despite discontinuation of the treatment. This observation confirms that in some cases during G-CSF treatment erythropoiesis and thrombopoiesis may improve in addition to the expected effect on neutrophils. While the patient remained in hematological remission, bone marrow examination revealed trilineage dysplasia. This finding suggests that the hematological remission in this patient may not have resulted from a recovery of non-clonal hematopoiesis of a normal clone, but may have derived instead from the monoclonal hematopoiesis of a neoplastic clone. PMID- 7545638 TI - Mutagenicity of bile and pancreatic juice from patients with pancreatico-biliary maljunction. AB - We attempted to detect mutagenic activity in bile and pancreatic juice from patients with biliary tract disease using the spore rec assay and wild (H17) and mutant (M45) strains. Three bile samples out of 5 obtained from patients with pancreatico-biliary maljunction showed positive reaction in the spore rec assay, and all contained a high level of amylase activity, while 300 microliters of bile samples obtained from 10 control patients without pancreatico-biliary maljunction did not show any positive reaction. Moreover, 300 microliters of the in vitro mixture of bile with an equal volume of pancreatic juice also showed a positive reaction after treatment for 12 days at 37 degrees C or for 10 min at 100 degrees C, suggesting that they were very stable and long-acting in vivo. These data suggest that possible mutagens might be formed by the mixing of bile with pancreatic juice regurgitated into the biliary tract, and that there might be a relationship to biliary tract cancer which often accompanies pancreatico-biliary maljunction. PMID- 7545639 TI - The YAG laser and Wallstent endoprosthesis for palliation of cancer in the esophagus or gastric cardia. AB - The need for frequent retreatment is a disadvantage of using endoscopic laser therapy (ELT) alone for palliative treatment of esophageal carcinoma. In this prospective study, therefore, we investigated the potential and feasibility of combining ELT with a self-expanding metallic stent (Wallstent). Twelve patients received ELT followed by stent placement (stent group) and were compared with 39 patients receiving ELT alone (ELT group). Swallowing ability was similar in the two groups. About one-third of the patients who had a short life expectancy, did not appear to benefit from stenting, whereas the interval between retreatments was prolonged by a factor of 2-4 in the remaining patients. Median survivals were 5.5 (range 1.0-23.5) months in patients with stents, and 4.5 (range 1.2-24.6) months in patients without stents. There were few complications related to stenting. In one patient, technical problems caused stent dislodgement into the stomach. Another stent patient died of hemorrhage from an untreated tumor in the stomach, but it was considered unlikely that the bleeding was caused by the stent. In conclusion, this preliminary trial suggests that a Wallstent endoprosthesis, used in combination with laser treatment, may become a valuable tool for prolonging the dysphagia-free interval in selected patients. PMID- 7545640 TI - Relation between markers for viral hepatitis and clinical features of Japanese patients with hepatocellular carcinoma: possible role of alcohol in promoting carcinogenesis. AB - To assess the relationship between hepatitis virus markers and the clinical features of hepatocellular carcinoma (HCC), we measured markers for hepatitis B virus (HBV) and hepatitis C virus (HCV) in 88 Japanese patients with HCC. Twelve (14%) patients were HBsAg-positive and 67 (76%) were anti-HCV-positive (both c100 3 and c11/c7). HCV-RNA was detected in 8 (38%) of the 21 anti-HCV-negative patients by PCR, so that 75 patients (85%) were infected with HCV. Of the HBsAg negative patients infected with HCV with no history of blood transfusion, the mean age of the alcoholics (consumption > 80 g ethanol daily for at least 10 years) was lower than that of the non-alcoholics (60 years vs. 65 years, P < 0.05). Among the HBsAg-negative and anti-HCV (or HCV-RNA)-positive patients with a history of blood transfusion, the mean interval between the time of blood transfusion and the diagnosis of HCC in the alcoholics was shorter (21 years) than that in the nonalcoholics (27 years), but the difference was not statistically significant. We conclude that infection by both HCV and HBV may play a role in the development of HCC, and that alcohol consumption may promote carcinogenesis. PMID- 7545643 TI - Gene expressions of oxytocin and oxytocin receptor in cumulus cells of human ovary. AB - Oxytocin (OT) has been detected in mammalian granulosa-luteal cells during the early stages. The purpose of this study was to explore gene expressions of OT and OT receptor (OTR) in human cumulus cells. Cumulus cells enclosing a mature oocyte were obtained from 6 women undergoing clinical in vitro fertilization and embryo transfer programs. OT and OTR gene expressions were investigated by employing reverse transcription polymerase chain reaction and reverse transcription polymerase chain reaction/single-strand conformation polymorphism methods. OT gene expression in the cumulus cells was positive in 5 women and weakly positive in the remaining patient. The structure of OT mRNA in the cumulus cells was equivalent to that in human hypothalamus. OTR gene expression was also observed in the cumulus cells. This study is the first to describe the simultaneous expression of both OT and OTR genes in human cumulus cells. It is suggested that local OT plays some important roles in fertility through modification of the micro-environment around the oocyte. PMID- 7545642 TI - Effects of beta-1 integrins in the process of implantation. AB - The expression and function of beta 1 integrins on human decidual cells were investigated. Flow cytometric analysis revealed that the cultured decidual cells expressed a high level of the beta 1 subunit on the cell surface. Mouse blastocysts attached to and spread onto cultured human decidual cells. Attachment of the blastocysts was a necessary prerequisite for the further outgrowth of trophoblasts. The addition of a monoclonal antibody recognizing the beta 1 subunit to the cultured decidual cells did not affect the rates of hatching and attachment of blastocysts. The outgrowth of embryos on decidual cells was inhibited by the addition of the anti-beta 1-subunit antibody in a dose-dependent manner. Furthermore, exposure of decidual cells to the anti-beta 1-subunit antibody significantly inhibited the extent of outgrowth of trophoblasts, implying that blastocyst attachment and outgrowth is mediated by different mechanisms. These observations suggest that beta 1 integrins on decidual cells may be involved in the process of blastocyst development and differentiation after attachment. PMID- 7545644 TI - Combined hepatocellular and cholangiocarcinoma: proposed criteria according to cytokeratin expression and analysis of clinicopathologic features. AB - We herein evaluated 36 cases of combined hepatocellular and cholangiocarcinoma (cHCC-CC) (including 29 surgically resected and seven autopsy cases) by the immunohistochemical methods of anticytokeratin antibodies 7 and 19, and then analyzed the clinicopathologic features by comparing cHCC-CC with ordinary hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The results indicated that even if mucin production could not be confirmed, nine cases with HCC areas that showed a histological resemblance to CC also showed immunohistological biliary differentiation. Therefore, we advocate that these HCC with biliary differentiation based on an immunohistochemical analysis should thus be included in the criteria of cHCC-CC in broad terms. Regardless of the extent of mucin production, the cHCC-CCs as indicated by an immunohistochemical analysis are considered to have a similar background to that of ordinary HCCs regarding such factors as the average age, male:female ratio, hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody (HCVAb) positivity, alpha-fetoprotein level, and the presence of cirrhosis. However, cHCC-CCs tend to metastasize to many organs and the lymph nodes, and, as a result, have a poor prognosis. PMID- 7545645 TI - Expression of cyclin D1 protein in centrocytic/mantle cell lymphomas with and without rearrangement of the BCL1/cyclin D1 gene. AB - Centrocytic/mantle cell lymphoma (CC/MCL) is a morphologically defined B-cell non Hodgkin's lymphoma characterized by a distinctive immunophenotype, BCL1/cyclin D1 (PRAD1) gene rearrangements, and, most recently, by overexpression of cyclin D1. Even using multiple breakpoint probes for BCL1 (MTC, p94PS) and cyclin D1, however, only approximately 70% of CC/MCL have a rearrangement consistent with a t(11;14) (q13;q32). To determine whether the type of molecular translocation affects the degree of cyclin D1 expression and to evaluate lymphomas diagnosed as CC/MCL but lacking molecular evidence of a BCL1 or cyclin D1 translocation, 16 CC/MCL and four cases of small lymphocytic lymphoma/B-CL1 (SLL/B-CLL) were stained using an anti-cyclin D1 antibody. All cases with a cyclin D1 translocation detected by Southern blotting techniques as well as four of the five CC/MCL without a documentable translocation showed nuclear cyclin D1 protein expression. There was no apparent correlation between staining intensity and the precise site or presence of a detectable translocation. Cases with a mantle zone growth pattern showed infiltration of the cyclin D1 positive cells into reactive follicular centers. None of the four SLL/B-CLL showed cyclin D1 expression. These findings show overexpression of the cyclin D1 protein in virtually all CC/MCL independent of the type or presence of a documentable BCL1 or cyclin D1 molecular rearrangement. The mechanism for cyclin D1 overexpression in the cases without a documentable rearrangement and the relationship of cyclin D1 overexpression to the pathogenesis of mantle cell neoplasia remain uncertain. PMID- 7545641 TI - Changes in prevalence of anti-HCV antibodies associated with preventive measures among hemodialysis patients and dialysis staff. AB - The latest prevalence of hepatitis C virus (HCV) infection was evaluated in 548 Japanese patients undergoing hemodialysis, and 216 members of the hospital dialysis staff. An assay of anti-HCV antibodies was performed using both first- and second-generation immunoenzymatic tests, and anti-HCV specificity was confirmed with a second-generation recombinant immunoblot assay. Of 548 hemodialysis patients, 90 (16.4%) were positive with first-generation, and 166 (30.3%) with second-generation tests. These rates were significantly higher than those for either hospital staff members (0.5% and 2.3%; p < 0.01) or healthy blood donors (0.98% and 1.30%; p < 0.001). Patients with a history of blood transfusion tended to have a higher positivity rate for anti-HCV than did the non transfused group (35.4% vs. 25.7% with second generation tests; p < 0.05). Positivity for anti-HCV was related to the duration of hemodialysis. Only a few patients seroconverted during the 2-year, 5-month period between the previous and present studies. Although hemodialysis patients remain a high-risk group for HCV infection, the prevalence of anti-HCV antibodies has decreased recently thanks to the use of erythropoietin for renal anemia, the universal screening of blood donors for anti-HCV antibodies, and improvements in infection control measures for this virus. PMID- 7545648 TI - Immunoregulation and drug treatment in chronic relapsing experimental allergic encephalomyelitis in the Lewis rat. AB - Chronic relapsing experimental allergic encephalomyelitis (CR.EAE) was induced by immunizing Lewis rats with total guinea-pig spinal cord (GPSC) tissue emulsified in enriched complete Freund's adjuvant (CFA). The proliferative responses of draining inguinal and popliteal lymph node cells to GP.MBP, purified protein derivative (PPD) and concanavalin A (ConA) appeared significantly modulated according to the clinical state of the animals. Responses appeared significantly decreased in both lymphoid compartments during the recovery periods compared with that during relapses. Therapeutic treatment of CR.EAE with cyclosporin and different lysolecithin derivatives, such as ET-18-OCH3, SRI 62-843 and MLS 266 337, starting at the spontaneous remission of the first disease bout, could suppress the manifestation of further relapses. Whereas cyclosporin only delayed the onset of the disease relapse until discontinuation of treatment, all lysolecithins showed a curative effect in most animals. Plasma corticosterone levels measured at different time points in placebo, cyclosporin and MLS 266-377 treated rats showed a strong correlation with the clinical state of the animals. High corticosterone levels were detected during stages of acute paralysis, whereas a decrease to normal levels was noted during each recovery phase. PMID- 7545647 TI - Environmental and occupational exposure to manganese: a multimedia assessment. AB - Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic additive used in Canada since 1976 as an anti-knock agent in unleaded gasoline. Its combustion leads to the emission of Mn oxides, especially Mn3O4. Since no study has assessed the potential risk of chronic exposure to low concentrations resulting from these emissions, the present investigation was undertaken to assess the level of environmental and occupational exposure of the human population. The multimedia exposure of two groups of workers (garage mechanics and blue-collar workers) potentially exposed to different levels of Mn from the combustion of MMT was assessed using personal air samplers, a dietary compilation, water samples at their places of residence, an epidemiological questionnaire and blood and hair samples. Results show that garage mechanics were exposed on average to higher atmospheric Mn at work (0.42 microgram/m3) than the blue-collar workers (0.04 microgram/m3). However, the contribution of atmospheric Mn to the total absorbed dose was less than 1%, and well below the standards established for occupational or environmental exposure; food contributes more than 95% of the multimedia dose. The average whole blood Mn concentrations were similar for the two groups (0.67 0.76 microgram/100 ml) and fall within the normal adult range. The average hair Mn concentrations were significantly higher for the garage mechanics (0.66 microgram/g) than for the blue-collar workers (0.39 microgram/g). The contribution of exogenous Mn versus endogenous Mn is questioned. As judged by the governmental standards or criteria for occupational and non-occupational environments, the current Mn levels in food, water and air may not cause any problems for the workers. PMID- 7545646 TI - Strontium 89 in the treatment of bony metastases. PMID- 7545649 TI - Simultaneous initiation of degranulation and inhibition of leukotriene release by soman in human basophils. AB - Previous studies noted that the serine esterase inhibitor, soman, could induce histamine release from human basophils. To investigate the mechanisms by which soman causes histamine release (a preformed mediator), we also examined its ability to induce leukotriene release (a newly synthesized mediator) from basophils. We found that no leukotriene release followed activation with soman, while histamine release was usually greater than 70%. In addition, soman and diisopropyl-fluorophosphate were found actively to suppress low level spontaneous leukotriene release as well as ongoing leukotriene release induced by anti-IgE antibody. Soman (0.3 mM) was able to stop leukotriene release as rapidly as the calcium chelator, EDTA. In a series of control experiments, it was noted that soman did not influence the metabolism of LTC4 to LTD4 or LTE4 (for which little metabolism occurred), eliminating the possibility that reduced LTC4 release could have resulted from its enhanced metabolism. Therefore, using one compound (soman), basophils could be simultaneously activated to degranulate while having the pathway leading to leukotriene release actively suppressed. These results provide further evidence that histamine and leukotriene release are independent pathways resulting from the activation of basophils. PMID- 7545650 TI - Interferon-gamma/tumour necrosis factor-alpha synergism as a mechanism for enhanced nitric oxide production following in vivo administration of muramyl dipeptide (MDP) to mice. AB - The aim of the present study was to investigate the role of muramyl dipeptide (MDP) in triggering host non-specific defence mechanisms, namely its influence upon the L-arginine-dependent biotransformation pathways leading to formation of nitric oxide (NO). MDP was applied subcutaneously to mice as one or three daily injections, 300 micrograms MDP each. Production of NO by peritoneal macrophages from these animals or the capability of their splenocytes to render virgin macrophages for enhanced NO production was assayed. It has been found that macrophages from MDP-treated animals produce considerably enhanced amounts of NO and that the splenocytes secrete soluble factors providing all the signals required for induction of NO biosynthesis. Introduction of anti-IFN-gamma or anti TNF-alpha antibodies into these splenocyte supernatants resulted in complete suppression of inducible NO production by macrophages. PMID- 7545651 TI - [Palliative tumor therapy in the elderly]. PMID- 7545652 TI - [Extrasystole]. PMID- 7545653 TI - Two distinct patterns of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models. AB - We established an in vitro peritoneal dissemination model using six ovarian cancer cell lines and cultured mesothelial cells. Ovarian cancer cells were classified into two types, invasive or adhesive, on the basis of their interaction with the mesothelial cell monolayer. The ovarian cancer cell lines derived from mucinous cystadenocarcinoma, poorly differentiated adenocarcinoma and undifferentiated carcinoma, which belonged to the invasive type, began to invade beneath the mesothelial monolayer from several hours after seeding in vitro, expelling the mesothelial cells at the periphery and forming colonies directly on the dish surface. On the other hand, cancer cell lines of clear cell carcinoma, which belonged to the adhesive type, showed colony formation with adhesion on the mesothelial monolayer even 18 h after seeding. Invasive-type cell lines invaded into the mesothelial monolayer at various rates in vitro, and the degree of invasiveness showed good correlation with the degree of peritoneal dissemination in vivo after intraperitoneal injection of cancer cells into nude mice. Adhesive-type cells showed rather higher dissemination rates in vivo. Microscopic observation of in vivo peritoneal dissemination at one day after inoculation also revealed two patterns of peritoneal involvement similar to those in vitro. In the in vitro model, anti-integrin alpha 2- and beta 1-antibodies inhibited the infiltration of invasive-type cells into the mesothelial monolayer, but did not affect colony formation by adhesive-type cells on the monolayer, indicating that invasion by both cell types was mediated by different molecules. This in vitro model is thought to be useful for analysis of the molecular mechanisms of peritoneal dissemination. PMID- 7545654 TI - Characterization of human pancreatic adenocarcinoma cell line with high metastatic potential in SCID mice. AB - The CD44 molecule and CD44 isoforms are expressed on some malignant tumours and it has been suggested that their expression may correlate with tumour spread. Human pancreatic carcinoma cell line (HPC-4) expressing CD44 was established from a patient with adenocarcinoma of pancreas. This line showed a rapid growth in vitro, several chromosome abnormalities and surface expression of some adhesion molecules (ICAM-1, LFA-3, beta 1-chain of VLA integrins, VNR). Xenotransplanted HPC-4 cells were able to grow rapidly in SCID mice as subcutaneous tumour, leading to 100% mortality within 3-5 weeks when 1 x 10(5)-1 x 10(7) cells were inoculated. Spontaneous metastases in the liver, lung, spleen and kidney of SCID mice were observed. Interestingly enough, HPC-4 cells in vivo and ex vivo also expressed HLA-DR molecules, but these were rapidly lost upon culture in vitro. It is suggested that the appearance of HLA-DR may be the result of interaction of the tumour with a local environment of the host, while CD44 expression may explain the rapid growth and occurrence of distant metastases in SCID mice. The ability of HPC-4 cells to form spontaneous metastases in SCID mice may prove to be a potentially interesting model of human carcinoma for testing new treatment modalities. PMID- 7545655 TI - Antiangiogenic substance(s) in a tumor cell line with low metastatic potential originating from the BALB/c mouse liver. AB - Two cell lines were established from liver cells (BALB/c mouse) exposed to benzo(a)pyrene: one was highly metastatic (G-5) and the other poorly metastatic (G-1) to the lung when subcutaneously implanted. However, there was no difference in lung colonization between G-1 and G-5 cells when they were intravenously injected. When G-1 cells were subcutaneously inoculated on one side of the back of mice followed by a challenge on the other side with G-5 cells 10 days later, the growth of the latter tumor was inhibited and the number of metastatic nodules in the lung was reduced. The functional vascular volume of G-1 tumor was less than the G-5 one. In mice bearing G-1 tumors, the neovascularization of intradermally inoculated G-5 cells was reduced. The conditioned medium from G-1 culture contained an inhibitory activity on the growth of endothelial cells from calf pulmonary artery. The inhibitory substance(s) was heat-stable, trichloroacetic acid-soluble, nondialyzable and resistant to various proteinases. The present results imply that G-1 cells produce an antiangiogenic substance(s), probably a polysaccharide(s), which inhibits the angiogenesis required for growth and metastasis of the G-5 tumor. PMID- 7545656 TI - Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. AB - BACKGROUND: The progression of tumor growth requires the recruitment of new blood vessels. It has been suggested that the degree of neovascularization would correlate with clinical prognosis. The purpose of the present study was to ascertain whether tumor vascularization correlated with clinical outcome in cases of primary squamous cell carcinoma of the head and neck (SCCHN). METHODS: In tumor biopsies from 48 patients, microvessel density was determined by immunohistochemistry based on polyclonal antibodies against factor VIII related endothelial antigen. Computerized image analysis was used to evaluate the staining intensity per histologic area. The degree of staining was quantitated and expressed as microvessel density, low and high microvessel density subgroups being compared with regard to survival. RESULTS: Median survival was 10 months in the subgroup with very low microvessel density scores, as contrasted to 69 months in the remainder with high scores (p = 0.08). Neither the patient's age, TNM status, clinical stage, nor histologic grade was related to microvessel density. Among the patients who eventually died of SCCHN (n = 23), there was a subgroup of patients with complete response to radiotherapy. This subgroup had significantly higher microvessel density and longer survival than did the patients who responded poorly or not at all to radiotherapy. CONCLUSION: The findings suggest that in SCCHN the degree of vascularization might be used as a predictor of response to radiotherapy. PMID- 7545657 TI - [The problem of radiogenic and chemotherapy-induced mucositis of the mouth and and oropharynx exemplified by accelerated radiochemotherapy with carboplatin in patients with inoperable squamous epithelial carcinomas of the head-/neck area. Heidelberg experiences]. AB - Despite numerous treatment measures mucositis of the mouth and pharynx due to radiochemotherapy frequently remains refractory to therapy. In most cases high doses of pain medications are till required. However, mucositis as a strong early reaction may be controllable by limiting cancer therapy. Within the current framework of accelerated radiochemotherapy with carboplatin, 50 patients with inoperable squamous cell carcinomas of the head and neck were followed from 1992 to 1994. Acute toxicity was documented from the first through eighth week after starting therapy. From the fifth week on, the degree of mucositis found was > 3 (WHO scale) in 24 patients. The extent of mucositis in 5 patients required interrupting therapy for 10 days on average. In 14 cases the average stay in hospital had to be prolonged by 10.2 days because of severe inflammation. In all, the average duration of mucositis after the end of the therapy amounted to 9.6 weeks. Twenty patients required bypass feedings with transnasal stomach tubes or percutaneous gastrostomy (PEG) tubes that were later removed. In addition, the incidences of dysphagia, xerostomia, hoarseness, skin reactions, nausea or vomitus and myelotoxicity were recorded. Descriptions of the supportive care concepts used at the University of Heidelberg are given and the supportive care concepts available scientific literature is updated. PMID- 7545658 TI - Time- and dose-related changes in the expression of substance P in salivary glands in response to fractionated irradiation. AB - PURPOSE: The expression of different neuropeptides in the innervation of submandibular and parotid glands of the rats was examined 2 and 5 days after initiation of radiation treatment as well as 10 and 180 days following the termination of irradiation. METHODS AND MATERIALS: The irradiation was given on 2 or 5 consecutive days with daily doses of 4-8 Gy up to a total dose of 20-40 Gy. Immunohistochemical methods were used for the demonstration of substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and, the rate limiting enzyme in the catecholamine synthetic pathway, tyrosine hydroxylase (TH). The content of SP was also analyzed by the use of radioimmunoassay (RIA). RESULTS: In the parenchyma of both the submandibular and the parotid glands of control animals as well as after 2 days of irradiation treatment, a few nerve fibers showing SP-like immunoreactivity (LI) were observed. A marked increase in the expression of SP in the innervation of the parenchyma in both glands was observed 10 days after cessation of radiation treatment. The number of stained nerve fibers and the intensity of fluorescence in the fibers seemed to be dose dependent because the group subjected to a total dose of 40 Gy displayed a more pronounced staining intensity than that treated with 30 Gy. These results were supported by the RIA analysis. One hundred eighty days after treatment no obvious differences in SP-expression were seen between control and irradiated animals. No acute and long-term alterations were seen with regard to the other peptides and TH. CONCLUSIONS: These results suggest that specific dose- and time-dependent changes in the expression of SP in the parenchyma of both submandibular and parotid glands occur in response to fractionated irradiation. The observations add further aspects to the tissue differences in physiological response and sensitivity to irradiation. PMID- 7545660 TI - Plasma growth hormone, insulin-like growth factor I, and insulin-like growth factor binding proteins in pigs with divergent genetic merit for postweaning average daily gain. AB - Characteristics of growth hormone (GH), IGF-I, and IGF-binding proteins (IGFBP) were studied in gilts sampled from lines of pigs selected for either fast (line F, n = 14) or slow (line S, n = 14) postweaning ADG. Repeated blood samples were obtained from gilts (approximately 55 kg BW) during a period of feed deprivation and again during refeeding. Averaged across time, the difference in mean plasma GH concentrations of F and S gilts was not significant (7.7 vs 6.4 ng/mL; P > .20) during feed deprivation, and frequently, height, and amplitude of GH pulses did not differ (P > .25) for F and S pigs. Overall, F gilts had greater concentrations of plasma IGF-I than S gilts during feed deprivation (217.3 vs 145.1 ng/mL; P < .03). Across line, plasma IGF-I decreased (P < .01) during feed deprivation. Average GH did not differ (P > .40) for F and S gilts during the refeeding period. Average plasma IGF-I tended (P = .05) to be greater in F gilts than in S gilts during refeeding. Consistent with changes over time during feed deprivation, plasma IGF-I averaged across line increased (P < .01) in response to refeeding. Averaged across time (0 and 48 h refeeding), activity of IGFBP-2 (singlet band at 34 kDa) did not differ significantly (P = .17) in F and S gilts. However, there was a tendency (P = .13) for a line x time interaction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545659 TI - Is substance P a mediator of radiation slaloadenitis? PMID- 7545663 TI - Interaction of calmodulin with its binding domain of rat cerebellar nitric oxide synthase. A multinuclear NMR study. AB - The intercellular messenger nitric oxide is produced through the action of nitric oxide synthases, a class of enzymes that is regulated by calcium-calmodulin (CaM). In this work, the interaction of CaM with a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods. Cadmium-113 NMR studies showed that binding of the cNOS peptide increased the affinity of CaM for metal ions and induced interdomain cooperativity in metal ion binding as earlier observed for complexes of CaM with myosin light chain kinase (MLCK) peptides. By using specific isotopically labeled [13C]methyl-Met and selenomethionine-substituted CaM in two-dimensional proton-detected 13C and 77Se NMR studies, we obtained evidence for the involvement of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobic surface areas on CaM, and they are also involved in the binding of other target proteins. A nitroxide spin-labeled version of the cNOS peptide caused broadening only for NMR resonances in the N-terminal half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM. pH titration experiments of CaM dimethylated with [13C]formaldehyde show that Lys-75 (and Lys 148) experience a large increase in pKa upon peptide binding; this indicates an unraveling of part of the helical linker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fashion to CaM. PMID- 7545662 TI - RNA chaperones and the RNA folding problem. PMID- 7545661 TI - Utero-placental vascular development and placental function. AB - The rate of fetal growth and subsequent birth weight are major determinants of postnatal survival and growth. Because the placenta is the organ through which respiratory gases, nutrients, and wastes are transported between the maternal and fetal systems, its primary function is to supply the metabolic substrates necessary to support fetal growth. Placental growth and development, therefore, are critical for normal fetal growth and development. During the last half of gestation in mammals, growth of the fetus is exponential, whereas utero-placental growth slows or ceases. Nevertheless, unless placental transport capacity keeps pace with the continually increasing demands of the fetus, fetal growth will be compromised. Studies over the last two decades have shown that placental transport capacity does indeed keep pace with fetal growth. This increase in placental function can be accounted for primarily by continual increases in placental (uterine and umbilical) blood flows, associated with increased placental vascularity. Placental vascular growth and development, in turn, are probably regulated by angiogenic factors produced by the placental tissues themselves. These placental angiogenic factors are produced primarily by the maternal placental tissues, are heparin-binding, and seem to be related to the fibroblast growth factor family. Further elucidation of the factors responsible for placental growth and vascular development is critical for an improved understanding of uteroplacental-fetal interactions, which result in delivery of a healthy offspring. PMID- 7545664 TI - Participation of acetylpseudouridine in the synthesis of a peptide bond in vitro. AB - Uracil, uridine, and pseudouridine were acetylated by refluxing in acetic anhydride, and the products of acetylation were incubated with a synthetic peptide (1-21) that corresponds to the N-terminal 21 amino acid residues of human myelin basic protein. Peptide bond formation, at the N alpha terminus in peptide 1-21, was obtained with acetyluracil and acetylpseudouridine, but not with acetyluridine. Transfer of an acetyl group from acetyluracil and acetylpseudouridine depended on acetylation in the N-heterocycle. X-ray crystallographic analysis definitively established N-1 as the site of acetylation in acetyluracil. Mass spectrometry of the acetylation products showed that one acetyl group was transferred to peptide 1-21, in water, by either acetyluracil or acetylpseudouridine at pH approximately 6. Release of the acetyl group by acylaminopeptidase regenerated peptide 1-21 (mass spectrometry) and automated sequencing (for five cycles) of the regenerated (deacetylated) peptide demonstrated that the N terminus was intact. The findings are discussed in the context of a possible role for pseudouridine in ribosome-catalyzed peptidyltransfer, with particular reference being made to similarities between the possible mechanism of acyl transfer by acetyluracil/pseudouridine and the mechanism of carboxyl transfer by carboxylbiotin in acetyl CoA carboxylase. The possibility that idiosyncratic appearance of a wide range of acyl substituents in myelin basic protein could be related to a peculiar involvement of ribosomal pseudouridine is mentioned. PMID- 7545665 TI - Peptides which bind to E-selectin and block neutrophil adhesion. AB - E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties. PMID- 7545667 TI - Silencer elements modulate the expression of the gene for the neuron-glia cell adhesion molecule, Ng-CAM. AB - The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5' end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5'-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development. PMID- 7545666 TI - Both extracellular immunoglobin-like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28. AB - The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain. PMID- 7545668 TI - Universal minicircle sequence binding protein, a CCHC-type zinc finger protein that binds the universal minicircle sequence of trypanosomatids. Purification and characterization. AB - Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5'-GGGGTTGGTGTA-3'). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single stranded DNA as well as single-stranded RNA but not double-stranded or four stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed. PMID- 7545669 TI - Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli. AB - Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD. PMID- 7545670 TI - Elements controlling the expression and induction of the skin hyperproliferation associated keratin K6. AB - The suprabasal keratin 6 (K6) is remarkable among the keratins as, in addition to being constitutively expressed in different stratified epithelia, it is induced in epidermis under hyperproliferative conditions, such as benign or malignant tumors, psoriasis, and wound healing. In addition, this keratin is also induced in skin treated with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid (RA). These characteristics make the study of K6 regulatory elements an especially interesting issue, in particular because these elements could be useful in designing gene constructs for the therapy of skin diseases. We have analyzed by mobility shift and footprinting experiments the cell type-specific enhancer of the bovine K6 beta gene (Blessing, M., Jorcano, J. L., and Franke, W. W. (1989) EMBO J. 8, 117-126) and have identified an AP-2-like element, two AP-1 elements (one of them composite), and a retinoic acid-responsive element (RARE). Mutagenesis experiments and cotransfections with retinoic acid receptors show that the RARE mediates enhancer activation by RA. Chloramphenicol acetyltransferase assays show that under normal culture conditions, the AP-1 element retains most of the enhancer transcriptional activity, while the RARE and AP-2 are weakly active. However, following RA treatment, the AP-1 element is repressed and the RARE is activated, resulting in an overall stimulation of the enhancer by RA in the BMGE+H cells used in our study. These results explain in part the complex and sometimes contradictory response of keratin 6 to hyperproliferative stimuli. PMID- 7545671 TI - Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites. AB - Activation of p70s6k in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells with the immunosuppressant rapamycin blocks phosphorylation of four of these sites (Thr229, Thr389, Ser404, and Ser411), whereas phosphorylation proceeds in the remaining three sites (Ser418, Thr421, and Ser424). If rapamycin is added postserum stimulation, the pattern of phosphorylation is qualitatively similar except that Ser411 is still highly phosphorylated. The inhibitory effect of rapamycin on serum-induced p70s6k activation and the phosphorylation of Thr229, Thr389, Ser404, and Ser411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycin-FKBP12. Wortmannin treatment pre- or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70s6k activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate that there are two kinase signaling pathways that must converge to activate p70s6k and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition. PMID- 7545672 TI - The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase. AB - Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The Vmax of ATP hydrolysis (approximately 30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O (2,4,6-trinitrophenyl)-adenosine-5'-triphosphate and adenosine 5'-(beta, gamma imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease. PMID- 7545673 TI - Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors. C-C CKR1, a receptor for macrophage inflammatory protein-1 alpha/Rantes, is also a functional receptor for MCP3. AB - Monocyte chemotactic protein-3 (MCP3) is recently identified and molecularly cloned C-C chemokine that is chemotactic for and activates a great variety of inflammatory cell types. MCP3 has been reported to interact with several C-C chemokine receptors, which can be simultaneously or selectively expressed on leukocyte subpopulations. In order to isolate receptor(s) for MCP3, a cDNA library was constructed using mRNA from a human NK-like cell line, YT. These cells showed high affinity binding sites for 125I-MCP3 and migrated in response to MCP3. A chemokine receptor cDNA clone, designated YT4, was sequenced and found to be identical to the known C-C CKR1 or macrophage inflammatory protein-1 alpha (MIP1 alpha)/Rantes receptor. YT4 cDNA was subcloned into a mammalian expression vector, and stable transfectants were prepared using the embryonic kidney cell line 293. The transfectants (YT4/293) showed high affinity binding for 125I-MCP3 in addition to specifically binding 125I-MIP1 alpha and 125I-Rantes. All three C C chemokines were able to cross-compete for binding sites on YT4/293 cells and induced directional migration of YT4/293 cells in vitro, with MCP3 being the most potent chemoattractant. MCP3, MIP1 alpha, and Rantes were equally able to cross attenuate the migratory response of YT4/293 cells to one another. In contrast, MCP1 and MIP1 beta had very limited capacity to compete for MCP3 binding on YT4/293 cells and had only a minor attenuating effect on MCP3-induced migration. Since MCP3 has been reported to use MCP1 receptor(s), our results with transfected 293 cells expressing only C-C CKR1 clearly establish that C-C CKR1 is also a functional receptor for MCP3. PMID- 7545674 TI - Identification of a variable region within the cytoplasmic tail of the IL-2 receptor beta chain that is required for growth signal transduction. AB - Interleukin-2 (IL-2) regulates numerous biological events, including T lymphocyte proliferation. Interleukin-2 receptor (IL-2R)-mediated signaling is triggered by ligand-induced heterodimerization of the IL-2R beta and gamma c subunits, which results in the activation of signaling intermediates that are associated with either IL-2R beta or gamma c. Previous mutagenesis studies of the IL-2R beta cytoplasmic tail demonstrated that the partially conserved box 1 and box 2 motifs and specific tyrosine residues are critical for growth signaling. By deletion and alanine scanning mutagenesis, another set of residues that are critical for IL-2R mediated signaling has now been identified. These residues lie within the divergent 35-amino acid "spacer" region separating box 1 and box 2. The role of this receptor subregion in early phases of IL-2R signaling was evaluated using BA/F3 stable cell lines expressing three functionally impaired mutants from this region. All three cell lines displayed substantially diminished growth responsiveness to IL-2. Receptor-mediated STAT factor activation, IL-2R beta phosphorylation, and Janus kinase activation were also markedly impaired. These findings indicate that this variable spacer region, which we have termed the V box, is essential for the initiation of IL-2R-mediated signal transduction. PMID- 7545675 TI - Identification of a putative Syp substrate, the PDGF beta receptor. AB - Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes. PMID- 7545677 TI - Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region. AB - Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238 243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL 6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids. PMID- 7545676 TI - Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix. AB - We show in this report that two v-src substrate proteins, p130Cas and cortactin, become tyrosine-phosphorylated during integrin-mediated cell adhesion to extracellular matrix substrata and upon cell attachment onto immobilized anti integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine or through antibodies against major histocompatibility complex. It also does not take place when adhesion-mediated reorganization of the actin cytoskeleton is inhibited with cytochalasin D. Tyrosine phosphorylation of p130Cas and cortactin coincides with tyrosine phosphorylation of focal adhesion kinase during integrin-mediated cell adhesion but is independent of cell adhesion in v-src-transformed cells. The tyrosine-phosphorylated sites in p130Cas and cortactin may serve as binding sites for proteins containing Src homology 2 domains, as is the case with two other integrin-regulated docking proteins, focal adhesion kinase and paxillin. Thus, these results suggest that ligand binding of integrins regulates the tyrosine phosphorylation state of multiple docking proteins. These proteins may mediate anchorage dependence of growth; their misregulation in v-src-transformed and other tumorigenic cells may be responsible for the anchorage independence of such cells. PMID- 7545678 TI - Identification and characterization of an interleukin-3 receptor-associated 110 kDa serine/threonine kinase. AB - We recently reported that interleukin-3 (IL-3) stimulation of the murine IL-3 responsive cell line, B6SUtA1, results in the rapid phosphorylation of the beta subunit of the IL-3 receptor (IL-3R), not only on tyrosine residues but on serine/threonine (Ser/Thr) residues as well. Since this occurred even at 4 degrees C, it suggested that a Ser/Thr-specific kinase might be closely associated with the IL-3R. To test this possibility, IL-3R complexes were isolated with anti-IL-3R (alpha IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the presence of a 110-kDa protein that was heavily phosphorylated in vitro on serine and threonine residues and that bound selectively to gamma-ATP-Sepharose beads. Moreover, this protein, which was not the 110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine phosphorylated in response to IL-3 and was specifically labeled in vitro with azido-[32P]ATP. These data, together with in vitro kinase inhibitor studies, suggest that an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa Ser/Thr kinase may be constitutively associated with the IL-3R and activated following IL-3 stimulation. A comparison of IL-3R and erythropoietin receptor complexes suggests that this 110-kDa protein may be preferentially associated with the IL-3R. PMID- 7545679 TI - Identification of a new acute phase protein. AB - We have previously reported mouse SIP24 protein as a secreted inducible protein produced by quiescent Balb/c 3T3 cells. SIP24 can be produced in response to many factors, including serum, basic fibroblast growth factor, prostaglandin F2 alpha, phorbol ester, and dexamethasone. Here we present evidence to show that SIP24 is the product of mouse 24P3 mRNA. The 24P3 cDNA was originally cloned from an SV40 transformed quiescent mouse primary kidney cell culture, and it has been classified as a new member of the lipocalin protein family. We show that the SIP24/24P3 protein and mRNA increase dramatically in mouse serum and liver during the acute phase response induced by turpentine injection. Injection of mice with dexamethasone caused a modest increase of SIP24/24P3 mRNA in the liver. Tissue distribution studies revealed that SIP24/24P3 is mainly expressed in liver during the acute phase response. SIP24/24P3 was also detected in the brain and the uterus. In mouse BNL (Balb/c normal liver) cells, the production of SIP24/24P3 is stimulated by tumor necrosis factor alpha, which is a major regulator of the expression of other acute phase proteins. From its pattern of regulation, we conclude that SIP24/24P3 is a new type 1 acute phase protein. PMID- 7545681 TI - Biosynthesis of GlyCAM-1, a mucin-like ligand for L-selectin. AB - L-selectin, a member of the selectin family of leukocyte-endothelial adhesion proteins, mediates the initial attachment of lymphocytes to lymph node high endothelial venules during lymphocyte recirculation. One of the endothelial associated ligands for L-selectin is GlyCAM-1, a mucin-like glycoprotein, which presents novel sulfated, sialylated and fucosylated O-glycans. In order to understand the generation of these glycans, we have examined the biosynthesis of GlyCAM-1 in lymph node organ culture. Using peptide-specific antibodies, lectins, and recombinant L-selectin, we detected the following species of GlyCAM-1: unglycosylated (< 28 kDa); modified with GalNAc only (28-33 kDa); modified with sialic acid, fucose, and sulfate but lacking L-selectin reactivity (40-50 kDa); and mature (L-selectin-reactive) ligand (50-60 kDa). Pulse-chase labeling at 15 degrees C suggested that GalNAc is added in a pre-Golgi compartment. Treatment with brefeldin A almost completely blocked sulfation, indicating that this modification occurs in the trans-Golgi network. Two distinct sialylation events occurred in the presence of brefeldin A, while fucosylation was partially blocked. We conclude that sialylation precedes both fucosylation and sulfation during biosynthesis. This ordering will help to identify the critical acceptor structures recognized by lymph node glycosyltransferases and sulfotransferases. PMID- 7545680 TI - Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody. AB - Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells. PMID- 7545682 TI - Regulation of the Fas apoptotic cell death pathway by Abl. AB - Relatively little is known about oncogene involvement in the regulation of Fas mediated apoptosis. Inhibition of Fas-induced cell death by the bcl-2 oncogene has been demonstrated to be only partial. In light of a growing body of evidence for the Abl kinase as a negative regulator of cell death, we sought to determine whether Abl expression could protect against Fas-mediated cell death. To address this question, we utilized two separate strategies. In the first, we expressed human Fas in K562, a chronic myelogenous leukemia cell line, which constitutively expresses bcr-abl and examined the effects of Fas ligation in these cells. Fas positive K562 transformants (K562.Fas) were found to be protected against Fas mediated cell death. However, down-regulation of Bcr-Abl protein levels in K562.Fas cells using antisense oligonucleotides targeted to bcr-abl mRNA rendered these cells highly susceptible to Fas-induced death. In the second approach we utilized a Fas-positive HL-60 cell line, which we transfected with a temperature sensitive mutant of v-Abl. HL-60.v-Ablts transfectants were found to be protected from Fas-induced apoptosis at the permissive but not the restrictive temperature for the Abl kinase. Taken together, these observations identify the Abl kinase as a negative regulator of Fas-mediated cell death. Since Abl was also found to block apoptosis mediated by ceramide, a recently proposed downstream effector of the apoptotic pathway initiated by Fas, we propose that Abl exerts its protective effects downstream of the early Fas-initiated signaling events. PMID- 7545683 TI - Association of 75/80-kDa phosphoproteins and the tyrosine kinases Lyn, Fyn, and Lck with the B cell molecule CD20. Evidence against involvement of the cytoplasmic regions of CD20. AB - CD20, a non-glycosylated cell-surface protein expressed exclusively on B lymphocytes, is one of a family of 4-pass transmembrane molecules that also includes the beta chain of the high affinity receptor for IgE. The precise function of CD20 is unknown, although in vitro effects of CD20-specific antibodies on resting B cells indicate that it is able to transduce an extracellular signal affecting the G0/G1 cell cycle transition. Previous studies have demonstrated that CD20-initiated intracellular signals involve tyrosine kinase activation and that CD20 is tightly associated with both serine and tyrosine kinases. Here, analysis of CD20-associated molecules has revealed that CD20 is associated with the Src family tyrosine kinases p56/53lyn, p56lck, and p59fyn and with 75/80-kDa proteins phosphorylated in vivo on tyrosine residues. Mutagenesis of CD20 was performed to define regions of CD20 involved in intermolecular interactions. Mutants were analyzed in the human T lymphoblastoid cell line Molt-4, in which ectopically expressed wild-type CD20 associated with p59fyn, p56lck, and 75/80-kDa phosphoproteins. Deletion of major portions of the cytoplasmic regions of CD20 did not abolish its association with either p75/80 or tyrosine kinases. The interaction between CD20 and the Src-related kinases is therefore likely to be independent of CD20 cytoplasmic domains and may occur indirectly. The interaction may be mediated by the p75/80 phosphoproteins, which were found to be tightly associated with the Src family kinases isolated from the CD20 complex. PMID- 7545684 TI - Suramin inhibits C6 glioma-induced angiogenesis in vitro. AB - Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-micron thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-microns pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently form networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiogenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor derived growth factor release and/or endothelium-mediated matrix proteolysis. PMID- 7545685 TI - Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the alpha IIb beta 3-integrin in melanoma cells. AB - In metastatic B16a murine melanoma cells, alpha IIb beta 3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, alpha IIb beta 3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and alpha IIb beta 3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the alpha IIb beta 3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells. PMID- 7545686 TI - Monoclonal antibody HFD9 identifies a novel 28 kDa integral membrane protein on the cis-Golgi. AB - We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers. PMID- 7545688 TI - Production and characterisation of monoclonal and polyclonal antibodies to different regions of the cystic fibrosis transmembrane conductance regulator (CFTR): detection of immunologically related proteins. AB - We have raised mouse monoclonal antibodies to eight synthetic peptides corresponding to different regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) and rabbit polyclonal antisera to beta-galactosidase fusion proteins which encompass three different regions of CFTR. Immunoblot, immunoprecipitation, immunofluorescence and immunocytochemical experiments demonstrate that, in addition to recognising CFTR, these antibodies recognise one or more immunologically related proteins with a similar molecular mass, calcium responsiveness and tissue distribution to CFTR. PMID- 7545689 TI - Mammary epithelial cell differentiation in vitro is regulated by an interplay of EGF action and tenascin-C downregulation. AB - Expression of the extracellular matrix glycoprotein tenascin-C in the mammary gland is associated with cellular proliferation and cell motility during organogenesis and tumorigenesis. Because the source and the regulation of tenascin-C in these tissues are unclear, we have used tenascin-C cDNA, FITC immunofluorescence and immuno-precipitation to examine tenascin-C expression of mammary epithelial cells. Using several mammary epithelial cell lines we could show that tenascin-C can be produced and secreted by epithelial cells. However it was found that tenascin-C synthesis was inversely correlated with the polarized epithelial phenotype. Among three mouse mammary epithelial cell clones, tenascin C expression was most abundant in HC-11 cells, the least differentiated cell type. Expression levels were high during the growth phase but were nearly abolished when cells were grown to confluence and induced to express milk proteins. Downregulation of tenascin-C by EGF apparently commits HC-11 cells to respond to lactogenic hormones and consequently, hormone induced levels of beta casein mRNA decreased significantly when HC-11 cells were grown on a tenascin-C substrate. On the other hand, TGF-beta, another growth factor involved in coordinated growth and differentiation of the mammary gland in vivo was found to be a very potent inducer of tenascin-C. The generation of fully polarized and tight epithelium affected the levels of tenascin-C expression. In contrast to HC 11 cells, which do not form epithelial domes in vitro, highly polarized and dome forming EpH4 and Fos-ER cells nearly lacked tenascin-C. Similarly, induction of dome formation in the rat mammary stem cell line Rama 25 by the differentiation inducer dimethylsulfoxide caused a loss of TN-C-transcripts. The inability of Fos ER cells to develop domes in the presence of soluble tenascin-C also suggests its interference with induction and maintenance of mammary epithelial cell differentiation. PMID- 7545687 TI - Caenorhabditis elegans cyclin A- and B-type genes: a cyclin A multigene family, an ancestral cyclin B3 and differential germline expression. AB - We have cloned cDNAs for Caenorhabditis elegans cyclins A1, B and B3. While cyclins A1 and B are most closely related to either A- or B-type cyclins of other species, cyclin B3 is less related to these cyclins. However, this cyclin is most similar to the recently identified chicken cyclin B3. Our identification of a Caenorhabditis homolog demonstrates that cyclin B3 has been conserved in evolution. Cyclin A1 is a member of an A-type multigene family; however the cyclin A1 cDNA only recognizes a single band on northern blots. A single-sized RNA is also observed for the cyclin B3 cDNA. In contrast, three different transcripts are observed for the cyclin B cDNA. Based on our analyses using RNAs from germline-defective mutants and from populations enriched for males, one cyclin B transcript is specific to the paternal germline. The two other cyclin B transcripts, as well as the cyclin A1 and cyclin B3 transcripts, are most abundant in the maternal germline and are only present at low levels in other tissues. Moreover, the 3' untranslated regions of each Caenorhabditis cyclin cDNA possess several copies of potential translational control elements shown in Xenopus and Drosophila maternal cyclin mRNAs to function during oogenesis and early embryogenesis. PMID- 7545690 TI - Inhibition of neuronal nitric oxide synthase by 7-nitroindazole: effects upon local cerebral blood flow and glucose use in the rat. AB - The novel nitric oxide synthase inhibitor 7-nitroindazole (7-NI) is relatively specific for the neuronal isoform of the enzyme and in this study we have used this compound to investigate the physiological role of perivascular nitric oxide containing nerves in the cerebrovascular bed. Following injection of 7-NI (25 or 50 mg/kg, i.p.), cerebral blood flow and glucose utilization were measured in the conscious rat using the fully quantitative [14C]iodoantipyrine and 2 [14C]deoxyglucose techniques, respectively. Neither dose of the drug produced any change in arterial blood pressure, confirming a lack of effect upon the endothelial isoform of the enzyme, although there was a pronounced decrease in heart rate (-28% by 10 min postinjection). Throughout the brain 25 mg/kg 7-NI i.p. resulted in decreases in blood flow of between -20% in the hippocampus and 58% in the substantia nigra. Increasing the dose to 50 mg/kg resulted in a further generalized decrease, to almost -60% in parts of the thalamus and hippocampus, but in every animal this higher dose of 7-NI also produced randomly distributed areas of relative hyperaemia, which were most commonly found in those areas where the most intense hypoperfusion was otherwise in evidence. Despite these changes in blood flow, in all but a very few areas of the brain no significant decrease in glucose use was measured at either of the two doses of 7 NI. Thus despite the greater specificity of 7-NI for neuronal nitric oxide synthase, the cerebrovascular effects of the drug in vivo are very similar to that reported for the arginine analogues. However, these data do suggest that nitric oxide-releasing neurones in the brain may have an important role to play in the regulation of cerebral blood flow. PMID- 7545692 TI - Effects of central inhibition of nitric oxide synthase on focal cerebral ischemia in rats. AB - We have investigated whether central inhibition of nitric oxide synthase (NOS) could modify the tissue damage of focal cerebral ischemia produced by occlusion of the middle cerebral artery (MCA) in rats. NG-Nitro-L-arginine methyl ester (L NAME) was administered intracerebroventricularly at two doses 15 min prior to occlusion of the MCA, as well as 4 and 24 h following occlusion. After the injection of L-NAME, the catalytic activity of the constitutive NOS, considered to be mainly neuronal, was effectively suppressed in the subcortical gray matter bilaterally, but not in the ischemic territory. Seven days after the MCA occlusion, the brains were evaluated for histopathologic damage. High-dose administration of L-NAME (120 micrograms/kg 15 min prior to MCA occlusion, followed by 150 micrograms/kg 4 and 24 h after occlusion) produced an enlargement of the infarct area and increased the volume of ischemic damage. These results indicate that extensive inhibition of NOS by a central route can increase the cerebral infarct size in focal ischemia even if NOS is not inhibited in the ischemic tissue and suggest that NO may also play a potentially beneficial role as well as a neurodestructive role in the pathophysiological mechanisms of focal cerebral ischemia. PMID- 7545693 TI - Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials. AB - An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV 2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance. PMID- 7545691 TI - The role of neuronal nitric oxide synthase in regulation of cerebral blood flow in normocapnia and hypercapnia in rats. AB - The nitric oxide synthase (NOS) inhibitors, nitro-L-arginine, its methyl ester, and N-monomethyl-L-arginine, have been shown to attenuate resting CBF and hypercapnia-induced cerebrovasodilation. Those agents nonspecifically inhibit the endothelial and neuronal NOS (eNOS and nNOS). In the present study, we used a novel nNOS inhibitor, 7-nitroindazole (7-NI) to examine the role of nNOS in CBF during normocapnia and hypercapnia in fentanyl/N2O-anesthetized rats. CBF was monitored using laser-Doppler flowmetry. Administration of 7-NI (80 mg kg-1 i.p.) reduced cortical brain NOS activity by 57%, the resting CBF by 19-27%, and the CBF response to hypercapnia by 60%. The 60% reduction was similar in magnitude to the CBF reductions observed in previous studies in which nonspecific NOS inhibitors were used. In the present study, 7-NI did not increase the MABP. Furthermore, the CBF response to oxotremorine, a blood-brain barrier permeant muscarinic agonist that induces cerebrovasodilation via endothelium-derived NO, was unaffected by 7-NI. These results confirmed that 7-NI does not influence eNOS; they also indicated that the effects of 7-NI on the resting CBF and on the CBF response to hypercapnia in this study were solely related to its inhibitory action on nNOS. The results further suggest that the NO synthesized by the action of nNOS participates in regulation of basal CBF and is the major, if not the only, category of NO contributing to the hypercapnic CBF response. PMID- 7545695 TI - Altered relation between circulating levels of insulin-like growth factor-binding protein-1 and insulin in growth hormone-deficient patients and insulin-dependent diabetic patients compared to that in healthy subjects. AB - To investigate whether previously reported increased levels of insulin-like growth factor-binding protein-1 (IGFBP-1) in GH-deficient patients only reflect decreased levels of insulin or are elevated in relation to insulin, diurnal profiles of IGFBP-1 and insulin were determined in plasma from patients with GH levels below 0.2 microgram/L throughout 24 h (n = 23) and compared to profiles from patients with insulin-dependent diabetes mellitus (IDDM; n = 9) and healthy subjects (n = 12). Samples were drawn using a continuous withdrawal technique at 20-min intervals. Levels of IGF-I were determined in one morning sample. As in healthy subjects, serum IGFBP-1 displayed a diurnal variation in GH-deficient as well as in IDDM patients, with lowest levels in the afternoon and evening and a rise with maximum levels during the night and morning. Fasting and 24-h mean levels of IGFBP-1 were significantly higher in GH-deficient patients [61 +/- 12 (P < 0.01) and 39 +/- 6 micrograms/L (P < 0.01), respectively] and IDDM patients [72 +/- 18 (P < 0.01) and 45 +/- 9 micrograms/L (P < 0.01), respectively] compared to those in healthy subjects (27 +/- 4 and 18 +/- 2 micrograms/L, respectively). Fasting levels of IGFBP-1 correlated to 24-h mean values of IGFBP 1 in all groups separately and in the combined group (r = 0.931; P < 0.001). An inverse relationship was found between IGFBP-1 and insulin in GH-deficient patients, both between 24-h mean values (r = -0.756; P < 0.001) and between fasting values (r = -0.721; P < 0.001). Corresponding values for healthy subjects were r = -0.548; P = 0.065 and r = -0.712; P < 0.01, respectively, whereas in IDDM patients the relationship was nonsignificant. Moreover, in GH-deficient patients, the diurnal mean levels of IGFBP-1 were inversely correlated to IGF-I (r = -0.477; P < 0.05) and body mass index (r = -0.450; P < 0.05). When insulin was taken into account, a tendency for a negative correlation between IGFBP-1 and IGF-I (P = 0.054) remained, whereas the relationship to body mass index disappeared. However, IGFBP-1 levels were elevated in relation to insulin levels in GH-deficient patients compared to healthy subjects (F = 48.7; P < 0.001 and F = 32.5; P < 0.001, diurnal mean values and fasting values, respectively). The majority of the IDDM patients had values in the same range as the GH-deficient patients.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545694 TI - Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids. AB - Insulin-like growth factor binding proteins (IGFBPs) serve as transport proteins for the IGFs and modulate their biological effects. In order to evaluate the relative contribution of IGFBP-5 to the IGF binding capacity of serum and other biological fluids and to study more accurately the in vivo regulation of serum IGFBP-5, we have developed an RIA for IGFBP-5 using recombinant human IGFBP-5 as the antigen, tracer, and standard. The validity of the IGFBP-5 RIA was established from the following data: 1) recombinant human IGFBP-5 and purified human bone-derived IGFBP-5 inhibited the binding of tracer to the antisera in an identical manner; 2) the immunoreactive IGFBP-5 in serum co-migrated with recombinant human IGFBP-5 in both sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance gel filtration chromatography; 3) none of the other known IGFBPs (IGFBP-1, -2, -3, -4, and -6) exhibited significant cross reactivity; 4) the addition of IGF-I or IGF-II to the samples did not affect the recovery of IGFBP-5; and 5) the recovery of the exogenously added IGFBP-5 to serum was greater than 90%. In order to further validate our IGFBP-5 RIA, measurements of IGFBP-5 were carried out in the conditioned media samples collected from human bone cells treated with effectors that are known to influence IGFBP-5 production. Treatment of human bone cells with bone morphogenetic protein-7, which increases IGFBP-5 production, caused an increase in IGFBP-5 protein levels; dexamethasone, which decreases IGFBP-5 production, caused a decrease in IGFBP-5 protein levels measured in the conditioned medium by this RIA. Application of this RIA to the measurement of the IGFBP-5 level in human serum revealed that the circulating level of IGFBP-5 in healthy women decreased from 664 +/- 108 ug/L in prepubertal girls to 222 +/- 42 ug/L in women ages 61-85 yr (P < 0.001). There was also a small but significant decrease in the mean serum level of IGFBP-5 in women ages 23-39 yr compared with the level in prepubertal girls (589 +/- 66 vs. 664 +/- 108 ug/L, P < 0.05). Additional studies with this assay may provide an insight into the physiological mechanism that regulates IGFBP-5 production in vivo and the potential role of this protein as an IGF transport protein in serum and other biological fluids. PMID- 7545696 TI - The measurement of growth hormone bioactivity in patient serum using an eluted stain assay. AB - We have adapted the MTT-ESTA bioassay for human GH (hGH) to measure the lactogenic bioactivity of the hormone in human serum. This highly quantitative in vitro colorimetric bioassay is based upon the reduction of a tetrazolium salt, 3 [4,5-dimethyl-thiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), to its formazan by lactogen-activated Nb2 cells. Relatively high concentrations of human serum (1-10%) modified responses to the hormone in a complex manner. As the serum effects varied between samples, it proved impossible to adapt the bioassay by the conventional approach of using a lactogen-depleted serum as a representative matrix. However, as the Nb2 cells were exceptionally sensitive to hGH, the serum effects could be diluted out. We adopted a dilution strategy by which all samples of human serum were included in the bioassay at a concentration of 0.625% or less. A valid assay was obtained, as judged by the criteria of parallelism between diluted samples and hGH standards, and recoveries of spiked samples that were close to 100%. Hormonal specificity was achieved with the use of a highly specific anti-PRL antiserum. A within-assay precision of between 2-5% over the dose range of 0.03-0.96 microgram hGH/L was attained. As only highly diluted samples could be used, the sensitivity of the clinical bioassay was 1.2-2.4 micrograms hGH/L. The between-assay precision was estimated to be 11% and 9% at initial hGH concentrations in serum of 4.8 and 19.2 micrograms hGH/L, respectively. By exploiting the high sample capacity of the eluted stain bioassay system, we followed the changes in bioactivity and immunoactivity of hGH in multiple timed samples after stimulation of hGH secretion in an adult by GHRH. Systematic and progressive changes were observed in the bioactive/immunoactive ratios. Analogous changes were observed after insulin-induced hypoglycemia in a child with short stature. We speculate that the changes in the bioactive/immunoactive ratios reflect alterations in the proportions of the isoforms of hGH in the circulation after acute stimulation. PMID- 7545697 TI - Serum insulin-like growth factors (IGFs) and IGF binding proteins 1, 2, and 3 in children with chronic renal failure: relationship to height and glomerular filtration rate. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. AB - Serum levels of insulin-like growth factor I (IGF-I), IGF-II, and IGF binding protein 1 (IGFBP-1), IGFBP-2, and IGFBP-3 were measured in 94 children with chronic renal failure (CRF). The results were compared with their respective age dependent normal ranges, and the relationship with height and residual glomerular filtration rate (GFR) was examined. Each IGF and IGFBP was quantified by specific RIA. Serum IGF-I and IGF-II levels were in the normal range throughout their entire childhood in the vast majority of cases. The mean age-related IGF-I (0.07 +/- 0.14 SD score) and IGF-II levels (0.06 +/- 0.11 SD) were similar. Age-related IGF-II but not IGF-I levels showed a weak inverse linear correlation with residual GFRs (r = -0.24, P < 0.02). Mean age-related IGFBP-1 serum levels (1.04 +/- 0.09 SD) were slightly elevated, whereas mean age-related serum IGFBP-2 levels (3.25 +/- 0.20 SD) and serum IGFBP-3 levels (2.61 +/- 0.12 SD) were markedly elevated. Significant inverse correlations were found between GFRs and age-related IGFBP-1 (r = -0.42, P < 0.001), IG-FBP-2 (r = -0.56, P < 0.001), and IGFBP-3 (r = -0.28, P < 0.005), but the increase in IGFBP-2 with declining GFR was relatively more pronounced than the respective increase in IGFBP-1 and IGFBP 3. The correlation between age-related IGF-I and relative height in prepubertal children with CRF (n = 54, r = 0.43, P < 0.001) was lower than in prepubertal controls (n = 68, r = 0.67, P < 0.001), and the slope of the regression line was significantly less steep, indicating that the normal relationship between IGF-I and height is disturbed in CRF. The normal relationship between IGFBP-3 and height was disrupted in CRF. Forward stepwise regression analysis revealed that height in CRF is correlated with IGF-I and inversely correlated with IGFBP-2. We conclude that the imbalance between normal IGFs and excessive IGFBP serum levels in CRF plays a pathogenic role in the growth failure of these children. PMID- 7545698 TI - Regulation of the insulin-like growth factors and their binding proteins by glucocorticoid and growth hormone in nonislet cell tumor hypoglycemia. AB - Hypoglycemia in patients with nonislet cell tumors is often secondary to overexpression of tumor insulin-like growth factor (IGF) II. In these patients the formation of serum complexes between IGFs, IGF binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) is impaired. An 87-yr-old woman with nonislet cell tumor hypoglycemia resulting from a localized fibrous tumor of the pleura was treated for 97 days with graded doses of prednisolone (30, 10, and 5 mg/day) followed by GH (1, 4, 8, 4, and 2 U/day). Both prednisolone and GH alleviated the hypoglycemia, concomitantly with increases in IGF-I, IGFBP-3, and ALS levels. Pretreatment serum IGFBP-2 and IGFBP-6 levels were greatly elevated, but as glucose normalized with treatment, only IGFBP-2 decreased, showing an inverse correlation with glucose (r = 0.716). IGFBP-1 gave a variable pattern not clearly related to blood glucose. Both treatments caused a redistribution of serum IGFBP 3 from binary- to ternary-complexed forms. However, only prednisolone improved the ability of IGFBP-3 to bind ALS in vitro. Prednisolone also suppressed IGF-II, the effect being confined to pro-IGF-II forms. Compared with normal IGF-II, pro IGF-II inhibited ALS binding to IGFBP-3 in vitro. Although prednisolone and GH reverse hypoglycemia by different mechanisms, with only prednisolone suppressing tumor IGF-II secretion, both increase the formation of ternary IGF-IGFBP-3 complexes. We conclude that the failure of serum IGFBP-3 and tumor IGF-II to complex with ALS is a primary cause of hypoglycemia in nonislet cell tumor hypoglycemia. PMID- 7545699 TI - Estrogen- but not androgen-dominant human ovarian follicular fluid contains an insulin-like growth factor binding protein-4 protease. AB - Estrogen-dominant follicular fluid (FFe) and granulosa-luteal cell conditioned media, in contrast to androgen-dominant FF (FFa), contain barely detectable levels of insulin-like growth factor binding protein-4 (IGFBP-4) by ligand binding techniques. The current study was designed to evaluate the possibility of an IGFBP-4 protease in FFe, which may alter the affinity of IGFBP-4 for insulin like growth factors (IGFs), rendering IGFBP-4 undetectable by ligand binding techniques. FFe and FFa were obtained from regularly menstruating women, and FFe was also obtained during in vitro fertilization procedures. Mixing experiments were performed by using human recombinant IGFBP-4 or IGFBP-4 in nonpregnancy serum (NPS) as substrate and FF as the source of the putative protease. Incubation of NPS at 37C for 5 h in the presence of FFe resulted in the reduction of IGFBP-4 to barely detectable levels when analyzed by Western ligand blotting, with no change occurring in the levels of the other binding proteins present in NPS. In contrast, incubation of FFa with NPS under similar conditions had no effect on the levels of IGFBP-4. The disappearance of IGFBP-4 when NPS was mixed with FFe exhibited optimal pH-dependence at pH 7-9. Inhibition of the putative protease by aprotinin, ethylenediaminetetraacetic acid, and 1,10-phenanthroline supports its identification as a metalloserine protease. Western immunoblot analysis detected the presence of a proteolytic fragment of approximately 17-18 kDa after incubation of recombinant IGFBP-4 in the presence of FFe but not in the presence of FFa. Similar incubation of other recombinant human IGFBPs did not reveal their degradation, further suggesting that the protease in FFe is specific for IGFBP-4. These data demonstrate the presence of an IGFBP-4-specific metalloserine protease in FFe but not in FFa, and they suggest that proteolytic cleavage may be responsible for effectively decreasing levels of inhibitory IGFBP 4 and thus increasing bioavailability of IGF peptides within estrogen-dominant follicles. The importance of this mechanism may lie in providing the dominant follicle with more available IGFs to synergize with gonadotropins in stimulating estradiol production and in inhibiting this synergy in androgen-dominant and atretic follicles. PMID- 7545700 TI - Requirement of RNA synthesis for pathfinding by growing axons. AB - The effects of actinomycin D were studied in cultured grasshopper embryos at different stages of development by following the outgrowth patterns of identified neurones known as aCC, pCC, and Q1. When administered at stages occurring before 31% of embryonic development, actinomycin D (0.05-0.10 microM for 24-48 hours) prevented axon extension, whereas it did not affect the development of the nervous system in embryos older than 34% of development. At 31-34% of development, actinomycin D perturbed pathfinding of aCC without blocking axon extension. Thus, only 22% of the aCCs (n = 271) in embryos treated with actinomycin D extended an axon along the intersegmental nerve as in control embryos. In the remaining embryos, aCC failed to turn into the intersegmental nerve root; its growth cone remained in the longitudinal connective, above or below the turning point. Neurones of the group caudal to the intersegmental nerve root could extend along either the anterior or posterior commissure of the next posterior segment. In contrast to the observations made with aCC, only 1.2% of pCC (n = 166) and 0.0% of Q1 (n = 45) in embryos treated with actinomycin D showed axon growth along aberrant pathways. The position of the growth cones of most pCCs and all Q1s observed were in various points along their normal pathway. Both pCC and Q1, as a population, showed an extension rate significantly lower than that of their control counterparts. The effect of actinomycin D on aCC pathway choice was probably mediated by inhibition of RNA synthesis, because incorporation of uridine into RNA was reduced by 40%. The labelling of several monoclonal antibodies (1C10, 3B11, 7F7) that recognise surface glycoproteins (lachesin, fasciclin I, and REGA-1) involved in nervous system development of grasshopper embryos was suppressed. Our results suggest that the navigation of some axons along different pathways requires the synthesis of new mRNA. PMID- 7545701 TI - Nitric oxide-synthesising neurons in the central subnucleus of the nucleus tractus solitarius provide a major innervation of the rostral nucleus ambiguus in the rabbit. AB - We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents. PMID- 7545702 TI - Origin of ascending projections to the nuclei of the lateral lemniscus in the big brown bat, Eptesicus fuscus. AB - The nuclei of the lateral lemniscus in the echolocating bat, Eptesicus fuscus, are large and highly differentiated. In each nucleus, different characteristic response properties predominate. To determine whether the dissimilar response properties are due in part to differential ascending input, we examined the retrograde transport from small deposits of horseradish peroxidase (HRP) or HRP conjugated with wheat germ agglutinin (WGA-HRP) in the nuclei of the lateral lemniscus. The intermediate nucleus (INLL) and the two divisions of the ventral nucleus (VNLL) receive almost exclusively monaural input from the anteroventral and posteroventral cochlear nuclei and from the medial nucleus of the trapezoid body. Lesser inputs originate in the lateral nucleus of the trapezoid body and the ventral periolivary area. Although the three monaural nuclei of the lateral lemniscus all receive input from the same set of nuclei, and from the same identified cell types in the cochlear nucleus, there is a difference in the relative proportions of input from these sources. The dorsal nucleus (DNLL) receives input mostly from binaural structures, the lateral and medial superior olives and the contralateral DNLL, with only a minor projection from the cochlear nucleus. The lateral and medial superior olives project bilaterally; the bilateral projection from the medial superior olive is unusual in that it is found in only a few mammalian species. The results show a segregated pattern of binaural projections to DNLL and monaural projections to INLL and VNLL that is consistent with the binaural response properties found in DNLL and the exclusively monaural response properties found in INLL and VNLL. The differences in response properties between monaural nuclei, however, are not due to input from different nuclei or cell types but may be influenced by differing magnitudes of the constituent ascending projections. PMID- 7545703 TI - Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: a Golgi study. AB - The prenatal developmental histories of layer I, fibrous (white matter), and protoplasmic (gray matter) astrocytes have been studied in the human neocortex by the rapid Golgi method. The developmental route followed by each of these astrocytes is a distinct process which evolves from a specific precursor, occurs at a different time, and is linked to a specific event. The differentiation of layer I astrocytes is linked to the neocortex external glial limiting membrane (EGLM), that of fibrous astrocytes to the early white matter vascularization and maturation, and that of protoplasmic astrocytes to the late gray matter ascending vascularization and maturation. At the start of development, three glial precursors are established in the neocortex: 1) original radial neuroectodermal cells with nuclei above the primordial plexiform layer (PPL) by losing their ependymal and retaining their pial attachments become early astrocytes of layer I and EGLM components; 2) neuroectodermal cells with nuclei below the PPL that retain their pial and ependymal attachments become type I radial glial cells which are committed to the guidance of neurons and the early EGLM maintenance; and, 3) neuroectodermal cells that lose their pial but retain their ependymal attachment are transformed into type II radial glial precursors. By progressively losing their ependymal attachment, type II radial glia precursors become freely migrating cells, establish vascular contacts, and differentiate into fibrous astrocytes (and into oligodendrocytes?) throughout the subplate, developing white matter, and paraventricular regions. After the formation of the gray matter, additional layer I astrocytes are needed for the EGLM late prenatal and postnatal maintenance because type I radial glia cells start to regress and to reabsorb their EGLM endfeet. A late ependyma-to-pia migration of glial precursors progressively repopulates layer I with additional astrocytes and establishes the ephemeral subpial granular layer (SGL) of Ranke. From the 15th week of gestation to the time of birth, late astrocytes of layer I lose their EGLM attachments, migrate freely into the maturing gray matter, establish vascular contacts, and differentiate into protoplasmic astrocytes. The protoplasmic astrocytes of the gray matter evolve from transformation of layer I astrocytes rather than from radial glia cells as is generally believed. PMID- 7545704 TI - Human recombinant stem-cell factor induces melanocytic hyperplasia in susceptible patients. AB - BACKGROUND: Recombinant human stem-cell factor (SCF), a cytokine acting on hematopoietic progenitor cells, has potential for the treatment of several hematologic and oncologic disorders. In a hematology-oncology phase I trial of SCF, several patients had cutaneous hyperpigmentation at the SCF subcutaneous injection sites. OBJECTIVE: Our purpose was to investigate the pathogenesis of this hyperpigmentation phenomenon. METHODS: Skin biopsy specimens were obtained before, at the completion of, and after SCF therapy and were processed for histology, immunohistology, and electron microscopy. RESULTS: Skin at the site of SCF injection had an increased number of melanocytes, increased melanocytic dendrite extension, and melanin as compared with noninjected tissue. Immunohistochemical stains revealed an increase in staining with melanocyte specific monoclonal antibodies HMB-45 and NKI/beteb, and a monoclonal antibody to the receptor for SCF, c-kit. CONCLUSION: Subcutaneous injection of SCF results in hyperplasia of melanocytes. SCF may be useful in the treatment of melanocytopenic disorders, but caution may be necessary in patients with disorders of melanocyte proliferation. PMID- 7545706 TI - Histopathologic aspects of cutaneous metastatic disease. AB - In cutaneous metastatic disease the histologic pattern may be specific or nonspecific. Carcinoma cutis most often shows only the nonspecific pattern of an adenocarcinoma, a squamous cell carcinoma, or an undifferentiated metastatic tumor. However, certain clinical and histologic characteristics may suggest a primary tumor site and type. In addition, special histologic stains and the use of immunohistochemical and electronmicroscopic techniques may also prove valuable in the diagnosis of carcinoma cutis and other cutaneous metastatic diseases. PMID- 7545705 TI - Short-contact anthralin treatment augments therapeutic efficacy of cyclosporine in psoriasis: a clinical and pathologic study. AB - BACKGROUND: Psoriasis is characterized by immune activation and increased epidermal proliferation. Cyclosporine acts by reducing T lymphocyte numbers and lymphokine production. Anthralin inhibits keratinocyte proliferation. OBJECTIVE: We investigated whether topical anthralin would augment clearing of psoriasis produced by systemic cyclosporine. METHODS: Twelve patients with psoriasis were treated with cyclosporine (5 mg/kg per day). Patients applied anthralin only to plaques on half of their body. They were treated until a remission or maximum benefit was achieved. Disease activity was assessed by a severity index and quantitative histopathologic markers. RESULTS: Of the 12 patients, the skin of five cleared within 10 weeks irrespective of anthralin use. The other seven (slow responders) continued treatment for a mean of 18 weeks. Slow responders had a significantly lower severity index, a thinner epidermis, fewer CD8+ cells, and fewer proliferating keratinocytes on the anthralin-treated side than on the non anthralin-treated side. CONCLUSION: The combination of cyclosporine and topical anthralin is effective in patients who are slow to respond to cyclosporine alone. PMID- 7545707 TI - Proliferating cell nuclear antigen staining in deep-penetrating nevi. PMID- 7545708 TI - Clinical and histopathologic analysis of the relationship between lichen planus and chronic hepatitis C. AB - A prospective clinical investigation of 45 patients with lichen planus (LP) demonstrated a significant association between LP and chronic hepatitis C. Anti hepatitis C virus (HCV) antibodies were found in 17 (37.8%) of the 45 LP patients. This was significantly higher than in the controls. This higher prevalence of anti-HCV antibodies was found equally in both male and female patients in the three types of LP; cutaneous only type, mucous only type, and both cutaneous and mucous type. Most of the patients with positive anti-HCV antibodies had abnormal values of transaminase enzymes and/or a past history of chronic hepatitis. Histological and immunohistological investigations of three cases with LP and chronic hepatitis C demonstrated some morphologic similarities between these two diseases. Histopathologic findings of both LP and chronic hepatitis C were based on a T lymphocytic infiltrate with keratinocyte or hepatocyte damage. The degrees of infiltrating cells positive to UCHL-1, MX-panB, Leu-7, and human leukocyte antigen (HLA)-DR antibodies in the chronic hepatitis C lesions seemed to be similar to those in the LP lesions. These results may support a possible relationship between LP and chronic hepatitis C and the hypothesis that LP may be associated with chronic liver diseases as a result of a cytotoxic attack on the hepatocytes. PMID- 7545709 TI - Treatment of granuloma annulare with tranilast. AB - Three cases of granuloma annulare which did not exhibit a self-limited course were treated with tranilast at the dose of 300 mg/daily. The treatment resulted in the resolution of skin lesions within three months of administration. Although spontaneous resolution is often observed in granuloma annulare, tranilast may provide an alternative therapy for the treatment of cases resistant to spontaneous healing. PMID- 7545710 TI - Multiple isoforms of guinea pig decay-accelerating factor (DAF) generated by alternative splicing. AB - Human decay-accelerating factor (DAF, CD55), which is one of the regulators of complement activation (RCA), prevents complement activation on homologous cell membranes, resulting in a functional discrimination between self cells and invading microorganisms. DAF is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein and has been identified at the molecular level only in primates. We have isolated guinea pig DAF cDNA clones from a spleen library and identified six different classes. All encode the same four short consensus repeat domains with 58% amino acid sequence identity to human DAF, but show variability in the C terminal region. Alternative splicing of two optional exons generates transmembrane, GPI-anchored, and secreted forms of guinea pig DAF, and differential usage of splice sites in the single exon composed of internally quintuplicated sequences generates variable Ser/Thr-rich regions. Multiple isoforms were expressed ubiquitously in all tissues and cells tested. Similar variability in the Ser/Thr-rich region has been reported in human membrane cofactor protein (MCP), another membrane inhibitor of the RCA family. There seems to be a common ancestral sequence in DAF and MCP consisting of a Ser/Thr-rich region, but obviously multiplication occurred independently, indicating the importance of variability of the Ser/Thr-rich region for the effective regulation of complement activation. PMID- 7545711 TI - Molecular cloning and chromosomal localization of the mouse decay-accelerating factor genes. Duplicated genes encode glycosylphosphatidylinositol-anchored and transmembrane forms. AB - Regulation of complement activation is essential in the prevention of damage to autologous tissue. This activity is mediated by the presence of specific complement regulatory proteins on the surface of host cells. In humans, one molecule involved in this regulation is a 70-kDa glycoprotein that has been designated decay-accelerating factor (DAF). We present the full-length cDNA sequence and chromosomal localization of the mouse genetic homologue of the human DAF gene. Interestingly, two classes of cDNA clones were obtained that, rather than representing alternately spliced mRNAs, were derived from two separate but closely related linked genes. Both genes encoded proteins with an amino-terminal signal sequence, followed by four short consensus repeats and a domain rich in serine and threonine. Hydrophilicity plots and alignment with human DAF predicted that one gene encoded a glycosylphosphatidylinositol-anchored form of mouse Daf with 64% nucleotide and 47% amino acid identity to human DAF. The product encoded by the second gene was predicted to have an alternate amino-terminal signal sequence and carboxyl-terminal membrane-spanning and cytoplasmic domains. The two mouse Daf genes share 85% nucleotide and 78% amino acid identities, and have been designated Daf-glycosylphosphatidylinositol and Daf-transmembrane to reflect the two alternate mechanisms of membrane attachment. mRNA expression analysis indicated that the two mouse Daf genes were differentially expressed in the adult mouse. Chromosome localization studies mapped the mouse Daf genes to chromosome 1, where they segregated with the C4-binding protein (C4bp) gene. PMID- 7545712 TI - Domains 1 and 4 of vascular cell adhesion molecule-1 (CD106) both support very late activation antigen-4 (CD49d/CD29)-dependent monocyte transendothelial migration. AB - We investigated the role of the 6 domain (6D) and 7 domain (7D) forms of human VCAM-1 as counter-receptors for the alpha 4 beta 1 integrin (VLA-4) in monocyte migration induced by C5a. Across Chinese hamster ovary (CHO) cell monolayers transfected with VCAM-6D or VCAM-7D, monocyte migration was not inhibited by treatment of monocytes with mAb to CD18. Addition of mAb to alpha 4 to the CD18 mAb inhibited monocyte migration by 90% across CHO VCAM-6D and CHO VCAM-7D. mAbs to domain 1 (4B9) or domain 4 (GH12) of VCAM-1 each inhibited migration across CHO VCAM-7D partially, when monocytes were also treated with anti-CD18 mAb. When the VCAM-1 mAbs were combined, migration of these monocytes across CHO VCAM-7D was further inhibited to the same degree as with mAbs to alpha 4 plus CD18. IL-1 treated human umbilical vein endothelium (HUVE) supported CD18-independent, VLA-4 mediated monocyte migration to C5a. A mAb to domain 1 of VCAM-1 almost completely inhibited the CD18-independent migration across HUVE activated with IL-1 for 2 h or 20 h, but was less inhibitory when HUVE was treated with IL-1 for 5 h. However, when mAbs to domain 1 and domain 4 were combined, CD18-independent migration was inhibited completely under all conditions tested. These results suggest that either domain 1 or domain 4 of VCAM-1 can mediate VLA-4-dependent monocyte transendothelial migration, that VLA-4 interaction with these two domains can account for all of the VLA-4-mediated migration, and that the expression of VCAM-1 variants on HUVE depends partly on the duration of IL-1 activation. PMID- 7545713 TI - IL-13 down-regulates CD14 expression and TNF-alpha secretion in normal human monocytes. AB - CD14, a glycosylphosphatidylinositol (GPI)-linked protein expressed on monocytes and neutrophils, regulates monocyte-lymphocyte interactions and serves as the LPS receptor. We showed previously that IL-4 down-regulates the expression of human CD14 by acting at the transcriptional level. We now investigate whether CD14 expression could also be regulated by IL-13, another member of the chromosome 5 cytokine gene family. IL-13 dose-dependently inhibited CD14 expression on human monocytes. By contrast, expression of CD23 and CD11b was enhanced strongly. Down regulation of CD14 involved neither shedding nor activation of endogenous GPI anchor-cleaving enzymes. Indeed, soluble CD14 was not increased in the supernatants of IL-13-stimulated monocytes, and expression of CD55/DAF, another GPI-linked protein, was unaffected by IL-13. CD14 transcript levels were reduced sixfold in IL-13-treated monocytes. These results suggest that IL-13 down regulates membrane CD14 by suppressing CD14 RNA expression. IL-13-dependent down regulation of CD14 resulted in the inhibition of CD14-mediated events. Indeed, CD14-mediated release of TNF-alpha was inhibited markedly (approximately 75%) in monocytes stimulated with LPS (100 ng/ml) after a 72-h preincubation with IL-13. However, IL-13 also directly inhibited monokine secretion, because it blocked PMA induced, CD14-independent TNF-alpha release. Down-regulation of CD14 and TNF alpha secretion may play a major role in the anti-inflammatory effects of IL-13 on LPS-stimulated monocytes. PMID- 7545715 TI - Hepatitis C-related chronic liver disease among asymptomatic blood donors in the north west of England. AB - In the first 19 months of screening, the North Western Regional Transfusion Centre (RTC) tested 224,000 consecutive blood donors for antibody to hepatitis C virus (anti-HCV) by second generation enzyme immunoassay (EIA). Of these, 366 repeatedly reactive samples were referred for confirmatory testing at Manchester Public Health Laboratory (PHL). There, the initial EIA was repeated, together with two further EIAs. All the referred samples were subjected to a confirmatory line immunoblot (RIBA-II). Reverse transcription followed by the polymerase chain reaction (RT-PCR), in order to detect viral RNA, was performed on selected samples. Among the donors, 61 accepted offers for medical review and were assessed for risk factors, clinical findings and results of standard liver function tests. Of these donors, 53 proceeded to liver biopsy. The overall prevalence of confirmed positive donors was 0.04%. Main risk factors identified included intravenous drug abuse in 31 (51%) donors and prior blood transfusion in 12 (20%) but a risk factor was not apparent in 11 (18%). Viraemia, detected by RT PCR, could be predicted with a high degree of accuracy by means of the readily available and simpler screening and confirmatory tests (EIA and RIBA-II). Established chronic hepatitis was demonstrated in 90% of the liver biopsies. A trend towards worsening histological findings accompanied increasing concentrations of serum transaminase. Even so, many donors with normal transaminase values had abnormal biopsies including those showing chronic active hepatitis (CAH). These findings indicate that a substantial proportion of previously unrecognised asymptomatic persons with established chronic liver disease exists among North Western blood donors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545714 TI - IL-6 stimulates vitronectin gene expression in vivo. AB - We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6. PMID- 7545716 TI - 2nd International Symposium on Benign Prostatic Hyperplasia Therapy. L'Aquila, Italy, May 20, 1995. PMID- 7545717 TI - Benign prostatic hyperplasia (BPH): when and whom to treat? PMID- 7545718 TI - Benign prostatic hyperplasia (BPH) and sexuality. PMID- 7545719 TI - Open prostatectomy. PMID- 7545720 TI - Transurethral prostatectomy (TURP): still the gold standard? AB - 1. The indication for treating BPH can be established very precisely today. It is important to select the alternative procedures with a good workup for individual therapy. 2. A large volume prostate with proven obstruction is an indication for TURP or open prostatectomy, still the best choice of treatment with the best longterm results concerning all obstructive parameters. In future there may be less surgery due to the other possibilities but teaching and handling the gold standard should not be forgotten in using alternative techniques. 3. Especially for high risk patients there are minimal invasive methods so that a permanent catheter in a patient should be an exception. 4. Also with new medications we expect better results. Prostate selective alpha-blockers and reduction inhibitors probably also in combination of both may be sufficient therapy for many patients. It has to be examined whether minimal invasive instrumental treatments like transurethral thermotherapy can be compared with medication. The most important result of the discussion in the last years was improving the clinical workup for finding the indication for surgery. Too many new instrumental methods appeared at the same time and we are in a period of selection and investigation to know which technique can be compared with the gold standard of TURP. Also in using minimal invasive therapy the indication must be very clear because also the new techniques are not totally free from complications. PMID- 7545721 TI - Endocrine therapy for benign prostatic hyperplasia in the 90's. AB - Endocrine therapy by means of castration for benign prostatic hyperplasia was introduced already in the middle of the 19th century. The technique was never popularized and was abandoned following the introduction of safe surgical techniques. In the second half of this century, small series of various endocrine treatments have been reported, mainly using progestational agents. The hormone dependency of the prostate is unique, since testosterone itself is not very active on the prostate cells but has to be converted to 5 alpha dihydrotestosterone, which is almost ten times as effective an androgen in the prostate cell. By blocking this conversion, highly specific antiandrogenic effect will be obtained in the prostate but not in other organs of the body. The first 5 alpha-reductase inhibitor, finasteride, has proven effective in reducing prostate DHT. In large clinical trials, it has shown to reduce prostate size, improve urinary flow and reduce symptom score, statistically significantly better than placebo. The effect is sustained over at least 3 years. In a double-blind, randomized, placebo-controlled study over 2 years, patients were similarly improved in the finasteride group, whereas they deteriorated in the placebo group. This indicates that finasteride is able to halt the progression of the natural course of benign prostatic hyperplasia. Benign prostatic hyperplasia, generally believed to be a stromal disease, is potentially dependent on estrogens for its development. By blocking aromatization of testosterone to estrogen in the prostate cells, a hypothetical beneficial effect on the disease process should be gained. Results from phase II-studies have been promising. However, in placebo controlled studies, aromatase inhibitors did not perform better than placebo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545722 TI - BPH alpha blocker therapy. PMID- 7545723 TI - Transurethral microwave heating, current status and future perspectives. PMID- 7545724 TI - Quality of life, voiding symptoms and uroflowmetry in healthy males and benign prostatic hyperplasia (BPH) patients. PMID- 7545725 TI - Transurethral needle ablation of the prostate (TUNA). A new treatment of benign prostatic hyperplasia using interstitial radiofrequency energy. PMID- 7545726 TI - Laser therapy: to combine the best of the new with the best of the old. PMID- 7545727 TI - Prostatic stents--how good are they? PMID- 7545729 TI - To have a better quality of life. PMID- 7545728 TI - The urodynamic assessment of benign prostatic hyperplasia obstruction. PMID- 7545730 TI - I-PSS and type of treatment. PMID- 7545731 TI - Benign prostatic hyperplasia. Age and type of treatment. PMID- 7545732 TI - Benign prostatic hyperplasia: associated pathology. PMID- 7545733 TI - Cost benefit assessment of surgical therapy for benign prostatic hyperplasia in the DRG ERA. PMID- 7545734 TI - Benign prostatic hyperplasia (BPH) therapy: when? how? why? the take-home message. PMID- 7545735 TI - Benign prostatic hyperplasia (BHP) and bothersome voiding symptoms. PMID- 7545736 TI - Hydromorphone and metabolite pharmacokinetics in children. PMID- 7545737 TI - Diversity and modulation of ionic conductances in leech neurons. AB - A complete understanding of animal behavior at the cellular level requires detailed information on the intrinsic biophysical properties of neurons, muscles, and the synaptic connections they make. In the past 10 to 15 years, electrophysiological studies of leech neurons have revealed a diverse array of voltage-gated ionic conductances distinguished by their pharmacological sensitivity to classic ion channel blockers. Voltage-clamp studies have provided new information about the kinetics and voltage-dependence of Na+ conductances, several K+ currents, including IA, IK and IK(Ca.), and high- and low-voltage gated Ca2+ conductances. These studies showed that the action potentials of most leech neurons result from the usual sequence of permeability changes to Na+, K+, and Ca2+ ions. They also added insight as to the role played by particular combinations of conductances in providing individual neurons with electrical properties appropriate for the particular information they encode. Evidence is accumulating on the modulatory actions fo endogenous neurotransmitters such as FMRFamide, serotonin, and octopamine on motor behaviors in the animal. Parallel studies suggest that changes in behavior can be explained, at least in part, by the alteration of firing patterns of selected neurons and muscles resulting from modulation of multiple ion conductances. This makes the leech exceptionally attractive for neuroethological studies because it is one of the simplest organisms in which the methods of psychology and neurobiology can be combined. Information gathered from this animal will therefore increase our understanding regarding general principles underlying the cellular basis of behavior. PMID- 7545738 TI - The pseudocalanolides: structure revision of calanolides C and D. AB - Nmr spectra of synthetic structures corresponding to those initially reported for natural compounds calanolide C [1] and calanolide D [2] showed some subtle differences from those of the natural products. Further analysis has resulted in revision of the structures of the natural compounds, now renamed pseudocalanolides C [3] and D [4]. The absolute stereochemistry of pseudocalanolide C was established as [6S, 7S, 8R] using the modified Mosher's method. PMID- 7545739 TI - Tau protein concentrations in cerebrospinal fluid of patients with dementia of the Alzheimer type. AB - Tau protein concentrations were measured in the CSF of 23 patients with dementia of the Alzheimer type (DAT), 36 patients with multi-infarct dementia (MID), and 23 control subjects. Tau protein concentrations were significantly higher in patients with DAT than in controls (P < 0.001) and patients with MID (P < 0.001). A significantly positive correlation between CSF tau protein and glucose concentrations (r = 0.79, P < 0.001) and evolution of disease (r = 0.47, P < 0.05), and a negative correlation with Folstein's mental state examination test (r = -0.73, P < 0.001) were found in patients with DAT. PMID- 7545740 TI - A case of recurrent headache and neurological deficit. PMID- 7545741 TI - Bacterial adsorption to fetuin and mucin pellicle. AB - The ability of Actinomyces viscosus (A. viscosus) WVU 627 to attach to hydroxyapatite (HA) beads treated with either bovine fetuin or bovine mucin was studied. All preparations used were blocked with bovine serum albumin prior to incubation with [3H] thymidine-labeled A.viscosus cells. The quantity of fetuin or mucin adsorbed on the HA beads was determined by use of [3H] fetuin or [3H] mucin. Approximately 6 micrograms protein of [3H] fetuin and 20 micrograms of protein of [3H] mucin adsorbed to 5 mg of HA beads at saturation, respectively. The presence of fetuin or mucin on HA beads promoted attachment of the organism. However, HA beads treated with human whole saliva as a positive control promoted A.viscosus attachment more effectively than HA beads treated with these glycoproteins. Attachment of two additional strains (B 236 and B 25) of A. viscosus to HA beads was also promoted by these glycoproteins. The number of A.viscosus cells which attached to fetuin-treated HA beads was dose-dependent, except for strain B 25. For all three A.viscosus strains tested, attachment to the experimental pellicle with mucin was dose-dependent. These findings suggest that the use of these newly developed bacterial adhesion assay systems may be effective for elucidating bacterial adhesion mechanisms. PMID- 7545742 TI - Evidence of meningioma infiltration into cranial nerves: clinical implications for cavernous sinus meningiomas. AB - Anatomical and biological studies of cavernous sinus meningiomas help us understand the biological heterogeneity of these tumors. The question of whether cavernous sinus meningiomas infiltrate cranial nerves is clinically important because of the effect on treatment planning. In the authors' experience of treating 36 patients with cavernous sinus meningiomas, tumor invasion into a cranial nerve was documented in two patients in whom a cranial nerve was resected during the cavernous sinus dissection. In both patients, histological examination using hematoxylin and eosin and bodian stains showed infiltration of the cranial nerves by a benign meningioma which, to the best of the authors' knowledge, is a condition previously unreported. This histological finding of meningioma invasion into a cranial nerve demonstrates the biological heterogeneity of cavernous sinus meningiomas and raises concern about the invasive character of meningioma. Because not all tumor cells can be identified radiologically or by direct visualization at surgery, occult tumor infiltration predisposes a patient to recurrence despite the best neurosurgical efforts. Evidence of cranial nerve infiltration by meningioma suggests that, in some circumstances, cavernous sinus dissection in the hope of total removal of a meningioma may be futile and, in the long term, may provide no advantage over treatment options with lower morbidity. PMID- 7545744 TI - Repair of critical size rat calvarial defects using extracellular matrix protein gels. AB - In this study the authors examined the capacity of gels of reconstituted basement membrane, laminin, and type I collagen to mediate repair of critical size defects in rat calvaria. Although autografts are widely used to repair bone defects caused by trauma or surgical treatment of congenital malformations, neoplasms, and infections, an adequate quantity of graft is not always available. Allogenic bone is readily available, but its use is associated with an increased incidence of nonunion, fatigue fracture, and rejection. Biologically active, purified components of basement membranes, which have been shown to promote osteogenic differentiation and angiogenesis in vitro and type I collagen (the major constituent of bone extracellular matrix) can be formed into native isotonic space-filling gels. In this study critical size calvarial defects were created in retired male Sprague-Dawley rats. Thirty-six animals were divided into seven groups. Group 1 (control) received no treatment for the defects. Group 2 animals were implanted with methylcellulose. Groups 3, 4, 5, and 6 were implanted with gels of type I collagen, reconstituted basement membrane, or laminin, respectively. The last group of three animals (Group 7) was implanted with 100 micrograms of type I collagen gels (identical to Group 3) and sacrificed at 20 weeks following a single CT scan to determine if complete healing could be obtained with this method given sufficient time. Except for rats in the type I collagen group that was evaluated by multiple computerized tomography (CT) scans biweekly from 2 to 12 weeks, bone repair was evaluated using CT at 12 weeks. Healing was quantified using three-dimensional reconstruction of CT. Following the final CT scan in each experimental group, animals were sacrificed, and a sample of tissues was evaluated by conventional histology. Animals treated with type I collagen gels showed 87.5% repair of the area of the defects at 12 weeks and 92.5% repair by 20 weeks. Increasing the gel volume 1.5 x accelerated complete repair to 3 months. Murine-reconstituted basement membrane and laminin gels induced 55.5% and 46.3% repair, respectively, at 3 months. In untreated control animals 7% repair of the area of the defects showed at 3 months. Histological analysis confirmed new bone formation in partial and completely healed defects. Bioengineered native collagen gels may have wide applicability for bone repair as an alternative bone graft material alone, in combination with autograft or marrow aspirate, or as a delivery system for osteogenic growth factors. PMID- 7545745 TI - Poster competitions. Another way to increase university-service interchange. PMID- 7545743 TI - Inhibition by 5'-methylthioadenosine of cell growth and tyrosine kinase activity stimulated by fibroblast growth factor receptor in human gliomas. AB - Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5'-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor- or platelet-derived growth factor stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible. PMID- 7545746 TI - Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes. AB - The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure. PMID- 7545747 TI - Increased expression of the beta 1, alpha 5, and alpha v integrin adhesion receptor subunits occurs coincident with remodeling of stress-deprived rabbit anterior cruciate and medial collateral ligaments. AB - The biomechanical, biochemical, and morphological properties of the anterior cruciate and medial collateral ligaments are dramatically altered in response to deprivation of normal physical forces and joint motion. Integrin adhesion receptors are known to play important roles in the tissue remodeling that occurs in the course of normal wound repair. We propose that integrins play a similar role in the remodeling of the extracellular matrix in stress-deprived periarticular ligaments. This study tests the hypothesis that altered expression of integrins on ligament fibroblasts accompanies this remodeling. The left knees of 15 New Zealand White rabbits were surgically immobilized in acute flexion and the right knees served as controls (no operation). The anterior cruciate and medial collateral ligaments were harvested at 1, 3, 5, 9, or 12 weeks after immobilization. Sections from the ligaments were immunostained with monoclonal antibodies specific for the integrin subunits beta 1, alpha 5, alpha 6, and alpha v, as well as with a negative control antibody. Fibroblasts within both the stress-deprived anterior cruciate and medial collateral ligaments demonstrated markedly increased staining for the beta 1, alpha 5, and alpha v subunits, as compared with the controls. The increased staining was greatest at 9 weeks in the anterior cruciate ligament and at 12 weeks in the medial collateral ligament. Western blot study of ligament proteins extracted with sodium dodecyl sulfate demonstrated an increased amount of beta 1 subunit protein in both ligaments from knees that were stress deprived for 9 and 12 weeks, as compared with the control ligaments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545748 TI - CD44v6 expression in non-Hodgkin's lymphoma: an association with low histological grade and poor prognosis. AB - The family of CD44 glycoproteins has diverse functions in cell-cell and cell matrix interactions. The standard form of CD44 is of importance in the dissemination of lymphoma, whereas the clinical significance of the variant exon v6-containing forms of CD44 (CD44v6) is not known. The expression of different forms of CD44 was investigated by using antibodies against the constant part of CD44 (CD44c) and CD44v6 in 56 primary and 17 recurrent non-Hodgkin's lymphomas and correlated with several clinicopathological parameters and with prognosis. Fifty-seven per cent of the primary non-Hodgkin's lymphomas expressed CD44v6 and 73 per cent expressed the constant epitope. Expression of both CD44c and CD44v6 was associated with low histological grade of malignancy. CD44c expression was associated with a low cellular proliferation rate as assessed by DNA flow cytometry. Of several factors tested, high expression of the variant from v6 was the only factor that was associated with unfavourable recurrence-free survival (P = 0.04). We conclude that CD44v6 is associated with a low histological grade, but, on the other hand, with an unfavourable outcome, which suggests that the combination of CD44v6 and histological grading may form a particularly strong prognostic parameter in non-Hodgkin's lymphoma. PMID- 7545750 TI - Absorption enhancement of a hydrophilic model compound by verapamil after rectal administration to rats. AB - The use of verapamil as an absorption enhancer for the paracellular route in-vivo was studied using FITC-labelled dextran (molecular weight 4000) (FD-4) as a hydrophilic model compound for transport enhancement. The kinetics of FD-4 after intravenous doses of 1 or 10 mg could be described by a two-compartment model with a systemic clearance of approximately 2 mL min-1 and a terminal plasma half life of approximately 36 min. Rectal administration to rats, performed as a rectal infusion of 10 mg FD-4 together with 7 mM verapamil, resulted in a 10-fold increase in the percentage of the dose absorbed over a 5-h period compared with the control and a 6-fold increase compared with a bolus administration, although the total amount absorbed remained relatively low (approx. 3% maximum). Large inter-animal variation in effect values were noted. The data indicate that although verapamil is able to enhance the absorption of hydrophilic compounds in vivo, practical application of verapamil for this purpose doses not seem feasible. PMID- 7545749 TI - [Conformational analysis of peptide antagonists in the design of pseudopeptide and non-peptide antagonists]. AB - The comparative conformational analysis of four Substance P antagonists (NK1-) having different chemical structures allowed to formulate an hypothesis for a peptidic NK1 pharmacophore. The salient features of this pharmacophore (three aromatic groups and two carbonyl functions) belong to a beta-turn conformation. Thus, this kind of conformation might be the basis for the design of newer pseudo peptidic and non-peptidic NK1 antagonists. PMID- 7545751 TI - Development of a colon delivery capsule and the pharmacological activity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in beagle dogs. AB - A peroral dosage form was examined to deliver recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the colon in beagle dogs. A new gelatin capsule with its inside surface coated with ethylcellulose was prepared for this purpose. RhG-CSF was dissolved with propylene glycol and was filled in the capsule. Several kinds of ethylcellulose-gelatin capsules with an ethylcellulose layer of thickness 46 to 221 mm were used. The capsule was filled with propylene glycol solution containing fluorescein as an absorption marker, castor oil derivative and citric acid. The hardness of the capsule was tested after the gelatin layer was dissolved using a hardness tester and was dependent on the thickness of the ethylcellulose layer of the capsule. The time, Tmax, at which plasma fluorescein level reaches its maximum following oral administration of ethylcellulose capsules was used as a parameter for the in-vivo disintegration time of the ethylcellulose capsule into the colon. Capsules of thickness 84 mm with a Tmax of 4-6 h were filled with rhG-CSF solution containing fluorescein and were administered to dogs. After administration, blood samples were collected for 96 h and the blood total leucocyte (BTL) counts were measured as a pharmacological index of rhG-CSF. The maximum BTL count appeared at 10 h then gradually decreased and returned to its normal level at 48 h. These results suggest the usefulness of ethylcellulose capsules for the delivery of rhG-CSF to the colon and the possibility of a new oral rhG-CSF dosage form has been elucidated. PMID- 7545752 TI - Pattern and surgery for oesophageal carcinoma in Saudi Arabia. AB - Forty-three patients with carcinoma of the oesophagus are presented, 30 of whom were male and 13 female, with ages ranging from 42 to 88 years. Mean duration of symptoms was 4.6 months. Of patients, 65.2% were in stage III and 30.2% in stage IIA. Stages I and IV were encountered in one patient each. Twenty-five were Saudis and 14 non-Saudis. Of the Saudi patients, 64.3% were from the Qaseem region. 48.8% had subtotal oesophagectomy with neck anastomosis and 51.2% had limited oesophagectomy with intrathoracic anastomosis. The mortality rate was 11.6% The 5-year survival rate was 33.3%. We found sex, histology, stage of disease and extent of oesophageal resection influence long-term survival. PMID- 7545753 TI - Transurethral resection of the prostate in 539 patients at a district general hospital. AB - This study audited length of hospital stay, mortality and morbidity associated with transurethral resection of the prostate (TURP) in a district general hospital over a 4-year period between 1990 and 1993. Five hundred and thirty-nine TURPs (409 benign and 130 malignant; median age 69 years) were carried out, which represented a twofold increase over the 4-year period. Although the mean duration of stay was 5 days, 92.5% of patients had a hospital stay of less than 7 days. One (0.2%) death occurred in a patient with ischaemic heart disease on the first postoperative day. The commonest cause of morbidity and delayed discharge from hospital was presence of outflow symptoms in nine (1.7%) patients. Transurethral resection of the prostate is a safe procedure in an ever-increasing elderly population with concomitant medical illness, but improved identification of 'at risk' patients is required in order to further lower the morbidity associated with this procedure. PMID- 7545754 TI - Evaluation of serum and seminal plasma markers in the diagnosis of canine prostatic disorders. AB - Serum and seminal plasma concentrations or activities of acid phosphatase (AP), prostate specific antigen (PSA), and canine prostate specific esterase (CPSE) were measured in normal dogs, dogs with benign prostatic hyperplasia (BPH), dogs with bacterial prostatitis, and dogs with prostatic carcinoma to determine if these assays would be of value in differentiating dogs with prostatic carcinoma from normal dogs, and dogs with other prostatic disorders. In addition, tissue sections of prostatic adenocarcinomas were stained with antiprostatic AP, anti CPSE, and anti-PSA antibodies to determine if these would be suitable immunohistochemical markers of prostatic carcinoma. Prostate-specific antigen was not detected in canine serum or seminal plasma. Serum and seminal AP activities did not differ significantly between normal dogs and those with prostatic diseases, or among dogs with different prostatic disorders. Serum CPSE activities were significantly higher in dogs with BPH than in normal dogs. Mean serum CPSE activities in dogs with BPH, bacterial prostatitis, and prostatic carcinoma were not significantly different from each other. Slight to moderate immunohistochemical staining of canine prostatic adenocarcinomas was noted for prostatic AP and PSA; most tumors did not stain for CPSE. These results show that proteins of prostatic origin appear in the serum of dogs as a result of prostatic pathology, especially BPH. Canine prostatic adenocarcinoma does not appear to be associated with significant increases in CPSE or AP activities, possibly because of down-regulation of these enzymes by prostatic carcinoma cells. It is also possible that failure to detect significant differences resulted from limited statistical power for some groups and pairwise analyses because of the small number of dogs evaluated. PMID- 7545755 TI - Abrogation of experimental systemic lupus erythematosus and primary antiphospholipid syndrome with intravenous gamma globulin. AB - OBJECTIVE: To evaluate the effect of intravenous gamma globulin (IVGG) treatment on the immunological and clinical manifestations of experimental systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS). METHODS: BALB/c mice were actively immunized with anti-DNA (MIV-7) monoclonal antibodies (Mab) or anticardiolipin (aCL, CAM) Mab, to induce experimental SLE and primary APS, respectively. Eight weeks after immunization the mice were treated for 6 weeks with IVGG (whole molecule), F(ab')2, or Fc fragments. The following studies were carried out: autoantibody profile (ELISA), clinical manifestations including erythrocyte sedimentation rate (ESR), white blood cell and platelet count, immunoglobulin deposits in the kidneys, and fetal resorptions. The presence of antiidiotypic activity to anti-DNA and aCL antibodies in the IVGG was determined by inhibition studies employing the F(ab')2 as inhibitor. RESULTS: Following treatment with IVGG or IVGG F(ab')2, a complete clinical remission, manifested as normal ESR and leukocyte counts, and lack of proteinuria or immunoglobulin deposits in the kidneys in the mice with experimental SLE, normal activated partial thromboplastin time, and fetal resorption rate in the mice with experimental primary APS was achieved. Autoantibody titers in the mice decreased to within normal levels. Treatment with Fc fragments had no effect upon those variables. Inhibition studies pointed to the presence of antiidiotypic activity to anti-dsDAN and aCL antibodies in the IVGG preparation. CONCLUSION: Treatment with IVGG can lead to clinical and immunological remission in mice with experimental SLE and primary APS. This effect may be carried out through manipulation of the idiotypic network and neutralization of pathogenic autoantibodies. Our results may justify the use of IVGG in patients with SLE and/or APS. PMID- 7545756 TI - Vasculitis complicating granulocyte colony stimulating factor treatment of leukopenia and infection in Felty's syndrome. AB - Recombinant myeloid growth factors have been increasingly used in recent years to combat induced and disease associated neutropenia. Their application in the management of Felty's syndrome with intercurrent infection has raised concern that resultant neutrophilia and activation of a diverse array of polymorphonuclear cell functions may have an adverse effect on the rheumatoid disease process. We describe a patient with Felty's syndrome receiving short term treatment with recombinant human granulocyte colony stimulating factor (GCSF), who then developed acute renal failure in conjunction with leukocytoclastic vasculitis and presumptive gout. We address the issue of "adding fuel to the fire" and review reported implications of GCSF in induction of vasculitis. PMID- 7545757 TI - On withholding nutrition and hydration in the terminally ill: has palliative medicine gone too far? A reply. AB - Patients who are dying of cancer usually give up eating and then stop drinking. This raises ethical dilemmas about providing nutritional support and fluid replacement. The decision-making process should be based on a knowledge of the risks and benefits of giving or withholding treatments. There is no clear evidence that increased nutritional support or fluid therapy alters comfort, mental status or survival of patients who are dying. Rarely, subcutaneous fluid administration in the dying patient may be justified if the family remain distressed despite due consideration of the lack of medical benefit versus the risks. Some cancer patients who are not imminently dying become dehydrated from reversible conditions such as hypercalcaemia. This may mimic the effects of advanced cancer. These conditions should be sought and fluid replacement therapy should be given along with the specific treatments for the condition. PMID- 7545758 TI - Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase. AB - RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase. PMID- 7545759 TI - Decreased growth of established human prostate LNCaP tumors in nude mice fed a low-fat diet. AB - BACKGROUND: Geographic variation in the incidence of clinically detected prostate cancer is considerable, with a 120-fold greater incidence in the United States than in China. The incidence of latent prostate cancer, however, shows little variation worldwide, with approximately 30% of men older than age 50 years having microfocal disease (determined by autopsy). Some epidemiologic studies have suggested that a high intake of dietary fat may constitute a risk factor for the development of advanced prostate cancer. PURPOSE: We studied the influence of dietary fat content on the growth of tumors established in athymic nude mice with androgen-sensitive, human prostatic adenocarcinoma cells (LNCaP cells). We also investigated whether manipulation of dietary fat content altered prostate specific antigen (PSA) production by these tumors. METHODS: Tumors were induced in nude mice by subcutaneous injection of 10(6) LNCaP cells. Both the American Type Culture Collection (ATCC) LNCaP cell line and a more androgen-responsive subline derived from it (i.e., the Harris LNCaP cell line) were used. Mice were fed a 40.5-kcal% fat diet at the time of tumor cell injection. Three weeks later, after measurable tumors were formed, the animals were assigned to receive diets with one of the following fat contents: 40.5, 30.8, 21.2, 11.6, or 2.3 kcal% fat. Food intake, animal weights, and tumor volumes were recorded weekly; serum PSA and testosterone levels were measured at the termination of the study. Post hoc multiple comparisons were made using the Student-Newman-Keuls procedure. Two sided tests of statistical significance were used to evaluate pairwise comparisons. RESULTS: Tumor growth rates, final tumor weights, and ratios of final tumor weights to animal weights were substantially greater in groups that continued to receive a 40.5-kcal% fat diet than in groups whose diets were changed to 2.3 kcal%, 11.6 kcal%, or 21.2 kcal% fat (all P values < .04). Comparison of these parameters among the 2.3-kcal%, 11.6-kcal%, and 21.2-kcal% dietary fat groups did not reveal any statistically significant differences. No statistically significant differences were noted in total ingested calories, animal weight gain, serum testosterone levels, or histopathologic characteristics of the tumors among the tested dietary groups. Serum PSA levels were highest in the 40.5-kcal% fat group and lowest in the 2.3-kcal% fat group (evaluated only for ATCC LNCaP cells; P < .05). CONCLUSIONS: Reduction of dietary fat substantially slows the growth of tumors established from human prostatic adenocarcinoma cells in a murine xenograft model. A positive association persists between tumor volumes and serum PSA levels even after extreme modification of dietary fat content. PMID- 7545760 TI - Innervation of cutaneous structures in the mouse hind paw: a confocal microscopy immunohistochemical study. AB - The normal innervation of structures in mouse foot pads was investigated with immunohistochemistry and confocal microscopy. Nerves were visualized by incubating Zamboni fixed, thick, frozen sections with antibodies to protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and protein zero. The antibodies were localized using cyanine 3.18 labeled anti-rabbit gamma globulin. PGP 9.5 immunolocalization showed dense nerve bundles at the base of the foot pad with branches to larger blood vessels, sweat glands and epidermis. Sweat gland tubules were surrounded by numerous sudomotor axons; single fibers accompanied the sweat duct toward the skin's surface. Nerve bundles containing myelinated and unmyelinated axons ran through and around the centrally located sweat gland cluster to end in free nerve endings and Meissner's-like corpuscles at the apex of the foot pad. Other bundles running parallel to the epidermis gave arcuate branches that supplied epidermis on the sides of the pads with a rich nerve network, principally with free nerve endings that often reached the most superficial cell layers of epidermis. Calcitonin gene-related peptide-immunoreactive (-ir) nerves were distributed to dermis and epidermis in lower density than PGP 9.5-ir fibers. Substance P-ir fibers were less numerous; most terminated as free endings in deeper layers of epidermis. Vasoactive intestinal peptide-ir nerves almost exclusively innervated sweat glands, ducts and blood vessels, but not epidermis. The mouse hind paw has potential to serve as a model system for investigations of functional and morphological changes that affect peripheral and autonomic nerves under diverse experimental conditions. PMID- 7545762 TI - Alterations in leukocyte adhesion molecule expression after burn injury. AB - OBJECTIVE: To determine if thermal injury alters the expression of leukocyte adhesion molecules. DESIGN: This is a controlled experimental animal study. MATERIALS AND METHODS: Partial thickness burns were created on the backs of New Zealand White rabbits. At 30 minutes, 2 hours, 4 hours, and 24 hours after burn, skin was harvested for immunohistochemistry. Monoclonal antibodies were used to study changes in intercellular adhesion molecule 1 (ICAM-1), E-selectin, and leukocyte CD11a. Staining was graded on a scale of 0 to 4. MEASUREMENTS AND MAIN RESULTS: ICAM-1 was significantly decreased at 24 hours after burn (p < 0.007, Wilcoxon signed rank test). CD11a was increased at 30 minutes (p < 0.02), 2 hours (p < 0.02), and 24 hours (p < 0.006). E-selectin was increased at 2 hours (p < 0.03). CONCLUSION: Thermal injury alters the expression of leukocyte adhesion molecules. PMID- 7545761 TI - Abnormal expression and glycosylation of the large and small isoforms of myelin associated glycoprotein in dysmyelinating quaking mutants. AB - The relative expression of large (L) and small (S) isoforms of the myelin associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants. PMID- 7545763 TI - Adjuvant recombinant human growth hormone stimulates insulin-like growth factor binding protein-3 secretion in critically ill trauma patients. AB - The early catabolic phase of severe injury is associated with acute growth hormone (GH), insulin-like growth factor-1 (IGF-1), and insulin-like growth factor binding protein-3 (IGFBP-30 deficiency. The metabolic half-life of circulating IGF-1 is prolonged by its binding to IGFBP-3. The role of this binding protein in nutritionally repleted multiple-injury patients has not been previously evaluated. We have measured plasma levels of these polypeptides and nitrogen (N) balance in 18 adult (15 males/3 females; mean age, 45 years), severely injured, hypermetabolic, and highly catabolic trauma patients within 48 to 60 hours after injury, when they were receiving maintenance fluids without calories or N and during 6 days of total parenteral nutrition (TPN). Before instituting TPN, the patients were randomized to receive (group H, n = 9) or not to receive (group C, n = 9) daily recombinant human growth hormone (rhGH), 0.15 mg/kg IM. Adjuvant rhGH significantly increases plasma levels of GH, IGF-1, IGFBP 3, and insulin. In addition, it shows better improvement in N balance. The bioavailability of IGF-1 is increased, as indicated by the decrease in IGFBP 3:IGF-1 ratio. A significant correlation between IGF-1 and IGFBP-3 levels is present in the trauma patients who received TPN and rhGH. A GH/IGF-1/IGFBP-3 axis that closely regulates the metabolic status of the patient is established in trauma. PMID- 7545764 TI - Effects of nitric oxide synthase inhibition on regional blood flow in a porcine model of endotoxic shock. AB - The results of early studies suggest that nitric oxide (NO) synthesis inhibition may be therapeutic in sepsis, but recent data indicate that NO inhibition may be harmful. This study investigates the effects of NO synthesis inhibition with N nitro-L-arginine methyl ester (NAME) on regional blood flow following endotoxemia. Anesthetized, instrumented swine were randomly divided into four groups. Controls received normal saline resuscitation (NSR) at 1 cc/kg/min beginning at T0. The lipopolysaccharide group (LPS) received NSR and Escherichia coli LPS, 200 micrograms/kg at T0. The LPS+NAME group received NSR and LPS at T0, plus NAME (50 micrograms/kg/min) starting at T1. The NAME group received only NSR and NAME. Hemodynamic data, regional blood flow, and gastric intramucosal pH (pHi) were measured hourly. LPS increased renal and carotid blood flow consistent with a hyperdynamic state. Mesenteric blood flow was decreased. Treatment of endotoxic animals with NAME decreased renal and carotid blood flow. Mesenteric blood flow and gastric pHi were improved by NAME. NO inhibition in endotoxic shock results in decreased carotid and renal blood flow, by decreasing cardiac output. Mesenteric blood flow and perfusion were improved; however, this requires further study for validation. PMID- 7545765 TI - [Adhesion molecules and leukocyte functions]. AB - The normal inflammatory response to infections requires the peripheral leukocytes to migrate across the blood vessels to the site of infection in response to chemotactic factors released in the site. The importance of cell to cell adhesion between the leukocytes and vascular endothelial cells in the leukocyte migration across the blood vessels was substantiated by the finding of a recently defined heritable disease called leukocyte adhesion deficiency (LAD). LAD is a disorder characterized by recurrent bacterial infections, impaired pus formation and wound healing, persistent periodontitis, and an abnormally high peripheral leukocyte count. The profound pathogenesis of the LAD has been defined as impairments in adhesion-dependent leukocyte functions such as chemotaxis due to a lack of leukocyte cell-surface expression of adhesion molecules of LFA-1, Mac-1 and p150/95, the beta 2-integrin. Very recently, a new disorder having almost the same clinical features and leukocyte dysfunctions as the LAD but normal expression of the beta 2-integrin was identified. The disease lacks cell-surface carbohydrate components, the sialyl Lewis antigens which are the ligands of another kind of adhesion molecule of selectins expressed on the vascular endothelial cells. The presence of these two diseases indicates that the leukocyte locomotion requires cell to cell adhesion through both the beta 2 integrin and selectins. PMID- 7545766 TI - [Clinical evaluation of enzyme immuno assay systems for HCV antibody--synthetic peptide antigen and recombinant antigen]. AB - To evaluate the frequency of the false positive results, we examined 168 patients for the presence of HCV using two HCV antibody assay systems, Synpep HCV-EIA (Synpep) and Abbott EIA II (Abbott). The results obtained by the two methods were significantly different in 22 patients. Cases in 17 of these patients, the results were positive with Abbott but negative with Synpep, and there were no clinical signs or delectable virus RNA. However, in 2 cases, the results were markedly positive with Abbott and weakly positive with Synpep. The presence of virus RNA and the increase of transaminase were observed in one case but both were noted in the other case. The serum of these two patients reacted with the C33C and C22-3 regions in RIBA II. We observed another 2 cases in which the elevation of the cut-off index with Synpep preceded that with Abbott at the early stage of acute hepatitis C. We also compared the cut-off index with the histology activity index (HAI) score determined by liver biopsy. The average cut-off index with Synpep was proportional to the HAI score in the range between 0 and 13. Based on the cut-off index/HAI score relationship, we suggest that patients with inactive chronic hepatitis show a Synpep cut-off index less than 11. PMID- 7545767 TI - ICG-enhanced digital angiography and photocoagulation of choroidal neovascularization in age-related macular degeneration. AB - Choroidal neovascular membranes are often poorly defined on fluorescein angiography because of fluorescein leakage or blockage of hyperfluorescence by overlying hemorrhage, lipid, turbid fluid, or pigment. Indocyanine green (ICG) is a highly protein-bound dye in the near infrared portion of the spectrum. Therefore, ICG remained in and around the neovascular membrane and enhanced the visualization of certain membranes poorly defined with fluorescein. ICG penetrated through the overlying turbid tissue, and improved the visualization of the underlying choroidal neovascular membrane. Using an infrared angiography system, the authors obtained 21 ICG-angiograms with suspected choroidal neovascularization, and compared them to fluorescein angiograms. In 5 of the 21 eyes, occult choroidal neovascularization was well delineated on the ICG angiograms. In 2 eyes, we were able to detect a well-defined choroidal neovascular membrane underlying a subretinal hemorrhage. In 12 of the 21 eyes with choroidal neovascular membrane, we performed argon-green laser photocoagulation applying the overlay technique of the ICG angiogram to red-free photo or the early fluorescein angiogram, and evaluated the effect of full coverage laser treatment. PMID- 7545768 TI - Current approaches in patients with ventricular tachyarrhythmias. AB - The article has summarized the studies and ongoing trials looking at the significance and treatment of ventricular tachyarrhythmias. In most instances, the presence of these arrhythmias is associated with an increased risk of future arrhythmic events. Electrophysiologic studies are helpful in risk stratification in patients with coronary artery disease but can be misleading in the setting of dilated cardiomyopathy and often produce nonspecific results in patients with HCM. The need for an invasive electrophysiologic study is crucial in the diagnosis of certain ventricular arrhythmias that are amenable to cure with radiofrequency catheter ablation, such as idiopathic ventricular tachycardia and BBR-VT. The correct approach for patients with SVT not amenable to catheter ablation remains to be determined. In deciding whether to use a device or drug therapy, however, one should take into consideration the degree of left ventricular dysfunction and the overall health status of the patient. For example, device implantation clearly reduces sudden death in patients with severe left ventricular dysfunction but may not change total mortality because these same patients may die of congestive heart failure. Device therapy might be more cost-effective for patients with less severe depression of left ventricular function. PMID- 7545769 TI - Depletion of myelin-basic-protein autoreactive T cells by T-cell vaccination: pilot trial in multiple sclerosis. AB - In a pilot trial, eight patients with multiple sclerosis were matched to control patients and received T-cell vaccination with irradiated T cells reactive to myelin basic protein (MBP) to deplete circulating MBP-reactive T cells. In the 2 years before and after vaccination, exacerbations decreased in five vaccinated patients (numbers 1, 2, 6-8) with relapsing-remitting disease from sixteen to three, respectively, and from twelve to ten in their matched controls. Magnetic resonance imaging showed a mean 8.0% increase in brain lesion size in the vaccinated patients compared with a 39.5% increase in the controls. Lesions and/or relapses worsened in three cases after vaccination in association with reappearance of circulating MBP-reactive T cells. PMID- 7545770 TI - Treatment of schizophrenia. PMID- 7545771 TI - Effects of inhibition of nitric oxide synthesis on TNF alpha serum levels in E. coli endotoxemic rats. AB - We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNF alpha serum levels in rats inoculated with E. Coli endotoxin (30 mg/kg i.v.) Pre-treatment of endotoxemic rats with NG-monomethyl-L arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNF alpha serum levels. By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modify serum TNF alpha. These results suggest that, in E. Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNF alpha synthesis. PMID- 7545772 TI - Enhancement of FK-506 activities by ganglioside (GM3) in vivo. AB - Influence of FK-506 and ganglioside (GM3) on acquired immunity to Hymenolepis nana reinfection was examined in BALB/c mice. Treatment of mice with FK-506 abolished the acquired immunity to challenge infection with eggs of H. nana when the agent was injected inraperitoneally at daily doses of 9.0 to 11.0 mg/kg (but not 8.5 mg/kg) on days -1, 0, 1, 2, and 3 relative to the challenge. Intravenous injection of GM3 at a daily dose of 10.0 mg/kg could not produce an immunosuppressive effect on the acquired immunity. Combination treatment with 8.5 mg/kg/day FK-506 and 10.0 mg/kg/day of GM3 inhibited the acquired immunity. These results suggest that GM3 will be a good candidate for a clinical supplementary immunosuppressive agent. PMID- 7545773 TI - Endothelium-dependent vasorelaxant effect of trilinolein: mediated by nitric oxide and cyclic GMP. AB - At concentrations ranged from 0.1 nM to 1 microM, trilinolein concentration dependently relaxed the phenylephrine-induced constriction of isolated rat aorta. Concentration-response curves for the interaction between trilinolein and phenylephrine showed that trilinolein was unlikely a competitive antagonist of phenylephrine. The vasorelaxant effect of trilinolein was dependent on the presence of intact endothelium. Both NG-nitro-L-arginine methyl ester and methylene blue antagonized this vasorelaxant effect. L-arginine partially reversed the effect of L-NAME on trilinolein. Linoleic acid had no vasorelaxant effect. We concluded that trilinolein is an endothelium dependent vasorelaxant and the underlying mechanism could be a stimulation of the nitric oxide and cyclic GMP pathway. PMID- 7545774 TI - Hepatitis C virus infection and genotypes in Japanese hemophiliacs. AB - Liver function and antibodies to hepatitis C virus and to human immunodeficiency virus-1 were examined in 195 Japanese patients with hemophilia. One hundred and seventy-three were positive for antibody to HCV and 61 for antibody to human immunodeficiency virus-1. In 63 patients, we examined HCV genotypes according to the double polymerase chain reaction method. Forty cases (63%) were infected with hepatitis C virus with a single genotype, including type 1a in five, type 1b in 21, type 2a in seven and type 2b in seven; 16 (25%) were infected with double genotypes, including types 1a + 1b in 14, types 1b + 2a in one and types 1b+2b in one; and four (6%) were infected with triple genotypes, including types 1a + 1b + 2b in two. Genotype could not be determined in three patients by this method. In the 191 nonhemophiliac patients with chronic hepatitis C, HCV genotyping was as follows: type 1a in 0, type 1b in 121, type 2a in 40 and type 2b in 10 of 171 cases (89.5%) with single infection and types 1b + 2a in five and types 2a + 2b in one of six (5.5%) with double infection. In the remaining 14 patients, genotype could not be determined. Frequent transfusion of domestic and/or imported coagulation factor concentrates probably caused the high incidence of HCV infection with rare or mixed genotypes in Japanese hemophiliacs. PMID- 7545775 TI - Long interval between HCV infection and development of hepatocellular carcinoma. AB - A high prevalence of HCV infection has been reported in patients with hepatocellular carcinoma. The progression from acute transfusion-associated hepatitis to hepatic cirrhosis and hepatocellular carcinoma has been suggested in several studies to be very long. We have investigated the prevalence of anti-HCV and the interval between HCV infection and hepatocellular carcinoma among 191 consecutive patients with cirrhosis and liver-cell carcinoma. Serum samples from 191 patients with cirrhosis and hepatocellular carcinoma, consecutively diagnosed in our hospital between 1988 and 1993, were tested for serological markers of HBV and HCV infection. One hundred and forty-eight patients (77.5%; 95% confidence interval (c.i): 76% to 80%) were anti-HCV positive by 2nd generation enzyme immunoassay (confirmed by 2nd generation recombinant immunoblot assay) and 152 patients (79.5%; 95% c.i: 76% to 80%) were anti-HCV positive by 3rd generation enzyme immunoassay, while only 14 (7.4%; 95% c.i: 5% to 10%) were HBsAg positive. Of the 29 anti-HCV positive patients with previous transfusion, the interval between the date of blood transfusion and the diagnosis of hepatic cirrhosis was 24 +/- 12.5 years and that of hepatocellular carcinoma was 26.8 +/- 12.4 years. These results confirm the high prevalence of HCV infection in patients with hepatocellular carcinoma and the slow sequential progression from HCV infection through cirrhosis and hepatocellular carcinoma. PMID- 7545776 TI - Procathepsin E and cathepsin E. PMID- 7545777 TI - Membrane-associated metalloproteinase recognized by characteristic cleavage of myelin basic protein: assay and isolation. PMID- 7545778 TI - The role of maternal serum alpha-fetoprotein, human chorionic gonadotrophin and oestriol in the antenatal screening of Down's syndrome. AB - The use of maternal age alone to identify pregnant mothers at risk of a fetus with Down's syndrome has recently been supplemented by maternal serum screening using biochemical markers such as alpha-protein, human chorionic gonadotrophin and oestriol. These tests have been reported to increase the sensitivity of antenatal detection of such fetuses from 35% to 67% with a false positive rate of 5%. However, these maternal serum markers may be affected by maternal weight, the smoking history of mothers and diabetes mellitus. Furthermore, such sensitivities are achieved only when gestational age is assessed accurately by ultrasound. Many further studies need to be carried out before the introduction of maternal serum screening into routine obstetric practice in Singapore. These include studies on the incidence of Down's syndrome in the local population, studies on the distribution of these serum markers in the second trimester of pregnancy, sensitivities and positive predictive values of such a test in the local population as well as the socio-economic implications of implementing such a screening test in the local obstetric population. PMID- 7545779 TI - First year's experience with an acute pain service--University Hospital Kuala Lumpur. AB - An Acute Pain Service (APS) was started in University Hospital, Kuala Lumpur by the Department of Anaesthesiology in October 1992 for more effective control of postoperative pain. The main modalities of treatment included patient controlled analgesia (PCA) using morphine or pethidine with PCA devises, epidural opiate analgesia (EOA) using tramadol or fentanyl/bupivacaine mixture and subcutaneous administration of morphine or pethidine. Five hundred and fifty-one patients were managed in the first year, with an overall patient satisfaction score of 83%. The majority (98.5%) of them were after abdominal or major orthopaedic surgery. Eighty per cent of patients scored < 3 on the verbal numeric pain scale, where 0 is no pain and 10 is the worst imaginable pain, on the first postoperative day. Nausea and vomiting was an unpleasant side effect in 20% of patients. PMID- 7545781 TI - B-cell non-Hodgkin's lymphoma presenting as a primary renal tumour in a child. AB - A 6-year-old boy presented with a unilateral renal tumour thought to be a Wilms' tumour. He had started a treatment but review of the histology and immunohistochemical studies proved the tumour to be a primary renal B-cell lymphoma. This case illustrates the importance of an open-minded approach in the histological examination of small round cell tumours in the kidney and immunohistochemical studies in such cases are strongly advocated. PMID- 7545780 TI - Recombinant human granulocyte-macrophage colony-stimulating factor in septic neutropenic pediatric cancer patients: detection of circulating hematopoietic precursor cells correlates with rapid granulocyte recovery. AB - Cycling intensive chemotherapy currently used to treat pediatric solid tumors induces severe neutropenia. Prolonged neutropenia is a major risk factor for septic death which occurs in up to 5% of febrile or septic neutropenic episodes. We treated 18 neutropenic pediatric cancer patients (eight females, 10 males) during 30 febrile and/or septic episodes with single daily doses of E. coli derived non-glycosylated recombinant human granulocyte-macrophage colony stimulating factor (rh-GM-CSF, 5 micrograms per kg of body weight). The cytokine was administered for a median period of 6.5 days (2-12 days). Analysis of circulating hematopoietic progenitor cells was performed at day 1 (baseline) and day 5 of rh-GM-CSF treatment and included flow cytometric CD34 analysis as well as the methylcellulose-based clonogenic assay. Prompt hematopoietic recovery and resolution of septic problems was observed in all children. The counts of leukocytes (WBC), absolute neutrophils (ANC), and platelets (PLT) rose above 1,000/microL, 1,000/microL, and 50,000/microL within 4 days (0-9), 5.5 days (2 13), and 6 days (0-14), respectively. Faster granulocyte recovery and improved recruitment of circulating hemopoietic precursors was observed in children with detectable amounts (> 0.1%) of CD34-positive mononuclear cells prior to rh-GM-CSF treatment. We conclude that, to some extent, the efficacy of rh-GM-CSF treatment in neutropenic cancer patients is influenced by the hematopoietic recovery status on the progenitor cell level. Although they respond more slowly to the treatment, patients without circulating CD34-positive progenitor cells may gain most from growth factor therapy. Rh-GM-CSF can be safely administered to febrile and/or septic neutropenic children treated for cancer. PMID- 7545782 TI - [The reliability and the significance of serum amylase levels in the diagnosis of the pancreatic injury]. AB - To clarify the diagnostic reliability and significance of serum amylase levels (SAL) in the patients with pancreatic injury (PI), we reviewed 67 PIs. The elapsed time between injury and arrival of the hospital (ETAI) in the patients with normal SAL (1.3 +/- 0.2 hours, mean +/- SE) was significantly shorter than those with hyperamylasemia (5.8 +/- 0.9 hours). SAL on arrival significantly correlated to ETAI in the patients with type I (contusion) and type III injury (ductal injury). Among 45 patients who had arrived over 3 hours after injury, none showed normal SAL. Regardless of severity of PIs, one third of the patients showed normal SAL on arrival within 3 hours after injury. Among 23 patients treated conservatively, SAL in 14 patients (60.9%) normalized within 48 hours after injury, and these patients had no complications related to PIs. Three of remaining 9, who had prolonged hyperamylasemia over 48 hours, had pancreatic ductal branch injury or pseudocysts. This frequency was significantly different compared to that in the patients whose SAL normalized within 48 hours after injury. In conclusion, SAL is unreliable and insignificant to diagnose PIs within 3 hours after injury. Not to overlook the PIs serologically, it is important to determine SAL over 3 hours after injury especially in the patients having stable vital signs and to whom PIs are strongly suspected clinically. PMID- 7545783 TI - Glucocorticoids regulate the secretion of a 21kDa-IGF-binding protein by sheep adipose tissue explants. AB - Using a solution phase assay we have demonstrated that sheep adipose tissue explants secrete insulin-like growth factor binding proteins (IGFBPs) when cultured in serum-free medium over a 24 h period. Further, we demonstrate that secretion of IGFBP(s) is inhibited (up to 50%) by incubation of the cultures in the presence of 10(-8) M dexamethasone. This inhibitory effect is overcome when insulin (10 ng/ml) and ovine growth hormone (100 ng/ml) are incubated together (but not separately) with glucocorticoid. Further characterisation of this IGF binding activity by high performance size exclusion chromatography and Western ligand blot analysis indicated that under our culture conditions sheep adipose tissue explants secrete one predominant 21 kDa IGFBP and it is this BP which is hormonally regulated as described above. We discuss our results in the context of endocrine/paracrine/autocrine control of adipose tissue metabolism and differentiation. PMID- 7545784 TI - Major and minor epitopes on the self antigen mouse cytochrome c mapped by site directed mutagenesis. AB - Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved. PMID- 7545785 TI - Antigen-binding sites dominate the surface properties of IgG antibodies. AB - A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties. PMID- 7545786 TI - Nitric oxide. No endothelial NO. PMID- 7545787 TI - Hypertension in mice lacking the gene for endothelial nitric oxide synthase. AB - Nitric oxide (NO), a potent vasodilator produced by endothelial cells, is thought to be the endothelium-dependent relaxing factor (EDRF) which mediates vascular relaxation in response to acetylcholine, bradykinin and substance P in many vascular beds. NO has been implicated in the regulation of blood pressure and regional blood flow, and also affects vascular smooth-muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Abnormalities in endothelial production of NO occur in atherosclerosis, diabetes and hypertension. Pharmacological blockade of NO production with arginine analogues such as L nitroarginine (L-NA) or L-N-arginine methyl ester affects multiple isoforms of nitric oxide synthase (NOS), and so cannot distinguish their physiological roles. To study the role of endothelial NOS (eNOS) in vascular function, we disrupted the gene encoding eNOS in mice. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure. PMID- 7545788 TI - Interaction of the erythropoietin and stem-cell-factor receptors. AB - Mutations in the KIT transmembrane protein-tyrosine kinase receptor affect erythropoiesis, resulting in fewer committed late progenitors (colony-forming unit erythroid, CFU-E) in the fetal liver. As the survival and proliferation of CFU-Es depend absolutely on erythropoietin (EPO), these results suggest that CFU Es cannot proliferate or mature further unless both the KIT and EPO receptor signalling pathways are functional. How KIT affects proliferation or differentiation of CFU-Es is not clear. Here we show that the KIT ligand SCF (for stem-cell factor) can replace EPO in supporting the growth and survival of HCD57 cells, an EPO-dependent erythroid-progenitor cell line expressing high levels of KIT. SCF supports the proliferation of 32D cells that express KIT only if they also express the EPO receptor. In HCD57 cells, SCF rapidly induces tyrosine phosphorylation of the EPO receptor, and KIT physically associates with the extended box 2 region in the cytoplasmic domain of the EPO receptor. Our results indicate that KIT may activate the EPO receptor by tyrosine phosphorylation to induce further proliferation and maturation of CFU-Es. PMID- 7545789 TI - Nitric oxide synthase activity is inducible in rat, but not rabbit alveolar macrophages, with a concomitant reduction in arginase activity. AB - Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 x 10(6) cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of 3H-citrulline (NO synthase activity) and 3H-ornithine (arginase activity) were determined. During incubation of rat alveolar macrophages with 3H-arginine clear amounts of 3H-citrulline and 3H ornithine (3.8 and 4.6% of the added 3H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra /intracellular of 17 and 70 for 3H-citrulline and 3H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of 3H citrulline was increased about 30-fold and this was accompanied by a reduction in 3H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mumol/l). Inhibition of NO synthase by NG-monomethyl-L arginine (L-NMMA, 100 mumol/l) in LPS treated alveolar macrophages reduced the formation 3H-citrulline by more than 90% and restored the 3H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of 3H citrulline and 3H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545790 TI - [Help with self-care]. AB - Instructions for self-treatment, whether printed, presented via computer or by audiovisual means, are effective in the management of phobias, panic disorder, other anxieties, depression, bulimia nervosa, obesity, alcohol problems, nicotine abuse, myocardial infarction, AIDS, compliance problems and the counseling of patients' relatives. A lasting improvement has been shown for up to 7 years. The mechanisms of effective self-change are discussed. PMID- 7545791 TI - Evidence for bidirectional changes in nitric oxide synthase activity in the rat striatum after excitotoxically (quinolinic acid) induced degeneration. AB - Nitric oxide, a gaseous inter- and intracellular messenger, is thought to mediate neurotoxicity via excitatory amino acid receptors which may contribute to the pathogenesis of a variety of neuronal diseases. Excitotoxin lesions induced by quinolinic acid were made unilaterally in the rat striatum to study biochemically, light- and electron microscopically the possible involvement of the nitric oxide synthesizing enzyme nitric oxide synthase in degeneration processes. 5 days after quinolinic acid injection nitric oxide synthase activity in the striatum was elevated to 196.5% (P < 0.005% as compared to controls). There was no requirement of Ca2+ for the enzyme activity measured indicating that the elevation is due to the inducible isoform of nitric oxide synthase. Parallel to the depletion of neurons by quinolinic acid a massive gliosis was seen. Whereas quiescent astroglial cells in the normal striatum did not show any light microscopically detectable nicotinamide adenine dinucleotide phosphate diaphorase reaction, reactive astroglia revealed a substantial labeling distributed over the cell body and their stellar processes. Within the lesion and, particularly, close to the needle tract the number of microglia/macrophages labeled by isolectin B4 increased dramatically. Reactive microglial cells macrophages, situated along the needle tract and characterized by a pseudopodic or a globular shape, contained highest staining activity. At the ultrastructural level only disintegrated, if any, neuronal perikarya were seen five days after quinolinic acid injection while numerous reactive glial cells were observed. Reactive astroglia showed nicotinamide adenine dinucleotide phosphate diaphorase activity by displaying a substantial labeling of the nuclear envelope and endoplasmic membranes. Occasionally stained mitochondria were encountered. Globular-shaped (ameboidal) microglia near the needle tract were rich in phagocytotic debris and, apart from formazan-positive endomembranes, their plasmalemma was often nicotinamide adenine dinucleotide phosphate diaphorase stained. Additionally, in those cells regions of highly electron-dense puncta were seen which differ sharply from other cytoplasmic areas. Such sand-like accumulations of nicotinamide adenine dinucleotide phosphate diaphorase positive grains have never been observed in other cell types, indicating a special type of nitric oxide synthase representation, possibly that of the inducible isoform. PMID- 7545792 TI - The effect of nitric oxide synthase inhibition on quinolinic acid toxicity in the rat striatum. AB - Neurons containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and acetylcholinesterase in the striatum are spared in Huntington's disease. It has been claimed that these neurons are also spared after intrastriatal injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid. In the present study the effects of intrastriatal injection of quinolinic acid (15, 30 and 60 nmol) on neurons containing NADPH diaphorase and acetylcholinesterase were examined in rats. Neurons identified histochemically were counted in whole striatal sections at the level of the injection site and at 400 microns intervals anterior and posterior to the injection site. There was a dose-related reduction in the total number of NADPH diaphorase-containing neurons counted in these levels, but only a mild loss of acetylcholinesterase-containing neurons. Acetylcholinesterase-positive neurons were observed near the injection site following administration of all doses. The effects of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (50 mg/kg, i.p. twice daily for seven days), on quinolinic acid (30 nmol. day 5)-induced toxicity were also investigated. Striatal sections were stained for NADPH diaphorase-, nitric oxide synthase- and acetylcholinesterase-containing neurons and cells were counted in whole striatal sections at the level of the injection site and at four levels posterior to the injection site. Nitric oxide synthase activity was measured in striatal homogenates. NG-Nitro-L-arginine methyl ester did not protect against or potentiate the loss of NADPH diaphorase-, nitric oxide synthase- or acetylcholinesterase-containing neurons or the loss in nitric oxide synthase activity. Acute intrastriatal injection of quinolinic acid may not be a suitable model for Huntington's disease and a role for nitric oxide in quinolinic acid induced toxicity is not supported in this model. PMID- 7545793 TI - Evidence for N-methyl-D-aspartate and AMPA subtypes of the glutamate receptor on substantia nigra dopamine neurons: possible preferential role for N-methyl-D aspartate receptors. AB - The present studies utilized extracellular single-unit recordings in chloral hydrate-anesthetized rats to evaluate the contribution of N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtypes of glutamate receptors to the excitatory effects of glutamate on substantia nigra dopamine neurons. Iontophoretic administration of NMDA, AMPA and glutamate increased the firing rate and amount of burst-firing of dopamine neurons. Iontophoretic application of the NMDA antagonist (+/-)-3-(2 carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP) inhibited the excitatory effect of NMDA and glutamate, but not that of AMPA. Iontophoretic application of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX), inhibited the excitatory effect of AMPA and glutamate, but not that of NMDA. CPP produced a greater antagonism of the glutamate excitation than did NBQX. In addition, CPP, but not NBQX, reduced the firing rate and burst-firing of a subpopulation of DA neurons. These data indicate that both NMDA and AMPA receptors are present on substantia nigra dopamine neurons and suggest that NMDA receptors may be more sensitive than AMPA receptors to endogenous glutamate and that a tonic glutamate tone, acting via NMDA receptor stimulation, may modulate the firing rate and burst-firing activity of some dopamine neurons. PMID- 7545794 TI - Embryonic hypothalamic expression of functional glutamate receptors. AB - Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamate's critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl 4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545795 TI - Contacts between serotoninergic fibres and dorsal horn spinocerebellar tract neurons in the cat and rat: a confocal microscopic study. AB - Contacts between serotoninergic nerve fibres and dorsal horn dorsal spinocerebellar tract neurons were analysed in order to investigate the morphological basis of actions of serotonin upon dorsal spinocerebellar tract neurons. In a series of experiments dorsal spinocerebellar tract neurons were labelled with intracellularly injected rhodamine-dextran in the cat. The neurons were monosynaptically excited by group II muscle afferents and cutaneous afferents and were identified by antidromic activation following stimuli applied in the cerebellum. In the second series of experiments dorsal spinocerebellar tract neurons were labelled by retrograde transport of Fluorogold injected into the cerebellum in the rat. In both series, serotoninergic fibres were labelled by using a specific anti-serotonin antiserum and were revealed by immunofluorescence. Appositions between the serotoninergic fibres and the cells were inspected with a dual channel confocal microscope. The merged images obtained with the two channels of the microscope were viewed in single optical planes 2 microns apart and in rotated three-dimensional reconstructions. Serotoninergic nerve fibres were found in apposition to cell bodies of all feline dorsal spinocerebellar tract neurons (n = 7) and of 75% of rat dorsal spinocerebellar tract neurons (n = 90). The numbers of putative contacts on cell bodies varied between less than 100 and nearly 300 (mean 160) in the cat and between about five and 30 in the rat. Contacts with dendrites of feline neurons were seen on 96% of 72 dendrites within 300 microns from soma and on 91% of 23 dendrites at distances of 300-500 microns. The number of such contacts varied from less than five to 150 on a single dendrite within these ranges of distances. Their total number within 100 microns from the soma was comparable or exceeded the number of contacts on the soma. PMID- 7545796 TI - Time-dependent changes in iron levels and associated neuronal loss within the substantia nigra following lesions within the neostriatum/globus pallidus complex. AB - The substantia nigra and globus pallidus (two iron-rich brain areas) receive a substantial innervation from the neostriatum, a considerable amount of which is GABAergic. Because of this anatomic relationship and the finding that prevention of GABA degradation in these two areas decreases their histochemical levels of iron, GABAergic transmission/metabolism may be important in regulating brain iron levels. Therefore, the present study investigated the effects of denervation of striatal/pallidal inputs to globus pallidus/substantia nigra on iron levels and associated pathologic changes in globus pallidus/substantia nigra. Adult male Sprague-Dawley rats received unilateral ibotenic acid infusions resulting in comprehensive lesions of the entire neostriatum/globus pallidus complex, or of either the anterior neostriatum or the posterior neostriatum/globus pallidus. Animals were killed at one week or one month following surgery. Between one week and one month postlesioning, comprehensive neostriatum/globus pallidus lesions induced a progressive decrease in substantia nigra volume, as well as a progressive increase in both substantia nigra zona reticularis iron staining and substantia nigra iron concentration. By one month following neostriatum/globus pallidus lesions, a marked 73% loss of substantia nigra zona reticularis neurons occurred in association with a 65% increase in glial cell numbers within zona reticularis. Compared to comprehensive neostriatum/globus pallidus lesions at the one month postlesion time point, more restricted anterior neostriatum and posterior neostriatum/globus pallidus lesions induced a less severe atrophy of the substantia nigra, a small (anterior neostriatum lesions) to moderate (posterior neostriatum/globus pallidus lesions) increase in substantia nigra zona reticularis iron staining, and either no zona reticularis neuronal loss (anterior neostriatum lesions) or limited zona reticularis neuronal loss selectively within areas of increased iron staining. These results suggest that destruction of striatal/pallidal innervation to the substantia nigra's zona reticularis induces a disruption of zona reticularis iron homeostasis, resulting in a redistribution and/or accumulation of iron in the zona reticularis and consequent zona reticularis of the substantia nigra neurodegeneration. The results further suggest that loss or dysfunction of striatonigral/striatopallidal GABAergic neurons in several neurodegenerative diseases (including Hallervorden-Spatz syndrome, progressive supranuclear palsy, multiple system atrophy, and Parkinson's disease) may result in an increase or redistribution of nigral iron to cause loss of substantia nigra neurons. PMID- 7545797 TI - Compression of the facial nerve caused increased nitric oxide synthase activity in the facial motor nucleus. AB - Nitric oxide synthase activities in the facial motor nucleus were studied in rats after unilateral compression of the facial nerve. Using a radiometric assay which measured the total soluble nitric oxide synthase activities in the facial motor nucleus and the surrounding tissues, it was found that nitric oxide synthase activities were markedly increased during facial paralysis that resulted from compression of the facial nerve. The subsequent decrease in nitric oxide synthase activities between postoperative days 20 and 40 coincided with the recovery of facial functions. In contrast, staining with NADPH-diaphorase histochemistry revealed that the diaphorase activities in the facial motor neurons were markedly increased between days 20-40 when the total activities as measured biochemically were in decline. However, staining of the vascular endothelium was increased on postoperative day 7 when the total activity was high. It is suggested that the increase in total nitric oxide synthase activities immediately after facial nerve compression may be predominantly endothelial. Since the increase in neuronal NADPH-diaphorase reactivity coincided with the recovery of facial functions, increased neuronal nitric oxide synthase may be a contributing factor to the restoration of facial innervation. The results of this study show that biochemical measurements of soluble nitric oxide synthase activities in tissue homogenates and NADPH-diaphorase histochemical staining in tissue sections may represent two distinct populations of nitric oxide synthase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545798 TI - Neurophysiological changes of spiny neurons in rat neostriatum after transient forebrain ischemia: an in vivo intracellular recording and staining study. AB - The spontaneous activities, evoked postsynaptic potentials and membrane properties of spiny neurons in rat neostriatum were compared before, during and after 5-8 min ischemia using intracellular recording and staining techniques in vivo. Severe forebrain ischemia was induced with the four-vessel occlusion method. Approximately 2.5 min after the onset of ischemia the baseline membrane potential quickly depolarized to -20 mV and remained at this level during ischemia. Repolarization began within 2 min after recirculation. The onset of ischemic depolarization was directly related to the severity of ischemia and its latency was inversely related to brain temperature. Spontaneous firing and membrane potential fluctuation of spiny neurons ceased immediately after ischemia and slowly recovered several hours after recirculation. No neuronal hyperactivity was observed up to 7 h after recirculation. Cortically evoked inhibitory postsynaptic potentials and late depolarizations disappeared earlier after ischemia and recovered later following recirculation than the initial excitatory postsynaptic potentials. Membrane input resistance of spiny neurons was significantly increased but the time constant remained the same following recirculation. The rheobase and spike threshold of spiny neurons were significantly increased and the repetitive firing evoked by depolarizing current pulse was suppressed shortly after recirculation. The results of the present study indicated that the spontaneous activity and evoked postsynaptic responses of spiny neurons are suppressed and the excitability of spiny neurons is decreased after transient ischemia. The polysynaptic responses are more sensitive to ischemia than the monosynaptic ones. PMID- 7545799 TI - Developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor properties and expression in the rat hippocampal formation. AB - The developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor properties in rat hippocampus were evaluated with quantitative autoradiography of ligand binding and in situ hybridization performed in adjacent sections with antisense oligonucleotides for AMPA receptor subunits (GluR1-3, flip and flop splice variants). Specific 3H-AMPA binding in different hippocampal subfields increased between postnatal day 7 and 15 and was higher in CA3 during the postnatal period when compared to adult levels. This effect was mostly due to high levels of high affinity binding sites in cell body layers during the developmental period. By contrast, autoradiograms of 3H-AMPA binding predominantly to the low affinity binding sites indicated an absence of these sites in cell body layers and the overall levels of binding exhibited little overshoot compared to adult levels during the developmental period. The changes in binding of the antagonist of the AMPA receptor, 6-nitro-7 cyanoquinoxaline-2,3-dione were markedly different from those for the high affinity AMPA binding sites but quite similar to those for the low affinity sites. The binding was extremely low at postnatal day 7 and increased rapidly between postnatal day 7 and 15 and slowly between postnatal day 15 and adult. Low levels of binding were observed in the cell body layer at every postnatal age. The changes in expression of messenger RNAs for the different subunits of the AMPA receptors were well correlated with the modifications in high affinity AMPA binding sites measured in the cell body layers also exhibiting an increased expression of the receptors at the transcriptional level during the developmental period as compared to adult levels. The relative expression of the GluR2 subunits decreased during the postnatal period and the time course for this reduction paralleled that for the increased vulnerability of hippocampal pyramidal neurons to a variety of insults. The results indicate that both the messenger RNAs for the subunits and the AMPA receptors exhibit increased levels of expression during the postnatal period compared to adult levels. They also suggest that nascent receptors might bind AMPA with high affinity before their insertion in membranes into functional receptors that have low affinity for agonists and high affinity for antagonists. The changes in subunit composition of the receptors during the postnatal period may have important implications for mechanisms of plasticity as well as of neuropathology. PMID- 7545800 TI - The ionic channel of neural nicotinic acetylcholine receptors is funnel-shaped. AB - Membrane currents evoked by iontophoretically applied acetylcholine were recorded from non-dissociated neurons of rat superior cervical ganglion using the whole cell patch-clamp recording method. Blocking effects produced by a series of specially synthesized organic compounds, the blockers of the open channel of ganglionic nicotinic acetylcholine receptors, used as tools, were studied, and dimensions of the channel were deduced from correlation between the sizes of the blocking molecules and their blocking activities. Two channel cross-profiles were found, small and large, as approximated by the rectangles with the most probable dimensions 5.8 x 8.0 A and 7.0 x (8.4 - 9.0) A, at intra- and extracellular sides of the channel, correspondingly, in addition to the 6.1 x 8.3 A (medium) cross profile found in our earlier work and localized between the two above. These findings indicate that the channel is funnel-shaped in the area where the open channel blockers are bound. The binding site is localized at the level of the medium cross-profile or between it and small cross-profile. The small cross profile is probably a selectivity filter. PMID- 7545801 TI - Distribution of nitric oxide synthase containing elements in the feline submandibular gland. AB - Combined nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunocytochemistry were used to study the distribution of nitric oxide synthesizing elements in the cat submandibular gland. A large number of thin varicose fibres, with intense staining for both markers, were seen around or in close contact with the acini. Some of the stained nerve fibres were associated with intra- and interlobular salivary ducts and blood vessels. All neurones in the submandibular ganglia showed intense staining for both NADPH-d and NOS. The epithelial layer of the salivary ductal branches and the endothelial lining of blood vessels were NOS immunonegative but NADPH-d positive. Our results suggest that NO might act as a neurotransmitter in the regulation of blood flow and secretion in the submandibular salivary gland. PMID- 7545802 TI - Selective accumulation of strontium-89 in metastatic deposits in bone: radio histological correlation. AB - The systemic administration of 89Sr has proven effective in the palliation of painful osseous metastases. Biodistribution studies with the gamma-emitter 85Sr suggest that both its uptake and retention are increased in bone metastases, where increased mineral turnover takes place. To study the pattern and nature of this process further, bones containing metastatic deposits were obtained from three patients who had previously been treated with 148 MBq of 89Sr. The bones were cut into 0.5-1.0 cm sections. The cut surfaces which faced together were marked with India ink, and adjacent sections were submitted for histology and autoradiography. Strontium deposition and retention were observed in regions which exhibited significant osteoblastic activity, mostly in areas adjacent to metastatic deposits, but also in subchondral and endosteal locations, as well as in an area corresponding to a pathological fracture with callus formation. With these exceptions, strontium deposition was not observed in histologically normal bone or within the marrow. Our findings demonstrate directly the selective nature of accumulation and retention of 89Sr and confirm previous clinical impressions. PMID- 7545803 TI - Angiogenesis in endometrial hyperplasia and stage I endometrial carcinoma. AB - OBJECTIVE: To evaluate angiogenesis in endometrial hyperplasia and stage I endometrial carcinoma, and to investigate the relationship between angiogenesis and tumor grade and depth of invasion. METHODS: Three groups of patients were analyzed: control patients who underwent hysterectomy for benign conditions (n = 19), patients with endometrial hyperplasia (n = 24), and patients with stage I endometrial carcinoma (n = 34). All hysterectomy specimens were stained immunohistochemically for factor VIII-related antigen as a sensitive and specific marker for vascular endothelium. Areas close to the deepest myometrial invasion or those with the highest grade of endometrial hyperplasia and the highest angiogenic intensity were selected. Three fields (x 400) were selected for each slide, and the mean microvessel count was calculated. Statistical analysis included Mann-Whitney U test or Kruskal-Wallis analysis of variance and Dunn post hoc procedure. P < .05 was considered significant. RESULTS: A significant difference was found between the microvessel count of controls versus the group with complex endometrial hyperplasia (median 21, range 16-80, versus median 38, range 20-130; P < .05). Microvessel counts of complex endometrial hyperplasia were significantly higher than those of simple hyperplasia (median 25, range 16 42; P < .05) and significantly lower than counts of endometrial carcinoma (median 77.5, range 19-189; P < .05). Microvessel counts in complex hyperplasia were not significantly different than those of noninvasive stage I endometrial carcinoma (median 38, range 20-130, versus median 44, range 19-119; P = .5). In cases of stage I endometrial carcinoma, a higher number of microvessels was noted in specimens with myometrial invasion than in those without myometrial invasion (median 44, range 19-119, versus median 83, range 19-189; P < .01). A higher number of microvessels was noted in cases with grade 2 than in those with grade 1, stage I endometrial carcinoma (median 44, range 19-98, versus median 96, range 63-189; P < .001). CONCLUSION: Complex endometrial hyperplasia and endometrial carcinoma are angiogenic. Furthermore, in stage I endometrial carcinoma, greater depth of invasion and higher tumor grade are directly correlated with angiogenic intensity. PMID- 7545804 TI - A multidirectional sonographic approach to elevated amniotic alpha-fetoprotein or positive acetylcholinesterase. AB - OBJECTIVE: To evaluate the combined transvaginal and transumbilical ultrasonographic approach in cases of elevated amniotic fluid (AF) alpha fetoprotein (AFP) and/or acetylcholinesterase in the second trimester. METHODS: Nine pregnant women with normal sonographic results were referred because of elevated AF AFP and/or acetylcholinesterase and screened using a transvaginal and transumbilical 6.5-MHz ultrasound transducer. All women were at 17-21 weeks' gestation. The fetal skull and the vertebral column were scanned in both perpendicular and tangential planes for maximal exploitation of the axial resolution of the transducer, in both directions and in two to three planes. RESULTS: Despite increased AF AFP and/or present acetylcholinesterase, no fetal malformation was detected in any of the nine cases. Open neural tube defects were not detected by the diagnostic technique. All nine women were delivered of term healthy neonates without malformations. CONCLUSION: Our preliminary data suggest that targeted sonographic screening combining transvaginal and transumbilical approaches may reliably rule out an open neural tube defect larger than 0.3 mm. This may influence the decision-making process when termination of pregnancy is considered in cases of high AF AFP and/or positive acetylcholinesterase. PMID- 7545805 TI - The oncogenic LIM protein Rbtn2 causes thymic developmental aberrations that precede malignancy in transgenic mice. AB - RBTN2 is activated by the chromosomal translocation t(11;14) (P13;p11) in some T cell leukaemias. Histologically similar T cell tumours develop with long latency in transgenic mice when either CD2 or thy1.1 promoters control rbtn2 expression. During the asymptomatic period, perturbation of T cell differentiation occurs in the thymus. The major anomalies present during this phase are an increase in the percentage of large thymocytes lacking CD4 and CD8 markers and also of small thymocytes express both the T cell marker CD3 and the B cell-specific form of CD45. These abnormal T cell populations can be clonal and thus a primary result of aberrant expression of the LIM-protein Rbtn2 is alteration of T cell differentiation preceding overt malignancy. These data provide a biological explanation for the role of Rbtn2 in tumorigenesis and presumably RBTN2 expression in T cells after the translocation t(11;14) in children has the same effect. PMID- 7545806 TI - H19 expression and tumorigenicity of choriocarcinoma derived cell lines. AB - Certain embryonal tumors demonstrate a loss of heterozygosity at the parentally imprinted region of chromosome 11p15.5. It has been hypothesized that this implicates a tumor suppressor gene at this locus. The human H19 gene maps to 11p15.5, is expressed in fetal tissues including the placenta and is paternally imprinted. Here we show that the abundance of H19 transcripts in cells of two choriocarcinoma derived cell lines (JAr and JEG-3) differs greatly. While JAr cells express high levels of H19 RNA, the expression of H19 in JEG-3 cells is much lower than that of normal trophoblasts. Cells of these two cell lines were subcutaneously injected into nude mice with subsequent tumor formation. A fivefold increase in the H19 RNA level was measured in tumors derived from JEG-3 cell lines as compared to these cells before injection. However this increase in H19 RNA did not alter the clonogenicity in soft agar nor the growth rate of the cells derived from these tumors as compared to the original JEG-3 cells. Nevertheless, the cells retaining the elevated level of H19 transcripts were more tumorigenic than the original cells. We propose that there is a selection of cells expressing high levels of H19 from the total JEG-3 cell population during the microevolution of tumor formation. These observations, together with our previous publications on H19 expression in human cancers, do not support the notion of a tumor suppressor role for the H19 gene. PMID- 7545807 TI - The acute promyelocytic leukaemia-associated PML gene is induced by interferon. AB - The PML protein concentrates within discrete nuclear structures known as nuclear bodies, also called NDs or PODs, which contain several proteins including the interferon (IFN)-inducible SP100 product. The function of these structures remains elusive. We and others have shown recently that they represent specific targets for adenovirus and herpes simplex virus. This prompted us to investigate whether PML, like SP100, might be induced by IFN and to explore the role of PML in viral infection. Here we report that PML mRNA levels increase rapidly in response to interferon treatment. This accumulation of PML transcripts is a primary IFN response since it does not require de novo protein synthesis. The IFN induced activation of the PML gene is accompanied by enhanced protein expression as revealed by immunolabelling. Both the intensity of the staining and the number of labelled structures increased upon interferon exposure. To probe the role of PML in IFN action, we compared the antiviral state established by alpha interferon in embryonic fibroblasts (EFs) derived from null mutant mice for PML and from wild-type control mice. The resistance to viral infection conferred by IFN-alpha was identical in both PML+/+ and PMLm/m fibroblasts indicating that PML is not an essential mediator of the antiviral effect of interferon. We also noted that DNA-binding factors are normally activated by IFN in PMLm/m cells. PMID- 7545808 TI - Epitopes in the 19kDa fragment of the Plasmodium falciparum major merozoite surface protein-1 (PfMSP-1(19)) recognized by human antibodies. AB - The antibody response to two different epitopes located in the C-terminal 19kDa fragment of the Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) has been studied using a competitive ELISA based on the inhibition of monoclonal antibody (MoAb) binding by serum samples. Sera from children aged three to eight years who suffered clinical symptoms of malaria, or were partially immune with an asymptomatic infection, and from adults all living in The Gambia, West Africa were tested. The results suggest that the antibody response to MSP-1(19) has a role in naturally-acquired immunity in Gambian individuals. PMID- 7545809 TI - [Molecular aspects of invasiveness and metastasis]. AB - Tumor progression involves the emergence of cell variants with increased proliferative and invasive potentialities. The acquisition of the invasive and metastatic behavior is associated with modulation of cell-cell and cell-substrate interactions. Tumor cells have to dissociate from the primary tumor and migrate through the basal lamina and the surrounding stroma before reaching the vessels. An aberrant expression of some growth factors and their cognate receptors, may contribute to an increase malignancy of tumor cells. We have postulated than such growth factors could be involved in the early events of metastatic spreading by altering cell interactions within a tumor, including proliferation, scattering and migration of tumor cells. In the rat bladder carcinoma NBT-II cell experimental model, we have shown that FGF-1 is a multifunctional factor during tumor progression; FGF-1 acts as a mitogenic factor, a scatter factor, an angiogenic factor, an inducer of matrix degradating enzymes and a tumorigenic factor. NBT-II cells producing constitutively FGF-1 are more invasive, tumorigenic and metastatic than non-producing cells. However, we have shown that within a tumor, FGF-1 producing cells are not dominant in vivo but rather confer by a community effect an "en bloc" behavior to the whole cell collective. This effect could be established either directly by a paracrine mechanism or indirectly by other induced factors. We provide evidence for a novel concept in tumor biology: tumor progression may result from a community effect mediated by a growth/scatter factor produced by a minority of the carcinoma cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545810 TI - Characterisation of Ca(2+)-dependent inwardly rectifying K+ currents in HeLa cells. AB - The whole-cell configuration of the patch-clamp technique was used to examine K+ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10(-7) mol/l free Ca2+, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K+ by isomolar Na+, greatly decreased inward currents and shifted the reversal potential as expected for K+ selectivity. The inwardly rectifying K+ currents exhibited little or no apparent voltage dependence within the range of from -120 mV to 120 mV. A square-root relationship between chord conductance and [K+] at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg2+ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K+ were: K+ (1.0) > Rb+ (0.86) > Cs+ (0.12) > Li (0.08) > Na+ (0.03). Slope conductance ratios measured at -100 mV were: Rb+ (1.66) > K+ (1.0) > Na+ (0.09) > Li (0.08) > Cs+ (0.06). K+ conductance was highly sensitive to intracellular free Ca2+ concentration. The relationship between conductance at 0 mV and Ca2+ concentration was well described by a Hill expression with a dissociation constant, KD, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba2+ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the KD for the blockade was analysed using a Woodhull type treatment, locating the ion interaction site at 19% of the distance across the electrical field of the membrane and a KD (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mumol/l and 3.5 mumol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K+ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at -120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5-10 mumol/l), histamine (1 mmol/l) or bradykinin (1-10 mumol/l) to cells dialysed with low-buffered intracellular solutions induced K+ currents showing inward rectification and a lack of voltage dependence. PMID- 7545811 TI - Decreased renal haemodynamic response to inhibition of nitric oxide synthase in subtotally nephrectomized rats. AB - To assess the renal haemodynamic response to manipulations of the nitric oxide (NO) system, we examined subtotally nephrectomized (SNX) rats and control rats (CON) 28 days after their operation. Bolus infusions of the NO synthase inhibitor NG-nitro-L-arginine (L-NA) were given intravenously at doses of 2 mg/kg and 10 mg/kg. Blood pressure was measured intra-arterially, glomerular filtration rate was measured by inulin clearance and fractional changes in renal blood flow (RBF) were determined by a Doppler flow probe. Both doses of L-NA caused a similar and dose-dependent increase in mean blood pressure in both SNX and CON rats. In contrast, the decrease in RBF and the increase in the renovascular resistance index (RVRI) was less in SNX rats as compared to CON rats (RBF = -70.1 +/- 2.2% of baseline vs -52.7 +/- 5.2%, P < 0.01; RVRI = +177 +/- 9% of baseline vs +243 +/- 24%, P < 0.05). These changes were not affected by autonomic blockade (hexamethonium), or by blockade of the angiotensin II receptor (Losartan). The exogenous NO donor sodium nitroprusside (0.5 and 1.5 micrograms.kg-1.min-1) lowered mean blood pressure to a similar degree in SNX and CON rats; in contrast, RVRI decreased less in SNX rats (86.9 +/- 9.2% of baseline) than in CON rats (68.2 +/- 4.6%, P < 0.05). We conclude that the reaction of the renal vasculature to manipulations of the NO system is altered in the SNX rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545812 TI - Characterization of two distinct Cl- conductances in fused human respiratory epithelial cells. II. Relation to cystic fibrosis gene product. AB - The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl- conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (Vm) and resistance (Rm) were recorded as well as their response to substitution of 90% of bath Cl- by isethionate (delta Vm,ISE), by I- (delta Vm,I), or by other halide anions. Fused CF cells had significantly (P < 0.05) higher control Vm values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than fused non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2+ ionophore A23187 with an increase in the Vm response to Cl- substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca(2+)-stimulated Cl- conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased delta Vm,ISE and delta Vm,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl- conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in Rm remains unexplained. Stimulation of the Ca(2+)-regulated Cl- conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545813 TI - [Hyperamylasemia and its origin in acute cholecystitis]. PMID- 7545814 TI - Prognostic significance of immunohistochemical proliferation markers (Ki-67/MIB-1 and proliferation-associated nuclear antigen), p53 protein accumulation, and neovascularization in clinical stage A nonseminomatous testicular germ cell tumors. AB - Histopathologic features alone fail to reliably stratify patients with clinical Stage A nonseminomatous germ cell tumors of the testis into groups with high and low risk for occult metastatic disease. Previous flow cytometric studies at Indiana University demonstrated a significant correlation between high proliferative activity and metastatic disease. The current study evaluated the prognostic significance of immunohistochemical markers related to tumor proliferation and aggressiveness in a consecutive series of clinical Stage A nonseminomatous germ cell tumors patients who underwent retroperitoneal lymph node dissection. Archival material of the orchiectomy specimens of 62 patients (45 pathologic Stage A, 17 with metastatic disease) was reviewed and immunohistochemically stained for Ki-67 antigen (MIB-1), proliferation-associated nuclear antigen (PC10), p53 protein (Pab1801), and Factor-VIII-related antigen (neovascularization). Staining with MIB-1 was significantly higher in the metastatic group (mean 80.2%, standard deviation [SD] 15.5) than in pathologic Stage A cases (66.3%, SD 27.9; P = 0.0032) and was predictive of metastatic status with a sensitivity of 82% and specificity of 69%. In this study, no patient with a MIB-1 value less than 52% had metastases. Proliferation-associated nuclear antigen and p53 staining correlated with MIB-1 values (R = 0.63 and 0.55, respectively) but did not correlate with metastatic status. Tumor angiogenesis was also not predictive of metastatic status. Assessment of proliferation rates using MIB-1 antibody in clinical Stage A nonseminomatous germ-cell-tumor patients may prove helpful in predicting metastatic status.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545815 TI - Expression of cytokeratin 7 in simultaneous mucinous tumors of the ovary and appendix. AB - Cytokeratin 7 (CK-7) is a simple epithelial keratin that may be used to investigate the site of origin of adenocarcinomas. In fact, CK-7 is present in ovarian epithelial neoplasms but is generally absent in colonic carcinomas. This pattern of CK-7 expression may aid in elucidating the genesis of mucinous tumors occurring simultaneously in the ovary and appendix, accompanied by psuedomyxoma peritonei. Five such cases were immunostained with anti-CK-7, and all showed a concordant staining pattern of the appendiceal, ovarian, and peritoneal lesions. Two cases showed a negative reaction for CK-7 and thus would appear to represent ovarian and peritoneal metastases from an appendiceal primary tumor. Three cases were CK-7 positive, and the nature of these mucinous lesions remains open to debate; they may either represent independent primary tumors or originate from the appendix. For comparison, five Stage I mucinous borderline tumors of the ovary and their normal appendices were also stained with anti-CK-7. These ovarian tumors were all CK-7 positive, whereas the appendices were negative. It is concluded that CK-7 is capable of distinguishing a group of tumors that can reliably be classified as primary appendiceal neoplasms metastatic to the ovaries and peritoneum. PMID- 7545816 TI - Frequency distribution of CD34 expression. PMID- 7545817 TI - Estradiol and chlordecone (Kepone) decrease adenosine 3'5'-cyclic monophosphate concentrations in the ovariectomized immature rat uterus. AB - Adenosine 3'5'-cyclic monophosphate (cAMP) has been repeatedly shown to mimic some actions of estrogen in the rat uterus. However, the relationship between estrogens and uterine cAMP remains controversial. The effect of chronic exposure (3 days) to a biologically potent, long-acting estrogen, estradiol benzoate (EB), or the xenoestrogen chlordecone (Kepone), which has a long half-life in the circulation, was examined in ovariectomized immature rats. Both compounds, when administered in doses that provided equal increases in uterine weight, produced equivalent decreases in uterine cAMP content. Although the decrease in cAMP was apparent within 48 hr, it was more pronounced at 72 hr. There was no reduction in cAMP produced in response to direct stimulation of uterine adenylyl cyclase by forskolin, indicating that loss of the enzyme was not a factor in the lowering of cAMP content. The pure anti-estrogen ICI-182,780, in a dose-dependent fashion, prevented the action the estradiol benzoate and chlordecone, suggesting that the lowering of cAMP was dependent on an estrogen receptor. The physiological significance of reduced uterine cAMP with chronic estrogen treatment remains to be determined. PMID- 7545818 TI - Relevance of mediators to cardiac parameters of isolated anaphylactic guinea-pig heart. AB - The release of histamine, eicosanoids and catecholamines were measured after induction of anaphylaxis in isolated guinea-pig hearts. The concentration-time profile of these mediators was compared with changes of cardiac parameters. The histamine and catecholamine levels of the coronary effluent were determined at 10 s intervals; thromboxane and prostacyclin levels at 60 s intervals. The release of histamine and norepinephrine were maximum between 20 and 30 s after the antigen challenge and decreased rapidly within 60 s. Thromboxane and prostacyclin increased to a maximum after 3 min and declined slowly within 10 min. The rise in histamine release was correlated with tachycardia. The release of thromboxane was correlated with the increase of coronary perfusion pressure. Cimetidine inhibited the tachycardia and clemastine reduced bradyarrhythmia. The inhibition of lipoxygenase and cyclooxygenase also reduced the rise in the perfusion pressure. These data suggest that different mediators are time-dependently involved in anaphylaxis-induced cardiac changes. PMID- 7545819 TI - Effects of an anti-beta-endorphin serum on tonic immobility in rabbits. AB - The involvement of endogenous beta-endorphin (beta-EP) in tonic immobility (TI) was evaluated in rabbits following the intracerebroventricular (ICV) administration of a specific antibody. Rabbits were tested twice with a 1-week intersession interval. For each animal, results of Session 1 were used as the baseline. Six hours before the beginning of Session 2, a bilateral ICV injection of specific anti-beta-EP serum or aspecific gamma-globulin (vehicle) was performed. Results showed that pretreatment with anti-beta-EP serum increased TI duration, whereas no change occurred in the vehicle group. In a parallel experiment we evaluated TI duration in the presence of formalin pain: TI increased in animals pretreated with aspecific gamma-globulins and decreased in the animals pretreated with anti-beta-EP serum. Results suggest that the beta-EP system acts to limit TI duration, but that this effect is reversed by persistent pain. PMID- 7545820 TI - Gender and estrous cycle effects of the 5-HT1A agonist, 8-OH-DPAT, on hypothalamic serotonin. AB - Effects of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.04, 0.25, or 1.0 mg/kg), on hypothalamic serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), and their ratio were determined in adult male rats and in diestrous, proestrous, and estrous female rats. Consistent with its action at the somatodendritic 5-HT1A autoreceptor, 8-OH-DPAT decreased the 5 HIAA/5-HT ratio, but the decrease was least evident in proestrous females and in males. Similar to hypothalamic tissue, there was also a decline in the 5-HIAA/5 HT ratio in the hippocampus after treatment with 0.25 mg/kg 8-OH-DPAT. When ovariectomized rats were treated with oil or estradiol benzoate followed 48 h later by oil or progesterone, 0.25 mg/kg 8-OH-DPAT produced a decrease in the hypothalamic 5-HIAA/5-HT ratio in every group except those rats treated with progesterone without estrogen priming. Treatment with estradiol benzoate increased hypothalamic 5-HIAA, and both progesterone and 8-OH-DPAT reduced the metabolite to the level of the ovariectomized control. These results suggest that both estrogen and progesterone contribute to an estrous cycle modulation of the 5 HT1A somatodendritic autoreceptor. PMID- 7545821 TI - A PET study of D2 and 5-HT2 receptor occupancy induced by risperidone in poor metabolizers of debrisoquin and risperidone. AB - The hydroxylation of the new antipsychotic drug risperidone to its main, active metabolite 9-hydroxyrisperidone is catalyzed by the hepatic cytochrome P450 enzyme CYP2D6, and cosegregates with the polymorphic hydroxylation of debrisoquin. We have previously examined central D2 dopamine and 5-HT2 receptor occupancy after 1 mg risperidone orally in three healthy subjects who were extensive metabolizers (EM) of debrisoquin, using positron emission tomography and the radioligands [11C]raclopride and [11C]NMSP. In this study, the same experimental design was repeated in two healthy poor metabolizers (PM) of debrisoquin to compare the D2 and 5-HT2 receptor occupancy induced by risperidone in EM and PM. The two PM had much higher plasma concentrations and longer elimination half-lives of risperidone than the three EM. Plasma concentrations of the sum of risperidone and 9-hydroxyrisperidone partly overlapped among the EM and PM. D2 receptor occupancy was 50% and 54% in the two PM, as compared to 40%, 43% and 55% in the EM. 5-HT2 receptor occupancy was 63% and 73%, as compared to 45%, 56% and 68% in the EM. These findings support the view that the active 9 hydroxyl metabolite of risperidone contributes to the in vivo effects of risperidone in humans, and thus partly counterbalances the marked variability in the disposition of risperidone. PMID- 7545823 TI - This path has pictures. PMID- 7545822 TI - [Kawasaki disease]. AB - Kawasaki disease (KD), first described in Japan in 1967 by Dr. Tomisaku Kawasaki, is an acute multi system vasculitis of infancy and early childhood characterised by high fever, rash, conjunctivitis, inflammation of the mucous membranes, erythematous induration of the hands and feet and cervical lymphadenopathy. Synonyms for Kawasaki disease include "Kawasaki syndrome" and "mucocutaneous lymph node syndrome" (MCLS, MLNS, MCLNS). Kawasaki disease was initially presumed to occur only in Japan; but now this disease is known in the whole world. The first cases in the United States were reported in Hawaii in 1976. In poland 5 cases were recognized, and first time described in 1981. The etiology of Kawasaki disease remains unknown. Toxic, allergic and immunologic causes have been suspected, but most investigators favor an infectious cause or an immune response to an infectious agent. Among classes of microorganism suspected of causing Kawasaki disease were bacteria, leptospires, fungi, rickettsiae and a number of viruses. Recently, there has been considerable interest in the possibility, that Kawasaki disease is caused by RETROVIRUSES. Although the disease is generally benign and self-limited, about 20% of children develop coronary artery aneurysms. In 5% of cases, giant aneurysm/more then 8 mm/develop, predisposing the patient to acute coronary artery thrombosis, myocardial infarction and sudden death. This is the most serious complication of KD. Other manifestations of hearth involvement, include pericarditis, myocarditis, myocardial failure and mitral regurgitation. Besides this many other clinical findings are commonly noted in KD; such as: pneumonia, diarrhea, arthritis, aseptic meningitis, otitis media, obstructive jaundice, hydrops of gallbladder and others.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545824 TI - [Drug therapy in bronchial carcinoma]. AB - In the treatment of bronchogenic carcinoma approaches vary depending upon whether the carcinoma in question is defined as a small cell or a non-small cell lung cancer. Small-cell lung cancer in the majority of cases must be seen as a systemic disease even with an early diagnosis. Because of this, chemotherapy is the dominant form of treatment. For patients with limited disease radiotherapy and surgery are additionally recommended as potentially curative measures, and for those with extensive disease, surgery and radiotherapy may serve as palliative treatment. Chemotherapy generally consists of a combination of two or more cytostatic drugs. As a rule 4 to 6 treatment cycles are administered. Maintenance therapy appears to be of little value. In case of tumor relapse, new cytostatic combinations can be attempted or the cytostatic regimen which was originally successful can be reintroduced. Whether or not a tumor responds to a particular chemotherapy is apparent after the first cycle of treatment. When the tumor shows no reduction in small-cell lung cancer, the treatment regimen can immediately be changed. The question of possible intensification of induction chemotherapy has yet to be clarified by clinical trials. The data gathered thus far, however, suggest that there is no measurable improvement in survival rates when chemotherapy is intensified beyond standard practice. In the case of non small cell lung cancer, the disease is predominantly characterized by locally limited tumor growth, so that radiotherapy and surgery are initially the preferred forms of treatment. Systemic therapy in non-small cell lung cancer has thus been mainly reserved for the stage of tumor dissemination (stage IV). For these patients chemotherapy has proved generally to have a purely palliative effect which is of limited duration. Recent clinical trials indicate, however, that better results can be obtained when chemotherapy is applied in stage III. These encouraging results stem from a number of clinical studies, in which polychemotherapy containing cisplatin (with or without radiotherapy) was applied preoperatively to initially inoperable stage III non-small cell lung cancer patients. It must be noted, however, that up until now these positive results have been achieved mainly in uncontrolled clinical investigations which must be confirmed by larger controlled trials. PMID- 7545825 TI - [Clinical aspects, diagnosis and drug therapy of hyperthyroidism]. AB - Graves' disease and toxic uni- or multinodular goiter are the most frequent causes of hyperthyroidism. Graves' disease is caused by thyroid stimulating immunoglobulins which are directed against the TSH receptor of thyroid follicular cells. Graves' disease affects more females than males and is associated with diffuse goiter and a rapid appearance of symptoms and signs of hyperthyroidism. Patients with Graves' disease are on average younger than patients with toxic nodular goiter. The diagnosis of Graves' disease is usually easy, particularly if signs of endocrine opthalmopathy are present. Toxic nodular goiter is seen more often in older patients with pre-existing goiters. Symptoms and signs of hyperthyroidism often appear only slowly. Hyperthyroidism in these older patients can be oligosymptomatic. Older patients should therefore be investigated for the presence of hyperthyroidism, even if they present only a few symptoms or signs which could suggest this diagnosis. The development of ultrasensitive TSH assays has simplified the diagnosis of hyperthyroidism and made the TRH-test, often used in the past, almost superfluous. At the present time, it is practically always possible to differentiate between Graves' disease and toxic nodular goiter as the cause of hyperthyroidism on the basis of clinical and laboratory findings alone, and in many cases thyroid scintiscans are therefore no longer necessary. A patient with newly diagnosed Graves' disease is treated with antithyroid drugs (carbimazole or PTU) for one year. If hyperthyroidism persists after this one year of antithyroid drug treatment, or if it recurs, another year of therapy with carbimazole or PTU is indicated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545826 TI - [Surgical and nuclear medicine treatment of hyperthyroidism]. AB - Radioiodine and surgery are the therapeutic tools of choice for the definitive treatment of patients suffering from hyperthyroidism. The decision on the therapy to choose for an individual patient will depend on several different factors: etiology of the hyperthyroidism, size of the goiter, age and general medical considerations, availability of radioiodine therapy or of an experienced endocrine surgeon, and the patient's own preferences. It is important for the general practitioner attending and advising a patient to have a basic knowledge of both therapeutic modalities. An overview of indications, results and risks of the radioiodine and surgical therapy is presented. PMID- 7545827 TI - Transurethral microwave thermotherapy (TUMT) for benign prostatic hyperplasia (BPH)--our first 100 cases. AB - One hundred consecutive cases treated by Transurethral Microwave Thermotherapy (TUMT) since October 1991 were analysed to assess its efficacy and safety. Out of these, 28 were in urinary retention. Patients were selected based on Madsen Symptom Score (MSS), Uroflowmetry, Transrectal Ultrasound Scanning (TRUS) plus biopsy and flexible cystoscopy. In the non-retention group, symptomatic improvement was 81%; mean MSS dropped from 13.6 to 2.6 at one year. Objective improvement was less marked: mean peak urine flowrate (PFR) (+45%), mean residual volume (-63%) and mean prostatic volume (-15%). 8.3% had failed TUMT requiring TURP. In the retention group, 79% was able to void freely after TUMT. Fourteen percent underwent TURP. Based on given criteria, the overall response rate for MSS and PFR averaged 71% at 3 months, 72% at 6 months and 84% at 1 year. Sixty seven percent of patients who responded to a phone interview were satisfied with TUMT treatment. Minimal morbidity was encountered: temporary retention for non retention group (24%), UTI (9%), haematuria (7%), impotence (2%) and fistula (1%). There was no treatment-related death. The results showed that TUMT is a viable alternative and safe treatment of BPH. PMID- 7545828 TI - Palliation of advanced gastric cancer by laparoscopic gastrojejunostomy. AB - Palliation of unresectable gastric carcinoma by conventional gastrojejunostomy is associated with significant morbidity and mortality. Laparoscopic gastrojejunostomy may be an alternative as it can achieve the same results with less morbidity. This is a case report of a 68-year-old man with an obstructed, unresectable gastric carcinoma that has metastasized to the lungs and liver. Attempt at laser boring was unsuccessful. Laparoscopic assessment revealed an immobile tumour fixed to the pancreas. A totally intraabdominal laparoscopic gastrojejunostomy was fashioned with endoGIAs (USSC Norwalk, CT). Operation time was 85 minutes. He was able to tolerate feeds on the 3rd postoperative day (POD), diet on the 5th POD and was discharged on the 8th POD. Satisfactory palliation at home was achieved for ten weeks before death. Laparoscopic gastrojejunostomy is thus a good option for palliation of obstructed advanced gastric cancer. PMID- 7545829 TI - Ion Channels and Genetic Diseases. Proceedings of the Society of General Physiologists 48th Annual Symposium. Woods Hole, Massachusetts, 7-11 September 1994. PMID- 7545830 TI - Correlating ion channels with disease using genetic linkage analysis. PMID- 7545831 TI - Endoscopic extraperitoneal lumbar sympathectomy. AB - From June 24, 1993, until November 9, 1993, eight sympathectomies were performed by extraperitoneal endoscopy for treatment of Sudeck atrophy. Seventy-five percent of the patients were satisfied with the result of the intervention. A follow-up after 4 months shows that four patients are free of pain. Two are satisfied, but some pain remains. In two cases, the intensity of the pain remains unchanged but the character of the pain has changed. This new technique is safe and offers the well-known advantages of minimal invasive surgery. Moreover, this endoscopic approach opens perspectives for the exploration of the entire retroperitoneum. PMID- 7545832 TI - [Pharmacotherapeutic use of antipruritic drugs in skin diseases]. AB - Pruritus is a predominant and serious phenomenon of various skin diseases. As mediators of pruritus, histamine, serotonin (hydroxytryptamine), eicosanoids, neuropeptides, cytokines and proteases are known. The symptomatic treatment of pruritus is variable. Glucocorticoids are well known antipruritic drugs. H1 antihistaminics are used frequently. But the efficacy of these drugs is questionable in dogs. Additionally, mast cell stabilisators, unsaturated fatty acids (i. e. primrose oil, fish oil) and other antipruritic agents (local anesthetics) are used. PMID- 7545833 TI - [Mammary carcinoma of the female dog: clinical relevance of the immunohistochemical demonstration of micrometastases in the regional lymph nodes]. AB - 77 formalin-fixed and paraffin-embedded mammary carcinomas and the regional lymph nodes of bitches were examined by immunocytochemical technique, all of them free of metastases in routine HE-staining. To compare the two methods, two serial sections were cut of three parts of the lymph nodes. One was used for incubation with the antibody AE1, which reacts with cytokeratin subtype I/A, the other for staining with HE. From 60 of the 77 bitches we received information about the operation and the follow up study period (surgical method, new tumours, survival rate and causes of death). These data were compared with the immunocytochemical results of the lymph nodes. In 84.4% of all 77 lymph nodes tumour cell embolism and/or micrometastases were detected. Comparing the two methods, we found that with HE-staining it was only possible to detect two thirds of all micrometastases containing more than 50 tumor cells. Smaller micrometastases were suspicious in a few cases, the majority could not be detected at all. In the follow up study there was evidence of a better prognosis for bitches with tumours detected in an early stage of growth and treated with radical mastectomy. This was independent of a positive or negative result of tumour cells in lymph nodes. The presence of cancer cells in lymph nodes was only important, if either the tumour was treated in an advanced stage or only single complexes or tumour nodes were extirpated. These dogs often showed metastases in the lung. PMID- 7545834 TI - Developmental neurotoxicity of chlorpyrifos: cellular mechanisms. AB - Chlorpyrifos, one of the most widely used pesticides, exhibits greater toxicity during development than in adulthood. We administered chlorpyrifos to neonatal rats in apparently subtoxic doses that caused no mortality and little or no weight deficits and examined developing brain regions (cerebellum, forebrain, brainstem) for signs of interference with cell development. One-day-old rats given 2 mg/kg sc of chlorpyrifos showed significant inhibition of DNA synthesis in all brain regions within 4 hr of treatment; equivalent results were obtained when a small dose (0.6 microgram) was introduced directly into the brain via intracisternal injection, indicating that the actions were not secondary to systemic toxicity. Inhibition of DNA synthesis was also seen at 8 days of age; however, at this point, there was regional selectivity, with sparing of the cerebellum. Between 1 and 8 days of age, brain regions develop wide disparities in cholinergic innervation; accordingly, we tested whether the effect of chlorpyrifos was mediated through cholinergic actions on nicotinic receptors known to mediate inhibition of DNA synthesis. Pretreatment with mecamylamine caused a decline in DNA synthesis by itself, but nevertheless prevented the effect of chlorpyrifos. Additionally, chlorpyrifos administration at 1 day of age caused an even larger inhibition of protein synthesis throughout the brain; the effect was distinct from that on DNA synthesis, as it diminished substantially by 8 days of age and did not develop any regional selectivity. The effects of chlorpyrifos on DNA and protein synthesis were not secondary to generalized cell damage or suppression of cell metabolism, as evidenced by maintenance of normal ornithine decarboxylase activities. These results indicate that low doses of chlorpyrifos target the developing brain during the critical period in which cell division is occurring, effects which may produce eventual cellular, synaptic, and behavioral aberrations after repeated or prolonged subtoxic exposures. PMID- 7545835 TI - The tolerability and pharmacology of interleukin-6 administered in combination with GM-CSF or G-CSF in the rhesus monkey. AB - The tolerability and potential target organ toxicity of rhIL-6 administered subcutaneously (s.c.) with rhGM-CSF or rhG-CSF were investigated in healthy nonhuman primates. Fifteen Rhesus monkeys were randomized to receive one of the following five regimens: rhIL-6, rhGM-CSF, rhG-CSF, rhIL-6 and rhGM-CSF, or rhIL 6 and rhG-CSF. Each cytokine was administered s.c. once daily at 20 micrograms/kg/day for 30-31 days. Marked increases in blood leukocyte counts (predominantly neutrophils) were observed in the rhGM-CSF and rhG-CSF treatment groups, but only a mild trend toward increased WBCs was observed with rhIL-6 alone. Platelet counts increased 1.7- to 2.2-fold in the rhIL-6 and rhGM-CSF groups. All regimens were well tolerated. RhIL-6, alone or in combination with either CSF, had no significant toxic effects at the dosages tested. Minimal to moderate bone marrow hyperplasia was observed in all except rhIL-6-treated animals, which correlated well with peripheral blood increases in WBCs. RhIL-6 treated animals demonstrated increased fibrinogen concentrations and erythrocyte sedimentation rates, decreased serum albumin/globulin ratios, and increased serum alpha-2-macroglobulin concentrations. Increased synthesis of acute-phase proteins was not observed in the other groups. Combining rhIL-6 with rhGM-CSF or rhG-CSF may reduce the rhIL-6-mediated acute-phase response while maintaining the desirable hematopoietic effects of the stimulating factors. PMID- 7545836 TI - Capillary blood versus arterial or venous blood for tacrolimus monitoring in liver transplantation. AB - Tacrolimus has been found to be useful in clinical solid organ transplantation. A very careful monitoring of the tacrolimus levels and dose adjustments are essential, at least in the immediate post-liver transplantation, situation. However, quite often after liver transplantation, patients have limited venous access for daily monitoring of tacrolimus levels. When the blood is sampled from a multilumen central venous catheter, used also for intravenous administration of tacrolimus, falsely elevated concentrations of tacrolimus have been observed. The present study examines the concentration of tacrolimus in capillary blood samples obtained from finger stick and compares its concentrations in simultaneously drawn samples from arterial line, peripheral venous puncture, and multilumen centrally placed venous catheter from the port used for tacrolimus infusion and the port not used for tacrolimus infusion. Ten adult post-liver transplantation recipients were studied. Whole blood concentration of tacrolimus in capillary blood was comparable to that of arterial blood, as well as to that of peripheral venous blood samples (r2 = 0.99; P = 0.72). Concentrations of tacrolimus in venous blood drawn from the port of the multilumen catheter used for intravenous tacrolimus infusion were 3-23 times higher (P = 0.0015), while the concentrations of venous blood drawn from the port not used for tacrolimus infusion were 1.7-4.5 times higher (P = 0.016), as compared with arterial, capillary, or peripheral venous whole blood concentrations. PMID- 7545837 TI - Newborn baboon serum anti-alpha galactosyl antibody levels and cytotoxicity to cultured pig kidney (PK15) cells. PMID- 7545838 TI - [Ilomedin (Iloprost) and Buerger disease]. AB - Three patients with Buerger's disease, ischaemic ulcers and pain, accepted treatment with ilomedin, a synthetic stable prostaglandin, as a supplement to standard treatment. The patients were treated intravenously, with ilomedin for six hours daily for 28 days. One patient had total healing of his ulcers just after treatment. The ulcers of the other patients showed almost complete healing about two months after treatment. However, the ulcers then returned to their previous size. Ilomedin was then used as preoperative treatment prior to amputation in order to facilitate healing after amputation. Our conclusions are that we now have a promising drug, ilomedin, in the treatment of patients with Buerger's disease, ulcers and pain, but controlled studies are necessary. PMID- 7545840 TI - [Prostate-specific antigen (PSA)]. PMID- 7545841 TI - [Molecular forms of prostate-specific antigen and their clinical significance]. AB - PSA is a proteolytic enzyme produced in the prostatic epithelium and secreted into the seminal fluid. PSA can also be a constituent of the serum even under apparently normal conditions. In many cases of prostate cancer (PCa), increased serum concentrations are found. Elevated serum levels, however, are also observed in benign prostatic hypertrophy (BPH), thus limiting the specificity of serum PSA as a marker for prostate cancer. Recently, subfractions of PSA ("free PSA"; PSA antichymotrypsin complex) were analyzed in order to gain a higher specificity of PSA as a tumor marker. The present paper describes biochemical features and clinical significance of the various PSA subfractions, pointing out an improved discrimination of PCa and BPH by evaluating the ratio "free PSA": total PSA. PMID- 7545839 TI - Neuroendocrine peptides in the prostate. AB - Circulating androgens are required for normal growth and maintenance of function of the prostate. However, the prostate also contains neuroendocrine peptides, found either in nerve terminals or in prostatic neuroendocrine cells, which are likely to regulate prostate growth or function. The neuronal peptides are likely to participate in the regulation of the synthesis and secretion of prostatic secretory products. While the function of the neuroendocrine cells is undefined, there is evidence for growth-regulating effects of several neuroendocrine cell peptides. Since neuroendocrine differentiation has been correlated with tumor grade and poor prognosis in prostate cancer, the peptide products of the neuroendocrine cells may influence cancer cell replication as well. Recent evidence in other tissues suggests that peptide hormone receptor second-messenger systems may interact with steroid receptors to modulate their actions. These findings raise the possibility that prostatic neuroendocrine peptides may modulate the response of prostate to androgens. PMID- 7545843 TI - [Can the tumor stage of prostate carcinoma be determined with the aid of prostate specific antigen?]. AB - Prostate-specific antigen (PSA) is known to correlate with tumor grading, tumor volume, and lymph node and osseous metastases. If PSA concentrations exceed 20 ng/ml, the risk of extracapsular tumor extension increases greatly, and above 50 ng/ml organ-confined disease is extremely rare. Due to a considerable overlap in PSA concentrations between various tumor stages, the prediction of tumor stage with PSA alone is barely possible. In combination with other preoperative diagnostic findings, e.g. digital rectal examination or histological grade in multiple biopsies, the predictive value of PSA in terms of tumor stage determination can be increased to a large extent. PMID- 7545842 TI - [Value of determining prostate-specific antigen for early detection or prostatic carcinoma]. AB - The use of prostate-specific-antigen in the early detection of prostatic carcinoma combined with digital-rectal examination results in a 2-3 times increase in prostatic carcinoma detection rate. 2/3 of PSA detected prostatic carcinoma are organ confined vs 40% of those which are detected by digital-rectal examination. 15-35% of all operated localized prostatic carcinoma have a normal PSA. A biopsy is indicated in men with a life expectancy of more than 10 years when the PSA value is above 10 ng/ml and/or digital-rectal examination is suspicious. This concerns only 2% of all men at the age older than 50 years. In 90% of all men older than 50 years the PSA is normal as well as the digital rectal examination. In 4% of these the result of PSA and digital-rectal examination is false negative that means 4% have prostatic carcinoma. However, repeated digital-rectal examination and PSA determination on a yearly basis detects most of these overlooked prostatic carcinoma which still are organ confined in about 90% of the cases. In men with a minimal elevated PSA-value of 4 10 ng/ml, 25% will have a prostatic carcinoma regardless of the finding on digital-rectal examination. The indication to do a biopsy can be specified by the use of age specific PSA cut-off-levels and most likely in future by determining the free PSA vs the complex-bound PSA. Controversy exists about the usefulness of PSA-density and PSA-velocity. PMID- 7545844 TI - [The value of prostate-specific antigen in therapy follow-up of prostatic carcinoma]. AB - The prostate specific antigen (PSA) has gained great importance for the diagnosis and the treatment of prostate carcinoma since its isolation from prostate tissue in 1979. PSA is produced and secreted almost only by the prostatic tissue. After radical prostatectomy PSA changes from a tissue specific marker to a tumor specific parameter. At least 30 days after radical prostatectomy the PSA serum level should decline nondetectable values. A high incidence for a local recurrence, distant metastases, or a incomplete resection of the prostate is a still present PSA serum value. After a radiation or androgen deprivation therapy the PSA value is a prognostic marker. A good prognosis can be expected if the PSA level decreases after androgen deprivation therapy to values below 4 ng/ml. In contrast to that increasing PSA levels indicate a local or distant recurrence. Progression of the tumor without an increase of PSA values is possible but rare. It could be shown in several studies, that the determination of PSA values is sufficient for routine treatment control and that other methods could therefore be omitted. PMID- 7545845 TI - [Different determination methods make interpretation of prostate-specific antigen more difficult]. AB - The availability of numerous different assays for the determination of prostate specific antigen (PSA) hs created substantial problems in the interpretation of PSA concentrations. Presently more than 40 assays are commercially promoted within the German market. The majority of the recently released assays claim the commonly used reference range (< 4 ng/ml) although this has no always been verified. Some companies entirely avoid the specification of reference ranges, others derive the data from very small cohorts. Reference ranges established with sera of young males or even with an unknown proportion of female sera are not valid in assessing the specificity of PSA assays to detect prostate cancer among male individuals between 50 and 80 years of age. Some companies recommend not to apply their assay for diagnostic purposes limit its use to the follow-up of patients previously diagnosed but to as having prostate cancer. This warning usually remains just as unknown to the urologist as the name of the assay used. Since PSA concentrations may vary in identical samples by a factor of 2, depending on the assay used, the clinician interpreting the results needs to be aware of the method applied and must have detailed information about the assay specific reference range. Without this information, PSA loses its valuable diagnostic and prognostic features. Apart from avoidable worries on the patient's part, evidence of prostate cancer may be missed or unnecessary biopsies may be performed. PMID- 7545846 TI - [Perioperative and postoperative complications of pelvic lymphadenectomy and radical prostatectomy in 320 consecutive patients]. AB - To examine the intra- and postoperative morbidity of radical retropubic prostatectomy we analyzed the first 320 consecutive patients with clinical stages T1b, T2a-c and negative lymph nodes by frozen section. Patient age varied from 42 to 75 years (mean 63.5 years). In 74.7% the estimated blood loss was less than 1500 ml. With a preoperative autologous blood collection program the intraoperative blood requirement for homologous blood units was only 15%. Intraoperative complications included rectal injuries with vesical rectal fistulas in 2.5% and ureteral injuries in 1.6%. Within the perioperative period the mortality rate was 0.9%. At 12 months after surgery 199 of 218 men (90.9%) were continent, 5.1% had minimal urinary incontinence, and only 4.6% had urinary incontinence grade III. Postoperatively, PSA (prostate-specific antigen) decreased to < 0.5% in 90.4% of the patients after radical prostatectomy. At 12 months after operation PSA was < 0.5 ng/ml in 83.4%. We conclude that radical retropubic prostatectomy is a safe procedure for the curative treatment of localized prostate cancer. PMID- 7545847 TI - [Congenital agenesis of the vas deferens and cystic fibrosis]. AB - Aspermia caused by absence of the vas deferens is well known in cystic fibrosis. It has been suggested that otherwise healthy males with congenital bilateral absence of the vas deferens (CBAVD), which was previously considered a distinct genetic entity, have an increased frequency of CF gene mutations. CBAVD is now considered to be a mild form of cystic fibrosis. We report the case of an azoospermic man who had undergone exploratory scrototomy because of aplasia of the epididymis and vas deferens. Genetic screening for cystic fibrosis revealed a compound heterozygote for CFTR mutations delta F 508 and R 117 H. PMID- 7545848 TI - [Telemetry during swimming in risk evaluation of heart patients in rehabilitation]. AB - Swimming differs from other forms of exercise due to its additional hydrostatic and thermal burden. It was investigated whether additional pathologic findings in comparison to history and standard exercise tests can be obtained by holter monitoring during swimming. Symptoms and exercise electrocardiogram were compared with the holter ECG during swimming in 125 patients divided into 3 groups with different diagnoses and severity of cardiac diseases. In a considerable percentage of patients ischemic changes and severe rhythm disturbances were found only during swimming with further diagnostic and therapeutic consequences, though patients with moderate and severe angina and with significant ischemic signs in the exercise test were excluded and mainly patients with slight or absent symptoms were evaluated predominantly. Thus, since swimming is a favorite leisure time occupation also in patients with diseases of heart and circulation, holter monitoring during swimming is of diagnostic importance in the rehabilitation of these patients. PMID- 7545849 TI - [Kinin antagonists in the treatment of acute stenosing laryngotracheitis in children]. AB - The treatment of 97 infants aged from 3 months to 2 years included: nasotracheal intubation (56 patients); i. v. injection, drops (77 patients) or aerosol inhalations (20 cases) of trasylol, kontrikal or gordox; inhalations of epsilon aminocaproic acid (12 cases); glucocorticoids (locally, inhalations, laryngeal drop infusions); local and oral bioantioxidants; noradrenaline for local vasoconstriction. The addition of antikinin medication to the complex of pathogenetically differentiated means which are also proteolytic enzyme inhibitors reduced the frequency and duration of tracheal intubation, duration of laryngeal stenosis by half. Systemic and local administration of antikinin drugs is advocated starting on the first post-diagnosis days, except cases of advanced acute inflammation in the presence of infiltrates in the cavum infraglotticum when antikinin, antihistamine and vasoconstrictors proved ineffective. PMID- 7545850 TI - A study on lymphocyte activation in maedi-visna virus induced pneumonia. AB - The stage of activation of bronchoalveolar lavage fluid (BALF) lymphocytes and peripheral blood lymphocytes (PBL) from maedi-visna virus (MVV) infected (n = 7) and control (n = 7) sheep was investigated by assessing four parameters of lymphocyte activation; lymphocyte size and complexity, loss of CD5+ T cells, expression of cell surface interleukin-2 receptor (IL-2R) and expression of DR and DQ MHC Class II molecules. BALF lymphocytes from MVV-infected animals had a significant loss of CD5+ lymphocytes (P < 0.05) and upregulation of DR and DQ MHC Class II molecules compared with controls, consistent with BALF lymphocyte activation. No changes in cell size and complexity or expression of IL-2R were observed. No evidence of PBL activation was detected. These findings suggest an impaired BALF lymphocyte activation during MVV infection. PMID- 7545851 TI - The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma. AB - An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa. PMID- 7545852 TI - Detection of specific T cell reactivity in sheep infected with Mycobacterium avium subspecies silvaticum and paratuberculosis using two defined mycobacterial antigens. AB - A 30 kDa antigen (P30) from Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and a 40 kDa (P40) antigen from Mycobacterium avium ssp. silvaticum (M. a. silvaticum) were employed in two different assays to measure the cell-mediated immune reactivity of ovine peripheral blood lymphocytes. In lymphocyte stimulation assays, proliferative responses to the P30 were observed only with lymphocytes from sheep inoculated with live M. a. paratuberculosis or M. a. silvaticum. Although this antigen was not subspecies-specific it differentiated between animals given live organisms and those inoculated with an inactive lysate. The P40 protein from M. a. silvaticum showed subspecies specificity by eliciting in vitro responses only with lymphocytes derived from sheep inoculated with live M. a. silvaticum. Similar results were obtained using an interferon-gamma release assay which proved to be a more rapid and sensitive system. PMID- 7545853 TI - Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes. AB - Despite the extensive molecular information on serum-derived human hepatitis B viruses (HBV), liver-derived replicative HBV genomes have remained largely uninvestigated. We have examined the sequences of the entire core antigen (nucleocapsid) of liver-derived HBVs in 15 different hepatoma patients. Bona fide mutations, rather than subtype polymorphism, have been identified based on the high-frequency occurrence of structural differences from wild type at the highly evolutionarily conserved positions, instead of at the positions known to contain genetic heterogeneity among different isolates from different geographic locations. The distribution of these naturally occurring mutations of HBV core gene appears to be nonrandom and is found predominantly within three major (I, IV, and V) and four minor domains (II, III, VI, and VII). In general, domain IV mutations correlate with domain V mutations. The replicative HBV DNAs tend to accumulate a higher number of mutated core domains than the integrated HBV DNAs. At the domain level, there is no significant difference in HBV core mutation frequencies between the liver tumors and the adjacent nontumorous livers. Strikingly, domains I, III, and V coincide with three major known T cell epitopes within the core protein in acute and chronic hepatitis B patients. Furthermore, these domains coincide with HLA class II-restricted T cell epitopes, rather than with the conventional HLA class I-restricted epitopes of cytotoxic T lymphocytes. Our results support the hypothesis that HBV core antigen variants can accomplish immunoevasion via accumulated escape mutations. In addition, they also provide a potential molecular explanation for the maintenance of persistent infection of human hepatitis B virus in chronic carriers. PMID- 7545854 TI - Characterisation of foscarnet-resistant strains of human immunodeficiency virus type 1. AB - Foscarnet is a broad-spectrum viral DNA polymerase inhibitor active in vitro and in vivo against human immunodeficiency virus type 1 (HIV-1). Strains of HIV-1 resistant to foscarnet were selected by in vitro passage in increasing concentrations of drug. Reduced susceptibility to foscarnet was evident at the levels of both HIV-1 replication and reverse transcriptase. Biologically cloned, foscarnet-resistant strains with distinct genotypes were hypersensitive to zidovudine, azidodeoxyuridine, nevirapine, and R82913 but had unchanged susceptibility to zalcitibine and didanosine. The reverse transcriptase of foscarnet-resistant strains had unique substitutions Glu89-Lys, Leu92-Ile, or Ser156-Ala, the third being associated with six polymorphic changes. Introduction of these mutations into wild-type HIV-1 by site-directed mutagenesis confirmed their role in foscarnet resistance. In the three-dimensional structure of the reverse transcriptase enzyme these amino acids are located close to the template strand of the template primer and far away from the putative pyrophosphate binding site, suggesting that the mechanism by which HIV-1 becomes resistant to foscarnet is indirect. Foscarnet resistance is thus likely to be mediated through an altered interaction of the mutant enzyme with the template strand of the template primer which distorts the geometry of the polymerase active site and thereby decreases foscarnet binding. PMID- 7545855 TI - National Hospital Discharge Survey: annual summary, 1993. AB - This report presents statistics on the utilization of non-Federal short-stay hospitals based on data collected through the National Hospital Discharge Survey from a national sample of the hospital records of discharged inpatients. Estimates are provided by the demographic characteristics of patients discharged, geographic region of hospitals, conditions diagnosed, and surgical and nonsurgical procedures performed. Measurements of hospital use include frequency, rate and percent of discharges and days of care, and average length of stay. PMID- 7545856 TI - Molecular and clinical findings in Austrian cystic fibrosis patients with mutations in exon 11 of the CFTR gene. AB - In order to determine the heterogeneity of mutations in exon 11 of the cystic fibrosis transmembrane conductance regular (CFTR) gene in Austrian cystic fibrosis (CF) patients, we analysed 207 non-delta F508 chromosomes by direct sequencing of PCR-amplified genomic DNA. A total of four previously described point mutations present on 14/207 (6.8%) non-delta F508 chromosomes were detected: G542X, G551D, R553X, and R553Q. The second CF mutation was delta F508 in most patients, W1282X (a nonsense mutation in exon 20) in one and a currently unknown non-delta F508 mutation in another case. One patient was documented to be homozygous for G542X. The proportion of non-delta F508 chromosomes among total CF chromosomes is 45% in Austria and 32% in the world population. In our population the mutations G542X, G551D, and R553X were found on 3.9% (1.7%), 1.9% (0.9%) and 0.5% (0.2%) of non-delta F508 chromosomes (of total CF chromosomes), respectively. The average worldwide frequencies of these mutations are higher: 7.1% (2.4%), 4.8% (1.6%), 2.1% (0.7%) of non-delta F508 chromosomes (of total CF chromosomes screened), respectively. A comparison of the allele frequencies in Austria with those detected in neighbouring countries reveals some notable differences. In a subsequent retrospective analysis we found that all nucleotide changes, identified by direct sequencing, can be detected by denaturing gradient gel electrophoresis (DGGE). The lack of any false positive or false negative result suggests that DGGE is a convenient and reliable screening method for point mutations. PMID- 7545857 TI - [New in Austria approved specialty drugs and new knowledge regarding currently available specialty drugs. Uroflo]. PMID- 7545858 TI - Topographic involvement of the striatal efferents in basal ganglia of patients with adult-onset motor neuron disease with basophilic inclusions. AB - This report concerns the topographic immunohistochemical analysis of the putamen, globus pallidus (GP) and substantia nigra (SN) of two patients with adult-onset motor neuron disease with basophilic inclusions (MND/BIs), seven patients with sporadic classic amyotrophic lateral sclerosis (sporadic ALS) and five neurologically normal individuals. The striatal efferent terminals of the GP and SN were visualized immunohistochemically using antibodies to met-enkephalin (MEnk) and substance P (SP). In specimens from patients with sporadic ALS and normal subjects there was intense immunostaining for MEnk and SP throughout the external and internal segments of the GP, respectively. By contrast, a marked reduction of MEnk- and SP-positive striatal efferents was seen in the ventrocaudal portions of both GP segments from the MND/BIs patients. Moreover, while MEnk-positive striosomes was readily detected in the putamen of normals and sporadic ALS patients, there was significant reduction in MEnk immunoreactivity, and no evidence of striosomal organization in the putamen of MND/BIs patients. In addition, whereas the SN of patients with sporadic ALS expressed SP, the ventrolateral SN portion of the MND/BIs patient tested had reduced immunoreactivity. The present findings on patients with MND/BIs may represent a reflection of the topographic striatum degeneration in this disease and appear to provide additional evidence for the heterogeneity of MND. PMID- 7545859 TI - The proximal peripheral nervous system is a major site of demyelination in experimental autoimmune encephalomyelitis induced in the Lewis rat by a myelin basic protein-specific T cell clone. AB - Experimental autoimmune encephalomyelitis (EAE) was induced in the Lewis rat by the passive transfer of a cytotoxic CD4+ T cell clone specific for the 72-89 peptide of guinea-pig myelin basic protein (MBP). Histological studies on rats with neurological signs showed that inflammation was present in the proximal peripheral nervous system (PNS), namely the spinal roots, as well as in the central nervous system (CNS). The main sites of demyelination were the spinal roots in the PNS, and the spinal cord root entry and exit zones in the CNS. The major involvement of the proximal PNS in autoimmune disease directed at MBP is in marked contrast to EAE induced by immunisation with myelin proteolipid protein, where the inflammation and demyelination are restricted to the CNS. These findings may have implications for the human inflammatory demyelinating diseases including multiple sclerosis, in which MBP is a putative target antigen. PMID- 7545860 TI - Serum bleomycin-detectable iron in patients with thalassemia major with normal range of serum iron. AB - "Free" iron, a potentially radical-generating low mass iron, and not found in normal human blood, was increased in the serum of blood-transfused thalassemia major patients seen in the Yangon General Hospital, Yangon, Myanmar (Burma). The low mass iron was detected by the bleomycin assay. Fifty-one blood samples were analyzed (from 28 males and 23 females). High "free" iron was detected in 47 sera samples from thalassemia patients. Serum ferritin, which reflects the body store iron, was higher than the normal range (10-200 ng/ml) in 49 patients. On the other hand, serum iron of 39 sera samples fell within the normal range (50-150 micrograms/dl). Four were less than 50 micrograms/dl and eight were more than 150 micrograms/dl. Almost all the patients' sera of normal or higher serum iron level contained "free" iron. Thus, almost all the sera from thalassemic patients from Myanmar contain bleomycin-detectable iron, even when serum iron is within the normal range. In developing countries where undernutrition is prevalent (serum albumin in these patients was 3.6 +/- 0.4 g/dl, P < 0.0001 vs. control value of 4.0 - 4.8 g/dl), normal serum iron does not preclude the presence of free iron in the serum. PMID- 7545861 TI - Virological and serological characterization of asymptomatic blood donors positive for anti-hepatitis C virus antibody. AB - To study the virological and serological characteristics of asymptomatic hepatitis C virus (HCV) carriers, 165 blood donors positive for antibody against HCV proteins by the second generation assay, were analyzed for their clinical backgrounds, serological reactivity against antigens derived from HCV by recombinant immunoblot assay, and the amount and genotype of HCV by the polymerase chain reaction. Compared with blood donors having abnormal levels of alanine aminotransferase (ALT), sera from the donors with normal levels of ALT reacted less frequently against NS4 antigens (anti-5-1-1: 34.4% vs. 54.5%, P = 0.0609; anti-c100-3: 34.4% vs. 56.1%, P < 0.05). Also the positivity for antibodies against these antigens were more frequent in sera from donors with genotype 1b HCV-RNA than other genotypes (anti-5-1-1: 61.0% vs. 23.5%, P < 0.01; anti-c 100-3: 61.0% vs. 26.5%, P < 0.01). The prevalence of each genotype in blood donors with normal ALT levels was different from that in patients with advanced liver disease (P < 0.05), genotype 1b being less and genotype 2a being more frequent. The number of HCV-RNA copies/0.5 ml in donors with normal ALT was 10(7.9 +/- 1.0) (n = 27) and that in patients with chronic liver disease was 10(7.4 +/- 0.8) (n = 116), the difference being statistically significant (P < 0.05). In conclusion, the results of this study suggest that asymptomatic blood donors carrying HCV have the serological and virological characteristics different from the patients with advanced liver disease. PMID- 7545862 TI - [Dye leakage from choroidal neovascularization with indocyanine green angiography]. AB - The ultrastructure of experimentally induced choroidal neovascularization was studied in correlation with dye leakage in indocyanine green (ICG) infrared fluorescence angiography. Newly formed vessels which demonstrated leakage of ICG extended into the subretinal space without enclosure of retinal pigment epithelium (RPE), and the endothelial cells were immature. Choroidal neovascularization which did not demonstrate leakage of ICG was enclosed by RPE without retinal detachment, and the endothelial cells were mature. The newly formed vessels with immature endothelium in the subretinal space that were covered with multiple layers of RPE demonstrated no leakage. These results show that ICG leaks form choroidal neovascularization which has immature vessels that are not enclosed by RPE and that extend into the subretinal space. PMID- 7545863 TI - High prevalence of hepatitis C virus among urban and rural population groups in Egypt. AB - Hepatitis C is a major health problem for Egypt. The aim of this study was to determine the seroprevalence of antibodies to hepatitis C virus among different population groups living in urban and in two different rural areas (Suez Canal and North Sinai) of Egypt. Secondary objectives were to study the possible association between multiple blood transfusions, haemodialysis or Schistosomiasis and the seroprevalence of antibodies to hepatitis C. A seroprevalence of hepatitis C virus in the urban blood donor population of 14.5% was found, confirming other reports. In the two rural areas of the Suez Canal and the North Sinai the seroprevalence was 14.4% and 15.5% respectively, showing a comparable seroprevalence in these three different populations. The seroprevalence was 70.4% in haemodialysis patients, 7.7% in health care workers, and 75.6% in thalassaemic children, thus a seroprevalence among multitransfused or haemodialysed patients comparable to the one described in many other countries. Schistomiasis does not seem to play a role in the seroprevalence of this disease in Egypt. PMID- 7545864 TI - MR imaging of bone marrow in children with osteosarcoma: effect of granulocyte colony-stimulating factor. AB - OBJECTIVE: Granulocyte colony-stimulating factor (GCSF) is used to stimulate myeloid cell production and function in children undergoing chemotherapy for osteosarcoma. We hypothesize that GCSF can cause reconversion of marrow from fatty to hematopoietic and that this change can be detected by MR imaging at sites away from the primary tumor. This benign effect of treatment should not be confused with tumor spread. MATERIALS AND METHODS: MR images of marrow of the affected and contralateral limbs were retrospectively reviewed for 16 patients with osteosarcoma of the femur or tibia; nine of these patients had received GCSF. A grade was assigned to marrow signal intensity at sites away from the tumor, and findings before and after treatment were compared. The validity of MR image interpretation was assessed by comparing the signal intensity of marrow with the histologic appearance of marrow at 19 resection margins. RESULTS: Changes consistent with reconversion were seen on MR images in seven of nine patients who had received GCSF in addition to chemotherapy and in none of seven patients who had received chemotherapy alone. The difference in proportions was statistically significant (p = .006; Fisher's exact test, two tailed). The histologic appearance of marrow at the resection margins agreed with the interpretation of the short-Tl inversion recovery sequence in all cases (100%). CONCLUSION: The findings suggest that GCSF causes changes in the MR imaging appearance of marrow. Histologic correlation supports the hypothesis that these changes are attributable to reconversion from fatty to hematopoietic marrow. Awareness of this finding is important to avoid false-positive diagnosis of marrow metastases. PMID- 7545865 TI - MR of cerebral Whipple disease. AB - A case of cerebral Whipple disease is reported. MR findings of the brain are discussed in relation to neuropathologic lesions reported previously. Repeated MR investigations may serve as a valuable tool to evaluate long-term efficacy of treatment in cerebral Whipple disease. PMID- 7545866 TI - Ki-67 antigen expression in hepatocellular carcinoma using monoclonal antibody MIB1. A comparison with proliferating cell nuclear antigen. AB - To evaluate the prognostic significance and clinicopathologic correlation of proliferative activity in patients with hepatocellular carcinoma, Ki-67 antigen expression was examined using immunohistochemical staining with monoclonal antibody MIB1. Seventy-two patients (65 men, 7 women; age range 24-77 years, mean, 52 years) having hepatocellular carcinoma surgically resected were studied. Tumor and nontumorous tissues were stained with monoclonal antibody MIB1 with microwave oven pretreatment. Tumor and nontumor MIB1 (T-MIB1 and NT-MIB1) scores were assessed by counting the positive staining nuclei per 1,000 cells. The T MIB1 score ranged from 5-630 per 1,000 cells (mean +/- standard deviation [SD] = 145 +/- 162). It was found to be significantly higher in less well-differentiated tumors (Edmondson's grades III and IV) than in well-differentiated ones (Edmondson's grades I and II) (P = .017). The T-MIB1 score was also higher in nonencapsulated tumors than in encapsulated ones, although it did not reach statistical significance (P = .069). It had no influence on tumor size, tumor invasiveness, the background disease in the nontumorous livers, patients' HBsAg status, or serum alpha-fetoprotein levels. Diseases in the nontumorous livers or patients' HBsAg status had no influence on the NT-MIB1 scores. When the tumors were stratified into two groups with T-MIB1 score < or = 20 and T-MIB1 score > 20, those patients with score < or = 20 had significantly longer disease-free survival (DFS) than those with scores > 20 (median DFS: 34 months and 4.7 months, respectively; P = .011). In addition, MIB1 and PCNA were closely correlated (P < .01). The authors conclude that proliferative activity in hepatocellular carcinoma, as defined by MIB1 immunohistochemical analysis, is significantly related to tumor cellular differentiation. It is also a potentially valuable prognostic factor in patients with this tumor. PMID- 7545867 TI - Supernumerary chromosome marker (1) in a developmentally delayed child. AB - A 15-month-old boy with mild developmental delay and several minor anomalies was found to be mosaic 46,XY/47,XY ,+mar(1). The marker r(1) was a small de novo ring identified by FISH with a painting type DNA probe. PMID- 7545868 TI - Previously apparently undescribed syndrome: shallow orbits, ptosis, coloboma, trigonocephaly, gyral malformations, and mental and growth retardation. AB - We describe 2 children with severe ptosis, trigonocephaly, broad nasal bridge, and major brain malformation. A total of 8 children have been reported who share most of these findings. Two of the individuals have had identical pericentric inversions involving chromosome 2p12-q14. These cases appear to represent a unique malformation syndrome. PMID- 7545869 TI - L206W mutation of the cystic fibrosis gene, relatively frequent in French Canadians, is associated with atypical presentations of cystic fibrosis. AB - Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Over 400 mutations have been reported at this locus. Although severe forms of cystic fibrosis are usually associated with pancreatic insufficiency, pulmonary dysfunction, and elevated sweat chloride, there is a wide range of phenotypes, including congenital absence of the vas deferens, observed with some of the milder mutations. The L206W mutation, which was first identified in patients from South France, is relatively frequent in French Canadians from Quebec. In this report, we document the atypical form of cystic fibrosis associated with this mutation, in a cohort of 7 French Canadian probands. PMID- 7545870 TI - Eleven Polish patients with microcephaly, immunodeficiency, and chromosomal instability: the Nijmegen breakage syndrome. AB - We report on 11 patients from 8 independent families (3 pairs of sibs) with a complex clinical pattern including microcephaly, peculiar "bird-like" face, growth retardation, and, in some cases, mild-to-moderate mental deficiency. Most of the patients have recurring respiratory tract infections. One girl has developed B-cell lymphoma. A detailed anthropometric study of 15 physical parameters, including 3 cephalic traits, was performed. It was possible to study the chromosomes of PHA-stimulated lymphocytes in all of the patients. We found structural aberrations with multiple rearrangements, preferentially involving chromosomes 7 and 14 in a proportion of metaphases in all individuals. Profound humoral and cellular immune defects were observed. Serum AFP levels were within normal range. Radioresistant DNA synthesis was strongly increased in all 8 patients who were hitherto studied in this respect. Our patients fulfill the criteria of the Nijmegen breakage syndrome, which belongs to the growing category of ataxia telangiectasia-related genetic disorders. In light of the increased predisposition to malignancy in this syndrome, an accurate diagnosis is important for the patient. PMID- 7545871 TI - Microcephaly/lymphedema and terminal deletion of the long arm of chromosome 13. PMID- 7545872 TI - Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. AB - Mast cells play an essential role during development of inflammation after chemical and immunological insults and have been implicated in tissue fibrosis and angiogenesis. The exact contribution of mast cells to these conditions is largely unknown. In this study, we found that a potent angiogenic and mitogenic polypeptide, basic fibroblast growth factor (bFGF), is localized to the majority of mast cells from normal skin and lung and in tissue samples characterized by fibrosis, hyperplasia, and neovascularization. Using specific antibodies to mast cell tryptase, tissue macrophage, and bFGF, we demonstrate that cytoplasmic bFGF immunoreactivity is localized to 96.8 +/- 9.6% of tryptase-positive cells in human fibrotic lung tissue (n = 10), 82.3 +/- 6.9% of tryptase-positive cells in rheumatoid synovia (n = 6), and 93.1 +/- 4.8% of tryptase-positive cells in skin hemangioma (n = 5). Moreover, these tryptase-positive cells comprise a major portion (86 to 97%) of nonvascular cells exhibiting cytoplasmic bFGF staining in these tissues. In contrast, macrophage-like cells contribute less than 10% of the bFGF-positive cells in the same samples. The specificity of the immunostaining results was supported by the finding that cultured human mast cells (HMC-1) express both bFGF mRNA and protein. Our data indicate that mast cells, a primary source of heparin, also serve as a significant source of a heparin-binding growth factor, bFGF, in these disease processes. These observations suggest that mast cells may contribute to these pathological conditions by releasing this polypeptide. PMID- 7545873 TI - Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. AB - Elevated expression of intercellular adhesion molecule-1 (ICAM-1) as well as E- and P-selectin occurs on the vascular endothelium in a number of disease states and is thought to play an early critical role in the adhesion of circulating leukocytes to the endothelium. The goal of the present study was to investigate the immunolocalization of these molecules in the retina and choroid of postmortem human tissue sections from nondiabetic and diabetic subjects. Whereas ICAM-1 was localized primarily within the choriocapillaris of nondiabetic subjects, immunoreactivity in diabetics was significantly elevated throughout the choroidal vasculature and within retinal blood vessels (P < 0.05). In the choroid, P selectin was most prominent in veins of the nondiabetic, whereas in diabetics, P selectin was significantly elevated in arteries (P < 0.001) and veins (P < 0.05) and, in some cases, was also observed in choriocapillaris. P-selectin immunoreactivity was not observed in the retina of any subject. E-selectin immunoreactivity was not observed in choroid or retina in any subjects. Neutrophil numbers per square millimeter of tissue were significantly elevated in diabetic choroid (P < 0.05) and retina (P < 0.001). Our results demonstrate that ICAM-1 and P-selectin are constitutively expressed in the normal choroid and are upregulated in the choroidal vasculature in diabetes, but only ICAM-1 was upregulated in the retina of diabetic subjects. Increased cell adhesion molecule expression may contribute to the retinal and choroidal microangiopathy observed in diabetics by enhancing leukocyte adhesion and consequently the incidence of capillary obstruction and endothelial cell injury. PMID- 7545874 TI - Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. AB - To elucidate the deposition of advanced glycation end products (AGEs) in aortic atherosclerosis, aortic walls were obtained from 25 autopsy cases and examined immunohistochemically and immunoelectron microscopically with a monoclonal antibody specific for AGEs, 6D12. Among the autopsy cases, atherosclerotic lesions were found in the aortas of 22 cases and were composed of diffuse intimal thickening, fatty streaks, atherosclerotic plaques, and/or complicated lesions. In these cases, intracellular AGE accumulation was demonstrated in the intimal lesions of aortic atherosclerosis in 12 cases. Compared with the diffuse intimal thickening, intracellular AGE accumulation was marked in the fatty streaks and atherosclerotic plaques. Immunohistochemical double staining with 6D12 and monoclonal antibodies for macrophages or muscle actin or a polyclonal antibody for scavenger receptors demonstrated that the AGE accumulation in macrophages or their related foam cells was marked in the diffuse intimal thickening and fatty streak lesions and that almost all macrophages and macrophage-derived foam cells possessed scavenger receptors. Immunoelectron microscopic observation revealed the localization of 6D12-positive reaction in lysosomal lipid vacuoles or electron-dense granules of the foam cells. These results indicate that AGE accumulation occurs in macrophages, smooth muscle cells, and their related foam cells. PMID- 7545875 TI - Experimental autoimmune insulitis. Induction by T lymphocytes specific for a peptide of proinsulin. AB - Type I diabetes, an autoimmune disease that occurs in humans and animals, is characterized by the destruction of insulin-secreting islet beta-cells of the pancreas. Antibodies directed toward multiple islet protein can be detected before diagnosis of type I diabetes; however, the identity of the inciting autoantigen(s) that targets beta-cells for destruction has not been defined. Autorecognition of many self-proteins by CD4+ T lymphocytes is restricted by the products of class II immune response genes of the major histocompatibility complex (MHC), and in human type I diabetes such a MHC association has been described. The present study uses a rat MHC class II (RT1.Bl) peptide binding motif to predict potentially autoreactive CD4+ T cell epitopes in two key islet beta-cell constituents: the enzyme glutamic acid decarboxylase (GAD) and the insulin precursor hormone proinsulin (PI). Seventeen-amino-acid-long peptide fragments of GAD and PI containing the binding motif were synthesized and used to generate peptide-specific, MHC class II-restricted, CD4+ T cell lines. Once established, the T cell lines specific for rat islet GAD and PI were adoptively transferred to naive, MHC-compatible rats. At 10 days after transfer, insulitis had developed in rats receiving PI-specific T cells, whereas no insulitis was observed in pancreata of rats receiving GAD-specific T cells. Of particular interest is the finding that the pathogenic T cell epitope identified in PI spans the endogenous cleavage site between the B-chain and C-peptide of insulin. Moreover, the PI-specific T cells were able to react specifically with material produced in vitro by a rat insulinoma cell line. These results demonstrate that pathogenic T cell epitopes can be located in portions of molecules that are subsequently degraded during normal enzymatic processing. As PI is found highest concentrations in the beta-cells of pancreatic islets, it is possible that this molecule and not its individual degradation products (ie, insulin and C-peptide) might serve as an autoantigen in the pathogenesis of type I diabetes. PMID- 7545876 TI - Bracken fern-induced bladder tumors in guinea pigs. A model for human neoplasia. AB - We have induced tumors by feeding guinea pigs with a diet containing 25 or 30% dried bracken fern for 100 or 150 days. A high incidence of bladder tumors was obtained. All but one animal had preneoplastic or neoplastic lesions after 4 months; after one year, 24 or 25 exposed animals had carcinoma. Bladder tumors obtained were essentially pure transitional cell carcinomas, although 4 cases (7% of the exposed animals and 10% of the 39 transitional cell carcinoma observed) showed areas of focal squamous metaplasia. Immunohistological detection of cytokeratins 10, 13, and 18 confirmed the transitional nature of these tumors. Tumor development can be followed by ultrasonography and cytology. Bladder tumors arose through several steps. Dysplasia and preneoplastic hyperplasia were seen after 4 months and papillary carcinomas appeared after 6 months, whereas muscle invasive carcinomas required 1 year. Thus this model reproduces the full spectrum of preneoplastic and neoplastic bladder lesions observed in humans. Interestingly, when tumors were induced in older guinea pigs, none of them progressed to a muscle-invasive stage. This phenomenon should provide the opportunity to study the molecular mechanisms associated with these two different growth patterns, a major issue in understanding human bladder tumor progression. PMID- 7545877 TI - In pursuit of the "holy grail": recombinant allergens and peptides as catalysts for the allergen-specific immunotherapy. AB - This presentation reviews recent progress in knowledge of (i) molecular biology and immunology of allergens and (ii) the methods for the modulation of allergen specific immune response, with special emphasis on our grass pollen allergens, particularly Pao p IX AL, used as a model. The last few years have seen tremendous progress in molecular characterization of allergens, leading to synthesis of a number of recombinant allergens. Furthermore, studies of the structure-activity relationship of allergens have led to the delineation of the epitopes of these allergens and of widespread cross-reactivities among diverse allergens. In view of the requirement for extensive pre-clinical studies, the application of the recombinant allergens and epitopes in the clinical realm has just begun. Studies in murine systems suggest that these allergens and/or their epitopic peptides downregulate allergen-specific immune responses de novo. The potential and drawbacks of the recombinant allergens and peptides for improving the existing immunotherapeutic approaches and for developing new approaches to specific immunotherapy are discussed. PMID- 7545878 TI - Immunoglobulin E regulation in humans, 1989-1994. PMID- 7545879 TI - Multiplicity of cross-reactive epitopes on Bet v I as detected with monoclonal antibodies and human IgE. AB - Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen (Betula verrucosa), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization. PMID- 7545880 TI - IgE autoantibodies in atopic dermatitis--occurrence of different antibodies against the CH3 and the CH4 epitopes of IgE. AB - Levels of "free" anti-IgE autoantibodies and IgE/anti-IgE immune complexes were measured in the sera of patients with atopic dermatitis before and after treatment, psoriasis patients, and nonatopic controls. In this measurement, we used two monoclonal antibodies with distinct in vitro functions (LE 27, BSW 17), directed against the epsilon CH3 and CH4 domains of the IgE Fc-fragment, in a novel immunobinding assay. In patients with atopic dermatitis, elevated levels of "free" anti-IgE antibodies and IgE/anti-IgE immune complexes were detected in comparison to psoriasis patients and controls. In addition, there was a positive correlation between total IgE and the amount of IgE/anti-IgE complexes detected by LE 27 (r = 0.7; P < 0.001) or BSW 17 (r = 0.64; P < 0.001) in patients with atopic dermatitis. In contrast, an inverse correlation was observed between total IgE and "free" anti-IgE antibodies (r = -0.34; P < or = 0.05) in atopic dermatitis. However, serum levels of anti-IgE autoantibodies before and after therapy in patients with atopic dermatitis did not differ, and levels of anti-IgE antibodies did not correlate with clinical severity, as evaluated by an established clinical scoring system. Our data clearly indicate that significantly elevated amounts of anti-IgE antibodies could be observed in patients with atopic dermatitis, which are directed against different epitopes on the IgE molecule. It is tempting to speculate that these autoantibodies exert different effects of IgE receptor-bearing effector cells and may play an important role in IgE regulation. PMID- 7545881 TI - The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors. AB - The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+ and CD8+ T cells and CD19+ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A2) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4+ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4+ and CD8+ T cells were stimulated by soluble low-affinity IgE receptor (Fc epsilon RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A2, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A2 cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc epsilon RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545882 TI - Melon sensitivity shares allergens with Plantago and grass pollens. AB - Possible associations between allergy to pollen and that to food allergens were studied in 262 patients sensitized to pollen. Forty-four patients (16.7%) showed some allergic symptoms after testing with fruits and vegetables, melon being the food most frequently involved (24 patients), followed by sunflower seed (12 patients). Skin testing was done by the prick method with natural fruit or vegetable, and also with commercial food extracts. We found in our region that the distribution of sensitivity to pollens in the group of patients with allergy to fruits or vegetables does not coincide with the prevalence in pollen-allergic subjects in general, since in the first group--subjects allergic to food--there was a major prevalence of allergy to Plantago (P < 0.01). In particular, in the group of subjects allergic to melon, the prevalence of sensitivity to grass and especially to Plantago was larger than in pollen-allergic subjects in general (P < 0.05 and P < 0.001, respectively). The use of fresh food produced better results than commercial extracts. A positive skin test to fresh melon closely correlated with positive CAP results. CAP inhibition experiments were carried out, and we found that Dactylis and Plantago extracts inhibited the binding of the melon-positive pool to solid-phase melon. The results suggest the existence of common antigenic epitopes in melon and Plantago pollen, and in melon and grass pollen. PMID- 7545883 TI - Cytokeratins as a marker for epicardial formation in the quail embryo. AB - Several techniques have been used to visualize the migration pattern of the epicardial cells from the proepicardial organ over the myocardial surface. As the epicardial cells contain keratin tonofilament bundles, we have incubated 92 whole mount quail hearts with an anti-keratin antibody. This immunohistochemical method showed that the complete epicardial covering of the embryonic heart is preceded by the formation of three epicardial rings. The epicardial rings are formed on the outer myocardial surface in the grooves that separate the cardiac segments from each other. We have also documented timing and patterning of isolated epicardial islands. They are not encountered at random over the myocardial surface, but only along the edge of the advancing epicardial front border and in two defined future epicardial ring areas on the ventral side of the outflow tract. The epicardial islands suggest that in the quail free-floating parts of epicardium can attach to the myocardium. Characteristics of the surface of the myocardium at the transitional zones between the cardiac segments, as well as the three-dimensional remodelling of the heart during cardiac morphogenesis seem to play a role in the pattern in which the epicardium eventually completely ensheaths the myocardial surface. Congenital heart defects are often related to malpositioned transitional zones that dictate the pattern of epicardial outgrowth. As the embryonic position of the epicardial rings is mirrored in the pattern of the main arterial stems, the coronary vascularization pattern might be altered in congenitally malformed hearts as well. PMID- 7545884 TI - Immunohistochemical localization of connexin 43 in the developing tooth germ of rat. AB - Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360-376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum intermedium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and predontoblasts was accumulated at cell surfaces facing the predentine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545885 TI - Clonidine and cirazoline inhibit activation of nicotinic channels in PC-12 cells. AB - Clonidine and cirazoline bind with high affinity to a nonadrenergic site in the brain stem, the so-called imidazoline I1 receptor. Our aim was to determine the mechanism by which these receptors act and their possible linkage to signal transducing heterotrimeric G-proteins. We examined the effects of clonidine and cirazoline on PC-12 cells, a neuronal cell line that is reported to possess the I1 site and have no alpha 2-adrenoceptors. In undifferentiated PC-12 cells loaded with the Ca2+ indicator dye fura-2, clonidine and cirazoline (10-100 microM) inhibited the increase in [Ca2+]i produced by nicotine (10 microM). This inhibition was not reversed by yohimbine (100 microM), and adrenaline and BHT 920 were ineffective at 100 microM. This effect was not inhibited by pretreatment with pertussis toxin (24 hours, 100 ng/ml) and not modulated by pretreatment with IBMX (100 microM). The nicotine-induced increase in [Ca2+]i is apparently due to Ca2+ entering via the intrinsic ion channel of the nicotinic acetylcholine receptor. Clonidine and cirazoline inhibited the inward current produced by nicotine (10 microM) as measured by the whole cell patch-clamp technique in differentiated PC-12 cells, recorded at a holding potential of -60 mV. In agreement with the results found with fura-2, inhibition of inward current was concentration dependent and not blocked by yohimbine (100 microM) or mimicked by adrenaline (100 microM). Pretreatment of PC-12 cells with pertussis toxin or infusion of GDP-beta-S (2 mM) via the patch pipette did not alter the inhibition of the nicotine-induced inward current by clonidine or cirazoline. Clonidine and cirazoline, but not adrenaline, displayed [3H]phencyclidine from Torpedo electroplaque membranes enriched in nicotinic acetylcholine receptors in a concentration-dependent manner (10-100 microM). Taken together, these results suggest that clonidine and cirazoline inhibit Na+ and Ca2+ entry through the nicotinic acetylcholine receptor via a nonadrenergic mechanism that is independent of G-proteins and cyclic nucleotides, presumably by direct blockade of the intrinsic ion channel of the nicotinic acetylcholine receptor. PMID- 7545886 TI - No relationship of I1- and I2-imidazoline binding sites to inhibitory effects of imidazolines on ligand-gated ion channels. An investigation in the adrenal medulla and in neuroblastoma cells. PMID- 7545887 TI - [Evaluation of hemoglobin dextran 10-benzene-tetracarboxylate, oxygen transporters, using a model of guinea pig intestine]. AB - With the aim of assessing of dextran-benzene-tetracarboxylate hemoglobin as an oxygen carrier, we studied histological changes in the intestinal loop. The intestinal tissue being very sensitive to hypoxia, in the anesthetized guinea pig, an innervated loop was vascularly perfused with open-flow during one hour at zero hematocrit. To estimate the capacity of hemoglobin solution to oxygenate this tissue, we observed the mechanical and histological changes in the organ and the arterio-venous difference in PO2, oxyhemoglobin, deoxyhemoglobin and we compared them with human albumin, Tyrode and non-modified hemoglobin. The PO2 arterio-venous differences were 51.9 +/- 7.1 torr (m +/- SEM) for Tyrode, 40.2 +/ 6.4 torr for albumin solution, 113.7 +/- 6.5 torr for non-modified hemoglobin and 123.1 +/- 7.9 torr for dex-BTC-Hb. Compared to albumin and Tyrode solutions, hemoglobin solutions transferred more oxygen to tissues. The desaturation of dex BTC-Hb was significantly superior (p < 0.05) to the one non-modified hemoglobin. The structure of jejunal villi when perfused with a hemoglobin solution, remained almost normal and the loop was still active. Nevertheless, non-modified hemoglobin leaked from the vessels to the lumen and caused oedema and a rupture of overlapping epithelium at the tip of the villi. With dex-BTC-Hb, such histological modifications were less significant. With albumin and Tyrode, all villi were totally necrosed and the loop was completely inert. We have demonstrated that dextran-benzene-tetracarboxylate hemoglobin had the ability to maintain the tissue alive thank to its good capacity to release oxygen and its satisfactory vascular persistence. PMID- 7545888 TI - Advantage--FK 506: reduced chronic rejection for lung transplant recipients. PMID- 7545889 TI - Clinical trial of tacrolimus versus cyclosporine in lung transplantation. AB - BACKGROUND: A prospective clinical trial was undertaken to compare the efficacy of tacrolimus (FK 506) versus cyclosporine as the primary immunosuppressive agent after lung transplantation. METHODS: Between October 1991 and May 1994, 133 single-lung and bilateral-lung recipients were randomized to receive either cyclosporine (n = 67) or tacrolimus (n = 66). The two groups were similar in age, sex, and underlying disease. RESULTS: One-year and 2-year survival rates were similar in the two groups, although the trend was toward increased survival with tacrolimus. Acute rejection episodes per 100 patient-days were fewer (p = 0.07) in the tacrolimus group (0.85) than in the cyclosporine group (1.09). Obliterative bronchiolitis developed in significantly fewer patients in the tacrolimus group (21.7%) compared with the cyclosporine group (38%) (p = 0.025), and there was greater freedom from obliterative bronchiolitis over time for patients receiving tacrolimus (p < 0.03). Significantly more cyclosporine-treated patients (n = 13) required crossover to tacrolimus than tacrolimus-treated patients to cyclosporine (n = 2) (p = 0.02). The switch to tacrolimus controlled persistent acute rejection in 6 of 9 patients. The overall incidence of infections was similar in the two groups, although bacterial infections were more common with cyclosporine (p = 0.0375), whereas the risk of fungal infection was higher with tacrolimus (p < 0.05). CONCLUSIONS: This trial demonstrates the advantage of tacrolimus in reducing the risk of obliterative bronchiolitis, the most important cause of long-term morbidity and mortality after lung transplantation. PMID- 7545890 TI - Isolated single-lung perfusion: a study of the optimal perfusate and other pharmacokinetic factors. AB - BACKGROUND: Isolated single-lung perfusion with doxorubicin hydrochloride was shown to be effective in clearing experimental sarcoma lung metastases in the rat. The best perfusate to be used for isolated lung perfusion and factors affecting the final lung concentration of doxorubicin are the subject of the present study. METHODS: In experiment 1, 60 animals were randomized to undergo isolated left lung perfusion with doxorubicin with six different perfusates (n = 10 per group): saline, low-potassium-dextran, 5% albumin, 6% hetastarch, 5% buffered albumin, and 6% buffered hetastarch. Five animals served as negative controls. After perfusion, the lung wet to dry ratio and final lung doxorubicin concentration were determined. In experiment 2, 60 animals underwent isolated left lung perfusion with either 80 micrograms/mL or 320 micrograms/mL of doxorubicin. Animals were perfused at either 0.5 mL/min or 1 mL/min and for 2, 6, or 10 minutes. At the end of the perfusion period, the left lung doxorubicin concentration was measured. Statistical analysis included analysis of variance, the Duncan test for multiple comparisons, and multiple linear regression analysis. Significance was defined as a p value of less than 0.05. RESULTS: In experiment 1, perfusion with 6% buffered hetastarch resulted in the lowest lung wet to dry ratio, significantly different from all groups except the controls. Perfusion with low-potassium-dextran solution led to the highest final lung concentration of doxorubicin. In experiment 2, a model to predict final lung doxorubicin concentration was constructed: Log (final lung concentration) = 1.9 + 0.0071.P + 0.186.T, where P is the measured perfusate concentration of doxorubicin, and T is the time of perfusion in minutes. The R2 was 0.91 and p, less than 0.001. The dose of doxorubicin per kilogram of animal body weight, the dose of doxorubicin per square meter of body surface area, the total amount of doxorubicin delivered, and the rate of perfusion did not meet the criteria to enter the equation. CONCLUSIONS: Isolated lung perfusion experiments should use 6% buffered hetastarch as the perfusate. The perfusate doxorubicin concentration and the duration of perfusion are the only factors determining the final lung concentration of doxorubicin. In lung perfusion experiments, the dose of chemotherapy is not as important as the perfusate concentration and the duration of the perfusion. Animals should be perfused at a lower rate so the lungs are exposed to less doxorubicin without changing the final lung concentration. PMID- 7545891 TI - Role of selectins in myocardial ischemia-reperfusion injury. AB - During reperfusion of ischemic myocardium, there is a well-orchestrated interplay between the coronary vascular endothelium and the circulating neutrophils. This interplay involves the initial slowing or "rolling" of neutrophils along the endothelium during the early moments of reperfusion, followed by firm attachment and amplification of the neutrophil response, and culminating with the diapedesis of neutrophils into the myocardial parenchyma where neutrophil-myocyte interaction contributes to the necrotic process. The selectins are glycoproteins that play a key role in the early phases of neutrophil adherence and activation. There are three members of the selectin family: P-selectin on endothelial cells and platelets, L-selectin on neutrophils, and E-selectin on endothelium. Monoclonal antibodies directed specifically toward these selectins and their associated ligands (eg, Sialyl Lewisx) not only substantiate their role in the dynamic process of neutrophil-mediated reperfusion injury but also offer a unique therapeutic opportunity to interfere with this cascade of inflammatory events. PMID- 7545894 TI - Tricyclic CNS active agents by intramolecular Oxa-Pictet-Spengler reaction. AB - Mitsunobu inversion of the (S)-configurated lactate (S)-7, which is prepared in four steps starting from (S)-tyrosine, leads to the (R)-configurated lactate (R) 7. The key step in the transformation of the enantiomeric lactates (S)-7 and (R) 7 into the benzomorphan analogous tricycles (R,S)-16a,b, (S,R)-16a,b, (S,S)-22, and (R,R)-22 is an intramolecular Oxa-Pictet-Spengler reaction: The amides (S) 13, (R)-13, (S)-19 and (R)-19, in which the carbonyl moiety-masked as an acetal is linked to the 2-phenylethanol moiety, are cyclized to give the tricyclic amides (R,S)-15, (S,R)-15, (S,S)-21, and (R,R)-21, respectively. In a concentration of 100 microM both enantiomers of 16a, 16b, and 22 are not able to compete with 3H-(+)-MK 801 for the phencyclidine binding sites of NMDA receptors. In vivo, only (R,S)-16b and (S,S)-22 exhibit weak sedative and analgesic activity. PMID- 7545892 TI - Coronary artery endothelial function after myocardial ischemia and reperfusion. AB - BACKGROUND: The consequences of ischemia-reperfusion injury on myocytes has been studied intensely, and previous investigations of methods of myocardial protection during global and regional ischemia have focused on resultant alterations in myocardial function. However, the coronary artery endothelium is also vulnerable to damage, and only recently have investigators been able to assess coronary endothelial function. METHODS: This review examines some aspects of coronary flow abnormalities that occur after ischemia and reperfusion. In addition, we summarize recent data that address the hypothesis that injury to the coronary artery endothelium may contribute to the pathophysiology of global (and regional) cardiac ischemia and reperfusion. RESULTS: It appears that ischemia and reperfusion selectively injure a component in the receptor/G-protein complex linking receptor-stimulus coupling to the activation of nitric oxide synthase. Further, oxygen radicals may contribute to this injury. Recent investigations demonstrate that oxygen radicals impair the receptor/G-protein complex specific to the nitric oxide signal transduction pathway rather than causing global receptor/G-protein dysfunction. CONCLUSIONS: The understanding of endothelial cell function and the elucidation of the nitric oxide pathway should further clarify our understanding of the pathogenesis of endothelial reperfusion injury and coronary vasospasm and contribute to the development of effective therapeutic interventions. PMID- 7545893 TI - The role of bradykinin and nitric oxide in the cardioprotective action of ACE inhibitors. AB - BACKGROUND: The angiotensin-converting enzyme inhibitor ramiprilat has been previously demonstrated to protect myocardium from ischemia/reperfusion injury. The objective of these investigations was to examine the roles of bradykinin, angiotensin II, and nitric oxide in the cardioprotective effects of ramiprilat. METHODS: Anesthetized, open-chest rabbits were instrumented for production of myocardial ischemia (30 minutes) and subsequent reperfusion (120 minutes), after which myocardial infarct size was measured. Animals were treated intravenously with either saline solution, ramiprilat (50 micrograms/kg), the bradykinin2 receptor antagonist HOE 140 (1 microgram/kg), ramiprilat + HOE 140, angiotensin II (2.5 ng.kg-1.min-1), the angiotensin II receptor antagonist losartan (20 mg/kg), ramiprilat + angiotensin II, the nitric oxide synthase inhibitor NG-nitro L-arginine methyl ester (100 micrograms.kg-1.min-1), or ramiprilat + NG-nitro-L arginine methyl ester. RESULTS: Among all treatment groups myocardial infarct size was reduced significantly below saline control only by ramiprilat (-54%) and ramiprilat + angiotensin II (-37%). Pretreatment with HOE 140 or NG-nitro-L arginine methyl ester abolished the cardioprotective effect of ramiprilat. Neither stimulation nor antagonism of angiotensin II receptors altered infarct size from the saline control level. Also, when isolated neonatal rat cardiomyocytes were exposed to hypoxia/reoxygenation, ramiprilat (100 mumol/L) and bradykinin (10 nmol/L) improved cell viability (approximately 60%), and the protective effect of both agents was reversed by administration of HOE 140 (10 mumol/L). CONCLUSIONS: These results indicate that the in vivo cardioprotective effect of ramiprilat can be abolished by antagonizing bradykinin receptors or inhibiting nitric oxide synthase, and that the effect is not related to angiotensin II receptor activity. The potential bradykinin-sparing property of ramiprilat may promote increased bradykinin-stimulated nitric oxide production leading to cardioprotection. Part of the cardioprotective effects of ramiprilat/bradykinin/nitric oxide may occur locally as demonstrated by the in vitro results using isolated cardiomyocytes. PMID- 7545895 TI - Ontogeny of circadian dentinogenesis in the rat incisor. AB - Periodic growth increments are found universally in the dentine of animals. The goal here was to determine when and how circadian dentine growth increments develop ontogenetically. A total of 97 rat pups, obtained from 13 mothers of the Wistar strain, were injected with nitrilotriacetato lead at appropriate intervals to chronologically label the dentine. Labelled rat pups were killed 5-50 days after birth. Histological transverse sections of the demineralized dentine were obtained from the maxillary incisors of the pups and stained with haematoxylin. Two types of increments were observed in the circumpulpal dentine. The first type was a deeply stained circadian band which appeared at intervals of about 16-24 microns. The other was a faintly stained, narrow, ultradian increment, at intervals of approx. 6-8 microns. The circadian increments were first detected during the second to third week after birth and were observed consistently afterwards. Before the circadian increments developed, the ultradian increments were predominant. These two rhythms seemed to be independent of each other and sometimes coexisted in one section. Thus, the two types of dentine increments may be due to two independent mechanisms. PMID- 7545896 TI - The angioarchitecture of the rat mandibular joint synovium. AB - The synovium (or synovial membrane) lines all intra-articular surfaces of synovial joints except for the articulating surfaces. This study describes the angioarchitecture of the synovium of the adult rat temporomandibular joint using microvascular corrosion casts and scanning electron microscopy. To corroborate findings, India ink-injected, thick-sectioned specimens (200 microns) and haematoxylin and eosin-stained tissue sections (7 microns) were analysed. Rostrally, the synovial membrane was fed by several layers of vessels branching towards the superficial lining of highly convoluted capillary loops. Arterioles were rare, and venules organized in large plexuses. The membrane thinned out in the caudal direction. Villi were present on the surface. They were flat, with a core of two parallel linear feeder vessels, one arteriole and one venule, which were interconnected by capillary loops. At the periphery of the articulating surfaces, the vessels of the synovial layer were flatter. Caudally, the synovial membrane of the richly vascularized retrodiscal pad showed many synovial folds. Feeder vessels were directed rostrally and branched in all directions. The number of subdivisions depended on the size of the synovial fold. Again, venules were organized in plexuses and lining capillaries were convoluted. The densely capillarized synovial membrane with its folds and villi is considered well adapted to serve synovial fluid production and joint dynamics. PMID- 7545897 TI - The angioarchitecture of the rat mandibular joint bilaminar zone. AB - In mammals, temporomandibular-joint articular disc attachments have a bilaminar pattern. The three-dimensional angioarchitecture of the bilaminar zone in the adult rat was described using scanning election microscopy of microvascular corrosion casts (47 specimens) and light microscopy of Indian ink-injected, cleared, thick-sectioned temporomandibular joints (6 specimens). The bilaminar zone had an axial core of feeder vessels composed primarily of flat venules which were organized in plexuses. Arterioles were few and slender. In both laminae, there were usually three branching levels until vessels approached the surface of the lamina, where a dense capillary meshwork was formed. Both laminae ended abruptly at the periphery of the avascular disc with a single, slightly undulating marginal vessel. This marginal vessel, which faced the avascular disc, was definitely larger in diameter than the other superficial capillaries and was rather a postcapillary venule than a capillary. Functionally, this marginal venule might be important in sustaining nutrition of the avascular disc centre by allowing bidirectional blood flow during jaw movements. PMID- 7545898 TI - Electron-microscopic and immunohistochemical studies of Langerhans cells and Thy 1-positive cells in mouse tongue epithelium subjected to local hyperthermia. AB - Local hyperthermia via skin has been used to treat cancer but may suppress local immune responses as a side-effect. To examine effects of heat on immunologically responsive cells in oral mucosa, mouse tongues were heated by an implant system at 43 degrees C for 20 min. The densities of Langerhans cells and Thy-1-positive cells rapidly increased within 3 h after the treatment, then returned to a normal level after 7 days. Electron microscopy confirmed that Langerhans cells in the tongue epithelium formed clusters with lymphocytic cells, suggesting an active response to the hyperthermia. PMID- 7545899 TI - Comparison of four fixatives for routine splenic histology and immunohistochemical staining for group II avian adenovirus. AB - Four different fixatives, A, B, C, and D, were compared for use in routine histology of splenic tissue and for immunohistochemical staining to detect group II avian adenoviral antigen. Sixteen pheasants were experimentally infected with type II adenovirus; spleens from each bird were harvested, divided, and treated with each of the four fixatives. All four fixatives were adequate for routine histology. Fixative B was significantly more sensitive for immunohistochemistry than the three other fixatives, and it also detected the viral antigens at the highest dilutions of the primary antibody. PMID- 7545900 TI - Information processing subsystems. Channel and net. The matter-energy routes for information. PMID- 7545902 TI - Interaction of mistletoe toxic lectin-I with sialoglycoproteins. AB - The binding properties of mistletoe toxic lectin-I (ML-I) with sialo-N- and O glycans were investigated by quantitative precipitin and precipitin inhibition assays. Human alpha 1-acid glycoprotein reacted strongly with ML-I, precipitating over 82% of the lectin nitrogen tested, while the precipitability of its asialo product decreased by 30%. Native fetuin precipitated 50% of the ML-I added, and its reactivity was reduced by 20% after desialylation. On the contrary, the poor reactivity of rat sublingual sialoglycoprotein with ML-I increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein-lectin interactions were inhibited by NeuAc alpha 2-->3/alpha 2- >6Gal beta 1-->4Glc and/or Gal beta 1-->4Glc (NAc) residues. From the above results, it is concluded that ML-I is specific for sialic acid. However, sialic acid of some O-glycans also acts as masking molecule as the precipitability of rat sublingual and bovine submandibular glycoproteins with ML-I increased after desialylation. PMID- 7545901 TI - The 33-kDa C-terminal domain of Raf-1 protein kinase exhibits a Ras-independent serum- and phorbol ester-induced shift in gel mobility. AB - Experiments were carried out to determine Raf-1 protein kinase domain fragments which exhibit a characteristic electrophoretic mobility shift noted with Raf-1 protein kinase in response to serum and phorbol ester (PMA) treatment of serum deprived NIH 3T3 cells. Epsilon-epitope tagged 84 kDa Raf-1 holoenzyme (HR epsilon), as well as the epsilon-epsilon pitope tagged 35 kDa N-terminal (RI epsilon), 33 kDa mid-portion (RII-epsilon), and 33 kDa C-terminal (RIII-epsilon) fragments of Raf-1 were overexpressed in NIH 3T3 cells. The overexpressed HR epsilon exhibited a serum- and PMA-induced shift in gel mobility similar to that noted with endogenous Raf-1. The C-terminal RIII-epsilon fragment exhibited a similar shift in gel mobility while the electrophoretic mobility of the N terminal RI-epsilon fragment remained unchanged. These results suggest that modification(s) within the 33 kDa C-terminal portion of Raf-1 which occur independently of association with Ras may be responsible for the band shift observed with serum and PMA treatment of serum-deprived NIH 3T3 cells. PMID- 7545903 TI - Isolation of active ribozymes from an RNA pool of random sequences using an anchored substrate RNA. AB - To analyze the structure-function relationship of a ribozyme, RNA molecules with cleavage activities were isolated from a pool of RNAs that have a region of 14 random nucleotides for catalysis by a novel in vitro selection method. Active ribozymes from the pool were selected using a substrate RNA anchored to an agarose bead. After 7 or 8 selection cycles, the pool of the selected molecules showed cleavage activity against the substrate RNA. Structures and catalytic activities of the individual selected RNA molecules were then analyzed by cDNA cloning, in vitro transcription and cleavage reactions. All the active ribozymes were found to catalyze the same sequence-specific cleavage reaction as does the hammerhead ribozyme and contain the conserved nucleotide sequences of the hammerhead ribozymes. PMID- 7545904 TI - TPO and IL-3 induce overlapping but distinct protein tyrosine phosphorylation in a myeloid precursor cell line. AB - TPO is a recently cloned cytokine which appears to play a central role in megakaryopoiesis and platelet production. It has been shown to be the ligand for c-mpl, a member of the hematopoietic receptor superfamily. We examined intracellular tyrosine phosphorylation events induced by TPO and compared them with events stimulated by IL-3, a pleiotropic cytokine which also has activity on the megakaryocyte lineage. The overall pattern of tyrosine phosphorylation stimulated by TPO and IL-3 in myeloid precursor cells revealed an overlapping but not identical pattern reflecting their distinct but partially redundant biological effects. We identify Shc and Vav as intracellular targets of TPO induced tyrosine phosphorylation. Moreover, we demonstrate that the tyrosine kinase Jak2 is phosphorylated after TPO stimulation. Whereas phosphorylation of these proteins was induced by both cytokines, phosphorylation of Jak1 was induced only by IL-3 and not by TPO, distinguishing the signal transduction of the two cytokines on a molecular level. PMID- 7545905 TI - SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties. AB - We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65 degrees C. The pH optimum ranges from 6.5-7.0. DNA binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea. PMID- 7545906 TI - The effect of phosphorylation on the antigenic reactivity of p53 in cultured human keratinocytes. AB - The detection of p53 in human keratinocytes is dependent on the specific anti-p53 monoclonal antibody that is used. Differences in antibody recognition are postulated to be due to the masking or exposure of particular epitopes in different conformations of p53. This study addresses the role of phosphorylation on p53-epitope accessibility in human keratinocytes. Keratinocytes were treated with the phosphatase inhibitor, okadaic acid, to determine the effect of inhibiting cellular phosphatases on p53 phosphorylation and epitope recognition. These studies suggest there is a correlation between the level of p53 phosphorylation and the antigenic reactivity of certain p53 epitopes in human keratinocytes. We also examined the ability of the catalytic subunits of protein phosphatase 1 and 2A to dephosphorylate p53 derived from human keratinocytes in vitro. These data suggest that PP2A may be the phosphatase that acts on p53 in cultured human keratinocytes. PMID- 7545907 TI - Members of a family of Drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs. AB - A polymerase chain reaction-based method was used to generate a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. We identified five cDNAs that code for antennal proteins containing six cysteines in a conserved pattern shared with known moth antennal proteins, including pheromone binding proteins. Another cDNA codes for a protein related to vertebrate brain proteins that bind hydrophobic ligands. In all, we describe seven antennal proteins which contain potential signal peptides, suggesting that, like pheromone binding proteins, they may be secreted in the lumen of olfactory hairs. The expression patterns of these putative odorant-binding proteins define at least four different subsets of olfactory hairs and suggest that the Drosophila olfactory apparatus is functionally segregated. PMID- 7545908 TI - Sequence analysis of the mutation at codon 834 and the sequence variation of codon 837 of c-abl gene. PMID- 7545909 TI - Analysis of K-ras, p53 and c-raf-1 mutations in beryllium-induced rat lung tumors. AB - Beryllium (Be) metal and several of its analogues have been shown to be carcinogenic in rats. In addition, workers employed at Be processing plants have been shown to have a slight excess of lung cancer. In this study, a single inhalation exposure to Be metal produced a 64% incidence of lung tumors in the F344/N rat. The most frequent tumor type observed was adenocarcinoma. These Be metal-induced lung carcinomas were examined for genetic alterations in the K-ras, p53, and c-raf-1 genes. DNA isolated from lung neoplasms was analyzed by PCR amplification and direct DNA sequence analysis, immunohistochemical analysis and Southern blot analysis. No K-ras codon 12, 13 or 61 mutations were detected in 24 lung tumors by direct sequencing. Using a more sensitive K-ras codon 12 mutation selection assay, K-ras codon 12 GGT-GTT transversions were detected in two of 12 adenocarcinomas. These results suggest that activation of the K-ras protooncogene is both a rare and late event, possibly stemming from genomic instability during the progression of some Be-induced rat adenocarcinomas of the lung. No mutant p53 nuclear immunoreactivity was observed in any Be-induced tumor. Because immunohistochemical analysis of the p53 protein only detects missense mutations, exons 5-8 of this gene were also analyzed by direct DNA sequencing. In order to perform the p53 sequence analysis, it was necessary to first characterize and sequence the p53 intron sequences flanking exons 5-8 and their splice sites. Details of this expanded intron DNA sequence information are given here. No mutations were detected within exons 5-8 of the p53 gene. No rearrangement of the c-raf-1 protooncogene was detected by Southern blot analysis. These results indicate that the mechanisms underlying the development of Be-induced lung cancer in rats do not involve gene dysfunctions commonly associated with human non-small cell lung cancer. PMID- 7545910 TI - Molecular cloning of Drosophila TFIID subunits. AB - Transcription initiation factor TFIID is a multisubunit complex containing a TATA box-binding factor (TFIID tau/TBP) and associated polypeptide factors (TAFs) with sizes ranging from M(r) approximately 20,000 to > 200,000. As a result of direct promoter interactions, TFIID nucleates the assembly of RNA polymerase II and other initiation factors into a functional preinitiation complex. Although the native TFIID complex mediates both basal and activator-dependent transcription in reconstituted systems, TBP itself is competent for only basal transcription. Thus, TAFs are essential cofactors for regulated transcription. The complementary DNAs encoding the p230 (M(r) 230,000), p110 and p85 subunits of TFIID have recently been cloned. Here we report the molecular cloning and characterization of the p62, p42, p28 and p22 subunits. These participate in a network of heterogeneous protein-protein interactions within TFIID. Sequence similarities between p62/p42 and the histones H4/H3, respectively, suggest that these subunits have a functional relationship with chromatin. PMID- 7545911 TI - Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus. AB - A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046-related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another. PMID- 7545912 TI - XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development. AB - We have isolated a Xenopus homeodomain sequence, XNkx-2.5, which shows significant similarity to mouse Nkx-2.5 and to the Drosophila tinman gene product. In Drosophila, tinman is required for formation of the heart and visceral mesoderm structures. In situ hybridization studies show that XNkx-2.5 is expressed in the heart region during early Xenopus development and later is also expressed in gut tissue. The observed similarity of sequences and expression patterns suggests that the regulatory mechanisms underlying heart formation may be conserved between distant species. PMID- 7545913 TI - Isolation of two isoforms of the PAX3 gene transcripts and their tissue-specific alternative expression in human adult tissues. AB - We have isolated two isoforms of cDNA clones from the human PAX3 gene, a candidate gene responsible for Waardenburg syndrome type I (WSI) as well as a gene associated with development of alveolar rhabdomyosarcoma. The gene product is considered to be one of transcription factors, and the two cDNA clones isolated, termed PAX3A and PAX3B, were generated by alternative splicing. The transcripts coded 215 and 206 amino acids, respectively, and shared 196 amino acids at the NH2 end. The amino acid sequence in the common region (residues 1 196) showed a 100% identity with that of exons 1-4 of the mouse Pax-3 gene. However, both of the PAX3 cDNAs lacked the DNA sequence corresponding to the paired-type homeodomain of the mouse Pax-3 gene. Analysis of gene expression in human adult tissues by reverse transcriptase polymerase chain reaction (RT-PCR) revealed tissue-specific expression of this gene. PAX3B was expressed in most of the tissues examined, but the PAX3A type of transcript was detected only in the cerebellum, esophagus, and skeletal muscle. PMID- 7545914 TI - Non-phosphorylating GAPDH of higher plants is a member of the aldehyde dehydrogenase superfamily with no sequence homology to phosphorylating GAPDH. AB - Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NADP specific, EC 1.2.1.9) operates in the cytosol of autotrophic eukaryotes where it generates NADPH for biosynthetic processes from photosynthetic glyceraldehyde 3 phosphate exported from the chloroplast by the phosphate translocator. Here we report the first cloning and characterization of cDNAs encoding complete polypeptide chains of nonphosphorylating GAPDH from pea and maize by using oligonucleotide probes derived from amino acid sequences determined for the purified enzyme. Unexpectedly, nonphosphorylating GAPDH cannot be aligned with the well-known sequences of phosphorylating GAPDH, but shares about 30% amino acid identity with various specialized and non-specialized aldehyde dehydrogenases (ALDHs) of eubacteria and eukaryotes. A phylogenetic analysis of this ALDH superfamily reveals a complex evolutionary pattern with numerous major branches carrying genes from eubacteria, eukaryotes, or both, encoding enzymes that are specific or non-specific for particular aldehyde substrates. This topology suggests a concomitant emergence of multiple substrate specificities from non-specialized ALDH during an early evolutionary phase of intense metabolic diversification. Although unrelated at the sequence level, non-phosphorylating aldehyde dehydrogenases and phosphorylating GAPDH resemble one another with respect to catalytic hydride transfer and covalent thiol ester formation. Whether or not this reflects an ancestral relationship can only be decided when crystallographic data for ALDH enzymes have become available. PMID- 7545915 TI - Complete RNA editing of unspliced and dicistronic transcripts of the intron containing reading frame IRF170 from maize chloroplasts. AB - The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA. PMID- 7545916 TI - Bidirectional transcription from the human immunoglobulin VH6 gene promoter. AB - The human immunoglobulin (Ig) heavy chain VH6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promoter are very active in non-B cells. In experiments designed to characterize regions upstream of the transcriptional start site that are important for promoter function, we produced a series of deletion constructs, including one containing sequences between -74 and -146. Surprisingly, this fragment had promoter activity in both orientations. Inspection of the VH6 promoter sequence indicated that there was a possible TATA box in the proper orientation upstream of the imperfect octamer. The -74 to -146 fragment functioned as a promoter in the reverse orientation in three B cell lines and in non-B (HeLa) cells, with a much higher level of activity seen in the HeLa cells. To determine if the promoter could work in both directions simultaneously, reporter genes were positioned up- and downstream of a VH6 promoter fragment. Reporter gene activity was found for both genes in B cells and HeLa cells. Using a reverse transcriptase-polymerase chain reaction procedure (RT PCR), we found a transcript corresponding to sequences upstream of the VH6 promoter in RNA from both the lymphoblastoid cell line ML-1, which actively transcribes the VH6 promoter, and the REH cell line, which does not. No transcripts were found in the KB epithelial cell line. Two or three mRNA 5' ends were found that mapped between -137 to -143 from the authentic VH6 transcription site, 31-37 nucleotides upstream of the putative TATA box. Inspection of the sequence upstream of the VH6 promoter demonstrated the presence of an open reading frame capable of coding for 96 amino acids. The VH6 promoter represents the second Ig promoter with bidirectional activity. PMID- 7545917 TI - A peptide derived from the amino terminus of endothelial-monocyte-activating polypeptide II modulates mononuclear and polymorphonuclear leukocyte functions, defines an apparently novel cellular interaction site, and induces an acute inflammatory response. AB - Endothelial-monocyte-activating polypeptide II (EMAP II) is a novel mediator isolated from conditioned medium of methylcholanthrene A-induced tumor cells which modulates properties of endothelial cells, mononuclear phagocytes (MPs), and polymorphonuclear leukocytes (PMNs) in vitro and induces an acute inflammatory response in vivo. A synthetic peptide comprising 15 residues from the N-terminal region (residues 6-20) was shown to induce directional migration of MPs and PMNs, with half-maximal effect at approximately 200-250 pM, whereas a peptide from the C terminus of EMAP II, as well as other irrelevant peptides, were without effect. Modulation of cellular phenotype by EMAP II-derived peptide was suggested by peptide-induced elevation of cytosolic free calcium concentration in fura-2-loaded MPs and PMNs and by stimulation of peroxidase release in PMNs. Consistent with these in vitro data, EMAP II-derived N-terminal peptide-albumin conjugates injected into the mouse footpad elicited inflammatory cell tissue infiltration, whereas albumin alone or EMAP II-derived C-terminal peptide conjugated to albumin incited little response. Binding of 125I-labeled EMAP II-derived peptide (residues 12-20) to MPs was saturable (Kd approximately 200 pM) and was blocked in a dose-dependent manner by the addition of intact EMAP II and unlabeled EMAP II-derived peptides (residues 6-20 and 12-20), whereas interleukin 1, tumor necrosis factor, formyl-methionyl-leucinyl-phenylalanine, or irrelevant peptides were without effect. Cross-linking of 125I-EMAP II-derived peptide (residues 12-20) by disuccinimidyl suberate to human MPs demonstrated a band, approximately 73 kDa, on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 125I-EMAP II-derived peptide also demonstrated specific binding to human PMNs and murine RAW cells. These data indicate that the N-terminal region of EMAP II defines a biologically active locus of the molecule which interacts with target cells via a potentially novel cellular receptor. PMID- 7545918 TI - Genetic variation in swine influenza virus A isolate associated with proliferative and necrotizing pneumonia in pigs. AB - A new antigenic variant of H1N1 swine influenza virus A (Sw/QC/5393/91 [QC/91]) has been found to be associated with porcine proliferative and necrotizing pneumonia. Analysis of its genomic RNA by T1 oligonucleotide mapping revealed that considerable genomic divergence exists between QC/91 and the swine influenza viruses currently circulating in North American swine herds. Analysis of the nucleotide sequence of the HA1 region of the hemagglutinin RNA of QC/91, in comparison with those of most common H1N1 human and swine influenza A viruses, showed the presence of multiple point mutations. Two amino acid substitutions appeared to be located in antigenic sites Sb and Ca. This correlates with antigenic variations demonstrated between A/NJ/8/76, A/Sw/WI/49/76, and Quebec isolate A/Sw/QC/5393/91 of swine influenza virus A. Another mutation was responsible for the loss of a glycosylation site, which may have also affected the antigenicity. The other mutations seem to have been accumulated progressively over time. This significant constancy in the fixation of mutations with time suggests that genetic diversity of these viruses may best be interpreted as the result of drifts in the population of circulating swine influenza viruses in Quebec. PMID- 7545919 TI - Isolation and nucleotide sequence analysis of human T-cell lymphotropic virus type II in Spain. PMID- 7545920 TI - Human rheumatoid factors with restrictive specificity for rabbit immunoglobulin G: auto- and multi-reactivity, diverse VH gene segment usage and preferential usage of V lambda IIIb. AB - To determine the molecular and functional properties of human rheumatoid factors (RF), we established stable hybridomas and Epstein-Barr virus-transformed B cell lines from the synovial fluid or peripheral blood of three patients with rheumatoid arthritis and one patient with systemic lupus erythematosus. 17 cell lines were obtained that produced high-titer immunoglobulin M (IgM) RF that reacted exclusively with rabbit but not human IgG or IgG of other mammalian species. Certain anti-rabbit IgG RF also had specificity for other mammalian antigens (Ag), including cytoskeletal proteins and intracellular proteins found in HeLa cells, as well as for Ag present in an extract prepared from the cell wall of group A streptococci. 13 of the 17 RF contained lambda-type light (L) chains, of which 12 were classified serologically as members of the lambda-L chain variable region (V lambda) subgroup, designated V lambda III. The heavy chain V region (VH) and V lambda sequences of nine of these IgM lambda RF were determined at the cDNA level. Five VH genes in three VH families were used by these antibodies (Ab), including VH1 (dp21/1-4b and dp10 [51p1]/hv1051), VH3 (dp38/3-15 and dp77/13-21), and VH4 (dp70/4-4b). The deduced V gene-encoded amino acid sequences of the lambda chains of these IgM lambda RF confirmed their serological classification as lambda III, and they were further classified as members of the relatively uncommon V lambda III subgroup, designated V lambda IIIb. Based on cDNA analyses, nine were the product of three different V lambda III b germline genes. Two such genes, designated hsiggll150 and hsiggll295, were cloned and sequenced from genomic DNA. Unique combinations of these VH and V lambda III b genes could be related to distinctive patterns of reactivity among the IgM lambda RF. Although the VH and V lambda regions of these Abs were expressed primarily as germline-encoded sequences, four of nine multireactive Abs had extensive V region mutation, indicative of an Ag-driven process. The finding that lambda IIIb L chains are preferentially found among anti-rabbit IgG RF, and that some of these Ab have specificity for other protein, cellular, and bacterial Ag, provides new insight into the pathogenesis of RA and related diseases. PMID- 7545922 TI - Identification of mutations in DNA polymerase beta mRNAs from patients with Werner syndrome. AB - Werner syndrome (WS) is a rare autosomal recessive disorder characterized by prematurely aged appearance. Genetic linkage analysis has placed the relevant gene in subchromosomal band 8p12. DNA polymerase beta gene has been mapped to chromosome 8p12-11 and thought to be involved in DNA repair and possibly in recombination. Somatic cells from WS patients exhibit chromosomal instability, a markedly reduced replicative life span and slow growth. The functions of DNA polymerase beta gene and its position prompted us to examine this gene in WS patients. We have found the novel DNA polymerase beta cDNA species in blood samples from WS patients, which contain 107 bp insertions or 87 bp deletions in the catalytic domain of DNA polymerase beta. These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS. PMID- 7545923 TI - Sequence, expression, and mapping of a rat Mhc class Ib gene. PMID- 7545921 TI - Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. AB - Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts. The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection. To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts. Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages. Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment. A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced. An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences. The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis. The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria. Both the A2 protein of Leishmania donovani and the S antigen of P. falciparum are stage specific and developmentally expressed in mammalian hosts. PMID- 7545924 TI - Adaptation of hepatitis C virus for persistent infection in patients with acute hepatitis. AB - Nucleotide sequencing was used to analyze amino acid substitutions in the putative envelope 1 (E1) and envelope 2/nonstructural 1 (E2/NS1) regions of hepatitis C virus (HCV) to clarify a viral mechanism of persistent infection in three patients with acute hepatitis C who developed chronic hepatitis and two patients with chronic hepatitis. The HCV RNA titer in serum decreased markedly after the onset of acute hepatitis and then re-elevated. During this period, the substitution rate in the E2/NS1 region (especially in the hypervariable region located in the N-terminus) was significantly higher in patients with acute hepatitis than in patients with chronic hepatitis (P < 0.05). When patients with acute hepatitis C became persistent HCV carriers, the substitution rate decreased to the level seen in patients with chronic hepatitis. The amino acid substitution rate in the E1 region in the acute phase was similar to that found in the chronic HCV carrier state. These observations suggest that rapid substitution of the amino acid sequence in the hypervariable region of the E2/NS1 region may be one of the mechanisms of persistent HCV infection. PMID- 7545926 TI - Molecular epidemiology of HIV-1 in Madrid. AB - Thirteen HIV-1 isolates from patients of different risk groups in Madrid (Spain) have been analyzed at the genetic level. Two distinct lineages of subtype B have been detected among the HIV-1 circulating in this area: one was related to SF 2/RF strains, whereas the other consists of a more heterogeneous group related to reference strain III-B. Variants of each lineage appeared to circulate preferentially within a risk group: III-B among intravenous drug users, and RF/SF 2 among male homosexuals. PMID- 7545925 TI - Expression patterns of novel genes during mouse preimplantation embryogenesis. AB - Little is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two-cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two-cell and two-cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190-1201, 1992) to isolate a panel of seven cDNA clones. Three cDNAs had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras-related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1-3, were found. The two Class 1 genes displayed an actin-like pattern, with a gradual decline in expression during oocyte maturation and through the two-cell stage, followed by increases at the eight-cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one-cell stage and then declined abruptly between the one- and two-cell stages; an increase then occurred at the eight-cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one-cell stage, with only a very slight increase in synthesis at either the eight cell or blastocyst stage. PMID- 7545927 TI - Mitochondrial transfer RNA genes of the yeast Candida parapsilosis. AB - Fifteen tRNA-encoding genes were mapped on the linear mitochondrial (mt) DNA of the yeast, Candida parapsilosis, and their sequences determined. The gene order and gene sequences indicate that the mt genome of this yeast belongs to a group clearly different from the already known group of linear mt DNAs of yeast. PMID- 7545929 TI - Characterization of a C alpha gene of swine. AB - The cDNA sequence encoding the constant region of the porcine IgA heavy chain as well as the exon-intron structure of the germline gene, have been determined. A cDNA clone (1A1) spanning the CH3 domain and part of the CH2 domain was isolated from a porcine mesenteric lymph node cDNA library. Clone 1A1 was aligned with a PCR-generated DNA fragment encompassing the CH1 domain through the 5' end of the CH3 domain to derive the complete cDNA sequence. Comparison with other mammalian C alpha heavy chains (hinge regions excluded) indicated that the deduced amino acid sequence of porcine C alpha is most homologous with the human C alpha subclasses (> 70%), followed by mouse C alpha (61%) and a consensus sequence of the 13 rabbit C alpha heavy chains (59%). The greatest sequence homology was found among the CH3 domains in all species. A striking feature of porcine C alpha is its short six amino acid hinge which like other mammalian IgAs, is encoded with the CH2 domain. Sequence analysis of germline C alpha, generated by PCR from liver or sperm DNA, revealed an exon-intron organization similar to other mammalian C alpha genes. Genomic Southern blot data are consistent with the presence of a single C alpha gene within the porcine genome. Data obtained in these studies will be valuable in pursuing the use of swine as a model in immunological research. PMID- 7545928 TI - Longitudinal analysis of hepatitis C virus infection and genetic drift of the hypervariable region. AB - Hepatitis C virus (HCV) infections in a cohort of chimpanzees were studied retrospectively. All animals had been inoculated intravenously with materials derived from a single-source chimpanzee plasma implicated in non-A, non-B hepatitis, prepared by extensive ultracentrifugation. Anti-HCV and HCV RNA were monitored by the confirmatory line immunoassay and by an RNA-capture polymerase chain reaction method, respectively. In a chronically infected chimpanzee, HCV RNA was detectable after 32 days and throughout the acute phase, dropped transiently below detection level, and became detectable again. In 3 other chimpanzees with acute resolving infections, HCV RNA was detected 7-11 days after inoculation and became permanently undetectable after alanine aminotransferase normalization. Various anti-HCV profiles were detected among the chimpanzees. Analysis of the hypervariable region in E2/NS1 in 7 chimpanzees suggested genome stability on transmission, revealed different mutation frequencies during chronic infection, and suggested the importance of immune selection during chronic HCV infection. PMID- 7545930 TI - Stat3 and Stat4: members of the family of signal transducers and activators of transcription. AB - The deduced amino acid sequence of two members of the signal transducers and activators of transcription (STAT) family from the mouse are described. Comparison with the deduced protein sequence of the two previously described genes (Stat91 and Stat113), discovered because of their activation as transcription factors after interferon-induced tyrosine phosphorylation, shows several highly conserved regions, including the putative SH3 and SH2 domains. The conserved amino acid stretches likely point to conserved domains that enable these proteins to carry out the several required functions they are known and proposed to carry out. While Stat1 and Stat3 are widely expressed, Stat4 expression is restricted to testis, thymus, and spleen. Antiserum to Stat3 detects a major approximately 92-kDa protein and a minor approximately 89-kDa protein, while antiserum to Stat4 precipitates one major protein of approximately 89 kDa. PMID- 7545931 TI - Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR). AB - A method for direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus was developed, based on reverse transcription of the viral RNA coupled to DNA amplification by polymerase chain reaction. A set of primers was designed from Lelystad virus sequence within ORF 7 encoding nucleocapsid protein. From seven Spanish field isolated strains the 312 bp amplified fragment was cloned and sequenced. Alignment with Lelystad virus sequence revealed a 96 97% homology. A maximum sensitivity of 6.7 TCID50 was achieved with the reported procedure in experimentally infected swine alveolar macrophages cultures. The sensitivity obtained in crude clinical samples from experimentally infected 3 weeks old pigs was approximately 10(2) TCID50. High specificity for the PRRS virus was demonstrated for the method, as none of the seven common swine virus assayed rendered DNA amplification product. PMID- 7545933 TI - [Cloning and sequencing of promoter and signal sequence coding regions from Bacillus subtilis]. AB - Promoter and signal sequence coding regions from B. subtilis were cloned in E. coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector. The Sau3A digested chromosome DNA was ligated with BamHI digested pGPB14. The ligated mixture was used to transform E. coli C600. Ampicillin and erythromycin resistant clones were selected. Recombinant plasmids were isolated from double resistant transformants. Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E. coli cells resistant to different concentrations of ampicillin. The recombinant plasmids were transformed and showed the same secretion function in B. subtilis. The amount and localization of beta-lactamase were determined in transformed E. coli and B. subtilis. The results indicate that the beta-lactamase activities of E. coli mainly in periplasm while the enzyme produced by B. subtilis secreted extracellularly. 10 of the cloned fragments were sequenced by Sanger's dideoxy chain termination method. The sequencing data show that all fragments contain promoter, ribosome binding site and signal sequence coding region. PMID- 7545934 TI - Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector. AB - The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus. PMID- 7545935 TI - The organization of the gene for the functionally dominant alpha-amino-3-hydroxy 5-methylisoxazole-4-propionic acid receptor subunit GluR-B. AB - The murine gene encoding the GluR-B subunit of alpha-amino-3-hydroxy-5 methylisoxazole-4-propionic acid receptors was characterized with respect to exon intron organization, transcriptional start site, alternatively spliced transcripts, and adenosine to guanosine substitutions between gene and complementary DNA sequence. The GluR-B gene spans > 90 kilobase pairs and harbors 17 exons. Transcription appears to initiate approximately 430 nucleotides upstream of the translational start codon, with no intron in the 5'-untranslated region of the gene. Four alternatively spliced mRNAs are generated from the primary GluR-B transcript, two containing the modules Flip and Flop, and another two with alternate C-terminal coding sequence. The major GluR-B mRNAs in murine brain, 4 and 6 kilobase differ in the length of their 3'-untranslated region. PMID- 7545936 TI - The utility of SATA satellite DNA sequences for inferring phylogenetic relationships among the three major genera of tilapiine cichlid fishes. AB - The SATA satellite DNA family of sequences, composed of three size variants of approximately 237, 230, and 209 bp, is conserved in the genomes of tilapiine and haplochromine cichlid fishes. In the present study we examined the utility of the SATA sequences for inferring phylogenetic relationships among the three major genera of tilapiine fishes, Oreochromis, Sarotherodon, and Tilapia. Hybridization of the monomer SATA repeat to genomic DNA of representative cichlid species established conservation of the sequence in the African tilapine and haplochromine lineages and its absence from other cichlid lineages. Bootstrapped DNA parsimony and neighbor-joining analyses of derived consensus sequences revealed two distinct clades, one containing the mouthbrooding genera Oreochromis and Sarotherodon, and the other containing the substrate spawning genus Tilapia. These results are consistent with recent independent studies using mitochondrial DNA and establish the utility of the SATA satellite DNA family for phylogenetic reconstruction. Concerted evolution of the SATA sequences was also demonstrated within the tilapiine tribe. PMID- 7545932 TI - Analysis of the core and E1 envelope region sequences of a novel variant of hepatitis C virus obtained in Indonesia. AB - Hepatitis C virus (HCV) is currently classified into 6 major types, HCV-1 through -6, each of which can be further divided into a few subtypes, e.g., HCV-1a, -1b, 1c, etc., on the basis of sequence variation of the viral genome. The core and E1 envelope regions of HCV genome were amplified from sera of Indonesian patients using reverse transcription-polymerase chain reaction. Sequence analysis of both core and E1 regions followed by molecular evolutionary phylogenetic analysis identified a novel sequence variant of HCV-1 (Td-6). Antibodies in the serum from which Td-6 was isolated reacted only marginally to the core protein of HCV-J, a representative strain of HCV-1b, despite strong antibody response against a mixture of the core, NS3 and NS4 proteins of HCV-1a. The possible mechanism for the diminished reactivity of the antibodies in the serum to the core protein of HCV-J is discussed. PMID- 7545937 TI - Sequence of an exon of the canine p53 gene--mutation in a papilloma. AB - Exon 8 of tumour suppressor gene p53 was sequenced in domestic dogs. Ten nucleotide differences were revealed in a comparison with the feline sequence. A mutation ACT-->TCT (threonine-->serine) in codon 284 was detected in a papilloma of the oral mucosa. PMID- 7545938 TI - Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene. AB - The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific "bush" that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421-469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197: 1-139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. PMID- 7545941 TI - The genomic structure encoding human initiation factor eIF-5A. AB - A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%. PMID- 7545940 TI - Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli. AB - When nutrients become limiting, many bacteria differentiate and become resistant to environmental stresses. For Escherichia coli, this process is mediated by the sigma s subunit of RNA polymerase. Expression of sigma s was induced by homoserine lactone, a metabolite synthesized from intermediates in threonine biosynthesis. Homoserine lactone-dependent synthesis of sigma s was prevented by overexpression of a newly identified protein, RspA. The function of homoserine lactone derivatives in many cell density-dependent phenomena and the similarity of RspA to a Streptomyces ambofaciens protein suggest that synthesis of homoserine lactone may be a general signal of starvation. PMID- 7545939 TI - Spatial discontinuities in human immunodeficiency virus type 1 quasispecies derived from epidermal Langerhans cells of a patient with AIDS and evidence for double infection. AB - A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 quasispecies was observed for epidermal Langerhans cells purified from skin patches taken from a patient with AIDS soon after death. Each patch presented a unique collection of sequences, distinct from those of juxtaposed patches or those derived from the other leg. Infection of Langerhans cells by virus from underlying T cells in the dermis might explain this partition. The analysis revealed the presence of two distinct cocirculating viral strains, indicating double infection. PMID- 7545942 TI - Nucleotide sequence of the genes encoding the canine herpesvirus gB, gC and gD homologues. AB - The nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB, gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses. PMID- 7545943 TI - Human microsomal triglyceride transfer protein large subunit gene structure. AB - Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of the multifunctional enzyme protein disulfide isomerase and a unique, large 97-kDa subunit. MTP is required for the assembly and secretion of very low density lipoproteins and chylomicrons by the liver and intestine, respectively. In vitro, MTP catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between phospholipid surfaces. We have characterized the gene encoding the large subunit of human MTP. It contains 18 exons and spans approximately 55-60 kb. Fluorescent in situ hybridization localized this gene to band 4q24 of chromosome 4. A (CA)n repeat polymorphic marker, which may be useful for investigating a link between the MTP gene and genetic defects in lipid metabolism, was identified in intron 10. Sequence analysis of the 5' flanking region of the gene revealed potential sites which may bind transcriptional factors and control MTP expression. PMID- 7545944 TI - Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain. AB - Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species. PMID- 7545946 TI - Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants. AB - Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr-TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3'-terminal region of the cr TMV RNA preceding the tRNA-like structure was shown to form six potentially stable pseudoknots. PMID- 7545945 TI - In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop. AB - The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS. PMID- 7545948 TI - Synthesis of ribosomal proteins during growth of Streptomyces coelicolor. AB - Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [35S]-methionine, separated by two dimensional polyacrylamide gel electrophoresis, and quantified using the BioImage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coli beta-galactosidase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHI fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHI fragment. PMID- 7545947 TI - Phylogenetic analysis of the stress-70 protein family. AB - The eukaryotic cyto-/nucleoplasmatic 70-kDa heat-shock protein (HSP70) has homologues in the endoplasmic reticulum as well as in bacteria, mitochondria, and plastids. We selected a representative subset from the large number of sequenced stress-70 family members which covers all known branches of the protein family and calculated and manually improved an alignment. Here we present the consensus sequence of the aligned proteins and putative nuclear localization signals (NLS) in the eukaryotic HSP70 homologues. The phylogenetic relationships of the stress 70 group family members were estimated by use of different computation methods. We present a phylogenetic tree containing all known stress-70 subfamilies and demonstrate the usefulness of stress-70 protein sequences for the estimation of intertaxonic phylogeny. PMID- 7545949 TI - A set of 99 cattle microsatellites: characterization, synteny mapping, and polymorphism. AB - Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites--HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15--as well as the microsatellite found in the kappa-casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at least 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle. PMID- 7545950 TI - Production and nucleotide sequence of an inhibitory human IgM autoantibody directed against platelet glycoprotein Ia/IIa. AB - Human B-cell lines were derived by limiting dilutions of Epstein-Barr virus (EBV) transformed peripheral B cells from a patient with an autoantibody against glycoprotein (GP)Ia/IIa, and manifesting defective collagen-induced platelet aggregation and a bleeding problem. Antibody-producing clones were selected for their reactivity with whole platelets or with affinity-purified GPIa/IIa by enzyme-linked immunosorbent assay (ELISA). One of these cell lines, selected for further evaluation, produced an IgM (E3G6) that interfered with platelet aggregation responses. Polymerase chain reaction (PCR) amplifications with two different sets of primers specific for human kappa-chains resulted in the rescue of a unique and identical sequence. The same was true for the mu-chain, from which it was concluded that the cell line was monoclonal. Further analysis showed that the kappa variable domain sequence is similar to the germline gene A30, to 2E7, an anti-GPIIb human autoantibody, and to HF2-1/17, a systemic lupus erythematosus (SLE)-associated broad-specificity human autoantibody. Thus, the specificity of our antibody, E3G6, appears to be determined by the mu-chain, the sequence of which is encoded by a VHIII gene segment strongly homologous to the germline gene DP-77, by a D gene that is not homologous to any of the germline D genes reported to date, and by JH4 gene segment that is germline. All four mutations versus DP-77 are in CDRs, and result in amino acid substitutions, which implies that E3G6 may have been derived from an antigen-driven response. PMID- 7545951 TI - Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man. AB - Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster. PMID- 7545952 TI - Arylamine N-acetyltransferase in Balb/c mice: identification of a novel mouse isoenzyme by cloning and expression in vitro. AB - Three genes encoding arylamine N-acetyltransferase were identified in Balb/c mice. All three genes were cloned from genomic DNA, sequenced and expressed in a bacterial expression system. Two of the genes corresponded to Nat-1 and Nat-2 which have been previously identified in A/J and C57B1/6 strains of mice (Martell et al., 1991). The new gene, designated Nat-3, can be distinguished from the other mouse Nat genes both by specific amplification using PCR and by restriction endonuclease digestion. The products of all three genes are demonstrated to catalyse acetylation of aminofluorene and anisidine following expression in Escherichia coli. PMID- 7545953 TI - The 1993-94 Genethon human genetic linkage map. AB - In 1992, we described a second-generation genetic linkage map of the human genome. Using 1,267 new microsatellite markers, we now present a new genetic linkage map containing a total of 2,066 (AC)n short tandem repeats, 60% of which show a heterozygosity of over 0.7. Statistical linkage analysis based on the genotyping of eight large CEPH families placed these markers in the 23 linkage groups. The map includes 1,266 intervals and spans a total distance of 3690 centiMorgans (cM). A total of 1,041 markers could be ordered with odds ratios greater than 1000:1. About 56% of this map is at a distance of 1 cM or less from one of its markers. PMID- 7545954 TI - A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. AB - A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate BRCA1 susceptibility alleles. The mutations include an 11 base pair deletion, a 1-base pair insertion, a stop codon, a missense substitution, and an inferred regulatory mutation. The BRCA1 gene is expressed in numerous tissues, including breast and ovary, and encodes a predicted protein of 1863 amino acids. This protein contains a zinc finger domain in its amino terminal region, but is otherwise unrelated to previously described proteins. Identification of BRCA1 should facilitate early diagnosis of breast and ovarian cancer susceptibility in some individuals as well as a better understanding of breast cancer biology. PMID- 7545955 TI - TLC1: template RNA component of Saccharomyces cerevisiae telomerase. AB - Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for chromosome stability. Because of the nature of DNA replication, telomeres require a specialized mechanism to ensure their complete duplication. Telomeres are also capable of silencing the transcription of genes that are located near them. In order to identify genes in the budding yeast Saccharomyces cerevisiae that are important for telomere function, a screen was conducted for genes that, when expressed in high amounts, would suppress telomeric silencing. This screen lead to the identification of the gene TLC1 (telomerase component 1). TLC1 encodes the template RNA of telomerase, a ribonucleoprotein required for telomere replication in a variety of organisms. The discovery of TLC1 confirms the existence of telomerase in S. cerevisiae and may facilitate both the analysis of this enzyme and an understanding of telomere structure and function. PMID- 7545956 TI - Isolation and functional analysis of the promoter of the bovine serum albumin gene. AB - The bovine serum albumin (bSA) promoter has been cloned from bovine genomic DNA using the polymerase chain reaction. In common with other albumin promoters, this promoter functions efficiently in the differentiated rat hepatoma cell line H4II and not in the its dedifferentiated derivative, H5. Analysis of 5' deletions of the bSA promoter after transient transfection into H4II has revealed that a short construct containing the HNF1 binding site and TATA box functions efficiently but requires the presence of the more upstream sequences to achieve full activity Footprint analysis of the promoter reveals seven sites of DNA protein interaction extending from -31 to -213. One of these sites, extending from -170 to -236, whose deletion results in a four fold increase in promoter activity. This site has not previously been reported in other albumin promoters and is bound by the C/EBP-like family of proteins. PMID- 7545957 TI - Centromeric repetitive sequences in Arabidopsis thaliana. AB - Two highly repetitive DNA sequences have been cloned from Arabidopsis thaliana, ecotype Columbia, and were characterized by molecular and cytological analyses. These two sequences belong to the same repeat family with 180-bp basic unit, being tandemly organized in clusters. Pulsed field gel electrophoresis showed that this repeat sequence family forms at least seven clusters from ca. 100 to 1200 kb in length and ca. 3500 kb in total. Fluorescent in situ hybridization to somatic metaphase cells with the monomeric repeat unit as a probe clearly revealed that this repeat family is located at the centromeric regions of all chromosomes. It was also shown that this repetitive sequence is closely associated with limited parts of heterochromatic blocks on the centromeric regions which are visible distinctly at meiotic prophase from leptotene to diakinesis. Furthermore, this sequence hybridized preferentially to both polar sides of five bivalent chromosomes at the first metaphase. These results suggest that the repetitive sequences of this family were derived from the regions very close to the centromeres or on the centromeres themselves. PMID- 7545958 TI - Pyruvate dehydrogenase deficiency caused by a 33 base pair duplication in the PDH E1 alpha subunit. PMID- 7545959 TI - Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush). AB - Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5' external spacer region (5' ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5' ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3-6 kb from the 18S. Sequencing of a 609-b segment of the 5' ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5' ETS using the polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545963 TI - Hepatitis C transmission in a hemodialysis unit: molecular evidence for spread of virus among patients not sharing equipment. AB - Three cases of simultaneous seroconversion to hepatitis C virus (HCV) in a hemodialysis unit initiated the investigation of the viral strains of 14 seropositive patients in the unit by nucleotide sequencing. The results showed that five patients had been infected with the same viral strain, and indicated that two other patients were sharing a second strain. Transmission was not related to blood transfusions and not associated with the dialysis machines, but occurred between patients treated on the same shift. The number of cases was higher than expected from the serological data. Thus, spread of virus may occur at high frequencies in environments where parenteral routes are made accessible, in spite of rigorous preventive measures. This may raise concern that non transfusion associated spread of HCV may be present and unnoticed in several hospital settings. PMID- 7545961 TI - The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10. AB - Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process. PMID- 7545960 TI - The dichotomous size variation of human complement C4 genes is mediated by a novel family of endogenous retroviruses, which also establishes species-specific genomic patterns among Old World primates. AB - The human complement C4 genes in the HLA exhibit an unusual, dichotomous size polymorphism and a four-gene, modular variation involving novel gene RP, complement C4, steroid 21-hydroxylase (CYP21), and tenascin-like Gene X (RCCX). The C4 gene size dichotomy is mediated by an endogenous retrovirus, HERV-K(C4). Nearly identical sequences for this retrotransposon are present precisely at the same location in the long C4 genes from the tandem RCCX Module I and Module II. Specific nucleotide substitutions between the long and short C4 genes have been identified and used for diagnosis. Southern blot analyses revealed that HERV K(C4) is present at more than 30 locations in the human genome, exhibits variations in the population, and its analogs exist in the genomes of Old World primates with species-specific patterns. Evidence of intrachromosomal recombination between the two long terminal repeats of HERV-K(C4) is found near the huntingtin locus on chromosome 4. It is possible that members of HERV-K(C4) are involved in genetic instabilities including the RCCX modules, and in protecting the host genome from retroviral attack through an antisense strategy. PMID- 7545962 TI - Myocyte enhancer factor 2 (MEF2) binding site is essential for C2C12 myotube specific expression of the rat GLUT4/muscle-adipose facilitative glucose transporter gene. AB - We have cloned and characterized the rat GLUT4 gene in order to identify the cis DNA elements responsible for tissue-specific GLUT4 expression. In this study, a variety of luciferase reporter gene constructs were transiently transfected into C2C12 myoblasts and myotubes as a model for skeletal muscle differentiation. These data identified a 103-base pair fragment, located from -522 to -420 relative to the transcription initiation site, that was sufficient to account for GLUT4 C2C12 myotube-specific expression. This fragment was operationally defined as an enhancer since it conferred myotube-specific expression in the context of both the minimal native GLUT4 or the heterologous thymidine kinase promoters in an orientation-independent manner. Further, mutagenesis of this fragment demonstrated that a sequence analogous to the muscle creatine kinase myocyte enhancer factor 2 (MEF2) binding site (-466 and -457) was required for transcriptional activation. Electrophoretic mobility gel shift assays demonstrated specific binding activity to the GLUT4 MEF2 sequences which directly correlated with functional expression. Although this element was capable of directing myotube-specific expression when cloned as multiple copies into luciferase reporter gene constructs, the MEF2 sequence alone was insufficient to enhance GLUT4 expression. These data demonstrated that GLUT4 muscle-specific expression is conferred by a 103-base pair DNA sequence located between -522 and 420 of rat GLUT4 gene. This region encompasses a MEF2 binding site which was necessary, but not sufficient, for transcriptional activation. PMID- 7545964 TI - Size polymorphism of chromosomes bearing gp63 genes in Leishmania of the braziliensis complex: indication of a rearrangement of the gp63 genes in L. braziliensis and L. peruviana. PMID- 7545965 TI - A phylogeny of the genus Nocardia deduced from the analysis of small-subunit ribosomal DNA sequences, including transfer of Nocardia amarae to the genus Gordona as Gordona amarae comb. nov. AB - According to phylogenetic analyses of nearly complete small-subunit ribosomal DNA sequences, the genus Nocardia should not comprise the two species Nocardia petroleophila and Nocardia amarae. N. amarae should be reassigned to the genus Gordona as Gordona amarae. All of the other Nocardia species form a monophyletic unit, closely related to species of the genus Rhodococcus. It is proposed to revive the name 'CMN' to comprise the genera Corynebacterium, Tsukamurella, Mycobacterium, Gordona, Rhodococcus and Nocardia that form a well identified and monophyletic unit. They are all characterized by a cell wall chemotype IV with mycolic acids. PMID- 7545966 TI - Isolation of 28 new porcine microsatellites revealing polymorphism. PMID- 7545967 TI - [Nucleotide sequence of DNA isolated from protein cores of rosette-like structures (elementary chromomeres) of mouse interphase chromosomes]. AB - Data are presented on the primary nucleotide sequence of 15 DNA fragments cloned from protein cores of rosette-like structures of mouse interphase chromosomes. According to data of a computer analysis, these DNA fragments represent protein noncoding regions of interphase chromosomes, contain nucleotide sequences causing DNA strand bending, and contain a large amount of cis-regulatory transcriptional elements and sites closely related to sites of DNA topoisomerase II cleavage. Five clones demonstrated partial length homology with satellite DNA, short and long interspersed sequences, and extrachromosomal DNA from mouse thymocytes. Six clones showed homology with sites of initiation of replication of viruses, yeast, and mice. Six clones were found to contain consensus sequences for non-homologous recombination in somatic cells and six clones contained consensus sequences of human VTR1.1 loci. PMID- 7545969 TI - Isolation and characterization of 21 novel expressed DNA sequences from the distal region of human chromosome 4p. AB - We have established an approach to the isolation of expressed DNA sequences from a defined region of the human chromosome. The method relies on the direct screening of cDNA libraries using pooled single-copy microclones generated by a laser chromosome micro-dissection in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) procedure. We applied this method to the distal region of human chromosome 4p (4p15-4pter), which contains the Huntington disease (HD) and the Wolf-Hirschhorn syndrome (WHS) loci. Twenty-one nonoverlapping and region-specific cDNA clones encoding novel genes were isolated in this manner. Ten of 21 clones were subregionally assigned to 4p16.1-4pter, and the remainder mapped to the region proximal to 4p16.1. Northern blot and reverse transcription followed by the PCR (RT-PCR) analysis revealed that 16 of these 21 clones detected transcripts in total RNA from human tissues. Our method is applicable to other chromosomal regions and is a powerful approach to the isolation of region-specific cDNA clones. PMID- 7545968 TI - Diversity of cytochrome P450 genes in the mosquito, Anopheles albimanus. AB - Degenerate oligonucleotide primers were designed for conserved regions of cytochrome P450 proteins of the CYP4 family and were used to amplify cDNA or genomic DNA from different strains of the New World malaria vector, Anopheles albimanus (Weidemann). The PCR products were cloned, sequenced and compared to each other and to members of the P450 family CYP4. Seventeen new P450 genes were identified in five CYP4 subfamilies. Five of the ten PCR products of genomic DNA were shown to contain a short (60-79 bp) intron at the same position as introns in the Drosophila CYP4D2 and CYP4E1 genes. PMID- 7545970 TI - Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle. AB - Several members of the sarcomeric myosin heavy chain (MHC) gene family have been mapped in the human genome but many of them have not yet been identified. In this study we report the identification of two human skeletal MHC genes as fast IIa and IIx MHC based on pattern of expression and sequence homology with the corresponding rat genes in the 3'-translated and untranslated regions. The distribution of these two gene products as well as that of the beta/slow MHC gene was analyzed in human skeletal muscles by in situ hybridization. The distribution of beta/slow, IIa, and IIx MHC transcripts defines three major muscle fiber types expressing a single MHC mRNA, i.e., either beta/slow, IIa, or IIx MHC mRNA, and two populations of hybrid fibers coexpressing beta/slow with IIa or IIa with IIx MHC mRNA. Fiber typing by ATPase histochemistry shows that IIa MHC transcripts are more abundant in histochemical type IIa fibers, whereas IIx MHC transcripts are more abundant in histochemical type IIb fibers. PMID- 7545972 TI - Genetic and antigenic variability of HIV type 1 in Brazil. AB - Six Brazilian strains of human immunodeficiency virus type 1 (HIV-1) were isolated from infected individuals residing in different regions of Brazil between 1987 and 1989. Phylogenetic analysis based on an 860-base pair env fragment, including V3, V4, V5, and the beginning of gp41, classified the Brazilian strains significantly in genotype B, with interhost distances between 5.9 and 13.1% (mean value, 10%). Amino acid sequence analysis of the V3 loop revealed that three strains contained the North American/European GPGR motif as the tip of the loop whereas in the other three strains proline (P) was substituted by tryptophan (W), methionine (M), or phenylalanine (F). A consensus peptide, Bra-cons, was designed containing GWGR as the tip of the loop. Serological reactivity to the Bra-cons peptide and other V3 peptides (MN, SF2, HBX2, RF, MAL, ELI, Z6, and a Cote d'Ivoire peptide, CI-cons) was compared for 114 HIV-1-positive sera from Rio de Janeiro. Sixty-nine sera (60.5%) reacted with peptides belonging to genotype B, of which 10 sera also reacted with peptides belonging to genotype A (n = 7) and D (n = 3). Eighteen sera (15.8%) had binding antibodies to the Bra-cons peptide. A high number of sera (n = 43; 37.7%) had no antibodies to any of the V3 peptides tested. This result suggests that HIV-1 variants with aberrant V3 loops may circulate in Rio de Janeiro. PMID- 7545971 TI - Developmental expression of the genes encoding transforming growth factor alpha and its receptor in the hypothalamus of female rhesus macaques. AB - Studies in female rats have shown that transforming growth factor alpha (TGF alpha) stimulates release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation, and that expression of the TGF alpha gene in the hypothalamus increases during both the initiation of normal puberty and after hypothalamic lesions that induce sexual precocity. Since blockade of epidermal growth factor receptors (EGFR), which mediate TGF alpha actions, delayed the normal timing of puberty, it was postulated that TGF alpha/EGFR contributes to the neuroendocrine process that underlies the initiation of normal female puberty. The present study was undertaken to examine the hypothesis that hypothalamic expression of the TGF alpha gene and its receptor changes in relation to the stage of sexual development in nonhuman primates, and to determine whether these changes are accompanied by corresponding alterations in LHRH gene expression. DNA fragments complementary to the coding regions of the rhesus monkey TGF alpha, EGFR and LHRH genes were cloned by reverse transcription polymerase chain reaction (RT-PCR), sequenced and used to prepare monkey-specific antisense RNA probes. A quantitative RT-PCR was developed in which the cloned sequences were utilized to prepare RNA standards for the quantitation of tissue mRNA levels. Both TGF alpha and EGFR mRNA levels in the medial basal hypothalamus and preoptic area of female monkeys were elevated during neonatal life (1 week to 6 months of age), when FSH secretion is also high, decreased during juvenile development (8-18 months of age), when secretion of both FSH and LH is low, and markedly increased during the expected time of puberty (30-36 months of age). No such changes were observed in either the cerebellum or the cerebral cortex, two brain regions irrelevant to neuroendocrine reproductive control. In contrast to the pronounced alterations in hypothalamic TGF alpha/EGFR gene expression observed during sexual development, LHRH mRNA levels did not vary significantly during this time. Hybridization histochemistry revealed the presence of both TGF alpha and EGFR mRNAs in cells scattered throughout the hypothalamus, but more predominantly in the median eminence, suprachiasmatic nuclei, optic chiasm and cells along the wall of the third ventricle. These results demonstrate that increases in TGF alpha and EGFR gene expression, specific to the neuroendocrine brain, occur during developmental phases in which gonadotropin output is also elevated--most noticeably at the time of puberty.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7545974 TI - Sequence organization of the human chromosome 2q telomere. AB - The terminal 240 kb of a human 2q telomere region was cloned in two overlapping yeast artificial-chromosomes (YACs). This DNA contains a region of low-copy subtelomeric repeats (within 50 kb of the 2q telomere), a segment of DNA duplicated on distal 8p23 (100 kb from the 2q telomere), and a region of single copy DNA (230 kb from the 2q telomere). Two CpG islands are present in the DNA segment duplicated on distal 8p23. RecA-assisted restriction endonuclease cleavage of genomic DNA samples revealed a potential 55 kb chromosome length polymorphism at the 2q telomere. This work provides telomeric closure of maps for human chromosome 2q, demonstrates a novel, subtelomere-specific DNA duplication, and will permit detailed molecular and cytological studies of this human telomere region. PMID- 7545973 TI - High transposition rates of Osvaldo, a new Drosophila buzzatii retrotransposon. AB - Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids. PMID- 7545976 TI - Ovary-specific expression of a gene encoding a divergent alpha-tubulin isotype in Xenopus. AB - We are investigating the structure and regulation of alpha-tubulin genes expressed in amphibian oocytes. We have characterised here a gene, X alpha T207, that produces a major alpha-tubulin mRNA of Xenopus laevis ovary. X alpha T207 mRNA was not detected in other frog tissues and its production may therefore be a key identifying feature of ovarian differentiation. In comparison to the tubulin isotypes so far described in mammals and Xenopus, the alpha-tubulin encoded by X alpha T207 is divergent in overall amino acid sequence, particularly in the N terminal region between residues 39-50. This pattern of divergence is also displayed by the ovary-specific alpha-tubulin gene of Drosophila, D alpha 4, although the two genes do not appear to be orthologous. The development of specialised microtubular structures and activities in oocytes, eggs and early embryos may then be correlated with the expression of a divergent alpha-tubulin isotype in a wide range of organisms. To understand the basis of the ovary specific expression of X alpha T207 we examined the transcriptional activity of wild type and mutant promoters after their microinjection in Xenopus oocytes. Only 65 bp upstream of the initiation site were required for full activity of the X alpha T207 promoter, and an element fitting the Y-box consensus was involved in controlling the efficiency of initiation. Previous oocyte injection experiments have implicated the Y-box in the oocyte-specific transcription of genes that are also expressed in other cell types, so its involvement in the oocyte-restricted expression of X alpha T207 further suggests that transcription factors recognising the Y-box normally regulate gene expression during oocyte development. Since a Y-box also occurs in the D alpha 4 promoter, our results suggest that in both organisms oocyte-specific expression of a divergent alpha tubulin could be achieved by a common mechanism. PMID- 7545975 TI - Evolution of H3N8 equine influenza virus from 1963 to 1991. AB - The antigenic properties of H3N8 influenza viruses isolated from outbreaks of equine influenza in Sweden between 1979 and 1991 have been studied in hemagglutination inhibition tests with polyclonal and monoclonal antisera, and antigenic drift of the virus has been demonstrated. To clarify the basis of the antigenic drift, amino acid sequences of the globular head regions (HA1) of the hemagglutinin membrane glycoproteins of virus strains from 1979, 1984, 1988 and 1990 have been deduced from the nucleotide sequences of the hemagglutinin genes, and the sequence information has been used to construct a phylogenetic tree of H3N8 equine influenza strains. Several strains from previous studies have been included to give a clearer picture of viral evolution in an international context. PMID- 7545977 TI - HIV type 1 variation in World Health Organization-sponsored vaccine evaluation sites: genetic screening, sequence analysis, and preliminary biological characterization of selected viral strains. WHO Network for HIV Isolation and Characterization. AB - A laboratory network has been established by the World Health Organization (WHO) to systematically isolate and characterize HIV strains from different parts of the world, and to obtain information and reagents that would facilitate HIV vaccine development. Sixty-three HIV-1 isolates obtained from 224 specimens collected during 1992-1993 in Brazil, Rwanda, Thailand, and Uganda were characterized in this initial study. Virus strains were first genetically subtyped using three different screening methodologies: PCR-gag fingerprinting, RNase A mismatch, and heteroduplex mobility assay (HMA). In addition, selected viruses were sequenced in V3 (52 strains), C2-V3 (42 strains), gp120 (15 strains), and/or gp160 (8 strains) regions of their envelope genes. These studies identified viruses belonging to different sequence subtypes in the four countries: 16 subtype B and 1 subtype C strains in Brazil, 13 subtype A strains in Rwanda, 15 subtype E and 2 subtype B strains in Thailand, and 3 subtype A and 13 subtype D strains in Uganda. Comparison of sequence data with results from the genetic screening efforts identified the HMA as a rapid and reliable method for sequence subtype determinations. The majority of strains were collected from persons documented to have recently seroconverted to HIV-1 positivity, and most strains were found to have slow replication and low cytopathic characteristics and to be non-syncytium-inducing (slow/low-NSI phenotypes) in vitro, which, in many cases, correlated with the corresponding genotype and charge of the V3 loop amino acid sequences. This collection of HIV strains is presently being characterized immunologically and serologically, including neutralization assays, to define whether there are immunological correlates of the sequence subtypes. Identification of potential immunotypes would be of considerable importance for the further development of HIV vaccines. PMID- 7545978 TI - NEX-1: a novel brain-specific helix-loop-helix protein with autoregulation and sustained expression in mature cortical neurons. AB - Mutations affecting peripheral nervous system development in Drosophila and mouse indicate that neural differentiation depends on the coordinated expression of cell type-specific bHLH proteins. We have identified a novel bHLH gene, termed NEX-1, which is expressed exclusively and abundantly in the mammalian central nervous system. By in situ hybridization, induction of the rodent NEX-1 gene coincides with the generation of postmitotic neurons and parallels overt neuronal differentiation and synaptogenesis. Unexpected for bHLH proteins, NEX-1 may play a role in neuronal function throughout adult life. High levels of its mRNA are sustained in mature pyramidal neurons of the hippocampus, cerebellum and several neocortical areas previously associated with learning and memory formation. When ectopically expressed in PC12 cells, NEX-1 transactivates the promoter of its own gene. This suggests that positive autoregulation stabilizes the activity of NEX-1 and its target genes in subsets of mature neurons. PMID- 7545979 TI - Nucleotide sequence of a 28-kbp portion of rice mitochondrial DNA: the existence of many sequences that correspond to parts of mitochondrial genes in intergenic regions. AB - The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants. PMID- 7545981 TI - Measuring and reporting the concentration of acetaldehyde in human breath. AB - Most of the acetaldehyde generated during the metabolism of ethanol becomes tightly bound to endogenous molecules such as haemoglobin, amino acids and certain phospholipids. Free acetaldehyde passes the blood-brain barrier and traces of this toxic metabolite are excreted through the lungs and can be detected in the expired air. The blood/air partition coefficient of acetaldehyde at 34 degrees C, the average temperature of end-expired air, is about 190:1. Because of various problems associated with measuring acetaldehyde in blood samples, several research groups have instead investigated the analysis of acetaldehyde in breath which offers an indirect and alternative approach for clinical and research purposes. However, care is needed when interpreting the results of breath acetaldehyde measurements, because of the possibility of local formation from microflora inhabiting the upper airways and mouth. The concentration of acetaldehyde exhaled in breath after drinking alcohol demonstrates large inter-individual differences depending on various genetic (racial) and environmental factors. Moreover, acetaldehyde is an endogenous metabolite and even without drinking any alcohol the concentrations expelled in breath span from 0.2 to 0.6 nmol/l, with higher levels observed in smokers and abstinent alcoholics. Breath acetaldehyde concentration reached between 5 and 50 nmol/l in European subjects who drank a moderate dose of ethanol (0.4-0.8 g/kg), with the highest values seen in smokers. The concentration of breath acetaldehyde in Japanese subjects after drinking alcohol reached between 200 and 500 nmol/l at the peak. These much higher levels follow because a large proportion of Orientals (40-50%) inherit an inactive form of the low Km mitochondrial isoenzyme of aldehyde dehydrogenase (ALDH2). The highest concentration of breath acetaldehyde were seen in healthy Caucasians who drank a small dose of alcohol (0.25 g/kg) after taking the alcohol-sensitizing drug calcium carbimide, which blocks the action of ALDH isozymes. During the most intense acetaldehyde-flush reaction, breath acetaldehyde reached between 200 and 1300 nmol/l, but even these abnormally high concentrations did not interfere with the analysis of ethanol in breath by means of non-specific infrared analysers currently used in many countries for testing drinking drivers. PMID- 7545980 TI - Oligoclonality and skewed T cell receptor V beta gene segment expression in in vivo activated human intestinal intraepithelial T lymphocytes. AB - Intraepithelial intestinal T lymphocytes (IEL) bearing alpha beta or gamma delta T cell receptors (TCR) are positioned to serve as a first line of defense against enteric pathogens. To investigate whether intestinal IEL are subject to antigenic selective forces distinct from that influencing (xp T cells in the peripheral blood (PBL), we performed a comparative analysis of V beta gene segment usage in IEL and PBL of immunologically normal donors by quantitative PCR. Primers for 22 different human TCR V beta gene segments of V beta gene segments families were used to analyze the repertoire of TCR beta chain transcripts in colonic IEL (c IEL), in corresponding colonic lamina propria lymphocytes (c-LPL), and in peripheral blood lymphocytes. In each of the three individuals examined, a limited number (1-4 out of 22) of TCR V beta families predominated and accounted for more than 50% of the total beta chain transcripts from c-IEL, whereas in PBL and c-LPL a more even distribution of V beta gene families was observed. The dominating V beta gene families were V beta 2, V beta 3, V beta 6, V beta 8 and V beta 14. In one individual, V beta 3 comprised more than 40% of the entire repertoire of c-IEL beta chain transcripts. Sequence analysis of the predominant V beta 3 family in this individual revealed identical sequences in 13 of 17 clones analyzed. Human alpha beta TCR+ c-IEL could not be driven to proliferate or exhibit cytotoxic function in vitro however, PCR analysis for detection of lymphokine mRNA revealed constitutive production of several lymphokines known to exert trophic effects on intestinal epithelial cells and pro-inflammatory activities. Taken together, the striking degree of oligoclonality may indicate that the intraepithelial intestinal immune system is targeted to a limited set of hitherto unknown self- or foreign antigens present in the intestine and orchestrates intramucosal inflammatory and regenerative processes. PMID- 7545982 TI - Interpreting the evidence on brief interventions for excessive drinkers: the need for caution. AB - Two recent reviews of the evidence on brief interventions in the alcohol field reach highly favourable conclusions regarding their effectiveness and cost effectiveness. However, both can be criticized on three grounds: (1) they pay insufficient regard to important differences within the family of brief interventions; (2) they do not sufficiently emphasize the crucial distinction between brief interventions among treatment seekers and non-treatment seekers; and (3) they arrive at over-optimistic and uncritical conclusions. While not wishing to dampen enthusiasm for the potential of brief interventions in reducing alcohol-related harm, the present article argues that, if the evidence is not to be misinterpreted by policy makers and purchasers of services, differences between brief interventions must be borne in mind and analyses of effectiveness in the treatment-seeking population must be clearly separated from those in the area of opportunistic interventions in the non-treatment-seeking population. The evidence for the effectiveness of opportunistic brief interventions is much stronger than for brief interventions in specialist settings for those seeking help, where their most prudent application should be restricted to patients with problems of relatively low severity. PMID- 7545983 TI - Assessment of zinc nutritional status of pellagra patients. AB - The objective of the present study was to assess zinc nutritional status in alcoholic patients with pellagra using plasma, hair, urine and nail zinc levels, as well as the zinc tolerance test. The study was conducted on 81 patients, 73 males and eight females. Zinc parameters were compared with those of 84 individuals with no apparent disease aged 23-45 years. Plasma zinc levels were lower in patients with pellagra than in the controls (P < 0.01). The results of the zinc tolerance test showed that: (1) basal zinc levels were 69.7 +/- 16.8 micrograms/100 ml in pellagrins and 82.3 +/- 34.0 micrograms/100 ml in the controls (P < 0.01); (2) after 1 h the increase in plasma levels was similar in the pellagrin and control groups; (3) during the second hour the increase was more marked in the controls (P < 0.01), and the same was observed during the third and fourth hours (P < 0.05). Urinary zinc excretion (mg/24 h) was higher in pellagrins (P < 0.01). Zinc concentration in hair and toenails did not differ between pellagrins and controls. We conclude that pellagrins present zinc deficiency as demonstrated by plasma and urine zinc levels and by their abnormal response to the zinc tolerance test. We suggest that hair and nail zinc levels should not be used to assess zinc nutritional status in patients with pellagra. PMID- 7545984 TI - Memory functioning in social drinkers: a study of event-related potentials. AB - Verbal memory functioning was assessed in light and heavy social drinkers by recording event-related potentials in subjects instructed to memorize lists of words using either repetition or elaborative learning strategies. Although overall performance did not differ significantly between light and heavy drinkers, heavy drinkers showed reduced within-list organization compared with light drinkers. There were statistically significant differences in the event related potential indices of memory functioning elicited during complex elaborative processing which were consistent with decreased within-list organization in heavy drinkers. PMID- 7545985 TI - The effect of ethanol ingestion on killing of Streptococcus pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis by rat neutrophils. AB - The precise effects of chronic ethanol ingestion on the role of neutrophil function in pneumococcal pneumonia have not been fully delineated. In this study, the bactericidal capacity of polymorphonuclear leucocytes (PMNL) from rats pair fed an ethanol-containing liquid diet or a liquid control diet was compared both in vitro and in vivo. The PMNL were allowed to phagocytose several bacterial species in vitro, and intracellular killing after 1h was determined by plate counts. There was no significant difference in killing of Streptococcus pneumoniae types 6B or 37, Staphylococcus aureus or Staphylococcus epidermidis by PMNL from ethanol-fed and pair-fed rats (E-PMNL and P-PMNL, respectively). However, E-PMNL killed significantly less of Streptococcus pneumoniae types 10A, 14 and 19F. To corroborate these results in vivo, rats were infected transtracheally, and quantitative lung counts were performed 1 week post infection. Streptococcus pneumoniae types 6B and 37, as well as Staphylococcus aureus, were effectively cleared from the lungs of both groups of rats. Streptococcus pneumoniae types 10A, 14 and 19F, however, were cleared well from the lungs of pair-fed but not ethanol-fed animals. These data suggest that chronic ethanol ingestion induces a strain-specific deficit in neutrophil bactericidal activity against certain S. pneumoniae which does not extend to commonly encountered staphylococci. PMID- 7545987 TI - Effects of various cytochrome P-450 inducers on testosterone metabolism and several monooxygenase activities in Sprague-Dawley (SpD), high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats. AB - Hydroxylation of testosterone (TST) has been shown to be regio- and stereo specific for a number of cytochrome P-450 isoenzymes. Three rat lines [Sprague Dawley (SpD), high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS)] were tested for this enzymatic specificity after treatment with phenobarbital, clofibrate, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile. These compounds are known to induce cytochrome P-450 2B, 4A, 1A and 3A1, respectively, in the rat. Induction efficiency was established by using the usual enzyme activities specific for these P-450s (pentoxyresorufin, lauric acid, ethoxyresorufin and nifedipine oxidase). Five mono hydroxylated TST metabolites were separated using a sensitive HPLC procedure. The hydroxylation of TST was found to be significantly different between the lines even in the uninduced state. The formation of the metabolites of TST, hydroxylated on 2 alpha or 7 alpha or 16 alpha positions and oxidated on carbon 17 (delta 4), was found to be significantly increased in SpD rats when compared with the HAS-LAS lines (P < 0.0001 in each case). When the HAS-LAS lines were compared, the quantity of 2 alpha and 16 alpha hydroxylated metabolites was found to be significantly lower in LAS rats (P < 0.05). These differences persisted, although in the opposite direction, after 3-methylcholanthrene (P < 0.01 for both 2 alpha and 16 alpha) and phenobarbital induction (P < 0.01 for 2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545986 TI - Cue-exposure to alcohol-associated stimuli reduces autonomic reactivity, but not craving and anxiety, in dependent drinkers. AB - A controlled trial was conducted of cue-exposure with dependent drinkers in treatment. All subjects were engaged in an insight-oriented therapy programme, and responses to an alcohol-associated, compared with a neutral, stimulus were assessed at the beginning and end of treatment. Compared with a control group, which did not receive intervening cue-exposure sessions, subjects who received such interventions manifested reductions in heart rate, salivation and arousal responses to the alcohol-associated, compared with the neutral, stimulus. They did not, however, show similar reductions in subjective estimates of craving and anxiety. These results and the desynchrony in reductions in cue-reactivity across response domains are discussed in terms of their implications for cue-exposure in treatment and recent theorectical conceptualizations of the relationship between autonomic reactivity, craving and drinking behaviour. PMID- 7545988 TI - Treatment, health promotion and alcohol controls and the decrease of alcohol consumption and problems in Ontario: 1975-1993. AB - We report information on trends in alcohol problems, alcohol consumption, alcohol availability and prevention measures in Ontario for the period 1975-1993. Problem measures show substantial declines over the period. Measures of consumption have declined as well, but not as much as problem measures. Economic availability (relative price) has remained stable, while physical availability has increased. Prevention measures show substantial increases over the period. PMID- 7545989 TI - 'J-shaped' relationship between drinking during pregnancy and birth weight: reanalysis of prospective epidemiological data. AB - Worldwide epidemiological studies on the effects of drinking during pregnancy on birth weight and prematurity were surveyed. Far more studies have reported no significant effects on birth weight than have reported significant decreases. Statistical analyses of the means from the prospective studies in this area indicated that both maternal smoking and alcohol consumption during gestation are associated with a significant decrease in birth weight. The effect of smoking is three times greater than the effect of alcohol. When the data were stratified by smoking status, maternal alcohol consumption did not have a significant effect on birth weight for non-smokers, but among smokers there was a significant linear trend with a threshold for decreased birth weight at about an average of two drinks per day. There was also a significant pattern of increased birth weight associated with low levels of alcohol consumption, suggesting an inverted 'J shaped' function between drinking during pregnancy and birth weight. PMID- 7545991 TI - The Windsor Clinic Alcohol Withdrawal Assessment Scale (WCAWAS): investigation of factors associated with complicated withdrawals. AB - A new scale for measuring the severity of the alcohol withdrawal syndrome has been developed. The Windsor Clinic Alcohol Withdrawal Assessment Scale (WCAWAS) has more objective criteria than its parent scales and has high inter-rater reliability and concurrent validity. This article describes the development of the WCAWAS and its use in an investigation of which factors were associated with complicated withdrawals in 142 consecutive patients admitted for detoxification. Multivariate analyses showed that drinking behaviour variables and MCV were successful at predicting complicated withdrawals. The WCAWAS was not successful at predicting which patients developed convulsions. Further studies are required to see if the WCAWAS is more successful at predicting which patients will develop visual hallucinations. The WCAWAS is useful as a teaching aid and in following the clinical course of patients and their response to treatment. PMID- 7545990 TI - Cytochrome P-450 2E1 in rat liver, kidney and lung microsomes after chronic administration of ethanol either orally or by inhalation. AB - In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contents and activities were tested in liver, kidney and lung from Wistar rats after the following treatments (1) oral administration of a 10% ethanol solution for 4 weeks; (2) pair fed controls; (3) oral administration of a 5% acetone solution for 1 week; (4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activity was measured using chlorzoxazone as substrate and CYP2E1 content was measured using Western blot analysis. In addition, the cellular distribution of CYP2E1 was studied in liver, lung and kidney by immunohistochemistry. Basal liver CYP2E1 was 10-20 times lower in lung and kidney than in liver. Inhalation was clearly the most efficient way of inducing CYP2E1, probably due to the continuous and high alcohol exposure. Among the organs tested, lung appeared to be the tissue least sensitive to induction even after ethanol inhalation, suggesting the absence of local induction. After ethanol intoxication, immunostaining was increased in the centrilobular region of the liver, in the alveolar cells of the lung and in the proximal convoluted tube of the kidney. The CYP2E1 activities decreased to control values in the three tissues tested, within 24 h after cessation of intoxication. PMID- 7545994 TI - Partnerships for success: identifying and empowering leaders to meet members' needs. PMID- 7545993 TI - Drug abuse and intoxication in alcoholics. AB - The frequency of drug, alcohol and combined alcohol/drug intoxication in forensic autopsy material is described. Alcoholics had frequently abused drugs; the cause of death was drug intoxication in 12%, combined alcohol/drug intoxication in 13%, and alcohol intoxication only in 4%. The manner of death was different in the various groups as it was almost equally stated as accident, suicide and unascertainable, respectively, with only drugs involved, whereas accidents were the predominant features in the combined alcohol/drug intoxication among alcoholics. In controls, accidents predominated on both types of intoxication. The alcoholics seemed to be more 'deliberately' choosing suicide with drugs, while both alcoholics and controls need more information with regard to the dangers of mixing alcohol and drugs. The frequency of combined alcohol and drug intoxication was much higher in alcoholics. PMID- 7545992 TI - Antibodies made against a formaldehyde-protein adduct cross react with an acetaldehyde-protein adduct. Implications for the origin of antibodies in human serum which recognize acetaldehyde-protein adducts. AB - Acetaldehyde, the major metabolite of ethanol, reacts with lysine and other free amino groups on proteins to form acetaldehyde-protein adducts. The presence of antibodies which recognize such acetaldehyde-protein adducts in sera from alcoholics has been attributed to an immune response to such adducts. Complicating this conclusion is the finding that sera from non-alcoholic control subjects also contain antibodies which recognize acetaldehyde-protein adducts. In the current research we sought to determine whether antibodies which recognize epitopes formed by the reaction of a protein with acetaldehyde can be formed in response to a protein modified with a structurally related protein adduct. We modified lysine residues on apolipoprotein (apo) B-100 with acetaldehyde and formaldehyde under reducing conditions, to form epsilon-N-methyl- and epsilon-N ethyl-lysine residues, and with acetic anhydride to form epsilon-N-acetyl-lysine residues, and made antibodies against these modified proteins in guinea-pigs. In ELISA assays antibodies made against methylated apoB-100 (Me-apoB) cross-reacted effectively with ethylated apoB-100 (Et-apoB), while antibodies made against acetic anhydride-modified apoB-100 did not cross-react. We conclude that methyl lysine shares one or more immunoreactive epitopes with ethyl-lysine, and that antibodies which recognize acetaldehyde-modified proteins can be formed in response to formaldehyde-modified proteins. We demonstrate that sera from both alcoholics and non-drinkers contain antibodies which recognize Me-apoB and Et apoB and that the titres of these antibodies are comparable. These data raise the possibility that some human serum antibodies which recognize acetaldehyde modified protein epitopes may have been made against formaldehyde-modified protein epitopes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7545995 TI - Environmental design, work, and well being: managing occupational stress through changes in the workplace environment. AB - The physical environment can be an important contributor to occupational stress. Factors that contribute to stress and other negative outcomes include: lack of control over the environment, distractions from coworkers, lack of privacy, noise, crowding, and environmental deprivations (such as lack of windows and aesthetic impoverishment). The design of "salutogenic" environments requires not only the elimination of negative stress inducing features, but also the addition of environmental enhancements, including such factors as increased personal control, contact with nature and daylight, aesthetically pleasing spaces, and spaces for relaxation alone or with others. Salutogenic environments also take into consideration positive psychosocial "fit," as well as functional fit between people and environments. At the heart of the current interest in the work environment are two major concerns: organizational productivity and employee well being. PMID- 7545996 TI - Myofascial pain syndrome: early recognition and comprehensive management. AB - Fewer conditions in clinical practice have generated more controversy and debate than those that cause musculoskeletal pain and dysfunction. The term myofascial pain refers to a syndrome of musculoskeletal discomfort. Myofascial pain is a significant physical disorder responsible for long term disability. Early diagnosis of myofascial pain syndrome and circumspect treatment without using narcotic and non-narcotic analgesics results in successful outcomes. The goal of treatment should always be function oriented, set at the beginning of a treatment program, and modified at periodic intervals. Concurrent psychosocial stressors in the home and at work should be identified and treated appropriately. PMID- 7545997 TI - Tuberculosis: preventing occupational transmission to health care workers. AB - 1. The anticipated reduction in the incidence of tuberculosis (TB) failed to consider four major factors: the advent of human immunodeficiency virus (HIV); the development of multi-drug resistant TB; the decline in public health services and limited access to health care for those in congregative facilities; and the increase in immigration of persons from countries with high prevalence of TB. 2. Some strains of multi-drug resistant TB are resistant to all anti-TB medications and seriously hamper infection control strategies. Unfortunately, MDR-TB is transmitted in the same manner as drug susceptible organisms and studies show comparable infection rates. 3. The role of the occupational health nurse in preventing workplace exposure to TB combines education, disease prevention, early detection, and treatment modalities to maximize the opportunity for a healthy work environment for health care workers. PMID- 7545998 TI - Questions from practice: a basis for research. AB - Occupational health nurses in clinical practice are in an excellent position to identify unanswered questions that affect the health and well being of employees. Once these questions have been asked, the occupational health nurses may proceed with structured research to find answers. The research begins with a thorough review of existing literature to learn the background of the issue and clearly define a research question. This question is then framed conceptually to guide the study. The theoretical framework may be supported or refuted by the research project. The study method is dictated by the research question and the theoretical framework. Two basic research methods are intervention and descriptive studies. Intervention studies, with treatment and control groups, attempt to show differences between groups when one receives a certain treatment and the other group does not. Descriptive studies generally survey attitudes or activities, then test for associations. Data analysis is determined by the type of study and type of data collected. Descriptive statistics generate frequencies and correlations; inferential statistics yield stronger information about associations of the variables. Interpretation of research findings must include consideration of threats to validity. Internal validity allows the investigator to state with confidence that the intervention was responsible for the difference between the groups. External validity allows for generalizability of the findings to other populations. The purpose of nursing research is to advance the discipline of nursing. Research refines nursing theories and guides practice. It is through research that occupational health nurses gain confidence to alter procedures and provide interventions that have been shown to be effective. PMID- 7545999 TI - Use of back belts to prevent low back injury. PMID- 7546000 TI - Automating internal audit data. PMID- 7546001 TI - Magnetic resonance spectroscopy in childhood AIDS encephalopathy. AB - Twenty-five children with acquired immunodeficiency syndrome (AIDS) underwent cranial magnetic resonance imaging and proton magnetic resonance spectroscopy. Patients were divided into 2 groups based on clinical parameters: encephalopathy and nonencephalopathy. N-acetyl aspartate/creatine ratios were compared between the 2 groups and to control data. Spectra were obtained for 2 volumes of interest: the basal ganglia region and the white matter. The mean basal ganglia region ratio for the AIDS encephalopathy patients (n = 8) was 1.12 and the ratio for the AIDS nonencephalopathy patients (n = 17) was 1.48. The ratio for the 9 controls was 1.57. The encephalopathy group had a significantly lower ratio than both the control (P < .001) and the AIDS nonencephalopathy group (P < .002). The mean white matter ratio for the encephalopathy group (n = 8) was 1.47 and for the AIDS nonencephalopathy group (n = 13) was 1.82 with a control (n = 6) ratio of 1.82. The encephalopathy patients had a lower white matter ratio than the nonencephalopathy (P < .05) patients but the ratio was not different than controls (P < .11). It is concluded that N-acetyl aspartate/creatine ratios are reduced in childhood AIDS encephalopathy and proton magnetic resonance spectroscopy may be helpful in defining brain human immunodeficiency virus-1 infection. However, further longitudinal studies are necessary to determine the sensitivity and specificity of this technique. PMID- 7546002 TI - MRI in neurofibromatosis type I: using fluid-attenuated inversion recovery pulse sequences. AB - Cranial magnetic resonance imaging results of 14 patients with neurofibromatosis type I were examined with T2-weighted fluid-attenuated inversion recovery pulse sequences, as well as conventional T2-weighted spin-echo sequences. Definition was better in 62 of 79 lesions or groups of lesions on fluid-attenuated inversion recovery images than on T2-weighted spin-echo images. The lesions were demonstrated not only in the brainstem, cerebellum, globus pallidus, and cerebral white matter, but also in the hippocampus, pulvinar thalami, and splenium of the corpus callosum. The latter 3 lesions have not been demonstrated or emphasized in previous studies. It is concluded that fluid-attenuated inversion recovery imaging is more effective in detecting multiple lesions in patients with neurofibromatosis type I than conventional T2-weighted spin-echo imaging. PMID- 7546003 TI - MRI, MRA, and neurodevelopmental outcome following neonatal ECMO. AB - Cranial magnetic resonance imaging (MRI) of 31 newborn infants treated with venoarterial cardiopulmonary bypass for severe but reversible respiratory failure, revealed major focal parenchymal lesions in 7 of 31 infants (23%) and demonstrated abnormal enlargement of extra-axial and ventricular cerebrospinal fluid spaces in 16 of 31 (51%). No preferential left versus right lateralization of focal injury was observed in conjunction with right common carotid artery and jugular vein ligation. No statistically significant relationships were found between major brain lesions on MRI scans and the clinical characteristics of the pre-extracorporeal membrane oxygenation (ECMO), ECMO, and post-ECMO course. Major focal brain lesions were significantly associated with an asymmetric cerebrovascular response to carotid ligation of the right versus left middle cerebral arteries as detected by magnetic resonance angiography (P < .05). Enlarged cerebrospinal fluid spaces were not significantly related to the presence of parenchymal MRI lesions, but were associated with lower Bayley neurodevelopmental scores for mental (MDI) and psychomotor evaluations (PDI) at 6 and 12 months (P < .05). It is concluded that asymmetries of cerebral vascular adaptation detected by magnetic resonance angiography after ECMO may be associated with major brain lesions revealed by MRI. Thereafter, the presence of enlarged cerebrospinal fluid spaces on MRI is associated with a poor shortterm developmental outcome. PMID- 7546004 TI - Maturational trends of EEG-sleep measures in the healthy preterm neonate. AB - Five physiologic groupings of 45 EEG-sleep measures were acquired from serial 24 channel EEG-sleep recordings (i.e., sleep architecture, continuity, EEG spectral, phasic, and autonomic measures), utilizing 129 studies on 56 healthy preterm infants from 28 to 43 weeks postconceptional age (PCA) who were neurodevelopmentally normal on follow-up. Regression analyses chose the least number of measures that best reflected maturation. Four of 45 variables (i.e., spectral alpha energy during quiet sleep, total spectral EEG energy, arousal number during active sleep, and percentage of EEG discontinuity) most significantly explained brain maturation in neonates < 36 weeks PCA. Three of 45 variables (i.e., spectral theta and beta energies during active sleep and spectral alpha energy during quiet sleep) were most representative after 36 weeks PCA. Spectral EEG energies were the strongest indicators of maturation compared with other measures, particularly in near-term neonates. PMID- 7546005 TI - Proton magnetic resonance spectroscopy in children with acute central nervous system injury. AB - Single voxel proton magnetic resonance spectroscopy (1H-MRS) was used in 30 infants and children with acute central nervous system injuries to determine the value of changes in specific metabolite ratios in predicting outcome. The mean age of all patients was 38 +/- 52 months and the mean time of study after insult was 7 +/- 5 days. 1H-MRS was determined in the occipital gray and parietal white matter (8 cm3 volume, STEAM sequence with TE = 20 ms, TR = 3,000 ms). Data were expressed as ratios of different metabolite peak areas including N acetylaspartate (NA), choline-containing compounds (Ch), creatine and phosphocreatine (Cr), and lactate (Lac). Statistically significant differences were observed when patients with good/moderate (G/M) outcomes (n = 17; mean age: 46 months) were compared to patients with bad outcomes (n = 10; mean age: 26 months). NA/Cr and NA/Ch were significantly lower in the bad outcome group (NA/Cr = 1.15 +/- 0.38; NA/Ch = 1.18 +/- 0.52) compared to the G/M group (NA/Cr = 1.41 +/- 0.28, P < .05; NA/Ch = 1.98 +/- 0.81, P < .01). Lactate was present in 80% of bad outcome patients and in none of the G/M group (P < .0001). Using a linear discriminant analysis and combining 4 clinical variables (Glasgow Coma Scale score, initial pH and glucose, number of days unconscious at time of 1H-MRS) allows classification of 94% of patients into their correct outcome group. Use of spectroscopy variables (NA/Cr, NA/Ch, Ch/Cr, presence of lactate) alone correctly classified 81% of patients. The combination of clinical and 1H-MRS variables correctly classified 100% of patients. Our findings suggest that 1H-MRS adds information which, in combination with clinical examination, may be useful in outcome assessment in children with serious acute central nervous system injury. PMID- 7546006 TI - Proton magnetic resonance spectroscopy of brain in congenital myotonic dystrophy. AB - Volume selective proton magnetic resonance spectroscopy of brain was performed on a 1.5 T magnet in 5 patients with congenital muscular dystrophy and compared to the results in 46 healthy children and 1 healthy adult. Peaks of N-acetyl aspartate, choline, and creatine but not lactate, were observed in both groups on proton magnetic resonance spectroscopy. Spectroscopy of controls revealed an increase with advancing age in the ratio of N-acetyl aspartate/choline and N acetyl aspartate/creatine and a decrease in the choline/creatine ratio. In patients with congenital myotonic dystrophy, the N-acetyl aspartate/choline ratio did not increase with advancing age, but the N-acetyl aspartate/creatine ratio did. The choline/creatine ratio decreased with advancing age, which matched the results of controls. At any age older than 4 years, the N-acetyl aspartate/choline and N-acetyl aspartate/creatine ratios were lower in patients with congenital myotonic dystrophy than in controls. The choline/creatine ratio did not differ between congenital myotonic dystrophy and controls. These results suggest that in patients with congenital myotonic dystrophy N-acetyl aspartate decreases and there exists a developmental disorder of neurons in brain. PMID- 7546007 TI - Immunohistochemistry for a bifunctional protein in patients with peroxisomal disorders. AB - Immunohistochemical studies using antisera against bifunctional protein, a beta oxidation enzyme, were performed on liver, kidney, and brain tissue specimens from patients with peroxisomal disorders and from controls to investigate the distribution and development of peroxisomes. Bifunctional protein-positive granules were not found in patients with Zellweger syndrome or neonatal adrenoleukodystrophy, whereas positive immunoreactivity was observed from 8 and 6 weeks gestation in the liver and kidney, respectively, and in the brain, from 23 25 weeks in the brainstem neurons and from 12-14 weeks in the white matter glia, in controls. Bifunctional protein immunoreactivity then increased with gestation in the brain. These results suggest that bifunctional protein immunohistochemistry is useful for the detection of peroxisomes, which are closely related to neuronal maturation and gliogenesis in premyelination in human brain development. PMID- 7546008 TI - Infarction of basal ganglia associated with California encephalitis virus. AB - A child developed acute hemiparesis due to infarction of basal ganglia and internal capsule. Pleocytosis of cerebrospinal fluid and elevated immunoglobulin M antibodies suggest that California encephalitis virus infection caused the stroke. PMID- 7546009 TI - Glutaric acidemia type II: neuroimaging and spectroscopy evidence for developmental encephalomyopathy. AB - Glutaric acidemia type II is associated with neonatal hypoketotic hypoglycemia, metabolic acidosis, profound hypotonia, progressive cardiomyopathy, and early death. Deficiency of either electron transfer flavoprotein or electron transport flavoprotein:ubiquinone oxidoreductase leads to intramitochondrial accumulation of metabolites of compounds oxidized by enzymes that transfer electrons to flavoprotein. No detailed results of antemortem neuroimaging or magnetic resonance spectroscopy have been described previously. We investigated a patient with typical neonatal onset glutaric acidemia type II without obvious dysmorphogenesis or renal malformations. Cranial tomographic scan revealed hypoplastic temporal lobes and marked widening of the sylvian fissures ("bat wing" appearance). Cranial magnetic resonance imaging documented underdeveloped frontal and temporal lobes with delayed myelination and hypoplasia of the corpus callosum. 31P-Magnetic resonance spectroscopy of muscle was grossly abnormal with a very low energy state consistent with mitochondrial dysfunction. 1H-Magnetic resonance spectroscopy of brain revealed elevated intracerebral lactate concentration and abnormally high choline/creatine ratio suggestive of dysmyelination. These findings constitute the first in vivo evidence of a developmental encephalomyopathy in glutaric acidemia type II. PMID- 7546010 TI - Transient dystonia of infancy, a result of intrauterine cocaine exposure? AB - Intrauterine cocaine exposure has been associated with multiple transient and permanent neurologic sequelae. Although dystonic reactions have been reported in cocaine users, infantile dystonia following intrauterine exposure has not. We describe 4 infants testing positive for cocaine metabolite at birth with subsequent transient dystonic reactions, beginning at 3 hours to 3 months of age and persisting for several months. PMID- 7546012 TI - Brain tumors associated with infantile spasms. AB - Two patients with brain tumors associated with infantile spasms are reported. Both infants displayed typical clinical features of infantile spasms, comprising tonic spasms manifesting in series and hypsarrythmia. In Patient 1, magnetic resonance imaging revealed a tumor in the hypothalamic region, suggestive of hypothalamic hamartoma. In Patient 2, cranial computed tomography and magnetic resonance imaging indicated the existence of a primary brain tumor with calcification in the right temporal lobe. Adrenocorticotropic hormone therapy combined with clonazepam relieved seizures in both infants. In Patient 1, resection of the hypothalamic tumor is impossible because the tumor lacks a stalk. In Patient 2, pathologic investigation of removed tumor tissue demonstrated mixed-oligoastrocytoma. It is suggested that focal lesions, like those in our patients, are involved in the development of infantile spasms. PMID- 7546013 TI - Spinal muscular atrophy with oculomotor palsy, epilepsy, and cerebellar hypoperfusion. AB - We report a boy with spinal muscular atrophy (SMA) type 1, who developed novel clinical and neuroradiologic features, indicating the broad spectrum of this degenerative disease. Widespread lesions in the central nervous system were disclosed by magnetic resonance imaging and single photon emission computed tomography (SPECT) studies. In particular, scattered regions of hypoperfusion demonstrated by SPECT suggested multisystem involvement including the cerebellum. The novel features of this patient were complex partial seizures and oculomotor palsy. PMID- 7546011 TI - Impairment of respiratory rhythmogenesis and sequelae of bacterial meningitis. AB - A 9-year-old boy with respiratory disturbance associated with medullary lesions after pneumococcal meningitis is reported. Although he lives a normal daily life, he cannot cough or sneeze. A polysomnographic study revealed a low respiration rate and an irregular respiratory rhythm not only during REM sleep but also during slow wave sleep, and marked desaturation during sleep. Respiratory function tests including CO2 response revealed normal values. Magnetic resonance imaging demonstrated bilateral small lesions in the medulla. This patient is unusual because respiratory rhythm is impaired, without decreased ventilatory capacity or CO2 response, supporting the possibility that rhythmogenetic respiratory neurons are located in a limited area of the human medulla. PMID- 7546014 TI - Congenital myopathy with ringlike distribution of myonuclei and mitochondria and accumulation of nemaline rods. A variant of centronuclear myopathy? AB - Histopathologic and ultrastructural findings in a muscle biopsy performed on an 11-year old boy with congenital hypotonia, weakness, respiratory insufficiency requiring chronic ventilatory support, and a probable X-linked inheritance are presented. The muscle biopsy disclosed a peculiar, ringlike arrangement of mitochondria and myonuclei in most muscle fibers. Accumulations of nemaline rods were present in approximately 10-15% of fibers. We believe that our patient represents a variant of myotubular/centronuclear myopathy. The histochemical findings suggest disturbance in developmental migration of nuclei and mitochondria probably due to impaired function of the cytoskeleton. PMID- 7546015 TI - Stimulation of group II phospholipase A2 mRNA expression and release in an immortalized astrocyte cell line (DITNC) by LPS, TNF alpha, and IL-1 beta. Interactive effects. AB - Astrocytes are immunoactive cells in brain and have been implicated in the defense mechanism in response to external injury. Previous studies using cultured glial cells indicated the ability of astrocytes to respond to bacteria endotoxin and cytokines, resulting in the release of phospholipase A2. In this study, we examined the interactive effects of lipopolysaccharides (LPS), interleukin 1 beta (IL-1 beta) and tumor necrosis factor (TNF alpha) to stimulate phospholipase A2 (PLA2) in an immortalized astrocyte cell line (DITNC) with many properties of type I astrocytes. Northern blot analysis using oligonucleotide probes derived from the cDNA encoding the rat spleen group II PLA2 indicated the ability of DITNC cells to respond to all three factors in the induction of gene expression and the release of PLA2. After an initial lag time of 2 h, PLA2 release was proportional to time, reaching a plateau by 12 h. This event occurred at a time period preceding any signs of cell death. Cycloheximide at 1.25 microM completely inhibited cytokine-induced PLA2 release. When suboptimal amounts of TNF alpha were added to the DITNC culture together with IL-1 beta or LPS, a synergistic increase in the induction of PLA2 release could be observed. On the other hand, combination of IL-1 beta and LPS resulted only in an additive increase in PLA2 release. Antibodies to IL-1 beta and TNF alpha completely neutralized the effects of these two agents on PLA2 release. However, neither antibody was able to inhibit the PLA2 release induced by LPS, suggesting that the effect of LPS was not complicated by the release of IL-1 beta or TNF alpha. Taken together, results show that the immortalized astrocyte cell line (DITNC) can be used for studies to elucidate the molecular mechanism underlying the cytokine signaling cascade and subsequent induction of PLA2 synthesis. PMID- 7546016 TI - HU-211, a nonpsychotropic cannabinoid, improves neurological signs and reduces brain damage after severe forebrain ischemia in rats. AB - The purpose of the present study was to examine the dose-response relationship and the therapeutic time window for the synthetic nonpsychotropic cannabinoid (HU 211) as a neuroprotective agent in transient, severe forebrain ischemia in the rat. Adult Sprague-Dawley rats were subjected to 20 min common carotid artery occlusion (CCAo) 24 h after coagulation of both vertebral arteries. Thirty minutes after the onset of CCAo, rats received an iv injection of HU-211 2, 4, or 8 mg/kg in HPCD (n = 12, 18, and 11, respectively), or the appropriate vehicle (n = 20). Neurological signs were scored daily for 3 d following ischemia. A significant improvement (p < 0.01, Kruskal-Wallis nonparametric test, followed by Mann-Whitney U-test, p < 0.05) of neurological deficits by the 4 mg/kg dose of HU 211, was observed 24, 48, and 72 h after insult. On the third day post-CCAo, the rat brain was taken for histopathological evaluation of the CA-1 sector of the hippocampus. Counts of viable neurons in the hippocampal CA1 field showed significantly more live cells in the HU-211 (4 mg/kg) treated animals (P < 0.001, ANOVA followed by Duncan's post-hoc test, p < 0.05). The drug was equally effective when given 30 and 60 min after ischemia, but neuroprotection was no longer significant after 3 h. We suggest that HU-211 may be a potential treatment for postischemic brain damage in human beings. PMID- 7546017 TI - Nordihydroguaiaretic acid and RHC 80267 potentiate astroglial injury during combined glucose-oxygen deprivation. AB - Membrane phospholipid degradation has been proposed to play a key role in hypoxic ischemic brain injury. We tested the hypotheses that both nordihydroguaiaretic acid, a phospholipase A2 and lipoxygenase inhibitor, and RHC 80267, a diacylglycerol lipase inhibitor, would decrease the release of [3H]arachidonic acid metabolites from prelabeled cultures of astroglia subjected to combined glucose-oxygen deprivation and that these inhibitors would also decrease astroglial injury during combined glucose-oxygen deprivation. Both nordihydroguaiaretic acid and RHC 80267 significantly inhibited the release of [3H]arachidonic acid metabolites during combined glucose-oxygen deprivation. This suggests that two separate enzymic pathways, the phospholipase A2 pathway and the phospholipase C/diacylglycerol lipase pathway, contribute to the release of astroglial [3H]arachidonic acid metabolites during combined glucose-oxygen deprivation. However, both of these lipase inhibitors increased astroglial cell death during combined glucose-oxygen deprivation, probably due to inhibition of arachidonic acid release. We speculate that arachidonic acid release may be a mechanism of astroglial self-preservation during combined glucose-oxygen deprivation. PMID- 7546018 TI - Modulation of signal transduction in rat synaptoneurosomes by platelet activating factor. AB - The potential involvement of platelet activating factor (PAF, 1-O-alkyl 2-O acetyl-sn-glycero-3-phosphocholine) in aggravation of ischemic brain injury has been recently postulated. Reported evidences in support of this thesis include increases of brain PAF concentration during ischemia and the neuroprotective effect exerted by PAF antagonists. In this article, we demonstrate that several PAF-mediated biochemical responses in synaptoneurosomes in vitro resemble these observed previously in ischemic brain and are widely acknowledged as the potentially causal factors in this pathology. In synaptoneurosomes prepared from rat hippocampus, 10 nM PAF caused an observable elevation of intracellular calcium as measured by fluorescence Fura-2A probe. A similar elevation of synaptoneurosomal [Ca2+]i was evoked by 1 mM glutamate treatment. As an effect of calcium entry after PAF application, a translocation of protein kinase C (PKC) toward plasma membranes was demonstrated by 3H-labeled phorbol-binding method. It was followed by an increase of 50 kDa proteolytic fragment of the enzyme (PKM) recognized on Western blots with anti-PKC antibody. Incubation of synaptoneurosomes in the presence of calcium chelators abolished these effects of PAF and significantly decreased the content of PKC in the membranes. Furthermore, PAF treatment markedly attenuated the receptor- and postreceptor-activated cAMP accumulation in synaptoneurosomes. The decrease of cAMP level seems to be secondary to the PAF-induced calcium entry with subsequent activation of cAMP specific phosphodiesterase, since it was completely blocked by IBMX, a potent inhibitor of this enzyme. Our observations indicate that PAF in a concentration found in ischemic brain can elevate [Ca2+]i and potentiate calcium-dependent intracellular signalling in synaptoneurosomes in vitro, including PKC translocation/activation and proteolysis, followed by IBMX-sensitive inhibition of cAMP production. The relative contribution of these events to ischemic brain injury is currently under extensive investigation. PMID- 7546019 TI - Interrelationship between the functional state of the central nervous system and lipid peroxidation level in brain following craniocerebral trauma. AB - The interrelationship between the functional state of the CNS and brain metabolism was studied in animals following craniocerebral trauma. Lipid peroxidation and antioxidant activity of rabbit brain were investigated a day after craniocerebral trauma. An increase in conjugated dienes, TBA-reactive products (TBARP), and fluorescent Schiff bases (SchB), and a sharp decrease in antioxidant activity in all brain regions investigated were established. The possibility of regulation of the disturbed processes by artificial alteration of the functional state of the CNS was shown. It was found that the day after stimulation of injured brain by amphetamine (phenamin) or piracetam, the values of lipid peroxidation and antioxidant activity came to the same levels as for controls. On the contrary, the depression of the CNS by phenobarbital (luminal) was not accompanied by such positive reactions. The stimulants did not possess antioxidant activity in vitro experiments on a model system. Thus, it was shown that following craniocerebral trauma, the lipid peroxidation process can be regulated by modification of the functional state of the CNS alone without special antioxidant therapy. PMID- 7546020 TI - The problems of alcohol abuse. PMID- 7546021 TI - Value of performing a chest radiograph in patients with diagnosis of "clinical sepsis". AB - In any infant admitted with diagnosis of "clinical sepsis", a chest radiograph is commonly obtained as a routine work-up. The purpose of this study was to establish the relationship between an abnormal chest radiograph and "clinical sepsis" in a population of infants. The clinical records and chest radiographs of 81 infants, (less than 3 month old) were reviewed. The temperature, white blood cell count, respiratory signs, symptoms, and chief complaints were recorded and compared with positive or negative chest radiographs. A statistically significant correlation with abnormality in the chest radiograph was not established. Four patients, (31%), with any respiratory signs had abnormal chest radiographs, whereas only 13, (19%) asymptomatic patients did. It was concluded that chest radiographs do not add useful information to the evaluation of a febrile infant who does not have clinical findings of pulmonary diseases. However, due to the limited population studied, it seems appropriate to continue the current recommended practice of ordering chest radiographs in febrile infants 3 months old or less. PMID- 7546022 TI - Total colectomy and "J" pouch ileo-anal pull through without temporary diverting ileostomy in the management of ulcerative colitis. AB - Anal sphincter saving operations are currently the procedures of choice for most patients with ulcerative colitis. In this study we review the experience of The Mayaguez Medical Center, Department of Surgery with 18 patients who underwent a total abdominal colectomy, rectal mucosectomy and "J" pouch ileoanal pull through without a temporary diverting ileostomy in the management of ulcerative colitis. The complications that are associated with this procedure in our patients are: wound infection, postoperative leak, early stricture and intestinal obstruction. Only one patient died and it was due to a massive pulmonary embolism. Finally, we conclude that this procedure should be reserved for well nourished, psycho logically stable young patients who are willing to tolerate the complications associated with it. PMID- 7546024 TI - Varicella (purpura fulminans variant) in an adult diagnosed at autopsy. AB - We are presenting a 70 years old man with purpura fulminans secondary to Varicella infection. A brief review of literature is done and mechanism of disease are briefly discussed. PMID- 7546025 TI - Thoracic spine osteochondroma causing spinal cord compression. An unusual cause of paraparesis. AB - A case of a thoracic osteochondroma with compression of the spinal cord is reported. The neuroimaging studies are described. PMID- 7546026 TI - Neuronal intestinal dysplasia: a role for surgery? AB - Neuronal intestinal dysplasia (NID) is a poorly understood colonic motility disorder with characteristic histopathological findings and clinical presentation. It is often associated with Hirschsprung's disease (HD) and can constitute a cause of failure of clinical improvement after adequate resectional pull-through surgery. Other conditions associated with NID are: Chronic Intestinal Pseudo-obstruction (CIPO), anorectal malformations and Multiple Endocrine Neoplasia (MEN) II syndrome patients. To increase the diagnostic yield of NID the pathologist should be aware and use histochemistry evaluation of the rectal biopsy specimen in patients with history of constipation or unexplained bouts of diarrhea. Adequate sampling of the temporary proximal colostomy done to HD patients should be examined for NID pathological changes. Treatment has centered around the clinical picture with most cases managed medically with prokinetic agents, colonic irrigations, and bowel cathartics until improvement and normalization of histology occur. There is evidence of progressive maturation of the enteric nervous system with time. Surgery is indicated for patients with severe clinical deterioration after failed medical management. PMID- 7546027 TI - Hantavirus infection: a rare disease which you should be aware. AB - Hantavirus infection is caused by viruses classified in the Hantavirus genus, of the Bunyaviridae family. Recently a new hantavirus has been recognized. Specific endemic areas has been identified, however a new outbreak was identified in the southwestern of the United States. The principal vectors are rodents. Human infection occur by aerosol from rodent urine, saliva, feces and rodent bites. The infection classically is manifested clinically by fever, hemorrhage and renal failure. The recent outbreak was associated with acute respiratory illness without renal involvement. The treatment is with intravenous Ribavirin. Specific recommendations for prevention and control are presented here in. PMID- 7546028 TI - [From paternalism to democracy: beneficence--person and current bioethics in Puerto Rico]. PMID- 7546023 TI - Adolescent student's compliance with testicular self examination. AB - To determine what adolescents students know or feel about testicular cancer, (TC) and the testicular self examination, (TSE), technique, 127 males students (mean age 16.14 years) were given a 21 item questionnaire. The role of the primary physician and the role of written and visual educational material were assessed for effectiveness in increasing the regular performance of monthly testicular self examination by male students at a Health Education Course at Cervantes Public School at Bayamon, P.R. Out of 127 male student, only 2.4%, (n = 3), initially knew about the symptoms of TC. Analysis of the attitude and knowledge questions revealed that respondents were not sure about general concepts in TC and, were not sure that this cancer could be self diagnosed. This study pretends to assess the effects of modeling and guiding practice as components within a comprehensive testicular self examination education program for adolescents male students. Three groups were studied from 10th, 11th, and 12th, grade. The variables investigated were knowledge of testicular cancer, attitudes toward testicular cancer, and frequency of self reported testicular self examination. The participants then received written material plus a physician's lecture on TC and TSE. Upon follow up, 87.5%, (n = 113) of the students reported performing monthly TSE, (P < 0.001), a statistically significant increase in performance. Although 20.3% (n = 26) reported having a physical examination by a physician within the previous 3 months, less than 4.7%, (n = 6) reported being taught TSE by a physician. Physicians need to educate males about testicular cancer and its early detection. PMID- 7546029 TI - Limits of natural science: brain research and computers. AB - The criterion that pure natural science can only investigate objective phenomena which can be observed by independent observers sets certain limits to our scientific understanding of brain functions. The methods and the present state of brain research and of computer development are described. The limitations of brain research are discussed by comparing the properties of brains and computers. At least for the time being we do not know of any natural scientific--i.e. physical or chemical--method which allows the objective measurement of consciousness, sensations, and emotions. PMID- 7546030 TI - Flavipucine and brunnescin, two antibiotics from cultures of the mycophilic fungus Cladobotryum rubrobrunnescens. AB - Two antimicrobial metabolites were isolated from submerged cultures of Cladobotryum rubrobrunnescens, a mycophilic fungus growing on a Inocybe species. One of the compounds proved to be identical to flavipucine (2), an antibiotic previously isolated from Aspergillus flavipes (Casinovi et al., 1968) and from a Macrophoma species (Sassa T. and Onuma Y. (1983), Agric. Biol. Chem. 47, 1155 1157). The other metabolite, brunnescin (1), is a new tetrasubstituted furan derivative which exhibits antibacterial, antifungal and cytotoxic effects. PMID- 7546031 TI - The role of phosphatidylglycerol as a functional effector and membrane anchor of the D1-core peptide from photosystem II-particles of the cyanobacterium Oscillatoria chalybea. AB - The intrinsic polypeptide D1, isolated from photosystem (PS) II-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents. Purification was demonstrated by Western blotting and amino acid sequencing. By carrying out D1-immunization in rabbits a polyclonal monospecific D1-antiserum was obtained. For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcholine (PC) on PSII-oxygen evolution was analysed in reconstitution experiments. In these experiments purified photosystem II (PSII)-particle preparations were treated with the enzyme phospholipase A2 and supplemented with lipid emulsions. We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation. A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex. Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions. A covalent binding seems not probable. The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting. The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1. This has led us to the conclusion that the phospholipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane. Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation. The high turn-over of D1 and the spatial separation of the synthesis- and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes. PMID- 7546032 TI - Antifungal compounds induced in the dual culture with Phytolacca americana callus and Botrytis fabae. AB - In order to investigate new metabolites which are only induced in a plant callus infected by a pathogenic fungus, dual cultures with combinations of 10 species of fungi and 6 plant cell lines from different species were established. Among the combinations tested, the methanolic extract of a dual culture consisting of a plant cell line, Phytolacca americana and a fungus, Botrytis fabae showed a marked antifungal activity to Cladosporium herbarum. The main active constituent of this extract was identified to be phytolaccoside B (Pls B) by the spectroscopic analyses. PMID- 7546034 TI - 1H NMR and calorimetric measurements on rabbit eye lenses. AB - The dynamic properties of water molecules in the rabbit lens were studied by proton nuclear magnetic resonance line shape analysis, measurements of relaxation times as a function of temperature and calorimetric measurements. The experiments prove, as already suggested by other authors, that there are two types of water in the lens of rabbit eyes, namely bound unfreezable hydration water and bulk freezable water. Line shape analysis and relaxometry showed, that this two types of water exist in two different environments, which may be identified as the nucleus and the cortex of the lens. The line shape analysis showed furthermore that water molecules in the rabbit lens has a common spin lattice relaxation time (T1), but two different transverse relaxation times (T2A and T2B). The tentative model of fast water exchange on the T1 time scale and slow water exchange on the T2 time scale, was used to explain experimental proton relaxation data of the rabbit lens. An estimation for this exchange rate kex by comparing it to the relaxation times is given (T1(-1) << kex << T1(-1)). It has also been shown by a calorimetric measurements, that the lenses can be easily under-cooled to temperatures well below the freezing point of water. The achievable maximum undercooling temperature of the lens is a function of the cooling rate KC, therefore it has to be considered as an experimentally adjustable parameter which is not characteristic for the investigated sample. Thus it must be noted that any previous discussions about the specific value of the temperature of water crystallisation in biological systems need to be carefully reconsidered. PMID- 7546033 TI - 5-hydroxy-3-vinyl-2(5H)-furanone--a new inhibitor of human synovial phospholipase A2 and platelet aggregation from fermentations of a Calyptella species (basidiomycetes). AB - 5-Hydroxy-3-vinyl-2(5H)-furanone, a potent and selective inhibitor of human synovial phospholipase A2 was isolated from fermentations of a Calyptella species. Its structure as identified by spectroscopic methods is identical to PA 147, an antibiotic previously isolated from a streptomycete. 5-hydroxy-3-vinyl 2(5H)-furanone inhibits the aggregation of human and bovine platelets stimulated by different inducers and exhibits weak antimicrobial activities. PMID- 7546035 TI - Secretion of neutral and acid DNases in cultivated human lymphocytes after incubation with DNA; possible consequences for inhalation anesthesia. AB - After incubation with DNA human lymphocytes release neutral and acid DNase activities into the culture medium; the release depends on DNA concentration and time of cultivation. The electrophoretic mobility of the released neutral DNase activity is in accordance with DNase I and the electrophoretic mobility of the released acid DNase activity with DNase II. The released DNase activities do not originate from dead cells and are not influenced by blast cell formation. The anesthetic halothane can inhibit the released neutral and acid DNase activities. Inhalation anesthesia can possibly disturb the correlation between DNA and DNases in human blood. PMID- 7546036 TI - Periodic current injection (PCI)--a new method to image steady-state membrane potential of single neurons in situ using extracellular voltage-sensitive dyes. AB - A new method is described which allows to image the steady-state distribution of membrane potential of single neurons in situ. The method consists of staining the tissue with an extracellular voltage-sensitive dye (Di-4-ANEPPS) and impaling a single neuron with a microelectrode. After focusing the imaging system onto the cell a large series of images are taken with a CCD camera at the appropriate excitation wavelength of the voltage-sensitive dye while the neuron's membrane potential is shifted by a periodic current injection (PCI). Afterwards two groups of images are averaged separately: those images while the cell was at rest and those images while the cell was hyperpolarized. After subtraction of these averaged images, the resulting difference image shows only the membrane potential of the cell which was altered periodically. The success of the method is demonstrated on leech cells in intact ganglia. If applied to cells with a basically two-dimensional arborization pattern, the decrease of activity in the difference image in areas further away from the injection site should relate to a decrease in membrane potential according to the passive electrotonic properties of the cell under study. PMID- 7546037 TI - Kinetics of metabolism in human kidney transplants measured by dynamic 31P NMR spectroscopy. AB - The technique of ex vivo 31P NMR spectroscopy has been used for the investigation of metabolic turnover in 15 cadaveric human kidneys obtained from brain-dead donors for transplantation. Measurements were carried out at 1.5 T time dependently during regular hypothermic storage in histidine-tryptophan-alpha ketoglutarate solution. The minimum delay between explanation and spectroscopy was 2 h 41 min. In one case of a discarded organ the measurements could be extended over a period of 161 h 35 min. A detailed kinetic model describing monoexponential ischemic phosphate turnover, which accounts for various interrelations of the NMR visible metabolites, has been derived. Averaged velocity constants of the decays of nucleotides and phosphomono- and -diesters ranged from 0.0047 to 0.294h-1 at approximately 4 degrees C. Intracellular pH decreased monoexponentially with an averaged velocity constant of 0.31 h-1. PMID- 7546038 TI - New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases. AB - Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of omega-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability to 0 degree C. Lactate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazoniumpentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3 alpha, 20 beta Hydroxysteroid dehydrogenase reacts at 0 degree C with the ethyl homologue and slowly with the propyl compound. The butyl- and pentyl analogues do not inactivate at 0 degree C. Tests with 14C-labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3 Acetylpyridinium)butyl 2'-phospho-adenosine diphosphate, a structural analogue of NADP+, was prepared by condensation of adenosine-2,3-cyclophospho-5' phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid with 2':3'-cyclonucleotide-3' phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehydrogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP+-dependent dehydrogenases the compound has been shown to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase. PMID- 7546039 TI - A study on tea aroma formation mechanism: alcoholic aroma precursor amounts and glycosidase activity in parts of the tea plant. AB - We have shown in molecular basis that alcoholic tea aroma is mainly formed by endogenous enzymatic hydrolysis of glycosidic aroma precursors during manufacturing. Amounts of alcoholic aroma precursor and glycosidase activity in each part of the tea shoot (Camellia sinensis var. sinensis cv Yabukita and a hybrid of var. assamica & var. sinensis cv Izumi) were indirectly measured by means of a crude enzyme assay. The aroma precursors were abundant in young leaves and decreased as the leaf aged. Glycosidase activity also decreased as leaves aged, but was high in stems. PMID- 7546040 TI - Enzymatic and electrochemical reactions of catalase immobilized on carbon materials. AB - It has been shown that catalase immobilized on graphite and soot undergoes an oxidation reduction transformation. Some results on the effect of the potential sweep rate, temperature and pH on this transformation are presented. On the basis of the relationship established, it has been proved that the redox transformation is related to the iron in the heme of the active center of the enzyme and takes place with a transfer of one electron. PMID- 7546041 TI - Molecular interactions of quinidine with phospholipid bilayers. AB - Quinidine (QUIN) is one of the most important and efficient antiarrhythmic drugs (AAD). It belongs to class I, which are the drugs that exert their action at the level of the sodium channels in the membrane of the myocard. Several hypotheses support the idea that the molecular mechanism of action of the AAD is via nonspecific interactions with phospholipids sited in the neighborhood of the channels. In order to probe the validity of these hypotheses, QUIN was made to interact with the phospholipids dimyristoylphosphadidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). These interactions were performed in a hydrophobic and a hydrophilic medium under a wide range of molar ratios. The resulting products were analyzed by X-ray diffraction. QUIN solutions were also made to interact with DMPC liposomes, which were studied by fluorescent spectroscopy. Finally, human erythrocytes which were incubated with QUIN solutions were observed by scanning electron microscopy. The results of these experiments proved that QUIN indeed interacted with phospholipid bilayers. PMID- 7546042 TI - Membrane association of the Rieske iron-sulfur protein. AB - The mode of membrane attachment of the Rieske iron-sulfur proteins from cytochrome b6f complex of pea thylakoids and from cytochrome bc1 complex of yeast mitochondria has been studied using biochemical approaches. The relative sensitivity of the Rieske protein to trypsin in the thylakoid membrane shows that all trypsin sites of the Rieske protein are on the lumen side of the thylakoid membrane. In contrast to cytochrome f the chloroplast Rieske protein was extracted from thylakoids using chaotropic agents (NaSCN, urea), an alkaline pH and relatively low concentrations of Trinon X-100. The cytochrome bc1 complex Rieske protein from mitochondrial membranes of yeast was also released by NaSCN and alkaline treatment. The results presented here led us to the conclusion that the mitochondrial and chloroplast Rieske proteins are extrinsic and that their association with the rest of the complex involves hydrophobic interactions. PMID- 7546043 TI - Antioxidant profile of mono- and dihydroxylated flavone derivatives in free radical generating systems. AB - A number of free radical generating systems were used to investigate the antioxidant properties and structure-activity relationships of a series of monohydroxylated and dihydroxylated flavones. Ortho-dihydroxylated flavones showed the highest inhibitory activity on enzymic and non-enzymic microsomal lipid peroxidation as well as on peroxyl radical scavenging. Most flavones were weak scavengers of hydroxyl radical, while ortho-dihydroxylated flavones interacted with superoxide anion generated by an enzymic system or by human neutrophils. This series of compounds did not exert cytotoxic effects on these cells. Scavenging of superoxide and peroxyl radicals may determine the antioxidant properties of these active flavones. PMID- 7546044 TI - Antioxidant protection of egg lecithin liposomes during sonication. AB - When model membranes are prepared by ultrasonic treatment of polyunsaturated phospholipids, radical production can induce a partial degradation of the polyunsaturated fatty acyl chains and the formation of lipid hydroperoxides. A suitable antioxidant employed during liposome preparation is able to protect them against lipid peroxidation. This work contains the results of studies on egg lecithin liposomes with incorporated antioxidants that were supposed to play the protective role mentioned. As it has been shown the antioxidant compounds used ensured a 40-60%, i.e., satisfactory protection of liposomes after 30 min sonication. Possible practical applications are discussed. PMID- 7546045 TI - Pharmacokinetic interaction between 4'-epidoxorubicin and the multidrug resistance reverting agent quinine. AB - The serum and red blood cell (RBCs) disposition of epirubicin (EPR) after intravenous bolus injection without and with coadministered quinine ( QUI) was investigated in patients undergoing a cyclic chemotherapy with EPR. QUI possesses a statistical significant influence on the EPR serum concentrations and, as a consequence, on the pharmacokinetic parameters for the initial distribution phase of EPR. Within the first 15 min after administration, EPR was distributed from the central compartment distinctly faster in compare to the control, when QUI was preadministered (t(1/2) = 6 min for the control group and t(1/2) = 3 min with QUI; -46%, p < 0.05). Yet, in the beta-phase when drug-elimination predominates, no statistical significant influence of QUI in regard to EPR serum and RBC concentrations could be observed. Half-life of elimination was 0.5 h for the control group and 8.6 h for the QUI group (-10%). The mean initial serum concentration (co) was reduced significantly by QUI from 7359 +/- 506 ng/ml to 4351 +/- 1682 ng/ml (-42%, p < 0.005). Furthermore, QUI caused a reduction of the serum bioavailability of EPR (expressed as AUC(o-24)-values) from 3404 +/- 1008 ng/ml x h to 2359 +/- 1073 ng/ml x h (-31%, p < 0.05). Vd and Vdbeta were increased at about 90% and the mean total body clearance was accelerated from 45.3 to 1487 ml/min, but due to the large standard deviations the calculated difference for these parameters was not statistically significant. In the observed time interval of 24 h, the red blood cell coefficient of distribution k(rbc) of EPR was lower if QUI was coadministered (k(rbc) = 1.25 +/- 0.12 for the control group k(rbc) = 1.15 +/- 0.13 under QUI; p < 0.04). The results point out that QUI induces an accelerated distribution of EPR from the blood into the tissue and that QUI additionally may have influence on the red-blood cell partitioning of EPR. PMID- 7546046 TI - Comparison on reactivity of Fe(III) and Al(III) compounds in the presence of hydrogen peroxide: its relevance to possible origin for central nervous system toxicity by aluminum ion. AB - The iron(III) compounds with several aminocarboxylate chelates containing an aryl or furan substituent exhibit high activity in enhancement of the reactivity of hydrogen peroxide, leading to facile hydroxylation at benzene ring, and to degradation of furan ring, but no such activity was observed for the corresponding Al(III) compounds. These results were interpreted in terms of the molecular orbital consideration, and lack of the activity of the Al(III) complexes was attributed to lack of electrophilic nature of the peroxide adduct due to the absence of a d-orbital; this may explain the fact that there were no tumors in Al-NTA(nitrilotriacetic acid)-treated rats. Based on the facts observed in this study, the decreased function of iron(III) ions for synthesizing neurotransmitters in the brain was assumed to be one of the possible origin for the neurotoxicity by injection of the Al(III) salts in vivo. PMID- 7546047 TI - Effects of juvenile hormone analogues (JHA) on the development of Trypanosoma cruzi. AB - Juvenile hormone analogues were tested for their lytic activity on Trypanosoma cruzi Chagas, 1909 blood tripomastigotes cultivated in vitro. The results indicated that the carbamate 4 and the phenoxyphenol derivative 1 are considered good candidates for blood sterilization. The compounds were also assayed for inhibition of development of parasites in infected mice showing a moderated degree of activity. PMID- 7546048 TI - Image analysis quantification in hormone receptor assay of mucinous carcinoma of the breast. Comparison with biochemical analysis. AB - Mucinous carcinoma (MC) of the breast is characterized by abundant extracellular mucin and by variable epithelial cellularity. Since some MCs are extremely hypocellular, we questioned the validity of biochemical (BIO) assays in these tumors. We analyzed paraffin-embedded tissue from 34 cases of MC of the breast for quantitative estrogen receptor (ER) and progesterone receptor (PR) using immunohistochemistry (IHC) on the Cell Analysis System CAS-200. Of the 34 cases, 31 (91%) were positive for ER, whereas 18 (53%) were positive for PR. In 21 cases the quantitative ER and PR were assayed biochemically by a dextran-coated charcoal method. Using the BIO results as the "true" values, the sensitivity of IHC for ER and PR was 100% and 78%, and the specificity was 13% and 64%, respectively. The low specificity of the values obtained by IHC was attributed to the fact that eight cases were "falsely" false positive (negative by BIO and positive by IHC) for ER and/or PR. Review of the histologic patterns of all 21 cases showed that 7 of the 8 falsely false positive cases were significantly hypocellular (epithelial cellularity 5-20%) as compared to the remaining cases (epithelial cellularity 20-75%). We conclude that immunohistochemical analysis of ER/PR status using image analysis in MC is a sensitive method, with the ability to detect receptor content when their concentration might be too low to be demonstrated by the conventional method as a result of sparse cellularity. PMID- 7546049 TI - Correlation between ploidy status, Erb-B2 and p53 immunohistochemical expression in primary breast carcinoma. AB - In the present study we evaluated the prognostic significance and correlation between ploidy status, lymph node status, estrogen-progesterone receptor status and the expression of Erb-B2 and p53 protein in 77 primary breast carcinomas. Quantitation of DNA ploidy was determined on Feulgen-stained touch imprints with an image analyzer, whereas localization of the immunohistochemical reaction of Erb-B2 and p53 protein was evaluated in paraffin-embedded tumor specimens with microscopy. The DNA histogram was diploid in 17 cases, poly/tetraploid in 24 and aneuploid in 36. We observed no correlation between ploidy status and hormone receptor content or lymph node status. The expression of Erb-B2 protein was observed exclusively in the membrane and in the cytoplasm of neoplastic cells and was uniformly distributed. Overexpression was observed in 89.2% of cases. Aneuploid tumors intensively expressed the oncogene in 20.3% of cases. A statistically significant correlation was observed between lymph node metastasis and Erb-B2 overexpression. The expression of p53 protein was expressed as nuclear staining in 17.6% of the cases, with variable intensity, mainly in ductal histotypes. Among these, 62% were aneuploid. Lymph node status and steroid receptor status did not correlate significantly with p53. From these data we conclude that DNA ploidy and Erb-B2 and p53 expression correlate with cell proliferation and differentiation and therefore may identify breast carcinoma patients with more aggressive disease. PMID- 7546050 TI - Comparison of DNA measurement performed by flow and image cytometry of embedded breast tissue sections. AB - Tissue sections are important for morphometric studies, such as the analysis of architectural pattern of tissues as well as nuclear morphology and chromatin texture describing DNA distribution in nuclei. Tissue sections are generally not recommended for use in DNA ploidy measurements due to possible errors based on sectioned and overlapped nuclei. However, image cytometry (IC) on tissue sections has the advantage of preserved architecture and selective sampling of nuclei. This is particularly important for the analysis of specimens with small areas of diseases tissue or with a variety of histologic patterns in the same region, such as premalignant changes or in situ tumors. To evaluate the potential of measurements on breast tissue sections in the estimation of DNA ploidy status, we compared IC and flow cytometry (FC) measurements on formalin-fixed breast cancer tissue from 51 paraffin-embedded blocks. For each specimen the DNA index and ploidy were determined by both methods. DNA indices of IC and FC corresponded roughly in 66% of cases. Agreement in ploidy was achieved in about 80% (40/51) of cases, where some of the discrepancies could be explained by the differences between invasive cancer and ductal carcinoma in situ (DCIS) illustrated by IC. Both methods agreed highly in aneuploid cases. However, about 50% of FC diploid cases were aneuploid by IC. Despite significant shortcomings of measurements obtained on archival tissues, meaningful data can be obtained not only by FC but also by IC performed on tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546051 TI - Knowledge-guided segmentation and morphometric analysis of colorectal dysplasia. AB - The histopathologic grading of colorectal adenomatous dysplasia is a subjective process. In this study, automated image analysis using knowledge-guided software was used to quantitatively assess colorectal glandular characteristics. Cases of histologically normal mucosa and of low, moderate and high grade dysplasia were examined using the technique. A total of 19 morphometric and densitometric features were measured on each gland. These included gland shape, epithelial area, nuclear stratification and nuclear optical density. Discriminant analysis of the data revealed those morphometric features which provided the best discrimination between the various histologic groups. The area of epithelium occupied by nuclei was the strongest discriminating variable, and this, in combination with a discriminant function derived from the remaining variables, was used to plot cases in bivariate sample space. The plots revealed that the data for normal glands were consistently well separated from dysplastic gland data. Data sets belonging to the various grades of dysplasia showed varying degrees of separation, depending which two histologic groups the discriminant function was based on. This study showed that automated image analysis of complex histologic scenes is possible using knowledge-guided segmentation and that it can provide useful data for the objective classification of colorectal dysplasia. PMID- 7546052 TI - Flow cytometric characterization of proliferation-associated nuclear antigen (p105) during the cell cycle in normal lymphocytes and promyelocytic leukemia cells (HL-60). AB - Bivariate flow cytometric analysis of p105 expression and DNA content was performed in human lymphocytes and promyelocytic leukemia cells (HL-60). We also employed a new method of detecting DNA strand breaks associated with apoptosis by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase (TdT). Phytohemagglutin stimulated proliferating lymphocytes express p105 maximally after 48 hours, similar to HL-60 cells in exponential growth phase. Antigen expression in G1 was notably heterogeneous in G1 phase of both cell types and highest in M-phase cells treated for 6 hours with vinblastine. However, the p105-DNA ratio changed very little. Cycloheximide did not affect P105 expression. Methotrexate decreased p105 expression. Camptothecin and teniposide induced apoptosis, but apoptotic cells still expressed p105. Dual-parameter measurement also demonstrated that TdT positive apoptotic cells expressed p105 at a higher level than apoptotic TdT negative cells. The data on drug treatment suggest that expression of p105 may be useful in monitoring chemotherapeutic effects but not as a marker of cell death from apoptosis. PMID- 7546053 TI - Nuclear texture in poorly differentiated small round cell tumors. Image analysis study of fine needle aspiration material. AB - Gradient analysis and pattern spectrum decomposition based on mathematical morphology concepts were used to explore nuclear texture patterns in a pool of 108 cells obtained by fine needle aspiration of five undifferentiated small round cell tumors of childhood, including one case each of Wilms' tumor, neuroblastoma, lymphoblastic lymphoma, Ewing's sarcoma and rhabdomyosarcoma. The aim of the study was to determine the presumptive value of nuclear pattern to correctly allocate each isolated cell to each of the five patients. The cells were examples of five histogenetically different tumors, all undifferentiated and with a close microscopic resemblance to one another. High gradient structures (heterochromatin euchromatin and nuclear membrane edges) were estimated by a difference-of-boxes filter, and pattern spectrum decomposition was obtained by successive openings and closings performed on the input gray tone image. One important feature of these procedures was that no prior selection by thresholding of the structures to be studied was required, thus obviating subjective bias. Percentages of correctly allocated cells by canonical analysis ranged from 70.0% (rhabdomyosarcoma) to 92.9% (Ewing's sarcoma). Although the five cases could be distinguished using seven texture variables, this does not imply generalization of the results for the differential diagnosis of these tumors. Nonetheless, the possibility that undifferentiated small round cells present distinctive nuclear patterns when studied by sensitive image analysis techniques is suggested by our results. PMID- 7546054 TI - Classifying cervical cells using a recurrent neural network by building basins of attraction. AB - This paper describes the result of classifying cervical cells employing a novel way of using a Hopfield-style neural network for classification. This method could be used as part of an automated cervical screening system. Rather than storing the exemplars (training elements) as stable points, a connection matrix is determined, using perceptron-type learning, such that the exemplars are placed in basins of attraction. The exemplars from different classes of cells are placed in different basins of attraction, with usually more than one basin per training class. A presented element is then classified by the basin of attraction it falls into. Input to the classification consists of one case of feature vectors derived from the cervical cell images; in another case, input consists of the images themselves. Good results were obtained using this technique. PMID- 7546055 TI - A hybrid neural and statistical classifier system for histopathologic grading of prostatic lesions. AB - Neural network and statistical classification methods were applied to derive an objective grading for moderately and poorly differentiated lesions of the prostate, based on characteristics of the nuclear placement patterns. A partly trained multilayer neural network was used as a feature extractor. A hybrid classifier system using a quadratic Bayesian classifier applied to these features allowed grade assignment consensus with visual diagnosis in 96% of fields from a training set of 500 fields and in 77% of 130 fields of a test set. PMID- 7546056 TI - Quantitative estimation of mast cells in Hodgkin's disease and reactive lymphoid/hyperplasia in aspiration cytology smears. PMID- 7546058 TI - Effect of driving speed on reaction time during motorway driving. AB - Effects of driving speed (70, 90 and 110 km/h) on subsidiary auditory reaction time were studied during car driving on a motorway with a speed limit of 110 km/h. Driving distance was held constant at about 200 km. Twenty-four subjects participated in a repeated-measurement design. Reaction time was found to be slower at 70 km/h than at 110 km/h. Before and after the driving session, the subjects' simple reaction time, mood, and alertness were measured in the laboratory. No significant differential after-effects of driving speed were found on any of these measures, although subjects rated themselves as less energetic towards the end of their journey when driving at the former compared to the latter speed. PMID- 7546057 TI - Nonfatal farm injuries in Ontario: a population-based survey. AB - A population-based mail survey of 2,000 farms was conducted to identify rates and patterns of nonfatal agricultural injury in Ontario. Crude, age-adjusted, and stratum-specific rates of farm injury were calculated using data from the returned questionnaires. Patterns of farm injury and its treatment were described by person, place, and time. Response to the survey was 74% (1,364 of 1,842 farms representing 4,110 farm persons). The crude rate of farm injury was 5.8 per 100 persons per year (95% CI: 5.1, 6.5). Common mechanisms of injury included injuries related to the use of farm machinery, overexertion from lifting, accidental falls, and injuries that occurred while working with farm animals. High injury rates were observed in the male 31-40 age group (12.2 per 100 persons per year). Spouses of farm owner-operators (1.7 per 100 persons per year) and their children (2.0 per 100 persons per year) reported lower rates than expected. Most injured persons attributed no factor or "carelessness" as the principle cause of the injury event. Less than 10% of injuries were reported to the provincial workers' compensation board. We conclude that young adult male farmers have the highest rates of injury and warrant targeting by injury control programs. Data from workers' compensation boards have limited utility in the surveillance of most farm injuries in Ontario, in light of their low rate of reporting. The frequency with which inattention or carelessness is attributed to the injury event (as opposed to environmental factors, which might be changed) suggests that injury control programs must address this gap in understanding among farmers, who clearly are vulnerable to traumatic injury. PMID- 7546059 TI - Trends in highway safety: effects of an aging population on accident propensity. AB - Aging of the United States population has a potentially adverse effect on highway safety. A key question is whether the known deterioration of driving skills with aging will be compensated by other factors, especially improved learning and attitudinal experiences of more recent cohorts. We investigate effects of driver age, cohort, and gender on accident propensity and evaluate confounding effects of year, location, and lighting. The accident propensity of different groups of drivers is measured, using a database of two-vehicle accidents, by the ratio of the number of at-fault drivers of a specific group to the corresponding number of not-at-fault drivers. Logistic regression modelling determines the statistical significance of the findings. The analysis reveals the following statistically significant effects: (a) middle-aged drivers are safer than younger drivers who, in turn, are safer than older drivers; (b) female drivers are safer on average than male drivers; (c) younger female drivers are safer than younger male drivers; (d) older male drivers are safer than older female drivers; (e) more recent cohorts of older drivers are safer than more distant cohorts; and (f) more distant cohorts of younger drivers are safer than more recent cohorts. We conclude that driver cohort provides a plausible explanation for many of the measurable, time-related accident trends that are observed. Accordingly, older drivers will be safer in the future because they will have acquired basic driving skills and attitudes in increasingly more automobile-dominated times. At the same time, the cohort effect appears small relative to other time related effects, notably aging, and older drivers will continue to be a high-risk component of the driving population and to require special consideration in driver education and licensing and in highway design and operations. PMID- 7546060 TI - The relationship of age and state license renewal policies to driving licensure rates. AB - We employ regression analysis to examine the relationship of state driver's license renewal requirements, population characteristics, and driving conditions to state driving licensure rates. Licensure rates are examined for the years 1985 through 1989. We separately examine all-age and senior licensed drivers. We find that license renewal requirements as well as population characteristics and driving conditions have systematic relationships to licensure rates. In particular, license renewal tests are associated with lower senior driving licensure rates. PMID- 7546063 TI - The effects of motorcycle helmets upon seeing and hearing. AB - This study assessed the effects of motorcycle helmets upon seeing and hearing by having 50 riders operate over a test route, changing lanes in response to an audible signal under three helmet conditions: none, partial coverage, and full coverage. Half of the subjects were assessed for the degree of head rotation during the lane changes, while the other half were assessed for hearing threshold (decibel level at which they first responded to the signal). Results showed that subjects in the vision study increased the degree of head rotation in proportion to the vision restriction imposed by the helmets, though not to the full extent of the restriction. However, individual differences in head rotation far exceeded the effect of variation in helmets. Subjects in the hearing study evidenced no differences in hearing threshold across the three helmet conditions. The authors conclude that the effects of helmets upon the ability to see and hear are, at most, far too small to compromise the safety benefits offered by head protection. PMID- 7546062 TI - Rollovers on sideslopes and ditches. AB - National data were analyzed to define the nature and importance of the problem of ran-off-road vehicles that rollover on slopes and ditches. Also, data from two specialized data bases were used to obtain insight on vehicle orientation and driver maneuvers in such crashes. This crash type was found to be the leading cause of ran-off-road driver fatalities, accounting for about one-fourth of this total. Highway factors associated with this crash type are identified to assist in the effort to define specific roadway locations where highway design countermeasures may be appropriate. Specifically, attention should be given to the outside of horizontal curves on rural two-lane roads. Vehicle dynamics computer simulations appear to offer the most promising approach to obtain needed insight on rollover risks for specific slope and ditch geometric combinations. Vehicle trajectory information found in this study should be useful in the design and interpretation of needed follow-on simulation studies. Vehicle trajectories in slope rollovers were found to be both different from, and more complex than, fixed object crashes. PMID- 7546061 TI - Personal and behavioral predictors of automobile crash and injury severity. AB - The purpose of this paper is to develop a statistical model explaining the relationships between certain driver characteristics and behaviors, crash severity, and injury severity. Applying techniques of categorical data analysis to comprehensive data on crashes in Hawaii during 1990, we build a structural model relating driver characteristics and behaviors to type of crash and injury severity. The structural model helps to clarify the role of driver characteristics and behaviors in the causal sequence leading to more severe injuries. From the model we estimate the effects of various factors in terms of odds multipliers--that is, how much does each factor increase or decrease the odds of more severe crash types and injuries. We found that driver behaviors of alcohol or drug use and lack of seat belt use greatly increase the odds of more severe crashes and injuries. Driver errors are found to have a small effect, while personal characteristics of age and sex are generally insignificant. We conclude with a discussion of our modeling approach and of the implications of our findings for appropriate traffic safety interventions and future research. PMID- 7546064 TI - Perceived risk and modal choice: risk compensation in transportation systems. AB - A transportation mode choice analysis is performed that examines behavioral responses to perceived risk in the choice of mode for daily commute trips. This methodology provides a technique for examining, by means of disaggregate individual level data, risk-compensating effects in transportation systems. Various measures of perceived risk are examined for explaining modal choice. Other studies have described how safety regulations have resulted in increases in "driving intensity." This study defines one component of driving intensity to be the increased probability of commuting by automobile. The results show that modal shifts occur when risk perceptions for a given mode are reduced. To demonstrate potential risk-compensating effects within the transportation system, an estimate of changes in accident fatalities due to commuting is derived using rough estimates of fatalities per person-mile travelled. It is shown that a given change in the perceived risk of commuting by automobile results in a less than proportionate change in net commuting fatalities. The relative magnitude is dependent on how objective reductions in risk translate into perceived reductions in risk. This study also shows that perceived safety improvements in bicycle transportation have an aggregate elasticity value that is greater than one. This means that bicycle safety improvements attract proportionately more people to bicycle commuting (i.e. a 10% increase in safety results in a greater than 10% increase in the share of people bicycle commuting). PMID- 7546065 TI - The safety value of guardrails and crash cushions: a meta-analysis of evidence from evaluation studies. AB - Evidence from 32 studies that have evaluated the safety effects of median barriers, guardrails along the edge of the road, and crash cushions (impact attenuators) is summarized by means of a meta-analysis. Two hundred and thirty two (232) estimates of safety effects are included in the meta-analysis. The presence of publication bias is tested by means of the funnel graph method. For most subsets of the data, no evidence of publication bias is found. Weighted mean estimates of safety effects are computed by means of the logodds method. Median barriers are found to increase accident rate, but reduce accident severity. Guardrails and crash cushions are found to reduce both accident rate and accident severity. The effects of guardrails and crash cushions on accident rate have been less extensively studied than the effects on accident severity. Current estimates of the effects on accident rate are highly uncertain because of methodological shortcomings of available studies. The effects of guardrails on accident severity are found to be quite robust with respect to study design and the number of confounding variables controlled in each study. In general, random variation in the number of accidents is the most important source of variation in study results. PMID- 7546066 TI - Compatibility considerations for low-mass rigid-belt vehicles. AB - A number of staged impacts performed by our group with the aid of a test device representing a low-mass vehicle (LMV) indicates that a rigid-belt body (RBB) is a valid means for providing adequate occupant safety also for LMVs in the strict sense (curb mass less than 600 kg). The RBB concept raises the problem of compatibility, however. Ideally, the deformability of car front structures should increase with increasing vehicle weight in order to ascertain compatibility. Published data on frontal deformation characteristics substantiate in contrast that conventional cars today exhibit an opposite behaviour. To evaluate the compatibility properties of ultrastiff LMVs, two crash experiments were performed along with a theoretical model analysis. An LMV with a mass of 680 kg (including batteries, 50% mass of two dummies, instrumentation) designed according to the RBB concept and a conventional care of 1320 kg--(equivalent loading conditions as LMV)--were crashed at 56 km/h in a frontal direction against a deformable barrier (FMVSS 214). Furthermore, a mathematical model was based on estimated deformation characteristics of conventional vehicles to predict intrusion distances into the FMVSS barrier in hypothetical frontal crashes with 56 km/h. The results indicate that due to its low mass an LMV does not represent an excessive compatibility problem for other car occupants in spite of the stiff RBB characteristics. PMID- 7546068 TI - Behavioural characteristics and involvement in different types of traffic accident. AB - Recent research has shown risk of road traffic accident involvement to be associated with the tendency to commit driving violations, fast driving, and a lack of thoroughness in decision making. It has also been shown that three broad types of accident can be used to categorise more than 70% of drivers' brief written descriptions of their accidents. These categories are "shunts" (rear-end collisions), right-of-way violations, and loss-of-control accidents. Furthermore, for each accident type the role of the reporting driver may be defined as active (striking) or passive (struck) depending on the position of his/her vehicle. This paper reports two studies examining the extent to which measures of tendency to commit violations (measured by the Driver Behaviour Questionnaire (DBQ)), frequency of fast driving (measured by the Driving Style Questionnaire (DSQ)) and thoroughness in decision making (measured by the Decision Making Questionnaire (DMQ)) are associated specifically with involvement in each of the main accident types. The results showed that a high DBQ-violation score was associated with accidents in general, both active and passive, and specifically with active loss of-control accidents and passive right-of-way accidents. High DSQ-speed and low DMQ-thoroughness were associated with active but not passive accidents, high DSQ speed with active right-of-way violations, and low DMQ-thoroughness was specifically associated with active shunts and active right-of-way violations. The implications of these results are discussed. PMID- 7546067 TI - The nature and source of the head injuries sustained by restrained front-seat car occupants in frontal collisions. AB - The paper examines the types of head injury sustained by restrained front-seat car occupants in frontal collisions. Injuries are classified into soft tissue, diffuse and focal brain injuries and facial bone or skull fractures. Survivors seldom sustain focal injuries although these are common amongst fatalities. The contact sources within the car are described. Intruding structures and high crash severities are typically associated with high rates of the more severe injuries from steering wheel contact, although some are sustained with intrusion below 11 cm. Low-speed impact testing on nondeployed airbag-equipped wheels is suggested. Toughened glass windscreens are overrepresented amongst those sustaining injuries from glazing materials. Test procedures to reduce injuries from pillar contacts should take account of the dynamic effects of an intruding pillar. Contacts with objects outside the car caused higher rates of severe fractures and brain injury; however, the total numbers are greater from interior contacts. PMID- 7546069 TI - Some implications of driver training for road accidents in Gaborone. AB - In many African countries, including Botswana, the driving schools and instruction given to pupils are uncontrolled. Thus, this paper presents the results of an investigation into the level of professional training offered by the driving schools in Gaborone. A total of 400 randomly selected drivers completed questionnaires on their training, accident records, and L-tests. Male trainee drivers received more instruction than females, and there was some indication that the pass rate on L-tests was lower for female drivers. Also, the pickup van forms the majority of the national vehicle population. It accounts for almost 40% of the accidents but is presently not used for training in the commercial driving schools. It is concluded that the quality of professional instruction in driving schools needs improvement so that learners can be encouraged to take training from these institutes. PMID- 7546072 TI - Field effectiveness of two restraint systems: the 3-point manual belt versus the 2-point motorized-shoulder/manual lap belt. AB - The research question addressed in this paper is whether or not occupant death, injury, and ejection outcomes differ between vehicles equipped with 3-point manual belts versus 2-point motorized-shoulder/manual-lap (motorized/manual) belt systems. Census crash data sets for the states of Washington and Texas, and the Fatal Accident Reporting System (FARS) were subset to provide data on front outboard occupants of Ford Escorts involved in crashes in calendar years 1981 1991. Logistic regression analyses showed that occupants of vehicles equipped with the motorized/manual system experienced 11.7% to 26.4% fewer K- or A-level injuries than occupants of vehicles equipped with the 3-point system. Similar analyses of FARS data showed lower ejection rates for occupants of vehicles with the motorized/manual system in both rollover and nonrollover crashes. The installation of the motorized/manual system provided a substantial safety benefit over the manual 3-point system in the time periods examined. PMID- 7546071 TI - Lower extremity fractures in motor vehicle collisions: the role of driver gender and height. AB - In a previous study it was noted that there was a higher incidence of lower extremity fractures among women drivers. Analyses were based on a linkage between trauma registry and police crash report data. The present study addresses the issue of whether the differences noted are attributed to driver gender or are merely a reflection of differences in driver height. An inverse association was noted between driver height and the incidence of lower-extremity fractures. Those shorter than average (5'7") for this population had a 64% increase in lower extremity fracture, which can be mainly attributed to ankle/tarsal injuries. Thus, the incidence of these injuries appears to be a function of driver height, with an increase among shorter drivers, most of whom are women. PMID- 7546070 TI - Emergency department and hospital admissions and deaths from traffic injuries in Barcelona, Spain. A one-year population-based study. AB - The purpose of the study was to assess the one-year incidence of traffic injuries among residents 14 years of age and older, as well as their distribution by age, gender, road-user category, place of occurrence, and severity of the injury. A one-year survey was conducted in the emergency departments in Barcelona, Spain, based on a multistage cluster sampling of 8-hour shifts stratified by time of the year, day of the week, and time of day. All death certificates (E810-829) for residents in this same age group were also included. Information was collected prospectively in EDs by specially trained nonstaff interviewers. The baseline estimate of incidence of injuries was 1,037 per 100,000 population. The highest incidence was for ages 20-24 and 15-20 years. Incidence rank-ordered road-use categories were: motorbike occupants, passenger car occupants and pedestrians, the first category accounting for almost half of all injuries. There were 108 admissions and 15 deaths per 1,000 incident cases. Case-admissions ratios increased with age and were highest for pedestrians, who also accounted for the highest case-fatality ratios and ISS mean scores. The study offers the first description of the epidemiology of traffic injuries in a large southern-European city. Although overall age and gender incidence patterns were similar to those described in other western countries, the striking contribution of motorcycle injuries has not been previously pointed out, underscoring the urgent need to implement effective strategies to reduce injury risk associated with motorcycles and mopeds. PMID- 7546073 TI - Developing policies for automated speed enforcement: a survey of Michigan opinions. AB - This paper describes select results of a pilot test of automated speed enforcement devices (ASEDs) in Michigan. A mail response survey of drivers (1,209 respondents) showed the general public favors use of ASEDs in select situations, particularly in school zones, in areas where traffic enforcement is dangerous for police, for heavy trucks, and in construction zones. The survey also showed opposition to ASED use on freeways, on bridges, and on "all roads." In general, observed speeders and persons who reported having multiple citations in the previous two years were in greater opposition to the use of ASEDs than the general population. PMID- 7546074 TI - Rejoinder to Hemenway. PMID- 7546075 TI - Influence of fatigue in nonfatal nighttime car accidents involving young male drivers. PMID- 7546076 TI - Rejoinder to Mannering. PMID- 7546078 TI - Rejoinder to Field and Clarke. PMID- 7546079 TI - A cost-effective alternative to conventional pediatric pouching. PMID- 7546081 TI - A clinical tool: updated readings on tissue interface pressures. PMID- 7546080 TI - Hypergranulation tissue: a nontraumatic method of management. AB - Granulation tissue above the periwound area is usually considered an impediment to wound healing. Although there is very little in the literature regarding hypergranulation tissue, the fact that there are numerous treatments by various wound clinicians demonstrates the recognition of its presence as a clinical problem. The paucity of published information prompted the authors to design a study to collect objective data on a treatment method they had found useful in their practices. This article explores the issue of hypergranulation and offers a nontraumatic method of management. A prospective non-controlled correlational study was undertaken with ten patients and twelve wounds using a polyurethane foam dressing to reduce hypergranulation tissue. The results demonstrated a significant decrease in height of 2 mm of granulation tissue from initial measurements to measurements taken two weeks later (p < 0.01). PMID- 7546082 TI - Case studies: considering dressing options. PMID- 7546085 TI - Healthcare reform--"do no harm". PMID- 7546083 TI - Bladder elimination options: an addendum. PMID- 7546084 TI - "Single payer" health system. PMID- 7546077 TI - The need for emergency department data in highway safety management systems. PMID- 7546086 TI - Case study: Harlequin fetus, a disease of cornification. AB - The Ichthyosis are a family of genetic skin diseases characterized by dry, thickened, scaling skin. Harlequin Fetus is the most rare and severe form of congenital ichthyosis. It is inherited in an autosomal recessive pattern. In this particular case study a Harlequin Fetus, who is now an 8 year old child, is described from birth to present. His skin care is detailed for others to understand that the diseases of cornification can be managed successfully with meticulous and consistent care. PMID- 7546087 TI - Conducting a group research project: learning from our mistakes. PMID- 7546088 TI - Patient education: patient willingness vs. nursing skill. PMID- 7546089 TI - In this era of managed care and reform, many uncertainties remain about how to best care for patients. PMID- 7546090 TI - Evaluation of two wound measurement methods in a multi-center, controlled study. AB - Methods for measuring wound size and healing have ranged from simple measurement with a ruler to sophisticated automated image analysis. As part of a multicenter, double-blind evaluation of a growth factor for wound healing, we evaluated the predictability and accuracy of two measurement systems. Four hundred and fifty paired comparisons (900 observation points) of lower extremity ulcers of either diabetic or venous stasis origin were evaluated weekly for at least four weeks. Wound size was determined by computer digitization of either color slide photographs (photo method) or acetate tracings (tracing method). Measurements of wound surface area for both methods were very similar, with a correlation coefficient of 0.97. The standard deviation of the methods, stratified by wound size and study center, were low (10 percent to 20 percent). Inter-site variability accounted for only 5.4 percent of the total variability noted in these observations. We have found that both the photo method and the tracing method may be useful in large, multi-center clinical trials when measurements of wound size are utilized to evaluate responsiveness to therapy. PMID- 7546091 TI - Methods of measuring wound size: a comparative study. AB - The lack of an accepted method of accurate and objective measurement of wound dimensions is a major obstacle to the assessment of effective wound management regimes. This study compares three different wound volume measurement techniques (filling the lesion with saline, molding of a dental impression material and a computer vision method based on image processing and the Structured Light technique) in terms of accuracy, precision and practicability in a clinical environment. Three groups of hospital staff, doctors, nurses and technicians, repeatedly measured a set of 6 different models of wounds. Measuring wound volume by filling it with saline produces results with standard deviations between 9 percent and 18 percent of the actual volume. Dental impression material performs better, between 5 percent and 16 percent, but is difficult to apply and time consuming to use. Apart from the advantage of providing instant optical records of wounds, the image processing method produces more reliable volume measurements with a standard deviation of between 3 percent and 15 percent. The results demonstrate that the computer based method yields the most reproducible results with a minimum of inter-observer error but the method is not applicable for undermined, very deep and very large wounds. PMID- 7546092 TI - The attempt to replace the hands of caring for patients with therapeutic medical technology--a disturbing trend. PMID- 7546093 TI - OBRA regulations for wound and skin care in a skilled care facility: review how you care for your residents. PMID- 7546095 TI - DMERC update: support surface policy released for comment. PMID- 7546094 TI - Infection control in the hospital: the role played by cross-contamination and the ICU. PMID- 7546096 TI - The legal view of healthcare reform: liability in the era of finite resources. PMID- 7546097 TI - Wound healing assessment: the clinical utility of wound healing rates. PMID- 7546098 TI - Clinical study: assessing the performance and skin environments of two reusable underpads. AB - The authors wanted to compare the performance and skin environment provided by a recently introduced reusable underpad (Test Group underpad) to an underpad already well established in the absorbent products market (Reference Group underpad). A controlled, prospective, randomized multi-centered clinical trial was conducted in 18 facilities (6 hospitals, 7 home health agencies, and 5 skilled nursing facilities) and completed by 107 patients. The majority of the patients had a Braden score near 13, were 70 to 80 years of age, and weighed 130 to 138 lbs. The Test Group underpad ranked more favorably overall and in all eight categories of daily assessment. These differences were statistically significant (p < 0.05) in the Total Score and in four of the eight individual categories: Keeping Skin Dry, Keeping Clothing Dry, No Pooling of Fluid, and Patient Comfort. The Test Group underpad also ranked more favorably with statistical significance (p < 0.05) in three of the assessment categories at the conclusion of the study: Pad Absorbency, Pad's Ability to Wick Moisture, and Pad's Ability to Keep Skin Dry. Investigators also noted that 57 patients (53%) exhibited some measure of improvement by the end of the study. Of these, 45 (79%) had been assigned to the Test Group. The Test Group underpad demonstrated better absorbency, kept patients' skin, clothing and bed linens drier, exhibited less pooling of fluid and resulted in higher patient comfort. As a result, it may have contributed to a more beneficial skin environment, allowing for prevention of preexisting skin integrity problems and enhancement of healing. PMID- 7546099 TI - Predictors of time to healing deep pressure ulcers. AB - Time to healing analysis methods (Kaplan-Meier time until healing curves) were used to compare time to healing deep pressure ulcers as a function of patient and wound characteristics at baseline and after two weeks of treatment. Time to healing was significantly reduced in patients who had a good nutritional status. Patients who were alert and coherent were also found to heal more expediently; however, mental status was not independently predictive of time to healing in the multivariable model. Larger wounds took longer to heal (median 20 days) than smaller wounds, but the difference was not statistically significant. After two weeks of treatment, ulcers in patients who were 60 to 70 years old, who had a good nutritional status at baseline and whose ulcers reduced at least 39 percent in size after two weeks, were found to heal much more expediently. Cox regression models showed that these factors were independently predictive of time until healing (Likelihood ratio statistic on 5 DF = 26.485, p < 0.001). Clinical assessments, both at baseline and regular intervals thereafter, may predict treatment outcome of full-thickness pressure ulcers. PMID- 7546100 TI - The role of the pedorthist in the prevention and management of diabetic foot ulcers. PMID- 7546101 TI - Conflicting points of view regarding the use of povidone-iodine. PMID- 7546102 TI - "The perils of povidone-iodine use". PMID- 7546103 TI - Quantifying wound fluids for the clinician and researcher. PMID- 7546104 TI - Restraint free environment for the elderly. PMID- 7546105 TI - A method of catheter change for patients with a spastic urinary sphincter. PMID- 7546106 TI - Diapers and underpads, Part 1: Skin integrity outcomes. PMID- 7546108 TI - Traumatic brain injury, pregnancy, and related management: a case study. PMID- 7546107 TI - Diapers and underpads, Part 2: Cost outcomes. AB - A randomized clinical trial was conducted at three acute care facilities between August and December 1993 to compare cost outcomes between patients managed with diapers or with underpads. The 166 patients were divided into five groups: polymer or nonpolymer underpads, polymer or nonpolymer diapers, and cloth underpads. Among other results, the average number of nonpolymer underpads used (3.4) compared to other products used (1.4 polymer underpads, 1.1 cloth underpads, 1.0 nonpolymer diapers, 1.0 polymer diapers) quantified nurses' perceptions that nonpolymer pads were not as effective as their polymer counterparts in absorbing incontinence. The nonpolymer underpad was, however, the least expensive of the products studied, even taking into account the volume of product used. The nonpolymer underpad group also took the most time for cleanup. The cloth underpad group consistently used all of the linen items, suggesting that complete bed changes were necessary. The polymer underpad group consistently used the least linen, suggesting that the incontinence episodes were successfully contained by the product. When all costs were combined for time (at $13.50 hourly aide wage), linen use and product type, there was a statistically significant difference between the five treatment groups (F 9.3, p < 0.0000). Total costs (product, linen and staff time) for diaper products were higher than either the polymer or cloth underpad groups. Recommendations, based on both parts 1 and 2 of this study are that the polymer diaper is the product of choice for the ambulatory patient, and the polymer underpad is the product of choice for the bedridden patient. The nonpolymer underpad should no longer have a place in incontinence management. PMID- 7546109 TI - Cranberry juice and its impact on peri-stomal skin conditions for urostomy patients. AB - In urostomy patients, peristomal skin problems are common and may stem from alkaline urine. Cranberry juice appears to acidify urine and has bacteriostatic properties, and is widely recommended for the reduction of urinary tract infections. Therefore, it is hypothesized that drinking cranberry juice might also prevent and/or improve skin complications for urostomy patients. To test this hypothesis, pH measurements of the skin around the stoma and of the urine of 13 urostomy patients were taken before and after instituting a regimen of drinking 160 to 320 g of cranberry juice each day for an average period of six months. Results showed an improvement in skin condition from 6 patients with erythema, maceration or pseudoepithelial hyperplasia at the beginning of the study to 2 patients with maceration or PEH. The average pH of the urine taken from the patients' pouches decreased a statistically significant amount from 8.0 to 7.3 (p = 0.0277), yet unexpectantly, the average pH of the fresh urine increased a statistically significant amount from 5.8 to 6.2 (p = 0.0178). Other results were not statistically significant. The authors conclude that while drinking cranberry juice did not appear to acidify the urine as expected, improvements were still seen in the skin conditions of the study participants, suggesting that drinking cranberry juice does positively impact the incidence of skin complications for these patients. PMID- 7546111 TI - Research & writing basics: elements of the abstract. AB - Writing an abstract is a challenging skill that requires precision and care. Criteria for well-formulated abstracts and abstract guidelines for 2 types of articles (empirical studies and reviews or theoretical articles) as well as a description of the content of a structured abstract are presented. Details were gleaned from a review of the literature including the American Medical Association Manual of Style, Eighth Edition and the Publication Manual of the American Psychological Association, Fourth Edition. A good abstract is like a crystal: it is a clear, sharp synthesis that elucidates meaning for the reader. PMID- 7546110 TI - A clinician's guide to common dermatology problems. PMID- 7546112 TI - The chronic wound pain experience: a conceptual model. AB - Chronic wound pain is experienced to a greater or lesser degree with most types of chronic wounds, yet research to date on this subject has been extremely limited. This article presents an empirically and inductively derived model, including a theoretical definition of the chronic wound pain experience (CWPE) and its subcomponents (noncyclic acute wound pain, cyclic acute wound pain and chronic wound pain). The model relates the CWPE to the nursing process and to nursing diagnoses and proposes intervention schemes to optimize patient outcomes. Avenues for future research are delineated. PMID- 7546113 TI - Assessing clinical efficacy of a hydrocolloid/alginate dressing on full-thickness pressure ulcers. AB - An absorbent hydrocolloid/alginate spiral dressing and a hydrocolloid secondary dressing were used in the management of 30 patients with 30 exuding State III and IV pressure ulcers. After a mean treatment time of 12.9 days (SD 6.5), all wounds had a significant increase in the amount of granulation tissue/epithelium and a decrease in the amount of devitalized tissue (p < 0.05). Wounds that underwent wide surgical debridement prior to the study were covered with 15 percent fibrin slough at study entry versus 39 percent for non-debrided wounds (p < 0.05). The dressing combination facilitated wound contraction and removal of fibrin slough in ulcers that were surgically debrided prior to the study. Ulcers which had not been surgically debrided expanded as autolytic debridement reduced the amount of fibrin slough/necrotic tissue present at the wound bed (Mean: 17.6 percent, p < 0.05). The absorbent spiral dressing helped manage exudate, was easy to use and comfortable for the patients. The average time between dressing changes in these exuding wounds was 1.56 days (SD = 0.95). Use of air-fluidized bed or mattress was found to significantly reduce wear time of the dressing (p < 0.01). Further studies are needed to confirm short-term, and evaluate long-term effects of this dressing combination on healing and debridement. PMID- 7546114 TI - The effects of non-thermal pulsed electromagnetic energy on wound healing of pressure ulcers in spinal cord-injured patients: a randomized, double-blind study. AB - The objective of this randomized, double-blind study was to determine if non thermal pulsed electromagnetic energy treatment significantly increases the healing rate of pressure ulcers in patients with spinal cord injuries. Subjects included volunteers admitted to a Veteran's Administration Hospital in New York over a 2 year period and consisted of 30 male spinal cord-injured patients, 20 with Stage II and 10 with Stage III pressure ulcers. Subjects were given non thermal pulsed high-frequency electromagnetic energy treatment for 30 minutes twice daily for 12 weeks or until healed. The percentage of pressure ulcers healed was measured at one week. Of the 20 patients with Stage II pressure ulcers, the active group had a significantly increased rate of healing with a greater percentage of the ulcer healed at one week than the control group. After controlling for the baseline status of the pressure ulcer, active treatment was independently associated with a significantly shorter median time to complete healing of the ulcer. Stage III pressure ulcers healed faster in the treatment group but the sample size was limited. For spinal cord-injured men with Stage II pressure ulcers, active non-thermal pulsed electromagnetic energy treatment significantly improved healing. PMID- 7546115 TI - Fluid handling properties of hydrogel dressings. AB - The fluid handling properties of four hydrogel dressings were examined in a laboratory-based study to determine their ability to absorb liquid from or donate liquid to a range of standard test substrates simulating wound tissue in various states of hydration. The tests suggest that three of the dressings are likely to be effective fluid-donating agents, and one of the dressings has a dual ability to donate or absorb liquid depending upon moisture content and nature of the substrate to which it is applied. These observations are discussed in the context of the clinical use of hydrogel dressings. PMID- 7546117 TI - Obtaining informed consent. PMID- 7546118 TI - Venous stasis ulcers: a review. AB - Venous stasis ulcers are one of the most common and perhaps the most costly of the vascular disorders. The etiology of venous ulcer formation has not been fully described; however, venous obstruction or valvular incompetence is almost always present. Manifestations of chronic venous insufficiency include mild to severe swelling, aching to frank leg pain, leg heaviness, dilated superficial veins and/or dilated tributaries of the saphenous vein system, skin changes and ultimately ulceration. The need for diagnostic testing may be minimal if the ulcer typifies the usual venous ulcer in persons recognized to be at high risk. Management includes both non-surgical and surgical management, wound debridement and electrical stimulation. Early management of venous stasis ulcers will improve outcomes. However, even with successful initial wound management, the recurrence rate approaches 70 percent. Much of this high recurrence rate can be related to the patient's inability to change life style and incorporate elastic bandage regimens and to poor tissue quality at the ulcer site. The management of venous ulcers should be regarded as a lifetime process. PMID- 7546116 TI - Evaluation of three nonwoven sponges in the debridement of chronic wounds. AB - A total of 15 evaluable patients were entered into a non-randomized evaluation with a primary goal to determine the efficacy of three approved nonwoven sponges, an 8 x 8 mesh with 64 apertures per square inch, a 13 x 13 mesh with 169 apertures per square inch, and a 19 x 8 mesh with 152 apertures per square inch, in the debridement of necrotic and fibrotic tissue. Patients were assigned to one of three sponge categories and then followed on a twice-a-week basis for four weeks. At each visit, the wound status was evaluated for the presence of necrotic and fibrotic tissue in the wound. Wounds were cleansed with sterile saline solution, and a saline moistened nonwoven fluffed sponge was applied to the wound surface and covered with a secondary wound dressing. Dressings were changed twice a day. The results suggest that all products were effective in debridement of nonviable tissue, but the best overall results were obtained with the 8 x 8 mesh product which more effectively debrided the wounds than the nonwoven sponges with the smaller apertures. The study results suggest that open mesh 100 percent cotton nonwoven sponges are effective in debriding nonviable tissue with minimal damage to viable tissue when appropriately used and not allowed to desiccate. PMID- 7546121 TI - Vignette: eczema, ichthyosis & psoriasis. PMID- 7546120 TI - Treatment of leg ulcers with cultured epithelial autografts: clinical study and case reports. AB - Leg ulcers are a significant medical problem in the United States. The number of affected patients at any given time is staggering. Treatment modalities have widely varied and are often unsuccessful. We evaluated the effectiveness of cultured epithelial autografts in the treatment of leg ulcers due to various etiologies that had failed other standard therapy. Eighty-six ulcers in 36 patients were treated with cultured epithelial autografts after full evaluation of their ulcers. The ulcers were grouped by etiology and healing was assessed by time to healing, number of ulcers healed and mean number of graft applications. The results achieved in the past five years have shown that cultured epithelial autografts are highly effective in treating chronic leg ulcers. Eighty percent of venous insufficiency ulcers healed in 5.7 weeks. Similar results were seen with ulcers of other etiologies including arterial insufficiency, pyoderma gangrenosum, scleroderma, and vasculitis. Positive results were seen in cases where other treatment methods had failed and seems independent of systemic corticosteroids. PMID- 7546123 TI - Can prevention eliminate caries? AB - There are four main factors involved in the carious process: at-risk tooth structure, plaque flora, fermentable carbohydrates, and time. Based on our knowledge of the carious process, four main preventive strategies have been developed over the years, namely, fluorides, fissure sealing, dietary choice, and plaque control. Fluorides are having a major impact on smooth-surface caries; hence, strategies combining fluorides and fissure sealing are very effective. However, use of fissure sealing is still problematic. Changing dietary practices with a view to reducing dental caries seems to be having little impact on a global scale. Plaque control, as practiced routinely by the majority of people, is not sufficient to result in caries reductions. Deprivation and poverty are strongly associated with high caries levels. Although the preventive strategies currently available are likely to result in lower caries levels for many, for logistical reasons and because of factors associated with deprivation and poverty, caries is likely to remain a major public health problem in most communities for the foreseeable future. PMID- 7546119 TI - Pressure and shear: their effects on support surface choice. AB - Many support surfaces are available, each with advantages and disadvantages, that claim to relieve or reduce the external forces contributing to the development of pressure ulcers. This article describes pressure and shear, and what a patient support surface must do to relieve those forces. Pressure is the vertical force of the weight of the patient's body on the surface. Shear is a horizontal force that causes the bony prominence to move across the tissue as the skin is held in place, and results from patient movement, nurse movement of the patient, and bed movement. There are seven basic requirements a support surface must meet in order to prevent pressure and shear: The surface must (1) conform to bony prominences without resistance, (2) not have significant memory, (3) allow patient immersion, (4) not bottom out, (5) relieve shear caused by patient movement, (6) prevent skin maceration, and (7) provide patient comfort. Six types of support surfaces are listed along with a summary of each surface's theoretical ability to deal with the forces described in this article. Understanding the physical forces that contribute to pressure ulcers, and each support surface's theoretical ability to relieve these forces, should make it easier to choose an appropriate support surface for each patient. PMID- 7546122 TI - Teamwork in prevention: possibilities and barriers to integrating oral health into general health. AB - While the term "teamwork" has been used in preventive dentistry for decades, little work has been done in the area of integrating oral health into general health via participation in primary health care or interdisciplinary teams. Oral health professionals must be prepared at the preprofessional and professional levels with the knowledge and skills required to become effective members and leaders of collaborative teams. This paper describes guiding principles for successful team development that are derived from the experience of other disciplines. As oral health professionals take a more active part in interdisciplinary collaborative teams, they must be prepared for roles beyond that of clinician. Five additional roles and examples of their importance to the prevention of oral disease and conditions are discussed. PMID- 7546124 TI - Objectives and review of the international milk fluoridation program. PMID- 7546125 TI - Feasibility of milk fluoridation and trends in dental caries of children in China. AB - The aim of the study is to investigate the feasibility of milk fluoridation as a vehicle for caries prevention, based on the current epidemiological status and its trends of dental caries in preschool children in the area of the Haidian District of Beijing, where the indications exist: Fluoride level in drinking water is low (0.2-0.3 ppm), and water fluoridation and other systemic uses of fluorides are unlikely. From 75.9 to 79.7% of 3-6-year-old children were identified as having high caries experience (dmft 4.29-4.35). It was found that 60% of the preschool children who attended kindergarten were 3-6 years old, and 40% of the 1-2-year-olds were living with their families. There is a local dairy to produce milk for the population living in this district. The amount of fresh milk produced daily is about 50,000-60,000 kg. A special kind of milk with vitamins A and D is also available for the preschool children. Data available from the Community MCH Centre indicated that the percentage of breast-feeding is 12% only. The first choice by parents for artificial feeding to the babies is fresh or powdered milk. Therefore, the breast-feeding project started in 1992. It is recommended that mothers' milk should be provided to the babies for at least four months after birth, when a specially prescribed milk for the babies, produced by the dairy, will be provided on a daily basis. The cost of milk is cheaper than others. In this connection, a five-year project on milk fluoridation as a pilot study at the community level for caries prevention of preschool children is now planned. The project is supported by BDMF, WHO, and Chinese MOPH. PMID- 7546126 TI - The effect of fluoridated milk on bovine dental enamel. AB - The concept of intra-oral cariogenicity tests, using naturally accumulated plaque, was pioneered by Koulourides (Koulourides et al., 1974). Tests rely on changes in microhardness of dental enamel after exposure to substrates. We used this model, with significant modifications, to determine the possible caries protective effect of fluoridated milk. Custom-made cast chrome intra-oral appliances were made to fit the lower arches of volunteers. Four removable, highly polished 3 x 4 mm gauze-covered bovine enamel blocks were slotted into the appliances. These were worn for 48 h so that plaque would build up. The enamel was color-coded with composite (Kerr Kolors, Kerr, Romulus, MI) to ensure error free removal and immersion extra-orally in the coded test substrates. PMID- 7546127 TI - Some operational aspects of school-milk fluoridation in St. Helens, Merseyside, UK. AB - St. Helens is a small industrial town situated about 20 km east of Liverpool. It lies in an area of social deprivation and, by UK standards, dental caries experience is high (e.g., dmft at 5 years = 2.8; DMFT at 12 years = 2.7). Water fluoridation is an important part of the government's strategy for improving oral health in such areas; however, in large parts of St. Helens, implementation of water fluoridation is complicated by reason of the multiple sources of water supply. The aims of the St. Helens study are therefore to examine the technical, organizational, and legal aspects of the fluoridation of school milk as an alternative public health approach. In the UK, children attending nursery units (kindergartens) from ages 2-4 years and infant schools from ages 4-7 years are eligible for 189 mL of milk to be consumed each day at school. These two schemes are funded or subsidized by the Departments of Health (UK) or the European Community, respectively. A preliminary review of the possibility of using school milk as a vehicle for fluoride has been published recently (Jones et al., 1992). The current paper will review progress over the past 12 months, including the response of schools, dairies, and other organizational considerations. PMID- 7546128 TI - Caries reduction by milk fluoridation in Bulgaria. AB - The aim of the present study has been to investigate the caries-reducing effect of a community-based milk fluoridation project. In those Kindergartens where fluoridated milk had been delivered on a regular basis, the estimated consumption corresponded to a daily intake of 200 mL milk containing 1 mg F- (5 ppm F-) for periods of 180-200 days per year. A total of 204 six-year-old children, randomly selected, was examined at baseline and three and a half years after the start of the project. A statistically significant reduction in the prevalence of dental caries was obtained for those children who had received fluoridated milk on a regular basis. In six-year-old children, the reduction in the dmft figure was 40% and in the DMFT figure, 90%. Despite the deficiencies in the design of this study (dictated by local circumstances), the results confirm the findings of other studies on the caries-reducing effect of fluoridated milk. The results also confirm that the earlier in the child's life the consumption of fluoridated milk starts, the better the effect. PMID- 7546130 TI - Saliva stimulants and the oral health of geriatric patients. AB - Root-surface caries (RSC) has been recognized as a specific and important dental disease. Significant advances have been made in the pathology and microbiology of RSC, and the need to standardize the guidelines for recording RSC data has been recognized. Researchers have emphasized the increasing impact RSC will have on the geriatric population, especially since the methods to treat and prevent this disease are limited. The purpose of this study was to investigate the possibility of limiting RSC in a Veterans Administration (VA) patient population, using polyol-containing saliva stimulants that were voluntarily consumed by residents of a VA Medical Center (VAMC) over a period of from six to 30 months. Another aim was to study the effect of this program on the gingival health of periodontal patients. PMID- 7546131 TI - The value of anti-caries and anti-plaque dentifrices at a community level. AB - Although dentifrices (in a modern sense) were first introduced at the turn of the century, it was not until the advent of fluoride-containing compounds that any of therapeutic value was produced. In the early post-war years, formulation compatibility difficulties occurred, with the result that none was demonstrated to have clinically proven efficacy until stannous fluoride was successfully incorporated in the "Crest" formulation of 1955. Thereafter, sodium monofluorophosphate (SMFP) appeared, to be followed by organic and acid-phosphate fluoride preparations, most of which were shown to produce clinical benefit over placebo controls. Since the late 1970's, however, such studies have been deemed unethical in many countries in view of the almost universal availability of efficacious fluoride dentifrices. While an NaF formulation was the first to be tested (albeit unsuccessfully) by Bibby (1945), there was a considerable time period before the arrival of both stable and effective silica-based systems in the early 1980's. Results of studies have varied, but the largest ever double blind NaF/SMFP head-to-head dentifrice trial yet published (Stephen et al., 1994) has indicated a 6.4% significant benefit in favor of NaF (in silica) over SMFP. With respect to the effect of dentifrice fluoride levels, above 1000 ppm dose response caries reductions have been obtained, but the efficacy of preparations containing 500 ppm F or less has yet to be demonstrated. In relation to root caries, Jensen and Kohout (1988) reported a 41.4% DFS significant reduction in subjects who used a NaF/silica dentifrice at 1100 ppm F, over a one-year period. PMID- 7546129 TI - The effects of non-fluoridated and fluoridated milk on experimental caries in rats. AB - The aim of the present investigation was to determine the caries-protective potential of non-fluoridated and fluoridated milk and to compare the efficacy of different compounds of fluoride as additives to milk. OM rats were maintained in three experiments in a frequency-controlled feeding machine of Konig et al. (1968) or, during one study, in metabolic cages with diet MIT 200 for three weeks. They received (1) milk with Ca-Fluoride [solubilized by KA1-Sulfate], (2) NaF, (3) NaMFP, and (4) Na-Silicofluoride. Controls were supplied with water or NaF solution of the same concentration of 10 or 15 ppm F. In addition, raw milk was provided ad libitum for the rats in a preliminary test. At the beginning and the end of the experiments, the pH of milk, its fluoride concentration, the body weight gain, the caries score, the fluoride concentration of the outermost enamel surface, the percentage of the interproximal bacteria, and the fluoride ingestion and excretion were determined. The raw milk significantly reduced the animal caries score by around 40%. This effect was lower but reproducible under programmed feeding with milk of a reduced fatty content (1.5%). The addition of Ca-Fluoride, which was not totally ionized (6.5 ppm F), reduced the caries score again by around 40%. Increasing concentrations of NaF (5, 10, 15 ppm F), Na Silicofluoride, or NaMFP showed similar caries-inhibiting effects without remarkable influence of the fluoride dosage used. The percentage of streptococci ranged from 30 to 60 in the fluoridated milk and control groups as well. The increasing fluoride deposition in the enamel reflected the various fluoride dosages offered. The rats receiving non-fluoridated milk or distilled water had a significantly higher incidence of dental caries than those receiving fluoridated milk. The permanent availability of fluoride during the animal tests caused a higher caries-inhibiting effect than in clinical human studies reported. PMID- 7546132 TI - Tooth-friendly sweets--a reliable alternative. PMID- 7546133 TI - Evaluating the social impact and effectiveness of four-year "Love Teeth Day" campaign in China. AB - A massive campaign on "Love Teeth Day" (LTD) has been celebrated nationwide each year in China since 1989. As announced in an official document, nine government and non-government organizations have jointly issued a circular designating that the 20th of September of each year be kept for the Love Teeth Festival in this country. The main activities were planned and conducted by the National Committee for Oral Health, which was set up in 1988. It aims to motivate the people's awareness of dental self-care, participation, and to promote community involvement in oral health education programs. For feedback, two types of questionnaires were designed and sent to the public and the organizers, respectively, after each campaign, and then returned to the office for data processing. The findings from a four-year study indicated that: (1) The activities started from three municipalities, 29 capitals of provinces, and some large cities (1989), and spread to most cities in the urban area, and about 300 counties in the rural area (1992); (2) 14,000 dental professionals and health workers participated in 1989 as information providers, and increased to 40,000 in 1992; (3) oral health knowledge has increased to 76.2% (1992) from 37% (1989); and (4) the people's will in dental prevention was strengthened. PMID- 7546134 TI - Early diagnosis and prevention of oral cancer and precancer: report of Symposium III. AB - Oral precancer encompasses several conditions and lesions. Among those entities included in the concept are leukoplakia, erythroplakia, lichen planus, and submucous fibrosis. For prevention, knowledge about etiologic and pathogenetic factors is imperative. It is well-known that excessive consumption of tobacco and alcohol has a bearing on the development of oral leukoplakia and probably also of erythroplakia. However, among leukoplakias, the idiopathic or cryptogenic type probably shows the most serious malignant potential. Involved in the development of such lesions may be general nutritional aspects, e.g., proper utilization of vitamin A. It is also well-known that iron deficiency has been linked to Plummer Vinson's syndrome, showing a precancerous trait. Among factors involved in the pathogenesis of lichen planus is probably mental stress. Thus, stress factors and related neurological components have been linked to the immunological system. Lifestyle factors, such as nutrition, tobacco, and alcohol, and also mental environment may be of importance for the development of oral precancer and cancer. PMID- 7546135 TI - Salt fluoridation and general health. AB - Salt fluoridation is a systemic form of fluoride supplementation, leaving it to the consumer whether he wants fluoride supplements or not, but thereafter not requiring special dependability for daily compliance. Most German drinking water has low fluoride concentrations. The estimated fluoride intake in German children is between 100 and 300 micrograms/day, and in adults, between 400 and 600 micrograms/day. Male subjects have higher mean intakes than females. From 70 to 90% of the salt intake of 10 to 13.5 g/day in German adults comes from commercially prepared foods. This leaves about 1 to 4 g of salt to be added as table salt at the individual level and to become the source of supplementary fluoride. To increase fluoride intake by at least 500 micrograms/d, and to prevent an additional intake of more than 3000 micrograms/day, it may be necessary to have salt at a fluoride level of around 500 micrograms/g or to include one commercial food to be prepared with fluoridated salt, e.g., bread. A salt fluoride concentration of 250 micrograms/g does not present a risk of dental fluorosis. However, clear recommendations about systemic fluoride supplementation must be given as long as there are fluoride tablets, fluoride-rich mineral waters, and fluoridated table salt available simultaneously. Persons at risk for hypertension from salt consumption require different means of fluoride supplementation. By and large, in areas of low drinking water fluoride, fluoridated table salt has the potential to become a means of systemic supplementation comparable with drinking water fluoridation. PMID- 7546136 TI - Fluoridated salt in France. AB - Since November 28, 1985, fluoridated salt at a concentration of 250 mg KF per Kg has been authorized in food in France. It was introduced in the market at the end of 1986 and represented, in 1993, more than 60% of the market of salt. The effect of this Public Health measure will be shown. The efficiency is evaluated in epidemiological studies concerning DMF in the whole of France and in terms of safety by measurement of urinary excretion of fluoride in children who consume potassium fluoride in the salt and fluoride tablets. Last, the prevention policy in France will be described, including the use of fluoridated salt. PMID- 7546137 TI - Global aspects of HIV pandemic. AB - Twice a year, the World Health Organization publishes reports on AIDS from member countries. At the most recent publication, 190 countries reported that they had no AIDS cases. The other countries reported 611,589 AIDS cases. The introduction will give a brief account of the HIV epidemiology, pointing out the areas where a rapid worsening is taking place. PMID- 7546139 TI - Trends in prevention--promotion of oral health within general health care... Possibilities and limitations in preventive dentistry. Proceedings of the 4th World Congress on Preventive Dentistry. Umea, Sweden, September 3-5, 1993. PMID- 7546138 TI - Periodontal problems related to HIV-1 infection. AB - The prevalence of periodontal diseases in HIV-infected infected persons is unresolved. While numerous reports have been published, the data are conflicting in part due to different populations studied, lack of consensus criteria for disease, study location, and biased samples. This presentation will be a collation of information available for the diagnosis and treatment of HIV/AIDS associated periodontal diseases. The use of "HIV" is no longer accepted as a diagnostic designation. Instead, the diagnostic categories of atypical gingivitis (erythematous gingival banding), necrotizing gingivitis, necrotizing periodontitis, and necrotizing stomatitis and distinguishing characteristics will be presented. It is essential that a distinction be made between those periodontal lesions that may occur in seropositive and seronegative individuals and those which appear to have more specific signs and symptoms associated with HIV infection and with immunosuppression in general. A simplified algorithm has been developed to help differentiate between periodontal diseases specific to the HIV-positive individual and those in the general population. Additionally, the grid may also be used to distinguish the different periodontal diseases known to be associated with HIV infection. PMID- 7546140 TI - Are current models for preventive programs sufficient for the needs of tomorrow? AB - We can be proud of our accomplishment in the prevention of dental caries. Many children in developed countries are now caries-free; however, by age 50, fewer than 5% of the population is caries-free. The drop in the prevalence of caries among specific age groups is undoubtedly due to widespread exposure to fluorides. However, caries protection by fluorides is not absolute; if caries attack continues throughout life, the prevalence of caries will increase. All available evidence strongly suggests that ambient levels of fluoride in the mouth play a critical role in protection against caries. It is clear that novel methods of delivery, such as sustained-release technology, will be developed to enhance the protective effect of fluoride, utilizing amounts of fluoride significantly less than those currently in use. The problems of preventing dental caries in developing countries are much more severe than those facing Western countries. The prevalence of caries appears to be increasing in no small measure due to the introduction of Western diets. Administration of fluoride through conventional routes is either impractical or inordinately expensive. It appears sensible, therefore, to direct preventive measures through dietary constituents associated with caries. Fluoridation of sugars appears to be highly practical and safe way to reduce the prevalence of caries in such communities. Fluoride and iodide are currently added to salt in many parts of the world. If we are to reduce the prevalence of caries effectively in all countries and in all age groups, current methods of prevention will have to be greatly enhanced, and/or effective additional approaches will need to be developed. PMID- 7546141 TI - Strategies in the design of preventive programs. AB - Dental caries is mostly recorded at the cavity level only. A reduced mean number of cavities in new age cohorts is often thought of as a result of prevention of the disease, dental caries. However, what is measured is rather our success in controlling the disease in such a way that prevalence of its more severe manifestations (cavities) can be reduced in children. Caries lesions and periodontal breakdown are cumulative with age and progress steadily in all populations. Thus, caries is the predominant reason for tooth loss in almost all age groups. The low prevalence and skewed distribution of dental caries make several fluoride programs less (if at all) cost-effective. Rather than considering a "whole population strategy" as opposed to a "high-risk strategy", it is argued that they should go hand in hand. However, the high-risk strategy may appear to have an unfavorable ratio of benefits to costs. So far the available literature shows no evidence that we have tests which, with sufficient predictive power, can identify groups or individuals of "high risk". It is therefore concluded that a population strategy should be maintained and further developed with emphasis on oral hygiene, because it influences norms and behavior. More knowledge about the pathogenesis of oral disease is needed before we can develop truly cost-effective strategies for the prevention of caries and periodontal breakdown. PMID- 7546142 TI - Quality assurance in prevention. AB - The successful prevention of caries in Sweden, as in many other countries, is an example of what might he obtained when a systematic and complete program is adopted. Fluoride programs, mechanical cleaning of the teeth, and improved nutrition and eating habits have been the most important fundamentals for the positive results. A diminished use of tobacco, with a reduced frequency of periodontitis as one of the consequences, has been successful in some groups in the population but less so in others. There is, however, evidence now in Sweden of a less active preventive approach concerning caries prophylaxis among some parents today than a generation ago. Thus, there are obvious reasons to continue with oral preventive programs, preferably integrated with general health programs. For acceptable quality in preventive programs to be obtained, concrete goals must be formulated. These should be based upon scientific findings. Those in the society who should be given priority in the program must be identified by epidemiological studies. Systems for follow-up of the preventive measures should be integrated into the programs when they are initiated. Total quality assurance programs should include parameters which describe the resources and the process needed to obtain the formulated goals. The qualifications of those involved in the process are among the most important resources for success. PMID- 7546143 TI - The public's oral health: the gaps between what we know and what we practice. AB - Health for All by the Year 2000 is a theme developed in 1979 by the World Health Organization. The theme includes oral health and is being promoted throughout the world. The advances in dental research make it possible to improve oral health and, concomitantly, general health. With the appropriate use of science-based, preventive regimens, dental caries and periodontal diseases can be prevented or controlled. Further, major risk factors for oral and lip cancer are known; thus, it is possible to reduce the incidence of these diseases. Available technologies, however, are useful only when they are used by appropriate user groups as recommended. The gaps between what is known about preventing oral diseases and what is practiced are often extensive. This presentation explores differences between scientific knowledge about risk factors for oral diseases and preventive regimens and procedures, public and professional knowledge and practices, and professional support for improved oral health through the application of primary preventive procedures, often referred to as "science transfer". Barriers to the adoption of preventive regimens and practices are discussed. Strategies using health education and health promotion for narrowing these gaps are presented. PMID- 7546144 TI - Strategies for application of scientific findings in prevention. AB - Dental research in the last 50 years has accomplished numerous significant advances in preventive dentistry, particularly in the area of research in fluorides, periodontal diseases, restorative dentistry, and dental materials, as well as craniofacial development and molecular biology. The transfer of scientific knowledge to clinical practitioners requires additional effort. It is the responsibility of the scientific communities to transfer the fruits of their findings to society through publications, conferences, media, and the press. Specific programs that the International Association for Dental Research (IADR) has developed to transmit science to the profession and the public have included science transfer seminars, the Visiting Lecture Program, and hands-on workshops. The IADR Strategic Plan also has a major outreach goal. In addition, the Federation Dentaire Internationale (FDI) and the World Health Organization (WHO) have initiated plans to celebrate World Health Day and the Year of Oral Health in 1994. These are important strategies for the application of scientific findings in prevention. PMID- 7546145 TI - Immunosuppressive drugs and their effect on experimental tumor growth. AB - The effect of cyclosporin (CyA), FK 506, and mycophenolate mofetil (MPM) on tumor growth was investigated using syngeneic mouse colon carcinoma 38. Mice were laparotomized and the tumor cells were injected into the portal vein to establish liver metastasis. The animals were grouped as follows: groups A-1, B-1, and C-1 were given CyA [15 mg/kg body weight (BW)], FK 506 (0.15 mg/kg BW), and MPM (100 mg/kg BW), respectively, 30 min before tumor inoculation and daily for 5 days by gavage; groups A-2, B-2, and C-2 were given CyA (30 mg/kg BW), FK 506 (0.3 mg/kg BW), and MPM (200 mg/kg BW), respectively, with the same dose timing; and groups A-3, B-3, and C-3 received CyA (30 mg/kg BW), FK 506 (0.3 mg/kg BW), and MPM (200 mg/kg BW), respectively, on the 7th post-tumor inoculation day and on the following 5 days. The mean tumor diameter in groups A-1 and A-2 was greater than that in the control group and in groups C-1 and C-2 at 3 weeks (P < 0.05). The mean tumor numbers in groups A-1 and A-2 were greater than those in the control group and in groups C-1 and C-2 at 4 weeks (P < 0.05). With in vitro MTT assay, all three drugs acted cytostatically on tumor cells with a higher concentration (10(-6)-10(-4) mol/l), while no cytostatic effect was noted with CyA at a lower concentration (10(-9)-10(-7) mol/l).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546146 TI - Superoxide scavenging activity in experimental liver transplantation. AB - Superoxide dismutase (SOD) activity was evaluated by measuring superoxide scavenging capability with the aid of an electron spin resonance (ESR) spin trapping method in a swine orthotopic liver transplantation (OLT) model. The animals were divided into two groups, depending on the length of the survival periods: the short survival group (n = 8) survived less than 6 days and the long survival group (n = 15) 6 days or longer. SOD activity was significantly lower in the short survival group than in the long survival group after reperfusion (P < 0.01). During the period of cold preservation, a minimal change in SOD activity was noted, regardless of the length of preservation. Serum aspartate aminotransferase (AST) levels after reperfusion and serum lactate dehydrogenase (LDH) levels 1 h after reperfusion were significantly higher in the short survival group than in the long survival group (P < 0.01 and P < 0.05, respectively). The difference in polymorphonuclear leukocytes (PMN) was significantly greater in the short survival group at 1 h after reperfusion (P < 0.01). The authors conclude that superoxide scavenging activities in the graft reflect the magnitude of reperfusion injury, which can be a reliable parameter for the estimation of graft outcome. PMID- 7546147 TI - Long-term effects of intrasplenically transplanted adult hepatocytes and fetal liver in hyperbilirubinemic Gunn rats. AB - We performed adult hepatocyte transplantation (HCTx) and fetal liver transplantation (FLTx) into the spleens of hyperbilirubinemic Gunn rats in congenic combination and we compared the long-term effects of these procedures for as long as 12 months. Proliferative activity of intrasplenic hepatocytes was evaluated using antiproliferating cell nuclear antigen (PCNA) immunohistochemical staining. The serum total bilirubin levels (T. Bil) significantly decreased from 7.16 +/- 0.25 mg/dl to 4.38 +/- 0.60 mg/dl 2 months after HCTx and gradually decreased thereafter until 12 months after transplantation (3.23 +/- 0.37 mg/dl, P < 0.05 vs preoperative value). The T. Bil change after FLTx was similar to that of HCTx: 7.22 +/- 0.24 mg/dl before FLTx, and 4.92 +/- 0.24 and 3.06 +/- 0.47 mg/dl, 2 and 12 months after FLTx (P < 0.05), respectively. Bilirubin glucuronides, which were not detectable in the bile from untreated Gunn rats, appeared in considerable amounts 4 months after HCTx and FLTx (27.5% and 36.0% of total bile, respectively). PCNA labeling indices of intrasplenic hepatocytes (4.9% +/- 0.9% and 3.7% +/- 0.7%, 6 months after HCTx and FLTx, respectively) were slightly higher than those of normal hepatocytes (1.0% +/- 0.1%) in the host liver. In conclusion, both adult and fetal rat hepatocytes transplanted into the spleen in congenic combination functioned for at least a year in terms of bilirubin glucuronidation. The spleen is considered to be one of the optimal grafting sites for hepatocytes, with nearly lifelong significant function and proliferative activity. PMID- 7546149 TI - Characterization, quantification, and localization of passenger T lymphocytes and NK cells in human liver before transplantation. AB - Quantification and localization of the main lymphocyte populations were studied in the livers of normal (n = 8) and brain dead (n = 8) subjects. Cytometric analysis performed on mononuclear cell suspensions obtained from liver biopsies was compared to an automatic image analysis of immunostained sections. The overall number of liver associated lymphocytes was in the usual range of peripheral blood content (2 to 9x10(9) cells). Phenotypic analysis showed predominant NK and CD8+ cells that highly expressed class II antigen and CD25 and CD69 activation markers. Quantitative mapping of these activated lymphocytes revealed their preferential localization in the portal tract and the perisinusoidal area as compared to the pericentrolobular zone, especially in donor livers. This strategic localization could suggest a possible early cooperation between donor lymphocytes and initial infiltrating cells from the recipient and could explain the special immunological status of allografted livers. PMID- 7546150 TI - Standardized quick en bloc technique for procurement of cadaveric liver grafts for pediatric liver transplantation. AB - This paper describes a quick procedure for cadaveric liver graft retrieval during multiple organ harvesting. The technique is based on minimal preliminary dissection, absence of in situ direct portal perfusion, and en bloc removal of the liver and pancreas, with an aortic patch encompassing the coeliac trunk and superior mesenteric artery. The results of 110 pediatric liver transplantations with 109 organs harvested using this technique are reported. There were no graft harvesting injuries. The liver graft primary nonfunction rate was 4.5% (5/110). The 3-month retransplantation rate was 10%. The actual patient survival rates were 93% at 3 months and 90% at 1 year; actual graft survival rates were 85.5% and 78%, respectively. The technique described was at least as safe as conventional procedures. A major advantage of the procedure is its flexibility, which allows for the easily combined procurement of other organs (whole pancreas and intestine). PMID- 7546148 TI - Intraportal delivery of immunosuppression to intrahepatic islet allograft recipients. AB - Local delivery of immunosuppressive agents may dampen local alloreactive events with avoidance of systemic toxicity. We investigated the innovative strategy of intraportal (IPO) delivery of three immunosuppressive agents in streptozotocin diabetic rat recipients of islet allografts (Lewis to Wistar-Furth) transplanted intrahepatically. IPO budesonide (BUD, 240 or 360 micrograms/kg per day), a potent steroid, and cyclosporin (CyA, 2 or 4 mg/kg per day) did not prolong graft mean survival time [MST +/- standard deviation (SD)] as compared to nonimmunosuppressed recipients. Fourteen days of IPO FK 506 (0.16 mg/kg per day) significantly increased MST as compared with untreated controls (49 +/- 29 vs 7 +/- 1 days, P < 0.01) and was more effective than intravenous (IV) FK 506 (17 +/- 7 days, P < 0.01). When FK 506 was given for 28 days, the benefit of IPO over IV delivery was reaffirmed (MST 81 +/- 32 vs 34 +/- 4 days, P < 0.01). The potential for toxicity was lessened by lower mean systemic levels in the IPO group as compared to the IV group (1.3 +/- 0.6 vs 3.5 +/- 0.9 ng/mg, P < 0.02). The strategy of continuous IPO FK 506 was effective in the prevention of rejection of intrahepatic islet allografts. PMID- 7546151 TI - Hypertension two years after renal transplantation: causes and consequences. AB - The incidence of hypertension 2 years after renal transplantation and the possible causes of hypertension were studied retrospectively. A group of 93 patients treated with cyclosporin (CyA), azathioprine (Aza), and/or prednisolone (Pred) were compared to a group of 31 patients treated with Aza and Pred. There were more patients with hypertension in the CyA group (73%) than in the Aza group (58%). Hypertension before transplantation predisposed to hypertension after transplantation. After transplantation, hypertension was most common among patients with polycystic kidney disease (46%), chronic glomerulonephritis (67%), and diabetes (71%). The accumulated immunosuppressive medication (CyA/Pred) did not affect the occurrence of hypertension. Hypertensive patients had significantly poorer graft function than did normotensive patients (serum creatinine level 229 mumol/l vs 162 mumol/l, P < 0.01). The 10-year graft survival was markedly impaired in the group with hypertension (42% vs 65% for normotensives, P < 0.05). The 10-year patient survival was 59% vs 79% (P = NS). The study further confirms the frequent finding that hypertension has a negative effect on graft and patient survival rates. PMID- 7546152 TI - Regeneration of ATP in kidney slices after warm ischemia and hypothermic preservation. AB - The current shortage of cadaveric kidneys may be alleviated to some degree by increasing our capabilities to use less than ideal donor kidneys, such as those from non-heart-beating donors. These kidneys are often exposed to no flow (ischemia) for varying lengths of time. Full utilization of these kidneys may require better methods of organ preservation that could reverse existing ischemic injury. This may conceivably require that, during preservation, energy stores (ATP) lost during warm ischemia be recharged. This would required continuous perfusion. Using a kidney slice model, we investigated the effects of simulated hypothermic machine perfusion with the UW gluconate perfusate on the capability of rabbit kidneys exposed to warm ischemia to regenerate ATP. After 30 min of warm ischemia, ATP content was low (0.2 mumol/g wet weight) but increased to 0.7 0.9 mumol/g wet weight after 24 h of simulated machine perfusion at 4 degrees C. After an additional 2 h of rewarming (37 degrees C in oxygenated Krebs Henseleit buffer), the slice ATP content increased to about 1.0 mumol/g wet weight (similar to kidneys not exposed to warm ischemia) when the antioxidants desferrioxamine and N-2-(mercaptopropionyl) glycine were included in the preservation media. Significantly less ATP was present without the antioxidants. After 60 min of warm ischemia, less ATP was regenerated after 24 h of simulated machine perfusion (about 0.4 mumol/g wet weight) than after 30 min of warm ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546153 TI - Rat heart-aorta cluster transplantation: a novel model to study transplant rejection. AB - The purpose of this study was to develop a microsurgical cluster model of heart plus entire thoracic aorta transplantation and to compare it to the isolated model of heart transplantation as a tool to study transplant rejection. Thirty six syngeneic (DA x DA and Lew x Lew) and allogeneic (DA x PVG and DA x Lew) cluster heart-aorta transplants were compared to 43 syngeneic and allogeneic isolated heart grafts. Graft survival, recipient survival and histological data on myocardial and aortic tissues were assessed. There was no statistically significant difference in graft survival between the two models studied (P > 0.05). In the cluster transplants, the aortic component was spared the severity of acute rejection noted for the myocardial counterpart. In conclusion, the results demonstrated that the cluster model was technically feasible and highly reproducible. Additionally, it was possible to apply this model to the study of experimental allograft rejection using novel immunosuppressants. The success of the cluster model in strongly mismatched transplant strain combinations underscores its potential for application in slower rejection combinations, making it particularly suited for chronic rejection studies. The inherent capacity for sampling a broader range of vessel sizes in one animal makes the cluster model more suitable than the isolated models of aorta or heart for application to experimental protocols. PMID- 7546154 TI - Heterozygous protein C deficiency and dysfibrinogenemia acquired by liver transplantation. AB - Orthotopic liver transplantation is now a successful treatment for end-stage liver diseases. Since most components of the coagulation system are synthesized by liver parenchymal cells, there is always a risk of genetic defects of hemostasis being transmitting by liver transplantation. Some coagulation factor defects, such as protein C deficiency, do not induce abnormalities in routine coagulation tests and, thus, go undetected before organ procurement. We report the first case, to our knowledge, of the transmission of heterozygous protein C deficiency, an autosomal recessive genetic defect, associated with dysfibrinogenemia, an autosomal dominant trait, by liver transplantation. Both the recipient and the donor presented with severe thrombotic complications. This case shows that potentially morbid genetic defects can be transmitted by organ transplantation, and it emphasizes the difficulty associated with organ procurement criteria, particularly for liver transplantation, in which routine blood tests appear insufficient for determining whether or not organs can or should be procured from a given donor. PMID- 7546155 TI - Liver transplantation in patients with Budd-Chiari syndrome. AB - Patients with Budd-Chiari syndrome (obstruction of the hepatic veins) and associated hepatic insufficiency may be candidates for orthotopic liver transplantation (OLT). In our series of 405 OLT patients, 3 were transplanted due to Budd-Chiari syndrome (0.7%). The indication for liver transplantation in these patients was severe hepatic insufficiency (chronic in two and acute in the third one). Morphologic study of the obstructions revealed apparently different causes, including thrombi, membranous webs in hepatic veins, and hydatid cyst compression. The surgical technique employed in these transplantations was similar to that for other etiologies. Due to its implications for the future course of OLT, it is important to determine the exact etiology of Budd-Chiari syndrome in the pretransplant period and to treat the patients with early and long-term anticoagulant therapy to avoid syndrome recurrence. PMID- 7546156 TI - N-acetylcysteine failed to improve early microcirculatory alterations of the rat liver after transplantation. AB - The application of radical scavengers reduces reperfusion injury of liver grafts despite the natural occurrence of cellular defense mechanisms enabling the cell to tolerate moderate oxidant stress without further cell damage. The glutathione peroxidase mechanism of the liver serves to reduce hydroxyl radical-induced lipid peroxidation by releasing reduced glutathione from intracellular stores. There is evidence that the application of cysteine-providing amino acids for glutathione synthesis could maintain or even increase liver glutathione. Therefore, the purpose of this study was to evaluate the effect of N-acetylcysteine (NAC) on oxidative stress-induced reperfusion injury after liver transplantation. This was done by applying intravital microscopy. Livers from female Sprague-Dawley rats weighing 220-260 g were stored for 20 h in University of Wisconsin (UW) solution and transplanted orthotopically using the cuff technique. Donors were given 150 mg/kg body weight NAC i.v. or placebo in a blind, random fashion 6 h prior to harvesting, followed by two injections of 50 mg/kg body weight, 4 and 2 h before explantation. In additional experimental groups, recipients were given a bolus of 83 mg/kg body weight NAC or placebo at the beginning of the recipient operations, 1 min prior to reperfusion, and 60 min after surgery. Ninety minutes after transplantation, intravital microscopy was applied and five liver lobules were recorded for 30 s after injection of acridine orange, a fluorescent leukocyte marker. Sinusoidal perfusion, sinusoidal width, and leukocyte adhesion, as well as reduced and oxidized glutathione, were determined in all livers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546158 TI - Treatment of IgA deficiency in liver transplant recipients with human breast milk. AB - IgA deficiency is associated with high mortality (42% at 120 days) following liver transplantation (OLTx). Most of the mortality has been associated with enteric infections. Mother's milk, or human breast milk (HBM), is a rich source of IgA that is considered to have beneficial effects in terms of protection from microbial translocation and enteric infections. Two IgA-deficient OLTx recipients were given HBM orally for 10 days perioperatively. HBM was given in order to replenish intestinal IgA. Both patients had an excellent infection-free post operative course. IgA levels in the serum rose from 5 to 10 mg/dl in one patient and from 7 to 30 mg/dl in the other. No complications from HBM administration were observed. We conclude that HBM can be used in IgA-deficient liver transplant recipients to reduce the risk of infectious complications in the post-operative period. PMID- 7546157 TI - Influence of bile on cyclosporin absorption from microemulsion formulation in primary liver transplant recipients. AB - We analysed the absorption, after oral application, of a new galenic form of cyclosporin A (CyA-NOF) in liver-grafted patients (n = 12) during the 1st week (days 2-4) after transplantation. Pharmacokinetic profiling was performed with an open or clamped T tube in situ or with the T tube absent. The pharmacokinetic parameters of CyA-NOF were influenced by T tube clamping and bile diversion. The highest AUC, Cmax and earliest Tmax values were found in patients without a T tube in situ, indicating that absorption of CyA-NOF in patients during the early course after liver transplantation is not bile-independent. CyA-NOF, at a dose of 7.5 mg/kg, was enterally absorbed with appropriate AUC and Cmax levels. Patients receiving a starting dose of 7.5 mg/kg were successfully maintained on CyA-NOF during the subsequent clinical course. PMID- 7546159 TI - Detection of suprahepatic caval stenosis following liver transplantation and treatment via balloon-expandable intravascular stent. PMID- 7546160 TI - What do people expect from their doctors? AB - Surprisingly few people seem to worry about the technical competence of doctors. What they worry about is their doctor's ability to understand the patient as a person and provide the right guidance. Financial, legal or managerial techniques seem powerless to ensure that this demand is met. The solution should be sought within the medical profession itself. PMID- 7546161 TI - Motorcycles for nurses in rural health posts of Senegal. AB - The role of senior nurses at health posts in Senegal's Nioro District was strengthened by supplying them with motorcycles financed by contributions from the general public through the committees running the posts. One vehicle was purchased each month, and the allocation sequence was determined by drawing lots. PMID- 7546163 TI - Epidemiology in action. Participants of the Liverpool Epidemiology Programme. AB - An outline is given of some international schemes aimed at strengthening epidemiological capabilities. Particular reference is made to the Liverpool Epidemiology Programme, dedicated to the exchange of ideas between trainers and health workers. Its activities have resulted in an expanding worldwide network of support for the development of training programmes and materials. PMID- 7546162 TI - Schistosomiasis control through rural health units. AB - In Egypt the main effort in the campaign against schistosomiasis involves providing free diagnosis and treatment through primary care facilities, especially rural health units. The prospects for improving these services are considered below. PMID- 7546164 TI - Not by drugs alone: the fight against parasitic helminths. AB - With particular reference to parts of eastern and southern Africa the authors outline the dire effects of parasitic worms on people in the tropics and subtropics. Helminth infections can be controlled with drugs but in the long term a more comprehensive approach is required, dealing with poverty, health care and education, living conditions, sanitation and water supplies. PMID- 7546165 TI - How to combat nosocomial infection in developing countries. AB - Hospital-acquired infections constitute a significant problem throughout the world. Ways of dealing with them are reviewed below, with particular reference to conditions in the developing countries. PMID- 7546167 TI - Inadvertent supply of substandard drugs. PMID- 7546166 TI - Smoking: easy to start but so hard to stop! PMID- 7546168 TI - Health care crisis in the USA. PMID- 7546170 TI - Who benefits from foreign aid? PMID- 7546169 TI - Educating deaf children in Togo. PMID- 7546171 TI - Nutritional guidance for mothers of anaemic infants. PMID- 7546172 TI - Training young volunteers in health promotion. PMID- 7546173 TI - Decentralization of psychiatric care in Burkina Faso. PMID- 7546174 TI - Sex education in Indian schools. PMID- 7546176 TI - Radical reforms for primary health care in New Zealand. AB - Financial, social and political forces have combined to produce radical reforms in New Zealand's health system. These are characterized by decentralized management; the integration of services that have traditionally been kept separate, such as the hospital and the community, and public and personal health; and focusing on the population rather than institutions as the basis of the system. These changes are accompanied by a shift of emphasis from secondary towards primary health care. PMID- 7546175 TI - The right objectives in health care planning. AB - In Dar es Salaam, United Republic of Tanzania, the traditional epidemiological approach to health service planning has been superseded by a process-oriented approach. The implications for managers are discussed below. PMID- 7546177 TI - Cost-effective outlays for better health outcomes. AB - The marginal return on health expenditure in high-income countries is low, whereas that in low-income countries is comparatively high. However, there is no clear evidence that higher health expenditure is the most cost-effective way of improving health in low-income countries; consideration should be given to the alternative of raising educational levels. PMID- 7546178 TI - Double think--a reply. PMID- 7546179 TI - Gathering data on oral mucosal diseases: a new approach. AB - A new approach to gathering epidemiological data on oral mucosal diseases has been tested with encouraging results in Sri Lanka. Its main advantage is that it does not depend on the examiner's ability to make a diagnosis, so it can be carried out by non-professionals such as dental students. PMID- 7546180 TI - Informatics: the key to efficiency. AB - In India a computer-based national health management information system is being implemented by linking more than 450 districts on a network. This and other actions in the field of informatics technology could significantly raise the efficiency of the country's health sector by making decisions more logical, speeding them up and monitoring their impact, and could help to improve the utilization of scarce resources. PMID- 7546181 TI - Patients deserve nurses: what you must know as a nurse. PMID- 7546184 TI - Supreme Court says nurses may be supervisors. PMID- 7546183 TI - Update! Innovate! The keys to nursing continuing education. PMID- 7546182 TI - Hanging on to nursing practice. PMID- 7546187 TI - "I just want to be a nurse"! PMID- 7546186 TI - Getting involved in lawmaking is easy. PMID- 7546185 TI - HIV/AIDS--voluntary testing for pregnant women. PMID- 7546188 TI - Rape: nurses trained to take evidence. PMID- 7546189 TI - A need for standardization of the anti-endothelial-cell antibody test. PMID- 7546190 TI - The immunology and developmental biology of the chicken. PMID- 7546192 TI - Th1 and Th2 subsets: paradigms lost? AB - The T helper 1 (Th1)/Th2 model has provided a valuable framework to investigate and explain many immune reactions and now pervades current thinking on the regulatory role of T cells. However, individual T cells and clones display remarkable diversity in their cytokine profiles, collectively forming a continuous spectrum in which Th1 and Th2 cells may be only two of the possible extreme phenotypes. For these reasons, Anne Kelso argues that cytokine-producing T cells cannot be classified into discrete subsets. PMID- 7546191 TI - Signal transduction in the activation of mast cells and basophils. PMID- 7546193 TI - Consequences of IgE/CD23-mediated antigen presentation in allergy. AB - Allergen-specific IgE antibodies are responsible for the generation of immediate type hypersensitivity reactions. However, as described here by Geert Mudde, Roy Bheekha and Carla Bruijnzeel-Koomen, IgE may also be involved in the uptake and processing of allergens. Such IgE-mediated antigen presentation may lead to a continuous (over) activation of the immune system due to high titers of IgE and the presence of large numbers of allergen-specific Th2 cells. In addition, it may be a cause for the advance of disease from a 'single allergy' to 'multi-allergy' syndrome. PMID- 7546194 TI - The role of IL-12 in the induction of organ-specific autoimmune diseases. AB - The concept that T cells are subdivided into T helper 1 (Th1) and Th2 subsets was recently extended to suggest that Th1 cells contribute to the pathogenesis of several organ-specific autoimmune diseases, whereas Th2 cells inhibit disease development. Here, Sylvie Trembleau and colleagues examine the role of interleukin 12 (IL-12), a key cytokine guiding the development of Th1 cells, in the induction of autoimmune diseases, and discuss potential immunointervention strategies based on administration of IL-12 antagonists. PMID- 7546195 TI - Direct antimicrobial activity of T cells. AB - T cells are generally thought to contribute to antimicrobial activity either by releasing lymphokines, which recruit and activate other cell types, or by major histocompatibility complex (MHC)-restricted lysis of infected host cells. Recently, it has become apparent that T cells can also mediate antimicrobial activity by direct interaction with microbial targets. Such interactions, which can be either antigen specific or nonspecific, occur in the apparent absence of MHC restriction and do not require the presence of other host cells. Microbial targets recognized by T cells include fungi, parasites and bacteria. Here, Stuart Levitz, Herbert Mathews and Juneann Murphy discuss the direct antimicrobial activity of T cells and speculate on its in vivo relevance. PMID- 7546197 TI - B-cell superantigens: implications for selection of the human antibody repertoire. AB - For several decades, B-cell interactions with antigens were thought to occur only through a clonal activation mechanism, in which the hypervariable regions of the immunoglobulin receptor are exclusively involved in ligand binding. However, an additional mode of interaction can occur, whereby molecules, termed B-cell superantigens, can bind human B cells bearing immunoglobulin receptors of a given variable (V)-gene family. This mechanism requires contributions from regions outside the conventional hypervariable loops and results in a B-cell response of increased magnitude. Here, Moncef Zouali reviews recent in vitro and in vivo observations on human B-cell superantigens in the context of the current consensus of B-cell development, and discusses the implications of these novel concepts with respect to pathogenesis. PMID- 7546198 TI - T-cell receptor signal sequences. PMID- 7546199 TI - Sequence similarity between beta-cell autoantigens. PMID- 7546201 TI - Restricted diversity in the T-cell repertoire. PMID- 7546196 TI - IgY: clues to the origins of modern antibodies. AB - IgY is the functional equivalent of IgG in birds, reptiles and amphibia, but many aspects of its biology are poorly understood. Recent studies have increased awareness of the genetics and functions of this molecule, and have revealed its position as the ancestor of the uniquely mammalian antibodies IgG and IgE. Here, Greg Warr, Kathy Magor and David Higgins review current knowledge of IgY structure, function and expression in the context of the evolutionary role of this primitive immunoglobulin. PMID- 7546202 TI - Immunology research in the UK: a scientific audit. PMID- 7546203 TI - Lactoferrin: a multifunctional immunoregulatory protein? PMID- 7546200 TI - Cellular mechanisms in the pathogenesis of bronchial asthma. PMID- 7546204 TI - Immunosenescence: ageing of the immune system. PMID- 7546205 TI - Costimulation and the regulation of antimicrobial immunity. AB - The regulated expression of costimulatory molecules is a major factor limiting T cell responses to self-antigens. However, the development of effective antimicrobial immunity requires that these molecules be induced on a variety of tissues, but most notably on macrophages. Here, Paul Kaye discusses the regulation of costimulatory molecules on macrophages and suggests that microbial interference in this process has important implications for immune regulation. PMID- 7546206 TI - The origin of CD4-CD8-TCR alpha beta+ thymocytes: a model based on T-cell receptor avidity. AB - Ralph Budd and Philip Mixter present a hypothesis suggesting that CD4-CD8-TCR alpha beta+ cells arising in the normal thymus result from a high-avidity T-cell receptor (TCR) signal bordering on negative selection. In normal mice, this subset is likely to be gradually deleted but, in the absence of Fas, these cells persist in lpr mice. The model they describe makes several predictions regarding the nature of this unusual T-cell subset. PMID- 7546207 TI - Antagonists or altruists: do viral mutants modulate T-cell responses? AB - The ability of altered peptide ligands (APLs) to modify or downregulate T-cell responses in vitro has generated much interest. Recently, it was proposed that viral variants may contain mutations in cytotoxic T lymphocyte (CTL) epitopes that allow mutual antagonism, and that these combine to downregulate the host immune response. Here, Miles Davenport discusses some inherent problems with this simple model and proposes a closer evaluation of the effects of viral competition on host-viral interactions. PMID- 7546208 TI - Bone marrow transplantation across HLA barriers by increasing the number of transplanted cells. AB - Throughout the 1970s, graft-versus-host disease (GVHD) was uniformly lethal in recipients of HLA-mismatched bone marrow. This major obstacle was overcome in 1980 by the introduction of rigorous T-cell depletion prior to transplantation into patients with severe combined immunodeficiency. However, in leukemia patients, the benefit of preventing GVHD was offset by graft rejection or graft failure. In this article, Yair Reisner and Massimo Martelli discuss how this problem may be overcome by intensification of the conditioning protocol in conjunction with a major increase in the dose of transplanted stem cells. PMID- 7546209 TI - Unraveling the neuroimmune mechanisms for the HIV-1-associated cognitive/motor complex. AB - Infection of the brain with human immunodeficiency virus 1 (HIV-1) often leads to the devastating loss of mental faculties. Surprisingly, HIV-1 elicits such brain dysfunction without significantly infecting neurons, astrocytes and oligodendroglia. The target for HIV-1 in the brain is the macrophage, which usually functions as a phagocytic, antigen-presenting and immune-regulatory cell. How can these cells produce such serious cognitive and motor brain impairments? Here, Hans Nottet and Howard Gendelman propose that HIV-1 penetrates the blood brain barrier inside differentiating macrophages, which become immune activated once inside the brain, and secrete high levels of neurotoxins. Chronic, subclinical disease results by astrocyte regulation of macrophage effector functions. Ultimately, endogenous control mechanisms break down, leading to motor and mental impairments in some affected subjects. PMID- 7546211 TI - Heterogeneity of organ-specific autoimmune diseases. PMID- 7546210 TI - High endothelial venules (HEVs): specialized endothelium for lymphocyte migration. AB - High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. Here, Jean-Philippe Girard and Timothy Springer highlight the unique properties of HEV endothelium, discuss the molecular mechanisms controlling HEV specialization and review evidence suggesting that HEVs could play an important role in the pathogenesis of chronic inflammatory diseases. PMID- 7546212 TI - Heat treatment of Corynebacterium ammoniagenes leads to aeration dependent accumulation of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate. AB - 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) identified as a new bacterial oxidative stress substance (Ostrovsky D. et al. (1993) Biochem. J., 295, 901-902) was shown to accumulate in Corynebacterium (Brevibacterium) ammoniagenes cells aerobically cultivated in peptone-yeast extract-glucose broth on heating for 1 hour at 45 degrees C. The enzyme(s) responsible for MEC biosynthesis is evidently oxidized for activation and is completely loosing its activity on anaerobic incubation at this temperature in an hour. Salt stress or drying did not provoke the MEC biosynthesis. PMID- 7546213 TI - Possible involvement of prostaglandin H synthase-1 induction in the differentiation of U-937 cells. AB - The promyelocytic human cell line U937, cultured in the presence of TPA and/or vit. D3, differentiates to monocytes and to macrophage-like cells. A potent stimulus for differentiation is represented also by colony stimulating factor-1 (CSF-1). Since this factor is a strong inducer of PGH synthase in human monocytes, we have investigated whether this event may be connected to the differentiation of U937. We have found that TPA, in the presence of serum, increased the production of thromboxane B2 (TXB2) 4-5 fold, while DMSO, which induced differentiation to neutrophils, was not active. Here we report studies indicating that the effect of protein and RNA synthesis inhibitors on prostanoid production, in cells incubated in the presence of CSF-1 (or FCS), can be correlated with an inductive event carried out by the growth factor, as demonstrated by the use of Western and Northern blotting procedures. However, while in human monocytes PGH-s and its mRNA are absent in controls and are expressed at high levels in CSF-1 stimulated cells, in U937 cells exposed to TPA, PGH-s mRNA was clearly detected by Northern blots, but its translation product was expressed at low level, and cells generated low amounts of TXA2 (13% of maximal production). After incubation with CSF-1 (or FCS) mRNA levels were only slightly modified, but large amounts of TXA2 accumulated in the medium. We have interpreted these findings by suggesting that CSF-1 is capable not only of regulating the expression of the gene encoding PGHs, but also of acing translationally to regulate the expression of its mature mRNA. PMID- 7546214 TI - Okadaic acid interferes with the maturation of human fetal kidney in serum-free culture. AB - A chemically-defined culture model has been established in our laboratory, enabling the study of the respective effects of growth factors on the developing kidney. Since this system involves the use of defined medium unsupplemented with serum, hormones or glucose, these limited conditions induce a decrease in DNA synthesis. Addition of insulin and transferrin significantly restores this important cellular function. The objective of the present study was to verify the influence of okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, in order to determine whether the latter modulate insulin action on human fetal kidney. Okadaic acid was found to decrease 3H-thymidine incorporation below control (L-15) levels in a dose-dependent manner. The influence of 0.01 microM okadaic acid could be detected as early as the 4th h of culture, and was progressive until the 48th h; at 0.4 microM, the effect was more dramatic with maximum inhibition obtained between 8 and 12 h. Moreover, at both concentrations, the drug completely abolished the stimulating effect of insulin plus transferrin over a 5-day period. Following removal of the toxin after 24 h of culture, DNA synthesis after 5 days still remained at levels observed in the presence of okadaic acid and insulin plus transferrin on day 1. This result indicates that the inhibitory influence of okadaic acid remained at the same level once the drug was removed, and that this effect is irreversible. In addition, okadaic acid at 0.4 microM induced cell death and morphological changes possibly associated with apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546215 TI - Vanadium (IV) inhibits calmodulin-stimulated skeletal muscle myosin light chain kinase activity. AB - Vanadium, believed to be an essential trace metal, exhibits numerous biological effects. Using electron spin resonance spectroscopy, we have demonstrated that vanadyl, vanadium (IV), the predominant intracellular form of vanadate (vanadium V), binds to calmodulin in the presence of physiological concentrations of magnesium, extending earlier work which showed competitive binding of vanadyl and calcium to calmodulin. In the presence of a magnesium-containing buffer, vanadyl does not lead to calmodulin activation of the calmodulin-dependent enzyme, rabbit skeletal muscle myosin light chain kinase; in the presence of calcium, vanadyl is a potent inhibitor of the calmodulin-activated form of the kinase. Thus, vanadyl can potentially interfere with some of the intracellular actions of calcium, presumably via binding to calmodulin. This observation deserves consideration in view of the potential clinical application of vanadium treatment to mimick insulin action and lower blood glucose. PMID- 7546217 TI - Nitric oxide stabilizes the Mo(V) oxidation state of dimethyl sulfoxide reductase from Rhodobacter sphaeroides without inhibiting enzyme activity. AB - Dimethyl sulfoxide reductase from Rhodobacter sphaeroides is isolated in an oxidized, Mo(VI) containing form. Both nitric oxide and reduced ascorbate carried out a one electron reduction of the enzyme with formation of stoichiometric amounts of EPR active Mo(V). Nitric oxide also caused a one electron oxidation of reduced, Mo(IV) enzyme. Mo(V) formation was accompanied by appearance of absorbance peaks at 387 and 528 nm. Neither nitric oxide nor ascorbate inhibited the enzyme nor did either compound support enzyme turnover. Both nitrite plus ascorbate and nitroxyl anion (NO-) induced a previously reported rhombic EPR signal (g1 = 1.994, g2 = 1.982, g3 = 1.966) which exhibited superhyperfine coupling to an exchangeable proton (A1 = 1.25 mT, A2 = 0.85 mT, and A3 = 1.0 mT). On the other hand, NO(g) induced an axial signal with g perpendicular = 1.982 and g parallel = 1.961 in which there is no evidence of superhyperfine coupling. Thus, ascorbate, nitric oxide, and nitric oxide donors induce and stabilize Mo(V) formation in dimethyl sulfoxide reductase without inhibiting enzyme activity. The resemblance between NO and the natural N-oxide substrates of this enzyme suggest that the Mo(V)-NO complex may be a transition state analog of the enzyme substrate complex. PMID- 7546216 TI - Physiological mechanisms regulating the conversion of selenite to elemental selenium by Bacillus subtilis. AB - We have demonstrated that the common soil bacterium, Bacillus subtilis, reduces selenite to an insoluble and much less toxic product--the red form of elemental selenium. Reduction was effected by an inducible system that appears to deposit elemental selenium between the cell wall and the plasma membrane. Glucose and sucrose supported selenite reduction. Although malate and citrate supported growth, no significant reduction of selenite occurred, indicating the importance of the redox state of the culture substrate. Selenite reduction in the millimolar concentration range (i.e., cultures supplemented with 1 mM selenite) was not affected by a ten-fold excess of nitrate or sulfate--compounds that serve as alternate electron acceptors and antagonize selenite reduction by anaerobic bacteria. Similarly, nitrite and sulfite did not significantly affect the rate or extent of selenite reduction. B.subtilis was able to grow and produce selenium (Se degree) at selenite concentrations ranging from 0.6 microM to 5 mM (50 ppb to 395 ppm selenium). At the lowest selenite concentration tested, 50 ppb selenium, B.subtilis removed 95% of the selenite from the liquid phase. The results suggest that selenite is reduced via an inducible detoxification system rather than dissimilatory electron transport. The findings establish the potential utility of B.subtilis for the bioremediation of selenite-polluted sites. PMID- 7546218 TI - Dietary energy restriction-induced modulation of protein kinase C zeta isozyme in the hamster pancreas. AB - Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C (PKC) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C-terminally directed anti-PKC zeta antibody detected an 81 kDa band in the pancreases of control and energy-restricted hamsters. Syrian golden hamsters were fed energy-restricted diets formulated such that the hamsters received 90% (10% energy restriction (ER)), 80% (20% ER), or 60% (40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC zeta isozyme levels by 40-75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC zeta isozyme expression was decreased by 75-80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC zeta isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC zeta levels between the dietary ER groups (10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC zeta in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC zeta signal transduction mechanisms in normal pancreatic physiology. PMID- 7546219 TI - Effects of genetic background on tumorigenesis in p53-deficient mice. AB - Mice with disrupted germline p53 alleles have been engineered by us and others and have been shown to have enhanced susceptibility to spontaneous tumors of various types. We monitored a large number of p53-deficient mice (p53+/- and p53 /-) and their wild-type littermates (p53+/+) of two different genetic backgrounds (129/Sv and mixed C57BL/6 x 129/Sv) up to 2 yr of age. p53+/- and p53-/- 129/Sv mice show accelerated tumorigenesis rates compared with their p53-deficient counterparts of mixed C57BL/6 x 129/Sv genetic background. The tumor spectra of the two strains of mice are similar except that almost half of 129/Sv p53-/- males develop malignant teratomas, whereas these tumors are rarely observed in C57BL/6 x 129/Sv mice and never in 129/Sv p53+/- males. In the study reported here, we further characterized the lymphomas that arose in the p53-nullizygous mice and found that over three-quarters of the lymphomas were of thymic origin and contained primarily immature (CD4+/CD8+) T-cells, whereas the remainder originated in the spleen and peripheral lymph nodes and were of B-cell type. The high incidence of early-onset lymphomas in the nullizygous mice makes these animals a good lymphoma model, whereas the heterozygous mice may be a useful model for Li-Fraumeni syndrome, a human inherited cancer predisposition. PMID- 7546220 TI - Environmental health science research and human risk assessment. AB - Environmental health science research, with its focus on fundamental science and disease prevention, is important for the development of rational and cost effective public health and regulatory policies related to environmental protection. Environmentally related diseases are preventable, yet they impose a major burden on society in terms of human suffering and costs related to health care. Similarly, the expenditure of hundreds of billions of dollars for regulatory compliance is a major economic concern. There is considerable debate regarding current regulatory risk assessment practices for environmental agents. Implicit in all risk assessment schemes is the need to extrapolate from high exposure studies to low-exposure situations and from known risks in rodents to probable risks in people. Both extrapolations are fraught with uncertainties. These uncertainties are accommodated in risk-assessment schemes by the incorporation of arbitrary "safety factors" and other default approaches. Since these factors are not derived experimentally, they may overestimate or under estimate actual risks. Risk-assessment methodology, its relevance to the human condition, and its use in protecting human health will greatly improve when our expanding knowledge of the basic biology of environmental effects is incorporated into toxicological testing and risk-assessment schemes. Moreover, exciting opportunities now exist to advance our understanding of the environmental and genetic bases of many common diseases and to design effective prevention and intervention strategies to combat their development. This report discusses some of the current opportunities and challenges. PMID- 7546221 TI - Molecular genetic basis of renal carcinogenesis in the Eker rat model of tuberous sclerosis (Tsc2). AB - We have recently identified on rat chromosome 10q a germline mutation in the tuberous sclerosis gene (Tsc2), the gene predisposing to renal carcinoma (RC) in the Eker rat. The homozygous mutant condition is lethal at around the 13th day of fetal life. In heterozygotes, RCs invariably develop in the first year of life. Histologically, RCs develop through multiple stages from early preneoplastic lesions (i.e., phenotypically altered tubules) to adenomas. The wild-type allele mutation has been found even in the earliest preneoplastic lesions, fitting Knudson's two-hit hypothesis and supporting the hypothesis that Tsc2 is a tumor suppressor gene. In this study, homozygous deletion of the Ink4 homologue on rat chromosome 5q was observed in 14 of 24 (58%) RC-derived cell lines. This may represent involvement of a second tumor suppressor gene, contributing to tumor progression. Considering previous results of studies of homozygous deletion of the Ifn alpha gene in five of 24 cases (21%) and the Ifn beta gene in one of 24 cases (4%), the order of the genes may be Ink4-Ifn alpha-Ifn beta. Microsatellite instability was not observed in 26 Eker rat tumors. PMID- 7546222 TI - Allelic loss at the tuberous sclerosis 2 locus in spontaneous tumors in the Eker rat. AB - Somatic events leading to the inactivation of tumor suppressor genes often involve chromosomal alterations that can be detected as loss of heterozygosity (LOH). In the Eker rat, spontaneous tumors of the kidney, uterus, and spleen develop as a result of a germline mutation of the tuberous sclerosis 2 (Tsc2) gene. We examined the pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker-derived tumors. Although most renal and uterine tumors (primary and cell lines) displayed LOH, splenic hemangiosarcomas did not. Although the presence of normal tissue may account for some of this difference, the possibility exists that an alternative mechanism, such as subtle mutation or gene dosage effects, may be involved during splenic tumorigenesis. Northern analysis confirmed that LOH resulted in loss of the wild-type transcripts for the Tsc2 gene. Thus, the inactivation of both alleles plays an important role in renal and uterine tumor development, in keeping with Knudson's two-hit hypothesis. In addition, renal tumors that retained the wild-type allele also did not express the normal transcript, suggesting that the remaining Tsc2 alleles had acquired subtle mutations resulting in loss of gene function. PMID- 7546223 TI - Predictions of rodent carcinogenicity testing results: interpretation in light of the Lave-Omenn value-of-information model. AB - The recent National Institute of Environmental Health Sciences/National Toxicology Program Carcinogen Prediction Challenge elicited a valuable array of predictions of the carcinogenicity of chemicals tested in the lifetime rodent bioassay. The data warrant additional analyses of the similarities and differences of the predictive methods. We provide here analyses of the sensitivity, specificity, and false-positive/false-negative tendencies of the different sets of predictions. Our value-of-information model provides guidance to testing agencies and regulatory agencies in determining the social value of additional information and setting up the framework for assessing the social consequences of different test strategies and nontest predictive methods. These considerations deserve attention in the second round of the Carcinogen Prediction Challenge. PMID- 7546224 TI - Strain-dependent differences in DNA synthesis and gene expression in the regenerating livers of CB57BL/6J and C3H/HeJ mice. AB - C3H/HeJ (C3H) mice are approximately 50-fold more susceptible to liver-tumor induction than C57BL/6J (B6) mice. This difference is susceptibility is a consequence of allelic differences in hepatocarcinogen sensitivity (Hcs) genes that control the growth of preneoplastic lesions in the liver. We have shown previously that these two strains differ in their responses to partial hepatectomy, which acts as a promoter of hepatocarcinogenesis in B6 mice but not in C3H mice. To determine whether there are also strain-specific differences in normal regulation of hepatic growth, we compared liver regeneration in C3H and B6 mice at the levels of DNA synthesis and gene expression. Partial hepatectomy induced a cascade of controlled events resulting in the regeneration of the liver to its original mass 11 d after surgery. We observed a two-fold greater level of DNA synthesis in C3H mice relative to B6 mice during the first peak of DNA synthesis, which occurred 35 h after hepatectomy in both strains. While the c-fos transcript was readily induced in both strains, there was a reduction in the expression of the late response genes E2F1 and dihydrofolate reductase in the livers of B6 mice when compared with the expression of these transcripts in the livers of C3H mice. The differential regulation of E2F1 between B6 and C3H mice may indicate that the Hcs genes and E2F1 function in the same signal transduction pathway of normal growth control. PMID- 7546225 TI - Mutational spectra in the lacl gene in skin from 7,12-dimethylbenz[a]anthracene treated and untreated transgenic mice. AB - Transgenic mice carrying the bacterial lacl gene in a lambda shuttle vector were used to isolate and characterize background and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutations in skin. Adult male mice were treated once topically with either DMBA or acetone or were left untreated. Seven days later, DMBA treatment had significantly increased the mutant frequency in the skin (mean +/- SEM, 36 +/- 3 x 10(-5)) versus in vehicle-treated (6.4 +/- 1.2 x 10(-5)) and untreated mice (7.1 x 1.0 x 10(-5)). At least 10 mutants from each of three DMBA treated and three untreated mice were selected for DNA sequence analysis. In each case, the entire 1080-bp target gene was sequenced. Base-pair substitutions predominated (86 of 96 mutations), although frameshift and deletion mutations were also detected. Twelve percent of the mutants carried more than one mutation. In controls, the mutations were predominantly GC-->AT transitions (26 of 42), and no AT-->TA transversions were recovered. In contrast, in the DMBA-treated mice, AT-->TA transversions represented 42% of the mutations (23 of 54) and GC-->AT transitions accounted for only 11%. The AT-->TA transversions occurred mostly at 5'-CA sites. This class of mutation has been recovered frequently in ras genes from DMBA-treated mice and probably represents an early event in carcinogenesis (Nelson MA et al., Proc Natl Acad Sci USA 89:6398-6402, 1992). Our present results are consistent with the types of DNA damage induced by DMBA. The observation of different mutant frequencies and spectra in treated and control mice demonstrates the utility of this approach in the study of mutagenesis in vivo. PMID- 7546228 TI - Evaluation of cryopreservation techniques for goat embryos. AB - A total of 410 goat embryos were divided at random into 9 groups. Cryoprotectants (glycerol, ethylene glycol or dimethylsulfoxide) were added by a 3-step procedure using increasing concentrations of cryoprotectant (0.5 M; 1 M; 1.5 M) in PBS at 10 min intervals. After freezing and thawing, each cryoprotectant was removed by 3 methods: the classic 3-step procedure (cryoprotectant 1 M-10 min; 0.5 M-10 min; PBS alone-10 min); the same procedure, but with sucrose added to the first 2 steps (sucrose 0.25 M and cryoprotectant 1 M-10 min; sucrose 0.25 M and cryoprotectant 0.5 M-10 min; PBS alone-10 min); and a 2-step procedure with sucrose alone (sucrose 0.25 M-10 min; PBS alone-10 min). Each removal protocol was performed for embryos in each cryoprotectant. The viability of the embryo was evaluated by its capacity to subsequently develop during 48 h in vitro culture. For morulae the development rate of the embryos was significantly higher when they were frozen with ethylene glycol than when dimethylsulfoxide or glycerol was used (P < 0.05). For blastocysts the development rate was the same whether they had been frozen with ethylene glycol or dimethylsulfoxide, and was significantly lower when they were frozen with glycerol (P < 0.05). Among the 3 removal procedures tested, the 3-step procedure with sucrose gave the best development rate and differed significantly from the classic 3-step procedure (P < 0.05). PMID- 7546229 TI - Relationships between microflora and caecal fermentation in rabbits before and after weaning. AB - Some microbiological and biochemical parameters of caecal content were studied in 15- to 49-d-old rabbits that were slaughtered sequentially. The ammonia level did not differ before weaning (11.5 mmol/L on average) (P = 0.41) or after weaning (7.4 mmol/L on average) (P = 0.19) but decreased by 40% (P < 0.001) between days 29 and 32. The level of pH decreased linearly (P < 0.001) throughout the period studied. The Escherichia coli counts decreased up to weaning (P < 0.001) and was then not significantly affected by age (P = 0.12). The total volatile fatty acid (VFA) concentration increased between days 15 (8.2 mmol/L) and 25 (33.9 mmol/L) (P < 0.05) and then levelled off below 40 mmol/L. Molar proportions in propionate and in branched-chain fatty acid (BCFA) and valeric acid were high at day 15 but decreased when the animals began to eat solid feed. The C3/C4 ratio reversed at weaning (3.8 on day 15 and 0.5 on day 49) whereas the acetic acid proportion was not affected by age (P = 0.19). High counts of anaerobic microflora were found between 15 and 22 d of age (10(11) bacteria/g of caecal content, on average) and did not change significantly according to the age (at about 10(10) bacteria/g), from day 29 until the end of the experiment (P = 0.29). Amylolytic flora had a similar evolution at a slightly lower level. In contrast, under our breeding conditions cellulolytic microflora slowly colonized the caecum and remained at a low level. The discriminant analysis revealed relationships between ages, intestinal microflora and fermentation parameter; the colibacilli flora was associated with mother-fed animals and amylolytic flora which was linked to BCFA and valeric acid, while the cellulolytic flora was associated with animals older than 4 weeks and linked to the production of C2, C3, C4 and ammonia. PMID- 7546227 TI - [Adaptation of rumen fermentation to monensin]. AB - Adaptation of rumen fermentation to monensin feeding has been studied with rumen fistulated sheep receiving a daily dose of 30 mg of monensin for a period of 21 d followed by a 28 d period during which 60 mg doses were administered. The ration consisted of 300 g of hay and 300 g of concentrates, fed at 9.00 h and 16.00 h. Monensin was placed in the rumen as an aqueous suspension, just prior to the morning feeding. Monensin infusion was preceded and followed by a period during which no monensin was infused. The following rumen fermentation parameters were determined: methane production, pH, volatile fatty acids (VFA) molar proportions, total volatile fatty acid concentration, lactate and ammonia concentrations and in sacco degradability of hay. Rumen gas expelled through the fistula was collected for 6 h per day and analysed. Total VFA concentration, molar proportions of individual VFA, pH, lactate and ammonia concentration were determined on rumen contents, sampled just prior the administration of monensin and 2 and 6 h later. In vitro incubations of 3 h were carried out with rumen fluid, sampled 1 h after feeding. In vivo and in vitro methane production was decreased by monensin feeding. The molar proportion of propionate in the rumen was increased, while acetate and butyrate percentages were lowered. The total VFA and ammonia concentrations were also decreased by monensin, but pH values were increased. In vitro production of propionate was stimulated by monensin administration and methanogenesis decreased. The organic matter in sacco degradability was not affected, probably because of the time difference between the introduction of bags and monensin in the rumen. These modifications of rumen fermentation persisted as long as monensin was given, indicating that in this experiment, there was no adaptation to the ionophore. PMID- 7546230 TI - Peroral administration of triiodothyronine (T3) affects absorption of immunolactoglobulins in calves. AB - On the basis of our studies which demonstrated that T3 is a natural milk-borne component of cow mammary secretions, this study investigated the influence of T3 (and thyroxine, T4) on the serum Ig level (used here as an indicator of intestinal absorption). Forty healthy calves were given a single dose of either T3 or T4 with the first colostrum meal 6 h following birth. Blood samples were taken before and 42-50 h after hormone administration. The T4 treatment resulted in metabolic changes that were reflected by an increase in blood glucose, triglycerides (TG), non-esterified fatty acids (NEFA), and a decrease in total serum proteins (TP). Ig levels were reduced from 27.5 milligrams (controls) to 20.6 milligrams. The T3 treatment caused an increase in serum TG and FFA (p < 0.01), and, in contrast to T4, an increase in TP and Ig (p < 0.001). These results indicated that peroral administration of T3, but not T4, could exert a positive effect on the transfer of immunolactoglobulins in the neonatal calf intestine. The contrasting hormonal effects are likely attributable to different responses of intestinal cells to T3 and T4. PMID- 7546226 TI - The impact of glutathione s-transferase M1 and cytochrome P450 1A1 genotypes on white-blood-cell polycyclic aromatic hydrocarbon-DNA adduct levels in humans. AB - Carcinogenic polycyclic aromatic hydrocarbons (PAHs) form DNA adducts via a complex metabolic activation pathway that includes cytochrome P450 (CYP) 1A1, whereas intermediate metabolites can be detoxified by conjugation through pathways including glutathione s-transferase M1 (GSTM1). PAH-DNA adducts can be measured in peripheral white blood cells (WBCs) and should reflect the net effect of competing activation and detoxification pathways and DNA repair as well as exposure. We have previously shown that WBC PAH-DNA adducts measured by an enzyme linked immunosorbent assay (ELISA) were associated with recent, frequent consumption of charbroiled food among 47 nonsmoking wildland fire-fighters who provided two blood samples 8 wk apart. In the investigation reported here, which was performed in the same population, we measured the association between the GSTM1 null genotype, which results in loss of enzyme activity, and PAH-DNA adduct levels, hypothesizing that subjects with this genotype would have higher levels of DNA adducts because of their decreased ability to detoxify PAH metabolites. However, PAH-DNA adduct levels were nonsignificantly lower in subjects with the GSTM1 null genotype (n = 28) compared with other subjects (n = 19) (median 0.04 fmol/microgram DNA vs 0.07 fmol/microgram DNA, respectively, P = 0.45, Wilcoxon rank-sum test). Adduct levels were also lower in the nine subjects heterozygous or homozygous for the CYP1A1 exon 7 polymorphism (which codes for a valine rather than isoleucine and is thought to be associated with greater CYP1A1 activity) compared with the 38 wild-type subjects (P = 0.12). In the entire group, there was a positive association between consuming charbroiled food and PAH-DNA adduct formation (r = 0.24, P = 0.02, Spearman rank-order correlation). This association was weaker in the subgroup of subjects with the GSTM1 null genotype (r = 0.03, P = 0.84) and stronger among the remaining subjects (r = 0.57, P = 0.0002). These results suggest that the GSTM1 null genotype and CYP1A1 exon 7 polymorphism are not associated with increased susceptibility for PAH-DNA adduct formation in peripheral WBCs measured by ELISA in nonsmoking populations. PMID- 7546231 TI - Endocrine bases of lactational anoestrus in the sow. AB - Parturition in the sow is followed by a period of anovulation which is prolonged by lactation. Follicular development and luteinizing hormone (LH) secretion are depressed during the last month of pregnancy. After parturition, LH secretion increases but is again inhibited by the establishment of lactation. Lactating sows are submitted to stimuli originating from the young, whose intensity culminates 3-14 d post-partum (pp), and to high nutrient requirements for milk production. The inhibitory effects of sucklings are imposed during the first 3 d pp and seem to be mediated by the action of opioids at the hypothalamic level. The nutritional deficit constitutes an additional inhibitory factor. As lactation continues, LH secretion progressively increases. A further rise in LH occurs at weaning. Variations in follicle-stimulating hormone (FSH) profiles are less marked. The divergence observed between LH and FSH might be explained by different mechanisms of control; FSH secretion depends mainly on ovarian inhibition whereas LH secretion depends mainly on factors related to lactation. Folliculogenesis progressively resumes during lactation and follicles acquire the ability to respond to the weaning-associated stimuli and begin preovulatory growth. Hormones modified by lactation, such as prolactin, insulin, growth hormone and insulin-like growth factor I, may influence folliculogenesis directly at the ovarian level or via modifications of gonadotrophin secretions. In conclusion, the inhibition of the hypothalamo-pituitary-ovarian axis during lactation is mainly due to suckling-induced neuroendocrine reflexes. We hypothesize that the nutritional deficit becomes relatively more important during the third and fourth weeks pp. PMID- 7546233 TI - [Autoimmune asthenospermia induced by an epididymal protein, ovine prealbumin (oPES)]. AB - The motility of ejaculated spermatozoa (% of progressive spermatozoa) has been evaluated visually on 26 adult rams. Half the animals were immunized with a secretory epididymal protein (prealbumin epididymal-specific (PES) ovine) injected according to immunizing protocols (Freund or Hunter adjuvant) intraperitoneally or intradermally (Freund or Hunter adjuvant) or intramuscularly (Hunter adjuvant or PES only). The other animals received the same product without the protein (controls). The product resulted in a strong asthenospermia which parallels the transient presence of anti-PES antibodies in the seminal plasma. A study with 64 ewes showed that the fertility of 10 immunized rams that had recovered sufficient forward motility to ensure fertilization, did not differ from that of control rams. PMID- 7546232 TI - [Effect of progesterone on ovulation length and duration of the ovarian cycle induced by the male effect in the Barbarine ewe and the local Tunisian goat]. AB - Two experiments were conducted on 30 Barbarine ewes (Exp 1) and 40 local Tunisian goats (Exp 2) in seasonal anoestrus (May). In both experiments, half of the females received 20 mg of progesterone intramuscularly just before male introduction (day 0). Blood samples were taken at 4 h intervals from the time of introduction of the ram, for the determination of the time of the preovulatory LH surge in response to the 'ram effect' (Exp 1) and once a day from days 1 to 8 to determine plasmatic progesterone concentrations (Exp 1, Exp 2). The ovulation rate was determined by coelioscopy at days 4 and 9, and oestrus behaviour was monitored twice daily in each experiment. In the ewe, progesterone delayed the time of the preovulatory LH surge (58.8 +/- 10.1 vs 20.5 +/- 10.7 h, P < 0.001), and suppressed hypofunctional corpora lutea (0/15 vs 7/14, P < 0.001). The induced ovulation rate was not significantly different between control and treated females (1.50 +/- 0.52 vs 1.26 +/- 0.46 respectively). Only 1 female in the control group and 2 in the treated group showed oestrus behaviour at the induced ovulation. Suppression of short cycles by progesterone treatment allowed synchronization of oestrus between days 17 and 20 compared to days 14 and 23 in the control group (P < 0.001). In the goat, all induced ovulations in the control group were followed by a short cycle. In the progesterone-treated group, 3 out of 20 goats developed abnormal induced corpora lutea (P < 0.001). First oestrus occurred between days 1 and 8 in control group and between days 2 and 3 in treated goats. Treatment with progesterone increased the percentage of females showing oestrus at induced ovulation (100 vs 35%, P < 0.001). The ovulation rate at first oestrus was increased in treated goats (1.85 vs 1.35, P < 0.001). In conclusion, 20 mg progesterone administered at day 0 is efficient at preventing the occurrence of short ovarian cycles. After ovulation induced by the male effect, the percentage of females in oestrus, and the induced ovulation rate are increased by progesterone treatment. PMID- 7546234 TI - Protein kinase C is not necessary for beta-casein gene induction by prolactin in HC11 mouse mammary cells. AB - HC11 mouse mammary cells cultured in the presence of insulin, cortisol and prolactin for 24 h accumulated beta-casein mRNA. When the specific inhibitor of protein kinase C, GF 109203 X, was added to the medium with the hormones, the accumulation of beta-casein mRNA was unaltered, although the protein kinase C activity was almost completely suppressed. This suggests that protein kinase C is not strictly necessary for prolactin to induce milk protein gene expression. PMID- 7546235 TI - Effect of vitamin E supplementation on immune status and alpha-tocopherol in plasma of piglets. AB - Twelve (Yorkshire) gilts were assigned to 2 dietary fat supplement groups starting at 57 d of gestation. Group 1 received no fat and Group 2 was supplemented with 5% Canola oil. Each group was supplemented with 0.1 ppm Se and 22 IU of DL-alpha-tocopherol acetate/kg of feed. Colostrum (d 0) and milk (7, 14, 21 and 28 d post partum) were sampled from gilts. At farrowing 3 piglets from each gilt of both groups were injected with alpha-tocopherol at birth (500 IU) and at 7 and 14 d (1,000 IU) of age and 3 piglets were injected with saline and used as control. Blood samples were taken from the newborn piglets at birth and at 7, 14, 21, 28 and 35 d of age. alpha-Tocopherol concentration in the colostrum of gilts was significantly higher than in the milk. Plasma alpha-tocopherol concentrations and antibody titres to Key-hole limpet haemocyanin of piglets injected with vitamin E were significantly higher than the control piglets. Vitamin E injected piglets had significantly higher alpha-tocopherol concentrations in spleen, liver, kidney, heart, lung and hip muscle than the control piglets. PMID- 7546236 TI - Coculture of ovine zygotes fertilized in vivo or in vitro and positive effect of CZB medium on the development of in vitro fertilized zygotes. AB - The development and quality of ovine zygotes derived from in vivo (IVOF) or in vitro fertilization (IVF) were compared after coculture on sheep oviductal cells. The same criteria were used to evaluate the coculture of IVF zygotes in CZB medium for 2 d followed by 199 medium for 6 d (CZB-199 coculture) or in 199 medium for 8 d (199 coculture). A higher overall developmental rate to blastocyst stages was obtained with IVOF (65.7%) than with IVF (23.2%) zygotes. More IVF zygotes reached blastocyst stages in CZB-199 (36.7%) than in 199 coculture (22.9%). The morphological aspect did not differ significantly between IVOF and IVF or between 199 and CZB-199 blastocysts. Histological examination revealed no significant difference in the pyknotic and mitotic indices and mean number of cells in the trophoblast and in the inner cell mass of hatched blastocysts between IVOF and IVF or between CZB-199 and 199 cocultures. According to criteria used in this study, the quality of blastocysts was equivalent, independently of fertilization or coculture systems. The use of CZB medium during the first cleavages increases the proportion of blastocysts. PMID- 7546238 TI - Suppression of natural killer cell activity by monocytes following immunotherapy for recurrent spontaneous aborters. AB - PROBLEM: Natural killer (NK) cell activity has previously been shown to decrease in normal pregnancy as compared with the nonpregnancy state. The purpose of this study was to determine NK cell activity in recurrent aborters and to investigate the kinetics of NK cell activity following immunotherapy. METHODS: Recurrent aborters (N = 17) were immunized with husbands' mononuclear cells (1 x 10(8)) twice during the early stage of current pregnancy. NK cell activity of recurrent aborters as well as that of normal pregnant (N = 12) and nonpregnant (N = 6) women (controls) was determined by 51Cr release assay. Monocytes were depleted from the mononuclear cell fraction and its effect on the NK cell activity was determined as well. RESULTS: At around 5 wk of gestation, NK cell activity in recurrent aborters before treatment was significantly higher (28.0 +/- 5.1%) than that in normal pregnancy (18.9 +/- 4.3%) (P < 0.01). Following immunotherapy, NK cell activity of recurrent aborters (N = 13) who maintained their pregnancy decreased significantly (21.7 +/- 8.9%) (P < 0.05). In contrast, NK cell activity of recurrent aborters (N = 4) who aborted their current pregnancy did not decrease. Depletion of monocytes resulted in a significant increase in NK cell activity (P < 0.05). CONCLUSIONS: This study suggests that the immunotherapy induces suppression of NK cell activity which may contribute for the maintenance of pregnancy. Moreover, monocytes may be involved in this suppression. PMID- 7546237 TI - An 80-kDa syncytiotrophoblast alloantigen bound to maternal alloantibody in term placenta. AB - PROBLEM: We have shown that most of the IgG present on term syncytiotrophoblast, membrane, microvesicles is bound to an 80 kDa protein antigen (R80K). METHODS: Microvesicles were prepared from term human placenta, and the IgG eluted at pH3. RESULTS: When IgG antibody was eluted at pH3 and reacted with acid-treated vesicles of other placentae, the alloantibody always bound to the preparation from which it was obtained, but only to about 10% of acid-treated preparations from other placentae. A similar polymorphic protein found in association with IgG antibody was found in term horse placentae. Cross-reactivity of the antibodies between species was not found. Using binding of labelled antibody, complement dependent cytotoxicity and FACS two-color analysis, the human polymorphic antigen was present on peripheral blood monocytes and B-lymphocytes. The R80k antigen on intact microvesicles was resistant to trypsin, but after acid elution of IgG, trypsin released a soluble 50 kDa fragment which reacted with the acid-eluted IgG antibody. CONCLUSION: The presence of antibodies to R80K in all term placentae studied, including first pregnancies, suggests that development of this alloantibody may be a normal requirement for successful pregnancy. PMID- 7546240 TI - Clinical significance of anti-GM3 antibodies in recurrent pregnancy loss with elevated level of antiphospholipid antibodies. AB - PROBLEM: The ganglioside-GM3 neutralizes the reactivity of antiphospholipid antibodies (APLs) to phospholipids in vitro. The question of whether anti-GM3 antibodies might exert influence in APLs-positive recurrent pregnancy loss patients who are undergoing prednisolone and aspirin (PSL/ASA) treatment was investigated. METHOD: The anti-GM3 antibody assay of sera was accomplished by ELISA. Sera of 56 patients with recurrent pregnancy loss, including 30 APLs positive cases given PSL/ASA treatment, were examined. RESULTS: Patients positive for IgG or IgM type anti-GM3 antibodies constituted 13/30 (43%) of the APLs positive group as compared with only 2/26 (8%) of those who were negative (P < 0.01). In pregnant women with APLs-positive treated with PSL/ASA, live births occurred in only 6/13 (46%) patients with detectable anti-GM3 antibodies, while in 16/17 (94%) who tested negative for anti-GM3 antibodies (P < 0.01). CONCLUSIONS: This observation suggests the possibility that presence of anti-GM3 antibodies may be an indicator for determining the prognosis in recurrent pregnancy loss with elevated level of APLs. PMID- 7546242 TI - Mammalian fertilization: egg and sperm (glyco)proteins that support gamete adhesion. PMID- 7546241 TI - Gonadotropin releasing hormone (GnRH) agonist induces down-regulation of the CD3+CD25+ lymphocyte subpopulation in peripheral blood. AB - PROBLEM: To test whether GnRH agonist could alter in vivo human immune cells and whether the alteration is related to the success of pregnancy in an in vitro fertilization-embryo transfer (IVF-ET) program. METHODS: Thirty-six infertile patients were enrolled under the long protocol of GnRH agonist (buserelin acetate) and superovulation with gonadotropin from our IVF-ET program. Peripheral B cells, NK cells, CD4+ and CD8+ T cells, and the expression of CD69, CD25, HLA DR, and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. RESULTS: B cells, NK cells, CD8+ T cells, and CD71+ T lymphocyte subpopulations were not changed throughout the whole course of treatment. CD4+ T cell and CD25+ T cell subpopulations were significantly down-regulated when the GnRH agonist was used for approximately 2 wk. CD3+CD69+, CD3+CD25+, and CD3+DR+ lymphocyte subpopulations were increased at 7 days (during implantation) and at 14 days after embryo transfer in pregnant patients, but not in patients who failed to get pregnant. CONCLUSIONS: The GnRH agonist had a transiently immunosuppressive effect on CD4+ and CD25+ T cells, but CD69+, CD25+, and HLA-DR+ T cells were activated during and after successful implantation. PMID- 7546243 TI - A monoclonal antibody to an early pregnancy factor-induced suppressor factor (EPF S1) disrupts implantation in mice. AB - PROBLEM: The importance of EPF during pregnancy has been established previously but the importance of the EPF-induced suppressor factor EPF-S1 in pregnancy has to date been unaddressed. Investigations were therefore conducted in order to study this. METHOD: Monoclonal antibodies to EPF-S1 were produced, and one antibody, designated R2T gamma, was characterized. Mated mice were passively immunized with R2T gamma and the effect on implantation determined. RESULTS: Characterization of anti-EPF-S1 R2T gamma revealed that it cross-reacted with EPF S1 of different MHC restriction but not with EPF or EPF-S2. When injected into mated mice on days 1 to 4, R2T gamma had no effect on pregnancy but when injections continued to day 5, pregnancy was affected; the number of embryos implanted on day 7 were significantly less than the number of corpora lutea counted, signifying embryonic loss. CONCLUSION: These studies show that anti-EPF S1 R2T gamma disrupts implantation in mice when injected on days 1 to 5 of pregnancy but not when injected on days 1 to 4, demonstrating that EPF-S1 exerts its effects around the time of implantation. PMID- 7546239 TI - Diagnostic and prognostic significance of anticardiolipin antibodies in patients with recurrent spontaneous abortions. AB - PROBLEM: The role of ACA in unexplained RSA is controversial. In the present study, diagnostic and prognostic aspects were investigated. METHOD: One hundred five nonpregnant patients with primary, 29 with secondary RSA, and 209 controls were investigated for IgG-ACA. Follow-up studies were done during pregnancy in 76 individuals. IgM-ACA were tested in a subset of patients. RESULTS: Elevated ACA levels were significantly more frequent in both patient groups (26 and 24%) than in controls (16%). However, there was no correlation of ACA with various parameters including pregnancy outcome. In ACA-positive patients with successful pregnancy a significant decrease of ACA values during pregnancy was observed, while ACA remained high in aborting patients. IgG- and IgM-ACA correlated well. CONCLUSIONS: Although the data from nonpregnant RSA patients does not allow diagnostic or prognostic conclusions to be drawn, sequential testing of ACA positive individuals provides the possibility to foresee pregnancy outcome. PMID- 7546244 TI - In vivo immunosuppressive effects of recombinant ovine interferon-tau (trophoblastin): r.oTP (r.oIFN-tau) inhibits local GVH reaction in mice (PLN assay), prevents fetal resorptions, and favors embryo survival and implantation in the CBA/J x DBA/2 mice combination. AB - PROBLEM: Ovine trophoblastin protein, be it natural or recombinant (oTP,r.oTP), a member of the tau interferon family (r.oIFN-tau), has been shown to possess immunosuppressive properties in vitro. It acts as a cytostatic agent across species. Indeed, it was immunosuppressive when tested on human and murine lymphocytes in a variety of in vitro immune assays, as it is also on syngenic (ovine) lymphocytes. METHODS: In the present paper, we first verified that this property to act across species also occurred in vivo assays; r.oTP was able to down regulate a local GVH reaction assay (PLN assay) in mice. We then took advantage of these properties of r.oTP to investigate its in vivo effects during murine pregnancy as there is no ovine equivalent of the murine CBA/J x DBA/2 resorption prone mating combination. RESULTS: When given in the postimplantation period, r.oTP drastically boosted resorptions in the CBA/J x DBA/2 matings, as did murine recombinant gamma interferon. However, the same r.oTP treatment in the peri-implantation period resulted in a reduction in resorptions in this spontaneous abortion system. CONCLUSION: The data suggested that r.oTP might have acted more by favouring implantation and embryo survival than by preventing the resorption process itself. The mechanisms possibly underlying these effects, as well as the putative uses of r.oTP evolving from these data, are discussed. PMID- 7546246 TI - Anthropological considerations relevant to the maturation of the immune response in humans. AB - PROBLEM: The reason for the postnatal maturation of the immune response in humans was explored from the anthropological standpoint in order to gain some insight into how development in modern humans evolved and how it might have affected the immune system. METHOD: The estimation of the length of gestation in modern humans if it had followed the same developmental pattern as in the other hominids and the higher primates was calculated by extrapolation from the lengths of gestation and the physical characteristics of these other primates compared to those of modern humans. RESULTS: These calculations show that the gestation time of modern humans would be 3-18 months longer than the 9 months that it is if modern humans had followed a linear evolutionary development comparable to that of the other primates. The key difference between modern humans and the other primates is the rapid and extensive development of the brain that occurs in utero and in the first 12-18 months postnatally in modern humans. CONCLUSION: The first 12-18 months postnatally in humans follows the embryonic/fetal pattern of development, whereas that of other primates does not. The maturation of the immune response in humans occurs during both the intrauterine and extrauterine phases of the embryonic/fetal pattern of development. PMID- 7546247 TI - Characterization of latent transforming growth factor-beta from human seminal plasma. AB - PROBLEM: Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor beta (TGF-beta) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF-beta represents a family of 25-kDa homodimeric proteins linked with disulfide bonds. TGF-beta associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF-beta versus inactive latent form of of TGF-beta, and mechanism of the TGF-beta activation in human seminal plasma remain to be elucidated. PURPOSE: To characterize seminal plasma latent form of TGF-beta, including its concentration, and the mechanism underlying the activation of TGF-beta. METHOD: Gel filtrations on ACA-34 and Biogel P-60 were used to fractionate seminal plasma. TGF-beta was measured by enzyme immunoassay using antibodies specific for TGF-beta 1 and TGF-beta 2, respectively. Radioreceptor assay with recombinant human [125I]-TGF-beta 1 was applied to qualitatively identify TGF-beta 1. Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF beta 1. RESULTS: Human seminal plasma contained both TGF-beta 1 and TGF-beta 2, predominantly in latent form. The total concentration of TGF-beta 1 averaged 238 ng/ml versus an average of 18 ng/ml for TGF-beta 2. The in vitro activation or release of TGF-beta 1 from latent TGF-beta 1 was achieved only at acidic pH of < 4.0, and was time and temperature dependent. At pH 3.7 and 37 degrees C, a significant activation of latent TGF-beta 1 was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF-beta 1 remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenyl-methylsulfonyl fluoride, E-64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF-beta 1 in human seminal plasma with recombinant human TGF-beta 1. CONCLUSION: Human seminal plasma TGF-beta is biologically activated from high molecular weight latent TGF-beta by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF-beta that may immunologically protect the integrity of sperm. PMID- 7546245 TI - A cautionary note. PMID- 7546248 TI - Prevention of human recombinant interleukin-1 beta (rhIL-1 beta) embryolethality with progesterone or indomethacin. AB - PROBLEM: Bropirimine and tilorone were found in earlier studies to be embryolethal when administered to Crl:TUC(SD)spf (TUC) rats on gestation day 10. Progesterone or indomethacin could, at least partially, prevent this effect. The immunomodulators appeared to mimic the luteolytic effects of PGF2 alpha, resulting in a shutdown in progesterone release by the corpora lutea, followed by a disruption in maternal support to the pregnant uterus and embryolethality. Since bropirimine has been shown to induce interleukin-1, and since this cytokine has been found to increase PGF2 alpha levels in human decidual cells, the decision was made to investigate whether human interleukin-1 beta might act in an analogous manner to bropirimine and tilorone. METHOD: Bropirimine (400 mg/kg, p.o.) or rhIL-1 beta (20, 30, or 40 micrograms/kg, s.c.) was administered on gestation day 10 to Crl:CD[BR] (CD) or TUC rats, alone and in combination with progesterone (2 mg/kg/day, s.c.) or indomethacin (0.6 mg/day, s.c., days 9-11). On gestation day 14 the dams were killed and their uterine contents examined. RESULTS: rhIL-1 beta (30-40 micrograms/kg) was embryolethal when administered to CD or TUC rats on gestation day 10. Progesterone or indomethacin coadministration prevented, at least partially, the embryolethality seen when rhIL-1 beta was administered (30 micrograms/kg) to TUC rats. CONCLUSION: Evidence was obtained in support of the hypothesis that interleukin-1 is involved in the embryolethal actions of the immunomodulators bropirimine and tilorone. PMID- 7546249 TI - Leukocyte subpopulation changes in rats with autotransplanted endometrium and the effect of danazol. AB - PROBLEM: This study examines immune cell populations in rats with autotransplanted endometrium and determines the effect of danazol on leukocyte subsets. METHODS: As an experimental model of endometriosis, an autologous endometrial segment was implanted in the rat peritoneum. We used flow cytometry to analyze lymphocyte subsets in the peripheral blood (PB) and peritoneal fluid (PF) of the following groups of rats: no treatment, sham operation, endometrial implantation, endometrial implantation treated with danazol, and normal rats treated with danazol. RESULTS: The natural killer (NK) cell population was decreased in both the PB and PF of rats with autotransplanted endometrium. Moreover, NK cells increased in a dose-dependent manner following danazol administration. Surgery itself increased the number of peritoneal macrophages as compared with the untreated group. This elevation was suppressed partially by endometrium-implantation and was attenuated by subsequent administration of danazol in a dose-dependent fashion. CONCLUSIONS: These data suggest that ectopic endometrial cells may release immunosuppressive factors. This is the first documentation that rats with autotransplanted endometrium show the same immunologic changes as humans with endometriosis, and establishes the utility of this model for the study of endometriosis. PMID- 7546250 TI - Comparative analysis of the immunophenotypes of decidual and peripheral blood large granular lymphocytes and T cells during early human pregnancy. AB - PROBLEM: The functional role of the leukocytes in the decidual is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified. METHOD: A comparative analysis of the phenotypes of decidual and peripheral blood large granular lymphocytes (LGLs) and T lymphocytes in early human pregnancy was performed on decidual tissue and blood samples obtained from ten patients at therapeutic abortion. RESULTS: Whereas most of the decidual LGLs were found to have a CD56bright++ phenotype, most of the peripheral blood NK cells (90%) showed the classical CD56dim+ phenotype, and only a small proportion were CD56bright+ cells. Another striking difference was found in the expression of very late antigen 1 (VLA-1, CD49a): Almost all the decidual CD56bright++ LGLs, but virtually none of the peripheral blood CD56+ NK cells expressed this antigen. Further differences were found in the expression of CD16, CD44, CD45RA, CD54, and CD57. There were also differences in phenotype between T cells derived from decidual tissue and those derived from peripheral blood. Approximately 31% of the CD3+ decidual T cells expressed VLA-1, but this antigen was virtually absent on peripheral blood T cells. A further difference was seen in the expression of HLA DR. This activation antigen was found on 32 +/- 13% of the decidual T cells but only 8 +/- 5% of the peripheral blood T cells. Additionally, the proportion of cells expressing CD38 was higher among decidual than peripheral blood T cells. CONCLUSION: The findings suggest that both decidual LGLs and a subset of decidual T cells are activated and possibly play a role in the control of trophoblast growth and placental development. PMID- 7546251 TI - Immunohistochemical studies of the adult human ovary: possible contribution of immune and epithelial factors to folliculogenesis. AB - PROBLEM: Formation of primordial follicles in adult ovaries could be a cryptic process limited to relatively small areas of the ovarian cortex and occurring during a certain stage of the menstrual cycle. Such an event may require a specific milieu provided by factors involved in developmental processes, i.e., morphoregulatory molecules and macrophages. METHOD: Adult human ovaries were investigated by immunohistochemistry for surface epithelium and granulosa cell markers (cytokeratin 18 and MHC class I), immune system-related morphoregulatory molecules (Thy-1 glycoprotein and N-CAM), and macrophage phenotypes (CD14, CD68, and MHC class II). RESULTS: In some ovaries 300-500 microns areas of surface epithelium were overgrown by tunica albuginea, descended into the stroma, and apparently fragmented into individual small (20-40 microns) follicle-like cell nests. Differentiation of the surface epithelium was accompanied by macrophages and Thy-1 glycoprotein. Small segments of surface epithelium showed N-CAM and a lacked MHC class I expression. In such segments, clear spherical germ-like cells migrated into the deeper stroma, associated with the microvasculature, and eventually aggregated with follicle-like cell nests. CONCLUSIONS: Our data suggest that surface epithelium may be involved in the formation of some primordial follicles in adult ovaries. This process, and further follicular fate, may require a precise interplay of immune system related morphoregulatory molecules and macrophages. PMID- 7546252 TI - Progress in a Moscow children's burn unit: a joint Russian-American collaboration. AB - A joint Russian-American paediatric burn programme involving Childrens Hospital No. 9 in Moscow and Project HOPE in Millwood, Virginia emerged from the efforts of burn professionals from both countries in caring for a group of children seriously burned as a result of the train-pipeline catastrophe that occurred in June 1989 in the Ural Mountains. This paper describes the burn unit and its activities during the years 1985-93 and includes: (1) a general description of the physical and administrative structure of the unit; (2) the demography of burn admissions; (3) clinical activities; (4) a comparison of the clinical results of the years before the institution of the combined programme (1985-89) with those achieved during the first 4 years of the combined collaboration (1990-93). Among the important changes that have occurred since the onset of the combined programme are: (1) overall reduction in the crude burn mortality rate; (2) decrease in burn deaths in all burn size groups; (3) dramatic reduction in the length of stay of children with the deepest burns; (4) marked improvement in the take of skin grafts applied to burn wounds and an almost total elimination of complete skin graft failures. PMID- 7546253 TI - Increased physiological dead space/tidal volume ratio during exercise in burned children. AB - Exercise testing enables the simultaneous evaluation of the cardiovascular and respiratory systems' ability to perform gas exchange. The physiological responses to exercise have not been previously reported in the postburn child. This investigation was designed to evaluate residual cardiopulmonary impairment in patients convalescing from severe burns. Spirometry, lung volumes and exercise stress testing were completed on 40 children with a mean time postburn injury of 2.6 +/- 1.9 years and mean burn size of 44 +/- 22 per cent TBSA. Respiratory variables studied during exercise included expired volume, tidal volume and respiratory rate, and physiological dead space/tidal volume (VD/VT) ratios. Stress testing revealed an increased VD/VT ratio consistent with uneven ventilation-perfusion relationships. The data indicate that patients who survive thermal injury may not regain normal cardiopulmonary homeostasis. PMID- 7546254 TI - Regional skin blood flow in deep burn wounds: a preliminary report. AB - Local skin blood flow (LSBF) using the Walker's deep burn rat model was studied on the first three postburn days using the carbon-14 iodoantipyrine ([14C]IAP) perfusion method. The radioactive [14C]IAP (12.5 microCi) was infused through the femoral vein over a period of 30 seconds and the blood samples were collected by a free flow from the femoral artery at 5-s intervals to evaluate the concentration of the isotope, in the blood. At the conclusion of the infusion, the rats were guillotined and biopsies were obtained from the burned skin, unburned skin from burned rats and skin from sham control rats to assay the isotope in the skin. The LSBF was calculated from the skin tissue and plasma radioactivity data using Jay's equation. The results showed significant decreases of the skin blood flow in the deep burn wound with 4.05 +/- 1.16, 5.31 +/- 1.32 and 4.77 +/- 2.48 ml/100 g/min as compared to the LSBF of unburned skin 10.27 +/- 1.49, 12.39 +/- 2.05, 14.79 +/- 1.85 ml/100 g/min on postburn days 1, 2 and 3 (P < 0.05). The blood flow of the control group skin was 11.5 +/- 1.97 ml/100 g/min (P < 0.05). There were also significant differences of LSBF among burn wounds on postburn days 1, 2 and 3 (P < 0.05). Pathological study of the deep burn wound showed that more of the blood flow was in the subcutaneous adjacent areolar tissue, than in the deep reticular dermis, and only a little occurred in the upper reticular dermis occasionally.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546255 TI - Pain in burn patients. AB - While severe pain is a constant component of the burn injury, inadequate pain management has been shown to be detrimental to burn patients. Pain-generating mechanisms in burns include nociception, primary and secondary hyperalgesia and neuropathy. The clinical studies of burn pain characteristics reveal very clear cut differences between continuous pain and pain due to therapeutic procedures which have to be treated separately. Some of the main features of burn pain are: (1) its long-lasting course, often exceeding healing time, (2) the repetition of highly nociceptive procedures which can lead to severe psychological disturbances if pain control is inappropriate. Pharmaco-therapy with opioids is the mainstay for analgesia in burned patients, but non-pharmacological techniques may be useful adjuncts. Routine pain evaluation is mandatory for efficient and safe analgesia. Special attention must be given to pain in burned children which remains too often underestimated and undertreated. More educational efforts from physicians and nursing staff are necessary to improve pain management in burned patients. PMID- 7546256 TI - Aetiology of burn injuries in childhood in Casablanca, Morocco: epidemiological data and preventive aspects. AB - A retrospective epidemiological study is reported concerning burn injuries in 775 children hospitalized at the unit of burn care of Casablanca between 1985 and 1993. The mean age was 3 years and 8 months; 69.9 per cent of the patients were below 5 years old. The male to female ratio was 1.43 to 1.0. The most common agents were scalding liquids (69.1 per cent) followed by flames (15.2 per cent). The authors underline through this report that preventive measures depend closely on the regional, environmental and sociocultural specificity. PMID- 7546257 TI - A study of cytokines in burn blister fluid related to wound healing. AB - This report indicates that retention fluid from blisters of partial skin thickness burns, which contains relatively large amounts of cytokines and growth factors, stimulates the wound healing process. Although epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) levels were low, relatively large amounts of cytokines including platelet derived growth factor (PDGF), interleukin (IL-6) and transforming growth factor (TGF) alpha were present and these exercised stimulatory effects on wound healing. TGF beta, which plays an important role in collagen metabolism and in scar formation, was also detected. Contrary to our expectations, IL-1 alpha and beta, both of which initiate inflammation, were detected at relatively low levels whereas IL-8 levels were rather high. Various cytokines were shown to coexist in a balanced state in the retention fluids, suggesting that epithelialization might be regulated via a cytokine network operating on the wound surface. The growth of keratinocytes in culture significantly increased with the addition of 1 per cent or more of blister fluid to the medium. PMID- 7546258 TI - Effect of glycerol on intracellular virus survival: implications for the clinical use of glycerol-preserved cadaver skin. AB - Glycerol has long been used for the preservation of skin allografts. The antimicrobial activity of glycerol has not been fully documented. This paper reports the results of an investigation of a model studying the effect of glycerol on the inactivation of intracellular viruses. Two viruses--herpes simplex type I (HSV-1) and poliovirus--were cultured within human dermal fibroblasts. These intracellular viruses were incubated with 50 per cent, 85 per cent and 98 per cent glycerol at 4 degrees C and 20 degrees C for 4 weeks. Each week, the cultures in glycerol and controls in fibroblast maintenance medium were assayed for virus infectivity by examining the ability of harvested viruses to infect further fibroblasts. At 4 degrees C, 85 per cent glycerol could not fully inactivate intracellular HSV-I or poliovirus even after 4 weeks; 98 per cent glycerol inactivated intracellular HSV-I (after 3 weeks) but could not fully inactivate intracellular poliovirus after 4 weeks. At 20 degrees C, 85 per cent glycerol inactivated intracellular HSV-I (within 1 week) but could not fully inactivate intracellular poliovirus after 4 weeks; 98 per cent glycerol inactivated intracellular HSV-I (within 1 week) and inactivated intracellular poliovirus (after 2 weeks). It is suggested that, on the basis of this study, glycerol can reduce intracellular virus infectivity but that its effects are very dependent on concentration, time and temperature such that we would recommend that allograft skin be exposed to 98 per cent glycerol for a minimum of at least 4 weeks at a minimum temperature of 20 degrees C before clinical use. PMID- 7546259 TI - Activity changes of ATP synthase and adenylate kinase in rat-liver mitochondria during the early phase after burn injury. AB - Using succinate as a substrate, the activities of ATP synthase and adenylate kinase in liver mitochondria of male Sprague-Dawley rats 30 min after 20 per cent full skin thickness burns were increased in comparison with the sham burn rats, suggesting that these two enzymes might contribute to the increased rates of ATP formation and oxidative phosphorylation coupling during the early phase after burn injury. PMID- 7546260 TI - A review of skin meshers. AB - Before the widespread clinical acceptance of mesh skin grafting, expansion of full thickness skin had already been reported. The senior authors, who performed the first mesh skingrafts, have tried several variations of instruments starting with a flat block, later using a roller device with staggered cuts (Mesh Dermatome type I), and further a roller with continuous cuts and a grooved carrier (Mesh Dermatome II). Now interchangeable rollers for different expansions (Zimmer Skin Mesher) and several other genuine meshers are available. Other innovations from the literature are mentioned. This article gives a classification and details of the different models. The basic principles are reviewed. A general formula is presented, allowing objective comparison of skin meshers. PMID- 7546261 TI - A formula to calculate blood cross-match requirements for early burn surgery in children. AB - Excessive bleeding from the burn site accompanies burn surgery, and blood transfusions are therefore an essential and expensive ingredient. A formula, based on the total surface area to be involved in surgery (i.e. burn plus donor area) and the patient's total blood volume, has been in use in our hospital for calculating blood cross-match volumes, but has not been scientifically evaluated. Prospectively the predicted blood loss, based on the formula in 111 consecutive paediatric burns undergoing surgery, was compared with the actual blood loss as measured using a gravimetric method. Provided that an accurate surface area to be involved in surgery is interposed into the formula, there was a statistically highly significant correlation between the predicted and actual blood loss. Invoking this simple formula may help in decreasing unnecessary cross-matching, and discarding, of blood products. PMID- 7546262 TI - An alternative method of skin grafting: the scalp microdermis graft. AB - Although microskin grafting has been used successfully to treat major burns, when the donor skin areas are inadequate, it is still not in popular use because of the difficulties of mincing and floating procedures. Floating is expected to produce more microskin patches with the dermal side upwards than with the epidermal side upwards. Another problem is that many microskin patches will be lost in the container during floating. This problem may be solved by preparing the microskin from the second layer scalp skin. This will be a graft of hair follicle cells, which can be sowed on the wound no matter which side is upwards. Grafting was carried out on well-prepared granulation beds, or on the fat surface after tangential excision, or on the bed after fascial excision of the burn. Allograft and Biobrane were used as cover dressings. The combination of fascial excision and allograft overlay gave an acceptable result. This can be an alternative method of skin grafting for burns with very limited donor skin areas when the scalp skin is not burned. PMID- 7546263 TI - A 3-year epidemiological review of burn unit admissions in Dublin, Ireland: 1988 91. AB - A retrospective multifactorial epidemiological study of all patients admitted to the Burns Unit, St James's Hospital, Dublin during a 3-year period from January 1988 to December 1990 was undertaken. One hundred and twenty patients were admitted. All patients were aged over 14 years. The mean patient age was 48.2 years. Males accounted for 56 per cent of admissions. The mean percentage body surface area (%BSA) burned was 24.9 per cent. Flame was the cause of burns in 60 per cent of cases and produced the most extensive lesions. The home was the most common site of injury. Almost half the patients admitted from residential institutions sustained their burns in hot baths. Those aged over 60 years sustain smaller burns but are at increased risk from thermal injury. Twenty-one per cent of burns were caused by open fires used for heating the home. The mean time in hospital was 49.9 days. Twenty-three per cent of patients died as a result of their injuries. We have identified those living in residential institutions to be at increased risk from scald burns and suggest methods by which they may be protected. PMID- 7546264 TI - Exuberant granulation tissue mimicking vascular tumours associated with burns. AB - A case of simultaneous development of multiple vascular neoplasms in a patient with a previous history of burns and lymphoblastic lymphoma is reported. Microscopic examination revealed angiomatosis made up of diffuse capillary proliferation. We speculate that endogenous factors could have played an important role in the development of these neoplasms. We discuss the clinical and histological differential diagnosis of the case presented: a disseminated variant of lobular capillary haemangioma, Kaposi's sarcoma, bacillary angiomatosis and hyperplastic granulation tissue. PMID- 7546265 TI - Pelvic abscess induced by a methicillin-resistant Staphylococcus aureus from haematogenous spread via the CVP line in a burn patient. AB - A 38-year-old female patients was found accidentally to have a positive culture of MRSA from a routine CVP catheter tip culture 1 week after she had complete wound closure. She was recovering from a partial skin thickness burn covering 42 per cent TBSA on the trunk and extremities. Fever and hip pain developed abruptly 1 week later when she was ready for discharge from hospital. Magnetic resonance imaging (MRI) of the pelvis disclosed an intramuscular abscess. Open drainage was performed and pus culture yielded a MRSA with the same sensitivity profile as the previous CVP tip culture. Vancomycin 500 mg every 6 h was used for 3 weeks until the drain culture disclosed a negative result, and a follow-up MRI indicated a loss of the abscess space. Follow-up at an outpatient clinic 3 months later showed that the patient remained symptom free. In this patient haematogenous dissemination was the most likely route of pelvic abscess formation. It should be remembered that MRSA infection is not always only a local problem, especially in the immunocompromised condition of burn injury. PMID- 7546266 TI - Extensive thrombosis of the caval venous system after central venous catheters in severely burned patients. AB - Central venous thrombosis is a rare but extremely dangerous complication following central venous catheter placement. Two cases of massive central venous thrombosis in severely burned patients are reported. The clinical course, diagnostic problems and treatment options are described and discussed. From these cases we conclude that central venous catheter thrombosis should be included early in the differential diagnosis of trunk and extremity oedema in patients with indwelling central venous catheters and that thrombolytic therapy is safe and effective even in the severely burned patient. PMID- 7546268 TI - Recent references. PMID- 7546267 TI - Stenosing tenosynovitis from migrated skin staples. PMID- 7546269 TI - Intraoperative ultrasonography of the liver. AB - Intraoperative ultrasound of the liver is a versatile and valuable adjunct to surgical inspection and palpation. The technique allows visualization of lesions that cannot be detected by routine cross-sectional imaging studies, allowing for more efficient and effective patient management. Demonstration of the proximity and the interrelationship of lesions to surrounding important vascular structures can be invaluable to the surgeon in choosing the most appropriate therapeutic option and in planning segmental resections. Specially developed intraoperative ultrasound probes can be used on standard diagnostic ultrasound B-mode machines. Electronic linear and curved-array scanners are most commonly used, with a 5-MHz frequency being optimal for liver imaging. Additional intraoperative uses for liver ultrasound include ultrasound-guided tumor cryoablation, ethanol injection, biopsy guidance, and laparoscopic ultrasound. PMID- 7546270 TI - CT-pathology correlation of pulmonary tuberculosis. AB - Pulmonary tuberculosis is a chronic granulomatous disease characterized radiologically by nodules, air-space consolidation, propensity for cavitation, and calcification, and pathologically by caseating granulomas or pneumonia, and a great propensity for fibrosis and dystrophic calcification. Primary tuberculosis typically appears as air-space consolidation with hilar or mediastinal lymphadenitis. Postprimary tuberculosis appears most commonly as nodular and linear opacities at the lung apex. CT findings of early bronchogenic spread of postprimary tuberculosis are centrilobular 2- to 4-mm nodules or branching linear structure, 5- to 8-mm poorly defined nodules, lobular consolidation, cavities, and thickening of interlobular septa. Centrilobular nodules or branching linear lesions and poorly defined nodules on CT scan correspond to caseation materials filling the bronchioles, and centrilobular air-space consolidation with caseation necrosis. Poorly defined nodule or lobular consolidation usually consists of central caseation necrosis and peripheral nonspecific inflammation. Cavitation usually occurs at the centrilobular area and may progress to a larger coalescent cavity. With antituberculous therapy, resolution typically occurs from the peripheral portion of the poorly defined nodule or lobular consolidation and results in varying degrees of fibrous bands, bronchovascular distortion, emphysema, and bronchiectasis. Miliary tuberculosis appears as well-defined randomly distributed nodules on CT scan that correspond with nodules consisting of central caseation necrosis and peripheral epitheloid and fibrous tissue. Analysis of CT images on the basis of pathologic correlation is helpful in understanding the morphology of pulmonary tuberculosis. PMID- 7546273 TI - Progression of HIV infection is associated with HLA-DQ antigens in Caucasians and African Americans. AB - In our previous work with human leukocyte antigen (HLA) association in human immunodeficiency virus (HIV) infection, African Americans (Afr Ams) and Caucasians (Caucs) exhibited HLA markers that were associated with protection or disease. The present study was designed to establish if HLAs were associated with the severity of HIV infection and progression to AIDS in Afr Am and Cauc adults. The frequency of serologically determined antigens (Ags) in the regional control population was compared to the HIV-infected population and the HIV-infected slow progressors were compared to rapid progressors by race. chi 2 analysis with Bonferroni adjustment, Kaplan-Meier survival analysis, linear logistic regression, Cox model of proportional hazards and standardized deltas were applied as applicable. Immune parameters were monitored over a mean follow-up period of 23 +/- 2 months for Afr Ams (n = 35) and 25 +/- 5 months for Caucs (n = 24). A better prognosis in the HIV+Afr Am group was associated with HLA-DQ1 with a risk ratio of 0.295. In the HIV+Cauc group, a preferable prognosis was associated with HLA-DQ3 with a risk ratio of 0.11, and a poor prognosis was associated with HLA-DQ2 with a risk ratio of 7. Afr Am haplotypes that appeared to have the greatest association with rapid progression of HIV infection were A69(28)-B40 and related haplotypes as well as B12-DR14(6). Cauc haplotypes with the strongest association with rapid and slow progression of HIV infection were A28-B17-DR9 and A30(19)-B67, respectively. The DR Ags of at least one haplotype that led to rapid progression in both races were associated with DQ9(3). An 'immune response' gene (DQ region) may control the progression of HIV infection in adults. The rapidly progressive DQ-associated peptide might block the progression of HIV if given as a novel vaccine. PMID- 7546272 TI - Melanocyte-stimulating hormone: a regulator of human melanocyte physiology. AB - Although the melanocyte-stimulating hormone (MSH) is well-recognised as a pigmentary hormone in animals, its role in human pigmentation is still a matter for conjecture, not least because cultured human melanocytes have proved to be relatively refractory to the peptide. However, recent work has shown that human melanocytes can respond to MSH peptides and the related adrenocorticotropic hormone. While the pigmentary responses are the most studied, they are by no means the only effects of these peptides on human melanocytes. This article reviews recent work on the responses of human melanocytes to MSH peptides and demonstrates that these peptides may be key regulators of human melanocyte physiology. PMID- 7546271 TI - Comparative studies on vascular endothelium in vitro. 2. Hypoxia: its influences on endothelial cell proliferation and expression of cell adhesion molecules. AB - Recent studies have presented evidence that the processes of hypoxaemia and reperfusion are involved in several pathogenetic mechanisms of atherosclerotic lesions. The ability of hypoxaemia to activate circulating white blood cells (WBCs) and enhance WBC-endothelial cell (EC) interactions is suspected to be a major factor in deleterious processes in the blood vessel wall. Various groups have suggested that cell adhesion molecules (CAMs), such as ICAM-1, VCAM-1 and E selectin and their leukocyte ligands are involved in intercellular activities of the relevant cell types. We studied the effects of different oxygen tensions, simulating normoxic conditions, hypoxia and hyperoxia in vitro with the help of an umbilical vein EC model in order to determine the effects of oxygenation on CAM presentation on vascular ECs with and without further cytokine and endotoxin (lipopolysaccharides; LPS) stimulation. Semiquantitative analysis of ICAM-1, E selectin and VCAM-1 was performed using cell enzyme immunoassay techniques. The presentation of ICAM-1, E-selectin and VCAM-1 remained on the whole unaffected by both hypoxia and hyperoxic conditioning after both 7 and 24 h. Stimulation of ICAM-1 by cytokines and LPS was only marginally influenced by the oxygen tension. Cytokine induction of E-selectin was not affected after 7 h and was even reduced under hypoxia, compared to the control culture after 24 h, while stimulation was increased by hyperoxia. VCAM-1 was reduced in both the hypoxic and hyperoxic culture, while being maximally stimulated by cytokines and LPS after 7 h. In general, an effect of hypoxia was not found without any further stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546274 TI - In vitro proteolysis of the red cell membrane in patients with HIV infection. AB - It has been suggested that acquired abnormalities of the red cell membrane due to various injuries [azidothymidine (AZT) therapy, immunoglobulin coating of red cells, differentiation abnormalities of erythroid precursors] contribute to the onset of anaemia in HIV-infected patients. In vitro proteolysis of erythrocyte membrane proteins is regarded as a molecular marker of membrane damage induced in vivo by different agents. We therefore investigated in vitro proteolysis of ghosts derived from red blood cells of 30 HIV-infected patients. Considered collectively, there was no significant increase in in vitro proteolysis in ghosts from anaemic HIV patients. However, a significantly higher degree of in vitro self-digestion of RBC membrane proteins was evident in HIV-infected patients with spleen enlargement, but not in splenomegalic patients suffering from liver cirrhosis. Neither AZT therapy nor the presence of a positive direct antiglobulin test seemed to be directly associated with increased in vitro protein breakdown. The results seem to suggest damage of the red cell membrane in HIV infection, induced by injuries on red cells during their prolonged retention inside an enlarged spleen, while it seems unlikely that AZT therapy or immunoglobulin coating of red cells play major roles in red cell damage. PMID- 7546277 TI - Biology, diagnosis and therapy of mastocytosis. PMID- 7546276 TI - Detection by polymerase chain reaction of all common Mycoplasma in a cell culture facility. AB - The identification of cell cultures contaminated with organisms from the class Mollicutes has led us to examine the effectiveness of polymerase chain reaction (PCR) for detecting these organisms in genomic DNA. We developed a previously identified nested PCR primer set and compared its ability to detect Mycoplasma with that of a commercially available PCR kit for detecting Mycoplasma. We found that although the commercial system detected and identified a few of the most common Mycoplasma species, the primer set (GPO-1, GPO-2, MGSO) detected the presence of all the common Mycoplasma species and many of the rare mycoplasma species previously encountered in tissue culture. PMID- 7546275 TI - Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages. AB - Systemic sclerosis (SSc) is a generalized autoimmune disorder characterized by immunological abnormalities, microvascular dysfunction, and tissue fibrosis. This study evaluated the inflammatory processes occurring in skin of 7 patients with SSc of recent onset (average disease duration of 10 +/- 3 months) to assess the involvement of monocytes/macrophages during the early stages of SSc. All SSc skin biopsies displayed inflammatory microvascular endothelial cell activation and fibrosis. Increased numbers of infiltrating inflammatory leukocytes were present in affected skin of recent onset SSc (p < 0.01) mainly consisting of CD14 positive monocytes/macrophages (p < 0.02). CD3 T lymphocytes were only slightly elevated in SSc skin (84 +/- 39) compared to normal (51 +/- 12), but the differences were statistically not significant. The ratio of CD14/CD3 cells was substantially higher in affected skin of recent onset SSc (4.0 +/- 2.0) than in normal skin (1.4 +/- 0.5, p < 0.01). Monocytes/macrophages, therefore, are the predominant infiltrating mononuclear cell in skin lesions of recent-onset SSc. These results strongly suggest that CD14-positive monocytes/macrophages play an important role during the early stages of SSc pathogenesis. PMID- 7546279 TI - Role of mast cells in atherosclerosis. PMID- 7546278 TI - Mast cells and basophils in adverse reactions to drugs used during general anesthesia. PMID- 7546281 TI - Role of mast cells and basophils in gastrointestinal inflammation. PMID- 7546280 TI - What are human heart mast cells for? PMID- 7546282 TI - Role of mast cells in arthritis. PMID- 7546283 TI - Role of mast cells and basophils in asthma. PMID- 7546284 TI - Mast cells and tumor-associated angiogenesis. PMID- 7546285 TI - Role of mast cells and basophils in rhinitis. PMID- 7546286 TI - Role of basophils and mast cells in acute and late reactions in the skin. PMID- 7546287 TI - Activation of Hox gene expression by Hoxa-5. AB - The Hox genes are expressed during embryonic development and have a role in specifying antero-posterior positional information. The genes are arranged in four clusters and a colinear relation exists between a gene's position in the cluster and its anterior boundary of expression. Genes with more anterior boundaries are also expressed earlier than genes with more posterior boundaries. Hox genes encode transcription factors; therefore, a model for the coordinate regulation of the genes within the Hox clusters is that Hox gene products regulate their own expression. To test this model, an inducible promoter was used to direct expression of exogenous Hoxa-5 in F9 embryonal carcinoma cells and the effect on endogenous Hox gene expression was measured using RNase protection assays. The production of Hoxa-5 from the expression vector activated a transient and simultaneous expression of other upstream and downstream genes of the same Hox cluster and genes from other clusters. There was an 8-hr delay between the peak of expression from the Hox vector and the endogenous Hox gene response, suggesting that Hox proteins activate other Hox genes indirectly or require additional factors which, in F9 cells, they also induce. PMID- 7546288 TI - Expression of the rat, S-100-related, calcium-binding protein gene, p9Ka, in transgenic mice demonstrates different patterns of expression between these two species. AB - p9Ka (also known as mts1/18A2/calvasculin/CAPL) is a member of the S-100-related family of small, calcium-binding proteins. Previous studies suggest apparent discrepancies between the expression of the p9Ka gene in rat, mouse, and human tissues. Here we demonstrate that the natural p9Ka gene is expressed at lower levels in mouse than in rat, and that, in mouse but not in rat, p9Ka mRNA is more highly expressed in cells of lymphoid origin. Transgenic mouse strains express rat-p9Ka transgenes in a gene copy-number-dependent manner. The rat p9Ka transgene mRNA shows the same tissue distribution in several lines of transgenic mice, a distribution that is characteristic of the rat, from which the transgenes were derived. These results show that there is a difference in the pattern of expression of the same gene in two closely related species, and that the pattern of expression found in rat is specified by the DNA in the rat gene itself. PMID- 7546289 TI - Use of the teleost saccule to identify genes involved in inner ear function. AB - The vertebrate inner ear sensory epithelia contain different types of hair cells and supporting cells. The teleost saccule is anatomically similar to the mammalian saccule and is primarily involved in the detection of translational acceleration and orientation with respect to gravity. To facilitate molecular studies of the teleost saccule cDNA libraries were constructed from microdissected Lepomis macrochirus (bluegill sunfish) saccular maculae. To our knowledge, this is the first report of cDNA libraries constructed from the saccule. In one instance, a non-polymerase chain reaction-based method of amplifying a mRNA population from limited amounts of starting tissue was employed that allowed construction of cDNA libraries from nanogram amounts of tissue mRNA. Conventional cDNA libraries were constructed from the sunfish saccular maculae as well. These cDNA libraries enriched in hair cell and supporting cell transcripts should facilitate molecular biological studies of inner ear sensory epithelia. As an example of their utility, efforts to identify tyrosine kinases expressed in the saccular endorgan using low-stringency hybridization screening of these cDNA libraries and the partial sequence of a cDNA found to encode an erbB-2-related tyrosine kinase are also reported. PMID- 7546290 TI - Molecular cloning of a cDNA encoding human spermine synthase. AB - We have isolated and sequenced cDNA clones that encode human spermine synthase (EC 2.5.1.22). The total length of the sequenced cDNA was 1,612 nucleotides, containing an open reading frame encoding a polypeptide chain of 368 amino acids. All of the previously sequenced peptide fragments of human and bovine spermine synthase proteins could be located within the coding region derived from the cDNA. An unusual sequence of AATTAA apparently signaled the initiation of polyadenylation. Sequence comparisons between human spermine synthase and spermidine synthases from bacterial and mammalian sources revealed a nearly complete lack of similarity between the primary structures of these two enzymes catalyzing almost identical reactions. A modest similarity found was restricted to a relatively short peptide domain apparently involved in the binding of decarboxylated S-adenosylmethionine, the common substrate for both enzymes. The apparent lack of an overall similarity may indicate that spermine synthase, the enzyme found only in eukaryotes, and spermidine synthase with more universal distribution, although functionally closely related, have evolved separately. PMID- 7546291 TI - The human type II 17 beta-hydroxysteroid dehydrogenase gene encodes two alternatively spliced mRNA species. AB - The isozymes of the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family are responsible for the formation of the 17 beta-hydroxysteroids delta 5 androstene-3 beta,17 beta-diol, testosterone, 17 beta-estradiol, and dihydrotestosterone from their corresponding 17-ketosteroid precursors, thus playing a pivotal role in the formation of active sex steroids in both steroidogenic and peripheral target tissues. To clone the type II 17 beta-HSD gene, the full-length cDNA type II 17 beta-HSD was used as probe to screen a human leukocyte genomic DNA library. The type II 17 beta-HSD gene contains seven exons and spans > 40 kbp. The type II 17 beta-HSD gene encodes two alternatively spliced mRNAs that give rise to the previously identified type IIA 17 beta-HSD protein of 387 amino acids, as well as to a related 291-amino-acid type IIB 17 beta-HSD protein of unknown function. RNA blot analysis revealed the presence of a major 1.45-kb transcript that is abundant in placenta and endometrium. The mRNA cap site has been localized in a region between 179 and 167 nucleotides upstream of the ATG start codon by RNase protection and S1 nuclease mapping analyses. Cloning of the 17 beta-HSD type II gene provides us with the tools to study its transcriptional expression. PMID- 7546292 TI - The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c. AB - The two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, are major integral membrane proteins of the lysosomes. They also occur in the plasma membrane, where they have been discovered independently as principal lactosaminoglycan-bearing glycoproteins and as tumor antigens. Avian LAMP-2 has recently been shown to be encoded by at least three transcripts resulting in variant transmembrane and cytoplasmic domains (Hatem et al., 1995). We report isolation and characterization of chicken genomic clones indicating that the three transcripts are the result of alternative splicing of a single LAMP-2 gene. Only a single LAMP-2, homologous to chicken LAMP-2a, has been described in mammals. To ascertain whether multiple forms of LAMP-2 also occur in mammals, we cloned cDNAs encoding LAMP-2 variants homologous to avian LAMP-2b and LAMP-2c from mouse brain cDNA libraries. Thus, the family of LAMP-2 proteins is conserved from bird to mammals and the diversity is generated by alternative splicing of a single LAMP-2 gene. PMID- 7546293 TI - Promoter binding factors regulating cyclin B transcription in the sea urchin embryo. AB - Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors that bind between -200 and -90 bp from the start of transcription. PMID- 7546298 TI - [The prospects and tasks of health in the Americas]. PMID- 7546300 TI - The Function of Sleep. Proceedings of an international symposium. Ravello, Italy, May 28-31, 1994. PMID- 7546296 TI - [Ultramicro-ELISA for measuring tetanus antitoxin in human serum]. AB - A solid-phase enzyme immunoassay (ELISA) for measuring tetanus antitoxin activity in human serum is described; the assay is based on a combination of the indirect method and ultramicro analysis. This rapid test, which has the capacity to analyze 78 blood samples per reagent plate (at a volume of 10 microL of diluted serum per sample), is proposed as an alternative to the traditional mouse bioassay system based on the neutralization of a known dose of tetanus toxin. Results from both tests showed a high correlation in the lineal regression analysis (r = 0.99; CI95%: 0.985 to 0.993). It is recommended that the ultramicro ELISA assay be used in the field to evaluate tetanus toxoid vaccines and to identify hyperimmune plasmas suitable for producing antitetanus immunoglobulin. PMID- 7546301 TI - EEG slow waves and sleep spindles: windows on the sleeping brain. AB - Slow waves and sleep spindles are prominent features of the EEG in non-REM sleep and some of the neurophysiological mechanisms underlying their genesis have been elucidated. In humans, slow-wave activity in non-REM sleep increases and EEG activity in the frequency range of sleep spindles decreases when wakefulness prior to sleep is varied from 2 to 40 h. The opposite changes are observed in the course of sleep, even when sleep is scheduled out of phase with the circadian rhythm of sleep propensity. Within non-REM sleep episodes the association between slow waves and sleep spindles is bi-phasic: both activities are correlated positively at the beginning and end of non-REM sleep episodes whereas in the middle part of non-REM sleep episodes high values of slow-wave activity coincide with low levels of spindle activity. An extension of wakefulness enhances the rise rate of slow-wave and spindle activity at the onset of sleep. Since macroscopic slow waves and sleep spindles both are dependent on hyperpolarization and synchronization of neurons in thalamo-cortical and cortical circuits, the sleep deprivation induced changes in these EEG activities may be related to reduced activating input to thalamo-cortical and cortical neurons, local facilitation of their hyperpolarization or facilitation of their synchronization. The precise regulation of slow-wave and spindle activity as a function of the duration and intensity of prior sleep and wakefulness demonstrates that these EEG oscillations are accurate indicators of non-REM-sleep homeostasis and suggests that they are fundamental to the sleeping brain. PMID- 7546295 TI - Tissue-specific alternative promoters regulate the expression of the two sarco/endoplasmic reticulum Ca-ATPase isoforms from Artemia franciscana. AB - The sarco/endoplasmic reticulum Ca-ATPase gene from Artemia franciscana is transcribed into two mRNAs of 4.5 and 5.2 kb that code for protein isoforms differing at their carboxyl terminus. Northern blot assays and anchored polymerase chain reaction (PCR) experiments have shown that these two mRNAs also differ at the initial part of their 5' untranslated region. The 5.2-kb mRNA specific 5' untranslated region is present as an independent exon whose transcription is regulated by a promoter different from the one previously described that regulates the expression of the 4.5-kb mRNA. The nucleotide sequence of the 5.2-kb mRNA promoter and the transcription initiation site have been determined. These results suggest that the expression of the two protein isoforms is regulated in A. franciscana at the transcription initiation step, in contrast with the vertebrates sarco/endoplasmic reticulum Ca-ATPase genes 1 and 2 which have unique promoters for transcription of the two isoforms encoded by each gene. PMID- 7546299 TI - A functional role for REM sleep in brain maturation. AB - The biological function of REM sleep is defined in terms of the functions of neural processes that selectively operate during the REM sleep state. The high amounts of REM sleep expressed by the young during a period of central nervous system plasticity suggest that one function of REM sleep is in development. The phenomenon of activity-dependent development has been clearly shown to be one mechanism by which early sensory experience can affect the course of neural development. Activity-dependent development may be a ubiquitous process in brain maturation by which activity in one brain region can influence the developmental course of other regions. We hypothesize an ontogenetic function of REM sleep; namely, the widespread control of neuronal activity exerted by specific REM sleep processes help to direct brain maturation through activity-dependent developmental mechanisms. Preliminary tests of the hypothesis have been conducted in the developing feline visual system, which has long been known to incorporate information derived from visual experience in establishing neuronal connectivity. We find that suppression of REM sleep processes by an instrumental REM deprivation procedure results in a significant enhancement of the effects of altered visual experience by monocular occlusion. Bilateral brainstem lesions that selectively block the occurrence of ponto-geniculo-occipital (PGO) waves are sufficient to produce similar results. These data indicate that the propagation of phasic influences during REM sleep interacts with other processes subserving neural development. This source of influence appears not to derive from the environment but rather stems from an intrinsic source of genetic origin. Examination of the neural activity associated with PGO waves in the lateral geniculate nucleus reveals a distribution of facilitatory influence markedly different from that induced by visual experience. We conclude that REM sleep directs the course of brain maturation in early life through the control of neural activity. PMID- 7546297 TI - [HIV infection and its effects on tuberculosis endemic in Chile]. PMID- 7546294 TI - Caenorhabditis elegans UBC-1, a ubiquitin-conjugating enzyme homologous to yeast RAD6/UBC2, contains a novel carboxy-terminal extension that is conserved in nematodes. AB - The RAD6/UBC2 gene from Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme involved in DNA repair, induced mutagenesis, and sporulation. Here we report the isolation and characterization of the Caenorhabditis elegans RAD6 homolog designated ubc-1. Ubc-1 encodes a 21.5-kD protein that shares considerable identity with RAD6 (66%) as well as with other RAD6 homologs, including Schizosaccharomyces pombe rhp6+ (70%), Drosophila melanogaster Dhr6 (83%), and the two human homologs HHR6A and HHR6B (84% and 83%, respectively). However, UBC-1 is distinct in being the only known RAD6 homolog, other than RAD6 itself, with a carboxy-terminal extension. Analysis of UBC-1 homologs from C. briggsae and Ascaris suum show that the presence of the carboxy-terminal extension is conserved in nematodes. When constitutively expressed from the yeast promoter ADH1, ubc-1 complements the DNA repair functions in a S. cerevisiae rad6 delta mutant. Surprisingly, ubc-1 fails to complement the sporulation function of RAD6, despite its possession of an acidic carboxy-terminal tail. C. elegans UBC-1 is capable of forming a thiol-ester bond with ubiquitin, but, unlike RAD6, is unable to transfer ubiquitin to histone H2B in vitro. Both cis and trans splicing are involved in the maturation of the ubc-1 transcript. The presence of the SL2 trans-splice leader in the ubc-1 transcript suggests that ubc-1 may be co transcribed as part of a polycistronic message. PMID- 7546302 TI - The function of sleep is to regulate brain excitability in order to satisfy the requirements imposed by waking. AB - A hypothesis is proposed which suggests that the requirements imposed by waking on a day to day basis dictates the way organisms sleep. This generates 'somnoprints' which are characteristic patterns of sleep directly related to events of waking. REM sleep is suggested to be the phase which contends with waking requirements by manifesting levels of excitability which have the dual function of generating the phase and maintaining it for period durations which are determined by the functional requirements imposed by the moment. Since the brain cannot maintain long periods of excitation, it always alternates with SWS for homeostatic reasons. The hypothesis includes the justification for the diversity of chemical signals regulating sleep, by suggesting that waking activities generate different needs which in turn generate different chemical signals and activate different set of neuronal nets who increase their excitability levels to maintain sleep through varying pathways. PMID- 7546303 TI - Processing of learned information in paradoxical sleep: relevance for memory. AB - After a short review of the post-learning paradoxical sleep (PS) deprivation effects and of the PS changes induced by learning, we present a set of electrophysiological and behavioural experiments showing that: (1) processing of relevant information is possible during PS; (2) new associations can be formed during PS; (3) previously learned information can be reprocessed during PS; and (4) the effects of information processed during PS can be transferred to the awake state and be expressed in behaviour. Altogether, these results support the idea that dynamic processes occurring during post-learning PS can contribute to the effectiveness of memory processing and facilitate memory retrieval in wakefulness. PMID- 7546304 TI - The function of fetal/neonatal rapid eye movement sleep. AB - The hypothesis is put forward that rapid eye movement (REM) sleep in early life serves as (1) an indicator for the degree of brain maturation and (2) the promoter of further brain development. This hypothesis, although not exclusive, differs (a) from the theory of Roffwarg et al. that REM sleep substitutes for 'wakefulness' during the period (early life) in which wakefulness is limited, (b) from the theory of Crick and Mitchson, i.e., the 'unlearning' hypothesis of REM sleep, (c) from the theory of Jouvet, i.e., that REM sleep is a time for genetic read-out and (d) from the theory of Freud, i.e., that dreams fulfil our wishes (in other words, activation of neuronal systems that were disproportionally activated during wakefulness). PMID- 7546305 TI - Sleep states and memory processes. AB - Evidence for the involvement of rapid eye movement (REM) sleep or paradoxical sleep (PS) with memory processing continues to accumulate. In animals, there is continuing evidence of relatively small, vulnerable paradoxical sleep windows (PSWs) following successful acquisition. These PSWs, which manifest as increases in PS over normal levels, appear to exhibit shorter latencies to onset when the amount of material presented during acquisition is increased. Prevention of the PSW results in memory deficits. In humans, there is now evidence that different types of tasks are differentially sensitive to rapid eye movement sleep deprivation (REMD). Memory for declarative or explicit types of tasks appear not to be affected by REM sleep loss, while memory for cognitive procedural or implicit types of material are impaired by REMD. Using post training auditory stimulation during REM sleep, memory enhancement of the procedural material is also possible. The memory for a fine motor task appears to be sensitive to post training stage 2 sleep loss. The important neural structures are generally not yet identifiable, although the hippocampus would appear to be important for place learning in the Morris water maze. PMID- 7546307 TI - The sequential hypothesis of the function of sleep. AB - In addition to modulatory roles concerning bodily functions, sleep is assumed to play a main processing role with regard to newly acquired neural information. Elaboration of memory traces acquired during the waking period is assumed to require two sequential steps taking place during slow wave sleep (SWS) and eventually during paradoxical sleep (PS). This view is suggested by several considerations, not the least of which concerns the natural sequence of appearance of SWS and PS in the adult animal. While the involvement of PS in memory processing is well documented, the involvement of SWS is supported by the results of baseline and post-trial EEG analyses carried out in rats trained for a two-way active avoidance task or a spatial habituation task. Together with control analyses, these data indicate that the marked increase in the average duration of post-trial SWS episodes does not reflect the outcome of non-specific contingent factors, such as sleep loss or stress, but is related to memory processing events. Several considerations have furthermore led to the proposal that, during SWS, after a preliminary selection step, the first processing operation consists in the weakening of non-adaptative memory traces. The remaining memory traces would then be stored again under a better configuration during the ensuing PS episode. This view is in agreement with several relevant features of sleep, including the EEG waveforms prevailing during SWS and PS, as well as the ontogenetic sequence of appearance of SWS and PS. Some theoretical considerations on the role of sleep are also in agreement with the sequential hypothesis. More recent data indicate that the learning capacity of rats is correlated with several baseline EEG features of sleep and wakefulness. They include the average duration of PS episodes and of SWS episodes followed by wakefulness (longer in fast learning rats), and the waking EEG power spectrum of fast learning rats whose output is more balanced in the frequency range below 10 Hz than in slow learning and in non-learning rats. Additional EEG data suggest that fast learning rats may accomplish 'on line' processing of newly acquired information according to a sequence of events not dissimilar from the one proposed by the sequential hypothesis. PMID- 7546306 TI - REM sleep and neural nets. AB - The broad features of rapid eye movement (REM) sleep are reviewed. Memory storage in the brain is probably quite unlike that in a digital computer, being distributed, superimposed and robust. Such memory systems are easily overloaded. If the stored memories share common features, random stimulation often produces mixed outputs. Simulations show that such overloading can be reduced by a process we call 'reverse learning'. We propose that this process is what is happening in REM sleep and that it explains in an unforced manner the condensation commonly found in dreams. Evidence for and against the proposed theory is discussed and several alternative theories are briefly described. The absence of REM sleep in the Enchidna and in two species of dolphins (that have relatively large brains) suggests that REM may allow the brain to be smaller than if REM were lacking. PMID- 7546309 TI - Brain organization and sleep function. AB - A view of brain organization and sleep function is presented. Sleep is hypothesized to begin at the neuronal group level. Sleep results in the use and thus maintenance, of synapses that are insufficiently stimulated during wakefulness thereby serving to preserve a constancy of a synaptic superstructure. It is further hypothesized that sleep at the neuronal group level is regulated by the production of substances whose rate of production or catabolism is synaptic use-dependent. If sufficient number of neuronal groups are in a sleep state (also called disjunctive state) then the perception of sleepiness occurs. Coordination of neuronal group sleep results from humoral and neuronal projection systems previously linked to sleep regulation. The theory presented is unique in that it: (a) hypothesizes an organizational level at which sleep occurs; (b) hypothesizes that sleep is neuronal--use-dependent, not wakefulness-dependent; (c) hypothesizes that sleep first occurs in evolution when complex ganglia evolved; and (d) hypothesizes the both non-rapid eye movement sleep (NREMS) and REMS serve the same function of synaptic reorganization. The theory is consistent with past theories of sleep function, yet provides a fundamentally new paradigm for sleep research. PMID- 7546308 TI - Possible mechanisms for reducing memory confusion during sleep. AB - Confusion readily occurs in memory processes between patterns that overlap substantially. Possible mechanisms are considered that might operate in an automatic manner to reduce such confusion. One such mechanism is the recall of patterns in a distorted way, so that they are enriched with greater activity in distinctive elements of experienced patterns than in overlapping elements. Selective consolidation based on such enriched patterns would reduce confusion in long-term recall and might benefit discrimination learning. It is shown how automatic algorithms could achieve this through a process with two phases. In the first phase, somewhat analogous to slow-wave sleep, it is necessary for the normal tendency of the nervous system to learn correlations of associated activity to be disabled. The second phase must occur while the cells most active in the first phase are relatively inexcitable. Enriched patterns would be generated during this phase through recall, which might be triggered by bursts of activity such as occur in rapid eye movement sleep. Selective consolidation would take place during the second phase. If such processes do occur in the nervous system, it seems likely that they would have evolved to occur during sleep. PMID- 7546310 TI - An integrative analysis to sleep functions. AB - Various studies suggest that some sleep functions, especially some slow wave sleep functions, are indispensable in mammals and related to brain regulation. It has been proposed that two of these functions are the adjustment of emotional balance and the processing of acquired emotional memories. During waking, the gradual accumulation of various randomly learned emotional memories in the limbic structures would inevitably imbalance and disorganize emotional behaviors. Although the emotional balance can be restored during waking by the ascending NA, DA, ACh and 5-HT systems, their roles in memory retention and emotional regulation may sometimes be dissociated and their adjustment of the emotional balance can only be a transient effect. On the other hand, the function of slow wave sleep for emotional adjustment can be long-lasting and is in agreement with its function on the processing of emotional memories. As a result, these sleep functions become indispensable in preventing the emotional imbalance inevitably caused by the accumulation of emotional memories. The effects of rapid eye movement sleep on memory and emotional regulation are just opposite to those of slow wave sleep. Low vigilance is required as premise for sleep to accomplish these indispensable functions. PMID- 7546311 TI - Mind and consciousness during sleep. AB - The epistemological-methodological constraints for an approach to dream as an object of scientific-experimental investigation are pointed out, along with the suggestion of a new memory paradigm, that utilizes the linguistic convention ('word play') proposed by Wittgenstein. A model of dream generation is proposed, in which mnemonic materials activated from long-term memory stores enter the dream production system (bottom-up process); such materials are elaborated and interpreted by a top-down process. A circular feedback activity, operating between these two components, is able to activate additional mnemonic materials. Some experimental results, concerning the incidence of dream recall in different stages of sleep, show evidence of the continuity of dreaming activity throughout the period of sleep. The relationships between dream and consciousness/awareness are then considered. Experimental data suggest that consciousness, interpreted as awareness (phenomenal awareness; meta-awareness; self awareness), can be present along the whole sleep/dream process, even if many modifications of meta-awareness and of self-awareness are possible. Some hypotheses on the relationship between sleep and dreaming are formulated. PMID- 7546312 TI - One possible function of sleep: to produce dreams. AB - Among the various functions of sleep, one is particularly significant for our mental life: that of producing dreams. This specifically human experience, which takes place only during sleep, is closely related with neurophysiological events. The coincidence between mental and biological events raises a complex epistemological question concerning the close relationship between dreams, viewed as a subjective experience and sleep, viewed as a vegetative experience which is possible to define from a behavioural, electrical, neurochemical and neurobiological viewpoint. In this presentation, the author criticizes Hobson and McCarley's internal generator theory, according to which the pons activates the limbic structures responsible for the recovery of memory in dreams, on account of physiological and psychological reasons. The author puts forward a cognitive hypothesis, which considers dreams as a symbolic process of elaborating, interpreting and reorganizing in narrative sequences all the material accumulated in the memory during waking hours. The author proposes, therefore, a psychoanalytical model of dreaming, in which dreams constitute a way of representing the individual's inner world with internal objects related with one another and with the Self. PMID- 7546313 TI - The function of sleep. Comments on the symposium and an attempt at synthesis. PMID- 7546314 TI - Sleep for development or development for waking?--some speculations from a human perspective. AB - The issue of the relationship between sleep and development could be posed in the following terms: (1) does sleep have a function for development? and (2) which is the specificity of sleep function during development? Is it possible to assess critical ages of emergence and decline of specific sleep functions? The results of recent investigations related to the so-called ontogenetic hypothesis for the function of rapid eye movement (REM) sleep will be reviewed; suggestions are put forward concerning the possible role of non-rapid eye movement (NREM) sleep. Because of the difficulties to provoke long-lasting sleep deprivation in humans during development, two different approaches were used. The results of one set of analyses concerned the secretion of growth hormone during sleep under normal and pathological conditions and the relationship between sleep organization and nutritional supply utilisation in infants and children. The second approach aimed at investigating the long-term development of children suffering from sleep abnormalities at earlier ages. Furthermore, the role of dreaming during development will be discussed. The data summarized here only partly support the function of sleep during development; we would like to underscore the difficulty to dissociate the function of sleep from that of waking. PMID- 7546315 TI - Phylogeny and the function of REM sleep. AB - Phylogenetic studies in placental and marsupial mammals have demonstrated three major correlates of increased REM sleep time across these species. These are high amounts of non-REM sleep time, safe sleep conditions and immaturity at birth. While these variables explain approximately 30% of the variance in REM sleep time across these orders, these relations are violated when animals other than placentals are included. Birds are small, many have safe sleeping situations and are certainly immature at birth, yet they have less REM sleep than the vast majority of mammals. The echidna is immature at birth, has high amounts of non REM sleep and safe sleeping conditions, yet has been reported to have no REM sleep. Our recent studies in the echidna indicate that REM and non-REM sleep did not evolve sequentially, but rather evolved as a differentiation of a primitive state which held the seeds of both sleep states. The echidna sleeps with an activated brainstem and EEG synchronized forebrain. Future studies of sleep phylogeny need to compare the behavior of key neuronal groups across the sleep cycle, since these results indicate that EEG variables and sleep state durations may given an inadequate picture of the nature of brain activity during sleep. PMID- 7546316 TI - Is sleep fundamentally different between mammalian species? AB - An avenue to investigate the functions of sleep is the comparison of sleep in different species, particularly in closely related ones and in species with extreme specializations. The features which are usually investigated are the occurrence of both sleep stages non-REM sleep and REM sleep, their amount per 24 h, the duration of the non-REM-REM sleep cycle and the daily distribution of sleep relative to the light-dark cycle of the environment. Recently also sleep homeostasis has been included, because it is now well established that mammalian species can compensate for sleep loss both by an increase in sleep duration as well as by intensifying non-REM sleep. The occurrence of EEG slow-wave activity has served as a measure for sleep intensity. The capacity to sleep more intensely enables animals to react more flexibly to sleep loss. The comparison of mammalian species has revealed striking similarities in the way sleep is regulated which indicates common underlying mechanisms. PMID- 7546317 TI - Functional consequences of sustained sleep deprivation in the rat. AB - Sleep deprivation disrupts vital biological processes that are necessary for cognitive ability and physical health, but the physiological changes that underlie these outward effects are largely unknown. The purpose of the present studies in the laboratory rat is to prolong sleep deprivation to delineate the pathophysiology and to determine its mediation. In the rat, the course of prolonged sleep deprivation has a syndromic nature and eventuates in a life threatening state. An early and central symptom of sleep deprivation is a progressive increase in peripheral energy expenditure to nearly double normal levels. An attempt to alleviate this negative energy balance by feeding rats a balanced diet that is high in its efficiency of utilization prolongs survival and attenuates or delays development of malnutrition-like symptoms, indicating that several symptoms can be manipulated to some extent by energy and nutrient consumption. Most changes in neuroendocrine parameters appear to be responses to metabolic demands, such as increased plasma catecholamines indicating sympathetic activation. Plasma total thyroid hormones, however, decline to severely low levels; a metabolic complication that is associated with other sleep deprivation induced symptoms, such as a decline in body temperature to hypothermic levels despite increased energy expenditure. Metabolic mapping of the brain revealed a dissociation between the energy metabolism of the brain and that of the body. Sleep deprivation's effects on cerebral structures are heterogeneous and unidirectional toward decreased functional activity. The hypometabolic brain structures are concentrated in the hypothalamus, thalamus and limbic systems, whereas few regions in the rest of the brain and none in the medulla, are affected. Correspondence can be found between some of the affected cerebral structures and several of the peripheral symptoms, such as hyperphagia and possible heat retention problems. The factor predisposing to mortality is a decreased resistance to infection. Lethal opportunistic organisms are permitted to infect the bloodstream, which presumably results in a cascade of toxic-like reactions. Host defense is thus the first system to fail. There is neither fever nor marked tissue inflammatory reactions typical of infectious disease states, suggesting that sleep deprivation is immunosuppressive. Each of the four abnormalities identified--(1) a deep negative energy balance and associated malnutrition; (2) heterogeneous decreases in cerebral function; (3) low thyroid hormone concentrations; and (4) decrease resistance to infection--can be viewed as having an early origin during the sleep deprivation process to signify the foremost pathogenic situation to which the other abnormalities might be secondarily related.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7546318 TI - Sleep deprivation in the rat by the disk-over-water method. AB - Chronic sleep deprivation may be required to reveal the most serious physiological consequences of sleep loss, but it usually requires strong stimulation which can obscure the interpretation of effects. The disk-over-water method permits chronic sleep deprivation of rats with gentle physical stimulation that can be equally applied to yoked control rats. A series of studies with this method has revealed little or no pathology in the control rats. The deprived rats show a reliable syndrome that includes temperature changes (which vary with the sleep stages that are lost); heat seeking behavior; increased food intake; weight loss; increased metabolic rate; increased plasma norepinephrine; decreased plasma thyroxine; an increased triiodothyronine-thyroxine ratio; and an increase of an enzyme which mediates thermogenesis by brown adipose tissue. The temperature changes are attributable to excessive heat loss and an elevated thermoregulatory setpoint, both of which increase thermoregulatory load, and the other changes are interpretable as responses to this increased load. This pattern indicates that sleep serves a thermoregulatory function in the rat. The sleep deprived rats also show stereotypic ulcerative and hyperkeratotic lesions localized to the tail and plantar surfaces of the paws, and they die within a matter of weeks; the mediation of these changes is unresolved. PMID- 7546319 TI - Energy conservation and sleep. AB - Phylogenetic and ontogenetic associations between sleep and endothermy are consistent with the hypothesis that sleep evolved in conjunction with endothermy to offset the high energetic cost of endothermy. The electrophysiological and thermoregulatory continuum of slow wave sleep, circadian torpor and hibernation substantiates a primordial link between sleep and energy conservation. Sleep constitutes a circadian and circannual rhythm of hypometabolic adaptation to biospheric energy cycles that is usually entrained through light-mediated suppression of melatonin secretion. When energy stores decline, energy is conserved by lowering Tb proportionally during sleep or by increasing the daily duration of sleep. PMID- 7546320 TI - Sleep function(s) and cerebral metabolism. AB - The function(s) of sleep would probably be better understood if the metabolic processes taking place within the central nervous system (CNS) during sleep were known in greater detail. The general pattern of the energy requirements of the brain during sleep is now outlined. Brain energy metabolism dramatically decreases during slow wave sleep (SWS) whereas, during rapid eye movement sleep (REMS), the level of metabolism is similar to that of wakefulness. However, these modifications of the energy metabolism, in good agreement with intracerebral recordings of neuronal firing, do not help in identifying the function(s) of sleep, since they are in line with several theories of sleep function(s) (protection, energy conservation, brain cooling, tissue restitution). On the other hand, several studies of brain basal metabolism suggest an enhanced synthesis of macromolecules such as nucleic acids and proteins in the brain during sleep. However, up to now, these data remain scarce and controversial. As a consequence, the research in the field of the brain metabolism during sleep has now come to a turning point, since the confirmation of sizeable cerebral anabolic processes would provide an outstanding argument in favour of the restorative theory of sleep. In this case, a hypothesis, based on clinical findings and preliminary metabolic data, might be further proposed. The putative biosynthetic processes would not equally benefit all the components of the CNS but would primarily be devoted to the maintenance of an optimal synaptic function. PMID- 7546321 TI - Muramyl peptides and the functions of sleep. AB - Muramyl peptides (MPs) are bacterially derived sleep factors which stimulate slow wave sleep. In the neonate, MPs are capable of inducing quiet sleep and suppressing active sleep. Given that active sleep is thought to be important for brain development during this period, the possibility that changes in the availability of MPs in the neonate may affect brain development was examined. Rat pups were given muramyl dipeptide (MDP) twice daily for the first 14 days post partum. It was hypothesised that MDP would stimulate quiet sleep at the expense of active sleep as has been shown in the young rabbit. There was no effect on neonatal levels of quiet sleep or active sleep. There was, however, a variety of effects, apparently unrelated to the sleep functions of MDP. These effects were changes in adult learning, serotonin metabolism and brain anatomy. The function of sleep in the mediation of the sleep-independent effects of MPs was examined, in particular the opposite effects of MDP on host immunity depending on the sleepiness of the host. In this light sleep does indeed serve to mediate the effects of MPs and it is speculated that many of the effects observed here may change in response to sleep levels in individuals. The notion that autonomic signalling between brain and spleen is more efficient during sleep is presented as an example of a function of sleep that may modulate the immunological effects of MPs. PMID- 7546323 TI - Observations on delta homeostasis, the one-stimulus model of NREM-REM alternation and the neurobiologic implications of experimental dream studies. AB - We first review the concept of delta homeostasis as formulated in our 1974 model and as developed quantitatively by Borbely and colleagues in several versions of the two-process model. We illustrate difficulties in the application of this quantitative model to the negative delta rebound in the rat and we put forward additional evidence that the negative rebound is a pathological rather than a homeostatic response to sleep deprivation. We next review experiments on rats in which the waking metabolic rate of limbic structures was increased by blockade of the NMDA-gated cation channel with ketamine and MK-801. As predicted by the 1974 homeostatic model, NREM delta increased during subsequent sleep. However, it remains to be shown that this powerful effect is actually caused by the metabolic change and that it is an intensification of physiological sleep rather than a non specific increase in EEG slow waves caused by neurotoxicity. We then outline our one-stimulus model of NREM/REM alternation. In this model NREM sleep is induced by periodic (neuroendocrine?) pulses. These pulses increase delta EEG amplitude and density, depress arousal level and inhibit neural activity. When the strength of the pulsatile stimulus falls below a critical level, REM emerges as neuronal escape. Last, we discuss the neurobiologic implications of two robust findings in experimental dream studies: the relation of dream reports to arousal and the consistent failure of controlled studies to demonstrate qualitative differences between NREM and REM mentation. PMID- 7546322 TI - Sleep as neuronal detoxification and restitution. AB - The classical 'hypnotoxin theory' was followed by extensive search for an endogenous sleep substance. Brain tissues and body fluids of sleeping and sleep deprived animals contained active sleep-inducing factors like the sleep-promoting substance (SPS). Uridine and oxidized glutathione (GSSG), two components of SPS, seem to regulate physiological sleep differentially. Uridine may facilitate the inhibitory neurotransmission at the synaptic level of the GABAA-uridine receptor complex. In contrast, GSSG may inhibit the excitatory neurotransmission at the synaptic level of the glutamate receptor. Thus, the two SPS components promote sleep by exerting a complementary action on the two major neurotransmitter systems in the brain that have mutually reciprocal functions. Further, among multidimensional functions of sleep, uridine may contribute to recover the activity of neurons, while glutathione may counteract excitotoxic events. Hence sleep at the behavioral level is a process of neuronal restitution and detoxification at the cellular level. Such a concept can be regarded as a modern version of the Ishimori-Pieron's hypnotoxin theory proposed early in this century. PMID- 7546324 TI - Influence of ethanol on the salsolinol excretion in healthy subjects. AB - This report describes the change of salsolinol (SAL) levels in the urine of healthy subjects after ethanol ingestion. A new rapid method for SAL extraction from urine sample and a GC-MS assay were used to study the urinary SAL excretion in a group of adult males (n = 14) and females (n = 13) with and without ingestion of ethanol. The results of this study show that the urinary SAL output of all subjects is significantly influenced by the intake of ethanol. A decrease in the SAL level was found in the urine of most of the volunteers (n = 17), whereas only eight subjects showed an increase in the SAL level after administration of ethanol. PMID- 7546325 TI - Prolonged treatment with carbamazepine increases the stimulatory effects of ethanol in mice. AB - Carbamazepine (CBZ) has been used in the treatment of alcohol withdrawal (AW). However, cases of induction of euphoric feelings when mixed with alcohol have been reported. We verified whether CBZ could potentiate ethanol stimulatory effects in animals. Two groups of mice were injected with saline (group I) or 2 g/kg ethanol (group II) IP, for 20 days. On the next day, each group was divided into two subgroups that received either a single dose of CBZ (10 mg/kg) or vehicle IP, followed, 30 min later, by saline or ethanol injection. Locomotor activity was measured. Acute CBZ did not change locomotor activity of ethanol treated mice. Treatment with CBZ or vehicle continued for 6 days. Finally, on the 28th day, 30 min after the last CBZ or vehicle injection, an ethanol challenge was given to group II and a saline injection to group I. The results showed a significant potentiation of ethanol stimulatory effects by chronic CBZ treatment. Data indicated that CBZ should be cautiously administered to alcohol-dependent patients. PMID- 7546326 TI - Determination of (R)- and (S)-salsolinol sulfate and dopamine sulfate levels in plasma of nonalcoholics and alcoholics. AB - We have developed a new method to determine free as well as sulfoconjugated salsolinol (SAL), separated into both enantiomers, and free and sulfoconjugated dopamine in human blood plasma. Among the group of nonalcoholics (R)-SAL (mean +/ SEM: 0.24 +/- 0.07 ng/ml) was found in all blood samples and (S)-SAL in 1 out of 20 (0.08 ng/ml). Ethanol loading induced a rise of both enantiomers as well as of dopamine whereby (S)-SAL was detected in the plasma of 13 subjects only [(R)-SAL: 0.79 +/- 0.24 ng/ml; (S)-SAL: 0.49 +/- 0.15 ng/ml; DA: 8.84 +/- 0.75 ng/ml]. The later finding favors the notion of an enzymatic formation of (S)-SAL. In alcoholics, (R)-SAL and (S)-SAL were elevated at the day of admission for detoxification [(R)-SAL: 0.65 +/- 0.82 ng/ml; (S)-SAL: 0.35 +/- 0.05 ng/ml] and normalized after several months, suggesting intoxication marker characteristics [month 6: (R)-SAL: 0.24 +/- 0.14 ng/ml; (S)-SAL: 0.20 +/- 0.05 ng/ml]. Patients with alcoholic parents had lowered (R)-SAL and (S)-SAL levels compared with family history negative alcoholics, suggesting genetic association of disturbance of the SAL biosynthesis and alcoholism. Among the personality traits, suicidality was linked with low (R)-SAL and (S)-SAL concentrations in contrast to novelty seeking, impulsivity, and harm avoidance scores. The scores on the self-rating anxiety scale correlated positive with (R)-SAL. These findings suggest trait marker characteristics of salsolinol. PMID- 7546327 TI - Early handling can attenuate adverse effects of fetal ethanol exposure. AB - The effects of early handling on physiological and hormonal responses of rats exposed to ethanol prenatally were studied. Male and female Sprague-Dawley rats from prenatal ethanol (E), pair-fed (PF), and ad lib-fed control (C) prenatal treatment groups were either handled (H) or nonhandled (NH) during the preweaning period and tested in adulthood. Early handling eliminated the deficit in preweaning weight gain observed in E compared to PF and C offspring. In adulthood, early handling eliminated the increased hypothermia observed in E and PF compared to C males following an ethanol challenge (2.0 g/kg, IP). In addition, H males displayed marginally less hypothermia overall than NH males. In contrast, handling accelerated the return to preinjection temperature in PF and C females but had no effect on E females. There were no significant differences among E, PF, and C rats in corticosterone (CORT) responses to ethanol challenge (1.5 g/kg, IP), but both males (marginally) and females in the H condition displayed higher CORT levels overall than NH rats. Early handling also eliminated the increased peak CORT response to restraint stress in E compared to C females, but did not affect the more prolonged elevation of CORT in E compared to PF and C females. There were no differences among E, PF, and C females in hippocampal type I and type II glucocorticoid receptor density or affinity. However, binding affinity of type II receptors was slightly but significantly increased in H compared to NH females. Together, these data indicate that early handling may modulate or attenuate some, but not all, of the adverse effects of fetal ethanol exposure on offspring growth and physiological responsiveness. PMID- 7546329 TI - [3H]MK-801 binding in various brain regions of rat lines selected for differential alcohol sensitivity. AB - N-Methyl-D-aspartate (NMDA) receptors are sensitive to ethanol at concentrations relevant to intoxication. To ascertain possible involvement of NMDA receptors in differential ethanol sensitivity between alcohol-sensitive ANT (alcohol nontolerant) and alcohol-insensitive AT (alcohol-tolerant) rat lines, characterization of a noncompetitive NMDA antagonist [3H]MK-801 binding to brain membranes was carried out. Saturation analyses of [3H]MK-801 binding to cerebrocortical, hippocampal, and cerebellar synaptosomal membranes revealed no statistically significant differences in either the affinity constant (Kd) or binding site density (Bmax) between the rat lines. Autoradiographic analysis of [3H]MK-801 binding to ANT and AT brain sections revealed a regionally heterogenous distribution of binding, without any detectable differences between the AT and ANT sections whether these were prepared from the brains of acutely ethanol-treated or nontreated animals. Glutamate, glycine, or the two in combination greatly increased [3H]MK-801 binding to brain membranes. In extensively washed crude cerebrocortical membranes, the maximal effect (Emax), but not potency (EC50) of glycine to increase [3H]MK-801 was slightly greater (p < 0.01) in the ANT than AT rats. The effects of glutamate or glutamate in the presence of saturating concentration of glycine (30 microM) were not significantly different between the two lines. Association parameters (t1/2 and Beq values) of [3H]MK-801 to its cortical binding sites were also similar. These results do not indicate any clear qualitative difference in [3H]MK-801 binding to NMDA receptors or in its modulation by glutamate and glycine between the ANT and AT rat lines. PMID- 7546328 TI - Nitric oxide synthase activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig: ontogeny and in vitro ethanol exposure. AB - Decreased nitric oxide (NO) formation, resulting from inhibition of NO synthase (NOS), may be important in the pathogenesis of ethanol central nervous system teratogenesis. The objectives of this study were to determine the ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig, and to test the hypothesis that direct exposure to ethanol inhibits NOS activity in these brain regions at selected developmental ages. NOS activity was quantitated by an optimized radiometric assay. The ontogeny study demonstrated that NOS activity in the hippocampus and frontal cortex was not fully developed prenatally, and apparently increased during postnatal life to attain adult level of activity at postnatal day > 60. In the cerebellum, NOS activity increased during prenatal life to an apparent maximum in the mature near term fetus at gestational day 63 (term, about 68 days), and then apparently declined during postnatal life to attain adult level of activity. In vitro ethanol exposure (25-100 mM) did not affect NOS activity in the hippocampus, frontal cortex, or cerebellum at any developmental age studied. These data indicate that, although the ontogeny of NOS activity varies between brain regions, ethanol does not directly affect NOS activity in the developing guinea pig. The effects of acute and chronic in utero ethanol exposure on NOS activity in these brain regions are currently being investigated. PMID- 7546330 TI - Effects of aminotriazole on ethanol, water, and food intake and on brain catalase in UChA and UChB rats. AB - Aminotriazole (AT), a catalase inhibitor, was administered to UChA (low ethanol consumer) and UChB (high ethanol consumer) rats. Ethanol, water, and solid food intake were measured during basic, treatment, and posttreatment periods. The effects of AT on brain catalase activity and acetaldehyde recovered during incubation of brain homogenates with ethanol were also studied in rats of both strains. Results showed that AT decreased voluntary ethanol intake in UChB rats, and also diminished the consumption of food by rats of both strains. No strain difference in brain catalase activity and acetaldehyde recovered during ethanol incubation was observed. The results suggest that AT effect on ethanol consumption is secondary to a reduction in the appetite for calories and not related to its catalase blocking effect. PMID- 7546331 TI - Volitional consumption of ethanol by fawn-hooded rats: effects of alternative solutions and drug treatments. AB - Behavioral and neurochemical measures of brain 5-hydroxytryptamine (5-HT) function in the Fawn-Hooded rat are abnormal relative to outbred strains of rats. Fawn-Hooded rats freely drink large amounts of 10% ethanol in the presence of water and have been proposed to be an animal model for studies related to alcoholism. In this study, Fawn-Hooded rats were given solutions of ethanol increasing in concentration from 3% to 30% (w/v in tap water) over 10 days with tap water in a second drinking tube and a third tube left empty. The solutions of ethanol that produced maximal drinking with a preference (ml ethanol/ml total fluid) near 50% ranged from 5% to 13%, which became the fixed individual concentrations for each rat. After a 5-day baseline period the rats were offered a solution in the third drinking tube of either 0.5% aspartame or chocolate Ultra SlimFast (diluted with water 2:1). The chocolate drink, but not aspartame, significantly reduced the consumption of alcohol by 73%. For the drug experiments, the rats were given successive 4-day periods of: baseline drinking; drug or saline injections b.i.d.; and a posttreatment period. Neither ipsapirone, a 5-HT1a partial agonist, nor naltrexone injected inhibited the intakes of ethanol solutions. Treatment with 2.5 mg/kg of amperozide, a 5-HT2 antagonist, decreased the consumption of ethanol by 38%, but also caused a decrease in consumption of food. These results show a pattern of drinking of increasing concentrations of ethanol different than other strains of rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546334 TI - Amphetamine-induced hyperactivity: differences between rats with high or low preference for alcohol. AB - This study determined the relationship between ethanol intake and spontaneous and amphetamine-induced locomotor activity. Locomotion was studied in high-preferring (HP; > 70% of total fluid intake consumed as alcohol) and low-preferring (LP; < 20% of total fluid intake consumed as alcohol) male Wistar rats with free access to water and a 6% (v/v) ethanol solution for 3 weeks. Following an alcohol-free 3 week period, the animals were tested for spontaneous motor activity for 1 h. One week later, locomotion was recorded in the same activity boxes following a subcutaneous injection with d-amphetamine sulfate (1 mg/kg). For determination of plasma levels of corticosterone, blood samples were taken immediately after each of the two tests for locomotor activity. There was no difference between HP and LP rats with regard to spontaneous locomotor activity. Neither were there any differences in plasma levels of corticosterone between the groups. Amphetamine stimulated locomotion in both HP and LP rats, but to a significantly greater extent in HP animals. Both groups had higher blood levels of corticosterone after the amphetamine test than after the drug-free test, but the corticosterone increase was significantly larger in the HP than in the LP rats. These data indicate that the same neural substrate (e.g., the mesocorticolimbic dopamine system) may mediate important aspects of both ethanol drinking and amphetamine responsiveness. Individual differences in the properties of this substrate may account for the finding that ethanol drinking and amphetamine responsiveness covary. A possible explanation for this association may be that prior consumption of ethanol sensitizes the neural substrate responsible for amphetamine-induced hyperactivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546333 TI - Acute ethanol and selected growth suppressor transcripts in regenerating rat liver. AB - To study the effects of acute ethanol on regenerating rat liver, the mRNA transcript levels of growth suppressor genes (prohibitin, TGF beta-1 and p53) were measured by Northern blot analysis during the G0, G1, and early S phases of compensatory growth after 70% partial hepatectomy (PH) in adult male rats. Selected animals were gavaged with either ethanol (3 g/kg) or glucose and underwent PH 1 h later. Other animals were either sham operated or underwent PH without gavage. Prohibitin and p53 transcripts were increased in relative abundance (as measured by an increase in band intensity) near the G1/S boundary (8-12 h post-PH) following both glucose and ethanol gavage. A transient increase in prohibitin transcripts at 0.5-1 h post-PH was found to be characteristic of glucose and nongavaged rats. Ethanol gavage significantly increased the relative abundance of prohibitin transcripts at 0.5-2 h post-PH. An increase in the TGF beta-1 transcripts at 4 h post-PH was found in the glucose and nongavaged rats. Ethanol gavage resulted in variable TGF beta-1 transcript expression near hepatectomy (0 h); however, mean differences were not statistically significant. Sham operation had no effect on the mRNA transcripts of the selected genes during the time periods sampled. These results and previous work suggest that the mitoinhibitory effects of acute ethanol exposure may occur via modulation of growth suppressor and proto-oncogene expression. PMID- 7546335 TI - Glutamate and N-methyl-D-aspartate binding sites in the guinea pig hippocampus: ontogeny and effect of acute in vitro ethanol exposure. AB - The objectives of this study were to characterize the ontogeny of the L-glutamate (glutamate) and N-methyl-D-aspartate (NMDA) binding sites in the developing guinea pig hippocampus, and to determine the effect of acute in vitro ethanol exposure on these binding sites. Specific [3H]glutamate binding and NMDA sensitive [3H]glutamate binding were determined using a guinea pig hippocampal synaptic membrane preparation (HSMP). To characterize the ontogeny of the density (Bmax) and affinity (Kd) of the glutamate and NMDA binding sites, saturation analysis was conducted on HSMP of guinea pigs at gestational day (GD) 50 (immature fetus; term, GD 68), GD 62 (mature, near-term fetus), postnatal day (PD) 13 (neonate), and PD > 60 (adult). To examine the effect of ethanol on the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 50, GD 62, PD 13, and PD > 60 was incubated with ethanol (0-100 mM), followed by determination of specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding. To determine the effect of 50 mM ethanol on the Bmax and Kd of the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 62 and PD > 60 was incubated with 0 or 50 mM ethanol followed by saturation analysis. The Bmax values of the hippocampal glutamate and NMDA binding sites were greater at GD 62 and PD 13 compared with GD 50 and PD > 60, but there was no change in the Kd of the binding sites throughout development.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546332 TI - 4-Methylpyrazole, an alcohol dehydrogenase inhibitor, exacerbates alcohol-induced microencephaly during the brain growth spurt. AB - Whether alcohol-induced microencephaly occurs as a result of the effect of alcohol or acetaldehyde remains an unanswered, yet important, question. The present study addressed this issue by using an alcohol dehydrogenase (ADH) inhibitor, 4-methylpyrazole (4-MP), that works by blocking the metabolism of alcohol to its primary metabolite acetaldehyde, thereby prolonging the actions of alcohol while minimizing the generation of acetaldehyde. Four groups of artificially reared Sprague-Dawley rat pups were treated with alcohol treatment (3.3 g/kg EtOH or isocalorically matched control formula from postnatal days 4 through 9) and 4-MP administration (IP, 50 mg/kg or saline). A suckle control group was introduced to control the effects of the artificial rearing procedure. On postnatal day 10, all pups were perfused. Alcohol in combination with 4-MP treatment produced a marked microencephaly, as assessed by brain weights or brain to body weight ratios, compared with other artificially reared groups. The peak BACs in the pups that received both alcohol and 4-MP were increased at least twofold compared with those that received alcohol alone. These findings indicate that 4-MP is an effective nontoxic ADH inhibitor and that microencephaly is associated with BAC levels. Most importantly, these results support the hypothesis that alcohol is a causative agent for alcohol-induced microencephaly and implicates the importance of functional ADH activity in attenuating alcohol induced neuroteratogenicity. PMID- 7546337 TI - Cocaethylene-induced lethality in mice is potentiated by alcohol. AB - Mice of the heterogeneously bred HS line were concurrently administered intraperitoneal injections of either 95, 75, 60, or 48 mg/kg cocaethylene or 48, 38, or 30 mg/kg cocaethylene in conjunction with the non-lethal dose of 6.0 g/kg (20% w/v) alcohol. Results indicate that alcohol administration significantly potentiated cocaethylene-induced lethality. This observation suggests that alcohol is capable of enhancing the lethal effects of cocaethylene. Results are discussed in terms of observations of sudden death in humans who abuse cocaine and alcohol. PMID- 7546336 TI - Effect of chronic maternal ethanol administration on glutamate and N-methyl-D aspartate binding sites in the hippocampus of the near-term fetal guinea pig. AB - The objective of this study was to determine the effect of chronic maternal administration of ethanol on hippocampal L-glutamate (glutamate) and N-methyl-D aspartate (NMDA) binding sites in the near-term fetal guinea pig. Starting on gestational day (GD) 2, pregnant guinea pigs received one of the following oral treatments up to and including GD 62 (term, about GD 68): 4 g ethanol.kg maternal body weight-1.day-1; isocaloric sucrose and pair-feeding; or water. Maternal blood ethanol concentration was determined at 1 h after the daily ethanol dose on GD 59. Fetuses were studied at GD 63 (mature, near-term fetus). Fetal body weight and brain weight were determined. The density (Bmax) and affinity (Kd) of the glutamate and NMDA binding sites in the fetal hippocampus were measured using a radioligand membrane binding assay; saturation analysis was conducted on hippocampal synaptic membrane preparation (HSMP). Maternal blood ethanol concentration on GD 59 was 269 +/- 111 (SD) mg/dl (59 +/- 24 mM). There was no maternal or embryonic fetal lethality in any of the three treatment groups, and ethanol treatment did not affect maternal body weight gain compared with sucrose or water treatment. Fetal brain weight, but not body weight, was decreased in the ethanol treatment group compared with the sucrose and water treatment groups. The Bmax values of the glutamate and NMDA binding sites were decreased in the ethanol treatment group compared with the sucrose and water treatment groups; there was no difference in the Kd values of the glutamate and NMDA binding sites among the three treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546338 TI - Evidence that the amygdala is involved in the inhibitory effects of 5-HT3 receptor antagonists on alcohol drinking in rats. AB - Two 5-HT3 receptor antagonists, tropisetron (1 and 10 ng) and ondansetron (10 and 100 ng) were tested for effects on ethanol drinking in Wistar male rats after bilateral microinjection into the amygdala. The animals had limited access (2 h/day) to the 10% (v/v) ethanol solution, food and water were available ad lib during the scheduled access period. Both drugs caused a decrease in ethanol drinking. Tropisetron (1 and 10 ng) decreased ethanol intake during the first hour of access. The lower dose (10 ng) of ondansetron was more effective than the higher (100 ng) dose. The finding implicates amygdaloid 5-HT3 receptors in the mechanism of ethanol intake in Wistar rats. PMID- 7546341 TI - Epidemiological studies on cystic echinococcosis in China--a review. AB - In the four decades from 1951 to 1990, the six provinces or autonomous regions (Xinjiang, Gansu, Qinghai, Ningxia, Xizang and Nei Monggol) reported a total of 26,065 surgical cases of hydatid disease, most of which were reported in the recent decade. About one third of the patients was children and adolescents under 15 years old. So far, cystic hydatid infections of local origin have been confirmed in 22 provinces, autonomous regions and municipalities in the whole nation. Findings of X-ray examination and real-time B-mode ultrasonography in agricultural and pastoral areas of Xinjiang, Gansu, Qinghai, Ningxia and Xizang showed that the morbidity rate of hydatidosis in human population varied between 0.5% and 4.5%. The main animal intermediate host in all these regions is sheep, the morbidity of which varied between 3.3% and 90%. The infection rate of adult Echinococcus granulosus in dogs varied between 7% and 71%. The high-risk period for humans contracting hydatid disease is the pre-school age. Direct contact of children with dogs and ingestion of water, vegetables and foods contaminated by worm eggs are the chief mode of transmission for human hydatidosis. The common practices of home slaughter and of feeding dogs on offal containing hydatid cysts facilitate the life cycle of the parasite. On the basis of hydatid control efforts for several years, the Ministry of Public Health promulgated officially the "1992-1995 National Programme for Hydatid Disease Control" in April, 1992, and pilot studies in which the control of hydatidosis is composed of extensive health education, sanitation of slaughtering and management and deworming of dogs are being established. Cystic echinococcosis has wide distribution in China and is a major public health problem in hyperendemic areas, poses a great threat against people's health and influences the development of livestock husbandry. This problem has received great attention from the medical and veterinary departments. In the recent decade great efforts have been made in the epidemiology, parasitology and clinical treatment of echinococcosis, resulting in a better understanding of the disease and procedures effective in bringing about control of the disease. PMID- 7546340 TI - In vitro effect of dithiocarbamate pesticides and of CaNa2EDTA on human serum dopamine-beta-hydroxylase. AB - Serum dopamine-beta-hydroxylase (DBH) inhibition has been reported in lead workers treated with CaNa2EDTA and in alcoholic patients repeatedly treated with the alcohol aversive drug Disulfiram. The mechanism of inhibition involves Cu++ chelation at the active site of DBH. The effect of CaNa2EDTA and Disulfiram on serum DBH has been compared to the effect of dithiocarbamate pesticides in vitro for the possible use of serum DBH determination for the biological monitoring of workers exposed to these pesticides. Most dithiocarbamates inhibit human serum DBH at micromolar concentrations (range of I50, 0.027-1.6 mumol/L). The inhibitory potency increased from methyl- and dimethyl dithiocarbamates to diethyl dithiocarbamates up to the most potent ethylene bisdithiocarbamates. The I50 of CaNa2EDTA was 3.8 mumol/L, higher than those of dithiocarbamates. Copper addition to the test system reactivated at stoichiometric concentrations dithiocarbamate-inhibited DBH indicating that both base line values and percent of inhibition can be calculated in a single blood sample. Results suggest that serum DBH determination could be useful in case of acute poisoning involving high doses of dithiocarbamate pesticides. PMID- 7546339 TI - Oxidative stress during antituberculous therapy in young and elderly patients. AB - Using allantoin (ATN) as a marker for reactive oxygen species (ROS), oxidative stress during antituberculous (anti-TB) therapy was compared in 10 young and 9 elderly patients. Before treatment, ATN plasma concentrations in patients were similar to that of volunteers. Administration of a combination of isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA) increased plasma ATN in both groups of patients. ATN concentrations (M +/- SE) at six hours were higher (P < 0.05) in elderly than in young patients on day one, 8.22 +/- 1.50 vs 1.89 +/- 0.98 microgram/mL); day 30, (5.85 +/- 0.82 vs 0.87 +/- 0.57 microgram/mL; and day 90, (4.84 +/- 1.24 vs 0.52 +/- 0.50 microgram/mL). Because total amount of ATN excreted was similar in both groups on the three occasions, more ATN was formed in elderly than young patients. In conclusion, there was more oxidative stress in elderly than young patients. It is thereby suggested that Anti-TB drugs induce formation of ROS and elderly patients are at a greater risk of toxicity probably because of poor antioxidant mechanisms. PMID- 7546342 TI - Pesticidal impact on the protein metabolism of freshwater field crab, Oziotelphusa senex senex (Fabricius). AB - The total protein increased in the gills and decreased in the muscle of the freshwater field crab Oziotelphusa senex senex at days 1 and 2 on exposure to lethal concentrations and at days 1 and 10 to sublethal concentrations of furadan, endosulfan, chlorpyrifos, and a mixture of these three in a 100:10:1 ratio. The increase in the gill protein was greater on exposure to the sublethal concentrations than to the lethal concentrations while the decrease in the muscle protein was greater on exposure to the lethal concentrations than to the sublethal concentrations. In the hepatopancreas, the protein content decreased on exposure to the lethal concentrations, but, in contrast, increased on exposure to the sublethal concentrations. These results clearly indicate that changes in the protein content are not only organ-dependent but also concentration-dependent, i.e., lethal versus sublethal. Irrespective of the changes in the total protein, the levels of free amino acids and the activities of protease, alanine and aspartate aminotransferases, and glutamate dehydrogenase increased in all the three organs of the crabs exposed to the lethal and sublethal concentrations, (more in lethal than in sublethal) and increased at a greater rate over time of exposure. Ammonia toxicity, measured by an increase in the hemolymph ammonia and a decrease in the urea, was also observed at the lethal concentrations of all the three pesticides. The ammonia and urea levels increased in the crabs exposed to the sublethal concentrations. Although the effect of each pesticide on the protein metabolism was similar, the degree of toxicity was the lowest on exposure to furadan, intermediate on exposure to endosulfan and chlorpyrifos, and cumulative on exposure to a mixture of the three pesticides. PMID- 7546345 TI - Studies on the effects of Deltamethrin on sodium net transport through the in vivo amphibian skin. AB - The action of micromolar concentrations of Deltamethrin on sodium net transport through the in vivo skin of the South American toad Bufo arenarum was studied. The effect of pure ethanolic insecticide solutions and commercial formulations when applied on the mucosal surface was assayed. Deltamethrin provoked a concentration-independent inhibition; the highest inhibition was found at the lowest concentrations. At highest concentrations of the insecticide the JnNa was not altered. PMID- 7546344 TI - Copper-induced changes in the urea uptake and urease activity in the cyanobacteria Anabaena doliolum and Anacystis nidulans: interaction with sulphur containing amino acids. AB - Copper-induced changes in the urea uptake and urease activity have been investigated in the cyanobacteria Anabaena doliolum and Anacystis nidulans. Copper, at and above 5 mumol/L concentration, inhibited urea uptake and urease activity systems in both the cyanobacteria in a concentration dependent manner. However, the urea uptake and urease activity systems in A. nidulans appeared slightly more tolerant to copper than than of A. doliolum. The inhibitory effect of copper on urea uptake and urease activity was mitigated by sulphur containing amino acids (cystine and cysteine), however, methionine could not do so, indicating the involvement of sulfhydryl (-SH) groups in the assimilation of urea in cyanobacteria. PMID- 7546347 TI - Uptake, depuration and subcellular distribution of cadmium in various tissues of Perna viridis. AB - Green-lipped mussels (Perna viridis) were collected from a local mariculture site and placed in pre-cleaned sea water tanks containing 0, 0.2, 0.5 and 1.3 micrograms/ml CdCl2. The level of Cd in the gill, viscera and gonad was measured. The concentration of Cd in the 3 tissues increased linearly over 4 days' of exposure. The rate of uptake depends on the concentration of Cd in the water. In the 3 tissues studied, the maximum rate of uptake (Vmax) was highest in the gill (500 micrograms/g dry weight/day), followed by viscera (100 micrograms/g dry weight/day) and gonad (56.8 micrograms/g dry weight/day). The majority of the Cd taken up was bound to a fraction of the heat-stable proteins similar to metallothioneins. The rate of Cd depuration from the tissues was poor. There was no change in Cd concentration over 8 days' depuration in clean water. PMID- 7546346 TI - Effects of silica on serum phospholipid, lipid peroxide and morphological characteristics of rat lung. AB - The effects of instilled silica have been studied on the serum-phospholipid (PL), lipid peroxide (LPO) and histopathology of rat lung up to 140 days from the first day of instillation. Silica induced relatively higher serum-PL throughout the experiment. The level of LPO also increased appreciably. They presented positive linear correlation. The early lesion was acute alveolitis with silica particles. These lesions became silicotic nodules on the 30th day, which then were enlarged gradually and fused by fibrosis. Alveolar macrophages (AM) were activated and surface structure was damaged. These results indicate that instilled silica can induce lipid peroxidation of cell membrane and selective accumulation of lung PL. PMID- 7546348 TI - Biorhythms and the biological clock involvement of melatonin and the pineal gland in life and disease. PMID- 7546349 TI - The last of the Titans? Some reflections on what the future holds for British neurosurgeons. PMID- 7546343 TI - Adsorption and uptake of cadmium by Azolla pinnata: kinetics of inhibition by cations. AB - A. pinnata showed a remarkable ability of taking up Cd from the external medium. Of the total Cd taken up by the test plant, surface adsorption was about 90%. Cd adsorption did not occur at a constant rate, however, an equilibrium was reached in 2 h. The uptake of Cd occurred at a constant rate. Test cations, including heavy metals (Ca, Mg, K, Na, Ni, Fe, Cu and Zn), inhibited adsorption as well as uptake of Cd. Cd adsorption was competitively inhibited by Cu, Fe and Zn, whereas Ca, Mg, K, Na and Ni caused non-competitive inhibition. Similarly, Cd uptake was competitively (Ca, Mg, Fe) or non-competitively (Na, K, Ni, Zn) inhibited by cations. Inhibition of Cd uptake by Cu was not wholly competitive. PMID- 7546350 TI - Biographical sketch. Lindsay Symon. PMID- 7546351 TI - Transoral surgery: some lessons learned. AB - The experience gained over the last decade has provided some further understanding of the rare, but potentially fatal problems associated with pathology at the craniovertebral junction. Some original concepts of the disease in the area have been challenged; new evidence has been provided to explain our alternative theories. The transoral procedure as a surgical tool is now well established. It is, however, technically demanding, and requires careful anatomical study and 'hands-on' workshop training before the novice will be competent. PMID- 7546352 TI - Surgical and non-surgical treatment of symptomatic intracranial meningiomas. AB - The results of treatment of intracranial symptomatic meningiomas from a personal consecutive series are presented. Thirty-three patients were retrospectively reviewed following surgical treatment and 11 patients were treated prospectively with gestrinone, a synthetic antiprogesterone drug. Comparative results suggest that research and future advances in treatment will be non-surgical. The time has come for neurosurgeons to recognize this reality. PMID- 7546353 TI - Neural injury repair: hope for the future as barriers to effective CNS regeneration become clearer. AB - In this review the author outlines the early history of clinical and scientific research upon the inability of the CNS in man to successfully regenerate following injury. As we proceed into the 21st Century we have gained a far greater understanding of the molecular biology, pathology and other factors that lead to the adult CNS being non-supportive and indeed actively inhibitory to axonal regrowth. On the basis of these recent advances in knowledge, the author outlines possible therapeutic approaches that may enable more effective CNS regeneration to be accomplished in the future. PMID- 7546354 TI - The physiology of the cerebrovascular parasympathetic innervation. AB - The parasympathetic innervation to the cerebral vessels has only recently begun to be investigated in detail and to be understood in terms of the control of brain function. Much of the experimental work has been motivated by the potent vasoactive potential of the parasympathetic fibres, which can produce profound increases in cerebral blood flow (CBF) when electrically stimulated. This paper first outlines the principal anatomical and biochemical evidence for the origins and pathways of the parasympathetic fibres and the basis of their influence on CBF. Next, studies performed to elucidate their physiological function are reviewed, and finally their possible functional significance is discussed in relation to normal and pathophysiological brain function and their possible involvement in the trigeminovascular system. PMID- 7546356 TI - Microsurgical treatment of arteriovenous malformations in critical areas of the brain. AB - Between June 1981 and November 1989, 56 patients who had arteriovenous malformations (AVMs) in critical areas of the brain, in deeply placed sites or were of large size were excised using microsurgical techniques. In 44 cases one stage surgery was used and in 10 cases deliberate surgical staging was performed. In four cases routine postoperative angiography revealed residual AVM. Further treatment was surgical in two cases, and focused radiation in two to achieve angiographically confirmed excision or obliteration of the AVM. Referral was following haemorrhage in 41 cases, progressive neurological deficit in nine cases and epilepsy in six cases. Forty-three patients made a good recovery, seven were fair, three had a poor result and three died. Three patients developed a new or increased deficit following surgery. Seven of nine patients who presented with neurological deficit improved. Seven of nine patients who had epilepsy and who had not bled are free of epilepsy, and are not receiving anticonvulsants on long term follow-up. No new cases of long-term epilepsy have occurred following surgery. Microsurgical excision of cerebral AVMs even in critical areas of the brain carries a better outcome than the natural history of conservatively treated lesions. Surgery should be considered not only following haemorrhage, but also in cases presenting with progressive neurological deficit or epilepsy. PMID- 7546357 TI - The continuing challenge of acoustic neurinomas (1949-1993). AB - Experience gained in the treatment of acoustic neurinomas over a period of 43 years is presented. This is divided into three eras based on the prevailing knowledge and techniques. Advances in diagnosis, anaesthesia and microsurgical techniques have reduced the mortality to 4% in large tumours and to less than 1% in small tumours. With large tumours, facial nerve preservation has been possible only in 20% of patients. Large tumours continue to predominate, thus providing a formidable challenge. Saving life and the functions of the lower cranial nerves still form the main goal in the majority of patients who come with large tumours. PMID- 7546358 TI - Recovery of vision following transsphenoidal surgery for pituitary adenomas. AB - The degree of recovery of vision is reported in 67 patients who underwent transsphenoidal surgery for pituitary adenomas compressing the visual pathways. There were no deaths, but 14 patients experienced postoperative complications. Overall, 88% showed visual improvement, 7% were unchanged and 4% were worse after surgery. PMID- 7546355 TI - Blood supply, blood flow and autoregulation in the adenohypophysis, and altered patterns in oestrogen-induced adenomatous hyperplasia. AB - The article addresses the fascinating topic of blood flow in the normal and the adenomatous anterior pituitary gland. This is considered in conjunction with the anatomy and physiology of the area and the neuro secretion. The mechanisms of the control of the development and growth of the prolactinoma and the changes in the microcirculation of the tumour are discussed. It is felt that its altered microvascular pattern and in particular the influx of the systemic low dopamine blood through the supplementary and newly developed blood supply plays an important role in the natural history of these tumours. PMID- 7546359 TI - The pathophysiology of cerebral vasospasm. PMID- 7546360 TI - So-called intracranial germ cell tumours: are they really of germ cell origin? AB - We have studied 139 cases of intracranial germ cell tumours up to the beginning of 1993, 63.3% of which showed monotypic histological patterns and 36.7% were shown to be mixed tumours. All these cases underwent surgery followed by radiation and/or chemotherapy. All cases of choriocarcinoma died within 2 years. Cases of yolk sac tumour (endodermal sinus tumour) and embryonal carcinoma also showed poor results. Mature teratoma had a 5-year survival rate (5 YSR) and 10 year survival rate (10 YSR) of 92.9% each. Immature teratoma and malignant teratoma showed a 5-YSR and 10-YSR of 75.0% each. Germinoma showed a 5-YSR of 94.7% and a 10-YSR of 91.2%. All these results may bring into question the validity of the germ cell theory, since germinoma, which should be the most undifferentiated according to the theory, was the most benign and choriocarcinoma and yolk sac tumour (endodermal sinus tumour) which should be the most differentiated, were the most malignant in the follow-up study. Therefore, germ cell tumours may not originate from one single type of cells (primordial germs cells), except for germinoma. The embryonic cells of various stages of embryogenesis may perhaps be misplaced in the bilaminar embryonic disc and become involved in the stream of lateral mesoderm at the time of the primitive streak formation and be carried to the future cranial area to be wrongly enfolded into the brain at the time of the neural tube formation. The following law may be propounded: Tumours composed of cells resembling the cells appearing in the earlier stages of embryogenesis (ontogenesis) are more malignant than those resembling the cells appearing in the later stages of embryogenesis (ontogenesis). PMID- 7546361 TI - Cerebral arterial spasm--a clinical review. AB - A review of the literature on cerebral vasospasm after aneurysmal subarachnoid haemorrhage (SAH) has shown that angiographic vasospasm occurs in 67.3% of cases when angiography is timed for the highest likelihood, and delayed ischaemic deficit or symptomatic vasospasm in 32.6%. The presence of vasospasm has a marked effect on overall outcome of SAH, and the outcome of delayed ischaemia itself is in about one-third death and in one-third permanent deficit. Management with fluid loading or induced hypertension and with calcium antagonists has been reported widely for both prevention and treatment, and can reduce the incidence and improve the outcome of vasospasm. Other forms of treatment including tissue plasminogen activator, aminosteroids and transluminal angioplasty also appear useful. In spite of these improved therapeutic possibilities, large numbers of patients are still being reported in whom no specific treatment is used. PMID- 7546362 TI - Hearing preservation in bilateral acoustic neurinomas. AB - Bilateral acoustic neurinomas are found in almost all patients with neurofibromatosis 2 (NF2). With better knowledge of the disease and development of diagnostic procedures, an increasing number of patients have been detected at an early stage of the disease. Preservation of hearing in these cases has become more feasible. Between 1979 and 1992, 110 acoustic neurinomas in 74 NF2 patients have been resected in this department. All tumours but 11 were completely removed. In 11 cases deliberate subtotal tumour removal was performed: bilaterally in one deaf patient for brainstem decompression, and unilaterally in nine patients with the aim of hearing preservation. In seven of these nine cases hearing was preserved. The total rate of hearing preservation was 36%. Considering only those patients with good preoperative hearing and small tumours, the rate of hearing preservation was 58%. Facial nerve preservation was 92%. Nerve reconstructions were performed in cases of facial nerve damage, with good results in most of the cases. The importance of early detection of the disease and early removal of tumours is stressed. Surgical decision making based on the author's personal experience in dealing with these cases is discussed, and some cases are illustrated. PMID- 7546363 TI - When and how to treat serious candidal infections: concepts and controversies. PMID- 7546365 TI - Chancroid: new developments in an old disease. PMID- 7546364 TI - Brucellosis: current epidemiology, diagnosis, and management. PMID- 7546366 TI - Cholangitis: pathogenesis, diagnosis, and treatment. PMID- 7546367 TI - Septic thrombophlebitis of major dural venous sinuses. PMID- 7546368 TI - An approach to acute fever and rash (AFR) in the adult. PMID- 7546370 TI - Enteropathogenic and enteroaggregative strains of Escherichia coli: clinical features of infection, epidemiology, and pathogenesis. PMID- 7546369 TI - Acute and chronic mastoiditis: clinical presentation, diagnosis, and management. PMID- 7546371 TI - BCG immunization: review of past experience, current use, and future prospects. PMID- 7546372 TI - Immunization in adults in the 1990s. PMID- 7546373 TI - Outpatient management of HIV infection in the adult: an update. PMID- 7546374 TI - Antimicrobial prophylaxis in patients undergoing solid organ transplantation. PMID- 7546375 TI - Prophylaxis in bowel surgery. AB - The busy colon and rectal surgeon deals daily with a sea of bacteria. Using good surgical judgment as well as time-honored techniques and innovative equipment the postoperative results are generally good. The role that appropriately administered efficacious antibiotics play in this scenario should not be underestimated and can only be realized when historical controls are evaluated. The results of these studies of antibiotic bowel preparation suggest that many different approaches may be equally effective in reducing infection after elective colonic resection. Certain features, however, appear to be common to most of the studies. 1 Oral antibiotic regimens with both aerobic and anaerobic activity (e.g., neomycin/erythromycin base) were used. 2 The oral agents were given in limited doses the day before operation. 3 Addition of systemic antibiotic agents without broad-spectrum coverage to the oral regimen generally did not improve the results. 4 Use of broad-spectrum parenteral antibiotic agents alone was associated with a lower infection rate than the use of systemic agents having only limited coverage. 5 Addition of a broad-spectrum parenteral antibiotic to the oral antibiotics may further reduce the postoperative infection rate. 6 Parenteral or oral antibiotics should be administered only for short periods of time during the perioperative period. Since the general acceptance of the approach outlined above, infection rates have decreased and the number of clinical studies reported has drastically decreased. The authors do feel, however, that there is a need for further study to outline possible benefits of other appropriate regimens (34). PMID- 7546376 TI - Update on diagnosis of Clostridium difficile-associated diarrhea. PMID- 7546377 TI - Pathways from hematopoietic stem cells to thymocytes. AB - A long-standing debate has been whether commitment to the T cell lineage occurs exclusively following thymus colonization, or whether prethymic T lineage restricted progenitors exist. Recently, the analysis of murine fetal blood for the presence of hematopoietic progenitor cells has led to the identification of a T lineage committed precursor population (designated prothymocytes). Fetal blood prothymocytes lack multipotent progenitor potential as shown by the fact that they fail to reconstitute B lymphocyte, myeloid and erythroid lineages. In addition to prothymocytes, fetal blood also contains a phenotypically distinct, pluripotent progenitor population which can reconstitute both T and B lymphocytes as well as myeloid and erythroid lineages. The identification of a circulating, T lineage restricted precursor population, which is also found in the blood of fetal athymic mice, provides strong evidence that T lineage commitment can precede thymus colonization. The thymus is not, however, exclusively colonized by prothymocytes. Under appropriate developmental conditions, multipotent precursor activity for non-T lineages such as B lymphocytes and thymic dendritic cells can be revealed within the intrathymic precursor pool. Moreover, evidence has been accumulated for a common progenitor for T cells and natural killer cells which may be distinct from multipotent intrathymic progenitors. PMID- 7546378 TI - Positive selection of T cells. AB - In the past year, significant technical developments have provided the opportunity to investigate the more mechanistic features of positive selection. Major progress has been made in determining the structure and function of the early pre-T cell receptor, in defining cell types that mediate positive selection, and in analyzing the contribution of MHC and co-receptors to CD4/CD8 lineage commitment. The most revealing studies have been those addressing the role of peptides in thymic selection. PMID- 7546380 TI - Cytokine production and requirements during T-cell development. AB - Cytokines have been presumed to play an important role during T-cell development, but until recently formal proof has been missing. Most cytokine-knockout mice show apparently normal intrathymic T-cell development. The exception to this is IL-7, as both IL-7 and IL-7R alpha knockout mice show abnormally low thymic cellularity, making IL-7 the first cytokine shown to be necessary for normal intrathymic T-cell development. PMID- 7546379 TI - Intrathymic and extrathymic clonal deletion of T cells. AB - Clonal elimination accounts for self-tolerance induction in the thymus and also affects mature T cells responding to exogenous antigens in the periphery. Recent evidence on the microenvironments, cell-cell interactions and signalling requirements for clonal deletion of immature and mature T cells is discussed. PMID- 7546381 TI - Positive and negative selection events during B lymphopoiesis. AB - Early in B-cell development, large numbers of cells have to be generated, each of which expresses only one type of B-cell receptor (i.e. Ig) on its surface. This is achieved by the surface expression of a pre-B cell receptor containing a mu heavy chain/surrogate light chain which differentially provides signals for two responses of precursor B cells at this stage of development. On the one hand, it signals inhibition of further rearrangements of variable heavy chain to diverse joining heavy chain loci to achieve allelic exclusion at the heavy-chain locus. On the other hand, it signals proliferative expansion by factors between 20 and 100. Later in B-cell development, tolerance to autoantigens must be established and maintained. Tolerance is achieved by developmental arrest and induction of secondary light-chain gene rearrangements in those IgM+ immature B cells that are reactive to autoantigens presented in the primary B-cell generating organs. Even later in development, when mature surface (s)IgM+/sIgD+ B cells encounter autoantigens presented to them in the periphery, either deletion or anergy of the autoantigen-reactive cells occurs. Anergic cells have a sIg-dependent, sIg proximal defect in signaling and are short-lived. Anergy can be broken in vitro by polyclonal activation via ligation of CD40 in the presence of IL-4. A small part of the remaining immature B cells not reactive to autoantigens are selected to become mature, antigen-reactive sIgM+/sIgD+ B cells. Molecules which might guide such positive selection of B cells still remain to be identified. PMID- 7546382 TI - Life and death during lymphocyte development and function: evidence for two distinct killing mechanisms. AB - During their development and after their functional activation most lymphocytes die, either because they fail to receive a survival signal or because they are actively killed. Recent experiments on Bcl-2 and Fas/Apo-1 have provided evidence that these two death processes are subject to distinct genetic control. PMID- 7546383 TI - Extrathymic T cell differentiation. AB - In the mouse, the gut mucosa is a major site of extrathymic differentiation of T cells. Recent data in this past year show that this process differs from the main thymic differentiation pathway not only in its location, but also in its use of costimulatory molecules, signal transduction modules, and mechanisms of repertoire selection. The thymus exerts an influence on the expansion of the extrathymically differentiated gut intraepithelial lymphocytes that appears to be varied in nature, including acting as a source of TCR- progenitors. All gut intraepithelial lymphocytes, whatever their extrathymic or thymic site of differentiation, have common features of activated and specialized cytotoxic cells. Other T cells may differentiate extrathymically, in particular in the liver; these later cells appear to have a very restricted, probably autoreactive repertoire, and also display natural killer cell features. PMID- 7546384 TI - B-cell stimulation. AB - Recent studies have identified CD40 ligand (CD40L) as the critical membrane expressed molecule responsible for T cell dependent B-cell activation. CD40L co operates with various cytokines to induce B-cell activation, proliferation, and immunoglobulin isotype switching. Some antigens, however, can also stimulate B cell activation and isotype switching in the absence of CD40L or T cells. Recent studies have suggested that cytokines derived from non-T cells, such as natural killer cells, macrophages and mast cells, are responsible for isotype switching in T cell independent responses. PMID- 7546385 TI - Somatic hypermutation. AB - For the generation of secondary response antibodies, immunoglobulin genes are subjected to hypermutation. Cells expressing antibodies with higher affinity are then selected by antigen. Recent clues to the mechanism of hypermutation come from experiments using transgenic mice enabling analysis of the controlling cis acting elements and the intrinsic features of the hypermutation, dissociated from the effects of antigenic selection. PMID- 7546386 TI - Single T cell probes for antigen/MHC expression. AB - T-cell receptor binding to the antigen/MHC ligand on the surface of antigen presenting cells is the central event in T-cell activation. Methods have been developed for measuring this recognition event in single T cells and have allowed their use as 'probes' for antigen/MHC expression. Single cell assays have allowed analysis of early events in T-cell activation, and of unknown ligands recognized by the T-cell receptor. PMID- 7546387 TI - New methods to predict MHC-binding sequences within protein antigens. AB - The identification and analysis of MHC-binding sequences within protein antigens, and ultimately the ability to predict them, is central to immunology. Recent advances have revealed increasingly complex MHC-binding motifs and allows prediction of sequences that bind to both classes of MHC molecules. The systematic characterization of binding motifs for all human MHC alleles is now possible and will facilitate the design of peptides for therapeutic intervention. PMID- 7546388 TI - Detection and isolation of rare cells. AB - Cells of biomedical interest are often present in very small numbers (i.e. they are rare), despite their functional significance. The analysis and isolation of previously inaccessible rare cells, such as peripheral hematopoietic stem cells, fetal cells in maternal blood, residual tumor cells or antigen-specific lymphocytes, has now become feasible through the development of new methods. Today, these techniques allow the detection, isolation and analysis of cells less frequent than one in a million. Not many problems in immunology would require higher resolution. PMID- 7546389 TI - Recent advances in differential display. AB - Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes. Methodological modifications have since been introduced to streamline the techniques. The major effort has centered on how to eliminate false positives as approached from a variety of angles, ranging from RNA sample preparation, northern blot confirmation, primer length variation, to better experimental design. PMID- 7546390 TI - Analysis of immunoglobulin gene rearrangements in single B cells. AB - The polymerase chain reaction allows the characterization of RNA and DNA sequences in samples as small as a single cell. The recent development of amplification systems designed to isolate rearranged immunoglobulin genes from single B lineage cells has provided a powerful tool to investigate various aspects of B-cell development. PMID- 7546391 TI - Lymphocyte development. PMID- 7546392 TI - Immunological techniques. PMID- 7546393 TI - Protein tyrosine kinases in the initiation of antigen receptor signaling. AB - Intracellular responses to antigen receptor engagement involve the activation of protein tyrosine kinases and the tyrosine phosphorylation of cellular proteins, including components of the antigen receptor. Phosphorylation of two tyrosine residues within an 18 amino acid segment of the cytoplasmic domain of antigen receptor subunits, and the subsequent association of either the Syk or Zap protein tyrosine kinase, has recently been shown to be required for successful antigen receptor signal propagation. The recent finding that distinct primary human immunodeficiencies result from mutations in genes encoding two non transmembrane protein tyrosine kinases underscores the importance of this class of enzyme in antigen receptor signal transduction. PMID- 7546394 TI - Regulation of immune function by protein tyrosine phosphatases. AB - CD45 is a leukocyte transmembrane protein tyrosine phosphatase that functions in lymphocyte activation by increasing the kinase activity of Src family members. In contrast, an intracellular protein tyrosine phosphatase that contains Src homology 2 domains, SHP, is required to inactivate receptors. Recent studies have examined the mechanisms of interaction and regulation of these enzymes. PMID- 7546395 TI - Cytokine receptor signaling mechanisms. AB - Recent studies have dramatically advanced our understanding of the post-receptor signaling machinery engaged by members of the cytokine receptor superfamily. Proximal components of this signaling machinery are members of a novel class of protein tyrosine kinases termed JAKs. A working model proposes that ligand induced dimerization of cytokine receptor subunits triggers the catalytic activation of the associated JAKs, which then couple the receptor to downstream signaling pathways. Ongoing research efforts are also yielding information regarding two potentially interactive pathways of nuclear signaling activated by cytokine receptor occupancy. These pathways are initiated by the activation of the Ras to mitogen-activated protein (MAP) kinase cascade and by the tyrosine phosphorylation of a newly defined set of transcription factors, the signal transducers and activators of transcription (STATs). Collectively, these results begin to provide a molecular basis for the long-observed phenomena of cytokine pleiotropy and redundancy. PMID- 7546396 TI - Immediate-early genes induced by antigen receptor stimulation. AB - Immediate early genes induced by triggering of the TCR frequently encode proteins that act in signal transduction cascades. Recent advances concerning several immediate-early proteins have been made, including signal-induced regulation of NF-kappa B by I kappa-B, the role of Nur77 in T-cell selection and apoptosis, and the function of PAC-1 in regulating the Ras/ERK pathway. PMID- 7546397 TI - Transcriptional regulation of the IL-2 gene. AB - The past two years have seen significant advances in our understanding of IL-2 gene transcription. Many of the relevant transcription factors have been identified, the intracellular mechanisms regulating their functions are being elucidated, and the multiple roles of calcineurin are beginning to be appreciated. PMID- 7546398 TI - The cell biology of CTL killing. AB - A great deal is known about the immunology of cytotoxic T lymphocyte killing, but much less is known about the cell biology of this process. Recent work has begun to elucidate the mechanisms that control lytic-protein secretion, and reveals that some unusual features of these secretory processes might explain some of the important features of killing by cytotoxic T lymphocytes. PMID- 7546399 TI - T cell independent antigens. AB - T cell independent antigens type 2 (TI-2), which are represented predominantly by polysaccharide antigens, stimulate humoral antibody responses in the absence of T cell help. We and others have recently reported that natural killer cells and/or natural killer cell derived lymphokines may provide a form of 'help' that is necessary for the induction and maintenance of TI-2 responses. Two natural killer cell derived lymphokines, interferon-gamma and granulocyte-macrophage colony stimulating factor, show synergistic stimulatory activity in inducing Ig secretion in B cells stimulated by a multivalent ligand that mimics TI-2 antigens. The recent finding that natural killer cells have receptors for various classes of polysaccharides supports a role for these cells in regulating responses to TI-2 antigens. PMID- 7546400 TI - CD23 and B-cell activation. AB - The past year has seen the publication of significant new findings on the regulation of CD23 expression, the precise interaction of CD23 with CD21 and its functional consequences. Moreover, new advances have been made in unravelling the biochemical network of events downstream from the triggering of CD23 in human B cells. Analyses of the properties of CD23-deficient mice have demonstrated a link between IgE regulation and CD23 in vivo. PMID- 7546401 TI - T helper cell differentiation in immune response. AB - Significant progress has been made in understanding the maturation of antigen specific CD4+ T helper cell responses. Recent progress centers on the network of cytokines, accessory molecules, and cell types that shapes the differentiation of distinct T helper cell subsets. Use of transgenic and knockout mice and well characterized in vivo models have helped clarify the interdependence or independence of many of these complex factors. PMID- 7546403 TI - Molecular mechanisms underlying functional T-cell unresponsiveness. AB - Evidence from tissue culture experiments and in vivo studies indicates that certain forms of antigen presentation render T cells unresponsive to subsequent antigenic stimulation. Great progress has been made recently in understanding the induction, maintenance, and in vivo relevance of this unresponsive state, called clonal anergy. PMID- 7546402 TI - Mouse NK1+ T cells. AB - Mouse NK1+ T cells constitute a special subset of alpha beta TCR+ T cells that express natural killer surface receptors and are thought to play an immunoregulatory role because of their unique ability to secrete IL-4 within minutes of primary activation. The recent discovery that they recognize non polymorphic MHC class I like ligands encoded by CD1 family genes sheds a new light on unusual aspects of their development as well as on some of the possible ways in which they might influence the regulation of the T-helper cell (types 1 and 2) classes of immune responses. PMID- 7546404 TI - Activation-induced death of mature T cells in the regulation of immune responses. AB - Deletion of self-reactive clones of immature thymocytes by activation-induced death (AID) is thought to be the primary mechanism for the establishment of self tolerance in the T-cell compartment. Recent evidence suggests that a genetically distinct but analogous process of AID in mature T cells is important in regulating peripheral immune responses. AID of peripheral T cells requires the expression of functional Fas and Fas ligand by the T-cell population. As qualitatively similar signals from the TCR are responsible for both T-cell expansion in inflammation and T-cell elimination by AID, regulating the balance between these opposing functions plays a crucial role in successful responses to pathogens and tumors while minimizing autoimmunity. PMID- 7546406 TI - Regulation of lymphocyte functions by nitric oxide. AB - Current interest in nitric oxide is derived from its numerous biological roles. Immunologically, apart from the well known role of nitric oxide in anti-microbial and autoimmune diseases, nitric oxide is now emerging as a potential powerful mediator of T-cell responses. Evidence now shows that nitric oxide can both enhance and suppress T-cell functions and that some subsets of T-cell clones can be activated to produce nitric oxide. PMID- 7546405 TI - Analysis of lymphocyte costimulation in vivo using transgenic and 'knockout' mice. AB - The use of transgenic technologies in the functional evaluation of the contributions of costimulatory pathways to T-cell activation in vivo has recently undergone a rapid expansion. During the past two years, mice deficient in costimulatory molecules and their receptors have been generated. These mice have revealed novel and critical in vivo functions of costimulatory pathways and have provided valuable models in which to test therapeutic strategies involving costimulatory pathway blockade. Transgenic mice constitutively expressing costimulatory molecules have provided insights into their role in peripheral tolerance. PMID- 7546407 TI - T-cell regulation of macrophage function. AB - Macrophage function has long been known to be controlled by activated T cells. As the cytokines that control macrophage activation are defined, it is becoming apparent that macrophage 'activation' is more subtle than previously appreciated. Moreover, recent work has shown the dependence of T-cell development on macrophages, from the costimulation of T cells by macrophage surface molecules such as the B7 family, to the modulation by monokines of T-cell phenotype and growth, including IL-1, IL-10, IL-12 and IL-15. Combinations of cytokines and costimulators have revealed some of the processes by which an apparently dichotomous T-cell response can regulate the subtle diversity of the immune response. PMID- 7546408 TI - Lymphocyte activation and effector functions. PMID- 7546410 TI - Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient. AB - OBJECTIVE: To investigate the phylogenetic relationship of HIV-1 proviral long terminal repeat (LTR) variants present in postmortem samples of lymph node, spleen, lung, dorsal root ganglion and spinal cord as well as in the peripheral blood of an HIV-1-infected patient dying with AIDS. DESIGN AND METHODS: Postmortem tissues were studied by a combination of histology, cell culture and molecular analyses. The patient had a stable CD4 count of 10 x 10(6)/I during the 12 months preceding death. A 540 base-pair fragment of the LTR including U3/R/U5 was amplified using polymerase chain reaction on proviral DNA from the five postmortem tissues and peripheral blood mononuclear cells obtained 2 months prior to death. The population of viral variants was determined by sequencing at least five plasmid clones of the amplicons. The relationship between the variants present in different body sites was investigated using molecular phylogeny methods. RESULTS: HIV-1 was present in all organs analysed and correlated with the presence of abnormal histology. Genetic variation leading to divergence from the consensus sequence was more frequently present in characterized transcription factor binding sites within the LTR (P < 0.0001) although the HIV-1 LTR quasispecies in the different body sites showed similar, relatively low levels of divergence (intra-organ median heterogeneity ranging from 0.0094 to 0.017). Phylogenetic analysis showed that the spinal cord and dorsal root ganglion harboured an LTR population genetically distinct from that present in other organs and more closely related to a previously characterized neurotropic strain of HIV (strain JRcsf). CONCLUSION: The independent clustering of HIV-1 LTR variants present in spinal cord and dorsal root ganglion shows that HIV-1 LTR evolution can occur in a compartmentalized fashion. The data show that the LTR is an important region to analyse in sequence variation studies of HIV since it may play a role in nervous tissue adaptation of HIV-1 and neuropathogenicity. Outgrowth of HIV-1 LTR variants that are most fit for the utilization of tissue specific transcription factors can occur in the nervous tissue. PMID- 7546409 TI - Preventive therapy for tuberculosis in HIV infection: the promise and the reality. PMID- 7546412 TI - In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from high-risk HIV-negative intravenous drug users. AB - OBJECTIVE: To study type 1 and type 2 cytokine patterns in HIV-negative high-risk intravenous drug users (IVDU). DESIGN: We investigated interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6 and IL-10 production by phytohaemagglutinin (PHA)-stimulated and unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-negative high-risk IVDU, HIV-negative controls and HIV-positive subjects. METHODS: Cytokine production was measured in supernatants using enzyme linked immunosorbent assay (ELISA) in 10 HIV-negative high-risk IVDU, 25 HIV negative controls, and 12 HIV-positive IVDU. We also determined spontaneous in vitro immunoglobulin (Ig) G and IgM production. RESULTS: HIV-negative high-risk IVDU showed increased IFN-gamma and decreased IL-4, IL-10 and IL-2, although the latter was not significant compared with HIV-negative controls. Further, HIV negative high-risk IVDU had reduced IgG production and impaired IgM-IgG switch. CONCLUSIONS: The reduced IL-2 and IL-4 production suggest an impaired CD4+ T-cell function in HIV-negative high-risk IVDU. The increased IFN-gamma production along with the decreased type 2 cytokine profile is consistent with the hypothesis that protective immunity against HIV may reside in type 1 responses and cell-mediated immunity. PMID- 7546411 TI - A genetic analysis of HIV-1 from Punjab, India reveals the presence of multiple variants. AB - OBJECTIVE: To determine the extent of HIV-1 genetic variation in Indian patients. DESIGN: To avoid any bias in selecting viral variants, HIV-1 DNA was amplified directly from the peripheral blood mononuclear cells of patients and sequenced. Genetic similarity between Indian sequences and other geographic isolates was analysed by phylogenetic analysis algorithms. METHODS: A fragment encompassing the C2/V3-V5 regions of HIV-1 gp120 was amplified from the lymphocyte DNA of 12 Indian patients. Multiple clones from each patient were sequenced. Nucleotide sequences encompassing about 650 base pairs were aligned for the Indian and other geographically distinct isolates. Inter-isolate relationships were analysed by means of distance, parsimony and neighbour-joining algorithms. RESULTS: Nucleotide sequence comparisons showed low interpatient variation. Amino-acid comparisons revealed a high degree of homology between Indian sequences in this study and those studied earlier. On distance and parsimony trees, most of the Indian sequences clustered together as subtype C. However, sequences from three patients also showed significant homologies and phylogenetic clustering outside of subtype C. CONCLUSIONS: The predominant strain of HIV-1 in India belongs to subtype C and little interpatient nucleotide sequence divergence in the majority of cases suggests recent spread of HIV-1 in this region. This study also presents the first evidence for non-C subtypes in the Indian population with two epidemiologically linked samples remaining unclassified for any existing env subtype. The presence of variant subtypes in Indian patients sheds light on the transmission routes of HIV-1 to India and emphasizes the need to include these sequences in vaccine development strategies. PMID- 7546413 TI - Virus-specific antibody production and polyclonal B-cell activation in the intestinal mucosa of HIV-infected individuals. AB - OBJECTIVE: To examine possible changes in mucosal B-cell activation status. DESIGN: To examine the frequency and isotype distribution of total and HIV specific antibody-secreting cells (ASC) in the intestinal mucosa of HIV-infected individuals. METHODS: Mucosal lymphocytes were obtained by enzymatic treatment of duodenal pinch biopsies and the numbers of ASC were assayed with the enzyme linked immunospot technique. RESULTS: High numbers of HIV-specific ASC were found in the intestine of all HIV-infected individuals despite low levels of HIV specific blood ASC. All HIV-infected individuals had large numbers of intestinal immunoglobulin (Ig) A-ASC against the HIV envelope glycoprotein gp160. Eight out of nine patients also had HIV gp160-specific intestinal IgG-ASC. These HIV specific ASC were detected irrespective of disease stage, route of infection, or levels of circulating CD4+ T cells. HIV-specific ASC were found in peripheral blood from patients with CD4+ T cells > or = 100 x 10(6)/l blood, but in none of three patients with low CD4+ T-cell counts. The frequencies of virus-specific ASC in the blood were on average 100-fold lower than that observed within the intestinal mucosa. Mucosal polyclonal B-cell activation was evident in HIV infected individuals, as documented by significantly elevated numbers of Ig secreting cells (ISC) in all three major Ig classes; on average, seven-, five- and 20-fold numbers of IgA, IgG and IgM-ISC compared with healthy controls. Furthermore, substantial numbers of ASC reacting with unrelated antigens such as dog albumin and keyhole limpet haemocyanin were detected in HIV-infected patients. Interestingly, patients with CD4+ T cells < 100 x 10(6)/l blood displayed large numbers of HIV-specific intestinal ASC even though total numbers of ISC, including ASC reactive to unrelated antigens, were decreased. CONCLUSIONS: The large numbers of virus-specific ASC found in the intestine of HIV-infected individuals may be a consequence of local replication of HIV-1 resulting in a continuous antigen stimulation. The persistence of strong intestinal anti-HIV responses even at late stages of disease suggest that the mucosal B-cell responses are functionally intact throughout the disease. Furthermore, these results suggest that there is no correlation between HIV specific ASC numbers and polyclonal B-cell activation. These observations indicate that intestinal B-cell activation is profoundly disregulated in HIV infected individuals. PMID- 7546415 TI - Recurrent transient neurological deficits in advanced HIV infection. AB - OBJECTIVES: To report the occurrence of a syndrome of recurrent neurological deficits in advanced HIV disease and to discuss its management and prognosis. DESIGN: Retrospective case study. SETTING: A regional treatment centre for HIV infected individuals in northwest England. MAIN OUTCOME MEASURES: Transient neurological deficit was defined as a focal neurological deficit of rapid onset which resolved completely within 24 h. Frequency, character and duration of episodes were recorded. Prior illnesses, CD4 count, changes in drug therapy and brain imaging investigations were also documented. RESULTS: Seven cases with recurrent transient neurological deficits were identified among 748 patients over a 10-year period. Six had a CD4 cell count < 50 x 10(6)/l. The episodes lasted between 1 and 12 h and resolved completely without lasting sequelae. Dysphasia and hemiparesis were the most common presentations. Recurrent episodes for each patient tended to follow a similar pattern. None had computed tomographic evidence of cerebral infarction or clinical evidence of completed stroke. Prognosis was varied and not evidently altered by the episodes of neurological deficit. Three patients may have benefited from treatment with migraine prophylactics. CONCLUSION: A syndrome of recurrent transient neurological deficits may occur in advanced HIV disease. We believe that in some cases this may be due to local cerebral vasospasm, comparable to a classic migraine aura. PMID- 7546414 TI - Lymphoid tissues targeting of liposome-encapsulated 2',3'-dideoxyinosine. AB - OBJECTIVE: To improve the pharmacokinetics and lymphoid tissues targeting of 2',3'-dideoxyinosine (ddI) by encapsulation in liposomes. METHODS: The pharmacokinetics and tissue distribution of free and liposome-encapsulated ddI were determined in C57BL/6 mice following intravenous and subcutaneous administration of a single bolus dose (3 mg ddI/kg). RESULTS: Intravenous administration of liposome-encapsulated ddI greatly reduced the systemic clearance of the anti-HIV agent. The elimination plasma half-life of ddI incorporated in 112 and 83 nm liposomes was 46 and 14 times higher than that of the free drug, respectively. The tissue distribution profile of liposomal lipids clearly showed that the use of liposomes allows efficient targeting of lymph nodes and macrophage-rich tissues (spleen and liver) for at least 24 h following intravenous injection. In contrast, the accumulation of liposomes in these tissues was much lower following subcutaneous administration. CONCLUSION: Incorporation of ddI in liposomes greatly improved the pharmacokinetics of the anti-HIV agent after intravenous injection. The use of liposomes could represent a convenient approach to targeting lymphoid tissues. Strategies aimed at improving drug retention within liposomes should further enhance and prolong drug delivery to lymphoid organs. PMID- 7546416 TI - Relation between stage of disease and neurobehavioral measures in children with symptomatic HIV disease. AB - OBJECTIVE: To study the relationships between stage of HIV disease, reflected by CD4+ lymphocyte percentages and p24 antigen levels, and HIV-associated central nervous system (CNS) abnormalities, measured by computed tomography (CT) brain scan ratings and neurobehavioral tests. DESIGN: Consecutive case series. SETTING: Government medical research center. PATIENTS: Eighty-six previously untreated children with symptomatic HIV-1 disease. RESULTS: CD4% measures correlated significantly with overall CT brain-scan severity ratings (r = -0.45; P < 0.001) as well as with its component parts (cortical atrophy, white matter abnormalities, and intracerebral calcifications); they were of comparable magnitude for vertically and transfusion-infected children. CD4% measures were also associated with the general level of cognitive function (r = 0.32; P < 0.005). Furthermore, patients with detectable serum p24 antigen levels (n = 39) had CT brain scans that were more abnormal than patients with undetectable p24 levels (n = 20; CT abnormality ratings of 21.3 versus 35.9; P < 0.02); similar differences were found for the cortical atrophy and calcification ratings. p24 levels also correlated with the overall CT brain-scan severity rating (r = 0.34; P < 0.01). CONCLUSIONS: Degree of CT brain-scan abnormality and level of cognitive dysfunction were significantly associated with the stage of HIV-1 disease, as reflected by either CD4 leukocyte measures or elevations of p24 antigen. The relation between the CT brain-scan lesions and markers of HIV disease (both CD4 and p24) suggest that these CNS abnormalities are most likely associated with HIV-1 infection, and further support the hypothesis that the interaction between systemic disease progression and CNS manifestations is continuous rather than discrete. PMID- 7546417 TI - Maternal HIV infection and infant mortality in Malawi: evidence for increased mortality due to placental malaria infection. AB - OBJECTIVES: To examine the relationship between maternal HIV infection, placental malaria infection, and infant mortality as a first step in investigating the possibility of increased vertical transmission of HIV due to placental malaria infection. DESIGN: Retrospective analysis of data from a cohort study of mothers and infants in rural Malawi conducted from 1987 to 1990. METHODS: Pregnant women in Malawi were enrolled in a study examining chemoprophylaxis during pregnancy. At delivery, placental malaria infection status was determined. Infants born into this study were visited every 2 months for the first 2-3 years of life. Deaths were investigated using a standardized 'verbal autopsy' interview. Maternal serum collected during pregnancy was tested for antibodies to HIV-1 by enzyme-linked immunosorbent assay with Western blot confirmation. RESULTS: Overall, 138 (5.3%) of 2608 women in the study were HIV-1-seropositive. Infant mortality rates were 144 and 235 per 1000 live births for children born to HIV-seronegative and HIV seropositive women, respectively (P < 0.001). In a multivariate model, the odds of dying during the post-neonatal period for an infant born to a mother with both placental malaria and HIV infection was 4.5 times greater than an infant born to a mother with only placental malaria, and between 2.7 and 7.7 times greater (depending on birthweight) than an infant born to a mother with only HIV infection. CONCLUSIONS: This study strongly suggests that exposure to both placental malaria infection and maternal HIV infection increases post-neonatal mortality beyond the independent risk associated with exposure to either maternal HIV or placental malaria infection. If confirmed, malaria chemoprophylaxis during pregnancy could decrease the impact of transmission of HIV from mother to infant. PMID- 7546418 TI - The predictive value of changes in serologic and cell markers of HIV activity for subsequent clinical outcome in patients with asymptomatic HIV disease treated with zidovudine. AB - OBJECTIVE: To determine if serologic marker responses to zidovudine treatment during the first year of antiretroviral therapy could predict subsequent HIV disease progression independently of absolute CD4 lymphocyte responses. METHODS: We conducted a case-control study in patients with asymptomatic HIV disease, who were initiating zidovudine therapy in a randomized, prospective trial. A total of 102 patients who progressed to AIDS or advanced AIDS-related complex and 177 randomly selected controls matched by baseline CD4 cell count and duration of follow-up had serum samples (from prior to and at 8, 16, 32 and 48 weeks of zidovudine treatment) assayed for acid-disassociated HIV p24 antigen, beta 2 microglobulin (beta 2M), neopterin, soluble interleukin (IL)-2 receptor, soluble CD4 protein and soluble CD8 protein. RESULTS: Median time to event for cases was 20.2 months; median follow-up on study was 35.4 months for controls. After controlling for absolute CD4 count at baseline, increased baseline serum concentrations of HIV p24 antigen, beta 2M, neopterin, and soluble IL-2 receptor were highly predictive of increased risk of HIV disease progression. In a multiple logistic regression model, controlling for baseline marker values, change in beta 2M consistently added independent value to change in CD4 count in predicting subsequent risk of disease progression. CONCLUSIONS: Monitoring serum immunologic markers, in particular beta 2M, in addition to absolute CD4 lymphocyte counts prior to and within the first 4 months after initiating dideoxynucleoside therapy can increase the accuracy of estimations of subsequent long-term risk of clinical HIV disease progression. This information may be useful to clinicians and patients who are making decisions about initiating or changing antiretroviral therapy. PMID- 7546419 TI - Sexual behaviour and HIV epidemiology: comparative analysis in France and Britain. The ACSF Group. AB - OBJECTIVE: France and Britain have similar size populations, yet the incidence of AIDS is threefold higher in France. The objective of this study was to compare data from two surveys recently performed in the two countries, in order to determine whether differences in sexual and drug-use behaviour could explain the different epidemiological patterns. DESIGN: Cross-sectional random sample surveys of France and Britain. RESPONDENTS: In France, 20,055 persons aged 18-69 years and in Britain, 18,876 persons aged 16-59 years were interviewed in 1990-1991. The following indicators were compared in the respondents aged 18-59 years: prevalence of homosexual experience and injecting drug use, number of sexual partners, prevalence of sexual practices, condom use and sex with prostitutes, age of sexual partners. RESULTS: Very similar results were found for the prevalence of male homosexual partnerships. Slightly higher numbers of lifetime partners were reported by French than British men, but no difference was found for recent periods. Anal intercourse and sex with prostitutes was more frequent among heterosexual French people than British people. Condom use was more systematic in Britain than in France. CONCLUSION: Only small differences were found between the two countries, although prevalence of risk indicators were higher in France. These differences combined with early development of prevention policies in Britain, together with the timing of virus introduction, may contribute to differences between the epidemics in the two countries. PMID- 7546421 TI - AIDS knowledge, risk behaviors, and factors related to condom use among male commercial sex workers and male tourist clients in Bali, Indonesia. AB - OBJECTIVES: To describe the AIDS/sexually transmitted diseases (STD) knowledge and risk behaviors, and to determine factors related to condom use among male commercial sex workers (CSW) and male tourist clients in Bali, Indonesia. DESIGN: Individual survey interviews were conducted with a sample of 80 male CSW and 100 tourist clients in the Kuta area of Bali. METHODS: Survey interviews included detailed questions on AIDS/STD knowledge, sexual behavior, and psychosocial measures related to risk taking. RESULTS: Most of the male CSW had heard about AIDS and other STD, although some misconceptions about transmission and risks of different sexual practices remained. Both oral and anal intercourse with tourists were common and condom use was far from consistent. Factors related to condom use for anal intercourse with tourist clients were condom beliefs, self efficacy, susceptibility to STD infection, and STD knowledge. Tourist clients were mainly from Australia and Europe and many had paid for sex in other parts of Indonesia as well as in high prevalence countries. Knowledge of AIDS/STD was very good among the tourists and previous experience with STD was common. Factors related to condom use with male CSW were condom beliefs and self efficacy. CONCLUSION: There is a very active and mobile group of male CSW and tourist clients present in Bali. Interventions with these men are needed due to the low level of knowledge about AIDS among CSW, their experience with STD and STD symptoms, and their level of risky sexual behavior. PMID- 7546420 TI - The social dynamics of HIV transmission as reflected through discordant couples in rural Uganda. AB - OBJECTIVE: To describe the role of men and women as sources of HIV transmission and to estimate HIV incidence among discordant couples resident in diverse rural communities in Uganda. SETTING: Rakai, a rural district in Uganda, East Africa. METHODS: A population-based cohort study, which has been conducted as annual serological and behavioral surveys since 1989. Community clusters were stratified into trading centers on main roads, intermediate trading villages on secondary roads and agricultural villages off roads. In the 1990 survey round, serological data were available for 79 discordant and 411 concordant HIV-negative couples aged 13-49 years. The present analysis examines sex-specific seropositivity associated with place of residence and the incidence of seroconversion among discordant couples between 1990 and 1991. RESULTS: Seventy-nine discordant couples were followed; the HIV-positive partner was male in 44 couples (57%) and female in 35 couples (43%). There was marked variation in the sex of the seropositive partner by place of residence: women were the HIV-positive partner in 57% of couples from trading centers, 52% from intermediate villages, and 20% from agricultural communities (P < 0.008). Condom use was higher in discordant couples in which the man was the uninfected partner (17.1%) rather than the woman (9.5%). HIV-positive women, but not HIV-positive men, reported significantly more sexual partners and more genital ulcers than seronegative individuals of the same sex. Seroincidence rates among men and women in discordant relationship were 8.7 and 9.2 per 100 person-years (PY), respectively, which was much higher than in concordant seronegative couples (men, 0.82; women, 0.87 per 100 PY). CONCLUSIONS: In this Ugandan population, men are the predominant source of new infections in rural villages. Risk factors and preventive behaviors vary with the sex of the infected partner, and seroconversion rates are similar in both sexes. PMID- 7546422 TI - The HIV/AIDS epidemic in Jamaica. AB - OBJECTIVE: To describe the HIV/AIDS epidemic in Jamaica. METHODS: Data from the national surveillance system for HIV infection and AIDS based in the Epidemiology Unit, Ministry of Health, were reviewed. These include case reports; HIV screening of blood donors, migrant farmworkers and US visa applicants; sentinel surveillance among antenatal clinic (ANC) attenders and sexually transmitted disease (STD) clinic attenders; and various serosurveys. RESULTS: A total of 669 AIDS cases were reported in Jamaica from December 1993 representing a cumulative AIDS case rate of 28 per 100,000 population. Since 1987 the annual AIDS case rate doubled every 2 years with 69% of individuals having died with AIDS. Heterosexual transmission predominates with the cumulative adult AIDS male-to-female case ratio declining from 2.8:1 in 1988 to 1.9:1 in 1993. A total of 55 children with AIDS account for 8.2% of all cases. The HIV infection rate per 1000 in 1993 was 3.8 among blood donors, 1.4 among ANC attenders and significantly higher among STD clinic attenders (men 6%, women 2.7%), homosexuals (9.6%), female prostitutes (12%) and individuals with repeat STD infections (10%). Consistent condom use increased from 27% in 1989 to 47% in 1993. CONCLUSIONS: HIV infection was introduced into Jamaica from abroad through several different routes including the Jamaican homosexual community, migrant farmworkers, female prostitutes, and informal commercial importers. HIV transmission is well established locally and is spreading more rapidly in Western Jamaica and along the North Coast, which may reflect increased sexual activity associated with tourism. Although awareness of AIDS and HIV is high and condom use has increased considerably, there are no grounds for complacency concerning the HIV/AIDS epidemic in Jamaica. PMID- 7546423 TI - Predictors of repeat HIV testing among gay and bisexual men. AB - OBJECTIVE: To examine the prevalence and predictors of repeat HIV testing. DESIGN, SETTING AND PARTICIPANTS: Cross-sectional data from two random household based and bar-based samples of gay/bisexual men in two medium-size cities (Tucson, Arizona and Portland, Oregon) with substantial numbers of AIDS cases, in 1992 (n = 2602). MAIN OUTCOME MEASURE: The prevalence and predictors of repeat testing among men who reported being HIV-tested at least once but not being HIV positive (n = 1583). RESULTS: In total, 51% of the sample had been tested three or more times, and 15% were tested more than once every 6 months. Men with higher risk were more likely to be repeatedly tested, although oral but not anal risk was a significant predictor of repeat testing in regression analyses. Men who did not know the HIV status of their primary partner were less likely to be repeatedly tested. Men who perceived that social norms favored secondary prevention, specifically adherence to medical recommendations for the treatment of HIV infection, and who communicated more often about testing were more likely to be repeatedly tested. CONCLUSIONS: Policy and clinical recommendations for repeat testing must be based on consideration of the complexity and multi-faceted nature of repeat testing. For some individuals, repeat testing may play a legitimate role in HIV prevention by reinforcing safe behavior and providing confirmation of HIV-negative status. However, for others repeat testing may indicate a need for different or more intensive interventions to encourage safe sex. PMID- 7546424 TI - The usage and costs of health services for HIV infection in Australia. AB - OBJECTIVE: To describe patterns of health-service usage and the resulting costs in 1992-1993 for Australian men. DESIGN: A prospective survey, stratified by phase of illness. SETTING: Hospital and community-based care. PATIENTS: A total of 128 homosexual men: 20 in phase 1 (CD4+ count > or = 500 x 10(6)/l), 31 in phase 2 (CD4+ count < 500 and > or = 200 x 10(6)/l), 30 in phase 3 (CD4+ count < 200 x 10(6)/l), and 47 in phase 4 (AIDS). MAIN OUTCOME MEASURES: Mean monthly service usage rates and costs. RESULTS: Health-service utilization increased and became more hospital-based as illness worsened; the main exception was use of antiretroviral drugs, which peaked in phases 2 and 3. Hospital admission was rare before diagnosis of AIDS. Hospital bed-days per patient per month averaged 3.3 for AIDS patients until the final 3 months of life increasing to 15.8 in the 3 months before death. Mean monthly costs (in 1992-1993 Australian dollars) were $331 [95% confidence interval (CI), 218-455] in phase 1, $667 (95% CI, 540-836) in phase 2, $1372 (95% CI, 1044-1776) in phase 3, and $4615 (95% CI, 3456-5985) for AIDS patients until the last 3 months of life and $13,308 (95% CI, 10,538 16,516) in the 3 months before death. Drugs comprised 57% of total costs in phase 1, but only 30% of costs for patients with AIDS, whereas hospital bed-days comprised 10% of phase 1 costs and 60% of AIDS costs. CONCLUSIONS: Health-care utilization and resulting costs increased with severity of illness, and were particularly high for AIDS patients in the 3 months before death. Service utilization patterns and components of costs varied between each phase. PMID- 7546425 TI - Hospital costs of treating haemophilic patients infected with HIV. AB - OBJECTIVE: To calculate the costs of treating HIV-infected haemophilic patients. DESIGN: Two-year retrospective study of hospital-based resource use and costs, from April 1991 to March 1993. SETTING: Haemophilia Centre and Haemostasis Unit, Royal Free Hospital and School of Medicine, London, UK. PATIENTS: Sixty patients infected with HIV between October 1979 and July 1985. RESULTS: During the 2-year period a total of 1668 hospital visits were made by patients. The mean number of episodes per patient-year (PY) was 0.6 inpatient admissions, 11.5 outpatient visits and 1.8 day cases. The mean cost per PY was 32,528 pounds, with the majority of this spent on clotting factor concentrate products and haemophilia inpatient admissions (81%). A mean cost for HIV-related treatment of 6050 pounds was estimated. The additional cost incurred in switching this group of haemophilic patients from intermediate-purity factor concentrate to high-purity products was 8614 pounds per PY. When clotting factor concentrate and expenditure on haemophilia-related inpatient admissions were excluded, the mean cost of treating HIV infection per PY was 6065 pounds, varying with CD4+ count (< or = 50 x 10(6)/l, 13,093 pounds; 51-200 x 10(6)/l, 6521 pounds; 201-500 x 10(6)/l, 2848 pounds; > 501 x 10(6)/l, 1497 pounds). CONCLUSIONS: CD4+ count may be used as a marker of costs of HIV infection. The HIV-related cost estimates can be used for the planning of current and future hospital-based care in the National Health Service in the United Kingdom. The switch from intermediate-purity factor concentrate to high-purity products has increased the mean HIV-related cost per PY of treating haemophilic patients infected with HIV. PMID- 7546426 TI - HIV risk behavior among Peace Corps Volunteers. AB - OBJECTIVE: To describe HIV risk behaviors among Peace Corps Volunteers (PCV) and to examine correlates of sexual risk behaviors. METHOD: Cross-sectional data were collected from 1242 randomly selected PCV serving in 28 countries in 1991. PCV reported the frequency of specific risk behaviors in self-administered questionnaires, which were completed anonymously and returned to the Centers for Disease Control and Prevention. RESULTS: Non-sexual HIV risk behaviors were rarely reported by PCV. Sixty-one per cent of the 1080 PCV who answered questions about sexual behavior during their Peace Corps service reported having at least one sex partner. Sixty per cent of PCV had another PCV partner, 39% had a host country national partner, and 29% had a non-PCV expatriate partner. Overall, less than one-third (32%) of unmarried PCV used condoms during every episode of sexual intercourse; more frequent use was reported in relationships with non-steady and (for male PCV) host-country national partners. Among male PCV, condom use was positively related to lower alcohol use and the belief that HIV was a problem in the host country. Female PCV reporting more condom use with male partners were younger and had fewer partners than those reporting less use. CONCLUSION: These data indicate that PCV are at risk for acquiring HIV through unprotected vaginal intercourse. All persons who become sexually active with new partners while travelling or living abroad should be encouraged to use condoms consistently. PMID- 7546427 TI - Anonymous HIV surveillance with risk factor elicitation at Scotland's largest prison, Barlinnie. AB - OBJECTIVE: To determine prevalence of HIV infection and risk behaviours among male inmates of Her Majesty's Prison (HMP) Barlinnie, Glasgow, Scotland on 8-9 September 1994. DESIGN: Cross-sectional study: voluntary, anonymous HIV surveillance (using saliva samples) of all inmates and linked self-completion risk-factor questionnaire. SUBJECTS: Of 1073 prisoners available to participate, 985 (92%) completed a risk-factor questionnaire and 982 salivettes were received for testing, of which 978 were tested for HIV antibodies (four were dry samples); 928 questionnaires passed logical checks for consistency. MAIN OUTCOME MEASURES: HIV prevalence on saliva testing, related risk behaviours and ratio of overall-to disclosed HIV prevalence. Proportion of all inmates who have ever injected drugs, ever injected inside prison, started injecting inside, ever had acute hepatitis, had a recent personal HIV test (since January 1993). RESULTS: Nine saliva samples [eight injecting drug users (IDU), one recognized other risk] out of 978 were HIV antibody-positive (three presumptively from known HIV-infected inmates). Overall HIV prevalence was estimated at 1% compared with a known prevalence of 0.4%, giving an overall-to-disclosed HIV prevalence ratio of 2.6 in HMP Barlinnie in September 1994. A higher proportion of prisoners from Glasgow (48%) than elsewhere (19%) were IDU. Year of first injection was also different by residence with 23% of Glasgow IDU having first injected after 1988 compared with 45% of IDU from elsewhere, mainly West and South Scotland. Half the IDU inmates reported having injected while incarcerated and 6% had started to inject while incarcerated. Ten per cent of all prisoners and 20% of IDU inmates had had a personal HIV test since January 1993. Logistic regression showed that there was a significant deficit of recent HIV test uptake by Glasgow residents (odds ratio, 0.5; 95% confidence interval, 0.27-0.89), that IDU were more likely to have had treatment for a sexually transmitted disease, and that IDU who had injected inside and those whose injecting career began prior to 1989 were more likely to have had acute hepatitis. CONCLUSION: A consistent harm-reduction policy is needed across prisons in the United Kingdom to avoid transmission of blood-borne viral infections. Drug injecting inside prison is common, a proportion of IDU inmates having first injected drugs while in prison, and much higher rates of hepatitis have been reported in association with injecting while incarcerated compared with that for IDU who only injected outside prison. PMID- 7546428 TI - Fluconazole-resistant oral candidosis in HIV-infected patients. PMID- 7546429 TI - Sorivudine (BV-ara-U) for the treatment of complicated refractory varicella zoster virus infection in HIV-infected patients. PMID- 7546430 TI - Failure of atovaquone in the treatment of cerebral toxoplasmosis. PMID- 7546431 TI - Angiolymphoid hyperplasia with eosinophils in HIV infection. PMID- 7546432 TI - Fatal Castleman's disease and pulmonary Kaposi's sarcoma in an HIV-seropositive woman. PMID- 7546433 TI - Feto-maternal transfer of zidovudine: study on the perfused human placental cotyledon system. PMID- 7546434 TI - Recurrent pseudotumor cerebri in an HIV-positive patient. PMID- 7546435 TI - Progressive multifocal leucoencephalopathy in AIDS: treatment with cytosine arabinoside. PMID- 7546436 TI - HIV-1 viraemia and splenectomy. PMID- 7546437 TI - Biological phenotype of HIV-1 and transmission. PMID- 7546438 TI - Savings associated with immediate confirmation of initial high quantitative enzyme immunoassays for HIV. PMID- 7546439 TI - [Current trends in the preservation of frozen fruits, as markers of hygienic quality of food]. AB - Spore germination and growth rate of pathogens can be greatly reduced by cooling right after harvest. Most tropical or subtropical crops, and even some temperate, fruits undergo from chilling injury (CI) at temperatures considerably above their freezing point. Several procedures have been designed to increase tolerance to CI. Prestorage temperature conditioning can reduce CI in subsequent cold storage. Intermittent warming interrupting the chilling period by periods at nonchilling temperatures may become a practical treatment to prolong the storage of chilling sensitive crops. Modified atmospheres extend the postharvest life of fruit by reducing their respiration rate. The maintenance of fruit in good physiological condition may result in considerable disease resistance. A direct effect against pathogens is also possible at concentrations of O2 lower than 1.8% or concentrations of CO2 higher than 10%. PMID- 7546440 TI - [Biological control in the preservation of pome fruits]. AB - Fresh fruits are susceptible of be attacked by several pathogenic fungi after harvest due to both their high water and nutrients content and their loss of most of the intrinsic resistance that protected them over their development while attached to the plant. Most rot pathogens can be controlled by various methods such as refrigeration, controlled atmospheres and fungicides. Biological control strategies are emerging as promising alternatives to the use of synthetic fungicides. Several factors must be considered for the selection of biocontrol agents to be used against postharvest fruits diseases. Survivability of the antagonist is a major factor to determine its usefulness. Antagonists must survive and be effective after their exposure to both postharvest treatments and storage conditions. Several antagonistic microorganisms have been found that can effectively inhibit postharvest diseases. Just as there is a diversity among microorganisms, there is also a diversity of mechanisms by which they operate. Although in most cases these mechanisms have not been satisfactorily elucidated, they are likely to involve antibiosis, nutrient competition, stimulation of host defense, predation and parasitism. In many cases, probably more than one mechanism operate. The marketing of some of these antagonists may be feasible and they could be an alternative to synthetic pesticides. PMID- 7546441 TI - [Food preservation through combined processes]. AB - Food preservation by combined processes is based on the combination of two or more existing preservation methods with the objective of developing milder preservation procedures. Currently two combined processes (CP) deserve a special attention, the preservation of food by high pressures (HP) and the preservation of food with the combined use of heat and ultrasounds under pressure (Mano-Thermo Sonication). In the preservation by HP, the food, at room temperature or at very mild temperature, is held during relatively long periods under very high pressures (100-1000 MPa) to inactivate its enzymes and/or microorganisms. This procedure has proved to be effective to inactivate vegetative cells but much less effective to inactivate most enzymes and bacterial spores. Several kinds of food preserved by this method have already been launched into the market. In Mano Thermo-Sonication (MTS Process) microorganisms and enzymes are inactivated by a combined heat/ultrasounds treatment under pressure. By this method, the lethality of heat treatments at the same temperature is highly increased. Therefore, the intensity of heat treatments can be drastically reduced. Heat resistance of spores is reduced by a factor of 1/10 and that of enzymes and vegetative cells is reduced by a factor of 1/50 approximately. The applicability of this procedure is currently being investigated. PMID- 7546442 TI - Safety aspects of "sous vide" products and prevention of microbial risks. AB - The diversity and quantities of vacuum packed and vacuum cooked prepared meals and menu components are rapidly growing on the European market. Because of the minimal heat processing, high water activity, absence of preservatives, and the use of many different often exotic ingredients, these products have a high risk potential. For this reason, Anglo-Saxon governments and industries are very sceptical about the safety of "sous vide" products. Industrial practice, as well as parallel tests and recent studies on inoculated packs have shown that, in many cases, it is more a potential than a real risk. Quality is the primary concern for "sous vide" products, and safety must be guaranteed by the application of the Hazard Analysis Critical Control Point (HACCP) concept, along with an adequate combination of different inhibitory factors (temperature of heating and cooling, pH, incorporation of starter cultures, bacteriocins and some particular enzymes). PMID- 7546443 TI - [Microbiological aspects of sparkling wine processing]. AB - The production of cava (sparkling wine produced according to the rules of the cava appellation, in i.e. Catalonia and La Rioja, Spain) involves several microbial factors such as growth, fermentation and death of yeast cells (Saccharomyces cerevisiae). Ethanol tolerance, flocculation and aroma characteristics of yeast cells as major selection factors for the production of cava are discussed in this review. PMID- 7546444 TI - [Sherry wine microorganisms]. AB - Sherry wine presents, during all its wine-making and aging process, a great diversity of yeast and bacteria, as well as in the wine itself; its particular wine-making system, with traditional and legal additions to correct the acidity and to get a final alcoholic content of 15%, originates a selection of accompanying microorganisms. Species of the genera Kloeckera, Candida, Saccharomyces, Pichia, Hansenula and Saccharomycodes, have been isolated during the fermentation process in different proportions. This fact confirms that, besides S. cerevisiae, strains of S. chevalieri and S. fermentati have an important role in the fermentative process, and that the film-forming Saccharomyces have great activity in the fermentation. The biological aging of the Sherry wine, carried out by S. cheresiensis, S. beticus, S. feduchii and S. rouxii, has been studied in "finos" and "manzanillas". Different species and percentages in both wines have been described. PMID- 7546445 TI - [Instrumental technics for the detection of microorganisms in brewing]. AB - Although modern production techniques keep microorganism levels very low in both finished beer and in beer process, there is still concern about early detection and prevention of accidental proliferation of brewery spoilage microorganisms. Traditional microbiological techniques can detect such low levels, but no results are obtained before 3 to 6 days of incubation. That is why rapid or even "instant" techniques are a main objective of research in the brewing industry. In this work, some instrumental techniques (DEFT, MMCF, impedance, pH, immunology and PCR based techniques, bioluminometry) are described and considered from the point of view of their regular use in brewery Microbiological Quality Assurance Laboratories. Real case experiences are brought forward. PMID- 7546446 TI - [Applications of molecular biology in the wine industry]. AB - Population dynamics of natural and inoculated industrial wine fermentations have been studied by using a simple molecular biology technique based on mitochondrial DNA restriction analysis profile. The predominance of the inoculated strain in the inoculated fermentations is obvious. A genetic transformation system has been developed for an industrial wine yeast strain named T73. By using this technique, different fungal hydrolases in this industrial strain have been expressed. Problems and benefits of the application of recombinant DNA techniques in wine yeast strains are also discussed here. PMID- 7546447 TI - [Effectiveness of modified atmospheres against psychrotrophic pathogenic microorganisms in proteinaceous food]. AB - Modified atmosphere packaging (MAP) of proteinaceous raw foods (meat, poultry and fish) extends their shelf-lives. It is well established that modified atmospheres (MA) inhibit the psychotropic aerobic Gram-negative bacteria, the main spoilage microflora of proteinaceous raw foods stored under refrigeration. Several researchers have warned about the possible growth of food poisoning microorganisms on them. Considering the minimal growth temperatures of pathogens, this review only deals with Aeromonas hydrophila, Clostridium botulinum, Listeria monocytogenes and Yersinia enterocolitica. C. botulinum produces its toxin in many different atmospheres, but it is unable to grow at temperatures below 3.3 degrees C, and its production rate of the toxin at temperatures below 4.5 degrees C is very low, to the extent that fish can be spoiled before the toxin is detected. Therefore, the control of the storage temperature of MAP fish seems to be indispensable to assure the absence of botulinal toxin. With regard to the other pathogens, vacuum is the atmosphere that may support more readily its growth; the higher the CO2 concentration in the atmosphere, the lower the growth rate is. Some investigations have shown that the growth rates of the psychotropic pathogens in MAP are lower than those of the spoilage flora. It has been shown also that A. hydrophila and L. monocytogenes growth rates are lower under MA than under aerobic storage. In relation to Y. enterocolitica, more investigations should be carried out in order to clear up its behaviour, because the available data in the literature are still confusing and sometimes even contradictory. In conclusion, there are no evidences that support the concern about MAP of proteinaceous raw foods representing a greater hazard than its conventional storage under air. PMID- 7546448 TI - [Escherichia coli, other Enterobacteriaceae and additional indicators as markers of microbiologic quality of food: advantages and limitations]. AB - The 93/43 European Union directive assigns to the food and catering industries the main responsibility for an integrated safety and quality assurance strategy in the food chain. Relying on hazard analysis, followed by design and adoption of control of all critical points and practices ("HACCP"). Hiatus-free compliance with such HACCP-based Codes of Good Practices is to be assessed by monitoring, recording results on process performance charts and gauging such data against experimentally established, attainable and maintainable references ranges ("standards"). Marker microorganisms are a major analytical tool for validating compliance in the sense of the EU directive. They should be expertly chosen amongst microbes usually present in food so that their, whose presence in quantities exceeding predetermined levels point to a lack of microbiological integrity of a food product. This may encompass (i) the potential presence of taxonomically, physiologically and ecologically related pathogens, markers are called index organisms; or else (ii) a lack of process integrity; in this case, markers are termed indicator organisms. The classical index organism was E. coli, introduced in the 1980's to monitor drinking water supplies. It is still used as an appropriate marker to assess the bacteriological safety of raw foods. In the 1920's the coli-aerogenes ("coliform") group was adopted as an indicator to validate the adequate processing, i.e. pasteurization of dairy products. Since the 1950's the entire Enterobacteriaceae taxon is preferred for the latter purpose because it is better defined in determinative sense and includes more organisms of significance. In some food and water supplies, processed for safety, more vigorous or more resistant organisms than the Gram-negative rods are reliable supplementary markers. These include Enterococcus spp., spores of the Clostridium genus, and bacteriophages of E. coli and Bacteroides fragilis mimicking the fate of enteric viruses under particular ecological conditions. Population surveys conducted by the authors provided ranges for epsilon-factors. Those factors were defined as the proportion between colony forming units (cfu) numbers of index organisms and the pathogenic agent to whose potential occurrence they are expected to point. Epsilon factor values obtained for thermotropic Enterobacteriaceae in relation to Salmonella spp. allow the calculation of the probability that the pathogen has been reliably eliminated by the processing of initially contaminated raw materials, when cfu's of the marker organisms remain below a reference range previously fixed. PMID- 7546449 TI - [Enteroinvasive Escherichia coli. Pathogenesis and epidemiology]. AB - Enteroinvasive Escherichia coli (EIEC) is an intestinal pathogen causing enteritis, with a similar pathogenic mechanism to that of Shigella, which causes an epithelial invasion of the large bowel leading to inflammation and ulceration of the mucosa. The patients often develop the symptoms of bacillary dysentery. The EIEC strains are atypical in their biochemical reactions and may ferment lactose late or not at all, are lysine decarboxilase negative, and non motile. In addition, most EIEC strains express somatic antigens which are either strongly related or identical to Shigella antigens. EIEC invasion is mediated by a large plasmid (140 MDa) coding for the production of several outer membrane proteins involved in invasiveness. These strains have been isolated with some regularity in South America, the Extreme Orient, and Eastern Europe. In Spain the incidence of enteroinvasive E. coli is extraordinarily low (0.2%), the serogroup O124 being the most frequently isolated. EIEC enteritis has been associated to sporadic cases occurring in travellers. Occasional outbreaks related to ingestion of contaminated water or food and person to person have been reported. PMID- 7546451 TI - The biochemical identification of carrier state in mothers of sporadic cases of X linked recessive ichthyosis. AB - X-linked recessive ichthyosis (XLI) is an inherited inborn error of metabolism due to steroid sulfatase (STS) deficiency. The STS activity was studied in 13 families that were referred to the Genetic Department, General Hospital of Mexico City, as being affected by ichthyosis. The study was specially focused on five apparently on familial cases and their mothers, in order to identify carrier status and provide adequate genetic counseling. STS activity was determined in leucocytes using 7-[3H]-dehydroepiandrosterone sulfate as substrate. None of the XLI patients showed STS activity (pmol/mg protein/h), four mothers had an activity compatible with a carrier state (0.19 +/- 0.02 vs 0.66 +/- 0.14 males or 0.90 +/- 0.30 females pmol/mg protein/h, p < 0.001) and only one mother showed a normal pattern, indicating that her son had a de novo mutation. It is important to determine the STS activity in the propositus mother of apparently non familial cases of XLI to identify the carrier state and provide and accurate genetic counseling, as most of these seem to correspond to inherited cases. PMID- 7546450 TI - [Enterotoxigenic, verotoxigenic, and necrotoxigenic Escherichia coli in food and clinical samples. Role of animals as reservoirs of strains pathogenic for humans]. AB - Toxigenic Escherichia coli of human and animal origin have been classified into three categories: enterotoxigenic E. coli (ETEC), verotoxigenic E. coli (VTEC), and necrotoxigenic E. coli (NTEC), ETEC are a major cause of infant diarrhoea in less-developed countries and frequently cause colibacillosis in domestic animals. Human ETEC strains may synthesize LT-I and/or STa enterotoxins and they may possess the colonization factors CFA/I to CFA/IV; porcine strains synthesize LT I, STa and/or STb, and possess the colonization antigens K88, P987, K99 or F41; and bovine strains are usually STa producers harbouring on the bacterial surface K99 or F41 colonization factors. There is a high host-specificity, because of that ETEC from animals are not pathogen for humans. VTEC strains may produce three mainly types of verotoxins (VT1, VT2, VT2vp1) that are functionally and structurally related to the shiga toxin. The VTEC of human and bovine origins produce VT1, VT2 or both, whereas VT2vp1 is elaborated by E. coli that cause edema disease in swine. The VTEC strains belonging mainly to serotypes O157:H7 or H-, O26:H11 and O111:H-, are now considered to be the major cause of two human syndromes of hitherto unknown cause: hemorrhagic colitis and hemolytic uremic syndrome. Most outbreaks of VTEC infection occurred in USA, Canada and United Kingdom during the last ten years and have been linked to consumption of undercooked ground beef, and, to a lesser extent, to the drinking of unpasteurized milk. Thus, the principal reservoir of VTEC is the intestinal tract of cattle. By contrast, it is presumed that human beings are the major reservoir of ETEC, and that contaminated water is a principal vehicle for transmission of ETEC infections. NTEC strains are able to elaborate two types of cytotoxic necrotizing factors (CNF1 and CNF2). Strains of human origin usually produce CNF1, whereas bovine NTEC generally synthesize CNF2. NTEC strains are not responsible for food-associated outbreaks of gastroenteritis, but CNF1 and CNF2 are very good markers of the source of food contamination. PMID- 7546452 TI - Direct carrier detection for severe haemophilia A: application to families with no available affected male. AB - Haemophiliae A is a common hereditary disorder of blood coagulation resulting from deficiency of factor VIII. Mutation analysis is the factor VIII gene has been hampered by the large size of the gene and the heterogeneity of molecular defects. In severe haemophiliae A, the most efficient methods of screening for point mutations can detect the lesions in 50 percent of cases only; this is explained by the recent finding (5) of an intragenic inversion that disrupts the factor VIII gene. Since this anomaly could not be characterized by these methods, Lakich et al. have also described a Southern blotting assay that allows a direct determination of the mutation. The use of this assay should greatly increase the feasibility and accuracy with which carrier detection and prenatal diagnosis can be made, as illustrated by the analysis of families with no available affected male that we present here. PMID- 7546454 TI - Costello syndrome: the natural history of a true postnatal growth retardation syndrome. AB - We present the natural history of a female child with Costello syndrome from birth to the present age of 12 years. The diagnostic clinical signs combined with a true postnatal growth retardation are illustrated. PMID- 7546453 TI - "C" trigonocephaly syndrome with diaphragmnatic hernia. AB - We report on a 6-year-old girl with C-trigonocephaly syndrome and diaphragmatic hernia. She is severely mentally retarded and shows the characteristic findings of this syndrome, including trigonocephaly, unusual facial features, especially intra-oral anomalies, low set and dysplastic ears, cardiac anomaly and neonatal hypotonia. Following our presentation at the 5th European meeting of dysmorphology in Strasbourg, P. Meinecke brought to our attention a case of C trigonocephaly who died in the neonatal period from complications of a diaphragmatic hernia. Another case of C-trigonocephaly without diaphragmatic hernia was communicated to us by D. Lacombe. We report these three observations and present a review of 26 alleged cases. PMID- 7546456 TI - The Williams Syndrome Association 6th National/International Professional Conference. San Diego, California, 1994. Abstracts. PMID- 7546455 TI - Anal atresia and abdominal wall defect as unusual symptoms in EEC syndrome. AB - In this report we document the concurrence of a large omphalocoele and anal atresia in a female newborn with EEC syndrome (Ectodermal dysplasia-Ectrodactyly Cleft palate). Up to now, the association anal atresia-EEC syndrome has been noted in 4 other unpublished patients indicating that anal atresia may constitute another true manifestation of this autosomal dominant syndrome with variable expression. PMID- 7546457 TI - Fragile X boys: evolution of the mental age in childhood. Preliminary data on 10 prepubertal boys. AB - In this report we present data on the longitudinal evolution of the mental versus the chronological age in 10 fragile X boys diagnosed before the age of 6 years and compare these findings to the longitudinal evolution in children with Down syndrome (6 patients) and Williams syndrome (4 patients). The present findings suggest that the evolution of the velocity of development is more decreased in fra(x) boys compared to the two other groups. PMID- 7546458 TI - Enhancing patient care: case management and critical pathways. AB - This paper describes the processes involved in case management and in developing critical pathways as a mechanism to improve customer focused care. Critical pathways can be used to manage the care of patients in settings as varied as acute care hospitals, tertiary care facilities and primary health care areas. Advantages and disadvantages of case management and the critical pathways method of management are outlined from a nursing perspective. PMID- 7546459 TI - Nursing texts and lesbian contexts: lesbian imagery in the nursing literature. AB - Using textual analysis, this paper locates and explicates the images of lesbians that have been presented in introductory nursing texts. The author argues that these images, when presented as 'text', adopt a veil of objectivity and neutrality. Textbooks have power and authority, they legitimize the opinions of authors which are often unsupported by evidence. In the case of lesbians, the use of language in texts effectively re-pathologizes that which has been de pathologized for over 20 years. Implications for scholarly practice are drawn from the findings of this paper. PMID- 7546460 TI - For euthanasia: ask not-why euthanasia? PMID- 7546461 TI - Rank-ordering: a suitable method for nursing research. AB - Rank-ordering is an easy to use and easy to analyse method of organising comparative data for judgement. It is a method that can be used in nursing research whenever the opinions of people are sought. As it is very difficult to find a simple, practical guide for this procedure, data from a study on patients' perception of their privacy in hospital are used to illustrate the analysis and presentation of rank-ordered data. PMID- 7546462 TI - Against euthanasia: palliative care rather than euthanasia. PMID- 7546463 TI - Euthanasia and practice: counselling and certification. PMID- 7546464 TI - Incontinence has different meanings for different people. AB - This study used a multi-method approach to examine older patients' attitudes to, and feelings about, their incontinence. The results indicate that patients did not necessarily accept incontinence as a normal aspect of the ageing process and that they used various strategies to cope with incontinence. Some patients found incontinence too stressful to discuss but most welcomed health professionals' initiation of discussion and saw such consultation as contributing to the effectiveness of continence management. Further study is needed to identify the reasons why some patients who have little professional assistance have positive attitudes to their incontinence and others do not. PMID- 7546465 TI - Effects of argon laser irradiation and acidulated phosphate fluoride on root caries. AB - PURPOSE: To determine the effects of argon laser irradiation (ArI) and topical acidulated phosphate fluoride (APF) treatment on artificial caries formation in root surfaces. MATERIALS AND METHODS: After soft tissue debridement and fluoride free prophylaxis, the teeth were divided into quarters and acid-resistant varnish was applied, leaving windows of sound root surface exposed. Each tooth received four separate treatments: (1) Control-mesiobuccal quarter; (2) ArI only- mesiolingual quarter; (3) ArI followed by APF- distobuccal quarter; (4) APF followed by ArI-distolingual quarter. ArI was for 10 seconds at 2W (100J/cm2) and APF treatment was for 4 minutes. After artificial lesion formation, sections were prepared and evaluated with polarized light. Mean lesion depths were determined and compared (ANOVA & DMR for a paired design). RESULTS: Mean lesion depths were: 347 +/- 41 microns--controls; 263 +/- 32 microns--ArI only; 158 +/- 21 microns- ArI followed by APF; and 149 +/- 17 microns -APF followed by ArI. Lesion depths were significantly different (P < 0.05) between the control group and each treatment group, and between ArI only group and either combined APF and ArI groups. No lesion depth difference (P > 0.05) was present between the combined APF and ArI groups. Argon laser irradiation significantly enhanced the resistance of root surfaces to demineralization. Combination of APF treatment with argon laser irradiation provided added protection against a constant artificial caries attack. PMID- 7546466 TI - Indirect composite preparation width and depth and tooth fracture resistance. AB - PURPOSE: To evaluate the influence of preparation width and depth on the fracture resistance of teeth restored with one type of indirect composite restoration. MATERIALS AND METHODS: Variations of depth and width were superimposed on a preparation of dentin design and dimensions with other variables including tooth size and morphology being controlled. Indirect composite restorations were constructed and placed in accordance with manufacturer's instructions. The restored teeth were subjected to compressive loading with fracture being induced by application of a 4 mm steel rod to the specimens in a universal testing machine, at a cross-head speed of 1 mm/minute. RESULTS: Preparation depths of 3 to 4 mm were found to be preferable, but when the prevalence of severe tooth fracture was assessed, the shallower preparation depth was found to be indicated. Cavity depths of one-third and one-half bucco-lingual width were evaluated, the fracturing forces for the group with the narrower width being statistically lower. PMID- 7546467 TI - Penetration of a light-cured glass ionomer and a resin sealant into occlusal fissures and etched enamel. AB - PURPOSE: To evaluate the penetration of a light-cured glass ionomer and a resin sealant into occlusal fissures and etched enamel. MATERIALS AND METHODS: Forty eight maxillary and mandibular caries-free premolars scheduled for extraction for orthodontic reasons were isolated, the occlusal surfaces subjected to prophylaxis and acid-etched with orthophosphoric acid prior to the application of the VariGlass VLC glass ionomer and Concise resin sealants. The teeth were extracted, two longitudinal median sectiors from each tooth were ground to a thickness of 80 100 microns, and the sealant penetration into the fissures evaluated. The sections were placed in nitric acid to dissolve the enamel so the lengths of the tags which had penetrated into the etched enamel could be measured at different sites on the walls of the fissures. RESULTS: Both sealants adapted well to the fissures but penetrated deeper into shallow, open fissures than into deep, constricted fissures. The VariGlass VLC tags into etched enamel were generally longer than the Concise projections. PMID- 7546468 TI - Acidogenicity of high-intensity sweeteners and polyols. AB - PURPOSE: To evaluate the plaque pH responses to sucrose, fructose, xylitol, sorbitol, acesulfame-K, aspartame, and saccharin at equal sweetness levels (equivalent to 10% sucrose) and water using an indwelling plaque pH telemetry system. MATERIALS AND METHODS: Eight adult panelists used each sweetener once in a Latin square study design. Plaque was allowed to accumulate for 3-6 days before each challenge period, and the panelists fasted for 12 hours prior to the 2 minute test rinse. Plaque pH was monitored for a 2-hour period after the rinse exposure. The parameters examined were area of the curves under pH 5.5 (pH X Time), pH changes from baseline, lowest pH attained and time below pH 5.5. RESULTS: The the high-intensity sweeteners (aspartame, saccharin and acesulfame K) and the polyols (sorbitol and xylitol) were all non-acidogenic and were not significantly different from each other while both sucrose and fructose were highly acidogenic. PMID- 7546469 TI - Enamel rehardening by bread consumption. AB - PURPOSE: To investigate the in situ rehardening effect by white or whole-meal bread on softened enamel. MATERIALS AND METHODS: Twelve volunteers wearing orthodontic removable appliances participated in this study. The intraoral test was chosen for measuring microhardness of enamel slabs inserted into the dental appliance. Average microhardness of enamel was calculated at three stages: At start (baseline), after exposing the enamel slabs to a citrus beverage (pH 3.4) for 30 minutes in vitro as a softening agent, followed by intraoral exposure for a duration of 5 minutes to stimulated secreted saliva by parafilm chewing (control), or to 15g white- and wholemeal-bread mastication respectively (experimental). RESULTS: A significant rehardening of the softened enamel surfaces took place following mastication of the two bread samples and salivary secretion. The differences in the rehardening potentials of the bread samples as compared to that of saliva were not significant. From the conditions chosen for the experiment, there was no sufficient evidence to support the hypothesis that bread eating with calcium and phosphate concentrations at the existent levels will afford a significant greater rehardening than saliva alone. PMID- 7546470 TI - Effect of mouthguard bleaching on enamel surface. AB - PURPOSE: To evaluate in vitro the effect of 10% carbamide peroxide in a mouthguard on the shear bond strength of resin composite to bleached enamel surfaces and morphological changes in these surfaces. MATERIALS AND METHODS: Thirty anterior extracted were divided into a control and a test group. Plastic trays were made for each tooth and the test group treated with Opalescence bleaching gel and incubated at 37 degrees C humid environment for 8 hours. The bleaching agent was washed and the trays replaced with water-soaked cotton wool for another 16 hours in the incubator. This procedure was repeated every day for 21 days. The control group was just treated with the water-soaked cotton wool. Selected teeth from both groups were evaluated with the SEM and 72 hours after the bleaching treatment, Scotchbond 2/Silux Plus rings were bonded to the enamel surfaces in both groups and sher bond strength testing performed. RESULTS: Bleaching created some enamel porosity and significant reduction in the resin composite shear bond strength. PMID- 7546471 TI - Tuberculosis infection in U.S. Air Force dentists. AB - PURPOSE: To determine if U.S. Air Force dentists have a significantly higher prevalence of infection from M. tuberculosis than a similar no-dentist group. MATERIALS AND METHODS: A written survey instrument was sent to all active duty Air Force dentists and lawyers. The survey asked individuals to voluntarily and anonymously give information regarding positive tuberculin skin testing which was subsequently treated with antituberculosis medication. Only positive responses which occurred during the time the respondent was practicing as a dentist or lawyer in the Air Force were counted. Significant exposures were considered to have occurred in those persons who were evaluated by a physician and actually put on a course of antituberculosis medication. RESULTS: The dentists returned 82.7% of the 1256 surveys sent, of which 2.37% indicated a significant exposure. The lawyers returned 79.6% of 1321 surveys, of which 1.47% were positive for significant exposure. Chi-square analysis indicated no significant difference between the two groups (P = 0.14). PMID- 7546472 TI - Intra-oral bonding of 4-META/MMA-TBB resin to vital human dentin. AB - PURPOSE: To investigate and compare the in vivo and in vitro tensile bond strengths of a 4-META/MMA-TBB adhesive resin (Superbond C&B, C&B-Metabond) to human dentin. MATERIALS AND METHODS: Caries-free teeth present on patients and scheduled for extraction for orthodontic reasons were used. Flat dentin surfaces were prepared and a 4-META/MMA-TBB resin applied. After 10 minutes, the teeth were extracted. Extracted caries-free teeth were also used and treated similarly. Tensile bond strength was evaluated and all specimens were sectioned immediately after bond testing, after polishing the debonded area or after treating the surfaces with HCl acid. RESULTS: Data were analyzed statistically, and no significant differences could be determined between third molars and first premolars, and in vivo and in vitro specimens. Further, tensile stress fractured specimens were examined under scanning and transmission electron microscopy. Widths (+/- 5 microns) of hybrid layers and impregnation of adhesive monomers into 10-3 solution demineralized vital human dentin substrates were essentially similar to those observed in extracted samples. The adhesive resin penetrated tubules to form resin tags, unimpeded by intra-tubular fluid under physiologic pressure in intra-orally prepared specimens. A hybrid layer formed on resin tags to seal against microleakage and pulpal attack by bacteria and their by-products. An acid-resistant hybrid layer was also observed in enamel. Finally, hydroxyapatite crystals that were encapsulated by polymerized 4-META/MMA-TBB resin was observed at the bases of resin-reinforced hybrid layers in the human dentin substrates. This observation is consistent with earlier findings and appears to be essential in maintaining durable dentin bonds. PMID- 7546473 TI - Microleakage of "surface-sealing" materials. AB - PURPOSE: To test several dental materials for their sealing properties by subjecting restored teeth to thermal cycling. MATERIALS AND METHODS: Ten teeth per group were assigned at random to six experimental and six control groups. In the experimental groups a resin composite was surface-sealed with IRM, glass ionomer, amalgam, dentin bonding agent and resin composite, experimental hybrid cement, or zinc oxide and eugenol. These six surface-seal materials were placed in bulk in the control group. After cavity preparation, restoration, cut back and application of the surface-seal material, the teeth were subjected to thermal cycling, imbedded and sectioned. A score of 0-3 was given to the amount of dye leakage observed. RESULTS: Zinc oxide and eugenol demonstrated the most leakage, whereas the resin composite system TPH had the least. The results of this in vitro study render the recommendation of zinc oxide and eugenol as a surface-seal material questionable. PMID- 7546474 TI - Nitrous oxide sedation: understanding the benefits and risks. AB - Nitrous oxide and oxygen is widely used for conscious sedation in dental offices. It can have benefits for patients who experience dental anxiety or are medically compromised and gain from the oxygen enrichment and stress reduction. Clinicians benefit when patients are cooperative and satisfied. There are a few patients for whom nitrous oxide would not be an optimum therapy. Chronic occupational exposure to low levels of nitrous oxide has several reproductive and health risks. Reducing the ambient concentrations is important. PMID- 7546476 TI - Comparison of the anticaries efficacy of dentifrices containing fluoride as sodium fluoride or sodium monofluorophosphate. AB - The dominance of the marketplace by dentifrices containing fluoride as sodium monofluorophosphate and sodium fluoride has given rise to great academic and commercial interest regarding the comparison of those two fluoride species. Since the 1970's, several comparative clinical trials have been conducted to investigate the relative anticaries efficacy of dentifrices containing these two agents, generally giving rise to equivocal results. Recently, attention has been turned to investigative efforts which employ information derived from the collective body of relevant comparative clinical studies available in the literature to establish an omnibus conclusion concerning this issue. Beginning with the sign test of Beiswanger & Stookey (1989), and continuing through the meta-analyses of Johnson (1993) and Proskin (1993), this line of research remains vital today. The recent publication of two studies (Marks et al, 1994; Stephen et al, 1994) previously documented in the literature only in abstract form (Conti et al, 1993; Stephen et al, 1993) has given rise to the need for the present meta analytic reconsideration of the available data. As in the previous meta-analyses, the quantitative results obtained depended on the studies which provided data for the calculations. However, as also indicated in previously-documented meta analyses, the clinical interpretation of the results, which was based on the guidelines published by the American Dental Association (Council on Dental Therapeutics, 1988), did not vary according to study selection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546475 TI - Surface morphology and chemical characterization of abrasion/erosion lesions. AB - PURPOSE: To describe the dentin surface morphology of abrasion/erosion lesions and to chemically characterize in vivo samples of sclerotic dentin. MATERIALS AND METHODS: Baseline polyvinylsiloxane impressions of eight in vivo caries-free lesions were taken. Dentin was collected from retention grooves for FTIR photoacoustic spectroscopic analysis. The cavity preparation was etched for 30 seconds with 37% phosphoric acid, rinsed, and dried. Impressions were taken of the etched surfaces. Epoxy resin dies were made of baseline and etched impressions, sputter-coated and examined at x1000 with the SEM. RESULTS: Lateral dentin tubule orientation was observed at gingival margins and on occlusal walls. Open cross-sectional tubules were seen at the depth of the groove. The mineral/protein ratio in the FTIR/PAS spectra of in vivo unetched sclerotic dentin samples suggested an increased mineral content. PMID- 7546478 TI - Dental vacuum system with skirt technology. AB - A heat-sterilizable vacuum system (Legend) featuring a skirt technology that confines the user's hand to the sterile instruments during initial hose coupling and during patient treatment is described. Because the user does not grasp the non-sterile vacuum hose connector and hose, plastic barriers and mandatory disinfection of the hose connector, hose, and instrument holders after each patient are not necessary. The vacuum system described promotes asepsis by using sterile vacuum instruments, encourages sound asepsis practice, prevents cross contamination, and is cost-effective. PMID- 7546477 TI - Resin-modified glass ionomer materials. A status report for the American Journal of Dentistry. AB - The resin-modified glass ionomer materials are hybrid materials of traditional glass ionomer cement with a small addition of light-curing resin, and hence exhibit properties intermediate to the two, with some characteristics superior to conventional glass ionomer materials. Generally, they have the advantages of both such as adhesion to tooth structure, esthetics, fluoride release and rapid hardening by visible light. Issues surrounding other physical properties, such as the mechanism of adhesion, sensitivity to water, long-term wear and strength, need to be addressed and substantiated. There are other considerations, which are not features of the conventional materials, but are of importance with the resin modified glass ionomer cements i.e. light-curing, which require further research to provide evidence of their behavior. The future for this group of materials is unknown, but the currently available materials appear to be promising. PMID- 7546479 TI - A method for microleakage evaluation along the dentin/restorative interface. AB - PURPOSE: To present a simple non-abrasive protocol in conjunction with the silver staining method, enabling unobstructed visualization of well-defined microleakage patterns within the demineralized dentin-restorative interface. MATERIALS AND METHODS: Class V preparations were made with an occlusal margin on bevelled enamel and a gingival margin on dentin/cementum. After restoration with All Bond 2/P-50 and Scotchbond 2/Silux, storage in water at 37 degrees C for 7 days and thermocycling 300 times between 5 degrees and 55 degrees C, the specimens were placed in silver nitrate solution. After silver fixation, demineralization and clearing in methyl salicylate, silver stain location was scored ordinally. In addition, a three dimensional portrait of leakage pathways was illustrated using photographic and computer digitization. RESULTS: The Wilcoxon signed-rank test showed that the extent of silver penetration before and after acid demineralization was not significantly different (P > 0.05). This study also confirmed that microleakage is not uniform along the circumference of a restorative margin. Although the protocol is insensitive to microleakage occurring solely within enamel, the predominant concern over that occurring within dentin, in particular along the cervical restorative border, renders it a serviceable alternative for future investigation of the integrity of the marginal seal from clinically restored in vivo specimens. PMID- 7546480 TI - Effect of long-term water storage on dentin bonding. AB - PURPOSE: To determine the effect of long term water storage on assembly bond strength and interfacial integrity of resin composite bonded to dentin. MATERIALS AND METHODS: Four groups, each containing 10 molar teeth, were established to measure the shear strength of All-Bond 2 and Amalgambond bonded P50 resin composite assemblies at 24 hours and after 6-month water storage at 37 degrees C. An additional four groups of similarly bonded Class V restorations were subjected to the same immersion periods. Marginal leakage was determined using eosin dye and the restoration interface was observed using polyvinyl siloxane impressions to determine gap location. RESULTS: The shear strength values (MPa) for All-Bond 2 were 32.68 +/- 7.12 and 23.29 +/- 5.77 at 24 hours and 6 months respectively. The mean values for Amalgambond were 26.03 +/- 5.94 and 12.90 +/- 6.90. The difference between the values at different immersion times was statistically significant (P < 0.05). There was no evidence of dye leakage in any of the teeth. No gaps were found at the margins of the restorations correlating with the absence of microleakage. Gaps were found commonly at the floor of the Amalgambond restorations. PMID- 7546481 TI - Removal of enamel surface stains with at-home vital bleaching. AB - PURPOSE: To report the results obtained with an at-home bleaching product in the removal of enamel surface stains. MATERIALS AND METHODS: An individualized vacuum formed mouthguard was fabricated and scalloped so that the terminal portion of the mouthguard ended approximately 1.0 mm from the gingiva. The patient was instructed to brush his teeth with Rembrandt Whitening Toothpaste prior to and after using the Rembrandt Gel Plus bleaching gel. In addition, the patient was informed as to the proper method of placing the gel in the mouthguard, and inserting the mouthguard into his mouth. The mouthguard was to be worn for 2-3 hours a day for a 2-week period. RESULTS: At the end of 2 weeks, the enamel surface stains were removed through the bleaching process. PMID- 7546483 TI - Effect of dentin moisture and storage time on resin bonding. AB - PURPOSE: To evaluate the effects of dentin moisture and two different storage times on the shear bond strength of resin composite bonded to dentin with Scotchbond Multi-Purpose Adhesive. MATERIALS AND METHODS: The occlusal surfaces of 60 extracted human molars were reduced to provide flat dentin surfaces. After being hand-finished using wet 400- and 600-grit SiC papers, the teeth were randomly divided into four groups of 15 specimens each. The teeth were etched, rinsed, and then either blotted with gauze which left a visibly moist surface or dried with compressed air. The primer and adhesive were applied according to the manufacturer's instructions and resin composite cylinders were bonded to the teeth. Two of the groups (one moist, one dry) were stored for 24 hours in 37 degrees C distilled water while the other two groups were stored for 90 days in 37 degrees C distilled water. At the end of their storage times, the specimens were loaded to failure in shear at 0.5 mm/minute. RESULTS: Mean bond strength values and standard deviations in MPa were: Dry dentin at 24 hrs, 13.30 +/- 5.1; Dry dentin at 90 days, 13.07 +/- 3.8; Moist dentin at 24 hours, 13.64 +/- 4.9; Moist dentin at 90 days, 15.58 +/- 5.3. No statistically significant differences were found for bond strengths to dry and to moist dentin for either storage time. Time had no significant effect for either the dry dentin group or the moist dentin group. PMID- 7546482 TI - Marginal ridge strength of Class II tunnel restorations. AB - PURPOSE: To test the strength, at the marginal ridge, of the tunnel preparation vs the Class II traditional box preparation when restored with composite or glass ionomer (GI). MATERIALS AND METHODS: Eighty-four extracted maxillary molars stored in normal saline and thymol were randomly divided into six groups of 14 each (determined by pilot study where alpha = 0.05, beta = 0.2). Group A and C were tunnel preparations. Group B and D were traditional Class II preparations. Group E-whole tooth (negative control), and Group F-tunnel preparation unrestored (positive control). Group A and B were restored with Scotchbond 2/P-50 composite. Group C and D were restored with Ketac Fil-GI. The marginal ridge of each tooth was loaded at 0.5 mm/minute on the Instron. A loading rod produced a contact point of 1.0 mm in diameter. RESULTS: The mean compressive loads (kg) required for fracture were: (A): 42.2 +/- 11.9, (B): 53.1 +/- 10.7, (C): 52.0 +/- 10.9, (D): 23.8 +/- 8.4. (E): 79.1 +/- 16.1, (F): 27.0 +/- 10.6. A significant difference was found between whole teeth (E) and all other groups (P < 0.05). The Newman-Keuls test showed a significant difference between Class II composite (B) and tunnel composite (A) (P < 0.05), between tunnel GI (C) and tunnel composite (A) (P < 0.05) but no difference between tunnel GI (C) and Class II composite (B). PMID- 7546484 TI - Wear resistance of resin cements. AB - PURPOSE: To evaluate the in vitro wear resistance of four resin cements and a glass ionomer cement using a three-body wear test. MATERIALS AND METHODS: Each cement was evaluated as a direct restorative material as well as in conjunction with an inlay. All restorations were placed into cylindrical shaped cavity preparations generated on the flattened occlusal surface of human extracted molars. The bulk wear resistance of a cement was determined when tested as a direct restorative material. RESULTS: The wear resistance of all cements was greater when tested in conjunction with a resin composite inlay restoration. Although wear of a cement is dependent upon the method of testing, it was greater when combined with a less wear resistant resin composite inlay material. All of the cement materials used in this study exhibited less wear resistance than the resin composite inlays with which they were used. This in vitro wear testing system can be used as a screening test for the cement materials before a clinical study. PMID- 7546485 TI - The four generations of dentin bonding. AB - The achievement of an adhesive bond between enamel and dentin and restorative materials has been an objective for generations of dental research workers. Dentin bonding agents have been classified into generations, with earliest generations showing unreliable bond strengths. Some currently available systems show in vitro shear bond strengths to dentin which are similar to the bond strength to enamel, this being considered an ideal property. Alongside other previously recognized ideal properties, appropriate criteria for state-of-the-art dentin bonding systems at the present time may be considered to include the ability of systems to operate in moist environments and be technique insensitive. PMID- 7546486 TI - Laboratory evaluation of the Gluma 3-step bonding system. AB - PURPOSE: To determine in vitro (1) the shear bond strengths (SBS) of the modified Gluma system to enamel and dentin; (2) the qualitative and the quantitative microleakage (ML) of Class V preparations on the cemento-enamel junction (CEJ) and in dentin, respectively, restored with the Gluma system; and (3) the effect of the system's components on enamel and dentin by scanning electron microscopy. MATERIALS AND METHODS: Seventy-five extracted human maxillary permanent central incisors and 75 permanent first and second molars were embedded in brass tooth cups with cold-cure acrylic resin. The facial surfaces of the anterior and the occlusal surfaces of the molar teeth were ground wet on 180- followed by 600-grit silicon carbide paper. Demarcated sites on the surfaces were treated according to the manufacturer's instructions and Pekafill composite was bonded to the treated surfaces. Fifteen specimens prepared on enamel were removed 1 minute after cure (A) and the SBS determined. The remaining specimens were removed 15 minutes after final cure, stored in saline at 37 degrees C for 24 hours (B), for 1 week without (C) and with temperature cycling (D), and for 4 weeks (E). Similar regimens were used for dentin, groups F, G, H, I and J. The SBS were determined and expressed in MPa. The data were analyzed by ANOVA, Student-Newman-Keuls and t-tests. For the qualitative ML evaluation, Class V cavities were prepared on the CEJ of 30 human premolars and restored with Gluma/Pekafill. After thermocycling in 0.5% basic fuchsin, the teeth were sectioned and ranked according to the degree of microleakage. Class V cavities were prepared on the facial root surfaces of 15 human premolars and restored. The ML was determined quantitatively by a spectrophotometric dye-recovery method and expressed as microgram dye/restoration. RESULTS: The mean +/- SD of the SBS in MPa were: A: 14.6 +/- 2.2; B: 21.2 +/- 1.6; C: 20.5 +/- 1.4; D: 24.2 +/- 2.0; E: 22.8 +/- 3.7; F: 7.0 +/- 2.0; G: 16.1 +/- 4.0; H: 15.6 +/- 3.7; I: 10.9 +/- 3.3; J: 14.4 +/- 3.3. The quantitative ML was 1.00 +/- 0.39 microgram dye/restoration. The lowest SBS were obtained on specimens tested after 1 minute. Temperature cycling had a significantly adverse effect on the SBS to dentin (P = 0.0003). Etch patterns conducive to bonding were produced on the enamel. The resin system penetrated into the dentin tubules. PMID- 7546487 TI - The outcome of a clinical trial of a dentin bonding system. Justice or injustice? AB - PURPOSE: To review the findings and to question the outcome of a clinical evaluation of a dentin bonding system. MATERIALS AND METHODS: The study was a split mouth, single blind (to patient), randomised within patient, clinical trial of Tripton used in conjunction with Opalux and the ultrafine compact-filled, experimental visible light cured resin composite M221784 (ICI Dental) in the restoration of mixed (enamel/dentin), caries free buccal-surface cervical lesions in vital, permanent canine and premolar teeth. The design of the trial was based on the then current American Dental Association (ADA) Clinical Protocol Guidelines for Dentin and Enamel Adhesive Materials. Restorations were placed as dictated by a predetermined randomised number scheme with each patient recruited to the study receiving at least one pair of matched restorations, comprising one restoration of each resin composite. RESULTS: The study reported was discontinued at 1 year subsequent to a 17% loss rate between 6 and 12 months after placement, with most of the restorations lost having been placed in what has subsequently become shown to be relatively unfavorable situations. It is suggested that the outcome of clinical trials of the type considered may warrant review in the light of new knowledge and understanding, and that regulatory bodies setting clinical protocol guidelines must give careful consideration as to whether the criteria for this type of trial should be more restrictive or required to include a representative range of clinically relevant situations. PMID- 7546489 TI - Improved classification of dippers by individualized analysis of ambulatory blood pressure profiles. AB - A decrease (dip) in blood pressure during sleep occurs in normal people and in patients with uncomplicated essential hypertension. Failure to identify such a dip suggests additional pathology, which makes the identification of "nondippers" important. Rigid definitions of nocturnal time periods (eg, night is defined as lasting from 22:01 to 06:00) to identify dippers and nondippers has been used for over a decade. However, these definitions may not correspond to actual sleep patterns, and thus may lead to faulty interpretations. We investigated whether or not an analysis of ambulatory blood pressure (BP) profiles according to the patients' individual reported awake/sleep pattern would result in an improved categorization of dippers and nondippers. Four groups of patients were investigated: normotensive volunteers, borderline hypertensive patients, essential hypertensive patients, and renal transplant recipients. In all four groups, blood pressure (systolic and diastolic) decreased to a greater degree when the individual reports were employed, compared to the fixed patterns. For systolic BP this difference (individualized v fixed 06:01 to 22:00 day and 22:01 to 06:00 night) amounted to 17.6 +/- 5.0 v 13.2 +/- 5.4 mm Hg for normotensive subjects, 18.5 +/- 6.0 v 11.7 +/- 8.6 mm Hg for borderline hypertensive subjects, 17.7 +/- 10.6 v 12.9 +/- 10.4 mm Hg for essential hypertensive patients, and 8.6 +/- 11.3 v 6.5 +/- 9.8 mm Hg in renal transplant patients (all P < .05). Individualized awake/sleep reports resulted in a better classification of dippers and nondippers, since misclassifications due to divergent sleep patterns (mainly going to bed late) were avoided. PMID- 7546488 TI - Essential hypertension predicted by tracking of elevated blood pressure from childhood to adulthood: the Bogalusa Heart Study. AB - It is well known that blood pressure (BP) levels persist over time. The present investigation examines tracking of elevated BP from childhood to adulthood and its progression to essential hypertension. In a community study of early natural history of arteriosclerosis and essential hypertension, a longitudinal cohort was constructed from two cross-sectional surveys > 15 years apart: 1505 individuals (56% female subjects, 35% black), aged 5 to 14 years at initial study. Persistence of BP was shown by significant correlations between childhood and adulthood levels (r = 0.36 to 0.50 for systolic BP and r = 0.20 to 0.42 for diastolic BP), varying by race, sex, and age. These correlations remained the same after controlling for body mass index (BMI). Twice the expected number of subjects (40% for systolic BP and 37% for diastolic BP), whose levels were in the highest quintile at childhood, remained there 15 years later. Furthermore, of the childhood characteristics, baseline BP level was most predictive of the follow-up level, followed by change in BMI. Subsequently, even at ages 20 to 31 years, prevalence of clinically diagnosed hypertension was much higher in subjects whose childhood BP was in the top quintile: 3.6 times (18% v 5%) as high in systolic BP and 2.6 times (15% v 5.8%) as high in diastolic BP, compared to subjects in every other quintile. Of the 116 subjects who developed hypertension, 48% and 41% had elevated childhood systolic and diastolic BP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546490 TI - The effect of sleep intervals on analysis of 24-h ambulatory blood pressure data. AB - Twenty-four hour blood pressure exposure and circadian blood pressure variability may be important predictors of hypertensive end-organ damage. Since sleep is a major determinant of circadian blood pressure decline, day/night blood pressure changes may be affected by sleep intervals. The current study compares 24-h blood pressure results with and without adjustment for patient-specific sleep intervals. Forty male hypertensive patients (22 with renal insufficiency and 18 with normal renal function) underwent 24-h blood pressure study with SpaceLabs 90207 monitors. They also filled out sleep questionnaires to evaluate time awake and asleep during the day and night intervals. Data was analyzed with and without adjustment for daytime asleep and nighttime awake intervals. The percent change in systolic, diastolic, and mean arterial pressure as well as heart rate was not different statistically or clinically with or without adjustment for sleep intervals. Nevertheless, there was a progressive increase in the day versus night percent change in these hemodynamic parameters with adjustment for sleep intervals. When we used arbitrary cut-offs to define "non-dipper", ie, less than 10% drop in a given hemodynamic parameter, and used patient-specific sleep intervals, clinically and statistically different numbers of "non-dippers" were seen versus sleep unadjusted data (19 v 12, using diastolic BP, P < .01). In conclusion, it may not be necessary to adjust for sleep intervals when relating 24-h blood pressure and day/night blood pressure variability to hypertensive end organ damage. However, arbitrary cut-offs to define "non-dippers" may be greatly and capriciously affected by sleep/awake intervals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546491 TI - Effects of actual versus arbitrary awake and sleep times on analyses of 24-h blood pressure. AB - Investigators conducting hypertension trials with ambulatory blood pressure (BP) monitoring have been analyzing study results using arbitrary times for day (wakefulness) and night (sleep). We prospectively evaluated the impact of using arbitrary times instead of patient reported awake and sleep times on mean 24-h, awake, and sleep BP, BP loads, and the awake-sleep BP difference in 50 subjects. Daytimes and nighttimes were derived from popular, arbitrary times reported in the literature. Compared to actual awake and sleep periods, arbitrary day and night division caused no significant differences in the mean awake and sleep BPs. However, limits of agreement for BP values derived for the actual and arbitrary times of wakefulness and sleep were substantial especially during sleep (awake systolic BP, -4 to 7 mm Hg; awake diastolic BP, -2 to 4 mm Hg; sleep systolic BP, -12 to 7 mm Hg; and sleep diastolic BP -7 to 4 mm Hg). Sleep BP loads (proportion of BPs > 120/80 mm Hg) were altered by greater than 10% in 20% to 30% of the subjects, depending on choice of time schedule. These data demonstrate that the calculation of BP and BP load during sleep may be altered by use of arbitrary, rather than actual, times of wakefulness and sleep in 24-h studies of ambulatory BP. PMID- 7546492 TI - How should the diurnal changes of blood pressure be expressed? PMID- 7546493 TI - Renin-angiotensin-aldosterone system, RR interval, and blood pressure variability during postural changes in borderline arterial hypertension. AB - This study aimed to examine the changes in RR interval and blood pressure (BP) variability and humoral factors during postural changes in borderline arterial hypertension. Twenty-nine patients (44 +/- 3 year; BP, 145 +/- 3/84 +/- 3 mm Hg) with borderline hypertension and a control group of 38 subjects (35 +/- 2 year; BP, 123 +/- 3/70 +/- 2 mm Hg) underwent power spectrum analysis of RR interval and BP (low frequency (LF), 0.05 to 0.15 Hz; high frequency (HF), 0.15 to 0.40 Hz) in the supine and standing positions. Concentrations of plasma renin activity, angiotensin II, and aldosterone in supine and standing positions had been determined in the hypertensive group. Borderline hypertensives are characterized by higher oscillations of systolic and diastolic BP, but not of RR interval in the supine position versus the control group. Low frequency and HF components of systolic and diastolic BP expressed as absolute data are also significantly higher in borderline hypertensives. Moreover, standing tended to increase the LF/HF ratio of both RR interval and BP variability compared to controls. The standing position was able to further activate the LF but not the HF component of BP variability. In borderline hypertension renin release during postural changes correlated well with the decrease in the power of the HF vagal component of RR interval variability (r = -0.70, P < .001) and with the increase of the LF component of diastolic blood pressure variability (r = 0.43, P = .03). In conclusion, our results indicate that in borderline arterial hypertension, LF and HF oscillations of BP are already significantly increased at rest.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546494 TI - Variations in magnesium and zinc in hypertensive patients receiving different treatments. AB - We studied the influence of captopril, atenolol, and verapamil on serum and intraerythrocyte concentrations of magnesium and zinc in 30 normotensive control subjects (12 men and 18 women, aged 30 to 65 years, mean +/- SD 45.76 +/- 12.15 years) and 30 patients with untreated mild or moderate essential hypertension (14 men and 16 women, aged 30 to 65 years, mean +/- SD 49.50 +/- 13.58 years). Ten each of the hypertensive patients were treated with captopril, atenolol, or verapamil. Physical examination and biochemical analyses (serum Mg and Zn) were done in all participants at baseline, and in patients after 3 and 6 months of treatment. The results were compared according to a nested design with Neumann Keuls test. We found no significant differences between controls and patients in serum and intraerythrocyte concentrations of Zn at the start of the study, although there was a significant decrease in serum Zn in patients after 3 (P < .01) and 6 months (P < .001) of treatment, regardless of the drug used. This decrease was thought to be attributable to the zincuric effect of captopril or to dietary measures, or both. Intraerythrocyte Zn was not significantly affected by antihypertensive treatment. Serum and intraerythrocyte concentrations of Mg were significantly lower (P < .001) in hypertensive than in normotensive subjects, and serum Mg in patients treated with verapamil was significantly lower (P < .05) than after treatment with captopril or atenolol. Serum Mg concentration was related directly with serum concentrations of high density lipoprotein cholesterol (r = 0.4043, P < .05). We conclude that supplementation with Mg may benefit patients with hypertension. PMID- 7546495 TI - Effects of alcohol moderation on blood pressure and intracellular cations in mild essential hypertension. AB - It is known that moderation of alcohol intake reduces blood pressure, although the exact mechanism has not yet been established. To clarify the hypotensive mechanism of alcohol reduction, we evaluated the change in cellular magnesium and sodium metabolism during alcohol reduction in mild hypertensive patients. We measured intraerythrocyte sodium and magnesium, intraplatelet free magnesium concentrations, and erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) in 17 mild essential hypertensive patients regularly consuming more than 40 g/day of alcohol, before and after 4 weeks of alcohol reduction, and 12 age-matched nondrinking hypertensives. Intraerythrocyte magnesium (P < .01) and intraplatelet free magnesium (P < .05) concentrations were significantly lower in drinkers than in nondrinkers. In drinkers, advice to reduce alcohol intake for 4 weeks resulted in a reduction in self-reported alcohol consumption from 461.7 to 71.6 g/week, a significant fall in both supine systolic blood pressure (136.3 +/- 10.8 to 130.8 +/- 11.3 mm Hg, P < .001) and supine diastolic blood pressure (85.1 +/- 8.6 to 82.6 +/- 8.7 mm Hg, P < .05). The fall in mean blood pressure correlated positively with the reduction in weekly alcohol consumption. Intraerythrocyte magnesium and Kos were increased (P < .05, P < .01, respectively), while intraerythrocyte sodium was decreased (P < .01). The increase in intraerythrocyte magnesium correlated negatively with the fall in mean blood pressure and positively with the increase in Kos, which correlated negatively with the decrease in intraerythrocyte sodium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546496 TI - Effect of dietary counselling on blood pressure and arterial plasma catecholamines in primary hypertension. AB - There is still a need of support for nonpharmacologic treatment of uncomplicated, mild-to-moderate essential hypertension. We investigated whether a low sodium based diet implemented by a nutritionist could lower blood pressure and affect sympathetic activity. Middle-aged, otherwise healthy men with never-treated essential hypertension (n = 95) were randomized to an intervention group, a blood pressure control group, and a time control group. The intervention group was advised to use less sodium chloride in their diet, and if necessary, less saturated fat and decrease body weight. They attended regular clinic visits as did the blood pressure control group. After 1 year, the intervention group had achieved on average 72 mmol/24 h lower urinary sodium excretion (P < .001) and a decrease in body weight of 2.7 +/- 0.5 kg (P < .001). Both supine and standing mean blood pressure were on average 8 to 10 mm Hg lower after intervention compared with the two control groups (P < .001). Arterial plasma epinephrine, measured in all 40-year-old subjects (n = 30), decreased in parallel in all three groups (P < .05), indicating some habituation to the invasive procedure and clinic visits. However, the decrease in norepinephrine was significant (P < .001) only in the intervention group; it correlated with the weight loss (r = 0.76, P < .05) and was significantly higher (P < .05) than in both control groups. These results suggest that broad dietary advice (ie, low intake of sodium chloride, saturated fat and energy), implemented by a nutritionist, may have a significant blood pressure lowering effect and a favorable sympathicolytic effect in uncomplicated, mild-to-moderate essential hypertension. PMID- 7546497 TI - Differential effects of felodipine and nifedipine on 24-h blood pressure and left ventricular mass. AB - 1,4-Dihydropyridine calcium antagonists, which have a rapid onset and short duration of action, tend to increase sympathetic activity, which may limit the regression of left ventricular hypertrophy when used for the treatment of hypertension. This study compares the effects of the shorter-acting formulation of nifedipine (PA) with longer-acting felodipine (ER) on 24-h blood pressure and left ventricular mass in patients with uncomplicated essential hypertension. Subjects were randomly allocated to receive nifedipine (n = 52) or felodipine (n = 56) over 8 weeks at increasing doses until the sitting office diastolic blood pressure (DBP) was < 90 mm Hg. An ambulatory blood pressure (ABP) recording and echocardiogram were performed at baseline and upon completion of the study. At the end of the dosing interval, felodipine lowered office DBP (mm Hg) by -18 +/- 12/14 +/- 1 compared to -14 +/- 2/11 +/- 1 for nifedipine (P < or = .05). Mean 24 h ABP was reduced (P < .001) by felodipine (-15 +/- 1/-10 +/- 1 mm Hg) and nifedipine (-15 +/- 1/-9 +/- 2 mm Hg). However, nifedipine caused an apparent biphasic response with felodipine reducing (P < .05) blood pressure more in the early afternoon compared to nifedipine. Left ventricular mass index was significantly reduced (P < .01) by felodipine (-6 +/- 1 g/m2), but not by nifedipine. Once-daily administration of felodipine achieves a more consistent control of blood pressure compared to twice-daily nifedipine and may be associated with a greater reduction in left ventricular mass. PMID- 7546498 TI - Time-dependent effect of isradipine on the nocturnal hypertension in chronic renal failure. AB - Nocturnal hypertension is frequently observed in chronic renal failure and contributes to the risk of target organ damages. We assessed whether antihypertensive therapy may restore a nocturnal blood pressure (BP) fall in this condition. A sustained-release oral formulation (SRO) of isradipine was used, and the possible differences in the response to morning nu evening dosing were also investigated. Sixteen hypertensive patients with chronic renal failure due to parenchymal kidney disease were studied after 2 weeks of single-blind placebo runin. According to the double-blind, randomized, cross-over design, they received 5 mg isradipine SRO at 08:00, or at 20:00 for 4 weeks, separated by a single-blind placebo period of 2 weeks. A 24-h BP monitoring at 10-min intervals was carried out at the end of each treatment using a SpaceLabs 90207 instrument. Under placebo, blunt BP profiles were observed, whereas HR showed a mean nocturnal fall of 17.4%, which remained unaltered after isradipine. Both isradipine treatments were equally effective in reducing the mean 24-h BP levels. However, the evening regimen showed a more pronounced effect during the night. The mean nocturnal fall in systolic/diastolic BP represented 4.8/8.7% and 7.5/10.9% of the corresponding daytime mean after morning and evening dosing, respectively. Only the evening administration reset the normal synchronization of the 24-h BP and HR profiles. Our findings demonstrate that antihypertensive treatment may restore a nocturnal BP fall in renal patients. An evening regimen of isradipine SRO seems more apt than a morning regimen to obtain this therapeutic goal. PMID- 7546499 TI - The efficacy and tolerability of enalapril in a formulation with a very low dose of hydrochlorothiazide in hypertensive patients resistent to enalapril monotherapy. AB - The antihypertensive efficacy and tolerability of formulations of enalapril and low (12.5 mg) and very low (6 mg) doses of hydrochlorothiazide (HCTZ) were compared with enalapril and placebo. Four hundred and two patients with mild to moderate essential hypertension were treated with 20 mg enalapril for 8 weeks. Patients (n = 296) with persistent supine diastolic blood pressure > 95 mm Hg after enalapril monotherapy were randomized to receive enalapril/placebo (group I), 6 mg enalapril/HCTZ (group II), or 12.5 mg enalapril/HCTZ (group III) for another 8 weeks in a double-blind design. The mean reductions in blood pressure were significantly larger in groups II and III compared to group I, 7.3 (95% CI, 9.0; -6.2), 7.7 (-9.2;-6.3), and 4.1 (-5.9;-2.9) mm Hg, respectively P < .01 for groups II and III compared to group I). No difference in side effects was observed between the three groups. A very low dose of 6 mg HCTZ acts synergistically when given together with enalapril, but is devoid of adverse metabolic effects. PMID- 7546500 TI - High-fructose feeding elicits insulin resistance without hypertension in normal mongrel dogs. AB - This study was undertaken to characterize blood pressure (by continuous blood pressure recording), renal hemodynamics, and excretory function in high-fructose fed insulin-resistant dogs. We fed 10 mongrel dogs for 28 days with a normal sodium diet containing 60% of the calories either as fructose (n = 6) or dextrose (n = 4). Fructose-fed dogs developed insulin resistance by the 21st day of the experimental diet, as estimated by the mean glucose concentrations (in arbitrary units, AU) during the final hour of the insulin suppression test (640.3 +/- 57 AU fructose-fed dogs upsilon 397.5 +/- 24.7 AU dextrose fed dogs; P < .05). Neither of the groups showed any change in body weight, or in fasting plasma levels of glucose or insulin. There was no difference in mean arterial pressure between the groups before or during either diet, nor did we find any important alterations in renal function in these animals. We conclude that insulin resistance can be induced by a high-fructose diet in the dog. However, it is not accompanied by either hypertension or alteration in renal function. These findings emphasize the importance of continuously recording blood pressure under resting conditions and suggests that in the fructose-fed dog, insulin resistance does not appear to lead directly to hypertension. PMID- 7546501 TI - Altered beta-adrenergic regulation of Na-K-Cl cotransport in cultured smooth muscle cells from the aorta of spontaneously hypertensive rats. Role of the cytoskeleton network. AB - To verify the hypothesis of Na-K-Cl cotransport (COTR) involvement in ion transport abnormalities as revealed in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR), we compared the rate of ouabain insensitive, bumetanide-inhibited 86Rb influx in quiescent and growing cultures of VSMC from the aorta of SHR and normotensive (BN.lx) rats and its regulation by the cAMP signaling system. Basal COTR was not altered in quiescent cells from SHR but was decreased by 30% to 40% (P < .02) in growing SHR VSMC as compared to BN.lx rats. In quiescent BN.lx VSMC, isoproterenol inhibited COTR by 50% and induced cell shape transition of > 90% of cells, resulting in the appearance of rounded VSMC with arborized cytoplasms. In contrast, isoproterenol elicited cell shape transition in only 50% of quiescent SHR VSMC and did not modify COTR. In growing cells, it decreased COTR by 85% to 95% and altered cell morphology in > 95% of VSMC without differences between SHR and BN.lx rats. Neither inhibitors of protein kinase A (H-89 and KT-5720) nor an inhibitor of phosphoprotein phosphatase (okadaic acid) affected cell shape transition and COTR suppression in isoproterenol-treated VSMC. The COTR suppression and cytoplasm arborization was also demonstrated by the addition of cytochalasin B, a disintegrator of microfilament bundles, and staurosporine, an inhibitor of protein kinase C. The effects of these compounds on COTR and SHR and BN.lx VSMC morphology were not different. The calmodulin antagonist R24571 decreased COTR by 60% to 70% in quiescent BN.lx VSMC and did not modify this carrier in SHR VSMC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546502 TI - Endothelin synthesis by porcine inner medullary collecting duct cells. Effects of hormonal and osmotic stimuli. AB - Previously, we have shown in porcine inner medullary collecting duct (IMCD) cells that endothelin (ET), probably in an autocrine fashion, suppresses arginine vasopressin (AVP)-induced synthesis of cAMP and thereby, may modify the action of AVP on IMCD fluid transport. In the present study we investigated the effects of various stimuli including extracellular tonicity on ET synthesis in porcine IMCD cells in culture. IMCD cells produced ET in a saturationlike time-dependent manner over a period of 24 h. Neither AVP (10(-7) mol/L), bradykinin (10(-7) mol/L), nor atrial natriuretic peptide (10(-7) mol/L) affected basal ET synthesis of IMCD cells at extracellular isotonicity (323 mOsm/kg H2O). The calcium ionophore A23187 (10(-7) mol/L) increased ET production by 38% within 2 h (P < .05). Preincubation for 48 h with increased osmolality in the incubation media from 323 to 600 mOsm/kg H2O by raising the concentrations of 1) NaCl (n = 6), 2) urea (n = 6), or 3) NaCl+urea (n = 6) increased ET synthesis from a control value of 225 +/- 25 pg/mg cell protein/2 h in isotonic medium to 1) 555 +/- 13 pg/mg cell protein/2 h (P < .01), 2) 354 +/- 18 pg/mg cell protein/2 h (P < .05), and 3) 448 +/- 22 pg/mg cell protein/2 h (P < .05), respectively, in hypertonic media. These data suggest that increases in papillary osmolality are associated with enhanced ET synthesis possibly involving a calcium-dependent process and attenuating AVP-dependent fluid absorption in a short-loop feedback fashion. PMID- 7546503 TI - Endogenous sodium pump inhibitors in human urine. Further identification of inhibitors of Na-K-ATPase. AB - We investigated the presence of endogenous Na-K-ATPase inhibitor(s), ie, ouabain like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (> 30 g NaCl/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M(r) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross reacted with a digoxin antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K ATPase inhibition and Ki-values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respectively. Mass-spectroscopy (MS) showed M(r) of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546504 TI - Hypertension-induced dysfunction of circulation in hemorrhagic shock. AB - Arterial blood pressure and plasma concentration of Na+, K+, glucose, and urea were studied in patients with essential hypertension during hemorrhagic shock. All 15 patients had established essential hypertension, and all had massive hemorrhage and shock. The control group included 37 normotensive patients in hemorrhagic shock. There were no differences in blood volume loss and age between the hypertensive and normotensive groups. A significant reduction (P < .001) of systolic and diastolic blood pressure as well as plasma concentration of Na+ and K+ were observed in hypertensive patients with hemorrhagic shock as compared with normotensive patients in hemorrhagic shock. Hypertensive patients in shock were found to have significantly elevated (P < .001) plasma glucose, urea, and heart rate compared with normotensive patients in shock. The hypertensive group mortality was significantly (P < .001) higher (80%) than the normotensive group in shock (24.3%). These data suggest that essential hypertension induces alterations in regulation and modulation of peripheral resistance, Na+/K+ membrane exchange, and the metabolism of glucose and urea during hemorrhagic shock. This heterogeneity of dysfunction induced by essential hypertension during hemorrhagic shock is probably caused by a change in the mechanism (diminish) of oxygen consumption at the cellular level, which results in an increase in nitric oxide production (which is O2 dependent), and may play an important role in hypertension and hemorrhagic shock. PMID- 7546506 TI - Sudden cardiac death. AB - This article uses a biologic model of sudden cardiac death to identify structural abnormalities that serve as the substrate for sustained arrhythmias and functional changes that are transient and necessary for triggering an arrhythmia. The biologic framework is a valuable tool to use in understanding the relationship between structure and function and in organizing patients' clinical presentations and outcomes. PMID- 7546505 TI - Resistance to insulin-mediated glucose uptake and hyperinsulinemia in women who had preeclampsia during pregnancy. AB - Plasma glucose and insulin responses to a 75-g oral glucose load, and the steady state plasma insulin (SSPI) and glucose (SSPG) concentrations after an infusion of somatostatin, insulin, and glucose, were determined 2 months after delivery in 26 women; 13 who had a normal pregnancy and 13 who developed preeclampsia. The plasma glucose response to oral glucose was not different in the two groups, but the plasma insulin response was significantly greater (P < .02) in those who had been preeclamptic. Although the mean (+/- SE) SSPI concentrations during the infusion study were similar in the two groups (51 +/- 2 v 56 +/- 2 microU/mL), the SSPG concentrations were significantly higher (P < .02) in those who developed preeclampsia (160 +/- 17 v 119 +/- 17 mg/dL). Thus, when studied 2 months after delivery, women who developed preeclampsia were relatively insulin resistant and hyperinsulinemic when compared to those who had an uncomplicated pregnancy. PMID- 7546507 TI - Emergent neurologic events. AB - Because the spectrum of severity of neurologic events ranges from minor to life threatening, and many neurologic reasons exist to seek health care, this article focuses on some of the more common pathologies. A general assessment plan is presented that may be adapted for the specific problems that individual patients present. Pain is discussed in relation to the nociceptor transmission of painful stimuli. An example of pain from a neurogenic shock-induced migraine headache is discussed from pathogenesis, presentation, and intervention viewpoints. Alterations in cerebral blood flow, cerebrovascular accidents, and subarachnoid hemorrhage are reviewed from a pathophysiologic perspective. PMID- 7546508 TI - Environmental emergencies. AB - Recreational activity sometimes leads to injury caused by environmental conditions, such as lightning. A familiarity with the effects of electricity, lightning, hyperthermia, and poisoning is essential for providers of care to the critically injured. This article discusses the pathophysiology and treatment of injuries caused by the environment. PMID- 7546509 TI - Pediatric emergencies. AB - The care of acutely ill children presents a unique challenge for health care professionals. Nurses often are required to make a quick assessment of the child's condition, even when the child is frightened or uncommunicative. This article addresses the most common of a multitude of illnesses seen in children and organizes them according to organ systems and common final pathways within a system. PMID- 7546510 TI - Sudden infant death syndrome. AB - Sudden infant death syndrome (SIDS) is a perplexing and troubling problem for health care professionals and the community. This article discusses the epidemiology of SIDS and nursing interventions to meet the needs of families affected by SIDS. PMID- 7546511 TI - The role of nursing assessment of traumatic events in sudden injury, illness, and death. AB - The trauma nurse plays a critical role in the assessment of the trauma patient. This assessment spans from the time of the patient's arrival at the hospital to discharge into the home and rehabilitation setting. It is important for nurses to be knowledgeable about trauma assessment, mechanism of injury, and the high risk and frequent complications that threaten the trauma patient. PMID- 7546513 TI - The beginnings. AB - Trauma care became more specialized in the 1980s, and the "other side" of trauma became evident. The experiences of health care professionals revealed that patients were not the only victims of trauma. Patients' families and friends, caregivers, and the community also experienced the devastating psychosocial effects of trauma. This article describes the multidisciplinary team approach at Hartford Hospital, Connecticut, that was designed to address the needs of all trauma victims. PMID- 7546512 TI - Clinical forensic nursing: a new perspective in the management of crime victims from trauma to trial. AB - Forensic nursing is one example of an innovative expansion of the role nurses will fill in the health care delivery system of the future. Because current policies of advocacy programs mandate the inclusion of criminal justice and health care providers, it is especially timely to propose that the forensic nursing specialist be placed in the trauma treatment environs to serve as a valuable link in interagency cooperation, ensuring that human needs and medicolegal interests are served. Because most emergency personnel and prehospital care providers ordinarily have only secondary interests in forensic matters, the motivated and skilled forensic nurse can serve as an invaluable resource for the criminal justice system, the hospital, and the patient. PMID- 7546514 TI - After care bereavement program. AB - Every year, 200 to 250 patients suffer the sudden death of a loved one in the emergency department at Hartford Hospital. Many families are not prepared for the grief they will experience and are left to grieve their loss alone. Recognizing the needs of these families led to the development of the Trauma Support After Care Program, which is described in this article. PMID- 7546517 TI - After care program volunteer: a personal reflection. AB - The After Care Program at Hartford Hospital is a two-sided experience for those who have been cared for and provide care for others as well. This article reveals the experience of an After Care Program volunteer who received its support after a traumatic loss. PMID- 7546516 TI - After care program II: a volunteer's perspective. AB - This article presents a volunteer's reflections on the Trauma Support After Care Program at Hartford Hospital. The support given to families who have suffered a tragic loss is described, as is the satisfaction a volunteer experiences in providing support for both the program and the families. PMID- 7546515 TI - After care program volunteer: a staff nurse's experience. AB - A staff nurse who assisted in comforting families through the Trauma Support After Care Program at Hartford Hospital shares her experiences in this article. Her initial hesitation regarding family care and follow-up is described, and her eventual success as a volunteer helping others cope with loss is highlighted. PMID- 7546518 TI - Staff support. AB - Staff support is essential for the individuals who care for patients with severe injuries. The loss of a colleague led to the development of the coping strategies described in this article. It is hoped that the experiences shared in this article will aid others in creating their own specific strategies for coping within hospitals or in other situations. PMID- 7546519 TI - Support of families who had a loved one suffer a sudden injury, illness, or death. AB - The sudden injury, illness, or death of a loved one leaves family members unprepared to deal with the stress of the situation. Hospital staff members should have the special knowledge and skills needed to support both the patient and his/her family. This article provides useful information for ensuring that the support process is healthy for all those involved. PMID- 7546520 TI - "Death-telling" research project. AB - An unexpected death is a traumatic event for everyone affected by it, especially family members. The manner in which a family is told that a death has occurred has a profound effect on their grief and bereavement process. The task of the health care professional working with these patients and their families is to assist their healing by being skilled in the "death-telling" process. This article focuses on the project that developed a teaching tool and videotape used to educate individuals about the proper way to inform families of the death of a loved one. PMID- 7546521 TI - Pediatric trauma support program: supporting children and families in emotional crisis. AB - During a traumatic situation, families often are unable to gather the strength and resources they need to cope. Family roles change, and the ability of individuals to deal with daily problems often diminishes. By helping children feel comfortable with the concept of death, one can work toward a greater understanding of death throughout society. This article explores appropriate, family-oriented support to provide to those enduring trauma, and support for staff members who must deal repeatedly with these emotional experiences. PMID- 7546523 TI - Future horizons for the victims of trauma. AB - The prevention of injuries and the future treatment of trauma victims are exciting and uncertain prospects. Continuous advances in the development of inclusive trauma care systems, technical surgical achievements, and patient centered redesign are important factors to consider. The integration of effort will be a recurrent theme in injury prevention and trauma care. PMID- 7546522 TI - Organ and tissue donation: rights and responsibilities. AB - Critical care staff members have an obligation to facilitate the rights of individuals and their families regarding organ and tissue donation. These rights can be promoted through collaboration between a hospital and an organ procurement organization. Collaboration and education will help organ and tissue donation become part of institutional norms, and health care professionals will be able to provide the option of donation as part of the continuum of care. PMID- 7546524 TI - Cross-cultural customs and beliefs related to health crises, death, and organ donation/transplantation: a guide to assist health care professionals understand different responses and provide cross-cultural assistance. AB - All individuals can expect to encounter some form of crisis and loss. Responses to these experiences are not universal and will be influenced by a person's cultural background and religious beliefs. This appendix provides information about the different responses to health crises, death, and organ donation and transplantation as practiced by various religious and cultural groups. PMID- 7546525 TI - Systemic cardiovascular response in hemodialysis without and with ultrafiltration with membranes of high and low biocompatibility. AB - In order to test whether dialyzer membrane biocompatibility influences systemic cardiovascular function, we treated 8 hemodialysis patients (4 men and 4 women, aged 24-73 years) with a low-biocompatible (cuprophane) and a high-biocompatible (polyacrylonitrile) membrane in a randomized double-blind crossover protocol using bicarbonate hemodialysis without ultrafiltration for the first 60 min and with ultrafiltration for the remaining treatment time. Left ventricular function and systemic hemodynamics were assessed noninvasively at baseline and during treatment by Doppler echocardiography combined with external subclavian artery pulse trace calibrated with oscillometrically measured brachial artery blood pressures. There was no significant difference in the cardiovascular response to the 2 membranes, neither during isolated hemodialysis nor when ultrafiltration was added. Mean arterial pressure increased 10% (p < 0.001) during isolated hemodialysis and returned to baseline levels with ultrafiltration. The cardiac index decreased 22% (p < 0.001) during ultrafiltration, due to the greater decrease in left ventricular stroke index (30%, p < 0.001) than increase in heart rate (9%, p < 0.05). Total peripheral resistance increased 10% (p < 0.05) during isolated hemodialysis and a further 19% (p < 0.01) when ultrafiltration was added. Hence, profound cardiovascular alterations were observed during hemodialysis treatment; however, these changes were not related to the biocompatibility of the membranes. PMID- 7546526 TI - Pre-/postdilution hemofiltration. AB - Hemofiltration creates the best conditions for toxin removal and cardiovascular stability in the treatment of chronic renal failure patients. The increase in hematocrit due to erythropoietin, the blood flow rate and the necessary volume of substitution fluid limit the post- or the predilution hemofiltration. The technical progress made now offers the possibility to routinely and safely treat patients with pre-/postdilution hemofiltration. When adjusting the substitution flow rate to the blood flow rate, small-molecule clearances are higher than those in hemodialysis and are close to those in hemodiafiltration. PMID- 7546529 TI - Thrombotic thrombocytopenic purpura in pregnancy: successful treatment with plasma exchange. Case report and review of the literature. AB - Thrombotic thrombocytopenic purpura (TTP) is a rare syndrome which presents typically with thrombocytopenia, microangiopathic hemolytic anemia, central nervous system symptoms, fever, and renal abnormalities. The diagnosis of TTP in pregnancy previously carried a poor prognosis and a high fetal mortality when presenting early in gestation. This case report describes the earliest presentation of TTP in pregnancy (6 weeks of gestation) we could identify in the literature treated successfully with a prolonged course of plasma exchange. The differential diagnosis and the pathogenesis of TTP in pregnancy are reviewed. Therapeutic options and data regarding the removal of pregnancy-related hormones by plasma exchange are presented. PMID- 7546528 TI - Intraperitoneal fluid volume changes during peritoneal dialysis in the rat: indicator dilution vs. volumetric measurements. AB - In order to validate the single injection RISA (125I human serum albumin) indicator diluation technique for assessing the alterations in intraperitoneal (i.p.) dialysate volume (IPV) which occur vs. time [V(t)] during peritoneal dialysis (PD), the RISA dilution technique was compared to V(t) determinations using a direct volume recovery method in Wistar rats. Sixteen milliliters of either 1.36 or 3.86% Dianeal or 0.9% NaCl were used as dialysis fluids in exchanges lasting between 1 and 360 min. Approximately 4% (4.41 +/- 0.59 (SE; n = 8) for 1.36% Dianeal and 4.07 +/- 0.72 (n = 4) for 3.86% Dianeal) of the RISA dose given intraperitoneally was lost from the dialysate during the first 1(-1.5) min after instillation, conceivably due to rapid tracer adsorption to peritoneal surfaces. Following the initial instant tracer loss and RISA dilution due to a residual volume (3.07 +/- 0.18 ml; n = 12), RISA disappeared at a fractional rate (FDR) of 2.10 +/- 0.14 x 10(-3) min-1 and 1.67 +/- 0.09 x 10(-3) min-1, during the first 30 min for 1.36 and 3.86% Dianeal, respectively. The overall FDR was 1.33 +/- 0.10 x 10(-3) and 0.707 +/- 0.082 x 10(-3) min-1 for 1.36% Dianeal (0 150 min) and 3.86% Dianeal (0-360 min), respectively, while the overall (0-150 min) FDR for the NaCl exchanges was 1.40 +/- 0.21 x 10(-3) min-1. These values correspond to RISA clearances out of the peritoneal cavity (KE) of 29.2 +/- 1.8, 22.1 +/- 1.6, and 25.7 +/- 2.4 microliter x min-1 for 1.36 and 3.86% Dianeal and 0.9% NaCl, respectively. The KE value for 3.86% Dianeal was significantly (p < 0.05) lower than for the two dialysates with lower osmolality. The slightly enhanced FDR of RISA during the first 30 min was partly due to the presence of nonprotein-bound free iodine in the RISA preparation used, and also to an enhanced disappearance of albumin during the first portion of the dwell. V(t) data from individual experiments using the RISA dilution technique (RISA curves) were analyzed by computer-aided nonlinear least-squares regression analysis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7546527 TI - Treatment of sepsis-associated severe acute renal failure with continuous hemodiafiltration: clinical experience and comparison with conventional dialysis. AB - The syndrome of sepsis-associated severe acute renal failure is a frequent component of sepsis-induced multiorgan failure. Continuous hemofiltration techniques are often used in its dialytic management but little is known about their impact. The aim of this study is to define the biochemical and clinical impact of continuous hemodiafiltration (CHD) in the management of this syndrome and to retrospectively compare it to that of conventional dialysis. A prospective, cohort study and retrospective comparison with historical controls was conducted at an intensive care unit (ICU) of a tertiary institution. Eighty seven consecutive septic patients with acute renal failure were treated by continuous hemodiafiltration and 40 consecutive similar patients by conventional dialysis. All new cases of severe acute renal failure with sepsis were treated by means of continuous hemodiafiltration. Historical controls were treated by means of conventional dialysis. Illness and sepsis severity were assessed on admission and prior to initiation of treatment. Biochemical variables were assessed daily. Outcome was measured as discharge from the ICU, duration of oliguria and discharge from hospital. Of the 87 patients treated by hemodiafiltration, 86 had multiorgan failure, 71 (81.6%) septic shock and 52 (59.8%) bacteremia/fungemia. Their APACHE II score on admission was 29.9 and their mean organ failure score prior to treatment was 4.3. Hemodiafiltration resulted in a significant fall in mean urea and creatinine levels within 24 h and in the correction of acidosis. The mean alveolar-arterial gradient fell from 276 to 211 mm Hg (p < 0.02) within 24 h of therapy. Complications were few and mostly related to vascular access.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546530 TI - The Leros PIKPA asylum. Deinstitutionalisation and rehabilitation project. AB - A three-year deinstitutionalisation and rehabilitation pilot intervention project was implemented at Leros PIKPA for people with severe learning disabilities. Initial conditions at the asylum were appalling. Residents suffered severe deprivation, extreme institutionalisation, and violation of basic human rights. Intervention involved professionals from different disciplines, and involved residents, their families, care staff, the institution, and the local community. As a result, resident care and adaptive behaviour has started to improve. Communication between residents and families has increased. Owing to training and sensitisation, care staff's poor resident-management practices and negative attitudes toward disabled people have changed. Living and hygienic conditions have been upgraded and building renovation is under way. Asylum administration and the local community have been sensitised to residents' needs. Eleven residents have moved to the project's pilot community home in Athens; two others now live with foster families. The results suggest that deinstitutionalisation and rehabilitation can be successfully initiated even in residential institutions of the severest kind. PMID- 7546531 TI - The children of Leros PIKPA. PMID- 7546532 TI - The psychosocial dynamics of change at Leros PIKPA asylum. AB - The pilot intervention project at Leros PIKPA asylum was resisted at all levels of its implementation. Resistance ranged from implicit and passive to explicit and hostile. It took the form of strong rejective attitudes, defensiveness, and repressed emotions of guilt, shame and fear. It was apparent in efforts to delay, obstruct or reverse the progress of the project and undermine the work of intervention team members. It was evident in the interactions of the intervention team with all parties involved in implementing the project: medical professionals and welfare services, the state and local administrations, the central and local administrations of PIKPA staff, the local community, and the asylum residents' families and relatives. It is argued that this resistance to change is due to the interplay of cultural/attitudinal, psychodynamic and socio-economic factors that are not unique to Leros or Greece. The analysis of the psychosocial processes involved in resistance to changing the Leros PIKPA asylum may offer insights to the deinstitutionalisation of custodial settings in general. PMID- 7546533 TI - Change in care staff's attitudes towards people with learning disabilities following intervention at the Leros PIKPA asylum. AB - The pilot intervention project at Leros PIKPA asylum sought to change the management of residents by decreasing care staff's tendency to view people with learning disabilities with attitudes of custodial segregation (e.g. "they should all live in institutions") and categorisation (e.g. "they are all alike"). To that end, staff were trained on the job in resident management and participated in seminars, sensitisation groups, clinical case presentations and visits to model rehabilitation units. To investigate whether staff attitudes towards people with learning disabilities had been influenced, staff members responded to a questionnaire of such attitudes, in March 1991 (initial assessment) and a year later (reassessment). Factor analyses of attitude ratings at initial assessment revealed a strong negative attitudinal construct, "Rejective and custodial segregation--Categorisation", according to which institutionalisation was the only appropriate type of care for people with learning disabilities, regardless of individual differences. At reassessment, however, this factor had split into two independent ones, "Rejective and custodial segregation" and "Categorisation", suggesting that the staff had understood that custodial care did not apply to all persons with learning disabilities indiscriminately. Reassessment factors further indicated that staff had comprehended the appropriateness of alternative care forms--community and home care. Analysis of Categorisation and Custodial segregation attitude ratings showed that only staff's tendency to view the disabled as "all alike" (Categorisation) had decreased at reassessment. The latter change and the structural change revealed by the factor analyses imply that staff's attitudes underwent a small but significant modification. PMID- 7546534 TI - Mortality among institutionalised people with learning disabilities in Greece: a 30-year survey at the Leros PIKPA asylum. AB - The life expectancy of institutionalised people with learning disabilities is shorter than that of the general population. Data on population dynamics in institutions for such people are vital for planning purposes. Mortality can be considered a crude measure of quality of health care. Mortality data on the 914 admissions to the Leros PIKPA asylum for children and young adults with learning disabilities and associated problems in the years 1961-91 were reviewed. The overall crude mortality rate was 59.2 deaths per 1000 person-years. Twenty-two per cent of the deaths occurred within a year after admission. Age-specific mortality rates were particularly high for those aged one to four years, and declined thereafter. Male residents had lower mortality than female residents in almost all of the age groups. Compared with sex- and age-specific mortality data for the general population of Greece, the observed rates were 20-150 times higher but still comparable to those reported for people with more severe learning disabilities in institutions in other countries. Lifetable analysis by length of stay showed that male residents had a statistically significant higher probability of survival than female residents, which could not be attributed to age-related differential mortality. Culture-specific differential admission criteria may account for the observed sex difference. PMID- 7546535 TI - [Pharmacologic profile of n-palmitoylglycine. Its effect on reserpine and haloperidol catalepsy]. AB - N-palmitoyl glycine (PG), synthesized by the mixed anhydrides method, displayed nootropic effects (in the "step-down" test) as well as an anti-immobility action in the forced swimming test, in mice. However no anticonvulsant effects were obtained, even at high dose (300 mg/Kg, po), in the maximal electroshock or against the convulsions induced by pentetrazol. In the study reported in this paper PG potentiated the reserpine or the haloperidol catalepsy in rats. The potentiation of the haloperidol catalepsy was antagonized by imipramine which displayed antiglutamatergic and antimuscarinic properties. On the other hand at 150 mg/Kg (ip), a dose which potentiated the haloperidol catalepsy, PG stimulated the dopaminergic function in the striatum. These results could therefore constitute a first clue suggesting a stimulation, by PG, of the cholinergic and GABAergic interneurons, by glycinergic potentiation of the NMDA receptors in the striatum. PMID- 7546537 TI - In vitro release of ketoprofren and ibuprofen from viscoelastic gels. AB - Gels of viscoelastic type formed by isopropylmyristate, Tween 60 and water are prepared in the presence or not of antiinflammatory agents such as Ketoprofen and Ibuprofen. In vitro release studies evaluated by using an absorption simulator were performed in order to determine the diffusion rate of these drugs from prepared gels. The obtained results showed a higher rate diffusion compared with the release from commercial products containing these drugs. PMID- 7546536 TI - [The regulation of drug prices in Italy]. AB - Medicinal products price regulation in Italy has been discussed. The financial law n. 537/93 has deeply modified the criteria utilised to define the industrial medicaments prices. The authors analysed the implications derived from the variation from a fixed price system to a system based on the media of the european drug prices. PMID- 7546539 TI - Synthesis of (+/-)-1-amino-6,7,8,8a-tetrahydro acenaphthene with possible central dopaminergic activity. AB - We describe the non stereoselective synthesis of (+/-)-1-amino-6,7,8,8a tetrahydroacenaphthene (14), a novel compound that belongs to the acetanaphtene group, that is presented as a rigid non hydroxylated 2-aminoindan which has a structural disposition of a dopaminergic pharmacophore that possess a phenylethylamine fragment. Intracerebroventricular administration of this compound induces an increase in urinary volume and sodium excretion in conscious rats. The renal actions of 14 were blocked by haloperidol pretreatment, suggesting that 14 acts centrally through a dopaminergic mechanism. PMID- 7546538 TI - Kojic acid production from cocoa juice by Aspergillus flavus entrapped in calcium alginate. AB - Sixteen microorganisms of Aspergillus strains were screened for production of kojic acid using cocoa juice as carbon source. Only Aspergillus flavus ATCC 9179 was found to produce the acid in low yield (22 mg/ml). Calcium alginate immobilization of the cells was used under optimum conditions to maximize the yield of kojic acid (60 mg/ml). Cultures were incubated in the medium with 50% of cocoa juice added in pulses of 8 ml each every 96 hours, and 4% methanol, pH 3.5, 150 rpm, 26 degrees C for three weeks. The incubations were monitored by thin layer and high pressure liquid chromatography. Kojic acid was extracted from the culture broth by organic solvent, concentrated and crystallized. The chemical identity of kojic acid was determined by HPLC, MS, 1H- and 13C-NMR spectroscopy. PMID- 7546540 TI - [New strategies for the treatment of schizophrenia: agonists for DA or glycinergic autoreceptors?]. PMID- 7546541 TI - [Patents in the pharmaceutical field: history and development of the institution]. AB - The aim of this work it is to highlight the relevance of many Domestic and European Laws concerning Property Right for Patents. A special attention is devoted to Patents of Drugs by means of detailed doctrinal references and also in the light of the current European Procedures. PMID- 7546542 TI - Evaluation of some arylhydrazones of p-aminobenzoic acid hydrazide as antimicrobial agents and their in vitro hepatic microsomal metabolism. AB - Benzoic acid p-amino-[(substituted phenyl/pyridyl) methylene] hydrazide derivatives were synthesised by interaction of p-aminobenzoic acid hydrazide with various aromatic aldehydes. The structures of the compounds were elucidated by use of their UV, IR, 1H-NMR and mass spectral data. These compounds were also evaluated for antimicrobial activity. The in vitro hepatic microsomal metabolism of benzoic acid p-amino-[(4-fluorophenyl)methylene]hydrazide (2d), a selected prototype from these compounds was also carried out. PMID- 7546543 TI - HPLC method for the quantitation of cispentacin enantiomers in rat urine. AB - The two enantiomers of cispentacin, an antifungal antibiotic, are determined by reversed phase HPLC, after derivatization with Marfey's reagent, in 24 h urine samples collected from rats treated sc and iv with 20 mg/kg cispentacin racemate obtained by synthesis. The application range of the method is 25-250 mg/L for each enantiomer with a precision of 4.0-9.0%. Comparison with an authentic sample of natural origin (-)-cispentacin indicated that (-) enantiomer is excreted unchanged in smaller percentage than (+) enantiomer, which is almost completely eliminated as such. PMID- 7546544 TI - [A lesson on the purity of pharmaceutical products derived from biotechnology: eosinophilia-myalgia syndrome]. PMID- 7546546 TI - Antimalarial sesquiterpenes from tubers of Cyperus rotundus: structure of 10,12 peroxycalamenene, a sesquiterpene endoperoxide. AB - Activity-guided investigation of Cyperus rotundus tubers led to the isolation of patchoulenone, caryophyllene alpha-oxide, 10,12-peroxycalamenene and 4,7-dimethyl 1-tetralone. The antimalarial activities of these compounds are in the range of EC50 10(-4)-10(-6) M, with the novel endoperoxide sesquiterpene, 10,12 peroxycalamenene, exhibiting the strongest effect at EC50 2.33 x 10(-6) M. PMID- 7546545 TI - PCR amplification using psoralen biotinylated primers: PAGE analysis and direct chemiluminescent detection. PMID- 7546547 TI - Cytotoxic biflavonoids from Selaginella willdenowii. AB - Bioactivity-guided fractionation of the leaves of Selaginella willdenowii afforded three known biflavones, 4',7"-di-O-methylamentoflavone, isocryptomerin and 7"-O-methylrobustaflavone, that were significantly cytotoxic against a panel of human cancer cell lines. Non-cytotoxic isolates were also obtained, namely, amentoflavone, bilobetin, robustaflavone and 2",3"-dihydroisocryptomerin, a new dihydrobiflavone. The structure for the new biflavonoid was unambiguously assigned by a combination of spectroscopic methods. PMID- 7546548 TI - Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence. AB - Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit. PMID- 7546550 TI - A bidesmosidic hederagenin hexasaccharide from the roots of Symphytum officinale. AB - A new bidesmosidic triterpenoidal saponin 3-O-[beta-D-glucopyranosyl-(1-->4)-beta D-glucopyranosyl-(1-->4)-alpha-L - arabinopyranosyl]-hederagenin-28-O-[alpha-L rhamnopyranosyl- (1-->4)-beta-D- glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, was isolated from the roots of Symphytum officinale. The structure was assigned by chemical methods and spectral analysis (1H, 13C, DEPT, NMR, EI-MS and FAB-MS) including 1H-1H COSY, 1H-13C COSY and HOHAHA. The prosapogenin of this saponin is also a new compound. PMID- 7546549 TI - Eucalyptone from Eucalyptus globulus. AB - A new cariostatic compound named eucalyptone was isolated from the leaves of Eucalyptus globulus. The structure of this compound was elucidated by spectroscopic methods. PMID- 7546551 TI - Two flavone 2'-glucosides from Scutellaria baicalensis. AB - Two new flavone glucosides, 5,2',6'-trihydroxy-6,7,8-trimethoxyflavone 2'-O glucoside and 5,2',6'-trihydroxy-6,7-dimethoxyflavone 2'-O-glucoside were isolated from the aqueous methanol extract of the roots of Scutellaria baicalensis. From the extract, seven phenolics, 5,7,2',6'-terahydroxyflavone, 5,7,2',5'-tetrahydroxy-8,6'-dimethoxyflavone, skullcapflavone II, baicalin, baicalin methyl ester, wogonin 7-glucuronide and 3,5,7,2',6' pentahydroxyflavanone were also isolated. PMID- 7546552 TI - Clausenol and clausenine--two carbazole alkaloids from Clausena anisata. AB - Two new carbazole alkaloids, designated as clausenol and clausenine, were isolated from an alcoholic extract of the stem bark of Clausena anisata. Their structures were established as 1-hydroxy-6-methoxy-3-methylcarbazole and 1,6 dimethoxy-3-methyl carbazole, respectively, from physical and chemical evidence and synthesis. Clausenol was found to be active against Gram-positive and Gram negative bacteria and fungi. PMID- 7546553 TI - 4-Quinolinone alkaloids from Dictyoloma peruviana. AB - The stem-bark of Dictyoloma peruviana yielded two new piperidino [1,2-a] 4 quinolinones, dictyolomide A and dictyolomide B. Their structures were established by NMR spectroscopy. PMID- 7546554 TI - Flavonol glycosides from Nitraria retusa. AB - The new flavonol trioside, isorhamnetin 3-O-4Rham-galactosylrobinobioside and five known flavonol glycosides, isorhamnetin 3-robinobioside, isorhamnetin 3 rutinoside, isorhamnetin 3-galactoside, isorhamnetin 3-glucoside and free isorhamnetin were isolated from the leaves and young stems of Nitraria retusa and characterized by UV and NMR spectroscopy. Isorhamnetin 3-xylosylrobinobioside was also tentatively identified. PMID- 7546555 TI - Plant transglutaminases. AB - The identification procedures, the characteristics and the potential function of the recently detected plant transglutaminases, are discussed in the light of the knowledge of animal transglutaminases. The enzyme has been studied occasionally in lower organisms (bacteria, fungi and green algae) and more extensively in Angiosperms. PMID- 7546556 TI - Biochemically active sesquiterpene lactones from Ratibida mexicana. AB - Bioactivity-directed fractionation of the methanol extract of the roots of Ratibida mexicana resulted in the isolation of two bioactive sesquiterpene lactones, isoalloalantolactone and elema-1,3,11-trien-8,12-olide. Both compounds caused a significant inhibition of the radicle growth of Amaranthus hypochondriacus and Echinochloa crus-galli, exerted moderate cytotoxic activity against three different solid tumour cell lines and inhibited significantly the radial growth of three phytopathogenic fungi. Isoalloalantolactone also caused the inhibition of ATP synthesis, proton uptake and electron transport (basal, phosphorylating and uncoupled) from water to methylviologen, therefore acting as a Hill's reaction inhibitor. The lactone did not affect photosystem I but inhibited photosystem II. The site of inhibition of isoalloalantolactone is located in the span of P680 to QA redox enzymes because the uncoupled electron transport from water to silicomolybdate and, from DPC to DCIP are inhibited approximately to the same extent. PMID- 7546557 TI - Purification and properties of four monocot lectins from the family Araceae. AB - Four new monocot lectins from the tubers of araceous plants, namely, Arisaema consanguineum Schott (ACA), A. curvatum Kunth (ACmA) and Sauromatum guttatum Schott (SGA) from the tribe Areae, and Gonatanthus pumilus D. Don (GPA) from the tribe Colocasieae have been purified by affinity chromatography on asialofetuin linked amino activated silica beads. These lectins possess similar physicochemical and biological properties. All the lectins gave a single peak on HPLC size exclusion and cation exchange columns, and a single band on PAGE, (pH 4.5). In SDS-PAGE, all the lectins gave a single band corresponding to a subunit of M(r) 1,3000. All the lectins yielded multiple peaks on anion-exchange column, multiple bands on non-denatured PAGE (pH 8.3) and a family of bands on isoelectric focusing. The lectins agglutinate rabbit, rat and sheep red blood cells (RBCs) but are inactive towards human ABO erythrocytes. The haemagglutination activity of these lectins is inhibited by asialofetuin only, while simple sugars/derivatives including chitin, porcine mucin and fetuin did not react. In serological studies against rabbit anti-SGA serum, all four lectins produced immunoprecipitin lines. The lectins within each tribe were identical but the lectins belonging to the tribe Areae were only partially identical to the lectins from the tribe Colocasieae. PMID- 7546558 TI - New bioactive adjacent bis-THF annonaceous acetogenins from Annona bullata. AB - Five new adjacent bis-THF annonaceous acetogenins, 32-hydroxybullatacin, 31 hydroxybullatacin, 30-hydroxybullatacin, and (2,4-cis and trans)-28 hydroxybullatacinones, were isolated from the ethanolic extract of the bark of Annona bullata Rich. (Annonaceae). The absolute configurations of the above five compounds, as well as those of (2,4-cis and trans)-32-, 31-, and 30 hydroxybullatacinones and (2,4-cis and trans)-bulladecinones, previously isolated from the same extract, were defined by the application of the advanced Mosher ester [methoxy(trifluoromethyl)phenyl acetate or MTPA] methodology. The determination of the absolute configuration of C-20 of (2,4-cis and trans) bulladecinones to be S supports our hypothesis that the cyclization of the THF rings of (2,4-cis and trans)-bulladecinones starts from C-12 (the right side). The first five compounds listed above showed potent bioactivities in the brine shrimp lethality test (BST) and among six human solid tumour cell lines. PMID- 7546559 TI - Highly methylated agars with a high gel-melting point from the red seaweed, Gracilaria eucheumoides. AB - Highly methylated agars were isolated from the red seaweed, Gracilaria eucheumoides, harvested in Japan. One of the obtained agars formed a thermo reversible gel with a high melting point up to 121 degrees and was indicated to consist of a regularly repeating structure,-->3 6-O-methyl-beta-D-Gal(1-->4) 3,6 anhydro-2-O-methyl-alpha-L-Gal 1-->. PMID- 7546560 TI - Acylated triterpene saponins from Silene jenisseensis. AB - From the roots of Silene jenisseensis a new trans-p-methoxycinnamoyl triterpene saponin has been isolated along with its cis-p-methoxycinnamoyl isomer as an inseparable mixture. Their structures were established by chemical means and spectroscopic methods including 1D- and 2D-homonuclear and heteronuclear correlation NMR spectroscopy as 3-O-[beta-D-galactopyranosyl-(1 --> 2)-beta-D glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl-(1 --> 2)-alpha-L-rhamnopyranosyl (1 --> 2)-beta-D-4-O-trans-p-methoxycinnamoyl-fucopyranosyl] quillaic acid and its cis-isomer, respectively. They did not show any activity in the in vitro chemoluminescence granulocytes assay, but exhibited only a weak inhibitory effect in the cyclooxygenase inhibition assay. PMID- 7546561 TI - Steroidal glycosides from Allium macleanii and A. senescens, and their inhibitory activity on tumour promoter-induced phospholipid metabolism of HeLa cells. AB - A new polyhydroxylated cholestane trisdesmoside and a new spirostanol pentasaccharide, together with five known spirostanol saponins, were isolated from the bulbs of Allium macleanii, and two known spirostanol saponins were isolated from the bulbs of A. senescens. The identification and structural assignments of the steroidal glycosides were performed by spectroscopic analysis and hydrolysis. Furthermore, the isolated compounds were evaluated for inhibitory activity on 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32P incorporation into phospholipids of HeLa cells, which is recognized as an excellent primary screening test to identify new antitumour-promoter compounds. PMID- 7546562 TI - Flavonol sinapoyl glycosides from leaves of Thevetia peruviana. AB - From the leaves of Thevetia peruviana four new flavonol glycosides, kaempferol 3 glucosyl(1 --> 4) [6"'-sinapoylglucosyl] (1 --> 2) galactoside and 3-[2"" sinapoylglucosyl](1 --> 4)[6"'-sinapoylglucosyl](1 --> 2)galactoside and kaempferol and quercetin 3-[6"'-sinapoylglucosyl](1 --> 2)galactoside were isolated, together with the known compounds, kaempferol and quercetin 3 glucosyl(1 --> 2)galactoside. The glycosides were characterized from FAB mass, 1H and 13C NMR and UV spectral data. PMID- 7546564 TI - Accessing new resources for drug discovery. PMID- 7546563 TI - Molecular diagnostics for infectious diseases: new approaches and applications. PMID- 7546565 TI - Engineering gut flora of ruminant livestock to reduce forage toxicity: progress and problems. AB - The rumen bacterium Butyrivibrio fibrosolvens has been genetically modified to detoxify fluoroacetate (a poisonous component of trees and shrubs in Australia, Africa and Central America) and has been shown to persist when it is returned to the rumen. Such bacteria may save animals from poisoning and, therefore, reduce economic losses for livestock industries in those countries. The ability to make genetic changes to rumen bacteria raises important questions about their practicality, and about the environmental factors that must be considered before releasing modified strains. The fluoroacetate-detoxifying bacterium provides an important model by which these issues can be examined. PMID- 7546566 TI - Combinatorial peptide libraries in drug design: lessons from venomous cone snails. AB - Many present-day drugs are derived from compounds that are natural products, a traditional source of which is fermentation broths of microorganisms. The venoms of cone snails are a new natural resource of peptides that may have a pharmaceutical potential equivalent to those from traditional sources, particularly for developing drugs that target cell-surface receptors or ion channels. In effect, cone snails have used a combinatorial library strategy to evolve their small, highly bioactive venom peptides. The methods by which the snails have generated thousands of peptides with remarkable specificity and high affinity for their targets may provide important lessons in designing combinatorial libraries for drug development. PMID- 7546567 TI - Constrained peptides as binding entities. AB - Displaying proteins and peptides on genetic packages and selecting packages that display high-affinity binders allows large numbers of peptidyl compounds to be tested for binding to targets. Phage-displayed libraries of unstructured peptides (UPs) have yielded binders for some targets, but not for many others. Therefore, more attention is being paid to peptidyl compounds that have varied regions that are subject to conformational constraint. PMID- 7546568 TI - Progress in anti-HIV structure-based drug design. AB - The course of drug development for the treatment of HIV-1 infection and AIDS is being revolutionized by high-resolution structures of essential viral proteins. We survey the impact on drug design of the recently elucidated structural knowledge of two essential enzymes, reverse transcriptase and protease, and three new targets, the viral integrase and the gene regulatory protein-RNA interactions, Tat-TAR and Rev-RRE. PMID- 7546569 TI - Recent developments in retro peptides and proteins--an ongoing topochemical exploration. AB - Main-chain peptidomimetics based on peptide-bond reversal and inversion of chirality represent important structural alterations for peptides and proteins, and are highly significant for biotechnology; these modifications have been widely applied: the D-HIV-protease dimer cleaves only all-D substrate; an all-D hexapeptide opioid is able to produce analgesia following intraperitoneal administration. Antigenicity and immunogenicity can be achieved by metabolically stable antigens such as all-D- and retro-inverso-isomers of natural antigenic peptides. Isomers, including the retro- and retro-inverso- forms, of hybrid peptides derived from cercropin A and melittin, maintain antimicrobial activity. Therefore, an insight is provided into structure-activity relationships and the rational design of biologically important isomeric peptides. PMID- 7546570 TI - Transgenic plants as vaccine production systems. AB - Transgenic plants that express foreign proteins with industrial or pharmaceutical value represent an economical alternative to fermentation-based production systems. Specific vaccines have been produced in plants as a result of the transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing a bacterial antigen stimulated humoral and mucosal immune responses when they were provided as food. These results provide 'proof of concept' for the use of plants as a vehicle to produce vaccines. PMID- 7546571 TI - Biomass degrading enzymes from anaerobic rumen fungi. AB - Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous mammals. They produce a complete set of polysaccharide hydrolytic enzymes which efficiently degrade plant cell-walls. This article summarizes the present work on biomass degrading enzymes with special emphasis on their cellulases, xylanases, and esterases from both monocentric and polycentric anaerobic fungi. The possibility of the cellulosome or xylanosome-like high molecular mass complexes in the fungi are also discussed. PMID- 7546572 TI - Analysis of a mycotoxin gene cluster in Aspergillus nidulans. AB - Aspergillus nidulans has functioned as a model system for the study of fungal genetics since the 1950s. Application of methodologies ranging from Mendelian genetics to the most sophisticated molecular biological techniques have resulted in a detailed understanding of genes and pathways involved in primary metabolism, secondary metabolism and development in A. nidulans. We have taken advantage of this background in developing A. nidulans as a genetic system to study the molecular mechanisms regulating aflatoxin biosynthesis. Aflatoxin, a carcinogenic polyketide, is the product of a lengthy biochemical pathway found in the asexual spp., A. flavus and A. parasiticus. A. nidulans possesses most if not all of this pathway and produces sterigmatocystin, the penultimate precursor of the aflatoxin pathway. We have identified a approximately 60 kb cluster of genes in A. nidulans whose products are involved in sterigmatocystin biosynthesis. This cluster contains at least 20 genes proposed to encode both enzymatic activities and regulatory proteins. Our results have shown that at least some of these genes are functionally conserved between A. nidulans, A. flavus and A. parasiticus, and that they are regulated in similar ways. Further studies of sterigmatocystin regulation in A. nidulans should yield information transferable to studies of (i) secondary metabolism in other filamentous fungi and (ii) aflatoxin regulation in A. flavus and A. parasiticus in particular. PMID- 7546574 TI - Reduction columelloplasty. A new method in the management of the nasal base. AB - The nasal base is very complex secondary to the dynamic relationship between the skin, underlying muscle, and cartilaginous structures. Recently, we have employed a new method for refinement of the nasal base, which we name reduction columelloplasty. This procedure employs a full-thickness diamond-shaped tissue excision between the feet of the medial crura. The incision is carried down through the skin, depressor septi muscle, and adjacent soft tissue. Following en bloc removal, the defect is carefully closed in a vertical orientation, approximating the medial crura of the lower lateral cartilages. As in the open rhinoplasty approach, the scar produced by this technique is of negligible importance if it is closed using proper techniques of skin eversion and early suture removal. We have found this procedure to be an extremely reliable method for narrowing splayed medial crura, increasing the nasolabial angle, modifying the shape of the nares, and for increasing tip projection. PMID- 7546573 TI - Telluromethionine in structural biochemistry. AB - One of the fundamental problems in macromolecular crystallography is the availability of the suitable heavy-atom derivatives necessary to solve the phase problem. The ability to label a protein with a tellurium-containing amino acid (telluromethionine) at internal sites through the utilization of protein biosynthesis supplies x-ray crystallographers a convenient phasing vehicle and nuclear magnetic resonance (NMR) spectroscopists an internal probe with which to study structure/function relationships via Te-125 NMR spectroscopy. In this communication we demonstrate the partial incorporation of telluromethionine into E. coli dihydrofolate reductase (DHFR) with no apparent perturbations to activity or substrate binding. Enzyme containing two moles TeMet exhibited a specific activity of 42 units/mg and a 1:1 binding ratio with methotrexate. PMID- 7546575 TI - Reconstruction of caudal end of septum. A case for transplantation. AB - OBJECTIVE: To examine the results of complete removal and reconstruction of the caudal end of the septum with emphasis on long term success. DESIGN: A case series reviewing the surgical outcome of 45 consecutive cases of transplantations for severely deformed of absent caudal ends of the septum. After at least 18 months, all 45 patients were given a postoperative evaluation. SETTING: All procedures were performed at Mayo Clinic, Rochester, Minn. PATIENTS: Forty-five patients (36 male and nine females) ranged in age from 7 to 78 years, with a mean age of 38 years. Twenty-seven patients had severe caudal end deformities, and 18 patients had absent or flaccid caudal end. OUTCOME MEASURES: Subjective assessment included the opinion of the patient, rhinologic examination, and photographic analysis. Anterior mask rhinomanometry was performed to measure nasal resistance. Clinically noted complications were documented when present. RESULTS: Subjectively, 36 (80%) of the 45 patients reported "normal" nasal breathing, and seven patients (15.6%) reported improved nasal breathing. Objective preoperative and postoperative rhinomanometry data, available in 28 cases, was related to the subjective findings. The longevity of the caudal end transplant was confirmed by reviewing the data from the 33 patients who were followed up more than 1 year. Ninety percent of the patients were free of complications. CONCLUSION: Objective and subjective results showed excellent long term results. The caudal end of the nasal septum can be removed safely and reconstructed. PMID- 7546576 TI - Modifications of the Furlow palatoplasty (six- and seven-flap palatoplasties). AB - OBJECTIVE: To assess and compare the Furlow palatoplasty, a modified Furlow palatoplasty, and traditional palatoplasty methods. DESIGN: A retrospective analysis of the results. MAIN OUTCOME MEASURES: Cases were evaluated for operating time and complications, including oronasal fistula and blood loss. RESULTS: There were no fistulas in any of the 19 patients who were included in our study. The modified Furlow palatoplasty was found to increase operating time and blood loss. CONCLUSION: The use of the modified Furlow palatoplasty is feasible in select cases; however, this technique increases operating time and blood loss, which must be balanced against the theoretical advantages of increased palatal function with the Furlow palatoplasty. PMID- 7546577 TI - Esthetic refinements in forehead flap nasal reconstruction. AB - OBJECTIVE: To identify refinements in forehead flap nasal reconstruction that consistently provide better esthetic and functional results. DESIGN: Case series of patients undergoing forehead flap nasal reconstruction from July 1, 1987, to May 31, 1994. SETTING: University hospital ambulatory surgery department. PATIENTS: Thirty-two patients with various nasal defects. INTERVENTIONS: Modifications of currently accepted techniques of paramedian forehead flap nasal reconstruction, namely, flap harvest and contouring, W-plasty closure of the superior forehead donor site, and creation of soft-tissue triangles. The principles of open-structure rhinoplasty are incorporated into cartilaginous reconstruction of the nasal tip and columella. The alar rim is reconstructed with cartilage grafts placed at the nasal rim. MAIN OUTCOME MEASURES: Esthetic and functional results of nasal reconstruction were subjectively graded by three otolaryngologists (V.C.Q., D.A.S., and M.F.R.) and the patients. RESULTS: Improved esthetic and functional nasal reconstruction. The most common nasal subunits reconstructed were as follows: ala, 27 patients (84%); side-wall, 22 patients (69%); dorsum, 18 patients (56%), and tip, 15 patients (47%). The esthetic results ranged from average to excellent (3 to 5 on a scale of 5), the functional results ranged from improved to much improved over preoperative breathing (4 to 5). Two patients required unplanned surgical revisions. Forty seven percent of patients chose to undergo dermabrasion. Five patients required postoperative intradermal injection of triamcinolone acetonide (Kenalog). Three patients required preoperative tissue expansion. Flap or graft loss, infection, or hematoma did not occur. CONCLUSIONS: The predictability of the techniques in providing excellent results in patients undergoing nasal reconstruction decreases the need for revision procedures and helps the patient and the surgeon achieve the desired outcome. PMID- 7546579 TI - The epithelial 'turn-in' flap in nasal reconstruction. AB - OBJECTIVES: To evaluate the epithelial "turn-in" flap for reconstruction of the internal lining in full-thickness nasal defects. Highlighted are its indications, technique, complications, and clinical success, particularly in comparison with the other common modalities for reconstructing this nasal lining. Also, to determine how the random flap withstands vigorous thinning. DESIGN: A retrospective chart review and clinical follow-up of all patients with full thickness nasal defects of the nose. SETTING: The patient pool comes from a tertiary care center that draws from a large geographic area in the Northwest. Both urban and rural demographics are represented. PATIENTS: Fifty-six patients with large full-thickness nasal defects were reviewed, 18 of which were reconstructed with the epithelial turn-in flap. The causes of the original defect were varied. INTERVENTION: Full-thickness nasal defects were reconstructed in a layered fashion. The turn-in flaps were aggressively thinned to the subdermal plane to minimize flap bulk and airway compromise. MAIN OUTCOME MEASURES: Attention was made to the size and precise location of the defect, the mode of reconstruction, flap viability, and nasal function. RESULTS: Eighteen patients had turn-in flaps for reconstruction of the internal lining. Sixteen patients (89%) had 100% viability of their flaps. Seventeen (94%) reported normal nasal function. CONCLUSIONS: The epithelial turn-in flap is a robust flap that provides dependable and functional results. One of the major advantages of this flap over other methods is that often there is no donor site morbidity. For many select defects, the turn-in flap remains a method of choice for repair of the internal lining. PMID- 7546578 TI - The soft-tissue shaving procedure for removal of adipose tissue. A new, less traumatic approach than liposuction. AB - OBJECTIVE: To introduce the soft-tissue shaving cannula, a new, precise alternative to conventional liposuction that embodies an entirely different method of soft-tissue extraction and that appears to be less traumatic and more precise than methods currently used in liposuction. DESIGN: Nonrandomized, nonblinded comparison of the soft-tissue shaver and conventional liposuction devices in fresh cadavers, followed by clinical use of the liposhaver in selected patients undergoing cosmetic facial liposuction. INTERVENTIONS: Conventional liposuction devices and the soft-tissue shaving cannulas in fresh cadavers (< 8 hours old). A submental lipectomy and a melolabial fold liposhaving were performed in a clinical setting. OUTCOME MEASURES: Subjective evaluation by the operating surgeons. RESULTS: The fat was cleanly shaved and the contour result was even, without dimpling or asymmetry. CONCLUSIONS: Our early experiences suggest that this new liposhaving technique may offer a precise, less traumatic alternative to conventional liposuction. Fat can be shaved in an open fashion under direct vision. It does not rely on a vacuum seal. The soft-tissue shaving cannula shaves fat one layer at a time. The depth of each layer removed, the depth of soft-tissue injury, and the optimal settings and sizes for various procedures remain to be reported. PMID- 7546580 TI - The use of expanded polytetrafluoroethylene (Gore-Tex) in rhinoplasty. A 6-year experience. AB - OBJECTIVE: To determine the safety and efficacy of expanded polytetrafluoroethylene (Gore-Tex soft-tissue patch, W. L. Gore & Assoc Inc, Flagstaff, Ariz) as an implant in rhinoplasty. DESIGN: A retrospective study of 137 patients who underwent rhinoplasty including augmentation with Gore-Tex over a 6-year period. A review of the medical literature concerning the use of Gore Tex as an implant in the head and neck was also conducted. SETTING: Two major academic medical centers and two private office surgical centers. PARTICIPANTS: One hundred thirty-seven consecutive patients who received Gore-Tex implants in the course of rhinoplasty. INTERVENTION: Sixty-nine patients presented for primary rhinoplasty; the remaining 68 presented for revision surgery. All received Gore-Tex nasal implants to augment the nasal dorsum and/or base. The grafts ranged from 1 to 6 mm in thickness. Follow-up ranged from 6 to 80 months, with an average of 25 months. OUTCOME MEASURES: Clinically noted complications and patient satisfaction. RESULTS: Three (2.2%) of 137 grafts became infected and were removed. One graft was removed 5 months post-operatively because of excessive augmentation. None of the patients who underwent implant removal required subsequent augmentation. All 137 patients are pleased with their results. CONCLUSION: Gore-Tex is a safe and effective implant material to use in primary and revision rhinoplasty when augmentation is needed and autogenous material is not available or desirable. PMID- 7546581 TI - Composite graft survival. An auricular amputation model. AB - OBJECTIVE: To study the effects of corticosteroids and fibroblast growth factor on composite graft survival using a rabbit model of auricular amputation and reimplantation. DESIGN: Randomized, "blinded," placebo-controlled, prospective animal study. SETTING: Animal laboratory in tertiary care center. INTERVENTION: Amputation of the distal 2 cm of the rabbit ear as a composite graft and reimplantation with simple 6-0 prolene sutures. All animals underwent the same surgical procedure and were randomized into the following four groups: (1) surgical reimplantation alone; (2) 30 mg/kg intramuscular methylprednisolone sodium succinate for 5 days, starting immediately postoperatively; (3) topical basic fibroblast growth factor for 5 days postoperatively; and (4) delayed reimplantation with corticosteroids. In group 4, the ears of the animal were amputated, placed in iced saline containers for 90 minutes, and given 30 mg/kg intramuscular methylprednisolone for 5 days, with the first dose starting immediately prior to reimplantation. MAIN OUTCOME MEASURES: Percentage graft survival and histologic characteristics of viable and nonviable composite graft tissue. RESULTS: The groups that received corticosteroids and delayed reimplantation with corticosteroids had a statistically significant increase in percentage of graft survival compared with the control group (P < .003 and P < .006, respectively). The growth factor group showed no significant difference from the control group. CONCLUSION: Neovascularization occurred in the viable grafts, thus suggesting its role in graft survival. This study establishes the efficacy of corticosteroids in enhancing composite graft survival. PMID- 7546582 TI - Split-thickness skin graft donor site management. A randomized prospective trial comparing a hydrophilic polyurethane absorbent foam dressing with a petrolatum gauze dressing. AB - OBJECTIVE: Traditionally, skin graft donor sites have been covered with fine-mesh gauze dressings, and a dry eschar has been allowed to form. Newer dressings that can provide a moist wound environment may facilitate reepithelialization. We compared a hydrophilic semipermeable absorbent polyurethane foam dressing that provides a moist wound environment with a petrolatum gauze dressing for donor sites. DESIGN: Prospective randomized trial; follow-up at 14 days. SETTING: Department of head and neck surgery in a tertiary care center. PATIENTS: Sixty eight eligible patients received one of the two dressings. Harvested skin grafts were 0.375-mm (0.015-in) thick; donor site surface areas were recorded. At postoperative day 14, the dressings were removed, and wound epithelialization was scored: 1, none; 2, scattered or spotty; and 3, complete. Donor site and operative site pain intensities were assessed by a visual numeric scale: none (0) to the worst (100) experienced over the preceding 24-hour period. Pain scores were available for 58 patients. MAIN OUTCOME MEASURES: Dressings were compared based on these criteria: healing at 14 days, infection, and donor site and operative site pain. RESULTS: A healing score of 3 was seen in 37% (14/38) of patients with hydrophilic semipermeable absorbent polyurethane foam dressings and in 17% (5/30) of patients with petrolatum gauze dressings (P = .06) by day 14. Overall, however, mean healing scores were similar in both groups. Mean healing scores for the patients who received a hydrophilic semipermeable absorbent polyurethane foam dressing was 2.3 (SD = 0.6) vs 2.2 (SD = 0.6) for patients who received the petrolatum gauze dressing (P = .20). Numbers of days required for complete epithelialization in these groups were 20.6 (SD = 10.1) and 19.3 (SD = 5.1), respectively (P = .49). One infection occurred in the group who received the petrolatum gauze dressing. The mean maximum pain intensity scores were lower for those who were given the hydrophilic semipermeable absorbent polyurethane foam dressing on postoperative days 1 through 3 (P = .003, .03, and .04, respectively). Pain increased with a larger donor site surface area for the patients with the petrolatum gauze dressing but not for the patients with the hydrophilic semipermeable absorbent polyurethane foam dressing. CONCLUSIONS: The hydrophilic semipermeable absorbent polyurethane foam dressing appears to have potential advantages over the petrolatum gauze dressing; it produces less initial patient donor site discomfort and tends to produce more complete donor site healing by postoperative day 14. PMID- 7546583 TI - An anatomic study of the temporoparietal fascial flap. AB - OBJECTIVE: To clarify the neurovascular relationships in the temporoparietal fascial flap and to access its possible use as a sensate free of a pedicled flap. DESIGN: Anatomic dissections (gross) and examinations (histologic) were performed on 10 fresh cadaver heads. SETTING: Academic tertiary care facility, Boston, Mass. MAIN OUTCOME MEASURES: To determine the relationship of the sensory innervation (the auriculotemporal nerve) to the vascular supply (the superficial temporal artery) of the temporoparietal fascial flap, in addition to determining safe incisions and the level of flap elevation that will preserve the sensory supply to the flap. RESULTS: There is a consistent relationship of the auriculotemporal nerve to the superficial temporal artery allowing for auriculotemporal nerve preservation with standard flap elevation techniques and easy nerve identification in this cadaveric study. CONCLUSION: A clear understanding of the anatomic pattern allows for the potential creation of a sensate fascial flap or vascularized nerve graft that would add a potential additional dimension to this fascial flap. PMID- 7546584 TI - Use of the tunable dye laser to delay McFarlane skin flaps. AB - OBJECTIVE: To determine whether the argon tunable dye laser, used in a noncutting, hemocoagulative mode, improves skin flap survival as effectively as a surgical delay procedure. DESIGN: A physiologic, controlled trial was completed with three groups: acute control skin flaps, surgical delay skin flaps, and experimental laser delay skin flaps. The outcome variable was percentage of skin flap survival. SUBJECTS: Forty-four male Sprague-Dawley rats. INTERVENTIONS: McFarlane skin flaps were raised in all groups. The first group had no delay procedure, the second had the standard surgical delay, and the third group was lased at the periphery of the flap 2 weeks before raising the acute flap. RESULTS: The acute control group had survival of 85.7% +/- 1.5% (mean +/- SE) of skin flaps; surgical control group, 94.4% +/- 0.7%; laser delay group, 96.5% +/- 0.1%. The survival of the experimental group was better than the acute control group and was not different from the surgical delay group by the Scheffe multiple comparison test. CONCLUSIONS: This research may lead to a technique to reproduce the benefits of the delay phenomenon without the risks and cost of a surgical procedure. PMID- 7546585 TI - The holmium:YAG laser-assisted otolaryngologic procedures. AB - OBJECTIVE: To determine the effectiveness of the holmium: YAG (Ho:YAG) laser in otolaryngologic procedures that necessitate the ablation of osseous and soft tissue. DESIGN: Case series. SETTING: Lahey Clinic, Burlington, Mass. PATIENTS: Consecutive series of 37 patients; 29 with chronic sinusitis, five with chronic dacryocystitis, one with recurrent choanal stenosis, one with tracheopathia osteoplastica, and one with a sphenoid sinus mucocele. INTERVENTION: The Ho:YAG laser was used to assist in 37 procedures, including endoscopic sinus surgery, dacryocystorhinostomy, treatment of choanal stenosis, ablation of obstructive tracheopathia osteoplastica, and removal of a sphenoid sinus mucocele. MAIN OUTCOME MEASURES: Postsurgical success and complications, satisfaction of the patients, and the ability of the laser to remove tissue. RESULTS: Complications occurred in eight patients: intranasal or ethmoid scarring (four), persistent polyps (one), bleeding (one), stent dislodgment (one), and tracheitis (one). Three patients required revision surgery. None of the complications were related to use of the laser, although the laser may produce increased scarring. The laser was effective for osseous and soft-tissue ablation, but its usefulness was limited for hemostasis. CONCLUSIONS: The Ho:YAG laser can be used in otolaryngologic procedures when surgical access is difficult or when controlled, precise ablation of osseous tissue is necessary. PMID- 7546586 TI - Serum antibodies to heat shock protein 70 in sensorineural hearing loss. AB - OBJECTIVE: To identify the 68-kd target of antibody in serum samples from patients with idiopathic, progressive, bilateral sensorineural hearing loss. DESIGN: To purify target protein from renal extracts using gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis and to transfer to nitrocellulose membranes. The purified protein was digested with trypsin, and peptide fragments were separated by high-pressure liquid chromatography. RESULTS: One fraction obtained by high-performance liquid chromatography contained a peptide of 2776 molecular weight. The sequence of a stretch of 22 amino acids within this peptide was identical to that of amino acids 424 through 445 of heat shock protein 70 (HSP70). On Western blotting, monoclonal antibody directed against HSP70 (but capable of recognizing both constitutive HSP70 [HSC70] and stress-inducible HSP70) reacted with the purified 68-kd protein. We compared the reactivity of serum samples from six patients with idiopathic, progressive, bilateral sensorineural hearing loss, as well as monoclonal antibody to HSC70, and monoclonal antibody to HSP70 with renal extract. The pattern obtained suggested that patient antibodies are preferentially directed at HSP70. CONCLUSION: The target of antibody in serum samples from patients with idiopathic, progressive, bilateral sensorineural hearing loss is HSP70. PMID- 7546588 TI - Long-term follow-up of olfactory loss secondary to head trauma and upper respiratory tract infection. AB - OBJECTIVE: To determine the extent to which olfactory function can improve after loss induced by head trauma or a previous upper respiratory tract infection (URI) and the time for this improvement for more effective patient counseling. DESIGN: Patients initially evaluated at the University of Cincinnati (Ohio) Taste and Smell Center were reevaluated for olfactory loss with the University of Pennsylvania (Philadelphia) Smell Identification Test 1 to 5 years after initial testing. Changes in score on this test were used to indicate improvement in sensory function. Subjective information on olfactory ability and olfactory symptoms was also collected. SETTING: University-based tertiary care center. PATIENTS AND OTHER PARTICIPANTS: Forty-one patients with olfactory loss induced by head trauma (20) or previous URI (21). RESULTS: Seven (35%) of 20 patients with head trauma improved on the smell test by 4 points or more. Fourteen of 21 (67%) patients with a previous URI had improved scores of this magnitude or more. A statistically significant correlation was noted between the amounts of improvement and length of follow-up for URI patients. Thirteen of these patients also reported improved olfactory function. CONCLUSION: These findings for patients with head trauma are consistent with other reports of recovery of (or improvement in) olfactory function after trauma-induced loss. For patients with previous URI, these data indicate that improvement in olfactory function occurs, but the improvement may take several years. PMID- 7546587 TI - Postoperative medical management in single-stage laryngotracheoplasty. AB - OBJECTIVE: To determine whether it is safe and effective to avoid the use of neuromuscular relaxants in patients who have indwelling nasotracheal tubes after undergoing single-stage laryngotracheoplasty. DESIGN: Retrospective case series. SETTING: University-based referral center specializing in the surgical management of laryngotracheal stenosis and other pathologic conditions of the airway. PATIENTS: Referred sample of 104 consecutive patients (67 males, 37 females) undergoing single-stage laryngotracheoplasty for subglottic stenosis. INTERVENTION: Single-stage laryngotracheoplasty. MAIN OUTCOME MEASURES: Incidence of accidental extubation, use of neuromuscular relaxants, incidence of reintubation after planned extubation, duration of intubation, overall rate of successful airway expansion. RESULTS: One patient self-extubated without sequelae. One patient required a brief course of neuromuscular relaxants. The success rate (without further laryngotracheoplasty) for all patients was 86% (89/104). For the 25 patients operated on in 1992, with at least 1 year of follow up, the success rate was 92% (23/25). CONCLUSION: Single-stage laryngotracheoplasty can be done safely and effectively without using paralyzing agents in the postoperative period. This approach has certain advantages, which are discussed. PMID- 7546589 TI - Invasive Aspergillus sinusitis in pediatric bone marrow transplant patients. Evaluation and management. AB - OBJECTIVES: To evaluate the following: the incidence of invasive Aspergillus sinusitis (AS); the value of surveillance nasal cultures and screening radiologic studies in predicting AS; the clinical criteria used to decide on surgical biopsy in patients suspected of having AS; the surgical and medical management of AS; and the outcome of AS in the peritransplantation period of children who underwent bone marrow transplantation. DESIGN: Retrospective medical chart review. SETTING: Tertiary care children's hospital. PATIENTS: Eighty pediatric patients who underwent bone marrow transplantation for a variety of refractory malignant neoplasms or lymphohematopoietic disorders at the Children's National Medical Center, Washington, DC, from April 1, 1988, to September 30, 1993. INTERVENTION: Diagnostic surgical biopsies, surgical debridement, and treatment with amphotericin B. MAIN OUTCOME MEASURE: Resolution of AS and discharge from the hospital. RESULTS: Seventy-two patients had screening sinus radiographs, 27 of which showed abnormalities. Aspergillus sinusitis developed in three of the patients with abnormal screening radiographs. Fifty-eight patients had screening nasal cultures. One culture was positive for Aspergillus, and histopathologically proved AS developed in this patient. Twelve diagnostic biopsies were done in nine patients. Three biopsy specimens showed histopathologic evidence of AS. The three patients with AS were successfully treated with aggressive surgical and medical therapy and were discharged from the hospital. CONCLUSION: The incidence of AS was 4% (3/80) in the patients who underwent bone marrow transplantation. Screening radiographs, while not a good predictor of AS, have a role in evaluation of patients undergoing bone marrow transplantation to define preexisting sinus disease. Screening nasal cultures do not reliably predict AS. When AS is suspected and diagnostic biopsy is considered, the seven clinical criteria outlined in this article should be used. Survival of immunocompromised patients with AS requires early diagnosis and aggressive surgical and medical therapy. PMID- 7546590 TI - Laryngeal cryptococcus. Treatment with oral fluconazole. AB - We treated a case of laryngeal Cryptococcus neoformans infection in a glucocorticosteroid-dependent patient with chronic obstructive pulmonary disease. To our knowledge, this is the first report of successful treatment of laryngeal cryptococcus using oral fluconazole as a single agent. PMID- 7546591 TI - Pathologic quiz case 1. Furuncular myiasis mimicking clinical mastoiditis. PMID- 7546592 TI - Pathologic quiz case 2. Sinonasal undifferentiated carcinoma (SNUC). PMID- 7546593 TI - Should we differentiate subtypes of Alzheimer disease? PMID- 7546594 TI - Preliminary findings from the predictors study: utility of clinical signs for predicting disease course. AB - The aim of the Predictors Study is to develop a model of the progression of Alzheimer disease (AD) that can predict disease course in individual patients. This article reviews our recent work investigating the utility of extrapyramidal signs, psychotic symptoms, and age at onset of AD for predicting time to reach important disease milestones and the rapidity of disease progression. These clinical features of the disease, all easily ascertainable at a patient's initial visit, are powerful predictors of disease course. Our findings have implications for patient management as well as the planning and interpretation of clinical trials. PMID- 7546595 TI - Identification of fast and slow decliners in Alzheimer disease: a different approach. AB - As the body of knowledge about Alzheimer disease (AD) expands, more evidence is appearing to suggest the existence of different subtypes. The delineation of subtypes is important for a variety of reasons, and there are probably several ways to identify subtypes. One crude but functionally promising distinction is between patients with early onset of AD and those with late onset. In theory, such a distinction appears to be a simple one, but critical methodologic issues are involved in this dichotomy, because a key aspect is estimating the rate of progression of the disease in different subtypes. This article discusses potential problems with various measures of deterioration and illustrates how the application of a novel technique--the trilinear model--can address many of these problems and provide additional valuable information. The trilinear model is especially well suited for use in pharmacologic studies because of the additional insight that it can provide. PMID- 7546597 TI - Biologic differences between early- and late-onset Alzheimer disease. AB - Large neurons shrink and synapses are lost in the neocortex as a function of normal aging, but different parts of the central nervous system vary in susceptibility to age changes. Thus, early-onset disease may appear different from late cases due to premorbid age changes rather than because of different pathogenesis. PMID- 7546596 TI - Is early-onset Alzheimer disease a distinct subgroup within the Alzheimer disease population? AB - Although patients with Alzheimer disease (AD) share major clinical and neurohistologic features regardless of age of onset, the hypothesis that early onset AD comprises a distinct subgroup remains viable. Most studies addressing this hypothesis find quantitative differences between early- and late-onset AD patients. Early-onset AD is characterized by shorter survival, more rapid cognitive deterioration, greater frequency of language disturbance, more severe and widespread neurochemical abnormalities, and a greater density of neurohistologic lesions. In addition, both the chromosome 14 genetic abnormality and chromosome 21 amyloid precursor protein mutations appear restricted to early onset familial AD. Age of onset of AD subjects may be relevant to the design of clinical trials. For example, the efficacy of a drug that slows disease progression may be more easily demonstrated in subjects with early-onset disease. PMID- 7546598 TI - A comment on age at onset as a subtype of Alzheimer disease. AB - Subtypes of Alzheimer disease (AD) can be approached from different ways. An intuitive and commonly proposed subtype relates to the age at onset of disease. Research on age at onset as a subtype has yielded conflicting results that may, in part, reflect different measurement approaches. Refinement of the measurement of change in AD should yield a clearer picture of subtypes, as would closer ties between neuropathologic and clinical work. Thus, it is suggested that future research should use measurement techniques that more closely characterize AD than do current approaches. In addition, progress would likely be through attempts to link clinical findings regarding rates of change to neuropathologic findings. PMID- 7546599 TI - Does early-onset Alzheimer disease constitute a distinct subtype? The contribution of molecular genetics. AB - Alzheimer disease (AD) is a clinical and pathologic diagnosis and refers to the findings of neurofibrillary tangles and amyloid plaques in the brain of a patient with dementia. Clinically it is recognized that there are familial and sporadic forms, with further division into those with presenile and senile onset. Clinical, neuroimaging, neuropathological, and neurochemical studies have attempted to identify differences between cases with an earlier and later onset, but have not identified a categorical biologic difference between the two groups. Recent advances in the molecular genetics of familial Alzheimer disease (FAD) and the discovery of defined genetic abnormalities have provided a robust approach to distinguishing between early- and late-onset cases within the group of autosomal dominant FAD. The precise biologic classification made possible by molecular genetic analysis of FAD provides a benchmark against which phenotypic differences can be assessed. This article argues that future studies will be able to contrast early-onset familial versus late-onset familial disease, and early-onset familial versus early-onset sporadic disease. Previous reports of phenotypic differences within AD may have been the result of including FAD within early-onset groups, though this remains to be established. PMID- 7546600 TI - Metal cation uptake by yeast: a review. AB - This review addresses metal uptake specifically by yeast. Metal uptake may be passive, active or both, depending on the viability of the biomass, and is influenced by a number of environmental and experimental factors. Uptake is typically accompanied by a degree of ion exchange and, under certain conditions, may be enhanced by the addition of an energy source. Intracellularly accumulated metal is most readily associated with the cell wall and vacuole but may also be bound by other cellular organelles and biomolecules. The intrinsic biochemical, structural and genetic properties of the yeast cell along with environmental conditions are crucial for its survival when exposed to toxic metals. Conditions of pH, temperature and the presence of additional ions, amongst others, have varying effects on the metal uptake process. We conclude that yeasts have contributed significantly to our understanding of the metal uptake process and suggest directions for future work. PMID- 7546601 TI - Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase. AB - Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids. PMID- 7546602 TI - In vitro electrochemical detection of wheat allergen using rat basophilic leukaemia (RBL-1) cells. AB - We have investigated an electrochemical method of detecting foods that cause an allergic reaction. Rat basophilic leukaemia (RBL-1) cells were sensitized with serum from a rat that was allergic to wheat. A sample containing the protein fraction of a food was added to the cells and incubated. The cells were immobilized on a membrane filter and attached to a basal-plane pyrolytic graphite electrode. When a potential was applied in the range 0-1.0 V relative to a saturated calomel electrode, an anodic peak current appeared at around 0.33 V. This peak current, attributed to serotonin, increased with time, and the maximum current (0.5 microA) was obtained 20-25 min of incubation. The response of the RBL-1 cells was specific to the protein fraction of wheat. The peak current increased linearly with increasing protein concentration in the range of 0.01-0.5 micrograms ml-1. These results suggest that the concentration of the protein bringing about the allergic reaction can be determined by cyclic voltammetry within 25 min. This method is more sensitive than the conventional skin tests. PMID- 7546603 TI - Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae. AB - The phytophathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 alpha by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 x 10(3)-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI-, HPII-). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases. PMID- 7546604 TI - Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose. AB - Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications. PMID- 7546605 TI - Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity. AB - Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N'-nitro-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-beta-alanyl-L-proline (PhAc-beta Ala-Pro) phthalyl-L-leucine (Pht-Leu) or phthalylglycyl-L-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-beta Ala-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The Km of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45 degrees C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold. PMID- 7546606 TI - Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW85. AB - Bacillus cereus strain UW85 produces an antibiotic, designated zwittermicin A, that is associated with the ability of UW85 to suppress damping-off disease of alfalfa (Medicago sativa) caused by the oomycete pathogen, Phytophthora medicaginis, in a laboratory bioassay. We have identified certain culture conditions that promote or suppress zwittermicin A accumulation by UW85. Maximum accumulation was detected in supernatants of trypticase soy broth cultures after sporulation, which is when cultures of UW85 provide the greatest suppression of damping-off on alfalfa. Inorganic amendments to trypticase soy broth cultures had the following effects on zwittermicin A accumulation and disease suppression: phosphate (50 mM or more) reduced zwittermicin A accumulation and disease suppression; ferric iron (0.25-1.0 mM) enhanced zwittermicin A accumulation and disease suppression; micronutrients (manganese, boron, copper, molybdenum, zinc) had no effect on zwittermicin A accumulation or disease suppression. Cultures of UW85 grown in chemically defined minimal medium supplemented with casein hydrolysate or grown in defined medium containing the minimal requirements for growth supplemented with five amino acids (Gln, Arg, Met, Phe, Ile) accumulated zwittermicin A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546607 TI - Regulation of scytophycin accumulation in cultures of Scytonema ocellatum. II. Nutrient requirements. AB - The effects of optimal sources and concentrations of major nutrients (supplying N, S, P, K+, Na+, Ca2+, Mg2+, and inorganic carbon) and organic buffers on growth and secondary metabolite accumulation in Scytonema ocellatum strain FF-66-3 were determined. Nitrate, phosphate, magnesium, and sulfur had no specific stimulatory or inhibitory effects on scytophycin accumulation within the range of concentrations that supported optimal growth. Calcium concentrations greater than those required for growth (0.1 mM) stimulated scytophycin accumulation. Sodium carbonate concentrations in excess of 0.25 mM strongly inhibited growth. Ammonium (2.5 mM) inhibited both growth and product formation. 3-[N Morpholino]propanesulfonic acid at 3-5 mM effectively controlled pH and facilitated both growth and product formation. PMID- 7546608 TI - The effect of pH and oxygen concentration on the formation of 3-ketodisaccharides by Agrobacterium tumefaciens. AB - The further optimization of 3-ketodisaccharide formation with sucrose, leucrose and iso maltulose was studied with special regard to pH and oxygen concentration in the reaction mixture with resting cells of Agrobacterium tumefaciens. It was found that the optimal pH values for the highest reaction rate and highest yield were different as the pH affected the stability of the 3-keto derivatives formed. A pH shift to 5.0 clearly reduced the enzymatic degradation of the 3-keto derivatives thus stabilizing them. The influence of constant oxygen concentrations on 3-ketosucrose formation was tested showing results not explicable with normal Michaelis-Menten kinetics. For each substrate a maximum of reaction rate and yield were obtained at very low oxygen concentrations. PMID- 7546609 TI - Anti-DNA.RNA antibodies: an efficient tool for non-isotopic detection of Listeria species through a liquid-phase hybridization assay. AB - This study was undertaken to evaluate the potential of a new approach using anti DNA.RNA monoclonal antibodies to detect Listeria in both pure culture and inoculated meat and meat products. A sensitive liquid-phase assay was first developed, based on the formation in solution of a hybrid between a 784-bp DNA probe, specific for the genus Listeria, and target rRNA. Monoclonal antibody and antisera raised against hybrid nucleic acids were then used in various immunoenzymatic assays to detect specific hybrids formed in solution. System 2, using a double sandwich enzyme-linked immunosorbent assay, and system 1, using a biotinylated probe, proved to be very effective. The method using biotin streptavidin complex, however, resulted in a higher background signal. System 2 described here, using unlabeled probe, was more effective. This strategy allowed the detection of as little as 2.5 pg target RNA from pure culture and 500 cells from inoculated meat homogenate, even in the presence of other contaminating bacteria. The assay was more sensitive and could be completed within 3 h, as opposed to several days when conventional culture methods were used. PMID- 7546610 TI - Biotransformation of dichloroaromatic compounds in nonadapted and adapted freshwater sediment slurries. AB - Nonadapted freshwater sediment slurries and sediment slurries adapted to dechlorinate 2,3-dichloropyridine (2,3-Cl2Pyd), 2,3-dichloroaniline (2,3-Cl2Anl), 2,3-dichlorophenol (2,3-Cl2PhOH), 3,5-dichloropyridine (3,5-Cl2Pyd), 3,5 dichloroaniline (3,5-Cl2Anl) and 3,5-dichlorophenol (3,5-Cl2PhOH) were studied to determine the rate, range and extent of biotransformation of structurally related compounds under anaerobic conditions. 2,3-dichloroanisole (2,3-Cl2Ans) and 3,5 dichloroanisole (3,5-Cl2Ans) were initially demethylated, producing 2,3-Cl2PhOH and 3,5-Cl2PhOH as intermediate transformation products. All other dichloroaromatic compounds examined were initially dechlorinated. The rates of dechlorination of 2,3-Cl2PhOH, 2,3-Cl2Anl, and 2,3-Cl2Pyd were significantly lower (5-15 times) in nonadapted sediment slurries compared to sediment slurries adapted to 2,3-Cl2Anl or 2,3-Cl2Pyd. In 2,3-Cl2PhOH adapted sediment, the rate of dechlorination of 2,3-Cl2PhOH was 15 times greater than in nonadapted sediment; however, the rates of dechlorination of 2,3-Cl2Anl and 2,3-Cl2Pyd were similar for 2,3-Cl2PhOH-adapted and nonadapted sediment slurries. In adapted and nonadapted sediment slurries, 2,3-Cl2PhOH, 2,3-Cl2Anl, and 2,3-Cl2Pyd were preferentially dechlorinated at the ortho, meta, and meta positions, respectively. Additionally, 2,3-Cl2Pyd adapted sediment slurries dechlorinated 2,3-Cl2PhOH and 2,3-Cl2Pyd at both ortho and meta positions. Rates of dechlorination of 3,5-Cl2PhOH, 3,5-Cl2Anl, and 3,5-Cl2Pyd were lower (2-4 times) in nonadapted sediment slurries compared to sediment slurries adapted to 3,5 Cl2Anl or 3,5-Cl2Pyd. In 3,5-Cl2PhOH adapted sediment, the rate of dechlorination of 3,5-Cl2PhOH was approximately 10 times greater than in nonadapted sediment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546611 TI - Effect of oxygen and storage conditions on the metabolic activities of polychlorinated biphenyls dechlorinating microbial granules. AB - The effect of oxygen and storage conditions on the metabolic activities, measured by volatile fatty acid (VFA) degradation and methane production, and by the dechlorinating activity of methanogenic polychlorinated biphenyl (PCB) granules, were studied. Incubation of the granules in air for different periods did not result in significant inhibition in volatile fatty acid degradation and methane production activities. The inhibitory effect of oxygen increased with increased length of exposure. The overall methanogenic activities, however, recovered after a 10-day incubation period in the absence of oxygen. Oxygen exposure did not cause any significant effect on the dechlorinating activity of the granules tested with a PCB mixture, Aroclor 1254. In 6 months, approximately 80% [based on the concentration (microM) of chlorine removed] of the Aroclor 1254 was dechlorinated even by granules exposed to oxygen for 168 h. Granules stored at room temperature (20 degrees C) appeared to be more active compared to the granules stored at 4 degrees C or -20 degrees C. Similarly, granules stored with a nutrient mixture, containing methanol, glucose and yeast extract showed higher metabolic activities. Our results demonstrate that the effect of oxygen exposure was not significant and was reversible. PCB granules could be stored at room temperature with an auxiliary carbon source in the presence of PCB without significant loss of activity. These properties make methanogenic PCB granules suitable candidates for practical use in PCB dechlorination and biodetoxification. PMID- 7546612 TI - Pseudomonas marginalis: its degradative capability on organic nitriles and amides. AB - Pseudomonas marginalis, capable of utilizing acetonitrile as the sole source of carbon and nitrogen, was isolated from an industrial waste site. P. marginalis metabolized acetonitrile into ammonia and acetate. The minimal inhibitory concentration values of different nitriles and amides for P. marginalis were in the range 5-300 mM. The bacterium was able to transform high-molecular-mass nitrile compounds and their respective amides into ammonia. The data from substrate-dependent kinetics showed that the Km and Vmax values of P. marginalis for acetonitrile were 33 mM and 67 nmol oxygen consumed min-1 (ml cell suspension)-1 respectively. The study with [14C]acetonitrile indicated that nearly 66% of the carbon was released as 14CO2 and 12% was associated with the biomass. The enzyme system involved in the hydrolysis of acetonitrile was shown to be intracellular and inducible. The specific activities of the enzymes nitrile aminohydrolase and amidase were determined in the cell-free extracts of P. marginalis. Both the enzymes could hydrolyze a wide range of nitriles and amides. The present study suggests that the biodegradation of organic nitriles and the bioproduction of organic acids may be achieved with the cells of P. marginalis. PMID- 7546613 TI - Enzymatic activity in the activated-sludge floc matrix. AB - The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3 month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass. PMID- 7546614 TI - Low HDL cholesterol: what are the prospects of increasing it? PMID- 7546615 TI - Helicobacter pylori, gastritis and gastric carcinoma. PMID- 7546616 TI - How many colon cancer genes? PMID- 7546617 TI - The genetic basis of paediatric heart disease. AB - This review focuses on recent advances in understanding the pathogenesis of paediatric heart disease and on the known single gene defects responsible for these diseases. Many paediatric cardiovascular diseases are heritable, have clinical manifestations in adult ages, are frequent in occurrence, and can have significant social and economic impact. Specific gene defects have been identified for hypertrophic and dilated cardiomyopathies, mitochondrial cardiomyopathies, Marfan's syndrome, Williams syndrome, familial supravalvar aortic stenosis, CATCH-22 syndrome and atrioventricular canal. Limited phenotypic response of the developing heart accounts for similar cardiovascular defects from differing gene defects. Although environmental factors affect expression of many of these genes, it is clear that single gene defects can be identified which cause paediatric cardiovascular disease. Interactions among cardiologists, cardiovascular surgeons, geneticists and basic scientists are vitally important in understanding the genetic basis of paediatric heart disease, its diagnosis and its therapy. PMID- 7546618 TI - Colorectal adenoma progression and genetic change: is there a link? AB - Focal neoplastic change occurs frequently within the colorectum. Yet, of the several hundreds of microadenomas that are likely to be present within an individual colorectum, only one or two will develop into a clinically diagnosable adenoma. In turn, only a fraction of adenomas will progress to malignancy. The risk that a particular microadenoma will end its natural history as a carcinoma varies according to clinical context. The risk is very low in familial adenomatous polyposis (FAP), but relatively high in hereditary non-polyposis colorectal cancer (HNPCC). This variation is governed by the timing and ordering of the underlying mutational events. In FAP, inactivation of the wild-type APC gene occurs early, whereas K-ras mutations are late events. The converse appears to apply in the case of sporadic adenomas. In flat adenomas, which are known to be relatively aggressive, K-ras mutations may not occur at all. In HNPCC, mutational events are accelerated as a result of defective DNA mismatch repair. The evolution of colorectal adenoma occurs through a variety of quite distinct genetic pathways. PMID- 7546619 TI - Oviductal function is critical for very early human life. AB - For normal fertilization, the ovum must be picked up from the ovarian surface or from the abdominal cavity into the ampulla. The rapid transport of gametes includes a complex reorganization of the oviductal smooth muscle electrical activity that precedes the mechanical activity. The 3-day stay at the ampulla isthmic junction requires both signals from the ovum to the oviduct and vice versa, supporting the ovum and regulating its to-and-fro movements. Oviductal fluid, a principal factor in tubal function, coats the newly fertilized egg, activates transcription and gives a signal for sperm fertility potential. Early blocks to embryo development in in vitro conditions, as compared to in vivo success, means that critical developments during the first cell cycles of embryonic life in the oviduct are actively regulated by oviductal embryotrophic factors. These have been used clinically in co-culture systems. Lytic factors are weak in human and other primates, predisposing to high incidence of tubal pregnancies, with considerable impact on medical practice. Diverse oviductal factors affect the incidence, infection being the most significant. Optimal oviductal function is necessary to provide a proper environment for early human life. PMID- 7546620 TI - Molecular and clinical aspects of inherited cardiomyopathies. AB - Hypertrophic cardiomyopathy (HCM) is phenotypically and genotypically a heterogeneous disease. Since 1989, four chromosomal loci have been identified for HCM and the genes residing on three of these have been identified as beta-myosin heavy chain (beta-MHC), cardiac troponin-T and alpha-tropomyosin. These genes code for sarcomeric proteins and exhibit the same phenotype, suggesting that HCM is a disease of the sarcomere. Over 40 missense mutations and one deletion of the beta-MHC gene have been identified. Similarly, missense mutations in the alpha tropomyosin gene and the cardiac troponin-T gene have been identified. From genetic studies, including de novo mutations, it is established that these mutations are indeed responsible for HCM. The molecular basis of the pathogenesis of the cardiac hypertrophy appears to be a compensatory response to the primary defect. In addition to providing a definitive presymptomatic diagnosis, studies correlating beta-MHC mutations with clinical prognosis suggest they have significant predictive value and can be helpful in genetic counselling and medical management. Dilated cardiomiopathies (DCM), the most common form of cardiomyopathies, have an estimated prevalence of nearly 40 per 100,000 individuals, and are the most common cause for cardiac transplantation in the United States. Familial dilated cardiomyopathy is thought to account for approximately 20% of the so-called cases of idiopathic DCM. PMID- 7546621 TI - Preface to the special section nitric oxide in medicine. PMID- 7546622 TI - Nitric oxide: biological mediator, modulator and effector. AB - Nitric oxide (NO) is synthesized from L-arginine by at least three isoforms of NO synthase enzyme (NOS). Once generated NO can interact with a number of molecular targets including haem proteins, enzymes, DNA, thiols, oxygen and superoxide. These reactions determine the profile of NO as a major biological mediator, modulator and effector molecule. PMID- 7546623 TI - Regulation and functional consequences of endothelial nitric oxide formation. AB - Since its discovery, endothelium-derived nitric oxide (NO) has become one of the most intensely investigated molecules in the field of cardiovascular physiology. Although initial investigations centred on the role of NO in mediating vasodilation and inhibition of platelet activation it has since become clear that this small, atypical signal molecule is also involved in regulating cell growth and proliferation as well as affecting the transcription of certain genes, the products of which have been implicated in the pathogenesis of such states as atherosclerosis and hypertension. Our understanding of the intracellular regulation of the NO synthases has also progressed and the constitutive endothelial enzyme is now known to be controlled by both intracellular Ca2+ and pH. In addition it would appear that this enzyme can also be upregulated in response to stimuli such as fluid shear stress and oestrogen. This review is intended to give the reader a glimpse of the multifaceted actions of endothelium derived NO. PMID- 7546624 TI - Nitric oxide in cardiovascular diseases. AB - Nitric oxide (NO), synthesized from L-arginine by a family of NO synthases (NOS), is a widespread biological mediator implicated in many physiological and pathophysiological processes, including a variety of cardiovascular diseases. Endothelium-derived NO, synthesized by a constitutive NOS, is involved in hypertension, atherosclerosis and certain heart diseases. In hypertension and atherosclerosis the role of NO is still controversial and seems to vary depending on the stage of the disease and model studied. In spontaneous hypertension, the production of NO is increased, but inefficacious, probably because of increased inactivation. In salt-induced hypertension NO production may be impaired. In atherosclerosis, an enhanced degradation of NO by superoxide radicals may explain the reduced endothelium-dependent relaxations. In pulmonary hypertension, the use of NO gas inhalation has been proposed as a future therapy for this condition. In the heart, NO regulates coronary flow and myocardial function; both functions are altered in coronary artery disease and cardiomyopathy. Nitric oxide synthesized by the inducible NOS takes part in several immunopathological diseases, such as endotoxin shock, which can particularly affect the heart. In endotoxaemia inducible NOS is overexpressed and the excess in NO production may account for the impaired cardiac performance of this condition. The overproduction of NO occurring in endotoxin shock is also responsible for the hypotension, catecholamine resistance and tissue damage characteristic of this disease. Treatment with inhibitors of NO synthesis is a promise for the future. PMID- 7546625 TI - Nitric oxide in hypertension and renal diseases. AB - Nitric oxide (NO) is intimately involved in the regulation of vascular tone, renal haemodynamics and sodium balance. The physiological actions of NO suggest important vascular and renal protective roles for NO. When produced in large amounts, however, NO may also mediate cytotoxic effects. Increasing evidence suggests that endothelial function, notably the NO pathway, may be compromised in hypertension. It is not known, however, whether changes in endothelial function are primary or secondary to the development of hypertension. In renal diseases evidence for both excessive and deficient activity of NO pathway has been found. Increased glomerular production of NO via inducible NO synthase (NOS) with potential cytotoxic consequences has been demonstrated in experimental acute glomerulonephritis. On the other hand, indirect evidence obtained by means of NOS inhibitors point out to an important renoprotective role for NO in renal diseases. NO may counteract disease progression in renal diseases by preventing glomerular microthrombi, maintaining renal perfusion and medullary oxygenation, and via its anti-inflammatory/antiproliferative effects. However, these beneficial effects of NO may be compromised (endothelial and/or tubular dysfunction) in chronic nephropathies resulting in an accelerated course of renal disease. In future, more specific inhibitors and activators of different NOS isoforms are needed to elucidate the role of NO in various renal diseases in detail, and for treatment strategies aimed at modifying the NO pathway. PMID- 7546626 TI - Nitric oxide in inflammation and immune response. AB - This short review deals with the role of a recently found signalling molecule, nitric oxide (NO), in inflammatory and immune responses. NO regulates inflammatory erythema and oedema and has cytotoxic action against micro organisms. In some instances (such as reperfusion injury) NO has cytoprotective properties. Production of large amounts of NO by activated macrophages accounts for their ability to suppress lymphocyte proliferation. NO synthesis in lymphocytes is questionable but cytokines secreted by activated lymphocytes regulate NO synthesis by macrophages. Constitutive NO synthase is activated in neutrophils in response to inflammatory stimuli and NO has diverse, often biphasic effects on neutrophil functions. Increased concentrations of nitrite and nitrate (metabolites of NO) are present in arthritic joints. NO is synthesized not only by migrated inflammatory cells but also by articular chondrocytes and inflamed synovial membrane. In the inflamed joint, NO regulates the synthesis of several inflammatory mediators and functions of inflammatory cells. In addition, NO seems to mediate some destructive effects of proinflammatory cytokines such as interleukin-1. In conclusion, NO regulates several humoral and cellular responses in inflammation, having both anti-inflammatory and proinflammatory properties depending on the type and phase of the inflammatory reaction. PMID- 7546627 TI - Nitric oxide in the central nervous system. AB - The majority of the data on nitric oxide (NO) in the central nervous system (CNS) relies on histochemical and immunohistochemical evidence concerning the distribution of the nitric oxide synthase (NOS), its inhibition by specific antagonists and its co-localization with the receptor enzyme guanylate cyclase (GC) in the same functional region. All three isoforms, endothelial (eNOS), neural (nNOS) and macrophage type inducible (iNOS), are of importance to the normal and pathological function of the CNS. In nNOS gene deleted mice eNOS seems to contribute to the maintenance of neuronal function. NO may contribute to synaptic plasticity as a retrograde mediator that is released by postsynaptic NMDA-receptor activation. Microglia contains membrane-bound inducible iNOS that may be important in host defence function. Glia and pericytes surrounding the blood vessels contain GC that is stimulated by NO released from endothelium and nerve endings. Excessive production of highly reactive NO may be responsible for the neurotoxicity mediated by NMDA receptors that contributes to the symptomatology of strokes and neurodegenerative diseases. Moreover, after initial stimulation by cytokines, large amounts of NO produced by iNOS in the microglia (brain-based macrophages) may cause cellular damage. PMID- 7546628 TI - Nitric oxide in the peripheral nervous system. AB - Nitric oxide (NO) is a neurotransmitter and neuromodulator in the central nervous system, but this small labile substance also seems to serve as a peripheral neurotransmitter. Abundant evidence is now available that NO, synthesized from L arginine by NO synthase (NOS), is a nonadrenergic noncholinergic relaxant transmitter of gastrointestinal smooth muscle. Electrically induced nonadrenergic noncholinergic relaxations are antagonized by NOS inhibitors in vitro and in vivo. In a bioassay superfusion system, the release of a substance with the pharmacological characteristics of NO from a gastrointestinal smooth muscle preparation was detected; also, indirect measurements (e.g. of the NO metabolite nitrite or of the co-product of its synthesis L-citrulline) suggest NO release. Immunohistochemistry with antibodies raised against the neuronal NOS showed immunoreactivity in cell bodies of neurones in the myenteric plexus and in nerve fibres in the muscular layer. These data suggest that nerve endings, innervating smooth muscle, are able to release NO that will penetrate the cells to induce relaxation (i.e. nitrergic neurotransmission). It is unlikely that NO as such is stored and it is generally accepted that it is synthesized on demand when the nerve endings are excited, although the possibility of the release of a NO containing molecule protecting it from degradation in the junction has been proposed. Other sources than neurones (interstitial cells, smooth muscle cells) for the NO involved in nonadrenergic noncholinergic inhibitory transmission have also been proposed. Using NADPH diaphorase as a marker for neuronal NOS, deficiency of the nitrergic innervation has been shown in isolated tissue from patients with infantile hypertrophic pyloric stenosis, achalasia and Hirschsprung's disease, suggesting that a lack of NO release might be involved in these disorders. Evidence in favour of nitrergic neurotransmission to smooth muscle has also been obtained in the respiratory and lower urinary tract, the corpora cavernosa and some blood vessels. PMID- 7546629 TI - Nitric oxide and airway disease. AB - Nitric oxide (NO) may play an important role in regulating airway function and in the pathophysiology of inflammatory airway diseases. Endothelium-derived NO may be important in regulating airway blood flow and, indirectly, plasma exudation. NO is the neurotransmitter of bronchodilator nerves in human airways and counteracts the bronchoconstriction due to cholinergic neural mechanisms. Inducible NO synthase (iNOS) is expressed in human epithelial cells in response to pro-inflammatory cytokines and oxidants, probably via activation of the transcription factor nuclear factor kappa B (NF-kappa B). There is increased expression of iNOS in the epithelium of asthmatic patients and in lung macrophages in bronchiectasis. This may account for the increased concentration of NO in the exhaled air of patients with inflammatory airways disease. Increased NO production in the airways may result in hyperaemia, plasma exudation, mucus secretion and indirectly increased proliferation of Th2 lymphocytes responsible for eosinophilic inflammation. Glucocorticoids inhibit the induction of iNOS in epithelial cells and reduce the elevated exhaled NO to normal values. Selective inhibitors of iNOS may be useful in the treatment of inflammatory airway diseases, such as asthma, in the future. PMID- 7546630 TI - Endogenous nitric oxide in cardiovascular disease and transplantation. AB - Nitric oxide (NO), derived from the vascular endothelium and other cells of the cardiovascular system, has important roles in physiological regulation of blood flow and may have pathophysiological functions in cardiovascular disease. The mechanisms involved in NO-induced vasodilatation and cytotoxicity are briefly reviewed in the context of inflammatory reactions and cardiovascular function. Although NO can hyperpolarize vascular smooth muscle, activation of the endothelium can induce hyperpolarization and vasodilatation by other means. Endogenous inhibitors of NO generated by leucocytes may compromise blood flow distribution after ischaemia and reperfusion injury. Chronic heart failure is associated simultaneously with impairment of endothelium-dependent vasodilatation and with excess production of NO via the inducible NO synthase (iNOS), although it is unclear whether the latter ameliorates or exacerbates ventricular dysfunction. Excess NO production is also one of the earliest signs of transplant rejection, and suppression of iNOS expression by immunosuppressant drugs such as cyclosporin A might be one means by which these drugs protect allografts. Disturbances in the activity of NOS isoforms in the artery wall also accompany the development of atherosclerosis, providing conditions propitious for vasospasm and thrombosis. Reversing the NO defects with therapeutic agents, including angiotensin converting enzyme (ACE) inhibitors, offers promise in protecting against some manifestations of vascular disease. PMID- 7546631 TI - Nitric oxide-based possibilities for pharmacotherapy. AB - The goal of nitric oxide (NO) based pharmacotherapy is to reach proper homeostasis of NO metabolism in the target tissue where endogenous production of NO is either too weak or excessively increased. In addition to the classic NO based therapy of cardiovascular conditions with nitrates, a variety of new therapeutic possibilities have emerged including sexual disorders, gastrointestinal system, immunology, tumour growth regulation and respiratory disorders. NO levels of target tissues can be affected directly by NO donors, or indirectly by increasing the level of L-arginine, a substrate of nitric oxide synthase (NOS). While increased production of NO by induceable NO (iNOS) by, for example, cytokines does not at present seem therapeutically meaningful, increased NO production by constitutive NOS (cNOS) may be involved in the beneficial effects of ACE inhibitors or oestrogens. NO production may be pharmacologically decreased by inhibition of expression of iNOS by glucocorticoids while both cNOS and iNOS derived NO production is inhibited by administration of false substrates, for example L-NAME. Additionally, the respiratory system and related vessels can be reached directly and more selectively by inhalation of pure NO gas. Possible problems in administering NO and perhaps some NO-donors include the toxic nature of the compound itself whereby vital enzyme systems may be inhibited and tissue damaging radicals formed. Future prospects of NO-based pharmacotherapy may feature selective ligands to different NOS isoforms and tissue selective donors that release NO in a controlled fashion. PMID- 7546632 TI - Interactions between endothelial secretogogues. AB - Among endothelial secretogogues prostacyclin (PGI2), nitric oxide (NO) and tissue plasminogen activator (t-PA) play a crucial role in maintaining thromboresistance, tone and structure of the vascular wall. Most receptor agonists, such as B2 kinin receptor agonists, or shear force produce a coupled release of all three secretogogues, and therefore interactions between them are to be expected. Essentially, PGI2 is a platelet suppressant, NO a vasodilator and t-PA a fibrinolytic agent. These and other properties of endothelial secretogogues supplement each other in protecting the cardiovascular system from injuries. It is not surprising that disturbances of the secretory function of endothelial cells are associated with atherosclerosis, diabetes, thrombosis or hypertension. Traditionally, PGI2, NO, t-PA or their substitutes are used individually for the treatment of peripheral arterial disease, angina pectoris or acute myocardial infarction. In light of recent findings, their joint administration can be advocated. For instance, NO donors will potentiate platelet suppressant action of PGI2 analogues, whereas exogenous PGI2 or TXA2 synthase inhibitors (i.e. following increase in endogenous PGI2) will abolish a paradox of prothrombotic action of t-PA or streptokinase. The replacement therapy with PGI2, NO or t-PA should match as closely as possible the physiologically coupled release of these secretogogues. PMID- 7546633 TI - Investigation of mortality and morbidity associated with severe self-injurious behavior. AB - A retrospective study was conducted to determine whether severe self-injurious behavior was associated with shortened lengths of life or greater sensory impairments for Minnesota Regional Treatment Center residents with developmental disabilities. The client records of 209 institutionalized individuals who died between January 1, 1980, and December 31, 1989, were reviewed. The data revealed that the 29 clients who exhibited severe self-injury did not live significantly shorter lives compared to matched cohorts without self-injury. However, those clients with severe self-injury were found to have a significantly higher incidence of vision and hearing impairments than did the matched cohorts. Implications of these findings were discussed. PMID- 7546634 TI - Prediction of cost, rates, and staffing by provider and client characteristics. AB - Cost of services to individuals with developmental disabilities in state institutions and private community agencies in South Dakota was related to provider agency characteristics, funding source, and client characteristics. Significant relations were found between cost and funding programs, total number of clients served by an agency, number of people in residential facilities, city population, and county unemployment rate. Funding source differences indicated that state-owned institutions were the most costly and community services funded solely by state funds were least costly. Among community-based agencies, cost bore a U-shaped relation to agency size, with intermediate-sized agencies being the least costly and large or small agencies being more costly. PMID- 7546635 TI - Replication and extended analysis of behavior state, environmental events, and related variables among individuals with profound disabilities. AB - Knowledge from previous studies pertaining to state behavior of individuals with profound and multiple disabilities and its relation to other environmental and physiological variables was replicated and extended. Behavior state and environmental data were collected over a 5-hour period for most of 66 students with profound disabilities from 21 educational settings. Results showed general consistency in state patterns and profile groupings with previous investigations, a strong relation between different state profiles and measures of development, and consistency of state patterns across CA levels. Transitional probabilities and z scores were used to confirm changes in state behavior of half of the subjects following primarily adult interactions. Results were discussed in relation to theoretical implications and intervention recommendations. PMID- 7546636 TI - Electrodermal orienting activity in children with Down syndrome. AB - The elicitation and habituation of electrodermal orienting responses to auditory stimuli of 19 children with Down syndrome and a control group, an age and sex matched sample of children without mental retardation were compared. Stimuli were ten 2-second 80 dB, 1000 Hz tones followed by a test tone (80 dB, 500 Hz). Children with Down syndrome gave electrodermal orienting responses of a lower amplitude than did the children in the control group. Both groups showed habituation of their responses. No significant differences were found between groups in either skin conductance levels or frequency of nonspecific responses. Overall, children with Down syndrome showed electrodermal hyporesponsivity in their orienting responses to the stimuli. PMID- 7546637 TI - Impact of size revisited: relation of number of residents to self-determination and deprivatization. AB - Research suggests that, contrary to common expectations, size does not have an impact on quality of care in residential facilities for people with mental retardation. In the present study conducted in Norway, the impact of number of residents on two quality of care measures--deprivatization and self-determination -was analyzed. The argument was made that the lack of positive findings in earlier research may have been due to the following reasons: (a) the living unit not the facility is the appropriate focus of analysis and (b) the effect of size is nonlinear and makes a difference only within a size range outside the range included in many earlier studies. Data from Norwegian facilities support these arguments: Living unit size had a substantial impact on self-determination and deprivatization in the 1 to 5 size range but not among larger units. PMID- 7546638 TI - Clinical and neurophysiological aspects of epilepsy in subjects with autism and mental retardation. AB - Clinical and neurophysiological findings for 28 patients with mental retardation, autism, and epilepsy were described. Correct classification of seizure type and epileptic syndrome (when possible), etiology, severity of autism and epilepsy, EEG findings, and neuroimaging findings were given. No particular epileptic syndrome was found to be more frequently correlated to autism, severity of autism was not correlated with a more pronounced tendency to develop seizures, and females with autism were more frequently affected by seizures than were males. In conclusion, the risk for epilepsy does not seem to be correlated to autism itself, but the same noxious event induces autism and epilepsy. The severity of epilepsy is strictly correlated with its etiopathogenetic mechanisms. PMID- 7546639 TI - Onset of speech-like vocalizations in infants with Down syndrome. AB - Canonical babbling of infants with and without Down syndrome was compared. Infants with Down syndrome and typically developing infants began canonical babbling in the first year of life, but the infants with Down syndrome began 2 months later. Once begun, their canonical babbling was less stable than that of typically developing infants. Age at onset of canonical babbling for the infants with Down syndrome was correlated with their scores at 27 months of age on the Early Social-Communication Scales. The results of this study suggest that Down syndrome influences vocal development in the first year of life and that early vocal development is related, possibly in combination with motoric and cognitive factors, to later social and communicative functioning of children with Down syndrome. PMID- 7546640 TI - Heightened concerns over the 1992 AAMR definition: advocacy versus precision. AB - We reiterated our earlier concerns (MacMillan et al., 1993) regarding the 1992 AAMR definition of mental retardation and replied to a defense of the definition (Reiss, 1994). Our major point here is that definitional precision should not be sacrificed to advance a particular ideological position. The result of such a compromise is a definition lacking precision and necessitating identification dependent upon assessment of behavioral dimensions for which there is currently no reliable measurement. We explored the varied purposes served by classification systems and criticized the new AAMR definition for lack of precision needed to serve research. In addition, we attempted to reduce confusion over the concepts of reliability, assessment, test bias, and use of factor analysis to establish the independence of adaptive skill domains. Some factual errors and incorrect representations in Reiss's article were pointed out. PMID- 7546641 TI - Additional support for the role of animal research in the study of human mental retardation. PMID- 7546642 TI - Some new aspects of molecular mechanisms of cyclosporin A effect on immune response. AB - A few protein targets were found to display a specific high-affinity interaction with the immunosuppressant cyclosporin A (CsA): cytosolic cyclophilins (CyP)A, B, C, D, E containing from 122 to 174 amino acid residues in a polypeptide chain, and secreted forms of CyP; CyP-40, 40-kDa CsA-binding polypeptide complexed with steroid receptor (SR); CyP-related 150-kDa receptor of natural killer (NK) cells; interleukin 8 (IL-8); actin; a family of molecular chaperones hsp70 and P glycoprotein (P-GP). All CyPs possess peptidyl-prolyl cis-trans isomerase activity (PPIase) and may serve as ATP-independent molecular chaperone proteins. The CsA-CyP complexes are specific inhibitors of Ca(2+)-and calmodulin-dependent protein phosphatase calcineurin (CaN). The inhibition of CaN blocks the activation of genes of IL-2, IL-2R, IL-4, etc. in T cells. In addition, immunosuppressive and/or antiinflammatory activity of CsA can be executed via CyP 40 and hsp 70 complexed with SR, and following the interaction with CyP-related receptor of NK and with IL-8. CsA binding to CyPC, P-GP and actin may throw light on the biochemical events leading to nephrotoxicity and graft vessel disease, two major side effects produced by CsA. The discovery of the interaction of human immunodeficiency virus type 1 (HIV-1) Gag protein with CyP and effective disruption of this interaction by CsA may be important for our understanding of the pathology caused by this immunosuppressive virus and will inspire therapeutic strategies to nip HIV in the bud. Bacterial immunophilins (ImPs) contribute to the virulence of pathogenic microorganisms. Elucidation of molecular mechanisms of microbial ImPs' action in the pathogenesis of bacterial infections may lead to new strategies for designing antibacterial drugs. PMID- 7546643 TI - Evaluation of Abbott TestPack RSV and an in-house RSV ELISA for detection of respiratory syncytial virus in respiratory tract aspirates. AB - The Abbott TestPack RSV was compared with an in-house RSV enzyme-linked immunosorbent assay (ELISA) for detection of respiratory syncytial virus (RSV) antigen. Nasopharyngeal specimens were obtained from 121 inpatients. RSV antigen was detected in 46 specimens by the Abbott TestPack, 42 of these being confirmed by the in-house RSV ELISA. Of the 75 specimens tested negative in the Abbott TestPack RSV, one was found positive by the in-house RSV ELISA. The sensitivity and specificity of the Abbott TestPack RSV versus the RSV ELISA were 98% and 95% respectively. PMID- 7546644 TI - Antimicrobial screening of zinc in the absence or presence of oleoresins and various resin acids. AB - Zinc and oleoresins are the main components of several wound dressings, and are also frequently used in root canal treatment. The in vitro antibacterial effects of zinc, six highly purified resin acids and two commercial oleoresins alone or combined in varying proportions were analysed. Oleoresins are composed of approximately 90% resin acids and the most common acids were included in this study. The antibacterial activity of the various chemicals was estimated using a Bioscreen robot analyser, which allowed 24 h kinetic documentation of bacterial growth. The bacteria employed were reference species commonly occurring on human skin or of oral origin. Zinc as well as the oleoresins and the pure resin acids all showed antibacterial activity when present in growth media, but the sensitivity of the bacteria varied. The presence of resin acids and oleoresins increased the antibacterial effect of zinc to varying degrees depending on the combination and the bacterial species tested. The results of the present study indicate that zinc, resin acids, or oleoresins alone, as well as combined, show antibacterial activity against selected aerobic Gram-positive and anaerobic Gram negative bacteria. PMID- 7546645 TI - Antibodies to Helicobacter pylori in dyspeptic patients, asymptomatic adults, and children from Costa Rica. AB - An enzyme immunoassay for the detection of serum IgG antibodies to H. pylori whole bacterial sonicate was used in a study of dyspeptic adult patients (n = 165), apparently healthy blood donors (n = 154), and children (n = 66) from Costa Rica, where a very high incidence of gastric carcinoma is observed. The mean antibody levels and frequency distributions were similar in the dyspeptic patients and the blood donors. Meanwhile lower antibody levels to H. pylori were more frequently observed in children than in adults, suggesting an age-dependent increase in seroprevalence, as described in studies from other populations. PMID- 7546646 TI - Fc gamma-receptor activity in the developing human placenta. AB - The expression of Fc gamma receptors (Fc gamma R) and annexin II in 20 placentae (range 8-27 weeks' gestation) and in 3 full-term placentae was studied using monoclonal antibodies (mAbs) and soluble immune complexes. Functionally active Fc gamma R was detected on the trophoblast, endothelial cells, and stromal cells (Hofbauer cells) from the 8th week of gestation. Fc gamma RI (mAb 32.2) and Fc gamma RIII (mAb 3G8) were detected only on Hofbauer cells, whereas Fc gamma RII (IV3 and C1KM5) were detected both on Hofbauer cells and endothelial cells. Fc gamma RIII (anti-Leu-11b) and the IgG-binding molecule annexin II (mAb B1D6) were expressed by Hofbauer cells, endothelial cells, and the trophoblast. There was some variation in staining among the different specimens, but the number of positive cells as well as the staining intensity increased from the first to the second trimester. In first trimester placenta, staining was localized both to the syncytio- and cytotrophoblast, with the strongest intensity in the cytotrophoblast and at the boundary between the two cell layers. In second and third trimester placenta, staining was localized to the syncytiotrophoblast. The localization and distribution of Fc gamma R on the trophoblast during ontogeny is of interest with regard to its presumed role in the transport of IgG from mother to fetus. PMID- 7546647 TI - Comparison of adherent and non-adherent staphylococci in the induction of polymorphonuclear leukocyte activation in vitro. AB - The ability to consume complement and activate neutrophils was investigated for staphylococci adherent to silicone surfaces and non-adherent staphylococci. Staphylococcus epidermidis strain ATCC 14990 and Staphylococcus aureus strain E 2371 were used in this study. The bacteria were allowed to adhere to silicone catheter segments for 2 h at 37 degrees C. Complement consumption was measured by reduction in serum haemolytic activity against sheep red blood cells. The induction of chemiluminescence was measured after opsonization of the staphylococci in 20% AB-positive human serum for 60 min at 37 degrees C. The bacteria consumed complement to approximately the same extent when adherent to the catheter segments, but more slowly in comparison with planktonic bacteria. When planktonic bacteria were compared, complement was consumed more quickly by S. epidermidis than by S. aureus. Measuring the induction of chemiluminescence by planktonic bacteria, S. epidermidis induced a lower response than S. aureus, while when adherent to the catheter segments the bacteria induced similar responses. These responses were only 15 to 20% of those induced by planktonic bacteria and only slightly higher than the spontaneous chemiluminescence by the neutrophils. Inter-strain variation was found, but all strains induced about the same low chemiluminescence when adherent to the catheter segments. The reduction in inflammatory response caused by adherence of staphylococci to catheter segments may interfere with phagocytosis and elimination of S. epidermidis during the early establishment of a foreign body infection. PMID- 7546648 TI - Tumor necrosis factor alpha plus interleukin 1 beta treatment protects granulocytopenic mice from Pseudomonas aeruginosa lung infection: role of an unusual inflammatory response. AB - We have recently demonstrated that treatment with interleukin 1 beta (IL-1 beta) plus tumor necrosis factor alpha (TNF alpha) protects granulocytopenic hosts from Pseudomonas aeruginosa aerosol challenge. In this study we characterized the inflammatory response induced by P. aerugionsa in granulocytopenic mice treated with 2,000 U IL-1 beta plus 2,000 U TNF alpha. Treatment with the nonsteroidal anti-inflammatory agent piroxicam abolished both the protective effect of cytokine treatment and the increase in myeloperoxidase (MPO) pulmonary activity. Histopathological studies revealed that, after aerosol challenge with P. aeruginosa, treatment with these cytokines induced migration and extravasation of mononuclear cells of immature appearance into the lung parenchyma. These cells contained MPO in their cytoplasm and displayed phagocytic capacity. Resident alveolar macrophages exhibited signs of activation and appeared in reduced numbers in bronchoalveolar lavage fluid. We suggest that the inflammatory response promoted by low TNF alpha plus IL-1 beta doses may be one mechanism responsible for protection of granulocytopenic hosts from P. aeruginosa aerosol challenge. PMID- 7546649 TI - Evaluation of a method for measurement of intracellular killing of Staphylococcus aureus in human neutrophil granulocytes. AB - A modified method for measurement of intracellular killing of Staphylococcus aureus in human neutrophil granulocytes is described. After phagocytosis of S. aureus the extracellular bacteria were sufficiently removed by repeated centrifugations and washings of the granulocytes. The optimal conditions for incubation of granulocytes for measurement of intracellular killing were found to be 37 degrees C in the presence of 5% CO2. Under these conditions, stable pH, the viability and the capacity of the granulocytes for superoxide anion generation were preserved. The number of intracellular viable bacteria was determined after lysis of the granulocytes, which should be done in H2O at pH 11 to ensure sufficient cell lysis. The kinetics and individual variation of the intracellular killing are described. The intra- or extracellular location of surviving bacteria was studied. After approximately 8 h incubation we observed intracellular growth of S. aureus followed by lysis of granulocytes and extracellular growth of bacteria. Consequently, the incubation period should not be extended beyond 5 to 8 h when the assay is used to study the effects of antibiotics on intracellular killing. PMID- 7546650 TI - The role of heparan sulphate-binding activity of Helicobacter pylori bacteria in their adhesion to murine macrophages. AB - The aim of this study was to determine the role of heparan sulphate (HS)-binding activity of Helicobacter pylori microbes in their adhesion to and ingestion by inflammatory peritoneal macrophages. Two H. pylori strains expressing sialic acid specific haemagglutinins but differing in the expression of heparan sulphate binding capacity were chosen for investigation. The attachment to an ingestion by macrophages of the H. pylori bacteria were estimated by ELISA using anti-H. pylori antibodies. The adhesion of both H. pylori strains could be inhibited by pretreatment of the bacteria with heparin (H), HS or fetuin, as well as by preincubation of the macrophages with heparinase or neuraminidase. However, detailed analysis of the data on the inhibition of bacterial adhesion to macrophages led to the conclusion that the attachment of H. pylori 25 bacteria, which expressed a high heparan sulphate binding, was mainly determined by HS binding structures. In contrast, the adhesion to macrophages of H. pylori bacteria 17874 microbes, which expressed a weak heparan sulphate binding, was more dependent on the exhibition of sialic acid-dependent haemagglutinins. The described variation in H. pylori bacterial surface structures mediating their adhesion to macrophages could suggest a similar variation in bacterial adhesion to stomach mucosa and maybe in the pathogenicity of H. pylori strains. PMID- 7546651 TI - Self-induced bacteremia. Case report. AB - We describe a patient with perforated appendicitis who postoperatively suffered repeated episodes of shaking chills and temperature spikes. Initial blood cultures yielded growth of Flavobacterium meningosepticum, Pseudomonas putida and Pseudomonas paucimobilis, and succeeding blood cultures growth of Pseudomonas acidoverans. These bacteria in combination led to a suspicion of self-inoculation of contaminated water through an intravenous catheter. Antibiotic treatment had no effect on the symptoms, which only ceased when the intravenous catheter was removed. PMID- 7546652 TI - p53 immunostaining in combined type small cell lung cancer. Brief report. AB - Abnormalities of the p53 oncogene in lung cancer have recently been reported to be more frequent in small cell lung cancer (SCLC) than in non-small cell lung cancer (non-SCLC), but their status in combined type SCLC is as yet unknown. In this study, immunohistochemical analysis using a polyclonal antibody against p53 protein was performed in 12 surgically resected specimens of combined type SCLC. Immunoreactivity of the p53 protein was found in 5 (42%) of the 12 cases, and the immunostaining pattern of the p53 protein in areas of the non-small cell carcinoma type was the same as in those of the small cell carcinoma type. Thus, it seems that the incidence of p53 abnormalities in combined type SCLC is slightly lower than in ordinary type SCLC. It is also suggested that abnormalities of the p53 oncogene in this histological type may not be a specific event related to the morphological difference between small cell carcinoma and non-small cell carcinoma. PMID- 7546653 TI - BCG as a vector for the construction of multivalent recombinant vaccines. PMID- 7546654 TI - Live Salmonella vaccines as a route towards oral immunisation. PMID- 7546655 TI - Live attenuated Shigella flexneri mutants as vaccine candidates against shigellosis and vectors for antigen delivery. AB - Up to now, no effective vaccine is available against shigellosis, a dysenteric syndrome caused by Shigella, a Gram-negative bacterium which invades the human colonic mucosa. About 40 years of research in the field have led to the conclusion that orally administered live Shigella vaccine strains are more effective in eliciting protection than killed bacteria given parenterally. Recently, the construction of promising new live attenuated vaccines has come with a better understanding of the fundamental determinants governing pathogenesis. As the development of new vaccine strategies requires knowledge of both pathogenesis and the immune response against infection, the current view of the pathogenic process of Shigella infection and the anti-Shigella immune responses elicited by the host are presented. The attempts in Shigella vaccine design are reviewed and, the future of these vaccines discussed. PMID- 7546656 TI - Listeria monocytogenes: a live vector able to deliver heterologous protein within the cytosol and to drive a CD8 dependent T cell response. AB - After an introduction on the entry and lifestyle of Listeria monocytogenes within mammalian eucaryotic cells, this chapter gives a brief overview of murine experimental listeriosis. Among the main characteristics of this murine model of infectious/pathogenic processes initiated by a facultative intracellular bacteria, we point out two main recent advances. One relates to Listeria monocytogenes-induced production of cytokines as local, and transient signals able to direct the immune responses along a type 1 pathway of CD4/CD8 T cell differentiation. The other relates (a) to the recognition of L. monocytogenes reactive CD8+ T lymphocytes as effectors able, once recruited within infected loci, to critically contribute to the complete clearance of the bacteria, and (b) to the recently recognized specificity of some of these CD8 lymphocytes in BALB/c mice. In this paper, we also briefly review (a) the readout assays presently used to monitor the outcome of the infectious/pathogenic processes and the related development and expression of immune responses induced by intravenous inoculation of wild-type virulent or attenuated L. Monocytogenes, (b) why all this information allows us to consider the use of L monocytogenes of attenuated virulence as relevant live recombinant vectors in order to deliver heterologous proteins to the class I processing and presentation pathway, and to induce CD8 T cells along the type 1 pathway. PMID- 7546657 TI - Recombinant adenoviruses as vaccines. AB - Over the past 15 years, advances in molecular biology have permitted in vitro and in vivo gene transfer into mammalian cells. Genetically engineered microorganisms are highly promising developments for gene therapy and the future of vaccines. Such vectors constitute genuine tools for high level expression of heterologous genes for both therapeutic and induced immunity applications. Several vector systems have emerged with different relative advantages and limits depending on the proposed application. Adenovirus (Ad) has gained our interest, and in this review we focus on its applicability as a vaccine vector. We describe its potentials, as well as some of the foreseen obstacles related essentially to its use in preventive medicine. PMID- 7546658 TI - Poxvirus-based vectors as vaccine candidates. AB - The safety issues surrounding the use of vaccinia-based vaccine candidates have been significantly addressed with the development of the NYVAC and ALVAC vectors. These vectors can be engineered to express multiple genes from the same pathogen or multiple genes from multiple pathogens. The use of these vectors to develop needed vaccines for human and veterinary medicine remains the focus of studies that are currently in progress. PMID- 7546659 TI - The use of picornaviruses as vectors for the engineering of live recombinant vaccines. PMID- 7546660 TI - Influenza virus vectors. AB - The establishment of reverse genetics methods to manipulate influenza virus genomes has allowed the generation of recombinant (transfectant) influenza viruses expressing foreign sequences. Strategies for the construction of influenza virus vectors include the insertion of foreign epitopes into influenza virus glycoproteins, the expression of polyproteins, and the rescue of bicistronic genes into infectious viruses. Influenza virus vectors have been obtained which express both B- and T-cell epitopes from different pathogens. These constructs have been shown to mount in immunized animals systemic and local antibody responses, and/or cytotoxic T-cell responses against the expressed epitope. The available evidence suggests that influenza virus vectors may be attractive candidates for the development of effective vaccines against different diseases. PMID- 7546661 TI - Immunogenicity of trypsin treated type 2 and type 3 poliovirus in rats. AB - Oral polio vaccine will encounter the proteolytic enzyme trypsin during administration but inactivated polio vaccine not. To investigate the effect on the humoral immune response, rats were immunized intramuscularly with trypsin treated type 2 and type 3 poliovirus. IgG and IgM responses were determined as well as the neutralizing antibody titer. It is shown that the immunogenicity of type 2 poliovirus, unlike that of type 3, is hardly affected by trypsin treatment. For type 3, trypsin treatment results in an increase in IgM and neutralizing response. The IgG response decreases after trypsin treatment. The results indicate that IPV formulations may be improved by the addition of trypsin treated type 3, as suggested by Roivainen and Hovi (J Virol 1987; 61: 3749-3753) but not by the addition of trypsin treated type 2 poliovirus. PMID- 7546662 TI - A simple serum-free freezing medium for serum-free cultured cells. AB - Because the presence of serum in cell culture raises safety problems for the production of biologicals, we have developed a serum-free medium (SFM) for the cryopreservation of animal cells. This medium is based of the SFM MDSS2, to which 10% dimethylsulfoxide (DMSO) and 0.1% methylcellulose or 3% polyvinyl pyrrolidone or no other additive than DMSO were added. Both, Vero and BHK-21 cells regularly cultivated in MDSS2, could be cryopreserved in the three serum-free freezing media and could be thawed without any cell loss. No differences could be found between the cells in the standard freezing medium (DMEM containing 10% fetal calf serum and 10% DMSO) and those frozen in SFM, with respect to cell growth and viability. In the case of the Vero cells no differences were observed with respect to their attachment. This medium represents the last step in fulfilling a complete serum-free animal cell based bioprocess. PMID- 7546663 TI - Differential susceptibility of transgenic PVR Tg-mice to poliovirus strains with varying neurovirulence in relation with monkeys. PMID- 7546664 TI - Points to consider on virus safe plasma products. PMID- 7546665 TI - Psychometrically matched tasks for assessment of hemispheric asymmetries of function. AB - One of the best methods for studying subtle lateralizing signs of brain dysfunction is the use of psychometrically matched tasks. The current study reports the development and psychometric matching of two pairs of measures of language and visual-spatial functioning. We expanded and modified four existing neuropsychological measures in order to develop tasks with a wide range of item difficulty that could be administered to subjects with impaired attention or extreme fatigue. Using 413 subjects, a Word Finding and a Dot Localization task were matched on their distribution of item difficulty and item-scale correlations. Measures of Similarities and Line Orientation were matched similarly. The psychometric matching for both pairs of tests was cross-validated using an additional 413 subjects. The matched measures of Word Finding and Dot Localization have been used in several studies to date and have proven valuable for identifying subtle lateralized impairment that might otherwise be obscured by psychometric artifacts. PMID- 7546666 TI - Stimulus and sex differences in performance of mental rotation: evidence from event-related potentials. AB - We examined stimulus and sex differences in reaction time (RT) and event-related potentials (ERPs) during mental rotation of letters and abstract designs (PMA figures). RTs replicated stimulus and angle effects found in previous studies, but no sex differences were found for either set of stimuli. ERP latency data showed women began stimulus evaluation earlier, and PMA rotations began later over smaller angles, whereas letter rotations began later over larger angles. ERP amplitude data replicated hemisphere, electrode, and angle effects found in earlier studies. Amplitude measures also showed greater involvement of anterior cortical areas for evaluation of letter figures and posterior right temporal lobe for PMA figures, and greater positivity of women's waveforms than men's over late evaluation and early rotation components. PMID- 7546667 TI - A redrawn Vandenberg and Kuse mental rotations test: different versions and factors that affect performance. AB - The available versions of the Vandenberg and Kuse (1978) Mental Rotations Test (MRT) have physically deteriorated because only copies of copies are available. We report results from a redrawn version of the MRT and for alternate versions of the test. Males perform better than females, and students drawn from the physical sciences perform better than students drawn from the social sciences and humanities, confirming other reports with the original version of the MRT. Subjects find it very hard to perform the MRT when stimuli require rotation along both the top/bottom axis and the left/right axis. The magnitude of effect sizes for sex (which account, on average, for some 20% of the variance) does not increase with increasing difficulty of the task. Minimal strategy effects were observed and females did not perform differently during the menstrual period as opposed to the days between the menstrual periods. Practice effects are dramatic, confirming other reports with the original MRT, and can also be shown to be powerful in a transfer for practice paradigm, where test and retest involve different versions of the MRT. Main effects of handedness on MRT performance were not found. PMID- 7546668 TI - An investigation of differences between three age groups in verbal and spatial task performance using the dual-task paradigm. AB - A dual-task paradigm with concurrent verbal and spatial tasks was used to investigate differences across three age groups for lateralized effects, magnitude of tapping interference, and cognitive task output. In addition, rescaled scores comparing verbal with spatial measures within and across age groups were analyzed to examine asymmetric age differences for right versus left hemisphere abilities. Results of the present dual task finger tapping investigation of three age groups showed all groups left lateralized for verbal tasks and right lateralized for spatial tasks. However, the magnitude of interference in tapping was significantly greater in paced nonideational shadowing tasks and during paced purposeful spatial tasks. Outcomes of exploratory analyses suggest a complex interplay between aptitude and the corresponding lateralized effects and cognitive effort to perform the task. PMID- 7546669 TI - The preparation of aiming movements. AB - Three experiments are reported in which subjects made rapid aiming movements to visual targets with their left and right hands. In Experiments 1 and 3, a precue protocol was employed. In the "simple" reaction time condition, subjects were precued with complete information concerning the target position. In the four choice condition, subjects were precued with partial information, indicating a subset of four possible target positions. In the eight-choice condition, advance information regarding the target position was entirely ambiguous. Results indicated that when subjects were provided with unspecific advance information concerning the position of the target, and thus were unable to partially prepare movements prior to the imperative stimulus, a left hand advantage for speed of initiation was obtained. When complete advance information was available, reaction times for the left and right hands were equivalent. The left hand advantage in choice conditions was eliminated when the accuracy of response execution was emphasized and subjects were afforded the opportunity for "on-line" preparation (Experiment 3). These data are discussed in relation to the role of the right hemisphere in the preparation of movement. PMID- 7546670 TI - Dual-task interference patterns reveal differential processing of upright and inverted faces. AB - The concurrent-task method was used to investigate the hemispheric locus of face encoding. In each of three experiments, 48 right-handed adults performed unimanual finger tapping while encoding whole or partial faces for subsequent recognition. Faces were upright in Experiments 1 and 2 but inverted in Experiment 3. Irrespective of stimulus orientation, face encoding disrupted left- and right hand tapping equally. Upright faces were less accurately recognized if learned during left-hand tapping than during right-hand tapping; inverted faces showed no lateralized interference. The results support tachistoscopic findings that indicate predominantly right-hemispheric processing of upright faces and bilateral processing of inverted faces. PMID- 7546671 TI - A comment on apperceptive agnosia. PMID- 7546672 TI - Sinusitis in patients with cystic fibrosis. AB - The management of sinus disease in children with cystic fibrosis is reviewed, based on a literature review and clinical experience in The Hospital for Sick Children, Toronto. Diagnostic and treatment approaches are discussed, with emphasis given to indications for surgical therapies. Great importance is given to the preservation of normal nasal anatomy whenever possible to minimize the possibility of iatrogenic injuries occurring, especially when revision surgery is required. PMID- 7546673 TI - Canine laryngeal transplantation: preliminary studies and a new heterotopic allotransplantation model. AB - While transplantation of the larynx may eventually be useful in post-laryngectomy reconstruction, three criteria must first be met before human transplants can be attempted: transplant viability must be high, immunosuppression must be safe and effective and functional recovery of the larynx must occur. To study these first two criteria, a total of 11 canine larynx transplants were performed: 3 autografts, 6 orthotopic allografts and 2 heterotopic allografts. The rationale and technical performance of these different transplant procedures are reviewed in detail. Orthotopic transplant recipients received cyclosporin A (CsA) while the heterotopic allograft recipients received RS-61443 and methylprednisolone in addition to CsA. Overall, 9 of 11 of the transplants remained viable. In contrast, all 3 autografted animals developed esophageal-cutaneous fistulas; 2 developed sepsis and were sacrificed on post-operative days (POD) 5 and 28, respectively. The third survived for 91 days and demonstrated a high degree of regeneration in the recurrent and superior laryngeal nerves of the transplant. Orthotopically transplanted dogs also had a high morbidity and perioperative mortality (5 of 6 animals). The single "long-term" survivor was treated with CsA alone, but developed complete transplant rejection on POD 33. The two heterotopic transplant recipients had no perioperative morbidity and the combination of CsA, RS-61443 and methylprednisolone given these latter animals was effective in the long-term prevention of rejection. One of these heterotopic recipients died of sepsis on POD 68 while the other remained alive and well on POD 168. Our present findings show that currently available microsurgical techniques allow experimental canine laryngeal transplantation to be done with significantly high transplant viability rates. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546674 TI - Impact of magnetic resonance imaging and computed tomography on the staging of laryngeal cancer. AB - This retrospective study quantitatively assessed the effects of magnetic resonance imaging (MRI) and computed tomography (CT) on the staging of laryngeal cancer. A blind comparison between CT and MRI was made in a group of previously untreated patients with squamous cell carcinomas of the larynx. From June 1992 to November 1993, 29 patients were eligible for study. Of these, 14 patients (48%) had supraglottic lesions, 11 patients (40%) had glottic lesions and 4 patients (14%) had both. No subglottic lesions were seen. The data suggest that clinical staging of laryngeal tumors is inadequate. MRI proved superior to CT for staging tumors, especially those confined to the supraglottis. Nevertheless, clinically staged T1 or T2 lesions could be adequately assessed by CT alone. Findings also indicate that MRI should be reserved for T3 or T4 clinically staged lesions. Furthermore, most nodal disease can be staged by CT. PMID- 7546675 TI - The Indian experience with immediate tracheoesophageal puncture for voice restoration. AB - A 3-year prospective study on primary tracheoesophageal puncture was carried out at Nanavati Hospital and Tata Memorial Hospital, Bombay, to assess its feasibility in a developing country and its success in vocal rehabilitation. We report our experience with primary tracheoesophageal puncture in 64 patients (57 males and 7 females) following surgical treatment for carcinoma of the pyriform fossa (45 cases) and endolarynx (19 cases). All patients underwent total laryngectomies with or without partial pharyngectomy and primary pharyngeal mucosal closure. Fifty-one patients underwent primary surgery followed by postoperative radiotherapy. At 3 months successful tracheoesophageal speech was achieved in 84% patients, although this percentage decreased to 67% at 9 months. There were no major complications. Successful vocal rehabilitation was related more to "patient factors" such as delays in seeking medical attention when prosthetic valves dislodged, improper use and care of the valves and inability to learn speech with the prosthesis. Overall, primary tracheoesophageal puncture was found to be feasible and should be used more extensively for vocal rehabilitation following laryngectomy in developing countries. PMID- 7546676 TI - A threshold decrease for electrically stimulated motor responses of isolated aging outer hair cells from the pigmented guinea pig. AB - When outer hair cells are isolated from guinea pig cochleas and are placed in normal Hank's medium, they exhibit aging as a slow tonic reduction in length and increase in diameter. During this time the lateral subsurface cisternae become progressively vesiculated and the optical density of the border seen under phase contrast microscopy decreases. A study of 65 outer hair cells was carried out using video imaging of this process. The base of each cell bonded to the Petri dish and the motility of the cuticular plate was recorded in two ways. To quantify the slow contraction of each preparation, the dimensions of the cell were measured from video replay. Displacements of the cuticular plate in response to an alternating electric field in line with the cell axis were also monitored using a video tracking technique. The amplitude of a 1 Hz stimulus required to cause a visually detectable motor response above baseline noise decreased as the cell degraded. Typically, fresh cylindrical cells exhibiting high optical contrast showed relatively small movements for field strengths up to 2 kVm-1. However, as the cell depolarized, the rigidity initially decreased and the cell could respond to field strengths down to 0.1 kVm-1 before cell death ultimately occurred. Such a threshold phenomenon in the isolated OHC has not been demonstrated directly until now. This result explains the variability of electromotility in aging in vitro preparations from the cochlea. PMID- 7546677 TI - Cervical metastasis from the unknown primary tumor. AB - A retrospective study was performed to examine the results of treatment for cervical lymph node metastases from an unknown primary between 1978 and 1988. We reviewed 66 patients treated with either surgery and/or radiotherapy (n = 41), radiation therapy and chemotherapy (n = 11), combined modality treatment with surgery (n = 8) or with management delayed to proven disease (n = 6). The male-to female ratio was 5.6 to 1, and the median age was 64 years (range, 15-89 years). Intercurrent death-corrected 5-year survival was 50%. The most important prognostic factors were M category and involvement of supraclavicular nodes. Primary tumor was eventually discovered in 12 patients during follow-up, 6 of whom received treatment with curative intent. The most common site of primary tumor was found to be the lung (n = 5). PMID- 7546679 TI - Amoxicillin concentrations in nasal secretions of patients with acute uncomplicated sinusitis and in paranasal sinus mucosa of patients with chronic sinusitis. AB - In this prospective randomized clinical study a total of 59 patients of both sexes (above 18 years of age) were enrolled. Thirty patients with acute sinusitis were randomly allocated to two treatment groups, one group receiving 1000 mg amoxicillin every 12 h for 10 days and the other group receiving 500 mg amoxicillin every 8 h for 10 days. The median concentration of amoxicillin in nasal secretions was 2.34 micrograms/ml in the 12-h administration group and 1.95 micrograms/ml in the 8-h administration group. Median bioavailability of antibiotic at 8-24 h did not show any statistical differences between the two treatment schemes [probability (Z) = 0.2]. Twenty-nine patients with chronic sinusitis were then randomly allocated to three groups, with patients receiving 1000 mg amoxicillin at 12, 8 or 6 h before nasal and/or sinus surgery was carried out. The mean amoxicillin concentrations in mucosal tissues removed intraoperatively ranged from 0.69 to 0.99 microgram/g samples. Statistical evaluation by analysis of variance did not show any statistically significant differences among the three treatment groups [probability (F) = 0.1705]. In all cases of acute and chronic sinusitis, amoxicillin concentrations exceeded minimum inhibitory concentration values for pathogens common in sinusitis. Our results indicate that 1000 mg amoxicillin administered twice daily produces tissue concentrations high enough to be clinically effective in patients with either acute or chronic sinusitis. PMID- 7546678 TI - Immunohistochemical and fine structural characterization of primary carcinoid tumors of the larynx. AB - Carcinoid tumors belong to the group of neuroendocrine tumors of epithelial origin, i.e., neuroendocrine carcinomas. These neoplasms usually occur in the gastrointestinal tract or bronchial system but are very rare neoplasms in the larynx. Since carcinoid tumors in this latter site may appear to be undifferentiated by light microscopy, they may possibly be misinterpreted and their neuroendocrine characteristics may remain unrecognized. Using immunohistochemical methods, three carcinoid tumors were studied and showed positive immunostaining for markers of epithelial origin (cytokeratins, epithelial membrane antigen, carcino-embryonic antigen) and, in particular, for markers of neuroendocrine differentiation (chromogranin, synaptophysin, neuron specific enolase). All tumors expressed calcitonin-, serotonin- and adrenocorticotropic-hormone-like immunoreactivity. In contrast, three poorly differentiated squamous cell carcinomas showed positive immunostaining for epithelial markers but did not show any immunoreactivity with markers of endocrine characteristics. Fine structurally, carcinoid tumor cells contained neurosecretory-type granules scattered throughout the cytoplasm. The present study demonstrated that (1) carcinoid tumors of the larynx possess distinct immunohistochemical characteristics that allow a clear classification, (2) it is advisable to use a battery of primary antibodies rather than rely on specificity and sensitivity of a single marker to establish diagnosis and (3) the fine structural demonstration of neurosecretory-type granules serves as a reliable adjunct to diagnosis. PMID- 7546680 TI - A comparative electron microscopic study of chemical techniques for preservation of auditory ossicles as homografts. AB - The ultrastructure of preserved ossicles at different shelf lives was compared. Homograft ossicles were harvested and preserved in 70% alcohol, 1:5000 Cialit (sodium-2-ethyl-mercuri-mercapto-benzoxazole-5-carboxylate), and buffered 4% formaldehyde at pH 7. The ultrastructure of each ossicle was studied by both scanning and transmission electron microscopy and was compared to that of fresh controls placed in 0.9 N saline solution. The results indicated clearly the superiority of 70% alcohol as a long-term preservative and 1:5000 Cialit as a short-term preservative. PMID- 7546681 TI - Effects of irradiation on the rat middle ear mucosa. A scanning electron microscopic study. AB - Adult Sprague-Dawley rats (n = 64) were used in this study to replicate in an animal model tissue change formed clinically during radiotherapy for the treatment of head and neck malignancy. Gamma irradiation was administered to both ears of each rat, using a cobalt-60 machine. A total dose of 30 Gy was administered, with 500 cGy/fraction given on 6 consecutive days to a time-dose fractionation value of 81. Animals were then sacrificed at sequential intervals. The tympanic bullae were removed and the mucosa was processed for scanning electron microscopy. Tissue findings indicated that irradiation might initially have no influence on the clearance activity of the middle ear. However, ultrastructural changes showed stagnation of secretion between cilia, falling off and different directions of the cilia, fused or collapsed cilia, and eustachian tube dysfunction (with mucosal edema). These findings possibly account for the occurrence of middle ear effusions in patients during and after radiotherapy for head and neck malignancy. It is our concept that insertion of a ventilation tube is necessary if abnormal mucociliary function appears, in order to clear and prevent the accumulation of any middle ear effusions and prevent further mucociliary dysfunction. PMID- 7546682 TI - Cell-surface glycoconjugate layer in the tubotympanic mucosa of the guinea pig as revealed by wheat germ agglutinin/gold labelling. AB - To evaluate the protective function of the mucous blanket (MB) against lectin substances, we examined at the ultrastructural level whether intraluminal colloidal gold-labelled wheat germ agglutinin (WGA) could enter the MB-covered epithelial cell surface of the guinea pig tubotympanic mucosa. Post-embedding staining with WGA/gold on thin tissue sections was done in parallel for comparison. The cell surface glycoconjugate of the eustachian tubal and transitional epithelium had a typical bilayered structure: the outer MB and the microvilli-associated glycocalyx (MAG), which were interposed by the interciliary fluid zone. In squamous epithelium of the distal middle ear, the MB adhered to the MAG, thereby forming a monolayered coat of glycoconjugates at the cell surface. In the pre-embedding staining, WGA/gold did not bind with the MB and MAG in the eustachian tube, and exclusively bound with MB in the transitional area. Direct binding was also found with MAG and the apical plasmic membrane in the squamous epithelium. These findings indicate that MAG is occluded by MB lined with the interciliary fluid zone for luminal access of lectin at the proximal lumina of the tubotympanic epithelium. It is also suggested that MB existing at two sites possesses a different WGA-binding capacity: shielding as a "dust cover" in the eustachian tube and entrapping as a "flypaper" against lectin in the transitional area of the middle ear. PMID- 7546683 TI - Vascular permeability changes associated with experimentally induced facial nerve lesions in the rabbit. AB - Changes in vascular permeability to sodium fluorescein following experimentally induced nerve lesions were examined in the rabbit facial nerve. Sodium fluorescein was injected intravenously as a permeability tracer and then localized by fluorescence microscopy. In control nerves, endoneurium showed only slight fluorescence while intense fluorescence was observed in the epineurium and perineurium. In nerves demonstrating edema and Wallerian degeneration, endoneurium was found to have an increased accumulation of tracer. This increased endoneurial vascular permeability in facial nerve lesions may explain nerve enhancement seen in gadolinium-enhanced magnetic resonance imaging in patients with facial nerve paralysis. PMID- 7546684 TI - The role of eye protection in work-related eye injuries. AB - PURPOSE/BACKGROUND: A recent survey of general hospitals by the Victorian Injury Surveillance System found that ocular trauma represented 15% of work-related injuries. As circumstances surrounding occupational eye injuries have been poorly documented previously, their associations to occupation, industry and work-safety practices, including safety eyewear use, need to be identified to develop appropriate preventive strategies for high-risk groups. DESIGN AND SETTING: From a prospective cross-sectional survey of all eye injuries treated at the Royal Victorian Eye and Ear Hospital, work-related cases were analysed for demographic, occupational and safety eye-wear information. Hospital-based data were supplemented by information from WorkCover Authorities and Labour Force statistics to derive incidence and cost estimates. RESULTS: There were 9390 eye injuries during the 18-month survey period; 42% (n=3923) of total and 29% (n=52) of penetrating ocular injuries occurred at work. The most frequently injured were metal, automotive and building trades workers grinding and drilling (41% of outpatients) and hammering (53% of penetrating eye injuries). Automotive workers had the highest frequency for penetrating injuries, and most were exposed to hammering and were also the least likely to wear safety eye-wear. CONCLUSION: Eye injuries are frequent (10% of work-related injuries) and highly preventable by the correct use of safety eye-wear, a cost-effective intervention that may result in cost savings of $59 million for work-type activities in the occupational and domestic settings in Australia each year. PMID- 7546685 TI - Patterns of visual loss associated with pituitary macroadenomas. AB - PURPOSE: To describe the patterns of visual loss associated with pituitary macroadenomas compressing the anterior visual pathway. METHOD: A prospective survey of 29 patients with pituitary macroadenomas who presented to the neurosurgical unit at St Vincent's Hospital, Melbourne. Selected patients had histologically verified pituitary macroadenomas, and visual defects (acuity, colour and/or field loss) consistent with anterior visual pathway compression. RESULTS: All patients had visual field defects detected on perimetry, and the majority were asymmetrical. Bitemporal defects were most common but field defects ranged from monocular defects to generalised constriction. Four patients (13.8%) did not report visual symptoms, and of those who had symptoms, blurred vision was the most common complaint. Ninety-six per cent of eyes had field loss, 56% had decreased colour vision, 46% had decreased acuity, 31% had optic disc pallor, and 2% had an ophthalmoplegia. CONCLUSIONS: Patients with visual pathway compression by pituitary macroadenomas may be asymptomatic despite having field defects. Perimetry is the most sensitive method of identifying compression, followed by colour vision, visual acuity, then the presence of optic atrophy. Automated static threshold perimetry appears to show early field defects better than manual kinetic perimetry. All patients with pituitary macroadenomas should have thorough ophthalmological examinations, including perimetry to document visual deficits secondary to compression. PMID- 7546686 TI - Hydroxyapatite orbital implants. Experience with 100 cases. AB - BACKGROUND: The hydroxyapatite (HA) intraorbital implant is a relatively new implant made from the porous skeleton of a coral species which allows fibrovascular ingrowth and therefore tissue integration. After fibrovascular ingrowth, a hole can be drilled in the implant and a motility peg inserted to increase movements of the prosthesis by coupling the implant to the prosthesis. METHOD: The records of the first 100 cases of HA intraorbital implants inserted by the one surgeon were analysed for complications, pain and nausea postoperatively, length of hospital stay, and further surgical procedures required. A series of acrylic implants inserted by the same surgeon was used for comparison. RESULTS: Twenty-five primary and 75 secondary HA implants were performed in patients ranging from six to 74 years of age. All were covered in donor sclera. Follow-up was three to 34 months (mean 16.9, median 17.0). Complications occurred in 15 patients and included too large an implant (seven cases) requiring surgical reduction, scleral exposure in three (repair required in one), an early small exposure of the coral in one case, late thinning of the conjunctiva and later exposure of the implant in one, and shallowing of the inferior fornix requiring mucous membrane grafting in three. No implants migrated, extruded or became infected. Of 80 patients beyond six months follow up, 28 (35%) had insertion of motility pegs, and six (7.5%) of these suffered minor complications related to the peg. Compared to patients having acrylic implants, the postoperative analgesic requirements and length of hospital stay were significantly greater for the HA patients. CONCLUSIONS: Hydroxyapatite intraorbital implants represent a significant advance over other implants and offer a more stable, safe alternative. They also offer the possibility of improved prosthesis motility. The additional cost of the implant, prolonged hospital stay and postoperative pain, should be considered in recommending such implants to patients either as primary or secondary implants. PMID- 7546687 TI - Interrater agreement in oculo-kinetic perimetry--a screening test for glaucoma. AB - In a study at the Flinders Medical Centre, South Australia, 24 glaucomatous eyes with known field defects were tested by three examiners of different backgrounds using oculo-kinetic perimetry, a screening test for glaucoma. The interrater agreement was excellent; only in one case did results differ. Seventeen eyes had a defective result, but in six cases the test was normal. Both patients and untrained staff find this test easy to do, but the apparent high false-negative rate is cause for concern and needs further evaluation. Oculo-kinetic perimetry may well be better used as a complementary test by those opticians doing tonometry and by general practitioners who currently rarely screen for glaucoma. PMID- 7546688 TI - Clinical detection of unilateral keratoconus. AB - PURPOSE: To determine the incidence of true unilateral keratoconus on the basis of computerised corneal topography in a group with clinically diagnosed unilateral keratoconus. METHODS: Retrospective review of 295 patients with keratoconus identified 51 patients with a provisional diagnosis of unilateral keratoconus. Thirty-one of these patients were re-examined clinically and 21 patients met the criteria for a clinical diagnosis of unilateral keratoconus. Computerised corneal topographic analysis (Tomey TMS) was then performed. RESULTS: In a group of 21 patients with clinically diagnosed unilateral keratoconus, computerised corneal topographic analysis identified 14 (67%) patients with bilateral keratoconus and seven (33%) patients with true unilateral keratoconus. Contact lens wear had no significant influence (P=0.76) on the topographical diagnosis of keratoconus in the clinically unaffected fellow eye. The estimated incidence of true unilateral keratoconus in the cohort of 295 patients was 4%. CONCLUSIONS: Computerised corneal topography improves sensitivity in detection of true unilateral keratoconus. PMID- 7546689 TI - The Albury-Wodonga syndrome. A tale of two cities. AB - PURPOSE: An epidemic of acutely painful eyes having a seasonal restriction and an apparent response to topical indomethacin, is described. METHODS: From patients seen in a rural private practice, the clinical picture of this acute syndrome and the use of indomethacin drops is described. RESULTS: A favourable response to topical indomethacin in 21 patients is documented. CONCLUSION: Topical indomethacin is worthy of further trial and investigation in the management of the Albury-Wodonga syndrome, harvester's eye, Christmas eye or seasonal corneal ulcer. PMID- 7546690 TI - Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments. AB - BACKGROUND: To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. METHODS AND RESULTS: Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. CONCLUSIONS: Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium. PMID- 7546691 TI - Blepharophimosis syndrome: association with colobomatous microphthalmos. AB - OBJECTIVE: To highlight the association of colobomatous microphthalmos with blepharophimosis syndrome. RESULT: We present a case of blepharophimosis syndrome associated with bilateral optic disc, retinochoroidal and iris colobomas, and microphthalmos, in a Caucasian boy. Inheritance in this case was autosomal dominant from the maternal side. CONCLUSION: Colobomatous microphthalmos is an infrequent ocular abnormality in the blepharophimosis syndrome. Ophthalmologists should be aware of this association. PMID- 7546692 TI - Sectorial iris infarction caused by meningococcal septicaemia. PMID- 7546693 TI - Late bacterial scleritis following beta irradiation presenting as orbital cellulitis. PMID- 7546694 TI - Sustained-release intravitreal ganciclovir pellets in the treatment of cytomegalovirus retinitis in the acquired immunodeficiency syndrome. PMID- 7546695 TI - The case against treating glaucoma. PMID- 7546696 TI - Ganglion cell death in glaucoma: pathology recapitulates ontogeny. AB - I review some improvements in our knowledge about the death of retinal ganglion cells in glaucoma during the last 20 years. These include the realisation that glaucoma damage precedes its detection by perimetry, the fact that the lamina cribrosa is a major site of axonal injury to ganglion cells, and the association between regional structure of optic nerve head connective tissue and the pattern of glaucoma damage. The selective susceptibility of larger retinal ganglion cells and its functional significance are described. Apoptosis is the mode of cell death in at least some ganglion cells in experimental glaucoma. This supports a theory that retrograde axonal transport failure leads to loss of trophic factor influence on ganglion cells, causing them to initiate their own suicide. As a consequence of this theory, two therapeutic avenues are suggested for prevention of glaucoma injury and cell death: delivery of trophic factors and manipulation of ganglion cell genetic expression of controlling influences over programmed cell death. PMID- 7546697 TI - Macular holes. Pathogenesis, natural history and surgical outcomes. AB - Idiopathic full-thickness macular holes (FTMH) are a common cause of usually uniocular reduced visual acuity. The accepted pathogenesis has macular hole formation proceeding in stages from an impending hole to a FTMH, with visual acuity deteriorating to less than 6/60 in 85% of cases. Recent surgical advances in the treatment of FTMH, with excellent anatomic and visual results, have changed the surgical indications for both impending and established full thickness macular holes. PMID- 7546698 TI - High-throughput microplate format for producing and screening riboprobes from bacterial cells. PMID- 7546699 TI - Effect of encapsulated ammonium persulfate on DNA banding in sequencing gels. PMID- 7546700 TI - Nonradioactive assay for sequence-specific DNA binding proteins. AB - A nonradioactive functional assay was developed to quantitate DNA binding proteins. The assay was designed to allow the use of 96-well microplates for high sample throughput. We show that the assay can measure sequence-specific DNA binding of purified proteins as well as DNA binding activity present in whole cell extracts. By using a temperature-sensitive DNA binding protein, we demonstrate that DNA binding as measured by this novel assay correlates with the biological function of the protein. PMID- 7546701 TI - Sensitive bioassay for the detection and quantification of angiotensin II in tissue culture medium. AB - We have developed a sensitive, high-throughput bioassay to quantify angiotensin II in culture medium. Using Chinese hamster ovary cells that stably express a transfected angiotensin II receptor as target cells, we demonstrated that an agonist-stimulated myelin basic protein kinase response can be used as a basis of quantitative bioassay for angiotensin II. The assay permits detection of as little as 10 pg of angiotensin II in 1 mL of medium and offers an excellent alternative to HPLC and radioimmunoassay. This approach may also be applicable for quantification of other peptide hormones or growth factors produced by cell cultures. PMID- 7546702 TI - Polyethylene glycol-mediated bacterial colony transformation. AB - A moderately efficient and quick method of bacterial colony transformation is described. Plasmid DNA was added to bacteria suspended in a solution of polyethylene glycol/calcium chloride (PEG/CaCl2). After a brief incubation and heat shock, the cells were directly plated. Transformation efficiencies up to 8.6 +/- 1.28 x 10(6) transformants per microgram of pUC18 were obtained. We have found that the reverse of the transformation process could also take place. Suspending a bacterial pellet harboring the plasmid of interest in PEG/CaCl2 results in the release of the plasmid DNA, and thus indirectly lends support to the transformation process. PMID- 7546703 TI - Lipofection reagents prepared by a simple ethanol injection technique. AB - Cationic liposomes are being utilized for the delivery of DNA into mammalian cells both in vitro and in vivo. In this report, we describe a rapid, simple injection method for the preparation of cationic liposomes that requires no special equipment. We demonstrate that, using this injection method, liposomes prepared with a commercially available cationic lipid (dimethyldioctadecylammonium bromide, DDAB) or a synthetic cationic cholesterol derivative, along with a neutral lipid (dioleoyl-L-alpha phosphatidylethanolamine, DOPE), effectively transfect a variety of cell lines in vitro. The transfection activity of liposomes prepared by this method was comparable to that obtained with liposomes prepared by the standard evaporation/sonication procedure and to that of a commercial reagent, Lipofectamine. PMID- 7546704 TI - Site-directed mutagenesis using thermostable enzymes. AB - The two-primer method for site-directed mutagenesis facilitates the mutation of targets in double-stranded DNA. We have encountered difficulties using the original method for the mutagenesis of DNA cloned into pBluescript vectors, which is possibly due to the presence of regions of secondary structure that cannot be efficiently copied by the enzymes used. We report a modification of the two primer method using the thermostable VentR DNA polymerase and Thermus aquaticus ligase, allowing an increase in reaction temperatures. The modified method is functional with the pBluescript family of vectors. PMID- 7546705 TI - Semiautomated quantitative detection of loss of heterozygosity in the tumor suppressor gene p53. AB - The most frequently altered gene in diverse tumor types is the tumor suppressor gene p53. Typically, normal function is inactivated by point mutation of one allele and deletion of the other. Therefore, loss of heterozygosity (LOH) of intragenic polymorphic markers is a strong indication for p53 involvement in a cancerous lesion. This study shows that a highly polymorphic short tandem repeat (STR) within intron 1 of p53 is an excellent marker for quantitative evaluation of LOH in tumor samples, whose multicolor, fluorescently tagged PCR products are analyzed and quantitated on an automated DNA sequencer. The range of error was analyzed in detail. Discrete allelic profiles were obtained following amplification of DNA from microdissected cell samples of patients with urogenital tumors. By calculating qLOH, the relative allele ratio of a tumor compared with healthy tissue, a quantitative expression for the LOH is obtained. PCR-based tumor DNA typing using fluorescent STR primers and automated analysis provides an enhanced level of accuracy and sensitivity required for routine analysis in clinical practice, where large numbers of tumor samples have to be processed. PMID- 7546706 TI - Identification of conserved sequences for PCR primer design by multiple alignments of dot matrix plots. PMID- 7546707 TI - ImageCalc: a Microsoft Windows application for quantitative image analysis and comparison. AB - In this report we describe a Microsoft Windows-based computer program (ImageCalc) for the analysis of gels and autoradiograms and for computation of stand-alone images and images that are related to each other (i.e., images with the same image parameters). The program is able to subtract, add, multiply and divide constant values or full images from another image. It measures the intensity of (part of) an image in two ways: (i) by calculation of the total intensity and the average intensity/pixel and (ii) by line scanning. The program allows importation of all images that are stored in an 8-bit uncompressed format and saves them in a Microsoft Windows bitmap format. Densitometric analysis of gels and autoradiograms, digitized using an ordinary optical scanner, is illustrated for glycosaminoglycans separated by electrophoresis on agarose gels and stained with Azure A silver, and for [32P]phosphatidic acid, separated by thin-layer chromatography. Another application of the program is the analysis of multiple related images that are resolved in time or for which different emission or excitation wavelengths are used (fluorescence microscopy). As an example, the change of cytosolic [Ca2+] is demonstrated in cultured human skeletal muscle cells after stimulation with acetylcholine. PMID- 7546708 TI - Quantifying immunoblots with a digital scanner. PMID- 7546709 TI - CARD: a drawing tool for RNA secondary structure models. AB - A graphical editor was developed to create publication-quality representations of RNA secondary structure models. A user-defined model can be gradually assembled from structural elements such as helix segments and loops. The type of structural element to be drawn is chosen from a menu. Its nucleotide sequence has to be entered from the keyboard. Afterwards, drawings can be manipulated by moving, deleting, rotating, copying or changing the structural elements separately. An example of a secondary structure model is given for a complete 18S rRNA molecule. PMID- 7546710 TI - DNAdraw: a program for preparing DNA and protein data for publication. AB - DNAdraw is a Macintosh program designed to prepare DNA and protein data for publication. In additional to providing standard ways of highlighting data, e.g., fonts, styles and shading, DNAdraw has special features for formatting sequence data and for handling aligned sequence data. Output for the program is to a PostScript printer. A simple example is presented to illustrate DNAdraw formatting and highlighting, and an example for aligned data is given. This new version of the program that has been written for other machines is the first to run under a Graphical User Interface (GUI) and incorporates many new features. PMID- 7546711 TI - A potent, cost-effective RNase inhibitor. AB - The performance characteristics of a potent new RNase inhibitor have been evaluated, as have applications for its use in molecular biology. PRIME Inhibitor is a protein, not related to the commonly available human placental RNase inhibitors (HPRI). It has a high specific activity, enhanced temperature stability, broad reaction pH range and significantly greater cost-effectiveness than commercial HPRI. PRIME Inhibitor is suitable for use in in vitro transcription, in vitro translation, first- and second-strand cDNA synthesis, preparation of RNA and mRNA, and reverse transcription-polymerase chain reaction. PMID- 7546712 TI - Quick and easy spreading technique for electron microscopy of DNA. PMID- 7546714 TI - Detection of transgenic animals without cell culture using fluorescence in situ hybridization. PMID- 7546713 TI - Section adherence to glass slides from polyester wax-embedded tissue for in situ hybridization. PMID- 7546715 TI - Screening of homozygous transgenic mice by comparative PCR. PMID- 7546716 TI - Inhibition of PCR by mineral oil exposed to UV irradiation for prolonged periods. PMID- 7546717 TI - Direct measurement of transfection efficiency in transient transfection assays. PMID- 7546718 TI - Buffer modification allows labeling of oligonucleotides at both the 5' and 3' termini. PMID- 7546719 TI - Increased yield of small DNA fragments purified by silica binding. PMID- 7546720 TI - Facile extraction and purification of filamentous fungal DNA. PMID- 7546721 TI - Use of filtered pipet tips to elute DNA from agarose gels. PMID- 7546722 TI - Dried template DNA, dried PCR oligonucleotides and mailing in 96-well plates: LDL receptor gene mutation screening. PMID- 7546723 TI - Simple and efficient method for the preparation of nuclear extracts. PMID- 7546724 TI - Isolation of RNA from Mycobacterium tuberculosis using a nitrogen decompression chamber. PMID- 7546725 TI - Synthesis and purification of milligram quantities of short RNA transcripts. PMID- 7546726 TI - Application of the immunoblot technique for quantitation of protein levels in cardiac homogenates. PMID- 7546727 TI - The critical period for long-term potentiation in primary sensory cortex. PMID- 7546728 TI - Surfing the DNA databases for K+ channels nets yet more diversity. PMID- 7546729 TI - When more is less: pathogenesis of glutamine repeat neurodegenerative diseases. PMID- 7546730 TI - Cerebral cortex: function and development. PMID- 7546731 TI - Systems and molecular genetic approaches converge to tackle learning and memory. PMID- 7546732 TI - Wiring by fly: the neuromuscular system of the Drosophila embryo. PMID- 7546733 TI - Postmigratory neural crest cells expressing c-RET display restricted developmental and proliferative capacities. AB - c-RET is an orphan receptor tyrosine kinase essential for enteric neurogenesis in mice and is involved in several human genetic disorders. RET is also one of the earliest surface markers expressed by postmigratory neural crest cells in the gut. We generated anti-RET monoclonal antibodies to isolate such cells. We find that RET+ cells are antigenically and functionally distinct from neural crest stem cells (NCSCs) characterized previously. Unlike NCSCs, which are RET- and MASH1-, most RET+ cells express MASH1. Moreover, unlike NCSCs, which are multipotent and have high proliferative capacity, many RET+ cells generate only neurons following a limited number of divisions. This behavior is observed even in the presence of glial growth factor, a polypeptide that suppresses neuronal and promotes glial differentiation by NCSCs. These data provide direct evidence for the existence of committed neuronal progenitor cells and support a model of neural crest lineage diversification by progressive restriction of developmental potential. PMID- 7546734 TI - Patterns of excitation and inhibition evoked by horizontal connections in visual cortex share a common relationship to orientation columns. AB - Combined optical imaging and electrophysiological techniques were used to assess directly the functional nature of long-range excitatory and inhibitory synaptic interactions between orientation columns in area 17 of ferret visual cortex. A significant correlation was found between the layout of iso-orientation columns and the pattern of evoked synaptic inputs between cortical sites: the largest amplitude inhibitory and excitatory synaptic responses were evoked in single neurons when stimulation and recording electrodes were located in orientation columns sharing the same angle preference. Both excitatory and inhibitory synaptic responses decreased in amplitude when stimulation and recording electrodes were located in orientation columns with orthogonal angle preferences. Changing the stimulus intensity altered the balance of evoked excitation and inhibition without changing the columnar specificity of inputs. These results directly demonstrate that horizontal connections modulate both excitatory and inhibitory synaptic interactions between iso-orientation columns. PMID- 7546735 TI - Pax3: a paired domain gene as a regulator in PNS myelination. AB - Pax3 RNA is expressed in neural crest when Schwann cell (SC) precursors migrate to the PNS. Pax3 RNA and SC markers were monitored in sciatic nerves of mice during development and nerve repair. An inverse correlation was observed between expression of Pax3 RNA and myelin basic protein (MBP). Inverse correlation was also observed in SC primary cultures. Treating cultures with forskolin, an adenylate cyclase agonist, repressed Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and elevated MBP. Subsequent microinjection with Pax3 expression vector elevated Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and repressed MBP. Thus, Pax3 is likely involved in the differentiation pathway to myelinating SCs. Pax3 repressed a 1.3 kb MBP promoter fragment in cotransfection assays, suggesting that it represses MBP transcription. PMID- 7546736 TI - Identification of a gephyrin binding motif on the glycine receptor beta subunit. AB - The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix. PMID- 7546737 TI - Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. AB - While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function. PMID- 7546738 TI - Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors. AB - We show that beta forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage. PMID- 7546739 TI - Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. AB - Sympathetic neurons from perinatal rat pups extend only a single axon when maintained in culture in the absence of glia and serum. Exposure to recombinant osteogenic protein-1 (OP-1) selectively induces the formation of dendrites that correctly segregate and modify cytoskeletal and membrane proteins and form synaptic contacts of appropriate polarity. OP-1 requires nerve growth factor (NGF) as a cofactor, and, in the presence of optimal concentrations of NGF, OP-1 induced dendritic growth from cultured perinatal neurons is comparable to that observed in situ. Sympathetic neuroblasts that had not formed dendrites in situ also responded to OP-1 in culture, indicating that OP-1 can cause de novo formation as well as regeneration of dendrites. These data imply that specific signals can regulate the development of neuronal shape and polarity. PMID- 7546740 TI - Activation of light-dependent K+ channels in ciliary invertebrate photoreceptors involves cGMP but not the IP3/Ca2+ cascade. AB - The activation of light-dependent K+ channels in ciliary photoreceptors from Pecten was investigated using intracellular dialysis of putative messengers and modulators. Neither elevated [Ca2+] nor BAPTA changed the membrane current in the dark or the light response. IP3 and the antagonists heparin and decavanadate were similarly ineffective, indicating that in these cells the IP3/Ca2+ signaling pathway is not crucial for phototransduction. By contrast, 8-Br-cGMP and cGMP induced an outward current accompanied by an increase in membrane conductance; 8 Br-cAMP was ineffective. The identity between the cGMP-induced and the light induced currents is suggested by the following: both are carried by K+ and blocked by 4-AP, and both show outward rectification. In addition, guanine cyclic nucleotides depressed the photoresponse and induced single-channel currents in excised patches of light-sensitive membrane. These light-dependent channels therefore appear to represent a link between the families of cyclic nucleotide gated channels and voltage-dependent K+ channels. PMID- 7546741 TI - Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. AB - Cyclic nucleotide-gated ion channels of retinal photoreceptors and olfactory neurons are differentially activated by ligands that vary only in their purine ring structure. The nucleotide selectivity of the bovine rod cyclic nucleotide gated channel (cGMP > cIMP >> cAMP) was significantly altered by neutralization of a single aspartic acid residue in the binding domain (cGMP > or = cAMP > cIMP). Substitution by a nonpolar residue at this position inverted agonist selectivity (cAMP >> cIMP > or = cGMP). These effects resulted from an alteration in the relative ability of the agonists to promote the allosteric conformational change associated with channel activation, not from a modification in their initial binding affinity. We propose a general mechanism for guanine nucleotide discrimination, in common with that observed in high affinity GTP-binding proteins, involving the formation of a pair of hydrogen bonds between the aspartic acid side chain and N1 and N2 of the guanine ring. PMID- 7546742 TI - A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide gated channel from rod photoreceptor. AB - The cyclic nucleotide-gated channel from rod photoreceptors is composed of two distinct subunits (alpha and beta). The properties of the alpha subunit, which can form functional channels by itself, are modified by coexpression with a homologous polypeptide, designated the beta subunit. However, the alpha subunit from rod photoreceptor membranes copurifies with a 240 kDa protein that is significantly larger than this putative beta subunit. We now demonstrate by peptide sequencing and by cloning and functional expression of cDNA that the 240 kDa protein represents the complete beta subunit with an unusual bipartite structure. The N-terminal part is essentially identical to a glutamic acid-rich protein (GARP), whereas the C-terminal part is highly homologous to the previously cloned human "beta subunit." Expression of the complete beta subunit in HEK 293 cells results in a polypeptide with the same apparent molecular weight as the 240 kDa protein of the native rod channel. Coexpression of the alpha subunit with the full-length beta subunit yields hetero-oligomeric channels with properties characteristic of the native channel. PMID- 7546743 TI - Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons. AB - The site of action potential initiation in substantia nigra neurons was investigated by using simultaneous somatic and dendritic whole-cell recording in brain slices. In many dopamine neurons, action potentials were observed first at the dendritic recording site. Anatomical reconstruction showed that in these neurons, the axon emerged from the dendrite from which the recording had been made. Action potentials showed little attention in the dendritic tree, which in dopamine neurons was shown to be due to recruitment of dendritic sodium channels and may be related to the dendritic release of dopamine. We conclude that in substantia nigra neurons, the site of action potential initiation, and thus the final site of synaptic integration, is in the axon. As the axon can originate from a dendrite, up to 240 microns away from the soma, synaptic input to the axon bearing dendrite may be privileged with respect to its ability to influence action potential initiation. PMID- 7546744 TI - Role of BCL-2 in the survival and function of developing and mature sympathetic neurons. AB - Sympathetic neurons, when placed in culture during the period of naturally occurring cell death, will die by apoptosis when deprived of nerve growth factor (NGF). In this system, the mRNA levels of the BCL-2 family members decrease after NGF deprivation and during apoptosis. Sympathetic neurons from BCL-2-deficient mice died more rapidly after NGF deprivation than neurons from wild-type littermates. Sympathetic neurons of adult animals are relatively independent of NGF for survival. If sympathetic neurons are maintained in vitro for several weeks, loss of acute trophic factor dependence develops with a time course similar to that seen in the intact animal. Examination of neurons from BCL-2 deficient mice showed that BCL-2 expression is not required for the development of trophic factor independence. Therefore, BCL-2 is an important regulator of the survival of sympathetic neurons after NGF deprivation during the period of naturally occurring programmed neuronal death, but BCL-2 is not involved in the development of trophic factor independence in mature sympathetic neurons. PMID- 7546745 TI - Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. AB - In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking. PMID- 7546746 TI - Calcium control of transmitter release at a cerebellar synapse. AB - The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated presynaptic Ca2+ entry by altering external Ca2+ levels and by blocking Ca2+ channels with Cd2+ or with the toxins omega-conotoxin GVIA and omega-Aga-IVA. For all of the manipulations, other than the application of omega-Aga-IVA, the relationship between Ca2+ influx and release was well approximated by a power law, n approximately 2.5. When omega Aga-IVA was applied, release appeared to be more steeply dependent on Ca2+ (n approximately 4), suggesting that omega-Aga-IVA-sensitive channels are more effective at triggering release. Based on interactive effects of toxins on synaptic currents, we conclude that multiple types of Ca2+ channels synergistically control individual release sites. PMID- 7546747 TI - Calcium-dependent transmitter secretion from fibroblasts: modulation by synaptotagmin I. AB - Following endocytic uptake of acetylcholine (ACh), CHO fibroblasts exhibit Ca(2+) dependent spontaneous quantal ACh release and depolarization-evoked ACh release, as detected by a whole-cell voltage-clamped myocyte in contact with the fibroblast. CHO fibroblasts transfected with synaptotagmin I, an integral membrane protein of synaptic vesicles, showed a reduced spontaneous quantal ACh release and an enhanced Ca(2+)-evoked ACh release, as compared with control cells. Biochemical and ultrastructural studies of endocytic activity using horseradish peroxidase as a marker further confirmed the inhibitory action of synaptotagmin I on spontaneous vesicular exocytosis and on elevated exocytosis induced by Ca2+. Through inhibition of exocytosis at the resting intracellular concentration of Ca2+ and removal of the inhibition upon depolarization-induced Ca2+ entry, synaptotagmin I could enhance the efficiency of excitation-secretion coupling. PMID- 7546748 TI - Transient and persistent phosphorylation of AMPA-type glutamate receptor subunits in cerebellar Purkinje cells. AB - We generated a polyclonal antibody, 12P3, specifically recognizing rat AMPA-type glutamate receptor (GluR) subunits phosphorylated at Ser-696 of GluR2 or at the homologous sites in GluR1, GluR3, and GluR4. Using 12P3, we demonstrate that a brief exposure of a rat cerebellar slice to AMPA leads to transient phosphorylation of the GluR subunits in Purkinje cell dendrites. Persistent phosphorylation over 30 min was obtained when exposure to AMPA was preceded by a 15 min perfusion of the slice with 8-bromo-cGMP, dibutyryl-cGMP, or calyculin A but not phorbol 12,13-diacetate. These results indicate that Ser-696 of GluR2, or the corresponding sites in other AMPA receptor subunits, is a specific site at which phosphorylation takes place when AMPA-type GluRs are activated by agonists, especially under the influence of certain second messenger activities. PMID- 7546749 TI - Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. AB - The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer approximately hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerve terminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsin on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated. PMID- 7546750 TI - Ion fluxes associated with excitatory amino acid transport. AB - Flux of substrate and charge mediated by three cloned excitatory amino acid transporters widely expressed in human brain were studied in voltage-clamped Xenopus oocytes. Superfusion of L-glutamate or D-aspartate resulted in currents due in part to electrogenic Na+ cotransport, which contributed 1 net positive charge per transport cycle. A significant additional component of the currents was due to activation of a reversible anion flux that was not thermodynamically coupled to amino acid transport. The selectivity sequence of this ligand activated conductance was NO3- > 1- > Br- > Cl- > F-. The results suggest that these proteins mediate both transporter- and channel-like modes of permeation, providing a potential mechanism for dampening cell excitability, in addition to removal of transmitter. PMID- 7546751 TI - Synergistic regulation of a neuronal chloride current by intracellular calcium and muscarinic receptor activation: a role for protein kinase C. AB - Using perforated patch recordings in combination with intracellular Ca2+ ([Ca2+]i) fluorescence measurements, we have identified a delayed Ca(2+) dependent Cl- current in a mammalian sympathetic ganglion cell. This Cl- current is induced by the synergistic action of Ca2+ and diacylglycerol (DAG) and is blocked by inhibitors of protein kinase C. As a result, the current can be induced by acetylcholine through the conjoint activation of nicotinic receptors (to produce a rise in [Ca2+]i) and muscarinic receptors (to generate DAG). This demonstrates an unusual form of synergism between the two effects of a single transmitter mediated via separate receptors operating within a time scale that could be of physiological significance. PMID- 7546752 TI - Total intravenous anesthesia for children undergoing brief diagnostic or therapeutic procedures. AB - STUDY OBJECTIVE: To compare the quality of anesthesia with propofol, ketamine, or etomidate in children undergoing brief diagnostic or therapeutic procedures. DESIGN: Retrospective study. SETTING: University hospital. PATIENTS: 971 pediatric oncology patients undergoing brief diagnostic or therapeutic procedures outside the operating room during a one-year period. INTERVENTIONS: Total intravenous anesthesia was administered primarily with ketamine, etomidate, or propofol for oncology-related procedures such as bone marrow aspiration, lumbar puncture, radiologic imaging, and radiation therapy. Quality assurance data were collected for all patients, including anesthetic technique, dosage, and the occurrence of specific adverse events during anesthesia and recovery periods. MEASUREMENTS AND MAIN RESULTS: There were 279 anesthesia-related occurrences, comprised almost entirely of five specific events: vomiting, hypoxemia (SpO2 less than 94%), tachycardia, agitation, and myoclonus. Ketamine was associated with vomiting (14.6%), agitation (15.0%), and tachycardia (19.5%). Etomidate was also associated with vomiting (9.9%) and agitation (1.2%). Hypoxemia was rare except in the propofol group (15.7%) and was easily managed with supplemental oxygen, but occasionally required manually assisted ventilation via face mask. In patients receiving propofol vomiting (0.5%) and agitation (1.2%) were rare. CONCLUSIONS: Anesthesia with propofol, ketamine, or etomidate is safe and efficacious for children undergoing brief procedures. Propofol is associated with a decreased incidence of postanesthetic agitation and vomiting. Its association with respiratory depression is confirmed. PMID- 7546753 TI - Cardiovascular reflexes during anesthesia induction and tracheal intubation in elderly patients: the influence of thoracic epidural anesthesia. AB - STUDY OBJECTIVES: To determine whether thoracic epidural anesthesia performed prior to general anesthesia provides hemodynamic protection from the stress of laryngoscopy and tracheal intubation; to access the autonomic reflex response to epidural anesthesia, general anesthesia, and airway stimulation. DESIGN: Randomized unblind, controlled study. PATIENTS AND SETTING: 20 elderly (over 60) patients scheduled for colonic or gastric surgery at a university medical center. INTERVENTIONS: All patients (n = 10, in each group) underwent a standardized anesthesia induction sequence that included fentanyl 2 micrograms/kg, thiopental sodium 3 to 5 mg/kg (up to loss of eyelid reflex), and vecuronium 0.1 mg/kg followed by laryngoscopy and tracheal intubation. Before general anesthesia, thoracic epidural anesthesia was performed with plain 1% lidocaine in the epidural group. Preoperatively, baroreflex function was assessed by the Valsalva maneuver and the cough test. Spectral analysis of heart rate (HR) variability was performed before as well as during anesthesia. MEASUREMENTS AND MAIN RESULTS: There were no differences between the two groups in basal hemodynamics autonomic reflex status. Thoracic epidural anesthesia (median upper level at T2, median lower level at L2) was associated with stable hemodynamics, preservation of baroreflex sensitivity, and increased ratio of low to high frequency (LF/HF) components of HR variability, suggesting withdrawal of vagal activity. In both groups, general anesthesia induction was associated with decreased total HR variability and tracheal intubation was followed by increased LF/HF ratio, reflecting cardiac sympathetic activation. Patients with thoracic epidural anesthesia presented significant attenuation of the maximal rise in mean arterial pressure, and the increase in HR tended to be lower although not significantly. CONCLUSIONS: Thoracic epidural blockade combined with general anesthesia was associated with preserved baroreflex function, and it afforded hemodynamic protection during laryngoscopy and tracheal intubation. PMID- 7546754 TI - Desflurane controls the hemodynamic response to surgical stimulation more rapidly than isoflurane. AB - STUDY OBJECTIVE: To compare the control of hemodynamic response to surgical stimulus of desflurane to that of isoflurane. DESIGN: Prospective randomized study. SETTING: Operating room of a major U.S. teaching hospital. PATIENTS: 59 ASA status I, II, and III patients 18 to 80 years of age and were undergoing orthopedic or intra-abdominal surgical procedures of 1 or more hours in duration. INTERVENTIONS: Group 1 (n = 29) received desflurane in oxygen (O2) for their surgical procedure. Group 2 (n = 30) received isoflurane in O2 for their surgical procedure. Thiopental sodium 4 mg/kg and fentanyl 3 micrograms/kg provided induction; vecuronium 0.1 mg/kg facilitated intubation. Prior to incision the volatile anesthesia drug was titrated to maintain systolic blood pressure (SBP) within 20% of preinduction (baseline) values. Any time after incision, an SBP increase greater than 20% of baseline was treated with a 30% increase in inspired anesthetic concentration for 3 minutes, or until SBP was within 10% of baseline. Another three 30% increases were allowed at 3 minute intervals to return SBP to 10% of baseline. If four 30% increases did not return SBP to 10% of baseline, additional fentanyl up to 5 micrograms/kg or labetalol in 5 mg increments was given. MEASUREMENTS AND MAIN RESULTS: Measurement of hemodynamics and anesthetic concentration occurred every 2 minutes prior to skin incision and every 5 minutes thereafter. Measurement of hemodynamics and anesthetic concentration occurred every minute during treatment of blood pressure (BP) response to surgical stimulus. Desflurane allowed for more rapid control of BP response to surgical stimulus median 2 minutes (range 1 to 12 minutes) for desflurane versus 6 minutes (range 1 to 12 minutes, p = 0.011). The desflurane group required fewer 30% incremental anesthetic increases than the isoflurane group (1.8 versus 2.5, p = 0.016) to control increased SBP. End tidal/inspired drug concentration ratios were closer to unity in the desflurane patients both before (0.94 versus 0.80) and after (0.86 versus 0.70) changes in drug concentration to treat increased SBP. CONCLUSIONS: Anesthetic depth can be more rapidly titrated with desflurane compared to isoflurane. Alveolar/inspired concentration ratio approaches unity more rapidly with desflurane anesthesia. PMID- 7546755 TI - Propofol-based anesthesia as compared with standard anesthetic techniques for middle ear surgery. AB - STUDY OBJECTIVE: To determine if a total intravenous (i.v.) technique with propofol and fentanyl is superior to isoflurane anesthesia in patients undergoing middle ear surgery. DESIGN: Prospective, randomized study. SETTING: Inpatient otolaryngology service at a university medical center. PATIENTS: 102 ASA status I and II nonobese patients with no significant history of diabetes, chronic cholecystitis, neuropathy, or neuromuscular disorders that could produce delayed gastric emptying. INTERVENTIONS: Patients were admitted to the study and randomly divided into three equal groups. I.V. administration of thiopental sodium 5 mg/kg for induction of anesthesia followed by 60% air/oxygen (O2) with isoflurane 1% to 2% end-tidal for maintenance anesthesia (group 1). The same anesthetic was given as above, with the addition of droperidol 25 mcg/kg given after induction (group 2). I.V. administration of propofol 2 mg/kg for induction of anesthesia followed by propofol 50 to 250 mcg/kg/min for maintenance anesthesia. All groups received fentanyl 3 mcg/kg i.v. after induction. MEASUREMENTS AND MAIN RESULTS: Surgical duration, induction, maintenance, and total anesthesia times were recorded in addition to eye opening and extubation. Intergroup comparisons of postoperative nausea, vomiting, and pain were done, as were recovery scores using the Steward system. Patients receiving propofol had significantly less nausea than those receiving isoflurane only (4 of 34 versus 12 of 34, p < 0.05) as well as vomiting (2 of 34 versus 8 of 34, p < 0.05). Immediate recovery scores were significantly better for propofol compared with the isoflurane/droperidol group. Recovery at 30 minutes was also faster with propofol compared with isoflurane or isoflurane/droperidol (5.7 +/- 0.1 min versus 5.1 +/- 0.2 min and 5.2 +/- 0.2 min, p < 0.05). CONCLUSIONS: Propofol-fentanyl seems to be a better anesthetic than isoflurane-fentanyl in reducing the incidence of nausea and vomiting after middle ear surgery. Through the addition of droperidol to the isoflurane anesthetic seemed as effective, emergence from anesthesia was slower. For middle ear surgeries producing emesis, propofol-based anesthetics produced a rapid emergence with less nausea and vomiting. PMID- 7546756 TI - The incidence of aspiration associated with the laryngeal mask airway: a meta analysis of published literature. AB - STUDY OBJECTIVE: To determine the incidence of pulmonary aspiration with the laryngeal mask airway (LMA). DESIGN: A meta-analysis of all published literature on the LMA to September 1993. MEASUREMENTS AND MAIN RESULTS: All 547 publications were reviewed and coded, and those observational studies in which the LMA was the main form of airway management were analyzed. Pulmonary aspiration was defined as either the presence of bilious secretions or particulate matter in the tracheobronchial tree or, if bronchoscopy was not performed, a postoperative chest radiograph with infiltrates present on preoperative chest radiograph of physical examination. In the study population, there were 3 cases of aspiration in 12,901 patients, and when combined with four independent reports excluded from the detailed analysis, this gave a final incidence of 2 in 10,000. Ten confirmed pulmonary aspiration events from published case reports showed that most cases had one or more predisposing factors. No death of permanent disability occurred. CONCLUSIONS: The evidence to date suggests that the pulmonary aspiration with the LMA is uncommon and comparable to that for outpatient anesthesia with the face mask and tracheal tube. Meticulous attention to selection of low-risk patients and appropriate operative procedures and avoidance of light anesthesia should reduce the incidence even further. PMID- 7546757 TI - Computerized axial tomography to define the distribution of solution after stellate ganglion nerve block. AB - STUDY OBJECTIVE: To define the spread of local anesthetic after C6 stellate ganglion nerve block using computerized axial tomography (CAT). DESIGN: Prospective, open descriptive study. SETTING: Outpatient pain consult center. PATIENTS: 10 ASA status I patients undergoing stellate ganglion nerve blocks for sympathetically maintained pain. INTERVENTIONS: Radiocontrast and local anesthetic was given in 5 ml increments to 20 ml total volume for C6 stellate ganglion nerve blocks in eight patients and C7 in two patients. MEASUREMENTS AND MAIN RESULTS: CAT scanning was performed at baseline and after 5, 10, 15, and 20 ml of injectate was administered. Cervical level and pattern of injectate spread was recorded after each increment. Neck pressure above C6 did not promote caudal spread. One half of the injections were beneath prevertebral fascia. Injections on top of the fascia spread more diffusely around C6. All injections in high volume reached the medial aspect of T1 around the head, not neck, of the first rib. CONCLUSIONS: Solutions injected for C6 stellate ganglion nerve block concentrate medial to the stellate ganglion at T1. Thus, they must produce upper extremity sympathectomy by a mechanism other than contact with the ganglion. PMID- 7546758 TI - Cesarean section in a pregnant patient with an anterior mediastinal mass and failed supradiaphragmatic irradiation. AB - Nodular sclerosing Hodgkin's disease stages IA and IIA are the most common presentation of this disease during pregnancy. Patients presenting with late Hodgkin's disease with failed irradiation for cesarean section present a unique challenge. When this presentation occurs, a voluntary interruption of pregnancy is recommended. Upper body irradiation during the second trimester of pregnancy is recommended as well. We report a case involving a pregnant patient at 34 weeks' gestation presenting for cesarean section with a symptomatic anterior mediastinal mass occupying over 50% of the thoracic diameter. The anesthetic management was performed using continuous spinal. PMID- 7546759 TI - Major conduction anesthesia in a patient with Klippel-Trenaunay Syndrome. AB - The successful management of major conduction anesthesia in a patient with Klippel-Trenaunay syndrome is discussed. This case illustrates that major conduction anesthesia can be safely used if proper imaging studies are obtained, if one is aware of the underlying disease process, and if there is no port wine lesion in the dermatomal area corresponding to the spinal segment where the needle is to be inserted. PMID- 7546760 TI - Arterially misplaced Swan-Ganz catheter in a hypotensive and hypoxic COPD patient. AB - Arterial misplacement of the Swan-Ganz catheter occurs occasionally and usually can be easily detected. However, in some special clinical settings, the problem may become more complicated. We report a case of chronic obstructive pulmonary disease in which, because of severe hypoxemia, systemic hypotension, and pulmonary hypertension, conventional methods failed to recognize the misplacement, until confirmed with blood gas analysis. PMID- 7546761 TI - Hyskon induced pulmonary hemorrhage. AB - Hyskon, a distension medium composed of dextran 70 in 10% dextrose in water, is often used during hysteroscopic procedures. Coagulopathy and pulmonary edema are the most commonly reported side effects encountered during its use. We present the first case report in which a healthy patient developed pulmonary hemorrhage from Hyskon use. The major side effects of Hyskon are discussed, as are the possible etiologies of the reported complication and the implications for anesthesiologists. PMID- 7546762 TI - Pharmacologic drugs for controlled hypotension. AB - Due to the risks of transfusion reactions and the transmission of infectious diseases, there has been increased interest in measures to limit intraoperative blood loss and avoid the need for homologous transfusion. Controlled hypotension is one technique that has been used to limit intraoperative blood loss. Several drugs have been used alone or in combination for controlled hypotension, including the inhalational anesthetics, direct acting vasodilators such as nitroglycerin and nitroprusside, beta adrenergic antagonists, and calcium channel blockers. Various drugs available to the clinician for controlled hypotension are reviewed. PMID- 7546763 TI - Intrauterine pregnancy and aortic valve replacement. AB - Cardiopulmonary bypass (CPB) does not appear to cause excessive maternal risk, but the potential for fetal complications is of great concern. In general, operative intervention should be delayed until at least the second trimester. When this is not possible, ethical issues arise and a clash of maternal autonomy versus "fetal rights" ensues. This conflict is further complicated by maternal status changes that may accompany valvular disease or develop after CPB. The case described herein summarizes and discusses these conflicts. PMID- 7546764 TI - Davy comes to America: Woodhouse, Barton, and the nitrous oxide crossing. AB - In the final decade of the eighteenth century, a new method of medical treatment appeared in England when physician Thomas Beddoes developed a systematic application of Joseph Priestley's "factitious airs", or gases, to treat consumptive patients. Supported by peers such as Erasmus Darwin and using applications designed for him by James Watt and other inventors, Beddoes combined technological innovation and gas inhalation in an attempt to cure his patients. Late in the decade Beddoes hired young Humphry Davy as his assistant; Davy quickly added nitrous oxide to the armamentarium. The prominent group Davy assembled to help him test nitrous oxide and record their experiences has seldom been equaled in medical history as a research population. Davy left the Pneumatic Institution in March 1801 and joined the staff of the infant Royal Institution in London, but news of the nitrous oxide soon reached American. Physician James Woodhouse, a professor of chemistry at the University of Pennsylvania since July 1795, left in early 1802 for England, where he met Davy. A few years later one of his chemistry students preserved an account of the extensive nitrous oxide trials that took place in Woodhouse's classroom throughout 1807. In that work William Barton discussed and replicates Davy's research and agrees with his conclusions. Such intermittent experimentation continued in the United States and Europe until Horace Well's public demonstration of ether inhalation in January 1845. This paper describes how nitrous oxide inhalation survived in America through the work of Woodhouse and Wells. Traveling showmen like Samuel Colt and Gardner Quincy Colton demonstrated the gas' effects at popular lectures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546765 TI - Pulmonary edema following naloxone administration in a healthy orthopedic patient. PMID- 7546766 TI - Propofol and alfentanil mixture for outpatient surgery. PMID- 7546767 TI - Vascular endothelial growth factor mRNA increases in alveolar epithelial cells during recovery from oxygen injury. AB - Destruction of pulmonary endothelial cells is characteristic of hyperoxic lung injury. During recovery from hyperoxia, pulmonary endothelial cells proliferate to regenerate the vascular endothelium. Vascular endothelial growth factor (VEGF) is a peptide growth factor that is mitogenic specifically for endothelial cells. We hypothesized that VEGF messenger RNA (mRNA) increases during recovery from acute hyperoxic lung injury. Adult rabbits were exposed to 100% oxygen for 64 h and allowed to recover in air for 0, 1, 3, and 5 days. In situ hybridization showed increased VEGF expression in alveolar epithelial cells beginning at 1 day recovery. By 3 days recovery the message was in alveolar epithelial cells throughout the lung. Compared with alveolar epithelial cells, little or no expression was noted in large vessel endothelial cells, airway cells, or smooth muscle cells. Combined in situ hybridization for VEGF and immunostaining for macrophages and other mesenchymal cells found no VEGF message in those cell types. Isolated alveolar macrophages had no detectable VEGF message. Cells expressing VEGF mRNA were enriched in alveolar type II cell preparations from recovering lung. Double in situ hybridization for VEGF and surfactant protein-C (SP-C) showed co-expression in a population of type II cells, but with an inverse relationship: cells with abundant VEGF mRNA did not have abundant SP-C mRNA. Type II cells in vitro expressed VEGF message, but only when the SP-C message abundance was relatively low. We conclude that alveolar type II cells express increased VEGF mRNA during recovery from acute hyperoxia. These findings are consistent with a role for VEGF in regulating microvascular endothelial repair after oxidant injury. PMID- 7546768 TI - Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. AB - Gram-negative sepsis is the most common cause of the adult respiratory distress syndrome (ARDS). Lipopolysaccharide (LPS) when administered in vivo produces pathophysiologic changes similar to those seen in ARDS. The pathogenesis of these changes is mediated in part by oxidative stress. We demonstrate that LPS induces high mRNA levels of the stress-inducible gene heme oxygenase-1 (HO-1) in the rat lung. Increased HO-1 mRNA levels correlate with increased HO-1 protein and enzyme activity. Immunohistochemical analyses of lung tissues from rats treated with LPS reveal abundant HO-1 expression in inflammatory and bronchoalveolar epithelial cells. We further examined the molecular regulation of HO-1 gene expression after exposure of RAW 264.7 macrophage cells to LPS in vitro. These cells respond to LPS with increased HO-1 mRNA expression and HO-1 gene transcription. Transcriptional activation of the mouse HO-1 gene by LPS is mediated by a 5' distal enhancer fragment located approximately 4 kbp upstream from the transcription site. Electrophoretic mobility shift assays show increased activator protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS treatment. Mutation of the AP-1 binding site in this enhancer fragment completely abolishes HO-1 gene activation while mutation of CCAAT/enhancer-binding protein (C/EBP) binding site exerts negligible effect, suggesting that the AP-1 family of transcription factors plays a critical role in regulating HO-1 gene activation following LPS treatment. Furthermore, upstream phosphorylation events modulate this AP-1-dependent expression of the HO-1 gene after LPS treatment. PMID- 7546769 TI - Alterations of ambient oxygen tension modulate the expression of tumor necrosis factor and macrophage inflammatory protein-1 alpha from murine alveolar macrophages. AB - Tissue injury that occurs as a result of ischemia and subsequent reperfusion is characterized by endothelial cell injury, edema formation, and the influx of inflammatory leukocytes. Two macrophage-derived proinflammatory cytokines which may play a critical role in cellular injury and leukocyte recruitment/activation that occurs in the setting of ischemia-reperfusion injury are tumor necrosis factor alpha (TNF) and macrophage inflammatory protein-1 alpha (MIP-1 alpha). To determine if modulation of ambient oxygen tensions in vitro alters the expression of proinflammatory cytokines from activated macrophages, murine alveolar macrophages (AMO) were cultured in various combinations of ambient oxygen concentrations, then the supernatant fluid and cell pellet assayed for the presence of TNF and MIP-1 alpha messenger RNA (mRNA) and protein. We demonstrated that conditions of anoxia (95% nitrogen/5% CO2) or hyperoxia (95% oxygen/5% CO2) independently resulted in the increased expression of both TNF and MIP-1 alpha mRNA and protein from lipopolysaccharide (LPS)-stimulated AMO, as compared with cells cultured in room air. The specific culture condition of anoxia (x 6 h) followed by hyperoxia (x 18 h) produced the greatest increases in both TNF and MIP-1 alpha, suggesting that when following a period of anoxic priming, oxygen stress results in exaggerated cytokine production. A period of at least 4.5 to 6 h of anoxia prior to hyperoxic exposure was found to be the minimal time required for anoxic priming. Furthermore, the coincubation of LPS-treated AMO with dimethyl sulfoxide (DMSO) attenuated the anoxia-hyperoxia-induced increases in TNF and MIP-1 alpha mRNA by 23% and 34%, respectively. These findings suggested that alterations in ambient oxygen tension can regulate the expression of TNF and MIP-1 alpha from activated AMO, and that oxidant-related cytokine production may represent an important mechanism by which inflammation occurs in the clinical settings of ischemia-reperfusion injury and hyperoxia. PMID- 7546770 TI - Eicosanoid production in rabbit tracheal epithelium by adenine nucleotides: mediation by P2-purinoceptors. AB - Adenosine triphosphate (ATP) acting through epithelial nucleotide receptors exerts multiple physiologic actions on airway mucociliary clearance and caliber. However, the effect of ATP on arachidonate metabolism in the airway remains unknown. In this study, the ability of ATP to regulate eicosanoid production was studied in vitro in full-thickness rabbit tracheal strips and separately in rabbit epithelial explant cultures. In the freshly isolated strips, ATP increased prostaglandin E2 (PGE2) release in a dose-dependent fashion, with an activation threshold at 10 microM ATP and a 3.5-fold increase in PGE2 output at 1 mM ATP. Epithelium removal decreased 1 mM ATP-evoked PGE2 release by 68%. Reverse-phase, high-pressure liquid chromatography (HPLC) of media from 3H-arachidonic acid incubated epithelial explants exposed to 1 mM ATP demonstrated increased output of the cyclooxygenase products PGE2 and prostaglandin F2a (PGF2a). Other identifiable eicosanoids did not increase. The concentration-response for ATP induced PGE2 release by explants was similar to that of tracheal strips. PGE2 release by 1 mM ATP was 27% of that elicited by ionomycin (10 microM) and was markedly inhibited by indomethacin (10 microM). Purinoceptor agonist-stimulated PGE2 release by the epithelium yielded a rank order of potency of uridine triphosphate (UTP) > or = ATP > 2-methylthio-ATP (2MeSATP) >> alpha,beta methyleneadenosine-5'-triphosphate (AMP-CPP) > or = adenosine. These results indicate that ATP, acting primarily through an epithelial P2-purinoceptor similar to the P2a subtype, stimulates eicosanoid metabolism in rabbit airway epithelium via the cyclooxygenase pathway, producing PGE2 as the predominant species. PMID- 7546771 TI - Downregulation of lysyl oxidase in cadmium-resistant fibroblasts. AB - Lysyl oxidase, a copper-dependent metalloenzyme, plays a central role in crosslinking of collagen and elastin in the extracellular matrix. Notably, lung lysyl oxidase activity is markedly stimulated in rats exposed to cadmium vapors. To further understand the mechanism of cadmium toxicity, the mRNA expression, synthesis, post-translational processing, and catalytic activity of lysyl oxidase were examined in cadmium-resistant (CdR) cells and the cadmium-sensitive Swiss mouse 3T3 cells from which they were derived. These CdR cells synthesized and accumulated markedly elevated levels of metallothionein, a known marker for cadmium resistance, whereas the expression of lysyl oxidase was reduced considerably. In comparison to the parental, cadmium-sensitive cells, the suppression of enzyme production in the CdR cells was seen at the mRNA level, at the levels of intracellular proprotein production and mature enzyme secreted into the medium, and in terms of total enzyme activity in the culture. The presence of cupric chloride in the culture medium during the incubation of the CdR cells for 16 h significantly enhanced lysyl oxidase activity accumulating in the medium, suggesting that lysyl oxidase deficiency in CdR cells may be related to abnormal copper metabolism. PMID- 7546772 TI - Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived granulocyte macrophage colony-stimulating factor. AB - The presentation and recognition of foreign antigen is the critical initial event in the development of local immunity. In the lung, antigen-presenting cell activity is largely attributable to pulmonary dendritic cells (DC) that are distributed along the airways and throughout the pulmonary interstitium in close proximity to overlying alveolar epithelial cells. To test the hypothesis that DC immunostimulatory activity might be locally regulated by overlying alveolar epithelial cells, we evaluated the ability of rat type II alveolar epithelial cells to influence the capacity of purified rat pulmonary DC to stimulate T-cell proliferation in an allogeneic, mixed leukocyte reaction. We found that alveolar epithelial cells greatly enhanced the ability of dendritic cells to induce T-cell proliferation. This effect on DC immunostimulatory activity was mediated by a soluble factor preferentially secreted from the basolateral epithelial cell surface. Alveolar epithelial cultures were found to express mRNA for granulocyte macrophage colony-stimulating factor (GM-CSF), and blocking antibodies against GM CSF partially neutralized the effect of epithelial cell-conditioned media on DC stimulatory activity, indicating that the effect was due at least in part to alveolar epithelial cell-derived GM-CSF. Through the polar secretion of GM-CSF, alveolar epithelial cells may play an important role in creating distinct immunologic environments within the lung. PMID- 7546773 TI - Catalase and glutathione reductase protection of human alveolar macrophages during oxidant exposure in vitro. AB - Because alveolar macrophages generate and release reactive oxygen metabolites but also contain antioxidative enzymes, they have the potential of either damaging or protecting tissues. We investigated the relative role of the hydrogen peroxide (H2O2)-scavenging antioxidative enzymes in H2O2 disposal and cell protection using freshly isolated (5 h ex vivo) and overnight (24 h ex vivo) cultured human alveolar macrophages. Cell protection was assessed on the basis of maintenance of cellular high-energy phosphates, leakage of intact nucleotides into the extracellular medium, and appearance of the nucleotide catabolic products xanthine, hypoxanthine, and uric acid. To investigate the relative importance of catalase and the glutathione redox cycle, the experiments were conducted in cells pretreated with amino-triazole (ATZ) to inactivate catalase or with 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase. Catalase, glutathione peroxidase, and glutathione reductase activities did not change significantly during overnight culture of the cells. Both freshly isolated and cultured cells consumed exogenous H2O2 mainly by the catalase-dependent pathway. When the cells were exposed to H2O2 (100 microM), catalase and the glutathione redox cycle equally participated in maintaining cellular high-energy nucleotides. However, when cultured cells were exposed to formylated peptide (FMLP) (10(-7) M), the glutathione redox cycle was responsible for the maintenance of high energy nucleotides. Furthermore, in both exposures, the glutathione redox cycle was more important in maintaining cell membrane integrity and preventing nucleotide leakage from the cells. Immunocytochemical labeling showed that catalase was primarily localized in the peroxisomal compartment of these cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546774 TI - Nitric oxide increases cellular glutathione levels in rat lung fibroblasts. AB - Nitric oxide (NO) has been demonstrated to play a protective role in cell injury. In this study, we have explored the effect of NO and two NO donors (sodium nitroprusside [SNP] and isosorbide dinitrate [ISDN]) on cellular glutathione (GSH) levels in a rat lung fibroblast cell line (RFL6 cells). SNP and ISDN significantly increased cellular GSH in RFL6 cells (5 x 10(-4) M SNP: 21.9 +/- 3.6 nmol/10(6) cells and 5 x 10(-3) M ISDN: 27.6 +/- 1.7 nmol/10(6) cells versus control: 13.2 +/- 0.4 nmol/10(6) cells; P < 0.05). The stimulatory effect of SNP and ISDN on GSH was first seen at 6 h and peaked at 12 to 24 h. A similar increase in GSH was observed in RFL6 cells exposed to 400 ppm NO for 7.5 h (NO: 20.5 +/- 3.4 nmol/10(6) cells versus control: 11.9 +/- 2.4; P < 0.05). SNP and ISDN also increased cellular GSH in bovine pulmonary artery smooth muscle cells (BPSMC) and bovine pulmonary artery endothelial cells (BPAEC). Buthionine sulfoximine (BSO) (0.01 mM), an inhibitor of the GSH synthetic enzyme gamma glutamyl cysteine synthetase, blocked the increase in GSH in RFL6 cells seen with both SNP and ISDN. In BPAEC, exposure to NO donors for 24 h stimulated glutamate uptake (SNP: 441 +/- 19 pmol/10 min/10(6) cells and ISDN: 677 +/- 48 pmol/10 min/10(6) min/10(6) cells versus control: 222 +/- 9 pmol/10 min/10(6); P < 0.05). This effect paralleled the increase in GSH. In RFL6 cells, only SNP increased glutamate uptake after 24 h of incubation. In summary, NO and NO donors increase cellular GSH in RFL6 cells, BPAEC, and BPSMC. The mechanism of this effect is unclear but may involve upregulation of the normal GSH synthetic pathways. This observation may explain in part the protective effect of NO seen in some cell culture systems and may contribute to a protective effect against oxidant injury in vivo. PMID- 7546775 TI - Inhibition of acetylcholine-evoked Cl- currents by 14-membered macrolide antibiotics in isolated acinar cells of the guinea pig nasal gland. AB - Recent clinical and experimental studies demonstrated that 14-membered macrolides suppressed fluid secretion from respiratory tract mucosa. The cellular mechanisms of acetylcholine (ACh)-induced electrolyte secretion in the acinar cell isolated from the guinea pig nasal gland were investigated by using a microfluorimetric imaging method and a patch-clamp whole-cell recording. The ACh-induced increase in intracellular Ca2+ concentration measured by the fura-2 method was little affected by three types of macrolides, josamycin (JM), erythromycin (EM), and roxythromycin (RXM). The ACh-evoked ionic currents were not inhibited by a 16 membered macrolide, JM, whereas both 14-membered macrolides, EM and RXM, significantly inhibited membrane conductance, especially inward currents, in a concentration-dependent manner. The ACh-induced Cl- current isolated by the ionic substitutional experiments was remarkably inhibited by EM and RXM. The order of inhibition was RXM > EM >> JM. Thus, 14-membered macrolides showed a direct inhibition of the Cl- conductance activated by ACh. PMID- 7546776 TI - Interleukin 1 beta (IL-1 beta) and the IL-1 beta-alpha 2-macroglobulin complex upregulate the platelet-derived growth factor alpha-receptor on rat pulmonary fibroblasts. AB - Fibroblasts are the primary proliferating cell type in pulmonary fibrosis. We previously showed that inorganic, fibrogenic particles alter the platelet-derived growth factor (PDGF) receptor system on rat lung fibroblasts (Bonner, J. C., et al. 1993, J. Clin. Invest 92:425-430). In lung fibroblasts, PDGF is the most potent proliferative cytokine, and the responses to PDGF isoforms depend on the relative amounts of two PDGF receptors (PDGF-R alpha and PDGF-R beta). Interleukin 1 beta (IL-1 beta) production by lung macrophages is increased following exposure to fibrogenic particles. We have examined the role of IL-1 beta in regulating the lung fibroblast PDGF receptor system. IL-1 beta induced a 10-fold increase in the number of binding sites for [125I]PDGF-AA, caused a 2 fold increase in affinity of [125I]PDGF-AB, but it had no effect on [125I]PDGF-BB binding. PDGF-R alpha gene expression was increased 5-fold after 4 h of IL-1 beta treatment. IL-1 beta increased the proliferative and chemotactic response to PDGF isoforms in the following order of potency: AA > AB > BB. IL-1 beta was tested for its ability to cause increased [125I]PDGF-AA binding when complexed to its binding protein, alpha 2-macroglobulin (alpha 2M). IL-1 beta bound covalently to fast methyl-amine-activated alpha 2M (alpha 2M-MA). IL-1 beta-alpha 2M-MA or alpha 2M-MA alone possessed minimal activity for inducing an increase in [125I]PDGF-AA binding. However, treatment of the IL-1 beta-alpha 2M complex with thioredoxin, which released bioactive IL-1 beta that was covalently bound to alpha 2M, maximally increased [125I]PDGF-AA binding to the same extent as free IL 1 beta. These results indicate that the fibroblast response to PDGF isoforms is modulated by a complex interaction involving IL-1 beta, alpha 2M, and thioredoxin, all of which are produced in vivo by activated macrophages. PMID- 7546777 TI - Insulin-like growth factor-I (IGF-I) regulates IGFBP-3 and IGFBP-4 by multiple mechanisms in A549 human adenocarcinoma cells. AB - The insulin-like growth factors (IGF-I and IGF-II) participate in the control of cell proliferation in normal and neoplastic lung cells. To examine the role of IGF binding proteins (IGFBPs) in modulating IGF actions in lung, we examined the production and regulation of IGFBPs from A549 cells, a human adenocarcinoma derived lung cell line. Ligand blot and immunoblot analysis of conditioned media (CM) from A549 cells demonstrated IGFBP bands of relative molecular mass (M(r)) approximately 39-43,000 (IGFBP-3), 34,000 (IGFBP-2), 30,000 (IGFBP-1), and 24,000 (IGFBP-4). IGFBP-3 abundance in A549 cell CM increased following exposure to IGF I and IGF-II (3.0- and 1.8-fold, respectively) without a change in IGFBP-3 transcript abundance, suggesting IGFBP-3 is post-transcriptionally regulated. Cycloheximide almost completely abrogated the IGF-I-stimulated increase in CM IGFBP-3, suggesting that ongoing protein synthesis is necessary for the IGF-I stimulated increase in IGFBP-3 abundance. Increases in IGFBP-3 occurred by at least two mechanisms, through activation of the type 1 IGF receptor and by a type 1 IGF receptor independent mechanism. The increase in IGFBP-3 was due, in part, to activation of the type 1 IGF receptor because blocking type 1 IGF receptor activation with an antibody (alpha IR3) diminished the IGF-I-induced increase in IGFBP-3 and insulin, at doses that stimulate the type 1 IGF receptor, increased IGFBP-3 abundance. The increase in IGFBP-3 was partially independent of type 1 IGF receptor activation because [QAYL]-IGF-I, an analog of IGF-I that binds the type 1 IGF receptor but not IGFBP-3, was less potent than IGF-I in stimulating IGFBP-3 abundance, and IGF-II, which binds IGFBP-3 normally, but binds the type 1 IGF receptor with lower affinity than IGF-I, was nearly equipotent to IGF-I in its stimulation of IGFBP-3 accumulation at low concentrations. These results suggest that ligand binding decreases IGFBP-3 clearance or increases IGFBP-3 accumulation in CM. IGF-I decreased IGFBP-4 abundance in A549 cell CM without decreasing IGFBP-4 mRNA transcripts and without increasing the amount of cell associated IGFBP-4. To determine whether the decrease in IGFBP-4 was due to increased degradation, cell-free CM was incubated with and without IGF-I, and IGFBP-4 abundance measured by ligand and immunoblot analyses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7546778 TI - Cellular infiltration and eicosanoid synthesis in brown Norway rat lungs after allergen challenge. AB - Allergen challenge of sensitized Brown-Norway (BN) rats results in increased excretion of cysteinyl-leukotrienes (cLTs) in bile. It is unclear whether this reflects an increased capacity of lung cells to synthesize 5-lipoxygenase products, and, if so, which cells are of primary importance. We have examined the effects of allergen challenge on the capacity of a mixture of isolated lung cells from ovalbumin (OA)-sensitized BN rats to synthesize LTs and other eicosanoids. Cells were isolated by enzymatic digestion of lung tissue before and either 6 or 24 h after challenge of sensitized rats with either OA or saline. A23187-induced synthesis of eicosanoids by these cells was measured using high-pressure liquid chromatography. OA challenge resulted in a significant influx of neutrophils into the lungs and a significant increase in the synthesis of 5-lipoxygenase products, in particular LTB4, by lung cells after 6 h. There was a positive correlation between the percentage of neutrophils in unfractionated lung cells and the amounts of LTB4 produced by these cells. OA challenge had little or no effect on the production of cLTs and the cyclooxygenase product 12-hydroxy-5,8,10 heptadecatrienoic acid. There was a significant increase in the infiltration of eosinophils into the lungs 24 h after OA challenge but no increase in the production of cLTs by lung cells at this time, suggesting that eosinophils from BN rats are unlikely to be the major site for the production of these substances. This was confirmed in experiments with partially purified eosinophils obtained from Sephadex-treated rats. In contrast, cLTs were major products of arachidonic acid metabolism by alveolar macrophages from BN rats. We conclude that allergen challenge results in an increased capacity of lung cells to synthesize 5 lipoxygenase products, in particular LTB4. Macrophages, rather than eosinophils, may be an important site for the synthesis of cLTs in BN rat lungs. PMID- 7546779 TI - Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice. AB - A murine pulmonary infection model utilizing intratracheal inoculation of Cryptococcus neoformans was used to analyze cytokines produced in response to opportunistic pathogens acquired via the respiratory tract. The specific question asked was whether early cytokine secretion in lung-associated lymph nodes (LALN) would predict whether this organism would be cleared from the lung. Lung colony forming units (CFU) were analyzed in two strains of mice over 12 wk, and lung clearance was found to be strain dependent. C.B-17 mice reduced their lung CFU burden between day 7 and day 14 of infection, had significantly higher in lung CFU than C.B-17 mice. The capacity of cells from lungs and LALN to secrete cytokines was significantly different between the strains when assessed at day 7 and day 14 after inoculation. When compared with sensitive C57BL/6 mice 7 days after infection, resistant C.B-17 mice demonstrated (1) increased interferon gamma secretion by LALN cells in vitro in response to media alone, heat-killed cryptococci, and the T cell mitogen concanavalin A and (2) increased interleukin (IL)-2 secretion by both LALN and lung cells in response to concanavalin A. IL-4 and IL-10 were comparable or undetectable in both mouse strains, whereas IL-5 was significantly higher in all lung cell cultures of C57BL/6 mice. Thus, an early regional Th1 immune response in C.B-17 mice correlated with resistance to the organism, whereas the absence of this response in C57BL/6 mice correlated with susceptibility. PMID- 7546780 TI - Transforming growth factor beta 1 downregulates the platelet-derived growth factor alpha-receptor subtype on human lung fibroblasts in vitro. AB - Fibroblasts are the central target cell in pulmonary fibrotic diseases, and their proliferation is mediated largely by platelet-derived growth factor (PDGF) isoforms secreted by activated lung macrophages. Several other macrophage-derived cytokines that are increased during fibrogenesis, including interleukin-1 beta and transforming growth factor-beta 1 (TGF-beta 1), could potentially modulate the mitogenic and chemotactic activity of PDGF by altering the expression of cell surface PDGF receptors on fibroblasts. The PDGF receptor system on fibroblasts from a variety of tissues shows heterogeneous responses to TGF-beta 1. Lung fibroblasts have not been investigated in this regard. TGF-beta 1 downregulated the gene expression of the 6.5 kb PDGF-alpha receptor (PDGF-R alpha) transcript in normal human lung fibroblasts in a concentration-dependent fashion that was maximal at 3 ng/ml TGF-beta 1; this corresponded with a decrease in cell-surface PDGF-R alpha as measured by radioligand binding assays using [125I]PDGF-AA. The TGF-beta 1-induced down-regulation of the PDGF-R alpha gene was rapid (maximal suppression by 2 h post-treatment) and preceded the decrease in cell-surface alpha-receptor (maximal reduction by 6 h post-treatment). TGF-beta 1 treatment did not alter the rate of PDGF-R alpha mRNA degradation following the inhibition of transcription using actinomycin D, indicating that TGF-beta 1 increases PDGF-R alpha transcription. Scatchard analysis of saturation binding data showed that TGF-beta 1 decreased the number of [125I]PDGF-AA binding sites 5-fold without affecting receptor affinity. [125I]PDGF-AB binding sites were downregulated approximately 25%, and the number of [125I]PDGF-BB binding sites was not changed by TGF-beta 1 treatment, indicating that the PDGF-beta receptor was not affected. TGF-beta 1 reduced the mitogenic and chemotactic response to PDGF-AA by > 90%, whereas these biologic response to PDGF-AB and PDGF-BB were inhibited 50% to 80%. The proliferative and chemotactic responses of fibroblasts during tissue remodeling or during lung fibrosis are likely controlled by a complex network involving PDGF isoforms and cytokines that modify the PDGF receptor system. PMID- 7546781 TI - Integrated backscatter and digital acquisition during myocardial contrast echocardiography: is there an advantage over conventional echocardiography for intracoronary injections? AB - This study was designed to answer the question of whether, despite their theoretic superiority, integrated backscatter imaging (IBS) and digital data acquisition (DA) offer any advantage over conventional echocardiography (CE) during quantitative myocardial contrast echocardiography. In vitro experiments were performed (1) to determine the microbubble concentration versus videointensity relationships for CE and IBS and (2) to define the relationship between flow through and microbubble transit rates for CE and IBS. These data were stored on videotape. In vivo experiments were performed whereby microbubbles were injected into the left anterior descending artery at different flow rates in 14 dogs and IBS and CE data were stored both in digital format and on videotape. Although the level of compression did not affect the microbubble concentration versus videointensity plots during IBS compared with CE, in practical terms the mean transit rate, peak intensity, and area under the curve were not affected by the level of compression for both forms of imaging as long as the postprocessing used for CE imaging was linear and the microbubble dose was small. In addition, although DA resulted in higher peak intensity and area under the curve compared with storage on videotape because of its broader dynamic range, the correlation between these measurements was excellent with both forms of image storage. We conclude that, although differences exist between CE and IBS and between Da and analog acquisition, these differences do not significantly affect the derivation of parameters from time-intensity plots during myocardial contrast echocardiography when contrast material is injected into a coronary artery. PMID- 7546782 TI - Myocardial contrast echocardiography in experimental coronary artery occlusion with a new intravenously administered contrast agent. AB - A new intravenously administered ultrasound contrast agent was studied in eight dogs during intermittent coronary artery occlusion. The area of the myocardial contrast defect was compared with that of the acute wall motion abnormality induced by coronary occlusion. A close correlation was found between these two independent measures of acute myocardial ischemia. The peak change in myocardial intensity during coronary occlusion was significantly less than for the same segment before ischemia and for a remote nonischemic segment. This new, intravenously administered ultrasound contrast agent can be used to evaluate the spatial distribution of hypoperfused myocardium and should therefore prove valuable in the clinical evaluation of ischemic syndromes. PMID- 7546783 TI - Cardiac cycle-dependent gray-level variation is not distorted by abnormal septal motion after cardiac surgery: a transesophageal videodensitometric study in humans. AB - Variation in cyclic (systolic-to-diastolic) echodensity has been demonstrated to be a reliable index of preserved myocardial function. Paradoxic septal motion can be observed frequently after cardiac surgery in the absence of any functional impairment. The aim of the study was to evaluate whether regional cycle-dependent gray-level variation in the septum is affected by abnormal septal motion after cardiac surgery. Ten patients undergoing cardiac surgery for coronary artery bypass grafting were evaluated by continuous transesophageal echocardiographic monitoring from the transgastric approach, both before and after surgery. In each patient septal motion was assessed qualitatively as normal or paradoxic. Images were digitized off-line and cyclic gray-level variation was measured in each patient by means of dedicated software. By selection, all patients exhibited normal septal motion and thickening at baseline. After surgery, five patients showed a paradoxic septal motion (group I), whereas in the remaining five patients (group II) septal motion remained normal. Percent area change, measured with the floating center-of-mass reference system, was similar in the two groups both before (I = 42% +/- 7% versus II = 44% +/- 13%; difference not significant) and after surgery (I = 39% +/- 8% versus II = 40% +/- 1%; difference not significant). Cyclic gray-level variation was also similar in the two groups, both at baseline (group I = 61% +/- 16% versus group II = 68% +/- 18%; difference not significant) and after surgery (50% +/- 13% versus 57% +/- 16%; difference not significant).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546784 TI - Dobutamine and dipyridamole stress echocardiography in patients with a low incidence of severe coronary artery disease. AB - The aim of this study was to determine the relative sensitivity, specificity, accuracy, and tolerance of dobutamine and dipyridamole stress echocardiography in patients with a lower likelihood of severe coronary artery disease. Previous comparative studies, which included patients with a history of myocardial infarction or a high incidence of coronary artery disease, showed both methods to have similar and acceptable diagnostic accuracy. To assess the role of these agents in evaluating patients with a lower likelihood of significant coronary artery disease, a lower-risk group was selected by excluding patients with known coronary artery disease, myocardial infarction, unstable angina, or strongly positive stress test results. Dobutamine and dipyridamole stress echocardiographic studies were performed in random order, before coronary angiography. Of the 46 patients enrolled (31 men and 15 women), 24 had atypical chest pain or none at all. Coronary angiography revealed no significant disease in 22 (48%), single-vessel disease in 11 (24%), and multivessel disease in only 13 patients (28%). Dobutamine and dipyridamole stress echocardiography were equally well tolerated, with identical accuracy (76%) that was maintained in patients with atypical symptoms. This confirms the usefulness of both dobutamine and dipyridamole stress echocardiography in evaluating patients with suspected coronary artery disease and extends this role to a lower-risk group for severe disease who often have atypical symptoms. The choice of which agent is used should reflect an institution's experience. PMID- 7546785 TI - Normal changes in left ventricular filling and hemodynamics during dobutamine stress echocardiography. AB - During high-dose dobutamine infusion, there is a dose-dependent increase in cardiac output, a reduction in systemic vascular resistance, a reduction in left ventricular size, an increase in the mitral A wave velocity, a reduction in the E/A velocity ratio, and a reduction in the isovolumic relaxation time, with little change in the rate-corrected isovolumic relaxation time. Left ventricular cavity obliteration is common. This information may be useful in defining the mechanism of hypotension commonly seen during dobutamine stress echocardiography. It is speculated that the diastolic behavior of the left ventricle during dobutamine stress echocardiography, especially the isovolumic relaxation time, may provide an additional indicator of early myocardial ischemia, but this needs to be confirmed by larger independent studies. PMID- 7546786 TI - Patterns of pulmonary venous and transmitral flow velocity in patients with hypertrophic cardiomyopathy. AB - This study determines the characteristics of left atrial filling from the pulmonary veins in 80 patients with hypertrophic cardiomyopathy by transthoracic echocardiography. Patients in sinus rhythm (n = 72) showed a typical triphasic pattern with a systolic phase (J) and diastolic phase (K) and a retrograde surge of flow during atrial contraction. In sinus rhythm, three characteristic patterns of pulmonary venous flow in hypertrophic cardiomyopathy were observed: (1) an increased peak J wave relative to the K wave with J/K ratios greater than 1 (n = 43; 60%), (2) equal peak J and K phases (n = 7; 10%), and (3) decreased J wave relative to K ratios and J/K less than 1 (n = 22; 30%). The J/K ratio correlated inversely with the E/A ratio (r = -0.64; p < 0.01). In eight patients with atrial fibrillation, left atrial filling occurred primarily in diastole (K phase). Combined use of mitral and pulmonary venous flow may be optimal for the noninvasive detection of abnormal left ventricular filling in patients with hypertrophic cardiomyopathy. PMID- 7546787 TI - Transesophageal echocardiographic evaluation of mitral regurgitation in hypertrophic cardiomyopathy: contributions of eccentric left ventricular hypertrophy and related abnormalities of the mitral complex. AB - This study was designed to evaluate the contribution of eccentric left ventricular hypertrophy and its related organic and spatial abnormalities of the mitral complex to the occurrence of mitral regurgitation in patients with hypertrophic cardiomyopathy We selected 45 consecutive patients with systolic mitral regurgitation by color Doppler echocardiography and performed transesophageal echocardiography in all patients. Eighteen patients were in the obstructive group and 27 patients were in the nonobstructive group of hypertrophic cardiomyopathy with asymmetric septal hypertrophy. Twenty subjects without any cardiac disorders served as the control group. The maximum area of mitral regurgitation was significantly greater in the obstructive group than in the nonobstructive group. Mitral regurgitation appeared more frequently during pansystole in the two groups with hypertrophic cardiomyopathy, particularly in the obstructive group. Mitral valve prolapse was observed in 20 (44%) of the 45 patients with hypertrophic cardiomyopathy. Distances between the posterior papillary muscle and anterior or posterior mitral anulus were significantly smaller in the two groups with hypertrophic cardiomyopathy than in the normal control group. In the obstructive group, the length of the anterior mitral leaflet and the thickness of the rough zone of the anterior mitral leaflet at mid diastole were significantly greater than in the other groups. Systolic anterior motion was observed in all patients with obstructive cardiomyopathy and contact between the interventricular septum and the anterior mitral leaflet during early diastole was observed in 17 of the 18 patients in the obstructive group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546788 TI - Substitution of left ventricular outflow tract diameter with prosthesis size is inadequate for calculation of the aortic prosthetic valve area by the continuity equation. AB - It remains uncertain whether prosthetic ring size should be used interchangeably with measured left ventricular outflow tract (LVOT) diameter in the continuity equation to estimate the aortic prosthetic valve area by transthoracic Doppler echocardiography. To determine the difference in area caused by this substitution, the area of the prosthetic valve was calculated in 143 patients with aortic bioprostheses by use of the standard continuity equation with the measured LVOT diameter (LVOT method) and then with the bioprosthetic size (size method). Compared with known in vitro prosthetic valve areas, the LVOT method (r = 0.86; standard error of the estimate +/- 0.16 cm2; p < 0.001) was more accurate than the size method (r = 0.74; standard error of the estimate +/- 0.40 cm2; p < 0.001). The prosthetic valve area estimated by the size method overestimated the area estimated by the LVOT method by an average of 15% +/- 23% (p < 0.001). This difference in area between the two methods has increased with the interval since implantation of the bioprosthesis (p = 0.01). It is concluded that prosthetic size should not be used instead of LVOT diameter during calculation of aortic prosthetic valve area. This restriction is particularly important in patients with older bioprosthesis. PMID- 7546789 TI - Echocardiographic evaluation of the coronary sinus. AB - The purpose of this study was to compare transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE) in the evaluation of the coronary sinus and its blood flow. Forty patients were studied by TTE and TEE. The distal coronary sinus and its right atrial communication could be identified in 21 of 40 by TTE, and in all patients by TEE. Coronary sinus diameter measurement at the right atrial communication was possible by TTE in 16 of 40, and in all patients by TEE (maximal diameter 6 to 14 mm, mean 9 +/- 2). Flow velocity measurement by pulsed Doppler was possible in 25 of 40 patients (63%) by TEE, and in none by TTE. The flow velocity pattern was similar to central vein flow velocity, with systolic and diastolic antegrade waves, and a small retrograde end diastolic wave. The coronary sinus cross-sectional area was measured in 5 patients by intravascular ultrasound. It varied in size and shape during the cardiac cycle, reaching a maximum (0.3 to 1.5 cm2) at end diastole, and decreasing by 40% to 70% at end systole. TEE is superior to TTE in the evaluation of the coronary sinus and its blood flow velocity. However, because of the variability in cross sectional area size and shape, measurement of coronary sinus blood flow may be inaccurate. PMID- 7546790 TI - New miniaturized versus conventional biplane transesophageal transducers: recent clinical experience in adults. AB - A subset of patients have substantial discomfort on examination with transesophageal echocardiography with the conventional probe, whereby the dimensions of the probe play a decisive role. Miniaturized biplane transducers have recently become available (2 x 32 channels, dimensions 9.5 x 8.7 mm, and circumference approximately 30% less than the conventional echoscope) and allow ultrasound examination at 3.5, 5.0 and 7.0 MHz. A prospective study was carried out in 90 patients to compare difficulties on insertion of the probe, subjective evaluation by the patient during examination, and the two-dimensional image, as well as Doppler and color-coded Doppler quality of the miniaturized biplane versus the conventional probe. In 62 patients, intubation of the esophagus proved less difficult with the smaller instrument and more difficult in nine cases. Seventy-six patients reported that they suffered less discomfort on use of the narrow instrument. Concomitant parasympatholytic medication was needed with the smaller probe in seven cases and 17 times with the conventional probe. As anticipated, quality of the two-dimensional image attained by the miniaturized probe was lower. With transmit/receive frequency of 7.0 MHz, however, image resolution was excellent in the near field of 5 cm and nearly equivalent to that of the conventional probe (5.0 MHz). Pulsed-wave and continuous wave Doppler and color-coded Doppler information from both probes was similar in quality. Whenever examination with a conventional transesophageal transducer promises to be difficult, or when sedation is contraindicated because of a severe illness or respiratory insufficiency, transesophageal echocardiography should be considered with a smaller biplane probe at higher transmit-receive frequencies. PMID- 7546791 TI - Ventricular interdependence during Valsalva maneuver as seen by two-dimensional echocardiography: new insights about an old method. AB - Two-dimensional echocardiography was used in 15 normal volunteers to assess left (LV) and right ventricular (RV) responses, as well as their interdependence, during the Valsalva maneuver. During the strain phase, LV and RV areas decreased progressively, the RV area decreasing more than the LV area. Immediately after strain release, the RV end-diastolic area increased suddenly and dramatically to 143.3% +/- 9.4% of its baseline value, whereas the LV end-diastolic area decreased further. This transiently overloaded right ventricle and associated septal shift changed LV shape and further reduced the LV cross-sectional area. Thus the resulting momentary drop in the stroke LV area may contribute, along with pulmonary blood pooling, to the abrupt systemic blood pressure drop characteristic of phase III seen in normal subjects. Real-time imaging with echocardiography during respiratory maneuvers is feasible for clinical use. Its application in patients with congestive heart failure might bring further understanding of LV and RV interrelationships in the failing heart. PMID- 7546792 TI - Giant blood cyst of the aortic valve. AB - Beyond infancy, blood cyst of the aortic valve is not known to occur. We describe a 16-year-old girl who had aortic valve stenosis and regurgitation and giant blood cyst of the aortic valve. Serial echocardiograms over a 12-year period demonstrated gradual enlargement of the cyst. PMID- 7546793 TI - A rare complication of aortic valve endocarditis: diagnosis with transesophageal echocardiography. AB - Rupture of the membranous interventricular septum resulting in a left ventricular to right atrial shunt is a rare complication of endocarditis. Early recognition of this complication is essential because these patients require early surgical intervention. We report a case of a left ventricular-right atrial shunt complicating aortic valve endocarditis in which transesophageal echocardiography was used to accurately diagnose the lesion before surgery. We will discuss the role of transesophageal echocardiography in the diagnosis of this lesion and will review the existing literature pertaining to this rare complication. PMID- 7546794 TI - Right ventricular diastolic collapse without hemodynamic compromise in a patient with large, bilateral pleural effusions. AB - A 22-year-old woman was admitted to the hospital with large bilateral pleural effusions and venous thromboembolism. Echocardiography revealed right ventricular diastolic collapse (RVDC) without physical signs of cardiac tamponade. This echocardiographic abnormality disappeared after thoracentesis. The results of this case report would suggest that pleural effusions were responsible for the echocardiographic finding of RVDC. Presence of RVDC in patients without clinical evidence of cardiac tamponade should alert physicians to look for pleural effusion. Echocardiographic reevaluation after thoracentesis should precede pericardiocentesis. PMID- 7546795 TI - Primary left ventricular mural endocarditis diagnosed by transesophageal echocardiography. AB - Primary left ventricular mural abscess was detected by transesophageal echocardiography and was confirmed at necropsy in a 44-year-old woman with Staphylococcus aureus bacteremia and cerebrovascular embolism. In two occasions, transthoracic echocardiography failed to show the mural abscess in this patient. Because of the aggressive nature of primary mural endocarditis, early use of transesophageal echocardiography is recommended in patients with Staphylococcal bacteremia and suspected endocarditis even in the absence of valvular abnormalities detectable by the transthoracic approach. PMID- 7546796 TI - Inverted left atrial appendage mimicking a left atrial mass during mitral valve repair. AB - This case report describes the appearance of a new homogenous mass seen in the left atrium on intraoperative transesophageal echocardiography after mitral valve repair. This "mass" was the inverted left atrial appendage. Echocardiographers need to recognize normal variants, such as an inverted left atrial appendage, to avoid misdiagnosis. PMID- 7546797 TI - Identification of a malpositioned atrial pacemaker lead across a patent foramen ovale by transesophageal echocardiography. AB - Transesophageal echocardiography was used for defining the unusual left atrial location of a malpositioned atrial pacemaker lead. This malpositioning occurred in a 75-year-old woman who had a small atrial septal aneurysm and a patent foramen ovale. Neither the precise location of the pacemaker lead tip nor the course of the lead was apparent by radiographic or transthoracic echocardiographic techniques. This report extends the potential utility of transesophageal echocardiography for the identification of malpositioned atrial pacemaker leads. PMID- 7546798 TI - Massive cerebral infarction caused by paradoxical embolism: detection by transesophageal echocardiography. AB - Two days after coronary artery bypass, a 75-year-old woman had sudden dense left sided hemiplegia. A transthoracic echocardiogram showed a suspicious mass in the right atrium and right ventricle, which was confirmed by transesophageal echocardiography. Transesophageal echocardiography further demonstrated an elongated mass across a patent foramen ovale. Deep femoral venous thrombosis and massive cerebral infarction were also noted by Doppler ultrasonography and head computed tomographic scanning, respectively. Paradoxical embolization was thought to be the cause of the stroke. PMID- 7546799 TI - Retransplantation of the lung. A single center experience. AB - While lung retransplantation remains the only therapeutic option in early or late graft failure, its value is viewed controversially. Of 134 patients undergoing pulmonary transplantation in our institution, 13 patients underwent 14 redos following heart-lung transplantation (n = 3), bilateral lung transplantation (n = 5), and unilateral lung transplantation (n = 5). Indications for retransplantation were acute graft failure (n = 2), persistent graft dysfunction (n = 3), airway complications (n = 2), and chronic graft failure (n = 7). Prior to retransplantation, six patients had been in stable respiratory failure, the remaining eight patients were on mechanical ventilation or extracorporeal membrane oxygenation (n = 2). Four patients died, 19, 43, 142, and 683 days following retransplantation due to pneumonia (n = 2), early onset of obliterative bronchiolitis (n = 1), and pulmonary embolism (n = 1). There was no correlation between mortality and intubation prior to re-operating, timing of operation, donor cytomegalovirus (CMV) status, or type of operation. Postoperative need for intensive care treatment was prolonged in patients undergoing acute retransplantation (P < 0.05). Actuarial 1- and 2-year survival rates were calculated at 77 and 64%. This was slightly lower than in the overall population following primary isolated lung transplantation (83 and 80%). Actuarial freedom from obliterative bronchiolitis (stage 3) at 1 and 2 years was calculated at 88 and 27% (primary grafts: 88% vs 72%; P < 0.05). Retransplantation is a realistic option in early and late graft failure after lung transplantation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546800 TI - Mechanical ventricular assistance for the failing right ventricle after cardiac transplantation. AB - Right ventricular failure secondary to elevated pulmonary vascular resistance (PVR) following orthotopic cardiac transplant is a complication with a high mortality; and patients with high resistance are often not accepted on transplant waiting lists. We describe six cases of right ventricular failure after cardiac transplant managed by right ventricular assist device (RVAD), four of whom died and two patients who survived following life-threatening complications. PMID- 7546801 TI - Factors determining survival in resected N2 lung cancer. AB - This retrospective study was based on 237 patients with non-small cell lung cancer (NSCLC) and nodal N2 disease. All accessible mediastinal lymph nodes (LN) were removed and classified according to their anatomical location in LN chains. The pulmonary resections performed were: pneumonectomy (n = 187), lobectomy (n = 44) and segmentectomy (n = 4). There was solitary nodal chain involvement by metastasis in 141 cases, two chains in 72 cases and three or more in 24; "skip" metastases were present in 26.6%. N2 disease would have been missed in 45 cases of single chain involvement (31.9%) if routine removal of mediastinal nodes had not been performed. The overall 5-year survival rate was 18.8%. Survival was not influenced by site, size or extension (T) of tumor, tumor histology or the presence of vascular invasion. The prognosis was significantly worsened by the presence of microscopic residual disease (30 cases) and of satellite nodules (23 cases). Survival was significantly improved when metastases involved a single LN chain (26.3 versus 8.3%, P = 0.0003). The location and number of involved nodes in the chain, "skip" metastases and the presence of extracapsular spread of carcinoma did not influence the prognosis. Routine mediastinal LN dissection is necessary to improve survival and for classification of lung cancer. Anatomic description allows better understanding of N2 disease which is not a contraindication to surgery when a gross complete resection can be achieved. PMID- 7546802 TI - Preoperative chemotherapy and immunochemotherapy for locally advanced stage IIIA and IIIB non small cell lung cancer. Preliminary results. AB - From January 1991 to November 1993, 110 patients with histologically confirmed stage IIIA and IIIB non-small cell lung cancer (NSCLC), were seen at our Institution. Our study was designed to evaluate whether redirection to surgery of otherwise unresectable patients may be obtained by preoperative therapy. Forty nine patients were considered eligible for neoadjuvant treatment. Thirty-two (Group I) were treated with two or three cycles of cisplatin, vinblastine and mitomycin C and 17 (Group II) received two cycles of cisplatin, VP16, alpha 1 timosine and interferon. The overall response rate was 81.2% for Group I and 88.7% for Group II. Downstaging was predictive of resectability (P < 0.05). Forty one patients (83.6%) underwent thoracotomy with 37 (75.5%) radical resections. Conservative techniques (bronchovascular reconstruction) (22 cases) were preferred over pneumonectomy (2 cases). The resectability rate was 84% for Group I and 87% for Group II (P = NS). Treatment-related complications were minor, with no deaths. Postoperative complications occurred in two cases in each group (7.4% and 14.3%). There was no histologic evidence of tumor in three patients. Two-year survival was 75% for Group I and 55% for Group II (P = NS). To date 35 patients who had complete resection are alive, and free of disease. We conclude that preoperative chemotherapy produces high response and resectability rates in both stage IIIA and IIIB unresectable NSCLC; radical resection using a conservative technique is possible in patients who are otherwise unresectable; no local recurrence occurred after radical resection; no significant differences were demonstrated between the two protocols. PMID- 7546803 TI - Concomitant open heart surgery and pulmonary resection for lung cancer. AB - From 1979 to 1993, 79 patients underwent pulmonary resection for lung cancer and a concomitant cardiac operation using extracorporeal circulation. There were 75 men and 4 women with a mean age of 65 years (range 52-77). Cardiac procedures consisted of coronary artery bypass grafting (CABG) in 69 patients (three redos), aortic valve replacement in 7 (2 combined with CABG), mitral valve repair in 1 (combined with CABG) and other in 2. In CABG cases the mean number of distal anastomoses was 4.0. Pulmonary resection included bilateral lobectomy in 1 patient, sleeve lobectomy in 3, pneumonectomy in 6, bilobectomy in 5, lobectomy in 60 and segmental resection in 4. Postoperatively 52 patients were stage I (65.8%), 18 stage II (22.8%) and 9 stage III a. Histology was squamous cell carcinoma in 48 patients (61%) and adenocarcinoma in 24 patients (30%). The hospital mortality was 6.3% (n = 5). Re-exploration for bleeding was necessary in seven patients. Follow-up was complete for all patients. The estimated mean survival for all patients (including hospital deaths) was 58 months. Two- and five-year survival rates were 62% and 42% with 45 and 22 patients, respectively, under surveillance. Lung cancer accounted for 64% of the late deaths. We conclude that pulmonary resection for lung cancer in patients undergoing a concomitant cardiac operation can be performed safely with low operative morbidity and mortality and good long-term survival. PMID- 7546804 TI - Surgical management of ventricular septal defect with aortic valve prolapse: clinical considerations and results. AB - Aortic valve prolapse is found in over 5% of children with ventricular septal defect (VSD). Although this association occurs mostly with doubly committed subarterial VSDs, in this study the predominant type of VSD was perimembranous. In order to determine the need and timing for surgery and whether the anatomical features of septal defect may influence clinical management and outcome in this lesion, we reviewed our experience with 28 consecutive patients, operated on for VSD with prolapsed aortic valve cusp, with or without aortic regurgitation. Twenty-two patients had a perimembranous VSD and six had doubly committed VSD. Aortic regurgitation was trivial or absent in nine patients, mild in ten and moderate to severe in nine. Associated cardiac anomalies were present in 18 patients, all having perimembranous VSD, and included right ventricular outflow tract (RVOT) obstruction (n = 6), discrete subaortic membrane (n = 4) or both (n = 8). None of these patients had more than moderate aortic regurgitation. The patients underwent surgical closure of the septal defect between the ages of 1.5 and 34 years of age (median = 7). Sixteen patients having mild or trivial aortic regurgitation underwent closure of the VSD only, and 12 patients underwent VSD closure with aortic valvuloplasty. Valvuloplasty was required more often in doubly committed VSDs (66%) and in the perimembranous type without associated anomalies (100%), and significantly less often in the presence of RVOT obstruction, subaortic membrane or both (22%). At follow-up (up to 5 years, mean 18 months), the grade of aortic regurgitation was unchanged in 11 and decreased in 5 patients undergoing closure of the VSD only.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546805 TI - Mid-term results of the modified Senning operation for cavopulmonary connection with autologous tissue. AB - The aim of this study was to determine the results and mid-term outcome of a modified Senning technique using autologous tissue for total cavopulmonary connection. The study involved 31 children, 8 with tricuspid atresia and 23 with complex congenital heart disease. In this operation, a flap of autologous atrial free wall tissue was used to tunnel inferior vena caval blood to the pulmonary arteries. An additional Damus-Kay-Stansel operation was required in 9 patients with subaortic obstruction. RESULTS: the early mortality rate was 16% (5 out of 31 patients) and there were four late deaths. COMPLICATIONS: Pleural effusions were encountered in 17 patients, of whom 4 had a concomitant pericardial effusion. Diaphragmatic paralysis was diagnosed in five patients, one of whom underwent surgical plication. Median hospital stay was 26 days. The 1- to 5-year actuarial survival was 68.6%. Follow-up ranged from 10 months to 7.1 years, mean 3.2 years. A serious atrial arrhythmia was diagnosed in one patient and another one died, possibly from rhythm disorders. Exercise tolerance and quality of life has improved in all but one of the survivors. Although follow-up is short, we have thus far witnessed a low incidence of hemodynamic and rhythm disturbances with this modification of the cavopulmonary connection. PMID- 7546806 TI - Prophylaxis in cardiac surgery. A controlled randomized comparison between cefazolin and cefuroxime. AB - In a prospective randomized two center trial, short-term prophylaxis with cefuroxime (CFX) in 189 patients was compared with cefazolin (CFZ) in 196 patients submitted to elective cardiac surgery. A total of 3 g was administered over 24 h in both groups. One major adverse reaction with CFX was noted. Patients were prospectively screened by infectious disease nurses for surgical wound and secondary infections. Sternal wound infections occurred in eight patients treated with CFX and all were minor. One patient from this group eventually died of infectious causes. In the CFZ-treated patients two major and six minor wound infections occurred requiring extensive debridement in two. Secondary infections occurred less frequently in the CFX group (13.2 per 100) than in the CFZ group (16.8 per 100) with two infection-related deaths in the CFX and one in the CFZ group. The most commonly identified organisms were Staphylococcus aureus and a variety of gram-negative organisms. No major differences were observed between the CFX and CFZ groups. Short-term administration of 3 g CFZ or CFX in this study could not demonstrate the advantage of one of the antibiotics used over the other in terms of clinical outcome, incidence or site of infection or organisms identified. The 24 h administration of 3 g CFZ or CFX provided suboptimal prophylaxis for wound infection or secondary infections in patients undergoing elective open heart surgery. PMID- 7546807 TI - Surgery in native valve endocarditis: indications, results and risk factors. AB - Seventy-nine patients (mean age 49 years) underwent valve replacement or repair for active (58.2%) or healed (41.8%) native valve endocarditis between 1976 and 1992. The most common indication for surgery was congestive heart failure (73.4%), followed by multiple systemic emboli (21.5%). Emergency operation was necessary in 27.8% of the cases. Operative mortality was 3.8% (3 patients) and late mortality 15.1% (12 patients). Streptococci were the most common infecting agents (41.8%), followed by Staphylococcus aureus (11.4%). No organisms were isolated in 27 cases (34.2%). Follow-up spanned 379.8 patient-years with a maximum of 15.8 years. Fifteen late valve-related events (periprosthetic leak, recurrent endocarditis, thrombo-embolic events and hemolysis) and 20 other late complications (anticoagulant-related hemorrhage, arrhythmias or congestive heart failure) occurred in 22 patients. The linearized rate for all late complications is 5.8% per patient-year. The influence of eight preoperative variables on overall mortality and late valve-related complications was assessed: age, valve(s) affected, active or healed infection, bacteriology, annular abscess, emergency or elective surgery, preoperative renal function and NYHA class. Only Staphylococcus aureus (P = 0.0012) was a significant predictor of late valve related complications. Furthermore, no difference in survival or in valve-related complications was found between the active and healed infections. PMID- 7546808 TI - Late results of mitral valve replacement with Carpentier-Edwards high profile bioprosthesis in young adults. AB - Carpentier-Edwards (C/E) high profile supra-annular porcine bioprostheses were implanted in 163 patients between September 1982-February 1987 in the mitral position due to rheumatic disease. The patients' ages ranged between 15 and 58 years (mean: 36.5 +/- 11.5). One hundred-two of the patients (62.5%) were female. The hospital mortality was 9.8% (16/163). The total cumulative follow-up period was 1093 patient-years (p-y) with a mean of 7.3 +/- 3.5 p-y (range 6 months-12 years). Linearised occurrence rates of the late complications were as follows: Thromboembolism 0.82%/p-v, anticoagulant-related bleeding 1.0%/p-y, prosthetic valve endocarditis 0.09%/p-y. Actuarial survival rates for 5 and 12 years were 87% +/- 6% and 76% +/- 9%, respectively, with 95% confidence limits. Freedom from structural deterioration was 92% +/- 5% for 5 years and 44% +/- 11% for 12 years. Freedom from all valve-related complications was 81% +/- 7 for 5 years and 26% +/ 9 for 12 years. In conclusion, this prosthesis provides satisfactory clinical performance in young population despite the high risk of degeneration with good prognosis and better tolerability of reoperation. PMID- 7546809 TI - Complete sternectomy for metastatic carcinoma with reconstruction using a latissimus dorsi musculocutaneous flap. AB - A 67-year-old man was hospitalized because of anterior chest pain and bulging. Histopathologic examination of a biopsy specimen revealed metastatic adenocarcinoma. For the metastatic sternal tumor a complete sternectomy was performed at all layers, following which the chest wall defect was reconstructed using no artificial materials but only a latissimus dorsi musculocutaneous flap. Postoperative respiratory function tests gave satisfactory results. This case suggests that, provided the organs within the thoracic cavity can be protected, solid or semi-solid stabilization achieved with artificial materials may not always be necessary, even in cases undergoing extensive sternectomy. PMID- 7546810 TI - The freedom of atherosclerosis of intramyocardial coronary arteries: is reduction of mural stress the only factor? PMID- 7546811 TI - [Treatment of complicated Parkinson disease with a solution of levodopa-carbidopa and ascorbic acid]. AB - We prescribed a solution of levodopa-carbidopa and ascorbic acid (LCAAS) to 21 Parkinsonian patients with motor complications. Eight patients continued the treatment for a mean period of 16.8 months, experiencing substantial increases in the number of hours with good functional capacity. Bothersome symptoms such as dystonia and akathisia in off periods disappeared in all cases in which they had been present and LCAAS was tolerated (in 6 of the 8 patients who continued in the study and in 4 who abandoned treatment late). Intake of other anti-Parkinsonian drugs was reduced. Thirteen patients abandoned the study, citing exacerbation of biphasic dyskinesia as the main reason. We conclude that LCAAS is a useful therapy in some Parkinsonian patients whose motor complications are not managed with conventional drug treatment. Screening of patients is probably of utmost importance to ensure that LCAAS is not administered to patients who already suffer intense biphasic dyskinesia. PMID- 7546812 TI - [Clinical application of botulinum toxin]. PMID- 7546813 TI - [Festooning of the corpus callosum in chronic hydrocephalus]. PMID- 7546814 TI - [Myokimia induced by gold salts]. AB - We report a 44 year old male suffering from rheumatoid arthritis that developed myokymias when treated with gold salts. Myokymia is a clinical phenomenon characterized by involuntary repetitive contractions of muscle fibres, which give an undulating appearance to the overlying skin, last a few seconds, and with a typical electromyographic activity described as myokymic discharges. The difference between the single twitch of the fasciculation and the tetanic contraction of myokymia may be evident only on electromyography in difficult cases. Neurological complications associated with gold are infrequent, and they include myokymia, peripheral neuropathy, Guillain-Barre syndrome, cranial nerves paralysis and encephalopathy. PMID- 7546815 TI - [Nodular subependymal heterotopia and epilepsy]. AB - Neuronal migration disorders may manifest as epilepsy alone and this is usually the case in nodular subependymal heterotopia, of which we present 5 cases. We consider this entity to be a well-defined epileptic syndrome because it is found nearly exclusively in women and is characterized by nearly constant seizures which start in the second or third decade of life, familial aggregation of cases, a clinical and EEG profile that suggests a temporal focus and the absence of associated cognitive or motor deficits. Seizures are usually controllable with medication. PMID- 7546816 TI - [Cerebral cavernous angiomas in pregnancy. Two cases and a review of literature]. AB - Whether pregnancy increases the risk of bleeding of cavernous angioma, as it does with other types of cerebral vascular malformation, is not known at present. We monitored the pregnancies of two patients with cerebral cavernous angiomas. One patient, whose long-standing epilepsy had until then been considered cryptogenic, with seizure occurring every 3 to 5 years, remained asymptomatic throughout her pregnancy which ended in spontaneous abortion. The parietal cavernous angioma was partially calcified and the tissue showed signs of earlier hemorrhage. The first symptom in the second patient was first-trimester hemorrhage from inside and around the angioma which was located in the optic chiasm. The lesion was fully excised in both patients. We review the literature and analyze the mechanisms that may be implicated in the clinical presentation during pregnancy of this type of cerebral vascular malformation. PMID- 7546817 TI - [Myasthenia gravis and non-Hodgkin's lymphoma]. AB - We describe 2 patients with myasthenia gravis and non-Hodgkin's lymphoma outside the thymus gland, in whom the two diseases progressed at different rates. Diagnosis of myasthenia gravis was based on clinical signs and compatible neurophysiologic studies, specifically by high acetylcholine antireceptor titers in the first patient and a positive Tensilon test in the second. In the first patient the clinical and serological signs of the two diseases progressed similarly. The association of these two diseases may have been the result of an underlying immunological disorder favoring their appearance, or of an immune response, caused by the lymphoma involving postsynaptic nicotinic acetylcholine receptors. PMID- 7546818 TI - [Norman's cerebellar atrophy. A case diagnosed by clinical signs and neuroimaging]. AB - We present a patient with early non progressive ataxia in whom improvement was slower in motor signs than in psychic activity. MR showed cerebellar atrophy, more severe in the vermis than in the hemispheres. Clinical features and brain images pointed to a primary degeneration of the granular layer of the cerebellum, as described by Norman. PMID- 7546819 TI - [Hypophyseal apoplexy]. PMID- 7546820 TI - [Generalized rigidity and dementia secondary to hypopituitarism]. PMID- 7546821 TI - Radiotherapy in addition to radical surgery in rectal cancer. AB - In order to lower unacceptably high local failure rates after surgery reported as curative for rectal cancer, perioperative radiotherapy has been extensively investigated. The collected information from a number of controlled trials indicates that the proportion of local recurrences is reduced to less than half when radiotherapy at moderately high doses is given preoperatively. This reduction in local failure rates is not seen after postoperative radiotherapy, even if higher doses have been used. Possibly, there is also a slight positive influence on survival from preoperative radiotherapy. Improved survival has been seen also in trials using postoperative radiotherapy, but then only when combined with chemotherapy. With proper dose planning, sufficiently high doses can be given preoperatively with little if any increase in postoperative mortality. Thus, although published knowledge is still rather limited, a properly planned preoperative radiotherapy seems to inflict small bowel and other complication rates, that are less than when radiotherapy of similar efficacy against the tumour is given postoperatively. PMID- 7546822 TI - Nicotinamide and other benzamide analogs as agents for overcoming hypoxic cell radiation resistance in tumours. A review. AB - Oxygen deficient hypoxic cells, which are resistant to sparsely ionising radiation, have now been identified in most animal and some human solid tumours and will influence the response of those tumours to radiation treatment. This hypoxia can be either chronic, arising from an oxygen diffusion limitation, or acute, resulting from transient stoppages in microregional blood flow. Although clinical attempts to overcome hypoxia have met with some success, the results have been far from satisfactory, and efforts are still being made to find better methods. Extensive experimental studies, especially in the last decade, have shown that nicotinamide and structurally related analogs can effectively sensitise murine tumours to both single and fractionated radiation treatments and that they do so in preference to the effects seen in mouse normal tissues. The earliest studies suggested that this enhancement of radiation damage was the result of an inhibition of the repair mechanisms, as was well documented in vitro. However, recent studies in mouse tumours have shown that the primary mode of action actually involves a reduction in tumour hypoxia. More specifically, these drugs prevent transient cessations in blood flow, thus inhibiting the development of acute hypoxia. This novel discovery led to the suggestion that the potential role of these agents as radiosensitizers would be when combined with treatments that overcame chronic hypoxia. The first attempt to demonstrate this combined nicotinamide with hyperthermia and found that the enhancement of radiation damage by both agents together was greater than that seen with each agent alone. Similar results were later seen for nicotinamide combined with a perfluorochemical emulsion, carbogen breathing, and pentoxifylline, and in all these studies the effects in tumours were always greater than those seen in appropriate normal tissues. Of all the analogs, it is nicotinamide itself which has been the most extensively studied as a radiosensitizer in vivo and the one that shows the greatest effect in animal tumours. It is also an agent that has been well established clinically for the treatment of a variety of disorders, with daily doses of up to 6 g being considered reasonably safe and associated with a low incidence of side effects. This human dose is equivalent to 100-200 mg/kg in mice and such doses will maximally sensitize murine tumours to radiation. These findings have now resulted in phase I/II clinical trials of nicotinamide, in combination with carbogen breathing, as a potential radiosensitizing treatment. PMID- 7546823 TI - Bladder preservation--a prospect for patients with urinary bladder cancer. PMID- 7546824 TI - Serum levels of cytokines and soluble cytokine receptors in patients with metastatic renal cell carcinoma or malignant melanoma receiving IL-2/interferon alpha combination therapy. AB - Elevated serum levels of soluble tumour necrosis factor receptor (sTNFR-55) and (at a lesser degree) of sTNFR-75 were found in most of the patients with metastatic renal cell carcinoma (RCC) or malignant melanoma (MM) before immunotherapy and with further increase during treatment (intravenous infusions of interleukin-2 [IL-2] and subcutaneous interferon-alpha [IFN-alpha]). In the majority of the patients with MM the pretreatment serum levels of IL-2 and soluble IL-2 receptor (sIL-2R) were increased, whereas fewer patients with RCC presented with increased serum levels of IL-2 and sIL-2R. Twelve days' treatment with IL-2/IFN-alpha, with a rest on days 6 and 7, resulted in a consistent further increase in the serum levels of sIL-2R and sTNFRs. In most patients the increase of sIL-2R and sTNFRs lasted for at least 3 weeks after treatment discontinuation. The clinical significance of the increase remains unknown. PMID- 7546825 TI - High cell proliferation activity determined by DNA flow cytometry and prognosis in epidermoid lung carcinoma. AB - DNA aneuploidy and high cell proliferation activity determined by DNA flow cytometry are known to be associated with poor prognosis in many human malignancies. The aim of the present study was to determine the long-term prognostic value of DNA ploidy and cell proliferation activity in lung cancer. The material consisted of samples from 290 paraffin-embedded lung cancers (171 epidermoid carcinomas, 62 small-cell carcinomas, 26 adenocarcinomas, and 31 others), of which DNA ploidy and cell proliferation activity as defined by S phase fraction (SPF) was determined by DNA flow cytometry. DNA aneuploidy was found in 61% of the tumours and the mean (+/- SD) SPF was 13.6 +/- 9.5%. DNA aneuploidy was associated with a high T-stage (p < 0.05) in epidermoid carcinoma, but not in small-cell carcinoma or adenocarcinoma. DNA aneuploidy and high SPF were associated with poor survival in epidermoid carcinoma. However, only SPF (relative risk (RR) = 1.80), N-(RR = 2.46) and M-stages (RR = 2.17) were statistically significant predictors of survival according to multivariate analysis. Regression-tree analysis showed that the prognostic value of SPF was restricted to localized (M0-stage) epidermoid carcinoma. Neither DNA aneuploidy nor SPF was associated with survival in small-cell lung carcinoma. In conclusion, SPF is an independent prognostic factor in epidermoid lung carcinoma. PMID- 7546827 TI - Papers from the 5th Scandinavian Breast Cancer Symposium. Reykjavik, Iceland, May 28-June 1, 1994. PMID- 7546826 TI - Severe complications after high-dose methotrexate treatment. PMID- 7546828 TI - Chronobiology. Implications for cancer chemotherapy. AB - Experimental studies have documented that both the toxicity and the antitumor activity of many cancer drugs are time dependent. Early clinical trials have confirmed this observation for several drugs. The basic concepts of chronobiology and its application to pharmacology are reviewed. As an example, clinical trials of circadian fluoropyrimidine delivery are reviewed. Other clinical results are presented in table form. The mechanisms pertinent to the circadian time dependence of fluoropyrimidines are discussed. PMID- 7546829 TI - Role of surgery in invasive breast cancer. AB - Surgery is still the single most important treatment modality for breast cancer. Even if some patients with early stage cancer are treated, and in a clinical sense cured, by surgery alone, adjuvant local and systemic treatments are required for most patients for the best possible tumor control. Surgery must be compatible and timed with other treatment modalities. It helps provide local control, which in some cases is enough for cure but in others is limited to being a means of palliation. Surgery provides the clinical team with material for diagnosis, for prognostication and for help in clinical decision making. The choice of the surgical procedure and its performance should be handled by surgeons, knowledgeable and interested in the field in order to obtain the best possible functional and cosmetic results. An algorithm for deciding the appropriate surgical treatment of the breast is presented. Surgical treatment of the axilla is controversial. Current debate indicates that exploration of the axilla can be safely omitted in some patient groups, but more research is needed. Other important areas for clinical surgical research are use of immediate breast reconstruction, choice of management when margins are involved with tumor growth after breast-sparing operations, and surgical approach to tumors which have been treated with chemotherapy preoperatively. PMID- 7546830 TI - Clodronate and other bisphosphonates as supportive therapy in osteolysis due to malignancy. AB - Clodronate, one of the most investigated bisphosphonates, has been clinically utilised for over 10 years in malignancy. It is the most used, most effective and safest drug in the treatment of hypercalcaemia. It inhibits lytic bone destruction, prevents bone fractures and relieves bone pain. Supportive clodronate therapy may even reduce hypercalcaemia mortality and the morbidity caused by osteolysis. These results have stimulated studies on the patients' quality of life. New methods for the measurement of bone resorption, such as the degradation product of type I collagen (ICTP), may improve the possibility of monitoring the effect of clodronate. Comparative studies with different bisphosphonates in hypercalcaemia and long-term controlled trials using bisphosphonates as supportive therapy in osteolysis due to malignancy are reviewed. PMID- 7546831 TI - Development of antiemetic therapy in cancer patients. AB - Patients still consider nausea and vomiting to be severe adverse consequences of cancer chemotherapy. The physiology of chemotherapy-induced nausea is generally unknown, but the finding that high doses of metoclopramide induce the antiemetic effect by antagonizing 5-HT3 receptors, has evoked increased interest in serotonin as a possible neurotransmitter. This has led to development of more selective 5-HT3 antagonists, such as ondansetron, granisetron and tropisetron, with improvement of antiemetic therapy, especially in patients receiving cisplatin-based chemotherapy. The efficacy of serotonin antagonists is further optimized by the addition of steroids and the dopamine D2 antagonist metopimazine. Many questions in antiemetic treatment are still unanswered and future trials should focus on patients receiving multiple-day chemotherapy or multiple cycles of chemotherapy and on patients resistant to initial antiemetic therapy. PMID- 7546832 TI - Differential expression of oestrogen regulated genes in breast cancer. AB - Pathological endpoints such as tumour size, lymph node status and vascular invasion remain the most useful guides in selecting treatment strategies for breast cancer. There is a need, however, to further investigate the molecular mechanisms that determine the properties of an individual tumour e.g., hormone responsiveness and probability of metastasis. While numerous prognostic factors have now been identified few have contributed to defining clinical response to therapy. Oestrogen-regulated genes are likely to be important since they not only define a functional oestrogen receptor, but alterations in their expression might provide insights into the mechanisms involved in tumour progression and loss of endocrine sensitivity. Recently an oestrogen responsive gene, pLIV1, has been isolated and shown to be expressed in ER+ disease where it appears to predict nodal involvement. The present paper describes aspects of its regulation and discusses the potential role of this and other genes in the development of endocrine resistance. PMID- 7546833 TI - Interactions between the immune system and breast cancer. AB - The functional and prognostic significance of lymphocytic infiltration of breast carcinomas has remained unclear. Using primary cultures we have demonstrated that lymphocytes could stimulate the growth of breast cancer epithelium in about half of the cases tested. The growth stimulation was subsequently shown to be strongly correlated with expression of MHC class I by the tumour cells. Furthermore, preliminary data suggest that carcinomas with a mixed population of MHC class I positive and -negative cells were associated with a higher incidence of lymph node metastases and increased relapse rate compared with tumours that were homogeneously MHC class I-positive or -negative. The interactions between breast cancer and the immune system are clearly complex and the results suggest that the nature of these interactions can be some extent be determined by the level and pattern of MHC class I expression by the tumour cells. PMID- 7546834 TI - Expression and regulation of cyclin genes in breast cancer. AB - Cyclins, the regulatory subunits of cyclin-dependent kinases, control passage through key check-points within the cell cycle. Since dysregulated expression and function of cyclins can lead to loss of normal growth control some of these genes are oncogenes. We have studied cyclin gene expression, regulation and function in breast cancers. Induction of cyclin D1 is an early event in mitogenic stimulation of breast cancer cells by growth factors and steroids. Furthermore, inhibition of cyclin D1 expression is an early response to growth inhibition by antioestrogens. Ectopic expression of cyclin D1 in T-47D breast cancer cells demonstrated that cyclin D1 is rate-limiting for progression through G1 phase and is sufficient for growth arrested cells to complete the cell cycle. Since this gene is frequently overexpressed in human breast cancers it may contribute to the development and progression of some breast carcinomas. PMID- 7546835 TI - Chromosome 17q-linkage seems to be infrequent in Icelandic families at risk of breast cancer. AB - Eight Icelandic families with multiple cases of breast cancer, and 17 pairs of sisters diagnosed by the age of 50 were analysed for linkage to markers around BRCA1 on chromosome 17q. The sister-pairs are thought to represent a wider population as compared to the larger high-risk families. Tumours were also analysed for LOH involving BRCA1. In accordance with a proposed tumour suppressive function of BRCA1, and high prevalence of LOH in 'linked' tumours, the paired sisters' tumours were assayed for double LOH events with common alleles retained. No such pair was observed, and LOH events were seemingly randomly distributed at a 38% frequency. This indicates that most or all pairs are due to other genes than BRCA1 or sporadic involvement. Of the eight high-risk families, only one showed convincing evidence of 17q-linkage. Therefore, BRCA1 mutations seem to be a minor explanation of familial risk of breast cancer in Iceland. PMID- 7546836 TI - TP53 abnormalities and genetic instability in breast cancer. AB - TP53 abnormalities in breast carcinomas and inherited TP53 changes in breast cancer patients and in Li-Fraumeni-like families were looked for. Tumours were screened for mutations in the TP53 gene by means of the PCR-CDGE method followed by PCR and direct sequencing. Allelic loss was determined by polymorphic markers, by comparing normal and tumour DNA. Abnormal protein expression was examined by immunohistochemical staining. TP53 abnormalities in the tumours were examined in relation to genetic instability, clinical data and family history. Genetic instability was studied by detection of oncogene amplification, allelic loss, karyotype analysis and fluorescent in situ hybridization, FISH. Our studies showed that TP53 abnormalities were significantly associated with amplification of the erbB2 oncogene and allelic loss on chromosome 17. Chromosomal abnormalities were also significantly more common in tumours with TP53 abnormalities. Looking at clinical data we found significant association between TP53 abnormalities and poor prognosis. PMID- 7546837 TI - High-dose therapy with autologous bone marrow stem cell support in primary and metastatic human breast cancer. A review. AB - A dose-response relationship has been demonstrated for metastatic human breast cancer. This increased response using moderately increased doses is generally not translated into an improved survival. The use of high-dose therapy to selected patients with metastases/recurrence responding to conventional doses of polychemotherapy may lead to an improved survival tail. Conventional doses of polychemotherapy in the adjuvant setting will reduce the relative mortality by around 25% 10 years after primary diagnosis. The use of high-dose therapy supported by autologous bone marrow stem cells may be markedly more effective in the adjuvant setting, especially to high-risk patients, compared with standard polychemotherapy. Several randomized studies are being planned or have already started in order to answer different aspects of this issue. PMID- 7546838 TI - The role of radiotherapy in breast conserving treatment. AB - Radiotherapy in breast conserving procedures is instrumental for reducing the frequency of a breast relapse, and may even improve survival. The treatment can be associated with morbidity, that is not negligible. A high-quality technique is needed in order to achieve an optimal therapeutic ratio. PMID- 7546839 TI - Breast-conserving surgery without radiotherapy. AB - Radiotherapy effectively prevents local recurrences, but on the other hand still over 80% of non irradiated women are free from local relapse 5 years after breast surgery aiming at radical removal of the primary tumor. So far, there are no indications that radiotherapy to the breast after breast conservation saves lives. The marginal monetary cost for prevention of one local recurrence is about $41,000. Thus, one clinically relevant question is if there is a group of women where the benefits of radiotherapy after breast conservation is outweighed by psychosocial and medical side-effects. Unfortunately, we still lack information from several areas, which makes a detailed cost-benefit analysis uncertain today. The problem situation would change: 1) If we knew more about the pathogenesis of local recurrences and thus also could undertake preventive measures other than radiotherapy. 2) If there was more information on risk factors for local recurrence, and we could identify a low-risk group that could be spared treatment. 3) If there were more valid empirical information on the psychosocial side-effects--both of a local recurrence and of the radiotherapy. 4) If the long term--positive and negative--effects of radiotherapy were better quantified. Until we have new information, postoperative radiotherapy after breast conservation remains the standard. PMID- 7546840 TI - Breast reconstruction. AB - Breast reconstruction after mastectomy for breast cancer has usually been performed as a delayed procedure, often not less than one year after completion of postoperative radiation therapy and cytotoxic chemotherapy. The reason for delaying the procedure has been the increased risk of wound complications after the adjuvant therapy. The reconstruction has been even further delayed for patients with increased risk of recurrence in order to avoid a reconstruction 'in vain' for a patient, who will succumb to the disease within a couple of years after the reconstruction. The breast reconstruction can also be performed immediately in conjunction with the mastectomy. The rather slow acceptance of this procedure has mainly been for practical reasons. The mastectomies are usually performed by general surgeons. The operation has not been centralized to larger hospitals, and the patients usually want to have the operation performed as soon as possible. It is therefore often difficult to arrange a joint operation by the general and the plastic surgeons. In addition, suspicions have been raised that enlargement of the dissection area could spread cancer and increase the risk of local metastases. The same types of operation can be used for delayed and immediate breast reconstruction. There are basically two possible ways of creating a new breast mound. One entails transferring muscle, subcutaneous fat and skin into the area of reconstruction in sufficient quantity to shape a breast mound. The other possibility is to shape the breast mound mainly by inserting a prosthesis, similar in consistency to breast tissue into the area of reconstruction. For practical reasons the implant has to be covered by a sufficient amount of soft tissue, and for this purpose a number of pedicled flaps can be used. The implant most commonly used is made of silicone gel and covered by a membrane to prevent the gel from oozing into the surrounding tissues. A recent innovation is an implant with textured surface in order to diminish capsule formation around the prosthesis. PMID- 7546841 TI - Assessment of quality of life in breast cancer. AB - Effective treatment for breast cancer can produce a reasonably good ten-year survival rate in comparison to many other cancer sites. Nevertheless, the potential psychological, sexual and physical dysfunction caused by both the diagnosis and treatments can have a deleterious impact on the quality of a woman's life. The range of possible treatments may have similar outcomes in terms of response and survival, but can produce very different effects on emotional well-being. Therefore, monitoring quality of life in breast cancer should be a mandatory part of follow-up in clinical trials. Data derived from various studies of quality of life can also be used to assist the doctor and patient in decision making about treatment options. Furthermore, assessment of quality of life can help identify those patients who might profit from psychosocial interventions. In this paper some of the instruments used to assess quality of life in breast cancer will be discussed. PMID- 7546842 TI - Adaptive significance of amylase polymorphism in Drosophila. X. Analysis of alpha amylase activity of two amylase variants in individual Drosophila subobscura flies. AB - Experiments were designed to estimate the variation of alpha-amylase activity in Drosophila subobscura individuals homozygous for AmyS and AmyF alleles at the Amy locus. The measurements of enzyme activities in six groups of male progenies determined in each individual have shown that degree of variation differs between S and F strains. Variability of amylase activity among male progenies of S strain was substantially greater, which also had significantly higher specific amylase activity than in F strain. The analysis of amylase activity variance showed that this variance among the males is 40 times, and among the females 2.7 times smaller in F than in S strain. PMID- 7546843 TI - Analysis of mouse hemoglobin alpha-chain locus mutation induced by N-ethyl-N nitrosourea. AB - In a mouse specific-locus test in stem-cell spermatogonia of mice after treatment with N-ethyl-N-nitrosourea (ENU), a mutation at the hemoglobin alpha-chain locus was obtained. In an original mutant, the mutation was detected as a change in band density using cellulose acetate electrophoresis, e.g. the slow moving band was much higher than that of normal F1 mice. Yet, migration of the mouse bands was similar. In isoelectric focusing, the migration of a band toward cathode was observed, indicating that the variant hemoglobin is caused by mutation at an alpha-globin chain. This mutation was found to be heritable and viable in homozygous condition from a genetic test on the mutant, suggesting that this mutation would be due to a base-pair change. According to the usual nomenclature of mutation at the hemoglobin locus, the allele symbol of this mutation was tentatively designated as Hbacc3. PMID- 7546844 TI - Suppressor of hairless, the Drosophila homologue of RBP-J kappa, transactivates the neurogenic gene E(spl)m8. AB - Suppressor of Hairless[Su(H)], the Drosophila homologue of RBP-J kappa is a novel type of sequence-specific DNA binding protein without known motifs, and highly conserved in various organisms. Su(H) regulates peripheral nervous system (PNS) development. Recently Su(H) was suggested to participate in the Notch-mediated signal transduction pathway. We show here that the Su(H) protein binds to TGTGGGAA sequence located 616 base-pairs upstream of the transcription initiation site of the Enhancer of split [E(spl)]m8 gene which is mapped to the terminus of the genetic cascade of the neurogenic genes. Su(H) transactivates the E(spl)m8 promoter not only in cultured Drosophila cells but also in vivo. The present study bridges the biochemical gap between Notch and E(spl) in the neurogenic gene cascade including Delta, Notch, deltex, Su(H), Hairless and E(spl). PMID- 7546845 TI - Chromosomal localization of the rat erythropoietin receptor gene by fluorescence in situ hybridization. AB - Erythropoietin stimulates proliferation and differentiation of erythroid progenitor cells by binding to a specific membrane receptor, erythropoietin receptor. By using the genomic clone derived from a rat cosmid library, the rat erythropoietin receptor gene was assigned to chromosome 8q24 by fluorescence in situ hybridization. PMID- 7546846 TI - Molecular characterization of a family of tandemly repetitive DNA sequences (pYS family) in the genus Hemitaxonus (Hymenoptera: Tenthredinidae). AB - Distribution of a family of tandemly repetitive sequences in Hemitaxonus japonicus (pYS family) was investigated among eight sawfly species and host races in the genus Hemitaxonus. High copy numbers of the repetitive sequences were detected in the two host plant races of H. japonicus, race Polystichum polyblepharum (race PP) and race P. tripteron (race PT), and H. sasayamensis, whereas a low copy number in H. minomensis. Comparison of the nucleotide sequences of the basic repeat units demonstrated a high degree of homology among the four tested species with high AT-rich sequences. Minor repeat units shorter than the consensus basic repeat unit of pYS family were found only in races PP and PT of H. japonicus. Analyses of their nucleotide sequences showed the occurrence of the host race specific sub-repeat units. PMID- 7546847 TI - [Computer use in voice analysis: clinical applications]. AB - Computers play an important role in voice analysis both in research and in the clinic. We have revised some basic concepts in signal digitization and some of the options present in the different voice analysis systems as: intensity and fundamental frequency measurement, perturbation calculation (jitter and shimmer) and spectrographic analysis. Clinical applications are outlined in conclusion. PMID- 7546848 TI - [Standard parameters of distortion products]. AB - In this study we have analyzed the incidence, range, frequency spectrum and threshold of distortion products in a sample of 232 normal hearing person. 116 younger adults, 58 female ranging in age from 18 to 30 years (mean age 23.1) and 58 males ranging in age from 18 to 32 (mean age 23.7) were included in the study. In all the ears studied, the presence of distortion products in a normal ear were found with regularity in the frequency region between 700 to 6,000 Hz. Higher incidence occurred in younger people and women but was not statistically notable. There is a direct correlation between distortion products amplitude and its primary intensity. Distortion products amplitude increases progressively from low (770 Hz) to high frequencies (6,000 Hz). The amplitude of distortion products and its mean value is higher in women, in all frequencies, except in the 700 to 1,000 Hz region. Input/output function had a mean of 50 dB SPL at 1 kHz, 43.8 at 2 kHz, 38.4 at 4 kHz and 33.5 at 6 kHz. PMID- 7546849 TI - [Active duration: new parameters in quantitative assessment of post-caloric nystagmus]. AB - This study describes a new parameter for quantifying the post-caloric nystagmus, the active duration, or the total periods where appropriate nystagmic response existed due to caloric stimulation. On a pathological population of 211 cases, the new parameter records a diagnostic sensitivity of 56% for the canal paresis and 59% for the directional preponderance, improving the sensitivity levels corresponding to the duration of the response according to classical criterion. Being easy to calculate, the active duration parameter is considered to be appropriate for the quantity valoration of the post-caloric nystagmus by means of computerized technics. PMID- 7546850 TI - [The modified transcochlear approach to the petroclival area and the prepontine cistern: indications, techniques and results]. AB - This paper presents the modification made for a transcochlear approach which would permit reaching the pertoclivis area and proeponten cistern. The disadvantage, inconvenience of the approach used on 5 patient is discussed here. PMID- 7546851 TI - [Our experience in the surgery of otosclerosis]. AB - A retrospective study of 613 operations on 495 patients with otosclerosis was made in the Department of Otorhinolaryngology of Hospital General Universitario of Alicante between 1974 to 1992. The clinical-pathological characteristics of this series were studied. Statistical analysis of the results was made. The improvement in air-bone gap for conversational frequencies was 27 dB. Closure of the postoperative air-bone gap for conversational frequencies was 7.6 dB and the overall percentage of closure of this gap was 80.5%. Better auditory results were obtained with partial platinectomy than with stapedectomy. Nothing was inserted between the prosthesis and the vestibule, permitting a shorter, easier operation with no occurrence of perilympathic fistule. PMID- 7546852 TI - [The selection of cervical vessels recipients in reconstructive surgery of head and neck with free flaps]. AB - One of the most important parameters for success in free tissue transfer to the head and neck are a proper and careful recipient vessel dissection and meticulous vascular microanastomotic technique. The principle that govern vessel positioning in the head and neck are more complex than those used in extremity free flap transfers. In this report the vascular selection as well as technical details are discussed, based on the literature data and our own experience in 20 patients. PMID- 7546853 TI - [Laryngocele: clinical and therapeutic study of 60 cases]. AB - Sixty patients with laryngocele were diagnosed in the last seven years: 25 of them without previous laryngeal pathology (group A) and the rest 35 with laryngeal or pharyngeal cancer (group B). The laryngocele was unilateral in the majority of the 25 patients of group A (68%). The internal laryngoceles were the most common type (63%). The initial symptom was hoarseness in 56% of these patients and 20% developed, in their evolution, an acute respiratory distress. The diagnosis was clinical in 16 patients and radiological in the rest. Endoscopically marsupialization with laser-CO2 was performed to remove internal laryngoceles. The mixed and external laryngoceles were completely removed via an external cervical approach without the need to perform any thyrotomy. The diagnosis was radiological by CT in the 35 patients of the group B. In 30 of them the CT was performed to evaluate the local extension of the laryngeal or pharyngeal cancer before its treatment. Supraglottic carcinoma was the most common laryngeal tumor (50%). The anatomic relationship between laryngocele and laryngeal cancer was ipsilateral to each other only in 50% of the patients. In the other 5 patients, no laryngocele was found in the radiological study previous to the treatment of the laryngeal or pharyngeal cancer. The diagnosis was made after chemotherapy and/or radiotherapy treatment. PMID- 7546854 TI - [Analysis of rearrangements in the first exon of c-myc oncogene in squamous cell carcinoma of the head and neck]. AB - Activation of c-myc has been implicated in the origin of different human tumors, and can be produced by diverse mechanisms as amplifications or rearrangements. We examined 55 squamous cell carcinomas of the upper aerodigestive tract for rearrangements involving the first exon of c-myc, which plays a regulatory role in the c-myc expression. Polymerase chain reaction (PCR) amplification of different fragments of c-myc exon 1 was performed using four oligonucleotide primers that correspond to consecutive sequences from the 5' end of the c-myc exon 1, combined with one oligonucleotide that corresponds to the 3' end. Amplified c-myc PCR products appear as single bands of 580, 495, 417 and 333 base pairs on the electrophoretic analysis. Only in one case a rearrangement involving the first exon of c-myc was found. We concluded that gene rearrangement is not a common mechanism of activation of c-myc in squamous cell carcinomas of the head and neck. PMID- 7546855 TI - [Adaptable and portable cephalometric devices: a new method in obtaining cephalometrics]. AB - Cephalometric radiographic explorations are widely recommended in the diagnosis of the obstructive sleep apnea syndrome (OSAS). In order to improve their diagnostic value, its practice in the upright and supine position has been proposed. A rigorous practice of cephalometric requires the employment of cephalometric devices in order to assure a correct positioning of the head and provide reliable X-ray pictures, comparable within reasonable accuracy over a period of time. The commercialized cephalostats are often expensive due to the structural conditions needed for their development and usually they not allow the simultaneous practice of upright and supine cephalometric. In this paper we describe a cephalometric adaptable device, developed by our research group, that may solve such problems. PMID- 7546856 TI - [An outpatient study in ENT (otorhinolaryngology) emergencies at a general hospital]. AB - ORL emergencies attended at in a general hospital for a year were studied from 1 7-1990 to 30-6-1991. 168,145 emergencies were received of which 3,101 (1.8%) were attended by an otorhinolaringologist. The age, sex, arrival time, aetiology, treatment, admissions and the admissions where AEP (Appropriateness Evaluation Protocol) criteria was applied were thoroughly examined. The most numerous pathologies presented were infectious inflammations (41%), hemorrhages (21%), and foreign bodies (18.5%). But the most frequent aetiologies were epistaxis (16.3%), otitis media (14.8%) and otitis externa (14.8%). Most patients arrived generally between 9-12 hrs. (21.3%), and 17-20 hrs. (25.4%). The average age was 38.8 years old, though there was a significant difference depending on the pathology presented. Infectious inflammations emergencies were usually the youngest age group (average age 32) and tumoral processes the eldest (average age 60), 5% of the cases were admitted. These admissions accounted for only 17.9% of total admissions. Because AEP criteria was employed 97.5% of admissions presented inclusion criteria. PMID- 7546857 TI - [Simultaneous bilateral facial paralysis]. AB - Simultaneous bilateral facial paralysis (SBFP) is very rare, but it can present itself as the first complaint in a wide spectrum of diseases and a comprehensive evaluation must be completed. Bilateral Bell's Palsy can be diagnosed only when all other possible causes have been excluded. The prognosis and treatment for SBFP is dependent upon the underlying etiology. We present four cases of SBFP seen in our department which were finally diagnosed as leukemic infiltration, sarcoidosis, Bell's Palsy and demyelinating polyneuropathy (probably Guillain Barre syndrome). The most important etiologies of the SBFP and the management of this entity are discussed. PMID- 7546858 TI - [Neurinoma of the primary facial nerve]. AB - Facial nerve neurinomas are unusual tumors which originate fin the Schwann cells of the nerve sheath. Diagnosis of these tumors has improved in the last years due to radiological and clinical advances. Here presented is a case study of a patient with neurinoma of the primary facial nerve which clinically manifested itself as a recurrent facial paralysis. A high resolution T-C scan was sufficient for tumour identification, treatment consisted in a middle fossa approach and tumour removal followed by hypoglossa-facial anastomosis. PMID- 7546859 TI - [Case presentation of malacoplakia of the middle ear]. AB - Malakoplakia is a chronic inflammatory process usually located in the urinary bladder but it may occasionally affect other organs. It is a rarely found in ENT cases. Such a case was diagnosed and treated in our department. Possible pathogenesis is also discussed. PMID- 7546860 TI - [Meningitis and cerebrospinal liquid fistula as a complication from the surgery of endolymphatic sac]. AB - A 28 years old woman diagnosed of Meniere's disease was surgically treated with a drainage from endolymphatic system and a valve placed. Six years after surgery, the patient presented a bacteria meningitis an a cerebrospinal fluid leak to mastoid was found. New surgery was needed. Complications of endolymphatic system surgery, its treatment and frequency are discussed. This case is a very rare. PMID- 7546861 TI - [Subdural empyema induced by frontal sinusitis]. AB - Nowadays the complications arising from sinusitis are rare. However its seriousness can even cause death. Intracranial complications are much more uncommon than the orbital ones. However, slight symptoms can appear, which may disjunct the correlation between clinical-radiology and the severity of the infection. The diagnosis is fundamentally based on the TAC, and early treatment with intravenous anti-biotherapy should be implemented, being followed by drainage surgery of the sinus and empyema. We have presented a case which evolved favourably and revised all the literature pertaining to it. PMID- 7546862 TI - [Solitary naso-sinusal fibrous tumor]. AB - Here we present two clinical cases of solitary naso-sinusal fibrous tumour. This tumour has a mesenchymal origin and has an evident pleural location, with well defined immunohistochemical and ultrastructural characteristics. The clinical manifestation is an apparent benign tumour with an inclination to local aggressiveness. The most effective treatment seems to be surgical removal, but given the low number of cases mentioned (there are only eight cases reported in international literature), and the tendency to eventual recurrence, called for cobalt therapy after surgery. The evolution of clinical cases is irregular. PMID- 7546863 TI - [Carotid paraganglioma: a case report]. AB - Carotid paraganglioma are tumors arising from chemoreceptor paraganglioma cells. These cells have proved to have an embryologic origin from the neural crests. Due to their low incidence these tumors are not an habitually found in daily practices. We present a case of carotid body paraganglioma diagnosed by clinical exploration, CT scan, magnetic resonance imaging and magnetic resonance angiography which was successfully treated by surgery. The diagnostic and therapeutic difficulties present in these tumors are discussed. PMID- 7546864 TI - [Pleomorphic adenoma of the minor salivary gland]. AB - Minor salivary gland tumors represent about 10-15% of salivary gland tumors. The most frequently seen histological type are pleomorphic adenomas, which are usually located at the hard palate. Clinical and pathological features of 7 cases are reviewed. PMID- 7546865 TI - Supporting problem drug users: improving methadone maintenance in general practice. PMID- 7546866 TI - Bad backs, good policy? PMID- 7546867 TI - Differences between general practices in hospital admission rates for self inflicted injury and self-poisoning: influence of socioeconomic factors. AB - BACKGROUND: Self-inflicted injury and self-poisoning are major causes of hospital admission of young adults throughout the United Kingdom, while in Scotland, suicide is the leading cause of death each year in persons aged 15 to 40 years. General practitioners are in a unique position in that they may have contact with the patient before the attempted suicide and later play a supportive role along with other health service and social work professionals. AIM: This study set out to examine the differences between 72 general practices in Tayside in the hospital admission rates for self-inflicted injury/poisoning among their patients, and the extent to which these differences were related to the socioeconomic conditions prevailing in the patients' areas of residence and in the areas in which practices were located. The study also aimed to examine the agents of self-inflicted injury/poisoning most commonly used by different age groups. METHOD: Details of admissions to hospitals in Tayside for self-inflicted injury/poisoning, from 1991 to 1993 inclusive, were obtained from a national, hospital inpatient discharge summary scheme, loaded onto a microcomputer and analysed using standard commercial software. Data from the 1991 census at the postcode sector level and death registrations summaries were obtained from the office of the registrar general for Scotland. The deprivation categories used were based on 1991 census data. RESULTS: In the study period, 52% of hospital admissions for self-inflicted injury/poisoning were of patients aged 30 years or over. Overall, the number of admissions of females exceeded that of males by 26%. Annual hospital admission rates for self-inflicted injury/poisoning per 1000 registered patients averaged over the Tayside practices correlated closely with the male unemployment rate in, and deprivation category of, the postcode sector in which the practice was located. Rates ranged from an average of 1.1 admissions per 1000 registered patients per year in sectors with less than 5% male unemployment of 4.6 where male unemployment was 15% or over. Similarly, the admission rates ranged from 1.1 per 1000 registered patients per year in the most affluent sectors to 3.3 in the most deprived sectors. The proportion of older patients who used sedatives, hypnotics and tranquillizers to poison themselves was greater than that of younger patients; analgesics, such as paracetamol, were the agents most commonly used for self-poisoning by the younger age groups. CONCLUSION: The routine monitoring at national level of hospital admission rates for self-inflicted injury/poisoning, using established computerized information systems, would enable the identification of those practices that have a relatively high proportion of such admissions. This would provide a starting point for the identification of pilot sites for the development of protocols for offering multi-agency support to high-risk groups of patients. PMID- 7546868 TI - Audit of preventive activities in 16 inner London practices using a validated measure of patient population, the 'active patient' denominator. Healthy Eastenders Project. AB - BACKGROUND: Reliable comparison of the results of audit between general practices and over time requires standard definitions of numerators and denominators. This is particularly relevant in areas of high population turnover and practice list inflation. Without simple validation to remove supernumeraries, population coverage and professional activity may be underestimated. AIM: This audit study aimed to define a standard denominator, the 'active patient' denominator, to enable comparison of professional activity and population coverage for preventive activities between general practices and over time. It also aimed to document the extent to which computers were used for recording such activities. METHOD: A random sample of people in the age group 30-64 years was drawn from the computerized general practice registers of the 16 inner London general practices that participated in the 'healthy eastenders project'. A validation procedure excluded those patients who were likely to have died or moved away, or who for administrative reasons were unable to contribute to the numerator; this allowed the creation of the active patient denominator. An audit of preventive activities with numerators drawn from both paper and computerized medical records was carried out and results were presented so that practices could compare their results with those of their peers and over time. RESULTS: Of the original sample of 2331 people, 25% (practice range 13%-37%) were excluded as a result of the validation procedure. A denominator based on the complete, unexpurgated practice register rather than the validated active patient denominator would have reduced the proportion of people with blood pressure recorded within the preceding five years from 77% to 61%, recording of smoking status from 68% to 53% and recording of cervical smears from 80% to 66%. Only 53% of the last recordings, within the preceding five years, of blood pressure and only 54% of those of smoking status were recorded on the practice computer. In contrast, 82% of recorded cervical smears were recorded on computer. CONCLUSION: The active patient denominator produces a more accurate estimate of population coverage and professional activity, both of which are underestimated by the complete, unexpurgated practice register. A standard definition of the denominator also allows comparisons to be made between practices and over time. As only half of the recordings of some preventive activities were recorded on computer, it is doubtful whether it is advisable to rely on computers for audit where paper records are also maintained. PMID- 7546869 TI - Relationship between the provision of counselling and the prescribing of antidepressants, hypnotics and anxiolytics in general practice. AB - BACKGROUND: The provision of counselling in general practice is increasing, despite uncertainty concerning its effectiveness. Furthermore, the relationship between counselling and prescribing of antidepressants, hypnotics and anxiolytics in general practice is not known. AIM: This study set out to assess the relationship between provision of counselling and prescribing of antidepressants, hypnotics and anxiolytics in general practice. METHOD: An observational, cross sectional study of general practices in Oxfordshire Family Health Services Authority was undertaken. Practices were surveyed on the availability of counselling services. The quantity and cost of prescribing of psychotropic drugs over one year (April 1992 to March 1993) were compared for practices with different levels of counselling provision. RESULTS: Of the 82 (96%) respondents, 74 (90%) referred patients for counselling; of these 74 practices, the highest levels of prescribing, in terms of number of items and net ingredient cost, were seen in those practices that employed a counsellor working on the premises. The lowest levels of prescribing were seen in those practices that referred their patients to a counsellor not working on the practice premises. CONCLUSION: The relationship between the provision of counselling and the level of prescribing of antidepressants, hypnotics and anxiolytics is complex. In this study lower levels of prescribing of these drugs in practices with higher provision of counselling were not observed. PMID- 7546871 TI - Women's decisions about whether or not to take hormone replacement therapy: influence of social and medical factors. AB - BACKGROUND: General practitioners have been described as reluctant to prescribe hormone replacement therapy although its use is widely discussed and advocated in the popular media. AIM: This study set out to identify women's perceptions of media coverage of hormone replacement therapy; the people influencing women's decisions about therapy and women's sources of information; their general practitioners' attitudes to therapy; and women's experiences of the primary health care team in relation to hormone replacement therapy. METHOD: In 1993, a postal questionnaire survey was undertaken of 1649 women aged between 20 and 69 years registered with eight general practices in Stockton-on-Tees. RESULTS: A total of 1225 women (74%) returned questionnaires. Women considered that the media portrayed mainly positive images of hormone replacement therapy. A substantial minority of women found the media information unhelpful (30%) or felt that it was incorrect (17%). General practitioners and practice nurses were most frequently considered to be the most important people in helping women decide about taking therapy, but relatives and friends were also important; 41% of women named no one. The media, friends and relatives were most commonly cited as the main sources of information about therapy. Of the women who had discussed hormone replacement therapy with their general practitioner, 65% felt that their general practitioner was in favour of its use for relieving menopausal symptoms. Two thirds of women felt they had had enough time and information when discussing hormone replacement therapy with their general practitioner and/or practice nurse. CONCLUSION: Women did not seem to perceive any resistance from their general practitioner to the prescribing of hormone replacement therapy. Although women gathered information about therapy from sources other than their doctor, doctors have an important role, as providers of the therapy, in listening to women and helping them to make their own decision about whether or not to take hormone replacement therapy. PMID- 7546870 TI - Influence of sex of general practitioner on management of menorrhagia. AB - BACKGROUND: There is an assumption that men and women general practitioners adopt different practice styles in the management of gynaecological disorders. However, there is little evidence to support this view. AIM: A study was undertaken to compare the practice styles of men and women general practitioners in the management of menorrhagia. METHOD: The study took place in 73 general practices in the Oxford Regional Health Authority area. A total of 348 patients who consulted 74 men general practitioners and 43 women general practitioners with a complaint of menorrhagia were recruited into the study and completed postal questionnaires nine and 18 months after entry into the study. Main outcome measures were men and women general practitioners' awareness of patients' treatment preferences, treatment received by patients, patients' involvement in treatment decisions and patients' satisfaction with treatment received. RESULTS: There were no statistically significant differences in treatment received by patients of men and women general practitioners. Fewer patients consulting women general practitioners were referred to a gynaecologist compared with patients consulting men general practitioners (56% versus 64%) and fewer underwent surgery (39% versus 47%). More patients consulting women general practitioners reported participation in treatment decisions (63% versus 53%) but more patients consulting men general practitioners were satisfied with the care they received (66% versus 55%). Again, these differences were not statistically significant. CONCLUSION: Although some indications of sex-associated differences in practice style were found in this study, the similarities in practice styles of men and women general practitioners were more striking than the differences. PMID- 7546872 TI - Does 48 hours' bed rest influence the outcome of acute low back pain? AB - BACKGROUND: Bed rest is a traditional treatment for back pain, yet only in recent years has the therapeutic benefit of this been questioned. AIM: The aim of this pilot study was to ascertain whether or not 48 hours' bed rest had an effect on the outcome of acute low back pain. METHOD: The study was conducted as a randomized controlled trial to compare a prescription of 48 hours' strict bed rest with controls; the control subjects were encouraged to remain mobile and to have no daytime rest. Nine general practitioners from practices in the West Midlands recruited patients in the age range 16-60 years who presented with low back pain of less than seven days' duration, with or without pain radiation. The outcome measures assessed were: change in straight leg raise and lumbar flexion after seven days, Oswestry and Roland-Morris disability scores after seven days and 28 days, and time taken from work. RESULTS: Forty two patients were recruited: 20 were allocated to bed rest and 22 as controls. Compared with the bed rest group the control group had statistically better Roland-Morris scores at day seven (P < 0.05) but not at day 28. At day seven, there were no statistically significant differences between groups in straight leg raise or lumbar flexion measurements although the control group had a better mean lumbar flexion than the bed rest group. The improvement in disability scores at day seven compared with day one was similar for the two groups but more of the control group had fully recovered (defined as scores of one or zero on the Roland-Morris disability scale and five or less on the Oswestry disability scale) by day seven. Remaining mobile did not appear to cause any adverse effects. The number of days lost from work in both groups was equal. A large number of self-remedies and physical therapies were recorded by subjects from both groups. CONCLUSION: The results of this pilot study did not indicate whether bed rest or remaining mobile was superior for the treatment of acute low back pain; however, the study sample was small. Subjects in the control group possibly fared better as they appeared to have better lumbar flexion at day seven. It appears that 48 hours' bed rest cannot be recommended for the treatment of acute low back pain on the basis of this small study. Large scale definitive trials are required to detect clinically significant differences. PMID- 7546873 TI - Confidentiality of medical records: the patient's perspective. AB - BACKGROUND: The development of modern information technology and the increasing amount of multidisciplinary teamwork in primary health care mean that the principle of patient confidentiality is becoming difficult to uphold. The debate about confidentiality so far has paid little attention to patients' views. AIM: A qualitative study was undertaken to explore patients' expectations and attitudes concerning confidentiality of patients' medical records in general practice. METHOD: Semi-structured interviews were carried out with 39 patients from one general practice. RESULTS: Patients' expectations diverged considerably from actual practice. The majority of interviewees felt that administrative and secretarial staff should not have access to medical records. Some patients had reservations about a doctor not directly involved in their care having access to their records. They were unaware of the fact that practice staff had ready access to their medical records. Interviewees had particular concerns about recording of nonmedical information in their records, and the confidentiality of computerized records. CONCLUSION: Assumptions of shared doctor-patient definitions of confidentiality, at least in this practice, would be misplaced. It is suggested that explicit negotiations about what is recorded in patients' records would go some way to addressing the discrepancies identified in this study. PMID- 7546875 TI - Proactive, nurse-run asthma care in general practice reduces asthma morbidity: scientific fact or medical assumption? AB - Nurse-run asthma care in general practice in the United Kingdom has become extremely common, particularly since the introduction of the 1990 contract for general practitioners, but there have been few controlled trials of the clinical effectiveness of this approach to asthma care. A study attempted to compare the outcome of asthma care over three years in two similar practices when one practice provided proactive, nurse-run care and the second continued with a traditional (reactive) approach, and to examine the process of care when such changes were introduced. Despite the investment of considerable resources, statistically significant differences could not be shown between the two practices using a comprehensive variety of outcome measures. This could be interpreted as meaning that nurse-run asthma care may be ineffective, but the negative outcome is much more likely to reflect difficulties in the manner in which the intervention practice sought to develop its service and in the research process. There were extensive methodological problems leading to a potential type 2 error. A randomized controlled trial of nurse-run asthma care would now be difficult to conduct, and so it may be necessary to accept nurse-run asthma care without definitive proof of its clinical effectiveness. PMID- 7546874 TI - Role of Helicobacter pylori in gastrointestinal disease: implications for primary care of a revolution in management of dyspepsia. AB - The majority of patients with dyspepsia are managed in general practice. However, most of the literature on Helicobacter pylori and its association with gastrointestinal disease has originated from secondary care. This review summarizes the role of H pylori in dyspepsia from the perspective of primary care and suggests a new strategy for the management of dyspeptic patients in this setting. Recent meta-analyses and consensus statements have supported the use of eradication therapy as first-line treatment of peptic ulceration. Studies from primary care have supported the use of eradication therapy in patients who have H pylori related peptic ulcer disease and require long-term H2-antagonist medication, on both clinical benefit and cost-effectiveness grounds. Of the many regimens proposed for the eradication of H pylori, the best evidence supports a triple combination of bismuth, metronidazole and tetracycline. Regimens using proton pump inhibitors may be more acceptable to patients but lack good evidence from trials. Use of a positive serum enzyme-linked immunoabsorbent assay for H pylori antibodies as a criterion for endoscopic investigation has been shown to result in a 23% reduction in endoscopic workload. Further research should answer questions of importance to general practitioners, such as the role of eradication therapy in patients with nonulcer dyspepsia and the effectiveness of eradication of H pylori in the prevention of gastric cancer. PMID- 7546876 TI - GP workload in nursing homes. PMID- 7546877 TI - Delay in cancer diagnosis. PMID- 7546878 TI - Pneumococcal sepsis in a splenectomized patient. PMID- 7546879 TI - Warfarin in stroke prevention. PMID- 7546880 TI - Urinary tract infection in children. PMID- 7546881 TI - [Late complications of chemoradiotherapeutic combinations: fundamental aspects and clinical experience]. PMID- 7546882 TI - [Late complications of radiotherapy. Radiofibrosis is reversible]. PMID- 7546883 TI - [Cancers of the esophagus. Role of surgery in the management of cancer of the esophagus]. PMID- 7546884 TI - [Cancers of the esophagus. Combined modality therapy by radiotherapy, chemotherapy with or without surgery]. PMID- 7546885 TI - [Cancers of the breast. Primary non-surgical treatments of so-called "operable" cancers of the breast]. PMID- 7546886 TI - [Cancers of the breast. First line chemotherapy for cancers of the breast]. PMID- 7546887 TI - [Cancers of the breast. Conservative treatment of locally advanced cancers of the breast by radiotherapy alone]. PMID- 7546888 TI - [ORL brachytherapy. Focus on brachytherapy for squamous cell carcinoma of the oral cavity and pharynx]. PMID- 7546889 TI - [ORL brachytherapy. Brachytherapy of epidermoid carcinoma of the oral cavity]. PMID- 7546890 TI - [ORL brachytherapy. Brachytherapy of squamous cell carcinoma of the oropharynx]. PMID- 7546891 TI - [ORL brachytherapy. Salvage brachytherapy in ORL cancers (oral cavity, oropharynx, nasopharynx)]. PMID- 7546892 TI - [Hyperthermia: principles, techniques. Current role in the treatment of cancer]. PMID- 7546893 TI - [Contribution of biology to the therapeutic decision: cancer of the breast in 1995]. PMID- 7546894 TI - [3-dimensional targeted radiotherapy. Definition and procedure]. PMID- 7546895 TI - [Correspondence between pre- and intra-radiotherapeutic data]. PMID- 7546896 TI - [A critical reading and interpretation of the results of phase III trials and meta-analysis]. PMID- 7546897 TI - [Quality of life measures in oncology]. PMID- 7546898 TI - [Late complications of radiotherapy: a century-old challenge?]. PMID- 7546899 TI - [Late complications of radiotherapy. Physiopathological aspects of radiation induced fibrosis]. PMID- 7546900 TI - [Late complications of radiotherapy. How to score the late toxicity of radiotherapy]. PMID- 7546901 TI - [Late complications of radiotherapy. Role of the time factor]. PMID- 7546902 TI - Susceptibility of human monocytes to HIV type 1 infection in vitro is not dependent on their level of CD4 expression. AB - Monocytes from HIV-seronegative persons were analyzed for CD4 expression and susceptibility to infection with HIV-1 on the day of isolation and following 1, 2, and 7 days in culture. Although surface CD4 was readily detected on freshly isolated monocytes, these cells were relatively resistant to infection. After 1 to 2 days in culture, when surface expression of CD4 had decreased over 90% to near background levels, cells became susceptible to infection with HIV-1. CD4 expression on monocytes cultured for 7 days was more than four times higher than that on freshly isolated cells, and the cultured cells were fully permissive to infection. These observations suggest that the differing susceptibility of monocytes and monocyte-derived macrophages to infection with HIV-1 is not simply proportional to the level of surface CD4 expression. PMID- 7546903 TI - Contribution of hypervariable domains to the conformation of a broadly neutralizing glycoprotein 120 epitope. AB - Three of the five hypervariable domains (V1-V3) of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 have previously been shown to be dispensable for antigenic epitopes recognized by broadly neutralizing monoclonal antibodies. In this study, the influence of the V4 and V5 domains on an epitope recognized by a broadly neutralizing human monoclonal antibody, 1.5e, was investigated. In contrast with the V1, V2, and V3 domains of gp120, the V4 and V5 domains were found to be critical for binding to both CD4 and 1.5e. Our results suggest that V4 and V5 are in structurally less flexible regions of gp120 than V1, V2, and V3 and raises the question of whether variable domains V4 and V5 are also indispensable for other broadly neutralizing antibodies in the same class as 1.5e. PMID- 7546904 TI - Preparation and characterization of human HIV type 1 neutralizing reference sera. AB - Reference neutralizing antibody (NA) reagents are needed for laboratories to be able to compare results of neutralization assays that will be used to monitor HIV 1 vaccine recipients. In an effort to establish such reference reagents two asymptomatic, seropositive patients were identified with medium to high amounts of cross-reactive NA activity against a number of HIV-1 strains. Sera obtained from each individual at three or four sequential phlebotomies were pooled, and the two pools were each distributed in > 3000 aliquots into glass ampoules and lyophilized, and the ampoules were flame sealed. An HIV-1 antibody-negative reference serum was prepared in a similar fashion after pooling serum from four individuals. Ampoules were tested for uniformity of fill, sterility, moisture content, residual oxygen, stability, infectivity, and presence of antibody. An international collaborative study was conducted to determine the potency of the samples in six laboratories, each using their own neutralization assays and reagents. The results indicated reasonable consistency between laboratories and that both sera have sufficient titers against a variety of strains for use as reference reagents. These reference sera have been included in the World Health Organization (WHO) AIDS Reagent Project and are available through the three AIDS reagent repositories. PMID- 7546905 TI - Frequency of provirus-bearing CD4+ cells in HIV type 1 infection correlates with extent of in vitro apoptosis of CD8+ but not of CD4+ cells. AB - Lymphocytes from HIV-1-infected subjects undergo massive apoptosis when cultured in vitro, and this phenomenon might reflect pathogenetic mechanisms leading to immune dysfunction in vivo. However, (1) lymphocyte death is not restricted to CD4+ cells but seems to involve predominantly CD8+ cells, and (2) the same phenomenon occurs in other viral infections. Furthermore, it is not known whether a relationship exists between the HIV-1 burden and this type of cell death. In this work we sought to determine whether the HIV-1 provirus load correlates with the propensity to apoptosis of CD4+ and CD8+ cells. We studied 10 HIV-1-infected patients with CD4+ cell counts above 500/mm3 and free of concomitant infections. We correlated the frequency of HIV-1-infected CD4+ cells with the extent of culture-induced apoptosis as well as with the phenotype of the apoptotic lymphocytes. We found that the magnitude of apoptosis correlated with the frequency of HIV-1-infected CD4+ cells (p = 0.0007), and that increasing viral load and apoptosis were associated with a shift to the selective death of CD8+ cells. Our data support the view that, in addition to CD4+ cell killing, another immunopathogenic effect of HIV might be that of priming CD8+ cells to apoptosis. In vivo, this could eventually lead to the exhaustion of the cytotoxic T cell compartment. PMID- 7546906 TI - Localization of human immunodeficiency virus Rev in transfected and virus infected cells. AB - The rev gene product of human immunodeficiency virus (HIV) is obligatory for viral replication. Rev interacts specifically with a structured RNA sequence within the viral genome termed the REV response element (RRE). Although the importance of Rev for the expression of viral proteins is well documented, its functional mechanism remains unresolved. Previous studies identified Rev in the absence of RRE to be a nuclear protein localized primarily within the nucleoli. To extend our understanding of the role of Rev in viral replication, immunolocalization studies of Rev and other nuclear components were carried out in transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV. In both types of cells, Rev-like immunoreactivity was distributed both in the nucleoplasm and cytoplasm. Within the nucleus, Rev immunoreactivity was not evenly distributed but was present within focal concentrations. In transfected cells that were double labeled for Rev and SC-35, which labels a known component of spliceosomes, the foci of Rev labeling were distinct from the "speckles" labeled by SC-35, although Rev foci and speckles were often juxtaposed. In addition, morphological changes in the three dimensional network of speckles were observed in both transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV-1 and HIV-2. Our observations are consistent with the proposed dual role of Rev in mRNA transport and splicing. PMID- 7546907 TI - Decreased thymidine kinase levels in peripheral blood cells from HIV-seropositive individuals: implications for zidovudine metabolism. AB - Azidothymidine (zidovudine, AZT) used for treatment of HIV infection blocks the viral reverse transcriptase after phosphorylation by cellular enzymes. The first step in this reaction is the formation of AZT monophosphate, primarily catalyzed by host cytoplasmatic thymidine kinase (TK1). The activity of TK1 was determined in extracts of PHA-stimulated peripheral blood mononuclear cells (PBMCs) from 20 healthy volunteers and 49 HIV-infected patients at different stages of disease. In both groups we found a large intra- and interindividual variation of TK activity. Because TK1 expression is cell cycle regulated the proportion of stimulated cells was determined in the samples and the median thymidine kinase activity calculated. It was 3.0 pmol/mg/min x % S phase in the HIV-seronegative group and 1.1 pmol/mg/min x % S phase in HIV-infected individuals. The difference in thymidine kinase activity is statistically significant (p = 0.0001). The concentration of TK1 protein in the same extracts was also determined by immunoblotting. A positive correlation (r = 0.74) was observed between TK activity and amount of TK1 protein. The reason for this downregulation of TK is still unknown but may be related to the anergy observed in lymphocytes from HIV infected persons. The reduced capacity for intracellular phosphorylation of AZT in HIV-infected individuals may be an important factor in the emergence of clinical AZT resistance and should also be accounted for in testing AZT resistance in vitro with PBMCs from healthy blood donors. PMID- 7546908 TI - Identification of human T cell lymphotropic virus type IIa infection in the Kayapo, an indigenous population of Brazil. AB - Human T cell lymphotropic virus type II (HTLV-II) infection is endemic in a number of indigenous populations in North, Central, and South America. In the present study we have employed serological and molecular methods to identify HTLV II infection in Indian communities in the Amazon region of Brazil. Sera (1324) from 25 different Indian communities were analyzed by ELISA and Western blot. One hundred and four samples (7.8%) from a number of culturally distinct and geographically unrelated populations were found to have reactivities consistent with HTLV-II infection. Of these, 67 were from the Kayapo Indian communities, which had an overall seroprevalence rate of greater than 30%. In addition, high seroprevalence rates were observed in three other communities, the Munduruku, Arara do Laranjal and the Tyrio, suggesting that there are additional foci of endemic infection in the Amazon region. In the Kayapo, seroprevalence rates tended to increase with age, supporting the importance of sexual transmission of the virus, and family studies demonstrated that vertical transmission is also an important route of infection. Restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of a region of the env gene demonstrated that the Kayapo are infected with the HTLV-IIa subtype. Moreover, nucleotide sequence analysis of the LTR demonstrated that this belonged to a distinct HTLV-IIa phylogenetic group. The identification of HTLV-IIa in the Kayapo is, as far as we are aware, the first identified endemic focus of infection by this subtype of HTLV-II in the Americas. PMID- 7546909 TI - Frequent detection of antibodies against HTLV antigens in patients with AIDS related non-Hodgkin lymphoma. AB - We studied the prevalence of anti-HTLV-I/II antibodies in 22 patients with AIDS related non-Hodgkin lymphoma (NHL), 453 HIV-1-infected patients without lymphoma (194 of whom were diagnosed as having AIDS), and 6 HIV-1-positive and 75 HIV-1 negative patients with Hodgkin lymphoma. The frequency of serological reactivity against HTLV antigens was significantly higher in the AIDS patients with lymphoma than in those without (8 of 22, 36.4% vs. 20 of 194, 10.3%-p = 0.0027). One of the HIV-1-positive and none of the HIV-1-negative patients with Hodgkin lymphoma showed anti-HTLV-I/II reactivity. Four of the eight seropositive NHL patients showed antibodies directed against HTLV-II recombinant antigens when tested for serological discrimination in a Western blot assay. A PCR study of PBMCs from the only patient with NHL still alive at the time of the study showed HTLV-II specific sequences in the genomic DNA. These data suggest that HTLV-II or a closely homologous retrovirus infects a high proportion of patients with AIDS associated NHL. PMID- 7546910 TI - Human foamy virus DNA forms and expression in persistently infected Dami megakaryocytic cells. AB - We have characterized human foamy virus (HFV) proviral DNA and determined HFV expression in a persistent infection model, the Dami megakaryocytic cell line. Molecular studies were performed on parental persistently infected cells (Dami P), as well as on derived clones (Dami-Cl). We report that in these nonlytic and non-HFV producer cells, viral DNA was found to be integrated into the cellular genome and that the few free proviral forms detected in Dami-P cells were deleted in their 5' LTR. Our molecular analysis indicates the presence of undeleted 5' LTR forms in the integrated provirus within a proviral population mainly composed of deleted forms. In addition, the deletion in the bel1 trans-activator gene, previously described by Saib et al., was found to be highly predominant. However, in 5-iodo-2'-deoxyuridine treated Dami-Cl cultures, virus production occurred, providing evidence for the presence of complete viral genome. Analysis of HFV expression in Dami-Cl cells, by Northern blot and immunoprecipitation, shows that the most striking difference between cytolytic and persistent HFV infection was the lack of expression of structural viral proteins, in contrast with Bet protein expression, which is maintained. Our data suggest that the Bet protein could be involved in the maintenance of viral persistency and that the persistently infected Dami system provides a suitable model for clarifying its function. PMID- 7546911 TI - Evaluation of murine leukemia virus infection as a model for thrombocytopenia of HIV/AIDS: mechanism of thrombocytopenia and modulation of thrombocytopenia by thrombopoietin. AB - Infection of mice with the murine leukemia virus (LP-BM5) was evaluated as a model for the thrombocytopenia of HIV/AIDS. Percent 35S incorporation into platelets, platelet size, platelet count, platelet-associated immunoglobulins (PAIgG), and megakaryocyte size and number were evaluated over a period of 3-9 weeks postinfection (PI). Thrombopoietin from human embryonic kidney cells was administered to mice 9 weeks PI, and similar indices of platelet production were measured 2, 3, and 4 days after treatment with a biological preparation of thrombopoietin (thrombocytopoiesis-stimulating factor, or TSF). Platelet counts decreased in a time-dependent fashion (p = 0.0006) following infection, reaching a nadir at 8 weeks PI (82% of control values). Percent 35S incorporation into platelets also decreased over the 9-week period (p = 0.0001), falling to 63% of control values by week 9. Additionally, platelet volume increased in a linear fashion (p = 0.01), rising to 105% of control values by week 9. No changes in PAIgG were noted over the 9-week period. Megakaryocyte numbers in the femoral marrow were decreased at 8 weeks PI (p = 0.02, 78% of control values), while increased mean megakaryocyte size (p = 0.007, 116% of controls) was noted in the same animals. Increased numbers of naked megakaryocyte nuclei were observed at 3 weeks PI (p < 0.05, 208% of control values). Administration of 2 U/mouse of a highly purified preparation of TSF to virus-infected, thrombocytopenic mice resulted in increased thrombocytopoiesis, as compared to human serum albumin treated, virus-infected controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546912 TI - Passive immunization of rhesus macaques against SIV infection and disease. AB - To evaluate the role of humoral immunity against simian immunodeficiency virus (SIV), we tested whether passive immunization with plasma from SIVmac251 vaccine protected or healthy infected animals would protect rhesus monkeys against intravenous infection with ten 50% animal infectious doses of the cell-free homologous virus. The challenge dose of this SIVmac251 virus stock had previously caused persistent infection in all (21 of 21) nonimmunized controls. A plasma pool was obtained from a donor that had been immunized with an inactivated whole SIVmac251 vaccine produced in human T cells. This plasma pool contained low levels of SIVmac binding and neutralizing antibody but had a high titer of antibodies recognizing human cell proteins. Given 4 or 18 hr before intravenous challenge, this plasma completely protected three of eight recipients from infection and delayed virus detection in one recipient. The five unprotected animals had only a transient or undetectable p27 antigenemia and low virus load in their PBMCs, and all survived at least 7 months after infection. By contrast, no protection was observed in 6 monkeys given inactivated, pooled plasma or purified immunoglobulin (Ig) from healthy SIVmac251-infected animals. This plasma pool and the Ig preparation contained high levels of SIV-binding and neutralizing antibody but no reactivity to human cellular components. Five of the six recipients had persistent antigenemia after challenge and four died acutely from simian AIDS in 4-7 months. These studies suggest that passive transfer of antibody to human cellular antigens can confer protection against SIVmac whereas passive transfer of neutralizing antibodies without human cellular antibodies does not protect against the homologous virus and may enhance infection. PMID- 7546913 TI - Genetic construction and in vitro characterization of SIVsmmPBj14-1.9 noninfectious particles. PMID- 7546914 TI - Antisense phosphorothioate oligodeoxynucleotides alter HIV type 1 replication in cultured human macrophages and peripheral blood mononuclear cells. AB - The use of antisense oligodeoxynucleotides as antiviral drugs to combat HIV-1 infection may offer an alternative to traditional pharmacological therapies. We compared the effects of two 28-mer antisense phosphorothioate oligodeoxynucleotides [PS-oligo(dN)] with non-sequence-specific controls on HIV-1 replication in long-term human monocyte/macrophage and PBMC cultures. The anti rev PS-oligo(dN) was complementary to the messenger RNA (mRNA) sequences derived from the overlapping region of the HIV-1 regulatory genes tat and rev, while anti gag targeted the translational initiation site of the gag mRNA. In vitro cytotoxicity of the PS-oligo(dN) was evaluated at concentrations ranging from 0.1 to 10.0 microM for a period of 20 days. Cell survival was 100% at 0.1 microM, but decreased to 5% at 10.0 microM in relation to the untreated control cultures. Our data demonstrate that replication of both the T cell-tropic and macrophage-tropic HIV-1 strains in primary cells can be inhibited by PS-oligo(dN) in a sequence specific and dose-dependent manner at concentrations achievable in vivo. However, the sequence-dependent antiviral activity of the utilized PS-oligo(dN) was limited to a window of specificity at concentrations between 0.25 and 1.0 microM. PMID- 7546915 TI - Human endogenous retrovirus K does not encode mouse mammary tumor virus-related antigens in human breast carcinomas. PMID- 7546916 TI - Nucleotide sequence of the HIV-2 EHO genome, a divergent HIV-2 isolate. PMID- 7546917 TI - [MR angiography (MRA)]. PMID- 7546918 TI - [Molecular basis of inherited muscular diseases]. PMID- 7546919 TI - [The clinical usefulness of botulinum toxin type A for spasmodic torticollis and facial spasm]. AB - We studied the clinical effectiveness of botulinum toxin type A for spasmodic torticollis and that for facial spasm by multicenter, non-blinded study. The freeze-dried crystalline botulinum toxin type A (AGN 191622; Allergan Inc., Irvine, CA) was injected into the hyperactive muscles and the clinical course was followed for 22 weeks. Repeated injections were done, if necessary, with an interval of 4 weeks. The toxin was highly effective in both disorders. In spasmodic torticollis, clinical severity improved in 38 (63.3%) and the global improvement was seen in 39 (65.0%) out of 60 patients. Subjective improvement was seen in 56 (93.3%). In facial spasm, 52 (92.9%) out of 56 patients improved after the treatment. Unfavorable reactions, mainly consisting of neck muscle weakness and dysphagia in torticollis and facial weakness in facial spasm, were mostly due to the excessive action of the toxin. They were usually mild and transient. No patients discontinued the trial because of side effects. Botulinum toxin injection is a very useful and safe method for the symptomatic treatment of spasmodic torticollis and facial spasm. PMID- 7546920 TI - [Sphingolipid changes in rat cerebral cortex during focal ischemia--how does ceramide accumulate in an ischemic condition?]. AB - Levels of ceramide, sphingomyelin, cerebroside and gangliosides were determined in rat cerebral cortex during focal ischemia produced by middle cerebral artery occlusion. Ceramide began to increase at 6 hours of ischemia and increased to 4.5 folds at 96 hours. Amino-linked fatty acids in increased ceramide were composed solely of non-hydroxy fatty acids, and stearic acid was the most prominent. Sphingomyelin, whose amino-linked fatty acids were mostly stearic acid, decreased in a time-dependent manner and became about a half of controls at 96 hours. Hydroxy fatty acid linked cerebroside decreased at and after 6 hours of ischemia, whereas significant decrease of non-hydroxy fatty acid linked cerebroside occurred only at 96 hours of ischemia. There were no measurable changes in the levels of gangliosides. The results suggested that ceramide was produced in the cerebral cortex by the breakdown of sphingomyelin during ischemia. PMID- 7546922 TI - [Repetitive discharges of proximal origin in a patient with carcinomatous sensory neuropathy]. AB - Repetitive discharges of a motor unit potential were observed during ulnar nerve conduction studies in a patient with pathologically verified carcinomatous sensory neuropathy. The repetitive discharges in this patient had a latency similar to that of the F-wave latency. The first discharge was followed by discharges identical in configuration every 3 to 4 msec. These discharges responded to all of the suprathreshold stimuli, had a constant configuration, and exhibited jitter in their latency. When paired stimuli were applied, the second stimulus collided with the first discharge, leaving the afterdischarges behind at the previous position. Based on these electromyographic findings, the origin of these repetitive discharges is believed to have been the proximal part of a motor axon or a neighboring demyelinating lesion, since degenerative changes have been demonstrated in ventral roots as well as dorsal root ganglia in this disease. PMID- 7546921 TI - [A case of astrocytoma of corpus callosum presented diagnostic dyspraxia]. AB - A case of astrocytoma whose first clinical presentation was diagnostic dyspraxia was reported. A 38-year-old right-handed male experienced funny motion of his left hand triggered by voluntary movement of his right hand. One day, he tried to insert a coin into the vending machine with his right hand, then the left hand was against the other. One month after that event, he experienced headache and vertigo. On admission, there were no abnormal findings on neurological examination. On neuropsychological examination, he was cooperative, well orientated and attentive, and there were no callosal disconnection symptoms. Frontal lobe function tests were slightly impaired. T1-weighted MRI demonstrated irregular mixed signal intensity mass lesion extending from the genu to the body of the corpus callosum and the cingulate gyrus. This lesion was slightly enhanced with Gd-DTPA. Biopsy was performed and histological diagnosis was fibrillary astrocytoma. After irradiation and chemotherapy, he was discharged from the hospital without evident neurological deficit. About 20 cases of diagnostic dyspraxia have been reported and almost all of them were caused by cerebro vascular disease. This is the first case of brain tumor who presented diagnostic dyspraxia. PMID- 7546923 TI - [Glioblastoma multiforme with liver metastasis--case report]. AB - Extracranial metastasis of glioblastoma is rare. This is an autopsy case report of a patient with glioblastoma multiforme found to have metastasized to the liver. A 42-year-old woman was admitted with a chief complaint of headache. Physical and neurological examinations on admission showed no abnormalities. CT and MRI demonstrated a tumor in the left parietooccipital region with invasion into the subependymal area of the left lateral ventricular trig-one. A cerebral angiogram showed tumor staining in the same area. Subtotal tumor resection was performed uneventfully. The microscopic diagnosis was glioblastoma multiforme. Postoperatively, the patient underwent whole brain and local irradiation, and intra-arterial ACNU infusion therapy. One month later, she developed low back pain, probably due to spinal dissemination. Postmortem examination showed local recurrence of the tumor and subarachnoidal dissemination not only in the base of the skull but in the lower spinal cord. Tumor was also observed in the liver, but no lung or lymph node metastasis was detected. Metastasis to the liver in this patient is believed to have occurred via the anastomosis between the vertebral and portal venous system. PMID- 7546924 TI - [Electrophysiologic evaluation of entrapment neuropathy caused by scar formation in recurrent motor branch of the median nerve]. AB - A unique case of mononeuropathy was encountered in a 20-year-old man with isolated involvement of the recurrent motor branch of the right median nerve following a skiing accident one year previously. There was also isolated involvement of the right thenar muscles innervated by the median nerve. Sensation was intact in the right hand and fingers. Conventional conduction studies in the right median nerve yielded no abnormal findings except a reduction in the amplitude of the muscle action potential. However, insertion of a needle electrode into the abductor pollicis brevis muscle revealed some delayed motor unit potentials following electrical stimulation at the wrist. We performed surgery and found the right recurrent motor branch to be entrapped by fibrous scar tissue 3 to 4 mm distal to branching from the median nerve. Direct recordings made from the nerve trunk using a tungsten microelectrode with supramaximal stimulation at the elbow revealed reduced amplitude of the compound nerve action potential distal to the site of entrapment. We concluded that scar tissue resulting from the previous injury had compressed the recurrent motor branch, causing this unique neuropathy. PMID- 7546925 TI - [Cauda equina cavernous hemangioma associated with hydrocephalus--case report]. AB - Spinal cavernous hemangioma is rare, and it is extremely rare for cavernous hemangioma to develop in the cauda equina. There has been only one report of hydrocephalus associated with cavernous hemangioma in the cauda equina. We report a case of cavernous hemangioma in the cauda equina diagnosed on the basis of the headaches due to hydrocephalus. A 67-year-old man was being treated for Parkinson's disease because of tremor of both upper extremities for several years. In December 1991 he complained of occasional headaches. On February 15, 1992 the headaches became severe and frequent, with nausea and vomiting, and his gait became unsteady. Four days later he came to our hospital. Neurological examination revealed fine finger tremor and truncal ataxia. Computerized tomography scanning and magnetic resonance imaging of the head revealed ventricular enlargement, but there were no mass lesions obstructing the cerebrospinal fluid pathway. Lumbar puncture at the L3-L4 level yielded bloody cerebrospinal fluid, and the pressure had increased to 410 mmH2O. Cerebral angiography showed no abnormal findings. Magnetic resonance imaging of the lumbar spine demonstrated an intradural tumor at the level of vertebral body L2. Spinal angiography showed no evidence of abnormal vascularity in the mass at the L2 level. On March 10, 1992, laminectomy at three levels, L1 to L3 was performed, and a well-defined blueberry-like intra-cauda equina tumor 1 cm. in diameter, was removed. One spinal nerve root passed through the tumor. The pathological diagnosis was cavernous hemangioma. After removal of the tumor, the patient's headaches improved, and a follow-up computerized tomography scan six months later showed normal ventricle size.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546926 TI - [A case of multiple myeloma complicated by acute transverse myelopathy due to epidural abscess]. AB - A 51-year-old man developed fever and lumbago followed by rapidly progressive bilateral sensory disturbance below the 9th thoracic spinal cord level, flaccid paraplegia, urinary obstruction and constipation. Based on radiological examinations and laboratory findings, a diagnosis of transverse myelopathy due to epidural abscess was made. A series of MRI studies revealed multiple abscess formation in the paravertebral muscles. Hypergammaglobulinemia with M protein was observed continuously, and further examination revealed multiple myeloma in the early stage. Since it has been reported that several different immunosuppressive mechanisms precede the development of bone lesions in multiple myeloma, these mechanisms may have played an important role in the rapid progression of the abscess in this patient. Multiple myeloma is not only important as one of the disorders underlying epidural abscess of unknown etiology, but important in predisposing to severe infection as a result of the immunosuppressive mechanisms present starting in the early stage of the disease. PMID- 7546927 TI - [Syringomyelia associated with tentorial meningioma]. AB - A 28-year-old male was admitted to our hospital with the complaints of numbness of the left upper limb and gait disturbance. Neurological examination disclosed slight left dysmetria, truncal ataxia and sensory disturbance at the dermatome of C8 and Th1. CT and MRI scans showed a large mass lesion in the left posterior fossa, ventricular dilatation, cavum septi pellucidi and cavum Vergae, empty sella, cervical syringomyelia and left tonsilar herniation. The tumor which attached to cerebellar tentorium was totally removed and a histological diagnosis of meningothelial meningioma was made. Postoperative MRI scan demonstrated a disappearing of syringomyelia with the improved tonsillar herniation. Association of syringomyelia with brain tumor is relatively rare, so its pathogenesis was discussed. PMID- 7546928 TI - [Magnetic resonance imaging in Tay-Sachs disease]. PMID- 7546929 TI - [A 86-year-old woman with dementia, gait and speech disturbance, and right hemiparesis]. AB - We report a 86-year-old woman who developed dementia, gait disturbance, speech disturbance, and right hemiparesis. The patient was well until March of 1979 when upon wakening up on one morning she noted slurring of her speech and weakness in her left upper and lower extremities. These symptoms cleared up during the next several months, however, she noted weakness in her left leg again in May 1985. In 1988, her posture became stooped and she walked in small steps. In 1990, she developed memory disturbance and difficulty in naming. In March 1993, she developed weakness in her right hand; she was treated with aspirin and amantadine HCl, however, she deteriorated during the next two week period, and was admitted to our hospital on March 27, 1993. On admission, she appeared alert, however, she could not answer verbally to questions; she could only utter unintelligible sounds. Apparently she was markedly demented. Her blood pressure was 170/98 mmHg, and general physical examination was unremarkable. Cranial nerves were grossly normal except for marked non-fluency in her word expression. She could not stand or walk, and apparently her right upper and lower extremities were paralyzed with some contracture. Deep reflexes were normally active without asymmetry. Chaddock sign was positive bilaterally. Sensory examination was difficult. Pertinent laboratory examination included WBC 13,000/microliters, BUN 152mg/dl, creatinine 3.75mg/dl, CRP 20.1mg/dl; a chest X-ray film revealed pneumonic shadow in the upper and the middle right lung fields. Cranial CT scan revealed multiple lacunar infarctions in both basal ganglia and cerebral white matters; periventricular lucency was also noted. She was treated with antibiotics and intravenous fluid. Acid-fast bacilli were recovered from sputum, and she was transferred to another hospital for the treatment of pulmonary tuberculosis. After its treatment she returned to our hospital on July 8, 1993, when her condition was complicated with aspiration pneumonia. On admission, she was semicomatose, and no intelligible words were heard. Right facial paresis of the central type was noted. She was unable to stand or walk, and her right upper and lower extremities were paretic. Deep reflexes were increased with extensor toe sign on the right. She was treated with chemotherapy and intravenous fluid, however, her clinical course was complicated with respiratory as well as urinary tract infections. She developed cardiac as well as renal failure and expired on September 25, 1993.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7546930 TI - [Approach to human glioma cells using antisense molecules]. PMID- 7546931 TI - [Therapy of malignant brain tumors by using apoptosis]. PMID- 7546932 TI - [Gene therapy of glioma using cytokine]. PMID- 7546933 TI - [p53 gene mutations in brain tumors]. PMID- 7546934 TI - [Dose-response relationship in the treatment of cervical dystonia with botulinum toxin type A (AGN 191622)--a phase II study]. AB - Injection of botulinum toxin type A has been the treatment of choice for spasmodic torticollis for several years. Although previous reports demonstrate its effectiveness and safety, the treatment strategy has been empirical. The present study, using the freeze-dried crystalline botulinum toxin type A (AGN 191622; Allergan Inc., Irvine, CA), aimed to compare the efficacy among three treatment groups divided into low, medium and high dosage levels. Fifty-one patients who entered the study were grouped into low-dose (60 units/session), medium-dose (120 units/session) and high-dose (240 units/session) groups. Two patients (one in low-dose group and the other in high-dose group) were excluded from the assessment of efficacy because they dropped out in the early phase of the study. One experienced worsening of an existing psychosis and the other developed an acute respiratory infection. Injection sites were decided individually by palpation. If the clinical response was not satisfactory four weeks after an injection, the patient was re-injected with the same dose of toxin. The follow-up period was 14 weeks from the initial injection. The results showed that the high-dose group improved more than the other groups in the parameters of severity of symptoms and subjective benefit (p = 0.000). Also, fewer injections were required in the high-dose group to achieve substantial clinical benefit. Although the mean reduction in Tsui's score was not statistically significant among the groups, the "marked improvement" was seen more frequently in the high-dose group (p = 0.033). Unfavorable adverse effects including excessive weakness and dysphasia were always mild and transient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546935 TI - [Experimental study on effects of omental transposition in cats with spinal cord injury]. AB - The effects of autogenous omental transposition on spinal cord injury in cats were examined using a cord crush injury model produced by a modification of Allen's weight-dropping technique. Twenty-three cats were divided into two groups: an omental transposition group, and a control group. The animals were sacrificed at 1, 2, 3, and 4 weeks after injury. The injured cords were examined histologically using India ink perfusion, morphometry of the spinal cord cavities and also by somatosensory evoked potential (SEP) studies. After 1 week, hemorrhagic necrosis, cavitation and demyelination of almost the same extent and severity were noted in both groups. These changes diminished with time in the omental transposition group, but tended to persist in the control group. In the omental transposition group, fibrous coats of Goldsmith were newly formed on the injured cord between the incised dura edges. After 3 weeks, the fibrous coat was thickened with active proliferation of fibroblasts and collagen fibers. India ink particles were noticed within the fine vessels of the omentum, fibrous coat and injured cord at 4 weeks. No India ink particles were found in the control group even after 4 weeks. In the somatosensory evoked potentials, biphasic or triphasic waves noted before injury were absent immediately after trauma. At 3 and 4 weeks, 3 of 5 cats with the omentum showed reappearance of the waves, whereas no wave reappearance was noted in the controls. These results suggest that autogenous omental transposition helps to accelerate the healing process after crush injury to the spinal cord. PMID- 7546936 TI - [Effects of 5-HT1A receptor agonist and 5-HT1B receptor agonist on single and paired rats' behavior]. AB - Serotonin (5-hydroxytryptamine, 5-HT) receptors as well as dopamine receptors are important in connection with schizophrenia. In this study we evaluated the effects of the 5-HT1A receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 8 OH-DPAT) and 5-HT1B receptor agonist (1-(3-trifluoromethylphenyl)piperazine, TFMPP) on the single and paired rats' movement distance in an open-field. Generally animals are gregarious in their natural setting, so the presence of another companion might alter the effects of the drugs. Therefore we devised a video analysis system to pick up the two rats' movements individually through two CCD video cameras and objectively recorded two rats' movement for 30 minutes. Experimental rats were injected with 8-OH-DPAT (0.05, 0.25, 1.25, 6.25 mg/kg, s.c.) or TFMPP (0.12, 0.5, 2.0, 8.0 mg/kg, i.p.) and the control rats were injected with saline. In the single cases experiments, the rats were put alone into the open field after the injection. In the paired cases experiments, they were put into the open field with a companion rat after the injection. In the 8 OH-DPAT experiment, the movement distance of single cases showed dose dependent increase tendency and that of the 1.25 mg/kg group and the 6.25 mg/kg group showed significant increase, but that of paired cases did not show that tendency, on the contrary, the movement distance of 0.05 mg/kg group showed significant decrease. In the TFMPP experiment, the movement distance of 2.0 mg/kg groups showed significant increases in the single and the paired cases. These findings suggest that both 5-HT1A receptors and 5-HT1B receptors affect the rats' movement distance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546937 TI - [Ubiquitin immunoreactivity in the central nervous system of gracile axonal dystrophy (GAD) mouse]. AB - In order to know the relationship of ubiquitin and axonal degeneration in the central nervous system (CNS) of the gracile axonal dystrophy (GAD) mutant mouse, the immunocytochemical study was performed in the spinal cord, medulla oblongata and brain of normal and GAD mice at 4, 9, 18 and 32 weeks of age. The polyclonal antibodies of ubiquitin were used for this study. The results were as follows: Many ubiquitin-positive dot-like structures (DS) were first observed in the gracile nucleus affected primarily with axonal degeneration. They extended to the gracile fasciculus on the dorsal part of spinal cord in accordance with the dying back type degeneration. The second-order neurons at Clarke's nucleus were also affected slightly later stages and revealed ubiquitin-positive DS along the posterior spinocerebellar tract and the white matter of certain lobes of cerebellum. In 18th week, the transneuronal degeneration started from the distal axonal ends of the primary sensory neurons came up to the some parts of cortical neurons through the secondary neurons in the thalamus. The ubiquitin-positive DS were detected on the ventral surface of the medulla oblongata and increased progressively in number along the corticospinal (pyramidal) tract of spinal cord which consists of the descending fibers derived from the cortical neurons. These findings suggest that abnormal proteins in the degenerating axons were ubiquitinated rapidly before they accumulated in the preterminal axons whose neuron stems are far away from the place, and that GAD mouse would be a useful animal model to know the mechanism(s) of the naturally occurring transneuronal degeneration from the distal axonal ends of both ascending sensory neurons and descending pyramidal neurons in the CNS. PMID- 7546938 TI - [L-DOPA-producing primary fibroblasts genetically modified with a retrovirus vector system]. AB - Although intracerebral grafting has become a new strategy for the treatment of Parkinson's disease, many problems related to the grafts remain. We focused on primary skin fibroblasts as grafts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing the cDNA of human tyrosine hydroxylase (TH) (pLTHSNL) or cytomegalovirus promoter (pCTHSNL) as a foreign promoter, and catecholamine production and release by these genetically modified fibroblasts, were analyzed in vitro immunocytochemically and by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The cells were supplemented with biopterin (BH4; (6R)-L-erythro-tetrahydrobiopterin), a cofactor required for TH activity, and they produced and released L-DOPA into the culture medium. When exposed to the combination of a foreign promoter and BH4, L-DOPA production increased in a time-dependent manner, and was unaffected by the number of cell-passages or the duration of liquid-nitrogen freezing. This suggests that the foreign gene (THcDNA)-containing retrovirus vector had integrated into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with THcDNA using this retrovirus vector system appear to be a promising graft for transplantation and gene therapy of Parkinson's disease in the future. PMID- 7546939 TI - [A case of very slowly progressive, high-cervical spondylotic myelopathy presenting with symmetric deep sensory deficits in the palms]. AB - We report a case of very slowly progressive, high-cervical spondylotic myelopathy with symmetrical deep sensory deficits in the palms. A 76-year-old man began to feel tingling sensation in the second fingers of the bilateral hands 30 years prior to admission. The abnormal sensation spread from the first to the third fingers, and subsequently all over the palms. He noticed intermittent sharp pain in the dermatomes of C4 and 5 bilaterally from his late sixties, and later he developed clumsiness of fine finger movements. In recent years he experienced stiffness in the thighs while walking. On neurological examination, there was a mild sensory deficits in light touch over the bilateral palms, while perception of temperature and pain was normal. Vibration sense was severely and position sense mildly impaired. Discriminative sensation, including graphesthesia, stereognosis, two-point discrimination and texture recognition, was severely impaired over the bilateral palms. On the other hand, all modalities of sensation were normal in the lower limbs. Gross motor dysfunction, such as weakness of limbs, amyotrophy or gait disturbance, was not present. He did not show limb ataxia, but the dexterity of his fingers was severely impaired. Deep tendon reflexes were mildly increased except for the Achilles tendons that were hyporeactive. Plantar responses were flexor bilaterally. Nerve conduction study revealed giant F waves and H reflexes by stimulations of the median and ulnar nerves bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546940 TI - [A case of myogenic intractable pain due to thalamic hemorrhage relieved by Vim Vo thalamotomy]. AB - A 58-year-old female patient with myogenic intractable pain due to thalamic hemorrhage which was relieved by stereotactic Vim-Vo thalamotomy was reported. She had an intractable pain, involuntary movement and deep sensory disturbance in her right arm. We performed stereotactic Vim-Vo thalamotomy using electrophysiological technique to relieve her involuntary movement and myogenic intractable pain. The electrophysiological recordings suggested the followings: (1) The destructive lesion by thalamic hemorrhage was located on from posterior border of Vim nucleus to the upper half of Vc nucleus. (2) Most of Vim neurons remained as intact state. (3) Thalamic electrical activities were relatively enhanced in the Vim nucleus. From these data, we can deduce that the deep sensory inputs reach the border area of Vim nucleus and Vc nucleus in the human thalamus. IMP-SPECT scans were performed before and after the operation. IMP-SPECT scan before operation showed us the contra-lateral cerebellar low perfusional state (crossed cerebellar diaschisis). This crossed cerebellar diaschisis disappeared after the operation. This result suggested that the interaction of cerebellum and Vim nucleus was very close. The above mentioned speculation was more compatible to Jones & Hirai's new parcellation of the human thalamus than to Hassler's. PMID- 7546941 TI - [A case of pure alexia whose writing ability with eyes closed was superior to that with eyes open]. AB - We compared the writing ability between eyes open (looking at one's writing) and eyes closed (without looking) in a case of pure alexia. The patient is a 84-year old right handed man who developed pure alexia following an infarction in the distribution of the left posterior cerebral artery. He showed right homonymous hemianopsia, slight memory disorder and slight anomia. He could not read any kinds of letters and words at all though he could categorize letters: Kana (phonograms), Kanji (morphograms) and Arabic numbers, and he could distinguish the real letters from false ones. He could achieve almost 60-70% of Kana and Kanji dictation, though it was not perfect. He was often confused and could not continue writing correct letters once interrupted. He wrote poorly when looking at what he was writing. So we compared the ability of dictation with his eyes closed, and that with his eyes open. We found the former way never caused confusion and got better results, which was statistically significant (Fisher's exact test (p < 0.05)). It seemed that his visual feedback of his own writing rather caused trouble in his writing. Thus we concluded that input (reading) process and output (writing) process work simultaneously and bi-directionally. Slight impairment of writing in pure alexia patients might be caused by the effect of impairment of input (reading) process, which is simultaneously connected with the output (writing) process. PMID- 7546942 TI - [A 56-year-old woman with parkinsonism and dementia with the age of onset at 41 years]. AB - We report a 56-year-old woman with parkinsonism and dementia who died of respiratory failure. The patient was well until the age of 41 when she noted insidious onset of difficulty in moving around. Soon after, she noted tremor in both her hands and gait disturbance. She received stereotaxic right thalamotomy when she was 46-year-old; after thalamotomy, improvement was noted in her tremor, rigidity, and in gait. However, a few months later, she started to experience motor fluctuations with worsening of her symptoms in the afternoon. This worsening was temporarily relieved by increasing her levodopa/benserazide dose. She started to show visual hallucination and agitation when she was 54-year-old. Her symptoms had progressively become worse with marked motor fluctuations and she was admitted to our hospital when she was 56-year-old. On admission, she was alert and general physical examination was unremarkable. Neurologic examination revealed that she was disoriented to time and place; memory was markedly disturbed and calculation was poor. Hasegawa dementia scale was 7/30. Higher cerebral functions appeared intact. She showed masked face, small voice, and some dysphagia. Other cranial nerves were intact including ocular movements. She was unable to walk by herself; when supported she walked in small steps with marked disturbance in the righting reflex. Mixed rigidity and Gegenhalten was noted in her four limbs and in the neck. Tremor was absent. She showed marked akinesia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7546943 TI - Tooth morphology and archaeology: a problem solving exercise. AB - A knowledge of tooth morphology is often a valuable aid in forensic science but another less well-known application is the analysis of skeletal remains from archaeological sites, particularly when these involve children. This article describes one such case. PMID- 7546944 TI - A penny in the primate space. PMID- 7546945 TI - The role of fluoride dentifrices in a national strategy for the oral health of children. AB - The Department of Health's recent publication 'An Oral Health Strategy for England' proposes a number of targets for improving the oral health of children by the year 2003. It recognises that the prevalence and severity of dental caries have decreased considerably in England over the last 25 years but, water fluoridation apart, it suggests no specific measures to ensure that this improvement continues. There is evidence to suggest that in many regions this will not be sufficient, and that in the medium term a more coordinated approach involving government, the profession and commerce is required. PMID- 7546946 TI - Dietary fluoride supplements. PMID- 7546947 TI - Informed consent and the practice of good dentistry. PMID- 7546948 TI - A survey of in-dwelling intravenous cannula use in general dental practice. AB - A study to ascertain the use of in-dwelling intravenous cannulae in general dental practice was performed. A self-completion questionnaire was distributed to the 149 dental practitioners registered with the Cleveland Family Health Services Authority. A total of 131 dentists (88%) completed and returned the questionnaire: 58% of practitioners were practising either general anaesthesia (GA) or intravenous sedation (IVS) and 59% of these practitioners routinely used an in-dwelling intravenous cannula to administer the anaesthetic or sedative agents. This was supervised by a medically qualified anaesthetist in 100% of GA cases and 55% of IVS cases. Sixty per cent of respondents had received didactic instruction at undergraduate level on gaining intravenous access, but 72% of these stated that the teaching had been inadequate. Seventeen per cent of the practitioners had never received formal training in gaining intravenous access. These results suggest the need for formal practical instruction in obtaining intravenous access may have to be addressed. PMID- 7546949 TI - Cross-infection control in dental laboratories. AB - The attitudes to cross-infection control of 800 dental laboratories registered with the Dental Laboratories Association have been surveyed. Considering the topicality of the subject material and the need for careful cross-infection control within and beyond the dental surgery, the response rate of 22% (175) was disappointingly low. As a result of the low response rate, the results should be seen as a guide only. Forty-nine percent of respondents had a cross-infection policy and of those with no policy 64% intended to implement one in the future. Thirty percent of laboratories receive known undisinfected work from the dental surgery. Of those items disinfected on arrival at the laboratory, those most frequently disinfected are dental impressions (77%) and dentures (51%). The most popular chemicals used for disinfection are household bleach, chlorhexidine and glutaraldehyde. Forty-four percent of the respondents generally (90% or more of the time) wear gloves when handling dental work received and opened in the laboratory. Seventy-four percent wear protective eye spectacles when trimming or polishing prostheses. Sixty-one percent used no disinfectant in the pumice and 93% did not disinfect the polishing instruments, eg wheels and mops. Forty-six percent had a policy for immunisation of staff against Hepatitis B. PMID- 7546950 TI - An unusual sequela to an inferior dental block injection. AB - This report describes an unusual case of blanching of the facial and palatal soft tissues following the administration of an inferior dental block injection. Possible anatomical explanations for the clinical observations are discussed. PMID- 7546951 TI - Lateral thinking: the management of missing upper lateral incisors. AB - Absence of the maxillary lateral incisor creates an aesthetic problem which can be managed in various ways. The condition requires careful treatment planning and a consideration of the options and outcomes following either space closure or prosthetic replacement. Recent developments in restorative dentistry have warranted a re-evaluation of the approach to this clinical situation. Factors relating both to the patient and to the teeth, including the presenting malocclusion and the effect on the occlusion, are considered. This review considers the possible options: no treatment; orthodontic space closure with canine modification; space maintenance and replacement of the missing tooth with denture, bridge (adhesive and conventional), or implant. PMID- 7546952 TI - Putting Yiamouyiannis into perspective. AB - Few names crop up so often in the context of water fluoridation as that of John Yiamouyiannis, an American who has campaigned hard to stop this public health measure not only in the United States but in other countries such as Britain. During 1993, Yiamouyiannis was given considerable air time by the BBC, appearing on its prestigious You and yours and Nature programmes where he denigrated the efforts of local health authorities to reduce tooth decay by means of fluoridating their water supplies. His dramatic claims about fluoridation--in particular, that it increases people's risk of dying of cancer--must understandably alarm those who take them at face value. So just who is John Yiamouyiannis? And do his allegations hold up? This review of his career is based on publicly documented records and statements. PMID- 7546953 TI - Child dental health survey. PMID- 7546954 TI - School dental screening. PMID- 7546955 TI - The study of informatics. PMID- 7546956 TI - Changes in dental caries experience of 12-year-old school children in two Somerset schools: a review after an interval of 30 years. AB - The examination of 12-year-old children attending schools in central Somerset, where similar examinations were conducted 5, 11, 15 and 30 years previously, has indicated that the caries experience of the children in these schools is continuing to decrease. However, the annual rate of change over the past 5 years is less than that of the previous 6-year period. The average DMFT, which was 5.36, 3.44, 2.77 and 1.05 30, 15, 11 and 5 years previously, is now 0.63. The major reduction between 1982 and 1988 was in the average number of filled teeth per child and this trend has continued. Although the proportion of children who are free from any experience of caries has continued to rise to 72%, the number with active caries has remained at 6% over the last 5-year period. PMID- 7546957 TI - The influence of dental gloves on the setting of impression materials. AB - The increased wearing of gloves during dental treatment has created new problems for general dental practitioners. One of these is the retarded set or total inhibition of addition silicones caused by natural latex gloves. This study was designed to test different gloves and impression materials in common use. Eight latex gloves, selected with different properties representing all possible types, together with four non-latex gloves, made of neoprene, styrene-butadiene, polyethylene and polyvinylchloride, were tested. As a control, mixing with bare hands and with maize starch was carried out. The viscosity changes of five types of impression materials (polyvinylsiloxanes, condensation silicones, alginates, polyether and polysulphides) were measured with a Zwick hardness tester to give the Shore A hardness. No impression materials other than polyvinylsiloxanes were affected by pure latex gloves and those only by two brands. All other impression materials can be mixed and handled with gloved hands without affecting their setting time. The retarded set of polyvinylsiloxanes caused by natural latex gloves is not so widespread as had previously been thought. PMID- 7546958 TI - The changing pattern of day-care treatment for children in a London dental teaching hospital. AB - Day-stay general anaesthesia facilities may be employed to provide comprehensive dental care for children who are unable to accept treatment using alternative methods of patient management. This approach to delivery of care has been shown to be cost-effective, acceptable and efficaceous. This study was set up to investigate changes in the pattern of care provided in day-care facilities at a London dental teaching hospital over a 20-year period--1972 to 1992. The results show that the use of the facilities at this centre has increased with time, with a greater increase amongst older children in recent years. Although the main use is still for the treatment of caries, oral surgery procedures, especially those related to orthodontic treatment, have increased by 200% between 1982 and 1992. This correlates with the increase in older age groups being treated. Details of mean numbers of teeth restored and extracted for each age group are presented for 1992. They indicate, for the younger age groups, not only the high treatment needs of patients referred to the hospital, but also suggest a trend for more radical treatment planning in order to avoid further general anaesthesia. Readers are reminded that, despite its advantages, general anaesthesia should be avoided if possible. PMID- 7546959 TI - Mandibular reconstruction and rehabilitation in childhood. AB - The management of a case of osteosarcoma of the mandible in an 7-year-old girl is presented. A variety of reconstructive techniques were used following surgical ablation of the tumour, including the use of a revascularised free bone flap and osseointegrated endosseous implants. PMID- 7546960 TI - The skills of the parrot? AB - The talking parrot can be given information by voice and then reproduce that information verbatim, without necessarily understanding it. I submit that the teaching, assessment and examination of dental students requires little more of them than the skills of the parrot. PMID- 7546961 TI - World War II: the Army Dental Corps in the Far East. AB - August 15 is the anniversary of VJ Day--Victory in Japan. This article, the first of a 3-part series dedicated to remembering the efforts of the Army Dental Corps during the second world war, reproduces the personal experiences--some horrifying, some desperate, some funny--of those dentists who did their service in the Far East and who experienced VJ Day at first hand. PMID- 7546962 TI - The electronic revolution. PMID- 7546963 TI - Fifty years on: Colin Davis interviews Eric Cooper. Interview by Colin Davis. AB - Just over 50 years ago, Eric Cooper was released from the gloomy fortress castle of Colditz after almost 4 years as a prisoner of war. He recently retired to a small village near Yarmouth on the Isle of Wight where his garden looks out over a great sweep of cornland with the long line of the chalk downs in the distance. It is impossible to imagine two more contrasted pictures and I was eager to find out about the old warrior's odyssey as we sat sipping sherry and looking out to that incomparable view. PMID- 7546964 TI - Specialist dental training. PMID- 7546965 TI - Survey of children's dental health. PMID- 7546966 TI - Student debt. PMID- 7546967 TI - Student debt. PMID- 7546968 TI - Reducing anxiety in new dental patients by means of leaflets. AB - Fifty adults, consecutive attenders at a dentist who was new to them, were selected. They completed the Spielberger State Anxiety Inventory before and after they had read one of two different leaflets in the waiting room. One, the informative leaflet, included information about pain control and stop signals. A comparison leaflet did not include this information but acknowledged, sympathetically, that many people are nervous of dental treatment. The leaflet also provided some explanations for that fear. Both leaflets were designed, according to published evidence, to be easily read and understood. The layout drew attention to the most important points. State Anxiety decreased significantly only in the patients who read the informative leaflet (P < 0.01). This occurred before the patients met their new dentist. Those patients given the informative leaflet rated it as much more helpful than those given the comparison leaflet (P = 0.007). PMID- 7546969 TI - Some factors affecting the standards of radiography in general dental practice. AB - This study had three aims. The first was to examine the influence of legislative and educational factors on facilities and equipment in dental practices, the second to find out if these and practice size affected standards and the third to assess the degree to which technique and other faults not connected with processing accounted for inadequate diagnostic quality in intra-oral radiography. Dentists from 62 practices returned questionnaires about the sizes of their practices, facilities and equipment for radiography and relevant updating, training and education of their staff. The quality of 305 radiographs taken in a sample of 11 practices was assessed. Ten examples of good radiographic practice were used to evaluate standards in the general practices. The 'good practice score' ranged from 2 (two practices) to 10 (one practice) with a mean of 6.2. Practices whose principals had engaged in the recommended educational activities scored more highly than those that had not (P < 0.05), and the mean scores in practices of two or more dentists were higher than in single-handed (P < 0.05). Of the 305 radiographs, the dentists, without the use of criteria to assess them, rejected 13.8%; with criteria they rejected 39.3% (P < 0.05). The researcher rejected 63.9% (P < 0.05). All films were processed under controlled conditions and it is likely that reject rates would have been higher under normal processing conditions. It is concluded that the use of criteria to assess radiographs and acceptance of postgraduate opportunities, particularly by single-handed practitioners, would improve the conduct of radiography. Further improvements might be gained by orienting updating courses and materials towards behavioural change. PMID- 7546970 TI - The use of a reinforced glass-ionomer cermet for the restoration of primary molars: a clinical trial. AB - The development of adhesive restorative materials has led to more conservative cavity design with greater reliance being placed upon the bond of a material with tooth tissue for retention of the restoration. Glass-ionomer cements may offer particular advantages but have yet to achieve the durability reported for amalgam. This study reports on the results of a 2.5-year prospective clinical trial comparing the durability of two glass-ionomer cements, a conventional material (Ketac Fil) and a metal reinforced cermet (Ketac Silver) in the restoration of Class II lesions in primary molars. Forty-six pairs of restorations were assessed in 37 children. The failure rate of Ketac Fil, 23%, was significantly lower than that of Ketac Silver, 41% (P < 0.05). The median survival time of the Ketac Fil restorations was significantly greater, 25.3 months, than that of the Ketac Silver restorations, 20.3 months (P < 0.05). These values may be an underestimate of the true longevity of both restoration types as many of the restorations survived intact at the censor date. Neither the age of the child nor the tooth restored influenced the durability of the restoration. The deterioration in both marginal integrity and anatomic form of the Ketac Silver restorations was significantly greater than the Ketac Fil restorations (P < 0.05). The durability of Ketac Silver was such that it cannot be recommended for use in restoring carious primary molars. PMID- 7546971 TI - The growth of new dental practices established in the North West of England and North Wales between 1985 and 1990. AB - This study examined the growth of 52 new dental practices established between 1985 and 1990 in the North West of England and North Wales. Growth was measured from the number of estimates (equivalent to a course of treatment) submitted by the practice to the Dental Practice Board since the practice first opened. Values ranged from less than 1000 estimates up to 12,000 estimates during the first 24 months; the modal value was between 2000 and 2999. The number of estimates was not associated with the resident population to dentist ratio for the surrounding locality. PMID- 7546972 TI - NHS dentistry--the road to a better future. AB - Dentistry in this country is approaching a major cross roads. Although not everyone will take the same route, now is the time to examine the options and identify a way ahead for the majority. This article seeks to examine the history which has led to the numerous changes and pressures evident in dentistry today; it examines the various components and argues that these lead inexorably to certain conclusions. PMID- 7546973 TI - Consultant services in restorative dentistry. AB - Since restorative dentistry was established as a specialty in the UK in 1973, consultant appointments in the field have largely centred around the dental teaching hospitals with a considerable shortage of posts in the district general hospitals. This article explains the growing demand for restorative consultants and calls for the expansion of the hospital restorative dental service. PMID- 7546974 TI - The benefits of beneficence: rewards of hospice volunteering. AB - Hospice volunteering can provide many benefits to the volunteer as well as to the hospice program. Identification of these benefits, which may be characterized as being of the body, mind, and spirit, is helpful in the recruitment of new volunteers as well as in the retention of current ones. It is important for hospice programs to find ways to assist volunteers to attain these rewards. PMID- 7546975 TI - Strengthening the family unit through the healing power of massage. PMID- 7546976 TI - Recognizing depression. PMID- 7546977 TI - Children's descriptions of their feelings and what they found helpful during bereavement. AB - Although clinicians have described the value of support groups in assisting children mourn the death of someone close, little is known about helping children with bereavement. By understanding what helps children mourn, nurses can better facilitate a healthy bereavement process. The purpose of this mini-ethnographic study, which used observation and participation procedures, was to identify what 16 children found helpful during their bereavement. Initially, the children's expressions of feelings were demonstrated both verbally and through art work. The patterns of the children's emotions were labeled as affective distress, somatic complaints, and for the boys, disruptive behavior. The children then described their mourning processes as being facilitated by out-of-door activity, yelling, and talking to others (e.g. God, mom, grandmother or grandfather). PMID- 7546978 TI - Engendering hope in the chronically and terminally ill: nursing interventions. AB - Nurses assume a primary role in the care of chronic and terminally ill individuals in their homes and are in a strategic position to foster or hinder hope. Using a descriptive survey design, home health care nurses and hospice nurses were asked to rate proposed hope interventions as to use and effectiveness in facilitating hope in their chronically ill and terminally ill clients. One hundred and fifty-eight registered nurses, representative of six hospice agencies and six home care agencies in a Midwestern state, completed the Hope Intervention Questionnaire. Provision of comfort and pain relief emerged as the most effective and most frequently used hope interventions by both the home health care nurses and the hospice nurses. The top 15 interventions, though ranked in slightly different order, were the same for hospice nurses and the home health care nurses and were reflective of the multi-dimensions of hope. The findings of this study have practical importance to nursing practice as they provide a framework for selecting strategies to foster hope in the chronically ill and terminally ill population. PMID- 7546979 TI - Nebulized morphine for hospice patients. PMID- 7546980 TI - Nebulized morphine: another point of view. PMID- 7546981 TI - Pain management and parenteral opioids: an update. PMID- 7546982 TI - Synthesis of an N-methyldehydroalanine-containing fragment of microcystin by combination of solid phase peptide synthesis and beta-elimination in solution. AB - A new method for the synthesis of dehydroalanine (delta Ala)-containing peptides has been developed by combining solid phase peptide synthesis (tert butyloxycarbonyl/HF-chemistry) with solution synthesis. A sequence from cyanobacterial hepatotoxin microcystin, Ac-D-gamma-Glu-[N-Me-delta Ala]-D-Ala-Leu amide was chosen as a model peptide. The precursor for the synthesis of the dehydroalanine-containing peptide, Ac-D-gamma-Glu-[N,S-diMeCys]-D-Ala-Leu, was synthesized on a solid phase followed by sulfonium salt formation on the resin. The resulting S,S-dimethylated peptide was cleaved from the resin with liquid HF. The HPLC-purified S,S-dimethylated cysteine-containing precursor peptide was subjected to beta-elimination in solution catalysed by DBU (1,8 diazabicyclo[5.4.0]undec-7-ene) in methanol. The final product, Ac-D-gamma-Glu-[N Me-delta Ala]-D-Ala-Leu amide, was purified by HPLC, and analysed by mass spectrometry and 1H NMR spectroscopy. The stability of the model peptide under acidic, neutral and basic conditions has been studied. PMID- 7546983 TI - Reinjection of thallium for detection of viable myocardium: why not do it immediately? PMID- 7546984 TI - Echocardiographic detection of congenital heart disease in the fetus: present and future. PMID- 7546985 TI - Anticoagulants in pregnancy. PMID- 7546986 TI - Myocardial infarction resulting from coronary artery dissection in an adolescent with Ehlers-Danlos syndrome type IV due to a type III collagen mutation. AB - Ehlers-Danlos syndrome encompasses a group of inherited disorders of connective tissue, some of which are characterised by abnormalities of collagen metabolism. The chromosomal location, identified genes and biochemical defects, inheritance pattern, and clinical features for the various known subtypes are outlined. Prenatal diagnosis is possible for types IV, VI, VIIA1, and VIIA2. An unusual presentation of type IV Ehlers-Danlos syndrome in a 16 year old boy with an anterior myocardial infarction resulting from dissection of the left anterior descending coronary artery is reported here. A clinical diagnosis of type IV Ehlers-Danlos syndrome was made subsequently and confirmed by the reduced production, impaired secretion, and abnormally slow electrophoretic migration of type III collagen, indicating an underlying mutation in the COL3A1 gene. This patient represents the first case of type IV Ehlers-Danlos syndrome with symptomatic coronary artery dissection. PMID- 7546987 TI - Efficacy of long-term anticoagulant treatment in subgroups of patients after myocardial infarction. AB - OBJECTIVE: To investigate the efficacy of long term oral anticoagulant treatment in subgroups of patients after myocardial infarction. DESIGN: Analysis of the effect of anticoagulant treatment in subgroups of hospital survivors of myocardial infarction based upon age, gender, history of hypertension, previous myocardial infarction, smoking habits, diabetes mellitus, Killip class, anterior location of infarction, thrombolytic therapy, and use of beta blockers. SUBJECTS: Participants of a multicentre, randomised, double blind, placebo controlled trial that assessed the effect of oral anticoagulant treatment on mortality as well as cerebrovascular and cardiovascular morbidity in 3404 hospital survivors of acute myocardial infarction. MAIN OUTCOME MEASURES: The effect of anticoagulant treatment on recurrent myocardial infarction, cerebrovascular events, and vascular events (the composite endpoint of reinfarction, cerebrovascular event, and vascular death). RESULTS: Long term anticoagulant treatment was associated with a reduction in mortality of 10% (95% confidence interval -11% to 27%), recurrent myocardial infarction of 53% (41% to 62%), cerebrovascular events of 40% (10% to 60%) and vascular events of 35% (24% to 45%). Treatment effect with respect to recurrent myocardial infarction was comparable among all subgroups of patients. Although treatment effect appeared to be somewhat smaller in females than in males (-11% v -45%), and in patients with diabetes compared to those without (-14% v -42%) with respect to vascular events, none of these differences reached statistical significance. In multivariate analysis, more advanced age, previous myocardial infarction, diabetes mellitus, and heart failure during admission were independently associated with increased incidence of cardiovascular complications. CONCLUSIONS: The relative benefit of long term anticoagulant therapy in survivors of myocardial infarction is not modified by known prognostic factors for cardiovascular disease. PMID- 7546988 TI - Neutralising antibodies after streptokinase treatment for myocardial infarction: a persisting puzzle. AB - OBJECTIVE: To determine the development of titres of streptokinase (SK) neutralising antibodies after a single dose of SK, to establish when titres decrease to levels at which a second dose might be effective. DESIGN: Analyses of blood samples taken from patients at intervals after SK administration. SETTING: Australian public hospital. PATIENTS: 104 patients with acute myocardial infarction who were treated with SK and 27 controls who were not. OUTCOME MEASURE: SK neutralising antibodies were measured once in each of the 27 controls and on 166 occasions in the 104 treated patients. RESULTS: Titres of SK neutralising antibodies rose after SK administration but returned to control levels by 2 years. CONCLUSIONS: SK might be effective again as a thrombolytic agent as early as 2 years after a single dose. These results are at variance with most previously published data and the reasons for this are not clear. Data evaluating patency rates after standard doses of streptokinase in patients with increased titres of neutralising antibodies are necessary before re-exposure to streptokinase can be recommended. PMID- 7546989 TI - Long-term prognostic significance of M mode echocardiography in young men after myocardial infarction. AB - OBJECTIVE: To evaluate the power of measurements of left ventricular size and function for predicting long term (82 month) mortality by performing echocardiography in 97 men who had survived an acute myocardial infarction. SETTING: University hospital specialising in cardiology. PARTICIPANTS: 97 consecutive male patients who had survived a myocardial infarction. MAIN OUTCOME MEASURES: The additive prognostic value of functional measurements to that provided by primary risk factors (smoking habits and lipoprotein levels), radiological heart size, exercise capacity, and number of major coronary arteries with haemodynamically significant stenoses was evaluated. An echo index was calculated from three echocardiographic variables (yielding one score point each if: left ventricular diameter at the end of diastole (LVDD) > or = 5.7 cm, left ventricular fractional shortening < or = 24%, and E point-separation (EPSS) > or = 10 mm). MAIN OUTCOME: 17 cardiac deaths occurred during follow up. RESULTS: Univariate analysis showed that treatment with loop diuretics for heart failure (P < 0.01), LVDD (P < 0.01), left ventricular diameter at the end of systole (LVDS) (P < 0.001), left atrial diameter (P < 0.001), fractional shortening (P < 0.05), and echo index (P < 0.001) were all associated with cardiac death. Angiographically determined regional wall motion disturbances (P < 0.005) and angiographic ejection fraction (P < 0.001) were also associated with cardiac death, as was the number of major coronary arteries with significant stenosis (P < 0.05). When all significant echocardiographic variables from univariate analysis were entered into Cox proportional hazards survival analysis, LVDS and left atrial diameter contributed independently to the prediction of cardiac death. If angiographic data were also entered into the model, the echo index made an independent contribution to the prediction of cardiac death. CONCLUSIONS: Among young male patients with a previous myocardial infarction, a simple M mode echocardiographic examination can identify high and low risk patients and improve the prediction of cardiac death made from clinical information, exercise test, chest x ray and angiographically determined ejection fraction. PMID- 7546990 TI - Early reactivation of ischaemia after abrupt discontinuation of heparin in acute myocardial infarction. AB - Intravenous heparin after thrombolytic therapy for acute myocardial infarction is an effective, widely used treatment. Six cases of acute myocardial infarction are reported with early disease reactivation following the abrupt discontinuation of heparin infusion three days after alteplase thrombolysis and concomitant aspirin therapy. Immediate reinfusion of heparin resulted in regression of symptomatic ischaemia in all six patients. The activated partial thromboplastin time values, determined four hours before the discontinuation of heparin therapy, were within the therapeutic range in five of the six patients, and no difference was found in the values obtained one hour after the reinfusion of heparin (P = 0.065). PMID- 7546991 TI - Influence of plaque morphology on the mechanism of luminal enlargement after directional coronary atherectomy and balloon angioplasty. AB - OBJECTIVE: To relate the mechanism of luminal gain after directional atherectomy and balloon angioplasty to the morphological characteristics of the coronary lesions, assessed by intravascular ultrasound imaging. DESIGN: Intravascular ultrasound imaging was performed before and after the revascularisation procedure to assess the contribution of wall stretching and plaque reduction in luminal gain. SUBJECTS: 32 patients undergoing balloon angioplasty and 29 undergoing directional coronary atherectomy. MAIN RESULTS: The main luminal area in vessels treated by balloon angioplasty increased from 1.51 (SD 0.30) to 3.91 (1.09) mm2 (P < 0.0001) with a concomitant increase in total vessel area from 11.44 (2.73) to 13.07 (2.83) mm2 (P < 0.0001). Therefore stretching of the vessel wall accounted for 68% of the luminal gain while plaque reduction accounted for the remaining 32%. This mechanism ranged from 45% in non-calcific plaques to 81% in echogenic plaques. The main luminal area in vessels treated by directional atherectomy increased from 1.49 (0.32) to 4.68 (1.73) mm2 (P < 0.0001), with a concomitant increase of total vessel area from 13.61 (4.67) to 15.2 (4.04) mm2 (P = 0.006). Thus stretching of the vessel wall accounted for 49% of the luminal area gain and plaque reduction for the remaining 51%. The presence of calcium influenced the relative contribution of these two mechanisms to the final luminal gain after directional atherectomy, since in calcific plaques stretching of the vessel wall accounted for only 9% of the luminal gain as compared to 56% in non calcific plaques. After balloon angioplasty there was greater evidence of coronary dissections (32% v 3% after directional atherectomy, P < 0.01) and plaque fissure (60% v 0%, P < 0.01). Plaque fissure was more frequently seen in echolucent and concentric lesions, whereas dissections prevailed in echogenic and eccentric lesions. CONCLUSIONS: Intravascular ultrasound imaging may allow the assessment of acute changes in lumen and vessel wall after revascularisation procedures, and help in evaluating the potential effect of the structure and morphology of coronary lesions on the mechanism of luminal enlargement. PMID- 7546992 TI - Effects of beta receptor antagonists in patients with clinical evidence of heart failure after myocardial infarction: double blind comparison of metoprolol and xamoterol. AB - OBJECTIVE: To evaluate whether xamoterol, a partial agonist, would improve exercise time more than metoprolol in patients with mild to moderate heart failure after a myocardial infarction. DESIGN: Single-centre double blind randomised parallel group comparison of metoprolol 50-100 mg and xamoterol 100 200 mg twice daily. PATIENTS: 210 patients aged 40-80 years (173 men) with clinical evidence of heart failure early after a myocardial infarction. 106 were given metoprolol and 104 xamoterol. MAIN OUTCOME MEASURES: Exercise test results and performance at three months; the exercise test, quality of life, and clinical assessments at baseline (5-7 days after the infarction) and after 3, 6, and 12 months. RESULTS: Exercise time increased at three months by 22% in the metoprolol group and 29% in the xamoterol group, but with no significant difference between the groups. Patients taking xamoterol showed overall non-significantly higher mean values of exercise time achieved with higher heart rates at rest and exercise. Improvements in quality of life, clinical signs of heart failure, and New York Heart Association functional class were seen in both treatment groups over one year, with minor benefits of xamoterol on breathlessness, peripheral oedema, and functional class. Eighteen patients taking metoprolol and 22 taking xamoterol withdrew from the study during one year, with a low mortality, reinfarction rate, and progress of heart failure in both treatment groups. Mean dose from baseline to 3 months was 135 mg metoprolol and 347 mg xamoterol. CONCLUSION: beta 1 Receptor antagonists with or without partial agonist activity are safe to use in mild to moderate heart failure after a myocardial infarction. Exercise tolerance, quality of life, and clinical signs and functional class of heart failure improve, and few patients show deterioration in their condition. Exercise tolerance is no better with xamoterol than metoprolol. PMID- 7546993 TI - Atrial arrhythmias and pacing after orthotopic heart transplantation: bicaval versus standard atrial anastomosis. AB - BACKGROUND: Right and left atrial configuration is more normal when the donor left atrium is anastomosed to a recipient left atrial cuff with direct anastomoses of the donor and recipient vena cavas on the right side. The right atrium and sinus node may be less disturbed by the technique of bicaval anastomosis than by the standard procedure. OBJECTIVE: To compare the incidence of atrial arrhythmias and pacing after bicaval and standard anastomoses. METHODS: 75 patients had heart transplants between January 1991 and December 1993. The notes were reviewed. Nine patients who died within the first 30 days were excluded from further analysis (seven patients with standard anastomoses, one with bicaval anastomosis, and one with a hybrid technique). RESULTS: 66 patients survived for more than 30 days. Thirty five patients had standard anastomoses and 31 bicaval anastomoses. Atrial tachyarrhythmias (atrial fibrillation, atrial flutter, atrial tachycardia, or supraventricular tachycardia) occurred on four days in three patients in the bicaval group compared with 27 days in 13 patients in the standard group (P = 0.009). The relative risk of atrial tachyarrhythmias with standard anastomosis was 5.52 (P = 0.015) compared with that of bicaval anastomosis. Atrial tachyarrhythmias requiring treatment occurred less often in the bicaval group (four episodes in three patients in the bicaval group and eight episodes in four patients in the standard group), and fewer patients with a bicaval anastomosis required temporary pacing (pacing on 20 days in 10 patients in the bicaval group, but pacing on 49 days in 16 patients in the standard group) and late permanent pacing (no patients in the bicaval group and three patients in the standard group), although these differences were not statistically significant. Patients in the bicaval group were discharged from hospital sooner than those in the standard group (mean 24.1 v 29.1 days, P = 0.024). CONCLUSIONS: The technique of bicaval anastomosis, in addition to theoretical advantages from maintaining a more normal atrial configuration, has a lower incidence of postoperative atrial tachyarrhythmias, may reduce the need for pacing, and allows earlier discharge from hospital. PMID- 7546994 TI - Squatting revisited: comparison of haemodynamic responses in normal individuals and heart transplantation recipients. AB - BACKGROUND: Squatting produces a prompt increase in cardiac output and arterial blood pressure which is accompanied by an immediate decrease in heart rate and forearm vascular resistance. The rise in cardiac output and blood pressure has been attributed to augmented venous return from compression of leg veins, while the decreases in heart rate and forearm vascular resistance are probably due to activation of cardiopulmonary and arterial baroreflexes. Haemodynamic patterns in nine normal men and six heart transplant recipients during 2 min of squatting were examined to determine the role of cardiac innervation in the mediation of these responses. METHODS: Stroke volume was monitored by ensemble averaged thoracic impedance cardiography and blood pressure was determined with an Ohmeda fingertip plethysmograph. These techniques provided continuous measurements which were capable of detecting transient and non-steady state changes. Forearm blood flow was measured with venous occlusion plethysmography. Measurements were obtained after 3 min of quiet standing, immediately after squatting, and at 20, 60, and 120 s of sustained squatting. RESULTS: Both groups exhibited similar increases in stroke volume index (normal individuals 10.5 ml/m2; heart transplant recipients 10.3 ml/m2) and mean arterial pressure (normal individuals 8.5 mm Hg; heart transplant recipients 5.0 mm Hg) which were sustained throughout squatting. Each group also showed an initial decrease in peripheral resistance (normal individuals 3.6 units; heart transplant recipients 7.7 units) followed by a return to baseline values after 20 s. Heart rate decreased in normal individuals (10 beats/min) but was unchanged or minimally increased (2 beats/min) in heart transplant recipients. Forearm vascular resistance was conspicuously decreased in normal individuals (47.8 units) but only minimally (20.9 units) and not significantly in heart transplant recipients. CONCLUSIONS: The major haemodynamic responses to squatting (increased cardiac output and blood pressure) are similar in normal individuals and heart transplant recipients. These responses are primarily due to augmented venous return and are not altered by cardiac denervation. Both groups also exhibited a transient decline in peripheral vascular resistance which is most likely mediated by arterial baroreflexes activated by the acute rise in arterial blood pressure. The absence of a significant decrease in forearm vascular resistance in heart transplant recipients suggests that this response is partially mediated by cardiopulmonary or ventricular baroreflexes or that local forearm flow mediated vasodilatation remains impaired after heart transplantation. PMID- 7546995 TI - Relation between QT and RR intervals in patients with bradyarrhythmias. AB - OBJECTIVE: To investigate the relation between QT and RR intervals in the sick sinus syndrome or high degree atrioventricular block. PATIENTS: 32 patients with episodes of prolonged RR intervals (> or = 2.6 s) on Holter electrocardiographic recordings. DESIGN: QT and RR intervals were measured manually every 100 to 150 beats on electrocardiographic strips reprinted from the Holter tape over 24 hours. The slope of the QT/RR relation was determined by the linear regression equation for RR intervals < or = 1.4 s (slope 1) and > 1.4 s (slope 2). RESULTS: Slope 2 (0.0068 (0.0030)) was significantly lower than slope 1 (0.0824 (0.0059), P < 0.0001) in the overall patient population. Slopes 1 and 2 were significantly lower (P < 0.001) in the 23 patients with QT intervals at the preceding RR interval of 1 s (QT1s) of < 0.44 s (0.0692 (0.0053) and 0.0019 (0.0030), respectively) than in the nine patients with QT1s intervals > or = 0.44 s (0.1159 (0.0091) and 0.0194 (0.0055), respectively). Slopes 1 and 2 correlated positively with QT1s interval in all patients. CONCLUSIONS: The QT/RR relation was comparatively flat when the RR interval was prolonged. Patients with prolonged QT intervals showed exaggerated prolongation of the QT interval with prolonged cycle lengths when compared with patients with normal QT intervals. PMID- 7546996 TI - Assessment of magnetic resonance velocity mapping of global ventricular function during dobutamine infusion in coronary artery disease. AB - BACKGROUND: Magnetic resonance imaging (MRI) is a versatile technique for examination of the cardiovascular system but only recently has assessment of myocardial ischaemia in coronary artery disease (CAD) become possible, for example by demonstrating abnormalities of regional ventricular contraction during stress. Global ventricular function during stress was assessed by MRI of aortic flow, which has not been previously attempted. DESIGN: Variables measured by MRI reflecting the effect of ischaemia on global ventricular function during dobutamine stress were correlated with thallium-201 myocardial perfusion tomography. PATIENTS: 10 normal controls and 25 patients with CAD. SETTING: Tertiary cardiac referral centre. METHODS: Novel MRI sequences and analysis systems were used to measure the following variables during staged dobutamine infusion to 20 micrograms/kg/min: stroke volume, cardiac output, cardiac power output, peak flow, peak flow acceleration, aortic back flow, and flow wave velocity. Heart rate, blood pressure, double product, and maximum tolerated dobutamine dose were also measured. Multiple regression analysis was used to compare changes during stress with 201TI tomography. RESULTS: All parameters except for stroke volume and diastolic blood pressure increased in the controls. In the patients with CAD a significant relation was shown between the extent of reversible ischaemia and the change in peak flow acceleration (P < 0.00001), peak flow (P = 0.002), cardiac power output (P = 0.036), maximum dobutamine dose (P = 0.039), and systolic blood pressure (P = 0.04). Peak flow acceleration accounted for 58.4% of the variation in reversible ischaemia, and after allowing for this, only cardiac power output remained independently predictive adding a further 4.2% to the model (adjusted r2 = 0.626). A decrease in peak flow acceleration with an increase in dobutamine infusion indicated moderate or severe ischaemia (chi 2 = 10.2, P = 0.017). CONCLUSION: MRI may be used to assess variables of aortic flow during stress, which includes acceleration with high temporal resolution. Peak flow acceleration was the most sensitive indicator of the effect of ischaemia on global ventricular function. PMID- 7546997 TI - Familial dilated cardiomyopathy: a worse prognosis compared with sporadic forms. AB - OBJECTIVE: To establish the time of onset of dilated cardiomyopathy (DCM) by review of annual chest x rays, which are obligatory in Hungary. DESIGN: A retrospective survey of chest x rays of a cohort of confirmed cases of DCM, to assess time of onset of cardiomegaly. Clinical course was compared by follow up over a mean of six years from the time of diagnosis. SUBJECTS: 240 patients with DCM (31 familial, 209 non-familial). Diagnosis was made by echocardiography in all cases and confirmed by coronary angiography and heart biopsy in some cases. MAIN RESULTS: At diagnosis, the mean age of the patients was 31.8 years in the familial group and 39.6 years in the non-familial group (P < 0.05). The time between the onset of cardiomegaly (cardiothoracic ratio > 0.45) and clinical diagnosis was 8.0 and 10.1 years respectively (P < 0.05). The six year survival was 6% in the familial group and 23% in the non-familial group (P < 0.05). CONCLUSIONS: The familial form of DCM is the more malignant form: it occurs at an earlier age and progresses more rapidly than non-familial DCM. PMID- 7546998 TI - Left ventricular morphology and diastolic function in uraemia: echocardiographic evidence of a specific cardiomyopathy. AB - OBJECTIVE: To see whether cardiac morphological and functional abnormalities in uraemic patients are determined by high blood pressure or if they are an expression of a specific cardiomyopathy. DESIGN: Cross sectional study. SETTING: City general hospital in Italy. SUBJECTS: 35 uraemic patients receiving haemodialysis (17 men, 18 women; mean age 60.3 (11.2); mean duration of dialysis 52 months) were selected from the 64 patients in Venice who were receiving dialysis; subjects with diabetes, haemochromatosis, valvar dysfunction, regional dyskinesias, and pericarditis were excluded. 19 control normotensive subjects (6 men and 13 women), matched for age. MAIN OUTCOME MEASURES: Echocardiographic measurements of left atrium, left ventricular end diastolic and end systolic volume, aortic root diameter, posterior wall and interventricular septum thickness, left ventricle mass index, and ejection fraction in controls and in patients according to whether they were normotensive (five men, eight women) or hypertensive (12 men, 10 women) on 48 hour ambulatory monitoring; left ventricular diastolic function by Doppler ultrasonography. RESULTS: Mean systolic and diastolic pressures, daytime systolic and diastolic pressures, and night time systolic and diastolic pressures were significantly higher in the hypertensive patients than in the normotensive patients. The normotensive patients had similar blood pressures to the controls. Left ventricular mass correlated significantly with the mean diastolic pressure and mean night time systolic and diastolic pressures. Parathyroid hormone concentrations were similar in the two groups of patients. Diastolic relaxation was impaired to the same degree in the two groups of patients. Parameters of diastolic function showed no relation to left ventricular mass, which was significantly higher in the hypertensive than in the normotensive patients. CONCLUSIONS: Uraemia is likely to induce specific changes in the relaxation properties of the myocardium. These changes are responsible for the impaired diastolic function independently of blood pressure, degree of hypertrophy, and metabolic changes, which suggests the existence of a specific cardiomyopathy. Hypertension remains a determinant of left ventricular mass. PMID- 7546999 TI - Implication of anterior septal malalignment in isolated ventricular septal defect. AB - OBJECTIVE: The aim was to define the long term prognosis of isolated ventricular septal defect (VSD) with anteriorly malaligned outlet septum. DESIGN: Cohort study. SETTING: University hospital, tertiary medical care centre. PATIENTS: Between July 1986 and June 1993, 63 patients were studied with an isolated VSD and anteriorly malaligned outlet septum (59 perimembranous; 4 muscular outlet). MAIN OUTCOME MEASURES: The diagnosis of septal malalignment, aneurysmal transformation, right ventricular obstruction, subaortic ridge, and aortic valve prolapse was based on echocardiographic criteria, then confirmed by angiography in 33 patients and by surgery in 28. An actuarial curve for each event was obtained by Kaplan-Meier non-parametric analysis and the significance was examined by log-rank test. RESULTS: Aneurysmal transformation decreased the size of the VSD in 52% of the patients, but was also associated with the appearance of subaortic ridge (p < 0.05). Progressive obstruction in the right ventricle was observed in 51%, more often in those without aneurysmal transformation (p < 0.05). Aortic valve prolapse was quite common whether or not aneurysmal transformation occurred (33% and 23%, respectively). This was attributed to the location of the VSD and the anterior malalignment of the outlet septum. Surgery was performed in 28 patients at a median age of 50 months because of significant left to right shunt (n = 5), or the development of obstruction in right ventricle (n = 9), aortic valve prolapse (n = 3), or combinations (n = 11). The presence of subaortic ridge per se was not considered to be a surgical indication. CONCLUSIONS: Anteriorly malaligned VSDs have variable presentation. Careful echocardiographic evaluation is needed to identify various combinations of progressive right ventricular obstruction, aneurysmal transformation, subaortic ridge, or aortic valve prolapse. In extreme cases a patient may have a pathology complex comprising right ventricular outflow obstruction, subaortic ridge, aortic valve prolapse, and anteriorly malaligned VSD. PMID- 7547000 TI - Assessment of right ventricular regional contraction and comparison with the left ventricle in normal humans: a cine magnetic resonance study with presaturation myocardial tagging. AB - OBJECTIVE: Right ventricular regional contractility has been thought to be difficult to assess precisely. Cine magnetic resonance imaging with presaturation myocardial tagging was employed to quantitate the contraction of the right ventricular free wall and to identify normal performance compared with the left ventricle. METHODS: Nine normal volunteers, aged 27-39 years, were examined in a 1.5 Tesla superconductive magnet, and short axis and four-chamber sections at the mid-ventricular level were imaged with cine magnetic resonance sequences. Tags, applied at end diastole as two parallel black lines, intersected the mid-portion of the free wall, dividing it into upper, centre, and lower segments in the short axis section, and anterior, middle, and posterior segments in the four-chamber section. From a series of cine magnetic resonance images at 50 ms intervals over a cardiac cycle, end diastolic, and early, mid-, and end systolic images were chosen for calculation of the endocardial, epicardial, and mean percent fractional shortening (%FS) in the six segments. RESULTS: There was (1) a gradual increase in %FS in systole in both sections (P < 0.001, < 0.005); (2) a poor transmural gradient of contractility; (3) a predominance of meridional shortening (whole length, mean end systolic %FS (SD): short axis, 17.4 (3.1)%; four-chamber, 30.1 (4.1)%; P < 0.001) in contrast to dominant circumferential shortening in the left ventricular lateral wall; (4) lower predominance of contractility in the short axis section (P < 0.001), and a middle dip of contractility in the four chamber section (P < 0.005). CONCLUSIONS: Heterogeneity of contractility was closely correlated with the myocardial fibre architecture, and with wall stress determined by its thickness and curvature. It was proved that right ventricular regional function could be analysed non-invasively using cine magnetic resonance imaging with myocardial tagging. PMID- 7547001 TI - Antenatal diagnosis of congenital heart disease and Down's syndrome: the potential effect on the practice of paediatric cardiology. AB - OBJECTIVE: To predict the effect of antenatal ultrasound screening for congenital heart disease and maternal serum screening of Down's syndrome on the practice of paediatric cardiology and paediatric cardiac surgery. DESIGN: A retrospective and prospective ascertainment of all congenital heart disease diagnosed in infancy in 1985-1991. SETTING: One English health region. PATIENTS: All congenital heart disease diagnosed in infancy by echocardiography, cardiac catheterisation, surgery, or necropsy was classified as "complex", "significant", or "minor" and as "detectable" or "not detectable" on a routine antenatal ultrasound scan. RESULTS: 1347 infants had congenital heart disease which was "complex" in 13%, "significant" in 55%, and "minor" in 32%. 15% of cases were "detectable" on routine antenatal ultrasound. Assuming 20% detection and termination of 67% of affected pregnancies, liveborn congenital heart disease would be reduced by 2%, infant mortality from congenital heart disease by 5%, and paediatric cardiac surgical activity by 3%. Maternal screening for Down's syndrome, assuming 75% uptake, 60% detection, and termination of all affected pregnancies, would reduce liveborn cases of Down's syndrome by 45%, liveborn cases of congenital heart disease by 3.5%, and cardiac surgery by 2.6%. CONCLUSIONS: Screening for congenital heart disease using the four chamber view in routine obstetric examinations and maternal serum screening for Down's syndrome is likely to have only a small effect on the requirements for paediatric cardiology services and paediatric cardiac surgery. PMID- 7547002 TI - Emergency coronary artery stenting for coronary dissection complicating diagnostic cardiac catheterisation. AB - Dissection of a coronary artery is a recognised and significant complication of diagnostic coronary angiography that often requires emergency coronary artery bypass grafting. A coronary stent was used to treat this complication. This technique has potential advantages in terms of speed of reperfusion and availability in centres performing diagnostic angiography without surgical cover on site. PMID- 7547003 TI - Left ventricular thrombi in a patient with the antiphospholipid syndrome. AB - A 41 year old woman with the antiphospholipid antibody syndrome presented with a cerebral embolus. This was caused by a mobile left ventricular thrombus that later resolved. There was an additional old left ventricular thrombus. Left ventricular thrombi such as these have not been previously described in this syndrome, and may have been under diagnosed. PMID- 7547004 TI - Right sided pectoral implantation of an "active can" transvenous implantable cardioverter-defibrillator with single right ventricular lead. PMID- 7547005 TI - Lingual haematoma after treatment with alteplase (recombinant tissue plasminogen activator) for acute myocardial infarction. PMID- 7547006 TI - Antibiotic prophylaxis in permanent pacemaker implantation. PMID- 7547007 TI - The case for concomitant carotid and coronary artery surgery. PMID- 7547008 TI - The difficulties of assessing right ventricular function. PMID- 7547009 TI - The angiotensin converting enzyme gene in cardiovascular disease. PMID- 7547010 TI - Atrial fibrillation: the last challenge in interventional electrophysiology. PMID- 7547011 TI - Cardiopulmonary resuscitation with active compression-decompression. PMID- 7547012 TI - Is aspirin safe for patients with heart failure? PMID- 7547013 TI - Reversible ischaemia in hypertrophic cardiomyopathy. AB - Atypical and typical chest pains are common symptoms in patients with hypertrophic cardiomyopathy. Some of these chest pains seem to be caused by ischaemia. It is difficult to objectively demonstrate ischaemia in hypertrophic cardiomyopathy. The first line treatment for chest pain considered to be ischaemic in patients with hypertrophic cardiomyopathy is the use of either a beta blocker or calcium blocker. Septal myectomy can be effective in patients with symptoms refractory to conventional treatment but is associated with significant morbidity and mortality. Recently dual chamber pacing has been advocated in such patients. In some cases dual chamber pacing alleviates chest pain in hypertrophic cardiomyopathy by an anti-ischaemic action, presumably by reducing the left ventricular outflow tract gradient and perhaps by causing an associated decrease in left ventricular outflow tract gradient and perhaps by causing an associated decrease in left ventricular end diastolic pressure. PMID- 7547014 TI - Selection factors for the use of thrombolytic treatment in acute myocardial infarction: a population based study of current practice in the United Kingdom. The European Secondary Prevention Study Group. AB - OBJECTIVES: To identify and rank the factors that currently limit the use of thrombolytic treatment in patients admitted to hospital with acute myocardial infarction. DESIGN: Weighted sampling study with retrospective data retrieval from clinical records. SETTING: All hospitals within the Trent region providing acute general medical services. PATIENTS: Random sample of 420 patients admitted during February-April 1993 who had acute myocardial infarction as the main discharge diagnosis. MAIN OUTCOME MEASURES: Treatment odds ratios (and 95% confidence intervals (CI)) for the use of thrombolysis in patient groups defined by relevant clinical characteristics. RESULTS: The patient population was older and less likely to have ST segment elevation on the initial electrocardiogram than patients entered into the randomised trials of thrombolysis. Thrombolytic treatment was given to 49% of patients (SE 2.4%). After controlling for negative associations with a history of stroke (treatment odds ratio 0.18 (95% CI 0.04 to 0.53)) and peptic ulcer (odds ratio 0.52 (95% CI 0.26 to 1.01)) use of thrombolysis decreased with increasing patient age. This was particularly noticeable for those aged > 74 years (odds ratio 0.17 (95% CI 0.05 to 0.51)) relative to those aged < 65 years. Thrombolysis was less likely to be used in patients with ST depression (odds ratio 0.22 (95% CI 0.11 to 0.41)) or bundle branch block (odds ratio 0.18 (95% CI 0.07 to 0.44)) than in those with ST elevation on the initial electrocardiogram. Delay from symptom onset to admission was more than 12 h in 15% of patients. CONCLUSIONS: The patient population admitted to hospital with acute myocardial infarction differs in several respects from the samples that have been included in the trials of thrombolysis. The main factors limiting wider use of thrombolysis are diagnostic uncertainty at admission and delayed presentation. Perceived clinical contraindications to treatment are of lesser importance. There is evident reluctance to use thrombolytic treatment in older patients, who were substantially under represented in the clinical trials. PMID- 7547015 TI - Early remodelling of coronary stenoses after thrombolytic treatment in patients with acute myocardial infarction. AB - OBJECTIVE: To assess the frequency of early remodelling of coronary stenosis morphology after thrombolytic treatment in patients with acute myocardial infarction. DESIGN: Coronary angiograms were analysed by a computerised edge detection analysis system. Coronary stenosis severity was measured and morphology classified as smooth or complex. PATIENTS: Coronary arteriograms were obtained approximately 90 min and 24 h after thrombolytic treatment from 40 patients with acute myocardial infarction. MAIN RESULTS: Stenosis morphology was complex in 22 patients (65%) and smooth in 11 (32%) 90 min after thrombolysis. The morphology of 11 (50%) complex coronary stenoses and three (27%) smooth stenoses had changed at 24 h (P < 0.05). The transition from complex to smooth was associated with a reduction in stenosis severity from 65 (4)% to 51 (5)% (P < 0.05). The stenosis severity was 63 (4)% and 60 (5)% in those with persistently complex morphology, and 56 (7)% and 50 (5)% in those with persistently smooth morphology at 90 min and 24 h respectively (NS). CONCLUSIONS: Transition of morphology from complex to smooth within 24 h is common. This transition is associated with a reduction in stenosis severity of a degree greater than that found in persistently smooth stenoses over the same interval. 50% of stenoses are smooth at 24 h. PMID- 7547016 TI - Metoprolol treatment for two years after coronary bypass grafting: effects on exercise capacity and signs of myocardial ischaemia. AB - OBJECTIVE: To evaluate whether prophylactic treatment with metoprolol for two years after coronary artery bypass grafting improves working capacity and reduces the occurrence of myocardial ischaemia in patients with coronary artery disease. METHODS: After coronary artery bypass grafting, patients were randomised to treatment with metoprolol or placebo for two years. Two years after randomisation, a computerised 12-lead electrocardiogram was obtained during a standardised bicycle exercise test in 618 patients (64% of all those randomised). RESULTS: The median exercise capacity was 140 W in the metoprolol group (n = 307) and 130 W in the placebo group (n = 311) (P > 0.20). An ST depression of > or = 1 mm at maximum exercise was present in 34% of the patients in the metoprolol group and 38% in the placebo group (P > 0.20) and an ST depression of > or = 2 mm at maximum exercise was present in 11% in the metoprolol group and 16% in the placebo group (P = 0.09). The median values for maximum systolic blood pressure were 200 mm Hg in the metoprolol group and 210 mm Hg in the placebo group (P < 0.0001), while the median values for maximum heart rate were 126 beats/min in the metoprolol group and 143 beats/min in the placebo group (P < 0.0001). The occurrence of cardiac and neurological clinical events two years postoperatively among exercised patients was comparable in the treatment groups. CONCLUSIONS: Treatment with metoprolol for two years after coronary artery bypass grafting did not significantly change exercise capacity or electrocardiographic signs of myocardial ischaemia. PMID- 7547017 TI - Diastolic aortic pressure rise during percutaneous transluminal coronary angioplasty: an index of left ventricular systolic dysfunction. AB - OBJECTIVES: To investigate the relation between diastolic aortic pressure response and left ventricular systolic dysfunction during percutaneous transluminal coronary angioplasty. BACKGROUND: The abnormal diastolic blood pressure rise during exercise in patients with coronary artery disease probably reflects left ventricular systolic dysfunction rather than the number of stenosed coronary arteries. METHODS: Aortic blood pressures and left ventricular systolic function indices were estimated in 26 patients with single proximal stenosis of the left anterior descending coronary artery both before and during angioplasty. RESULTS: During coronary angioplasty all patients presented an increase in diastolic aortic pressure (P << 0.001), 8-12s before intracoronary electrocardiographic changes. During acute ischaemia there was a decrease in left ventricular ejection fraction (P << 0.001) and stroke volume (P << 0.001) and an increase in end systolic volume (P << 0.001) and left ventricular end diastolic pressure (P << 0.001). No statistically significant changes were observed in systolic blood pressure or heart rate. The aortic diastolic pressure increase was correlated with the decrease in ejection fraction (r = -0.95, P << 0.001) and with the increases in end systolic volume (r = 0.86, P << 0.001) and left ventricular end diastolic pressure (r = 0.85, P << 0.001). CONCLUSIONS: The rise in diastolic aortic pressure during percutaneous transluminal coronary angioplasty occurs earlier than intracoronary electrocardiographic changes and is related to ischaemic left ventricular systolic dysfunction. PMID- 7547018 TI - Non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility. AB - OBJECTIVE: To assess a non-invasive test for endothelial dysfunction, an important early event in the atherogenic process. METHODS: Using high resolution ultrasound, the accuracy of detecting small changes in vessel diameter was assessed using phantom "arteries", and the same equipment was then used to measure flow mediated dilatation in the brachial artery of 40 healthy adults aged 22-51 years, studied on four occasions; intervals between scans were 1-2 days, 1 2 weeks, and 2-4 months. RESULTS: Differences between pairs of phantom "arteries" with diameters 0.1-0.2 mm apart were correctly estimated in 162 of 264 cases (61%); no measurement by any of four independent observers was > 0.1 mm in error, and the mean error was 0.04 mm. For in vivo scans, the overall coefficient of variation for flow mediated dilatation was 1.8% (1.6% for women, 1.9% for men, P = 0.18). In 34/40 subjects (85%), all values for flow mediated dilatation were within 2.5% of the overall mean for each subject. A nested analysis of variance showed the expected between patient variability, and also significant day to day variation, but little between weeks or months. Using these data to generate power function analyses, we calculated that for individuals, an improvement in flow mediated dilatation of 4-8% is significantly greater than natural variability. In clinical trials, a mean improvement in flow mediated dilatation of at least 2% would usually be required to detect a treatment benefit, with much larger subject numbers needed for a parallel group compared to a crossover trial design. CONCLUSIONS: Vascular responses to endothelium dependent and independent stimuli in systemic arteries can be studied non-invasively in man. Subjects should be studied on at least two occasions before and after any intervention, to optimise the chance of showing a significant effect from any potentially beneficial therapy. PMID- 7547019 TI - Atrial and ventricular function after cardioversion of atrial fibrillation. AB - OBJECTIVE: Previous studies on atrial recovery after cardioversion of atrial fibrillation have not taken into account new knowledge about the pathophysiology of transmitral and transtricuspid flow velocity patterns. It is possible to shed further light on this problem if atrioventricular inflow velocity, venous filling pattern, and atrioventricular annulus motion are recorded and interpreted together. DESIGN: Prospective examinations of mitral and tricuspid transvalvar flow velocities, superior caval and pulmonary venous filling, and mitral and tricuspid annulus motion were recorded using Doppler echocardiography. Examinations were performed before and 24 hours, 1 month, and 20 months after cardioversion. SETTING: Tertiary referral centre for cardiac disease with facilities for invasive and non-invasive investigation. PATIENTS: 16 patients undergoing cardioversion of atrial fibrillation in whom sinus rhythm had persisted for 24 hours or more. RESULTS: Before conversion there was no identifiable A wave in transvalvar flow recordings. The total motion of the tricuspid and mitral annulus was subnormal and there was no identifiable atrial component. Venous flow patterns in general showed a low systolic velocity. After conversion, A waves and atrial components were seen in all patients and increased significantly (P < 0.01) with time. There was a similar time course for the amplitude of annulus atrial components, an increased systolic component of venous inflow, an increased A wave velocity, and a decreased E/A ratio of the transvalvar velocity curves. The ventricular component of annulus motion was unchanged. Changes in general occurred earlier on the right side than the left. CONCLUSIONS: This study indicates that, in addition to the previously known electromechanical dissociation of atrial recovery that exists after cardioversion of atrial fibrillation, there may also be a transient deterioration of ventricular function modulating the transvalvar inflow velocity recordings. Function on the right side generally becomes normal earlier than on the left. Integration of information from transvalvar inflow curves, annulus motion, and venous filling patterns gives additional insight into cardiac function. PMID- 7547020 TI - Prospective study of left ventricular function after radiofrequency ablation of atrioventricular junction in patients with atrial fibrillation. AB - BACKGROUND: In patients with drug resistant incessant supraventricular tachycardia, radiofrequency induced ablation of the atrioventricular junction and pacemaker implantation have hitherto been considered a treatment of last resort. OBJECTIVE: To assess the short and long term effects of ablation of the atrioventricular junction on systolic and diastolic left ventricular function in patients with atrial fibrillation with and without impaired left ventricular function. PATIENTS: 29 patients (19 men; mean age 65 (SD 7) years (range 50-76)) undergoing ablation of the atrioventricular junction for drug refractory atrial fibrillation were examined a mean of 2, 65, and 216 days after ablation of the bundle of His. MAIN OUTCOME MEASURES: Left ventricular ejection fraction and early filling deceleration times (Edec) were assessed by Doppler echocardiography after 1 to 2 hours of ventricular pacing at a rate of 80 beats/minute. RESULTS: In 14 patients with a left ventricular ejection fraction < 50% left ventricular ejection fraction increased significantly from 32% (11%) to 39% (11%) (65 days) and 45% (11%) (216 days) (P < 0.001); Edec increased from 142 (46) ms to 169 (57) ms (65 days) and 167 (56) ms (216 days) (P < 0.05). In 15 patients with an ejection fraction > or = 50% at the initial examination no significant change in systolic function was observed. CONCLUSIONS: In patients with left ventricular dysfunction long term improvement of systolic and diastolic left ventricular function was seen after ablation of the atrioventricular junction for rate control of atrial fibrillation. This procedure had no adverse effects on normal left ventricular function. PMID- 7547021 TI - Perinodal slow potential as a local guide for transcatheter radiofrequency ablation of atrioventricular nodal reentrant tachycardia: therapeutic efficacy and electrophysiological mechanisms of success. AB - BACKGROUND: A specific local indicator in the Koch's triangle could be critical to the complication-free treatment of atrioventricular nodal reentrant tachycardia by transcatheter radiofrequency ablation. Recording of perinodal slow potential reflects a slow conduction area, and probably indicates the location of the slow pathway component of the circuit. Specific ablation of the slow pathway would carry the least risk of atrioventricular block. METHOD AND RESULTS: Guided by the mapped perinodal slow potential, atrioventricular nodal reentrant tachycardia was successfully eliminated in all of 55 consecutive patients in one session. Fifty two patients (94.5%) had confirmed slow potential at the final success sites. Despite the good result, the underlying electrophysiological mechanisms of early success from slow-potential-guiding catheter ablation were heterogeneous: selective slow pathway eradication in 31 patients (56.4%, group A), selective slow pathway modification in 18 patients (32.7%, group B), inadvertent fast pathway damage in six patients (10.9%, group C). Group B patients had the preservation of dual atrioventricular nodal pathways, adequate atrio-Hisian delay, fast pathway facilitation, and a higher frequency of inducible, single non-conducted nodal echo (15/18, 83.3% v 6/31, 19.4% in group A, P << 0.001). The upper communicating path of the circuit was implicated as another site of radiofrequency destruction. Three recurrences were documented in follow up study. However, reablation by the same approach caused complete atrioventricular block in one patient (1.7%, 1/58 procedures). None of the local characteristics of ablation sites was an independent predictor of procedure outcome. CONCLUSIONS: Perinodal slow potential is not a specific slow pathway indicator in transcatheter radiofrequency ablation of atrioventricular nodal reentrant tachycardia. Multiple strategic sites of the reentry circuit may be damaged through similar local signals. PMID- 7547022 TI - Determination of left ventricular mass in systemic hypertension: comparison of standard and signal averaged electrocardiography. AB - OBJECTIVE: To investigate the quantitative relationship, if any, between signal averaged electrocardiographic variables and echocardiographically determined left ventricular mass in hypertensive subjects. DESIGN: Cohort analytic prospective study. SETTING: University hospital. SUBJECTS: 50 hypertensive subjects selected consecutively from inpatients. Patients older than 75 years, with underlying cardiac disease, with inconclusive echocardiograms with bundle branch block, or in atrial fibrillation were excluded. INTERVENTIONS: Antihypertensive therapy involving 41 patients was continued. MAIN OUTCOME MEASURES: Left ventricular mass calculated in accordance with the standards of the Penn convention. Thirteen criteria derived from combinations of signal averaged electrocardiographic X, Y, and Z Frank orthogonal leads, including voltage criteria, duration, and time voltage integrals of the QRS complex. Four widely used standard electrocardiographic criteria for detection of left ventricular hypertrophy. RESULTS: There was no difference in the values for any of the electrocardiographic variables between patients with (n = 29) and without left ventricular hypertrophy (n = 21). The time-voltage integral of QRS in the horizontal plane was the best signal averaged variable related to left ventricular mass (r = 0.33, P = 0.019); however, the correlation with Rodstein voltage was stronger (r = 0.46, P = 0.0009). A positive correlation was also found between left ventricular indexed mass and Rodstein voltage (r = 0.43, P = 0.0019). Stepwise regression analysis revealed Rodstein voltage as the only predictor of indexed mass (P = 0.0019), and Rodstein voltage (P = 0.0022) and body weight (P = 0.011) as the only independent correlates of left ventricular mass. CONCLUSIONS: The relation between electrocardiographic variables and left ventricular mass or indexed mass is of limited value; signal averaged orthogonal leads do not improve this assessment compared with standard electrocardiographic leads. PMID- 7547023 TI - Late systemic desaturation after total cavopulmonary shunt operations. AB - OBJECTIVES: To assess the medium-term results of total cavopulmonary shunt operations in children with left atrial isomerism, interrupted inferior vena cava, and complex congenital heart defects. BACKGROUND: Creation of a total cavopulmonary shunt provides very good interim palliation for children with interrupted inferior vena cava and complex congenital heart disease; however, longer term results after this operation have not been reported. METHODS: Detailed follow up of six children who underwent creation of a total cavopulmonary shunt at a tertiary referral centre. RESULTS: There were no early or late deaths. Oxygen saturations at discharge ranged from 89% to 92% (mean 90%). At last follow up (mean 4.7 years) saturations at rest ranged from 73% to 81% (mean 77%) (P < 0.05). All patients underwent exercise stress testing. At peak exercise oxygen saturations ranged from 62% to 87% (mean 71.5%) and during recovery from 68% to 85% (mean 78%). Cardiac catheterisation was performed in five patients with saturations of less than 80% at rest or peak exercise. No patient had pulmonary arteriovenous fistula. Systemic venous to hepatic venous collaterals were documented in four patients. These were localised below the diaphragm in three and above the diaphragm in one patient. The collateral vessel was successfully embolised in three of these patients, with a rise in resting oxygen saturations from 6 to 10% (mean 7%). CONCLUSIONS: The development of systemic venous to hepatic venous collaterals is a common complication in patients who undergo a total cavopulmonary shunt operation. This can lead to significant desaturation at rest and during exercise. Detailed angiographic studies of the infradiaphragmatic system veins is required for diagnosis. Transcatheter embolisation of such vessels gives good relief. PMID- 7547025 TI - Ventricular tachycardia during exercise testing as a predictor of sudden death in patients with chronic chagasic cardiomyopathy and ventricular arrhythmias. AB - OBJECTIVE: To verify the prognostic value of exercise induced ventricular arrhythmias in patients with chagasic cardiomyopathy. METHODS: 69 consecutive patients (37 male, 32 female; age range 21-67 years) with chronic chagasic cardiomyopathy and ventricular arrhythmias (more than 10 ventricular premature complexes per hour) were evaluated during treadmill exercise testing, using the Bruce protocol. Protocol end points were peak heart rate or presence of sustained ventricular tachycardia. MAIN OUTCOME MEASURE: Sudden cardiac death. RESULTS: 44 patients (group I) developed ventricular tachycardia during exercise testing (five sustained and 39 non-sustained), and 25 did not (group II). After a follow up of 24 (SD 15) months sudden cardiac death occurred in seven patients in group I and in none in group II (P < 0.05). CONCLUSIONS: Ventricular tachycardia on exercise testing is significantly associated with sudden cardiac death in patients with chronic chagasic cardiomyopathy and ventricular arrhythmias. PMID- 7547024 TI - Right ventricular filling in dilated cardiomyopathy. AB - PURPOSE: To assess right ventricular filling in dilated cardiomyopathy. PATIENTS: 32 patients with dilated cardiomyopathy and 24 healthy controls. METHODS: Stroke distances were measured by pulsed Doppler echocardiography at left ventricular outflow and left and right ventricular inflow. The inflow tract dimensions of both ventricles and the outflow tract dimension of the left ventricle were measured from two dimensional images. Right and left sided atrioventricular (AV) ring excursions were measured by M mode echocardiography at the tricuspid and mitral rings. Stroke volume was derived as stroke distance multiplied by left ventricular outflow tract area. Total stroke distances were calculated as the sum of AV valve Doppler stroke distances and ring excursion. The effective orifice areas of the two AV valves were thus defined as stroke volumes divided by total stroke distance. RESULTS: Total tricuspid stroke distance was normally less than mitral (6.0 (1.7) v 7.6 (1.7) cm, P < 0.05), implying that effective orifice area of the tricuspid valve was consistently greater (6.6 (1.6) v 4.5 (0.8) cm2, P < 0.01). Total tricuspid ring excursion was normally more than mitral (2.30 (0.30) v 1.62 (0.22) cm, P < 0.01). Total tricuspid stroke distance in dilated cardiomyopathy was also less than mitral (7.8 (2.4) v 9.7 (2.8) cm, P < 0.05). Tricuspid stroke distance was significantly increased in patients with dilated cardiomyopathy compared with that in healthy controls (P < 0.05 v controls), though stroke volume was much smaller (26 (10) v 63 (11) ml, P < 0.01) so that tricuspid effective orifice area was reduced to less than half normal (2.7 (1.2) cm2, P < 0.01). Total tricuspid ring long axis excursion was more than mitral (1.37 (0.6) v 0.74 (0.21) cm, P < 0.01). Right ventricular end diastolic inflow dimension was increased compared with that in healthy controls (3.9 (0.7) v 2.8 (0.5) cm, P < 0.01), correlating inversely with tricuspid effective orifice area (r = -0.71, P < 0.01). Total tricuspid ring excursion was bimodally distributed as a low amplitude group (less than 1.6 cm, n = 23) and a high amplitude group (more than 1.6 cm, n = 9), in which the interval P2 to onset of tricuspid flow was much longer (100 (35) v 50 (14) ms, P < 0.01). CONCLUSIONS: Enlargement of the right ventricular inflow tract in dilated cardiomyopathy, especially to more than 5 cm, is accompanied by a progressive decrease in effective tricuspid orifice area, sometimes to less than 1 cm2 and increased inflow velocities. Right ventricular relaxation was incoordinate in 28% of the patients studied. These disturbances of right ventricular filling are likely to compromise overall cardiac function independently of left ventricular disease. PMID- 7547026 TI - A study of spontaneous echo contrast in patients with rheumatic mitral stenosis and normal sinus rhythm: an Indian perspective. AB - OBJECTIVE: To study the incidence of spontaneous echo contrast in left atrium of Indian patients with rheumatic mitral stenosis in normal sinus rhythm and to define its relations. SUBJECTS: Transthoracic and multiplane transoesophageal echocardiographic studies were performed in 89 consecutive patients with rheumatic mitral stenosis who were in normal sinus rhythm. RESULTS: Spontaneous echo contrast in the left atrium was seen in 57.3% of patients on multiplane transoesophageal echocardiography and in only 5.6% on transthoracic echocardiography. The mean mitral valve area was 1.07 (SD 0.33) cm2 and 1.32 (0.45) cm2 (P = 0.004), mean left atrial size was 4.27 (0.67) cm and 3.91 (0.5) cm (P = 0.029), mean diastolic pressure gradient was 12.64 (5.69) mm Hg and 10 (5.5) mm Hg (P = 0.049), and absence of mitral regurgitation was seen in 45% and 23% of patients respectively (P = 0.1). Among patients with spontaneous echo contrast, 31% had either left atrial/appendage thrombus or a history of embolism, upsilon 0% in patients without spontaneous echo contrast (P < 0.0001). CONCLUSIONS: There is a high incidence of spontaneous echo contrast in the left atrium in Indian patients with rheumatic mitral stenosis in normal sinus rhythm on multiplane transoesophageal echocardiography. These patients are likely to embolise or form thrombi in the left atrium. The presence of spontaneous echo contrast is also associated with significantly smaller mitral valve area, larger left atrium, and higher mean diastolic mitral pressure gradient. PMID- 7547027 TI - Single catheter approach for occlusion of a patent arterial duct with a Rashkind double umbrella. AB - OBJECTIVES: To determine the benefits of using a single venous catheter and a single angiogram during catheter occlusion of a patient arterial duct with the Rashkind double umbrella compared with those of venous and arterial catheters and multiple angiograms. DESIGN: Retrospective review of case notes. PATIENTS: 103 consecutive patients. The long sheath could not be advanced adequately in two patients. 101 patients had 104 implantations. Median (range) age was 35 (7-549) months and median (range) weight 13 (7-62) kg. Fifty four implantations were performed using the venous and arterial method and 50 using the venous only method. RESULTS: Median procedure times (70 v 90 min), number of angiograms (one v four), and angiographic dye volume used (2 v 7 ml/kg) were significantly reduced using the venous only method compared with those of the venous and arterial method. There was no significant difference in fluoroscopy time (venous only 9 v venous and arterial 10 min). CONCLUSIONS: Considerable improvements can be made in the technique of catheter closure of patent arterial ducts using the Rashkind double umbrella without compromising outcome using venous cannulation alone and a single angiogram, rather than venous and arterial cannulation and multiple angiograms. reduced risk to arteries from cannulation, The benefits are reduced radiation exposure, reduced risk to arteries from cannulation, shorter procedures, and lower equipment costs. PMID- 7547028 TI - The earliest site of atrial activation in patients with isomeric appendages. AB - BACKGROUND: The sinus node is known to be duplicated in hearts with bilateral right appendages, but its site is uncertain when both appendages are of morphologically left pattern. OBJECTIVE: To determine the earliest site of activation of the atria, and to assess this site of activation relative to the anticipated location of the sinus node in patients with isomeric atrial appendages. STUDY DESIGN: Electrophysiological recordings by epicardial mapping during operations through a median sternotomy. PATIENTS: Since 1987, 44 consecutive patients with isomeric right appendages and 23 with isomeric left appendages. RESULTS: In 77% of the patients with isomeric right appendages, the site of earliest activation was superiorly located at the junction of one or other atrium and a superior caval vein; in other words, in the anticipated site of a sinus node. In contrast, an inferior site of earliest activation at the junction of an atrium with an hepatic vein was most common in patients with isomeric left appendages (56%). The site of earliest activation was not related to the veno-atrial junctions in six patients with isomeric right appendages (14%), nor in five with isomeric left appendages (22%). Moreover, in six patients with isomeric right appendages (13%), and three with isomeric left appendages (13%), additional sites of earliest activation were observed when the dominant site was suppressed. The locations of the earliest activation observed by epicardial mapping did not always accord with those expected from the preoperative electrophysiological examination, nor did they always match the anticipated site of the sinus node as documented by previous histological investigations. CONCLUSIONS: Epicardial mapping showed marked variation in functional arrangement of the earliest atrial activation. This information could be of future use when planning surgical procedures. PMID- 7547029 TI - Percutaneous transluminal coronary angioplasty in patients 70 years of age or older: 12 years' experience. AB - OBJECTIVE: To evaluate the short and long term results of coronary angioplasty in patients aged 70 years and older and identify the determinants of long-term survival. DESIGN: A retrospective analysis of clinical, angiographic, and procedure related variables on a consecutive series of patients. PATIENTS: 163 patients aged 70 years and older (mean (range) age 73 (70-83) years; 63% men) who underwent a first coronary angioplasty procedure between 1981 and 1993. RESULTS: Procedural success was achieved in 82% of patients. Four patients (2%) died, three (2%) had a myocardial infarction, and five (3%) underwent emergency coronary artery bypass surgery. Complete follow up data were available for all patients (median (range) 35 (2-146) months). During the follow up period 16 patients (10%) died, two (1%) suffered non-fatal myocardial infarction, and 12 (7%) underwent elective coronary artery bypass surgery. A second angioplasty procedure was performed in 24 patients (15%). The cumulative probability of survival was 90.7% at 1 year and 83.4% at 5 years. Survival free from myocardial infarction, bypass surgery, and repeat angioplasty at 1 and 5 years was 68.2% and 56.0%, respectively. Proportional hazards regression analyses identified incomplete revascularisation as the only independent predictor of poorer overall survival (P = 0.04) and event free survival (P < 0.001). At census, of the 143 survivors, 75 (52%) were asymptomatic, 58 (41%) had mild angina, and only 10 (7%) complained of grade III or IV angina. Some 112 patients (78%) improved by at least two angina grades. CONCLUSION: Coronary angioplasty can be performed safely in the elderly and provides good symptomatic relief and favourable long-term outcome. Complete revascularisation may not be necessary if the primary goal is to achieve symptomatic relief, but incomplete revascularisation is associated with poorer long-term survival. PMID- 7547030 TI - Like father like son? Sons of patients of European or Indian origin with coronary artery disease reflect their parents' risk factor patterns. AB - OBJECTIVE: To investigate the extent to which risk factor patterns associated with coronary artery disease (CAD) in patients of Indian origin and in those of North European origin undergoing coronary angiography for suspected angina were reflected in their apparently healthy sons aged 15-30 years. DESIGN: Prospective study in which risk markers were measured in patients of Indian origin and in matched European patients undergoing angiography and in their sons. SETTING: Patients attending a regional cardiac centre and their families. PATIENTS: 102 consecutive male patients of Indian origin undergoing diagnostic coronary angiography for suspected angina and 89 of their sons aged between 15 and 30 years; 102 age matched male European patients and 82 sons. MAIN OUTCOME MEASURES: Father son correlations for risk markers predicting the severity of parental CAD; differences in mean levels of these markers between young males of Indian origin and those of North European origin. RESULTS: Lp(a) lipoprotein, total cholesterol, and serum insulin were independent predictors of the severity of CAD in patients of Indian origin and in those of North European origin. In both groups, there was strong correlation between paternal and filial serum insulin (r = 0.41 Indian origin, r = 0.49 North European, P < 0.001), Lp(a) lipoprotein (r = 0.44 Indian origin, r = 0.48 North European, P < 0.001), and total cholesterol (r = 0.39 Indian origin, r = 0.45 North European, P < 0.001) concentrations, and the risk factor profiles of the sons were predictive of CAD severity in their fathers. Sons of patients of Indian origin had significantly higher serum insulin (Indian origin 14.3 mU/l v North European 8.4 mU/l, P = 0.002) and Lp(a) lipoprotein (Indian origin 19.1 mmol/l v North European 10.5 mmol/l, P = 0.001) concentrations than sons of patients of North European origin. CONCLUSIONS: Apparently healthy young men aged 15-30 years from either ethnic community already reflect risk marker patterns associated with coronary artery disease in their parents, both for genetically determined factors such as Lp(a) lipoprotein and environmentally influenced factors such as insulin and cholesterol. Health promotion measures aimed at reducing the prevalence of CAD should include the adolescent and young adult populations, particularly those with a family history of CAD, or who are from ethnic communities in which this diagnosis is prevalent. PMID- 7547032 TI - Reversibility of tachycardia-induced cardiomyopathy after radiofrequency ablation of incessant supraventricular tachycardia in infants. AB - Tachycardia-induced cardiomyopathy developed in a 3 month old infant with permanent junctional reciprocating tachycardia, which was incessant despite medical treatment. The patient underwent transcatheter radiofrequency ablation. There were no complications and 8 months after the procedure the patient was symptom free without medication. PMID- 7547031 TI - Factors determining case fatality in myocardial infarction "who dies in a heart attack"? AB - OBJECTIVE: To examine the determinants of case fatality in the first major ischaemic heart disease event (heart attack) after screening. METHODS: Prospective study of 7735 middle aged men drawn from general practices in 24 British towns. RESULTS: During 11.5 years follow up there were 743 major ischaemic heart disease events of which 302 (40.6%) were fatal within 28 days of onset. Previous definite myocardial infarction or stroke and age at time of event were most strongly associated with case fatality. In men with no previous myocardial infarction or stroke, after adjustment for a range of risk factors, antihypertensive treatment (odds ratio (OR) = 1.97, P < 0.05), arrhythmia (OR = 1.93, P = 0.06), increased heart rate (OR = 2.03, P = 0.06), and diabetes (OR = 2.61, P = 0.07) were associated with increased case fatality. High levels of physical activity (OR = 0.53, P < 0.05) and moderate drinking (16-42 units/week) (OR = 0.61, P < 0.05) were associated with lower case fatality, although moderate drinking was not associated with a lower incidence of major ischaemic heart disease events. Current smoking, serum total cholesterol, and systolic blood pressure were not significantly associated with case fatality. In men with previous myocardial infarction or stroke, arrhythmia and to a lesser degree antihypertensive treatment, moderate or heavy drinking, and diabetes were associated with higher case fatality. CONCLUSION: These findings suggest that physical activity may be an important modifiable factor influencing the incidence of ischaemic heart disease and the chance of survival in men without a previous heart attack or stroke. Arrhythmia, increased heart rate, diabetes, and treatment for hypertension are also areas of concern. PMID- 7547033 TI - Fifty years of tachycardia. PMID- 7547034 TI - Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4. AB - The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody. PMID- 7547035 TI - Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha. AB - A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The recombinant GP Ib alpha fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 micrograms of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib alpha fragment. PMID- 7547036 TI - Isolation of p10 gene from Bombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system. AB - A homologue of Autographa californica NPV (AcNPV) p10 gene was identified and cloned from Bombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign genes, the p10 promoter is not so strong as the polyhedrin promoter. PMID- 7547037 TI - Volume preloading, spinal hypotension and caesarean section. PMID- 7547038 TI - Liposomal local anaesthetics. PMID- 7547039 TI - Volume preloading is not essential to prevent spinal-induced hypotension at caesarean section. AB - We have compared the protective effect of 1000 ml preload with 200 ml preload of crystalloid solution, administered during the 10 min before spinal anaesthesia was induced, in 60 healthy women with no fetal compromise undergoing elective Caesarean section. The spinal anaesthetic was managed identically in both groups by an anaesthetist who was unaware of the volume of fluid administered. A prophylactic infusion of ephedrine 60 mg in Hartmann's solution 500 ml was given according to maternal arterial pressure. Hypotension was defined as > or = 30% reduction from baseline or < 90 mm Hg, and was treated by i.v. ephedrine bolus doses. There was no significant difference in ephedrine requirements between the two groups or in the incidence, severity or duration of hypotension: 10 women in the 1000-ml group, five episodes lasting > or = 3 min compared with nine women in the 200-ml group, four lasting > or = 3 min. There was no difference between neonates in each group. We have now abandoned the routine of preloading before regional anaesthesia. PMID- 7547040 TI - Visceral pain during caesarean section: effect of varying dose of spinal amethocaine. AB - The aim of this study was to determine how varying the dose of spinal amethocaine influences the incidence of visceral pain associated with exteriorization of the uterus after delivery. In a double-blind study, we examined the incidence of visceral pain in 40 parturients undergoing Caesarean section under spinal anaesthesia with amethocaine 8, 10, 12 or 14 mg in 2 ml of 5% glucose. The dose of spinal amethocaine was found to influence the incidence of visceral pain associated with exteriorization of the uterus after delivery. The incidence of visceral pain was lower in the 12-mg (P < 0.05) and 14-mg (P < 0.01) groups than in the 8-mg group, although there were no differences in maximum spread of analgesia and circulatory changes between the four groups. This study suggests that a slightly higher dose of spinal amethocaine (12-14 mg) is preferable for Caesarean section. PMID- 7547041 TI - Quantitative evaluation of tourniquet leak during i.v. regional anaesthesia of the upper and lower limbs in human volunteers. AB - Although it is accepted that during i.v. regional anaesthesia (IVRA) local anaesthetic can leak under the tourniquet into the systemic circulation, no published study has evaluated this leak quantitatively. In volunteers, during two random sessions, we have simulated IVRA using standard techniques with a radiolabelled compound which is chemically similar to lignocaine and has comparable tissue distribution (0.1 mg of HIDA labelled with 100 muCi of 99mTc in 40 ml of saline). The decrease in radioactivity was measured with a gamma camera for the 20 min of tourniquet inflation and for the 20 min of washout after cuff deflation. While the tourniquet was inflated, the leak for the lower limb (mean 29 (SD 8) %) was significantly greater (P < 0.004) than the leak for the upper limb (15 (5) %). Moreover, in each of 10 volunteers, the leak was always greater for the lower than the upper limb. During the first 3 min after tourniquet deflation the loss of radioactivity was 58 (8) % of the maximal amount for the upper limb and 39 (8) % for the lower limb (P < 0.001). As the leak under the tourniquet was significantly greater for the lower than the upper limb, we conclude that IVRA for the lower limb can be associated more frequently with a shorter duration of successful anaesthesia and/or failure. PMID- 7547042 TI - A multifactorial analysis to explain inadequate surgical analgesia after extradural block. AB - A multivariate analysis of inadequate extradural analgesia was carried out prospectively on 1051 patients undergoing lumbar extradural anaesthesia for surgery performed on structures innervated by T10-S5. Ninety-six patients (9%) experienced pain during surgery. Age, extradural fentanyl, diazepam sedation and duration of surgery had no significant influence. We found some weak evidence that the type of surgery affects the risk of feeling pain. The probability of pain increased with increasing weight, except in overweight women, and was significantly greater for both shorter and taller patients, relative to patients of average height. The probability of pain decreased with increasing dose of local anaesthetic, increasing spread of extradural analgesia, addition of adrenaline, and fentanyl or thiopentone sedation. In conclusion, patient-, surgery- and anaesthesia-related factors influence the risk of inadequate extradural analgesia. If such factors are taken into account, an increase in the success rate may be anticipated. PMID- 7547043 TI - Evaluation of an amethocaine gel preparation for percutaneous analgesia before venous cannulation in children. AB - We have evaluated the efficacy and safety of a preparation of 4% amethocaine gel in alleviating the pain of venous cannulation in children. In an initial open study of 148 children, clinically acceptable anaesthesia was achieved in 92% of cases. The preparation was then compared with 5% EMLA cream in a single-blind study in 94 patients using an application time of 40 min. We found clinically acceptable conditions in 85% of patients receiving amethocaine gel compared with 66% in the EMLA group. There were no significant adverse effects noted in each group, although 37% of those children treated with amethocaine gel showed localized erythema at the application site. The results suggest that amethocaine gel has greater efficacy and a faster onset time than EMLA cream when used for this purpose in children. PMID- 7547044 TI - Peritonsillar infiltration with low-dose tenoxicam after tonsillectomy. AB - We have compared the effect of peritonsillar infiltration with tenoxicam 5 mg and placebo on postoperative pain after tonsillectomy. Fifty patients undergoing bilateral elective tonsillectomy under general anaesthesia were allocated randomly to receive peritonsillar infiltration with tenoxicam 5 mg in 8 ml of normal saline (4 ml per tonsil) or normal saline only, before tracheal extubation. Median time to first request for morphine (30 min in each group, P = 0.83), cumulative morphine requirements from 0 to 2 h after surgery (two and one doses, P = 0.50), and from 2 to 24 h after surgery (one dose in each group, P = 0.17) were similar. There were no significant differences between groups in VAS scores at rest or when drinking 100 ml of water at any time. The power of detecting a reduction in VAS scores of 20 mm was 90% at the 5% significance level. PMID- 7547045 TI - Comparison of desflurane and isoflurane in anaesthesia for dental surgery. AB - We studied 50 ASA I-II patients, aged 18-65 yr, undergoing elective orofacial surgery. Anaesthesia was induced with fentanyl and propofol, and maintained with 66% nitrous oxide in oxygen and either desflurane or isoflurane to compare recovery characteristics and cardiovascular stability. Cardiovascular responses to induction, intubation and incision were similar with both agents, although the increase in heart rate in response to intubation was less marked in the desflurane group. Maximum end-tidal concentrations of desflurane required were 4.0-10.6% (mean 6.8%) compared with maximum isoflurane concentrations of 1.1-2.3% (mean 1.6%). Mean duration of anaesthesia was 46 (SD 17.9) min (range 25-89 min) in the desflurane group and 41 (11.5) (23-60) min in the isoflurane group. Times to extubation were 6.7 (2.1) (3-10) min and 11.3 (4.1) (5-23) min, to eye opening 6.8 (2.2) (3-11) min and 12.7 (6.9) (7-37) min, to stating date of birth 9.0 (2.3) (4-12) min and 15.0 (6.9) (8-39) and to discharge from the recovery room 45 (11.6) (22-80) min and 64 (20.9) (28-134) min, for the desflurane and isoflurane groups, respectively (all P < 0.0001). No serious complications occurred in any patient. PMID- 7547046 TI - Effect of temperature and cardiopulmonary bypass on the auditory evoked response. AB - We have recorded auditory evoked potentials before and during cardiopulmonary bypass in 10 adult patients undergoing cardiac surgery under moderate hypothermia to 27-28 degrees C. The immediate effect of bypass was a small decrease in latency and increase in amplitude of the early cortical response. We also studied two adults and two children during profound hypothermia with circulatory arrest during cardiopulmonary bypass. Reduction in core temperature to 25 degrees C resulted in an increase in latency and amplitude of the brain stem responses; below this temperature the amplitude decreased but latency continued to increase until the auditory evoked response trace became completely flat between 21 and 19 degrees C. These changes were reversible on rewarming. PMID- 7547047 TI - Recovery after electroconvulsive therapy: comparison of propofol with methohexitone anaesthesia. AB - We have studied prospectively 39 patients receiving a course of electroconvulsive therapy (ECT) for major depressive disorder; they were allocated randomly to receive either propofol or methohexitone for anaesthesia. Recovery after the third ECT treatment was assessed by finger tap and digit symbol substitution tests at 15, 30, 45, 60 and 90 min after induction. Seizure duration (median (interquartile range)) was shorter with propofol (24 (10) s) than methohexitone (29 (17) s) (P = 0.08). There was no significant difference in psychometric recovery for drug type, duration of the seizure or initial severity of depression. These results suggest that the more rapid recovery rates noted with propofol in other procedures are not evident after electrically induced seizures. PMID- 7547048 TI - Anaesthetic experience of vertical banded gastroplasty. AB - We have reviewed data from 200 patients who underwent weight-reducing surgery in a district general hospital. Mean age was 38.5 yr (range 14-60 yr) and mean body mass index (BMI) 45.7 kg m-2 (range 30.0-86.8 kg m-2). There was one death, which resulted from pulmonary embolus 3 weeks after surgery. Upper abdominal surgery of this type is justified in the morbidly obese and problems were not encountered that would exclude its practice from non-specialized units. PMID- 7547049 TI - Genotype and phenotype relationships for mutations in the ryanodine receptor in patients referred for diagnosis of malignant hyperthermia. AB - Anaesthesia-induced malignant hyperthermia (MH) may be caused by specific gene defects in the skeletal muscle ryanodine receptor. We have studied the frequency of occurrence of the C1840T mutation, analogous to the porcine mutation, and three mutations associated both with MH and central core disease (G7301A, C487T and C1209G). We investigated skeletal muscle specimens from up to 137 patients testing negative and 101 patients testing positive for MH susceptibility by the North American MH Group protocol. The presence or absence of the mutations was determined by polymerase chain reaction and restriction enzyme digestion. The frequencies of occurrence of the C1840T and C487T mutations were 2% and 1%, respectively, in MH-positive subjects and were the only two mutations identified. One subject with central core disease did not have any of the three mutations examined associated with this disorder. Therefore, the porcine and central core disease-associated mutations examined in the ryanodine receptor account for a small proportion (approximately 3%) of MH-positive diagnoses. The mutations examined did not occur in any of the MH-negative patients, supporting an association between defects in the ryanodine receptor and a positive diagnosis for MH.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547050 TI - Biodistribution of liposomes after extradural administration in rodents. AB - We have mapped over 24 h the biodistribution of 99mTc-labelled multilamellar and small unilamellar liposomes in rabbits and rats by scintigraphic imaging after extradural injection. Multilamellar vesicles formed a depot at the site of injection; small unilamellar vesicles spread immediately along the extradural space and entered the systemic compartment 30 min after injection. Well delineated liver and kidney labellings were seen after 24 h. The use of 3H cholesterol-labelled small unilamellar vesicles suggested hepatic capture of intact liposomes with sizes averaging 0.05 microns drained unmodified into the systemic circulation through the extradural lymphatics. These results have led to the selection of multilamellar vesicles (0.1-15 microns size range) for clinical trials using liposome-associated local anaesthetics. PMID- 7547051 TI - Biodistribution of liposome-associated bupivacaine after extradural administration to rabbits. AB - After one extradural injection of 0.25% bupivacaine 0.3 ml and 3H-bupivacaine 0.005 mCi in multilamellar liposomes, no systemic radioactivity (plasma, liver, heart muscle) was obtained for 1 h, and the labelling was less than that of systemic distribution of plain bupivacaine for the following 3 h. In contrast, radioactivity in the lumbar spinal nerves peaked in the first hour and remained higher than that of plain bupivacaine for 4 h. No radioactivity was measured in cerebrospinal fluid. Small unilamellar vesicles incorporating 3H-cholesterol did not significantly label spinal nerves and central nervous structures indicating that the mode of action of liposomal bupivacaine did not involve uptake by nerve structures. Rapid uptake of radioactivity by spinal nerves suggested exchange of bupivacaine between liposomes and nerve sheaths. PMID- 7547052 TI - Anaesthetic agents decrease the activity of nitric oxide synthase from human polymorphonuclear leucocytes. AB - Nitric oxide (NO) inhibitors reduce the threshold for anaesthesia. We have investigated the action of anaesthetic agents on human nitric oxide synthase (NOS) activity. Thiopentone reduced mean NOS activity to 36.6 (SD 8.9) % of control at 100 mumol litre-1 (P < 0.001) and 50.9 (20.3) % at 1 mmol litre-1 (P < 0.05). Ketamine showed similar effects, with activity reduced to 67.0 (17.6) % (P < 0.05) and 57.7 (8.5) % (P < 0.001) at 100 mumol litre-1 and 1 mmol litre-1, respectively. Etomidate 100 mumol litre-1 did not significantly alter activity (88.2 (8.1) %) but 1 mmol litre-1 did (60.6 (10.4) %, P < 0.005). Halothane also caused a significant decrease in NOS activity at all concentrations. This effect was specific as other enzymes were unaffected. We conclude that anaesthetic agents have a profound effect on NOS activity and as inhibition of NO release augments anaesthesia, we suggest that this may play a role in the mechanism of anaesthesia in humans. PMID- 7547053 TI - Absorption of nitrogen dioxide and nitric oxide by soda lime. AB - Inhaled nitric oxide (NO) is used for the treatment of pulmonary hypertension as a selective pulmonary vasodilator. However, NO is oxidized rapidly to the more toxic nitrogen dioxide (NO2). Elimination of NO2 from inspired gas is essential for safe clinical use NO. We therefore investigated the efficacy of soda lime in absorbing NO2 from NO2-containing gases. Commercially available soda limes (Soda sorb and Wako lime-A), were exposed to the following six gas mixtures containing NO and NO2 in a hypoxic carrier gas for 20 min: No. 1: NO 40 ppm; No. 2: NO 35 ppm and NO2 5 ppm; No. 3: NO 30 ppm and NO2 10 ppm; No. 4: NO 20 ppm and NO2 20 ppm; No. 5: NO 10 ppm and NO2 30 ppm; and No. 6: NO2 40 ppm. Both types of soda lime completely absorbed the NO2 in all samples when it was present (Nos 2-6). NO concentration in these gas mixtures was reduced by an amount equal to the NO2 absorbed by soda lime. NO was absorbed minimally when NO2 was not present in the mixture. Nitrite was detected from the Wako lime-A granules exposed to the test gas by the chemical analysis. These findings suggest that soda lime completely absorbs NO2 by chemical neutralization, but NO is absorbed as simultaneously absorbed NO2 only where NO and NO2 coexist. Therefore, we conclude that soda lime is useful for NO2 absorption during NO inhalation therapy but NO monitoring from a point distal to the soda lime is required for precise control of inspired NO concentration. PMID- 7547054 TI - Testing the porphyrinogenicity of propofol in a primed rat model. AB - We evaluated the porphyrinogenicity of propofol in a rat model. After a pilot study had been conducted to determine an optimal dose, 48 fasting male Sprague Dawley rats were allocated randomly to six groups. The animals in groups 1-3 received saline i.p. In groups 4-6, the animals were given allylisopropylacetamide (AIA). Twelve hours later, animals in groups 1 and 4 received saline, groups 2 and 5 were given propofol 150 mg kg-1 i.p., followed by 75 mg kg-1 3 h later, and groups 3 and 6 received phenobarbitone 50 mg kg-1 i.p. and 25 mg kg-1 i.p. The animals were anaesthetized and killed 3 h after the second drug bolus and we measured the concentration of cytochrome P450, total porphyrin content and the activity of delta-aminolaevulinic acid synthase (ALAS) in the liver. Urinary delta-aminolaevulinic acid (ALA) and porphobilinogen (PBG) concentrations were measured. Analysis of variance and the t test with Bonferroni's correction were used to compare data. The hepatic cytochrome P450 concentration in the non-primed groups varied from 28.1 to 31.1 nmol g-1; administration of AIA decreased this to 20.1-20.9 nmol g-1. Total hepatic porphyrins were between 0.78 and 1.22 nmol g-1 in the non-primed groups and between 2.71 and 3.54 nmol g-1 in the AIA-primed groups. Hepatic ALAS activity was 29.2 and 35.5 nmol h-1 g-1 in groups 1 and 2. In the primed saline group, ALAS activity was measured at 134.5 nmol h-1 g-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547055 TI - Mechanism of the differential sensitivity in the rat adductor and abductor laryngeal muscles to a non-depolarizing neuromuscular blocker. AB - We have compared the neuromuscular blocking effects of tubocurarine at pre- and postsynaptic sites in the lateral cricoarytenoid muscle (LCA) (one of the adductor muscles of the vocal cords) and in the posterior cricoarytenoid muscle (PCA) (sole abductor muscle of the vocal cords) of the rat. Fine wire electrodes were inserted into both muscles and evoked compound electromyographic (EMG) responses measured by supramaximal stimulation of the recurrent laryngeal nerves. End-plate potentials (EPP), mean quantal content and carbachol sensitivity to tubocurarine in both muscles were measured using intracellular microelectrodes. Tubocurarine produced a concentration-dependent reduction in the EMG responses, EPP amplitude, mean quantal content and carbachol sensitivity. The LCA muscle was more resistant to tubocurarine than the PCA muscle in EPP amplitude, mean quantal content and carbachol sensitivity, suggesting unequal pre- and postsynaptic sensitivity for inhibition of elicited acetylcholine release, reduction in EPP amplitude and loss of evoked muscle action potentials. Examination of muscle fibre composition demonstrated that the LCA muscle contained a significantly higher fraction of slow twitch muscle fibres than PCA muscle. However, the sizes of the fibres were similar in both muscles. We conclude that the mechanism of unequal sensitivity to a non-depolarizing neuromuscular blocker in the LCA and PCA muscles may be explained by differential sensitivities at the pre- and postsynaptic sites of the neuromuscular junction. PMID- 7547056 TI - Pharmacokinetics of analgesics, sedatives and anaesthetic agents during cardiopulmonary bypass. AB - CPB produces changes in the blood-gas partition coefficient dependent on the prime used and temperature. Fortunately, the overall effect for hypothermic CPB and a crystalloid prime is only +2%. A volatile agent started during hypothermic CPB takes longer to equilibrate and agents already in use need to re-equilibrate, potentially changing the depth of anaesthesia, until equilibration is complete. As these agents are metabolized to a small degree and washout is fast, the duration of action is not prolonged after CPB. PMID- 7547057 TI - Effects of intraoperative N-acetylcysteine in orthotopic liver transplantation. AB - N-acetylcysteine (NAC) is an antioxidant agent which has been shown to benefit patients with fulminant hepatic failure. We have examined its effect in patients with chronic liver disease undergoing orthotopic liver transplantation by giving NAC during operation. In a prospective, randomized, double-blind, placebo controlled study of 50 patients, NAC appeared to induce mild vasodilatation, improve oxygen delivery and consumption, and reduce base deficit, but data interpretation was difficult. There were no significant effects on mortality, morbidity or postoperative graft function. PMID- 7547058 TI - Accidental intrathecal insertion of an extradural catheter during combined spinal extradural anaesthesia for caesarean section. AB - The combined spinal-extradural technique is used to provide analgesia and anaesthesia in obstetric anaesthetic practice. The accidental insertion of an extradural catheter into the dural opening made previously by the spinal needle is thought to be a theoretical risk. We report a case during combined spinal extradural anaesthesia for Caesarean section in which this complication occurred. PMID- 7547059 TI - Pregnancy complicated by aortic dissection: caesarean delivery during extradural anaesthesia. AB - We report a case where dissection of the aorta occurred in pregnancy; only medical management was undertaken. Delivery was by Caesarean section during extradural anaesthesia and was accomplished safely several weeks after the dissection. The aetiology, association with pregnancy, diagnosis and management of acute dissection of the aorta are discussed. PMID- 7547060 TI - An evaluation of methods of increasing the flow rate of i.v. fluid administration. AB - I have evaluated in vitro methods of increasing the flow rate of clear fluids through an i.v. cannula at room temperature. These included, alone and in combination: increasing the height of a gravity-fed system; increasing the i.v. cannula diameter, manual compression of the lower drip chamber and the use of pressure bags. Flow rate was measured using a uroflowmeter, which was found to be reliable and reproducible. The most effective methods of increasing flow were the use of a 14-gauge cannula rather than a 16-gauge cannula, which resulted in a 50% increase, and the use of a 300-mm Hg pressure bag with automatic adjustable pressure regulator, which doubled the flow rate. The combination of these two tripled the overall flow to nearly 600 ml min-1. Manual compression of the drip chamber, despite producing peak pressures of more than 100 cm H2O, was an inefficient method of improving flow compared with an external pressure bag. PMID- 7547061 TI - Thiopentone anaesthesia at Pearl Harbor. AB - A wartime embargo on casualty figures and an imprecise contemporary editorial contributed to the persisting belief that a grossly excessive mortality rate from barbiturate anaesthesia for surgery of the injured occurred after the Japanese attack on the American bases in Hawaii in December 1941. From accounts by surgical staff and official hospital records which have become available through US Freedom of Information legislation, it is clear that the rumoured death rate from this cause has been greatly exaggerated. PMID- 7547062 TI - Malignant hyperthermia and compartment syndrome. PMID- 7547063 TI - Malignant hyperthermia and compartment syndrome. PMID- 7547064 TI - Another complication of the combined extradural-subarachnoid technique. PMID- 7547065 TI - Ovarian hyperstimulation syndrome. PMID- 7547066 TI - Animal toxins. PMID- 7547067 TI - Postoperative analgesia for arthrolysis of the elbow joint. PMID- 7547068 TI - Retrograde cannulation of the jugular vein. PMID- 7547069 TI - Comparison of ropivacaine and bupivacaine for extradural analgesia. PMID- 7547070 TI - Mivacurium and prolonged neuromuscular block. PMID- 7547071 TI - Preoperative fasting for paediatric anaesthesia. PMID- 7547072 TI - Hypothermia during liver transplantation. PMID- 7547073 TI - Neurotoxicity of 5% lignocaine. PMID- 7547074 TI - Psychological characteristics and patient-controlled analgesia. PMID- 7547075 TI - Expanding the applications of patient-controlled techniques. PMID- 7547076 TI - Inhibition of tumor cell growth by a human B-cell line. AB - An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20+, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time- and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy BM binding. Lytic activity of Hairy-BM was found only against Breast-M. PMID- 7547077 TI - Inhibition of interferon-gamma and interleukin-2 production from lymphocytes stimulated with food antigens by an anti-allergic drug, Tranilast, in patients with food-sensitive atopic dermatitis. AB - N(3',4'-dimethoxycinnamoyl) anthranilic acid (Tranilast) inhibits antibody mediated hypersensitivity reactions, and is an effective drug for patients with bronchial asthma or allergic rhinitis. Interferon-gamma (IFN-gamma) production of ovalbumin (OA)-stimulated peripheral blood mononuclear cells (PBMCs) from hen's egg-sensitive patients with atopic dermatitis (AD) was significantly higher than those of healthy controls. Tranilast inhibited this IFN-gamma production. Moreover, interleukin-2 (IL-2) production of OA-stimulated PBMCs from hen's egg sensitive patients with AD was also inhibited by Tranilast. Our results suggest that Tranilast can be used to the patients with food sensitive AD. PMID- 7547078 TI - Use of recombinant interferon-alpha in human immunodeficiency virus (HIV) infected individuals. AB - RATIONALE AND OBJECTIVE: Interferon alpha (IFN-alpha) has anti-retroviral activity and is a possible HIV infection-limiting factor. The aim of this work is to prevent or delay disease progression in asymptomatic Human Immunodeficiency Virus (HIV) carriers. DESIGN AND INTERVENTIONS: Recombinant IFN alpha-2b (3 x 10(6) IU 3 times weekly) was compared to no treatment (control) in a randomized trial. Endpoints were: (i) appearance of any CDC group IV symptoms and (ii) disease progression (which excluded shifts to group IVC2 or reversible IVA, or IVB). The trial lasted from October 1987 to February 1992. SETTING: The trial was performed at the "Santiago de las Vegas" sanatorium, a specialized institution for the care of HIV-infected and AIDS patients. POPULATION: Subjects were anti HIV-1 seropositive, Western blot-confirmed, asymptomatic (CDC group II), or with generalized lymphadenopathies (CDC group III). The groups had 79 (control) and 71 (IFN) patients. MAIN RESULTS: Long-term IFN-alpha treatments significantly reduced the proportion of patients who shifted to any group IV (control: 46/79; IFN: 14/71; p < 0.001) or developed AIDS (control: 27/79; IFN: 12/71; p < 0.05). IFN also delayed progression to AIDS (95% confidence interval for 0.5 probability of progression) from 67-83 to 116-180 months after infection. The IFN group had significantly less opportunistic infections and non-infectious complications. CD4 cell count and hemoglobin decreased in the control but not in the IFN group. Fewer IFN-treated patients developed positive serum HIV antigen detection. CONCLUSION: IFN alpha treatment during the early stages of infection seems to be beneficial to the patients. PMID- 7547079 TI - Proliferative responses towards native, heat-denatured and pepsin-treated ovalbumin by peripheral blood mononuclear cells from patients with hen's egg sensitive atopic dermatitis. AB - In order to clarify the mechanism of food-antigen recognition, the proliferative responses of peripheral blood mononuclear cells (PBMCs) to native, heat-denatured or pepsin-treated ovalbumin (OA) were investigated in 16 hen's egg-sensitive patients with atopic dermatitis (AD). Seven of them had hypersensitivity to boiled hen's egg and others had not. The responses of PBMCs to heat-denatured OA were lower than those to native OA in the patients without hypersensitivity to boiled hen's egg. However, there were no differences of the responses of PBMCs between heat-denatured OA and native OA in the patients with hypersensitivity to boiled hen's egg. Moreover, the reduction of the responses of PBMCs to pepsin treated OA was recognized in six out of seven patients. The primary structure of OA did not change by heating or pepsin treatment according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the secondary structure of OA changed in connection with the reduction of the responses of PBMCs to denatured OA. In addition, we demonstrated the suppressive effect of anti-HLA-DR and anti-HLA-DQ monoclonal antibodies on the proliferative response of PBMCs to OA. The results suggested that the proliferative responses of PBMCs to OA were restricted by HLA-DR or HLA-DQ in hen's egg-sensitive patients with AD. PMID- 7547080 TI - Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies. AB - Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA) mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10(11) of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells. PMID- 7547081 TI - Antitumor effects of human recombinant macrophage colony-stimulating factor against rat brain tumors. AB - The tumoricidal effects of M-CSF were examined using two subcutaneously transplanted rat brain tumor cell lines, 9L and T9 gliomas. In rats treated with high-dose M-CSF (16 million U/kg administered for 4 days a week for 3 weeks), 9L glioma growth was inhibited by 81.9% following subcutaneous (s.c.) injection and by 70.5% after intraperitoneal (i.p.) injection and T9 glioma growth was inhibited by 69.2% after i.p. injection. After short-term treatment with high dose M-CSF (32 million U/kg administered s.c. for 6 consecutive days, 9L glioma growth was inhibited by 82.1%. All these inhibitory effects differed significantly compared with the respective untreated control groups. However, treatment with low-dose M-CSF (1.6 million U/kg administered s.c. for 4 days a week for 3 weeks) showed no significant effects against 9L and T9 glioma growth compared with the untreated controls. No significant effects of M-CSF against cell proliferation, measured as PCNA expression, were observed in any group. Significant hematopoietic effects on the leukocyte counts were observed only in the groups treated with high dose M-CSF. These results suggest that M-CSF at a high dose which produces hematopoietic effects on peripheral leukocytes inhibits the growth of gliomas. This inhibitory effect may have been due to a tumoricidal mechanism of M-CSF that depended on the production or release of some hematopoietic soluble factors, but was independent of PCNA expression by the tumors. PMID- 7547082 TI - Transfer factor prevents relapses in herpes keratitis patients: a pilot study. AB - Transfer Factor is a dialysable moiety obtained from immune lymphocytes. It has been successfully used for the treatment of several viral infections including labial and genital herpes. In the present study, thirty-three patients with low immune response to HSV antigens and suffering from herpes ocular infections were orally treated with HSV-specific transfer factor (TF). Their relapse index was reduced from 20.1 before treatment to 0.51 after TF administration, with only 6/33 patients relapsing. Although this is not a placebo-controlled-randomized study, the results suggest that TF specific for HSV antigens may be efficacious for preventing relapses of ocular herpes infections as has been the case with genital and labial localisations. PMID- 7547083 TI - Semi-synthesis of polymyxin-B conjugated ovalbumin: evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-alpha synthesis. AB - A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations. PMID- 7547084 TI - In vivo effects of recombinant human lymphotoxin on human medulloblastoma xenograft: enhancement of antitumor activity of etoposide. AB - The authors investigated the antitumor activities of rHuLT alone and in combination with etoposide on human meduloblastoma xenografts growing subcutaneously in nude mice. Intravenous administration of rHuLT (1.0 x 10(5) U/kg, 5.0 x 10(5)U/kg, 2.5 x 10(6)U/kg, three times a week for three weeks) suppressed medulloblastoma growth depending on the dose. However, the highest dosage caused serious side effects. Combining rHuLT (intravenously, 5.0 x 10(5)U/kg, three times a week for three weeks) with etoposide (intraperitoneally, 20mg/kg, once a week for three weeks) increased the antitumor activity without causing serious toxicity. Microscopically, tumor specimen showed thrombosed tumor vessels and massive necrosis 3 weeks after rHuLT treatment. Ultrastructural examination revealed that 120 minutes after the administration of rHuLT alone, disruption of interendothelial junctions was evident, and that the endothelial cells were destroyed at 240 minutes. Concentration of etoposide in tumor tissue peaked 30 minutes after intraperitoneal administration, and then decreased with time. When etoposide was administered in combination with rHuLT, the concentration of etoposide in tumor tissue after 60 to 240 minutes was significantly higher than when etoposide was given alone, and the area under the concentration versus time curve was also greater for the tumors of mice with combination treatment. The findings suggest that the proper combination of rHuLT and etoposide may have synergistic antitumor activities. Histological changes suggest that increased concentrations of etoposide within the tumor after combination therapy may occur due to increased vascular permeability and/or decreased etoposide clearance which is the result of blood statis in the tumor vasculature. PMID- 7547085 TI - The pharmacokinetics of tenidap sodium: introduction. PMID- 7547086 TI - Single and multiple dose pharmacokinetics of tenidap sodium in healthy subjects. AB - 1. The absorption, protein binding, clearance and absolute bioavailability of tenidap sodium were studied after single and multiple dosing. 2. Thirteen healthy male volunteers received a single 120 mg oral dose of tenidap sodium and a 20 mg intravenous infusion of deuterated tenidap ([D3]-tenidap) on day 1. This was followed by a 6-day washout period (days 2-7) and then further daily doses of oral tenidap sodium 120 mg for 21 consecutive days (days 8-28) with an additional 20 mg intravenous infusion of [D3]-tenidap on day 28. Twelve subjects were eligible for pharmacokinetic evaluation. 3. Following multiple oral doses, the half-life of tenidap is approximately 23 h. 4. Following single and multiple dose administration, the absolute bioavailability is 85%. 5. Systemic clearance of [D3]-tenidap was 29% greater on day 28 than on day 1 indicating a significant increase in intrinsic clearance (CLint) of tenidap since protein binding of tenidap in plasma did not change during the study. Consistent with the increase in systemic clearance, the half-life of [D3]-tenidap decreased and the ratio of AUC(0,24h) day 28/AUC day 1 following oral dosing was less than one. Tenidap is subject to extensive hepatic metabolism, so the increase in CLint may indicate that tenidap induces its own metabolism. 6. Steady-state was achieved by the eleventh day of dosing. Since numerous studies in patients with rheumatoid arthritis have shown that multiple dosing with tenidap is clinically efficacious, this suggests that the pharmacokinetic differences observed between the first and twenty-first day of multiple tenidap dosing do not influence the clinical response. PMID- 7547087 TI - Pharmacokinetics of tenidap sodium administered with food or antacid in healthy volunteers. AB - 1. The effects of food and antacid on the pharmacokinetics of tenidap were investigated in this randomised, 3-way cross-over study. 2. Twenty-one healthy young men, mean age 27.4 years, received single oral doses of tenidap sodium 120 mg at weekly intervals after either an overnight fast, with food or with 20 ml of the antacid Maalox (aluminum hydroxide 1.8 g and magnesium hydroxide 1.2 g). Plasma samples collected immediately before and up to 96 h after each tenidap dose were assayed for tenidap using a validated h.p.l.c. method. The assay data were used to determine the pharmacokinetic parameters of tenidap in each group. 3. Co-administration of tenidap with food produced a statistically significant delay in the rate of absorption (tmax, 4.4 h) (P < 0.001). There was no statistically significant change in Cmax. However, co-administration with the antacid significantly decreased both the mean rate and extent of absorption of tenidap compared with the fasting state: AUC, 420.93 micrograms ml-1 h (antacid), 476.31 micrograms ml-1 h (fasting) (P = 0.026); Cmax 14.3 micrograms ml-1 (antacid), 18.0 micrograms ml-1 (fasting) (P = 0.001); tmax 4.5 h (antacid), 2.9 h (fasting) (P < 0.001). Neither food nor the antacid had any effect on the elimination of tenidap. These changes in tmax are unlikely to be of any clinical significance owing to the long half-life of tenidap. 4. Treatment was well tolerated. Only two adverse events were reported that were considered by the investigator to be related to tenidap. There were no reports of laboratory or cardiovascular abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547088 TI - Cimetidine does not alter the clearance or plasma binding of tenidap in healthy male volunteers. AB - 1. An open-label, placebo-controlled study was conducted to examine the effect of cimetidine on the steady-state pharmacokinetics of tenidap. 2. Twenty-four healthy volunteers received tenidap sodium (120 mg) each morning throughout the study. On day 14 plasma levels of tenidap had reached steady state; half of the subjects were treated concomitantly with cimetidine 800 mg at night, while the other half received placebo. Allocation of cimetidine or placebo was randomised. Plasma profiles of tenidap were measured on days 14 and 16. 3. The addition of cimetidine did not significantly affect the Cmax, tmax, or lambda z of tenidap. The AUC(0,24h) increased by 4% between days 14 and 16 in the cimetidine treatment group, compared with a decrease of 2% in the placebo group. This small difference is statistically significant (P = 0.047), but it is not considered to be clinically relevant. 4. It is concluded that concomitant administration of cimetidine does not significantly affect the pharmacokinetics of tenidap at steady state. PMID- 7547089 TI - Tenidap sodium decreases renal clearance and increases steady-state concentrations of lithium in healthy volunteers. AB - 1. An open-label, randomised placebo-controlled study was conducted to determine the effects of tenidap sodium, a novel, cytokine-modulating anti-rheumatic drug, on the steady-state concentrations and renal clearance of lithium carbonate. 2. Eighteen healthy male volunteers received 450 mg lithium carbonate twice daily for 15 days and once on day 16. On days 9-16 subjects also received either placebo or 120 mg day-1 tenidap 2 h prior to the morning dose of lithium. 3. Following a single dose of tenidap, the renal clearance of lithium decreased significantly by 0.36 l h-1 (-23%) compared with the clearance in the placebo group, which increased by 0.18 l h-1 (+14%). Steady-state serum lithium levels increased after a single dose of tenidap by 0.069 mEq l-1 (+13%), and in the placebo group levels increased by 0.003 mEq l-1 (+0.5%); this difference was not significant. 4. After 7 days' continuous administration of tenidap, the renal clearance of lithium had decreased by 0.38 l h-1 (-25%), compared with the placebo group in which clearance had increased by 0.16 l h-1 (+12%). The steady state serum concentration of lithium increased by 0.208 mEq l-1 (+39%) in the tenidap group and by 0.063 mEq l-1 (+10%) in the placebo group. Both of these differences were significant. 5. Two subjects who received lithium plus tenidap experienced gastrointestinal side effects, compared with none of those who were administered lithium plus placebo. 6. It is recommended that serum lithium levels be monitored when tenidap and lithium are administered concomitantly. PMID- 7547090 TI - Effect of tenidap sodium on the pharmacodynamics and plasma protein binding of warfarin in healthy volunteers. AB - 1. An open-label, randomised study was performed to assess the effect of tenidap sodium on the pharmacodynamics and plasma protein binding of warfarin. 2. Fourteen healthy male volunteers received either a single oral dose of 120 mg tenidap sodium or matching placebo capsules from days 11 to 36. A single oral dose of 0.75 mg kg-1 warfarin was administered on days 1 and 32. 3. The mean prothrombin AUC(1,120h) value between baseline and day 32 increased from 1692.4 +/- 234.5 s h to 1769.3 +/- 218.0 s h in the group given tenidap, and decreased from 1747.6 +/- 289.4 s h to 1708.1 +/- 236.8 s h in the placebo group. 4. Tenidap caused a slight delay in the normalisation of prothrombin times following the second dose of warfarin on day 32 compared with the first dose on day 1. This was significant at 36, 48, 72 and 96 h but not at 120 h after administration of warfarin. 5. The mean percentage of unbound warfarin in the tenidap group (0.08% +/- 0.09) was significantly different (P = 0.047) from that in the placebo group (-0.03% +/- 0.10) but this was not considered to be clinically meaningful. 6. These data indicate that prothrombin times should be monitored during concomitant administration of tenidap and warfarin. PMID- 7547091 TI - The effects of age and gender on the pharmacokinetics of tenidap sodium in patients with rheumatoid arthritis and osteoarthritis. AB - 1. This open-label study was conducted to evaluate the effects of age and gender on the pharmacokinetics of tenidap sodium in patients with either rheumatoid arthritis (RA) or osteoarthritis (OA). 2. A total of 145 male and female patients, 80 with RA (aged 22-91 years) and 65 with OA (aged 45-83 years) each received a single dose of 120 mg tenidap sodium. Pharmacokinetic parameters were estimated from individual tenidap plasma concentration-time curves determined up to 120 h post-dose. Tenidap plasma protein binding was determined in the youngest and oldest age groups for both RA and OA patients. 3. In RA patients, age and gender did not significantly affect the disposition of tenidap or the percentage of unbound tenidap in plasma. Only for tmax were statistically significant effects in the analysis of covariance observed and these were attributed to very high tmax values in two female patients. Likewise, in patients with OA, age and gender did not significantly affect the pharmacokinetics of tenidap, although patients with larger body weights had lower Cmax values. 4. Pharmacokinetic parameters were not significantly different between RA and OA patients, except for t1/2, where two outlier RA patients had low values that caused the mean value to be significantly lower in RA (24 h) than in OA (26 h) patients. Pharmacokinetic parameters for pooled values from RA and OA patients were as follows: t1/2 = 25 h, AUC = 538.4 micrograms ml-1 h, Cmax = 21.9 micrograms ml-1, tmax = 3.2 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547092 TI - The effect of tenidap sodium on the disposition and plasma protein binding of phenytoin in healthy male volunteers. AB - 1. The effects of tenidap sodium 120 mg day-1 at steady state and placebo on the plasma protein binding and pharmacokinetics of phenytoin were compared in this randomised, double-blind, placebo-controlled, parallel-group study, involving 12 healthy young men, conducted over 34 days. 2. Single oral doses of phenytoin 200 mg were given on days 1-3 and 29-31, and intravenous phenytoin, 250 mg infused over 20 min, was given on days 4 and 32. Tenidap (120 mg day-1), or matching placebo, was administered as single oral daily doses from days 8 to 34 inclusive. 3. The plasma protein binding of phenytoin was determined immediately before oral phenytoin administration on days 1 and 29. Pharmacokinetic parameters were estimated from the serum phenytoin concentration-time curves derived on days 4 and 32 following the phenytoin infusions. The differences between the pre- and post-treatment mean percentage of unbound plasma phenytoin and mean pharmacokinetic parameters were compared between treatment groups. 4. Tenidap sodium 120 mg day-1, at steady state, increased the percentage of unbound phenytoin in plasma by approximately 25%, but did not significantly affect AUC(0,48h) or Cmax. 5. Since tenidap increases the percentage of unbound phenytoin in plasma, when monitoring phenytoin plasma concentrations free concentrations of phenytoin should be considered. 6. Tenidap was well tolerated throughout the study. PMID- 7547093 TI - Tenidap sodium does not alter the clearance or plasma protein binding of tolbutamide in healthy male volunteers. AB - 1. This randomised, double-blind, parallel group study in 12 healthy young men compared the effects of tenidap sodium 120 mg day-1, at steady state, with placebo on the plasma protein binding and clearance of tolbutamide. 2. Each subject received a 1000 mg intravenous infusion of tolbutamide given over 5 min on day 1 of the study, and again on day 30 following 22 days of successive tenidap or placebo administration. 3. The percentage of unbound tolbutamide in plasma was determined immediately before each infusion. Mean pharmacokinetic parameters (system plasma clearance, terminal phase rate constant, apparent volume of distribution at steady state) of tolbutamide were derived from individual tolbutamide plasma concentration-time curves generated after infusion. The within group day 30 minus day 1 differences were compared between treatment groups. 4. Tenidap was shown to have no statistically or clinically significant effects on any of the parameters assessed. These results indicate that tenidap does not induce or inhibit the P450IIC9 isozyme which metabolises tolbutamide and that tenidap does not displace tolbutamide from plasma protein binding sites. 5. Both tenidap and tolbutamide were well tolerated. No severe treatment-related adverse events were reported, no subject withdrew from the study, and there were no reports of treatment-related laboratory abnormalities, or significant variations in vital signs. PMID- 7547094 TI - Effect of tenidap sodium on digoxin pharmacokinetics in healthy young men. AB - 1. The effects of tenidap sodium and placebo on digoxin pharmacokinetics were compared in 14 healthy young men, in a double-blind, parallel-group study lasting for 24 days. 2. Subjects were administered digoxin alone for the first 10 days and digoxin plus tenidap 120 mg day-1 or placebo for the remaining 14 days. 3. Changes in the means between day 10 (digoxin monotherapy) and day 24 (combined therapy) for renal clearance, area under the plasma concentration-time curve during the dosing interval, and the minimum and maximum plasma digoxin concentrations did not differ significantly between the tenidap and placebo groups. There was a small but statistically significant increase (0.5 h) in the time taken to reach maximum plasma digoxin concentration following 14 days' continuous tenidap co-administration compared with placebo, but this was not considered to be clinically meaningful. 4. Co-administration of tenidap and digoxin was well tolerated. No subject withdrew from the study during combination treatment. Treatment-related adverse events were of mild to moderate severity and were reported by four subjects on digoxin monotherapy, four on tenidap and digoxin, and by two on digoxin and placebo. Those reported by the tenidap group predominantly affected the gastrointestinal system and were mild in severity. There were no reports of laboratory test abnormalities or cardiovascular abnormalities related to combined digoxin and tenidap administration. 5. The results of this study indicate that, in healthy young men, co-administration of tenidap with digoxin does not have any apparent clinically significant effects on the pharmacokinetic profile of digoxin, and the treatment is well tolerated. PMID- 7547095 TI - An investigation into the effect of tenidap sodium on the pharmacokinetics of a combined oral contraceptive. AB - 1. The effects of tenidap sodium and placebo on the pharmacokinetics of a combined oral contraceptive (Microgynon 30) were evaluated in 18 healthy premenopausal women in a double-blind, cross-over study lasting two menstrual cycles. 2. Tenidap (120 mg day-1) or placebo was given for 11 days, starting within 4 days of menstruation and Microgynon 30, containing levonorgestrel (150 micrograms) and ethinyloestradiol (30 micrograms), was administered on day 10 of tenidap therapy. 3. The mean maximum plasma levonorgestrel concentrations (Cmax), time to Cmax (tmax) and area under the plasma time-concentration curves (AUC(0,t)) did not differ between subjects given tenidap or placebo. The Cmax, tmax and AUC(0,t) values for ethinyloestradiol did not differ between tenidap and placebo recipients. Only the ethinyloestradiol Cmax showed a significant difference (P = 0.02) between menstrual cycles 1 and 2 (252.9 pg ml-1 and 271.3 pg ml-1, respectively). 4. Co-administration of tenidap and Microgynon 30 was well tolerated and no subject withdrew from the study because of side-effects. There were no side-effects considered to be related to tenidap and no clinically significant laboratory abnormalities were considered to be related to treatment. 5. The results of the study suggest that the pharmacokinetics of the oestrogen and progestin components of the oral contraceptive Microgynon 30 are unlikely to be affected by concomitant administration of tenidap. PMID- 7547096 TI - The effect of tenidap on the anti-hypertensive efficacy of thiazide diuretics in patients treated for mild to moderate hypertension. AB - 1. This randomised, placebo-controlled, double-blind, parallel-group study was conducted to assess the effect of tenidap sodium 120 mg, a novel anti-arthritic cytokine modulating drug, on the hypotensive efficacy of the thiazide diuretics hydrochlorothiazide or bendrofluazide. 2. Twenty-three male and female patients, aged 41-78 years, with mild to moderate, uncomplicated, essential hypertension controlled with thiazide diuretic therapy, received either a single daily dose of tenidap sodium 120 mg or matched placebo for 22 days in addition to their diuretic therapy. Changes between baseline and endpoint in supine and standing systolic and diastolic pressures and pulse rate were compared between treatment groups. 3. Daily treatment with tenidap reduced the anti-hypertensive efficacy of the thiazide diuretics. Blood pressure tended to increase marginally and the increase in mean standing diastolic pressure observed with tenidap was significantly greater than the change in the placebo group. All pressures tended to decrease in the placebo group and all endpoint measurements were within 7 mm Hg of baseline in both groups. 4. Treatment-related side effects of mild to moderate severity were reported in two subjects receiving tenidap, but in neither case was treatment discontinued. Two subjects receiving placebo also experienced side effects considered to be treatment-related and both were withdrawn from the study. 5. The results of this study suggest that tenidap may be given to patients treated for mild to moderate essential hypertension controlled with thiazide therapy; however, the patient's blood pressure should be regularly monitored. PMID- 7547097 TI - The effect of tenidap on the anti-hypertensive efficacy of ACE inhibitors in patients treated for mild to moderate hypertension. AB - 1. A randomised, placebo controlled, double-blind, parallel group study was conducted to assess the effect of tenidap sodium, a novel cytokine modulating drug, on the stable hypotensive response to the angiotension converting enzyme (ACE) inhibitor enalapril in subjects with mild to moderate, uncomplicated, essential hypertension. 2. Twenty-four male and female hypertensives, aged 33-77 years, received either 120 mg tenidap sodium or matched placebo daily for 22 days concomitantly with enalapril. 3. Mean endpoint supine and standing, systolic and diastolic pressures remained within 10% of baseline in each treatment group. However, the endpoint values were marginally above baseline during double-blind treatment with tenidap and marginally below baseline in the group receiving placebo. The increases in supine and standing systolic pressures in the tenidap group differed significantly from the changes in the placebo group. There were no significant differences between groups in changes in pulse rate. 4. Gastrointestinal side effects of mild to moderate severity attributed to treatment with tenidap were experienced by five subjects, one of whom was withdrawn during the third week of treatment. One subject receiving placebo was withdrawn because of a moderate headache attributed to study treatment. 5. The results of this study suggest that treatment with tenidap may interfere with the anti-hypertensive efficacy of ACE inhibitors. It is recommended that blood pressure should be monitored when tenidap is administered concomitantly with an ACE inhibitor. PMID- 7547099 TI - Spondyloarthropathies. PMID- 7547098 TI - Lack of photosensitising potential of tenidap, a novel anti-rheumatic agent. AB - 1. The potential of orally administered tenidap sodium, a novel anti-rheumatic agent under investigation for the treatment of rheumatoid arthritis and osteoarthritis, to cause phototoxic reactions was investigated. 2. Twenty-four healthy volunteers entered a randomised, double-blind study in which they received tenidap sodium 40 mg or 120 mg or placebo daily for 7 days. The minimal erythema dose was determined using wavelengths of 305-460 nm for 3 days prior to drug administration and on the last 3 days of dosing. Appropriate clinical and laboratory tests were performed before, during and after the dosing period. 3. Tenidap did not have a statistically significant effect on immediate or delayed photosensitivity. No drug-related side effects were reported and there were no clinically significant adverse findings from the laboratory tests. 4. At the dosage and duration of tenidap sodium used, a photosensitising potential has not been demonstrated. PMID- 7547100 TI - Infectious arthritis and immune dysfunction. PMID- 7547101 TI - Osteoarthritis and crystal deposition diseases. PMID- 7547102 TI - HLA-B27 and its subtypes in world populations. AB - Twenty-five years ago, HLA-B27 was recognized as a new HLA specificity. It is present throughout Eurasia but is virtually absent among the genetically unmixed native populations of South America, the Bantus and Sans (Bushmen) of equatorial and southern Africa, and the aborigines of Australia. B27 is a serologically determined specificity that encompasses nine different allotypes (subtypes), B*2701 to B*2709. Ankylosing spondylitis or related spondyloarthropathies thus far have been documented in individuals with any of the following five subtypes: B*2701, B*2702, B*2704, B*2705, or B*2707. PMID- 7547103 TI - Structural polymorphism and function of HLA-B27. AB - Over the past year, new subtypes have been described and significant advances have been made in understanding peptide binding to HLA-B27. These developments help in interpreting how antigen presentation is modulated by HLA-B27 polymorphism, in determining the similarities and differences in T cell antigenicity among subtypes, and in finding the basis of HLA-B27 evolution. New insights into the pathogenetic link between enteric bacteria and HLA-B27 come from demonstration of a direct relationship between gut flora and joint inflammation in transgenic rats and from involvement of HLA-B27 in modulating bacterial invasion of mammalian cells. The association between HLA-B27 and spondyloarthropathy may be related to antigen-presenting specificity, but a different mechanism, presentation of a B27-derived peptide by class II antigens, may contribute to the pathogenesis of acute anterior uveitis, emphasizing the need for a comprehensive understanding of the structure, antigenicity, and function of HLA-B27. PMID- 7547104 TI - Epidemiologic, clinical, and therapeutic aspects of reactive arthritis and ankylosing spondylitis. AB - The role of microbes in the pathogenesis of various arthritides continues to be the focus of keen interest. In the study of genetic factors that predispose to spondyloarthropathies, HLA-B27 transgenic animal models present exciting new research possibilities. The basic principles in the management of these arthritides have not changed, but an increasing number of patients are being treated with disease-modifying antirheumatic drugs when the response to nonsteroidal anti-inflammatory drugs is inadequate. PMID- 7547105 TI - Enteropathic arthritis, Whipple's disease, juvenile spondyloarthropathy, uveitis, and SAPHO syndrome. AB - During the past year, concepts of spondyloarthropathy and associated gut abnormalities have been further confirmed. In addition, the evolution of spondyloarthropathy to ankylosing spondylitis seems to be determined by the underlying gut inflammation. Other factors present early in spondyloarthropathy that affect the long-term prognosis of the disease have been determined. HLA-B27 has an impact on the severity of spondyloarthropathy. Its presence is also highly associated with uveitis. Paradoxically, HLA-B27+ uveitis is usually benign, with a good prognosis. A subgroup of patients can, however, have severe posterior uveitis; immunosuppressive drugs have been used to treat this disorder. PMID- 7547106 TI - Etiopathogenesis of reactive arthritis and ankylosing spondylitis. AB - Advances in molecular biology have brought more refined techniques to the study of the structure and function of HLA molecules and the biology of arthritogenic bacteria. Site-directed mutations in HLA and bacterial genes have focused attention on amino acids that are playing a critical role in the dynamic interaction of host and pathogen. The role of HLA-B27 in conferring disease susceptibility is being evaluated in both human disease and in B27 transgenic animal models. The continued interchange between clinical research in the spondyloarthropathies and the in vitro systems analyzing the immunology and microbiology of these diseases has maintained a high level of interest in this field of arthritis research. PMID- 7547107 TI - Rheumatic fever and poststreptococcal reactive arthritis. AB - Rheumatic fever is a catastrophic illness in many parts of the world, particularly in developing nations, where the incidence has been estimated to be between 10 and 15 million new cases each year. In the United States, rheumatic fever had become a rarity, having virtually disappeared by the mid 1960s. Of increasing concern, however, was the abrupt rise in the incidence of rheumatic fever in the United States in the mid 1980s, with reported "outbreaks" in middle class communities in five cities and two military camps. Recently, a number of cases of poststreptococcal reactive arthritis have been reported. On close examination, however, these are most likely alternate clinical presentations of rheumatic fever. It is widely accepted that rheumatic fever occurs following an overactive immune response by a genetically susceptible host to oropharyngeal infection with group A beta-hemolytic streptococci. Nevertheless, details of pathogenesis at a level allowing more effective intervention remain obscure. The question of pathogenesis holds a deep interest, because rheumatic fever is one of the few autoimmune diseases with a known infectious etiology. PMID- 7547108 TI - Bacterial arthritis. AB - A number of experimental models of bacterial arthritis demonstrated that immune factors, especially directed cytokines, play an important role in cartilage destruction. The most important studies in bacterial arthritis were a review of the clinical manifestations of gonococcal arthritis and two reports of the use of polymerase chain reaction to detect Neisseria gonorrhoeae DNA in synovial fluid. Polymerase chain reaction may be an important diagnostic test in culture-negative cases and may be very helpful in understanding the pathophysiology of gonococcal arthritis. PMID- 7547109 TI - Osteomyelitis. AB - The complexities of osteomyelitis make its diagnosis and treatment challenging. Current trends emphasize early diagnosis and aggressive treatment. Imaging has improved, with nuclear scans and magnetic resonance imaging, and technique modifications have enhanced the specificity of these tests. Treatment depends on thorough debridement of necrotic bone and tissue, accurate cultures and administration of culture, and sensitivity-specific antibiotics. Antibiotic delivery has expanded to include effective oral agents and local agents mixed with polymethylmethacrylate or a biodegradable substance. Success rates in treating this disease have improved with the use of a systematic approach, making outcome more predictable. PMID- 7547110 TI - Lyme disease. AB - In the United States, Lyme disease is the most common arthropod-borne infection. The majority of cases are reported from the Northeast, the Midwest, and California, which are areas with established foci of Borrelia burgdorferi. Advances in the understanding of the epidemiology of Ixodes ticks, the established vector for transmission of Lyme disease, provided strategies to reduce tick populations and the risk of acquiring disease. Genotypic diversity of subspecies of B. burgdorferi has been associated with differences in disease expression. New methods for detection of B. burgdorferi have expanded understanding of the pathogenesis of Lyme disease and provided clues into the mechanisms responsible for persistent symptoms. A safe vaccine for the prevention of Lyme disease in humans has been developed, and clinical vaccine efficacy trials are currently under way. PMID- 7547111 TI - Mycobacterial and fungal infections of bone and joints. AB - The number of cases of tuberculous bone or joint infection reported annually in the United States has been rising, but it decreased slightly in 1993. Management of skeletal tuberculosis is a complex and evolving issue that requires knowledge of the treatment of drug-resistant organisms. Nontuberculous mycobacteria also may cause skeletal infections, which are often located in tenosynovium or osteoarticular components of the hand or wrist but may occur at other skeletal sites, particularly when there is underlying immunosuppression. Identification of the organism and determination of its drug sensitivities are crucial for providing optimal therapy. Fungal infections of bone or joint can be difficult to treat, but the availability of fluconazole and itraconazole has extended our therapeutic options for some mycoses. PMID- 7547112 TI - Viral arthritis including HIV. AB - Viruses are attractive candidates for infectious etiologic agents or cofactors in the development of rheumatic diseases. The epidemic of HIV infection and the recognition of "emerging viruses" continues to fuel interest in the possible role of viruses in the pathogenesis of diseases without defined etiologies. During 1994, roles for parvovirus B19 in vasculitis and erosive rheumatoid arthritis were entertained. We were reminded that rubella infection may present with polyarthritis. Our understanding of the rheumatic disease manifestations of hepatitis C virus infection was broadened to include polyarthritis. A possible role for herpesviruses in Sjogren's syndrome continued to be explored without definite resolution. Paramyxoviruses were offered as an agent in the development of Paget's disease. The retroviruses continued to attract attention because of rheumatic disease syndromes in AIDS patients and the ability of retroviruses to latently infect the host and alter host immune responses. This review highlights efforts made in the past year to elucidate the role of viral infection in rheumatic disease. PMID- 7547113 TI - Cartilage matrix molecules in serum and synovial fluid. AB - The search continues for constituents of articular cartilage in the serum and synovial fluid of patients with arthritis. This work is driven by the hope that such "markers" will be useful in the diagnosis and management of rheumatic diseases. Absent information about the kinetics of these molecules continues to hinder interpretation of their concentration. Recent progress includes recognition of markers that appear to be specific for diseased cartilage and identification of factors that may distinguish between reversible and irreversible disease. PMID- 7547114 TI - Assessment outcomes of clinical drug trials in osteoarthritis. AB - The management of osteoarthritis has been limited to symptomatic treatment to decrease pain and improve function without modifying the underlying disease process. New insights into the structure, physiology, and function of cartilage, however, have led to the development of promising approaches that might ultimately modify the underlying disease process. Over the past year, a number of therapeutic trials in osteoarthritis were reported in the literature. These studies provide an opportunity to review methodologic challenges in clinical trials of osteoarthritis. PMID- 7547115 TI - Metabolic and endocrine disease and arthritis. AB - Many metabolic and endocrine disorders have effects on the musculoskeletal system, either due to primary changes in bone and collagen or resulting in secondary arthritic and bone changes. Disorders of ossification include heterotopic ossification, diffuse idiopathic skeletal hyperostosis (DISH), and the spondyloarthropathies. The early stages of heterotopic ossification have been analyzed using immunohistochemical techniques to trace the distribution of noncollagenous proteins. Several radiographic features of DISH and ankylosing spondylitis were discussed, including rib hyperostosis and syndesmophytes. Hypertrophic osteoarthropathy with associated arthritis was described in seven patients with hepatic failure and transplantation. Remarkable improvement in arthritis followed transplantation. The inverse relationship between osteoarthritis and osteoporosis continues to be studied, and it has been hypothesized that genetics play a major role in determining the rate of bone turnover, resulting in, for example, "bone formers." Both growth hormone excess and deficiency lead to changes in the incidence of osteoarthritis. Finally, one study documented the improved outcome in joint replacements in hemophiliac patients. PMID- 7547116 TI - Gout, uric acid metabolism, and crystal-induced inflammation. AB - Serum triglyceride levels are significantly higher and serum high-density lipoprotein cholesterol levels are lower in patients with gout compared with healthy individuals. Whereas increased serum triglyceride levels exist intrinsically in gout, serum uric acid concentration correlates inversely with insulin sensitivity and positively with serum triglycerides. Interaction of monosodium urate crystals with granulocyte-macrophage colony-stimulating factor and with tumor necrosis factor-activated neutrophils favored the production of interleukin-1 over that of interleukin-1-Ra, resulting in a proinflammatory imbalance. Interaction of the crystals with iron or tyrosine kinase may modify their inflammatory response and can be an important modulating mechanism in gouty arthritis. E-selectin is a specific marker for synovial fluid soluble endothelial activity and is increased in the synovial fluid of patients with gouty arthritis, as well as in that of patients with other inflammatory arthritides. Similarly, E selectin was found to be high in joints with monosodium urate crystal-induced synovitis. In addition, synovial fluid levels of interleukin-8 were found to be high in gout, rheumatoid arthritis, and osteoarthritis. PMID- 7547117 TI - Calcium pyrophosphate crystal deposition disease and other crystal deposition diseases. AB - "Tumoral" calcium pyrophosphate dihydrate (CPPD) crystal deposition in the ligamentum flavum is rare and can lead to compression myeloradiculopathy in the cervical spine and to spinal canal stenosis in the lumbar spine. CPPD crystal deposition disease is rarely associated with Bartter's syndrome and hypomagnesemia. Intramuscular corticotropin has proved effective in the treatment of acute episodes of pyrophosphate arthritis in patients with multiple medical illnesses in whom nonsteroidal anti-inflammatory drugs are contraindicated. IgG binding to CPPD crystals enhances neutrophil activation (by increasing intracellular cytoplasmic calcium levels) and seems to play a greater role in pyrophosphate than in urate crystal-induced inflammation. Transforming growth factor-beta 1 stimulates intracellular pyrophosphate generation by articular chondrocytes. The effect is inhibited by probenecid, an anion transport blocker, through interfering with the active transport of intracellular pyrophosphate to cartilage matrix where crystals form. Calcific tendinitis associated with underlying cortical bone erosions is an uncommon manifestation of apatite crystal deposition disease. Closed-needle tidal irrigation proved beneficial in two patients with Milwaukee shoulder syndrome. Two patients with nodular subcutaneous cholesterol crystal deposition are described. PMID- 7547118 TI - [Peritoneal mesothelioma]. AB - Peritoneal mesothelioma is a quite rare tumor with an incidence of one or two cases per million inhabitants; approximately in the 30-45% of the cases diagnosed it is associated with the corresponding pleural mesothelioma. A predisposing factor is a previous occupational exposure to materials containing asbestos. Clinical symptomatology and instrumental findings may be confusing. Diagnosis is often possible only after direct vision and histologic examination during laparoscopy or laparotomy but it is always tardy, whereas therapy is still unsuccessful. Surgical excision and radiotherapy are less effective than intraperitoneal chemotherapy which seems to lengthen the average survival rate. PMID- 7547119 TI - [Choledochoduodenostomy in the treatment of benign disease of the common bile duct: evaluation of long-term results]. AB - The Authors report their experience in the treatment of 36 patients managed by side-to-side choledochoduodenostomy for common bile duct disease and followed for a period of five years. The main indication to surgery was a choledochal dilatation more than 1.5 cm. The surgical technique consisted in a single layer side-to-side choledochoduodenostomy with absorbable suture according to Roessner. There was no operative mortality and few post surgical complications (six patients, 15.78%) were registered; 25 patients (69.45%) were completely asymptomatic at five years, while in six cases (16.66%) significant symptoms were observed. In one case a biliary gastropathy with no metaplasia was documented. PMID- 7547120 TI - [Large cystic polyps of the stomach]. AB - The Authors report the case of a 83 year old woman with large cystic polyps of the stomach. Gastric cystic polyps are polypoid lesions which may develop following functional disorders such as increased mucosal stimulation by gastrin, or excessive retention of gastric secretions. The incidence of these polyps is variable, depending on their being underestimated since they are not always macroscopically visible. The case here reported is very interesting not only for the number of lesions (over 50), but also for their size (5-25 mm in diameter). PMID- 7547121 TI - [Video laparoscopic cholecystectomy: our experience]. AB - The Authors report their experience of laparoscopic cholecystectomy (VLC) from October 1993 to May 1994. Fourty patients underwent laparoscopic cholecystectomy for gallstone disease; two patients required laparotomic conversion for Mirizzi's syndrome, while complications were observed in two cases. Only one case required laparotomy in third post-operative day for duodenal electrocautery injury. Diagnostic procedures and surgical technique are also reported. On the basis of their experience, the Authors conclude that mini-invasive surgery doesn't mean less difficult surgery, but a good knowledge of the anatomy and a thorough experience of biliary open and reparative surgery are necessary before laparoscopic cholecystectomy is performed. PMID- 7547122 TI - [Complete regression of Barrett's esophagus after drug therapy]. AB - Barrett's esophagus is defined as a disorder in which the normal stratified squamous mucosa of the esophagus is replaced by columnar epithelium. Patients with Barrett's esophagus are at risk to develop an adenocarcinoma of the esophagus. Pathologic gastroesophageal reflux is correlated to the disease and therapeutical options must aim to stop this noxa in order to prevent columnar metaplasia, and subsequent dysplasia and/or neoplasia. The Authors report the case of a patient in whom the complete regression of the metaplasia was observed after medical therapy. PMID- 7547123 TI - [Cholelithiasis and diabetes]. AB - The occurrence rate of diabetes was analyzed in 3370 consecutive patients admitted and surgically treated for biliary lithiasis at the I Department of Surgery-University of Rome "La Sapienza"-between 1959 and 1993. The incidence of such association was found to be overlapping with that of normal population. The natural history and the outcome of patients surgically treated for biliary lithiasis do not seem to be burdened by a concurrent diabetes. Diabetes was in turn found to be correlated with a more frequent presence of stones in the biliary tract. Possible causes of this correlation are discussed. The need in these patients of a more accurate diagnostic approach and specific therapeutic solutions is stressed. PMID- 7547124 TI - [Laparoscopic treatment of hepatic cysts]. AB - Laparoscopic treatment of simple hepatic cysts is reported. Recent indications to conservative surgical treatment for this benign disease are considered and different therapeutic options are analyzed. Surgical technique adopted in two cases observed is illustrated. In conclusion, the Authors consider laparoscopic treatment as an effective method for a mini-invasive surgical approach. PMID- 7547125 TI - [Rehabilitation of the colostomy patient]. AB - The Authors report their experience concerning the rehabilitation of patients with temporary or definitive colostomy. Mechanical and psychosocial implications as well as different rehabilitative methods are discussed and literature data are reviewed. Surgery should play a major role in this rehabilitation programme, either in terms of prevention or definitive treatment; nonetheless only through a multidisciplinary approach these patients will achieve a better quality of life. PMID- 7547126 TI - [The Trabucco tension-free hernioplasty: our experience and technical considerations]. AB - The Authors report their experience in the management of 30 patients undergone tension free hernioplasty according to Trabucco for inguinal hernia. Results and some modifications of the original technique are discussed. PMID- 7547127 TI - [Surgery and blood transfusion]. AB - Advances in surgery and the extension of the surgical indications have amplified the problems due to hemorrhage, main side effect, and its treatment, which in the past consisted primarily in whole blood transfusion. Such practice, however, involves the surgeon in a series of problems due to shortage of blood donors, religious beliefs and most of all the risks related to the transfusion practice itself. Apart from early and late reactions, the risk of transmission of infective diseases, post-transfusional immunodepression and legal problems must be pointed out. Recently, to solve these problems, the indications to blood transfusion have been restricted to severe hypovolemic shock and severe untreatable hypoxia; the separate use of blood components has been privileged; and autologous blood transfusion techniques like pre-deposit for donation, normovolemic preoperative hemodilution and intraoperative autologous transfusions have been used. It's mandatory that the surgeon keeps in date with the Transfusional Medicine progresses working in strict collaboration with the Transfusional Services to best protect the good health of the patients he has in care. PMID- 7547128 TI - [Embolic complications in atrial fibrillation. Data of the Studio Italiano Fibrillation Atriale (SIFA)]. PMID- 7547129 TI - [New technologies and carotid surgery]. PMID- 7547130 TI - Surgical management of reflux esophagitis associated with duodenal ulcer. AB - The prevalence of associated reflex esophagitis and the effects of surgery for ulcer on coexistent esophagitis were assessed in 687 patients operated on for duodenal or pyloric ulcer. Eighty-one patients (12%) had a preoperative endoscopic diagnosis of reflux esophagitis. The association resulted to be more frequent in patients with pyloric or bulbar stenosis than in patients without stenosis (20% vs 5%, p = 0.000001). Billroth II gastric resection, performed in most cases, caused the healing or the improvement of esophagitis in 95% of cases as demonstrated at endoscopy 6 months after surgery, particularly in 98% of patients with pyloric or bulbar stenosis and in 80% of patients without stenosis (p = 0.05). Therefore, gastric resection, eliminating the main pathogenetic factors of reflux esophagitis associated with pyloric or bulbar stenosis (gastric acid hypersecretion, impaired gastric emptying), assures the healing of esophagitis in most cases. However, anomalies in Lower Esophageal Sphincter function might play an important role in the pathogenesis of reflux esophagitis in duodenal ulcer patients without stenosis. In these patients, on the basis of manometric and pH monitoring data, it may be useful to associate an anti-reflux procedure or a duodenal diversion with a gastric resection in presence of Lower Esophageal Sphincter hypotonia. PMID- 7547131 TI - [Tracheal resection-anastomosis in carcinoma of the thyroid infiltrating the airways. A case report]. AB - The Authors report a case of thyroid carcinoma with tracheal involvement. Since the neoplastic invasion of the trachea was 3 cm long total thyroidectomy with sleeve tracheal resection and immediate reconstruction with end-to-end anastomosis were performed. The diagnostic and therapeutic approach is discussed and the Literature data reviewed. PMID- 7547132 TI - [Octreotide in surgery]. PMID- 7547134 TI - [Our experience with the treatment of duodenal perforation with suture]. AB - The Authors report their experience in the treatment of perforated duodenal ulcer retrospectively analysing 42 patients treated with simple suture on emergency. Morbidity was 30.9% and mortality (operative and postoperative) was 16.6%, both significantly influenced by the age of the patients, the associated medical disease and the presence of shock. Results of the follow up concerning 33 patients are also reported: in 57.5% of the cases simple suture combined with medical therapy was able to resolve the peptic disease. The analysis of the results shows that the procedure is indicated for acute duodenal ulcers and for elderly patients with associated diseases. PMID- 7547133 TI - [Surgical treatment of primary carcinoma of the female breast in geriatric age. Retrospective studies of 190 cases]. AB - On the basis of a review of 190 cases of breast cancer in elderly patients, the Authors discuss clinical and anatomopathological features for a proper surgical strategy. After an accurate evaluation of the operative risk and stage of the neoplastic disease, conservative surgical techniques or as less demolitive as possible, i.e. quadrantectomy with axillary lymphadenectomy, Madden or Patey's modified mastectomies, with respect for oncological radicality, are recommended in the elderly. The importance of an early diagnosis in reducing the frequency of locally advanced neoplasms (typical in the elderly) which if operable require Halsted's procedure, is furthermore emphasized. PMID- 7547135 TI - [Calculosis of the appendix: report of a case]. PMID- 7547136 TI - [Clinical and therapeutic features of MEN II]. AB - Multiple Endocrine Neoplasia type 2 is a genetically transmitted poliendocrinopathy, in which pheochromocytoma (PCC), medullary thyroid carcinoma (MTC) and parathyroid disease may occur and coexist with different rates. Although the clinical behaviour of each component is well known and usually predictable, controversies still exist on the most appropriate therapeutical strategy when patients exhibit synchronous lesions; many elements may influence this choice: the evidence of bilateral (PCC) or multicentric (MTC) lesions, the evaluation of their malignancy, the adoption of therapies other than surgery (alpha receptor blockers in PCC). In this paper the Authors report their experience in diagnosing and treating MEN 2; it is based on 5 patients operated from 1972 to 1993 and on the screening of their relatives for thyroid and/or adrenal lesions. The individual manifestations of the disease, with regard to time and extent of organ involvement, are considerably variable; however, an aggressive approach of MTC, even in presence of a synchronous PCC, as well as a unilateral adrenalectomy (with a close follow up of the patient) when surgical exploration does not show an involvement of the remaining gland, is suggested. PMID- 7547137 TI - [Peritoneal echinococcus: report of a case]. AB - The Authors report a case of peritoneal echinococcal cyst and discuss the pathogenetic and clinical aspects as well as the diagnostic and therapeutic approaches of such a particular localization. They underline the need of a radical surgical treatment in order to definitively eliminate the parasite and the disease. PMID- 7547138 TI - [Treatment of differentiated carcinoma of the thyroid and its limitations]. AB - The Authors report their experience in the management of differentiated thyroid cancer (DTC) with extrathyroidal extension including nodal and distant metastases. Twelve patients were included in the review: 11 of them were previously and elsewhere considered not curable. On the contrary, integrated treatment (surgery, radioiodine and radiation therapy) allowed to control the disease in 10 patients with a survival ranging from 14 years to 29 months. Cytopathologic analysis, in the two patients who died, showed a differentiated thyroid cancer with anaplastic areas. Integrated treatment, therefore, represents the treatment of choice for advanced DTC only in total absence of anaplastic cells. PMID- 7547139 TI - [A case of xanthogranulomatous pyelonephritis. Diagnostic and therapeutic problems]. AB - The use of the modern diagnostic imaging techniques allows in most cases a diagnosis of nature of the lesions involving parenchymal organs. The authors report a case of renal xantogranuloma, rare pseudotumoral inflammatory pathology, treated by radical extended nephrectomy on the basis of a pre-operative diagnosis of malignant tumor. Despite the risk of false positives, in front of a renal mass whose nature is sufficiently characterized by the imaging techniques, the routine use of more invasive investigations such as arteriography and percutaneous needle biopsy seems not justified. PMID- 7547140 TI - [Intrahepatic cholangiojejunostomy on the third segment in non-operable neoplasms of the hepatic hilum]. AB - The authors report their experience in 19 cases of primitive or secondary tumor of the hilus of the liver which couldn't benefit from radical operation. Palliative treatments--to resolve the jaundice (which is often the only responsible for a short-term exitus)--include surgical (intrahepatic bilio digestive derivations) and non surgical procedures (PTDB, percutaneous transtumoral intubation). Considering the ephemeral results of the latter, the Authors take peripheral derivative operations into account and particularly the Soupault and Couinaud technique (intrahepatic colangiojejunostomy) performed in 7 patients with satisfying results. It is an easy and quick technique--because of constant anatomical situation of the left intrahepatic biliary distribution and of the superficial location of the duct of the III segment--which allows a lasting biliary decompression with a good residual life. PMID- 7547141 TI - [Gynecomastia: diagnosis and therapy update]. AB - The Authors report their experience in the management of gynaecomastia. Physiologic, pathologic and iatrogenic causes of the lesion, as well as possible interfering mechanisms, and diagnostic protocol are examined. When there is no spontaneous regression or when the precipitating factors are eliminated surgery is the therapeutical choice, also because it helps in resolving the related psychological problems affecting these patients. PMID- 7547142 TI - [Secondary prevention of stroke in non-valvular atrial fibrillation, study SIFA]. PMID- 7547143 TI - Effects of high levels of selenite and Escherichia coli on enrichment of Salmonella and detection of Salmonella by polymyxin-cloth enzyme immunoassay. AB - The effect of various concentrations of selenite on the recovery of Salmonella typhimurium and Escherichia coli was examined in a rich medium containing 0.5% cholate. For heat-injured S. typhimurium, longer preincubation periods were required for recovery in higher concentrations of selenite. When 0.7% selenite was added to 6-h preincubated cultures of heat-injured cells, S. typhimurium was fully recovered while E. coli, whether heat-injured or uninjured, did not recover when present at numbers at less than 2 x 10(7) colony forming units (CFU). The polymyxin-cloth enzyme immunoassay detected S. typhimurium grown from a small number (as low as 1 CFU) of cells in the presence of a large excess of E. coli. PMID- 7547144 TI - Prevalence of Bacillus cereus in selected foods and detection of enterotoxin using TECRA-VIA and BCET-RPLA. AB - Enterotoxigenic Bacillus cereus was detected in cooked foods (17), rice noodles (3), wet wheat noodles (2), dry wheat noodles (10), spices (8), grains (4), legumes (11) and legume products (3). One hundred ninety-four (42.3%), 70 (15.3%) and 23 (5.2%) of the 459 presumptive B. cereus colonies isolated from PEMBA agar were identified as B. cereus, Bacillus thuringiensis and B. mycoides, respectively. B. cereus isolates were examined for growth temperature, pH profile and enterotoxin production using both TECRA-VIA and BCET-RPLA kits. One hundred seventy-eight (91.8%) and 164 (84%) of the strains were enterotoxigenic as determined using TECRA-VIA and BCET-RPLA, respectively. Eighty-two (50%) of the enterotoxigenic strains were capable of growing at 5 degrees C, and 142 (86.6%) grew at 7 degrees C within 7 days of incubation. The enterotoxigenic strains did not grow at pH 4.0 but 69 (42.0%) of the strains were able to grow at pH 4.5 within 7 days at 37 degrees C. The isolates were resistant to ampicillin (98.8%), cloxallin (100%) and tetracycline (61.0%), and susceptible to chloroamphenicol (87%), erythromycin (77.4%), gentamycin (100%) and streptomycin (98.7%). PMID- 7547145 TI - Lactic acid inhibition of the growth of spoilage bacteria and cold tolerant pathogens on pork. AB - The antibacterial effects of a 3% solution of lactic acid at 55 degrees C were assessed, by examining aerobic bacterial growth on artificially-inoculated pork fat and lean tissue. Discs of fat or lean tissues, each of 10 cm2 surface area, were aseptically excised from pork Longissimus dorsi muscle and inoculated with the cold tolerant pathogens Listeria monocytogenes 4b Scott A no. 3, Yersinia enterocolitica 0:4,32 or Aeromonas hydrophila ATCC 7966, or with the wild type spoilage bacteria Pseudomonas fragi or Brochothrix thermosphacta. After inoculation, each meat disc was immersed in water or lactic acid for 15 s and aerobic bacterial growth followed during 15 days of storage at 4 degrees C. P. fragi and B. thermosphacta grew on both fat and lean, but the pathogens grew on fat tissue only and A. hydrophila did not survive on lean. Lactic acid reduced all test bacteria on fat to below detectable levels within 4 days of treatment and no bacteria could be recovered from acid-treated fat surfaces for the remainder of the 15-day storage interval. Bacteria attached to lean were generally more resistant to lactic acid. In some instances the acid was bacteriostatic (P. fragi, L. monocytogenes) while in others the population declined at a greatly reduced rate as compared with a similar population on fat (B. thermosphacta, Y. enterocolitica). A. hydrophila was equally sensitive to lactic acid on lean and fat. Depending upon the tested strain, tissue type and storage time, maximum reductions in the number of bacteria recovered from acid treated pork ranged from 1 to 8 log cycles. The high bactericidal efficacy of lactic acid applied to pork fat was attributable to a low tissue pH, which varied from 3.49 to 4.41 during the 15 days of aerobic storage. PMID- 7547146 TI - Enhancement of antigen-specific antibody production by extracellular slime products from slime-forming Lactococcus lactis subspecies cremoris SBT 0495 in mice. AB - The effect of extracellular slime products (ESP) produced by Lactococcus lactis subspecies cremoris SBT 0495 on antigen specific antibody production was studied in mice. ESP contained 48.5% protein, 15.4% neutral sugar, and 1.1% of phosphorus. The optimum dose of ESP was between 100 to 500 micrograms per mouse. ESP administered intraperitoneally (200 micrograms per mouse) enhanced the production of specific antibody in mice. These results indicate that ESP may act as an adjuvant. PMID- 7547147 TI - Phenotypically based taxonomy using API 50CH of lactobacilli from Nigerian ogi, and the occurrence of starch fermenting strains. AB - One-hundred and twenty isolates of lactic acid bacteria isolated from ogi and three traditional cereal-based alcoholic beverages from Nigeria, together with 18 reference strains from Swedish sour doughs, and 50 type-and reference strains of mainly Lactobacillus, were phenotypically classified on their fermentation ability of 49 carbohydrates, including soluble starch. Data were examined by Jaccard Coefficient (SJ), Simple Matching Coefficient (SSM), and unweighted pair group algorithm with arithmetic averages (UPGMA). Seven major clusters were defined at the 82% SJ-similarity level (corresponds to the SSM-level of 91%). Three were identified as Lactobacillus plantarum or L. plantarum-like (together 41% of the ogi isolates). The others were obligately heterofermentative; Cluster 7 was identified as L. confusus (11% of the ogi isolates). Three minor clusters were identified as L. murinus, L. agilis or L. gallinarium, and Leuconostoc mesenteroides subsp. mesenteroides, respectively. The phenotype of the L. plantarum isolates varied within wide limits. Seventeen isolates possessed starch fermenting capacity. Nine of these were identified as L. plantarum or L. plantarum-like (isolated from ogi); one was identified as Leuconostoc mesenteroides subsp. mesenteroides, and the rest were unidentified non-clustering strains. PMID- 7547148 TI - A method and medium for the electrical detection of Listeria spp. from food. AB - The development of a liquid medium for the detection of Listeria spp. by capacitance monitoring of food samples previously enriched in UVM 1 broth is described. Rapid growth of Listeria monocytogenes was shown to occur in liquid media with selectivity based on antibiotics found in Oxford agar. The final capacitance medium contained higher concentrations of the Oxford selective agents than Oxford agar and did not require the esculin/ferric ammonium citrate reaction to be observed. The medium relied upon the ability of Listeria spp. to induce a greater than 30% change in capacitance within 30 h. When run in parallel with the Listeria spp. test samples of a large food company, the method gave far fewer false-positive results than Fraser broth. PMID- 7547149 TI - Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus. AB - Streptococcus thermophilus 347 isolated from yogurt produces a bacteriocin referred as thermophilin 347. The bacteriocin was evidenced in the neutralized, filtered and catalase treated culture supernatant fluid of the producer strain. After partial purification, thermophilin 347 exhibited a bactericidal effect against Listeria monocytogenes and several closely related lactic acid bacteria. The activity of thermophilin 347 was lost after protease treatment but was maintained after heating at 100 degrees C for 1 h; after autoclaving at 121 degrees C for 15 min the activity was reduced by 50%. SDS-PAGE of partially purified thermophilin 347 was used to detect bacteriocin activity corresponding to an apparent molecular mass between 2.5 and 6.2 KDa. PMID- 7547150 TI - Glucono-delta-lactone and citric acid as acidulants for lowering the heat resistance of Clostridium sporogenes PA 3679 in HTST working conditions. AB - The heat resistance of Clostridium sporogenes PA 3679 spores has been studied to establish the influence of acidification with glucono-delta-lactone (GDL) and citric acid on the thermal resistance parameters (DT and z) of this microorganism and to compare their effect with phosphate buffer and natural asparagus as reference substrates. A reduction in DT values was observed in asparagus puree as the acidification level increased with both acidulants although this effect was more evident at the lower treatment temperatures studied (121-127 degrees C). Citric acid was more effective for reducing the heat resistance of spores than GDL at all of the temperatures. The reduction in pH diminished the value of the z parameter, although it was necessary to lower the pH to 4.5 to obtain a significant reduction. PMID- 7547151 TI - Fecal recovery following oral administration of Lactobacillus strain GG (ATCC 53103) in gelatine capsules to healthy volunteers. AB - Recovery of the suggested probiotic strain Lactobacillus GG in feces was studied after oral administration. Lactobacillus GG was given to 20 healthy human volunteers for 7 days in gelatine capsules with daily doses of 1.6 x 10(8) cfu and 1.2 x 10(10) cfu. All the volunteers in the higher dose group had detectable numbers of Lactobacillus GG in their feces during the test period. The strain was detected in feces of all the volunteers after 3 days of administration. No effect was observed on the total number of fecal lactobacilli. Fecal detection of the strain may facilitate dose-response studies and provide a useful tool in dietary studies utilizing the strain in foods or food-type products. PMID- 7547152 TI - ACE inhibitors after myocardial infarction. PMID- 7547153 TI - Gynaecomastia in bodybuilders. PMID- 7547154 TI - Does encouraging good compliance improve patients' clinical condition in heart failure? AB - The aim of this study was to determine whether improved compliance by intensive medication counselling, given by a pharmacist, to elderly patients with chronic stable heart failure can influence both objective and subjective measures of heart failure. Elderly patients were randomly allocated to receive a 3-month counselling programme, or no counselling. Measures recorded at the beginning and end of the study included; submaximal 6-minute exercise tests, visual analogue scores of breathlessness, the Nottingham Health Profile, and clinical signs of heart failure. Compliance was measured by a tablet count and medication knowledge assessed by means of a questionnaire. There was no significant difference between the groups in their initial level of compliance, medication knowledge or other assessments. Compliance improved for the counselled group by 32% (P < 0.001) but remained unchanged for controls. Medication knowledge improved for the counselled group only. The 6-minute exercise test improved by 20 metres from a baseline of 137 m for the counselled group (P < 0.005) but worsened by 22 m for the control group (P < 0.01). Distance to breathlessness improved for the counselled patients and worsened for controls. In contrast bodyweight, jugular venous pressure and Nottingham Health Profile scores did not change significantly for either group. Peripheral and pulmonary oedema scores improved for the counselled group (P < 0.01) but remained unchanged for controls. A small improvement was seen in the visual analogue scores (P < 0.05) for the counselled group only. Improved compliance due to intensive medication counselling had a small but measurable beneficial effect on objective measures of heart failure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547155 TI - Surgical correction of gynaecomastia in bodybuilders. AB - Gynaecomastia is a common and well-recognised side-effect of anabolic steroid abuse in athletes. A staging system is proposed and a technique of excision under local anaesthetic described. Careful selection of cases and the use of meticulous technique can achieve good cosmetic results, without risk of recurrence of gynaecomastia. PMID- 7547156 TI - Gynaecomastia in association with minocycline. PMID- 7547157 TI - First-line treatment in acute non-dysenteric diarrhoea: clinical comparison of loperamide oxide, loperamide and placebo. UK Janssen Research Group of General Practitioners. AB - The use of antimicrobial agents for the treatment of acute diarrhoea has become more common with the introduction of quinolone compounds, which are active against most types of bacterial pathogens. Despite the fact that such drugs have been used for empirical therapy or even for prophylaxis, current opinion would restrict their use to specific groups of patients who are likely to show particular benefit from them. Non-specific therapy seems a more appropriate initial treatment for cases of acute, non-dysenteric diarrhoea. Clinical trial data are presented here comparing the effects of loperamide oxide 1 and 2 mg to those of placebo and loperamide 2 mg in this condition. All the drug preparations were significantly superior to placebo, in particular reducing the time to complete relief of symptoms to about 24 hours, as opposed to 45 hours on placebo treatment. Of these preparations, loperamide oxide 1 mg is to be preferred, as it produces fewer constipation-like episodes after treatment. The introduction of loperamide oxide 1 mg represents a useful advance in the non-specific treatment of acute, non-dysenteric diarrhoea. PMID- 7547158 TI - Does haemostatic therapy improve the prognosis in upper gastrointestinal bleeding? AB - A study was conducted of 816 patients with peptic ulcer haemorrhage, comparing outcome before and after the introduction of endoscopic therapy. The control group comprised 505 patients admitted with bleeding due to benign peptic ulcer over a 5-year period before endoscopic therapy, and 311 patients after introduction of endoscopic therapy were studied prospectively. The two groups were well matched for age, sex, shock, endoscopic findings and use of ulcerogenic drugs. The introduction of endoscopic therapy was associated with a reduction in surgical intervention and mortality rates for gastric and duodenal ulcer. The beneficial effects of endoscopic therapy appear to be due to a reduction in the need for surgical intervention in patients with an ulcer base visible vessel. The authors suggest that endoscopic injection therapy may result in an improved outcome from peptic ulcer haemorrhage. Adrenaline injection treatment seems to be the treatment of choice in view of its simplicity, low cost and availability. PMID- 7547159 TI - Adult dengue haemorrhagic fever at Kuala Lumpur Hospital: retrospective study of 102 cases. AB - A retrospective study involving 102 adults with dengue haemorrhagic fever (DHF) was conducted to investigate the demographic aspect, clinical presenting features, laboratory investigations, complications, and mortality associated with the disease. The clinical diagnosis of DHF was in accordance with WHO recommendations. Epistaxis, gingivitis, haematemesis and gastritis were among the common complications. Platelet levels tended to decline from a higher value on admission (mean 67,000/mm3) to lower levels on subsequent days, with the lowest (mean 61,000/mm3) being on day 6 of the fever. Hyponatraemia (46.8%) was commonly observed. Morbidity of DHF was significant (29.4%) but the case fatality rate remained low (2.0%) in our adults, suggesting that adults are less likely than children to suffer from shock syndrome. PMID- 7547160 TI - A comparison of prescribed medication in learning disability patients before and after resettlement from hospital into the community. AB - The use of prescribed medication was examined in 34 people with learning disability who underwent a planned resettlement from a hospital into the community. No significant differences were found in the number of subjects receiving antipsychotic, antimuscarinic, antidepressant, and anticonvulsant medication, lithium preparations, and non-psychoactive medication, just prior to discharge and 1 year after discharge. This study indicates that long-stay hospitalised adults with moderate, severe or profound mental retardation can successfully be relocated into the community--in spite of suffering from behavioural and major psychiatric disorders--with the aid of appropriate medication, psychiatric follow-up, and community resources. PMID- 7547161 TI - Improving outcome after acute myocardial infarction: what is the role of ACE inhibitors? AB - Acute myocardial infarction continues to be the number 1 killer in industrialised countries. While the more widespread use of thrombolytic therapy has made a dramatic impact on patient survival, changes in long-term prognosis after discharge from hospital have not improved radically and 5-year mortality remains at over 30%. The single most important determinant of survival in the long term is left ventricular function. The process of ventricular dilatation and remodelling begins early after infarction. While such changes may initially go unrecognised clinically, without intervention progressive functional impairment will ensue and the majority of patients will develop signs and symptoms of heart failure, which carries a worse prognosis than many forms of cancer. ACE inhibitors act on both the haemodynamic and neurohormonal mechanisms in heart failure. Several large-scale clinical trials have clearly demonstrated that early use of these agents in stable patients who are not hypotensive or in cardiogenic shock can reduce significantly the development of heart failure and death. A strategy for the early initiation of ACE inhibitor therapy is proposed to improve survival in AMI patients. PMID- 7547162 TI - Heart failure in the elderly patient. AB - Both prevalence and incidence of heart failure rise with age. Diagnosis of heart failure in the older patient may be difficult because of atypical symptoms or the acceptance of symptoms as manifestations of old age. Heart failure is not a diagnosis but a syndrome. Echocardiograms should be obtained in most elderly patients to aid diagnosis and assessment. Loop diuretics, taking into account the altered homoeostasis of old age and presence of co-morbidity, are the mainstay of symptomatic treatment. ACE inhibitors are likely to benefit survival, although formal trials have failed to include many older patients. Digoxin and direct vasodilators are less well tolerated in elderly patients. PMID- 7547163 TI - Current aspects in the management of pneumonia. AB - Changes are occurring in our approach to the management of patients with pneumonia due to changes in our understanding of the pathogenesis of pneumonia, changes in the causative pathogens, and changes in the investigative techniques and therapies available. This article reviews the management of patients with pneumonia, highlighting some of these areas. PMID- 7547164 TI - Haemolytic anaemia complicating Epstein-Barr virus infection. AB - Hepatitis is a well-recognised complication of Epstein-Barr virus (EBV) infection that usually resolves spontaneously. Jaundice occasionally results from the unusual complication of autoimmune haemolytic anaemia rather than hepatitis. Two adult patients are described with infectious mononucleosis-related hepatitis who also developed significant haemolysis, a situation that could have led to diagnostic delay and a deleterious outcome. Haemolysis should be considered in all patients who become jaundiced after infectious mononucleosis. The management of this potentially fatal complication is discussed. PMID- 7547165 TI - Severe reversible arterial spasm with ergotamine. AB - A patient is described with marked unilateral arterial spasm of the proximal vessels and severe ischaemia of the left foot as a result of excessive use of ergotamine tartrate. Treatment with intravenous heparin infusion followed by chemical sympathectomy was successful. PMID- 7547166 TI - Diffuse hepatic haemangiomatosis in the elderly. PMID- 7547167 TI - Acute appendicitis presenting with surgical emphysema and pneumomediastinum. AB - A case is reported of a young man presenting with abdominal pain and surgical emphysema of the neck. A chest x-ray revealed pneumomediastinum but other investigations excluded oesophageal rupture. He was found to have a complicated acute appendicitis. The history and likely causes of this unusual presentation of a common complaint are reviewed. PMID- 7547168 TI - Persistent electrocardiographic changes after flecainide overdose. AB - Flecainide acetate is a potent class 1C antiarrhythmic agent, overdosage with which is rare. An overdose is reported in a patient on long-term therapy, resulting in life-threatening arrhythmias and prolonged electrocardiographic abnormalities. PMID- 7547169 TI - Platelet storage pool deficiency in pregnancy. PMID- 7547170 TI - Median nerve entrapment after elbow dislocation: a new radiological sign. PMID- 7547171 TI - Recurrent bilateral shoulder dislocation resulting from dyskinesia associated with dopaminergic stimulation therapy. AB - An 80-year-old woman with Parkinson's disease developed recurrent bilateral shoulder dislocations. The dislocations occurred during dopaminergic treatment which induced severe dyskinesia. The inability to ensure immobilisation of the limb indicates a conservative approach. PMID- 7547172 TI - Pituitary microadenoma in Turner's syndrome. AB - A case of Turner's syndrome with a pituitary microadenoma is reported. Various implications of the diagnosis are discussed. PMID- 7547173 TI - Membrane potential of smooth muscle cells of human placental chorionic vessels. Comparative effects of MgCl2 and MgSO4. AB - Magnesium has been shown to produce various effects on vascular smooth muscle, in that its absence elicits an increase in muscle tone and increases sensitivity to a number of stimulatory agonists. In the present study, the influence of MgCl2 and MgSO4 was investigated in human placental chorionic muscle cells (from arteries and veins with or without endothelium), especially on the membrane potential, Um. Classically, Um was obtained from microelectrodes inserted into smooth muscle cells through the endothelium or directly. In arteries with endothelium, the smooth muscle cells were depolarized by MgSO4 (threshold: 6 mM) and MgCl2 (threshold: 8 mM). In endothelium-free arteries, the smooth muscle cells were also depolarized by MgSO4 (threshold: 8 mM) and MgCl2 (threshold: 6 mM). Identical results were obtained with veins, but thresholds were different. The different thresholds may indicate that MgCl2 influences the cell membrane potential directly, while MgSO4 interferes first with the endothelial cells, which may act as an intermediary between the Mg2+ ions and the membrane of the smooth muscle cells. PMID- 7547174 TI - Effect of magnesium on secretion of platelet-derived growth factor by cultured human umbilical arterial endothelial cells. AB - Conditioned media were prepared by incubating human cultured umbilical arterial endothelial cells for 48 h in magnesium (Mg) sufficient (900 microM) and deficient (100 microM) conditions. Minimum essential media (MEM) are designated as [900]- and [100]-MEM, respectively. After the incubation, a portion of the [100]-MEM media was adjusted from 100 to 900 microM magnesium ([100-900]-MEM). Smooth muscle cells were incubated with the three media and their growth rates were determined by [3H]-thymidine incorporation and cell counting. The growth rate in [100-900]-MEM was significantly higher than in [900]- or [100]-MEM. When platelet-derived growth factor (PDGF) was neutralized by the addition of a mixture of anti-PDGF-AA and -BB, [3H]-thymidine incorporation in [100-900]-MEM decreased by 12.5 per cent, but only by 4.9 per cent in [900]-MEM. These results indicate that magnesium deficiency increases the secretion of PDGF by endothelial cells. This is also supported by the results of the radioimmunoassay for PDGF-BB; the quantity of PDGF in the magnesium-deficient media was greater than in the magnesium-sufficient media. PMID- 7547175 TI - The effects of magnesium deficiency on DNA and lipid synthesis in cultured human umbilical arterial endothelial cells. AB - This study investigated the effects of magnesium deficiency on thymidine incorporation and lipid synthesis in human umbilical arterial endothelial cells. To study [3H]thymidine incorporation, the cells were exposed for 12 to 96 h to experimental media containing decreasing magnesium concentrations of 237, 118 and 5 microM. A magnesium concentration of 949 microM was used as control. At 48 h of exposure to the experimental media, magnesium was restored to 949 microM in half of the magnesium-deficient cultures. To determine [14C]acetic acid distributions among cholesterol, phospholipid and triglyceride, the cells were treated with decreased magnesium media at the levels mentioned above for 12 to 48 h. The results showed that [3H]thymidine incorporation into DNA was inhibited by magnesium deficiency. The inhibition was augmented correspondingly with decreased magnesium concentrations and increased exposure periods. When magnesium in the medium was enhanced to normal level, incorporation was no longer inhibited. Incorporation of [14C]acetic acid into phospholipid was inhibited, its incorporation into triglyceride was stimulated, but its incorporation into cholesterol was not affected by magnesium deficiency. We suggest (i) that with the restoration of adequate magnesium, the inhibiting effect of magnesium deficiency on thymidine incorporation is reversible; (ii) that magnesium deficiency can result in a redistribution of [14C]acetic acid between phospholipid and triglyceride. PMID- 7547176 TI - Triglyceride-rich lipoproteins from magnesium-deficient rats are more susceptible to oxidation by cells and promote proliferation of cultured vascular smooth muscle cells. AB - An important characteristic of hyperlipaemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins (TGRLP). The present investigation was performed to determine the susceptibility of TGRLP isolated from magnesium-deficient rats to metal ion and cell dependent peroxidation and the effect of these lipoproteins on cultured vascular smooth muscle cells (VSMC). TGRLP were isolated by sequential ultracentrifugation from plasma of control and magnesium-deficient rats fed for 8 d on adequate or magnesium-deficient diets. Magnesium-deficient animals were hypertriglyceridemic as compared to control rats. After exposure to oxidative stress in vitro, Cu2+ or activated macrophages, conjugated diene production was significantly higher in TGRLP isolated from magnesium-deficient rats. Culture of VSMC with TGRLP from magnesium-deficient rats resulted in more important cell growth than with lipoproteins isolated from control animals. After incubation with VSMC, TGRLP from magnesium-deficient rats showed higher conjugated diene production than those from control rats. These results support the atherogenic properties of magnesium deficiency, which is accompanied by hyperlipaemia and which affects two important linked pathways in atherosclerosis, lipoprotein peroxidation and VSMC proliferation. PMID- 7547177 TI - Effect of dietary magnesium on the susceptibility of mice to infection by protozoan parasites of the Apicomplexa and Mastigophora phyla. AB - Severe magnesium deficiency protects mice against infections by Plasmodium Spp. and Babesia hylomysci which invade mature erythrocytes. By contrast severe magnesium deficiency does not protect against parasite infections by P. berghei which invades reticulocytes, Toxoplasma gondii which invades macrophages, and Trypanosoma brucei which lives free in blood. The results indicate that the infectious response depends on the severity of magnesium deficiency and on the parasite species. The decrease in red blood cell magnesium and increased oxidant stress are possible explanations for the protective effect of magnesium deficiency. PMID- 7547178 TI - Plasma, cytosolic and membrane magnesium content in renal insufficiency. AB - Plasma magnesium, cytosolic (lymphocytic) and membrane (erythrocytic) magnesium concentrations were determined in 15 controls with normal renal function compared to 12 patients with renal insufficiency (chronic glomerulonephritis, serum creatinine 2.5 +/- 0.8 mg/dl, mean +/- SD). Plasma magnesium concentrations were determined by atomic absorption spectroscopy (AAS), cytosolic free magnesium with the fluorescent indicator Mag-Fura-2, and membrane magnesium by AAS, referred to membrane protein content which was determined according to Bradford's method. Plasma magnesium was 0.91 +/- 0.08 mmol/litre in controls versus 0.95 +/- 0.09 mmol/litre in renal-insufficient patients. Cytosolic free magnesium content was 2.38 +/- 0.75 mmol/litre in controls and 2.61 +/- 0.35 mmol/litre in the renal insufficient group. In the renal-insufficient group membrane magnesium concentrations were found to be significantly increased as compared to the control group (2.85 +/- 0.62 vs 0.53 +/- 0.22 mmol/g membrane protein, mean +/- SD, P < 0.05). The results show that cell membranes may be of special importance in renal insufficiency in avoiding magnesium overload of the cell and in keeping free cytosolic magnesium stores stable. PMID- 7547179 TI - Salicylate poisoning: two-dimensional J-resolved NMR urinalysis. AB - Identification of a case of acute salicylate intoxication using 300 MHz 1H NMR spectroscopy of a urine sample is reported. It has been achieved by using a combination of a one-dimensional experiment with water presaturation and a two dimensional homonuclear J-resolved experiment. By these means, lysine and the three major metabolites of acetylsalicylic acid have been assigned in the crude urine. The results are compared with those obtained at 600 MHz and with classical biochemical methods. The use of this method for routine diagnosis in biological analysis is discussed. PMID- 7547180 TI - Preliminary observations of transverse relaxation rates obtained at 3 tesla from the substantia nigra of adult normal human brain. AB - The transverse relaxation rates R2 and R2* were measured at 3 T in the substantia nigra of the midbrain of adult normal human controls. The relaxation rate arising from magnetic inhomogeneities, R2', was calculated from the relationship R2* = R2 + R2'. No significant differences were found for any parameters between left and right side substantia nigra regions, however, a significant inverse correlation was found between R2 and R2' (r = 0.70, p = 0.035). Water diffusion, in the presence of paramagnetic metal ions, may play a role in determining the relative contributions to reversible and irreversible transverse relaxation and may explain the inverse correlation demonstrated here. PMID- 7547181 TI - A 31P NMR investigation into the effects of repeated vascular occlusion on uterine metabolites, intracellular pH and force, in vivo. AB - Little is known about the metabolic effects of ischaemia on high energy phosphates in vivo in smooth muscle. We have developed a method for reversibly occluding the uterine artery, which allows simultaneous measurement of uterine metabolites using 31P NMR spectroscopy, and intra-uterine pressure, in vivo during ischaemia. We have investigated the effects of repeated ischaemia on metabolites, intracellular pH and contractions in anaesthetized rats. Occlusion produced an immediate drop in uterine blood flow and decreased contractions. Although contractions recovered upon reperfusion after both occlusions, the contractile activity was less after the second period of occlusion, suggesting less resistance after a prior ischaemic period. Significant falls in [ATP] and [phosphocreatine] and an increase in [P(i)] occurred during both occlusions. These were all reversed within 30 min of reperfusion. There was a large drop in intracellular pH produced by occlusion, which was rapidly reversed upon reperfusion. The changes in metabolites and intracellular pH were similar during the repeated ischaemic period, to those occurring during the first ischaemic period suggesting no alteration in energy production or utilization had occurred, with prior exposure to ischaemia. The significance of these results to the functioning of the uterus in labour is briefly discussed. PMID- 7547182 TI - Long-term observation of in vivo 31P NMR spectra in carbon tetrachloride intoxicated rabbit liver using implanted wireless surface coil. AB - Phosphorus-31 NMR spectra in rabbit liver were successively observed for more than 5 months in a chronic CCl4 intoxication model using implanted wireless surface coils. The quality of the obtained NMR spectra was satisfactory and there was sufficient time resolution to follow the dynamic changes that occurred during a fructose tolerance test. The implanted coil did not lead to any serious adverse effects such as infection or liver dysfunction. During an 18-week administration of CCl4, the area of the phosphodiester peak significantly decreased. The ratio of P(i)/beta-ATP was transiently elevated and gradually returned to the control level. In the fructose tolerance test, the increase in phosphomonoester and the decrease in P(i) and beta-ATP after i.v. fructose loading were apparent, but the degrees in these changes became smaller during the CCl4 intoxication. This wireless surface coil was useful for consecutive and non-invasive 31P NMR observations of the liver to follow chronically treated animal models over a period of several months. PMID- 7547183 TI - 1H MR visible lipids in colon tissue from normal and carcinogen-treated rats. AB - Metabolic characteristics of colon mucosa, submucosa, muscularis and tumour specimens from four control (n = 105) and nine carcinogen (azoxymethane)-treated (n = 91) Sprague-Dawley rats were investigated by ex vivo 1H MRS. Ninety-seven per cent of pure mucosa samples (n = 59) yielded spectra with narrow lipid resonances (chemical shift delta of -(CH2)n-, 1.3 ppm; linewidth at half-height v1/2, 30-50 Hz). Eighty-two per cent of control mucosa samples with histologically proven submucosa contamination (n = 11) and 46% of control cross sections (containing mucosa, submucosa and muscularis; n = 57) yielded spectra with broad lipid resonances (delta-(CH2)n-, 1.5 ppm; v1/2, 80-100 Hz) identical to those of adipose tissue surrounding rat colon. Thirty per cent of tumour samples (n = 10) yielded spectra with narrow lipid resonances while 70% contained no significant amount of MR visible lipids. We conclude that (i) lipids giving rise to broad resonances are in the heterogeneously distributed adipocytes of submucosa, (ii) lipids giving rise to narrow resonances are within the mucosa in an unknown structural environment, and (iii) the type and distribution of lipids in human and rat colon are similar. Tumours contained significantly more taurine than pure control mucosa (n = 15; p < 0.004) and pure mucosa containing aberrant crypt foci (putative preneoplasm, n = 36; p < 0.002). Our results suggest that the rat colon is a good model for 1H MR investigations of human colon carcinogenesis. PMID- 7547184 TI - Signal changes in gradient echo images of human brain induced by hypo- and hyperoxia. AB - The effect of hypoxia (inspired oxygen fraction, FiO2 of 10% and 16%) and hyperoxia (FiO2) of 100%) on gradient echo images of the brain using long echo times was investigated in six healthy volunteers (age 24-28 years). Different flip angles were used with an FiO2 of 10% to assess the importance of saturation effects. The total cerebral blood flow was measured by a phase mapping technique during normoxia as well as hypoxia (FiO2 of 10% and 16%) and hyperoxia (FiO2 of 50% and 100%). High relative signal changes were found, independently of the flip angle, with FiO2 of 10%. With a flip angle of 40 degrees the values of delta R2* for cortical grey matter, central grey matter, white matter and the sagittal sinus were 0.79, 0.41, 0.26 and 3.00/s; with a flip angle of 10 degrees the corresponding values were 0.70, 0.37, 0.24 and 3.15/s. The total cerebral blood flow increased by 41% during inhalation of 10% O2 and decreased by 27% during 100% O2; no flow changes were seen during moderate changes in FiO2. It is concluded that flow effects play a minor role for fMRI signal strength in this application since (i) they did not abolish the signal changes caused by changes in blood oxygenation during hyper- and hypoxia, (ii) the observed signal changes were closely related to the changes in arterial oxygen saturation during hypoxia and (iii) the signal changes were little affected by changing the flip angle from 40 degrees to 10 degrees. PMID- 7547185 TI - Transmural distribution of 2-deoxyglucose uptake in normal and post-ischemic canine myocardium. AB - This investigation was performed to determine (i) whether 31P spatially localized 31P NMR spectroscopy could be utilized to determine the transmural distribution of 2-deoxyglucose (2DG) uptake in the in vivo canine heart and (ii) whether transmural 2DG uptake would be affected by a preceding ischemic insult. 2DG was infused and the accumulation of 2-deoxyglucose-6-phosphate (2DGP) was monitored (by means of spatially localized 31P NMR) in control hearts, in pharmacologically hyperperfused hearts, and in hearts subjected to four (5 min) occlusions of the left anterior descending coronary artery. Myocardial blood flow was measured with radioactive microspheres. In control hearts, subendocardial (ENDO) 2DGP contents were significantly higher than those in the subepicardium (EPI) being 3.8 +/- 0.3 and 2.2 +/- 0.2 mumol/g, respectively; the ENDO/EPI ratio of 2DGP was 1.70 +/- 0.21. During hyperperfusion blood flow increased approximately four-fold but 2DGP accumulation was not altered. ATP levels in post-ischemic myocardium were significantly decreased (ENDO more than EPI) and 2DGP accumulation in each layer was increased (p < 0.01 vs control); however, the ENDO/EPI ratio of 2DGP was not altered. 2DG infusion induced a marked elevation of blood insulin and norepinephrine levels. These data demonstrate that in the presence of high blood levels of 2DG and insulin: (i) 2DGP accumulation can be measured in the in vivo canine heart; (ii) in normal hearts 2DG uptake is more pronounced in the inner layers of the left ventricular wall (this transmural 2DG uptake gradient is not due to subendocardial hypoperfusion); and (iii) 2DG uptake is greater in the post ischemic heart but the ENDO/EPI gradient of 2DG uptake is not altered indicating that the more severe ischemic insult in the subendocardium does not result in a disproportionate increase in 2DG uptake in that region of the myocardium. Although 2DG uptake patterns in this model most probably reflect those of glucose (at comparable glucose and insulin levels), quantitative extrapolations with regard to the rate of glucose uptake are not possible from the present data. PMID- 7547186 TI - Experimental allergic encephalomyelitis in non-human primates: MRI and MRS may predict the type of brain damage. AB - Volume-localized proton spectroscopy and T2-weighted MRI were performed on 23 monkeys with experimental allergic encephalomyelitis (EAE). The purpose of this study was to determine the relationships between temporal changes in lesion activity (measured on T2-weighted MRI), MRS [N-acetyl aspartate (NAA), creatine (CR), choline (CHO)], and the histologic definition of disease determined post mortem. Animals were scanned in the same areas of the brain once a week before and after sensitization to myelin basic protein (BP). Histologic lesion types were predicted by a combination of preceding MRI and MRS measurements. Acutely fatal EAE lesions were large and monophasic as visualized by MRI, and increased CHO (p < 0.02, n = 16) and CHO/CR ratio (p < 0.001, n = 16) were detected by MRS at disease onset. Chronic EAE lesions were preceded by multiple inflammatory attacks as visualized by MRI and consistently low levels of NAA (p < 0.02, n = 13) and NAA/CR (p < 0.01, n = 13) which occurred after the initial attack. MRI negative brain regions (from animals that were sensitized to BP) were associated with low CHO/CR (p < 0.1, n = 5). The temporal correlation of MRI lesion activity and absolute MRS proton metabolites shows promise for predicting the subsequent duration and histologic type of lesions in EAE in non-human primates. PMID- 7547187 TI - Simultaneous recording of EEG, DC potential and diffusion-weighted NMR imaging during potassium induced cortical spreading depression in rats. AB - Electrophysiological recording during in vivo NMR imaging for correlation of electrophysiological events with changes in NMR parameters has, until now, not been satisfactory. Here we present a method that uses specially prepared calomel electrodes for the continuous monitoring of both cortical steady potential and EEG during in vivo NMR imaging studies of experimental animal models. In order to demonstrate the reliability of this approach we elicited single episodes of cortical spreading depression in rats by intracortical potassium acetate injection, using an intracerebral micropipette. During the passage of DC potential shifts and associated reduction in EEG amplitude, hyperintense regions, reflecting the progression of cortical spreading depression over the cortex, were visible in diffusion-weighted images of the rat brains. No susceptibility artifacts due to the electrodes and micropipette were visible in the NMR data. Thus, we have demonstrated under controlled experimental conditions, that electrophysiological recording with calomel electrodes can be performed simultaneously with diffusion-weighted NMR imaging. The described method enables further NMR investigations of phenomena which are detectable by electrophysiology. PMID- 7547188 TI - 31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation. AB - The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547189 TI - Diffusion coefficients of ATP and creatine phosphate in isolated muscle: pulsed gradient 31P NMR of small biological samples. AB - Measurements of the intracellular diffusion coefficients (Di) of ATP and creatine phosphate (PCr) in stable, isolated preparations of skeletal muscle were made by means of pulsed field gradient (PFG) 31P NMR. Experiments used a PFG NMR probe specifically designed for small, superfused biological samples. This provided a magnetic field gradient in the z axis of up to 195 G/cm with minimal eddy currents. DiATP and DiPCr in white (fast, glycolytic) skeletal muscle from goldfish (Carassius auratus) were determined to be 2.48 +/- 0.33 and 3.49 +/- 0.33 x 10(-6) cm2/s, respectively, at 25 degrees C and a diffusion time of approximately 19 ms. For comparison with Di values, diffusion coefficients of ATP and PCr also were measured in solutions of ionic composition similar to that of fish muscle cytosol. The in vitro diffusion coefficients of ATP and PCr were 3.54 +/- 0.11 and 5.28 +/- 0.08 x 10(-6) cm2/s, respectively, at 25 degrees C. PMID- 7547190 TI - Water-suppressed one-dimensional 1H NMR chemical shift imaging of the heart before and after regional ischemia. AB - This study tests the hypothesis that brief periods of ischemia result in an increase in myocardial lipids during early reperfusion. We conducted 1H NMR spectroscopy to serially measure myocardial lipids before and after regional ischemia. Localized 1H NMR spectra (spatial resolution of 1.25 mm) were obtained using a one-dimensional chemical shift imaging technique. Two regions, the subendocardium and the subepicardium, were estimated by summing spectral areas from three slices (3.75 mm). Two groups of dogs that underwent a 45 min ischemia and 4 h reperfusion were studied: a group in which the myocardium beneath the surface coil underwent ischemia and reperfusion; and a group in which the ischemic event was distant from the tissue under the surface coil. Microsphere measurements showed significant blood flow reductions in the subepicardium and subendocardium in the ischemic zones during coronary occlusion. Flow returned to baseline values during reperfusion. In the ischemic zone group, the subendocardium, the triglyceride resonance areas decreased by 24% (p < 0.05) during reperfusion. However, subepicardial triglyceride areas were unchanged. Subendocardial creatine areas were also unchanged. The non-ischemic zone group subendocardial triglycerides decreased by 33% (p < 0.05) following ischemia and reperfusion in the remote region. In contrast to the ischemic group, the subepicardial triglyceride resonance areas decreased by 42% (p < 0.05). Subendocardial creatine areas were unchanged. These data show that triglycerides of the ischemic-reperfused subendocardium do not increase during 4 h of reperfusion. Furthermore, they show that the triglycerides resonance areas of the non-ischemic region decrease following remote ischemia and reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547191 TI - In vivo 31P MRS: absolute concentrations, signal-to-noise and prior knowledge. AB - Absolute metabolite concentrations have been estimated for nucleoside triphosphate and P(i) from in vivo 31P MR measurements using ISIS localization in a rat tumour model, and the results have been compared to those obtained from acid extracts of the tumours. The aim of the experiment was to assess the performance of four different spectral analysis techniques used for absolute quantitation. The spectral analysis techniques used were two frequency domain methods (peak area integration and Lorentzian fitting--FITSPEC) and two time domain methods (VARPRO and HLSVD). The spectra were acquired in blocks so that the degradation in performance of the four spectral analysis methods with decreasing signal-to-noise ratio (SNR) could be compared and referenced. This and the inclusion of a sophisticated method incorporating prior knowledge yields a more realistic and comprehensive protocol than previously published comparisons. The results suggest that VARPRO is the method of choice for quantitative analysis of tumour 31P MR spectra, giving the most reliable results at low SNR. PMID- 7547192 TI - Human papillomaviruses: their clinical significance in the management of cervical carcinoma. AB - Studies have shown a strong association between certain human papillomaviruses and the development of cervical carcinoma and its precursor lesions. The oncogenic potential of papillomaviruses has been clearly demonstrated in both laboratory animals and cultured cells. Recent advances in our understanding of viral pathogenesis have provided insights into the natural history of papillomavirus infection and subsequent development of neoplasia. A more thorough understanding of the molecular mechanisms responsible for viral oncogenesis will facilitate the development of novel preventive and therapeutic strategies to prevent and treat papillomavirus-associated cervical neoplasias. Strategies under current investigation are focusing on the induction of effective humoral and cell mediated immunity, the expression of HPV gene products, and cofactors that interact with HPV gene products to affect cell transformation. As a result of these investigative efforts, prophylactic HPV capsid vaccines and other gene therapies may soon become clinically available. PMID- 7547193 TI - Clinical trials Referral Resource. Clinical trials at the medicine branch of the NCI--Part I. PMID- 7547194 TI - Antibody therapy appears promising for cryptococcal infections. PMID- 7547195 TI - Pre-antibiotic treatments spur modern fungal infection research. PMID- 7547196 TI - NCI's cancer information systems--bringing medical knowledge to clinicians. AB - The National Cancer Institute's computerized information systems have been designed to help physicians cope with the information explosion by translating the medical literature into usable forms. Systems developed by the NCI's International Cancer Information Center provide access to a comprehensive source of bibliographic citations on cancer research (the CANCERLIT database) and to current, peer-reviewed syntheses of state-of-the-art clinical information on cancer (the PDQ database). This article describes how the PDQ databases were developed and are kept up-to-date, and how physicians and other health professionals can access PDQ and other NCI information by computer and fax. PMID- 7547197 TI - Tomotherapy: making radiation therapy more precise and target-specific. PMID- 7547198 TI - Current combined treatment of high-grade osteosarcomas. AB - The evaluation and treatment of osteosarcoma have evolved considerably over the past 2 decades, with corresponding dramatic improvements in prognosis. In large part, the improved outlook is attributable to intensive multiagent adjuvant chemotherapy, with better imaging modalities and reconstructive techniques also playing an important role. The current standard treatment for nonmetastatic osteosarcoma includes neoadjuvant chemotherapy, limb-sparing "wide" surgical resection, and reconstruction of the defect. Approximately 80% of patients are spared amputation. Limb salvage should be considered when an adequate surgical margin is attainable without significant neurovascular compromise and the surgical reconstruction is likely to provide better function than amputation. Because a good chemotherapeutic response is an important favorable prognosticator, much recent attention has been focused on predicting response preoperatively to allow closer surgical margins. Aggressive surgical treatment of pulmonary metastases has also been of measurable benefit. PMID- 7547199 TI - Health care costs: market forces and reform. AB - The cry for health care reform has diminished somewhat. Nevertheless, more changes are forthcoming. For the foreseeable future, health care will demand a rising share of resources. Earlier estimates put the proportion of health expenditures to Gross Domestic Product at almost 17% by the year 2000. Although there is no magic number regarding this proportion, its relative level in the United States strongly suggests that changes need to take place. It would appear that the population prefers that changes be instituted through the private sector, and not through extensive government involvement and regulation. PMID- 7547200 TI - Morbidity of contemporary radical retropubic prostatectomy for localized prostate cancer. AB - Complication rates in 1,000 consecutive patients who underwent radical retropubic prostatectomy for clinically localized prostate cancer between November 1989 and January 1992 were assessed and compared to complication rates in a historical group of patients operated on by primarily the same surgeons prior to 1987. In the contemporary series, there were no operative deaths, only 22% of patients required blood transfusion, and only six (0.6%) patients suffered rectal injuries. Early complications, including myocardial infarction, pulmonary embolism, bacteremia, and wound infection, occurred in less than 1% of patients. Vesical neck contracture, the most common late complication, developed in 87 patients (8.7%). At 1 year post-surgery, 80% of patients were completely continent, and fewer than 1% were totally incontinent. PMID- 7547201 TI - Role of laparoscopic techniques in colorectal cancer surgery. AB - Laparoscopic intestinal resection is a relatively new application of endoscopic technology that has evolved as a direct result of the successes and benefits seen with laparoscopic gallbladder surgery. Currently acceptable and feasible laparoscopic intestinal resections include those for diagnostic procedures, fecal diversion, Crohn's disease, diverticulitis, familial polyposis, rectal prolapse, and palliative colorectal cancer surgery. However, the efficacy of laparoscopic resection for curative cancer surgery remains a topic of much debate. Issues surrounding curative laparoscopic oncologic resection include the ability to perform an acceptable oncologic resection, the question of morbidity and mortality compared to conventional surgery, and the problem of port site recurrences. Thus, at present, curative laparoscopic oncologic surgery must be conducted within the framework of a prospective, randomized clinical trial, which includes full informed patient consent. PMID- 7547202 TI - Why oncologists should get involved in practice guideline development. ASCO Health Services Committee Program. PMID- 7547203 TI - Biologic and clinical advances in multiple myeloma. AB - Recent advances in our understanding of the cellular and molecular derangements involved in multiple myeloma are beginning to be translated into novel therapeutic approaches. Growth factors, specifically interleukin-6, appear to be critical for disease progression, and interruption of autocrine and paracrine loops has been achieved, with resultant inhibition of myeloma cell growth. Oncogenes, tumor-suppressor genes, and cell-survival genes have all been found to be dysregulated in some myeloma patients. The implications of acquisition of the multidrug resistance phenotype are just beginning to be understood. High-dose therapeutic regimens with bone marrow or peripheral stem-cell rescue are being studied in an attempt to produce a cure. Autologous marrow and peripheral blood stem-cell transplantation are better suited to the older myeloma patient population than is allogeneic marrow transplantation, and have also yielded promising results. PMID- 7547204 TI - Current management of acute lymphoblastic leukemia in adults. AB - Intensive remission chemotherapy followed by post-remission consolidation and maintenance therapies has achieved complete remission rates of 75% to 90% and 3 year survival rates of 25% to 50% in adults with acute lymphoblastic leukemia (ALL). These results, although promising, are still less favorable than those achieved in childhood ALL. However, various novel experimental and clinical approaches show promise for improving cure rates. Also, specific therapies directed at high-risk subgroups with ALL are beginning to emerge. Detection of specific chromosomal abnormalities at diagnosis identifies patients who are at risk of failing to achieve remission, as well as those who are likely to have short, intermediate, or prolonged disease-free intervals after successful remission induction. Such prognostic information may, ultimately, be used to assign risk categories and to individualize post-remission therapy. PMID- 7547205 TI - Clinical trials referral resource. Clinical trials at the surgery branch of the NCI. PMID- 7547206 TI - Ebola haemorrhagic fever. PMID- 7547207 TI - Dengue and dengue haemorrhagic fever. PMID- 7547208 TI - Veterinary public health--zoonoses control. Outbreak of infection with Salmonella enteritidis phage type 21 associated with shell eggs. PMID- 7547209 TI - Viral haemorrhagic fever. Management of suspected cases. PMID- 7547210 TI - Expanded programme on immunization. Diphtheria immunity in the adult French population. PMID- 7547211 TI - Intracellular metabolites of mercaptopurine in children with lymphoblastic leukaemia: a possible indicator of non-compliance? AB - As part of a programme assessing the pharmacokinetics of oral thiopurines given for lymphoblastic leukaemia, we assayed intracellular metabolites of mercaptopurine in children from all over the United Kingdom who were given a standard dose of the drug. The metabolites we measured, thioguanine nucleotides and methylmercaptopurines, are products of two competing metabolic pathways and would be expected to show an inverse correlation. A total of 327 children from 17 centres in the UK were studied. All were on the same therapeutic schedule of mercaptopurine. All had been on an unattenuated full protocol-directed dose (at least 75 mg m-2) for a minimum of 7 days before assay. There was a very wide variation in the concentration of the two metabolites measured; the thioguanine nucleotides ranged from 0 to 1255 pmol per 8 x 10(8) red cells (median 289, lower quartile 210, upper quartile 377) and the methylmercaptopurine metabolites ranged from 0 to 46.3 nmol per 8 x 10(8) red cells (median 5.18, lower quartile 2.31, upper quartile 11.59). The anticipated negative correlation was not apparent, but the ratio between the two was not randomly distributed. No child had both metabolite concentrations in the upper quartiles, but in 32 (10%) children the concentration of both metabolites was in the lower quartile. Of the 32, only one metabolite was detected in four and none at all in six. The most likely explanation for these findings is that a minority of children with lymphoblastic leukaemia fail to take oral mercaptopurine either totally or intermittently. The extent of the problem is unknown, but we suspect it may be clinically important in at least 10% of patients. PMID- 7547212 TI - Endocrinological and clinical evaluation of exemestane, a new steroidal aromatase inhibitor. AB - The androstenedione derivative, exemestane (FCE 24304), is a new orally active irreversible aromatase inhibitor. Fifty-six post-menopausal advanced breast cancer patients entered this study to evaluate the activity of four low exemestane doses in reducing oestrogen levels. The drug's tolerability and clinical efficacy were also assessed. Exemestane was orally administered to four consecutive groups at daily doses of 25, 12.5, 5 and 2.5 mg, and the changes in oestrogen, gonadotrophins, sex-hormone binding globulin and dehydroepiandrosterone sulphate levels were evaluated. Drug selectivity was studied by measuring 17-hydroxycorticosteroid urinary levels. After 7 days of treatment, mean oestrone and oestradiol levels had decreased by respectively 64% and 65% (a decrease which was maintained over time); in the 2.5 mg group, oestrone sulphate levels also decreased by 74%. Gonadotrophin levels were significantly higher, whereas no changes in the other serum hormone levels or any interference with adrenal synthesis were detected. Treatment tolerability was satisfactory: nausea and dyspepsia were reported in 16% of patients. The overall objective response rate was 18%. In conclusion, exemestane is effective in reducing oestrogen levels at all of the tested doses and shows interesting clinical activity. PMID- 7547213 TI - Comparative efficacy of a single oral dose of ondansetron and of buspirone against cisplatin-induced emesis in cancer patients. AB - Buspirone, an agonist of the 5-HT1A subtype of serotonin receptors, has shown antiemetic activity in animal models. However, in cancer patients treated with cisplatin, ondansetron, given either i.v. (one 8-mg dose 30 min after cisplatin) or orally (one 16-mg dose at the end of cisplatin infusion) was superior (P < 0.001) to buspirone (60 mg p.o. at the end of cisplatin and 60 mg p.o., 30 min later), in all parameters of antiemetic efficacy. These results are in favour of 5-HT3 receptors, but against the participation of 5-HT1A receptors in acute emesis associated with cisplatin chemotherapy. PMID- 7547214 TI - Paclitaxel (Taxol) in relapsed and refractory ovarian cancer: the UK and Eire experience. AB - The purpose of our study was to investigate the efficacy and toxicity of paclitaxel in patients with relapsed or refractory epithelial ovarian cancer in the context of a large multicentre study performed in the UK and Eire. Patients with previously treated epithelial carcinoma of the ovary or fallopian tube who fulfilled the eligibility criteria were entered in the study. Eligibility criteria included: measurable or evaluable disease; Eastern Cooperative Oncology Group (ECOG) performance status 0-2; up to three prior chemotherapy regimens, one of which had to contain a platinum agent; adequate haematological, renal and hepatic function; and no significant cardiac history. Patients received either 175 mg m-2 or 135 mg m-2 paclitaxel. The lower dose was administered to patients who had received more than two prior chemotherapy regimens. Paclitaxel was given by i.v. infusion over 3 h every 21 days. Response was assessed at three-cycle intervals or earlier if required. A total of 155 patients were registered for the study in the UK of whom 140 were eligible for response and toxicity evaluation, and 12 patients were assessed for toxicity only. Hair loss was the most frequently reported toxicity, with 74% (119/152) of patients reporting grade 3 alopecia. The most frequently reported serious toxicity was neutropenia, with 49% (74/152) of patients experiencing neutropenia grade 3 or 4. The response rate was 16% [two complete responders (CR), 20 partial responders (PR)], the median duration of response was 275 days and median survival was 244 days. Paclitaxel is active in relapsed and platinum-resistant epithelial ovarian cancer. It is well tolerated and can be given in an out-patient setting. The UK and Eire experience is very similar to that of US investigators in this group of patients. Further work is required to assess the optimal use of the drug in both first- and second line therapy. PMID- 7547215 TI - Effect of interleukin 2 on urinary excretion of degradation products of prostacyclin and thromboxane A2 in patients with ovarian cancer. AB - We studied the effect of intraperitoneal recombinant interleukin 2 (rIL-2) on the production of prostacyclin (PGI2) and thromboxane A2 (TxA2) in six patients with metastatic ovarian malignancy. Time-span urine samples collected before and after 17 intraperitoneal instillations of IL-2 (6 x 10(5) IU m-2) were assessed for 2,3 dinor-6-keto-prostaglandin F1 alpha (dinor-6-keto; a metabolite reflecting the in vivo product of PGI2) and 2,3-dinor-thromboxane B2 (dinor-TxB2; a metabolite reflecting the production of TxA2). Analysis was by high-pressure liquid chromatography, followed by radioimmunoassay. Recombinant IL-2 administration was accompanied by a significant rise (85%; P < 0.02) in the output of dinor-6-keto within the first 2 h, and this elevation persisted for up to 6 h. Moreover, output of dinor-TxB2 also rose; this rise (30%) was significant (P < 0.02) 6 h after the instillation. These effects may, in some yet unknown manner, prove significant in the anti-cancer action of rIL-2. PMID- 7547216 TI - Treatment interruptions and complications with two continuous hepatic artery floxuridine infusion systems in colorectal liver metastases. AB - Continuous hepatic artery floxuridine infusion benefits patients with colorectal liver metastases. Implanted infusion pumps are more expensive but may result in fewer treatment interruptions than when using an external pump connected to a port. We have assessed device-related complications, treatment interruptions and added nurse interventions in 95 patients undergoing a total of 959 treatment cycles via either implanted pump (64 patients) or port (31 patients). Compared with the implanted pump, the port was associated with a significant increase (P < 0.003) in catheter blockage (24/31 vs 2/64 patients), treatment interruption (15/265 vs 12/694 treatments) and added nurse intervention (80/265 vs 20/694 treatments). Survival in patients with colorectal liver metastases is limited and the complications of treatment should be kept to a minimum. An implanted subcutaneous infusion pump offers the benefit of a 3-fold lower incidence of treatment interruption and a 30-fold lower incidence of catheter blockage than when continuous infusion chemotherapy is given via an external infusion device. PMID- 7547217 TI - Prognosis after salvage treatment for unselected male patients with germ cell tumours. AB - Long-term outcome of salvage treatment was reviewed in 67 unselected male patients relapsing during or after their primary cisplatin-based chemotherapy for metastatic germ cell tumours. Seven patients underwent only surgery and/or radiotherapy as curatively intended salvage treatment. Thirty-five patients (52%) had a complete or partial response to salvage treatment, 20 (57%) of whom relapsed again. With a median follow-up of 90 months (range 3-143 months) 20 patients (30%) are alive with no evidence of disease, 15 continuously disease free and five currently disease-free. The 5 year survival from start of salvage treatment is 37% for the group as a whole. Multivariate analysis identified age < or = 35 years, complete response to primary treatment and a relapse-free interval > 3 months as independent predictors of favourable outcome of salvage treatment. A group of patients with these good-risk factors (42%) had a 5 year survival of 72% compared with the remaining patients (58%) with a 5 year survival of only 11%. Whereas patients with good-risk features may be adequately managed by conventional salvage treatment, the remaining patients carry a very poor prognosis and require innovative and more aggressive approaches. PMID- 7547218 TI - Plasma chromogranin A marks emesis and serotonin release associated with dacarbazine and nitrogen mustard but not with cyclophosphamide-based chemotherapies. AB - Chromogranin A (CgA) is present in high concentrations in enterochromaffin cells, where it is co-localised with serotonin in the storage granules. Plasma CgA has been reported to mark emesis and serotonin release associated with cisplatin treatment. However, it is not known whether plasma CgA could be an indicator of emesis and of serotonin release in patients receiving non-cisplatin chemotherapies. Therefore, in this study we evaluated, in cancer patients, the temporal relationships between the increases in plasma CgA and urinary 5 hydroxyindoleacetic acid (5-HIAA) and the development of vomiting following dacarbazine, nitrogen mustard and cyclophosphamide treatments. Metoclopramide was used as antiemetic. With dacarbazine, nitrogen mustard and cyclophosphamide the median time to the onset of emesis was 2.3, 2.8 and 5.3 h and the duration of intense emesis was 3, 2 and 6 h respectively. Plasma CgA and urinary 5-HIAA increased after dacarbazine- and nitrogen mustard-based chemotherapies, with maximal increases between 4 and 6 h after initiation of drug infusion. The time course for the increases in plasma CgA paralleled that of urinary 5-HIAA and the period of intense emesis. A highly significant (P = 0.0009) positive correlation (r = 0.68) was found between the increases in plasma CgA and in urinary 5-HIAA. Cyclophosphamide treatment was not associated with increases in plasma CgA and in urinary 5-HIAA, despite inducing emesis; this indicates that the increases in CgA and 5-HIAA after dacarbazine and nitrogen mustard are not due to the act of vomiting per se. In summary, plasma CgA is a marker of serotonin release (most likely from enterochromaffin cells) after dacarbazine and nitrogen mustard-based chemotherapies, exocytosis being the most likely mechanism for the release of serotonin. Serotonin released from enterochromaffin cells seems to trigger the emetic response to dacarbazine and nitrogen mustard; however, cyclophosphamide may release serotonin from a different pool (enteric serotonin neurons and/or CNS serotonin?). PMID- 7547219 TI - Cervical cancer in Kerala: a hospital registry-based study on survival and prognostic factors. AB - The survival experience of 452 cervical cancer patients registered during 1984 by the hospital registry of the Regional Cancer Centre, Trivandrum, Kerala, India, is described in this paper. Eighty per cent of the patients completed the prescribed treatment, which was predominantly radiotherapy. The vital status of each patient was established by scrutiny of case records and by reply-paid postal enquiries. The observed survival rates were estimated by the Kaplan-Meier method and prognostic factors were assessed using Cox's proportional hazards regression analysis. The overall 5 year observed survival rate was 47.4% (95% CI, 41.6 52.9%). Socioeconomic status, performance status and the clinical stage of disease emerged as independent predictors of survival. Low survival was associated with advanced stages of disease, low socioeconomic status and poor performance status. The problems in studying survival from cancer in developing countries and the strategies used to improve follow-up rates in India are discussed. It is stressed that trends in survival rates may be used to evaluate cancer control programmes in developing countries in the absence of reliable mortality statistics and, even when mortality data are available, survival rates are valuable comparative statistics. Earlier detection by improving the awareness of the population and the physicians will improve survival rates, but a more effective and prudent approach would be to prevent invasive cervical cancer, and thereby reduce mortality, by implementing feasible and effective screening programmes in India. PMID- 7547220 TI - Haemoccult test properties according to type and number of positive slides in mass screening for colorectal cancer. AB - Despite encouraging results from recent studies, there is still no consensus to undertake mass screening using the Haemoccult test in the general population. The success of mass screening for colorectal cancer depends among other things on Haemoccult test properties. In on-going screening programmes, the Haemoccult test consists of six slides and a test is considered positive if at least one slide is coloured. The aim of this work was to study the influence of the type and number of positive slides on the Haemoccult test's positive predictive value and characteristics of screened lesions. This work focuses on 63,958 first tests in a mass screening programme in Calvados (France) among people aged 45-74 years. There was a linear relation between the positive predictive value for cancer or an adenoma larger than 1 cm and the number of positive slides (P < 10(-4)). The positive predictive value for cancer or large adenoma was significantly higher when 4-6 slides were positive (44.3%) than when only 1-3 were positive (19.1%) (P < 10(-4)). In this latter group, the subjects in whom tumours were detected were younger and had significantly less extensive cancers. Borderline tests (no slides positive and at least one slide with a blue coloration confined to the edges) had a positive predictive value for cancer or an adenoma larger than 1 cm no different to that of tests with 1-3 positive slides. Subjects with borderline results were markedly younger than the others and had less extensive cancers and rectal localisation more often than the others. Our results suggest that (1) increasing the number of positive slides required to declare a test positive leads to an increase in the positive predictive value but is not to be recommended because of the sensitivity of the test and (2) considering borderline Haemoccult tests as positive in on-going and future mass screening campaigns would allow an increase in the sensitivity of the test, especially for rectal cancer and low extensive tumours without any decrease in its positive predictive value. PMID- 7547221 TI - Trends of skin cancer in the Canton of Vaud, 1976-92. AB - Trends in incidence and mortality for basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and cutaneous malignant melanoma (CMM) for the period 1976-92 were analysed using data from the Cancer Registry of the Swiss Canton of Vaud. Among the 12,473 cases registered, 63% were basal cell carcinomas, 25% squamous cell cancers, 9% cutaneous malignant melanomas and 3% other miscellaneous histological types. Age-standardised incidence rates increased substantially for all histological types considered, from 44% increase for BCC in males to a more than 3-fold increase for SCC in females, with only signs of a levelling off in 1991-92, following a peak of incidence rates in 1986-90. From 1976-80 to 1991-92 CMM incidence increased by approximately 80% in both sexes. In 1991-92, age standardised (world) incidence rates per 100,000 were 69.3 for basal cell, 29.1 for squamous cell cancers and 11.5 for melanomas in males, and, respectively, 62.2, 18.0 and 12.3 in females. With respect to mortality, in males rates increased for both non-melanocytic cancer (> 40%) and CMM (> 53%) whereas in females CMM, BCC and SCC rates remained approximately stable over the calendar periods examined. In 1991-92, age-standardised mortality rates per 100,000 were 2.6 for melanoma and 0.7 for other skin cancers in males, and, respectively, 1.6 and 0.2 in females. Upward trends in incidence were also present, and relatively homogeneous across, various age groups examined. However, SCC and CMM levelled off over the last period, and some decline was apparent in males below age 45. Separate analysis by anatomical site showed substantial increases in the head and neck for SCC and BCC, and in the trunk for CMM. In 1991-92, middle-aged women had almost equalled male incidence rates of BCC and SCC. A female excess of CMM incidence seemed to have disappeared since 1981-86. The increase in skin cancer incidence thus continued in this population up to the late 1980s, with a plateau only after 1990. PMID- 7547222 TI - Dose- and time-response for breast cancer risk after radiation therapy for benign breast disease. AB - Exposure of the breast to ionising radiation increases the risk of breast cancer, especially among young women. However, some issues remain controversial, for instance the shape of the dose-response curve and the expression of time-related excess. The main purpose of this report was to examine the dose-response curves for radiation-induced breast cancer formulated according to radiobiological target theories. Another purpose was to analyse the time-related excess of breast cancer risk after exposure when dose and age at first exposure were held constant. Breast cancer incidence was analysed in a cohort of 3090 women diagnosed with benign breast disease during 1925-61 (median age 37 years). Of these, 1216 were treated with radiation therapy. The dose range was 0-50 Gy (mean 5.8 Gy). The incidence rate as function of dose was analysed using a linear quadratic Poisson regression model. Cell-killing effects and other modifying effects were incorporated through additional log-linear terms. Additive and multiplicative models were compared in estimating the time-related excess. The analysis, which was based on 278 breast cancer cases, showed a linear dose response relationship at low to medium dose levels with a cell-killing effect of 5% Gy-1 (95% confidence interval 2-9%). For a given absorbed dose and age at first exposure the time-related excess was proportional to the background rates with a suggestion that the excess remains throughout life. PMID- 7547223 TI - Meta-analysis of chemotherapy in head and neck cancer: individual patient data vs literature data. PMID- 7547224 TI - The genetics of breast and ovarian cancer. AB - A number of genes are known to be involved in inherited susceptibility to breast and/or ovarian cancer. In the context of high-risk families the most important genes are BRCA1 on chromosome 17q, which is associated with a high penetrance of both breast and ovarian cancer, and BRCA2 on chromosome 13q, which causes a high risk of breast cancer but a lower risk of ovarian cancer. Other high-risk cancer genes that confer increased risks of breast or ovarian cancer in addition to other cancers include the hereditary non-polyposis colorectal cancer genes and the TP53 gene, which causes breast cancer as part of the Li-Fraumeni syndrome. The predisposing mutations in these genes are relatively rare in the population. More common genes which are associated with an increased, but lower, risk of breast cancer are the ataxiatelangiectasia gene and the HRAS1 gene. This paper reviews recent progress in mapping and cloning of these susceptibility genes, and provides estimates of the cancer risks associated with each gene and the frequency of predisposing mutations. PMID- 7547225 TI - Evidence for mutual interdependence of epithelium and stromal lymphoid cells in a subset of papillary carcinomas. AB - We have correlated the morphological features of 30 human thyroid carcinomas with the cellular localisation of insulin-like growth factor 1 (IGF-1) mRNA and IGF-1 receptor peptide using in situ hybridisation with digoxigenin-labelled oligoprobes and immunohistochemistry. Four of the five follicular carcinomas studied showed a consistent, uniform, strong positivity for IGF-1 mRNA in tumour cells compared with weakly positive surrounding normal follicular tissue and negative stroma. The majority of papillary carcinomas showed weak to moderate epithelial positivity for IGF-1 mRNA and negative stroma. Immunohistochemistry for IGF-1 receptor showed moderate positivity confined to the tumour epithelial cells in both follicular and the majority of papillary carcinomas. However, in a subgroup of papillary carcinomas characterised by a diffuse stromal lymphoid infiltration (n = 5), the stromal cells showed a much stronger reactivity for IGF 1 mRNA than the tumour or background thyroid, and the tumour cells showed a uniformly high level of immunoreactivity for IGF-1 receptor. These results are compatible with the growth of the papillary carcinoma in these cases being the result of a symbiotic relationship between the stromal lymphoid cells and the tumour epithelium with the lymphoid cells responding to an antigen produced by the tumour cells and the tumour cells responding to a growth factor produced by the lymphoid infiltrate. We suggest that this mechanism may be important in other tumours regularly associated with a widespread lymphoid infiltrate. PMID- 7547226 TI - Urokinase and macrophages in tumour angiogenesis. AB - Recent studies have shown that elevated levels of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) in breast cancer correlate with an increased risk of a reduced relapse-free survival time and shortened overall survival times. Urokinase PA and PAI-1 are independent prognostic indicators for breast cancer. The fact that plasminogen activators are indispensable for tube formation of microvascular cells and that they may induce angiogenesis in vitro strongly suggests a role for uPA and PAI-1 in tumour neovascularisation. Because macrophages and tumour cells produce uPA, we postulate a close collaboration between tumour cells and tumour-associated macrophages in angiogenesis. To investigate how uPA levels and macrophage counts in tumour tissue correlate with angiogenesis, we counted microvessels and determined uPA levels and macrophage content in 42 primary invasive breast carcinomas. Using light microscopy, we highlighted the vessels by staining their endothelium cells immunocytochemically for CD31 and factor VIII and the macrophages for CD68. After obtaining tumour tissue extracts, we determined the uPA and PAI-1 levels by ELISA. A positive correlation between microvessel density, vascular invasion, uPA level, macrophage content and proliferation rate was found. PMID- 7547227 TI - Expression of basic fibroblast growth factor and its receptor in human pancreatic carcinomas. AB - We examined the expression of basic fibroblast growth factor (FGF) and FGF receptor by immunohistochemistry in 32 human pancreatic ductal adenocarcinomas. Mild to marked basic FGF immunoreactivity was noted in 19 (59.4%) of the 32 tumours examined, and 30 (93.3%) of the tumours exhibited a cytoplasmic staining pattern against FGF receptor. The tumours were divided into two groups according to the proportion of positively stained tumour cells: a low expression group (positive cells < 25%) and a high expression group (positive cells > or = 25%). No statistically significant difference in tumour size, differentiation, metastases or stage was found between the low and high basic FGF expression groups. However, a significant correlation was found between FGF receptor expression level and the presence of retroperitoneal invasion, lymph node metastasis, and tumour stage. In addition, low FGF receptor expression was significantly associated with a longer post-operative survival as compared with high FGF receptor expression, whereas there was no significant difference in post operative survival between the low and high basic FGF expression groups. Increased expression of FGF receptor is correlated with the extent of malignancy and post-operative survival in human pancreatic ductal adenocarcinomas. Thus, overexpression of FGF receptor may prove to be a more useful prognostic marker than basic FGF expression level in pancreatic cancer patients. PMID- 7547228 TI - DNA aneuploidy in early breast cancer. AB - High-resolution flow cytometric (FCM) DNA analysis was performed on 148 unfixed, frozen tissue samples from four groups of early breast cancers: invasive carcinomas (ICs) with predominance of carcinoma in situ (DCIS) (group I), small clinical cancers < or = 15 mm (group II), node-negative, clinical cancers (group III) and small screening-detected cancers < or = 15 mm (group IV). The median tumour size was 12 mm. The aim of the study was to support, with a larger sample, our recent findings with respect to DNA ploidy pattern in the selected group of ICs with predominance of DCIS (group I). Similar results to this group were found for both the small clinical cancers and the node-negative cancers, with respect to frequency of DNA aneuploidy (79% and 90%), DNA index (DI) distribution, intratumoral DNA heterogeneity and S-phase fraction. A high frequency of DNA hyperdiploid clones was found, in particular related to highly differentiated tumours. A significant difference was found compared with the screening-detected cancers, which were characterised by a much lower frequency of DNA aneuploid samples (49%) and may represent a biologically specific group of low-malignant, slowly growing tumours. Associations were shown between histological grade and DI subclasses, and between lymph node status and DNA diploidy/aneuploidy, whereas DI was not correlated with tumour size. The DNA ploidy findings in this series of early cancers are concordant to our own results from preinvasive lesions as well as those reported from series of more advanced cancers. PMID- 7547229 TI - Detection of polypeptides associated with the histopathological differentiation of primary lung carcinoma. AB - Two-dimensional polyacrylamide gel electrophoresis combined with a non-enzymatic sample preparation technique is useful for analysing clinical tumour material. Using these techniques, we analysed the relationship between the histopathological findings in primary lung malignancies and the expression of a number of unidentified polypeptides that were detected in the molecular weight region 20-35 kDa. In this study 45 cases of primary lung cancer (PLC) (21 cases of adenocarcinoma, ten cases of squamous cell carcinoma, five cases of large-cell carcinoma, one case of adenosquamous cell carcinoma, five cases of small-cell carcinoma and three cases of carcinoid tumour) were examined. For reference, a human diploid fibroblast cell line (W138) and normal peripheral lymphocytes were used. Sixteen polypeptides were judged to be associated with histopathological features. These polypeptides seem to be valuable as differentiation markers. The simultaneous evaluation of these polypeptides and some other proliferation markers (e.g. PCNA, PCNA 'satellite', Numatin/protein B23 and lamin B) seems to clarify the characteristics of each case of PLC. Furthermore, it is possible to classify PLC based on the two-dimensional electrophoresis findings, and this classification of PLC is suggested to reflect the biological features of the tumour more precisely than that based only on morphology. PMID- 7547230 TI - Potentiation of temozolomide-induced cytotoxicity: a comparative study of the biological effects of poly(ADP-ribose) polymerase inhibitors. AB - Four poly(ADP-ribose) polymerase (PADPRP) inhibitors [3-aminobenzamide, benzamide, 3,4-dihydro-5-methoxyisoquinolin-1(2H)-one (PD 128763) and 8-hydroxy-2 methylquinazolin-4(3H)-one (NU1025)] were compared with respect to their effects on a number of biological end points. The following parameters were assessed: their ability to inhibit the enzyme in permeabilised L1210 cells; their ability to potentiate the cytotoxicity of temozolomide (including the cytotoxicity of the compounds per se); their ability to increase net levels of temozolomide-induced DNA strand breaks and inhibit temozolomide-induced NAD depletion. PD 128763 and NU1025 were equipotent as PADPRP inhibitors, and 40- and 50-fold more potent than benzamide and 3-aminobenzamide respectively. All the compounds acted in a concentration-dependent manner to potentiate the cytotoxicity and increase DNA strand break levels in cells treated with temozolomide. There was an excellent correlation between the potency of the compounds as PADPRP inhibitors and their effects on cell survival and DNA repair. Temozolomide treatment caused a decrease in cellular NAD levels, and this was abolished by the PADPRP inhibitors. In conclusion, the new generation of PADPRP inhibitors are at least 50-fold more effective than 3-aminobenzamide as chemopotentiators, and can be used at micromolar rather than millimolar concentrations in intact cells. PMID- 7547231 TI - Efficacy of intravenous delta-aminolaevulinic acid photodynamic therapy on rabbit papillomas. AB - Endogenously induced protoporphyrin IX (PPIX), a metabolite of delta aminolaevulinic acid (ALA), has been evaluated as a photosensitising agent for destruction of papillomas in cottontail rabbit papillomavirus-infected Dutch belted and New Zealand rabbits. Three factors were evaluated: (1) relative retention ratio of drug in normal tissue, papilloma and plasma over time; (2) tissue tolerance to treatment factors; and (3) efficacy of treatment protocol. Three drug doses of ALA were examined: 50, 100 and 200 mg kg-1. Actual PPIX concentrations in tissue and plasma were determined spectrophotofluorometrically. The optimal treatment time occurred 3-6 h post ALA injection. The highest PPIX concentration ratio between papilloma and normal skin was 6:1. Different light doses were investigated, using an injection to exposure interval of 3 h and an irradiance of 100 mW cm-2 at a wavelength of 630 nm. Efficacy without risk of significant damage to normal skin was obtained using 100-200 mg kg-1 ALA and 40 60 J cm-2. A long-term (3 months) cure rate of 82% was obtained with a single treatment, provided that papilloma depth did not exceed 8 mm, volume was not more than 1000 mm3 and the plasma concentration of PPIX immediately before exposure was above 500 micrograms ml-1. The short time between injection and treatment and high efficacy, together with PPIX disappearance from plasma and tissue within 24 h, make injected ALA a highly attractive drug for photodynamic therapy. PMID- 7547232 TI - Effect of endogenous and exogenous EGF on the growth of EGF receptor hyperproducing human squamous cell carcinoma implanted in nude mice. AB - The effect of epidermal growth factor (EGF) on the biological behaviour of human tumours in vivo is still controversial. We investigated the effect of EGF on the growth of an EGF receptor-hyperproducing human epidermoid carcinoma, A431 tumour, and on a human small-cell lung carcinoma, H69 tumour, without detectable EGF receptor by using sialoadenectomised (sialex) mice as an endogenous EGF suppressed animal model. The plasma EGF concentration in the sialex athymic mice was significantly lower than that in the sham-operated mice (P < 0.05). After exogenous EGF replacement with an implanted minipump, the plasma EGF concentration was significantly increased in both groups (P < 0.05). There was no significant difference between the body weight growth curves of sialex and sham operated mice with and without EGF treatment. The tumour weight of A431, both estimated and measured in sialex mice, was significantly lower than that in sham operated control mice (P < 0.05), and the growth of A431 tumour was significantly increased by exogenous EGF treatment (P < 0.05). Mitotic activity of these tumours detected by immunohistochemical staining for incorporated bromodeoxyuridine indicated a mitosis-stimulatory effect of endogenous and exogenous EGF on A431 tumours. In contrast to these findings on A431 tumours, a growth-stimulatory effect of endogenous and exogenous EGF was not observed in the H69 tumour. These results suggest a growth-promoting effect of physiological levels of endogenous EGF on EGF receptor-hyperproducing human tumours in vivo. PMID- 7547233 TI - Oxygen dependence of cellular uptake of EF5 [2-(2-nitro-1H-imidazol-1-yl)-N (2,2,3,3,3-pentafluoropropyl)a cet amide] : analysis of drug adducts by fluorescent antibodies vs bound radioactivity. AB - The present studies were initiated to quantitate the oxygen dependence of bioreductive metabolism-induced binding of EF5, a pentafluorinated derivative of the 2-nitroimidazole, etanidazole. Two different assays were compared: first, radioactive drug incorporation into cell lysates, which provides a direct measure of drug metabolism or uptake; second, monoclonal antibody detection of cellular macromolecular adducts of EF5 after whole cell permeabilisation and fixing. The antibodies (a single clone designated ELK3-51) were conjugated with the fluorescent dye Cy3, with fluorescence determined by fluorescence microscopy and flow cytometry. For the two cell lines tested (V79 Chinese hamster fibroblasts and 9L rat glioma), the oxygen dependence of binding was found to be the same for the two techniques. Using the antibody binding technique, the fluorescence signal was highly reproducible between experiments, resistant to light or chemical bleaching and stable over time following cell or tissue staining. Flow cytometric analysis of cells from rat 9L tumours treated with EF5 in vivo or in vitro showed a distribution of fluorescent signal which was very compatible, on both a relative and absolute basis, with the in vitro results. Our results indicate that immunofluorescent techniques provide a quantitative assay for bioreductive drug adducts, and therefore may be able to measure the absolute oxygen concentration distribution in cell populations and tissues of interest. PMID- 7547234 TI - Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide]. AB - One of the most sensitive hypoxia detection methods is based on the observation that binding of nitroimidazoles to cellular macromolecules occurs as a result of hypoxia-dependent bioreduction by cellular nitroreductases. Nitroimidazole binding techniques provide measurements of hypoxia to virtually any degree of spatial resolution and with a multiplicity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome conjugated monoclonal antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the radiation-resistant phenotype of 9L epigastric tumours grown in our laboratories can be associated with the presence of hypoxic cells. PMID- 7547235 TI - MDM2 overexpression is rare in ovarian carcinoma irrespective of TP53 mutation status. AB - Somatic mutations in TP53 are seen in many human cancers. In addition, the protein product of the wild-type TP53 can be sequestered by the protein MDM2 (murine double minute 2). This protein is commonly overexpressed in human sarcomas and gliomas, usually as a result of gene amplification. In this study, 43 ovarian carcinomas (OCs) were analysed for aberrations in the TP53 gene by immunohistochemistry (IHC), loss of heterozygosity (LOH) or mutation analysis. The MDM2 gene and its product was studied by Southern blotting and IHC. Over 50% of the OCs studied showed mutations in TP53 by either direct sequencing (19/36, 53%), positive IHC (23,43, 53%) or both, whereas 0/32 had amplification of MDM2 and only 1/37 tumours had positive IHC using the anti-MDM2 antibody IF-2. The solitary example of positive IHC in this series was seen in a mixed mullerian tumour with sarcomatous differentiation and was not accompanied by MDM2 DNA amplification. These results support previous data showing that around 50% of OCs have mutations in TP53 and in addition, suggest that MDM2 is not amplified in OC, but the presence of sarcomatous features in mixed mullerian tumours may result in positive immunohistochemistry with IF-2. PMID- 7547236 TI - Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver. AB - Nitroimidazole markers of tumour hypoxia bind to normoxic liver and the question has been raised whether this is due to low oxygen concentration or microregional activity of specialised nitroreductases. To answer this question, the binding patterns of the 2-nitroimidazole, pimonidazole, were compared following perfusion of surgically isolated rat livers in anterograde and retrograde directions. Perfusion at low flow rates in anterograde or retrograde directions can be used intentionally to alter oxygen gradients without altering enzyme distributions. Perfusion by means of the portal vein (anterograde direction) produced pimonidazole binding in the pericentral region of liver similar to that observed for pimonidazole binding in vivo. A complete reversal of this binding pattern occurred when the isolated liver was perfused by way of the central vein (retrograde direction). In this case, pimonidazole binding occurred in the periportal region. The extent and intensity of binding in the periportal region during perfusion in the retrograde direction was similar to that in the pericentral region during perfusion in the anterograde direction. It is concluded that low oxygen concentration rather than the non-homogeneous distribution of nitroreductase activity is the primary determinant of 2-nitroimidazole binding in liver. PMID- 7547237 TI - Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models. AB - This study reports on the development of a liposomal formulation of vincristine with significantly enhanced stability and biological properties. The in vitro and in vivo pharmacokinetic, tumour delivery and efficacy properties of liposomal vincristine formulations based on sphingomyelin (SM) and cholesterol were compared with liposomes composed of distearoylphosphatidylcholine (DSPC) and cholesterol. SM/cholesterol liposomes had significantly greater in vitro stability than did similar DSPC/cholesterol liposomes. SM/cholesterol liposomes also had significantly improved biological properties compared with DSPC/cholesterol. Specifically, SM/cholesterol liposomes administered intravenously retained 25% of the entrapped vincristine after 72 h in the circulation, compared with 5% retention in DSPC/cholesterol liposomes. The improved retention properties of SM/cholesterol liposomes resulted in plasma vincristine levels 7-fold higher than in DSPC/cholesterol liposomes. The improved circulation lifetime of vincristine in SM/cholesterol liposomes correlated with increased vincristine accumulation in peritoneal ascitic murine P388 tumours and in subcutaneous solid A431 human xenograft tumours. Increased vincristine delivery to tumours was also accompanied by increased anti-tumour efficacy. Treatment with SM/cholesterol liposomal formulations of vincristine resulted in greater than 50% cures in mice bearing ascitic P388 tumours, an activity that could not be achieved with the DSPC/cholesterol formulation. Similarly, treatment of mice with severe combined immunodeficiency (SCID) bearing solid human A431 xenograft tumours with SM/cholesterol vincristine formulations delayed the time required for 100% increase in tumour mass to > 40 days, compared with 5 days, 7 days and 14 days for mice receiving no treatment or treatment with free vincristine or DSPC/cholesterol formulations of vincristine respectively. PMID- 7547238 TI - The effect of blood flow modification on intra- and extracellular pH measured by 31P magnetic resonance spectroscopy in murine tumours. AB - Intra- and extracellular pH (pHi and pHe) were measured simultaneously by 31P magnetic resonance spectroscopy (MRS) in CaNT tumours before and after blood flow modification. Before modification, pHi was 7.1 +/- 0.09 (n = 11) and pHe [measured with an MRS-visible extracellular marker, 3-aminopropyl phosphonate (3 APP)] was 6.7 +/- 0.05 (n = 8). Chemical shift imaging and localised MRS experiments showed that the 3-APP signal was only from the tumour, not surrounding tissue. After modification by vascular occlusion, independent of whether tumours were maintained at room temperature (22-24 degrees C) or kept warm (33-35 degrees C), there was a decrease in pHi and pHe with pHi decreasing to a greater extent. Qualitatively similar results were found using flavone acetic acid (FAA) as a blood flow modifier; only four out of nine tumours responded to FAA. Concomitant with the reduction of the pH gradient after modification was a decrease in the phosphorylation state of the adenine nucleotides measured either as ATP/Pi by MRS or [ATP]/[ADP][P(i)] in tumour extracts. These results indicate that the intracellular uptake of chemotherapeutic drugs which are dependent on the transmembrane pH gradient will not be enhanced in cells made ischaemic as a result of vascular shutdown. PMID- 7547239 TI - Growth rates or radiobiological hypoxia are not correlated with local metabolite content in human melanoma xenografts with similar vascular network. AB - Investigations were carried out on two lines of human melanomas (MF; n = 12 and EE; n = 13) xenografted in nude mice. The tumours were characterised by a similar vascular supply but showed a pronounced difference in the rate of volume growth and in the radiobiologically hypoxic fraction. The distribution of ATP, glucose and lactate in the tumours was investigated using quantitative bioluminescence and single photon imaging. Concentrations of the metabolites were obtained as global values for the entire tumour mass, in regions with densely packed, structurally intact tumour cells ('viable zones'), in areas with necrosis, stromal cells and fibrous material ('necrotic zones') and in adjacent normal tissue. In all melanomas investigated glucose concentrations were significantly lower and lactate concentrations were significantly higher than in normal tissue. In contrast, no significant differences for ATP were detected. ATP and glucose concentrations were significantly less in necrotic than in viable tumour zones, whereas lactate concentrations were nearly equal in these tumour parts. Corresponding results were obtained in central versus peripheral tumour zones. There was no dependency of global or regional metabolite concentrations on tumour size within the volume range 110-1470 mm3. Based on this lack of dependency, metabolic concentrations were averaged over the whole tumour size range. Metabolite concentrations were not significantly different either globally or regionally between the two tumour entities investigated, a finding which held true for all three metabolites registered. Thus, metabolite distributions apparently mirror the similarity in vascularity of MF and EE melanomas rather than reflecting intrinsic properties with regard to tumour growth rates or susceptibility to radiation. PMID- 7547240 TI - DT-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents? AB - DT-diaphorase (DTD) is an important enzyme for the bioreductive activation of the new alkylating indoloquinone EO9. In preclinical studies, EO9 has shown selective anti-tumour activity against solid tumours and under hypoxic conditions. The levels of three reductive enzymes have been determined in three types of human solid tumours, together with corresponding normal tissues and normal liver. DTD enzyme activities were measured in tumour extracts using 2,6 dichlorophenolindophenol (DCPIP) and NADH as substrates; cytochrome P450 reductase or cytochrome b5 reductase activities were assessed with cytochrome c and NADPH or NADH respectively. DTD activity was highest in non-small-cell lung (NSCLC)-tumours (mean 123 nmol DCPIP min-1 mg-1), followed by colon carcinoma (mean 75 nmol min-1 mg-1) and squamous cell carcinoma of the head and neck (6 fold lower than NSCLC). DTD activity was very low in normal liver and normal lung (4-6 nmol min-1 mg-1), while the levels in normal colon mucosa or normal mucosa of the head and neck region were in the same range as the corresponding tumours. The levels of the two other reductive enzymes, cytochrome P450 reductase (CP450R) and cytochrome b5 reductase (Cb5R), were 5 to 25-fold lower than those of DTD in all the tissues, except for normal liver, in which DTD was 2 to 4-fold lower. The degree of variation found for DTD (range 4-250 nmol min-1 mg-1), was not observed for these enzymes (CP450R, 0.8-7.8 nmol cytochrome c min-1 mg-1; Cb5R, 3.5-27.6 nmol min-1 mg-1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547241 TI - Effects of transforming growth factor beta-1 on growth-regulatory genes in tumour derived human oral keratinocytes. AB - This study examined the effect of transforming growth factor beta-1 (TGF-beta 1) on c-myc, RB1, junB and p53 expression together with pRb phosphorylation, in carcinoma-derived and normal human oral keratinocytes with a range of inhibitory responses to this ligand. Amplification of c-myc was observed in eight of eight tumour-derived cell lines and resulted in corresponding mRNA expression. The down regulation of c-myc expression by TGF-beta 1 predominantly reflected growth inhibition by TGF-beta 1, but in two of eight tumour-derived cell lines which were partially responsive to TGF-beta 1 c-myc expression was unaltered by this ligand. While RB1 mRNA levels were unaltered by TGF-beta 1, the ligand caused the accumulation of the underphosphorylated form of the Rb protein in all cells irrespective of TGF-beta 1-induced growth arrest. junB expression was up regulated by TGF-beta 1 in cells with a range of growth inhibitory responses. All cells contained mutant p53. TGF-beta 1 did not affect p53 mRNA expression in both tumour-derived and normal keratinocytes and there was no alteration in p53 protein levels in keratinocytes expressing stable p53 protein following TGF-beta 1 treatment. The data indicate that TGF-beta-induced growth control can exist independently of the presence of mutant p53 and the control of Rb phosphorylation and c-myc down-regulation. It may be that TGF-beta growth inhibition occurs via multiple mechanisms and that the loss of one pathway during tumour progression does not necessarily result in the abrogation of TGF-beta-induced growth control. PMID- 7547243 TI - Polymorphic epithelial mucin (MUC-1)-containing circulating immune complexes in carcinoma patients. AB - Circulating immune complexes (CICs) containing polymorphic epithelial mucin (PEM/MUC-1) were found in sera of 24.5% of 151 primary breast carcinoma patients and 18-21.4% of patients with advanced ovarian (n = 56) and breast carcinomas (n = 61), 37% of patients with benign breast tumours, but in only 2.1% of 96 healthy individuals. The incorporation of PEM into CICs affects the detection of circulating PEM in commercial immunoassays such as the CA 15-3 assay, as suggested by a negative correlation between levels of PEM-containing immune complexes (PEM-CICs) and CA 15-3 values, and confirmed by isolation of PEM from CA 15-3-negative sera containing high levels of PEM-CICs. The amounts of PEM masked by human antibodies correspond to significant values of the CA 15-3 assay when monitoring patients for carcinoma. Most antibodies in PEM-CICs were of IgG class, suggesting their specific nature to the PEM epitopes. PMID- 7547242 TI - In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody. AB - The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR). PMID- 7547244 TI - Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. AB - Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR) sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-x8) fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds. with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-x8)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5 fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools. PMID- 7547245 TI - Transience of cervical HPV infection in sexually active, young women with normal cervicovaginal cytology. AB - Human papillomavirus DNA was detected in cervical specimens from 366 sexually active young women with cytomorphologically normal cervices using the polymerase chain reaction. In 93% (25/27) of initially infected women, the same viral type was not detected upon re-examination four menstrual cycles later. These results suggest that the majority of HPV infections in young women are transient. PMID- 7547246 TI - A microdissection approach to detect molecular markers during progression of prostate cancer. AB - To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate. PMID- 7547248 TI - The prognostic value of morphometry in advanced epithelial ovarian cancers. AB - The relationship between morphometric and clinical data was assessed in a series of 60 advanced ovarian carcinomas. Morphometric parameters included nuclear area, nuclear perimeter, shortest and longest nuclear axis, roundness coefficient, volume percentage of epithelium (VPE) and mitotic index. All patients had at least 5 years of follow-up. Univariate survival analysis showed that FIGO stage (P < 0.001), VPE (P < 0.001), mean nuclear area (P < 0.001) and size of residual tumour (P < 0.001) are significantly associated with survival. When the response rate of these patients to cisplatin combination chemotherapy was evaluated, variables with good prognostic outcome were residual tumour size (P = 0.01), mean nuclear area (P = 0.0006) and s.d. of nuclear area (P = 0.0019). We conclude that morphometric parameters are able to support diagnostic and therapeutic decisions. PMID- 7547247 TI - p53-independent death and p53-induced protection against apoptosis in fibroblasts treated with chemotherapeutic drugs. AB - Many recent studies have implicated p53 in the cellular response to injury and induction of cell death by apoptosis. In a rat embryonal fibroblast cell line transformed with c-Ha-ras and a mutant temperature-sensitive p53 (val135), cells were G1 arrested at the permissive temperature of 32 degrees C when overexpressed p53 was in wild-type conformation. In this state cells were resistant to apoptosis induced by etoposide (at up to 50 microM) or bleomycin (15 microU ml 1). Cells at 37 degrees C with overexpressed p53 in mutant conformation were freed from this growth arrest, continued proliferating and showed dose-dependent increases in apoptosis. This death is independent of wild-type p53 function. Control cells containing a non-temperature-sensitive mutant p53 (phe132) were sensitive to both etoposide and bleomycin after 24 h at 32 degrees C and 37 degrees C, indicating that the results are not simply due to temperature effects on pharmacokinetics or DNA damage. Our data show that induction of a stable p53 mediated growth arrest renders these cells much less likely to undergo apoptosis in response to certain anti-cancer drugs, and we conclude that the regulatory role of p53 in apoptosis is influenced by the particular cellular context in which this gene is expressed. PMID- 7547249 TI - Infrequent CDKN2 (MTS1/p16) gene alterations in human primary breast cancer. AB - Changes which lead to excessive cyclin production or to loss of cell cycle inhibition by proteins such as p16/MTS1 may release breast tumour cells from the constraints of cell division. In order to establish the frequency of MTS1/p16 gene alteration and its relation with genetic damage to the p53 and cyclin D1 genes, we have studied these gene abnormalities in 164 human primary breast cancers and in six breast cancer cell lines. Two breast cancer cell lines and one primary tumour showed a homozygous deletion of exon 2 of the MTS1 gene. Using single-strand conformation polymorphism and subsequent sequencing analysis, one tumour showed an alteration at codon 67 (CCC-->CTC; Pro to Leu). Another tumour showed a mutation at codon 98 (without amino acid change) with an additional polymorphism at codon 140. This polymorphism was also found in 13 other tumour samples, but has no effect on (disease-free) survival. From these data we conclude that the occurrence of CDKN2 (p16/MTS1) mutation in primary breast cancer is a rare event and is not likely to be involved in human breast tumour carcinogenesis and progression. PMID- 7547250 TI - Metallothionein expression in human breast cancer. AB - Metallothioneins are ubiquitous low molecular weight proteins characterised by high cysteine content and affinity for binding heavy metals. Abnormal metallothionein function and expression have been implicated in various disease states, including neoplasia. The aim of this study was to investigate metallothionein expression in human breast carcinoma. Sections of routinely fixed and processed blocks of tumour from 100 consecutive cases of primary operable breast carcinoma were stained for metallothionein using a recently developed monoclonal antibody and a standard immunohistochemical technique. Expression was scored on the basis of microscopical assessment of percentage of tumour cells staining. One patient was lost to follow-up and excluded from the study. A significant association (P < 0.0001) was observed between metallothionein expression and tumour type, with low levels being observed in tumours of good prognostic type. There was also a significant association with local recurrence (P < 0.02) and a significant difference (P < 0.02) in both survival and disease free interval between tumours showing low and high levels of expression, the latter indicating a poor prognosis. No relationship was observed with patient age, tumour size, lymph node stage, histological grade, vascular invasion, menopausal status or oestrogen receptor status. The assessment of metallothionein expression in human breast cancer appears to provide prognostic information and may have important implications for understanding its development. PMID- 7547251 TI - Gene methylation of oestrogen and epidermal growth factor receptors in neoplastic and perineoplastic breast tissues. AB - Oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) gene methylation was evaluated in neoplastic and perineoplastic breast tissues from 20 patients. In both tissues, ER gene methylation was inversely correlated with protein levels, while EGFR gene methylation was not. A preferential ER gene hypomethylation was found in neoplastic tissues, suggesting a significant role in neoplastic transformation. PMID- 7547252 TI - Are Japanese and European gastric cancer the same biological entity? An immunohistochemical study. AB - To examine the suggested biological difference between Japanese and British gastric cancers, immunohistochemistry was used to demonstrate eight markers of biological activity in a matched series of 40 Japanese and 33 British cases. There were no differences in the proportions of Japanese and British tumours positive to epidermal growth factor, epidermal growth factor receptor, transforming growth factor alpha, cripto or p53. A significantly greater proportion of British tumours were positive to c-erbB-2 whilst a significantly greater proportion of Japanese tumours were positive to nm23. British tumours had a significantly greater mean proliferating cell nuclear antigen proliferation index than Japanese tumours. These differences could be clinically significant. PMID- 7547253 TI - Immunohistochemically detectable bcl-2 expression in colorectal carcinoma: correlation with tumour stage and patient survival. AB - The bcl-2 gene encodes for a mitochondrial membrane proto-oncoprotein, the expression of which is known to suppress programmed cell death (apoptosis). In the present study the prognostic value of bcl-2 proto-oncoprotein was immunohistochemically investigated in a series of 104 colorectal carcinomas. The bcl-2 staining patterns were semiquantitatively assessed and correlated with the pTNM stage, Dukes' classification, lymphocytic infiltration (Jass classification) and tumour grade as well as parameters not associated with prognosis (gender, age, tumour site, histological tumour type). Statistical analysis was carried out using the Kaplan-Meier method, the log-rank test, hazard ratios and their confidence intervals. Fifty-five out of 104 cases completely lacked immunohistochemical bcl-2 expression. Fewer than 5% of bcl-2-positive cells were found in 25, 5-50% in 17 and more than 50% in five cases. The extent of bcl-2 expression by tumour cells decreased significantly with respect to increasing tumour size (P < 0.05), decreasing lymphocytic infiltration (P < 0.05) and chance of poor clinical outcome (P < 0.05), but not with worsening of Dukes stages. In multivariate analysis (Cox regression model) bcl-2 expression remained as an independent prognostic parameter with Dukes' classification as stratification factor (P < 0.001). Although the biological functions of bcl-2 protein are not yet well understood, our results provide further evidence that bcl-2 oncoprotein appears to be associated with favourable clinical outcome. Thus immunohistochemical bcl-2 phenotyping of colorectal carcinoma may contribute in future to the clinical management of these patients. PMID- 7547254 TI - Altered patterns of retinoblastoma gene product expression in adult soft-tissue sarcomas. AB - Altered expression of the retinoblastoma (RB) tumour-suppressor gene product (pRB) has been detected in sporadic bone and soft-tissue sarcomas. Earlier studies, analysing small cohorts of sarcoma patients, have suggested that these alterations are more commonly associated with high-grade tumours, metastatic lesions and poorer survival. This study was designed to re-examine the prevalence and clinical significance of altered pRB expression in a large and selected group of soft-tissue sarcomas from 174 adult patients. Representative tissue sections from these sarcomas were analysed by immunohistochemistry using a well characterised anti-pRB monoclonal antibody. Tumours were considered to have a positive pRB phenotype only when pure nuclear staining was demonstrated, and cases were segregated into one of three groups. Group 1 (n = 36) were patients whose tumours have minimal or undetectable pRB nuclear staining (< 20% of tumour cells) and were considered pRB negative. Patients with tumours staining in a heterogeneous pattern (20-79% of tumour cells) were classified as group 2 (n = 99). The staining of group 3 (n = 39) was strongly positive with a homogeneous pRB nuclear immunoreactivity (80-100% of tumour cells). pRB alterations were frequently observed in both low- and high-grade lesions. Altered pRB expression did not correlate with known predictors of survival and was not itself an independent predictor of outcome in the long-term follow-up. These findings support earlier observations that alterations of pRB expression are common events in soft-tissue sarcomas; nevertheless, long-term follow-up results indicate that altered patterns of pRB expression do not influence clinical outcome of patients affected with soft-tissue sarcomas. It is postulated that RB alterations are primary events in human sarcomas and may be involved in tumorigenesis or early phases of tumour progression in these neoplasias. PMID- 7547255 TI - Continuous carboplatin infusion during 6 weeks' radiotherapy in locally inoperable non-small-cell lung cancer: a phase I and pharmacokinetic study. AB - A phase I study was performed in 21 patients with previously untreated, locally inoperable, non-small-cell lung cancer (NSCLC) with ambulatory continuous carboplatin infusion together with continuous thoracic irradiation over 6 weeks. A dose range for carboplatin of 15 mg m-2 day-1 during the last 21 days (first level), during the last 31 days (second level), or during 6 weeks of the radiation period (third level) and thereafter 20 or 25 mg m-2 day-1 during 6 weeks of radiation (fourth and fifth level) was used. The total radiation dose was 60 Gy given as 2 Gy day-1 for 5 days week-1. The first three patients received radiotherapy without carboplatin. WHO grade III/IV leucopenia and thrombocytopenia occurred in the last two dose levels in two out of six and one out of six patients with 20 mg m-2 day-1 respectively, and in all three patients with 25 mg m-2 day-1 (dose-limiting toxicity). One local infection around the port and a subclavian vein thrombosis occurred. Radiation toxicity of the lung and oesophagus did not seem to be influenced by carboplatin treatment. Out of 21 patients one had a complete response (CR), ten partial response (PR), six stable disease (SD) and four progressive disease (PD). Total (TPt) and ultrafilterable plasma platinum (UPt) were measured in the last three dose levels with atomic absorption spectrophotometry with Zeeman correction. The mean (s.d.) level for TPt for 6 weeks at 15, 20 and 25 mg m-2 day-1 was 0.76 (0.15), 0.78 (0.19) and 0.90 (0.22) mg l-1 for UPt 0.10 (0.03), 0.12 (0.02) and 0.20 (0.03) mg l-1 respectively. TPt concentration levelled off after 3 weeks. The mean (s.d.) CLTB for UPt was 281 +/- 21 ml min-1 and correlated with glomerular filtration rate (r = 0.61, P = 0.03). As estimated with the sigmoid Emax model defined by the Hill equation the percentage reduction in platelets correlated with the area under the curve for UPt (r = 0.77). The maximum tolerable dose of carboplatin with concomitant continuous 60 Gy radiotherapy is 25 mg m-2 day-1; the recommended dose for phase II or III studies is 20 mg m-2 day-1 day for 6 weeks. PMID- 7547257 TI - Why Europe is so vital to nurses. Interview by Jo Barr. PMID- 7547256 TI - Bryostatin 1, a novel antineoplastic agent and protein kinase C activator, induces human myalgia and muscle metabolic defects: a 31P magnetic resonance spectroscopic study. AB - Bryostatin 1, a novel antineoplastic agent and protein kinase C (PKC) activator, has been found to induce myalgia (muscle pain) 48 h after administration in clinical trials. This is the dose-limiting toxicity and has restricted the duration of therapy in phase I trials. To investigate the mechanisms and try to increase toleration of the drug, we studied calf muscle metabolism of 14 patients at rest and during exercise and subsequent recovery using 31P magnetic resonance spectroscopy (MRS) before and 4 h, 48-72 h and 1-2 weeks following bryostatin therapy. In resting muscle there was a significant (P < 0.001) increase in the phosphodiester/adenosine 5'-triphosphate (PDE/ATP) ratio 48 h post bryostatin and in patients with myalgia compared with pre-bryostatin control studies. Following exercise, patients with myalgia showed significantly slower phosphocreatine (PCr) and ADP recovery half-time (P < or = 0.05) suggesting impaired mitochondrial (oxidative) energy production, possibly due to a direct effect on the mitochondria or secondary to reduced blood flow. The apparent proton efflux rate following exercise was significantly reduced 4 h after bryostatin (P < or = 0.05), suggesting reduced blood flow. The rate of post-exercise reoxygenation was studied in four patients by near-infrared spectroscopy 4 h post bryostatin. In three of these the rate was reduced, consistent with reduced muscle blood flow. Bryostatin 1 appeared to cause a long-lasting impairment of oxidative metabolism and proton washout from muscle, consistent with a vasoconstrictive action. Thus these studies provide evidence for two mechanisms of the dose-limiting toxicity for bryostatin. Prospective studies on the use of vasodilators to improve the tolerance of the drug should be carried out. PMID- 7547258 TI - Girl infanticide rife in India. PMID- 7547259 TI - NHS proves family unfriendly. PMID- 7547260 TI - The health of tomorrow's adults. PMID- 7547261 TI - Advances in hormone replacement therapy. AB - Thousands of women in the UK undergo hormone replacement therapy each year. Many of them experience side-effects and choose to discontinue treatment. In this article, the authors review some of the products currently available and the new approaches to therapy that are under investigation. PMID- 7547262 TI - Using workshops to implement supervision. AB - Clinical supervision is both topical and novel among many practice disciplines so it is important to consider how to take such an important initiative forward. This article describes a series of workshops designed to achieve this aim. The article focuses on the participants' perceptions of clinical supervision and its relevance to their practice environment. The authors also suggest ways to assist clinical staff further in this area. PMID- 7547263 TI - Nurses' pay is back on the agenda. PMID- 7547264 TI - Bionursing: explaining falls in elderly people. AB - In the second article in our series on bionursing, the authors focus on the integration of theory and practice. Using a case history to illustrate the point, the authors look at how bionursing can help nurses identify the causes of unexplained falls in elderly people, and avoid further harm occurring to the patient. PMID- 7547265 TI - Earn your stripes. PMID- 7547266 TI - Never going to be easy: giving bad news. PMID- 7547267 TI - 'Nurses must have right to speak out'. PMID- 7547268 TI - Living in the village. PMID- 7547269 TI - Called to the bars. PMID- 7547270 TI - Compromise or cave-in? PMID- 7547271 TI - Winning hearts and minds. PMID- 7547272 TI - Reducing the risk of eye contamination. AB - The recent case of an Italian nurse waiting for compensation after contracting HIV from a splash to her eye (1) has highlighted the serious risk of contracting infection through the eye conjunctiva. In this article, the author suggests nurses need to take more notice of recommendations and guidelines that they should wear eye protection when performing procedures likely to cause splashes to the eye. PMID- 7547273 TI - The nurses' pay Review Body. PMID- 7547274 TI - Helping adults cope with abuse as children. AB - This article describes the experience of a co-facilitator in planning and developing a closed therapeutic group, within a mental health unit, for female survivors of sexual abuse. The author discusses the process of setting up a group of this type, and by reviewing a typical group programme, explains the benefits of this approach. PMID- 7547275 TI - Vocational training: its role in nursing. AB - The use of occupational standards in professional health care curricula is currently of great importance following support for their use from the NHS Executive and the employment department. In the first of two articles reviewing the role of National Vocational Qualifications (NVQs) in nursing education, the author explains the background and development of NVQs, and the introduction and function of the Care Sector Consortium, identifying the links that now exist with nursing education. PMID- 7547276 TI - Teaching and promoting testicular self-examination. PMID- 7547278 TI - Personal tutors have a role to play. PMID- 7547277 TI - Nothing to declare. PMID- 7547279 TI - Is there life outside the NHS? PMID- 7547280 TI - Advancing in confusion. PMID- 7547281 TI - Thermus silvanus sp. nov. and Thermus chliarophilus sp. nov., two new species related to thermus ruber but with lower growth temperatures. AB - Strains of Thermus silvanus sp. nov. and strains of Thermus chliarophilus sp. nov. were isolated from the hot spring at Vizela in northern Portugal and the hot spring at Alcafache in central Portugal, respectively. The strains of T. silvanus produce orange-red-pigmented colonies and have an optimum growth temperature of about 55 degrees C, while the strains of T. chliarophilus produce yellow pigmented colonies and have an optimum growth temperature of about 50 degrees C. The strains of both species are catalase negative. These species can be distinguished from each other and from Thermus ruber by biochemical characteristics, fatty acid composition data, and 16S rRNA gene sequence data. Our phylogenetic analysis showed that strains VI-R2T (T = type strain) and ALT-8T belong to the T. ruber line of descent. The type strain of T. silvanus is strain VI-R2 (= DSM 9946), and the type strain of T. chliarophilus is strain ALT-8 (= DSM 9957). PMID- 7547282 TI - Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. AB - The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range. The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis. The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA. We identified a total of five genomic species, including the previously described species Rhizobium ciceri. All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R. ciceri. Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles. The complete 16S rRNA sequences of strains representing the two major groups, R. ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches. Both of these strains belonged to the Rhizobium loti Rhizobium huakuii branch; R. ciceri UPM-Ca7T was closely related to R. loti, and strain UPM-Ca36T was clearly separated from R. ciceri and closely related to R. huakuii. Thus, genomic species 2 could be distinguished from R. ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics. Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain. PMID- 7547283 TI - Phylogenetic analysis of the genera Cellulomonas, Promicromonospora, and Jonesia and proposal to exclude the genus Jonesia from the family Cellulomonadaceae. AB - The 16S rRNA gene sequences of eight Cellulomonas species, two Promicromonospora species, and Jonesia denitrificans were determined, and these sequences were compared with the sequences of about 50 representatives of the Arthrobacter line of descent in the order Actinomycetales. We found that in spite of its current assignment to the family Cellulomonadaceae, J. denitrificans branches outside the radiation of this taxon and cannot be considered a member of it. The two Promicromonospora species do not cluster separately from Cellulomonas species and are more closely related to Cellulomonas species than to each other. PMID- 7547284 TI - Phylogenetic analysis of mycolic acid-containing wall-chemotype IV actinomycetes and allied taxa by partial sequencing of ribosomal protein AT-L30. AB - The phylogenetic relationships among 30 mycolic acid-containing wall chemotype IV actinomycete strains and 12 strains belonging to allied taxa were examined by determining the amino acid sequences of the ribosomal AT-L30 proteins of these organisms. Sequencing 20 N-terminal amino acids of AT-L30 preparations revealed that the members of the genera containing mycolic acid-containing actinomycetes form two clusters; the first cluster contains the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella, and the second cluster contains the genera Corynebacterium and Mycobacterium. The genus Nocardia was placed in a clade containing the genus Rhodococcus. The data showed that Tsukamurella paurometabolum is closely related phylogenetically to the genus Gordona. The phylogenetic clusters identified were entirely consistent with the proposal of Goodfellow that the family Nocardiaceae should encompass the mycolate-containing, cell wall type IV actinomycete genera Nocardia, Rhodococcus, Gordona, and Tsukamurella. The genera Actinomyces and Micrococcus exhibited AT-L30 amino acid sequence characteristics intermediate between those of actinomycetes and those of typical eubacteria. The genera Nocardia, Gordona, Mycobacterium, Actinoplanes, and Micromonospora were each a taxon that consisted of phylogenetically coherent species. In contrast, the genera Rhodococcus and Corynebacterium are taxa that consist of phylogenetically distantly related species. In general, my results are consistent with previous 16S rRNA sequencing results, but significant differences were also found. My data, together with previous AT-L30 sequencing data, show that phylogenetic relationships among taxa can be determined by using markers other than the ribosomal gene sequences. PMID- 7547285 TI - Paenibacillus (formerly Bacillus) gordonae (Pichinoty et al. 1986) Ash et al. 1994 is a later subjective synonym of Paenibacillus (formerly Bacillus) validus (Nakamura 1984) Ash et al. 1994: emended description of P. validus. AB - A polyphasic study in which we performed an amplified ribosomal DNA restriction analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, a gas chromatographic analysis of methylated fatty acids, pyrolysis mass spectrometry, a random amplified polymorphic DNA analysis, a phenotypic analysis, and an analysis of the levels of DNA binding of Paenibacillus gordonae and Paenibacillus validus strains (including both type strains) showed that these organisms form a homogeneous group and that the names P. gordonae and P. validus are therefore synonyms. P. validus has nomenclatural priority, and an emended description of this species is given; the type strain is strain LMG 11161 (= ATCC 43897). PMID- 7547286 TI - Chloroflexus aggregans sp. nov., a filamentous phototrophic bacterium which forms dense cell aggregates by active gliding movement. AB - Two strains of thermophilic photosynthetic bacteria, designated MD-66T (T = type strain) and YI-9, were isolated from bacterial mats in two separate hot springs in Japan. These new isolates were phenotypically similar to Chloroflexus aurantiacus in some respects. They were thermophilic filamentous photosynthetic bacteria that grew well at 55 degrees C either anaerobically as photoheterotrophs or aerobically as chemoheterotrophs. They exhibited gliding motility, produced bacteriochlorophylls a and c, contained chlorosomes, and required thiamine and folic acid as growth factors. However, isolates MD-66T and YI-9 had the ability to rapidly form mat-like dense aggregates of filaments, an ability which has not been observed in any C. aurantiacus strain. Carbon source utilization tests revealed that unlike C. aurantiacus, the new isolates did not utilize acetate, citrate, ethanol, or glycylglycine. An analysis of the carotenoid components revealed that isolates MD-66T and YI-9 contained mainly gamma-carotene and OH gamma-carotene glucoside fatty acid esters. These isolates also contained only trace amounts of beta-carotene, which is a major carotenoid component (28.4% of the total carotenoids) in C. aurantiacus. The results of DNA hybridization studies suggested that the new strains were genetically distinct from C. aurantiacus (levels of similarity, 9 to 18%), and 16S rRNA sequence comparisons showed that strain MD-66T was related to C. aurantiacus at a similarity level of 92.8%. On the basis of our data, we propose that a new Chloroflexus species should be created for our new isolates; the name of this new species is Chloroflexus aggregans, and the type strain is strain MD-66 (= DSM 9485). PMID- 7547287 TI - Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. AB - The results of a phylogenetic and chemotaxonomic analysis of the genus Micrococcus indicated that it is significantly heterogeneous. Except for Micrococcus lylae, no species groups phylogenetically with the type species of the genus, Micrococcus luteus. The other members of the genus form three separate phylogenetic lines which on the basis of chemotaxonomic properties can be assigned to four genera. These genera are the genus Kocuria gen. nov. for Micrococcus roseus, Micrococcus varians, and Micrococcus kristinae, described as Kocuria rosea comb. nov., Kocuria varians comb. nov., and Kocuria kristinae comb. nov., respectively; the genus Nesterenkonia gen. nov. for Micrococcus halobius, described as Nesterenkonia halobia comb. nov.; the genus Nesterenkonia gen. nov. for Micrococcus halobius, described as Nesterenkonia halobia comb. nov.; the genus Dermacoccus gen. nov. for Micrococcus nishinomiyaensis, described as Dermacoccus nishinomiyaensis comb. nov.; and the genus Kytocossus gen. nov. for Micrococcus sedentarius, described as Kytococcus sedentarius comb. nov. M. luteus and M. lylae, which are closely related phylogenetically but differ in some chemotaxonomic properties, are the only species that remain in the genus Micrococcus Cohn 1872. An emended description of the genus Micrococcus is given [corrected]. PMID- 7547288 TI - Taxonomy and phylogeny of industrial solvent-producing clostridia. AB - We performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as Clostridium acetobutylicum strains, by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. The 55 strains examined exhibited a good correlation between their biotypes and DNA fingerprints, which allowed us to divide them into nine groups. The DNA fingerprints of the nine groups differed markedly, but within each group the DNA fingerprints exhibited a high level of similarity. To determine the phylogenetic relationships of the nine groups, we performed a 16S rRNA gene sequence analysis. The results of a comparative analysis of the partial sequence corresponding to positions 830 to 1383 (Escherichia coli numbering) of the 16S rRNA gene indicated that the nine biotype groups could be assembled into four taxonomic groups. The complete 16S rRNA sequences of strains representing these groups were determined. Our phylogenetic analysis revealed that the amylolytic type strain C. acetobutylicum ATCC 824 (taxonomic group I) was only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90 to 90.5%). The strains belonging to taxonomic groups II, III, and IV, represented by C. acetobutylicum NCP 262, "Clostridium saccharoperbutylacetonicum" N1-4, and C. acetobutylicum NCIMB 8052T (T = type strain), respectively, were closely related (levels of sequence similarity, 98.2 to 98.9%). C. acetobutylicum NCIMB 8052T exhibited a level of similarity of 100% with the type strain of Clostridium beijerinckii. Reclassification of the saccharolytic solvent-producing strains is necessary, and possible names for the four taxonomic groups are discussed. PMID- 7547289 TI - Bradyrhizobium liaoningense sp. nov., isolated from the root nodules of soybeans. AB - Seventeen strains of extra-slowly growing (ESG) soybean rhizobia isolated from root nodules of Glycine soja and Glycine max growing in five provinces (Liaoning, Heilongjiang, Shanxi, Hubei, and Anhui) in the People's Republic of China were compared with 48 reference strains belonging to the genera Bradyrhizobium, Rhizobium, and Agrobacterium by performing a numerical analysis of 191 phenotypic features. Our results showed that all of the ESG strains examined clustered closely in the genus Bradyrhizobium but were separated from Bradyrhizobium japonicum at the species level and that they could be differentiated from Rhizobium and Agrobacterium species at the genus level. On the basis of the results of our numerical taxonomy analysis, a genomic DNA G & C content analysis, DNA-DNA hybridization experiments, a partial 16S rRNA sequence analysis, a serological analysis, an N and C content analysis, and an N/C ratio analysis of members of the three groups of soybean rhizobia, we propose the name Bradyrhizobium liaoningense sp. nov. for the ESG strains; the type strain of this species is strain 2281. PMID- 7547290 TI - Phylogenetic inferences and taxonomic consequences of 16S ribosomal DNA sequence comparison of Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina and reclassification of V. eurihalina as Halomonas eurihalina comb. nov. AB - The phylogenetic positions of the moderately halophilic bacteria Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina were determined by PCR amplification of rRNA genes and direct sequencing. The resulting data were compared with data for other bacteria obtained from 16S rRNA sequence databases. C. marismortui, V. eurihalina, and D. salina clustered phylogenetically within the gamma subclass of the Proteobacteria and are closely related to other species on the Halomonas-Deleya branch. C. marismortui belongs in the family Halomonadaceae and has the characteristic 16S rRNA signatures defined for this family, including the distinctive cytosine residue at position 486 found in all members of the Halomonadaceae. V. eurihalina is closely related to the type species of the genus Halomonas, Halomonas elongata, and we formally propose that V. eurihalina should be transferred to the genus Halomonas as Halomonas eurihalina comb. nov. The type strain of this species is strain F9-6 (= ATCC 49336). D. salina is not as closely related to other species belonging to the Halomonas-Deleya complex, but is more closely related to Halomonas elongata than to Deleya aquamarina, the type species of the genus Deleya. A polyphasic approach will be necessary to determine the natural taxonomic positions of the species belonging to the genera Halomonas and Deleya, as well as C. marismortui, V. eurihalina, Halovibrio variabilis, and Paracoccus halodenitrificans. PMID- 7547291 TI - Phylogenetic analysis of the genus Corynebacterium based on 16S rRNA gene sequences. AB - The 16S rRNA gene sequences of 30 strains representing 23 validated Corynebacterium species and 7 currently non-valid Corynebacterium species were determined. These sequences were aligned with the sequences of other Corynebacterium species and related actinomycete taxa. A comparative sequence analysis revealed that there is considerable phylogenetic depth and internal structure in the genus Corynebacterium. Turicella otitidis and the amycolate species Corynebacterium amycolatum were located at the periphery of the genus Corynebacterium. It was evident that the species of the genus Corynebacterium form a monophyletic association and, together with other chemotype IV and mycolic acid-containing taxa (including the genera Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella), form a natural suprageneric group. PMID- 7547292 TI - Heterogeneity within human-derived centers for disease control and prevention (CDC) coryneform group ANF-1-like bacteria and description of Corynebacterium auris sp. nov. AB - Recently, Centers for Disease Control and Prevention coryneform group ANF-1 bacteria were described as Corynebacterium afermentans, and group ANF-1-like bacteria were described as Turicella otitidis. Over a 1.5-year period 10 strains of a previously undescribed, gram-positive, rod-shaped organism that was not partially acid fast and resembled ANF-1-like bacteria were isolated from different pediatric patients with ear infections. These previously undescribed coryneform bacteria exhibited a distinct colony morphology and consistency, had a carbon source utilization pattern distinct from the carbon source utilization patterns of C. afermentans and T. otitidis, had a cell wall based on meso diaminopimelic acid, contained mycolic acids, and had DNA G+C contents of 68 to 74 mol%. A 16S rRNA gene sequence analysis revealed that these clinical isolates are members of the genus Corynbacterium and that they are distinct from C. afermentans and T. otitidis. On the basis of phenotypic and phylogenetic evidence we propose a new species, Corynebacterium auris, for these Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria. The type strain is strain DSM 44122 (CCUG 33426). PMID- 7547293 TI - Phylogeny of the genus Corynebacterium deduced from analyses of small-subunit ribosomal DNA sequences. AB - We determined almost complete small-subunit ribosomal DNA sequences of 50 reference strains belonging to the genera Corynebacterium, Rhodococcus, and Gordona and compared these sequences with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum parsimony methods), as well as a bootstrap analysis, were used to assess the robustness of each topology which we obtained. The results of comparative phylogenetic analyses confirmed that the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Tsukamurella, and Turicella form a monophyletic taxon within the phylum containing the high-G+C-content gram-positive bacteria. The genus Corynebacterium appeared to be a monophyletic unit whose members could be divided into four major clusters. The validity of the genus Turicella is doubtful since members of this genus clearly belong to the genus Corynebacterium. The variability of chemotaxonomic characteristics within the genus Corynebacterium suggests that small-subunit ribosomal DNA sequence analysis is probably the most straightforward method for confirming that a bacterium belongs to this genus. PMID- 7547294 TI - Halobaculum gomorrense gen. nov., sp. nov., a novel extremely halophilic archaeon from the Dead Sea. AB - A novel extremely halophilic archaeon was isolated from the Dead Sea. This isolate is rod shaped and, like Halobacterium sodomense, requires a relatively low level of sodium ions for growth and a very high level of magnesium; optimal growth occurs in the presence of 0.6 to 1.0 M Mg2+. The new strain resembles members of the Halobacterium saccharovorum-Halobacterium sodomense-Halobacterium trapanicum group in many physiological properties. However, the polar lipid composition of this organism is characteristic of representatives of the genus Haloferax; a sulfated diglycosyl diether is present, and the glycerol diether analog of phosphatidylglycerosulfate is absent. The G+C content of the DNA is 70 mol%. We found that on the basis of 16S rRNA sequence data our new isolate occupies a position intermediate between the position of the Halobacterium saccharovorum group and the position of the genus Haloferax and is sufficiently different from the previously described members of the Halobacteriaceae to justify classification in a new species and a new genus. We propose the name Halobaculum gomorrense gen. nov., sp. nov. for this organism; the type strain is strain DSM 9297. PMID- 7547295 TI - Phylogenetic analysis of the genera Alteromonas, Shewanella, and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and proposal of twelve new species combinations. AB - Small-subunit ribosomal DNA sequences were determined for 17 strains belonging to the genera Alteromonas, Shewanella, Vibrio, and Pseudomonas, and these sequences were analyzed by phylogenetic methods. The resulting data confirmed the existence of the genera Shewanella and Moritella, but suggested that the genus Alteromonas should be split into two genera. We propose that a new genus, the genus Pseudoalteromonas, should be created to accommodate 11 species that were previously Alteromonas species, including Pseudoalteromonas atlantica comb. nov., Pseudoalteromonas aurantia comb. nov., Pseudoalteromonas carrageenovoa comb. nov., Pseudoalteromonas citrea comb. nov., Pseudoalteromonas denitrificans comb. nov., Pseudoalteromonas espejiana comb. nov., Pseudoalteromonas haloplanktis comb. nov. (with two subspecies, Pseudoalteromonas haloplanktis subsp. haloplanktis comb. nov. and Pseudoalteromonas haloplanktis subsp. tetraodonis comb. nov.), Pseudoalteromonas luteoviolacea comb. nov., Pseudoalteromonas nigrifaciens comb. nov., Pseudoalteromonas rubra comb. nov., and Pseudoalteromonas undina comb, nov., and one species that previously was placed in the genus Pseudomonas, Pseudoalteromonas piscicida comb. nov. We propose that P. haloplanktis (type strain, ATCC 14393) should be the type species of the genus Pseudoalteromonas. At this time the emended genus Alteromonas is restricted to a single species, Alteromonas macleodii. PMID- 7547296 TI - Natronococcus amylolyticus sp. nov., a haloalkaliphilic archaeon. AB - The alpha-amylase-producing haloalkaliphilic archaeon Natronococcus sp. strain Ah 36T (T = type strain) was isolated previously from a Kenyan soda lake, Lake Magadi. Most cells of strain Ah-36T occurred in irregular clusters, and the colonies were orange-red. The polar lipids of this organism were composed of C20, C20 and C20, C25 derivatives of phosphatidylglycerol and phosphatidylglycerophosphate. Phosphatidylglycero-(cyclo-) phosphate, which is characteristic of Natronococcus occultus, was not detected. The complete nucleotide sequence of the 16S rRNA gene revealed that the closest relative of strain Ah-36T is N. occultus ATCC43101T (level of similarity, 96.4%), an extremely halophilic archaeon. However, strain Ah-36T did not exhibit a significant level of DNA homology to N. occultus ATCC43101T, which represents the only previously described species in the genus Natronococcus. We describe a new species for strain Ah-36T, for which we propose the name Natronococcus amylolyticus. PMID- 7547297 TI - Campylobacter hyointestinalis subsp. lawsonii subsp. nov., isolated from the porcine stomach, and an emended description of Campylobacter hyointestinalis. AB - The taxonomic relationships of seven isolates obtained from porcine stomachs (the "CHY" group), which resembled (but were distinct from) the type strain and other reference strains of Campylobacter hyointestinalis, were examined by using phenotypic and genomic methods. The phenotypic characteristics and ultrastructure of the new organisms were characteristic of Campylobacter species, although they could be distinguished from all previously described taxa. A numerical analysis of 38 phenotypic characters revealed that the new isolates formed a distinct group at a similarity level of 90.1% and could be clearly distinguished from reference strains representing 20 related taxa, principally species and subspecies belonging to the genera Campylobacter, Arcobacter, and Helicobacter. DNA-DNA hybridization studies revealed that the porcine stomach strains were genomically homogeneous (levels of relatedness, 84 to 90%), although the levels of DNA homology with type and reference strains of C. hyointestinalis were relatively high (56 to 71%). Differences in the DNA base compositions of the CHY group and C. hyointestinalis strains were also observed. Our data indicate that the new porcine isolates should be considered members of a subspecies of C. hyointestinalis, for which we propose the name Campylobacter hyointestinalis subsp. lawsonii subsp. nov. The type strain is strain CHY 5 (= LMG 14432 = NCTC 12901 = CCUG 34538). The description of C. hyointestinalis is emended accordingly, and a description of Campylobacter hyointestinalis subsp. hyointestinalis subsp. nov. is given. PMID- 7547298 TI - Streptomyces costaricanus sp. nov., isolated from nematode-suppressive soil. AB - A new bacterial strain, strain CR-43T (T = type strain), which was isolated from tropical soil and was previously shown to have antinematodal and antibiotic properties, is described. The name Streptomyces costaricanus is proposed for this organism. The generic placement of strain CR-43T was based on its typical morphology, its production of LL-diaminopimelic acid, and its fatty acid composition. To clarify the taxonomic position of strain CR-43T, it was compared with the type strains of similar Streptomyces species. The results of a number of biochemical tests and a profile analysis of the hydrolyzable fatty acids indicated that CR-43T differs from previously described species. Strain CR-43 (= ATCC 55274 = NRRL B-16897) is the type strain of S. costaricanus sp. nov. PMID- 7547299 TI - Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus". AB - Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms. PMID- 7547300 TI - Description of Thermoanaerobacter brockii subsp. lactiethylicus subsp. nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp. finnii comb. nov., and an emended description of Thermoanaerobacter brockii. AB - A strictly anaerobic, thermophilic, gram-positive, spore-forming cubacterium designated strain SERB 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92 degrees C. The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 microns) with peritrichous flagella. The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomporphic and filamentous (length, 15 microns) in old cultures. Growth occurred at temperatures of 40 to 75 degrees C, and optimum growth occurred at temperatures between 55 and 60 degrees C. The fermentable substrates included glucose, fructose, galactose, mannose, cellobiose, maltose, sucrose, lactose, D-xylose, D-ribose, mannitol, pyruvate, and starch. The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2. The DNA base composition was 35 mol% G+C. The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99%. The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of > 70% were obtained, these organisms belong to the same species. Therefore, we propose that T. finnii should be reclassified as a subspecies of T. brockii, Thermoanaerobacter brockii subsp. finnii comb. nov. This automatically creates Thermoanaerobacter brockii subsp. brockii. We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T. brockii, Thermoanaerobacter brockii subsp. lactiehylicus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547301 TI - Haloanaerobium lacusroseus sp. nov., an extremely halophilic fermentative bacterium from the sediments of a hypersaline lake. AB - A new extremely halophilic chemoorganotrophic bacterium (strain H200T [T = type strain]) was isolated from the hypersaline sediments of Retba Lake in Senegal. This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram negative, obligate anaerobe that grew optimally at 40 degrees C in the presence of 180 to 200 g of NaCl per liter. The DNA base composition was 32 mol% guanine plus cytosine. The fermentation products from glucose were ethanol, acetate, H2, and CO2. Yeast extract was required for growth. The fermentable substrates included D-fructose, galactose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, D-mannitol, glycerol, and Casamino Acids. On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species. The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species. We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus. PMID- 7547302 TI - Transfer of Streptococcus adjacens and Streptococcus defectivus to Abiotrophia gen. nov. as Abiotrophia adiacens comb. nov. and Abiotrophia defectiva comb. nov., respectively. AB - We performed this study to determine the 16S rRNA sequences of the type strains of Streptococcus adjacens and Streptococcus defectivus and to calculate the phylogenetic distances between these two nutritionally variant streptococci (NVS) and other members of the genus Streptococcus. S. adjacens and S. defectivus belonged to one cluster, but this cluster was not closely related to other streptococcal species. A comparative analysis of the sequences of these organisms and other low-G+C-content gram-positive bacteria revealed that the two NVS species formed a distinct cluster and were only remotely related to the Aerococcus and Carnobacterium clusters. The highest level of homology (93.7%) was found between S. adjacens and Carnobacterium divergens. Carnobacterium species have meso-diaminopimelic acid in their cell walls, but S. adjacens and S. defectivus have L-lysine as the diamino acid at position 3 in their peptidoglycan tetrapeptides. On the basis of our findings and the results of previous phenotypic studies, we propose that the NVS species should be placed in a new genus, the genus Abiotrophia, as Abiotrophia adiacens comb. nov. and Abiotrophia defectiva comb. nov. PMID- 7547303 TI - Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. AB - The ixodid tick Ixodes persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23S-5S rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23S rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hermsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afzelii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16S rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16S rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia miyamotoi sp. nov. for this spirochete; strain HT31 is the type strain. PMID- 7547304 TI - Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR. AB - We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70 degrees C reverse transcription extension step permitted improved read through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup. Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed. PMID- 7547305 TI - Characterization of Lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy. AB - A novel obligately intracellular bacterium, ileal symbiont intracellularis, which was obtained from the intestines of pigs with proliferative enteropathy disease, was grown in pure cocultures with tissue cultures of rat cells. An examination of the 16S ribosomal DNA gene sequence revealed that the isolates which we obtained are members of the delta subdivision of the Proteobacteria and that the sequences of these organisms exhibit a level of similarly of 91% with the sequence of Desulfovibrio desulfuricans ATCC 27774. These isolates were homogeneous and differed in cellular morphology, acid fastness, phenotype, electrophoretic protein profile, and habitat from Desulfovibrio species. On the basis of the results of an integrated study of the phenotype and genotype of a consistent morphological entity found in particular porcine cells and associated with a well defined clinical condition, we concluded that these bacteria belong to a previously undescribed genus and species, for which we propose the name Lawsonia intracellularis gen. nov., sp. nov. A species-specific recombinant DNA probe was cloned previously, and this probe was used to identify the bacterium in tissue culture cells and in the ileal epithelia of pigs with proliferative enteropathy disease. Coculture of the organism with a rat enterocyte cell line allowed us to designate strain NCTC 12656 the type strain and to describe the new genus and species. The organism which we cultured is pathogenic for pigs and causes proliferative enteropathy lesions in their ilea and colons, and Koch's postulates were fulfilled for this organism. PMID- 7547306 TI - Genome comparisons in the yeastlike fungal genus Galactomyces Redhead et Malloch. AB - The G+C contents of the DNAs of 41 strains belonging to the genus Galactomyces Redhead et Malloch were determined by the thermal denaturation method. Melting profiles revealed that the DNAs of these strains are heterogeneous. Four groups were recognized on the basis of this heterogeneity. However, DNA similarity values, which were calculated by using DNA-DNA reassociation kinetics, revealed that the strains could be divided into six subgroups. Strains belonging to the same subgroup exhibited high levels of DNA similarity (84 to 100%). The members of two subgroups, corresponding to Galactomyces citri-aurantii and Galactomyces reessii, exhibited low levels of DNA similarity with the members of the other subgroups (20 to 27%). The members of the four remaining subgroups, which contained only strains previously identified as Galactomyces geotrichum, exhibited intermediate levels of reassociation (41 to 59%). Some combinations of phenotypic characteristics correlated with the subgroups; a key based on phenotypic characteristics that can be used to distinguish the subgroups is presented. PMID- 7547307 TI - 16S rRNA gene similarities indicate that Hallella seregens (Moore and Moore) and Mitsuokella dentalis (Haapsalo et al.) are genealogically highly related and are members of the genus Prevotella: emended description of the genus Prevotella (Shah and Collins) and description of Prevotella dentalis comb. nov. AB - Because of similarities in the cellular fatty acid compositions of Hallella seregens and Mitsuokella dentalis, we determined the 16S rRNA gene sequences of the type strains of these species to assess their relationship. A very high level of sequence relatedness (approximately 99.8%) was found between H. seregens and M. dentalis, indicating that these species are genealogically closely related. A comparative sequence analysis revealed that these two species are members of the genus Prevotella and are phylogenetically remote from Mitsuokella multiacidus (the type species of the genus Mitsuokella), which was found to be a member of the Sporomusa subbranch of the Clostridium subphylum of the gram-positive bacteria. On the basis of our phylogenetic findings, we propose that M. dentalis should be reclassified as Prevotella dentalis comb. nov. PMID- 7547308 TI - Reclassification of Micrococcus agilis (Ali-Cohen 1889) to the genus Arthrobacter as Arthrobacter agilis comb. nov. and emendation of the genus Arthrobacter. AB - Phylogenetic evidence derived from a 16S ribosomal DNA analysis indicated that the type strain of Micrococcus agilis, DSM 20550 (= ATCC 966 = CCM 2390), is less closely related to the type species of the genus Micrococcus, Micrococcus luteus, than to the type species of the genus Arthrobacter, Arthrobacter globiformis, and related Arthrobacter species. The phylogenetic position of M. agilis is supported by the presence of peptidoglycan variation A3 alpha and by the presence of MK 9(H2) as the major isoprenolog, a characteristic also found in strains of A. globiformis, Arthrobacter crystallopoietes, Arthrobacter atrocyaneus, Arthrobacter citreus, Arthrobacter aurescens, Arthrobacter ilicis, Arthrobacter ureafaciens, Arthrobacter oxydans, Arthrobacter histidinolovorans, and Arthrobacter nicotinovorans. The last six species and M. agilis are characterized by the presence of threonine in the interpeptide bridge of the peptidoglycan. Threonine has not been found in the peptidoglycans of other Arthrobacter species or in members of the genus Micrococcus. Despite the fact that a morphological life cycle is not known, these data support the proposal that M. agilis should be transferred to the genus Arthrobacter as Arthrobacter agilis comb. nov. PMID- 7547309 TI - Wigglesworthia gen. nov. and Wigglesworthia glossinidia sp. nov., taxa consisting of the mycetocyte-associated, primary endosymbionts of tsetse flies. AB - The primary endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) are harbored inside specialized cells (mycetocytes) in the anterior region of the gut, and these specialized cells form a white, U-shaped organelle called mycetome. The P-endosymbionts of five tsetse fly species belonging to the Glossinidae have been characterized morphologically, and their 16S ribosomal DNA sequences have been determined for phylogenetic analysis. These organisms were found to belong to a distinct lineage related to the family Enterobacteriaceae in the gamma subdivision of Proteobacteria, which includes the secondary endosymbionts of various insects and Escherichia coli. These bacteria are also related to the P-endosymbionts of aphids, Buchnera aphidicola. Signature sequences in the 16S ribosomal DNA and genomic organizational differences which distinguish the tsetse fly P-endosymbionts from members of the Enterobacteriaceae and from the genus Buchnera are described in this paper. I propose that the P endosymbionts of tsetse flies should be classified in a new genus, the genus Wigglesworthia, and a new species, Wigglesworthia glossinidia. The P-endosymbiont found in the mycetocytes of Glossina morsitans morsitans is designated the type strain of this species. PMID- 7547310 TI - Evidence for the placement of the gram-negative Catonella morbi (Moore and Moore) and Johnsonella ignava (Moore and Moore) within the Clostridium subphylum of the gram-positive bacteria on the basis of 16S rRNA sequences. AB - Comparative 16S rRNA analysis was used to determine the phylogenetic positions of Catonella morbi and Johnsonella ignava, which are members of two monospecific genera of gram-negative anaerobic bacilli isolated from human gingival crevices. Both of these genera were found to belong to cluster XIVa (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) of the Clostridium subphylum of gram-positive bacteria. Within this cluster, which contains several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species, C. morbi and J. ignava formed two distinct lines that were separate from all other taxa. Our findings support the separate generic status of the genera Catonella and Johnsonella and show that these genera do not belong to the family Bacteroidaceae but instead belong to the gram-positive Clostridium subphylum. PMID- 7547311 TI - A new group (type 3) of Mycobacterium celatum isolated from AIDS patients in the London area. AB - We describe a new group (type 3) of the recently proposed species Mycobacterium celatum isolated from eight patients with AIDS in London, England. Sequences of genes coding for 16S rRNA (EMBL accession no. Z46664) showed a divergence of 17 bases from M. celatum type 2 reference isolates and a divergence of 7 bases from M. celatum type 1 reference isolates. A reference strain is available (NCTC 12882). PMID- 7547312 TI - Replacement of NCTC 4175, the current type strain of Proteus vulgaris, with ATCC 29905. Request for an opinion. AB - The current type strain of Proteus vulgaris, NCTC 4175 (= ATCC 13315), differs substantially from typical strains of this species both biochemically and chemotaxonomically. DNA relatedness studies revealed that strains previously classified as P. vulgaris belong to six genomospecies. One of these genomospecies contains strains that are negative in indole, salicin, and esculin reactions (biogroup 1) and has been named Proteus penneri. A second genomospecies, which is most frequently isolated from human urine, contains typical P. vulgaris strains that are positive in indole, salicin, and esculin reactions (biogroup 2). The members of the remaining four genomospecies are indole positive and negative in salicin and esculin reactions (biogroup 3). Of 36 biogroup 3 strains studied, only strain NCTC 4175T (T = type strain) and one other strain, CDC 1732-80, belong to genomospecies 3. To retain NCTC 4175 as the type strain of P. vulgaris would restrict this species to these two strains, whose origins are unknown. This would mean that hundreds of strains for which the description of P. vulgaris was written and which have been representatives of this species for the past 50 years would have to be renamed as members of a new species. To prevent this confusion, we request that biogroup 2 reference strain ATCC 29905 (= CDC PR1) replace NCTC 4175 as the type strain of P. vulgaris. PMID- 7547313 TI - The 1st International Congress on Pediatric Pulmonology. Nice, France, June 2-5, 1994. PMID- 7547314 TI - Penicillin-resistant Streptococcus pneumoniae and acute pulmonary infections: prevalence and severity. PMID- 7547315 TI - Nitric oxide inhalation. PMID- 7547316 TI - Target diseases for the newly developed technology in respiratory care. PMID- 7547317 TI - Clinical lessons from an experimental model of ARDS. AB - The oleic acid (OA) lung injury model is one of the most common laboratory methods for studying the acute respiratory distress syndrome (ARDS) in experimental animals. Many features of the model mimic the pathophysiology of ARDS in humans, despite obvious differences in pathogenesis. Thus, the OA lung injury model has been used extensively to study the consequence of lung injury and to evaluate potential new treatment strategies. PMID- 7547318 TI - Acute respiratory distress syndrome (ARDS) in children: Multicenter Collaborative Study of the French Group of Pediatric Intensive Care. PMID- 7547319 TI - Mechanical ventilation of children with the adult respiratory distress syndrome. AB - Approximately 2-5% of admissions to pediatric intensive care units are due to diseases which develop into the adult respiratory distress syndrome (ARDS). About 8% of intensive care patient days are involved in treating patients with ARDS. ARDS is associated with approximately a third of deaths in the pediatric intensive care unit. Overall mortality is 50-70%. Frustratingly, little improvement in outcome has occurred over the past decade. The outcome predictors which have been developed to date have been unreliable for individual patients who may have been treated with alternative respiratory support modalities. The following is a review of current ventilatory management of ARDS, including the promising new modality of high-frequency oscillatory ventilation. PMID- 7547320 TI - Is there a role for inhaled nitric oxide in pediatric ARDS? AB - Despite advances in ventilator management and use of extracorporeal lung support, mortality related to ARDS in pediatric patients has not been reduced over the past 20 years. Progressive respiratory failure, due to evolution of the primary illness or to complications of ventilator therapy, significantly contributes to poor outcome. ARDS is characterized by severe ventilation-perfusion mismatch and by pulmonary hypertension. Because of their side effects which affect systemic hemodynamic status or worsen intrapulmonary shunting, intravenous vasodilator trials have been of limited interest. Nitric oxide (NO) has been recognized as a gas with vasodilator properties. In neonates studies have shown that inhaled NO may have an important role in the therapy of persistent pulmonary hypertension. Inhaled NO in adults with severe ARDS has been shown to reduce pulmonary hypertension without producing systemic vasodilation. This reduction of pulmonary vascular resistances may reduce pulmonary edema formation, decrease vasoconstrictor response to cardiotonic agents, and improve biventricular function. In addition, arterial oxygenation seems to be increased by improved matching of ventilation with perfusion. Improvement of oxygenation with inhaled NO suggests that use of lower tidal volumes and FIO2 may be more successful. Until now, there are no published studies regarding NO administration in ARDS affecting nonneonatal pediatric patients. However, the results obtained in adults and newborns suggest that inhaled NO may be a useful adjuvant therapy of ARDS in children, possibly in association with other therapies. Even in adults it remains unclear whether therapy with inhaled NO can reduce morbidity and mortality. Prospectives and randomized studies are essential to assess the real utility of inhaled NO in ARDS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547321 TI - Chest X-ray in children: use of phosphor plates. PMID- 7547322 TI - Thoracic helical CT in pediatric patients. PMID- 7547323 TI - Diagnostic procedures of GER in childhood lung disease. AB - Respiratory diseases are frequently related to gastroesophageal reflux (GER). In the absence of classic symptoms like vomiting, silent GER can only be ruled out by further studies. Esophageal pH monitoring of long duration (18 to 24 hr) is now recommended as the technique of choice in infants and children with atypical presentation of GER. Mechanisms of GER pathway are complex and may provoke chronic pneumonia or wheezing; pH monitoring cannot be considered an "all or nothing" exam. Several esophageal pH profiles have been described in various respiratory diseases. PMID- 7547324 TI - Empiric antibiotic treatment of acute bacterial infections of the lower respiratory tract in children in an era of changing etiology. PMID- 7547325 TI - Mortality during acute bacterial infections of the lower respiratory tract in children. PMID- 7547326 TI - Resurgence of tuberculosis in children. PMID- 7547327 TI - BCG policies in Europe. PMID- 7547328 TI - Immunological aspects of pulmonary tuberculosis in children. AB - In recent years new information has been collected about the immunological responses to pathogenic mycobacteria. More information on cellular and molecular responses of cells in murine and human tuberculosis has been produced and T cells' role in the production of selected cytokines has been clarified. Studies in mice have provided insight into the phases of the T cell response to virulent M. tuberculosis, the role of various T cell subsets, and the repertoire of antigens recognized by these cells. However, despite this new information, some of which has been confirmed in humans, large gaps remain in our knowledge about the immune response to this infection, particularly concerning cellular or molecular mechanisms involved in acquired protection. Even if some extrapolations from adult data can be made, large gaps in our knowledge exist on the potential immune defects in young infants who are prone to develop tuberculosis soon after infection. PMID- 7547329 TI - Efficacy of BCG immunization against tuberculosis in children. PMID- 7547330 TI - The value of flexible bronchoscopy in childhood pulmonary tuberculosis. PMID- 7547331 TI - Treatment of pulmonary tuberculosis in children: new trends. PMID- 7547332 TI - Older and newer challenges of tuberculosis in children. AB - Tuberculosis (TB) in children represents one of the best examples of what we have termed an "adultosis"--a disease inflicted by adults on children and not by children on adults--and which can be acquired by direct contact or by the indirect result of various types of abuses, such as social ones. The same socioeconomic and political failures that have allowed TB and HIV to remain uncontrolled in many countries have also contributed to the emergence of multiple drug resistant (MDR) M. tuberculosis. These older and newer challenges of TB in children and some approaches to their resolution are briefly discussed here. PMID- 7547333 TI - Nontuberculous pulmonary infections in pediatric AIDS. PMID- 7547334 TI - Natural history of childhood asthma. PMID- 7547336 TI - Role of eosinophils in childhood asthma inflammation. AB - The serum levels of eosinophil cationic protein (ECP) are raised in asthmatic children and indicate an eosinophil involving inflammatory process of the lungs. ECP measurements in serum may be used to monitor the severity of asthma in children and the efficacy of treatment with inhaled corticosteroids as well as to monitor allergen exposure. From these rather indirect studies we suspect that the inflammatory process in childhood asthma is the same as that seen in adults and involves the eosinophils. PMID- 7547335 TI - Update in allergy testing in childhood asthma: how do you know whether you are successfully controlling the patient's inflammation? AB - The levels of serum eosinophil cationic protein (ECP) in asthmatic patients have been shown to be increased in acute and undertreated asthma as a result of inflammation. ECP is released during in vitro clotting of peripheral blood. The exposure of the atopic individual to an offending allergen stimulates the activation of the blood eosinophils and their release of ECP into serum. Serum ECP levels reflect avoidance of the allergen, and successful treatment of asthma inflammation with corticosteroids cause a reduction of the inflammation in the lung. When individual patients with asthma are followed, the level of serum ECP can be used (1) as an early indicator of the degree of inflammation, (2) for monitoring the efficacy of corticosteroid therapy, and (3) for possible noncompliance with treatment. PMID- 7547337 TI - Clinical diagnosis of infant asthma. PMID- 7547338 TI - Assessing nocturnal asthma in children. PMID- 7547339 TI - From pathophysiological mechanisms to pharmacological treatment of childhood asthma. PMID- 7547340 TI - Risk factors for airway hyperresponsiveness in childhood asthma. PMID- 7547341 TI - Early acquisition of sensitization in childhood asthma. AB - Recent research indicates that the key events involved in the development of lifelong allergic sensitization to environmental antigens occur during infancy and early childhood. This process involves interactions between mutually antagonistic populations of allergen-reactive T-helper lymphocytes, which occur around the time of initial allergen exposure. PMID- 7547342 TI - Environmental control in childhood asthma. PMID- 7547343 TI - Treatment of severe asthma in childhood. PMID- 7547344 TI - Clinical and radiological approach of pulmonary diseases in HIV infection in children. PMID- 7547345 TI - The risk of anesthesia in the asthmatic child. PMID- 7547346 TI - Associations and interactions between the esophagus and the lower respiratory tract. AB - There are important interactions between the esophagus and the lower respiratory tract (LRT). These occur because of their physical proximity and the functional activities. Malfunctions or lack of coordination between these organs leads to serious human illness such as asthma, aspiration pneumonia, and gastroesophageal reflux. PMID- 7547347 TI - Pathophysiology and mechanisms of gastroesophageal reflux in childhood asthma. PMID- 7547348 TI - Management of GER in childhood asthma. PMID- 7547349 TI - Food allergy and asthma. AB - Food allergy (FA) is one of the causes of atopic dermatitis (AD), of acute urticaria, of reactions of the gastrointestinal tract, and of acute systemic anaphylaxis, but its role in asthma appears to be less clear. The prevalence and incidence of subjects with food-induced wheezing have not been well studied. In addition, the number of subjects with proven food-induced wheezing by double blind, placebo-controlled oral food challenge (DBPCOFC) has been small. At the moment wheezing is considered unusual in food-hypersensitive subjects, and wheezing as the unique symptom of FA is rare. Furthermore, most cases of food induced asthma have been observed in children. Food allergy may trigger allergic respiratory symptoms through two main routes: ingestion or inhalation. Children with asthma, who are allergic to foods, present some particular features such as AD and a related significantly elevated total serum IgE level. Alternatively, FA may occur in patients who are "high IgE responder" and more prone to become sensitive to many allergens, including foods. Therefore, children with asthma and a history of AD and/or elevated total serum IgE level should be carefully assessed for FA. We have shown that a significant proportion of children with IgE mediated cow's milk allergy experienced asthma following DBPCOFC with cow's milk. PMID- 7547350 TI - Molecular biology of cystic fibrosis: state of the art. PMID- 7547351 TI - Relationships between gene mutations and clinical features in cystic fibrosis. PMID- 7547352 TI - Bacterial infection in cystic fibrosis (CF): special reference to mycobacteria and Burkholderia cepacia. PMID- 7547353 TI - Aerosol administration of antibiotics. AB - Antibiotics can be aerosolized to the lower respiratory tract of patients with pulmonary infections. With attention to stability of the antibiotic, its ability to be aerosolized, and particle size of the aerosol, rational design of an antibiotic aerosol treatment regimen is possible. The choice of nebulizer for aerosol antibiotic administration depends not only on drug stability under the nebulization conditions, but practical considerations such as ease of use in the home and expense. In general, a greater volume of respirable aerosol is generated by an ultrasonic device in comparison to the jet nebulizers. Of the factors affecting antibiotic delivery to the lower respiratory tract, the patients and their disease are the most important. This patient effect is not directly amenable to medical intervention, but can be controlled through measurement of antibiotic concentrations in respiratory secretions, seeking a target estimated from the best available in vitro data. PMID- 7547354 TI - Mycobacterium avium intracellulare infection in children with AIDS. PMID- 7547355 TI - Nonantibiotic inhaled therapy in cystic fibrosis: new data. PMID- 7547356 TI - Invasive and noninvasive ventilatory assistance in cystic fibrosis. PMID- 7547357 TI - Energy balance and nutritional support in cystic fibrosis. PMID- 7547358 TI - Heart-lung and bilateral lung transplantation in children with cystic fibrosis. AB - Bilateral lung and heart-lung transplantation are indicated for cystic fibrosis children with end-stage lung disease. This article discusses the current criteria for inclusion-exclusion, surgical techniques, and results. PMID- 7547359 TI - Developmental aspects of pulmonary defenses in children. AB - It is now generally accepted that maturational immaturity of lung defense system(s) exists in the young infant and child which, together with other maturational deficiencies of immunologic systems, contribute to their undue susceptibility to infections, many of which are associated with pulmonary infections. The alveolar macrophage (AM), of central importance to lung defense, has been studied extensively in the neonatal rabbit by our group and in the human, by others. Collectively, the results indicate small numbers of morphologically and functionally immature AMs prior to birth followed by a dramatic increase within the first 24 hr. This increase coincides with the large release from type II cells of surfactant, which not only may be involved in the functional maturation of developing AMs but also, if present in large quantities, may lead to decreased activity of these cells. There is also an age-related increase in chemotactic and microbicidal activity of AMs during maturation. Deficiencies in bone marrow pools, complement and cytokines (IFN-gamma, TNF alpha) as well as IgG isotype switching are known to occur in the neonate which may contribute to increased susceptibility to infection and which are amenable to immune interventions. PMID- 7547360 TI - Progress toward the development of a respiratory syncytial virus vaccine. PMID- 7547361 TI - Pertussis: the new acellular vaccines. PMID- 7547362 TI - There's no place like home: evaluation of obstructive sleep apnea in the child's home. PMID- 7547363 TI - Why should infants with sleep apneas and apparent life-threatening events be recorded polygraphically? PMID- 7547364 TI - The use of nasal CPAP in children. PMID- 7547365 TI - Development of the surfactant system. AB - Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes that lines and stabilizes alveoli. Surfactant-specific proteins are early markers of the embryonic lung. Precursors of type II pneumocytes appear to be committed early and are able to pursue their differentiation program in a relatively autonomous way. Despite this potential, surfactant ontogeny and storage actually take place relatively late in the course of fetal development. Surfactant accumulation is, therefore, delayed until a certain stage after which it is stimulated by a number of factors. Recent investigations have pointed out the possible role of paracrine mechanisms in the determinism of this control, especially with regard to retinoids and TGF-beta. PMID- 7547366 TI - Surfactant replacement therapy. AB - Surfactant replacement therapy for treatment or prevention of the respiratory distress syndrome (RDS) has been studied intensively over the past decade. Randomized controlled trials have demonstrated a reduction in the odds of neonatal death of about 40% and of pulmonary air leaks of 35 to 70% depending upon the type of surfactant used. Prophylaxis or very early treatment is superior to later treatment, especially for the very preterm (< 28 week) infant. Natural (derived from animal lungs) surfactants have a more rapid onset of action than synthetic surfactants and may also provide better long-term benefits, but further comparative trials will be needed to demonstrate this conclusively. Surfactant treatment should not be viewed as a substitute for prenatal steroid therapy to enhance fetal lung maturity; the treatments are synergistic. PMID- 7547367 TI - Patient-triggered ventilation. PMID- 7547368 TI - Stroke and hypertension. Antihypertensive therapy withdrawal. AB - The effect of antihypertensive therapy withdrawal in acute ischaemic stroke patients was assessed in order to establish possible correlations between blood pressure values and the modified Rankin Scale (mRS) outcome. One hundred and twelve consecutive patients with acute ischaemic stroke and hypertension were treated with relatively similar regimen. Seventy-two hours following the onset of stroke, systolic blood pressure decreased to < or = 180 mmHg in 110 patients. Antihypertensive therapy was randomly discontinued in 59 patients. Clinical outcome and mortality were compared in patients with and without antihypertensive therapy withdrawal, no statistically significant differences (p > 0.05) between the two groups being found. Statistically significant positive correlations (p < 0.05) between mRS outcome and the decrease of systolic and diastolic blood pressure values were in patients treated with anticoagulant and antihypertensive therapy. Statistically significant negative correlations (p < 0.05) between mRS outcome and low levels of diastolic blood pressure were noted in patients who discontinued antihypertensive therapy. Our findings emphasize the need to associate antihypertensive and anticoagulant therapy in acute ischaemic stroke patients with hypertension. Spontaneous decrease of diastolic blood pressure values showed poor outcome. The management of hypertension associated with acute ischaemic stroke should be reconsidered. PMID- 7547369 TI - Neuromyotonia in alcoholic patients with gastrectomy. AB - Four patients with severe muscle cramps were reported. In two of them the electrical signs of neuromyotonia were demonstrated. All the patients had undergone gastrectomy and all were immoderate consumers of alcohol. The neuromuscular disorder was reversible in all the patients. PMID- 7547370 TI - Stroke: therapeutic window. PMID- 7547371 TI - Immunohistochemistry in the diagnosis of dystrophinopathies. AB - Sixty-four muscle biopsies obtained from patients with Duchenne muscular dystrophy, limb-girdle dystrophy, congenital muscular dystrophy, or who referred for diagnosis were examined with histochemical methods for dystrophin staining with antidystrophin antibodies. Six atypical cases in whom the dystrophin expression was inconsistent with both clinical and morphopathological pattern were selected: one case of Duchenne muscular dystrophy in whom dystrophin was partially represented, one case referred for investigation in whom dystrophin was absent, despite the lack of clinico-morphological signs supporting the diagnosis of Duchenne disease, two cases of congenital muscular dystrophy in whom dystrophin was absent and one patient with the same diagnosis in whom dystrophin was present, but quantitatively reduced. A patient with limb-girdle muscle dystrophy, in whom immunohistochemical investigation was necessary for the diagnosis is also reported. In conclusion, the immunohistochemical investigation using antidystrophin labelled antibodies of muscle biopsies obtained from patients with various types of muscular dystrophy is mandatory. PMID- 7547372 TI - Melodic intonation therapy in the verbal decoding of aphasics. AB - Melodic Intonation Therapy is a well-known method exploring the verbal encoding of aphasics but within this study, it was used to investigate the verbal decoding (the auditory comprehension). Two separate groups, 240 cases each, were investigated, the former with MIT and the latter with other therapy methods. Each group included three subgroups according to the three frequent types of aphasia (Wernicke, Broca and Anomia). The method of semantic fields was associated to treat the second group since it is usually used in the treatment of aphasics with auditory decoding disturbances. All patients were tested twice, before and after therapy. PMID- 7547373 TI - Habituation of orienting reaction in therapy-resistant partial epilepsy. AB - A polygraphic study on resistance to habituation of the somatic, autonomic and EEG components of the orienting reaction elicited by a repetitive auditory stimulus was performed in 66 epileptics with therapy-resistant partial seizures (TRPS) and in 135 matched subjects in two control groups. The study showed a significantly higher resistance to habituation of the orienting reaction in epileptics with TRPS vs. the normal subjects of the control group I. A significantly higher resistance to habituation was also noted vs. the epileptics with therapy-controlled partial seizures of control group II. The correlational analysis of data showed that the severity of these habituation disturbances in epileptics with TRPS depended on the patients' age, type of electroclinical seizures, pretrial seizure frequency, type of resting EEG, administered treatment (no. of administered antiepileptic drugs/patient), daily dose, as well as on the serum level of these drugs. The multivariate regression analysis showed that the most significant predictive variables for the habituation disturbances were the pretrial seizure frequency, type of electroclinical seizures, daily dose of administered antiepileptic drugs and their serum level. The data also evidenced that the antiepileptic treatment improves the interictal habituation disturbances, the effect being more marked as the treatment was more sustained. The above-mentioned habituation changes in epileptics with TRPS should be ascribed to some disturbances in nervous excitability. PMID- 7547374 TI - Amino acid content of the brain in rats focal penicillin induced epilepsy. AB - The initial objective of the present study was to investigate the role of excitatory and inhibitory amino acids in generalized as compared to focal epilepsy, both forms being induced by the same convulsant agent, i.e. penicillin. Our attempts to obtain in the rat the generalized epilepsy, constantly induced in cats by systemic administration of penicillin, were unsuccessful. This is probably due to the rudimentary development of the cerebral cortex in rodents as compared to the feline cortex. The tentative conclusion was drawn that the cortex is the brain structure mainly involved in the genesis of petit mal seizures. Penicillin was applied to the cortex of 40 white Wistar rats and the electrical cortical activity was registered. The concentrations of glutamate, aspartate, glycine, GABA and serine were determined in the cerebral cortex, the brain stem and the cerebellum. The same amino acids were determined in the brain of 20 controls. No significant changes in the amino acid contents were obtained in the cerebral cortex. In the brain stem the glutamate level was significantly increased while the glycine content was markedly decreased. These findings are consistent with the involvement of the brain stem structures in seizure activity. PMID- 7547375 TI - The dystrophin gene and its product--a view. PMID- 7547376 TI - Lipofuscin and ceroid pigments in aging and brain pathology. A review. I. Biochemical and morphological properties. PMID- 7547377 TI - Anticardiolipin-antibodies in stroke and in other neurological disorders. AB - Anticardiolipin antibodies (ACL-A) are acquired antiphospholipid antibodies characteristically found in patients with systemic lupus erythematosus or related autoimmune diseases. Several reports have shown that there may be an association between ACL-A and various neurological disorders, in particular cerebral ischemia. Using a micropin enzyme linked immunosorbent assay we measured the levels of ACL-A in the sera of 225 unselected patients with various neurological disorders. The prevalence of ACL-A in the whole group was 4.0% (9/225). However, the prevalence in patients with ischemic cerebrovascular disorders was 9.1% (5/55). With one exception (thrombocytopenia was found more often in ACL-A positive cases) there was no difference with respect to the prevalence of risk factors for stroke and associated diseases between ACL-A-positive and ACL-A negative patients with TIA/stroke. High titers of ACL-A were also found in a few patients with epilepsy (n = 2), migraine (n = 1), and intracranial meningioma (n = 1). In patients with ischemic cerebrovascular disorders search for ACL-A may help to identify patients with a possibly higher risk of thrombosis. PMID- 7547378 TI - Acetazolamide therapy evaluation in haemorrhagic stroke. AB - The influence of acetazolamide in patients with hemorrhagic stroke was assessed in 54 patients in comparison with the influence of other therapies in 68 patients included in a control group. Modified Rankin Scale and mortality rate were evaluated at three different moments: onset, 72 hours and control (3 weeks-one month from the onset). A better outcome was seen when acetazolamide was given. Mortality rate was significantly lower in the group of acetazolamide. This therapy may be safely used in haemorrhagic stroke, especially when hydrocephalus is associated. PMID- 7547379 TI - Lumbosacral tumors with a retroperitoneal development. AB - Neoplasms are relatively rare in the lumbosacral region. The present study presents two cases with tumors in the lumbosacral region, a chordoma and a tumor with multinucleated giant cells which further showed a presacral development. Some common aspects of the clinical and radiologic symptomatology were evidenced as well as the specific characteristics of each separate tumor in our cases. The diagnosis was possible due to the modern radiologic equipment. PMID- 7547380 TI - Down-regulation of Langerhans cell protein kinase C-beta isoenzyme expression in inflammatory and hyperplastic dermatoses. AB - The family of protein kinase C (PKC) isoenzymes plays a fundamental part in signal transduction, and thereby regulates important cellular functions, including growth, differentiation, cytokine production and adhesion molecule expression. In lesional psoriatic skin, Ca(2+)-dependent PKC activity, PKC-beta protein and epidermal Langerhans cell (LC) PKC-beta immunostaining are significantly decreased, indicating activation and subsequent down-regulation of PKC. Whether these changes occur in other inflammatory/hyperplastic dermatoses is, however, unknown. We examined PKC-alpha and PKC-beta expression in normal skin, psoriasis, cutaneous T-cell lymphoma (CTCL), lamellar ichthyosis, non bullous ichthyosiform erythroderma, atopic dermatitis, urushiol-induced allergic contact dermatitis, and sodium lauryl sulphate (SLS)-induced irritant contact dermatitis. Cryostat sections were stained for PKC-alpha and PKC-beta, and the LC marker CD1a, using an immunoperoxidase technique and specific monoclonal antibodies. Double-labelling studies, in normal skin, revealed co-expression of PKC-beta and CD1a by epidermal LCs. Analysis of the number of PKC-beta+ and CD1a+ epidermal LCs, in diseased compared with normal skin, revealed three categories: (i) in psoriasis and CTCL, the PKC-beta+ epidermal LC number was significantly reduced, whereas the CD1a+ epidermal LC number was unchanged; (ii) in allergic and irritant contact dermatitis, both PKC-beta+ and CD1a+ epidermal LCs were significantly reduced in number; and (iii) in atopic dermatitis, the PKC-beta+ epidermal LC number was normal, and CD1a+ epidermal LCs were significantly increased in number. Moreover, the ratio of epidermal LC PKC+/CD1a+ was reduced in all the dermatoses studied, suggesting activation of PKC-beta, with subsequent down-regulation. Within the dermis, increased PKC-beta staining of infiltrating cells was observed in all the conditions studied except lamellar ichthyosis and non-bullous ichthyosiform erythroderma. These data indicate that: (i) down regulation of LC PKC-beta occurs in a variety of inflammatory and hyperplastic skin disorders, and is not unique to psoriasis, and (ii) the pattern of epidermal LC PKC-beta and CD1a expression varies among the diseases studied. In mice, PKC activation induces LC migration. Thus, down-regulation of epidermal LC PKC-beta associated with reduced CD1a+ epidermal LCs in allergic and irritant contact dermatitis suggests that PKC-beta may transduce the signal for migration of LCs from human epidermis. PMID- 7547381 TI - In situ detection of retinoid-X receptor expression in normal and psoriatic human skin. AB - Increasing evidence suggests that the retinoid-X receptors RXR(-alpha,-beta, gamma) play a crucial part in regulating the transcriptional activity of several steroid hormone receptors, including 1,25-dihydroxyvitamin D3 receptors (VDR) and retinoic acid receptors (RAR-alpha,-beta,-gamma). We developed a new technique for immunohistochemical in situ detection of RXR receptors, and investigated the localization of RXR-alpha in normal and psoriatic human skin using a recently raised corresponding specific antibody. RXR-alpha positive cells related to the skin were phenotyped by sequential sections and a double-labelling procedure for the simultaneous demonstration of this nuclear receptor and cell membrane antigens, as well as cytokeratin 10, HLA-DR and vimentin. Our findings indicate that: (i) RXR-alpha is strongly expressed in normal and psoriatic human skin; (ii) most of the cell types in normal human skin, including keratinocytes, melanocytes, fibroblasts and skin immune cells such as Langerhans cells, reveal strong nuclear immunoreactivity for RXR-alpha, with less cytoplasmic staining; (iii) altered levels or distribution of RXR-alpha in the skin do not appear to be involved in the genesis of psoriasis vulgaris, but subepidermal and subcellular distribution suggest a function of RXR-alpha in the transition from proliferation to differentiation in epidermal keratinocytes; (iv) expression in the hair follicle points to a contribution from RXR-alpha to hair growth. PMID- 7547382 TI - Properties of the carcinoma-associated antigen MH 99/KS 1/4 in normal and transformed human keratinocytes: regulation of synthesis, molecular cross-linking and ultrastructural localization. AB - A 38-kDa cell-surface glycoprotein defined by monoclonal antibody MH 99 is markedly increased in many epithelial tumours. In normal human skin, it is a characteristic marker for germ-cell phenotypic tissues. Although the gene encoding the MH 99 antigen has recently been cloned, and several histological and biochemical studies have been performed, the biological function of this interesting antigen still remains unknown. In the present study, we examined the synthesis of MH 99 in keratinocyte populations showing different in vitro differentiation capacity. Normal keratinocytes, spontaneously immortalized keratinocytes (cell line HaCaT), three SV-40-transformed keratinocyte lines (130, 425, and HaSV), and two squamous cell carcinoma lines (SCL-1 and SCL-2), were compared. Radioimmunoprecipitation revealed the highest levels of synthesis in cell populations with the least differentiation. This was paralleled by an increase of MH 99 synthesis in normal keratinocytes cultured in low concentrations of Ca2+ and by an increase of MH 99 synthesis during subculture of normal keratinocytes. Both phenomena were paralleled by an opposite behaviour of a differentiation marker. Molecular cross-linking and subsequent immunoprecipitation led to a decrease of the MH 99 signal, but an increase of a high molecular weight protein signal was seen. After cleavage of the crosslinker, the MH 99 signal reappeared, whereas the signal of the large protein remained unchanged. Thus, the MH 99 antigen may be associated with a high molecular weight protein on the cell surface, supporting the suggestion of a receptor-like function. Phosphorylation of the molecule could not be detected. Immunoelectron microscopy revealed homogeneous distribution on the cell surface, but cells of the same culture exhibited clear differences in their MH 99 expression. A concept for MH 99 regulation in normal and transformed human keratinocyte populations in vitro is proposed, showing that the synthesis of MH 99 is inversely correlated with cell differentiation. The association with a high molecular weight protein supports the suggestion that the MH 99 antigen interacts with other molecules. PMID- 7547383 TI - Immunohistochemical characterization of keratin expression in clear cell acanthoma. AB - The nature of clear cell acanthoma has not been clarified, although many hypotheses have been proposed, including a benign neoplasm derived from epidermis or the acrosyringium, or a non-specific dermatosis. In this study, seven cases of clear cell acanthoma were analysed by immunohistochemical techniques, using various monoclonal antikeratin antibodies, and antibodies against filaggrin, involucrin and epithelial membrane antigen. Different immunoreactivities were noted between clear cell acanthoma and a normal eccrine gland, including the acrosyringium. Immunoreactivities of clear cell acanthoma were almost identical to those of normal epidermis, although some antibodies gave a different staining pattern between clear cell acanthoma and normal epidermis. The expression of cytokeratins in psoriatic epidermis has been reported to change as a result of abnormal differentiation or maturation. Clear cell acanthoma showed a similar staining pattern to inflammatory dermatoses such as psoriasis vulgaris, lichen planus and discoid lupus erythematosus. We speculate that clear cell acanthoma is a localized form of inflammatory dermatosis rather than a neoplasm. PMID- 7547384 TI - Expression of the 27-kDa heat shock protein in human epidermis and in epidermal neoplasms: an immunohistological study. AB - The 27-kDa heat shock protein (HSP27) is a member of the small heat shock protein (HSP) family. In addition to its putative function in thermotolerance, this protein may play a part in the regulation of cell growth and differentiation. This study was conducted to assess the significance of the expression of HSP27 in human epidermis and in cutaneous neoplasms. Sixty-two biopsy samples from normal human skin and from inflammatory and neoplastic skin diseases were investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue sections, using a monoclonal antibody specific for HSP27. In normal human epidermis, HSP27 is expressed in the upper epidermal layers with a cytoplasmic staining pattern. The basal cell layer does not express detectable amounts of HSP27. In hair follicles, staining is mainly confined to the outer root sheath and to the infundibular epithelium. Melanocytes, dermal fibroblasts and endothelial cells do not express detectable amounts of HSP27. HSP27 could not be detected in fetal skin until the 20th week of gestation. Tumour cells in basal and squamous cell carcinomas do not express significant amounts of HSP27. In solar keratoses, seborrhoeic keratoses, human papillomavirus (HPV)-induced hyperproliferative lesions and inflammatory skin conditions, HSP27 expression largely resembles the pattern observed in normal human skin. HSP27 is expressed in a differentiation related pattern in normal human epidermis and hyperproliferative disorders of the epidermis. We conclude that HSP27 may be regarded as a marker of differentiation in epidermal keratinocytes. Absence of HSP27 in the upper epidermal layers may be a marker for epidermal malignancy. PMID- 7547385 TI - Expression of stem cell factor in cutaneous mastocytosis. AB - Stem cell factor has recently been identified as a potent growth factor for bone marrow stem cells, melanocytes and mast cells. In order to evaluate its possible role in human mastocytosis, skin lesions from 13 patients with urticaria pigmentosa and five patients with mastocytomas, and normal skin specimens from five healthy donors were studied by immunohistochemistry, using polyclonal and monoclonal (hkl-12) antibodies against stem cell factor, and a monoclonal antibody (YB5.B8) against its receptor, the c-kit proto-oncogene product. Stem cell factor expression was noted in all sections studied, with an equal distribution pattern for both antibodies, but a weaker intensity with the hkl-12 reagent. Cytoplasmic staining was noted in keratinocytes, Langerhans cells, sweat gland ductal lining cells, mast cells, endothelial cells and spindle-shaped dermal stromal cells. An intense, diffusely granular reaction pattern was noted in all cells, except for a sparse, coarsely granular pattern in mast cells and stromal cells. In urticaria pigmentosa, staining was weaker in keratinocytes, but more prominent in Langerhans cells. In all sections, toluidine blue-positive mast cells and TA 99-positive basal epidermal melanocytes were the only cells to react with the c-kit antibody. Mastocytomas and urticaria pigmentosa lesions thus exhibit different patterns of stem cell factor expression. However, a possible pathogenetic role of this factor in mastocytosis remains to be determined. PMID- 7547386 TI - Differences in the expression of pemphigus antigens during epidermal differentiation. AB - Epidermal desmogleins with molecular weights of 130/140 kDa (Dsg3 or PVA) and 150/160 kDa (Dsg1 or DGI) are recognized by autoantibodies from patients with pemphigus vulgaris (PV) and pemphigus foliaceus (PF), respectively. In order to understand the histogenesis of both types of pemphigus, we studied the expression patterns of Dsg1 and Dsg3 during stratification of cultured keratinocytes. Monolayers of cultured normal human keratinocytes demonstrated uniform intercellular staining with PV sera. The staining pattern was distinct from the focal staining with PF sera observed only in the stratified areas. Both Dsg1 and Dsg3 proteins and their mRNA were expressed by the monolayers, whereas no production of Dsg2 (HDGC) mRNA was found. The relative ratio of Dsg3 to the total desmogleins, as determined by density on immunoblotting, decreased as the cultured keratinocytes stratified. In the completely stratified keratinocytes cultured on collagen membrane, Dsg1 became predominant, with subsequent reduction of PV antigen expression. The relative decrease of Dsg3 (PVA) during epidermal differentiation might be responsible for the induction of suprabasal acantholysis in PV. PMID- 7547387 TI - Eicosapentaenoic acid-induced changes in membrane fluidity and cell adhesion molecules in cultured human keratinocytes. AB - The effects of eicosapentaenoic acid (EPA, 20:5n-3) on the lipid dynamics of cultured epidermal keratinocytes and their expression of cell adhesion molecules were investigated, and were compared with those of arachidonic acid (AA, 20:4n 6). When keratinocytes were treated with 3 micrograms/ml of EPA or AA for 72 h, these compounds were found to be incorporated into the cells. EPA-induced lipid changes were distinguished by a significant increase in the cellular content of n 3 polyunsaturated fatty acids, whereas AA treatment resulted in an increase in the cellular content of n-6 arachidonic acid. These changes in fatty acid composition were accompanied by an increase in cellular membrane fluidity, which was evaluated by the diffusion coefficient, using the method of fluorescence recovery after photobleaching (FRAP) [from 1.77 +/- 0.34 x 10(-8) cm2/s untreated to 2.23 +/- 0.35 x 10(-8) cm2/s EPA-treated (P < 0.001), and 2.16 +/- 0.35 x 10( 8) cm2/s AA-treated (P < 0.001)]. Intercellular adhesion molecule-1 (ICAM-1) was induced on the keratinocyte membrane in the presence of tumour necrosis factor alpha and interferon-gamma, and pretreatment with EPA or AA further enhanced the expression, almost to an equal degree, as estimated by flow cytometry (P < 0.05). These results indicate that the modulation of ICAM-1 expression does not seem to be EPA-specific, but is presumably a consequence of increased membrane fluidity due to the increased levels of unsaturated fatty acids of both the n-3 and n-6 series in the membrane. PMID- 7547388 TI - Effects of retinoids on glycosaminoglycan synthesis by human skin fibroblasts grown as monolayers and within contracted collagen lattices. AB - Fibroblasts grown within contracted collagen lattices synthesize substantially less glycosaminoglycans than fibroblasts grown as monolayers on a plastic substrate. [3H]glucosamine incorporation into hyaluronate was reduced by 70%, and incorporation into sulphated glycosaminoglycans was reduced by 40%. However, incorporation into heparan sulphate and chondroitin sulphates was reduced by 14 and 49%, respectively, resulting in a substantial change in the proportions of the individual glycosaminoglycans. On the basis of [3H]glucosamine incorporation, hyaluronate constituted 80% of the total glycosaminoglycans synthesized in monolayer cultures, but only 67% in collagen lattice cultures. Incorporation of 35SO4 into chondroitin sulphates was reduced by 22%, whereas no change was observed in heparan sulphates following culture within collagen lattices. Exposure of the fibroblast cultures to retinoic acid (10(-6) mol/l) and retinyl propionate (2 x 10(-6) mol/l) resulted in a decrease in the incorporation of [3H]glucosamine into hyaluronate by up to 41% in monolayer cultures, and 25% in collagen lattice cultures. The retinoids stimulated the incorporation of [3H]glucosamine into heparan sulphate by up to 72%, and chondroitin sulphates by up to 30%, whereas 35SO4 incorporation remained essentially unaltered. Only modest changes in the incorporation of both isotopes into fibroblast sulphated glycosaminoglycans were observed following exposure to the retinoids in lattice cultures. Q-Sepharose ion-exchange chromatography at pH 2.0 revealed that there was no change in the degree of polymer sulphation of either chondroitin sulphate or heparan sulphate isolated from collagen lattice cultures compared with monolayer cultures. Retinoic acid (10(-6) mol/l) treatment did, however, reduce the degree of polymer sulphation of heparan sulphates and chondroitin sulphates in both monolayer and lattice cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547389 TI - Influence of the extracellular matrix on fibroblast responsiveness to phenytoin using in vitro wound healing models. AB - Recent reports indicate that the topical administration of phenytoin to cutaneous wounds can promote repair. However, isolated skin cells (keratinocytes and fibroblasts) in vitro have varied in their response to phenytoin, giving rise to apparently contradictory results. We have examined how the structure of the extracellular matrix in which human dermal fibroblasts are grown in vitro can influence the response of these cells to phenytoin. The results indicate that, when fibroblasts are embedded within freely-contracting, relaxed, types I collagen matrices, they are insensitive to phenytoin treatment. However, if fibroblasts are grown in collagen matrices which are nonretracting and under tension, phenytoin (5-50 micrograms/ml) significantly (P < 0.01) stimulates cell proliferation, and inhibits collagenase activity in a dose- and time-dependent manner. The fact that the effects of phenytoin on dermal fibroblasts are biphasic and influenced by the surrounding matrix may help to explain why in vitro investigations with phenytoin give rise to inconsistent data. It also suggests that the matrix alterations which accompany wound healing may modulate the effects of phenytoin on dermal fibroblasts. PMID- 7547390 TI - Changes in epidermal proliferation and differentiation in allergic and irritant contact dermatitis reactions. AB - Allergic and irritant contact dermatitis are similar clinically, histologically and on immunohistochemistry. In the present investigation, we assessed whether study of the recruitment of cycling epidermal cells, and the expression of keratin 16 and involucrin, are of use in differentiating between the response to contact allergens and the response to the irritant detergent sodium lauryl sulphate. Both allergic and irritant challenges induced epidermal proliferation, and the expression of keratin 16 and involucrin, but the dynamics were different. Two and 3 days after challenge, a highly significant difference between the allergic and irritant reactions was observed with respect to involucrin expression assessed by MON-150 staining. PMID- 7547391 TI - In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients. AB - The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients. PMID- 7547393 TI - The pathogenesis of hidradenitis suppurativa: a closer look at apocrine and apoeccrine glands. AB - We undertook a retrospective pathological study of 118 skin resection specimens from 101 patients with hidradenitis suppurativa. Follicular occlusion was identified in all the specimens, regardless of disease duration (1 month to 18 years), but was not noted in the axillary and inguinal skin of controls. We therefore regard follicular occlusion as an early and important feature in the pathogenesis of the disease. The presence of apoeccrine glands in axillary skin provided an in vivo model to directly observe the effects of follicular occlusion on follicle inflammation and apocrine gland destruction. In the majority of cases, active folliculitis was associated with apocrinitis and apocrine destruction, whereas apoeccrine glands, which drain directly on to the epidermal surface, appeared intact and non-inflamed. These observations provide direct evidence in an in vivo model that follicular occlusion by keratinous material, with subsequent active folliculitis and secondary destruction of the skin adnexae and subcutis, occur as an integral step in the pathogenesis of hidradenitis suppurativa. PMID- 7547392 TI - Clinical, ultrasound and hormonal markers of androgenicity in acne vulgaris. AB - Androgenic stimulation of sebaceous glands is an important factor in the development of acne. We examined 36 females (aged 14-34 years), selected because none had received oral contraceptives, anti-androgen therapy, or systemic antibiotics during the previous year, or isotretinoin therapy, prior to their participation in the study. Subjects were divided into groups on the basis of acne severity, as follows: physiological, mild and moderate. Only two patients had polycystic ovaries on ultrasound examination. Seven patients had irregular menses; none had evidence of hirsutism. We found that the severity of acne, based on the acne grade, was highly correlated with the inflammatory lesion count, and less correlated with the sebum excretion rate. Either acne grade or inflammatory lesion count could be related to some of the five androgenic hormone determinants; free testosterone (TESTOS), delta 4 androstenedione (DELTA 4), sex hormone binding globulin (SHBG), dehydroepiandrostenedione sulphate (DHEAS) and dihydrotestosterone (DHT). Multiple linear regression analysis determined the best model for predicting ACNE scores as involving DELTA 4 and DHEAS (positive effects), and SHBG (negative effect), P < 0.005, R2 = 0.36). In none of the patients were the levels of DHEAS or SHBG outside the normal range. The findings in the two patients with polycystic ovaries did not differ significantly from those in the remainder of the patients. PMID- 7547394 TI - Pharmacokinetics of topical hydrocortisone at plasma level after applications once or twice daily in patients with widespread dermatitis. AB - Percutaneous absorption of hydrocortisone was measured by determining plasma cortisol during dexamethasone suppression in 26 patients with widespread atopic dermatitis. The first and second days of treatment with applications of 1% hydrocortisone cream twice daily were studied separately in two groups of six patients. Plasma cortisol levels rose after the first two applications, reaching a maximum in 24 h. The levels then began to fall, indicating possible restoration of the skin barrier. In two other groups of seven patients, the second application was made with a cream base alone. Two types of cream base were studied, one with 60% water and the other with 30% water. With the base containing 30% water, a brief increment was seen after 2 h. On the basis of this pharmacokinetic study, treatment of acute dermatitis could be intensified by applying hydrocortisone cream twice a day on the first day, but from the second day onward one application a day seems to be sufficient. PMID- 7547395 TI - Protection of children against sunburn: a survey of parental practice in Leicester. AB - The incidence of melanoma in the U.K. is increasing more rapidly than that of most other malignant tumours. Sunburn in childhood increases the risk of malignant melanoma in later life and it is therefore essential that protection of children is improved if primary prevention of melanoma is to be effective. We asked 238 parents in Leicester how they protected their children against sunburn, how often their children suffered sunburn, and whether they had heard of malignant melanoma. Although most (80%) had heard of melanoma, 47% did not regularly ensure that their children used a sunblock lotion, and only 34% regularly protected them from the midday sun. Forty-eight per cent of parents stated that their children burned at least once a year. New approaches to public education about melanoma may be needed to improve the protection of children against sunburn. PMID- 7547396 TI - Transverse leukonychia of toenails due to repeated microtrauma. AB - Twelve women were seen with multiple transverse bands on the nail plates of the great toes. The bands were separated by normal nail, and had the same contour as the distal edge of the lunula. This disorder, which is usually bilateral, results from pressure on the free edge of the nail plate when it is not trimmed short. PMID- 7547398 TI - Laser therapy of childhood haemangiomas. AB - A total of 37 haemangiomas were treated in 28 patients by means of the flashlamp pumped pulsed dye laser (FPDL). Twenty-nine haemangiomas were classified as superficial, and eight as mixed type. With FPDL treatment, excellent or good results were obtained in nearly 60% of superficial haemangiomas and 40% of mixed haemangiomas. In three of four patients with mixed type haemangiomas good results were obtained by means of Nd: YAG laser irradiation. In two patients, small superficial haemangiomas were removed by argon laser coagulation or CO2 laser vaporization, respectively. These results indicate that many children benefit from laser therapy of haemangiomas. Side-effects were rare. The FPDL is the treatment modality of choice for superficial haemangiomas, and the Nd: YAG laser for thicker lesions. Based on our results and the results reported in the literature, early laser therapy of childhood haemangiomas can be recommended. PMID- 7547397 TI - Dapsone versus topical immunotherapy in alopecia areata. AB - Twenty-seven patients with severe alopecia areata were treated with dapsone. The results of a mean treatment duration of 10 +/- 0.5 months are reported, and compared with the results of long-term topical immunotherapy obtained previously at the same institute. The efficacy of dapsone proved to be markedly inferior to that of topical immunotherapy. The percentage of patients showing regrowth of hair during treatment with dapsone was comparable with the occurrence of spontaneous regrowth of hair reported in the literature. PMID- 7547399 TI - Photodynamic therapy of epithelial skin tumours using delta-aminolaevulinic acid and desferrioxamine. AB - Photodynamic therapy (PDT) uses photosensitizing drugs, such as porphyrins, and light for cancer treatment. In the present clinical study we employed topical application of the porphyrin precursor delta-aminolaevulinic acid (ALA) in combination with desferrioxamine (df) for the induction of endogenous porphyrin synthesis. Irradiation was performed with a light source consisting of a halogen lamp with a red filter and a fibreoptic device. Irradiances ranged from 50 to 300 mW/cm2. We treated 49 patients with this PDT regimen. In 32 patients we treated a total of 34 superficial basal cell carcinomas (BCCs) and 22 nodular BCCs, in nine patients 43 solar keratoses, and in eight patients 10 lesions of Bowen's disease. After a single treatment, 30 (88.2%) of the superficial and seven (31.8%) of the nodular BCCs, 35 of 43 (81.4%) solar keratoses, and three (30%) of the Bowen's disease lesions showed complete remission. The post-treatment observation period was up to 20 months. Repeat therapy was required in six nodular and four superficial BCCs. Biopsies were obtained before treatment, and at variable intervals after treatment. Topical PDT of skin tumours with ALA-df-induced porphyrins is effective in the management of epithelial skin tumours. PMID- 7547400 TI - Adjuvant high-dose intravenous gammaglobulin in the treatment of pemphigus and bullous pemphigoid: experience in six patients. AB - At present, initial high-dose prednisone is the treatment of choice for patients with pemphigus and bullous pemphigoid. To reduce the risks associated with long term corticosteroid treatment, other immunosuppressants are often given as steroid-sparing agents. Occasionally, the dose of steroids cannot be reduced. In this study, we report six patients with pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid, in whom the daily corticosteroid dose could only be tapered to acceptable, effective, maintenance levels following treatment with high-dose intravenous gammaglobulin. PMID- 7547401 TI - Demodicidosis or rosacea: what did we treat? AB - We report a 75-year-old man with a fulminant rosacea-like eruption, suggestive of demodicidosis. Multiple Demodex folliculorum mites were found in facial scales and pustules and, on histological examination, in the infundibulum of pilosebaceous follicles and in the dermis. Intradermal mites were surrounded either by polymorphonuclear granulocytes and histiocytes, or by a granulomatous infiltrate containing foreign-body giant cells, which had phagocytosed the parasites. Complete recovery, with disappearance of facial mites, was achieved by treatment with a combination of oral and topical metronidazole, although this drug is not known to be miticidal. PMID- 7547403 TI - Late-onset focal dermal elastosis: clinical and histological features. AB - We describe two patients with lesions clinically resembling pseudoxanthoma elasticum and histologically exhibiting focal elastosis, with normal-appearing elastic fibres in the mid- and deep dermis. We consider that these skin lesions represent a previously undescribed entity, whose pathogenesis appears to be related to the ageing process. PMID- 7547402 TI - Pigmented pedunculated malignant melanoma. A rare variant of nodular melanoma. AB - We report a 44-year-old man who presented with a rapidly growing, deeply pigmented, pedunculated tumour. The lesion was diagnosed as a pedunculated malignant melanoma, a rare variant of nodular malignant melanoma. The clinical presentation of this tumour and its histological features are discussed. PMID- 7547404 TI - Paget's disease of the scrotum: report of a case with underlying carcinoma of the prostate. PMID- 7547405 TI - Multinucleate cell angiohistiocytoma: treatment with argon laser. AB - Multinucleate cell angiohistiocytoma mainly affects middle-aged women, and usually presents as grouped reddish-brown papules. Histopathological features include the presence of multinucleate cells in the reticular dermis, and numerous dilated dermal vessels. The lesions are benign, and may persist for years. Treatment is needed for cosmetic reasons. We report the successful use of the argon laser to treat two patients who had multinucleate cell angiohistiocytoma. PMID- 7547406 TI - Methotrexate for the treatment of severe mucocutaneous lupus erythematosus. AB - We report three women with severe, chronic mucocutaneous lupus erythematosus who showed a significant improvement during oral methotrexate therapy; the improvement was sustained after the drug was discontinued. Their disease had previously been refractory to a wide range of conventional systemic immunosuppressive therapies. Other treatments were progressively withdrawn, and control was maintained, for a variable length of time, with methotrexate alone, or with a small dose of prednisolone. PMID- 7547407 TI - Congenital agminated segmental naevi. AB - We report a 39-year-old woman with agminated segmental naevi on the right side of her face. The lesions were present at birth, and she had no other congenital abnormalities. PMID- 7547408 TI - Successful surgical treatment of severe pretibial myxoedema. AB - We report a 46-year-old man with severe and long-standing pretibial myxoedema, who responded well to local surgical treatment. This improvement has been maintained over a 12-month period, perhaps because of concomitant treatment with octreotide. PMID- 7547409 TI - A case of Behcet's disease aggravated by gingival infection with methicillin resistant Staphylococcus aureus. AB - We report a case of Behcet's disease aggravated by gingivitis and carious teeth infected with methicillin-resistant Staphylococcus aureus. Recurrent severe ulcers in the mouth, and on the genitalia and legs were closely linked with the infection, and dramatically improved after extraction of the carious teeth and administration of systemic vancomycin hydrochloride. PMID- 7547410 TI - Acrokeratosis paraneoplastica (Bazex syndrome) occurring with acquired ichthyosis in Hodgkin's disease. AB - We report a patient who exhibited two paraneoplastic skin disorders, acrokeratosis paraneoplastica and acquired ichthyosis, in association with Hodgkin disease. Treatment of the Hodgkin's disease resulted in rapid improvement of both skin disorders, confirming a causal relationship. The simultaneous presence of two paraneoplastic skin disorders suggests a common pathogenetic factor. Transforming growth factor alpha, produced by tumour cells, may play a part in the aetiopathogenesis of hyperproliferative paraneoplastic skin conditions. PMID- 7547411 TI - Recurrent chancriform pyoderma: report of a case with tongue lesions. Is Staphylococcus aureus implicated? AB - We report a case of recurrent chancriform pyoderma. Staphylococcus aureus was isolated from only one of seven lesions. Immunological responses to S. aureus, as measured by peripheral neutrophil function, were normal. We question the notion that chancriform pyoderma represents an abnormal immune response to a primary staphylococcal infection. To our knowledge, this is only the third reported patient with lesions affecting the tongue. PMID- 7547412 TI - Very short duration therapy with oral terbinafine for fingernail onychomycosis. PMID- 7547413 TI - Bilateral symmetrical pilar leiomyomas on the breasts. PMID- 7547414 TI - Classic (non-AIDS-related) Kaposi's sarcoma in a Japanese patient, successfully treated with alpha-2b-interferon. PMID- 7547415 TI - Neonatal dermal erythropoiesis associated with severe rhesus immunization: amelioration by high-dose intravenous immunoglobulin. PMID- 7547416 TI - Congenital alternate nail dystrophy. PMID- 7547417 TI - Toxic epidermal necrolysis associated with severe hypocalcaemia, and treated with cyclosporin. PMID- 7547418 TI - Pityriasis lichenoides in a family. PMID- 7547419 TI - Rapid laboratory confirmation of Fabry's disease: a reminder. PMID- 7547420 TI - Evaluation of nurse prescribing in a dermatology unit. PMID- 7547421 TI - Skin nodules in palmar fasciitis and polyarthritis associated with carcinoma. PMID- 7547422 TI - Serum lipoprotein (a) levels in Behcet's disease. PMID- 7547423 TI - Bullous skin reaction induced by omeprazole. PMID- 7547424 TI - Clinical and biological effects of balneotherapy with selenium-rich spa water in patients with psoriasis vulgaris. PMID- 7547425 TI - Matrix and cell interactions in connective tissue. British Connective Tissue Society Meeting. Manchester. Abstracts. PMID- 7547427 TI - Strain differences in vulnerability of hippocampal neurons to transient cerebral ischaemia in the rat. AB - Strain differences in the vulnerability of hippocampal neurons to an ischaemic insult were investigated in Sprague-Dawley, Wistar and Fischer 344 rats. Transient global brain ischaemia was produced for 5 minutes by a combination of bilateral carotid artery occlusion and oligaemic hypotension (40 mmHg) induced by exsanguination. The number of viable neurons in the CA1 subfield was counted under a light microscope 7 days after the ischaemic insult. The density of viable neurons in the CA1 subfield of normal rats was around 150 cells/mm of CA1 length in all the strains examined; after global brain ischaemia, this parameter in Sprague-Dawley, Wistar and Fischer 344 strain rats was approximately 110, 120, and 70, respectively. The results suggest that the hippocampal CA1 neurons of Fischer 344 strain rats are more vulnerable to ischaemic insult than those of the other strains. There were many F344 rats (8/21) which showed atypical vasculature patterns in the posterior region of the circle of Willis, suggesting less blood flow through anastomosis from basilar artery to posterior cerebral artery and/or ill-balanced blood flow between left and right hemispheres. These anatomical variations in the circle of Willis may in part contribute to the strain differences in the vulnerability to cerebral ischaemia. PMID- 7547426 TI - The mesothelial cell and its role in asbestos-induced pleural injury. PMID- 7547428 TI - Growth retardation in pregnancy: experimental model in the rabbit employing electrically induced thermal placental injury. AB - Intrauterine growth retardation involves haemodynamic modifications in the uterus and placenta. In order to study intrauterine growth retardation we obtained an electrically induced thermal placental injury in a rabbit experimental model. The effects on weight of fetuses, placentas and single organs were recorded and statistically analysed. Fetal growth retardation was recorded in 71% of fetuses exposed to 15 mA direct current per 40 seconds, and resulted in only 7.9% of normal fetuses. The data presented in this study have shown a retardation of fetal growth particularly evident in the liver weight with a relative sparing of brain development. Kidney and heart do not present a statistically significant reduction in growth. This growth retardation model is simple and readily reproducible. PMID- 7547429 TI - Assessment of hepatobiliary function and placental barrier integrity in pregnant rats by combination of bromosulphophthalein and fluorescein isothiocyanate plasma disappearance tests. AB - The permeability of the placental barrier to bromosulphophthalein (BSP) is believed to be very low. Whether this property is shared by other cholephilic organic anions, such as fluorescein isothiocyanate (FITC), is not known. When BSP was injected (140 mumol/kg body wt) into the left jugular vein of non-pregnant or pregnant female rats (at the 21st day of pregnancy), a similar and rapid plasma disappearance was observed during the first few minutes; afterwards, a slower disappearance phase was found. This phase was different in these groups, that is, a longer retention of BSP in the maternal bloodstream of pregnant rats was accompanied by a slower BSP output into bile. It was impossible to demonstrate the presence of BSP in fetal blood or the placenta by colorimetric methods. These results are consistent with the modifications occurring in the hepatic handling of BSP during pregnancy together with a marked impermeability of the placenta to the dye, at least in the mother-to-fetus direction. After intravenous FITC (10 mumol/kg body wt) administration to the mother, the compound was rapidly transferred into both the maternal bile and the fetal blood. Thereafter, FITC refluxed back from the fetal-placental compartment to the maternal blood as soon as the maternal liver reduced its plasma concentrations, which were first higher (approximately threefold) and then similar to those found in fetal blood. The reversible retention of FITC by the fetal-placental compartment accounts for a significant delay in both FITC bile output and plasma disappearance as compared with those observed in non-pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547430 TI - Different effect of cyclosporin A on arthritides induced by a muramyl dipeptide analogue or the complete adjuvant in rats. AB - Muramyl dipeptide-Lys(L18) given by daily subcutaneous injection for 14 days induced arthritis in male Lewis, Fischer and nude rats, to a lesser degree in the last two than in the first strain. On the other hand, a single intradermal administration of Freund's complete adjuvant induced arthritis in Lewis and Fischer but not nude rats. Cyclosporin A (CsA) co-administered for 14 days markedly exacerbated the muramyl dipeptide-Lys(L18) induced arthritis (MIA) in rats of all three strains, but completely blocked the development of the adjuvant induced arthritis (AIA) in Lewis rats. Furthermore, AIA was transferred to recipient Lewis rats through spleen cells obtained from the donors with AIA, whereas MIA was not. Antibodies against type II collagen and DNA were not detected in sera from MIA or AIA rats. These data show a clear difference between MIA and AIA in the pattern of development of arthritis and suggest that delayed hypersensitivity reactions may be involved in the pathogenetic mechanisms of AIA, but not in MIA. PMID- 7547431 TI - Detection of herpesvirus DNA in cottontop tamarins: no association with colitis. AB - Colitis occurs spontaneously in the cottontop tamarin; it shares similar clinical, endoscopic and histological features with, and has a similar response to treatment as, human ulcerative colitis. An association between human ulcerative colitis and the presence of DNA from multiple herpesviruses (human herpesvirus 6 (HHV-6), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in the colon has been described. Intestinal herpesvirus infection may be involved in the pathogenesis of this condition. In a pilot study, coded full thickness colonic specimens from 27 cottontop tamarins were examined for the presence of HHV-6, CMV and EBV DNA using the nested polymerase chain reaction. The sections represented a spectrum of disease activity ranging from histologically normal bowel to severe colitis. A low prevalence of DNA from these viruses was found, which bore no relation to the presence of inflammation. Although these herpesviruses may have a pathogenetic role in human ulcerative colitis, they are not associated with colitis in this animal model. PMID- 7547432 TI - Induction of osteoclast characteristics in cultured avian blood monocytes; modulation by osteoblasts and 1,25-(OH)2 vitamin D3. AB - It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro. PMID- 7547433 TI - The characterization of a rabbit model of inflammatory bowel disease. AB - The absence of a simple, clinically relevant, animal model of inflammatory bowel disease (IBD) hampers research into this disease. In this study, colitis was induced in rabbits by intracolonic installation of 2,4,6-trinitrobenzene sulphonic acid (TNB) in 25% ethanol. Rabbits were killed from zero hours to 6 weeks and their colons examined. Rabbits were examined by endoscopy at weekly intervals. A single dose of TNB in ethanol produced dose dependent inflammation and ulceration, which at its optimum (40 mg) resulted in cobblestoning, strictures, and bowel wall thickening. The damage score at endoscopy was consistent with the score on macroscopic examination of the colon. Histopathological features of inflammation and ulceration observed in all animals that received 40 mg TNB included crypt abscesses, ulceration, crypt architectural distortion and, occasionally, granulomas and pseudopolyps. These changes, which are similar to those observed in IBD, persisted for 6 weeks. No lasting abnormalities were observed in control animals treated with TNB in saline, with ethanol alone, or with saline only. Histopathological similarity and the prolonged duration of inflammation, compared to other models, make this a suitable model for investigating inflammation in the colon. Furthermore, the model is accessible to endoscopy which adds to its value in experimental studies. PMID- 7547434 TI - Ebola virus transmission. PMID- 7547436 TI - Serotonin causes acute gastric mucosal injury in rats, probably via 5HT1D receptors. AB - 5-HT-induced acute gastric mucosal injury was assessed in rats by using 5HT1, 5HT2, 5HT3, 5HT4 or muscarinic receptor related drugs. Rats were treated with antagonists i.p. and 30 minutes later either vehicle, 5-HT (20 mg/kg) or other agonists were administered s.c. The stomachs were removed 4 hours after the last injection and mucosal integrity was assessed by light microscopy using a histological ulcer index (HUI). The HUI was found to be significantly increased following 5-HT administration (1.57 +/- 0.3) when compared with controls (0.14 +/ 0.1). 5HT1 agonist 5-carboxamidotryptamine (20 mg/kg) produced acute gastric erosion and increased the HUI (P < 0.05). The HUI in the animals receiving 5-HT1D agonist sumatriptan (7 mg/kg) was found to be 1.62 +/- 0.24. 5HT2 antagonist ketanserine (2.5-15 mg/kg), 5HT3 antagonist ondansetron (1-5 mg/kg), 5HT4 antagonist DAU 6285 (1-10 mg/kg) and atropine (1.5-30 mg/kg) exerted no effect whereas 5HT1/2 antagonist metitepine (0.05-0.5 mg/kg) caused a dose dependent inhibition of the effect of 5-HT. The results from this study demonstrate that 5 HT causes acute gastric mucosal injury and this injury is probably due to the activation of the 5-HT1D receptors. PMID- 7547435 TI - Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. AB - The potential of aerogenic infection by Ebola virus was established by using a head-only exposure aerosol system. Virus-containing droplets of 0.8-1.2 microns were generated and administered into the respiratory tract of rhesus monkeys via inhalation. Inhalation of viral doses as low as 400 plaque-forming units of virus caused a rapidly fatal disease in 4-5 days. The illness was clinically identical to that reported for parenteral virus inoculation, except for the occurrence of subcutaneous and venipuncture site bleeding and serosanguineous nasal discharge. Immunocytochemistry revealed cell-associated Ebola virus antigens present in airway epithelium, alveolar pneumocytes, and macrophages in the lung and pulmonary lymph nodes; extracellular antigen was present on mucosal surfaces of the nose, oropharynx and airways. Aggregates of characteristic filamentous virus were present within type I pneumocytes, macrophages, and air spaces of the lung by electron microscopy. Demonstration of fatal aerosol transmission of this virus in monkeys reinforces the importance of taking appropriate precautions to prevent its potential aerosol transmission to humans. PMID- 7547437 TI - Histomorphometry of long bone growth plate in swimming rats. AB - In exercises involving running, muscle power and gravitational forces act together to affect bone mass in accordance with Wolff's law. However, the direct effect of muscle activity on bones in non-weight-bearing activities, such as swimming, has not been explored. Previous data indicate that swimming exerts a positive effect on bone growth and development in young rats. We performed a histomorphometric study on the effect of swimming on the growth plate and subepiphyseal area of young adult rats. The experiments were carried out on 28 12 week-old albino Sabra rats. One group of 14 rats was trained to swim 1 hour/day, 5 days a week, for 12 weeks. Another group of 14 rats served as controls. The proximal femur and humerus of each animal were examined histomorphometrically. There was an increase in the subepiphyseal cancellous bone trabecullae of the femur. In the growth plate there was an increase in the number of column cells and proliferative cells. These changes were more pronounced in the femur than the humerus. We conclude that swimming induces an increase in subepiphyseal cancellous bone in young adult rats by enhancing growth plate activity. PMID- 7547438 TI - Effects of cyclosporin A on some accessory cells of rat thymus. AB - We used immunohistochemistry with monoclonal antibodies (TRPM1, TRPM2) and histochemistry (acid phosphatase (AcP)) to investigate the effects of cyclosporin A (CsA) on macrophages and interdigitating cells (IDCs) in adult rat thymus after 21 days of treatment, and 21 days after stopping treatment. We also studied the development of IDCs and macrophages in 2, 6, 12, 20 and 30-day-old rats after 21 days of CsA administration to the pregnant mothers. In adult rats after 21 days of CsA treatment, IDCs were absent and only a small number of macrophages were present in the cortex; 21 days after stopping treatment the distribution of IDCs and macrophages had become similar to that in normal adults. The AcP+ macrophages in treated adult rats disappeared, as shown by immunohistochemistry, 21 days after CsA treatment and were again present, similarly to control animals, 21 days after stopping treatment. Therefore CsA causes the thymus medulla of adult rats to disappear and also a significant decrease in the macrophage population. We also found that while in normal rat neonates the thymus has the features of the adult thymus by the 12th day, in neonates from CsA treated mothers this did not appear until the 30th day. CsA treatment to pregnant rats delays thymus development in the young animals but does not cause persisting morphological alterations. This last finding was similar to that observed in adult rats 21 days after the end of CsA treatment. PMID- 7547439 TI - Pathologic studies and comparison of the virulence of herpes simplex virus type 2 from Okinawa, Japan and Chiang Mai, Thailand. AB - The virulence of four herpes simplex virus type 2 (HSV 2) strains (K1-K4) isolated in Okinawa, Japan was investigated, and compared with four strains (C1 C4) from Chiang Mai, Thailand and a standard laboratory strain SAV. Virulence was tested on BALB/c and C57/black mice. After viral inoculation intraperitoneally, the distribution with the passage of time of the virus in the brain and other organs was also studied using the polymerase chain reaction (PCR) method and immunohistochemistry. Generally, Okinawan HSV 2 (K1, K3 and K4) were less virulent than Chiang Mai strains (C1-C4). Among the Okinawan strains, K2 was the most virulent, but slightly less so than C3 and C4. All four Chiang Mai strains (C1-C4) and one Okinawan strain (K2) were more virulent than SAV strain. The virulence of K3 was very weak and no animals died from the intraperitoneal inoculation. In the brain, viral DNA from each strain was demonstrated at 1-9 days after inoculation by the PCR method. However, K3 strain was detected in the brain only between one day and 3 days after virus inoculation, and not after day 5. Immunohistochemically, the virus antigen was first demonstrated around the 3rd ventricle at one day after viral inoculation, then strongly at the ventral hypothalamus and the temporal lobe at 3 days after viral inoculation, and slightly in the frontal lobe, hippocampus, pons and cerebellum (on day 5 after inoculation). Furthermore, in Kupffer cells in the liver and macrophages in the spleen, numerous viral antigens were demonstrated from one to 9 days after viral inoculation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547440 TI - Cell proliferation and apoptosis in isoprenaline-induced sialosis in the rat submandibular glands. AB - Regression of submandibular acinar cell hyperplasia after withdrawal of the mitogenic stimulus induced by isoprenaline was studied in male Wistar rats. Intraperitoneal administration of 39 mg of isoprenaline, in divided doses over a 9-day period, resulted in a marked increase in gland size and weight. The proportional volume occupied by acinar cells increased. These changes were associated with acinar cell proliferation and an increase in the mitotic index and cytoplasmic: nuclear ratio. Thus, both hyperplasia and hypertrophy clearly contributed to gland enlargement. Granular and other duct cells appeared to be unaffected by isoprenaline administration. Following cessation of the drug, the mitotic index reached a peak value by the second day but thereafter declined rapidly. This change was matched by a marked rise in the apoptotic cell index, the latter reaching a maximum by the fourth day. By the end of the second week, glands had returned to normal with respect to size and weight and neither hyperplasia nor apoptosis could be detected histologically. These results confirm that apoptosis is involved in the regulation of submandibular salivary gland size. PMID- 7547441 TI - Expression of xenobiotic metabolizing enzymes in tumours of the urinary bladder. AB - The cytochromes P450, epoxide hydrolase and glutathione S-transferases are several of the major groups of enzymes involved in the metabolism of xenobiotics and these enzymes may have a role in influencing the response of tumours to anti cancer drugs. In this study the cell specific expression of individual xenobiotic metabolizing enzymes has been investigated using immunohistochemistry in primary transitional cell tumours of the urinary bladder. The cytochromes P450 CYP1A, CYP2C and CYP3A, were present in 68, 28 and 68% of tumours respectively and the expression of CYP1A correlated with bladder tumour grade (P = 0.03). Epoxide hydrolase was identified in 84% of tumours while the alpha, mu and pi forms of glutathione S-transferase were expressed in 56, 72 and 52% of tumours respectively. In normal bladder epoxide hydrolase and glutathione S-transferase pi were the main enzymes expressed while there was no expression of CYP2C. PMID- 7547442 TI - In situ proliferating status of mononuclear cells in rat small bowel grafts. AB - Rat small bowel transplantation was performed in three groups: syngeneic graft, Lewis to Lewis; allogeneic graft, Brown Norway to Lewis; and allogeneic graft with the immunosuppressant FK506 (1 mg/kg). Standard histology and bromodeoxyuridine (BrdU) labelling immunohistochemistry were applied to evaluate the in situ spontaneous proliferating status of the mononuclear cells in the graft lymphoid tissue. In untreated allograft, BrdU-labelling index (BLI) in lamina propria lymphocytes increased significantly on the 5th post-operative day (POD), when no sign of rejection was detected in the villous architecture, compared with isograft (11.8 +/- 2.0% vs 1.9 +/- 0.5%, P < 0.05). Changes in the BLI of lamina propria lymphocyte were also detected on the 3rd and 7th POD, although there were statistically less significant than on the 5th POD. In intraepithelial lymphocytes the changes in BLI were more probably caused by ischaemic damage than immunological reaction. In Peyer's patches and mesenteric lymph nodes, elevation of BLI was specific to rejection like that seen in lamina propria lymphocytes. By administration of FK506, both the elevation of BLI and the histopathological changes were suppressed successfully except for a transient elevation of BLI in Peyer's patches and mesenteric lymph nodes on the 3rd POD. PMID- 7547443 TI - Alterations of surfactant lipid turnover in silicosis: evidence of a role for surfactant-associated protein A (SP-A). AB - Type II cell hypertrophy with surfactant accumulation in the lung is a common observation in silicosis. Mechanisms leading to these alterations are poorly understood. By using silica dusts and alveolar fluids from saline and silica exposed sheep, we explored four different pathways of surfactant turnover in vitro: (1) synthesis and (2) secretion of lipids by rat type II cells; and dipalmitoylphosphatidylcholine (DPPC) uptake/reuptake by (3) type II cells and (4) alveolar macrophages. Silica had no direct specific effect on type II cell lipid metabolism. Alveolar fluids from both saline and silica exposed animals induced several alterations compared to control medium: (a) an increase in lipid synthesis (60 to 130%, P < 0.05); (b) a decrease in lipid secretion (25 to 70%, P < 0.05); (c) a 50 to 75% increase in DPPC reuptake by type II cells (P < 0.05); (d) a 65 to 75% decrease in DPPC uptake by alveolar macrophages (P < 0.05). DPPC uptake by in vivo silica exposed alveolar macrophages was reduced. Alterations of surfactant lipid metabolism induced by alveolar fluids from silicotic animals was more pronounced than in those treated with control fluids. Anti SP-A antibodies significantly suppressed most of the alveolar fluid induced effects on surfactant turnover. From these in vitro data, silica-induced type II cell hypertrophy seems to result from an increase in lipid synthesis activity and an imbalance in the lipid secretion/reuptake ratio. PMID- 7547445 TI - Vascular medicine: an alternative approach to arterial disease. AB - The National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) is leading a nationwide initiative to develop comprehensive programs that integrate clinical care, education, and research in vascular disease by developing vascular centers designed to provide comprehensive patient care through a multidisciplinary approach. Only a few medical centers have a dedicated vascular medicine specialty. To date, 11 centers nationwide have received NIH support devoted to creating vascular centers and promoting vascular medicine as a specialty. In June 1992, Northwestern University/Northwestern Memorial Hospital received one of these awards. The NIH recognizes the complex medical problems in patients with vascular disease. The resulting necessity for care by multiple physicians has traditionally led to duplication and fragmentation of services. Until recently, vascular surgery programs have focused primarily on the surgical treatment of vascular disease. This approach does not address risk reduction and the diverse care needs of the population of patients with vascular disease. PMID- 7547444 TI - Morphological and morphometric study of atrial specific granules and other secretory components in dogs experimentally infected with Trypanosoma cruzi. AB - Changes in blood volume can induce morphometric and morphological alterations in the secretory complex of the myoendocrine cells due to the stretching of atrial walls. These alterations were studied by electron microscopy, using dogs infected intraperitonially with Trypanosoma cruzi and necropsied during the acute phase of the infection when congestive heart failure was present. Several changes were observed in the myoendocrine cells of the heart: hypertrophy and hyperplasia of rough endoplasmic reticulum and Golgi complex, increase in telenuclear secretory complex, increase in fusion of type B atrial specific granules (ASG), decrease of the total number of ASG, enlargement of the maximum diameter of type A ASG and a relative increase in the number of type B ASG. These alterations suggest a larger secretory activity of the atrial myoendocrine cells with a larger secretion of atrial natriuretic peptide (ANP). PMID- 7547446 TI - Postoperative lower extremity bypass surveillance: beyond ankle arm blood pressures. AB - Surveillance is essential to the postoperative follow-up of lower extremity bypass grafts. Early, intermediate, and late thrombosis place the patient's limb at risk, so detection of problems before the graft fails is critical. Because contrast angiography is not routinely performed for surveillance, most vascular surgeons rely on history, physical examination, and noninvasive vascular studies (NVS) to assess perfusion to the lower extremity after bypass grafting. These NVS include ankle/brachial waveforms, blood pressures, and indexes before and after exercise. The purpose of this study is to report our findings with duplex color flow ultrasonography (DCU) to examine lower extremity bypass grafts. According to our protocol, we monitor lower extremity grafts with ankle/brachial Doppler pressures, analogue waveforms, and lower extremity exercise when possible. These NVS are performed by nurses in the vascular laboratory before the patient is discharged from the hospital, at least twice during the first year, and then annually. DCU is also performed at least two times during the first year and then annually. If the study results are abnormal or if the patient has symptoms, testing is usually repeated. When abnormalities persist contrast angiography may be warranted. We have detected anatomic and hemodynamic changes in lower extremity bypasses by use of our protocol. By adding DCU to ankle/brachial blood pressures, we have identified aneurysmal dilation, diffuse atherosclerosis, focal narrowing, arteriovenous fistulas caused by unligated venous branches, retained venous valves, and disease progression proximal or distal to the graft.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547447 TI - Defining vascular nursing: a survey of vascular nurses. AB - For the purpose of defining the specialty of vascular nursing, a survey was sent to the nursing membership of the Society for Vascular Nursing. Nurses in all geographic locations were included, and the survey was accompanied by a self addressed, stamped return envelope. A total of 873 nurses were surveyed. Respondents were encouraged to give the survey to other vascular nurses who were not members of the Society for Vascular Nursing. The initial mailing resulted in 276 responses. A second mailing provided an additional 224 responses for a total response of 500. Fifty-six of the 500 respondents (11.2%) were nonmembers. Response rate of members was 50.9% (444 of 873). Demographic information was obtained regarding age, years in nursing and vascular nursing, sex, employment type, and location. In addition to demographic information, the survey asked the respondent to identify a definition or definitions of vascular nursing. There was no restriction on space or content. In a small pilot study, "defining the specialty" was the only question asked. However, on the basis of responses to the pilot, the form was refined to request a description of the skills necessary to be a vascular nurse. A total of 73 definitions were identified on the basis of coded responses. The range in number of definitions given on any survey was 0 to 13. Essential skills listed by any one respondent ranged in number from 0 to 14. A total of 90 skills were identified. The purpose of this study is to identify the definitions of vascular nursing given by vascular nurses. In addition, the skills identified as necessary will be elaborated on.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547448 TI - The registered nurse first assistant role in surgical wound closure: an integrated review. AB - Knowledge of suturing techniques, suturing materials, and the stages of wound healing is essential for obtaining good operative results and avoiding wound infections and scarring. An extensive literature search was undertaken, and a resultant integrated review of the studies was assembled. The literature deals specifically with information on methods of wound closure, wound healing, suturing techniques, and suturing materials in the coronary, surgical, and vascular surgery population. It is hoped that the information provided will give the practicing registered nurse first assistant and other surgical nurses a working knowledge of some of the best methods for the closure of skin wounds, and how best to apply those methods in practice. PMID- 7547450 TI - Position statement: tobacco abuse. The Society for Vascular Nursing. PMID- 7547449 TI - The use of tables, illustrations, and graphs for effective research presentation. AB - Presenting research findings is an essential component of the research process. Visual aids such as tables, illustrations, and graphs enhance the process of presenting research results. Use of these types of visual aids allow the presenter to be more effective and to capture the audience's full attention. PMID- 7547451 TI - Visceral ischemia. PMID- 7547452 TI - The editors' labours: separating the wheat from the chaff. AB - From the author's standpoint, the editor is viewed as 'one who separates the wheat from the chaff and then prints the chaff'! Since its inception, Pediatric Neurosurgery has tried to provide the reader with the guarantee that only the 'wheat' is published. This paper will examine the policies of the journal's Editorial Board and in particular outline the review process for submitted manuscripts. In addition to certain operational items, the paper will consider how the 'peer' in 'peer review' is identified, the grading instruments for paper acceptance, the likelihood that the readers and peer reviewers agree on manuscript quality, and how authors should view failure. The Society is firmly committed to our journal. Our individual responsibilities begin with paper preparation and then its presentation at the annual meeting. Thereafter, it is important for the membership to appreciate that their participation, as either author and/or reviewer, is critical for the continued success of our journal. PMID- 7547453 TI - Reduction cranioplasty. AB - Reduction cranioplasty can greatly improve the quality of life for selected patients with severe macrocephaly and can significantly diminish some of the difficulties in the long-term chronic care of others. Because of differences in age, cranial morphology and surgical goals, the surgical plan must be tailored to the individual patient. Three techniques for reduction cranioplasty, with the advantages and disadvantages of each, are described. Four patients, representing the spectrum of severe macrocephaly and also the problems associated with reduction cranioplasty, are presented. Surgical indications, tactical considerations and risks are discussed. PMID- 7547454 TI - Intracranial pressure in single-suture craniosynostosis. AB - The indications for surgical correction of craniosynostosis in which there is involvement of only one of the cranial vault sutures have traditionally been based upon the cosmetic merits of the deformity alone. Whilst it is now appreciated that intracranial hypertension is commonly associated with the more complex forms of craniosynostosis, this aspect has not previously been addressed in detail among cases of single-suture craniosynostosis. This retrospective study reports our experience of overnight subdural intracranial pressure monitoring in 74 children with premature closure of a single cranial suture. A single coronal suture was involved in 37 patients, the sagittal suture in 25 and the metopic suture in 12. Intracranial pressure was raised in 13 (17%), borderline in 28 (38%) and normal in 33 (45%). Elevated intracranial pressure was seen more commonly where a midline suture was involved (sagittal or metopic) than when a single coronal suture was fused. We conclude that intracranial hypertension occurs in a significant proportion of children with single-suture craniosynostosis and suggest that this factor should be borne in mind during the initial assessment of these children so as to enable timely intervention where required and appropriate counselling of parents. PMID- 7547455 TI - Normal sutural fusion and the etiology of single sutural craniosynostosis: the microspicule hypothesis. AB - Single sutural craniosynostosis is a disorder wherein a calvarial suture fuses prematurely, resulting in an abnormally shaped head. Children afflicted are normal neurologically, and the bone bridging the sutures is normal histologically. The mechanism of normal or pathologic sutural fusion is unknown. These facts prompted the authors to reexamine normal sutural anatomy and the concepts of skull growth in an animal model. Histochemical staining to identify osteoblasts and osteoclasts and tetracycline labeling were performed on neonatal rabbit calvarial sutures. Osteoblasts are found on all bony surfaces including the sutural edges, but do not extend across the sutural space. Thus the periosteum per se does not bridge the suture. Osteoclasts are found only in the diploic space. Thus, there is no mechanism to remove bone at/or within the suture. Tetracycline labeling revealed immense bone production at the suture as compared with dural and periosteal surfaces. Microscopic spicules of bone bridging the suture were identified. Based upon the above observations, we propose a new hypothesis for the mechanism of normal and pathologic sutural fusion. Bony microspicules normally and intermittently form and bridge the suture. As there is no mechanism to remove these spicules, we propose that normal mechanical forces cause them to fracture. A spicule that fails to fracture functions as a scaffold, upon which more bone is deposited, resulting in sutural fusion. Single sutural craniosynostosis results when a normal process occurs prematurely. PMID- 7547457 TI - Acquired Chiari-I malformation and hydromyelia secondary to a giant craniopharyngioma. AB - Hydromyelia, or hydrosyringomyelia is frequently associated with the Chiari I malformation of the cerebellar tonsils. Descent of the cerebellar tonsils is considered a congenital anomaly with a few reports of 'acquired' Chiari I malformation. We report a patient with a giant craniopharyngioma and hydrocephalus who at presentation had a concomitant Chiari I malformation and hydromyelia. The patient underwent gross total resection of the tumor and, with no further treatment, demonstrated spontaneous resolution of the Chiari I malformation and hydromyelia during the postoperative period. This suggests that the Chiari I malformation and the resulting hydromyelia were 'acquired', and were caused by an intracranial mass effect. This provides further evidence for an associative mechanism of cerebellar tonsillar descent and the development of hydromyelia. PMID- 7547456 TI - Syringomyelia of the distal spinal cord in children. AB - The syndrome of syringomyelia in children has been extensively described in the literature and is said to most commonly involve the cervical and thoracic spinal cord. We here present two children who had an unusual constellation of signs and symptoms, characterized by bowel and bladder disturbance in one, and in the other with intermittent paroxysmal severe pain and cramping and flexion of the lower extremities accompanied at times by episodes of incontinence. Both were found on magnetic resonance imaging (MRI) to have a syrinx localized to the most distal cord. This was not associated with Arnold-Chiari malformation, trauma, tumors or any other form of spinal dysraphism. Both patients underwent placement of a syringomyelia-peritoneal shunt with complete resolution of symptomatology. Postoperative MRI revealed a complete collapse of the syringomyelia cavity. In those children presenting with bowel or bladder disturbances, associated or not with intermittent pain, flexion attacks, or cramping in the lower extremities, the differential diagnosis of a syringomyelia of the distal cord should be part of the clinical considerations. PMID- 7547458 TI - Cost analysis of continuous intrathecal baclofen versus selective functional posterior rhizotomy in the treatment of spastic quadriplegia associated with cerebral palsy. AB - The purpose of the study was to analyze the relative cost of selective functional posterior rhizotomy (SFPR) and continuous intrathecal baclofen in the treatment of children with severe spastic quadriplegia related to cerebral palsy. No attempt was made to analyze the efficacy of the two types of treatment. Nine children with spastic quadriplegia secondary to cerebral palsy in whom continuous intrathecal baclofen was attempted were matched as closely as possible with a group of 10 patients with spastic quadriplegia out of a total of 100 children who had undergone SFPR in the same time period. Clinical care flow charts were created to identify the various points of contact with members of the health care team, so that cost points could be identified and costs calculated. The cost per patient up to 1 year a after treatment CDN$ 64,163.10 for patients with implanted pumps for continuous intrathecal baclofen versus CDN$ 16,913.54 for SFPR. When adjustments were made to exclude costs and savings associated with research protocols, the average for the baclofen group decreased to approximately CDN$ 63,000, with minimal change for the SFPR group. The higher cost per patient on baclofen was related to the cost associated with screening patients who did not go on to have implantation of a continuous infusion pump, and to additional hospitalization for complications in the baclofen group. It is cautioned that this cost analysis was based on the experience at British Columbia's Children's Hospital, and the results may not be generalizable to other institutions or to other patient populations. PMID- 7547459 TI - Myelinoclastic diffuse sclerosis presenting as a mass lesion in a child with Turner's syndrome. AB - A rare case of myelinoclastic diffuse sclerosis (MDS), occasionally referred to as Schilder's disease, is reported in a child with Turner's syndrome. The child originally presented with a 3-week history of nausea, vomiting and frontal headaches. Magnetic resonance imaging showed a large, contrast-enhancing, right frontal lobe mass which was ultimately resected uneventfully. Complete laboratory investigations and pathological evaluation of the resected specimen verified the case to be MDS. The clinical presentation, laboratory evaluation, imaging characteristics and diagnosis are discussed in this review of the disease. The importance of including demyelinating diseases in the differential diagnosis for newly discovered mass lesions in the pediatric population is underscored by this case. PMID- 7547460 TI - Intracranial hypertension after cranial vault decompression for craniosynostosis. AB - A 3-year-old boy with chronic papilledema, bilateral coronal synostosis and Pfeiffer syndrome underwent an orbitofrontal advancement. One month postoperatively, his papilledema had resolved, however, his vision progressively deteriorated over the ensuing 3 months to legal blindness. He had no symptoms or signs of increased pressure. Multiple imaging studies were unremarkable except for dilatation of the optic nerve sheaths. A lumbar puncture revealed intracranial hypertension, which resolved after treatment with a lumboperitoneal shunt. Intracranial hypertension may persist after craniofacial reconstruction. In patients who have progressive neurological deterioration after craniofacial reconstruction, direct measurement of intracranial pressure is indicated, despite a lack of clinical signs or symptoms of intracranial hypertension and normal imaging studies. PMID- 7547461 TI - Intracranial hemangiopericytomas in children. AB - Hemangiopericytomas (HPs) are rare tumors, about 10% of which occur in children. Since 1988, 2 children with intracranial HP have been seen at the University of Texas M.D. Anderson Cancer Center. The patients presented at ages 2 weeks and 1 month. Both were treated with surgery alone. We also review 4 cases previously reported in the literature; at presentation, those patients were ages 2 days to 9 months. Infantile HPs have a better prognosis than do those that occur in adults, and an improved outcome is also evident for intracranial lesions. Tumors that occur in the neonatal period may be treated with surgery alone; however, those that occur after the neonatal period may be more likely to recur. PMID- 7547462 TI - Pathogenesis of craniosynostosis. PMID- 7547463 TI - Differential requirements for segment polarity genes in wingless signaling. AB - The segment polarity genes wingless and engrailed are required throughout development of Drosophila. During early embryogenesis, these two genes are expressed in adjacent domains, in an inter-dependent way. Later, their expression is regulated by different mechanisms and becomes maintained by auto-regulation. To dissect the genetic requirements for the initial signaling between wingless and engrailed expressing cells, we have previously used a transgenic Drosophila strain that expresses wingless under the control of the heat shock promoter (HS wg). Focusing on the later phases of wingless and engrailed regulation, we have now extended these studies, using embryos carrying various combinations of segment polarity mutations and the HS-wg transgene. We confirm some of the existing models of regulation of the expression of wingless and engrailed. In addition, we find that HS-wg embryos require engrailed for induction of ectopic endogenous wingless expression. Signaling from engrailed cells to this novel wingless expression domain is dependent on hedgehog but also on porcupine. We further demonstrate a novel requirement for hedgehog in maintenance of expression of engrailed itself. PMID- 7547464 TI - Race: a Drosophila homologue of the angiotensin converting enzyme. AB - We report the isolation and characterization of a putative angiotensin converting enzyme (ACE) in Drosophila, called Race. General interest in mammalian ACE stems from its association with high blood pressure; ACE has also been implicated in a variety of other physiological processes including the processing of neuropeptides and gut peristalsis. Mammalian ACE is a membrane associated zinc binding protease that converts angiotensin I (A I) into angiotensin II (A II). A II functions as a potent vasoconstrictor by triggering a G-coupled receptor system in the smooth muscles that line blood vessels. Drosophila Race is composed of 615 amino acid residues, and shares extensive sequence identity with mammalian ACE over its entire length (over 42% overall identity and greater than 60% similarity). Evidence is presented that Race might correspond to a target of the homeobox regulatory gene, zerknullt (zen). Soon after zen expression is restricted to the dorsal-most regions of the embryonic ectoderm, Race is activated in a coincident pattern and becomes associated with the amnioserosa during germ band elongation, shortening and heart morphogenesis. After germ band elongation, Race is also expressed in both the anterior and posterior midgut, where it persists throughout embryogenesis. Race expression is lost from the dorsal ectoderm in either zen- or dpp- mutants, although gut expression is unaffected. P-transformation assays and genetic complementation tests suggest that Race corresponds to a previously characterized lethal complementation group, 1(2)34Eb. Mutants die during larval/pupal development, and transheterozygotes for two different lethal alleles exhibit male sterility. We propose that Race might play a role in the contractions of the heart, gut, or testes and also suggest that Hox genes might be important for coordinating both developmental and physiological processes. PMID- 7547465 TI - Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate. AB - In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord. PMID- 7547466 TI - The role of fs(1)K10 in the localization of the mRNA of the TGF alpha homolog gurken within the Drosophila oocyte. AB - A critical step in Drosophila dorsoventral patterning is the movement of gurken mRNA from the anterior cortex of the oocyte to the oocyte's anterodorsal corner at stage 8 of oogenesis. Such movement is dependent on fs(1)K10. It has been proposed that fs(1)K10 mediates gurken mRNA movement by down-regulating gurken mRNA levels, thus ensuring that gurken mRNA does not saturate its receptors located in the oocyte's anterodorsal corner. In contradiction to this model, we show here--both genetically and immunocytochemically--that GRK protein levels are lower in the anterodorsal region of fs(1)K10 mutant oocytes than in the anterodorsal region of fs(1)K10+ oocytes. From this and other data, we propose a more direct role for fs(1)K10 in the gurken mRNA localization process. PMID- 7547467 TI - Localization of Pbx1 transcripts in developing rat embryos. AB - Recently, a new family of homeodomain proteins has emerged, that includes extradenticle, ceh-20, Pbx1, Pbx2 and Pbx3. The Pbx family has been shown to modulate the biological activities of the Hox proteins. We demonstrate here by in situ hybridization that Pbx1 transcripts are present in many embryonic tissues. Highest levels of Pbx1 expression in the developing embryo, from 12 to 20 days post coitum, are found in neuronal tissues, including brain, spinal cord and ganglia. In addition, Pbx1 transcripts are also detectable in the gut, lung, olfactory epithelium and kidney. The expression pattern of Pbx1 overlaps with that of many of the Hox gene products and is consistent with them acting in parallel to regulate common target genes. PMID- 7547468 TI - Patterns of conservation and divergence at the even-skipped locus of Drosophila. AB - The even-skipped (eve) gene of Drosophila melanogaster has been intensively studied as a model for spatial and temporal control of gene expression, using in vitro and transgenic techniques. Here, the study of eve is extended, using evolutionary conservation of DNA sequences. Conservation of much of the protein, and of known regulatory elements, supports models for eve function and regulation that have previously been advanced, and extensive conservation found in noncoding sequences predicts that functional elements exist that have yet to be defined. In contrast, a part of the protein implicated in transcriptional repression has diverged extensively while preserving overall amino acid composition, highlighting potentially essential features of this domain. Also, the basal promoter has diverged extensively, indicating evolutionary flexibility of promoter function. PMID- 7547470 TI - 1(2)gl gene regulates late expression of segment polarity genes in Drosophila. AB - To analyse the possible roles of Drosophila tumour suppressor genes, 1(2)gl and 1(2)gd, in differentiation programmes of imaginal cells, we investigated their interactions with two segment polarity genes, viz., cubitus interruptus Dominant (ci-D) and engrailed (en), by examining their patterns of expression in tumourous imaginal discs of 1(2)gl4 or 1(2)gd1 homozygous larvae. While the 1(2)gd1 mutation did not have much effect, the areas of expression of ci-D and en in the tumourous discs of 1(2)gl homozygous larvae were significantly increased and the anterior-posterior compartment boundary was no longer identifiable. To examine if the loss of en expression compartment boundary in 1(2)gl tumourous discs was due to overproliferation of the posterior compartment cells or due to a deregulated expression of en in the anterior compartment cells, 1(2)gl4 homozygous cell clones were generated in 1(2)gl4 enlacZ/++ background. A distinct X-gal staining in 1(2)gl homozygous clones in the anterior compartment in wing imaginal discs or in adult wings confirmed deregulated ectopic expression of en in 1(2)gl mutant anterior compartment cells. We suggest that 1(2)gl is involved in regulating post embryonic expression of segment polarity genes. PMID- 7547469 TI - Degradation of maternal string mRNA is controlled by proteins encoded on maternally contributed transcripts. AB - In Drosophila, maternal string mRNAs are stable for the first few hours of development, but undergo specific timed degradation at the cellularisation stage. To determine whether the proteins that control this degradation are maternally or zygotically transcribed, we have used in situ hybridisation to determine the fate of maternal string transcripts in mutant embryos which lack individual chromosome arms. Our data indicate that maternal string mRNA persists for the normal period in all these mutants. Using alpha-amanitin to inhibit zygotic transcription we have shown that degradation of maternal mRNA is unaffected. Therefore, the proteins required to activate the degradation of string mRNA are encoded on a maternally contributed mRNA. We discuss possible models to explain the degradation pathway. PMID- 7547471 TI - Isolation and embryonic expression of Xel-1, a nervous system-specific Xenopus gene related to the elav gene family. AB - We have identified a new member of the elav gene family in Xenopus laevis. This gene, Xel-1, like the other elav-related genes, encodes a putative RNA-binding protein that contains three RNA Recognition Motifs and is solely expressed in the nervous system. Xel-1 is most likely the Xenopus homologue of Hel-N1, one of the three known human genes related to elav. Xel-1 is not expressed in early neural precursors but rather in differentiating neurons of the central nervous system, as well as in the cranial and the spinal ganglion cells. Xel-1 thus appears to be an early differentiation marker for both the central and the peripheral nervous system of Xenopus laevis. PMID- 7547472 TI - Dorso-ventral and rostro-caudal sequential expression of M-twist in the postimplantation murine embryo. AB - M-twist is the murine homolog of the Drosophila twist gene which is a zygotic target for maternal genes that establish embryonic dorso-ventral polarity and is necessary for mesoderm formation. We recently showed that before gastrulation, M twist transcripts are detected in morulae and blastocysts, then in extra embryonic tissues of early implanted mouse embryos before the onset of gastrulation, and we suggested that M-twist might be involved in embryonic polarity (Stoetzel et al., submitted). Here, using in situ hybridization on whole mount embryos, we present the expression pattern of M-twist from primitive streak stage up to 10.5 days p.c. In implanted embryos, M-twist is first expressed in extra-embryonic tissues, then in embryo proper around egg cylinder stage within some embryonic ectodermal cells of the primitive streak. Slightly later, scattered cells within the amniotic cavity apparently detached from the primitive streak also express the gene. Then, M-twist transcripts accumulate in head mesenchyme, the first aortic arches, somites and lateral mesoderm and, as development proceeds, successively the second, third and fourth branchial arches, the anterior limb buds and, finally, the posterior limb buds. Thus M-twist expression in implanted embryos occurs first along a dorso-ventral gradient pattern until the headfold stage, then it is gradually observed along the rostro caudal axis of the embryos as development procedes in the mesodermal cell layer and in neural crest cell derivatives. In addition, we show the existence of some previously undescribed subsets of scattered cells that express M-twist and thus might participate in murine embryo development. PMID- 7547473 TI - Hoxc-9 mutant mice show anterior transformation of the vertebrae and malformation of the sternum and ribs. AB - To clarify the function of the Hoxc-9 gene, we have introduced a mutation into the Hoxc-9 locus by gene targeting. Skeletal analysis of homozygotes revealed that anterior homeotic transformation occurred from the 10th thoracic vertebrae to the first lumbar vertebra. Bending and fusion of the ribs were observed. Eight or nine pairs of ribs were attached to the sternum. The sternum showed an abnormal pattern of ossification. Phenotypes of the mutant mice resembled those of the Hoxc-8 mutant mice. The data suggested that a functional interaction between the Hoxc-8 and Hoxc-9 proteins is involved in segmental determination. Moreover, changes in the Hoxc-8 gene expression pattern suggested that the Hoxc-9 gene regulates expression of the Hoxc-8 gene. PMID- 7547474 TI - Organization of Wolbachia pipientis in the Drosophila fertilized egg and embryo revealed by an anti-Wolbachia monoclonal antibody. AB - Cytoplasmic incompatibility (CI) in Drosophila is related to the presence of Wolbachia, an intracellular microorganism found in many species of insects. In order to study the intracellular localization of Wolbachia in eggs and embryos, we have purified the bacteria from fly embryos and subsequently generated a monoclonal antibody (Mab Wol-1) specific for Wolbachia. Indirect immunofluorescence staining using Wol-1 reveals that during mitosis, Wolbachia are localized near spindle poles and centrosomes. Double label immunofluorescence experiments using anti-tubulin and anti-Wolbachia antibodies show that Wolbachia co-localize with centrosomal microtubules throughout the cell cycle. Direct interactions between the bacteria and centrosome-organized microtubules are implied from seven observations: (1) throughout the mitotic cycle, the position and movement of Wolbachia precisely mimic the behavior of the centrosome and apparently associated with centrosome-organized microtubules; (2) Wolbachia segregate equally to each spindle pole during mitosis; (3) Wolbachia do not associate with spindle microtubules during mitosis; (4) Wolbachia located in the egg cortex localize to the domains of cytoplasm organized by microtubules during blastoderm formation; (5) polar body nuclei that lack centrosomes but contain associated microtubules do not contain Wolbachia; (6) Wolbachia no longer associated with yolk nuclei, following differentiation and loss of centrosomes; (7) during pole cell formation, Wolbachia co-localize with the centrosome on the apical side of the nucleus as pole cells form. Quantitative data indicates that no Wolbachia growth occurs during the preblastoderm period even though rapid nuclear, and subsequent cellular, proliferation takes place during this same period. This indicates that Wolbachia are under strict growth regulation by the host suggesting that host factors play a role in regulating growth of Wolbachia in the egg. Further cellular and molecular studies of the extensive, global interactions between host and symbiont observed in this egg should provide important new insights into the evolution of host/symbiosis and the cell biology of cytoplasmic incompatibility. PMID- 7547475 TI - Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers. AB - We tested the possibility of inducing ectopic expression of the gene Engrailed-2 (En-2) in the rhombomeres of the 2-days-old chick embryo. The experimental procedure consisted of grafting En-2-expressing neuroepithelium (prospective isthmocerebellum) from a quail or a mouse embryo into different rostrocaudal levels in the hindbrain of a host chick embryo. The graft replaced a given excised host rhombomere, the rostral and caudal limits of which were either also excised or left intact in different experiments. Induction of En-2 occurred in the host hindbrain, but only when the graft did not contact with host interrhombomeric boundaries and only in the alar plate of the rhombomeres immediately contacting the graft. Long survival experimental embryos showed that induced rhombomeres give rise to a cerebellar phenotype in their transformed alar plates. We thus demonstrate here a pluripotential state of the early rhombomeres as well as a possible role of the interrhombomeric limits as barriers to morphogenetic influences. PMID- 7547476 TI - Inhibition of cell fate in Drosophila by Enhancer of split genes. AB - The neurogenic genes of Drosophila act during many different times and places during development. It is thought their role is to repress cell fate within a group of equivalent cells and thus allow the singling out of discrete numbers of precursors. Amongst the genes at the neurogenic locus, Enhancer of split is a family of seven related genes that encode proteins containing the basic helix loop-helix motif characteristic of transcriptional regulators. Previous functional analyses of these genes have relied on deletions which eliminate many other genes. We have ectopically expressed two of the Enhancer of split basic helix-loop-helix genes, m5 and m8, to test their effect on the determination of the precursor cells of adult sensory organs. Ectopic expression of m5 or m8 before bristle precursor division results in loss of sensory bristles from all parts of the adult fly. Ectopic expression after bristle precursor division produces bristles with aberrant cuticular structures. We have also tested the effect of reducing Enhancer of split gene function using mitotic recombination and show that this de-represses the neural fate and produces supernumerary sensory bristle neurons. We conclude that the Enhancer of split basic helix-loop helix genes inhibit neural fate during the selection of neural precursors, and that they also play a role in restricting the neuronal fate to one of the four progeny cells of the bristle precursor. PMID- 7547477 TI - Expression and targeting of Syrian hamster prion protein induced by heat shock in transgenic Drosophila melanogaster. AB - To evaluate the fruit fly as a model for studying neurodegenerative diseases caused by prions, transgenic flies were generated by introducing the Syrian hamster prion protein (SHaPrP) gene into the Drosophila melanogaster germ line by P element-mediated transformation. Nine transgenic lines were isolated; induction of transgenes that had been placed under the control of the Drosophila heat shock promoter, hsp 70, resulted in the synthesis of full-length SHaPrP. The relative molecular weight of the recombinant protein was lower than that of authentic SHaPrP due to incomplete processing of Asn-linked CHOs. To determine the cellular localization of SHaPrP, Drosophila Schneider line 2 cells were transfected with the same constructs used for fly transformation. Heat shock induced SHaPrP was anchored to the surface of S2 cells by a glycolipid, demonstrating that the carboxy-terminal glycolipidation signal of SHaPrP is recognized by this evolutionarily distant host. When SHaPrP was synthesized in transgenic flies constitutively by subjecting them to heat pulses continuously, no difference in their lifespans compared with controls was detected. Furthermore, expression of SHaPrP for 20 days did not produce protease resistant SHaPrP, which is the major and possibly only component of the infectious prion. In contrast to transgenic mice overexpressing SHaPrP, which develop a profound neuromyopathy, no disease phenotype was associated with expression of SHaPrP over the entire lifespan of transgenic flies. PMID- 7547478 TI - Dual organisation of the Drosophila neuropeptide receptor NKD gene promoter. AB - Neuropeptide function in the peripheral and central nervous systems has been described in mammals as well as in insects. We previously reported the cloning of the neuropeptide receptor NKD, a Drosophila melanogaster homologue of the mammalian tachykinin receptors. This receptor is expressed during Drosophila embryonic development and in the adult fly. Use of the NKD promoter region to drive beta-galactosidase expression in transgenic flies reveals a bipartite promoter organisation: the distal region controls NKD expression in neurosecretory cells of the central nervous system during late embryogenesis, whereas the proximal region is responsible for transient expression in peripheral nervous system during late embryogenesis, whereas the proximal region is responsible for transient expression in peripheral nervous system precursor cells early in development. This early NKD expression, first restricted to the sensory organ precursor cell, an atonal positive cell, is abolished in the ato1 mutant. In addition, we show that the proneural protein atonal, in association with daughterless, transactivates the NKD promoter in Schneider S2 cells via the proximal E box NKDE2. Furthermore, heterodimers of atonal and daughterless interact with this E box in gel shift assay. PMID- 7547480 TI - How to achieve comprehensive teamwork in your pediatric dental office through total quality management (TQM). AB - Teamwork is considered the key to success in any organization. Total Quality Management (TQM) is a systematic process that optimizes quality and copes with change. The process includes customer satisfaction, as the ultimate criterion of quality; worker empowerment; and reliance on statistical process, rather than inspection and continuous improvement. Managers not only adopt new techniques but also a new philosophy, rethinking their roles to become leaders and mentors instead of bosses. Fourteen points, developed during several decades, are considered the cornerstone of quality and teamwork, and they can apply to what we do in the pediatric dental office. Together, these principles should be viewed as an ongoing process that can take up to five years or more and should be managed with the same emphasis as other systems in your office. The dentist, or leader, implements and takes part in the process, which will improve morale by increasing team spirit. PMID- 7547479 TI - Murine Wnt-11 and Wnt-12 have temporally and spatially restricted expression patterns during embryonic development. AB - The Wnt gene family encodes a set of signalling molecules implicated in the development of a wide range of organisms. We have recently cloned partial cDNA sequences of murine Wnt-11 and Wnt-12. Here, we describe the spatio-temporal expression patterns of both genes during mouse embryogenesis. Wnt-11 expression is first detected within the truncus arteriosus from 8.25 dpc. By 9.5 dpc, Wnt-11 expression is detected in the somites at the medial junction of the dermatome and the myotome. Wnt-11 transcripts are also detected in limb bud mesenchyme from the time the bud is first visible. Wnt-12 is detected in the apical ectodermal ridge from 10.5 dpc. The implications of these expression patterns are discussed. PMID- 7547481 TI - Palatal expansion with a total denture. AB - A case report of a pediatric patient who had posterior crossbite is presented. She was diagnosed with hypophosphatasia and had primary tooth loss. A total denture was constructed for aesthetics and function. For the correction of posterior cross bite, a palatal screw was placed in the upper denture. The upper archs successfully expanded to a proper relationship. PMID- 7547482 TI - Root canal treatment of primary molars with infected pulps using calcium hydroxide as a root canal filling. AB - In the two case reports, pulpectomy is described as the treatment of choice for a necrotic pulp. A calcium hydroxide paste was used as root canal filling. Its simple application and subsequent good resorption during by the eruption of the permanent tooth strengthened that choice. PMID- 7547483 TI - Modulation of occlusal plane during puberty. AB - The biological significance of the occlusal plane is of tremendous importance in the study of the etiology, diagnosis and treatment of malocclusion. The purpose of this investigation was to correlate the changes on the occlusal plane of patients in treatment with functional appliances and the subjects with no need of treatment during the prepubertal and pubertal period. Data on the space between the upper and lower occlusal lines-Interocclusal Distance (IOD) was obtained periodically. Some remarks about which changes are naturally provoked or not are very useful in the treatment of malocclusions and the evaluation of the occlusal plane in cases that do not need treatment. PMID- 7547484 TI - Radiographic diagnosis of the normal alveolar bone height in the primary dentition. AB - Epidemiological studies on the prevalence of marginal alveolar bone loss (ABL) in children and adolescent are often based on a single range of measurements from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC distance). For individual diagnosis however, the clinician must take in consideration that the CEJ-ABC distances in the primary dentition increase with age, and significantly differ among sites. The purpose of the present study is to present the range and cumulative probabilities of the CEJ-ABC distances, at the primary cuspid-molar area, to be utilized for the individual diagnosis of ABL on bite wing radiographs of the primary dentition. In bite-wing radiographs from 316 children aged 4 to 12 years, the normal CEJ-ABC distances were measured in 0.1 mm increments at 2007 sites; mesial surfaces of the primary molars, and distal surfaces of the primary molars and cuspids. A positive correlation was found between the mean CEJ-ABC distance per patient and age. Most of the measurements were < 2 mm, and measurements between 2 to 3 mm were found, in low percentages after age 9, at the distal surfaces of teeth 53, 63, 54, 64, 73 and 83 and the mesial surfaces of teeth 54, 64. PMID- 7547485 TI - Clinical evaluation of the retention and wear of a light-cured pit and fissure glass ionomer sealant. AB - The purpose of this study was to evaluate the 12-month retention and wear of an experimental light-cured glass ionomer for pit and fissure sealing. A total of 25 patients 7-14 years-old were selected from the Pediatric Dentistry Clinic at the University of Texas Health Science Center at San Antonio. The patients resided in areas without fluoridated water. Each tooth was isolated with cotton rolls, dried with oil-free compressed air and GC Dentin Conditioner applied with a small disposable brush to the pits and fissures for 20 seconds. The teeth were rinsed with water and gently air dried using an air/water syringe. The teeth were not desiccated. GC experimental glass ionomer for pit and fissures was used. The powder/liquid ratio was dispensed at 1.4 gm/1.0 gm (one level spoonful of powder to one drop of liquid). The powder was mixed for 15-20 seconds. An explorer was used to apply the mixed sealant to the tooth and teased into all pits and fissures. The sealant was then cured for 20 seconds. Occlusion was corrected after the sealant was light-cured. Immediately, a color slide and a vinyl polysiloxane impressions were taken. The impressions were poured in epoxy resin. Color slides, impressions and epoxy models were also made at 3, 6, 9 and 12-month recalls. A total of 95 sealants were placed and follow-up for 12 months. The results showed that with the clinical visual inspection all sealants were present at 3 and 6 months postoperatively. At 12 months, only 20% of the sealants were clinically evident.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547487 TI - Periodontal findings in cases of incisor cross-bite. AB - The effect of cross-bite on periodontal health in the anterior teeth was investigated in 40 teenage subjects. The results have shown a relationship between the cross-bite in the anterior teeth and periodontal health as assessed by Plaque Index, Gingival index and pocket depth measurements. The periodontal status of the teeth in cross-bite revealed a more unfavorable condition compared to the well-aligned teeth. This was observed in both males and females. However, pocket depth on the lingual site for both sexes does not reach a significant difference between the cross-bite and the control teeth. PMID- 7547486 TI - The relationship between dental caries in the primary dentition and anti S. mutans serum antibodies in children with Down's syndrome. AB - The purpose of this study was to evaluate the status of dental caries in seventy five subjects with Down's Syndrome, ages two to eighteen years and to evaluate the relationship between dental plaque or caries experience and serum antibody titers against S. mutans and S. mitis. These antibodies were examined since an enhanced host response to S. mutans might be responsible for the low caries prevalence in Down's Syndrome. Antibody titers were determined using the enzyme linked immunosorbant assay (ELISA) and titers were expressed in ELISA units. The frequency of all subjects who were caries-free was 46.1%, while 61.4% of the subjects under five years were caries-free. The average DMFT score in the subjects of Hellman Dental Age group I (primary dentition) was 15.9. The highest frequency of caries occurred on occlusal surfaces, followed by proximal surfaces of the upper incisors and smooth surfaces on incisors. The severity of dental caries in the subjects with Down's Syndrome was bipolar and could be categorized as either mild or severe. In the subjects having a primary dentition, a significant positive correlation was found in the relationship between the Original Caries Severity Score (OCSS) and the plaque score. There was also a significant positive correlation between OCSS and IgM antibody titer to S. mutans. In contrast, no correlation between antibody titers to S. mitis and these variables was found. It is not clear if these antibodies are protective and responsible for the reduced caries rate seen in Down's Syndrome. PMID- 7547488 TI - Accidental swallowing of a gold cast crown during orthodontic tooth separation. AB - Accidental swallowing of a gold cast crown that became loose after separation with brass wire for orthodontic band placement, is reported. The intervention to remove the ingested crown subsequent to its location in the GI tract on immediate radiographic examination of chest and abdomen included endoscopic examination. Since the crown had past distal to the duodenum it was decided to keep a watch on its movement by daily radiographic examination. The crown passed with excreta on the 5th day uneventfully. Literature on ingestion of foreign bodies of dental origin is reviewed. Possible emergencies and their management is discussed. PMID- 7547489 TI - Compound composite odontoma associated with an impacted cuspid. AB - This case report involved the removal of a compound composite odontoma from a fourteen year-old male patient at an urban dental school. The pathology report did indeed reveal the tissue mass was that of a compound composite odontoma. These lesions usually occur in the second decade of life, in the anterior maxilla or mandible, with no gender or race predilection. Three types of odontomas are identified, including are: compound composite odontoma, complex composite odontoma, and ameloblastic fibro-odontoma. The etiology of these lesions is unknown, although environmental (trauma, infection) and hereditary influences play a role. Treatment consists of complete enucleation and curettage followed by histological verification by an oral pathologist. PMID- 7547490 TI - Interceptive approach to tooth eruption abnormalities: 10 year follow-up of a case. AB - A child affected by lack of eruption of the upper right primary cuspid from the presence of a compound odontoma was followed from 3 to 13 years of age. The early removal of the obstacle to eruption and the correct control of the case evolution allowed for a satisfactory alignment of the permanent teeth by means of minimal therapeutical intervention. PMID- 7547491 TI - Williams syndrome. AB - Williams syndrome is a rare syndrome. It usually can be diagnosed first in dental clinics because of the typical oral and facial features of the syndrome. The aim of this article is to report this 8 year old Turkish boy with Williams syndrome, and to revise the dental features of these special patients and the dental treatment procedures that can be followed for these patients. PMID- 7547492 TI - Dental manifestations of inbreeding. AB - This study evaluated the prevalence of genetic dental defects of children within different levels of inbreeding in Borj Salhi, Tunisia. One hundred and twenty one children six to nineteen years old were studied. Results were compared to Tunisian Pediatric Dentistry Society report. The findings showed high prevalence of dental structure anomalies (16%) and malocclusions (28%). It was concluded that genetic factors are responsible for a statistically significant portion of the dental anomalies. PMID- 7547493 TI - Application of image analysis to the study of skin granulomas. AB - Histologically- and immunohistochemically- stained paraffin sections of a well documented collection of infectious skin reactions were studied using a BIOCOM 200 image analysis system. Examples of quantitative evaluation of dermal fibrosis and collagen isotypes pattern, as well as morphometric demonstration of cutaneous vessels or tissue macrophage changes were shown. In addition, the topography of specifically immunostained cell populations was specified in different granulomatous reactions. There was a loss of cytokine positive signal on paraffin sections during the tissue processing. Immunohistochemistry and image analysis were shown to be complementary techniques when applied to pathological material. However, technical improvements will be necessary before routine application of the techniques to the study in situ of the role of cytokines and remodelling enzymes. PMID- 7547494 TI - Expression of c-erbB-2 and p53 in colorectal adenocarcinoma. AB - Expression of c-erbB-2 and p53 protein was analysed retrospectively by immunohistochemistry in formalin-fixed tissue samples from 293 patients with colorectal adenocarcinoma. There was a significant positive relationship between c-erbB-2 and p53 expression (P < 0.0001). Co-overexpression of c-erbB-2 and p53 tended to be increased in tumours with infiltrative growth (P = 0.08) and higher S-phase fraction (P = 0.085). In combined survival analysis, patients with tumours in both c-erbB-2 positivity and p53 negativity had a more favourable outcome (P = 0.03). Multivariate analysis revealed that p53 overexpression was significantly associated with poor prognosis independent of c-erbB-2 expression, DNA-ploidy, S-phase fraction, growth pattern and Dukes stage (P = 0.002). We conclude that there is an inter-reaction between the two oncoproteins in the tumour development and that the overexpression of p53 proteins may be a powerful prognostic predictor in colorectal adenocarcinoma. PMID- 7547495 TI - Heterogeneity of the chromatin fine structure in DNA-diploid breast cancer cells. AB - The chromatin fine structure of Feulgen-stained DNA-diploid tumour cells was studied by means of image cytometry in fine-needle aspiration biopsies of 115 breast cancer patients. The investigation was focused on diploid, several subtypes of polyploid, and aneuploid tumours. Only the clearly separable diploid peaks of these tumours were measured cytometrically by an OPTIMAS-based image analysis system, which led to a set of 93 primary cytometric features in each diploid subpopulation. The results demonstrate that the diploid tumours are clearly different to the diploid compartments of various subtypes of polyploid and aneuploid tumours. Between those diploid subpopulations from polyploid tumours, chromatin structure differences also exist. A higher degree of similarity between polyploid and aneuploid tumours, compared to diploid, became evident. The actual structural differences between the diploid subpopulations indicate both increasing variances within the peak and increasingly coarse and contrasted, irregular chromatin structure with the occurrence of tetraploid, octoploid, and aneuploid DNA-histogram peaks. Therefore, the structural effects of an increasing genomic instability seems not to be restricted to the non diploid cell populations. PMID- 7547496 TI - Quantitative immunohistochemistry using the CAS 200/486 image analysis system in invasive breast carcinoma: a reproducibility study. AB - We evaluated the intra- and inter-observer reproducibility of quantitative immunohistochemical (IHC) analyses using the Cell Analysis Systems (CAS) 200/486 image analyzer of Estrogen Receptor (ER), Progesterone Receptor (PR), proliferation-associated nuclear protein (Ki67), HER-2/neu (c-erbB-2) protein over-expression and cathepsin D (CD) in 20 randomly-selected invasive breast carcinomas. Qualitative analysis of IHC Epidermal Growth Factor Receptor (EGF-R) was also assessed in this study for comparative purposes. Duplicate blind assessments by the same observer showed excellent correlations for all quantitative IHC features (P < 0.001; P = 0.004 for neu). However, the immuno quantitative analyses results between the 3 different operators showed lower correlation coefficient values, thus being less reproducible. This resulted in systematic differences and bias between the observers. This was also clear from the overall agreement between the 3 observers which was 70% for ER, 70% for PR, 56% for Ki67, 79% for c-erbB-2 and 75% for CD. The qualitative visual assessments of EGF-R, expressed as either positive or negative, showed a 75% agreement between observers and 85% intra-observer agreement (comparable to quantitative digital image processing results). The same results were obtained with kappa statistics. A further analysis of the factors causing the lack of reproducibility was performed. For quantitative IHC, segmentation of stored and retrieved digitized images was quite reproducible between and within well-trained observers. However, variation between different fields of vision of one and the same section showed large variations for most cases. Therefore, differences in sampling of fields within a section appeared to be the major cause of lack of reproducibility between observers, although segmentation differences still added slightly to the inter-observer variations. Accordingly, a strict sampling protocol of fields of vision is mandatory to obtain reproducible quantitative IHC results. It is clear from the present study that so-called random (but in fact, at convenience) selection of fields of vision for measurement is not a sufficient guarantee of adequacy of the sampling. PMID- 7547497 TI - Flow cytometric analysis of c-myc oncoprotein in non-small-cell lung carcinoma: comparison with immunohistochemical results. AB - c-myc oncoprotein expression was evaluated in 36 non-small-cell lung carcinomas using immunohistochemical and flow cytometric analysis. Formalin fixed and paraffin embedded material was used. The same monoclonal antibody (mouse anti human c-myc) was employed both for immunohistochemistry and flow cytometry. For the immunohistochemical evaluation we calculated the percentage of stained cells on 200 neoplastic cells. c-myc oncoprotein was measured using flow cytometry by linking the monoclonal with a secondary FITC-conjugated antibody; for each sample 20,000 events were analysed and the percentage of cells positive for green fluorescence was calculated. DNA content was obtained by propidium iodide staining. Results showed a high correlation between immunohistochemical and cytometric data, suggesting that flow cytometry could be used as an alternative to immunohistochemistry in evaluating nuclear antigens. Moreover, using flow cytometry information on DNA content can be obtained simultaneously. PMID- 7547498 TI - Flow cytometric DNA analysis of fine-needle aspirates of prostatic benign lesions. AB - Fine-needle aspiration samples from 54 consecutive patients suffering from prostatic benign lesions were analyzed for both cytological diagnosis and DNA cell content, by means of standard cytological criteria and flow cytometry, respectively. Of these patients, 13 displayed prostatitis: three being classified as acute prostatitis, 7 as chronic and 3 as granulomatosous prostatitis. All cases had a diploid DNA cell content, with a mean percentage of S-phase cells of 1.8 +/- 2.7%. Although no major differences were detected between patients with and without prostatitis, the former group had a slightly higher proportion of S phase cells (1.4 +/- 2.2% vs 2.7 +/- 3.7%). Moreover, within those patients with prostatitis, acute prostatitis cases showed the highest proliferation rates although differences did not reach statistical significance. PMID- 7547499 TI - The nonstructural proteins of the autonomous parvovirus minute virus of mice interfere with the cell cycle, inducing accumulation in G2. AB - The nonstructural (NS) proteins of the autonomous parvovirus minute virus of mice (prototype strain) are involved in viral DNA replication and in the regulation of parvoviral and heterologous promoters. By constructing cell lines having integrated the NS coding sequence under the control of an inducible promoter, we were able to demonstrate that NS proteins are toxic, once expressed in the transformed cells. Cell killing appears after several days of NS expression, suggesting that NS toxicity involved cellular factors. In this paper, we show that NS proteins are cytotoxic and interfere with the cell cycle in proliferating cells only NS expression is innocuous in resting cells, whereas in growing cells, it induces the accumulation of G2 cells. This cytostatic effect is enhanced upon neoplastic transformation, which sensitized the cells to NS killing. Moreover, as clones resistant to NS toxicity undergo no alteration of their cycle, this cytostatic effect of NS proteins could be an early step on the way to cell killing. These observations strongly suggest that NS toxicity involves cellular factors associated with the regulation of the cell cycle. PMID- 7547501 TI - Bcl-2 and thermotolerance cooperate in cell survival. AB - Apoptosis is a normal physiological process of cell death that can also be experimentally induced by a variety of cytotoxic stimuli. The protein product of the oncogene bcl-2 is an effective inhibitor of apoptosis in mammalian cells, including that caused by exposure to heat. Transient heat resistance can be induced by prior exposure of cells to mild heat treatment. This thermotolerant state is believed to be mediated by an increase in the steady-state concentration of one or several heat shock proteins. Using both morphological and long-term end points, i.e., the appearance of apoptotic cells as assessed by nuclear morphology and clonogenic cell survival, we show that bcl-2 expression confers on thermotolerant cells an even greater resistance to heat-induced cell death. bcl-2 expression does not alter either the steady-state or heat-induced expression of heat shock proteins in cells, nor is bcl-2 itself a stress-inducible protein. The results suggest bcl-2 and the thermotolerant state act via independent mechanisms to inhibit apoptosis and provide evidence that two separate pathways can collaborate to promote cell survival. PMID- 7547500 TI - Genetic analysis of growth inhibition by GAL4-L kappa B-alpha in Saccharomyces cerevisiae. AB - I kappa B proteins bind to and regulate Rel/NF- kappa B transcription factors. We showed previously that a fusion protein (GAL4-p40) containing the DNA-binding domain of GAL4 and sequences of chicken l kappa B-alpha (p40) inhibits growth in the yeast Saccharomyces cerevisiae. We now show that p40 must be bound to DNA to inhibit yeast growth, p40 proteins, bound to DNA either as GAL4 or LEXA fusion proteins, inhibit yeast growth. In contrast, p40 proteins that cannot bind to DNA, such as full-length p40, a GAL4-l kappa B fusion protein containing a mutant GAL4 DNA-binding domain, and a fusion protein (GAD-p40) containing the transcriptional activation domain of GAL4 fused to p40, each failed to inhibit cell growth. As with GAL4-VP16, GAL4-p40 needs a functional cellular ADA2 gene to exert its growth-inhibitory effect in S. cerevisiae. Using a high copy suppression strategy, we have isolated three S. cerevisiae genes that restore normal growth to yeast expressing GAL4-p40 or LEXA-p40. We have termed these rescuing genes collectively as SIK genes, for "Suppressors of 1 kappa B." Expression of the SIK genes specifically suppresses the growth-inhibitory activity of GAL4-p40 and LEXA-p40 because SIK gene expression cannot block GAL4 VP16-mediated growth inhibition in S. cerevisiae. SIK1 encodes a novel protein that contains a COOH-terminal repeat that has been found in many microtubule binding proteins. SIK2 encodes NH2-terminal acetyltransferase, and SIK3 encodes the yeast ribosomal S4 protein. None of the SIK proteins binds directly to p40 sequences in vitro, suggesting that the SIK proteins are likely to act downstream of the direct point of growth inhibition by GAL4-p40. Our results may be useful for devising strategies for identifying vertebrate inhibitors of l kappa B proteins and of other proteins that inhibit growth in S. cerevisiae. PMID- 7547502 TI - A secreted serine protease can induce apoptosis in Pam212 keratinocytes. AB - The epidermal keratinocyte cell line Pam212 undergoes spontaneous apoptosis in culture, providing an in vitro model for the early steps of epidermal differentiation. Pam212 cells exhibit characteristics of basal keratinocytes, committed for the transition to the spinous layer of the epidermis. Bcl-2 can regulate the differentiation of these cells by negatively regulating several genes that have been implicated in apoptosis. We show evidence that a serine protease activity, secreted by the Pam212 cells, could induce apoptosis in Pam212 and several other cell lines. This activity might be regulated via the bcl-2 pathway. We suggest that this serine protease could either directly, via binding and/or cleavage of a serine protease-activated receptor, or indirectly, via the cleavage of an unknown protein, activate the signaling for apoptosis in Pam212 cells. Alternatively, this secreted serine protease could reenter the cell and start a proteolytic cascade reaction that leads to cell death. This is based on the induction of apoptosis in several cell lines by the partially purified serine protease activity, and the minimal effect of protein synthesis inhibition on Pam212 apoptosis. We propose that in vivo, a two-step mechanism controls keratinocyte apoptosis and differentiation. The basal cells of the epidermis contain all of the necessary proteins required for apoptosis, as well as the repressor protein Bcl-2. As Bcl-2 levels go down, the cells commit to terminal differentiation. A serine protease, secreted from these cells, then induces the death process. This second step enables the cells to undergo apoptosis and continue the process of terminal differentiation. PMID- 7547503 TI - FGF-8 isoforms differ in NIH3T3 cell transforming potential. AB - We previously identified Fgf-8 as a frequently activated gene in tumors from mouse mammary tumor virus-infected Wnt-1 transgenic mice, suggesting that Fgf-8 is a proto-oncogene. We further determined that multiple, secreted protein isoforms that differ at their mature amino termini are encoded by alternatively spliced mRNAs transcribed from the gene. We now present evidence that there are differences in the potency of NIH3T3 cell transformation displayed by three of the FGF (fibroblast growth factor)-8 isoforms. We find that stable transfection of a cDNA for the FGF-8b isoform leads to marked morphological transformation of NIH3T3 cells and rapid tumorigenicity of the transfected cells in nude mice. In contrast, transfection of a cDNA for the FGF-8a or FGF-8c isoform results in moderate morphological changes in the NIH3T3 cells, and the transfected cells are weakly tumorigenic in nude mice. All three transfections result in cells that express comparable amounts of Fgf-8 mRNA and that produce the FGF-8 protein isoforms. The morphological changes observed in NIH3T3 cells can be reproduced by the addition of recombinant FGF-8 protein isoforms to the culture medium. Therefore, these results indicate that there are differences in the potency of transformation of NIH3T3 cells by FGF-8 protein isoforms and suggest that these FGF-8 isoforms may have different in vivo functions. PMID- 7547504 TI - Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells. AB - The transforming growth factor-beta (TGF-beta) superfamily is a group of secreted growth factors that appears to play a central role in mesenchymal differentiation, including cartilage and bone formation. The present study examines the role of one member of this family, vgr-1, also called bone morphogenetic protein-6, in mesenchymal cell differentiation. This factor may be considered as a prototype for the largest subgroup of related factors within the TGF-beta superfamily, the function of which has as yet been poorly defined. vgr-1 has been localized previously to hypertrophic cartilage and has been shown to induce endochondral bone formation in vivo. To further characterize the role of vgr-1 in bone and cartilage differentiation, we stably transfected the pluripotent mesenchymal cell line ROB-C26 with a vector to overexpress vgr-1. Overexpression of this factor did not affect cell shape or morphology, but it enhanced osteoblastic differentiation in vitro and altered cellular responsiveness to retinoic acid. Furthermore, the extracellular matrix produced by these vgr-1-overexpressing cells induced ectopic bone formation in vivo and osteoblastic differentiation in vitro, similar to the matrix produced by C26 cells treated with retinoic acid. The osteoinductive effect of the matrix from vgr-1-overexpressing cells was blocked using a neutralizing vgr-1 antibody but not with a neutralizing TGF-beta 1 antibody, indicating that vgr-1 alone was required for this osteogenic effect. In contrast, the osteoinductive effect of matrix from retinoic acid-treated cells was blocked with both vgr-1 and TGF-beta 1 antibodies, suggesting that TGF-beta 1 may act prior to vgr-1 during osteoblastic differentiation. We further demonstrated that osteoinduction by vgr 1 was dependent on presentation of vgr-1 within the matrix, because the osteoinductive effect of matrix from vgr-1-overexpressing cells could not be mimicked with the addition of soluble vgr-1 to parental C26 cells. Finally, overexpression of MyoD within the C26 cells overexpressing vgr-1 converted the cells to myoblasts, indicating that vgr-1 had induced early osteoblastic. PMID- 7547505 TI - Mutually exclusive expression of two dominant-negative helix-loop-helix (dnHLH) genes, Id4 and Id3, in the developing brain of the mouse suggests distinct regulatory roles of these dnHLH proteins during cellular proliferation and differentiation of the nervous system. AB - The dominant-negative helix-loop-helix (dnHLH) proteins Id1 and Id2 have been implicated in the regulation of cell proliferation and differentiation in myogenesis, neurogenesis, and/or hematopoiesis. To further investigate the functional role of dnHLH proteins, we have performed in situ hybridization analysis on serial sections of mouse embryos from days 9.5 to 17.5 postcoitus to establish the spatial and temporal expression patterns of Id3 (HLH462) and Id4, a recently isolated fourth member of the mammalian dnHLH gene family. Id3 transcripts are present throughout embryogenesis and are found in neural cells as well as in cartilage primordia and in epithelial cells lining a variety of organs. The spatial expression pattern of Id3 overlaps considerably with the previously determined pattern of Id1. Id4 expression, which is up-regulated during embryogenesis, is restricted to specific cells of the central and peripheral nervous system. Within the detection limits of in situ hybridization, Id4 and Id3 expression is mutually exclusive in neural precursor cells of the developing brain, suggesting distinct regulatory functions for these dnHLH proteins during neurogenesis. PMID- 7547506 TI - Rapid activation and down-regulation of protein kinase C alpha in 12-O Tetradecanoylphorbol-13-acetate-induced differentiation of human rhabdomyosarcoma cells. AB - Human rhabdomyosarcoma RD cells express the myogenic regulatory factors MyoD and myogenin but differentiate spontaneously very poorly. Prolonged treatment of RD cells with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13 acetate (TPA) induces growth arrest and myogenic differentiation as shown by the accumulation of alpha-actin and myosin light and heavy chains, without affecting the expression of MyoD and myogenin. In this study, we show that short-term phorbol ester treatment of the cultures is sufficient to trigger myogenic differentiation but not growth arrest. Furthermore, PKC inhibitors, such as staurosporine or calphostin C, prevent TPA-induced differentiation but not cell growth arrest. These data suggest that the two events are mediated by different pathways; a possible interpretation is that the activation of one or more PKC isoforms mediates the induction of differentiation, whereas the down-regulation of the same or different isoforms mediates the growth arrest. To address the mechanism whereby TPA affects cell growth and differentiation in RD cells, we first analyzed PKC isoenzyme distribution. We found that RD cells express the alpha, beta 1, gamma, and sigma PKC isoenzymes. Only the alpha isoform is exclusively found in the soluble fraction, but it translocates to the membrane fraction within 5 min of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to both the soluble and the particulate fractions and are down-regulated after long-term TPA treatment. By immunofluorescence analysis, we show that the PKC alpha down-regulation is specific for those cells that respond to TPA by activating the muscle phenotype. We propose that TPA-induced differentiation in RD cells is mediated by the transient activation of PKC alpha, which activates some of the intracellular events that are necessary for MyoD and myogenin transacting activity and for the induction of terminal differentiation of RD cells. By contrast, the constitutively active beta 1 and sigma are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-induced cell growth arrest. PMID- 7547507 TI - Increased glycosylation of beta 1 integrins affects the interaction of transformed S115 mammary epithelial cells with laminin-1. AB - The effect of transformation on the expression and the functions of beta 1 integrins was studied using an in vitro cell transformation model. S115 mammary epithelial tumor cells undergo transformation into tumorigenic fibroblastoid cells in the presence of steroids. Transformation was found to reduce the attachment and the spreading of S115 cells on laminin-1 but not fibronectin. Adhesion of S115 cells to laminin-1 was inhibited in the presence of an antibody against the beta 1 integrin subunit. Both nontreated and transformed S115 cells expressed at least two putative laminin-1-binding beta 1 integrins at the same level. In transformed cells, however, the mature integrin subunits appeared to be structurally altered, showing a slower electrophoretic mobility. Treatment with N glycosidase-F and tunicamycin abolished this mobility difference, suggesting that the presence of complex-type N-linked oligosaccharides was responsible. Detailed enzymatic analysis of the oligosaccharides present on the beta 1 subunits revealed that the difference in glycosylation is, at least partially, due to poly N-lactosaminoglycan chains on beta 1 integrin from transformed cells. Removal of this difference in glycosylation by either cleavage of the polylactosaminoglycan chains with endo-beta-galactosidase or inhibiton of complex-type glycan formation with swainsonine repeatedly enhanced the spreading of transformed cells on laminin-1. Thus, the increased size of complex-type oligosaccharides on beta 1 integrin may affect cell-laminin-1 interactions. Similar changes may contribute to the altered adhesion of cancer cells during the invasion and metastasis. PMID- 7547508 TI - Modulation of markers associated with tumor aggressiveness in human breast cancer cell lines by N-(4-hydroxyphenyl) retinamide. AB - The retinoid N-(hydroxyphenyl) retinamide (4-HPR) appears to be a promising tool for chemoprevention of breast carcinoma, and clinical trials to evaluate its effect are in progress. However, its action on tumor cells has remained largely undefined. We report here that 4-HPR induced apoptosis and/or differentiation in breast cancer cell lines, independent of hormone receptor status and retinoic acid receptor expression, although it was slightly more efficient in inhibiting proliferation of estrogen receptor-positive cells. 4-HPR up-modulated expression of several differentiation markers (class 1 HLA, laminin, and beta 1 integrin chain) and down-regulated expression of molecules associated with tumor progression, including the p185/HER2 oncoprotein, the epidermal growth factor receptor, and the M(r) 67,000 laminin receptor. These data suggest that 4-HPR could exert a beneficial effect by inhibiting cell proliferation and modulating breast tumor aggressiveness. PMID- 7547509 TI - Regulation of cathepsin D gene expression in HL-60 cells by retinoic acid and calcitriol. AB - Cathepsin D (ctsd) is a lysosomal acid protease found in neutrophils and monocytes. We investigated whether differentiating agents increase the expression of ctsd mRNA in HL-60 cells. Treatment with either retinoic acid or calcitriol enhances the steady-state levels of ctsd mRNA in a dose-dependent manner. The stimulation by retinoic acid requires new protein synthesis. Pretreatment with retinoic acid enhances the response of the ctsd gene to prostaglandin E2. To determine whether the effects of retinoic acid and calcitriol are associated with differentiation, we pretreated Hl-60 cells for 120 h with inducers of granulocytic differentiation (lithium chloride, DMSO, and retinoic acid) and monocytic differentiation (calcitriol, sodium butyrate, and phorbol ester). Lithium chloride and DMSO do not significantly affect ctsd mRNA expression, and none of the granulocytic inducers alters the subsequent response of the ctsd gene to calcitriol. All of the monocytic inducers stimulate ctsd mRNA, and both calcitriol and sodium butyrate significantly potentiate the subsequent response to retinoic acid. Transcription initiation of the ctsd gene occurs at one major and several minor sites and is unaffected by treatment with retinoic acid and calcitriol or pretreatment with other differentiating agents. Although differentiation appears to influence ctsd mRNA expression, calcitriol and retinoic acid stimulate ctsd gene expression via mechanisms that are independent of their role in differentiation. PMID- 7547510 TI - Cytokine regulation of the liver transcription factor hepatocyte nuclear factor-3 beta is mediated by the C/EBP family and interferon regulatory factor 1. AB - Three distinct hepatocyte nuclear factor-3 (HNF-3) proteins (alpha, beta, and gamma) regulate the transcription of numerous liver-enriched genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of proteins known to be critical for determination events in embryogenesis. We have demonstrated previously that two binding sites in the -184 HNF-3 beta promoter are recognized by widely distributed factors and that there is also a critical autoregulatory site, we identified a binding site for a cell-specific factor, LF H3 beta, that may function in restricting HNF-3 beta gene expression to hepatocytes. Our present study demonstrates that members of the C/EBP and proline and acidic amino acid-rich subfamilies of basic region leucine zipper transcription factors bind the LF-H3 beta site, and cotransfection of HepG2 cells shows that these factors are able to activate an HNF-3 beta promoter reporter construct. The LF-H3 beta-C/EBP binding sequence also confers HNF-3 beta promoter stimulation in response to interleukin (IL)-1 and IL-6. Upstream of this HNF-3 beta proximal promoter region, an IFN-stimulated response element core sequence ( 231 to -210) was found that mediates transcriptional induction by IFN-gamma but not IFN-alpha. Gel mobility supershift assay demonstrates that an IFN-gamma induced protein-DNA complex is disrupted by an antibody specific for interferon regulatory factor-1/interferon-stimulated gene factor-2. Consistent with this finding, we observed that IFN-gamma induction requires ongoing protein synthesis. Surprisingly, the effect of the three cytokines (IL-1, IL-6, and IFN-gamma) in combination as assayed by the same model is not synergistic. HNF-3beta joins the C/EBP family on the list of liver-enriched transcription factors, the expression of which is modulated by cytokines. PMID- 7547511 TI - Transient inhibition of protein synthesis induces the immediate early gene VL30: alternative mechanism for thapsigargin-induced gene expression. AB - Induction of gene expression in response to calcium ionophores or thapsigargin, which inhibits the calcium-ATPase responsible for sequestering intracellular calcium, has frequently been attributed to direct stimulatory events subsequent to the elevation of intracellular free calcium. VL30 is a murine gene that is transcriptionally induced in response to a large array of mitogenic and transforming stimuli. We have shown previously that an enhancer element within the VL30 promoter region is dependent upon cotreatment with thapsigargin or calcium ionophore for a full-scale induction of gene expression. In this report, we demonstrate that both thapsigargin and calcium ionophores induce a transient inhibition of protein synthesis in Rat-1 cells transfected with a VL30 enhancer driven reporter construct. Recovery of protein synthesis is facilitated by cotreatment with epidermal growth factor or phorbol esters. Furthermore, treatment with cycloheximide or DTT, which inhibit protein synthesis without altering intracellular calcium levels, can substitute for thapsigargin or ionophores in stimulating VL30 gene expression. These results suggest that the stimulatory effects of thapsigargin and calcium ionophores on VL30 expression may be mediated, at least in part, by the ability of these agents to initiate stress responses associated with inhibition of protein synthesis. PMID- 7547512 TI - Regulation of chicken vimentin gene expression by serum, phorbol ester, and growth factors: identification of a novel fibroblast growth factor-inducible element. AB - Vimentin is a cytoskeletal protein that belongs to the intermediate filament protein family. It is normally expressed in cells of mesenchymal origin and is developmentally as well as cell cycle regulated. Multiple silencer elements as well as unique antisilencer element are responsible for regulating the chicken vimentin gene. The silencer elements bind a protein of M(r) 90,000 (the silencer protein), whereas the antisilencer element binds a protein of M(r) 110,000 120,000 (the antisilencer protein). In this study, we examined the effect of serum, phorbol ester, transforming growth factor beta, and fibroblast growth factor of gene expression and identify the regions in the 5'-end of the chicken vimentin gene responsible for induction. The binding activity of both the silencer and the antisilencer proteins are affected by 12-O-tetradecanoylphorbol 13-acetate treatment, whereas the antisilencer element is inducible by fibroblast growth factor. PMID- 7547513 TI - The role of cancer registries. PMID- 7547514 TI - Is the clinical oncology information network (COIN) the answer? PMID- 7547515 TI - Radiotherapy for prostate cancer: the changing scene. PMID- 7547516 TI - Management of localized prostate cancer: an epidemiological perspective. AB - Prostate cancer is an important and increasing source of male morbidity and mortality. In the absence of any primary preventative strategy, medical approaches to control it will concentrate on attempts at cure in localized disease and effective palliation otherwise. Observational epidemiological studies suggest that, in practice, differences in the effectiveness of aggressive and conservative approaches will be small, but may yet be worthwhile in selected groups of men. However, the confounding and biases inherent in all observational epidemiology mean that the data available from this source is insufficiently certain or precise either to make treatment recommendations for individuals, or to quantify relative benefits to inform health policy. Randomized trial data has not suggested any overwhelming benefit for any one treatment modality, but the five published trials have been small and lacked the statistical power to demonstrate potentially important differences. Aggressive management aimed at cure should be evaluated in adequately designed randomized trials in comparison with expectant medical management ('watchful waiting'). The trials currently planned or under way should be supported enthusiastically by all centres with an interest in management of prostate cancer. PMID- 7547517 TI - Transforming growth factor beta: relevance to radiotherapy. PMID- 7547518 TI - The costs of hyperfractionation: an economic analysis incorporating consideration of waiting time for treatment. AB - Multiple fractions per day are being used increasingly in clinical radiotherapy. There are sound radiobiological reasons for this, but the approach uses more time on treatment machines than conventional radiotherapy. The effect of using hyperfractionated schedules for a small minority of radically treated patients has been assessed using a previously published model. The analysis shows that profitability is severely affected by pure hyperfractionation, and that a schedule with a 2-week gap could, by increasing the number of patients it would be possible to treat palliatively, actually increase profitability. Waiting times for treatment could increase, sometimes to clinically unacceptable levels, for patients in all categories. PMID- 7547519 TI - Simultaneous carboplatin and radiotherapy for all stages of head and neck squamous cell carcinoma. AB - The study investigated the toxicity and efficiency of the concomitant administration of radiotherapy and carboplatin to patients with head and neck carcinomas. Sixty-three patients with head and neck squamous cell carcinomas, other than nasopharyngeal cancer and Stage I (UICC) laryngeal cancers, were treated by external radiotherapy and four courses of carboplatin at a dose of 100 mg/m2 per week. In two patients, only three courses were possible due to renal toxicity. In the other 61 patients, toxicities were self-limiting and no patient required interruption of carboplatin administration. No patient required discontinuation of radiotherapy because of acute toxicity. Of 61 evaluable patients, a complete response (CR) was obtained in 11.5% and a partial response (PR) in 60.7% at 40 Gy. In 41 patients treated to 65 Gy (including two patients with maxillary sinus carcinoma, who were treated by debulking surgery), CR was obtained in 76.9% and CR+PR was 100% at the end of treatment. The actuarial survival rate of the 63 patients at 2 years was 69.2%, with a median follow-up period of 24.4 months. One of 12 patients who received salvage surgery after radical radiotherapy has died due to poor wound healing after the surgery. The schedule was safe, providing a weekly check of serum samples was possible. It is likely that the rate of local control and vocal cord preservation in laryngeal tumours might improve if concurrent carboplatin is used. Careful follow-up is required to determine the long-term effect of concomitant carboplatin administration. PMID- 7547520 TI - The effect of head and neck irradiation on taste dysfunction: a prospective study. AB - Taste loss is a major cause of morbidity in patients undergoing head and neck irradiation. In a prospective study, 26 patients undergoing radical head and neck irradiation at the Royal Marsden Hospital, Sutton, and the Queen Elizabeth Hospital, Birmingham, were assessed for taste loss and xerostomia. Taste was tested using a subjective questionnaire and by objective taste testing with a series of solute solutions (sucrose, sodium chloride, urea and hydrochloric acid) at increasing concentrations, to determine the threshold level of taste sensation, both before and after radiotherapy. Xerostomia was assessed using a patient questionnaire. The volume of tongue and parotid contained within the high dose volume of the radiation treatment field was determined for each patient and correlated with the degree of objective and subjective taste loss as well as the degree of xerostomia. The results have shown that both objective (r = 0.59; P = 0.0016) and subjective taste loss (r = 0.78; P = 0.0001) was significantly associated with the proportion of tongue, but not parotid, contained within the radiation treatment field. The data gave no evidence to suggest any relationship between recovery of taste loss and volume of parotid or tongue irradiated. However, recovery of subjective taste loss, 1 month after completing radiotherapy was seen in two patients, both of whom had been treated using a wedge pair technique to avoid the contralateral area of the tongue. Changes in xerostomia were significantly correlated with the proportions of both tongue (r = 0.54; P = 0.004) and parotid (r = 0.82; P = 0.0001) within the radiation treatment fields.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547521 TI - An audit of craniospinal irradiation for medulloblastoma in Newcastle 1970-1992. AB - The case notes were reviewed of 55 patients treated with craniospinal irradiation for cerebellar medulloblastoma during the period 1970-1992. Twenty patients treated by various techniques before 1978 had a survival at both 5 and 10 years of 33%. Thirty-five patients treated from 1978 onwards were irradiated using a standard technique, which is described; their actuarial disease free survival was 59% at 5 years and 47% at 10 years. Our results are similar to those reported from other centres. The recent literature is reviewed. Irradiation of the whole craniospinal axis (CSA) is necessary for disease control, but the optimum dose of radiation is still disputed. It is likely to be in excess of 25 Gy but less than 35 Gy to the whole CSA, and 50 Gy or greater to the posterior fossa. The role of adjuvant chemotherapy is still not proven. The clinical significance of the dose inhomogeneity across the junction between the cranial and spinal fields, and the effect of feathering, are uncertain. PMID- 7547522 TI - Skin care during radiotherapy: a survey of UK practice. AB - A postal survey of skin care during radiotherapy throughout UK centres has shown considerable variability in practice between units. No scientific study has established the value of many of the treatments advocated and suggestions for a more rational approach to the care of irradiated skin are made. In particular, the continued use of common topical applications for the management of dry and moist desquamation is questioned. PMID- 7547523 TI - Measurement of irradiated small bowel volume in pelvic irradiation and the effect of a bellyboard. AB - From June 1992 to January 1993, 51 consecutive patients with pelvic cancers treated with radiotherapy alone, or surgery plus post-operative radiotherapy, were included in a study of the effectiveness of a false table-top (bellyboard) in displacing small bowel loops out of anteriorposterior (AP) and posterioranterior (PA) opposing pelvic fields. The small bowel was opacified with barium. The volume of small bowel within the radiotherapy fields in the prone position with a bellyboard was reduced by 134-300 ml (29%-61%) when compared with that in supine position, and by 136-216 ml (28%-50%) when compared with that in prone position without a bellyboard, depending on the type of previous surgical procedures. The differences were highly statistically significant and likely to be clinically significant. The bellyboard we used is simple, economical, convenient and well tolerated. Its use is recommended, especially when AP and PA pelvic fields are used. PMID- 7547524 TI - Split course radiotherapy for bladder cancer in elderly unfit patients. AB - Between 1980 and 1987, 89 patients with T1-T4 carcinoma of the bladder were treated with a split course of external beam radiotherapy. All patients were felt to be unsuitable for a prolonged course of radical radiotherapy because of age and/or poor general health. The intention was to provide local control. The majority of patients were treated with 10-12 fractions, with a mean dose of 45 Gy (range 31.5-57.7), within a mean overall time of 48 days. Of the 89 patients only 62 were assessable with a median follow-up of 110 months (range 64-157). In the remainder, poor health, poor performance status, or early (< 3 months) death did not allow assessment of tumour control. Complete remission was achieved in 28/89 (31%) patients. This was highly dependent on T stage: 56% T1/T2, 30% T3a/T3b, 5% T4. Median survival for patients with Stage T1/T2, T3a/T3b and T4 disease was 22, 10 and 8 months respectively. Acute grade 1-2 radiation reactions occurred in 60% of these patients, and only 5/62 (8%) experienced grade 2-3 late complications, assessed according to the RTOG scoring system. No patient had more severe treatment related morbidity. PMID- 7547525 TI - Massive cutaneous large cell T-cell non-Hodgkin's lymphoma of the pinna. AB - We report a patient with a cutaneous large cell T-cell non-Hodgkin's lymphoma of the pinna, a condition that has not been reported before. Despite the large dimensions of the tumour, the patient's ear was restored by electron therapy. PMID- 7547526 TI - Non-Hodgkin's lymphoma of the uterine cervix: a report of three patients. AB - Three patients with primary non-Hodgkin's lymphoma of the uterine cervix are described. The results of their treatment and a review of the international literature would support the statement that more patients with localized primary non-Hodgkin's lymphoma of the uterine cervix can be cured with radiotherapy, or with a combination of chemotherapy and irradiation. PMID- 7547527 TI - Extraosseous osteogenic sarcoma of the mediastinum occurring in a Chinese patient. AB - Osteogenic sarcoma can occur rarely in soft tissues, predominantly of the proximal lower limbs. The mediastinum is a less common site, with only five reports in the literature. We report a case occurring in a young Chinese man and review the literature. PMID- 7547528 TI - Radiotherapy in a district general hospital. PMID- 7547529 TI - The UNOS Scientific Renal Transplant Registry. United Network for Organ Sharing. AB - 1. The number of cadaveric transplants performed each year at United States transplant centers has increased very little, from 7,200 in 1988 to 8,100 in 1993. Living-donor transplants increased during the same period from 1,656 to 2,562. 2. The recipient and donor populations have aged since UNOS began collecting data. In 1988, 39% of first-cadaver transplant recipients were over age 45 compared with 45% in 1993. During the same period, the percentage of cadaver kidneys from donors over age 45 increased from 16% to 26%. 3. Recipients over age 60 or under age 19 had 65% 3-year graft survival rates compared with 70% for those in the intervening age groups (p < 0.001). As many as 60% of graft failures after the first year were accounted for by deaths with a functioning graft when the recipient was over 60 compared with less than 15% when the patient was under age 30 (p < 0.01). Rejection caused 45% of graft failures after the first year for recipients under age 45 but only 17% in those over 60 (p < 0.01). When deaths were censored in the graft survival calculation, recipients over age 45 had the highest 3-year survival rate of 79% compared with 72% for those aged 6 18 (p < 0.001). 4. The 3-year graft survival rate for kidneys from donors over age 60 was 55% and from donors aged 46-60 or under 5, it was 58%. Both results were significantly poorer than the 75% survival rate achieved using kidneys from 19 to 30 year-olds. 5. The racial distribution among first-cadaver kidney transplants has been relatively stable between 1988 and 1993, with 60% Whites, 23% Blacks, 8% Hispanics, 3% Asians, and the remainder, other groups. One-year graft survival rates were consistently 3-5% lower for Blacks than for other races (p < 0.001), and the difference increased to 12% by 3 years. The poorer survival rates for Blacks were unaffected by the donor's race. Blacks had the highest patient survival rates. More than 38% of first year failures and 47% of later failures in Blacks were due to rejection, compared with 31% for Whites in both periods (p < 0.01). When deaths were censored from the survival calculation, the graft half-life increased from 11 to 15 years for Whites but only from 5 to 6 years for Blacks. 6. Diabetes became the most prevalent disease among first cadaver transplant recipients, accounting for 28% of the 1993 activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547530 TI - Lung transplantation at the University of Pittsburgh: 1982 to 1994. AB - Lung transplantation is a growing modality of treatment for patients with end stage lung disease. In our program, survival has improved significantly in recent experience. Progress in candidate and donor selection, allograft preservation technique, recipient surgery, and postoperative management combine to reduce recipient morbidity and mortality. Although the tailored antibiotic treatment has significantly reduced the risk of bacterial pneumonia within 2 weeks after operation, infection is still a major cause of death for long-term recipients. Extensive studies need to be continued to understand the pathogenesis of OB and to establish the treatment for OB. PMID- 7547531 TI - Cardiac transplantation at the University of Pittsburgh: 1994 update. AB - During the past 14 years, 746 hearts have been transplanted at the University of Pittsburgh. Immunosuppression has evolved from a CsA-based protocol with or without lympholytic prophylaxis to a tacrolimus-based protocol. Tacrolimus offers comparable survival to the conventional immunosuppression but is associated with lesser side effects and lower recurrent rejection. It also serves as an effective rescue agent for intractable rejection in patients receiving CsA-based immunosuppression. However, the shortage of donors, chronic rejection, and the morbidity associated with chronic use of nonspecific immunosuppression remain the major limitations in clinical transplantation. Currently, research at the University of Pittsburgh focuses on the induction of donor-specific transplantation tolerance in order to eliminate the morbidity associated with nonspecific immunosuppression and on left ventricular-assist devices as a bridge and/or an alternative to cardiac transplantation. PMID- 7547532 TI - Cardiac transplantation at the Cleveland Clinic Foundation--the first 10 years. AB - Cardiac transplantation in adults currently achieves a 90% one-year survival. Current policies of matching, rejection therapy, and infection prophylaxis have neutralized some variables that previously had been associated with increased risk. Small size or young age continues to be a high risk situation. In our recent experience 2 factors have emerged that were not risk factors previously: ischemic duration and female gender. PMID- 7547534 TI - Liver transplantation for viral hepatitis: the experience of the hepatobiliary center at Hospital Paul Brousse. AB - The prevalence of HBV recurrence after liver transplantation is higher in patients with viral B cirrhosis than in patients with viral B-D cirrhosis or fulminant hepatitis B and is related to the presence of HBV replication prior to transplantation. Long-term passive anti-HBs immunoprophylaxis is the best current way for prevention of HBV reinfection and improved long-term survival. The rate of recurrence of HCV infection is high, reaching 85%, and the rate of HCV hepatitis in the graft is approximately 75%. In most cases, HCV hepatitis leads to chronic hepatitis. The severity of graft hepatitis is related to the level of viremia at the time of the hepatitis and to the genotype 1b. The methods of prevention of HCV infection after liver transplantation are yet to be found. The treatment of graft HCV infection with interferon should be well evaluated and given cautiously. Other antiviral treatments without an immunostimulating effect are needed. Finally, patients transplanted for hepatitis B without HBV replication, receiving posttransplant long-term anti-HBs immunoprophylaxis, and those transplanted for HCV cirrhosis have a 5-year survival similar to other groups of transplanted patients. Patients belonging to these groups can be considered as candidates for transplantation. Patients with active HBV replication should be included in specific trials for prevention of HBV reinfection. PMID- 7547535 TI - Liver transplantation at California Pacific Medical Center, San Francisco, California. AB - A number of modifications in patient selection, operative technique, and immunosuppressive management have greatly contributed to the success of the liver transplant program at CPMC. Graft rejection and the timely detection of EBV infection are ongoing problems in hepatic transplantation that are foci of active research in our field. To address these issues, our group is investigating the activity of cytokines and adhesion molecules using sophisticated molecular techniques, and we are developing a sensitive assay for EBV markers in blood. These and other projects currently in progress will continue when we move our liver transplant program to Stanford University Medical Center in January 1995. PMID- 7547536 TI - Liver transplantation at Mount Sinai Hospital. AB - Liver transplantation is an exciting field of research in which evolution in surgical techniques and immunosuppression and preservation have dramatic effects on overall patient survival. Our research at Mount Sinai is focused on preventing recurrent disease, optimizing the use of new immunosuppressive agents, and improving graft function. It is our hope that our research will eventually make it possible for us to overcome the supply-demand disparity and permit us to ask "When should this patient be transplanted?" rather than the very difficult question, "Who should be transplanted?" PMID- 7547537 TI - Auxiliary partial orthotopic liver transplantation for acute liver failure: the Hannover experience. AB - APOLT has been developed both to enable the native liver to regenerate in acute liver failure and to avoid the risks of long-term immunosuppressive therapy. We present our experience with APOLT in 4 patients with acute liver failure. The patients were 33, 18, 5, and 34 years of age, respectively. The causes of hepatic failure were: one HELLP syndrome, one paracetamol intoxication, and 2 causes which remained undetermined. All patients were in coma before transplantation. First symptoms had occurred 13, 2, 30, and 20 days prior to APOLT, respectively. In all cases, segments II and III of the recipient's own liver were resected before implanting the auxiliary liver orthotopically. The auxiliary graft consisted of segments II and III in 2 cases and of II, III, and IV in the other 2 patients. The auxiliary graft showed good initial function in all cases. All 4 patients are alive 5 years, 15 months, 6 months, and one month after transplantation. In the last patient, an arterial thrombosis of the graft on the 5th postoperative day required retransplantation. The immunosuppressive therapy could be stopped in 3 cases and the graft was removed in 2 patients 15 and 40 days after APOLT, respectively. This shows that APOLT represents an effective treatment for patients with acute liver failure, which enables restoration of native liver function. Thus, APOLT should be considered in every patient with acute and potentially reversible liver failure in whom a transplantation is indicated. PMID- 7547533 TI - Hepatic transplantation at the University of Pittsburgh: new horizons and paradigms after 30 years of experience. PMID- 7547538 TI - UCLA liver transplantation: analysis of the first 1,000 patients. AB - Graft and patient survival rates for Black patients were higher than for any group. This may be due to the younger age distribution among Black transplant recipients versus other races at UCLA. Graft and patient survival for Asian patients were significantly lower than for any other group. However, this result was not totally accounted for by the rapid recurrence of disease in hepatitis B patients. Patients with a positive flow cytometry crossmatch had significantly lower first and second graft survival rates due to early graft loss. Patients with PRA of more than 10% had a higher proportion of positive flow crossmatches. However, as a group, patients with more than 10% PRA did not demonstrate decreased graft survival. Consideration should be given to prospectively flow crossmatching the more than 10% PRA group. Patients with zero-DR mismatches had better survival than patients with one- and 2-DR mismatches. Prospective HLA matching in OLT patients is not currently done. PMID- 7547539 TI - Liver transplantation in the United States: results from the National Pitt-UNOS Liver Transplant Registry. United Network for Organ Sharing. AB - The growth in liver transplantations recorded by the Pitt-UNOS Liver Transplant Registry since October 1987 continues, and in 1993 the rate of increase was greater than it had been in recent years. This is in spite of the fact that the net growth of new centers was smaller in 1993 than in any previous year examined. Pediatric recipients in 1993 were compared with those in previous years, and no significant differences were found for sex, race, or age. In contrast to prior years, the majority of pediatric recipients in 1993 awaited transplantation at home. The most common indication for liver transplantation in children was biliary atresia, although the proportion of recipients with this primary liver disease decreased slightly in 1993. Significant increases were noted in the proportions of pediatric recipients with fulminant liver failure, and hepatoblastoma. Significantly fewer children received ABO-incompatible livers in 1993 compared with prior years, part of which may be a function of the increasing use of living-related donors. Many of the characteristics examined for adult recipients had different distributions in 1993 than in prior years. The proportion of White recipients declined in 1993, due to increases among Black and Hispanic recipients. The mean and median ages of adult recipients continued to increase because of the increasing proportion of recipients aged 60 and over. The proportion of adult recipients awaiting transplantation outside of the hospital continued to increase in 1993. The increase in the proportion of recipients with positive CMV serology is likely due to the increasing age of the recipients in 1993. A smaller proportion of multiorgan transplantations was performed in 1993, due to the elimination of procedures involving only the liver and pancreas. Alcoholic cirrhosis was replaced by hepatitis non-A, non-B, or C as the most common reason for LTX. The proportions of recipients with fulminant liver failure and malignancies, indications for poorest survival, declined significantly in 1993. The cumulative probability of surviving for 6 years after initial transplantation was 0.70 (without retransplantation = 0.58) for pediatric recipients.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547540 TI - Living-unrelated renal donor transplantation: the UNOS experience, 1987-1991. United Network for Organ Sharing. AB - 1. LURD transplants were associated with excellent one-year graft survival of 92%. This survival was superior to that for cadaver transplants performed during the same period. 2. High-risk groups for LURDs are children (age < 18 years) and repeat transplant recipients. Both groups were associated with significantly decreased graft survival. 3. The effects of HLA matching and donor-specific transfusions are not significant. 4. Because of the critical shortage of donor organs and the increasing waiting time for renal transplantation, the use of LURDs can be recommended as a means to expand the number of available organs. PMID- 7547541 TI - Pediatric renal transplantation at the University of Minnesota: the cyclosporine years. AB - Overall mortality was low (3.5%) in 199 children who received 217 transplants and quadruple immunosuppression. Graft survival was better in patients on quadruple immunosuppression, compared with historical patients on standard immunosuppression. When censored for deaths with a functioning graft, cumulative graft survival beyond one year was significantly better in patients on quadruple immunosuppression. Acute rejection was more common in CAD recipients and in retransplant recipients. Acute rejection was also more common in patients with FSGS, compared with other causes of ESRD. Preemptive transplants were not associated with a higher incidence of acute rejection. Late acute rejection episodes tended to be associated with a CsA dose < 5 mg/kg/day at one-year posttransplant. Chronic rejection was the major cause of graft loss. Risk factors for graft loss within the first posttransplant year were acute tubular necrosis and an initial CsA dose of < 5 mg/kg/day. Risk factors for graft loss after the first posttransplant year were late onset of the first acute rejection episode and a panel reactivity of > 25% at the time of transplant. PMID- 7547542 TI - Renal transplantation at Allegheny General Hospital with special reference to the use of extreme-age cadaveric donors. AB - We report our experience with 25 living-donor and 427 cadaveric-donor kidneys followed for over 6 years. Patient survival exceeds 90% at 5 years for all recipients. Overall one-year and 5-year graft survival rates were 82.7% and 65.5%, respectively. Approximately one-third of CAD were of extreme age (eg, < or = 10 and > or = 60 years old). One-year and 5-year GS rates of this group were 72.2% and 54.8%, respectively. Kidneys from elderly donors probably should be given to age-matched recipients. Infant en bloc kidneys had good GS. They also adapted well their growth and function to adult recipients and should be used more frequently to alleviate the organ shortage problem. HLA mismatches, transplant PRA level, original kidney disease, and immunosuppressive regimens significantly affected the GS. Race, sex, cytomegaloviral antibody states, unacceptable antigens, length of pretransplant dialysis, and CAD cold ischemic time had no significant effect on GS. PMID- 7547543 TI - Thirty years of renal transplantation in Helsinki. AB - During triple-drug immunosuppression, consisting of azathioprine, steroids, and cyclosporine, the number of acute rejections was low. Close monitoring of the patient and the graft during the early posttransplant weeks with regular cytological and histological analyses was the cornerstone of early diagnosis and a favorable outcome of acute rejection. Genetic homogeneity of our patient population, third-party transfusion program, and use of well HLA-matched kidney grafts may have been additional factors behind the low rate of acute rejection. Acute rejection within the first three postoperative months did not predispose the renal graft to chronic rejection in the long term. The histologically determined allograft damage index was a reliable predictor of future graft survival. Recipients with systemic diseases such as diabetes and amyloidosis, as well as elderly patients, could be transplanted safely with results only slightly inferior to those achieved with primary kidney disease. Results in diabetic recipients have steadily improved, encouraging the continuation of critical evaluation of the patients' pretransplant status and the preference for cadaveric transplants. Renal transplantation in small children yielded superior patient and graft survival rates. We consider the use of living donors justifiable in these cases. The five million population of Finland offers an ideal size for a transplant center. Continuous education of medical personnel and the general public is crucial for the cadaver transplant program. PMID- 7547545 TI - Renal transplantation from cadaver donors over 60 years old. AB - The influence of donor age on the short- and long-term outcome of cadaveric kidney transplantation was analyzed at our institution. During a 6-year period, 34 and 806 patients underwent kidney transplantation from cadaver donors over or less than age 60, respectively. Graft and patient survivals were compared throughout follow-up and herewith reported. In addition, main medical and surgical complications among recipients of elderly cadaver donors are detailed. PMID- 7547547 TI - Simultaneous kidney and pancreas transplantation at the San Raffaele Scientific Institute: clinical experience and results. AB - Pancreas and kidney transplantation is performed in uremic IDDM patients to cure end-stage renal failure and diabetes. Seventy-two simultaneous kidney-pancreas transplantations were performed at our Institution between July 1985 and November 1994. All transplants were performed using heart-beating cadaver donors. The first 25 patients received 26 segmental pancreas according to Dubernard (KPS), whereas the last 46 patients received a whole, bladder-drained pancrea according to Sollinger (KPW). Mean pancreas cold and warm ischemia times were 294 +/- 14 and 44 +/- 2 minutes, respectively, in the KPS group and 660 +/- 37 and 40 +/- 8 minutes, respectively, in the KPW group. Twelve (48%) KPS patients and 19 (41%) KPW patients had postoperative pancreas surgical complications: vascular thrombosis led to graft failure in 5 KPS patients (20%) and 2 KPW patients (4%) (p = 0.01). Pancreatic fistula, hemorrhagic complications, and duodenum-bladder leakage were the surgical complications observed more frequently. Six KPS patients (24%) and 8 KPW patients (17%) underwent reintervention as a consequence of surgical complications. Fifteen KPS patients (60%) and 30 KPW patients (65%) experienced an acute kidney rejection episode, which was steroid-resistant in 14 KPW and 2 KPS patients. The actuarial survival rates for simultaneous kidney pancreas recipients at one and 4 years were 92% and 84%, respectively, for KPS recipients, and 95% and 88%, respectively, for KPW patients. Kidney actuarial survival rates at one and 4 years were 96% and 76% respectively, for group KPS, and 93% and 89%, respectively, for KPW patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547544 TI - Renal transplantation at the University of Pittsburgh: the impact of FK506. AB - 1. In an unselected adult renal transplant population, FK506 as the primary immunosuppressive agent yielded one- and 2-year actuarial patient survival rates of 95% and 93% and one- and 2-year actuarial graft survival rates of 89% and 83%, respectively. Forty-nine percent of successfully transplanted patients were weaned off steroids. 2. In pediatric renal transplant patients, FK506 has been associated with 100% one- and 3-year actuarial patient survival rates and 98% and 85% one- and 3-year actuarial graft survival rates, respectively. Sixty-two percent of successfully transplanted patients were taken off prednisone, with dramatic improvements in height. 3. FK506 has been used successfully in rescuing 70-74% of adult or pediatric renal transplant patients with an acute rejection that failed conventional therapy. 4. Kidney/bone marrow transplantation under FK506 therapy has been successfully performed without graft-versus-host disease and with routine augmentation of chimerism. 5. The side effects of FK506 included nephrotoxicity, neurotoxicity, and diabetogenicity; they were comparable to those seen with CsA. 6. FK506 is an important new addition to the immunosuppressive armamentarium in renal transplant patients. PMID- 7547546 TI - Twenty-five years of transplant experience at Policlinico University Hospital of Milan. AB - 1. CsA treatment played a major role in the improvement of renal transplantation in the third era of our experience for every class of recipient. Many of the most recent patients involved higher risks than in previous eras. 2. The type of donor (LD or CD) did not make a significant difference in patient or graft survival rates up to 4 years. However, the survival rate of LD kidney transplants was better in later follow-ups than that of CD transplants. Pediatric and older kidneys can be used safely, although a lower survival rate is achieved by these grafts. 3. The sex and the side (right or left) of the donor kidney does not affect the outcome of transplantation. 4. In both multiorgan and single-kidney donor origin, there was no difference in graft survival. As all MOD kidneys were flushed with UW Solution and all SKD kidneys were flushed with EC Solution, we can assume that there is no difference in graft outcome, whichever of these solutions is employed. 5. The limited availability of CD grafts has been overcome by using marginal kidneys and adopting extracorporeal microvascular techniques. The results compared well with those of normal allografts. 6. The transplant outcome is penalized by expected higher mortality in older patients. And in pediatric patients there is a higher rate of graft failure due to rejection. 7. Retransplanted grafts under CsA have a better success rate than those using conventional therapy. 8. Extrarenal pathology and the actual risk ratio for CsA treated recipients still need to be defined by future follow-ups. 9. Our results in renal transplantation, particularly from CDs, could be defined as good in comparison to those of other large transplant centers. 10. The main problem remains that the supply of CD kidneys has decreased in recent years, whereas the demand is increasing, perhaps due to the opening of other transplant centers, an inadequate policy for organ procurement, and ineffective legislation, the last being essential to promote kidney transplantation. PMID- 7547548 TI - Pancreas transplantation: the Nebraska experience. AB - In summary, we believe that combined PKT can be performed safely and effectively in the absence of uremia, thereby providing the potential for arresting the progression of diabetic complications prior to the development of ESRD. Furthermore, performing solitary PTx prior to the need for a kidney transplant can be accomplished with morbidity and results comparable to PKT. Rehabilitation potential tends to favor patients undergoing either solitary PTx or preemptive PKT. In selected IDDM patients without end-stage diabetic nephropathy, we believe that solitary PTx or PKT are effective treatment options and need not be considered as preemptive, especially in view of increasing waiting times and the variable progressive nature of diabetic complications. As results continue to improve, solitary PTx may offer a potential solution to the growing number of IDDM patients awaiting kidney transplantation in the United States. Ultimately, the role of solitary PTx in the treatment of IDDM will be determined by long-term studies documenting the prevention or arrest of secondary diabetic complications. In the short-term, improvement in quality of life and rehabilitation potential makes preemptive PKT or solitary PTx important therapeutic alternatives for consideration. PMID- 7547549 TI - Bone marrow transplantation for chronic myelogenous leukemia: clinical outcomes. AB - Marrow transplant is potentially curative therapy for CML. Allogeneic sibling donor marrow transplant may be the therapy of choice for younger patients early in the course of disease. Early transplant is an important influence on disease free survival and relapse after related-donor transplant therapy, although additional patient characteristics influencing outcome can be identified and may have cumulative adverse effects. The 5-year disease-free survival of patients transplanted within one year of diagnosis and without signs of advanced disease is greater than 65%. Significant problems remain, however, including early mortality (primarily from infection, pneumonia, and pneumonitis) and relapse of CML following transplant, including late relapse occurring more than 5 years posttransplant. For patients without a matched, related donor, unrelated-donor marrow transplant may be a treatment option and can result in successful outcome for patients with CML. Relapse following unrelated-donor marrow transplant is rare. However, the use of an unrelated donor is associated with significant toxicity, including early mortality, engraftment failure, and ongoing morbidity and mortality associated with acute and chronic graft-versus-host disease. For patients who lack an available matched-related or -unrelated donor, autologous marrow transplant has been developed as an alternative approach to therapy. Long term survival following autologous marrow transplant is possible and may even approach the survival for allogeneic related-donor recipients, although cure of disease is not achieved. PMID- 7547550 TI - Unrelated-donor marrow transplants: the experience of the National Marrow Donor Program. AB - As of December 1994, more than 3,000 marrow transplants had been accomplished using unrelated donors provided by the National Marrow Donor Program. With more than 1.5 million donors listed in the registry, over 60% of patients now find an HLA-A,B,DR phenotypic match at the initial search. The HLA types of these patients are biased toward the common Caucasian haplotypes, but the likelihood of identifying donors for non-Caucasian patients has improved with time. Analysis of the first 462 transplants showed disease-free survival (DFS) at 2 years to be approximately 40% in good-risk patients and 20% in poor-risk patients. Chronic myelogenous leukemia transplanted within the first year after diagnosis had 45% DFS. Some recent reports from individual transplant centers demonstrate results closer to those obtained with sibling donors, while a limited retrospective comparison suggests that unrelated-donor transplants are at least equivalent to and probably better than autologous transplants. A single HLA mismatch at A, B, or DR can be tolerated, but results are better with phenotypic identity. The most recent NMDP analysis has also identified younger donors and male donors as favorable variables in evaluating one-year survival of unrelated-marrow recipients. PMID- 7547551 TI - Nonheart-beating donors: the Maastricht experience. AB - The growing gap between the number of organs available for transplantation and patients on waiting lists demands additional donor sources. Nonheart-beating (NHB)-donor programs are known to increase the number of kidneys available. The group of potential NHB donors is very diverse and therefore 4 categories have been identified. At the University Hospital Maastricht, a NHB-donor program was implemented in 1980 to harvest kidneys after an in situ perfusion technique. In order to evaluate the function of kidney grafts from NHB donors, a retrospective multicenter study on the NHB-donor kidneys transplanted until 1992 was performed, using a control group of kidneys from heart-beating (HB) donors. The short-term results showed more delayed function (DF) in the NHB-donor group accompanied by higher serum creatinine levels at one-month posttransplant. The long-term outcome, however, was equal in both groups showing similar graft and patient survival rates up to 5 years. Apart from the type of donor, HB or NHB, only the number of HLA-DR mismatches could be identified as a potential risk factor for delayed graft function. NHB donors contribute considerably to reducing the gap between offer and demand in kidney transplantation and transplant centers should include NHB donors in their procurement programs. Meanwhile, efforts should be made to improve the short-term posttransplant graft function. Together with reducing DF, searching for valuable tools in viability assessment should also be an objective. Past viability tests never found broad clinical use but might be important in the optimal and safe usage of the potential NHB donor pool. PMID- 7547553 TI - Current world opinion regarding the use of living donors. PMID- 7547552 TI - Transplantation 1994: the year in review. AB - Progress during 1994 has yielded important positive effects on the clinical practice of transplantation. During the span of one generation, organ transplantation has moved from an experimental endeavor to an established therapeutic modality. Technical advances have improved the safety of the surgical enterprise, such that the major focus of research is now immunosuppressive therapy. Although tacrolimus has been approved for use in liver transplantation, ongoing trials seek to establish its role in the immunosuppressive arsenal. Undoubtedly, the introduction of mycophenolate mofetil and Neoral in the coming year, and probably sirolimus shortly thereafter, will broaden the spectrum of immunosuppressive agents, thereby improving transplant results. The recently completed multicenter studies not only demonstrated effective cooperative efforts by the transplant community, but also offered new insights into the more efficient design of future clinical trials. Although rejection remains the major barrier to transplant success, increasingly it is recognized that improved outcomes demand a decreased spectrum of iatrogenic side effects that markedly increase morbidity. Thus, strides in immunosuppressive management must be evaluated both by mitigation of rejection episodes and by quality of life measures. The ultimate goal of achieving transplant tolerance is now being addressed in clinical studies. There is no universal agreement concerning the role of chimerism as a strategy for tolerance induction; preclinical studies suggest that refined donor-type antigens or selectively enriched donor-cell infusions represent more likely tolerogenic stimuli. The challenge of inducing immunologic tolerance remains an elusive goal as we approach the new millennium. PMID- 7547554 TI - More or less living donation? PMID- 7547555 TI - The influence of organ supply and demand pressures upon the "primum non nocere" principle in living-donor transplantation. PMID- 7547556 TI - Live-organ donation and transplantation. PMID- 7547557 TI - The use of living donors. PMID- 7547558 TI - Current opinion: living donation and organ transplantation. PMID- 7547559 TI - Living-kidney donation: safety by procedure. PMID- 7547560 TI - The use of living donors. PMID- 7547561 TI - Living-donor transplantation--a view from Australia. PMID- 7547562 TI - Living-related and unrelated renal donation: the University of Wisconsin perspective. PMID- 7547563 TI - A positive view of living donors for transplantation. PMID- 7547564 TI - High graft survival with spousal and 2-HLA-haplotype mismatched sibling-donor transplants. PMID- 7547565 TI - Kidney transplantation using living donors in Japan: how the donors feel. PMID- 7547566 TI - Thoracic organ transplants in the United States: a report from the UNOS/ISHLT Scientific Registry for Organ Transplants. United Network for Organ Sharing. International Society for Heart and Lung Transplantation. AB - 1. The frequency of heart and lung transplantation has increased dramatically over time. The number of heart transplants increased 170% between 1968 and 1981. Exponential increases began in 1982 and increased through 1990, as evidenced by a 1,835% growth in the number of procedures performed during those years. In more recent years, heart transplant numbers have leveled off, with only an 8% increase in the last 3 years. Lung transplant procedures have grown significantly in the short period of time between 1987 (n = 18) and 1993 (n = 669). 2. Since the beginning of the ISHLT Registry in 1968 the number of thoracic transplant programs has increased 7,767%, from 3 to 236. 3. The most frequently reported indications for thoracic transplantation include: coronary artery disease (43.4%) for heart, cystic fibrosis (39.5%) for double-lung, emphysema/COPD (40.2%) for single-lung and primary pulmonary hypertension (38.3%) for heart-lung. 4. The majority of heart transplant recipients are at least 18 years old (89.5%), male (78.2%), and White (83.8%). The majority of lung transplant recipients are at least 18 years old (93.7%), female (53.2%), and White (91.3%). 5. One-year survival over time has almost doubled for all types of thoracic transplantation, with increases from 47.7% in 1968-79 to 81.6% in 1993 for heart; 35.3% in 1987 to 67.1% in 1993 for lung; and 40% in the early 1980s to 73% in 1993 for heart-lung. 6. Long-term 10-year survival rates were 33.5% for heart and 5.6% for heart-lung transplant recipients. Five-year survival for lung recipients was 37%. 7. Male-to male donor-to-recipient gender-match heart transplant patients exhibited slightly higher survival (4-5%) than other match pairs from one to 5 years posttransplant. Female-to-male combinations in lung transplant recipients exhibited slightly higher survival (4-8%) at 3 years than other match pairs. 8. There was no significant difference in 5-year survival for donor hearts procured from local, intraregional, or interregional sources. Lungs procured from interregional sources exhibited an 8-10% advantage over local and intraregionally procured organs at 3 years posttransplant. PMID- 7547567 TI - Kidney transplantation--the living donor. PMID- 7547568 TI - Use of kidneys from living-related or unrelated donors: pro. PMID- 7547569 TI - Living-organ donation: time for a donor charter. PMID- 7547570 TI - Donor factors. AB - 1. Although left kidneys had a 2% higher graft survival rate at one-year posttransplant, the survival rates for left and right kidneys were comparable at 2, 3, and 4 years after transplantation. Kidneys transplanted en bloc were shown to have a 6% lower graft survival rate than either left or right kidneys. 2. Male donor kidney had a 4% higher rate of graft survival than female donor kidneys at both one and 4 years posttransplant. 3. Kidneys obtained from Black donors had a 4% lower graft survival rate at one-year posttransplant, and a 7% lower graft survival rate at 4 years after transplantation than White donor kidneys. White, Hispanic, and Asian donors all had comparable rates of graft survival at one, 2, and 3 years posttransplant. Black donor kidneys also had a significantly shorter half-life; 6.7 years, compared with 7.1 years for kidneys from Asian donors, 9.7 years for Hispanic donors, and 8.6 years for White donors. Blacks continued to make up a small fragment of the total donor pool, accounting for less than 10% of all cadaveric donor kidneys. 4. Kidneys from type O donors had a 5% higher graft survival rate at 4 years posttransplant when compared to AB kidneys and had a 2% higher 4-year graft survival rate than either type A or B kidneys. 5. Pediatric (younger than 5 years of age) donor kidneys had a 10% lower graft survival rate than kidneys from donors between 6 and 45 years of age. Kidneys from donors over 60 years of age had an 11% lower one-year graft survival rate than donors between 6 and 45 years of age, and a 19% lower survival rate at 4 years posttransplant. Survival rates decreased with increasing donor age; kidneys from donors between 46 and 60 years of age had a 5% lower graft survival rate at one year, and a 9% lower rate of graft survival at 4 years posttransplant when compared to "middle aged" donors. The poorest graft survival was observed for kidneys from donors over 60 years of age; 10% and 19% lower graft survival at one and 4 years posttransplant, respectively, compared to donors between 6 and 45 years of age. 6. Kidneys from trauma donors had a 4% higher survival rate at one-year posttransplant, and a 6% higher survival rate at 4 years posttransplant when compared to kidneys from nontrauma donors. Kidneys obtained from victims of motor vehicle accidents, traumatic suicides, and assaults (gunshot wounds and stabbings) had the highest rate of graft survival.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547571 TI - Pediatric renal transplantation. AB - 1. Approximately half of the pediatric patients received grafts from their parents and half from cadaver donors. Pediatric recipients accounted for only 5% of cadaver-donor transplants. 2. Pediatric patients differed from adults in the incidence of primary disease, principally in the occurrence of diabetes (almost no juvenile diabetics had progressed to renal failure compared with 30% among adults). Obstructive uropathy and dysplasia were the primary diseases for about 37% of pediatric patients compared with 3% in adults. 3. Pediatric patients under the age of one had a markedly lower graft survival with cadaver donors. Such patients had much higher survival rates with parental donor kidneys. 4. Pediatric retransplants from parents had a graft survival comparable to adult grafts. Regraft survival in children given cadaveric kidneys were significantly lower than adult grafts. 5. Patients under the age of 18 with no diuresis on the first day or who required dialysis in the first week had a markedly lower graft survival rate compared with that of adults with comparable early dysfunction. 6. Kidneys from donors under 2 years old yielded a 67% one-year graft survival rate in infants, but an 87% survival in patients 16-18 years of age at 6 selected individual centers. These centers, that had performed more than 20 infant-donor renal transplants, achieved results comparable to those of transplants using grafts from older donors in recipients aged over 11 years. 7. Pediatric patients who received grafts mismatched for 5- and 6-HLA antigens had a significantly lower graft survival rate than those with better matches. 8. Black patients ages 11-18 years old had a lower graft survival rate than White patients in the same age group. PMID- 7547572 TI - Primary disease effects and associations. AB - 1. Although graft survival for most primary disease processes are similar at one year, significant divergence occurs by 5 years. ALP, IGA, and PC had the highest 5-year graft survival rates (72.8%, 71.2%, and 68.5%, respectively) whereas HTN and NS, the lowest (51.8% and 46.0%, respectively). 2. When primary diseases are grouped by pathogenic, pathophysiologic, and clinical similarities, the group of diseases with systemic manifestations had the lowest 5-year graft survival (55%), and the group including cystic and inherited diseases had the highest 5-year graft survival (69%). Black recipients had a predominance of "systemic" primary diseases (57%). 3. Despite having overall lower graft survival than Whites (p < 0.00001), there was no significant difference between Black and White 3-year graft survival for recipients with PC, ALP, IGA, and SLE. 4. PC recipients enjoyed excellent long-term graft survival (69%). Black recipients with PC had a 5-year graft survival rate of 64.6%. Recipients with PC had decreased posttransplant dialysis need, decreased early rejection rate, and better HLA matching than most other recipients. 5. Recipients with SLE as their primary disease had among the highest fraction of grafts lost to rejection (45.4% of all grafts lost) and the highest pretransplant sensitization rate (59.6%). 6. Recipients with HTN as their primary disease had overall lower 5-year graft survival (58% versus 63% in Whites, 44% versus 47% in Blacks), a lower rate of early allograft function (10% versus 12%, p < 0.00001), and more posttransplant dialysis needs (28.8% of patients requiring dialysis vs 23.5%, p < 0.00001) than recipients without HTN. Blacks with HTN had the lowest long-term graft survival (44.4%) of any other single group. 7. IDDM patients who expressed DR3 and/or DR4 alleles had significantly higher graft survival than patients without these DR groups. Whites expressing DR3 and DR4 and DR3 or DR4 alleles had better overall HLA matching (p < 0.001) and graft survival (75.4% and 70.7% versus 58.5% and 65.1%, p < 0.00001) than Blacks with similar DR expression. 8. SPK recipients had better 5-year graft survival than KAT recipients (66.2% versus 54.6%, p < 0.000001). This effect is most likely due to the selection of "better" lower-risk patients for SPK grafts. PMID- 7547573 TI - Renal retransplantation. AB - 1. In 1984, second graft survival rates were 10% lower than first grafts, but in 1992, the survival difference was reduced to 1%. Multiple grafts in 1984 were 23% lower than first grafts, but showed only a 7% difference in 1992. In 1992, 12% of kidney grafts were performed into second graft recipients and 3% into multiple graft recipients. 2. If first grafts survived one to 12 months posttransplant, the second graft survival was less than if they had survived longer than 12 months, as seen in many previous analyses. Here we showed that patients with a first graft duration of one to 12 months had a higher incidence of sensitization than patients with a first graft duration of more than 12 months. This may indicate that immunization was the cause of failure more frequently among those patients who rejected earlier than later. 3. Since 1989, the interval between first graft rejection and second graft transplantation was not a factor in second graft survival. A strong correlation was noted between high PRA and interval to regrafting. This probably reflects the increasing difficulty in finding negative crossmatch donors as PRA increases. 4. Repeat mismatches for HLA-DR were deleterious to second grafts, although repeat mismatches for HLA-A,B were not, confirming earlier studies (1,5). HLA-A,B,DR mismatches correlated well with second and multiple transplant outcomes. 5. Patients receiving second cadaver donor transplants had the same graft survival regardless of whether the first graft was another cadaver donor or a living-related one. On the other hand, second living-related donor transplants had a higher graft survival rate if the first graft had also been from a living-related rather than a cadaver donor (p < 0.05). This suggests that it would be advantageous if the first graft came from a living-related donor with a cadaver donor as the second graft, rather than the reverse situation. 6. Urine production on the first postoperative day was a very strong indicator of subsequent graft survival, particularly for second and multigraft patients. Failure to diurese on the first day resulted in a second graft survival of 60% at one year compared with 80% for those that diuresed on the first day. 7. Similarly, dialysis requirements were a major factor in predicting subsequent graft survival. For second graft patients who required dialysis, one-year graft survival was 63%, compared with 84% if no dialysis was needed. 8. The fraction of patients who had insulin dependent diabetes for first grafts was 27%, 15% for second grafts, and 9% for multiple grafts.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547574 TI - HLA matching effect: better survival rates and graft quality. AB - 1. HLA matching remains a major factor in kidney transplantation. Much of the total graft failures can be eliminated through better HLA matching. 2. Very early effects of HLA matching can be seen with the requirement of dialysis within one week. 3. Even among kidneys which are functioning at the beginning of each period, more frequent rejection treatment with increasing numbers of A,B,DR mismatches was observed. 4. Among functioning kidneys, HLA matching affects the quality of kidney function, as reflected in the serum creatinine levels during all periods. 5. Immunological graft failures (regardless of cause) are strongly associated with HLA mismatching. 6. The effect of HLA matching was similar at centers with high or low overall graft survival rates. 7. The fraction of zero A,B,DR mismatches has increased dramatically in recent years. However, this is a large difference in the numbers between centers and OPOs. 8. Preformed cytotoxic antibodies to HLA tend to force a higher degree of matching for the A and B loci, resulting from T-lymphocyte crossmatching. 9. Because of the linkage of the 3 HLA loci (A, B, and DR), matching for one often results in matching for 2 or 3 of the loci. 10. Chronic glomerulonephritis patients having DR1 had superior graft survival rates than patients without DR1. 11. HLA frequencies in IDDM, hypertensive nephropathy, CGN and PC were significantly different from controls in many Class II specificities and some Class I specificities. PMID- 7547575 TI - Refinement of permissible HLA mismatches. AB - Cadaver-donor transplants performed before 1987 that had survived for more than 7 years despite having all 6-A,B,DR antigens mismatched were used to develop a list of permissible mismatches. When these permissible mismatches were applied to an entirely different set of cadaver-donor grafts performed after 1987, patients with permissible mismatches had a higher graft survival than those with an equivalent number of immunogenic mismatches. In addition, a new list of permissible mismatches was produced from living-donor transplants which survived for more than one year despite having mismatches. When this list made from living donor transplants was checked against cadaver-donor transplants, patients with one-permissible-A,B,DR mismatch had a projected 10-year graft survival equivalent to that of zero-A,B,DR mismatched transplants. Patients with one immunogenic mismatch had a significantly lower graft survival. Patients with only permissible mismatches had a higher graft survival than those with immunogenic mismatches and equivalent to patients with one immunogenic mismatch. It was projected that the percentage of patients who can benefit from a zero-A,B,DR mismatched transplant by national sharing can be doubled if one permissible-mismatched grafts are also shared. Even in local pools, the use of permissible-mismatched grafts can be projected to improve 10-year graft survival rates from the current 41% to 52%. PMID- 7547576 TI - International Cell Exchange, 1994. AB - 1. We summarize typings of 40 cells for Class I antigens and 20 cultured cell lines for Class II antigens through the International Cell Exchange in 1994. Serologic Class II typings were compared with DNA typings for the same 20 cells. Two hundred eighty-one laboratories participated in the monthly Class I Serum Exchange. One hundred nineteen serology laboratories and 74 DNA laboratories reported Class II specificities on a monthly basis. 2. The average detection levels, as well as the high detection levels, were determined for 16 A-locus and 27 B-locus antigens. Mean detection rates of 95% or greater average detection were obtained for 12 A-locus and 10 B-locus antigens. Lower than 80% agreement was calculated for one A-locus antigen (A74) and 7 B-locus (B46, B48, B61, B67, B73, B75, B77) antigens. 3. We compared discrepancy rates of 10 A-locus and 7 B locus antigens typed 3 times or more. The false-negative discrepancy rates, i.e. how often the antigen was missed, were greater for more of the B-locus specificities than for the A-locus antigens. B62, having the highest false positive rate, tended to be overassigned. The discrepancy rates, especially the false-negative rate, for B70 were shown to decrease over a 7-year period. 4. In 1994, 8 laboratories attained records of total no misses for all analyzed antigens. Twelve laboratories had final records of only one discrepancy, and 5 laboratories had impressive perfect records (zero false negatives and false positives) for their yearly antigen reports. 5. Retyping of 12 Class I and 8 Class II reference cells showed improved detection of antigens. Results of a donor typed 4 times over 11 years demonstrated marked improvement, nearly doubling for A33, B38, and B75. Two cells first typed in 1991, then retyped in 1994, showed improved detection for Class II splits by serology and DNA typing. 6. We updated the list of sequenced Class I Exchange cells. Seven new cells were added as well as confirmatory sequence data for A*2403 to the A9.3 (cell 762) and a new B*0705 (cell 715) for previously listed cells. Variants detected in the exchanges for Class I (DT/B7x40, B15, B16, B5x53) and Class II (DRB1*1111, DQB1*02, DR1, DR2) specificities were discussed. Eleven rare or unusual Class II haplotypes were studied in 1994. 7. The mean detection levels were determined for 9 broad DR and 4 DQ specificities by serology and compared to those attained for the respective generic (low resolution) DNA typing.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547577 TI - Pancreas transplant results in the United Network for Organ Sharing (UNOS) United States of America (USA) Registry compared with non-USA data in the International Registry. AB - As of 1994, more than 6,300 pancreas transplants were reported to the IPTR. More than 4,300 were performed in the USA, including more than 3,000 since the inception of the UNOS Registry in October 1987. The BD technique was used for 96% of USA cases but only for 64% of non-USA cases. In the overall analysis of USA BD cadaveric pancreas transplants reported to the registry by August 1994 (n = 3,000), patient survival and pancreas graft function survival rates were 91% and 72% at one year, 87% and 67% at 2 years, and 84% and 62% at 3 years, respectively. When the USA data for BD cases were analyzed according to the 3 major recipient categories: SPK, (n = 2584); PAK (n = 241); and PTA (n = 175)], patient survival rates were no different (91%, 91%, and 90% at one year, respectively), but pancreas graft survival rates were significantly higher in the SPK than in the PAK and PTA categories (76%, 47%, and 48%, at one year, respectively). In the SPK group, kidney graft survival rate at one year was 85%. Although the overall results were not as good for non-USA as for USA pancreas transplants, this was probably because the results with non-BD techniques were not as good as when BD was used, and in Europe, more than one-third of the cases were by techniques other than BD. The patient, pancreas, and kidney graft survival rates at one year for BD SPK transplants in Europe (n = 579) and other non-USA locations (n = 66) were similar to those in the USA; in Europe they were 92%, 78% and 84%, respectively. The graft survival rates for solitary pancreas transplants, however, were higher in the USA. Outcomes were also compared according to whether induction immunotherapy in USA recipients included ALG/ATG, OKT3, or neither. In the SPK category, there was no difference among the protocols, with one-year graft survival rates being 76% in the ALG/ATG (n = 1,130), 78% in the OKT3, (n = 927) but 69% in the Neither (n = 294) group (which had a significantly lower graft survival). In the PAK category, the use of OKT3 (n = 49) was associated with lower graft survival rates than when ALG/ATG (n = 154) or neither (n = 37) were given (33%, 51%, and 48% at one year, respectively). In the PTA category, the use of ALG/ATG (n = 109) was associated with significantly higher one-year graft survival rates than when OKT3 (n = 55) or neither (n = 8) were used (52%, 46%, and none, respectively).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547578 TI - International sera exchange analyses in relation to DNA sequences: summary report from 1990 to 1994. AB - One hundred ninety-five sera were tested against over 7,000 cells originating from approximately 100 laboratories in the world during the past 5 years. This collaboration was useful to compare local data with data from other laboratories, although the local panel of cells varied from 30 to 200. In analyzing the reactions of the sera, it was noted that antibodies that react cleanly to well defined antigens can usually be defined by a single unique amino acid residue. Antisera which appear to be definable by several amino acids (a motif) have reactivities which are more variable. PMID- 7547579 TI - Cytomegalovirus antibody status and kidney transplantation. AB - 1. First cadaver-donor recipients had a 7% death rate if the kidney originated from a CMV+ donor compared with 5% if the kidney came from a CMV-donor. This 2% difference was highly significant (p < 0.001). Graft survival rates were correspondingly 2-3% lower as a result of deaths. 2. This same trend was noted in the 1991-1993 period as in the 1988-1990 period. 3. Increased incidence of deaths in D+/R+ transplants was most frequently statistically significant when patients were divided by early function. 4. Death rates in diabetics rose from 7% in D-/R- combinations to 13% in D+/R- patients (p < 0.001). Patients with other diseases did not show as marked an effect. 5. Among kidneys from living-related donors, there was no noticeable effect of donor CMV status. Thus, for these donors, no precautions need be taken regarding CMV status. Spousal-donor transplants had a higher graft survival if a D-/R- rather than other combinations were used. 6. The incidence of CMV positivity was slightly higher in Black patients compared with Caucasians, in females compared with males, and increased progressively with age. CMV positivity increased in patients with multiple grafts, presumably because transplanted patients increased in CMV positivity. Among patients who were on dialysis, the CMV positivity was higher than in donors of cadaver organs. There was a substantial difference in CMV positivity in different areas of the country, ranging from 36% in Ohio to 65% in Washington. 7. The HLA matching effect was greater than the CMV effect, justifying its use as the main prospective factor in kidney allocation. CMV prophylaxis is needed for patients receiving kidneys from CMV+ donors. The data indicate that, whenever possible, CMV+ donor kidneys should not be used for CMV- IDDM patients. PMID- 7547580 TI - Multifactorial analysis of renal transplants reported to the United Network for Organ Sharing Registry: a 1994 update. AB - 1. From a multivariate log-linear analysis of 57,303 renal transplants between 1988 and 1994, the top 10 factors influencing one-year and 3-year cadaveric graft survival rates were ranked as follows: [table: see text] 2. Center effects accounted for 30% and 28% of all assignable variations in one-year and 3-year outcomes, respectively. Although center variation dominated 32 other variables, most factors were relatively independent of transplant center. 3. Novel to our own multifactorial analyses of the UNOS Kidney Transplant Registry were 6 pretransplant factors (recipient pretransplant dialysis, pregnancy, PRA technique, donor disposition and preservation, and ABO compatibility). Survival rates over the various combinations of these new factors were not significantly different. 4. For the first time in our multivariate analyses, 4 posttransplantation factors (delayed graft function, rejection episodes prior to discharge, induction and maintenance drug therapies) were included in the log linear model. It is noteworthy that graft survival in both transplant periods was seriously imperiled following delayed graft function or rejection prior to discharge, yet the accounting for these pseudo-outcome variables did not alter the influence of the remaining 31 transplant factors. Finally, maintenance drug therapies strongly influenced short-term outcomes but did not influence long-term results, except for a noteworthy trend toward increased survival rates for FK506 therapy. PMID- 7547581 TI - Summary of 1994 report of center-specific graft and patient survival rates. PMID- 7547582 TI - World Transplant Records--1994. Patients who currently have functioning transplants. PMID- 7547583 TI - Worldwide Transplant Center Directory. Kidney Transplants. PMID- 7547584 TI - Worldwide Transplant Center Directory. Bone Marrow Transplants. PMID- 7547585 TI - Worldwide Transplant Center Directory. Heart Transplants. PMID- 7547586 TI - Worldwide Transplant Center Directory. Heart-Lung Transplants. PMID- 7547587 TI - Worldwide Transplant Center Directory. Lung Transplants. PMID- 7547589 TI - Worldwide Transplant Center Directory. Liver Transplants. PMID- 7547590 TI - Worldwide Transplant Center Directory. Pancreas Transplants. PMID- 7547588 TI - The UNOS OPTN waiting list: 1988 to 1994. United Network for Organ Sharing. Organ Procurement and Transplantation Network. AB - 1. On November 30, 1994, the combined waiting list contained 37,138 registrations. This number represents an 11.4% annual increase in registrations from 33,352 registrations as of December 31, 1993. This percentage of annual increase in registrations is the lowest since 1988. Kidney registrations account for 73.2% of all registrations. It should be noted that the number of registrations increases every year, but the percentage of annual increase tended to decline. 2. In general, the median waiting times for kidney, kidney-pancreas, liver, heart, and lung registrants have increased since 1988. For pancreas registrants, the median waiting times tended to decrease. Overall, heart-lung registrants have the longest median waiting time, whereas liver registrants have the shortest median waiting time, among all registrants on the combined waiting list. Registrants waiting for a kidney-pancreas transplant have a shorter median waiting time than registrants waiting for a kidney or pancreas transplant alone. 3. Kidney, liver, heart, and lung registrants who were listed in 1992 with blood type O waited longer than those with other blood types. Of kidney-pancreas and pancreas registrants, those who had previous transplants waited longer than those who did not. Blacks on the kidney, kidney-pancreas, liver, and heart waiting lists waited longer than patients of other races. Median waiting times varied among different age groups, for different organs. 4. The reported deaths on the kidney waiting list increased each year since 1988, but the percentage of deaths among all kidney registrants in a selected year remained within a range of 3.3 3.8%. Similarly, the number of deaths on the liver, heart, and lung waiting lists increased yearly, whereas the percentage of deaths on these waiting lists remained static over the years. For liver, heart, and lung, the percentage rates of death were 8.5%, 13.7%, and 11.5%, respectively. 5. Among registrants removed from the waiting lists for transplantation, about 58% of the removals were for kidney transplants, 20% for liver transplants, and 16% for heart transplants. Kidney-pancreas and liver transplant rates were higher than those of other organs. Kidney-pancreas transplant rates were higher than those of kidney or pancreas alone. Heart transplant rates were lower than those of kidney-pancreas and liver, but higher than those of other organs. Note that the kidney-pancreas and liver transplant rates have declined over the years. PMID- 7547592 TI - Worldwide Transplant Center Directory. Islet Transplants. PMID- 7547591 TI - Worldwide Transplant Center Directory. Kidney-Pancreas Transplants. PMID- 7547593 TI - Worldwide Transplant Center Directory. Small Bowel Transplants. PMID- 7547594 TI - Worldwide Transplant Center Directory. Multi Transplants. PMID- 7547595 TI - Worldwide Organ Procurement Organization Directory. PMID- 7547596 TI - Report from the International Bone Marrow Transplant Registry and the Autologous Blood and Marrow Transplant Registry--North America. AB - Bone marrow transplants are an effective treatment for many life-threatening diseases. Considerations for use include potential for cure, availability of a suitable donor, feasibility of using autologous stem cells, and risks of transplant-related mortality. Not addressed in this article, but also important, is consideration of the relative efficacy of conventional therapy in specific settings. The IBMTR and ABMTR offer a unique resource for examining the role of allogeneic and autologous transplants in cancer treatment. PMID- 7547597 TI - Occurrence of cancers in immunosuppressed organ transplant recipients. AB - The findings in this study emphasize the need for lifetime follow-up of organ transplant recipients. Although a high percentage of posttransplant tumors are low-grade malignancies that are readily amenable to treatment, cancer has become a major cause of death in patients otherwise successfully treated by transplantation (6). An Australasian study of the causes of death of patients who survived for at least 10 years with a functioning renal allograft compared to those on dialysis for at least 10 years without transplantation, or with a failed transplant, showed that the proportions of deaths caused by cancer were 26%, 1%, and 3%, respectively. Nonetheless, the future holds promise. Attempts are being made to modify the present blunderbuss attack on the immune system with more specific methods of control. Much work is currently being done to induce states of immune unresponsiveness directed specifically, and only, at the foreign antigens of the allograft. Hopefully these efforts will eliminate the need for long term or intense immunosuppressive therapy and the problem of posttransplant malignancies will be relegated to a footnote in the history of organ transplantation. PMID- 7547598 TI - A genetic profile of Romany (Gypsy) subethnic group from a single region in Slovakia. AB - The data presented here are on population structure and genetic markers (ABO, RH, MN, HP) in two series of so-called Slovak Romany subethnic groups from a single region (Gemer) in the Southeastern part of Slovakia. The results demonstrate that favourable conditions have existed for population genetic mechanisms operating in isolated populations, namely genetic drift and inbreeding. In addition, an attempt was made to compare the observed data with those available for other Romany populations and for Slovaks. PMID- 7547599 TI - Red cell isoenzyme polymorphism in the East Slovakian population. AB - The population of East Slovakia was studied for the following red cell isoenzymes: acid phosphatase (ACP), phosphoglucomutase (PGM), adenosine deaminase (ADA) and esterase D (ESD). The gene frequencies observed were as follows: ACP1*A = 0.3330, ACP1*B = 0.6143, ACP1*C = 0.0527, PGM1*2 = 0.2673, ADA*2 = 0.0721, ESD*2 = 0.1061. No significant differences were found between East Slovakia gene frequencies in this study and those of Herzog [1992] on inhabitants of Prague and those of Bambuchova [1985] on the Bratislava population. PMID- 7547601 TI - alpha-1-Antitrypsin (Pi) polymorphism in Serbia: deviation of Pi M subtype distribution from the Hardy-Weinberg equilibrium. AB - The distribution of the alpha 1-antitrypsin (Pi) phenotypes and subtypes was investigated in a population sample of 1060 unrelated individuals from Serbia (Yugoslavia). The allele frequencies estimates were: Pi*M1: 0.702; Pi*M2: 0.183; Pi*M3: 0.088; Pi*Z: 0.013, Pi*S: 0.007; Pi*P: 0.004; Pi*F: 0.003. The observed phenotype frequencies differed very significantly from those expected assuming H.W. equilibrium (chi 2 = 49.51, p < 0.0005). The deviation from equilibrium involved the three Pi*M subtypes: an excess of Pi*M1, Pi*M2 and Pi*M3 homozygotes was found, with the corresponding decreased number of M1M2 and M1M3 heterozygotes. The possible significance of this finding is discussed. PMID- 7547600 TI - Distribution of ApoBII, MCT118 (D1S80), YNZ22 (D17S30), and COL2A1 Amp-FLPs (amplified fragment length polymorphisms) in Caucasoid population of Slovakia. AB - Amp-FLPs are simple and rapid tools for genetic characterization of both individuals and populations. This paper presents allele frequencies of four Amp FLPs (ApoBII, MCT118, YNZ22, and COL2A1) based on the analysis of more than 100 unrelated Caucasoid Slovaks. The proportion of heterozygotes observed and expected, and the probability that two individuals taken at random from the population would be identical in a given polymorphism (PI), was determined for each Amp-FLP. PMID- 7547602 TI - Serum protein polymorphism in Chuetas (Majorcan Jews)--GC, A2HS, ORM, ITI and HP. AB - A sample of 140 Chuetas (descendants of Majorcan Jews) were typed for the GC, A2HS, ORM, ITI and HP serum proteins. Studies on ORM and ITI markers have not yet been reported in other Jewish populations. The allele frequencies obtained were: GC*1 = 0.610; A2HS*1 = 0.787; ORM*F1 = 0.339; ORM*S = 0.497; ITI*1 = 0.581; ITI*2 = 0.414; HP*2FS = 0.625; HP*1S = 0.230; HP*1F = 0.135. Some rare variants were found in polymorphic frequencies (ORM*S1 = 0.043; ORMS*S2 = 0.096; A2HS*10 = 0.015). These results have been compared with those found in other Jewish and non Jewish European populations. The relatively high frequency of the HP*2FS allele and the presence of ORM*S1 and ORM*S2 variants in Chuetas show the Jewish origin of this population. The frequencies of GC and A2HS in Chuetas are similar to those found in other surrounding non-Jewish populations. ITI results are similar to those found in the two European populations studied. HP frequencies suggest a Spanish admixture. PMID- 7547603 TI - Distribution of ACP1, AK1 and ALAD polymorphisms in northern Portugal. AB - Red cell acid phosphatase (ACP1), adenylate kinase 1 (AK1), and aminolevulinate dehydratase (ALAD) polymorphisms were studied in a population sample from Northern Portugal. The gene frequency estimates found were: ACP1*A = 0.2825, ACP1*B = 0.6625, ACP1*C = 0.0547, ACP1*R = 0.0003 (N = 1517); AK1*1 = 0.9778, AK1*2 = 0.0213, AK1*3 = 0.0009 (N = 1081); ALAD*1 = 0.9094, ALAD*2 = 0.0906 (N = 1043). PMID- 7547604 TI - Distribution of group specific component (GC) and transferrin (TF) subtypes in populations of Sri Lanka. AB - A random sample of 503 individuals from five endogamous groups of Sri Lanka was studied for the genetic polymorphism of the group specific component (GC) and transferrin (TF) using isoelectric focusing. Both systems showed statistically significant heterogeneity among the five main populations of the island. The GC allele frequencies of Malays are significantly different from those of the other four populations (Sinhalese, Tamils, Moors and Burghers). However, the TF system shows less variation, since only the Moors show a significant heterogeneity compared to Tamils and Burghers. The frequencies found in the present study are very different from those reported for the populations of the Indian mainland. PMID- 7547605 TI - Rhesus polymorphism in New Caledonia. I. Genetic structures of three local populations. AB - Rhesus phenotypes are presented for a total of 2,052 individuals belonging to three sympatric populations of New Caledonia: Kanaks (Melanesians), Wallisians (Polynesians) and Europeans born in New Caledonia. The maximum-likelihood gene frequency estimations reveal a significant deviation from Hardy-Weinberg equilibrium for the Kanak and Wallisian samples. As suggested on statistical grounds, this disequilibrium can be attributed to antigene mistypings, frequently encountered for this system. This hypothesis leads to new estimations of Rhesus haplotype frequencies for the two samples. They reveal a high degree of genetic similarity (89.6%) between Kanaks and Wallisians, with little or no evidence of admixture with Europeans from New Caledonia. The latter are genetically close to southern Europeans in having a very high R1 frequency. PMID- 7547607 TI - Some genetic markers in Valachian (Olachian) Gypsies in Slovakia. AB - ABO, RH, MN, Kell, P, Lutheran, Lewis, HP, GC, ACP, PGM1, ADA and ESD markers were studied in 122 Valachian Gypsies from Vinodol in West Slovakia. The Valachian Gypsies represent about 5% of the total number of Gypsies (400,000) living in the Czech and Slovak Republic. The results show that their gene pool differs greatly from the one obtained for other Gypsy populations. Since Valachian Gypsies form an endogamous isolate with a high degree of inbreeding, genetic drift and founder effect might have contributed to this difference. PMID- 7547606 TI - Rhesus polymorphism in New Caledonia. II. Genetic comparison with other Oceanians. AB - Rhesus haplotype frequencies computed for 804 and 546 Wallisians from New Caledonia are compared to the data collected for 13 Oceanian samples belonging to the same Austronesian linguistic family. Genetic distances are computed between the 15 populations and used for a principal coordinate analysis. Kanaks are genetically close to Fidjians, while the Wallisian sample share a high genetic similarity with the Tonga islanders. The results obtained for the whole area including the Wallis homeland for the Wallisian sample indicate a tight relationship between geographical and genetic differentiations in the Pacific, supported by a high correlation coefficient between the two distance matrices. However, the observed patterns are better explained by the history of migrations reconstructed from archaeological and linguistic data than by a pure isolation-by distance model. PMID- 7547608 TI - Transference and counter-transference variations in the course of the cognitive analytic therapy of two borderline patients: the relation to the diagrammatic reformulation of self-states. AB - The cognitive-analytic therapy of two patients with borderline personality disorder is described. Patients and therapists completed post-sessional questionnaires which yielded measures of transference and counter-transference. The relation of changes in these to the sequential diagrammatic reformulation of the patients' self-states is considered. PMID- 7547609 TI - Family therapy with 'invisible families'. AB - This paper describes a therapeutic model for working with children in the care system who have severe behavioural problems. The model is an extension of consultation work and was developed from clinical material. It links some of the theories about the internal conflicts of children to their family history and ways of consulting with the professional network, to give a coherent framework for action. The advantages and shortcomings of this model are discussed and some preliminary results are reported. PMID- 7547610 TI - Migration, traumatic bereavement and transcultural aspects of psychological healing: loss and grief of a refugee woman from Begameder county in Ethiopia. AB - Post-traumatic stress disorder (PTSD), grief and bereavement may be manifested in a variety of ways in different populations and cultures. Following is the description of an Ethiopian immigrant woman from the county of Begameder whose baby died during the long exodus from Ethiopia and who, because of the different environmental conditions in 'the new land', could not undergo traditional purification rituals. Subsequently she suffered various cultural signs and symptoms of PTSD due to complicated bereavement and, on top of that, was seen as 'impure' by both her family and herself. For two years her symptoms were attributed to a severe form of bronchial asthma and she did not respond to treatment. When eventually brought to psychiatric attention, she was erroneously diagnosed as suffering from psychosis and treated inappropriately. Accurate anamnesis, combined with adequate counselling, provided the correct diagnosis, and a combination of supportive psychotherapy, traditional healing and purification rituals resulted in a resolution of the syndrome. Thirty months of follow-up showed that the results of the treatment were stable and satisfactory. The specific aspects of cultural manifestations of grief and mourning, the meaning of hallucinations not in the context of psychosis, purification rituals, the role of traditional healing among immigrants from a totally different culture, and the difficulties that helpers may have interpreting and making sense of the immigrants' behaviour and complaints are discussed. PMID- 7547611 TI - The effects of varying information content and speaking aloud on auditory hallucinations. AB - The aim of this study was to investigate why requiring hallucinating schizophrenic subjects to read aloud produces large reductions in reports of auditory hallucinations. In Expt 1 hallucinating subjects (N = 9) were required to sort cards quietly into one, two, four, 13 and 26 piles. It was shown that the large reductions in the reports of hallucinations produced by reading aloud could not be accounted for in terms of the information content of the task. In Expt 2 the subjects (N = 7) were required to place the cards into one or two piles quietly or whilst saying the colour of the card aloud. Sorting cards into two piles whilst saying the colour of the card produced the largest reductions in the reports of hallucinations. It was concluded that it was the requirement to make overt motor and verbal response that produced the large reductions in reports of auditory hallucinations in the reading-aloud task. PMID- 7547612 TI - The role of the father in parental postnatal mental health. AB - Contrary to idealized images of parenthood, the months immediately following childbirth are characterized by enhanced rates of parental mental illness and filicide. In this paper we consider fathers and their role in the postnatal well being of the parturient couple. The process of becoming a father is influenced by not only the man's internal and external experiences of 'father', but also the woman's, since the new infant's relationship with father is mediated by mother and her conjugal relationship with father. When a couple's prenatal relationship is based on a system of shared parental constructs involving a denigrated father and overvalued mother and a concomitant intolerance of the idea of a creative (Oedipal) couple, they will experience particular difficulties in adjusting to the new family configuration. In working with individuals or couples who have experienced post-partum psychological difficulties it is important to remember that not only maternal imagos but also paternal and conjugal ones need to be addressed. PMID- 7547613 TI - Why do psychiatrists neglect religion? AB - This paper analyses a number of possible reasons why modern psychiatry has neglected the therapeutic effects of religious beliefs. The gap which exists between psychiatry and religion is a relatively recent phenomenon and is partly related to psychiatry's progress in elucidating the biological and psychological causes of mental illness, rendering religious explanations superfluous. In addition, it is often assumed that religious attitudes are inevitably linked with phenomena such as dependence and guilt which are frequently seen as undesirable. Psychiatrists and psychologists tend to be less religiously orientated than their patients, which may further increase the professional's idea that religious beliefs are associated with disturbance. However, it has long been suspected that a positive relation exists between religion and mental health, and recently, the psychology of religion has provided empirical support for this idea. Psychiatry faces the challenge to accommodate this evidence into theory and practice. PMID- 7547614 TI - A case study of female genital self-mutilation in schizophrenia. AB - A case of deliberate genital self-mutilation in a woman with familial schizophrenia is presented. Such behaviour, though well recognized in females with severe personality disorders, is extremely rare in female psychotics. The genital mutilation may be partially understood as a consequence of delusionally motivated action against a background of low self-esteem, premorbid body-image preoccupation, forced early sexual activity and hence profound ambivalence towards adult sexuality. Dealing with this constructively was hampered by a cognitive style characterized by impaired reasoning and reality testing. Tests of reasoning, judgement and reality testing showed deficits, and computed tomography revealed dilatation of the left frontal ventricular system. Both neuropsychological and psychodynamic factors appeared to be of relevance in this case. PMID- 7547615 TI - Male genital self-mutilation: a case report. AB - A review of the literature on self-mutilation of male genitalia and/or auto castration reveals that they are infrequent and usually related to psychoses or toxic states. In this report the authors present a case of a 17-year-old non psychotic Japanese male who mutilated his genitalia under clear consciousness because of a conflict over his frequent masturbation. PMID- 7547617 TI - Pancreatic enzyme supplementation in acute pancreatitis. AB - This study evaluates the effect of oral pancreatic enzyme supplements on pain, analgesic requirement and the incidence of complications in patients with acute pancreatitis. This double blind, prospectively randomised placebo controlled study included 23 patients. Pain was monitored using a visual analogue scale; the analgesic requirement was assessed with a numerical score. No significant differences were noted between the median (range) pain scores of patients who received placebo: 22 (17.1-58) and those who received enzymes: 23 (11.3-63). Hospital stay was 7 (5-10) days in patients on placebo and 8 (6-24) days in the enzyme group (p = 0.069). Analgesic requirements were: placebo 20 (6-60) and enzymes: 16 (0-63) (p = 0.56). This study has shown no beneficial effect of oral pancreatic enzyme supplements in the initial management of patients with acute pancreatitis. PMID- 7547618 TI - Hepatocellular carcinoma arising in non-cirrhotic haemochromatosis. AB - Hepatocellular carcinoma arising in a patient with genetic haemachromatosis, without cirrhosis, has only been described once previously. We present a patient with a 15 year history of genetic haemachromatosis who underwent resection of a hepatocellular carcinoma in a liver with normal architecture. PMID- 7547619 TI - Cryotherapy of liver tumours--a practical guide. AB - The use of cryotherapy for the treatment of some unresectable liver tumours has been clearly established as a therapeutic option. Intra-operative ultrasound has enhanced the process by enabling the surgeon to identify hepatic lesions and to allow visualisation of the freezing process to ensure that the cryolesion will include the tumour mass. The purpose of this paper is to provide a practical guide to surgeons who wish to perform cryotherapy of liver tumours. Patient selection and anaesthetic considerations are important. The surgeon should be able to deal with the complications of cryotherapy, particularly the intra operative haemorrhage which may arise from cracking of the hepatic parenchyma as the iceball thaws. Follow-up is based on tumour marker assay and imaging of the liver and repeat cryotherapy can be considered for selected cases. PMID- 7547620 TI - Repeated hepatic dearterialization for unresectable liver metastases from gastric cancer: review of five cases. AB - A novel method of repeated hepatic dearterialization was evaluated in five patients with multiple metastases from gastric cancer in both hepatic lobes. After gastrectomy with extensive lymph node dissection (R2/3), all patients underwent implantation of a vascular occluder around the hepatic artery. Cannulation of the hepatic artery was added for later chemotherapy. The hepatic artery was occluded repeatedly for 1 hour twice daily in combination with intrahepatic infusion of anticancer drugs for as long as possible. Three of five patients demonstrated marked tumour regression with unexpectedly long survival (16 months in two patients and one still alive at 15 months). Carcinoembryonic antigen (CEA) levels decreased to almost normal in four patients who had initially high levels. The present experiences seems to indicate that long survival can be hoped for in patients with advanced gastric cancer with unresectable liver metastases. PMID- 7547621 TI - Palliative resection of pancreatic adenocarcinoma. A survey of British surgeons. AB - A survey was carried out by postal questionnaire of the attitudes of British surgeons to pancreatic resection as palliation for ductal adenocarcinoma of the pancreas. Replies from 24 surgeons related to experience in over 700 resections. The incidence of estimated residual local disease after resection was median 12.5 percent, range 0-35 percent. Half (12) of the surgeons felt that pancreatic resection with residual macroscopic disease was justified. Only 3 (12.5 percent) surgeons accepted that palliative resection in the presence of liver metastases was sometimes justifiable. Further evidence is required of improved quality of life after resection before the majority of surgeons will accept palliative resection in the management of pancreatic ductal adenocarcinoma. PMID- 7547622 TI - Gall bladder tumour, choledochal cyst and an anomalous pancreatico-biliary junction. AB - This report highlights the association of a tumour in an acalculous gall bladder with an anomalous pancreatico-biliary junction (PBJ) and a type IVa choledochal cyst. Cholecystectomy and Rouxen-Y hepatico-jejunostomy (RHJ) was performed after division of the common bile duct (CBD) and excision of the dilated segment. The details of the case are presented and the role of an abnormal PBJ in gall bladder carcinogenesis is discussed. PMID- 7547623 TI - ERCP following laparoscopic cholecystectomy: a safe and effective way to manage CBD stones and complications. AB - The efficacy of ERCP in detecting and treating post-laparoscopic cholecystectomy problems was examined in a series of consecutive patients undergoing directed examination of the biliary tree over a two-year period. Three major diagnostic groups were identified: leaks and bile duct injuries (n = 9), retained common bile duct stones (n = 18), and post-cholecystectomy pain (n = 13). These diagnostic groups differed in degree of abnormal bilirubin (p = .004) and time between surgery and ERCP (p = .0005). Diagnosis of a post-operative complication was successful in 92% of attempted cases. Therapy was successful in 92% of attempted cases. Three patients developed mild pancreatitis as a result of ERCP. This series underscores the efficacy of a multi-disciplinary approach to problems which occur after laparoscopic cholecystectomy. PMID- 7547624 TI - Clinical and enzymatic investigation of induction of oxygen free radicals by ischemia and reperfusion in human hepatocellular carcinoma and adjacent liver. AB - Serum concentration of thiobarbituric acid (TBA) reactants in the hepatic vein were measured before and after transient dearterialization of the liver in five human subjects bearing unresectable hepatocellular carcinoma (HCC). During 1 hour of the occlusion of the hepatic artery, change in TBA reactants level was slight. However, the mean value of TBA reactants in 1 hour after the reflow increased to 1.50 +/- 0.11 nmol/ml (mean +/- S.E.) and was significantly higher (p < 0.05) than those before hepatic dearterialization (1.28 +/- 0.11 nmol/ml) and just before the release of occlusion (1.32 +/- 0.09 nmol/ml). Further, two endogeneous scavenger enzymes, superoxide dismutase (SOD) and catalase (CAT), and one of the major sources of oxygen free radicals, xanthine oxidase (XOD) were measured in human untreated HCC and the corresponding adjacent liver tissue. The results demonstrated an increase in SOD in 81.8% (9/11) of HCC, and a decrease in CAT in 72.7% (8/11) of HCC when compared with the corresponding adjacent liver tissue. The mean value of SOD in HCC was significantly higher (66.8 +/- 6.5 vs 52.8 +/- 3.8 U/mg protein; p < 0.05), and that of CAT was significantly lower (22.6 +/- 2.4 vs 36.0 +/- 6.1 U/mg protein; p < 0.05) than those in liver tissue. All of nine HCC samples had a significantly lower activity of XOD (6.4 +/- 1.9 vs 20.3 +/- 3.4 pmol/minute/mg protein; p < 0.01) than the corresponding liver tissue. There was no obvious relation between the content of SOD and CAT in HCC, or in liver tissue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547626 TI - Liver resection under ischemia: inflow occlusion or total hepatic isolation. PMID- 7547625 TI - U-tube in high bile duct obstruction. PMID- 7547627 TI - Is a conservative approach justified in penetrating liver injury? PMID- 7547628 TI - Narrow versus wide diameter portacaval H-graft shunts. PMID- 7547629 TI - Drug therapy for acute bleeding from portal hypertensive gastropathy. PMID- 7547630 TI - Loss of heterozygosity on the X chromosome in human breast cancer. AB - The analysis of loss of heterozygosity (LOH) in tumours can be a powerful tool for mapping the sites of tumour suppressor genes in the human genome. A panel of breast cancer patients was assembled as pairs of tumour and lymphocyte DNA samples and LOH studies carried out by Southern hybridisation with polymorphic loci mapping to the X chromosome with appropriate controls. Deletion mapping revealed a high frequency of small regionalised deletions, defining at least three independent regions, one of which is particularly well mapped to a 500 kb stretch of DNA in the distal portion of the pseudoautosomal region of Xp. A second region has been identified within the pseudoautosomal region close to the pseudoautosomal boundary, and there is a third discrete site of loss on distal Xq. Perturbations of sequences at these regions represent independent events in a number of patients. This study represents the first detailed analysis of LOH on the X chromosome in human breast tumours, the results of which indicate that at least three regions of this chromosome are involved in the disease. PMID- 7547631 TI - Cytogenetic studies of breast carcinomas: different karyotypic profiles detected by direct harvesting and short-term culture. AB - Chromosome analysis was performed on samples from 85 consecutive patients with breast cancer by one or more of three different methods: direct harvest, culture after mechanical disaggregation, and culture after collagenase digestion. Metaphases suitable for karyotyping were obtained in 70% of the cases; direct harvest yielded metaphases in 29% and cultures without and with digestion in 40% and 59%, respectively. Chromosomal abnormalities were detected in 37 cases. Cells judged to be phenotypically abnormal in culture were twice as likely to reveal chromosomal aberrations as normal-looking cells. Eight cases showed multiclonal abnormalities. Significant differences were detected in the karyotypic profile depending on the method used. With direct harvest, the yield of complex chromosomal changes was 87%, compared to 44% after culture of digested tissue (P < 0.01), and also polyploidy was more common in direct-harvested samples. Detailed karyotypic analysis was possible in 29 primary tumors. The chromosomes most frequently involved were 1, 3, 7, 11, 16, and 17. Recurrent structural abnormalities were der(1;16)(q10;p10), i(1)(q10), del(6)(q21), and del(1)(p22). Breakpoints clustered to the centromere regions of chromosomes 1, 3, 11, 15, and 16 and to the short arms of chromosomes 7, 17, and 19. Seven of twenty-nine fully analyzed cases had a family history of breast cancer, and changes of chromosomes 1, 3, and 15 seemed to be more common in these cases. There was an association between karyotype and survival: The 3 year survival was 63% in patients with complex karyotypic changes and 92% in those without complex changes. PMID- 7547632 TI - Loss of heterozygosity on chromosome arms 5q, 11p, 11q, 13q, and 16p in human testicular germ cell tumors. AB - To identify common regions of deletion in human testicular germ cell tumors (TGCTs), we have screened tumors from 33 patients for loss of heterozygosity (LOH) using Southern blot analysis with 39 polymorphic markers covering 21 chromosome arms. Losses in more than 2 tumors and occurring at a frequency of > 10% were found on chromosome arms 5q, 11p, 11q, 13q, and 16p, the highest being on chromosome arm 5q (19%). It is suggested that tumor suppressor genes on 5q among others may be involved in testicular tumorigenesis and that LOH in this region requires further investigation. No losses were found on 12q and 17p despite the fact that the most common cytogenetic abnormality in TGCTs is an i(12p) and that the TP53 gene on 17p is the most frequently mutated gene in human cancers. The level of allelic imbalance varied considerably from one chromosome region to another (0-80%) and did not generally reflect the pattern of LOH. It tended to be high in overrepresented regions of the genome, 1q, 7p, and 12p. The tumor from one patient had a seminomatous component and a less differentiated component. We provide evidence for a common origin of both components and show that it is likely that this tumor has progressed from the seminoma to the less differentiated histology. PMID- 7547633 TI - In situ hybridization analysis of the Y chromosome in gonadoblastoma. AB - Gonadoblastoma is a rare tumor arising in the streak gonads of about 30% of 46,XY sex-reversed females. Because gonadoblastoma develops only in patients who have Y chromosome material and dysgenetic gonads, it has been hypothesized that positive expression of a gene (or genes) on the Y chromosome (GBY) is involved in the etiology of the tumor. To examine the Y chromosome directly in tumors, we performed nonisotopic in situ hybridization of a biotin-labeled Y-specific probe for the DYZI locus on formalin-fixed, paraffin-embedded sections of tumor samples from four different patients. After hybridization to DYZI, the Y chromosome was found to be present in all gonadoblastoma foci in the four patients studied, and the gonadoblastoma foci showed an average of 85% cell nuclei positive for the Y chromosome on tissue sections. Normal male and female control tissues showed an average of 78% and 0% positive nuclei, respectively. One patient with bilateral gonadoblastoma had previously been shown to be mosaic, with a 45,X/46,XY karyotype in lymphocytes, skin fibroblasts, and cultures from both gonads. Examination of sections of this patient's gonads showed 79% positive nuclei within the gonadoblastoma foci, whereas the nontumor stromal tissue had 19% positive nuclei. These results indicate that, in this mosaic gonad, tumor foci developed only from cells that had a Y chromosome. Our results support the hypothesis that there is a GBY locus on the Y chromosome and that the Y chromosome is retained in the gonadoblastoma foci during the development of the tumor. PMID- 7547634 TI - Allelic imbalance in gastric cancer: an affected site on chromosome arm 3p. AB - In order to detect regions of DNA containing tumor suppressor genes involved in the development of gastric cancer, we performed an allelotype study on 78 gastric adenocarcinomas from a population composed largely of Texan Hispanics and Anglos, two ethnic groups that have a ratio of incidence rates of gastric cancer of approximately 2:1. In total, 42 microsatellite markers were employed, which detected at least one site per arm of each autosome in the human genome. These included several markers linked to known tumor suppressor genes (TP53, APC, DCC, RB1, and BRCA1). Sites showing quantitative allelic imbalance (AI) greater than 30% were located on 3p (36%), 11q (31%), 12q (38%), 13q (33%), 17p near TP53 (74%), and 17q near BRCAI (32%). Among the 22% of cases showing microsatellite instability (MI), a subset (4 of 17) showed instability at 59% or more of sites tested. No ethnic bias was detected in cases showing MI or in cases with AI at sites with rates of AI above 30%. Tumors of the intestinal subtype were significantly more likely than diffuse tumors to show AI at DI3S170 (P = 0.01). A deletion map of chromosome arm 3p was prepared for tumors with AI at D3S1478. These data indicate that a tumor suppressor gene on chromosome arm 3p is involved in the development of a subset of gastric cancers. PMID- 7547635 TI - Molecular analysis of genetic changes in ependymomas. AB - Ependymomas are glial cell-derived tumors. They are, in contrast to other gliomas (astrocytomas, oligodendrogliomas, and oligoastrocytomas), ill-defined with respect to the genes and chromosomal segments important in their tumorigenesis. In this study, we extensively screened 17 ependymomas for genetic changes characteristic of other gliomas. Allelic loss was detected on chromosome arm 22q in three tumors; on chromosome 10 in two tumors; on chromosome arm 17p in two tumors; and on chromosome arms 6q, 9p, 13q, and 19q, each in one tumor. No allelic losses were found on chromosome arms 1p and 16q. None of the tumors had EGFR gene amplification. In each case, the chromosomal segment affected by the deletion included the region known to harbor a tumor suppressor gene important in glioma tumorigenesis. We conclude that ependymomas resemble the other glial neoplasms with respect to type and location of the chromosomal changes involved. Given the relatively infrequent occurrence of these genetic changes, ependymomas should be considered genetically as low-grade gliomas. PMID- 7547636 TI - Loss of chromosome 17 loci in prostate cancer detected by polymerase chain reaction quantitation of allelic markers. AB - Using a polymerase chain reaction/microsatellite marker system, we demonstrated that 6 of 22 (27%) clinical stage B (early) primary prostate tumors showed loss of heterozygosity at one or more of five loci on chromosome 17. The sensitivity of this study was increased by use of a PhosphorImager and statistical analysis of replicate tumor-normal DNA pairs. Two patients showed tumor-specific interstitial loss at a locus in close proximity to the familial breast cancer gene BRCA1. These findings suggest that genes on the proximal long arm of chromosome 17 play a pivotal role in the early development of at least a subset of prostatic tumors. PMID- 7547637 TI - Genomic DNA and messenger RNA expression alterations of the CDKN2B and CDKN2 genes in esophageal squamous carcinoma cell lines. AB - The genes CDKN2B (MTS2) and CDKN2 (MTS1) encoding the proteins p15 and p16 are both located on chromosomal band 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2 and CDKN2B belong to a family of cyclin-dependent kinase 4 inhibitors (INK41) and control cell proliferation during the G1 phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancers. To investigate whether CDKN2B and CDKN2 are involved in esophageal tumorigenesis, we studied homozygous deletion, intragenic mutation, and messenger RNA (mRNA) expression of CDKN2 and CDKN2B in nine esophageal squamous cancer cell lines. Polymerase chain reaction (PCR) amplification revealed that five of the nine cell lines (55%) manifested homozygous deletions of CDKN2B, CDKN2, and/or flanking loci on chromosomal band 9p21. Reverse transcriptase-PCR (RT-PCR) was used to examine CDKN2 and CDKN2B mRNA in the nine cell lines. Lack of CDKN2 and CDKN2B mRNA correlated perfectly with homozygous deletion involving these genes. No subtle intragenic mutations of CDKN2B or CDKN2 were detected by DNA sequencing of their entire coding sequences in any cell lines lacking homozygous deletion. Two of the cell lines manifested homozygous deletions excluding CDKN2; one of these two deletions also excluded CDKN2B. These results suggest that inactivation of CDKN2B and CDKN2 may contribute to the malignant phenotype in esophageal cells and that homozygous deletion may be the predominant mechanism for inactivation of CDKN2B and CDKN2. Alternatively, a gene or genes adjacent to CDKN2B/CDKN2 may constitute the target(s) of deletion at this locus. PMID- 7547638 TI - Patterns of loss of heterozygosity at loci from chromosome arm 13q suggests a possible involvement of BRCA2 in sporadic breast tumors. AB - Loss of heterozygosity (LOH) at loci from chromosome 13 is frequently observed in breast cancer. Chromosome 13 contains at least two cancer genes, the well characterized RB1 gene located at 13q14 and the breast cancer-susceptibility gene, BRCA2, recently localized to 13q12. To investigate the possible involvement of BRCA2 in sporadic breast tumors, we looked at LOH at eight microsatellite (CA)n markers distributed along chromosome 13 in a panel of 59 primary breast carcinomas. We show that some LOH does not include the RB1 locus and is associated with the BRCA2 gene region. PMID- 7547639 TI - Loss of heterozygosity in the tuberous sclerosis (TSC2) region of chromosome band 16p13 occurs in sporadic as well as TSC-associated renal angiomyolipomas. AB - Angiomyolipomas (AMLs) are renal tumors that occur both sporadically and in association with tuberous sclerosis (TSC). TSC is an autosomal dominant disorder characterized by hamartomatous lesions in multiple organs. Two TSC loci are recognized: TSC1 on 9q34 and TSC2 on 16p13. Loss of heterozygosity (LOH) at the TSC1 and TSC2 loci in lesions from TSC patients has recently been reported. Lesions that are not associated with TSC have not been previously examined for LOH at the TSC loci. We analyzed 29 renal angiomyolipomas from patients without a history of TSC. Three tumors demonstrated LOH on 16p13. This is the first report indicating that mutations in TSC2 occur in tumors of patients who do not have TSC. We also found LOH on 16p13 in 5 of 8 TSC-associated AMLs. Two of these tumors were from a single patient and demonstrated different regions of LOH. These findings support the hypothesis that the TSC2 gene functions as a tumor suppressor. PMID- 7547640 TI - Increased platelet phospholipase A2 activity in schizophrenia. AB - The intracellular enzyme phospholipase A2 (PLA2) plays an essential role in the breakdown of membrane phospholipids, which regulate the physicochemical properties of the cell membrane. In the brain PLA2 has been reported to influence receptor function and signal transduction. Regarding dopaminergic neurons, data from animals experiments and from binding studies suggest that PLA2 activation reduces dopaminergic neurotransmission. In the present study we investigated intracellular PLA2 in platelets from 31 DSM-III-R paranoid schizophrenic patients (15 neuroleptic-naive) compared to 31 healthy individuals and to 31 non schizophrenic psychiatric controls, both matched to the schizophrenics by age and sex. Platelet PLA2 activity was significantly increased in schizophrenics as compared to healthy and to psychiatric controls. Neuroleptic treatment reduced significantly the enzyme activity. Our findings in platelets suggest an accelerated breakdown of membrane phospholipids in schizophrenia. An accelerated phospholipid breakdown has also been reported in the frontal cortex from schizophrenic patients. Further studies should clarify whether increased PLA2 in the brain, as observed in platelets, could contribute to a frontal dysfunction in schizophrenia. PMID- 7547641 TI - Gender differences in disability: a comparison of married patients with schizophrenia. AB - Gender differences in disability constitute a fertile area of research, as disabilities need to be measured and evaluated in the social context which defines role expectations and consequently the role performance. This paper reports on the differences in disability in married patients with schizophrenia, as marital status is an important determinant of role expectation. The study sample constituted 30 married patients, of both sexes, who satisfied DSM-III criteria for schizophrenia, and were living with their spouse at the time of assessment. Disability was evaluated using the DAS (modified version). The findings indicated that women were more disabled than men on many of the evaluation parameters (p < 0.05); there was also a strong correlation between negative symptoms and disability variables in both the sexes. While a correlation between PSE syndromes and disability variables was seen in the case of males, the relationship was not seen in females. Stepwise regression also revealed that negative symptoms predominated among the factors associated with global disability in both sexes. Most of the reports in the literature reveal that women are less disabled than men. The findings of this study, that women are more disabled than men, is discussed in the context of the social conditions prevailing in India. PMID- 7547642 TI - Impact of childbirth on a series of schizophrenic mothers: a comment on the possible influence of oestrogen on schizophrenia. AB - From a series of 180 admissions to a Mother-Baby Unit 45 patients with diagnoses of schizophrenia were identified. Demographic data together with clinical information were noted, and each file was rated using the OPCRIT programme. Two mutually exclusive groups were derived from OPCRIT, a 'narrow group' (n = 16), corresponding to subjects satisfying Feighner's criteria for schizophrenia, and a 'broad group' (n = 21), corresponding to those meeting ICD10 but not Feighner criteria. In addition to significant differences in age on admission, employment, home circumstances, ethnicity and age of onset of illness, there was a marked contrast between broad and narrow groups in course of illness post partum. 43% of the broad group experienced an acute illness episode after delivery compared with none of the narrow group, a contrast not attributable to differences in clinical state or treatment during pregnancy. These data suggests that childbirth may exert a differential effect on the course of illness in severe and more benign forms of schizophrenia, and that more severe schizophrenic illnesses may not be influenced by the changes associated with childbirth, such as the fall in oestrogen levels. PMID- 7547643 TI - The development of the concept of akathisia: a historical overview. AB - This paper traces the history of 'akathisia' and related syndromes, and examines the important studies that have helped shape our current understanding of the concept. Even though the term has come to be used synonymously with drug-induced akathisia, its origin was in the pre-neuroleptic era, and it is still often used to describe syndromes not related to medication. The literature clearly distinguishes restless legs syndrome (RLS) from akathisia. The complexity of the akathisia syndrome has increasingly become manifest, and a number of sub-types have been described. Recent attempts have been made to operationalize its diagnostic criteria and understand its pathophysiology. Akathisia due to non neuroleptic drugs, in particular the serotonin-specific reuptake inhibitors (SSRIs), has also received much attention. The development of newer psychopharmacotherapeutic drugs, with different side-effects profiles, has made this focus pertinent and timely. PMID- 7547644 TI - Primitive (developmental) reflexes, tardive dyskinesia and intellectual impairment in schizophrenia. AB - Primitive reflexes, also known as higher cerebral, developmental or release reflexes, are present in foetal and infant life, and are found in certain organic brain diseases. They are normally regarded as non-localising signs of cerebral immaturity or dysfunction which are uncommon in the normal population. The main aims of this study were to find out whether recent reports of an association between primitive reflexes and severity of cognitive impairment in dementia and between primitive reflexes and tardive dyskinesia in schizophrenia could be replicated in a younger population of schizophrenic patients. Forty-eight schizophrenic patients (mean age 51 years) were assessed for primitive reflexes, involuntary movements and cognitive function, and 58% exhibited at least one primitive reflex and 23% at least two. No association was found between primitive reflexes and cognitive impairment or between primitive reflexes and tardive dyskinesia. These results fail to support the hypothesis that the presence of primitive reflexes in some schizophrenic patients indicates a vulnerability to tardive dyskinesia and intellectual decline with advancing age, but long-term prospective studies would be required to test this hypothesis adequately. Nevertheless, these findings support the notion of neurodevelopmental or neurodegenerative brain disease in at least a proportion of patients with schizophrenia. PMID- 7547645 TI - Assessing the predictive value of teacher reports in a high risk sample for schizophrenia: a ROC analysis. AB - We examined teachers' reports to determine early patterns of school behavior that identify children who were diagnosed schizophrenic in adulthood. High risk subjects (n = 207) in the Copenhagen High Risk for schizophrenia project were examined on a 25-item questionnaire completed by teachers. A likelihood ratio approach was used to create a classification index relating school report to DSM IIIR diagnoses made 31 years later. A standard receiver operating characteristic (ROC) analysis was used to evaluate the efficacy of the classification indices for distinguishing preschizophrenics from children who later developed schizotypal disorder, other nonpsychotic disorders, and no mental illness. Different items predicted schizophrenia in males and females. To a significantly greater degree than their controls, preschizophrenic males were lonely and rejected, behaved inappropriately, were disciplinary problems, emotionally high strung, and were more likely to have repeated a grade; preschizophrenic females were nervous and passive. Both preschizophrenic males and females were judged by teachers to be vulnerable to future psychotic problems. These findings are consistent with our preliminary findings, as well as with other research. They highlight the ability of teachers to identify school behaviors that may be used to select future schizophrenics for early intervention. PMID- 7547646 TI - A preliminary study of externally and self-ordered task performance in schizophrenia. AB - Frontal lobe dysfunctions are assumed to be involved in the cognitive problems of schizophrenia. The inability to plan or organize behavior, however, may be the result of an inability to process information (i.e., attention disorder) as well as of an incapacity of information processing (i.e., overstimulation). Wiegersma et al. (1990) described impaired performance in frontal lobe patients on self ordered tasks (Randomization Span and Sequential Pointing span) but intact performance on externally ordered tasks (Digit Span and Missing Item scan). These four tasks were used in the present study in which ten schizophrenic patients and ten matched controls participated. The schizophrenic patients exhibited significant impairments in the self-ordered as well as in the externally ordered tasks. It was concluded that the impairment of schizophrenic patients may be caused by overstimulation rather than by attention disorders. PMID- 7547647 TI - Gender and the use of neuroleptics in schizophrenia. Further testing of the oestrogen hypothesis. AB - According to the oestrogen hypothesis, the lower need for neuroleptic drugs in female schizophrenics is caused by the antidopaminergic effect of oestrogens. Due to the decreased oestrogen production during the menopause, this 'neuroleptic' effect is lost and the need for neuroleptic drugs increases in female schizophrenics. This hypothesis was tested by studying a sample of 1097 schizophrenic patients (DSM-III-R), who were discharged from hospital and followed for three years. Prescribed daily doses of neuroleptic drugs were recorded and converted to chlorpromazine equivalents. Males had higher daily doses of neuroleptics than females; this gender difference, however, was only significant in middle-aged groups. Daily doses were also associated with age at the onset of illness, duration of illness and clinical status. Results did not support the oestrogen hypothesis: In females, there was no consistent increase in daily doses of neuroleptics after menopause age. Results are discussed from the viewpoint of testosterone secretion, body weight and smoking habits, which are gender-bound and thus can, at least in part, explain gender differences in the use of neuroleptic drugs in schizophrenic patients. It is pointed out that the age at the onset of illness and the duration of illness should be taken into account when the effects of gender and age on the need for neuroleptics are studied. PMID- 7547648 TI - Sydenham's chorea and schizophrenia: a case report. AB - The following is a case report of a patient with Sydenham's chorea who later developed schizophrenia. Autopsy examination of this patient revealed mineral deposits in the basal ganglia. The deposition of minerals, especially iron, within subcortical brain structures has been associated with dopaminergic abnormalities, schizophreniform symptoms, and abnormal movement disorders. The psychosis these patients experience is sometimes resistant to treatment with traditional neuroleptics. A CT or MRI scan may prove useful in screening those patients with Sydenham's chorea that develop psychotic symptoms. PMID- 7547649 TI - Psychotic symptoms in schizophrenics during chronic fluvoxamine treatment. A report of two cases. PMID- 7547650 TI - Normalization of auditory brainstem responses resulting from improved clinical symptoms in schizophrenia. PMID- 7547651 TI - Effects of haloperidol on CSF glutamate levels in drug-naive schizophrenic patients. PMID- 7547652 TI - 1994 ASRA Lecture. The pathogenesis of neuropathic pain. PMID- 7547653 TI - Regional anesthesia and chronic pain management in the 1920s and 1930s. The influence of the American Society of Regional Anesthesia. AB - BACKGROUND AND OBJECTIVES: Physicians in the 1920s and 1930s began to treat patients with chronic pain syndromes using regional anesthetic techniques for both temporary and permanent block of pain pathways. The founding of the American Society of Regional Anesthesia (ASRA) in 1923 provided a unique venue for the dissemination of information concerning regional anesthesia for both surgery and chronic pain management. METHODS: The growth of chronic pain management on a national basis was assessed by using the Quarterly Cumulative Index to the Medical Literature to trace the distribution of information on regional anesthesia. From the Minutes of Meeting of the American Society of Regional Anesthesia, presented papers and the discussion that followed were analyzed. RESULTS: Inquiries into regional anesthesia during the 1920s and 1930s predominantly dealt with technique. For the first time pain papers were listed under the regional anesthesia heading in the Index. The papers presented at ASRA meetings during the period helped develop the use of regional anesthesia for both chronic pain management and surgical anesthesia. CONCLUSIONS: The ASRA was instrumental in bringing together physicians interested in regional anesthesia and pain management. During the 1930s physician anesthetists came to predominate as the organization's officers and members and helped translate the work of the ASRA into a part of the knowledge required to be a specialist physician in anesthesia. PMID- 7547654 TI - Aging reduces the reliability of epidural epinephrine test doses. AB - BACKGROUND AND OBJECTIVES: The authors recently determined that intravascular injection of an epinephrine test dose reliably produced an increase in heart rate > or = 20 beats/min in young individuals. However, aging is associated with a significant reduction in beta-adrenergic responsiveness. This study was designed to determine whether aging decreases the magnitude of heart rate (HR) increase after intravascular injection of epinephrine. METHODS: Heart rate and systolic blood pressure were recorded during randomized and double-blind injections of 3 mL lidocaine plain or lidocaine with 10 or 15 micrograms epinephrine in 30 elective surgical patients between 21 and 81 years old. RESULTS: Increasing age was associated with smaller increases in HR after intravascular injection of epinephrine. The reduction in HR increase was statistically significant after 10 micrograms (P = .006, r2 = .24), but not after 15 micrograms (P = .25, r2 = .05) of epinephrine. Heart rate increases were of greater magnitude for patients younger than 40 years old (P = .01 for 10 micrograms epinephrine, and P = .03 for 15 micrograms epinephrine). One patient (of 11) over 60 did not respond to the 15 micrograms test dose with tachycardia. Aging did not alter systolic blood pressure increases after 10 or 15 micrograms epinephrine (P = .27, r2 = .04 and P = .4, r2 = .03, respectively). CONCLUSIONS: Aging is associated with a reduction in the magnitude of HR increase after intravenous injection of epinephrine. The results suggest that to detect an intravascular injection in healthy people, a test dose containing 10 micrograms epinephrine will suffice before the age of 40. However, even 15 micrograms epinephrine will not be totally reliable in older patients, owing to decreased beta-adrenergic responsiveness. PMID- 7547655 TI - Systemic vascular uptake of epinephrine from the lumbar epidural space in parturients. AB - BACKGROUND AND OBJECTIVES: To assess the systemic vascular uptake of epinephrine from the lumbar epidural space in parturients during epidural anesthesia for cesarean delivery. METHODS: Thirty-eight healthy patients were randomly divided into two equal groups. In one group, bupivacaine-epinephrine (5 micrograms/mL) mixture was used to produce lumbar epidural anesthesia to a T6 level; in the other group, bupivacaine 0.5%. Maternal venous plasma epinephrine and norepinephrine concentrations were measured before anesthesia (baseline), at a T6 sensory level and at delivery; and 10, 20, 30, and 50 minutes after delivery. Statistical comparisons were made using nonparametric analysis of variance at P < .05. RESULTS: In the bupivacaine-epinephrine group, the plasma epinephrine concentration increased approximately fourfold at T6 sensory level compared to the baseline value or to the corresponding value in the bupivacaine group (P < .01), and it remained elevated significantly for 20 minutes after delivery. In the bupivacaine group, no significant changes occurred in the epinephrine concentrations. In both groups, plasma norepinephrine concentration increased at T6 level approximately 1.8-fold compared to the baseline value (P < .01), with no intergroup differences. CONCLUSIONS: Epinephrine is absorbed from the epidural space and raises the maternal circulating epinephrine concentration. Maternal plasma norepinephrine concentration rises during lumbar epidural anesthesia regardless of epinephrine use. PMID- 7547656 TI - Does continuous intravenous infusion of low-concentration epinephrine impair uterine blood flow in pregnant ewes? AB - BACKGROUND AND OBJECTIVES: Bolus intravenous injection of epinephrine can decrease uterine blood flow. This study examined the effects of intravenous infusion of epinephrine on uterine blood flow in the gravid ewe. METHODS: Maternal and fetal vascular catheters and a maternal electromagnetic uterine artery flow probe were implanted in 10 near-term gravid ewes. After recovery, saline, 0.125% bupivacaine, 0.125% bupivacaine with 1:200,000 epinephrine, 0.125% bupivacaine with 1:400,000 epinephrine, and 0.125% bupivacaine with 1:800,000 epinephrine were infused into the maternal superior vena cava. Drugs were infused at 10 mL/h for 30 minutes and then at 20 mL/h for an additional 30 minutes. Animals also received an intravenous bolus of epinephrine 15 micrograms. Throughout all infusions, maternal heart rate, systemic and pulmonary blood pressures, uterine blood flow, cardiac output, and acid-base balance were measured, as well as fetal heart rate, blood pressure, and acid-base balance. RESULTS: Epinephrine 15 micrograms decreased uterine blood flow to 68 +/- 14% of baseline (mean +/- SD). Infusion of all solutions had no effect on any measured hemodynamic variable. CONCLUSIONS: In gravid ewes, intravenous infusion of < or = 1.67 micrograms/min epinephrine altered neither maternal hemodynamics nor uterine blood flow. To the extent that sheep data can be extrapolated to humans, these results suggest that continuous intravenous infusion of epinephrine in local anesthetic solutions is safe if the epidural catheter should enter a blood vessel during the infusion. PMID- 7547657 TI - Perceptions of side effects following axillary block used for outpatient surgery. AB - BACKGROUND AND OBJECTIVES: The study evaluated patient perceptions of side effects related to axillary block (AB) and how they might influence a patient's decisions about subsequent ABs. METHODS: A questionnaire concerning the occurrence and duration of side effects following AB and patient preference for a subsequent AB was completed and returned by each of 1,149 patients. All ABs were performed using a 22 gauge short bevel needle. In all 86.5% had orthopedic surgery, with the remainder vascular access procedures. Three AB techniques and two anesthetic solutions were used. RESULTS: Patient-reported incidences of side effects were bruising, 19.1%; soreness, 39.7%; persistent numbness, 11.0%; and nausea, 10.8%. Bruising was reported more frequently by female patients, those undergoing orthopedic procedures, and those between 20 and 30 and over 80 years of age. Soreness was reported more frequently in orthopedic procedures and by those aged 20 to 30. Technique used did not affect the incidence of side effects or patient willingness to have a repeat AB. Ninety-three percent expressed willingness to have another AB; this was affected by whether the patient experienced a side effect, the duration of the side effect, and by the number of side effects experienced. Those between the ages of 20 and 40 were less willing to have another AB. CONCLUSIONS: Even though 51% of the patients reported at least one side effect, 93% would choose to have another AB, indicating a high overall satisfaction with AB anesthesia, which significantly decreased as the number and duration of side effects increased. PMID- 7547658 TI - Effect of anesthetic technique on acute formalin-induced pain in mice. AB - BACKGROUND AND OBJECTIVES: The aim of this study was to determine if anesthetic technique influences the degree of acute formalin-induced pain in mice. METHODS: Mice were randomized to receive either combined spinal/general anesthesia or general anesthesia prior to a subcutaneous injection of formalin in a hindpaw. The mice in the combined spinal/general anesthesia group received halothane anesthesia and subarachnoid lidocaine. The mice in the general anesthesia group received halothane anesthesia only. After the mice recovered from anesthesia, paw licking behavior was recorded for 1 hour. A statistical comparison was made using analysis of variance and the Kolmogorov-Smirnov test. RESULTS: The mice in the combined spinal/general anesthesia group (n = 20) had a significantly shorter time to first paw lick (mean +/- SD) after formalin injection compared to the mice in the general anesthesia group (n = 20) (16.1 +/- 4.5 vs. 22.1 +/- 6.3 min, P = .002). No difference was observed in the number of paw licks in the mice receiving either combined spinal/general anesthesia or general anesthesia (40 +/- 15 vs. 44 +/- 18, P = .47). No difference was observed in the duration of paw licking in either group (231 +/- 76 vs. 237 +/- 124 s, P = .84). CONCLUSIONS: The administration of a pre-emptive subarachnoid lidocaine block in the combined spinal/general anesthesia group did not reduce the degree of formalin-induced paw licking behavior. Combined spinal/general anesthesia did not decrease acute formalin-induced pain in mice. PMID- 7547659 TI - Plasma concentrations of bupivacaine in celiac plexus block. AB - BACKGROUND AND OBJECTIVES: Following stellate ganglion block, systemic absorption of local anesthetics is rapid. Pharmacokinetic data for local anesthetics following other blocks, such as celiac plexus blocks, are lacking. METHODS: Plasma concentrations of bupivacaine in venous blood samples following celiac blocks were measured in 10 patients using a high-performance liquid chromatography technique; 40 mL plain bupivacaine 0.25% was administered. RESULTS: Celiac plexus block resulted in maximum plasma concentrations of 0.7-2.5 mg/L bupivacaine (mean, 1.5 +/- 0.6 mg/L). In 3 of 10 patients plasma concentrations above 2 mg/L occurred. The maximum concentrations were reached 10 30 minutes after the injection (17 +/- 8 minutes). No clinical signs of central nervous system toxicity occurred. All patients showed hemodynamic stability following the blocks. CONCLUSIONS: Maximum plasma concentrations of bupivacaine occur rather late following celiac blocks compared to stellate ganglion or intercostal blocks. The rather high plasma concentrations of bupivacaine indicate the need for appropriate clinical monitoring. PMID- 7547660 TI - Does sympathetic ganglionic block prevent postherpetic neuralgia? Literature review. AB - BACKGROUND AND OBJECTIVES: To examine specifically the role of sympathetic block in the prevention of postherpetic neuralgia by its application in the treatment of acute herpes zoster. METHODS: Data sources included a Medline search and cross referencing of articles and text books. A total of 84 references were reviewed. Peer-reviewed articles were selected according to their relevance to the subject and originality. The data were critically analyzed by the author with the specific intention of avoiding bias. RESULTS: The opinion of the medical community is divided on the role of sympathetic block in preventing postherpetic neuralgia because of the lack of controlled trials and the conflicting retrospective reports as to its effectiveness. While many reports promote the early use of sympathetic blocks during acute herpes zoster to prevent postherpetic neuralgia, others deny their value. CONCLUSIONS: Considering the degree of uncertainty, and the seriousness of postherpetic neuralgia, sympathetic block in addition to treatment with acyclovir should be considered early during acute herpes zoster. Large controlled trials are needed to provide the necessary scientific evidence. PMID- 7547661 TI - Importance of the needle bevel during spinal and epidural anesthesia. AB - BACKGROUND AND OBJECTIVES: The needle bevel has an important effect on the path that the needle takes as it traverses tissue. Many anesthesiologists, especially trainees, are unaware of the magnitude of this effect. There is a need for a model, constructed from readily available materials, that can be used to demonstrate the bevel effect and to assess various strategies for accurately advancing the needle. METHODS: Styrofoam blocks were prepared. The following needles were studied: 25-gauge Quincke, 22-gauge Quincke, 18-gauge Quincke, 24 gauge Sprotte, 25-gauge Whitacre, 22-gauge Tuohy, and 17-gauge Tuohy. The effect of bevel orientation on deflection from midline was measured. Representative results were photographed. RESULTS: The bevel has a significant effect on the path of Quincke and Tuohy needles. The deflection is more pronounced for thinner needles. Pencil-point needles show much less deflection than Quincke needles. CONCLUSIONS: Styrofoam blocks provide a model for teaching the basics of spinal and epidural anesthesia. By utilizing styrofoam the trainee can master needle control and gauge the effect of bevel orientation. PMID- 7547662 TI - Selective musculocutaneous nerve block and infraclavicular brachial plexus anesthesia. Case report. AB - BACKGROUND AND OBJECTIVES: Regional anesthesia of the upper extremity may be achieved by the infraclavicular approach to the brachial plexus. METHODS: Advantages of this approach include profound anesthesia of the upper extremity with minimal risk of complications. RESULTS: Isolated block of the musculocutaneous nerve may result by this approach if biceps muscle contractions are accepted as evidence of brachial plexus location by peripheral nerve stimulation. CONCLUSIONS: Stimulation of the musculocutaneous nerve in the infraclavicular region results in biceps muscle contraction. Inadequate anesthesia of the upper extremity may result due to exiting of the musculocutaneous nerve outside the axillary sheath in this region. Evidence of more distal stimulation (finger/wrist flexion) improves the likelihood of successful block of the brachial plexus by the infraclavicular route. PMID- 7547663 TI - Long-term brachial plexus anesthesia using a subcutaneous implantable injection system. Case report. AB - BACKGROUND AND OBJECTIVES: Continuous brachial plexus anesthesia with local anesthesia has been used since 1946 for prolonged surgical procedures, in postoperative pain relief, and in sympathetic nerve block. The benefit or effectiveness of this technique in the relief of sympathetically maintained pain has yet to be established. METHODS: The following case report describes the placement of an axillary catheter connected to a port for the self-administration of local anesthesia to provide brachial plexus anesthesia in a 43-year-old woman suffering from sympathetically maintained pain. RESULTS: The administration of local anesthesia through this injection system resulted in complete pain relief without motor impairment of the arm. The device functioned for 118 days, making physical therapy easier. An infection occurred at the entry site, requiring the removal of the port. CONCLUSIONS: Controlled studies need to be done to establish the safety and efficacy of this form of therapy. PMID- 7547664 TI - Combined general and epidural anesthesia for a patient with Takayasu's arteritis. Case report. AB - BACKGROUND AND OBJECTIVES: The case is presented of a 44-year-old woman with type I Takayasu's arteritis, undergoing total abdominal hysterectomy. METHODS: Her previous symptoms were related to subclavian and vertebral artery lesions that were treated surgically, and to right carotid stenosis (amaurosis fugax 1-year before the operation) that was not treated. Peripheral pulses were present and blood pressure monitoring was not a problem (invasive intra-arterial pressure monitoring was used). Neurologic monitoring was considered to be mandatory in this case and a computerized electroencephalography monitor was used, both to confirm the adequacy of anesthesia and, more importantly, to monitor unilateral cerebrovascular events. RESULTS: The patient underwent surgery with combined epidural and general anesthesia, without any complications. The epidural block was used throughout the early postoperative period for analgesia. CONCLUSIONS: The management of patients with Takayasu's arteritis requires a knowledge of the location and pathophysiology of vascular lesions. PMID- 7547665 TI - Unintentional combined epidural and subdural block. Case report. AB - BACKGROUND AND OBJECTIVES: Unintentional subdural block, while attempting an epidural anesthetic, is a rare but known complication. The authors describe a radiologically confirmed case of an unintentional combined epidural and subdural block while attempting to perform an epidural block for transurethral resection of prostate in a middle-aged man. METHODS: Loss of resistance to air injection was used for identifying the epidural space prior to catheter placement. Following the epidural injection of 20 mL of a 1.5% lidocaine and 0.5% bupivacaine mixture, the patient developed rapid, intense, prolonged, and extensive bilateral motor block up to C2 level. RESULTS: The patient had aphonia and respiratory paralysis requiring endotracheal intubation and controlled ventilation for 3 hours. CONCLUSIONS: Postoperative radiologic examination revealed the spread of the dye in both epidural and subdural spaces, causing the extensive sensory and motor block. PMID- 7547666 TI - Huber needle and Tuohy catheter. PMID- 7547667 TI - Backache after spinal anesthesia. PMID- 7547668 TI - Transient motor deficit without sensory loss following subarachnoid sufentanil. PMID- 7547669 TI - Response to postblock epidural hematoma causing paraplegia. PMID- 7547670 TI - Interpleural block for acute combined cervical and thoracic herpes zoster. PMID- 7547671 TI - Pre-emptive epidural analgesia may prevent phantom limb pain. PMID- 7547672 TI - Misalignment of the spinal needle during the spinal epidural combined anesthesia. PMID- 7547673 TI - Side-effects during patient-assisted epidural analgesia. PMID- 7547674 TI - Cutaneous application of ethyl chloride spray. PMID- 7547675 TI - Comment on combined spinal-epidural-general anesthesia. PMID- 7547676 TI - Modelling thymic functions in a cellular automaton. AB - Along the lines developed by Celada and Seiden, for simulating an immune system by means of cellular automata, we have constructed a 'thymus' where T cells undergo positive and negative selection. The populations thus 'matured' have been analyzed and their performance has been tested in machina. The key feature of this thymus is to allow chance meeting and possible interaction between newly born T cells and antigen presenting cells. The latter represent both the epithelial and the dendritic cells of the biological organ and are equipped with MHC molecules that can accommodate selected self peptides. All possible specificities are represented among the virgin T cells entering the thymus, but this diversity is drastically reduced by the time they exit as mature elements. In the model organ the fate of T cells, i.e. whether they will undergo proliferation or apoptosis, is governed by their capacity to recognize MHCs and the affinity of this interaction. Crucial parameters turn out to be the concentration of presenting cells, the number of types of MHC per cell, the 'size of self' in terms of the number of different peptides and their prevalence. According to the results, events in the automaton can realize unforeseen cooperations and competitions among receptors, depending upon the interaction order and frequency, and ultimately determine the rescue or the killing of thymocytes. Thus the making of the mature T repertoire has a random component and cannot be completely predicted. PMID- 7547677 TI - Up-regulation of monocytic IL-10 by tumor necrosis factor-alpha and cAMP elevating drugs. AB - It is well established that endotoxin [lipopolysacharide (LPS)] induces pro inflammatory cytokine production in monocytes, which is followed by secretion of the anti-inflammatory cytokine, IL-10. IL-10 down-regulates inflammatory response [tumor necrosis factor (TNF)-alpha, IL-1, IL-6, IL-8] as well as IL-10 synthesis itself. We wondered whether pro-inflammatory cytokines such as TNF-alpha may be involved in the regulation of human IL-10 synthesis. TNF-alpha induced de novo IL 10 mRNA expression in a dose-dependent manner but no IL-10 protein in human peripheral blood mononuclear cells. Furthermore, LPS-induced IL-10 gene and protein expression was significantly inhibited by neutralizing anti-TNF-alpha mAb. On the basis of these results, we conclude that TNF-alpha is involved in the up-regulation of its antagonist IL-10. Paradoxically, drugs that effectively inhibit expression of TNF-alpha via the elevation of intracellular cAMP level (iloprost, pentoxifylline, prostaglandin E2 and N6,2-O-dibutyryl cAMP) augmented the endotoxin-induced IL-10 synthesis at both protein and mRNA levels. In order to provide a basis for the analysis of the transcriptional regulation of the human IL-10 gene, we isolated a fragment of the human IL-10 gene containing 1308 bp of the 5' non-coding sequence. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation, including a cAMP responsive element which could explain the cAMP-mediated effects. The lack of a NF-kappa B-like binding site in the human sequence suggests a NF-kappa B-independent mechanism of TNF-alpha-induced IL-10 gene activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547678 TI - Co-activation of naive CD4+ T cells and bone marrow-derived mast cells results in the development of Th2 cells. AB - Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This assumption was substantiated using neutralizing anti-IL-4 antibodies which abolished the BMMC mediated Th2 development in all cases. Addition of IL-12, a cytokine that was shown to antagonize the Th2-promoting effect of IL-4 in vivo, could not inhibit the development of IL-4-producing T cells, but gave rise to a T cell population which produced relatively high amounts of IL-4 and IFN-gamma. Since BMMC represent the in vitro equivalent of mucosal mast cells these data suggest that IgE-activated mucosal mast cells can bias an emerging T cell dependent immune response towards a Th2 dominated reaction by the initial production of IL-4. PMID- 7547679 TI - Tumor necrosis factor-alpha up-regulates Bcl-2 expression and decreases calcium dependent apoptosis in human B cell lines. AB - Group I and Epstein-Barr virus-negative Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface Ig receptors or after exposure to a calcium ionophore. We show here that tumor necrosis factor (TNF)-alpha protects these B cell lines against Ca(2+)-dependent apoptosis. Protection was associated with up-regulation of bcl-2 mRNA and protein expression. The increase of Bcl-2 expression induced by TNF-alpha was inhibited by chelerythrine, a specific inhibitor of protein kinase C (PKC), suggesting that Bcl-2 expression was dependent on PKC activation. Furthermore, we show that phorbol esters and cyclosporin A (CsA), which prevent Ca(2+)-dependent apoptosis, up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expression is controlled by calcineurin since we have shown that FK506 but not rapamycin had the same effect on Bcl-2 expression, whereas okadaic acid, an inhibitor of phosphatases 1, 2A and 2C, was ineffective. These data provide direct evidence that TNF-alpha prevents Ca(2+) dependent apoptosis by a Bcl-2-dependent mechanism mediated by PKC. PMID- 7547680 TI - Contrast between class I and class II MHC-mediated differentiation of a CD4+CD8+ T cell line: implications for lineage commitment. AB - Experiments in transgenic mice have demonstrated that thymocyte differentiation into the mature CD4+ helper or CD8+ cytotoxic T cell lineage is ultimately dependent upon the specificity of the TCR for class II or class I MHC molecules respectively. However, the initial mechanistic events involved in this process remain unclear. To address this issue, we have expressed a TCR specific for an ovalbumin peptide and the Kb class I MHC molecule in the DPK CD4+CD8+ precursor T cell line. This cell line originally expressed a TCR specific for a pigeon cytochrome c peptide and class II MHC molecules and has been shown previously to differentiate into CD4+CD8- cells upon recognition of antigen in vitro or thymic epithelial cells in vivo. Surprisingly, we find that recognition of either class I or class II MHC by these cells initiates differentiation into the CD4+ lineage and induces down-regulation of recombination associated genes. Unlike recognition of class II MHC, however, recognition of class I MHC does not induce full maturation. These results support a model in which (i) commitment to the CD4 lineage occurs prior to positive selection and (ii) CD4 lineage commitment is associated with a requirement for activation by a class II MHC-specific TCR in order to complete differentiation. PMID- 7547681 TI - Novel mAbs reveal potent co-stimulatory activity of murine CD27. AB - Members of the tumor necrosis factor receptor (TNFR) family are emerging as important molecules implicated in the regulation of proliferation, differentiation and survival of T and B lymphocytes. Among these receptors is CD27, the function of which has thus far only been studied in the human system, where it amplifies the T cell proliferative response induced by TCR triggering. We report here the generation of mAbs to murine CD27, by an efficient method involving the use of transfected Armenian hamster fibroblasts. Previous analysis had already indicated that murine CD27 mRNA is uniquely expressed in lymphoid cells. As determined with one of the newly developed antibodies, murine CD27 is expressed on the great majority of both alpha beta and gamma delta T lymphocytes, on a small population of peripheral B cells, and on a very small subset of B220+ cells in the bone marrow. This distribution largely corresponds to that in the human system. However, unlike human CD27, which is primarily expressed in mature, medullary thymocytes, murine CD27 is found on all thymocytes, except a subset of CD4-CD8- precursors. Upon cross-linking, anti-CD27 mAb amplified the proliferative response of purified T lymphocytes to suboptimal stimulation with concanavalin A at least 4-fold. This indicates that such mAbs can mimick ligand binding and demonstrates that CD27 also acts as a potent co-stimulatory molecule in the murine system. PMID- 7547682 TI - The nature of the immunogen determines the specificity of antibodies and T cells to selected peptides of the 38 kDa mycobacterial antigen. AB - We report in this study that the nature of the injected immunogen or infection of mice can change the specificity of antibodies against different epitopes of the mycobacterial 38 kDa antigen. Following immunization with a selection of 14 peptides, only p65-83 and p285-304 resulted in antibodies binding to the whole antigen, whereas the recombinant antigen induced antibodies to p201-220 and p230 249, which are non-immunogenic following peptide immunization. Killed tubercle bacilli stimulated antibodies exclusively against the N-terminal 1-20 peptide, whilst infection with live tubercle bacilli led to antibodies recognizing non continuous epitopes, demonstrable by binding to the 38 kDa protein, but to none of the tested peptides. The most striking finding is that protein immunization failed to induce antibodies to p65-83, although this epitope is immunodominant for T cells, irrespective of the mode of sensitization. Since this peptide contains non-overlapping B (69-75) and T (75-81) cell stimulatory cores, it appears that T help dedicated to the adjacent B epitope following peptide immunization may have shifted in favour of B cells reacting either with distantly located linear epitopes p1-20 and p201-220 following immunization with the whole protein, or to conformational epitopes following tuberculous infection of mice. The mechanisms which can deviate T help from one to another B cell specificity could involve regulation of antigen processing, liable to the nature of the immunogen. The described results suggesting distinct functional pairing between T and B epitopes, but with limited fidelity, may have general implications for the design of subunit vaccines. PMID- 7547683 TI - IL-4-induced B cell migration involves transient interactions between beta 1 integrins and extracellular matrix components. AB - IL-4 has previously been shown to stimulate motile responses in murine B lymphocytes. This was studied as acquisition of motile morphology and migration through filters in microchemotaxis chambers. In this paper, we investigated IL-4 stimulated migration of B cells into gels of native collagen fibers, which may be a more physiologically relevant assay. When IL-4 was present in the gel and/or in the medium above, B cells were able to invade the collagen gel. Migration was dependent on the dose of IL-4 and was optimal after 45 h of incubation. It appeared that IL-4 acted by inducing both chemokinesis and chemotaxis. Fibronectin (FN) was found to be an important factor for B cell locomotion, since low concentrations of FCS or FN in the gel matrix greatly improved migration. B cell locomotion was inhibited by antibodies specific for beta 1, alpha 4 and alpha 5 integrins, indicating the presence of integrin-extracellular matrix (ECM) interactions in lymphocyte motility responses. Migration was not associated with an up-regulation of beta 1, alpha 4 or alpha 5 integrins. The adhesion between substrate and cells is likely to be of low affinity, since IL-4-stimulated, as well as non-stimulated B cells, did not adhere to ECM-coated culture wells. Our data suggest that transient interactions between integrins and the ECM matrix may favour B cell migration. PMID- 7547684 TI - Regulation of IFN-gamma mRNA production in murine natural killer cells. AB - We have previously reported that large, presumably in vivo activated, B cells stimulate murine natural killer (NK) cells to secrete increased levels of IFN gamma. In order to further understand the mechanism of IFN-gamma induction, we compared the regulation of IFN-gamma mRNA production after stimulation of NK cells with either B lymphocytes or phorbol myristate acetate (PMA)+ionomycin. Here we show that stimulation of NK cells by either stimuli results increase in IFN-gamma mRNA, albeit with different kinetics. Although the induction requires new RNA synthesis, we could not detect increased transcription of the IFN-gamma gene after stimulation. Measurement of the rate of mRNA degradation after IFN gamma mRNA has accumulated demonstrates that this mRNA is more stable than IFN gamma mRNA from unstimulated NK cells. Together, these results suggest that the increase in IFN-gamma mRNA and protein in NK cells, stimulated by B cells or PMA+ionomycin, results from stabilization of pre-existing IFN-gamma message. Our results also suggest that induction of the factor which stabilizes the mRNA, although as yet unknown, requires new RNA synthesis. PMID- 7547685 TI - Characterization of a 50 kDa surface membrane protein on thymic stromal cells as an important factor for early T cell development. AB - We previously reported that the nude mouse-derived splenic T cell clone, N-9F, exhibits a proliferative response to the SL10.3 thymic epithelial cell clone. In the present study we generated an Armenian hamster mAb, HS9, specific for SL10.3, which inhibited the N-9F's proliferative response to SL10.3. We performed thymocyte repopulation experiments using fetal liver cells and 2'-deoxyguanosine treated thymic rudiments. After 14 days of culture, donor fetal liver cells proliferated and differentiated to CD4+CD8+ and CD4-CD8+ with some CD4+CD8- cells in the host thymic rudiments. However, most of the thymocytes remained at a CD4 CD8- immature stage in the presence of HS9 and the cell recovery was reduced to 30% of the control. Immunohistostaining and flow cytometry studies revealed that HS9 reacted with stromal cells of fetal thymus at the earliest from day 14 gestation. Neither thymocytes nor lymph node T cells were stained with HS9. HS9 antigen was distributed not only on thymic subcapsular and cortical stromal cells, but also on peripheral B cells in adult mice. The antigen that HS9 detected was found to be a 50 kDa surface membrane protein on thymic stromal cells. On the other hand, the 50 kDa molecule is associated with two other molecules of 80 and 100 kDa on the B cells. These data indicate that the HS9 antigen may have an important role for early T cell development, especially at a stage from CD4-CD8- to CD4+CD8+, and may have some unknown function on B cells. PMID- 7547686 TI - Differential sensitivity of human naive and memory CD4+ T cells for dexamethasone. AB - Human CD45RA+ ('naive') and CD45RO+ ('memory') CD4+ T cells were compared with respect to their sensitivity to dexamethasone (DEX). In three different activation pathways, i.e. (i) immobilized anti-CD3, (ii) immobilized anti-CD3 plus soluble anti-CD28 and (iii) soluble anti-CD2 plus soluble anti-CD28, naive CD4+ T cells appeared more sensitive to DEX than memory CD4+ T cells. In the anti CD3 system this difference in sensitivity was apparent at a suboptimal DEX concentration. Addition of anti-CD28 rendered the cells largely insensitive to DEX, indicating that the CD28 pathway is less dependent of the DEX-sensitive transcription factor AP-1. However, the alternative pathway of T cell activation through CD2/CD28 triggering was highly sensitive to DEX when naive cells were studied; in the case of memory cells, at least a 10-fold higher DEX concentration was needed to achieve a comparable inhibition. The strong inhibitory effect of DEX on naive CD4+ T cells stimulated via the alternative pathway was completely abrogated by activation of protein kinase C (PKC) with phorbol myristate acetate. Our data suggest that at least two different mechanisms contribute to DEX resistance, i.e. CD28 triggering and PKC activation, which may occur more effectively in memory cells making them less sensitive to DEX. PMID- 7547687 TI - Definition of a human T cell epitope from influenza A non-structural protein 1 using HLA-A2.1 transgenic mice. AB - Previous results from this laboratory demonstrated that the dominant influenza A epitope recognized by HLA-A2.1-restricted cytotoxic T lymphocytes (CTL) from HLA A2.1 transgenic mice was the matrix protein 1 (M1) peptide epitope that is immunodominant in human CTL responses. However, analysis of a large number of CTL lines revealed a subset of influenza A/PR/8/34-specific murine CTL that recognized an HLA-A2.1-restricted epitope distinct from M1. Using recombinant vaccinia viruses encoding different influenza gene segments, the epitope recognized by these CTL was shown to be derived from A/PR/8 non-structural protein 1 (NS1). Because these CTL did not recognize targets infected with the A/Alaska/6/77 strain of influenza, candidate peptide epitopes were synthesized based on sequences that included an HLA-A2.1-specific binding motif, and that differed between A/PR/8 and A/Alaska. All of these CTL recognized a nonamer and a decamer peptide which contained a common eight amino acid sequence and two distinct sets of binding motif residues. However, the nonamer peptide was able to sensitize CTL for half-maximal lysis at 80- to 2500-fold lower doses than either the octamer or decamer. The homologous peptide derived from A/Alaska NS1 contained conservative amino acid changes at positions 4 and 8, and was not recognized at any tested concentration, although it bound with higher affinity to HLA-A2.1 than the peptide from A/PR/8. The A/PR/8 NS1 nonamer epitope was also recognized by human influenza A-specific CTL derived from two individuals. These results substantiate the general utility of HLA class I transgenic mice for the identification of human CTL epitopes for other pathogens. PMID- 7547688 TI - T cell activation through Thy-1 is associated with the expression of a surface protein (p100) on a subset of CD4 cells. AB - Thy-1 molecules, which lack a transmembrane domain, can nonetheless induce T cell activation; it has thus been suggested that a separate transmembrane molecule associated with Thy-1 is required for signal transduction. We have previously characterized a transmembrane protein with an Mr of 100,000 (p100), which is non covalently bound to two glycosyl-phosphatidylinositol (GPI)-linked molecules, Thy 1 and ThB. p100 is selectively expressed on the T cell surface and divides peripheral CD4 cells into two subpopulations. This differential expression on CD4 cells allowed us to investigate the role of p100 in signal transduction through Thy-1 molecules. Here we report that only p100+ CD4 cells proliferate and release cytokines in response to cross-linkage of Thy-1, although both p100+ and p100- CD4 cells strongly express Thy-1 on their surfaces. Control stimulation by anti CD3 antibodies or concanavalin A induces identical thymidine uptake by the two CD4 cell populations. Interestingly, these two populations of CD4 cells had different cytokine release profiles after activation through CD3: only p100+ CD4 cells released high amounts of IL-2 and IFN-gamma, whereas both populations released IL-4. p100 expression correlates with the induction of homotypic aggregation of T cells after Thy-1 triggering. p100 is associated with kinase activity (fyn and lck), and phosphorylated proteins of 90, 59, 57 and 33 kDa co precipitate with Thy-1 only in p100+ CD4 cells. Altogether, these data suggest that p100 is involved in signal transduction through Thy-1. p100 expression by activated CD4 cells in vivo may be relevant to the proposed function of Thy-1 as an accessory signaling molecule in cell activation. PMID- 7547689 TI - The protein tyrosine kinase p56lck regulates TCR expression and T cell selection. AB - The role of the protein tyrosine kinase (PTK), p56lck, in T cell development was evaluated by mating TCR transgenic mice with transgenic mice that expressed lckF505, a constitutively activated form of p56lck which is under the control of the lck proximal promoter element. The TCR transgenic mice expressed either a receptor specific for the male antigen presented by Db (H-Y TCR) or a receptor specific for pigeon cytochrome c peptide presented by I-Ek class II MHC molecules (AND TCR). The lckF505 transgene caused lower TCR expression in immature CD4+CD8+ thymocytes from normal and TCR transgenic mice. Consistent with the conclusion that activated p56lck causes lower TCR expression, the PTK inhibitor, herbimycin A, was able to restore TCR expression to normal levels in CD4+CD8+ thymocytes from TCR/lckF505 doubly transgenic mice. However, despite lower TCR expression, calcium mobilization was only moderately reduced in CD4+CD8+ thymocytes from H-Y TCR/lckF505 doubly transgenic mice. Furthermore, negative selection of CD4+CD8+ thymocytes expressing the H-Y TCR occurred efficiently in H-Y TCR/lckF505 doubly transgenic male mice despite lower TCR levels. By contrast, analysis of H-Y TCR/lckF505 and AND TCR/lckF505 doubly transgenic mice showed that positive selection in these mice was reduced by 4- to 5-fold by the lckF505 transgene. The smaller proportion of cells that were positively selected in doubly transgenic lckF505 mice expressed normal levels of TCR but higher levels of the appropriate CD4 or CD8 co-receptor molecule. These results indicate that the positive selection of thymocytes is regulated by the enzymatic activity of p56lck. PMID- 7547690 TI - A CD4+ T cell line-secreted factor, growth promoting for normal and leukemic B cells, identified as thioredoxin. AB - In this study, a B cell growth stimulatory factor, constitutively secreted by a human CD4+ T cell hybridoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltration and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymidine incorporation in normal and leukemic (B CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithiothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30-50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin disulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified approximately 100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was identical to the previously published sequence of human Trx from HTLV-1 transformed T cells, adult T cell leukemia-derived factor/Trx.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547691 TI - T cell-induced B cell blasts differentiate into plasma cells when cultured on bone marrow stroma with IL-3 and IL-10. AB - B lymphocytes activated by T cells in secondary lymphoid organs mature into plasma cells after migration into the medullary cords of these organs, mucosal lamina propria or bone marrow. To analyze each step leading to plasma cell generation, we set up a two-step culture system of purified tonsillar B cells. In a primary stage, B cell blasts were generated by co-culturing B cells with an irradiated T cell clone activated with immobilized anti-CD3. In a secondary step, culturing these blasts on bone marrow stromal cells (BMSC) induced them to secrete large amounts of IgG, as well as some IgM and IgA. Other fibroblast-like cell lines were less efficient at sustaining the differentiation of blasts into Ig-secreting cells, suggesting that these are specific properties of BMSC. Addition of IL-3 and IL-10 further stimulated IgG secretion by B cell blasts cultured on BMSC, mostly the IgG1 subclass. These two cytokines probably acted through different pathways, as (i) the effect of IL-3 but not IL-10 was dependent upon prolonged T cell pre-activation and (ii) their combined stimulatory effect was additive. B cell blasts cultured on BMSC with a combination of IL-3 and IL-10 differentiated into non-proliferating plasma cells as determined by poor thymidine incorporation, typical cellular morphology, intense expression of intracytoplasmic Igs, very high levels of surface CD38 and lack of surface CD20. PMID- 7547692 TI - The perinatal presence of antigen (p-azophenylarsonate) or anti-mu antibodies lead to the loss of the recurrent idiotype (CRIA) in A/J mice. AB - The immune response of A/J mice against p-azophenylarsonate (Ars)-keyhole limpet hemocyanin (KLH) is characterized by the dominance, late in primary and during the secondary, of a recurrent idiotype called CRIA, encoded by a canonical combination of Ig gene segments. In this study, A/J mice were given Ars coupled to deaggregated human gamma globulins (dHGG) within 24 h after delivery. The offsprings from these mice were then exposed as adults to Ars-KLH. These animals developed an unusual immune response. The level of anti-Ars antibodies was nearly normal but a dramatic shift in repertoire was observed: the cross-reactive idiotype which is the hallmark of the anti-Ars response in A/J mice was completely absent. The idiotype could be recovered by injection of anti-idiotypic antibodies alone, with no need of lipopolysaccharide coupling. Therefore the presence of antigen at birth can lead to a strong perturbation of idiotype selection. Similar results were obtained with neonatal treatment using anti-IgM antibodies. After recovery of suppression, A/J mice can mount an anti-arsonate response of normal level but devoid of the dominant idiotype. PMID- 7547693 TI - HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA-1, EBNA-2 and BZLF 1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1. AB - B lymphocytes immortalized with EBV in vitro, lymphoblastoid cell lines (LCL), express eight EBV-encoded proteins, EBV nuclear antigens -1 to -6 (EBNA-1 to -6), and latent membrane proteins 1 and 2 (LMP 1 and 2). After appropriate stimulations of blood lymphocytes from seropositive individuals, MHC-restricted cytotoxic T cells (CTL), which lyse LCL cells, can be generated in vitro. Such CTLs can recognize EBNA-2 to -6, and LMP 1 and 2, but not EBNA-1-derived peptides presented on the cell surfaces. We posed the question whether this exceptional feature of EBNA-1 is due to lack of MHC class I binding peptides. A computer search for 11 human leukocyte antigen (HLA) alleles showed that EBNA-1 has a lower number and lower proportion of relevant binding motifs to several alleles than EBNA-2 to -6 and LMP 1 and 2. The low motif numbers in EBNA-1 is in line with its apparent failure to generate a CTL response, and it may be the consequence of immunoselection allowing the existence of EBV genome-carrying B cells in the immunocompetent hosts. The binding capacities of synthetic peptides of EBNA-1 and -2 and of the immediate early lytic cycle protein BZLF-1 to HLA A0201 (A2) and HLA-B7 molecules were tested in an MHC stabilization assay. The peptide transporter-deficient T2 line, which expresses a low level of HLA-A2 and its HLA-B7 transfectant subline, were used for this purpose because specifically bound peptides elevate the surface expression of these MHC molecules. Of five synthetic nonamer EBNA-1 peptides which include the relevant binding motif, four bound to A2. In a series of 20-amino acid-long overlapping EBNA-1 peptides none showed binding to A2, while eight peptides bound to B7. Two 20-amino acid-long EBNA-2 and seven BZLF-1 peptides were identified as A2 binders, and four EBNA-2 and eight BZLF-1 peptides bound to B7. Thus, we have exclude the possibility that the inability of the EBNA-1 protein to generate HLA-restricted CTLs could be due to the lack of HLA class I binding peptides in its sequence. The finding that several EBNA-1 peptides could bind to these two HLa molecules does not, however, necessarily reflect the natural situation because the peptides may not be processed and/or transported to the cell surfaces. We have stimulated lymphocytes of healthy donors with relevant HLA types with the autologous LCL.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547694 TI - Transgenic mice expressing MHC class II molecules with truncated A beta cytoplasmic domains reveal signaling-independent defects in antigen presentation. AB - The thymic development and peripheral activation of CD4+ T cells are critically dependent upon interactions with MHC class II molecules on the surface of antigen presenting cells (APC). In vitro studies involving transfection of cell lines with mutant MHC molecules have demonstrated that the cytoplasmic domains of class II molecules can be required for efficient antigen presentation. To address the role of class II cytoplasmic domains in physiological, non-transformed APC and in vivo immune responses, we have generated transgenic mice which express only truncated class II A beta molecules lacking the 13 membrane distal residues. In vivo, CD4+ T cell development and immune responses to conventional protein antigens, parasitic infections and skin grafts were indistinguishable between control and transgenic mice. Nevertheless, in vitro, APC from transgenic mice poorly stimulate T cell hybridomas and wild-type in vivo-primed T cells. Neither class II-mediated induction of B7-1 expression nor homotypic aggregation were diminished in transgenic B cells, suggesting that both cAMP and tyrosine kinase signaling pathways remain intact despite truncation of the A beta cytoplasmic domain. Furthermore, chemically-fixed cells from transgenic animals are impaired in their antigen presenting capacity. Thus, in contrast to previous studies with cell lines transfected with truncated class II molecules, these results suggest that signaling-independent mechanisms account for the defective in vitro antigen presenting capacity of physiological APC expressing truncated A beta proteins. PMID- 7547696 TI - The pathogenic human monoclonal anti-DNA that induces experimental systemic lupus erythematosus in mice is encoded by a VH4 gene segment. AB - Systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human anti-DNA IgM mAb that was derived from a patient with cold agglutinin disease. The latter anti-DNA mAb expresses the common idiotype (Id) designated 16/6 Id. The original human hybridoma 16/6 that secreted an IgM antibody that bound ssDNA and carried the 16/6 Id had switched in culture to secrete an IgG molecule. Herein we show that the IgG 16/6 antibody contains the previously reported characteristics of the original IgM 16/6 mAb: it expresses the 16/6 Id and is capable of inducing experimental SLE in susceptible mouse strains. The identify of the IgG 16/6 anti-DNA mAb to the original IgM mAb was shown both by serological techniques and at the T cell level. The human IgG 16/6 mAb was found to be encoded by a germline gene from the human VH4 gene family, with high similarity to the germline gene VH4.21 that was previously shown to code for anti DNA antibodies isolated from SLE patients. The VH4.21 germline gene was found to also code for most antibodies with cold agglutinin activity that were isolated from patients with cold agglutinin disease. PMID- 7547697 TI - Cytolytic T lymphocytes displaying natural killer (NK)-like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production. AB - Natural killer (NK) cells express surface receptors for defined groups of HLA class I alleles. The specific interaction between these receptors and HLA class I molecules expressed on target cells results in inhibition of NK-mediated target cell lysis. In this report, we analyzed whether similar mechanisms were operating in cytolytic T lymphocytes (CTLs) capable of lysing NK-sensitive target cells. T cell clones were screened for their ability to lyse K562 target cells. The selected clones expressed either gamma delta or alpha beta TCR. The majority of these clones failed to lyse the HLA class I+ R8/15375 cell line; however, upon addition of the previously described A6-136 (IgM) or 6A4 F(ab')2 anti-HLA class I mAbs, target cells were efficiently lysed. Lysis of autologous phytohemagglutinin blasts in the presence of anti-HLA class I mAbs occurred primarily with TCR gamma delta+ CTLs. Recognition of HLA class I molecules on target cells implies the expression of NK-related specific receptors in CTL clones. Indeed, phenotypic analysis of > 300 CTL clones with NK-like activity revealed that 28% expressed p58 molecules (specific for HLA-C alleles) while 30% expressed CD94 molecules (specific for the Bw6 specificity). These receptor molecules were found to function as inhibitory receptors, as revealed by the effect of anti-p58 or anti CD94 mAbs (of IgG isotype) on the lysis of the Fc gamma R+ K562 target cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547695 TI - Role of macrophages and dendritic cells in primary cytotoxic T lymphocyte responses. AB - The successful induction of class I restricted cytotoxic T lymphocytes (CTL) responses with soluble non-replicating antigens relies upon vehicles which deliver antigen in vivo appropriately to antigen presenting cells (APC), which for CTL may be dendritic cells (DC). In this study, we have followed the distribution of liposomes and their incorporated antigen and compared the efficacy of splenic macrophages (Mo) and DC at inducing primary CTL responses in vitro. Our results show that whereas liposomes locate predominantly in the splenic red pulp and marginal zone locations, some of their soluble antigen contents redistribute to the central white pulp, comprising mainly DC and T cells. Such antigen redistribution was most apparent following administration of pH-sensitive liposomes. In comparisons of the APC activity of Mo and DC taken at various times post-injection, DC were always superior to Mo. However, if Mo were depleted prior to antigen exposure, DC were ineffective APC for CTL induction. Furthermore, addition of supernatant from OVA-liposome treated Mo to naive DC-T cell cultures in vitro resulted in OVA-specific T cell responses. These studies indicate a role for Mo in enhancing the antigen presenting function of DC. PMID- 7547699 TI - Mechanisms of immune tolerance induction through the thymic expression of a peripheral tissue-specific protein. AB - A major process through which the immune system becomes tolerant to self proteins involves the deletion of self reactive cells in the thymus. However, T cells reactive to peripheral tissue-specific proteins can escape this deletion and become tolerized in the periphery by a variety of mechanisms. We report here, contrary to expectation, that the pancreas-specific protein, elastase I, is also expressed at a low level in the thymus, and that this thymic expression contributes to tolerance induction. To study the mechanism of this tolerance induction, we utilized a double transgenic mouse model. In these mice the expression of a model protein, SV40 T antigen, is directed by the elastase I promoter and hence parallels elastase I expression in the pancreas and thymus. These mice were crossed with mice transgenic for a TCR specific for T antigen, so the majority of thymocytes and T cells in these mice express the transgene. In double transgenic mice we find that thymic expression of T antigen results in anergic thymocytes which also show a reduction of Th1 activity with no decrease in Th2 activity. These functional characteristics persist in peripheral T cells, but there is also a depletion in the number of T antigen reactive T cells in lymph nodes. Chimeras were constructed which directly demonstrated that the thymus is the site of tolerance induction and that the tolerizing element is thymic epithelium. We propose that the loss of Th1 activity as a consequence of the thymic epithelium being encountered by tissue-specific proteins results in the functional tolerization of CTL in vivo, despite the fact that CTL are fully functional in vitro. In this way autoimmune destruction is contained. Thymic expression of peripheral proteins may therefore be an additional way in which tolerance to peripheral proteins can be achieved. PMID- 7547698 TI - TNF-alpha regulates IL-4-induced Fc epsilon RII/CD23 gene expression and soluble Fc epsilon RII release by human monocytes. AB - We examined the regulatory effects of TNF-alpha on IL-4-induced gene expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) in human monocytes and IL-4-induced soluble Fc epsilon RII (sFc epsilon RII) release from monocytes. IL 4-induced Fc epsilon RII expression on the surface of monocytes was reduced by TNF-alpha as early as 1 day after culture and the effect of TNF-alpha increased with prolonged culture. The present analysis was designed to examine whether or not TNF-alpha could suppress IL-4-induced Fc epsilon RII mRNA expression and enhanced IL-4-induced sFc epsilon RII release. The addition of TNF-alpha to monocyte cultures with IL-4 significantly reduced Fc epsilon RII expression on the surface of monocytes and significantly increased sFc epsilon RII release from monocytes. Over time, there was an inverse relationship between the disappearance of cell surface Fc epsilon RII and the appearance of sFc epsilon RII in culture supernatants. Fc epsilon RII mRNA expression in monocytes cultured with IL-4 was not affected by TNF-alpha when examined at 6 h after cultivation. When the cells were cultured with TNF-alpha for more than 24 h, however, TNF-alpha down regulated IL-4-induced Fc epsilon RII mRNA levels. This correlated with the kinetics of down-regulation of IL-4-induced Fc epsilon RII expression on the surface of monocytes by TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547700 TI - Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: identification of forms increased in cancer patients' sera. AB - Normal human sera contained 10-60 ng/ml of soluble membrane cofactor protein (MCP, CD46) whereas sera of > 50% of the cancer patients contained > 60 ng/ml. MCP purified by immunoaffinity chromatography from both normal and cancer patients' sera consisted of three bands of 56, 47 and 29 kDa on SDS PAGE/immunoblotting. The upper two components were increased in cancer patient sera. The 56 and 47 kDa soluble forms served as a cofactor for factor I-mediated cleavage of C3b. MCP expressed on Chinese hamster ovary (CHO) cells protects host cells from human C3 deposition and complement-mediated cytolysis, especially by activation of the alternative pathway. In this same assay system, exogenously added soluble MCP also protected untransfected CHO cells; however, its potency was much less than that of the endogenous membrane form. For example, 8 micrograms/ml of soluble MCP was equal to 10(4) copies/cell of the expressed MCP. Recombinant soluble forms possessed similar activity to the naturally occurring soluble forms and high doses (> 150 micrograms) blocked Arthus-like reaction induced in guinea-pigs by anti-Forssman antibody. These data establish that soluble forms of MCP are present in human sera that possess cofactor activity and their concentrations, especially the 56 and 47 kDa forms, are increased in sera of cancer patients. High doses of the recombinant soluble forms may be therapeutically useful for suppressing inflammatory responses. PMID- 7547701 TI - Regulation of B cell precursor proliferation by aminopeptidase A. AB - The BP-1/6C3 molecule expressed by early B lineage cells and some stromal cells has been identified as aminopeptidase A (APA). We have previously demonstrated that IL-7 selectively induces BP-1/APA expression by pre-B cells coincident with their growth. Here we directly demonstrate that BP-1 is preferentially expressed by the proliferating subpopulation of normal B cell progenitors. Furthermore, when non-adherent BALB/c bone marrow cells were incubated with IL-7 in the presence of purified BP-1 antibody, B cell precursor proliferation was markedly inhibited. Modulation of the BP-1/6C3 antigen did not occur in the presence of the BP-1 antibody, but APA enzymatic activity was significantly inhibited. The 6C3 antibody, which recognizes a different epitope on the APA molecule, had no effect on either B cell precursor proliferation or APA enzyme activity. We hypothesized that neutralization of APA by the BP-1 antibody results in inhibition of IL-7 driven B cell precursor proliferation. However, when isolated 14.8+ bone marrow cells were cultured with IL-7 in the presence of the BP-1 antibody, no inhibition of proliferation occurred. This data suggested that the effect of the BP-1 antibody might be related to the action of APA on peptides in the marrow microenvironment which are not present in cultures of isolated B cell precursors. The addition of irradiated non-adherent bone marrow cells to the 14.8+ cell cultures restored the inhibitory effect of the BP-1 antibody. Based on these observations, we propose that APA cleaves a small peptide which serves as a natural inhibitor of B cell precursor proliferation. PMID- 7547702 TI - Interactions among polymorphic and conserved residues in MHC class II proteins affect MHC-peptide conformation and T cell recognition. AB - The structural basis for MHC-restricted T cell recognition of the N-terminal peptide of myelin basic protein (MBP Ac1-11) presented by two mouse class II alleles, Ak and Au, was examined, focusing on the roles of A beta chain polymorphic residues 38 beta (in the beta sheet) and 61 beta (in the alpha helix) in controlling the responses of panels of Ak- and Au-restricted T cell hybridomas. Despite the conservative nature of the substitutions at 38 beta (k = Val, u = Leu) and 61 beta (k = Trp, u = Tyr), transfectants expressing Ak or Au proteins carrying allelic substitutions at 38 beta and/or 61 beta gave dramatically reduced T cell responses. The modest reduction in peptide binding detected using a biotinylated MBP peptide analog appear insufficient to explain the reduced responses, suggesting that changes at 38,61 beta create conformational changes in the MHC-peptide complex. The impact of allelic substitutions at 38,61 beta on T cell responses is also modulated by other residues differing between Ak and Au. To explore the structural basis for these phenomena, protein models were developed of the Ak, Au and 38,61 beta mutant proteins using self-consistent ensemble optimization methodologies. Substitutions of the alternative allelic residue at 38 beta and/or 61 beta, which are in van der Waals contact, change the configuration of this region of the peptide-binding groove, and potentially might affect the conformation of the bound peptide and its hydrogen-bonding to residue 61 beta. The models predict that this region of the groove is markedly altered by allelic differences at A beta residue 9 beta (k = His, u = Val) which determine the position of the side-chain of Tyr30 beta, adjacent to residues 38 beta and 61 beta. Thus, interactions among polymorphic and conserved residues control the antigen presentations functions of MHC class II proteins. PMID- 7547703 TI - Activation of natural killer cells by the mAb YTA-1 that recognizes leukocyte function-associated antigen-1. AB - The mAb YTA-1, which brightly stains CD3-CD16+ large granular lymphocytes (LGL)/natural killer (NK) cells and CD8+ T cells by immunofluorescence, is specific for leukocyte function-associated antigen (LFA)-1. Some mAbs recognizing the LFA-1 alpha chain (CD11a) or LFA-1 beta chain (CD18) inhibited the binding of YTA-1 to peripheral blood mononuclear cells. YTA-1 mAb could be chemically cross linked to 170 and 96 kDa molecules, whose molecular weights correspond to those of LFA-1 alpha and beta respectively. YTA-1 bound to COS-7 cells co-transfected with CD11a and CD18 cDNAs, but not to untransfected cells. Reactivities of YTA-1 to K562 cells transfected with LFA-1 alpha and beta (CD11a/CD18) cDNAs and to CHO cells transfected with Mac-1 (CD11b/CD18) or p150, 95 (CD11c/CD18) cDNAs strongly suggest that YTA-1 recognizes either LFA-1 alpha or an epitope formed by a combination of LFA-1 alpha and beta. Treatment of fresh CD3-CD16+ LGL with YTA-1 augmented cytolytic activity and induced proliferation. F(ab')2 fragments of YTA 1 augmented NK cytotoxicity, indicating that the NK activating signal was transmitted through LFA-1 without involvement of Fc gamma receptor III. In contrast, the other mAbs against LFA-1 could not activate NK cells. These results collectively indicate that YTA-1 recognizes a unique epitope of LFA-1, which is involved in activation of fresh NK cells. PMID- 7547704 TI - T cells from unprimed mice respond to the self antigen heme, in a class II restricted manner, at a frequency similar to alloresponses. AB - Heme is a non-protein autoantigen which stimulates potent proliferative responses by T cells from unprimed mice of some strains. These studies show that T cells responding to heme in primary responses are predominantly CD4+, classically I-A restricted, and use diverse TCR characterized by the expression of distinct V, D and J gene segments. These characteristics distinguish heme from superantigens and mitogens which exhibit degenerate MHC restriction and, in the case of superantigens, restricted V gene usage. Using limiting dilution analysis these studies also show that the potent primary response of H-2s mice reflects a high frequency (0.26-0.45%) of heme responsive T cells in the periphery, comparable to the frequency of alloresponsive T cells reported by others in primary mixed lymphocyte reactions. In contrast, heme responsive T cells occur at -10-fold lower frequency in unprimed H-2d mice (0.03%). To determine the antigen recognized by heme reactive T cells, the mass spectra of peptides eluted from the high responder haplotype, I-A(s), were examined. These indicated a markedly different molecular weight distribution of peptides isolated from cells grown in the presence of heme, compared with those from cells grown in its absence. This suggests that heme mediates the expansion of diverse T cells in the peripheral repertoire by a mechanism similar to that for allogeneic responses in which the profile of naturally processed peptides bound to the MHC class II molecule is changed. PMID- 7547705 TI - Immunoprotection against systemic candidiasis in mice. AB - We have previously described an immunosuppressive B cell mitogenic (ISM) protein, p43, produced by Candida albicans, which plays an important role in the survival of the microorganism in the host. The N-terminal amino acid sequence of p43 was found to be different from all amino acid sequences registered in updated protein databanks. Immunization of BALB/c mice with p43 partially neutralized the biological effects of this protein, namely depletion of bone marrow pre-B and B cells, the increased numbers of total and large B and CD4+ lymphocytes, and the non-specific polyclonal response of splenic IgG2a-, IgG2b- and IgM-secreting plaque forming cells. Immunization of BALB/c mice with p43 fully protected the mice against the fungal infection. In contrast, immunization with C. albicans sonicates (Cs) was not protective. Our data indicated that specific antibodies against p43 protected, whereas those against Cs facilitated C. albicans infection. Thus, the ratio between anti-p43 and anti-Cs antibody titres was much lower in the non-protected mice (Cs-immunized and control non-immunized) than in p43-immunized mice. Moreover, passive administration of specific anti-p43 antibodies significantly protected against fungal infection, whereas passive administration of specific anti-Cs antibodies markedly increased the susceptibility to C. albicans infection. These observations are discussed on the basis of alternative approaches of immunointervention. PMID- 7547706 TI - Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus. AB - Listeria monocytogenes spends most of its intracellular life cycle in the cytosol of the infected eucaryotic cells. Within this cellular compartment originates the endogenous pathway of antigen processing and presentation. We thus assumed that recombinant L. monocytogenes expressing an heterologous protein, the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV), should be able to induce antigen-specific CD8+ T cells in vivo. The LCMV nucleoprotein gene was inserted in phase with the sequence coding for the putative signal sequence of the hemolysin of L. monocytogenes in order to target its secretion into the cytosol of the infected cell. The ability of this recombinant bacterium to induce LCMV-reactive CD8+ T cells was then monitored in BALB/c mice. The immune status of the immunized BALB/c mice was studied on the seventh day after a single i.v. injection of a sublethal dose of the recombinant bacteria: (i) cytotoxic CD8+ T cells were detected in liver; (ii) using in vitro re-stimulation with PMA and ionomycin, secondary cytotoxic CD8+ T cells were detected in spleen; (iii) an early inflammatory reaction dependent on the presence of CD8+ T cells occurred in the footpad after intraplantar inoculation of live LCMV; and (iv) mice were protected against an otherwise lethal intracerebral LCMV challenge; the protection was accompanied by elimination of the virus. When the immune status of the immunized hosts was monitored for a longer period post-immunization, the balance between immune protection and immunopathology described for the anti-LCMV immune responses was observed; two phases of protection were detected, flanking a transitory phase of exacerbation of the lymphocytic choriomeningitis disease (weeks 2-5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547708 TI - Antigens released at schizont burst stimulate Plasmodium falciparum-specific CD4+ T cells from non-exposed donors: potential for cross-reactive memory T cells to cause disease. AB - In an individual experiencing the first attack of malaria, symptoms of disease can occur at very low parasitemia. T cells and cytokines have been implicated in the etiology of disease symptoms, and others and ourselves have shown that T cells from non-exposed individuals can be stimulated by malaria parasites. Here, we show that nine from 11 blood samples, naturally infected with malaria parasites, could stimulate proliferation of a malaria-specific T cell clone derived from a non-exposed donor. T cells were able to respond to infected blood at a parasitaemia as low as 0.000003% (comparable to the level at which individuals can first experience symptoms of malaria) and secrete cytokines implicated in pathology. Antigens capable of stimulating T cells are expressed throughout the blood stage, but are specifically released at the time of schizont rupture. While most TCR V beta genes are expressed during the T cell response of naive donors to malaria parasites, processing of parasite antigens is blocked by chloroquine and monensin, and activation of 36 of 41 malaria-specific clones tested was restricted by defined MHC class II allelic antigens, strongly suggesting that parasites do not act as mitogens nor as superantigens. The clones react to various commonly encountered pathogenic and non-pathogenic microbes. These data support the concept that activation of cross-reactive memory T cells by malaria parasites contributes to disease symptoms in individuals experiencing their first attack of malaria. PMID- 7547707 TI - Sequence analysis and antigen binding characteristics of Ig SCID Ig+ mice. AB - SCID mice as they age may develop a limited number of functional B and T cell clones. VH and VL gene sequences of hybridoma antibodies derived from the spleens of five adult SCID Ig+ mice revealed nine clonally unrelated, i.e. derived from different precursor lymphocytes, groups of Ig. Of these, four used the VH7183 family, one Q52, three J558 and one VH3609. VH7183 was seen in excess over J558 despite a 5-fold larger number of J558 family members in the mouse genome. Preferential use of D proximal VH genes along with reduced N region addition in the heavy chain VDJ joins of these antibodies suggest derivation from a population of lymphocytes with characteristics of the perinatal repertoire. All of the hybridomas produced kappa light chains of several different VL family groups. Sequences of 23 VH and 22 VL genes of hybridomas derived from one mouse (no. 45) showed that they were all clonally related, differing only in somatic mutations. Autoreactivity of these antibodies to cellular antigens [including (U1)RNP-specific A protein and SmB] was analyzed, and shown to correlate with the geneology and specific mutations within members of the clone. The geneological tree derived from sequence data permitted a study of the microevolution of a clone of B cells and showed that as it developed there was a tendency toward increased strength of self-reactive binding as well as alterations in specificity toward these autoantigens. Analysis of replacement:silent mutation ratios within members of this clone reflected limited affinity maturation in mouse 45 and none for another mouse, no. 58. These data are consistent with the progressive differentiation of B cells in an immunodeficient state in the absence of clonal competition with B cells of similar antigen specificities as they populate the spleen. PMID- 7547709 TI - Identification and functional characterization of mouse CD29 with a mAb. AB - The beta 1 integrin subfamily, alternatively called very late activation antigen (VLA), has been implicated in various cellular functions. In this study, we generated a mAb against the mouse beta 1 subunit (CD29) to examine the functional property of mouse VLA proteins. After immunization with affinity-purified mouse VLA-4 (alpha 4 beta 1), a hamster mAb, HM beta 1-1, was established by screening mAb that reacted with alpha 4-negative neuroblastoma C1300. The antigen defined by HM beta 1-1 was widely distributed in various mouse cell lines and HM beta 1-1 immunoprecipitated a 110-120 kDa protein common to VLA-1 and VLA-6, indicating that HM beta 1-1 recognizes the beta 1 subunit of mouse integrins. We then examined the inhibitory effect of HM beta 1-1 on VLA-dependent cell adhesion and activation. HM beta 1-1 blocked the adhesion of mouse tumor cell lines to extracellular matrix proteins including collagen, laminin and fibronectin. Moreover, splenic T cell proliferation induced by anti-CD3 mAb and allogeneic mixed lymphocyte response were strongly inhibited by HM beta 1-1 in combination with an anti-LFA-1 mAb. We conclude that HM beta 1-1 reactive with mouse CD29 can inhibit VLA-dependent cellular functions and, thus, would be useful for studying the physiological role of beta 1 integrins in vivo. PMID- 7547710 TI - Cytokine dependence of V gamma 3 thymocytes: mature but not immature V gamma 3 cells require endogenous IL-2 and IL-7 to survive--evidence for cytokine redundancy. AB - It has previously been described that V gamma 3 cells can proliferate extensively in vitro in the presence of different cytokines. Here, the role of cytokines in the maintenance of V gamma 3 cells in the thymus has been determined. Culture of fetal thymocytes in cell suspension for 24 h showed that, whereas immature TCRlowHSAhigh V gamma 3 cells remained viable, all mature TCRhighHSAlow V gamma 3 cells died. These cells died by apoptosis since protein synthesis was required and flow cytometric analysis as well as DNA gel electrophoresis showed that the DNA was degraded to oligonucleosomal bands. Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetal thymocytes rescued V gamma 3 cells from dying. Addition of IL-1, IL-3, IL-5, IL-6, IL-9, TNF-alpha or IFN-gamma was without effect. Phenotypic analysis showed that the alpha-chain of the IL-2 receptor (IL 2R alpha) was expressed by part of the immature V gamma 3 thymocytes, all mature V gamma 3 cells expressed the beta-chain of the IL-2 receptor (IL-2R beta). Addition of anti-IL-2R beta mAb to fetal thymic organ culture (FTOC) resulted in a moderate reduction of the cell number of mature V gamma 3 thymocytes. Addition of anti-IL-2R alpha, anti-IL-4 or anti-IL-7 mAb had no effect. The cell number of mature V gamma 3 cells was highly reduced when both anti-IL-2R beta and anti-IL-7 mAb were added to FTOC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547711 TI - A common mutation in the hominoid class I A-locus IFN-responsive element results in the loss of enhancer activity. AB - Despite the observed coordinate expression of HLA-A and -B antigens in somatic tissues, there is growing evidence that the A and B class I genes are differentially regulated at the transcriptional level. Previous studies indicate that this may be related to locus-specific structural differences in certain enhancer elements. We have recently examined the 5' proximal regulatory regions of the A and B homologs in the higher non-human primates and found pronounced differences between the loci. Sequence analysis shows the B-promoters are more homogeneous, whereas the A-locus promoters are divergent among the various species examined. The differences in A- and B-promoters is exemplified by the regulatory element referred to as the IFN-responsive element (IRE). While the B locus IRE is conserved among all primates examined, the A-locus IRE are divergent and reveal different sequences in the human/chimpanzee, gorilla and orang-utan. In reporter gene bioassays, the B-locus IRE exhibited an enhancer activity in response to induction with IFN-beta and IFN-gamma. In contrast, all the variants of the A-locus IRE found in hominoid primates were unresponsive to IFN. One base substitution shared by all the primate IREs proved to be inactivating. These results provide a molecular basis of how duplicated gene loci encoding structurally and functionally similar antigen-presenting molecules may become differentially responsive to physiological stimulus. PMID- 7547712 TI - Sequence heterogeneity of murine acquired immunodeficiency syndrome virus: the role of endogenous virus. AB - A defective murine leukemia virus is the causative agent of murine acquired immunodeficiency syndrome (MAIDS). We have cloned cDNAs from both virus infected and non-infected cells using the PCR methods with primers corresponding to the franking sequence of the unique p12 gag gene. Sequence analysis of these cDNA clones revealed: (i) the presence of endogenous virus related to MAIDS virus in C57BL/6 mice, (ii) B cell lineage specific expression of endogenous virus and (iii) extensive heterogeneity of MAIDS virus recovered from virus infected cells due to the recombination of the related viruses (defective pathogenic virus, ecotropic virus and endogenous virus). These findings suggest that the creation of virus variants in infected cells may play an important role in virus pathogenesis and escape from immune attack during the development of MAIDS. PMID- 7547713 TI - Stability of Th1 and Th2 populations. AB - Using an in vitro model for the development of IFN-gamma-producing (Th1) and IL-4 producing (Th2) cells from CD4+ T lymphocytes expressing a transgenic TCR, we show that IL-12 and IL-4 are the most potent stimuli for the differentiation of naive T cells to effector populations. When combinations of cytokines are present during T cell priming, the effect of IL-4 is dominant. Furthermore, differentiated Th1 cells can be converted into IL-4 producers by exposure to IL 4, but the Th2 phenotype is not reversible. The stability of Th2 populations may limit the ability to regulate Th2-dominant responses in pathologic situations. PMID- 7547715 TI - Defects in the structure and expression of the genes for the T cell marker Rt6 in NZW and (NZB x NZW)F1 mice. AB - Rt6 is a T cell-restricted GPI-anchored membrane protein and a member of the family of mono(ADP-ribosyl)transferases. One of the two murine Rt6 genes is deleted in NZW mice. This finding is reminiscent of the deletion of one of the TCR beta genes in the same mouse strain and it is an intriguing possibility that these gene deletions arose by a common genetic mechanism. The Rt6 locus retained by the NZW mouse (designated Rt6-1) is polymorphic among inbred strains of laboratory mice. The NZW mouse shows several strain-specific restriction fragment length variants in this Rt6 locus and five amino acid substitutions occur in the predicted native Rt6 polypeptide of the NZW mouse relative to the corresponding polypeptides of NZB and BALB/c mice. Whereas transcript levels of the two Rt6 genes appear to be normal in spleen and intestine of NZB mice, the corresponding tissues of NZW mice show reduced levels of transcripts from the Rt6 locus retained in this mouse strain. Moreover, reduced levels of Rt6 mRNA also occur in spleen and intestine of (NZB x NZW)F1 hybrid animals, indicating that F1 animals have inherited a dominant factor from the genetic background of the NZW mouse, resulting in low levels of Rt6 expression. It is conceivable that the alterations in the Rt6 genes of the NZW mouse and/or the factor(s) affecting defective Rt6 expression constitute part of the genetic contribution of the NZW mouse to the autoimmune lupus-like disease in (NZB x NZW)F1 animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547716 TI - Effects of a static magnetic field on haemoglobin structure and function. AB - The exposure of mice to static magnetic fields (MF) of different strengths (1000 4000 G) for 10 min was studied. The effects of these magnetic fields on the function of haemoglobin (Hb), and its conformational stability, auto-oxidation kinetics, bioenergetics and viscosity, and those of different Hb derivatives were investigated. The fractions and concentrations of the inactive Hb pigments (such as methaemoglobin, carboxyhaemoglobin and sulfohaemoglobin) and the active Hb (in the HbO2 form) were determined using a newly developed multi-component spectrophotometric method. The direct effect of magnetic fields of relatively high strengths (3500 and 4000 G) led to different Hb conformations, accompanied by changes in intermolecular interactions represented by the slope of the eta sp/C = F(C) lines and Huggins' constant K', while no measurable change in the intrinsic viscosity [eta] of Hb was observed. These results indicate a lack of changes in the dimensions and shape of the Hb molecule. Study of the kinetics of oxyhaemoglobin auto-oxidation revealed decreases in the auto-oxidation reaction rate of 2-5.9% and 10-17%, under the effect of static MFs of strengths 1000-2500 G and 3500-4000 G, respectively. PMID- 7547714 TI - Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells. AB - Our recent studies on an autoantibody-transgenic mouse line demonstrated that peritoneal B-1 cells are responsible for autoimmune symptoms. However, whether B 1 cells in the peritoneum are generally involved in the pathogenesis of autoimmune disease remains controversial. To test the possible involvement of peritoneal B-1 cells in autoimmune symptoms of autoimmune-prone NZB mice, we eliminated the peritoneal cells by hypotonic shock with repeated i.p. injection of distilled water every 7 days into neonatal or 8-week-old NZB mice. By this treatment, B-1 cells, which self-renew within the peritoneal cavity, are expected to be preferentially eliminated, while other peritoneal cells can be easily supplied from bone marrows after this treatment. Indeed, in distilled water treated old NZB mice, the number of B-1 cells decreased in spleen as well as in lamina propria of the gut but the numbers of conventional B cells and T cells did not change. Moreover, the production of autoantibodies against erythrocytes significantly decreased and the occurrence of autoimmune hemolytic anemia was reduced in 12-month-old treated NZB mice. Similarly, the elimination of peritoneal cells of NZB/NZW (NZB/W) F1 mice by water injection decreased anti-DNA IgG antibodies in the sera and reduced the pathological changes of the kidney. These results suggest that peritoneal B-1 cells may be a source of autoantibody producing cells in autoimmune diseases of NZB and NZB/W F1 mice. PMID- 7547717 TI - Molecular studies of glycogen phosphorylase b from bovine liver. AB - Two active isoforms of bovine liver phosphorylase with distinct subunit composition have previously been purified (Camara Artigas, A., Baron, C. and Parody-Morreale, A. Prot. Express. Purif. 1994, 5, 157), one showing three SDS PAGE polypeptide bands (molecular mass = 97, 55 and 40 kDa) and the other showing just one (molecular mass = 97 kDa). A molecular mass of 200 kDa has been determined for the native enzymes by gel filtration. Amino acid analyses have been performed in both cases, giving the same results which are similar to those obtained with other phosphorylases. SDS-PAGE experiments at different concentrations of the three-band enzyme have suggested a 1:1:1 stoichiometry between the polypeptides. The pyridoxal-5'-phosphate site is located in the 55 kDa polypeptide and the phosphorylation site in the 40 kDa one. These polypeptides can be generated from the three-band enzyme by tryptic attack in the presence of glycogen without loss of enzyme activity. In the absence of glycogen, 55 kDa and 38 kDa polypeptides are generated, with a significant decrease in activity. We conclude that the three-band enzyme is a dimer composed of an intact monomer and a broken one. The lyotropic salt activation site of the enzyme is near the amino terminal group. PMID- 7547718 TI - FTIR and NMR studies on the hydration of a high-M(r) subunit of glutenin. AB - The hydration behaviour of a purified high-M(r) subunit of glutenin has been studied using Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. The water-insoluble protein was examined in an unalkylated form with intermolecular disulfide bonds, and in a reduced and alkylated (unpolymerized) form. Hydration produced a marked increase in chain mobility, especially above a threshold water content of about 37% w/w. NMR experiments also showed that some parts of the chain were held in a much less mobile state, even at higher water contents. Little difference could be seen between alkylated and unalkylated subunits, implying that NMR is sensitive to localized motions, but not to any restrictions imposed by disulfide bridges close to the chain ends. FTIR spectra of the protein films have shown that increasing hydration enables changes to occur in favour of a more extended and beta-sheet-type structure. The changes in secondary structure are very noticeable at water contents corresponding to the NMR mobility threshold. The behaviour is influenced by intermolecular interactions. beta-sheet formation is enhanced by the presence of disulfide bonds in the unalkylated samples. There is little evidence of beta structure (sheet or extended chain) either in the dry state, where protein protein interactions are strongest, or in dilute acetic acid solution, where the interactions are weakest. The balance between protein-protein and protein-water hydrogen-bonding interactions therefore appears to influence the formation of beta-sheet and extended chain structures, and these may in turn affect the elasticity of high M(r) subunits. PMID- 7547719 TI - The glass-transition behaviour of wheat gluten proteins. AB - The glass-transition behaviour of four hydrated wheat gluten proteins (alpha gliadin, gamma-gliadin, omega-gliadin and high-molecular-weight (HMW) subunits of glutenin) was studied using differential scanning calorimetry (DSC). By fitting the data to the Gordon-Taylor equation, which has previously been used to describe the plasticization of polymers by diluents, the glass-transition temperatures (Tg) for the dry proteins were found by extrapolation. The values for Tg were within the range 397-418 K. Values for the heat capacity increment delta Cp at Tg for the plasticized proteins were also determined and ranged from 0.29-0.47 J g-1 K-1 with no dependence on water content. The differences in glass transition behaviour of the proteins are discussed in relation to their secondary structure. PMID- 7547720 TI - Biosynthesis and characterization of hydroxybutyrate-hydroxycaproate copolymers. AB - Most polyhydroxyalkanoates (PHAs) reported to date fall into one of two broad classes: either hydroxybutyrate-hydroxyvalerate copolymers (typified by the PHA produced by Alcaligenes eutrophus), or hydroxyoctanoate-rich heteropolymers (typified by the PHA produced by Pseudomonas oleovorans). Few reports of copolymers rich in hydroxybutyrate (HB), but containing a minor proportion of a co-monomer with a higher carbon number than valerate, have appeared. Here we report on the biosynthesis and characterization of HB-rich polymers containing 2 4 mol% of hydroxycaproate (HC) units, as well as a terpolymer containing HC and hydroxyoctanoate (HO) units. These polymers were produced in good yield by Comomonas testosteroni, Bacillus cereus and an unidentified third organism when grown on caproate or octanoate. The minor co-monomers were found to be rejected from the PHB crystallites by X-ray analysis and by quantitative analysis of the melting point depression. The greatly reduced melting point, coupled with the retention of a high degree of crystallinity, could make these materials attractive as melt-processible thermoplastics. PMID- 7547721 TI - Spectroscopic studies of Manduca sexta and Sesamia nonagrioides chorion protein structure. AB - The secondary structure of Manduca sexta and Sesamia nonagrioides chorion proteins has been studied in intact chorions using laser-Raman and Fourier transform infra-red (FTIR) spectroscopy and in a solution containing extracted and reassembled chorion proteins using circular dichroism (CD) spectroscopy. Laser-Raman and IR spectra suggest the predominance of antiparallel beta-pleated sheet structure in intact chorion proteins of both Lepidoptera species. The bands at 1673, 1674 cm-1 (amide I) and 1234-1238 cm-1 (amide III) in the laser-Raman spectra can best be interpreted as resulting from abundant antiparallel beta pleated sheet structure. Analysis of the amide I band suggests that chorion proteins consist of 60-70% antiparallel beta-pleated sheet and 30-40% beta-turns. Supporting evidence for the prevalence of antiparallel beta-pleated sheet in chorion proteins was supplied using FTIR spectroscopy by the observation of a very intense absorption band at 1635 cm-1 (amide I) and of a weak band at 1530, 1525 cm-1 (amide II) from chorions of both species. Surprisingly, analysis of the CD spectra of extracted and reassembled chorion proteins suggests that, in solution, they retain a regular secondary structure most probably dominated by beta-pleated sheet. We therefore suggest that the prominent regular beta-sheet structure of chorion proteins may exist in solution and dictate the aggregation and polymerization process in vivo. PMID- 7547722 TI - Intermediate filament structure of alpha-keratin in baboon hair. AB - High-quality photographic recording of X-ray diffraction data from the hard alpha keratins has mainly been limited to that from porcupine quill. Earlier diffraction patterns of hair have shown very few reflections. In the present study, extensive sets of high-quality data have been obtained using a synchrotron source and an image plate detector, revealing information about the internal structure, both axial and lateral. The diffraction patterns of hair from six different baboons have been examined. The meridional axial diffraction data reveal evidence of the superposition of three and possibly four separate lattices. Despite considerable overlapping, sufficient reflections unique to each lattice have been observed. These lattices comprise an infinite one of 46.76 nm, together with finite lattices of 19.67, 27.20 and 7.27 nm. These patterns reveal information regarding the fibrillar structure of the hair fibres, namely that it consists basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. There is also an indication of the presence of another set of cylinders of smaller diameter, possibly comprising the microfibrils of keratin. PMID- 7547723 TI - Penetration of ciprofloxacin into bone: a new bioabsorbable implant. AB - The implantation of antibiotic-containing cement beads became standard adjuvant local antibiotic therapy of chronic osteomyelitis. The new developed bioabsorbable drug carrier system of polyglycolic acid with the antibiotic Ciprofloxacin was tested in vitro and in vivo. The goal of this study was to determinate the penetration depth of Ciprofloxacin into bone cortex and marrow. Two monofil, PGA cylinders, 3.2 x 5 mm in size, containing 3 mg of Ciprofloxacin each, were implanted into the proximal part of both femora of 18 rabbits (New Zealand white). After sacrifice, the concentration of Ciprofloxacin in micrograms/g cortex and marrow was measured with high-performance liquid chromatography in relation to the distance from the test material at day 2, as well as at 1, 2, 3, 4, and 6 weeks postimplantation. For a distance up to 5 mm, marrow levels of the drug exceeded cortical levels at day 2 (5000 to 240 micrograms/g). At a distance of 5-10 mm, cortex levels were similar to marrow levels after 2 weeks and were higher than marrow levels at week 3. This observation could be made at a distance between 10 and 15 mm only after 2 days. Later, marrow concentrations again exceeded that in cortex. At a distance of more than 15 mm, antibiotic levels were low and approximated. After 6 weeks, at 5 mm distance, a bactericidal drug concentration of about 2 micrograms/g in bone marrow could be measured. Drug penetration into cortical bone in bactericidal concentrations of about 2 micrograms/g was achieved at up to 30 mm in the first few days.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547725 TI - Novel antiadhesion barrier does not prevent anastomotic healing in a rabbit model. AB - Intraperitoneal adhesions following surgical procedures cause considerable morbidity. Hyaluronic acid/carboxymethylcellulose (HA/CMC) films have been shown to be effective agents in decreasing adhesion formation. However, when there is an inadvertent leak of bowel contents into the peritoneum due to incomplete anastomosis, adhesion formation about a defect in order to prevent further leakage and to promote healing of the wound is important for the prevention of morbidity and mortality. The purpose of this study was to determine if an antiadhesion film (HA/CMC) impairs these potentially beneficial adhesions to bowel anastomoses, thus predisposing them to enteric leaks with subsequent peritonitis. Sixty-four rabbits were divided in two groups, each undergoing a complete or partial (90% anastomosis to simulate anastomotic leak) large bowel anastomosis. Half of each of the above groups were treated by wrapping a HA/CMC film over the anastomosis and the other half were untreated controls. These two subgroups were then further divided equally and sacrificed at either 7 or 14 days for evaluation of anastomosis integrity and strength. The average anastomotic bursting pressures did not change significantly between those groups treated with HA/CMC when compared to untreated controls at 7 or 14 days or in the complete or partial anastomosis group (Student's t test). Adhesion formation to the anastomosis was not impaired in either group independent of HA/CMC film application. This study suggests that while HA/CMC film has been shown to decrease adhesions in other models, healing of a rabbit colonic anastomosis even in the presence of an anastomotic defect takes place, further suggesting that the stimulus for adhesion formation can overcome the antiadhesion properties of HA/CMC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547724 TI - Longitudinal forces acting at side-to-end and end-to-side anastomoses when a knitted polyester arterial prosthesis is implanted in the dog. AB - In a previous study, which investigated the tensions at the proximal and distal end-to-end anastomoses of a vascular prosthesis, no significant differences were found in the longitudinal forces between the two anastomotic sites after different periods of implantation. The present follow-up study was devised to study the longitudinal forces at a proximal side-to-end and a distal end-to-side anastomosis using a warp knitted polyester prosthesis implanted as a canine thorocoabdominal bypass. The external surface of the prosthesis contained a specially printed "tension indicator" design, which enabled changes in length to be assessed photographically at the two anastomoses at the time of implantation and at sacrifice. The longitudinal force at each site was then calculated using a relationship obtained experimentally between the total longitudinal force and the changes in length of the virgin graft in vitro. The in vitro measurements on the prosthesis were performed using a computer-controlled laser calibration system. Although the results showed a tendency toward a gradual loss in longitudinal force at both anastomoses over 7 months in situ, statistical analysis showed no significant difference in the longitudinal force at the two anastomotic sites after any period of implantation. Histopathological and textile analysis also showed that the characteristics of the prostheses were similar at both anastomotic sites. This does not mean, however, that the intramural stress concentrations experienced by the two sites were the same. While comparing the level of the longitudinal forces measured in this study with those observed in the previous thoracic aorta study using end-to-end anastomoses, no significant differences were observed due to the different types of anastomoses. It would appear that the longitudinal force is not one of the main etiological factors that cause the formation and localization of anastomotic failure. PMID- 7547726 TI - Comparative efficacy of nonsteroidal anti-inflammatory drugs and anti-thromboxane agents in a rabbit adhesion-prevention model. AB - A variety of nonsteroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit postsurgical peritoneal adhesion formation in a number of animal models. A rabbit uterine horn adhesion model was used to directly compare several commonly used NSAIDs of different chemical classes in a single animal study to evaluate their ability to prevent adhesion formation. The effect of thromboxane inhibitors on adhesion prevention was also evaluated. Each of the NSAIDs tested (tolmetin, ibuprofen, aspirin, and indomethacin) showed significant and comparable efficacy. In this same study, imidazole, a thromboxane synthetase inhibitor, also showed significant efficacy. In a second study, ridogrel, an inhibitor of thromboxane synthetase as well as a thromboxane A2 receptor blocker, also showed significant efficacy in reducing peritoneal adhesion severity. These results further support the view that NSAIDs act to prevent adhesions through a common mechanism. In addition, thromboxane A2 inhibitors were also shown to be efficacious in adhesion prevention, suggesting that platelets may play a substantial role in adhesion formation. PMID- 7547727 TI - Noninvasive diagnosis of cardiac allograft rejection: the effect of procainamide. AB - The surface electrocardiogram (ECG) has been used as a noninvasive technique for the diagnosis of cardiac allograft rejection. Alteration in conduction, R-wave amplitude, and rhythm have been associated with rejection. These ECG findings are modulated by the myocyte sodium channel, but are inconsistent and occur only during severe rejection episodes. The purpose of this study was to (1) characterize changes in cardiac electrophysiology during allograft rejection using the highly sensitive intramyocardial electrocardiogram and (2) determine whether pharmacological sodium channel blockade with procainamide enhances subtle ECG changes. Nine mongrel dogs underwent heterotopic heart transplantation in which four intramyocardial leads (one anteriorly and posteriorly on each ventricle) were attached. Leads exited to a subcutaneously placed ECG block which was transcutaneously accessed posttransplant to record direct intramyocardial electrocardiograms. Six animals were treated with procainamide, while three were not and served as controls. Daily measurements included the QRS, QT, and QTc intervals and the R-wave amplitude. Endomyocardial biopsies were performed weekly and also when significant decline in ECG amplitude occurred. Detailed ECG interval analysis failed to establish any correlation between conduction and rejection, even in the procainamide-treated group. Intramyocardial amplitude analysis, however, had a sensitivity of 100% and a specificity of 86% for the diagnosis of rejection. The results indicate that intramyocardial ECG interval analysis is not predictive of rejection even when prolonging conduction with procainamide. Amplitude analysis, however, remains an accurate noninvasive means for the early detection of cardiac allograft rejection and should allow more selective use of endomyocardial biopsy. PMID- 7547728 TI - A model of unilateral pulmonary lobar transplantation. AB - Unilateral lung transplantation has become an accepted treatment for patients with end-stage pulmonary disease. Donor shortage, however, is a major limitation, with up to 87% of patients dying of their pulmonary disease while awaiting transplantation. This is especially true in neonatal and pediatric patient populations. The use of organ segments from cadaveric or living donors may provide a solution. The purpose of this study, therefore, was to evaluate the function and hemodynamic response to pulmonary lobar transplantation using a swine model. Five transplants were performed for acute study, while 10 were performed for 6-week survival. The left lower lobe was harvested from a 70- to 75 kg donor animal. The lobe was then transplanted into a 20 to 25-kg recipient following left pneumonectomy. Graft function was determined by pulmonary arterial and venous blood gas analysis. Cardiac output, pulmonary pressure, and pulmonary vascular resistance were measured under two experimental conditions: (1) baseline and (2) with the right pulmonary artery occluded, forcing the entire cardiac output through the lobar graft. All grafts showed excellent acute and long-term function with regard to gas exchange. The lobar grafts, however, were characterized by high pulmonary vascular resistance both acutely and 6 weeks post transplant. Contralateral pulmonary artery occlusion resulted in hemodynamic instability and right heart failure. No animal was able to be solely supported by the lobar transplant for more than one hour. These results have prompted a bilateral lobar transplant model and current studies are in progress. PMID- 7547729 TI - Doderlein's bacillus: friend or foe? PMID- 7547730 TI - The natural history of vulvar intraepithelial neoplasia. PMID- 7547733 TI - Audit of 114 non-neoplastic vulvar biopsies. AB - OBJECTIVE: The purpose of this study was to show the benefits and limitations of vulvar biopsy in the setting of a multidisciplinary clinic specialising in non neoplastic diseases of the vagina and vulva. DESIGN: One hundred and fourteen vulvar biopsies were reviewed and classified according to the classification of the International Society for the Study of Vulvar Diseases. RESULTS: The histological diagnoses were lichen sclerosus 25%, lichen simplex chronicus 35%, non-erosive inflammatory dermatoses comprising psoriasis, spongiotic dermatitis, dermatophytosis and psoriasiform dermatitis 13%, erosive vulvitis and lichen planus 9%, nonspecific inflammation 6%, miscellaneous 9% and normal 4%. CONCLUSIONS: Biopsies in cases of lichen sclerosus were useful for confirmation of clinical diagnosis and to exclude early invasive malignancy. In lichen simplex chronicus, biopsies helped exclude an underlying dermatosis requiring specific treatment. Psoriasis, spongiotic dermatitis, dermatophytosis and excoriated lichen simplex chronicus posed a common clinical differential diagnosis of the reddened vulva. The eroded vulva often proved a diagnostic problem clinically and histologically. The clinical syndrome of vestibulitis did not have a specific histological picture, and biopsies showed nonspecific inflammation, mild hyperplasia or were normal. No case of squamous cell hyperplasia was diagnosed and the place of this diagnosis in the ISSVD classification needs review. PMID- 7547731 TI - Diagnosis, prevention and management of ovarian hyperstimulation syndrome. AB - The overall incidence of clinically important (moderate to severe) OHSS ranges from 1% to 10% of IVF cycles, but only a small proportion (0.5% to 2%) of the cases are severe. In extreme but rare cases, secondary complications such as deep vein thrombosis, respiratory distress and acute hepato-renal failure may occur. The main risk factors are the presence of polycystic ovaries, high ovarian response to superovulation therapy, the use of hCG to trigger the ovulatory process or for luteal phase support, and the endogenous production of hCG by an early pregnancy. The pathogenesis of OHSS is unknown, although the predominant biochemical mediator is thought to be the renin-angiotensin system. Ovarian stimulation should always be carefully monitored to identify those women at risk. In IVF cycles, the hCG injection should be withheld if the risk is judged to be too great. Some women will benefit from a policy of proceeding to collect oocytes, but electively cryopreserving any resulting embryos, thus allowing the ovarian stimulation cycle not to be wasted. The administration of albumin at the time of oocyte collection will reduce the chance of severe OHSS occurring. If a decision is made to proceed with oocyte recovery and embryo transfer, it may be advisable to give 5000 IU of hCG, rather than 10,000 IU, as the ovulatory trigger. Progesterone, and not hCG, should be given in the luteal phase. Women developing mild or moderate OHSS should be kept under outpatient surveillance to detect the minority that may progress to severe OHSS. Those with severe OHSS should be hospitalised for fluid and electrolyte management. Paracentesis under ultrasound guidance is recommended where there are tense ascites, but further surgical intervention should rarely be undertaken and only when there is good clinical evidence of ovarian torsion or haemorrhage. PMID- 7547732 TI - The chronically symptomatic vulva: aetiology and management. AB - OBJECTIVE: To determine the causes and management of chronic vulval symptoms and to compare the findings in patients first presenting to a gynaecologist with those in patients first presenting to a dermatologist. DESIGN: A prospective study of 144 patients, approximately half each being referred to a gynaecologist and a dermatologist. Diagnosis was based on clinical history, vulvoscopy, vulval biopsy and bacteriology. Biopsies were examined by a histopathologist experienced in dermatopathology and gynaecological pathology. RESULTS: The two patient groups were similar in both range and frequency of conditions. The commonest cause of chronic vulval symptoms was dermatitis, which was found in 64% of our patients. Dermatitis occurred alone in 55% and was found in association with histological evidence of human papilloma virus (HPV) in a further 9%. These patients responded to simple dermatological methods, mainly topical corticosteroids. Histopathological evidence of HPV was encountered in only 23% of our patients, and of these 36% also demonstrated dermatitis on biopsy. Most responded to topical corticosteroids. Another 7% had lichen sclerosus, and all responded to potent topical corticosteroid. The remaining 15% demonstrated a range of diagnoses, including psoriasis, dysaesthetic vulvodynia, vulval intraepithelial neoplasia (VIN) and chronic candidiasis. The majority of patients had a corticosteroid responsive dermatosis rather than a gynaecological condition. CONCLUSIONS: The majority of patients with a chronically symptomatic vulva who present to either a gynaecologist or a dermatologist have a dermatological condition that responds to simple dermatological treatments. We believe that the presence or absence of the human papilloma virus is not relevant to most patients with a chronically symptomatic vulva and treatments should not be aimed at eradicating this virus. Histopathologists and gynaecologists who have focused on gynaecological disorders have often missed simple dermatological conditions that are easily treatable. PMID- 7547735 TI - The management of recurrent genital herpes infection in pregnancy: a postal survey of obstetric practice. AB - OBJECTIVE: To determine clinical practice amongst obstetricians in the UK in the antepartum and intrapartum management of pregnant women with recurrent genital herpes infection. METHODS: All Members and Fellows of the Royal College of Obstetricians and Gynaecologists resident in the UK were sent a questionnaire requesting information concerning their management of pregnant women with recurrent genital herpes infection. RESULTS: There was a 76% response rate to the questionnaire. Of the 1201 obstetricians who responded, only 369 (31%) admitted to having a formal policy governing the management of herpes in pregnancy within their unit. However, regular screening was advocated by 718 (60%), of whom 463 (64%) performed regular antenatal swabs for viral culture. At the time of presentation in labour 974 obstetricians (81%) routinely examined the genitals for evidence of a recurrence. When asked in what circumstances caesarean section would be considered an appropriate method of delivery in women with genital herpes infection, 1107 (92%) felt that visible active lesions at the time of labour was sufficient. However, when the membranes had been ruptured for more than four hours in the presence of genital lesions, only 678 (56%) considered this an indication for caesarean section. Caesarean section was more likely to be considered appropriate in this situation by obstetricians who performed antenatal screening (chi 2 = 30.38, P < 0.0001). Five hundred and ninety-six obstetricians (50%) felt that a positive viral culture obtained at antenatal screening from the most recent occasion prior to presentation in labour was an indication for caesarean section, although of this group 192 (32%) said they did not perform antenatal screening by viral culture. The reporting of a recurrence by the patient without visible evidence of disease was considered an appropriate indication for caesarean section by 438 respondents (36%). Maternal request for caesarean section regardless of recurrences at delivery was considered an acceptable indication for operative delivery by 745 obstetricians (62%). CONCLUSIONS: 1. There seems to be little agreement amongst obstetricians in the UK regarding the management of recurrent genital herpes infection in pregnancy. 2. The management possibilities are reviewed and suggestions are made for a more cohesive approach to the problem. PMID- 7547734 TI - Management of female genital mutilation: the Northwick Park Hospital experience. AB - OBJECTIVE: To outline the problems associated with female genital mutilation and to highlight the need for deinfibulation before delivery. DESIGN: A review of women attending a newly established African Well Woman Clinic. Age at infibulation, gravidity of clinic attenders and adequacy of introitus for management of labour were assessed. SETTING: Northwick Park Hospital, Harrow, Middlesex. SUBJECTS: Fifty women attending a newly established African Well Woman Clinic, of whom 13 were nulliparous, 14 were primigravid and 23 were multigravid. RESULTS: The average age at which infibulation had occurred was 6.7 years. At the time of clinic attendance the mean age of pregnant and nonpregnant patients was 26 and 23.3 years, respectively. Of the 14 primigravid patients, only 50% had an adequate introitus to allow management of the first and second stages of labour. Five had deinfibulation performed antenatally or at delivery. Ninety-three percent of the primigravid patients and 74% of the multigravid patients had a vaginal delivery. CONCLUSIONS: We believe that the Northwick Park Hospital management policy for infibulated women closely mirrors the cultural practices in Somalia. The policy also improves obstetric management of infibulated patients. Twenty-six percent of referrals were of nonpregnant women, and this practice is to be encouraged. PMID- 7547736 TI - Selective miscarriage of Down's syndrome fetuses in women aged 35 years and older. AB - OBJECTIVE: To estimate the fetal loss of Down's syndrome fetuses between the time of chorionic villus sampling (10 weeks gestation) and the time of amniocentesis (16 weeks gestation) and term in women aged 35 years and older. DESIGN: The age specific prevalence rates in the first trimester of Down's syndrome were estimated using the Danish cytogenetic register in combination with results from four published studies. These were compared with the reported prevalence at the time of amniocentesis and at birth. SUBJECTS: 5927 singleton pregnancies undergoing chorionic villus sampling (71 cases of Down's syndrome and 5856 unaffected cases). This was combined with published data on a further 231 cases of Down's syndrome and 16,620 unaffected cases. MAIN OUTCOME MEASURES: Age specific prevalences at the time of chorionic villus sampling. Proportion of pregnancies lost between the time of chorionic villus sampling and the time of amniocentesis and term. RESULTS: Thirty-two percent of Down's syndrome pregnancies are lost between the time of chorionic villus sampling (10 weeks) and the time of amniocentesis (16 weeks) and 54% are lost by term. CONCLUSIONS: The high fetal loss rates of Down's syndrome between the time of chorionic villus sampling and term introduce problems when evaluating first trimester screening tests with respect to their effective detection rates at term. A recommendation for quoting term risks is made. PMID- 7547737 TI - Transabdominal cervicoisthmic cerclage in the management of recurrent second trimester miscarriage and preterm delivery. AB - OBJECTIVE: To evaluate the role of transabdominal cervicoisthmic cerclage (TCC) in the management of recurrent mid-trimester pregnancy miscarriage and preterm delivery. DESIGN: An observational study of 50 women with history of recurrent second trimester miscarriage and preterm delivery in whom TCC was performed. SETTING: A tertiary referral centre for high risk obstetrics. RESULTS: TCC was performed on 51 occasions in 50 women. One had TCC performed in two successive pregnancies. Prior to the procedure, they had experienced 36 first trimester miscarriages and 163 later pregnancy losses. These included 152 midtrimester miscarriages and 11 neonatal deaths after preterm delivery. There had been 4 survivors after preterm delivery. The total number of pregnancies after TCC was 61 with three women losing one pregnancy and subsequently having a successful pregnancy. Of these women 44 now have had a successful outcome. Six have not had a successful outcome. Eight women have had two successful consecutive pregnancies after TCC. The fetal survival was 85.2% post-procedure. CONCLUSIONS: TCC is a procedure that may be used in a highly selected group of women with anatomical defects of the cervix and previous failed transvaginal cerclage resulting in recurrent second trimester pregnancy loss and preterm birth. Strict selection criteria were applied as TCC is an invasive procedure. Although TCC is not widely used our study suggests it may be the procedure to consider in women who have had recurrent second trimester pregnancy losses and preterm births associated with cervical damage. PMID- 7547738 TI - Elaboration of stem villous vessels in growth restricted pregnancies with abnormal umbilical artery Doppler waveforms. AB - OBJECTIVE: To assess the elaboration of placental stem villous vessels from pregnancies complicated by intrauterine growth restriction (IUGR) with absent end diastolic flow velocity detected prior to delivery in the umbilical artery. DESIGN: Comparison between IUGR and control groups of the distribution, in 15 microns increments of 600 randomly chosen stem vessel profiles (post-fixation diameter 10-160 microns) identified by immunohistochemical localisation of alpha smooth muscle actin in the vessel media. SETTING: Clinical teaching hospital and university anatomy department. SUBJECTS: Paraffin-fixed blocks obtained from placentas of eight pregnancies complicated by IUGR and eight gestational age matched controls. RESULTS: The distribution of the stem villous vessels in the IUGR placentas, as assessed by the mean vessel diameter in each case, did not differ from the controls (mean vessel diameter 31.8 microns [SD 2.4] vs 29.6 microns [2.3]; P = 0.13). In five IUGR cases alpha-smooth muscle actin positive cells (myofibroblasts) were identified within the stroma of nonmuscularised peripheral (mature intermediate and terminal) villi, but in none of the controls. CONCLUSIONS: Our data do not support the theory that IUGR with absent end diastolic flow velocity in the umbilical artery is due to a selective loss of small stem villous vessels. The increased impedance in this condition may be conferred more distally within the nonmuscularised capillaries of the peripheral villi. PMID- 7547739 TI - Obstetric antecedents of surgically treated obstetric brachial plexus injuries. AB - OBJECTIVE: To determine whether the anatomy of an obstetric brachial plexus lesion (OBPL) is causally related to the preceding obstetric history. DESIGN: Anatomical classification of the OBPL during reconstructive neurosurgical treatment in consecutive infants who had surgery for OBPL between 1986 and 1994 and relating these findings with the characteristics of the preceding birth. SETTING: De Wever Hospital, Heerlen, The Netherlands. SUBJECTS: All infants who had surgical treatment for OBPL between 1 April 1986 and 1 January 1994 (n = 130). RESULTS: An Erb's C5-C6 injury was preceded more frequently by a difficult breech birth (19/26 cases or 73%). In contrast, the more extensive forms of Erb's palsy classified as a C5-C7 injury or a total palsy with a C5-T1 injury were observed more frequently after complicated cephalic birth (52/59 or 88%, and 43/45 or 96%, respectively). The extent of anatomical damage as expressed by the incidence of an avulsion of one or more spinal nerves was 18/26 (69%) in Erb C5 C6, 13/59 (22%) in Erb C5-C7 and 21/45 (47%) in total C5-T1 palsy. CONCLUSION: The Erb's C5-C6 palsy, occasionally bilateral and/or complicated by phrenic nerve injury, was the most frequent form of OBPL after a breech birth. The more extensive form of Erb's palsy and the total palsy were observed more frequently after delivery in a cephalic presentation. The pure form of Erb's palsy and the total palsy were characterised by a higher incidence of nerve avulsions than the extensive form of Erb's palsy. PMID- 7547740 TI - Sonographic, stereological and Doppler flow velocimetric assessments of placental maturity. AB - OBJECTIVE: To determine whether sonographic assessments of increasing placental maturity are associated with changes in the theoretical diffusing capacity of the organ and with changes in vascular resistance. DESIGN: Placental maturity was assessed sonographically in 10 organs: five were classified as completely immature and five as completely mature. Doppler flow velocimetric recordings were obtained shortly before delivery performed by elective caesarean section. Placental biopsies taken immediately after delivery were analysed stereologically. RESULTS: No differences were observed in either placental weight or volume, but the morphometric diffusing capacity of the villous membrane was 40% higher in the mature than in the immature placentas. Doppler indices did not alter with increasing placental maturity. CONCLUSIONS: Normal term placentas differ significantly in their diffusion characteristics, even when organ weight and volume remain constant. Umbilical vascular resistance may be heavily influenced by the number of parallel circulatory units offered by the placental lobules. PMID- 7547741 TI - The mortality and morbidity associated with umbilical cord prolapse. AB - OBJECTIVE: To examine the management of cord prolapse and its morbidity and mortality. DESIGN: Retrospective study of consecutive babies born after cord prolapse, identified using the Oxford Obstetric Data System, and those with registered handicap, identified by the Oxford Region Register of Early Childhood Impairments. SETTING: District maternity hospital managing more than 6000 deliveries annually. SUBJECTS: One hundred and thirty-two babies born after the identification of cord prolapse in the John Radcliffe Hospital between January 1984 and December 1992. MAIN OUTCOME MEASURES: Survival rates, condition at birth assessed by Apgar scores at 1 and 5 minutes and blood gas values on cord blood samples, and incidence of major handicap at three years of age. RESULTS: The incidence of cord prolapse was 1 in 426 total births. There were six stillbirths and six neonatal deaths. One baby died as a result of birth asphyxia. The uncorrected perinatal mortality rate was 91 per 1000. Of 120 survivors, only one baby was known to suffer a major neurological handicap. Electronic cardiotocographs aided the diagnosis of cord prolapse in 41% of cases. Apgar scores were better with a shorter diagnosis to delivery interval, but cord gas results did not correlate well with Apgar scores or the diagnosis to delivery interval. CONCLUSIONS: Cord prolapse occurs with a relatively stable incidence in this population irrespective of changes in obstetric practices. Despite the high incidence of ominous cardiotocographs, low Apgar scores and acidaemia on blood gas analysis, the fetal outcome is not as poor as might be expected and mortality is predominantly attributable to congenital anomalies and prematurity rather than birth asphyxia. PMID- 7547742 TI - Pregnancy ultrasound and childhood cancer: a second report from the Oxford Survey of Childhood Cancers. PMID- 7547743 TI - Misoprostol in the management of spontaneous abortion. PMID- 7547744 TI - Progestogen therapy and prevention of functional ovarian cysts during pituitary desensitisation with GnRH agonists. PMID- 7547745 TI - Intra-vesical foreign body: an unusual cause. PMID- 7547746 TI - Follow up and outcome of isthmic pregnancy located in a previous caesarean section scar. PMID- 7547747 TI - Expectant management of twin pregnancy with single fetal death. PMID- 7547748 TI - Local research ethics committees: hindrance or help? PMID- 7547749 TI - Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. PMID- 7547750 TI - Neonatal respiratory morbidity and mode of delivery at term: influence of timing of elective cesarean section. PMID- 7547751 TI - Neonatal respiratory morbidity and mode of delivery at term: influence of timing of elective caesarean section. PMID- 7547752 TI - In vitro fertilisation and gamete intrafallopian transfer: an integrative analysis of research, 1987-1992. PMID- 7547753 TI - Uterine rupture during therapeutic abortion in the second trimester using mifepristone and prostaglandin. PMID- 7547754 TI - Mouth operated mucus extractors: safe to use? PMID- 7547755 TI - Changes in the male to female ratio at different stages of life. PMID- 7547756 TI - The current use of exogenous surfactants in the newborn. PMID- 7547757 TI - The extremely preterm infant. PMID- 7547758 TI - A multicentre comparative study of 17 experts and an intelligent computer system for managing labour using the cardiotocogram. AB - OBJECTIVES: To investigate 1. whether an intelligent computer system could obtain a performance in labour management comparable with experts when using cardiotocograms (CTGs), patient information, and fetal blood sampling and 2. whether experts could be consistent and agree in their management of labour. SUBJECTS: An intelligent computer system and 17 clinicians experienced in fetal monitoring from 16 centres in the UK. DESIGN: Fifty cases with complete intrapartum CTGs and clinical data were reviewed by each expert and the system independently on two occasions, at least one month apart. Each CTG was scored in 15 min segments according to a protocol and estimates of the cervical dilatation and fetal scalp blood pH were given when requested. MAIN OUTCOME MEASURES: Consistency and agreement in the recorded scores, agreement and timing of cases recommended for caesarean sections, fetal blood sampling rates, intervention in cases with poor outcome and intervention in cases with good clinical outcome. RESULTS: The system: 1. Agreed with experts well and significantly better than chance (67.33%, kappa = 0.31, P << 0.001). 2. Was highly consistent (99.16%, kappa = 0.98, P << 0.001) when used by two operators independently. 3. Recommended no unnecessary intervention in cases with normal delivery and good condition (cord artery pH > 7.15, vein pH > 7.20, 5 min Apgar > or = 9 and no resuscitation). This was better than all but two of the experts. 4. Recommended delivery by caesarean section in 11 cases; at least 15 of the 17 experts in each review also recommended caesarean section delivery in these cases. The majority did so within 15 min of the system and two-thirds did so within 30 min. 5. Identified as many of the birth asphyxiated cases (cord arterial pH < 7.05 and BDecf > or = 12, and Apgar score at 5 min < or = 7 with neonatal morbidity) as the majority of experts and one more than was acted upon clinically. The experts were found to be consistent and to agree. There was good agreement in the cases and the timing of caesarean section recommendations. The majority of experts did not recommend operative intervention in cases which had a normal delivery and good outcome, but did recommend operative interventions in 10 of 12 cases delivered with cord arterial pH < 7.05. However, in one of the cases delivered with birth asphyxia, 14 of the 17 experts and the system failed to recommend intervention. CONCLUSIONS: The system's performance was found to be indistinguishable from the experts' in the 50 cases examined, but it was more consistent. This demonstrates the potential for an intelligent computer system to improve the interpretation of the CTG and decrease intervention. Furthermore, the good performance of most experts in this study demonstrates the potential effectiveness of the CTG and raises important questions regarding why the CTG has fallen short of expectations in current practice. PMID- 7547759 TI - Blues and depression during early puerperium: home versus hospital deliveries. AB - OBJECTIVE: To investigate whether women who give birth at home are less prone to mood disturbances during the early puerperium than those who give birth in hospital. DESIGN: A prospective study of 303 pregnant women who registered for antenatal care. SETTING: The antenatal clinic at St Joseph's Hospital, Veldhoven, The Netherlands, and five antenatal consultation programmes of local midwives working in the surrounding region. SUBJECTS: Three hundred and eighty-two consecutive caucasian women registering for antenatal care were approached. Of these, 303 consented to participate and 293 completed the study. MAIN OUTCOME MEASURE: The predictor variable was the way in which the women gave birth: spontaneous vaginal parturition at home or in hospital as follows: spontaneously; vaginal parturition after stimulation with medication; vaginal parturition with forceps/vacuum extraction; or caesarean section. The outcome variables were blues and depression. The occurrence of blues was assessed at 4 weeks postpartum, using Pitt's criteria. The occurrence of depression was assessed at 4 weeks postpartum using the Research Diagnostic Criteria. The possible confounding effects of a set of obstetrical and psycho-social variables relating to the early puerperium were investigated using logistic regression analysis. RESULTS: Of the 293 women who completed the study, 52% gave birth at home. Significantly more nullipara gave birth in hospital. Parturition occurred where it had been planned in 77% of women; referral occurred later on in pregnancy in 11% and during labour in 12%. Nullipara had to be referred significantly more often than multipara. In general, there was no difference in the incidence of blues and depression between women who gave birth at home and those who gave birth in hospital. Obstetric factors were not related to the occurrence of blues or depression in the early puerperium. CONCLUSIONS: Women who gave birth in hospital are no more prone to postpartum mood disturbances, such as blues and depression, than women who give birth at home. PMID- 7547760 TI - HLA-G deletion polymorphism and pre-eclampsia/eclampsia. AB - OBJECTIVE: To investigate a HLA-G deletion polymorphism in pre-eclamptic pedigrees and the general population. DESIGN: A population association study of HLA-G genotypes from pre-eclamptic/eclamptic patients and control groups. SETTING: Analyses undertaken in the School of Biological Sciences, Macquarie University. Patients were from Royal Women's Hospital, Melbourne, and controls were from Westmead Hospital and Macquarie University, Sydney. SUBJECTS: One hundred and ninety-six individuals, consisting of 29 pre-eclamptic/eclamptic (PE/E) patients, 13 individuals born of a PE/E pregnancy, 46 blood relatives of PE/E patients, 21 husbands of PE/E patients, 25 women normotensive in first pregnancy, 15 husbands of women normotensive in first pregnancy and 47 staff and students of Macquarie University. RESULTS: Genotypic and gene frequencies were not significantly different in the seven groups examined. CONCLUSION: There is no detectable relationship between susceptibility to pre-eclampsia or being born of a pre-eclamptic pregnancy and HLA-G genotype. PMID- 7547761 TI - Nitric oxide synthase activities in placental tissue from normotensive, pre eclamptic and growth retarded pregnancies. AB - OBJECTIVE: To measure nitric oxide synthase activity in tissues from the placenta, placental bed and umbilical cord at delivery in normal and complicated pregnancies. DESIGN: A prospective blinded study. SETTING: The obstetric departments of three London teaching hospitals. SUBJECTS: Samples of whole placenta, dissected stem villous arteries, umbilical cord vessels and the placental bed of the uterus were collected at delivery and assayed for nitric oxide synthase activity. Samples of placenta were taken from ten normotensive, six pre-eclamptic and eight growth retarded pregnancies, and stem villous arteries from a further seven normotensive pregnancies. RESULTS: There was minimal placental bed nitric oxide synthase activity in each group. Placental villous homogenates from pregnancies complicated by pre-eclampsia and fetal growth retardation had significantly lower activities of nitric oxide synthase than those from normotensive women with appropriately grown babies. There were no significant differences in calcium dependent or calcium independent nitric oxide synthase activities in the umbilical vein and artery in the normal or in the pre eclamptic groups. However, there was significantly more calcium dependent than calcium independent nitric oxide synthase in the umbilical veins in all groups. CONCLUSIONS: Local nitric oxide production in the placental bed of the uterus is unlikely to contribute substantially to the low resting vascular tone in the uteroplacental circulation. However, a relative deficiency of placental nitric oxide in pregnancies complicated by fetal growth retardation and pre-eclampsia may contribute to the development of the high impedance fetoplacental circulation found in these conditions. PMID- 7547762 TI - Presentation and management of choriocarcinoma after nonmolar pregnancy. AB - OBJECTIVE: To ascertain the mode of presentation and treatment outcome for women with choriocarcinoma after a nonmolar pregnancy. DESIGN: Retrospective analysis of case records between 1985 and 1994. SETTING: A referral centre for trophoblastic disease. SUBJECTS: One hundred women with choriocarcinoma: 62 after a live birth, six after a live birth preceded by a molar pregnancy and 32 after a nonmolar abortion. RESULTS: Choriocarcinoma after nonmolar pregnancies represent 17% of the total gestational trophoblastic tumours requiring treatment. Vaginal bleeding was the commonest symptom in all groups, but symptoms from metastatic disease were important in the group presenting after a live birth. Metastatic disease was present in 31% of cases after live birth and 43% post-abortion. The median interval between the antecedent pregnancy and choriocarcinoma was five and six months, respectively. High risk multi-agent chemotherapy was required in 82% and 60% of cases, respectively. The mortality rate was significantly higher after a live birth than a nonmolar abortion (21% vs 6%). CONCLUSIONS: Treatment of choriocarcinoma after a live birth is associated with an unacceptably high mortality rate. Vaginal bleeding is an important early symptom and a pregnancy test should be performed if it persists after usual medical treatment. PMID- 7547763 TI - A comparison of colour Doppler sonography and the pelvic arteriogram in assessment of patients with gestational trophoblastic disease. AB - OBJECTIVE: To assess whether colour Doppler sonography can replace pelvic arteriography in the assessment of patients with gestational trophoblastic disease. DESIGN: An observational study in which patients with gestational trophoblastic disease were assessed with both pelvic arteriography and colour Doppler sonography. SETTING: The oncology unit of the Department of Obstetrics and Gynaecology, University of Hong Kong. The unit is the main tertiary referral centre for treatment of trophoblastic diseases in Hong Kong. SUBJECTS: Thirty-two consecutive women referred over an 18 month period. MAIN OUTCOME MEASURES: The site of localisation of the tumour and the size of the tumour as assessed by both methods. RESULTS: Eleven patients had a spontaneous fall of beta-hCG titres and did not have pelvic arteriograms performed. The remaining 21 patients had both investigations performed. Four patients had no evidence of abnormal pelvic vessels either on colour Doppler sonography or on pelvic arteriography. Their raised beta hCG titres were due to metastatic disease elsewhere. In the remaining patients the location of the tumour vessels was found to be concordant between the two methods of imaging. Measurements of the mean diameter of tumour bulk by the two methods also agreed well (r = 0.93, P < 0.0001). Pelvic arteriogram over estimated the size of the tumour in two patients by 2 and 3 cm, respectively, but this did not alter the risk categories of the patients. CONCLUSION: Colour Doppler sonography is a noninvasive technique that can replace pelvic arteriography in the assessment of patients with trophoblastic diseases. PMID- 7547764 TI - Ovarian cancers related to minimal access surgery. AB - OBJECTIVE: To review the clinical features of women with ovarian cancer on whom minimal access surgery has been performed and to determine guidelines for the safe use of minimal access surgery for adnexal masses. DESIGN: Postal survey of members of the British Gynaecological Cancer Society (BGCS) and retrospective case review. RESULTS: BGCS consultants used ultrasound scanning (70%) and serum CA-125 estimations (53%) prior to adnexal mass surgery. The membership felt that adnexal masses with solid elements, diameter greater than 8 cm, multi-ocular or bilateral cysts or increased blood flow on Doppler scanning should not be operated on by minimal access surgery. A positive family history was also considered to be a contraindication. A total of 29 cases of ovarian cancer were identified on whom MAS had been performed. The incidence of cases was 4.1% of referrals to a tertiary referral centre (the Royal Marsden Hospital). The median age of patients was 37 years (range 20 to 68 years) and 13 had State I cancers. The mean delay between diagnosis and staging was 6.5 weeks. CONCLUSION: Guidelines on the use of minimal access surgery in the management of adnexal masses need to be agreed. Women who have an ovarian cancer diagnosed whilst having minimal access surgery should have an accepted staging procedure. We do not recommend the routine use of minimal access surgery for the treatment of ovarian cancer outside a trial. PMID- 7547765 TI - The effects of gemeprost on the second trimester fetus. AB - OBJECTIVE: To determine whether the use of gemeprost is associated with histological changes in the second trimester fetus. SETTING: Histopathology department of a university hospital. DESIGN: Retrospective comparison of histological features in fetuses aborted following maternal administration of gemeprost, with those in fetuses after spontaneous miscarriage. OUTCOME MEASURES: Degree of tissue fragmentation; other histological abnormalities. RESULTS: Significantly greater fragmentation of the liver was found in fetuses exposed to gemeprost (P = 0.046). Nonsignificant effects were found for brain (P = 0.082) and heart (P = 0.183), and no effect was seen on the kidney, adrenal and lung. No other significant histological differences were found between the two groups. CONCLUSIONS: This study is the first to document an effect of gemeprost on the fetus, but confirmation is required in further studies. Other implications are discussed. PMID- 7547766 TI - Hormone replacement therapy, sleep quality and psychological wellbeing. AB - OBJECTIVE: To examine the effect of hormone replacement therapy upon sleep quality and duration in postmenopausal women. DESIGN: Randomised, single-blind, placebo-controlled trial. SETTING: Sleep research laboratory. SUBJECTS: Thirty three healthy postmenopausal women. INTERVENTIONS: Continuous 0.625 mg conjugated equine oestrogens with 0.15 mg cyclic norgestrel taken for 12 days per 28 day cycle. MAIN OUTCOME MEASURES: Occurrence of vasomotor symptoms, polysomnographic sleep stage measures, Stanford sleepiness questionnaire, Crown-Crisp experiential index and the cognitive failures questionnaire. RESULTS: Hormone replacement therapy results in an improvement in menopausal symptoms but not in parameters of sleep quality. Despite this, certain measures of psychological wellbeing showed significant improvement in the hormone replacement therapy group. CONCLUSION: Hormone replacement therapy results in a measurable improvement in physical and psychological welfare, the latter being independent of improvement in sleep quality. PMID- 7547767 TI - Burch colposuspension: a 10-20 year follow up. AB - OBJECTIVE: To review the outcome of women who underwent Burch colposuspension 10 to 20 years ago and to assess factors which affect long term success. DESIGN: Longitudinal retrospective study. SETTING: Urogynaecology Unit, St George's Hospital, London. SUBJECTS: One hundred and nine women with genuine stress incontinence. MAIN OUTCOME MEASURES: Survival analysis of subjective and objective cure of stress incontinence. RESULTS: Cure of incontinence following Burch colposuspension is time-dependent, with a decline for 10 to 12 years when a plateau of 69% is reached. Factors adversely affecting cure are previous bladder neck surgery (logrank test P = 0.02), pre-operative weight greater than 80 kg, intra-operative blood loss more than 1000 ml and the development of post operative detrusor instability. Post-operative complications included de novo detrusor instability (14.7%), long term complaints of voiding difficulty with objective recovery at the final follow up (22%) and recurrent urinary tract infection (4.6%). CONCLUSIONS: Long term follow up after colposuspension is necessary to assess sequelae. We suggest that new continence procedures should be followed up for 5 to 10 years. PMID- 7547768 TI - How acute is the acute shortage of oocyte donors in the UK? Results of a British national survey. PMID- 7547769 TI - Obstetric pelvimetry in the UK: an appraisal of current practice. PMID- 7547770 TI - A successful pregnancy in a patient with homocystinuria and a previous near-fatal postpartum cavernous sinus thrombosis. PMID- 7547771 TI - Disappearance of fetal choroid plexus cysts during the second trimester in cases of chromosomal abnormality. PMID- 7547772 TI - Should perinatal postmortems be carried out by specialist pathologists. PMID- 7547773 TI - A classical dilemma. PMID- 7547774 TI - Fetal nuchal translucency: ultrasound screening for fetal trisomy in the first trimester of pregnancy. PMID- 7547775 TI - Fetal nuchal translucency: ultrasound screening for fetal trisomy in the first trimester of pregnancy. PMID- 7547776 TI - Fetal nuchal translucency: ultrasound screening for fetal trisomy in the first trimester of pregnancy. PMID- 7547777 TI - Low vision: a parochial view. PMID- 7547778 TI - Transscleral drainage of subretinal fluid revisited. PMID- 7547779 TI - Are gram negative bacteria involved in HLA-B27 associated uveitis? PMID- 7547780 TI - How effective is an integrated approach to low vision rehabilitation? Two year follow up results from south Devon. AB - AIMS: A survey was undertaken to assess the effectiveness of an integrated approach to the provision of low visual aids (LVAs) in south Devon over a 2 year follow up period. This integrated approach includes the assessment of patient needs by low vision therapists, followed by the provision of suitable LVAs, with particular emphasis on training in their use. METHODS: A total of 125 patients were selected at random from the 445 patients seen in the low vision clinic at Torbay Hospital in the year 1991. These patients were sent questionnaires relating to the service over a 2 year period. Questionnaires from 111 patients were analysed at 1 year and 75 questionnaires together with 46 clinical reassessments, after 2 years. RESULTS: Using a similar questionnaire to one used in a previous study in the UK from a unit where LVA training was not provided, not only was a higher rate of satisfaction found with the services provided, but also the LVAs dispensed were used more frequently. The majority of the LVAs provided were of the simple, inexpensive variety and wastage was very low. CONCLUSIONS: It was concluded that this integrated approach to low vision rehabilitation with emphasis on training in the use of less complex LVAs exceeds the performance of other types of service that rely on the dispensing of more complex LVAs. PMID- 7547781 TI - Prospective, randomised, controlled trial comparing suture needle drainage and argon laser drainage of subretinal fluid. AB - AIMS: This study was designed to compare suture needle drainage (SND) with argon laser drainage (ALD) of subretinal fluid. METHODS: A prospective, randomised, controlled, clinical trial was carried out on 93 patients undergoing external drainage of subretinal fluid during scleral buckling surgery for rhegmatogenous retinal detachment. The incidence of successful drainage, incidence of subretinal haemorrhage, incidence of retinal incarceration, and incidence of retinal perforation were determined. RESULTS: The success rate was 97.9% in the ALD group and 84.8% in the SND group (difference-13.1%, 95% CI-26.4% to 2.0%). The incidence of clinically significant subretinal haemorrhage was 4.3% in the ALD group compared with 28.3% in the SND group (difference 24.0%, 95% CI 7.6% to 40.4%). The incidence of incarceration was comparable in each group. There were no cases of retinal perforation. CONCLUSIONS: The use of ALD was associated with a higher rate of successful SRF drainage, and a lower incidence of clinically significant subretinal haemorrhage than SND. ALD is preferred when an endolaser is available. PMID- 7547782 TI - Non-retinovascular leakage in diabetic maculopathy. AB - BACKGROUND: Diabetic macular oedema is the leading cause of vision deterioration in diabetic retinopathy. Extracellular fluid within the retina, which distorts the retinal architecture, was assumed to be strictly of retinal vasculature origin. However, there is some experimental evidence supporting clinical observations suggesting a possible role of the retinal pigment epithelium (RPE). An unusual form of diabetic maculopathy is presented in which the RPE and the subretinal space play the main role. METHODS: Fluorescein angiograms of 1850 non proliferative diabetic retinopathy (NPDR) patients were examined. Nineteen eyes (14 patients, 1% of NPDR patients) met the criteria-mainly having minimal diabetic retinopathy with only a few microaneurysms and no clinically significant macular oedema (CSMO). Early phase angiograms were compared with late phase angiograms. RESULTS: It was found that in all 19 eyes the area of diffuse RPE late phase leakage was spread around the macular area. No cystic changes or cystoid macular oedema were present in any of the eyes and the visual acuity was 6/10 or better in all the eyes. CONCLUSIONS: Possible changes occur in the RPE that may be responsible for the late leakage in NPDR patients-namely, diabetic retinal pigment epitheliopathy. These changes are associated with a breakdown of the outer blood-retinal barrier, consisting of leakage through RPE cells. No focal or diffuse leakage across the RPE has been reported in the literature related to the fluorescein angiograms in diffuse diabetic maculopathy. PMID- 7547783 TI - Comparison of flow velocity of ophthalmic artery between primary open angle glaucoma and normal tension glaucoma. AB - AIMS: This study describes an investigation of the relation between the change of intraocular circulatory dynamics and visual field defect between primary open angle glaucoma (POAG) and normal tension glaucoma (NTG). METHODS: The method was to determine the blood flow velocity of the ophthalmic artery using colour Doppler imaging and quantitative peak systolic flow velocity, mean envelope velocity, end diastolic velocity, and Pourcelot resistivity index (RI) were determined. The visual field was examined using program 30-2 of the Humphrey field analyser: the mean deviation (MD) and corrected pattern standard deviation (CPSD) were calculated as indices for visual field defects. There were 25 patients with POAG and 25 patients with NTG. RESULTS: There were no significant differences in all the four indices of the ophthalmic arterial flow and in both of visual field indices between patients with POAG and those with NTG. However, NTG patients alone showed a statistically significant correlation between RI and MD. CONCLUSION: The results suggest the vascular resistance of the ophthalmic artery may be associated with the development of visual field defect in NTG patients. PMID- 7547784 TI - Strict control of glycaemia: effects on blood flow in the large retinal vessels and in the macular microcirculation. AB - AIMS: The purpose of this study was to investigate the effect of instituting strict diabetic glycaemic control on the retinal macular microcirculation and to compare this effect with that observed in the main retinal veins. METHODS: In 28 insulin dependent diabetic patients with poor glycaemic control a regimen of strict diabetic control, consisting of four daily insulin injections was instituted and maintained for 6 months. Retinal haemodynamics were investigated in the macular microcirculation by the blue field simulation technique and in the major retinal veins by a combination of bidirectional laser Doppler velocimetry and monochromatic fundus photography. Progression of diabetic retinopathy was assessed from fundus photographs taken at baseline and at the end of the study. RESULTS: Institution of strict diabetic control resulted in a significant increase in leucocyte velocity in the macular circulation (p = 0.013). No significant difference in this increase was observed between eyes that showed progression (n = 8) and no progression (n = 20) of retinopathy during the study. Significant correlations were found between relative changes over time of blood flow measured in the main retinal veins and relative changes of leucocyte velocity determined in the macular microcirculation at 2 months (p = 0.008) and 6 months (p = 0.001) but not at 5 days (p = 0.49). In the eight eyes that showed progression of retinopathy, the product of leucocyte velocity and density at baseline was significantly higher than normal (p < 0.05). During the length of this study, this product was also significantly higher in the eight eyes that showed retinopathy progression than in the 20 eyes that did not show progression (p = 0.005). CONCLUSION: Our results suggest that increased flow in the macular microcirculation may be associated with progression of retinopathy, thus supporting the hypothesis that increased blood flow may play a role in the development of diabetic microangiopathy. Although there are correlations between the changes detected in the macular microcirculation and those measured in the main retinal vessels, there are also differences which need to be further investigated in order to better understand pathogenetic mechanisms. PMID- 7547785 TI - Macular blood flow velocity in sickle cell disease: relation to red cell density. AB - AIMS/BACKGROUND: While the retinal lesions of sickle cell retinopathy have been well documented, their pathogenesis remains unclear. The purpose of this study was (1) to compare macular blood flow velocity in patients with sickle cell disease and controls, and (2) to determine in sickle cell patients the relation between macular blood flow velocity and red blood cell density. METHODS: Macular blood flow velocity was measured in 18 patients with stable sickle cell disease and 45 normal controls using blue field entoptoscopy. Red blood cell density was determined by the phthalate ester density method. RESULTS: There were no significant differences between patients and controls for leucocyte velocity. However, in the sickle cell patients leucocyte velocity in the macular capillaries was significantly negatively associated with greater range of red blood cell density (p < 0.002 and p < 0.04 for right and left eyes, respectively). CONCLUSION: These results suggest that in sickle cell patients heterogeneity of the density of the red blood cells may slow down macular capillary blood flow. PMID- 7547786 TI - Severe course of cutaneous melanoma associated paraneoplastic retinopathy. AB - BACKGROUND: Melanoma associated retinopathy (MAR) is a paraneoplastic syndrome in metastatic cutaneous melanoma presenting with nightblindness, light sensations, mild visual loss, and reduced b-waves in the electroretinogram (ERG). METHODS: A patient with MAR was followed for a period of 25 months with repeated examinations including visual field testing and recording of standard electro oculography, standard ERG, and photopic On and Off responses. RESULTS: A male patient with a very severe course of MAR is described. In addition to the clinical signs seen in previous patients, severe visual deterioration and vitreous inflammation were the predominant signs. The vitreous inflammation resolved after systemic corticosteroid therapy. Nightblindness and the reduced b waves in the ERG remained unchanged during the follow up period. However, further visual deterioration and paracentral scotomas developed. Dark adaptation was markedly abnormal. Photopic On responses were reduced, but Off responses were preserved. Antibodies against retinal bipolar cells were isolated from blood samples of this patient. CONCLUSION: Vitreous inflammation may mask the diagnosis of MAR. ERG findings indicate that the more severe and progressive course is the result of local retinal changes and not progressive generalised retinal degeneration. PMID- 7547787 TI - High dose intravitreal ganciclovir for CMV retinitis: a shelf life and cost comparison study. AB - BACKGROUND: It was previously found that high dose intravitreal ganciclovir provided superior treatment of cytomegalovirus retinitis compared with intravenous treatment. This study examined the stability and solubility of the ganciclovir solution to determine the shelf life of prepared solution, and compared the cost of intravitreal with intravenous therapy. METHODS: For the solubility studies high performance liquid chromatography was used to determine the ganciclovir concentration in various solutions. Measurements were taken of freshly made 20 mg/ml solution, the same solution stored at room temperature or frozen for 10, 17, and 24 days, after the solution was filtered, and after it was heated at 56 degrees C. For the cost comparison analysis the cost of 22 patients treated exclusively with intravitreal high dose ganciclovir was compared with cost estimates for the same patients treated with a standard intravenous therapy regimen over the same time. RESULTS: There was little variation in the concentration of ganciclovir regardless of the storage conditions, suggesting that the 20 mg/ml solution was very stable. The heating and filtering experiments suggest that maximum solubility was achieved both in the freshly prepared and thawed frozen stored solution. The total cost of the intravitreal treatment was Aus $172,435 and the estimate of intravenous treatment was Aus $490,521. This represents a total saving of $318,086 (65%), or $14,458 per patient. The average number of weeks of treatment for each patient was 27.9 (613 weeks/22 patients), so the saving per patient per year was $29,946. CONCLUSION: High dose intravitreal ganciclovir therapy may be administered in a very cost effective way, which along with its acceptability, safety, and clinical efficacy make it an attractive method of treatment of CMV retinitis. PMID- 7547788 TI - Excimer retreatment for scarring and regression after photorefractive keratectomy for myopia. AB - AIMS/BACKGROUND: Scarring associated with regression of refractive effect can occur after photorefractive keratectomy (PRK) for myopia. The experience of treating these complications is reported. METHODS: Eighteen of 285 eyes (6.3%) were retreated with the excimer laser. Age, sex, preoperative primary treatment keratometry, pre-primary treatment, pre-retreatment and post-retreatment spherical equivalents, best corrected and uncorrected visual acuities were recorded and analysed. RESULTS: At 6 months post-retreatment, the mean spherical equivalent was -2.07 dioptres (D) (SD 4.60 D). This spherical equivalent persisted in eyes followed for 12 months (-2.85D, SD 4.09 D). Nine of 18 eyes (50%) had uncorrected visual acuity of 6/12 (20/40) or better. If retreatment was undertaken within 6.5 months of the initial PRK, then scarring was likely to recur (p = 0.035). Nine of 10 eyes (90%) which had a retreatment spherical equivalent less than two thirds of their primary treatment spherical equivalent were within plus or minus 1.25D from emmetropia after retreatment. Four of 11 eyes (36%) followed for 12 months after retreatment rescarred with further regression. CONCLUSION: The data showed that eyes with scarring and regression of myopia should not be treated with PRK within 6 months of the initial procedure. Eyes with the highest percentage of regression towards their initial myopia tend to have a poor response to retreatment. PMID- 7547789 TI - Scar tissue orientation in unsutured and sutured corneal wound healing. AB - AIMS: This study aimed to evaluate stromal wound healing morphology in short term unsutured compared with sutured corneal wounds, to define regional variation in healing within radial keratotomy wounds. METHODS: Stromal scar tissue orientation (fibroblast and collagen fibre orientation) was analysed in unsutured and adjacent sutured keratotomy wounds in monkeys, 2 to 9 weeks after surgery, using light and transmission electron microscopy. RESULTS: At 2 to 4 weeks, scar tissue orientation was transverse to the wound edge in unsutured wounds, but sagittal in sutured wounds. At 5 to 9 weeks, a reorientation of scar tissue sagittal to the wound was seen in the unsutured wounds, proceeding from the posterior to anterior wound regions. In sutured wounds, a scar tissue reorientation transverse to the wound was seen, proceeding from the anterior wound region in a posterior direction. CONCLUSIONS: Within the same cornea, sutured and unsutured wounds showed opposite patterns of healing. Sutured wounds initially healed more slowly, but obtained pseudolamellar continuity over time. In contrast, healing of unsutured wounds was characterised by an early approximation towards lamellar repair that was followed by an ineffective reorganisation of the scar. This latter pattern of healing, that may be associated with a variable weakening of the wound, may relate to the clinical findings of unpredictability and/or progression of refractive effect following radial keratotomy. PMID- 7547793 TI - Temporary prism treatment of acute esotropia precipitated by fusion disruption. PMID- 7547790 TI - Feasibility of laser targeted photo-occlusion of ocular vessels. AB - AIMS/BACKGROUND: Neovascularisation occurs in many major ocular diseases such as diabetes, age-related macular degeneration, and sickle cell disease. Laser photocoagulation is typically used to obliterate the vessels but it also causes severe damage to adjacent normal tissues. This is a very significant limitation especially in the treatment of choroidal neovascularisation which often covers large areas of the posterior pole and the fovea. A method, laser targeted delivery, has been developed capable of releasing drugs locally and non invasively in the choroidal or retinal vasculature. This method could be used to target a photo-sensitiser to neovascular membranes and cause their selective occlusion by irradiating them. The targeting properties of the method promise to yield a treatment for neovascularisation that does not damage adjacent tissues and thus preserves vision. The purpose of the present study was to test the feasibility of occluding ocular vessels with this method. METHOD: The iris vessels of the albino rat were chosen because the treatment could be assessed unequivocally and followed with time. Aluminium phthalocyanine tetrasulphonate was encapsulated in heat sensitive liposomes and administered systemically. The iris vessels were irradiated with a yellow laser to raise their temperature to 41 degrees C, cause a phase transition in the liposomes and thereby locally release the photosensitiser. The laser was also used to excite the released photosensitiser and cause occlusion. The effect was monitored immediately and for 8 months thereafter. Controls for the effect of the laser and the unencapsulated drug were conducted. RESULTS: The results demonstrated that occlusion can be achieved and sustained for the period of follow up. The controls showed that the effect was not due to heat or to the activation of the low dose of free drug. CONCLUSION: These preliminary findings indicate that laser targeted photo occlusion is a promising new method for the treatment of neovascularisation. PMID- 7547792 TI - Strategies for the management of microbial keratitis. PMID- 7547791 TI - Immunohistochemical evidence of neuronal and glial differentiation in retinoblastoma. AB - BACKGROUND: The study sought to investigate the histogenesis of retinoblastoma. METHODS: One hundred specimens of retinoblastomas were examined along with those of 18 astrocytic gliomas and 15 medulloblastomas to compare similarities of glial differentiation in retinoblastoma and the two types of brain tumour. Employing avidin-biotin immunoperoxidase technique, antibodies were applied against neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 protein (S-100). RESULTS: Most rosettes and fleurettes, and some undifferentiated cells in retinoblastomas were NSE positive, but GFAP and S-100 negative. GFAP and S-100 positive cells in retinoblastomas were detected mostly in well differentiated glial cells which were interpreted as reactive or non-neoplastic cells. Some of the GFAP and S-100 positive cells in retinoblastomas were defined as tumour cells that resembled neoplastic astrocytes in astrocytic gliomas and medulloblastomas. CONCLUSION: Retinoblastoma may arise from primitive bipotential or multipotential cells capable of neuronal and glial differentiation. PMID- 7547794 TI - Alpha fetoprotein as marker for a case of orbital yolk sac tumour. PMID- 7547795 TI - Lobar emphysema and atelectasis syndrome, a nosological unity. AB - Shrinking and atelectatic pulmonary processes--atelectasis syndrome--and lobar emphysema show morphological similarities: congenital malformations of the bronchial structure, external compression of the bronchi as well as inflammatory processes. Ventilation mechanics determine whether in the described processes an atelectasis or an emphysema manifests itself clinically. Usually, one finds over expansions in connection with atelectasis syndrome and atelectases together with lobar emphysema. Both processes can be reversible, if we are not dealing with bronchial malformations. Longer lasting processes as well as acute, life threatening forms require operation. The belief that atelectasis syndrome and lobar emphysema constitute a nosological unity is illustrated by our cases of 9 patients with atelectasis syndrome and 16 patients with lobar emphysema. PMID- 7547796 TI - Characteristics of continuous esophageal pH-metering in infants with gastroesophageal reflux and apparent life-threatening events. AB - Episodes of apnea during the first year of life have been classified with the term "Apparent Life-Threatening Events" (ALTE). Gastroesophageal reflux (GER) has been accepted to be one of the factors which can favor ALTE. The aim of this work is to study the continuous 24 h gastroesophageal pH-metering (fundamentally the Reflux Index [RI] and the Area Under Curve [AUC] parameters in three different periods of time: total pH-metering, pH-metering excluding the first two post prandial hours, and pH-metering during sleep time, for 24 infants, 14 with ALTE and 10 without it). Between these two groups (with and without ALTE) there were no significant differences in the total pH-metering concerning the RI (2 +/- 1.2 vs 5.6 +/- 7.5 p > 0.05) and concerning the AUC (24.9 +/- 14.4 vs 67.4 +/- 84 p > 0.05), but in the analysis of the sleep period, the RI (p < 0.05) and the AUC (p < 0.01) both showed significant differences in the group of infants without ALTE. These results confirm that pH-metering study during sleep is the procedure of choice for recognizing infants with ALTE. PMID- 7547797 TI - Does hyperamylasemia in choledochal cyst indicate true pancreatitis? An experimental study. AB - Patients with choledochal cyst often have repeated attacks of abdominal pain accompanied by hyperamylasemia, and they may be diagnosed as having acute pancreatitis. However, the attacks generally tend to subside in a short period by conservative treatment, and evidence of pancreatitis is rarely observed at the time of surgery. Choledochal cyst is commonly associated with pancreatobiliary maljunction, and high concentrations of pancreatic enzymes in bile are usually observed. When the bile duct pressure increases due to obstructive cholangitis, pancreatic enzymes in bile may regurgitate into the blood stream. Cholangiovenous reflux of amylase might cause hyperamylasemia. In order to investigate the mechanism of hyperamylasemia by cholangiovenous reflux, canine pancreatic juice or bile from a patient with choledochal cyst was injected into the obstructed common bile duct in dogs. The pancreatic enzymes in bile could readily enter into the blood stream at the pressure level of 15 mmHg or more in the bile duct. The peak amylase level in the thoracic lymph was observed to be more than 4 times higher than that in the blood serum, and the lymph flow during 30 minutes increased significantly from 8.1 to 20.4 ml at the bile duct pressure level of 20 mmHg. The reflux of amylase in bile into the blood stream via both the hepatic vein and thoracic duct might result in hyperamylasemia in the patients with choledochal cyst. PMID- 7547798 TI - Characteristics of choledochal cysts in neonates and early infants. AB - Thirty-six infants with choledochal cyst consisting of 28 babies less than 12 months of age and 8 between 13 and 24 months of age, were analyzed. Characteristics of cases were as follows: (1) they were usually of the cystic type, (2) a huge abdominal mass and jaundice with alcoholic stool were typically found, (3) no symptom suggesting acute pancreatitis was observed, (4) amylase levels in bile were usually low, despite the presence of pancreatobiliary malunion, while other pancreatic enzymes in bile commonly showed a high concentration, and (5) primary cyst excision and biliary reconstruction was the treatment of choice, and could be safely performed with favorable outcomes even in babies less than 1 year of age. PMID- 7547799 TI - Partial or near-total pancreatectomy for nesidioblastosis? AB - We report on 11 consecutive cases of nesidioblastosis successfully managed, in a 22-year period from 1972-1993 at The Children's Hospital, Birmingham, England. In the pre-operative period all patients were managed by constant glucose administration ( > 10 mg/kg/min) and hyperglycaemic agents such as diazoxide, glucagon, growth hormone and somatostatin either singly or in combination. Seven patients underwent partial pancreatectomy, 2 of whom needed a subsequent near total resection; 4 others had a near-total pancreatectomy as the primary procedure. The 5 patients who have had partial pancreatectomies are healthy and on no regular medication. Of the 6 patients who had near-total pancreatectomy 3 require insulin for diabetes mellitus and 3 are on pancreatin for pancreatic exocrine deficiency. We recommend partial pancreatectomy as the first operation in the treatment of nesidioblastosis. PMID- 7547800 TI - Medical treatment of postappendectomy intraperitoneal abscesses in children. AB - Treatment of postappendectomy intraperitoneal abscesses is classically surgical (drainage). We report a retrospective study (1988 to 1991) of our experience in providing exclusively medical treatment for 11 such isolated abscesses in children. The abscess was detected 7 times within the 10 days following appendectomy. Ultrasonography localized the abscess 7 times in the right iliac fossa and 4 times in Douglas' pouch. In 4 cases it was larger than 5 cm in diameter. Parenteral antibiotic therapy associated ticarcillin and clavulanic acid 6 times and piperacillin, tobramycin and metronidazole 5 times. Drug efficacy was evaluated by clinical signs (fever, pain, ileus) and ultrasonography. In one case, an associated ileus led to reoperation at day 4 of treatment. In the other 10 cases, clinical signs disappeared during the first week of treatment. At day 7, parenteral was replaced by oral antibiotic therapy (metronidazole 7 times, amoxicillin-clavulanic acid 3 times) continued until the abscess was no longer visualized on ultrasonography (5 times at 3 weeks, 5 times at 1 month). Medical treatment of isolated postappendectomy abscesses in children would thus appear to be a logical choice in the absence of an associated ileus. PMID- 7547802 TI - Non-malignant tumors that can mimic cancer during the neonatal period. AB - TOPIC: Neonatal tumors include a varied group of neoplasias. Histologically non malignant tumors must be differentiated from cancer in these newborns, since their management and evolution is completely different. METHODS: We have reviewed all the non-malignant neonatal tumors (NMNT) seen during a 21 year period at The Pediatric Oncology Unit of "La Fe" Children's Hospital (Valencia, Spain). Congenital malformations and easily-recognizable tumors such as epidermic or dermoid cysts, lipomas, capillary and cutaneous hemangiomas, lymphangiomas, and small nevi lesions were excluded. During the same period, we have diagnosed 40 cases of neonatal cancer. RESULTS: Forty patients were found to have NMNT: 15 teratomas, which in 10 cases were sacrococcygeal, 8 solid hemangiomas, 4 mesoblastic nephromas, 4 infantile myofibromatosis, 3 follicular ovarian cysts, and 6 miscellaneous tumors. Epidemiologic and etiopathogenic factors, clinical manifestations, treatment and evolution were evaluated. One cardiac teratoma was a post-mortem finding. In our series, with a median follow-up of 8 years and 4 months, 38 of 39 neonates are still alive with a very low incidence of sequelae, 36 of them with no evidence of tumor. PMID- 7547801 TI - Routine bilateral exploration for inguinal hernia in infancy and childhood. AB - Whether to explore the contralateral side is a real question regarding the management of unilateral inguinal hernias in infants and children. The surgeon can easier make a decision if he knows the true incidence and prevalence of the bilateral involvement in different ages. During the years 1982 to 1991 the authors could find 138 contralateral hernias developed after unilateral herniorrhaphy in 2554 children (5.4%). Fifty-five percent of these children (76 patients) were younger than 1 year of age, 74% of them (102 patients) were younger than 3 years of age at the time of the initial repair. The interval between the operations was less than 1 year in 54% of the cases. To get more precise data, we explored 148 children younger than 3 years of age bilaterally during 1992. The result was positive in 104 cases (70.3%). The bilateral involvement was most frequent under the first 6 months of life (83.5%), then it dropped gradually. We found a patent processus vaginalis on the asymptomatic side in 74.3% of girls and in 61.1% of boys. The overall contralateral involvement of unilateral hernias was 64.8%, independently of which side the hernia had appeared on. The patent processus vaginalis can be regarded as a precursor of indirect hernias, so the contralateral exploration can prevent the development of a later hernia. The authors conclude that bilateral exploration is mainly justified during infancy, but in case of girls they suggested applying it until three years of age. PMID- 7547803 TI - Efficiency of the anorectal manometry for the diagnosis of Hirschsprung's disease in the newborn period. AB - We have used a continuous perfusion probe for manometry in newborns. We carried out 95 pressure recordings in 56 patients labeled as delayed passage of meconium longer than 24 h. When the relaxation reflex of the anal canal was not present after rectal stimulation with a distensible balloon, the tests were repeated weekly up to the first month of life. Thereafter, suction biopsies were carried out to confirm the final Hirschsprung's disease (HD) diagnosis. The amplitudes of the fluctuating waves in HD subjects were higher than those in non-HD patients. Although the procedure could provide false positive results, no possibilities exist for false negative results as proved in our study. All the five cases with HD were diagnosed by our manometric procedure as well as by histochemistry. The method is advantageous and profitable for the diagnosis of HD in case of delayed passage of meconium or distal functional obstruction of the anal canal. PMID- 7547804 TI - Polypoid formations at the anal margin in infants less than two years old. AB - Polypoid formations in the anal canal and at the anal margin are sometimes observed in the newborn and in infants less than 2 years old. Unlike hamartomatous polyps, which are commonly reported in infantile proctology, these growths are rare in early childhood. Only by performing a pathological anatomy examination is it possible to determine the nature of the numerous affections encountered to assess prognosis and to decide on suitable therapeutic management. PMID- 7547805 TI - Sensation in the anal region and rectum after surgery for anorectal malformations. AB - Sensation of the anal region and rectum after surgery for anorectal malformations was investigated in 32 patients (17 with high type, 6 with intermediate type and 9 with low type anomaly), aged 5 to 16 years. All patients were examined as to whether or not they could appreciate sensations of touch, pain, heat and cold in the circumanal skin and in the anal canal. Rectal sensation and compliance were also examined by anorectal manometry. All patients had normal sensation in the circumanal skin, but less than half of them had normal sensation in the anal canal. Patients with normal bowel control showed a significantly higher incidence of touch sensation in the anal canal than those with fecal incontinence or constipation. It was suggested that a sensation defect of the anal canal is related to a disturbance of discrimination between flatus and feces. The results of rectal sensation and compliance showed that reduced elasticity of the neorectum and low rectal compliance were important factors of fecal incontinence after surgery for anorectal malformations. PMID- 7547806 TI - Posterior sagittal anorectoplasty: functional results of primary and secondary operations in comparison to the pull-through method in anorectal malformations. AB - BACKGROUND: Control of defaecation after surgical correction of high and intermediate types of congenital anorectal malformations is difficult. The posterior sagittal approach with careful reconstruction of the external sphincter is postulated to give a better outcome than the pull-through operation. The functional results of these procedures performed in one centre were evaluated in order to identify prognostic factors. MATERIAL AND METHODS: Between 1979 and 1992 66 patients with high or intermediate congenital anorectal anomalies were treated in our centre. In 16 patients a pull-through operation (Kiesewetter-Rehbein) was performed. After 1984, the posterior sagittal anorectoplasty (PSARP) (Pena and deVries) was used in 35 patients as the primary operation. In 22 patients a PSARP operation was done as a secondary procedure, in one third of these patients after a previous pull-through operation in our centre. The functional results were analysed in retrospect. RESULTS: Sixty operations in 53 patients could be evaluated. The overall continence rate was 34%. After the pull-through operation six out of 15 patients (40%) were continent, after a primary PSARP 10 out of 25 (40%) and after a secondary PSARP operation five out of 20 patients (25%). Patients with a sacral defect were continent only in 16% as compared to 44% of the patients with a normal sacrum. Sex was also relevant: 67% of the girls were continent, compared to 30% of the boys. Ten out of 11 girls (90%) with a normal sacrum became continent. CONCLUSION: The PSARP for high and intermediate anorectal malformations does not give better functional results than the pull through operation. The prognosis is determined by other factors than the type of operation, notably sex and the presence or absence of sacral defects. PMID- 7547807 TI - Cultured epidermal autograft in the management of critical pediatric burn patients. AB - We treated 4 patients with CEA at "La Paz" Children's Hospital between July 1992 and September 1993. All of them had acute flame burns. The total body surface area (TBSA) of burn injury ranged from 75% to 85% with an age range of two to five years. Before placement of CEA homografts and biosynthetic materials were used to prepare the wound bed. CEA take ranged from 40% to 90%. One of the children died after polyethylene glycol intoxication when the exposed areas were nearly totally covered. Two more have started rehabilitation therapy and the last patient was grafted just two months ago. PMID- 7547808 TI - A case of a mediastinal neurenteric cyst demonstrated by prenatal ultrasound. AB - We report the case of a posterior large mediastinal neurenteric cyst demonstrated by prenatal ultrasound (US). Postnatal chest radiography and CT studies demonstrated a posterior, large cystic mediastinal mass associated with vertebral abnormalities including scoliosis, hemivertebrae and anterior spina bifida. Because postnatally respiratory distress developed and rapidly progressed, an emergency operation was performed 12 hours after delivery. The cyst was excised via right posterolateral thoracotomy. Postoperatively, respiratory distress regressed. This is the third reported case of a neurenteric cyst demonstrated by prenatal ultrasound. PMID- 7547812 TI - Unusual distribution of enamel hypoplasia in an 11-year-old child with Proteus syndrome. AB - An 11-year-old girl with Proteus syndrome attended our dental department because her teeth were sensitive to cold stimuli and because of the poor appearance of her anterior teeth. Extraoral examination revealed several of the previously reported craniofacial features of Proteus syndrome: facial and skull asymmetry, exostoses of the nasal bridge, and mandibular prognathism. Intraoral examination revealed enamel hypoplasia of primary and permanent teeth. The distribution of hypoplasia was unusual in that only teeth on the right side of the mouth were affected, and also in that only the distal half of the crown of the maxillary right permanent central incisor was affected. Behavioural problems necessitated treatment under general anaesthesia; the affected primary teeth were extracted and the hypoplastic permanent teeth were restored, relieving the patient's symptoms and greatly improving aesthetics. PMID- 7547811 TI - Imperforate anus, malrotation and Hirschsprung's disease: a rare association. AB - The association of imperforate anus with anomalies of rectal innervation is very rare. Moreover, malrotation only rarely accompanies these malformations. We report 2 patients with this unusual triple association and its impact on their management. Retrospectively both patients presented at birth with the same radiological picture of distended distal colon and right-sided small bowel location. We discuss the importance of a systematic search for associated intestinal malformations in cases of imperforate anus in order to avoid serious complications and to choose the optimal operative strategy. PMID- 7547810 TI - Spheric duodenal duplication in a 13-year-old child. AB - Duodenal duplications are rare. Modern imaging procedures demonstrate the relation of the duplication to the duodenum and the adjacent organs. Thus the surgical procedure can be selected preoperatively. Endoscopic ultrasound is an excellent technique for visualizing a duodenal duplication. A cystic duodenal duplication measuring 6 cm in diameter was resected in a 13-year-old boy. PMID- 7547813 TI - Candida infection of a persistent palatal ulcer in a 20-month-old child. AB - A 20 month-old infant presented with a painless, persistent lesion on the hard palate which had been present for 6 months. The lesion was aggressive in appearance, approximately 2 cm in diameter with raised, rolled edges, and there was what appeared to be granulation tissue present together with some necrotic slough in its base. It was considered to represent a traumatized area with superimposed secondary Candida infection. A direct smear of the lesion was positive for Candida. Antifungal therapy, and an attempt to avoid further irritation by advising the mother not to give the child a feeding bottle whenever possible, resulted in complete healing of the lesion. There has been no recurrence in 2 years. PMID- 7547809 TI - Childhood actinomycosis: report of two cases. AB - The authors report two cases of actinomycosis in children: one thoracic and the other retroperitoneal. They emphasize the difficulties of diagnosis before the stage of parietal extension with cutaneous fistula and characteristic yellow granular discharge. These difficulties are due to: The rarity of visceral actinomycosis, particularly in children. The lesion has a similar appearance to that of a tumor; an extensive pre-operative work-up is mandatory (ultrasound, computed tomogram scan, repeated ultrasound-guided needle biopsy), although this work-up may not necessarily lead to the correct diagnosis. A surgical biopsy will often confirm the diagnosis, provided the diagnosis has been previously considered. The necessity of using very specific tests for correct identification of the organism. Therefore, in a case of pseudo-inflammatory pseudotumor, visceral actinomycosis must be considered in order to guide microbiological and pathological studies, although this diagnosis is rare. Once the diagnosis has been made, prolonged treatment with penicillin is effective and complete recovery is generally obtained. PMID- 7547814 TI - A plastic screw-cover embedded in the hard palate of an infant aged 13 months. PMID- 7547815 TI - History of the International Association of Paediatric Dentistry. Part 3: Samuel D. Harris and some early pressures for international developments. PMID- 7547816 TI - Between-meal eating, toothbrushing frequency and dental caries in 4-year-old children in the north of Sweden. AB - Two hundred and forty-nine 4-year-old children were examined for dental caries, and data were collected on frequency of toothbrushing, use of fluorides, and intake of nine different snack products. Dental caries experience of children who brushed once or twice daily with parental help was significantly lower than that of children who brushed irregularly. The intake of snacks was high. Buns and cakes, ice cream, and sweet beverages were consumed more often than sweets. Children who had high snack intakes and brushed irregularly had significantly higher caries experience than those with low snack intakes and regular toothbrushing. Therefore irregular toothbrushing was shown to potentiate the impact of frequent snacking. PMID- 7547817 TI - Resin bonding efficacy of Gluma 2000 to dentine of primary teeth: an in vitro study. AB - The aim of this study was to investigate (i) the effect of Gluma 2000 conditioning solution on the dentine of primary teeth, (ii) the penetration of Gluma 2000 adhesive resin into the etched dentine, (iii) the shear bond strength of a composite resin restorative material (Pekafill) bonded with Gluma 2000 to dentine, and (iv) the depth of penetration of adhesive resin into the etched dentine as a function of the conditioning time. Dentine surfaces conditioned for 15, 30, 60 and 120 seconds were examined by scanning electron microscopy. The smear layer was totally removed and, with the longer conditioning times, a fine crystalline precipitation, presumably calcium oxalate, was formed. After Gluma 2000 bonding of composite resin and dissolution of the teeth the resin interface showed excellent penetration into the etched dentine. Shear bond strengths of composite resin bonded with the adhesive resin were similar following conditioning for 15, 30, 60 and 120 seconds. Microphotographs of debonded specimens indicated that the mode of failure was cohesive within the resin. The depth of penetration of resin into etched dentine was determined by scanning electron microscopy, and a linear relationship was found between the thickness of the resin-impregnated layer and conditioning time. It was therefore concluded that depth of resin penetration and shear bond strength are not correlated. The study shows that the Gluma 2000 bonding system produces reliable adhesion of composite resin to dentine of primary teeth. PMID- 7547818 TI - Children's ratings of dental injection and treatment pain, and the influence of the time taken to administer the injection. AB - Three hundred and eight children aged 3-16 years (mean age 10.2), who were undergoing routine dental treatment, recorded on visual analogue scales their ratings of pain associated with injection and treatment. The injection pain scores were examined for their relationship to age, gender, time taken to administer the injection and injection type. The treatment pain scores were compared between groups who had teeth either extracted or restored, and between groups assessed by the operator as having total or partial anaesthesia. A significant inverse correlation was found between subjective injection pain and injection duration. The difference in treatment pain scores was significant between groups assessed by the operator as having total or partial anaesthesia. Inferior dental nerve blocks were rated significantly more painful than buccal infiltrations. Age, gender, and the operative procedure performed had no statistically significant relationship to the injection pain scores or treatment pain scores. The visual analogue pain scale was found to be unsuitable for use by children under 7 years of age. PMID- 7547819 TI - An evaluation of four methods of assessing the behaviour of anxious child dental patients. AB - The aim of this study was to evaluate four behavioural scales which have been used to assess anxious children during dental treatment: The Frankl (F), Houpt (H), Visual Analogue (VAS) and Global Rating (GR) scales. The study measured the agreement on three of the scales (F, H and VAS) between two judges expert in the management of anxious children and also between four other dentists. The operating dentists scored each visit using the fourth scale (GR). Twenty-nine anxious children aged 3-16 years who had been referred to a specialist clinic were included in the study, and 64 separate visits were recorded on videotape. The two-judge panel evaluated the children's behaviour on all 64 visits, and the four-judge panel evaluated a 12-visit segment. Comparisons within and between the two-judge and four-judge panels showed close agreement on the Visual Analogue and Houpt scales but not on the Frankl scale. There was significant correlation between the Global Rating scale and the Visual Analogue, Houpt and Frankl scales. PMID- 7547820 TI - A clinical trial comparing two doses of oral temazepam for sedation of paediatric dental patients. AB - A study was carried out to compare the effectiveness of two doses of temazepam elixir for the behaviour management of paediatric dental patients. Twenty-two children aged 2-5 years (mean 4.06 years), classified as ASA Class I and II (American Society of Anesthesiologists Classification), were assigned to receive either 0.3 or 0.5 mg/kg temazepam elixir in a double-blind randomized cross-over study. All treatment sessions were videotaped and rated independently by three paediatric dentists for the degree of crying, movement, sleep and overall behaviour at 15-minute intervals while being treated. The results showed no statistically significant differences between the two doses. PMID- 7547821 TI - Recovery of inulin from Jerusalem artichoke (Helianthus tuberosus L.) in the small intestine of man. AB - The recovery of inulin, a naturally occurring beta (2-->1)-fructan isolated from Jerusalem artichoke (Helianthus tuberosus L.), in the small intestine of man was studied in ileostomy subjects. The ileostomists were given a low-dietary-fibre diet based on white wheat bread and virtually free of inulin, and the same diet with the addition of 10 g and 30 g inulin product respectively, and the recovery and mean transit time (MTT) of inulin were estimated by tracking inulin in ileal effluent. The recovery of inulin was approximately 87% at both ingestion levels. MTT was 4.9 (SE 0.6) h at an intake of 10 g inulin product decreasing to 3.4 (SE 0.3) h at an intake of 30 g inulin product. A significant change in the fructose:glucose ratio of inulin from ingestion (4.1) to recovery in ileal effluent (4.5-4.7) and a lower recovery of the glucose residue than of the fructose residue of inulin indicate that the low-molecular-weight inulins are more sensitive to hydrolysis than the high-molecular-weight fragments. The loss of inulin during passage through the small intestine is presumably due to hydrolysis by either acids or enzymes and to microbial degradation by the microflora permanently colonizing the distal small intestine. The concentrations of lactic acid (LA) and short-chain fatty acids (SCFA) in frequently collected ileal effluents on the control day were approximately 6 nmol/l and approximately 55 mmol/l respectively. During periods with inulin ingestion the concentration of LA increased to 18-26 mmol/l (P < 0.052), while the concentration of SCFA ran converse and decreased to 18-32 mmol/l (P < 0.023). The osmotic loads (68 and 204 mosmol/l) associated with the ingestion of inulin product caused minor malabsorption of low-molecular-weight sugars. PMID- 7547822 TI - Influences of dietary and intraduodenal lipid on alertness, mood, and sustained concentration. AB - The effects of intraduodenal and dietary lipid on alertness, mood and performance in a task requiring sustained attention were investigated in two studies. The first experiment compared the effect of duodenal infusion of either 100 g/l Intralipid (8.36 kJ/min) or isotonic saline (9 g NaCl/l) in paired studies carried out on two non-consecutive days on five male volunteers. Two consecutive 3 h infusions, one of lipid, the other saline, were given blind on each day using a crossover design. Analysis of variance indicated that lipid significantly reduced alertness (P < 0.05) and affected the speed and accuracy of performance in a sustained attention task (P < 0.05). A second experiment compared the effects on eight male volunteers of two isoenergetic lunches of similar appearance, taste and protein content but differing fat and carbohydrate (CHO) contents (fat energy:CHO, 64:18 v. 7:76). Alertness was lower (P < 0.05) and responses to stimuli in a sustained attention task were slower after the high-fat meal than after the low-fat meal (P < 0.05). In conclusion, infusion of lipid into the small intestine, and the substitution of fat for carbohydrate while keeping energy and protein constant in a lunch, both cause an enhanced postprandial decline in alertness and concentration. This may be related to the presence of lipid in the small intestine. PMID- 7547824 TI - Dietary assessment methods. PMID- 7547823 TI - A randomized double-blind controlled calcium supplementation trial, and bone and height acquisition in children. AB - There is limited information relating Ca intake to bone and height acquisition among Oriental children who consume little or even no milk. The present controlled study investigated the acquisition of bone mass and height of Chinese children with an initial Ca intake of approximately 567 mg/d who were supplemented to about 800 mg/d. Eighty-four 7-year-old Hong Kong Chinese children underwent an 18-month randomized, double-blind, controlled Ca-supplementation trial. The children were randomized to receive either 300 mg elemental Ca or a placebo tablet daily. Bone mass of the distal one-third radius was measured by single-photon absorptiometry, lumbar spine and femoral neck were determined using dual-energy X-ray absorptiometry. Measurements were repeated 6-monthly. Baseline serum 25-hydroxycholecalciferol concentration and physical activity were also assessed. Baseline Ca intakes of the study group and controls were respectively 571 (SD 326) and 563 (SD 337) mg/d. There were no significant differences in baseline serum 25-hydroxycholecalciferol concentration (P = 0.71) and physical activity (P = 0.36) between the study and control groups. After 18 months the study group had significantly greater increases in lumbar-spinal bone mineral content (20.9 v. 16.34%; P = 0.035), lumbar-spinal area (11.16 v. 8.71%; P = 0.049), and a moderately greater increment in areal bone mineral density of the radius (7.74 v. 6.00%; P = 0.081) when compared with the controls. The results confirm a positive effect of Ca on bone mass of the spine and radius but no effects on femoral-neck and height increase. A longer trial is warranted to confirm a positive Ca effect during childhood that may modify future peak bone mass. PMID- 7547825 TI - Effects of three microbial probiotics on postprandial porto-arterial concentration differences of glucose, galactose and amino-nitrogen in the young pig. AB - Postprandial kinetics of porto-arterial concentration differences of glucose (G), galactose (Gal), L-lactic acid (LA) and amino-N (AN) were studied in the piglet after the ingestion of 10(7) colony-forming units (cfu) Sporolactobacillus P44 (SP), or 10(6) cfu Bacillus cereus IP5832 (AC), or 10(6) cfu of a combination of Lactobacillus acidophilus, L. fermentum and L. brevis (AB)/g feed. Sixteen fistulated piglets (portal vein and brachiocephalic trunk; mean body weight 22 (SD 2) kg) were used. The diet was based on skimmed milk (320 g/kg), barley (300 g/kg), wheat bran (110 g/kg), maize (100 g/kg) and lactose (70 g/kg). The postprandial blood kinetics, four measurements per animal at 1-week intervals, were studied for 6 h after the ingestion of test meals of 400 g basal diet (BD) or this diet supplemented with the bacteria (SP, AC and AB respectively). Areas of porto-arterial concentration differences (APACD) of G, Gal and LA were not influenced by the bacteria supplements. APACD of AN was significantly higher after the ingestion of the SP diet than that estimated for BD. PMID- 7547826 TI - Dietary change after smoking cessation: a prospective study. AB - A population sample of 375 men and women cigarette smokers were recruited to take part in a prospective study of smoking cessation to test the hypothesis that stopping smoking is associated with an increased consumption of the essential fatty acid linoleic acid, which explains the concomitant reduction in risk of coronary heart disease. Diet was assessed using a 10 d weighed record in 301 smokers at baseline, 153 at 4-month follow-up, of whom twenty-six had quit smoking, and 122 at 1-year follow-up, of whom twenty had quit. Compared with continuing smokers, those who had quit at the 4-month follow-up (mean 10 and 13 weeks for men and women respectively) had statistically significant increases in body weight (5%), energy intake (13%), total dietary fat (24%), all specific types of dietary fat (26% polyunsaturated fat, 26% linoleic acid, 30% eicosapentaenoic acid, 23% monounsaturated fat and 22% saturated fat) and vitamin E intake (19%). The foods which appeared to contribute to increases in energy and fat intakes at the 4-month follow-up were vegetable oils and polyunsaturated margarines, processed meats and meat pies. By follow-up at 1 year (mean time since quitting 31 and 41 weeks for men and women respectively) there were no detectable differences in energy and total fat intakes. However, intakes of eicosapentaenoic acid and pteroylglutamate (folate) were statistically significantly higher in the quitters compared with the continuing smokers (37% for eicosapentaenoic acid and 16% for folate). We conclude that the short-term increase in dietary intake of linoleic acid, which is not sustained by 1 year, cannot explain the reduction in risk of coronary disease following smoking cessation. PMID- 7547827 TI - An in vitro procedure for studying enzymic dephosphorylation of phytate in maize soyabean feeds for turkey poults. AB - An in vitro method was developed to predict inorganic P release from maize soyabean poultry feeds containing supplemental phytase (EC 3.1.3.8), and to quantify the effect of acid phosphatase (EC 3.1.3.2), fungal protease (EC 3.4.23.6) and Aspergillus niger cellulase (EC 3.2.1.4) on phytate dephosphorylation. Pepsin (EC 3.4.23.1) and pancreatin digestion periods were preceded by a 30 min pre-incubation at pH 5.25 to simulate digestion in the crop of poultry. Pancreatin digestion was carried out in dialysis tubing, with a ratio of about 1:25 (v/v) between the digesta and dialysing medium, to simulate gradient absorption from the duodenum. The feed:water ratio was kept within physiological limits and a constant proportion of feed weight to digestive enzymes was maintained. There was a linear response to increasing dosages of phytase up to 1000 phytase units (FTU)/kg feed, and to increasing phosphate concentration in feeds. In vivo validation was performed with growing turkeys (1 3 weeks) fed on diets containing 12 g Ca/kg and 0, 500 or 1000 FTU phytase/kg in a factorial arrangement with 0, 1, 2 or 3 g supplemental phosphate/kg (from KH2PO4). After a simple transformation (variable/in vitro P = f (in vitro P)), amounts of P hydrolysed from feed samples by in vitro digestions correlated with 3-week body-weight gain (R 0.986, P < 0.0001), toe ash (R 0.952, P < 0.0001), feed intake (R 0.994, P < 0.0001) and feed efficiency (R 0.992, P < 0.0001). The dephosphorylating ability of phytase in vitro was significantly enhanced (P < 0.05) by the addition of acid phosphatase. Fungal acid protease and Aspergillus niger cellulase also enhanced the dephosphorylation process in vitro. PMID- 7547828 TI - Diet selection in sheep: the role of the rumen environment in the selection of a diet from two feeds that differ in their energy density. AB - The effect of the energy density (ED) of feeds offered as a choice on the diet selection of sheep, and the relationship between the rumen environment and the diet selected from feeds of different ED were investigated in two experiments. In the first experiment two feeds, L and H, and their mixture M (3:1 w/w) were formulated. All feeds had similar calculated metabolizable protein:metabolizable energy (ME) ratios, but differed in ED (7.4, 8.1 and 10.1 MJ ME/kg fresh feed for L, M and H respectively). The feeds were offered ad lib. either singly or in paired choices (L/M, L/H and M/H; n6 per treatment) to growing sheep. Although the rate of live-weight (Lwt) gain on feed H was higher than on feeds L or M, and the daily rate of feed intake lower, the sheep on feed choices did not consume only feed H. Instead they selected a mixture of both feeds offered, such that the total amount of H consumed per kg fresh feed was similar on choices L/H and M/H. The rate of Lwt gain of sheep on choices L/H and M/H was not different from that achieved on feed H alone. In the second experiment the choice L/H was offered to fistulated sheep (10 months of age, mean Lwt 57.5 kg) in an 8 x 8 Latin square, with 7 d periods. Treatments were infusions into the rumen (total volume 1 litre) over 4 h on days 1-4 of each period of acid (HCl; Acid 1, 400; Acid 2, 300 and Acid 3, 200 mmol/l), alkali (NaOH; Alk 1, 316; Alk 2, 212 and Alk 3, 109 mmol/l) and control (NaCl; Con 1, 315 and Con 2, 209 mmol/l). Infusate osmolalities (mOs/kg) were 795 (Acid 1), 585 (Acid 2, Alk 1 and Con 1), 390 (Acid 3, Alk 2 and Con 2) and 200 (Alk 3). Infusion treatment significantly affected the diet selection of the sheep (P < 0.05) according to the osmolality of infusate, but not according to rumen pH. During infusions intake of feed H tended to decline with increasing treatment osmolality, whereas intake of L remained constant. The effects on diet selection and feed intake were of a short duration with no carry over effects.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547830 TI - A stable isotope study of zinc kinetics in Irish setters with gluten-sensitive enteropathy. AB - The short-term kinetics of Zn turnover were studied in Irish setters with gluten sensitive enteropathy and control dogs following intravenous injection of 0.25 mg 96.5% enriched 70ZnCl2. The 70Zn enrichment of serum was found closely to obey two-compartment kinetics and the derived two-compartment decay equation was used to calculate the size and turnover of the two initial rapidly exchanging pools of body Zn. In normal Irish setters isotopic Zn initially equilibrates with a pool (a) of size 1.27 (SD 0.46) mumol/kg and then with a second pool (b) of size 6.83 (SD 1.72) mumol/kg. The fractional turnover of pool (b) was approximately one eighth that of pool (a). Enteropathic dogs showed no reduction in the size of either rapidly exchangeable Zn pool, reduction in serum Zn concentration or abnormality in Zn balance and hence these results do not support the possibility of an underlying Zn deficiency in this disorder. PMID- 7547829 TI - Maternal essential fatty acid patterns during normal pregnancy and their relationship to the neonatal essential fatty acid status. AB - Although essential fatty acids (EFA) and their longer chain, more unsaturated derivatives play a major role during pregnancy, hardly any information is available with respect to the course of the maternal EFA status during an uncomplicated pregnancy and its relationship to the neonatal EFA status. Therefore, a longitudinal study was started in which 110 pregnant women gave repeated blood samples from the 10th week of gestation until delivery. After birth a blood sample from the umbilical vein and a maternal venous blood sample were collected as well, and 6 months after delivery a final blood sample from the mother was taken. The absolute (mg/l) and relative (% total fatty acids) amounts of the fatty acids in plasma phospholipids were determined. The total amounts of fatty acids increased significantly during pregnancy. This pattern was similar for the individual fatty acids and fatty acid families. The relative amount of linoleic acid (18:2n-6) did not change during pregnancy, whereas the relative amount of arachidonic acid (20:4n-6) decreased. Despite maternal mobilization of docosahexaenoic acid (22:6n-3, DHA), suggested by a temporary increase in the DHA status until 18 weeks gestation, the DHA status steadily declined thereafter. This pattern was associated with a progressive increase in the DHA deficiency index in maternal blood throughout pregnancy and resulted in a sub-optimal neonatal DHA status. The overall maternal EFA status also declined steadily during pregnancy. Therefore, the question arises whether the mother, under the prevailing dietary conditions, is able to meet the high fetal requirement for EFA. PMID- 7547831 TI - Effect of dietary magnesium level on urinary and faecal excretion of calcium, magnesium and phosphorus in adult, ovariectomized cats. AB - Dietary Mg restriction is generally considered to contribute to the prevention of struvite urolithiasis in cats, but its effects on faecal and urinary excretion of Ca and P have not been systematically investigated. The present study seeks to fill the gap. In a 4 x 4-week crossover study, ovariectomized cats were fed on purified diets containing either 0.40, 0.79, 1.59 or 3.17 mmol Mg/MJ (0.19, 0.38, 0.76 and 1.52 g Mg/kg diet). Increasing the dietary Mg level from 0.40 to 3.17 mmol Mg/MJ by the addition of extra MgCO3 raised urinary and faecal excretion of Mg from 0.14 to 0.68 mmol/MJ and from 0.28 to 1.66 mmol/MJ respectively. The 8 fold increase in Mg intake significantly raised urinary excretion of Ca from 0.06 to 0.09 mmol/MJ. Apparent absorption and urinary excretion of P were depressed by 13 and 25% respectively when the dietary Mg level was raised from 0.40 to 3.17 mmol/MJ. A dietary level of 0.40 mmol Mg/MJ (0.19 g Mg/kg diet) was found to be sufficient to maintain Mg balance in the adult ovariectomized cats fed on the purified diet. PMID- 7547832 TI - Dietary phosphorus restriction to half the minimum required amount slightly reduces weight gain and length of tibia, but sustains femur mineralization and prevents nephrocalcinosis in female kittens. AB - The effects of dietary P restriction to half the recommended minimum level on growth, bone and renal mineralization and urinary composition were studied in female kittens. In two separate experiments, 8-week-old weanling kittens were fed on purified diets containing either 4.6 or 9.2 mmol P/MJ (2.8 or 5.6 g P/kg diet). In the second experiment there was an additional low-P diet in which the Ca concentration was reduced from 9.5 to 4.8 mmol/MJ (7.5 v. 3.8 g Ca/kg diet). P restriction slightly but systematically reduced weight gain (to a maximum of 16%) and growth of the tibia (by 1-4%); the former effect was statistically significant (P < 0.05) between the ages of 15 and 20 weeks in Expt 1 only, and the latter did not reach statistical significance at any time point (P > or = 0.13). No adverse effect of P restriction was found on mineralization of femur at the age of 39 weeks. Kidney Ca concentrations were significantly lowered (Expt 1, 6 v. 20 mumol/g dry weight, P < 0.001; Expt 2, 7 v. 16 mumol/g dry weight, P < 0.01) in cats fed on the low-P diets, this effect not being affected by the dietary Ca:P ratio. Urinary P concentration was significantly depressed (by 50 96%) after feeding the low-P diets (P < 0.001). P intake did not influence P, Ca and Mg retention during the period of 15 to 39 weeks of age. PMID- 7547833 TI - A recipe-based, diet-planning modelling system. AB - In a recent article in the British Journal of Nutrition, Sklan & Dariel (1993) presented a method for diet planning employing a mixed-integer programming algorithm for meeting nutritional requirements at minimum costs for institutions or individuals. They recognized that most food items are generally consumed in whole units and as such they are represented as integer variables. However, as in most previous studies, they derived the minimum cost diets by optimizing over purchased food items. The present paper presents a computer-assisted, diet planning modelling system for individuals by optimizing over recipes instead of food items. This is accomplished by restricting the integer programming solutions to those bundles of food that represent reasonably popular meal recipes. The modelling system is composed of three main components: recipe data entry, database management, and the model. The recipe data entry component creates and stores recipes. It also provides nutritional analysis of the recipes. The database management component creates and maintains several databases necessary to build the modelling data file. The modelling component solves the user specified model. Currently, the model component can solve for the optimal diet by minimizing cost or minimizing cooking and preparation time. The optimal diet is prepared to satisfy the recommended nutritional guidelines for a predefined group of individuals for 1 week. The system currently has 895 popular recipes found in Hawaii. Diet plans generated using this modelling system with differing objectives are discussed and compared. PMID- 7547834 TI - Effect of dietary protein quality on energy metabolism in growing chickens. AB - A slaughter experiment was carried out to study the effect of dietary protein quality on maintenance energy requirements and energy costs for protein accretion and fat deposition in fast-growing broiler-type male chickens. Three isonitrogenous (200 g crude protein/kg DM) and isoenergetic (14 kJ metabolizable energy (ME)/g DM) semipurified diets based on soyabean meal unsupplemented (diet S) or supplemented with 20 g L-lysine/kg (diet SL) or 2 g DL-methionine/kg (diet SM), in order to promote a decrease or an increase in growth rate respectively, were selected and given at four feeding levels (ad lib. or restricted to 40, 28 and 18 g DM/d, on average) to 10-d-old fast-growing male broiler-type chicks for 2 weeks. Both the efficiency with which ME was used to support growth (kg) and the maintenance requirements (MEm) significantly decreased inversely to the biological value of the dietary protein (kg = 0.660, 0.600 and 0.572; MEm = 597, 522 and 464 kJ/kg W0.75 per d, for diets SL, S and SM respectively). The partial efficiencies of use of ME for protein accretion (kp) or fat deposition (kf) were also inversely related, the former increasing with the quality of the protein offered. An alternative procedure was used to try to overcome the statistical problems inherent in the partition of ME between fat and protein. PMID- 7547835 TI - Effect of dietary protein concentration on responses to Escherichia coli endotoxin in broiler chickens. AB - The effect of dietary protein concentration on stress responses against injection of Escherichia coli lipopolysaccharide (LPS) was studied in male broiler chickens. Chickens (7 d of age) were fed on a 100 (low-protein; LP) or 300 g protein/kg (high-protein; HP) diet for 2 weeks. LPS was injected intraperitoneally every 2 d during the final 6 d, or once 16 h before the end of the experiment, at a concentration of 900 micrograms/chick. The LPS injection did not affect body-weight gain, feed intake, gain:intake ratio, or plasma Fe concentration. The single injection of LPS reduced plasma Zn concentration, but the repeated injections did not. Feeding the HP diet increased the response of plasma Zn concentration to the single injection of LPS. Plasma albumin concentration was reduced by LPS injection. Feeding the HP diet resulted in a higher plasma alpha 1-acid glycoprotein (AGP) concentration than feeding the LP diet, in chicks untreated with LPS. An increase in plasma AGP concentration observed after LPS injection in chicks fed on the LP diet was greater than that seen in chicks fed on the HP diet. No significant changes in plasma AGP concentration in response to repeated injections of LPS were observed in chicks fed on the HP diet. Plasma interleukin-1 (IL-1)-like activity was greater in chicks fed on the LP diet than in those fed on the HP diet, when LPS was injected. The response of plasma IL-1-like activity to the single injection of LPS in chicks fed on the LP diet was the greatest among the treatment groups. These results suggest that acute-phase responses to LPS injection are much greater in chicks fed on a LP diet than in those fed on a HP diet, and multiple injection of LPS weakens the responses. PMID- 7547836 TI - Do breeds of pig differ in the efficiency with which they use a limiting protein supply? AB - An experiment was designed to test the prediction that two genetically-very different kinds of pigs would retain the same amounts of protein in their bodies when given the same allowances of the same feed for the same period of time, when these allowances were limiting for both. An allowance of 33.957 kg of a feed with 12.76 MJ metabolizable energy (ME) and 189 g crude protein (N x 6.25)/kg feed was given to Large White x Landrace (LW x) and Chinese Meishan (CM) female pigs over three different periods of time: (1) ad lib. (AL) with the time taken by individual pigs to consume the allowance being a variable, (2) over a period of 7 weeks (H) and (3) over a period of 9 weeks (L). In addition, in a fourth treatment, pigs of both breeds received the same allowance but supplemented with starch also over a period of 7 weeks (HS). The performance of the pigs on treatment AL was affected by pig breed, with CM pigs gaining protein at a slower, and lipid at a faster, rate than LW x pigs. On treatments L, H and HS the average amounts of protein retained were 2.693 and 2.655 kg for the LW x (n 15) and CM (n 15) pigs respectively (SED 0.106 kg). There was a statistical indication that the LW x pigs may have been more efficient on L, and less efficient on HS, than the CM pigs but we have been unable to propose any biological reason for such an effect, if it was in fact a real one. Thus, the efficiency with which ideal protein was utilized was close to being constant, and apparently at its maximum, for the two breeds. However, although CM pigs had the same protein gain, and the same live weights, on the same feed allowances as the LW x pigs, they gained significantly more lipid. This was attributed in part to their digesting their feed better and in part to their having a lower energy requirement for maintenance through a lower level of physical activity. Given that these two very different kinds of pigs use limiting protein with the same efficiency, it is suggested that it is safe to make the assumption in models of pig growth that the material efficiency of using limiting protein is constant across genotypes of pig. PMID- 7547837 TI - Differences in propionate-induced inhibition of cholesterol and triacylglycerol synthesis between human and rat hepatocytes in primary culture. AB - Propionate is a short-chain fatty acid formed in the colon and supposedly involved in the cholesterol-lowering effect of soluble fibre. To explore the underlying mechanism(s) of this fibre action, we have used human hepatocytes in primary culture to study the effects of propionate on hepatic lipid synthesis. Initial experiments with mevalonate and mevinolin, a competitive inhibitor of hydroxymethylglutaryl (HMG)-CoA reductase (EC 1.1.1.88) were performed to evaluate basic regulatory mechanisms in these cells; results were compared with those obtained with rat hepatocytes. Incubation for 24 h with mevalonate caused a similar, concentration-dependent inhibition of [14C]acetate incorporation into cholesterol in human and rat hepatocytes. Likewise, mevinolin (100 mumol/l) inhibited the formation of cholesterol from radiolabelled acetate by about 80% in cells from both species. Propionate inhibited cholesterol as well as triacylglycerol synthesis from [14C]acetate with a similar concentration dependency in rat hepatocytes. Fifty percent inhibition was obtained at a propionate concentration of only 0.1 mmol/l. This propionate-induced inhibition was not affected by a 100-fold excess of unlabelled acetate. Human hepatocytes were much less susceptible in this respect: propionate concentrations of 10-20 mmol/l were required to obtain similar inhibitory effects in these cells, i.e. values greatly exceeding reported portal propionate concentrations in humans. The results suggest the existence of differences in the regulation of hepatic cholesterol (and triacylglycerol) synthesis between human and rat liver cells. These results do not support the hypothesis that the fibre-induced decrease in plasma cholesterol concentration in man is mediated by a direct effect of propionate on hepatic cholesterol synthesis. PMID- 7547838 TI - Effect of propionate on fatty acid and cholesterol synthesis and on acetate metabolism in isolated rat hepatocytes. AB - In the present study the actual role of propionic acid in the control of fatty acid and cholesterol synthesis was investigated in isolated liver cells from fed rats maintained in the presence of near-physiological concentrations of glucose, glutamine and acetate. Using 3H2O for lipid labelling, propionate appears as an effective inhibitor of fatty acid synthesis and to a lesser extent of cholesterol synthesis, even at the lowest concentration used (0.6 mmol/l). Butyrate is a potent activator of both synthetic pathways, and the activating effect was not counteracted by propionate. Using 1-[14C]acetate, it was observed that propionate at a moderate concentration, or 1 mmol oleate/l, are both very effective inhibitors of 14C incorporation into fatty acid and cholesterol. This incorporation was drastically inhibited when propionate and oleate were present together in the incubation medium. The net utilization of acetate by rat hepatocytes was impaired by propionate, in contrast to oleate. 1-[14C]butyrate was utilized at a high rate for fatty acid synthesis, but to a lesser extent for cholesterol synthesis; both processes were unaffected by propionate. Intracellular citrate concentration was not markedly depressed by propionate, whereas it was strongly elevated by butyrate. In conclusion, propionate may represent an effective inhibitor of lipid synthesis when acetate is a major source of acetyl-CoA, a situation which is encountered with diets rich in readily fermentable fibres. The present findings also suggest that propionate may be effective at concentrations close to values measured in vivo in the portal vein. PMID- 7547839 TI - Taurocholic acid adsorption during non-starch polysaccharide fermentation: an in vitro study. AB - The association of radiolabelled taurocholic acid with the solid fraction of a faecal fermentation mixture was measured. A human faecal inoculum was incubated with [24-14C]taurocholic acid and several non-starch polysaccharide sources (pectin, wheat bran, ispaghula (Plantago ovata) husk and seed), glucose or a substrate-free control. Portions of fermentation mixture were taken at 0, 3, 6, 21 and 24 h and centrifuged to acquire a supernatant fraction and a pellet containing the fermentation residue. 14C was measured in supernatant fractions and pellets at all time points. Volatile fatty acids (VFA) were measured at 0 and 24 h to confirm bacterial growth. Radioactivity in the pellet increased over time for all substrates. Glucose resulted in the greatest incorporation of taurocholic acid into the pellet, followed by pectin. At 24 h the proportion of the total radioactivity found in the pellet was 92% for glucose, 79% for pectin, 60% for wheat bran, 59% for ispaghula seed, 53% for ispaghula husk and 26% for the control (mean of duplicates). Glucose and pectin produced the greatest quantity of VFA at 24 h. VFA production was highly correlated with radioactivity in the pellet (r0.976, P < 0.005). These results suggest that the bile acid binding capacity of a faecal culture mixture may be strongly influenced by the fermentability of the available substrate and hence related to bacterial metabolic activity. PMID- 7547840 TI - Effect of oat saponins and different types of dietary fibre on the digestion of carbohydrates. AB - The effects of oat saponins (a mixture of avenacosides A and B) and dietary fibre (cellulose and guar gum) on the disaccharidase activities in the proximal small intestine of the rat were investigated. The influence of avenacosides A and B on the activity of disaccharidases and alpha-amylase (EC 3.2.1.1) was also studied in vitro. In vivo, oat diets with three avenacoside contents (negligible, normal and twice normal) were used. No significant differences in sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20), trehalase (EC 3.2.1.28) and lactase (EC 3.2.1.21) activities were found between the oat groups after 19 d feeding. The rats that were given cellulose tended to have higher disaccharidase activities compared with the other groups. The avenacosides inhibited the lactase activity significantly in vitro while no or small effects on the other disaccharidases were found. In contrast, the in vitro hydrolysis of starch by alpha-amylase was increased in the presence of saponins, probably due to their detergent effect. Thus, the in vitro studies showed that the avenacosides could influence the enzyme activities. In vivo, these effects are probably minor due to the low avenacoside concentrations found in oats. PMID- 7547844 TI - Comparing energy expenditure between groups of people with different body masses. PMID- 7547841 TI - Comparative study of the fermentative characteristics of inulin and different types of fibre in rats inoculated with a human whole faecal flora. AB - It is known that the physico-chemical characteristics of fibre modify their fermentation characteristics in the colon. Previously we showed the varying effects of inulin and different types of fibre on the hepatic and intestinal xenobiotic-metabolizing enzymes (XME) in initially germ-free rats inoculated with a human, methanogenic, whole-faecal flora (Roland et al. 1994). The aim of the present work was to assess whether or not these effects could be related to differences in production of fermentation metabolites (gases excreted in vivo and caecal metabolites) due to the different compositions of fibre. The different types of fibres were analysed with regard to their solubility and their composition of neutral monomers and uronic acids. Inulin was totally soluble, carrot (Daucus carota), cocoa (Theobroma cacao) and wheat bran were partially soluble; pea (Pisum sativum) and oat were nearly totally insoluble. Uronic acids were found mostly in carrot and cocoa fibre. Glucose was present as the main neutral monomer in each fibre type. Xylose was found also in wheat bran, pea and oat fibres, and arabinose was found in wheat bran. Inulin consumption led to high levels of H2 production but no CH4 production, to a 4-fold greater caecal concentration of butyrate than with the other fibres and to a decrease in caecal pH. Conversely, rats fed on carrot or cocoa fibre produced a large amount of CH4 but no H2 and generated a different profile of short-chain fatty acids (SCFA). The lowest amounts of gases and SCFA were found in rats fed on wheat bran, pea and oat fibre. We observed a relationship between the caecal concentration of SCFA and the activity of hepatic glutathione-S-transferase (EC 2.5.1.18) but no direct link was shown between the other XME and the fermentation profile. PMID- 7547842 TI - Exogenous and endogenous nitrogen flow rates and level of protein hydrolysis in the human jejunum after [15N]milk and [15N]yoghurt ingestion. AB - Milk and yoghurt proteins were 15N-labelled in order to measure the flow rate of exogenous N during digestion in the human intestine. After fasting overnight, sixteen healthy volunteers, each with a naso-jejunal tube, ingested either [15N]milk (n 7) or [15N]yoghurt (n 9). Jejunal samples were collected every 20 min for 4 h. A significant stimulation of endogenous N secretion was observed during the 20-60 min period after yoghurt ingestion and the 20-40 min period after milk ingestion. The endogenous N flows over a 4 h period did not differ between the groups (44.3(SEM 6.5) mmol for milk and 63.5(SEM 5.9) mmol for yoghurt). The flow rates of exogenous N indicated a delayed gastric emptying of the yoghurt N compared with N from milk. The jejunal non-protein N (NPN) flow rate increased significantly after milk and yoghurt ingestion due to an increase in the exogenous NPN flow rate. The NPN fraction of exogenous N ranged between 40 and 80%. The net gastro-jejunal absorption of exogenous N did not differ significantly between milk (56.7(SEM 8.5)%) and yoghurt (50.9(SEM 7)%). The high level of exogenous N hydrolysis is in accordance with the good digestibility of milk products. Fermentation modifies only the gastric emptying rate of N and does not affect the level of diet hydrolysis, the endogenous N stimulation or the digestibility rate. PMID- 7547843 TI - The thermic effect of food in normal-weight and overweight pregnant women. AB - A defective thermic response to food may be an energy-sparing adaptation in both obesity and pregnancy. To evaluate the combined effect of obesity and pregnancy on postprandial thermogenesis, the thermic effect of food was assessed for a 240 min period following a high-carbohydrate meal and a typical mixed meal in nine normal-weight non-pregnant, eight overweight non-pregnant, eight normal-weight pregnant and six overweight pregnant women using indirect calorimetry. A test meal that provided 60% of each subject's measured daily requirement for basal metabolism was used. Pregnant women were studied during weeks 30-35 of gestation. Neither obesity nor pregnancy altered the thermic effect of food, although the response to the mixed meal was greater (P < 0.01) than that to the high carbohydrate meal in all cases. The mean responses for the high-carbohydrate and mixed meals were 26.9 (SD 6.0) and 30.1 (SD 6.2) % baseline energy expenditure respectively, and 7.4 (SD 1.6) and 8.3 (SD 1.6) % of the meal energy load respectively. Obesity and pregnancy were associated with hyperinsulinaemia (P < 0.005) following both test meals, suggesting that postprandial thermogenesis was not altered by insulin resistance in this group. The incremental glucose response was elevated (P < 0.001) in the pregnant women following both test meals; overweight women tended to have a greater incremental glucose response following the high-carbohydrate meal, but it was not significant (P = 0.065). These results do not provide evidence of an impaired thermic response to food in either overweight or third trimester pregnant women. PMID- 7547845 TI - Determination of digestible energy values and fermentabilities of dietary fibre supplements: a European interlaboratory study in vivo. AB - The performance of methods to determine energy conversion factors for dietary fibre (DF) supplements and fermentability (D) values of their non-starch polysaccharides (NSP) was investigated. Heats of combustion, digestible energy (DE) and D values were determined on five DF supplements in five European laboratories on five separate occasions. In each instance the DF supplements were fed to juvenile male Wistar rats at two doses, 50 and 100 g/kg basal diet, for 3 weeks with food and faeces collected in the 3rd week. Among-laboratory variations in heats of combustion (delta Hc) were < 2%. DE values (kJ/g dry weight) at the upper and lower doses respectively were: 10.4 and 9.9 for a high-methoxyl apple pectin, 9.5 and 9.4 for a sugar-beet DF supplement, 12.2 and 12.7 for soyabean DF supplement, 3.8 and 4.0 for maize bran, and 0.3 and 0.3 for Solka-floc cellulose. Variations among laboratories, among occasions and among animals were < 1, < 2 and < 2.5 kJ/g respectively. The among-occasion: among-laboratory variance ratio for DE was 0.5, suggesting the method performed equally well in all laboratories. There was no evidence of learning of fatigue or fatigue in the performance of the method. D values were also independent of dose and at the high and lower doses were: pectin 0.92 and 0.95, sugar-beet NSP 0.68 and 0.68, soyabean NSP 0.86 and 0.88, maize bran 0.17 and 0.18, cellulose 0.07 and 0.06. Among-laboratory variance tended to increase with decreasing fermentability and ranged from 0.03 to 0.18. The DE and D data were not significantly different from a previously proposed relationship DE = 0.7 x delta Hc x D, where delta Hc is the heat of combustion of the supplement. We conclude that while the among-laboratory variation in the D of difficult-to-ferment NSP is too large for the reliable prediction of energy value the method for the direction determination of DE is both reproducible and repeatable, that DE is independent of dosage of DF supplement up to 100 g/kg diet, and that it is safe to discriminate between energy values with a precision of 3 kJ/g. The conversion of both DE and D to net metabolizable energy for the purpose of food labelling, tables and databases is described. PMID- 7547846 TI - Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study. AB - Five European laboratories tested a simple in vitro batch system for dietary fibre fermentation studies. The inoculum was composed of fresh human faeces mixed with a carbonate-phosphate buffer complex supplemented with trace elements and urea. Five dietary fibre sources (cellulose, sugarbeet fibre, soyabean fibre, maize bran and pectin) were used by each laboratory on three occasions to determine pH, residual non-starch polysaccharides (NSP) and short-chain fatty acid production during fermentation. Cellulose and maize bran degradabilities were very low (7.2(SE 10.8) and 6.2 (SE 9.1)% respectively after 24 h), whereas pectin and soyabean fibre were highly degraded (97.4 (SE 4.4) and 91.1 (SE 3.4)% respectively after 24 h). Sugarbeet fibre exhibited an intermediate level of degradability (59.5 (SE 14.9)%). Short-chain fatty acid production was closely related to NSP degradation (r 0.99). Although each variable was ranked similarly by all laboratories, some differences occurred with respect to absolute values. However, the adaptation of donors to the experimental substrates was not an influential factor. Interlaboratory differences could be reduced either by adding less substrate during incubations or using less-diluted inocula. In vitro fermentations with inocula made from human faeces and from rat caecal contents gave similar results. There was a close correspondence between the data obtained in the present experiment and those previously published in in vivo studies in the rat using the same fibres. The in vitro batch system tested during the present study provides a rapid means of obtaining quantitative estimates of the fermentation and the estimation of the energy content of new sources of dietary fibre. PMID- 7547847 TI - Biochemical validation of a self-administered semi-quantitative food-frequency questionnaire. AB - The present study is a biochemical validation of a food-frequency questionnaire (FFQ) with optical reading, i.e. containing food portion photographs to help to assess quantities. Forty-four healthy subjects, non-smokers and not taking vitamin supplements, were recruited for the study. After completion of the questionnaire, subjects were asked to keep a 7 d weighed dietary record (7DR). Three 24 h urine samples were collected on 3 different days over the week of food recording for the analysis of urea-N, P and K. On the 4th day of food recording, blood was collected for determination of alpha-tocopherol, beta-carotene and ascorbic acid. N, P and K determined in urines and from 7DR were significantly correlated (Spearman rank correlation test), r values being 0.77, 0.57 and 0.42 respectively. The correlations with the FFQ were significant only for N (r 0.45) and P (r 0.39). Blood ascorbic acid and beta-carotene concentrations correlated with dietary intake when determined from 7DR (both r 0.44), but not when determined from FFQ. No correlation was found for alpha-tocopherol. The data obtained seem to prove the validity of the FFQ in defining eating patterns in terms of some nutrients, but not vitamins, at least as far as non-supplemented subjects are concerned. The way in which foods were grouped in the questionnaire could account for these results. PMID- 7547848 TI - Dietary factors affecting the maximum feed intake and the body composition of pre ruminant kid goats of the Granadina breed. AB - An experiment was carried out with kid goats of the Granadina breed to identify the dietary factors affecting voluntary feed intake of the kid goat and those that additively could determine its body composition. The animals used were from birth to 61 d of age, fed ad lib. on different milk replacers containing 200, 240 and 280 g crude protein/kg DM and 200, 240 and 280 g fat/kg DM, thus giving nine dietary treatments. The utilization of the milk replacers and the animals' body composition were determined by balance and slaughter trials. There were significant positive effects of protein concentration of the milk replacers on component digestibilities, energy metabolizability, feed intake, empty-body weights, empty-body composition and protein and fat retention. The concentration of fat in the milk replacers also had a significant positive effect on the digestible and metabolizable energy concentration of the diets and on fat retention. The relationships existing between feed intake and diet composition (concentration of digestible protein, metabolizable energy and digestible protein:metabolizable energy ratio) as well as between empty-body composition or protein and fat retention and diet composition, were examined. From these it was deduced that feed intake was significantly influenced by the digestible protein concentration of the diets. The higher the digestible protein concentration the higher the feed intake up to a maximum digestible protein concentration value. As the digestible protein concentration of the diets was the dietary factor which significantly influenced feed intake, this also significantly influences the body composition and the protein and fat retention. The protein concentration of the feed at which metabolizable energy intake in these animals would be greatest was estimated to be 347 g/kg DM. PMID- 7547849 TI - Intake, digestion and small intestinal protein availability in sheep in relation to ammoniation of wheat straw with or without protein supplementation. AB - The effects of ammoniation of wheat straw with or without supplementation of protein sources of either high (casein) or relatively low (potato protein) rumen degradability on intake and digestion were studied with four sheep in a 4 x 4 Latin square design. Rations offered were: (1) untreated wheat straw (UWS), (2) ammoniated wheat straw (AWS), (3) AWS supplemented with 3.2 g casein/kg live weight (W)0.75 per d (AWSC) and (4) AWS supplemented with 3.9 g potato protein/kg W0.75 per d (AWSP). Straw was offered ad lib. and all rations were supplemented with sugarbeet pulp and a mineral mixture. NH3 treatment increased intake and digestion. Supplementation of AWS with potato protein increased total digestible organic matter intake (DOMI) compared with AWS whereas supplementation with casein did not affect total DOMI. Protein supplementation of AWS significantly reduced rumen digestion of cellulose, and when the supplementation was with casein it reduced rumen digestion of neutral-detergent fibre and hemicellulose also. This lower rumen digestion was compensated by a higher proportion of digestion occurring in the hindgut for hemicellulose (P < 0.05 for AWSC, P > 0.05 for AWSP), but not for cellulose. Across all rations, rumen fluid volume increased with increasing cell-wall intake. The efficiencies of microbial protein synthesis were (average of three different methods of estimation) 23.3, 26.2, 34.8 and 31.7 g N/kg apparently-rumen-degraded organic matter for UWS, AWS, AWSC and AWSP respectively. The difference between UWS and AWS was not significant, but values for AWSC and AWSP were significantly higher than that for AWS. The rumen digestion of feed amino acid-N (AA-N) was significantly higher for AWSC than for the other rations. The apparent small-intestinal digestion of AA-N and N was significantly higher for AWSP than for the other rations. The true small intestinal digestion values were 0.86, 0.84 and 0.68 for AA-N, N and non-protein N respectively. Ileal endogenous losses of AA-N were approximately 6 mg/g duodenal non-protein dry-matter flow. Linear relationships were observed between DOMI and N balance and truly absorbed AA-N, indicating that DOMI could have been limited by small-intestinal amino acid availability. Regression of N balance v. truly absorbed AA-N resulted in an estimate of net efficiency of utilization of truly absorbed AA-N or 0.54. PMID- 7547850 TI - Lipid peroxidation, prostacyclin and thromboxane A2 in pigs depleted of vitamin E and selenium and supplemented with linseed oil. AB - In a 2 x 2 balanced factorial experiment the biochemical effects on pigs of two dietary factors were investigated. The first factor was alpha-tocopherol and Se supplementation and the second factor was supplementation with alpha-tocopherol stripped linseed oil. In pigs fed on diets depleted of alpha-tocopherol and Se, increases in concentrations of markers of lipid peroxidation (4-hydroxynonenal and hexanal) were observed. However, skeletal myopathy was only observed in those pigs fed on diets depleted of alpha-tocopherol and Se and supplemented with oil. In those pigs, increased lipid peroxidation was observed in heart and supraspinatus muscle. The plasma concentration of thromboxane B2 was increased in pigs fed on diets depleted of alpha-tocopherol and Se, suggesting an increased tendency towards platelet aggregation. However, this change was reversed in pigs depleted of alpha-tocopherol and Se, but supplemented with oil. This may have been a consequence of loss of arachidonic acid, the substrate for thromboxane formation, as a result of lipid peroxidation. PMID- 7547851 TI - Effects of dietary fatty acid composition on basal and hormone-stimulated hepatic lipogenesis and on circulating lipids in the rat. AB - Thirty male rats were randomly assigned to one of three dietary groups in which the source of dietary fat was either a mixed oil, maize oil or fish oil. Effects of dietary fatty acid composition on in vitro rates of [U-14C]glucose incorporation into hepatic total lipids and into hepatic triacylglycerol were measured under basal, insulin (4 nM)-, gastric inhibitory polypeptide (GIP; 6 nM) and insulin + GIP (4 nM + 6 nM)-stimulated conditions. Effects of the three diets on postprandial plasma triacylglycerol, cholesterol, insulin and GIP concentrations were also measured. The fish-oil diet decreased rates of basal glucose incorporation into hepatic total lipids (P < 0.05) and hepatic triacylglycerol, (P < 0.01) compared with the mixed-oil diet. The presence of insulin + GIP in the incubation medium stimulated glucose incorporation into hepatic total lipids in the maize-oil (P < 0.01) and fish-oil groups (P < 0.05), as well as into hepatic triacylglycerol in the maize-oil group (P < 0.005). In addition, the fish-oil diet decreased postprandial plasma triacylglycerol levels compared with both other dietary groups (P < 0.05 both cases), and the mixed-oil diet markedly increased postprandial plasma insulin levels compared with the other dietary groups (P < 0.001). PMID- 7547852 TI - Digestive adaptations of rats given white bread and cooked haricot beans (Phaseolus vulgaris): large-bowel fermentation and digestion of complex carbohydrates. AB - Two experiments were carried out to examine the short (1-3 d) and medium (14 d) term adaptations of rat large-bowel (LB) fermentation to alterations in substrate supply brought about by including cooked haricot beans (Phaseolus vulgaris) in diets based on white bread. Changes in organic matter (OM) flow from the ileum occurred within 1 d and were stable for 14 d but the pattern of caecal short chain fatty acids took much longer to stabilize with considerable increases in butyrate between 1 and 3 d and up to 14 d. This suggests that the metabolic activity of the LB microflora may take a considerable time to stabilize after an abrupt change in substrate supply. Despite an almost fourfold change in OM supply to the LB, the proportion of this OM apparently fermented in that organ (0.46) remained fairly constant. None of the apparent resistant starch measured in the diets could be detected in faeces. Dietary non-starch polysaccharides (NSP) were extensively fermented with similar values (0.82) for both bread and bean NSP and there was little indication of any interaction between the two diet components on NSP fermentation. An attempt was made to fractionate ileal and faecal OM to provide a basis for a quantitative model of LB stoichiometry. PMID- 7547853 TI - Absorption and endogenous excretion of phosphorus in growing broiler chicks, as influenced by calcium and phosphorus ratios in feed. AB - Absorption and endogenous excretion of P by male broiler chicks (14-29 d old) were quantitatively evaluated at different Ca:P ratios (1, 1:1; 2, 1.5:1; 3, 2:1; 4, 2.5:1) in four groups given experimental diets ad lib. The P content was the same in all diets. An isotope-dilution technique was used to determine endogenous faecal and renal excretion. Ca and P retentions in the whole body were estimated according to the comparative slaughter technique. P absorption was calculated from retention and endogenous excretion. Absorption and endogenous excretion of P amounted to (mg P/d per chick): 304, 270, 160 and 158; and 135, 109, 31 and 30 in groups 1, 2, 3 and 4 respectively. Widening of the Ca:P ratio in the feed limited the P absorption. Availability of feed P amounted to (%): (1) 66, (2) 57, (3) 32 and (4) 30, and the amounts of absorbed P retained were (%): (1) 56, (2) 60, (3 and 4) 81. The increasing Ca concentration in the feed showed a greater effect on P absorption than on P retention. The ratios of relative retention to relative endogenous excretion of absorbed P were: (1) 1.27, (2) 1.50, (3 and 4) 4.26. PMID- 7547854 TI - Zinc absorption in adult humans: the effect of iron fortification. AB - The effect of Fe fortification on the absorption of Zn was studied by radioisotopic labelling of single meals, followed by measurements of whole-body retention of 65Zn at 14 d after intake. Healthy adult volunteers participated in the study. Weaning cereal, wheat bread and infant formula, foods that are all frequently Fe-fortified, were evaluated in the study. The amounts of Fe added as FeSO4 were similar to the levels in commercial products in Europe and the USA, and were 200 or 500 mg Fe/kg (weaning cereal), 65 mg Fe/kg (white wheat flour) and 12 mg Fe/l (infant formula). For comparison, Zn absorption was measured in the same subjects, from identical test meals containing no added Fe. No statistically significant differences were found when Zn absorption from the Fe fortified test meals was compared with that from non-Fe-fortified test meals. Fractional Zn-absorption values from Fe-fortified v. non-fortified meals were 31.1 (SD 11.9) v. 30.7 (SD 7.0)% (weaning cereal; 200 mg Fe/kg), 37.7 (SD 16.6) v. 30.2 (SD 9.9)% (weaning cereal; 500 mg Fe/kg), 36.5 (SD 14.4) v. 38.2 (SD 18.1)% (bread; 65 mg Fe/kg flour) and 41.6 (SD 8.1) v. 38.9 (SD 14.5)% (infant formula; 12 mg Fe/l). The addition of Fe to foods at the currently used fortification levels was thus not associated with impaired absorption of Zn and the consumption of these Fe-fortified foods would not be expected to have a negative effect on Zn nutrition. PMID- 7547856 TI - Standard free energy change for the hydrolysis of the alpha, beta phosphoanhydride bridge in ATP. PMID- 7547855 TI - The effect of the gel-forming liquid fibre on feeding behaviour in man. AB - A novel substance called liquid fibre (LF) has been developed which gels in the stomach and dramatically delays gastric emptying. The prolonged stomach distension LF causes would be expected to reduce food intake. The present study tested whether LF affected psychological factors connected with eating behaviour and short-term food intake. Paired studies were carried out on seventeen healthy but overweight volunteers (ten male, seven female) with body mass indices of 24 34 kg/m2 who were non-restricted eaters. On one occasion (randomized) they took drinks of LF (300 ml each) at 09.05, 11.55 and 18.00 hours, and on the other they took placebo drinks. Subjective feelings were assessed by visual analogue scales. The amount of food consumed at an appetizing pre-selected meal presented at 12.15 hours was measured covertly. Food diaries were kept until 16.00 hours on the following day. The visual analogue scales indicated that LF reduced hunger and the amount of food desired, and increased fullness, all of which would be expected to cause a reduction in food intake. However, there were no differences in the amount or type of food eaten at the appetizing test-meal (6073 v. 5824 kJ, P = 0.41). Food eaten later in the day was significantly delayed by LF (7.0 v. 5.9 h, P = 0.030), and the amount tended to be reduced (4328 v. 5439 kJ, P = 0.088). The energy consumed on the following day also tended to be lower after LF (3802 v. 4737 kJ, P = 0.130). This suggests that gastric distension is a relatively unimportant influence on eating behaviour when non-restricted eaters are presented with an appetizing meal and that intestinal factors seem more important for prolonging satiety and reducing subsequent food intake. PMID- 7547857 TI - Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd. AB - A novel experiment has been used to show proximity relationships between sites on the surface of the cytochrome bd quinol oxidase of Escherichia coli. The artificial protease iron (S)-1-[p-(bromoacetamido)benzyl]-EDTA (Fe--BABE) was conjugated to selected reactive cysteines placed in subunit I or subunit II, with the aim of identifying amino acid residues within approximately 12 A of each site of attachment. The protease was activated with H2O2 and ascorbate for a few seconds, and hydrolysis products were isolated and analyzed by N-terminal sequencing. Among other results, we found that residue 39 of subunit II is near residue 255 of subunit I in the putative quinone-binding domain (Q loop) of the oxidase. Since this technique is insensitive to the nature of the amino acid side chains, it should prove generally valuable in revealing spatial relationships both within and between subunits in complex proteins where high-resolution structural information is not available. PMID- 7547858 TI - Reconstitution of the second step in NO synthesis using the isolated oxygenase and reductase domains of macrophage NO synthase. AB - Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves the enzyme into a dimeric oxygenase fragment that contains heme, tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconstituted system. Between 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, considerably higher than the stoichiometry obtained with native iNOS (0.5 NADPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and oxygenase domains each retain their separate catalytic functions but interact to catalyze only the second step of NO synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547860 TI - Interfacial properties of the polyheme cytochrome c3 superfamily from Desulfovibrio. AB - In order to compare the interfacial behavior of the polyheme cytochromes c which belong to the cytochrome c3 superfamily, the monomolecular film technique was used to determine whether and how these metalloproteins interact with (phospho)lipids). Measurements of the variations of surface pressure and surface potential versus time have shown differences in their penetration capacity into phosphatidylcholine, dicaprin, and phosphatidylglycerol films. The Desulfovibrio vulgaris Hildenborough cytochrome with 16 hemes (Hmc) and Desulfovibrio desulfuricans Norway tetra- and octaheme cytochromes c3, which have been assumed to be soluble periplasmic molecules, may be considered as extrinsic membrane proteins, unlike the D. vulgaris Hildenborough cytochrome c3 (Mr 13 000). The interfacial properties are discussed in terms of the available three-dimensional structural data, the electrostatic potential calculation, and the results obtained by hydrophobic cluster analysis of the cytochrome sequences. The very different behavior of the two cytochromes c3 (Mr 13 000) enlightens the role of a particular surface loop in the interaction with a model membrane. A functional interpretation is proposed assuming that the D. vulgaris Hildenborough Hmc and both cytochromes c3 (Mr 13 000) and (Mr 26 000) from the Norway strain might provide the link between periplasmic hydrogen oxidation and cytoplasmic sulfate reduction. PMID- 7547861 TI - Electron transfer from the tetraheme cytochrome to the special pair in the Rhodopseudomonas viridis reaction center: effect of mutations of tyrosine L162. AB - The structure of the photosynthetic reaction center (RC) from Rhodopseudomonas viridis is known to high resolution. It contains a firmly bound tetraheme cytochrome from which electrons are donated to a special pair (P) of bacteriochlorophylls, which is photooxidized upon absorption of light. Tyrosine at position 162 of the L-subunit of the reaction center (L 162 Y) is a highly conserved residue positioned halfway between P and the proximal heme group (c 559) of the cytochrome. By specific mutagenesis this residue was exchanged against the amino acids phenylalanine (F), glycine (G), methionine (M), leucine (L), tryptophan (W), threonine (T), and histidine (H). All mutants were expressed in Rps. viridis using a recently established transformation system [Laussermair & Oesterhelt (1992) EMBO J. 11, 777-783]. They were shown biochemically to synthesize all four subunits of the RC (cytochrome, subunits L, M, and H) and to assemble them correctly into the membrane. The structures of two mutants (L 162 F and L 162 T) were determined and found not to differ significantly from the wild type structure. All mutants grew photosynthetically. The absorption spectrum of all the mutants is the same as in WT, but the redox potential of P and of c-559 was changed by the mutations. The kinetics of electron transfer from the heme group to the special pair were measured in chromatophores by flash absorption. As found earlier in the wild type (Y) several exponential components were needed to fit the data.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547859 TI - Laser-flash kinetic analysis of the fast electron transfer from plastocyanin and cytochrome c6 to photosystem I. Experimental evidence on the evolution of the reaction mechanism. AB - The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin (Pc) and cytochrome c6 (Cyt) to photooxidized P700 in photosystem I (PSI) has been studied by laser-flash absorption spectroscopy using a number of evolutionarily differentiated organisms such as cyanobacteria (Anabaena sp. PCC 7119 and Synechocystis sp. PCC 6803), green algae (Monoraphidium braunii), and higher plants (spinach). PSI reduction by Pc or Cyt shows different kinetics depending on the organism from which the photosystem and metalloproteins are isolated. According to the experimental data herein reported, three different kinetic models are proposed by assuming either an oriented collisional reaction mechanism (type I), a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer (type II), or rearrangement of the reaction partners within the complex before electron transfer takes place (type III). Our findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionary replacement of the ancestral Cyt by Pc, as well as the appearance of the fast kinetic phase in the Pc/PSI system of higher plants, would involve structural modifications in both the donor protein and PSI. PMID- 7547862 TI - Catalysis by hamster dihydroorotase: zinc binding, site-directed mutagenesis, and interaction with inhibitors. AB - Hamster dihydroorotase is the central domain of a trifunctional protein which has been cloned, overexpressed, and purified from Escherichia coli. Using the cDNA encoding the dihydroorotase domain, site-directed mutagenesis of amino acid residues conserved between species has enabled identification of three ligands of zinc at the catalytic site as His15, 17 and 158. The underlined amino acids of the nonapeptide sequence Ile12-Asp13-Val14-His15-Val16-His17- Leu18-Arg19-Glu20 from hamster are conserved between dihydroorotases from 8 species. It is proposed that the residues Asp13-His15-->ZnII form a triad at the active site and that Arg19, for which even the conservative mutation Arg19-->Lys yields an inactive enzyme, is involved in substrate binding. Site-directed mutagenesis of the conserved His186-->Ala yielded a mutant enzyme with a reduced affinity for 65Zn2+. The Km for dihydroorotate (DHO) increased from 4.0 to 11 microM, while the Vmax decreased from 1.2 to 0.53 mumol min-1 (mg of protein)-1, implicating this residue in only a minor way with binding of DHO and in catalysis. The mutation Asp230-->Glu resulted in a 14-fold increase in Km and a 16-fold decrease in Vmax, indicating involvement of this conserved residue in both binding and catalysis. The mutation Lys239-->Gly increased the Km for DHO 110-fold with a 2 fold increase in Vmax, suggesting that this residue may form a hydrogen bond with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547864 TI - Potent 2'-amino-2'-deoxypyrimidine RNA inhibitors of basic fibroblast growth factor. AB - Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505 510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets. Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases. We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands to a potent angiogenic factor, basic fibroblast growth factor (bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(14) molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC-3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2'-amino-2' deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycytidine) binds to bFGF with an apparent dissociation constant (Kd) of 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Disassociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10( 3) s-1 (t1/2 = 5.9 min). The calculated value for the association rate constant (kon = k(off)/Kd) was 5.6 x 10(6) M-1 s-1. Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditions with KdbFGF/Kdprotein values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [125I]bFGF binding to both low-affinity sites (ED50 approximately 1 nM) and high-affinity sites (ED50 approximately 3 nM) on CHO cells expressing transfected FGF receptor-1.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547863 TI - Structural and dynamic characterization of the phosphotyrosine binding region of a Src homology 2 domain--phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches. AB - Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pocket of the C-terminal SH2 domain of phospholipase C gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and possibly the Arg 18 guanidino groups. PMID- 7547865 TI - 1H NMR investigation of the electronic and molecular structure of the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus. Identification of Asp 14 as a cluster ligand in each of the four redox states. AB - The molecular and electronic structure of the four-iron cluster of the ferredoxin (Fd) from the hyperthermophilic archaeon, Pyrococcus furiosus, Pf (which has only three Cys in the cluster binding consensus sequence), has been investigated by 1H NMR in order to determine the identity of the noncysteinyl cluster ligand in each of the four redox states [Gorst, C. M., Zhou, Z. H., Ma, K., Teng, Q., Howard, J. B., Adams, M. W., & La Mar, G. N. (1995) Biochemistry 34, 8788-8795], and to characterize the electron spin ground state for the reduced cluster which at 10 K exhibits an unusual predominant S = 3/2 ground state [Conover, R. C., Kowal, A. T., Fu, W., Park, J. -B., Aono, S., Adams, M. W. W., & Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541]. It is demonstrated that a combination of 1D and 2D NMR tailored to relaxed resonances allows the location of four hyperfine shifted and paramagnetically relaxed spin systems which dictates that all four cluster ligands are amino acid side chains, rather than a solvent water/hydroxide at the unique non-Cys ligation site. Three of the ligands could be sequence-specifically assigned to the three Cys residues (positions 11, 17, and 56) in the consensus sequence for cluster binding, hence identifying the fourth ligand as Asp 14. It is concluded that the identification of Asp ligation to a 4Fe cluster is readily achieved in the reduced, but not in the oxidized cluster of Fd. Analysis of the relaxation properties and pattern of the hyperfine shifts in Pf Fd reveals very strong similarities to other Fds with S = 1/2 ground states, leading to the conclusion that the S = 3/2 ground state is not detected in solution at ambient temperatures, and this in independent of the redox state of the two remaining Cys residues in the protein (positions 21 and 48). However, the electron self exchange rate for 4Fe Pf Fd is significantly slower than for other 4Fe Fd with complete Cys ligation. Changes in the pattern of hyperfine shifts between oxidized and reduced clusters for the four ligands in Pf Fd reveal that the most significant variation occurs for the Asp 14 orientation, suggesting that the altered Asp orientation may "gate" the electron transfer. PMID- 7547866 TI - Three-dimensional solution structure of the cyanide adduct of a Met80Ala variant of Saccharomyces cerevisiae iso-1-cytochrome c. Identification of ligand-residue interactions in the distal heme cavity. AB - The 1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/Cys102Ser) in the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOEs. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 +/- 0.11 and 1.32 +/- 0.14 A for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-6419]. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding "distal cavity". The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins. PMID- 7547867 TI - NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer. AB - Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N- 1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants. PMID- 7547868 TI - Calcium-dependent solvation of the myristoyl group of recoverin. AB - Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene 8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation. PMID- 7547869 TI - Positive cooperativity with Hill coefficients of up to 6 in the glutamate concentration dependence of steady-state reaction rates measured with clostridial glutamate dehydrogenase and the mutant A163G at high pH. AB - Glutamate dehydrogenases from many sources display nonclassical kinetic behavior suggestive of allosteric interaction among the six subunits of the hexamer. A three-dimensional structure now potentially offers a framework for explaining the basis of such behavior in clostridial glutamate dehydrogenase, and this paper offers evidence of extreme, all-or-none cooperativity in the binding of glutamate by this enzyme. A site-directed mutant of clostridial glutamate dehydrogenase in which Ala163 in the glutamate binding site is replaced by glycine displays a markedly sigmoid dependence of reaction rate on glutamate concentration (S0.5 = 200 mM), with a Hill coefficient of 3.4 when assayed at pH 10.5 with 1 mM NAD+. Under the same conditions the wild-type enzyme gave no measurable rate with glutamate concentrations in the range normally used for kinetics (0-100 mM) but gave a steep rise in reaction rate from 600 to 1200 mM glutamate. At pH 9.0, where the wild-type enzyme has previously been shown to be "inactive" in a standard assay, a study extending to much higher glutamate concentrations again revealed a sigmoid dependence, with a Hill coefficient of 5.4 and an S0.5 at 150 mM glutamate. With the mutant A163G the apparent cooperativity was less, with a Hill coefficient of 2.3, and the affinity for glutamate was higher, with S0.5 of 7 mM. Both proteins gave normal hyperbolic dependence on glutamate concentration at pH 7 and pH 8. At pH 9 and with saturating glutamate, both enzymes showed a hyperbolic dependence of the rate on NAD+ concentration. The NAD+ concentration, however, affected the observed degree of cooperativity with varied glutamate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547870 TI - Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core. AB - The structure of [14-38]Abu, a variant of bovine pancreatic trypsin inhibitor (BPTI) with only the 14-38 disulfide bridge intact, has been analyzed by two dimensional 1H and 1H-15N NMR. Except for the 18-24, 29-35 antiparallel beta sheet, residues in all regions of the molecule give two exchange cross peaks for each 1H; for one residue, Gly 37, three exchange cross peaks are observed. The presence of exchange cross peaks indicates that the residues sample conformations that interconvert on a time scale of milliseconds or longer. Over 90% of the NMR spectra have been assigned, including backbone and side chain atoms and their exchange cross peaks. Analyses of chemical shifts, chemical exchange, hydrogen isotope exchange, and NOEs indicate that [14-38]Abu at pH 4.5 and 1 degree C is an ensemble of interconverting conformations, in all of which the 18-24, 29-35 antiparallel beta-sheet is native-like and intact. Outside the antiparallel beta sheet, residues undergo local order/disorder transitions. The stable structure of [14-38]Abu is not in the vicinity of the 14-38 disulfide bond but rather is in the slow-exchange core. NOE analysis indicates that the main tertiary interactions involve hydrophobic contacts with the rings of Tyr 21, Tyr 23, and Tyr 35. As a model for early folding intermediates, the structure of [14-38]Abu suggests that BPTI folding is initiated by stabilization of a turn existing in the unfolded protein and involves both local and nonlocal hydrophobic interactions. PMID- 7547871 TI - Comparative studies of the monomeric and filamentous actin-myosin head complexes. AB - The functional and structural properties of the monomeric and filamentous actin myosin head (S1) complexes were compared under strictly controlled conditions which avoid the S1-induced polymerization of monomeric actin. Under these conditions, monomeric (G) and filamentous (F) actin were found to activate S1 Mg(2+)-ATPase by 3- and 120-fold, respectively, in the presence of a 5-fold excess of actin over S1. Using the change in fluorescence intensity of pyrene-G actin induced by S1 binding in the presence of various nucleotide analogues, we discovered that the ternary G-actin-S1-AMPPNP complex could not be formed. Moreover, the association constants of G-actin to S1-ADP or of ADP to the G-actin S1 complex were at least an order of magnitude lower than in the filamentous state. Such a low affinity between G-actin and the S1-nucleotide intermediates can reasonably explain the lack of ATPase activation by the monomeric complex. Analysis of the G-actin-S1 interface by chemical cross-linking and limited proteolytic experiments showed that, in the monomeric complex, S1 interacted almost exclusively by its positively charged segment 636-642 with the patch of negative residues located on the actin flexible loops 1-7, 20-28, and 90-100. Moreover, the variation in the cross-linking pattern and in the proteolytic susceptibility of S1 segment 636-642 demonstrated that this electrostatic interface was different in the monomeric and the filamentous complexes. Taken together, the results suggested that the G-actin-S1 interaction encompasses only a small fraction of the strong as well as of the weak F-actin-S1 interface. The monomeric complex would in fact resemble more the collision complex which takes place early in the F-actin-S1 interaction. PMID- 7547872 TI - Functional significance of the binding of one myosin head to two actin monomers. AB - The functional significance of the interaction of one myosin head (S1) with two actin monomers was investigated by comparing the properties of the cross-linked monomeric and filamentous actin-S1 complexes. S1 was cross-linked to monomeric actin (G-actin) either in the absence or in the presence of DNase I by 1-ethyl-3 [3-(dimethylamino)propyl]carbodiimide. The binary G-actin-S1 and ternary DNase I G-actin-S1 complexes were then purified by a combination of ion exchange and gel filtration columns. Both the binary and the ternary complexes were characterized by negligible, though different, Mg(2+)-ATPase activities of 0.018 and 0.006 s 1s(-1), respectively. Using fluorescence, light-scattering, and ultracentrifugation experiments, we found that only the binary G-actin-S1 complex could be polymerized in the presence of 2 mM MgCl2. Electron microscopic analysis of the cross-linked filamentous complex showed fully decorated filaments with the arrowhead pattern characteristic of the non-covalent complex in the rigor state. Such a 100% cross-linked F-actin-S1 complex exhibited a Mg(2+)-ATPase activity of 6.2 +/- 0.8 s-1, slightly lower than the maximum velocity of the non-cross-linked complex of 8.6 +/- 0.8 s-1, but comparable to the 6.9 +/- 0.6 s-1 obtained for a partially (35%) cross-linked complex. These results implied that the activation of S1 ATPase by actin requires the interaction of S1 with a second actin monomer within the thin filament. They also suggested that the full activation of the filamentous complex is not dependent on the degree of saturation of the thin filament by myosin. PMID- 7547874 TI - Control of the depth of molecules within membranes by polar groups: determination of the location of anthracene-labeled probes in model membranes by parallax analysis of nitroxide-labeled phospholipid induced fluorescence quenching. AB - The location of anthracene-labeled molecules incorporated into model membranes was measured by fluorescence quenching. The depth of the anthracene group was calculated from the degree of quenching by lipids carrying a nitroxide at different depths, using the parallax analysis (Chattopadhyay & London (1987) Biochemistry 26, 39-45). A series of anthracene derivatives was examined in order to determine what polar functional groups would anchor at the membrane surface, and at what depth anchoring would occur. An anthracene with only a methyl group was not anchored at the membrane surface, but derivatives with polar or charged groups did anchor near the membrane surface as demonstrated by a shallower anthracene depth. Based on anthracene depths, protonated primary amine, secondary amine, and hydroxyl groups appear to be located 15-16 A from the center of the membrane. A quaternary amino locates more shallowly, at 18 A from the bilayer center. A protonated carboxyl group is slightly deeper, at 14 A from the center of the bilayer. Ester groups are found to be weakly anchoring, having a location dependent on the structure of the molecule to which they are attached. In methyl 9-anthracenepropionate, the ester group is located about 13 A from the bilayer center. Anthracene esters attached to cholesterol or cholesterol esters showed various depths. An anthracene ester attached to the tail of cholesterol was located 1-6 A from the center of the bilayer for a cholesterol derivative, but at 12 A from the bilayer center for a cholesterol oleate derivative.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547873 TI - Release of biological activities from quiescent fibronectin by a conformational change and limited proteolysis by matrix metalloproteinases. AB - We reported that specific biological activities are confined to three domains of the fibronectin (Fn) molecule [Fukai et al. (1991) J. Biol. Chem. 266, 8807; Fukai et al. (1993) Biochemistry 32, 5746]: the potent ability to stimulate the adipocyte differentiation of ST-13 cells is in the amino-terminal fibrin-binding (Fib 1) domain (referred to as Fib 1 domain activity); the RGD-dependent activities that stimulate NIH-L13 cell migration and inhibit adipocyte differentiation are in the central cell-binding (Cell) domain (Cell domain activity); and the activity that stimulates cell migration in a RGD-independent manner is in the carboxyl-terminal fibrin-binding (Fib 2) domain (Fib 2 domain activity). Human plasma Fn which was purified without exposure to a denaturant, such as urea, exhibited no Fib 1, Fib 2, or Cell domain activity. By exposure to urea or surface adsorption, Fn showed Cell domain activity but not those of the Fib 1 and Fib 2 domains. Whether the cryptic domain activities are disclosed or not depended on whether or not the responsible domains were irreversibly exposed from confined environments of Fn structure as confirmed by light-scattering measurement and enzyme immunoassay using domain-specific monoclonal antibodies. We then investigated the action of matrix metalloproteinases (MMPs) in liberating the Fib 1, Cell, and Fib 2 domain activities. Matrilysin released only the Cell domain activity. In contrast, stromelysin, collagenase, and especially gelatinase A additionally liberated the Fib 1 and Fib 2 domain activities. The Fib 1, Fib 2, and Cell domains acquired much higher activities when they were freed from linkage with adjacent domains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547875 TI - 4-4-20 anti-fluorescyl IgG Fab' recognition of membrane bound hapten: direct evidence for the role of protein and interfacial structure. AB - The surface forces apparatus was used to identify the molecular forces that control the interactions of monoclonal 4-4-20 antifluorescyl IgG Fab' fragments with fluorescein-presenting supported planar bilayers. At long range, the electrostatic force between oriented Fab' and fluorescein monolayers was controlled by the composition of the protein exterior surrounding the antigen combining site rather than by the overall protein charge. The measured positive electrostatic potential of the Fab' monolayer at pH > pI(Fab') was consistent with the structure of the exposed Fab' surface in which a ring of positive charge at the mouth of the antigen-combining site dominates the local electrostatic surface properties. Substantial differences in the electrostatic forces measured with denatured Fab' further demonstrated that the measured electrostatic surface properties and the consequent long-range interaction forces are controlled by the protein surface composition. At short range, the strength of the Fab'-mediated adhesion was modulated not only by the length of the fluorescein tether but also by membrane hydration. Steric hydration barriers at the membrane surface reduced the adhesion strength in proportion to their range of influence. These results provide direct evidence that long-range protein interactions with immobilized ligands are controlled by both the protein and the membrane surface compositions, while short-range, specific binding is modulated by both the protein structure and the membrane interfacial properties. PMID- 7547876 TI - Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles. AB - Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity. PMID- 7547878 TI - Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide. AB - Nitric oxide (NO) generated by diethylamine nonoate (DEA/NO), an NO donor, readily oxidized the free sulfhydryl group of human serum albumin (HSA) as well as the sulfhydryl groups of reduced glutathione (GSH) and dithiothreitol (DTT) at pH 7.4 and 37 degrees C. Under anaerobic conditions, the major products of the oxidation of HSA thiol by NO were the sulfenic acid (RSOH) of HSA and nitrous oxide (N2O). The stoichiometry for this reaction, viz., 1 mol of HSA sulfhydryl oxidized to 1 mol of N2O produced, is consistent with a net two-electron oxidation of the protein thiol to a sulfenic acid. The sulfenic acid product of HSA was shown to react with dimedone and GSH, two known reactions of sulfenic acids. In contrast, anaerobic oxidation of GSH and DTT by NO gave a stoichiometry close to the expected ratio of 2:1 (sulfhydryl oxidized to N2O produced) for the oxidation of these thiols to their disulfides and N2O. Under aerobic conditions, significant fractions of the sulfhydryl groups of HSA, GSH, and DTT were oxidized to their respective thionitrites, presumably by N2O3. Thionitrite formation was not observed in the absence of oxygen. The production of HSA-sulfenic acid by NO, as well as by other oxidizing agents such as H2O2 and peroxynitrite, followed by its reaction with circulating GSH or L-Cys may account for the mixed disulfides of HSA observed in plasma. PMID- 7547880 TI - Inactivation of steroid sulfatase by an active site-directed inhibitor, estrone-3 O-sulfamate. AB - Steroid sulfatases are responsible for the hydrolysis of 3beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pKa values of the enzymatic reaction and pKa of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pKa of tyrosine (9.7) and the pKa of histidine will allow the roles that (6.8) are similar to the pKa values of inactivation, these amino acid residues may play a role in the catalytic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547879 TI - Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3. AB - We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547877 TI - Modulation of the substrate specificity of the mammalian phosphatidylinositol 3 kinase by cholesterol sulfate and sulfatide. AB - The substrate specificity of the purified, mammalian phosphatidylinositol 3 kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed. PMID- 7547881 TI - Glutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase. AB - Pyrroline-5-carboxylate, an intermediate in the biosynthesis and degradation of glutamate, proline, and ornithine, acts as a strong reversible inhibitor of glucosamine-6-phosphate synthase, competitive with respect to glutamine. Proton magnetic resonance spectroscopy shows that, under these conditions, pyrroline-5 carboxylate exists in rapid equilibrium with glutamate gamma-semialdehyde (0.05%). The observed variation of Ki with pH is consistent with inhibition by this rare species. Glutamate gamma-semialdehyde is expected to react reversibly with a cysteine residue at the active site, identified by earlier inactivation studies, to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. The apparent Ki value of glutamate gamma-semialdehyde is approximately 3 x 10(-8) M. PMID- 7547882 TI - Trypsin specificity increased through substrate-assisted catalysis. AB - Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis. PMID- 7547884 TI - Crystal structure of a peptidyl pyridinium methyl ketone inhibitor with thrombin. AB - The crystal structure of a complex between a bivalent peptidyl pyridinium methyl ketone inhibitor and human alpha-thrombin has been solved and refined at 2.0 A to an R factor of 0.18. The inhibitor, (D)cyclohexylalanine-Pro-Arg-(CH2N+C5H4CH2CO) (Gly)4-Asp- Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-cyclo-hexylalanine-(D)Glu (coded P596), which forms a reversible covalent complex with thrombin, is highly potent with a Ki = 4.6 +/- 1.0 x 10(-14) M, lower than that of recombinant hirudin. The N-terminal, active-site-directed portion of the inhibitor is linked to the fibrinogen recognition exosite binding portion by a tetraglycine segment. The strong electron-withdrawing effect provided by the permanent positive charge on the pyridinium nitrogen makes the arginyl carbonyl carbon more susceptible to nucleophilic attack. In the crystal, a covalent P596-thrombin complex is observed. The electron density surrounding the active site portion and the pyridinium of the inhibitor is very well defined, clearly showing the existence of a covalent bond between the Ser195 O gamma and the now tetrahedral carbon of the inhibitor. The decreased binding ability of thrombin inhibitors containing N terminal acetylation is discussed as is the effect of replacing the P3 (D)phenylalanine with (D)cyclohexylalanine. The electron density surrounding the remainder of the inhibitor is generally well defined, the exceptions being the C terminal (D)Glu, the highly flexible tetraglycine linker, and some of the solvent directed side chains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547883 TI - Specificity and orientation of trigonal carboxyl esters and tetrahedral alkylphosphonyl esters in cholinesterases. AB - We have examined the specificity of planar carboxyl and tetrahedral phosphonyl esters for mouse cholinesterases and have delineated the orientation of these ligands in the enzyme active center. The approach involved altering acyl pocket dimensions by site-specific mutagenesis of two phenylalanines and varying ligand size and enantiomer presentation. Substrate catalysis rates by wild type acetylcholinesterase (AChE) of acetyl-, butyryl-, and benzoylthiocholine diminished with increasing size of the acyl moiety. In contrast, substitution of the acyl pocket phenylalanines giving the mutants F295L and F297I of AChE yielded more efficient catalysis of the larger substrates and a specificity approaching that of butyrylcholinesterase. Extension from planar substrates to enantiomerically pure organophosphonates allowed for an analysis of enantiomeric selectivity. We found that AChE reactions are 200-fold faster with the Sp than the Rp enantiomer of of cycloheptyl methylphosphonyl thiocholine. Upon the acyl pocket size being enlarged, the Rp enantiomer became more reactive while reaction with the Sp enantiomer was slightly reduced. In fact, the F297I mutant displayed inverted stereospecificity. A visual correlation with the kinetic data has been developed by docking the ligands in the active site. Upon placement of the phosphonyl oxygen in the oxyanion hole and the leaving group being directed out of the gorge, the Rp, but not the Sp, enantiomer engendered steric hindrance between the alkoxyl group and the acyl pocket. Replacing F297 with Ile accommodated the bulky alkoxyl group of the Rp isomer in the acyl pocket, allowing similar orientations of the phosphonyl oxygen and the leaving group to the Sp isomer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547885 TI - Molecular characterization of a cyclosporin A-insensitive cyclophilin from the parasitic nematode Brugia malayi. AB - The cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosporin A (CsA) to varying degrees. We have isolated a cDNA clone encoding a novel cyclophilin from the human filarial parasite Brugia malayi. This gene possesses an N-terminal domain homologous to cyclophilins from diverse phyla (49 60% amino acid sequence identity) and a hydrophilic C-terminal domain. The cyclophilin domain was overexpressed in Escherichia coli and found to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 7.9 x 10(6) M-1 s-1. A histidine residue in lieu of tryptophan in the highly conserved CsA-binding site suggests that B. malayi cyclophilin is more closely related to the cyclophilin-like proteins described recently from natural killer (NK) cells, plants, and the 40 kDa cyclophilins from mammals. In accordance with the histidine-containing CsA-binding domain, the B. malayi enzyme was relatively insensitive to inhibition by CsA, since an IC50 value of 860 nM (compared to 19 nM for human cyclophilin A) was determined. PMID- 7547886 TI - Effects of temperature and glycerides on the enhancement of Agkistrodon piscivorus piscivorus phospholipase A2 activity by lysolecithin and palmitic acid. AB - The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer. PMID- 7547887 TI - Insertion of diphtheria toxin in lipid bilayers studied by spin label ESR. AB - The pH dependence of the insertion of diphtheria toxin into bilayers of dioleoylphosphatidylglycerol (DOPG) has been studied by using ESR spectroscopy of spin-labeled phosphatidylglycerol with the reporter group at either the 5 position or the 14-position of the sn-2 chain (5-PGSL and 14-PGSL, respectively). At neutral pH, addition of diphtheria toxin has little effect on the ESR spectra of either spin label in large unilamellar vesicles of DOPG. At acidic pH, the outer hyperfine splitting of the 5-PGSL label is increased, and a second component corresponding to lipids whose chain motion is selectively restricted appears in the spectra of the 14-PGSL label, in the presence of diphtheria toxin. The motionally restricted component of 14-PGSL has a large outer hyperfine splitting (2Amax approximately 61 G) and corresponds to spin-labeled lipids the chains of which are in direct contact with the membrane-penetrant part of the inserted toxin. This restricted component is present, although to a lesser extent, in vesicles containing 90% of the zwitterionic lipid dioleoylphosphatidylcholine and displays a limited selectivity for negatively charged relative to zwitterionic spin-labeled phospholipids. The fraction of lipids which are motionally restricted by the toxin increases with decreasing pH, titrating in DOPG vesicles with an apparent pKa of approximately 6.1. The outer hyperfine splitting of the 5-PGSL label titrates with an apparent pKa of approximately 5.5, suggesting that this might be preferentially sensitive to a later stage in the insertion of the toxin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547888 TI - Lateral organization of liquid-crystalline cholesterol dimyristoylphosphatidylcholine bilayers. Evidence for domains with hexagonal and centered rectangular cholesterol superlattices. AB - The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole fractions (CMFs). The most notable of these occurred at CMFs of 0.15, 0.33, and 0.67. Deviations were also frequently observed at CMFs of 0.12, 0.20, 0.25, and 0.40. Merocyanine 540 reproducibly reported deviations at CMFs of 0.15 and 0.33 and frequently reported values close to 0.12, 0.20, and 0.25. In absorbance (turbidity) and light scattering versus CMF plots, well-defined kinks were observed at CMFs of 0.16, 0.33, 0.52, and 0.67. The occurrence of kinks or other deviations at those particular CMFs is most readily explained in terms of a superlattice model previously developed to explain the lateral distribution of pyrenylphospholipids in bilayers [Somerharju, et al. (1985) Biochemistry 24, 2773-2781; Virtanen, J. A., et al. (1988) J. Mol. Electron. 4, 233-236]. This model is based on the assumptions that (i) each cholesterol molecule replaces a single acyl chain in a hexagonal lattice, (ii) cholesterol molecules, because of their larger size, perturb the lattice, (iii) this perturbation is minimized when the cholesterol molecules are maximally separated from each other, and (iv) the maximal separation is achieved when the cholesterol molecules form a hexagonal or centered rectangular superlattice. All detected critical CMFs, except that at CMF 0.67, are predicted by the model, thus strongly supporting its validity. The critical CMF at 0.67 is a limiting case, which can be accounted for by assuming that cholesterol and phospholipid molecules form alternating rows, i.e., formation of a cholesterol superlattice with rectangular symmetry. As predicted by the superlattice model, composition-driven order-to-disorder transitions occur between the critical CMFs, as indicated by increased data scatter and sample fluctuations in those regions. Another important prediction of the superlattice model is that domains with different cholesterol superlattices should coexist at most cholesterol concentrations. Such domains do not have to be extensive to account for the critical events observed here; rather, they are expected to be dynamic entities of limited size. It is very likely that such microscopic domains with distinct cholesterol superlattices also coexist in biological membranes. This is expected to have remarkable effects on both the structure and functions of these membranes. PMID- 7547889 TI - Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA. AB - We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5 methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions. PMID- 7547890 TI - Maduropeptin: an antitumor chromoprotein with selective protease activity and DNA cleaving properties. AB - Maduropeptin (MDP) is a recently isolated antitumor antibiotic, consisting of an enediyne-containing chromophore embedded in a highly acidic protein. This holoantibiotic damages duplex DNA in vitro, producing a mixture of single- and double-strand breaks at selected sites. The chromophore, isolated as the methanol adduct from the protein-containing holoantibiotic, exhibits the same selectivity and cleavage chemistry as the chromoprotein complex. Preliminary evidence suggests that the primary DNA breaks involve 4'-H abstraction from the deoxyribose sugars at the cleavage sites. Unlike most other enediyne antitumor antibiotics, DNA strand scission is not bioreductively induced by MDP or the methanol adduct of the chromophore. This was also observed for the C1027 chromophore. DNA cleavage is inhibited in the presence of certain cations (Ca2+, Mg2+) as was observed with the kedarcidin chromophore. 1H NMR spectroscopy studies on the methanol adduct of the maduropeptin chromophore in the presence of calcium chloride provide clues regarding its activation and give insight as to the regions of the chromophore important for DNA binding. Our results suggest that the solvent artifact of the chromophore may in essence be a prodrug and it regenerates the parent chromophore as in the holoantibiotic prior to cleaving DNA. As with kedarcidin and neocarzinostatin, maduropeptin exhibits a high affinity for histones, in vitro, cleaving them to low molecular mass peptides. Histone H1, the most opposite in net charge, is cleaved most readily. This latter activity may serve to disrupt the chromatin superstructure in vivo, prior to exposing the DNA to the chromophore. PMID- 7547891 TI - Trans effects on cysteine ligation in the proximal His93Cys variant of horse heart myoglobin. AB - Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547892 TI - Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: symmetry of the carbonyl interactions and close equivalence of the QB vibrations in Rhodobacter sphaeroides and Rhodopseudomonas viridis probed by isotope labeling. AB - The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were 18O on both carbonyls and 13C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The QB-/QB spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for a number of bands attributed to vibrations of QB and QB-. The vibrational modes of the quinone in the QB site are compared to those of ubiquinone in vitro, leading to band assignments for the C = O and C = C vibrations of the neutral QB and for the C***O and C***C of the semiquinone. The C = O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm-1 for both species. This demonstrates symmetrical and weak hydrogen bonding of the two C = O groups to the protein at the QB site. In contrast, the C = C vibrations are not equivalent for selective labeling at C1 or at C4, although they both contribute to the approximately 1617-cm-1 band in the QB-/QB spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C = C mode of QB does not involve displacement of the C4 carbon atom, while the motion of C1 is not hindered. Further analysis of the the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C4 carbonyl. The FTIR observations provide compelling evidence for almost identical conformation and identical interactions of the ubiquinone in the QB binding site of Rb. sphaeroides and Rp. viridis in contrast to the X-ray structures, which yield different descriptions for the hydrogen-bonding pattern of QB binding. In the semiquinone state, the bonding interactions of the C***O groups are also symmetrical and the C***C are inequivalent at C1 and C4. However, the interactions are almost the same in the RCs of both species. PMID- 7547893 TI - PhoE signal peptide inserts into micelles as a dynamic helix-break-helix structure, which is modulated by the environment. A two-dimensional 1H NMR study. AB - Proteins that are destined for export out of the cytoplasm of Escherichia coli cells are synthesized as precursor proteins with N-terminal extensions or signal sequences, which are essential for translocation of the protein across the inner membrane. Signal sequences contain very little primary sequence homology, and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of signal sequences, we have studied the signal peptide of PhoE (MKKSTLALVVMGIVASASVQA) by two-dimensional nuclear magnetic resonance and circular dichroism in different membrane mimetic environments. The secondary structure of the PhoE signal peptide was analyzed via interresidue nuclear Overhauser enhancement measurements, chemical shifts of backbone protons, and by measuring amide proton exchange. The membrane mimetic environments studied were trifluoroethanol (TFE) and micelles of sodium dodecyl sulfate (SDS) or dodecylphosphocholine (DPC). In all systems alpha-helix formation was observed. In TFE, the alpha-helix stretches from the positively charged N-terminus to Ser18. In SDS and DPC micelles, the N- and C-terminal alpha helical half are separated from each other by a kink at the Gly12 position, with the helical content being higher at the N-terminus and lower at the C-terminus. In zwitterionic DPC micelles, the C-terminal region has a less regular or more flexible structure compared to SDS. The insertion of the PhoE signal peptide into the hydrophobic environment of the micelles was demonstrated by the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547894 TI - Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors. AB - The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+) ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4 benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+) induced calpain activation and a fusion process inhibited by annexin V. PMID- 7547895 TI - Kinetics of nucleotide-induced changes in the tryptophan fluorescence of the molecular chaperone Hsc70 and its subfragments suggest the ATP-induced conformational change follows initial ATP binding. AB - The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment. PMID- 7547896 TI - A designed heterotrimeric coiled coil. AB - Principles that guide folding of coiled coils were tested by designing three peptides that preferentially associate with each other to form a heterotrimeric coiled coil. The core positions of the designed helices contained residues that promote formation of trimeric coiled coils. Ionic interactions were employed to mediate heterospecificity, and negative design was used to favor formation of the heterotrimer over alternative arrangements. A program was written to select sequences that maximized the number of attractive interhelical interactions in a parallel heterotrimer and the number of repulsive electrostatic interactions in alternative species. Solution studies indicate that an equimolar mixture of the three peptides forms a helical trimer with high specificity and stability. These results validate the principles used to guide the design and suggest that the heterotrimer may serve as a useful, autonomous trimerization domain. PMID- 7547897 TI - Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates. AB - The equilibrium denaturation of an Escherichia coli-derived recombinant murine interleukin-6 (mIL-6) was studied using fluorescence and circular dichroism spectroscopy. The urea-induced unfolding of mIL-6 at pH 4.0 can be described by a two-state unfolding mechanism based on the superimposibility of the CD and fluorescence unfolding transitions. Assuming a two-state mechanism and a linear dependence of the free energy of unfolding on denaturant concentration, a value of 6.9-9.0 kcal/mol was calculated for the free energy of unfolding in the absence of denaturant [delta GU(H2O)]. However, when GuHCl was used as a denaturant at pH 4.0, a biphasic unfolding transition was observed. This unfolding transition has a distinct midpoint occurring at 2.5 M GuHCl, which is indicative of the formation of stable folding intermediates. Similar intermediate folded species were also observed at pH 7.4 when either urea or GuHCl were used as denaturants. The intermediate folded states of mIL-6 exhibited a tendency to aggregate, as judged by the concentration dependence of their fluorescence characteristics. The fluorescence emission maximum of mIL-6 at pH 7.4 in the presence of 1.5 M GuHCl, for example, was blue-shifted from 343 nm at a protein concentration of 50 micrograms/mL to 336 nm at 500 micrograms/mL. Intermediate formation at pH 4.0, using 10 mM sodium acetate buffer and urea as the denaturant, was facilitated by the addition of 0.4 and 0.8 M salt, where the salt was either NaCl or GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547898 TI - Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant. AB - The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site. PMID- 7547899 TI - Hydroxybenzoyl-CoA reductase: coupling kinetics and electrochemistry to derive enzyme mechanisms. AB - Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint potential of the enzyme active site, and the kinetic parameter, V/K. By coupling electrochemistry and steady-state kinetics, mechanistic information could be obtained that could not be determined by either method alone. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found to be: apparent V/Km = Vmax/(Km(1 + exp[nF/RT(E - E(o)e)])), where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E(o)e is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics with electrochemistry yielded the E(o)e (-350 mV vs NHE) for HBCR and maximum values under optimal redox conditions for kcat and kcat/Km (9 s-1 and 1.8 x 10(5) M-1 s 1, respectively). In addition, theory was developed that could distinguish a single two-electron transfer mechanism from one involving two successive one electron transfers. HBCR was found to be in the latter class. Interestingly, the derived mechanism for HBCR is similar to that of the Birch reduction, the classical organic chemical reaction for reductive dehydroxylation of phenolic compounds. The methodology described here represents a novel approach that should help elucidate the mechanisms of other oxidoreductase and redox-activated enzymes. PMID- 7547900 TI - Role of binding energy with coenzyme A in catalysis by 3-oxoacid coenzyme A transferase. AB - Succinyl-CoA:3-oxoacid coenzyme A transferase (EC 2.8.3.5), which catalyzes the reversible conversion of succinyl-CoA and acetoacetate into acetoacetyl-CoA and succinate through a covalent enzyme thiol ester intermediate, E-CoA, utilizes binding energy from noncovalent interactions with CoA to bring about an increase in kcat/KM of approximately 10(10)-fold. The approximately 40-fold stronger binding of desulfo-CoA (KI = 2.7 +/- 0.7 mM) compared to desulfopantetheine (KI = 110 +/- 15 mM), both of which inhibit competitively with respect to acetoacetyl CoA, shows that binding to the nucleotide domain of CoA at the active site provides ca. -2.2 kcal/mol of binding energy to stabilize noncovalent complexes with the enzyme. This is much smaller than the ca. -8.9 kcal/mol that the nucleotide domain contributes to the stabilization of the transition state and the ca. -7.2 kcal/mol that it contributes to stabilizing the E-CoA intermediate [Fierke, C. A., & Jencks, W. P. (1986) J. Biol. Chem. 261, 7603-7606]. This shows that most of the approximately 10(6)-fold increase in kcat/KM that is brought about by binding to this domain is in kcat, which is increased by a factor of about 10(5). Binding to the central pantoic acid domain of CoA is stronger in the transition state than in the Michaelis complex by ca. -3.4 kcal/mol; this corresponds to an additional increase in kcat of approximately 350-fold. Covalent enzyme thiol esters analogous to E-CoA but containing the short-chain CoA analogues N-acetylaletheine (NAA) and N-acetylcysteamine (NAC) are more stable than the enzyme thiol ester containing pantetheine (E-Pant) by approximately 3.5 and approximately 4.8 kcal/mol, respectively. Thus, interactions between the pantoic acid domain of CoA and the active site destabilize E-CoA by approximately 4.8 kcal/mol, approximately 1.3 kcal/mol of which arises from interaction with the amide group of the pantoic acid domain and approximately 3.5 kcal/mol of which arises from interaction with other portions of the pantoic acid domain. E Pant is more reactive toward acetoacetate and succinate by a factor of approximately 10(7) than E-NAA and E-NAC. This shows that the destabilization caused by these interactions in E-CoA is relieved in the transition state, in which binding to the pantoic acid moiety is strongly favorable with delta delta G approximately -5.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547901 TI - Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase. AB - Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5 (3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate. PMID- 7547903 TI - Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 2. Reactions of the galactosyl-enzyme intermediate with alcohols and azide ion. AB - Velocities for the synthesis of beta-D-galactopyranosyl derivatives by transfer of the galactosyl group from beta-galactosidase to seven alkyl alcohols, glucose, and azide ion have been determined as the difference in the velocities for beta galactosidase-catalyzed cleavage of 4-nitrophenyl beta-D-galactopyranoside to give 4-nitrophenoxide anion (v PNP) and hydrolysis of this compound to give D galactose (v Gal). Rate constant ratios kROH/ks (M-1) for partitioning of the galactosylated enzyme between reaction with alkyl alcohols and solvent determined by this method are in good agreement with values of kROH/ks (M-1) determined by analysis of alcohol inhibition of enzyme-catalyzed hydrolysis of the corresponding alkyl beta-D-galactopyranosides. Absolute rate constants kROH (M-1 s-1) for reaction of alkyl alcohols with the galactosylated enzyme intermediate were calculated from the corresponding rate constant ratio kROH/ks (M-1) and ks = 710 s-1. A Bronsted parameter of (beta nuc) ROH = -0.19 +/- 0.10 was determined from the second-order rate constants for the reactions of alcohols with the galactosylated enzyme. The large difference between (beta 1g)kcat/Km = -0.75 +/- 0.14 for cleavage of alkyl beta-D-galactopyranosides to form the galactosylated enzyme and (beta nuc)ROH = -0.19 for the reverse synthesis reaction requires that the equilibrium constants for galactosyl group transfer from alkyl beta-D galactopyranosides to the enzyme increase sharply with decreasing pKa of the alkyl alcohol leaving group. These data give beta eq = -0.56 +/- 0.05 for the reaction of alkyl beta-D-galactopyranosides with ethanol to form ethyl beta-D galactopyranoside and alkyl alcohol. Several effects that lead to this increased ease of cleavage of alkyl beta-D-galactopyranosides with decreasing basicity of the alkoxy group are discussed. A second-order rate constant of kGlc = 1.2 x 10(4) M-1 s-1 was determined for reaction of glucose with the galactosylated enzyme. The relatively low reactivity of glucose is surprising, because an earlier observation that the galactosylated enzyme complex generated by the cleavage of lactose undergoes release of glucose and synthesis of allolactose at nearly equal rates suggests that the binding of glucose to the galactosylated enzyme should be partly irreversible and that it takes place near the encounter controlled limit. The data suggest a significant stabilization of nonproductive complexes formed by binding of glucose to the galactosylated enzyme. beta Galactosidase catalyzes the hydrolysis of beta-D-galactopyranosyl azide, but not the synthesis of this compound by reaction of azide ion with the galactosylated enzyme.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7547902 TI - Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 1. Bronsted parameters for cleavage of alkyl beta-D-galactopyranosides. AB - Seven substituted alkyl beta-D-galactopyranosides 1-OR have been prepared and shown to be fair to excellent substrates for hydrolysis catalyzed by beta galactosidase (Escherichia coli, lac Z). Bronsted parameters of (beta 1g)k3 = 0.49 +/- 0.13 and (beta 1g)kcat/Km = -0.75 +/- 0.14, respectively, were determined at pH 8.6 for k3 (s-1), the first-order rate constant for cleavage of enzyme-bound 1-OR, and kcat/Km (M-1 s-1), the second-order rate constant for reaction of the free enzyme and 1-OR. There is a weak correlation between log Km and the pKa of the alkyl alcohol leaving group, which is attributed to stabilization of the Michaelis complex by hydrophobic interactions between the enzyme and electron-withdrawing halogen substituents at the alkoxy leaving group. These binding interactions are probably both productive and expressed in the value of kcat/Km and nonproductive and expressed in the value of k3. The negative values of beta 1g are inconsistent with enzymatic catalysis of endocyclic cleavage of the glycosidic bond. The values of beta 1g for enzyme-catalyzed cleavage of alkyl beta-D-galactopyranosides lie between those observed for the spontaneous (beta 1g approximately -1.25) and specific-acid-catalyzed (beta 1g approximately 0) cleavage of acetals, and these pathways are therefore excluded for the enzyme-catalyzed reaction. Removal of the metal cofactor Mg2+ from the enzyme causes a approximately 0.2 unit decrease in (beta 1g)k3 for beta galactosidase-catalyzed cleavage of 1-OR. The interpretation of this change in beta 1g is unclear. The Bronsted coefficients for the beta-galactosidase catalyzed reaction are consistent with participation by an essential catalytic residue in concerted general-acid catalysis of cleavage of the glycosidic bond of 1-OR and/or stabilization of developing negative charge at the alkoxy oxygen by interaction with the magnesium ion cofactor. PMID- 7547904 TI - Functional properties of the individual thioredoxin-like domains of protein disulfide isomerase. AB - The two thioredoxin-like domains of human protein disulfide isomerase (PDI) have been produced in bacteria as individual soluble, folded protein molecules, and their functional properties have been compared to those of intact PDI. The two individual domains were very similar in their functional properties, and there were no indications of synergy between them, so it is unlikely that they have intrinsically different functions in PDI. Both domains efficiently introduced disulfide bonds into unfolded model proteins and peptides but were less efficient than PDI with folded substrate protein molecules. Relative to PDI, neither domain had substantial activity in catalyzing disulfide bond isomerization. This pattern of activities is very similar to that of the bacterial catalyst DsbA and probably reflects similarities in the catalytic mechanisms of these proteins. The differences in activity between PDI and its thioredoxin-like domains suggest that other features of the PDI molecule are also required for its complete range of thiol-disulfide exchange activities. PMID- 7547905 TI - In vivo participation of a high potential iron-sulfur protein as electron donor to the photochemical reaction center of Rubrivivax gelatinosus. AB - We have found that the only high redox potential electron transfer component in the soluble fraction of Rubrivivax gelatinosus TG-9 is a high-potential iron sulfur protein (HiPIP). We demonstrated the participation of this HiPIP in the photoinduced electron transfer both in vivo and in vitro. First, the addition of HiPIP to purified membranes enhanced the rate of re-reduction of the photooxidized reaction center. Second, the photooxidation of HiPIP was observed in intact cells of Ru. gelatinosus TG-9 under anaerobic conditions by EPR and absorption spectroscopies. Analysis of flash-induced absorption changes showed that the equilibration of positive equivalents between the reaction center and HiPIP occurs in less than 1 ms after flash excitation. The complete re-reduction of the photooxidized reaction center is achieved in tens of milliseconds. The turnover of a cyt bc1 is also involved in this reaction, as shown by a slow electrogenic phase of the membrane potential linked to this process. PMID- 7547906 TI - NIH shift in the hydroxylation of aromatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea. Evidence against an arene oxide intermediate. AB - The migration of deuterium and hydrogen was observed in the aromatic hydroxylation of specifically deuterated, monosubstituted benzenes catalyzed by ammonia monooxygenase of Nitrosomonas europaea. The phenolic products of the hydroxylation of aromatics containing ortho-/para-directing substituents (F, Cl, Br, I, OH, NH2, CH3, CH2CH3, and OCH3) were primarily para-phenols. In contrast, with aromatics containing meta-directing substituents (NO2 and CN), the phenolic products were a more even mixture of meta-and para-phenols. ortho-Fluorophenol was the only ortho-phenolic product observed. The nature of the products suggested that the reaction involved an enzyme-specific, electrophilic addition to the aromatic ring so as to favor hydroxylation at either the meta- or para positions. With the fluoro-, chloro-, and bromobenzene substrates, the values for the migration and retention of deuterium during hydroxylation (NIH shift) were nearly identical when the deuterium was either at the site of hydroxylation or at an adjacent site, indicating a possible common intermediate. The values of the NIH shift with the nitrobenzene substrate were significantly lower when the deuterium was at the site of hydroxylation than at an adjacent site, indicating the operation of a direct loss mechanism. The present results suggest that the aromatic hydroxylation involved a radical or carbocation intermediate which decayed, without the formation of an arene oxide, to form phenolic products with the accompanying direct loss of deuterium at the site of hydroxylation or the shift of the deuterium to an adjacent site. PMID- 7547907 TI - Noncooperativity of biotin binding to tetrameric streptavidin. AB - Streptavidin tetramers have been separated according to their biotin content by anion exchange chromatography. Biotin-free and biotin-saturated streptavidin were coincubated. Streptavidin at intermediate ligation levels, i.e., with one, two, or three molecules of bound biotin, accumulates over time. A steady state distribution of ligation levels is reached after 2 days. When biotin was allowed to redistribute starting from homogeneous populations containing two molecules of biotin per tetramer, a similar steady state distribution of ligation levels was observed, thereby demonstrating an equilibrium distribution. Quantification of this equilibrium indicates that biotin binds to streptavidin with no cooperativity. PMID- 7547908 TI - Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. AB - Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD+ by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD+. These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalyst, His444'. A pKa of 5.1 was estimated for Cys44 in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S. Ferricyanide stoichiometrically reoxidizes the FADH2 of both altered forms of the enzyme. PMID- 7547909 TI - Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides. AB - The cysteines that comprise the active site disulfide in lipoamide dehydrogenase have been individually mutated to a serine residue to give the altered enzymes, C44S and C49S, making it possible to study the redox behavior of the FAD in the absence of the disulfide. The redox potential of the FAD in C44S and C49S was 379 and -345 mV, respectively, at pH 7.0, 25 degrees C. A plot of the redox potential as a function of pH for C49S gave slopes of 57 mV/pH from pH 5.0 to 7.9 and 10 mV/pH from pH 7.9 to 8.8. The plot of the redox potential as a function of pH for C44S gave slopes of 70 mV/pH from pH 5.0 to 7.9 and 4 mV/pH from pH 7.9 to 8.38. The change in the slope at pH 7.9 is associated with the ionization (pKa) of the FADH2 to FADH- in the reduced form of both enzymes. These determinations show that the redox potential of the FAD in C49S, in C44S, and in wild type enzyme is modulated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide, and that the flavin is bound more tightly to the oxidized forms of these enzymes than to the reduced forms. The redox potentials of these enzymes determined using NADH and NADPH at pH 7.6, 25 degrees C are as follows: C44S, -350 mV, -369 mV; C49S, -328 mV, -353 mV, respectively. Thus, pyridine nucleotide binding raises the redox potential of the flavin, showing that both substrates bind more tightly to the reduced form of the enzymes, as well as tighter binding of NADH to the enzymes than that of NADPH. Kd values for the binding of NADH and NADPH to oxidized C44S and C49S were determined in pre-steady state kinetics at pH 7.6 and 25 degrees C, which were monophasic when NADPH was the reductant and biphasic with NADH. The binding constants for NADPH were 660 microM for C44S and 500 microM for C49S; using NADH, the binding constants were 137 microM for C44S and 23 microM for C49S. Fluorescence and absorbance spectrophotometry were used to determine the binding of NAD+ to the oxidized forms of the enzymes as 275 microM and 270 microM for C44S and C49S, respectively. PMID- 7547910 TI - Time-resolved fluorescence investigations of the interaction of the voltage sensitive probe RH421 with lipid membranes and proteins. AB - Fluorescence lifetimes and fluorescence anisotropy decays of the voltage sensitive styryl-pyridinium dye RH421 have been measured in the presence of dimyristoylphosphatidylcholine vesicles, the water soluble enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase (rubisco), and Na+,K(+)-ATPase-containing membrane fragments. The effect of an intramembrane electric field on the photophysical properties of the dye was investigated by the binding of the hydrophobic ion tetraphenylborate (TPB) to the membrane. TPB was found to significantly increase the average fluorescence lifetime and the order of membrane-bound dye. The increase in fluorescence lifetime is consistent with reorientation of the dye further into the membrane interior. The increase in order may be attributed to an electric field-induced alignment of the dye molecules. From the values of the rotational diffusion constant experimentally determined, the expected response time of the dye to an applied electric field can be calculated for a reorientational mechanism to be on the order of tens of nanoseconds. Experiments with rubisco showed that the dye interacts strongly with the protein. In this case, the dye is so tightly bound that it has almost no independent motion and rotates virtually solely with the protein. The rate of rotational motion of the dye in the presence of Na+,K(+)-ATPase-containing membrane fragments is similar to that in pure lipid membranes. The order parameter of the dye in the Na+,K(+)-ATPase membrane fragments is close to the maximum value. This is most probably due to the high density of protein molecules, which restricts the range of motion of the dye. PMID- 7547911 TI - The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is alpha-mannopyranose. AB - Recently, the novel C-glycosidic linkage of a hexopyranosyl residue to the indole ring of tryptophan residue 7 of human RNase U(s) was reported [Hofsteenge, J., Muller, D. R., de Beer, T., Loffler A., Richter, W. J., & Vliegenthart, J. F. G. (1994) Biochemistry 33, 13524-13530]. Identification of this monosaccharide is a prerequisite for studies of its biosynthesis and its biological relevance. Using vicinal proton-proton coupling constants and rotating-frame nuclear Overhauser enhancements, ewe demonstrate that the C-linked substituent is alpha mannopyranose. Furthermore, the nuclear magnetic resonance (NMR) data indicate that the mannopyranose moiety in a glycopeptide derived from RNase U(s) adopts several conformations on the NMR time scale. PMID- 7547912 TI - Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli. AB - Human phenylalanine hydroxylase (hPAH) contains three tryptophan residues (W120, W187, and W326). All three tryptophan residues were mutated to phenylalanine either as single mutants or in combination, and one tryptophan was also mutated to isoleucine. The mutant enzymes were expressed in Escherichia coli and purified as fusion proteins with maltose-binding protein and a linker region containing a recognition site for the serine protease factor Xa. After cleavage by factor Xa, all mutants were purified to homogeneity, and the kinetic and spectroscopic properties of the proteins were studied. All the proteins had high catalytic activities, but the affinity for phenylalanine was increased for the W1201 and W120F mutants, and decreased for the W187F and W326F mutants. Using steady-state fluorescence spectroscopy, the contributions of the individual tryptophan residues to the total intrinsic fluorescence of the protein were estimated. On the basis of measurements of mutants containing only one tryptophan, it was calculated that W120, W187, and W326 account for approximately 61, 13, and 26% of the total tryptophan fluorescence of hPAH, respectively, while the positions of the emission maxima (335.5-336.5 nm) and the widths at half-height (55-60 nm) of the emission spectra of the individual tryptophans were rather similar. After incubation with phenylalanine, the quantum yield of wild-type hPAH increases by 15%, and the emission maximum is shifted from 336.5 to 347 nm. This effect is mainly due to changes in the W120 emission. On the basis of fluorescence quenching studies, this amino acid is the most surface-exposed of the tryptophan residues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547913 TI - Second-site suppressor mutations for the Asp-66-->Cys mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli. AB - Asp66 is the only essential acidic residue in the putative hydrophilic loop2-3 region of the transposon Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)] [Yamaguchi, A., Nakatani, M., & Sawai, T. (1992a) Biochemistry 31, 8344-8348]. Escherichia coli cells producing a D66C mutant of TetA(B) showed no tetracycline resistance. A spontaneous second-site revertant was isolated from the cells carrying the D66C mutant gene, which showed moderate resistance to tetracycline [minimum inhibitory concentration (MIC), 50 micrograms/mL]. The entire sequencing of the revertant genes revealed two secondary mutations, i.e., the codon 40 of GCT (Ala)-->GAT (Asp) and T-->G at 17 bases upstream from the initiation codon of the tetA gene. There was a T-->G mutation at position -17, which was a mutation in the tet promoter/operator region, which caused a decrease in TetA(B) production. The full expression of the A40D/D66C double and the A40D single mutants, which were constructed by site-directed mutagenesis, was deleterious for cell growth. The -17T-->G mutation mitigated the deleterious effect of these mutants through reduction of expression. The -17T-->G single mutation introduced into the wild-type tet gene did not affect the level of resistance, although the expression was significantly reduced. Intact cells carrying the A40D/D66C and 17T-->G/A40D/D66C mutant plasmids showed a reduced level of tetracycline accumulation due to active efflux, whereas no significant tetracycline uptake was observed in inverted membrane vesicles prepared from these mutant-producing cells. The A40D mutation is located at opposite side of the membrane to the D66C mutation in the putative secondary structure of TetA(B). The second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. PMID- 7547914 TI - Mixed disulfide intermediates during the reduction of disulfides by Escherichia coli thioredoxin. AB - The reduction of disulfides by thioredoxin involves a two-step mechanism. The first step features an intermolecular attack of Cys32 of thioredoxin on the disulfide with formation of a protein mixed disulfide and release of 1 equiv of thiol. The second step involves intramolecular breakdown of the mixed disulfide intermediate via attack of Cys35 with concomitant formation of the oxidized protein and release of a second equivalent of thiol. Study of mixed disulfide intermediates for Escherichia coli thioredoxin is exceedingly difficult because the second step is highly favorable. We have studied these intermediates via two approaches. First, Cys35 can be mutated to the similar but chemically nonreactive residue serine. This precludes breakdown of the intermediate. Second, "mass action trapping" techniques can be used because the second step of the mechanism is first-order in the forward direction and second-order in the reverse direction. This has yielded a thermodynamic breakdown of the reaction into its two component steps. Results for reaction of thioredoxin and 2-hydroxyethyl disulfide indicate that about half of the free energy change for the entire process is associated with the first step. Comparison with a small molecule cysteine analog suggests that significant interactions stabilize the mixed disulfide intermediate. Two-dimensional NMR analysis of the C35S thioredoxin 32C beta-mercaptoethanol mixed disulfide shows packing interactions between the mixed disulfide moiety and Trp31 and Ile75. Additionally, studies with C35S thioredoxin show that substitution of the cysteine residue slightly perturbs the equilibrium for the first step in the reaction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547915 TI - Functional expression and subcellular localization of a high-Km hexose transporter from Leishmania donovani. AB - We have used expression in Xenopus oocytes to characterize a new hexose transporter from the parasitic protozoan Leishmania donovani. This transporter utilizes the hexoses glucose, fructose, and mannose as substrates. A substrate saturation curve for 2-deoxy-D-glucose reveals a very high Km, estimated to be approximately 150 mM. Immunolocalization of the protein with an antibody directed against the COOH terminus indicates that the transporter is present primarily in the parasite plasma membrane but is not detectable in the flagellar membrane. Since this protein is expressed in the insect stage promastigotes but not in the intracellular amastigotes, it may be specialized to function following an insect sugar meal when the concentrations of sugars surrounding the parasite are high. PMID- 7547916 TI - Spectroscopic and lipid binding studies on the amino and carboxyl terminal fragments of Locusta migratoria apolipophorin III. AB - The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (approximately 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species approximately 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of approximately 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547918 TI - Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes. AB - The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547917 TI - Membrane-bound c-type cytochromes in Heliobacillus mobilis. In vivo study of the hemes involved in electron donation to the photosynthetic reaction center. AB - The amount of heme per photosynthetic reaction center (RC) was examined in whole cells of Heliobacillus mobilis, and a stoichiometry of 5-6 hemes c and 1-3 hemes b per RC was found. Virtually the full complement of heme was seen to be functionally connected to the pool of electron donors to the photosynthetic RC. The kinetic parameters of electron transfer between reduced c-type hemes and the photooxidized primary donor P798+ were studied in whole cells and membrane fragments. The in vivo half-times of electron donation (50% with t 1/2 = 110 microseconds, 50% with t 1/2 = 600 microseconds) were seen to slow down to half times in the range of several and several tens of milliseconds following disruption of cells. A severe conformational alteration or a change in the identity of the donating heme is discussed. Redox titrations of the flash-induced absorption changes performed on whole cells in the presence of mediators yielded the following redox midpoint potentials: P798, Em = +240 mV; heme c553, Em = +190, +170, and +90 mV for the heme components oxidized after the first, second, and third flash, respectively. The results demonstrate that the pool of c553 hemes donating electrons to the RC is heterogeneous and that it consists of either several distinguishable cytochromes or multiheme cytochromes or both. The number of hemes reduced and the kinetics of heme rereduction after flash-induced oxidation were found to depend strongly on the degree of anaerobicity in the interior compartment of the cell. A model rationalizing the obtained results in terms of a set of differing redox components is proposed. PMID- 7547919 TI - Kinetics of nucleotide binding to pyruvate carboxylase. AB - The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547920 TI - Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. AB - In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4 fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements. PMID- 7547921 TI - Characterization of calponin binding to actin. AB - Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding of [14C]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variable and dependent on ionic strength. Below 110 mM ionic strength, the stoichiometry of binding was 1:1. As the ionic strength was increased above 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 calponin per 2 actin monomers. At physiological ionic strength, the binding exhibited a small degree of positive cooperativity and was adequately described by a single class of binding sites with an association constant of 6 x 10(6) M-1. The affinity decreased to 20% of this value in the presence of ATP. Irrespective of the ionic strength, actin formed bundles when saturation with calponin exceeded about 30%. Measurements of the rate of association were complicated by this bundling, but the upper limit for this reaction was placed at 10(6) M-1 s-1. The addition of calponin to actin-caldesmon complexes caused displacement of the caldesmon. PMID- 7547922 TI - Properties of troponin C acetylated at lysine residues. AB - We have studied the properties of rabbit skeletal troponin C (TnC) fully acetylated at its lysine residues (AcTnC). Acetylation causes a decrease in thermal stability of both domains of TnC in the absence of Ca2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal domain monitored by far-UV circular dichroism is decreased by 16.3 degrees C. In the presence of 1 mM CaCl2, no cooperative unfolding can be detected up to 90 degrees C for either TnC or AcTnC. At 25 degrees C, CD spectra show that AcTnC has a slightly lower alpha-helix content in the absence of Ca2+, but higher in the presence of Ca2+ as compared to unmodified TnC. Acetylation causes a 3.5-fold increase in affinity for Ca2+ at the low-affinity sites and a 2-fold decrease at the high-affinity sites. Polyacrylamide gel electrophoresis under nondissociating conditions (no SDS, no urea, pH 8.6) indicates that acetylation has little effect on the apparent affinity of TnC for troponin I; however, the binding of the acetylated peptides corresponding to the N-terminal domain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enhanced, the altered properties of the N-terminal domain being predominantly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetylation increases TnC's affinity for myofibrils in the presence of Ca2+ approximately 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of myofibrils containing AcTnC is shifted to lower Ca2+ concentrations, consistent with the higher Ca2+ affinity of AcTnC at the low-affinity sites. These data indicate that positively charged lysine side chains, especially those located in the N-terminal domain, modulate TnC's structural stability and interactions with Ca2+ and other troponin components. PMID- 7547923 TI - Interaction of nucleotide-free Hsc70 with clathrin and peptide and effect of ATP analogues. AB - The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences in the effect of bound ATP and ADP on the interaction of hsc70 (constitutive hsp70) with its protein substrates. In the present study, we investigated the interaction of protein substrates with nucleotide-free hsc70 and with hsc70 with bound ATP analogues. We found, first, that nucleotide-free hsc70 appeared to interact differently with different substrates. Specifically, nucleotide-free hsc70 behaved much more like hsc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dissociated from nucleotide-free hsc70 in contrast to its very slow binding to and dissociation from hsc70-ADP. On the other hand, nucleotide-free hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrome c peptide dissociated very slowly from nucleotide-free hsc70 compared to its rapid dissociation from hsc70 ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very little further effect on the properties of the nucleotide-free hsc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of analogues. PMID- 7547925 TI - Photodynamic and radiolytic inactivation of ion channels formed by gramicidin A: oxidation and fragmentation. AB - Ion channels formed by the peptide gramicidin A in planar lipid membranes have been reported to react very sensitively upon irradiation of the membrane by ionizing radiation (radiolysis), by UV light (photolysis), or by visible light in the presence of appropriate photosensitizers (photodynamic inactivation). In all three cases the effect is due to the presence of the four tryptophan residues of the pentadecapeptide. Modifications of these amino acids--due to an interaction with free radicals formed upon water radiolysis or due to light absorption--have been found to reduce the membrane conductance by many orders of magnitude. The present study was intended to correlate functional changes, observed at the level of single ion channels, with changes of the molecular structure identified by mass spectrometry. About 98% of the inactivated channels showed a single-channel conductance of virtually zero, while about 2% of the channels present before irradiation are converted to a state of reduced conductance (and reduced lifetime). On the structural level, irradiation in the presence of the photosensitizer Rose Bengal was found to produce oxidation and fragmentation of the peptide at the positions of the tryptophan residues. Our results provide evidence that the main effect of radiolysis, or of photodynamic treatment, is the cleavage of the peptide backbone leading to immediate closure of an open ion channel. PMID- 7547924 TI - Interaction of a fluorescent paclitaxel analogue with tubulin. AB - To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3 (dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3 (dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel binding site on tubulin. PMID- 7547926 TI - Molecular interactions of ether-linked phospholipids. AB - Earlier studies have shown that ether phospholipids display phase-forming properties distinct from those of their ester phospholipid counterparts. Dihexadecylphosphatidylcholine (DHPC) forms an interdigitated bilayer when fully hydrated, and dihexadecylphosphatidylethanolamine (DHPE) is observed in the inverted hexagonal phase (HII) at elevated temperatures. In contrast, the acyl lipid analogues display these phases only under more extreme conditions. In the present study, we examine fully hydrated mixtures of DHPC and DHPE by X-ray diffraction and differential scanning calorimetry and describe the temperature- composition phase diagram for the binary phospholipid system, DHPC/DHPE. Addition of 7 mol % DHPE to DHPC abolishes the ability of DHPC to form an interdigitated bilayer gel phase (L beta I), whereas 10 mol % DHPC destabilizes the HII phase favored by DHPE by elevating (to > 100 degrees C) or removing the L alpha-->HII transition. Evidence for bilayer gel phase separation occurring in DHPC/DHPE mixtures is obtained. In conclusion, it is found that small amounts of the appropriate phospholipid can seriously compromise the formation of the L beta I and HII phases. PMID- 7547927 TI - Binding of prenylated and polybasic peptides to membranes: affinities and intervesicle exchange. AB - The small GTP-binding protein G25K and the protein K-Ras 4B contain prenyl groups (geranylgeranyl and farnesyl, respectively) that are thioether linked to a C terminal cysteine which is methylated on its alpha-carboxyl group. These proteins, like many other prenyl proteins, also have a string of basic residues near their C-termini. A series of prenylated peptides based on the C-terminal sequences of human brain G25K and human K-Ras 4B were synthesized and analyzed for their membrane binding affinities. G25K peptides containing an N-terminal N acetyltryptophan group were studied because their binding to membranes containing a trace of dansylated phospholipid could be detected by fluorescence resonance energy transfer. The G25K peptide lacking a prenyl group and a C-terminal methyl ester did not detectably bind to vesicles, and binding was enhanced by more than 500-fold if the peptide was geranylgeranylated. For the farnesylated peptide, methylation of the C-terminus increased membrane affinity by at least 60-fold if the vesicles contained phosphatidylserine and by 3-fold if they lacked this acidic lipid. The geranylgeranylated and methylated G25K peptide remains irreversibly attached to vesicles over several minutes only if the vesicles contain phosphatidylserine, whereas the corresponding nonmethylated or farnesylated and methylated peptides dissociate rapidly (less than a few seconds) from neutral or anionic vesicles. Farnesylation of the nonmethylated K-Ras 4B peptide enhances its affinity to vesicles containing acidic phospholipids (phosphatidylglycerol or phosphatidylserine) by 70-fold, and methylation leads to an additional dramatic (150-fold) increase in membrane affinity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547928 TI - Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol. AB - Direct effects of ethanol on the interaction of cytosolic lipid transfer proteins with ligands are not known. In this study, recombinant liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2) were used in conjunction with a series of fluorescent fatty acid probe molecules to compare the relative dielectric properties of the ligand binding sites and to examine the effects of ethanol in vitro on ligand interaction with these proteins. L-FABP and SCP-2 exhibited broad but distinct ligand specificities. Although NBD-stearic acid bound with high affinity to both proteins, emission spectra showed that the relative dielectric constant of the ligand binding site in SCP-2 was significantly lower than in L-FABP, 2 vs 24. Furthermore, affinities of L-FABP for NBD-fatty acid probes were NBD-stearic acid > NBD-lauric acid >>> NBD hexanoic acid, NBD-acetic acid. In contrast, SCP-2 bound only NBD-stearic acid with a Kd of 0.23 microM and Bmax of 0.98 mol/mol. This observation of SCP-2 specifically binding the fluorescent NBD-stearic acid was confirmed with RdB stearic acid and the naturally fluorescent cis-parinaric acid, both of which had similar affinities and stoichiometries. Ethanol in vitro had no effect on L-FABP NBD-stearic acid binding. However, ethanol at physiological concentrations (25 mM) dramatically inhibited NBD-stearic acid binding to SCP-2. In conclusion, the data show that both L-FABP and SCP-2 specifically bind fluorescent fatty acids. However, the ligand binding sites of L-FABP and SCP-2 differed dramatically in their dielectric properties and their sensitivity to ethanol. PMID- 7547929 TI - Fatty acid flip-flop in phospholipid bilayers is extremely fast. AB - The rate of movement of fatty acids (FA) across phospholipid bilayers is an important consideration for their mechanism of transport across cell membranes but has not yet been measured. When FA move undirectionally across phospholipid bilayers, the rapid movement of un-ionized FA compared to ionized FA results in transport of protons. We have previously used this property to show that FA move spontaneously ("flip-flop") across the bilayer of small unilamellar vesicles within approximately 1 s (Kamp & Hamilton, 1992, 1993). This work extends the time resolution of this assay into the millisecond time range by use of stopped flow fluorometry. In small unilamellar vesicles (diameter, approximately 25 nm) at neutral pH, flip-flop of all fatty acids studied (lauric, myristic, palmitic, oleic, and stearic) was > or = 80% complete within 5-10 ms. In large unilamellar vesicles (diameter, approximately 100 nm), the same fatty acids exhibited fast flip-flop but with a measureable rate (t 1/2 = 23 +/- 12 ms). The calculated pseudounimolecular rate constant of the un-ionized FA (kFAH) approximately 15 s 1. There was no dependence of the flip-flop rate on the fatty acid chain length or structure. We also monitored the rate of desorption and transbilayer movement of (anthroyloxy)stearic acid in small unilamellar vesicles. Whereas previous studies suggested slow flip-flop of this FA analogue, the present studies suggest that (anthroyloxy)stearic acid flip-flops rapidly and that earlier studies did not truly measure the transbilayer movement step. These findings further support the view that proteins are not required for translocation of FA across cell membranes. PMID- 7547930 TI - Constitutive activation of opsin: interaction of mutants with rhodopsin kinase and arrestin. AB - Mutation of Gly90, Glu113, Ala292, and Lys296 in the visual pigment rhodopsin constitutively activates the protein for activation of the G protein transducin. Three of these mutations have been shown to cause two different human diseases. Mutation of Gly90 and Ala292 results in complete night blindness, and mutation of Lys296 results in the degenerative disease retinitis pigmentosa. We show here that the mutants not only constitutively activate transducin but are also constitutively activated for phosphorylation by rhodopsin kinase. In addition, the phosphorylated mutants are shown to bind tightly to the inhibitory protein arrestin in a reaction that quenches the activity toward transducin. Thus the same mutations that result in constitutive activation of transducin also result in constitutive phosphorylation by rhodopsin kinase and binding of arrestin to inhibit the activity. This implies that the same conformational change may be responsible for activation of transducin and rhodopsin kinase. It also suggests that degeneration of photoreceptor cells in retinitis pigmentosa results indirectly from the activated state of the receptor, perhaps as a consequence of phosphorylation and persistent binding of arrestin. PMID- 7547932 TI - Agonist-induced desensitization and phosphorylation of human 5-HT1A receptor expressed in Sf9 insect cells. AB - The human 5-HT1A receptor was expressed in Sf9 insect cells to examine desensitization as manifested by agonist-induced uncoupling from G proteins and second messengers. New binding sites were detected after infection of cells with the 5-HT1A receptor-bearing baculovirus. 5-HT1A receptor agonists caused inhibition of cAMP accumulation that could be attenuated by specific receptor antagonists. Brief pretreatment with 5-HT resulted in (1) an uncoupling of receptor from G proteins as evidenced by a loss of high-affinity agonist binding sites and a diminished ability of the receptor to increase incorporation of AA GTP into endogenous Go alpha-like G proteins, (2) a decreased ability of the receptor to inhibit cAMP accumulation, and (3) increased phosphorylation of the 5 HT1A receptor on serine and threonine residues. Phosphorylation occurred in the presence of a number of cyclic nucleotide dependent kinase inhibitors, and desensitization of the cAMP response occurred in the presence of H-7 and also in cells with prolonged exposure to PMA. Both phosphorylation and desensitization were markedly attenuated by 100 nM and 1 microM heparin and demonstrated similar time courses and concentration-response relationships. Those results demonstrate a close association between agonist-induced desensitization and phosphorylation of the 5-HT1A receptor in Sf9 cells through a pathway that mainly does not involve protein kinase A or C and might involve a G protein-linked receptor kinase. PMID- 7547931 TI - An acidic motif within the third intracellular loop of the alpha2C2 adrenergic receptor is required for agonist-promoted phosphorylation and desensitization. AB - The alpha 2C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. To investigate the role of this region, we utilized site-directed mutagenesis to delete these 16 amino acids as well as to substitute them with glutamine, thereby conserving size but not charge. The wild-type and mutated alpha 2C2 receptors were permanently expressed in CHO cells. Neither substitution nor deletion of this region affected receptor expression, agonist or antagonist binding affinities, guanine nucleotide sensitive formation of the high-affinity agonist-receptor-G protein complex, or functional coupling of the receptor to Gi. We considered that since alpha 2C2 agonist-promoted desensitization is due to phosphorylation by the beta-adrenergic receptor kinase (or a related kinase), that this region may be important for establishing the acidic mileau required by this kinase. Therefore, the consequences of 30 min of agonist preexposure on subsequent alpha 2C2-mediated inhibition of adenylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha 2C2 receptor underwent approximately 52% functional desensitization and a approximately 40% loss of high-affinity binding after such exposure. In contrast, deletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correlated with those obtained in whole cell phosphorylation experiments. Cells expressing each receptor were incubated with [32P]orthophosphate and exposed to agonist, and receptors were purified by immunoprecipitation. The deletion and the substitution mutant receptors underwent agonist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15%, respectively, relative to wild-type alpha 2C2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547933 TI - N7-cyanoborane-2'-deoxyguanosine 5'-triphosphate is a good substrate for DNA polymerase. AB - The 5'-triphosphate of the boronated nucleoside analog N7-cyanoborane-2' deoxyguanosine (7bdGTP) was synthesized, and a series of experiments was initiated to assess the potential of the compound to serve as a substrate for DNA polymerases. We show here that 7bdGTP can be incorporated into DNA by Sequenase. The resulting hemiboronated extension products are resistant to cleavage by treatment with either DMS and heat or a number of restriction enzymes. Further, in the polymerase chain reaction, 7bdGTP can be utilized as a substrate for Taq polymerase. Finally, by kinetic analysis, we have found that 7bdGTP is a more efficient substrate for exonuclease-free Klenow than normal dGTP. Thus, the introduction of a cyanoborane moiety to the N7 position of dGTP results in a nucleotide that is accepted in lieu of normal dGTP by a number of DNA polymerases. PMID- 7547934 TI - Relationship between in vivo activity and in vitro measures of function and stability of a protein. AB - The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein. PMID- 7547936 TI - Cooperative assembly of signal recognition particle RNA with protein SRP19. AB - Signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of secretory proteins to the lipid bilayer of the endoplasmic reticulum. SRP contains the protein SRP19, which is an important structural and functional component, believed to promote the assembly of the particle. We have purified the human SRP19 protein to homogeneity from recombinant bacteria which overexpress the polypeptide, and have studied details of the binding to SRP RNA via gel mobility shift and RNase sensitivity assays. SRP19 interacts with two SRP RNA conformers with different affinities such that the more compact RNA species is bound more avidly. Furthermore, binding was found to be highly cooperative. Binding constants and Hill coefficients were determined for several mutant SRP RNAs in which individual RNA helices were deleted. These results confirmed that both SRP RNA helices 6 and 8 are important for SRP19 binding. Enzymatic RNA structure probing of a 150-nucleotide mutant SRP RNA fragment and of the corresponding RNA-SRP19 complex showed that cooperativity may be due to protein induced conformational changes in the large domain of the SRP RNA. Finally, SRP19 bound specifically not only to SRP RNA but also to the A-form of Escherichia coli 5S ribosomal RNA, thereby indicating structural similarities between these two RNA molecules. PMID- 7547935 TI - Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular Serratia marcescens endonuclease. AB - The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent dye labeled duplex strands was prepared by affinity chromatography. Cleavage experiments carried out under single hit conditions demonstrate that the enzyme shows preferences for GC rich regions in dsDNA, in particular d(G).d(C)-tracts, and avoids cleavage of d(A).d(T)-tracts. There is a correlation between cleavage at a given position in one strand with cleavage at the same position in the other strand of the duplex. ssDNA cleavage occurs at somewhat different preferred sites than observed in dsDNA. On dsDNA, the Serratia nuclease produces a very different cleavage pattern compared to bovine pancreatic DNase I, with the notable exception that both enzymes avoid d(A).d(T)-tracts. In general, the Serratia nuclease compared to DNase I is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ionic strength or in the presence of DMSO, it becomes more nonspecific. Addition of urea, however, makes the enzyme more selective than observed under standard conditions. From these results which were confirmed by the results of cleavage experiments with synthetic oligodeoxynucleotides, we conclude that the Serratia nuclease like DNase I is sensitive to global features of the DNA, for example, the width of the minor groove. In addition, localized sequence-dependent interactions between substrate and nuclease determine whether a site is cleaved preferentially. Some of these interactions seem to be the same for ds- and ssDNA. PMID- 7547937 TI - NMR spectroscopic analysis of the DNA conformation induced by the human testis determining factor SRY. AB - The conformation of an eight base pair DNA oligonucleotide duplex bound to the human testis determining factor SRY and the orientation of the protein domain within the complex have been analyzed by a variety of NMR methods which permit the selective observation of protons attached to 12C nuclei in the presence of uniformly enriched 13C/15N protein. Qualitative analysis of nuclear and rotating frame Overhauser enhancement spectra at multiple mixing times indicates that the conformation of the SRY-bound DNA is distinct from that of A- and B-DNA, in agreement with the recent three-dimensional structure determination of the complex [Werner, M. W., Huth, J. R., Gronenborn, A. M., & Clore, G. M. (1995) Cell 81, 705-714]. Selective observation of intermolecular NOEs between protein and DNA indicates that partial intercalation of a protein side chain occurs between two adenine bases in the DNA octamer. The analysis of structural features by NMR for this unusual DNA conformer and the orientation of the protein domain on the DNA is discussed. The structural features of the DNA complexed to SRY are remarkably similar, but not identical, to those of DNA complexed to the TATA binding protein (TBP). PMID- 7547938 TI - Membrane lipid alkyl chain motional dynamics is conserved in Sarcina ventriculi despite pH-induced adaptative structural modifications including alkyl chain tail to tail coupling. AB - Sarcina ventriculi, an anaerobic Gram-positive bacterium, adapts to increasing temperature, the presence of organic solvents, or the lowering of the pH of its growth medium by joining the tails of membrane lipids from opposite sides of the bilayer, forming transmembrane, bifunctional fatty acid species. Since this is done to offset the increase in membrane mobility caused by these perturbations, it is of interest to determine whether the motional (dynamic) properties of membrane lipid alkyl chains are conserved. In this study, conservation of the motional time scales of the alkyl chains of total membrane lipids from Sarcina ventriculi cells grown at different pH values was demonstrated using proton nuclear magnetic resonance (NMR) spectroscopy. The NMR longitudinal relaxation times (T1) of the protons in the bulk methylene groups were measured for lipids from cells grown at pH 3.0 and 7.0. These measurements indicated that the temperature profile of the T1 relaxation behavior for the methylene protons from these two different preparations was the same. Analysis of the data from T1 measurements indicated that the thermal barrier for relaxation is the same in both lipid systems. This is only true if the pH of the sample on which the measurement is being made is adjusted to the same value as that at which the corresponding cells were cultured. It is clear from this latter observation that the state of protonation of the lipid head groups is a contributor to the overall motional freedom of the membrane lipid components. The correlation times (tau c) of characteristic lipid alkyl chain motion were estimated to be approximately 10( 10) s.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547940 TI - Structural behavior of the CpG step in two related oligonucleotides reflects its malleability in solution. AB - We report on the determination of the solution structure of two sequence-related oligonucleotides, d(GTACGTAC)2 and d(CATCGATG)2. Results have been obtained by using a combined approach of (a) two-dimensional NMR, including proton and phosphorus experiments, (b) restrained molecular mechanics performed with sugar phase angle, backbone epsilon angle, and NOE distances as input, and (c) back calculation refinements against the NOE spectra at various mixing times. The two oligonucleotides adopt the B-DNA structure with, however, noticeable differences centered on their core sequence and especially the CpG step. Due to the permutation of its flanking residues, the CpG step modifies its twist values and backbone epsilon value; globally, the CpG step appears more flexible within the tetranucleotide TCGA than ACGT. The solution structure of d(GTACGTAC)2 differs from the previously reported X-ray structure, which was found to be A-form throughout [Takusagawa, F. (1990) J. Biomol. NMR 3, 547-568]. On the other hand, in the X-ray structure of d(CCAACGTTGG)2 [Prive et al. (1991) J. Mol. Biol. 217, 177-199] the structure of the ACGT sequence is similar to that found in solution d(GTACGTAC)2. Similarly, the central TCGA tetranucleotide of d(CATCGATG)2 presents a solution structure analogous to that observed on the X-ray structures of both d(CGATCGATCG)2 [Grzeskowiak, et al. (1991) J. Biol. Chem. 266, 8861-8883] and d(CGATCGmeATCG)2 [Baikalov, et al. (1993) J. Mol. Biol. 231, 768-784]. At the end we discuss the possible biological significance of the particular structures exhibited by the ACGT and TCGA tetranucleotides. PMID- 7547939 TI - Evidence for the importance of weakly bound water for matrix metalloproteinase activity. AB - The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated. In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates. Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme. The former reflects the hydrophobic nature of the enzyme-substrate interaction and the effect can be corrected for by using the solvent to water partition coefficient of the substrate in the mixed solvent systems. The catalyzed hydrolysis of substrate, corrected for the effect of hydrophobic partitioning, was demonstrated to be sixth order in water for gelatinase A and third order in water for stromelysin 1. Variation in water concentration did not produce saturation even at concentrations close to 55.5 M. The results indicate that weakly bound water molecules are essential to mediate the interaction between substrate and enzyme. The sensitivity of these enzymes to water concentration could be an important mechanism for regulating catalytic activity in vivo. PMID- 7547941 TI - Sequence-specific hydrolysis of yeast tRNA(Phe) mediated by metal-free bleomycin. AB - Bleomycin A2 (BLM) was found to mediate sequence specific hydrolysis of tRNA(Phe) in the absence of added metal ions. BLM A2 promoted phosphodiester bond hydrolysis 3' to the pyrimidine residue at all resolved Py-Pu sites not involving modified bases, as demonstrated by high-resolution electrophoretic analysis of 5' and 3'-32P-end-labeled substrates. The reaction proceeded with surprising facility, approaching efficiency that of oxidative strand scission mediated by the FeII.BLM A2 complex. By the use of a number of BLM congeners, as well as a study of the time, temperature, and salt dependence of the hydrolysis, it was shown that in many respects the hydrolytic reaction parallels the oxidative degradation of RNA and DNA mediated by metallobleomycins. Thus, in contrast to the well-characterized oxidative degradation of DNA and RNA by bleomycin studied for two decades, the present report documents the ability of certain metal-free bleomycins to mediate RNA hydrolysis. PMID- 7547942 TI - Molecular dynamics simulation of the solution structures of Ha-ras-p21 GDP and GTP complexes: flexibility, possible hinges, and levers of the conformational transition. AB - Unconstrained molecular dynamics simulations of the GDP and GTP complexes of Ha ras p21 protein are performed in aqueous environment for 500 ps, using the GROMOS force field. The solvated structures are mutually compared as well as to the X ray structures [Tong, L. A., de Vos, A. M., Milburn, M. V., & Kim, S. H. (1991) J. Mol. Biol. 217, 503-516; Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsh, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The simulations show areas of flexibility, with deviations from the original structures. The parts that show differences between the two solvated forms are those from residues 12 to 17, 25 to 38, 41 to 51, 57 to 73, 99 to 112, and 120 to 152, coincident with areas of flexibility. Some of these areas also show differences between the X-ray structures and are part of loops on the surface of the protein. Many of the residues in the ends of these loops undergo dihedral transitions during the solvation process. Of all the dihedral transitions observed, 62% occur around the ends of these loops. This suggests that the ends of the areas from 12 to 17, 25 to 38, and 57 to 73 are the hinge points of the conformational transition between the GTP and the GDP forms. The study of the nucleotide interactions in the solution forms shows that residues 29, 30, and 35 establish contacts with the gamma-phosphate and the sugar ring of the GTP and thus these contacts could be proposed as the possible levers of the conformational transition that accompanies GTP hydrolysis. PMID- 7547943 TI - Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase. AB - Cytochrome c peroxidase (CcP) reacts with peroxide to form compound I, an intermediate that has an oxy-ferryl iron center and a stable indolyl radical at Trp 191. During the normal catalytic cycle, the oxy-ferryl heme and the Trp 191 radical are reduced by sequential electron transfers from ferrous cytochrome c (Cc). To investigate the role of protein structure in these electron transfer reactions, mutagenesis was used to replace Trp 191 with Phe. The Trp 191-->Phe enzyme [CcP(MI,F191)] reacts with peroxide to form an oxy-ferryl iron center and a transient porphyrin radical. The reaction of Cc from horse and yeast with peroxide-oxidized CcP(MI,F191) was characterized under transient and steady-state conditions. The rate of ET from Cc to the oxy-ferryl heme of CcP(MI,F191) was decreased by at least 10,000-fold relative to the CcP(MI) parent. This effect was observed at 20 and 100 mM ionic strength, with both yeast and horse cytochrome c as the substrate. Thus, Trp 191 is a critical component of all pathways that permit rapid reduction of the oxy-ferryl heme by Cc under these conditions. The reaction of the porphyrin radical with Cc was difficult to characterize, owing to the short half-life of this intermediate. The oxidation of Cc by this intermediate had a maximum rate constant of 32 s-1 at pH 6.0, 25 degrees C. Circumstantial evidence suggests that the porphyrin radical is not directly reduced by Cc, but is instead reduced via a protein-based radical intermediate. The steady-state activity of the mutant enzyme was 300-600-fold lower than the CcP(MI) parent, but kcat is 7-20 times greater than the rate constant for reduction of the oxy-ferryl heme under all conditions examined. Thus, the oxy ferryl heme is not reduced to the ferric state under steady-state conditions. Transient changes in the absorption spectrum further indicate that steady-state oxidation of Cc2+ by CcP(MI,F191) occurs via reaction of peroxide with the oxy ferryl enzyme. PMID- 7547944 TI - pKa of the protonated Schiff base and aspartic 85 in the bacteriorhodopsin binding site is controlled by a specific geometry between the two residues. AB - The structure and function of the light-driven proton pump bacteriorhodopsin appear to be determined by the exact geometrical conformation of specific groups in the retinal binding site, including bound water molecules. This applies to the pKa values of the protonated Schiff base, which links the retinal chromophore to Lys216, and to Asp85. In the present work we show that the geometrical constraints imposed by the ring structures of several synthetic retinals can induce substantial changes in the pKa values of the Schiff base and of Asp85. Thus, the artificial pigments derived from 13-demethyl-11,14-epoxyretinal (2) and 13-demethyl-9,12-epoxyretinal (3) show protonated Schiff base pKa values of 8.2 +/- 0.1 and 9.1 +/- 0.1, respectively, as compared with 13.3 in the native (all trans-retinal) pigment. We also suggest that in both systems the pKa of Asp85 increases from 3.2 in the native bR to above 9. Analogous, though smaller, effects are obtained for artificial bR pigments derived from 12,14-ethanoretinal (4), 11,13-propanoretinal (5), 11,13-ethanoretinal (6), and p-(CH3)2N-C6H4-HC = CH-C(CH3) = CH-CHO 7. The effects of geometry on the pKa values (those on Asp85 being more pronounced) are attributed to the disruption of the original, well defined, structure in which the Schiff base and its Asp85 counterion are bridged by bound water molecules. These results are the first to show that it is possible to modify the pKa values of the Schiff base and Asp85 in appropriate artificial pigments, without inducing intrinsic pKa changes in the chromophore or introducing a mutation in the protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547945 TI - Time-resolved titrations of the Schiff base and of the Asp85 residue in artificial bacteriorhodopsins. AB - Deprotonation/protonation processes involving the retinal Schiff base and the Asp85 residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pKa values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp85, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pKa values of the above two residues are substantially modified: 13-CF3 bR, pKa (Schiff base) = 8.2 +/- 0.2; 13-demethyl 11,14-epoxy bR, pKa (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pKa (Asp85) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pKa (Asp85) congruent to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp85 moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547946 TI - ENDOR and special TRIPLE resonance spectroscopy of QA.- of photosystem 2. AB - ENDOR and special TRIPLE spectroscopies have been used to study the electron spin density distribution and hydrogen bonding of the plastosemiquinone anion radical, QA.-, of photosystem 2. The semiquinone radical was made accessible to ENDOR through the use of exogenous cyanide, which decouples the radical from the ferrous iron of the photosystem 2 ferroquinone acceptor complex [Sanakis, Y., et al. (1994) Biochemistry 33, 9922]. H2O/D2O exchange was used to assign hyperfine couplings to hydrogen-bonded protons, and orientation-selected special TRIPLE spectroscopy has revealed the orientation of hydrogen bonds relative to the quinone ring. Methyl group resonances have also been assigned. ENDOR spectra of the decylplastosemiquinone anion radical in vitro are presented for comparison. This shows that interaction with the protein leads to changes in the electron spin density distribution and the hydrogen bond orientation; both hydrogen bonds are parallel to the quinone ring plane in vitro, whereas QA.- has one parallel and one perpendicular to the plane. These results are discussed in the light of previous ENDOR studies of the ubiquinone radical QA.- of Rhodobacter sphaeroides and the predicted structure of the QA-binding region of photosystem 2. PMID- 7547947 TI - Intermolecular electron transfer from substrate-reduced methylamine dehydrogenase to amicyanin is linked to proton transfer. AB - Within the methylamine dehydrogenase-amicyanin complex, intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) and copper. The ET reactions from two chemically distinct reduced forms of TTQ were studied. The quinol form of TTQ was generated by reduction by dithionite. An aminoquinol form of TTQ, in which an amino group displaces the carbonyl oxygen, was generated by reduction by the substrate methylamine. Thermodynamic analysis of the ET reactions suggested that the ET event was rate-limiting for the redox reaction between quinol TTQ and copper, but not for the ET reaction from aminoquinol TTQ to copper. Solvent kinetic isotope effect studies indicated that proton transfer was involved in the rate-limiting reaction step for the ET from the substrate reduced enzyme, but not the dithionite-reduced enzyme. Solvent deuterium kinetic isotope effects of 1.5 and 12.2 were obtained, respectively, for the ET reactions from dithionite-reduced and substrate-reduced methylamine dehydrogenase. These results demonstrate that application of ET theory to the analysis of thermodynamic data for the intermolecular protein ET reactions can potentially be used to distinguish between true ET reactions and those which are gated or attenuated by adiabatic events. Kinetic models are presented to explain how the incorporation of the substrate-derived amino group into TTQ may alter the rate limiting step for ET. PMID- 7547948 TI - Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure. AB - Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547949 TI - Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments. AB - Solution X-ray scattering experiments have been carried out on recombinant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase- and peptide-binding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptide-binding fragment (residues 388 554). Modeling based on steady-state values of radii of gyration (Rg's) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments while Hsc70 and the 60 kDa fragment are prolate and relatively elongated. Rg values decrease significantly in the presence of MgATP relative to their values in the presence of MgADP (delta Rg approximately 4-5 A) for Hsc70 and the 60 kDa fragment; in contrast, they are essentially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of Rg for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transition to the ATP-induced Rg occurs significantly more rapidly than ATP hydrolysis while the reverse transition to the larger Rg value does not occur before product release. Altogether, the solution scattering data support a model in which a conformational change in Hsc70 (presumably to the low-peptide-affinity state) is predicated on ATP binding while the reverse transition is predicated on product release. PMID- 7547950 TI - Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. AB - The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin. PMID- 7547951 TI - Crystal structure of interleukin 10 reveals an interferon gamma-like fold. AB - The crystal structure of recombinant human interleukin 10 (rhIL-10) has been determined by X-ray crystallography at 2.0 A resolution. Interleukin 10 is a dimer composed of identical polypeptide chains related by a 2-fold axis. The molecule is predominantly alpha-helical. The main-chain fold resembles that of interferon gamma (IFN-gamma) in which the structural integrity of each domain is dependent on the intertwining of helices from each peptide chain. Comparison of rhIL-10 and IFN-gamma reveals differences in helix lengths and orientations of the 2-fold related domains. Interleukin 10 and IFN-gamma contain several conserved residues in their internal cores which suggest a possible "fingerprint" for detection of other members of this fold. PMID- 7547952 TI - Structure of the calcium ion-bound gamma-carboxyglutamic acid-rich domain of factor IX. AB - We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX. PMID- 7547953 TI - Study of human calcitonin fibrillation by proton nuclear magnetic resonance spectroscopy. AB - The fibrillation of human calcitonin (hCT) has been investigated by NMR in aqueous solution. The time course of proton one- and two-dimensional NMR spectra of hCT (80 mg/mL at pH 2.9) was measured during the fibrillation. It showed a gradual broadening of the peptide peaks, followed by a rapid broadening and subsequent disappearance of the peaks. The gradual broadening can be attributed to equilibrium between monomer and associated hCT, whereas the rapid broadening can be attributed to formation of aggregates and to gelation of the peptide solution. All the peptide peaks did not broaden and disappear simultaneously. Peaks of residues in the N-terminal (Cys1-Cys7) and central (Met 8-Pro23) regions broadened and disappeared faster during the gradual broadening than those in the C-terminal region (Gln24-Pro32). Moreover, in the N-terminal and central residues, peaks of Cys1, Leu4,9, Met 8, Tyr12, Asp15, and Phe16,19,22 disappeared faster than those of Asn3,17, Ser5, Cys7, Gln14, Lys18, and His20. Hydrogen deuterium exchange of amide protons indicated the formation of hydrogen bonds caused by association of hCT molecules. The amphiphilicity of the peptide appears to be important for the hCT association. PMID- 7547954 TI - Resonance Raman studies of Escherichia coli cytochrome bd oxidase. Selective enhancement of the three heme chromophores of the "as-isolated" enzyme and characterization of the cyanide adduct. AB - Cytochrome bd oxidase is a terminal bacterial oxidase containing three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be at a dinuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinone. One of the unique functional features of this enzyme is its resistance to high concentrations of cyanide (Ki in the millimolar range). With the appropriate selection of laser lines, the ligation and spin states of the b558, b595, and d hemes can be probed selectively by resonance Raman (rR) spectroscopy. Wavelengths between 400 and 500 nm predominantly excite the rR spectra of the b558 and b595 chromophores. Spectra obtained within this interval show a mixed population of spin and ligation states arising from b558 and b595, with the former more strongly enhanced at higher energy. Red excitation wavelengths (590-650 nm) generate rR spectra characteristic of chlorins, indicating the selective enhancement of the d heme. These rR results reveal that cytochrome bd oxidase "as isolated" contains the b558 heme in a six-coordinate low-spin ferric state, the b595 heme in a five-coordinate high-spin (5cHS) ferric state, and the d heme in a mixture of oxygenated (FeIIO2 <--> FeIIIO2-; d650) and ferryl-oxo (FeIV = O; d680) states. However, the rR spectra of these two chlorin species indicate that they are both in the 5cHS state, suggesting that the d heme is lacking a strongly coordinated sixth ligand.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547955 TI - Identification and spectral characterization of the external aldimine of the O acetylserine sulfhydrylase reaction. AB - The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547956 TI - Effects of hydrostatic pressure on the kinetics reveal a volume increase during the bacteriorhodopsin photocycle. AB - A protein structural change in the photocycle of the proton pump, bacteriorhodopsin, detected earlier in the M photointermediate by diffraction, consists mainly of changes at the cytoplasmic surface that include an outward tilt of the cytoplasmic end of helix F. Such a conformational rearrangement would result in greater exposure of the interhelical cavity to the medium, increased binding of water, and thus an increase in volume. In order to correlate the structural change with the kinetics of the photoreaction cycle, we measured the effects of hydrostatic pressure between 1 bar and 1 kbar on the rate constants of the photocycles of wild type bacteriorhodopsin and the D96N mutant. Combining the results provided all of the activation volumes and, therefore, the changes of volume in the various states after the K photointermediate is formed. There is an approximately 32 mL/mol volume increase after deprotonation of the retinal Schiff base to the extracellular side, during the M1 --> M2 reaction, that is not reversed until well after its reprotonation from the cytoplasmic side. The magnitude of this volume increase is about as predicted by the increase of the lattice constant in the M state. It occurs in the photocycle at the proposed reprotonation switch, supporting the idea that this conformation change is what alters the accessibility of the Schiff base from one membrane side to the other. Additionally, we observe a large positive (approximately 50 mL/mol) activation volume for proton exchange between D96 and the Schiff base of the wild type protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547957 TI - Ligand binding and protein dynamics in cupredoxins. AB - Type 1 copper sites bind nitric oxide (NO) in a photolabile complex. We have studied the NO binding properties of the type 1 copper sites in two cupredoxins, azurin and halocyanin, by measuring the temperature dependence of the ligand binding equilibria and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6) 10(2) s). In both proteins, we find nonexponential kinetics below 200 K that do not depend on the NO concentration. Consequently, this process is interpreted as geminate recombination. In azurin, the rebinding can be modeled with the Arrhenius law using a single pre-exponential factor of 10(8.3) s-1 and a Gaussian distribution of enthalpy barriers centered at 22 kJ/mol with a width [full width at half-maximum (FWHM)] of 11 kJ/mol. In halocyanin, a more complex behavior is observed. About 97% of the rebinding population can also be characterized by a Gaussian distribution of enthalpy barriers at 12 kJ/mol with a width of 6.0 kJ/mol (FWHM). The pre-exponential of this population is 1.6 x 10(12) s-1 at 100 K. After the majority population has rebound, a power-law phase that can be modeled with a gamma-distribution of enthalpy barriers is observed. Between 120 and 180 K, an additional feature that can be interpreted as a relaxation of the barrier distribution toward higher barriers shows up in the kinetics. Above 200 K, a slower, exponential rebinding appears in both cupredoxins. Since the kinetics depend on the NO concentration, this process is identified as bimolecular rebinding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547958 TI - Structural basis for actomyosin chemomechanical transduction by non-nucleoside triphosphate analogues. AB - Methylation of 2-[(2,4-dinitrophenyl)amino]ethyl triphosphate (dNOTP) was found to abolish its ability to support actin sliding in the in vitro motility assay. A comparative study of the interaction of myosin subfragment 1 (S1) and actoS1 with methylated (MdNOTP) and non-methylated dNOTP was undertaken. Both analogues were shown to be substrates for S1 NTPase in the presence of K+/EDTA, Ca2+, or Mg2+, although their rates of hydrolysis in the presence of the divalent cations were significantly greater than that occurring for ATP. However, actin had only a marginal effect on the rate of hydrolysis of MdNOTP, in sharp contrast to its effect on the hydrolysis of dNOTP and ATP which were quite similar. Moreover, while dNODP is able to form stable ternary S1 complexes with orthovanadate (Vi) or berylium fluoride (BeFx), whose formation results in increased thermal stability of S1, the methylated diphosphate analogue was unable to do so. These differences can be related to methylation-induced changes in the conformation of dNOTP indicated by molecular-modeling approaches. These studies suggest that methylation prevents the specific interaction of the aryl ring of dNOTP with S1 in the adenine binding region necessary for the formation of the force-producing intermediate (M. D. P*) during the S1 Mg(2+)-NTPase cycle. PMID- 7547959 TI - Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity. AB - A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547960 TI - Rapid plasmenylethanolamine-selective fusion of membrane bilayers catalyzed by an isoform of glyceraldehyde-3-phosphate dehydrogenase: discrimination between glycolytic and fusogenic roles of individual isoforms. AB - Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser & Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N-ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate which satisfies the mathematical constraints imposed by the observed rates of fusion of synaptic vesicles with the presynaptic membrane in vivo. PMID- 7547961 TI - Interactions between dystrophin glycoprotein complex proteins. AB - The organization of the dystrophin glycoprotein complex (DGC) was studied by investigating interactions between its components. For this purpose, mouse dystrophin and syntrophin-1 (alpha-syntrophin) sequences were expressed as chimeric fusion proteins and used in overlay binding experiments to probe gel blots of purified rabbit muscle DGC. In order to identify the DGC proteins that bind to different regions of dystrophin, the amino-terminal 385 amino acids, the unique carboxy-terminal domain (amino acids 3266-3678), and the adjacent cysteine rich region of dystrophin homologous to alpha-actinin (amino acids 3074-3265) were expressed as separate fusion proteins. The cysteine-rich sequences of dystrophin predominantly bound adhalin (gp50) and to full length dystrophin suggesting that these sequences may also be important to dystrophin dimerization. The carboxy-terminal domain sequences strongly bound all of the DGC syntrophins and weakly, adhalin, while the amino-terminal sequences of dystrophin bound none of the proteins of this complex. Fusion proteins containing alpha-syntrophin sequences bound not only to dystrophin but also to all three DGC syntrophins, adhalin, and gp35. The interactions identified here were used to refine the existing model of DGC organization to make it consistent with the current data. PMID- 7547962 TI - Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation. AB - Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2 fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation. PMID- 7547963 TI - Tryptophans 231 and 234 in protein C report the Ca(2+)-dependent conformational change required for activation by the thrombin-thrombomodulin complex. AB - Human protein C circulates as both single- and two-chain zymogens. Activation by the physiological activation complex, thrombin-thrombomodulin, generates the anticoagulant enzyme, activated protein C. Ca2+ binding to the protease domain of protein C is accompanied by 5.5 +/- 0.2% quenching of intrinsic fluorescence that correlates with the conformational change required for the rapid activation by the thrombin-thrombomodulin complex. To map which Trp residues report this Ca2+ binding, candidate Trp residues at positions 84, 115, 145, 205, 231, and 234 were changed individually to Phe within a protein C deletion mutant lacking the Gla domain (GDPC). Of these, the Trp to Phe mutation at position 231 (W231F) eliminated the Ca(2+)-induced fluorescence quenching, and the Trp 234 to Phe mutation (W234F) increased the maximum quenching in protein C to 9.4 +/- 0.4%. Upon Ca2+ binding, the fluorescence emission intensity of the W231F mutant was increased 3.4% +/- 0.6%. The Kd for this site (84 +/- 20 microM) was similar to that of GDPC (Kd = 39 +/- 4 microM). To compare the properties of single- and two chain protein C, we replaced the Lys156-Arg157 dipeptide cleavage site in protein C with Thr and Gln to form GDPCKR/TQ. GDPCKR/TQ and the two-chain form of protein C were activated at the same rate with the thrombin-thrombomodulin complex, they exhibited similar Ca2+ dependence for both activation and fluorescence quenching, and these enzymes had the same chromogenic activity. In contrast to the zymogen form, activated human Gla-domainless protein C did not undergo a Ca(2+)-induced fluorescence change. These results indicate that the environment of Trp 231 and 234 within the Ca2+ binding loop of the protein C zymogen are perturbed by Ca2+ binding to the zymogen. PMID- 7547964 TI - The roles of factor VII's structural domains in tissue factor binding. AB - Factor VIIa binds to tissue factor in one of the initial steps of blood clotting. In order to determine the role of the various domains of the factor VII molecule in this interaction, we made several chimeric factor VII proteins using recombinant DNA techniques. The molecules have factor IX domains substituted into factor VII and vice versa. The domains exchanged were the 4-carboxyglutamic acid plus aromatic stack domain (gla), the first epidermal growth factor-like domain (Egf-1), the second epidermal growth factor-like domain (Egf-2), and the catalytic domain. Using tissue factor-coated microtiter wells, competition binding studies with 125I-labeled factor VIIa indicated factor VIIa's Kd is 4.2 nM. Employing the same microtiter plate assay, koff and kon were determined and yielded a Kd of 1.5 nM. The results of competitive binding experiments and activation assays using chimeric proteins indicated the interaction between factor VIIa and tissue factor involves direct contact between tissue factor and factor VIIa's Egf-1 domain and catalytic domain. On the other hand, the gla and Egf-2 domains, while necessary for optimal binding, may merely impart structure to the rest of the molecule. However, either one or both of the latter domains might contribute a relatively small amount of energy to direct binding. PMID- 7547965 TI - Thiol ester cleavage-dependent conformational change in human alpha 2 macroglobulin. Influence of attacking nucleophile and of Cys949 modification. AB - Cleavage of the thiol ester that exists between the side chains of Cys949 and Gln952 in human alpha 2-macroglobulin (alpha 2M) destabilizes the native conformation and leads to a large-scale conformational change that results in exposure of the receptor binding domain and to changes in electrophoretic mobility and sedimentation coefficient. The basis of this destabilization of the alpha 2M native conformation following thiol ester cleavage is not understood. We have extended observations that chemical modifications of the newly-formed SH in thiol ester-cleaved alpha 2M can slow the rate of conformational change in an attempt to determine the factors that influence the kinetic stability of the native conformation. Using changes in the fluorescence of alpha 2M-bound 6-(p toluidino)-2-naphthalenesulfonic acid, we have determined the rate constant for conformational change in human alpha 2M, following thiol ester cleavage by ammonia, methylamine, or ethylamine, both in the absence and in the presence of an SH-modifying group, methyl methanethiosulfate. The influence of bait region cleavage in half of the alpha 2M tetramer on this rate has been examined by comparing the properties of native alpha 2M with those of I-form alpha 2M. The properties of two recombinant alpha 2M variants, C929S and C949Q, have also been examined. We found that the stabilizing effects of Cys949 and Gln952 modification were synergistic and optimal for S-thiomethylation in conjunction with methylamine cleavage of the thiol ester. Modification of Gln952 in the absence of SH modification was destabilizing.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547966 TI - Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin. AB - The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1 antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin. PMID- 7547968 TI - The nature of the transition state for enzyme-catalyzed phosphoryl transfer. Hydrolysis of O-aryl phosphorothioates by alkaline phosphatase. AB - There has been much speculation that enzymes change the nature of the transition state for phosphoryl transfer from the dissociative transition state observed in solution reactions to an associative transition state at the enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) for nonenzymatic and enzymatic reactions, provided that the specificity of the enzyme's binding site does not perturb the dependence of rate on the intrinsic reactivity of a series of substrates. The shallow binding groove of Escherichia coli alkaline phosphatase (AP) and its wide specificity suggest that this enzyme may be suited for such an approach. A second requirement of this approach is that the actual chemical step is rate-limiting. Comparisons of the reactions of aryl phosphorothioates and aryl phosphates support the previous conclusion that a nonchemical step limits kcat/KM for reactions of aryl phosphates, but suggest that the chemical cleavage step is rate-limiting for the aryl phosphorothioates. We therefore determined the dependence of the rate of AP catalyzed cleavage of a series of aryl phosphorothioates on the intrinsic reactivity of the substrates. The large negative values of beta leaving group = 0.8 for the enzymatic reaction (kcat/KM) and -1.1 for the nonenzymatic hydrolysis reaction suggest that there is considerable dissociative character in both the enzymatic and nonenzymatic transition states. Despite the wide specificity of AP, certain substrates deviate from the LFER, underscoring that extreme care is required in applying LFERs to enzymatic reactions. The large negative value of beta leaving group suggests that AP can achieve substantial catalysis via a transition state with dissociative character. PMID- 7547967 TI - Electron transfer reactions between aromatic amine dehydrogenase and azurin. AB - Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed that the apparent Km for azurin decreased with increasing ionic strength. These results are the opposite of what was observed for the reaction between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyanin, despite the fact that in both cases the pairs of redox proteins are each acidic proteins. It was further demonstrated that azurin does not function as an effective electron acceptor for MADH, and that amicyanin does not function as an effective electron acceptor for AADH. Thus, while the two TTQ enzymes each use type I copper proteins as physiologic electron acceptors, there is a strong specificity for which copper protein serves as a redox partner. The kinetic parameters for the electron transfer reactions from reduced AADH to oxidized azurin were determined by stopped-flow spectroscopy. Different results were obtained depending upon whether AADH was reduced chemically with dithionite or with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (kET) at 30 degrees C were 4 and 102 s-1, respectively. Kinetically determined values of Kd also differed by a factor of 2.4. These data suggest that the incorporation of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent kET. The interaction between AADH and azurin was also quantitated using an ultrafiltration binding assay. This yielded a Kd of 300 microM for the AADH--azurin complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547969 TI - Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation. AB - Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles. PMID- 7547970 TI - Site-directed mutagenesis of tyrosine-71 to phenylalanine in Citrobacter freundii tyrosine phenol-lyase: evidence for dual roles of tyrosine-71 as a general acid catalyst in the reaction mechanism and in cofactor binding. AB - Tyr71 is an invariant residue in all known sequences of tyrosine phenol-lyase (TPL). The substitution of Tyr71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for beta elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these substrates exhibit kcat values reduced by 10(3)-10(4)-fold, while the kcat/Km values are reduced by 10(2)-10(3)-fold, compared to wild-type TPL. However, for substrates with good leaving groups (S-(o-nitrophenyl)-L-cysteine,beta-chloro-L alanine, and O-benzoyl-L-serine), Y71F TPL exhibits kcat values 1.85-7% those of wild-type TPL. Y71F TPL forms very stable quinonoid complexes with strong absorbance at 502 nm from L-phenylalanine, tyrosines (L-tyrosine, 3-fluoro-L tyrosine, and [alpha-2H]-3-fluoro-L-tyrosine), and S-alkylcysteines (S-methyl-L cysteine, S-ethyl-L-cysteine, and S-benzyl-L-cysteine). The time courses of the formation of quinonoid intermediates in these reactions are biphasic. The slow phase shows a dependence on concentration of PLP and is due to the cofactor binding steps, while the fast phase is due to the amino acid alpha-deprotonation and reprotonation steps. The rate constants for the fast phase of the reactions of Y71F TPL with L-phenylalanine and S-methylcysteine are similar to those for alpha-deprotonation or reprotonation steps in the reactions of wild-type TPL. The PLP binding constant of Y71F TPL is estimated to be 1 mM by spectrophotometric titration, compared to 0.6 microM for wild-type TPL.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547971 TI - Effect of cysteine to serine mutations on the properties of the [4Fe-4S] center in Escherichia coli fumarate reductase. AB - Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154, and Cys158 are replaced individually by Ser have been constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters have been investigated. The Cys154Ser and Cys158Ser FrdB mutations result in enzymes with negligible activity that have largely dissociated from the cytoplasmic membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters. In contrast the Cys148Ser and Cys151Ser FrdB mutations result in functional membrane bound enzymes that are able to support growth under anaerobic and aerobic conditions. EPR studies of these mutants indicate that all three of the constituent Fe-S clusters are assembled, and the redox and spectroscopic properties of the [2Fe-2S] and [3Fe-4S] clusters are unchanged compared to the wild-type enzyme. In both mutants the [4Fe-4S] cluster is assembled with one non-cysteinyl ligand, and the available data suggest serinate coordination. The physicochemical consequences are perturbation of the intercluster spin interaction between the S = 1/2 [4Fe-4S]+ and S = 2 [3Fe FS]0 clusters and a 60-mV decrease in redox potential for the [4Fe-FS]2+,+ cluster in the FrdB Cys148Ser mutant, and a S = 1/2 to S = 3/2 spin state conversion for the [4Fe-4S]+ cluster and a 72-mV decrease in redox potential for the [4Fe-4S]2+,+ cluster in the FrdB Cys151Ser mutant. Taken together with the previous FrdB Cys to Ser mutagenesis results [Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schroder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965-8969; Manodori, A., Cecchini, G., Schroder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703-2712], the results provide strong support for the proposal that all three clusters are located in the FrdB subunit with Cys57, Cys62, Cys65, and Cys77 ligating the [2Fe-2S] cluster, Cys148, Cys151, Cys154, and Cys214 ligating the [4Fe-4S] cluster, and Cys158, Cys204, and Cys210 ligating the [3Fe-4S] cluster. The role of the low potential [4Fe-4S] cluster in mediating electron transfer from menaquinol to the FAD active site is discussed in light of these mutagenesis results. PMID- 7547972 TI - Aldehyde dehydrogenase activity of Drosophila melanogaster alcohol dehydrogenase: burst kinetics at high pH and aldehyde dismutase activity at physiological pH. AB - The ability of Drosophila alcohol dehydrogenase (D-ADH) to catalyze the oxidation of aldehydes to carboxylic acids has been re-examined. Prior studies are shown to have been compromised by a nonenzymic reaction between the aldehydic substrates and amine-containing buffers, e.g., glycine or Tris, and an amine-catalyzed addition of aldehyde to NAD+. These reactions interfere with spectrophotometric assays for monitoring aldehyde oxidation and obscure the nature and scope of D ADH-catalyzed aldehyde oxidation, particularly at physiological pH. Use of nonreactive buffers, such as pyrophosphate or phosphate, and 1H NMR spectroscopy to monitor all the components of the reaction mixture reveals the facile dismutation of aldehydes into equimolar quantities of the corresponding acids and alcohols at both neutral and high pH. At high pH, dismutation is accompanied by a small burst of NADH production to a steady-state concentration ( < 10 microM) that represents a partitioning between NADH dissociation and aldehyde reduction. The increase in A340 is therefore not a direct measure of the aldehyde oxidation reaction, and the resulting kinetic values cannot be compared to those for alcohol dehydrogenation. The present results for D-ADH, combined with data from the literature, establish that aldehyde oxidation, manifest as dismutation, is a widespread property of alcohol dehydrogenases with potential physiological importance in alcohol metabolism and aldehyde detoxification. PMID- 7547973 TI - Probing enzymic transition state hydrophobicities. AB - Hydrophobic interactions are important in numerous biological processes; however, the nature and extent of hydrophobic interactions in nonaqueous enzymology remain poorly defined. We have estimated the free energies of enzyme--substrate hydrophobic interactions for a model reaction catalyzed by subtilisin BPN'(from Bacillus amyloliquefaciens) in various solvents. Transition state stabilization of subtilisin in water has contributions from both ground state destabilization of hydrophobic substrates and intrinsic enzyme--substrate hydrophobic interactions. Both contributions are evident even in hydrophobic organic solvents and can be modified by protein engineering of the enzyme's binding site, as well as by changing the hydrophobicity of the reaction medium. We have also developed a method to estimate the hydrophobicity of the enzymic transition state involving systematic variation of the substrate and solvent hydrophobicities. The observed binding pocket hydrophobicities were directly affected by replacing the Gly166 residue, located at the back of hydrophobic S1 binding pocket of subtilisin BPN', with more hydrophobic amino acids such as alanine and valine. Thus, the observed S1 binding pocket hydrophobicities of the wild-type, G166A, and G166V mutants were measured to be 1.2, 1.8, and 2.6 log P units, respectively. Our method of calculating effective binding pocket hydrophobicity was found to be applicable to other enzymes, including horseradish peroxidase and alpha-chymotrypsin. Measurements of the binding pocket hydrophobicities have significant implications toward tailoring enzyme function in aqueous as well as nonaqueous media. PMID- 7547974 TI - Acid-base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies. AB - The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible. PMID- 7547975 TI - Examining the structural and chemical flexibility of the active site base, Lys 258, of Escherichia coli aspartate aminotransferase by replacement with unnatural amino acids. AB - The active site base, Lys-258, of Escherichia coli aspartate aminotransferase (AATase) was mutated to a unique cysteine for chemical modification in a cysteine free AATase [Quint; Gloss et al. (1992) Biochemistry 31, 32-39]. Two homolysine analogues (gamma-thiahomolysine and gamma-dithiohomolysine) and the carboxylate series [glutamate, (carboxymethyl)cysteine, and (carboxyethyl)cysteine] were prepared. The kcat and kcat/KM values for amino acids exhibited by the 258-homo Lys AATases are ca. 1000-fold less than those of Quint. However, the kcat/KM values for keto acids are only reduced by 2.5-10-fold. The 258-side-chain carboxylates bind amino acids 1-4 orders of magnitude more tightly than Quint, but have first-order catalytic rate constants 5-7 orders of magnitude less than Quint. The 258-homo-Lys AATases stabilize the E.PLP vs the E.PMP species by approximately 3 kcal/mol relative to Quint; the 258-side-chain carboxylate variants stabilize E.PMP vs E.PLP by 5.8 kcal/mol relative to WT. The C alpha-2H kinetic isotope effects for the reactions of 258-homo-Lys and 258-side-chain carboxylate AATases demonstrate that transaldimination is equally or more rate determining than C alpha proton abstraction for both sets of enzymes. The values of kcat/KM calculated for the 258-side-chain carboxylate variants from the Bronsted plot of Toney and Kirsch [(1989) Science 243, 1485-1488] are in close agreement with those observed. This "tethered" Bronsted analysis shows that the earlier reported poor reactivity of carboxylates in chemical rescue is due to electrostatic exclusion from the active site. PMID- 7547978 TI - Characterization of mammalian C-CDC25Mm exchange factor and kinetic properties of the exchange reaction intermediate p21.C-CDC25Mm. AB - This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25Mm (C CDC25Mm), isolated in pure form as recombinant protein from Escherichia coli. Formation of a complex with nucleotide-free H-ras p21 could be analyzed on native gel electrophoresis by combining C-CDC25Mm and p21.GDP, as the result of the fast separation of GDP from p21. Therefore, in order to obtain highly purified heterodimer in preparative amounts, p21.GDP and C-CDC25Mm were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25Mm-mediated nucleotide exchange on p21 depended on the nature of the bound nucleotide (6 times faster for p21.GDP than p21.GTP) but was uninfluenced by the nature of the free nucleotide acting as second substrate. No saturating concentration of p21.GDP could be determined by measuring the C-CDC25Mm-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the Km and the kcat of the reaction were calculated to be > 11 microM and > 0.8 s-1, respectively. The dramatic increase in the nucleotide exchange rate was found to be almost exclusively based on the strong stimulation of the "off-rate". The "on-rate" of the nucleotide on the p21.C-CDC25Mm complex was similar for GDP and GTP and was little increased vs that on p21 alone. The observation that the apparent affinity of both nucleotides for the p21.C-CDC25Mm complex was lower than for p21 alone stressed the great affinity of this complex, whose Kd was calculated to be approximately 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25Mm differs for a number of specific properties. PMID- 7547977 TI - Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist. AB - The murine agouti gene encodes for a novel 131 amino acid protein. The sequence includes a 22 residue putative secretion signal, an internal basic region, and a C-terminal domain containing 10 cysteines. Agouti has been found to antagonize the binding of certain pro-opiomelanocortin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), to the murine melanocortin-1 receptor (MC1-R). We report the purification of a secreted murine agouti to homogeneity by a two step procedure from baculovirus-infected Trichoplusia ni (T. ni). The protein is glycosylated and exhibits competitive, high-affinity antagonism (Ki = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals that agouti exists in a monomer--dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indicate that the protein is highly stable to thermal denaturation. Enzymatic digestion to probe disulfide bond arrangement yielded a discrete C-terminal (Val 83-Cys 131) domain. The isolated highly cysteine-rich C-terminal domain retains alpha-MSH antagonism equipotent with mature agouti. This bioactive domain contains all 10 cysteines which exhibit sequence homology when aligned with several conotoxins. PMID- 7547976 TI - Biosynthesis and palmitoylation of endothelial nitric oxide synthase: mutagenesis of palmitoylation sites, cysteines-15 and/or -26, argues against depalmitoylation induced translocation of the enzyme. AB - The presence of myristoylated endothelial nitric oxide synthase (eNOS) in cytosolic fractions of bovine aortic endothelial cells (BAEC) suggests that N myristoylation of eNOS is not sufficient for membrane localization. Therefore, we examined if posttranslational palmitoylation was another molecular signal for the membrane attachment of eNOS. Metabolic labeling showed incorporation of [3H]palmitic acid into the membrane-bound, but not the cytosolic, form of eNOS. Fatty acid analysis demonstrated the labeled fatty acid incorporated into eNOS was palmitate, linked via a hydroxylamine-labile thioester bond. Biosynthesis and turnover studies show that the turnover of palmitate is much faster than the protein itself. However, the rates of palmitoylation and depalmitoylation were not affected by bradykinin or ionomycin treatment of BAEC. To examine the contribution of palmitoylation to the membrane association of eNOS, we mutated cysteine-15 and -26. Both mutations markedly diminished palmitoylation of eNOS, but did not significantly alter its membrane association. Additionally, [3H]palmitic acid was not incorporated into nonmyristoylated mutant eNOS (G2A eNOS), suggesting that myristoylation is necessary for subsequent palmitoylation of the enzyme. Taken together, our data imply that palmitoylation does not play a major role in membrane association of eNOS and other amino acid sequences, in conjunction with N-myristoylation, are necessary and sufficient for membrane association of the enzyme. PMID- 7547979 TI - Tissue factor gene transcription in serum-stimulated fibroblasts is mediated by recruitment of c-Fos into specific AP-1 DNA-binding complexes. AB - Serum stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of the protease cascade leading to blood coagulation. In this study, we demonstrate that two AP-1 DNA binding elements located 200-220 bp upstream of the transcription start site are both necessary and sufficient to confer serum inducibility to the TF gene promoter in fibroblasts. Analysis of AP-1 DNA-binding complexes indicates that the predominant form of AP-1 activity in quiescent cells consists of an unidentified Fos-related protein and JunD. While c-Fos is notably absent from these preexisting complexes, serum stimulation results in the rapid entry of c Fos into the TF AP-1 DNA-binding complexes. A similar induction of c-Fos DNA binding activity occurs in cells treated with recombinant growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). Importantly, overexpression of JunD and c-Fos abrogates the requirement for serum in the stimulation of TF promoter activity in fibroblasts. Together, these data indicate that the entry of c-Fos into heterodimeric AP-1 DNA-binding complexes with JunD is a key event underlying serum-stimulated transcription of the TF gene in fibroblasts. PMID- 7547980 TI - The role of 2'-hydroxyl groups in an RNA-protein interaction. AB - The role of the 2'-hydroxyl group in RNA--protein interaction has been investigated using MS2 coat protein and its hairpin RNA operator as a model system. Derivatives of the MS2 translational operator were prepared where individual riboses were replaced by deoxyribose and their binding affinities to MS2 coat protein were determined. Only 1 (U-5) out of 15 positions tested reduced protein affinity by 1.6 kcal/mol. A variety of other 2'-modifications were tested at this position to understand the role of this particular 2'-hydroxyl group. Normal binding of the U-NH2 variant and weaker binding of the U-O-methyl variant are consistent with the ability of these functional groups to provide a hydrogen bond donor. This is also supported by recent crystallographic data which indicate a possible interaction between the 2'-hydroxyl of U-5 and the carboxylate group of glutamate 63 [Valegard et al. (1994) Nature 371, 623-626]. Complementary experiments introducing riboses into a DNA hairpin confirm the putative protein contact, and also identify a requirement for riboses in the two upper base pairs of the hairpin. Several arguments suggest these riboses are required to maintain an A-form helix in this region of the binding site. A minimum requirement of four 2'-hydroxyl groups for wild-type coat protein binding has been determined, one of which is at the -5 position and other three in the upper stem in any combination.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547981 TI - Correlation between cytotoxicity and DNA binding of polypyridyl ruthenium complexes. AB - The cytotoxicity of chloropolypyridyl ruthenium complexes of structural formulas [Ru(terpy)-(bpy)Cl]Cl, cis-[Ru(bpy)2Cl2], and mer-[Ru(terpy)Cl3] (terpy = 2,2':6'2"-terpyridine, bpy = 2,2'-bipyridyl) has been studied in murine and human tumor cell lines. The results show that mer-[Ru(terpy)Cl3] exhibits a remarkably higher cytotoxicity than the other complexes. Moreover, investigations of antitumor activity in a standard tumor screen have revealed the highest efficiency for mer-[Ru(terpy)Cl3]. In a cell-free medium, the ruthenium complexes coordinate to DNA preferentially at guanine residues. The resulting adducts can terminate DNA synthesis by thermostable VentR DNA polymerase. The reactivity of the complexes to DNA, their efficiency to unwind closed, negatively supercoiled DNA, and a sequence preference of their DNA adducts (studied by means of replication mapping) do not show a correlation with biological activity. On the other hand, the cytotoxic mer-[Ru(terpy)Cl3] exhibits a significant DNA interstrand cross-linking, in contrast to the inactive complexes which exhibit no such efficacy. The results point to a potential new class of metal-based antitumor compounds acting by a mechanism involving DNA interstrand cross linking. PMID- 7547982 TI - Recognition of DNA interstrand cross-links of cis-diamminedichloroplatinum(II) and its trans isomer by DNA-binding proteins. AB - Recognition and processing by cellular proteins of DNA modified by platinum complexes have been suggested to be relevant to the mechanism of their antitumor activity. Platinum complexes form on DNA various mono- and bifunctional adducts. It has already been described by other authors that intrastrand cross-links formed on DNA by antitumor cis-diamminedichloroplatinum(II) (cisplatin) between neighboring purine residues are recognized by several DNA-binding proteins. In contrast, these proteins do not recognize the intrastrand cross-links formed on DNA by cisplatin or its clinically ineffective trans isomer (transplatin) between nonadjacent base residues. An eventuality heretofore not addressed is that DNA interstrand cross-links (ICLs) of platinum compounds may be recognized by and bound to DNA-binding proteins. DNA probes of 110 base pairs (bp) were constructed containing five equally spaced ICLs of cisplatin or transplatin. These ICLs were formed at specific sites at which these adducts are preferentially formed in natural DNA. Gel electrophoresis mobility shift and competition assays with these probes were used to investigate the specific recognition and binding of the calf thymus HMG1 protein to the DNA ICLs of both platinum isomers. The ICL of antitumor cisplatin was recognized by and bound to the HMG1 protein with a similar affinity as the 1,2-intrastrand d(GpG) cross-link of this drug. The protein binding to the ICL is selective for the DNA modification by cisplatin, but not by chemotherapeutically inactive transplatin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547983 TI - Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants. AB - The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane. PMID- 7547984 TI - Burkitt lymphoma Daudi cells contain two distinct farnesyltransferases with different divalent cation requirements. AB - Farnesylation is a lipid posttranslational modification required for the biological function of several signaling proteins including the Ras oncoprotein where this modification is required for malignant transformation. Here we report the identification of two distinct farnesyltransferases (FTases) in Burkitt lymphoma Daudi cells. Separation of Daudi cell cytosolic fractions by ion exchange chromatography resulted in two peaks (FTases I and II) that, on gel filtration, show molecular masses of 90 000 and 250 000 Da, respectively. Immunoblotting experiments showed that FTase I is composed of an alpha/beta heterodimer of about 50 000 Da each. FTase II contained a beta-subunit that is immunologically indistinguishable from the beta-subunit of FTase I and the previously reported human and rat brain FTase but contained an alpha-subunit that reacted poorly with a rat brain anti-alpha-antibody. As in rat brain FTase, Daudi FTases I and II both required magnesium for enzymatic activity. However, their zinc requirements differed. In the absence of Zn2+, FTase I had little activity (10%) whereas FTase II had 30% of its maximum activity (maximum activity obtained in the presence of Zn2+). Furthermore, whereas both FTases I and II were potently inhibited by KB-Ras C-terminal Cys-Val-Ile-Met tetrapeptide mimics, only FTase I but not FTase II required zinc for peptide binding and inhibition. PMID- 7547985 TI - Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity. AB - Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems. PMID- 7547987 TI - High-pressure-assisted reconstitution of recombinant chloroperoxidase. AB - An expression vector containing a T7 promoter and an OmpA signal sequence followed by the DNA sequence of mature chloroperoxidase from the fungus Caldariomyces fumago has been transformed into Escherichia coli. This construct gave high-level expression of apochloroperoxidase when induced with isopropyl thiogalactopyranoside. The nonglycosylated apoenzyme was secreted into periplasmic space. The recombinant apochloroperoxidase was expressed at a level representing about 2% of the total cellular protein. Before conversion to holoenzyme, the apochloroperoxidase was denatured in 8 M urea and partially purified by DEAE chromatography. Maximum yields of holoenzyme were obtained when the denatured apochloroperoxidase, dissolved in a refolding buffer containing iron protoporphyrin IX, calcium ions, and oxidized glutathione, was subjected to high pressure (207 MPa) at -12 degrees C and then allowed to refold at atmospheric pressure and room temperature. The recombinant holoenzyme was characterized by absorption and CD spectroscopy and tested for halogenation and peroxidation activity. The yield of active holochloroperoxidase was about 5% when high-pressure treatment was used as part of the reconstitution process. In the absence of pressure treatment, holoenzyme was formed at about the 1% level. The holochloroperoxidase preparations which resulted from high-pressure treatment showed, upon return to atmospheric pressure, a considerably higher content of native-like secondary structure compared to the nonpressurized preparations. These experiments show that active recombinant chloroperoxidase molecules can be produced, and prove that glycosylation is not a mandatory requirement for chloroperoxidase refolding. PMID- 7547986 TI - Purification and characterization of a secreted arginine-specific dibasic cleaving enzyme from EL-4 cells. AB - A secreted dibasic cleaving peptidase capable of converting dynorphins into Leu enkephalin-Arg6 was purified from the medium of EL-4 mouse thymoma cells. The enzyme is a novel metalloendopeptidase with a neutral pH optimum (6.9) and a molecular weight of approximately 130 000. The dibasic cleaving enzyme was completely inhibited in the presence of 20-50 mM amine buffers, 0.1 mM EDTA, 0.5 mM 1,10-phenanthroline, 0.5 mM N-ethylmaleimide, and 1mM DTNB. Unlike the Kex2 family of proteases, Ca2+ did not activate the endopeptidase, but high concentrations (1 mM) of metal ions such as Cu2+, Ni2+, Zn2+, and Co2+ completely inhibited the enzyme. Inhibition was not seen with 0.2 mM TLCK, 1 mM DTT, and 1 mM PMSF. The enzyme will cleave Arg-Arg and Arg-Lys bonds, but not Lys-Arg or Lys Lys bonds in identical environments, and no aminopeptidase or carboxypeptidase activity was seen. The size of the substrate does not seem to be a determining factor, since dynorphin A(1-12) is cleaved at a rate similar to prodynorphin B(228-256) containing 29 amino acids. The identity of the residues on either side of the cleavage site influences the rate of processing, as noted by different rates of cleavage for the same size peptides dynorphin A(1-13) vs dynorphin A(1 9) vs beta-neoendorphin. The presence of proline in the P3' (alpha-neoendorphin), P4' (dynorphin A(1-11)), or P5' (bovine adrenal medulla dodecapeptide) position does not prevent cleavage, but neurotensin and its (1-11) fragment containing both P2 and P2' proline residues are not cleaved. PMID- 7547988 TI - Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme. AB - Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547989 TI - Double-stranded damage of DNA.RNA hybrids by neocarzinostatin chromophore: selective C-1' chemistry on the RNA strand. AB - Glutathione-activated neocarzinostatin chromophore generates bistranded lesions in the hybrid formed by yeast tRNA(phe) and DNA complementary to its 31-mer 3' terminus. To elucidate the chemistry of the RNA cleavage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequence r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was studied as substrates. In addition to cleavage at both U residues, major damage was produced in the form of an abasic site at the U residues. Evidence for abasic site formation on the RNA strand was obtained from sequencing-gel analysis and measurement of uracil base release. Initial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites. The site containing the substitution was not a target for cleavage or abasic site formation, and the particular T residue, staggered two nucleotides in the 3' direction on the complementary DNA strand, was cleaved significantly less. These studies were valuable in identifying the DNA ds partner of the RNA attack site. Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal 32P label at the 3' end of the RNA strand. Substitution of deuterium for hydrogen at the C-1' position of the U residues led to a substantial isotope effect (k1H/k2H = 3) upon the formation of the RNA abasic lesion and the RNA cleavage products, providing conclusive evidence for selective 1' chemistry. On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was also true for the T residue in an oligodeoxynucleotide analogue of the RNA strand. Chemical mechanisms to account for the RNA cleavage and abasic site formation via C-1' hydrogen abstraction are proposed. PMID- 7547991 TI - A single binding mode of activated enediyne C1027 generates two types of double strand DNA lesions: deuterium isotope-induced shuttling between adjacent nucleotide target sites. AB - It has been previously reported that the potent enediyne antitumor antibiotic C1027 chromophore produces in DNA restriction fragments double-strand lesions at the sequence GTTA1T/ATA2A3C (damage positions are numbered), involving A1 and A3 [Xu, Y.-j., Zhen, Y.-s., & Goldberg, I. H. (1994) Biochemistry 33, 5947-5954]. Using oligodeoxynucleotide substrates, an additional double-strand lesion has been found within this sequence to involve A1 and A2. The lesions, which include strand breaks and abasic sites, are due to hydrogen atom abstraction from C4' at A1 and from C5'at A3 or C1' at A2 by the diradical species of activated drug. Lesions at A2 or A3 are always part of double-strand lesions. The drug radical center involved in attack at A2 or A3 is readily quenched by solvent methanol so as to produce a single-strand lesion at A1. By using methanol containing carbon bound deuterium, there is a substantial isotope effect on the quenching reaction, resulting in enhanced double-strand lesion formation. In the absence of methanol, almost all damage at A1 belongs to double-strand lesions. There is a considerable flexibility of the drug radical attacking A2 or A3, such that the presence of deuterium at C1' of A2 results in substantial shuttling of the attack to the C5' of the neighboring nucleotide A3. These data strongly suggest the presence of a single mode of binding of activated drug but one which permits the drug diradical center attacking A2 or A3 to have considerable leeway in target selection. Quantitative affinity cleavage binding analysis is consistent with this proposal. PMID- 7547990 TI - Phosphorylation mechanism of nucleoside diphosphate kinase: 31P-nuclear magnetic resonance studies. AB - The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. 31P chemical shifts were measured on both native and denatured enzyme. In the enzymatically phosphorylated enzyme denatured by 9 M urea or 7 M guanidine hydrochloride, the NDP kinase phosphohistidine signal appeared between the signals of N delta and N epsilon free monophosphohistidines used as reference compounds and added to the sample. A signal with the same intermediate position was also observed in the pronase digest of the alkaline-denatured phosphorylated enzyme. However, when phosphohistidines of the phosphorylated synthetic peptide pGlu-His-Gly were taken as references, the NDP kinase and the N delta peptide phosphohistidine signals were shown to be identical, providing evidence that phosphorylation occurs on the N delta of the active site histidine residue. Moreover, the rate of hydrolysis of the histidine-bound phosphate is in agreement with a modification at the N delta position. Phosphorylation of the NDP kinase by phosphoramidate provided a result similar to that of the enzymatic phosphorylation. In both cases, phosphorylation could not be detected on any amino acid other than histidine. Particularly, no phosphoserine residue was observed. PMID- 7547992 TI - Expression of the testis-specific histone H1t gene: evidence for involvement of multiple cis-acting promoter elements. AB - The histone H1t gene is expressed exclusively in testis primary spermatocytes. Previous studies indicate that accumulation of H1t mRNA occurs only in primary spermatocytes in normal rats and in transgenic mice bearing the rat H1t transgene. In this study, DNA sequences of human, monkey, mouse, and rat H1t genes were compared and found to be almost identical in the proximal promoter region extending from the H1/AC box through the TATAA box. In addition to conserved elements common to replication-dependent H1 promoters, the H1t promoter contains a unique TE element, and sequences within this element may contribute to enhanced expression of the gene in primary spermatocytes. Two imperfect inverted repeat sequences designated TE1 and TE2, that are located within the larger TE element, overlap a central GC-rich region and bind specifically to nuclear proteins derived from primary spermatocytes. Protein interactions characterized by methylation interference and UV cross-linking experiments indicate that a complex of proteins with a molecular mass of approximately 180 kDa binds TE1. The GC-rich region in H1t and in some replication dependent histone H1 promoters contains an Sp1 consensus sequence. Although the H1t/TE element that contains the GC-rich region binds nuclear proteins, it does not appear to bind Sp1 obtained from cell populations enriched in primary spermatocytes as determined by electrophoretic mobility supershift assays using polyclonal anti-Sp1 antibodies. PMID- 7547993 TI - Nucleosomes reconstituted in vitro on mouse mammary tumor virus B region DNA occupy multiple translational and rotational frames. AB - The mouse mammary tumor virus acquires a highly reproducible chromatin structure when integrated into cellular DNA. Previous studies have suggested that the LTR is arranged as a series of six phased nucleosomes, that occupy specific positions on the LTR. On the basis of nucleosome reconstitution studies using DNA from the B region of the LTR, it has been argued that this sequence directs a uniquely positioned nucleosome. Here we demonstrate in vitro that reconstituted B region nucleosomes adopt at least five distinct translational positions in two rotational frames on a 206 bp fragment of DNA. We have resolved an initial reconstitute into its component species using nondenaturing gel electrophoresis, and precisely mapped the positions of each species using a hydroxyl radical footprinting assay. To confirm the nucleosome positions determined with the hydroxyl radical assay, nucleosome boundaries were mapped using exonuclease III. Comparison of the results from the hydroxyl radical footprinting and exonuclease III assays revealed a symmetrical pattern of overdigestion by exonuclease III which made unequivocal determination of nucleosome boundaries dubious. We conclude that the general use of exonuclease III to map the positions of nucleosomes may lead to incorrect assignment of position, and that assignment of position through the determination of the nucleosome pseudo-dyad from hydroxyl radical footprinting data represents a superior method of analysis. PMID- 7547996 TI - Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. AB - The muscle-type acetylcholine (ACh) receptor has the composition alpha 2 beta gamma delta. The subunits are arranged quasisymmetrically around a central, ion conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the alpha M2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substituted-cysteine-accessibility method to alpha M1. We analyzed 15 consecutive residues, starting at alpha Pro211 at the extracellular end of M1. Wild-type alpha contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant alpha subunits, together with wild-type beta, gamma, and delta subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: alpha Y213C was susceptible to MSTEA added in the presence or the absence of ACh, alpha P211C, alpha I215C, alpha V216C, alpha N217C, and alpha I220C were susceptible in the absence of ACh, and alpha V218C was susceptible in the presence of ACh. These results imply that M1 is exposed in the channel, and its exposure changes during gating or desensitization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7547995 TI - Species-specific microhelix aminoacylation by a eukaryotic pathogen tRNA synthetase dependent on a single base pair. AB - We report here that tyrosyl-tRNA synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA synthetase and aligns with the prokaryotic tyrosyl tRNA synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region. The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr). Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices. However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair. We show that P. carinii tyrosyl-tRNA synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr. In contrast, the enzyme does not charge E. coli microhelixTyr. Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P. carinii tyrosyl-tRNA synthetase. Conversely, we found that E. coli tyrosyl-tRNA synthetase can charge an E. coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair. The results demonstrate that the common structural framework of homologous tRNA synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation. We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA synthetases. PMID- 7547994 TI - Dissociation kinetics between a mouse Fc receptor (Fc gamma RII) and IgG: measurement by total internal reflection with fluorescence photobleaching recovery. AB - Total internal reflect with fluorescence photobleaching recovery (TIR-FPR) has been used to examine the dissociation kinetics between monomeric mouse IgG and a mouse Fc receptor (moFc gamma RII) reconstituted into substrate-supported planar membranes. IgG1, IgG2a, and IgG2b exhibited similar dissociation kinetics, whereas IgG3 did not bind. The fluorescence recovery curves for the IgG-moFc gamma RII interactions were best described by two reversible components (1.4 s-1, 66% and 0.06 s-1, 18%) and an irreversible component ( < 0.01 s-1, 16%). The kinetic parameters for a mouse anti-dinitrophenyl (DNP) IgG1 antibody were equivalent in the absence and presence of saturating amounts of DNP-glycine, demonstrating that possible allosteric changes which might occur in IgG1 upon hapten binding do not appreciably affect the kinetic characteristics of moFc gamma RII binding. The fluorescence recovery curves for polyclonal mouse IgG Fc were similar to those for intact IgG, showing that decreasing the size of the IgG 3-fold does not alter the dissociation rate. The dissociation kinetics of IgG1 decreased considerably in a low ionic strength buffer, indicating that the IgG1 moFc gamma RII interaction has significant electrostatic components. PMID- 7547997 TI - Ripple phase in asymmetric unilamellar bilayers with saturated and unsaturated phospholipids. AB - In a solution of phosphate-buffered saline (PBS), unilamellar bilayers with saturated phosphatidylcholines in one leaflet and negatively charged, unsaturated phospholipids in the other leaflet were observed in the ripple phase at room temperature using atomic force microscopy (AFM). This is the first observation of the ripple phase in asymmetric bilayers. Sodium and phosphate, components of PBS, were found to be necessary for the formation of the ripple structure in the asymmetric bilayers composed of dipalmitoylphosphatidylcholine (DPPC) and 1 palmitoyl-2-oleoylphosphatidylglycerol (POPG), demonstrating a dependency for specific ions for this phase. These results indicate that the two leaflets of a bilayer are closely coupled to give rise to such a long range and complicated morphology. PMID- 7547998 TI - Structure of the human steroidogenic acute regulatory protein (StAR) gene: StAR stimulates mitochondrial cholesterol 27-hydroxylase activity. AB - Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by enhancing the metabolism of cholesterol into pregnenolone. We determined the organization of the StAR structural gene, mapped to 8p11.2. The gene spans 8 kb and consists of seven exons interrupted by six introns. The 1.3 kb of DNA upstream from the transcription start site directed expression of a luciferase reporter gene in mouse Y-1 adrenal cortical tumor cells but not in BeWo choriocarcinoma cells. Reporter gene expression in the Y-1 cells was increased more than 2-fold by 8-Br-cAMP, indicating that the 1.3 kb DNA fragment contains sequences that confer tissue-specific expression and cAMP regulation. The sequence of a related StAR pseudogene, mapped to chromosome 13, lacks introns and has an insertion, numerous substitutions, and deletions. Expression of StAR in COS-1 cells cotransfected with cholesterol 27-hydroxylase (P450c27) and adrenodoxin resulted in a 6-fold increase in formation of 3 beta-hydroxy-5 cholestenoic acid, demonstrating that StAR's actions are not specific to steroidogenesis but extend to other mitochondrial cholesterol-metabolizing enzymes. PMID- 7547999 TI - X-ray structure and catalytic mechanism of lobster enolase. AB - Enolase prepared from lobster tail muscle yielded trigonal crystals with one 47 kDa subunit per asymmetric unit. X-ray data were collected on the apoenzyme at 2.4 A resolution and on a complex with Mn2+ and the inhibitor phosphoglycolate at 2.2 A resolution. The corresponding cDNA was amplified from a library of lobster muscle cDNA, and a sequence corresponding to residues 27-398 was determined. It is highly homologous to other enolases, including yeast enolase for which an X ray structure is available. Yeast enolase was used as a starting point for crystallographic refinement, which led to models of lobster enolase having R factors below 22% and good stereochemistry. These models are very similar to yeast enolase; they have the same fold with a beta 3 alpha 4 N-terminal domain followed by an atypical alpha/beta barrel. Lobster apoenolase and the ternary complex differ only in the position of three mobile loops. In the complex, a single Mn2+ ion is seen ligated to three carboxylates and three water molecules. Phosphoglycolate binds near, but not directly to, the metal. His 157, which belongs to one of the mobile loops, is in contact with the C2 atom of the ligand. A water molecule hydrogen-bonds to the carboxylate of the ligand and to those of Glu 166 and Glu 209. We suggest that His 157 is the base that abstracts the C2H proton, whereas the water molecule is part of a proton relay system keeping the substrate in the carboxylic acid form where the pKa of the C2H group is low enough for proton transfer to His 157. The resulting catalytic mechanism is different from those proposed on the basis of the yeast enzyme X-ray structures, but it fits with earlier biochemical and spectroscopic data. PMID- 7548000 TI - Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction. AB - The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Dano, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface. PMID- 7548001 TI - Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus. AB - Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur. The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF Tu has a higher affinity for GDP than GTP. This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant. Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca. 2-3-fold less strongly bound. Caged GTP is bound about 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP. The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity. PMID- 7548003 TI - Kinetics of pore formation by an antimicrobial peptide, magainin 2, in phospholipid bilayers. AB - The kinetics of the pore formation by magainin 2, an antimicrobial peptide from Xenopus laevis, in lipid vesicles was investigated. The pore formation was estimated by the efflux of a fluorescent dye, calcein, from large unilamellar vesicles composed of egg yolk phosphatidylglycerol. The time courses of the dye release were well-described by a novel model in which the peptide molecules translocate from the outer to the inner monolayer by forming a pore. The concentration dependence of the leakage rate suggested that the pore consists of pentameric magainin. The obtained kinetic parameters estimate that, at a lipid-to peptide molar ratio of 117, 9 pores with a lifetime of 40 microseconds open per second per vesicle in the initial phase. The apparent deactivation of the pore with increasing time can be ascribed to the reduced peptide density in the outer leaflet due to the translocation. Incorporation of phosphatidylcholine destabilized the pore, indicating the importance of anionic lipids in the stable pore formation. PMID- 7548002 TI - The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1. AB - The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as a nucleotide-free dimeric Ran.RCC1 complex. This model is sufficient to describe all experimental data obtained, so that no additional reaction steps must be assumed. All the rate and equilibrium constants for the four-step mechanism have been determined either experimentally or from a simultaneous theoretical fit to all experimental data sets. The affinities of RCC1 to Ran.GDP and Ran.GTP are similar (1.3 x 10(5) and 1.8 x 10(5) M-1, respectively) and are high enough to allow formation of the ternary complex under appropriate concentration conditions. In the absence of excess nucleotide and at low Ran concentrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleotides (GDP or GTP) to Ran in the corresponding ternary complexes are reduced by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the ternary complexes leads to a dramatic increase in the dissociation rate constants by similar orders of magnitude (from 1.5 x 10(-5) s(-1) to 21 s(-1) for GDP) and thus to facilitated nucleotide exchange. The quantitative results of the kinetic analysis suggest that the exchange reaction does not per se favor the formation of the Ran.GTP complex, but rather accelerates the formation of the equilibrium dictated by the relative affinities of Ran for GDP/GTP and the respective concentrations of the nucleotide in the cell. The extent of Ran.GTP formation in vivo can be calculated using the constants derived. PMID- 7548004 TI - Kinetics and inhibition of lipid exchange catalyzed by plasma cholesteryl ester transfer protein (lipid transfer protein). AB - The cholesteryl ester transfer protein-catalyzed cholesteryl ester transfer is inhibited by two compounds identified by a large-scale screening of cholesterol backbone-containing molecules. Kinetic analysis shows that U-95,594, an amino steroid, inhibits competitively the cholesteryl ester transfer protein-catalyzed transfer of both cholesteryl esters and triglycerides, as well from high-density lipoproteins as from synthetic microemulsions. In contrast, U-617, an organomercurial derivative of cholesterol, inhibits competitively the transfer of cholesteryl ester from either donor but is without any effect on triglyceride transfer. In addition to the rapid, competitive inhibition of cholesteryl ester transfer, U-617 also slowly and reversibly reacts with cholesteryl ester transfer protein to produce an additional 10-fold decrease in cholesteryl ester transfer activity but, again, without effect on triglyceride transfer. PMID- 7548005 TI - Xestoquinone activates skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2. AB - Xestoquinone isolated from a sea sponge Xestospongia sapra inhibited both Ca2+ and K(+)-(EDTA) ATPase of skeletal muscle myosin. The inhibition was abolished in the presence of dithiothreitol. Xestoquinone reacted with 2-mercaptoethanol, a sulfhydryl (SH) compound. Unlike N-ethylmaleimide, a well-known SH reagent, modification of 2 mol of SH groups per myosin by xestoquinone caused a marked increase in the actomyosin ATPase activity. Kinetical analysis of stimulatory effects of xestoquinone indicates a decrease in the actin concentrations which gives half of the maximum velocity (Vmax) of actomyosin ATPase reaction without affecting the Vmax, suggesting an increase in the affinity of myosin for actin. N Ethylmaleimide can still modify both the SH1 and SH2 groups after modification of 2 mol of SH groups by xestoquinone. Xestoquinone modified myosin SH groups which caused changes in the tryptophan fluorescence intensity and circular dichroism. These results suggest that xestoquinone modifies the specific SH groups in myosin distinct from SH1 and SH2, resulting in activation of actomyosin ATPase. It is also suggested that xestoquinone strengthens the interaction between actin and myosin through conformational change in the myosin molecule. PMID- 7548007 TI - Effects of perchlorate on depolarization-induced conformational changes in the junctional foot protein and Ca2+ release from sarcoplasmic reticulum. AB - Perchlorate is one of the most potent activators of skeletal muscle excitation contraction (E-C) coupling reported in the literature, but the detailed mechanism of its action remains to be elucidated. In an attempt to further resolve the mode of perchlorate action, the effects of increasing concentrations of perchlorate on the voltage-dependent (T-tubule-mediated) and voltage-independent portions of Ca2+ release were investigated using the isolated triad model. Low concentrations of perchlorate (< or = 10 mM) activated SR Ca2+ release only when the T-tubule moiety was chemically depolarized. Higher concentrations of perchlorate (30-100 mM), on the other hand, produced significant activation of SR Ca2+ release, regardless of whether or not the T-tubule was depolarized. In order to gain further insights, we monitored the conformational change in the junctional foot protein (JFP), which presumably is an important intermediate step in E-C coupling [Yano, M., El-Hayek, R., & Ikemoto, N. (1995) J. Biol. Chem. 270, 3017-3021], using the fluorescently labeled triad preparation. Again, low concentrations of perchlorate (< or = 10 mM) produced a preferential activation of voltage dependent protein conformational change, while higher concentrations of perchlorate produced significant activation of voltage-independent protein conformational change. An increase in the ryanodine binding by perchlorate occurred only in the higher concentration range where the voltage-independent protein conformational change was activated. These results suggest that perchlorate activates E-C coupling by acting on at least two different steps: at lower concentrations, on the T-tubule-to-JFP signal transmission step; at higher concentrations, on the JFP directly. PMID- 7548008 TI - Heterogeneity of antidepressant binding sites on the recombinant rat serotonin transporter SERT1. AB - Antidepressant drugs block the uptake of serotonin into serotonergic nerve terminals and blood platelets. Here, binding of the tricyclic antidepressant [3H]imipramine to the recombinant rat serotonin transporter SERT1 expressed in human embryonic kidney cells was found to be nonhomogeneous. Scatchard analysis and competition experiments revealed the existence of two distinct antidepressant binding sites. At site 1, [3H]imipramine binding was strictly sodium-dependent with an apparent KD of approximately 10 nM. In contrast, [3H]imipramine binding to site 2 occurred also in the absence of sodium and exhibited a lower affinity. Binding of the nontricyclic antidepressant [3H]citalopram was observed only at site 2. The natural substrate of this carrier, serotonin, competitively inhibited antidepressant binding at both sites; however, its affinity to site 2 was approximately 5-fold lower. These data provide a molecular explanation for the distinct pharmacological actions of different antidepressants. PMID- 7548006 TI - Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II. AB - We cloned the full-length cDNA for the cytoplasmic myosin II regulatory light chain (RLC) from a stage 1-2 Xenopus oocyte library. The Xenopus RLC is 94% identical to the chicken smooth muscle myosin RLC. All of the protein kinase C and myosin light chain kinase phosphorylation sites are conserved. Using trifluoperazine [Trybus, K. M., Waller, G. S., & Chatman, T. A. (1994) J. Cell Biol. 124, 963-969], we removed the RLC of smooth muscle myosin and replaced it with recombinant Xenopus RLCs. The wild-type Xenopus RLC substitutes for the gizzard RLC in actin-activated ATPase and in vitro motility assays. We made alanine substitutions of the two residues phosphorylated by myosin light chain kinase, Ser-19 and Thr-18. All of the myosin hybrids, regardless of their mutations or phosphorylation, have similar K+EDTA ATPase activities. As expected, the T18A, S19A hybrid had no actin-activated ATPase, whereas the T18A hybrid phosphorylated on Ser-19 had an actin-activated ATPase similar to that of wild type hybrids phosphorylated only on Ser-19. The actin-activated ATPase of myosin phosphorylated only on Thr-18 is approximately 15-fold lower than that of myosin phosphorylated on Ser-19. Phosphorylation of either Ser-19 or Thr-18 permits the formation of filaments. Remarkably, in the gliding filament assay, myosin phosphorylated only on Thr-18 moves actin filaments at velocities similar to myosin phosphorylated on Ser-19 or both Thr-18 and Ser-19. PMID- 7548010 TI - Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis. AB - The structure of the ca. 250 amino acid hormone binding domain of the human estrogen receptor (hER-LBD), expressed in E. coli and purified as a complex with estradiol, has been probed by selective proteolysis, with analysis of the protein fragments both by classical methods (SDS-PAGE and Edman N-terminal sequencing) and by mass spectrometry (HPLC-coupled electrospray ionization mass spectrometry (LC/ESI-MS)). Rapid cleavage by several proteases (trypsin, chymotrypsin, thermolysin, and Asp-N endoproteinase) is observed within a localized region (residues 297-303) at the N-terminus. In contrast, proteolytic scission at the C terminus is less localized and more progressive; initial cuts by trypsin, chymotrypsin, thermolysin, V8, and Asp-N proteinases are observed to occur in the region 553-571, followed by further cleavage with thermolysin (548) and trypsin (548, 531, and 529). Thus, N304 and K529 define the protease-resistant N- and C termini of a core structure for this domain that appears to contain the elements sufficient for ligand binding. The remaining segment of this domain (530-553), which is known to embody elements essential for ligand-modulated transcription activation (AF-2), is likely a surface-exposed region that, through these studies, is shown to be accessible to proteases. Only a single region within the 26 kDa ligand-binding core (N304-K529) has been identified as being readily accessible to proteases; rapid proteolysis using the proteases trypsin, chymotrypsin, and thermolysin, is localized to residues 465-468, with cleavage occurring at residues K467, L466, and both T465 and S468, respectively. The flexibility implied by the cuts in this internal 465-468 region suggest that the hER-LBD may actually consist of two subdomains. These proteolysis studies provide a substantially refined view of the conformational nature of the human estrogen receptor ligand binding domain. PMID- 7548009 TI - Probing the molten globule state of alpha-lactalbumin by limited proteolysis. AB - Limited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548012 TI - The effect of manganese on the oxidation of chemicals by lignin peroxidase. AB - It has recently been discovered that lignin peroxidase isozyme H2 (LiPH2) has the ability to oxidize Mn2+ (Khindaria et al., 1995). Furthermore, at pH 4.5, the physiological pH of Phanerochaete chrysosporium, LiPH2 oxidizes Mn2+ at a much faster rate (25 times) than veratryl alcohol (VA). The ability of Mn2+ to act as a redox mediator for indirect oxidations catalyzed by LiPH2 was therefore investigated. In the presence of physiologically relevant levels of oxalate and Mn2+, the rate of LiPH2-catalyzed oxidation of all substrates studied was dramatically increased. Up to 10-fold stimulations were observed compared to the rates of oxidation of substrate in either the presence or absence of VA. We propose that the stimulation is due to the ability of LiPH2 to oxidize Mn2+, producing the strong oxidant Mn3+, at a high rate. The rates of oxidation of the substrates showed a hyperbolic dependence on Mn2+ in the presence of oxalate, a chelator which was required for maximal activity. The oxalate dependence of the oxidation rates correlated well with the concentration of the 1:1 complex of Mn(2+)-oxalate. The relative concentrations of the substrates and H2O2 and the rate constants for their reactions with Mn3+ determined which chemical was oxidized by the enzymatically produced Mn3+. The importance of the ability of Mn(2+)-oxalate to stimulate the oxidation of chemicals by LiPH2 is discussed. PMID- 7548013 TI - Effects of trifluoroethanol on the conformations of peptides representing the entire sequence of bovine pancreatic trypsin inhibitor. AB - The effects of the cosolvent trifluoroethanol on the conformations of four peptides representing the entire sequence of bovine pancreatic trypsin inhibitor (BPTI) have been measured by CD and NMR. No substantial amounts of helical conformations were induced in one peptide with four proline residues dispersed throughout its sequence, and there were no substantial effects on its average conformational properties or on the interactions between neighboring residues that are normally evident. The other three peptides became helical, although not completely, over their entire lengths. There was a reasonable correlation between the induced content of alpha-helix and the predicted helical propensities of all four peptides. Only one of these peptides is helical in native BPTI; the other two are extended beta-strands. The latter two have an intrinsic propensity for helix formation, but a greater propensity for beta-sheet formation in folded proteins. PMID- 7548014 TI - Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium. AB - Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548011 TI - Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin. AB - We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548015 TI - Cleavage processing of angiotensin-converting enzyme by a membrane-associated metalloprotease. AB - Angiotensin-converting enzyme (ACE) is synthesized as a type 1 ectoprotein. It is released from the cell surface by a proteolytic cleavage-secretion process which is enhanced by treatment of the cells with phorbol esters. Here, we report the development of an in vitro cell-free assay system for the cleavage-secretion, its characterization, and the identification of a potent inhibitor of this process. Membranes prepared from ACE89 cells secreted the testicular isozyme of ACE (ACET) in a temperature- and time-dependent fashion. As expected, the in vitro secreted ACET lacked the membrane-anchoring carboxy-terminal tail of the cell-associated ACET. The in vitro secretase activity was resistant to high salt extraction and to inhibitors of serine, chymotrypsin, trypsin, cysteine, aspartate, and elastase type proteases. However, the activity was sensitive to metal ion chelators and to a synthetic hydroxamic acid derivative, compound 3, a known inhibitor of certain metalloproteases. Compound 3 very efficiently blocked both basal and phorbol ester-stimulated ACET secretion by ACE89 cells. The inhibition was rapid, dose dependent, and reversible, and ACET synthesis, glycosylation, and transport were not affected. Cleavage-secretion of ACET in transiently transfected HeLa cells was also inhibited by compound 3. Finally, in vitro cleavage-secretion of the other isozyme of ACE, ACEP, by membranes isolated from rabbit lungs was strongly inhibited by compound 3. These results indicate that the cleavage-secretion of both isozymes of ACE is carried out by an integral membrane metalloprotease which is specifically inhibited by compound 3. PMID- 7548016 TI - Cloning of the murine counterpart of the tumor-associated antigen H-L6: epitope mapping of the human and murine L6 antigens. AB - The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by mAb 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution of the single H-L6 residue Leu122 with Ser resulted in the H-L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548017 TI - An independent role of cytochrome c-550 in cyanobacterial photosystem II as revealed by double-deletion mutagenesis of the psbO and psbV genes in Synechocystis sp. PCC 6803. AB - Cytochrome (cyt) c-550 and the 33 kDa protein are two extrinsic components that function in maintaining oxygen evolution in cyanobacterial cells. Deletion of either of the two components has been shown to result in cyanobacterial phenotypes that are still capable of photoautotrophic growth albeit with a reduced rate. In order to study the function of cyt c-550 in cyanobacterial photosystem II (PSII) and its possible interaction with the 33 kDa extrinsic protein, we constructed a mutant lacking both cyt c-550 and the 33 kDa protein by inactivating the psbV and psbO genes simultaneously in a cyanobacterium, Synechocystis sp. PCC 6803. The resultant double-deletion mutant was unable to grow photoautotrophically and showed almost no oxygen-evolving activity (less than 10% of the wild type). This residual activity was also lost rapidly upon illumination, suggesting an increased sensitivity of the mutant cells toward photoinhibition. Thermoluminescence measurements indicated that the mutant virtually cannot undergo normal charge accumulation (S-state transitions) leading to oxygen evolution. Herbicide-binding and Western blot analyses showed that the mutant accumulates the PSII complex to an extent of only 20% of that in wild-type cells. Combined with previous results, the present results indicated that cyt c 550 supported oxygen evolution in the single-deletion mutant lacking the 33 kDa protein alone and vice versa. Thus, both cyt c-550 and the 33 kDa protein function independently in maintaining cyanobacterial oxygen-evolving activity in vivo, and both of them are required for the optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548019 TI - Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase. AB - The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5' phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme. To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10 formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo. PMID- 7548018 TI - Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated. AB - Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy. PMID- 7548020 TI - Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia. AB - Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0 7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia. PMID- 7548021 TI - Inhibition and inactivation of vanadium bromoperoxidase by the substrate hydrogen peroxide and further mechanistic studies. AB - Hydrogen peroxide, which is a substrate of vanadium bromoperoxidase (V-BrPO), has been shown to be a noncompetitive inhibitor of V-BrPO. Hydrogen peroxide inhibition increases with increasing pH. The inhibition is reversible under the conditions of the initial steady-state kinetic experiments. Analysis of the inhibition constants (KiiH2O2, KisH2O2) versus H+ concentration indicates that an ionizable group with a pKa between 6.5 and 7 is involved in the inhibition. The origin of the oxygen atoms in the dioxygen produced by the V-BrPO-catalyzed bromide-assisted disproportionation of hydrogen peroxide has been shown through H2(18)O2 labeling experiments to originate from the same molecule of hydrogen peroxide. V-BrPO-catalyzed bromination is shown to be an electrophilic (Br+) as opposed to a radical (Br.) process. The stoichiometry of H2O2 consumed to MCD reacted or to O2 produced is reported. The concentration of hydrogen peroxide also affects the competition of dioxygen formation during MCD bromination; competitive dioxygen formation is strongly enhanced at high pH. Turnover of V BrPO under conditions of very high hydrogen peroxide concentration leads to irreversible inactivation at pH 4 and pH 5. Much less inactivation occurs during turnover at long reaction times at higher pH (> pH 6), and the inactivation can be fully reversed by subsequent addition of vanadate. PMID- 7548022 TI - Catalytic and potentiometric characterization of E201D and E201Q mutants of Trypanosoma congolense trypanothione reductase. AB - Trypanothione reductase is a member of the structurally and functionally well characterized family of flavoprotein reductases, which catalyze the reduced pyridine nucleotide dependent reduction of their disulfide, peroxide, or metal ion substrates. Trypanothione reductase is found in a wide variety of Trypanosoma species, where the enzyme serves physiologically to protect the organism from oxidative stress and assists in maintaining low intracellular levels of hydrogen peroxide. The redox potential of the flavin and the hydride ion transfer reaction of the pro-S hydrogen of NADPH to N5 of FAD have been proposed to be influenced by the presence of a conserved Lys-Glu (K60-E201) ion pair at the bottom of the nicotinamide binding pocket. We have evaluated this hypothesis by making modest substitutions for both the Lys and Glu residues using site-directed mutagenesis. Replacement of the K60 residue with an arginine led to a poorly expressed, and completely inactive, enzyme. Replacement of the Glu201 residue with either a glutamine (E201Q) or an aspartate (E201D) residue led to expressed enzymes which could be readily purified in > 20 mg amounts using protocols developed for the WT enzyme, and which had significant residual trypanothione-reducing activity. These enzymes have now been characterized to determine their redox potentials, catalytic activities, and nucleotide specificities. Relative to the WT enzyme, both E201D and E201Q exhibit ca. 5% of WT trypanothione-reducing activity using NADPH as reductant, but significantly enhanced quinone reductase activity. The oxidase activity of both mutants is enhanced by over 50-fold compared to that of the WT. The redox potential of the WT enzyme has been determined to be -273 mV, while both the E201D and E201Q exhibit more positive redox potentials (-259 and 251 mV, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548023 TI - The tryptophan synthase alpha 2 beta 2 complex: kinetic studies with a mutant enzyme (beta K87T) to provide evidence for allosteric activation by an aminoacrylate intermediate. AB - To investigate the mechanism by which the tryptophan synthase beta subunit accelerates the cleavage of the indole-3-glycerol phosphate catalyzed by the alpha subunit (alpha reaction), kinetic experiments were carried out with wild type and mutant forms of the alpha 2 beta 2 complex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schiff base between aminoacrylate and pyridoxal phosphate at the beta site. To test this hypothesis, we investigated a mutant form of the alpha 2 beta 2 complex having the lysine-87 of its beta subunits replaced by threonine. The mutant alpha 2 beta 2 complex (K87T) exhibits normal activity for the alpha reaction but fails to catalyze formation of L-tryptophan from L-serine and indole (beta reaction). However, the mutant enzyme can form a Schiff base intermediate with L-serine at the beta site. Using a "chemical rescue" method, we converted K87T L-serine intermediate to an aminoacrylate intermediate. Steady state kinetic studies reveal that the aminoacrylate derivative exhibits a 7-fold enhancement in kcat/Km for the alpha reaction relative to that of the L-serine derivative of the mutant or the wild-type enzyme in the absence of L-serine. Rapid kinetic data show that the aminoacrylate derivative of the mutant enzyme exhibits a 6-fold increase in the rate constant for the indole-3-glycerol phosphate cleavage reaction. In addition, rate constants for the reverse reaction and product release steps are also altered. Together, these changes lead to a decrease in Km and an increase in kcat.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548024 TI - Substrate specificity and kinetic studies of nodulation protein NodL of Rhizobium leguminosarum. AB - All lipo-chitin oligosaccharides identified from Rhizobium leguminosarum carry an O-acetyl moiety on C6 of the nonreducing terminal N-acetylglucosamine residue. Previously, we have shown that purified NodL protein, using acetyl-CoA as acetyl donor, in vitro acetylates N-acetylglucosamine, chitin oligosaccharides, and lipo chitin oligosaccharides. In this paper, the enzymatic properties and substrate specificity of NodL protein were analyzed, using a spectrophotometric assay to quantify NodL transacetylating activity. NodL functions optimally under alkaline conditions. Transacetylating activity has a broad temperature optimum between 28 and 42 degrees C. NodL protein is stable for at least 15 min up to 48 degrees C. Glucosamine, chitosan oligosaccharides, terminally de-N-acetylated chitin derivatives, and cellopentaose were identified as acetyl-accepting substrates for NodL protein. Quantitative substrate specificity studies show that chitin derivatives with a free amino group on the nonreducing terminal residue are the preferred substrates of the NodL protein. Our results strongly indicate that the nonreducing terminally de-N-acetylated chitin oligosaccharides produced by the NodC and NodB enzymes are the in vivo acetyl-accepting substrates for NodL protein. PMID- 7548025 TI - Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes. AB - The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone. PMID- 7548026 TI - Structure of the 70-kDa soluble lytic transglycosylase complexed with bulgecin A. Implications for the enzymatic mechanism. AB - Bulgecins are O-sulfonated glycopeptides that are able to enhance the antibacterial activity of beta-lactam antibiotics. The 70-kDa soluble lytic transglycosylase (SLT70) from Escherichia coli forms a specific target of these compounds. Using X-ray crystallography, the three-dimensional structure of a complex of SLT70 with bulgecin A has been determined to 2.8-A resolution and refined to an R factor of 19.5%. The model contains all 618 amino acids of SLT70 and a single molecule of bound bulgecin, located in the active site of the enzyme. The glycopeptide inhibitor is bound in an extended conformation occupying sites analogous to the B, C, and D subsites of lysozyme. Upon binding of bulgecin, the three-stranded antiparallel beta-sheet in the C domain shows a pronounced shift toward the inhibitor. In subsite D, the proposed catalytic residue Glu478 forms a hydrogen bond to the hydroxymethyl oxygen of the proline part of bulgecin and interacts electrostatically with the proline NH2+ group. These interactions, in addition to the interactions observed for the 2-acetamido group of the N-acetylglucosamine residue bound in subsite C, may explain the strong inhibition of SLT70 activity by bulgecin, suggesting that bulgecin acts as an analogue of an oxocarbonium ion intermediate in the reaction catalyzed by SLT70. The structure of the SLT70--bulgecin A complex may be of assistance in the rational design of novel antibiotics. PMID- 7548027 TI - Luminal calcium regulates the inositol trisphosphate receptor of rat basophilic leukemia cells at a cytosolic site. AB - Hormones, growth factors, and other stimuli can generate Ca2+ spikes and waves by activation of the phosphoinositide (PI) pathway. The sources of these Ca2+ signals are inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ stores. Here we use a rapid perfusion apparatus to measure the release of 45Ca2+ from permeabilized rat basophilic leukemia (RBL) cells to investigate the regulation of IP3-mediated Ca2+ release by cytosolic and luminal Ca2+. At 200 nM IP3, Ca2+ release was potentiated by an increase in the cytosolic Ca2+ concentration. This potentiation by Ca2+ was nearly absent at 500 nM IP3. Previous studies in smooth muscle cells and neurons showed an inhibition of Ca2+ release above 300 nM Ca2+. In contrast, no such inhibition was observed in RBL cells. When assayed at low cytosolic Ca2+ concentrations, IP3-mediated release was steeply dependent upon luminal Ca2+ concentration. At high luminal Ca2+ concentration, 1 microM IP3 released most of the stored Ca2+ even in the complete absence of cytosolic Ca2+. However, at high cytosolic Ca2+ concentrations (890 nM), IP3-mediated release was no longer steeply dependent upon the luminal Ca2+ concentration. Furthermore, high concentrations of BAPTA inhibited IP3-mediated release in the absence of cytosolic Ca2+. This suggests that a rapid and local luminal Ca2+ feedback is generated by luminal Ca2+ ions binding to cytosolic sites of the same channel or closely associated channels. This "luminal Ca2+ feedback" can be initiated by an increase in the concentration either of IP3, of cytosolic Ca2+, or of luminal Ca2+. It is likely that "luminal Ca2+ feedback" is exploited by cells in both the initiation and termination of Ca2+ spikes. PMID- 7548028 TI - Copper(II) inhibition of electron transfer through photosystem II studied by EPR spectroscopy. AB - EPR spectroscopy was applied to investigate the inhibition of electron transport in photosystem II by Cu2+ ions. Our results show that Cu2+ has inhibitory effects on both the donor and the acceptor side of photosystem II. In the presence of Cu2+, neither EPR signal IIvery fast nor signal IIfast, which both reflect oxidation of tyrosinez, could be induced by illumination. This shows that Cu2+ inhibits electron transfer from tyrosinez to the oxidized primary donor P680+. Instead of tyrosinez oxidation, illumination results in the formation of a new radical with g = 2.0028 +/- 0.0002 and a spectral width of 9.5 +/- 0.3 G. At room temperature, this radical amounts to one spin per PS II reaction center. Incubation of photosystem II membranes with cupric ions also results in release of the 16 kDa extrinsic subunit and conversion of cytochrome b559 to the low potential form. On the acceptor side, QA can still be reduced by illumination or chemical reduction with dithionite. However, incubation with Cu2+ results in loss of the normal EPR signal from QA- which is coupled to the non-heme Fe2+ on the acceptor side (the QA(-)-Fe2+ EPR signal). Instead, reduction of QA results in the formation of a free radical spectrum which is 9.5 G wide and centered at g = 2.0044. This signal is attributed to QA- which is magnetically decoupled from the non-heme iron. This suggests that Cu2+ displaces the Fe2+ or severely alters its binding properties. The inhibition of tyrosinez is reversible upon removal of the copper ions with EDTA while the modification of QA was found to be irreversible. PMID- 7548029 TI - Direct measurement of lipid peroxidation in submitochondrial particles. AB - The susceptibility of the polyunsaturated fatty acid parinaric acid (cis-PnA) to peroxidative damage with concomitant loss of its fluorescent character can be used to detect lipid peroxidation in a direct and sensitive way. The procedure, originally developed to measure peroxidation in lipid vesicles and erythrocyte membranes, has been adapted for the study of submitochondrial particles. Optimal conditions for the concentrations of cis-PnA (0.8 mol %), mitochondrial membrane (100 microM membrane phospholipid), and the radical generating system (50 microM NADH and 10 microM:1 mM Fe(III)-ADP) were established. In the absence of peroxidation inducing compounds, a stable fluorescent signal can be detected. Upon addition of NAD(P)H and ADP-Fe(III), lipid peroxidation starts, and the observed fluorescence decrease is a measure of peroxidation. Both NADH and NADPH were able to induce lipid peroxidation in submitochondrial particles in the presence of an iron chelate. The use of NADH resulted in higher rates of peroxidation compared with NADPH at the same concentration. Whereas the rate of NADH-induced lipid peroxidation was maximal at very low NADH concentrations (2.5 microM) and decreased when the concentration became higher, the NADPH-induced lipid peroxidation reaches saturation at 100 microM. NADH-induced lipid peroxidation in submitochondrial particles from different rat tissues (heart, skeletal muscle, and liver) resulted in a clear difference in peroxidation rates. The highest rates were observed in heart submitochondrial particles, while the lowest rates were obtained in submitochondrial particles derived from liver. Skeletal muscle submitochondrial particles showed intermediate rates of lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548030 TI - Secondary electron transfer processes in membranes of Heliobacillus mobilis. AB - Picosecond transient absorption difference spectroscopic experiments were performed on membranes of the antenna/reaction center complex of Heliobacillus mobilis to study the electron transfer processes. Particular emphasis was placed on the blue spectral region, where the difference spectra of iron-sulfur centers and quinones are significantly different. Spectra were measured at room temperature in the wavelength region from 400 to 470 nm and from 630 to 730 nm. Laser excitation was into the 788 nm Q gamma band of the bacteriochlorophyll g of the reaction center complex. Global analysis in both wavelength regions reveals three kinetic components. A 25 ps phase originates from the decay of the excited state of antenna to form the primary charge-separated state P798+A0-; a 600 ps component is assigned to the electron transfer from the primary electron acceptor A0 to a secondary electron acceptor; a nondecaying component on the time scale measured represents the formation of the secondary charge-separated state. When the secondary electron acceptors were reduced by adding dithionite at pH 11, the 600 ps component disappeared. Only a 25 ps component and a constant were observed in the 630-730 nm region. The 25 ps component is assigned to the excitation decay in the antenna and the formation of P798+A0-, just as in the nonreduced sample. In the reduced sample, the P798+A0- state does not decay on the time scale measured. In the 400-470 nm region, the same kinetic behavior was observed. The absorption difference spectra of the primary and the secondary electron acceptor were constructed from different charge-separated states.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548031 TI - Effect of ionic strength on the kinetic mechanism and relative rate limitation of steps in the model NADPH-cytochrome P450 oxidoreductase reaction with cytochrome c. AB - Although the kinetic mechanism of the NADPH-cytochrome P450 oxidoreductase (P450R) reaction with cytochrome c3+ has been determined at 850 mM ionic strength [Sem, D.S., & Kasper, C. B. (1994) Biochemistry 33, 12012-12021], this mechanism is no longer valid at lower ionic strength. At 850 mM ionic strength, the mechanism is two-site ping-pong, and reaction at the electron acceptor site is itself ping-pong. As the ionic strength is decreased below 850 mM, the initial velocity profiles begin to show curvature when cytochrome c3+ is the varied substrate. These data are consistent with a mechanism that is still two-site ping pong, but now with random sequential binding of two molecules of cytochrome c3+ at the electron acceptor site. Decreasing ionic strength also causes a change in rate-limiting steps, with (V/K)cytc and (V/K)NADPH increasing while Vmax decreases (below 500 mM ionic strength). These results are consistent with favorable ionic interactions being important for binding NADPH and cytochrome c3+ and with product (NADP+) release becoming the rate-limiting step in Vmax at low ionic strength. Vmax decreases significantly at higher ionic strength (> 500 mM), while (V/K)NADPH decreases only slightly. The DV isotope effect is largest (2.4) at 500 mM ionic strength but decreases at both low and high ionic strength as steps other than hydride transfer become more rate-limiting. D(V/K)NADPH also decreases at both low and high ionic strength, but to a lesser extent than DV.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548032 TI - Kinetics of the interaction between the human factor VIIIa subunits: effects of pH, ionic strength, Ca2+ concentration, heparin, and activated protein C catalyzed proteolysis. AB - Coagulation factor VIIIa consists of a heterotrimer in which the A2 subunit is bound to the A1/A3C1C2 dimer. The dissociation of this complex causes the spontaneous and reversible decay of factor VIIIa activity. In order to characterize the kinetics and affinity of the interaction between A2 and A1/A3C1C2, as well as the influence of different parameters on the interaction, the subunits were chromatographically separated and reassembled in a BIAcore instrument (Pharmacia Biosensor). In the binding experiments, A2 was free in solution, whereas A1/A3C1C2 was immobilized on the dextran surface by direct coupling or captured on an immobilized monoclonal anti-C2 antibody. At our chosen standard condition (pH = 6.0, I = 0.12, and [Ca2+] = 2 mM), the association rate constant, dissociation rate constant, and resulting equilibrium dissociation constant were ca. 1.4 x 10(4) M-1s-1, 2.1 x 10(-4)s-1, and 16 nM, respectively. Increasing the ionic strength or Ca2+ concentration resulted in both slower association and faster dissociation. At 0.3 M NaCl or 25 mM Ca2+, the dissociation constant was > 1 microM. This implies that electrostatic forces involved in the interaction contribute at least one-fourth of the total binding energy. Increasing pH caused a similar effect, yielding a dissociation constant of ca. 0.9 microM at pH 7.5. In those cases where the equilibrium dissociation constants had been determined from solution phase experiments [Fay, P. J., & Smudzin, T. M. (1992) J. Biol. Chem. 267, 13246-13250; Lollar, P., Parker, E. T., & Fay, P. J. (1992) J. Biol. Chem. 267, 23652-23657], these constants agreed well with our results.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548033 TI - Structural basis of antibody cross-reactivity: solution conformation of an immunogenic peptide fragment containing both T and B epitopes. AB - A synthetic octadecapeptide with the amino acid sequence of residues 23-40 of toxin alpha from Naja nigricollis, cyclized with a disulfide bridge between residues 23 and 40, induces antibodies that cross-react with toxin alpha. We report a structural analysis of this peptide in aqueous solution using NMR spectroscopy and molecular modeling. Structures compatible with the 151 obtained NMR distance restraints were generated using a random simulated annealing protocol followed by restrained high-temperature dynamics and energy minimization. The generated structures are compared with that of the corresponding sequence in the native toxin. The two stretches 23-28 and 37-40 adopt a canonical beta-strand structure in the toxin but are disordered in the peptide. The region 28-36 is ordered in both the peptide and the toxin. Residues 28-30 and 34-36 adopt beta-strand structures in the toxin but loop structures in the peptide. Residues 30-33 form a reverse turn in both the peptide and the toxin. Residues Val-27, Trp-28, Ile-35, and Ile-36 form a hydrophobic cluster. The similar, reverse-turn fold of residues 30-33 in the peptide and the toxin may be associated with the immunogenic cross-reactivity. PMID- 7548034 TI - Preferential cross-linking of matrix-attachment region (MAR) containing DNA fragments to the isolated nuclear matrix by ionizing radiation. AB - The sequences that anchor DNA, matrix-attachment regions (MARs), can be identified by their specific and preferential binding to the nuclear matrix. This microenvironment may be hypersensitive to the formation of ionizing radiation induced DNA damage, including DNA-protein cross-links (DPC). To examine the induction of DPC at or near MARs, we developed an in vitro binding assay by using nuclear matrices isolated from murine erythroleukemia cells by high-salt extraction of DNase I-digested nuclei. The cross-linking of nuclear matrix protein to DNA fragments containing kappa-immunoglobulin (kappa-Ig) or an hsp 70 MAR was studied. Fragments of pBR322 of similar size to the MAR-containing fragments served as non-MAR controls. Two types of experiments were conducted: type A in which nuclei were irradiated and nuclear matrices were isolated and assayed for the binding of exogenous 32P-labeled DNA fragments, and type B in which mixtures of isolated nuclear matrices and [32P]DNAs were irradiated and assayed for binding. Poly(dAT) served as a competitor in the binding assays, because it eliminated nonspecific binding of DNA to the nuclear matrix and revealed the radiation-induced increase in tightly bound DNA. When nuclear matrices were isolated from irradiated nuclei (0-200 Gy) and incubated with the kappa-Ig MAR fragment in the absence of poly(dAT) (type A experiments), much nonspecific, non-dose-dependent binding was observed. With poly(dAT) in the incubation mixture, a dose-dependent decrease (p < 0.001) in the binding was revealed, indicating a radiation-induced loss of available binding sites, perhaps due to the cross-linking of endogenous sequences. The pBR322 fragment did not show a similar loss of binding sites. Irradiation of mixtures of isolated nuclear matrices and end-labeled fragments (type B experiments) allowed the study of radiation-induced cross-linking of exogenous fragments to the matrices. If poly(dAT) was present during irradiation, nonspecific binding was eliminated; however, no significant increase (p = 0.5) in the specific binding of the DNA to the nuclear matrix was observed. In contrast, if poly(dAT) was added after irradiation, in addition to the elimination of nonspecific binding, a radiation dose-dependent increase in binding was revealed for both the kappa-Ig MAR and the hsp MAR (p < 0.001), but not for either of the pBR322 fragments. The results indicate that the specific interaction of MARs with proteins of the nuclear matrix provides a radiation-sensitive substrate for the formation of DNA-protein cross-links. PMID- 7548035 TI - The trinucleotide repeat sequence d(CGG)15 forms a heat-stable hairpin containing Gsyn. Ganti base pairs. AB - To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), electrophoretic mobility, chemical modification, and P1 nuclease studies were performed with a single-stranded (ss) oligonucleotide containing (CGG)15 [ss(CGG)15]. The results suggest that ss(CGG)15 forms a hairpin with the following features: (i) a stem containing Gsyn. Ganti base pairs; (ii) at > or = 200 mM K+, CGG repeats on the 5' portion of the stem base-paired to GCG repeats on the 3' side (referred to as the (b) alignment); and (iii) heat stability (Tm = 75 degrees C in low ionic strength). At < or = 100 mM K+, dimethyl sulfate reactions indicated that the hairpin in the (b) alignment was in equilibrium with another structure, presumably a hairpin in the alternative (a) alignment (CGG repeats on the 5' portion of the stem base-paired to CGG repeats on the 3' portion of the stem). Molecular dynamics simulations suggested that the loop region of the (a) alignment contained two guanines stacked on top of one another. The same guanines in the (b) alignment were base-paired in a syn-anti arrangement. We propose that the stability of the loop partially determines the stem alignment. PMID- 7548036 TI - Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy. AB - Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model. PMID- 7548037 TI - Nonnative capping structure initiates helix folding in an annexin I fragment. A 1H NMR conformational study. AB - A 21-residue peptide, P1AQFD5ADELR10AAMKG15LGTDE20D, corresponding to the (helix A)-loop motif of the second repeat of human annexin I, was synthesized and studied by 2D proton NMR. The conformational properties of the peptide were characterized at different temperatures in pure aqueous solution and in a TFE/H2O (1:4 v/v) mixture. In pure aqueous solution, the peptide adopts a preferred conformation, comprising both elements of native and nonnative structures. A high alpha helix content is present in the DADELRA segment, which corresponds to an initiation site in the middle of the native alpha helix sequence. At the N terminus flanking region, a particular nonnative folding is revealed by the J(NH CH alpha) coupling constants and a set of unusual NOE connectivities which correspond to a helix interrupt at the first D residue. Addition of relatively small amount of TFE restores the native helix fold at the C-terminus but not at the N-terminus. On the contrary, the nonnative N-terminus structure is clearly stabilized by TFE. Our data indicate that this structure comprises (i) an Asp5-x x-Glu8 N-terminal capping box, as recently named by Harper and Rose [Harper, E. T., & Rose, G. D. (1993) Biochemistry 32, 7605-7609], (ii) a (i,i + 3) Asp7-x-x Arg10 salt bridge, and (iii) a hydrophobic cluster centered on Phe4 which mainly interacts with Leu9 but also with Ala2, Ala6, and Ala12 in a dynamic way. This structure is rather stable since it is still observed at 293 K in aqueous solution and 313 K in the presence of TFE. It constitutes a very potent initiation site of the alpha helix structure. This is, however, a nonnative structure involving highly conserved residues in the whole annexin family and thus may play an important role in the folding pathway as a transient "compacting helper". PMID- 7548038 TI - Structure of the tetraheme cytochrome from Desulfovibrio desulfuricans ATCC 27774: X-ray diffraction and electron paramagnetic resonance studies. AB - The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazao et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure. PMID- 7548039 TI - Contribution of the FAD binding site residue tyrosine 308 to the stability of pea ferredoxin-NADP+ oxidoreductase. AB - The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners. PMID- 7548040 TI - Unique binding of a novel synthetic inhibitor, N-[3-[4-[4 (amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N-allylglycine methanesulfonate, to bovine trypsin, revealed by the crystal structure of the complex. AB - Trypsin and N-[3-[4-[4-(amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N allylglycine methanesulfonate (1), a newly designed and orally active synthetic trypsin inhibitor, were cocrystallized. The space group of the crystal is P2(1)2(1)2(1) with cell constants a = 63.74 A, b = 63.08 A, and c = 69.38 A, which is nearly identical to that of the orthorhombic crystal of guanidinobenzoyltrypsin. The structure was refined to a crystallographic residual R = 0.176. The refined model of the 1-trypsin complex provides the structural basis for the reaction mechanism of 1. On the basis of the present X-ray results, it is proposed that the potent inhibitory activity of 1 is mainly due to the formation of an acylated trypsin through an "inverse substrate mechanism" and its low rate of deacylation. PMID- 7548041 TI - Circular dichroism spectroscopy of three tyrosine-to-phenylalanine substitutions of fd gene 5 protein. AB - Circular dichroism spectroscopy was used to study mutants of phage fd gene 5 protein (Y26F, Y34F, and Y41F) in which three of the five tyrosines, Tyr-26, Tyr 34, and Tyr-41, were individually substituted with phenylalanine. The tyrosine 229 nm CD bands of the wild type, Y26F, and Y41F gene 5 proteins decreased in magnitude during complex formation with either fd ssDNA or poly[d(A)]. However, the Y34F gene 5 protein showed no decrease in the 229 nm band during titrations of these nucleic acids. This suggested that Tyr-34 of the wild type gene 5 protein dominated the 229 nm CD changes upon binding to single-stranded DNA. Titrations of poly[d(A)] or fd ssDNA with wild type, Y26F, Y34F, or Y41F gene 5 proteins resulted in perturbations of the nucleic acid near-UV CD bands, specific for the particular nucleic acid, but similar for all four proteins (in 2 mM sodium phosphate buffer, pH 7.0). For both nucleic acids, the addition of protein beyond a certain [protein monomer]/[nucleotide] ratio (0.25 for poly[d(A)] or 0.33 for fd ssDNA) resulted in a partial reversal of the CD change of the nucleic acid. These data are interpreted to mean that, in addition to the two well-known n = 4 and n = 3 stoichiometric modes of binding, there is a third mode of binding in which the nucleic acid is in limited contact with the protein. As shown by salt dissociation studies of complexes with poly[d(A)], the binding affinities, K omega, of the proteins were in the order: wild type > Y26F >> Y34F > or = Y41F (for the n = 4 binding mode in 0.1-0.2 M NaCl). Our data indicate that Tyr-34 plays a more important role in forming a complex with ssDNA than is apparent in current models of the g5p.ssDNA complex. We suggest that the hydroxyl moieties of Tyr-34 and Tyr-41 are both somehow involved in stabilizing the interface of bound protein dimers. PMID- 7548042 TI - Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant. AB - The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor. PMID- 7548043 TI - Reexamining the Mossbauer effect as a means to cleave DNA. AB - The ability of a DNA-bound Mossbauer isotope to absorb resonant gamma-radiation and subsequently, upon decay, induce DNA double-strand breaks by emission of low energy Auger electrons was examined with a simple plasmid DNA cleavage assay. This mechanism was postulated by Mills et al. [Mills et al. (1988) Nature 336, 787] in the observed ablation of tumor cell growth with 57Fe(III)-bleomycin and Mossbauer radiation. The observed linearization of supercoiled pAA15 plasmid DNA upon treatment with five or more 57Fe(III)-bleomycin per plasmid precluded its use for testing Mossbauer effect induced cleavage. An alternative 57Fe-DNA binding complex, [57Fe(phen)2(DPPZ)](PF6)2.H2O (57(1)), was synthesized and found to tightly bind DNA (K = 9.8 x 10(5) M-1) yet not induce DNA nicks or cuts at loadings of less than 500/pAA15 plasmid. Mossbauer irradiation of 57(1)/pAA15 samples under frozen and solution conditions does not result in observable linearization of the plasmid DNA over control samples. Some linearization is observed in all irradiated samples but is attributed only to the photoelectric effect. PMID- 7548044 TI - Formation of novel hydrophobic complexes between cationic lipids and plasmid DNA. AB - An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548045 TI - The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. AB - Transfer free energy measurements of amino acids from water to the osmolytes, sucrose and sarcosine, were made as a function of osmolyte concentration. From these data, transfer free energies of the amino acid side chains were obtained, and the transfer free energy of the peptide backbone was determined from solubility measurements of diketopiperazine (DKP). Using static accessible surface evaluations of the native and unfolded states of ribonuclease A, solvent exposed side chain and peptide backbone areas were multiplied by their transfer free energies and summed in order to evaluate the transfer free energy of the native and unfolded states of the protein from water to the osmolyte solutions. The results reproduced the main features of the free energy profile determined for denaturation of proteins in the presence of osmolytes. The side chains were found collectively to favor exposure to the osmolyte in comparison to exposure in water, and in this sense the side chains favor protein unfolding. The major factor which opposes and overrides the side chain preference for denaturation and results in the stabilization of proteins observed in osmolytes is the highly unfavorable exposure of polypeptide backbone on unfolding. Except for urea and guanidine hydrochloride solutions, it is shown that all organic solvents (e.g., dioxane, ethanol, ethylene glycol) and solutes (osmolytes) for which transfer free energy measurements have been determined exhibit unfavorable transfer free energy of the peptide backbone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548046 TI - Prolyl isomerase as a probe of stability of slow-folding intermediates. AB - Catalysis of slow folding reactions by peptidyl prolyl cis-trans isomerase (PPI) provides estimates of stabilities of intermediates in folding of normal and mutational variants of yeast iso-2 cytochrome c. A two-state model postulating a rapid preequilibration of intermediates with the unfolded protein is employed to calculate the stabilization free energy of the intermediate from the catalytic efficiency (kcat/Km) of PPI toward slow folding species. Stability measurements have been made for two distinct slow-folding intermediates: the absorbance detected (IIS) and fluorescence-detected (IIIS) intermediates. Mutation-induced changes in the stability of the intermediates and in the activation free energy for slow folding are compared to changes in equilibrium thermodynamic stability. The results show that (1) for iso-2 the absorbance-detected intermediates (IIS) are slightly more stable than the fluorescence-detected intermediates (IIIS), (2) most mutations have different effects on equilibrium stability and the stability of the IIS or IIIS intermediates, and (3) for both slow folding reactions the mutation-induced changes in the activation free energy are small compared to the magnitude of the activation free energy barrier. Differential effects of mutations on equilibrium stability and the stability of intermediates provides a means of assessing the sequence-encoded structural specificity for folding. Mutations with different effects on intermediate stability and equilibrium stability change the encoded folding information and may alter folding pathways and/or lead to different three-dimensional structures. Identification of mutations which stabilize a folding intermediate relative to the native conformation provides an empirical approach to the design of thermodynamically stable forms of folding intermediates. PMID- 7548047 TI - The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle. AB - During the L-->M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base from D96 in the M-->N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH > 8, where according to independent evidence the L<==>M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the pKa of the protonated Schiff base, and now D85 becomes protonated as in the wild type.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548048 TI - Structure and isomerization of an intrastrand cisplatin-cross-linked octamer DNA duplex by NMR analysis. AB - The anticancer platinum compound cis-Pt(NH3)2Cl2 (cisplatin) forms covalent cross linked adducts with DNA, with the intrastrand didentate adduct between two adjacent guanines being the major product. The platinum atom is coordinated at the N7 positions of adjacent guanines. The duplex consisting of d(CCTG*G*TCC) and its complement d(GGACCAGG), where G*G* stands for the cisplatin cross-linked lesion site, has been analyzed by 1D- and 2D-NMR spectroscopy and its structure solved by the NOE-restrained refinement procedure with the aim to understand the structural distortion associated with the lesion. The refined duplex is unwound (approximately -21 degrees) and kinked (approximately 58 degrees) toward the major groove at the G*G* site, and the minor groove is significantly widened. The deoxyriboses of the G4* and G5* nucleotides are of the N-type (C3'-endo) and S type (C2'-endo) conformations, respectively. The two guanine bases adopt the R configuration (the alpha/beta angles being 112 degrees/290 degrees, respectively), such that the G5*H8 proton (upfield at 8.19 ppm) senses the ring current shielding effect of the G4* base (G4*H8 at 8.76 ppm). The G4*.C13 base pair is perturbed significantly, consistent with the lack of detection of its imino proton. The intrastrand Pt-G*pG* cross-link is metastable in the present DNA duplex. The molecule is slowly converted into a more stable interstrand didentate adduct (between G4 and G9) promoted by the presence of the nucleophilic chloride ion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548049 TI - Alternate view on thermal stability of the DNA duplex. AB - A concept, based on the changes in internal pressure of water due to addition of salts, has been used to interpret the thermal stability of the DNA duplexes. Dependence of thermal stability of duplexes on the salt concentrations has also been described in terms of variations in the internal pressure of water observed in presence of ionic components. The thermal stabilities at high external pressures (up to 200 MPa) compared to those at atmospheric pressure are linearly related to the corresponding variations in internal pressures at several salt concentrations. PMID- 7548050 TI - Evidence for a methylammonium-binding site on methylamine dehydrogenase of Thiobacillus versutus. AB - The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ. PMID- 7548051 TI - Interaction of NADPH-adrenodoxin reductase with NADP+ as studied by pulse radiolysis. AB - The reduction of flavin in NADH--adrenodoxin reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced directly the flavin of the reductase to form a blue semiquinone of the enzyme. Subsequently, the semiquinone decayed by dismutation to form the oxidized and fully reduced forms of the enzyme with a second-order rate constant of 4.4 x 10(4) M-1 s-1. In the presence of equimolar NADP+, the decay of eaq- accompanied an absorption increase at 400 nm, the spectrum of which, formed transiently, is identical to that of NADP radical (NADP.). Subsequently, the transient species decayed concomitantly with the formation of the semiquinone. The rate constant in the formation of the semiquinone was independent of the concentration of the enzyme (6.1 x 10(4) s-1 at pH 7.5). From these results, it is concluded that eaq- reacts with NADP+ bound to the enzyme to form NADP. initially, and subsequently, an electron flows from the NADP. to the flavin by an intracomplex electron transfer. A similar result was obtained in the reaction of CO2- or N-methylnicotinamide radical with the NADP(+)-adrenodoxin reductase complex. These results suggest that the nicotinamide moiety of NADP+ bound to the enzyme is accessible to the solvent and masks the flavin completely. PMID- 7548052 TI - Solution structures of psoralen monoadducted and cross-linked DNA oligomers by NMR spectroscopy and restrained molecular dynamics. AB - We have used two-dimensional 1H NMR spectroscopy to determine the solution structures of the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furanside monoadducted (MAf) and the photoisomeric HMT interstrand cross-linked (XL) DNA oligonucleotide d(5'-GCGTACGC-3')2. The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for both the MAf and XL from both A form and B-form dsDNA starting structures. The root-mean-square (rms) deviations of the coordinates for the 40 structures for the MAf and XL were 1.12 and 1.10 A, respectively. The rmsd of the MAf with respect to the XL is 2.20 A. The local DNA structure is distorted in both adducts, with the helix unwound by 34 degrees and 25 degrees for the MAf and XL, respectively, and an overall helical repeat of 11 base pairs, caused by intercalation of the HMT. The MAf is a photochemical intermediate on the path to interstrand XL. Considerable local structural distortion is induced by both adducts, but the DNA returns to B-form structure within three base pairs of the damage site. There is no significant bend in the helix axis of either the MAf or the XL. We have evaluated the accuracy of the two major methods of converting NOESY data into interproton distances, the isolated spin-pair approximation (ISPA) and the complete relaxation rate matrix analysis (RMA). Both methods were evaluated by comparing the resulting calculated interproton distances generated to known covalently fixed distances in the HMT. The overall structures were evaluated by checking their agreement with biophysical evidence from non-NMR techniques. Only the modified RMA method gave correct interproton distances. PMID- 7548053 TI - Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices. AB - D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides. PMID- 7548054 TI - Structure-function analysis of the adherence-binding domain on the pilin of Pseudomonas aeruginosa strains PAK and KB7. AB - The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue synthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal sequence of the PAK pilin protein (residues 128-144) contains the adherence binding domain. Another pilin strain, KB7, has been cloned and sequenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, Pathogenesis and Evolving Biotechnology, pp 343 351, American Society for Microbiology, Washington, DC]. The C-terminal 17 residue sequence of the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compared to the PAK sequence. In this study, the interactions between the A549 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-terminal 17 residue synthetic peptide of the pilins. The ability of these peptide analogs to inhibit the binding of the biotinylated PAK pili to A549 cells was assessed. Six PAK amino acid side chains (Ser131, Gln136, Ile138, Pro139, Gly141, and Lys144) and nine KB7 side chains (Ala130, Thr131, Thr132, Val133, Asp134, Ala135, Lys136, Arg138, and Pro139) were found to be important in mediating the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pili to A549 cells. This suggests that the interactions between the pilin ligand and the A549 cell surface receptors are dependent on the conformation mediated by the disulfide bridge (Cys129 and Cys142). The residues considered to contribute to bacterial adherence are referred to as the "adhesintope". Four PAK and three KB7 side chains were located in a structurally more rigid region of the disulfide bridged peptide as revealed by two-dimensional NMR studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The structural aspects of the pilin-receptor interactions related to the mapped adhesintope sequences are discussed. The dissimilarities between the PAK and KB7 adhesintopes may suggest that compensatory mutations could occur among different pilin strains so as to allow the pilin adhesins to interact with the same receptor. PMID- 7548055 TI - The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization. AB - The Azotobacter vinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S]2+,+ cluster in each subunit. It is shown to be functional in reconstituting an O2 stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV-visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S]2+,+ cluster is coordinated exclusively by cysteine residues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g = 2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable temperature MCD, resonance Raman, and redox properties (Em = -262 +/- 10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S]2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe-S-C-C dihedral angles are close to 180 degrees and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizes in an orthorhombic space group with unit cell dimensions a = 135 A, b = 135 A, and c = 38 A and that there are at least two dimers per asymmetric unit. PMID- 7548056 TI - 1H NMR solution structure of an active monomeric interleukin-8. AB - The solution structure of a monomeric form of interleukin-8 (IL-8) has been solved using 1H NMR spectroscopy. The chemically synthesized nonnatural analog [IL-8 (4-72) L25 NH-->NCH3] has the same activity as that of native IL-8. Thirty structures were generated using the hybrid distance geometry and simulated annealing protocol using the program X-PLOR. The structure is well-defined except for N-terminal residues 4-6 and C-terminal residues 67-72. The rms distribution about the average structure for residues 7-66 is 0.38 A for the backbone atoms and 0.87 A for all heavy atoms. The structure consists of a series of turns and loops followed by a triple-stranded beta sheet and a C-terminal alpha helix. The structure of the monomer is largely similar to the native dimeric IL-8 structures previously determined by both NMR and X-ray methods. The major difference is that, in the monomeric analog, the C-terminal residues 67-72 are disordered whereas they are helical in the two dimeric structures. The best fit superposition of the backbone atoms of residues 7-66 of the monomer structure on the dimeric IL-8 structures showed rms differences of 1.5 and 1.2 A respectively. The turn (residues 31-35), which is disulfide linked to the N-terminal region, adopts a conformation in the monomer similar to that seen in the dimeric X-ray structure (rms difference 1.4 A) and different from that seen in the dimeric NMR structure (rms difference 2.7 A).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548057 TI - Intermolecular interactions between protein C inhibitor and coagulation proteases. AB - Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60 insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases. PMID- 7548058 TI - Concurrent reduction of iodine and oxidation of EDTA at the active site of horseradish peroxidase: probing the iodine binding site by optical difference spectroscopy and steady state kinetic analysis for the formation of active enzyme I(+)-EDTA ternary complex for iodine reductase activity. AB - Horseradish peroxidase (HRP) catalyzes the reduction of iodine to iodide by EDTA with pseudocatalatic degradation of H2O2 to O2 (Banerjee et al., (1986) J. Biol. Chem. 261, 10592-10597; and Banerjee (1989) J. Biol. Chem. 264, 9188-9194). The reduction of iodine (I+) is dependent on EDTA concentration and is blocked by spin trap, DMPO, indicating the involvement of free radical species in the reduction process. Incubation of EDTA with both HRP and H2O2 results in the appearance of triplet ESR signal of spin-trapped EDTA radical (aN = 15 G), indicating its one-electron oxidation to a nitrogen-centered monocation radical (N-N+). The latter oxidizes H2O2 to evolve O2 and regenerate EDTA. In the presence of I+, a ternary complex of compound I-I(+)-EDTA is formed, which generates compound II-I. complex and both nitrogen-centered dication radical (N(+)-N+) through intermolecular electron transfer from EDTA nitrogens. Compound II-I. complex is further reduced similarly by another molecule of EDTA to form ferric enzyme, I-, and (N(+)-N+).(N(+)-N+) the oxidation product of EDTA, which may be released from the active site and, being more reactive, oxidizes H2O2 to O2 at a faster rate to regenerate EDTA. The existence of (N(+)-N+) is suggested from the similarity of its ESR signal with that of single nitrogen-centered monocation radical (N-N+). EDTA degradation by oxidative decarboxylation due to two-electron oxidation from the same or both nitrogen, atoms is not evident, and EDTA concentration remains the same throughout the reactions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548059 TI - Spectroscopic investigation of Tet repressor tryptophan-43 upon specific and nonspecific DNA binding. AB - The F75 Tet repressor mutant (F75 TetR) contains a single tryptophan residue located at position 43 in the operator recognition alpha-helix. Previous studies [Hansen, D., & Hillen, W. (1987) J. Biol. Chem. 262, 12269-12274] have shown that the fluorescence intensity of this residue is dramatically reduced upon operator binding. In order to determine the origin of this quenching and the role of Trp 43 in the binding mechanism, we have investigated its fluorescence properties upon F75 TetR binding to a tet operator containing 76 bp DNA fragment (specific binding) and to sheared calf thymus DNA (nonspecific binding). Trp-43 steady state fluorescence intensity was quenched by 72% upon specific binding and by 45% upon nonspecific binding. These fluorescence intensity decreases were not accounted for by similar decreases in the respective fluorescence lifetimes. The apparent quenching calculated from the average lifetimes was about 0.33 in both binding modes. This shows the presence of a static quenching process, clearly favored upon specific binding as compared to nonspecific binding. This is consistent with stacking interactions between Trp-43 and the DNA bases, as suggested by molecular graphics [Baumeister, R., Helbl, V., & Hillen, W. (1992) J. Mol. Biol. 226, 1257-1270]. The equilibrium constant between nonfluorescent and fluorescent tryptophan residues was 5 times higher upon binding to specific DNA than to nonspecific DNA. The preferential static quenching of Trp-43 in the specific complex suggests that stacking interactions might contribute to the specific binding mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548060 TI - Inhibition of peptidyl-prolyl cis/trans isomerase activity by substrate analog structures: thioxo tetrapeptide-4-nitroanilides. AB - The ubiquitous cyclophilins belong to peptidyl-prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). They are able to catalyze the cis/trans isomerization about peptidyl-prolyl amide bonds. The mode of action of human cytosolic cyclophilin (Cyp18cy) has been studied on substrate analog tetrapeptide-4 nitroanilides containing the thioxo peptidyl-prolyl bond. Five peptides of the general structure Ala-Xaa-psi [CS-N]-Pro-Phe-NH-Np (Xaa = Gly, Ala, (S)-2 aminobutyric acid, Phe, and Leu) containing the thioxo peptidyl-prolyl bond were synthesized. The kcat values for the chymotryptic cleavage of 4-nitroanilide bond of the thioxo tetrapeptide-4-nitroanilides ranged from 1.7 to 9.0 s-1 and were sufficiently high to analyze the conformational equilibria by isomer-specific proteolysis. The rate constants of the cis/trans isomerization of the thioxo peptidyl-prolyl bond were found to be 25-100-fold lower due to the O/S substitution. Cyp18cy binds both thioxo peptides and oxo peptides in similar manner in the active center but cannot utilize the sulfur analogs as substrates. Instead, competitive inhibition occurs, which was further characterized for Ala Gly-psi[CS-N]-Pro-Phe-NH-Np. The inhibition was nearly independent of the pH value in the range of pH 4.5-9, exhibiting apparent Ki values ranging from 200 to 600 microM. In comparison to Ala-Gly-trans-psi[CS-N]-Pro-Phe-NH-Np, the cis thioxo peptide Ala-Gly-cis-psi[CS-N]-Pro-Phe-NH-Np was found to possess an approximately 30-fold higher affinity for the active site of the enzyme. Thus, in the presence of stoichiometric amounts of Cyp18cy, the total amount of Ala-Leu cis-psi[CS-N]-Pro-Phe-NH-Np in solution, detectable by isomer-specific proteolysis, was considerably enhanced. PMID- 7548061 TI - Expression of saxiphilin in insect cells and localization of the saxitoxin binding site to the C-terminal domain homologous to the C-lobe of transferrins. AB - Saxiphilin is a plasma protein from the bullfrog (Rana catesbeiana) that is homologous to transferrin. Most known transferrins contain two binding sites for Fe3+/HCO3-, one in each of two homologous domains called the N-lobe and C-lobe. However, native saxiphilin does not bind Fe3+ but stoichiometrically binds one molecule of the neurotoxin saxitoxin (STX) with a dissociation constant (KD) of approximately 0.2 nM. To pursue structural analysis of the STX binding sites, cDNA encoding saxiphilin was used to construct a baculovirus expression vector that directs synthesis and secretion of a approximately 92-kDa recombinant saxiphilin protein (R-sax) in cultured insect cells. Culture medium harvested from infected cells contained 25-67 pmol of [3H]STX binding sites/mL with a KD of 0.22 nM. The kinetics and pH dependence (pK0.5 = 5.4) of [3H]STX binding to R-sax are similar to native saxiphilin, implying proper folding and functional activity. Another baculovirus expression vector was constructed to encode a deletion mutant of saxiphilin consisting of the first 20 N-terminal residues containing the secretory signal sequence spliced to the C-terminal, 361-residue fragment homologous to the C-lobe domain of transferrins. This vector directed the secretion of a approximately 38-kDa derivative of saxiphilin (C-sax) that was recognized by antisaxiphilin antibody. C-sax also exhibited [3H]STX binding activity with a lower affinity KD of approximately 0.9 nM, a 4-fold faster dissociation rate for [3H]STX than native saxiphilin, and a pH dependence (pK0.5 = 5.7) similar to R-sax (pK0.5 = 5.4).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548062 TI - Sequence specificity of alkylation for a series of nitrogen mustard-containing analogues of distamycin of increasing binding site size: evidence for increased cytotoxicity with enhanced sequence specificity. AB - The covalent sequence specificity of a series of nitrogen mustard-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two, or three pyrrole-amide units. Previous characterization of the biological profile of the series revealed an increase in cytotoxicity for each corresponding increase in the number of pyrrole units, while showing poor cross-link formation in isolated and cellular DNA. Examination of the sequence specificity revealed that BAM produced guanine-N7 lesions in similar manner to other conventional nitrogen mustards. The monopyrrole BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM. However, alkylation of adenines was also seen that was found to be minor groove adenine-N3 lesions. The dipyrrole and tripyrrole conjugates did not produce detectable guanine-N7 alkylation but only alkylated in AT tracts. In addition, the tripyrrole conjugate preferentially alkylated only a subset of those sites alkylated by the monopyrrole and dipyrrole conjugates. Two sites, 5'-TTTTGG and 5'-TTTTGA, confirmed as guanine-N3 and adenine-N3 lesions, respectively, were strongly alkylated by the tripyrrole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies comparing distamycin and the tripyrrole conjugate showed identical non covalent recognition of AT-rich sites. Hence, the drug that possessed the most enhanced sequence specificity for alkylation was also the most cytotoxic of this series. PMID- 7548063 TI - Effects of bizelesin (U-77779), a bifunctional alkylating minor groove agent, on genomic and simian virus 40 DNA. AB - Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic DNA of BSC-1 cells, as well as intracellular and purified simian virus 40 (SV40) DNA, were examined. Alkaline sucrose sedimentation analysis indicated a nonrandom distribution of heat-labile damage in BSC-1 cell genomic DNA with frequencies of 1-60 lesions/10(6) base pairs (bp) for bizelesin concentrations from 10 to 400 nM, respectively. Extrapolation of these data suggested that, at 0.15 nM bizelesin, approximately 10(2) adducts per cell may be sufficient to inhibit cell growth by 90% (D10). While the frequency of bizelesin adducts in intracellular SV40 DNA was comparable to that in genomic DNA, higher levels of lesion formation are observed with purified SV40 DNA. Chromatin structure has little effect on localization of bizelesin adducts since treatment of either infected cells or purified SV40 DNA reveals a similar pattern of drug induced damage. Bizelesin adduction sites (mapped on the SV40 genome as thermally induced strand breaks at 50-100 bp resolution) are found in regions centered at 4200, 3900, 4700, and approximately 5200. The location of these regions of intense bizelesin bonding coincides with the sites of potential cross-links predicted using the 5'-T-(A/T)4-A-3' sequence. The analysis of bizelesin adducts at the sequence level in the 3943-4451 SV40 DNA fragment indicated that 40% of total damage was in potential cross-linking sites and an additional 35% in the 5' A-(A/T)4-A-3' monoalkylating sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548064 TI - Structural factors contributing to the hydrophobic effect: the partly exposed hydrophobic minicore in chymotrypsin inhibitor 2. AB - The structural basis for the stability of a partly solvent-exposed hydrophobic minicore, formed by the residues Leu51, Val57, and Phe69 in the reactive loop of the serine protease inhibitor chymotrypsin inhibitor 2 (CI2), was analyzed from the stability of 17 mutant proteins, in which side chain methylene groups were deleted or rearranged. The mutations destabilize the protein by 0.3-4.8 kcal/mol, an average of 0.6 kcal per removed methylene group. Double mutant cycles show significant interaction between individual pairs of side chains. There is an excellent linear correlation (r = 0.995) between the free energy of unfolding relative to wild type (delta delta GU-F) and the packing density nC (the number of methyl and methylene groups within 6 A of the removed atoms). delta delta GU-F correlates more weakly with changes in solvent-accessible surface area upon mutation. The correlation improves when the change in solvent-accessible surface are upon mutation is separated into distinct contributions from polar atoms that are able to hydrogen bond to solvent (delta AHB) and from nonpolar atoms (delta AHP). There is also a correlation, however, between packing density and changes in surface area. Elsewhere in CI2, delta delta GU-F for mutations in the buried hydrophobic core correlates best with packing density, whereas in the exposed surface of the alpha-helix, the best correlation is with change in surface area.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548065 TI - Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain. AB - The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. Its ligand-binding domain consists of seven cysteine-rich ligand-binding repeats, each approximately 40 amino acid residues long. Ligand-binding repeats occur in other members of the LDL receptor (LDLR) gene family and in a number of functionally unrelated proteins. As a first step toward an understanding of the structure and function of LB repeats, we have expressed the amino-terminal ligand-binding repeat (LB1) of the human LDLR as a recombinant peptide (rLB1) and have determined its disulfide-pairing scheme. Oxidative folding of rLB1 yielded a single isomer which contained three disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG C7) made to LB1 in the native LDLR, suggesting that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly folded structure. Disulfide bond connections were established using two separate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide bond which connected Cys(IV) and Cys(VI). In the second approach, disulfide bonds were sequentially reduced with tris(2-carboxyethyl)phosphine and the resulting cysteine residues alkylated with iodoacetamide. An analysis of peptides which contained two cysteinylacetamide residues, derived from a single reduced disulfide bond, showed that Cys(I) and Cys(III) were disulfide-bonded and confirmed the presence of a disulfide bond between Cys(IV) and Cys(VI). We infer that the remaining disulfide bond bridges Cys(II) and Cys(V).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548066 TI - 2'-fluoro-2'-deoxy-D-arabinoflavin: characterization of a novel flavin and its effects on the formation and stability of two-electron-reduced mercuric ion reductase. AB - With the goal of generating a novel fluorine-containing flavin analogue with a reduction potential the same as normal flavin, 2'-fluoro-2'-deoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD forms, UV-visible spectral properties are similar to those of normal flavins, and tight binding to riboflavin binding protein and mercuric ion reductase occurs with very similar spectral changes. The reduction potential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1' methylene group insulates the redox-active isoalloxazine from the 2'-fluorine. With the intent of using the analogue as a fluorine NMR probe of the active site environments of two-electron-reduced mercuric ion reductase, apoenzyme was reconstituted and its behavior under reducing conditions examined. Whereas with normal enzyme, addition of two electrons gives rapid formation of a charge transfer species where FAD remains oxidized and a disulfide is reduced to a thiol/thiolate pair, with the 2'-FaFAD enzyme, addition of two electrons gives rapid reduction of the flavin followed by slow transfer of electrons to the disulfide with very little development of the typical charge-transfer absorption. Analysis of crystal structure data suggests that having the fluorine in the alternate arabino stereochemistry places it much nearer the flavin-proximal cysteine/cystine sulfur, where it may inhibit both electron transfer from reduced flavin and the charge-transfer interaction between reduced thiolate and FAD. PMID- 7548067 TI - A bacterial luciferase reaction with a negative temperature coefficient attributable to protein-protein interaction. AB - A yellow fluorescent protein (YFP) present in a strain of bioluminescent bacteria is shown here not only to modify the color and intensity of the emission, as already known and attributed to the interaction of YFP with a luciferase intermediate, but also remarkably to confer a negative temperature dependence to the in vitro system. The in vitro bioluminescence decay rate is actually independent of temperature in the range 5-25 degrees C, at approximately 1 microM YFP concentration. Several hypotheses are considered to explain this effect, based either on inactivation of YFP itself at higher temperatures or on its binding equilibrium with the luciferase intermediate. The first hypothesis is favored. Fluorescence anisotropy measurements show that YFP loses its chromophore at higher temperatures, but this alone cannot account for the negative temperature dependence. Gel chromatography shows the existence of an inactive YFP dimer, and the formation of more dimer at higher temperatures cannot be ruled out but is unlikely in our experimental conditions. Conformational changes may contribute to YFP inactivation. To our knowledge, there is no prior example of an enzymatic reaction in which the rate is slower at higher temperatures, within a physiological range. PMID- 7548068 TI - The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA. AB - The integration host factor (IHF) of Escherichia coli is a small, sequence specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA. PMID- 7548069 TI - Highly purified photosynthetic reaction center (PscA/cytochrome c551)2 complex of the green sulfur bacterium Chlorobium limicola. AB - The photosynthetic reaction center (RC) complex that forms a homodimer of core and cytochrome c subunits was isolated from Chlorobium limicola f. thiosulfatophilum, strain Larsen. The complex showed only two subunit bands at 68 (PscA core) and 21 kDa (cytochrome c551) on SDS-PAGE analysis, indicating the complete deletion of the light-harvesting bacteriochlorophyll a (BChl a) protein as well as the iron-sulfur protein. It contained 27 +/- 3 molecules of BChl a, 7 +/- 1 Chl-670, 3 +/- 1 carotenoids, and 1.6 +/- 0.1 c-type hemes per the primary electron donor P840. The complex showed a light-induced charge separation and recombination between P840 and the acceptor Chl-670 at 77 K as follows: P840*Chl 670-->P840+Chl-670(-)-->P840TChl-670-->P84 0 Chl-670. Pigment compositions and their function in the (PscA/cytochrome c551)2 complex were studied by absorption, circular dichroism, and fluorescence spectroscopy. PMID- 7548071 TI - Domains of Mnt repressor: roles in tetramer formation, protein stability, and operator DNA binding. AB - The Mnt repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of gene regulatory proteins. Proteolytic cleavage of Mnt with chymotrypsin reveals that it consists of two structural domains. Both domains are required for high-affinity operator binding. The N domain (residues 1-51) is dimeric and binds weakly but specifically to operator DNA. The C domain (residues 52-82) forms an independent alpha-helical, tetramerization domain and, by itself, has no DNA binding activity. In intact Mnt, the N and C domains help to stabilize each other against denaturation but appear to be linked rather flexibly. Assays of the half operator affinities of Mnt and the isolated N domain indicate that binding to adjacent half-sites in the whole operator is stabilized by protein-protein contacts between N domains in addition to protein-protein contacts between C domains. PMID- 7548070 TI - Sequence-specific covalent modification of DNA by cross-linking oligonucleotides. Catalysis by RecA and implication for the mechanism of synaptic joint formation. AB - Oligodeoxynucleotides (ODNs) were conjugated to chlorambucil and used as affinity labeling reagents to study joint molecule formation by the Escherichia coli recombinase recA. Chlorambucil is a bifunctional nitrogen mustard which alkylates the N-7 position of guanine in the major groove of double-stranded DNA (dsDNA). Incoming ODNs at least 30 nucleotides long cross-linked to a long homologous duplex DNA in the presence of recA and ATP gamma S. Efficient cross-linkage to the complementary recipient strand of the joint occurred preferentially at guanines positioned 5' relative to the appended chlorambucil group. The pattern of recipient strand alkylation was identical to that observed within a protein free duplex and indicated that strand exchange had occurred prior to alkylation. Modification of the outgoing homologous strand of the joint was less efficient and spanned a 15-20 nucleotide long region offset to the 3' side of the tethered chlorambucil. Alkylation of both recipient and outgoing strands in the same joint molecule occurred with low frequency. By contrast, no affinity alkylation of the displaced strand was observed within a synthetic D-loop. These reaction patterns suggest that the incoming ODN approaches from the minor groove of the duplex to yield a poststrand exchange joint in which the major groove of the newly formed heteroduplex harbors the outgoing strand in an unpaired state. No evidence was obtained for the involvement of a triple-stranded DNA intermediate in recombination. PMID- 7548072 TI - Repair of UV damage in actively transcribed ribosomal genes. AB - Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active ribosomal genes in mouse Friend erythroleukemia cells. Ribosomal genes (rDNA) are multicopied, but only a fraction is transcriptionally active (or transcriptionally "poised"). Selective psoralen binding was used to separate the active fraction, which has an open chromatin structure, from inactive rDNA. EcoRI digestion was used to selectively release the active fraction from nuclei for DNA repair studies. UV dose response curves indicate there is no significant bias for CPD formation in either strand of both types of rDNA chromatin. More importantly, there was no evidence for transcription repair coupling in the individual strands of active and total rDNA. Indeed, over an 8 h period (one cell-cycle), repair of CPDs was almost nonexistent in either strand of active and total rDNA. Furthermore, the fraction of each chromatin structure remains constant during these repair times, suggesting that chromatin rearrangements observed during excision repair of nonnucleolar chromatin do not occur following UV damage of rDNA. However, CPDs are removed efficiently from the transcribed strand of the constitutively expressed c-abl gene (transcribed by Pol II), demonstrating that these cells are capable of transcription repair coupling. PMID- 7548073 TI - The tissue-specific nuclear matrix protein, NMP-2, is a member of the AML/CBF/PEBP2/runt domain transcription factor family: interactions with the osteocalcin gene promoter. AB - The nuclear matrix protein, NMP-2, was originally identified as an osteoblast specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt 441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML 1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding runt domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/runt domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/runt domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a runt domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene. PMID- 7548074 TI - Probing a potassium channel pore with an engineered protonatable site. AB - Blockade by intracellular cations reduces outward conduction of K+ in inward rectifier K+ channels. Mutations of residue 171 in the second transmembrane (M2) segment of the ROMK1 channel have been found to affect the affinity for blockade by intracellular Mg2+ and polyamines. In the present study, we examined the mechanism by which this residue mediates blockade by placing a proton acceptor (histidine) at this position. The results allow us to draw two conclusions. First, the side chain of residue 171 is located in the ion conduction pore about halfway across the transmembrane voltage drop. Second, its side chain comes into close contact and interacts electrostatically with a blocking ion. PMID- 7548075 TI - Interaction of alpha-lactalbumin with phosphatidylglycerol. Influence of protein binding on the lipid phase transition and lipid acyl chain mobility. AB - The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes. PMID- 7548076 TI - Multifunctional activity of the extracellular domain of the M-type (180 kDa) membrane receptor for secretory phospholipases A2. AB - M-type (180 kDa) receptors for secretory phospholipases A2 (sPLA2s) are thought to mediate some of the physiological effects of group I sPLA2, including smooth muscle contraction and cell proliferation. The M-type sPLA2 receptor is a large glycoprotein composed of several distinct extracellular domains which belongs to the C-type lectin superfamily. This receptor binds with high affinity both pancreatic group I and inflammatory group II sPLA2s as well as various sPLA2s purified from snake venoms. This paper shows that the rabbit M-type sPLA2 receptor is a multifunctional protein which is able to promote cell adhesion on type I and IV collagens most probably via its N-terminal fibronectin-like type II domain. It also shows that binding of sPLA2s to a recombinant soluble form of this receptor is associated with a noncompetitive inhibition of phospholipase A2 activity. PMID- 7548077 TI - Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516. AB - The phosphorylation of threonine residues in the catalytic core of several protein kinases is important for the functional integrity of these enzymes. The corresponding residues of cGMP-dependent protein kinase I alpha (cGMP kinase) are Thr-514 and/or Thr-516. The in vivo phosphorylation and functional role of these residues was studied. cGMP kinase was overexpressed and purified as a catalytically active and inactive enzyme in Sf9 insect cells and in Escherichia coli, respectively. The enzymological and physicochemical properties of the Sf9 cGMP kinase were indistinguishable from that of the purified bovine lung enzyme. The cysteines of cGMP kinase including Cys-518 were labeled with vinylpyridine. Amino acid sequencing and mass spectroscopy of the labeled peptides showed that Thr-516 was phosphorylated in the enzyme purified from Sf9 cells but not in that from E. coli. The functional importance of phosphothreonine-516 was investigated by substitution of Thr-516 by alanine (T516A) or by glutamate (T516E). Expression in insect cells of the T516A mutant resulted in a protein lacking detectable kinase activity, whereas the T516E mutant retained basal phosphotransferase activity. In E. coli, the exchange of Thr-516 by glutamate did not lead to the synthesis of a catalytically active enzyme. These results demonstrate that phosphothreonine-516 of cGMP kinase is crucial for the formation of an enzymatically active protein kinase. PMID- 7548078 TI - Mechanism of the conformational change in sodium pump reported by eosin. AB - The conformational change in sodium pump reported by eosin has been reinvestigated because of apparent discrepancies between results obtained in other laboratories with this reversibly-bound, fluorescent reporter group and results obtained in our laboratory with covalently bound fluorescein. The study demonstrates that both probes report the same reaction. Data obtained by following the conformational change in unphosphorylated enzyme with either fluorescein or eosin can be superimposed and fit by an equivalent site mechanism [Smirnova, I. N., & Faller, L. D. (1995) Biochemistry 34, 8657-8667] with the same numerical values of the rate constants for the conformational change and of the dissociation constants for the transported ions. The result is synthesis of data from several laboratories obtained with different probes over more than a decade into a simple and quantitative interpretation of Na+ and K+ interaction with the enzyme and regulation of a protein conformational change that explains the stoichiometry of transport. Therefore, the study supports both the hypothesis that conformational changes function in transport and our proposal that the ion binding sites in one conformation of the enzyme are identical and independent. A corollary to be tested in the future is that ions are transported by a concerted mechanism. PMID- 7548079 TI - Effect of chloride on the thermal reverse reaction of intermediates of iodopsin. AB - Among the intermediates in the bleaching process of iodopsin, a chicken red sensitive cone visual pigment, the batho and meta I intermediates (batho and meta I) formed at low temperatures revert to the original iodopsin by thermal reactions [Yoshizawa & Wald (1967) Nature 214, 566-571; Imamoto, Imai, Yoshizawa, & Shichida (1994) FEBS Lett. 354, 165-168]. In order to elucidate the relationship between Cl- binding to iodopsin and these reverse reactions, we have prepared a sample of iodopsin whose Cl(-)-binding site is vacant (anion-unbound iodopsin) and compared the thermal reactions of its batho and meta I intermediates with those of Cl(-)-bound (native) and nitrate-bound iodopsins. The reverse reaction from batho is observed in both Cl(-)-bound and anion-unbound iodopsins, while the reaction from meta I is observed only in Cl(-)-bound iodopsin. These results indicate that Cl- binding is indispensable for the reverse reaction from meta I, but not from batho. The reverse reaction from meta I has been further investigated as a function of Cl- concentration, and the dissociation constant of Cl- in meta I is estimated to be approximately 20 mM. This value is about 200 times larger than that of iodopsin (0.1 mM), and close to the physiological Cl- concentration in photoreceptor cells, suggesting that Cl- could be released from the protein moiety during the bleaching of iodopsin. PMID- 7548080 TI - X-ray absorption spectroscopy comparison of the active site structures of Phanerochaete chrysosporium lignin peroxidase isoenzymes H2, H3, H4, H5, H8, and H10. AB - The iron heme and its immediate environment can provide information that is pivotal to our understanding of the structural and mechanistic features that confer unusual properties to the heme peroxidases. X-ray absorption spectroscopy (XAS), which is ideally suited for the investigation of the local environment and electronic structure of the heme iron of hemeproteins, has been used to characterize a variety of lignin peroxidase and manganese-dependent peroxidase isoenzymes produced by the white rot fungus Phanerochaete chrysosporium. The data suggest no differences within the error in the first coordination shell of iron for the isoenzymes H2, H3, H4, H5, H8, and H10 examined in this study. The pyrrole nitrogens are at a distance of 2.05 +/- 0.015 A, and the proximal histidine nitrogens are at 1.93 +/- 0.02 A, while the sixth ligands are located at 2.17 +/- 0.03 A. Significant differences are observed in higher coordination shells which may be related to conformational differences in the heme. PMID- 7548081 TI - Dynamics of tryptophan binding to Escherichia coli Trp repressor wild type and AV77 mutant: an NMR study. AB - Binding of L-tryptophan to Escherichia coli trp repressor wild type (WT) and AV77 mutant was studied by 1H NMR spectroscopy. Ligand binding to the proteins resulted in changes in line widths and chemical shifts of ligand resonances, but no changes in the coupling constant were observed. Line width and chemical shift changes of the H4 L-tryptophan proton were monitored as a function of temperature and ligand and protein concentrations. For the WT repressor, the H4 proton displays slow exchange at low temperatures (20-35 degrees C), while fast exchange occurs in the range from 45 to 65 degrees C. From 35 to 40 degrees C, the range of intermediate exchange, lines are broadened beyond detection. For the AV77 mutant, the intermediate and fast exchange regions are shifted at least 5 degrees C to higher temperatures. Line shapes of L-tryptophan H4 proton resonances were simulated using a general expression based on McConnell's modified Bloch equations for a two-site exchange. From the simulations, an exchange frequency (upsilon exch) of about 3000 Hz was obtained at 45 degrees C for WT and about 1000 Hz for AV77 mutant. The activation energy for the process is 32.7 kcal K-1 mol-1 for the WT and 29.1 kcal K-1 mol-1 for AV77. At 45 degrees C, the dissociation and association rate constants (k-1 and K+1, respectively) were calculated to be 2.0 x 10(3) s-1 and 9.9 x 10(6) M-1 s-1, for the WT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548082 TI - Crystal structure of glycyl endopeptidase from Carica papaya: a cysteine endopeptidase of unusual substrate specificity. AB - Glycyl endopeptidase is a cysteine endopeptidase of the papain family, characterized by specificity for cleavage C-terminal to glycyl residues only and by resistance to inhibition by members of the cystatin family of cysteine proteinase inhibitors. Glycyl endopeptidase has been crystallized from high salt with a substrate-like inhibitor covalently bound to the catalytic Cys 25. The structure has been solved by molecular replacement with the structure of papain and refined at 2.1 A to an R factor of 0.196 (Rfree = 0.258) with good geometry. The structure of the S1 substrate binding site of glycyl endopeptidase differs from that of papain by the substitution of glycines at residues 23 and 65 in papain, with glutamic acid and arginine, respectively, in glycyl endopeptidase. The side chains of these residues form a barrier across the binding pocket, effectively excluding substrate residues with large side chains from the S1 subsite. The constriction of this subsite in glycyl endopeptidase explains the unique specificity of this enzyme for cleavage after glycyl residues and is a major component of its resistance to inhibition by cystatins. PMID- 7548083 TI - N-terminus and lysine side chain pKa values of melittin in aqueous solutions and micellar dispersions measured by 15N NMR. AB - Melittin (MLT) is a 26-amino acid cytolytic peptide from the Apis mellifera honey bee. It is known to exist as an alpha-helical tetramer, as an alpha-helical monomer, or as a monomeric random coil depending on solvent conditions. The charge state of MLT is believed to be a major factor in determining its aggregation properties and its interaction with lipids. Several, contradictory, indirect measurements of the pKa values of the three lysine groups in MLT have been reported. In the present study, high-resolution 15N NMR at 50.6 MHz was used to directly measure the pKa values of the amino groups of the Gly-1, Lys-7, Lys 21, and Lys-23 residues of MLT. Specifically, the pH dependence of MLT 15N chemical shifts was measured separately for the isotopically enriched backbone nitrogen of Gly-1 and the side chain nitrogen atoms of Lys-7, Lys-21, and Lys-23 at a MLT concentration of 1.2 mM and a temperature of 23 degrees C. Measurements were made for MLT in potassium phosphate buffer, in neat water, and in 1 myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) lipid micelles. The experiments showed for MLT tetramer in aqueous phosphate buffer that the amino nitrogen of Gly-1 has a pKa of 8.15, and that the Lys-7, Lys-21, and Lys-23 side chain nitrogen atoms have pKa values of 10.21, 10.03, and 10.24 respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548084 TI - Tetramer-dimer equilibrium of phosphofructokinase and formation of hybrid tetramers. AB - Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site for the substrate fructose-6-phosphate. At a given KSCN concentration, the activity varies with the concentration of protein as expected from a simple equilibrium between active tetramers and inactive dimers. The equilibrium constants for the dissociation of a tetramer into dimers have been determined in 0.4 M KSCN for the wild-type enzyme and the noncooperative mutant T125S, the hypercooperative mutant E148A-R152A, and the inactive mutant D127S. The stability of the tetrameric structure is decreased by the mutations E148A R152A that are in the interface and increased by the mutation T125S that does not belong to it. There could be an inverse correlation between the cooperativity of the saturation by fructose-6-phosphate (in absence of any effector) and the stability of the interface that contains its binding site. Hybrid tetramers can be formed upon reassociation of a dimer from an active phosphofructokinase (wild type, T125S, or E148-R152A) with a dimer from the inactive D127S mutant, and their stability and cooperativity toward fructose-6-phosphate have been measured without purifying them. The results indicate that the formation of a hybrid interface involves some flexibility of the two dimers and that the allosteric coupling between distant sites could be related to the plasticity and instability of the interactions across this interface. PMID- 7548085 TI - Prothymosin alpha: a biologically active protein with random coil conformation. AB - Prothymosin is an acidic protein with an unusual amino acid composition. Though its exact function is not yet known, its high evolutionary conservation and wide tissue distribution suggest an essential biological role. Its physical state, which is controversially discussed in previous publications, was investigated using small-angle X-ray scattering, dynamic light scattering, mass spectrometry, and circular dichroism (CD). Our results unequivocally demonstrate that prothymosin is a monomer under physiological conditions. The protein adopts a random coillike conformation but exhibits persistence of direction and curvature. No regular secondary structure is detectable by CD. The Stokes radius, Rs = 3.07 nm, and the radius of gyration, RG = 4.76 nm, are 1.77 and 3.42 times larger, respectively, than those expected for a compactly folded protein consisting of 109 amino acid residues. A remarkable amount of secondary structure is formed only in the presence of trifluoroethanol at low pH. The finding that a biologically active protein molecule with 109 amino acid residues adopts a random coil conformation under physiological conditions raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides. PMID- 7548086 TI - Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. AB - 2D and 3D heteronuclear NMR methods have been used to characterize the structure of hen egg-white lysozyme in a partially folded state using uniformly 15N-labeled protein. This state is formed by the denaturation of the protein in 70% trifluoroethanol (TFE)/30% water (v/v) and is characterized by substantial helical secondary structure in the absence of extensive tertiary interactions. 15N-filtered 3D NOESY and TOCSY experiments have allowed the sequential assignment of resonances for all but 2 of the 126 main chain amide nitrogen atoms and of the majority of main and side chain proton resonances. The conformation of the polypeptide chain was characterized by analysis of the pattern of NOEs, H alpha chemical shift perturbations, 3J(HN, H alpha)-coupling constants, and hydrogen exchange protection. These NMR parameters are highly complementary and are consistent with a model for the TFE state in which six regions of the polypeptide chain are substantially ordered in helical conformations. The structure in different regions however, shows different levels of persistency. Five of the helices exhibit significant protection of amide hydrogens against exchange with solvent and are located in regions of the polypeptide which are helical in the native state. By contrast, helical structures of greater flexibility are observed both as extensions to the native-like helices and as a nonnative structure in the region of the molecule which forms the C-terminal part of the beta-sheet in the native state. No specific structural preferences are detected in regions corresponding to the long loop and to the N-terminal part of the beta-sheet of native lysozyme. A combination of local features of the polypeptide chain, including the predicted propensities of residues for helix formation and for their participation in N- and C-terminal helix capping interactions, allows the conformational behavior of the polypeptide chain of hen lysozyme to be rationalized for this partially folded state. The analysis implies that the nonnative structures are a result of interactions which are local to the polypeptide chain. These, and the highly persistent native-like structures, give insight into species which form early during folding. PMID- 7548087 TI - Kinetic mechanism of kinesin motor domain. AB - The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed by a decrease, which indicates that two isomerizations are induced by nucleotide binding. The ATPase mechanism is fitted by a six-step reaction: [formula: see text] where, T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; K(T) and K(D) are states in rapid equilibrium with the free nucleotide. A set of kinetic constants for 20 degrees C 50 mM NaCl is K1 = 2 x 10(4) M-1, k2 = 200 s-1, k3 = 9 s-1, k5 = 0.01 s-1, and K6 = 2 x 10(-5) M. Values of K1 and K6 are estimates for mant-ATP and mant-ADP, respectively. ADP dissociation is the rate-limiting step. The rate constant for a decrease in fluorescence for the transitions from the high fluorescence K.T state to the low fluorescence K.D state is equal to k3, the rate constant of the hydrolysis step measured by quench flow experiments. The decrease could occur in step 3 or step 4 if k4 > k3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548088 TI - Mechanism of microtubule kinesin ATPase. AB - A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k2 = 200 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = 25 s-1 (50 mM NaCl, 20 degrees C). k2 was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-ADP. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for ADP by mixing K.ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.D state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 12 +/- 2 s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of ADP is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a dimer as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations. PMID- 7548089 TI - Interaction of brain spectrin (fodrin) with phospholipids. AB - Binding of brain spectrin to frozen and thawed liposomes was studied, using a pelleting assay, as a function of lipid composition. Saturable binding was observed for all lipid mixtures that included aminophospholipids, as well as for the total lipid of synaptic plasma membranes. Binding was strong and saturable, with dissociation constants in the nanomolar range. There were two pH optima at ca. 6.0 and 7.5 and a sharp ionic strength optimum, corresponding to physiological solvent conditions. No competition could be detected with extraneous globular proteins, serum albumin, and hemoglobin. The results imply a strong, direct interaction between brain spectrin and the neuronal plasma membrane in the cell. PMID- 7548091 TI - Discovering new dimensions. PMID- 7548092 TI - 20 years of community nursing service: Memphis Sexual Assault Resource Center. PMID- 7548090 TI - Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain. AB - ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose chromatography, and trace amounts of contaminants were removed by passing through a MonoQ column. The yield was 20 mg from a 500 mL culture of E. coli. The purified ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275 nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive ATP, 3H at adenine but not 32P at gamma-phosphate was retained by the protein on gel filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding to the ncd motor domain is tight, although there is an equilibrium between the protein and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the absence of microtubules, but microtubules activated the turnover to a similar extent to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of mantATP increased at 0.005 s-1, which is likely to reflect the release of endogenous ADP and incorporation of mantATP into the protein. The fluorescence intensity of the ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing the mantADP release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548093 TI - A volunteer's story: parents reaching out. Interview by Anita Napier. PMID- 7548095 TI - Depending on the kindness of strangers: organ donation in the '90s. PMID- 7548094 TI - Educating the community on advance directives. TNA Committee on Ethics & Human Rights public service program successful. PMID- 7548096 TI - Health care changes may bring increased liability risk for nurses. PMID- 7548097 TI - Evolving across disciplines. PMID- 7548098 TI - The ethics of embryo experimentation. PMID- 7548099 TI - Ethical issues in the case of surgical repair of cleft palate. AB - There are some advantages to prospective randomized clinical trials (PRCT) to resolve some limited clinical problems. But, when this method is used to determine the best surgical procedure to close the palatal cleft space, there are strong ethical considerations that cannot be overcome. There are two basic problems. The first is having the surgeon perform surgical procedures which he/she does not believe is the treatment of choice or that can be performed as skillfully as others, even after demonstrations. Secondly, this method does not consider the theoretical aspect that many clefts within the same cleft type are different in the relative size of cleft space to size of soft tissue available for closing the cleft space, thereby creating different degrees of scarring. Different outcomes to the same surgery must, therefore, result irrespective of the surgeons' skills or treatment plans. Retrospective research studies, whether they involve one or more institutions, have been and are still very valuable in improving the knowledge base of all areas of cleft palate habilitation. PMID- 7548101 TI - Poly-(L-lactic) acid membranes in palatal surgery in beagle dogs: clinical and histologic evaluation. AB - This article reports on the histologic findings from a larger study that was designed to investigate whether the attachment of scar tissue to underlying bone, which is normally found after palatal surgery, can be prevented by using biodegradable poly-(L-lactic) acid membranes. Von Langenbeck's procedure was simulated in 12-week-old beagle dogs without clefts. In one group normal wound healing was allowed. In two groups, membranes were inserted immediately after surgery or 3 weeks thereafter. Sham and control groups were also included. Histologic evaluation was carried out at regular intervals. Reports have been published on other aspects, such as clinical wound healing, contraction and maxillary arch development in beagle dogs following this treatment. After direct implantation of membranes, wound healing was retarded. Disintegration of the membranes started soon after implantation and remaining particles were surrounded by a fibroblastic sheath and a fibrous capsule. At sites where membrane particles persisted, attachment of the scar tissue to the underlying bone by Sharpey's fibers was prevented. PMID- 7548102 TI - Axial configuration of the velopharyngeal valve and its valving mechanism. AB - The variability of the normal velopharyngeal (VP) closure mechanism was studied by investigating VP anatomy in relation to its closure mechanism in 60 patients. The axial configuration of the VP isthmus, as observed in axial CT scans at rest, was found to be correlated with VP function in terms of its closure patterns in speech as observed by nasendoscopy. A flat VP isthmus was found to be closed mainly in the anteroposterior direction, forming the coronal closure pattern. A deep VP isthmus is closed by movement of the velum and medial movement of the lateral pharyngeal walls, forming the circular closure pattern. A flat VP isthmus occurs when the hamuli are posteriorly located and the muscular slings, therefore, open more posteriorly. We conclude that posterior insertion of the velar muscles on to the skull base results in a flatter, larger VP axial configuration, whereas an anterior insertion results in a VP axial configuration that is deeper and less flat. A flat VP isthmus contracts mainly in an anteroposterior direction, exhibiting a coronal closure pattern, while a deep or round VP isthmus contracts centripetally, exhibiting a circular closure pattern. Variability of the VP valving mechanism is of anatomic and not of functional origin. PMID- 7548100 TI - Onlay silicone and hydroxyapatite-tricalciumphosphate composite (HAP-TCP) blocks interfere with nasal bone growth in rabbits. AB - We investigated how a new type of synthetic porous hydroxyapatite ceramic (HAP TCP) acts when it is implanted in growing membranous bone. Seventy-six New Zealand White infant male rabbits (4-week-olds) were used. Rabbits received HAP TCP block or silicone block implantation in their right nasal bone. The left nasal bone was used for a sham. Serial or cross-sectional examinations by morphometry, radiology, blood biochemistry, and histology were carried out. Both the HAP-TCP and silicone groups exhibited no systemic growth disturbance in terms of morphometry and blood biochemistry. Dual energy x-ray absorptiometry (DEXA) revealed, however, a decrease in the bone mineral content (BMC) of the right nasal bone in the silicone group. Histology revealed a superior affinity of HAP TCP to bone tissue than that of silicone. When a HAP-TCP block was implanted under the periosteum it bonded directly to bone tissue. However, sinking of the implants into the bone tissue were noted both in the HAP-TCP and the silicone groups in longitudinal observation. These results suggest that although HAP-TCP has superior affinity to bone tissue, this by itself is not enough sufficient reason to believe that HAP-TCP can be effectively applied during the growth period. PMID- 7548103 TI - Relationships between integrated oral-nasal differential pressure and velopharyngeal closure. AB - Oral-nasal differential pressures are derived measures that incorporate both active (e.g., articulatory) and passive (e.g., nasal structure) components. This study was designed to examine integrated oral-nasal differential pressures in speakers with different levels of velopharyngeal closure. Integrated oral-nasal differential pressure data were obtained from 20 noncleft adults with normal speech and 166 speakers with repaired palatal clefts. Velopharyngeal competency for the cleft subjects, as determined by aerodynamic assessment, ranged from adequate to grossly incompetent. Results of the data analysis indicate that integrated pressures are not maintained at a consistent level across all groups. This lack of consistency across all degrees of velopharyngeal opening may reflect the flexibility, as well as structural limitations, of a speech pressure regulating system. PMID- 7548104 TI - Guided bone regeneration in calvarial bone defects using polytetrafluoroethylene membranes. AB - Guided bone regeneration is defined as controlled stimulation of new bone formation in a bony defect, either by osteogenesis, osteoinduction, or osteoconduction, re-establishing both structural and functional characteristics. Bony defects may be found as a result of congenital anomalies, trauma, neoplasms, or infectious conditions. Such conditions are often associated with severe functional and esthetic problems. Corrective treatment is often complicated by limitations in tissue adaptations. The aim of the investigation was to compare histologically the amount of bone formed in an experimentally created parietal bone defect protected with one or two polytetrafluoroethylene membranes with a contralateral control defect. A bony defect was created bilaterally in the parietal bone lateral to the sagittal suture in 29 6-month-old male Wistar rats. The animals were divided into two groups: (1) In the double membrane group (n = 9), the left experimental bone defect was protected by an outer polytetrafluoroethylene membrane under the periosteum and parietal muscles and an inner membrane between the dura mater and the parietal bone. (2) In the single membrane group (n = 20), only the outer membrane was placed. The right defect was not covered with any membrane and served as control. The animals were killed after 30 days. None of the control defects demonstrated complete or partial bone regeneration. In the single membrane group, the experimental site did not regenerate in 15 animals, partially in four, and completely in one. In the double membrane group, six of the experimental defects had complete closure with bone, two had partial closure, and one no closure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548105 TI - Relationships between cleft severity and attractiveness of newborns with unrepaired clefts. AB - Perceptions of attractiveness of newborns with unrepaired clefts were investigated by using a range of photographs with clefts of differing severity as ordered by a rule of thumb system commonly adopted by surgeons. The investigation involved a combination of factors such as completeness, unilaterality or bilaterality, and palate involvement. Orderings of attractiveness by surgeons, nursing staff, and adult groups unfamiliar with clefts were remarkably consistent. In addition, the responses reflected the rule of thumb system with ranked preferences of the raters being significantly related to the severity of cleft impairment. The scale devised could be used for comparative treatment outcome studies requiring assessments of initial infant attractiveness. PMID- 7548107 TI - Craniofacial deformities associated with juvenile hyperthyroidism. AB - Three juvenile cases of thyrotoxic craniosynostosis are reported. Two patients had small heads with posterior slanted foreheads. Skull expansion was performed in one patient. Various degrees of ocular proptosis and shallow orbits without enlargement of the extraocular muscles were seen in all cases. Zygomatic repositioning for zygomatic hypoplasia and exophthalmos was performed in two patients. The facial skeleton was involved in thyrotoxic craniosynostosis in these children, and their clinical features seemed to resemble craniofacial dysostosis. PMID- 7548106 TI - Follow-up of electromyographic and cephalometric findings in patients with unilateral cleft lip and palate after fifteen months of continuous wearing of a special removable appliance. AB - A 15-month follow-up study was conducted on 13 children with unilateral cleft lip and palate. The patients presented with short upper lip, abnormal lip seal, and maxillary retrusion. All had undergone surgery in childhood. Cephalometric and electromyographic measurements were made prior to the start of treatment, 4 months into treatment, and after 15 months of continuous wear of a special removable appliance. The cephalometric measurements on paired comparison showed major improvement in both the sagittal maxillary and dentoalveolar positions. The electromyographic activity of the superior orbicularis oris muscle demonstrated no significant changes over the treatment period. Improvement in the sagittal maxillary and dentoalveolar positions seems to suggest that the elimination of the restrictive effect of the superior orbicularis oris muscle is a good therapeutic approach to promote normal growth potential in children with cleft lip and palate. PMID- 7548108 TI - Triopia: craniofacial malformation with prosencephalic duplication. AB - A case of ocular duplication with complex craniofacial and central nervous system anomalies is described. The anomaly is termed triopia because the child's most overt and distinguishing feature was three eyes: the left orbit contained two globes with independent ocular adnexa; the right orbit contained one normal appearing and functioning globe. Computer assisted medical imaging was used to define, in vivo, the intra- and extracranial soft and hard tissue anomalies: the cerebral hemisphere ipsilateral to the ocular duplication was also duplicated. Possible bases for this anomaly include duplication of primordia for the eye and secondary prosencephalon. PMID- 7548109 TI - Periodontal and prosthetic treatment of a cleft lip and palate patient: a case report. AB - Although cleft lip and palate patients are usually treated by a multidisciplinary team involving physicians and dentists, their periodontal condition may be over looked. Crowded or malpositioned teeth, hypertrophic gingiva, orthodontic appliances, and prosthetic replacements can impede proper plaque removal and thus perpetuate periodontal disease. It is important to incorporate periodontal treatment into the comprehensive treatment as early as possible. This case report discusses the periodontal surgical procedures involved in eliminating a residual ridge defect and the fitting of the final prosthetic reconstruction. Also, the importance of the identification and management of periodontal conditions characteristic of cleft lip and palate patients before and after surgical, orthodontic, and prosthetic rehabilitation will be emphasized. PMID- 7548110 TI - Predominance and improvement of velopharyngeal function in patients without complete closure in blowing. PMID- 7548111 TI - Cleft lip and palate reconstruction. PMID- 7548114 TI - Hepatic uptake and intracellular processing of LDL in rainbow trout. AB - The process of receptor-mediated endocytosis in poikilothermic vertebrates such as salmonid fish have not been subjected to much research, compared to the detailed studies done in mammalian systems. We have investigated the hepatic uptake and intracellular processing of low-density lipoprotein (LDL) in rainbow trout liver. After intravenous injection of the [125I]tyramine-cellobiose ([125I]TC) -labelled lipoprotein, the liver was perfused and cells isolated or fractionated by differential centrifugation and isopycnic centrifugation in Nycodenz gradients. We found that LDL was mainly endocytosed by parenchymal cells of the liver. Cell fractionation experiments showed that LDL was localized sequentially in three groups of organelles of increasing density. Initially, LDL was localized in small, slowly sedimenting endosomes before being transferred to denser endosomes (prelysosomes) and finally to dense lysosomes. The lysosomes were identified by three lysosomal marker enzymes. Degradation products formed from [125I]TC-labelled LDL could also be detected in prelysosomal vesicles. In vitro experiments with cultured trout hepatocytes demonstrated that intracellular processing of [125I]TC-LDL in these cells could be suppressed by endocytic and lysosomal inhibitors. The catabolism of LDL in rainbow trout therefore follows the endocytic-lysosomal pathway described for many macromolecules in mammalian cells. PMID- 7548115 TI - Palmitoleate formation by soybean stearoyl-acyl carrier protein desaturase. AB - Palmitoyl (hexadecanoyl)-acyl carrier protein (ACP) was found to be an alternate substrate for recombinant soybean stearoyl (octadecanoyl)-ACP delta 9-desaturase. The fatty acid product was identified as palmitoleic acid ((Z)-hexadec-9-enoic acid), which suggests that the locus of desaturation was fixed with respect to the carboxyl, not methyl, end of each substrate acyl group. The apparent specificity factor (Vmax/Km) of the desaturase for stearoyl-ACP was > 100-fold larger than for palmitoyl-ACP, mostly because of the difference in Vmax for the two substrates. PMID- 7548112 TI - The different effects of sphingosine on diacylglycerol kinase isozymes in Jurkat cells, a human T-cell line. AB - We studied the effect of sphingosine on the activities of soluble and membrane bound isozymes from Jurkat cells using combinations of different substrates (arachidonoyl- and didecanoyl DGs) and assay methods (octylglucoside mixed micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isoforms, which could be distinguished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-80 kDa DGK antibody. We confirmed the presence of arachidonoyl DG-specific DGK in membranes, though this isozyme was not activated by sphingosine. We detected in the cytosol at least two species of sphingosine-activatable and non-activatable DGK isoforms, the major species being the 80 kDa DGK. We postulate that both or either of the two soluble DGKs may be the target of the sphingosine action. PMID- 7548113 TI - Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line. AB - Sphingosine is known to regulate a variety of cellular functions through protein kinase C-dependent or independent pathways. In an attempt to investigate differential functions of diacylglycerol kinase (DGK) isozymes, we tested the effect of sphingosine on DGK operating in intact Jurkat cells, a human T-cell line. We found that phosphatidic acid (PA) synthesized from endogenous diacylglycerol (DG) and exogenously added short-chain DGs like dioctanoylglycerol were markedly enhanced by approx. 20 microM sphingosine. Further studies such as the use of protein kinase C down-regulated cells, mass measurements of cellular DGs, analysis of molecular species of PA and the effect of exogenous DG on the conversion of endogenous DG to PA suggested that sphingosine directly activated cellular DGK having a broad specificity toward DG molecular species. PMID- 7548116 TI - Phospholipase A2-induced stimulation of A549 lung adenocarcinoma cell line proliferation. AB - Phospholipase A2 (PLA2) activity was found in the cytosolic fraction of the A549 human lung adenocarcinoma line. This PLA2 had a molecular mass of approximately 70 kDa as assessed by gel filtration chromatography and required submicromolar concentrations of calcium concentrations for optimal activity. These characteristics are consistent with the cytosolic PLA2 recently reported in other cell types, such as U937 cells. We have now demonstrated that A549 cell PLA2 (PLA2 activity: 1 unit/ml) partially purified by gel filtration stimulated proliferation of A549 cells by 50% after 3 days of culture. Similarly, porcine pancreatic PLA2 (0.1 unit/ml) also promoted proliferation of A549 cell cultures by 42%. Furthermore, A549 cell PLA2 stimulated prostaglandin E2 release (approx. 7-fold increase). Both PLA2s lost activity when treated with p-bromophenacyl bromide. Neither porcine pancreatic PLA2 nor A549 cell PLA2 reversed the inhibitory activities of dexamethasone and indomethacin on cell growth. These results suggest that both of these PLA2s stimulate A549 cell growth, and that this is likely to be mediated by increased eicosanoid production. PMID- 7548117 TI - Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex. AB - The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex. PMID- 7548119 TI - Mobilization of acyl chains from endogenous cellular phospholipids into cholesteryl esters during foam-cell formation in mouse peritoneal macrophages. AB - We previously showed that phosphatidylserine-containing liposomes (phosphatidylserine liposomes) are effectively metabolized and induce foam-cell formation in cultured macrophages (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). When cultured mouse peritoneal macrophages were incubated with phosphatidylserine liposomes of different cholesterol content, cholesteryl esters were formed in the cells in proportion to the cholesterol content of the liposomes added to the culture. Cholesteryl esters were formed only when the cells took up more than a certain amount of cholesterol (approx. 160% above the basal cell level). Fatty acyl moiety derived from the exogenous phospholipids were utilized preferentially for cholesteryl ester formation. Once cholesterol as an acceptor is exhausted within cells, the acyl chain may be utilized for triacylglycerol formation. Not only exogenous lipid acyl chains but also endogenous phospholipid acyl chains were also utilized for cholesteryl ester and triacylglycerol formation under the present conditions, since the radioactivity in phospholipids was incorporated into both cholesteryl esters and triacylglycerols in macrophages whose endogenous phospholipids had been prelabeled with radioactive fatty acids. Among phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol served as the acyl donor, whereas sphingomyelin and cardiolipin did not. Mobilization of acyl chains from endogenous phospholipids to the neutral lipid fraction was also observed during foam-cell formation induced by acetyl low-density lipoprotein. The substance(s) produced within cell in the process of foam-cell formation may trigger the acyl chain mobilization from endogenous phospholipids. PMID- 7548118 TI - Transport and uptake of retinol during chicken oocyte growth. AB - Most, if not all, components found in the yolk of a chicken egg are extracted from the plasma compartment during the rapid growth phase of the oocyte. Uptake of the major yolk constituents, very-low-density lipoprotein and vitellogenin, has been shown to be mediated by a specific receptor in the plasma membrane of the oocyte (Barber, D.L., Aebersold, R., Sanders, E.J. and Schneider, W.J. (1991) J. Biol. Chem. 266, 18761-18770). In the current study, we sought biochemical evidence for the uptake into oocytes of a minor but biologically very important component, the vitamin retinol. For transport in serum, retinol is bound to retinol-binding protein (RBP), which in turn forms a complex with transthyretin (TTR). In order to gain insight into the biochemical details of transport of the vitamin, we have identified, purified and characterized RBP, TTR, and RBP-TTR complexes from chicken serum and yolk. The results demonstrate that both serum and yolk contain the tertiary retinol-RBP-TTR complexes as well as free RBP and TTR. Western blots of yolk collected from oocytes at different stages of growth show that both RBP and TTR, but not albumin, are more abundant at early stages relative to total yolk protein. In addition, we find both RBP and TTR in endocytic clathrin-coated vesicles of the oocyte. Our results support the hypothesis that retinol, which must be imported by the oocyte for proper embryonic development, is internalized by the chicken oocyte bound to its serum protein-transport complex. PMID- 7548120 TI - Infrared spectroscopic study of the interaction of diacylglycerol with phosphatidylserine in the presence of calcium. AB - The interaction of 1,2-dipalmitoylglycerol (DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in aqueous dispersion in the presence and in the absence of Ca2+ by using Fourier transform infrared spectroscopy (FT-IR) and 45Ca(2+)-binding. FT-IR showed that DPG increased the phase transition of DPPS and induced a rigidification of the DPPS/DPG-Ca2+ complex. In the absence of Ca2+, the incorporation of DPG produced an increase in the proportion of dehydrated carbonyl groups in the mixture of DPPS plus DPG whereas, in the presence of Ca2+, DPG suppressed the solid-solid phase transition of phosphatidylserine-Ca2+ complexes. The phosphate band of DPPS was analyzed using a multivariate statistical analysis, indicating that DPG induced a higher dehydration of the PO2- group in the presence of subsaturating Ca2+ concentrations. Even very low concentrations of DPG, such as 2 mol%, already produced a significant effect. In the presence of both DPG and Ca2+, dehydration of DPPS increased, so that full dehydration was reached at a DPPS/Ca2+ molar ratio of 2.94 instead of 2.04 as observed for pure DPPS. However, the stoichiometry of the binding of Ca2+ to DPPS was not significantly altered by the inclusion of DPG as revealed by 45Ca(2+)-binding experiments, indicating that, in this situation, full dehydration of the PO2- groups of DPPS was reached when approx. 2 out of every 3 molecules of DPPS were binding Ca2+. The effects reported here for the interaction of DPG with DPPS may be significant for a number of biological situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes or the activation of protein kinase C, where the dehydration effect produced by diacylglycerols may explain, at least in part, their effects. PMID- 7548121 TI - Intralysosomal glycerophospholipid catabolism in liver: hydrolysis of amino alcohol-containing phospholipids and their metabolites. AB - Liver lysosomes were isolated from untreated rats and rats pretreated with Triton WR-1339. Purified lysosomes were also separated into lysosomal matrix and membrane fractions. With freshly prepared and frozen biological material, the lysosomal catabolism of various stereospecifically radiolabeled amino alcohol containing glycerophospholipids and their potential metabolites was studied. Basically there was no qualitative difference in the formation of phospholipid metabolites in both preparations: after long-term incubation, free fatty acids, lysophospholipids, acyl-free phosphodiesters were detected, and to a far lesser extent, amino alcohol-containing phosphomonoesters and only traces of free amino alcohols. These findings indicate the presence of lysosomal phospholipases A as well as C and lysophospholipase(s), with pH optima of about 4.5, and they clearly exclude phospholipase D activity. Unfractionated lysosomes and their soluble as well as particulate subfractions were not capable of hydrolysing the acyl-free amino alcohol-containing phosphodiesters. These compounds must therefore be considered one of the end products of the intralysosomal catabolism of amino alcohol-containing phosphoglycerides. They are presumably cleared from the lysosomal compartment by an as yet unknown transport system in the lysosomal membrane. In liver, the extralysosomal site of their (Mg(2+)-dependent) hydrolysis seems to be the plasma membrane. By contrast, hydrolysis of glycero-3 phosphate and the amino alcohol-containing phosphomonoesters was catalysed in the lysosomal compartment, with a pH optimum of about 5.0, although at considerably lower rates than that of glycero-2-phosphate, a model substrate for lysosomal acid phosphatase. PMID- 7548122 TI - n-6 and n-3 fatty acid accumulation in thp-1 cell phospholipids. AB - The human monocytic leukemia cell line THP-1 is depleted of the long-chain n-6, AA, when compared to human monocytes. This reflects the low availability of this FA in the growth medium generally used for cultured cells. The effects of AA, as well as EPA, supplementation of THP-1 cells on the incorporation of these FA in cell PL, especially in PC and PE, was investigated. In addition the incorporation of labeled AA in PL from THP-1 cells was compared to that in human monocytes. Measurements were done through HPLC separation of PL, detected by UV absorption and radioactivity, FA analysis by GC and characterization of PC subclasses by FAB MS. Marked differences were observed in the incorporation of the two FA in cell PL, particularly two PC subclasses, and in the accumulation in individual PL after supplementation of THP-1 cells. Accumulation of AA and EPA in THP-1 cells appeared to be mutually independent. The incorporation of AA was also quite different in THP-1 from that in monocytes. Thus, characterization of the FA content in lipids of cultured cells is an essential requirement for optimal utilization of these cells. PMID- 7548123 TI - Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A I-containing subpopulations. AB - Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the sample's endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed. PMID- 7548124 TI - Comparison of apo A-I-containing subpopulations of dog plasma and prenodal peripheral lymph: evidence for alteration in subpopulations in the interstitial space. AB - To study in vivo reverse cholesterol transport, dog plasma and lymph apo A-I containing subpopulations were compared by two-dimensional electrophoresis. Charge and size of subpopulations were similar in plasma and lymph, but the distribution of subpopulations varied considerably. An increase in pre-beta and pre-alpha particles in lymph suggests these changes are a reflection of in vivo reverse cholesterol transport. PMID- 7548125 TI - Transport of competing Na and K ions by (222) C10-cryptand, an ionizable mobile carrier: effects of pH and temperature. AB - The kinetics of the electroneutral exchange of competing sodium and potassium with protons across the membrane of large unilamellar vesicles (LUV) were determined at two pH values when transport was induced by the simultaneous presence of (222)C10-cryptand and FCCP (proton carrier) at various temperatures. The aim of the present work was to quantify the pH-dependent enthalpies of an ionizable mobile carrier affinities for competing alkali cations, and to focus on the effects of pH and temperature on the competitive transport selectivity of the carrier for K+ over Na+ ions. At any given temperature and pH, the apparent pH dependent affinity of (222)C10 was higher for K+ than for Na+. The enthalpy of this affinity for K+ was significantly lower than that for Na+, whereas it varied similarly with the pH (delta H(KpHmK) = 32.8 and 37.0 kJ/mol, and delta H(KpHmNa) = 47.9 and 52.9 kJ/mol at pH 7.8 and 8.8, respectively). When using a kinetic model, the pH effect on these parameters was discriminated (delta H(KmK) = 37.9 kJ/mol and delta H(KmNa) = 53.9 kJ/mol). The pH-dependence of the delta H(KpHm) of the cations could therefore theoretically be shown to arise from the temperature-induced changes in the ionization of the buffer dissolved in the aqueous phases and of the amine groups of the binding cavity of the carrier. The K/Na competitive transport selectivity (Sc(K/Na)) of (222)C10 increased linearly with the K+ concentration. It decreased hyperbolically with increasing concentration of Na+ while being independent of pH at any given temperature. In equimolecular ionic mixtures, Sc(K/Na) varied from 2.2 to 3.0 when temperature rose from 20 degrees C to 35 degrees C (delta H(Sc(K/Na)) = 15.6 +/- 0.5 kJ/mol). The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes. PMID- 7548128 TI - Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin. AB - It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux. However, less information is available about effects of verapamil on drug phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs. Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes. An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil. Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism. Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids. It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane. PMID- 7548126 TI - Lateral domain heterogeneity in cholesterol/phosphatidylcholine monolayers as a function of cholesterol concentration and phosphatidylcholine acyl chain length. AB - Mixed monolayers of cholesterol and phosphatidylcholines having symmetric, different length acyl chains (10 to 16 carbons each) were prepared at the air/water interface. The partitioning of a fluorescent probe, NBD-cholesterol at 0.5 mol%, among lateral domains was determined by epifluorescence microscopy. The mixed monolayers had cholesterol concentrations of 20, 25, or 33 mol%, and in all these monolayers, lateral domain heterogeneity was observed within a defined surface pressure interval. This surface pressure interval was highly influenced by the phosphatidylcholine acyl chain length, but not by the cholesterol content of the mixed monolayer. The characteristic surface pressure, at which the line boundary between expanded and condensed phases dissolved (phase transformation pressure), and the monolayer entered an apparent phase-miscible state, was about 20 mN/m for di10PC and decreased as a linear function of the phosphatidylcholine acyl chain length to be about 2.5 mN/m for di16PC. During initial compression of the monolayers, the sizes of the condensed phases were generally larger, and to some extent heterogeneous with respect to the size distribution, as compared to the situation in monolayers which had experienced a compression/expansion cycle, which took them above the phase transformation pressure. This suggest that the domains observed during initial compression were not equilibrium structures. This study has demonstrated that both the cholesterol content and the phosphatidylcholine acyl chain length markedly influenced the properties of laterally condensed domains in these mixed monolayers. Since the possibility for the formation of attractive van der Waals forces between cholesterol and acyl chains increase with increasing acyl chain length, and since the phosphocholine head group is similar in all systems examined, the observed differences in domain shapes, properties, and stability most likely resulted from differences in van der Waals forces. PMID- 7548127 TI - Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes. AB - Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [14C]ascorbate to [14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes. PMID- 7548130 TI - Osmotic stress sensitizes sterol-free phospholipid bilayers to the action of Amphotericin B. AB - We have tested the ability of Amphotericin B to form ion channels/defects in osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. We found that sterol-free LUV exhibit greatly increased sensitivity to AmB channel formation in the soluble oligomer state (> 0.5 microM) under modestly hypoosmotic conditions (< 100 delta mosM). These vesicles are completely insensitive under isoosmotic conditions. The related antibiotics, Amphotericin B methyl ester and Nystatin showed almost no activity under hypoosmotic conditions in the absence of sterol. This difference may be attributable to differences in solution oligomeric states. Experiments with KCl and CaCl2 internal buffers demonstrate that these sterol-free AmB membrane disruptions are highly selective for monovalent cations (K+) over anions (Cl-), ruling out massive lysis or unselective membrane defects caused by osmotic pressure. Thus, AmB seems to be acting as a 'molecular harpoon', an expression coined to describe substances which can selectively target osmotically stressed, strained or highly curved membranes. These results may provide a rationale for AmB's reported anti-HIV activity and reported activity against sterol-free small unilamellar vesicles (highly curved membranes) as well as the reduced activity of liposomal drug delivery systems toward cholesterol-containing and sterol-free membranes (fewer soluble oligomers). PMID- 7548129 TI - The cationic lipid stearylamine reduces the permeability of the cationic drugs verapamil and prochlorperazine to lipid bilayers: implications for drug delivery. AB - The therapeutic activity of a wide variety of drugs is significantly improved when their longevity in the circulation is extended by encapsulation in liposomes. To improve the retention of cationic drugs in liposomes, we have investigated the effect of the cationic lipid stearylamine on the permeability of the calcium channel blocker verapamil and the antipsychotic drug prochlorperazine, both of which are also multidrug resistance modulators. Both drugs were efficiently incorporated into liposomes composed of DSPC/cholesterol that possessed a transmembrane pH gradient (inside acidic). However, the efflux of the loaded drugs was relatively rapid (i.e., 50% of the encapsulated verapamil was released after 4 h at 37 degrees C), despite the presence of a 3 unit pH gradient (pHi = 4.0, pHo = 7.5). Drug retention within the liposomes was improved by increasing the magnitude of the transmembrane pH gradient to approx. 5 units (pHi = 2.0, pHo = 7.5). Further improvements in drug retention were achieved by the addition of 10 mol% of the cationic lipid stearylamine in the DSPC/cholesterol liposomes. The combination of the 5 unit pH gradient and stearylamine resulted in increases of the retention of verapamil and prochlorperazine by approx. 20- and 5-fold, respectively. Calculation of the permeability coefficients for the charged (cationic) and neutral forms of the drugs indicated that the neutral forms of both drugs were approx. 10(4)-fold more permeable than were the cationic forms of the drugs. Further, the presence of stearylamine reduced the permeability coefficient for the cationic species of the drugs by approximately an order of magnitude, but had no effect on the neutral species of the drugs. The efflux curves observed for both verapamil and prochlorperazine could be mathematically modeled by assuming that the primary influence of stearylamine was on the development of a positive surface charge density on the inner monolayer of the liposome. Taken in sum, these results indicate that stearylamine is effective at decreasing the leakage of cationic drugs from liposomes, and may prove to be a valuable component of liposomal drug formulations. PMID- 7548131 TI - Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H , 13C- and 31P-NMR spectroscopy. AB - We report the results of a comparative study of the molecular order and dynamics of phosphatidylcholine (PC) bilayer membranes in the absence and presence of cholesterol, ergosterol and lanosterol, using deuterium (2H) nuclear magnetic resonance (NMR) of deuterated phospholipid molecules, in addition to solid state 13C and 31P-NMR. Using dimyristoylphosphatidylcholines (DMPCs) specifically labeled at positions 2', 3', 4', 6', 8', 10' and 12' of the sn-2 chain together with the perdeuterated 2-[2H27]DMPC derivative, the order profile for 9 of the 13 methylene groups of the sn-2 chain was established at 25 degrees C for DMPC, DMPC/cholesterol, DMPC/ergosterol and DMPC/lanosterol membranes, at a fixed sterol/phospholipid mol ratio of 30%, and in the presence of excess water. The overall ordering effects were found to be ergosterol > cholesterol >> lanosterol. Transverse relaxation (T2e) studies of these systems indicated that while for DMPC, DMPC/cholesterol and DMPC/ergosterol the relative relaxation rates were in qualitative agreement with models which assume cooperative motions of the bilayer molecules as the main relaxation mechanism, those in DMPC/lanosterol were anomalously high, suggesting alterations of lipid packing. Using dipalmitoylphosphatidylcholine (DPPC) deuterated at the trimethylammonium group of the choline moiety, we found that the differential ordering and motional effects induced by the sterols in the acyl chains were also reflected in the headgroup, both in the gel (L beta) and liquid-crystalline phases. 13C and 1H spin dynamics studies of these systems, including cross-polarization, rotating frame longitudinal relaxation and dipolar echo relaxation rates showed that the mobility of the different regions of the phospholipid molecules in the binary lipid systems were inversely correlated with the ordering effects induced by the sterols. A novel combination of C-D bond order parameters (obtained by 2H-NMR) and 13C-1H cross polarization rates confirmed these results. The effects of the same sterols at the same molar proportion on the unsaturated lipid 1 [2H31]palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (2H31-POPC) at 25 and 35 degrees C were different from those observed on DMPC and showed ordering effects which are largest for cholesterol, while ergosterol and lanosterol produced significantly smaller effects. Transverse relaxation studies indicate that while cholesterol does not perturb cooperative motions in POPC, both ergosterol and lanosterol do. Again, high-resolution solid state 13C-NMR studies support the conclusions of the 2H-NMR experiments.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7548132 TI - Generation of giant protoplasts of Escherichia coli and an inner-membrane anion selective conductance. AB - Established methods were modified and combined to generate unilamellar giant protoplasts in order to study the electric events on the cytoplasmic membrane of Escherichia coli with patch-clamp technique. The activities of many types of conductances were encountered, one of them is characterized here. This channel conductance is 109 pS in 150 mM KCl; it prefers anions, it is highly voltage dependent and is blocked by micromolar concentrations of anthracene-9-carboxylic acid. PMID- 7548133 TI - Charge determination of membrane molecules in polymer-supported lipid layers. AB - A method for two-dimensional micro-electrophoresis and charge determination of fluorescence-labeled membrane molecules in lipid layers is presented. Therefore, the labeled molecules are dissolved in a lipid monolayer which acts as a fluid matrix. The essential part of the sample preparation is an aqueous polymer film composed of agarose onto which the layer is transferred by Langmuir-Blodgett technique. The induced force of an applied electric field leads to a drift of the charged fluorescent molecules. The mobility is determined by an analysis of the steady-state bleach profile which is produced by continuous fluorescence micro photolysis of a rectangular area of the monolayer. Testing a variety of amphiphilic molecules, measurements yielded values of zero, plus or minus one elementary net charge within a margin of error. The experimental set-up described here can be used for lateral separation, enrichment and isoelectric focusing of membrane components. PMID- 7548135 TI - Corrigendum to "Cloning of a cGMP-gated cation channel from mouse kidney inner medullary collecting duct" [Biochim. Biophys. Acta 1236 (1995) 197-200]. PMID- 7548134 TI - cDNA cloning of MCT1, a monocarboxylate transporter from rat skeletal muscle. AB - PCR was used to amplify the coding region of CHO MCT1 cDNA. This was then used to screen a rat skeletal muscle cDNA library which lead to the isolation of a full length cDNA encoding MCT1 from rat. The cDNA derived amino acid sequence shows 94% and 86% identity to CHO and human MCT1, respectively. PMID- 7548136 TI - Real-time PET analysis of metastatic tumor cell trafficking in vivo and its relation to adhesion properties. AB - Although a number of studies have indicated that highly metastatic cells tend to adhere more to target endothelium in vitro than low or non-metastatic cells, direct evidence about the correlation between cellular adhesiveness and organ disposition of the cells has not been obtained. Using positron emission tomography (PET), we have developed a novel technique that enables the non invasive detection of the real-time tumor cell trafficking. The present study shows the correlation between trafficking of murine large cell lymphoma RAW117 and the adhesion properties of the cells in vitro. Cells accumulated in the liver time-dependently, and accumulation of RAW117-H10, liver metastatic subline cells, was more intense than that of RAW117-P, the parental cells, indicating that the metastatic potential is correlated with the in vivo accumulation of the cells in the target tissue. To examine whether the adhesion properties of the cell membrane determine the cell trafficking, we performed PET analysis after altering the adhesion properties on the cell membrane by means of cellular protein kinase C modulation, since the modulation of this enzyme is known to alter the surface adhesion molecules, i.e., those of the integrin superfamily. The treatment of RAW117-P with 12-O-tetradecanoylphorbol 13-acetate, which caused augmentation of adhesion to hepatic sinusoidal microvessel endothelial cells (HSE) in vitro, enhanced the hepatic accumulation of the cells in vivo. On the contrary, treatment of RAW117-H10 with the protein kinase C inhibitor H-7, 1-(5 isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, which reduced the adhesion activity of the cells to HSE, suppressed their accumulation in the liver, although the suppression was observed only during the first 30 min after administration of the cells. These data suggest that the adhesion properties of metastatic lymphoma cells are critical for the accumulation of these cells in the target tissue. PMID- 7548137 TI - Effect of substrates and pH on the intestinal Na+/phosphate cotransporter: evidence for an intervesicular divalent phosphate allosteric regulatory site. AB - Intervesicular divalent phosphate-induced inhibition of the intestinal brush border membrane Na+/phosphate cotransporter was examined using Na(+)-dependent phosphate uptake, substrate-induced tryptophan fluorescence quenching, and the apparent pKa values for substrate-induced conformational changes. In right-side out (RSO) reconstituted proteoliposomes, only monovalent phosphate inhibited Na(+)-dependent phosphate uptake in the absence of pre-equilibration. Addition of divalent phosphate to inside-out (ISO) proteoliposomes resulted in 80 +/- 5% inhibition of Na(+)-dependent phosphate uptake in the absence of pre equilibration. The nature of divalent phosphate-induced inhibition of cotransporter function was examined using cotransporter partial reaction assays based on substrate-induced conformational changes reported as changes in tryptophan fluorescence. Na+ but not K+ induced a quenching of tryptophan fluorescence with a K0.5 of 25 mM and an apparent Hill coefficient of 1.8. Monovalent phosphate (difluorophosphate) induced a further quenching of tryptophan fluorescence with a K0.5 of 53 microM. Divalent phosphate (monofluorophosphate) had no effect on tryptophan fluorescence, but inhibited the difluorophosphate-induced quenching of tryptophan fluorescence. The Na+ to Na++ divalent phosphate (monofluorophosphate) conformation and the Na+ to Na++ monovalent phosphate (difluorophosphate) conformations were compared using tryptophan quench reagents. These transitions had different apparent pKa values and different phenylglyoxal sensitivities consistent with monovalent phosphate and divalent phosphate interacting with the cotransporter at separate sites. PMID- 7548138 TI - Examination of the molecular mechanism of SH reagent-induced inhibition of the intestinal brush-border membrane Na+/phosphate cotransporter. AB - SH residues on the rabbit intestinal brush-border membrane Na+/phosphate cotransporter were examined using a variety of SH specific reagents, proteolytic digestion and HPLC separation of SH-labeled cotransporter, and partial reaction assays. Of the seven SH-containing peptide fragments on the non-denatured non reduced cotransporter six peptides were labeled: five SH-containing peptides were labeled with acrylodan or IAF (iodoacetamidofluorescein) and three peptides were labeled with IAEDANS. One SH-containing peptide was labeled with IAEDANS or fluorescein maleimide only. Selective SH labeling conditions employing acrylodan and IAEDANS were used to identify the environments of these SH-containing peptides in the native cotransporter. The nature of SH reagent-induced inhibition of Na(+)-dependent phosphate uptake was examined using substrate-induced conformational changes, and substrate-induced changes in IAEDANS and acrylodan fluorescence of the SH-labeled Na+/phosphate cotransporter. The results indicate that five of the SH-labeled peptides sense the Na(+)-induced conformational change, three peptides sense the Na++ difluorophosphate-induced conformational change, and one peptide senses only the Na++ monofluorophosphate-induced conformational change. Five of the SH-labeled peptides are passive participants in the substrate-induced conformational changes with only SH 51 involved in cotransporter function. Alkylation of SH 51 resulted in a cotransporter conformation which differed from the substrate-mediated conformations and was characterized by increased monofluorophosphate sensitivity. PMID- 7548139 TI - Na-dependent glutamate transport in high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells. AB - Na-dependent glutamate (Glu) influxes in the red blood cells of normal low K (LK), high K and high glutathione (HK/HG), and high K and low glutathione (HK/LG) dogs were compared. The ranges of the influxes in LK, HK/HG and HK/LG cells were 1.0-63, 62-174 and 1.3-26 mumol/1 cells per h, respectively. Some LK and HK/LG dogs had red blood cells with extremely low Glu influxes. In cells with extremely low Glu influxes, however, there were clear Na-dependent Glu influxes. In LK, HK/HG and HK/LG cells, the Km of Na-dependent Glu influx with respect to the medium Glu concentration were 17, 20 and 19 mM, respectively, and the half maximal activation (K1/2) with respect to medium Na concentration was 39, 40 and 42 microM, respectively. By the addition of harmaline, a hallucinogenic alkaloid, the Vmax in LK cells was not affected and the Km was increased, while the Vmax was decreased and the Km increased in HK/HG and HK/LG cells. The Ki value with harmaline by means of Dixon plot in LK cells was 5.2 mM, against 1.8 and 1.9 mM in HK/HG and HK/LG cells, respectively. These results suggest that the difference in the Na-dependent Glu influxes between 2 HK groups was due to the varying quantity, not the quality, of the transporter. PMID- 7548140 TI - Protein kinase C-dependent regulation of L-arginine transport activity in Caco-2 intestinal cells. AB - The regulation of plasma membrane L-arginine transport activity was investigated in differentiated and undifferentiated states of the human intestinal cell line, Caco-2. The sodium-independent, leucine-insensitive uptake of L-arginine measured in this study has been assigned by us previously to system y+ in Caco-2 cells. Treatment of cells with serum-free media containing epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated system y+ arginine transport activity in Caco-2 cells. Transport upregulation by these growth factors or by TPA was blocked by cycloheximide or the PKC inhibitor chelerythrine. Arginine uptake was diminished during the course of differentiation, attributable to a reduction in the transport system y+ capacity (Vmax) with no change in apparent affinity (Km). TPA stimulated arginine uptake required at least 3 h of continual exposure, and increased the membrane's transport capacity (Vmax) in both undifferentiated and differentiated cells. TPA elevated the diminished transport Vmax of differentiated cells TPA to the elevated Vmax value associated with undifferentiated cells. We conclude that upregulation of arginine transport is part of a pleiotropic response to EGF/TGF alpha, and that this involves PKC and de novo synthesis of polypeptides associated with system y+ transport activity. PMID- 7548141 TI - Effect of starvation on the activity of the mitochondrial tricarboxylate carrier. AB - The effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35-40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the Vmax of this process decreases while there is no change in the Km. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein. PMID- 7548142 TI - The effect of bilayer composition on calcium ion transport facilitated by fluid shear stress. AB - Passive calcium ion permeability across liposome bilayers is increased during exposure to fluid shear forces attainable in the mammalian vasculature. In this study, liposomes prepared from three different lipid mixtures (phosphatidylcholine alone; phosphatidylcholine and cholesterol; a mixture of anionic and cationic phospholipids plus cholesterol) are exposed to uniform shear stress in a rotational viscometer. Liposome permeability to calcium ion is estimated from continuous measurement of free intraliposome calcium ion concentration using a fluorescence technique. Calcium ion permeability in the absence of fluid force and susceptibility to shear-induced permeability modulation are positively correlated with estimated bilayer compressibility. Fluid shear forces are presumed to influence bilayer packing and modulate defect formation in proportion to bilayer compressibility. Bilayer defects produced by fluid forces may increase liposome permeability. PMID- 7548143 TI - Electrophoretic evidence for the impairment of complexes of the respiratory chain during iron/ascorbate induced peroxidation in isolated rat liver mitochondria. AB - The impairment of the complexes of the respiratory chain was studied in isolated rat liver mitochondria under the conditions of an iron/ascorbate-mediated oxidative stress. Using blue native electrophoresis technique the NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome-c oxidoreductase, cytochrome oxidase and ATP synthetase were separated from mitochondrial samples at different stages of peroxidation and quantified by densitometry. In the second dimension the protein complexes were separated into their individual subunits by Tricine/SDS electrophoresis. In relation to the time course of lipid peroxidation protein losses were moderate in the exponential phase and enhanced towards plateau phase of TBARS formation, when the intensity of staining for the native complexes became reduced by 84%, 69%, 63% and 24% for complexes I, III, V and IV, respectively, and a high molecular aggregation band as a putative marker of oxidative stress was formed. The decline of overall staining by 23%, a decrease in trichloroacetic acid precipitable protein and the formation of acid soluble primary amines suggest the occurrence of fragmentation or degradation processes. Apparently, the impairment of the respiratory chain complexes during peroxidation was not reflected in altered electrophoretic mobilities or specific losses of protein subunits of these innermitochondrial membrane components. PMID- 7548144 TI - Pore kinetics reflected in the dequenching of a lipid vesicle entrapped fluorescent dye. AB - Pore formation in lipid vesicle membranes can be monitored by the fluorescence signal F(t) arising from the induced release of a self-quenching dye in the course of the elapsed efflux time t. We present a basic theoretical analysis of pertinent experimental data allowing the quantitative evaluation of information on the pore kinetics and mechanism. This implies an investigation of the 'dynamic' quenching factor Qt exhibited by that fraction of dye which is still being retained inside the liposomes at t. It is shown how Qt depends on the mode of release which could be 'all-or-none' or more gradual as expressed by a parameter rho < or = 1 (related to the pore lifetime), i.e., the average dye retention factor in a vesicle after a single pore opening. A fit to measured values of Qt at a sufficient extent of efflux may be applied in order to determine rho. Then the pore formation rate per liposome, va(t), can be derived from the registered F(t). We give a practical demonstration of the procedures with carboxyfluorescein-loaded phosphatidylcholine liposomes of two different sizes to which the wasp venom peptide mastoparan X had been added. PMID- 7548145 TI - A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane. AB - In earlier studies, the fluorescence probe 1-(4-(trimethylamino)phenyl)-6 phenylhexa-1,3,5-triene (TMA-DPH) was shown to interact with living cells by instantaneous incorporation into the plasma membrane, according to a water (probe not fluorescent)/membrane (probe highly fluorescent) partition equilibrium. This made it interesting both as a fluorescence anisotropy probe for plasma membrane fluidity determinations and as a quantitative tracer for endocytosis and intracellular membrane traffic. In order to ascertain the limiting concentrations for its use in these applications, we performed a systematic study of its fluorescence properties (intensity, lifetime, anisotropy) in the plasma membrane and in endocytic membranes of intact L929 mouse fibroblasts. Some of the experiments were repeated on mouse-bone-marrow-derived macrophages and on phospholipidic LUV to confirm the results. Rather unexpectedly, it was observed that: (i) the incorporation of TMA-DPH into the membranes, monitored by UV absorption measurements, remained proportional to the probe concentration over the wide range explored (5 x 10(-7) M-2.5 x 10(-5) M); (ii) however, concerning fluorescence, quenching effects occurred in the membranes above certain critical concentrations. These effects were shown to result from Forster-type resonance auto-transfer; (iii) strikingly, the critical concentrations were considerably higher in early-endocytic-vesicle membranes than in the bulk plasma membrane. It was established that membrane fluidity was involved and this was confirmed by the parallel study on phospholipidic vesicles. Potential applications of these properties as a novel approach for evaluating membrane fluidity are suggested. PMID- 7548146 TI - The A1 agonist CCPA reduced bisoxonol-monitored membrane potential depolarization elicited by high K+ in cerebrocortical nerve endings. AB - In this study the effect of the A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA) on bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol) monitored membrane potential in cerebrocortical nerve endings was evaluated. CCPA (30, 100 and 300 microM) caused a dose-dependent decrease of high K(+)- and veratridine-induced membrane depolarization. This decrease was counteracted by the A1-specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (30-100 microM). On the contrary, the A2 receptor antagonist 9-chloro-2-(2-furanyl)-5,6 dihydro-1,2,4-triazolol-[1,5-c]quinazol ine-5- imine (CGS 15943) was unable to interfere with the lowering effect exerted by CCPA (100 microM) on K(+)-elicited membrane depolarization. Finally, the A2 receptor agonist 2-[p-(2 carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS 21680) did not induce any modification of K(+)-induced membrane depolarization. The results of the present study suggest that K(+)-induced membrane depolarization in cerebrocortical brain nerve endings may be modulated by A1 receptors. PMID- 7548148 TI - The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole. AB - The yeast plasma membrane proton pumping ATPase (H(+)-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H(+)-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the H(+)-ATPase and is fungicidal. Glucose dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H(+)-ATPase to proceed through its reaction cycle. A strong correlation is observed between inhibition of medium acidification and H(+)-ATPase activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with beta-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H(+)-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H(+)-ATPase. These findings suggest that the plasma membrane H(+)-ATPase may serve as an important molecular target for antifungal intervention. PMID- 7548149 TI - Participation of a proton-translocating plasma membrane ATPase, acid phosphatase and alkaline phosphatase in ATP degradation by Aspergillus niger extracts. AB - Extracts of A. niger could catalyze sequential hydrolysis of the three phosphate moieties of the ATP molecule optimally at pH 2 and at pH 8. At pH 2 the hydrolysis was effected by an ATPase followed by acid phosphatase while at pH 8 alkaline phosphatase was the only involved enzyme. Separation of these three phosphate-hydrolyzing enzymes was achieved by Sephadex G-100 column chromatography. The A. niger ATPase seems to have two unique features. First, it was easily solubilized in distilled water and second it had optimum activity at pH 2. The activity of this enzyme was not affected on addition of Na+, K+ or Ca2+ to the assay reaction mixture. It was neither inhibited by sodium azide nor by potassium nitrate but inhibited by orthovanadate, DES, DCCD, Mg2+ and Pi. The substrate concentration-activity relationship was of the hyperbolic type. The enzyme had high specificity for ATP, was inert with ADP and its activity with GTP represented about 6% only of that obtained with equimolar amount of ATP. PMID- 7548147 TI - The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1. AB - Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973 27978). In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 nM in parent mIMCD 3 cells, P < 0.001). To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells. PMID- 7548150 TI - Activation of a Cl(-)-conductive pathway in primary cultures of rat inner medullary collecting duct (IMCD) cells under hypotonic stress. AB - In intracellular recordings with conventional microelectrodes on rat IMCD cells, we find that hypotonic stress depolarizes membrane voltage and decreases cell input resistance. Ion substitution experiments reveal that these effects are largely due to the activation of a prominent Cl- conductance. After block of this conductance with dideoxyforskolin a smaller concomitant increase in K+ conductance becomes detectable. PMID- 7548152 TI - A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation. AB - Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase. PMID- 7548151 TI - Understanding and increasing protein stability. AB - This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins. It considers the effects of organic solvents on stability and the special features of thermophilic proteins. The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example. Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents. One can also use chemical modification to modify protein stability and some of the main approaches are reviewed. The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis. The article concludes by summarizing the beneficial effects of certain additives on protein stability. PMID- 7548153 TI - Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c. AB - beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the derivatives modified at lysines 72, 73, and 79 which are discussed separately. The electron transfer activity of the beta-thiopropionyl cytochromes c with bovine heart cytochrome-c oxidase was decreased to extents dependent on the position of the modification. Aminoethylation, a secondary modification which reverses the charge change, restored the electron transfer rate to that observed with the unmodified cytochrome c, irrespective of the location of the primary modification. These results afford a direct experimental demonstration that alterations in kinetics with physiological electron transfer partners resulting from modifications which cause a change of the charge of surface side chains are solely due to the electrostatic effects. Of the many chemically modified cytochromes c prepared to date, the singly substituted beta-thiopropionyl cytochromes c are likely to be particularly useful as the thiol allows covalent linkage of any sulfhydryl reactive reagent to a well-defined location on the protein surface by a simple procedure, even when the secondary modifier is relatively unstable, a crucial advantage not otherwise readily achieved. PMID- 7548154 TI - Thermal unfolding of the cytotoxin alpha-sarcin: phospholipid binding induces destabilization of the protein structure. AB - The effect of membrane binding on the structure and stability of the cytotoxin alpha-sarcin has been studied by differential scanning calorimetry, Fourier transform infrared and fluorescence spectroscopic techniques. The thermal unfolding of alpha-sarcin in aqueous solution fits into a two-state transition characterized by a transition temperature (Tm) of 52.6 degrees C and a calorimetric enthalpy (delta Hcal) of 136 kcal/mol. Upon interaction with phosphatidylglycerol vesicles, alpha-sarcin undergoes conformational changes, as deduced from the FTIR and fluorescence emission spectra. These changes result in a decreased Tm and delta Hcal values for the thermal unfolding of phospholipid bound alpha-sarcin. The lower Tm value for lipid-bound alpha-sarcin is also observed at the level of secondary and tertiary structures, based on analyses of both the amide I' infrared spectrum and the tryptophan emission of the protein as a function of temperature, respectively. The results obtained indicate a protein destabilization promoted by the phospholipid interaction. PMID- 7548156 TI - Reaction mechanism for the inactivation of the quinoprotein methylamine dehydrogenase by phenylhydrazine. AB - Phenylhydrazine has previously been shown to be an irreversible inactivator of the tryptophan tryptophylquinone (TTQ) enzyme methylamine dehydrogenase [Davidson, V.L. and Jones, L.H. (1992) Biochim. Biophys. Acta 1121, 104-110]. Structure-reactivity correlations have been performed to elucidate the mechanism by which this inactivation occurs. The reactions of a series of p-substituted phenylhydrazines with methylamine dehydrogenase were examined. Correlation with electronic substituent effects was observed. A Hammett plot of the second order inactivation rate constants versus sigma p exhibited a positive slope. Plots of these rate constants against substituent constants which reflected either resonance or field/inductive parameters for each p-substituent indicated that the rate was primarily influenced by resonance electronic effects. A Bronsted plot of the inactivation rate constant against pKa of each substituted phenylhydrazine yielded a beta-value (slope) of 0.7. Based upon these results, a reaction mechanism is proposed for the inactivation of methylamine dehydrogenase by phenylhydrazines, and a structure is proposed for the putative transition state for the rate-limiting step in the overall processes of binding and adduct formation by phenylhydrazine. The relevance of these results to the process of imine formation between substrate amines and TTQ during the normal catalytic process is also discussed. PMID- 7548155 TI - Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins. AB - This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins. PMID- 7548159 TI - Prediction of denaturing tendency of organic solvents in mixtures with water by measurement of naphthalene solubility. AB - Addition of miscible organic solvents to water increases the solubility of naphthalene. The logarithm of the solubility is linearly dependent on the co solvent concentration, in an intermediate range. The relative solubilising effects of different solvents correlate well with their known tendency to denature proteins (using literature data for trypsin, cytochrome c, chymotrypsinogen, chymotrypsin, laccase and myoglobin). This is expected if denaturation occurs when the hydrophobic effect has been reduced by a characteristic extent for a given protein. Naphthalene solubility predicts denaturation as well as does the denaturation capacity model. PMID- 7548158 TI - Activation of chicken liver dihydrofolate reductase in concentrated urea solutions. AB - The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3 fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed. PMID- 7548160 TI - Mutation in the interdomain tether influences the stability and refolding of the enzyme rhodanese. AB - Rhodanese is a single polypeptide chain of 293 amino acids that is folded into two globular domains of nearly equal size that are connected by a 16 amino acid tether. Two amino acids, Val-Asp (VD), were inserted into the interdomain tether through site-directed mutagenesis to produce the new interdomain sequence, E145PSRPEPAIFKAVDTLNR. The purified mutant protein, when unperturbed, was virtually indistiguishable in all properties tested and gave a specific activity that was at least 90% of the WT. However, the tether mutant was considerably less stable to perturbation compared with the WT enzyme. The interdomain hydrophobic surfaces in the mutant were more easily exposed, and the formation of intermediate folding states was facilitated. The rate of unassisted refolding was slightly less for the mutant, and the yield of active enzyme was somewhat reduced. The mutation introduced a new V8 proteinase cleavage site, but this site was not accessible in the native mutant which was as resistant to proteolysis as the WT enzyme. However, perturbation with low concentrations of urea that could form folding intermediate(s), allowed facile cleavage of the mutant to give fragments that appeared to represent the individual domains. In addition, the perturbed mutant could be proteolyzed close to one end of the polypeptide, a position that is far from the site of mutation, and which was not readily cleaved in the WT enzyme or the native form of the mutant. These results indicate that mutation in the interdomain tether can have dramatic effects on the stability and conformational transitions of rhodanese. PMID- 7548161 TI - Simulations of electron transfer in the NADPH-bound catalase from Proteus mirabilis PR. AB - Catalase-bound NADPH both prevents and reverses the accumulation of compound II, an inactive form of catalase that is generated from the normal active intermediate form (compound I) when catalase is exposed to a steady flow of hydrogen peroxide. The mechanism for the regeneration reaction is unknown although NADPH could act either as a one-electron or a two-electron donor. Recently, a reaction scheme has been proposed in which the formation of compound II from compound I generates a neighboring radical species within the protein. NADPH would then donate two electrons, one to compound II for reduction of the iron and the other to the protein free radical. In this paper, we report calculations to find the dominant electron tunneling pathways between NADPH and the heme iron in the catalase from the peroxide-resistant mutant of Proteus mirabilis. Two major tunneling pathways are found which fuse together on Ser-196. It is suggested that the sequence Gly-Ser of the loop that divides the beta 5 strand is the key element for shielding a radical amino acid. PMID- 7548162 TI - Circular dichroism and Fourier transform infrared spectroscopic studies on the secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate carboxykinases. AB - The secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate (PEP) carboxykinases was quantitatively examined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies. From CD analyses, values of 24% alpha-helix and 38% beta-sheet were obtained for the E. coli enzyme, while the corresponding values for the S. cerevisiae PEP carboxykinase were 20% and 36%. Analysis of the amide I' infrared band indicated 20% alpha-helix and 36% beta-sheet for the S. cerevisiae enzyme, while for the E. coli protein values of 40% beta-sheet and between 9 and 36% alpha-helix could be inferred. It is concluded that the bacterial enzyme has more secondary structure elements than the yeast protein. No alteration of the CD or FTIR spectra was detected upon substrate or metal ion binding to any enzyme. PMID- 7548157 TI - Simultaneous existence of two types of ribosome-inactivating proteins in the seeds of Cinnamonum camphora--characterization of the enzymatic activities of these cytotoxic proteins. AB - Two types of ribosome-inactivating proteins (RIPs), camphorin and cinnamomin, have been purified from the seeds of Cinnamomum camphora. Camphorin is a type I RIP with a molecular mass of 23 kDa. Cinnamomin is a type II RIP having three isoforms, its molecular mass is around 61 kDa. The A- and B-chain derived from cinnamomin isoforms have similar mobilities in SDS-polyacrylamide gel. Camphorin and cinnamomin are both glycoproteins. In phylogenesis, it is particularly significant to find the simultaneous existence of type I and type II RIP as well as three isoforms of the latter in the same organ of a higher plant. The molecular mechanism of action of these two RIPs on mammalian ribosomes was demonstrated to be the specific RNA N-glycosidase activity. They modify 28S RNA among the four species of ribosomal RNAs. The cleavage site is the adenosine at position 4324 (rat liver 28S rRNA) embedded in the highly conserved 'R/S domain'. It is the first report that an intact type II RIP (cinnamomin) exhibits RNA N glycosidase activity. Additionally, camphorin and cinnamomin can cleave supercoiled double-stranded DNA into nicked and linear forms (Ling et al. (1994) FEBS Lett. 345, 143-146). PMID- 7548163 TI - Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris. AB - The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI 6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G. PMID- 7548164 TI - Comparison of primary structures and substrate specificities of two pullulan hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R 47. AB - Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively. PMID- 7548168 TI - Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization. AB - Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences. PMID- 7548166 TI - Effect of protamine on the solubilization of collagen-tailed acetylcholinesterase: potential heparin-binding consensus sequences in the tail of the enzyme. AB - Asymmetric acetylcholinesterase (AChE) contains three tetrameric sets of catalytic subunits disulfide-linked to structural subunits of a collagenic tail. This form is localized in the basement membrane zone of the neuromuscular junction, where it interacts with proteoglycans. It has been described that heparin-binding domains of many proteins contains clusters of basic residues. Here we show that protamine--a highly basic protein--specifically solubilizes asymmetric AChE from the rat neuromuscular junction, starting at 25 micrograms/ml and reaching a plateau at 250 micrograms/ml protamine. We also show that protamine was able to displace AChE bound to heparin-agarose. Two synthetic peptides corresponding to the sequence of the collagenic tail polypeptide also release the enzyme. Finally, we propose that two heparin-binding consensus sequences (-B-B-X-B-) are present in the tail of AChE. Our results indicate that clusters of basic residues are responsible for the interaction of the collagen tailed AChE with proteoglycans. PMID- 7548167 TI - Structural features of the hydroxy- and keto-disubstituted bile salts: human serum albumin binding. AB - The binding of keto- and hydroxy bile salts to human serum albumin, the identity of the bile salts binding sites and the identification of the amino acids present in these sites were studied. The keto bile salts cholanate-3-one (C3), cholanate 3,6-dione (C3-6) and cholanate-3-hydroxy-6-one (KHC) were found to quench the native fluorescence emission of albumin. This suggested that the tryptophan residue of human albumin (residue 214) is accessible to the keto bile salts and not to the hydroxy parent compounds. The binding of the keto bile salts was characterized by a simple population of binding sites with Ka ranging from 22 x 10(4) M-1 for the mono keto bile salt (C3) down to 4 x 10(4) M-1 for the hydroxy keto bile salt (KHC). The substitution of an oxo group at carbon C3 in C3-6 molecule for a hydroxy group (KHC) produce a significant decreasing of the interaction, suggesting that the hybridization state of the carbon at C3 in the steroid ring of the bile salt molecule is also an essential requirement for bile salts binding. It was found that bile salts are bound to the benzodiazepine binding site on human albumin (site II), producing a perturbation on site I, fatty acids and bilirubin binding site. The presence of only one substituent at C3 (oxo or OH) produce an important perturbation on the fatty acid binding sites, decreasing the polarity of the its microenvironment, while a little effect was observed for the dihydroxy and di-oxo-substituted BS, suggesting that the hydroxy substituents at C6, C7 and C12 do not interact in a significant manner with the fatty acid binding sites on HSA. The participation of specific amino acids in albumin-bile salt binding sites depends on the polar groups on the bile salt molecules as exemplified by the quantitatively different role of lysyl residues like those interacting with KHC, C3 and C3-6, and tyrosyl residue interacting with KHC. The following amino acids in human albumin were found to play a role in the bile salts-albumin interaction: lysyl 195 and 225, several arginyls, histidyl 146 and tyrosyl 411. PMID- 7548165 TI - Spectroscopic characterization of the alkylated alpha-sarcin cytotoxin: analysis of the structural requirements for the protein-lipid bilayer hydrophobic interaction. AB - alpha-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of alpha-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far UV circular dichroism analysis reveals the presence of residual secondary structure (beta-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the alpha-helix and turn contents and a concomitant decrease in the beta-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of alpha-sarcin with lipid membranes. PMID- 7548169 TI - Band-shifting through polypeptide beta-sheet structures in the cyanine UV-Vis spectrum. AB - If oxa- or thiacarbocyanine is introduced into an aqueous poly-L-lysine (PL) solution in a concentration higher than that of aggregation, then a shift of the absorption band of the cyanine monomer (M) can be observed in the UV-Vis spectrum, provided that the PL has a beta-sheet conformation. Other polypeptide aggregates with a high beta-sheet content exhibit this effect as well, whereas for PL with an alpha-helix conformation no spectral shift is observed. The force field optimized molecular models and the calculated interaction energies prove that the beta-sheet interacts significantly more intensively with the cyanine than the alpha-helix does. The quantum chemically calculated highest occupied and lowest unoccupied molecular orbital (HOMO-LUMO) energies of the cyanines and cyanine beta-sheet polypeptide complexes predict a M-shift to bathochromic frequencies in agreement with experimental findings. In the case of the measured M-shift to hypsochromic frequencies, the shift appears to be influenced by the presence of cyanine J-aggregates. The results open the way for a fast and simple method to identify polypeptide beta-sheet structures in biological and other systems containing polypeptides by using cyanine as a sensor. PMID- 7548170 TI - The role of histidines 26 and 33 in the structural stabilization of cytochrome c. AB - Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro 44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about. PMID- 7548172 TI - Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3 beta-thiol as a fatty acyl acceptor. AB - Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3 beta-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 beta-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin/thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The Km for thiocholesterol was 203.6 microM with a Vmax of 5.3 nmol thiocholesteryl ester formed/h per microgram. The Km for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 microM with a Vmax of 8.8 nmol cholesteryl ester formed/h per microgram. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3 beta-SH group of thiocholesterol forming a cholesteryl thioester. Although the 3 beta-SH group is not as good a terminal acceptor as the 3 beta-OH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester. PMID- 7548171 TI - The thermodynamics of association and unfolding of the 205-316 C-terminal fragment of thermolysin. AB - The 205-316 C-terminal fragment of thermolysin has been studied by differential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimental conditions. The effect of sample concentration at the different pH values on the calorimetric traces is consistent with a monomer-dimer equilibrium of the folded fragment, which undergoes thermal unfolding into individual fragments. Equilibrium sedimentation experiments at 10 degrees C and different pH values confirm the presence of the association equilibrium and provide the value of the dimerization constants. The global analysis of the calorimetric, heat capacity curves has been carried out by a multidimensional fitting to the model N2<-->2N<-->2U. The analysis leads to a complete thermodynamic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a partially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information currently available and compared with the energetics of unfolding of the shorter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lara, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases the relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment. PMID- 7548173 TI - Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: possible involvement of calcium/calmodulin. AB - The differential implication of protein kinase C (PKC) isozymes in antigen- or PMA-induced phospholipase D (PLD) activation was investigated in rat basophilic leukemia (RBL-2H3) cells. In [3H]oleic acid-labeled cells, both antigen (100 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (100 nM) produced a specific product of PLD activation, [3H]phosphatidylbutanol (PBut) in the presence of butanol. Pretreatment of cells with a selective PKC inhibitor, Ro31-8425 (1-5 microM) inhibited PMA-stimulated PLD activity by 85%. In contrast, the antigen stimulated PLD activity was much less sensitive to the inhibitor. RBL-2H3 cells express PKC alpha, beta, delta, epsilon and zeta isozymes and down-regulation of PKC by exposure to PMA (20 nM) for 1-2 h caused rapid decrease in PKC alpha and beta isozymes, leaving PKC delta, epsilon and zeta isozymes intact. Apparent decreases in the levels of PKC alpha and beta to about 50% were observed after adding 20 nM PMA for 1 h, when PMA-stimulated PLD activity was inhibited by up to 70%. Decrease in antigen-stimulated PLD activity was evident after 2 h PMA treatment, when PKC alpha and beta decreased by nearly 70%. These results suggest that in the antigen-mediated PLD pathway PKC may be implicated but not play such a great role as PMA-stimulated pathway which is mediated through PKC alpha or beta. Then, we have examined the involvement of calcium/calmodulin (CaM) in PLD activation by antigen, since the antigen-stimulated PLD activation showed the absolute requirement for extracellular calcium. Preincubation of RBL-2H3 cells with a CaM antagonist W-7 (20 microM) inhibited the antigen-stimulated PLD activity by 90%, but W-5, a chlorine-deficient analogue of W-7 that only weakly interact with CaM, caused little inhibitory effect. Another non-specific CaM antagonist, trifluoperazine (TFP) also inhibited PLD activation. These results suggest that calcium/CaM may be involved in the antigen-stimulated PLD activation. PMID- 7548174 TI - Fermentable carbohydrates exert a more potent cholesterol-lowering effect than cholestyramine. AB - The purpose of this work was to assess the respective role of bile acid excretion and of the end-products of cecal fermentations in the cholesterol-lowering effect of complex carbohydrates. The effects of two different fermentable carbohydrates (guar gum, beta-cyclodextrin), and sequestrant resin (cholestyramine) have been investigated in male Wistar rats. Guar gum and beta-cyclodextrin are broken down in the large bowel, with fermentation rich in propionic acid (37% against 26% for control), whereas cholestyramine did not enhance cecal fermentation. beta Cyclodextrin and guar gum were less potent than cholestyramine to enhance bile acids and sterol excretion. Nevertheless, fermentable carbohydrates exerted a more potent cholesterol-lowering effect than cholestyramine. beta-Cyclodextrin also depressed triacylglycerol-rich lipoprotein (TGRLP). Fermentable carbohydrates lowered cholesterol of LDL and HDL1 fractions. The induction of hepatic HMG-CoA reductase was practically proportional to rate of fecal steroid excretion. Moreover, with beta-cyclodextrin, hepatic HMG-CoA reductase induction was concomitant to a decrease in fatty acid synthase (FAS) activity. Thus, the cholesterol-lowering effect of fermentable carbohydrates could be related to a depressed lipogenesis, as well as to an accelerated removal of HDL1, in relation to an elevated hepatic demand of cholesterol. In conclusion, fermentable carbohydrates could favour cholesterol elimination and have a general lipid lowering effect by exerting more complex physiological effects than cholestyramine. PMID- 7548176 TI - Neutrophil release of arachidonic acid, oxidants, and proteinases: causally related or independent. AB - This investigation examined the concept that arachidonic acid (AA) serves as a second messenger in stimulation of the respiratory burst and degranulation of polymorphonuclear neutrophils (PMN). The main support for this idea is from observations that reagent AA, added to cell suspensions, stimulates the respiratory burst and degranulation and these events are blocked by PLA2 inhibitors. We verified that exogenously-added AA stimulated release of O2-, myeloperoxidase (MPO), and lysozyme (LZ), but this required amounts of AA which approximated the critical micellar concentration. This suggested that such administration of AA might act as an extracellular agonist, similar to particulate stimuli, rather than acting as a second messenger as might occur following mobilization of AA from cellular membranes. To investigate the role of fatty acids released by hydrolysis of cellular phospholipids, exogenously-added group I, II or III PLA2's were used to mobilize fatty acids from cellular membranes. Mole quantities of cell-associated free fatty acids were measured by negative ion chemical ionization gas chromatography/mass spectrometry. AA mobilization in response to exogenous PLA2 was dose- (0.1 to 10 U/ml PLA2) and time-dependent (peak at 1 to 2 min with a reduction by 4 min). Resting neutrophils contained < 10 pmol free AA/10(7) PMN; the receptor-mediated agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) alone did not increase these values. Exogenously-added PLA2 generated large quantities of free AA in control and fMLP-treated cells (462 +/- 122 and 2097 +/- 176 pmol/10(7) PMN, respectively); however, this did not induce O2-, nor did it augment the level of O2- stimulated by fMLP. Also, PLA2 caused no degranulation and did not alter degranulation induced by fMLP. PLA2 also did not alter O2- or degranulation responses in primed PMN. The data indicate that mobilization of AA from cellular phospholipids neither stimulates nor modulates the respiratory burst or degranulation of PMN. PMID- 7548175 TI - Relation between micellar structure of model bile and activity of esterase. AB - In a model bile solution composed of lecithin (L)-bile salt (B), the solubilization of lipid and the accessibility of enzyme to the lipid were examined by observation of EPR spectra and measurement of enzyme activity. The lifetime of the spin probe in the micellar phase was estimated to be approx. 1 microsecond by means of line shape analysis. Both population and lifetime increased with temperature and the molar ratio of lecithin to bile salt (L/B). The EPR data indicated that simple micelle of bile salt, mixed disk micelle of bile salt-lecithin, and multi-lamellar mixed disk micelle can exist in a model bile solution, depending on the L/B molar ratio across a range from 0 to 1.5. The maximal power of the mixed disk micelle to solubilize cholesteryl ester in the model bile at a L/B molar ratio of 1:1 was confirmed by EPR measurement of cholesteryl 12-DOXYL-stearate. Observation of the enzyme activity on a mixture of model bile and substrate at 37 degrees C revealed selective accessibility of cholesterol esterase (bovine pancreas) to mixed disk micelle, of cholesterol oxidase (Streptomyces cinnamomeus) to both simple and mixed disk micelle, and of pancreatic lipase (porcine pancreas) to both simple micelle and an oil droplet of substrate. The temperature-dependent activity of cholesterol oxidase to cholesterol in mixed disk micelle can be explained in terms of mesomorphic phase transition of lecithin side chains followed with fluidity of liquid crystal phase. Regarding phospholipase C from Bacillus cereus, though the selective accessibility to the micelles was not observed at 37 degrees C, a decrease in activity for mixed disk micelle could be found at lower temperatures. PMID- 7548177 TI - Antioxidant properties of demethyldiisoeugenol. AB - The antioxidant properties of demethyldiisoeugenol were investigated in this study using various models. Demethyldiisoeugenol inhibited Fe(2+)-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50 1.8 +/- 0.1 microM. Demethyldiisoeugenol was more effective than alpha tocopherol and BHT in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH). It also scavenged superoxide anion generated by xanthine/xanthine oxidase and peroxyl radical (ROO.) derived from 2,2'-azobis(2 amidinopropane)dihydrochloride (AAPH) in aqueous system with a stoichiometric factor of 1.3 +/- 0.2. Furthermore, it prevented conjugated-diene formation and apolipoprotein B (apo B) oxidation of LDL. However, demethyldiisoeugenol did not scavenge 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN)-derived peroxyl radical in hexane. It also did not chelate Fe2+, did not inhibit xanthine oxidase activity or possessed hydroxyl radical (.OH) scavenging activity. Experimental results indicate that demethyldiisoeugenol is a potentially effective antioxidant and can protect rat brain homogenate and LDL against oxidation. PMID- 7548178 TI - Expression of fatty acid binding protein in the liver during pregnancy and lactation in the rat. AB - Expression of the liver fatty acid binding protein (L-FABP) has been studied in the liver of pregnant and lactating rats. The L-FABP concentration found in the cytosol by immuno-enzymatic assay (ELISA) was consistently higher in the dams during the pregnancy and the lactation than in the age-matched virgin females. Paradoxically, a decrease in the L-FABP mRNA level occurred in the maternal liver during the last days of the gestation. This level remained low on days 7 and 14 of the lactation. Since the transcription rate of the L-FABP gene was unchanged in the maternal liver, these data suggest a post-transcriptional regulation of the L-FABP during pregnancy and lactation in the rat. The nutritional adaptations occurring during pregnancy and lactation are not involved in this regulation since a chronic maternal food-restriction failed to correct these modifications. The mechanism of this regulation is presently unknown, but possibilities include hormonally mediated effects. PMID- 7548179 TI - Nascent VLDL phospholipid composition is altered when phosphatidylcholine biosynthesis is inhibited: evidence for a novel mechanism that regulates VLDL secretion. AB - Previous work has shown that inhibition of phosphatidylcholine biosynthesis inhibits very low density lipoprotein (VLDL) secretion by causing a decrease in the number of particles in the Golgi but not in the endoplasmic reticulum of rat liver (Verkade et al. (1993) J. Biol. Chem. 268, 24,990-24,996). One explanation for this observation was that VLDL from choline deficient livers was degraded in a post-endoplasmic reticulum compartment. This hypothesis was supported by experiments in which choline deficient (CD) or choline supplemented (CS) rat hepatocytes were incubated +/- Brefeldin A. In the presence of Brefeldin A, VLDL secretion was blocked, but no difference was observed in the degradation of apolipoprotein B (apoB) within the CD or CS cells. If increased catabolism of apoB were occurring in the endoplasmic reticulum of CD hepatocytes, enhanced degradation of apoB in CD cells might have been expected. Inhibition of phosphatidylcholine biosynthesis also caused decreases in the phosphatidylcholine content of membranes of the secretory pathway. The lipids of nascent VLDLs from the lumina of endoplasmic reticulum and Golgi prepared from CD rat liver were relatively enriched in phosphatidylethanolamine and depleted of phosphatidylcholine when compared to samples from CS liver. The changes in nascent VLDL phospholipid composition mimicked that of the organelle membranes from which the VLDLs were isolated. Possibly the phospholipid composition of the organelles is a factor in determining the final phospholipid composition of VLDLs. One hypothesis is that when phosphatidylcholine biosynthesis is impaired, nascent VLDL is assembled incorrectly and degraded by a quality control protease in a post-endoplasmic reticulum compartment. PMID- 7548180 TI - Characterization and partial purification of phospholipase D from human placenta. AB - We report the existence in the human placenta of a phosphatidylcholine hydrolyzing phospholipase D (PLD) activity, which has been characterized and partially purified. Triton X-100 effectively solubilized PLD from the particulate fraction of human placenta in a dose-dependent manner. However, Triton X-100 caused decreasing enzyme activities. Maximum transphosphatidylation was obtained with 2% ethanol. The enzyme was found to have a pH optimum of 7.0-7.5 and an apparent Km of 33 mol% (or 0.8 mM). Ca2+ and Mg2+ was not required for the enzyme activity. Addition of phosphatidyl-4,5-bisphosphate, but not phosphatidylethanolamine, to the substrate mixture gave rise to a pronounced dose dependent increase in PLD activity (EC50 = 0.3 mol%), suggesting a regulatory role of this phospholipid in PLD action. The enzyme was inhibited by sodium oleate when partly or fully substituting for octylglucoside in the substrate mixture. The PLD activity was enriched 15-fold by solubilization and purification on a DEAE-Sepharose column. N-Ethylmaleimide (10 mM) markedly inhibited the purified enzyme, indicating the presence of free thiol groups on PLD. Sphingosine (20 microM) and (+/-) propranolol (53 microM) had no direct effect on PLD activity. The present results form the basis for further purification of a PLD from human tissue. PMID- 7548181 TI - Antioxidative activity of ubiquinol-10 at physiologic concentrations in human low density lipoprotein. AB - Ubiquinol-10 is a powerful lipid-soluble antioxidant found in cell membranes and lipoproteins in vivo. Its mechanism of action on lipid peroxidation has been determined in model and biological systems. Data concerning antioxidative activity of ubiquinol-10 in lipoproteins, however, are still controversial. The present work examines its role in the prevention of low density lipoprotein (LDL) oxidation, specifically its influence on a copper-mediated oxidative modification of human LDL in vitro. We found that ubiquinol-10 incorporated in LDL in subnormal concentrations (0.05-0.13 mol/mol LDL incorporated in comparison with 0.10-1.20 mol/mol LDL reported as normally in human LDL) slightly but not significantly decreased production of lipid peroxidation products (lipid peroxides, conjugated dienes, thiobarbituric acid-reactive substances) during the first hours of oxidation. The extent of apolipoprotein B modification (LDL fluorescence at 360/430 nm) was also decreased. Increasing the ubiquinol-10 concentration in LDL to 0.55-1.48 mol/mol LDL made it significantly more resistant to copper-mediated oxidation than native LDL. Adding the same amounts of either ubiquinone-10 or alpha-tocopherol to the LDL suspension had almost no effect on its oxidation. Ubiquinol-10 decreased alpha-tocopherol consumption during LDL oxidation and was consumed more rapidly than the latter. These results demonstrate that LDL ubiquinol-10 content is an important factor influencing LDL susceptibility to oxidation by copper and suggest that it represents the first line of defense against oxidative modification in human LDL. PMID- 7548182 TI - Total parenteral nutrition stimulates hepatic cholesterol synthesis in the rat. AB - Cholesterol synthesis was studied in parenterally fed rats, as compared to orally fed rats with or without saline infusion. Conditions of total parenteral nutrition (TPN) involved the intravenous infusion of a nutritive mixture containing 20% Intralipid as the lipid source (50% of non-protein energy) at the continuous rate of 2 ml per h, for five days. In rats maintained in isotopic steady state by daily injections of [3H]cholesterol, isotope dilution indicated that the endogenous plasma cholesterol input was significantly higher (+15%, P < 0.05) in TPN than in orally fed rats, which suggested a slight stimulation of whole body cholesterogenesis. Cholesterol synthesis was assessed in TPN and orally fed rats by the in vivo incorporation of [1,2-13C]- and [1-14C]acetate into hepatic and intestinal sterols, and by the activity of HMG-CoA reductase in microsomes isolated from liver and small intestine. Both methods demonstrated that TPN markedly stimulated the hepatic cholesterol synthesis, since the radioactivity of liver sterols was 6- to 10-fold higher, and the activity of HMG CoA reductase 5-fold higher, in TPN than in orally fed rats. Despite the weight reduction of the small intestine, by about 20% after TPN, the incorporation of exogenous [14C]acetate into intestinal sterols was similar in TPN and orally fed rats. As the liver and intestine are the main organs responsible for the appearance of endogenous cholesterol in plasma, it may be concluded that the increased endogenous plasma cholesterol input was mainly due to a strong stimulation of hepatic cholesterol synthesis in TPN rats. PMID- 7548183 TI - Age-related changes in the percentage of oleate in adipose tissue of male and female Fischer rats. AB - Fischer 344 rats showed sex difference in the percentage of oleate in lipids of the omental adipose tissue (Thorling, E.B. and Overvad, K. (1994) Nutr. Res. 14, 569-576). The development of this difference was studied with respect to time in rats maintained on laboratory chow, from the age of 3 weeks to 20 weeks. From the age of 3 weeks to 5 weeks the percentage of oleate increased slightly in both sexes. From the age of 5 weeks to 13 weeks the percentage (mean +/- S.D., n = 5) increased further in the female rats from 24.8 +/- 0.6% to 27.6 +/- 0.6%, whereas in the male rats it dropped from 22.9 +/- 0.4% to 20.2 +/- 0.6% at the same time points, respectively. In adult rats, age 20 weeks, the percentage of oleate was 28.6 +/- 0.3% and 19.6 +/- 0.5% for females and males, respectively. Castrated males partly maintained their juvenile level, being 21.1 +/- 1.1% fourteen weeks after castration at the age of 6 weeks. Oestrogen injections twice a week to the castrated rats increased their oleate percentage within the same period to 23.4 +/- 0.3%, partly reflecting the increase observed in the female rats. Stearic acid showed similar but less pronounced changes. The ratio oleic acid/stearic acid was constantly higher in the female than in the male rats, and this difference increased with age. The results of the present study suggest that these changes in percentage of oleate in adipose tissue lipids may partly have been caused by an effect of sex steroids on the delta-9-desaturase. PMID- 7548184 TI - Sexual dimorphism in the metabolic response to the calcium channel antagonists, diltiazem and clentiazem, by hyperlipidemic JCR:LA-cp rats. AB - The JCR:LA-cp rat is obese, insulin resistant, and hypertriglyceridemic. The obese male rats spontaneously develop atherosclerosis and ischemic myocardial lesions that are prevented by treatment with the calcium channel antagonist, nifedipine. Male and female JCR:LA-cp rats were treated with the calcium channel antagonist, diltiazem, and a closely related compound, clentiazem (at 30 mg/kg). Clentiazem, but not diltiazem, caused a significant increase in body weight of both sexes in the presence of decreased food consumption. Serum triacylglycerols were decreased by half by both drugs in male rats only, reflecting decreased very low-density lipoprotein (VLDL) secretion. Females did not respond with lower concentrations of triacylglycerol (although VLDL secretion rate was decreased) and showed increased concentrations of cholesterol in the high-density lipoprotein (HDL) fraction. Diltiazem-treated male rats showed decreased VLDL particle size, together with a shift to shorter-chain fatty acids in the triacylglycerols. This effect was not seen with clentiazem treatment. There was no effect on insulin and glucose metabolism in these insulin-resistant animals. Calcium channel antagonists have complex metabolic effects in the hypertriglyceridemic rats, with highly beneficial hypolipidemic effects in the males that are not seen in the females. The sexual dimorphism of these responses is sex linked, but appears not to be due to the steroid sex hormones. These results suggest caution in the chronic treatment of human females with these agents and the importance of detailed human studies in females and individuals with the insulin-resistant/hypertriglyceridemic/obese syndrome. PMID- 7548185 TI - Regulation of bradykinin-stimulated phospholipase C and arachidonic acid release by protein kinase A in MDCK-D1 cells. AB - Regulation of phospholipases C (PLC) and arachidonic acid (AA) release by cAMP dependent protein kinase (PKA) was investigated in MDCK-D1 cells. Bradykinin (BDK) was used to stimulate PLC and AA release, while arginine vasopressin (AVP), forskolin (FSK), isobutylmethylxanthine (IBMX) were used to increase cAMP levels and stimulate PKA. When cells were preincubated for 20 min with 10 microM FSK + 0.5 mM IBMX, and subsequently treated with 1 microM BDK or control medium for 40 min, the basal and BDK-stimulated PLC activity, measured as accumulated labelled inositol phosphate (InsP) after 40 min and inositol trisphosphate (InsP3) after 10 s, were significantly inhibited. In a parallel manner, FSK + IBMX also significantly decreased both basal and BDK-stimulated diacylglycerol (DAG) production. The basal and BDK-enhanced AA release into the media was also significantly inhibited by pretreatment with FSK + IBMX. In parallel experiments, H-89, a specific inhibitor of PKA, was preincubated for 60 min prior to addition of BDK and this resulted in a reversal of FSK+IBMX-induced inhibition of basal and BDK-stimulated PLC activity and AA release. An inhibitor of inositide hydrolysing PLC, U73122, (1 microM) was also found to blunt BDK-stimulated PLC activity and BDK-enhanced AA release which indicated that stimulation of AA release by the nonapeptide was second to PLC activation. The ionophore, A23187, (10 microM) greatly stimulated AA release and to a much lesser extent, PLC activity. Its effect on AA release however was not blocked by inhibiting protein kinase C (PKC) with staurosporine (SSP) and consequently did not notably involve the PLC-PKC cascade. Activation of PKA with FSK + IBMX was found to significantly inhibit the enhancement of AA release by ionophore. With 12-tetradecanoyl-phorbol 13-acetate (TPA) also present there was a synergistic increase in the A23187 stimulated AA release and activation of PKA under such conditions inhibited AA release to a similar extent though the synergistic effect remained. The results strongly suggest a role for PKA in the regulation of PLC activity and AA release in MDCK-D1 cells. Control of AA release by PKA, is mediated both by mechanisms which involve blunting of PLC activity and mechanisms which are downstream from the PLC-PKC cascade. PMID- 7548186 TI - Selected eicosanoids increase the proliferation rate of human colon carcinoma cell lines and mouse colonocytes in vivo. AB - Eicosanoids have been implicated in colon carcinogenesis, but their role remains unclear. The levels of PGE2 are elevated in colon cancer tissues and in blood draining colon tumors. The effect of eicosanoids on the proliferation of colonic cells is unknown. We studied the effect of several prostaglandins (PGs) and leukotriene (LT)B4 on the proliferation rate of the human colon adenocarcinoma cell lines SW1116 and HT-29 and of 16,16-dimethyl PGE2 (dmPGE2) on the colon of BALB/c mice. PGs E2, F2 alpha, I2, the methyl ester of PGE2, dmPGE2, and LTB4 (10(-10), 10(-8), 10(-6) M), administered for up to 72 h, stimulated cell proliferation in SW1116 cells and all but PGF2 alpha and PGI2 stimulated proliferation in HT-29 cells. The proliferative effect was time- and concentration-dependent. However, in SW1116 cells the response to PGs was 'bell shaped', being maximal at 10(-8) M, with the 10(-10) and 10(-6) M concentrations being less effective. In HT-29 cells, the addition of methyl groups to the PGE2 molecule increased the proliferative effect. None of these eicosanoids affected the distribution of these cells in the cell cycle or their rate of programmed cell death (apoptosis). dmPGE2 stimulated 3.6-fold the proliferation of colonocytes in normal BALB/c mice. This was determined by bivariate flow cytometric analysis of the expression of proliferating cell nuclear antigen (PCNA) in virtually pure populations of mouse colonocytes. dmPGE2 did not alter the cell cycle distribution of these cells. We conclude that several PGs as well as LTB4 stimulate the proliferation of human colon carcinoma cells in vitro, while dmPGE2 has a similar effect on mouse colonocytes in vivo. These findings raise the possibility that eicosanoids may contribute to colonic carcinogenesis by stimulating the proliferation rate of tumor cells in the colon. PMID- 7548187 TI - The transfer of docosahexaenoic acid from the yolk to the tissues of the chick embryo. AB - Changes in the amounts of the major fatty acids present in the lipids of the yolk complex and the embryo were delineated during embryogenesis of the chicken. The rates of transfer of palmitic, oleic, linoleic, linolenic and arachidonic acids from the lipids of the yolk complex were essentially identical. In contrast, docosahexaenoic acid (DHA) was preferentially transferred from the yolk complex at a rate which was significantly higher than that exhibited by the other major fatty acids. The rates of accumulation of both arachidonic acid and DHA in the lipids of the whole embryo were significantly greater than the rates observed for the C16 and C18 fatty acids, particularly between days 12 and 16 of the 21-day embryonic period. Analysis of the fatty acid composition of plasma lipid throughout development indicated that the triacylglycerol fraction contained relatively high proportions (up to approx. 14% w/w of total fatty acids) of DHA, but much lower proportions (approx. 3%) of arachidonic acid. In contrast, plasma phospholipid was enriched in arachidonic acid (up to approx. 18%), but contained much lower proportions (generally less than 3%) of DHA. A considerable amount of DHA was incorporated into adipose tissue triacylglycerol, so that by the time of hatching, the tissue represented a major store of this fatty acid. Over the hatching period, the amount of DHA in adipose triacylglycerol decreased dramatically, by up to 85%, whereas there was little or no change in the amounts of the other major fatty acyl components in this tissue. The amount of DHA as a component of brain phospholipid increased continuously throughout the developmental period studied. However, by the time of hatching, the amount of DHA in brain phospholipid represented less than 10% of the amount of this fatty acid originally present in the lipids of the yolk. PMID- 7548188 TI - Inhibition of microsomal cholesterol ester hydrolase by okadaic acid in isolated rat hepatocytes. AB - Okadaic acid, a potent and specific inhibitor of protein phosphatases 1 (IC50 10 20 nM) and 2A (IC50 0.05-2 nM) caused early and sustained inhibitions of microsomal cholesterol ester hydrolase activity in hepatocyte suspensions. The changes in the kinetic properties of the esterase and its response to exogenous alkaline phosphatase and cyclic AMP-dependent protein kinase after cell exposure to 1 microM or 1 nM okadaic acid differed markedly among themselves, which suggests the involvement of both protein phosphatases 1 and 2A in the regulation of the microsomal hydrolysis of cholesterol esters. Furthermore, the inhibitory effect of okadaic acid is likely to be independent of the dibutyryl-cyclic AMP promoted cell events leading to stimulation of esterase activity. PMID- 7548189 TI - Blood sphingomyelins from two European countries. AB - The objectives of this study were to isolate and analyse human blood sphingomyelins from two European populations which have significant differences in both the composition of dietary fat source and the rate of mortality from coronary heart disease. Blood samples have been taken from healthy adult volunteers from a Greek (Thessaloniki) and a French (Paris) region. The sphingomyelin fraction from red blood cells and plasma, separately, have been isolated by thin-layer chromatography and analysed by mass spectrometry after soft chemical ionisation with gas ammonia. The conditions used gave spectra with three discrete areas which correspond to ions coming from sphingoid bases, fatty acids and ceramides. Eight sphingoid bases have been detected in both red blood cells and plasma. Sphingosine was the major contributor. The rest of the sphingoid bases have 16 to 19 carbon atoms and 0 to 2 double bonds. Fourteen fatty acids, the same for red blood cells and plasma, have been detected with 16 to 26 carbon atoms and 0 to 2 double bonds. Palmitic acid was the most abundant fatty acid. Thirty eight ceramides have been detected in red blood cells and forty in plasma. Thirty six ceramides were common in red blood cells and plasma. Statistically significant differences (P < 0.01) have been detected between the two populations for some sphingoid bases, fatty acids and ceramides. Differences have also been found between the two populations in the degree of saturation of these sphingomyelin parts. These differences are attributed to (a) high monounsaturated fat, olive oil, consumption by Greeks and (b) other environmental and genetic factors. It is suggested that the detected differences between the two populations contribute to the different coronary heart disease rates observed among Greeks and French. PMID- 7548190 TI - Occurrence of a furan fatty acid in marine bacteria. AB - A fatty acid containing a furan ring was detected in the cellular lipids of marine bacteria, Shewanella putrefaciens, Marinomonas comunis, Enterobacter agglomerans, Pseudomonas fluorescens, etc., which were isolated from the intestinal liquor of fishes. Analytical data indicated that the fatty acid was 10,13-epoxy-11-methyloctadeca-10, 12-dienoic acid. Therefore, we propose that furan fatty acids detected in marine fish are derived not only from marine plants but also from intestinal bacteria of fishes. PMID- 7548191 TI - The human steroidogenic acute regulatory (StAR) gene is expressed in the urogenital system and encodes a mitochondrial polypeptide. AB - The first enzymatic step in the biosynthesis of steroid hormones occurs in the mitochondrial inner membrane and is dependent on the mobilization of cholesterol from cellular stores. We report on the isolation of a human cDNA which encodes a mitochondrial protein called steroidogenic acute regulatory (StAR) protein, implicated in transport of cholesterol into mitochondria. Nucleotide and predicted amino acid sequence analyses indicate that the human and murine polypeptides are highly conserved, sharing 87% identity with an overall homology of 92%. Analysis of the distribution of StAR mRNA transcripts in human tissues by Northern blotting reveals several mRNA species, the most abundant of which is a 1.8 kb mRNA transcript present in testes, ovaries and kidneys. Using in vitro translated protein, we demonstrate that the StAR gene product can be efficiently imported into exogenously added mitochondria. PMID- 7548192 TI - Formation and metabolism of 14,15-epoxyeicosatrienoic acid by human reproductive tissues. AB - Human granulosa-luteal cells cultured in the presence of arachidonic acid produced low levels of the epoxygenase metabolite 14,15-epoxy-5,8,11-(Z,Z,Z) eicosatrienoic acid (14,15-EpETrE) as determined by HPLC analysis and gas chromatography mass spectrometry. When authentic 14,15-[3H]EpETrE was incubated with these cells in the absence of serum it was metabolised initially to the dihydroxy derivative (14,15-dihydroxy-5,8,11-eicosatrienoic acid, 14,15-DiHETrE) and subsequently to a number of more polar metabolites as determined by HPLC. Fetal calf serum protected 14,15-EpETrE from metabolism for at least 2 h. A similar pattern of metabolism was obtained when 14,15-[3H]EpETrE was incubated with a human choriocarcinoma cell line (BeWo). Microsomes from this cell line converted arachidonic acid to a large number of radioactive metabolites including 14,15-DiHETrE and 11,12-DiHETrE although there was no evidence for the parent epoxides. These results extend earlier findings that human reproductive tissues produce epoxygenase metabolites, and demonstrate the rapid metabolism of these compounds by intact cells in the absence of serum. PMID- 7548193 TI - Effect of membrane environment on inhibition of acyl-CoA:cholesterol acyltransferase by a range of synthetic inhibitors. AB - The effect of the membrane environment of acyl-CoA:cholesterol acyl transferase (ACAT), an important intracellular enzyme of cholesterol metabolism, on the properties of a range of inhibitors of varying potencies was studied. ACAT activity from rat liver was solubilised with 3% deoxycholate (97% solubilised activity). After dilution into cholesterol/phosphatidylcholine liposomes (molar ratio 0.35), the assay of this reconstituted system showed linearity with protein and time. Saturation with oleoyl-CoA was achieved at 10 microM. Comparison of the potency of the ACAT inhibitors in the reconstituted assay and in a microsomal assay revealed a relationship between the lipid content of the assay and the inhibitory activity for potent inhibitors of ACAT (CI976, CL277,082, YMI7E and DuP128). This relationship was unrelated to lipophilicity of the drugs. Octimibate, lovastatin and progesterone, none of which is a potent ACAT inhibitor but which have all been described as ACAT inhibitors in the literature, all had low potencies in both assay systems. These results suggest that the lipid concentration must be taken into account when comparing potencies of ACAT inhibitors. The present data also indicate that some compounds which inhibit cholesterol esterification may do so by an indirect mechanism. PMID- 7548194 TI - Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters. AB - The effects of dietary oleic acid on cholesterol metabolism were investigated and compared with those of palmitic acid in hamsters. Addition of 5% oleic acid to a 0.1% cholesterol-supplemented diet decreased plasma total cholesterol, very low density lipoprotein (VLDL) cholesterol, and low density lipoprotein (LDL) cholesterol, increased hepatic LDL receptor activity, and decreased plasma cholesteryl ester transfer protein (CETP) activity in comparison with 0.1% cholesterol alone. In contrast, addition of 5% palmitic acid to a 0.1% cholesterol-supplemented diet increased total cholesterol and LDL-cholesterol, increased plasma CETP activity, and suppressed hepatic LDL receptor activity to a greater extent than 0.1% cholesterol alone. Neither oleic acid nor palmitic acid altered hepatic microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity, but oleic acid increased hepatic microsomal cholesterol 7 alpha-hydroxylase activity. These results suggest that dietary oleic acid inhibits the increases in total, VLDL-, and LDL-cholesterol induced by dietary cholesterol by preventing both LDL receptor suppression and increased CETP activity, whereas dietary palmitic acid augments the cholesterol-induced increases in total and LDL-cholesterol by both further suppression of LDL receptor activity and further stimulation of CETP activity. PMID- 7548195 TI - Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells. AB - Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 microM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxy arachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.1-0.8 microM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 microM all-trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all-trans-retinoic acid. When all trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all-trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR alpha activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR alpha heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic acid as is the same process caused by arachidonic acid. PMID- 7548196 TI - In vivo turnover of 1,2-dipalmitoylphosphatidylcholine and sphingomyelin in rabbit erythrocytes. AB - The in vivo turnover of both 1,2-dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) in rabbit erythrocytes was studied. DPPC, either 14C-labelled in the fatty acyl chain at the 2-position of the glycerol moiety or 3H-labelled in the choline's methyl group, and [N-methyl-14C]SM (bovine) were introduced into the membrane of freshly isolated rabbit erythrocytes by using phospholipid transfer proteins. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. Analysis of blood samples shows that both labels disappear from the circulating cells with the same rate, resulting in a half-time value of about 6.4-6.6 days. This result demonstrates that the loss of DPPC from the cells is due to transfer of intact molecules to the plasma and that a deacylation process is of no or minor importance as mechanism of renewal of DPPC. Labelled sphingomyelin, introduced into the rabbit erythrocyte membrane in a similar way, disappears from the circulating red cell with a half-time value of 15.5 days. This accounts for a daily replacement of the total SM pool by 3.2%. PMID- 7548197 TI - On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. AB - The interfacial activation of Candida antarctica lipase A (CALA) and B (CALB) has been investigated and compared with that of Humicola lanuginosa lipase (HLL). CALB displayed no interfacial activation towards p-nitrophenyl butyrate (PNPB) when exceeding the solubility limit of the substrate. No activation was observed towards p-nitrophenyl acetate (PNPA) at the addition of sodium dodecyl sulfate (SDS) nor in the presence of a solid polystyrene surface. The catalytic action of CALB was very different from that of Humicola lanuginosa lipase, which showed a pronounced interfacial activation with the same substrates. The basis for the anomalous behaviour of CALB is proposed to be due to the absence of a lid that regulates the access to the active site. In contrast to CALB, CALA expressed interfacial activation, but the activation was not as prominent as for Humicola lanuginosa lipase (HLL). The structural basis for the activation of CALA is unknown. PMID- 7548198 TI - Degradation of fluorescent and radiolabelled sphingomyelins in intact cells by a non-lysosomal pathway. AB - The aim of the present study was to investigate the role of the entitled neutral, sphingomyelinase in the non-lysosomal pathway of sphingomyelin degradation by intact cells (Spence et al. (1983) J. Biol. Chem. 258, 8595-8600; Levade et al. (1991) J. Biol. Chem. 266, 13519-13529). The uptake and degradation of sphingomyelin by intact living cells was studied using cell lines exhibiting a wide range of activity levels of acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this purpose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell lines, some of them derived from patients with Niemann-Pick disease (deficient in the acid, lysosomal sphingomyelinase) were incubated with radioactive, [oleoyl 3H]sphingomyelin or fluorescent, pyrene-sulfonylaminoundecanoyl-sphingomyelin. Either compound was taken up by a pathway which was not receptor-mediated and hydrolyzed by all intact cells, including those derived from Niemann-Pick disease patients. Moreover, their degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinase. The intracellular sphingomyelin degradation rates showed no correlation with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-Pick patients lacking the lysosomal sphingomyelinase, and having no detectable in vitro activity of the 'classical' neutral sphingomyelinase, were able to degrade the exogenously supplied sphingomyelins. Indeed, in vitro these cells were shown to exhibit neutral, magnesium- and dithiothreitol-dependent sphingomyelinase activities, that might contribute to the non-lysosomal pathway for sphingomyelin degradation to ceramide in intact cells. PMID- 7548199 TI - Substrate specificity of microsomal 1-acyl-sn-glycero-3-phosphoinositol acyltransferase in rat submandibular gland for polyunsaturated long-chain acyl CoAs. AB - Microsomal 1-acyl-sn-glycero-3-phosphoinositol (1-acyl-GPI) acyltransferase in the rat submandibular gland showed the highest specific activities for eicosanoid related polyunsaturated acyl-CoAs, such as arachidonoyl-, bishomo-gamma linolenoyl- and 5,8,11,14,17-eicosapentaenoyl-CoAs, with low Km values. High activities were also obtained with acyl-CoAs having long (more than 14 carbon atoms) and n - 6 unsaturated (more than 3 double bonds) acyl chains. This enzyme also utilized acyl-CoAs having trans-unsaturated or branched chains, but not short-chains, as substrates, although the activity levels for trans-unsaturated acyl-CoAs were lower than those for cis-unsaturated acyl-CoAs. Chronic administration of isoproterenol induced decreases of this enzyme activity and the content of arachidonic, bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids at the sn-2 position of phosphatidylinositol. These results suggest that enrichment of arachidonic acid in the sn-2 position of phosphatidylinositol is established by the high specificity and affinity of 1-acyl-GPI acyltransferase for arachidonoyl-CoA. On the other hand, the low level of bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids in the sn-2 position of phosphatidylinositol may be explained by their limited availability. PMID- 7548200 TI - Phospholipase A stimulation in tumor cells by subtoxic concentration of tert butyl hydroperoxide. AB - Previous studies have shown an increase in the intracellular free arachidonic acid content associated with a disturbance in phospholipid metabolism in P815 tumor cells exposed to subtoxic concentration of tert-butyl hydroperoxide. The present study was to determine the respective contribution of the major phospholipid-metabolizing enzymes that could be involved in this process. The enzymes (phospholipase A, lysophospholipase, acylCoA:lysophosphatidylcholine acyltransferase and acylCoA synthetase) were studied under their respective optimal conditions. When P815 cells were treated with 50 microM of tert-butyl hydroperoxide, a significant stimulation (x 2.5) of phospholipase A was observed after 15 min of treatment. The activity of the acyltranferase tended to be higher in cells treated by tert-butyl hydroperoxide while the other enzyme activities (lysophospholipase and acyl CoA synthetase) were not affected. t-BHP did not significantly induce higher levels of lipid peroxides in P815 cells. These results show that, in the tumor cell line P815, the disturbance of phospholipid and arachidonate metabolism induced by t-BHP is linked to phospholipase A, the activation of which seems independent of oxidative stress. PMID- 7548201 TI - Glutamate stimulates the formation of N-acylphosphatidylethanolamine and N acylethanolamine in cortical neurons in culture. AB - The formation of anandamide (N-arachidonoylethanolamine), N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [14C]ethanolamine, [U 14C]arachidonic acid, [3H]arachidonic acid, [32P]phosphate, [14C]stearic acid, or [3H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [14C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N acylethanolamine (0.06-0.55% of total radioactivity) and N acylphosphatidylethanolamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [32P]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D (Streptomyces chromofuscus) resulted in two compounds, one comigrating as phosphatidic acid and the other as N-acylethanolamine. Compound I could be labelled with [14C]stearic acid and [3H]myristic acid, but not with [3H]- or [14C]arachidonic acid. Exogenous [3H]anandamide was metabolised with a t1/2 of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced the older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED50 = 40 microM) the formation of both compounds. It is suggested that N-acylethanolamine and N acylphosphatidylethanolamine are formed in relation to the cytotoxicity induced by glutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachidonic acid. PMID- 7548202 TI - Effects of added l-carnitine, acetyl-CoA and CoA on peroxisomal beta-oxidation of [U-14C]hexadecanoate by isolated peroxisomal fractions. AB - (1) During peroxisomal beta-oxidation of [U-14C]hexadecanoate, at concentrations higher than 100 microM, long-chain 3-oxoacyl-CoA-esters and 3-oxobutyryl-CoA accumulate. Only 3-oxobutyryl-CoA accumulates at a low concentration of [U 14C]hexadecanoate. Accumulation of long chain 3-oxoacyl-CoA esters is most extensive when the supply of CoA can be considered limiting for beta-oxidation. (2) Added acetyl-CoA was found to inhibit peroxisomal beta-oxidation. This inhibition was not significantly relieved by added L-carnitine and carnitine acetyltransferase (EC 2.3.17). (3) Added L-carnitine, at concentrations below 0.2 mM, was found to stimulate peroxisomal beta-oxidation of [U-14C]hexadecanoate by up to 20%, causing the conversion of acetyl-CoA into acetylcarnitine. Higher concentrations of L-carnitine were progressively inhibitory to beta-oxidation. This effect was specific for L-carnitine as both D-carnitine and aminocarnitine neither caused stimulation at low concentrations, nor inhibition at higher concentrations. Added L-carnitine caused accumulation of acylcarnitines of chain lengths ranging from 4 to 16 carbon-atoms. The inhibition observed with higher concentrations of added L-carnitine is likely due to conversion of [U 14C]hexadecanoate into [U-14C]hexadecanoylcarnitine. (4) Low concentrations of added hexadecanoylcarnitine was shown to inhibit peroxisomal beta-oxidation by about 15%, while added acetylcarnitine did not inhibit at concentrations up to 100 microM. (5) These data are interpreted to indicate significant control being exerted on flux at the stage of thiolysis either directly by means of CoA availability, or indirectly by means of the rate of acetyl-CoA generation. PMID- 7548203 TI - Linoleic acid oxidation in the presence of amino compounds produces pyrroles by carbonyl amine reactions. AB - The reactions of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-LOOH) and its degradation product 4,5(E)-epoxy-2(E)-decenal with butylamine and lysine were studied to determine whether pyrrole derivatives isolated in model reactions were produced in complex systems involving hydroperoxides. Incubated reaction mixtures were studied by gas chromatography coupled with mass spectrometry or high performance liquid chromatography coupled with mass spectrometry (HPLC-MS), and some compounds were isolated by column chromatography or semipreparative HPLC, and identified by 1H- and 13C-nuclear magnetic resonance spectroscopy and MS. The reaction of epoxyalkenals with amino groups produced two types of pyrrole derivatives: 1-substituted 2-(1'-hydroxyalkyl)pyrroles and 1-substituted pyrroles. 1-Substituted 2-(1'-hydroxyalkyl)pyrroles were responsible for the development of color and fluorescence by a polymerization reaction, which implied the formation of dipyrrylmethanes and dipyrrylmethenes. 1-Substituted pyrroles were final products in these reactions and their determination might be used as an index of oxidative stress. The above reactions were also observed between 13 LOOH and amino compounds, and suggested that the pyrrole polymerization mechanism plays a role in the fluorescence observed by reaction of hydroperoxides and amino groups. PMID- 7548204 TI - Comparison of the hepatic uptake and processing of cholesterol from chylomicrons of different fatty acid composition in the rat in vivo. AB - The effect of the fatty acid composition of chylomicrons on the uptake and processing of the cholesterol they carry was investigated in the rat in vivo. Rats kept on a standard low fat pellet diet and tube fed a single dose of palm, olive, corn or fish oil (rich in saturated, n-9 monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids, respectively) were used to prepare [3H]cholesterol-labelled chylomicrons of different fatty acid composition. These were then injected intravenously into rats (kept on the standard diet), and the clearance of radioactivity from the blood, distribution in the plasma lipoprotein density fractions, uptake by the liver and appearance in the bile were studied. [3H]Cholesterol from fish and corn oil chylomicrons was cleared from the blood more rapidly than that from palm and olive oil chylomicrons. After 180 min the proportion of the radioactivity present in the plasma in high density lipoprotein (HDL) was less when the chylomicrons were derived from palm oil as compared to any of the other oils. Approx. 40% of the administered label was recovered in the liver after 180 min in all experiments. The percentage of the injected radioactivity secreted into bile during 180 min was significantly higher with corn and fish oil chylomicrons than with palm oil chylomicrons, with chylomicrons from olive oil in an intermediate position, and these differences were most pronounced between 60 and 120 min after administration of the label. These studies clearly demonstrate that the fatty acid composition of chylomicrons has important effects on the hepatic uptake and processing of the cholesterol they carry, with enrichment with polyunsaturated fatty acids leading to an increased rate of uptake and more rapid removal from the body via the bile as compared to enrichment with saturated or monounsaturated fatty acids. PMID- 7548205 TI - The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. PMID- 7548206 TI - Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA. AB - Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites within p220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique. In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites. Specific and quantitative localization of induced facultative fragments was achieved in as low as 1 ng of DNA digest corresponding to a peak intensity below 0.1 absorbance unit upon laser scanning. An ENU dose dependent increase in the intensity of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block at EcoRI (G/ATTC) and BamHI (G/GATCC) restriction endonuclease sites, respectively. To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was assessed in plasmid DNA established in various Escherichia coli strains as a function of post-treatment incubation time in the recovery medium. The repair is indicated by the gradual disappearance of the 7.1, 7.7, 11.9 and 5.5 kb facultative fragments within the wild-type and mutant E. coli strains. The ethyl-adducts within EcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficient E. coli with an estimated t1/2 of approximately 40 min. However, decreased overall rate and at least 2.2-times lesser extent of repair was observed in the repair-deficient (ada+ogt-) and (ada-ogt+) cells. No measurable repair was noticed in alkyltransferase defective double mutant (ada-ogt-) even after 2 h of post treatment incubation. The repair of ethyl-adducts at NotI site (GC/GGCCGC) in 5.5 kb facultative fragment occurred at a relatively faster rate (t1/2 of 27 min) in wild-type bacteria. A 1.5-fold slower repair of ethyl-adducts in BamHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G in NotI sequence. These results demonstrate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair in E. coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context. PMID- 7548207 TI - Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus. AB - Complementary and genomic DNAs coding for a Brassica napus chitinase have been cloned and sequenced. The genomic DNA contains one intron and encodes a 322-amino acid basic chitinase with a 20-amino acid N-terminal signal peptide followed by a 40-amino acid cysteine-rich domain, linked by a hinge region to the main domain of the enzyme. The sequence of the cDNAs is identical to the exon sequence deduced from the genomic DNA. A probe derived from this gene identified a 1.2-kb transcript present in high amount in roots, moderate in floral tissues and low in stems and leaves. The synthesis of these transcripts is regulated during development and is induced in roots by wounding and ethephon. This type of chitinase is encoded by two sequences in Brassica napus, as shown either by Southern hybridizations or by genomic amplification and sequencing using the polymerase chain reaction. These genes are homologous to one sequence found in the Brassica oleracea genome. PMID- 7548208 TI - Properties of elongation factor-2 fragments obtained by partial proteolysis. AB - Rat liver elongation factor eEF-2 was treated with endoproteinase Glu-C. Two major fragments were obtained, which were identified by N-terminal sequencing and purified. The larger one (F61) contained 554 residues including the N-terminal end, and after a second cleavage released a N-terminal peptide (F7) of 62 residues. The smaller one (F34) contained the other 303 residues including the C terminal end. F61 and F34, either isolated or after combination, were unable to catalyze protein synthesis. However, we show by fluorimetry that F61 could still interact with GTP and GDP. This fragment was was able to participate into a ternary complex with ribosome and GDP, but not with ribosome and a GTP analogue. It was unable to protect the ribosome against ricin-inactivation and to be phosphorylated by the eEF-2-specific Ca(2+)-calmodulin-dependent kinase, though it contained Trp221 and Thr56 involved in these reactions. On the other hand, F34 could be ADP-ribosylated in the presence of NAD+ and diphtheria toxin, but this fragment was apparently unable to bind to ribosomes. These results and those obtained with other proteinases are discussed in the light of the data published recently which show the existence of five different domains in the three dimensional structure of EF-G. PMID- 7548209 TI - Nucleotides of tRNA (Glu) involved in recognition by barley chloroplast glutamyl tRNA synthetase and glutamyl-tRNA reductase. AB - The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex with the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the recognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a number of species were used to determine the nucleotides involved in the recognition of the barley chloroplast tRNA(Glu) by the barley enzymes. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The chemically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA synthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chloroplasts if barley, Chlamydomonas reinhardtii, tobacco, cucumber, wheat and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia coli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 conserved bases and five of these bases, G10, A26, U34, U35 and A37 are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(Glu) from E. coli, bakers yeast and barley germ and the barley chloroplast glutamyl-tRNA(Gln) are not effective substrates for the barley chloroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences of these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yields seven common differences in the primary sequence. These 7 nucleotides, A7-U66, U29-A41, A53-U61, and U72 are expected to be required for recognition by the barley chloroplast glutamyl-tRNA(Glu) reductase. PMID- 7548210 TI - Triple helix directed psoralen adducts induce a low frequency of recombination in an SV40 shuttle vector. AB - Triple helix forming oligonucleotide directed psoralen adducts in a mammalian shuttle vector have been reported to be repaired efficiently in human cells. In this study we examined the role of intermolecular homologous recombination in triple helix targeted psoralen adduct repair. A simian virus 40 (SV40) shuttle vector carrying a mutated supF gene was treated with a triplex forming oligonucleotide psoralen conjugate and cotransfected into human cells with a second plasmid bearing the wild type supF gene. Recombinants with a reactivated marker gene were detected by an X-gal assay in indicator bacteria. We could observe a low frequency of psoralen adduct induced recombination indicating that recombination does not play a major role in triplex directed psoralen adduct repair. The implications for targeted mutagenesis by triple helix forming oligonucleotides are discussed. PMID- 7548211 TI - Isolation and characterization of two distinct cDNA clones encoding corn seed cysteine proteinases. AB - We obtained two cDNA clones encoding corn seed cysteine proteinases (CCP1 and CCP2). Sequence analysis showed that CCP1 consists of 371 amino acid residues, in a prepro-protein form, with two unique short insertions in the mature protein region that are not found in papain or other common CPs. CCP2 consists of 360 amino acid residues with a vacuole sorting signal in the pro-sequence region. An amino acid sequence similarity of 42% was found between the mature protein region of CCP1 and that of CCP2. Although CCP1 is highly homologous to pea 15a CP (72%) and Arabidopsis thaliana RD 19 CP (79%), both of which are known to be induced only when the plant is exposed to a dehydrated environment, it showed very low homologies to other known cysteine proteinases (38-43%). CCP2 showed as much as 87% and 89% identity to rice oryzain gamma and barley aleurain, respectively. We also observed that the CCP1 mRNA is expressed in ripened corn seeds, although its expression reaches a maximum 5 days after the onset of germination. On the other hand, the CCP2 mRNA is expressed only during germination, with maximum expression at the 3-day stage. These results suggest the presence of at least two cysteine proteinases playing differential roles in the corn seeds. PMID- 7548212 TI - Molecular cloning and sequence analysis of cDNA encoding plasma alpha-1 antiproteinase from Syrian hamster: implications for the evolution of Rodentia. AB - Complementary DNA clones encoding plasma alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) were isolated from Syrian hamster liver cDNA library and sequenced. The deduced amino acid sequence of putative reactive site (P3-P'3) was Ile-Pro-Met-Ser-Val-Pro, characteristic of alpha-1-antiproteinase of orthodox type (Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932). A molecular phylogenetic tree of all known orthologous proteins was constructed based on the synonymous substitution rate. The result shows that the hamster has branched off first before the divergence among mice, rats, and gerbils, and that the rabbit is the closest relative of the guinea pig which is separated from the rodents. Although this tree differs largely from the classical phylogeny based on the morphology (hamsters and gerbils belong to the same family, Cricetidae, and the guinea pig belongs to the order Rodentia), it lends support to recent concepts that the hamster and guinea pig differ, in a number of biochemical features, not only from each other but also from mice and rats, and that the guinea pig may belong to an order distinct from Rodentia. PMID- 7548213 TI - Isolation of cDNA clone encoding human homologue of senescence marker protein-30 (SMP30) and its location on the X chromosome. AB - We have isolated and characterized a cDNA clone encoding human homologue of senescence marker protein-30 (SMP30), a calcium binding protein also called regucalcin (RC). This clone (pHSMP6) has 1356 base pairs (bp) and contains an open reading frame of 897 bp, which encodes 299 amino acids. The estimated molecular weight of the deduced polypeptide is 33,250 and pI is 5.836. The homology of amino acid sequences between human homologue and rat SMP30 is 88.6%. Using pHSMP6 as a probe, the chromosomal location of the human homologue of SMP30 gene was determined. The results of regional mapping using a panel of 11 rodent human somatic hybrids indicated that the gene is located in the p11.3-q11.2 segment of the X chromosome. This gene thus could be a candidate for one of the X linked diseases mapped to this regions. PMID- 7548214 TI - Sequence and functional characterization of the terminal exon of the human insulin receptor gene. AB - We present 5.1 kb of the 3' noncoding region sequence of the human insulin receptor gene and identification of four functional polyadenylation domains responsible for 3'-end processing of the 5.4, 6.9, 8.0 and 9.4 kb human insulin receptor mRNA, respectively. The insulin receptor gene contains five putative polyadenylation sites (P1-P5), located 5160, 6502, 7488, 8945 and 8957 base pairs (bp) downstream from the translational initiation site. All putative polyadenylation sites are flanked by upstream AU rich and downstream GU rich regions which regulate mRNA stability and mRNA cleavage, respectively. Also, two RNA stem-loop structures have been identified. To determine its role on gene expression, a reporter gene was constructed containing various lengths of the insulin receptor 3' UTR and transiently transfected into COS 7 cells. A 539 bp fragment (4897-5436 bp downstream from the IR translational initiation site) inhibited CAT expression by 5-6 fold. Further downstream addition of 1169 bp of the insulin receptor 3' untranslated region enhanced gene expression by 2-fold. These studies provide evidence that the insulin receptor 3' untranslated region can modulate gene expression. PMID- 7548215 TI - cDNA sequence of the murine synaptonemal complex protein 1 (SCP1). AB - We isolated and sequenced cDNAs for the murine synaptonemal complex protein 1 (SCP1). The whole cDNA sequence displays respectively 93% and 90% identity with the previously reported rat and hamster cDNAs. We show, however, that the encoded amino acid sequence extends for an additional stretch of 51 residues at its amino terminal end. PMID- 7548216 TI - Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae. AB - The aim of this paper is to gather and complete data about four members of a new gene family. Mutation in SUR4 gene was originally selected as a suppressor of defects caused by mutations in RVS161 or RVS167 genes. Cloning and sequencing of the SUR4 gene were performed. The deduced protein contains six putative transmembrane domains. Sequence comparison revealed that two yeast genes, FEN1 and JO343, shared significant similarities with SUR4. Mutants for SUR4 and FEN1 have the same pleiotropic phenotype, including bud localization defects, resistance to an immunosuppressor and resistance to ergosterol biosynthesis inhibitors. The double inactivation of SUR4 and FEN1 genes is lethal. These data and other aspects implicating SUR4 in glucose metabolism, suggest an involvement of these genes in the dynamics of cortical actin cytoskeleton in response to nutrient availability. Moreover, the existence of a fourth homologous gene in C. elegans extends the family to pluricellular organisms. PMID- 7548217 TI - Molecular cloning and sequencing of the gene encoding a sheep arginine vasopressin type 1a receptor. AB - The gene for the sheep arginine vasopressin type 1a (V1a) receptor subtype was cloned from a genomic library. The deduced amino acid sequence shows characteristics of a G-protein coupled receptor and high sequence identity to human and rat V1a receptor sequences (81% and 73%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a tissue distribution consistent with a type V1a receptor. The genomic DNA (7.1 kb) contains a 1586 bp intron between the putative 6th and 7th transmembrane domains. PMID- 7548218 TI - Promoter analysis of the bovine gene for seminalplasmin. AB - In this study we mapped the transcriptional initiation site of the gene for seminalplasmin (SAP) by primer extension analysis, situated 125 nucleotides upstream of the translational initiation site of the SAP-specific mRNA. We showed that the TATA-box in position -30 of the SAP gene is part of a functional promoter. A 280 bp region of the 5'-flanking region exerted a strong positive effect on promoter activity. In this region we identified consensus sequences for the transcriptional control elements AP1, AP2, PEA3 and GATA. PMID- 7548219 TI - Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors. AB - We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes. PMID- 7548220 TI - Trans-activation of the wheat histone H3 promoter by Gal4 DNA-binding domain (1 94) in plant cells. AB - This is the first demonstration that Gal4 DNA-binding domain (1-94) itself has the ability to trans-activate an eukaryotic promoter in vivo. It significantly activated a wheat histone H3 core promoter, but slightly activated a CaMV 35S core promoter in transiently transfected tobacco cells when the Gal4-recognition sequences were put upstream of the core promoters. PMID- 7548221 TI - A high dose of the STM1 gene suppresses the temperature sensitivity of the tom1 and htr1 mutants in Saccharomyces cerevisiae. AB - A new gene (STM1; suppressor of tom1) of Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of a tom1 mutant, by increasing its gene dosage. The gene could also suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al. (1994) Mol. Gen. Genet. 245, 107-116) and was physically mapped in the region near PEP3 on chromosome XII R. The predicted gene product (29,999 Da) is basic and partially homologous to various histone H1. The level of the gene expression increased 2-fold when exposed to mating pheromone. PMID- 7548223 TI - Metabolism of L-arginine through polyamine and nitric oxide synthase pathways in proliferative or differentiated human colon carcinoma cells. AB - HT-29 Glc-/+ cells originate from a human colon adenocarcinoma. These cells have been selected in a glucose-free culture medium and switched back in a glucose containing medium. In this condition, they can spontaneously differentiate after confluency in enterocyte-like cells according to the activity of the brush-border associated hydrolase dipeptidyl peptidase IV. Since L-arginine can generate polyamines which are necessary for cellular proliferation and also differentiation, and nitric oxide with reported anti-proliferative property, the metabolism of this amino acid was examined in proliferative and differentiated isolated HT-29 cells. Proliferative HT-29 cells were characterized by micromolar intracellular concentration of putrescine and millimolar concentration of spermidine and spermine. In these cells, L-arginine is converted to L-ornithine and putrescine and to a minor part to nitric oxide and L-citrulline. Putrescine was taken up by HT-29 cells, leading to the production of a modest amount of spermidine. The diamine was slightly incorporated into cellular proteins and largely released in the incubation medium. The proliferative HT-29 cells take up spermidine and spermine but do not catabolize these polyamines and slightly released spermidine. Differentiation of HT-29 cells is not associated with change in intracellular polyamine content but is paralleled by an almost complete extinction of de novo synthesis of putrescine (due to a dramatic decrease of ornithine decarboxylase activity) and by a reduced release capacity of putrescine. In contrast, putrescine net uptake and incorporation into cellular proteins remained unchanged after differentiation. Furthermore, spermidine and spermine metabolism as well as the circulation of L-arginine in the nitric oxide synthase pathway were also not modified after differentiation. In conclusion, putrescine is the L-arginine-derived molecule, the metabolism of which is specifically and markedly modified when HT-29 cells move from proliferative to differentiated state. PMID- 7548222 TI - Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation. AB - The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved. PMID- 7548224 TI - Phosphorylation of eukaryotic elongation factor 2 in differentiating and proliferating HL-60 cells. AB - Ca(2+)-and calmodulin-dependent protein kinase III (CaM PKIII) phosphorylates eukaryotic elongation factor 2 (eEF-2) in HL-60 cells. Dephosphorylation of the factor in these cells is catalyzed by phosphoprotein phosphatase 2A alone. Differentiation of the HL-60 cells by all-trans retinoic acid resulted in a reduced growth rate and a marked decrease in the intracellular concentration of eEF-2. During differentiation the activity of the eEF-2 kinase is gradually reduced and reaches 10% of that found in undifferentiated cells 5 days after the onset of differentiation. The capacity to dephosphorylate phospho-eEF-2 remained unaltered in the growth-arrested cells. Differentiation without reduced proliferation was induced in the HL-60 cells by interferon-gamma. Under these conditions, differentiation had no effect on the cellular content of eEF-2 or the ability to dephosphorylate phospho-eEF-2. However, the differentiated cells showed a dramatic decrease in the specific activity of the eEF-2 kinase. The results show that the cellular content of eEF-2 varies with the rate of proliferation and that the activity of the eEF-2 kinase is high in undifferentiated proliferating cells and decreases upon differentiation even under conditions of an unaltered growth rate. PMID- 7548225 TI - Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells: comparison with the effect of Rab3AL peptide. AB - Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells. PMID- 7548226 TI - Transport of bimane-S-glutathione in human erythrocytes. AB - Export of glutathione S-conjugate of bimane (BSG) was studied in human erythrocytes. Characteristics of the BSG transport is similar to that of dinitrophenyl-S-glutathione (DNP-SG). BSG transport has two kinetic components, one of high affinity and low capacity (Km = 7.4 +/- 0.2 mumol/ml cells, Vm = 2.7 +/- 0.1 nmol/min per ml RBC) and another of low affinity and high capacity (Km = 242 +/- 8 mumol/ml cells, Vm = 9.6 +/- 1.6 nmol/min per ml RBC). BSG export is inhibited by vanadate (Ki = 65 +/- 6 microM) and fluoride (Ki = 11.4 +/- 0.8 mM). Activation energy of the transport is 67 +/- 7 kJ/mol. BSG transport is independent of membrane potential; its rate increases with pH in the pH range of 6-8, in line with the assumption that the anionic conjugate is cotransported with proton. BSG import to erythrocyte membrane inside-out vesicles is stimulated by ATP. Fluorimetric measurements of BSG export require low amounts of cells and may also be useful for other cell types as an alternative to studies of glutatione S conjugate transport using radioactive substrates. PMID- 7548227 TI - Effects of N-3554S, a polyprenyl phosphate, on B16-F10 mouse melanoma cells. AB - N-3554S, an optically active S-isomer of alpha-dihydrodecaprenyl phosphate, reduced the tumorigenicity of cultured B16-F10 mouse melanoma cells probably by affecting protein N-glycosylation. Accordingly, membrane glycoprotein samples were prepared from the melanoma cells cultured with or without N-3554S, and amounts and structures of N-linked sugar chains were determined. Analyses of the N-linked oligosaccharides released by hydrazinolysis from these samples and reduced with NaB3H4 revealed that the N-3554S-treated cells contain 1.5-1.8 times as much oligosaccharides as the control cells, and the relative amounts of high mannose-type and bi-, tri- and tetra-antennary complex-type sugar chains are almost the same between two samples. Western blot analysis, however, showed that binding of L-PHA, which binds to oligosaccharides with the GlcNAc beta 1- >6(GlcNAc beta 1-->2)Man structure, is significantly reduced in 90 K, 96 K, 140 K, 155 K and 180 K glycoproteins in N-3554S-treated cells. Immunoblot analysis showed that the 140 K glycoprotein could be a fibronectin receptor. It was also shown that N-3554S treatment enhances the adhesiveness of the cells to fibronectin. These results indicate that N-3554S affects N-glycosylation of membrane glycoproteins and alters the cell surface properties of B16-F10 cells. PMID- 7548228 TI - Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC K8. AB - We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5 isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required. PMID- 7548230 TI - Glutamine increases collagen gene transcription in cultured human fibroblasts. AB - We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane 2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression. PMID- 7548231 TI - Clusterin gene expression in apoptotic MDCK cells is dependent on the apoptosis inducing stimulus. AB - Clusterin (Apolipoprotein J, complement lysis inhibitor) is a widely expressed multifunctional glycoprotein. The expression of clusterin mRNA has been reported to be elevated in a broad spectrum of apoptotic or degenerative tissues. More recently, it was shown that within these tissues clusterin is expressed in the surviving rather than in the dying cells, and that clusterin gene expression is actually down-regulated in the apoptotic cells. We have studied the expression of the clusterin gene in apoptotic MDCK cells. Cell death was initiated by three different stimuli: application of the steroid hormone antagonist RU 486, activation of protein kinase C by the application of the phorbol ester TPA, and- since clusterin is involved in lipid and cholesterol transport--perturbation of cell membranes by cholesterol. We show that clusterin gene expression is repressed in cells undergoing apoptosis in response to the application of RU 486 and TPA, but is unchanged in cells in which apoptosis has been triggered by cholesterol treatment. PMID- 7548229 TI - Fibroblasts co-expressing tyrosinase and the b-protein synthesize both eumelanin and phaeomelanin. AB - Melanin synthesis in the mouse involves the interaction of many pigmentation loci. Tyrosinase, the product of the albino (c) locus, catalyses the first step of the pathway. The brown (b) locus protein has significant homology to tyrosinase and controls black/brown coat coloration, but its function is controversial. To investigate the function of the b-protein and its interaction with tyrosinase, we established cell lines expressing both tyrosinase and the b protein by transfecting tyrosinase-expressing fibroblasts with a b-protein expression vector. The tyrosinase-expressing parent line does not have L dopachrome tautomerase activity, but this enzyme is detectable in double transfectants as well as in fibroblasts expressing the b-protein alone. Cells expressing both proteins have a higher steady-state level of tyrosinase than fibroblasts expressing tyrosinase alone, and contain elevated levels of melanin intermediates. This is thought to result from interaction of tyrosinase with the b-protein. Only phaeomelanin is detectable in fibroblasts expressing tyrosinase alone, whereas double transfectants synthesise significantly more phaeomelanin and detectable eumelanin. PMID- 7548232 TI - Inhibition on platelet activation by shikonin derivatives isolated from Arnebia euchroma. AB - Acetylshikonin, teracrylshikonin, beta,beta-dimethylacrylshikonin and shikonin, isolated from Arnebia euchroma, inhibited collagen (10 micrograms/ml)-induced aggregation of washed rabbit platelets in a concentration-dependent manner with IC50 values of 2.1 +/- 0.2, 2.8 +/- 0.3, 4.2 +/- 0.5 and 10.7 +/- 0.7 microM, respectively. Acetylshikonin also inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA, 100 microM), U46619 (1 microM), platelet-activating factor (PAF, 3.6 nM) and thrombin (0.1 U/ml) in a concentration-dependent manner. The IC50 values of acetylshikonin on the inhibition of these four agonists-induced platelet aggregation were 3.1 +/- 0.4, 2.2 +/- 0.2, 8.0 +/- 0.6 and 12.7 +/- 1.0 microM, respectively. The thromboxane B2 formation caused by collagen, PAF and thrombin was inhibited by acetylshikonin, while formations of thromboxane B2 and prostaglandin D2 caused by AA were not inhibited. Acetylshikonin did not inhibit cyclooxygenase activity since it did not attenuate prostaglandin E2 formation after incubation of sheep vesicular gland microsomes with AA. Acetylshikonin suppressed both the rise of intracellular Ca2+ concentration and the generation of [3H]inositol monophosphate caused by these five aggregation inducers. Platelet cyclic AMP level was unaffected by acetylshikonin. These data indicate that acetylshikonin inhibits platelet activation by suppression of phosphoinositide breakdown. PMID- 7548233 TI - Atherosclerosis alters the composition, structure and function of arterial smooth muscle cell plasma membranes. AB - The object of this study was to examine changes in plasma membranes of arterial smooth muscle (ASM) during atherogenesis obtained from cholesterol-fed (2%) rabbits. A microsomal fraction highly enriched with plasma membrane markers was prepared by subcellular organelle fractionation from ASM freshly isolated from the thoracic aorta. The membranes were analyzed for unesterified (free) cholesterol (FC) content, membrane bilayer structural parameters (X-ray diffraction), phospholipid (PL) composition, and Na+/K(+)-ATPase activity and kinetics. Following 8 weeks on diet, membrane FC content increased 67.1%. Small angle X-ray diffraction demonstrated an increase in membrane hydrocarbon core electron density and an increase in overall lipid bilayer width (56-62 A). This increase in bilayer width was highly correlated with the membrane FC content (r = 0.992). Both membrane FC content And bilayer width independently correlated with time on cholesterol diet. The phospholipid profile of the membrane revealed a 16.4% increase in phosphatidylcholine (PC), 19.3% decrease in phosphatidylethanolamine (PE) and 62.8% increase in sphingomyelin (SM) content with no change in total PL content. Na+/K(+)-ATPase activity was decreased 52.2% (P < 0.005), and [3H]ouabain binding kinetics demonstrated a 27.6% decrease in maximum binding sites (Bmax) (P < 0.01) while the dissociation constant (Kd) remained unaltered. Membranes obtained from control ASM cells enriched with FC in culture demonstrated changes similar to those in atherosclerotic ASM membranes including an increase in membrane FC content, an increase in bilayer width, and a decrease in Na+/K(+)-ATPase activity with decreased ouabain Bmax. These data demonstrate marked compositional, structural and functional changes in ASM cell membrane characteristics in dietary atherosclerosis. These changes were highly correlated with cholesterol accumulation in the plasma membrane bilayer and were observed before the appearance of visible lesions. We suggest that these membrane defects may be linked with early atherogenesis. PMID- 7548234 TI - Cirrhosis of the human liver: an in vitro 31P nuclear magnetic resonance study. AB - Human livers with histologically proven cirrhosis were assessed using in vitro 31P NMR spectroscopy. Spectra were compared with those from histologically normal livers and showed significant elevations in phosphoethanolamine (PE) and phosphocholine (PC) and significant reductions in glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC). There were no significant differences in spectra from livers with compensated and decompensated cirrhosis. These results help to characterise the alterations in membrane metabolism in cirrhosis of the liver. PMID- 7548235 TI - The involvement of low-density lipoprotein in hemin transport potentiates peroxidative damage. AB - Hemin binds to isolated low-density lipoprotein (LDL) and thereby triggers LDL oxidation. In this study we investigated whether hemin can get together with LDL under physiological conditions. The relative affinity of three blood components to free hemin was as follows: RBCM < LDL < albumin. At physiological molar ratio of LDL/albumin all the hemin was bound to albumin. In molar excess of albumin over hemin, existing even under pathological conditions, albumin served as an efficient antioxidant for the plasma hemin-induced LDL oxidation. RBCM-embedded hemin, unlike plasma hemin, affected LDL: the mobile hemin was transferred from RBCM to LDL in the absence of albumin, whereas in the presence of albumin most of the mobile hemin finally reached the albumin but partially via LDL. Thus, a transient hemin is built up in LDL. This transient hemin triggered LDL oxidation which was not inhibited but rather promoted by albumin. The involvement of albumin in this oxidation was explained by its acting as a pump thereby increasing the transient hemin in LDL. It is suggested that increased membrane hemin level as in hemoglobinopathies and/or excess LDL in dyslipidemia provide conditions for hemin-induced LDL oxidation. PMID- 7548236 TI - Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects. AB - Plasma cholesterol level is controlled by various factors. In the present study, high plasma activity of cholesterol esterase was found to correlate with plasma total cholesterol and low density lipoprotein (LDL) cholesterol levels in normolipidemic human subjects. However, the cholesterol esterase is not elevated in plasma of patients with familial hypercholesterolemia. These observations suggest that cholesterol esterase level is not determined by plasma cholesterol level, but elevated cholesterol esterase may be causative in increasing plasma cholesterol and LDL. Additional experiments further demonstrated that cholesterol esterase can convert the larger and less-atherogenic LDL to the smaller and more atherogenic LDL subspecies in vitro. These results suggest that plasma cholesterol esterase contributes to the formation and accumulation of atherogenic lipoproteins, and thus is a major risk factor for premature atherosclerosis in normal human subjects. PMID- 7548237 TI - Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG. AB - A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction. PMID- 7548238 TI - Cholesterol sulfate is not degraded but does not accumulate in Epstein-Barr virus transformed lymphoid cells from patients with X-linked ichthyosis. AB - The metabolism of cholesterol sulfate (CS) was investigated in immortalized, Epstein-Barr virus-transformed lymphoid cell lines derived from normal individuals and patients affected with recessive X-linked ichthyosis (XLI). Normal lymphoid cells expressed arylsulfatase C and steroid sulfatase (including cholesterol sulfatase) activities, and these two sulfohydrolases showed the same enzyme properties as in other human cells, e.g., leukocytes or skin fibroblasts. XLI-derived lymphoid cell lines exhibited extremely deficient activity of both arylsulfatase C and steroid sulfatase. While normal and XLI intact, living lymphoid cells could take up exogenous radiolabelled CS through a non-receptor mediated process. XLI cells were completely unable to degrade CS to cholesterol. However, despite their defect in CS degradation, steroid sulfatase-deficient cells did not accumulate CS because of outflux of this sterol. The potential implications of these findings to the pathogenesis of increased CS content in plasma and epidermis of XLI patients are discussed. This study also demonstrates that immortalized lymphoid cell lines may represent a useful experimental model system for the study of XLI. PMID- 7548239 TI - Induction of hepatic CYP1A2 by the oral administration of caffeine to rats: lack of association with the Ah locus. AB - Caffeine was administered to male Wistar albino rats for two weeks at three concentrations, namely 0.1, 0.2 and 0.3%, and hepatic cytochrome P450-dependent mixed-function oxidase determined. Caffeine administration gave rise to a marked, dose-dependent increase in the O-deethylation of ethoxyresorufin and, to a lesser extent, in the O-depentylation of pentoxyresorufin. Erythromycin N-demethylase, p nitrophenol hydroxylase and lauric acid hydroxylase activities, as well as total cytochrome P450 content were unaffected by this treatment. Immunoblot analysis revealed that caffeine gave rise to a dose-dependent increase in the hepatic CYP1A2, and at the highest dose only, CYP2B apoprotein levels. Apoprotein levels of CYP3A and CYP2E1 were not modulated by the treatment with caffeine at all dose levels studied. Caffeine could not displace [3H]TCDD from the rat hepatic cytosolic Ah receptor. Computer analysis showed that caffeine is essentially a planar molecule with an area/depth ratio 4.8, characteristic of CYP1A substrates/inducers. Molecular modelling revealed that the caffeine molecule could orientate itself within the putative CYP1A2 active site so as to facilitate demethylation of the N-1, N-3 and N-7 positions. However, at physiological pH, the N-9 nitrogen atom is likely to be partially protonated, allowing it to participate in an electrostatic interaction with the negatively-charged glutamate 318-residue, favouring N-3 demethylation, the major pathway of metabolism in both humans and animals. In conclusion caffeine, being essentially planar, is an inducer of CYP1A2 in rat liver. PMID- 7548240 TI - Correlation between rat liver regeneration and mitochondrial energy metabolism. AB - The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation. PMID- 7548241 TI - [Comparison of the diagnostic value of CT and MRI in injuries of the cervical vertebrae]. AB - The aim of our study was to compare the diagnostic capacities of computed tomography (CT) and magnetic resonance imaging (MRI) in the diagnostic evaluation of acute cervical spinal column injuries. We examined 39 patients with cervical spine injury suspected either clinically or by plain radiography, or even confirmed. In 30 patients, 86 acute traumatic lesions were observed in the area of the cervical spine, 83% of which were retrospectively recognisable by CT and 95% on MRI films. In nine patients, no acute traumatic pathologic pattern could be found either by CT or by MRI or any other subsequently employed diagnostic methods. CT yielded 100% of the osseous acute traumatic findings, the degenerative lesions narrowing the spinal channel, and of the dislocations, but only 33% of the lesions of the longitudinal ligaments, 50% of intramedullary haemorrhages, 60% of paravertebral soft tissue haematomas, 83% of vertebral disc herniations of protrusions, and none of the six nonhaemorrhagic spinal cord contusions. Without exception, MRI revealed all the traumatic medullary and paravertebral soft tissue changes, dislocations, and spondylophytes narrowing the spinal channel, but only 50% of the C2-odonteous fractures, 89% of the transverse processes', and 92% of the vertebral lamina fractures. Basing on these results, after primary plain film radiograph imaging, the performance of MRI seems to be recommendable prior to CT in diagnostic evaluation of traumatic cervical spinal lesions, if possible with regard to the patient's clinical state and the global organization, unless immediate CT imaging of other body regions (i.e. of the head) is already being planned anyway, Nevertheless, MRI should not be abandoned within the overally framework of this disease pattern. PMID- 7548242 TI - [Conventional roentgen examination of the lung in fibrosing alveolitis- correlation with histological, clinical and biochemical findings]. AB - QUESTION: Is it possible to draw conclusions on aetiology, histology or clinical course, looking at the different radiomorphologic appearance and its changes in chest x-rays of patients with "fibrosing alveolitis"? MATERIAL AND METHOD: Retrospective correlation of chest x-rays with all relevant clinical and histological data of 33 patients. RESULTS: In all patients typical clinical signs with changes of lung function and typical histological changes were found. There was no strict correlation between single parameters and radiological findings. In the estimation of the clinical course, no correspondence between radiological findings and clinical data was found in only 18% of the parameters. CONCLUSION: The chest x-ray is an objective investigation which clearly shows the course of fibrosing alveolitis beside its undisputed function in diagnosing acute complications like infiltration, effusion or development of pneumothorax. PMID- 7548243 TI - [Pulmonary infiltrations in Sjogren syndrome--a radiodiagnostic study]. AB - We report on the radiological findings of 8 female patients with primary Sjogren's syndrome and pulmonary infiltrations. In the chest x-ray, all patients featured an interstitial diffuse bilateral reticular pattern which was most prominent in the bases. The apices of the upper lobes were not involved. There were no pleural effusions. The radiologic appearances changed only slowly. In three cases the lesions progressed to form a patchy confluent pattern. 4 of 8 patients developed within 24 months a nodal or extranodal lymphoma of low-grade malignancy. PMID- 7548244 TI - [De Anquin disease or spinous engagement syndrome]. AB - We report on a rare disorder called de Anquin's disease or spinous engagement or impingement syndrome. Low back pain in this specific syndrome probably combined with sciatica is caused by a hypertrophic spinous process in combination with a spina bifida occulta of an underlying vertebra which leads to compression of the neural structures beneath. Mostly the enlarged spinous process is seen at the level L5 and the spina bifida occulta at the level S1. Between 1981 and 1993 six patients were treated surgically after long-lasting periods of conservative therapy by resection of the hypertrophic spinous process combined with revision of the nerve roots and division of adhaesions. All patients had been reexamined clinically and radiologically. Up to now three of six patients are free of all symptoms, pain diminished in two others. The back pain situation is unchanged in only one patient. PMID- 7548245 TI - [Comparative imaging of HIV associated diseases of the hepatobiliary system in computerized tomography and cholangiography]. AB - With the incidence of AIDS being on the rise, diagnostic work-up of hepatobiliary disease in these patients is of increasing interest. 57 x-ray cholangiograms and computed tomographic studies of 13 AIDS-patients with clinical signs of cholangitis were reviewed. Nine patients had abnormal cholangiographic findings. Computed tomography of 8 patients revealed hepatobiliary disease in six cases. Of three patients with cholangiograms reflecting bile duct dilatation of neoplastic aetiology, CT was required in two for definite diagnosis. Cholangiography is the method of choice for diagnosing AIDS-associated cholangitis, whereas computed tomography compares favourably in depicting neoplastic disease of the hepatobiliary system. Follow-up of AIDS-associated cholangitis usually delineates unchanged radiologic findings. PMID- 7548246 TI - [Computerized tomography aspects of amiodarone poisoning]. AB - Amiodarone, an antiarrhythmic drug, may cause pulmonary toxicity with typical high-attenuation-pleuro-parenchymal lesions and increased attenuation of liver and spleen. Characteristic CT findings are diagnostically significant. PMID- 7548248 TI - [Ruptured intracranial dermoid as an incidental finding]. AB - This report presents a case of a ruptured intracranial dermoid accidentally found in a 51-year old patient. The tumour appears as a large lesion located in the posterior fossa. The CT and MRI studies identify fatty material into the CSF spaces providing intraventricular fat-fluid levels. These characteristic findings are indicative of the diagnosis. PMID- 7548247 TI - [Radioisomorphisms of sclerosing cholangitis]. AB - For differentiation of Primary Sclerosing Cholangitis (PSC) from AIDS-associated cholangitis (AAC), 95 CT or Cholangiography studies of 37 patients were reviewed and signs of biliary disease analysed. Only two each of PSC and AIDS-subjects were free from biliary lesions compatible with some kind of sclerosing cholangitis. Subgroup analysis revealed wide overlapping of morphological findings in PSC and AAC. We conclude that radioisomorphism of etiologically different forms of sclerosing cholangitis may indicate pathophysiologically similar courses. PMID- 7548249 TI - [Combination of rare anomalies of the cervical spine and peripheral skeleton]. AB - This paper presents a case report on a 59-year old patient of small stature with moderate mental retardation, strabismus and several rare skeletal anomalies. We found an os odontoideum, a dysontogenetic 3fold fusion of cervical vertebrae, multiple double-sided wrist bone dysplasias and brachytelephalangical digiti pedis I, but no anomalies of the heart or genitals. Clinical signs, biochemical parameters and results of x-ray and ultrasonic examination are demonstrated in this complex malformation syndrome that combines different anomalies known from other syndromes. PMID- 7548250 TI - [An unusual case of endometriosis]. AB - Endometriosis can be found in 10 to 20% of all menstruating women. Most often the lesions are localized in the pelvis, but in rare cases they can be found in almost every region of the body. The diagnostic clue is the typical history of premenstrual pain. Imaging can demonstrate the site and size of extra-pelvic lesions and an image-guided diagnostic puncture for histological diagnosis can be done. PMID- 7548251 TI - [Abdominal involvement in neurofibromatosis type 1]. AB - Neurofibromas are benign tumours that arise from neuroectodermal tissues. They may occur as solitary lesions, or multiple, then referred to as neurofibromatosis, or von Recklinghausen's disease. We report on a 27-year old patient with large involvement of many nerve trunks and plexus (elephantiasis nervosa), responsible for bowel obstruction and anuria. PMID- 7548253 TI - [Fistula carcinoma in chronic soft tissue infection]. AB - Squamous cell carcinoma without accompanying osteomyelitis is a rare complication in chronic soft tissue inflammation. We report on a patient with soft tissue squamous cell carcinoma developing only two years after a war accident. PMID- 7548252 TI - [Magnetic resonance tomography diagnosis of plexiform fibrohistiocytic tumor]. AB - A case report on a 30-year old female patient who underwent resection of a haemangiopericytoma followed by radiation therapy in 1988 is presented. 6 months ago a space-occupying lesion was detected within the radiation field, which was classified as a plexiform fibrohistiocytic tumour (PFT). On MRI PFT showed the similar signal intensities as the muscles in T1, as well as T2-weighted images. After gadolinium-DTPA no enhancement within the tumour could be measured. Based on its exclusively dermal and subcutaneous location, and its signal behaviour uncommon for a malignant soft tissue mass, PFT must be included in differential diagnosis in any examining for local tumour recurrence. PMID- 7548254 TI - [Estimating patient dose in computerized tomography]. AB - This paper presents a model for the estimation of the effective patient dose in computed tomography. It is demonstrated how equivalent organ doses for selected examination can be estimated on the basis of the scanner-specific axis dose and published conversion factors. Problems and limitations of this method are discussed. The determination of the effective dose from the organ equivalent dosis is explained. The explanations are illustrated by an exemplary calculation, and the doses of the most important CT examinations are summarised. PMID- 7548256 TI - [Optimizing pelvic MR diagnosis with a new gastrointestinal contrast medium]. AB - AIM: Optimization of pelvic MRI by a new concept of water-equivalent bowel contrast agent. METHODS: We performed contrast enhancement of the bowel by oral application of 1000 ml of an aqueous mannitol solution one hour before pelvic MRI. In addition we performed an enema by 250 ml of water in one patient. RESULTS: Oral and rectal contrast enhancement of the bowel was tolerated without complications by our patients. Pelvic MRI was improved by a better delineation of the bowel. CONCLUSION: The concept of water-equivalent bowel enhancement is promising. It must be assessed by further studies; in particular, it must be compared with the available concepts of positive and negative bowel contrast enhancement. PMID- 7548255 TI - [Expectations from digital imaging and archiving procedures]. PMID- 7548257 TI - [ROC analysis of image quality in digital luminescence radiography in comparison with current film-screen systems in mammography]. AB - AIM: To compare in mammography the image quality of digital luminescence radiography (DLR) to that of usual film screen mammography and xeromammography. MATERIALS AND METHODS: Three single emulsion film-screen combinations, one double coated high resolution film and xeroradiography, were tested for this purpose. In our phantom study the detectability of microcalcifications, fibrils and low contrast details were first of all studied separately. Image processing techniques were, for example, contrast variation by grey scale level windowing, "unsharp mask" filtering and regulatable edge enhancement. Phantom images were made and then the image quality was evaluated by observer performance study using a receiver operating characteristic (ROC analysis). RESULTS: Best results in respect of detection of microcalcifications and fibrils were found in xeroradiography, luminescent image plate and double-coated film-screen combination. These systems showed more favourable ROC curves than the single emulsion film-screen combinations. CONCLUSIONS: Our results indicate that image quality of digital images in the field of image processing is equal to that of conventional mammographic techniques and partially superior to detection of low contrast details. PMID- 7548258 TI - [Possible applications of ultrasound in corticotomy-callus distraction]. AB - Sonographic examination can yield additional information in the course of corticotomy-callus distraction. Hence, we focus on individual slices--which are different from the standardised ultrasound examination of the skeleton system--in the area of the corticotomy-callus distraction. In ultrasound examination the structure of corticalis in the field of corticotomy is scanned circularly. Depending on the point of time of the corticotomy between the structure of corticalis, the callus can be visualised sonographically. The callus distraction can be divided into three stages: I. echo-poor structure between the two ends of the corticalis, II. echo-rich structure between the two ends of the corticalis, III. area of corticalis in the area of distraction with (III a), visualised without (III b) break. The described method is an additional low-cost imaging procedure in diagnosis, for the patient free of radiation and stress, which can yield additional information during and after completed callus distraction (question of permissible emotional stress). The examination is reproducible. The x-ray-controls in the course of time are mandatory, because the sonographic examination cannot judge reliably the deviation of the axis in the area of callus distraction. PMID- 7548259 TI - [Changed guidelines by the federal medical office regarding quality dismissed. Caution in purchasing new enhancement screens]. PMID- 7548260 TI - Surgery in the very old patient: evaluation of factors linked to postoperative morbidity and mortality. AB - One hundred and sixty-seven patients over 79 years of age were studied prospectively in our Emergency Surgery Department in order to evaluate their outcome, and the possible existence of factors linked to morbidity and mortality. The most common indications for surgery were gallstones (22.1%), hernias (14.9%), colo-rectal cancer (13.7%), peptic ulcer (6.5%), gastric cancer (5.9%) and ischemic or hemorrhagic vascular diseases (13.1%). Emergency surgery was performed in 93 (55.6%) patients. Forty-nine patients (29.3%) developed 83 postoperative complications. The risk of morbidity was statistically higher in patients who had more than two associated diseases (p < 0.05) and received blood transfusions (p < 0.01). The mortality rate was 16.1%, and was significantly related to ASA scores > or = 4 (p < 0.001) and a high degree of intraoperative bacterial contamination (Classes III-IV) (p < 0.05). Compared to elective surgery emergency operations had a higher morbidity (33.3% vs 24.3%) and mortality (21.5% vs 9.4%), but the difference was not significant. Mortality/morbidity ratio was significantly higher in emergency, as compared to elective surgery (64.5% vs 38.8%, p < 0.001). PMID- 7548261 TI - Effects of different types of anti-aging dietary restrictions on age-related atrial natriuretic factor changes: an immunochemical and ultrastructural study. AB - The effects of two types of anti-aging dietary restrictions restrictions-60% diet restriction (DR) and every-other-day feeding (intermittent feeding, IF)-on age related changes in atrial natriuretic factor (ANF) metabolism were studied in male Sprague Dawley rats by standard radioimmunological procedures and transmission electron microscopy. Both diet restrictions decreased plasma ANP (atrial natriuretic peptide) levels significantly (12-month-old rats: ad libitum fed controls 96 +/- 17.3 pg/mL, IF 63 +/- 4.8 pg/mL, DR 74 +/- 14.4 pg/mL; 24 month-old rats: AL 109 +/- 6.3 pg/mL, IF 75 +/- 8.9 pg/mL, DR 65 +/- 9.1 pg/mL). Dietary restriction prevented the age-related increase in ANP concentration in both the right atrium (12-month-old rats: AL 0.25 +/- 0.033 microgram/mg, IF 0.22 +/- 0.023 microgram/mg, DR 0.30 +/- 0.044 microgram/mg; 24-month-old rats: AL 0.39 +/- 0.037 microgram/mg, IF 0.10 +/- 0.015 microgram/mg, DR 0.07 +/- 0.011 microgram/mg) and left atrium (12-month-old rats: AL 0.23 +/- 0.033 microgram/mg, IF 0.13 +/- 0.019 microgram/mg, DR 0.17 +/- 0.035 microgram/mg; 24-month-old rats: AL 0.44 +/- 0.037 microgram/mg; IF 0.07 +/- 0.009 microgram/mg, DR 0.07 +/- 0.011 microgram/mg). Endocrine cardiocytes of diet-restricted senescent rats could be readily distinguished ultrastructurally from cells of ad libitum fed controls due to a higher number of beta-atrial specific granules of lower electrondensity. In conclusion, anti-aging diet restriction regimens fully prevented the age-related increase in the hormone store in atrial tissue, and lowered plasma ANP levels. PMID- 7548262 TI - Repeated testing of normal elderly with the Boston Naming Test. AB - The Boston Naming Test is commonly viewed as a measure of language ability, particularly, confrontational naming. Its utility in detecting word-retrieval problems in clinical populations is well documented. However, studies which would explore information-processing mechanisms involved in BNT performance are not available. Results of our repeated testing of 122 subjects between the ages of 57 and 85 with the 60-item version of BNT and other measures over three annual probes revealed high stability of the BNT scores over time, which suggests a lack of the practice effect, whereas cross-sectional analysis demonstrated some decline in the BNT scores in subjects over 70 years of age. In spite of the high stability in BNT scores on repeated testing, the pattern of correlations between the BNT and measures tapping different cognitive domains shifted over time. Results suggest predominantly the verbal mode of information processing in BNT performance on the first probe, as opposed to visuo-spatial mode on the third probe in our sample of elderly individuals. PMID- 7548263 TI - Age and laxative use in hospitalized patients. A report of the "Gruppo Italiano di Farmacovigilanza nell'Anziano--GIFA". AB - Laxatives are frequently taken especially by older persons, who have multiple diseases and are disabled. Major differences exist among countries in the type of laxatives taken, and knowledge of the intake patterns is important because of the potential adverse effects associated with particular ingredients. Laxatives and factors related to their use are described in a sample of 3257 patients admitted to 58 hospitals of the Gruppo Italiano di Farmacovigilanza nell'Anziano (GIFA) in 1991. The mean age was 68.2 years, median 73 years, more than 30% were age > or = 80 years, and 48.7% were men. The overall prevalence rates of laxative use during hospital stay and prior to admission were 11.5% and 11.3%, respectively. Increasing age was independently associated with laxative use after adjusting for gender, activities of daily living, cognitive function, number of active medical problems, medications taken, Italian region and length of stay. After excluding the prescriptions for hyperammoniemia, lactulose was the most frequently taken laxative (prevalence: 6.7% during hospital stay and 4.3% prior to hospital admission), followed by anthranoid laxatives such as senna, cascara, rhein and aloe (1.9% in-hospital and 3.3% pre-hospital). Other less frequently taken laxatives were picosulfate, phenolphthalein, lactitol, glycerol, bisacodyl and docusate. These patterns differ substantially from those reported by surveys conducted in other countries. Further studies are needed to assess the risks and benefits related to the intake of specific laxative ingredients. PMID- 7548265 TI - Quality of care from the elderly person's perspective: subjective importance and perceived reality. AB - The aim of this study was to carry out a theoretically-based survey of elderly persons' perceptions of quality of care, in terms of their evaluations of actual care conditions, as well as the subjective importance they ascribe to these. The sample consisted of 428 elderly (> or = 60 years) persons, in four different care environments: a geriatric department (N = 51); persons receiving home nursing (N = 111); nursing homes (N = 111); and service homes (N = 154) in two Swedish cities. Data were collected by way of personal interviews, structured from the questionnaire, Quality from the Patients' Perspective (QPP). The QPP is theory based and consists of 40 items covering 17 factors. Each item was evaluated in two ways by the respondent: assessment of perceived reality, and evaluation of subjective importance (Likert scales). The results show that personal characteristics, such as age, sex, and self-rated health, had a limited effect on reports of what were regarded as highly important care characteristics, as well as on ratings of actual care conditions. Psychological well-being was strongly related to perceived reality ratings: a favorable well-being covaried with favorable ratings and vice versa. Considerable differences were noted in both types of ratings when the four types of care environments were compared. On the subjective importance scales, persons living in service homes reported lower scores, particularly on the scales designed to measure the medical-technical competence of the caregivers. On the perceived reality scales, the participants in service homes and the geriatric department scored lower.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548264 TI - Is late-life caloric restriction beneficial? AB - Caloric restriction initiated in young mice and rats results in increases in mean and median life span. When caloric restriction is implemented in older animals, an increase in life span is still observed; however, the magnitude of the increase is not as great as that observed in animals calorie restricted since they were young. Here we report the results of a pilot study in which caloric restriction was initiated in mature, older rats. Survival rates and terminal pathology were characterized and compared between a cohort of 17 continually ad libitum fed Long Evans rats and a cohort of 18 Long Evans rats, which were gradually introduced to 33% restriction in diet consumption at 18 months of age. No difference in the median life span was observed between the two groups. The data suggest there may be a level of maturity, or a stage in the aging process, after which caloric restriction no longer increases longevity. PMID- 7548267 TI - Aging, zinc and cell-mediated immune response. PMID- 7548268 TI - Zinc, human diseases and aging. AB - Zinc is one of the most important trace elements in the body for many biological functions; it is required as a catalytic component for more than 200 enzymes, and as a structural constituent of many proteins, hormones, neuropeptides, hormone receptors, and probably polynucleotides. Due to its role in cell division and differentiation, programmed cell death, gene transcription, biomembrane functioning and obviously many enzymatic activities, zinc is considered a major element in assuring the correct functioning of an organism, from the very first embryonic stages to the last periods of life. This biological role together with the many factors that modulate zinc turnover explains on one hand, the variety of clinical and laboratory signs resulting from its reduced bioavailability, and on the other, the high number of human pathologies characterized by alterations in the zinc pool. As zinc supplementation is efficacious in most of these conditions, it is regarded more as an oriented therapeutical support, than a simple dietary integrator. Furthermore, the relevance of zinc status to many age associated diseases and, according to experimental studies, the aging itself of the major homeostatic mechanisms of the body, i.e., the nervous, neuroendocrine and immune systems, places zinc in a pivotal position in the economy of the aging organism. PMID- 7548266 TI - Age-response effectiveness of gallopamil for the treatment of myocardial exertional ischemia. A medium-term randomized cross-over double-blind placebo controlled trial. AB - We evaluated the efficacy and safety of gallopamil 150 mg daily in middle-aged and elderly patients with stable exertional ischemia, using a medium-term randomized double-blind cross-over placebo-controlled trial. Twenty middle-aged patients (52.8 +/- 6 years; range 38-61 years) and 14 elderly patients (67.4 +/- 2.8 years; range 65-73 years) with stable exertional ischemia underwent a bicycle exercise test. After a run-in period, both groups received treatment with either placebo or gallopamil 50 mg tid for 28 days. At the end of this time, each patient crossed over to the alternate regimen. Gallopamil significantly reduced heart rate, blood pressure and rate pressure product (from 15.37 +/- 2.7 to 13.65 +/- 4.16 U x 10(-3); p < 0.01) in elderly patients at submaximal exercise, but had no effect in middle-aged patients (from 14.52 +/- 4.45 to 13.49 +/- 3.77 U x 10(-3); p = NS). At peak exercise, none of the hemodynamic parameters was modified with gallopamil in either group. At peak exercise, both middle-aged and elderly patients achieved rate-pressure products similar to those reached during placebo at higher work loads. Exercise duration and maximal work load significantly increased in both groups. Electrocardiographic signs of ischemia were favorably influenced by gallopamil in both groups (from 1.39 +/- 0.5 mm to 0.76 +/- 0.73 mm; p < 0.001 in the middle-aged patients and from 1.5 +/- 0.34 mm to 1 +/- 0.76 mm; p < 0.01 in the elderly patients).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548269 TI - The British Columbia Continuing Care system: service delivery and resource planning. AB - The Ministry of Health and Ministry Responsible for Seniors in British Columbia, Canada, has developed a comprehensive and integrated service delivery system for the care of the elderly and the disabled. This system has a single point of entry, and contains all of the major components of Long-Term Care and Home Care services under one administrative umbrella, the Continuing Care Division. This paper presents on overview of the Division's service delivery system and its planning framework. The latter provides a vehicle for decision makers to pro actively re-allocate resources from residential services to community and home based services within Continuing Care. PMID- 7548270 TI - Gene therapy in Europe. PMID- 7548272 TI - Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer. AB - The ability of lysosomal enzymes to be secreted and subsequently captured by adjacent cells provides an excellent basis for investigating different therapy strategies in lysosomal storage disorders. Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminidase (AGA) leading to interruption of the ordered breakdown of glycoproteins in lysosomes. As a consequence of the disturbed glycoprotein catabolism, patients with AGU exhibit severe cell dysfunction especially in the central nervous system (CNS). The uniform phenotype observed in these patients will make effective evaluation of treatment trials feasible in future. Here we have used fibroblasts and lymphoblasts from AGU patients and murine neural cell lines as targets to evaluate in vitro the feasibility of enzyme replacement and gene therapy in the treatment of this disorder. Complete correction of the enzyme deficiency was obtained both with recombinant AGA enzyme purified from CHO-K1 cells and with retrovirus-mediated transfer of the AGA gene. Furthermore, we were able to demonstrate enzyme correction by cell-to-cell interaction of transduced and nontransduced cells. PMID- 7548271 TI - Trans-complementation of E1-deleted adenovirus: a new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses. AB - Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. To achieve this goal, replication-deficient (E1-) adenoviruses (Ad) are promising vectors. We have previously demonstrated efficient CFTR gene delivery to the airways of cotton rats and rhesus monkeys using a replication-deficient adenovirus, Ad-CFTR. Here, we have investigated an important safety issue, the interaction between the vector and wild-type virus which can provide the missing E1 function in trans. We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats. However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited. To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome. We demonstrate that when cells are coinfected with wild-type virus and the new attenuated vector, the viral DNA containing the natural encapsidation sequences is preferentially packaged, leading to a rapid dilution of the recombinant virus. PMID- 7548273 TI - A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers. AB - Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue. PMID- 7548274 TI - Transduction of genes coding for a histocompatibility (MHC) antigen and for its physiological inducer interferon-gamma in the same cell: efficient MHC expression and inhibition of tumor and metastasis growth. AB - The mouse mammary carcinoma TS/A, of BALB/c (H-2d) origin, was transfected with the murine interferon-gamma (IFN-gamma) gene (Int. J. Cancer 55: 320, 1993). We used IFN-gamma transfectants as recipients for a second round of transfections with murine allogeneic class I histocompatibility (H-2b) genes that are modulated by IFN. Transfectants with either gene alone, as well as parent TS/A cells (TS/A pc), were used as controls. Only double transfectants expressed high levels of the allogeneic H-2b genes, while in H-2b single transfectants the expression was very low (but was induced by treatment with exogenous IFN-gamma). The tumorigenic potential of IFN-gamma or H-2b single transfectants was reduced in comparison to TS/A-pc. IFN-gamma+H-2Kb double transfectants were almost nontumorigenic, while IFN-gamma+H-2Db clones gave rise to tumors in about one-half of mice. The experimental metastatic ability of all IFN-gamma+H-2b double transfectants was very low. IFN-gamma single transfectants were known to induce a strong macrophage response in the host. The expression of allogeneic H-2 antigens added a T lymphocyte-mediated response that accounted for the lower tumorigenicity of double transfectants. These results show that it is possible to steer the immune response evoked by tumor cells for therapeutic purposes. Moreover, the high H-2 expression obtained in IFN-gamma+H-2b double transfectants suggests that single IFN-gamma transfectants are ideal recipients for all IFN-sensitive genes. This approach can be used also for other general-purpose inducers of gene expression. PMID- 7548275 TI - Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex. AB - Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%). PMID- 7548276 TI - The "bystander effect": association of U-87 cell death with ganciclovir-mediated apoptosis of nearby cells and lack of effect in athymic mice. AB - Cells expressing the herpes simplex-thymidine kinase (HS-TK) gene as a consequence of retroviral transduction, as well as TK-negative (TK-) bystander cells, can be killed by treatment with ganciclovir (GCV). In vitro, this "bystander effect," has been attributed to metabolic cooperation through gap junctions or to the uptake of apoptotic vesicles. We show that GCV treatment kills TK-negative U-87 glioma cells cocultured with cells that express TK (TK+) but that have lost the capacity for releasing retroviral particles. A photometric enzyme immunoassay identifies histone-associated DNA fragments, typical of apoptosis, in the cytosol of GCV-treated TK+ cells, and apoptotic features are also demonstrated by ultrastructural studies. Northern blot analysis and the reverse transcription polymerase chain reaction (PCR) show that connexin 43, a major constituent of gap junctions, is expressed in TK+ and U-87 cells. The size of U-87 tumors in nude mice subsequently injected with TK+ cells and GCV is not significantly different than in untreated animals; whereas, after injecting 1:1 mixtures of U-87 and TK+ cells, GCV treatment only causes a temporary regression of tumor growth. On the contrary, when the injected mixtures contain PA317.STK.SBA (a retroviral producer cell line that can transduce efficiently the HS-TK gene) and U-87 cells, tumors are destroyed effectively by GCV treatment. Thus, an experimental setting in which U-87 gliomas are matched with cells that are able to express, but not to transduce, the HS-TK gene indicates that the bystander effect kills U-87 cells in vitro by mechanisms associated with apoptotic death. In vivo, this effect is not sufficient to restrain the tumor growth taking place in immunodeficient animals. PMID- 7548277 TI - Overview of regulation of gene therapy in Europe: a current statement including reference to US regulation. PMID- 7548278 TI - Report on the 2nd annual meeting of the European Working Group on Human Gene Transfer and Therapy, London, November 18-21, 1994. PMID- 7548279 TI - International perceptions and approval of gene therapy. AB - Gene therapy is in clinical trials in a number of countries, raising the question of whether different ethical standards can be justified in different countries. One key issue is how divergent are the perceptions and bioethical reasoning of peoples around the world. An International Bioethics Survey with 150 questions, including 35 open ones, was developed to look at how people think about diseases, life, nature, and selected issues of science and technology, biotechnology, genetic engineering, genetic screening, and gene therapy. The mail response survey was conducted in 1993 among the public in Australia, India, Israel, Japan, New Zealand, Russia, and Thailand, and the same written survey was conducted among university students in Australia, Hong Kong, India, Japan, New Zealand, The Philippines, Russia, Singapore, and Thailand. Similar questions were included in an international high school education bioethics survey among high school teachers in Australia, Japan, and New Zealand. Further international comparisons to the United States and Europe are made. About three-quarters of all samples supported personal use of gene therapy, with higher support for children's use of gene therapy. The diversity of views was generally similar within each country. The major reasons given were to save life and increase the quality of life. About 5-7% rejected gene therapy, considering it to be playing God, or unnatural. There was very little concern about eugenics (0.5-2%), and more respondents gave supportive reasons like "improving genes," especially in Thailand and India. Support for specific applications was significantly less for "improving physical characters," "improving intelligence," or "making people more ethical" than for curing diseases like cancer or diabetes, but there was little difference between inheritable or noninheritable gene therapy. PMID- 7548280 TI - Gene therapy of human melanoma. Immunization of patients with autologous tumor cells admixed with allogeneic melanoma cells secreting interleukin 6 and soluble interleukin 6 receptor. PMID- 7548283 TI - Surveillance of antibiotic resistance in Neisseria gonorrhoeae and implications for the therapy of gonorrhoea. PMID- 7548282 TI - The third wave: HIV infection among heterosexuals in the United States and Europe. PMID- 7548281 TI - Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. AB - The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548284 TI - Management of protozoan infections in AIDS. The Jefferiss Wing Therapeutics and Protocols Group. PMID- 7548285 TI - Are you sure it's syphilis? A review of false positive serology. AB - This article on false positive serological reactions for syphilis reviews the rapid developments which have taken place in the serodiagnosis of syphilis in recent years since the advent of the AIDS epidemic. An overview of non-specific and specific treponemal serological tests in relation to acute and chronic biological false positive reactions is followed by closer consideration of syphilis serology in the context of HIV infection, pregnancy and other conditions which may produce false positive reactions. PMID- 7548286 TI - 'Grey case'. PMID- 7548287 TI - Testing for HIV antibody: a comparison of two services offered in a genitourinary medicine clinic. AB - Rates of screening for sexually transmissible infections in patients using different services provided by a genitourinary medicine clinic for testing for HIV antibodies are presented. Those patients whose primary reason for attending the clinic was HIV antibody testing and used the same day result (SDR) service were significantly less likely to be screened for other infections than those using the normal waiting time (NWT) service, (P < 0.00001). This was true for both males and females. Of those patients screened for other infections in the SDR and NWT groups 29% and 35% respectively were found to have a sexually transmitted infection. It would appear that an SDR service offers little benefit for the majority of patients as only a few patients would not have had an HIV antibody test had the SDR not been available. PMID- 7548289 TI - Cervical cytological screening in HIV-infected women in Dublin--a six-year review. AB - A retrospective study of the results of cervical cytological screening of HIV infected women attending an inner city ambulatory HIV clinic over a 6-year period between 1987 and 1992 was carried out. During this time a total of 165 HIV infected women attended for management of their HIV disease. The results of cervical cytological specimens (smears) were available in 136 (82.4%) women. The risk categories for HIV infection of these 136 women were intravenous drug use 110 (80.9%), heterosexual sex 24 (17.6%) and undetermined 2 (1.5%). Eighty-five (62.5%) of the 136 women were classified CDC group 2, 30 (22%) CDC group 3, and 21 (15.5%) CDC group 4 at the time of initial cytological screening. Forty-one (30.1%) women had mild dysplasia/CIN 1, 21 (15.4%) had moderate dysplasia/CIN 2 and 17 (12.5%) had severe dysplasia/CIN 3. The overall prevalence of dysplasia/CIN was 58.1%. Twenty-seven (34.2%) of the women with dysplasia/CIN had cytological evidence of human papillomavirus infection. No association between the clinical stage of HIV disease and the presence or degree of dysplasia/CIN was demonstrated. Women with cytological evidence of CIN were significantly more likely to have had genital warts than those with no evidence of CIN (OR 3.1, CI 1.1-10). In those women with cervical dysplasia who underwent colposcopic examination, CIN was confirmed in a high proportion of cases. The default rate from colposcopy, however, was high (35.4%). PMID- 7548288 TI - Self-treatment of female external genital warts with 0.5% podophyllotoxin cream (Condyline) vs weekly applications of 20% podophyllin solution. AB - Sixty women with genital warts were randomly allocated to treatment with either weekly application of 20% podophyllin solution or self-treatment with 0.5% podophyllotoxin cream twice daily for three days in weekly intervals. After a maximum of 4 treatment cycles a final assessment was carried out after 3 months. Primary clearance after termination of treatment was 82% for podophyllotoxin and 59% for podophyllin solution. After excluding relapses at the 3-month follow-up, final clearance for podophyllotoxin (71%) was significantly better (P < 0.05) than that for podophyllin solution (48%). The total frequency of warts eradicated was 94% with podophyllotoxin and 74% with podophyllin solution (P < 0.001). Local adverse effects were generally mild or moderate. Podophyllotoxin cream provides a mode of easy application for women with external genital warts and had in this study a significantly better effect than podophyllin solution. PMID- 7548290 TI - The activity of candidate virucidal agents, low pH and genital secretions against HIV-1 in vitro. AB - The effect of low pH, normally present in the female genital tract, on HIV viability was examined. HIV is more acid stable than previously reported with no substantial reduction in infectivity occurring until pH levels are reduced below 4.5. The virucidal activity of 3 topical spermicides and chlorhexidine was assessed in vitro using previously established and newly modified assay systems. None of the agents tested had a selectivity index (SI) greater than 5.2. Semen and cervical secretions were assessed for their ability to inhibit HIV-1. While no virucidal effect was found in the latter, seminal fluid was found to have significant activity against HIV-1 and a SI of approximately 50. PMID- 7548291 TI - Sexually transmitted diseases and human immunodeficiency virus infection among women with genital infections in Burkina Faso. AB - This study reports the prevalence of sexually transmitted diseases (STDs) among gynaecological outpatients presenting at the Bobo-Dioulasso Hospital (Burkina Faso) with genital infections and examines the factors associated with HIV infection in this population. Of 245 eligible non-pregnant women, 220 consented to participate in the study. Seventy-seven per cent had sexually transmitted infections. The most common were: Trichomonas vaginalis (28%), Chlamydia trachomatis (27%), bacterial vaginosis (20%), Candida albicans (17%), Neisseria gonorrhoeae (11%). The prevalence of HIV infection was 42% (95% c.i. 35.3, 48.3). Logistic regression analyses revealed Neisseria gonorrhoeae to be the only STD significantly associated with infection with HIV (P = 0.04). A sedimentation rate greater than or equal to 100 mm in the first hour was also associated with HIV infection (P < 0.001). Women consulting for genital infections constitute a high risk group for HIV infection and other STDs. Management of these women should focus on the early diagnosis and treatment of STDs. PMID- 7548293 TI - HIV seroprevalence in healthy blood donors in northeastern Zaire. AB - Despite high seroprevalence rates in some parts of Africa, there is notable variation in prevalence between population subgroups. To document changes and trends in HIV seroprevalence in northeastern Zaire, 1989 to 1992 blood donor data were reviewed. Overall, 2453 donors were tested with seropositivity varying from 2.8% in 1989 to 6.9% in 1992. The increase in seropositivity was significant for men (2.5 to 5.8%, P = 0.017) and for people residing in rural areas (2.0 to 6.1%, P = 0.0008) but not for women (5.4 to 8.6%, P = 0.15) nor for urban individuals (10.5 to 8.6%, P = 0.55). These findings suggest that: 1) HIV infection is spreading in previously less-affected population subgroups rather than increasing widely in the entire population, 2) the HIV epidemic could be reaching a plateau or endemic phase in northeastern Zaire, and, 3) continued blood donor screening and wise transfusion practices are needed. PMID- 7548292 TI - Clinical features and serum beta 2-microglobulin levels in HIV-1 positive and negative Tanzanian patients with tuberculosis. AB - Serum beta 2-microglobulin (beta 2M) rises in the later stages of HIV disease and has therefore been used to monitor progression to AIDS. However, little work has been done on patients co-infected with HIV and tuberculosis. We studied clinical features and serum beta 2-M in 35 Tanzanian patients treated for pulmonary tuberculosis (9 HIV-positive, 26 HIV-negative). The provisional WHO clinical definition of AIDS for use in Africa was fulfilled by 89% of the HIV-positive and 65% of the HIV-negative patients. Median serum beta 2-M on admission was slightly higher in HIV-positive (3.17 mg/l) than in HIV-negative (2.85 mg/l) patients. Serum beta 2-M fell during treatment in 17/24 (71%) of HIV-negative and 3/7 (43%) HIV-positive patients followed up for 6 months. We conclude that serum beta 2-M is frequently raised in active tuberculosis, and is therefore an unreliable indicator of the stage of HIV disease in co-infected patients. The WHO clinical definition of AIDS also proved unreliable in patients with tuberculosis. PMID- 7548294 TI - Salmonella typhimurium empyema in an AIDS patient. PMID- 7548295 TI - Kaposi's sarcoma in the kidney. PMID- 7548296 TI - Routine screening for Chlamydia trachomatis urethral infections in men. PMID- 7548297 TI - Chlamydial urethritis in heterosexual men attending a genitourinary medicine clinic: prevalence, symptoms, condom usage and partner change. PMID- 7548298 TI - Antenatal attenders and HIV testing. PMID- 7548299 TI - [Cyclospora: a pathogenic parasite in humans]. PMID- 7548300 TI - [Hereditary diseases and congenital malformations at the Unit of Medical Genetics of the University of Zulia. Years: 1983-1992]. AB - The Medical Genetics Unit at Universidad del Zulia (UGM-LUZ) gives counsel to patients with partial and total genetic diseases. Counseling is available for patients of both sexes and all ages, from public and private health centers and several medical specialities. In the present study an analysis of 4617 clinical records from families referred for genetic counseling to the UGM-LUZ is given. The study spans from January 1983 to December 1992. Fifty four (1.2%) of these histories correspond to pre-nuptial counseling, 773 (16.7%) pre-conceptional, 316 (6.8%) pre-natal and 3474 (75.3%) for diagnosis. A computerized system was developed, based on relational data base manager, that permits access with interactive Dbase type applications. A total of 5433 diagnoses were made. The most frequent causes of genetic diseases were chromosomal abnormalities (12.32%), mainly Down and Turner syndromes. Mendelian diseases occupied 14.45% of all cases, with Marfan and Noonan syndrome, Osteogenesis imperfecta. Duchenne-Becker muscular dystrophy and Incontinentia Pigmenti as the most frequent syndromes. Diseases that involve multifactorial inheritance, such as neural tube defects, accounted for 7.36% of all diagnosis. Effects of teratogenic agents such as german measles, radiations and others were detected in 3.96% of all cases. In 8.5% of the patients a hereditary factor was suspected. No definitive diagnosis was reached in 32.45% of all cases and 20.96% of the patients were normal. The need for data from other medical genetic centers is stressed. In this way the regional and national genetic diseases on morbidity can be known. PMID- 7548302 TI - [Role of environmental factors in the development of insulin-dependent diabetes mellitus (IDDM) in Venezuelan children]. AB - In view of the controversy surrounding the role of environmental factors, such as the presence of bovine albumin in milk, or viral infections, in the etiology of IDDM, a study was undertaken to determine the relationship between these events and the subsequent risk of developing IDDM. On 40 venezuelan diabetic children (< 18 y) and forty, age, sex and race-matched controls were studied at the same time. Parents of children completed a questionnaire on the infant's feeding habits, its environment and family history. The X2 method and the Fischer's exact test were used to analyze the results. We found that 20% of the controls, and 10% of IDDM (NS), were never breast-fed. In 95% of controls vs 65% of IDDM (p < 0.001), cow's milk was given exclusively from birth, or combined with breast feeding, 65% of IDDM and 60% of controls (NS) were breast-fed (alone or combined with milk substitutes) for more than three months. These results do not support the hypothesis that early exposure to breast milk substitutes increases the risk of IDDM in venezuelan children. The study revealed, however, that a family history of diabetes mellitus was present in 55% of IDDM vs 30% of controls (p < 0.05) and mumps infection before the onset of diabetes was recorded in 42.5% of IDDM in comparison with 12.5% of controls (p = 0.005). Other viral infections (rubella, chicken pox) had no statistical significance. The latter results suggest an association between a family history of diabetes mellitus and viral infections with the development of IDDM among this group of children. PMID- 7548301 TI - Low urinary dopamine excretion associated to low sodium excretion in normotensive Piaroa Amazonian ethnia compared to urban subjects. AB - The objective of this work was to compare urinary dopamine, noradrenaline, adrenaline, sodium and potassium excretion in a group of normotensive Piaroa Amazonic ethnia who do not use salt in their regular food intake, against a group of urban normotensive citizens known to have a high salt intake in their regular meals. Twenty adult normotensive Piaroa subjects living in the Amazonas forest, 11 men and 9 women, 23-72 years old, and 33 normotensive urban citizens, 25-70 years old, 17 men and 17 women, were included in the study. After a 10 min. rest, an average of three supine systolic (SBP) and diastolic (DBP) blood pressure recordings was obtained. Piaroas subjects SBP and DBP were 111.3 +/- 2.9 mmHg and 62.7 +/- 1.9 mmHg respectively; urban subjects SBP and DBP were 111.8 +/- 2.2 mmHg and 70.3 +/- 1.6 mmHg respectively. Supine heart rate was lower in Piaroas (58.0 +/- 1.8 beats/min) than in urban subjects (76.5 +/- 1.9 beats/min), p < 0.05. Sodium urinary excretion was much lower in Piaroas (12.6 +/- 5.2 mmol/24 h) when compared to urban subjects (210.7 +/- 24.5 mmol/24 h), p < 0.01. No difference was found in daily urinary potassium excretion between Piaroas and urban subjects (50.4 +/- 7.2 mmol/24 h vs 45.1 +/- 7.4 mmol/24 h). Urinary dopamine excretion was lower in Piaroas (314.7 +/- 40.1 micrograms/24 h) in comparison to urban subjects (800.4 +/- 59.2 micrograms/24 h), p < 0.05. Daily urinary noradrenaline and adrenaline excretion were 67.9% and 85.4% respectively lower in Piaroas than in urban subjects. In conclusion, lower amounts of sodium daily intake are associated to lower kidney dopamine production in Piaroas as compared to urban subjects. Apparently indigenous tribes might require less kidney dopamine synthesis to excrete the very small amounts of salt they consume in their regular food intake. The opposite was found in urban subjects; more kidney dopamine synthesis would be required for larger amounts of urinary sodium excretion. In this population, essential hypertension has been associated to a failure of the natriuretic mechanism triggered by dopamine onkidney tubules. PMID- 7548303 TI - [Effects of the administration of vitamin K on the activity of Factor II, VII and X in healthy newborns]. AB - The study was designed to know the effect of oral vitamin K (VK) treatment, on clotting factors II-VII-IX-X and the protein induced by VK absence from factor II (PIVCA II) on full term infants. Seventy healthy newborns were studied and each was randomly placed in one of two groups: Group A, newborns that received human milk and milk formula (mixed feeding)and group B, newborns that were exclusively breast fed. These groups were also divided in two subgroups: I received 2mg of VK1 orally and II (control) did not receive VK. Clotting activity of the coagulation factors and PIVCA II was determined from blood plasma obtained immediately after birth and 48 hours after VK administration. Basal activity of the factors analyzed was similar in all groups with values ranging from 25% to 40%. After 48 hours a significant increase in all factors studied and a decrease of PIVKA II was observed in those children who received oral VK. The results suggest that oral VK effectively increases VK dependent factors and prevents the risk of hemorrhagic disease in the newborn, with the advantage of being less traumatic and less risky to the infant than intramuscular VK. PMID- 7548304 TI - [Cervical intraepithelial neoplasia: Chlamydia trachomatis and other co-factors]. AB - The incidence of Chlamydia trachomatis (Ct) in patients with diagnosis of Cervical Intraepithelial Neoplasia (CIN) was studied in one hundred eighty patients. The Chlamydiazyme test was performed in all of them. Endocervical samples were taken from 103 patients with CIN and 77 women who sought medical attention for different gynecological reasons (CG). Twenty three tests (12.8%) were positive; 15 of them had CIN (14.6%) and 8 were from the control group (10.4%). It was found a statistical significant difference between NIC III and early intercourse, NIC III and age, NIC and vaginal douches and, among NIC and number of pregnancies and deliveries. There was not a significant difference among Ct and early intercourse, number of sexual partners, pregnancies, deliveries, vaginal douches, oral contraceptives (OC), and vaginal discharge. No statistically significant differences were found between NIC and number of sexual partners, and OC and vaginal discharge. The low incidence of Ct in patients with CIN does not mean that Ct does not play a role in the origin and development of the cervical pathology. PMID- 7548305 TI - [Hemoglobin and nutrient concentration in middle-class adolescents. Relationship with school performance]. AB - Concentrations of hemoglobin and serum nutrients that participate in the erythropoiesis (iron, folic acid and vitamin B12) were studied in 213 adolescents (112 male, 101 female) belonging to a medium income group that assisted at a private secondary educational institution. The purpose of the present work was to observe the hematologic and nutrient status in this group and its relationship with the academic achievement. The level of academic performance was determined using the Final mean grades (0 to 20 points scale) and the Academic Achievement index (A.A.I.) calculated as the ratio of approved courses over the total. A high prevalence of iron (16.6%) and folate (14.2%) deficiency was found in the total group with predominance in the female adolescents. It was noted a relationship between anemic men and A.A.I. (p < 0.05) and no other difference was observed between individuals with nutritional deficiency and their academic achievement. Therefore, when it was established as cut-off point 20 micrograms/L for ferritin, a good correlation was found between iron status and academic achievement index (r = 0.411, p < 0.01). The hematologic and biochemical data were correlated with academic parameters. A positive correlation was obtained between A.A.I. and hemoglobin, total iron binding capacity (TIBC) in males. When using Final mean grades, a positive correlation was shown with folic acid (males) and vitamin B12 (females). This work reveals a high incidence of iron and folate deficiency, specially in the female adolescent group. These findings may be due to a inadequate dietary intake combined with an increase of nutritional requirements and probably parasitic infestation. These factors may contribute to an impairement of the academic achievement. It will be important the assessment of iron and folate status of the adolescent for the normal and integral development of his cognitive and psychomotor functions. PMID- 7548306 TI - [Dyslipidemia and hyperinsulinemia in normoglycemic-obese relatives of patients with non-insulin dependent diabetes mellitus]. AB - To determine the most frequent dyslipidemias among first-degree relatives of NIDDM patients, and its association with their glucose-tolerance status and hyperinsulinemia, we have started to examine members of NIDDM pedigrees, according to American Diabetes Association guidelines for nuclear family studies. In a large family with 2 NIDDM siblings in the 2nd generation, and 4 siblings with NIDDM in the 3rd generation, we have evaluated 14 first degree relatives and also 15 sex and aged matched healthy control subjects without family history of diabetes. The NIDDM relative group presented BMI = 31.8 +/- 3.9 kg/m2, SBP = 128 +/- 18.2 mmHg, DBP = 84 +/- 12.7 mmHg. Both relatives and controls were subjected to a 2h 75g OGTT for glucose and insulin determinations. Although none of NIDDM relatives has IGT, both Glycemic Area (GA) and Insulin Area (IA) were greater (p < 0.01) in the NIDDM relative group. The Insulin/Glucose ratio was also higher (p < 0.01) at 0 and 120 min of OGTT, this might be indirect evidence of Insulin- Resistance. Fasting serum lipids in the NIDDM relatives were TG = 148 +/- 24mg/dl, T-Chol = 244 +/- 10.7mg/dl, HDL-C = 34.2 +/- 2.5mg/dl; lipids in the control group were TG = 84.8 +/- 10.1mg/dl, T-Chol = 167 +/- 10.2mg/dl, HDL-C = 44.4 +/- 2.6mg/dl. Electrophoretic pattern showed type IIa (30.7%) and IIb (61.5%) hyperlipidemias in the NIDDM relatives. In this group, there was a positive and significant association between basal insulin and DBP (r = 0.67; p < 0.01), and between DBP and both TG (r = 0.74; p < 0.01)) and VLDL-C (r = 0.58; p < 0.05). It was also obtained a negative association between basal insulin and HDL-C (r = -0.89; p < 0.001). These data suggest that hyperinsulinemia in association with lipid abnormalities could appear early (before the development of Impaired Glucose Tolerance and Diabetes) in first degree relatives of NIDDM patients. PMID- 7548308 TI - Myasthenia gravis beginning during pregnancy, partum or puerperium: a distinct clinical entity. PMID- 7548307 TI - [Gliomatosis cerebri. Ante-mortem diagnosis]. AB - Gliomatosis Cerebri is an unusual neoplastic entity that affects the central nervous system (C.N.S.); it consists of a marked proliferation of glial cells with variable degrees of differentiation in extensive areas of the brain and/or spinal cord. The process is primarily an infiltrative one rather than destructive. Most of the cases are diagnosed at autopsy. The authors report a new case of Gliomatosis Cerebri in a 46 year old woman, with a long clinical history- 15 years--of focal seizures; and during the year prior to her admission: behavioral changes, vertigo, urinary incontinence and frequent falls. C.N.S. imaging revealed extensive involvement of the white matter of both cerebral hemispheres. Stereotactic guided biopsy showed a moderately cellular astrocytic neoplasm. A review of the medical literature led to the conclusion that the present case constitutes one of the very few examples in which the diagnosis of Gliomatosis Cerebri was achieved ante-mortem. PMID- 7548309 TI - Kinetic models of odor transduction implemented as artificial neural networks. Simulations of complex response properties of honeybee olfactory neurons. AB - We present a formal model of olfactory transduction corresponding to the biochemical reaction cascade found in chemosensory neurons. It assumes that odorants bind to receptor proteins which, in turn, activate transducer mechanisms corresponding to second messenger-mediated processes. The model is reformulated as a mathematically equivalent artificial neural network (ANN). To enable comparison of the computational power of our model, previously suggested models of chemosensory transduction are also presented in ANN versions. In ANNs, certain biological parameters, such as rate constants and affinities, are transformed into weights that can be fitted by training with a given experimental data set. After training, these weights do not necessarily equal the real biological parameters, but represent a set of values that is sufficient to simulate an experimental set of data. We used ANNs to simulate data recorded from bee subplacodes and compare the capacity of our model with ANN versions of other models. Receptor neurons of the nonpheromonal, general odor-processing subsystem of the honeybee are broadly tuned, have overlapping response spectra, and show highly nonlinear concentration dependencies and mixture interactions, i.e., synergistic and inhibitory effects. Our full model alone has the necessary complexity to simulate these complex response characteristics. To account for the complex response characteristics of honeybee receptor neurons, we suggest that several different receptor protein types and at least two second messenger systems are necessary that may interact at various levels of the transduction cascade and may eventually have opposing effects on receptor neuron excitability. PMID- 7548310 TI - On the parameter estimation for diffusion models of single neuron's activities. I. Application to spontaneous activities of mesencephalic reticular formation cells in sleep and waking states. AB - For the Ornstein-Uhlenbeck neuronal model a quantitative method is proposed for the estimation of the two parameters characterizing the unknown input process, namely the neuron's mean input per unit time mu and the infinitesimal standard deviation per unit time sigma. This method is based on the experimentally observed first- and second-order moments of interspike intervals. The dependence of the estimates mu and sigma on the moments of the observed interspike intervals and on the neuronal parameters is clarified, and a comparison is made between the estimates based on the classical Wiener model and those yielded by the Ornstein Uhlenbeck model. Comprehensive tables are included in which the displayed values of mu and sigma have been calculated in terms of physiologically realistic pairs of first- and second-order moments. Our method is finally applied to interspike interval data recorded from neurons in the mesencephalic reticular formation of the cat during hypothetical sleep, slow-wave sleep stage, and wake stage. PMID- 7548311 TI - Limitations of the surface electromyography technique for estimating motor unit synchronization. AB - Motor unit synchronization was estimated from the surface electromyograms (EMG) of the first dorsal interosseus muscle of human volunteers by a simplified surface-EMG technique (Milner-Brown et al. 1973, 1975). Single motor units were identified from intramuscular recordings and were used to obtain a spike triggered average of the surface-EMG. The discharge rate of a reference motor unit was controlled at two levels (high and low), and the effect of motor unit activity on the surface-EMG estimate of synchronization was studied in 56 motor units. The surface-EMG estimate of motor unit synchronization was significantly higher when the reference motor unit discharged at the high rate than when it discharged at the low rate. A regression analysis indicated that the synchronization ratio calculated from the surface EMG was significantly correlated with the level of EMG activity in the muscle. Motor unit synchronization was also estimated from surface-EMG measurements that were derived by computer simulation. The simulation permitted manipulation of motor unit activity (discharge rate and recruitment) with a complete absence of synchrony among the units in the pool. The stimulated surface-EMG index was influenced by an artifact associated with signal rectification, and this effect changed non-monotonically with motor unit activity. Furthermore, the increase in the motor unit activity reduced the signal-to-noise ratio of the spike-triggered surface EMG average, and consequently decreased the sensitivity of the surface EMG index as an estimate of motor unit synchronization. We conclude that the simplified surface-EMG method (Milner-Brown et al. 1973, 1975) does not provide a useful index of motor unit synchronization due to its inability to accurately distinguish the synchronization from methodological effects related to a rectification artifact and variation in the signal-to-noise ratio. PMID- 7548312 TI - Development of simple fitness landscapes for peptides by artificial neural filter systems. AB - The applicability of artificial neural filter systems as fitness functions for sequence-oriented peptide design was evaluated. Two example applications were selected: classification of dipeptides according to their hydrophobicity and classification of proteolytic cleavage-sites of protein precursor sequences according to their mean hydrophobicities and mean side-chain volumes. The cleavage-sites covered 12 residues. In the dipeptide experiments the objective was to separate a selected set of molecules from all other possible dipeptide sequences. Perceptrons, feedforward networks with one hidden layer, and a hybrid network were applied. The filters were trained by a (1, lambda) evolution strategy. Two types of network units employing either a sigmoidal or a unimodal transfer function were used in the feedforward filters, and their influence on classification was investigated. The two-layer hybrid network employed gaussian activation functions. To analyze classification of the different filter systems, their output was plotted in the two-dimensional sequence space. The diagrams were interpreted as fitness landscapes qualifying the markedness of a characteristic peptide feature which can be used as a guide through sequence space for rational peptide design. It is demonstrated that the applicability of neural filter systems as a heuristic method for sequence optimization depends on both the appropriate network architecture and selection of representative sequence data. The networks with unimodal activation functions and the hybrid networks both led to a number of local optima. However, the hybrid networks produced the best prediction results. In contrast, the filters with sigmoidal activation produced good reclassification results leading to fitness landscapes lacking unreasonable local optima. Similar results were obtained for classification of both dipeptides and cleavage-site sequences. PMID- 7548313 TI - General relation between variance-time curve and power spectral density for point processes exhibiting 1/f beta-fluctuations, with special reference to heart rate variability. AB - Counting statistics in the form of the variance-time curve provides an alternative to spectral analysis for point processes exhibiting 1/f beta fluctuations, such as the heart beat. However, this is true only for beta < 1. Here, the case of general beta is considered. To that end, the mathematical relation between the variance-time curve and power spectral density in the presence of 1/f beta-noise is worked out in detail. A modified version of the variance-time curve is presented, which allows us to deal also with the case beta > or = 1. Some applications to the analysis of heart rate variability are given. PMID- 7548314 TI - Complex sensory-motor sequence learning based on recurrent state representation and reinforcement learning. AB - A novel neural network model is presented that learns by trial-and-error to reproduce complex sensory-motor sequences. One subnetwork, corresponding to the prefrontal cortex (PFC), is responsible for generating unique patterns of activity that represent the continuous state of sequence execution. A second subnetwork, corresponding to the striatum, associates these state-encoding patterns with the correct response at each point in the sequence execution. From a neuroscience perspective, the model is based on the known cortical and subcortical anatomy of the primate oculomotor system. From a theoretical perspective, the architecture is similar to that of a finite automaton in which outputs and state transitions are generated as a function of inputs and the current state. Simulation results for complex sequence reproduction and sequence discrimination are presented. PMID- 7548315 TI - A distributed-parameter model of the myelinated human motor nerve fibre: temporal and spatial distributions of action potentials and ionic currents. AB - A double cable model of the myelinated human motor nerve fibre is presented. The model is based on the nodal and internodal channels in a previous, two-component model of human motor axons (Bostock et al. 1991), added to a complex extended cable structure of nodal, paranodal and internodal segments. The model assumes a high-resistance myelin sheath and a leakage pathway to the internodal axolemma via the paranodal seal resistance and periaxonal space. The parameter values of the model were adjusted to match the recordings of threshold electrotonus in human motor fibres from Bostock et al. (1991). Kirchoff's current law was used to derive a system of partial differential equations for the electrical equivalent circuit, and numerical integration was performed with a fixed time increment and non-uniform spatial step sizes, in accordance with the complex structure of the fibre. The model calculations provide estimates of the spatial and temporal distributions of action potentials and their transaxonal and transmyelin components, both in different segments of the fibre and at different moments during action potential propagation. The distribution of transaxonal and transmyelin currents along the fibre and their contributions from different ionic channels are also explored. PMID- 7548316 TI - Integro-differential equations and the stability of neural networks with dendritic structure. AB - We analyse the effects of dendritic structure on the stability of a recurrent neural network in terms of a set of coupled, non-linear Volterra integro differential equations. These, which describe the dynamics of the somatic membrane potentials, are obtained by eliminating the dendritic potentials from the underlying compartmental model or cable equations. We then derive conditions for Turing-like instability as a precursor for pattern formation in a spatially organized network. These conditions depend on the spatial distribution of axo dendritic connections across the network. PMID- 7548317 TI - The ventricular to pulmonary artery connection. Introduction. PMID- 7548318 TI - Uses of homograft conduits for right ventricle to pulmonary artery connections in the neonatal period. AB - A significant number of congenital heart defects share the common characteristic that surgical interventions to either "repair" or palliate these defects generally require the use of nonautologous conduits to substitute for an absent right ventricle to pulmonary artery connection. Tetralogy of Fallot with pulmonary atresia, truncus arteriosus, transposition of the great arteries with ventricular septal defect and left ventricular outflow tract obstruction, and certain forms of double-outlet right ventricle are among the defects in which these conduits are used to create a pathway from the right ventricle to the pulmonary arteries. Such a conduit should ideally have growth potential, be nonthrombogenic, and function indefinitely. No such conduit has yet been devised, although efforts continue to be made with "tissue engineering" techniques to develop a conduit with one or more of these characteristics. In addition, when these conduits are used in the neonate, other characteristics including stiffness, conduit bulk, ease of passing sutures, and porosity are critically important to the success of a neonatal repair. We have found homografts to be the conduits which currently best fulfill the physical and handling requirements for neonatal applications in patients with defects in which autologous vascular tissue cannot easily make up for an absent right ventricle to pulmonary artery connection. The current discussion is therefore focused on the use of homograft conduits interposed between the right ventricle and pulmonary artery as part of neonatal reparative operations. PMID- 7548319 TI - Performance of right ventricle to pulmonary artery conduits after repair of truncus arteriosus: a comparison of Dacron-housed porcine valves and cryopreserved allografts. AB - Repair of complex congenital heart defects often requires the use of extracardiac conduits. These repairs can be technically accomplished with excellent early results. However, the long-term performance of various conduits is less than optimal. In this report, we examined the long-term outcome of xenograft valved Dacron conduits and cryopreserved allograft valved conduits used for reconstruction of the right ventricular outflow tract in young patients with truncus arteriosus. A retrospective review was performed on 222 patients who underwent primary surgical repair of truncus arteriosus between January 1975 and December 1994 using a xenograft valved synthetic conduit (group I; n = 175) or with a cryopreserved valved allograft conduit (group II; n = 47). Median age at repair was 121 days in group I and 70 days in group II; median weight of patients in both groups was the same (4.2 kg). Conduit-related early deaths occurred in 5.7% (10/175) of patients in group I and none in group II. In a cohort of age matched patients, actuarial freedom from conduit-related reintervention at 5 years follow-up was significantly better (P = .037) for the cryopreserved valved allograft conduits when compared with xenograft valved synthetic conduits. However, this difference was not apparent by 7 years. Multivariate analysis showed conduit size (P = .0005) as a significant predictor of early conduit related reintervention. This study shows that mid-term performance of cryopreserved allografts (which have the advantage of technical ease of insertion of availability of small sizes for use in neonates), is better than the xenograft Dacron conduits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548320 TI - Congenitally corrected transposition of the great arteries: ventricle to pulmonary artery connection strategies. AB - Congenitally corrected transposition of the great arteries is an uncommon congenital heart defect that may result in symptoms by virtue of associated cardiac anomalies. Ventricular septal defect, obstruction to pulmonary blood flow, tricuspid valve regurgitation, and conduction abnormalities may be found alone or in combination. In the presence of significant obstruction to pulmonary blood flow, repair generally requires the use of an extracardiac conduit from the morphologic left (pulmonary) ventricle to the pulmonary arteries in order to avoid surgically induced complete heart block. A combined atrial inversion by the Mustard or Senning technique and Rastelli operation has been applied in order to use the morphologic left ventricle in the systemic circulation, particularly when important systemic atrioventricular valve regurgitation is present. In this repair, a conduit is then placed from the morphologic right ventricle to the pulmonary arteries. An arterial repair may be used in preference to the Rastelli technique in the absence of pulmonary stenosis, avoiding the use of a conduit. Selection and timing of the optimal surgical repair is dependent on the specific knowledge of the anatomy in each individual patient. PMID- 7548321 TI - The right ventricle to pulmonary artery connection: when homografts are not always available. AB - Aortic or pulmonary homografts are currently the most popular conduits for correction of right ventricle to pulmonary artery (RV-PA) discontinuity. Problems of nonavailability or limited supply in developing countries, however, necessitate recourse to other forms of repair. Forty-nine patients of 72 with RV PA discontinuity underwent repair without use of a homograft at our institution over a 5-year period. Handmade pericardial valved conduits (n = 18), nonvalved tubes (n = 13), and nonconduit procedures like the REV operation (n = 11) and extended pericardial gusset (n = 5) were mainly used. The choice of procedure was governed by various factors including type of cardiac defect, pulmonary artery pressure and morphology, previous operation, and socioeconomic constraints. Extracardiac conduits do not provide a permanent solution to the problem of RV-PA discontinuity, and efforts to innovate and refine nonconduit procedures must continue. PMID- 7548322 TI - Valved conduits of bovine pericardium for right ventricle to pulmonary artery connections. AB - Between February 1991 and October 1994, 29 patients received right heart bovine pericardial valved conduits. Patients' ages ranged from 19 days to 18 years (mean 3.5 years), with 8 patients younger than 1 year of age. Ten patients had truncus arteriosus type II or III, 7 had pulmonary atresia with ventricular septal defect (VSD), 6 had transposition of the great arteries and pulmonary stenosis (PS), 3 had corrected transposition with VSD and PS, 2 had double-outlet right ventricle, and 1 had aortic atresia with normal left ventricle and VSD. Two hospital deaths (6.9%) occurred, but no late deaths. The postoperative serial echocardiographic studies showed a mean transconduit gradient of 16 mm Hg with a range of 2 to 37 mm Hg. All patients are in functional class I. Although the intermediate results are satisfactory, considering the late complications known from others conduits, long-term follow-up is necessary. PMID- 7548323 TI - The use of a glutaraldehyde-preserved ovine pulmonary valve, as a pulmonary valve substitute in infants. AB - Reconstruction of the right ventricular outflow tract with a valved conduit is often necessary to repair some forms of congenital heart disease. We have used a 12- to 13-mm sized glutaraldehyde-preserved lamb's (ovine) valve for this purpose. The valves were harvested from the local abattoir, dissected, and stored in glutaraldehyde at 4 degrees C for 2 weeks before use. The valve was placed in four infants, two with truncus arteriosus, one with aortic valve atresia and ventricular septal defect, and one with absent pulmonary valve syndrome. In three patients, the valve had to be replaced in the second year of life, and in one patient in the fourth year of life. All patients are alive and well at follow-up. In the short-term, this valve has functioned well and could be considered where commercially available conduits or homografts are not routinely available. PMID- 7548324 TI - The equine pericardial valved conduit and current strategies for pulmonary reconstruction. AB - In the period from 1976 to February 1995, 268 cases underwent extracardiac conduit repair (ECR), or direct reconstruction of the pulmonary artery. Of these 268 patients, 64 had a valved Dacron conduit, 143 received handmade valved equine pericardial conduits (Xenomedica), 24 had autologous fresh pericardial conduits, and 37 cases had pulmonary artery reconstruction by direct anastomosis without the use of tubular conduits. Hospital mortality in each group showed gradual improvement chronologically; 20.3% in the Dacron group, 9.8% in the equine conduit group, 4.2% in the autologous pericardial conduit group, and 5.4% in the direct anastomosis group, and the mortality rate was significantly higher in the Dacron group (P = .0261). Late death was seen in 7 of 51 (13.7%) in the Dacron group, 12 of 129 (9.3%) in the equine group, 1 of 23 (4.3%) in the autologous pericardial conduit, and none in the direct anastomosis group (P = .1263). Actuarial survival rates of the patients in the Dacron and equine conduit groups showed better survival in the latter without statistical significance: 75.0%, 83.2% at 5 years, and 69.7%, 80% at 10 years, respectively. However, actuarial survival rates among short-term survivors in the Dacron and equine conduit groups remained the same: 87.5%, and 88.7% at 10 years. Our current strategy in this entity is autologous tissue reconstruction of the pulmonary artery either by direct anastomosis or autologous pericardial conduit. Of 30 cases with transposed great arteries or truncus arteriosus, direct anastomosis could be performed in 19 (63.3%) since January 1992.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548326 TI - Ventricular to pulmonary artery connections: generalizations. AB - The basic information about surgically created ventricular to pulmonary artery connections is condensed into five general headings. The information is useful, but is no substitute for the technically competent, well-informed, thoughtful, and intuitive cardiac surgeon. PMID- 7548325 TI - Late results of pulmonary ventricle to pulmonary artery conduits. AB - The development of the extracardiac conduit has been one of the greatest advances in cardiac surgery. Conduits have decreased the mortality rate of several standard operations and have made possible the correction of numerous complex congenital cardiac anomalies that previously were uncorrectable. However, the ideal conduit has not yet been developed. We have reviewed the long-term (16 to 29 years) results of our early experience with this technique. PMID- 7548327 TI - Effect of rhG-CSF on the mobilization of CD38 and HLA-DR subfractions of CD34+ peripheral blood progenitor cells. AB - Several studies have demonstrated that both CD34+/CD38- and CD34+/HLA-DR- human hematopoietic progenitor cells have properties associated with hematopoietic stem cells. However, the kinetics of these two cell populations in human peripheral blood (PB) after priming with granulocyte colony-stimulating factor (rhG-CSF) has not been investigated. By using flow-cytometric analysis we have shown that administration of rhG-CSF to 14 patients eligible for peripheral blood progenitor cell (PBPC) transplantation led to an increment of CD34+/CD38+ and CD34+/HLA-DR+ cells in the PB that paralleled the increase of total CD34+ cells, indicating that such subpopulations are responsible for the major release of CD34+ cells. Furthermore, rhG-CSF priming led to a significant mobilization of fractions of more immature CD34+/CD38- and CD34+/HLA-DR- cells to the PB. In the leukapheresis preparations, the average frequency of CD34+ cells lacking the CD38 or HLA-DR antigens was low (5% and 30%, respectively), with little overlap between the CD38 and HLA-DR- subpopulations. In addition, the yield of each subset of CD34+ cells (CD34+/CD38 +/- and CD34+/HLA-DR +/-) in the PB correlated with the numbers in the collected material. The results of the present study indicate that administration of rhG-CSF causes a significant increase of CD34+/CD38 +/- and CD34+/HLA-DR +/- cells in PB, and that such cells can be then safely harvested by leukapheresis procedures. PMID- 7548328 TI - Establishment and characterization of a novel CD34-positive human myeloid leukemia cell line: MHH225 growing in serum-free culture. AB - A new human multilineage myeloid leukemia cell line, MHH225, has been established in our laboratory from the bone marrow of a 60-year-old patient suffering from acute megakaryoblastic leukemia (M7); it provides a unique model for studying the effect of biologic and chemical agents on the lineage specificity of a multipotent myeloid leukemia clone containing a mixed population of megakaryoblast, erythroblast, and myeloblast cells in a serum-free culture. Morphologically, all 225 cells are large blast cells with basophilic cytoplasm containing no granules, large round nucleus containing 2-3 prominent nucleoli, and fine chromatin structure and a large nuclear/cytoplasm ratio. The MHH225 cells are CD34+HLA-DR+CD33+CD13+ with 57.6%, 28.3%, and 7.8% of them being CD41+, glycophorin A+, and CD15+, respectively, and all lymphoid-specific antigens are negative. The karyotype analysis of MHH225 cells revealed a deletion of the short arm of chromosome 7: del(7)(p13)-, a whole-arm translocation between the long arms of chromosomes 9 and 21: t(9;21)(q10;q10), and a chromosome 11 with an elongated long arm due to duplication of chromosome 11 material as well as to translocation of part of chromosome 9 onto 11q+. Also, chromosome 21 was deleted in some metaphases or showed a ring formation in other metaphases. Utrastructurally, MHH25 cells display a strong platelet peroxidase activity in the nuclear envelope and the endoplasmic reticulum. The MHH25 cells have been grown exponentially without growth factors or conditioned media or serum only in RPMI1640 culture medium. None of the myelopoietic growth factors, i.e., interleukin-3, GM-CSF, G-CSF, erythropoietin, or interleukin-6, has any effect on the proliferation and differentiation of MHH25 cells. The two, hematopoietic inhibitory cytokines, interferon-alpha and tumor necrosis factor-alpha, have only minimal growth inhibitory effect. Stem cell factor showed only weak growth stimulatory effect on MHH225 cells but significantly inhibited chemotherapy induced apoptosis in these cells. The new cell line MHH225 should constitute a useful model for studying stem cell antigen (CD34)-positive human multilineage myeloid leukemia cells carrying a deletion in the short arm of chromosome 7 and an aberration in chromosome 11 and provide a unique tool for investigating human hematopoietic stem cell biology and its cytokine regulation in serum-free cultures. To our knowledge, the MHH225 cell line is the first human CD34-positive leukemia cell line growing in serum-free cultures to be established. PMID- 7548329 TI - Circulating hematopoietic progenitor cells in polycythemia vera: the in vivo effect of hydroxyurea. AB - The in vivo effects of hydroxyurea (HU) on circulating erythroid (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) in patients with polycythemia vera (PV) have been evaluated. HU induced a strong decrease of both BFU-E and CFU-GM in the first month of treatment. During the following 4 months of treatment the level of circulating progenitors remained at very low values, until the end of the period of observation. HU activity involved both erythroid and myeloid committed progenitors and both erythropoietin-stimulated (normal) and endogenous (derived from the abnormal PV clone) BFU-E. PMID- 7548330 TI - Immunophenotyping of B lymphocytes by multiparametric flow cytometry in bone marrow aspirates of healthy adults. AB - Establishing reference ranges by multiparametric immunophenotyping of mature B cells in bone marrow of healthy adults is of interest because the detection of bone marrow infiltration and persistence of light chain restriction, as well as discrimination between reactive and malignant lymphocytes are important applications of B-cell immunophenotyping. To determine the pattern of antigens as expressed by malignant mature B lymphocytes in the present study, bone marrow aspirates of healthy adults were investigated for the presence and percentage frequency of those antigens as defined for immunophenotyping of B cells by the REAL Classification. For this purpose, analysis of CD19-positive B lymphocytes by 'live gate' analysis was performed. The established two-color as well as three color stainings will serve as a basis for future investigations designed to test multiparametric analysis of B lymphocytes in bone marrow aspirates. All investigated antibodies stained with varying percentage frequency on B-cell subtypes, and no statistical significant difference was found between bone marrow aspirates of women and those of men. On the basis of this analysis, all the reported lineage antigen combinations are present both in malignant B lymphocytes and in normal B cells in considerable percentage frequency. These findings are of importance for follow-up investigations of patients with non-Hodgkin's lymphomas by multiparametric immunophenotyping. PMID- 7548331 TI - The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test. AB - The influences of time of storage of platelet-rich plasma (PRP), temperature of storage of PRP, platelet number in PRP, mean platelet volume in whole blood, sex, age, hemoglobin concentration, and different forms of PRP storage on platelet aggregation (PAG) tests, performed with epinephrine, collagen, arachidonate, and ristocetin by a four-channel aggregation profiler (Platelet Aggregation Profiler, Model PAP-4, Bio/Data Corporation, Hatboro, PA 19040, U.S.A.), were evaluated in four groups of subjects (52 men, 22 women, age range 20-85 years, hemoglobin concentration range 8.4-16.8 g/dl). The PRP was stored with or without packed cells, at room temperature or at 4 degrees C, for 0-6 h. The ideal platelet number of PRP for performing the PAG test fell between 150 and 500 x 10(9)/l. If the number was less than 150 x 10(9)/l, the result of PAG should be meaningless. No significant change was noted for up to 6 h when the PRP was stored either at room temperature or at 4 degrees C. Hemoglobin concentration and mean platelet volume did not affect the PAG. However, there was significant but weak correlation (p = 0.0125, r = 0.3696) between age and PAG when using arachidonic acid as the agonist. Men had significantly increased PAG when collagen and ristocetin were used as the agonists. The PRP was stored best at room temperature, without packed cells. In conclusion, to obtain the best result from a PAG test, the PRP should be kept without packed cells at room temperature for no longer than 6 h, and the platelet number should fall between 150 and 500 x 10(9)/l. PMID- 7548332 TI - Cardiac iron overload in thalassemic patients: an endomyocardial biopsy study. AB - Secondary heart failure induced by organ siderosis is the main cause of death in patients affected by thalassemia major. At present it cannot be predicted whether heart siderosis is correlated with iron overload and little is known about the real cardiac histological pattern of post transfusional hemochromatosis in patients with thalassemia major and intermedia. The study aim was to evaluate cardiac iron overload by non invasive and invasive techniques. Fifteen thalassemic patients were investigated and endomyocardial biopsy performed in ten revealed different grades of endomyocardial iron overload with histochemical positivity. Non invasive techniques are not able to furnish an exact picture of the cardiac hemochromatosis. There was a significant correlation between serum ferritin and myocardial iron grade. Patients with elevated ferritin levels and poor compliance to chelating therapy are at high risk of severe heart hemochromatosis. It was seen that endomyocardial biopsy is a useful tool in studying myocardial iron. PMID- 7548335 TI - The annual conference of the International Society for Environmental Epidemiology and the International Society for Exposure Analysis. Noordwijkerhout, The Netherlands, August 30-September 1, 1995. Abstracts. PMID- 7548333 TI - Magnetic resonance imaging reveals a markedly inhomogeneous distribution of marrow cellularity in a patient with myelodysplasia. AB - A 47-year-old male patient with myelodysplasia showed increasing values of serum lactate dehydrogenase (up to 3500 units/l) and an increasing blast count. Several biopsies (taken from the posterior iliac crest) revealed marked hypocellularity. In contrast, magnetic resonance imaging of the marrow demonstrated an inhomogeneous distribution of marrow with hypocellular and also large hypercellular areas not detected by cytological and histological analysis. A location for biopsy of hypercellular marrow was provided by T1-weighted and water selective magnetic resonance imaging. The findings in the patient were compared with those in a matched healthy volunteer. PMID- 7548336 TI - 11th International Symposium on Epidemiology in Occupational Health. Noordwijkerhout, The Netherlands, September 5-8, 1995. Abstracts. PMID- 7548334 TI - Primary plasma cell leukemia with extensive dense osteosclerosis: complete remission following combination chemotherapy. AB - Diffuse osteosclerotic myeloma is very rare, and primary plasma cell leukemia with extensive osteosclerosis is even more rare. We describe a 71-year-old man who presented with severe anemia and dense widespread osteosclerosis similar to the X-ray finding of myelosclerosis. His peripheral blood showed 40% plasma cells. Bone marrow examination revealed heavy plasma cell infiltration with marked myelofibrosis and myelosclerosis. Protein electrophoresis and immunoelectrophoresis demonstrated an M-protein of IgG-lambda type. He was treated with cyclophosphamide, vincristine, and prednisolone for 10 months. A complete remission was obtained, with disappearance of M-protein and circulating plasma cells and normalization of complete blood counts, bone marrow picture, and biochemical parameters, as well as complete regression of myelofibrosis and osteosclerotic lesions. Unmaintained complete remission lasted for more than 1 year and he survived for more than 22 months. Our case indicated that one must include in the differential diagnosis of an osteosclerotic lesion the possibility of multiple myeloma, and that combination chemotherapy can induce a complete remission in this disease. PMID- 7548337 TI - Counting isn't easy. PMID- 7548338 TI - How bad is categorization? PMID- 7548339 TI - Is cancer causation simpler than we thought, but more intractable? PMID- 7548340 TI - Life expectancy in four U.S. racial/ethnic populations: 1990. AB - Previous estimates of life expectancy in the United States have not corrected for biases in population and mortality data, and no study has examined life expectancy in U.S. Asian/Pacific Islander and American Indian populations. We used information on population undercounts by race/ethnicity in the census and on misclassification of race/ethnicity on death certificates to calculate life expectancy for black, white, American Indian, and Asian men and women in the United States in 1990. Correction for undercount and misclassification had little effect on life expectancy estimates for whites, but it substantially decreased estimates for American Indians and Asians. Asian men had life expectancies of 82.0 years and Asian women 85.8 years--the highest life expectancies reported for any population in the world and beyond the limit predicted by some current theories. PMID- 7548342 TI - History of depression as a risk factor for Alzheimer's disease. AB - Research regarding the possible association between Alzheimer's disease and a history of depression has been inconclusive. Using a case-control design, we assessed the strength of the association between reported history of depression and onset of Alzheimer's disease. We enrolled probable Alzheimer's disease cases (N = 294), who were ascertained and diagnosed by our Alzheimer's Disease Patient Registry, and randomly selected nondemented controls (N = 300) of similar age and gender from the same base population. The mean age (for cases) was 78.5 years. Informants provided data regarding history of depression. "Treated depression" was defined as depression for which a physician/psychologist consultation, medication, or hospitalization had occurred. Restricting treated depression to exclude primary loss or grief reactions, we found a modest association with Alzheimer's disease [odds ratio (OR) = 1.8; 95% confidence interval (CI) = 0.9 3.5] after adjusting for gender, age, education, and type of informant. When these data were stratified by depression onset year, we observed an odds ratio of 2.0 (95% CI = 0.9-4.6) for depression occurring more than 10 years before the onset of dementia symptoms, and an OR of 0.9 (95% CI = 0.2-3.0) for depression onset within 10 years of the onset of dementia symptoms. Thus, depressive episodes occurring well before dementia symptom onset appear to increase the risk of Alzheimer's disease. PMID- 7548341 TI - Dose-response and trend analysis in epidemiology: alternatives to categorical analysis. AB - Standard categorical analysis is based on an unrealistic model for dose-response and trends and does not make efficient use of within-category information. This paper describes two classes of simple alternatives that can be implemented with any regression software: fractional polynomial regression and spline regression. These methods are illustrated in a problem of estimating historical trends in human immunodeficiency virus incidence. Fractional polynomial and spline regression are especially valuable when important nonlinearities are anticipated and software for more general nonparametric regression approaches is not available. PMID- 7548343 TI - Uranium dust exposure and lung cancer risk in four uranium processing operations. AB - We examined the relation between uranium dust exposure and lung cancer mortality among workers employed in four uranium processing or fabrication operations located in Missouri, Ohio, and Tennessee. Among workers who had at least 30 years of potential follow-up, we identified 787 lung cancer cases from death certificates and matched one control to each case. Health physicists estimated individual annual lung doses from occupational exposure primarily to insoluble uranium compounds, using contemporary monitoring data. With a 10-year lag, cumulative lung doses ranged from 0 to 137 centigrays (cGy) for cases and from 0 to 80 cGy for controls. Health physicists assigned annual external radiation doses to workers having personal monitoring records. Archivists collected smoking information from occupational medical records. Odds ratios for lung cancer mortality for seven cumulative internal dose groups did not demonstrate increasing risk with increasing dose. We found an odds ratio of 2.0 for those exposed to 25 cGy and higher, but the 95% confidence interval of 0.20 to 20 showed great uncertainty in this estimate. There was a suggestion of an exposure effect for workers hired at age 45 years or older. Further analyses for cumulative external doses and exposures to thorium, radium, and radon did not reveal any clear association between exposure and increased risk, nor did dichotomizing workers by facility. PMID- 7548344 TI - Is the use of psychotropic drugs associated with increased risk of ischemic heart disease? AB - Earlier reports of associations between panic disorder, depression, and ischemic heart disease have not evaluated the role of the drugs used to treat these conditions. As part of a larger study on reducing cardiovascular disease in an entire community, we estimated the association between psychotropic drugs and ischemic heart disease in a large community-based population using a cohort study design. We defined exposure as current use of psychotropic medications at the time of the health interview. We determined ischemic heart disease by International Classification of Diseases, 9th revision, Clinical Modification, codes and by an epidemiologic algorithm using clinical diagnostic criteria. An elevated risk for clinically significant ischemic cardiac events was moderately associated with benzodiazepine use [relative risk (RR) = 2.0; 90% confidence interval (CI) = 1.1-3.9] and strongly associated with antidepressant use (RR = 5.7; 90% CI = 2.6-12.8), although the latter estimate was based on only six antidepressant users who had an ischemic event. PMID- 7548346 TI - Selecting an exposure lag period. AB - In epidemiology, there is an inclination to consider more credible the larger estimates of exposure effect. For example, higher relative risks or rate ratios are often emphasized as a criterion for choosing among various hypothesized exposure-lag values. An alternative criterion for this choice might be based on a goodness-of-fit measure. We present examples, based on hypothetical data, in which an exposure-lag parameter is estimated by trial and error fitting: we compare the behavior of the likelihood-ratio goodness-of-fit statistic obtained over the assigned values of the parameter with that of the relative risk. We show that there can be inconsistencies between the highest-estimate and likelihood based goodness-of-fit criteria. Concern about the validity of the highest estimate criterion prompts us to recommend that this criterion not be used for the estimation of exposure-weighting parameters, which should preferably be based on a priori biological knowledge or on goodness-of-fit criteria. PMID- 7548345 TI - Is the human immunodeficiency virus-related Kaposi's sarcoma epidemic coming to an end? Insights from the Tricontinental Seroconverter Study. AB - A decline in the proportion of Kaposi's sarcoma among AIDS cases since the 1980s has been attributed to changes in sexual behavior among homosexual men and a decreasing exposure to a hypothesized Kaposi's sarcoma cofactor. Recent studies have shown that the incidence rate of Kaposi's sarcoma has remained relatively stable, which seems to argue against the hypothesis of a declining exposure to the putative cofactor. To examine this paradox, we evaluated the incidence of Kaposi's sarcoma, using Cox proportional hazard analyses, and performed a simulation to compare incidences of different AIDS outcomes among 407 homosexual men with documented dates of seroconversion. Our data show that men who seroconverted early in the epidemic did not progress faster to Kaposi's sarcoma than men who seroconverted more recently. A lower incidence rate of Kaposi's sarcoma would be expected among the latter group if exposure to the hypothesized cofactor is decreasing over time. The percentage of Kaposi's sarcoma among incident AIDS cases decreased over the years following seroconversion, but not over calendar time. This study demonstrates that the decline in the proportion of Kaposi's sarcoma among AIDS cases should not be interpreted as a decline in the incidence of Kaposi's sarcoma and that there is no evidence that a hypothesized Kaposi's sarcoma cofactor is declining over calendar time. PMID- 7548347 TI - Effect of hypertensive diseases in pregnancy on birthweight, gestational duration, and small-for-gestational-age births. AB - We measured the impact of hypertensive disorders in pregnancy (chronic hypertension, pregnancy-induced hypertension, and eclampsia) on birthweight, gestational duration, and small-for-gestational-age births. We employed a retrospective cohort design using North Carolina birth certificate data for the years 1988-1990, with the analyses based on nearly 300,000 pregnancies. Women noted to have eclampsia during pregnancy had a substantially greater risk of delivering very-low-birthweight infants (birthweight < or = 1,499 gm; risk difference (RD) = 6.7%) and moderately-low-birthweight infants (1,500-2,499 gm; RD = 14.6%), and very preterm (gestational age < 33 weeks; RD = 7.1%) and moderately preterm (33-36 weeks; RD = 9.3%) births, compared with women without hypertension. Corresponding risk differences for these outcomes were 1.9% and 5.1% for very low and moderately low birthweight, and 1.6% and 3.0% for very preterm and moderately preterm for women with chronic hypertension. Risk differences were of similar magnitude for women with pregnancy-induced hypertension for these outcomes. Hypertensive disorders in pregnancy were associated with small-for-gestational-age infants, with risk differences of 5.1%, 3.5%, and 9.2% for chronic hypertension, pregnancy-induced hypertension, and eclampsia, respectively. The patterns of risk were similar for preterm births due to spontaneous onset of labor and those due to other precipitating events. We observed similar patterns for black and white women. Control for confounders through multivariable logistic regression models did not alter the observed patterns. In spite of the limited data quality, we found that hypertensive disorders in pregnancy substantially increase the risk of low birthweight, preterm births, and small-for-gestational-age births. PMID- 7548348 TI - Soft tissue sarcoma and non-Hodgkin's lymphoma in workers exposed to phenoxy herbicides, chlorophenols, and dioxins: two nested case-control studies. AB - We examined the effect of exposure to chemicals present in the production and spraying of phenoxy herbicides or chlorophenols in two nested case-control studies of soft tissue sarcoma and non-Hodgkin's lymphoma. Eleven sarcoma and 32 lymphoma cases occurring within an international cohort were matched for age, sex, and country of residence with 55 and 158 controls, respectively. Exposures to 21 chemicals or mixtures were estimated by three industrial hygienists who were blind to the subject's case-control status. Excess risk of soft tissue sarcoma was associated with exposure to any phenoxy herbicide [odds ratio (OR) = 10.3; 95% confidence interval (CI) = 1.2-91] and to each of the three major classes of phenoxy herbicides (2,4-dichlorophenoxyacetic acid, 2,4,5 trichlorophenoxyacetic acid, and 4-chloro-2-methylphenoxyacetic acid), to any polychlorinated dibenzodioxin or furan (OR = 5.6; 95% CI = 1.1-28), and to 2,3,7,8-tetrachlorodibenzo-p-dioxin (OR = 5.2; 95% CI = 0.85-32). Sarcoma risk was not associated with exposure to raw materials or other process chemicals. In the non-Hodgkin's lymphoma study, associations were generally weaker than those found in the study on sarcoma. These findings indicate that workers exposed to phenoxy herbicides and their contaminants are at a higher risk of soft tissue sarcoma. PMID- 7548349 TI - Effect of cigarette smoking and coffee drinking on time to conception. AB - To test the hypothesis that coffee consumption and cigarette smoking are associated with time to conception, we analyzed data collected from 1,341 primigravidas participating in the Child Health and Development Studies in the San Francisco Bay Area between 1959 and 1966. Women were interviewed in person about their reproductive and medical history, habits (smoking, coffee and alcohol consumption), and sociodemographic characteristics. Logistic regression models showed that after adjusting for covariates, women who smoked cigarettes had about one-half the fertility [odds ratio (OR) = 0.5-0.6] of nonsmoker-noncoffee drinkers for times to conception of 6 and 12 months, regardless of whether they drank coffee. On the other hand, coffee consumers who did not smoke did not have decreased fertility compared with nondrinkers who did not smoke (adjusted OR = 1.0-1.2). Coffee drinking did not increase the risk of delayed conception among smokers over the risk posed by the smoking exposure by itself (adjusted OR = 0.6 0.8). We observed an effect of smoking even at low doses (1-9 cigarettes per day). The present study adds to the evidence of an association between cigarette smoking and reduced fertility. PMID- 7548351 TI - Controlling for time-since-hire in occupational studies using internal comparisons and cumulative exposure. AB - Employees within an occupational cohort may demonstrate a more favorable mortality experience while maintaining employment than those who leave employment. At the same time, they may experience an apparent decline in health with time-since-hire. The time-since-hire effect may occur independently of exposure but may nevertheless result in groups categorized by cumulative exposure that are not comparable. Controlling for time-since-hire appears to solve this problem. To quantify the empirical bias in estimates of exposure effect due to confounding from time-since-hire, we analyzed two occupational cohorts using Poisson regression with and without adjustment for time-since-hire or time-since start-of-follow-up. In a cohort exposed to airborne arsenic, a strong dose response relation with respiratory cancer mortality had been established. In a cohort exposed to external, penetrating ionizing radiation, a weak and controversial dose-response relation had been reported. The parameter estimates relating exposure to disease from the models that explicitly adjusted for time since-hire or time-since-start-of-follow-up are within 10% of the estimates from models that did not. It appears, from this empirical analysis of two datasets, that occupational studies may not need to adjust explicitly for such time-related factors as time-since-hire or time-since-start-of-follow-up if these are implicitly controlled through other variables in the model. PMID- 7548350 TI - Sexual behavior, sexually transmitted diseases, and risk of cervical cancer. AB - To explore sexually transmitted diseases and sexual behavior as risk factors for cervical cancer, we analyzed data from a population-based case-control study of breast and cervical cancer in Costa Rica. Data from 415 cases of cervical carcinoma in situ, 149 cases of invasive cervical cancer, and 764 controls were included in the analysis. Multivariate analysis showed that lifetime number of sex partners, first intercourse before age 15 years, number of livebirths, herpes simplex virus type 2 seropositivity, and serologic evidence of previous chlamydial infection were predictors of carcinoma in situ. Serologic evidence of previous syphilis was not associated with carcinoma in situ. Predictors for invasive cervical cancer included lifetime number of sex partners, first intercourse before age 15 years, number of livebirths, serologic evidence of previous syphilis, herpes simplex type 2 infection, and chlamydial infection. Cigarette smoking, socioeconomic status, self-reported history of sexually transmitted diseases, and douching were not associated with either carcinoma in situ or invasive cervical cancer. PMID- 7548352 TI - Lower mortality for female-female twins than male-male and male-female twins in rural Senegal. AB - Twins have been registered prospectively for 12-22 years in 42 small villages in the Bandafassi area of Eastern Senegal. We studied 98 pairs of twins to test whether twins in opposite-sex pairs have higher postneonatal mortality than same sex twins. Neonatal mortality for twins was 41.3%; mortality for infants and for children under age 5 years was 53.0% and 66.8%, respectively. Neonatal mortality was identical for same-sex and opposite-sex twin pairs, but much higher for boys than girls [relative risk = 1.8; 95% confidence interval (CI) = 1.2-2.6]. There was clustering of double neonatal deaths for all types of twins. In the postneonatal period, female-female twins had lower mortality than other twin types. Twins had higher post-neonatal mortality as long as the co-twin was alive [mortality rate ratio (MR) = 2.6; 95% CI = 1.0-6.7]. Girls had excess mortality when the co-twin was of the opposite sex (MR = 4.3; 95% CI = 1.2-15.3), whereas there was no difference for boys. In conclusion, contact with a co-twin of the opposite sex increased child mortality for female twins. Our data are not sufficient to determine whether this difference is specific for girls or applies to boys as well. PMID- 7548353 TI - High birthweight as a predictor of prostate cancer risk. AB - In a follow-up study of a cohort of 50-year-old men who were born in 1913 and were living in Gothenburg, Sweden, we found an association between birthweight and prostate cancer. Of 366 men with known birthweight, there were 21 patients with prostate cancer. The incidence is about five times higher in the highest quartile of birthweight than in other birthweight groups. The findings indicate that aspects of the pre- and perinatal period may affect the risk of subsequent prostate cancer. PMID- 7548354 TI - Parental age and breast cancer mortality. AB - Because older ages of both mothers and fathers have been hypothesized by others to increase the subsequent risk of breast cancer in female babies, we analyzed the association between maternal and paternal age at birth and mortality from breast cancer in a cohort of 384,796 American women. Cox proportional hazards modeling accounted for age, family history of breast cancer in first-degree relatives, age at menarche, age at first pregnancy, and parity. We found little association between paternal age at birth and death from breast cancer. Although there was no clear linear trend for higher risk with increasing age of the mothers at birth, women born to mothers age 45 years or older had a relative hazard of 1.30 (95% confidence interval = 0.85-1.98), compared with women born to mothers under the age of 20 years. Although these findings are of little public health significance, they may indicate a hormonal profile in older mothers that predisposes female offspring to a higher risk of breast cancer in later years. PMID- 7548355 TI - An evaluation of the oral cancer control program in Cuba. AB - The Cuban oral cancer screening program, functioning since 1984, requires all subjects of > or = 15 years to have an annual oral visual inspection by dentists. It covered 12-26% of the target population annually. Less than 30% of the subjects with suspect lesions complied with referral. The program identified 16% of the 4,412 incident oral cancers in Cuba during 1984-1990. The proportion of stage I cases increased from 24% in 1983 to 49% in 1990, although staging information was available only for half of the cases. There was no change in oral cancer incidence and mortality in Cuba over the last decade that could be ascribed to the screening program. PMID- 7548357 TI - Prevalence of chronic diseases in alcohol abstainers. AB - We used data from the 1983 Italian National Health Survey, based on 58,462 adults ages 25 years and over who are representative of the general Italian population, to compare the prevalence of 16 major chronic diseases or groups of diseases in alcohol abstainers (ex- and never-drinkers combined) and current drinkers. We found elevated odds ratios among alcohol abstainers for diabetes, hypertension, myocardial infarction, other heart diseases, anemias, gastroduodenal ulcer, cholelithiasis, liver cirrhosis, urolithiasis, and renal insufficiency. PMID- 7548358 TI - Cancer incidence among female manual workers. AB - We investigated cancer incidence during the period 1970-1992 among unskilled Icelandic female workers who contributed to two pension funds for manual workers. We found an increase in the standardized rate ratios (SRRs) for all cancer with increasing time before the follow-up period began. We found no clear relation between cancer incidence and length of employment. The SRRs were higher for those who became members of the funds in 1977 or later than for those who started earlier: among these women, we found SRRs for all cancers, 1.36; cancer of the stomach, 1.49; cancer of the lung, 1.48; cancer of the cervix, 3.19; and cancer of the bladder, 6.00. PMID- 7548359 TI - Does number of children increase the rate of coronary heart disease in men? AB - We examined the association between number of children and coronary heart disease risk among men in two large prospective cohort studies. Data from the Framingham Heart Study comprised a group of 1,632 men who were followed for 30 years; data from the first National Health and Nutrition Examination Survey National Epidemiologic Follow-up Study included 2,584 men who were followed for 3-5 years. We found no relation between number of children and coronary heart disease incidence in these cohorts. This lack of association in men contrasts with the previous positive association found between pregnancy and coronary heart disease in women. PMID- 7548356 TI - Body mass and physical activity are important predictors of serum androgen concentrations in young healthy men. AB - The relative importance of body mass index (BMI), demographic, and life-style factors in predicting androgen and sex hormone-binding globulin (SHBG) concentrations of young healthy men has not been clearly established. We evaluated the role of age, BMI, height, habitual physical activity, education, smoking, coffee and alcohol intake as predictors of serum testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate, androstenedione, and SHBG concentrations in 100 healthy men 18-22 years old. BMI was the only independent predictor of dihydrotestosterone and dehydroepiandrosterone sulfate, and both BMI and physical activity were independent predictors of serum testosterone concentrations, whereas other variables did not appear to play a role of comparable importance. PMID- 7548360 TI - Determinants of serum lipid and lipoprotein concentrations in children. AB - We evaluated determinants of serum lipid and lipoprotein concentrations in 3,106 schoolchildren who participated in a community survey. We administered a brief questionnaire and measured height, weight, and fasting serum lipid concentrations. Family history of hypercholesterolemia and body mass index were strong determinants of lipid and lipoprotein levels. Results for specific foods were not always what would have been expected based on their contents of saturated fatty acids and cholesterol. Our findings point to the need for more detailed studies of the effects of specific foods, especially cheeses, milk, and whole grain bread, on the entire lipoprotein profile of children. PMID- 7548361 TI - Avoiding power loss associated with categorization and ordinal scores in dose response and trend analysis. PMID- 7548363 TI - The power of plague. PMID- 7548362 TI - Genetic-environmental interactions and low-level exposure to carcinogens. PMID- 7548364 TI - The European Union ponders the tasks of conducting epidemiologic research. PMID- 7548366 TI - Fractional expected numbers. PMID- 7548365 TI - A-bomb studies threatened. PMID- 7548367 TI - Black box epidemiology. PMID- 7548368 TI - Age at first and last births and risk of breast cancer. PMID- 7548369 TI - Toxicity of different nonsteroidal antiinflammatory drugs. PMID- 7548370 TI - Standardized mortality odds ratios. PMID- 7548371 TI - Feasibility of collecting maternal blood and hair samples in a case-control study of congenital anomalies. PMID- 7548372 TI - Partial enterocele (Richter's hernia). PMID- 7548373 TI - Effect of haemorrhagic shock and intraosseous resuscitation on plasma and urine catecholamine concentrations and urinary clearance in pigs. AB - OBJECTIVE: To evaluate the plasma and urinary catecholamine concentrations after episodes of haemorrhagic shock treated by intraosseous infusion of a small volume of hyperosmotic fluid, followed by whole blood, in a standard porcine model. DESIGN: Randomised open study. SETTING: University hospital, Norway. MATERIAL: 18 piglets. INTERVENTIONS: All animals were bled to an arterial systolic pressure of 60 mm Hg. After 30 minutes 100 ml hyperosmotic (2.4 mol/l) fluid (glucose/sodium chloride) was given either intraosseously (n = 6) or intravenously (n = 6). Ninety minutes later the animals were again bled to a systolic pressure of 60 mm Hg, and after 30 minutes an autologous whole blood transfusion was given by either intraosseous or intravenous infusion. Six animals acted as untreated controls. MAIN OUTCOME MEASURES: Changes in haemodynamic variables and plasma and urinary catecholamine concentrations. RESULTS: Two pigs in the control group died. The hyperosmotic infusion improved the circulatory performance significantly (p < 0.05). Thirty minutes after both bleeds the plasma catecholamine concentrations were increased by 10-15 times, but returned to the reference range in all groups 90 minutes after the hyperosmotic infusion. Two hours after the whole blood infusion the catecholamine concentrations of the treated animals were at baseline values, significantly lower than those of the controls. The corresponding urinary catecholamine concentrations of the controls were significantly increased. There were no differences between the two treatment groups. CONCLUSION: The catecholamine measurements indicate that there may possibly be protective renal excretion of excess shock-induced plasma catecholamines after acute haemorrhage. PMID- 7548374 TI - The operative peritoneal fibrinolytic response to abdominal operation. AB - OBJECTIVE: To measure changes in the fibrinolytic properties of human peritoneum during operation. DESIGN: Open study. SETTING: University hospital, UK. SUBJECTS: 20 patients undergoing elective operations for non-inflammatory disease. INTERVENTIONS: Peritoneum was biopsied at the beginning and end of operation. MAIN OUTCOME MEASURES: Peritoneal plasminogen activating activity (PAA) and the concentrations of tissue plasminogen activator (t-PA), urokinase, and plasminogen activator inhibitors 1 and 2 were measured at both time points. RESULTS: Peritoneal PAA was reduced over the time of the operation (p < 0.05) as was the concentration of t-PA (p < 0.05). The urokinase concentration rose significantly (p < 0.05), but plasminogen activator inhibitors 1 and 2 were not detected. CONCLUSIONS: Elective abdominal operation caused an immediate reduction in peritoneal PAA which seemed to be secondary to a reduced concentration of t-PA. Such a reduction in peritoneal fibrinolytic activity allows the early deposition of fibrinous deposits within the peritoneal cavity. PMID- 7548375 TI - Comparison for four techniques of catheter insertion in patients undergoing continuous ambulatory peritoneal dialysis. AB - OBJECTIVE: To compare four different ways of implanting catheters for continuous ambulatory peritoneal dialysis (CAPD) in an effort to reduce the incidence of complications. DESIGN: Retrospective study. SETTING: Teaching hospital, Taiwan. SUBJECTS: 166 Patients who had 180 catheters inserted between 1985 and 1993. INTERVENTIONS: 49 Catheters were inserted through midline incisions (in 24 of which the catheter was fixed with an additional suture) and 131 were inserted through paramedian incisions (in 88 of which the catheter was fixed with an additional suture). MAIN OUTCOME MEASURES: Morbidity, particularly the incidence of migration of the catheter and incisional hernia. RESULTS: 8/68 Catheters migrated in patients in whom no additional fixing suture had been used, compared with 2/112 in whom an additional suture had been used (p = 0.007). There were 4 incisional hernias in 49 midline, compared with 0/131 paramedian, incisions (p < 0.0001). Significantly more catheters had to be removed after midline than after paramedian incisions (35/49 compared with 56/131, p = 0.0008); chi square for independence 15.02, df 3, p = 0.0018. CONCLUSION: For the implantation of catheters for CAPD the paramedian incision is associated with significantly fewer complications than the midline incision and the incidence is even lower if the catheter is fixed to the lower peritoneum with an additional suture. PMID- 7548376 TI - Prevalence and risk factors of varicose veins in lower extremities: mini-Finland health survey. AB - OBJECTIVE: To study the prevalence and risk factors of clinically important varicose veins in the lower extremities. DESIGN: Cross-sectional study with self administered questionnaire. SETTING: A national health examination survey, Finland. SUBJECTS: 8000 people from 40 geographical areas representing the adult population aged 30 years and over. RESULTS: The prevalence of varicose veins diagnosed by a physician was 25% in women and 7% in men. Twenty-nine per cent of the men and 53% of the women with diagnosed varicose veins had been treated surgically. Age, height, body mass index (weight/height2), standing at work, and the number of births were expected risk factors associated with varicosities. Unexpectedly, signs of osteoarthrosis were positively, and diabetes negatively, associated with varicose veins. Urban dwelling and high income correlated positively with varicose veins treated surgically in women. CONCLUSIONS: Varicose veins in the lower extremities are common, and demand considerable health service resources. The aetiology of varicose veins is still incompletely understood and more research is needed to develop possible preventive measures. PMID- 7548377 TI - Conservative surgery in the treatment of hepatic hydatidosis. AB - OBJECTIVE: To examine the morbidity, mortality and recurrence rate after a modified conservative operation in the treatment of hepatic hydatidosis. DESIGN: Prospective open study. SETTING: University hospital, Greece. SUBJECTS: 67 Consecutive patients with hepatic hydatidosis, operated on between 1985 and 1990. INTERVENTION: The liver was mobilised and abdominal cavity isolated with pads soaked in 15% saline solution. Hydatid fluid was aspirated from the cysts which were widely deroofed, sterilised with 15% saline, and then oversewn with a braided absorbable suture. Drains were left in place and a third of patients also had omentoplasty (n = 22). MAIN OUTCOME MEASURES: Morbidity, mortality and recurrence rate. RESULTS: One patient died (1%), 4 developed complications (6%), and there were 3 recurrences (6%). It made no difference whether an omentoplasty was added or not. CONCLUSION: Conservative surgery achieves satisfactory results in the treatment of hepatic hydatidosis. PMID- 7548378 TI - Laparoscopic cholecystectomy without cholangiography: a study of 151 consecutive cases. AB - OBJECTIVE: To evaluate the safety of laparoscopic cholecystectomy without operative cholangiography. DESIGN: Open study. SETTING: Outpatient and private hospitals, Finland. SUBJECTS: 151 Patients (of whom all but the first 10 were consecutive) with symptomatic gallstones, or chronic or acute cholecystitis. INTERVENTIONS: Laparoscopic cholecystectomy and (in three patients) preoperative endoscopic retrograde cholangiography (ERC). MAIN OUTCOME MEASURES: Conversion to open operation and morbidity. RESULTS: Only 2 patients (2%) required conversion to open cholecystectomy (one bled and in one the anatomy was difficult) and 4 (3%) developed complications (bleeding, n = 2, bile collection, and subcutaneous emphysema as a result of malfunction of the carbon dioxide insufflator). Three patients underwent ERC after operation and two were found to have common duct stones. Both had been noted to have large cystic ducts at operation. CONCLUSION: Laparoscopic cholecystectomy can be done safely by experienced surgeons without operative cholangiography. PMID- 7548379 TI - Anastomotic breaking strength and healing of anastomoses in rat intestine with and without chronic radiation damage. AB - OBJECTIVE: To assess the influence of chronic radiation damage on anastomotic healing in the small bowel in rats. DESIGN: Controlled laboratory study. SETTING: University hospital, Sweden. MATERIAL: 90 male Sprague-Dawley rats. INTERVENTIONS: A short segment of the distal ileum was exteriorised and irradiated with a single dose (experimental group, n = 45) or exposed only (control group, n = 45). Twenty weeks later resection and anastomosis were done within this segment using 7/0 polypropylene. MAIN OUTCOME MEASURES: The anastomotic breaking strength, the amount of perianastomotic hydroxyproline, and the number of anastomotic complications. RESULTS: The breaking strength and the amount of perianastomotic hydroxyproline were higher in the irradiated than in the non-irradiated group. In contrast, anastomotic complications were significantly more common in irradiated animals. CONCLUSION: Anastomotic complications in irradiated intestine are not related to the amount of perianastomotic collagen or to breaking strength. PMID- 7548381 TI - Outpatient surgical treatment of anal fissure. AB - OBJECTIVES: To review our experience of treating anal fissures by lateral subcutaneous anal sphincterotomy in the outpatient clinic. DESIGN: Retrospective study. SETTING: University hospital, Israel. SUBJECTS: 108 of 112 Consecutive patients with anal fissures. INTERVENTIONS: All patients had failed to respond to a two week course of conservative treatment. They all underwent lateral subcutaneous anal sphincterotomy under local anaesthesia in either the outpatient clinic or the day-case operating theatre. MAIN OUTCOME MEASURES: Early and late morbidity, patient satisfaction, admission to hospital, and recurrence. RESULTS: 108 of the 112 were followed up for a mean of 20 months (range 2-56). 100 (93%) reported that the late results were good to excellent. Four patients bled immediately after the operation and required admission to hospital and four developed abscesses of which only one responded to conservative treatment; 14 patients complained of initial mild incontinence but in most this resolved over time; 92 patients (85%) had no complaints at late follow up, and only 3 reported either no improvement or recurrence; 10 reported slight soiling, and only one patient remained incontinent. CONCLUSION: Sphincterotomy is the operation of choice for patients with anal fissures that have not responded to conservative treatment, particularly as it can be done effectively and safely in the outpatient clinic under local anesthesia. PMID- 7548382 TI - Tracheolaryngectomy for papillary thyroid cancer. PMID- 7548380 TI - Timing of antibiotic treatment in non-perforated gangrenous appendicitis. AB - OBJECTIVE: To assess whether antibiotic treatment with cefuroxime and tinidazole started during the operation was as effective as treatment started before operation in patients with gangrenous non-perforated appendicitis. DESIGN: Prospective randomised study. SETTING: University hospital, Sweden. SUBJECTS: 114 patients with gangrenous, non-perforated appendicitis who had had antibiotics started before operation and 120 whose treatment was started during operation out of a total of 575 who presented with a presumptive diagnosis of appendicitis. MAIN OUTCOME MEASURES: Morbidity and mortality. RESULTS: There were no deaths, and the rates of infective complications were 1/114 (0.9%) and 3/120 (3%), respectively. The median hospital stay was four days in both groups. CONCLUSION: Antibiotic treatment started during the operation is not significantly worse at preventing infective complications in non-perforated, gangrenous appendicitis than treatment started before the operation. PMID- 7548383 TI - Multiple branchial cleft anomalies. PMID- 7548384 TI - Duodenal isolation for the treatment of perforation after endoscopic sphincterotomy. PMID- 7548385 TI - Acute lung damage after bilateral insertion of femoral intramedullary interlocking nails for metastatic bone disease. PMID- 7548386 TI - Colorectal carcinoma. PMID- 7548387 TI - Ultrastructure of cardiac myocytes in the greater bandicoot rat (Bandicota indica). AB - Cardiac myocytes in the left ventricle and atrium of the greater bandicoot rat (Bandicota indica) were examined by transmission electron microscopy. The fine structure showed typical features of mammalian cardiac myocytes. In atrial myocytes, however, the areas occupied by mitochondria were much smaller than that filled with myofibrils. The decrease in mitochondria and the abundance of myofibrils are thought to be an ultrastructural adaptation to the large body size of this species. Many conducting myocytes were observed in both atrium and ventricle. The atrial conducting myocytes were ultrastructurally different from the Purkinje fibers of the ventricle. We suggest that the abundance and the ultrastructural variation of conducting myocytes are related to the physiological aspects, such as body size, of this animal. PMID- 7548388 TI - Effect of chlormadinone acetate-pellet implantation on the volume of prostate, peripheral blood levels of sex hormones and semen quality in the dog. AB - Chlormadinone acetate (GS implant, CMA) pellet, a synthetic luteal hormone preparation, was subcutaneously implanted at 5, 10 and 20 mg/kg in four normal male dogs ranging in age from 3 to 10 years to determine the changes in the prostatic volume, peripheral plasma levels of sex hormones and semen quality. The plasma levels of CMA, LH, testosterone (T) and 5 alpha-dihydrotestosterone (DHT) were measured by radioimmunoassay. The prostatic volume was measured by computed tomography. The semen was collected by digital manipulation. The pellet was removed 26 weeks after implantation. The effects of CMA pellet implantation were examined during implantation and until 22 weeks after removal. The prostatic volume was reduced to 61 +/- 3 (mean +/- S.E., n = 4), 52 +/- 5 (n = 4) and 53 +/ 9 (n = 4)% of the preimplantation volumes in the 5, 10 and 20 mg/kg groups, respectively. The plasma CMA levels in the 10 mg/kg and 20 mg/kg groups peaked at 2 weeks, but were gradually decreased. At 22 weeks after removal of the pellet, the prostatic volume returned to 74-85% of the preimplantation volumes. The plasma LH levels tended to increase after implantation in all groups. The plasma T and DHT levels were slightly decreased in all groups. In the 10 and 20 mg/kg groups, the number of sperm and motility sperm was reduced, and the rate of abnormal sperm increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548389 TI - Determination of cardiac output by echocardiography. AB - To determine cardiac output using a non-invasive manner, we examined correlations between cardiac output values determined by ten different kinds of echocardiography and those determined by the thermodilution method. With respect to the M-mode method, one of the rotary ellipsoid approximation methods using a short axial section at the papillary muscular level, namely the GIBSON method, showed the highest correlation with the thermodilution method (r = 0.84; p < 0.01). Among the Doppler mode method, the highest correlation (r = 0.93; p < 0.01) was exhibited by the method in which the area of the luminal section of the outflow tract was accurately measured using a trace method on a short axial cross section of the tract flowing out of the left ventricle. These findings suggest that it is possible to determine cardiac output by echocardiography in the same was as with the thermodilution method. Moreover, non-invasive determination methods using echocardiography have been confirmed to be highly beneficial in clinical settings. PMID- 7548391 TI - Morphometry on proximal tubule of the kidney in rat pups from uninephrectomized mothers. AB - Effects of maternal uninephrectomy in rats, which was performed on day 5 of gestation, on development of proximal renal tubule of the pups were morphometrically investigated. One day after birth, both the proximal tubular length per unit volume (1 mm3) of whole kidney and total proximal tubular volume in the neonates from uninephrectomized mothers were significantly larger than those in the neonates from sham-operated mothers. Six weeks after birth, no significant differences in parameters of the kidney were observed between the pups from uninephrectomized and sham-operated mothers. These results suggest that maternal uninephrectomy in the rat accelerates the development of proximal renal tubule in the fetus and that the renotropic activity is not lasting during postnatal development of the kidney. PMID- 7548390 TI - Prevalence of upper respiratory tract disorders detected with a flexible videoendoscope in thoroughbred racehorses. AB - A total of 350 thoroughbred racehorses found clinically as having coughs or abnormal breath sounds underwent endoscopic examination of the upper respiratory tract at rest. As a result, abnormal endoscopic findings were obtained in all horses, demonstrating that thoroughbred racehorses with a clinical abnormality in the respiratory system necessarily had disorders in the upper respiratory tract. Whereas 11 types of disorder were observed, a combination of three different types of disorder were found in the highest frequencies in 3-, 4- and 5-year-old horses (27.8%, 29.2% and 29.7%, respectively). Among 6-year-olds or older horses, those having only one type of disorder accounted for the greatest proportion (47.6%). The prevalence of pharyngeal lymphoid hyperplasia or abnormalities of the epiglottis decreased with age. PMID- 7548392 TI - Characterization of rainbow trout C-polysaccharide binding proteins. AB - Two proteins were isolated from rainbow trout sera by affinity chromatography using C-polysaccharide-Sepharose 4B column. One was appropriate to the trout C reactive protein (CRP) that was reported previously (Murai et al. 1990. Dev. Comp. Immunol. 14: 49-58). The other was a newly found protein that had apparent molecular weight of 135,000 on native gradient polyacrylamide gel electrophoresis, and isoelectric points of 5.2-5.8. The N-terminal sequence (twenty amino acids) of the newly found protein was similar to CRPs from other species (e.g. 44% homology with plaice CRP). On electron microscope, the newly found protein was observed as pentagonal symmetry structure. PMID- 7548393 TI - Production and immunohistochemical characterization of specific antibody to oviductal fluid of miniature 'Shiba' goat. AB - Monoclonal antibodies were produced against the oviductal secretory substances in 'Shiba' goats and their nature was characterized. In the ampullary region, very strong immunoreactivity with one clone (EE-1) was localized within the cytoplasm of non-ciliated cells ("secretory cells"), whereas very little or no immunoreactivity was observed in the isthmus region. This reactivity pattern was observed in both estrous and luteal phases. Immunohistochemical analysis showed the antigen to be either absent or minimally present in other organs including the alimentary canal, liver, pancreas, lung, kidney, uterus, thyroid gland, heart, spleen and spinal cord, and in oviducts of other species such as hamster, sheep and cow. The immunoglobulin isotype of EE-1 was determined as IgM with a kappa light chain. The positive reactions were eliminated by treating deparaffinized sections with periodic acid or testicular hyaluronidase. Results obtained in this study suggest that ampullary secretory cells in 'Shiba' goat oviduct contain a species- and tissue-specific antigen containing hyaluronic acid or hyaluronidase-sensitive carbohydrates. PMID- 7548396 TI - Macroscopic observations of mammary glands and teats of Japanese serows, Capricornis crispus, with special reference to past gestation. AB - Mammary glands and teats were examined to estimate the reproductive history of Japanese serows. The presence of milk was an indicator of reproduction in the previous year. The thickness of the mammary glands is indicative of their function. The females whose mammary glands are more than 10 mm thick may be regarded as having delivered half a year before. The distance between teats is not always indicative of the function of the mammary glands. Animals with teats 10 mm or less in length and those with teats longer than 10 mm may be judged to be nulliparous and multiparous, respectively. PMID- 7548394 TI - Vascular pattern of a metastatic liver tumor in a dog:--a scanning electron microscopic study of resin casts. AB - Metastasis of a malignant mixed tumor occurring in the abdominal wall was found in the liver of a 15-year-old dog male. The vascular system of the metastatic lesion was examined by scanning electron microscopy using the resin cast technique. The existence of arterio-portal shunts was confirmed. The shunts arose from branches of the hepatic artery in retrograde or vertical direction and merged into the capillary plexus replacing the branches of the portal vein, forming the so-called thread and streaks vessel in the field of hepatic angiography. Vessels with the thread and streaks appearance histologically represented tumor emboli growing in the lumen of a large branch of the portal vein. There were capillaries running inside along the portal vein and forming a longitudinal dense network in the vein. Branches of the hepatic artery feeding the metastatic lesion were well developed and formed a coarse arterial network in their course. Tributaries of the hepatic vein from the lesion were also well developed. PMID- 7548395 TI - Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR. AB - For a rapid diagnosis of canine distemper virus (CDV) infection, the reverse transcription-PCR (RT-PCR) was carried out to detect CDV nucleoprotein (NP) gene from canine peripheral blood mononuclear cells (PBMCs). Two sets of primers were targeted to two regions of NP gene of CDV Onderstepoort strain. The NP gene fragments were well amplified by the RT-PCR in each of the RNA extracts from Vero cells infected with 6 laboratory strains of CDV including Onderstepoort strain, and from PBMCs of a dog experimentally infected with CDV. The amplified NP gene was detected in 17 of 32 samples from dogs which were clinically suspected for CDV infection at veterinary hospitals. No RT-PCR product was found in 52 samples from healthy dogs including 40 specific pathogen free beagles vaccinated with an attenuated live virus-vaccine for CDV and 12 stray dogs. The RT-PCR provides a fast, sensitive, and supplementary method for the diagnosis of CDV infection in dogs. PMID- 7548397 TI - Cranial and cerebral-ventricular landmarks for accurate stereotaxic approach to hypothalamic nuclei in the goat brain. AB - This paper describes a new stereotaxic coordinate system for the goat brain based on cranial landmarks. An osseous triangle (a-b-c) formed by the point of junction of the crista galli with the caudal ventral part of the frontal sinus septum (a), the external occipital protuberance (b), and the midsagittal projection of the external acoustic meatus (c), was measured using lateral radiographs and ventriculographs and showed a constant mathematical relation. The rostral angle was 20.3 +/- 1.0 (mean +/- SD) degree in 23 goats studied regardless of their cranial size which varied considerably from one animal to another. The hypotenuse length (a-b distance) was found to be a good predictor of the rostral nuchal position of the anterior commissure (AC) and the infundibular recess of the third ventricle (INF), by which the individual variance of the stereotaxic coordinates for a given hypothalamic structure could be compensated. The anterior-posterior distances from the external acoustic meatus was highly correlated with the a-b distance for AC (r = 0.88) and INF (r = 0.90). Using these cranial landmarks and the method outlined in this paper, uncertainty in coordinate values for AC and INF in the goat brain was reduced considerably in comparison to deviation observed when the ordinary Horsley-Clarke axis (Reid's plane) was employed. PMID- 7548398 TI - Hypothalamic structures of the goat on stereotaxic coordinates. AB - Serial brain sections of 50 microns thickness were made from celloidin embedded brains taken from adult male goats. They were stained by means of cresyl violet either with or without luxol fast blue, and the cellular and fiber patterns were examined both macro- and microscopically. Brain sections were transferred on to scanner and their images were recorded by computer. The brain maps were made with the aid of appropriate software and presented as a series of coronal graphics of the hypothalamus that cover the area from the mammillary tubercles to the level of the optic chiasm. The thalamus and the amygdala were also included. Reduction of the brain tissue during the histological procedure was analyzed by means of coronal and sagittal scanning by computer tomography (CT) imaging. It was related to the proportion of the gray and white matters in the encephalic area. The hypothalamus showed an 11% shrinkage. The stereotaxic location of the hypothalamic nuclei was estimated by compensating for the reduction rate of the area. The stereotaxic coordinates developed for goat brain were easy to apply and transfer to the computer coordinate system. PMID- 7548399 TI - Molecular cloning of cDNA for equine ovarian inhibin/activin beta A subunit. AB - cDNAs encoding equine inhibin/activin beta A subunit were isolated from an equine follicle cDNA library and characterized. Using primers based on the rat inhibin/activin beta A subunit cDNA sequence, a RT-PCR was performed to generate the probe for screening. Four positive clones were isolated. Analysis of the nucleotide sequence of these clones revealed that two pairs of identical clones were present, Eq-beta A-1 (0.9 kb) and Eq-beta A-2 (1.5 kb). Eq-beta A-2 clone contained a complete open reading frame encoding 426 amino acids. The deduced amino acid sequence of equine inhibin/activin beta A subunit showed high similarity (> 90%) to those of five other mammalian species. Northern blot analysis revealed that placenta from mare on day 180 of pregnancy contained a 1.5 kb inhibin/activin beta A subunit mRNA. PMID- 7548400 TI - Immunocytochemical localization of desmin in skeletal muscle of swine. AB - A porcine antibody was used to clarify the localization of desmin filaments in the swine longissimus muscle by post-embedding immuno-electron microscopy. The major site of desmin localization was the bundle of filaments connecting Z-discs of adjacent myofibrils. These desmin filaments were localized not only between but also within myofibrils. This indicates that there exist more complicated networks rather than simple bundles of desmin filaments running parallel to the Z discs. Clarification of desmin distribution within the myofibrils surrounding Z discs provides a basic concept for consideration of the combination and transformation of vimentin to desmin in developing human embryonic muscles. PMID- 7548401 TI - Comparison of extravascular lung water volume with radiographic findings in dogs with experimentally increased permeability pulmonary edema. AB - The relationship between extravascular lung water volume (ELWV) and chest radiographical findings was studied in general-anesthetized beagles. The dogs were experimentally injected with oleic acid to increase pulmonary vascular permeability. When the ELWV value in the dogs increased more than approximately 37% from the control value, their chest radiographs began to show signs of pulmonary edema. At this time, the chest X-ray density increased to 10% above the control level. PaO2 decreased, and PaCO2 increased after the administration of oleic acid. This clearly showed that the pulmonary gas exchange function was reduced following increasing ELWV. This comparison showed that probably the thermal-sodium double indicator dilution measurement of ELWV can detect slight hyperpermeability pulmonary edema that does not show on chest radiographs. The chest radiograph was therefore not suitable for the detection of slight pulmonary edema, because it did not show any changes in the early stages in hyperpermeability pulmonary edema. PMID- 7548402 TI - Quantification of feline immunodeficiency virus (FIV) proviral DNA in peripheral blood mononuclear cells of cats infected with Japanese strains of FIV. AB - The polymerase chain reaction (PCR) method was applied for measurement of the proviral DNA copy number of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMC) of cats experimentally and naturally infected with FIV. In experimentally infected cats except one cat infected with the Petaluma strain, FIV-specific DNAs were efficiently amplified with the PCR method under the conditions used in this study. In the naturally FIV-infected cats, the specific DNAs were also amplified. We established a quantitative method for measurement of proviral DNA copy number in PBMC from cats infected with TM2-type of FIV strains, and found that the number was variable among the six cats examined, ranging from 10(4.0) to 10(5.7) copies per 10(5) PBMCs. This method can be applicable to cats naturally infected with FIV of TM2-type. Proviral DNA quantitation developed here could be useful as an additional parameter to evaluate the relationships among the proviral load, immune response and development of the clinical symptoms, and to monitor efficacy of antiviral therapy in vivo. PMID- 7548403 TI - Effects of vitamin B2 on neutrophil functions in cattle. AB - Vitamin B2 was intramuscularly administered to Holstein cattle and the ensuing changes in peripheral blood neutrophil function were investigated. The neutrophil count displayed a significant increase at 1-2 days after administration, while nitroblue tetrazolium reducing activity and phagocytic bactericidal activity were enhanced at 1-4 days after administration in calves and at 1-6 days after administration in adult cows. The increases in the neutrophil count and the activation of neutrophil functions were observed, being manifested at dosages of 10 mg/kg or greater for calves and 5 mg/kg or greater for adult cows. PMID- 7548404 TI - Protective effects of prostaglandin E2 on the paraquat-induced constriction of the fetal ductus arteriosus in the rat. AB - Pregnant rats on day 21 of gestation received a subcutaneous injection of paraquat (25 mg/kg). Two hr later, some fetuses of these rats received a subcutaneous injection of prostaglandin E2 (PGE2). The caliber of the ductus arteriosus (DA) of fetuses was measured 1 hr later. The DA of fetuses of paraquat treated pregnant rats was significantly constricted compared with that of fetuses of control pregnant rats given physiological saline. However, the DA of PGE2 treated fetuses of paraquat-treated pregnant rats had a caliber comparable to that of control rats. The maternal plasma PGE2 level decreased following paraquat treatment. These results suggest that the decrease of maternal PGE2 level results in a constriction of the DA of fetuses. PMID- 7548406 TI - Effects of splenectomy on luteolysis in pseudopregnant rabbits. AB - The effects of splenectomy on luteolysis in pseudopregnant rabbits were observed. Pseudopregnancy was induced in the rabbits by mating with vasoligated mature males and injection of human chorionic gonadotrophin (D0). In these rabbits, the concentration of serum progesterone increased for 7 days after the induction of pseudopregnancy. In the control group (sham-operation), the concentration of serum progesterone returned to the level of the pre-ovulatory levels by 14 days of pseudopregnancy. On the other hand, in the splenectomized group, in which splenectomy was performed at D7, the serum progesterone concentration was maintained at the level of the functional luteal phase (D7) at least until 21 days of pseudopregnancy. These findings indicate that the spleen is concerned with luteolysis of the rabbit. PMID- 7548405 TI - Isolation and characterization of Staphylococcus aureus in rats trapped at restaurants in buildings in downtown Tokyo. AB - 165 (18.1%) out of 910 rats captured at restaurants in 14 buildings in downtown Tokyo were positive for Staphylococcus aureus. The 165 S. aureus strains isolated were biotyped into A, B, C, D, G, and untypable groups (UT1 and UT2). The UT1 was the most frequent (72 strains, 43.6%), followed by biotype G (33 strains, 20.0%). The strains were classified into coagulase types I, II, III, IV, V, VI, VII, VIII, and an untypable group. Coagulase type V was the most frequent (92 strains, 55.8%), followed by coagulase type VII (25 strains, 15.2%). Enterotoxins A, B, C, or D were produced by 35 strains. Enterotoxins A and B were the most frequent (13 strains each). Toxic shock syndrome toxin-1 was produced by 3 strains. 65 strains were resistant to ampicillin, 2 to oxytetracycline, and 1 to erythromycin. PMID- 7548407 TI - Assessment of the sedative effect of medetomidine and determination of its optimal dose in thoroughbred horses. AB - The present study was carried out to assessing the sedative effect of medetomidine and determining its optimal dose in thoroughbred horses. Excessive ataxia after sedative treatment is dangerous for horses. Therefore three doses which may cause sufficient sedation with only mild ataxia were examined. Response to stimulation and the severity of ataxia suggested that 7.5 micrograms/kg BW, i.v., is optimal. PMID- 7548408 TI - Analysis of Fasciola sp. antigen by enzyme-linked immunotransfer blot using sera from experimentally and naturally infected cattle. AB - Soluble polypeptides extracted from adult Fasciola sp. were analysed by SDS-PAGE, and their antigenicity was characterized by enzyme-linked immunotransfer blot (EITB) using sera from experimentally and naturally infected cattle. Polypeptides of adult Fasciola sp. with estimated molecular mass of 64-52 kDa, 38-28 kDa, 17 kDa, 15 kDa, 13 kDa and 12 kDa, were recognized by sera obtained in both early and late stages of infection. Furthermore, two polypeptides of more than 160 kDa were detected by sera only in the early stage of infection. The results of EITB using sera of dairy cows naturally infected with Fasciola sp. suggest that polypeptides of 64-52 kDa may be candidates as serodiagnostic antigen of bovine fascioliasis. PMID- 7548409 TI - Assignment of the bacterial agent of urinary calculus in young rats by the comparative sequence analysis of the 16S rRNA genes of corynebacteria. AB - Comparative 16S rRNA gene sequencing was used to assign four isolates of spontaneous urinary calculus in young laboratory rats. The phylogenetic relationships among the rat isolates and selected species of corynebacteria were also inferred. Based on the homology and evolutionary distance analysis, the 16S rRNA genes of the rat isolates were almost identical with that of Corynebacterium renale ATCC 19412. Also the results of the phylogenetic analysis showed a close relationship among the isolates and C. renale, but they were clearly different from C. pilosum, C. cystitidis, C. kutscheri and Rhodococcus equi. The results of the present study and previously published biochemical data demonstrate that the organism involving urinary infections in young rats is identified to be C. renale. PMID- 7548410 TI - Immunological cross-reaction between lactoferrin and transferrin. AB - Hardly any or only a weak immunological cross-reaction was found between native lactoferrin (Lf) and transferrin (Tf). However, when these iron-binding proteins were denatured with sodium dodecyl sulfate and dithiothreitol, a definite immunological cross-reaction was detected between them. These results indicate that although Lf and Tf are immunologically quite different from each other in their native forms, they have the common antigenic determinant(s) in their unfolded forms. PMID- 7548411 TI - A bioassay for bovine interleukin-1 by the A375 cell growth inhibition. AB - Usefulness of a human melanoma cell line A375 was evaluated for detecting bovine interleukin-1 (IL-1). The A375 cell growth was inhibited by culture supernatant of lipopolysaccharide-stimulated bovine peripheral blood mononuclear cells (LPSsup) in a dose dependent manner. A mixture of anti-human IL-1 alpha and beta antibodies suppressed 60% of this inhibitory activity and was confirmed to bind to about 23 k dalton peptides in the LPSsup by Western blotting. Although serum and bronchoalveolar lavage fluid from a healthy cow showed a low inhibitory activity, those from pneumonic cows showed the higher activities. These activities were also suppressed by anti-human IL-1 antibodies. These findings show the A375 cell growth inhibition assay can be a useful bioassay for bovine IL 1 (like) activity. PMID- 7548412 TI - Low level of immunoglobulin G2a subclass correlates with a deficiency in T helper cell function in LEC mutant rats. AB - Long-Evans Cinnamon (LEC) rats bear a congenital deficiency in CD4+8- thymocytes and consequently a deficiency in helper T cell function. This mutation is caused by a single recessive gene referred to as thid (T helper immunodeficiency). It has been reported that rat immunoglobulin(Ig) G2a subclass is a counterpart of the mouse IgG1. Serum IgG2a levels in LEC rats were ten-fold lower than those of normal rats. To identify a cause of low IgG2a levels in LEC rats, we made backcross rats, (F344 x LEC)F1 x LEC, and examined linkage to the thid mutation. The serum IgG2a levels of rats showing thid/thid phenotype were much lower than those of rats showing +/thid phenotype. This indicates that the thid mutation correlates with low level of IgG2a subclass. Furthermore, LEC rat B cells were shown to secret IgG2a normally when these were stimulated with LPS and IL-4, suggesting that a cause of low level of IgG2a was due to defect of T cell function but not due to B cell disfunction in LEC rats. These results confirm the idea that T-helper (Th) function is necessary for the class switch to IgG2a subclass in rats. PMID- 7548413 TI - Natural infection with attaching and effacing Escherichia coli and adenovirus in the intestine of a pigeon with diarrhea. AB - Attaching and effacing Escherichia coli (AEEC) and adenovirus infections in a 6 month old pigeon were found by retrospective histologic examinations. Histologically, numerous gram-negative bacilli covered the surface of the enterocytes of the ileum. Brush borders were not sharply defined on the apical enterocytes at the sites of bacterial attachment. Scanning electron microscope showed that much of the mucosal surface was heavily colonized by bacilli and that the microvilli were lost. Many intranuclear inclusions were seen in enterocytes from the duodenum to the cecum. On transmission electron microscopy, adenovirus like particles were observed in the nuclei of enterocytes. PMID- 7548414 TI - Suppression of canine parvovirus growth in CRFK cells by canine distemper virus. AB - The growth of canine parvovirus (CPV) in Crandell feline kidney cells was suppressed when canine distemper virus (CDV) was simultaneously inoculated. The both virus antigens were found in the same cells. The synthesis of capsid protein antigen of CPV decreased in the co-inoculated cells. The growth of CDV was not affected by co-inoculation with CPV. These results suggest that the CDV showed suppressive effects on the CPV growth in cells when they are inoculated simultaneously. PMID- 7548415 TI - Immunohistochemical studies on the development of TSH cells in the pituitary of Xenopus laevis larvae. AB - Since thyroid-stimulating hormone (TSH) producing cells are thought to play an essential role during metamorphosis, their development was immunohistochemically examined to reveal the appearance and topographical changes in the distribution in the hypophysis of Xenopus laevis tadpole from hatching to the end of metamorphosis. TSH-immunoreactive cells initially appeared at stage 49 (just the beginning of development of the pars nervosa in the hypophysis) as small clusters in the middle part of the pars distalis. They showed conspicuous changes in number during late premetamorphosis: their number reached a peak at stage 51, suddenly decreased at stage 52 (just before completion of the hypophysial histogenesis) followed by gradual increase until the end of metamorphosis. At later stages, they were restricted to the posterior half of the pars distalis. PMID- 7548416 TI - Superovulation induction by human menopausal gonadotrophin in rabbits. AB - Superovulation induction in laboratory animals is an important technique as a means of providing eggs for embryonic research. Superovulation induction by human menopausal gonadotrophin (HMG) has not been fully established in rabbits. In the present study, the relationship between the dosage of injected HMG and the superovulatory response including fertilization rate was studied. The most satisfactory result was obtained in the group in which 30 iu of HMG was injected subcutaneously three times at 24-hr intervals. The superovulatory response by this method was considerably better than that by conventional six injections of follicle-stimulating hormone (FSH) having been widely accepted. PMID- 7548417 TI - Production of term offspring by in vitro fertilization using old mouse spermatozoa. AB - We successfully produced offspring of old male BDF1 mice using in vitro fertilization. Although 7 old male mice (33 months of age) were infertile and revealed frequent degeneration in the seminiferous epithelia, 4 of them had spermatozoa in the caudae epididymides. The IVF rate of their sperm with eggs from young ICR mice was very high (82.0%). The production rate of offspring was also satisfactory (61.6%). The present study indicates that the IVF method is useful for producing offspring of aged male mice. PMID- 7548419 TI - Synergistic effects of insulin-like growth factor II (IGF-II) with leukemia inhibiting factor (LIF) on establishment of rat pluripotential cell lines. AB - To examine effects of IGF-II on establishment of pluripotential diploid cells from rat embryos, we cultured blastocysts in medium containing mouse LIF with or without IGF-II. Combination of mouse LIF (5,000 units/ml) and rat IGF-II (100 ng/ml) promoted growth of inner cell mass (ICM) and was effective for establishment of pluripotential cell lines derived from the ICM. The cell lines indicated colony forms different from the rat ES cell lines. However, they showed morphological alteration to adult-like tissue cells, formed embryoid body in suspension culture, and thus, seemed to retain a pluripotent characteristics. The rat IGF-II is useful for establishing of pluripotential cells efficiently. PMID- 7548420 TI - A novel repetitive sequence from Mycoplasma hyopneumoniae. AB - We have isolated a novel repetitive DNA element from Mycoplasma hyopneumoniae strain VPP11, the entire structure of which is distinct from those of prokaryotic transposons, insertion sequences or eukaryotic retroposons reported. Southern blot hybridization experiments indicate that at least eight copies of this element locate on the M. hyopneumoniae genome. The size of this repetitive sequence is 4,193 bp which includes 270- and 272-bp direct long terminal repeats at each terminus. The internal domain of this element defines three open reading frames. PMID- 7548418 TI - Protoporphyric disorder in livers of broiler chickens. AB - Two cases of protoporphyrin (PP) disorder detected in a 60- and 65-day old female chicken were described. The gross lesions were restricted to the livers which were enlarged and dark green to black in color. Histologically, dark brown granules were found in hepatocytes, Kupffer cells, macrophages, sinusoids, bile canaliculi, and bile ducts. These granules, as seen in smears and sections of livers, were red under a fluorescence microscope and exhibited bright birefringence with a centrally located dark Maltese-cross by polarized light. Ultrastructurally, these granules consisted of aggregates of needle-like crystals in a radial arrangement. Fluorometrically, extracted level of PP in the affected liver was determined to be 390.6 micrograms per gram of wet tissue. Spectrofluorometric scans of liver extract and PP standard were almost identical. PMID- 7548421 TI - Biochemical and antigenical characterization of tannin-protein complex degrading enterobacteria isolated from koalas, Phascolarctos cinereus. AB - Biochemical and antigenical characteristics of tannin-protein complex degrading enterobacteria (T-PCDE) isolated from Koalas, Phascolarctos cinereus, were investigated. T-PCDE had a specific profile of characteristics, and T-PCDE was distinguished from those of 12 type strains of Enterobacteriaceae used. PMID- 7548422 TI - Inhibition of viral multiplication in cells chronically infected with mouse hepatitis virus by antisense RNA against the polymerase gene. AB - Recently, we showed that the antisense RNA containing a hammerhead ribozyme sequence against the polymerase gene of mouse hepatitis virus (MHV) inhibited viral multiplication in acute infection [10]. In the present study, we examined the inhibitory effects of an antisense RNA on viral multiplication in chronic MHV infection. In cell line LR-2, in which the 926-nucleotide (nt) antisense RNA containing a ribozyme sequence against the polymerase gene was expressed constitutively at a high level, chronic MHV infection was established through the maintenance of infection over 100 days postinfection (d.p.i.). After 200 d.p.i., no infectious progeny virus was observed in the culture medium of chronically MHV infected LR-2 cells. Our present results showed that the anitsense RNA could also inhibit viral multiplication in chronic MHV infection. PMID- 7548423 TI - Incidence of brown dog ticks, Rhipicephalus sanguineus, at a kennel in Okayama Prefecture. AB - Twenty two adult dogs suffered from tick infestation at a kennel in Okayama Prefecture in the summer of 1994. Four of them had been introduced from U.S.A. Some dogs showed pyoderma, anemia, neutrophilia or eosinophilia. Neither Babesia gibsoni nor Hepatozoon canis was detected on the smear samples of peripheral blood. The ticks detected were morphologically identified as Rhipicephalus sanguineus. This case was a rare incidence of the tick in the other areas of Japan than Okinawa. PMID- 7548424 TI - Effect of enzymes on the growth of human and animal rotaviruses. AB - The growth of group A human, bovine, equine and porcine rotaviruses were enhanced by pretreatment of virus with pancreatin, trypsin, protease, alkaline phosphatase or pepsin and incorporation of these enzymes in maintenance medium. In contrast, alpha-amylase or lipase inhibited the growth of equine and porcine rotaviruses. The other enzymes, adenosine deaminase, lactase, lysozyme, ribonuclease or triose phosphate isomerase gave little or no change in the growth of all four rotaviruses. PMID- 7548425 TI - Effects of hindlimb cutaneous afferent inputs on spinal reflex recording from tail muscle motoneurons in the spinalized cat. AB - In 4 spinalized cats, the effects of afferent inputs from hindlimb cutaneous nerves (sural cutaneous nerve: Sur) on mono-and poly-synaptic reflex recorded from tail muscle motoneurons were studied before and after spinal lesioning at S2 3 level. Monosynaptic reflex was enhanced by ipsilateral Sur stimulation at short conditioning-test stimulus interval and this effect was not observed after spinal lesion of ipsilateral side of spinal cord. Polysynaptic reflex was inhibited by stimulation of Sur in both sides and this inhibitory effect was depressed by contralateral spinal lesioning. PMID- 7548426 TI - Lungworm, Filaroides osleri, infection in a dog in Japan. AB - A 7-month-old male Pomeranian had severe dyspnea for 2 weeks. The lateral bronchogram showed a stenosis of the trachea. Inspite of supportive therapy including supplemental oxygen, the dog died 5 days later. Six pedunculated nodules were recognized in the mucosal surface of the trachea at necropsy. The tracheal nodules were histopathologically granuloma characterized by many coiled parasites containing a little collagen fibers, lymphocytes, plasma cells and macrophages. Female parasites had a lot of embryonated eggs in the uterus. Immature worms were observed in the dilated lymph vessels of bronchial and bronchiolar wall in the lungs. The worms were identified as Filaroides osleri based on the parasitological examinations. PMID- 7548427 TI - Restriction endonuclease analysis of bovine herpesvirus type 1 isolates from calves with fatal encephalitis: comparison with vaccine virus. AB - Meningo-encephalitis in feedlot cattle sporadically occurred in the Tokachi area in northern Japan. The calves had been vaccinated intranasally with a mixed live vaccine (infectious bovine rhinotracheitis virus, bovine viral diarrhea-mucosal disease virus, and parainfluenza 3 virus) for which intramuscular inoculation was indicated. Two additional live vaccines, bovine adenovirus type 7 and bovine respiratory syncytical virus, had been inoculated simultaneously. Eleven isolates of bovine herpesvirus type 1 were plaque-purified from two brains with fatal encephalitis; their viral DNAs were examined by restriction endonuclease analysis (REA) using PstI and HindIII. The REA patterns of the virus clones were almost identical to those of the vaccine strains 758-43, suggesting that the isolates from this outbreak of fatal encephalitis originated in the abnormally administered vaccine. PMID- 7548428 TI - Protein A in Staphylococcus aureus isolates from pigs. AB - The presence and quantity of protein A in Staphylococcus aureus 147 isolates from the tonsils of healthy pigs were examined by three methods. Cell-bound protein A was detected in 71 (48%), 104 (71%) and 123 (84%) of 147 isolates by the slide hemagglutination test, microplate hemagglutination test and enzyme-linked immunosorbent assay (ELISA), respectively. Extracellular protein A was not detected in any isolates by the microplate hemagglutination test. When the quantity of cell-bound protein A in the isolates was determined by the ELISA, most of the isolates contained about 0.8 to 2.2 micrograms of protein A/ml in bacterial cell suspensions of a concentration of MacFarland No. 3. PMID- 7548429 TI - The antitumor activity of the DNA fraction from Mycobacterium bovis BCG (MY-1) for glioblastoma. AB - The antitumor activity of the DNA fraction extracted from Mycobacterium bovis BCG (MY-1) for glioblastoma was investigated in the experimentally produced brain tumor in rats. The tumor-bearing rats were given intralesional injection of 1 mg of MY-1 twice a week for three weeks, and were sacrificed for comparison with those of control rats. The main macroscopic features of the tumors treated with serial injections of MY-1 were cystic and destructive structures, which were histologically characterized by multiple microcysts containing macrophages. Furthermore, infiltration of leukocytes as well as the perivascular cuffing in the marginal area was observed. These findings suggested that the serial injections of MY-1 into the brain tumor have the therapeutic potential for glioblastoma. PMID- 7548430 TI - Invasion and viability of Campylobacter jejuni in experimentally contaminated Japanese quails' eggs. AB - In Japanese quail eggs experimentally immersed in a suspension of Campylobacter jejuni from human patients (Y6817, Y6878), strain Y6817 was recovered from 15 (18.8%) eggs among 80 between 0.5 hr and 72 hr after immersion. Invasion of the eggs by strain Y6878 was also seen in 10 (12.5%) of 80 eggs between 0.5 hr and 24 hr after immersion. When egg yolk was inoculated with both strains and held at 4 degrees C, the organisms were detectable for a longer time than the ones kept at 20 degrees C. On the other hand, when albumen was inoculated with both strains, the organisms died within 96 hr at 4 degrees C and within 24 hr at 20 degrees C. PMID- 7548431 TI - Immunohistochemistry products workshop: forum on the scientific and regulatory issues sponsored by the Food and Drug Administration. PMID- 7548432 TI - Differentiation of Langerhans cells from interdigitating cells using CD1a and S 100 protein antibodies. AB - The present study shows that Langerhans cells can be differentiated from interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S 100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells. PMID- 7548433 TI - Fixation and staining of planaria for histological study. AB - Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acid-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH. PMID- 7548434 TI - Changes in neutrophil granule protein and cytoplasmic fibrils in human acute myeloid leukemias. AB - Granule protein deficiencies in morphologically mature neutrophil cells of peripheral blood from human patients with acute myeloid leukemia was demonstrated using post-embedding immunocytochemistry. Abnormal immunoreactivity of granule proteins was detected in seven of nine patients. Decreased immunoreactivity patterns were found more for the primary granule markers elastase and myeloperoxidase than for the secondary granule marker lactoferrin. Leukemias with a predominant myeloid component, in contrast to those with a predominant monocytoid component, had more neutrophil cells showing immunodeficiencies for one or more granule markers. The proportion of neutrophil cells showing immunodeficiencies varied greatly for each granule marker; more variation was obtained for elastase, lactoferrin and myeloperoxidase than for lysozyme, possibly because lysozyme is a marker for both granule types. In addition, no correlation could be found between any of the immunoreactivity deficiencies for the neutrophil granule glycoproteins elastase, lactoferrin, lysozyme and myeloperoxidase and the abundance of a particular set of ultrastructural features in the circulating leukemic cells from any of the nine patients. Nonetheless, most of the immature myeloid cells from peripheral blood of leukemic patients showing neutrophil protein immunoreactivity abnormalities in one or more granule markers often and randomly displayed one or more unusual ultrastructural features. The clinical and pathological significance of neutrophil granule protein deficiencies and the abundance of fibrillar structures in malignant myeloid cells presently is uncertain. PMID- 7548435 TI - Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis. AB - Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Epon embedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility. PMID- 7548436 TI - A comparison of digoxigenin and biotin labelled DNA and RNA probes for in situ hybridization. AB - A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin. PMID- 7548437 TI - Effects of storing initiated 2-hydroxyethyl methacrylate solutions for embedding tissues for light microscopy: some practical implications. AB - The effects of storing 2-hydroxyethyl methacrylate (HEMA) solutions for embedding tissues for light microscopy were studied using three commercially available HEMA embedding kits: Technovit 7100, Technovit 8100, and JB-4. These HEMA solutions were examined at various times of storage over a period of one year using a panel of physicochemical techniques including gas chromatography, titration, viscosimetry, determination of the maximum polymerization temperature and the time required to reach the maximum temperature, and detection of degradation products of HEMA monomers by histochemical procedures. The quality of the resin blocks was examined by the observation of mini-folds in sections. Data obtained from these tests showed that the release of by-products as a result of the degradation of the HEMA monomer during storage of HEMA solutions does not occur. Development of cross-linking agents by transesterification of HEMA monomer was not detected either. Gradual decrease of the inhibitor concentration during storage proved to be the main cause of the reduction of shelf-life of HEMA solutions. Inconsistent tissue infiltration after storage may be due to decreased rates of tissue penetration as a result of HEMA chain lengthening. Guidelines for safe and economical handling of HEMA mixtures are given. PMID- 7548438 TI - Cell cycle perturbation by sodium butyrate in tumorigenic and non-tumorigenic human urothelial cell lines assessed by flow cytometric bromodeoxyuridine/DNA analysis. AB - The effect of sodium butyrate on cell proliferation was studied in eight human urothelial cell lines differing in transformation grade (TGr): Hu 1752 (mortal, TGr I); HCV29 (immortal and tumorigenic, TGr II); HCV29T, T24, T24A, T24B, Hu 961A and Hu 1703He (tumorigenic, TGr III). In all cell lines, except Hu 1752, addition of 4 mM sodium butyrate at 18 h after replating resulted in a significantly decreased population of adherent cells after a further 24-48 h. This might partially be explained by detachment of cells, probably mainly S phase cells, from the substrate in the lines HCV29, HCV 29T, Hu 961A and Hu 1703He. Flow cytometric DNA analysis of the adherent cell population showed that all TGr II and III urothelial cell lines were DNA aneuploid, and that butyrate perturbed the cell cycle distribution in these cell lines, mainly by a decrease of the S phase fraction. Flow cytometric bromodeoxyuridine (BrdUrd)/DNA analysis of continuously BrdUrd labelled cultures, using a 'washless' BrdUrd/DNA staining technique, showed that butyrate inhibited the G0/1-S phase transition, indicated by a delayed depletion of BrdUrd negative G0/1 cells in the cell lines HCV29, HCV29T, T24B, Hu 961A and Hu 1703He. BrdUrd/DNA analysis further showed that butyrate inhibited the G2M-G0/1 phase transition, indicated by a pronounced accumulation of BrdUrd positive G2M cells in the cell lines HCV 29T, T24B, Hu 961A and Hu 1703He. Microscopy of HCV29T and Hu 961A cells indicated that this block did not occur in mitosis. The parental cell line T24 and the cell line T24A did not show an accumulation of BrdUrd negative G0/1 cells or BrdUrd positive G2M cells like that occurring in the derived cell line T24B. PMID- 7548439 TI - An analysis of the growth of the retinal cell population in embryonic chicks yielding proliferative ratios, numbers of proliferative and non-proliferative cells and cell-cycle times for successive generations of cell cycles. AB - Growth curves of the retinal cell population of embryonic chicks were fitted by a branching-process model of cell population growth, thereby estimating the proliferative ratios and mean cell-cycle times of the generations of cell cycles that underlie retinal growth. The proliferative ratio determines the proportion of cells that divides in the next generation, so the numbers of proliferative and non-proliferative cells in each generation of cell cycles were obtained. The mean cell-cycle times determine the times over which the generations are extant. Assuming growth starts from one cell in generation 0, the proliferative cells reach 3.6 x 10(6) and the non-proliferative cells reach 1.1 x 10(6) by generation 23. The next four generations increase the proliferative cell numbers to 13.9 x 10(6) and produce 20.1 x 10(6) non-proliferative cells. In the next five generations in the end phase of growth, non-proliferative cells are produced in large numbers at an average of 13.9 x 10(6) cells per generation as the retinal lineages are completed. The retinal cell population reaches a maximum estimated here at 98.2 x 10(6) cells. The mean cell-cycle time estimates range between 6.8 and 10.1 h in generations before the end phase of growth and between 10.6 and 17.2 h in generations in the end phase. The retinal cell population growth is limited by the depletion of the proliferative cell population that the production of non-proliferative cells entails. The proliferative ratios and the cell-cycle time distribution parameters are the likely determinants of retinal growth rates. The results are discussed in relation to other results of spatial and temporal patterns of the cessation of cell cycling in the embryonic chick retina. PMID- 7548440 TI - Effect of ebrotidine on the density of antral G-cells in the gastric mucosa in the rat. AB - Two groups of male rats were treated orally for 60 days with ebrotidine and cimetidine, used as reference standard, respectively. The dose used of both drugs was 500 mg/kg. A third group, used as control, received 10 ml/kg of an aqueous agar suspension. After receiving the last dose, the animals were killed by inhalation of CO2 in a sealed chamber and the stomachs were removed for histological preparation. The purpose of this study was to count antral G-cells throughout the gastric mucosa by light microscope. A PAP system-associated antigastrin immunohistochemical method was used for cell identification. Cell density, with respect to their location in the gastric mucosa and the treatments given, was calculated using image analysis techniques. The results showed that ebrotidine did not cause any significant differences in the density of the population of these cells compared with the control group, while cimetidine induced a significant proliferation of antral G-cells. PMID- 7548441 TI - Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours. AB - We analysed the effects of nitrogen mustard (HN2) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN2 produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN2, the host cells in the treated tumours showed a significantly decreased G0/G1 peak and an increased G2/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G0/G1 peak coupled with relatively decreased proportions of S and G2/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN2 administration (P < 0.01). Thirty-two days after treatment with HN2, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P < 0.01). These results show that the administration in vivo of HN2 can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN2 treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells. PMID- 7548442 TI - Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration. AB - Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37 degrees C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O2) or the 0.01% O2 mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10(6) cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O2 were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10(6) cells/h) in 21% O2 was 62.5. This rate increased to 428.8 pg/10(6) cells/h at 0.01% O2, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O2 were too low to be measured by ELISA. However, between 10% and 0.01% O2, secretion rates increased from 5.0 to 376.0 pg/10(6) cells/h, a 75-fold increase. Therefore, at very low O2 levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm3. PMID- 7548443 TI - Influence of long-term limitation of cell proliferation on the cell cycle duration. AB - The hypothesis was tested that constant, long-term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L-929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 10(4) cells. No changes in the duration of the cell cycle were observed when, following long-term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence. PMID- 7548444 TI - Inhibitory effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on rat hepatocyte proliferation induced by 2/3 partial hepatectomy. AB - To better understand the mode of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced alterations in hepatic cell proliferation and the potential link to tumour-promoting activity, we investigated the effects of TCDD on the expression of certain key genes involved in liver cell growth and the effects of TCDD on induced hepatocyte cell proliferation. Gene expression analysis was conducted, by Northern blot hybridization, using RNA isolated from female Sprague Dawley rat livers collected at various times during a 14-day dosing period with TCDD known to produce alterations in cell proliferation. No major changes were observed in the expression of the transforming growth factors TGF-alpha and TGF beta or in oncogenes ras, src and myc. However, the expression of the transcription factors C/EBP, HNF-1 alpha and HNF-4 decreased after 14 days of TCDD treatment. To investigate how TCDD affects hepatic growth, cell proliferation analysis was conducted in rats stimulated to undergo hepatocyte proliferation following either 2/3 partial hepatectomy or lead nitrate treatment. Cell proliferation was quantified by means of immunocytochemical detection of Proliferating Cell Nuclear Antigen (PCNA). Fourteen days of pretreatment with TCDD caused an overall inhibition of hepatocytes in the growth fraction (G1, S, G2 and M) from 61 +/- 3% in the control-partial hepatectomy group to 41 +/- 3% in the TCDD-partial hepatectomy group. A periportal pattern of cell proliferation was observed in the TCDD-partial hepatectomy group as compared to the panlobular pattern of cell proliferation in the control-partial hepatectomy group. TCDD pretreatment also produced an inhibition of cell proliferation induced by the liver mitogen lead nitrate. TCDD-induced inhibition of hepatocyte proliferation could play a role in TCDD tumour promotion and hepatocarcinogenesis through the creation of a environment whereby preneoplastic cells continue to expand while normal hepatocyte proliferation is inhibited. PMID- 7548445 TI - Hormone gradients and cartilage cell kinetics. AB - We present a model of growth control in mammalian cartilage growth plates by hormones. The model is based on the distribution of insulin-like growth factors I and II (IGF-I and IGF-II) and their receptors, and assumes that a hormone receptor complex of IGF controls cells proliferation. A system of differential equations is derived and solved with simplifications in extreme cases, for the one-dimensional time independent case. Even if opposite extremes, such as proliferation control by factors extrinsic to the cell versus intrinsic to the cell, are assumed, similar distributions of hormones and proliferating cells are produced. Hence, choice between alternative models of growth control must be based on empirical observations. On the positive side, similarities between our model for cartilage growth and other models for differentiation and proliferation are evident and might be exploited for unifying these systems on an abstract level. PMID- 7548447 TI - Oral intake of selenium has no effect on the serum concentrations of growth hormone, insulin-like growth factor-1, insulin-like growth factor-binding proteins 1 and 3 in healthy women. AB - The administration of large doses of selenium (Se) to rats leads to reduced serum levels of somatotropin (growth hormone) and insulin-like growth factor-1 (IGF-1), followed by growth retardation. Similar experiments in humans have been contradictory. The effects of wheat Se and selenomethionine supplementation were investigated in healthy, Norwegian women. In study 1, the participants (n = 18) were given Se-rich bread with 100, 200 and 300 micrograms Se daily for 6 weeks. Initial serum Se concentration were 1.5 +/- 0.2 mumol/l (mean +/- SD). Serum Se increased in a dose-dependent manner in the three groups (p < 0.001). There was no effect on somatotropin and IGF-1 at any of the Se doses given. In study 2 (n = 24), the effects of 400 micrograms selenomethionine daily for 15 weeks were studied in a placebo controlled study. In the treatment group, serum Se concentrations increased by more than 100%. There was, however, no effect on serum somatotropin and IGF-1 concentrations, nor was there any effect on IGF binding proteins 1 and 3. Our results indicate that at normal or slightly increased intakes, Se has no effect on the serum concentrations of these two hormones in healthy individuals. PMID- 7548446 TI - Dual porphyria of coexisting variegata and cutanea tarda. AB - While porphyria cutanea tarda and porphyria variegata are independent diseases, we report on seven rare cases with a coincidence of these two different porphyrias in one individuum. The mutual clinical symptom was a cutaneous photosensitivity, which is a major symptom in porphyria cutanea tarda and a facultative one in porphyria variegata. Additionally, five patients had also experienced episodes of acute abdominal pain, which were in three cases accompanied by neurological symptoms, thus offering evidence for an acute hepatic porphyria, such as porphyria variegata. Determination of urinary porphyrin metabolites revealed a porphyria cutanea tarda-like excretion pattern with an elevation of uroporphyrin (mean 1134 nmol/24 h, range 563-4052, normal < or = 30) and heptacarboxyporphyrin (mean 389 nmol/24 h, range 64-830, normal < or = 4). In all patients, however, urinary coproporphyrin was also increased, reaching levels too high for porphyria cutanea tarda but typical for porphyria variegata (mean 1788 nmol/24 h, range 142-4168, normal < or = 120). Fecal porphyrin excretion also resembled the variegate-type with a high concentration especially of protoporphyrin (mean 628 nmol/g dry weight, range 401-1018, normal < or = 151), accompanied by an increase of coproporphyrin (mean 194 nmol/g dry weight, range 75-409, normal < or = 37). The urinary porphyrin precursors 5-aminolaevulinic acid and porphobilinogen were markedly elevated only in one patient, who was in an acute porphyric phase at the time of investigation. The activity of uroporphyrinogen decarboxylase in erythrocytes was considerably decreased in six of our cases (33-64%) and slightly diminished in the other one (83% of normal activity).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548448 TI - Performance of a direct, immunoseparation based LDL-cholesterol method compared to Friedewald calculation and a polyvinyl sulphate precipitation method. AB - The analytical performance of a direct, immunoseparation based LDL-cholesterol method (Genzyme Corporation) was evaluated on an ELAN analyser (Merck), and compared with the performance of routinely used methods (LDL-cholesterol estimated by the Friedewald equation, and LDL-cholesterol obtained after polyvinyl sulphate precipitation). Within-day coefficients of variation (CVs) were 0.79 to 2.51% for immunoseparation based LDL-cholesterol; the between-day CVs varied between 2.62 and 3.89%, i.e. within the recommended National Cholesterol Education Program (NCEP) goal of < 4%. A method comparison study, according to the National Committee for Clinical Laboratory Standards (NCCLS) EP9 P guidelines, was performed using fasting normo- and hypertriacylglycerolaemic as well as cholestatic sera. In fresh normotriacylglycerolaemic sera immunoseparation based LDL-cholesterol (y) and Friedewald LDL-cholesterol (x) values were identical as slope and intercept of the Passing & Bablok regression equation were not significantly different from one and zero, respectively (y = 1.006 x -0.107; N = 45). In contrast, immunoseparation based LDL-cholesterol (y) differed significantly from polyvinyl sulphate LDL-cholesterol (x) results (y = 0.922 x + 0.234; N = 103). Freezing normotriacylglycerolaemic sera (three weeks, 20 degrees C) resulted in a negative bias of -5.8% for the immunoseparation based LDL-cholesterol method, and in a positive bias of +5.3% for the polyvinyl sulphate method, compared to fresh specimens. Immunoseparated LDL-cholesterol was completely recovered up to at least 37.84 mmol/l serum triacylglycerols. We conclude that the immunoseparation based LDL-cholesterol method is a practical, not technically demanding technique well applicable within routine clinical laboratories.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548449 TI - The determination of alpha 1-microglobulin by means of an automated latex immunoassay. AB - Polystyrene (latex) particles coated with human anti-alpha 1 microglobulin antibodies are used in an automated "kinetic" assay for alpha 1-microglobulin in urine. For values below 12 mg/l, there was no significant difference between two kinds of standard, but above 12 mg/l the results depend on the origin of the alpha 1-microglobulin standard. Correlation between values obtained with both standards was good (r2 = 0.968). The method has a between-run imprecision (CV) of 11-16%. Comparison with two commercial enzyme immunoassays gave a bias of -10% to +40%, while two nephelometric methods differed by 0% to 23%, possibly because the standards used in these methods were of different origin. These results indicate the necessity of standardization of the determination of alpha 1-microglobulin. The detection limit of our method was 0.8 mg/l, enabling the application of the method for epidemiological investigations. PMID- 7548450 TI - Clinical utility of a wheat-germ precipitation assay for determination of bone alkaline phosphatase concentrations in patients with different metabolic bone diseases. AB - Bone alkaline phosphatase was evaluated by wheat-germ lectin precipitation in several clinical conditions. The study included 33 premenopausal healthy women, 46 postmenopausal apparently healthy women, 19 growing children, 24 patients with Paget's disease, 31 patients with primary hyperparathyroidism and 66 patients with hepatobiliary diseases. In postmenopausal women the mean T score (i.e.: the number of SD below or above the mean for premenopausal women) was 2.6 +/- 1.3 (SD) for bone alkaline phosphatase and 1.61 +/- 1.21 for total alkaline phosphatase (p < 0.001). The T score for bone alkaline phosphatase provided a better discrimination from normals for both Paget's disease (22.1 +/- 27.8 versus 12.8 +/- 16 p < 0.001) and primary hyperparathyroidism (8.2 +/- 4.3 versus 4.6 +/ 3.7 p < 0.005 for bone alkaline phosphatase and total alkaline phosphatase respectively). After treatment with intravenous bisphosphonate the percent decrease of bone alkaline phosphatase was larger than that of total alkaline phosphatase both in patients with Paget's disease (-46% versus -72% p < 0.01) and in patients with primary hyperparathyroidism (-21% versus -47% p < 0.02) and an estimate of the precision (delta mean/SD of the delta mean) for bone alkaline phosphatase was 1.9-3.7 times higher than that of total alkaline phosphatase. In twelve osteoporotic patients treated for six months with oral alendronate the decrease in bone turnover was detected with significantly higher precision with bone alkaline phosphatase than with total alkaline phosphatase (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548451 TI - Rapid and easy procedure for the determination of immunoglobulin class and light chain type of anti-lactate dehydrogenase antibodies in macro-lactate dehydrogenase. AB - We developed an easy to perform and rapid method for determination of the immunoglobulin class and light chain type of anti-enzyme antibodies present in macro-enzymes. The procedure is a combination of two routinely used laboratory kits, and it allows identification of the antibody involved within 1.5 hour. The applicability of the method was demonstrated for macro-lactate dehydrogenase. PMID- 7548452 TI - The recovery of proteolytic activity using the Salivette system. AB - The recovery of proteolytic activity from human saliva was investigated using the Salivette system. Comparison of activity measurements with those obtained from whole saliva indicated that the Salivette system allowed increased recovery of proteolytic activity suggesting minimal enzyme binding to the cotton roll. The significance of these results is that previous estimates of proteolytic activity in mixed saliva may not have been due to increasing release of proteases by stimulation of secretion, but by altered binding properties of the sampling system used. PMID- 7548453 TI - Improved measurement of transketolase activity in the assessment of "TPP effect". AB - The indirect estimation of thiamine levels in human blood by measuring thiamine pyrophosphate effect on erythrocyte transketolase activity is the method of choice in most clinical laboratories. We describe here an optimized, time-saving, and accurate method to determine the thiamine pyrophosphate effect in as many as 16 blood samples simultaneously. The method is based on a multi-point determination using a computer remote-controlled microplate reader. For multiple sample handling, three pooled reaction mixtures are freshly prepared and loaded onto a 96 well microtitre plate. A pre-written software is then initiated to remote-control the system. The data is retrieved and processed to calculate thiamine pyrophosphate effect by a self-written "macro" on a "Quattro-Pro" worksheet database. This method proves to be highly accurate (coefficient of variance: 2.7%), reproducible (coefficient of variance: 4.1%) and economical. PMID- 7548454 TI - Discordant results in the assay of cytoplasmic islet cell autoantibodies with ELISA and immunohistochemical techniques. AB - We evaluated 6 batches of a solid phase enzyme-linked immunosorbent assay (ELISA) Isletest-ICA kit commercially available for the determination of autoantibodies to pancreatic islet cells, and compared the results with those obtained by a standardized immunohistochemical method. Following the immunohistochemical determination of autoantibodies to pancreatic islet cells, sera from patients with insulin-dependent diabetes mellitus, both positive and negative for autoantibodies to pancreatic islet cells, were randomly selected and analysed by ELISA. Sera from healthy control subjects, as well as standards recommended by the International Diabetes Workshop (IDW) ICA (Autoantibodies to Pancreatic Islet Cells) Proficiency Program, were included. Of the sera testing positive for autoantibodies to pancreatic islet cells in the immunohistochemical assay, only 14 +/- 5% were found to give a positive reaction in the ELISA. Among the sera from healthy control subjects and pancreatic islet cell autoantibody-negative insulin-dependent diabetes mellitus patients, 25 +/- 7% and 1 +/- 1%, respectively, yielded false-positive readings for autoantibodies to pancreatic islet cells. These results clearly show that the ELISA test presently available does not reliably detect autoantibodies to pancreatic islet cells, even qualitatively. Thus, it cannot be used for screening subjects at risk of developing diabetes. PMID- 7548455 TI - Influence of bacteriuria on the determination of urinary albumin excretion rate. PMID- 7548457 TI - Behavioral response to pharmacological challenges: potentials and pitfalls. PMID- 7548456 TI - Evaluation and clinical application of the Enterotest for the determination of human biliary porphyrin composition. AB - The Enterotest string test is an easy and non-invasive method for sampling duodenal fluid, which has been successfully used for the analysis of duodenal microflora, as well as biliary bile acid and lipid composition. The method was evaluated for determination of porphyrins in duodenal bile in normal subjects and subjects with porphyria, following cholecystokinin induced gall bladder contraction; it is known that analysis of biliary porphyrins is more discriminatory for the diagnosis of asymptomatic porphyria than their analysis in faeces or urine. Moreover, serial analysis of bile from patients with erythropoietic protoporphyria may help in establishing their ability to secrete protoporphyrin in bile and to assess effects of treatment. The binding of various porphyrins to Enterotest strings was investigated by incubating pieces of the string in different human bile samples with low to very high porphyrin concentrations, followed by HPLC analysis of porphyrins both in the native bile and in extracts obtained from the strings. No differences between porphyrin composition in native bile and extracts were observed. Duodenal fluid obtained by means of the Enterotest from volunteers not receiving cholecystokinin showed large variations in porphyrin patterns not resembling those of native bile. Mesoporphyrin, a secondary porphyrin derived from protoporphyrin by bacteria, was often detectable. These data indicate that the duodenal content without cholecystokinin injection does not reflect biliary porphyrin composition. The presence of mesoporphyrin in the whole intestinal tract, but not in serum and bile, suggests that there is no enterohepatic circulation of secondary porphyrins. There was close agreement between the porphyrin ratios found with the standard duodenal intubation technique and the Enterotest, performed simultaneously in one healthy volunteer after induction of gall bladder contraction by cholecystokinin. From these experiments, it was concluded that fluid adsorbed to the Enterotest string after gall-bladder contraction can be used to determine biliary porphyrin composition. Since duodenal bile is diluted gall bladder bile, variable porphyrin concentrations were found when applying the Enterotest in combination with cholecystokinin in the same subject on successive days. However, porphyrin ratios, such as the protoporphyrin to coproporphyrin I ratio, were relatively constant. In subjects with symptomatic variegate porphyria, the Enterotest showed highly aberrant porphyrin patterns, with increased protoporphyrin to coproporphyrin I ratios and, in addition, the presence of some unknown porphyrins. A deviating biliary protoporphyrin/coproporphyrin I ratio in one patient appeared to be a useful diagnostic index for the presence of latent variegate porphyria (or variegate porphyria in remission).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7548458 TI - Event-related potentials and alpha synchronization in preadolescent boys at risk for psychoactive substance use. AB - Numerous studies have evaluated event-related potentials (ERPs) as biological indicators of the liability for alcoholism. This study extends that approach by investigating ERPs in boys at risk for other substance use disorders. Prepubertal (10-12 years) sons of fathers diagnosed with psychoactive substance dependence (n = 28) were compared to matched sons of nonaffected fathers (n = 26) on an auditory ERP oddball task. Multivariate analyses of variance applied to peak amplitude and latency measures indicated small to moderate between-groups differences at midline or parietal sites: N2 and P3 amplitude; P2, N2, P3, and Nc latency. This replicated P3 amplitude findings in alcoholism-risk studies, though the effect size was moderate. Analysis of event-related alpha power indicated significantly longer latency of alpha synchronization and oscillations of desynchronization in boys at risk. The alpha power findings were statistically the more robust of the measures applied. The role of neurocognitive factors in determining liability for substance use disorders is discussed. PMID- 7548459 TI - Visual ERPs evidence for enhanced processing of threatening information in anxious university students. AB - There is accumulating evidence that highly anxious individuals selectively attend to threatening information; however, contrary to expectations, there is no evidence of enhanced processing of threat stimuli in those individuals. We investigated this question by using a sample of 20 University students who were split into two groups consisting of 10 high-anxious and 10 low-anxious subjects according to Spielberger Trait Anxiety Inventory score (median STAI = 40). Without emphasizing speed, subjects were required to decide whether visually presented words (positive, neutral, or threat) matched semantically with previous priming sentences (threat or positive) displayed on a computer screen (altogether, two types of priming sentences and three types of probe words). There was a fixed interval of 1.1 seconds between priming sentences (SI) and probe words (S2) as well as between each priming sentence word. Response time and visual event-related potentials (ERPs) were recorded in both conditions. The results showed that, compared to the Low-Anxious group, the amplitude of N100 and P400 were enhanced for the High-Anxious group in the threat priming conditions. Low-anxious individuals showed almost identical processing of threat-related situations and positive situations. Furthermore, the P400 peak latency was shorter for emotional incongruous probes in high-anxious individuals. ERPs results of the experiment suggest that highly anxious individuals deploy more processing resources to threatening information. This bias in information processing occurs in the absence of any behavioral changes (indicated by reaction times). Attentional bias in anxiety therefore implies that threatening information is given a priority over other information and is more persistently activated in anxiety states. PMID- 7548460 TI - Stressful life events and EEG sleep in depressed and normal control adolescents. AB - EEG sleep measures in 35 depressed and 33 normal control adolescents were examined in relation to stressful life events occurring in the year before sleep studies. There was a significant interaction between stressful life events and diagnostic status for REM latency and total REM time. In the normal controls, the presence of stressful life events was significantly associated with reduced REM latency and increased total REM time. Among the depressed adolescents, there were no significant effects of stressful life events on REM latency or total REM time. The depressed adolescents with no stressful life events (n = 9) had significantly lower REM latency values compared to normal control adolescents with no stressful life events (n = 13)(61.7 +/- 50.0 vs. 132.1 +/- 79.0, p < or = .01). It appears that stressful life events influence at least some measures of adolescent sleep and should be considered in future controlled studies aimed at understanding sleep changes in adolescent depression. PMID- 7548461 TI - Clinical and chronobiological effects of light therapy on nonseasonal affective disorders. AB - Light therapy (bright or dim light) was given at different times (morning or evening) to 27 unmedicated patients with nonseasonal depression (according to DSM III-R criteria) and 16 normal volunteers. Circadian rhythms in body temperature were measured before and after light therapy. Bright light significantly improved clinical symptoms of depression, as measured by the Hamilton Rating Scale for Depression (HRSD), independent of the time of phototherapy. Dim light therapy had no effect on HRSD scores. Circadian rhythms of body temperatures in patients with affective disorder were more sensitive to the entraining effects of bright light than those of normal subjects, but these effects were not related to clinical improvement. Bright light exposure has an antidepressant effect on patients with nonseasonal depression, but the effect is unlikely to be mediated via the same circadian system that regulates body temperature. PMID- 7548462 TI - The WAGxDA rat: an animal model of cholinergic supersensitivity. AB - A heightened response to the muscarinic effects of acetylcholine appears to be involved in many of the symptoms associated with affective disorders. We have developed an animal model for cholinergic supersensitivity to study this involvement in more detail. Our findings on cholinergic supersensitivity and on behavioral despair in this model, the WAGxDA F1 hybrid, are reported here. Female WAGxDA rats show a heightened response to muscarinic agonist in a temperature depression test (TDT) and both males and females show an increased level of inherent despair in a Porsolt swim test; however, this cholinergic supersensitivity does not appear to be based on an increased density or affinity of cholinergic receptors. Other possible mechanisms are discussed. PMID- 7548463 TI - Plasma neurotransmitters, blood pressure, and heart rate during supine resting, orthostasis, and moderate exercise in dysthymic depressed patients. AB - Dysthymic depressed patients showed platelet-serotonin (pS) + plasma-free serotonin values greater than normal as well as plasma noradrenaline values lower than normal during supine resting period (0'). Conversely, no significant differences were observed in the 0' values of any other of the measured parameters: systolic, diastolic and differential blood pressure (SBP, DBP, DP), heart rate (HR), adrenaline (Ad), dopamine (DA), cortisol, and platelet aggregability between patients and controls. Although patients showed then normal DP reduction at orthostasis (1'), this was not prevented by atropine as it does in controls. Patients but not normals showed significant rises of DBP at orthostasis and exercise (5') periods, which were positively correlated with NA rises. On the contrary, the abnormally raised resting fS values registered in patients showed progressive and significant reductions throughout the test that were negatively correlated with DBP-NA values. Adrenaline did not show the normal 5'-fS peak. The above findings suggest that dysthymics show hypoactivity of the two branches of the sympathetic system (neural + adrenal) along with hyperparasympathetic activity. Furthermore, their low NA + high pS values contrast with the high NA + low pS registered in major depressed subjects. PMID- 7548465 TI - Lack of association between clinical symptoms and postmortem indices of brain serotonin function in Alzheimer's disease. PMID- 7548464 TI - Maintenance ECT for treatment of therapy-resistant paranoid schizophrenia and Parkinson's disease. PMID- 7548467 TI - Early intervention for schizophrenia: can the course of the illness be altered? PMID- 7548466 TI - Does vitamin E prevent tardive dyskinesia? PMID- 7548468 TI - Inversion of the hemispheric laterality of the anterior cingulate gyrus in schizophrenics. AB - The anterior cingulate gyrus (acg) is involved in mechanisms of attention and emotion, where the right hemisphere is considered to be dominant. One of the models for neuropsychological dysfunction in schizophrenia suggests an impairment in the balance of lateralized functions. Fourteen adult human female brains, having no macroscopic lesions, were used in this study. Seven brains came from female patients with clinical diagnoses of residual schizophrenia (DSM-III-R; APA 1987). Seven female brains were used as controls. Thirteen male brains were also studied, with the sole purpose of establishing the typicality of the female controls. All schizophrenic brains were age matched with control brains. Right laterality for weight (71.4%) and surface (85.7%) was observed in the acg of female control brains. The inversion of this laterality in a significant number of the schizophrenic cases was the most relevant finding in this study. PMID- 7548469 TI - Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. AB - This study tests the hypothesis that nicotinic cholinergic receptors, including those sensitive to the antagonist alpha-bungarotoxin, are decreased in the hippocampus of schizophrenics. The hypothesis is derived from the finding that alpha-bungarotoxin causes a defect in the inhibitory gating of auditory-evoked potentials in laboratory animals that resembles a defect in auditory sensory gating observed in schizophrenics. Nicotine transiently normalizes this psychophysiological deficit in schizophrenic patients. Postmortem brain tissue was obtained from eight schizophrenic and eight age-matched nonschizophrenic subjects. Sections of the hippocampus were labeled with [125I alpha-bungarotoxin and imagined by autoradiography. Binding of the nicotinic agonist [3H]-cytisine was determined in tissue homogenates. alpha-Bungarotoxin labeled a population of putative interneurons in the hippocampus, primarily in the dentate gyrus and the CA3 region of Ammon's horn. This labeling was significantly decreased in the tissue from the schizophrenic patients, with seven or eight patients below the range of the nonschizophrenic subjects. There was also a significant decrease in the binding of cytisine. The results were not related to generalized hippocampal cell loss, drug exposure at time of death, or smoking history. This initial study suggests that schizophrenic patients have fewer nicotinic receptors in the hippocampus, a condition which may lead to failure of cholinergic activation of inhibitory interneurons, manifest clinically as decreased gating of response to sensory stimulation. PMID- 7548470 TI - Selected quantitative EEG (QEEG) and event-related potential (ERP) variables as discriminators for positive and negative schizophrenia. AB - Heterogeneity is a major obstacle in the search for biological substrates in schizophrenia. The positive and negative distinction, even if too simplistic, may improve our understanding of underlying processes. Frontostriatal deficits have been related to negative symptoms, while dysfunction of the dominant temporal lobe appears more relevant to the generation of positive symptoms. Despite interactions between the subsystems, different neurophysiological profiles could be expected for patients predominantly affected at each of those levels. We performed discriminant analysis on 10 neurophysiological variables (hypothesis related) in schizophrenic patients grouped by positive or negative symptoms (PANSS), obtaining a discriminant that correctly classified the sample. The function was then tested in a new sample of patients with schizophrenia, affective psychoses, and controls, classifying subjects with 78% sensitivity and 85% specificity. Our findings suggest that predominantly negative and positive schizophrenics have different neurophysiological profiles, which are consistent with the hypotheses of hypofrontality and temporal lobe dysfunction, respectively. A linear relation between discriminant scores and PANSS ratings might reflect coexisting pathologies or compensatory interactions in the mixed subgroup. PMID- 7548472 TI - Schizophrenic patients with deficit syndrome have higher plasma homovanillic acid concentrations and ventricular enlargement. AB - In order to investigate the biological characteristics of deficit syndrome in schizophrenia (Carpenter et al 1988), we examined cerebroventricular ratios (CVRs) and plasma concentrations of homovanillic acid (HVA) in a group of schizophrenic inpatients with deficit syndrome (n = 20) and in a control group of age- and sex-matched schizophrenic inpatients without deficit syndrome (n = 20). Symptoms and intelligence levels were measured using the Brief Psychiatric Rating Scale (BPRS) and the Wechsler Adult Intelligence Scale (WAIS), respectively. Patients in the deficit group had significantly higher CVRs as well as significantly elevated plasma HVA concentrations when compared with patients in the nondeficit group. We also found that the mean total WAIS score in the deficit group was significantly lower than that in the nondeficit group. These findings suggest the biological heterogeneity of schizophrenia. Increased central dopaminergic turnover, as indicated by higher plasma HVA concentrations, may partially account for the pathogenesis of deficit syndrome. PMID- 7548471 TI - Decreased DOPAC in the anterior cingulate cortex of individuals with schizophrenia. AB - The dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was found to be decreased in the anterior cingulate cortex of individuals with schizophrenia compared with normal controls. The finding does not appear to be solely related to the presence of antipsychotic medications, age, postmortem interval, or freezer time. No changes in norepinephrine and its metabolites were found. PMID- 7548473 TI - Olfactory identification ability in pre- and postmenopausal women with schizophrenia. AB - Previously we reported olfactory deficits in young male but not female patients with schizophrenia. In the present report, olfactory identification ability in pre- and postmenopausal women with schizophrenia and normal control subjects was assessed using the University of Pennsylvania Smell Identification Test (UPSIT). Additionally, serum estradiol levels were measured on the same 2 days as smell testing was completed. Olfactory deficits were observed in pre- and postmenopausal women with schizophrenia but were more pronounced in the postmenopausal patients. Regarding estradiol levels, women with schizophrenia had lower estradiol levels than did normal control subjects. The findings of this study indicate that olfactory deficits do exist in women with schizophrenia and may be accentuated by estrogen depletion. PMID- 7548475 TI - Affective reactivity of language and the startle response in schizophrenia. PMID- 7548474 TI - Neuroleptic treatment and negative symptoms in deficit and nondeficit schizophrenia. PMID- 7548477 TI - [Anesthesia with low fresh gas flow in clinical routine use]. AB - Anaesthesia in low-flow techniques gains increasing interest. The possibility of cost reduction, widespread use of highly developed anaesthesia machines and monitors, and introduction of two new fluorinated inhalational anaesthetics with low solubility in human tissues encourage the use of low-flow anaesthesia techniques. Further advantages are improved climatisation of breathing gas and estimation or even measurement of the important parameter "oxygen consumption". The anaesthesia machines and inhalational anaesthetics currently available allow a safe use of low-flow techniques if safety requirements are complied with (tight circle system, monitoring of: inspired oxygen concentration, minute ventilation, airway pressure, transcutaneous oxygen saturation). Low-flow anaesthesia techniques using a fresh gas flow rate of 1 l/min can be performed with almost every anaesthesia machine. However, the use of multigas monitors, analyzing most parts of the breathing gas, facilitates the use of low-flow techniques. Multigas monitors and anaesthesia machines equipped with intermittent fresh gas delivery are recommended for the use of fresh gas flow rates close to the metabolic rate. Because of its physicochemical properties the new inhalational anaesthetic desflurane offers advantages for the use in low-flow anaesthesia techniques. PMID- 7548476 TI - [High frequency ventilation and low-flow: anesthesiologic aspects beyond conventional anesthesia and ventilation techniques]. PMID- 7548478 TI - [Capnography for bronchoscopy with rigid technique using high frequency jet ventilation (HFJV)]. AB - OBJECTIVES AND METHODS: Rapid bronchoscopy in general anaesthesia still has its precise indications, where the high frequency jet ventilation technique offers several advantages. The monitoring of ventilation, however, has been rather unsatisfactory up to date. We therefore studied capnography in 60 bronchoscopies during HFJV (rate: 100/min; I:E = 0.33; driving pressure: 0.08-0.14 MPa) using a rigid bronchoscope with a distally located sampling port. Continuous capnograms were recorded. End-tidal partial pressures of carbon dioxide (petCO2), however, were obtained from 2-3 single breaths by intermittently reducing the jet frequency to 10-12/min. After 6 min (MP1: whole group; n = 60) and 18 min of HFJV (MP2: n = 34 of this group) petCO2 values were regularly obtained and compared to pCO2 in synchronously drawn capillary blood samples (pcCO2). The jet driving pressure initially adjusted to body weight, however, was only corrected according to petCO2, aiming at 34 mmHg. RESULTS: During HFJV, sinusoidal capnograms permitted the identification of every single jet impulse. With instruments being passed through the bronchoscope, however, these curves were substantially distorted. Mean pcCO2 at MP1 (37.8 +/- 6.7 mmHg) and MP2 (37.2 +/- 6.7 mmHg) demonstrated normal ventilation to light hyperventilation. Differences from mean petCO2 obtained during low frequency breathing were 3.3 mmHg at MP1 and 4.4 mmHg at MP2 (p < 0.05). There were strong correlations between the individual pairs of pcCO2 and petCO2 from MP1 (r = 0.80) and MP2 (r = 0.75) as well as between the pairs of dpcCO2 and dpetCO2 from both MPs (r = 0.77). The accuracy of the ventilator setting according to petCO2 with reference to pcCO2 was 73% for MP1 and 74% for MP2 (sensitivity: 75%/79%; specificity: 72%/67%). CONCLUSIONS: Capnography in rigid bronchoscopy during HFJV proved a clinically applicable addition to monitoring. Its routine use is strongly recommended in interventional bronchoscopy. The true petCO2 values obtained by intermittent single low frequency jet breathing permit estimates of gas exchange sufficiently exact for clinical purposes and for adjustment of the ventilator setting. Wave forms of the continuously recorded capnogram during HFJV are a warning of impeded ventilation or airway obstruction and, thus, of the danger of barotrauma or hypoventilation. Besides contributing to patient safety, this monitoring method might improve the acceptance of HFJV for bronchoscopy. Furthermore, it can also be applied to rigid bronchoscopy with common ventilation. PMID- 7548481 TI - [Current developments in preclinical therapy]. PMID- 7548482 TI - [The value of preclinical thrombolysis in acute myocardial infarct]. PMID- 7548479 TI - [Behavior of cerebral blood flow velocity in conventional ventilation and superimposed high frequency jet ventilation]. AB - OBJECTIVE: Patients with increased intracranial pressure or vasospasm after subarachnoidal haemorrhage with decreased cerebral perfusion present a special problem on developing respiratory insufficiency, since kinetic therapy or extracorporal life support are contraindicated. Superimposed High Frequency Jet Ventilation (SHFJV) has been shown to be of benefit in ventilating patients with pulmonary insufficiency. The aim of this study was to evaluate if SHFJV could be safely applied in patients with critical cerebral blood flow; if so, SHFJV might be beneficial when pulmonary insufficiency occurs concomitantly. METHODS: The study was performed in 14 patients (3 with pulmonary insufficiency) applying first moderate hyperventilation (paCO2 31 to 36 mmHg) followed by increased hyperventilation (paCO2 27 to 30 mmHg) with CMV and SHFJV and measuring intracranial pressure (ICP), cerebral perfusion pressure (CPP) and blood flow velocity (BFV) of the middle cerebral artery. BFV of the middle cerebral artery which correlates closely to the cerebral blood flow, was measured continuously with transcranial Doppler ultrasound. RESULTS: CMV: Increased hyperventilation leads to a statistically significant increase in paO2 (121.3 to 147.2 mmHg, p < 0.05), SaO2 (98.5% to 99.2%, p < 0.05) and decrease in BFV (systole 115.9 to 89.6 cm/s, diastole 44.6 to 31.8 cm/s, p < 0.05). Heart rate, mean arterial blood pressure, ICP and ventilation parameters did not show any statistically significant differences. SHFJV: During SHFJV the parameters demonstrated similar patterns as during CMV. However, none of the changes were statistically significant (paO2 111.9 to 125.9 mmHg, SaO2 97.9 to 98.8, BFV systole 106 to 95 cm/s, diastole 52.7 to 42.4 cm/s, n.s.). After calculating the mean BFV according to the Markwalder formula to a standard paCO2 of 40 mmHg CMV and SHFJV were compared to one another. No statistical difference was seen between the two different ventilation techniques. CONCLUSION: In patients with increased ICP, pulmonary complications such as pneumonia or ARDS are frequently observed. Since there are indications that SHFJV is of benefit in pulmonary insufficiency, the study was conducted to demonstrate that SHFJV can be safely applied in patients with increased ICP. PMID- 7548480 TI - [Rates of awakening, circulatory parameters and side-effects with sevoflurane and enflurane. An open, randomized, comparative phase III study]. AB - OBJECTIVE: Sevoflurane is a "new" volatile inhaled anaesthetic currently undergoing phase III clinical trials in Europe and USA. Owing to the low blood solubility, rapid induction of anaesthesia and emergence from anaesthesia would be expected. In this study, we compared emergence times and haemodynamics in patients receiving either sevoflurane or enflurane. Furthermore, all adverse experiences were recorded, and the relationship to the drug administered was rated. METHODS: Thirty ASA physical status I and II patients were studied in an open, prospective and randomised clinical trial. Anaesthesia was induced with fentanyl, thiopentone and vecuronium for facilitating endotracheal intubation. Anaesthesia was maintained with sevoflurane or enflurane, 60% nitrous oxide in oxygen and additional doses of fentanyl (1-2 micrograms/kg/h). ECG, blood pressure (non-invasive), inspiratory and end-tidal concentrations of sevoflurane or enflurane were monitored continuously. At the end of surgery, administration of sevoflurane or enflurane and nitrous oxide stopped without tapering and emergence times were recorded. All adverse experiences which occurred until the third postoperative day were recorded and the relationship to the inhaled anaesthetic was rated as "none", "unlikely", "possible", "probable" or "highly probable". RESULTS: With the exception of the end-tidal concentration at the end of surgery and the mean inspiratory and end-tidal concentrations, which were higher for sevoflurane, the two patient groups were comparable. Pulmonary elimination was significantly faster and emergence time was significantly shorter (5 vs. 9 minutes) with sevoflurane. Emergence time did not correlate with the duration of anaesthetic exposure (MAC hours) for sevoflurane. There was no difference in the time courses of heart rate and mean arterial blood pressure between sevoflurane and enflurane. No adverse experiences with a "probable" or "highly probable" relationship to the inhaled anaesthetic were observed. CONCLUSION: Emergence time after inhalation anaesthesia depends on (alveolar) ventilation, blood-gas solubility coefficient and, at least for enflurane and isoflurane, on the dose applied (MAC hours). There is no positive correlation between emergence time and dose applied for sevoflurane. Due to the lower blood gas solubility coefficient (0.6-0.7 for sevoflurane vs. 1.8 for enflurane) pulmonary elimination is faster and emergence time is shorter with sevoflurane. Supplementing inhalation anaesthesia with fentanyl, there is no difference in the time courses of heart rate and mean arterial blood pressure between sevoflurane and enflurane. PMID- 7548485 TI - [Resuscitation by active compression and decompression]. PMID- 7548483 TI - [Significance of initial defibrillation by paramedics]. PMID- 7548484 TI - [Alternative vasopressor agents in cardiopulmonary resuscitation]. PMID- 7548486 TI - [Cyclopropane]. PMID- 7548487 TI - [Fournier's gangrene. Experiences and changes in the disease picture since its initial description]. AB - Fournier's disease mostly occurs in immunosuppressed men in the 5th to 7th decade of life. Bacteria from the urogenital or colorectal tract lead to a rapid spreading soft tissue infection. Painful scrotal or perineal swelling and a black spot as a sign of beginning necrosis are guiding symptoms. Involved bacteria are grampositive cocci, enterobacteriaceae and anaerobes. Main principles of therapy are immediate radical debridement and broad spectrum antibiotics. Sepsis renders the prognosis more infaust. PMID- 7548488 TI - [Comment on W. Sibrowski, M. Penner, C. Schneider: Autologous blood is safer than heterologous blood transfusion--fact or fiction?]. PMID- 7548489 TI - Public health, medical care, and mortality rates. AB - This study examined the association between public health spending and leading mortality rates in the United States. Results indicate that government spending on public health is significantly associated with improving the life chances of the population. Public health spending was consistently associated with reduced total mortality and with lower mortalities due to heart disease, cancer, and stroke--the leading causes of death in the United States. However, medical care expenditures on hospital care and prescription drugs were significantly associated with increased total mortality and mortalities due to heart disease, cancer, and stroke. The policy implication is to reorient the currently expensive, clinically based, treatment-focused medical care system toward a more cost-effective health care system oriented toward public health and prevention. A more balanced health care system, with concomitant emphasis on medical care and public health, should be established. PMID- 7548490 TI - Mental health services: critical component of integrated primary care and substance abuse treatment. AB - The documented high incidence of mental health disorders among individuals in substance abuse treatment argues for the importance of studying the provision of mental health services to this population. This survey documents how the Linkage Programs assembled an array of mental health services based on the assessed health care needs of clients. Specialty and nonspecialty personnel addressed the extensive mental health needs of ethnically diverse, multiproblem clients with substance abuse problems. The innovative use of nonconventional providers, however, did not eliminate the shortfall between the number of clients with mental health problems and the number who were referred to and received mental health services; nor did it enable all Linkage Programs to address the mental health needs of the most severely ill clients. There is a continuing need to forge relationships between mental health providers and integrated providers of primary care and substance abuse treatment. PMID- 7548491 TI - Impact of access and social context on breast cancer stage at diagnosis. AB - This study examined the impact of individual demographic characteristics (age, race/ethnicity, and type of reporting hospital), together with measures of social context, including area of residence socioeconomic status (SES), change in SES, and access to screening (area mammography capacity), on breast cancer stage at diagnosis among New York City residents diagnosed between 1980 and 1985. Three logistic regression models evaluated the impact of individual variables, social context variables, and both classes of variables combined on the outcome of having late-stage (regional or distant) compared to early-stage (local) cancer. The logistic regression models indicated that women aged 50 to 64 years were independently more likely to have late-stage cancer than those younger or older. Controlling for individual and social context variables, African American women were 25 percent more likely than White women to have late-stage breast cancer (p < 0.0001); higher odds seen in the individual model for Hispanic women disappeared after controlling for area SES. In the combined model, women diagnosed from public hospitals were 44 percent more likely to have late-stage disease than those diagnosed in nonpublic settings (p < 0.0001). In both the social context and combined models, area mammography capacity was a significant independent predictor of stage (p = 0.016); area-level SES, but not change in SES, was independently related to late-stage disease (p = 0.002). These data suggest that breast cancer control activities should more actively ensure adequate access to screening, particularly for minorities, populations living in socioeconomically disadvantaged areas, and those cared for in the public sector. PMID- 7548492 TI - Preterm delivery among women in the South Carolina Medicaid High Risk Channeling Project. AB - The purpose of this study was to determine the association of various demographic characteristics, medical risks, and prenatal interventions with birth outcomes of women in the South Carolina Medicaid High Risk Channeling Project. The study population consisted of 5,012 mothers who delivered between January 1989 and June 1991; each was matched with her infant. Logistic regression was used to calculate the odds ratios for the independent effects of various maternal factors on gestational age. Analyses of preterm delivery indicated that high-risk African American women were 60 percent more likely to deliver a preterm baby than high risk White women. The mean gestational age was 37.3 weeks for African American newborns and 38.2 weeks for White newborns. Prenatal nutritional education had a significantly positive association with gestational duration. This study confirms that high-risk African American women are more likely than high-risk White women to experience a preterm delivery. Further research is needed regarding the positive association between prenatal nutrition education and the prevention of preterm deliveries. PMID- 7548493 TI - Amebiasis. PMID- 7548494 TI - Injection drug use and human immunodeficiency virus infection. AB - In this paper we discuss the epidemiology and natural history of human immunodeficiency virus (HIV) infection in users of injection drugs. Use of injection drugs plays a central role in the HIV infection/AIDS epidemic in the United States, Europe, and many parts of the developing world. The significance of this role has been underappreciated until quite recently because of a number of factors. One factor has been systematic, albeit inadvertent, underreporting of cases of HIV disease and AIDS in drug injectors as a consequence of the initially narrow surveillance case definition for AIDS. A measure of this phenomenon has been the disproportionately larger increment of new cases in this population with each successive revision of the Centers for Disease Control and Prevention's surveillance case definition for AIDS. Other reasons for the underappreciation of the magnitude and consequences of HIV infection and AIDS in injection drug users include the lack of necessary diagnostic facilities in the institutions where drug users are often treated, high mortality rates among HIV-infected drug users for whom a diagnosis of AIDS has not yet been made, the severe marginalization of this population and its lack of advocates, and the localization of the initial epidemic in this population to certain geographic areas. PMID- 7548495 TI - Efficacy and tolerability of ciprofloxacin prophylaxis in adult household contacts of patients with cholera. AB - We conducted a randomized double-blinded study in Lima, Peru, to assess the tolerability and efficacy of a single 250-mg dose of ciprofloxacin in preventing diarrhea and Vibrio cholerae O1 infection among household contacts of bacteriologically confirmed index cases. Adult household contacts with negative baseline stool cultures were included. A total of 213 household contacts were evaluable. The study drugs were well tolerated in both groups. Ciprofloxacin did not prevent the acquisition of V. cholerae O1 infection nor the development of diarrhea. However, in a subgroup of 30 household contacts with positive baseline stool cultures a reduction in the bacterial load and a trend toward prevention of diarrhea were observed among ciprofloxacin recipients. When all household contacts were evaluated, a trend toward prevention of diarrhea was observed with the prophylactic regimen. Ciprofloxacin failed to prevent V. cholerae O1 infections during a period of low transmissibility. PMID- 7548496 TI - Ciprofloxacin for the treatment of cholera: a randomized, double-blind, controlled clinical trial of a single daily dose in Peruvian adults. AB - We conducted a randomized, double-blind clinical trial to compare ciprofloxacin (250 mg once a day for 3 days) with tetracycline (500 mg four times a day for 3 days) in terms of efficacy and safety in the treatment of moderate-to-severe cholera in Peruvian adults. The baseline characteristics of the groups were similar. A total of 202 patients (102 in the tetracycline group and 100 in the ciprofloxacin group) were included in the efficacy analysis. The clinical and bacteriologic efficacies of the two regimens were similar. The study drugs were well tolerated. We conclude that ciprofloxacin given once a day is as effective as the standard tetracycline regimen for the treatment of cholera in adults. The ciprofloxacin regimen may represent an alternative to the standard treatment in areas where Vibrio cholerae O1 strains that are resistant to commonly used antimicrobials are prevalent. PMID- 7548497 TI - Photo quiz. Familial Mediterranean fever (FMF) PMID- 7548498 TI - Attributable mortality of bacteremia associated with the Bacteroides fragilis group. AB - Uncontrolled studies have suggested that Bacteroides fragilis group bacteremia has an attributable mortality rate between 13% and 33%. To determine the true attributable mortality rate and the mortality risk ratio associated with bacteremia due to the B. fragilis group, we conducted a matched-pair study in which cases were matched to controls for age, gender, year of admission, principal discharge diagnosis, and types of major surgery by an investigator blinded to survival status. Cases and controls were comparable in demographic and clinical characteristics. Cases had a significantly higher mortality rate (28% vs. 8.7%, P = .002, McNemar's test), with an attributable mortality rate of 19.3% (95% CI, 8%-30%; P = .003) and a mortality risk ratio of 3.2. In a multivariate analysis, three clinical factors were independently correlated with mortality: the presence of B. fragilis group bacteremia (RR: 4.9; 95% CI: 3.7-6.0; P = .009), congestive heart failure (RR: 8.0; 95% CI: 6.6-9.3; P = .003) or chronic liver disease (RR: 6.3; 95% CI: 4.8-7.7; P = .01). Cases also had a 16-day-longer stay in the hospital (P = .0007, Wilcoxon's signed rank test) compared with controls. Thus, B. fragilis group bacteremia contributes significantly to morbidity and mortality. PMID- 7548500 TI - Bartonella henselae: etiology of pulmonary nodules in a patient with depressed cell-mediated immunity. AB - We describe an immunocompromised renal transplantation patient with opportunistic lung infection due to Bartonella henselae (formerly Rochalimaea henselae) and provide evidence suggesting transmission from a pet cat. Computed tomographic scans of the chest and lung biopsies provided material for diagnosis. The etiology was established by polymerase chain reaction and sequencing of a 16S ribosomal DNA segment from infected lung tissue. Histopathologic and serological evidence supported the molecular data. B. henselae was isolated from the blood of eight of the patient's many cats. The patient responded to prolonged therapy with doxycycline, and relapse did not occur during a 1-year follow-up. B. henselae joins a long list of pathogens that can cause lung infections in association with cell-mediated immunodeficiency states. Molecular methods are useful in diagnosis of this infection in light of the bacterium's fastidious growth characteristics. If an immunocompromised patient has lung nodules and a history of exposure to cats, B. henselae should be sought in biopsy specimens. PMID- 7548499 TI - The impact of infectious diseases on the health of U.S. troops deployed to the Persian Gulf during operations Desert Shield and Desert Storm. AB - An assessment was conducted of the impact of infectious diseases on the 697,000 U.S. troops deployed to the Persian Gulf during 1990-1991 in Operations Desert Shield and Desert Storm. The incidence of nonbattle injuries, including infectious diseases, during this conflict was lower than during previous wars involving U.S. military personnel. The major reported causes of morbidity were generally mild cases of acute diarrheal and upper respiratory disease. The most unexpected outcome was the lack of arboviral infections, particularly sandfly fever, and the occurrence among U.S. troops of 12 cases of visceral leishmaniasis due to Leishmania tropica. The fact that infectious diseases were not a major cause of lost manpower, in sharp contrast to the experience among military personnel in World War II, can be attributed to a combination of factors: the presence of a comprehensive infrastructure of medical care, extensive preventive medicine efforts, and several fortuitous circumstances. Beneficial conditions that may not be present in future conflicts in this region include isolation of most combat troops to barren desert locations during the cooler, winter months, which provided the least favorable conditions for transmission of arthropod-borne diseases. PMID- 7548501 TI - The role of anaerobic bacteria in recurrent episodes of sinusitis and tonsillitis. AB - Chronic and recurrent upper respiratory tract infections represent a significant clinical challenge. The causative organisms tend to be heterogeneous, involving both aerobes and gram-positive and gram-negative anaerobes. There is evidence that these mixed groups of bacteria interact synergistically, enhancing and prolonging the overall virulence of infection. The role of anaerobic bacteria, in particular their proposed ability to protect susceptible organisms by the production of beta-lactamases, has been the subject of intense speculation. The evidence of a significant role for anaerobic bacteria in recurrent episodes of tonsillitis and sinusitis is reviewed and the most appropriate antimicrobial strategies and possible future developments in diagnosis and therapy are discussed. PMID- 7548502 TI - Epidemiology of fungal infections: current perspectives and future directions. Introduction. PMID- 7548503 TI - Epidemiology of nosocomial fungal infections, with emphasis on Candida species. AB - Currently, about 180 hospitals participate in the National Nosocomial Infections Surveillance (NNIS) system. From January 1980 through April 1990, 27,200 fungal isolates causing nosocomial infections were reported from these hospitals; Candida species accounted for 19,621 (72.1%) of these isolates. Immunocompromised patients are at particularly high risk for candidemia. In patients with acute lymphocytic leukemia, treatment with vancomycin and/or imipenem appears to be an independent risk factor for candidemia; colonization of stool by Candida species may be another important predisposing factor in these patients. Rapid detection of invasive candidemia in these high-risk patients is particularly important to the improvement of rates of survival. Methods for rapid detection, such as the measurement of mannan (the major cell-wall polysaccharide of Candida), may be useful for diagnosing invasive candidiasis and for monitoring the response of this infection to antifungal therapy. Further studies of risk factors and the development of new methods for rapid diagnosis and monitoring should help decrease the morbidity and mortality associated with nosocomial fungal infections. PMID- 7548505 TI - Epidemiology of fungal infections: the promise of molecular typing. AB - As the incidence of invasive fungal disease-particularly nosocomial candidal infection-has increased significantly over the past two decades, molecular typing has become increasingly important for the development of rational infection control measures and therapeutic strategies. Numerous molecular methods have been used to subtype Candida species, including restriction endonuclease analysis of genomic DNA, Southern hybridization analysis, pulsed-field gel electrophoresis, and polymerase chain reaction-based approaches. Increasingly, typing techniques are being applied to other fungal organisms as well. Although an ideal epidemiological typing technique applicable to a wide range of fungal pathogens is not yet available, several molecular-typing methods may permit rapid, simple, and sensitive discrimination of specific strains among the most clinically important species of fungi. PMID- 7548504 TI - Nosocomial candidemia: risk factors and attributable mortality. AB - Over the past decade, the incidence of hospital-acquired bloodstream infections caused by Candida species has risen and the species associated with such infections have changed. The incidence of candidemia is dramatically higher in high-risk, critical-care units than in other parts of the hospital. Certain underlying physical conditions including acute leukemia, leukopenia, burns, gastrointestinal disease, and premature birth predispose patients to nosocomial candidemia. Independent risk factors include prior treatment with multiple antibiotics, prior Hickman catheterization, isolation of Candida species from sites other than the blood, and prior hemodialysis. In this article some of the challenges posed by the management of nosocomial candidemia are presented in three case studies. In addition, the results of several investigations of nosocomial candidemia at the University of Iowa Hospitals and Clinics are reviewed. PMID- 7548506 TI - Human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis in Sao Paulo, Brazil. AB - We conducted a prospective clinical and epidemiologic evaluation of 45 cases of human T lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Sao Paulo, Brazil. All enrolled patients had progressive chronic myelopathy and high titers of HTLV-I and HTLV-II antibodies, as determined by enzyme immunoassay and western blot. In 24 cases, the polymerase chain reaction (PCR) was performed so that HTLV-I could be distinguished from HLTV-II. The clinical and epidemiologic features of the patients from our study were similar to those of patients with HAM/TSP from other areas of endemicity for HTLV-I except that more patients in our study had received a blood transfusion prior to their illness. Despite the presence of HTLV-II virus in Brazil, all patients whose serum was tested by PCR were found to be infected with the HTLV-I virus. PMID- 7548507 TI - Use of interferon gamma and mebendazole to stop the progression of alveolar hydatid disease: case report. AB - We describe a 28-year-old female who presented with metastatic spread of a suspected occult tumor. The diagnosis of alveolar echinococcosis was established by needle biopsy of the liver and ELISA. The patient's clinical symptoms initially decreased after treatment with mebendazole, but the lesions progressed during the following 6 months. Albendazole was not available in Austria. Because the synthesis of interferon gamma (IFN-gamma) and IFN-alpha is markedly decreased in patients with echinococcosis, we treated our patient with IFN-gamma while continuing mebendazole therapy. This combination halted progression of the disease during the 12 months of follow-up. PMID- 7548508 TI - Joint infections due to Listeria monocytogenes: case report and review. AB - Joint infections of bone are unusual manifestations of listerial infection. Fourteen cases of septic arthritis due to Listeria monocytogenes have been previously described in adults. We report the 15th case of septic arthritis due to Listeria in which bilateral prosthetic hips in a renal transplant patient were involved. In addition, we review the literature on listerial joint infections. PMID- 7548510 TI - Invasive pneumococcal disease in central Australia. AB - Invasive pneumococcal disease remains a major problem in certain groups of people. We undertook a hospital-based review of all cases of invasive pneumococcal disease in central Australia over a 2-year period. We observed 79 cases of invasive disease in 78 patients. The incidence of invasive pneumococcal disease was highest in Aborigines under 5 years of age (1,025 cases per 100,000 population per year). The relative risk for Aborigines compared with non Aborigines was 31.6 (95% CI, 12.8-78.1). Pneumonia was the commonest disease observed (82% of patients). Eight patients died (10.1%), and all of these patients had identifiable risk factors for pneumococcal disease. Serotyping showed that all except two isolates were covered by the existing 23-valent pneumococcal vaccine. These data reveal that Aborigines in central Australia have the highest reported rate of invasive pneumococcal disease in the world. A vaccination program in central Australia should decrease admissions and deaths due to pneumococcal disease. PMID- 7548509 TI - Simian bites and bacterial infection. AB - Three patients with simian bites and resultant infection are described. The bacteriology of the wounds was diverse and included alpha-hemolytic streptococci and other streptococci in all wounds, enterococci, Staphylococcus epidermidis, and Enterobacteriaceae. A literature review revealed brief mentions of 132 cases of simian bites in some of which Bacteroides species, Fusobacterium species, and Eikenella corrodens were isolated. Infection, despite antimicrobial therapy, and complications, such as osteomyelitis and flexion contractures, occurred frequently. PMID- 7548512 TI - Bacteremia due to Clostridium difficile: case report and review of extraintestinal C. difficile infections. AB - We report the case of an 85-year-old woman who developed diarrhea and bacteremia caused by Clostridium difficile after a prolonged hospitalization. C. difficile is a major cause of antibiotic-associated diarrhea but only rarely has been reported to cause extraintestinal infection. Isolation of C. difficile from a wide range of specimens outside the intestinal tract has been reported and is not always associated with diarrhea or intestinal pathology. PMID- 7548511 TI - Acyclovir-induced neutropenia in an infant with herpes simplex encephalitis: case report. AB - A newborn infant whose condition was diagnosed as herpes simplex encephalitis and who had subsequent recurrences of skin disease had repeated episodes of neutropenia while receiving therapy with intravenous (30 mg/[kg.d]) or oral (30 mg/]kg.d]) acyclovir. The neutropenia did not recur when the dosage of oral acyclovir was reduced to 10 mg/(kg.d). This case represents the first well documented report of acyclovir-induced neutropenia. PMID- 7548513 TI - Clarithromycin-induced mania in two patients with AIDS. AB - Acute psychosis was observed in two patients with AIDS who were treated with clarithromycin for disseminated Mycobacterium avium complex infection. The psychosis resolved when treatment with clarithromycin was discontinued and recurred when it was resumed. An adverse response to clarithromycin therapy is a rare but curable cause of acute psychosis in patients with AIDS. PMID- 7548514 TI - Pneumothorax in patients with pulmonary tuberculosis. PMID- 7548515 TI - Mycobacterium genavense infection in a patient with AIDS who was successfully treated with clarithromycin. PMID- 7548516 TI - A brain abscess due to multiply-resistant Enterobacter cloacae successfully treated with meropenem. PMID- 7548517 TI - Use of amphotericin B lipid complex for treatment of disseminated cutaneous fusarium infection in a neutropenic patient. PMID- 7548518 TI - Pentamidine concentrations in a mother with AIDS and in her neonate. PMID- 7548519 TI - Two-day continuous bladder irrigation with amphotericin B. PMID- 7548520 TI - Pneumococcal soft tissue infections in patients with systemic lupus erythematosus. PMID- 7548521 TI - Drug-resistant pneumococcal meningitis in an American adult. PMID- 7548522 TI - Catheter-related bacteremia due to Pseudomonas paucimobilis in neutropenic cancer patients: report of two cases. PMID- 7548523 TI - Predictive value of acid-fast smears of sputum obtained by expectoration versus induction or bronchoalveolar lavage in patients with tuberculosis. PMID- 7548524 TI - Tuberculous pyomyositis: an unusual presentation of disseminated Mycobacterium tuberculosis infection. PMID- 7548525 TI - Myasthenia gravis and systemic lupus erythematosus in association with human immunodeficiency virus infection. PMID- 7548526 TI - Diarrhea associated with parenteral vancomycin therapy. PMID- 7548527 TI - Appendicitis due to Mycobacterium avium complex in an adolescent infected with the human immunodeficiency virus. PMID- 7548528 TI - Baker's yeast as adjunctive therapy for relapses of Clostridium difficile diarrhea. PMID- 7548529 TI - Proceedings of the 1994 meeting of the Anaerobe Society of the Americas. Marina del Rey, California, 29-31 July 1994. PMID- 7548531 TI - Molecular biology as a tool for taxonomy. AB - Bacterial taxonomy based on phenotypic properties has encountered several problems: many organisms grow to poorly under laboratory conditions to be studied; the same phenotypic property often arises independently in more than one branch of a phylogenetic tree; and phenotypic schemes sometimes become unwieldy. Thus, molecular approaches have found a niche in taxonomy. Measurement of DNA hybridization between strains is the single most definitive tool for defining a species. Data on sequences of DNA and amino acids can be used to infer phylogeny. The molecular phylogenetic approach is most useful at levels ranging from kingdom to species. Restriction fragment length polymorphisms or variability in random polymerase chain reactions often differentiates strains. In the majority of cases, different molecular approaches tend to confirm established taxonomic schemes for bacteria. However, discrepancies between standard taxonomy and molecular data are not rare, especially when dealing with anaerobes. The contemporary approach to taxonomy takes into account both standard and molecular data. PMID- 7548533 TI - Differentiation of Bacteroides gracilis, Bacteroides gracilis-like organisms, and Campylobacter species on the basis of dehydrogenase patterns. PMID- 7548530 TI - Anaerobes under assault: from cottage industry to industrialization of medicine and microbiology. AB - More than 100 years have elapsed since the initial discovery of anaerobiosis, the first anaerobic microbiological isolation, and the first clinical descriptions of human anaerobic infection. New species are still being discovered, the important role of anaerobes as normal flora and in the maintenance of health is becoming increasingly appreciated, and the development of resistance by anaerobes to virtually all antimicrobials has recently accelerated. Anaerobes and their products have been employed widely and at times have been associated with political evolution, such as the establishment of the state of Israel. Despite all this, the current era of recession and health care economizing threatens the field of anaerobic bacteriology with funding cutbacks and diminished recognition. In order to prevent stagnation in the field and its decline, anaerobists must remain committed and vigilant and must take an increasingly active stance in education, publication, and research. PMID- 7548532 TI - Analysis of Bacteroides species by pyrolysis mass spectrometry. AB - Two methods for the classification of isolates of the Bacteroides fragilis group were compared in a study of 133 clinical isolates and 10 reference strains. There was broad similarity between the pattern of clusters in pyrolysis mass spectrometry (PyMS), which reflects whole-cell composition, and that in conventional tests, which reflect nutritional interactions with the environment. A single large cluster was resolved by both approaches; this cluster comprised mainly strains identified as B. fragilis and was surrounded by a multiplicity of small clusters (42 in PyMS and 24 in conventional tests). Individual clusters corresponding to strains identified as Bacteroides thetaiotaomicron and Bacteroides vulgatus, respectively, were found by both techniques. However, for the remainder of strains, little correlation was evident between clustering in PyMS and that in conventional tests. Further comparison with data obtained by sodium dodecyl sulfate- polyacrylamide gel electrophoresis for this collection of strains showed equal levels of disagreement between classification based on whole cell protein and each of the classifications described here. In previous studies of other genera, PyMS-based and conventional test-based classifications showed good agreement. It appears that the B. fragilis group presents intractable problems in phenotypic characterization. PMID- 7548534 TI - Immunoblot typing of Bacteroides gracilis. PMID- 7548535 TI - Structure and function of Bacteroides fragilis capsular polysaccharides: relationship to induction and prevention of abscesses. AB - The capsular polysaccharide complex (CPC) of Bacteroides fragilis exhibits unusual biological properties. This polysaccharide complex promotes the formation of intraabdominal abscesses and, when administered systemically, can prevent abscess induction in a rat model of intraabdominal sepsis. Each of these biological properties is mediated by a T cell-dependent immune mechanism. The CPC consists of two distinct polysaccharides, PS A and PS B, each with repeating units that have positively charged amino groups and negatively charge carboxyl or phosphate groups. Analysis of these polysaccharides as well as of other charged polysaccharides of bacterial origin, before and after chemical modification, revealed that the oppositely charged groups are required for promotion of intraabdominal abscesses as well as for the distinct properties associated with the B. fragilis CPC and delineate one mechanism by which these biological responses occur. PMID- 7548536 TI - Isolation of enterotoxigenic Bacteroides fragilis from extraintestinal sites by cell culture assay. PMID- 7548537 TI - Enterotoxigenic Bacteroides fragilis. AB - Enterotoxigenic Bacteroides fragilis (ETBF) has been recently associated with watery diarrheal disease in livestock and young children. These strains of B. fragilis secrete an extracellular heat-labile protein toxin with a mess of approximately 20 kD. This toxin has been shown to have two major biological activities: stimulation of secretion in ligated intestinal segments in lambs and calves and alteration of the morphology of intestinal epithelial cell lines in vitro. In vivo the pathology of animal intestine exposed to an ETBF strain is disruption of the colonic epithelium with rounded, swollen surface epithelial cells. Similarly, in vitro intestinal epithelial cells derived from human colonic carcinomas become rounded and detach from neighboring cells after treatment with the ETBF toxin. These results suggest that the ETBF toxin is a secretory cytoskeleton-altering toxin. However, the contribution of the ETBF toxin to the pathogenesis of ETBF-associated diarrheal disease remains to be determined. PMID- 7548538 TI - Biological activity of Bacteroides lipopolysaccharide--reappraisal. AB - Lipopolysaccharide (LPS) was extracted from six species of Bacteroides by the phenol/water, petroleum/chloroform/ phenol, and Triton/magnesium methods. Yields and chemical analysis demonstrated that the products were different. Biological activity (endotoxicity) was assessed by the limulus amebocyte lysate (LAL) assay, induction of tumor necrosis factor (TNF) from human mononuclear leukocytes, and a lethality model with galactosamine-sensitized mice. Results showed that endotoxicity varied greatly depending on the species and the extraction method. LPS prepared by the phenol/water method was most endotoxic and that from Bacteroides fragilis had the greatest activity. Compared with Escherichia coli LPS, the phenol/water extract of B. fragilis was sevenfold more active in the LAL assay and marginally less active (five-to seven-fold) in the bioassay for TNF induction. However, when B. fragilis LPS was added to E. coli LPS, the induction of TNF was inhibited. In the mouse model, B. fragilis LPS was 5,000-fold less toxic. If the gastrointestinal tract is the source of the endotoxin in patients with systemic inflammatory response syndrome, then the obligately anaerobic Bacteroides species, which outnumber the facultative species such as E. coli by 1,000-fold, should not be overlooked. PMID- 7548540 TI - Effect of iron depletion on protein profiles of Bilophila wadsworthia. PMID- 7548539 TI - Antibiotic effects on bacterial viability, toxin production, and host response. AB - The efficacy of an antibiotic in human or experimental infection is presumed to be proportional to its in vitro antimicrobial activity, yet antibiotics having comparable in vitro activity may have markedly different efficacies in vivo. For example, we have reported that clindamycin is more efficacious than penicillin in experimental gas gangrene caused by Clostridium perfringens in animals. To explain these differences, we compared the dynamics of bacterial killing and suppression of toxin synthesis. In addition, we investigated the ability of clindamycin and penicillin to modulate lipopolysaccharide-induced cytokine production in human peripheral blood mononuclear cells. Our results suggest that clindamycin affects protein synthesis in both prokaryotic and eukaryotic cells. These data may, in part, explain why the efficacy of clindamycin is greater than that of penicillin and demonstrate that clindamycin may be important immune modulator. PMID- 7548542 TI - Polymerase chain reaction analysis of the Porphyromonas gingivalis collagenase gene. PMID- 7548541 TI - Molecular analysis of surface-associated enzymes of Porphyromonas gingivalis. AB - There is now increasing evidence that surface-associated enzymes, previously considered to be involved in intermediary metabolism or virulence, play a role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we report the molecular aspects of two such enzymes, the cysteine proteinase gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 base pairs that encodes a 49,000-M(r) protein of 445 amino acids. The gdh gene showed high homology (78.3%) with that of Clostridium symbiosum. Optimal codons accounted for 35.9% of the total codon usage, indicating high expression of this enzyme. These data are currently being used to carry out targeted mutagenesis, which was established here for gingivain. Conditions for targeted mutagenesis within the histidine domain of the catalytic site of gingivain using Tn 4351 was successfully achieved. Consequently, the catalytic functions, such as gingivain's capacity to hydrolyze the synthetic substrate alpha-benzoyl-arginine-4 nitroanilide, were disrupted. PMID- 7548545 TI - Survival of anaerobes in original specimens transported by overnight mail services. AB - Overnight mail delivery was evaluated for its effect on the recovery of facultative and anaerobic microbes in cultures of clinical specimens from patients. Ten clinical specimens, which were collected at different geographic locations and during different weather conditions, were cultured at the site and after overnight delivery to a distant laboratory. Forty-five facultative anaerobic isolates and 48 anaerobes were recovered. There was no significant difference in numbers of strains or relative quantities recovered in cultures of transported and nontransported specimens. With proper collection, transport, and inoculation of specimens, overnight delivery did not compromise recovery of clinically relevant microbes. PMID- 7548544 TI - Energetics of molecular hydrogen oxidation in the oral pathogen Campylobacter rectus. PMID- 7548543 TI - Comparative studies of the IgA1 protease genes from Haemophilus influenzae, Neisseria gonorrhoeae, and Prevotella melaninogenica. PMID- 7548546 TI - The AnaeroGen system: a noncatalytic alternative method for growth of clinical isolates of anaerobic bacteria. PMID- 7548547 TI - Role of the reference laboratory in susceptibility testing of anaerobes and a survey of isolates referred from laboratories in England and Wales during 1993 1994. AB - Methods for susceptibility testing of anaerobes and their patterns of susceptibility remain debatable. The responsibilities of the Public Health Laboratory Service Anaerobe Reference Unit include identification of referred isolates; development of methods; monitoring of the occurrence of anaerobic infections; performance of taxonomic studies; maintenance of a reference collection; and monitoring of the antibiotic susceptibility of clinical isolates. Selection of antibiotics for anaerobic infections is often based upon the findings of batch tests on referred isolates in specialized centers. If the information gathered is to be a reliable guide, there should be a structured rolling survey of clinical isolates from a wide range of hospital laboratories. Strains of Bacteroides, Prevotella, Clostridium, and Propionibacterium species and peptostreptococci referred to the Anaerobe Reference Unit during 1993-1994 were tested with the Etest to determine the minimum inhibitory concentrations of metronidazole, penicillin, amoxicillin/clavulanate, cefoxitin, imipenem, erythromycin, clindamycin, and tetracycline. Metronidazole susceptibility (generally) and penicillin resistance among Bacteroides species were consistent findings, and most isolates were susceptible to imipenem, but other results varied between and within species. PMID- 7548549 TI - Enzymatic profiles of Prevotella, Porphyromonas, and Bacteroides species obtained with the API ZYM system and Rosco diagnostic tablets. PMID- 7548548 TI - Update on the taxonomy and the clinical and laboratory characteristics of pigmented anaerobic gram-negative rods. AB - Pigmented anaerobic gram-negative rods are currently categorized as 17 species distributed in three genera: Prevotella, Porphyromonas, and Bacteroides. These organisms are often encountered in clinical specimens but are also found as part of the indigenous flora on various mucosal surfaces. Several studies are presently assessing the association of individual species with health and disease. For example, Porphyromonas gingivalis and Porphyromonas endodontalis are key putative pathogens in adult periodontitis and root canal infections, respectively. Porphyromonas asaccharolytica is prevalent in extraoral infections. The Porphyromonas species of animal origin have been isolated from infected bite wounds in humans. Isolates closely resembling Bacteroides levii have been recovered from various types of human infections. According to preliminary reports, Prevotella intermedia tends to be associated more often with periodontal disease than with a healthy oral cavity. In the laboratory, enzyme profiling facilitates the identification of these pigmented rods. Beta-Lactamase production is more common among prevotella species (30%-50%) than among Porphyromonas species (< 10%). PMID- 7548550 TI - Comparison of the use of oligonucleotide probes, 4-methylumbelliferyl derivatives, and conventional methods for identifying Prevotella bivia. PMID- 7548551 TI - Cellular fatty acid analysis in the differentiation of Clostridium in the clinical microbiology laboratory. AB - The identification of Clostridium species can be problematic for the diagnostic microbiology laboratory. The introduction of the MIDI Microbial Identification System (MIS) has enabled personnel in diagnostic laboratories to perform cellular fatty acid analyses on a variety of microorganisms. We used the Virginia Polytechnic Institute (Blacksburg, VA) Anaerobe Library (Moore Version 3.8) to perform analyses on 216 strains representing 18 species of Clostridium. The organisms were characterized with use of traditional biochemical methods that employ prereduced anaerobically sterilized media and other reference diagnostic methods. The MIS identified 86% of the strains correctly to the species level without the need for supplemental tests, and identified an additional 6% of the strains to species levels with the aid of a few supplemental tests. Only 3% of strains were identified by genus incorrectly. The cellular fatty acid patterns of selected, medically important clostridia were so distinctive that 100% of these species were identified correctly. The MIS has many practical benefits, including speed of identification, and few disadvantages (such as equipment cost) for the clinical microbiology laboratory. PMID- 7548553 TI - Rapid disk methods for the detection of esculin hydrolysis and urease activity in anaerobic bacterial strains. PMID- 7548552 TI - Use of fatty acid analysis (microbial identification system) for the identification of Clostridium difficile. PMID- 7548555 TI - Bilophila wadsworthia isolates from clinical specimens. PMID- 7548556 TI - Incidence and clinical relevance of anaerobic bacterial infections in patients with AIDS at an infectious diseases institute. PMID- 7548554 TI - Bacteroides levii-like organisms isolated from clinical specimens. PMID- 7548557 TI - Bacteriology of chronic otitis media, chronic sinusitis, and paranasal mucopyocele in Japan. AB - A bacteriologic study of cases of chronic otitis media, chronic sinusitis, and paranasal mucopyocele diagnosed at Gifu University Hospital was performed between 1989 and 1992. Bacteria were isolated from all of 13 samples of discharge from the ears of patients with chronic otitis media, all of 14 mucosal biopsy specimens from patients with chronic sinusitis, 8 of 10 aspirate samples from patients with chronic sinusitis, and 9 of 15 aspirates samples from patients with mucopyocele. Cultures often yielded polymicrobial growth, with an average of 3.1, 3.8, and 4.2 species per positive specimen from chronic otitis media, chronic sinusitis, and mucopyocele, respectively. The most commonly encountered anaerobes were Peptostreptococcus, Propionibacterium, and Prevotella species; the most common aerobes were Staphylococcus and Streptococcus species. One strain of Prevotella melaninogenica highly resistant to ampicillin and two strains of Peptostreptococcus resistant to cefaclor were found. PMID- 7548559 TI - Aerobic and anaerobic microbiology of bacterial tracheitis in children. PMID- 7548558 TI - Immune response to Fusobacterium nucleatum and Prevotella intermedia in patients with peritonsillar cellulitis and abscess. PMID- 7548560 TI - A retrospective review of cases of anaerobic empyema and update of bacteriology. AB - We conducted a retrospective study to update the bacteriology of 46 cases of anaerobic empyema that were originally studied between 1976 and 1993 at the Wadsworth Anaerobic Bacteriology Clinical Research Laboratory (Los Angeles). Anaerobic bacteriologic studies were completed for all 46 pleural fluid specimens, and aerobic bacteriologic studies were completed for 41 of these specimens. Thirty-seven clinical charts were available for review. A total of 161 anaerobic isolates (3.5 per patient) representing 64 species or groups were recovered. The most common isolates were as follows: Fusobacterium nucleatum (19); Prevotella oris-buccae group (13, 9 of which were P. oris); Bacteroides fragilis group (11, 4 of which were B. fragilis); pigmented Prevotella species (17, 8 of which were in the Prevotella intermedia-nigrescens group); Peptostreptococcus species (17, 9 of which were Peptostreptococcus micros); Eubacterium species (7); Lactobacillus species (8); Actinomyces species (7); and Clostridium species (7). Nineteen if the cases were of purely anaerobic etiology; of these, eight were caused by a single organism: F. nucleatum (five cases); B. fragilis (two cases); and Prevotella mangus (one case). Of the 45 aerobic isolates (1.1 per patient), viridans streptococci were most common (21 isolates), followed by group D nonenterococcal streptococcus (four isolates). Only nine gram negative rods (six enteric and three nonenteric organisms) and one Staphylococcus aureus isolate were recovered. The susceptibility to penicillin of 64 isolates was examined with the use of the spiral gradient method; 21 (33%) of these isolates were beta-lactamase positive (MICs ranged from 1.1 to > or = 54 micrograms/mL vs < or = 0.27 micrograms/mL for beta-lactamase-negative strains). PMID- 7548561 TI - Anaerobe-induced bacteremia in Italy: a nationwide survey. The Italian Anaerobe Study Group. PMID- 7548562 TI - Bacteremia due to a multicellular, spirally aggregated Clostridium strain. PMID- 7548563 TI - Leptotrichia sanguinegens sp. nov., a new agent of postpartum and neonatal bacteremia. PMID- 7548564 TI - Ventriculoperitoneal shunt infection due to anaerobic and facultative bacteria: case report. PMID- 7548565 TI - Clostridial disease of the gut. AB - Clostridia are an important cause of morbidity and mortality in humans and animals. Some of the most common clostridial infections are those of the gut. The primary infections in humans are Clostridium perfringens food poisoning and Clostridium difficile-mediated antibiotic-associated diarrhea and colitis. Less common but important infections include non-food poisoning C. perfringens nosocomial diarrhea and C. perfringens type C necrotizing jejunitis (pig-bel). C. perfringens is also the dominant cause of gastrointestinal infections in animals, although Clostridium septicum causing braxy in sheep, Clostridium colinum causing ulcerative enteritis is avian species, and Clostridium spiroforme causing enterotoxemia in rabbits are important exceptions. PMID- 7548566 TI - Clinical correlates of anaerobic bacteriology in peritonitis. AB - Recent surgical reports indicate that for patients with secondary bacterial peritonitis, surgeons do not routinely use the identification of the bacterial pathogens and determination of their antimicrobial susceptibilities in choosing antimicrobial therapy. Further, some surgeons advocate abandoning the routine practice of obtaining culture specimens from patients with complicated appendicitis, because the data from the clinical laboratory have not been found to have an impact on postoperative care. We review the rationale for continued surveillance of and implementation of bacteriological data in treatment of secondary peritonitis. We also describe in detail the anaerobic flora of secondary peritonitis, the unique susceptibility patterns of these organisms, and the specific virulence factors of anaerobes, particularly Bacteroides fragilis. The fact that clinical investigations sometimes result in treatment failure when gram-negative anaerobes are resistant to the antimicrobials used or when complete antimicrobial susceptibility data are not available emphasizes the need for accurate and early knowledge of the bacteriologic characteristic of the flora of the operative site. We emphasize the relationship of in vitro susceptibility of intraoperative isolates with clinical outcome. We propose a cooperative trial that would demonstrate that successful antimicrobial therapy should be based on the susceptibility of the flora of the operative site, which correlates with clinical outcome. PMID- 7548567 TI - Aerobic and anaerobic microbiology of gastrostomy-site wound infections in children. PMID- 7548568 TI - Prevalence of Clostridium difficile among healthy Chilean infants: evaluation by commercial enzyme immunoassay versus standard cytotoxin assay. PMID- 7548569 TI - Isolation of Clostridium difficile at a university hospital: a two-year study. PMID- 7548570 TI - Community-acquired Clostridium difficile-associated diarrhea. PMID- 7548571 TI - Recurrent Clostridium difficile infection in a patient with selective IgG1 deficiency treated with intravenous immune globulin and Saccharomyces boulardii. PMID- 7548572 TI - Anaerobes predominate among the vaginal microflora of prepubertal girls. PMID- 7548573 TI - Role of bacterial vaginosis in pelvic inflammatory disease. AB - Pelvic inflammatory disease (PID) is a frequent infection in sexually active young women and results in adverse sequelae, including tubal-factor infertility and ectopic pregnancy. In the 1970s investigations using culdocentesis demonstrated that anaerobic bacteria played an important role in the etiology of PID. This finding has subsequently been confirmed by studies utilizing laparoscopy and/or endometrial biopsy to obtain specimens directly from the upper genital tract (uterine cavity and fallopian tube) of patients with acute PID. Recently, several investigations have shown an association between bacterial vaginosis and the development of acute PID. The microorganisms associated with bacterial vaginosis include anaerobes such as Prevotella bivia, other Prevotella species, and Peptostreptococcus species. These studies that have demonstrated the presence of bacterial vaginosis-associated bacteria in addition to the sexually transmitted organisms Neisseria gonorrhoeae and Chlamydia trachomatis suggest that treatment of acute PID must be broad spectrum in nature and effective against anaerobic bacteria as well as N. gonorrhoeae and C. trachomatis. PMID- 7548576 TI - Foot infections in diabetic patients: the role of anaerobes. AB - The prevalence of anaerobic bacteria in cultures of specimens from foot infections in diabetic patients is dependent upon the method of obtaining the specimen, the care with which it is transported anaerobically, and the sophistication of the laboratory methods. The rate at which anaerobes are isolated with use of the best methods ranges from 74% to 95% of patients, but it is only 41% to 53% in clinical studies of patients with limb-threatening infection. The mean number of bacterial isolates from an infected foot ranges from 4.1 to 5.8, of which 1.2 to 2.6 isolates are anaerobic. Most anaerobic isolates are gram-positive, and Peptostreptococcus species are most common. Bacteroides species are the most common anaerobic gram-negative isolates. Treatment covering anaerobic bacteria is included in most empirical regimens, but the use of agents that are modestly active against anaerobic organisms (i.e., fluoroquinolones or trimethoprim-sulfamethoxazole) has also been successful. Surgical debridement and drainage are essential adjuncts to antimicrobial therapy and may assist in the control of anaerobic infection. Questions regarding management of such infections parallel those regarding management of polymicrobial intraabdominal infections, but to date, studies of the former have been much less sophisticated than those of the latter. PMID- 7548574 TI - The role of bacterial vaginosis and vaginal bacteria in amniotic fluid infection in women in preterm labor with intact fetal membranes. PMID- 7548575 TI - Bacteriology of skin and soft-tissue infections: comparison of infections in intravenous drug users and individuals with no history of intravenous drug use. AB - The bacteriology of cutaneous or subcutaneous abscesses (86 specimens) among intravenous drug users (IVDUs) was compared with the bacteriology of abscesses (74 specimens) in patients with no history of intravenous drug use (non-IVDUs). The IVDU abscesses yielded 173 aerobes and 131 anaerobes. Staphylococcus aureus was the most common aerobe isolated (50% of specimens yielded this isolate), followed by "Streptococcus milleri" (46%). The commonly encountered anaerobes were Fusobacterium nucleatum (17%), pigmented Prevotella species (22%), Peptostreptococcus micros (17%), Actinomyces odontolyticus (15%), and Veillonella species (13%). The non-IVDU isolates included 116 aerobes and 106 anaerobes. S. aureus was isolated from 53% of these specimens, followed by coagulase-negative staphylococci (19%), "S. milleri" (19%), and Streptococcus pyogenes (16%). The main groups of anaerobes were Peptostreptococcus species (35%), Bacteroides species (19%), and gram-positive bacilli (31%). Overall, 67% of the IVDU isolates were of oral origin, compared with 25% of the non-IVDU isolates. Of the specimens from IVDUs and non-IVDUs, 48% and 67%, respectively, yielded only aerobes, and 2% and 4%, respectively, yielded only anaerobes. Sixty-four percent of the patients had one or more beta-lactamase-producing organisms. PMID- 7548577 TI - Use of an anaerobic collection and transport swab device to recover anaerobic bacteria from infected foot ulcers in diabetics. PMID- 7548578 TI - The biochemical milieu of the host in the selection of anaerobic species in the oral cavity. AB - The periodontal pocket provides a unique structural site for studies on host/bacterial interactions. The pocket is colonized by a complex but characteristic anaerobic bacterial flora. Many new taxa have been described that have now been supported by comparative rRNA sequence analysis. Within recent years, several molecular approaches have been used to describe both cultivable and noncultivable species, and new genotypes have been reported. Microbial activity rather than the mere presence of a microorganism at a site must be a major factor in the process of disease development. Information on metabolic activities of species and identification of substrates are therefore essential to elucidate the complex interactions that are likely to occur in vivo. We have been using a variety of analytical procedures such as 13C substrate-enrichment nuclear magnetic resonance, 14C isotopic labeling experiments, enzyme assays, impedance measurements, and various chromatographic and electrophoretic procedures to study key species of this predominantly asaccharolytic flora. Results have so far indicated a major role for cationic and anionic acids as sources of energy, but the mechanisms of substrate processing may differ significantly between species. In this ecosystem, crevicular fluid is the likely source of nutrients for species. Components of this fluid appear to have a role in the selection of species in subgingival sites. PMID- 7548579 TI - Arbitrarily primed polymerase chain reaction analysis of periodontal pathogens: discriminative primers and genetic diversity. PMID- 7548580 TI - Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplification with the polymerase chain reaction. AB - The utility of the 16S ribosomal RNA-based polymerase chain reaction (PCR) for detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola was examined and compared with that of anaerobic culture. Primer pairs consisting of 20-27 nucleotides amplified 404- to 688-bp regions of 16S ribosomal RNA genes of these organisms. This method had a lower detection limit of 50 target cells in a background of 10(7) cells. Its specificity for B. forsythus, P. gingivalis, and T. denticola seemed high. The primers for A. actinomycetemcomitans, C. rectus, and P. intermedia cross-reacted with some closely related species but did not reveal amplification products in tests with more distantly related organisms. The primers for E. corrodens did not seem to cross-react with oral organisms. This PCR technique was sensitive, reproducible, and easy to perform. PCR-based amplification may prove valuable for the detection of some periodontal pathogens in crude subgingival specimens. PMID- 7548582 TI - Refractory periodontitis in a Colombian population: predominant anaerobic bacterial flora and antibiotic susceptibility. PMID- 7548581 TI - Microbiological study of juvenile periodontitis in Chile. PMID- 7548583 TI - Serum responses of cats with periodontal/gingival disease to members of the genus Porphyromonas. PMID- 7548584 TI - Occurrence of gram-negative black-pigmented anaerobes in subgingival plaque during the development of canine periodontal disease. PMID- 7548585 TI - Typing of 143 isolates from 10 species of Bacteroides by sodium dodecyl sulfate polyacrylamide gel electrophoresis of whole-cell proteins. PMID- 7548587 TI - An international study on the unification of nomenclature for typing Clostridium difficile. PMID- 7548586 TI - Ribotyping of Clostridium perfringens isolates. PMID- 7548588 TI - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of EDTA extracted cell-surface protein antigens is a simple and reproducible method for typing Clostridium difficile. PMID- 7548589 TI - Correlation of pyrolysis mass spectrometry and outer membrane protein profiles of Clostridium difficile. PMID- 7548590 TI - Antimicrobial susceptibility tests for anaerobic bacteria with use of the disk diffusion method. PMID- 7548592 TI - Evaluation of the Etest for determinations of antimicrobial spectrum and potency against anaerobes associated with bacterial vaginosis and peritonitis. PMID- 7548593 TI - Susceptibility results for the Bacteroides fragilis group: comparison of the broth microdilution and agar dilution methods. AB - The antimicrobial susceptibilities of members of the Bacteroides fragilis group were compared using the agar dilution and broth microdilution methods. A total of 455 B. fragilis group isolates were tested against 10 antibiotics. Significant disparity in susceptibility results for most antibiotics was observed between the two methods. Broth microdilution susceptibility results were most similar to agar dilution results when a twofold lower breakpoint was used. In addition, broth microdilution failed to detect resistance to some antibiotics when the recommended agar dilution breakpoint was used. MIC50, MIC90, and geometric mean MIC values for broth microdilution were consistently twofold to fourfold less than those for agar dilution. Susceptibilities of Bacteroides that are determined with use of broth microdilution may more accurately correspond to those determined with use of agar dilution if lower breakpoints are used for interpretation. PMID- 7548594 TI - Effect of choice of medium on the results of in vitro susceptibility testing of eight antibiotics against the Bacteroides fragilis group. AB - The susceptibility of isolates of the Bacteroides fragilis group to eight antibiotics was determined by the agar dilution method on three media. At intermediate breakpoints established by the National Committee for Clinical Laboratory Standards (NCCLS), only minimal differences in the susceptibility of each species to any of the antibiotics were documented among the three media. At NCCLS susceptible breakpoints, greater differences among media were found for cefoxitin and cefotetan than for the other drugs tested. Only minimal differences among the three media were evident in comparisons of MIC50, MIC90, and geometric mean MIC values for all antibiotics tested. Thus, use of any one of these three media does not significantly affect agar-dilution MIC results for the B. fragilis group. PMID- 7548591 TI - Evaluation of susceptibility of anaerobic organisms by the Etest and the reference agar dilution method. PMID- 7548595 TI - Induction of beta-lactamase production in the anaerobic microflora by cefoxitin. PMID- 7548596 TI - Antibiotic susceptibility profiles of Bacteroides fragilis and Bacteroides thetaiotaomicron in Japan from 1990 to 1992. AB - The antimicrobial susceptibility of clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron collected from December 1990 through November 1992 was determined by the agar dilution technique. Metronidazole, imipenem, and cefoperazone/sulbactam remained highly active against both organisms. Rates of resistance to those agents were 0, 2%, and 0.9% in B. fragilis and 0, 0.9%, and 3% in B. thetaiotaomicron, respectively. Cefoxitin and other cephamycins were active against B. fragilis; rates of resistance to these agents did not tend to increase. With the inclusion of these data, the variation of rates of resistance to several agents was summarized for each year from 1987 to 1992. Rates of resistance to imipenem decreased in 1991 and 1992 among isolates of B. fragilis (2.3% in 1991, 1.8% in 1992) and B. thetaiotaomicron (2.4% in 1991, 0 in 1992). Rates of resistance to cefoxitin in B. thetaiotaomicron varied from 10% to 38% during these 6 years, though the distributional peak of MIC values did not change. The rate of resistance to ofloxacin in B. fragilis increased from 42% in 1989 to 81% in 1992. The rate of resistance to ampicillin in B. thetaiotaomicron was 68% in 1992--approximately 30% lower than before. Mostly, however, the rates of resistance to the antimicrobial agents examined did not change significantly. PMID- 7548598 TI - Potential factors influencing carbapenem activity against Bacteroides fragilis group isolates. PMID- 7548597 TI - Activity of ampicillin/sulbactam, ticarcillin/clavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children. AB - The activity of 14 antimicrobial agents against 253 clinical isolates of anaerobic bacteria from pediatric infections was assessed by the agar dilution method. Fifty-eight percent of the isolates were from intraabdominal sites. The drugs tested were ampicillin/sulbactam, ticarcillin/clavulanate, ampicillin, sulbactam, piperacillin, cefoxitin, cefotaxime, cefoperazone/sulbactam, clarithromycin, azithromycin, erythromycin, clindamycin, metronidazole, and chloramphenicol. Ticarcillin/clavulanate was active against all isolates. Clarithromycin was the most active macrolide; combination of this agent with its 14-hydroxy metabolite did not result in synergy. Sixty-two percent of Bacteroides fragilis group isolates, 13% of B. fragilis isolates, and 22% of peptostreptococcal isolates were resistant to clindamycin at a concentration of 4 micrograms/mL. The distribution of these strains in clinical specimens and the patterns of antimicrobial susceptibility documented were different from the findings for isolates from adults in the Los Angeles area. PMID- 7548599 TI - beta-Lactamase production and penicillin susceptibility among different ribotypes of Prevotella melaninogenica simultaneously colonizing the oral cavity. PMID- 7548600 TI - Distribution of tetracycline and macrolide-lincosamide-streptogramin B resistance genes in anaerobic bacteria. PMID- 7548601 TI - Characterization of erythromycin resistance in Campylobacter (Wolinella) rectus. PMID- 7548602 TI - Comparison of the activity of meropenem with that of other agents in the treatment of intraabdominal, obstetric/gynecologic, and skin and soft tissue. AB - Meropenem, a new carbapenem with improved stability in the presence of human dehydropeptidase-I[1], was evaluated in three prospective, multicenter, randomized, controlled clinical trials in North America. We compared the in vitro activity of meropenem and conventional antimicrobial agents for the treatment of intraabdominal, obstetric/gynecologic, and skin or soft tissue infections as well as the responses of pathogens to all of these agents. The trials of the drug for intraabdominal infection were double blind, and those for the obstetric/gynecologic and soft tissue infections were open labeled. Overall, MICs of meropenem for pathogens were lower, and the pathogen response rates were at least comparable to those for the following comparative agents: clindamycin plus tobramycin (for intraabdominal infections); clindamycin plus gentamicin (for obstetric/gynecologic infections); and imipenem and cilastatin (for skin or soft tissue infections). Meropenem has high in vitro potency and covers a broad spectrum of anaerobic and aerobic pathogens. PMID- 7548604 TI - Factors affecting anaerobic dehalogenation of orthochlorophenol. PMID- 7548603 TI - Comparison of cefoxitin, cefotetan, and ampicillin/sulbactam as prophylaxis for polymicrobial infection in mice. PMID- 7548605 TI - Influence of light on the presence of Clostridium perfringens in caves. PMID- 7548606 TI - Use of sugarcane molasses for culture of Clostridium chauvoei. PMID- 7548607 TI - Overview of clinically important anaerobes. PMID- 7548608 TI - Biocompatibility of membranes used in the treatment of renal failure. AB - Haemodialysis membranes with a wide range of solute and hydraulic permeabilities are used clinically. Such membranes are manufactured from either cellulose or synthetic co-polymers and their biocompatibility is commonly characterized by the complement activation and white cell changes observed during their use. The cellobiosic unit may be modified by the partial or total replacement of the hydroxyl groups by diethylaminoethyl (Hemophan), acetate (cellulose acetate), triacetate (cellulose triacetate) or 2,5-acetate (Diaphan). We have undertaken a prospective study in which such renal membranes have been studied in terms of the complement activation and neutropenia produced with the aim of investigating the relationship between modification of the cellobiosic unit and the magnitude of neutropenia and complement activation, and the extent to which membrane base material influences these parameters, by comparing the changes observed in modified cellulose membranes with that for a synthetic membrane (polysulphone). Our findings show that, while the degree of substitution varies between < 1% and total substitution, there is no correlation between the numbers of hydroxyl groups replaced and alteration of complement activation and neutropenia. However, by modification of the cellobiosic unit it is possible to produce a membrane whose biocompatibility is similar to that of a membrane manufactured from a synthetic co-polymer such as polysulphone. PMID- 7548609 TI - Permeability and blood compatibility properties of chitosan-poly(ethylene oxide) blend membranes for haemodialysis. AB - Chitosan-poly(ethylene oxide) (PEO) blend membranes, using different molecular weights of PEO, were developed for improved permeability and blood compatibility. The equilibrium hydration increased from 44.7% for chitosan to 62.5% for chitosan PEO blend membranes when the molecular weight of PEO was 10,000 (10K) or higher. An increase in the hydration of PEO blend membranes was due to intermolecular association between PEO and chitosan chains. Scanning electron microscopy showed that chitosan-PEO membranes were highly porous with size ranging from 50 to 80 nm in diameter observed in membranes made with PEO10K. Electron spectroscopy for chemical analysis suggested an increase in PEO on the membrane surface with increasing molecular weight in the blend. The permeability coefficient of urea increased from 5.47 x 10(-5) cm2 min-1 in chitosan to 9.86 x 10(-5) cm2 min-1 in chitosan-PEO10K membranes. The increase in permeability coefficient could be either due to an increase in the hydrophilicity or the high porosity of the membranes. Although chitosan-PEO membranes did not prevent serum complement activation, platelet adhesion and activation were significantly reduced. Chitosan PEO blend membranes, therefore, appear to be beneficial in improving the permeability of toxic metabolites and in reducing the thrombogenicity for haemodialysis. PMID- 7548610 TI - Healing of mandibular defects with different biodegradable and non-biodegradable membranes: an experimental study in rats. AB - Membranes, clinically used to improve bone regeneration according to the osteopromotion principle, have primarily been made of expanded polytetrafluoroethylene (Gore-Tex Membrane). Recently, different types of biodegradable membranes have become available. This investigation explored the osteopromotive potential of 10 different biodegradable and non-biodegradable membrane materials. Scanning electron microscopy revealed quite different surface configurations of these membranes, even though some of them were chemically closely related. Standardized, transosseous, critical size mandibular defects were made bilaterally in adult rats and were randomly covered with the different types of membrane. After 6 wk of healing, evaluation was performed by light microscopy according to a histological scoring system. Varying degrees of bone healing were seen beneath the different membranes. Some of the membranes (such as Gore-Tex Augmentation Material, Millipore and Resolut 'long term') revealed a good osteopromotive effect, whereas others had little or no beneficial effects on bone healing. Certain membrane materials caused a pronounced inflammatory response in the surrounding soft tissue, while others displayed a low inflammatory reaction. The study shows that different membranes differ strongly in osteopromotive efficacy, even if seemingly chemically closely related. Furthermore, the study demonstrates that membranes developed primarily for periodontal regeneration purposes may not be adequate to promote bone healing. PMID- 7548611 TI - Immunohistochemical study of the soft tissue around long-term skin-penetrating titanium implants. AB - Bone-anchored percutaneous titanium implants have become a well-established clinical procedure with a low incidence of adverse reactions. However, passage through the skin leads to a breach in the barrier to exogenous pathogens. In the present study, monoclonal antibodies were used to investigate the distribution of lymphocyte subpopulations in the soft tissue around such implants. Eight biopsies from patients with clinically irritated skin, five from non-irritated and eight from skin without skin-penetrating implants were analysed. The number of immune cells was increased in the group of patients with skin penetration compared with patients without skin penetration. In the group with clinical irritation there was an increased level of B-lymphocytes compared with those without irritation. The data suggest that there is an immunological compensation for the mechanical loss in barrier function at these implants and that an antibody-mediated response is present at clinical signs of irritation. PMID- 7548612 TI - In vitro assessment of bioadhesion for periodontal and buccal drug delivery. AB - Bioadhesion could significantly improve oral therapeutics for periodontal diseases and mucosal lesions. This project was designed to examine the factors important to prolonged adhesion (adhesion time) in organ culture under standardized conditions. A wide variety of bioadhesives were tested in the model and the effect of mucin was also examined. Whilst many gels adhered for 1-5 h, others (chitosan and Eudispert) showed no retention loss over 4 d. Histologically, chitosan also showed excellent tissue wetting properties. For most materials, however, mucin significantly reduced adhesion times (P < 0.05). In conclusion, the absence of mucin, the control of gel hydration and swelling, and wetting characteristics were identified as key factors for prolonged adhesion. PMID- 7548613 TI - Implantation of sepiolite-collagen complexes in surgically created rat calvaria defects. AB - The response of osseous tissue to the implantation of sepiolite-collagen complexes has been studied. Sepiolite, sepiolite-collagen complex and 0.5% glutaraldehyde-treated sepiolite-collagen complex were implanted in created circular defects in rat calvaria. The tissue reactions were analysed using light, transmission and scanning electron microscopies. The patterns of bone growth were radiographically analysed and the bone activity was indirectly quantified by using a point-count method. The reaction against the three implanted materials is characteristic of a foreign body reaction with abundant macrophages and giant cells. Implanted products have been detected in macrophages, which suggest the involvement of phagocytosis in the resorptive process. Bone grew at the implantation sites originating excrescences or sometimes a thin bridge at the defect margins. The studied materials, after implantation in contact with bone tissue, did not produce any toxic effect or necrosis, allowing bone activity. PMID- 7548615 TI - Cell attachment to laser-induced AAm- and HEMA-grafted ethylene-propylene rubber as biomaterial: in vivo study. AB - With the purpose of improved tissue compatibility, ethylene-propylene rubber (EPR)-based vulcanizates have been surface grafted with acrylamide (AAm) and 2 hydroxyethyl methacrylate (HEMA) using CO2-pulsed laser as excitation source. Grafted surfaces were characterized by performing scanning electron microscopy combined with energy dispersive X-ray analysis and attenuated total reflectance infrared spectroscopy to study the surface morphology and grafting. Surface hydrophilicity (measured by water drop contact angle) increased for the grafted samples. Fractal type of morphology is formed by the grafted poly(AAm) and poly(HEMA) chains on the surface of EPR, which provides both hydrophilic and hydrophobic sites. In vivo tissue compatibility was assessed by implanting test samples in the deep intramuscular and peritoneal layers of rabbits. After 8 weeks of implantation, comparative results indicate that the adhesion of macrophages to EPR samples modified with AAm and HEMA, with no respiratory burst and cellular damage, is significantly lower than their adhesion on unmodified surfaces which show an activated state of the attached macrophages. Also, no acute or chronic inflammatory reaction was observed at the site of implantation and a thinner fibrous tissue capsule formed around the modified samples, whereas foreign body giant cells adhered to unmodified EPR. PMID- 7548616 TI - Surface modifications of alginate/poly(L-lysine) microcapsular membranes with poly(ethylene glycol) and poly(vinyl alcohol). AB - Three different surface modifications were conducted on the membranes of double layer alginate/poly(L-lysine) microcapsules with tosyl chloride-activated poly(ethylene glycol), cyanuric chloride-activated poly(ethylene glycol) and tosyl chloride-activated poly(vinyl alcohol), separately. All these surface modifications strengthen the microcapsular membranes. Of the three surface modified microcapsules, those treated with cyanuric poly(ethylene glycol) and tosylated poly(vinyl alcohol) have mechanical strength higher than that treated with tosylated poly(ethylene glycol). The permeabilities of the proteins were only slightly affected by the surface modifications. When IW32 erythroleukaemia cells were entrapped inside these microcapsules, cells proliferated to a density of (1.0-1.5) x 10(7) cells cm-3 after 7 d culturing compared with 2 x 10(6) cells cm-3 attained by free cell culturing. The intracapsular concentration of erythropoietin which was secreted by the IW32 cells accumulated to concentrations of 5-7 U cm-3. In addition, 3T3 cells were found to have a very low tendency to attach to the microcapsular membranes of all three surface-modified preparations. PMID- 7548617 TI - In situ polymerization of pyrrole in animal tissue in the formation of hybrid biomaterials. AB - Porcine pericardium was impregnated with pyrrole or its derivative, sodium 4-(3 pyrrolyl)butanesulphonate (SPBS), by soaking the animal tissue in the monomer. Subsequent in situ chemical polymerization of the monomer-rich tissue using FeCl3 as initiator produced black polypyrrole-tissue hybrid biomaterials. The rate and extent of polymerization was found to be greater in 0.5 M acetic acid than in Hepes-buffered saline (HBS) and also greater for SPBS than for pyrrole. However, better tissue integrity was obtained for polymerization in HBS. Histological examination showed that the monomers do not permeate through the entire tissue, restricting polymerization to the surface layers of the tissue. The samples so formed do not exhibit detectable electrical conductivity. PMID- 7548614 TI - Silicone rubber-hydrogel composites as polymeric biomaterials. VI. Transport properties in the water-swollen state. AB - Permeation of inorganic salts in water-swollen silicone rubber-hydrogel composites consisting of a silicone rubber matrix and lightly cross-linked particles of poly(2-hydroxyethyl methacrylate), poly(2-hydroxyethyl methacrylate co-methacrylic acid), poly(methacrylic acid), polyacrylamide or poly(acrylamide co-methacrylic acid) hydrogels was investigated. The results, together with earlier data on permeation of non-ionic low-molecular-weight substances through the composite materials, were evaluated in terms of the free-volume diffusion theory. It was found that the materials with water content exceeding a certain limit are highly permeable to the salts, and that, as regards permeation properties, they behave as homogeneous water-swollen hydrogels. The dependence of electrical conductivity of the water-swollen composites on the hydrogel phase content was measured, and the results are discussed in relation to other transport properties and to the structure of the materials. PMID- 7548618 TI - Chemokines, a family of chemotactic cytokines. AB - Chemokines are low-molecular-weight proteins that stimulate recruitment of leukocytes. They are secondary pro-inflammatory mediators that are induced by primary pro-inflammatory mediators such as interleukin-1 (IL-1) or tumor necrosis factor (TNF). The physiologic importance of this family of mediators is derived from their specificity. Unlike the classic leukocyte chemo-attractants, which have little specificity, members of the chemokine family induce recruitment of well-defined leukocyte subsets. Thus, chemokine expression can account for the presence of different types of leukocytes observed in various normal or pathologic states. There are two major chemokine sub-families based upon the position of cysteine residues, i.e., CXC and CC. All members of the CXC chemokine sub-family have an intervening amino acid between the first two cysteines; members of the CC chemokine sub-family have two adjacent cysteines. As a general rule (with some notable exceptions), members of the CXC chemokines are chemotactic for neutrophils, and CC chemokines are chemotactic for monocytes and a small sub-set of lymphocytes. This review discusses the potential role of chemokines in inflammation and focuses on the two best-characterized chemokines, monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, and interleukin-8 (IL 8), a member of the CXC chemokine sub-family. PMID- 7548619 TI - The role of phosphotyrosine signaling pathway in a parotid gland proliferation and function. AB - Tyrosine phosphorylation and the intracellular signaling processes associated with it have been the focus of intense study due to its importance in the regulation of biological processes as diverse as cell proliferation and cell differentiation. While much of what we now understand has been derived from the study of cell lines and tumor cells, the salivary glands provide a model to examine the effects of tyrosine kinases and tyrosine phosphatases in a normal differentiated tissue. This review will focus, therefore, on the role tyrosine kinases and phosphatases play in inducing the transition from stasis to active proliferation and their potential role in mediating secretory function of the salivary glands. PMID- 7548620 TI - Complement biosynthesis in the central nervous system. AB - Complement is an important effector arm of the human immune response. Binding of proteolytic fragments derived from activation of complement by specific receptors leads to responses as diverse as inflammation, opsonization, and B-cell activation. The importance of characterizing the expression and regulation of complement in the CNS is highlighted by growing evidence that complement plays a significant role in the pathogenesis of a variety of neurological diseases, such as multiple sclerosis and Alzheimer's disease. In vitro studies have demonstrated that astrocytes, the predominant glial cell type in the brain, are capable of expressing or producing a majority of the components of the complement system. Expression of many complement proteins synthesized by astrocytes is regulated by both pro- and anti-inflammatory cytokines, many of which are also produced by several cell types in the CNS. In addition to astrocytes, ependymal cells, endothelial cells, microglia, and neurons have recently been shown to synthesize various complement proteins or express complement receptors on their cell surfaces. Together, these studies demonstrate that several cell types throughout the brain have the potential to express complement and, in many cases, increase expression in response to mediators of the acute phase response. These studies suggest that complement may play a greater role in CNS immune responses than previously thought, and pave the way for better understanding of the dynamics of complement expression and regulation in vivo. Such understanding may lead to therapeutic manipulation of complement host defense functions in a variety of inflammatory and degenerative diseases in the CNS. PMID- 7548621 TI - Oral leukoplakia. AB - Leukoplakia has evolved as a clinico-pathologic concept over many years, with the current clinical designation being accepted worldwide. Reflective of the biology of leukoplakia is the concept of cellular atypia and epithelial dysplasia. Adding to a better understanding of leukoplakia in general has been the definition of relevant clinical subsets which, in some cases, includes etiology (snuff), while in other cases a verrucous clinical appearance will suggest a more aggressive anticipated behavior pattern. Tobacco usage, in many of its forms, remains the prime etiologic factor; however, other considerations also apply. More recently, the potential etiologic role of Candida albicans has been stressed, as well as its possible role in carcinogenesis. So-called oral hairy leukoplakia has been defined in relation to a possible Epstein-Barr viral infection, usually in the immunosuppressed patient. Other viruses, human papilloma virus in particular, have been implicated in leukoplakia, while genetic alterations involving tumor suppressor elements (p53) have also been investigated. Finally, the management of this common condition remains a variable and includes local, topical, and systemic therapies such as anti-oxidants, carotenoids, and retinoids. PMID- 7548622 TI - Biochemical composition of human saliva in relation to other mucosal fluids. AB - This paper describes several salivary components and their distribution in other mucosal secretions. Histatins are polypeptides which possess exceptional anti fungal and anti-bacterial activities, but are nevertheless present only in saliva. Proline-rich proteins (PRPs) are members of a closely related family, of which the acidic PRPs are found solely in saliva, whereas the basic PRPs are also found in other secretions. Mucins are a group of glycoproteins that contribute to the visco-elastic character of the mucosal secretions. Despite the similarities in their structure and behavior, mucins have distinct tissue distributions and amino acid sequences. Other salivary proteins are present in one or more mucosal secretions. Lysozyme is an example of a component belonging to an ancient self defense system, whereas secretory immunoglobulin A (sIgA) is the secreted part of a sophisticated adaptive immune system. Cystatins are closely related proteins which belong to a multigene family. Alpha-Amylase is a component that is believed to play a specific role in digestion, but is nevertheless present in several body fluids. Kallikrein and albumin are components of blood plasma. But whereas albumin diffuses into the different mucosal secretions, kallikrein is secreted specifically by the mucosal glands. The presence of these proteins specifically in saliva, or their distribution in other mucosal secretions as well, may provide important clues with respect to the physiology of those proteins in the oral cavity. PMID- 7548623 TI - Molecular mechanisms of dental enamel formation. AB - Tooth enamel is a unique mineralized tissue in that it is acellular, is more highly mineralized, and is comprised of individual crystallites that are larger and more oriented than other mineralized tissues. Dental enamel forms by matrix mediated biomineralization. Enamel crystallites precipitate from a supersaturated solution within a well-delineated biological compartment. Mature enamel crystallites are comprised of non-stoichiometric carbonated calcium hydroxyapatite. The earliest crystallites appear suddenly at the dentino-enamel junction (DEJ) as rapidly growing thin ribbons. The shape and growth patterns of these crystallites can be interpreted as evidence for a precursor phase of octacalcium phosphate (OCP). An OCP crystal displays on its (100) face a surface that may act as a template for hydroxyapatite (OHAp) precipitation. Octacalcium phosphate is less stable than hydroxyapatite and can hydrolyze to OHAp. During this process, one unit cell of octacalcium phosphate is converted into two unit cells of hydroxyapatite. During the precipitation of the mineral phase, the degree of saturation of the enamel fluid is regulated. Proteins in the enamel matrix may buffer calcium and hydrogen ion concentrations as a strategy to preclude the precipitation of competing calcium phosphate solid phases. Tuftelin is an acidic enamel protein that concentrates at the DEJ and may participate in the nucleation of enamel crystals. Other enamel proteins may regulate crystal habit by binding to specific faces of the mineral and inhibiting growth. Structural analyses of recombinant amelogenin are consistent with a functional role in establishing and maintaining the spacing between enamel crystallites. PMID- 7548624 TI - [Recovering the thread of life--destruction and reconstruction of the social world of patients after cancer of the colon]. AB - To find a lifeline again. The destruction and rebuilding of the social world of patients after cancer of the colon. This qualitative study arose from the concern of the Swiss Cancer Society (Schweizerische Krebsliga) to optimise the service of social work and home-nursing. 36 patients who lived in their own homes after treatment for cancer of the colon were interviewed. Transcripts were analysed, using the method and coding practice of "Grounded Theory" of Glaser, Strauss & Corbin. The results reported here show that the illness affected life to varying degree. The experience of cancer of the colon affected body image, activities of daily living, work, thought, emotion, social life and material circumstances. The process of continuing to live is affected by the state of health, quality of social relationships and financial security among other factors. Research which focuses on interactions in the context of their conditions and consequences is the hallmark of the interactionist social sciences. The theoretical and methodological premises of the interactionist school of Anselm Strauss generate knowledge which is particularly useful in relation to professional support of clients in the health and social services. PMID- 7548625 TI - [Caring for dying patients--and if they refuse?]. AB - Caring for dying patients...and if they say no? Caring for dying patients is a difficult and demanding task for nurses. One aspect of this theme, namely the question how nurses react to and feel about the desire of dying patients to omit nursing actions with the goal of improving their well-being, was studied in an clinic for internal medicine of a university hospital. The care of 4 dying patients was observed and 8 involved nurses were interviewed. A grounded theory approach was chosen for analysing the data. The results show under which circumstances dying patients expressed their wish to omit certain nursing actions and what actions were not wanted. Furthermore the paper describes: (1) factors which influenced the nurses activities, (2) nurses strategies and (3) the emotional consequences nurses activities had on themselves. From the results several conclusions could be drawn for the care of dying patients. The study shows that nurses can become aware of and reflect upon their dealing with patients' wish to omit nursing actions. PMID- 7548626 TI - [Bibliographies on Dorothea Orem's nursing theory]. AB - Bibliography on the subject of Dorothea Orem's nursing theory Dorothea Orem's theory is now well known in Switzerland. In many first level and continuing education courses it is taught, dealt with and critically examined. Various attempts are made to translate it into practice. This compilation shows, in the form of abstracts of various articles, how Orem's self-care-deficit theory is applied, discussed and reflected upon. Part of this paper compares Orem's theory with other nursing theories. This bibliography, like that already published in Pflege, Vol. 7, 1994, Nr. 3, was compiled in the context of the topic "Pain", as part of the Higher Professional Education Course, level 2, of the Advanced School of Nursing, Aarau. PMID- 7548627 TI - [The comprehension of health in the mirror of time]. AB - At any point of time, what we understand by the term "health" is influenced by the prevailing ideology and value judgements. The cosmological, anthropological perspective of antiquity became subordinated to the transcendental view of the middle ages. In modern times secularisation, naturalisation, specialisation, technology, have led to biologic orientation. One can see that behind each of the definitions of health listed (that of WHO, that offered by anthropologists, that derived from psychosomatic medicine and research, that of value concepts and that of quality of life), different pictures of the world which evoke different value/judgements and ideologies emerge. These determine to a considerable degree how one thinks and acts and they find expression in health care and professional nursing practice. The concept of health reflects human understanding of self and of the world and challenges us to employ a systematic, dynamic, historical approach. PMID- 7548628 TI - [How can the patient's home be changed into a work place for the nurse? Interaction theoretical note on the role of the professional nurse in ambulatory care]. AB - In home-care-situations nurses are confronted with the special problem of changing the house of the patients into a nurse's place of work and to play at this "stage" the role of professional nurses. The role-theory of Goffman is the background to outline some structural barriers for the performance of this role. In such and other care-giving-situations the nurse must be able to keep a necessary amount of role-distance, to regain professional autonomy. PMID- 7548629 TI - [Work psychological evaluation of the activity of counseling in community nursing. A pilot study]. AB - Working conditions of community health nurses were investigated with the assistance of patient education rating. The subject of the study was: "Is it possible for community health nurses to perform the activity of patient education completely? Are there conditions for patient education which allow only a partial activity? How do community health nurses experience the weak points at their work place in community health nursing?" Industrial psychology developed an activity rating system (TBS), which has been completed by the observations made by community health nurses during patient education and checked in interviews. The criteria of complete and incomplete activity and the experience of the community health nurses concerning patient education have been collected, analysed and interpreted. The analysis and rating of patient education carried out by community health nurses enables conclusions to be drawn about the whole system (structure of organisational activity and individuals approach). The unclear definition of the activity of patient education is the reason why the quality of the activity is insufficient. The role of the community health nurse is vague for herself, the employer and the patient. The organisational structure lacks professional support, which is indicated for community health nurses in the education of patients who have to deal with the challenges of illness. Inter connected organisational and personnel development is recommended, to motivate the staff, promote the efficiency and improve the quality of the work. PMID- 7548630 TI - [A check list for working conditions and work satisfaction of nurses in general hospitals--report on results]. AB - During a training on continuing education the nursing staff of the "Klinikum Bamberg" decided to start systematic research into career patterns, working conditions and work satisfaction. A checklist was developed. The study reports findings from 543 nurses in 4 german general hospitals. Leaving aside details about specific clinical areas it emerged a high agreement in positively evaluated working conditions and in negatively evaluated as well. Particularly positively evaluated areas are work-content, colleague in the wards, relationships with the immediate superiors. There was little homogeneity concerning working hours and range of duties. What was generally thought to need improvement was staffing level, workload, bed occupancy, hospital management/organization and salar. These findings are consistent with mostly all newer publications on this topic. They too are consistent with the professional self-awareness of the nursing staff inferrable from the career expectations: The most important object only rarely were in keeping with the traditional image of "service to humanity". Personal aims, such as money, were much more prevalent, but also to be able to give the best possible and dignific care. With respect to these findings we propose to do the following strategy: It no longer serves a useful purpose to continue research with variing methods of data collection and to confirm the already well known facts. It rather seems politically necessary to take the next steps: Using a standardized method of data collection, to get comparable data, based on this to establish the actual state of working conditions from the sight of the nursing staff. Finally to demand the changing of these conditions which need to be improved as the necessary framework for modern nursing practice. Our experience suggests that the check list is a suitable tool for the collection of these data. PMID- 7548631 TI - [Work satisfaction and working conditions of the nursing staff as the basis of internal interventions]. AB - Satisfaction and working conditions of nursing staff as a basis for internal interventions Can analyzing the working situation of nursing staff by means of a questionnaire contribute to improving working conditions and thus reduce fluctuation and increase work-satisfaction? This was the basic question of this investigation and aimed at creating an instrument that could help work out existing resources and strategies for possible interventions. Therefore, an anonymous questionnaire containing 114 questions concerning the current situation was presented to the nursing staff in two departments of internal medicine and one surgical department of a university hospital in southern Germany in April 1993. Analysis of the data of the different working fields (ordinary/intensive care, clinics) showed individual differences regarding resources and possible interventions. Furthermore, the assumption could be confirmed that by means of such a project the nursing management gains specific starting points for the preparation of change. The prospects of improving working conditions seem quite good, as these findings are based upon the statements of the staff concerned. It has to be emphasized that the nursing staff were able to identify themselves with the conclusions when the results were presented to them. PMID- 7548632 TI - [The history of medical nursing schools at the Vienna University (1812 to 1846) and at the Royal Charite Hospital in Berlin since 1832]. AB - The first examples of nurse training, beginning in Vienna in 1812 and in Berlin in 1832, demonstrate that research in nursing-history must explore primary sources which are still in existence, in order to counteract current cliche and perhaps even develop completely new insights into historical accounts. Nursing literature in Austria did not start will the textbook by Billroth (1st edition 1881), but with the textbook by Maximilian Florian Schmidt, same publisher, 50 years earlier. His professorship was not held in the University but in what would not be a technical school. The textbook by Schmidt, and not that by Franz Anton Mai (1782), provided the submission for the curriculum which Dieffenbach made available to the school opened in Berlin in 1832. Dieffenbach was given the title of "Professor" for his services to "the improvement of nursing" and not for his pioneer work in plastic and orthopedic surgery. It was not Dieffenbach's curriculum but the 1837 revision of the work of Gedike, who was not a pupil of Dieffenbach's, which was used by the ministery responsible for the Board of Trustees of the hospital, for the state controlled course of nursing education. This educational programme was not less optional than that statutorily controlled one from 1907 onwards. PMID- 7548634 TI - Interactive multivariate modelling of 4-amino-2-carboxytetrahydroquinolines as antagonists of the glycine-site directed NMDA receptor-ion channel complex. AB - Multivariate quantitative conformation-activity relationships of a series of 4 amino-2-carboxytetrahydroquinolines have shown that the biological activity profile (inhibition of (3H)glycine binding to rat cortical membranes and antagonism of depolarizations due to N-methyl-D-aspartic acid (NMDA) in a rat cortical slice preparation) depends on the molar refraction of the substituents, the total energy related to the nitrogen atom and the carbon atom of the -NHCOR1 bridge of the lead molecule (Figure 1), and the 1.4 nonvalency energy. Diagnostic statistics shows that the model assumptions are approximately satisfied. PMID- 7548635 TI - Structure-activity relationship studies of CNS agents. XII. 1-[4-(4-aryl-1 piperazinyl)butyl]-3,4-dihydro-2(1H)-quinolinones: new 5-HT1A, 5-HT2 and D2 ligands with a potential antipsychotic activity. AB - The synthesis of three 1-[4-(4-aryl-1-piperazinyl)butyl]-3,4-dihydro-2(1H) quinolinones (5-7) was described and the receptor binding profile (5-HT1A, 5-HT2, alpha 1, D1, D2) was determined. It was found that m-chloro (5) and o-methoxy (6) derivatives are potent antagonists of the 5-HT1A, 5-HT2 and D2 receptors. It was shown that compound 6 resembles very well some atypical antipsychotics and may be considered as a novel agent of this class of drugs. PMID- 7548633 TI - Synthesis and pharmacological evaluation of phenolic 2-methyl-4-phenyl-1,2,3,4 tetrahydroisoquinolin-4-ols as a new norepinephrine potentiator. AB - 2-Methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline-4-ol(PI-OH) (2a) and its derivatives form a new class of compounds possessing norepinephrine (NE) potentiating activity. As a new series of compounds, the isomeric 4-(4 hydroxyphenyl)- and 4-(3-hydroxyphenyl)-2-methyl-1,2,3,4- tetrahydroisoquinolin-4 ols (3a and 3b), and 4-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-4-ol (3c) were prepared and their NE potentiating activities were evaluated. The 4-(4 hydroxyphenyl) analogue (3a) had only moderate activity and the 4-(3 hydroxyphenyl) analogue (3b) possessed a slightly higher activity than PI-OH. The 4-(3,4-dihydroxyphenyl) analogue (3c) was the most active compound and the pD2 value was 7.71 +/- 0.06 (activity ratio; 26.9 fold) at a concentration of 3 x 10( 6) M. These results indicate the importance of dihydroxyphenylethanolamine moiety of 3c for the inhibition of NE uptake. PMID- 7548637 TI - The hydrolysis of N-methylisatin: on how a simple rate-pH profile may mislead. AB - Alkaline hydrolysis of the title isatin 4 in unbuffered solution obeys a rate law such that kobs varies; is directly proportional to [OH-]2 at pH 10 changes smoothly to kobs varies; is directly proportional to [OG-] by pH 13. As in the case of other isatins, this inflection in the pH profile is traced to ionisation of the substrate by addition of OH-; the UV spectrum of the anion has been recorded. Hydrolysis is also very sensitive to general base catalysis, which has mechanistic implications and would probably have prevented any development of this compound as a potential drug. PMID- 7548636 TI - Molecular modeling of 5-HT3 receptor antagonists: geometrical, electronic and lipophilic features of the pharmacophore and 3D-QSAR study. AB - If the geometrical pharmacophore of 5-HT3 receptor antagonists has been proposed by different authors, the electronic and lipophilic features of the ligands had to be precised. A 3-D QSAR study has enlightened the importance of three parameters derived from molecular electrostatic and molecular lipophilicity potentials. A multiple linear regression equation has been established. Its predictive character (non specific binding of 3[H]-ICS 205-930) has been tested with success for three different new ligands. PMID- 7548638 TI - A model of the serotonin 5-HT1A receptor: agonist and antagonist binding sites. AB - We built a model for the 5-HT1A receptor, using the 3D-structure of bacteriorhodopsin as a structural template. With the use of site-directed mutagenesis data, several potent 5-HT1A agonists, belonging to five different structural classes, and an aryloxypropanolamine antagonist, were docked into the receptor model. After docking, the surrounding of the ligands appeared to be in full agreement with previously reported SAR-data of 5-HT1A ligands. In this study, for the first time, an explanation for 5-HT SAR results is given in terms of interactions between ligands and amino acid residues. Also the selectivity of 8-OH-DPAT for the 5-HT1A receptor is accounted for. In our model the agonists and the antagonist interact with different residues on several helices. They all interact with the essential aspartic acid on helix III, that is known to bind all amines to receptors for biogenic amines. This partial overlap of the binding sites accounts for the antagonism of the class of aryloxypropanolamines and for the deviating SAR of this class of compounds when compared to agonists. PMID- 7548639 TI - [Molecular biology and future of internal medicine]. PMID- 7548640 TI - [Alpha tumor necrosis factor in central nervous system disease associated with HIV infection]. AB - The aim of the present study is to evaluate the relationship between the alpha tumor necrosis factor (TNF-alpha), interleukin 1 beta (IL-1 beta) and the neurological disease associated to the HIV-1 infection and different neurological manifestations (15 infections of the CNS and 11 AIDS-dementia complexes) and 14 from a control group. The mean value of TNF-alpha in CSF of patients with HIV-1 infection and AIDS-dementia complex was 19.8 +/- 30.6 pg/ml, superior to that of the control group (p < 0.05). The group of patients with HIV-1 and opportunistic CNS infection has a TNF-alpha value of 28.5 +/- 37.8 pg/ml, that is superior to that of the patients with the AIDS-dementia complex (TNF-alpha = 7.9 +/- 9.4 pg ml; p < 0.05). Within the group of patients with a CNS infection, the value of TNF-alpha was greater in those in the acute phase (44.2 +/- 42.4 pg/ml) than in those in the chronic phase (6.8 +/- 7.6 pg/ml; p < 0.05). The TNF-alpha in the CSF is a good marker of infection of the CNS in the HIV-1 infection. PMID- 7548641 TI - [Plasma cortisol and adrenal response to ACTH stimulation in diabetes mellitus with moderate decompensation]. AB - No significant difference was found between the mean plasma cortisol levels in 2 groups of diabetic patients (10 with non-severe metabolic decompensation and 10 well compensated). Likewise, there was not statistical difference on considering the diurnal Cortisol/nocturnal Cortisol quotients in both groups; these quotients besides showed the persistence of the circadian rythm. No valuable difference was seen in the adrenal response to ACTH stimulus. It was found direct correlation between glycemic levels and cortisol increments after stimulus in the total series of 20 patients (r = 0.44; p < 0.05). There were also significant correlations between diurnal and nocturnal Cortisol (r = 0.63; p < 0.01) and between Cortisol levels before and after stimulus (r = 0.68; p < 0.01). No differences were found in Cortisol levels on estimating the incidence of diabetic complications (retinopathy or nephropathy). All these data suggest the tendency to adrenal hyperactivity due to the diabetic decompensation through the action of the hypothalamic-pituitary axis. PMID- 7548642 TI - [Preoperative geriatric assessment in major surgery in the aged]. AB - The objective of this study is to investigate if a comprehensive geriatric assessment is useful for predicting morbimortality, functional impairment and the risk for institutionalization for elderly patients after major surgery. A prospective study was carried out at a tertiary hospital, between April and June 1993, including those elderly surgical inpatients for whom a preoperative assessment of the Geriatric Department was demanded. Beside traditional parameters of surgical risk (ASA clas, Goldman index, respiratory and nutritional risk), other medical, functional and social parameters were evaluated. A perioperative follow-up was made and a new functional evaluation one month after discharge. 49.5% of the patients presented perioperative complications and the mortality rate was 10%. 11% needed residential accommodation after discharge. Traditional surgical risks as well as previous functional capacity were predictors of perioperative morbimortality. But only previous functional capacity and nutritional status predict institutionalization. No predictors of functional impairment were found, with the exception of perioperative complications. PMID- 7548644 TI - [Bronchogenic cyst with atypical localization]. AB - Bronchogenic congenital cysts are uncommon. They most frequently develop into the lung parenchyma. Rarely, mediastinal site may occur due to intestinal origin. Bronchogenic cysts seldom arise in the posterior mediastinum. Computerized tomography of the chest allowed a diagnosis of a posterior mediastinal bronchogenic cyst in our patient with poor symptoms. Cyst aspiration and MRI (magnetic resonance imaging) also provide the diagnosis. Treatment is by surgical excision if symptoms occur and have good prognosis. PMID- 7548643 TI - [Study of associated risk factors and prevalence of heart diseases in patients with arterial hypertension]. AB - We studied the frequency of heart disease and association with other coronary risk factors in 243 consecutive patients (124 male and 119 female) suffering from arterial hypertension (Group HT). The mean age was 67.5 +/- 9.6 years. This group was compared to a group of 357 subjects (217 male and 140 female) without arterial hypertension (Group noHT) and mean age of 63.5 +/- 13 years. In our group the patients with arterial hypertension presented smoking habits in 35%, hypercholesterolemia in 22%, left ventricular hypertrophy (LVH) in 18%, alcoholic habits in 15%, hypertriglyceridemia in 12%, diabetes in 9% and hyperuricemia in 7%. 15% of the patients suffering from arterial hypertension turned out with coronary heart disease (62% angina and 38% myocardial infarction), 19% with atrial fibrillation and 13% with heart failure. Compared to the patients without hypertension we found significant statistical correlation with the age (67.5 +/- 9.6 HT and 63.5 +/- 13 no HT, p < 0.001), the LVH (18% HT and 4% no HT, p < 0.001) and number of coronary risk factors (2 +/- 1 HT and 1.1 +/- 0.9, p < 0.001). The percentage of people without cardiac disease is lower among the group with arterial hypertension (53% HT and 71% noHT, p < 0.001), showing as well a higher incidence of atrial fibrillation (19% HT and 11% noHT, p < 0.05) and heart failure (13% HT and 7% noHT, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548645 TI - [Amyloid intestinal pseudo-obstruction as initial manifestation of IgA multiple myeloma]. AB - Amyloidosis is a well known complication of Multiple Myeloma. Although involvement of the gastrointestinal tract is common in patients with Amyloid, severe symptoms are no frequent, nevertheless vascular deposits may produce gastrointestinal ischemia and bleeding and perforation. Injured mucosae of the intestinal well may produce malabsorption and the neuromuscular infiltration determine alterations of gastrointestinal motility. Although renal and cardiac disfunction is the most frequently cause of death in these patients, intestinal pseudo-obstruction bears a serious prognosis. We present a case of a patients who was operated because of a presumptive diagnosis of intestinal obstruction, which small bowel was infiltrated with Amyloid and which bone marrow demonstrated multiple myeloma. PMID- 7548646 TI - [Pericardial tamponade and carcinomatous lymphangitis as primary manifestation of lung neoplasm]. AB - Cardiac tamponade (CT) and Carcinomatous lymphangitis (CL) association as an initial clinical presentation of a neoplasm is very uncommon, creating diagnosis difficulties in the patient first evaluation. This paper reports one case of a male who was admitted in Emergency Department with clinical and radiological findings of heart failure. Following studies showed CT and CL secondary to a bronchial adenocarcinoma. Differential diagnosis is really important for its associated therapeutic implications because of the CT hemodynamic worsening situation due to the diuretic and vasodilators used in the treatment of heart failure. An echocardiography should be done because it is very useful for the initial evaluation of a heart failure of obscure origin. PMID- 7548647 TI - [Adverse reaction to cefonicid analogous to serum sickness]. AB - We report a case of a male who was treated with cephonicid because of a surgical complication. Serum-sickness like symptoms were diagnosed two weeks later. Medical references are discussed. PMID- 7548648 TI - [Follicular cancer of the thyroid: atypical presentation]. AB - Follicular thyroid carcinoma usually presents as a thyroid nodule, with rare cases showing metastatic disease at diagnosis. We present a patient with follicular thyroid carcinoma who was diagnosed as a large pelvic metastatic mass with a biochemical profile of subclinical hyperthyroidism. She was treated with local-field radiotherapy, since surgical resection was not possible and radioiodine therapy would be of little benefit due to its large volume. She is currently in continuous remission two years from diagnosis. PMID- 7548649 TI - [New etiopathogenic, diagnostic and therapeutic aspects in primary pulmonary hypertension]. AB - Primary Pulmonary Hypertension (PPH) is a rare disease of unknown aetiology that is diagnosed only when the underlying causing factors are undetermined. Usually is discovered in its late stage, when pulmonary vascular resistances are severely compromised and the pathologic changes already well developed, resulting in right heart failure and death within several years of the onset of symptoms. The data published in the last years have provided new insights into the disease knowledge. In this article currents concepts about aetiology, pathogenesis and open lung biopsy value are reviewed; clinical manifestations and diagnostic methods are described; the usefulness of medical treatment to improve quality of life in these patients, mainly anticoagulation and vasodilator therapy with high dose nifedipine, and how lung transplantation has increasingly become an option for selected patients with PPH, is also analysed. PMID- 7548650 TI - [HTLV-I AND HTLV-II in Spain]. AB - Up to October 1994, twenty-four HTLV-I and 113 HTLV-II infected persons have been identified in Spain. A third of HTLV-I carriers were immigrants coming from endemic regions. In contrast, most HTLV-II-infected persons were Spanish native drug users. Four cases of HTLV-I-associated myelopathy (TSP) were found, and three patients suffered lymphoma (ATL). A former drug user carrying HTLV-II infection developed a severe proximal myopathy. Studies performed on more than 100,000 blood donors allowed the identification of only two persons with HTLV-I infection. PMID- 7548651 TI - [Spontaneous pneumothorax in response to chemotherapy of metastatic germinal tumor]. PMID- 7548652 TI - [Acute confusion syndrome and thalamic infarction]. PMID- 7548653 TI - [Catamenial pneumothorax]. PMID- 7548655 TI - [Transitory monoclonal gammapathy and disseminated intravascular coagulation in Mediterranean boutonneuse fever]. PMID- 7548654 TI - [Atrioventricular block and Kearns-Sayre syndrome: prognostic implications]. PMID- 7548656 TI - [Acute pancreatitis as initial manifestation of lung carcinoma]. PMID- 7548657 TI - [Diagnosis of primary biliary cirrhosis in an 84-year-old man]. PMID- 7548658 TI - [Evaluation of family and community medicine residents]. PMID- 7548659 TI - [Medicosocial profile of the crucial caregiver. Is he/she an occult patient?]. AB - OBJECTIVE: To determine the prevalence of physical pathology (morbidity felt) and psychological unease (anxiety-depression) in crucial carers for disabled patients, and so establish a profile of them. DESIGN: An observational study. Analysis of two historical cohorts. SETTING: Primary care. Urban health centre. PATIENTS: The exposed cohort was made up of 54 carers for disabled patients, chosen at random from the records at our Centre. The non-exposed cohort (n = 54), matched for age, gender and educational level, was chosen at random from the consulting rooms. MEASUREMENTS AND MAIN RESULTS: By means of a questionnaire, descriptive variables were collected, i.e. the morbidity felt, consumption of medicine, psychological unease (Goldberg's anxiety-depression scale), perception of social support (modified Duke Unl), index of the effort of the carer (IEC) and the profile of the person being cared for. The "typical" carer was a 57-year old woman (CI 95: 54-61), with a low educational level, daughter or wife of the disabled person, who received external help in few cases (11%). Problems of health referred to by carers (Arthralgia, migraines, asthenia, feeling depressed and insomnia), are more frequent (p < 0.001) than in non-carers. 75% took some medication, as against 45% of non-carers (p < 0.001). 83% displayed anxiety and 63% depression, against 36 and 37%, respectively, in the non-exposed cohort. A high IEC implied greater risk of anxiety (p < 0.05) and depression (p < 0.001). CONCLUSIONS: The crucial carer has a greater prevalence of physical and psychological morbidity. A high IEC and a perception of low social support create the conditions for higher anxiety and depression. Caring for the carer needs to be integrated into care for the disabled patient. PMID- 7548660 TI - [Level of agreement in the interpretation of electrocardiograms]. AB - OBJECTIVE: To evaluate diagnostic concordance in the interpretation of electrocardiograms (ECG) between general practitioners and a referral cardiologist. DESIGN: Crossover and descriptive. SETTING: Seven General Medicine consulting rooms at Burgo de Osma Health Centre. PATIENTS AND OTHER PARTICIPANTS: 318 ECGs included and distributed at random among the doctors, from May 1992 to April 1993 (11 months). 28 were excluded for analysis. MEASUREMENTS AND MAIN RESULTS: The proportion of agreement observed (Po) and the Kappa (K) index were obtained. Po was > 90% in all diagnostic categories, except normality (76.5%). Overall K was 0.52 (C.I. 95%: 0.50-0.54); K = 0.79 in disturbances in frequency; K = 0.52 in diagnosis of normality; K = 0.46 in disturbances of rhythm; K = 0.38 in ventricular conduction disorders; K = 0.36 in repolarisation disturbances; K = 0.32 for disorders of the auricular-ventricular union; K = 0.17 for unspecific disorders and K = 0.07 for auricular disturbances. CONCLUSIONS: Overall concordance is acceptable, as it is for normality and rhythm and frequency disturbances; for the remaining diagnostic categories it was light or ordinary. The Primary Care team (PCT) in comparison with the cardiologist underdiagnosed the frequency and a-v union disorders and overdiagnosed the unspecific disorders. The PCT must improve in their diagnoses of greater prevalence and low K value. PMID- 7548661 TI - [Detection of visual acuity disorders and amblyopia in preschool children]. AB - OBJECTIVE: To call attention to the visual sharpness disorders which pre-school children on a determined paediatric list present. DESIGN: A descriptive study. SETTING: Riano Health Centre, Langreo, Asturias. PARTICIPANTS: Pre-school children aged 4 and 5 at the study date (1994). MEASUREMENTS AND MAIN RESULTS: A total of 155 children captured for the study through the nursing child programme were evaluated. Data were collected from their corresponding clinical histories. Evaluation was performed by Paediatrics and Ophthalmology specialists and second year interns in Family and Community Medicine at the Valle del Nalon hospital. To check ocular alignment, the Hirschberg test was used; and to explore visual sharpness, the Pigassou optotype was applied. Disturbance of ocular alignment was suspected in 40.6% of cases and pathology was confirmed in 58.3%; whereas in 44% of cases referral to the ophthalmologist was due to errors in the visualisation of the optotype, with 50% presenting pathology. Amblyopia was found in 5.4% of the total population studied. In the breakdown of ophthalmic pathology it was observed that compound astigmatism was the most common finding (25%). CONCLUSIONS: 1. The high prevalence of Amblyopia found underlines the importance of considering it as a priority objective in prevention programmes. 2. The Hirschberg test and early use of optotypes should be considered elementary routine exploration tests in the scheduled check-ups. 3. It would be useful to strengthen training within the population as a whole as well as improve the linking of Primary and Specialist Care. PMID- 7548662 TI - [Hospital admissions of children for conditions treatable by ambulatory care in the community of Valencia]. AB - OBJECTIVE: To describe the importance which ambulatory care sensitive conditions (ACSC) have for the child population of the Valencia Community during the period 1980 to 1988, taken from the Hospital Morbidity Survey (HMS). DESIGN: Descriptive, retrospective study. SETTING: Hospital discharges collected in the HMS from the Valencia Community. PATIENTS: Children under 15 years old. MEASUREMENTS AND MAIN RESULTS: The total percentage of hospital discharges from ACSC observed were 16, 13.2 and 10% for the provinces of Valencia, Castellon and Alicante respectively. The highest percentage was shown in the 1 to 4 year old age group (19%). The proportion of discharges for ACSC was higher in girls (15%) than in boys (13%). Children under one year old show the highest rates. Valencia has for the whole period the highest rate ratio than 1 of being hospitalised for ACSC. CONCLUSIONS: The magnitude of these conditions is important and suggest the possibility of their use as a monitoring instrument of certain health care activities. PMID- 7548663 TI - [Analysis of research in primary health care in the Murcia region (1988-1992)]. AB - OBJECTIVE: To analyse Primary Health Care (PHC) research in Murcia between 1988 and 1992, looking at the possible influence of regional PHC conferences (RC) on the production of scientific work, which might be presented to RC, national conferences (NC) or national reviews (NR), as well as the impact of personal factors. DESIGN: A crossover, retrospective and descriptive study. PARTICIPANTS: Publications in NR and presentations at Murcia PHC RC and at NC between 1988 and 1992. 176 works were analysed. There were two sources of information: a) documents on PHC done in the region, articles, editorials or letters to the editor in NR collected in the IME (Spanish Medical Index), or as oral contributions, papers or posters in PHC RC or NC; b) personal and professional data of the authors. MEASUREMENTS AND MAIN RESULTS: Documents: title, authors, sources, date, theme, type, work centre and describer. Authors: sex, age, work activity, work situation, place of work and number of jobs. Statistical treatment: Chi squared and variance analysis. Between 1988 and 1992 works presented to RC duplicated (16 to 39) and increased by six to NC (1 to 6) and NR (2 to 13). Health Centres (62% of studies) made the greatest scientific contribution. CONCLUSIONS: The celebration of RC has a positive impact on the number of articles sent to NR, but not on the presentation of work in NC. Increase in the number of PHC research works (18 in 1988 and 52 in 1992) does not imply higher proportional collaboration of nonmedical health staff. PMID- 7548664 TI - [Consensus process in the field of health inequalities. The Delphi technique]. AB - OBJECTIVE: An approach to finding the determinants of the risk of health inequality among foreign immigrants living in the city of Barcelona. DESIGN: A descriptive study done by interviewing a group of key informers, using the Delphi technique. SETTING: Barcelona city Health Region. PARTICIPANTS: 20 professionals from different sectors and levels of care were selected because of the relationship of their work area to the immigrant population. Primary Care, specialist, hospital, Family Planning and Public Health professionals took part from the health field; Primary Care, planning and non-governmental organisations from the social field. MEASUREMENTS AND MAIN RESULTS: A series of mailed questionnaires were used to elicit the participants' opinions and perceptions of the determinants of foreign immigrants' health inequalities. The final reply figure was 90%. Of the overall points granted to the agreed situations, 52.4% concerned aspects relating to their general living situation, 25.1% to health care and 22.4% to socio-cultural questions. CONCLUSIONS: The results show that, in addition to the health-care field, general living situation and socio-cultural questions play a fundamental part in determining foreign immigrants' health inequalities. We considered that the Delphi was a useful and rapid technique for obtaining qualitative information on the determinants of health inequalities and that it permitted us to contribute information which was complementary to other studies of inequalities which used classical health indicators. PMID- 7548665 TI - [Utilization of visits and analysis of the impact of programmed visits]. AB - OBJECTIVES: To discover the characteristics of the use of consultations with regards to frequency of, and reasons for, attendance. To measure the repercussion on on-demand attendance of including patients with chronic pathologies in the appointments system. PATIENTS: Two samples of clinical histories were extracted at random from two general medical lists; one sample of 125 histories in order to determine the characteristics of the previous year's consultations; another of 44 histories where the patient had used the appointments system, in order to quantify the attendances for one year before and one after their inclusion in the system. DESIGN: A descriptive retrospective study. SETTING: Fuentes Norte Health Centre (Zaragoza). MEASUREMENTS AND MAIN RESULTS: The average of consultations per inhabitant per year was 3.7.4.8% attended_11 times. Women attended more than men (4.8 times as against 2.6; p < 0.0005), as did those over 65 (6.9 times; p < 0.0005). The main reasons for an on-demand consultation were bureaucratic (20%) and acute respiratory infections (16.3%); and for consultation with appointment, Hypertension (35.3%) and Obesity (32.8%). The average of on-demand consultations went down from 6.3 to 3.5 (p < 0.0001) on introducing the patients into the appointments system; but the overall number of consultations for these patients went up from 6.3 to 10.5 (p < 0.0001) because of the general medical and nursing appointments. CONCLUSIONS: We must limit unnecessary bureaucratic requests and encourage self-care. The efficacy of scheduled consultations must be analysed; and those attending must be identified and dealt with. PMID- 7548666 TI - [Opinion of asymptomatic HIV-positive patients about their primary care physicians]. AB - OBJECTIVE: To find the opinion of asymptomatic HIV+patients about their Primary Care doctors. DESIGN: A descriptive, crossover study with an 11-item questionnaire, filled in by the patient him/herself anonymously and analysing: communication of being seropositive, frequency of attendance, confidence in the doctor and their competence in dealing with the patient. SETTING: AIDS Information and Prevention Centre, Alicante. PATIENTS: Consecutive sampling of the first 100 HIV+patients who attended voluntarily during the first 5 months of 1993 (from January 3 to May 18 inclusive). MEASUREMENTS: 60.2% (C.I. 95%, 69.9%- 50.5%) said they communicated their being seropositive to the doctor. Intravenous drug users communicated this fact more (72.9%), against 41% of the rest of the groups (p = 0.0016). 25% had little or no confidence in their doctor and 44% considered him/her incompetent or barely competent, with these two variables being related (r = 0.42; p = 0.0001). Communicating their condition to the doctor is only related to more frequent attendance at the clinic (p = 0.0001). The reasons for not doing so are due mainly to lack of confidence (30.8%) and "didn't think of it" (46.2%). The doctor's competence obtained the lowest points average, 2.6, with its Confidence limits sharing no value with frequency of attendance or confidence. CONCLUSIONS: An important percentage of patients do not communicate their condition, due basically to problems of confidentiality and rejection. There are doubts among HIV+patients interviewed about their doctor's competence, at the same time as they have little confidence in him/her. PMID- 7548667 TI - [Therapeutic relations between health professionals and patients. Experience of a work group]. PMID- 7548668 TI - [Effects on arterial pressure of the use of anti-inflammatory nonsteroidal agents]. PMID- 7548669 TI - [Do the users know where to seek emergency health care?]. PMID- 7548670 TI - [Efficacy of diet in the control of hypercholesterolemia]. PMID- 7548671 TI - [Influenza vaccination]. PMID- 7548673 TI - [Family physician's role in anorexia nervosa]. PMID- 7548672 TI - [Liquorice: drug or food?]. PMID- 7548674 TI - [Evaluation of undergraduate teaching: conclusions of the Work Group on Family Medicine and University. Alicante, June, 1994]. PMID- 7548675 TI - [Diarrhea associated with antibiotics and disease caused by Clostridium difficile]. PMID- 7548676 TI - The permeability of fibrin network developed in human plasma. AB - Fibrin network permeability has an important role in thrombosis and inflammation since it influences the rate of transport of macromolecules through the network by convection. The conditions of polymerization of fibrin determine the network permeability and this has been attributed to variability in fibrin fibre thickness. Inconsistencies between values for fibrin fibre thickness derived from turbidity and permeability were examined. Networks were developed from human plasma by the addition of thrombin and network polymerization was modified pharmacologically. Dextran (MW 70,000) and poloxamer 188 both increased, and lauryl sulphate decreased, network permeability and network turbidity. Network fibre thickness was consistently higher when derived from permeability than from turbidity. Network permeability was significantly more susceptible to pharmacological manipulation by these agents than network turbidity. These inconsistencies were attributed to variation in the arrangement of the network fibres such as inhomogeneity of network fibre distribution and to fibre aggregation or alignment. Collectively these factors prohibit the derivation of fibrin fibre thickness from permeability. The dimensionless permeability (network permeability/(fibre radius)2) was used as an index of network fibre arrangement and found to be readily modified pharmacologically. Physiological and pharmacological regulation of fibrin network permeability may be predominantly mediated through modification of fibre arrangement and not through fibre thickness. PMID- 7548677 TI - Platelet-derived microvesicles and activated platelets express factor Xa activity. AB - Activated platelets and platelet-derived microvesicles demonstrate procoagulant properties. It is known that following stimulation, negatively charged phospholipids and factor Va become located on their surfaces. The aim of this study was to see whether activated platelets and platelet-derived microvesicles also expressed some factor Xa activity on their surfaces in a system where factor Xa did not come from external sources. In order to study this question, flow cytometry, as well as the use of a chromogenic substrate to factor Xa and a clotting assay in a factor X depleted plasma, were applied. A prothrombinase assay was also applied using prothrombin, CaCl2 and a chromogenic substrate to thrombin. The platelets were gel-filtered or washed, suspended in Tris-buffered saline, and activated by calcium ionophore A23187 or the thrombin receptor agonist peptide SFLLRN. Microvesicles and activated platelets were separated by centrifugation. Flow cytometry using a monoclonal antibody against factor Xa demonstrated the presence of factor Xa on the surface of the activated platelets. In addition, platelet-derived microvesicles and activated platelets demonstrated factor Xa activity on their surfaces detected directly by splitting of the chromogenic substrate to factor Xa, or by the prothrombinase assay. The thrombin generation in the last assay could be inhibited by a selective factor Xa inhibitor (recombinant tick anticoagulant peptide (rTAP)), soybean trypsin inhibitor, and antithrombin III plus LMW-heparin, all inhibiting at the factor Xa level, as well as by leupeptin which also inhibited the thrombin-chromogenic substrate interaction as such.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548679 TI - Polymorphic variation in the human protein C (PROC) gene promoter can influence transcriptional efficiency in vitro. AB - The haplotype frequencies of two closely linked diallelic polymorphisms in the protein C(PROC) gene promoter were determined in the British population. In principle, differences in transcription efficiency between PROC promoter haplotypes could represent an additional risk factor in determining whether or not an individual already predisposed to venous thrombosis will come to clinical attention. In order to explore this postulate, transient transfection of human hepatoma cells with PROC promoter-luciferase reporter gene constructs was performed in vitro. In HepG2 cells, the T.....A haplotype exhibited at least a two-fold higher transcription efficiency than the C.....G haplotype. A sample of British protein C deficiency patients with recurrent venous thrombosis was then allelotyped by a combination of oligonucleotide discriminant hybridization and DNA sequencing. The frequency of the low expressing C.....G haplotype was found to be slightly yet not significantly higher in these patients (0.43) than in controls (0.35). PMID- 7548678 TI - Influence of changes in lifestyle on fibrinolytic parameters and recurrence rate in patients with venous thromboembolism. AB - A prospective, non-randomized, interventional study was carried out to evaluate the effect of changes in lifestyle on abnormal fibrinolytic parameters, and the influence of those, in turn, on the risk of recurrence among patients with venous thromboembolism. Patients with elevated plasminogen activator inhibitor 1 (PAI-1) levels or decreased release of tissue plasminogen activator (t-PA) antigen were given information and advice about improved diet, increased physical activity, weight loss and/or cessation of smoking. Totally 144 patients (119 with elevated PAI-1 and 25 with deficient t-PA release) were followed with repeated analyses of the fibrinolytic parameters for a median of 8 months after the initial advice, and 65% of the patients managed to execute at least one change in their lifestyle. The reduction of PAI-1 activity after one, two and three changes in lifestyle were -2.4, -10.0 and -14.0 AU/ml (-10, -30 and -48% of baseline activity), respectively. Those who accomplished two or three changes in their lifestyle had a greater chance of normalizing the PAI-1 level than those with no or only one change (P = 0.009). The effects of improved diet, weight loss and increased physical activity on the PAI-1 level were of a similar magnitude, -7.0 to -10.4 AU/ml (24-32% reduction). The effect on t-PA release was more difficult to evaluate due to the limited number of those patients. After a follow-up of 6 years (mean) after the first visit the patients were inquired about recurrent events, with a response rate of 86%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548681 TI - 3rd International Conference on Factor XIII: From Molecular Biology and Physiology to Clinical Experience. Marburg, Germany, 8-9 June 1995. Abstracts. PMID- 7548682 TI - [Principles and experiences for modeling chaotic attractors of heart rate fluctuations with artificial neural networks]. AB - The aim of the present paper was, using artificial neural networks, to identify chaotic attractors presumed to be responsible for heart rate fluctuations. Chaotic behaviour is based on low-dimensional deterministic processes which are highly sensitive to initial conditions and therefore of limited predictability. Chaotic attractors produce orbits in the phase space where the points are dense. Following transformation of measured heart rate date into the phase space, such heart rate prediction characteristics were found in 6 adult rabbits. A low dimensional deterministic model was designed by means of an artificial neural network [2*dimension of the time series +1) input neurons, 1 hidden layer, 1 output neuron and was successfully employed to predict the next 5 heart beats. Training of the neural network in terms of the prediction of the suspected chaotic orbits was dramatically improved by considering all possible prediction intervals within the prediction horizon. The trained models enabled the heart rate fluctuations to be distinguished during consciousness, under anaesthesia and additional vagal blockade. PMID- 7548680 TI - Assay of protein S in systemic lupus erythematosus. AB - Free protein S:Ag and protein S activity determined by clotting assay are often regarded as synonymous; however, in the study group of patients, abnormal protein S activity (PS:Act) results correlated poorly with abnormal free (PS:Ag(r2 = 0.164). Significantly none of the patients in whom lupus anticoagulant and low free protein S:Ag were detected were found to have low PS:Act. These results could not be explained by activated protein C resistance, suggesting that lupus anticoagulant may cause prolongation of the clotting time within the PS:Act assay and thus give falsely high results. As reduced levels of free protein S:Ag and/or protein S activity are considered risk factors of thrombosis in systemic lupus erythematosus, these findings question the suitability of the protein S activity assay for patients who may have phospholipid antibodies. PMID- 7548684 TI - [Microwave volumetry of the extremities: a technical measuring alternative to the noninvasive venous occlusion test]. AB - Venous occlusion plethysmography is a well-established noninvasive procedure for the investigation of venous outflow disorders. Various different sensor systems are available for the purpose, e.g. photoplethysmography, strain-gauge plethysmography and impedance plethysmography. The technique of microwave volumetry described in this paper is based on the interaction of the electromagnetic fields within the loss medium, tissue, and uses an air-filled resonant cavity as a sensor. As in the case of a microwave oven, this is excited by a high-frequency generator, but involves the use of far less power. As the volume of the measured objects (foot, arm) increases, the losses in the resonator also increase and can be analysed in a computer. The results can be represented in the form of a classical venous occlusion curve. PMID- 7548683 TI - Laser Doppler flowmetry--peripheral microcirculation during cessation of cerebral and cardiocirculatory function. AB - Laser Doppler flowmetry (LDF) is a noninvasive method to study the microcirculation. We studied the feasibility of the clinical use of LDF in the intensive care unit by continuously recording different LDF parameters during the cessation of cerebral and cardiocirculatory function. The LDF-Conc (concentration of red blood cells) reflected biological phenomena correlated with the heart rate and microvibration that could not be detected with conventional means (systemic blood pressure, peripheral oxygen saturation). PMID- 7548685 TI - Induration of the diabetic foot pad: another risk factor for recurrent neuropathic plantar ulcers. AB - Ten diabetic feet with polyneuropathy and a history of recurrent plantar ulcers were assessed for soft tissue abnormalities of the plantar pad, in comparison with non-diabetic control feet. Plantar pad thickness and hardness were measured at the metatarsal heads. In addition, joint mobility was assessed by measuring the passive dorsal flexion of the metatarsophalangeal joints, and peak plantar pressures were studied during walking. The results showed slightly less joint mobility and thinner plantar pads in the diabetics as compared with control subjects; however, the differences failed to reach statistical significance. Soft tissue hardness at the heel was also comparable in patients and controls. At the sites of previous plantar ulcers over the metatarsal joints, soft tissue was significantly harder than at other sites in the same feet or in the control feet; peak plantar pressure at two metatarsal heads was higher in the diabetic feet than in the control feet (p < 0.05). Induration of the periarticular soft tissue may predispose to recurrent foot ulcers in diabetics by decreasing the shock absorption capacity of the plantar pad. PMID- 7548686 TI - [Life expectancy indicator for re-sterilizable plastic materials]. AB - Semi-disposable products in which only part of the product is disposable while another part made of plastic is designed to be reusable, are an effective counter to the increasing masses of plastic waste generated by hospitals. The major problem with such products has been the fact that the resterilizable parts could withstand only a limited number of resterilizations and the user had no means of seeing when this number had been reached. To resolve this problem, the Life-Span Indicator was developed in cooperation with Beiersdorf AG. This indicator changes colour from blue to orange after about 100 sterilizations. It is based on an azo pigment diffused into a carrier polymer, POM. Repeated heating of the slow colour change of the pigment. Not all the details of the mechanisms involved are clear, but the indicator functions reliably and reproducibly. Since the pigment is inside the polymer, the colour-change reaction is not affected by mechanical pre cleaning or disinfection. The user can thus readily see how long he can use such products without risking malfunction. The number of resterilizations can be varied to suit the product, and the indicator can be shaped as required, for example as a nonseparable snap-in plug or a capable binder. PMID- 7548688 TI - Tests for genetic linkage and homogeneity. AB - This report concerns likelihood ratio tests in a heterogeneous model for linkage, where the recombination fraction has a binomial mixture distribution with an unknown proportion of unlinked families. We consider families of unequal sizes with known or unknown phase data. In both cases, the limit distributions of the linkage test statistics are a mixture of a mass at ) and of a X2/1 distribution in equal proportions and homogeneity test statistics tend to the supremum of Gaussian processes. The critical values of the homogeneity tests are simulated, and the power functions of the linkage and homogeneity tests are compared in a simulation study. PMID- 7548687 TI - Multivariate contingency tables and the analysis of exchangeability. AB - There are settings in the social and health sciences where it is natural to question whether a collection of discrete random variables is exchangeable. In this paper the inter-relationships between parameter symmetry, parameter invariance, and exchangeable discrete random variables are investigated within the log-linear models framework. We demonstrate how log-linear models can be used to formulate and test hypotheses of various forms of exchangeability, and to characterize departures from exchangeability. Conditions under which the observed cross-classification collapses into a lower dimensional cross-classification, while preserving the essential probability structure of the higher dimensional cross-classification, and the model structure of this lower dimensional cross classification are presented. The development is sufficiently general to allow for subsetting the variables into classes, which is important for some applications. For example, in studying the spatial clustering of periodontal disease, there is interest in studying differences among disease patterns between the upper and lower arches in terms of parameter symmetry, parameter invariance, and exchangeability. Cross-sectional periodontal disease data from a study of Pima Indians residing in the Gila River Indian Community are used to illustrate how log-linear models may be used to examine for exchangeability, and for specific departures from exchangeability. PMID- 7548689 TI - Marginal regression for repeated binary data with outcome subject to non ignorable non-response. AB - Using a model that accounts for non-ignorable non-response, we analyzed data from the Muscatine Risk Factor Study (Woolson and Clarke, 1984, Journal of the Royal Statistical Society, Series A 147, 87-99) on the effects of gender and age on obesity in schoolchildren. The methodology is related to that of Diggle and Kenward (1994, Applied Statistics 43, 49-93), except that the repeated data are binary, not continuous, and the non-response occurs in various patterns, not just dropouts. We found strong evidence that non-response was non-ignorable. In addition, we found that the proportion of children who were obese differed significantly with gender and increased with age. PMID- 7548690 TI - Use of historical marker data for assessing treatment effects in phase I/II trials when subject selection is determined by baseline marker level. AB - Although the primary focus of Phase I clinical trials is to assess clinical pharmacology and possible toxicities, any information on the potential effect of treatment would be useful in helping to determine priorities between treatments for further study. We consider the scenario where data are routinely collected on a marker of disease progression on all patients attending a clinic, but the trial is restricted to patients who have a marker level within a defined range at study baseline. Using a two-step approach to estimation, the marker histories are used to give predictions of marker values during trial follow up, assuming no treatment effect and adjusted for the regression to the mean effect, for those subjects selected for the trial. Comparison between the observed responses and the predicted values then forms the basis for moment-based estimation and hypothesis testing for the treatment effect. The method is easily extended to compare summary measures for repeated measurements during follow up in the trial to predicted summary measures. An example using CD4 cell counts in an AIDS study is given. PMID- 7548691 TI - Tests of random mating for a highly polymorphic locus: application to HLA data. AB - Testing for random mating at an HLA locus is a difficult problem because of the highly polymorphic nature of the HLA loci. We discuss some methodological issues and propose several tests. A simulation study is conducted to evaluate these tests. The single allele test and the shared allele test deal with small sample sizes by aggregating the data in different ways. The shared allele test is found to be a more powerful method of detecting non-random mating patterns involving a deficiency or an excess of similar genotypes than the single allele test. We show that random mating of couple at the genotype level implies the random mating of couple at the allele level. Several multi-allele approaches are proposed for large population-based data sets. Among them, the corrected allele-table test performs better than the generalized Wald test in terms of power and size. These methods are then applied to an HLA data set of Caucasian couples, and no solid evidence for non-random mating at the HLA A, B, and DR loci is found. PMID- 7548692 TI - Model selection for extended quasi-likelihood models in small samples. AB - We develop a small sample criterion (AICc) for the selection of extended quasi likelihood models. In contrast to the Akaike information criterion (AIC). AICc provides a more nearly unbiased estimator for the expected Kullback-Leibler information. Consequently, it often selects better models than AIC in small samples. For the logistic regression model, Monte Carlo results show that AICc outperforms AIC, Pregibon's (1979, Data Analytic Methods for Generalized Linear Models. Ph.D. thesis. University of Toronto) Cp*, and the Cp selection criteria of Hosmer et al. (1989, Biometrics 45, 1265-1270). Two examples are presented. PMID- 7548693 TI - Empirical estimation of a distribution function with truncated and doubly interval-censored data and its application to AIDS studies. AB - In this paper we discuss the non-parametric estimation of a distribution function based on incomplete data for which the measurement origin of a survival time or the date of enrollment in a study is known only to belong to an interval. Also the survival time of interest itself is observed from a truncated distribution and is known only to lie in an interval. To estimate the distribution function, a simple self-consistency algorithm, a generalization of Turnbull's (1976, Journal of the Royal Statistical Association, Series B 38, 290-295) self-consistency algorithm, is proposed. This method is then used to analyze two AIDS cohort studies, for which direct use of the EM algorithm (Dempster, Laird and Rubin, 1976, Journal of the Royal Statistical Association, Series B 39, 1-38), which is computationally complicated, has previously been the usual method of the analysis. PMID- 7548694 TI - Reference values obtained by kernel-based estimation of quantile regressions. AB - In this paper, we study the problem of estimating non-parametrically a quantile regression curve as it applies to computing reference values. We propose an automatic procedure that uses a symmetrized nearest-neighbor kernel estimator of conditional distributions. We also discuss ways of measuring the dispersion of quantile regression estimator. One is based on the asymptotic distribution of such quantiles, while the other relies on the bootstrap method. The results of a small simulation study show that the methods of the paper perform rather well even in a situation where a good parametric solution is available. As an example, we analyze a small part of a data set that was collected to establish reference values for blood velocity in different parts of the umbilical cord of human fetuse as they grow toward birth. PMID- 7548695 TI - Correction for covariate measurement error in generalized linear models--a bootstrap approach. AB - A two-phase bootstrap method is proposed for correcting covariate measurement error. Two data sets are needed: validation data for approximating the measurement model and data with a response variable. Bootstrap samples from both the data sets validation data are taken. Parameter estimates of the generalized linear model are calculated using expectations of the measurement model from the validation data as explanatory variables. The method is compared through simulation in logistic regression with the correction method proposed by Rosner, Willet, and Spiegelman (1991, Statistics in Medicine 8, 1051-1069). A real data example is also presented. PMID- 7548697 TI - Survival curves subjected to occasional insults. AB - We describe a parametric family of survival curves which can be used to model time to event data in systems subjected to occasional insults or shocks. Parameters control the location, magnitude, and duration of the effect for each insult. We use these models to describe the survival history of a large cohort of Drosophila that were transferred to new vials on a weekly basis as their nutrient medium deteriorated. A periodic model with fewer parameters exhibits a good fit to the data. PMID- 7548698 TI - On plotting renovated samples. AB - In this note we use the Buckley-James method for censored regression in the p sample problem where the samples are subject to right-censoring. The samples are reconstructed so as to remove the effect of censoring, and graphical procedures based on quantiles (such as boxplots) may then be used as a standard data analytic tool to describe the variable being measured. PMID- 7548696 TI - Relative potency of two preparations in two-way elimination of heterogeneity designs with multivariate responses. AB - For two preparations, administered in two-way elimination of heterogeneity designs, where response is multivariate, formulas are given for tests of hypotheses concerning the existence of a common relative potency. Point and interval estimation of relative potency are also considered. PMID- 7548700 TI - Randomization model methods for evaluating treatment efficacy in multicenter clinical trials. AB - This paper studies randomization model methods for analyzing data from a multicenter study comparing the effectiveness of two treatments. The Mantel Haenszel mean score statistic, which can be used for continuous or ordered categorical response variables, is shown to be a useful nonparametric alternative to standard linear model methods for testing the significance of the average treatment difference. In an extensive simulation study, the mean score test performs nearly as well as the optimal linear model methods when the normal theory assumptions are satisfied. A related estimator of the average treatment difference is also studied. This estimator, which is a weighted average of the center-specific mean differences, is analogous to the commonly used Mantel Haenszel estimator of the average odds ratio in stratified 2 x 2 contingency tables. The proposed estimator is equivalent to the fixed-effects analysis of variance estimator from the main effects model, and valid estimation of its variance is feasible under very general assumptions. PMID- 7548701 TI - Sequential monitoring of survival data with the Wilcoxon statistic. AB - When a spending function is used in sequential data monitoring of a clinical trial, it is important to know the information fraction at the times of interim analysis. In a maximum duration designed study, the information fraction is unknown when data are monitored, and it has to be estimated. The modified Wilcoxon statistic developed by Peto and Peto and modified by Prentice is often used to compare two survival curves in a clinical trial. We give guidelines for estimating the information fraction in a maximum duration trial when this statistic is employed. When there is a relatively low event rate or the survival time is approximately exponential, the information fraction for the Peto-Peto Prentice Wilcoxon statistic is very close to that of the popular logrank statistic. In other cases, it would be helpful to estimate the information fraction as a function of elapsed calendar time. We discuss both group sequential and continuous monitoring. PMID- 7548699 TI - Stopping a clinical trial very early based on unplanned interim analyses: a group sequential approach. AB - In the conduct of a clinical trial, unexpectedly high rates of toxicity may cause a researcher to want to terminate the trial early even though no formal stopping rule had been specified. The experience of one such clinical trial is used as an example of the ways in which group sequential methodology can be applied in deciding to stop the study, as well as in reporting the results of the clinical trial. This approach is then compared to a Bayesian analysis. PMID- 7548702 TI - Statistical analysis of passive surveillance disease registry data. AB - Passive surveillance disease data involve a registry of individuals who are at risk of disease and who are not under active follow up. The most serious limitations with such data are incomplete ascertainment of cases of disease and little or no follow-up information on patient vital status. This paper considers whether it is possible to estimate disease risk from such data and, if not, what additional information is required. In general, relative risks based on passive surveillance data will be biased even under the assumption that the probability of disease reporting and the hazard of death from other causes are the same for all individuals in the registry. However if the disease is rare, this bias is negligible. Methods are developed for estimating absolute disease incidence rates by combining passive surveillance data with a cohort study. Analytic approaches are proposed for the situations when death rates from all other causes are known and also unknown, and it is found that there is little loss in efficiency even if death rates are not known. There are considerable gains in efficiency for estimating absolute disease incidence rates by supplementing a cohort study with passive surveillance registry data compared to using the cohort study alone, especially if the exposure is rare and the cohort study is small relative to the size of the registry. Intuitively, the cohort data provides information about absolute rates of disease, while the passive surveillance data provides information about relative risks. The methods are applied to a registry of patients with an artificial heart valve that is at risk of breaking. PMID- 7548704 TI - On the reliability and precision of within- and between- population estimates of relative rate parameters. AB - Comparisons of individual- and aggregate-level analyses of data from a multigroup observational study are made using an exponential form relative rate model. Stratified, analytic random effects, and aggregate random effects analyses are studied. Estimating equations are developed to give a consistent estimation procedure across analyses and corresponding information matrices are compared. Simulation studies provide insight into the efficiency and bias of relative rate parameter estimates with respect to covariate dispersion, confounding, and covariate measurement error. PMID- 7548703 TI - Bayesian analysis of proportional hazards models built from monotone functions. AB - We consider the usual proportional hazards model in the case where the baseline hazard, the covariate link, and the covariate coefficients are all unknown. Both the baseline hazard and the covariate link are monotone functions and thus are characterized using a dense class of such functions which arises, upon transformation, as a mixture of Beta distribution functions. We take a Bayesian approach for fitting such a model. Since interest focuses more upon the likelihood, we consider vague prior specifications including Jeffreys's prior. Computations are carried out using sampling-based methods. Model criticism is also discussed. Finally, a data set studying survival of a sample of lung cancer patients is analyzed. PMID- 7548705 TI - A multiplicative random effects model for meta-analysis with application to estimation of admixture component. AB - A multiplicative random effects model is constructed for combining results from different studies. An EM algorithm is developed for the maximum likelihood estimator under the Empirical Bayes framework. The method is applied to estimation of the admixture component in admixed populations. The admixture estimates can be useful in genetic epidemiology and DNA fingerprinting. PMID- 7548706 TI - Hazard function estimation using B-splines. AB - A flexible parametric procedure is given to model the hazard function as a linear combination of cubic B-splines and to obtain maximum likelihood estimates from censored survival data. The approach yields smooth estimates of the hazard and survivorship functions that are intermediate in structure between strongly parametric and non-parametric models. A simple method is described for selecting the number and location of knots. Simulation results show favorable root mean square error compared to non-parametric estimates for both the hazard and survivorship functions. Three methods are given to calculate confidence intervals based on the delta method, profile likelihood, and bootstrap, respectively. The procedure is applied to estimate hazard rates for acquired immunodeficiency syndrome (AIDS) following infection with human immunodeficiency virus (HIV). Spline methods can accommodate complex censoring mechanisms such as those that arise in the AIDS setting. To illustrate, HIV infection incidence is estimated for a cohort of hemophiliacs in which the dates of HIV infection are interval censored and some subjects were born after the onset of the HIV epidemic. PMID- 7548708 TI - Discriminant analysis algorithm based on a distance function and on a Bayesian decision. AB - We propose a new algorithm for the allocation of an individual to one of several possible groups or populations. The algorithm enables us to define a finite partition over the sample space, based on distance function. This partition is used, jointly with the application of a standard Bayesian decision rule, to allocate individuals to the populations. The algorithm also provides a measure of the allocation confidence for each individual, in a similar manner to that of logistic regression. The error rates for classification are also computed using the leave-one-out method. Results are compared with those obtained with other discriminant analysis techniques previously reported: Fisher's linear discriminant function, the quadratic discriminant function, logistic discrimination, and others. PMID- 7548709 TI - Interim analyses in 2 x 2 crossover trials. AB - A method is presented for performing interim analyses in long term 2 x 2 crossover trials with serial patient entry. The analyses are based on a linear statistic that combines data from individuals observed for one treatment period with data from individuals observed for both periods. The coefficients in this linear combination can be chosen quite arbitrarily, but we focus on variance based weights to maximize power for tests regarding direct treatment effects. The type I error rate of this procedure is controlled by utilizing the joint distribution of the linear statistics over analysis stages. Methods for performing power and sample size calculations are indicated. A two-stage sequential design involving simultaneous patient entry and a single between period interim analysis is considered in detail. The power and average number of measurements required for this design are compared to those of the usual crossover trial. The results indicate that, while there is minimal loss in power relative to the usual crossover design in the absence of differential carry-over effects, the proposed design can have substantially greater power when differential carry-over effects are present. The two-stage crossover design can also lead to more economical studies in terms of the expected number of measurements required, due to the potential for early stopping. Attention is directed toward normally distributed responses. PMID- 7548707 TI - Semi-parametric estimation in failure time mixture models. AB - A mixture model is an attractive approach for analyzing failure time data in which there are thought to be two groups of subjects, those who could eventually develop the endpoint and those who could not develop the endpoint. The proposed model is a semi-parametric generalization of the mixture model of Farewell (1982). A logistic regression model is proposed for the incidence part of the model, and a Kaplan-Meier type approach is used to estimate the latency part of the model. The estimator arises naturally out of the EM algorithm approach for fitting failure time mixture models as described by Larson and Dinse (1985). The procedure is applied to some experimental data from radiation biology and is evaluated in a Monte Carlo simulation study. The simulation study suggests the semi-parametric procedure is almost as efficient as the correct fully parametric procedure for estimating the regression coefficient in the incidence, but less efficient for estimating the latency distribution. PMID- 7548711 TI - A random-effects model for analysis of infectious disease final-state data. AB - Ball (1986, Advances in Applied Probability 18, 289-310) presented an extension to the "General Epidemic Model" in which an individual's (random) infectious period could have any distribution whose Laplace transform could be specified. This paper describes the fitting of Ball's model to data on the final state of infection within households, and gives an intuitive mathematical derivation of the corresponding likelihood function. We extend the model in several ways, including an extension to allow for random-effects heterogeneity in disease transmission rate between individuals. We give an algorithm for the efficient numerical computation of maximum likelihood estimators of the transmission rates, and describe the assessment of goodness of model fit. The methodology is illustrated with recent survey data on outbreaks of Shigella sonnei in 102 households in Manchester, UK. The results are consistent with previous anecdotal evidence of the infectiousness and susceptibility of individuals within households as a function of age and sex. PMID- 7548713 TI - Dose-response models with covariates. AB - Two approaches are presented to incorporate covariate effects into dose-response models. The first approach postulates a parametric form for the dose-response function with the resulting parameters then assumed to depend on the covariates. The second approach is semi-parametric: it assumes that the logit ratio (the difference between the logit of success rate at the covariate level and that of the baseline level) can be expressed as a regression model in the covariate effects and the dose level. We present results of a simulation study which compares the efficiency of the two approaches. PMID- 7548710 TI - A group sequential procedure for all-pairwise comparisons of k treatments based on the range statistic. AB - In this paper, a group sequential procedure for all-private comparisons of the means of k independent normal populations with a common known variance is proposed. A repeated range test is defined and its critical points are tabulated. The power function is studied and minimum group size needed to achieve a desirable power level is provided. The test can also be converted into a set of repeated simultaneous confidence intervals from which all-pairwise comparisons can be made. An example of the application of this new interference method is given. PMID- 7548712 TI - A two-stage procedure for survival studies with surrogate endpoints. AB - A two-stage procedure for survival studies with surrogate endpoints is proposed. The objective of the procedure is to reduce the duration of a survival study relative to classical procedure. A surrogate endpoint is an event which is related to survival time and may occur earlier during follow up. In the first stage, all patients are followed to the primary endpoint in order to evaluate the strength of the relationship between the surrogate endpoint and survival. In the second stage, follow up is terminated on patients who reach the surrogate endpoint. Indirect inferences on the survival endpoint is now possible by virtue of the first stage analysis. We present methods for data collected in the two stage procedure, for estimating the survivorship function, S(t), and for comparing two treatment groups using a non-parametric permutation test. The methods are applied to the results of a study of resected lung cancer. PMID- 7548714 TI - Design and analysis of group sequential logrank tests in maximum duration versus information trials. AB - When monitoring a clinical trial with failure time data using the logrank test and the type I error spending function approach, the information time has to be estimated as a fraction of the maximum number of failures. In maximum duration trials, the denominator of this fraction is a random quantity and has to be estimated; besides, there are two candidates for the denominator, one under the null hypothesis of no treatment difference and the other under the specified alternative hypothesis. Either way, some adjustments are necessary in determining group sequential boundaries in order to maintain type I error at a desired significance level. As a consequence, the type I error spending function will be altered from the one chosen for the design, thus affecting the operating characteristics of the subsequent group sequential logrank tests. In maximum information trials, however, the maximum amount of information is fixed, and thus the estimate of the information time is always unbiased. The net effect is that computation of group sequential boundaries becomes straightforward, with a potential saving in study durations as compared to maximum duration trials. We will illustrate how adjustments are made in maximum duration trials to maintain type I error when the information times are estimated with the information horizons under the null and alternative hypotheses and present numerical explorations to compare robustness of two different estimates of the information times. We then propose a design procedure for maximum information trials and investigate the properties of maximum information trials for different group sequential boundaries. We also compare maximum information trials and maximum duration trials based on an example. PMID- 7548715 TI - Breast cancer in Swedish women before age 50: evidence of a dual effect of completed pregnancy. AB - We set out to detect a transient increase in risk of breast cancer following childbirth, the existence of which has been postulated, but for which empirical evidence is contradictory. Breast cancers and births occurring among the cohort of Swedish women born after 1939 were linked, yielding 3,439 cases and 25,140 age matched controls with at least two children. Within three years of their last childbirth, women had an estimated rate of breast cancer of 1.21 (95 percent confidence interval [CI] = 1.02-1.44) times that of women whose last birth was 10 or more years earlier, after adjustment for parity and age at first birth. Further analyses suggested that this effect reflected, in part, a small transient increase in breast cancer risk that lasts for about three years following completed pregnancy. The effect of age at first birth on breast cancer risk appears to be confounded by time since last birth; the parity-adjusted rate ratio for having a first birth at age 35 years or more compared with under 20 years is reduced from 1.72 (CI = 1.14-2.58) to 1.36 (CI = 0.88-2.09) on additional adjustment for time since last birth. A transient increase in breast cancer risk after childbirth appears thus appears to account for part of the effect of age at first birth on breast cancer risk. PMID- 7548717 TI - Registration of lung cancer in Scotland: an assessment of data accuracy based on review of medical records. AB - Lung cancer represents a major public health problem in Scotland. Cancer registration data permit the approximate incidence of this disease to be measured directly and the projected incidence to be modelled. Thus, in addition to epidemiologic studies and survival analyses, cancer registration data may be used for planning and monitoring relevant health services. Since the value of the data depends on their quality, we undertook a large-scale study of the accuracy of cancer registration data in Scotland. The medical records of a random sample of cancer registrations attributed to the year 1990 were sought. The sample contained 340 registrations of lung cancer, 309 (91 percent) of which had relevant medical records available for scrutiny. Registration details were reabstracted from available records and compared with data in the registry. Results revealed 19 discrepancies in identifying items of data (surname, forename, gender, and date of birth) involving 16 (5.2 percent) patients. Most were trivial and would not disturb record linkage. Discrepancy rates were found to be: 7.8 percent in postcode of residence at the time of diagnosis, 10 percent in 'anniversary date' (excluding differences of six weeks or less), 12.5 percent in histologic verification status; 4.2 percent in ICD-9 site code (the first three digits), and 15.5 percent in four digit ICD-O morphology code (excluding 'inferred' morphology codes). This relatively high level of accuracy gives weight to routinely published incidence figures and supports the use of these data for exploratory epidemiologic studies, assessment of health care needs, and calculation of survival. PMID- 7548716 TI - The University of Minnesota Cancer Prevention Research Unit vegetable and fruit classification scheme (United States). AB - High vegetable and fruit (V&F) intake has been associated with a lower risk of many cancers. However, the specific V&F, the active compounds present in V&F, and the dose at which they confer protection are unknown. Standard methods for assessing, classifying, and quantifying V&F exposures in epidemiologic studies have not been established. Differences among studies occur due to inherent differences among V&F, and across dietary assessment methods, study populations, etiologic hypotheses, and analytic methods. The V&F classification scheme presented here characterizes and quantifies V&F consumption for elucidating risk relationships, identifying chemopreventive compounds present in V&F, and facilitating identification of potential biomarkers of V&F intake. Broad criteria define which plant foods count as V&F. Formation of food groups is based on proposed biological mechanisms of action. Five main groups are included: Total V&F; Total Vegetables; Total Fruits; and two groups orthogonal to these -- the Botanical and Phytochemical groups. Subgroups are specified within each main group. V&F exposure is quantified as the absolute amount consumed (weight) or as the number of household servings. This classification scheme has public health applications and may be used to examine associations with chronic diseases other than cancer. PMID- 7548718 TI - Cancer mortality by educational level in Italy. AB - Social differences in health concern both ethics and science. From a public health point-of-view, one must assess actual differences and then try to find explanations. This was made possible for the first time for cancer in Italy via nationwide record-linkage between the 1981 census and the national death index. Over the subsequent six months after census, the study-base included 31,000 deaths for cancer and 18 million person-years at risk. Rate ratio (RR) were estimated through a Poisson regression model adjusted by age and geographic area of residence. Educational level was used as social level indicator. Profound social differences were evident for buccal cavity (RR = 3.10 for lowest cf highest educational level), esophagus (RR = 3.00), stomach (RR = 3.43), and larynx (RR = 3.30) among men, and for stomach (RR = 2.25) and uterus (RR = 1.76) among women. Colon (RR = 0.62) and pancreas cancers (RR = 0.65) presented an inverse relationship among men, as did colon (RR = 0.37), breast (RR = 0.56), ovary (RR = 0.45), and melanoma (RR = 0.62) among women. In conclusion, the Italian population at the beginning of the 1980s had large social differences in the risk of dying from cancer, confirming the patterns commonly found in such other countries as Great Britain, France, and New Zealand. Some dissimilarities, useful for hypothesis generation on the mechanisms of inequality, were evident, such as the generally highest social differences found among northern Italian men and among southern Italian women. PMID- 7548719 TI - Oral contraceptive use and breast cancer risk among African-American women. AB - Recent epidemiologic studies, most of them in predominantly White populations, have suggested that long duration of oral contraceptive (OC) use may increase the risk of breast cancer at young ages. We assessed the relationship of OC use to the risk of breast cancer in African-American women aged 25 to 59 years, using interview data from a multipurpose hospital-based case-control study. Five hundred and twenty-four cases hospitalized for invasive breast cancer were compared with 1,021 controls with nonmalignant conditions unrelated to OC use. Relative risks (RR) and 95 percent confidence intervals (CI) were estimated relative to a reference category of use for less than 12 months; potential confounders were controlled by multiple logistic regression analysis. Among women under age 45, three or more years of OC use was associated with an increased risk of breast cancer: the RR estimate was 2.8 (CI = 1.5-5.0) for three to four years of use, and declined to 1.5 (CI = 08.3.0) for 10 or more years of use. Recency and timing of use did not explain the observed association. Among women aged 45 to 59, OC use was associated with little or no increase in risk: the RR estimate for three or more years of use was 1.3 (CI = 0.7-2.4). The findings add to the evidence from studies of White women and a recent study of Black women which have suggested an increased risk of breast cancer at young ages for moderate or long duration use of OCs. PMID- 7548722 TI - Strenuous physical activity in young adulthood and risk of breast cancer (United States). AB - The epidemiologic data on the relation between strenuous physical activity and breast cancer are limited and inconsistent. Because risk of breast cancer may be influenced by ovarian function which, in turn is modulated by physical activity, the hypothesis that exercise may be associated with a reduced risk of breast cancer merits further investigation. We, therefore, conducted a large case control study in 1988-1991, and interviewed 6,888 women (17 to 74 years of age) with breast cancer in Maine, Massachusetts, New Hampshire, and Wisconsin (United States). Interviewed controls (9,539 women, 18 to 74 years of age) were selected randomly from lists of licensed drivers (for younger women) or from a roster of Medicare enrollees (for older women). We used multivariate adjusted odds ratios (OR) and 95 percent confidence intervals (CI) from logistic regression models to estimate relative risks between self-reported physical activity when 14 to 22 years of age and breast cancer. When compared with sedentary controls, women who reported any strenuous physical during ages 14 to 22 years had a modest reduction in the risk of breast cancer (OR = 0.95, CI = 0.93-0.97). However, those who exercised vigorously at least once a day had a 50 percent reduction in risk of breast cancer (OR = 0.5, CI = 0.4-0.7). These data support the hypothesis that women who are physically active have a reduced risk of breast cancer. PMID- 7548721 TI - Estimation and projections of stomach cancer trends in Italy. AB - Mortality data from official sources, and survival data from population-based cancer registries, are used for the estimation of incidence and prevalence of stomach cancer. Time trends of morbidity, survival, and mortality during the period 1970-90 are presented and analyzed. Incidence rates were decreasing during the considered period, but the rate of decrease was slowing down during the last decade. Almost stable rates, and even slightly increasing for women, were estimated for the youngest cohorts. Relative survival for stomach cancer was higher for women and for young ages; it was associated positively with period of diagnosis, and presented a significant South-North geographic gradient. Prevalence was estimated as decreasing during the period 1970-80, but increasing during the successive decade, due to both better survival and population aging. Projection of stomach cancer morbidity and mortality to the year 2000 showed that the disease should still be considered in Italy as a major public health problem. PMID- 7548720 TI - Reproductive factors and colon cancer: the influences of age, tumor site, and family history on risk (Utah, United States). AB - The Utah (United States) Population Database was used to evaluate the associations between reproductive factors and colon cancer risk and the impact that family history, age at diagnosis, and tumor site have on these associations. From the cohort of (White) women in the database, all first-primary cases of colon cancer (n = 819) and controls who had complete fertility information (n = 3,202) were examined. The majority of tumors (68.6 percent) among women diagnosed at age 64 years or less were in the distal segment of the colon, while among women 65 or older, the majority of tumors (55.7 percent) were proximal. Women diagnosed before age 65 had a lower risk of colon cancer with increasing numbers of liveborn children (odds ratio [OR] = 0.6, 95 percent confidence interval [CI] = 0.3-0.9 for women with five or more children compared with women with one or two children). A long interval between first and second births (first birth interval) was associated with increased risk of tumors in the distal segment of the colon (OR = 1.4, CI = 1.0-2.0) and among women diagnosed before age 65 (OR = 1.6, CI = 1.0-2.5); a longer, average birth-interval was associated with increased risk of proximal tumors (OR = 1.5, CI = 1.1-2.1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548723 TI - Formaldehyde and cancer morbidity among male employees in Denmark. AB - Formaldehyde, a genotoxic and potent animal carcinogen, is widespread in the working environment as well as in private homes. The risk for cancer morbidity in Denmark during 1970-84 was estimated from standardized proportionate incidence ratios (SPIR) among men whose longest employment had been held since 1964, at least 10 years before diagnosis, in 265 companies in which exposure to formaldehyde was identified. The results do not support the hypothesis that formaldehyde is associated with lung cancer (SPIR = 1.0, 410 cases). Significantly elevated risks were found for cancers of the colon (SPIR = 1.2, 166 cases), kidney (SPIR = 1.3, 60 cases), and sino-nasal cavities (SPIR = 2.3, 13 cases). For sino-nasal cancer, a relative risk of 3.0 (95 percent confidence interval = 1.4-5.7) was found among blue-collar workers with no probable exposure to wood dust, the major confounder. This study provides further evidence that occupational exposure to formaldehyde increases the risk for sino-nasal cancer. PMID- 7548724 TI - High bladder cancer mortality in rural New England (United States): an etiologic study. AB - An interview study of next-of-kin of 325 persons who died of bladder cancer and 673 individuals who died of other causes in Vermont and New Hampshire (United States) was conducted to assess reasons for the persistent pattern of elevated bladder cancer mortality for both genders in rural New England. There are some evidence of elevated risks for both leather and textile workers that rose to over twofold for workers who also lived near these industries and for persons with French-Canadian ancestry. Occupational exposures in the textile and leather industry may explain at least a portion of the excess bladder cancer risk in rural New England. PMID- 7548726 TI - Report on the monitoring study of Chemical Research in Toxicology. PMID- 7548725 TI - Cigarette smoking and alcohol consumption and the risk of pancreatic cancer: a case-control study in Shanghai, China. AB - Cancer of the pancreas has been rising in incidence in Shanghai, China since the early 1970s. In 1987-89, this malignancy ranked eighth in cancer incidence among men and ninth among women in Shanghai. To examine risk factors for this tumor in urban Shanghai, a population-based case-control study was conducted. Cases (n = 451) were permanent residents of Shanghai, 30 to 74 years of age, newly diagnosed with pancreatic cancer between 1 October 1990 and 30 June 1993. Deceased cases (19 percent) were excluded from the study. Controls (n = 1,552) were selected among Shanghai residents, frequency-matched to cases by gender and age. Cases and controls were interviewed about their demographic background and potential risk factors, including tobacco, alcohol and beverage consumption, diet, and medical history. Adjusted odds ratios (OR) and 95 percent confidence intervals (CI) were estimated using logistic regression models. Current cigarette smoking was associated with excess risk of pancreatic cancer in both men (OR = 1.6, CI = 1.1 2.2) and women (OR = 1.4, CI = 0.9-2.4). ORs increased significantly with number of cigarettes smoked per day, and with duration and pack-years of smoking. Risk increased three- to sixfold among those in the highest categories of cigarette consumption, while risk decreased with increasing years since smoking cessation. Former smokers who stopped smoking for 10 or more years had risks comparable to nonsmokers. PMID- 7548727 TI - Quantitative structure-activity relationships based on computer calculated parameters for the overall rate of glutathione S-transferase catalyzed conjugation of a series of fluoronitrobenzenes. AB - The present study describes quantitative structure--activity relationships (QSAR's) for the overall rate of conjugation of a series of fluoronitrobenzenes catalyzed by cytosolic glutathione S-transferases based on experimental data and outcomes of computer calculations. The natural logarithm of the rate of conjugation of the series of fluoronitrobenzenes correlates (r = 0.986) with the calculated energy (E) of their lowest unoccupied molecular orbital (LUMO) and also (r = -0.987) with the relative heat of formation (delta delta HF) for formation of the Meisenheimer complex of the fluoronitrobenzenes with a MeS- model nucleophile. In addition, the paper describes QSAR's for the chemical reaction of glutathione with the fluorinated nitrobenzenes both at pH 7.6 and at pH 9.9. These QSAR's are parallel to the one obtained for the enzyme catalyzed conversions. This indicates that in the overall reaction (both chemical and enzyme catalyzed) the interaction between the thiolate anion of glutathione and the fluoronitrobenzene leading to the Meisenheimer reaction intermediate is the rate-limiting step in overall conversion of these substrates. The parallel QSAR's of the chemical and enzymatic reaction also indicate that in the enzymatic reaction chemical reactivity parameters determine the overall outcome of catalysis and, in addition, that the chemical and enzymatic reactions proceed through a similar reaction pathway with comparable reaction intermediates. Additional results of the present study demonstrate that the regioselectivity of the glutathione conjugation cannot be explained on the basis of calculated characteristics of the LUMO of the fluoronitrobenzenes or the delta delta HF for the formation of their Meisenheimer reaction complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548729 TI - Quantum mechanical studies of the structure and reactivities of the diol epoxides of benzo[c]phenanthrene. AB - Benzo[c]phenanthrene has a crowded bay region that has been called a fjord region. As a result of the interaction between the atoms across the fjord region, it is a nonplanar molecule with a significant barrier between two helical structures. The crowding in the fjord region also affects the three-dimensional structure of the fjord region diol epoxide. Quantum mechanical studies have been performed to determine the structure and reactivities of the fjord region diol epoxides. Eight local minimum energy three-dimensional structures have been found for the trans diol of 1,2,3,4-tetrahydro-3,4-dihydroxybenzo[c]phenanthrene 1,2 epoxide. They can be characterized by three dichotomies: one between syn and anti, one between quasidiaxial and quasidiequatorial, and the third that depends on nonplanarity of the parent polycyclic aromatic hydrocarbon due to interactions in the crowded bay region, that we have named "in" and "out" based on the position of the epoxide oxygen relative to the distal ring. The structures with the epoxide oxygen on the same side of the saturated ring as the distal ring (in ) are more stable than the structures where the epoxide is on the opposite side (out-). The calculated lowest energy syn and anti structures for the diol epoxide of benzo[c]-phenanthrene are both in-quasidiequatorial, in agreement with experiment. Analysis of the results indicates that the electrostatic interaction across the fjord region could be responsible for the increased stability of the syn-in-quasidiequatorial structure compared to the syn-in-quasidiaxial structure and the stability of the in- structures in general when compared to the out- structures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548728 TI - Benzenediazonium ion derived from Sudan I forms an 8-(phenylazo)guanine adduct in DNA. AB - 1-(Phenylazo)-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14) is a liver and urinary bladder carcinogen in mammals. Sudan I forms benzenediazonium ion during cytochrome P-450 catalyzed metabolism. Calf thymus DNA was reacted with Sudan I activated by microsomal enzymes or with benzenediazonium ion in vitro, and the adducts formed were analyzed by the 32P-postlabeling technique. Both enrichment procedures (1-butanol extraction and nuclease P1 digestion) of this technique were employed for detection and quantitation of the DNA adducts formed. Cochromatographic analyses of adduct spots obtained by reaction with DNA or homopolydeoxyribonucleotides showed that the major Sudan I-DNA adduct was formed with deoxyguanosine. This adduct was also found in DNA directly reacted with benzenediazonium ion. The major Sudan I-DNA adduct was characterized by UV/vis absorbance spectroscopy as well as by the chromatographic properties of the adduct on cellulose or poly(ethylenimine)--cellulose TLC and HPLC. The characteristics are identical to those of the adduct synthesized from benzenediazonium ion and guanine, identified by mass, UV/vis, and 1H-NMR spectroscopy as 8-(phenylazo)guanine. The results suggest strongly that benzenediazonium ion derived from Sudan I reacts with DNA in vitro to form the stable 8-(phenylazo)guanine adduct. PMID- 7548731 TI - Acrolein mercapturates: synthesis, characterization, and assessment of their role in the bladder toxicity of cyclophosphamide. AB - Acrolein is the metabolite of cyclophosphamide (CP) believed to be involved in the bladder toxicity associated with this anticancer drug. The mechanism by which this extremely reactive intermediate is delivered to the bladder is not known. Glutathione (GSH) readily conjugates with acrolein, and the acrolein mercapturate S-(3-hydroxypropyl)-N-acetylcysteine (3-hydroxy-PrMCA) has been found in the urine of animals and man given CP. The objectives of this study were to prepare and characterize synthetic standards of the GSH acrolein adduct (3 oxopropyl)glutathione (3-oxoPrGSH), the acrolein mercapturates S-(3-oxopropyl)-N acetylcysteine (3-oxoPrMCA) and 3-hydroxyPrMCA, and the S-oxidation product of 3 oxoPrMCA (3-oxoPrMCA S-oxide). In addition, the release of acrolein from, and the bladder toxicity of, these conjugates was determined. 3-OxoPrGSH and 3-oxoPrMCA were prepared with a 99% yield by condensing acrolein with GSH and N acetylcysteine, respectively. 3-HydroxyPrMCA was prepared with a 63% yield by refluxing 3-chloropropanol and N-acetylcysteine in a basic medium. Oxidation of 3 oxoPrMCA with H2O2 was used to prepare 3-oxoPrMCA S-oxide. By decreasing the reaction time to 1 h, and adjusting the ratio of 3-oxoPrMCA to H2O2, the yield of 3-oxoPrMCA S-oxide was increased to 96%. The anhydrous aldehyde, 3-oxoPrMCA, afforded characteristic aldehydic proton resonances (1H NMR) in deuterated dimethyl sulfoxide. New resonances were observed in deuterated water, indicating a 75% hydration of the aldehyde to the corresponding geminal diol. This phenomenon was enhanced with 3-oxoPrMCA S-oxide where approximately 100% hydration of the aldehyde to the corresponding geminal diol was observed. When incubated at 25 degrees C in 100 mM potassium phosphate buffer containing 1 M KCl, pH 8.0, 3-oxoPrMCA released approximately 6% and 3-oxoPrMCA S-oxide released approximately 16-18% of the theoretical maximum yield of acrolein after 30 min, as indicated by an increase in absorbance at 210 nm and confirmed by trapping this aldehyde as a semicarbazone. There was less than a 2% yield of acrolein from 3-hydroxyPrMCA or 3-oxoPrGSH under similar conditions. At pH 7.4 the release of acrolein from 3-oxoPrMCA and 3-oxoPrMCA S-oxide was decreased by 50%. An assay where aldehydes are reacted with m-aminophenol in acid media produced fluorescence consistent with 72%, 46%, 23%, and 1% yields of acrolein from 3 oxoPrMCA S-oxide, 3-oxoPrMCA, 3-oxoPrGSH, and 3-hydroxyPrMCA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7548730 TI - Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen. AB - Chlorophyllin (CHL), a food-grade derivative of the green plant pigment chlorophyll, has recently been shown in this laboratory to be a potent inhibitor in vivo of hepatic aflatoxin B1 (AFB1)-DNA adduction and hepatocarcinogenesis (Breinholt et al. (1995) Cancer Res. 55, 57-62). We report here that CHL forms a strong noncovalent complex with AFB1 in vitro (dissociation constant (Kd) by Scatchard analysis = 1.4 (+/- 0.4) microM based on copper chlorin content), which may contribute to its anticarcinogenic activity. Kd values for the related porphyrins chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ratio analysis provided evidence that all porphyrins examined associate with AFB1 at an approximate one to one stoichiometric ratio. Energy minimization computer modeling of the complex indicates a favorable association energy of -20 kcal/mol, independent of oxidation state of the 8,9-double bond of AFB1. AFB1 incubated in vitro with liver microsomes in the presence of added CHL showed comparable levels of inhibition in the production of several phase 1 metabolites, including the postulated procarcinogenic metabolite AFB1 8,9-epoxide. Kinetic analysis of microsome-catalyzed AFB1-DNA adduction suggested a CHL inhibition constant near 10 microM chlorin. In vivo, addition of CHL to concentrated AFB1 solutions followed by gavage administration resulted in dose-dependent inhibition of hepatic AFB1-DNA adduction, whereas the same dosages of AFB1 and CHL incorporated into a single bolus of trout diet for gavage provided less protection at all CHL doses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548732 TI - Stereoisomeric selectivity of 2,3-dimercaptosuccinic acids in chelation therapy for lead poisoning. AB - The formation constants of lead chelates of the stereoisomers of 2,3 dimercaptosuccinic acid (DMSA) were determined from potentiometric titrations in the presence of the competing ligand, EDTA. The lead chelates formed at pH 7.4 with the stereoisomers of DMSA are the monomeric complexes PbL and HPbL. Formation of PbL and HPbL at pH 7.4 is independent of total concentrations of lead and DMSA present, and so is the concentration ratio of PbL:HPbL. Lead is completely chelated at pH 7.4 when the total concentration of ligand is equal to or greater than the total concentration of lead present. Lead tends to bind to a greater extent with rac- than with meso-DMSA, and the relative extent increases with an increase in the concentration ratio of ligand to lead and finally reaches a constant value of 45. The binding sites in the chelates, PbL, of the stereoisomers of DMSA are the two thiolate groups and one carboxylate group. rac DMSA also forms a dimeric complex Pb2L2 in which both carboxylate groups of the ligands participate in binding with lead ions. The formation constants of the lead chelates of rac-DMSA were invariably found to be larger than those of the corresponding of meso-DMSA chelates, because in all the lead chelates of the stereoisomers of DMSA formed in solution, rac-DMSA existed in staggered anti conformations, whereas meso-DMSA preferred a staggered gauche conformation with respect to carboxylate groups in the ligands. The potential of using ZnL2 of rac DMSA as a therapeutical lead chelator was assessed by considering its lead mobilizing ability and its ability to deplete endogenous zinc; on this basis it is predicted that ZnL2 of rac-DMSA is a better chelator than meso-DMSA for the treatment of lead poisoning. PMID- 7548733 TI - The influence of the p-alkyl substituent on the isomerization of o-quinones to p quinone methides: potential bioactivation mechanism for catechols. AB - Previously, we have shown that an additional bioactivation pathway for the hepatocarcinogen safrole (1-allyl-3,4-(methylenedioxy)benzene) exists which may contribute to its toxic effects: initial O-dealkylation of the methylenedioxy ring, forming the catechol, hydroxychavicol (HC, 1-allyl-3,4-dihydroxybenzene), 2 electron oxidation to the o-quinone (4-allyl-3,5-cyclohexadien-1,2-dione), and isomerization, forming the more electrophilic p-quinone methide (2-hydroxy-4 allylidene-2,5-cyclohexadien-1-one) [Bolton, J. L., Acay, N. M., & Vukomanovic, V. (1994) Chem. Res. Toxicol. 7, 443-450]. In the present investigation, we explored the effects of changing pi-conjugation at the 4-position on both the rate of isomerization of the initially formed o-quinones to the QMs and the reactivity of the quinoids formed from 4-propylcatechol (1), 2,3-dihydroxy 5,6,7,8-tetrahydronaphthalene (2), and 4-cinnamylcatechol (3). We selectively oxidized the catechols to the corresponding o-quinones or p-quinone methides and trapped these reactive electrophiles with glutathione (GSH). The GSH adducts were fully characterized by UV, NMR, and mass spectrometry. Microsomal incubations with the parent catechols in the presence of glutathione produced only o-quinone glutathione conjugates. However, if the trapping agent (GSH) was added after an initial incubation time, both o-quinone and p-quinone methide GSH conjugates were observed. The results indicate that extended pi-conjugation at the para position enhances the rate of isomerization of the o-quinone to the quinone methide. Thus the half-life of the o-quinones decreased in the following order: the o-quinone of 1 > 2 > HC > 3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548734 TI - Linear free energy relationships for reactions of electrophilic halo- and pseudohalobenzenes, and their application in prediction of skin sensitization potential for SNAr electrophiles. AB - Published kinetic data and linear free energy relationships for nucleophilic aromatic substitution reactions (SNAr reactions) are analyzed so as to derive a reactivity parameter (RP), defined as sigma sigma-(o,m,p) + 0.45 sigma*(ipso), quantifying the relative reactivities of SNAr electrophiles toward aniline in ethanol. It is shown that the dataset of Landsteiner and Jacobs, in which 20 SNAr electrophiles were classified on the basis of experimental evidence as either skin sensitizing and reactive to aniline or nonsensitizers and unreactive to aniline, can be equivalently classified on the basis of RP values and that predictive criteria can be defined as follows: sensitizing/aniline reactive: RP > 3.80; nonsensitizing/unreactive to aniline: RP < 3.65. These predictive criteria based on calculated RP values are applied to seven further SNAr electrophiles to predict which are sensitizers and which are not. There is close agreement between the predictions and the biological data. This validates the applicability of the reactivity parameter RP, derived from linear free energy relationships, to prediction of skin sensitization potential for SNAr electrophiles. More generally, the applicability of physical organic chemistry principles to structure-activity relationships in contact allergy is further demonstrated. PMID- 7548735 TI - Direct characterization of protein adducts of the lipid peroxidation product 4 hydroxy-2-nonenal using electrospray mass spectrometry. AB - Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2 nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins. PMID- 7548736 TI - Identification of N-acetylcysteine conjugates of 1,2-dibromo-3-chloropropane: evidence for cytochrome P450 and glutathione mediated bioactivation pathways. AB - The haloalkane 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen, mutagen, nephrotoxin, and testicular toxin. The identification of N-acetylcysteine conjugates of DBCP provides information on GSH mediated and cytochrome P450 mediated bioactivation pathways in the expression of DBCP-induced toxicities. N Acetylcysteine conjugates excreted in the urine of male Sprague-Dawley rats administered DBCP, C1D2-DBCP, C2D1-DBCP, C3D2-DBCP, or D5-DBCP (80 mg/kg) were purified by reverse-phase HPLC as their methyl ester derivatives and characterized by fast atom bombardment tandem mass spectrometry. These metabolites were also converted to tert-butyldimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) to facilitate the identification of N-acetyl-S-(2,3-dihydroxypropyl)cysteine (Ia), an apparent regioisomer of Ia, 2-(S-(N-acetylcysteinyl))-1,3-propanediol (Ib), N-acetyl-S-(3 hydroxypropyl)cysteine (IIa), and N-acetyl-S-(3-chloro-2-hydroxypropyl)-cysteine (III). Metabolites Ia, Ib, and III displayed quantitative retention of deuterium, an observation consistent with the formation of episulfonium ion intermediate(s) in their biogenesis. Mercapturate IIa retained three atoms of deuterium from D5 DBCP, and two atoms of deuterium from the dideuterio analogs (C1D2-DBCP and C3D2 DBCP), thus invoking P450 mediated formation of 2-bromoacrolein (2-BA) as an intermediate in the biogenesis of IIa. A mechanism is proposed in which conjugate addition of GSH to 2-BA, subsequent episulfonium ion formation, and addition of GSH afford 1,2-(diglutathion-S-yl)propanal. Glutathione mediated reduction is invoked to afford S-(3-hydroxypropyl)GSH which would be excreted in the urine as IIa. The quantitative retention of deuterium from C1D2-DBCP or C3D2-DBCP was indicative of isotopically sensitive branching of P450 metabolism at either C1 or C3 to afford 2-BA. C2D1-DBCP showed a 30% retention of 1 deuterium atom in IIa; the loss of the deuterium is consistent with 2-BA formation, whereas the retention of one deuterium atom is indicative of the formation of metabolite IIa through GSH conjugation of either 2,3-dibromopropanal or 2-bromo-3 chloropropanal. These data indicate that IIa is a marker metabolite for the potent direct-acting mutagen, 2-BA, or its metabolic precursors 2,3 dibromopropanal or 2-bromo-3-chloropropanal. Therefore, evidence has been presented for bioactivation of DBCP by glutathione and cytochrome P450 mediated mechanisms. PMID- 7548737 TI - Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies. AB - Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis. PMID- 7548738 TI - Stereoselective catalysis of a retro-Michael reaction by class mu glutathione transferases. Consequences for the internal distribution of products in the active site. AB - The reaction of glutathione (GSH) with trans-4-phenyl-3-buten-2-one (PBO) is readily reversible in aqueous solution with an apparent (pH-dependent) equilibrium constant at pH 8 of 6.4 x 10(2) M-1. Two class mu isoenzymes of GSH transferase from rat (M1-1 and M2-2) and two site specific mutants (M1-1/V9I and M2-2/I9V) catalyze the addition of GSH to PBO and the elimination of GSH from the two diastereomeric products (isomers A and B) of 4-(S-glutathionyl)-4-phenyl-2 butanone with varying degrees of efficiency and stereoselectivity, with the major kinetic product in the addition reaction (isomer A) being the preferred substrate for the elimination reaction. The kinetic stereoselectivity of the addition reaction and the steady-state kinetics of the elimination reactions with product isomers A and B are used to estimate internal stereochemical equilibrium constants in which product isomer B is predominant. This result is consistent with the internal equilibrium constants measured under conditions of enzyme in excess. The results can be used to construct reaction coordinate diagrams for the interconversion of central complexes in the enzyme-catalyzed reactions. The possible metabolic consequences of the reversibility of additions of GSH to alpha, beta-unsaturated carbonyl compounds are discussed. PMID- 7548739 TI - Specific disulfide formation in the oxidation of HIV-1 zinc finger protein nucleocapsid p7. AB - In vitro oxidation of the HIV-1 nucleocapsid protein p7 by the C-nitroso compound 3-nitrosobenzamide (NOBA) has been investigated. When reconstituted p7 was incubated with NOBA, three disulfide bonds were formed per molecule of p7, Cys 15 Cys 18, Cys 28-Cys 36, and Cys 39-Cys 49. These were identified using the proteolytic enzyme endoproteinase Lys-C and mass spectrometry. When the denatured protein (Apo-p7) was incubated with NOBA, a more random pattern of multiple S-S linkages was found. Oxidation of reconstituted p7 also occurred on treatment with cupric ions (Cu2+), and the same three major disulfide bonds were formed as in the reaction with NOBA. These results suggest the interpretation that the oxidation reaction occurs at the zinc-binding centers while zinc cations are still bound and that the two zinc fingers are not identical in their chemical properties. This latter point is consistent with the independent biological roles reported previously for the two fingers in the viral infection cycle. PMID- 7548740 TI - In vitro reaction with DNA of the fjord-region diol epoxides of benzo[g]chrysene and benzo[c]phenanthrene as studied by 32P-postlabeling. AB - The chemical reactivities of the optically-pure fjord-region syn- and anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxides (B[g]CDEs) toward DNA in vitro have been compared with those of the optically-pure fjord-region syn- and anti benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (B[c]PhDEs), using the standard 32P-postlabeling assay. The (+)-anti-, (+)-syn-, and (-)-syn-isomers of the two sets of diol epoxides showed similar extents of reaction with DNA, but the (-) anti-B[c]PhDE was 2.5 times more reactive toward DNA than the corresponding B[g]CDE isomer and was the most reactive of the eight diol epoxides studied. When the reactions of the B[g]CDEs with DNA were analyzed by the nuclease P1-enhanced method of 32P-postlabeling, the observed adduct levels were between 3 and 10 times lower than were obtained using the standard method of 32P-postlabeling. By analyzing by TLC and HPLC the 32P-postlabeled products of the reactions of the diol epoxides with synthetic polynucleotides, the relative reactions of the B[g]CDEs and B[c]PhDEs with guanine and adenine bases in DNA were determined. All four B[g]CDE isomers reacted with adenine residues in similar proportions to those seen for the B[c]PhDE isomers. Thus, the presence of an additional benzene ring on the benzo[c]phenanthrene structure, distant from the fjord region, does not radically alter the reactivity or base selectivity of the fjord-region diol epoxides, except in the case of the (-)-anti-isomer of benzo[g]chrysene. The reasons for the lower reactivity of this isomer compared with that of the corresponding isomer of benzo[c]phenanthrene are unclear.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548741 TI - Physicochemical mechanism of the interaction between cobalt metal and carbide particles to generate toxic activated oxygen species. AB - Hard metal alloys (or cemented carbides) are made of a mixture of tungsten carbide particles (WC, more than 80%) cemented in cobalt metal powder (Co, 5 10%). The inhalation of hard metal particles may cause an interstitial pulmonary disease, the mechanism of which involves an interaction between Co and WC particles. Some epidemiological data also suggest that hard metal dust can induce lung cancer in workers. In a macrophage culture model, butylated hydroxytoluene (1 mM) protected from the cytotoxicity of hard metal particles, suggesting a possible involvement of lipid peroxidation in the toxicity of these powders. In a biochemical system, a mixture of Co and WC particles, but not Co or WC alone, stimulated the production of thiobarbituric acid-reactive substances from arachidonic acid. Using a spin trapping system applied to aqueous particulate suspensions and electrochemical techniques, we present experimental evidence that the association of Co and carbide particles represents a specific toxic entity producing large amounts of activated oxygen species. The mechanism of this interaction proceeds through the oxidation of cobalt metal catalyzed at the surface of carbide particles and resulting in the reduction of dissolved oxygen. This physicochemical property of hard metal particles provides a new basis for interpreting their inflammatory action and their possible carcinogenic effect on the lung. PMID- 7548743 TI - Formation of N2-tetrahydrofuranyl and N2-tetrahydropyranyl adducts in the reactions of alpha-acetoxy-N-nitrosopyrrolidine and alpha-acetoxy-N nitrosopiperidine with DNA. AB - We studied the reactions with DNA of alpha-acetoxy-N-nitrosopyrrolidine (alpha acetoxyNPYR) and alpha-acetoxy-N-nitrosopiperidine (alpha-acetoxyNPIP) in order to obtain more information on adduct formation by metabolic activation via alpha hydroxylation of two cyclic nitrosamines, N-nitrosopyrrolidine (NPYR) and N nitrosopiperidine (NPIP). Enzyme hydrolysis and HPLC analysis of DNA that had been reacted with unlabeled, [14C]-, or [3H]alpha- acetoxyNPYR permitted the positive identification of N2-(tetrahydrofuran-2-yl)deoxyguanosine (THF-dG). It was identified by comparison of its UV spectrum and retention time to those of a standard, by conversion upon NaBH4 treatment to N2-(4 hydroxybutyl)deoxyguanosine, and by neutral thermal hydrolysis to 2 hydroxytetrahydrofuran (THF-OH). The levels of THF-dG in DNA exceeded that of other adducts of alpha-acetoxyNPYR. Reaction of alpha-acetoxyNPIP with DNA followed by enzyme hydrolysis and HPLC analysis resulted in the positive identification of two diastereomers of N2-(3,4,5,6-tetrahydro-2H-pyran-2 yl)deoxyguanosine (THP-dG) by comparison of their retention times and UV spectra to those of standards. The levels of THP-dG were similar to those of THF-dG formed from alpha-acetoxyNPYR. Neutral thermal hydrolysis of DNA that had been reacted with alpha-acetoxyNPIP produced 2-hydroxy-3,4,5,6-tetrahydro-2H-pyran (THP-OH). Studies on the mechanism of formation of THF-dG and THP-dG indicated that stable cyclic oxonium ion-derived electrophiles could be their major precursors. Our data provide the first evidence for the formation of cyclic oxonium ion-derived DNA adducts from cyclic nitrosamines and indicate some potential differences in DNA binding between alpha-acetoxyNPYR and alpha acetoxyNPIP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548742 TI - Reactions of alpha-acetoxy-N-nitrosopyrrolidine and alpha-acetoxy-N nitrosopiperidine with deoxyguanosine: formation of N2-tetrahydrofuranyl and N2 tetrahydropyranyl adducts. AB - The goal of this study was to compare the reactions of alpha-acetoxy-N nitrosopyrrolidine (alpha-acetoxyNPYR) and alpha-acetoxy-N-nitrosopiperidine (alpha-acetoxyNPIP) with deoxyguanosine (dG). alpha-AcetoxyNPYR and alpha acetoxyNPIP are stable precursors to the alpha-hydroxynitrosamines which are formed metabolically from NPYR and NPIP. These alpha-hydroxynitrosamines are believed to be the proximate carcinogens of NPYR and NPIP. NPYR and NPIP, although structurally similar, have remarkably different carcinogenic properties, and a comparison of the reactions of their metabolically activated forms with dG and ultimately DNA could provide insights on their mechanisms of carcinogenicity. Reactions of alpha-acetoxyNPYR and alpha-acetoxyNPIP with dG were carried out at 37 degrees C and pH 7.0. The products were analyzed by HPLC and characterized by their spectral properties and by comparison to standards. In each reaction, one of the major products was a new type of dG adduct: N2-(tetrahydrofuran-2- yl)dG (THF-dG) from alpha-acetoxyNPYR and N2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)dG (THP dG) from alpha-acetoxyNPIP. THF-dG was synthesized independently by reaction of either 2-chlorotetrahydrofuran or 2,3-dihydrofuran with dG. Similarly, THP-dG was prepared by reaction of 2-chloro-3,4,5,6-tetrahydro-2H-pyran with dG. The structures of THF-dG and THP-dG were established by their UV and 1H-NMR spectra. THF-dG was less stable than THP-dG, but could be readily converted to a stable derivative, N2-(4-hydroxybutyl)dG, by reaction with NaBH4. THF-dG and THP-dG were converted to dG and 2-hydroxytetrahydrofuran or 2-hydroxy-3,4,5,6-tetrahydro-2H pyran, respectively, upon neutral thermal or acid hydrolysis. This reaction was found to be reversible, with the adducts being produced in substantial amounts by reaction of 2-hydroxytetrahydrofuran or 2-hydroxy-3,4,5,6-tetrahydro-2H-pyran with dG. The latter reaction accounts for part of the THF-dG and THP-dG produced from the alpha-acetoxynitrosamines; stable oxonium ion-derived electrophiles may also be involved in the formation of THF-dG and THP-dG. Comparisons of the yields of various adducts in the reaction of alpha-acetoxyNPYR and alpha-acetoxyNPIP with dG showed some major differences. Whereas yields of THF-dG and THP-dG were similar, adducts formed from open chain diazonium ion or related intermediates were formed more extensively from alpha-acetoxyNPYR than from alpha-acetoxyNPIP. Adducts formed from enal products of the two nitrosamines were also different. Adduct formation as characterized in this study may account for some of the contrasting carcinogenic properties of NPYR and NPIP. PMID- 7548744 TI - Metabolism, bioaccumulation, and incorporation of diethanolamine into phospholipids. AB - Diethanolamine (DEA) is a major industrial chemical which has low acute toxicity, but, on repeat exposure, has significant cumulative toxicity. The present work suggests that the cumulative toxicity can be attributed to the fact that, unlike most small polar molecules, DEA accumulates to high concentrations in certain tissues following repeat exposure. The highest concentrations of DEA were seen in liver, kidney, spleen, and brain. Investigations described here have determined that DEA is metabolized by biosynthetic routes common to ethanolamine and is conserved, O-phosphorylated, N-methylated, and incorporated into phosphoglyceride and sphingomyelin analogues as the parent compound and as its N-methyl and N,N dimethyl derivatives. This is the first report of the conjugation of a xenobiotic headgroup with a natural ceramide to form aberrant sphingomyelins. DEA-derived phosphoglycerides constituted the majority of aberrant phospholipid following acute administration. On repeat administration, DEA bioaccumulated to plateau levels at approximately 8 weeks. This bioaccumulation was accompanied by an increasing degree of methylation and accumulation of aberrant sphingomylenoid lipids in tissues. Uptake and incorporation of DEA into ceramide derivatives in human liver slices were also demonstrated in the present studies. It is speculated that the cumulative toxicity observed on repeat administration of DEA to rats is caused in part by increasing levels of aberrant phospholipids derived from this unnatural alkanolamine. PMID- 7548745 TI - Metabolism of 5-(glutathion-S-yl)-alpha-methyldopamine following intracerebroventricular administration to male Sprague-Dawley rats. AB - 5-(Glutathion-S-yl)-alpha-methyldopamine [5-(GSyl)-alpha-MeDA] is a putative metabolite of the serotonergic neurotoxicants 3,4-(+/-) (methylenedioxy)amphetamine and 3,4-(+/-)-(methylenedioxy)methamphetamine. Glutathione (GSH) conjugates of several polyphenols are biologically (re)active. Therefore, as part of our studies on the role of 5-(GSyl)-alpha-MeDA in MDA mediated neurotoxicity, we determined the regional brain metabolism of 5-(GSyl) alpha-MeDA (720 nmol) following intracerebroventricular administration to male Sprague-Dawley rats. 5-(GSyl)-alpha-MeDA was rapidly cleared from all brain regions examined, and regional differences in the distribution of gamma-glutamyl transpeptidase (gamma-GT) correlated with the formation of 5-(cystein-S-yl)-alpha methyldopamine (5-[CYS]-alpha-MeDA). We also observed the formation of 5-(N acetyl-L-cystein-S-yl)-alpha-MeDA (5-[NAC]-alpha-MeDA) in all brain regions, indicating that the brain has the ability to synthesize mercapturic acids. Peak concentrations of 5-(NAC)-alpha-MeDA were found in the order: hypothalamus > midbrain/diencephalon/telencephalon > pons/medulla > hippocampus > cortex > striatum. In contrast to 5-(GSyl)-alpha-MeDA and 5-(CYS)-alpha-MeDA, 5-(NAC) alpha-MeDA was eliminated relatively slowly from the brain. Differences were also found in cystein conjugate N-acetyltransferase activity in microsomes prepared from the various brain regions, but little difference was observed in brain cytosolic N-acetyl-L-cysteine conjugate N-deacetylase activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548746 TI - Chloramphenicol oxamylethanolamine as an end product of chloramphenicol metabolism in rat and humans: evidence for the formation of a phospholipid adduct. AB - Chloramphenicol (CP) has been implicated as, although not proven to be, a causative agent of aplastic anemia in humans. Recent studies from our laboratory have presented evidence that CP-oxamylethanolamine was an end product of CP biotransformation in birds. Because this novel metabolic pathway has never been reported in other species, we have now expanded these investigations to rat and humans. [3H]CP was administered po (10 mg/kg) to adult male Wistar rats and to a human volunteer. Urine was collected and analyzed by HPLC and GC-MS for CP metabolite determination. In rat, the two most important metabolites in 0-24 h urine were CP-base and CP-acetylarylamine which together accounted for about 50% of the ingested radioactivity. The remainder was due to unchanged CP, CP-oxamic acid, CP-alcohol, CP-glucuronide, and CP-oxamylethanolamine. The presence of these end products was also demonstrated in man. CP-oxamylethanolamine represented 0.74% and 1.37% of the ingested radioactivity in rat and human urine samples, respectively. CP-oxamylethanolamine formation was confirmed in vitro with isolated rat hepatocytes, suggesting the involvement of liver in the production of this metabolite. The origin of CP-oxamylethanolamine has been investigated with the use of hepatic liver microsomes from phenobarbital-treated rats. The incubation of [3H]CP with this subcellular fraction led to the binding of a radiolabeled compound to the microsomal lipids, whereas no binding occurred when CP-oxamic acid was incubated with the microsomes. Enzymatic hydrolysis of the microsome lipid fraction with phospholipase D from Streptomyces chromofuscus released CP-oxamylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548747 TI - A predictive substrate model for rat glutathione S-transferase 4-4. AB - Molecular modeling techniques have been used to derive a substrate model for class mu rat glutathione S-transferase 4-4 (GST 4-4). Information on regio- and stereoselective product formation of 20 substrates covering three chemically and structurally different classes was used to construct a substrate model containing three interaction sites responsible for Lewis acid--Lewis base interactions (IS1, IS2, and IS3), as well as a region responsible for aromatic interactions (IS4). Experimental data suggest that the first protein interaction site (pIS1, interacting with IS1) corresponds with Tyr115, while the other protein interaction sites (pIS2 and pIS3) probably correspond with other Lewis acidic amino acids. All substrates exhibited positive molecular electrostatic potentials (MEPs) near the site of conjugation with glutathione (GSH), as well as negative MEP values near the position of groups with Lewis base properties (IS1, IS2, or IS3), which interact with pIS1, pIS2, or pIS3, respectively. Obviously, complementarity between the MEPs of substrates and protein in specific regions is important. The substrate specificity and stereoselectivity of GST 4-4 are most likely determined by pIS1 and the distance between the site of GSH attack and Lewis base atoms in the substrates which interact with either pIS2, pIS3, or a combination of these sites. Interaction between aromatic regions in the substrate with aromatic amino acids in the protein further stabilizes the substrate in the active site. The predictive value of the model has been evaluated by rationalizing the conjugation to GSH of 11 substrates of GST 4-4 (representing 3 classes of compounds) which were not used to construct the model. All known metabolites of these substrates are explained with the model. As the computer aided predictions appear to correlate well with experimental results, the presented substrate model may be useful to identify new potential GST 4-4 substrates. PMID- 7548748 TI - Heterogeneous DNA adduct formation in vitro by the acetylated food mutagen 2 (acetoxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine: a fluorescence spectroscopic study. AB - The food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) forms adducts to DNA guanine bases at the C-8 position. No other DNA adduction site has been verified for PhIP, nor has any experimental data been collected on the conformation of the PhIP-DNA covalent complex. To determine if multiple PhIP-DNA adduct species exist, or if PhIP-DNA adducts assume multiple conformations, 2 (acetoxyamino)-1-methyl-6-phenylimidazo[4,5-b]-pyridine (N-acetoxy-PhIP) was reacted with calf thymus DNA, followed by an evaluation of the resulting adduct complexes by fluorescence spectroscopy. Approximately 20% of the N-acetoxy-PhIP formed covalent complexes with DNA. Two major and several minor spots were observed by 32P-postlabeling, suggesting a minimum of two major adduct species. UV/vis spectra of the PhIP-modified DNA also showed heterogeneous formation of PhIP-DNA adducts. Fluorescence excitation and emission spectroscopy with or without fluorescence quenching (silver ion and acrylamide) was used to evaluate the number of adducts formed, and the low-resolution conformation of each adduct. Four adduct fluorophores were observed and assigned the nomenclature PAi, where "PA" denotes PhIP Adduct and i = 1-4 in order of fluorescence emission band energies, with 1 the highest and 4 the lowest energy, respectively. Excitation maxima for the adduct fluorophores ranged from 340 to 370 nm, and emission maxima ranged from 390 to 420 nm. The fluorescence from adduct PA1 was quenched by silver but not acrylamide, suggesting a helix-internal configuration. Adduct PA2 fluorescence was strongly enhanced upon silver binding but was not affected by acrylamide, also indicating that this adduct was internal. The fluorescence from adducts PA3 and PA4 was quenched by acrylamide but not silver; thus PA2 and PA3 were tentatively assigned as solvent-accessible. These data are the first suggesting heterogeneous formation of PhIP adducts to intact DNA, but we cannot as yet determine how many chemical species of adduct are formed or if a given species exists in multiple conformations. PMID- 7548750 TI - Interactions of nickel(II) with histones. Stability and solution structure of complexes with CH3CO-Cys-Ala-Ile-His-NH2, a putative metal binding sequence of histone H3. AB - Nickel(II) compounds are established human carcinogens, but the molecular mechanisms underlying their activity are only partially known. One mechanism may include mediation by nickel of promutagenic oxidative DNA damage that depends on Ni(II) binding to chromatin. To characterize such binding at the histone moiety of chromatin, we synthesized the peptide CH3CO-Cys-Ala-Ile-His-NH2 (L), a model of the evolutionarily conserved motif in histone H3 with expected affinity for transition metals, and evaluated its reactivity toward Ni(II). Combined spectroscopic (UV/vis, CD, NMR) and potentiometric measurements showed that, at physiological pH, mixtures of Ni(II) and L yielded unusual macrochelate complexes, NiL and NiL2, in which the metal cation was bound through Cys and His side chains in a square-planar arrangement. Above pH 9, a NiH-3L complex was formed, structurally analogous to typical square-planar nickel complexes. These complexes are expected to catalyze oxidation reactions, and therefore, coordination of Ni(II) by the L motif in core histone H3 may be a key event in oxidative DNA base damage observed in the process of Ni(II)-induced carcinogenesis. PMID- 7548749 TI - Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status. AB - It is postulated that the hepatotoxicity of valproic acid (VPA) results from the mitochondrial beta-oxidation of its cytochrome P450 metabolite, 2-propyl-4 pentenoic acid (4-ene VPA), to 2-propyl-(E)-2,4-pentadienoic acid ((E)-2,4-diene VPA) which, in the CoA thioester form, either depletes GSH or produces a putative inhibitor of beta-oxidation enzymes. In order to test this hypothesis, 2-fluoro-2 propyl-4-pentenoic acid (alpha-fluoro-4-ene VPA) which was expected to be inert to beta-oxidative metabolism was synthesized and its effect on rat liver studied in comparison with that of 4-ene VPA. Similarly, the known hepatotoxicant 4 pentenoic acid (4-PA) and 2,2-difluoro-4-pentenoic acid (F2-4-PA) were compared. Male Sprague-Dawley rats (150-180 g, 4 rats per group) were dosed ip with 4-ene VPA (0.7 mmol/kg per day), 4-PA (1.0 mmol/kg per day), or equivalent amounts of their alpha-fluorinated analogues for 5 days. Both 4-ene VPA and 4-PA induced severe hepatic microvesicular steatosis ( > 85% affected hepatocytes), and 4-ene VPA produced mitochondrial alterations. By contrast, alpha-fluoro-4-ene VPA and F2-4-PA were not observed to cause morphological changes in the liver. The major metabolite of 4-ene VPA in the rat urine and serum was the beta-oxidation product (E)-2,4-diene VPA. The N-acetylcysteine (NAC) conjugate of (E)-2,4-diene VPA was also found in the urine. Neither (E)-2,4-diene VPA nor the NAC conjugate could be detected in the rats administered alpha-fluoro-4-ene VPA. In a second set of rats (3 rats per group), total liver GSH levels were determined to be depleted to 56% and 72% of control following doses of 4-ene VPA (1.4 mmol/kg) and equivalent alpha-fluoro-4-ene VPA, respectively. Mitochondrial GSH remained unchanged in the alpha-fluoro-4-ene VPA treated group but was reduced to 68% of control in the rats administered 4-ene VPA. These results strongly support the theory that hepatotoxicity of 4-ene VPA, and possibly VPA itself, is mediated largely through beta-oxidation of 4-ene VPA to reactive intermediates that are capable of depleting mitochondrial GSH. PMID- 7548752 TI - Synthesis of N-(1-nitropyren-6-yl and 8-yl)-2'-deoxyribonucleosides. AB - A new type of 1-nitropyrene--DNA adduct via addition--elimination reaction was synthesized. Treatment of fluorinated 1-nitropyrene with 3'- and 5'-O-protected 2'-deoxyribonucleoside in dimethyl sulfoxide at 140 degrees C afforded N-(1 nitropyren-6-yl or 8-yl)-2'-deoxyribonucleoside. These DNA adducts resulted from addition of the exocyclic amino group of deoxynucleosides to the fluorinated carbon of the fluoro-1-nitropyrene following elimination of fluoride anion. This is the first report that describes the 1-nitropyrene-DNA adducts in which aromatic ring moiety of 1-nitropyrene is covalently linked to the exocyclic amino group of the deoxyribonucleoside. From our findings, we suggest that the addition elimination reaction may be responsible for the formation mechanism of the putative 1-nitropyrene--DNA adducts in vivo. PMID- 7548751 TI - Sulfotransferase-mediated activation of 7,8,9,10-tetrahydro-7-ol, 7,8 dihydrodiol, and 7,8,9,10-tetraol derivatives of benzo[a]pyrene. AB - Some hydroxymethyl-substituted polycyclic aromatic hydrocarbons have been shown to be converted to electrophilic, mutagenic, or tumorigenic sulfuric acid ester metabolites by cytosolic sulfotransferase activity in rodent liver. Likewise, certain types of aromatic compounds with a secondary alcoholic functional group at the benzylic position undergo metabolic activation through sulfonation. Enzymatic oxidation of benzo[a]pyrene produces such secondary alcohols as dihydrodiol and tetraol derivatives as primary metabolites. Sulfo conjugation of the benzylic hydroxy group of each of these metabolites is expected to generate an electrophilic sulfuric acid ester capable of covalently binding to DNA, which may contribute to mutagenesis and carcinogenesis by benzo[a]pyrene. Although the model benzo-ring secondary benzyl alcohol, 7-hydroxy-7,8,9,10 tetrahydrobenzo[a]pyrene, covalently bound to DNA and also exerted mutagenicity in the presence of rodent hepatic cytosols and 3'-phosphoadenosine 5' phosphosulfate, no such sulfotransferase-dependent activation was observed with dihydrodiol or tetraol derivatives of benzo[a]pyrene. Thus, it seems likely that appearance of the adjacent non-benzylic hydroxy functional group(s) in latter metabolites hinders the benzylic sulfonation in these molecules. PMID- 7548753 TI - Probing the mechanism of bioactivation of MPTP type analogs by monoamine oxidase B: structure-activity studies on substituted 4-phenoxy-, 4-phenyl-, and 4 thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridines. AB - Previous studies have shown that 4-benzyl-1-cyclopropyl-1,2,3,6 tetrahydropyridine is an excellent monoamine oxidase B (MAO-B) substrate (kappa cat/KM = 1538 min-1 mM-1) although the corresponding 4-phenyl analog displays MAO B inactivating properties only. This behavior led us to speculate that the pathway for the MAO-B catalyzed oxidation of these tetrahydropyridines may not necessarily proceed via an initial single electron transfer step as proposed by others but rather through an initial alpha-carbon hydrogen atom abstraction step. In the present studies we have examined the interactions of various 4-phenoxy-, 4 phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives, some of which bear substituents on the phenyl ring. The 4-thiophenoxy- and all of the 4-phenoxytetrahydropyridine derivatives proved to be substrates but not inactivators of MAO-B, while several of the 4-phenyltetrahydropyridine derivatives were inactivators but not substrates. A case of particular interest was 1-cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine, which displayed only substrate properties. The results are discussed in terms of two catalytic pathways, one of which involves partitioning of the proposed cyclopropylaminyl radical cation intermediate between cyclopropyl ring opening and proton loss while the second involves partitioning of the parent amine between an initial single electron transfer step, leading to cyclopropylaminyl radical cation formation and enzyme inactivation, and an initial alpha-carbon hydrogen atom abstraction step, leading to an allylic radical and dihydropyridinium product formation. PMID- 7548754 TI - The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver. AB - To determine whether benzidine is acetylated in dog, like rat, the metabolism of benzidine was assessed with dog and rat liver slices. Slices were incubated with 0.05 mM [3M]benzidine for 4 h. Media and cellular DNA were analyzed for acetylated benzidine metabolites and adducts. In rat, benzidine was rapidly converted to acetylated metabolites. At 1 h, benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine represented 5%, 23%, and 54%, respectively, of the total radioactivity in media. Within 2 h, 75% of the radioactivity was N,N' diacetylbenzidine. In dog, 45% of the radioactivity was present in metabolites more polar than benzidine by 4 h. No N-acetylated metabolites were observed in dog liver slice media. To identify acetylated benzidine DNA adducts, N (deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was prepared and identified by FAB MS. This nucleoside adduct was used to synthesize N-(deoxyguanosin-8-yl)-N acetylbenzidine and N'-(deoxyguanosin-8-yl)-N-acetylbenzidine. Nucleoside adducts from slices incubated with [3H]benzidine were analyzed by HPLC. With this method of analysis, the 3H-material did not correlate with the synthetic adduct standards. To improve sensitivity and identify liver adducts, a 32P-postlabeling method was developed. 2'-Deoxyguanosine 3'-monophosphate adduct standards of acetylated benzidine were prepared. 32P-Postlabeling analysis demonstrated that rat liver contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine after a 1- or 4-h exposure to benzidine. In contrast, no acetylated adducts were detected in dog. Results indicate that dog is a nonacetylator with respect to benzidine. The availability of acetylated benzidine nucleotide standards allowed unambiguous identification of N'-(3'-monophosphodeoxyguanosin-8-yl)-N acetylbenzidine as the adduct present in rat liver slices. These nucleotide adduct standards will be useful in subsequent studies in animals and human. PMID- 7548756 TI - Bromobenzene 3,4-oxide alkylates histidine and lysine side chains of rat liver proteins in vivo. AB - The hepatotoxic effects of bromobenzene (BB) are correlated with and generally ascribed to the covalent modification of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. Previous studies revealed that quinone as well as epoxide metabolites of BB form adducts to protein sulfur nucleophiles, that the quinone-derived adducts are more abundant by a factor of ca. 7, and that collectively these sulfur adducts account for only about 10% of the total protein covalent binding [Slaughter, D. E., and Hanzlik, R. P. (1991) Chem. Res. Toxicol. 4, 349-359]. To examine the possibility that metabolically-formed BB-3,4-oxide alkylates nitrogen nucleophiles on proteins under toxicologically relevant conditions in vivo, we synthesized standards of N tau-(p-bromophenyl)histidine (7) and N epsilon-(p-bromophenyl)lysine (8) as anticipated adduct structures and used them to guide a chromatographic search for their presence in hydrolysates of liver protein from BB-treated rats. While radio-LC chromatography and GC/MS provide unequivocal evidence for their presence, the amounts of 7 and 8 observed are very low ( < 1% of total covalent binding). The apparently small net contribution of epoxide metabolites to covalent binding of BB in vivo suggests the majority of binding may arise via quinone metabolites, but this should not be construed to imply that quinone adducts are necessarily more important toxicologically than epoxide adducts; in this context the identity of the protein targets is probably at least as important as the type of electrophilic metabolite involved. PMID- 7548755 TI - Characterization of a chemically reactive propranolol metabolite that binds to microsomal proteins of rat liver. AB - We have characterized a chemically reactive propranolol (PL) metabolite which binds to proteins in rat liver microsomes. During incubation with rat liver microsomes (1 mg of protein) fortified with an NADPH-generating system, 4 hydroxypropranolol (4-OH-PL) quickly disappeared from the reaction medium, but none of the possible metabolite peaks was detected under the high-performance liquid chromatographic conditions used. The consumption of 4-OH-PL depended on microsomes and NADPH. The reaction was not affected by inhibitors of cytochrome P450 or FAD monooxygenase, but was markedly diminished in the presence of cytosol and ascorbic acid. The effect of cytosol was inhibited by potassium cyanide but not by sodium benzoate or dimethyl sulfoxide, and was also not affected by heating at 60 degrees C for 30 min, suggesting that superoxide (SO) ion was involved in the reaction and that it was blocked by superoxide dismutase (SOD) present in the cytosol. Cu,Zn-SOD, purified from cytosol, effectively mimicked the suppressive effect of cytosol. Incubation of 4-OH-PL in an SO-generating system of xanthine and xanthine oxidase generated 1,4-naphthoquinone (1,4-NQ), which was identified by TLC, HPLC, and GC/MS. 1,4-NQ was also formed in microsomal incubates containing NADPH and small amounts of microsomes (below 0.1 mg of protein). These results indicate that 4-OH-PL is converted by SO, or some reactive oxygen species derived from it, to 1,4-NQ which binds to proteins and is one of the reactive metabolites of PL. PMID- 7548757 TI - The lipoic acid containing components of the 2-oxoacid dehydrogenase complexes mimic trifluoroacetylated proteins and are autoantigens associated with halothane hepatitis. AB - Anti-CF3CO antibodies, monospecific toward trifluoroacetylated proteins (CF3CO proteins), which are elicited in experimental animals and humans exposed to the anesthetic agent halothane, cross-react with an unknown protein of approximately 52 kDa, constitutively expressed in tissues of experimental animals and humans not previously exposed to the agent. Using anti-CF3CO antibody, the protein(s) of 52 kDa could be immunoprecipitated from solubilized rat heart homogenate. Two dimensional gel electrophoretic analysis revealed the presence of distinct major (P1, P2) and minor (P3, P4, P5) protein components with apparent molecular masses of 52 kDa. From each of the components P1 and P2, the amino acid sequences of three peptides were determined and found to exhibit 100% identity with the corresponding amino acid sequences of the E2 subunit of the rat 2-oxoglutarate dehydrogenase complex (OGDC). Additionally to the E2 subunit of OGDC, anti-CF3CO antibody also recognized on immunoblots the purified E2 subunit of the branched chain 2-oxoacid dehydrogenase complex (BCOADC) and protein X, a constituent of the pyruvate dehydrogenase complex (PDC), in a manner sensitive to competition by N6-(trifluoroacetyl)-L-lysine (CF3CO-Lys), 6(RS)-lipoic acid, and N6-(6(RS) lipoyl)-L-lysine (lipoyl-Lys). Furthermore, a discrete population of autoantibodies was identified in sera of patients with halothane hepatitis which could not discriminate between the lipoylated target epitope present on the E2 subunit of OGDC and epitopes on CF3CO-RSA, used as model for CF3CO-proteins. These data suggest that the autoantigenicity of these proteins in halothane hepatitis is based on the molecular mimicry of CF3CO-Lys by lipoic acid, the prosthetic group common to protein X and the E2 subunits of OGDC and BCOADC. PMID- 7548758 TI - Quantitative immunohistochemical analysis of 4-aminobiphenyl-DNA in cultured cells and mice: comparison to gas chromatography/mass spectroscopy analysis. AB - Two monoclonal antisera, 4C11 and 3C8, recognizing 4-aminobiphenyl (4-ABP)--DNA adducts were developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Both antisera are highly specific for 4-ABP-DNA and, at the highest concentration tested, do not recognize the DNA adducts of several other aromatic amines tested including 1-aminopyrene, 8-nitro-1-aminopyrene, and 6-nitro-1-aminopyrene. An immunohistochemical method for detecting adducts was developed in R52 cells, a mouse NIH3T3 cell line expressing high levels of cytochrome P450 1A2. Quantitation of fluorescence labeling indicated a dose related increase in staining in cells treated with 0, 6, 30, 60, and 300 microM 4 ABP. To apply the method to tissue samples, Balb/c mice were treated with 0, 4, 10, 20, 40, and 80 mg/kg 4-ABP and liver, bladder, and lung tissue analyzed by immunohistochemical staining of tissue sections. There was a dose-related increase in specific nuclear staining in liver and bladder tissues with no detectable staining in lung tissue. DNA from liver tissue was also analyzed by alkaline hydrolysis of 4-ABP, derivatization with pentafluoropropionic anhydride, and gas chromatography/mass spectroscopy analysis. A good correlation (r = 0.98, p < 0.0001) was found between DNA damage levels determined by the two methods. Based on adduct levels determined by GC/MS in both R52 cells and liver tissue, the immunohistochemical method has a limit of sensitivity of approximately 1 adduct/10(7-8) nucleotides. Immunohistochemistry should be useful for analysis of 4-ABP-DNA adducts in human tissue biopsies as well as exfoliated cells from the oral mucosa and urinary bladder. PMID- 7548759 TI - Simulation of lipid peroxidation in low-density lipoprotein by a basic "skeleton" of reactions. AB - A minimal kinetic model describing lipid peroxidation in low-density lipoprotein (LDL) has been set up. Models have been calculated by numeric integration of the differential equations describing this system consisting of seven reactions and eleven reactants in a single compartment. The model describes the usually observed behavior of the reaction system, showing that the crucial intermediate is the lipid peroxyl radical (LOO.). During different stages of the reaction, depending on the presence of antioxidants (alpha-tocopherol), different pathways in the reaction scheme become active. Simulation also demonstrates that tocopherol-mediated propagation can occur under certain conditions, i.e., a low rate of initiation. This, however, does not mean that tocopherol enhances lipid peroxidation in LDL, as without tocopherol the process would be much faster. Further extension of the basic model by inclusion of a hypothetical antioxidant leads to a model which is capable of describing Cu(2+)-induced LPO over the whole lag phase up to full propagation. PMID- 7548761 TI - Glucuronidation of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, a metabolically activated form of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, by phenobarbital-treated rats. AB - In the rat, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung tumors independent of the route of administration. To exert its carcinogenic potential, NNK must be metabolically activated. Like most nitrosamines NNK is activated by alpha-hydroxylation. The striking tissue specificity of tumor induction by nitrosamines has been primarily attributed to the efficient alpha hydroxylation of a particular nitrosamine by its target tissue. Two other factors which may contribute to this are the following: the relative capacity of different tissues to detoxify the alpha-hydroxynitrosamine and the preferential uptake of the active metabolite by the target tissue. In the present study we report the characterization of the O-glucuronide of 4 ((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone (alpha-hydroxymethylNNK Gluc). The formation of this glucuronide could either serve as a detoxification pathway or provide a stable transport form of the alpha-hydroxylated metabolite. In addition, the metabolism of NNK to a glucuronide of the alpha hydroxynitrosamine provides the first definitive evidence for the formation of alpha-hydroxymethylNNK. alpha-HydroxymethylNNK-Gluc was present in the urine of rats treated with phenobarbital (PB) and NNK. It was also formed by hepatocytes from PB-treated rats, accounting for 4% of the total metabolites in the media following incubation with 1 microM NNK. The data that support the identity of this metabolite as alpha-hydroxymethylNNK-Gluc are as follows. (1) Incubation of this metabolite with beta-glucuronidase resulted in the quantitative release of 4 hydroxy-1-(3-pyridyl)-1-butanone (HPB), the decomposition product of alpha hydroxymethylNNK.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548760 TI - Inhibition of 2,5-hexanedione-induced protein cross-linking by biological thiols: chemical mechanisms and toxicological implications. AB - n-Hexane is metabolized to the gamma-diketone 2,5-hexanedione (2,5-HD), a derivative that covalently binds to lysine residues in neurofilament (NF) protein to yield 2,5-dimethylpyrrole adducts. Studies comparing the pyrrole-forming potential and neurotoxic potency of gamma-diketones have demonstrated that pyrrolylation is an absolute requirement in the neuropathogenesis. Autoxidative cross-linking of pyrrolylated NF proteins occurs and is proposed as a second required event. In the present study, the role of nucleophilic thiols and amines in the pyrrole-mediated cross-linking reaction was investigated. When pyrrolylated ribonuclease was incubated with N-acetyllysine, N-acetylcysteine, or glutathione in physiologic buffer (pH 7.4) under air, pyrrole-to-pyrrole cross linking was inhibited only by the thiol-containing compounds. Stable thiol- pyrrole conjugates containing a bridge from the pyrrole ring at C-3 to the sulfur atom of the thiol were characterized by thermospray LC/MS and 1H-NMR spectroscopy. In contrast to low-molecular-mass thiols, SDS--PAGE studies indicated that, under the same incubation conditions, free thiols present in proteins did not undergo reaction with pyrrole adducts to form cross-links. Further experiments using a low-molecular-mass pyrrole derivative indicated that glutathione may also able to suppress pyrrole dimerization without conjugate formation, possibly via inhibition of a free radical-dependent mechanism. The results suggest the following: (1) 2,5-HD-induced protein cross-linking is mediated primarily by pyrrole-to-pyrrole bridging under physiologic conditions, and (2) glutathione and other low-molecular-mass thiols may inhibit the pyrrole dimerization reaction by two distinct pathways. These findings have significant implications for the mechanism of gamma-diketone neuropathy. PMID- 7548762 TI - Investigations of glutathione conjugation in vitro by 1H NMR spectroscopy. Uncatalyzed and glutathione transferase-catalyzed reactions. AB - Conjugation reactions of glutathione (GSH) and related thiols with diethyl maleate (DEM) and other alpha, beta-unsaturated carbonyl compounds have been investigated by 1H NMR spectroscopy. The products from the reaction with DEM and diethyl fumarate (DEF) are shown to be the diastereomers of S-(alpha,beta diethoxycarbonylethyl)glutathione. During the course of the reaction, DEM isomerized to DEF, and the rate of isomerization was dependent upon whether the solvent was 1H2O or 2H2O. The observed rate data exhibit apparent second order kinetic behavior. The reaction of maleate with GSH was considerably slower, and solvent-dependent isomerization was observed, while little reaction of fumarate with GSH was observed at pH 6.5. Reaction of DEM with N-acetyl-L-cysteine followed a similar course to that of GSH, and although L-cysteine reacted rapidly with DEM, it did not promote the isomerization of DEM. Reactions involving penicillamine and N-acetylpenicillamine were considerably slower. Conjugation reactions catalyzed by commercial GSH transferases and selected rat and human purified isoenzymes were also investigated. Of those isoenzymes studied, rat GSH transferase 4-4 was found to exert the greatest degree of stereo control in conjugation reactions with DEF. PMID- 7548763 TI - Type I benzophenone-mediated nucleophilic reaction of 5'-amino-2',5' dideoxyguanosine. A model system for the investigation of photosensitized formation of DNA-protein cross-links. AB - 5'-Amino-2',5'-dideoxyguanosine has been synthesized in order to investigate the intramolecular reactivity of an amino group toward the guanine radical produced by type I photosensitization mechanism. Benzophenone-mediated photosensitization of 5'-amino-2',5'-dideoxyguanosine in aerated aqueous solution results in the formation of a predominant cyclic nucleoside together with an unstable nucleoside precursor. The two modified nucleosides have been isolated by reverse phase high performance liquid chromatography and characterized by spectroscopic measurements including 13C and 1H NMR, fast atom bombardment mass spectroscopy, and UV absorption. The stable photoproduct has been identified as 9-oxa-2,4 diazabicyclo[4.2.1]non-2-en-7-ol, 3-amino- (1R-exo), whereas its precursor has been assigned as acetic acid, [(7-hydroxy-9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-3 yl)amino]oxo- (1R-exo). A reaction mechanism, involving nucleophilic addition of the sugar amino group to guanine radical intermediates, is proposed to explain the formation of the two photoproducts. PMID- 7548764 TI - The antioxidant effect of spermine NONOate in human low-density lipoprotein. AB - The nitric oxide (*NO) donor N-[4-[1-(3-aminopropyl)-2-hydroxy-2 nitrosohydrazino]butyl]-1,3- propanediamine, also referred to as Spermine NONOate (SNN), inhibited the copper(II) sulfate-initiated oxidative modification of human low-density lipoprotein (LDL) as measured by the formation of thiobarbituric acid reactive substances, conjugated diene formation, and changes in electrophoretic mobility. The presence of the nitronyl nitroxide 1-oxy-2-[p (trimethylammoniumyl)phenyl]-4,4,5,5- tetramethylimadazoline 3-oxide, a scavenger of *NO, antagonized the inhibitory activity of SNN. The inhibition of copper dependent LDL oxidation had a nonlinear dependence on SNN concentration. Low concentrations of SNN ( < 4 microM) were only poorly effective at inhibiting LDL oxidation; however, a dramatic enhancement of inhibition occurred above 4 microM SNN. This behavior was qualitatively different from that of butylated hydroxytoluene, a phenolic chain-breaking antioxidant, which exhibited an approximately linear concentration dependence in this system. Addition of 13[S (E,Z)]-hydroperoxy-9,11-octadecadienoic acid, a lipid hydroperoxide, to LDL diminished the antioxidant effect of 4 and 8 microM SNN, but not that of 12 microM SNN. SNN inhibited the depletion of alpha-tocopherol during both copper dependent and 2,2'-azobis(2-amidinopropane)-dependent oxidation of LDL. We propose that a direct reaction is occurring between NO and the lipid peroxyl radical, forming a lipid-nitroso adduct. Formation of this product would not only remove the lipid peroxyl radical, thus preventing chain propagation, but would also prohibit the regeneration of lipid hydroperoxide, thereby stopping further transition metal ion-dependent initiation. The difference in the kinetic behavior between SNN and conventional antioxidants can be explained by this effect. PMID- 7548765 TI - Free radical oxidation of (E)-retinoic acid by prostaglandin H synthase. AB - Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostaglandin H synthase was investigated employing ram seminal vesicle microsomes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5 phenyl-4-penten-1-yl hydroperoxide (PPHP)]- or arachidonic acid-dependent cooxidation by microsomal prostaglandin H (PGH) synthase as evidenced by UV spectroscopic analysis of reaction mixtures. Cooxidation of RA by microsomal or purified PGH synthase, using PPHP as substrate, was characterized by uptake of dioxygen which was first order with respect to enzyme concentration. Dioxygen uptake was inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O2 uptake was inhibited by the spin trap nitrosobenzene. ESR spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, characterized by hyperfine coupling constants of alpha H = 3.2 G and alpha N = 15.8 G. Reverse phase HPLC analysis of reaction mixtures demonstrated the formation of 4-hydroxy-RA, 5,6-epoxy-RA, 4-oxo RA, (13Z)-retinoic acid, and other geometric isomers which were identified on the basis of cochromatography with synthetic standards, UV spectroscopy, and/or mass spectrometry. Mechanisms are proposed for the hydroperoxide-dependent, PGH synthase-catalyzed oxidation of RA that are consistent with these results. PMID- 7548766 TI - Highly insect-resistant transgenic tobacco plants containing both B.t. and CpTI genes. AB - The cowpea trypsin inhibitor (CpTI) gene was synthesized according to its cDNA sequence using DNA synthesizer and confirmed by DNA sequencing. The CpTI gene and modified Bacillus thuringiensis (B.t.) delta-endotoxin gene were cotransformed to tobacco explants mediated by Agrobacterium tumefaciens. The integrations of B.t and CpTI genes were confirmed by PCR and Southern hybridization. Three kinds of transgenic plants were obtained: (1) containing CpTI gene, (2) containing B.t gene, (3) containing both CpTI and B.t genes. Bioassays showed that all these transgenic plants were toxic to the larvae of Heliothis armigera and that the tobacco plants containing both genes had enhanced toxicity to larvae by comparison with plants containing only CpTI or B.t gene. PMID- 7548767 TI - Construction, expression and the characterization of t-PA mutants with increased plasma half-life and resistance to inhibition by PAI-1. AB - Three t-PA mutants, t-PA del K1 (with deletion of K1 domain), t-PA del (296-302) (with deletion of PAI-1 binding site) and their combination mutant t-PA del (K1, 296-302), were constructed by DNA recombination and site-directed mutagenesis techniques. Then the three t-PA mutants were transiently expressed in COS-7 cells, and in addition, the combination mutant t-PA del (K1, 296-302) was stably expressed in CHO cells. The biological analysis of the expression products demonstrated that t-PA del (296-302) and t-PA del (K1, 296-302) had obtained the resistance to inhibition by PAI-1. In addition, the half-life of t-PA del (K1, 296-302) in rat plasma was increased 6 fold while the mutant affinity for fibrin was only slightly affected. Therefore, it was reasonable to consider that the mutant t-PA del (K1, 296-302) may become a potent candidate of new thrombolytic agent. PMID- 7548768 TI - Specific cleavage of two target sequences of the tobacco mosaic virus RNA by a di component ribozyme. AB - Two di-component ribozymes RZ34 and RZ13, the simplest forms of multi-component ribozymes, by ligating mono-ribozymes RZ3 and RZ4 and RZ1 and RZ3 were constructed, respectively. The in vitro results indicate that each di-component ribozyme is able to cleave two different TMV transcripts, with the specificity and efficiency similar to their mono-ribozyme counterparts. Possible effects on the specificity and efficiency by temperature and the positions of the component ribozymes in these di-component ribozymes were also studied. PMID- 7548769 TI - High-level expression of the human tumor necrosis factor-alpha. AB - Five expression plasmids for a total chemically synthesized gene of tumor necrosis factor-alpha (TNF-alpha) were constructed, which differ in two aspects: 1) distance (D) between the SD and the initiation codon ATG; 2) energy-delta G0f298 released to form the stable secondary structure in the translation initiation region. The plasmid with a (D) of 6bp has the lowest delta G0f298 (absolute value) and showed the highest expression level, up to 60% of the total bacterial proteins. Codons usage will impose much influence on expression level, so it is reasonable and understandable that the total chemically synthesized TNF alpha gene with the codons preferable to E. coli use gave much higher expression level than the sc-TNF-alpha in which only partial N-terminal codons were modified. PMID- 7548770 TI - Molecular cloning and expression of human granulocyte-macrophage colony stimulating factor in Escherichia coli system. AB - Human granulocyte-macrophage stimulating factor (hGM-CSF) is an important hematopoietic growth factor. hGM-CSF is able to stimulate hematopoietic progenitors to produce granulocytes, macrophages and eosinophils. hGM-CSF also shows the potential to treat myeloid leukemias. In order to prepare enough hGM CSF, a genetically engineered strain E. coli HB101/pZW.GM47 was constructed by DNA recombination technologies in the present study, and characterized by restriction analysis, DNA sequencing, SDS-PAGE, Western blotting and bioassays. The strain has been shown to produce a 14kDa mature hGM-CSF protein with expression level higher than 40% and a specific activity of 5.0 x 10(7) u/mg which shows a good potential for clinical applications. PMID- 7548771 TI - Effects of cassava starch on the property of artificial endosperm and on the germination of artificial seeds. AB - Effects of 4 polymers on the property of artificial endosperm and on the germination of artificial seeds were studied. Results showed that complexed endosperm made from 1% cassava starch and 1.5% alginate improve the ventilation and imbibition properties of artificial seeds made from alginate only and enhanced the germination rate of artificial seeds. PMID- 7548773 TI - Studies on kinetics of substrate utilization of hydrogen production from wastewater with immobilized cells of photosynthetic bacteria. AB - The kinetic characteristics of substrate utilization by immobilized cells of Rhodopseudomonas capsulata 386 and Rhodopseudomonas sp. D for H2 production was investigated. The results showed that substrate utilization did not proceed simultaneously with H2 evolution, H2 producing capacity of immobilized cells with agar was higher than that of alginate immobilized cells, but the appearance of maximum H2 producing activity was later than the last one. The kinetics of substrate utilization (glucose) by immobilized cells of strain D followed the first-order reaction, rate constant k value was 1.2 x 10(-2) L/h, analysis of macrokinetics indicated that substrate utilization by immobilized cells in H2 evolution process was governed by biochemical reaction other than diffusion transfer limitation, because of the Thiele modulus were 0.125 and 0.154 for immobilized cells with D = 3.6 mm and 4.4 mm particles, the corresponding effective factor were determined as 0.998 and 0.988, respectively. An immobilized cell bioreactor, fed by glucose and lactate, was employed for continuous H2 evolving system. It was found that hydrogen production were 0.659 L/d and 0.477 L/d in immobilized cells system of strain 386 and D when lactate used as H2 evolving substrate, while the volumetric H2 rate were all up to 1.0 L/L.d. PMID- 7548772 TI - A GOD electrode free from the influence of ethanol. AB - The influence of ethanol on GOD electrode was investigated. When enzyme electrode was made by the Sandwich method, 0.1% (V/V) ethanol had an obvious influence on the response of the electrode with 4.3% deviation. The influence increased with increasing ethanol content of the samples. This influence can be avoided by using a nylon mesh GOD electrode, which was successfully used for the determination of glucose in the range of 5-20 mM with ethanol content up to 9% (V/V). Such an electrode showed good reproducibility, stable response activity and long storage life ( > 30 days). PMID- 7548774 TI - Construction and expression of 75kDa readthrough protein gene from beet necrotic yellow vein virus. AB - Using DNA recombinant techniques, coat protein (CP) gene and 54kDa fragment from beet necrotic yellow vein virus (BNYVV) RNA2 were ligated to construct 75kDa readthrough protein gene. Comparing with wild-type 75kDa readthrough protein gene, four nucleotides of the constructed gene were replaced, including CP amber termination codon UAG to AUG. Correspondingly, two amino acids were changed. The temperature inducible expression vectors containing 75kDa readthrough protein gene or its 54kDa fragment were constructed and transformed into E. coli BL21 (DE3), respectively. The results of SDS-PAGE and Western blotting showed that (1) the 75kDa readthrough protein gene was expressed specifically by temperature induction and some smaller protein components, one of which is 37 kDa, were observed as well; (2) only a 37kDa protein was produced from the 54kDa fragment. PMID- 7548775 TI - Mean residence time of drugs administered non-instantaneously and undergoing linear tissue distribution and reversible metabolism and linear or non-linear elimination from the central compartment. AB - Based on disposition decomposition analysis (DDA), equations for the mean residence times (MRT) in the body are derived for a drug and its interconversion metabolite that undergo linear tissue distribution and linear or non-linear elimination from the central compartment after non-instantaneous administration of the drug. The MRT of the drug after non-instantaneous input can be related to the MRT of the drug after intravenous administration, the ratio of the total area under the plasma concentration-time curve of the drug after non-instantaneous administration to that after intravenous administration, the bioavailability of the drug, and the mean input time of the drug. Similar relationships also exist for the MRT of the interconversion metabolite after non-instantaneous input of the drug. The application of these equations to a non-linear reversible metabolic system is illustrated with computer simulations. PMID- 7548776 TI - Absorption of new HIV-1 protease inhibitor, KNI-272, after intraduodenal and intragastric administrations to rats: effect of solvent. AB - KNI-272 is a tripeptide drug that has a strong pharmacological potential for treating human immunodeficiency virus type 1 (HIV-1). We have already reported the pharmacokinetic characteristics of KNI-272 after intravenous and intraduodenal (ID) administrations to rats. In this study, KNI-272 was administered to rats as a solution and the effect of four kinds of solvent on the bioavailability (BA) of KNI-272 was determined using rats. The mixtures included propylene glycol (PG) and water (70% PG), a solution of PG (100% PG), a solution of Tween 80 (Tween 80), and a mixture of PG and HCO60, a polyoxyethylated, 60 mumol, castor oil derivative (PG:HCO60 = 7:3). After ID administration to rats at a dose of 50.0 mg kg-1, the mean peak plasma concentrations, Cmax, were 2.58 +/- 0.53 (SE) (70% PG), 3.28 +/- 0.51 (100% PG), 3.15 +/- 0.51 (Tween 80), and 4.66 +/- 0.68 micrograms mL-1 (PG:HCO60). The highest BA, 44.6%, was obtained after ID administration of KNI-272 dissolved in PG:HCO60. On the other hand, after intragastric (IG) administration of KNI-272 solution in which the drug was dissolved with PG:HCO60, the Tmax, the Cmax, and the BA were 1.25 +/- 0.60 h, 2.33 +/- 0.65 micrograms mL-1, and 24.2%, respectively. The Cmax and BA values were equal to half of the values obtained after ID administration of KNI-272 dissolved in the same solution. In this study, as the PG concentration in the solution increased and the other additives (Tween 80 and HCO60) were coadministered, the BA of KNI-272 after ID administration increased. These results suggest that, for the development of an oral dosage form of KNI-272, a non-ionic surfactant that dissolves in the duodenum or small intestine and that enhances the absorption of this drug from the gastrointestinal tract into the enterocytes is needed. PMID- 7548777 TI - Pharmacokinetics of methotrexate after intravenous and intramuscular injection of methotrexate-bearing positively charged liposomes to rats. AB - The pharmacokinetics and tissue distribution of methotrexate (MTX) were investigated after intravenous (IV) and intramuscular (IM) injection of free MTX (treatment I), freshly prepared MTX-bearing positively charged liposomes (large unilamellar vesicles), PLUVs (treatment II), and empty PLUVs mixed manually with free MTX (treatment III), 4 mg kg-1 as free MTX to rats, using HPLC assay. After 1 min IV infusion, the plasma concentrations of MTX (Cp), the area under the plasma concentration-time curve (AUC, 173 against 314 micrograms mL min-1), the terminal half-life (t1/2, 24.0 against 412 min), the mean residence time (MRT, 13.0 against 324 min), and the apparent volume of distribution at steady state (VSS, 289 against 3370 mL kg-1) were significantly larger, but the total body clearance (CL, 23.1 against 12.8 mL min-1 kg-1), the renal clearance (CLR, 8.38 against 3.09 mL min-1 kg-1), the non-renal clearance (CLNR, 14.6 against 9.56 mL min-1 kg-1), and the amount of MTX excreted in urine (Xu, 415 against 275 micrograms) were significantly lower in treatment II than in treatment I. This could be due to the fact that some of the MTX-bearing PLUVs were entrapped in tissues and the rest were present in plasma (larger MRT and Vss in treatment II), and MTX is slowly released from MTX-bearing PLUVs (longer t1/2 in treatment II). In the present HPLC assay, the concentrations of MTX represent the sum of free MTX and MTX in MTX-bearing PLUVs (larger Cp and AUC and slower CL in treatment II). Saturable formation of 7-hydroxymethotrexate from MTX was reported in rabbit blood and nonlinear disposition of MTX was also reported in rats and rabbits (lower Xu and CLR in treatment II). After 1 min IV infusion, some pharmacokinetic parameters of MTX, such as AUC, CL, CLR, CLNR, and Xu, were significantly different between treatments I and III, but nonetheless the differences were smaller than those between treatments I and II. After both IV and IM administration, the amount of MTX remaining per gram of tissue or organ in the kidney, stomach, small intestine, and large intestine was significantly smaller in treatment II than in treatment I.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7548778 TI - The bioavailability of cefpodoxime proxetil tablets relative to an oral solution. AB - The bioavailability of cefpodoxime proxetil tablets relative to an oral solution of cefpodoxime proxetil in a sucrose/alcohol/citric acid vehicle was studied in 11 healthy volunteers in a randomized, crossover study. Fasted subjects took one cefpodoxime proxetil 100 mg tablet or 50 mL of a 2 mg mL-1 cefpodoxime proxetil oral solution on two separate occasions. In a third study period, all subjects took a 100 mg dose of the oral solution with a high-fat meal to investigate the effect of food on cefpodoxime proxetil absorption from the oral solution. Serial blood samples were obtained over a 24 h period, and urine was collected for 48 h after dosing. Cefpodoxime concentrations in plasma and in urine were determined using HPLC methods. The bioavailability of cefpodoxime proxetil tablets relative to the oral solution was 82%, as determined from AUC ratios. There was no difference in the rate of cefpodoxime absorption between dosage forms. Food had no effect on the extent of drug absorption from the oral solution but did result in delayed absorption. These results suggest that complete dissolution of cefpodoxime proxetil is critical for optimal bioavailability. PMID- 7548780 TI - Pharmacokinetics and hepatic first-pass effect of (-)-carbovir in the anesthetized rat. AB - Studies were designed to allow an in vivo estimation of the hepatic extraction ratio and to test the hypothesis that the pharmacokinetic parameters of (-)-CBV are significantly different during anesthesia. (-)-CBV was administered as an i.v. bolus followed by i.v. infusion into either the portal (n = 3) or the jugular (n = 3) veins of anesthetized male Sprague-Dawley rats. These studies indicate that (-)-CBV had a very low hepatic extraction ratio, in agreement with previous (-)-CBV in situ liver perfusion studies. Additionally, anesthesia was shown to alter the pharmacokinetics of (-)-CBV by reducing the total body clearance of this drug. PMID- 7548779 TI - Time course of cyclosporine and ist motabolites in blood, liver and spleen of naive Lewis rats: comparison with preliminary data obtained in transplanted animals. AB - the time course of intravenously administered cyclosporine (1 mg kg-1) and its metabolite AM1, AM9, and AM1c were examined in the blood, liver, and spleen of naive Lewis rats. Cyclosporine concentration versus time data for all three tissues were qualitatively similar, following a biexponential model C = Ae-gamma 1t + Be-gamma 2t with maximum cyclosporine concentrations reached at 1 h. Whole blood cyclosporine clearance, terminal half-life, mean residence time, steady state volume of distribution, and hepatic extraction ratio (calculated from blood data) were similar to previously reported results. Cyclosporine in the liver showed the largest area under the concentration-time curve, mean residence time, and disposition and terminal half-lives. Spleen cyclosporine mean residence time and and terminal half-life were not significantly different from blood parameters. Metabolites AM1, AM9, and AM1c showed almost parallel time courses in all three tissues. The hydroxylated derivative AM9 was the major metabolite found in all tissues, with twofold greater levels in the liver compared to the blood and spleen. Slightly less AM1 was found in the liver relative to blood and spleen, where it was present in equal amounts. AM1c levels in the liver were not different from those in the spleen and were greater than observed for blood. The results obtained above were reflected in preliminary studies using liver transplanted rats treated with multiple doses of cyclosporine. Both blood and liver biopsy levels of CyA, AM1, and AM9 post-transplant showed twofold to fourfold decreases from day 3 ( samples taken 4 h post-CyA-dose) and concentrations were not significantly different from similarly sampled naive controls. More importantly, the metabolite/CyA ratios did not vary significantly between liver and blood in the two groups. For naive rats, and liver transplanted animals not undergoing rejection, changes in blood cyclosporine levels seem to predict variations in tissue concentrations. PMID- 7548781 TI - Methodology for using oral dose pharmacokinetic data to select drugs for prolonged release formulations and validation of the method using simulated data. AB - A computer aided method for selecting drugs as potential candidates for oral prolonged release formulations has been previously published. In order to decide whether trial formulations were warranted, prolonged release dosing was simulated to find all release rates and doses producing successful regimens in every subject for whom clinical pharmacokinetic data were available. Intravenous dose data were required because the models and their parameter values could not be assessed with oral doses. The new method uses model independent equations derived from rapidly absorbed oral doses to simulate the administration of prolonged release formulations. Using these equations, repetitive oral dosing regimens were reiteratively simulated to search for all successful release rates and doses in every available subject. Results were validated by comparison to those obtained with the published method using theophylline and hypothetical drugs, which included examples of flip-flop and vanishing exponentials. When the oral reference dose data were reliable, results were similar even when the model independent parameter values did not agree with those used to generate the oral reference dose data. Differences were observed only when the random errors in the oral reference dose data were large. If more than one model independent equation provided equivalent fits to the oral reference dose data, the final results were similar independent of which equation was employed. PMID- 7548782 TI - Pharmacokinetics of alpha-dihydroergocriptine in rats after single intravenous and single and repeated oral administrations. AB - The pharmacokinetics of alpha-dihydroergocriptine methane sulphonate in rats were investigated using an HPLC method for the detection of unchanged alpha dihydroergocriptine (DHEK) in plasma, organs (kidneys, heart, lungs, spleen, liver, and brain), and urine. The plasma profile of DHEK obtained after intravenous administration at a dose of 5 mg kg-1 (as base) of DHEK methane sulphonate showed a three compartment pharmacokinetic model with an elimination half-life of 6.78 h. The kinetics of DHEK after a single oral administration at a dose of 20 mg kg-1 (as base) showed two peaks: the second peak, at about 6 h, was probably due to an enterohepatic cycle. The disposition of DHEK consisted of an absorption half-life of 0.02 h, a distribution half-life of 2.15 h and an elimination half-life of 5.83 h. The pharmacokinetics of DHEK, after repeated oral administrations at the same dose, were similar to those after a single oral administration. The absolute bioavailability was 4.14% after a single oral administration and 3.95% after repeated oral administrations. The analysis of the organs showed that DHEK was rapidly absorbed and distributed in all tissues, mostly in lungs, kidneys, and liver, but it is interesting to observe that it also reached the brain. After repeated oral administrations plasma and tissue concentrations were similar to those obtained after a single administration; therefore it is possible to exclude the onset of autoinduction or accumulation phenomena of DHEK in rats' organs. Urinary excretion of the unchanged drug was low (0.38% of the administered dose in the intravenous route and 0.04% in the oral route), being in agreement with a low oral bioavailability and a rapid and extensive metabolism (first-pass effect). PMID- 7548783 TI - Pharmacokinetics of aditoprim in normal and febrile sheep. PMID- 7548785 TI - Changes of insulin and glucagon receptors following bile duct ligation and its release. AB - The specific bindings of insulin and glucagon to 4 x 10(4) g pellets containing hepatic plasma membrane were measured at 1 and 2 weeks after bile-duct ligation and 1 week after reduction of jaundice. At 1 week of jaundice, insulin binding was not decreased compared to control rats, however, glucagon binding 30(6)% was significantly lower than that 43(8)% in control rats (p < 0.01). At 2 weeks of jaundice, insulin binding 35(3)% was significantly lower than that 40(5)% in control rats (p < 0.05), and glucagon binding 26(4)% was also significantly lower compared to control rats 42(4)% (p < 0.01). The decreased insulin and glucagon receptors returned to nearly the levels of controls after 1 week's reduction of jaundice. These results suggested that the measurement of receptor binding may be useful as an indicator of the severity of hepatocyte injury. PMID- 7548784 TI - Growth inhibitory effects of pMC540 and merodantoin on established MCF-7 human breast tumor xenografts. AB - The effect of preactivated merocyanine 540 (pMC540) and one of its chemically synthesized active isolate merodantoin on established human MCF-7 human breast tumor xenografts was investigated. Preactivation is a novel photochemical method for the production of chemotherapeutic compounds that exert their biological effects independent of light. These compounds thus produced, are cytotoxic to human breast cancer cells in vitro and in vivo but only minimally cytotoxic towards normal cells. Nude mice bearing established breast tumors (with or without exogenous estradiol) received injections of pMC540 (250 mg/kg) or merodantoin (75 mg/kg) with or without concurrent treatment with tamoxifen. Treatment with pMC540 and merodantoin caused a 74% and 84% inhibition of tumor growth respectively. Combination of these drugs with tamoxifen did not produce a significant enhancement of growth inhibition. In the absence of exogenous estradiol, identical treatment with pMC540 and merodantoin resulted in 41% and 25% inhibition of tumor growth respectively. Both agents caused a significant (59%) inhibition of growth of estrogen independent human breast tumors established from MDA-MB-435 cells. These results show that photochemically generated novel compounds in pMC540 are effective in suppressing the growth of established human MCF-7 and MDA-MB-435 breast tumor xenografts. PMID- 7548786 TI - The effects of roxatidine on neuromuscular transmission. AB - We have investigated the effects of the H2 receptor antagonist roxatidine on the neuromuscular transmission by using the sciatic nerve-gastrocnemius muscle preparation of the rat in vivo. Roxatidine, administered by i.v. injection, potentiates the neuromuscular blockade induced by d-tubocurarine, pancuronium and aminoglycoside antibiotic, kanamycin. Moreover, the drug alone is capable of producing a blockade on the preparation stimulated at high frequency. The neuromuscular blockade induced by roxatidine is partially reversed by 4 aminopyridine but not by dimaprit. PMID- 7548787 TI - A diurnal variation in the tumorigenic response of mouse epidermis to a single application of the strong short-acting chemical carcinogen methylnitrosourea. A dose-response study of 1, 2 and 10 mg. AB - A total of 670 hairless mice (hr/hr Oslo strain, 50% females) were exposed to a single topical application of two doses of MNU dissolved in 100 microliters reagent grade acetone in order to study whether there really is a diurnal variation in the sensitivity of epidermal cells to the short-acting alkylating carcinogen methylnitrosourea (MNU). Three hundred and fifty-one mice were exposed in groups to a single application of 1 mg MNU at either 04:00, 08:00, 12:00, 16:00, 20:00 or 24:00 Central European time. A number of 287 mice were exposed in three groups to single application of 2 mg MNU at 8:00, 12:00 or 20:00. To look at the dose-response relationship we also treated an additional group of 32 mice with 10 mg MNU at 12:00. The mice were kept in plastic cages located in the same room in an animal department with controlled temperature, air flow, humidity and a constant light/darkness rhythm (07:30 - 19:30). The development of all types of skin tumors was observed and the results presented as tumor rates (percentage of tumor-bearing animals in relation to the number of animals alive a appearance of the first tumor related to time), and tumor yields (the cumulative occurrence of all skin tumors standardized for comparison of groups of 32 mice related to time). Most animals were examined once a week for 54 weeks, but those to which 10 mg MNU was applied were observed for only 34 weeks. Modern, well accepted statistical methods were used to analyse the significance of differences between the results. A diurnal variation in tumor production after a single application of 1 mg MNU was demonstrated with a relatively high tumor crop after application in the period from 24:00 to a peak at 08:00, and a lower crop at 12:00 to 20:00 with a trough at 16:00. When 2 mg MNU was applied, there was definitely a low tumor production after application at 12:00 compared to the two other times. There was a good and almost straight-line dose-response relationship after 1, 2 and 10 mg MNU. The results give a strong support to the hypothesis that there is a diurnal variation in the sensitivity of epidermal cells to the short-acting alkylating carcinogen MNU.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7548788 TI - P-170 glycoprotein expression in gastric and colorectal carcinomas and normal mucosa. An immunocytochemical study. AB - During the past few years it has become apparent that simultaneous resistance of tumour cells to a number of heterocyclic drugs (multidrug resistance) is often correlated with overexpression of a P-glycoprotein (P-gp or P-170). P-gp expression can be studied by molecular biology and immunohistochemical techniques. The latter provide a rapid, sensitive and specific screening method suitable for testing even a relatively small number of tumour cells like those obtained at biopsy. The aim of this study was to detect and localize the immunohistochemical expression of P-gp in normal and neoplastic gastrointestinal tissue using the Mab JSB-1 in formalin-fixed paraffin-embedded specimens. A particularly striking finding of our study was the consistent, prevalent higher expression of P-gp in the stomach than in the colon, with a higher percentage of immunostaining in normal than in neoplastic tissue. This is in agreement with the fact that not only is the prognosis known to be worse for stomach cancer, but also the response to treatment is lower. Further studies should be carried out to verify the possibility of making routine tests of this kind for the evaluation of multidrug resistance, to guide the selection of patients for treatment of cancer with chemotherapeutic drugs. PMID- 7548790 TI - Selenium-induced activation of brain Na(+)-K(+)-ATPase in rats. AB - The influence of selenium supplementation on the activity of Na(+)-K(+)-ATPase and on the degree of free radical generation was studied in brain tissue of rats. Selenium was administered for 5 months in drinking water, and was measured in plasma by the fluorometric method at the end of the experimental period. Animals were sacrificed and brain tissue homogenates were used for enzyme assay and for assessment of lipid peroxide formation. Brain tissue from rats who received selenium showed significantly increased Na(+)-K(+)- ATPase activity but also significantly decreased lipid peroxide farmation. Since this enzyme is known to be inhibited by oxygen free radicals, selenium supplementation appears to exert a beneficial effect on the Na(+)-K(+)-ATPase activity by preventing free radical induced damage. PMID- 7548789 TI - Generation of monoclonal antibodies utilizing the host anti-idiotypic network. AB - In accordance with Jerne's idiotypic network theory, it was attempted to generate antibodies utilizing the host's anti-idiotypic reactions. A murine anti-tumour monoclonal antibody was administered to rats; we examined the possibility of whether the animals would develop anti-idiotypic antibodies, the paratope of which would be the "internal image" of the tumour associated antigen that the original murine monoclonal antibody recognizes. These anti-idiotypic antibodies would generate a second generation of anti-idiotypic antibodies, which would have the same specificity as the immunogen murine antibody. It was found that, while all rats had no anti-tumour response prior to immunization with the anti-tumour murine monoclonal antibody, they all developed antitumour antibodies following administration. The latter observation came as a consequence of the development of anti-mouse immunoglobulin and anti-idiotypic antibodies. The animals were sacrificed and their spleen cells were fused with myeloma cells for the production of monoclonal rat anti-tumour antibodies. Finally, a monoclonal antibody was selected which appeared to recognize the same tumour-associated antigen as the originally administered murine antibody, as shown by cell-binding and competition assays, as well as immunohistochemistry. This new approach allows for "replication" of polyclonal and monoclonal antibodies without the need of the antigen and it may also serve as an immunotherapy strategy for the augmentation of the antitumour immune response of patients receiving monoclonal antibody treatment. PMID- 7548791 TI - Ex-vivo fine needle aspiration. A new method of xenografting non-small cell carcinoma of the lung. AB - Ex-vivo needle aspiration (xvFNA) has been rarely used to obtain viable tumor cells. It has been occasionally employed for short-term cultures. Xenografting of lung carcinoma in athymic nude mice provides a good animal model for the study of this neoplasm. Successful engraftment using conventional methods has been disappointing) low (d 40%). Enzymatic digestion of the tumor fragments to obtain cell suspension lowers viability. We postulated that xvFNA might provide readily available tumor cell suspensions for xenografting lung carcinoma and that it would provide a higher success rate of engraftment than the conventional techniques. We aseptically performed xnFNA in 35 cases of freshly resected non small cell carcinoma of the lung. These included 15 adenocarcinomas, 17 squamous carcinomas and 3 undifferentiated non-small cell carcinoma (UNSCC). Tumor cell suspensions were injected subcutaneously in athymic nude mice. Tumor necrosis in the aspirates ranged from 20-90% (median 60%). Gross evidence of engraftment was seen in 30 of 35 cases (85.7%) 1-19 weeks postimplantation (median 2 weeks). This was seen in UNSCC (3/3), squamous carcinomas (13/17) and adenocarcinomas (14/15). Xenograft sizes ranged from 5-34 mm (median 19 mm). They showed similar morphology to the primary tumors. Ex-vivo FNA used for harvesting lung carcinoma cells and their xenografting is an effective method for obtaining viable material for studying this neoplasm. PMID- 7548792 TI - Immunosuppression of mice by cyclophosphamide (CyP) and cyclosporin A (CsA) changes the course of HSV-1 infection and the distribution of viral DNA in target organs. AB - The effects of the immunosuppressive drugs cyclophosphamide (CyP), cyclosporin A (CsA) and the Langerhans cells immunomodulator OK-432, a bacterial cell wall preparation, on herpes simplex virus type-1 (HSV-1) infection in mice were studied. An increased mortality of HSV-1-infected mice was observed following intraperitoneal injection of both the pathogenic HSV-1 (Justin) and the apathogenic UL56-deleted HSV-1-M-LacZ strains. Intraperitoneal injection of CyP prior to infection with HSV-1 increased the susceptibility to infection of normally resistant mouse strains, including the inbred strain C57BL/6 and the outbred Sabra strain, with the virus reaching the pancreas, the spinal cord and the spleen. Polymerase chain reaction (PCR) to detect viral DNA in mouse organs following IP HSV-1 injection after treatment with CyP and/or OK-432 showed that CyP treatment prevented the virus from reaching the pancreas, while a combination treatment of both chemicals allowed HSV-1 to reach the pancreas. These studies indicate that treatment of mice with immunosuppressive drugs (CyP and CsA), which affect dendritic cells and T cells, prevents the migration of HSV-1 to the pancreas, while the immunomodulator OK-432, which induces dendritic cell activity, did not prevent virus migration to the pancreas. Lung samples were consistently positive for viral DNA following treatment with either CyP or CsA. PCR to detect viral DNA in mice injected with HSV-1 via the footpad route following pretreatment with CyP and OK-432 also through the footpad revealed the presence of HSV-1 DNA in the spinal cord on days 1, 3, and 5 post infection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548793 TI - Neuromuscular blocking drug pharmacodynamics after chronic exposure to H2- antagonists. AB - This study tested the hypothesis that chronic exposure to H2-antagonists may affect neuromuscular function. Cimetidine or ranitidine was administered to rats for twenty one days as subcutaneously implanted biodegradable pellets. Control rats received placebo pellets. Serum cimetidine and ranitidine concentrations ranged from 0.1 to 0.5 micrograms/mL over this period. On the study day, surgically prepared anaesthetized rats received either succinylcholine (SCh) or atracurium (ATr) to achieve complete paralysis of the tibialis anterior muscle. After recovery an infusion dose of each neuromuscular blocker was titrated to produce 50% muscle paralysis with each blocker. Exposure to cimetidine or ranitidine did not alter SCh or ATr dose to maximum paralysis or the time course of recovery from the initial paralysis. SCh or ATr infusion rates required to elicit 50% paralysis were also unaffected by cimetidine or ranitidine pretreatment. These results indicate that chronic exposure to cimetidine or ranitidine at human therapeutic concentrations does not affect the neuromuscular pharmacodynamics of SCh or ATr in rats. PMID- 7548794 TI - Early embryonic cells activate the alternative complement system. AB - Murine embryonic stem cells, embryonic carcinoma cells and pre-implantation embryos were found to be extremely sensitive to cytolysis by normal human serum as compared to matured cells. The cytolytic activity to embryonic cells was not removed by pre-absorption of serum with spleen lymphocytes. Conditions which block both complement activation pathways or, selectively, the alternative pathway completely abrogated the activity of human serum against embryonic cells whereas the activity was retained under conditions which block the classical complement pathway, indicating that embryonic cells activate the alternative complement system (ACS). The cytotoxic effect to murine embryonic cells was reproduced using syngeneic murine serum. Concerning the mechanism of ACS activation, the expression of regulators of complement activation and of membrane bound sialic acid was analysed. Embryonic cells express mRNA for Crry similarly to other cells but additionally express Cr2-transcripts not found in most adult cells. Embryonic cells have strikingly low levels of membrane-bound sialic acid compared to adult cells. PMID- 7548795 TI - Reversal of doxorubicin-resistance in solid tumors by clomipramine. AB - Resistance to cytotoxic treatment is a major obstacle to more successful cancer treatment, and approaches to overcome the drug resistance of tumors have received much attention in recent years. In the present in vivo study the psychotropic drug clomipramine was used as chemosensitizer in the doxorubicin (DOX)-resistant L 1210 cell line growing as solid tumors in mice. A significant reduction in the growth of DOX-resistant tumors was observed after treatment with clomipramine and DOX (p = 0.014, Kruskal Wallis-test; p = 0.006, Wilcoxon rank sum test). Thus, clomipramine might be useful in reversing DOX-resistance in solid tumors. PMID- 7548796 TI - Transcatheter arterial embolization for hepatocellular carcinoma with portal vein thrombosis. AB - In order to evaluate the possible benefits of transcatheter arterial embolization (TAE) in hepatocellular carcinoma (HCC) patients with peripheral portal vein thrombosis, 96 consecutive HCC cases with peripheral portal vein thrombosis were analysed. Of them, 35 cases received TAE and 61 cases did not. Most (77.8%) of the TAE-treated cases showed decreased alpha-fetoprotein (AFP) levels after treatment, but 57.1% of them suffered another rise in AFP levels and subsequently died. One patient (2.8%) developed progressive jaundice after TAE and died within 1 month, while four of the non-TAE cases died within 1 month after diagnosis. In general, TAE is safe for HCC patients with peripheral portal vein thrombosis. In addition, using Cox's regression model for multivariate survival analysis, serum total bilirubin (< or =, > 2 mg/dL; P = 0.0254), AFP (< or = 3155 ng/mL, > 3155 ng/mL; P = 0.0002) and treatments (TAE, non-TAE; P = 0.0059) were found to affect their prognosis. There was significant difference in survival between TAE and non TAE groups, the 6 month, 1 year and 2 year survival rates were 91.4 versus 62.3%, 51.4 versus 26.2% and 17.1 versus 4.9% (P = 0.0017). The median survival times of TAE and non-TAE groups were 10.3 versus 3.7 months, respectively. Though TAE only provided palliative treatment, it did prolong survival in HCC patients with peripheral portal vein thrombosis. PMID- 7548797 TI - Polarizing microscopy of partially dissolved gallstone powder: a simple technique for studying gallstone composition. AB - A quick and reliable method for estimating the proportion of constituents of a stone may be useful in determining the prevalence of chemical type of gallstones in different geographic areas or ethnic groups. Chemical and infrared spectroscopy estimation facilities are not commonly available in many parts of the world and visual inspection may not be reliable for scientific purposes. To investigate whether identification of microcrystalline solids in partially dissolved stone powder could accurately predict the chemical nature of the gallstone we undertook a blind study on 40 samples of gallstones and compared our results with those of visual inspection and quantitative infrared spectroscopy. There were 29 cholesterol stones (mean cholesterol 83.4 +/- 7.5%), six intermediate cholesterol stones (mean cholesterol 48.0 +/- 21.2%) and five pigment stones (mean cholesterol 14.3 +/- 4.3%) as determined by quantitative infrared spectroscopy. Microscopic examination of partially dissolved gallstone powder in ethanol correctly identified all 29 cholesterol gallstones (sensitivity 100%, specificity 92%), four of six intermediate cholesterol stones (sensitivity 67%, specificity 100%) and all five pigment stones (sensitivity 100%, specificity 97%). It also detected microspheroliths of calcium carbonate in 14 of 16 calcium carbonate containing gallstones (sensitivity 88%, specificity 100%). The chemical grouping of stones on the basis of microscopic examination was correct in 37 (93%) of 40 samples and was especially useful in identifying 'intermediate' cholesterol stones which cannot be recognized by visual inspection. Thus microscopic examination of powdered gallstones appears to be a simple and reliable method of determining gallstone composition. PMID- 7548798 TI - Interferon therapy for patients more than 60 years of age with chronic hepatitis C. AB - Nineteen patients aged > 60 years with chronic hepatitis C (CHC) received interferon (IFN) therapy and a complete response (CR) was achieved by five of them (26%). The incidence of CH with severe fibrosis in this elderly group was significantly higher than in another 52 patients with CHC who were < 60 years of age (the younger group; P < 0.05). There was no significant difference in the hepatitis C virus (HCV) genotype distribution between the elderly group and the younger group. However, the HCV-RNA titre was significantly higher in the elderly group than in the younger group (P < 0.05). There was no significant difference in the efficacy rate of IFN in the elderly and younger groups after standardization of the background factors. In the elderly group, the HCV-RNA titre was significantly lower in the patients achieving CR than in those with no response (P < 0.05). These data suggest that elderly patients with a low HCV-RNA titre can still respond well to IFN therapy. PMID- 7548799 TI - Overexpression of p53 in hepatocellular carcinomas: a clinicopathological and prognostic correlation. AB - Overexpression of the p53 tumour suppressor gene is one of the most common abnormalities in primary human cancers and appears to be a result of point mutation within a highly conserved region of the gene with subsequent encoding for a mutant, more stable protein. In the study, 71 surgically resected hepatocellular carcinomas (HCC) were examined to study the expression of the p53 gene, its relation with clinicopathological parameters and its prognostic significance. Using immunohistochemical detection for mutant p53 protein with monoclonal antibody PAb1801, p53 overexpression was found in 22 tumours (31%) but in none of the non-tumorous liver specimens. Overexpression of p53 was more frequent in tumours with poor cellular differentiation (P = 0.01), in tumours > 5 cm in diameter (P = 0.05), and in those with giant cells present (P = 0.03) and, less significantly, of massive type of Eggel's classification (P = 0.06). It did not have any significant correlation with hepatitis B or C status, background liver disease or serum alpha-fetoprotein levels, nor was it related to tumour invasiveness (venous permeation, direct liver invasion and tumour microsatellite formation). In addition, the presence of p53 mutant protein did not influence tumour recurrence or patients' survival rates. The data suggested that p53 mutation in HCC was associated with a later stage of oncogenesis. However, it was not apparently related to tumour invasiveness/aggressiveness and prognosis. PMID- 7548800 TI - Haemodynamic effects of enalaprilat on portal hypertension in patients with HBsAg positive cirrhosis. AB - It has been suggested that enalaprilat inhibits the renin-angiotensin-aldosterone system in plasma and tissue; it may therefore reduce portal vascular pressure owing to secondary hyperaldosteronism in patients with liver cirrhosis. In order to evaluate this concept, 20 patients with hepatitis B surface antigen (HBsAg) positive liver cirrhosis and portal hypertension received an intravenous infusion of 2.5 mg of enalaprilat. Wedged hepatic venous pressure, free hepatic venous pressure and cardiac index were measured before, immediately after, and then 15 min, 30 min and 1 h after intravenous enalaprilat infusion. The mean pressure gradient between wedged hepatic venous pressure and free hepatic venous pressure was significantly decreased, by 13% immediately after, 18% at 15 min, 23% at 30 min and 13% at 1 h after infusion of enalaprilat. Thirteen patients experienced a decrease of hepatic venous pressure gradient (HVPG) greater than 5 mmHg, another three 3-5 mmHg and the remaining four patients exhibited no significant change in HVPG. Systemic haemodynamic indices, including pulmonary arterial pressure, pulmonary capillary wedge pressure and central venous pressure, decreased significantly at 15 and 30 min after enalaprilat infusion (P < 0.01). Liver function, renal function and blood routine before and after enalaprilat infusion showed no significant change. There were no adverse effects during or after enalaprilat infusion. We conclude that enalaprilat infusion can quickly and safely reduce the hepatic venous pressure gradient in patients with HBsAg positive cirrhosis. PMID- 7548801 TI - Hepatoprotection in ethinylestradiol-treated rats is provided by tauroursodeoxycholic acid, but not by ursodeoxycholic acid. AB - Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) have been suggested as potential treatments for drug-induced cholestasis. It was therefore decided to study the effects of administration of UDCA or TUDCA on individual serum bile acid concentration, conventional liver tests and associated hepatic ultrastructural changes in ethinylestradiol-treated (EE) rats mg/kg per day). Control rats were treated s.c. with propylene glycol. EE-treated rats were randomly assigned to receive daily i.p. injections of placebo, TUDCA or UDCA. Four rats in each group were treated for 4 consecutive days, and a second four for 14 days. After 4 days of treatment, the serum levels of cholic acid and taurocholic acid were significantly increased in EE-treated rats. None of the conventional liver tests were significantly different among the four groups. After 14 days of treatment the serum levels of cholic acid, chenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, bilirubin, alkaline phosphatase and gamma glutamyltransferase were significantly raised in EE and EE plus UDCA treated rats. EE plus TUDCA treated rats, however, had no significant changes in these individual serum bile acids or conventional liver tests. The ultrastructure of livers from EE plus TUDCA treated rats was similar to those of controls. On the other hand, EE and EE plus UDCA rats both showed a significant reduction in sinusoidal microvilli. These results show that treatment of rats for 4 days with EE induces significant rises in the serum concentrations of two individual bile acids and that TUDCA protects against this.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548802 TI - Clinical evaluation of titration of hepatitis C virus core antibody and its subclasses. AB - Titrations of anti-hepatitis C core (anti-HCc) immunoglobulin G (IgG) antibodies and its subclasses were studied in 90 patients with acute and chronic hepatitis C virus (HCV) infection, including 27 patients who underwent interferon (IFN) therapy. The positivity rates for each anti-HCc subclass were as follows: 95.2% for IgG1, 12.0% for IgG2, 69.9% for IgG3 and 19.3% for IgG4. The total anti-HCc IgG titre correlated well with the IgG1, titre, indicating that IgG1 was the main virus-specific IgG. Changes of IgG1 production mainly contributed to fluctuations of the anti-HCc IgG titre and corresponded well to positivity for HCV-RNA during and after IFN therapy. IgG3 was detected prior to IgG1 during the early phase of acute hepatitis in some cases and also appeared with relapse after IFN therapy. The serial assay of anti-HCc subclasses showed the patients' humoral immune response to HCV infection, and might be useful for evaluation of anti-viral immunity influenced by IFN therapy. PMID- 7548803 TI - Rise of plasma myeloperoxidase during interferon therapy. AB - The plasma myeloperoxidase (MPO) level was evaluated using a specific radio immunoassay (RIA) for MPO in alpha 2b-interferon (IFN)-treated patients with chronic viral hepatitis. The plasma MPO was checked before and after the initial 2 weeks use of IFN at a dose of 6 x 10(6) U/day. The mean concentration of plasma MPO was found to be markedly higher after IFN therapy than that before the therapy (421.7 +/- 34.3 vs 242.9 +/- 23.0 ng/mL, P < 0.001). The plasma MPO negatively correlated with the granulocyte count (r = -0.37, P < 0.02) and the platelet count (r = 0.49, P < 0.01, while it positively correlated with serum alkaline phosphatase (ALP; r = 0.41, P < 0.03). The plasma MPO also showed a strong correlation with plasma polymorphonuclear granulocyte elastase (PMN elastase; r = 0.73, P < 0.001). Our study thus suggests that the increased release of MPO from destroyed granulocytes is responsible for the high concentrations of the plasma MPO in patients during IFN therapy, because the plasma MPO, PMN elastase and ALP abundant in granulocytes all increased in spite of a decrease in the granulocyte count. Granulocytopenia during IFN therapy may therefore be due to the increased destruction of granulocytes in addition to a direct suppression of the bone marrow by IFN. PMID- 7548804 TI - Haemodynamic effects of a combination of propranolol and clonidine in patients with post-hepatitic cirrhosis. AB - The haemodynamic effects of a combination of propranolol and clonidine were evaluated in 20 patients with post-hepatitic cirrhosis. Haemodynamic measurements were taken before and 30 min after an intravenous injection of 0.1 mg/kg of propranolol. Thereafter, each patient was given an oral dose of 150 micrograms of clonidine and re-measured 60 min later. In this series, eight patients were defined as 'non-responders' (a decrease in hepatic venous pressure gradient of < 10%) after propranolol treatment. Of both the responders and non-responders, propranolol caused expected decreases in the cardiac index and heart rate while mean arterial pressure remained unchanged. Of the propranolol responders, a significant decrease in hepatic venous pressure gradient was observed. After the addition of clonidine, in both the responders and non-responders there was a further decrease in hepatic venous pressure gradient with a concurrent drop in mean arterial pressure, but cardiac index and heart rate remained unaltered. In conclusion, the combination of propranolol and clonidine in post-hepatitic cirrhotic patients enhanced the reduction of portal pressure achieved by propranolol alone. The beneficial effects of the combination of the two in the reduction of portal pressure appeared to be similar in both the propranolol responders and non-responders. However, the drop in mean arterial pressure following the addition of clonidine may be hazardous to cirrhotic patients. PMID- 7548805 TI - Membranous obstruction of inferior vena cava in Taiwan. AB - Membranous obstruction of the inferior vena cava (MOVC) is a rare cause of Budd Chiari syndrome. When compared to the West, the incidence of MOVC was reported to be higher in the Orient, India and South Africa. From 1979 to 1993, 16 consecutive Chinese patients (mean age 50 years) with MOVC were retrospectively evaluated. The diagnosis in these MOVC patients was usually delayed with a mean lag time of 111 months. Most of the cases were detected by a delicate ultrasonographic examination of the abdomen. Segmental narrowing of the inferior vena cava in 11 patients was the predominant type of MOVC, while five experienced a membranous obstructive type. Only one of five surgery-treated patients had a post-operation survival > 5 years, while five out of the nine patients who received conservative treatment still survived after a 6-15 year follow up. Two patients received percutaneous angioplasty (PTA). One survived 4 years and the other expired 5 years after the PTA. Two patients (12%) developed a hepatocellular carcinoma in their disease course, and the incidence was lower than in previous reports from Japan and South Africa. The incidence of hepatitis B surface antigen in MOVC did not increase in our patients compared with the general population. In conclusion, the inferior vena cava should be carefully evaluated in an ultrasonographic examination of the abdomen to increase the diagnosis rate of MOVC. Surgical intervention should be carefully justified according to the patient's symptoms and signs, the types of obstructive lesions and the expertise of the surgeons. PMID- 7548806 TI - Diagnosing ascites: value of ascitic fluid total protein, albumin, cholesterol, their ratios, serum-ascites albumin and cholesterol gradient. AB - Ascitic fluid total protein, albumin, cholesterol, their ascites/serum ratios, serum-ascites albumin and cholesterol gradients were measured for their ability to differentiate cirrhotic, malignant and tuberculous ascites in 76 patients. The mean +/- s.d. ascitic fluid total protein, albumin, cholesterol, their respective ascitic fluid/serum ratios in cirrhotic ascites were lower than malignant and tuberculous groups (P < 0.001 for each). The difference between malignant and tuberculous groups was significant for ascitic fluid/serum total protein (P < 0.05) and ascitic fluid/serum albumin (P < 0.01) only. Mean serum-ascites albumin gradient in cirrhotics was higher than in the malignant and tuberculous groups (P < 0.001 for each). The difference between malignant and tuberculous groups was significant (P < 0.01). Mean +/- s.d. serum-ascites cholesterol gradient in cirrhotics was higher than that in malignant and tuberculous groups (P < 0.001 for each). The difference between malignant and tuberculous groups was also significant (P < 0.01). Both serum/ascitic fluid total protein less than 0.5 and ascitic fluid cholesterol less than 55 mg/dL had 94% diagnostic accuracy for differentiating cirrhotic from malignant and tuberculous differentiating cirrhotic from malignant and tuberculous ascites. Serum ascitic fluid albumin gradient greater than 1.1 g/dL, ascitic fluid/serum albumin less than 0.65 and ascitic fluid albumin less than 2 g/dL had diagnostic accuracy of 92, 92 and 91%, respectively. Ascitic fluid total protein had diagnostic accuracy of 88%. None of the tests was able to differentiate between malignant and tuberculous ascites. Measurement of ascitic fluid cholesterol concentration is a simple method of differentiating cirrhotic from non-cirrhotic ascites. PMID- 7548807 TI - Triple therapy for the eradication of Helicobacter pylori and reduction of duodenal ulcer relapse: comparison of 1 week and 2 week regimens and recrudescence rates over 12 months. AB - The aim of this study is to assess the relationship between Helicobacter pylori and the relapse of duodenal ulcer, and also to evaluate the differences in efficacy and side effects between 1 week and 2 week triple therapy. Sixty-two patients with active duodenal ulcer, which healed within 8 weeks of nizatidine treatment, were randomly allocated to one of two groups. Group 1 (n = 29) received no drugs, Group II (n = 33) received triple therapy for 1 week (IIa, n = 16) or 2 weeks (IIb, n = 17). Eleven patients whose ulcer did not heal after an 8 week nizatidine treatment period were randomly assigned into Group IIa (n = 5) and IIb (n = 6). Seven patients whose ulcer recurred after discontinuation of nizatidine were allocated to receive 2 weeks of triple therapy. All patients received endoscopy 6 weeks after entry, and again at 3, 6 and 12 months unless both ulcer recurrence and H. pylori infection were found. The frequency of ulcer relapse 6 weeks after the active duodenal ulcer had healed was 83% (24/29 in Group I, 13% in Group 11a and 14% in Group IIb. The cumulative rate of recurrence was significantly higher in Group I than in Group II (90 vs 30% at 12 months, P < 0.01). Ulcer relapse was associated with persistence of H. pylori infection (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548808 TI - Risk factors for ulcerative reflux oesophagitis: a case-control study. AB - A case-control study was undertaken to investigate the effects of smoking, alcohol consumption, use of non-steroidal anti-inflammatory and other analgesic medications and family and medical history on the risk of ulcerative reflux oesophagitis (URO). We recruited 191 cases with URO diagnosed at endoscopy, 162 hospital controls who had also undergone endoscopy and 140 community controls from the Adelaide metropolitan area. From these three groups of subjects, 134 case-community control pairs, Matched on age, sex and postcode of residence and 142 case-hospital control pairs, matched on age, sex, hospital and endoscopist, were formed. Elevated non-significant risks were found in those smoking at least 20 cigarettes per day relative to those who never smoked (relative risk = 1.9, 95% confidence interval: 0.9-3.9 in case-hospital control pairs; relative risk = 1.9, 95% confidence interval: 0.9-3.7 in case-community control pairs). There was no elevation in risk associated with the use of non-steroidal anti-inflammatory drugs, with alcohol consumption, factors related to medical and reproductive history, nor with family history except for paternal history of heartburn (relative risk = 2.5, 95% confidence interval: 1.2-5.4 in case-hospital control pairs; relative risk = 1.9, 95% confidence interval: 1.0-4.0 in case-community control pairs). With the possible exception of smoking, no other risk factors for ulcerative reflux oesophagitis related to lifestyle are apparent. PMID- 7548809 TI - Hospitalization of Aboriginal adults for digestive disorders in Western Australia, 1989-91. AB - Hospital admission rates for many gastrointestinal, hepatobiliary and pancreatic diseases were much higher in Aboriginals aged 15 to > 65 years than among the rest of the population of that age in Western Australia in 1989-91. Alcohol related conditions were particularly prominent: the relative rate (RR) for alcoholic gastritis was > 30; for acute alcoholic hepatitis in young adults > 20; for alcoholic cirrhosis at 30-64 years the RR was about 4 to > 10; the RR for haematemesis and melaena was > 3; for acute pancreatitis at 30-64 years the RR ranged from about 3 to 20. Admissions for cholelithiasis in Aboriginal males were 1.5-2 times as frequent as in other males; for Aboriginal females the RR was > 2; acute cholecystitis was much commoner in Aboriginal patients from 30 to 64 years of age than in other patients of the same age. Illnesses coded as 'non-infectious enteritis and colitis' were the commonest diagnostic category in the International Classification of Diseases (ICD 9) classification of digestive system disorders among Aboriginal patients; admissions for these conditions occurred at double to more than seven times the rates that occurred in the same age groups in non-Aboriginal patients. Many of these illnesses were probably due to undetected gastrointestinal infections and parasitic infestations. This study shows that Aboriginal adults have disproportionately high rates of morbidity from many diseases of the digestive system. The findings have important implications for clinical services as well as for the development of preventive and promotional health strategies for Aboriginal people. PMID- 7548811 TI - Dietary modulation of colonic mucosal urokinase activity in rats. AB - The amount and type of dietary fibre ingested influences colonic luminal characteristics, especially the concentration of carbohydrate fermentation products such as butyrate. This study aimed to assess whether diets supplemented with fibres of differing fermentability (delivering different amounts of butyrate to the colon) influence mucosal activities of urokinase and brush border hydrolases, and epithelial turnover. Groups of five rats were fed one of four diets containing low (2%), highly fermented (guar 10% or oat bran 10%) or slowly fermented fibre (wheat bran 10%) for 4 weeks. Activities of urokinase, alkaline phosphatase, dipeptidyl peptidase IV and maltase were measured in mucosal homogenates of proximal and distal colon and from rectum. Proliferative kinetics were assessed in distal and proximal colon by the metaphase arrest technique. Hydrolase activities were similar across all four dietary groups but a significant difference was found for urokinase (P = 0.014). This was due to a reduction in urokinase activities of > 30% at the three sites in the wheat bran group compared with the other groups. Of proliferative indices, only crypt column height differed across the groups (P = 0.038) and was highest in rats fed wheat bran and lowest in those fed the low fibre diet (P = 0.047). The proportion of mitoses in the top one-fifth of the crypt also differed across groups (P = 0.038) due to the high values in the distal colon of the low fibre group. Thus, addition of a slowly fermented (but not highly fermented) fibre to the diet of rats reduces net urokinase activity in large bowel mucosa and increases the life span of colonic epithelial cells without changing activities of brush border hydrolases. PMID- 7548810 TI - The role of colonoscopy in screening persons with family history of colorectal cancer. AB - First-degree relatives of colorectal cancer patients are at increased risk for developing colorectal neoplasms. In order to assess the potentiality of colonoscopy screening in this high-risk population, 213 asymptomatic family members (age range 30-69 years, mean 42.8 years) of those patients with colorectal cancer received colonoscopic examination at Chang-Gung Memorial Hospital from April 1992 to May 1994. Twenty-eight persons with 42 lesions (polyps or cancer) were identified, including 28 adenomas, nine hyperplastic polyps and five adenocarcinomas. The positive detection rate was 9.9% for adenoma and 2.3% for cancer. Colorectal neoplasms afflicted males more frequently than females (16.7 vs 5.7%, P < 0.05) and occurred less frequently in those < 40 years of age (5.5 vs 17.2%, P < 0.05). Forty-two per cent of the detected neoplastic lesions were beyond the reach of 60 cm flexible sigmoidoscopy and 36% of adenomas were < 0.5 cm in size and would be missed if patients were screened by air contrast barium enema. Cost analysis revealed that the charges of both screening colonoscopy and screening flexible sigmoidoscopy/air contrast barium enema were approximate. Colonoscopy also has a high acceptability and safety. It appears appropriate to use colonoscopy, rather than flexible sigmoidoscopy or air contrast barium enema, as an initial screening procedure for persons with a family history of colorectal cancer, especially those > 40 years of age. PMID- 7548812 TI - Barrett's oesophagus and Helicobacter pylori. AB - In order to demonstrate the presence of Helicobacter pylori in the metaplastic epithelium of Barrett's oesophagus and to evaluate its possible association with this entity, we examined 29 cases of Barrett's oesophagus where concomitant antral biopsies were also available. These cases were compared with an equal number of age and sex matched controls of uncomplicated reflux oesophagitis. H. pylori was present in 11 of 29 cases of Barrett's oesophagus (38%). No increase in the frequency of H. pylori antral gastritis was found in patients of Barrett's oesophagus compared to the control group of uncomplicated reflux oesophagitis. The positivity of Barrett's oesophagus for H. pylori correlated with the presence of H. pylori antral gastritis (P < 0.05), although in two cases of H. pylori positive Barrett's oesophagus antral biopsies were negative for H. pylori. No difference was found in the severity of inflammatory and dysplastic changes of H. pylori-positive and H. pylori-negative Barrett's oesophagus. Presence of H. pylori does not seem to alter the natural history of Barrett's oesophagus. PMID- 7548813 TI - How safe is endoscopic sphincter of Oddi manometry? AB - The safety of endoscopic manometry of the sphincter of Oddi was evaluated in a prospective survey of 158 consecutive procedures in 126 patients with either unexplained pain after cholecystectomy or idiopathic recurrent pancreatitis. The only complication was that of pancreatitis which was defined as the development of abdominal pain in association with a plasma amylase above the reference range. This occurred in 13 patients (8%) and was more frequent (P = 0.001) when the indication for the procedure was idiopathic recurrent pancreatitis (29%) than unexplained pain (6%). Pancreatitis was also more frequent (P = 0.02) in patients with abnormal manometry (14%) than in those with normal manometry (3%) and occurred at highest frequency (50%) in a subgroup of patients with idiopathic recurrent pancreatitis and sphincter stenosis (high sphincter basal pressure). All episodes of pancreatitis were mild with a median increase in hospital stay of 2 days; no patients died. The risk of pancreatitis after endoscopic manometry is relatively low but increases in patients with abnormal sphincter manometry, particularly those with idiopathic recurrent pancreatitis. PMID- 7548814 TI - Bowel cancer: watching over the family. PMID- 7548815 TI - Classification of gastric lesions associated with portal hypertension. AB - There are two gastric mucosal lesions which commonly occur in patients with portal hypertension; one is portal hypertensive gastropathy and the other is gastric varices. These lesions occasionally cause fatal haemorrhage. Several classifications for these lesions have been proposed, but none of them have been evaluated prospectively. We consider it better to classify portal hypertensive gastropathy into three stages including non-specific redness, specific mosaic pattern and red spots. Gastric varices may be classified according to form, location and mucosal lesions. Prospective trials are needed to obtain more practical evidence for the accurate classification of these gastric lesions. PMID- 7548816 TI - Clinical associations between thyroid and liver diseases. AB - The liver has an important role in thyroid hormone metabolism and the level of thyroid hormones is also important to normal hepatic function and bilirubin metabolism. Besides the associations between thyroid and liver diseases of an autoimmune nature, such as that between primary biliary cirrhosis and hypothyroidism, thyroid diseases are frequently associated with liver injuries or biochemical test abnormalities. For example, thyroid diseases may be associated with elevation of alanine aminotransferase and alkaline phosphatase, which is mainly of bone origin, in hyperthyroidism and aspartate aminotransferase in hypothyroidism. Liver diseases are also frequently associated with thyroid test abnormalities or dysfunctions, particularly elevation of thyroxine-binding globulin and thyroxine. Hepatitis C virus infection has been connected with thyroid abnormalities. In addition, antithyroid drug therapy may result in hepatitis, cholestasis or transient subclinical hepatotoxicity, whereas interferon (IFN) therapy in liver diseases may also induce thyroid dysfunctions. These thyroid-liver associations may cause diagnostic confusions. Neglect of these facts may result in over of under diagnosis of associated liver or thyroid diseases and thereby cause errors in patient care. It is suggested to measure free thyroxine (FT4) and thyroid-stimulating hormone (TSH) which are usually normal in euthyroid patients with liver disease, to rule out or rule in coexistent thyroid dysfunctions, and consider the possibility of thyroid dysfunctions in any patients with unexplained liver biochemical test abnormalities. It is also advisable to monitor patients with autoimmune liver disease or those receiving IFN therapy for the development of thyroid dysfunctions, and patients receiving antithyroid therapy for the development of hepatic injuries. PMID- 7548818 TI - Budd-Chiari syndrome due to pacemaker-induced thrombosis. AB - We report a unique case of Budd-Chiari syndrome caused by pacemaker leads-induced thrombosis. A 34 year old female patient was subjected to a permanent pacemaker insertion because of refractory paroxysmal supraventricular tachycardia attacks related to Wolff-Parkinson-White syndrome. Three years later, another pacemaker was re-implanted because of its dislodgement. Four episodes of skin infections at the implantation site were noted thereafter. The patient developed symptoms of abdominal pain and ascites 5 years after the second pacemaker implantation. Ultrasonography and computerized tomography of the abdomen revealed hepatomegaly with ascites and dilated inferior vena cava. An echocardiogram displayed thrombus formation in the superior vena cava, the right atrium and the inlet of the inferior vena cava into the right atrium. Inferior and superior venacavogram confirmed the above findings. With the impression that Budd-Chiari syndrome was caused by pacemaker-induced thrombus, we removed the pacemaker first and thoracotomy with thrombectomy was then performed. The clinical symptoms resolved after the operation. To our knowledge, this is the first case reported in the literature and this observation supported the thrombosis theory for membranous obstruction of inferior vena cava. PMID- 7548817 TI - A long-term survivor of ruptured hepatocellular carcinoma after hepatic resection. AB - We treated a patient who had previously undergone a hepatic resection for ruptured hepatocellular carcinoma (HCC) but developed a solitary peritoneal recurrence at the site of the incision 8 years and 9 months later. Since no other recurrence was evident, we resected the tumour. The primary tumour was 2.5 cm in size and histological examination revealed HCC without any histological risk factors for intrahepatic recurrence. The peritoneal tumour consisted of less differentiated cancer cells than those found in the primary tumour. The positive rates of Ki-67 were 10% in the primary tumour and 23.3% in the peritoneal recurrence. The DNA indexes in both tumours were considered to be identical. The comparison between the primary and peritoneal tumours suggested that the histological differentiation and proliferation activity can change after recurrence, in spite of consistent DNA ploidy contents. Clinically, a patient who undergoes a hepatic resection for ruptured HCC can survive for a long time, such as 10 years, if they have good liver function and small HCC without any histological risk factors for intrahepatic recurrence. However, since late recurrence is possible, a follow up for as long as 10 years is recommended. PMID- 7548819 TI - Management of symptomatic polycystic liver disease: laparoscopy adjuvant with alcohol sclerotherapy. AB - An 81 year old woman with extensive polycystic liver disease received laparoscopic unroofing and fenestration, followed by 95% ethanol sclerotherapy, resulting in almost complete disappearance of the cysts. Such a procedure can avoid complications of both the multi-segmental liver resections and extensive fenestrations, and untoward reactions from a large amount of alcohol. In addition, it can achieve a satisfactory result. It is suggested that this procedure could be a safe and effective alternative to traditional open surgery in selective patients. PMID- 7548821 TI - Expression of nodulin genes in plant-determined ineffective nodules of pea. AB - The mutant E135 (sym 13) of pea (Pisum sativum L.) forms a normal number of small white nodules that contain bacteroids, but these bacteroids lack nitrogenase activity. To evaluate the effects of the sym 13 gene on the expression of nodulin genes, cDNA clones for nodulins were isolated from pea nodules and the expression of nodulin genes in ineffective E135 nodules was compared with that in nitrogen fixing nodules on the wild-type parent, cv. Sparkle. Nineteen cDNA clones for nodulins, including ENOD2 and cDNAs for two distinct leghemoglobins (Lbs), were isolated from Sparkle nodules by a subtractive hybridization procedure. All the nodulin genes examined were expressed in nodules on both E135 and Sparkle plants. However, the level of expression of seven genes, one of which was an Lb gene that corresponded to PsN5, was significantly lower in E135 nodules. The levels of Lb apo-proteins, with the exception of Lb-III and Lb-IV, in E135 nodules resembled those in Sparkle nodules, but the level of heme in E135 nodules was lower than that in Sparkle nodules. Although the expression of the two Lb genes that corresponded to PsN5 and PsN120 in E135 nodules was slightly depressed by exogenous ammonia, the level of the PsN5 transcript was still lower than the control level in Sparkle nodules. Our results indicate that the plant gene sym 13 does not influence the induction of nodulin genes but does influence the level of the expression of some genes, one of which is a gene for Lb, as well as the level of heme. PMID- 7548820 TI - Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements. AB - Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles. PMID- 7548823 TI - A salt- and dehydration-inducible pea gene, Cyp15a, encodes a cell-wall protein with sequence similarity to cysteine proteases. AB - The pea (Pisum sativum) gene Cyp15a encodes a protein with sequence similarity to cysteine proteases. Expression of Cyp15a was investigated during pea seedling development and in response to environmental stress. Cyp15a shows increased transcription and elevated mRNA levels in plant tissues that are partially dehydrated or treated with 0.6 M mannitol. Cyp15a mRNA levels also increase in seedlings treated with 0.2-0.25 M NaCl or KCl. During development, Cyp15a mRNA levels increase within 6 to 12 h in cotyledons and axes during germination and continue to increase for at least 96 h. Illumination of dark-grown seedlings increased Cyp15a mRNA abundance in elongating and non-elongating stem tissues. GA and ABA, which modulate the abundance of many seed-localized cysteine proteases, did not significantly modulate Cyp15a mRNA levels in stems. The protein encoded by Cyp15a contains a typical amino-terminal secretory targeting domain. This domain is followed by a pro-sequence containing ca. 110 amino acids that is found in other cysteine proteases. Polyclonal antibodies, directed against CYP15a, recognized both the larger pro-form and the cleaved mature form of CYP15a on western blots. Immunolocalization assays indicated that both forms of the protein are located in cell walls of stem cortical cells. PMID- 7548824 TI - Intron length variation of the Adh gene in Brachyscome (Asteraceae). AB - Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5' three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3' end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon. PMID- 7548825 TI - Specificity in the immobilisation of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.). AB - A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H2O2 and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed. PMID- 7548826 TI - Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds. AB - Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to includes six putative membrane spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds. PMID- 7548822 TI - Evolution of the Group 1 late embryogenesis abundant (Lea) genes: analysis of the Lea B19 gene family in barley. AB - The highly conserved Group 1 late embryogenesis abundant (Lea) genes are present in the genome of most plants as a gene family. Family members are conserved along the entire coding region, especially within the extremely hydrophilic internal 20 amino acid motif, which may be repeated. Cloning of Lea Group 1 genes from barley resulted in the characterization of four family members named B19.1, B19.1b, B19.3 and B19.4 after the presence of this motif 1, 1, 3 and 4 times in each gene, respectively. We present here the results of comparative and evolutionary analyses of the barley Group 1 Lea gene family (B19). The most important findings resulting from this work are (1) the tandem clustering of B19.3 and B19.4, (2) the spatial conservation of putative regulatory elements between the four B19 gene promoters, (3) the determination of the relative 'age' of the gene family members and (4) the 'chimeric' nature of B19.3 and B19.4, reflecting a cross-over or gene-conversion event in their common ancestor. We also show evidence for the presence of one or two additional expressed B19 genes in the barley genome. Based on our results, we present a model for the evolution of the family in barley, including the 20 amino acid motif. Comparisons of the relatedness between the barley family and all other known Group 1 Lea genes using maximum parsimony (PAUP) analysis provide evidence for the time of divergence between the barley genes containing the internal motif as a single copy and as a repeat. The PAUP analyses also provide evidence for independent duplications of Group 1 genes containing the internal motif as a repeat in both monocots and dicots. PMID- 7548828 TI - VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots. AB - We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II. Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I. Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II-III, while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone. PMID- 7548827 TI - A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression. AB - ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor induced gene expression. PMID- 7548829 TI - Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana. AB - A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron. PMID- 7548831 TI - Characterization of the structure and determination of mRNA levels of the phenylalanine ammonia-lyase gene family from Populus kitakamiensis. AB - We isolated two new PAL genes, palg2b and palg4, from Populus kitakamiensis, palg2a and palg2b are clustered and palg2b encodes a polypeptide of 710 amino acids. The nucleotide sequence in the coding region of palg2b was 94.6% identical to that of palg2a. The promoter regions of palg1, palg2a and palg2b have several elements conserved among many phenylpropanoid biosynthetic genes. We measured the mRNA levels of the four PAL genes by S1 mapping using total RNA from stem tissues developing secondary xylem. Results showed that the transcript level of palg2b was higher than that of the other PAL genes. PMID- 7548830 TI - Activity of the yeast FLP recombinase in Arabidopsis. AB - The coding sequence for FLP recombinase, originally from the 2 mu plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations. PMID- 7548833 TI - Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. AB - We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy. PMID- 7548832 TI - Molecular cloning and expression of mRNAs encoding H1 histone and an H1 histone like sequences in root tips of pea (Psium sativum L.). AB - Two cDNA clones representing mRNAs, highly expressed in pea root tips, were isolated by mRNA differential display. Ribonuclease protection analyses showed different patterns of expression of these two messages in several pea tissues. Sequence analysis showed that the first clone, PsH1b-40, has 100% homology with a previously isolated H1 histone cDNA, PsH1b. However, it has an additional 30 nt at the 3' end which is absent in PsH1b, suggesting possible multiple polyadenylation sites in the same mRNA. The second clone, PsH1b-41, encodes a deduced 19.5 kDa protein of 185 amino acids with an isoelectric point of 11.5. The putative globular domain of the encoded protein showed 67-71% residue identity with globular domains of 28 kDa pea PsH1b H1 histone and Arabidopsis thaliana H1-1 H1 histone. It has 9 repeating motifs of (T/S)XXK. In the C terminal domain, there are four lysine-rich repeating motifs of SXK(T/S)PXKKXK which may be involved in chromatin condensation and decondensation. Southern blot analysis of nuclear DNA shows that PsH1-41 belongs to a multigene family. PMID- 7548834 TI - Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter. AB - A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I footprinted elements (SBF-1 boxes I, II, and III, 5' to 3') in the 5' region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-beta-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position -173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the -326 to -173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position -173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues. PMID- 7548837 TI - Induction of apoptosis by tumor suppressor genes and oncogenes. AB - The p53 tumor suppressor gene product, and the bcr-abl, bcl-2, and c-myc gene products all appear to influence the susceptibility of cells to apoptosis. In addition to the role p53 protein plays in mediating a cell cycle arrest in G1 following DNA damage, p53 also performs functions critical for removal of damaged cells by initiating apoptosis in certain physiological situations. Cells which express deregulated c-myc are sensitized to apoptosis following various growth suppressing stimuli and these observations have provided new insights into how apoptosis-suppressing genes such as mutant p53, bcl-2 and bcr-abl may cooperate during transformation and how they might influence the sensitivity of cells to radiation and chemotherapy. PMID- 7548835 TI - Developmental expression of a turgor-responsive gene that encodes an intrinsic membrane protein. AB - We previously reported that the pea (Pisum sativum) gene, Trg31, shows increased transcription and elevated mRNA levels in plant tissues which are dehydrated and lose turgor. The protein encoded by Trg31 is homologous to members of the MIP intrinsic membrane protein superfamily. Expression of Trg31 was characterized during pea seedling development and in transgenic tobacco using Trg31 promoter::Gus fusions. In pea, Trg31 mRNA abundance was highest in roots followed by flowers, stems and leaves. In roots, Trg31 mRNA levels were highest in non elongating regions and low in root tips. In dark-grown seedlings, Trg31 mRNA levels were high in stems and illumination caused mRNA abundance in stems to decrease. Histochemical analysis of transgenic tobacco expressing Trg31 promoter::Gus constructs showed high GUS activity in root to shoot and hypocotyl to cotyledon junctions and cotyledons in germinating seedlings. High activity was also observed in the leaf marginal meristem and trichomes. In more mature seedlings, Trg31 promoter activity was observed in the non-elongating portion of the root and in stems especially in the vascular tissue. A gradient of expression was noted in leaf to stem junction zones with highest expression in the younger tissues. Very high expression was observed in stems of flowers and other floral tissues including the calyx, corolla, style, ovules, pods and pollen. This expression pattern suggests that the Trg31 gene product may facilitate transport from sources, through transmitting tissues to sinks. PMID- 7548836 TI - Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.). AB - A cDNA coding for arginine decarboxylase (ADC, EC 4.1.1.19) has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.). The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively. When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity. The ADC activity expressed in S. cerevisiae was inhibited 99% by alpha DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity. No activity was detected in the untransformed S. cerevisiae, nor when it was transformed with an antisense ADC construct. This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S. cerevisiae is able to process correctly the protein. In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds. Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript. In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion. The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting. PMID- 7548838 TI - Induction of genes during apoptosis: examples from the immune system. AB - The immune system provides many examples of cell populations that are susceptible to the induction of apoptosis. Self-reactive immature cells are deleted by triggering apoptosis. Additionally, mature peripheral lymphocytes are induced to undergo apoptosis, particularly when hyperactivated. In the past few years, several genes have been linked to cell death in lymphoid cells. However, only a handful of these genes has been shown to be required for cell death to occur in the immune system. This review focuses on signals known to mediate apoptosis in the immune system and those genes demonstrated to be required for the induction of cell death. PMID- 7548839 TI - Cell death in health and disease: the biology and regulation of apoptosis. AB - Apoptosis is a morphologically stereotyped form of cell death, prevalent in multicellular organisms, by which single cells are deleted from the midst of living tissues. Recognition of the cellular corpses and their removal by phagocytosis occurs without disturbance to tissue architecture or function and without initiating inflammation. Apoptosis is regulable and is of fundamental importance to tissue development and homeostasis. Cellular susceptibility to apoptosis is determined by a variety of signals, of both extracellular and internal origin, including proliferative status. Dysregulated apoptosis is important in the pathogenesis of several important human diseases including neoplasia, and recognition of the defects involved is prompting development of new therapeutic strategies. PMID- 7548840 TI - The bcl-2 gene family. AB - Viability is a fundamental determinant of all cellular functions, and regulation of cell viability is important in shaping tissue differentiation and organism development. The genes in the bcl-2 family can profoundly influence cell viability. In addition to bcl-2, cellular genes in this family include bcl-x, bax, mcl-1, and A1 (from vertebrates), as well as ced9 (from Caenorhabditis elegans). Different members of the family exhibit a spectrum of activity, ranging from survival-enhancement of death-enhancement. Thus, the combinatorial effects of various bcl-2 family members may allow a fine level of control over the important function of cell viability. In addition, alterations in these genes may cause aberations in cell death and thus contribute to cancer. PMID- 7548842 TI - Implications of apoptotic cell death regulation in cancer therapy. AB - The process of programmed cell death, or apoptosis, is now recognized as an important component of multistep carcinogenesis. Traditionally, interventions used in the treatment of cancer patients were predicated on enhanced rates of proliferation in the tumor cells. It is now appreciated that tumor growth may, in many circumstances, be more a function of reduced rates of cell death rather than enhanced proliferation per se. Our understanding of the molecular regulation of apoptotic cell death is leading to the design and implementation of treatment strategies based on cell death induction. PMID- 7548843 TI - Elements of chromosome structure and function in fission yeast. AB - The investigation of fission yeast chromosome structure and function has moved rapidly over the past 10 years. The isolation of replication origins, telomeres and centromeres has allowed the development of minichromosomes, a yeast artificial chromosome (YAC)-like cloning system and investigations into chromosome segregation and behaviour during mitosis and meiosis. Many mutants have been isolated which are defective in chromosome segregation. The development of the fluorescent in-situ hybridization (FISH) technique for use in S. pombe has allowed the localization of centromeres and telomeres throughout mitosis and meiosis. In combination with indirect immunofluorescence to detect spindle and chromosomal proteins, the FISH technique should further advance our understanding of fission yeast chromosome structure and function. The recent discovery of a heterochromatin-like structure mediating transcriptional repression at centromeres reinforces the notion that fission yeast centromeres are similar to those of larger eukaryotes. Further characterization of such phenomena will accelerate the genetic dissection of this important chromosomal element. PMID- 7548844 TI - DNA structure checkpoints in fission yeast. AB - A DNA structure checkpoint can be defined as any checkpoint which responds to changes in the structure of the DNA either through the cell cycle, or in response to outside events such as DNA damage. Genetic analysis of DNA structure checkpoints in fission yeast has identified several distinct pathways responding to different circumstances. Three checkpoints have been identified which inhibit the onset of mitosis. (1) A radiation checkpoint which prevents mitosis after DNA damage. (2) A checkpoint linking S phase and mitosis (the S-M checkpoint) that prevents mitosis when DNA synthesis is incomplete. (3) A checkpoint linking G1 to mitosis (the G1-M checkpoint) that prevents the onset of mitosis in cells which are arrested in the G1 period of the cycle. A large number of genetic loci that are required for these checkpoints have been identified through mutant analysis, and the involvement of the relevant genes with the individual checkpoint pathways has been investigated. The largest class of checkpoint genes, known as the 'checkpoint rad' genes, are required for all the DNA structure checkpoints and the evidence suggests that they may also be involved in regulating DNA synthesis following precursor deprivation (hydroxyurea treatment) or when the replication fork encounters DNA damage. In this review, the available genetic and physiological evidence has been interpreted to suggest a close association between the 'checkpoint rad' class of gene products and the DNA-protein complexes that regulate and perform DNA synthesis. Biochemical evidence will be required in order to prove or disprove this hypothesis. PMID- 7548841 TI - Survival factors, intracellular signal transduction, and the activation of endonucleases in apoptosis. AB - Cell survival is normally mediated by factors in the extracellular environment, whereas genetic changes that constitutively activate intracellular survival pathways often occur in cancer. It is suggested that a Ras/Raf/MAP kinase dependent pathway is critical for cell survival. Apoptosis results from loss of these survival factors or deregulation of survival pathways. If protein kinase cascades mediate survival, then it is likely that phosphatases mediate apoptosis. Potential targets for dephosphorylation include regulators of ion homeostasis as these have been implicated in the regulation of endonucleases associated with apoptosis. Survival factors also modulate anticancer drug response and understanding these pathways may improve therapy. PMID- 7548845 TI - Cyclins of the fission yeast Schizosaccharomyces pombe. AB - Five cyclin-like genes, cig1, cig2/cyc17, mcs2, puc1 and cdc13, have been discovered in S. pombe to date. It is not yet clear what their functions are or even whether they are all involved with control of the cell cycle. Conflicting data for cig1 and cig2/cyc17 have obscured analysis of their function and cig1 remains largely uncharacterized, although clues to the role of cig2/cyc17 have emerged. There is genetic data available for the more distant cyclin homologue mcs2, which has an essential although as yet unspecified role. Puc1 may be involved in regulation of exit from the cell cycle. The first cyclin to be discovered, and the best understood, is cdc13 which with cdc2 promotes mitosis. Studies of the roles of cdc2 and cdc13 in the overall ordering of the cell cycle suggest that cdc13 and probably other cyclins are key regulators, maintaining the order of S phase and mitosis during the cell cycle. PMID- 7548846 TI - The control of septum formation and cytokinesis in fission yeast. AB - Our understanding of the control of cytokinesis is limited in comparison with our knowledge of the controls over the initiation of S phase or mitosis. Study of genetically tractable systems such as Schizosaccharomyces pombe are a useful way to address this problem, since mutants defective in regulation of cytokinesis have been identified. Cloning and analysis of the proteins they encode has begun to shed light upon how formation of the division septum is initiated and directed to the correct place in the cell. Some of these mutants may also be implicated in coordinating mitosis and cytokinesis. PMID- 7548850 TI - Cellular and molecular characterization of transdifferentiation in the process of morphallaxis of budding tunicates. AB - In the budding tunicate, Polyandrocarpa misakiensis, a bud consists of two epithelial sheets, of which the inner, atrial epithelium shows developmental multipotency. It contains pigment granules in the cytoplasm and expresses a few differentiation markers on the cell surface. During bud development, these features are lost and new differentiation markers appear in organ rudiments that arise from the atrial epithelium. This transdifferentiation of the multipotent epithelium requires at least one cycle of cell division. It may be triggered by endogenous retinoids, probably retinoic acid (RA). RA acts on mesenchymal cells, which then secrete proteases that would serve as an actual transdifferentiation factor of the atrial epithelium. PMID- 7548849 TI - Transdifferentiation of isolated striated muscle of jellyfish in vitro: the initiation process. AB - Fragments of striated muscle tissue of Anthomedusae can be isolated and cultured. Without further treatment the isolated muscle fragments maintain the differentiated state. When treated with enzymes degrading the adhering extracellular matrix, drugs activating protein kinase C or substances destroying the actin cytoskeleton, dedifferentiation and DNA replication are initiated and transdifferentiation to several new cell types occurs. Initiation of DNA replication seems to be correlated with a disturbance of cell-ECM interactions. If muscle fragments are combined with isolated ECMs, cell migration onto the grafted ECMs occurs and DNA-replication and transdifferentiation are initiated in those cells which adhere to both, the native and the grafted ECM. If, however, the cells can stretch into a monolayer and adhere entirely to either the native or the grafted ECM, DNA-replication is inhibited. Carbohydrate moieties seem to be involved in mediating these cell-substrate interactions. PMID- 7548848 TI - Pheromone communication in the fission yeast Schizosaccharomyces pombe. AB - Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation. PMID- 7548847 TI - Control of signal transduction and morphogenesis by Ras. AB - The single Ras homologue (Ras1) of S. pombe regulates two distinct processes: (1) Signal transduction through a MAP kinase-like protein kinase cascade in response to mating pheromones. In this pathway Ras1 interacts with the protein kinase Byr2 and leads to its activation in conjunction with a signal from the receptor coupled, heterotrimeric G protein. (2) Polarized cell growth both during the cell cycle and during directed cell extension towards a mating partner. Ras1 interacts with Ral1/Scd1, a putative guanine-nucleotide-exchange factor, which could activate Cdc42, Rho-like GTP-binding protein. Cdc42 may regulate the dynamics of the actin cytoskeleton. PMID- 7548851 TI - Transdifferentiation as a basis for amphibian limb regeneration. AB - Limb regeneration is a phenomenon occurring only in some urodeles. The process seems to be initiated by the dedifferentiation of the terminally differentiated cells. These cells differentiate, subsequently, to the tissues that comprise the limb, thus reconstructing the pattern of the missing limb part. In this paper we review and present evidence that certain cell types of the limb have the capacity to differentiate to different cell types than their original one by cellular metaplasia. This switch is called transdifferentiation. The focus of this review is the process of dedifferentiation which is the necessary prerequisite for differentiation, and the possible mechanisms involved. PMID- 7548852 TI - Transdifferentiation and retinal regeneration. AB - The neural retina of amphibians and chick embryos regenerates following damage. Retinal regeneration requires a change in the differentiated state of the cells of the pigmented epithelium of the retina to a neural progenitor phenotype. The molecular mechanisms that control the cell fate decision between these two very different cell types involves soluble growth factors of the fibroblast growth factor family, as well as components of the extracellular matrix. Recent experiments have also begun to detail differences in gene expression between the neural retina and the pigment epithelium that may be critical for their phenotypic distinction. PMID- 7548853 TI - From lens regeneration in the newt to in-vitro transdifferentiation of vertebrate pigmented epithelial cells. AB - Through studies to clarify the cellular origin of lens regeneration in the newt, the pigmented epithelial cells of the iris and the retina of many vertebrate species have been shown to possess a dormant potency to transdifferentiate into the lens. The method of in-vitro culture of pigmented epithelial cells has been optimized to enable detailed studies of the transdifferentiation process by molecular techniques. Growth factors and extracellular matrix components are found to be important in the control of the transdifferentiation process. New systems for in-vitro culture are introduced, while prospects for renewed in-vivo studies using newts are given. PMID- 7548854 TI - Hepatic transdifferentiation in the pancreas. AB - The differentiated state of specialized cells appears to be dependent on interactions between the extracellular microenvironment, cytoplasmic signals and DNA. Perturbations in these interactions lead to phenotypic alterations of the cell--referred to as transdifferentiation. Copper deficiency in rats leads to global acinar cell loss due to apoptosis possibly leading to perturbations in cell-cell interactions and the microenvironment. Acinar cell loss is associated with the proliferation of ductular epithelial and oval cells. Massive depletion of the acinar cell pool creates severe expansion pressure on oval and ductular cells to fill the vacuity. This probably causes a change in the commitment of these cells resulting in transdifferentiation into hepatocytes. Pancreatic hepatocytes exhibit all the morphological and functional properties of liver parenchymal cells. PMID- 7548855 TI - Transdifferentiation induced by gene transfer. AB - While for many tissues the differentiation process is well characterized, little is known about 'master switch' genes determining a specific differentiation pathway and having the potential to induce this process in a cell designed for a different differentiation pathway. Based on heterokaryon and 5-aza-cytidine induced hypomethylation experiments, the muscle determination gene MyoD1 was identified and isolated, which was shown to induce myogenic differentiation even in cells of ectodermal lineage. Since transdifferentiation studies could also be performed in drosophila in vivo by 'false' expression of developmental genes, it is tempting to speculate that experimentally induced transdifferentiation mimics processes during embryonic development and tissue maturation. PMID- 7548856 TI - Regulation of differentiation pathway of skeletal mesenchymal cells in cell lines by transforming growth factor-beta superfamily. AB - The skeletal mesenchymal cells including osteoblasts, chondrocytes, muscle cells and adipocytes are believed to be derived from the common mesenchymal progenitors. Using various cell lines, multipotential mesenchymal progenitors capable of differentiating into these cell lineages have been demonstrated to be present in the skeletal tissues. The developmental potential of the progenitors becomes more restricted according to their maturation stages in the specialized cell lineages. Among the various growth factors, bone morphogenetic protein (BMP), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, plays an important role in the regulation of the differentiation pathway of these mesenchymal cells. BMP-2 induces the undifferentiated mesenchymal progenitors to differentiate into osteoblasts, chondrocytes and adipocytes. BMP-2 also stimulates the committed osteo-chondroprogenitors to differentiate into more mature cell types, but inhibits myogenic cells to differentiate into myotubes. BMP-2 converts the differentiation pathway of C2C12 myoblasts into the osteogenic lineage associating with decreases in the expression levels of the muscle specific regulatory factors such as myogenin and MyoD. TGF-beta inhibits myotube formation in myogenic cell lines, but does not stimulate osteoblast differentiation. The TGF-beta superfamily plays important roles in the differentiation process of the skeletal mesenchymal cells. PMID- 7548857 TI - Synovial fluid lipids and apolipoproteins: a contemporary perspective. AB - Normal human synovial fluid contains extremely low concentrations of lipoproteins and apolipoproteins, in sharp contrast to those found in plasma. Increased amounts of cholesterol and other lipids have been found in the synovial fluid of a chronic inflammatory joint disorder, rheumatoid arthritis (RA). More recently, apolipoproteins AI, B and E have also been found in increased amounts in RA synovial fluid. Theories have been proposed to account for this increase in the amount of apolipoproteins and for the source of lipids and lipoproteins in normal synovial fluid; however, the mechanisms have not yet been established. Lipoproteins may play dual roles in synovial fluid: A functional one in normal synovial fluid and, as some suggest, a pathologic one in the abnormal synovial fluid of certain arthritic diseases. The recent data prompt the need to define synovial fluid lipids, lipoprotein particle subfractions and their constituent apolipoproteins, as well as their respective roles in synovial fluid. PMID- 7548858 TI - Flow conductivity of rat dermis is determined by hydration. AB - The flow rate of phosphate buffered saline through dermis was measured as a function of applied pressure. Hyaluronan and collagen, the two principal materials which confirm resistance to flow in dermis, were not lost from the tissue during the experiments which lasted up to two days. From Darcy's Law, the average flow conductivities were 2 to 6 x 10(-12) cm4/dyn x s and decreased with increasing applied pressure. We conclude that the tissue is compacted in proportion to the applied pressure during the flow experiments. The hydraulic flow conductivity is described mathematically as a function of compaction induced by the applied pressure. PMID- 7548859 TI - The pressure-flow relation in resting rat skeletal muscle perfused with pure erythrocyte suspensions. AB - In spite of numerous investigations of erythrocyte rheology, there is limited information about the influence of erythrocyte suspensions on whole organ pressure-flow relationships. In this study, we present whole organ pressure-flow curves for resting vasodilated gracilis muscle of the rat, in which the microanatomy and vessel properties have been determined previously. For pure erythrocyte suspensions from donor rats, the organ resistance increases only mildly with perfusion time (less than a 5% shift over a one-hour perfusion time), while in contrast, erythrocyte suspensions containing leukocytes show an increases of resistance near 100% over a period of 25 min. Variation in pressure flow curves in the muscle at the same arterial hematocrit between different rats is less than 15%. The pressure-flow relation for pure erythrocyte suspensions depends on hematocrit. Shear thinning is exhibited at high hematocrits, while Newtonian behavior is approached at arterial hematocrits below 15%. The whole organ apparent viscosity for pure erythrocyte suspensions (normalized by cell free plasma resistance) is a non-linear function of hematocrit; at physiological pressures, it reaches values comparable to those of apparent viscosities measured in rotational viscometers or in in vitro tube flow (diameters greater than 0.8 mm). The apparent viscosities estimated from the whole organ experiments tend to be higher than those measured in straight tubes under in vitro conditions. The pressure-flow curves for pure erythrocyte suspensions are shifted towards lower pressures than the curves for mixed suspensions of erythrocytes at the same hematocrit and with leukocytes at physiological cell counts. These acute experiments show that pure erythrocyte suspensions yield highly reproducible resistances in the skeletal muscle microcirculation with dilated arterioles. Relative apparent viscosities measured in vivo are higher than those measured in straight glass tubes of comparable dimesions. PMID- 7548860 TI - An LDA and flow visualization study of pulsatile flow in an aortic bifurcation model. AB - The structure of pulsatile flow in a rigid aortic bifurcation model was studied by means of a flow visualization technique and three-dimensional laser-Doppler anemometry. The model was made of glass, having the same shape as that of the average human aortic bifurcation. It was installed into a mock circulatory loop that generated physiological pulsatile flow. Flow separation was observed during accelerated and decelerated flow periods. Double helical flow existed inside the flow separation in the early accelerated flow period. In the decelerated flow period, disturbed flow appeared behind the separation zone. Flow was strongly disturbed during the back flow period, and then was gradually stabilized in the forward flow period. The flow separation and the disturbances released from the flow separation zone greatly influenced near-wall velocities along the lateral wall. The wave form of the near-wall velocity in the flow separation zone was much different from that observed in the aortic portion and behind the separation zone; for example, the magnitude of the negative peak velocity in the direction of the tube axis was larger than that of the positive one, and mean velocity in a cycle was very low. This abnormal phasic change of the near-wall velocity may be associated with atherogenesis. The three-dimensional velocity measurement is very useful for the detailed analysis of near-wall velocity patterns. PMID- 7548861 TI - Flow patterns in three-dimensional left ventricular systolic and diastolic flows determined from computational fluid dynamics. AB - A realistic model of the left ventricle of the heart was previously constructed, using a cast from a dog heart which was in diastole. Previous studies of the three-dimensional heart model were conducted in systole only. The purpose of this investigation was to extend the model to both systole and diastole, and to determine what the effect of a previous cardiac cycle was on the next cardiac cycle. The 25.8 cc ventricular volume was reduced by 40% in 0.25 seconds, then increased to the original volume in another 0.25 seconds and then allowed to rest for 0.25 seconds. Runs done with an ejection fraction of 60% showed little variation from one cardiac cycle to another after the third cardiac cycle was completed; the maximum velocity could vary by over 30% between the first and second cardiac cycles. In systole, centerline and cross-sectional velocity vectors greatly increased in magnitude at the aortic outlet. Most of the pressure drop occurred in the top 15% of the heart. The diastolic phase showed complex vortex formation not seen in the systolic contractions; these complex vortices could account for experimentally observed turbulent blood flow fluctuations in the aorta. PMID- 7548862 TI - Flow cytometric studies of platelet responses to shear stress in whole blood. AB - The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates ("large" refers to particles larger than 10 microns in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods. PMID- 7548863 TI - [Large equipment: precedent for autonomy?]. PMID- 7548864 TI - Radiographic evaluation of developing instability of the Mecron cementless, threaded acetabular prostheses. AB - PURPOSE: To evaluate radiographically the history of the Mecron acetabular prosthesis. MATERIAL AND METHODS: The pelvic radiographs of 350 consecutive patients with a smooth threaded acetabular prosthesis type Mecron were retrospectively evaluated (follow-up 1 to 6 years, mean 4 years). A demarcation zone seen at the medial side of the prosthesis was graded from 0 (absent) to 3 (severe). Prostheses with grades 0 and 1 were considered stable and those with grade 3 with or without migration were presumed to be unstable. RESULTS: At five years, 62 (62%) of the cups were unstable and only 23 (23%) were stable. The rate of migration was unacceptably high, and seen in 43 (43%) patients. Migration was only seen in prosthesis with a grade 3 demarcation. CONCLUSIONS: Demarcation visible on radiographs is an important sign of instability of the prosthesis. With the grading system it is possible to evaluate radiographically the history of the prosthesis. PMID- 7548865 TI - [Quantification of lymph node perfusion using color duplex ultrasonography]. AB - PURPOSE: 20 parameters were defined to differentiate benign from malignant lymphadenopathy using computer assisted analysis of the lymph node perfusion demonstrated by colour Doppler flow imaging. MATERIAL AND METHODS: In 112 lymph nodes a computer assisted analysis of 10 consecutive colour Doppler flow sonograms was performed after digital data transfer to a work station. Planimetric results, the colour coded Doppler shift frequency, changes of the perfusion during the heart cycle and the perfusion in the adjacent tissue were analysed using 20 parameters. RESULTS: Discriminant analysis resulted in 7 parameters which reclassified correctly 82% of the lymph nodes (sensitivity 84%, specificity 80%). 72% of the lymph nodes could be classified in the three groups reactive lymphadenopathy, lymph node metastasis and malignant lymphoma. CONCLUSIONS: Clinically known subjective signs of malignancy (increased size, low echogenicity, alterations of the perfusion) can be analysed quantitatively to differentiate benign from malignant lymphadenopathy. PMID- 7548866 TI - [Value of spiral CT in the diagnosis of nasolacrimal duct stenosis]. AB - PURPOSE: This study was to evaluate postdacryocystographic spiral CT in the diagnosis of stenoses of the nasolacrimal duct system. PATIENTS AND METHODS: Dacryocystography and postdacryocystographic spiral CT were performed in 11 patients with epiphora. RESULTS: Postdacryocystographic spiral CT results in detailed information about the nasolacrimal duct system. Localization, degree, and aetiology of stenoses can be determined more reliably than with dacryocystography alone. CONCLUSION: Postdacryocystographic spiral CT improves planning surgical or radiological interventions such as balloon dacryocystoplasty of the nasolacrimal duct. PMID- 7548867 TI - [CT angiography in the follow-up after carotid endarterectomy]. AB - PURPOSE: To prove the usefulness and clinical value of early CT angiography with maximum intensity projection after carotid endarterectomy: for demonstrating postoperative results. METHODS: In 16 patients with angiographically confirmed 20 high grade stenoses of internal carotid artery CT angiography was performed before and after carotid surgery within 7 days. The evaluation of successful surgery was related to complete plaque removal and patency of internal carotid artery after endarterectomy based on preoperative CT angiography. Classification of stenosis was performed according to the NASCET study. RESULTS: In 2/20 cases severe stenoses remained after surgery, which were demonstrated using CT angiography and confirmed by digital subtraction arteriography. 3/20 cases showed mild stenosis and 15/20 were normal. CONCLUSION: CT angiography is best suited to evaluate results after carotid surgery. The method enables delineation of complete removal of calcified plaque and patency of carotid artery. PMID- 7548868 TI - [Focal and diffuse lesions in dynamic MR-mammography of healthy probands]. AB - PURPOSE: To evaluate the range of normal contrast enhancement behaviour in dynamic MR mammography of healthy breast parenchyma, and to reduce false-positive results caused by incidental contrast enhancing lesions. MATERIALS AND METHODS: We performed a prospective two-armed study on 20 healthy premenopausal volunteers who were examined four times within one month (Group 1) or during four consecutive months (Group 2). Incidence, prevalence, morphology, and contrast enhancement pattern of lesions were evaluated. RESULTS: 82% (9/11 of Group 1) and 78% (7/9 of Group 2) showed at least one of a total of 60 contrast enhancing lesions. In 54% or 33% (Group 1 or 2), lesions with enhancement velocity beyond the malignancy threshold were found. 72% or 75% of lesions resolved completely during follow-ups. 69% of lesions were focal with irregular border. Time/signal intensity diagrams were almost always not suspicious; no wash-out phenomenon was seen. CONCLUSION: In MR mammography of premenopausal breast parenchyma, focal contrast enhancing lesions-even with enhancement beyond the malignancy threshold may occur without any underlying pathology. PMID- 7548869 TI - [Rupture of lateral ligaments of the ankle joint: MR imaging before and after functional therapy]. AB - PURPOSE: Documentation via MRI of the healing of ruptured lateral collateral ankle ligaments after functional therapy. PATIENTS AND METHODS: 35 patients with ankle sprain were examined by MRI and stress radiographs, 13 were operated afterwards, 22 patients underwent a functional conservative therapy and were examined by MRI and stress radiographs and second time after three months. RESULTS: MRI reports were correct in 12 of 13 operated cases. After conservative therapy we did not find any disrupted ankle ligament. MRI showed intact ligaments thickened by scar. CONCLUSION: MRI is able to show injuries of the lateral collateral ankle ligaments and demonstrates the healing by scar after conservative therapy. PMID- 7548870 TI - [1H-MR spectroscopic imaging in patients with clinically diagnosed Alzheimer's disease]. AB - PURPOSE: To detect regional differences in accompanying metabolic changes, 1H Magnetic Resonance Spectroscopic Imaging (MRSI) was performed in 16 patients with Alzheimer's disease (AD); the clinical diagnosis was based upon DSM-III-R and NINCDS-ADRDA guidelines. METHODS: In the hippocampal region metabolic maps of the local distribution of N-acetylaspartate (NAA), choline (Cho), creatine compounds (P(Cr)) and lactate were determined. Ratios of Cho/NAA, (P)Cr/NAA and Cho/(P)Cr calculated from selected hippocampal spectra were compared to those from healthy volunteers (n = 17). RESULTS: AD patients demonstrated an increase of Cho/NAA and (P)Cr/NAA ratios caused by increased choline compounds and decreased NAA. These alterations were observed in 11/12 cases in the hippocampal and in 7/12 in the temporo-occipital region. Hippocampal Cho/NAA ratios (0.56 +/- 0.19) were significantly elevated compared with controls (0.33 +/- 0.04; p < 0.0001). CONCLUSION: The observed elevation of choline compounds in the hippocampus supports the hypothesis that alterations in the cholinergic system play an important role in Alzheimer's disease. The observed reduction of NAA is due to neuronal degeneration. PMID- 7548871 TI - [Magnetic resonance tomography and localized proton spectroscopy in 2 siblings with Canavan's disease]. AB - We present the findings of magnetic resonance imaging (MRI) and localised 1H magnetic resonance spectroscopy (MRS) in two brothers with Canavan's disease, a rare autosomal recessive leukodystrophy. Urine specimens of one child were evaluated by MRS. All examinations were performed in the same whole body 1.5 T superconducting magnet. MRI revealed the typical pattern of leukodystrophy including a more severe demyelination in the older child. The younger brother showed additional high signal lesions in the globi pallidi on T2-weighted images. MRS of the brain had an elevated ratio of N-acetyl-aspartate (NAA)/phosphocreatin + creatin (Cr) while the ratio of Cholin/Cr was reduced. in urine spectroscopy the concentration of NAA was markedly increased. The ratio of NAA/creatin + creatinin was 880 +/- 10% mmol/mol (normal: 5-21 mmol/mol). Diagnosis of Canavan's disease was supported by gas chromatographic urine examination with an 80-100 fold elevation of NAA concentration. Hence, the diagnosis of Canavan's disease could be established by increased ratio of NAA/Cr and decreased ratio of Cho/Cr relation in brain spectroscopy and high NAA concentration in urine spectroscopy. PMID- 7548872 TI - [Accuracy of artificial neural networks in radiological differential diagnosis of solitary bone lesions]. AB - PURPOSE: To evaluate the usefulness of artificial neural networks (ANN) for differential diagnosis of bone tumours and tumour-like lesions. MATERIAL AND TECHNIQUE: Different ANN were designed to distinguish between 23 types of bone lesions and to classify these lesions as benign or malignant on the basis of 28 items of clinical and radiographic information. Training of the feed-forward networks with a back-propagation algorithm was performed using either 46 hypothetical examples or 115 real cases. The data base for testing the different ANN included 115 clinical cases (5 cases for each possible diagnosis), which were different from the examples used for training. The decision performance of the ANN was evaluated by means of ROC analysis. Results were compared to the performance of an experienced radiologist. RESULTS: The radiologist was significantly superior in finding the correct diagnosis (69/115 vs. 41/115; p < 0.001) and the area under his ROC curve (referring to the discrimination between benign and malignant lesions) was slightly larger than that of two different ANN (0.973 vs. 0.945 and 0.973 vs. 0.962 resp.; differences not significant). CONCLUSION: ANN may be potentially useful to distinguish between malignant and benign lesions, but their performance in differentiating between 23 diagnoses must be improved significantly. PMID- 7548873 TI - [Malignant neoplasms after kidney transplantation: value of an annual radiological screening program]. AB - PURPOSE: To evaluate the prevalence of malignant neoplasms after renal transplantation by means of a radiological screening programme and to determine the role of some clinical and demographic parameters concerning pathogenesis of these malignancies. MATERIAL AND METHODS: Between November 1992 and June 1994 in a prospective study 504 consecutive renal allograft recipients (331 m, 173 f) aged 51 +/- 13 years underwent routine abdominal ultrasound examinations including the renal transplant and p.a. and lateral chest x-rays once a year. RESULTS: This screening programme revealed 11 malignant neoplasms in 11 patients (2.2%). We detected 6 renal cell carcinomas (RCC) in the patient's native kidneys, two RCCs in two renal allografts, two non-Hodgkin-lymphomas in the liver and the renal allograft, respectively, and one ovarial carcinoma. Patients with renal cell carcinomas in the native kidneys were significantly older than allograft recipients without tumors. The presence of acquired cystic kidney disease (ACKD) seems to be an additional risk for the development of RCC. There were no significant differences in the time on dialysis, the time with functional renal allograft, and the immunosuppressive therapy. CONCLUSION: Yearly abdominal ultrasound screening including the renal allograft is a valuable tool for the early detection of neoplasms in asymptomatic renal allograft recipients. However, routine yearly chest x-rays should not be performed in renal allograft recipients without preexisting tumours. PMID- 7548874 TI - [Radiation exposure during digital hysterosalpingography: how much is due to fluoroscopy?]. AB - PURPOSE: To determine the proportion of radiation exposure from fluoroscopy during the performance of hysterosalpingography, using a digital image intensifier. MATERIAL AND METHOD: 20 women with primary or secondary sterility were examined. The total radiation dose was measured and the proportion due to fluoroscopy was determined. The use of a computer programme permitted accurate differentiation of the dose obtained by fluoroscopy and radiography. Total mean radiation dose was 256 cGy x cm2; fluoroscopy contributed 187 cGy x cm2. On average, fluoroscopy contributed 69% of total radiation dose. CONCLUSION: During hysterosalpingography with a digital image intensifier, fluoroscopy should be as brief as possible since it contributes most of the radiation dose. PMID- 7548876 TI - [Mammographic findings in lipodystrophy]. PMID- 7548875 TI - [Effects of viscosity, cannula size and temperature in mechanical contrast media administration in CT and magnetic resonance tomography]. AB - PURPOSE: Mechanical contrast media application is used to improve image quality and allow better quantitative assessment of the contrast media kinetics for CT as well as MRI. The pressure within the i.v. line system must be known to enable safe and quantitatively exact application. MATERIAL AND METHODS: Experimental setup to analyse the inline pressure, the storage volume as well as the exactness of delivery within the i.v. line system was prepared. Representative contrast media Ultravist 300, Ultravist 370, Magnevist and Conray 60 as well as 0.9% NaCl were measured with ten different flow rates from 0.1 ml/sec to 5.0 ml/sec using the six most common i.v. cannula sizes (22G-14G). RESULTS: System pressures between 38-60 psi (maximum allowed system pressure = 60 psi) are commonly obtained with standard sizes of i.v. cannulas, such as an 18 G cannula and flow rates > or = 2.5 ml/sec using Ultravist 300 (viscosity 13 mPa.s at 20 degrees C). Higher system pressures (> 25 psi) produce an exponential increase of the storage volume within the i.v. line system. The additional storage volume within the i.v. line tubing (d = 2.9 mm, 76 shore) is at 60 psi and 20 degrees C 5 ml/m, and at 37 degrees C 11 ml/m in addition to the volume of the tubing of 7 ml/m. CONCLUSION: Based on the measured data, both the minimum and the recommended sizes of the i.v. cannulas in respect of flow rate and viscosity of the used contrast media were compiled for system pressures at < or = 60 psi as well as < or = 38 psi. PMID- 7548877 TI - Embolisation of a cystic lymphatic malformation of the neck. PMID- 7548878 TI - [Therapy of coronary steal syndrome by percutaneous transluminal angioplasty]. PMID- 7548879 TI - [Mazabraud's syndrome]. PMID- 7548881 TI - [Double-sided or right-sided tracheal bronchus: chance findings in CT]. PMID- 7548880 TI - [CT imaging of chondrosarcomas in the head and neck region]. PMID- 7548882 TI - Pathogenesis aspects of arthrosis in histopathological and electronmicroscopical studies of articular cartilage, correlated to some immunological parameters. AB - Twenty-five biopsies of arthrosic cartilage with radiological correspondence, arthro, sic cartilage under study for Rheumatoid Arthritis (RA) and posttrauma cartilage as control-were examined using histopathological (HE, VG, PAS-Alcian, Gomori, Safranine O) and electronmicroscopical techniques. The arthrosic cartilage with radiological correspondence shows superficial and deep fissures, perichondrocytic gaps and modified reticulino-collagenic network at the histopathological examination. At the level of synovia-cartilage junction, we found some villous aspects of the synovia desquamating in the proximity of the affected cartilage. The investigated arthroses for RA presented some destructive fibrous modifications of the synovia similar to rheumatoid synovitis and associated with some dystrophic chondrocytic alterations. The ultrastructural affection was severe leading to cellular degeneration. The immunologically studied arthroses for RA had seric pathologic values regarding: circulating immune complexes (CIC) (mean = 67.08 +/- 1.45 U), Ig.M(mean = 358 +/- 3.02 UI/ml) and anti collagen antibodies (mean = 409.9 +/- 0.42 U). The synovial depletion of complementary fraction C3(mean = 42.3-1 mg%) as against the normal seric level (mean = 63.07 +/- 0.49 mg%) suggests an immune synovitis. Correlation of immunomorphopathological data emphasize that arthrosis coexists with a secondary synovitis following a primitive degenerative process and allows arthroses under study for RA to be separated from other degenerative rheumatism diseases. PMID- 7548883 TI - Histopathological and ultrastructural modifications of the arthrosis articular cartilage. AB - Thirty samples of articular cartilage taken during the operation from patients with incipient arthrosis, arthrosis with radiological modifications and arthrosis under study for Rheumatoid Arthritis (RA) were investigated using histopathological (HE, VG, PAS-Alcian, Gomori, Safranine O) and electronmicroscopic techniques. The control material was made of posttraumatic cartilage (Moore prosthesis). Histopathologically, the incipient arthrosis cartilage had superficial exfoliations associated with reduced saframinophilic tinctorial perichondrocytic activity. The arthrosic cartilage with typical radiological modifications was individualized at the synovia-cartilage junction by villous aspects of the synovia associated with perichondrocytic gaps, reduction of safraninophilia and modifications of reticuline-collagenic network. The arthrosic cartilage under study for RA revealed destructive fibrous modifications of the synovia and severe affection of the articular cartilage at synovia-cartilage junction. Electronmicroscopically, the ultrastructural affection was minimum in the incipient arthrosis cartilage developing to chondrocytic degeneration in arthrosis with radiological correspondent. Both histopathological and ultrastructural data emphasize the fact that arthrosis is associated with synovitis following a primitive degenerative process similar to rheumathoid synovitis in arthrosis under study for RA. PMID- 7548884 TI - Histoenzymological and ultrastructural studies of the macrophage-type pulmonary cell modifications under different chronic stasis conditions. AB - The optical histoenzymological and electronomicroscopical investigations were carried out en 90 pulmonary biopsies taken from patients under different chronic stasis conditions. To investigate macrophage-type cells we studied the reactions of acid phosphatase and non-specific esterase like enzyme markers, indicating the metabolic state activation degree of this cellular compartment. The ultrastructural examinations revealed the existence of two types of activated pulmonary macrophages. The former is the alveolar macrophage with a prevailing phagolysosome apparatus that pleads for phagocytic function and the latter is the interstitial macrophage with subcellular aspect corresponding to the synthesis and secretion function. The presence of these activated alveolar and interstitial macrophages, as well as their extremely increased number, plead for their direct participation in the appearance and aggravation of the morphophysiopathological disorders in the chronic stasis lungs. PMID- 7548885 TI - Ultrastructural features in two cases of congenital dyserythropoietic anemia type II. AB - Two patients with congenital dyserythropoietic anemia (CDA) type II were investigated in bone marrow as well as in blood cells. The majority of red cell precursors showed typical morphological abnormalities: high incidence of binucleated erythroblasts, normoblastic hyperplasia, nucleo-cytoplasmic dissociations, karyorrhexis of some nuclei, presence of synartesis phenomenon, invagination of cytoplasmic portions into the nuclear area and presence of marginal cisterna. The modified erythroblasts were present in the peripheral blood, too. The presence of Gaucher-like cells suggests an abnormal catabolism of the red cell precursors membranes. PMID- 7548887 TI - Cell death in normo- and teratogenesis. II. Cell death in teratogenesis. AB - The second part of the review presents the main facts and problems with respect to the role of cell death in teratogenesis (pathogenetic cell death). After mentioning the first approaches to the domain, reference is made (in order to avoid repetition) to the synthetic article published by Menkes et al. (1970), as to a series of investigations and results up to that date. Subsequently, more recent contributions are presented, first of all a series of results obtained in the Laboratory of Embryology and Teratology, Timiscara. The genetic background and the morphology of pathogenetic cell death are briefly discussed. As a conclusion, the scheme (published in 1970) of cell death linked teratogenesis is reproduced in a somewhat modified and extended way. PMID- 7548886 TI - Cell death in normo- and teratogenesis. I. Cell death in normogenesis. AB - This first part of the review deals with the main facts and problems related to the role of cell death in normal prenatal development. After an overview of the pioneering work in this field, the essential contributions brought during the last few decades are presented, with special stress upon the contribution brought by the team of the Laboratory of Embryology and Teratology, Timisoara. The morphology of cell death is analysed based upon the classification of Schweichel and Merker (1973). Finally, a detailed account is given on the recent, impressive advance with respect to molecular and molecular-genetic mechanisms underlying and determining the cell death process. PMID- 7548888 TI - Morphological aspects of endocrine cells in human fetal gastrointestinal mucosa. Microscopical, electronmicroscopical and immunohistochemical studies. AB - This paper describes the morphological features and distribution of endocrine cells in the human fetal gastrointestinal mucosa between the 8-9th and the 36th the weeks of gestation. We used techniques of argentic impregnation, techniques with chromogranine and neuron specific enolase, electronmicroscopical techniques and immunohistochemical techniques as well (PAP and ABC) with antibodies for G cells, EG cells, D cells, S cells CCK (I) cells and EC cells. The first argyrophilic and/or argentaffin cells appear in the fetal gut of 8-9 weeks old. We describe the appearance, distribution, morphology of the identified endocrine cells with immunohistochemical techniques and we discuss the possible morphofunctional interrelations between them, but also with the neighbouring structures like lymphoid tissue in ileum, colon and appendix in embryofetal period. PMID- 7548889 TI - Interference of some enzymatic modulators in the hepatic aggression induced by xenobiotics. AB - The aim of the paper was to compare the erythrocyte serum and hepatic chomogenate antioxidative factors in order to assess their involvement in the detoxification events. The catalase and superoxiddismutase levels, important factors of the cellular defence, were sensitivity modulated in an acute experiment on Wistar rats. Carbofuran was administered in a non-lethal dose (7 mg/b.w.) single or in the presence of certain antioxidative agents (Vitamin E, Caffeine, Aspirin) EDTA and Cysteine for their role in protecting membranes against oxidative damage. The erythrocyte parameters (SOD, Catalase) were well related to seric factors, especially ceruloplasmin level, with varied magnitudes. GGT a marker of hepatotoxicity and G1-DH, a mitochondrial marker, were in a good correlation with erythrocyte factors. The changes seem to modulate a transmembranary disturbance process, as in hepatocyte pictures. PMID- 7548890 TI - Ignore high-tech dentistry and perish. PMID- 7548891 TI - A combined porcelain onlay/amalgam restoration for an endodontically treated posterior tooth. AB - This article describes a new method of fabricating an aesthetic restoration for an endodontically treated posterior tooth. The tooth is prepared with minimal reduction of sound tooth tissue. Amalgam is condensed into the orifices of the root canals, the pulp chamber, and the remainder of the cavity. The tooth and the amalgam core are then prepared to receive a porcelain onlay. Additional retention is provided by 1 mm deep holes cut in the amalgam floor of the preparation which is then microetched. The porcelain onlay with its margins remote from the gingiva is bonded with dual-cure composite cement. The learning objective of this article is to familiarize the reader with this new procedure. PMID- 7548892 TI - Implant-protected occlusion. PMID- 7548893 TI - Use of bondable reinforcement fiber for post and core build-up in an endodontically treated tooth: maximizing strength and aesthetics. AB - Post/core placements in endodontically treated teeth are utilized as a means to retain a core or foundation for the final restoration. The question of whether a post placed in an endodontically treated tooth actually increases its strength is still being debated. Prefabricated metal posts have been used in most clinical situations in the past. Due to the reflection of metal through the remaining tooth structure, these posts present some concerns involving aesthetics. To address these concerns, this article presents a method of post fabrication using a bondable reinforcement fiber as the dowel post material. The learning objective of this article is to introduce this new post fabrication technique along with the materials utilized. A clinical case is used to present the technique. PMID- 7548894 TI - Regenerative treatment of periodontal defects utilizing a bioresorbable collagen membrane. AB - Barrier membranes composed of resorbable collagen have demonstrated potential for use in guided tissue regeneration (GTR) procedures for repair and regeneration of periodontal defects. This article reviews the rationale for use of a collagen membrane in GTR therapy and related clinical procedures. Characteristics of the material are outlined, and indications and contraindications for utilization are discussed. Two cases are presented to demonstrate details of these principles and techniques. The learning objective of this article is to familiarize the reader with a new absorbable collagen membrane and its application in the periodontal area. PMID- 7548895 TI - High tech abuse. PMID- 7548897 TI - Is PCR a useful tool for the diagnosis of tuberculosis in 1995? PMID- 7548896 TI - Fabrication of a metal-free ceramic restoration utilizing the monobloc technique. AB - This article presents a new technique which utilizes a porcelain core/crown unit, fabricated in the laboratory as a single component. The monobloc technique was developed by the author to replace the traditional metal post and core which prevents the transmission of light through porcelain crowns, creating a dark color effect. Between 1989 and 1992, a number of cases were successfully treated with several variations of the monobloc technique. Approximately 50 cases were completed with vitro-ceramic and followed up. The learning objective of this article is to introduce this novel technique. The article discusses the development of the technique, its advantages, disadvantages, and the potential failures. The clinical procedure is illustrated with several case presentations. PMID- 7548898 TI - Mycobacteria in HIV-infected patients in Buenos Aires. AB - SETTING: F. J. Muniz Hospital and Department of Phthisiopneumonology, in Buenos Aires. OBJECTIVE: To analyze bacteriological findings concerning tuberculosis and other mycobacteriosis, in association with HIV infection and AIDS. DESIGN: From June 1985 to December 1991, 2521 samples from 1259 HIV-seropositive and AIDS patients were analyzed: 1133 samples were of bronchopulmonary origin and the remaining 1388 of extrapulmonary origin. Drug susceptibility tests were performed using the proportions method. RESULTS: Mycobacterial disease was confirmed by culture in 240 of the 1259 HIV/AIDS patients (19%). Mycobacterium tuberculosis was isolated in 223 of these cases (92.9%) and M. bovis in two, while M. avium complex (MAC) strains were identified as the cause of disease in 14 patients (5.8%). In only one case was disease due to M. kansasii. Blood cultures were positive in 21.2% of these 240 cases. Resistance of M. tuberculosis to antituberculosis drugs was found in 9.4% of the 223 isolates. In only one case was multidrug resistance detected, in a patient who had received previous treatment. CONCLUSION: Smear examination, although less sensitive than in HIV negative patients, was still a simple and reliable tool for the rapid diagnosis of mycobacterial disease. Blood culture aided in the successful diagnosis of about half of the cases of disseminated tuberculosis and of all cases of MAC disease. An alarming spread of tuberculosis was detected among a group of HIV positive prisoners, and the possible emergence of multidrug resistance should be anticipated. PMID- 7548899 TI - Incidence of smear-positive pulmonary tuberculosis from 1981-83 in a rural area under an active health care programme in south India. AB - SETTING: Four villages in the K V Kuppam Block of North Arcot Ambedkar District, Tamil Nadu, south India. OBJECTIVE: To determine the prevalence and incidence of microscopy positive patients with pulmonary tuberculosis. DESIGN: The entire population of 22,847 was covered prospectively; the prevalence survey in 1981 was conducted through house to house interviews followed by collecting and examining sputum specimens by microscopy from symptomatic patients (cough for more than 2 weeks duration or other respiratory or systemic symptoms compatible with tuberculosis). The prevalence survey was followed by monthly visits for two consecutive years to obtain an accurate estimate of the incidence. RESULTS: The prevalence in the population among persons aged 10 years and above was 2.41 per 1000, the average annual incidence during 1981-83 was 1.11 per 1000 or 46% of the prevalence. The male:female ratio in the prevalence survey was 2.6:1, while in the incidence assessment it was 2.4:1. CONCLUSION: The results of the study provide valuable information on the relation between prevalence and incidence of sputum smear-positive cases which should help to better our understanding of the epidemiology of tuberculosis in a disadvantaged part of the world. The reliable estimate of incidence of the microscopy positive cases provided by the study should be very useful for assessing the efficacy of case finding in the District Tuberculosis Programme in India. PMID- 7548900 TI - The increase in tuberculosis notifications in England and Wales since 1987. AB - SETTING: A retrospective study of tuberculosis notifications in England and Wales published by the Office of Population Censuses and Surveys. OBJECTIVE: To analyse the rise in tuberculosis notifications in England and Wales by different sub groups over the time period 1982-91. DESIGN: The average annual percentage change in tuberculosis notification rates (per 100,000) for the groups were analysed by age, sex, standard region and disease site. RESULTS: The average annual increase in tuberculosis notifications from 1987-91 in females was +2.55 (95% confidence interval [CI] 0.5-4.7); males showed no significant change (-0.6%, 95% CI: -2.9 to +2.7). The increase was greater in younger subjects: 3.2% in females aged 25 44 (95% CI 0.9-5.5) and 4.3% in males aged 15-24 (95% CI 1.7-6.9). Only three standard regions showed an increase: the South East, the West Midlands, Yorkshire and Humberside. This increase was significant only in females from the South East, and was predominantly in non-respiratory tuberculosis. CONCLUSION: The rise in tuberculosis notification rates from 1987-91 has been predominantly in females, in those under 65, has occurred in 3 regions, and predominantly in non respiratory tuberculosis. The analysis highlights some of the inadequacies of the present notification system for tuberculosis. PMID- 7548901 TI - Laboratory reports of opportunistic and other mycobacterial infections and their relationship to notifications of tuberculosis in England and Wales. AB - SETTING: England and Wales, UK. OBJECTIVE: To investigate: (1) whether misclassification of opportunistic mycobacterial disease had contributed to the failure of tuberculosis notifications to continue declining; (2) whether laboratory reports of Mycobacterium tuberculosis complex infections could be used to validate trends in the tuberculosis notification system. DESIGN: Descriptive epidemiological study using laboratory reports of infections to the Public Health Laboratory Service Communicable Disease Surveillance Centre, Medical Research Council National Surveys of Tuberculosis and tuberculosis notifications to the Office of Population Censuses and Surveys. RESULTS: Compared to 1983, an extra 1% of tuberculosis notifications in 1988 were opportunistic mycobacterial disease inappropriately notified as tuberculosis. On the basis of the expected proportion of microbiologically confirmed tuberculosis infections, laboratory reporting was incomplete: for one laboratory report there were four notifications. CONCLUSION: Misclassification of opportunistic mycobacterial infection was estimated to account for only a small proportion of the excess tuberculosis notifications since 1988. This excess of tuberculosis notifications could be due to artefact, for instance because a greater proportion of cases are being notified. Laboratory reports of tuberculosis infections are of limited use in validating the recent trends in tuberculosis notification rate. At present changes in the level of reporting of laboratory isolates are likely to obscure genuine trends. PMID- 7548902 TI - Disease due to Mycobacterium kansasii in the Czech Republic: 1984-89. AB - SETTING: Endemic area of North Moravia, Czech Republic. OBJECTIVE: Evaluate the incidence of human disease due to Mycobacterium kansasii. The follow-up of some bacteriological and clinical features. DESIGN: A retrospective analysis of M. kansasii patients. RESULTS: M. kansasii was isolated from the sputum, tissue and other specimens obtained from 650 persons during the period 1984-89. In only 471 of them was this mycobacterium deemed to be the causative agent, predominantly of lung disease. The most typical radiographic finding in these patients was lung infiltration and/or thin-walled cavity. CONCLUSION: As in previous years the highest incidence of disease remains in an endemic area of North Moravia. The effects of treatment in follow-up patients were influenced not only by the antituberculosis regimen but also by a high frequency of associated diseases. Sputum conversion within 30 days was not affected by the presence or absence of a cavity. Authors consider water to be the source of infection. PMID- 7548904 TI - An uncontrolled trial of pefloxacin in the retreatment of patients with pulmonary tuberculosis. AB - OBJECTIVE: To study the role of Pefloxacin in the retreatment of pulmonary tuberculosis. DESIGN: 14 cases of sputum smear-positive tuberculosis were given pefloxacin in doses of 400 mg daily, along with isoniazid and thioacetazone plus additional drugs wherever indicated. They were followed and assessed by sputum smear microscopy and chest X-ray regularly for the total duration, which ranged from 15-18 months. RESULTS: 10 of the patients completed treatment and achieved a satisfactory response in the form of persistent sputum, negativity and radiologically quiescent disease. Two developed serious side-effects and had to discontinue medication. One patient failed on therapy as evidenced by persistent sputum positivity at 6 months. CONCLUSION: Pefloxacin may have a role in the retreatment of pulmonary tuberculosis along with other drugs, as a companion drug in selected cases. The other advantages are the low cost of treatment and high safety profile. PMID- 7548903 TI - A pilot study of antituberculosis combinations comparing rifabutin with rifampicin in the treatment of HIV-1 associated tuberculosis. A single-blind randomized evaluation in Ugandan patients with HIV-1 infection and pulmonary tuberculosis. AB - SETTING: This pilot study was conducted at the Joint Clinical Research Centre (JCRC) in Kampala, Uganda, where tuberculosis (TB) is an epidemic health problem aggravated by the HIV-1 pandemic. OBJECTIVE: To evaluate the feasibility of a larger phase III trial utilizing rifabutin as a substitute for rifampicin in short-course therapy for pulmonary TB. DESIGN: Single-blind randomized trial in 50 patients with new onset smear- and culture-positive pulmonary tuberculosis and HIV-1 infection. Comparison of daily, intermittently supervised 6-month treatment regimens of rifabutin versus rifampicin, together with isoniazid, ethambutol and pyrazinamide. RESULTS: Rifabutin- and rifampicin-containing regimens had comparable efficiency. However, rifabutin-treated patients had significantly more rapid clearance of acid-fast bacilli from sputum at 2 months (P < 0.05, Fisher exact test) and over the entire study period (P < 0.05, logrank test) than rifampicin-treated patients. The presence of cavitary disease was associated with a longer sputum conversion time for patients treated with either regimen. No major adverse events requiring dosage reduction or withdrawal of any study medication were seen in either treatment group. Mean absolute peripheral blood CD4 T lymphocyte counts increased by 28% from week 0 to week 12 in all subjects (334-427/microliters, respectively). An unexpected finding was the isolation of Mycobacterium africanum from 49% of the sputum cultures. This is the first report indicating a high prevalence of M. africanum in human TB in Uganda. CONCLUSION: Short-course antituberculosis regimens containing rifabutin or rifampicin are both safe and efficacious in the treatment of HIV-1 associated tuberculosis. Rifabutin-containing regimens were associated with earlier sputum smear and culture conversion. PMID- 7548905 TI - Rapid changes in thyroid function tests upon treatment of tuberculosis. AB - SETTING: Inpatient service and tuberculosis (TB) clinic of a public hospital. OBJECTIVES: (1) To test the hypothesis that an hepatic effect of antituberculosis drugs increases serum thyroxine-binding globulin (TBG); (2) to resolve conflicting reports on thyroid function in TB. DESIGN: Measurement of serum thyroid hormones, thyroid hormone binding (T3RU) and binding proteins (TBG, transthyretin [TTR] and albumin) in 38 patients with active TB and in 29 healthy tuberculin-positive controls, before and about 10 days into therapy. RESULTS: With therapy of TB (with isoniazid [INH], rifampin [RIF], ethambutol and/or pyrazinamide), TBG increased above control values and T3RU decreased (P < 0.001). These changes were weakly correlated with liver enzyme activities but did not predict clinical hepatitis, which developed in only 1 patient. T3 was initially subnormal in 61% of 38 TB patients, while T4, thyrotropin (TSH) and TBG were normal. T3, TTR and albumin, all negative acute phase reactants, increased towards normal by day 10 (P < 0.001). Thyroid function remained unaltered in 14 control patients taking INH, whereas T3RU decreased (binding increased) and T3 increased in 15 taking INH and RIF (P < 0.001). CONCLUSIONS: TB patients manifest the expected low T3 of non-thyroid illness, but, unlike most sick patients, usually have normal or increased serum binding of thyroid hormones. Chemotherapy further increases binding by increasing TBG, an effect probably due to RIF. PMID- 7548906 TI - Evaluation of serum KL-6 levels in patients with pulmonary tuberculosis. AB - SETTING: KL-6, a human MUC-1 mucin preferentially expressed on type II pneumocytes, is a sensitive serum marker for evaluating alveolar damage of interstitial pneumonia and pulmonary fibrosis. Some patients with pulmonary tuberculosis develop severe respiratory dysfunction caused by extensive pulmonary fibrosis, compensatory emphysema and fibrous pleural thickening. OBJECTIVE: To evaluate the clinico-pathological significance of KL-6 in pulmonary tuberculosis. DESIGN: Serum KL-6 levels were measured in sera from 57 patients with active pulmonary tuberculosis and 38 healthy controls by a sandwich-type enzyme-linked immunosorbent assay. Immunohistochemistry was performed by an avidin-biotin peroxidase complex method. RESULTS: KL-6 levels were significantly higher in the patients than in the healthy controls (518 +/- 693 [SD] vs 227 +/- 91 U/ml, P < 0.001) and increased significantly according to the extent of pulmonary lesions evaluated by chest X-ray (P < 0.001). There was a significant negative correlation between serum KL-6 levels and % vital capacity (VC) (r = 0.642, P < 0.05). KL-6 was strongly expressed on proliferated type II pneumocytes and cuboidal epithelial cells adjacent to thickened intralobular septa and pleura. CONCLUSIONS: In pulmonary tuberculosis, serum KL-6 originates from proliferated type II pneumocytes and cuboidal epithelial cells, and is a useful marker presenting the degree and extent of pulmonary fibroproductive lesions. PMID- 7548908 TI - Comparison of the ability of Mycobacterium avium, M. smegmatis and M. tuberculosis to invade and replicate within HEp-2 epithelial cells. AB - OBJECTIVE: Previous studies have demonstrated that mycobacteria can interact with epithelial cells, a property which can be important for establishing infection. In this study we investigated comparatively the ability of Mycobacterium avium, M. tuberculosis and M. smegmatis to invade and multiply within HEp-2 epithelial cells. DESIGN: The ability to invade and to multiply intracellularly in HEp-2 cells was examined using a virulent strain of M. avium, a virulent (H37Rv) and an attenuated (H37Ra) strain of M. tuberculosis and a strain of M. smegmatis. The locus responsible for M. avium invasion was also cloned in Escherichia coli and M. smegmatis. RESULTS: It was observed that M. avium invaded HEp-2 cells with greater efficiency than M. tuberculosis and M. smegmatis, while the H37Rv strain of M. tuberculosis was more efficient in invading HEp-2 than H37Ra and M. smegmatis. Both M. avium and M. tuberculosis were capable of multiplying within HEp-2 cells, while M. smegmatis was not. E. coli K12 and M. smegmatis were transformed with M. avium DNA. The invasive locus of M. avium provided E. coli K12 and M. smegmatis strains S5M101-1 and S5M101-2 with the ability to invade HEp 2 epithelial cells. Transformed M. smegmatis strains were able to grow intracellularly. CONCLUSION: 'Virulent' strains of M. avium and M. tuberculosis were shown to invade and to multiply within HEp-2 epithelial cells. This property was transferred to E. coli K12 and M. smegmatis by transformation with the invasive locus of M. avium. The ability of certain strains of mycobacteria to invade epithelial cells (bronchial, alveolar, intestinal) may represent an important phenotypic characteristic and could be directly related to pathogenicity. PMID- 7548907 TI - Preliminary evaluation of a Mycobacterium tuberculosis lipooligosaccharide (LOS) antigen in the serological diagnosis of tuberculosis in HIV seropositive and seronegative patients. AB - OBJECTIVE: To assess the value of detection of specific IgG or IgM antibodies against three polar glycolipids of Mycobacterium tuberculosis in HIV seropositive and seronegative patients with documented active tuberculosis. DESIGN: Using an ELISA test, concentrations of IgM and IgG antibodies against phenolic glycolipid Tb1 (PGL Tb1), 2.3 diacyl trehalose (DAT) and polar lipooligosaccharide (LOS) were measured in the serum of 36 patients with active tuberculosis: 16 tuberculosis patients were coinfected with HIV (TB HIV+), and 20 were HIV seronegative (TB HIV-). 104 control sera were tested: they were collected from 50 healthy blood donors and from 54 asymptomatic HIV seropositive patients. Specificity, sensitivity and predictive positive and negative values were calculated. RESULTS: For the LOS antigen tested, sensitivity, specificity and predictive positive value of specific IgG antibodies were 75%, 95% and 84% respectively among the 36 tuberculosis patients. No significant results were obtained either with the IgM against the LOS antigen or with IgG against the DAT and PGL Tb1 antigens. Significant sensitivity and positive predictive values were demonstrated only with the ELISA test measuring specific IgG against the LOS antigen. The sensitivity was greater in the TB HIV+ subgroup (94%) as compared with the TB HIV- (60%) subgroup. These results were not related to different potential factors such as the site of disease (pulmonary or extrapulmonary) or by the acid-fast bacteria load as detected by the smear microscopic (Ziehl-Neelsen) examination. CONCLUSIONS: The results suggest that of the 3 antigens tested, only the LOS antigen is a potential marker for detecting the development of tuberculosis in HIV patients. PMID- 7548909 TI - Humoral immune response in infants after BCG vaccination. AB - SETTING: Child welfare division of a university-based department of pediatrics in Turkey. OBJECTIVE: To determine specific IgM and IgG response after BCG vaccination. DESIGN: Prospective, randomized trial. METHODS: Serum samples of 66 infants were taken before and at 2, 4, 6 and 15 months after vaccination. 35 infants were vaccinated at the first month and 31 at the second month after delivery. An ELISA assay was performed using PPD antigen. RESULTS: IgM antibodies increased significantly (P < 0.05) during the post-vaccination period. On the other hand IgG antibodies decreased in the second month after vaccination. At the fourth month the IgG level began to rise continuously (P < 0.01). The infants who were vaccinated at the second month showed a higher IgM response at the second and fourth month than those vaccinated in the first month (P < 0.05), but the difference was not visible thereafter (P > 0.05). 52 infants (78%) were tuberculin positive 8 weeks after vaccination. CONCLUSION: Anti-PPD IgM and IgG levels rise progressively in BCG-vaccinated infants. PMID- 7548910 TI - Evaluation of a modified BACTEC method to study the activity of disinfectants against Mycobacterium tuberculosis. AB - SETTING: BACTEC radiometry could provide a useful tool for evaluating the effect of disinfectants on mycobacteria. OBJECTIVE: The aim of this study was to improve the recovery of mycobacteria in the BACTEC system and thereby develop a new method for disinfectant testing with Mycobacterium tuberculosis. DESIGN: Protein was added to BACTEC 7H12B media. Growth and the recovery of M. tuberculosis after exposure to disinfectants was measured using viable counts and BACTEC radiometry. RESULTS: Protein additives enhanced growth in 7H12B media. After serial dilution of inoculum, 7H12B plus 10% egg yolk recovered mycobacteria from a 10(-2) higher dilution than viable counts. After 5 min disinfection with 3% peroxygen compound (Virkon), 3% Virkon plus saline or 2% gluteraldehyde (Cidex) no mycobacteria were recovered using standard 7H12B. However, using 7H12B plus protein, growth was detected in all of these within 30 days. In contrast, viable counts detected growth with Cidex and Virkon after 5 min disinfection but not from Virkon in saline. Viable counts took 3-8 weeks to determine. CONCLUSION: Using this modified BACTEC system for the evaluation of disinfectant action on mycobacteria provides advantages in speed, sensitivity and ease of use when compared with viable counts. PMID- 7548911 TI - The supply of antituberculosis drugs: price evolution. AB - The cost of antituberculosis drugs and chemotherapy regimens has fallen sharply in the last 2 years. This evolution in the international market, although attenuated by local currency devaluation, should encourage the directors of national tuberculosis programmes to quantify their needs more clearly and to organise centralised purchasing of antituberculosis drugs from non-profitmaking suppliers offering quality controlled generic drugs at the lowest prices. PMID- 7548912 TI - A national computer-based surveillance system for tuberculosis notification in Singapore. AB - SETTING: The notification rate of tuberculosis (TB) among residents in Singapore has been declining at a mean rate of 5.6% per annum, from 307 cases per 100,000 population in 1960 to 54 cases per 100,000 population in 1992. A National TB Notification Registry was set up in 1958 using a manual card system, and was captured into a computer database from 1986. OBJECTIVE: To monitor epidemiological trends of TB in Singapore with more speed, versatility and analytical capabilities, a new microcomputer-based surveillance system was developed in 1993. DESIGN: The main software programmes used in this system were DBase IV (version 1.5) and Epi Info (version 5). These versions could use base memory interchangeably and were therefore incorporated into a single application DBase programme. Security features were incorporated into the programme. The TB database was linked to the National HIV Notification Registry to enhance surveillance of combined TB and human immunodeficiency virus infection (HIV). RESULTS: The system was able to track notifications and TB culture results, address letters and analyze data and enabled prompt dissemination of information. CONCLUSION: The authors believe that this system would enhance surveillance and provide timely information for national TB control programmes. However, the effectiveness of this system is dependent on an established notification structure with notifications for tuberculosis of sufficient completeness. PMID- 7548913 TI - Multiple intracranial tuberculomas mimicking brain metastases. AB - A 50-year-old woman was admitted to our hospital with upper gastro-intestinal tract (GIT) bleeding, and complaining of severe headache and recurrent generalized tonic clonic seizures for the last year. Physical examination showed no focal neurological deficits. The patient was diagnosed with multiple brain metastases with occult primary depending on computerised tomography (CT) findings. Biopsy of enlarged left axillary lymph nodes showed caseating tuberculoid lymphadenitis compatible with tuberculosis. Intracranial lesions resolved completely on antituberculosis treatment. PMID- 7548915 TI - Pathogenesis of cervical tuberculous lymphadenitis: pathways to anatomic localization. PMID- 7548914 TI - Detection of mycobacteria in sputum smears prepared by cytocentrifugation and sedimentation. AB - The efficacy of sedimentation combined with cytocentrifugation of sputa was examined by preparing smears for the detection of mycobacteria. The detection of acid-fast bacilli from sputum sediment by cytocentrifugation yielded 32 smears positive for acid-fast bacilli, out of which in 26 cases better results were obtained than by the conventional method. More bacilli were found in 20 cases and on 6 occasions solely the cytospin method was positive. The differences in the number of bacilli were 100-200-fold in 5 cases and 10-fold in 12 paired cases. The technique applied is suitable for making culture and smear from the same sediment. The sedimentation combined with cytocentrifugation increases the effectiveness of the detection of acid-fast bacilli in smears. PMID- 7548916 TI - Pathogenesis of tuberculosis: pathway to apical localization. PMID- 7548917 TI - Rifabutin in the treatment of newly diagnosed pulmonary tuberculosis. PMID- 7548918 TI - Non-compliance with tuberculosis treatment: patients and physicians. PMID- 7548919 TI - Lactose malabsorption and irritable bowel syndrome. Effect of a long-term lactose free diet. AB - Lactose malabsorption may induce abdominal symptoms indistinguishable from those of the irritable bowel syndrome (IBS), however the exact relationship between the two conditions and the optimal differential diagnostic workup are still to be defined. We prospectively studied the prevalence of lactose malabsorption (by means of a hydrogen breath test) and the clinical effect of a long-term lactose free diet in 230 consecutive patients with a suggested diagnosis of irritable bowel syndrome, no organic disease of the GI tract, and no history of milk intolerance. Lactose malabsorption was diagnosed in 157 patients (68.2%). In 48 (43.6%) of the 110 patients who complied with the diet symptoms subsided, in 43 they were somewhat reduced and in 17 they remained unchanged. Symptoms never fully subsided in lactose malabsorbers non-compliant with the diet or in normal lactose absorbers who adhered to a lactose-free regimen. Partial improvement was observed in 20% of these subjects. No relation was demonstrated between pre-trial symptoms and the outcome of the diet. The occurrence of symptoms during the lactose breath test strongly suggested a favorable response to diet, but did not help in predicting whether symptoms would subside or be reduced. Conversely, their absence during the test was not associated with an acceptable negative predictive value. The high prevalence of lactose malabsorption in the patients under study suggests that in Italy IBS and lactose malabsorption are frequently associated. A test for diagnosing lactose malabsorption should always be included in the diagnostic workup for IBS and a long-term lactose-free regimen recommended if the test is positive. PMID- 7548920 TI - Comparison of lactulose and inulin as reference standard for the study of resistant starch fermentation using hydrogen breath test. AB - Disaccharide lactulose is commonly used as a standard to quantitate the colonic fermentation of undigested sugars by means of H2 breath measurements. However, its high hydrogen production rate during fermentation may make it inappropriate for mimicking the fermentation of more complex carbohydrates, such as starch. Indigestible carbohydrates with a higher molecular weight might be more suitable than lactulose as a standard in H2 breath studies of starch digestibility. To test this hypothesis, we measured H2 breath in 8 healthy volunteers after a standard meal supplemented with 5 g or 10 g of lactulose or inulin, an indigestible oligosaccharide with an average degree of polymerization 4.5 times higher than that of lactulose. The results were then compared with those obtained after a standard meal containing a known amount (6.1 g) of resistant starch from high-amylose corn starch. Median H2 breath excretion per gram of reference carbohydrate was lower after the 5 g dose of inulin than after the 5 g dose of lactulose (19.1 vs 26.6 ppm x h x g-1; Wilcoxon's rank test p = 0.021) but similar after the two 10 g doses (inulin 22.4; lactulose 23.6; p = 0.234). Median H2 breath excretion per gram of resistant starch was significantly lower than that for both lactulose and inulin (p < 0.02), being 4.7 ppm x h x g-1. In vitro fermentation for 8 hrs with fecal homogenate showed similar mean hydrogen production rates for inulin and lactulose (30.5 vs 27.7 mL/mg fermented carbohydrate), and a significantly lower rate for starch (9.1 mL/mg) (n = 7; ANOVA p = 0.0007).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548921 TI - Caffeine clearance in subjects with constitutional unconjugated hyperbilirubinemia. AB - To investigate the behaviour of caffeine (CAF) in patients with Gilbert's Syndrome (GS), a combined oral loading test of caffeine and chenodeoxycholic acid was performed in 14 healthy subjects and 71 patients with GS. Indocyanine green (ICG) kinetics was tested in 50 subjects with GS and in all control subjects. Fasting serum bile acids (SBA) and clearance after CDCA loading were within normal range in normal and GS subjects. No significant difference in levels either of bilirubin or of SBA was observed in GS cases with normal (52 cases, 488 +/- 63 ml/min) or impaired (19 cases, 338 +/- 30 ml/min) caffeine clearance. Eleven GS cases showed altered ICG clearance. No correlation was found between bilirubin and bile acids, CAF or ICG. Fasting SBA were normal even in cases of CAF or ICG altered kinetics, thus excluding structural damage in unconjugated hyperbilirubinemia. CAF altered kinetics in 27% of GS cases may suggest multiple deficits in the hepatocellular metabolism, thus confirming the heterogeneity of this syndrome. PMID- 7548923 TI - Histopathology of chronic viral hepatitis: guidelines for a revised classification. AB - Over the last few years a need for reassessment of the classification of chronic hepatitis has emerged in both clinicians who diagnose and treat liver patients and histopathologists who evaluate liver biopsies. The introduction of an aetiologic terminology, the evaluation of inflammatory activity and necrosis (grading) as well as of fibrosis as the equivalents of a stage in the disease appear to be major requirements. Due to the present satisfactory knowledge of the natural history of hepatitis B, there is also a discussion on the advisability of a personalized classification of chronic hepatitis B, i.e. a classification based upon consideration of profiles (or phases) rather than rigid morphological categories. This paper is intended as a guideline with a view to a revised classification of chronic hepatitis. PMID- 7548922 TI - Strategies used by viruses to evade the immune response. PMID- 7548924 TI - Bile acid structure and intestinal absorption in the animal model. AB - A close structure-activity relationship exists between the transport of bile acids (BA) in the liver and intestine; hepatic and intestinal BA transport can be evaluated and compared by using perfused liver and perfused intestine in the rabbit. The passive intestinal absorption is limited to the unconjugated BA, which occurs throughout the small bowel and colon, and is conditioned by the apical membrane lipid composition. A higher diffusion component is found in the terminal ileum compared to the jejunum, and seems to be related to the higher cholesterol-to-phospholipid ratio of the ileal brush border membranes. The active transport system is well characterized and the brush border membrane receptor, cytosolic BA binding proteins and basolateral anion exchange protein have been identified. Recently, the ileal BA transporter has been cloned from the hamster and human ileum and the main cytosolic BA binding protein was cloned from the rat ileum. In the liver, the active transport predominates on the passive diffusion both for conjugated and unconjugated BA. The maximal transport capacity in the liver is tenfold higher than in the intestine, while the Km values are of the same order of magnitude, i.e. in the millimolar range. Neither system operates at its maximum transport rate with prevalent concentrations of BA in portal blood or luminal content. PMID- 7548925 TI - Clinical applications of otoacoustic emissions. PMID- 7548926 TI - Referral rates and cost efficiency in a universal newborn hearing screening program using transient evoked otoacoustic emissions. AB - Recently, a National Institutes of Health Consensus Statement recommended that all infants be screened for hearing prior leaving the birthing hospital using a two-stage screening process based on transient evoked otoacoustic emissions (TEOAEs). Although the value of identifying hearing loss before 1 year of age is widely recognized, the feasibility of universal newborn hearing screening using TEOAE is sometimes questioned because it is presumed that the technique has a high false positive rate and is not cost efficient. This paper presents new data for 4253 infants from an operational universal newborn hearing screening program using a TEOAE procedure that answers those arguments. PMID- 7548927 TI - Estimated noise distributions across stages of consciousness utilizing the Fsp quantification technique. AB - We used the Fsp technique to quantify the influence of changing stages of consciousness on the signal-to-noise ratio of the middle latency response (MLR). The MLR and polysomnographic activity were obtained from 10 subjects during a continuous 6-hour period. The Fsp statistic was applied to the MLR data in order to determine the number of scores above a specified criterion in each stage of consciousness and time window of analysis. Results demonstrated that the number of Fsp scores exceeding the criterion of acceptance decreased with increasing depth of sleep. The awake stage yielded the greatest proportion of scores above the criterion. The background noise data were analyzed separately. The distributions of noise differed as a function of stage of consciousness. PMID- 7548928 TI - Development and standardization of SCAN-A: test of auditory processing disorders in adolescents and adults. AB - This paper reports on the development and standardization of SCAN-A: Test of Auditory Processing Disorders in Adolescents and Adults. The standardization version of SCAN-A included six subtests; two filtered words (FW) subtests, two auditory figure-ground (AFG) subtests, a competing words (CW) subtest, and a competing sentences (CS) subtest. Studies included development of normative results on subjects at different ages, determination of subtests to retain in the final version, investigation of test-retest reliability, and a concurrent validity study comparing the results of SCAN-A to SCAN scores obtained on the same subjects. The final version of SCAN-A contains four subtests that take 20 minutes to administer. Raw scores can be converted to standard scores and percentile ranks. Cut-off scores are suggested for normal, questionable, and abnormal performance. PMID- 7548931 TI - Revised Communication Self-assessment Scale Inventory for Deaf Adults (CSDA). AB - The 125-item Communication Self-assessment Scale Inventory for Deaf Adults evaluates difficult communication situations, their importance to the client, communication strategies, and attitudes toward hearing loss. Each scale contains subscales, and the scales may be used independently of each other. The original 115-item scale was revised to improve internal consistency of some of the subscales and modify language in some items. Internal consistency reliability, test-retest reliability, and normative data are presented. The scales were presented to a group of prelingually deaf adults and adolescents who differed in the frequency of hearing aid use, primary mode of communication home, school, and work, and educational background. Subjects responded to some of the scales and subscales in a significantly different manner based on these parameters. PMID- 7548930 TI - Should the audiometric database analysis method (draft ANSI S12.13-1991) for evaluating the effectiveness of hearing conservation programs be accepted as a US national standard?. AB - The value of audiometric database analysis (ADBA) procedures for evaluating the effectiveness of hearing conservation programs (HCPs) was studied in a population of 82,195 workers. We rated the HCP using four ADBA procedures. Results showed that the HCP ratings ran the gamut from "unacceptable" to "acceptable" from year to year and from procedure to procedure. A mere 7 percent of the total population was eligible for ADBA analysis. This drastically reduced sample size precludes subgroup analyses of even a large cohort and likely could not be applied to the HCPs of most small- and medium-sized businesses. The low levels of agreement observed (weighted kappa = 15%-25%) between the ADBA procedures reflect poor validity. Generalizability of these results would be inappropriate when so few nonrandomly selected individuals contribute to the evaluation. In view of these problems, acceptance of the ADBA method as a national standard may not be warranted. PMID- 7548929 TI - Transient evoked otoacoustic emissions and pseudohypacusis. AB - The audiologic diagnosis of pseudohypacusis continues to challenge the clinical audiologist. The introduction of otoacoustic emissions (OAEs) to the test repertoire of the audiologist may prove valuable in the evaluation of pseudohypacusis. This report highlights five cases in which transient evoked otoacoustic emissions (TEOAEs) were used to cross-check the validity of subjective audiologic thresholds. Relationships of TEOAEs to other objective measures of audiologic threshold are shown to substantiate the value of the procedure for the diagnosis of pseudohypacusis. Suggestions for the use of OAEs in cases of pseudohypacusis are discussed. PMID- 7548932 TI - Improving the efficiency of speech audiometry: computer-based approach. AB - Speech audiometric measures were carried out on 981 patients using a computer based system that permitted a comparison between two modes of signal presentation, manual and automatic. The manual mode, designed to simulate the pace of live-voice testing, allowed the presentation of speech signals at a rate dictated by the patient's response. The automatic mode, designed to simulate tape player testing, fixed the interstimulus interval. A comparison of elapsed time for the completion of word recognition testing with phonetically balanced (PB) word lists showed that use of the manual mode resulted in increased efficiency. Testing time was reduced by an average of 22 percent. These results suggest that the clinical efficiency of speech audiometric testing can be enhanced by using a computer-based manual approach. The combination of digitally recorded speech stimuli with a computer-based manual approach may provide a useful compromise between the efficiency of live-voice testing and the signal consistency of magnetic-tape or compact-disc recordings. PMID- 7548933 TI - Developmental changes in static admittance and tympanometric width in infants and toddlers. AB - Longitudinal measures of peak-compensated static acoustic admittance and tympanometric width are reported for infants and toddlers from 6 months to 30 months of age, based on over 1600 assessments of 88 children during a 24-month period. The subjects were all African-American children in full-time day care. Significant age effects were observed, with younger children displaying lower static admittance values and wider tympanograms. The results of this investigation underscore the importance of age- and population-specific norms when using acoustic admittance measures to evaluate middle ear status in children. PMID- 7548934 TI - Acoustic-immittance characteristics of children with middle-ear effusion: longitudinal investigation. AB - The purpose of this investigation was to describe, in a longitudinal prospective study, the acoustic-immittance profile during sessions with effusion and during sessions without effusion in children with recurrent middle-ear effusion (MEE). The static-acoustic middle-ear admittance, tympanometric width (TW), tympanometric peak pressure (TPP), and ipsilateral acoustic reflex (IAR) were evaluated in 36 ears of 18 children with recurrent MEE and 24 ears of 12 children without a history of MEE. Recurrent MEE was operationally defined as MEE diagnosed by microtoscopy and/or pneumotoscopy at four or more sessions over the first year of investigation. Subjects in the recurrent MEE group were followed over a time span of 1.1 to 3.0 years with an average intersession interval of 3.0 months. The results revealed that MEE was present at 78.3 percent of the sessions. A pure-tone average (PTA) exceeding 25 dB HL was present at 80 percent of the effusion sessions in the recurrent MEE group. The false-alarm rate for each of the individual acoustic-immittance measures, especially the TPP and IAR, was markedly higher during the otoscopically normal sessions of the recurrent MEE group than in the control group. This suggests that even when MEE is absent at a particular session, recurrent episodes of MEE appear to alter the acoustic immittance characteristics of the middle ear. Negative findings on all or three of the four acoustic-immittance measures occurred in only 1 percent of the effusion sessions in the total recurrent MEE group as compared with 76 percent of the normal sessions in the control group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548935 TI - When paroxysmal positioning vertigo isn't benign. AB - The electronystagmographic auditory brainstem response and magnetic resonance imaging findings for a 33-year-old male with a 3.5 cm left vestibular schwannoma are presented. Of particular interest was the presence of an unusual positioning nystagmus following the Dix-Hallpike maneuver in the right head-hanging position. The patient demonstrated a nystagmus that was immediate in onset and not fatigueable upon repeated positioning. During positioning, the patient experienced a vertical bobbing sensation and dysphoria, but not rotational vertigo. Most importantly, the nystagmus had a predominant downbeating vertical component. the case illustrates the diagnostic significance of downbeating nystagmus elicited by the Dix-Hallpike maneuver. PMID- 7548936 TI - The effect of female body size on male mounting behaviour in Dermacentor variabilis and D. andersoni. AB - This study was undertaken to determine if body size of female ticks was an important factor in eliciting the mating behaviour of Dermacentor variabilis and D. andersoni males. Dummy female ticks (DFTs) representing varying sizes of feeding females were prepared from plastic beads. Size of DFT was shown to be important in determining both the time spent by males in contact with DFTs and the extent of male mating response released. Dermacentor variabilis males preferred the smaller-sized DFT, whereas D.andersoni males preferred the larger sizes. Males of both species were able to discriminate between size of DFT only when mounting sex pheromone (MSP) was present. Size of the DFT was more important than the composition of the MSP extract in determining the time males spent in contact with DFTs. Males of both species were sensitive to variations in concentration of extract, and spent 2-6 times longer in contact with DFTs when the concentration was optimal than when it was not. No significant difference in the male's mating response was seen in response to variation in concentration of heterospecific extract. Male mating response, i.e. the behaviour of the male as it progresses through the initial contact, climbing onto the dorsal surface and turning onto the venter, was shown not to be dependent upon how long the male spent in contact with the DFT. Male D. andersoni had a much lower mating response to DFTs than D. variabilis males, suggesting that some further stimulus may be required. PMID- 7548937 TI - A comparison of susceptibility to stocks of Trypanosoma simiae of Glossina pallidipes originating from allopatric populations in Kenya. AB - A colony of Glossina pallidipes which originated from Nguruman, Rift Valley Province, Kenya, was significantly more susceptible than a colony of the same species which originated from Shimba Hills, Coast Province, Kenya, to infection with a stock of Trypanosoma simiae CP 11 isolated from wild G. austeni in Coast Province, Kenya, irrespective of whether pigs or goats were used as infecting hosts. Male G.pallidipes from both the colonies were more susceptible than females to this T.simiae stock. Similarly, a G.pallidipes colony of Nguruman origin was significantly more susceptible than the colony of Shimba Hills origin to infection with another stock of T.simiae CP 813 isolated from wild G.pallidipes in Coast Province, Kenya, again irrespective of whether pigs or goats were used as infecting hosts. The susceptibility of the sexes of G.pallidipes from both the colonies to T.simiae CP 813 did not differ significantly when pigs were used as as infecting hosts, but male G.pallidipes from both the colonies were significantly more susceptible than female tsetse to this T.simiae stock when goats were used as infecting hosts. Nevertheless, if the observed differences in susceptibility of the two G.pallidipes colonies reflect transmission of trypanosomes by the two allopatric populations of tsetse in the field, then the epidemiology of simiae-trypanosomiasis probably differs between these two areas of Kenya. PMID- 7548938 TI - Analysis of gene flow in North American face fly (Musca autumnalis) populations. AB - Vertical polyacrylamide gel electrophoresis was used to resolve enzymes at ten putative loci in face flies Musca autumnalis De Geer, a colonizing, Palaearctic species established in North America for at least 210 generations. Flies were sampled in 1991 from six locations in Iowa, two in Maryland, two in Minnesota, two in Tennessee, three in New York, and three in Missouri. Nondirectional temporal variation in gene frequencies over a 4-year interval was detected at farms in central Iowa. Heterogeneity in allele frequencies was detected among farms in Iowa, Maryland, New York and Minnesota but not in Tennessee and Missouri. There were no consistent departures from random mating. Partitioning variances in gene frequencies showed that 58% of the variation occurred in populations among states and 43% between populations within states. Mean reproducing immigrants per population per generation was estimated to be eighteen flies. No regional genetic differentiation was detected, and there were no barriers to gene flow within or among diverse populations in the six states. Earlier data bearing on gene flow among and between Nearctic and Palearctic face fly samples were analysed and significant differentiation was not detected. PMID- 7548940 TI - The relationship between CDC light-trap and human-bait catches of endophagic sandflies (Diptera: Psychodidae) in the Peruvian Andes. AB - A study was carried out in the Peruvian Andes to test the suitability of CDC light traps for monitoring changes in the human-landing rate of endophagic phlebotomine sandflies, following house-spraying with pyrethroid insecticide. On four pairs of consecutive nights, sandflies were caught inside eight sprayed and eight unsprayed houses, either by human bait or by CDC light traps. The sandflies collected were Lutzomyia verrucarum (97%) and Lu.peruensis (3%), both probable vectors of Leishmania peruviana, and the species composition was unaffected by house-spraying. A non-linear relationship was detected between light-trap and human-bait catches, but the relationship did not diverge significantly from linearity within the range of sandfly abundance found in most houses in the endemic area (i.e. between 3 and 200 sandflies/house-night), and did not differ significantly between sprayed and unsprayed houses. However, light trap catches had a significantly lower proportion of blood-fed females in sprayed than in unsprayed houses, probably due to an insecticidal effect on post-blood-feeding behaviour. The proportion of Lu. verrucarum was significantly higher in light trap than in human bait catches, indicating that Lu.peruensis is either more anthropophilic or less phototropic than Lu.verrucarum. PMID- 7548939 TI - Effects of fenoxycarb on development and reproduction of the oriental cockroach, Blatta orientalis. AB - In a laboratory study, groups of third-instar Blatta orientalis nymphs were reared to adulthood in arenas containing fenoxycarb (48 mg a.i./m2) treated ceramic or plywood tiles. The reproductive capacity of the emergent adults was assessed by pairing each individual with two untreated individuals of the opposite sex. Oothecal production, oothecal hatch and the numbers of nymphs emergent from each hatched ootheca were monitored. Exposure to 1-day-old deposits of fenoxycarb reduced adult emergence by 45-75% in comparison with an untreated control treatment. Substantial (> 40%) mortality also resulted when nymphs were exposed to deposits up to 3 months old on plywood and up to 6 months old on ceramic. Exposure to fenoxycarb significantly extended the time taken to reach adulthood of males contacting 6-month-old deposits on both surfaces, and of females contacting 1-year-old deposits on ceramic. Adult females exposed as nymphs to fenoxycarb were unable to produce oothecae, except one female laid a non-viable ootheca. Untreated females paired with treated males produced large numbers of oothecae of normal appearance but very low viability, with only 0-7.1% hatching. With marked effects on both development and reproduction in B.orientalis, fenoxycarb is a promising agent for control of this species. PMID- 7548942 TI - Malaria infection potential of anopheline mosquitoes sampled by light trapping indoors in coastal Tanzanian villages. AB - Anopheline mosquito populations were studied during 1992 in seven villages south of Bagamoyo, coastal Tanzania, prior to malaria control intervention using insecticide treated bednets. To collect mosquitoes, CDC light traps were used in ten houses per village fortnightly for 12 months. Anopheles females were identified and checked by ELISA for the presence of malaria sporozoite antigen and source of bloodmeal. An.funestus peaked in June-July after the long rains. Three members of the An.gambiae complex had different seasonality: An.arabiensis, An.gambiae and small numbers of An.merus were collected. In most villages transmission was extremely high and perennial with the entomological inoculation rate reaching three to eleven infective bites per person per night in July and persisting at around 0.1 and 1 for most of the remainder of the year. Sporozoite infection rates within the An.gambiae complex ranged from 2% to 25%, with the peaks in January and July following the two rainy periods. An.funestus showed a similar pattern. The light traps were reliable, simple to operate, and proved to be satisfactory to study the mosquito vector population. PMID- 7548944 TI - Phlebotomine sandflies associated with a focus of cutaneous leishmaniasis in Valle del Cauca, Colombia. AB - A survey was made of the phlebotomine sandfly fauna of La Guaira, a village with coffee plantations near Cali, Colombia, from which cases of American cutaneous leishmaniasis had been reported due to Leishmania (Viannia) panamensis and Le. (V.) braziliensis. Among six species of sandfly collected on human bait, Lutzomyia youngi was most important in terms of biting nuisance. Lu.columbiana, Lu.lichyi and Lu.scorzai as well as Lu.youngi adults occurred throughout the year. Sandfly man-biting activity occurred throughout the night and was highest within 2 h of sunset. Despite its abundance in nocturnal samples, Lu.youngi was rarely taken in diurnal resting site collections. In contrast, Lu.lichyi was collected on tree-trunks during the day in large numbers and was the only species biting in daylight. The implications of these and other findings for leishmaniasis control measures in La Guaira are considered. PMID- 7548945 TI - Evaluation of deltamethrin-impregnated bednets and curtains against phlebotomine sandflies in Valle del Cauca, Colombia. AB - The effectiveness of bednets and curtains (nylon mesh 64 per cm2) impregnated with deltamethrin at 26 mg a.i./m2 in reducing the biting nuisance caused by three phlebotomine sandfly species: Lutzomyia columbiana, Lu.lichyi and the predominant Lu.youngi (Diptera: Psychodidae), was evaluated at La Guaira, a rural settlement in Valle de Cauca near Cali, Colombia. Pairs of volunteers collected sandflies under impregnated bednets, in rooms protected by impregnated curtains or in unprotected rooms in a randomized matched design. Collections were made in three houses per night on three consecutive nights, so that each house was sampled under each of the three treatments. This routine was repeated at 2-week intervals for 6 months. There was no significant difference between the overall numbers of sandflies collected in rooms with or without impregnated curtains. Only 0.14 sandflies/man-hour were caught on human bait under impregnated bednets, significantly fewer than the numbers collected on human bait outside the nets in the same room (1.91) or in unprotected rooms (3.29). In a second set of experiments carried out in La Guaira and the neighbouring community of Jiguales, the effect of deltamethrin impregnation was evaluated by comparing numbers of sandflies collected on human bait under treated and untreated nets. Significantly fewer were collected under the impregnated nets (0.25 v. 0.69/man-hour). Wild caught female Lu.youngi exposed to treated netting for 2 min in the laboratory all died with 24 h. The impact of deltamethrin-impregnated bednets was considered to be useful against Lu.youngi and other potential vectors of leishmaniasis in such communities. PMID- 7548946 TI - Field studies on the effect of cattle skin secretion on the behaviour of tsetse. AB - The effect of ox skin secretions (sebum) on the behaviour of tsetse flies, Glossina spp., was investigated in the field using electrified targets, some of which operated intermittently, and by direct observations of flies landing on treated and untreated cloth. As the off-period of an intermittently operating electrified target increased, the catch decreased both with and without the sebum present. Targets with sebum always caught more flies than targets without sebum, but there was no evidence to suggest that sebum increased the duration of stay on a target. Direct observations of flies on cloth targets revealed that for both species the presence of sebum reduced the duration of contact and for G.pallidipes the number of return contacts was increased. The results from direct observations were used to predict the number of repeat landings that would need to be made by flies in order to account for the catch of tsetse at intermittently electrified targets. PMID- 7548947 TI - The effect of host resistance on the metabolic rate of engorged females of Rhipicephalus evertsi evertsi. AB - This study investigated the effect of acquired resistance in guinea-pigs on the metabolic rate of adult females of the tick Rhipicephalus evertsi evertsi. Guinea pigs were subjected to three successive infestations of ticks and the rate of CO2 production (VCO2) measured in first and third infestation engorged females. Ticks which fed on resistant hosts showed a 52% decrease in mass compared to ticks that fed on naive animals. Reduction in mass was accompanied by a decrease in VCO2 (mlh-1) per tick but an increase in mass specific VCO2 (mlg-1h-1). However, both groups shared a single allometric relationship between body mass and metabolic rate (VCO2). We suggest that the differences in size rather than any factor directly relating to the mechanism of acquired resistance account for the differences in metabolic rate between ticks fed on naive and resistant guinea pigs. PMID- 7548941 TI - Comparison of sampling anopheline mosquitoes by light-trap and human-bait collections indoors at Bagamoyo, Tanzania. AB - The mosquito sampling efficiency of CDC miniature light-traps, relative to night biting collections, was evaluated indoors at two sites in coastal Tanzania. We found that the total number of anophelines captured overnight by light-traps (hung beside a bednet in use) was 1.23 times the number of anophelines captured by human-bait collections. This relationship was not affected significantly by changes in the mosquito density, order of trapping method, date of sampling, or number of household occupants. Malaria sporozoite rates were twice as high among mosquitoes captured by light-trap as compared to those captured by night-biting collection. This was attributed to the tendency of light-traps to capture a larger proportion of gravid mosquitoes, which also had high sporozoite rates. The differences in sporozoites rates according to abdominal stage indicates that unfed mosquitoes captured by light-traps may define more precisely the human biting activity and sporozoite rates as seen by night-biting collections. Our study shows that light-traps, when used in combination with night-biting collections, can be an effective and sensitive means for measuring human-biting activity and the sporozoite rate. PMID- 7548948 TI - Laboratory evaluation of desiccants and insecticidal soap applied to various substrates to control the deer tick Ixodes scapularis. AB - Desiccant and soap pest control products were tested against Ixodes scapularis nymphs on sod, pachysandra, landscaping stones and pinebark woodchips in a laboratory study. High mortality (91-100%) was obtained with the silica-based desiccant Drione and Safer's insecticidal soap (SIS) treatments except for SIS treated woodchips. SIS and Drione contain 0.2 and 1% pyrethrins, respectively. SIS may be absorbed by the woodchips and not picked up by the nymphs as mortality decreased to 78%. Diatomaceous earth and Dri-die reduced nymphs by only 10-41%; neither desiccant contains insecticides. Nymphal mortality increased 15-17% after 20 or 100 ml of water was sprayed over sod plugs treated with SIS or Drione. The addition of isopropyl alcohol (ROH) to SIS increased the efficacy of SIS against nymphal I.scapularis on treated sod plugs but not on treated landscaping substrates. Drione, SIS, SIS-ROH and chlorpyrifos 50WP treated landscaping stones and pinebark woodchips resulted in 88-95% and 72-96% nymphal mortality, respectively. Unexpectedly, these substrates and treatments affected tick movement as well. Effective pest control products applied to xeric landscaping substrates present in maintained beds and borders near wooded areas could deter tick movement and provide significant levels of tick control. PMID- 7548943 TI - Sugar meal sources for the phlebotomine sandfly Lutzomyia longipalpis in Ceara State, Brazil. AB - In the Brazilian village of Boqueirao do Renato Parente, Ceara State, where visceral leishmaniasis is endemic, significant variation in the abundance of phlebotomine sandflies between different types of vegetation was demonstrated by castor oil sticky trap collections. Population densities of Lutzomyia longipalpis (Lutz & Neiva) sandflies were higher in beans and maize crops than in the surrounding open and tree scrub. Significant relationships were detected between the abundances of sandflies and aphids, suggesting aphid honeydew as a potential food source. Sugar meal analysis, using high-performance anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) and the cold anthrone test, was used to demonstrate that significantly more sandflies fed on bean plants contaminated with aphids and honeydew than on uncontaminated beans. Furthermore, higher concentrations of sugars were detected in flies which fed on aphid/honeydew-contaminated beans, suggesting that sugar was more easily available and/or that honeydew/aphids act as phagostimulants for sandflies. Amongst wild sandflies collected from a bean field, significantly more female sandflies were 'sugar-positive' than males, though the sugar concentrations in positive sandflies were similar for both sexes. The concentrations of di- and trisaccharides in sandfly homogenates, honeydew extracts and phloem exudates were very low. PMID- 7548949 TI - Exposure to stable flies reduces spatial learning in mice: involvement of endogenous opioid systems. AB - Biting flies influence both the physiology and behaviour of domestic and wild animals. This study demonstrates that relatively brief (60 min) exposure to stable flies, Stomoxys calcitrans (L.), affects the spatial abilities of male mice. Stable fly exposure resulted in poorer subsequent performance in a water maze task in which individual mice had to learn the spatial location of a submerged hidden platform using extramaze visual cues. Determinations of spatial acquisition and retention were made with mice that had been previously exposed for 60 min to either stable flies or house flies, Musca domestica (L.). Mice exposed to stable flies displayed over one day of testing (six blocks or sets of four trials) significantly poorer acquisition and retention of the water maze task than either mice that had been exposed to house flies or fly-naive mice. This attenuation of spatial learning occurred in the absence of any evident sensorimotor or motivational impairments. The reduction in spatial abilities involved endogenous opioid systems, as the decreased performance resulting from stable fly exposure was blocked by pre-treatment with the prototypic opiate antagonist, naltrexone. These results indicate that relatively brief exposure to biting flies can lead to a decrease in spatial abilities which is associated with enhanced endogenous opioid activity. These results support the involvement of endogenous opioid systems in the mediation of the behavioural and physiological effects of biting fly exposure. They further suggest that decreases in spatial abilities and performance may be part of the behavioural consequences of biting fly exposure in domestic and wild animals. PMID- 7548950 TI - A study of the diapausing behaviour of Rhipicephalus appendiculatus and R. zambeziensis under quasi-natural conditions in Zambia. AB - The diapausing behaviour of Rhipicephalus appendiculatus was studied under quasi natural conditions in the Eastern Province of Zambia. Newly-moulted adults of ticks indigenous to the study area entered a behavioural diapause when exposed to daylengths below a critical photoperiod, provisionally a daylength between 11 h 20 min and 11 h 45 min. In the Eastern Province of Zambia the diapause was apparently not terminated by a long-day signal, but by a weakening of the photoperiodic maintenance of the diapause because of increasing age of the ticks. Adults of a reference stock indigenous to Kenya also entered a diapause when exposed to daylengths below the same threshold and maintained this diapause for the same length of time. Adults of a reference stock of Rhipicephalus zambeziensis indigenous to Zimbabwe did not enter a diapause when exposed to the same daylengths. The relevance of the findings is discussed in relation to the distributions of the two species. PMID- 7548951 TI - Anopheles arabiensis and An. gambiae chromosomal inversion polymorphism, feeding and resting behaviour in relation to insecticide house-spraying in Tanzania. AB - Differential responses of the mosquitoes Anopheles arabiensis and An. gambiae sensu stricto to house-spraying with DDT or lambda-cyhalothrin were evaluated in relation to chromosomal inversion polymorphism, feeding and resting behaviour of these malaria vectors in Tanzania. Blood-fed mosquitoes from pit traps outdoors, exit traps on windows and indoor-resting catches were identified cytogenetically and the chromosomal inversion frequencies compared between samples and species. Their outdoor-resting behaviour was assessed by a mark-release-recapture experiment and by determining the proportion of freshly blood-fed individuals in exit traps. The source of bloodmeals was analysed by an ELISA method. Endophagic females of An. arabiensis were more likely than those of An.gambiae to exit from a house on the night of blood-feeding. Only in one out of three villages was there evidence that chromosomally distinct individuals within a species had different preferences for resting sites. There were indications, but not conclusive evidence, that mosquitoes caught indoors or outdoors had a tendency to return to the same type of resting site. In villages sprayed with either insecticide, the mean age of the vector populations was greatly reduced, compared with those in the unsprayed villages. An.arabiensis females exited from DDT sprayed houses after blood-feeding, whereas with lambda-cyhalothrin those exiting were mostly unfed and there was a decline in the human blood index. The excitorepellency of DDT was perceived as a disadvantage, whereas lambda cyhalothrin apparently had more impact on malaria transmission by An.arabiensis. PMID- 7548953 TI - Activation of Anopheles gambiae mosquitoes by carbon dioxide and human breath. AB - Female Anopheles gambiae Giles mosquitoes were observed individually in a cage within a wind tunnel and their responses to pulses of carbon dioxide recorded on video tape. The range of concentrations tested revealed an 'activation' threshold concentration of carbon dioxide in the region of 0.01% above background. At this concentration, approximately 60% of the mosquitoes took off and flew upwind. Pulses of human breath, diluted with wind tunnel air to reproduce equivalent concentrations of carbon dioxide, elicited similar levels of response and the same 'activation' threshold concentration. These findings are discussed in relation to the activation of host-seeking mosquitoes. PMID- 7548952 TI - Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status. AB - Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus. PMID- 7548954 TI - Tick control: a standardized terminology. PMID- 7548955 TI - Improved medium for Aedes aegypti tissue culture. PMID- 7548956 TI - Cysteine residues modify the electrophoretic mobility of amplified esterases associated with insecticide resistance in Culex mosquitoes. PMID- 7548957 TI - Multiresidue determination of quinolone antibiotics using liquid chromatography coupled to atmospheric-pressure chemical ionization mass spectrometry and tandem mass spectrometry. AB - Liquid chromatography coupled to atmospheric-pressure chemical ionization mass spectrometry and tandem mass spectrometry (MS/MS) methods for the determination and quantification of four quinolone antibiotics were adapted from a published procedure for liquid-liquid extraction from catfish muscle and high-performance liquid chromatographic analysis. In-source collision-induced dissociation was used to optimize fragmentation to produce mass spectra consisting of the protonated molecule and two characteristic fragment ions of nearly equal intensity. Selected ion monitoring of three ions per quinolone yielded sensitive detection in catfish muscle extracts (estimated instrumental detection limits 0.8 1.7 ppb). The intensity ratios were used to confirm the presence of three of the quinolones based on the accurate agreement (< or equal to 10% deviation) between ratios derived from fortified catfish extracts and those from standard quinolones. MS/MS was used to increase the specificity and sensitivity of analysis. Scans using constant neural loss (CNL) of 18 mass units gave a sensitive response for the quinolones suggesting that CNL scans may be applicable to multiresidue screening for unknown quinolones. MS/MS with multiple reaction monitoring of the [MH - 18]+ transition for each quinolone yielded the highest sensitivity analysis in catfish extracts (estimated instrumental detection limits 0.08-0.16 ppb). PMID- 7548958 TI - Reverse isotope dilution analysis of 13CO2 using gas chromatography/isotope ratio mass spectrometry: application to the quantitative determination of 13CO2 released by human polymorphonuclear leukocytes. AB - We propose a new method for the quantitative determination of carbon dioxide released by a biological microgenerator: a suspension of human polymorphonuclear leukocytes (PMNL). This method is based on the reverse isotope dilution analysis by gas chromatography/isotope ratio mass spectrometry of 13CO2 released by PMNL in a controlled isotope abundance atmosphere containing 3% CO2. 13CO2 release is effective after PMNL stimulation in the presence of [13C]glucose, labeled on positions 1, 2 or 6. The validation of this method is carried out by the measurement of the isotope ratio 13CO2/12CO2 using known amounts of [13C]sodium hydrogen carbonate and the comparison with theoretical isotope abundances derived from various CO2 equilibria. Complete release of CO2 is achieved by the acidification of the medium. This method requires only few cells, displays high sensitivity and specificity and can be applied to the analysis of large series of samples using an automatic sample injector. In addition, this method can also be applied to other types of biological microgenerators of carbon dioxide. PMID- 7548959 TI - Tryptic mapping of human chorionic gonadotropin by matrix-assisted laser desorption/ionization mass spectrometry. AB - The potential of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for use in the identification of human chorionic gonadotropin (hCG) seized by law enforcement agencies is investigated. Analysis of untreated hCG revealed signals corresponding to the molecular ions of the intact hCG heterodimer and both its non-covalently linked subunits. Unfortunately, due to carbohydrate heterogeneity, the peaks are broad which makes accurate mass assignment, and consequently identification, difficult. Peptide mapping by MALDI-TOF following tryptic digestion gave sequence coverage of 59% and 52% for the alpha- and beta-subunits respectively. Nevertheless, the tryptic map was considered to provide unambiguous identification of hCG. This was confirmed by searching peptide-mass databases with the experimentally determined masses. Our data suggest that peptide mapping by proteolytic digestion followed by MALDI-TOF mass spectrometry is a suitable analytical technique for the identification of hCG seized by the legal authorities. PMID- 7548960 TI - Sequencing electroblotted proteins by tandem mass spectrometry. AB - Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization. PMID- 7548963 TI - Error-tolerant protein database searching using peptide product-ion spectra. AB - A method for matching proteins in databases with unknown samples using data derived from peptide product ion masses is described. The power of the method is due to the speed of analysis and to the ability to locate proteins in a database even when the experimental data show anomalies due to derivatization or post translational modification. PMID- 7548962 TI - The characterization of proteins and peptides by automated methods. AB - A suite of software programs called the BioToolBox is described. The primary purpose of the BioToolBox is to facilitate the characterisation of proteins and peptides using data acquired by IonSpray mass spectrometry and tandem mass spectrometry. As an example, the characterization of a 50 KDa protein (transcription termination factor-rho) is described. The programs which comprise the BioToolBox produce complementary information which is exchanged between the software modules. PMID- 7548964 TI - General disinfection guidelines. AB - Cleaning and disinfection of surfaces which have been in contact with animals, poultry or organic material is a vital element in controlling bacterial and viral diseases, and ensuring the wholesomeness and safety of foods. The thoroughness of pre-disinfection cleaning is the most important determinant of the efficacy of disinfection processes. Disinfectant users and officials responsible for the use of disinfectants must have clear goals and a sound plan of action. They must choose appropriate products, properly clean and prepare the site, and take the necessary steps to ensure the safety of animals, humans, equipment and the environment. They must also objectively evaluate the results of disinfection procedures. Safe and effective disinfectant strategies require an understanding of the actions and toxicological hazards of the chosen products, a clear plan of action, regulatory discipline, conscientious documentation, responsible supervision and post-disinfection testing. Disinfection procedures and policies must meet legal and environmental requirements and the changing expectations of society. PMID- 7548961 TI - High resolution matrix-assisted laser desorption/ionization time-of-flight analysis of single-stranded DNA of 27 to 68 nucleotides in length. AB - Mass spectra of single-stranded DNA oligonucleotides were acquired using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOFMS). Resolution enhancement using space-velocity correlation focusing allows for facile observation of different oligomers, the direct observation of individual DNA-metal adducts, and investigation of counter ion structure. PMID- 7548965 TI - Disinfection of trucks and trailers. AB - The author provides an outline of the basic procedure for cleaning and disinfecting road transport vehicles, with special attention to the types of cleaning agents which may be used. PMID- 7548967 TI - Disinfection of livestock production premises. AB - Livestock production premises (e.g. barns, and pens for lambing, calving, weaning and holding animals) encounter disease problems mainly associated with the housing of new-born and young animals, pregnant females and suckling mothers. The author describes a programme of cleaning and disinfection for use during the routine operation of livestock premises. A further programme is described for use in the event of an outbreak of an Office International des Epizooties List A disease, taking into account the particular problems associated with this type of disease. PMID- 7548966 TI - Aircraft and ship disinfection. AB - Shipment of animals, animal products and foodstuffs (for human or animal consumption) by sea and air is economically important. However, the possibilities of pathogen transmission represent a genuine danger, even if veterinary controls are performed continuously. Bacteria, viruses, fungi and parasites excreted by animals may contaminate surfaces and are protected by organic matter. Cleaning is an important step in the sanitizing process, and if this is performed well, more than 90% of microorganisms can be removed. Chemical disinfectants must be compatible with cleaning procedures, instruments and materials used previously, and should be without harmful effects on humans or animals. The author outlines the basic procedures and fundamental principles involved. PMID- 7548968 TI - Disinfection in the dairy parlour. AB - The dairy farmer has the responsibility of producing milk under clean and hygienic conditions, employing appropriate techniques to clean and disinfect the milking equipment and the milking parlour. The ability of raw milk to retain its quality under storage, and the safety of the product for the consumer, can both be directly related to the bacterial content of the milk. In most countries, bacterial content is one of the factors considered in determining the level of payment for milk. Cleaning and disinfection are complementary processes: neither process alone will achieve the desired end result. Milk with low bacterial and somatic cell counts cannot be produced unless milking equipment is effectively cleaned and disinfected between milkings and the cows are kept healthy. The author considers the various sources of bacterial contamination and increased somatic cell counts in raw milk, and describes the cleaning and disinfection practices recommended for the production of milk of good microbiological quality. PMID- 7548969 TI - History of disinfection from early times until the end of the 18th century. AB - The author describes and analyses the methods of disinfection in use until the end of the 18th century, i.e. before the scientific demonstration of the role of pathogenic microorganisms. These methods are classified into three categories: chemical (by derivatives of sulphur, mercury, copper, and also by alkalis and acids), physical (heating, fumigation, filtration, etc.) and biological (burial). The author concludes that, despite their empiricism, these methods were of great value to those responsible for controlling diseases of animals, as in some cases they were able to eradicate diseases while still ignorant of the causal mechanisms. PMID- 7548970 TI - History and future perspectives of the use of disinfectants in animal health. AB - Programmes for the prevention, control and eradication of animal diseases are closely interlinked in many ways. In the field of animal health over the last 100 years, surveillance, diagnostics, rapid response, regulatory and legal authorities, epidemiological investigations, together with guidelines for dealing with diseased animals and contaminated premises and materials, have proved to be the critical links in the chain of actions needed to prevent, control and eradicate diseases. Disinfection, as an integral part of the protection of animal health, has become more sophisticated and more effective over this period. The historical trends suggest that disinfection will be even more important in the future, and that disinfectants and procedures must be further developed to keep pace with new scientific findings, changing agricultural structures and contemporary social concerns. PMID- 7548971 TI - Modes of action of disinfectants. AB - The exact mechanism of action of a disinfectant is not easy to elucidate. The notion of 'target' in the bacterial cell, frequently evoked for the antibiotics, is not clear for disinfectants (except for some, e.g. chlorhexidine). In understanding the mode of action of a disinfectant, it can be difficult to distinguish the primary stage (characteristic of the mode of action) and the secondary stage (consequence of the action). The author describes the actions of disinfectants on the external membrane, cytoplasmic membrane and energy metabolism of cells; these actions include rupture of the membrane, loss of permeability and coagulation of the cytoplasm. PMID- 7548972 TI - Chemicals used as disinfectants: active ingredients and enhancing additives. AB - Active ingredients used in microbiocidal products in the European Union constitute some 250 chemical entities. Approximately 100 of these chemicals are commonly used in disinfectant products. The majority of these substances may be classified into distinct chemical groupings. A brief review of the chemical, physical and microbiological properties of each group is given, together with some indications of additives which may be used to enhance their properties, and factors which may detract from them. Some indications of usage areas are given. PMID- 7548973 TI - Testing disinfectants for efficacy. AB - Testing under laboratory conditions is undoubtedly useful in assessing the activity of disinfectants. However, such testing must be regarded as no more than a preliminary to field trials. This not only indicates the unreliability of laboratory tests, but also poses the wider problem of how any laboratory evaluation can be correlated with the field requirements. Many attempts have therefore been made to devise tests which are suitable to evaluate the in-use requirements of disinfection (e.g. on farms). The aim of these techniques is to record the end-point of a disinfection procedure on the surfaces in animal houses. In view of the need to standardise testing conditions in the field, the author provides details of the optimum time and place for taking samples under field conditions. Samples for culture media should be taken from the floor when dry. The various techniques (i.e. swabbing, agar cylinder, agar carrier, 'ready to-use' set) should provide information on the end-point of the disinfection. The desired end-points is approximately one viable bacterium per cm2 of surface area. The technique used should demonstrate whether the disinfection has been effective. PMID- 7548974 TI - Environmental considerations of disinfectants used in agriculture. AB - Using disinfectants in agriculture increasingly requires consideration of the indirect effects on the environment and human health. At present, only limited information is available on post-application effects of disinfectants, and such data therefore needs to be gathered in order to assess potential effects. In addition, a basis exists for establishing precautionary guidelines using current information. Optimum handling and application procedures require a knowledge of the specific features of each type of disinfectant. A need to establish product standards through efficacy tests has also emerged. The benefits of disinfectants for disease control must also be weighed against any environmental consequences. Emergency application of a disinfectant carries different implications from routine use on farms, and any large-scale application should undergo an environmental impact assessment. PMID- 7548975 TI - Special needs for disinfectants in food-handling establishments. AB - Freedom from microbial and foreign body contamination is essential in food production, and the principal means for controlling the surface route of contamination is sanitation (cleaning and disinfection). After the cleaning phase, disinfection plays a crucial role in further reducing microbial numbers and viability. However, disinfectants for use in the food industry may contaminate the product. Therefore, as well as being effective and suitable for factory use, such disinfectants must also be non-toxic and non-tainting. The author describes the usage criteria and operator safety requirements of disinfectants, together with methods to determine taint potential, toxicity and efficacy. Using these parameters, a limited number of disinfectants are judged suitable for general food industry use and these are compared. PMID- 7548976 TI - Niceritrol prevents the decrease in red blood cell 2,3-diphosphoglycerate and neuropathy in streptozotocin-induced diabetic rats. AB - Nerve ischemia/hypoxia has been linked to the pathogenesis of diabetic complications. Red blood cell 2,3-diphosphoglycerate is an important regulator of peripheral tissue oxygenation; however, the relationship between 2,3 diphosphoglycerate concentration and diabetic complications has not been studied in detail. This investigation focused on the relationship between red blood cell 2,3-diphosphoglycerate and diabetic neuropathy, by measuring motor nerve conduction velocity and sciatic nerve blood flow in streptozotocin-induced diabetic rats. The effect of treatment with niceritrol, a nicotinic acid derivative that acts as a vasodilator and reduces serum lipid concentrations, on 2,3-diphosphoglycerate concentration and diabetic neuropathy was also examined. Untreated diabetic rats had significantly lower concentrations of red blood cell 2,3-diphosphoglycerate, higher concentrations of serum total cholesterol and triglyceride, as well as reduced motor nerve conduction velocity and sciatic nerve blood flow, compared to untreated normal rats. Niceritrol prevented these abnormalities without correcting hyperglycemia in diabetic rats, but had no effect on these parameters in normal rats. Red blood cell 2,3-diphosphoglycerate concentration and motor nerve conduction velocity showed a positive correlation with sciatic nerve blood flow and 2,3-diphosphoglycerate, respectively. These observations suggest that ischemia/hypoxia plays an important role in the development of diabetic neuropathy, and that niceritrol has a therapeutic effect on this condition by improving endoneurial ischemia/hypoxia. PMID- 7548977 TI - The progression of retinopathy over 2 years: the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study. AB - This study examined potential risk factors for the incidence and progression of retinopathy in a large representative cohort of childhood onset insulin-dependent diabetic patients. Participants in the Epidemiology of Diabetes Complications (EDC) Study underwent a full clinical examination at baseline and again at a 2 year follow-up. Retinopathy status was ascertained using stereo fundus photographs graded according to the modified Airlie House System. The study population is based on a large cohort of childhood-onset insulin-dependent diabetic patients, seen within 1 year of diagnosis at the Children's Hospital of Pittsburgh between January 1950 and May 1980. A total of 657 subjects participated at baseline (1986-1988), with 80% of eligible survivors taking part in the follow-up examination. This report concerns risk factors associated with the progression of diabetic retinopathy over a 2-year period, and the interaction of these factors with the presence of nephropathy. Analyses showed that baseline diastolic blood pressure was significantly associated with the incidence of any retinopathy, while glycosylated hemoglobin, baseline severity of retinopathy, serum triglycerides, and, to a lesser extent, higher levels of low-density lipoprotein (LDL) cholesterol and fibrinogen were associated with the progression of retinopathy. Progression to proliferative retinopathy was related to higher LDL cholesterol, fibrinogen, serum triglycerides, albumin excretion rate, and glycosylated hemoglobin (GHb). Risk factors varied with the presence of nephropathy. In the absence of nephropathy, GHb was a significant predictor of progression, whilst this was not the case in the presence of nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548979 TI - Possible effect of angiotensin-converting enzyme inhibition on glomerular charge selectivity. AB - Angiotensin-converting enzyme (ACE) inhibitors are known to reduce urinary albumin excretion (UAE) in diabetic patients. Animal studies have shown that, besides diminishing the glomerular capillary pressure, ACE inhibitors might reduce albuminuria by influencing glomerular charge selectivity through glomerular preservation of heparan sulphate proteoglycan. In humans, an indirect measurement of glomerular charge selectivity can be obtained by calculating the glomerular charge selectivity index (SI), a clearance ratio of IgG/IgG4, two identically sized but differently charged molecules. The aim of the present study was to evaluate the effect of ACE inhibition on charge selectivity by comparing SI in type I (insulin-dependent) diabetic patients with microalbuminuria after 6 years of treatment either with or without captopril. Thirty-five of 45 patients participating in a prospective randomized study evaluating the effect of captopril in preventing the development of diabetic nephropathy were included in the present study, 17 being treated with captopril, 18 left as untreated controls. The selectivity index was calculated after measuring s-IgG, u-IgG, s IgG4, and u-IgG4. The results demonstrated a higher selectivity index in the captopril-treated group [1.21 (0.51-1.94) median (range)] compared to the control group [0.94 (0.31-1.87)], however, the difference was not statistically significant (p = 0.16). A negative correlation between the selectivity index and UAE was demonstrated in the captopril-treated group (r = -0.77; p = 0.0004), whereas the correlation in the control group did not reach statistical significance (r = -0.3; p = 0.2).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548981 TI - Components of variance for vibratory and thermal threshold testing in normal and diabetic subjects. AB - Quantitative sensory testing (QST) is commonly used in the assessment of diabetic neuropathy. However, little data are available on the reliability of tactile and thermal testing devices. Reproducibility of QST measures between centers has not been previously reported. This study was designed to validate QST testing procedures and determine if these devices are suitable for large scale multicenter clinical trials. Finger and toe vibratory (Vf, Vt) and thermal (Tf, Tt) thresholds were determined for ten normal individuals by a two-alternative forced-choice procedure using the Optacon Tactile Tester (OTT) and Thermal Sensitivity Tester (TST). Threshold measurements were reproducible between technologists and had a day-to-day coefficient of variation of Vf 20%, Vt 23%, Tf 41%, and Tt 95%. Thresholds were determined for 140 normal individuals at six centers. Mean threshold values between centers were not significantly different. Center-to-center coefficients of variation (CV) were Vf 44%, Vt 45%, Tf 47%, and Tt 87%. There was no significant difference in threshold measures with regard to sex, side studied, presence of calluses, or skin temperature. Vf thresholds significantly correlated with age (p < 0.01). There was no correlation between either vibratory or thermal thresholds in normal individuals, and nerve conduction velocities (NCV). Thermal and vibratory thresholds were determined for 98 diabetic patients. Diabetic subjects without clinical evidence of neuropathy were not significantly different from normal individuals, but diabetic patients with neuropathy had increased thresholds compared to normals (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548983 TI - Nd:Yag laser-assisted balloon angioplasty of superficial femoral artery occlusions in a diabetic population. AB - Laser-assisted balloon angioplasty (LABA), has potential as a treatment modality for re-establishing flow in occluded arterial segments. The objective of this retrospective review is to summarize the results of LABA of totally occluded superficial femoral arteries in 33 limbs among 32 diabetic patients. Indications for vascular intervention in this population included disabling claudication in 15 (45.6%), rest pain in 9 (27.2%), and tissue loss in 9 (27.2%) limbs. The review of preoperative arteriograms allowed for the division of limbs into three groups by occlusion length as follows; less than 5 cm, between 5 and 10 cm, and greater than 10 cm. Overall initial success rate was 21 of 33 (63.6%). Nine of ten (90%) occlusions less than 5 cm in length; six of nine (66.7%) occlusions between 5 and 10 cm in length; and six of 14 (42.9%) lesions greater than 10 cm in length were initially successfully recanalized. Overall cumulative patency rate at 1 year was 22.5%. For occlusion lengths less than 5 cm, cumulative patency rates at 1 year and 2 years were 66.4% and 33.2%, respectively. All lesions greater than 5 cm that were initially recanalized, uniformly re-occluded by 1 year. The number of complications (vessel perforation or dissection) increased as lesion length increased. The theoretic advantages of laser energy's ability to recanalize occlusive lesions are lost when coupled with the balloon angioplasty technique, especially in diabetics. Laser-assisted balloon angioplasty in its present form should be considered an investigational tool requiring further technologic refinements and controlled clinical trials. PMID- 7548982 TI - Occurrence of late specific complications in type II (non-insulin-dependent) diabetes mellitus. AB - The objective of the present study was to determine the occurrence of late specific complications, i.e., nephropathy, retinopathy, and autonomic neuropathy, in type II (non-insulin-dependent) diabetic subjects with a recent onset and with a disease duration of at least 5 years. The study design comprised of a population-based controlled cross-sectional survey of middle-aged type II diabetic subjects in the City of Tampere, Southwest Finland. The mean (SD) albumin excretion rate per 24 h was found to have increased in recently diagnosed diabetic subjects, i.e., 54 (111) mg (p < 0.0001), and in long-term diabetic subjects, 134 (479) mg (p < 0.0001), compared to nondiabetic controls, 16 (19) mg. Microalbuminuria (30 mg/24 h < or = albumin excretion rate < or = 300 mg/24 h) was detected in 8% of nondiabetic subjects and in 29% of recently diagnosed subjects and 27% of long-term diabetic subjects. The prevalence of clinical nephropathy (albumin excretion rate > 300 mg/24 h) was 7% in long-term and 4% in recently diagnosed diabetic subjects and zero in nondiabetic subjects. The differences between diabetic and nondiabetic subjects tested for microalbuminuria and clinical nephropathy were significant (p = 0.02-0.0001) exempting the difference between recently diagnosed female diabetic subjects and nondiabetic female subjects tested for clinical nephropathy. Seventy-five percent of biopsied diabetic subjects with an albumin excretion rate exceeding 100 mg/24 h were found to have diabetic glomerulosclerosis, while the rest had a normal finding. In long term diabetic subjects the prevalence of nonspecific, background and proliferative retinopathies were present in 40%, 31%, and 8%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548978 TI - Factors associated with impaired vibration perception in Mauritians with normal and abnormal glucose tolerance. Mauritius NCD Study Group. AB - Vibration perception threshold (VPT) was measured in 1185 Indian, Creole, and Chinese men and women in Mauritius, where the current prevalence of diabetes mellitus in adults aged 25-74 years is estimated to be 13%. Vibration perception threshold was measured using a biothesiometer, at seven sites in the hand, wrist, foot, and ankle, during a population survey in 76% of 574 known diabetic patients (KDM), 79% of 525 newly diagnosed diabetic patients (NDM), 18% of 1121 subjects with impaired glucose tolerance (IGT), and in 127 normal subjects. The association of VPT with glucose tolerance and other risk factors was assessed in order to identify individuals most at risk of foot ulceration and to determine whether risk factors and normal levels for VPT in these ethnic groups were consistent with those reported for Caucasians. After adjusting for age and height, geometric mean VPT at six of seven sites increased significantly with worsening glucose tolerance and increasing duration of diabetes in both men and women, VPT also increased significantly with level of fasting plasma glucose in men, but not women. Smoking and alcohol consumption had no effect on VPT, and body-mass index (BMI) was positively associated only at some sites. Chinese subjects had lower VPTs than Indians or Creoles. In multiple linear regression models, age, male gender, duration of diabetes, ethnic group, and height (lower extremity sites) were significantly associated with VPT among diabetic subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548980 TI - Long-term effects of antihypertensive treatment and good glycemic control on plasma lipids in diabetic rats. AB - The long-term effects of angiotensin-converting enzyme inhibitors (captopril and enalapril), calcium-entry blockers (diltiazem and nicardipine), and good glycemic control on plasma lipids and lipoproteins were studied in streptozotocin diabetic rats. Diabetic rats had increased plasma cholesterol, tryiglycerides, very-low density lipoprotein (VLDL), and low-density lipoprotein (LDL) cholesterol, and decreased levels of high-density lipoprotein (HDL) cholesterol than in normal rats. Compared to other antihypertensives, nicardipine seems to have a less beneficial effect on lipids and lipoproteins. However, it is only the good glycemic control that normalized these plasma lipids and lipoproteins in diabetic rats. This suggests that good glycemic control prevents dyslipidemia in diabetic rats. The observed beneficial effects of antihypertensives were unrelated to either food or water intake. PMID- 7548984 TI - Investigation of the effect of poorly controlled diabetes mellitus on erythrocyte life. AB - Erythrocyte half-life (Et-1/2) has been measured in 11 patients with poorly controlled blood sugar levels and compared with a normal control group to determine whether decreased red cell deformability, which occurs in diabetic patients, causes shortening of erythrocyte life or not. No difference was seen (Et-1/2 = 28.1 days in diabetic patients and 28.5 days in controls). There was also no correlation between Et-1/2 and glycosylated hemoglobin (HbA1) levels. It has been concluded that poorly controlled diabetes mellitus has no effect upon erythrocyte life. PMID- 7548985 TI - Hematuria in children and adolescents with insulin-dependent diabetes mellitus. AB - Hematuria is not described as a common finding in diabetic nephropathy, and may suggest nondiabetic renal disease. We reviewed the records of 59 children and adolescents with insulin-dependent diabetes mellitus referred to the Children's Kidney Center from 1983 to 1992. Fifty-two patients had clinical and/or biopsy evidence of diabetic nephropathy; 18/52 (35%) had microscopic hematuria at the time of referral. Patients with hematuria on presentation were referred for: hypertension (61%), proteinuria (61%), and decreased glomerular filtration rate (GFR) (11%). For patients without hematuria on presentation, reasons for referral included hypertension (79%), proteinuria (56%), and decreased GFR (3%). When comparing patients with and without hematuria, those with hematuria had a significantly longer duration of diabetes (12.8 +/- 3.1 versus 10.8 +/- 3.7 years, p < 0.05). The groups did not differ significantly with regard to age (18.3 +/- 1.8 versus 17.1 +/- 2.9 years), height (162.2 +/- 10.4 versus 159.3 +/- 11.3 cm), weight (63.9 +/- 10.9 versus 59.4 +/- 14.8 kg), systolic blood pressure (137.2 +/- 11.9 versus 133.2 +/- 13.2 mm Hg), diastolic blood pressure (85.6 +/- 7.6 versus 83.9 +/- 13.4 mm Hg), serum creatinine (1.0 +/- 0.18 versus 1.0 +/- 0.43 mg/dL), blood urea nitrogen (15 +/- 5 versus 13 +/- 4 mg/dL), glomerular filtration rate (117 +/- 34 versus 117 +/- 46 mL/min/1.73 m2), 24-h urine protein (2311 +/- 3862 versus 570 +/- 476 mg/day), or microalbuminuria (75 +/- 41 versus 34 +/- 35 micrograms/min). We detected a significant association between retinopathy and microscopic hematuria (sensitivity 47%, specificity 82%, p < 0.05), but no association between labstix proteinuria or sex and hematuria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7548986 TI - Laparoscopic versus conventional fundoplication. AB - The operative and short-term results of 29 patients who had an open fundoplication were compared to 36 patients who had a laparoscopic fundoplication. The operative time was 2.8 +/- 0.4 and 3.1 +/- 0.3 h for the open and laparoscopic procedure, respectively. The length of hospital stay was 9.2 +/- 0.7 days for the open and 1.8 +/- 0.2 days for the laparoscopic. There were five complications in the open group and one in the laparoscopic group. The follow-up period was 3-32 months and all patients had a barium swallow and esophago gastroduodenoscopy (EGD) for postoperative evaluation. All patients had resolution of esophagitis on follow-up endoscopy, were free of reflux symptoms, and no longer required antireflux medication. Laparoscopic Nissen fundoplication has an operative time that is comparable to the open procedure. The complication rate for laparoscopic fundoplication is acceptable. Patients undergoing a laparoscopic fundoplication have a shorter hospital stay compared to patients with an open procedure. The short-term outcome for the laparoscopic fundoplication group was excellent. PMID- 7548988 TI - Minimally invasive approach in Mirizzi's syndrome. AB - During a 4-year period (November 1990-September 1994), 1152 patients underwent laparoscopic cholecystectomy (LC). In five (0.4%) patients a cholecysto choledochal fistula (Mirizzi's syndrome type II) was diagnosed and a minimally invasive treatment (endoscopy-laparoscopy-interventional radiology) was attempted. The first two cases were converted to open surgery probably because of severe anatomical distortion and inadequate confidence in performing a laparoscopic choledochal repair. The last three patients were successfully treated by minimally invasive procedures. These data indicate that a minimally invasive treatment can be safely attempted through a multi-disciplinary approach in Mirizzi's syndrome. PMID- 7548987 TI - Laparo-vaginal treatment of uterine procidentia. AB - The following is a description of a combined vaginal and laparoscopic repair of vaginal eversion with uterine prolapse (procidentia). There are few procedures that seek to correct the condition while preserving vaginal function. Among them are (1) vaginal approach for sacrospinous fixation, (2) abdominal sling procedures, and (3) abdominal and laparoscopic approaches for promontorial fixation. We are presenting a technical report of a modified sling procedure done via laparoscopy. This technique achieves the functional vaginal reconstruction and avoids the potentially dangerous bleeding associated with the sacral fixation. It has also been our experience that the sacrospinous fixation technique affords limited visual exposure, and henceforth the laparoscopic vaginal sling procedure may be a better alternative. PMID- 7548989 TI - Minimizing trochar site herniation in laparoscopic cholecystectomy. AB - The technique for laparoscopic cholecystectomy (LC) was originally devised and described has remained unchanged because of its efficacy. LC involves a 10-mm trochar in periumbilical region, 10-mm trochar in the epigastrium, and two 5-mm trochars at the right anterior axillary line and right midclavicular line. The exposure and dissection provided by the instruments placed through these trochars is usually adequate and the necessity for additional ports is rare. Our technique modification eliminates the epigastric 10-mm port and replaces it with a 5-mm epigastric port and also eliminates one of the lateral 5-mm ports. Recent articles have reported a significant incidence of Richter's or incisional hernias at the trochar sites. Our modification of the standard technique has the advantage of eliminating a potential incisional hernia at the epigastric port site and further improvement of cosmetic result. PMID- 7548990 TI - The first 100 laparoscopic cholecystectomies in the Republic of Yemen. AB - Laparoscopic cholecystectomy (LC) has become a commonplace operation in most Western nations. We are reporting the results of the first 100 operations in the Republic of Yemen. Although the techniques and the results are similar to those previously described by others, developing nations have unique problems that can also be addressed satisfactorily by minimally invasive surgery. PMID- 7548991 TI - Laparoscopic microwave coagulo-necrotic therapy for hepatocellular carcinoma: a feasible study of an alternative option for poor-risk patients. AB - In spite of the development of techniques for liver resection, preoperative evaluation of hepatic functional reserve and some other therapeutic options such as transcatheter arterial embolization (TAE) and percutaneous ethanol injection therapy (PEIT), liver failure after surgery is sometimes observed. Some patients, who have small liver cancers (under 2 cm in diameter) are diagnosed by various tests, but hepatic resection is not an option for the subset who have lowered hepatic functional reserve. Conventionally, such cases have been treated by TAE and/or PEIT. For the tumors in segments 7 or 8, as termed by Couinaud, which are located just beneath the diaphragm and are difficult to investigate by ultrasonography (US), or to perform PEIT, we have recently applied laparoscopic microwave coagulo-necrotic therapy (LMCNT) with the guidance of US and in some cases, thoracoscopic MCNT (TMCNT), which is performed across the diaphragm to necrotize the tumor, again with the help of US. Here, we report some cases treated by this technique, and the intraoperative color doppler US, which helps evaluate the effectiveness of MCNT. PMID- 7548992 TI - Endoscopic clip-knot suturing technique: preliminary report of application in retroperitoneal ureterolithotomies. AB - A newly developed laparoscopic knot-substituting technique is presented and applied in cases of retroperitoneal laparoscopic ureterolithotomies. The authors substituted the difficult laparoscopic knotting techniques with the technique of using one or two clips to hold the sutures tight, which shortens and simplifies the endoscopic suturing. This technique is a great advantage, mainly in cases of major reconstructive laparoscopic operations. The technique and advantages of retroperitoneoscopy are discussed as well. PMID- 7548994 TI - Incarcerated paraumbilical incisional hernia and abscess--complications of a spilled gallstone. AB - Gallstones may fall into the peritoneal cavity during performance of cholecystectomy. They are more easily retrieved in an open operation. Some controversy exists as to what should be done with gallstones lost during laparoscopic cholecystectomy (LC) because complications of abandoned stones have been reported. This case report describes a patient who presented with an incarcerated hernia and an associated abscess cavity containing a large spilled gallstone, which on computed tomography scan suggested a possible abdominal wall tumor. Spilled stones may cause subsequent problems and should be removed whenever possible, but should not be an indication for conversion to open operation. PMID- 7548993 TI - Laparoscopic minilaparotomy Billroth I gastrectomy with extraperigastric lymphadenectomy for early gastric cancer using an abdominal wall-lifting method. AB - Laparoscopic gastrectomy with extraperigastric lymphadenectomy for early gastric cancer has never been performed because of technical difficulties attributable to the lack of appropriate techniques, the high cost of laparoscopic instruments, and the need for numerous disposable stapling devices. In order to solve these problems, we have designed a method of laparoscopic minilaparotomy using an abdominal wall-lifting method, and a patient with early gastric cancer (depth of submucosa) underwent by this laparoscopic minilaparotomy distal gastrectomy with extraperigastric lymphadenectomy. During his postoperative recovery, the patient requested no narcotic analgesic, and was discharged on postoperative day 14. PMID- 7548996 TI - Laparoscopy-guided percutaneous button gastrostomy in children after previous abdominal surgery. AB - Children with previous abdominal surgery have not been optimal candidates for percutaneous endoscopic gastrostomy (PEG) insertion due to the possibility of inadvertant injury to adjacent structures. Using laparoscopic guidance we successfully inserted a primary PEG button in two children who had multiple previous abdominal procedures. PMID- 7548995 TI - Video-assisted subxiphoid pericardiectomy. AB - We report a case of malignant pericardial effusion with chronic tamponade treated by video-assisted subxiphoid pericardiectomy in a 58-year-old female. The procedure was performed under local anesthesia through a 3.5-cm midline incision over the xiphoid process using a mediastinoscope, a 5-mm telescope connected to the video camera, and an endoscopic grasper and scissors. A large piece of pericardium (5 cm x 6 cm) over the right ventricle was excised. The patient obtained immediate relief and was discharged on the second postoperative day. Follow-up at 4 months showed no recurrence of effusion. Video assistance allows generous pericardiectomy, and as the rate of recurrence has been suggested to be directly related to the extent of resection, video-assisted subxiphoid pericardiectomy offers a theoretical advantage over the conventional approach, as more pericardium can be excised. We recommend that this technique receive further investigation. PMID- 7548997 TI - Laparoscopic surgery in pregnancy: report on two cases. AB - Laparoscopic cholecystectomy (LC) and laparoscopic appendectomy (LA) are well established surgical procedures. Although pregnancy initially was considered a contraindication to laparoscopic surgical procedures, successful reports of LC and LA during pregnancy have appeared in the surgical literature. Two cases are presented in this report, one LC and one LA, both in second-trimester pregnancies. Both pregnancies contained undisturbed and ended with spontaneous vaginal delivery of healthy infants. Laparoscopic surgery in pregnant patients is feasible and provides a reasonable alternative to the standard surgical approach. PMID- 7549000 TI - Ultramicrotomy of semiconductors and related materials. AB - Ultramicrotomy has found novel application in materials research using transmission electron microscopy, for both analytical and high resolution work. Specimens 20-50 nm in thickness with lateral dimensions of up to 100 microns may routinely be prepared, with the additional advantage of precise area location for cross-sectional samples from real devices (e.g., VLSI structures). Much of this work is possible through the availability of diamond knives with various included angles and lengths at reasonable cost. This paper reports on various applications of ultramicrotomy, in particular, lattice imaging of surfaces and interface regions from epilayers of II-VI compound semiconductors and related materials. Ultramicrotomed cross-sections have enabled modern electron beam imaging, diffraction and analytical techniques to be brought to bear near surfaces and across interfaces of multilayer structures, yielding high spatial resolution information on crystallography, defect structure and composition. PMID- 7548998 TI - Metabolic responses to cholecystectomy: open versus laparoscopic. PMID- 7548999 TI - Ultramicrotomy of nanocrystalline materials. AB - The methodology and one of the first attempts to produce transmission electron microscopic (TEM) specimens of nanocrystalline metals, alloys and ceramics by ultramicrotomy are presented. Samples of the pure elements Co, Pd; alloys of Y-12 at. % Fe, Al-7 at. % Ag and W-30 at. % Ga; and ZnO ceramic, were found to section successfully to varying degrees. Advantages of sections prepared through ultramicrotomy over ion beam methods include extensive electron-transparent regions of uniform thickness and absence of ion beam damage. Typical artefacts were observed (knife marks, tearing, pull-out, shear lamellae, section curling, and anodic dissolution) but did not impede TEM analysis significantly. A potentially important effect observed was that of a texture development upon sectioning of the Co and Pd samples. It is thought that this unusual phenomenon results from the extremely fine scale of the microstructure and the purity of the Co and Pd samples, and may be enhanced by frictional heating effects and the state of the knife edge. PMID- 7549001 TI - Electrochemical and microstructural study of oxide films formed electrochemically at microcrystalline Al-Fe-V-Si alloys. AB - A recent advance in metallurgical technology has been the application of rapid solidification techniques to Al alloy production. FVS0812 is the designation given to a microcrystalline Al-based alloy consisting of 8 wt% Fe, 1 wt% V and 2 wt% Si. It is a two-phase alloy, consisting of ca. 27 vol percent of approximately spherical Fe-V-Si-rich dispersoids in an essentially pure Al matrix. The high strength, low density properties of this advanced material, and other related alloys, have not yet been realized, however, due, in part, to the inability of the alloy to form a thick, adherent, abrasion-resistant outer surface oxide film, a feature readily achieved at conventional Al alloys by normal anodizing methods. The present research has involved an electro-chemical study of oxide film growth at the 812 alloy, with the specific goals being to seek an understanding of the origin of the oxide film growth problem and ultimately to propose alternative approaches to the formation of a thick, stable oxide film at this material. The techniques used in this research have included electrochemical methodologies such as cyclic voltammetry and electrochemical impedance spectroscopy. Crucial information has been obtained through transmission electron microscopy (TEM) of ultramicrotomed specimens. Experiments were carried out initially in neutral borate solutions to characterize the compact barrier oxide film formed in this environment and expected to be present beneath the porous oxide film formed in the normal sulfuric acid anodizing medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549002 TI - Development and application of a dry ultramicrotomy technique for the preparation of galvanneal sheet coatings. AB - The formability of galvanneal steel sheets used in the automotive industry is influenced by the presence and distribution of brittle and difficult to distinguish Zn-Fe intermetallics in the coating. Characterization of these intermetallics requires a high spatial resolution technique such as analytical transmission electron microscopy (ATEM). Sample preparation by ion milling is impossible due to iron redeposition, and traditional ultramicrotomy using water affects the coating chemistry. A technique based on dry ultramicrotomy has therefore been developed. To optimize the technique, different parameters (knife angle, cutting medium, thickness setting on the ultramicrotome, cutting speed) have been investigated for the preparation of galvanneal coatings and pure A1 sections. Results show that dry cutting does not affect the coating chemistry but shortens the life of the knife. Knife quality (cleanliness, sharpness and absence of defects) is a major factor to obtain good dry sections. The best results for the more ductile pure A1 are obtained with a 35 degrees knife whilst for the harder galvanneal coating it is recommended to use a 55 degrees knife. These results suggest that the sectioning mechanism for the harder material involves more a cleavage-fracture mechanism whilst a greater amount of shear is involved when sectioning relatively ductile A1. The optimum parameters for sectioning galvanneal coatings are established and results obtained by parallel electron energy loss spectrum imaging and energy dispersive X-ray spectrometry in the TEM are given. This study shows that with a good control of all the sectioning parameters it is possible to obtain good sections repeatedly and rapidly. PMID- 7549003 TI - Ultramicrotomy of industrial alumina. AB - Industrial materials, such as alumina, often pose difficulties in their preparation for TEM examination. Composite and particulate materials are particularly difficult to prepare using conventional thinning techniques, i.e., ion beam and chemical jet thinning. Ultramicrotomy (UM) can be used to produce TEM specimens with a uniform thickness and an unaltered composition. Some crystalline materials, i.e., alumina hydrate, were difficult to section due to conflict between the cutting direction and cleavage planes. Sectioning was successful when these two directions were mutually parallel or perpendicular. At other orientations shattering occurred. Microcrystalline particulate materials, i.e., calcined alumina, were sectioned successfully in particles < 70 microns in diameter. The phases found in industrial alumina particles were gamma, delta, theta, and alpha alumina. Gamma alumina consisted of fine-grained, equiaxed crystallites. The selected area electron diffraction (SAED) patterns indicated poor crystallinity with a distinct hexagonal texture. Delta and theta alumina appeared as an undifferentiated intermediary microstructure of elongated grains. The SAED patterns indicated poor crystallinity, but without a distinct texture. Alpha alumina was found to be a coarse-grained crystalline phase with high diffraction contrast. SAED patterns consisted of fine, randomly oriented spots. Considerable variation was observed in the distribution of phases. In some specimens, discrete particles of gamma and alpha predominated. In others, particles were a mixture of phases representative of the bulk composition. To characterise these samples, TEM of numerous whole particles was required. Ultramicrotomy was the only preparation technique capable of producing such samples. PMID- 7549004 TI - Ultramicrotomy of diamond films for TEM cross-section analysis. AB - Ultramicrotomy has been used to prepare TEM cross-sections of typical hard dielectric, semiconductor, and metal coatings, providing a critical capability in the study of structure-property relationships of thin films. Ultramicrotomy of thin film coatings requires meticulous attention to technique and handling. The sample to be microtomed must be very small, well bonded to the epoxy embedding medium, and precisely oriented. In this article we report the ability to microtome TEM cross-sections of diamond and cubic boron-nitride (cBN) coatings. PMID- 7549005 TI - EM-tomography of section collapse, a non-linear phenomenon. AB - Using back projection for reconstruction and tilt series of Epon or Lowicryl embedded and sectioned material, we demonstrated: (1) a reduction in thickness of 50% for Epon and 80% for Lowicryl sections, and (2) a non-uniform density distribution along the electron-optical axis in sections. The highest density was found at the vacuum exposed side of the section. The formvar side of the section showed a similar increase in density, but not to the same extent. Minimalization of electron exposure, even without pre-exposure, did not affect the reconstructed thickness, nor did it affect the non-uniform density distribution. However, parallax measurements showed that at 150K, collapse of Epon sections does not take place. For EM-tomography of plastic embedded material our findings imply that at the top and bottom portion of the sections the dimensions of the reconstructed structures are distorted, but that in the middle portion the dimensions are reliably retained. PMID- 7549006 TI - Comparative methodological investigations on the cytochemical localization of calcium in brain and inner ear of cichlid fish. AB - Four different methods for calcium precipitation are compared in the optic tectum and the inner ear of the cichid fish, Oreochromis mossambicus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and oxalate-pyroantimonate) produce fine grained deposits, often flocculent in the latter method. The fourth method (potassium-pyroantimonate) generates predominantly coarse-grained reaction product. The calcium content of the deposits is always proven with energy filtering transmission electron microscopy (EFTEM). In both tissues fine-grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and in addition in some mitochondria and at the cytoskeleton. The coarse-grained deposits of the potassium-pyroantimonate method have a more unspecific distribution. This is the only method which produces extracellular deposits in the inner ear, whereas in the optic tectum extracellular precipitates are always present except with the oxalate-pyroantimonate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of the fixation time up to 24 hours leads to an increase of the reaction product, which also becomes coarse-grained. These observations are corroborated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reaction product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the investigated tissue, but it is necessary to optimize a certain method for a given tissue. PMID- 7549007 TI - On the use of local statistical properties in focusing microscopy images. AB - Techniques to extract focus properties of microscopy images are many and varied. Many of them, however, rely on the summation of local statistical properties. Presented here is an examination of conventional focus determination algorithms, and a new method based on planar histograms of these local statistical properties. It is shown that this new method provides finer discernment of the focus properties of an image, and provides a means to extend the focus of an optical microscopy system. PMID- 7549008 TI - Participation of autonomic nervous system in the pathogenesis of Chagas disease. AB - Evidences accumulated over the last decade give adequate proof for the existence of circulating antibodies in Chagas disease which binds to beta adrenergic and muscarinic cholinergic receptor of lymphocytes and myocardium. The interaction of the antibodies with lymphocytes and cardiac neurotransmitter receptors behaving as an agonist, triggers in the cells intracellular signal transductions that alter the physiological behaviour of this cells. These events converted the cells in pathologically active cells. Thus, antibodies activating beta adrenergic receptors of T helper (Th) lymphocytes increase cAMP and releases PGE2 by T suppressor/cytotoxic (Ts/c) cell, inducing in this way, immunosuppression by simultaneous inhibition of Th and stimulation of Ts/c cell function. All these antibodies actions were mimetized by parasite's membranes. On the other hand, the interaction of antibodies against heart beta adrenergic and cholinergic receptors trigger physiologic, morphologic, enzymatic and molecular alterations, that leading to cardiac damage. The analysis of the prevalence and distribution of these antibodies shows a strong association with seropositive asymptomatic patients with autonomic dysfunction in comparison with those asymptomatic without alteration of the heart autonomic disorders; pointing to that the presence of these antibodies may partially explain the cardiomyoneuropathy of Chagas disease, in which the sympathetic and parasympathetic systems are affected. The deposit of autoantibodies on the myocardial neurotransmitter receptors, behaving like an agonist, could induced desensitization and/or down regulation of the receptors. This in turn, could led to a progressive blockade of myocardium neurotransmitter receptors, with sympathetic and parasympathetic dennervation, a phenomenon that has been described in the course of Chagas cardioneuropathy. PMID- 7549009 TI - [Changes in distribution and isoenzymatic expression of protein kinase C in B lymphocytes induced by alloantigenic stimulation]. AB - Protein kinase C comprises a family of distinct isoenzymes that are involved in signal transduction pathway triggered by activation of membrane receptor. These isoenzymes differ in their tissue distribution, activation requirements and substrate specificity. Recent reports have shown the regulation of PKC-isoenzymes expression in physiological and pathological models. Recently, we shown PKC participation in B cells allogeneic response to intact cells and solubilized alloantigen. Here, alloimmunization influence upon PKC isoforms expression on murine B cells was analyzed. Our data indicates that PKC beta is the main isoform expressed on B cells with a lesser amount of alpha, epsilon and zeta and a very small amount of delta isoform. When analyzing B cells from alloimmunized mice we observed an increment of isoforms a and e and a decrease of the beta isoforms while increasing the number of immunizations. An increase of membrane bound PKC enzyme was also observed. Furthermore with increasing number of immunizations a decrease in LPS induce proliferation was found. In contrary, while normal B cells showed non proliferation when stimulated with solubilized alloantigen, they displayed a strong proliferation when they were obtained from animals with 5-6 alloimmunizations. This results shown that alloimmunization induces changes in the distribution and expression of PKC isoenzymes that are correlated to changes in the biological response triggered by B cell stimulation. PMID- 7549010 TI - Inhibitory action of in vitro ethanol and acetaldehyde exposure on LHRH-and phorbol ester-stimulated testosterone secretion by rat testicular interstitial cells. AB - Ethanol and acetaldehyde have been shown to inhibit testicular steroidogenesis. However the mechanism(s) of signal transduction involved in their action is still unclear. We examined the possible involvement of phospholipid-sensitive, calcium dependent protein kinase (protein Kinase C, PK-C) in the intracellular mechanism of action of ethanol and acetaldehyde by stimulating testosterone production in rat testicular interstitial cells with LHRH and the phorbol ester PDBu, both of which activate PK-C at receptor (LHRH) and post-receptor (PDBu) sites. Ethanol (2000 mg %) inhibited 10(-7) M LHRH and 200 nM PDBu-stimulated testosterone production by 81 +/- 4.7% and 60 +/- 20.4%, respectively. Acetaldehyde (20 mg %) reduced the amount of testosterone produced by 10(-7) M LHRH and 200 nM PDBu by 59.4 +/- 1.2% and 52.5 +/- 5.4% respectively. Basal testosterone levels were unaffected by ethanol and reduced by acetaldehyde. However, the functional test of cell viability by preincubating cells with these doses of ethanol and acetaldehyde did not decrease their ability to respond appropriately to subsequent stimulation with LHRH, demonstrating that cell viability was unaffected by incubation with these drugs. The data presented here suggest that direct ethanol and acetaldehyde exposure results in a reduced ability of the testicular interstitial cells to respond to stimulation of PK-C pathway. PMID- 7549011 TI - [XXVI Annual meeting of the Argentine Society of Experimental Pharmacology. Mar del Plata, 26-30 November 1994. Abstracts]. PMID- 7549012 TI - Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA. AB - We present herein an improved method for the use of osmium ammine in a Feulgen type reaction for specific staining of DNA at EM level and an analysis of its Schiff-type reagent behaviour. The activation of osmium ammine to a Schiff-type reagent (so far routinely performed by bubbling with gaseous SO2) can be accomplished by adding S0(2)-generating chemicals as for light microscopy. When used after HCI hydrolysis on epon or acrylate sections, activated osmium ammine behaves like a Schiff-type reagent, and the DNA staining can be selectively and completely abolished by aldehyde blocking agents. This preparation has the advantage of eliminating the use of gaseous SO2 thus rendering the technique more widely available to laboratories which cannot handle gas cylinders containing SO2. We recommend the use of osmium ammine in 8N acetic acid and 40mM sodium metabisulfite for 1 h at 37 degrees C for epon sections, and in 0.2N HCl and 0.2M metabisulfite for 30 min at room temperature for acrylate sections. PMID- 7549014 TI - Cytofluorometric evidence for differential genome endoreplication in the cluster neurons of Lophius piscatorius L. (Osteichthyes, Lophiiformes). AB - Quantitative microfluorometric evaluation indicated that, in Lophius piscatorius, the DNA content of large neurons of a cluster located at the boundary between the medulla oblongata and the spinal cord varied from 4C to more than 5000C. This finding must be considered exceptional for the nervous system of Vertebrates. DNA contents were correlated to both nuclear and animal size. Utilization of AT and GC specific fluorochromes showed that the increase in DNA content is due to differential genome amplification involving GC-rich DNA sequences. PMID- 7549013 TI - Age-related changes in the density of muscarinic cholinergic M1 and M2 receptor subtypes in pyramidal neurons of the rat hippocampus. AB - Age-dependent changes in the expression of muscarinic M1 and M2 cholinergic receptors were assessed in the CA1 and CA3 fields of hippocampus using radioligand binding and autoradiographic techniques with [3H]-pirenzepine and [3H]-AF-DX 116 as ligands. Male Wistar rats of 2 months (young), 12 months (adult) and 27 months (old) of age were examined. Radioligand binding analysis revealed a significant decrease of the density of muscarinic M1 cholinergic receptors with increasing age and no change in muscarinic M2 cholinergic receptors. Autoradiographic evaluation of the number of silver grains developed within the cell body of pyramidal neurons of the CA1 and CA3 fields revealed a decrease of muscarinic M1 cholinergic receptors in the 27-month-old rats in comparison with younger cohorts and no changes in muscarinic M2 cholinergic receptors. These findings suggest that the reduction of muscarinic M1 sites noticed between rats of 2- and 12-months of age using radioligand binding techniques is probably dependent on the loss of hippocampal neurons rather than on the reduction of receptor density per neuron. Our data also indicate that appropriate morphological techniques associated with quantitative analysis may be useful in assessing age-dependent changes in the expression of neurotransmitter receptors by specific neuronal populations. The possible pharmaco-therapeutic relevance of the decreased expression of muscarinic M1 cholinergic receptors by pyramidal neurons of the hippocampus of old rats is discussed. PMID- 7549015 TI - S-100-immunoreactive nerves in the urinary bladder of the rat. AB - The innervation of the rat urinary bladder was investigated by light microscopy with use of S-100 antiserum and the peroxidase-antiperoxidase immunohistochemical technique according to Sternberger. The S-100 protein, a marker for myelinated nervous fibers, was mostly present in the muscle coat and the adventitia at the base of the bladder. Myelinated fibers were not observed in the epithelial layer, nor were ganglia found in the bladder wall. No differences in the amount and distribution of nerve fibers were found in full and empty bladders. Results were correlated with the analysis of the whole nervous component conducted by Ag staining according to Linder. PMID- 7549016 TI - Catecholamines in human dental pulp. A combined immunohistochemical and chromatographic study. AB - Pharmacological studies have suggested that nerve-released catecholamines may play an important role in the regulation of vascular tone and in the modulation of sensory nerve activity in animal teeth. We have used tyrosine hydroxylase immunohistochemistry to detect catecholamine-producing cells in human dental pulp and high performance liquid chromatography to identify and quantitate catecholamines in this tissue. Tyrosine hydroxylase-immunoreactivity was confined to a sub-population of nerve fibres that were mainly localized around blood vessels. Considerable concentrations of norepinephrine (17.8 +/- 3.75 pg/mg tissue) and much lower concentrations of dopamine and epinephrine (0.27 +/- 0.10 and 0.19 +/- 0.11 pg/mg, respectively) were measured in all samples examined. It is suggested that catecholamines in human dental pulp are exclusively contained in nervous structures that are mainly associated with blood vessels and that norepinephrine is the candidate neurotransmitter of these nerve fibres. These data provide the basis to further studies addressed to clarify the possible functions of catecholamines in human dental pulp during physiological as well as inflammatory situations. PMID- 7549017 TI - Morphological and histochemical changes caused by sodium dodecyl sulphate in the gills of giltheads (Sparus aurata, L.). AB - An investigation was made into the biological effects of the anionic detergent sodium dodecyl sulphate (SDS), on the gills of giltheads (Sparus aurata, L.). The fish were exposed to concentrations of 5, 8.5, 10 and 15 mg/l SDS. The surface tension acquired at each concentration was determined and the LC50 calculated. Serious morphological damage to the gills and changes in protein and carbohydrate molecules (demonstrated by histochemical techniques) were observed. The degree of these alterations was dependent upon the SDS concentration and the length of time of exposure. It is suggested that changes in the gills lead to respiratory dysfunction and that this may be one of the major causes of death. PMID- 7549018 TI - Ultrastructural localization of alkaline phosphatase activity in the proximal convoluted tubule cells of the gerbil Meriones crassus using a cerium-based method. AB - Histochemical activity of alkaline phosphatase in the proximal convoluted tubule of the gerbil Meriones crassus has been detected. The reaction product was strongly marked at both the apical surface and the basal part of the cells comprising the basal infoldings with their extended tips and the profiles of the membrane-bound bodies that were easily recognisable by their rounded shape. The reaction product was in the form of fine, uniform deposits in response to cerium chloride as a capture agent. Other cytoplasmic structures did not display these deposits. In addition, the study provided the application of levamisole which caused remarkable reduction of the enzyme activity. Using lead nitrate as a capture agent, the sections showed dense coarse, irregular clusters nearly at the same sites. Control experiments, processed in the absence of the substrate sodium beta-glycerophosphate did not demonstrate the presence of any deposits. The present study, which demonstrates for the first time the presence of alkaline phosphatase activity in the proximal convoluted tubule cells of such desert rodents, does not consider the relationship of the enzyme localization and the basal components of the cells including the nature of the membrane-bound bodies which are still under investigation. PMID- 7549019 TI - Acid phosphatase activity in mating type I and mating type II cell lines of Paramecium primaurelia. AB - The cellular acid phosphatase content, a marker enzyme for lysosomal activity, in Paramecium primaurelia mating type I and mating type II cells was determined by optical laser scanning microscopy. The naphthol AS-TR phosphatase-hexazotized pararosaniline method was used to visualize acid phosphatase activity by the light microscopy. Cell lines of both mating types were tested during culture life, from the early log phase to the death phase. The amount of acid phosphatase was higher in mating type II than in mating type I until the onset of the stationary phase, and then the values reversed. Indeed, during the log phase of growth, mating type II cells formed a higher number of food vacuoles, so that, by taking up a higher amount of bacteria, they sooner became deprived of food. It is suggested that, by lacking nutrients, their synthesis activities and acid phosphatase content were reduced as compared with mating type I cells. PMID- 7549020 TI - Structural and functional hierarchy of eukaryotic cilia and flagella. AB - There are now a variety of methods to investigate the morphofunctional aspects of eukaryotic cilia and cilia. These methods are useful for investigating the basic mechanism of eukaryotic axonemal mechanochemical function and understanding the function and interaction of its components. It is clear that the complex structure of eukaryotic axoneme requires the combination of all these techniques to unravel its mystery. The compositionally simple in vitro microtubule assays are crucial in investigating the functions of different dyneins within an axoneme. However, because such assays do not include other components of the axoneme and the important mechanical feedback present in a beating axoneme, reactivation of the entire structure will continue to play a basic role in the morphofunctional study of eukaryotic axonemes. PMID- 7549023 TI - Physician-assisted suicide, a symposium. Death made too easy or an act of profound compassion? PMID- 7549021 TI - Characterization of the tissue regression process in the uterus of older mice as apoptotic by the presence of Tp30, an isoform of terminin. AB - Whether old uteri that have undergone involution do so by an apoptotic mechanism was examined by the presence of known biochemical and morphological markers for programmed cell death. Terminin, a protein identified by an unique monoclonal antibody, has three forms, Tp-90, Tp-60, and Tp-30: Tp-90 (the 90 kDa form) is only present in growing and quiescent non-growing cells; Tp-60 (the 60 kDa form) is found in senescent cells; and finally, Tp-30 (the 30 kDa form) is found in cells committed to apoptotic death. Biochemical analysis of a protein, Tp30, previously identified as a marker for the commitment to programmed cell death, was performed with both young (5-month-old) and old (24-month-old) C57BL/6J mouse uteri. In addition to biochemical analysis of Tp30 presence in uterine tissue, propidium iodide (PI) staining and DNA framentation by nick-end labelling with fluorescence-conjugated UTP were used to characterize apoptosis-related changes in the chromatin organization of the nucleus. Results indicate that within the old uterus Tp-30 is indeed detected in the tissue extracts and was the major terminin band, while Tp-90 and Tp-60 were the major bands observed in extracts of the younger mouse uterus. The presence of Tp30 in the older uterine tissue suggests that the tissue regression which has occurred in the uterus of older mice may be apoptotic in nature. This suggestion is further supported by the demonstration of increases in the number of cells showing apoptotic morphology, i.e. positive staining with UTP reflecting the presence of nuclei with nicked DNA, localized exclusively in the uterine stroma of older females. The presence of DNA fragmentation, as reflected by UTP staining, was virtually absent from the young uteri. These data suggest that apoptosis may be a part of the cellular mechanism contributing to the regression of uterine tissue in the older female during involution, appearing as an age-dependent event. PMID- 7549022 TI - Capital punishment and the physician: the views of six Rhode Island physicians. PMID- 7549024 TI - The precipitating factor in coeliac disease. AB - In recent years, remarkable progress has been made in the elucidation of cereal protein structure and its relation to coeliac toxicity. Gluten proteins of wheat can be classified according to their primary structure into high-, medium- and low-molecular-weight (HMW, MMW, LMW) groups. Each of these groups contains two or three different protein types having partly homologous, partly unique, structural elements: chi- and gamma-type HMW subunits of glutenin (HMW group), omega 5 and omega 1,2-type gliadins (MMW group) and alpha-type gliadins, gamma-type gliadins and LMW subunits of glutenin (LMW group). Numerous proteins from the same type do exist with only a few modifications of the amino-acid sequence. The structure of the HMW and LMW group proteins can be divided into three and five domains, respectively. Most typical for each type and unique for cereals are the glutamine and proline-rich domains containing repetitive sequences (HMW group: domain B; LMW group: domain I). omega-type gliadins consist almost entirely of repetitive sequences. Rye and barley, closely related to wheat, have protein types homologous to those of wheat. Early investigations showed that wheat gluten and, in particular, the alcohol-soluble gliadin fraction contained the factor toxic for coeliac patients. Equivalent protein fractions of rye, barley and probably oats were also considered to be toxic. The effects of toxic proteins were not destroyed by digestion with pepsin, trypsin and pancreatin. In-vivo (instillation) testing established the toxicity of alpha-type gliadins, and in vitro (organ culture) testing of gliadin peptides demonstrated that the N terminal region (domain I) of alpha-type gliadins is involved in activating coeliac disease. The longest sequences common for toxic peptides were found to be -Pro-Ser-Gln-Gln- and -Gln-Gln-Gln-Pro-. Various in-vitro tests and two in-vivo studies on synthetic peptides support the importance of one or both of these sequences. They do not occur in non-toxic food proteins and are characterized by their ability to form a beta-turn conformation. Although these sequences are probably not sufficient for toxicity in themselves, and other amino-acid residues are additionally required, they could serve as the starting point for further investigation. PMID- 7549025 TI - The gluten-host interaction. AB - Work continues to progress in the unravelling of the molecular interactions involved in the pathogenesis of coeliac disease. The immunogenetics of the disease implicate certain HLA DQ alleles as necessary for subsequent disease development. These HLA molecules have been shown to be necessary in the binding and presentation of gliadin peptides to antigen-specific T cells. Current work is examining the precise HLA-antigen interaction that may lead to the development of antigen-blocking agents. The isolation of antigen-specific T cells has led to the confirmation of a toxic T-cell epitope of the gliadin protein (residues 31-49) and it would appear likely that additional toxic epitopes may be similarly characterized in the near future. No common TCR motifs have so far been detected, although these may become apparent as this work progresses. The gliadin peptide sequence, residues 31-49, has now been demonstrated to be toxic in vivo. Additional toxic T-cell epitopes may also be present within gliadins, but this identification of a toxic gliadin sequence for the first time raises the possibility of future manipulation of the wheat genome (and other toxic cereals) that could lead to the development of new graminae cereals with the properties of wheat, but which do not induce toxicity in patients with coeliac disease. PMID- 7549026 TI - The humoral immune system in coeliac disease. AB - IgA is transported into intestinal secretions to perform exclusion of luminal antigens. The prerequisites are antigen sampling by the Peyer's patch M cells, antigen processing by antigen-presenting cells, and presentation of antigenic peptides by HLA class II molecules to immunocompetent T-cells. The basis for intestinal immunity is the maturation cycle of specifically primed T and B cells from the gut-associated lymphoid tissue via mesenteric lymph nodes and peripheral blood back to the intestinal lamina propria. In coeliac disease, patients are sensitized against gluten and serum gliadin antibodies are often detected. Gliadin antibodies are also found in other gastrointestinal diseases, other disorders and in healthy individuals not carrying the coeliac disease-specific DQA/DQB alleles. On the other hand, serum reticulin and endomysium autoantibodies are both sensitive and highly disease-specific. Positivity in patients with normal jejunal morphology indicates latency of coeliac disease. These tissue autoantibodies are directed against fibroblast-derived extracellular matrix proteins. The immune system is involved in the amplification and perpetuation of the abnormalities of the intestinal mucosa in coeliac disease. The role of antibody in the pathogenesis remains unknown. The author hypothesizes gluten triggered autoimmune mechanism to be operative. PMID- 7549027 TI - T-cell responses and cellular immunity in coeliac disease. AB - Increasing evidence points to a direct role for T cells in the mediation of the coeliac intestinal lesion. There is good evidence for increased local T-cell reactivity, manifest as increased in T-cell activation in the lamina propria and T-cell proliferation in the epithelial compartment. A likely scenario is that gluten elicits antigen-specific responses by lamina propria T helper cells, probably of the Th1 (inflammatory-mediator) subtype, leading to secretion of pro inflammatory cytokines. Such cytokines may have direct effects on intestinal enterocytes, as well as mediating indirect effects by upregulation of MHC antigens and by enhancing the activity of cytolytic T cells. Although gluten specific IEL responses have not been demonstrated by intraepithelial T lymphocytes (IELs), increasing evidence suggests that IELs can act as cytolytic effector cells and hence are likely to exert enteropathic effects under the influence of pro-inflammatory cytokines. PMID- 7549028 TI - Morphology of the mucosal lesion in gluten sensitivity. AB - Gluten sensitivity is associated with a spectrum of mucosal lesions, arbitrarily termed pre-infiltrative, infiltrative-hyperplastic, flat-destructive and atrophic hypoplastic. Histologically and immunohistologically these lesions are all compatible with T-cell-driven events operative at a local mucosal level. They are classifiable either in terms of antibody titres (pre-infiltrative) (see Chapter 10) or by the characteristic disposition of IELs throughout the surface and crypt epithelium. From in-vivo challenges, it has been demonstrated: (i) that all these lesions comprise a dynamically interrelated series of events, culminating in the severe flat-destructive lesion; and (ii) that gluten evokes a dose-responsive infiltration of IELs (CD3+ CD8+ and TCR alpha beta + or gamma delta +) into the epithelium. Apart from that, little is known of the functions of IELs; it is possible they may have little to do with the evolving mucosal pathology of gluten sensitivity. Increasing work seems to support a view, proposed from this laboratory over 10 years ago, that the immune-mediated responses in jejunal tissue in gluten sensitivity arise in the lamina propria, in association with DR+ macrophages and an abundance of CD4(+)-activated lymphocytes. Many other inflammatory consequences flow from these interactions, involving activation of mast cells, eosinophils and neutrophils, elaboration of cytokines and other products of inflammation, and increased hyperpermeability of the microvasculature with upregulation of adhesion molecules. The result is a doubling of lamina propria volumes in the severe flat lesion. Evidence is also given to show that measurable changes in lamina propria inflammation occur with the infiltrative hyperplastic lesion. Symptomatology is not related to the degree of proximal mucosal pathology, but to the extent of the mucosal lesion. Data, although scanty, suggests that lesional pathology involves only 30-50% of the entire small bowel mucosa. Thus, most patients, irrespective of proximal mucosal damage, have latent (or asymptomatic) gluten sensitivity. Symptom development requires additional environmental triggers, of which infection is a major contributor. It should also be noted that, while these various environmental triggers may precipitate symptomatology, they do not advance the severity of the mucosal lesion. PMID- 7549029 TI - Coeliac disease in childhood. AB - Coeliac disease usually presents in infancy or early childhood with diarrhoea, vomiting and interference with weight gain and growth. Withdrawal of dietary gluten is followed by resolution of the symptoms and signs and restoration of normal weight gain and growth; the characteristic subtotal villous atrophy of the jejunal mucosa also recovers. Later re-introduction of dietary gluten will lead to a return of the jejunal mucosal abnormality in the majority and to clinical relapse in many but not all. The severity and timing of both are variable and 5% of children initially considered on clinical, biopsy and gluten response evidence to have coeliac disease appear to develop permanent tolerance to gluten, although mucosal relapse may occur years after the re-introduction of dietary gluten in a minority, emphasizing the need for long-term follow-up. Although a diagnostic and subsequent follow-up jejunal biopsy are necessary to confirm the diagnosis, anti gliadin IgA and IgG, anti-reticulum and anti-endomysium antibodies are now almost totally reliable in identifying children who have coeliac disease and are valuable in monitoring the adequacy of gluten withdrawal. Dietary compliance is frequently poor and regular supervision by a paediatric dietitian is needed; indeed, lifelong supervision to ensure gluten withdrawal is essential to reduce the chance of developing later gastrointestinal malignancy. PMID- 7549030 TI - Coeliac disease in adults. AB - Coeliac disease is a chronic disease characterized by small bowel villous atrophy which impairs nutrient absorption and improves on withdrawal of wheat gliadins and barley, rye and oat prolamins from the diet. Knowledge of the adult form of coeliac disease has greatly improved in recent years. Although this knowledge is not yet sufficiently widespread among referring clinicians, it has, over the past few years, allowed an increasing number of patients to be diagnosed with subclinical forms characterized by minor, transient or apparently unrelated symptoms. As a consequence, our views on the clinical and epidemiological aspects of this condition, the prevalence of which in the general population is believed to be close to 1 in 300, have changed and are still changing. Since it has been demonstrated that a strict gluten-free diet is protective against the complications of adult coeliac disease, it is important that even subclinical and silent forms are diagnosed and treated as early as possible. Non-invasive screening tests, such as anti-gliadin and anti-endomysium antibody estimation, should therefore be used systematically in groups considered to be at risk of coeliac disease. These include first-degree relatives of coeliac patients and patients with insulin-dependent diabetes mellitus, iron-deficiency anaemia, epilepsy with cerebral calcification, recurrent aphthous stomatitis and dental enamel hypoplasia. Other conditions will probably be identified in the near future. PMID- 7549031 TI - The major complications of coeliac disease. AB - Neoplasms constitute the major complication of coeliac disease, and high-grade T cell lymphoma of the small intestine (enteropathy-associated T-cell lymphoma) is the most common neoplasm in this category. HLA genotyping indicates that in patients with enteropathy-associated T-cell lymphoma have the coeliac disease associated DQA1*0501, DQB1*0201 phenotype, although additional HLA-DR/DQ alleles may represent risk factors for lymphoma development. Molecular biological and immunohistochemical studies have shown that the intestinal mucosa distant from the tumour contains clonal populations of small T cells, often of the same clone as the high-grade T-cell lymphoma. These findings suggest that enteropathy associated T-cell lymphoma arises in the setting of coeliac disease and evolves from reactive intraepithelial lymphocytes through a low-grade lymphocytic neoplasm to a high-grade tumour, which is usually the cause of the presenting symptoms. Most cases of chronic ulcerative enteropathy (ulcerative jejunitis) are probably part of the same disease process. If the ulceration occurs at a time when the neoplastic T-cells are of a low grade, morphological recognition of tumour cells in the ulcers may be impossible. Carcinoma of the pharynx and oesophagus, and adenocarcinoma of the small intestine, are increased in frequency in patients with coeliac disease. The increased risk of carcinoma of the oesophagus may be related to vitamin A deficiency. A number of reports have indicated an increased prevalence of various types of chronic hepatitis in patients with coeliac disease, but no coherent view of the cause of this association has emerged. Similarly, patients with coeliac disease have been reported to have various forms of fibrosing lung disease of uncertain causation. In recent years, there have been several reports, mainly from Italy, of a syndrome of epilepsy and bilateral brain calcification occurring in coeliac patients. The pathogenesis of this condition is not known and its prevalence in other communities is uncertain. Splenic atrophy occurs frequently in patients with coeliac disease and is related to the severity of the disease and degree of dietary control. Splenic atrophy predisposes to infection with capsulated bacteria, although mortality studies indicate that infection with these organisms is not a major cause of death in patients with coeliac disease. PMID- 7549032 TI - Dermatitis herpetiformis. AB - Dermatitis herpetiformis (DH) is a relatively rare skin disorder with an estimated incidence of 1:10,000 in the UK. It is characterized by urticarial plaques and blisters on the elbows, buttocks, and knees, although other sites may also be involved. The eruption tends to be persistent: only 10-15% of patients have spontaneous remission over a 25-year study period. The disease is characterized by the presence of IgA deposits in the upper dermis of uninvolved skin and the diagnosis should not be made in the absence of these deposits. Two thirds of patients have a small intestinal enteropathy with villous atrophy as seen in coeliac disease (CD). However, the remaining third also show evidence of a gluten sensitivity in the intestine, as judged by increased lymphocytic infiltration of the epithelium. Villous atrophy also ensues after gluten challenge in those patients with previous normal villous architecture. The initial treatment of the rash is with one of the following three drugs, dapsone, sulphapyridine or sulphamethoxypyridazine. However, the rash also clears with gluten withdrawal. It must be stressed, however, that the average time to achieve significant reduction in drug requirements is 6 months and it can be over 2 years before drugs are no longer required. On re-introduction of gluten the eruption recurs. Patients with DH have a high incidence of auto-immune disorders, thyroid disease, pernicious anaemia, and insulin-dependent diabetes, and should be screened for those diseases on a yearly basis. As with coeliac disease there is also an increased incidence of lymphoma and a gluten-free diet appears to protect patients from this complication. The mechanism by which gluten causes the skin lesions has still to be elucidated, but current investigations implicate lymphocytes and cytokines in the pathogenesis. The original hypothesis of an antigen-antibody reaction in the skin with complement activation causing the skin lesions, may not be correct. PMID- 7549033 TI - Coeliac disease research and clinical practice: maintaining momentum into the twenty-first century. AB - Recent research shows that each word in the definition of coeliac disease, permanent gluten sensitive enteropathy, must now be reviewed, revised or reinterpreted. Permanent--but there are now well-documented cases of acquired disease, and perhaps also partial recovery of gut gluten tolerance. Enteropathy- gluten sensitivity is expressed in a spectrum, with a mild form seen as normal architecture with high count of intraepithelial lymphocytes. Gluten--the provoking agent--Investigators are intensively working to identify the precise toxic sequence, and to establish how this will link in with new genetic information. Mechanism of sensitivity? or hypersensitivity?--Critical to this is new knowledge on the modulation and regulation of immunity to intestinal antigens, including gliadin. A hypothesis is presented, as to the pathogenesis of gluten-sensitive enteropathy, which combines concepts of oral tolerance and of the regulation of expression of delayed type hypersensitivity reactions in the gut mucosa. PMID- 7549034 TI - Stressed rats fail to expand the CD45RC+CD4+ (Th1-like) T cell subset in response to KLH: possible involvement of IFN-gamma. AB - Exposure to stressors effects various aspects of immune function, including the in vivo antibody response. We have previously reported that rats exposed to an acute session of inescapable tail shock (IS) show long-term reductions in anti KLH (keyhole limpet hemocyanin) IgM and IgG. The mechanisms responsible for this suppression are currently unknown. Previous work has suggested changes in CD4+ T cells could be important. We report here that exposure to IS results in a reduction in Con A-stimulated IFN-gamma levels in mesenteric lymphocytes and splenocytes taken immediately after IS termination. In addition, IS exposure prevents the KLH-induced increase in the number of CD45RC+CD4+ T cells (Th1-like) in both the mesenteric lymph nodes and the spleen 4 days after immunization. The failure of KLH to expand the CD45RC+CD4+ subset could be due to the stress induced reduction in IFN-gamma levels reported in cells taken at the time of immunization. Implications of these findings as a mechanism for the decrease in the in vivo antibody response previously reported is discussed. PMID- 7549035 TI - Spatial learning impairment in mice infected with Legionella pneumophila or administered exogenous interleukin-1-beta. AB - The effect of interleukin-1 beta (IL 1 beta) on spatial learning was examined. In one experiment, C57BL/6 mice were given daily injections (100 ng/mouse) of recombinant murine IL1 beta prior to training on the Morris water maze. In another experiment, mice were infected with a sublethal dose of a gram-negative bacterium (Legionella pneumophila; Lp). Mice rendered ill by the infection were given either anti-IL1 beta antibodies (100 micrograms/mouse) or saline and then trained on the water maze. Results indicated that (1) exogenous IL1 beta blocked acquisition of spatial learning, (2) Lp infection attenuated learning on this task, and (3) neutralizing circulating IL1 beta in Lp-infected mice normalized learning despite the continuation of the illness. The data indicate that cognitive impairment may be a component of cytokine-mediated sickness behavior. PMID- 7549036 TI - Pyrogens specifically disrupt the acquisition of a task involving cognitive processing in the rat. AB - In addition to changes in body temperature and other metabolic and physiological responses corresponding to immune activation, pyrogens can induce profound behavioral changes referred to collectively as sickness behavior. One feature of sickness behavior, sometimes reported in clinical settings, but rarely exposed to experimental analysis, is depressed cognitive functioning. The present series of five experiments sought to demonstrate the existence of specific cognitive deficits in rats, independently of any confounding performance effects of pyrogen injections. The behavioral task used, called autoshaping, consisted of presenting hungry Wistar rats with a stimulus (introduction of a retractable lever) that predicted food delivery. Control rats quickly learned to press the lever, although this response does not influence the probability of food delivery. When pyrogens (250 micrograms/kg lipopolysaccharide, 4 micrograms/rat interleukin-1 beta, or 300 mg/rat yeast) were injected to rats during acquisition of this task, they severely disrupted acquisition while the pyrogen was active. The same treatments were, however, without effect on performance when injected later, when performance had stabilized. It is argued that these results demonstrate specific, performance-independent effects of pyrogens on the cognitive processes needed for the acquisition of this task. The results are discussed in terms of the relationship between these effects and the cytokines induced in the brain by pyrogens, and in terms of the exact nature of the cognitive process likely to be affected. PMID- 7549037 TI - Peripheral catecholamines are involved in the neuroendocrine and immune effects of LPS. AB - There is evidence for bidirectional communication between the brain and the immune system. The immune system is subjected to neuroendocrine influences and reciprocally the hypothalamopituitary-adrenal axis is modulated by immune signals. Lipopolysaccharides (LPS), used to mimic infectious/inflammatory diseases, induce a series of stress markers, including modifications of monoaminergic transmission, enhancement of HPA axis activity, and decreased immune activity. In the present work we investigated the participation of peripheral catecholamines in the immune and endocrine responses to LPS in vivo. We studied the effects of LPS after chemical sympathectomy using 6 hydroxydopamine (6-OHDA), which does not cross the brain-blood barrier (BBB) in adults when peripherally injected. 6-OHDA administration was able to interfere with the effects of LPS on immune cells; however, the effects depended on the lymphoid tissue tested. In fact, the depression of mitogenesis induced by LPS was reversed by 6-OHDA in the spleen but not in the thymus. Moreover, 6-OHDA also interfered with the endocrine modifications induced by LPS. This neurotoxin completely or partially inhibited the effect of LPS on ACTH and corticosterone secretion, respectively. Taken together, these results clearly demonstrate that in vivo, the peripheral sympathetic nervous system participates in the immune and endocrine effects of LPS. PMID- 7549038 TI - Altered thyrotropic and somatotropic responses to environmental challenges in congenitally athymic mice. AB - Neonatal thymectomy or congenital absence of the thymus induces morphologic alterations in pituitary somatotrophs as well as in thyroid epithelium. It was therefore of interest to assess somatotropic and thyrotropic cell morphology and the corresponding serum hormone levels in athymic nude mice under basal and stressful conditions, taking as a reference their haired counterparts. Normal (+/+), heterozygous nude (nul+) and homozygous (nu/nu) CD-1 mice were subjected to either 1-h immobilization stress or 2-h cold stress. Serum levels of growth hormone (GH), thyrotropin (TSH), thyroxine (T4), and triiodothyronine (T3) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed lower basal levels of serum TSH, GH, and T3, but not T4, than their heterozygous littermates. Immunohistochemical assessment of somatotropic and thyrotropic cell populations revealed a normal morphology in the athymic animals. Immobilization stress induced a marked reduction in GH and TSH levels in normal mice but had only a weak effect in athymic animals. Two hours of cold exposure caused a comparable increase in serum TSH in normal and athymic animals, whereas the serum T4 and T3 response to cold was greater in the athymic nudes. Cold exposure drastically reduced serum GH levels in normal animals but had only a weak effect in the athymic mice. We conclude that congenital athymia in the mouse is associated with decreased basal levels of serum TSH and GH in the presence of a normal somatotroph and thyrotroph morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but rather, to an altered modulation of pituitary hormone secretion. PMID- 7549039 TI - Naloxone exacerbates intestinal and systemic anaphylaxis in the rat. AB - Following sensitization to ovalbumin (OA), male Wistar rats were pretreated with naloxone (20 mg/kg i.p.) and subjected to antigen challenge (3 mg OA i.p.). Naloxone exacerbated both systemic and intestinal anaphylaxis when injected 10 and 90 min before the antigen challenge. This was evidenced by a more pronounced drop in rectal temperature, higher hematocrit values, and by an enhanced elevation of basal short-circuit current (an indication of the secretory tone of the small intestine studied in Ussing chambers). Pretreatment with an equipotent does of methylnaloxone (200 mg/kg i.p.), a peripherally acting opiate antagonist, exacerbated the indices of intestinal anaphylaxis but had no apparent effect on indices of systemic anaphylaxis. Thus, our data strongly suggest that in the rat, components of the systemic hypersensitivity reaction are mediated through central opioid receptors, whereas the changes in gut function characterizing intestinal anaphylaxis are mediated through peripheral opioid receptors. PMID- 7549040 TI - Antitumor effect of keto-diepoxides isolated from the fungus Nattrassia mangiferae. AB - A novel keto-diepoxide Sch 49209 and its derivative Sch 50672, produced by the fungus Nattrassia mangiferae, inhibited tumor cell invasion through an artificial basement membrane. These compounds, at nontoxic concentrations, inhibited invasion of HT-1080 cells in a dose-dependent manner. The IC50 values for Sch 49209 and Sch 50672 were 0.75 and 8 microM, respectively, when cells were incubated with drugs for 5 h. Sch 49209 inhibited both tumor cell invasion and cell motility to the same extent under conditions that did not cause any apparent cytotoxicity. Sch 4209 and Sch 50672, however, inhibited the growth of ras transformed cells in a semisolid medium in a 5-day culture with IC50 values of 0.6 and 2.4 microM, respectively. They also inhibited the anchorage-dependent growth of pT24 cells in vitro with IC50 values of 0.5 and 0.9 microM for Sch 40209 and Sch 50672, respectively. Using the murine lung epidermoid carcinoma M27 cells implanted SC in mice as a model, we found both Sch 49209 and Sch 50672 inhibited the growth of this tumor at doses ranging from 2 to 10 mg/kg. These compounds also decreased the formation of spontaneous lung metastasis in this model. Sch 50672 inhibited the growth of human tumor xenografts, SW620 and A431, in athymic mode mice. Our data suggest that keto-diepoxides inhibit tumor growth and metastasis and that this activity may be due, in part, to anti-invasive activity. PMID- 7549041 TI - A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents. AB - V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding. PMID- 7549042 TI - Modulation of cisplatin resistance in acquired-resistant nonsmall cell lung cancer cells. AB - To obtain cisplatin (CDDP)-resistant cells possessing the clinically induced resistance phenotype, H-460 nonsmall cell lung cancer cells (NSCLC) were pulse treated with 20, 60, 80 microM CDDP for 1 h every week, respectively. Twelve months later, three CDDP-resistant cell lines (H-460/CDDP20, H-460/CDDP60, H 460/CDDP80) were obtained that exhibit different levels of CDDP resistance (6- to 22-fold), and the possible mechanisms of resistance were studied. These resistant cells were cross-resistant to carboplatin and melphalan, but not to adriamycin or 5-fluorouracil. CDDP resistance in these cell lines appeared to be stable even after 6 months of growth in cisplatin-free medium. There was no evidence of drug accumulation differences between parental and resistant cells. Although both intracellular glutathione (GSH) content and glutathione S-transferase (GST) activity increased 1.5- to 2.5-fold in the resistant cells, only a minimal reversal of drug resistance was observed after buthionine sulfoximine (BSO) treatment, which depleted intracellular GSH levels. An enhancement of DNA repair activity was found in the resistant cell lines and played the major role in the cisplatin resistance phenotype. Using H-460/CDDP80 cells as a model, addition of a nontoxic concentration of pentoxifylline (PTX) significantly enhanced CDDP induced cytotoxicity in a synergistic manner. Furthermore, more prominent reversal of CDDP resistance could be achieved when the resistant cells were pretreated with BSO, followed by PTX and CDDP combined treatment. This provides a rationale for combination therapy in refractory lung cancer using CDDP and two resistance modulators. PMID- 7549043 TI - Cytotoxic potential of monoalkylation products between mitomycins and DNA: studies of decarbamoyl mitomycin C in wild-type and repair-deficient cell lines. AB - Hypoxic regions in solid neoplasms have been associated with tumor recurrence and resistance to several cancer treatment modalities including radiation therapy. Various strategies have been designed to target these resistant cells, including the use of the bioreductive alkylating agent mitomycin C (MC), which exerts preferential cytotoxicity under hypoxic conditions in most cell lines. Analyses of the mechanism of action of MC indicate that this drug can form cross-links with DNA; it is currently thought that this bisadduct is the critical lesion responsible for inhibiting DNA synthesis. Computer-generated models suggest that the MC adduct fits snugly into the minor groove of B-DNA without imposing major distortion on the structure of the DNA molecule. To gain additional insight into the role of cross-linkage in the cytotoxicity of MC, we studied the analogue, decarbomoyl mitomycin C (DMC). The structure of DMC is identical to that of MC with the exception of the substitution of the carbamoyl group at the C-10 position by a nonalkylating hydroxyl group (-OH); this alteration would be expected to prevent DMC from forming bisadducts with DNA. In chemical systems, DMC produces only DNA monoadducts. If indeed it is the MC-DNA cross-links which are responsible for cell kill, one would predict DMC to be less cytotoxic than MC. However, tissue culture studies using DMC revealed that DMC is at least as toxic as MC to EMT6 mouse mammary tumor cells and to wild-type AA8 Chinese hamster ovary (CHO) cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549044 TI - O6-methylguanine-DNA methyltransferase in human brain tumors detected by activity assay and monoclonal antibodies. AB - O6-methylguanine-DNA methyltransferase (MGMT) activity was measured in 68 tissue samples taken from brain. A wide range in activity among samples was observed, with all nonmalignant samples showing transferase activity (Mer+) but approximately 15% of WHO grade II low-grade astrocytomas and WHO grade IV glioblastoma multiformes lacking activity (Mer-). On average, astrocytomas and glioblastomas showed less transferase activity than either nonmalignant tissue or meningiomas. Monoclonal antibodies specific for MGMT showed both cytoplasmic and nuclear staining of Mer+ brain tumor cells in culture but no staining of Mer- cells in culture. In pathology specimens from anaplastic astrocytomas, glioblastoma multiformes, and meningiomas, antibody staining revealed both cytoplasmic and nuclear MGMT, while one sample showed little or no MGMT-specific staining. These results help explain why nitrosoureas have been among the most successful agents in treatment of brain tumors and indicate the subcellular localization for the repair activity, which may be relevant to nitrosourea resistance. PMID- 7549046 TI - Effect of four disintegrins on the adhesive and metastatic properties of B16F10 melanoma cells in a murine model. AB - Four disintegrins, eristostatin, albolabrin, barbourin and echistatin, injected IV into C57BL/6 mice in combination with B16F10 murine melanoma cells, inhibited formation of experimental lung metastases with ID50s of 0.05, 1.0, 0.9, and 3.7 mumoles per mouse, respectively. When injected 1 h after tumor cells, albolabrin, echistatin and barbourin had the same antimetastatic activity, while eristostatin was not active. Eristostatin (IC50 7-8 nM) was more potent than echistatin (IC50 74-75 nM), barbourin (IC50 46-60 nM), and albolabrin (IC50 130-165 nM) as an inhibitor of murine platelet aggregation induced by ADP or tumor cells. Fibronectin was the best substrate for melanoma cell adhesion (95%), followed by laminin (47%) and vitronectin (24%). Albolabrin was the strongest and eristostatin the weakest inhibitor of cell adhesion to all substrata. Adhesion of melanoma cells to albolabrin, echistatin, and barbourin was partially inhibited by monoclonal antibody against mouse alpha v subunit. This antibody bound to B16F10 melanoma cells in suspension and inhibited binding of fluorescein isothiocyanate (FITC)-labeled disintegrins to these cells, being the most effective with FITC-labeled albolabrin. Our study suggests that a major contribution of eristostatin to inhibition of lung colonization is via preferential binding to platelet alpha IIb beta 3 integrin and blocking tumor cells interaction with platelets. A major contribution of albolabrin, barbourin and echistatin appears to be by interference with other integrin receptors on the tumor cell surface. Albolabrin appeared to inhibit RGD-dependent integrins containing alpha v subunit, such as alpha v beta 3 and alpha v beta 1. PMID- 7549045 TI - Reference listings in cancer research. PMID- 7549047 TI - Effects of resection of celiac and pyloric branches of vagus nerve on the interdigestive motor activity of the upper digestive tract and biliary tree. AB - It has been reported that interdigestive motor activities occur in the gallbladder and sphincter of Oddi as well as in the gastroduodenal tract and truncal vagus nerves modulate the gastroduodenal motility pattern. In the present study, the vagal branches which influence on the interdigestive motor activity of these organs were determined in conscious dogs. In the normal dog, interdigestive motor activities closely related to interdigestive migrating motor complex (IMC) in the gastric antrum and descending duodenum were recorded in the gallbladder and sphincter of Oddi. In dogs whose celiac branches of the vagus nerve were chronically resected, cycle periods of interdigestive motor activity in the stomach, duodenum, gallbladder and sphincter of Oddi significantly prolonged as compared with those in the normal dog but the interrelation of them persisted. Quiescent period in motility prolonged and the active period shortened. Resection of the pyloric branches had no influence on the interdigestive motor activities in all organs. The results imply that celiac branches of the vagus nerve modulate the interdigestive motor activity in the stomach, descending duodenum, gallbladder and sphincter of Oddi. PMID- 7549048 TI - Gastric myoelectrical activities in elderly human subjects--surface electrogastrographic observations. AB - To investigate the alteration of the gastric electrical activities by aging, we performed electrogastrography (EGG) on 13 asymptomatic young human subjects under 60 years old (the young group) and 12 subjects over 60 years (the elderly group). Surface EGG was recorded at fasting state for 30 minutes in each subject. We expressed the frequency distribution of surface EGG in each subject with the median value of frequency distribution (FMED) to represent the center of the electrical activities, and the interquartile range of frequency distribution (FIQR) as the indicator of regularity. FMED in the young group was 3.0: [2.9, 3.4] (median: [range]) cycles per minute (cpm), while it was 3.3: [3.0, 4.0] in the elderly group which showed statistically a significant difference between the groups (p = 0.01). FIQR in the young group was 0.5: [0.1, 1.8] and that in the elderly group was 0.55: [0.4, 1.7], which was not significantly different statistically. These data showed that aging increases the frequencies of the rhythm of surface EGG, without disturbing its regularity. This change may be a part of a spectrum of physiological changes accompanied with aging, and are unlikely to have clinico-pathological significance. PMID- 7549049 TI - Effects of NG-nitro-L-arginine on alpha-agonists-induced contraction of aortae from Wistar Kyoto rats and stroke-prone spontaneously hypertensive rats. AB - Differences in the influences of endothelium-derived nitric oxside (NO) on alpha agonists-induced contraction in the aortae of spontaneously hypertensive and normotensive rats were studied by blocking NO synthesis with NG-nitro-L-arginine (L-NNA). L-NNA potentiated the contraction induced by noradrenaline. The potentiation was smaller in the preparation from stroke-prone spontaneously hypertensive rats (SHRSP) than in the preparation from Wistar Kyoto rats (WKY). Similar potentiation was observed in the contraction induced by phenylephrine; the potentiation was also smaller in the preparation from SHRSP. alpha 2 agonists, clonidine and UK-14304 induced dose-dependent contraction only in the presence of L-NNA. The dose-response curves for alpha 2-agonists in SHRSP aorta were different from those in WKY aorta; the maximum tension was observed at the concentration of 10(-6) M in the preparation from WKY, while the contraction further increased up to 10(-4) M in the preparation from WKY. Noradrenaline, clonidine and UK-14304 but not phenylephrine induced relaxation which was blocked by L-NNA. The relaxation was impaired in the preparation from SHRSP in greater extent than that by acetylcholine. It is suggested that basic or noradrenaline stimulated NO release from endothelium decreased in the preparation from SHRSP and that alpha 2-adrenoceptor of both the endothelium and smooth muscle may be altered in the preparation from SHRSP. PMID- 7549051 TI - Surveying donor families: a comparison of two organ procurement organizations. AB - The primary purpose of this study was to identify variables that affect cadaveric donor family satisfaction with the donation process and coping with grief. A secondary purpose was to describe the variation of respondents from two different donor populations to establish whether donor families in one geographic region differ from donor families in another. A survey was mailed to 233 families over a 16-month period. Data analysis of returned surveys was based on frequency distributions, measures of association and factor analysis. Identical items in the Regional Organ Bank of Illinois (ROBI) Survey and the Hartford Transplant Center (HOPO) Survey were compared and tested for significance. Eighty-nine percent of the donor families understood what "brain death" meant, and 85% felt that the donation request was appropriate when it was made less than 6 hours after a diagnosis of brain death. Family members provided the most helpful information about donation to other family members 65% of the time. The two most important reasons for donation were "to make something positive come out of the death" and "family member's belief in helping others". These responses also ranked highest in the HOPO survey. The support of family, friends and religion were the most frequent coping mechanisms for grief. In comparing responses from ROBI and HOPO, significant differences were found on two items relating to agency procedure, but no significant differences were found when family responses were compared. Factors were identified which predicted the desire to donate, the likelihood of increased stress and a "sharing" factor that related to the wishes of the family member and further participation with the organ procurement organization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549050 TI - [Gastric emptying of semi-solid meal in normal man with special reference to relationship between plasma acetoaminophen concentrations and gastric emptying time]. AB - Correlation between gastric emptying time measured by a radioisotope method and plasma acetoaminophen level was studied in 11 preoperative early gastric cancer in semisolid meal. A significant correlation was found between both gastric emptying half-time and plasma acetoaminophen level at 30, 45 minutes after ingestion of meal, and plasma acetoaminophen level at maximum. We conclude that acetoaminophen analysis is useful to measure gastric emptying at semisolid meal. PMID- 7549053 TI - Rejection with duodenal rupture after solitary pancreas transplantation: an unusual cause of severe hematuria. AB - A common urologic complication after pancreas transplantation with bladder drainage is hematuria. However, hematuria is usually mild and self-limiting and rarely requires open surgical intervention. We report an unusual case of refractory hematuria that began 17 days after solitary pancreas transplantation due to severe rejection. The patient developed severe duodenal segment swelling with mucosal rupture that eventually required operative therapy. This case is illustrative of the diagnosis and management of gross hematuria after pancreas transplantation and demonstrates the limitations of cystoscopic techniques. This unusual complication must be considered in the differential diagnosis of refractory hematuria with pancreas allograft dysfunction and may represent a problem unique to solitary pancreas transplantation until better immunosuppressive strategies are available. PMID- 7549052 TI - Toxicity versus rejection--or why conversions between cyclosporine A and FK506 were performed after liver transplantation. AB - The introduction of cyclosporine A (CsA) and FK506 significantly improved the outcome of liver transplantation. However, the postoperative course and outcome of liver transplant recipients in still compromised by rejection, over immunosuppression-induced infection and immunosuppression-associated toxicity. In the present study, we evaluated the reason for conversion between immunosuppressive regimens in 121 patients, 60 treated with FK506 and 61 patients treated with CsA-based immunosuppression. Five patients treated primarily with CsA (8.3%) were converted to FK506 therapy because of refractory acute of chronic rejection within 12 months following liver transplantation (LTX). In 2 patients, conversion was performed after Re-LTX. In 4 of these 5 patients, rejection was successfully treated according to histological and laboratory investigations, while in the remaining patient, graft function improved with persisting histological evidence of chronic rejection. Moderate and severe neurologic symptoms during the early postoperative period, i.e. organic brain syndromes (OBS), seizures, hemiparesis, dysphasia, dysathria and cerebellar symptoms were observed in 21.3% of patients treated with FK506 and in 11.7% of patients treated with CsA (p = n.s.). Five patients treated primarily with FK506 were converted to CsA due to severe neurotoxicity. Early postoperative renal insufficiency was observed to a similar extent with 42.6% of FK506- and 36.7% of CsA-treated patients. 8.3% of FK506-treated patients and 11.7% of CsA-treated patients required hemodialysis (p = n.s.) There patients were converted from FK506 to CsA due to persisting renal insufficiency. Moderate and severe neurologic symptoms were observed more frequently under treatment with FK506 than CsA, and all conversions from FK506 to CsA (13.3%) were performed because of neuro- or nephrotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549054 TI - Long-term follow-up of hepatitis C virus (HCV) infection in liver transplant patients. AB - Chronic hepatitis represents a frequent event after orthotopic liver transplantation (OLT). To ascertain the influence of HCV infection on the clinical and histological outcome of these patients, we have investigated the long-term outcome of 22 patients with end-stage chronic liver disease undergoing liver transplant focusing the attention on the role of different HCV genotypes in determining recurrence and severity of post-OLT liver disease. For all patients blood samples taken before OLT and 3 months, 1, 2 and 3 years after OLT were tested for antiHCV antibodies by two different enzyme-linked immuno-assays and by recombinant immuno-blot II and for the presence and type of HCV RNA by nested PCR (5' untranslated region and core gene primers). Of the 16 pre-OLT antiHCV positive patients, 14 (87.5%) had recurrence of HCV infection while 2 cleared HCV. Pre-OLT genotype recurred in 11 of these 14 patients (2 genotype I) 8 genotype II - in 1 case associated with genotype III - and 1 genotype IV). Of the 6 pre-OLT antiHCV-negative patients, only 1 (16.6%) became persistently HCV infected, with genotypes I and II. The recurrence of genotype II strictly related with development of severe chronic hepatitis while genotype I and IV were associated with milder forms of liver disease and were more easily cleared. PMID- 7549057 TI - The optimal immunosuppressant after liver transplantation according to diagnosis: cyclosporine A or FK506? AB - Since we may soon be able to choose between primarily CsA- or FK506-based immunosuppression, it is important to establish the superior immunosuppressive agent for the individual patient. In the present study, 121 patients, 61 randomly assigned to FK506- and 60 assigned to CsA-based immunosuppression, were analyzed according to the primary diagnosis for liver transplantation. One-year patient survival was similar in all groups. However, the incidence and severity of acute rejection within the 1st year after transplantation was significantly higher in patients transplanted due to HCV disease who were receiving FK506 (58.8%) compared with those patients receiving CsA (27.8%; p < or = 0.05). Furthermore, the incidence of moderate and severe neurotoxicity was significantly higher during the 1st month after LTX in patients transplanted owing to HCV disease treated with FK506 (35.3%) compared with those patients receiving CsA (16.7%; p < or = 0.05). Irrespective of the immunosuppressive regimen, the incidence of early postoperative neurotoxicity was significantly lower in patients transplanted owing to HBV disease, alcoholic cirrhosis and various other liver diseases summarized than in patients transplanted due to HCV disease receiving FK506 therapy. During the 1st year, the incidence and severity of rejection in patients transplanted due to alcoholic cirrhosis and PBC was significantly lower in patients treated with FK506 (11.1% for both groups) compared with those patients receiving CsA (54.5% and 60.0%, respectively; p < or = 0.05. Furthermore, this was accompanied by a lower incidence of toxicity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549055 TI - Clinical importance of pre-morteum blood lymphocytes in cadaver donor tissue typing. AB - We have refined our immunomagnetic bead (IM bead) procedures to isolate pure and viable lymphocyte subpopulation from pre-morteum (PM) blood for cadaver donor HLA typing, preliminary and final crossmatches (XMs). The results of 1220 XMs were compared using T/B lymphocytes isolated either from PM blood or spleen/lymphnode (SPLN) tissue. IM bead technique was used to isolate T/B cells from PM blood and nylon wool column (NWC) technique was used to isolate T/B cells from SPLN. When we compared the outcome of 800 T-cell crossmatches using T cells from PM blood or SPLN of 5 separate cadaver donors, NWC TXMs tended to be more falsenegative for high PRA (> 10%, total 500 XMs) as well as low PRA (< 10%, total 300 XMs) did not reach statistical significance. In contrast, NW BXM (420 B XM) were found to be far more false negative than IM bead BXM regardless of the PRA of the patients. In order to ensure that NWC BXMs were indeed false negative, 23 sera with known anti-DR antibodies were BXMed where antigen-specific B cells were isolated by both the techniques. Our results showed that IM bead BXM identified the DR specificities greater than 90% of the time, the titers of ab specificities were stronger (1:8). In comparison, NWB cell XMs were weak (titers 1:2), and the false negative rate for some ab was as high as 73%. Using IM bead and NWC techniques we compared our turnaround time (TAT) for cadaver donor typing, preliminary and final XMs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549056 TI - Measurement of malondialdehyde as a marker of oxygen free radical production during renal allograft transplantation and the effect on early graft function. AB - We prospectively measured malondialdehyde (MDA), a marker of free radical oxygen damage during 44 renal transplant operations. When corrected for intra-operative changes in plasma volume, there was a significant increase in the ratio of MDA to total cholesterol (x10 3), from a median of 0.32 (0.24-0.44) (interquartile range) to 0.39 (0.31-0.50) at 30 minutes following reperfusion, p < 0.01 and to 0.36 (0.31-0.51) after 60 minutes, p < 0.01; whereas there was no intra-operative increase in MDA in 10 patients undergoing routine elective surgery, who acted as controls. The change in MDA/cholesterol ratio at both 30 and 60 minutes following reperfusion was greater in those patients with poor early graft function (serum creatinine > 250 umol/l at the end of the 1st post-operative week), mean 0.32 (sem 0.08) at 30 min and 0.32 (0.09) at 60 min, compared to those with good function (serum creatinine < 250 umol/l), 0.12 (0.05) and 0.10 (0.04) respectively, p < 0.05. This suggests that the products of oxygen free radical damage can be measured during renal transplantation, and that they may have an adverse effect on early graft function. PMID- 7549058 TI - Hypomagnesemia in renal transplant patients: improvement over time and association with hypertension and cyclosporine levels. AB - Hypomagenesemia is frequently encountered early after kidney transplantation, especially in patients receiving cyclosporine (CsA). However, there have been no studies addressing the natural history of this disorder in adult transplant recipients. We conducted this investigation to study the change in the prevalence of hypomagnesemia over time in renal transplant patients as well as to determine the factors associated with this change. Three patient groups were studied: 24 CsA-treated patients followed longitudinally at 1, 3 and 6 months post-transplant (Group 1a, 1b, 1c); 33 CsA-treated patients at least 2 years post-transplant (Group 2; mean follow-up 55 +/- 25 months); and 31 non-CsA-treated patients at least 2 years post-transplant (Group 3; mean follow-up 132 +/- 57 months). The following parameters were monitored: serum and urine magnesium levels; serum potassium; creatinine clearance; fractional excretion of magnesium; and trough CsA levels. In group 1 patients, longitudinal follow-up showed a significant linear trend for improvement in the serum magnesium over time (1.6 +/- 0.3, 1.7 +/- 0.2, 1.8 +/- 0.2 mg/dl; p = 0.0015) as well as a decline in the whole blood CsA level (316 +/- 103, 251 +/- 82, 194 +/- 67 ng/ml; p = 0.0015) at 1, 3 and 6 months, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549059 TI - Recurrence of hepatitis B virus cirrhosis and hepatocellular carcinoma: an indication for retransplantation? AB - Hepatitis B virus reinfection as well as recurrence of hepatocellular carcinoma are frequent complications in liver allograft recipients. This is the report of a patient who had two liver transplantations with a 6-year interval, both for the same indication-postnecrotic cirrhosis and liver cancer. Following first and second transplantation, hepatitis B reinfection occurred at 12 and 6 months, and allograft cirrhosis developed after 20 and 10 months, respectively. Intrahepatic recurrence of hepatocellular carcinoma was found after 69 months. Two years following retransplantation, the patient is tumor-free, and has normal graft function. In selected patients with hepatitis B-related liver disease, and hepatocellular carcinoma liver replacement is indicated when combined with effective immunoprophylaxis, and other adjuvant therapy. Late recurrence of both diseases is unusual, and retransplantation may come into question. PMID- 7549060 TI - HMGCoA reductase inhibitors and omega-3 fatty acids as adjunctive agents in maintenance immunosuppression after solid organ transplantation. AB - Omega-3 fatty acids have been shown to play a role in preventing development of both acute and chronic rejection. New evidence also suggests that HMGCoA reductase inhibitors may have an immunomodulatory effect and may decrease the incidence of rejection in both animal and human transplant models. Omega-3 fatty acids and HMGCoA reductase inhibitors may be important adjunctive agents in improving short-and long-term allograft function. PMID- 7549061 TI - Clinical features of nosocomial rotavirus infection in pediatric liver transplant recipients. AB - A retrospective survey of nosocomial rotavirus infection in pediatric liver transplant recipients was performed. Immunocompetent children with nosocomial infections served as controls. Co-pathogens were not identified. A total of 12 transplant cases and 12 controls could be evaluated. New onset vomiting occurred in 7/8 cases and 6/11 controls lasting an average of 2.8 days per case and 0.8 days per control (p < .05). New onset fever (>38 degrees C) was noted in 8/12 cases and 9/12 controls. New onset occult blood was noted in 7/11 cases and 1/12 controls (p < .01). A concomitant rise and fall in transaminases was noted in 5/12 transplant recipients. Eleven of the 12 were maintained on constant or increased immunosuppression doses without the development of fulminant disease. The presence of increased days of vomiting and occult blood in stools suggests that rotavirus causes a more invasive process in the intestinal mucosa of transplant recipients compared to immunocompetent children. However, the process remains self-limited despite the use of potent immunosuppressives. PMID- 7549062 TI - Efficacy of octreotide in controlling refractory diarrhea following bone marrow transplantation. AB - We report the use of octreotide in 10 patients with severe diarrhea, refractory to loperamide, following bone marrow transplantation (BMT). Five patients had regime-related toxicity (RRT) and 5 were suffering from acute intestinal graft versus host disease (GvHD). Complete responses were observed for all patients with RRT, with optimal response being observed in the patients with dose escalation to 250 micrograms tds. Only partial responses were observed in the patients with intestinal GvHD. Increased doses of systemically administered cyclosporin-A (CSA) were necessary to maintain therapeutic levels for 2 patients. Octreotide is very effective in controlling RRT diarrhea. It is less effective in the control of GvHD-related diarrhea. CSA levels require close monitoring, whether the drug is administered systemically or orally. PMID- 7549063 TI - Mechanisms of physiological fibrinolysis. AB - The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, which degrades fibrin. Two immunologically distinct plasminogen activators (PA) have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA mediated plasminogen activation is mainly involved in the dissolution of fibrin in the circulation, whereas u-PA mediated plasminogen activation mainly plays a role in pericellular proteolysis. Plasminogen activation is regulated by specific molecular interactions between its main components, such as binding of plasminogen and t-PA to fibrin, or to specific cellular receptors resulting in enhanced plasminogen activation, inhibition of t-PA and u-PA by plasminogen activator inhibitors (PAI) and inhibition of plasmin by alpha 2-antiplasmin. Controlled synthesis and release of PAs and PAIs primarily from endothelial cells also contributes to the regulation of physiological fibrinolysis. The lysine binding sites situated in the kringle structures of plasminogen play a crucial role in the regulation of fibrinolysis by modulating its binding to fibrin and to cell surfaces, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin. PMID- 7549064 TI - Plasminogen activators and plasminogen activator inhibitors: biochemical aspects. AB - Although this chapter does not represent a historical review, it will be clear how the biochemistry of t-PA, u-PA, PAI-1 and PAI-2 has evolved and where we stand in 1994. While the functional activities of the proteins were recognized at least three to four decades ago, highly purified preparations became available around 1980. In the mid-eighties the cDNAs of the proteins were cloned, representing a major breakthrough in the biochemistry of the four proteins. Amino acid sequences were derived from the nucleotide sequences, homologies with other proteins were recognized and larger amounts of (recombinant) proteins became available for research. In addition, mutant proteins were prepared by recombinant DNA technology, enabling investigation of structure-function relationships. This report is mainly based on the latter studies. Detailed information about three dimensional structures of the proteins and the mode of interaction with other macromolecules is still lacking. To obtain this information will be the goal for biochemists in the coming years. PMID- 7549065 TI - Receptors for plasminogen and t-PA: an update. AB - Over the past decade, the existence of cell-surface receptors for components of the plasminogen system, t-PA, u-PA, plasminogen and plasmin, has been demonstrated. Plasminogen receptors have been detected on virtually all cell types tested, and occupancy has also been demonstrated in biological settings. Characteristic features of plasminogen receptors include their relatively low affinity and their extraordinarily high density on many cells. These receptors recognize the lysine binding sites associated with the kringles of plasminogen. Plasminogen receptors include proteins with carboxyl-terminal lysine residues (enolase and annexin II are representatives) and nonproteins, such as gangliosides. Plasminogen binding to cells enhances plasmin activity by augmenting plasminogen activation, increasing the enzymatic activity of plasmin, and protecting plasmin for inactivation by inhibitors. t-PA receptors serve two major functions, clearance and cell-surface localization. The liver is the main organ for t-PA clearance; parenchymal, endothelial and Kupffer cells are all capable of t-PA uptake. Clearance receptors on these cells are heterogeneous and include ones which recognize the carbohydrate side chains of t-PA and ones which take up t-PA: PAI-1 complexes. Receptors which recognize free t-PA also mediate liver clearance, and alpha 2-MR/LRP is a representative of this latter category. Receptors that localize t-PA on cell surfaces serve a profibrinolytic function. Vascular endothelial cells are rich in such receptors, and annexin II is a representative of these t-PA binding sites. Circulating blood cells also bind t PA, and some of the sites on these cells are shared with plasminogen. Cells of neuronal origin are capable of binding t-PA with high affinity; and amphoterin, a protein involved in neurite outgrowth, may be a neuronal t-PA receptor. Overall, the plasminogen system is one of the most widely distributed and versatile of the cell surface-proteinase systems. By activating bound plasminogen by cell-bound plasminogen activators, the cell harnesses the broad proteolytic activity of plasmin. Cells can then utilize this activity to perform functions such as assisting in cell migration. PMID- 7549066 TI - Pathophysiology of fibrinolysis. AB - The observed association between abnormal fibrinolysis and a tendency toward bleeding or thrombosis demonstrates the (patho)physiological importance of the fibrinolytic system. Deficient fibrinolysis represents an important haemostatic abnormality associated with thrombosis. A decreased fibrinolytic activity may be due to a defective synthesis and/or release of tissue-type plasminogen activator (t-PA) from the vessel wall, to a deficiency or functional defect in the plasminogen or fibrinogen molecule or more frequently to increased levels of t-PA inhibitor. Alternatively, excessive fibrinolysis due to increased levels of t-PA, to alpha 2-antiplasmin deficiency or to plasminogen activator inhibitor-1 (PAI-1) deficiency, may result in bleeding tendency. PMID- 7549068 TI - The urokinase/urokinase-receptor system and cancer invasion. AB - u-PA binds with high affinity to its specific GPI-anchored receptor on the cell surface. The binding has at least two important consequences: (1) it enhances the rate of plasminogen activation on the cell surface; and (2) it focuses the u-PA proteolytic activity at the leading front of migrating cells. Several recent findings suggest that surface-bound u-PA is essential for the invasive ability of tumour cells, even if a picture is emerging indicating a concerted action with other proteases, like collagenases and cathepsin B (Kobayashi et al, 1992; Ossowski, 1992; Schmitt et al, 1992; (Dano et al, 1994). In some tumours, e.g. colon, breast and skin cancer, in situ hybridization studies have given an insight into the u-PA/u-PAR tumour biology showing a complex interplay between stromal and cancer cells Dano et al, 1994). u-PA, u-PAR, and PAI-1 tumour content are now well established prognostic factor in breast cancer. This body of knowledge could be used for theurapeutic purposes. For example, a large study with 671 patients has allowed the identification of node-negative patients which, according to their u-PA levels, would need adjuvant therapy (Foekens et al, 1992). Many other tumours, especially colorectal cancer, expect a direct clinical evaluation of u-PA, u-PAR and serpins as prognostic factors. PMID- 7549067 TI - Fibrinolysis and atherosclerosis. AB - The link between impaired fibrinolytic function and CHD has been reinforced considerably in the past couple of years. This has been achieved by a combination of epidemiological, clinical, cell biological and molecular biological studies. The molecular mechanisms for the identified associations between more established risk factors for atherosclerotic disease and impaired fibrinolytic function now need to be disentangled to promote the design of specific drugs that may pave the way for intervention. The possibility that some of the observed relations are epiphenomena should also not be disregarded. The concept of genotype-specific differences in the susceptibility of the individual to common metabolic disturbances needs to be examined in greater detail. Basic research on the role of fibrinolysis in atherosclerosis and its thrombotic complications should be given high priority, because the modulation of fibrinolytic function is likely to become an important approach to prevention. PMID- 7549069 TI - Physiological consequences of over- or under-expression of fibrinolytic system components in transgenic mice. AB - Studies with transgenic mice over- or under-expressing components of the fibrinolytic system, have revealed a significant role of this system in fibrin clot surveillance, reproduction, (vascular) wound healing, brain function, health and survival. The distinct phenotypes associated with single loss and the more severe phenotype associated with combined loss of plasminogen activator gene function suggest that through evolution, both plasminogen activators have evolved with specific but overlapping biological properties. Interestingly, the role of the fibrinolytic system in thrombosis and vascular wound healing became more apparent after challenging mice single deficiencies of plasminogen activators with an inflammatory, or traumatic challenge, respectively. It therefore seems warranted to examine possible consequences of loss of plasminogen activator gene function in other processes including atherosclerosis, neoangiogenesis, inflammatory lung and kidney disease and malignancy. The plasminogen activator knock-out mice with their thrombotic phenotypes are also valuable models to evaluate whether adenoviral mediated gene-transfer of wild-type or mutant plasminogen activator genes is able to restore normal thrombolytic function and to prevent thrombosis. Preliminary evidence suggests that impaired thrombolysis of t-PA deficient mice can be completely restored using adenoviral-mediated gene transfer of rt-PA (Carmeliet et al, 1994c). In addition, analysis of neointima formation in plasminogen activator deficient mice suggests that controlled reduction of fibrinolytic activity in the vessel wall might be beneficial for the prevention or reduction of restenosis. Whether this can be achieved with gene transfer methodologies remains to be defined. PMID- 7549070 TI - Thrombolytic therapy of acute myocardial infarction. AB - Considering the enormous increase in the use of thrombolytic therapy over the last decade, many of the early concepts of thrombolytic therapy have proved to be remarkably robust. Early and sustained restoration of coronary patency remains the ultimate goal. Streptokinase is still extensively used despite evidence that alteplase may, under some conditions, be more effective. Aspirin is of proven efficacy, heparin is important with alteplase but less so with streptokinase. The benefits of early thrombolysis, even if this means pre-hospital administration, have been repeatedly confirmed. On the debit side, more effective thrombolysis seems to go hand in hand with increased bleeding risk, and primary angioplasty seems to be emerging as a viable alternative in high risk patients. More effective regimens tend to be more complex, and the proportion of eligible patients receiving thrombolytic therapy is still relatively low. Better early diagnosis, by methods independent of the electrocardiogram, and simplified but effective treatment regimens using improved thrombolytic agents are likely developments in the near future. PMID- 7549071 TI - Thrombolytic therapy of non-cardiac disorders. AB - Patients with a recent (less than 10 days) proximal deep vein thrombosis of the leg or pelvis are candidates for thrombolysis as the major benefit over heparin seems to be the prevention of the postphlebitic limb, an aim which is still not proven in a satisfactory manner. Nonocclusive thrombi appear to lyse more readily than occlusive thrombi. For this indication the optimal dose regimens for the three thrombolytic drugs (streptokinase, urokinase, alteplase) are not established. Acute massive pulmonary embolism with hypotension or shock should be treated with thrombolytic drugs and, pending the outcome in the first hour, be considered for pulmonary embolectomy. Major acute pulmonary embolism with haemodynamic instability responds well to thrombolysis. Whether thrombolysis is superior to heparin in subacute intermediate pulmonary embolism has not been proven unequivocally in terms of mortality or clinically important endpoints. Systemic administration of thrombolytic drugs for peripheral arterial occlusion has been abandoned for catheter-directed and intraoperative intra-arterial repeated bolus or short-term infusions. The efficacy and safety of intravenous thrombolytic treatment following a major ischaemic stroke is presently being tested in large scale trials; its use must be restricted to experimental protocols. PMID- 7549072 TI - New thrombolytic agents and strategies. AB - Despite their widespread use in patients with acute myocardial infarction, all currently available thrombolytic agents suffer from a number of significant limitations, including resistance to reperfusion, the occurrence of acute coronary reocclusion, and bleeding complications. Several lines of research towards improvement of thrombolytic therapy are being explored, including strategies to enhance the fibrinolytic potency of plasminogen activators and to improve conjunctive antiplatelet or antithrombotic agents. Mutants and variants of plasminogen activators, chimeric plasminogen activators, and conjugates of plasminogen activators with monoclonal antibodies have been constructed, and plasminogen activators from animal or bacterial origin have been evaluated. Some of these new thrombolytic agents have shown promise in animal models of venous or arterial thrombosis and in pilot studies in patients with acute myocardial infarction. Such molecules include mutants of tissue-type plasminogen activator (t-PA) with prolonged half-life and/or resistance to protease inhibitors and staphylokinase. Antiplatelet strategies include the use of platelet glycoprotein IIb/IIIa receptor blocking agents, of thromboxane synthase inhibitors and endoperoxide receptor antagonists. Antithrombotic strategies include the use of selective inhibitors of thrombin, tissue factor or factor Xa. The efficiency and safety of these new agents in man will have to be carefully evaluated. PMID- 7549073 TI - Ischemic heart disease. PMID- 7549074 TI - New developments in the management of ischemic heart disease. Commentary. PMID- 7549075 TI - New aspects of risk factors for the development of atherosclerosis, including small low-density lipoprotein, homocyst(e)ine, and lipoprotein(a). AB - The risk factors for coronary artery disease have been expanded in recent years to include several clinically significant metabolic disorders. The small, dense low-density lipoprotein trait is one of the most common inherited coronary artery disease risk factors and recent reports describe the clinical use of low-density lipoprotein phenotyping for coronary artery disease risk determination, and for treatment in patients with established disease. Apolipoprotein E isoforms play a role in diet responsiveness and may explain approximately 12% of cases of myocardial infarction. Hypoalphalipoproteinemia appears to be a spectrum of overlapping disorders and is difficult to treat. Low-density lipoprotein oxidation may be affected by dietary sources of oxidized fat, and a recent antioxidation trial reported negative results. In the past year, homocyst(e)inemia was reported to play a significant role in coronary artery disease risk prediction and lipoprotein(a) phenotypes appear to clarify the risk of lipoprotein(a). PMID- 7549076 TI - New biochemical markers for myocardial cell injury. AB - New biochemical markers for myocardial cell injury that provide new diagnostic options have recently been introduced. The analytical sensitivity and specificity of the traditional creatine kinase and creatine kinase MB activity measurements have been improved by monoclonal anti-creatine kinase MB antibodies. An assay for creatine kinase MB subforms has proven helpful in early detection of myocardial infarctions and has the potential to guide admissions to coronary care units. Myoglobin was shown to add valuable information to the presenting electrocardiogram in patients with suspected myocardial infarctions. Early assessment of the success of reperfusion therapy can be performed by serial measurements of various markers. The more cardiospecific troponins appear to be advantageous parameters with which to quantify infarct size and to disclose perioperative myocardial infarctions. Troponin T detects minor myocardial cell injury in patients with unstable angina and allows stratification of patients into high- and low-risk categories. Future studies must define the role of the improved diagnostic markers on patient management and costs. PMID- 7549077 TI - The importance of early patency after acute myocardial infarction. AB - The importance of achieving rapid patency of the infarct-related artery during acute myocardial infarction has become well recognized. Early, sustained patency of the infarct-related vessel correlates with improved left ventricular function, better in-hospital outcomes, and lower mortality. Various strategies designed to improve early patency, including "prehospital" thrombolysis, use of an accelerated recombinant tissue plasminogen activator regimen, and immediate angioplasty have been studied. This paper reviews the importance of achieving early patency, the various strategies employed, and the evidence for their efficacy. Future directions in treatment of acute myocardial infarction are touched upon briefly. PMID- 7549079 TI - Thrombolysis in the era of the randomized trials. AB - Thrombolytic therapy in acute myocardial infarction is of established value in recanalizing the occluded coronary artery, reducing infarct size, and decreasing mortality. Here, we review the extensive information provided by large clinical trials on agent and patient selection, timing of treatment, adjuvant therapies, and complications. Early treatment is of prime importance. Tissue plasminogen activator is slightly superior to streptokinase, especially in young patients treated early. Intravenous heparin should be used in conjunction with tissue plasminogen activator, although its role when streptokinase is used is less clear. Aspirin, beta-blockers, and converting enzyme inhibitors are of proven value as adjunctive therapies; nitrates and magnesium are not. Newer antithrombotic and antiplatelet agents (eg, hirudin and glycoprotein IIb/IIIa receptor antagonists) may further improve results. PMID- 7549078 TI - Direct angioplasty for acute myocardial infarction. AB - As a primary treatment modality for acute myocardial infarction, direct percutaneous transluminal coronary angioplasty has traditionally been reserved for hemodynamically compromised patients and those with contraindications to thrombolytic therapy. However, both basic and clinical studies have recently pointed out the advantages of direct angioplasty with regards to infarct artery patency, ventricular function, and clinical outcome. This review highlights and summarizes recent work pertaining to the general application of direct angioplasty for the treatment of acute myocardial infarction. PMID- 7549080 TI - The role of coronary angiography after thrombolysis. AB - Intravenous thrombolysis has become a primary mode of reperfusion therapy for selected patients with acute myocardial infarction. The timing and potential benefits of cardiac angiography and coronary revascularization after thrombolysis in patients experiencing an uncomplicated myocardial infarction have been extensively studied, but remain clinically controversial. This review discusses the potential benefits and timing of acute and convalescent phase cardiac catheterization after thrombolytic therapy in patients with acute myocardial infarction. PMID- 7549081 TI - Antiplatelet, antithrombotic, and lytic therapy for unstable angina, and the appropriate role of revascularization. AB - The management of unstable angina continues to be a challenge for the clinician. Evidence is accumulating that inflammation plays an important role in plaque rupture and thrombosis. Thrombolytic treatment is not indicated in unstable angina, and in fact may even be harmful. The use of new platelet glycoprotein IIb/IIIa receptor antagonists and direct thrombin inhibitors offers great potential in the management of unstable angina. Balloon angioplasty for unstable angina is associated with a high major complication rate, which can be reduced by the use of these novel agents. New techniques such as directional atherectomy do not appear to be safer than balloon angioplasty. PMID- 7549082 TI - Coronary angioplasty versus coronary surgery in the management of angina. AB - Until 1977, coronary revascularization was only possible using open surgical techniques. Approximately 10 years after the introduction of coronary angioplasty, a series of major clinical trials comparing the outcome of patients treated by surgery and angioplasty were planned and executed. The results of these trials are now becoming available and show that in patients with symptomatic disease affecting one, two, or three vessels, angioplasty is a viable alternative to surgery. From a purely medical point of view, the advantages and disadvantages of the two techniques are so finely balanced that patients preference has become an important parameter in selection of a primary intervention. PMID- 7549083 TI - Intracoronary stents. AB - A number of significant milestones were achieved in the field of coronary stent technology in 1994. Two large, randomized multicenter studies demonstrated that stenting has a superior "antirestenosis" effect compared with balloon angioplasty. The application of intravascular ultrasound-guided high-pressure adjunct balloon dilatation for optimal stent expansion and the use of more potent antiplatelet agents improved bleeding complications, shortened hospital stays, and lowered the incidence of stent thrombosis despite reduced anticoagulation following stent placement. Ongoing development of new technologies, including drug-coated and radioactive stents that provide both mechanical support and inhibition of myointimal proliferation, will likely further reduce restenosis frequency. These promising developments will ensure an exciting future for coronary stents as a vital interventional cardiology modality. PMID- 7549084 TI - Atherectomy (directional, rotational, extractional) and its role in percutaneous revascularization. AB - After an initial wave of enthusiasm, atherectomy devices face a number of difficult issues today. The first two randomized studies comparing balloon angioplasty with directional atherectomy, the Coronary Angioplasty Versus Excisional Atherectomy Trial (CAVEAT) and the Canadian Coronary Atherectomy Trial (CCAT), showed no clinical benefit for atherectomy. Data from these trials, as well as other studies, suggest that the mechanism of lumen enlargement with atherectomy may be less beneficial than expected. A number of investigations are currently evaluating the benefit of even more aggressive debulking with directional coronary atherectomy, but in view of the increased incidence of acute complications, it is unlikely that this technique will increase its share in routine coronary angioplasty in the near future. Clinical evaluation of rotational and extractional atherectomy has not reached a conclusive phase yet, but results from registries and single-center observations show that these devices require adjunctive balloon angioplasty in a large proportion of cases, on the one hand, and that they do not solve the issue of restenosis, on the other. The difficulty in delineating the rationale for use of these devices highlights the urgent need for controlled and carefully designed device trials. PMID- 7549086 TI - Atherosclerotic heart disease in the elderly. AB - The elderly represent an increasingly important and challenging subset of the population of patients with ischemic heart disease. They are more likely to have comorbid conditions, atypical presentations, and unfavorable outcomes. Some of these features are undoubtedly related to the structural and functional changes in the cardiovascular system associated with aging. The available data suggest that standard pharmacologic, thrombolytic, and definitive revascularization techniques have important roles in the treatment of these patients, but have been underused. PMID- 7549087 TI - Intracoronary Doppler flow velocity measurements for the evaluation and treatment of coronary artery disease. AB - The functional significance of coronary artery disease is often inadequately revealed by the solely anatomic information provided by coronary angiography, especially with respect to intermediate-severity stenoses and the complex lesions that result from percutaneous coronary intervention. The clinical application of coronary flow velocity measurements using the Doppler flow wire during cardiac catheterization represents a promising means for providing immediate physiologic assessment of coronary artery lesion significance. Recent correlations with translesional hemodynamics and noninvasive perfusion imaging have confirmed the accuracy and reliability of Doppler translesional flow velocity assessment. The use of intracoronary flow velocity data for functional assessment can refine the selection of patients for revascularization. Furthermore, measurements of translesional and distal coronary flow velocity dynamics during procedures provide immediate data regarding the physiologic adequacy of the intervention. The information provided by the analysis of coronary blood flow using the Doppler guide wire can serve to improve the diagnostic and therapeutic capabilities of interventional cardiology. PMID- 7549085 TI - Platelet glycoprotein IIb/IIIa receptor inhibitors in ischemic heart disease. AB - The key role of platelets in the pathogenesis of ischemic heart disease has led to the development of new classes of agents to control platelet function. The platelet glycoprotein IIb/IIIa receptor mediates the final common pathway to platelet aggregation. Drugs that block the glycoprotein IIb/IIIa receptor potently inhibit platelet aggregation. Monoclonal antibodies, cyclic peptides, and peptide-derivative glycoprotein IIb/IIIa inhibitors have been developed. The monoclonal antibody Fab fragment, chimeric 7E3, has been shown to significantly reduce ischemic complications and clinical restenosis after high-risk angioplasty in the large-scale Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC) trial. A number of glycoprotein IIb/IIIa inhibitors have been tested in patients with unstable angina with similarly positive results, and initial trials in patients with acute myocardial infarction are also encouraging. Further evaluation of these agents in large scale trials is currently underway and should help determine the place and appropriate use of these agents in the clinical arena. PMID- 7549088 TI - Platelet-activating factor (PAF)-like phospholipids formed during peroxidation of phosphatidylcholines from different foodstuffs. AB - Previously, we reported that induction of peroxidation of synthetic phosphatidylcholines (PCs) containing a polyunsaturated fatty acid by Fe(2+)-EDTA in the presence of ascorbate resulted in the formation of four types of PCs with an sn-2-oxidatively fragmented acyl group, which had platelet-aggregating activity due to interaction with platelet-activating factor (PAF) receptors. These PCs were compounds with a short-chain monocarboxylate, omega hydroxymonocarboxylate, dicarboxylate, and dicarboxylate semialdehyde residue, respectively. In this study, we investigated the PAF-like lipids formed during peroxidation of PCs from hen egg yolk, salmon roe, sea urchin eggs, and krill in an FeSO4/EDTA/ascorbate system. The platelet-aggregating activities of these oxidized PCs were all inhibited by FR-900452, an antagonist of PAF. The activity of oxidized krill PC, which was equivalent of 89.8 +/- 8.8 pmol 16:0-PAF/mumol of starting PC, was about 5 times those of oxidized PCs from salmon roe and sea urchin eggs, and about 50 times that of oxidized hen egg yolk PC. The PAF-like phospholipids that had different combinations of long-chain alkyl or acyl groups with one of the above four types of short-chain acyl groups were identified by gas chromatography-mass spectrometry. The results indicated that foodstuffs that are rich in 1-O-alkyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine are potential sources of compounds with high PAF-like activity formed by deleterious lipid peroxidation. PMID- 7549089 TI - Antihypertensive effects of angiotensin fragments in SHR. AB - When angiotensin fragments, Val-Tyr and Angiotensin III (ANG III), with potent ACE inhibitory activity were intravenously administered to spontaneously hypertensive rat (SHR), a significant reduction of diastolic blood pressure was observed. After incubation of ANG III with SHR plasma, four fragments with ACE inhibitory activity, Val-Tyr (ANG (3-4)) (IC50 = 26.0 microM), Ile-His-Pro-Phe (ANG (5-8)) (11.6 microM), Tyr-Ile-His-Pro-Phe (ANG (4-8)) (457.5 microM), and Val-Tyr-Ile-His-Pro-Phe (ANG (3-8)) (6.55 microM), were confirmed to generate in SHR plasma. Compared the metabolic behavior of ANG II in SHR plasma with that in normotensive Wistar plasma, the initial degradation rate (3.07 nmol/ml/min) in Wistar plasma was about 2-fold higher than that in the SHR one (1.75 nmol/ml/min). PMID- 7549090 TI - Inhibitory effect of phosphorylated oligosaccharides prepared from potato starch on the formation of calcium phosphate. AB - The inhibitory effect of phosphorylated oligosaccharides, which were prepared from potato starch, on the formation of calcium phosphate in vitro were investigated. Phosphorylated oligosaccharides from potato were fractionated by ion-exchange chromatography into two fractions, PO-1 and PO-2. Fraction PO-1 was composed of maltotriose, maltotetraose, and maltopentaose to which one phosphate group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphate groups were attached. The average degree of polymerization of dephosphorylated PO-1 and PO-2 was evaluated to be 4.02 and 5.82, respectively. Fraction PO-2 was the main component having an inhibitory effect on calcium phosphate formation. In addition, among the phosphorylated monosaccharides, glucose-1,6-diphosphate and fructose-1,6 diphosphate were more effective inhibitors of the formation of calcium phosphate than glucose-6-phosphate and fructose-6-phosphate. These results suggest that the strength of the inhibitory effect might depend on the number of phosphate groups attached to each sugar molecule. PMID- 7549091 TI - Prodigiosin 25-C suppression of cytotoxic T cells in vitro and in vivo similar to that of concanamycin B, a specific inhibitor of vacuolar type H(+)-ATPase. AB - The effects of prodigiosin 25-C (PrG) which preferentially suppresses cytotoxic T cells (CTL), was examined in comparison with concanamycin B (CMB), a specific inhibitor of vacuolar type H(+)-ATPase (V-ATPase). PrG and CMB directly inhibited the cytotoxic function of CTL and neutralized acidic organelles of CTL in vitro. In addition, PrG or CMB was injected in C57BL/6 mice after immunization with an allogeneic mastocytoma, P815. PrG and CMB inhibited the killing activity of CTL against the tumor and reduced the population of CD8+ cells without affecting CD4+ and B220+ populations in the spleen. PrG and CMB had only a negligible effect on antibody production induced by sheep red blood cells (SRBC) and mitogenic responses of lymphocytes. These results suggest that PrG and CMB have similar immunosuppressive properties at least through their inhibitory effects on acidification of intracellular organelles required for the effective function of CTL. PMID- 7549093 TI - Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation. AB - Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation. The transformation efficiency (transformants per microgram plasmid DNA) decreased with increases of size of the DNA. However that of 2.0 x 10(3) transformants per microgram of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp. The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA). The transformation efficiency as high as 9.3 x 10(4) transformants per microgram of purified plasmid pUB110 was obtained, using a cell concentration of 7.6 x 10(10) cells/ml and DNA concentration of 4 micrograms/ml in buffer containing 0.3 M sucrose, 1 mM CaCl2, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 mus with a single squared pulse at 0 degrees C. The gene expression for antibiotic resistance after electroporation was completed within 1.5 h. The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis. PMID- 7549092 TI - Recognition system for dietary fatty acids in the rat small intestine. AB - Linoleic acid and oleic acid markedly increased the influx of 45Ca into isolated intestinal epithelial cells, and this increase reflected a rise in the intracellular calcium level. Methyl linoleate had no effect, while glutamic acid and somatostatin both inhibited the linoleic acid-induced influx of 45Ca. In addition, methyl linoleate had no effect, while glutamic acid inhibited linoleic acid-induced hormone-responsive pancreatic exocrine secretion. PMID- 7549094 TI - Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter. AB - The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction. PMID- 7549095 TI - Cloning and nucleotide sequence of the calmodulin-encoding gene (cmdA) from Aspergillus oryzae. AB - A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene. PMID- 7549096 TI - Defatting and desalting treatment of indonesian dried-salted fish: dietary effects on alpha-tocopherol and peroxide levels in the serum and liver of rats. AB - The main problem with dried-salted fish (DSF) products is lipid oxidation. PUFA of fish oil is very easily oxidized, and sodium chloride is known to be a pro oxidant. Many researchers have found that the products of lipid oxidation had negative effects on a variety of species, so we evaluated the effect of a desalting and defatting treatment on the lipid oxidation of Indonesian DSF. The dietary effect of untreated DSF, defatted DSF and desalted DSF on diarrhea, on the internal organs, on hepatic, serum, and urinary lipid peroxidation, and on hepatic and serum alpha-tocopherol were evaluated by using rats. The defatting treatment had a significant effect (p < 0.01) on reducing the lipid oxidation variables of the DSF sample and on protecting the rats from diarrhea. Compared with the rats in the casein group, these in the untreated DSF group had significantly higher values (p < 0.05) for hepatic, serum and urinary lipid peroxidation, but significantly lower values for hepatic and serum alpha tocopherol. No significant differences were observed between the rats fed with casein and defatted DSF. PMID- 7549097 TI - Kinetic analysis of yeast inactivation by high pressure treatment at low temperatures. AB - Inactivation of Saccharomyces cerevisiae by high pressure treatment from 120 to 300 MPa in the range of -20 to 50 degrees C followed pseudo first order reaction kinetics. The regression analysis of 43 inactivation rates showed that pressurization at sub-zero temperatures (-20 and -10 degrees C) enhanced the effects of pressure as pressurization at higher temperatures: i.e., pressurization at 190 MPa and -20 degrees C gave the same effect as pressurization at 320 MPa and room temperature. The results imply that high pressure treatment applied to food sterilization at lower temperatures has a greater effect with smaller pressure without destroying the original taste and flavor. Additional effects of sugars and salts on inactivation of yeast are also described. PMID- 7549098 TI - Prediction of stroke lesions in stroke-prone spontaneously hypertensive rats by glutathione peroxidase in erythrocytes. AB - The incipient timing of cerebral strokes in the stroke-prone spontaneously hypertensive rats (SHRSP) was biochemically determined by investigating the relationship between the glutathione peroxidase (GSH-Px) activity in erythrocytes and the extent of stroke lesions. When the blood pressure of SHRSPs was maintained at over 240 mmHg, the GSH-Px activity fell, and the body weight also decreased. In SHRSP whose GSH-Px activity in erythrocytes had dropped below 23 units/ml of blood, the incidence of cerebral strokes was 98% (n = 88/90). The hematocrit level did not change even after the GSH-Px activity had dropped to 23 units/ml of blood. The reduced GSH-Px activity in erythrocytes observed during continued hypertension was found to be due to a decrease in GSH-Px protein, and not to any inactivation of the enzyme, as evident from immunochemical titration. At the moment when the GSH-Px activity had dropped to 23 units/ml of blood, and the control diet was changed to one based on fish or a hydralazine treatment given, the activity recovered, and an increase in body weight and prolongation of the life-span were observed. It was deduced from these findings that tracing the GSH-Px activity in erythrocytes in SHRSP would serve as an indicator for predicting and prognosing stroke lesions. PMID- 7549099 TI - Stimulation of insulin action and stabilization of cell membrane in 3T3-L1 cells by glycinin acidic subunit A1a. AB - Glycinin acidic subunit A1a and its trypsinized product (A1a/Try) were found to potentiate insulin-mediated antilipolysis antagonized to isoproterenol in 3T3-L1 preadipocytes and adipocytes. A1a and A1a/Try protected 3T3-L1 preadipocytes from the cytotoxic action of trypan blue. Furthermore, the oxygen consumption in rat liver mitochondria that was accelerated during incubation, which implies an increase in the permeability of the inner membrane, was suppressed by the addition of A1a and A1a/Try. On the other hand, the chymotryptic product of A1a did not show such effects. These results suggest that specific amino acid alignment is required for the functions of A1a and that the insulin-stimulating action of A1a and A1a/Try is closely correlated with their stabilizing effect on cell membranes. PMID- 7549100 TI - Metal-characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1. AB - N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was a zinc metalloenzyme which contained 2.06-2.61 g.atom of Zn per mole of enzyme. The zinc atom was required for the catalytic activity and stability of the enzyme. The N terminal amino acid sequence of Pseudomonas sp. 5f-1 D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp. xylosoxydans A-6. PMID- 7549101 TI - Histamine synthesis by cells of the macrophage lineage in bone marrow of mice. AB - An injection of Escherichia coli lipopolysaccharide (LPS) increased the activity of histidine decarboxylase (HDC) in bone marrow (BM) cells of C3H/HeN mice much more than in C3H/HeJ mice, which are resistant to various effects of LPS. In WBB6/F1 (W/Wv) mice, which are genetically deficient in mast cells, HDC activity increased more than in C3H/HeN mice. Cultured BM cells of W/Wv mice spontaneously synthesized histamine in a HDC-dependent way. LPS caused a slight increase in HDC associated histamine synthesis by these cells. Treatment of the BM cells with murine recombinant granulocyte-macrophage colony stimulating factor (mrGM-CSF) increased the histamine synthesis. In addition, treatment with mrGM-CSF made the cells respond to LPS by a dose-dependent increase in HDC activity and histamine synthesis. Most dish-adherent BM cells that had been treated with both mrGM-CSF and LPS for 48 h were stained for nonspecific esterase and not for chloroacetate esterase, and had twice as much HDC activity as the nonadherent cells had. Immunocytochemical analysis of the BM cells of W/Wv mice treated with both mrGM CSF and LPS showed that HDC was in the cytoplasm of cells having Mac-1, a macrophage-differentiation antigen. These results suggest that cells of the macrophage lineage in the BM of mice synthesize histamine. PMID- 7549102 TI - Production of insulin-like growth factors and their binding proteins in primary cultures of rat liver parenchymal and nonparenchymal cells. AB - Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells. Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP 1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191-1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-1 into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu2cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-1. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu2cAMP, and triiodothyronine (T3), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-1 was increased by Bu2cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations. These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver. PMID- 7549103 TI - Nucleotide sequence and expression of alpha-glucosidase-encoding gene (agdA) from Aspergillus oryzae. AB - We have isolated an alpha-glucosidase(AGL)-encoding gene (agdA) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus niger gene as a probe. Southern hybridization analysis showed that the agdA gene is on a 5.0-kb ScaI fragment and there is a single copy in the A. oryzae chromosome. Comparison with the A. niger agdA gene indicated that the agdA gene contains three putative introns from 52 to 59 nucleotides long, and that it encodes 985 amino acid residues. The deduced amino acid sequence of A. oryzae AGL is 78% homologous with the A. niger AGL. The high degree of homology with the amino acid sequence bordering the putative catalytic residue of a number of AGL enzymes, and this enzyme suggests that Asp492 is a catalytic residue of A. oryzae AGL. The cloned gene was functional. Transformants of A. oryzae containing multiple copies of the cloned agdA gene showed a 6-16 fold increase in AGL activity. Like the Taka-amylase A and glucoamylase genes of A. oryzae, expression of the agdA gene was induced when maltose was provided as a carbon source, but expression was not induced by glucose. This result suggested that cis-element(s) involved in maltose induction may be also present in the agdA promoter region. PMID- 7549104 TI - Molecular cloning of the genes for pyruvate kinase of two bacilli, Bacillus psychrophilus and Bacillus licheniformis, and comparison of the properties of the enzymes produced in Escherichia coli. AB - The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5 phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different. PMID- 7549105 TI - Absorption of (-)-epigallocatechin gallate into the circulation system of rats. AB - The absorption of (-)-epigallocatechin gallate (EGCg) into the circulation system was studied in rats. EGCg was detected in rat plasma after an oral administration of 50 mg, the concentration of EGCg in the plasma reaching the highest level about an hour after dosing, and then decreasing quickly. PMID- 7549106 TI - 1'-Acetoxychavicol acetate as an inhibitor of phagocytosis of macrophages. AB - We screened extracts of edible plants for inhibitors of phagocytosis by peritoneal exudate macrophages. 1'-Acetoxychavicol acetate was isolated from the ethyl acetate extract of Languas galanga, and this compound strongly inhibited phagocytosis at an IC50 value of 1.2 microM with negligible effects on pinocytosis and cell viability. Target(s) of 1'-acetoxychavicol acetate was suggested to be downstream of the signal transduction pathway that is mediated by protein kinase C. PMID- 7549107 TI - Mutational analysis of the fic promoter recognized by RpoS (sigma 38) in Escherichia coli. AB - fic gene expression at the stationary phase is dependent on RpoS (sigma 38). For this paper, the fic promoter (P fic) mutants were isolated; C at the position -34 was changed into T. They did not show the stationary phase-specific expression dependent on RpoS in vivo. Furthermore, it was confirmed that they were recognized by E sigma 70 as efficiently as E sigma 38 in vitro. PMID- 7549108 TI - Segmental motion of subsite C of hen egg-white lysozyme: fluorescence depolarization studies of Kyn62-lysozyme as an active analogue. AB - The dynamical properties of subsite C of hen egg-white lysozyme were investigated using Kyn62-lysozyme as an active analogue. Time-resolved fluorescence depolarization studies showed that the segmental motion of kynurenine which was important in subsite C was described with two components of which the rotational correlation times were phi 1 = 150 ps and phi 2 = 1.4 ns, respectively. Although these two segmental motions retained 90% of motional freedom, the slower motion was completely restricted and the degree of freedom was lost to 40% during the interaction with oligomers of N-acetyl-D-glucosamine. PMID- 7549109 TI - Purification and properties of two chitinases from Streptomyces sp. J-13-3. AB - Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45 degrees C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi A and increase of Chi-B was observed in their productions during cultivation. PMID- 7549110 TI - Modification tolerability of soybean proglycinin. AB - Proglycinins containing multiple modifications, that is, with two kinds of single modifications, designed previously to improve food functions, were assessed for ability to form proper conformations. This was done to discover the modification tolerability of proglycinin. Based on our results, three new criteria are proposed for judging formation of proper conformations. Our results suggest that proglycinin molecules tolerate different combinations of single modifications without misfolding. PMID- 7549111 TI - Primary structure of an allergenic peptide occurring in the chymotryptic hydrolysate of gluten. AB - An allergenic peptide was isolated from the chymotryptic hydrolysate of gluten by gel filtration and HPLC. The primary structure of the peptide was determined as Ser-Gln-Gln-Gln-Gln- Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Gln Pro-Pro-Phe- Ser- Gln-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe. The amino acid sequence similarity shows that the peptide originated from a low molecular-weight glutenin chain. PMID- 7549112 TI - Excretion from rats of ketone bodies and methylmalonic acid in urine resulting from dietary vitamin B12 deficiency. AB - Ketone bodies were assayed in the urine of ten-week-old vitamin B12-deficient rats by a high-performance liquid chromatography. The urinary excretion of ketone bodies (352.5 +/- 68.3 mumol/day) as well as of methylmalonic acid was increased significantly by dietary vitamin B12 deficiency. PMID- 7549113 TI - Identification of the essential regions for LukS- and H gamma II-specific functions of staphylococcal leukocidin and gamma-hemolysin. AB - Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leudocidin and LukF and H gamma II for gamma-hemolysin. To identify the pivotal domain(s) of LukS and H gamma II responsible for their specific cytolytic activities, a series of chimeric genes (lukS/hlg2) were constructed and expressed in Escherichia coli. The results indicate that the C-terminal 24-residue and the N-terminal 57-residue segments of LukS and H gamma II, respectively, are the essential regions for the LukS and H gamma II functions. PMID- 7549114 TI - [Radiotherapy and Ewing tumor in children]. AB - Despite high local control rate in Ewing's sarcoma the exact indication of radiation therapy is still controversial as well as the choice of the target volume and the optimal dose of radiation. The importance of the quality of radiotherapy has been stressed in recently published data and has shown a significant impact on long term local control with adequate radiation therapy. The dramatic improvement of precision allowed by the conformal therapy and three dimensional dosimetry allow to expect a decrease of late effects expected for second malignancies. However, late sequellae and radio-induced osteosarcoma still remain the major side effects after radiotherapy. The authors discuss the results of the main trials on Ewing's sarcoma on the choice of dose and target volume. Surgery is still the preferred choice for small tumors if the foreseen outcome is identical. PMID- 7549115 TI - [Cytotoxic chemotherapy in elderly patients: present and future]. AB - Cancer in elderly people accounts for more than 50% of the malignant tumors treated per year in France and this population of patients has a rather high-life expectancy. Chemotherapy is active in these elderly patients but clearly more toxic than for young ones. The general tendency among the physicians to empirically reduce the doses is due to the known increased risk of unexpected toxicities. That is why there is such a large variety of conflicting opinions in the literature concerning the benefit and toxic effects of cytostatic drugs in the elderly. Therefore, it appears consistent to adjust chemotherapy regimen according to physiological criteria. Among them is biological age which is a better parameter than chronological age to describe the biological heterogeneity of this population of patients. Nakamura et al have published an interesting model for the calculation of biological age by principal component analysis using 11 easily measurable biological and clinical variables in a series of healthy elderly people. This kind of approach is not at present available for cancer patients but it allows to demonstrate that the chronological age is only one among many other age-related variables and is not sufficient to fully describe it. The variations in pharmacokinetic data are more frequent in the elderly than in younger people and this reflects age-related physiological heterogeneity. This factor is well taken into account in recently described population pharmacokinetic models, bayesian fittings and adaptative control which may represent promising approaches of cytostatics dose adjustments. Such models have been successfully developed in young patients receiving doxorubicin, methotrexate, melphalan and teniposide. They require a low number of blood samples to determine individual parameters and further adjust the doses, and are therefore of potential interest in old patients. Prospective studies are warranted in the future in order to recommend their use in the elderly. PMID- 7549116 TI - [Several transcription factors participate in the functioning of the alpha fetoprotein gene promoter]. AB - The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity. PMID- 7549117 TI - [Vertebral osteosarcoma. Review of the literature apropos of a case]. AB - Osteosarcoma of the vertebral column (OSV) is a rare tumor which represents 0.85% to 2% of all osteosarcomas. In 95% of the cases they manifest themselves through pains and 80% of other cases through neurological disorders. Usually located on lumbar vertebrae it can also be found on the rest of the vertebral column. Its radiologic aspect is one of lysis in 48% of cases but a condensation can also be met in 27% of cases. The differential diagnosis with an osteoblastoma is difficult and must be left in the hands of the pathologist who bases it on precise criteria (cellular pleomorphism, stroma, presence of giant cells...). The secondary osteosarcoma of the vertebral column represents 30% of all cases of OSV. The heterogeneity of the studies has made it difficult to quantify them. The prognosis of OSV is poor: survival average is of 15.3 months and relative risk of recurrence compared to a femoral lesion is of 3.9 months. PMID- 7549118 TI - [Value of inhibin as a tumor marker in granulosa cell tumors. Apropos of 6 new cases treated at the Gustave-Roussy Institute]. AB - Lappohn et al showed that inhibin can be used as a marker of granulosa cell tumors (GCT). Inhibin ia s glycoprotein specifically produced by ovarian granulosa cells. This peptide has been given in six women of 45 patients with a GCT who were referred for treatment or judgment in the Institut Gustave Roussy of Villejuif. Inhibin remained undetectable in four patients without evidence of disease. In one of them, inhibin level decreased progressively after surgical resection (for 10 months). Inhibin was also given in two patients with recurrence: in one case inhibin level was very high, and in one case it was barely detectable. In finding with other publications, inhibin can be used as a maker for reccurent disease, when patients are menopaused even when tumor does not produce steroids. PMID- 7549119 TI - [Efficacy of the tumor necrosis factor-alpha (rTNF-alpha) associated with interferon-gamma and chemotherapy in extracorporeal circulation in the limb in inoperable malignant melanoma, soft tissue sarcoma and epidermoid carcinoma. A 4 year experience]. AB - The authors review their experience of 4 years with isolated limb perfusion for the application of high dose TNF-alpha associated to IFN-gamma and melphalan for the treatment of regionally advanced tumours such as malignant melanoma, soft tissue sarcoma and epidermoid carcinoma. In malignant melanoma, the complete remission rate reaches 91%. In irresectable soft tissue sarcoma, this treatment when used as a neoadjuvant treatment saves the limb from amputation in 87.5% of the cases. Similar results are obtained for epidermoid carcinoma. With the regional application of high doses of TNF-alpha associated to chemotherapy and IFN-gamma, it has been possible to validate the concept of a strategy based on a dual targeting, that is the selective impact of the intratumoral vessels by TNF alpha and of the tumour cells by chemotherapy. This approach appears to be the treatment of choice for locally advanced tumours of the limbs. However, as a single therapy, this procedure should be considered in melanoma as an induction therapy, and in sarcoma, as a preoperative treatment. PMID- 7549120 TI - [Statistical study of a series of 672 cases of carcinoma of the uterine cervix. Results and complications according to age and modalities of treatment]. AB - The study bears on 672 infiltrating carcinomas of the cervix treated from 1977 until the end of 1991, by a radiosurgical combination (311 patients) or by exclusive irradiation (361 patients). The radiosurgical series includes mostly stages IB and II and patients under 50 years because of the therapeutic protocol. Most of the patients aged over 50 years and all stages III were treated by exclusive irradiation. External beam irradiation was most often performed in 4 fields by linear accelerator of 12 and 25 MeV. Uterovaginal brachytherapy used the technique of molds. In 55 cases, a complementary interstitial brachytherapy was applied on residual node. A computer dosimetry was made for each patient with calculation of the doses delivered to organs at risk and to node areas (points of calculation ICRU n degrees 38). The results at 5 years are as follows for the total series: locoregional control (LRC) 79%, specific survival (SS) 73%, overall survival 70%. For stage I, the LRC of the radiosurgical series is 92%, that of the series of exclusive irradiation 87% (no significant difference, neither for SS if we consider the tumoral size); For stage II, the LRC is 70% in the radiosurgical series and 79% in the series of exclusive irradiation. There is no difference if proximal stage II is compared. Conversely, for distal stage II, the difference is very significant in favour of exclusive irradiation (LRC 31%/77%, SS 26%/70%, p < 0.006). If we consider the results according to age, the difference for distal stage II comes mostly from patients under 50 years and especially those aged 40 years or under. For stage III, the LRC is 61% for patients over 50 years and 34% for those aged 50 years or under (p = 0.006). As the nodes, the results of surgical pieces and lymphadenectomy are studied. The patients under 40 years in stages II and III present more metastases than others (p = 0.003). Among the therapeutic factors, the dose rate and the treatment duration were particularly studied. A detailed study of the complications is made for the radiosurgical series as for the series of exclusive irradiation according to the French Italian glossary of complications as well as a study of the factors inducing them. PMID- 7549121 TI - [Comparison of anxiety and pain in two procedures of hematopoietic stem cell collection: cytapheresis and bone marrow collection]. AB - The aim of this study was to compare anxiety, pain and discomfort of cancer patients submitted to two procedures of hematopoietic stem cells collection: peripheral blood stem cells collection (PBSCC) or bone marrow collection (BMC). Patients, randomized (July 1993-February 1994), in view of autograft, to receive the first procedure or the second one, completed self-administered questionnaires before, during and after the procedure. Anxiety was evaluated by State-Trait Anxiety Inventory. Pain was assessed using visual analogical scale (VAS) and McGill Pain questionnaire. Before the procedure, in comparison with PBSCC patients (n = 40), BMC patients (n = 25) experienced more State-anxiety due to the procedure approach (p < 0.01) and more trouble or inconvenience for having to come and stay at the hospital (p < 0.0001). During the procedure, pain related to BMC, as assessed by VAS, is significatively higher than pain induced by PBSCC, whichever the access used (p < 0.001). The McGill total score is twice as high for BMC patients than for patients submitted to PBSCC with femoral catheter (n = 19). The latter patients significatively reported more pain than patients without femoral catheter (n = 21). At the discharge from hospital, 32% of BMC patients judged the procedure quite difficult vs 5% of PBSCC patients (p < 0.05). These results explain a higher acceptability of the peripheral blood stem cells collection. PMID- 7549122 TI - [Genetic alterations in localized cancers of the prostate: identification of a common region of deletion on the chromosome 18q]. AB - Prostate cancer is one of the most common malignancies in men. Few authors have attempted to identify consistent genetic alterations at the molecular level in adenocarcinoma of the prostate, but those most frequently reported are loss of heterozygosity (LOH) involving chromosome arms 8p, 10q, 16q, and 18q and inactivation of the TP53 tumor suppressor gene. In order to determine if alterations frequently found in other adenocarcinomas (breast, ovarian, colorectal), including losses of genetic material from chromosome arms 1p, 3p, 7q, 8p, 11p, 17p, 17q, and 18q, are also involved in prostate cancer, we examined 20 localized early-stage prostate tumors. We detected no mutations of the TP53 gene. Allelic losses were found from 7q (33%), 8p (50%), 10q (20%), and 18q (33%). Furthermore, as the first step toward isolating tumor suppressor genes on 18q, we used six polymorphic markers and identified a small common deleted region between the chromosome 18 centromere and the D18S19 locus. PMID- 7549123 TI - [Treatment of ifosfamide induced encephalopathy with methylene-blue]. AB - The authors report a case of encephalopathy after treatment with ifosfamide treated by a methylene blue infusion. This side-effect is rare but influenced by high doses and short infusions of this drug. Recently, a treatment of ifosfamide encephalopathy with methylene blue has been reported. This case report confirms the interest of such a treatment. PMID- 7549124 TI - Rising incidence of catheter-related bacteremia/fungemia. PMID- 7549125 TI - Urinary albumin fragments as a new clinical parameter for the early detection of diabetic nephropathy. AB - Urinary albumin fragments (uAF) from patients with NIDDM were analyzed as a possible factor in the early discovery of diabetic nephropathy before emergence of microalbuminuria. SDS-PAGE and immunoblot assay were employed for detection of uAF. Samples from 252 patients with NIDDM, 158 patients with non-diabetic diseases, and 48 healthy volunteers were examined; uAF were detected in 139 (55.2%), 94 (59.5%), only one (2.1%), respectively. In diabetic patients with normoalbuminuria, uAF were detected in 48 out of 159 cases (30.2%). Two years after the initial study, 3 of the 17 diabetic patients (17.6%) with normoalbuminuria and uAF showed micro- or macroalbuminuria. It was concluded that detection of uAF might be useful for the early detection of diabetic nephropathy in NIDDM. PMID- 7549126 TI - Effect of 14-membered ring macrolide therapy on chronic respiratory tract infections and polymorphonuclear leukocyte activity. AB - We studied the efficacy of the long-term administration of 14-membered ring macrolides in treating patients with diffuse panbronchiolitis (DPB) (34 patients) and bronchiectasis (BE) (40 patients). Oral administration of erythromycin (400 or 600 mg), roxithromycin (150 or 300 mg) or clarithromycin (200 or 400 mg) given daily for at least 2 months, was evaluated. The efficacy of erythromycin, roxithromycin, and clarithromycin in DPB was 19/24 (79%), 6/7 (86%), and 2/3 (67%), respectively. Efficacy of these agents in BE exceeded 50%. We determined the effect of these macrolides on the activity of polymophonuclear leukocytes (PMNs) obtained from healthy volunteers. There were no significant differences between the effects of these 14-membered ring macrolides and josamycin, a 16 membered ring macrolide which was previously found to be ineffective in treating DPB. Thus, the effectiveness of the 14-membered ring macrolides in treating DPB appears to depend on mechanism(s) other than alterations in PMN activity. PMID- 7549127 TI - Significance of urinalysis for subsequent kidney and urinary tract disorders in mass screening of adults. AB - To evaluate dipstick urinalysis as a predictor of subsequent kidney and urinary tract disorders in apparently healthy adults, we designed a cross-sectional, prospective, and retrospective study within a cohort. The severity of proteinuria was significantly (p < 0.01) associated with the amount of pathological casts, whereas hematuria without proteinuria was not. The frequency of subsequent serum creatinine increase (0.3 mg/dl/5 years or more) was significantly enhanced (from 0.4% to 7.3% along with the severity of proteinuria, whereas it was not related to the severity of hematuria. Patients who subsequently developed renal failure and glomerulonephritis exhibited hematuria (11.1-32.1%) less frequent than proteinuria (62.3-83.3%). Even those with renal tumors or stones showed infrequent (14.3-27.9%) hematuria. Thus, urine protein and occult blood have different implications in mass screening. PMID- 7549128 TI - Alpha 1-antichymotrypsin level in cerebrospinal fluid is closely associated with late onset Alzheimer's disease. AB - The levels of alpha 1-antichymotrypsin (ACT) in cerebrospinal fluid (CSF) from 66 sporadic Alzheimer's disease (AD) patients and 54 normal controls were measured by enzyme immunoassay and compared. There was no correlation (r = 0.259, n = 54) between the ACT level and normal aging. The levels of ACT were significantly higher in the total AD group (p < 0.01) than in the normal control group. Dividing AD patients into early onset AD (n = 27) and late onset AD groups (n = 39), the mean level of CSF ACT in the late onset AD group was significantly higher than that in the normal control group (p < 0.001) and that in the early onset AD group (p < 0.01). Thus, the level of ACT in CSF is closely associated with late onset AD. PMID- 7549129 TI - Clinical evaluation of catheter-related fungemia and bacteremia. AB - Forty-four patients with catheter-related infection admitted to Hokusho Central Hospital between 1985 and 1991 were studied retrospectively. The rate of catheter related fungemia or bacteremia to all corresponding cases of fungemia and bacteremia increased from 7.7% in 1985 to 28.8% in 1991. The isolated pathogens were Candida parapsilosis (8 strains), Candida tropicalis (6 strains), methicillin-resistant Staphylococcus aureus (MRSA) (6 strains), methicillin sensitive S. aureus (MSSA) (5 strains) and Streptococcus epidermidis (3 strains). Bacteremia occurred after catheterization of the femoral vein for a mean duration of 37 days. The period was significantly shorter than that after catheterization of the subclavian vein (56 days). The major isolates from the subclavian vein were Candida spp. (14/17, 82.4%), followed by MRSA (1/17, 5.9%) and MSSA (1/17, 5.9%), while isolates from the femoral vein were Candida spp. (6/16, 37.5%), MRSA (5/16, 31.3%) and MSSA (3/16, 20.8%). Catheter removal alone did not improve the clinical condition, particularly in MRSA bacteremia; the combination of antimicrobial therapy and removal of the catheter was necessary for a better prognosis. PMID- 7549130 TI - Infections in patients with liver cirrhosis and hepatocellular carcinoma. AB - Infections in immunocompromised hosts have been an important clinical problem. Patients with liver cirrhosis and/or hepatocellular carcinoma are at a high risk of infection due to multiple factors. Five hundred and two patients admitted with liver cirrhosis and/or hepatocellular carcinoma were evaluated for infection. The infection rate was not influenced by the etiology of hepatic diseases or the presence of hepatocellular carcinoma, however, it increased with the advance of clinical stages of liver cirrhosis and hepatoma. The respiratory tract and urinary tract were the most common sites of infection, being involved in 50% and 28% of cases, respectively. The major pathogens of respiratory tract infection were S. aureus, H. influenzae, and P. aeruginosa. Gram-negative bacteria was the common isolate from sputum and urine, and S. aureus was also common in gram positive bacteria. The infection rate was high in patients who died although infections could rarely be implicated as the direct cause of death. These findings should be a guide for the clinicians in treating patients with liver cirrhosis and/or hepatocellular carcinoma who exhibit signs of infection. PMID- 7549131 TI - Immunological study of anti-M2 in antimitochondrial antibody-negative primary biliary cirrhosis. AB - We investigated the specificity of various autoantibodies in antimitochondrial antibody (AMA)-negative patients with primary biliary cirrhosis (PBC). We examined sera from 144 patients with PBC, 17 of whom were AMA negative by indirect immunofluorescence. The AMA-negative group showed a significantly higher positivity for smooth muscle antibody, but not for antinuclear antibody, as compared with the AMA-positive group. IgG class anti-PDH by enzyme-linked immunosorbent assay (ELISA) were detected in 13% of the AMA-negative group. However, all PBC patients showed positive IgG, IgA, and/or IgM class anti-M2 to the four M2 proteins by immunoblotting. These results suggest that the detection of IgG and IgM class anti-PDH and that of antibodies to the four M2 proteins increases the positivity of this ELISA method, and that detection of IgG, IgA, and IgM class anti-M2 to the four M2 proteins by immunoblotting is useful in diagnosing AMA-negative patients with PBC. PMID- 7549132 TI - Characterization of the oxidative metabolism in lactate dehydrogenase A deficiency. AB - Recurrent rhabdomyolysis due to decreased glycolysis occurred during strenuous exercise in patients with lactate dehydrogenase A subunit (LDH-A) deficiency. We report the features of oxidative metabolism of four patients from two families in whom the severity of the disease differed. Enzyme activities of muscle LDH were decreased to < 5% of control. There was no difference in the gene abnormality. Maximal oxygen uptake in the patients with severe and mild symptoms was approximately 73% and 92% of control. Respiratory exchange ratio was increased to more than 1.0 during maximal exercise. These findings suggest that in these patients, the oxidative functions of glycogenolysis in which pyruvate is required for fuel of maximal oxidative metabolism are preserved, and that disease severity may be related to the degree of muscle oxidative capacity. PMID- 7549133 TI - Evaluation of lymph nodes on computed tomography images in epidermoid lung cancer. AB - This study was undertaken to design the optimal criteria for practical guidelines for lymph node metastasis evaluation in epidermoid lung cancer. Resected lymph nodes (n = 214) were analyzed by preoperative computed tomography (CT) images and postoperative histopathological findings. Definite criteria were defined by efficiency, and possible criteria by receiver operating characteristic (ROC) analysis. Statistical values of highest efficiency in 214 lymph nodes revealed that the minor axis offered the best criteria (minor axis 13 mm; efficiency 93%). ROC curve using the minor axis which showed the highest values of sensitivity and specificity in the optimal criteria (minor axis 9.3 mm; sensitivity 76%, specificity 76%). The criteria of the paratracheal lymph nodes were smaller than those of tracheobronchial and subcarinal nodes. In conclusion, it is desirable to evaluate metastasis of lymph nodes by the minor axis, and criteria should be considered changeable to accommodate the anatomical location in epidermoid lung cancer. PMID- 7549134 TI - An outbreak (159 cases) of transient thyrotoxicosis without hyperthyroidism in Japan. AB - We report an outbreak of transient thyrotoxicosis that occurred between June and September 1993, in Matsuyama City, Ehime Prefecture. One hundred fifty-nine cases of thyrotoxicosis were identified, all in individuals without goiter. Thyroid autoantibodies were absent, and the serum thyroglobulin level was depressed. Ultrasonography of the thyroid disclosed no abnormal findings, while scintigraphy of the thyroid with 99mTc revealed poor uptake. About ten days after the onset, the serum thyroid hormone levels were normalized. No significant elevation of serum viral antibodies was found. These observations suggest that a thyrotoxicosis factitia may have been the cause of the present outbreak. PMID- 7549135 TI - Changes in renal function with aging among Japanese. AB - Renal function was assessed in 329 inpatients who were presumably free of renal disease or water-electrolyte imbalance and the relationship between renal function and aging and sexual differences were investigated. Serum creatinine levels were slightly increased with aging, and were significantly higher in males than in females. Urinary creatinine excretion significantly decreased with aging; this decline was more pronounced in males. Urinary creatinine excretion was significantly higher in males. Creatinine clearance also decreased significantly with aging and this tendency was still observed after correction for body surface area. Sexual differences played no part in creatinine clearance. There was a significant increase in the serum beta 2-microglobulin level with aging. In addition, the Cer of the aged Japanese population was lower in comparison with western counterpart. The findings suggest the need for special consideration in conducting clinical tests or administering drugs to this segment of the population in Japan. PMID- 7549136 TI - Daytime hypertension and the effects of short-term nasal continuous positive airway pressure treatment in obstructive sleep apnea syndrome. AB - Daytime blood pressure (BP) in 31 male patients with obstructive sleep apnea syndrome (OSAS) was measured and the effects of nasal continuous positive airway pressure (CPAP) treatment on daytime BP were studied. Subjects were 48 +/- 10 (mean +/- SD) years old and weighed 80 +/- 13 kg. The mean systolic BP and diastolic BP were 135 +/- 15 mmHg and 88 +/- 14 mmHg, respectively and daytime hypertension was present in 12 (38%) subjects. Apnea index (AI) and the lowest oxygen saturation during sleep were significantly more severe in the hypertensive (HT) than in the non-hypertensive (NHT) patients (p < 0.05). AI was significantly correlated with diastolic BP (p < 0.05) and the mean and lowest oxygen saturation during sleep were significantly correlated with both systolic (p < 0.05) and diastolic BP (p < 0.01). After nasal CPAP treatment for two weeks, both systolic and diastolic BP were significantly reduced; the former from 135 +/- 15 mmHg to 126 +/- 10 mmHg (p < 0.005) and the latter from 88 +/- 14 mmHg to 78 +/- 6 mmHg (p < 0.001). These data form direct evidence that daytime hypertension is partially induced by OSAS and is reversible with nasal CPAP treatment. PMID- 7549137 TI - Autopsy findings of Addison's disease caused by systemic cytomegalovirus infection in a patient with acquired immunodeficiency syndrome. AB - We previously reported a case of Addison's disease associated with acquired immunodeficiency syndrome (AIDS) (Endocr J, 41:13, 1994). A 46-year-old man with hemophilia B and AIDS was diagnosed as Addison's disease. The positive cytomegalovirus (CMV) antigen in urine suggested that CMV adrenalitis may have caused the adrenal insufficiency. Despite treatment with ganciclovir, the patient died one year later. Autopsy findings revealed that the typical inclusions of CMV were seen in the lung, adrenal glands (both cortex and medulla) and small intestine. Here, we describe the subsequent clinical course and postmortem findings of this case. PMID- 7549138 TI - Plasma exchange in chronic inflammatory demyelinating polyradiculoneuropathy; different beneficial effects and their correlation to the clinical features. AB - We report 7 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who showed significant clinical improvement by plasma exchange (PE). Their clinical features were extensively varied but fulfilled the diagnostic criteria for CIDP. The patients could be divided into 2 subgroups based on the mode of effectiveness of PE; one group consisted of those who once achieved complete remission, but required highly frequent PEs for long term maintenance of remission and the others were those who showed persistent and complete remission with several sessions of PE alone. These findings raise issues about the long-term therapeutic goals to be achieved by PE for CIDP patients, particularly on supportive therapy other than PE, as well as the optimization of frequency and duration of PE. PMID- 7549139 TI - Phlegmonous colitis in a patient with liver cirrhosis, hepatocellular carcinoma and acute promyelocytic leukemia. AB - We describe a case of phlegmonous colitis occurring in a 70-year-old man with liver cirrhosis, hepatocellular carcinoma and acute promyelocytic leukemia. He developed an acute abdominal emergency and died during the first day of admission. Autopsy revealed a colon lesion characterized by suppurative inflammation associated with marked edema and hemorrhage in the submucosa. These findings were identical both macroscopically and microscopically to those of phlegmonous colitis. PMID- 7549140 TI - Acute myelomonocytic leukemia in a patient with multiple myeloma: evidence for different clonal origin. AB - A case of 77-year-old female with multiple myeloma (IgG-k) developed acute myelomonocytic leukemia (AMMoL) following a myelodysplastic stage after chemotherapy with melphalancyclophosphamide combinations for 6 years. The leukemic blast cells expressed both myeloid antigens (CD11b, CD13, CD14, CD15, CD33 and CD34) and T/B lymphoid antigens (CD2, CD4, CD22 and PCA1). Cytogenetic analysis revealed a chromosome deletion -7. Analysis of immunoglobulin genes showed the heavy chain genes in germ line configuration. These findings indicate that the AMMoL was a therapy-related stem cell leukemia and was a clonal origin genetically different from multiple myeloma irrespective of plasma cell phenotype. PMID- 7549141 TI - Aspergillus aneurysm of the middle cerebral artery causing a fatal subarachnoid hemorrhage. AB - A rare case of Aspergillus aneurysm of the central nervous system (CNS) leading to subarachnoid hemorrhage (SAH) is reported. An 83-year-old woman developed visual disturbance and headache. Computed tomographic scans showed no evidence of aneurysm or tumor in the intracranium. She suddenly died from SAH. Autopsy revealed massive SAH due to ruptured Aspergillus aneurysm of the middle cerebral artery. Aspergillus was suggested to have extended from the paranasal sinuses. Aspergillosis of CNS should be considered in patients with neurological symptoms such as visual disturbance and trigeminal neuralgia, especially in cases of the aged or immunocompromised. PMID- 7549142 TI - Magnetic resonance imaging of multiple brain abscesses of the bilateral basal ganglia. AB - A 64-year-old woman developed multiple brain abscesses of the basal ganglia associated with Klebsiella pneumoniae septicemia. Magnetic resonance (MR) images showed three different stages of the brain abscesses. The images of early cerebritis of this site mimicked lacunar infarctions or dilated Virchow-Robin spaces. The differentiation of the brain abscess from lacunae and dilated Virchow Robin spaces is discussed, together with the evolution of the brain abscesses on MR images. PMID- 7549143 TI - Improvement of hypothyroidism after glucocorticoid replacement in isolated adrenocorticotropin deficiency. AB - We report a 50-year-old female who suffered from reversible hypothyroidism accompanied by isolated ACTH deficiency. There were no findings indicating a complication of autoimmune thyroiditis. Replacement of maintenance dose of glucocorticoid not only led to improvement of thyroid function, but also caused a transient decrease in T3 and an increase in reverse T3, suggesting that chronic cortisol deficiency may impair thyroid function, and that the maintenance dose, as well as pharmacological doses of glucocorticoids may influence T4 deiodination. The findings of this case suggest that thyroid function should be re-evaluated to avoid unnecessary replacement of thyroid hormone, a few months after glucocorticoid replacement. PMID- 7549145 TI - Polymyositis associated with thymoma and the subsequent development of pure red cell aplasia. AB - A 53-year-old woman with polymyositis associated with thymoma subsequently developed pure red cell aplasia (PRCA). She was hospitalized because of fever and muscle weakness, and diagnosed as having polymyositis by muscle biopsy. Remarkable clinical improvement followed administration of prednisolone. Progressive anemia became evident, however, while prednisolone was being tapered. Erythroid aplasia and the presence of thymoma confirmed the diagnosis of PRCA. Further examinations revealed that cytotoxic T cells may play an important role in the pathogenesis of this case. PMID- 7549144 TI - Ruptured subcapsular giant hematoma of the spleen as a complication of chronic pancreatitis. AB - We present a 63-year-old man with chronic pancreatitis and the rare complication of giant subcapsular splenic hematoma. The hematoma showed no size reduction for 6 weeks. Then, the hematoma was infected with pseudomonas aeruginosa after the recurrence of the pancreatitis, and it finally ruptured. This case suggested that in cases of giant subcapsular splenic hematoma with chronic pancreatitis reductive pressure treatment should be administered as early as possible. PMID- 7549146 TI - Long-term observation of a case of pulmonary arteriovenous fistula with pulmonary hypertension. AB - A case of pulmonary arteriovenous fistula (PAVF) with pulmonary hypertension (PH) occurring in an adult woman is described. Resection of PAVF was not performed and she has been followed up for 5 years, receiving repeated right cardiac catheterization. We discuss the causal relationship of PH and the development of PAVF. PMID- 7549147 TI - Mexiletine for painful alcoholic neuropathy. AB - Five patients suffering from painful alcoholic neuropathy showed severe painful sensory disturbance in their extremities. Although their pain was not ameliorated by the typical usual analgesic agents, oral mexiletine (MX) therapy was remarkably effective for the pain (especially tingling and aching sensation) without major side effects. This study indicated that the minimum effective dose was 300 mg per day and the effective concentration of MX in plasma was 0.66 +/- 0.15 micrograms/ml in these patients. Thus, oral MX therapy can be a reliable treatment for pain in alcoholic neuropathy. PMID- 7549148 TI - Cushing's disease preceding sarcoidosis. AB - We present a 32-year-old-woman with Cushing's disease and sarcoidosis. She had been diagnosed as having Cushing's disease in 1985. She had transsphenoidal surgery followed by pituitary radiation. Subsequently her plasma cortisol level gradually decreased to normal range. In 1991, she manifested bilateral hilar lymphadenopathy on chest X-ray, erythema nodosum, subcutaneous nodules and granulomatous uveitis. From the histological findings of the skin lesions, the patient was diagnosed as having sarcoidosis. Interestingly, an inverse relationship between the disease activity of sarcoidosis and the level of serum cortisol in Cushing's disease was observed in the clinical course of this patient. Co-existence of Cushing's disease and sarcoidosis is very rare. Since the therapeutic significance of steroids in sarcoidosis has been well established, this case provides insight to the relationship between sarcoidosis and steroids. PMID- 7549149 TI - Thyroid anaplastic carcinoma producing granulocyte-colony-stimulating factor and parathyroid hormone-related protein. AB - A 65-year-old woman noticed a rapidly enlarging neck mass with tenderness and complained of dyspnea. She was diagnosed as having anaplastic thyroid carcinoma. She died of respiratory failure 14 days after admission. Marked leukocytosis and hypercalcemia were observed in the clinical course. Both serum granulocyte-colony stimulating factor and parathyroid hormone-related protein levels were elevated. The cancerous tissue was also immunohistochemically stained for both peptides. We conclude that the leukocytosis and hypercalcemia of this patient were induced by these two factors produced by the tumor. PMID- 7549150 TI - Hereditary hemorrhagic telangiectasia showing severe anemia which was successfully treated with estrogen. AB - Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease) is a bleeding disorder attributed to a vascular developmental abnormality. It is transmitted as an autosomal dominant trait. A 63-year-old female was admitted because of repeated episodes of severe anemia which resulted from bleeding of telangiectases in the gastric mucosa. Conventional therapies including endoscopical microwave coagulation and ethanol injection were not effective. The persistent anemia necessitated frequent blood transfusion. Estrogen was orally administrated and blood transfusion became unnecessary. Thus, estrogen therapy should be considered as one of the effective treatments for recurrent severe anemia due to hereditary hemorrhagic telangiectasia. PMID- 7549151 TI - Hemolytic-uremic syndrome due to deoxycoformycin: a report of the second case. AB - A hypercalcemic patient with adult T-cell leukemia was treated with deoxycoformycin (DCF) in a dose of 5 mg/m2 daily for three days. Several days later, severe thrombocytopenia appeared abruptly and then microangiopathic hemolytic anemia ensued. Subsequently, renal failure and hypertension developed, and the patient died seven weeks after DCF therapy. Needle necropsy of the kidney showed glomerular damage including swelling of endothelial cells, mesangiolysis and segmental collapse. This is the second reported case of hemolytic-uremic syndrome due to DCF. PMID- 7549152 TI - Exercise-induced angina with intermittent ST-segment elevation. AB - A 59-year-old man was admitted for evaluation of mild exertional angina in the morning. During graded treadmill exercise stress testing, the patient had reproducibly intermittent chest pains associated with ST-segment elevations in leads II, III and aVF. A baseline coronary angiogram showed a coronary narrowing of 90% in the middle segment of the right coronary artery. The coronary narrowing was immediately resolved by an intracoronary injection of nitroglycerin. This was a very rare case of variant angina with intermittent ST-segment elevations and chest pains which were reproducibly demonstrated during exercise and the recovery phase of treadmill testing. PMID- 7549153 TI - Targeting HIV with liposome-encapsulated antivirals. PMID- 7549154 TI - Additional tests to differentiate Arcanobacterium haemolyticum and Actinomyces pyogenes. AB - A commercially available biochemical test panel, commercially available diagnostic tablets and gas-liquid chromatography (GLC) of cellular fatty acids were used to find out whether Arcanobacterium haemolyticum and Actinomyces pyogenes could be further differentiated from each other. Xylitol and alpha methyl-D-glucoside fermentation, Voges-Proskauer reaction and tributyrate hydrolysis were found to be useful additional tests which differentiated Arc. haemolyticum and A. pyogenes. GLC analysis revealed major differences in the cellular 16:0, 18:2(9,12) and 18:1(9) fatty acid composition of the two species. Especially the Voges-Proskauer test available as diagnostic tablets can be easily performed in clinical microbiology laboratories, in addition to the tests now used to differentiate Arc. haemolyticum from A. pyogenes. PMID- 7549155 TI - Aflatoxin contamination of sorghum seeds during storage under controlled conditions. AB - Aflatoxins were estimated in two sorghum seed varieties (CSH-9 and AJ 140) collected in India and stored at three different temperature and humidity levels showed 20 degrees C and 73.5% relative humidity (RH) to be safe storage conditions. A maximum aflatoxin level was observed at 31 degrees C and 81.0% RH. Though the fungus grew well at 40 degrees C and all the humidity levels tested aflatoxin production was comparatively lower but reached hazardous level after 5 months of storage. Though all four aflatoxins were detected in both the seed samples, aflatoxin B1 and aflatoxin G1 were most predominant. PMID- 7549156 TI - Evidence of canary droppings as an important reservoir of Cryptococcus neoformans. AB - After preliminary results had shown the occurrence of Cryptococcus neoformans in canary droppings in southern Italy, the increasing epidemiological interest in cryptococcosis and the habitats of the agent led to more extensive studies. 180 samples of canary droppings were collected from pet shops and private households in two towns (Messina and Reggio Calabria) of southern Italy and culturally examined for C. neoformans. The examination was carried out with the help of the brown colour effect (BCE) specific of the C. neoformans colony on Guizotia abyssinica creatinine agar (Staib agar). 48 samples (26.6%) were positive; of these, 32/108 (29.6%) were from pet shops and 16/72 (22.2%) from private households. The colony growth was not mucous, i.e. microscopically the blastospores were found to be little or not encapsulated. All C. neoformans strains tested by the auxanographic method showed a weak assimilation of creatinine. The variety status of the isolates was tested with canavanine-glycine bromothymol-blue (CGB) agar; all strains tested (n = 420) were found to be C. neoformans var. neoformans. In the light of extensive studies on C. neoformans and the canary in the pre-AIDS era in Germany, the present observations in Italy ask for further basic research, epidemiological studies, and education of the public. PMID- 7549157 TI - Particle agglutination assay for detection of albumin and IgG binding cell surface components of Helicobacter pylori. AB - Human serum albumin (HSA), bovine serum albumin (BSA) and human IgG were immobilized on latex beads to detect cell surface components of Helicobacter pylori binding to BSA, HSA, and IgG by a particle agglutination assay (PAA). In a total of 32 H. pylori strains tested, 16 strains interacted with BSA and 12 strains with HSA; 24 strains expressed binding of human IgG. The specificity of the agglutination reaction was studied by a particle agglutination inhibition assay performed by pre-incubating bacterial cell suspensions in buffers containing homologous proteins, unrelated glycoproteins and a number of common monosaccharides. Treatment of H. pylori cells with heat and proteolytic enzymes abolished binding to microbeads with immobilized IgG and albumin. IgG-binding surface components from cells of H. pylori strain CCUG 17875 were effectively extracted by incubating a cell suspension with 10 mM EDTA, 0.015 M sodium phosphate buffer (pH 7.2), and by washing H. pylori cells with distilled water. However, attempts to extract a fraction rich in albumin-binding components were unsuccessful. We conclude that cells of various H. pylori strains express commonly IgG-binding proteins, and that albumin-binding is probably mediated by non-specific hydrophobic interactions. PMID- 7549159 TI - Thrombocytopenia, leukopenia and abnormal liver function tests in the initial phase of tick-borne encephalitis. AB - One-hundred-seventy-five patients in whom bone marrow puncture had been performed at the Department of Infectious Diseases, University Medical Centre, Ljubljana, during the period from 1988 to 1992, were analysed in a retrospective study. In five out of these 175 patients, tick-borne encephalitis virus infection was confirmed by serological tests and in all of them bone marrow puncture was performed in the initial phase of the illness as part of diagnostic work-up in a patient with a febrile illness accompanied by leukopenia and thrombocytopenia. Bone marrow examination revealed minimal reactive changes or normal findings. In addition to leukopenia and thrombocytopenia, abnormal liver function tests were also found in four patients. All these patients later developed meningoencephalitis, i.e. they had clinically typical tick-borne encephalitis with biphasic course of the illness. In the initial phase of tick-borne encephalitis in addition to well-known leukopenia, thrombocytopenia and abnormal liver function tests may be found. PMID- 7549158 TI - Association of enterohemolysin and non-fermentation of rhamnose and sucrose with Shiga-like toxin genes in Escherichia coli from calves. AB - Fecal Escherichia (E.) coli strains from calves were tested for simply detectable phenotypical features associated with Shiga-like toxin (SLT) genes. DNA hybridization with SLT-specific oligonucleotide gene probes (detection of genes for SLT-I and SLT-II) was the "gold standard" for the evaluation of Vero cell cytotoxicity, fermentation of several saccharides, beta-D-glucuronidase activity and production of alpha-hemolysin (alpha-Hly) or enterohemolysin (E-Hly). While SLTEC and non-SLTEC did not significantly differ in production of alpha-Hly, beta D-glucuronidase activity and fermentation of D-sorbitol, production of E-Hly and non-fermentation of L-rhamnose (Rha) and D-sucrose (Suc) were associated with SLT genes. Sensitivity and specificity of the E-Hly+ phenotype were 53% and 88% for identification of calf SLTEC. When three markers were combined to form the parameter ["E-Hly+ or (Rha- and Suc-)"], sensitivity was higher (65%) and specificity was almost the same (85%). Production of enterohemolysin and inability to ferment rhamnose and sucrose were more often associated with the SLT I gene than with SLT-II genes. Approximately 71% SLT-I+ E. coli were positive in the enterohemolysin assay. The test combination "E-Hly+ or (Rha- and Suc-)" and Suc-)" was most valuable for the presumptive identification of SLT-I+ E. coli (sensitivity 85%, specificity 83%). These data suggest that the phenotype "E-Hly+ or (Rha- and Suc-)" may be a helpful marker for the detection of SLT-I+ E. coli in SLTEC associated diarrhoea of calves. PMID- 7549160 TI - Bacteroides fragilis adheres to laminin significantly stronger than Bacteroides thetaiotaomicron and other species of the genus. AB - The laminin binding properties of eight species of the genus Bacteroides were examined using latex particle agglutination assay. B. fragilis was found to bind strongly to laminin, whereas all other species tested showed no or only weak laminin adherence. The pronounced differences in laminin binding activity between B. fragilis on the one side and B. thetaiotaomicron and B. ovatus on the other were determined to be statistically significant (p < 0.001 and p < 0.01, respectively). With regard to the relevance of laminin adherence for bacterial pathogenicity and invasiveness, our results give a possible explanation for the well-known finding that B. fragilis is the most frequently isolated pathogen in anaerobic bacteremia. PMID- 7549161 TI - Blood group phenotype determines lectin-mediated adhesion of Pseudomonas aeruginosa to human outer ear canal epithelium. AB - Pseudomonas aeruginosa is the most frequent bacterial pathogen causing acute diffuse otitis externa. In a recent prospective phase II study we demonstrated that lectin-mediated bacterial adhesion can be blocked by receptor-analogue carbohydrates in patients suffering from Pseudomonas aeruginosa-induced acute otitis externa. In this investigation, human ABO blood group antigens were analysed on outer ear canal epithelial cells with standard routine histological procedures by monoclonal antibodies for the blood groups A and B, and with Ulex europaeus I lectin for the blood group O, respectively. In all cases (n = 20) the blood groups could be shown immunohistologically. P. aeruginosa-specific adhesion and inhibition assays were performed in the presence of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), D-mannose and A-like substance. Outer ear canal tissue sections were incubated with P. aeruginosa (strain PA 60), presenting lectin-specificity for GalNAc. Sections from patients presenting with blood group A were closely settled with bacteria in the presence of non-specific GlcNAc, D-mannose and PBS however, GalNAc and A-like substance inhibited the microbial adhesion. Amongst others, P. aeruginosa present adhesion molecules (lectins) with specificity for GalNAc. Thus, the correlation between blood group A phenotype and P. aeruginosa-induced acute diffuse otitis externa was investigated. Statistical evaluation proved a highly significant association. These data support the hypothesis that P. aeruginosa lectins with GalNAc specificity apparently adhere to GalNAc moieties, representing the terminal blood group A-determinant and further indicate that patients presenting with blood group A may have a genetic disposition for this form of otitis externa. PMID- 7549162 TI - Bacteroides fragilis: an exogenous pathogen? AB - Thirteen strains of Bacteroides fragilis isolated from contaminated water or from cases of intestinal and non-intestinal infections were comparatively analyzed in order to detect a possible biological relationship among them. The observation of similar profiles among the two groups, regarding the capacity of inducing abscesses and some surface properties, may support the hypothesis that the species could also act as an exogenous pathogen. PMID- 7549163 TI - Intrathecal immune response in neuroborreliosis: importance of cross-reactive antibodies. AB - The intrathecal IgG response to Borrelia burgdorferi was evaluated in 35 patients with neuroborreliosis (NB). Samples were tested with and without preabsorption with Treponema phagedenis. Specific antibody concentrations in the CSF and serum were determined by ELISA. The antibody index (AIBb = QBb/QIgG) was calculated from the ratio between the CSF/serum quotients for specific antibodies (QBb) and total IgG (QIgG). Intrathecal synthesis of B. burgdorferi antibodies was demonstrated in 31 samples before and in 24 samples after preabsorption with T. phagedenis. The clonal distribution of intrathecally produced IgG antibodies was determined by isoelectric focusing combined with affinity blotting. B. burgdorferi-specific oligoclonal IgG bands occurring predominantly in the CSF were demonstrated in 32/35 patients. In 29/32 samples, the major proportion of these bands also reacted with T. phagedenis. Preabsorption of samples with T. phagedenis removed a considerable share of bands reacting with B. burgdorferi. In patients with neurosyphilis, intrathecal synthesis of antibodies with specificity for B. burgdorferi was demonstrated in 7/10 samples before, and in no sample, after preabsorption of cross-reactive antibodies. Due to the lower sensitivity in determining the AIBb (-20%), preabsorption of cross-reactive antibodies cannot be generally recommended. In all patients with suspected neuroborreliosis, but uncommon neurological symptoms and missing anamnestic data concerning a tick bite or erythema migrans, neurosyphilis should be excluded by a negative TPHA test. PMID- 7549164 TI - Small mammals as reservoir hosts for Borrelia in Russia. AB - In 1992-1993, a total of 29 isolates of Borrelia were obtained from small mammals captured in taiga forests of the Perm' region: 26 isolates were obtained from Clethrionomus glareolus and one each from C. rufocanus, Microtus oeconomus, and Apodemus sylvaticus. Isolation of Borrelia was performed by plating the animal material on BSK-II medium. Most isolates (86.2%) were obtained from the urinary bladder, the rest from the heart and spleen. According to the results of IFA with a set of monoclonal antibodies, isolates did not contain Borrelia burgdorferi sensu stricto. Preliminary data suggest that they can belong to B. garinii and B. afzelii. On the basis of the abundance of animals, it is suggested that C. glareolus, a background species among small mammals inhabiting the study region, is the main reservoir host for Borrelia. PMID- 7549165 TI - Memory complaints in chronic pain. AB - OBJECTIVE: In clinical practice, patients with chronic pain frequently report problems with memory functioning. This issue, however, has received little attention in the scientific literature. The present study was designed to investigate this common problem and to stimulate research interest in this neglected and important area. DESIGN: Self-reported memory problems were investigated in two groups of chronic pain patients--patients with pain from acceleration-deceleration automobile accidents (n = 56) and patients with pain from various work accidents (n = 27)--and two control groups involving medical/dental (n = 24) and psychotherapy patients (n = 20). SETTING: Private practice, chronic pain, rehabilitation psychology services. RESULTS: Our findings suggest that memory complaints are higher in patients with chronic pain than in medical/dental or psychotherapy patients. No differences were found between chronic pain groups. On more general measures of memory complaint, differences between pain patients and controls were attributed to the severity of patients' depression. On a questionnaire designed to be more specific to memory complaint in chronic pain patients, differences in memory complaint between pain patients and controls were found, even after the effects due to depression were statistically removed. Although pain patients often attribute their memory problems to codeine use and/or psychoactive medications, there was no support for this in the present study. CONCLUSIONS: Within the limitations of this study, these findings suggest that memory complaints may be related not only to depression but also to the presence of chronic pain. Further research in this area is needed. PMID- 7549166 TI - Memory for pain after nerve-block injections. AB - OBJECTIVE: To examine the accuracy of memory for pain in patients with chronic pain after injection of a local anesthetic and to investigate psychological factors hypothesized to coincide with distortion of memory. DESIGN: Consecutive patients receiving nerve-block injections recorded pain before the block, during the effect of the block, approximately 2 days after the block, and 2 weeks after the block. Memory for pain during the effect of the block was assessed 2 days and 2 weeks after the block for comparison with recorded pain ratings. SETTING: Outpatient, multidisciplinary pain-treatment center of a university medical center. PATIENTS: Forty-nine adult patients with chronic pain. INTERVENTIONS: Local nerve-block injections resulted in a significant, temporary reduction in pain, thus allowing patients to rate, and later recall, intensity of reduced pain. OUTCOME MEASURES: Subjective ratings of pain intensity (numeric rating scale) were compared with recalled pain intensity. Demographic variables and psychological self-report measures were administered at evaluation. RESULTS: Memory distortions are commonplace and are more likely to involve recollection of higher pain levels than were reported at the time of the injection. Psychological self-report measures did not identify subjects who experienced distorted memory for pain relief. CONCLUSIONS: An awareness of these distortions should lead health-care professionals to monitor and refer to patients' actual pain reports made during a treatment intervention rather than relying on patients' recall to gauge the efficacy of interventions. Memory distortions could influence avoidance behaviors implicated in the development of chronic pain by some theories. PMID- 7549167 TI - 0.0625% bupivacaine with 0.0002% fentanyl via patient-controlled epidural analgesia for pain of labor and delivery. AB - OBJECTIVE: To compare the utility of 0.0625% bupivacaine with fentanyl administered via patient-controlled epidural analgesia (PCEA) to a traditional continuous epidural infusion for pain of labor and delivery. DESIGN AND SUBJECTS: Forty-one women in established labor were randomized to receive either (a) 0.0625% bupivacaine with 2 micrograms/ml fentanyl via PCEA (demand dose = 3 ml, lockout interval = 6 min, background infusion = 6 ml/h, no 1 or 4 h limits) or (b) 0.125% bupivacaine with 2 micrograms/ml fentanyl via continuous epidural infusion (CEI) at 12 ml/h. Supplemental 0.25% bupivacaine (3 ml every 5 min, p.r.n., x 3) was administered for treatment of breakthrough pain upon patient request. The study protocol was double-blind and placebo-controlled. OUTCOME MEASURES: Visual analogue pain scores, motor strength, pinprick level of sensory analgesia and bupivacaine use were assessed by an anesthesiologist unaware of the individual patient's randomization to a particular study group. RESULTS: The cephalad extent of pinprick sensory analgesia was significantly lower during both the first (p < 0.03) and second (p < 0.03) stages of labor in patients receiving PCEA. However, visual analogue pain scores, intensity of motor blockade, and need for physician-administered supplemental bupivacaine were comparable in both groups. Patients receiving PCEA used 40% less bupivacaine per hour while achieving analgesia comparable to patients receiving CEI. CONCLUSIONS: The results of this study show that 0.0625% bupivacaine with 2 micrograms/ml of fentanyl is an effective analgesic combination when used via PCEA. PMID- 7549168 TI - Ketorolac versus fentanyl for postoperative pain management in outpatients. AB - OBJECTIVE: The purpose of this study was to compare the efficacy and safety of i.v. ketorolac and fentanyl for moderate to severe postoperative pain in patients undergoing elective surgery in an ambulatory surgery unit. DESIGN: A double-blind randomized trial. SETTING: An ambulatory surgery unit in a university-affiliated hospital. PATIENTS: Sixty-nine patients undergoing elective laparoscopy, inguinal hernia repair, or knee arthroscopy were enrolled. INTERVENTION: Patients were randomly assigned to receive intravenous ketorolac 30 mg (n = 38) or fentanyl 50 micrograms (n = 31) for moderate to severe postoperative pain. OUTCOME MEASURES: Pain, assessed using a 100-mm visual analog scale and a 5-point verbal pain scale; adverse effects, as well as vital signs were recorded every 15 min for 150 min or until discharge from the postanesthesia care unit, 6 and 24 h after discharge. RESULTS: Pain reduction on both visual analog and verbal scales was significantly greater with fentanyl than ketorolac at 15 min. In addition, the proportion of patients requiring remedication at the 15-min time point was significantly greater in the ketorolac group. However, there were no significant differences between fentanyl and ketorolac between 30 and 150 min after surgery. Notably, pain reduction was significantly greater with ketorolac on the verbal scale at the 6 h measurement. CONCLUSIONS: Ketorolac appears not be as effective as fentanyl in treating early postoperative pain. Although fentanyl still appears to be the drug of choice in the early postoperative period, the parenteral use of ketorolac was more effective during the later postoperative period in providing longer lasting analgesia. PMID- 7549169 TI - Moclobemide in chronic neuropathic pain: preliminary case reports. AB - OBJECTIVE: This trial aimed to study moclobemide, a reversible, Type-A selective monoamine oxidase inhibitor, in patients with chronic neuropathic pain. DESIGN: Pain clinic patients suitable for antidepressant therapy were treated for 2 months in an open label, uncontrolled trial with moclobemide 150-600 mg/day. OUTCOME MEASURES: Visual analog scores of pain and affective symptoms, a side effect checklist, and clinical global impressions of efficacy and tolerability. RESULTS: The trial was terminated after assessment of seven patients; partial analgesic activity was discernible only in one, and beneficial mood effects notable in two others. Significant adverse effects, particularly insomnia and GI disturbance, were reported by the majority of patients. CONCLUSION: Moclobemide appears to have limited efficacy in the treatment of neuropathic pain. PMID- 7549170 TI - Felbamate relieved trigeminal neuralgia. AB - OBJECTIVE: The analgesic efficacy of the novel anticonvulsant felbamate was evaluated in trigeminal neuralgia. PATIENTS AND SETTING: This trial was offered in a tertiary referral center to three outpatients with severe pain who had exhausted other medical options, yet did not wish to undergo surgery. INTERVENTIONS: Felbamate was prescribed as the sole analgesic for approximately 1 month. OUTCOME MEASURES: A visual analogue scale was utilized. RESULTS: Felbamate potently diminished the severe pain of trigeminal neuralgia and was well tolerated. The drug was withdrawn because of subsequent reports elsewhere of aplastic anemia and hepatic failure associated with it when used for epilepsy. CONCLUSIONS: Preliminary evidence suggested that felbamate was effective in relieving trigeminal neuralgia. Stabilization of neuronal membrane depolarization was the most likely mechanism of action. However, significant potential risks preclude further use of felbamate in the management of nonmalignant pain. Principles that have proven useful in screening for new anticonvulsant drugs might be relevant to the development of methods by which to search for new analgesic drugs. PMID- 7549171 TI - Subarachnoid neurolytic block under general anesthesia in a 3-year-old with neuroblastoma. AB - CASE REPORT: A 3-year-old boy with neuroblastoma complained of severe pain in the left lower extremity. Pharmacologic management had previously been attempted, but severe pain continued, and further upward titration was complicated by sedative effects. METHODS AND RESULTS: Because the focus of treatment had become the controlling of pain, a lumbosacral subarachnoid neurolytic block was performed under general anesthesia. One-time neurolysis was more acceptable to the family than a procedure like epidural analgesia, that requires greater management. Contrast medium was used to monitor the spread of the neurolytic. An epidural catheter was inserted during the neurolytic block procedure for possible future use. The short-term results were good--pain reports and opioid doses decreased greatly, although with increased incontinence. The boy had new abdominal distention and pain 5 days after neurolysis. Opioid doses and sedatives were increased. He died 3 days later. PMID- 7549172 TI - Relevance of epidurography and epidural adhesiolysis in chronic failed back surgery patients. AB - OBJECTIVE: Pain treatment in the chronic failed back surgery patient remains problematic. Defining the pathogenesis of the pain could be helpful in treatment. The assumption that epidural fibrosis and adhesions might play an important role in the origin of the pain is verified. DESIGN: We investigated 34 patients in whom peridural fibrosis was suspected. An epidural catheter was inserted via the sacral hiatus. Injections of contrast dye, local anesthetic, corticosteroid, and hypertonic NaCl 10% were carried out daily for 3 days. Spread of the contrast dye in the epidural space was evaluated after 10 and 20 ml injection volume. SETTING: Subjects were patients in a pain clinic of a university hospital in Belgium. PATIENTS: Chronic pain patients with failed back surgery syndrome were examined. Nerve pathology was demonstrated and epidural fibrosis suspected or proved with magnetic resonance imaging (MRI) examination. OUTCOME MEASURES: Improvement in the contrast filling defects of the epidural space were noticed during treatment and correlated with pain improvement. RESULTS: Filling defects were noted in 30 of the 34 patients investigated. After the third day an objective improvement of contrast spread was documented in 14 patients. In seven patients improvement in pain occurred for only a very limited period (1 month). Statistical analysis (chi square analysis) could not demonstrate that improvement of contrast spread was correlated with better pain behavior. In 16 patients no improvement in contrast spread could be visualized. Pain improvement occurred in only four patients and for a limited period of 1 month. Long-term results are even worse. CONCLUSION: Epidurography might confirm epidural filling defects for contrast dye in the patients with epidural fibrosis. A better contrast dye spread, assuming scar lysis, does not guarantee a sustained pain relief. A more direct visualization of the resulting functional changes after adhesiolysis as with epiduroscopy might be useful. PMID- 7549173 TI - In response to article by Drs. Devulder et al. PMID- 7549174 TI - In response to article by Drs. Devulder et al. PMID- 7549175 TI - Pain treatment facilities referral selection criteria. PMID- 7549176 TI - In response to article by Drs. Sundblom et al. PMID- 7549177 TI - Back again? PMID- 7549178 TI - The time is now. PMID- 7549179 TI - Association between workers' compensation and outcome following multidisciplinary treatment for chronic pain: roles of mediators and moderators. AB - OBJECTIVE: To determine whether the tendency for chronic pain patients who receive Workers' Compensation to show a poorer response than non-compensated patients to pain treatment can be accounted for by mediating factors; to assess whether moderating factors can distinguish subgroups of Workers' Compensation recipients who react very poorly to treatment from compensated patients who respond well. DESIGN: Outcome study based on archives. SETTING: Multidisciplinary pain treatment center. PATIENTS: Of 214 patients, 158 had complete data. OUTCOME MEASURE: Blind ratings of narrative discharge summaries written by the Pain Treatment Center staff. RESULTS: A significant negative relationship between receiving Workers' Compensation and outcome was mediated by a pessimistic belief in the ability to return to former occupation. Moderator effects showed that Workers' Compensation recipients with high initial pain and a history of pain related surgery fared worse than any other group. Moreover, Workers' Compensation recipients not characterized by high pain and a history of surgery responded as well as noncompensated patients. CONCLUSIONS: The inadequate response to pain programs shown by Workers' Compensation recipients may be partly understood in terms of well-defined mediating factors, which may admit to amelioration via clinical intervention. Moreover, Workers' Compensation patients should not be considered high risks for failure by sole virtue of their compensation status. Multifactor assessment methods may be needed to identify that portion of compensation recipients who are actually at appreciable risk for treatment failure so that appropriate adjustments in treatment regimen may be made. PMID- 7549180 TI - Phosphorylation of transcription factors and control of the cell cycle. AB - Protein phosphorylation has evolved as the most versatile posttranslational modification widely used by cells. Signal transduction pathways mediated by activation of MAP kinases and protein kinase C trigger the exit of cells from the quiscence (Go-->G1 transition). Indeed, binding of growth factors at the cell surface triggers their receptors, usually possessing a tyrosine kinase on the cytoplasmic side, to phosphorylate other molecules passing on the information sequentially to GRB2 protein, to p21ras, to c-Raf-1, to MAP kinase kinase, to MAP kinase, to p90rsk, to transcription factors. Activated PKC, MAP kinase, and pp90src can translocate to the nucleus where they phosphorylate a number of protein transcription regulators in a cell cycle-dependent manner or in response to cell stimulation for exit from quiescence. The cell cycle is mainly regulated by p34cdc2 or otherwise called cdc2 in association with cyclins B at G2/M and by Cdk2 in association with cyclins A, D1, and E at G1/S checkpoints; phosphorylation of histone H1 and lamins by cdc2 triggers chromosome assembly and nuclear envelope breakdown, respectively, as a prelude to mitosis. Cdc2 activities functioning as a G2/M regulator are controlled by its phosphorylation and dephosphorylation at Ser/Thr residues. MAP kinases might be the missing link in the chain connecting the Go to G1 transition with the cell cycle regulation, whereas phosphorylation of replication protein factors, retinoblastoma, and p53 might link the G1 to S transition with the control of DNA synthesis. A number of transcription factors are known to stimulate DNA replication, including p53, c Myc, AP-1, Oct-1, T-antigen; the DNA binding activities of all these proteins and their interaction with other transcription factors are controlled by phosphorylation. The nuclear import of several proteins including NF kappa B, Dorsal, glucocorticoid receptor, ISGF3, rNFIL-6, T antigen, and the kinases PKC, MAP, and p90rsk, are dependent on their phosphorylation at specific sites. Histone phosphorylation stimulated at discrete stages of the cell cycle or in response to cAMP or other stimuli might induce profound changes in chromatin organization. PMID- 7549181 TI - The retinoblastoma protein as a fundamental mediator of growth and differentiation signals. AB - The retinoblastoma gene (RB) is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Mouse models created through gene knock-out and transgenic methods were established for exploring and manipulating RB in vivo. These models and several other pieces of evidence have shown that the retinoblastoma protein (Rb) plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming more obvious in some biological systems: Rb sequesters the transcription factor of E2F to regulate entry of cell cycle but enhances the activities of another class of the transcription factors, exemplified by NF-IL6, to initiate terminal cellular differentiation. Thus, the Rb protein can serve as a mediator for extracellular signals of growth or differentiation. The fundamental question of why only limited cell types are susceptible to tumor formation when Rb expression is lost, however, remains unanswered at present. PMID- 7549183 TI - Rupture of the right atrium secondary to blunt cardiac injury. AB - Occult cardiac injury following blunt trauma is underestimated in frequency of occurrence. Recent reports indicate that it rivals thoracic aortic transection as the cause of death following traffic fatalities. The most common mechanisms include rapid cardiac rotation while the great vessels remain fixed, transfer of hydraulic pressure from the abdomen or extremities, and direct myocardial compression by the sternum. Presentation can be diverse and findings can include jugular venous distention, upper extremity cyanosis, unexplained hypotension, and muffled heart sounds. An enlarged cardiac silhouette is occasionally seen on roentgenographic evaluation. A surprisingly large number of these patients are noted to have no significant signs of external thoracic trauma. Possible mechanisms include rapid rotation and deceleration of the heart with fixation of the great vessels, transmission of a large venous pressure wave to the heart following abdominal or extremity compression, and direct compression of the heart by the sternum. Following prompt diagnosis and rapid surgical intervention, a large number of these patients can be expected to survive this otherwise fatal injury. PMID- 7549182 TI - Repair of mitral regurgitation from myxomatous degeneration in the patient with a severely calcified posterior annulus. AB - Severe posterior annular calcification poses a particular challenge to mitral valve repair. In a series of 252 mitral valve repairs for myxomatous degeneration performed between 1980 and 1993, 14 patients had a severely calcified posterior mitral valve annulus. Ages ranged from 61 to 81 years. Twelve patients were preoperative NYHA Class III or IV, and five patients required concurrent coronary artery bypass procedures. Operative techniques included complete resection of the calcified posterior annulus, resections of portions of the posterior leaflet with leaflet advancement, and placement of an annuloplasty ring. There were no operative deaths and all patients had a postoperative echocardiographic confirmation of relief from mitral regurgitation. During a mean follow-up time of 36 months (6 months to 8 years), there has been one late valve reoperation and only one late death, from thromboembolism in a patient with atrial fibrillation. These data indicate that even in the presence of severe calcification of the posterior mitral annulus, mitral valve repair for myxomatous degeneration can be performed with a low-operative risk and satisfactory long-term results. PMID- 7549184 TI - Preperitoneal placement of ventricular assist devices: an illustrated stepwise approach. AB - Improvement in left ventricular assist device (LVAD) implantation techniques may favorably impact on the significant perioperative morbidity and mortality that follows placement of these devices. Based on the difficulties faced and complications encountered during our 45 case experience, we developed a quick reference illustrated guide which outlines in detail the steps and considerations critical for successful LVAD implantation. PMID- 7549185 TI - Latissimus dorsi dynamic cardiomyoplasty: role of combined ICD implantation. AB - Latissimus dorsi cardiomyoplasty is a promising surgical therapy in some patients with congestive heart failure. Although the mortality in heart failure patients is attributable primarily to heart failure and ventricular arrhythmias, the mechanism of death after cardiomyoplasty is not well characterized. We describe the clinical course of a patient undergoing cardiomyoplasty and discuss the role of combined use with an implantable cardioverter defibrillator. A 39-year-old man with congestive heart failure due to a massive anterior wall myocardial infarction was evaluated for latissimus dorsi cardiomyoplasty. The patient was in NYHA Functional Class III due to heart failure. He did not have any significant exertional or rest angina. During a Naughton stress test, the patient could exercise for 10 minutes, achieving 4 METS. Pulmonary function study showed a peak V O2 of 22.1 mL/min per kg. Radionuclide angiography demonstrated that the anterior wall was akinetic with a left ventricular ejection fraction of 22%. Cardiac hemodynamic studies suggested moderate pulmonary hypertension, elevated wedge pressure, and suboptimal response to exercise. A Holter recording showed frequent ventricular extrasystoles. Cardiomyoplasty was preferred to heart transplantation because the patient did not have end-stage heart failure. Postoperatively, the patient required low doses of dopamine. He developed recurrent, sustained, and hemodynamically significant episodes of ventricular tachycardia. He was treated with a combination of amiodarone and procainamide. He died 2 days postoperatively with ventricular fibrillation. Ventricular arrhythmias are a major cause of death in patients with heart failure. Latissimus dorsi cardiomyoplasty appears to be a promising but unproven therapy in such patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549186 TI - Intraoperative coronary artery dilatation with angioscopic guidance. AB - The use of intraoperative balloon dilatation during coronary artery bypass surgery has been limited due to relatively unpredictable and potentially damaging results. The development of fiberoptic angioscopy permits safe visualization of the interior of coronary arteries and may be a valuable adjunct to intraoperative balloon dilatation. A 56-year-old male underwent four vessel coronary grafting with progressive intraoperative balloon dilatation of a second more distal stenosis of the left anterior descending coronary artery. Angioscopy was used to determine optimal balloon sizing and allowed visualization of associated intimal changes that occurred during the procedure, resulting in a successful outcome for this patient. PMID- 7549187 TI - The management of chylothorax/chylopericardium following pediatric cardiac surgery: a 10-year experience. AB - We reviewed the management of 25 cases of chylothorax/chylopericardium (CT/CP) in 24 patients (9 females, 15 male; 3 days to 11-years-old) following 1605 cardiothoracic procedures (incidence of 1.5%) between January 1984 and December 1993 at our institution. The surgical procedures preceding the occurrence of lymph leak included ligation of patent ductus arteriosus (6 patients), coarctation/double aortic arch repairs (3), complex intracardiac repairs (11), and systemic to pulmonary shunts (5). There were 3 CPs and 22 CTs. All of the patients were initially treated nonsurgically with diet modification using either total parenteral nutrition (TPN) or enteral low fat solid food or enteral elemental diet supplemented with intravenous lipid emulsion. Twenty-one cases (84%) responded to conservative therapy. Of those, 15 had TPN as the initial treatment; the average duration of lymph leak was 13.7 (range 7 to 30) days and the average maximal lymph leak was 39.4 (range 15 to 130) mL/kg per day. The other six cases had low-fat enteral diet as the initial treatment, four resolved completely. Two with high-central venous pressure had to be switched to TPN prior to complete resolution. The average duration of lymph leak in this subgroup was 30 (range 12 to 56) days with the average maximal lymph leak was 30.1 (range 8.5 to 59) mL/kg per day. Excluding these two cases, the average lymph leak of the rest of the group was very compatible to the TPN group of 15 days. Lymphocytopenia and hyponatremia were frequently seen during CT/CP (47.6% and 43%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549188 TI - Anomalous origin of the left coronary artery from the pulmonary artery in adults. AB - Between March 1986 and December 1994, four adult patients underwent surgery for anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) at the German Heart Institute Berlin. The patients, aged 27, 35, 54, and 60, were all females. ALCAPA was diagnosed 3 to 8 months before surgery. The patients presented with cardiac murmur (two patients), abnormal ECG pattern (two patients), arrhythmia (one patient), and acute myocardial infarction (one patient). During surgery a two coronary system was established either by Takeuchi operation (one patient), interruption of the anomalous vessel and aortocoronary saphenous vein bypass (two patients), or internal mammary artery bypass (one patient). There were no postoperative deaths, however, one patient had to be reoperated because of a recurrent shunt. Clinical improvement was observed in all four patients with disappearance of fatigue, angina, dyspnea, and ischemic ECG changes. Despite having this long-standing congenital heart defect, adult and elderly patients with ALCAPA may benefit from surgical intervention to establish a two coronary system. PMID- 7549189 TI - Geographical variations in the presentation of ruptured aneurysms of sinuses of valsalva: evaluation of surgical repair. AB - From 1981 to 1992, 13 male and 7 female patients underwent surgical correction for ruptured aneurysms of sinus of valsalva. A total surgical experience of 22 procedures including 2 reoperations is presented, accounting for 1.37% of open heart surgery for congenital heart disease at PGIMER Chandigarh. Ninety percent were in the 20- to 40-year age group. Forty-five percent of patients had symptoms of > 1-year duration (range 2 months to 20 years) and catastrophic onset of symptoms was noted in four (18%). All patients had localized aneurysms originating either in right coronary sinus (14 pts) or noncoronary sinus (8 pts). Sites of origin and rupture are detailed. Associated congenital abnormalities such as ventricular septal defect (VSD) (13 pts), aortic regurgitation (3 pts), and left superior vena cava and atrial septal defect (ASD) (1 pt each) were noted. The data pertaining to Oriental and Western groups of patients were analyzed, and the differences in age, mode of presentation, site of origin, rupture, and the spectrum of associated abnormalities were elucidated. The majority of the patients (86.4%) were operated by the Bicameral approach. Repair was tailored according to the extent and severity of the defect in the sinus of Valsalva and aortic valve annulus and also the presence and site of VSD. PMID- 7549190 TI - Retrograde cerebral perfusion with hypothermic blood provides efficient protection of the brain: a neuropathological study. AB - Retrograde cerebral perfusion is a method that is recently being used for protection of the brain during operations on the aortic arch. This method is useful but is said to provide a limited time for protecting the brain. We designed an experiment in dogs to investigate neuropathologically the effect of protecting the brain for 120 minutes under: (1) circulatory arrest (CA); (2) retrograde cerebral perfusion with moderately cooled blood (RCPMC); and (3) retrograde cerebral perfusion with deeply cooled blood (RCPDC). We calculated the number of the abnormal cells of 400 hippocampal neurons per dog light microscopically. The number was 199 +/- 23 (mean +/- 1 SD) in the CA group, 149 +/- 50 in the RCPMC group, and 72 +/- 33 in the RCPDC group. The difference between the CA group and the RCPMC group was not statistically significant (p < 0.05), but there was a significant difference between the RCPMC and RCPDC groups (p < 0.05). The degree of cerebral protection provided by retrograde cerebral perfusion for 120 minutes is not sufficient when using moderately cooled blood. If we use deeply cooled blood at a temperature of about 10 degrees C, we should obtain a sufficient degree of protection of the brain. PMID- 7549191 TI - Predictors of stroke after cardiac surgery. AB - From January 1, 1988 to September 30, 1993, 44 of 2,455 patients undergoing cardiac surgery for acquired heart disease at our institution sustained an intraoperative or postoperative cerebrovascular accident (CVA). Demographic data, atherosclerosis risk factors, past medical history, cardiac catheterization reports, and intraoperative findings were retrospectively reviewed. The highest rate of CVA was in the sub-group of patients undergoing simultaneous myocardial revascularization and carotid endarterectomy (18.2%). The lowest rate was in a group of patients who underwent aortic valve replacement (0.9%). Severe aortic arch atherosclerosis with the presence of atheromatous material or calcinosis at the cannulation site was identified intraoperatively in 43.2% of patients with neurological complications and in 5% of the group without CVA (x2 = 18.1, p = 0.0001). Of 44 patients with CVA, 13.6% had a history of preoperative completed stroke. CPB time was 90.1 +/- 4.9 min vs. 71.6 +/- 3.7 min (p = 0.004), and aortic cross-clamping time was 54.5 +/- 3.2 min compared to 39.8 +/- 2.7 min (p = 0.001) in groups with and without postoperative stroke, respectively. Hypertension was an independent risk factor of postoperative CVA (x2 = 9.5, p = 0.02), but age was not. Neurological complications correlated with high operative mortality (38.6%) and prolonged postoperative hospital stays (35.1 +/- 5.3). These data describe predictors for the development of post-cardiopulmonary bypass CVA and identify a high-risk subgroup for neurological events. The preoperative recognition of risk factors is an essential step toward the reduction of morbidity and mortality. PMID- 7549192 TI - Timing of tracheal extubation in adult cardiac surgery patients. AB - In this article, we examine 14 studies conducted from 1974 to 1994 on "early" endotracheal extubation (0 to 12 hours postoperatively) in adult cardiac surgery patients. Aspects reviewed include: criteria for patient selection; criteria for extubation; analyses of feasibility and safety; effects of anesthetic technique; and patient morbidity. Advantages and disadvantages of early or "fast-track" extubation are discussed as are directions for future research. Selection criteria varied among studies; patients were most commonly excluded because of severe, preexisting pulmonary disease or ventricular dysfunction. Based on the studies examined, however, at least 70% to 80% of adult patients would meet selection criteria. Three universal criteria were applied in all studies: (1) patient is awake and responsive; (2) adequate gas exchange while breathing spontaneously; and (3) cardiovascular stability. To facilitate early extubation in appropriately selected patients, the choice of anesthetic technique and postoperative sedation technique appears to be important. Anesthetic techniques based on inhalational anesthetic agents, supplemented by moderate doses of narcotics, are more appropriate than high-dose narcotic anesthesia for early extubation protocols. Postoperative sedation with propofol, which has a rapid offset of action, may be particularly advantageous. Every published investigation has concluded that early extubation is safe, feasible, and desirable. Morbidity and mortality have not been shown to be affected by early extubation. Anesthetic technique and the patient's medical condition are the two major factors to consider in accomplishing early extubation. PMID- 7549193 TI - Preconditioning and its potential role in myocardial protection during cardiac surgery. AB - Myocardial preconditioning is the phenomenon whereby a brief stress to the heart (e.g., ischemia, hypoxia, etc.) prior to a prolonged period of ischemia renders the heart more resistant to ischemic injury. The cardioprotective effects of preconditioning include reduced infarct size and reduced ventricular arrhythmias. Preconditioning also is associated with beneficial metabolic effects during the prolonged ischemia, effects that also are observed during intermittent cardioplegia. However, there are conflicting reports about the effects of preconditioning on postischemic ventricular function. Although adenosine is thought to be the endogenous mediator of ischemic preconditioning, there are some important differences between adenosine and ischemic preconditioning mediated cardioprotection. PMID- 7549194 TI - Modulation of coronary collateral angiogenesis: a canine model of neovascularization induced by chronic ischemia. AB - Coronary collateral development in response to chronic myocardial ischemia is modulated by the integrated release of a variety of growth factors. Protamine, a specific inhibitor of angiogenesis in vitro and in vivo, may attenuate coronary collateral development. The purpose of this investigation was to characterize a model of canine coronary collateral development and to ascertain the effects of protamine on collateral perfusion in response to chronic myocardial ischemia. Ischemia-induced coronary collateral enhancement in response to daily, repetitive, 2-minute left anterior descending (LAD) coronary artery occlusions over 22 consecutive days was demonstrated in control (saline treated) dogs by time dependent, significant (p < 0.05) increases in collateral blood flow to ischemic myocardium. Concomitant with increases in collateral perfusion, myocardial contractile function during LAD occlusion was progressively normalized, and percent coronary flow repayment was reduced successively. These indicators of collateral development were attenuated significantly by administration of subcutaneous protamine. The present findings demonstrate that a model of brief, repetitive, coronary artery occlusion in chronically instrumented dogs sensitively elicits extensive enhancement of the coronary collateral circulation in response to chronic myocardial ischemia. In contrast, protamine attenuates coronary collateral development through its actions as an inhibitor of angiogenesis. PMID- 7549195 TI - Bacterial infection of cardiomyostimulator abdominal pocket following cardiomyoplasty procedure: an original approach to preserve synchronous muscle stimulation. AB - Dynamic cardiomyoplasty (DC) represents a new technique in therapy for refractory heart failure. So far, DC has been applied to more than 500 cases worldwide but reports on postoperative complications and related management are still lacking. We present the case of a patient suffering from refractory chronic heart failure for which the DC procedure was applied also accompanied by the complication of an infection process at the cardiomyostimulator pocket that began 2 weeks postoperatively. Following trials with several unsuccessful conservative approaches, an original procedure was developed to temporarily retain the implanted stimulation system, while at the same time maintain the synchronous contractions of the wrapped muscle. Finally, reimplantation of the pacing system was achieved with a low-risk procedure, effective cardiac assistance was preserved, and the infection process was arrested 3 years following DC. PMID- 7549196 TI - A simplified technique for repair of an injured internal mammary artery. AB - Injury to the internal mammary artery (IMA) during harvesting or the construction of sequential anastomosis can be troublesome and may force the surgeon to abandon the use of this valuable conduit. Since July 1991, we have utilized a simple technique for repair of a damaged IMA anywhere along its length. This involves leaving the IMA as a pedicled in situ graft and anastomosing a small piece of vein graft to an arteriotomy at the site of injury. The IMA is then used as originally planned, usually to the left anterior descending, and the vein graft can be used for a second vessel or simply tied. The technique was used in 12 patients, all of whom had an uncomplicated postoperative course. PMID- 7549198 TI - MR cholangiopancreatography: comparison between two-dimensional fast spin-echo and three-dimensional gradient-echo pulse sequences. AB - The purpose of our study was to perform a prospective comparative analysis of three-dimensional (3D) steady-state free precession (SSFP) and two-dimensional (2D) fast spin-echo (FSE) imaging in the evaluation of 26 patients with suspected bile duct obstruction. SSFP and highly T2-weighted FSE sequences were obtained for each patient in multiple planes. Both sequences were reviewed independently and results were compared with findings from direct cholangiography (n = 17) or from a combination of sonography and CT (n = 9). The extrahepatic bile duct [EHBD] and intrahepatic bile duct [IHBD] were dilated in 32% and 54% of patients, respectively. The EHBDs were visualized in 44% of patients with SSFP, versus in 96% with FSE. One or more IHBD segments were seen in 42% of the SSFP sequences and in 100% of the FSE sequences. A portion of, or the entire, pancreatic duct was seen in 23% of the SSFP sequences and in 65% of the FSE sequences. Our findings lead us to conclude that T2-weighted FSE sequences are superior to SSFP sequences in visualizing the biliary tree and pancreatic duct and that they should replace gradient-echo sequences in MR cholangiopancreatography. PMID- 7549197 TI - Transient increased segmental hepatic enhancement distal to portal vein obstruction on dynamic gadolinium-enhanced gradient echo MR images. AB - The purpose of the present study was to demonstrate the frequency of occurrence of transient increased segmental hepatic enhancement distal to portal vein obstruction in patients with a lobar (main branch) portal vein obstruction. MR images of all patients with main and lobar branch portal vein obstruction examined by dynamic gadolinium enhanced gradient echo MR images between December 1990 and July 1994 were reviewed retrospectively. All studies included T2 weighted imaging, T1-weighted spoiled gradient echo 'fast low angle shot ([FLASH])', and postgadolinium enhanced FLASH imaging at 1, 45, and 90 sec and 10 min. Fourteen patients were identified with portal vein obstruction which included: six with main portal and right and left branch occlusion, six with right lobar, and two with left lobar. In the six patients with main portal vein obstruction, enhancement on 1-sec postgadolinium FLASH images was homogenous (three patients), diffusely heterogeneous (two patients), or peripherally hyperintense (one patient). In eight of eight patients with isolated obstruction of the right or left lobar portal vein, transient-increased segmental enhancement distal to portal vein occlusion was observed on immediate postcontrast images. Relatively high signal intensity of the involved segments was present on 1-sec images and liver parenchymal enhancement became more homogeneous by 45 to 90 sec in all cases. In conclusion, transient-increased segmental enhancement occurred in eight of eight patients with isolated right or left portal vein occlusion. We postulate that this effect occurs due to increased hepatic arterial blood flow in the presence of portal vein obstruction. PMID- 7549199 TI - Oral superparamagnetic contrast agent (ferumoxsil): tolerance and efficacy in MR imaging of gynecologic diseases. AB - The purpose of this study was to determine the tolerance and the efficacy of the oral contrast agent ferumoxsil in the assessment of gynecologic diseases. Twenty patients underwent MR imaging at 1.5 T. T1-weighted spin-echo (SE) and T2 weighted fast SE images were obtained before and after ingestion of 600-900 mL of the superparamagnetic negative contrast agent ferumoxsil. No side effects were observed. No statistically significant increase in artifact generation was present in the postcontrast images. The efficacy in bowel marking was significant for the small bowel (P = .0001) and the cecum (P < .01), but not significant in all sequences for the sigmoid. In the postcontrast images, delineation of the uterus, the right-sided adnexa, the lymph nodes, and the pathologic lesions was significantly better (P < .01), but not all sequences showed an improvement in delineation of the other pelvic organs. After administration of ferumoxsil, the level of confidence in diagnosis was significantly higher (P = .0001), but no change in diagnosis was made from the pre- to the postcontrast images. We found the oral contrast agent ferumoxsil to be well tolerated and effective in marking the bowel and in delineating the normal organs as well as the pathologic lesions on pelvic MR images. PMID- 7549200 TI - A segmented K-space velocity mapping protocol for quantification of renal artery blood flow during breath-holding. AB - Two important prerequisites for MR velocity mapping of pulsatile motion are synchronization of the sequence execution to the time course of the flow pattern and robustness toward loss of signal in complex flow fields. Synchronization is normally accomplished by using either prospective ECG triggering or so-called retrospective gating. However, if the studied vessel moves periodically in space as a result of respiratory motion, as in the case of renal arteries, a second synchronization with respect to the vessel motion in space may be necessary. One method to overcome this problem is to use the segmented k-space technique, in which the entire data acquisition can be made within a breath-hold by the sampling of several phase-encoding lines within a small time window during each heart cycle. The aim of this study was to investigate the performance of a segmented k-space velocity mapping protocol for renal artery flow determination. The protocol uses 16 phase-encoding lines per heart beat during 16 heart cycles and gives a temporal velocity resolution of 160 msec. Comparison with a conventional ECG-triggered velocity mapping protocol was made in phantoms as well as in volunteers. In our study, both methods showed sufficient robustness toward complex flow in a phantom model. In comparison with the ECG technique, the segmentation technique reduced vessel blurring and pulsatility artifacts caused by respiratory motion, and average flow values obtained in vivo in the left renal artery agreed between the two methods studied.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549201 TI - Single breath-hold volumetric imaging of the heart using magnetization-prepared 3 dimensional segmented echo planar imaging. AB - We show the feasibility of single breath-hold volumetric imaging of the heart using a three-dimensional (3D) segmented echo planar imaging (EPI) pulse sequence. Fifteen healthy subjects were evaluated using three magnetization preparation schemes: (a) a driven equilibrium T2-weighted preparation for bright blood and dark myocardium; (b) a STEAM magnetization preparation for dark blood; and (c) fat suppression for coronary artery imaging. An interleaved EPI trajectory encoding six echoes per interleave with a 1090 Hz/pixel readout bandwidth was used to collect a 126 x 256 matrix in 22 heartbeats with data acquisition windows per cardiac cycle of 71-285 msec for 8-32 sections. Multiplanar reconstructions could be used if thin (1-3 mm) sections were acquired. Breath-hold volumetric imaging with 3D segmented EPI holds promise for rapid volumetric evaluation of cardiac anatomy. PMID- 7549202 TI - Influence of bolus volume and dose of gadolinium chelate for first-pass myocardial perfusion MR imaging studies. AB - First-pass MR myocardial perfusion measurements require a well-defined left ventricular (LV) blood pool input function. We used a peripheral intravenous (i.v.) injection of a gadolinium (Gd) chelate to obtain a well-characterized LV time-intensity curve. Using a strongly T1-weighted subsecond MR sequence, we performed cardiac MR imaging after administering an IV bolus injection of one of three different doses of the Gd chelate: a standard dose (0.1 mmol/kg, group I, n = 8); a low dose with two bolus volumes (0.01 mmol/kg, 1/10e bolus volume, group II, n = 7, and 0.01 mmol/kg diluted in saline, same bolus volume as group I, group III, n = 3); and an intermediate dose (0.05 mmol/kg, group IV, n = 5). Unlike in group I (high dose), in groups II and III (low dose), the LV curve had a well-defined first peak, followed by a downslope and a recirculation peak. With the intermediate dose (group IV), a saturation effect still remained on the LV curve. The signal intensity (SI) enhancement of the myocardium was respectively 580 +/- 77% at 0.1 mmol/kg, 362 +/- 95% at 0.05 mmol/kg, and at 0.01 mmol/kg, it was 184 +/- 33% in group II and 272 +/- 8% in group III. In conclusion, with subsecond T1-weighted MR imaging and a low dose of Gd chelate (i.e., 0.01 mmol/kg), the LV input function is a well-defined first step for MR perfusion modeling. PMID- 7549203 TI - Nonlinear excitation profiles for three-dimensional inflow MR angiography. AB - An RF excitation pulse for three-dimensional (3D) time-of-flight (TOF) MR angiography (MRA) with a nonlinear excitation profile was numerically calculated under the condition of uniform vessel signal across the excitation volume (slab), and the superiority of the optform pulse as compared with conventional RF pulses and TONE pulses was demonstrated. For this purpose we acquired MRA of the lower leg and of the carotid and vertebral arteries in a 30-year-old healthy volunteer. Although the flow velocity ranges in these two anatomic locations are different by about a factor of 10, in both cases the corresponding optform pulse provided the best signal homogeneity at the highest level. PMID- 7549204 TI - Variable-angle uniform signal excitation (VUSE) for three-dimensional time-of flight MR angiography. AB - A spatially asymmetric RF pulse that improves the uniformity of blood signal intensity and vascular contrast in three-dimensional (3D) MR angiography (MRA) is presented. The pulse, called variable-angle uniform signal excitation (VUSE), was designed to provide uniform signal response and improved contrast for blood flowing through a 3D imaging volume during a FLASH sequence. The VUSE excitation profile was optimized on the basis of the number of pulses seen by the blood, which varied with the velocity of through-plane flow, repetition time, and slab thickness with the maximum flip angle at the flow exit constrained at 90 degrees. The theoretical results show that the optimal RF pulse gives more uniformity for flow signal than does a linear ramp excitation profile or a Gaussian pulse combined with a presaturation pulse. After truncation and filtering of the VUSE pulse in the time domain, the general shape of the VUSE RF excitation profile is maintained, but the maximum flip angle is reduced. The arteries of the neck in a healthy volunteer were imaged with the VUSE pulse, a constant flip angle (flat) pulse, and a linear ramp pulse in flow-compensated 3D MRA sequences. The VUSE pulse produced the most uniform signal as evidenced by the lowest relative dispersion of signal along the left vertebral artery (18.0 versus 26.4 to 23.6 for the other studies). F-distribution tests also showed that the signal distribution obtained with the VUSE pulse in a 3D FLASH sequence was statistically different from that for the flat and the linear ramp pulses. PMID- 7549205 TI - Accuracy of MR phase contrast velocity measurements for unsteady flow. AB - The accuracy of MR phase contrast (PC) velocity measurements for unsteady flow has been quantitatively assessed. Spatially resolved velocity fields were measured in a long straight tube using a gated PC technique, and the resulting MR PC velocity data were compared with velocities derived from the analytic Womersley solution to the Navier-Stokes equations governing fluid flow. The overall root-mean-square (rms) difference between the measured and analytic velocities was 1.6 cm s-1 for nominally sinusoidal flow waveforms with peak velocities ranging from 51.6 cm s-1 to 59.8 cm s-1. This rms difference corresponded to 7.5% of the mean fluid velocity, which is similar to the cited accuracy of approximately 5% for MR PC velocimetry for steady flows. Linear regression between the PC velocity measurements and the velocities obtained using the analytic expression was highly significant (r2 = 0.997) and yielded a slope of 0.998, close to the expected value of 1. We conclude that the gated MR PC velocity measurements in unsteady flow are accurate. PMID- 7549206 TI - Partial volume effects in volume-localized phased-array proton spectroscopy of the temporal lobe. AB - MRS techniques can aide in confirming the location of seizure foci in temporal lobe epilepsy. N-acetyl aspartate (NAA), creatine plus phosphocreatine, choline containing compounds, and lactate are most often the clinically relevant metabolites in these studies. We examined the importance of partial volume effects from tissue heterogeneity in temporal lobe spectroscopy on the metabolite ratios. Our study shows that localized spectroscopy, using three different voxel sizes, centered on the anterior body of the hippocampus, produces significantly different values for the NAA to the creatine ratio. The spectroscopy was performed at 1.5 T using the PRESS pulse sequence and a phased-array coil system specifically designed for the temporal lobe. The data exhibits a clear trend of increasing NAA to creatine ratios with increasing voxel size. This trend demonstrates that partial volume effects can contribute to variation of NAA to creatine ratios in healthy subjects. PMID- 7549207 TI - Correlation between high-field T2-weighted MR imaging and histology of ischemic lesions in gerbil brain. AB - Global forebrain ischemia in the Mongolian gerbil is a common animal model for use in stroke research. We produced lesions of graded severity in gerbil brains (after prescreening by MR imaging) by performing 6-minute bilateral carotid artery occlusions while monitoring pericranial temperature with a temporalis muscle thermocouple probe and maintaining the temperature at 32 degrees C, 36 degrees C, or 40 degrees C. Lesion severity was scored 4 days after occlusion from findings on spin-echo images acquired at 7 T and from histologic scores. Statistically significant correlation was observed between the MR imaging score and brain temperature and between the MR imaging score and the area of the CA region of the hippocampus measured by histology. In addition, because prescreening with MR imaging revealed abnormalities in the hippocampus of some of the animals, and these animals were rejected from the study, the statistical significance of the result could be strengthened. PMID- 7549208 TI - Artifacts in functional magnetic resonance imaging from gaseous oxygen. AB - Unexpectedly large fluctuations in signal intensity were identified in the functional MRI (FMRI) of normal subjects breathing pure oxygen intermittently. To test the hypothesis that the signal changes were due to fluctuating concentrations of gaseous (paramagnetic) oxygen in the magnetic field, echo planar gradient echo images were acquired of a phantom contiguous to an oxygen mask through which pure oxygen was administered intermittently via plastic tubing. As a control, room air was administered intermittently or oxygen continuously in the same experimental protocol. Signal intensity changes of up to 60% temporally correlated with the administration of oxygen were produced in the phantom. In functional images prepared from the echo planar images, the signal intensity changes resulted in artifacts especially at interfaces in the phantom. The intermittent administration of pure oxygen during acquisition of data for FMRI may produce signal intensity changes that stimulate or obscure function. PMID- 7549209 TI - Comparison of T2 relaxation in blood, brain, and ferritin. AB - T2 was measured in samples of human blood and monkey brain over a field range of 0.02-1.5 Tesla, with variable interecho times, and was compared with previous data on ferritin solutions (taken with the same apparatus). 1/T2 in deoxygenated blood increased quadratically with field strength, as noted previously, but in brain gray matter the increase was linear, as also was the case in ferritin solution. In both deoxygenated blood and gray matter, 1/T2 increased with interecho time, but appeared to level off at times around 50 msec, as expected from the theory of diffusion through magnetic gradients. Diffusion times estimated by using the chemical exchange approximation were 3.4 msec for deoxygenated blood and 5.7 msec for the globus pallidus. The quadratic field dependence in blood is consistent with this same theory, but the linear dependence in brain tissue and in ferritin solutions remains unexplained. PMID- 7549210 TI - MR imaging of ventriculomegaly--a qualitative and quantitative comparison of communicating hydrocephalus, central atrophy, and normal studies. AB - Both communicating hydrocephalus and central atrophy cause ventricular dilatation. However, patients with hydrocephalus may require treatment. The aim of this study was to assess qualitatively and quantitatively the efficacy of MR imaging in the differentiation of communicating hydrocephalus from central atrophy. The midsagittal T1-weighted MR images of 33 patients with communicating hydrocephalus, 31 patients with central atrophy, and 23 normal subjects were evaluated qualitatively and quantitatively. This included configuration of the aqueduct; area of the septum pellucidum, third ventricle, and fourth ventricle; and morphology of the corpus callosum. Distal dilatation of the aqueduct was detected in 33.3% of patients with communicating hydrocephalus and in none of those with central atrophy. The corpus callosum was elevated in patients with communicating hydrocephalus when compared with that in patients with central atrophy. In conclusion, an analysis of midsagittal T1-weighted images has identified useful qualitative and quantitative criteria in the differentiation of communicating hydrocephalus from central atrophy. The configuration of the aqueduct with funneling at the fourth ventricular end strongly suggests the presence of communicating hydrocephalus rather than central atrophy alone. PMID- 7549211 TI - Effects of MR exposure on axonal outgrowth in the sympathetic nervous system of the chick. AB - The effects of MR exposure on the rate and specificity of sympathetic preganglionic axonal outgrowth were examined in the chick embryo. Embryos were exposed to a static magnetic field of 1.5 T for 6 hours, 64 MHz RF field pulses, and a switched magnetic field gradient of amplitude 0.6 G/cm for 4 hours. No significant difference in axonal outgrowth was observed between MR-exposed and control embryos. In addition, the distributions of several major extracellular matrix (ECM) molecules, laminin, fibronectin, and collagen IV, were examined. Immunostaining patterns of these ECM molecules during axonal outgrowth showed no difference between MR-exposed and control embryos. Our results suggest that the MR exposure conditions used in this study do not affect axonal outgrowth in the sympathetic nervous system of the chick. PMID- 7549212 TI - CSF-suppressed T2-weighted three-dimensional MP-RAGE MR imaging. AB - Fluid-attenuated inversion recovery (FLAIR) is a pulse sequence used for acquiring T2-weighted images of the brain and spine in which the normally high signal intensity of CSF is greatly attenuated. The CSF-suppressed T2-weighted contrast of this technique may be more sensitive to a variety of disorders than that of conventional T2-weighted imaging. The primary disadvantage associated with conventional spin-echo implementations of FLAIR is the relatively limited anatomic coverage that can be achieved in a reasonable imaging time. We developed and optimized a three-dimensional magnetization-prepared rapid gradient-echo (3D MP-RAGE) pulse sequence that combines CSF-suppressed T2-weighted contrast similar to existing FLAIR techniques with anatomic coverage characteristic of 3D imaging. A preliminary evaluation of the new sequence was performed by imaging healthy volunteers and patients with multiple sclerosis. PMID- 7549213 TI - Clearance of gadolinium chelates by hemodialysis: an in vitro study. AB - Although the gadolinium (Gd)-chelates currently approved for clinical use in the United States are considered "readily dialyzable," the actual clearance rates have not been published. The purpose of this study was to establish in vitro dialysis clearance rates of Gd-chelates to develop rational strategies for removing the agents with dialysis. Three agents, Gd-diethylenetriaminopentaacetic acid (Gd-DTPA), Gd-HP-DO3A and Gd-DTPA-BMA were diluted separately in plasma and saline and were dialyzed by using a clinical dialyzer unit with a commercially available capillary filter at rates of 0-300 cc/min. Predialyzer and postdialyzer concentrations were determined by inductively coupled atomic emission photometry. Urea and creatinine clearance rates also were determined. The clearance rate for Gd-chelates were considerably lower than that of urea and creatinine, which are generally considered benchmarks of dialysis efficiency. At 300 cc/min flow rates, the clearances were (clearance in cubic centimeters per minute with 95% confidence interval): Gd-DTPA-74 cc/min (68-80 cc/min) delta-HP-DO3A 67 cc/min (63-71cc/min); and Gd-DTPA-BMA 67 cc/min (63-71cc/min). In comparison, urea and creatinine were 180 cc/min (178-181 cc/min) and 142 cc/min (124-131 cc/min), respectively. There was no difference between clearance rates of Gd-chelates in saline and plasma, implying there was no protein binding. By using a first order kinetic model of dialysis time, more than 12.2 to 14.7 hours of dialysis would be necessary to remove 97% of the injected dose of Gd-chelate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549214 TI - MR assessment of posttraumatic flexion contracture of the elbow. AB - Preoperative assessment of posttraumatic flexion contracture of the elbow includes plain radiographs and tomograms, which are difficult to obtain in the coronal plane due to the contracture. We conducted this study to determine the usefulness of MR imaging in the work-up of these patients. Twelve patients with flexion contracture of the elbow were studied. In addition to standard spin-echo sequences, a sagittally acquired spoiled gradient-recalled echo 3D data set of the flexed elbow was obtained and reformatted coronally using a curved plane of reconstruction. The MR findings were compared to the plain films, tomograms and surgical results. MRI allowed identification of loose bodies that were sometimes poorly visualized, or not seen, on plain films, and demonstrated degenerative changes equally as well as tomograms. MR showed soft tissue abnormalities including capsular and collateral ligament thickening. Curvilinear reconstructions were helpful in the assessment of collateral ligaments in patients with severe contractures. We conclude that MR is useful in the evaluation of elbow flexion contractures, particularly in assessing soft tissue causes. PMID- 7549215 TI - Unusual MR features in a case of completely thrombosed giant aneurysm of the posterior cerebral artery. AB - An unusual case of completely thrombosed giant aneurysm of the left posterior cerebral artery, showing much more loss of signal on T2-weighted images compared with T2*-weighted images in the periphery of the thrombus is presented. Possible mechanism of this preferential signal loss on T2-weighted images is discussed. PMID- 7549216 TI - MR imaging and vascular access ports: ex vivo evaluation of ferromagnetism, heating, and artifacts at 1.5 T. AB - The purpose of our study was to assess ferromagnetism, heating, and artifacts associated with vascular access ports exposed to a 1.5-T MR system. Twenty-eight different vascular access ports were evaluated in this investigation. Ferromagnetism was determined by using two previously described techniques. Temperature changes were measured immediately before and after performing a pulse sequence on the vascular access ports for 60 min at a specific absorption rate of 3.1 W/kg. Artifacts were assessed in association with the use of a fast GRASS pulse sequence. None of the vascular access ports displayed ferromagnetism. Heating was 0.2 degrees C or less. The presence of artifacts varied, depending on the component materials. The lack of ferromagnetism and negligible heating indicates that MR imaging performed at 1.5 T or less may be conducted safely in patients with any of the vascular access ports tested. None of the associated artifacts produced by the vascular access ports is considered to pose a substantial problem for MR imaging. PMID- 7549217 TI - Coexistence of three competing microbial populations in a chemostat with periodically varying dilution rate. AB - Coexistence of three microbial populations engaged in pure and simple competition is not possible in a chemostat with time-invariant operating conditions under any circumstances. It is shown that by periodic variation of the chemostat dilution rate it is possible to obtain a stable coexistence state of all three populations in the chemostat. This is accomplished by performing a numerical bifurcation analysis of a mathematical model of the system and by determining its dynamic behavior with respect to its operating parameters. The coexistence state obtained in the periodically operated chemostat is usually periodic, but cases of quasi periodic and chaotic behavior are also observed. PMID- 7549218 TI - On the training and performance of high-order neural networks. AB - An extensive study was undertaken on the architecture, training, and properties of neural networks of order higher than 1. The formulation of the training of high-order neural networks as a nonlinear associative recall problem provides the basis for their optimal least squares training. The simplicity of the outer product rule motivates the study of the approximation of optimal least squares training by the outer-product rule and the effect of the network order on the efficiency of this approximation. Neural networks with composite key patterns are subsequently proposed as the natural generalization of neural networks of order higher than 1. The properties of this class of neural networks are revealed by studying their optimal least squares training and its relationship with the outer product rule. The performance of neural networks with composite key patterns is analytically evaluated by studying their capacity. The properties and performance bounds provided by the analytical study are verified through experimental results. PMID- 7549219 TI - Sinks, sources, and spotted owls: a territorial population model with continuous mortality and discrete birth. AB - A model is developed for the dynamics of a territorial population for which mortality and the search for suitable territories are treated as continuous processes and birth occurs as a discrete pulse once each year. It is shown that the trajectories converge either to a fixed annual cycle or to extinction for both groups as long as the individual reproductive success and survival rate for the territorial population exceeds that for those individuals not holding territories. Conditions that determine whether the population will persist are provided. PMID- 7549220 TI - Threshold parameters for epidemics in different community settings. AB - Threshold parameters of epidemic models play a central role in the assessment of proposed control strategies for infectious diseases. They have been determined for numerous standard epidemic models. This paper points out, with several examples, that threshold parameters depend on the social setting of the community and the variations in the behavior of the members of the community. Specifically, communities are considered in which individuals have fixed patterns of behavior or random patterns of behavior as well as communities of households with fixed or random patterns of behavior. A threshold parameter is computed for each of the different settings. Some comparisons are made to provide insights into the effects that social settings and behavior changes have on the threshold parameters. PMID- 7549221 TI - A stochastic model for multistage tumorigenesis in developing and adult mice. AB - A stochastic process model for one-, two-, and three-stage malignant transformation has been developed for embryonic and adult mice. The model has been used to study the influence of mutation rate, number of stages required for transformation, and number of stem cells at risk on the kinetics of spontaneous appearance of malignant tumors. As expected, tumors appeared earlier with fewer required mutational stages, higher mutation rate, and greater number of stem cells at risk. However, a notable observation was that tumor latency was more strongly influenced by number of stages and by stem cell number at lower mutation rates than at higher rates. This implies that tumor latency may be a less useful observation when the spontaneous mutation rate is high. In the future, the model will be applied to analysis of tumorigenesis experiments in transgenic mice with p53 genetic abnormalities, subjected to irradiation or chemical tumorigenesis at different stages of development. PMID- 7549222 TI - Effect of zatebradine, a novel 'sinus node inhibitor', on pulmonary function compared to placebo. AB - Zatebradine, a member of a novel class of drugs called 'sinus node inhibitors', is a specific heart rate lowering drug suitable for the treatment of stable angina pectoris. Animal studies showed that relatively high intravenous doses of zatebradine contracted guinea-pig airways by a histamine-like mechanism. Therefore, the objective of the present study was to assess the bronchopulmonary effects in asthmatic patients who showed a moderate to severe bronchial hyperreactivity towards histamine (PC20 FEV1 < or = 1 mg/ml). Moreover, it had to be established to what extent the bronchial responsiveness to inhaled salbutamol was retained and whether pulmonary effects were related to the severity of bronchial hyperreactivity. By means of a double-blind cross-over design, single oral doses of zatebradine (10 mg) or placebo were administered on two occasions with a washout phase of at least 3 days. Sixteen patients, four female and 12 male, with stable mild to moderate bronchial asthma (FEV1 less than 80% predicted) were selected. Their mean age was 54 years and the mean FEV1 was 1.831 (59% predicted). They showed a mean improvement in FEV1 of 27% 15 min after inhaling 200 micrograms salbutamol; the mean PC20 was 0.35 mg/ml. Following test drug intake, the respiratory and cardiac effects were assessed at regular time intervals up to 6 h after administration. In comparison to placebo, zatebradine induced small, but significant (P < 0.05), mean falls of 128 ml and 168 ml in FEV1 at 3 h and 6 h after drug intake. A large inter-individual variation in response was noted.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549223 TI - Addition of a bacterial alginate lyase to purulent CF sputum in vitro can result in the disruption of alginate and modification of sputum viscoelasticity. AB - Alginate is a large molecular weight exopolysaccharide present in the purulent airway secretions of cystic fibrosis (CF) patients. This polymer, produced by some of the opportunistic pathogens associated with the recurrent lung infections characteristic of CF, has been suggested to effect an increase in the viscoelastic properties of purulent CF airway secretions. We have investigated the use of an enzyme targeted at this exopolysaccharide, an alginate lyase obtained from a bacterial source, to disrupt its polymeric nature and effect a change in the rheological properties of CF sputum in vitro. Expectorated sputum samples obtained from hospitalized CF patients were found to contain 80-200 micrograms alginate per ml sputum with no measurable endogenous alginate lyase activity. Treatment with exogenous alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa resulted in the disruption of alginate and a decrease in sputum viscoelasticity in a small percentage of the samples tested. Similar treatment of these samples with recombinant human deoxyribonuclease I to cleave DNA present in purulent sputum and the use of alginate extracted from sputum as an alginate lyase assay substrate suggested that the inability of the exogenous alginate lyase to disrupt sputum alginate was not due to substrate inaccessibility or an unresponsive substrate. Concentrations of Ca2+ and Zn2+ in alginate lyase-resistant sputum samples, determined by metal ion analysis, were found to inhibit enzyme activity in studies using seaweed alginate as a substrate. High concentrations of Ca2+ and Zn2+ in sputum samples initially resistant to lyase activity could be reduced significantly in some samples by dialysis and these same samples acquired sensitivity to the lyase. Other sputum samples did not show reduced concentrations of Ca2+ and Zn2+ following dialysis and these samples remained lyase-insensitive. Together, these results suggest that bacterial alginate present within purulent CF sputum may be quite stable, that endogenous alginate lyase activities appear to be limited and that the in vitro addition of exogenous alginate lyase can lead to the disruption of alginate and a change in the viscoelastic properties of some purulent CF sputum samples. PMID- 7549224 TI - Hyperresponsiveness of the airways following exposure of guinea-pigs to racemic mixtures and distomers of beta 2-selective sympathomimetics. AB - Allergic bronchospasm in sensitized guinea-pigs was totally suppressed by acute subcutaneous infusion of rac-salbutamol (0.69 microgram/kg per min) for < 1 h. More prolonged infusion of rac-salbutamol induced a progressive susceptibility to inhaled antigen so that, by 48 h, animals collapsed and died following inhalation of antigen. In anaesthetized animals, acute infusion of rac-salbutamol (1.67 micrograms/kg per min) suppressed airway obstruction, an effect that can be attributed to beta 2-adrenoceptor activation by the eutomer (R-salbutamol). Acute intravenous infusion of the distomer (S-salbutamol) (1.67 micrograms/kg per min) induced hyperresponsiveness to histamine without having any effect upon airway calibre. It is suggested therefore that subcutaneous infusion of rac-salbutamol initially abrogates the bronchoconstrictor response to antigen because the bronchodilator action of the eutomer predominates over hyperreactivity attributable to the distomer. Conversion from protection to susceptibility was not determined by reduced beta 2-adrenoceptor activation since animals could be protected from a lethal response to antigen by inhalation of rac-isoprenaline or by subcutaneous injection of rac-salbutamol. The seeming progressive loss of efficacy of R-salbutamol may result from disproportionate accumulation of S salbutamol if, as in man, there is stereospecific metabolism of R-salbutamol. The capacity of S-salbutamol to evoke hyperresponsiveness is shared by S-isoprenaline and S-terbutaline and, as has been shown previously for rac-isoprenaline, the capacity of S-salbutamol to elicit hyperresponsiveness was not evidenced following section of the vagus nerves. No mechanism has yet been established which might account for this property of S-salbutamol or for other S-enantiomers of sympathomimetics. PMID- 7549225 TI - Pulmonary penetration of dirithromycin in patients suffering from acute exacerbation of chronic bronchitis. AB - The aim of this study was to evaluate the concentrations of dirithromycin, a new macrolide antibiotic, in bronchial secretions (BS), bronchial mucosa (BM), epithelial lining fluid (ELF) and serum in 25 patients with acute exacerbation of chronic bronchitis after a 5-day, once-daily, dirithromycin regimen. All patients received dirithromycin, 500 mg (two 250 mg tablets) given orally once daily at 08.00 fasted, for 5 consecutive days. They were divided into five groups (n = 5 in each group) according to sampling time (24, 48, 72, 96 and 120 h, after the last dose). Mean serum concentrations remained low throughout the study (0.44 microgram/ml at 24 h, 0.31 microgram/ml at 48 h, 0.33 microgram/ml at 72 h, 0.12 microgram/ml at 96 h and 0.11 microgram/ml at 120 h, respectively), although they were higher than the MICs for Moraxella catarrhalis for up to 72 h and than that for Streptococcus pneumoniae for up to 120 h after the last dose. By contrast, in all other samples, mean concentrations were higher than the MICs for many relevant respiratory pathogens for at least 3 days, and higher than that for S. pneumonia and M. catarrhalis for up to 120 h (mean concentrations measured 2.67, 2.15, 1.74, 0.27 and 0.17 micrograms/ml, respectively, in BS; 2.59, 2.59, 1.96, 0.41 and 0.27 micrograms/g, respectively, in BM; 2.21, 2.25, 1.57, 0.22 and 0.15 micrograms/ml, respectively, in ELF). These findings demonstrate that dirithromycin is concentrated in each of these potential sites of infection for up to 3 days after a 5-day course of therapy. Therefore, short-term therapy with dirithromycin may be useful for many respiratory infections. PMID- 7549226 TI - Pharmacological modulation of platelet-derived growth factor (B) mRNA expression in alveolar macrophages and adherent monocytes. AB - The macrophage profibrotic cytokine, Platelet Derived Growth Factor B [PDGF(B)], is thought to play a central role in orchestrating the fibrotic response in the pathogenesis of cryptogenic fibrosing alveolitis. In this study, we have asked if drugs that increase intracellular cAMP and are commonly administered to patients with lung disease have the ability to downregulate PDGF(B) mRNA. Incubation of human alveolar macrophages from healthy smokers in the presence of dibutyryl cAMP prevented the previously reported dexamethasone-induced increase in PDGF(B) mRNA (P < 0.05). Similarly, the combination of aminophylline (2.5 mM) and salbutamol (1 microM) prevented the adherence-dependent increase in PDGF(B) mRNA in adherent human peripheral blood monocytes (P < 0.05), whilst causing an increase in the mRNA expression of the cAMP-dependent gene c-fos (P = 0.059), and an increase in the intracellular concentration of cAMP (P = 0.05). Finally, the presence of a lower concentration of aminophylline (0.25 m) in conjunction with salbutamol (1 microM) also prevented the dexamethasone-induced increase in PDGF(B) mRNA in alveolar macrophages from healthy smokers (P < 0.05). Stimulation by these drugs was not associated with a change in the abundance of the mRNA of the house keeping gene, glyceraldehyde-3-phosphate dehydrogenase. We speculate that drugs, which increase intracellular cAMP, may provide a novel therapeutic avenue whereby PDGF(B) expression in patients with cryptogenic fibrosing alveolitis may be reduced. PMID- 7549227 TI - Effect of ibuprofen on thrombin-induced pulmonary edema in the rat. AB - The effect of ibuprofen on thrombin-induced pulmonary edema was studied in rats. Thrombin infusion produced a significant increase in lung weight, wet weight/dry weight ratio and relative lung water content, a rise in mean pulmonary arterial pressure and a fall in mean systemic arterial pressure. It also caused a progressive decrease in PaO2 and a continuous increase in pH and PaCO2. Administration of either the S-isomer or R-isomer of ibuprofen at doses of 5 mg/kg body weight prior to thrombin infusion resulted in significant reduction in lung weight, wet weight/dry weight ratio and water content. The wet weight/dry weight ratio and the water content were somewhat lower after infusion of the S isomer than of the R-isomer. Ibuprofen diminished the thrombin-induced increase in mean pulmonary arterial pressure and attenuated the early and late decrease in mean systemic arterial pressure caused by thrombin. Ibuprofen also stabilized thrombin-induced impairments in PaO2, PaCO2 and pH. The results thus indicate that ibuprofen effectively counteracts hemodynamic changes, stabilizes impairments in arterial blood gas variables and attenuates the increase in lung vascular permeability to protein with pulmonary edema caused by thrombin. The results also indicate a substantial R to S chiral inversion of ibuprofen in vivo in the rat. PMID- 7549228 TI - Pinacidil-induced relaxation in pulmonary arteries isolated from pulmonary hypertensive and normotensive rats and pre-contracted with different spasmogens. AB - Vasorelaxant responses to the potassium channel opening drug, pinacidil, were obtained on preparations of pulmonary artery and aorta taken from rats with pulmonary hypertension (induced by chronic hypoxia or monocrotaline) and pre contracted either submaximally with endothelin-1 (ET-1), PGF2 alpha, U46619 (thromboxane-mimetic) or noradrenaline (NA), or with 80 mM K+. In pulmonary artery, but not aorta, from pulmonary hypertensive rats the maximum relaxant response to pinacidil was increased, when compared with data in control rats, irrespective of the spasmogen used to precontract the tissues. The increase in maximum was associated with relaxation to below the tissue resting baseline and probably reflected the presence of inherent contractile tone in arteries from pulmonary hypertensive rats. In addition the potency (-log EC50) of pinacidil was increased in pulmonary arteries from pulmonary hypertensive rats but this was seen only in preparations contracted with ET-1 (30-fold increase) or NA (seven fold increase) and not in those contracted with PGF2 alpha, U46619 or K+. As a result, in ET-1 contracted preparations from pulmonary hypertensive rats pinacidil was 29-fold more potent on pulmonary artery than on aorta. To explain the increase in potency it is speculated that during the development of pulmonary hypertension the mechanism whereby ET-1 and NA contract pulmonary arteries may change from one in which Ca2+ influx plays only a minor role to one in which Ca2+ influx predominates, although no direct evidence to support this speculation has yet been obtained. PMID- 7549229 TI - Angiotensin II enhances responses to endothelin-1 in bovine bronchial smooth muscle. AB - Angiotensin II and endothelin-1 are putative mediators in asthma. In this study we have examined the effect of angiotensin II on endothelin-1-induced contractions in bovine bronchi and the receptor types involved in the response to these agonists. Angiotensin II alone is very low in potency, producing only small contractions. In the presence of angiotensin II 10(-7) or 3 x 10(-7) M, contractions evoked by endothelin-1 were markedly enhanced. The AII1-receptor antagonist, losartan, abolished this enhancement suggesting that angiotensin II exerts this effect via an AII1-receptor. The contraction evoked by endothelin-1 is mediated via an EtA-receptor subtype since the EtA-receptor antagonist FR139317 attenuated the response. This is offset by an inhibitory EtB-type receptor, resulting in a larger contraction when these receptors are desensitized. Indeed, the EtB-receptor agonist sarafotoxin S6c reversed methacholine-evoked tone in a concentration-dependent manner. In conclusion, angiotensin II potentiates contractions evoked by endothelin-1 in bovine bronchi. This may be a mechanism by which angiotensin II--which has little activity in bronchi--may evoke substantial changes in airway tone. Angiotensin II evokes this potentiation via AII1-receptors, whilst endothelin-1 evokes contraction via EtA receptors, an action which is offset by an inhibitory effect of EtB-receptors. PMID- 7549230 TI - Symposium on neonatal transfusion practices. PMID- 7549231 TI - Small volume red blood cell transfusions for neonatal patients. PMID- 7549232 TI - Massive transfusion in the neonate. AB - The neonate who undergoes massive transfusion is at risk for transfusion associated complications similar to the adult, but also faces some that are unique to the infant. By understanding the mechanics of the procedures that result in single or multiple blood volume exchange, the transfusion medicine physician can better assist his/her colleagues in the support of these patients. PMID- 7549234 TI - Blood component therapy in neonatal hemostatic disorders. PMID- 7549233 TI - Platelet transfusion therapy in newborn infants. PMID- 7549235 TI - Blood component and immunotherapy in neonatal sepsis. AB - The future role of IVIG remains unclear. Many, but not all, studies indicate efficacy in the prevention of late-onset disease in premature infants. The role of type-specific IVIG is evolving and may hold promise for both prevention and treatment of neonatal sepsis. Granulocyte transfusions, as adjuvant therapy in neonatal sepsis, seem to be beneficial. However, the difficulty and expense of collection, as well as the advent of colony-stimulating factors, have shifted the focus away from their routine use. Colony-stimulating factors present varied and exciting potential uses, including modulating neonatal hematopoiesis. Current studies are primarily aimed at understanding their effects on neonatal hematopoiesis. Future studies will need to expand on this knowledge and examine what effects they have on treating or preventing neonatal sepsis. PMID- 7549236 TI - The fetus as a recipient and donor of blood components. PMID- 7549237 TI - Transfusion-transmitted infections in the newborn. PMID- 7549238 TI - Noninfectious adverse effects of blood transfusion in the neonate. AB - There are many potential noninfectious complications that may be associated with the transfusion of blood products to the neonate. Most of these can be avoided by the careful preparation of the product according to current standards and the careful monitoring of the infant for metabolic changes during a transfusion. In general, there have been fewer systematic studies of transfusion-associated complications in neonates than in adults, possibly because of the difficulty of establishing a cause-effect relationship in the neonatal patient population. Such data, if available, would be useful for providing more effective blood product therapy to the neonate. PMID- 7549239 TI - [Changes in life expectancy and mortality in East Germany after reunification (1989-1992)]. AB - Whereas some arguments can be advanced suggesting that the life expectancy in east Germany should have declined directly after the fall of the Berlin Wall in 1989, other arguments suggest an increase. The aim of this study was to identify the actual developments and to explain the findings. Census data and mortality statistics from East and West Germany before unification were used to calculate standardized mortality ratios and life expectancies for various population groups. The differences in life expectancy between East and West were broken down according to age groups. The main finding was that the life expectancy of east German men declined in 1990 by 0.9 years, and only reached the 1989 level again in 1992. This was due solely to an increase in mortality for men under the age of 65. In 1990 and 1991, there were 3,400 more deaths among men under the age of 65 than would have been expected on the basis of the mortality rates of 1989. In contrast, the life expectancy of women hardly declined at all in 1990, and in 1992 it was already one year more than for 1989. The most important reasons for the increased numbers of deaths of men under the age of 45 were motor vehicle accidents, whereas ischaemic heart disease and cirrhosis of the liver were more significant for men between the ages of 45 and 65. Suicides did not increase after the fall of the Berlin Wall. It could be shown that the findings were not the results of artifacts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549240 TI - [Disability payments in diabetes mellitus in social security: what is necessary?]. AB - Since their publication in 1974 the recommendations of the "German Society for Public and Private Welfare" are regarded as competent guideline for the allocation of grants concerning the diet for diabetic subjects according to section 23 part 4, position 2 BSHG (Federal German Law for Social Security). The former nutrition concepts are compared to the current international conceptions regarding the diet of diabetic individuals. Possible variations concerning food plans are shown in model plans considering realistic habits of consumption. Our analysis justifies additional expenses limited to about 20 to 30% for insulin dependent diabetic subjects compared to an isocaloric regular diet. Additional expenses for obese non-insulin-dependent diabetic subjects are not necessary. There is an extremely slim portion allocated for food within the social security benefit according to section 22 BSHG. That will require an comprehensive supply of education for the realisation of the current recommendations for nutrition. The education programmes for compiling suitable food plans to remain within the scheduled cost limits need to be organized by regional carriers of social welfare, whereby public Health offices could make an important contribution. PMID- 7549241 TI - [Aspects of process quality in quality assurance regarding inpatient psychosomatic rehabilitation]. AB - Quality assurance in in-patient psychosomatic rehabilitation is performed basically by an external, general, structure-oriented and outcome-oriented programme of quality assurance conducted by statutory insurance bodies. In contrast, the differentiation of quality assurance in special fields of rehabilitation as well as the elaboration of the dimension of process in quality assurance have been neglected. Particular conditions of treatment in in-patient psychosomatic rehabilitation highlight the importance of the quality of procedure. Among these are the relatively longer duration of treatment, the particular dynamics and liability to disturbances of psychotherapeutic interaction, the cooperation of different therapeutic fields within a comprehensive plan of treatment and the specificity of treatment goals for each individual patient. Such a kind of quality assurance relating to procedure is made feasible by two types of structural elements of quality of procedure in in patient psychosomatic-psychotherapeutic treatment. Iterative structural elements of procedure (e.g. team conferences, supervision) serve to control the cooperation of the team by means of a random sample of patients. Sequential structural elements (e.g. admission conference, intermediate review conference, discharge conference) help in monitoring systematically the course of treatment in relation to each individual patient. As for the obtaining of information, sequential structural elements of assurance of quality of procedure make routine monitoring possible, whereas iterative elements create space for spontaneous notification of disturbances. PMID- 7549242 TI - [Formal uniform organization of quality parameters in public health--a methodological suggestion]. AB - The relevance of many suggestions for quality management of medical practice and health care is still unsatisfactory. The mass of all the existing suggestions for quality assurance and quality control made a homogeneous method even more difficult all efforts should be directed at finding quality parameters for the most frequent performances in basic care (family doctors). A uniform method and setting up an institute will be important for coordination between specialists, medical societies and relevant institution for exchanging experiences, practice and findings. OBJECTIVE: Suggestions for a homogeneous method to develop quality parameters of medical care. PMID- 7549243 TI - [Mercury burden in humans caused by housing in formerly mirrored rooms]. AB - 42 persons aged from 3 to 70 years, who have lived in rooms with mercury pollution (mercury in the air 1-70 micrograms/m3), were examined clinically and by bio-monitoring (blood and urine). There was no evidence of macro-mercurialism. 2 persons showed reversible clinical signs of mercury intoxication. The correlation between mercury level of the indoor air and blood or urine is presented, the clinical findings and threshold values are discussed. PMID- 7549244 TI - [Use of fluorides and dental visits in elementary school students of the Rems Murr district and conclusions for dental health--results of a parent survey (1993)]. AB - After a dental examination at school a representative sample of 1244 schoolchildren in the Rems-Murr district received a questionnaire which had to be answered by their parents at home. The questions dealt with the use of fluorides and dental consultation of the children. Forty-nine per cent of the questionnaires were returned. The survey shows that fluoride supplementation by tablets enjoys an acceptance of more than 90% in early childhood, but loses much of its importance in later years. Eighty-eight per cent of the pupils brush teeth with a fluoridated dentifrice and ca. 39% of the households use fluorinated salt. Fluorides are frequently combined in an inappropriate way. This indicates that parents do not always know exactly how to use the products or simply confuse them with others. Dentists do not reserve topical fluoride applications for selected caries risk groups, as is fixed by law, but offer them to all their patients. More than 90% of the children see a dentist at least once and about 70% at least twice a year. Many children with untreated deciduous teeth had visited their dentist only a few weeks or months previous to the examination at school. Probably many dentists reject treatment of deciduous teeth for economic reasons. Consumers have to be instructed more intensively about the use of fluorides and should receive more specific advice adapted to their individual caries risk. To improve treatment of deciduous teeth modifications in the sickness insurance refund tariff system are necessary. PMID- 7549245 TI - [Teaching/learning model for the social medicine course in human medicine at the Berlin Free University]. AB - Considering the discussions on the reform in medical education and the Free University of Berlin's plans for a reformed curriculum for medical studies, a new model in teaching and learning for the course "social medicine" was developed and tested. The model deals with students' interests in topics of social medicine and allows inductive access to matters of social medicine by means of exemplary learning. Teacher's task is to be moderator and organizer of learning processes. Experiences show that this procedure leads to improved interest in and more engagement towards the subject "social medicine" within the reach of the course. PMID- 7549246 TI - [Providing practice-oriented social medicine in medical education--a lecture concept]. AB - This article outlines the concept on which lectures in Social Medicine offered at the Institute for Occupational, Social and Environmental Medicine of the University of Erlangen-Nuremberg for the past three years are based. In addition to the fundamentals of Social Medicine, in our lectures we impart practical and career-oriented knowledge of the social security system and socio-medical diagnostics and how to compile medical assessments. We thus try to make it easier for the prospective physicians to later give help and advice to their patients about gaining social welfare benefits. Additionally we instruct the students in the basics of preventive medicine and the economics of medical activity. Consequently they recognise the importance of these topics for their later work in a clinic or as general practitioners. Our concept is in accordance with the intended modification of the German legislation governing the permission of doctors to practise medicine. Improvements are planned which will gear medical training better with the requirements of a medical practitioner as well as offering more intensive instruction in preventive medicine and the economy aspects of health care. PMID- 7549247 TI - [Blind alleys and misconceptions in public health]. AB - The concept of hygiene was created in the 19th century although Hippocrates had already conceived an influence of atmosphere, soil and water on human health. The concept of a public health organisation, however, is a fairly recent one. Environmental and social hygiene were the two poles of the new discipline that focussed on public health. However, the ideologies of capitalism, communism and socialism as well as of social darwinism and "survival of the elite" discredited social hygiene. The decline of totalitarianism was associated with a "loss of face" of state-controlled medicine, including social hygiene. Both the post-World War II German constitution and the previous German statutory health insurance ordinance had blocked it, and hence, no Federal bill on public health was carried. The consequences of this disregard of public health are poor protection by vaccination, a gap in compulsory notification and in epidemics control and high rates of nosocomial infections. Absolutely no development of the science of epidemiology was possible whereas that of medical microbiology is choked by the system now in existence. There is a great misconception within individual hygiene by identifying it merely with cleanliness. Hygiene became a synonym for cleanliness, although that had evolved during a long cultural sociological process centuries before hygiene was established. The modern evolution of the science of hygiene shows the danger that emphasis on healthy lifestyles or on environmental protection may result in regulations and finally in a tyranny that may threaten the liberty of human rights. The so-called "principle of concern" is an example of such irrationality because there is no sensible proportion between risk and expense. PMID- 7549248 TI - [Comment on H. Scherb and E. Weigelt: Statistical description of health complaints following pyrethroid exposure]. PMID- 7549249 TI - [Natural epidemiology of Lyme borreliosis with reference to clustered incidence of illnesses in the suburbs of a North Baden community]. AB - Human infection with Borrelia burgdorferi, the causative agent of Lyme disease, is unusually common in the "Scheelkopf" area of the town Bruchsal in north Baden (Germany), a situation which has led to considerable publicity and public concern. This study was carried out in order to clarify this situation by determining the prevalence of B. burgdorferi in both the free-living tick populations (Ixodes ricinus) and the rodent population from the "Scheelkopf" as well as from surrounding control areas. The polymerase chain reaction (PCR) was used to determine the presence of infection in whole tick preparations and in mouse bladders. The prevalence of B. burgdorferi in freeland ticks ranged from 19% to 44% and in mice from 6% to 29% depending on the area studied. The "Scheelkopf", with prevalences for ticks and mice of 33% and 10% respectively, was not significantly different from the control areas. Our results indicate that there is a high risk of human infection throughout the study area. This is probably related to the intensive use of the area for gardens and the related recreational behaviour of the human population in conjunction with the high rate of infection prevailing in I. ricinus. PMID- 7549250 TI - [The prevalence of viral hepatitis markers in the general population]. AB - The study objective was to measure the characteristic serological markers prevalence for viral hepatitis A, viral hepatitis B and viral hepatitis Delta in a representative group from apparent healthy general population. Viral hepatitis infections represent through their frequency and gravity a public health problem, in our country. A total of 860 subjects from 13 districts sided in the south part of the country were investigated. The sample were distributed by 9 age group. By using a certain work algorithm we examined the following serological markers: HBs Ag, anti HBs, AgHBe/antiHBe, AgHD/antiHD. For the serologic investigation we used ELISA test with Wellcozyme and Sanofi Diagnostics Pasteur Kits. The results of our study confirm high prevalence values for HAV and HBV (65.8% respectively 44.9%). We could estimate that 73.5% from the population has passed through either HAV infection or HBV infection and 29.1% to 37.7% has passed through both infections. The study also proved a very high HBs Ag carriage (10.5% to 30.9%). The HBe Ag were detected only in HBs Ag carrier children (4.5% to 13%). The presence of anti HBe was 31.4%. From the investigated persons 10% had hepatitis Delta virus infection. PMID- 7549251 TI - [The markers of hepatitis B, C and D viral infection in multiply transfused patients]. AB - OBJECTIVE: We aimed to assess the seroprevalence of HBV, HCV and HDV virus markers in multi-transfused patients from Cluj-Napoca. MATERIAL AND METHODS: Stored serum samples of 105 multi-transfused patients (25 children, 19 adults and 61 chronically hemodialyzed patients) have been tested for HBsAg, anti-HBs, total anti-HBc, anti-HCV, total anti-HDV by automated ELISA (Sanofi Diagnostics Pasteur kits). RESULTS: HVC infection has been observed in 4/25 (16%) children, 14/19 (74%) multi-transfused adults and 48/61 (79%) haemodialysis patients. 8/25 (32%) children, 17/19 (89%) adults and 47/61 (77%) haemodialysis patients had HBV infection markers. Anti-HDV have not been found in HBV infected multi-transfused children and adults, respectively. Only 2/47 (4.25%) HBV infected haemodialysis patients had HDV infection markers. The prevalence of double infection (HCV and HBV) was high (4%, 84.2% and 67.2% in children, adults and haemodialysis patients). The prevalence of viral hepatitis markers correlated to the amount of transfused blood, and in haemodialysis patients also correlated to the duration on dialysis. CONCLUSIONS: In multi-transfused patients from Cluj area, the prevalence of viral hepatitis markers is high. The double infection (HCV and HBV) is frequent, especially in adults. The prevalence of HDV infection markers in HBV infected patients is low, in contrast with previously reported results. PMID- 7549252 TI - [The prevalence of hepatitis C and B viral markers in patients with sexually transmitted diseases in Cluj]. AB - BACKGROUND: Recently, hepatitis C virus (HCV) have been recognised as being one of the most important cause of non-A, non-B hepatitis. Although parenteral transmission of HCV is wellknown, the risk of sexual transmission is still under debate. OBJECTIVE: This study was conducted to assess the HCV infection markers in sexually transmitted disease (STD) patients from an endemic area for hepatitis B virus (HBV) infection. Additionally, we studied the prevalence of HBV infection markers in these patients. PATIENTS AND METHODS: The sera of 121 patients with STD from Cluj-Napoca have been tested for antibodies to HCV (anti-HCV), Hbs antigen (Hbs Ag), antibodies to Hbc antigen (anti-HBc), by automated ELISA. RESULTS: Anti-HCV were detected in 26/121 (21.5%) patients: 73/121 (60.3%) patients presented HBV infection markers HBV infection markers (HbsAg and or anti HBc) have been observed in 22/26 (84.6%) HCV infected patients. CONCLUSIONS: Prevalence of HCV infection markers in STD patients from Cluj is high (21.5%). HBV infection markers have been detected in 60.3% of patients. In STD patients form Cluj HCV infection markers have been correlated to HBV infection markers. PMID- 7549253 TI - [The role of the hepatitis B, C and D viruses in the etiology of chronic hepatopathies in Cluj]. AB - OBJECTIVE: We studied the prevalence of viral hepatitis B, C and D markers in chronic hepatopathies from Cluj. MATERIAL AND METHODS: Sera of 297 patients with chronic hepatopathies (236 adults and 61 children) have been tested for viral hepatitis markers: HBsAg, anti-HBc, anti-HBs, HBeAg, anti-HBe, anti-HDV, anti HCV, by automated ELISA. RESULTS: HBV infection markers in 32% (adults) and 4.9% (children), and HDV infection markers in 11.8% (adults) and 26.3% (children). Double (HBV and HCV) and triple infection (HBV, HDV and HCV) were observed in 28.4% (adults), 4.9% (children), and 3.4% (adults), 0% (children), respectively. CONCLUSIONS: Hepatitis virus infection markers, especially HBV and HCV play an important role in the determinism of chronic hepatopathies from Cluj area, both in children and adults. PMID- 7549254 TI - [The malaria situation in Romania: 1980-1994]. PMID- 7549255 TI - [Human leptospirosis in Romania in 1985-1994]. PMID- 7549256 TI - [The evolution of rabies in Romania. The biological characteristics of the strains of the rabies virus isolated in Romania]. PMID- 7549257 TI - [The clinical and immunological correlations between the p24 antigenemia levels and those of anti-p24 antibodies in HIV-seropositive children]. AB - The influence of HIV I p24 antigen immune complexing with anti-p24 antibodies of the assessment of their respective levels in HIV-positive sera was studied. ELISA tests were used for evaluating anti-p24 and p24 antigenemia, with or without acid dissociation. We have observed that: 1. p24 antigenemia usually coexisted with low anti-p24 levels, an inverse correlation between these two parameters being traced; acid dissociation increased the percentage of p24 positive sera, especially when anti-p24 titers are low; 2. on contrary, after acid dissociation, p24 Ag remain undetectable in 55.56% of patients presenting high titers and 29.41% of those with low levels of anti-p24; acid dissociation do not increase anti-p24 titers. Whereas the first group of observations suggests that p24 Ag and anti-p24 Ab may be involved in immune complexes, the second set indicates that p24 Ag and Ab were not inevitably linked in such complexes. So, they may be indicative for two distinct biological phenomena. PMID- 7549258 TI - [The resistance to antibiotics and chemotherapeutic agents of Shigella strains isolated from HIV-seropositive children]. PMID- 7549259 TI - [Purulent meningitis with nonserotypable Haemophilus influenzae in infants and adults]. PMID- 7549260 TI - [Staphylococcus saprophyticus isolated in blood cultures from normal-weight newborn infants with septic arthritis]. AB - The paper deals with the identification of some Staphylococcus saprophyticus strains received by the Staphylococcus National Reference Centre, in the Cantacuzino Institute, Bucharest, Romania. The strains were isolated from blood cultures pre-elevated in a maternity in Bucharest from normoponderal newborns with septic arthritis. Recent data from literature concerning the infections caused by coagulase negative staphylococci in general, with special reference to those cause by S. saprophyticus are presented. The identification scheme used in the Cantacuzino Institute and the minimal microbiological diagnosis tests of S. saprophyticus recommended to the clinical laboratories by the International Society for the Taxonomy of Staphylococci and Micrococci are also described. PMID- 7549261 TI - [The evaluation of the immunofluorescence reaction in the study of Mycoplasma development]. PMID- 7549262 TI - [Ion Cantacuzino--the brilliant initiator of Romanian concerns in the area of cholera]. PMID- 7549263 TI - [Human myiases]. AB - This general review presents a synthesis concerning the human myiases recorded all over the world, with reference to their incidence within the temperate regions in North America and Europe, including Romania. The report deals with both entomology, pathology, epidemiology matters (notifying the hazard of world spreading) and therapeutics, prophylaxis ones, and 5 tables are also inserted that contain the flies the most involved in the human pathology. PMID- 7549264 TI - [An evaluation of the antidiphtheria protection in the general population]. AB - The assessment of the antidiphtheria protection in healthy population is common for a surveillance system within any National Program of Immunization. Our study is intending to measure the antidiphtheria immunity level in apparently healthy peoples living in the southern Romania. Our study were carried out on 8594 healthy subjects. Sample were stratified by nine age groups being representative for the above mentioned area. The titration of diphtheria antitoxin in our laboratory were performed by passive hemagglutination assay (PHA) and in vivo neutralization test (NT). In our study, we had considered as "protective immunity level" an antitoxin titers equal or above 1/640. The overall prevalence of protected peoples was 88.1% (C.I. 95%: 87.4%-88.8%). The prevalence of protected people against diphtheria decrease by age from 94.5% (1-9 years of age) to 82.4% (60 years and over). We think that overall, our national strategy for diphtheria control is quite efficient, but need to be sustained by specific actions concerning some high risk population groups. PMID- 7549265 TI - [The efficiency under epidemic conditions of the measles vaccination of 1st-grade pupils]. PMID- 7549266 TI - Radical surgery for gastric cancer in Singapore. AB - This study was conducted to define the pattern and results of gastric cancer surgery in Singapore by reviewing the epidemiological data from the Singapore Cancer Registry together with a review of a personal series of 182 patients operated on over a 14 1/2-year period. As in Japan and the West, the incidence of gastric cancer is decreasing in Singapore; it was 2.3% per year for men and 1.5% for women in the period 1968-1982. Nevertheless, the disease is still common and the respective age-standardized rates among the ethnic Chinese in Singapore of 37.3 and 15.4 per 100,000 per year remain higher than in the West but are only about half the rate observed in Japan. These rates in Singapore Chinese are also higher than those seen in the Chinese-Americans in the United States, but are lower than those of the Chinese of Shanghai, which suggests that lifestyle and diet influence the incidence of gastric cancer. In the series of 182 patients, gastric adenocarcinoma occurred in the antrum or body or the whole stomach in 78.5% and in the cardia in 21.5%. Presentation was late: stage I, 10%; stage II, 7.7%; stage III, 28%; and stage IV, 54.3%. Surgical resection was performed whenever technically possible (146 patients, 80%). In 86 patients, all macroscopic tumors were excised by a radical resection approximating the R2 type of resection, with only one operative mortality.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549267 TI - Association between elevated plasma granulocyte colony-stimulating factor and the degree of surgical stress in patients undergoing gastrointestinal surgery. AB - To characterize the changes in perioperative plasma granulocyte colony stimulating factor (G-CSF) and analyze the effect of surgical stress on its kinetics, 41 patients undergoing gastrointestinal surgery with varying degrees of surgical stress were examined. The plasma levels of G-CSF significantly increased immediately after the operation, probably in response to surgical injury. This elevation was much higher in the 15 esophagectomy patients, at 883 +/- 300 pg/ml on postoperative day (POD) O, than in the 14 gastrectomy patients, with a value of 233 +/- 151 on POD O, (P < 0.01) or in the 12 cholecystectomy patients, with a value of 64 +/- 41 on POD 1 (P < 0.01). These findings led us to conclude that G CSF levels increase significantly in the immediate postoperative period and are most likely associated with the degree of surgical stress. In addition, we studied the priming effect of G-CSF on polymorphonuclear leukocytes (PMNs). G-CSF enhanced PMN superoxide anion (O2-) production and luminol-dependent chemiluminescence (CL) induced by opsonized zymosan in a dose-dependent manner. A significant enhancement was seen in the G-CSF level (1 ng/ml) which was almost the same as the maximum G-CSF level in the esophagectomy patients. Furthermore, postoperative PMN activation occurred after the elevation of plasma G-CSF. Thus, we propose that elevated G-CSF may act as one of the mediators which activate PMN function postoperatively. PMID- 7549268 TI - Increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure. AB - The fact that thrombomodulin (TM) is released into the bloodstream from damaged vascular endothelial cells led us to hypothesize that plasma levels of soluble TM could be an indicator of the development of organ failure. In this study, we examined the changes in plasma levels of TM in 60 septic patients and 13 postsurgical patients, and investigated the circulating levels of interleukin 6 (IL-6) and polymorphonuclear leukocyte elastase (PMN-E) to determine the mechanism causing the excess liberation of TM. The arterial ketone body ratio (AKBR) was also measured as an indicator of the hepatocyte energy state. Of the 60 septic patients, 26 developed organ failure, 10 of whom died. In contrast, none of the postsurgical patients developed organ failure. The mean plasma level of TM was significantly higher in the septic patients who developed organ failure compared to those without organ failure (P < 0.001) or the postsurgical patients (P < 0.001). Furthermore, those patients whose plasma TM values became elevated over 6.0 ng/ml frequently developed complications. A positive correlation was also observed between the plasma TM levels and the IL-6 (P < 0.01) and PMN-E levels (P < 0.01). In contrast, a negative correlation was seen between the plasma TM levels and the AKBR (P < 0.01). These findings show that plasma TM could be a useful indicator of impending organ failure during sepsis. PMID- 7549269 TI - Expression of p53 protein related to human papillomavirus and DNA ploidy in superficial esophageal carcinoma. AB - We examined the p53 protein and human papilloma virus (HPV) by immunohistochemistry and DNA ploidy by cytofluorometry in paraffin-embedded esophageal carcinoma tissue specimens. Sixty-one patients with superficial esophageal carcinoma were operated on between 1983 and 1991 without any prior treatment. Immunostaining of the anti-p53 protein antibody (CM1) was positive in 32 carcinomas (52%). Patients with p53-positive tumors had a poorer outcome than those with p53-negative tumors (P < 0.05). In addition, patients with p53 positive tumors did not have any characteristic site of relapse. Only 5 of the 61 patients (8.2%) had HPV-positive tumors. One of these 5 carcinomas expressed both p53 protein and HPV. Three patients with HPV-positive tumors which had invaded the submucosal layer died of relapse. A determination of DNA ploidy revealed 30 patients with aneuploid tumors, 13 with polyploid tumors and 18 with diploid tumors. The outcome of the patients with aneuploid tumors was worse than that of the patients with diploid tumor (P < 0.05). p53 protein expression was not associated with DNA ploidy; however, the 16 patients who had both p53-positive and aneuploid tumors had a worse prognosis than patients with p53-negative and aneuploid tumors (P < 0.01). These findings suggest that p53 protein expression in conjunction with DNA ploidy may be a useful indicator in evaluating the prognosis of patients with superficial esophageal carcinoma. PMID- 7549270 TI - Evaluation of bone loss and the serum markers of bone metabolism in patients with hyperparathyroidism. AB - Bone loss and the serum markers of bone metabolism were studied in 22 patients with primary hyperparathyroidism and 108 patients with renal hyperparathyroidism. The parameters of bone loss were bone mineral density in the distal radius and lumbar vertebrae, measured by dual energy X-ray absorptiometry, and bone mass index (sigma GS/D) and the metacarpal index, in the second metacarpal bone, measured by the digital image processing method. Alkaline phosphatase (AIP), intact osteocalcin (OC), and the carboxyterminal propeptide of type I procollagen (PICP) were measured as serum markers of bone formation, while tartrate-resistant acid phosphatase (TRACP) and the carboxyterminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) were measured as serum markers of bone resorption. Bone loss and elevated markers of bone metabolism were observed both in patients with skeletal symptoms and in those without. Furthermore, the decrease in the cortical bone mass was more predominant than that of the trabecular bone. As markers of bone formation, AIP and OC seemed to be more sensitive than PICP, and as markers of bone resorption, ICTP appeared to be more sensitive than TRACP. Thus, a close correlation was observed between bone loss and the markers of bone formation and resorption. PMID- 7549271 TI - Postoperative alteration in the size of Dacron vascular prostheses implanted in the infrarenal abdominal aorta. AB - The degree and significance of postoperative alteration in the size of Dacron grafts in the infrarenal abdominal aorta was assessed by computed tomography. We studied a total of 41 knitted Dacron fabric grafts, comprising 28 Gelseal triaxial grafts and 13 Hemashield Microvel grafts, and 29 woven Dacron fabric (Veri-Soft Woven) grafts which were used to replace the infrarenal abdominal aneurysms. These three types of Dacron fabric dilated immediately after replacement to 1.4-1.5 times the manufactured size, but did not dilate further after the surgery. PMID- 7549273 TI - Ultrasonographic characteristics of different methods of inguinal hernia repair. AB - A randomized blind sonography estimation analysis of seven different methods of inguinal hernia repair was performed using a commercially available linear scanner with a 5-MHz transducer frequency. A total of 58 male patients with a mean age of 56 years who had been free of recurrence since undergoing a hernia repair were examined 3-12 years after surgery. The analysis was accomplished by three static and four dynamic indices. A five-grade scale was used to assess the operated side while control pattern images of the unoperated operated side, and of a control group of five men who had not undergone surgery, were assessed with four points. The score from the static ultrasound assessment ranked the methods of Lotheissen-McVay and Shouldice first and the methods of Marcy and Postempsky Halsted I second, while the score from dynamic indices ranked iliopublic tract repair and the methods of Marcy and Shouldice first and the method of Lotheissen McVay and bilateral preperitoneal prosthetic repair second. The highest aggregate score was demonstrated by the Shouldice method and the lowest by Bassini's method. The results of this study provide additional information which may be clinically useful in the context of the current state of inguinal hernia surgery. PMID- 7549274 TI - The Lichtenstein open "tension-free" mesh repair of inguinal hernias. AB - All standard methods of hernia repair involve suturing together tissues which are not normally in apposition. This violates the basic surgical principle that tissue must never be approximated under tension and thus accounts for an unacceptable number of failures. A total reinforcement of the inguinal floor with a sheet of suitable biomaterial and the employment of a "tension-free" technique is a more effective approach. Since June 1984, 4,000 primary inguinal hernias have been repaired on an outpatient basis and under local anesthesia at the Lichtenstein Hernia Institute by the open "tension-free" technique using Marlex mesh. The patients were followed from 1 to 11 years by physician examination. The follow-up rate was 87%. There were four recurrences. The causes of recurrence and how to avoid them are herein discussed. Three of the recurrences occurred at the pubic tubercle and were caused by placing the mesh in juxtaposition to the tubercle. This error has since been corrected by overlapping the mesh at the pubic bone. One recurrence was caused by a disruption of the lower edge of the mesh from the shelving margin of Poupart's ligament. The error here was the utilization of a patch that was too narrow and therefore under tension. It became apparent that a wider patch, fixed in place with an appropriate degree of laxity, was required. PMID- 7549272 TI - Screening for abdominal aortic aneurysm and occlusive peripheral vascular disease in Japanese residents. AB - To evaluate the prevalence of abdominal aortic aneurysm (AAA) and occlusive peripheral vascular disease (PVD) in Japanese residents, and to examine the correlations between these diseases and the risk factors of atherosclerosis, 348 residents of a village in central Japan aged between 60 and 79 years were screened. The screening for AAA was performed using ultrasonography (US) and that for PVD was performed by palpation and Doppler US. No AAA was found, and a right common iliac arterial aneurysm was detected in a 79-year-old man (0.3%). The mean diameter of the infrarenal abdominal aorta was 18.7 mm and an abdominal aorta of 25 mm or greater in diameter was seen in 16 participants (4.6%), all of whom need to be followed up. PVD was suspected in two patients (0.6%) with a low ankle brachial pressure index. Of a total of five patients diagnosed or suspected of having a common iliac arterial aneurysm or PVD, four (80%) had at least one risk factor for atherosclerosis. Thus, we conclude that Japanese residents with risk factors predisposing them to atherosclerosis such as hypertension, obesity, abnormal serum lipid levels, and a history of smoking should be selectively screened for AAA and PVD due to the low prevalence of these diseases and from the viewpoint of cost-effectiveness. PMID- 7549275 TI - Expression of the adult T-cell leukemia-derived factor, human thioredoxin, in the allotransplanted canine lung. AB - The relationship between the expression of adult T-cell leukemia-derived factor, human thioredoxin (ADF/TRX), and rejection in transplanted canine lungs was investigated in this study. Of a total 27 adult mongrel dogs, 24 underwent allotransplantation of the left lung with no immunosuppressant and the other three underwent autotransplantation of the left lung. Of the allotransplanted dogs, five were killed on postoperative day (POD) 1, five on POD 2, seven on POD 3, and seven on POD 5, while all three autotransplanted dogs were killed on POD 5. Histological examination was performed on the 24 allotransplanted left lungs (group A), 12 autologous right lungs (group B), and bilateral lungs of the three autotransplanted dogs (group C). The lung tissue was stained with anti-ADF antibody, and the high-ADF-producing cells (ADFh cells) in a randomly chosen field were counted as an index of ADF expression. As the signs of rejection in the group A lungs became more severe with time, the ADFh cells increased in number: 1.68 +/- 1.15, 6.08 +/- 3.44, 14.03 +/- 6.09, and 47.74 +/- 18.89, on PODs 1, 2, 3, and 5, respectively. However, the number of ADFh cells in the group B and group C lungs did not become significantly different from that on POD 1 in group A. These results suggested that ADF/TRX expression may be useful for the early diagnosis of rejection of transplanted lungs. PMID- 7549276 TI - Lymphatic vessel-to-isolated-vein anastomosis for secondary lymphedema in a canine model. AB - To design a more rational and effective surgical method of performing lymphatic venous anastomosis to treat secondary lymphedema of the lower extremities, the following experiments were conducted on three groups of dogs: group A underwent an end-to-side lymphatic node-to-vein anastomosis at the inferior vena cava; group B underwent a "burying" lymphatic vessel-to-vein anastomosis at the femoral vein; and group C underwent a burying lymphatic vessel-to-isolated-vein anastomosis at the femoral vein. In group C, the femoral venous segment was isolated by distal ligation and proximal valvuloplasty and the patency of the anastomosis was investigated by infusing yellow Microfils through the distal lymphatic vessel. The patency of the anastomosis was nil in group A by 10 days after the anastomosis, 40% in group B by 180 days; and 71.4% in group C by 180 days, respectively. Thus, we clinically applied the technique of lymphatic vessel to-isolated-saphenous-vein anastomosis in a patient with secondary lymphedema of the bilateral lower extremities. A satisfactory reduction in the size of the limbs was achieved and there has been no further recurrence of cellulitis in the 42 months since her surgery. This study shows that lymphatic vessel-to-vein anastomosis is an effective technique for the surgical management of secondary lymphedema, so long as the anastomosis is completely protected from any contact with blood. PMID- 7549277 TI - Malignant insulinoma causing liver metastasis 8 years after the initial surgery: report of a case. AB - We report herein a rare case of malignant insulinoma which recurred as multiple liver metastasis 8 years after the initial resection. The patient was a 51-year old Japanese man who originally presented in 1985 at the age of 43 years suffering from general malaise and syncope. The initial surgery in 1985 involved complete enucleation of a 15 x 13 mm insulinoma located in the uncus of the pancreas. Histopathologically, the tumor was diagnosed as a benign adenoma (insulinoma) which was immunohistochemically stained with only the anti-insulin monoclonal antibody. Macroscopically, there were no signs of either invasion or metastasis. During the subsequent 7 years, he did not show any symptoms or significant abnormality in laboratory data. However, in 1993, the patient again experienced syncope with hypoglycemia and hyperinsulinemia. Ultrasonography revealed multiple echogenic lesions in the liver and a second laparotomy confirmed multiple hepatic metastases from insulinoma, the histopathological findings of which were similar to those of the primary tumor from 8 years before. The patient is currently being treated with streptozotocin and 5-fluorouracil via a catheter in the hepatic artery. PMID- 7549278 TI - Acute left atrial thrombus causing cardiogenic shock following mitral valve replacement: report of a case. AB - We report herein the rare case of a 53-year-old woman who developed cardiogenic shock due to an acute left atrial thrombus following replacement of the mitral valve. A definitive diagnosis was not able to be made using precordial echocardiography because of the broad, flat shape of the thrombus; however, transesophageal echocardiography imaged the thrombus in detail. The patient was initially stabilized by percutaneous cardiopulmonary support after which a thrombectomy was successfully performed. PMID- 7549279 TI - The detection of coronary ostial lesions by intraoperative transesophageal echocardiography: report of a case. AB - Transesophageal echocardiography (TEE) is a valuable diagnostic tool for providing clear images of the proximal coronary arteries. We describe herein the case of an elderly man in whom dissection and an atherosclerotic plaque in the proximal coronary arteries were demonstrated by TEE during combined coronary artery bypass grafting and aortic valve replacement. Thus, retrograde cardioplegia was employed, whereby trauma to the coronary ostia was avoided. PMID- 7549280 TI - Malposition of a totally implantable venous access catheter in the azygous vein: report of a case. AB - We report herein the case of a 2-year-old girl with short bowel syndrome who developed chest pain 2 weeks after a totally implantable central venous access catheter had been surgically placed to facilitate total parenteral nutrition. A lateral chest X-ray and contrast flow study subsequently revealed that the catheter tip had been inserted into the azygous vein. Although this complication is very rare, it may be encountered more commonly with the increasing use of this type of venous access. Thus, we believe that a lateral chest X-ray should be routinely performed on all patients following catheter insertion to check for possible malposition of the tip in the azygous vein. PMID- 7549281 TI - Thoracoscopic resection of a solitary pulmonary lymphangioma: report of a case. AB - We present herein the rare case of a young man who was found to have a solitary tumor in the right upper lobe of his lung by a routine chest X-ray. The tumor was removed by thoracoscopic surgery, and pathological examinations confirmed the diagnosis of a primary lymphangioma of the lung. A brief review of the available literature on this extremely rare type of benign tumor follows the case report. PMID- 7549282 TI - Successful utilization of a video-assisted thoracic approach to repair Morgagni's hernia: report of a case. AB - We describe herein the successful utilization of a video-assisted thoracic surgical approach to repair Morgagni's hernia. The patient was a 62-year-old woman in whom a routine chest X-ray had revealed an asymptomatic mass, which was presumed to be a pericardial lipoma or Morgagni's hernia. The video-assisted thoracic surgical approach was combined with a right submammary minithoracotomy to successfully repair the hernia without performing a laparotomy. The patient's postoperative course was uneventful and she was discharged 14 days after surgery. Thus, we believe that video-assisted thoracic surgery may be a useful and effective method for repairing Morgagni's hernia. PMID- 7549283 TI - The use of an ileostomy connector to diminish the frequency of defecation prior to ileostomy closure in patients with a pelvic pouch. AB - A new method for allowing stool passage into the pelvic pouch before ileostomy closure to verify the defecation state and diminish stool frequency is reported herein. This was accomplished by fitting an ileostomy connector connecting the proximal and distal openings of the diverting loop stoma. The ileostomy connector was initially in place for 6 h a day, the length of time being gradually increased until it was able to be left in for 24 h a day over a 3-month period. The calculated daily frequency of stools decreased from 24 to 6 or 7 times, and the mean daily frequency immediately after ileostomy closure was 6.5 times. Physiological study also showed an improvement, with squeeze pressure increasing from 35 cmH2O to 116 cmH2O and the maximum tolerated volume increasing from 35 ml before, to 90 ml 3 months following the use of an ileostomy connector. Thus, we conclude that an ileostomy connector may be useful to predict postoperative functional outcome and its complications, and to diminish the frequency of defecation before ileostomy closure in patients with a covering loop stoma. PMID- 7549284 TI - Kawasaki disease. PMID- 7549285 TI - Editorial comment on phenylketonuria. PMID- 7549286 TI - Changes in breastfeeding practices in Norwegian maternity wards: national surveys 1973, 1982 and 1991. AB - Total breastfeeding rates in Norway increased from < 30% at 12 weeks in 1968 to > 80% in 1991. Concomitant changes in routines related to breastfeeding in all Norwegian maternity wards were examined in 1973, 1982 and 1991. Head midwives responding to identical survey questionnaires described "usual practices" in their wards. The considerable changes over these 18 years reflected international recommendations such as those summarized in WHO/UNICEF's 1989 Joint Statement and "Ten steps to successful breastfeeding". Undisturbed and prolonged contact between mother and baby became more common, as did more respect for the needs of the nursing couple, and more individualized care. In responses to open-ended questions, many respondents expressed a positive attitude towards these changes, while "clusters" of negative attitudes were also seen. As research on inborn infant behaviour and mother-infant interaction proceeds, the present survey suggests areas where further information may benefit both mothers and health workers. PMID- 7549288 TI - Healthy eating for infants--mothers' actions. AB - The aim of this study was to analyse the nutrient composition, with respect to energy density, protein, fat, carbohydrate, non-starch polysaccharides and certain micronutrients, of home-prepared infant foods and to compare the nutrient composition with the draft European Commission directive for baby foods, the published nutrient content of manufactured baby foods, and with breast milk. Two hundred and sixty-five samples of home-prepared weaning foods for infants aged 3 12 months were collected and chemically analysed. Many of the food samples were low in energy, protein, fat, iron, calcium and zinc and high in non-starch polysaccharides and sodium. Home-prepared weaning foods have some shortcomings in terms of nutrient composition. Advice from health care professionals needs to focus on these areas at the same time as presenting the nutritional merits of manufactured baby foods. PMID- 7549287 TI - Growth, fatty acid composition of plasma lipid classes, and plasma retinol and alpha-tocopherol concentrations in full-term infants fed formula enriched with omega-6 and omega-3 long-chain polyunsaturated fatty acids. AB - Full-term infants fed formula without dietary long-chain polyunsaturated fatty acids (LCF) exhibit significantly lower plasma LCP values than breast-fed infants. We studied prospectively two groups of healthy full-term infants fed conventional infant formula without LCP (F, n = 10) or the same formula enriched with both omega-6 and omega-3 LCP (LCP-F, n = 12). Anthropometric data were obtained and fatty acid (FA) compositions of plasma phospholipids, triglycerides and sterol esters as well as plasma retinol and alpha-tocopherol concentrations were determined at 5 days and 1, 2, 3 and 4 months of age. Gains in weight, length and head circumference did not differ between the two groups throughout the study period. Plasma FA values did not differ at 5 days of age. Between 1 and 4 months of age, plasma phospholipids of infants fed LCP-F consistently had significantly (p < 0.05) higher percentages of arachidonic acid (1 month: 9.7 (0.8) versus 7.0 (1.3) %wt/wt, 4 months: 8.7 (0.5) versus 6.6 (1.0) %wt/wt, median (interquartile range), LCP-F versus F) and docosahexaenoic acid (1 month: 2.9 (0.5) versus 1.6 (0.3) %wt/wt; 4 months: 2.9 (0.4) versus 0.9 (0.3) %wt/wt). Plasma retinol and alpha-tocopherol concentrations did not differ between the two groups throughout the study. We conclude that this form of LCP enrichment of formula for full-term infants effectively enhances plasma LCP contents without detectable adverse effects. The potential effects on functional outcome need to be studied carefully in prospective clinical trials. PMID- 7549289 TI - Chronic carbon monoxide poisoning in children. PMID- 7549290 TI - Nutrient intake and food consumption of adolescents and young adults with phenylketonuria. AB - Food and nutrient intake was assessed in 99 PKU patients (12-29 years old) by two food protocols (7 days and 4 days, respectively). Ninety-three patients completed at least one 7-day food record and 83 both records. Nineteen of 93 patients had already stopped taking the phenylalanine-free amino acid mixture (AAM), which is enriched with vitamins, minerals and trace elements. Plasma phenylalanine levels in this group were significantly higher than in patients who were still taking the AAM. Even without the AAM, protein intake still met the recommendations, but thiamin, riboflavin, folate, calcium and iron levels were below 80% of the US RDA in most patients. For those still taking the AAM, calorie, protein, vitamin and mineral intakes were above the recommendations. The diet was characterized by a low intake of fiber (median 14 (range 8-35) g/day), fat (27 (10-47) cal%) and cholesterol (75 (13-417) mg/day) as well as a high ratio of polyunsaturated/saturated fatty acids (0.7 (0.2-2.4)). Problems with dietary compliance in adolescents and young adults may lead to a combination of marginal nutrient intake and high phenylalanine levels. PMID- 7549291 TI - Congenital chylothorax with hydrops: postnatal care and outcome following antenatal diagnosis. AB - We consecutively managed 25 cases of fetal chylothorax with hydrops (pleuroamniotic shunting in 20/25 cases). Three of the 16 liveborn infants died before day 5 from malformations (n = 1) or complications of antenatal origin (n = 2). Eleven of the 13 survivors were treated in our unit. Four infants whose chylothorax had resolved before birth following antenatal shunting were delivered at term, and had no respiratory disease. Seven infants, whose chylothorax persisted, were delivered prematurely and required intensive respiratory care (with mechanical ventilation for a median duration of 34 days). The 11 infants were maintained on total parenteral nutrition for a median duration of 31 days. They were discharged home after complete clinical recovery at a median age of 64 days. Antenatal pleuroamniotic shunting may improve the prognosis of congenital chylothorax with hydrops. Chylothorax persisting at birth resolves progressively with medical management. PMID- 7549292 TI - Percutaneous central i.v. access in the neonate: experience with 535 silastic catheters. AB - We examined 535 central venous catheterizations performed in a neonatal intensive care unit. A total of 273 catheters were positioned using intra-atrial ECG monitoring. With an average indwelling time of 23 days, we noted one complication for every 153 indwelling catheter days. A diagnosis of sepsis was confirmed on 22 occasions (4.1%). Sepsis occurred more often in infants with a birth weight < 1000 g (6.9%) compared with infants > 1000 g (3.1%). The risk of phlebitis was highest when the saphenous vein was used as the puncture site. The lowest risk of phlebitis was when the basilic vein was chosen. Five cases of thrombosis, two of myocardial perforation and one intravascular catheter breakage were registered. Catheter placement under ECG monitoring proved to be a very suitable method for reducing the incidence of malpositioning. There were no side effects specific to the ECG method. The ECG method can be used safely and makes radiological control usually unnecessary. PMID- 7549293 TI - Continuous haemodynamic monitoring in children: use of transoesophageal Doppler. AB - A wide range of invasive and non-invasive techniques for monitoring the haemodynamic condition of critically ill patients is now available. A general reluctance on the part of paediatric intensive care specialists to use pulmonary artery thermodilution catheters and the need for constant realignment of hand held Doppler probes has necessitated the search for a technique which is relatively non-invasive and provides continuous information on the haemodynamic condition of critically ill paediatric patients. We sought to establish if transoesophageal Doppler fulfilled these criteria. Eleven children who had recently undergone cardiac surgery were studied. Median age was 39 months and weight 14.9 kg. Five simultaneous pairs of measurements of cardiac index (CI: thermodilution) and minute distance (MD: transoesophageal Doppler) were made, as a baseline, when each child was haemodynamically stable. Following a fluid challenge, five repeat pairs of measurements were made. The mean percentage changes for CI and MD were 16.4% (range 5.3-44%) and 16.6% (3.4-47.7%), respectively. The average coefficients of variation for measurements of CI and MD were 3.5% and 2.9%, respectively. The mean difference in percentage change between CI and MD was -0.5% (95% confidence interval for the bias -4% to 3%; limits of agreement -10.7 to +9.7%). Our study indicates that transoesophageal Doppler is reproducible, easy to use and provides clinically acceptable information when following changes in CI in haemodynamically stable paediatric patients. PMID- 7549294 TI - Iv gamma globulin treatment of Kawasaki disease in Japan: results of a nationwide survey. AB - The administration of i.v. gamma globulin (IVGG) for Kawasaki disease was investigated throughout Japan in 1993 by obtaining information from the pediatric departments of 2652 hospitals with more than 100 beds. A total of 1826 hospitals (68.9%) responded, reporting on 11,221 patients who were diagnosed during the survey period from January 1991 to December 1992. There were 8958 patients (79.8%) who received IVGG treatment. The most common treatment modality was 200 mg/kg (29.6%), followed by 400 mg/kg (18.7%) and 300 mg/kg (12.9%), all for 5 days. The distributions of total dose were: 1000 mg/kg or less, 45.7%; 1001-1500 mg/kg, 27.3%; and over 1500 mg/kg, 23.8%. For all patients to whom IVGG was administered, treatment was started in 53.8% by day 5 of illness and in 86.1% by day 7. The proportion of those with cardiac sequelae was higher in patients who were treated with IVGG, possibly due to the fact that those who were more severely affected were more likely to be treated with IVGG. PMID- 7549295 TI - Kawasaki disease in Sweden: incidence and clinical features. AB - The incidence and clinical pattern of patients with Kawasaki disease in Sweden and the outcome of treatment with i.v. immunoglobulin (Sandoglobulin) and aspirin were examined in a national prospective study over a 2-year period. Cases not referred to the study were identified by inquiry. Ninety-nine children were diagnosed as having Kawasaki disease. The annual incidence rate was calculated to be 2.9 per 100,000 in children younger than 16 years of age and 6.2 per 100,000 in children younger than 5 years of age. The median age of our patients was 2.2 years and the male-to-female ratio was 2.3:1. Cardiac ultrasonography revealed abnormalities in 33% of all patients, and 14% of cases in the prospective study had pronounced abnormalities. In most cases (91%) treatment had a prompt effect on fever and morbidity in general, and side effects were mild. Two infants, identified from the inquiry, died from rupture of an aneurysm in the coronary artery in the acute phase of the disease. The risk of cardiac involvement is obvious and emphasizes the importance of early diagnosis and treatment. PMID- 7549296 TI - Alanine- and glucose-based hypo-osmolar oral rehydration solution in infants with persistent diarrhoea: a controlled trial. AB - To evaluate the efficacy of a hypo-osmolar and a standard (World Health Organization) oral rehydration salt (ORS) solution in persistent diarrhoea, a randomized controlled clinical trial was conducted in 55 children. After a 1-day observation period the children were assigned to one of three solutions: standard ORS (WHO-ORS) (osmolality 311 mosmol/l), hypo-osmolar ORS containing L-alanine and glucose (osmolality 255 mosmol/l) and i.v. polyelectrolyte solutions (osmolality 293 mosmol/l) for ongoing replacement of stool loss for the next 4 days. Excellent acceptability of ORS (101-160 ml/kg body weight/day) by the children was observed. There were no significant differences in the total intake of solutions and food, and frequency of stools among the groups. Stool outputs were significantly less in infants receiving hypo-osmolar ORS than in those receiving WHO-ORS for 0-24 h (p = 0.04), 0-48 h (p = 0.01), 0-72 h (p = 0.04) and 0-96 h (p = 0.03). The results indicate a sufficient scope of ORS practice in persistent diarrhoea. Furthermore, we found that a hypo-osmolar ORS containing L alanine and glucose is as efficacious as an iv solution and more effective than WHO-ORS for replacement of ongoing stool loss in persistent diarrhoea. PMID- 7549297 TI - Localized proton magnetic resonance spectroscopy of cerebral abnormalities in children with carbohydrate-deficient glycoprotein syndrome. AB - Morphologic and metabolic abnormalities in six children aged 2-9 years with carbohydrate-deficient glycoprotein (CDG) syndrome were assessed by magnetic resonance imaging (MRI) and localized proton magnetic resonance spectroscopy (MRS). In all patients, MRI revealed pronounced cerebellar atrophy. Follow-up examinations in two patients suggested early onset and rapid progression in the first years of life. Further pathologies comprised Dandy-Walker malformation, atrophy of the pons, brain stem and olives, supratentorial frontotemporal cortical atrophy, slightly dilated ventricles and a small corpus callosum. Two patients presented with small cysts in the white matter. The prominent metabolic abnormality detected by proton MRS in five patients was a reduction in N acetylaspartate in white matter by more than 20%, indicating loss of vital neuroaxonal tissue. Further findings in white matter were glutamine and gamma aminobutyrate increases by a factor of 2. One patient with type III CDG syndrome showed the most severe alterations of metabolite concentrations. PMID- 7549298 TI - Gangliosides in children with autism. AB - Concentrations of the four major brain gangliosides, GM1, GD1a, GD1b and GT1b, biochemical markers of neuronal membranes, were determined in the cerebrospinal fluid (CSF) of 20 children with autism and in 25 controls. In addition, the gangliosides were determined in children with different forms of non-progressive neurological disorders lacking clinical features of autism. GM1, GD1a, GD1b and GT1b were significantly increased in patients with autism compared with age matched controls and children with non-progressive neurological disorders. The gangliosides have previously been shown to have a function in synaptic transmission and increased synaptic activity leads to added release of gangliosides. Our finding of increased CSF levels of gangliosides in autism suggests increased synaptic activity in this disorder. PMID- 7549299 TI - CPAP treatment of obstructive sleep apnoea and neurodevelopmental deficits. AB - Four boys aged 6-16 years with neurodevelopmental deficits were treated with CPAP for obstructive sleep apnoea. Their diagnoses were: Obesity with mild mental retardation, (2) attention deficit hyperactivity disorder, (3) epilepsy associated with left hemiparesis and (4) mild mental retardation due to fragile X syndrome. Previous therapeutic attempts, including adenotonsillectomy, amitriptyline and methylphenidate in our patients prior to CPAP treatment were unsuccessful. A follow-up period of 12-48 months demonstrated a number of clinical benefits such as improvement in sleep quality and daily arousal, and a decrease in the frequency of seizures and episodes of pneumonia. Polysomnographic studies indicated a significant improvement in sleep parameters such as apnoea frequency, awakenings, sleep efficiency and arterial oxygen saturation. Side effects were mild and readily alleviated. CPAP is a feasible therapeutic intervention in intractable obstructive sleep apnoea of childhood, even when associated with neurodevelopmental deficits. PMID- 7549300 TI - Development of stomach-ache and headache during middle childhood: co-occurrence and psychosocial risk factors. AB - Development of somatic symptoms and associations with psychosocial risk factors were investigated in a longitudinal study of Norwegian children aged 4-10 years. Complaints of stomach-ache only were associated with emotionally well-adapted children, and mothers with low education and high emotional support. Children complaining of headache only behaved well as preschoolers, showed a tendency towards high achievement motivation at school and their mothers were employed outside the home. Children with the co-occurrence syndrome seemed to constitute a separate entity. They differed from the others as the syndrome was associated with previous behavioural and emotional problems, current emotional disturbances and mothers with less support. Family demographic stability, further child health problems and school factors were not associated with the co-occurrence syndrome. PMID- 7549301 TI - Prevalence of chronic knee pain in children and adolescents in northern Finland. AB - A questionnaire was sent to 967 schoolchildren, in two age groups, in northern Finland in order to investigate the prevalence of chronic knee pain. A response rate of 88.5% was achieved. Adolescents (aged 14-15 years) had significantly more knee pain (p < 0.0001) than children (aged 9-10 years). The total prevalence of chronic knee pain at the time of the evaluation was 18.5% among adolescents and 3.9% among children. There was no significant difference in the prevalence of chronic knee pain between boys and girls in these age groups. Overweight was not a predisposing factor in chronic knee pain. PMID- 7549302 TI - Interleukin-6 and soluble interleukin-6 receptor in cord blood in the diagnosis of early onset sepsis in neonates. PMID- 7549303 TI - Enhancement of erythropoiesis by erythropoietin, bovine protein and energy fortified mother's milk during anaemia of prematurity. AB - Twenty-four premature infants, < 32 weeks gestational age, were randomly assigned in a double-blind, placebo-controlled trial to 6 weeks of treatment with either recombinant human erythropoietin (rHuEpo) 150 U/kg three times per week given sc (n = 12) or placebo (n = 12). The infants were fed a diet rich in protein (3.2 g/kg/day) and energy (130 kcal/kg/day) based on their own mother's milk fortified with bovine protein together with moderate iron supplementation (4 mg/kg/day). During the treatment (rHuEpo versus placebo) significant differences in mean (+/- SD) reticulocyte count (4.8 +/- 1.2 versus 2.7 +/- 1.4%; p < 0.01), mean packed red cell volume (PCV) (0.38 +/- 0.03 versus 0.34 +/- 0.04, p < 0.05) and mean haemoglobin concentration (12.6 +/- 1.1 versus 11.5 +/- 1.2 g/100 ml; p < 0.05) were found. Within the rHuEpo group, PCV and haemoglobin concentration remained unaltered from entry to 1 week after cessation of treatment whereas a significant decline was observed in the placebo group. No indications of iron deficiency were seen. We conclude that moderate doses of rHuEpo given to infants fed a diet rich in protein and energy are effective in ameliorating anaemia of prematurity. High iron supplementation does not seem to be essential for a significant erythropoietic response. No adverse effect attributable to rHuEpo was observed. PMID- 7549304 TI - Prader-Willi syndrome: effects of weight loss on sleep-disordered breathing, daytime sleepiness and REM sleep disturbance. PMID- 7549305 TI - Clinical features and lung function in 18-year-old adolescents with alpha 1 antitrypsin deficiency. PMID- 7549306 TI - Post-streptococcal acute glomerulonephritis in Turkey. PMID- 7549307 TI - Prevalence of and factors associated with childhood asthma in Hong Kong. AB - The prevalence of asthma and eczema in children between 3 and 10 years of age living in Hong Kong was determined by a questionnaire survey administered to the parents of 535 children in 1989. The prevalence of asthma was 6.0% (95% confidence limit (CI) 4.0-8.0%) and the prevalence of eczema 6.8% (95% CI 4.6 9.0%). Age, sex, parental smoking and breast feeding in early life did not show significant associations with asthma. However, four different measures were associated with the prevalence of asthma: the presence of eczema, high serum IgE level, high number of upper respiratory infections and a relatively high family income, with odds ratios of 7.6, 4.9, 4.5 and 2.7, respectively. PMID- 7549308 TI - Guidelines for optimal medical care of persons with Down syndrome. International League of Societies for Persons with Mental Handicap (ILSMH). AB - There are numerous clinical conditions observed in persons with Down syndrome, as described above, which should be taken into consideration in the course of their medical care and management. If provided with optimal medical services, pursuing specific evaluations and examinations, with a focus on preventive aspects and fostering well being in all areas of human functioning, the quality of life of individuals with Down syndrome can be enhanced significantly and their contribution to society substantial. PMID- 7549309 TI - Long-term follow-up of patients with IgG subclass or IgA deficiencies. PMID- 7549310 TI - An unusual cause of congenital ascites. AB - A newborn premature male with hydrops fetalis and massive congenital ascites is presented. The ascites recurred despite repeated paracenteses. The ascites was possibly due to a portal vein obstruction caused by malposition of the portal vein and malrotation of the small intestine. This is the first case report of congenital ascites associated with malposition of the portal vein. PMID- 7549311 TI - Failure of cefotaxime treatment in two children with meningitis caused by highly penicillin-resistant Streptococcus pneumoniae. AB - Two infants, aged 8.5 and 11 months, were admitted for meningitis caused by Streptococcus pneumoniae. Failure of cefotaxime led to the identification of highly penicillin-G-resistant strains. Minimum inhibitory concentrations (MICs) for penicillin were > 2 micrograms/ml, and cefotaxime MICs were 2 micrograms/ml. Both patients rapidly responded to a combination of i.v. imipenem and rifampicin. It is now mandatory to test in-vitro susceptibilities of Streptococcus pneumoniae to penicillin G and other beta-lactam agents when meningitis is diagnosed in infants. PMID- 7549312 TI - Recurrent abdominal pain in childhood. PMID- 7549313 TI - Achalasia with absent tear production. AB - We report the unusual and very rare association of achalasia with deficient tear production, which is probably an autosomal recessive inherited syndrome. The diagnosis of achalasia was confirmed by radiologic, endoscopic and endosonographic studies and the absent tear production by Schirmer's test. PMID- 7549314 TI - Seckel's syndrome associated with congenital cystic adenomatoid malformation of the lung. PMID- 7549315 TI - [Current evaluation of myopia correction with the excimer laser]. AB - Keratectomies with the excimer laser demonstrate extraordinarily smooth edges. Thermal and actinic side effects are negligible. Currently two techniques are in clinical use: the wide-field approach and the scanning-slit approach. A third one, the flying-spot approach is being tested in clinical studies. Postoperative refraction is mainly determined by the amount of correction. For corrections up to 6.0 D we found success rates of more than 90% and the refraction stabilizes during the first year after surgery. If the correction is higher, the success rate is decreased to 50% or less and the regression takes years. The complication rate is also dependent on the amount of correction: with corrections of more than 6.0 D it exceeds 1% and in corrections of 9.0 D and more, manifest corneal scars occur in more than 10% of the cases. Other complications are: eccentric ablation, IOP elevation due to steroid medication, and ptosis. Rheumatic diseases are absolutely contraindicated because of potential corneal melting ulcers, and subretinal neovascularization. In addition, corrections of more than 6.0 D are relatively contraindicated. More recent techniques like astigmatic PRK, LASIK, and hyperopic PRK are discussed. PMID- 7549316 TI - [Photorefractive keratectomy (PRK). The limits of our knowledge]. AB - The confines of our knowledge with respect to excimer laser photorefractive keratectomy are particularly manifest in two areas: the effects of the excimer on the corneal tissue, especially in the course of the wound healing process; and the optical aberrations caused by the laser treatment through structural alterations in a previously clear cornea. The criteria currently used to evaluate the outcome of excimer refractive surgery are not adequate. More stringent and nuanced assessments are needed to expand the frontiers of our knowledge. PMID- 7549317 TI - [Critical thoughts on current laser surgery of the cornea]. AB - OBJECTIVE: To analyze critically refractive surgery of the cornea by excimer laser and to compare laser surgery with other methods of treatment of refractive errors of the eye. MATERIAL AND METHODS: This analysis has to be restricted to a comparison of the treatment of myopia by keratotomy and photoablation with the ArF excimer laser. Correction of hypermetropia and of astigmatismus has to be left out, along with all the other methods to correct myopia, such as glasses, contact lenses, keratomileusis, epikeratoplasty, alloplastic implants, implantation of intraocular lenses with negative power, and replacement of the clear lens by an posterior chamber lens. The essential literature is screened. For intrastromal ablation with the picosecond Nd:YLF laser we relay on own experiences, also with the use of the ArF excimer laser we are not without own experiences. RESULTS AND STATEMENTS: For comparison of refractive surgery of the cornea not only the PERK study and the recommendations of the American Academy of Ophthalmology have to be considered, but also the newest developments in radial keratotomy such as two-step incision and reoperation with reopening of the keratotomy wounds. With these techniques the same precision can be reached as with the excimerlaser, and also higher myopias can be corrected. The dangers of the procedure, such as infection, perforation at surgery or laceration by contusion remain much larger. Intrastromal photoablation did not reach clinical maturity. Superficial photoablation is an almost safe procedure. A reduction of 3 D of myopia can be reached with satisfying precision, although higher myopias are still a problem. Pain following the ablation is considerable. Haze and disturbed vision at night can be present; infectious keratitis is rare, but possible. CONCLUSIONS: The critical fact of both procedures, keratotomy and photoablation with excimer laser, remains that healthy eyes are treated; therefore, even rare complications weigh much heavier than if sick eyes are treated. Because this is cosmetic surgery, the individuum asking for this type of procedure has to pay for on his own. Olson demands: "In determining when new technology is acceptable, we must consider the financial cost and the expected benefit to society. Is it an equitable tradeoff?" If we look at refractive surgery, especially laser photoablation, in the context of the needs for ophthalmic care of the whole world, then this type of surgery is out of proportion. The balance could be restored if, with every laser application, funds were given for third-world projects. Excimer-laser surgery may be justified insofar as the research with these lasers leads to useful therapeutic methods. PMID- 7549318 TI - [Corneal changes and corneal healing after keratomileusis in situ. In vivo studies using confocal slit-scanning microscopy]. AB - So far, no investigations of micromorphological changes in the human cornea after in situ keratomileusis have been published. In 10 eyes of five patients who had undergone in situ keratomileusis, wound healing processes were followed up with a newly developed confocal slit-scanning video-microscope. Post-operatively, alterations in the corneal endothelium and in the corneal stroma were found. Wide streaky distortions were observed within the stroma above and below the lamellar incision during the early days immediately after surgery. These distortions were probably induced by shearing stresses of the oscillating blade of the microkeratome. Small scattering centers were seen at the level of the lamellar incision, which are thought to reflect a very slight roughness of the interface. In the immediate postoperative period the corneal endothelium showed reversible inhomogeneitis similar to blebs after short-term contact lens wearing, which are thought to be due to a hypoxia during the surgery. The endothelial alterations and the streaky distortions within the stroma disappeared within 9 days after the surgery whereas the scattering centers regressed much more slowly and could still be observed 60 days after the surgery. The total amount of scattered light of the cornea, however, was not increased except in one patient. The results show that outside the level of the lamellar incision, in situ keratomileusis leads to only short-term alterations of the corneal micromorphology, and that confocal in vivo slit-scanning video-microscopy allows thorough examination of these alterations. PMID- 7549319 TI - [Keratomileusis in situ in high grade myopia. Initial results]. AB - Besides photorefractive keratectomy (PRK) with the excimer laser, different methods of intrastromal keratectomy are now available for refractive corneal surgery in myopic patients. The first results of keratomileusis in situ in high grade myopia are presented. Keratomileusis in situ was performed with a semiautomatic mechanical microkeratome in 15 amblyopic eyes with a refraction (spherical equivalent) between -12 and -25 D (mean -18.18 D). The principle of the method consists in a change of the anterior corneal curvature by resection of a refractive lenticulus out of the corneal stroma. The first step is the removal of a superficial corneal lamella consisting of epithelium, Bowman's layer, and superficial stroma. After this, the refractive lenticulus is resected out of the stroma, and the superficial lamella is sutured back to the cornea. The postoperative refraction (spherical equivalent) 4 weeks after the operation ranged between + 1.5 and -4.5 D (mean -1.37 D). Best corrected preoperative visual acuity was achieved again postoperatively within 4 weeks in most of the patients; in some patients the postoperative visual acuity even exceeded the preoperative values. After an uncomplicated healing process in 14 of the 15 eyes the cornea remained completely transparent in the optical zone, and the effect of treatment remained stable. Scar formation was observed in one eye only, where the lamellar cut was inadvertently performed in Bowman's layer and not in the stroma. Thus, keratomileusis in situ is a supplementary technique to conventional refractive surgery in high-grade myopia. Its advantages are quick optical rehabilitation and an almost scar-free optical zone, because the integrity of Bowman's layer is almost completely maintained with this technique. PMID- 7549320 TI - [Effect of corneal hydration and dehydration of donor tissue on the precision of the thickness in lamellar refractive keratoplasty]. AB - The influence of corneal hydration on the precision of plano lamellas and on the precision of lenticles modified with a "non-freeze" technique was studied in pig eyes. Ten pig eyes were dissected with a rotating lamellar microkeratome, and the discs were measured with a mechanical pachymeter. The hydration of the discs was measured in BSS and in dextran solution. A total of 160 corneal discs with different degrees of hydration were modified with a "non-freeze" technique on concave anf convex vacuum cylinders and measured with a photokeratoscope TMS. The discs increased in thickness within 3 h becoming 1.7 times as thick in BSS and 1.2 times as thick in dextran. After refractive dissection minor modifications were achieved with higher precision than were larger corrections. However, we achieved an undercorrection after all refractive dissections. Plano-discs for refractive modification should be taken from dehydrated corneoscleral discs or from intact eyes. Owing to mechanical factors during cutting and tissue hydration undercorrection of the intended change was observed. PMID- 7549321 TI - [Corneal reinnervation after lamellar refractive corneal surgery]. AB - In a retrospective study corneal sensitivity after refractive autokeratoplasty for correction of myopia was measured. Thirty-eight patients operated on for keratomileusis in situ and 18 patients for keratomileusis myopica were examined with the Draeger esthesiometer between 1 and 23 months after surgery. Three measurements points were on the refractive disc, two others on the untouched cornea. After keratomileusis myopica, the corneal reinnervation is significantly delayed compared to keratomileusis in situ. In the first group sensitivity is normal after 2 years and in the second group after 1 year. Even refraction and visual acuity depend on metabolism and reorganization of the tissue after surgery. Besides retarded cell repopulation and disturbed stromal metabolism- caused by toxic and cryopreservation effects--the dissection depth of the refractive ablation is responsible for these phenomena. PMID- 7549322 TI - [Special indications for photorefractive (PRK) and phototherapeutic (PTK) excimer laser keratotomies. A presentation of 5 cases]. AB - Five patients with specific indications for photorefractive and phototherapeutic keratectomy (PRK and PTK) by excimer laser at 193 nm were treated successfully: they had presented with epithelial dystrophy of the cornea, amyloid, von Szilly's scleroperikartitis, myopia after cataract extraction and before secondary implantation of an intraocular lens in the other eye, and myopia. In all cases it was possible to avoid extensive surgical procedures such as lamellar keratoplasty or intraocular lens exchange. There were no recurrences in the patients with superficial corneal diseases, and the patients who had undergone PRK were within 0.25 Dpt of the target acuity. The follow-up was between 8 months and 1 year. PMID- 7549323 TI - [Excimer laser phototherapeutic keratectomy (PTK) and modified bare sclera technique for treatment of pterygium]. AB - Forty-six pterygia were surgically removed using a modified "bare sclera" technique. Additionally, an excimer laser PTK was performed using the immersion method to smooth the irregular surface of the cornea. Follow-up was 6 to 10 months. We found 24 recurrences. In patients with pterygia of more than 4 mm who were from southern Europe, the Middle East, or Africa, the recurrence rate was 100%. In pterygia from 2 to 4 mm the recurrence rate was 50%; in pterygia up to the limbus no recurrences were noted. The excimer laser has no positive influence on pterygia recurrences that have been removed using the modified "bare sclera" technique. PMID- 7549324 TI - [Keratoplasty a chaud]. AB - Between 1980 and 1992 we performed 128 keratoplasties a chaud on 69 (54%) eyes with progressive bacterial and on 59 (46%) eyes with progressive herpetic keratitis. In 44 of the eyes the corneas were spontaneously perforated, and in 28 (22%) a descemetocele was present. Only grafts that had been followed for at least 1 year (3.2 +/- 2.4 years) were analyzed. During this time 88 (95%) of the severely threatened eyes were preserved. In the operated eyes visual acuity improved in 20 (54%) in the bacterial and in 36 (69%) in the herpetic group. The main complications were immunological allograft rejection, cataract progression and recurrence of herpes. Allograft rejection occurred in 29 (71%) of the bacterial grafts and remained irreversible in 18 (44%) cases. 30 (63%) eyes in the group with herpetic corneal lesions developed graft rejection, 19 (37%) of these rejections were irreversible. Cataract occurred in the postoperative course in 25 (61%) of the bacterial eyes and in 23 (44%) of the herpetic ones. Recurrence of herpes presented a specific problem in this group and appeared in 12 (23%) of the grafts during a follow-up of 1 month to 12 years (3.8 +/- 2.4 years). Our results demonstrate that keratoplasty a chaud is a valuable form of managing urgent pathological processes of the cornea and shortening the course of the disease. This procedure also allows satisfactory primary functional results in many cases, however, secondary re-keratoplasty and cataract surgery are necessary to achieve functional rehabilitation. PMID- 7549325 TI - [Long-term stability of astigmatism after suture correction in penetrating keratoplasty]. AB - Severe astigmatism after penetrating keratoplasty is frequently a major problem. If one uses a technique to adjust a single running 10-0 nylon suture in the early period after keratoplasty, the suture tension can be redistributed and astigmatism reduced. Eighteen eyes with an average of 8.8 D of postkeratoplasty astigmatism underwent suture adjustment. The mean keratometric astigmatism before suture removal was 3.1 D. After suture removal with an follow-up of 25 months, the mean corneal astigmatism was 3.2 D, only one eye did the suture break during adjustment. No infection, vascularization or rejection occurred. PMID- 7549326 TI - [Tenon-plasty and early keratoplasty in severe chemical eye burns]. AB - In severe eye burns with destruction of extensive areas of the conjunctiva and epibulbar tissue, Tenon plasty has proved to be successful. Because the epithelium fails to cover the denuded stroma, the corneal surface must be protected by an artificial epithelium. If necrolysis occurs, a tectonic keratoplasty must be performed early. The clinical courses of 12 patients with 14 very severely burned eyes are reported. In addition to Tenon plasty, early penetrating keratoplasies with large diameters 10-16 mm were performed up to 3 months after the accident. The follow-up time was between 6 and 34 months (mean 15.1 months). In five cases the Tenon tissue showed marked inflammation, and the keratoplasties developed large, persistent epithelial defects and had to be covered by conjunctiva. In the other cases it was possible to preserve a healthy epithelial layer by applying soft contact lenses. In 79% of the cases an endothelial graft rejection was observed. In about 50% the transplants were lost. Early keratoplasties are mainly for tectonic repair in severely burned eyes. Optical rehabilitation was achieved in only a few cases. PMID- 7549327 TI - [Long-term results of Tenon-plasty in treatment of severe chemical eye burns]. AB - From November 1986 to December 1993, 50 patients with 62 very severe eye burns were treated with Tenon plasty. Forty-one eyes showed widespread scleral ischemia, while 21 eyes had developed corneoscleral ulcerations. Surgery was done 3-126 days after the injury. A total of 210 quadrants was treated with Tenon plasty. Epithelization of the Tenon sheets was complete within 21 days in 79% and in all eyes up to 51 days. In all eyes corneoscleral ulcerations could be prevented or healed. In 48% isolated symblepharon formation or severe scarring of the fornices developed within 3 months after Tenon plasty. In these cases restoration of the conjunctiva was achieved by transplantation of autologous conjunctiva or nasal and buccal mucosa. Spontaneous epithelization of the burned cornea was achieved in 25.8%. In 36 eyes penetrating keratoplasty was performed. Sixteen (44.5%) grafts failed due to surface problems, while 12 (33.3%) grafts failed because of an endothelial immune reaction. Tenon plasty proved to be successful in preventing early necrolysis of the anterior eye segment in very severe eye burns. However, the prognosis for penetrating keratoplasty in these eyes is limited due to surface problems and immune reactions. PMID- 7549328 TI - [Visualization of keratocytes in the human cornea with fluorescence microscopy]. AB - There is little information about the state of human keratocytes in longterm cornea storage and the influence of these cells on the survival of the corneal transplant after keratoplasty. Up to now stromal keratocytes have not been taken into consideration in regard to the culture conditions in cornea banking. Therefore, a method was used, first described by Poole et al., who stained porcine corneas using the Live/Dead-Assay (Molecular Probes Inc.). After staining porcine and human corneal slices with the fluorogenic substance calcein AM and the fluorochrome ethidium homodimer, we examined the fluorescing cells to acquire more knowledge about the density, morphology and vitality of human keratocytes. Living cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, producing an intense, uniform, green fluorescence in living cells. Ethidium homodimer enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence on binding nucleic acids, thereby producing a bright red fluorescence in dead cells. The examinations were performed using a conventional fluorescence microscope and a confocal slit scanning microscope. Like in the porcine corneas, we found three keratocyte subpopulations in the human corneas, but there were some differences in morphology and patterns of staining between the two species in the central and posterior stroma. After long periods of cornea storage we found signs of cell degeneration like loss of cytoplasmic processes and an increasing number of dead cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549329 TI - [Pterygium excision with intra-operative administration of low dosage mitomycin C]. AB - Pterygium excision with postoperative instillation of mitoymcin C (MMC, 0.4 mg/ml) is encouraging because of the technical simplicity and low recurrence rates, but serious postoperative complications have been described (scleral necrosis, corneal perforation, glaucoma, cataract). The authors studied the efficacy and safety of pterygium excisions with a single intraoperative application of low-dose MMC (0.2 mg/ml). Pterygium excision (bare sclera technique) was performed in 18 patients with primary or recurrent pterygia. MMC (0.2 mg/ml) was applied intraoperatively (5 min) to the exposed sclera. Patients were followed up for signs of recurrence and complications. There were four recurrences (mean follow-up 13.8 months). Postoperative complications consisted of granuloma formation (n = 2). Delayed conjunctival healing was observed in all cases. Scleral necrosis, corneal complications, cataract, or glaucoma were not seen. Intraoperative application of MMC apparently reduces the recurrence rate after pterygium excision. The use of a 0.2 mg/ml concentration of MMC appears to be safe. PMID- 7549330 TI - [Predicting recurrent pterygium based on morphologic and immunohistologic parameters]. AB - BACKGROUND: Independently of the technique for excision used, pterygia recur quite frequent. We investigated whether recurrences can be predicted by means of histological and immunohistological parameters. MATERIALS AND METHODS: In a prospective clinical investigation the frequency of recurrent pterygia after excision and subsequent autologous limbal grafting was studied. The pterygia of 10 patients without recurrence (mean follow-up time 13 months) were compared retrospectively and semiquantitatively with the pterygia of 7 patients who had a recurrence. The following parameters were taken into consideration: (1) elastoid degeneration; (2) amount of vascularization; (3) morphological signs of "dry eye" (e.g., loss of goblet cells, beginning keratinization); (4) reactive inflammation; (5) amount of CD1a-, CD4-, CD8- and CD68-positive cells; (6) expression of PDGFalpha, PEDGFbeta, EGF, and laminin receptors. RESULTS: No appreciable differences between recurrent and nonrecurrent pterygia were found. CONCLUSION: As adjuvant therapeutic strategies like application of mitomycin C or beta-irradiation may lead to complications, it would be desirable it they could be limited to patients at risk. The histological and immunohistological parameters investigated probably do not allow prediction of pterygial recurrence. Consequently, they are not helpful in deciding whether adjuvant therapy should be started or not. PMID- 7549331 TI - [Amphotericin B--bioavailability in the cornea. Studies with local administration of liposome incorporated amphotericin B]. AB - Amphotericin B remains an important antifungal agent in the treatment of ocular mycosis. Since topical ocular application is limited because of ocular irritation and poor penetration, we studied the pharmacokinetics of amphotericin B encapsulated in unilamellar liposomes (AmBisome). One drop (20 microliters) of AmBisome or an equivalent concentration of amphotericin B was applied to rabbit eyes. Drug concentrations were measured 15, 60, 120 and 240 min following administration of the agents by HPLC in cornea and aqueous humor. The effect of intact (group A) and debrided corneal epithelium (group B) was also studied. Corneal amphotericin B levels were significantly higher (P < 0.01) after 15 min in animals receiving amphotericin B as compared to AmBisome in group A. At later time points no differences in the corneal drug levels were found, and the drug levels following AmBisome application were remarkably stable. Epithelial removal resulted in increased corneal drug levels following application of both amphotericin B preparations. Significantly higher drug levels were observed after free amphotericin B treatment at 15-60 min (P < 0.01). Drug levels in the aqueous humor did not differ between the two amphotericin B preparations and remained below therapeutically effective concentrations. These results suggest that topically delivered AmBisome provides stable corneal drug levels, but has the potential benefit of lowered ocular toxicity. PMID- 7549332 TI - [Endothelial cell loss after phacoemulsification and 3.5 vs. 5 mm corneal tunnel incision]. AB - This prospective study was performed to compare the central endothelial cell loss (ECL) after phacoemulsification and 3.5 with 5 mm temporal clear-corneal incision. Moreover, the influence of ultrasound time and power on postoperative endothelial cell density was evaluated. PATIENTS AND METHODS. Sixty-two patients (age 71 +/- 7.7 years) without corneal pathology were operated by phacoemulsification (Storz, Premiere) with a temporal, self-sealing, two-step clear-corneal incision followed by posterior chamber IOL implantation under viscoelastic (1% sodium hyal-uronate). Thirty-one one-piece plate-haptic foldable silicone IOLs (Chiron C10; Staar Surgical AA-4203) were implanted by an injector through a 3.5 mm incision (group A). The other 31 PMMA IOLs (Pharmacia 809P) were implanted through a 5 mm incision (group B). One surgeon performed all operations with the same technique (bimanual phacoemulsification in the capsular bag). Sutureless wound closure was performed in group A, while the wounds in patients of group B were closed with a single radial stitch. The central endothelial cell counts were recorded preoperatively, on days 2-5 and 6 months postoperatively using contact specular microscopy. RESULTS. The collective data revealed a cell loss of 7.9% (+/- 4.1 SD) on days 2-5 postoperatively and 6.7% (+/- 2.9) after 6 months. A direct linear relationship was found to exist between ultrasound time (UT) and ECL. In group A (7 patients) the ECL slightly increased from 3.5% in the first week postoperatively to 3.8% after 6 months, operated under UT < or = 1 min 30 sec. The ECL decreased from 8.2% to 6.4% after 6 months in group A (19 patients), operated under UT of 1 min 30 sec, and from 10.8% to 8.9% under UT of 2 min 31 sec-3 min 30 sec (5 patients). The ECL in group B decreased from 6.3% in the first postoperative week to 5.4% 6 months postoperatively, operated under UT < or = 1 min 30 sec (10 patients), from 8.1% to 7%, operated under UT of 1 min 31 sec-2 min 30 sec (14 patients), and from 11.2% to 10.4% under UT of 2 min 31 sec 3 min 30 sec (7 patients). CONCLUSION. Endothelial cell loss of 6.7% after phacoemulsification through a temporal clear-corneal incision compares favorably with other series in which cell loss was determined following cataract surgery with or without IOL implantation. Phacoemulsification and 3.5 mm clear-corneal incision evoked less ECL of 6.2% compared with phacoemulsification and 5 mm clear corneal incision with ECL of 7.3% after 6 months. PMID- 7549333 TI - [Glycoprotein D (5-23) specific Th2-T-cell line induces HSV-1 keratitis]. AB - BALB/c inbred Igh-1-disparate mice exhibit different susceptibility to the development of HSV-1 stromal keratitis (HSK), which may be due to the differential immune regulation. CD4+ T lymphocytes may be critical for the disease induction. A T-cell line (CD4+, T-cell receptor V beta 8+, interleukin 4+) specific for the N-terminal amino acids 5-23 of glycoprotein D from HSV-1 [gD(5-23)] was established from HSK susceptible C.AL-20 mice. HSK-resistant C.B 17 mice, and HSK-susceptible BALB/c mice were injected intraperitoneally with cells (5 x 10(5)/mouse) alone or combined with HSV-1 corneal inoculation (10(5) PFU, KOS strain). Control groups were injected with HSV-antigen-unrelated cells (PPD specific), or were only HSV-1 infected. Migration of the adoptively transferred gD(5-23) Th2 cells was analyzed by histology, by immunohistochemistry and by cell membrane labelling (PKH26). The transfer of gD(5-23) cells accelerated the disease onset (day 2, compared to day 7 without cells). The transfer of gD(5-23) cells increased the incidence of HSK (BALB/c 100%, C.B-17 20%) compared to mice that were only infected with HSV-1 (BALB/c 75%, C.B-17 0%). Keratitis was more severe in mice injected with gD(5-23) cells. In contrast, the transfer of PPD-specific cells did not influence the disease patterns. Mice injected with gD(5-23) cells and not inoculated with HSV-1 did not develop keratitis. The results suggest that CD4+ MHC class II, V beta 8+, IL-4 expressing T-cells (T helper 2) may be important for the induction of HSK.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549334 TI - [Scar formation in the cornea after photorefractive keratectomy]. AB - The morphologic correlate of the so-called subepithelial haze after photorefraktive keratectomy (PRK) has not yet been decided. Excimer laser keratectomy 100 microns in depth was performed in an eye that was scheduled for enucleation. Three months after surgery a dense scar was diagnosed with the slit lamp. After enucleation the cornea was investigated by light microscopy. The thickness of the scar was less than 1 micron in most sections with a maximum of 5 microns. This surprising finding explains the predominantly scattering power of the subepithelial haze, which results in a relatively good visual acuity of 20/30 to 20/25 in most clinical cases. PMID- 7549335 TI - [Differential value of various vital stains of corneal endothelium]. AB - Specular microscopy shows the integrity of the endothelial cell pattern, and vital staining methods allow the evaluation of endothelial viability. Dyes commonly used such as trypan blue enter the cells passively through the disturbed membrane. The uptake of fluorescein diacetate (FDA) is an active process. To compare the efficiency of the two methods we performed double stainings and examined the results. Pig cornea endothelium was stained by both trypan blue and FDA and vital and non-vital cells were compared. More non-vital cells were stained by FDA than by trypan blue, indicating that cells with membranes that are not permeable by trypan blue cannot metabolize FDA. This fact is important where estimating the endothelial viability of donor corneas. PMID- 7549336 TI - [Keratomileusis with a lamellar microkeratome and the excimer laser]. AB - In a combined prospective study with E. Arnott's department, intrastromal excimer keratomileusis on the lenticle stroma was performed in 15 eyes of 15 patients (follow-up 6 months). The primary keratectomy was performed using the lamellar automatic rotating microkeratome, followed by refractive modification by the excimer laser on the excised corneal discs. For the laser ablation, a repetition rate of 10 Hz and a fluence on the corneal plane of 180 mJ/cm2 was used. Following surgery, all treated eyes were subjected to guttae of Maxitrol q.i.d. for 12 weeks. Excimer laser intrastromal keratomileusis effectively combines corneal microsurgery and photoablation. The predictability of the refractive correction is better than with the other surgical keratomileusis techniques. PMID- 7549338 TI - [Morphologic papillar signs of high pressure glaucoma]. AB - PURPOSE: To examine the morphology of the optic disc in eyes with high-pressure open-angle glaucoma. MATERIAL AND METHODS: Using color stereo optic slides, 306 eyes of 183 patients with secondary open-angle glaucoma and 65 eyes of 35 patients with juvenile open-angle glaucoma (group I) were morphometrically examined and compared with 1119 eyes of 585 patients older than 40 years and suffering from with primary open-angle glaucoma (group II). RESULTS: The eyes of group I as compared to group II showed significantly (P < 0.0001) higher maximal intraocular pressure values and a significantly (P < 0.05) smaller optic disc and parapapillary atrophy. There was a tendency towards steeper and deeper cupping and a lower frequency of optic disc hemorrhages in group I than in group II. The optic disc shape did not vary significantly between the two groups. CONCLUSIONS: Minor parapapillary atrophy suggests high-pressure glaucoma. Barotrauma may be associated with a mostly diffuse damage of the optic nerve. The only slight enlargement of the parapapillary atrophy in the eyes with high-pressure glaucoma indicates that the development of the parapapillary atrophy may at least partially be independent of an elevation of intraocular pressure. The optic disc shape is not useful to identify eyes with high-pressure glaucoma. PMID- 7549337 TI - [High mean pallor value--a risk factor for deterioration of the visual field in glaucoma and ocular hypertension. A pilot study using the optic nerve head analyzer]. AB - Does the disc parameter "mean pallor value" suggest a risk for further deterioration of visual field defects? PATIENTS: In 10 patients with ocular hypertension and 23 patients with primary open-angle glaucoma (Octopus 201, Humphrey perimeter) disc pallor was calculated by double examinations with the ONHA in 1987-1988, and a long-term follow-up of the visual field was performed. In all patients we also acquired information about the IOP level and visual field during the observation time from the ophthalmologists. Only one eye per patient was evaluated after randomized selection. RESULTS: In ocular hypertensive patients only 1 of 10 eyes showed deterioration of the visual field. In the glaucoma group 4 eyes out of 23 showed an increase in visual field loss. The eyes with a tendency to deteriorate had a higher mean pallor value compared with the mean value of the corresponding group [ocular hypertensives: 0.40 compared to 0.31 +/- 0.07 (0.02); POAG: 0.50 +/- 0.06 (0.03) compared to 0.33 +/- 0.08 (0.02); mean value +/- SE (SEM)]. The difference was statistically significant in the glaucoma group; Wilcoxon, Mann and Whitney U-test; P < 0.005. CONCLUSIONS: Taking into consideration all risk factors, the mean pallor value might help additionally to prognosticate the risk of deterioration of the visual field as far as 5-year follow-up allows this preliminary conclusion. The pallor value alone is not suitable for the follow-up of glaucoma patients because of the influence of increasing lens opacities and changes in the video system of the ONHA over years. PMID- 7549339 TI - [Possibilities for imaging the retinal nerve fiber layer with the scanning laser ophthalmoscope]. AB - BACKGROUND: Documentation of the retinal nerve fiber layer using red free light is not established as a routine method. The reasons for this are the difficulties involved in taking the pictures and developing them, especially in glaucoma patients, and the need for maximum pupil dilation. The scanning laser ophthalmoscope (SLO) allows fast and easy documentation of the fundus with the possibility of simultaneous image control during the examination. PATIENTS AND METHODS: Nerve fiber layer images of 48 eyes of 25 patients taken with the argon blue light of the SLO were compared to conventional photographs of the nerve fiber layer with red-free light. In addition, we compared documentation of the nerve fiber layer using argon-blue or argon-green light in the SLO. RESULTS: The nerve fiber layer images obtained with the SLO were of good quality for all 48 eyes included in the study, whereas with conventional photography we could not obtain images in 9 eyes. There was a high correlation between the two methods in the detection of localized damage or zones with diffuse nerve fiber atrophy for the 39 eyes documented with both methods, and there was better image quality in some using the SLO. The use of blue light permits the nerve fiber layer to be seen more easily than with green light. CONCLUSIONS: The SLO allows nerve fiber bundle defects or diffuse atrophy to be detected similar to conventional fundus photographs using red free light sources. There is no further need to dilate the pupil maximally due to the confocal principle and the scanning technique. The smaller angle of view of the SLO requires fixation movements of the patient being examined for the detection of beginning peripheral nerve fiber bundle defects. PMID- 7549341 TI - [Ultrasound biomicroscopy findings in various forms of glaucoma]. AB - Ultrasound biomicroscopy is a new imaging technology that uses high-frequency ultrasound and thus allows depiction of the anterior portion of the globe in microscopic resolution. It is independent on the clarity of the optical media and allows, for the first time, a non-invasive demonstration of the structures of the posterior chamber and their relationship to each other. Therefore, it is especially suitable for diagnosis and research on the various forms of glaucoma. Our examinations were performed with an ultrasound biomicroscope (UBM Humphrey Model 840) with a 50 MHz transducer. This allows resolution of structures up to 50 microns. Thirty-nine patients with different forms of glaucoma were examined: POAG, pigmentary glaucoma, pseudoexfoliation glaucoma, pupillary block, plateau iris syndrome, ciliolenticular block, congenital glaucoma, traumatic glaucoma and situations after glaucoma surgery. The following findings could be demonstrated: configuration of the anterior chamber angle region, iris curvature, ciliary body and lens position, differentiation between solid and cystic tumors, traumatic changes and trabeculectomy openings. With this information ultrasound biomicroscopy offers additional details for the differential diagnosis and treatment of various forms of glaucoma. PMID- 7549340 TI - [Sensitivity and specificity of the Heidelberg retinal tomograph for imaging nerve fiber bundle defects in glaucoma patients with localized visual field defects. Clinical study]. AB - In order to verify whether nerve fiber bundle defects can be detected by the confocal technique of the Heidelberg Retina Tomograph (HRT), we compared the localization of localized visual field defects with the localization of the expected corresponding nerve fiber bundle defect. We examined 105 patients: 49 with primary open-angle glaucoma (POAG), 26 with low-tension glaucoma (LTG) and 10 with ocular hypertension (OH), and as a control-group 20 healthy eyes. The glaucoma stage had been defined by examination of the central visual field with the Octopus Perimeter 201 program 31 or 32 and/or the Humphrey Perimeter GG program. In healthy eyes, eyes with OH and POAG stage I (n = 19), no nerve fiber bundle defect could be found. In stage II (n = 15) and stage III (n = 14) we found nerve fiber bundle defects in all patients with POAG and LTG (except for one patient with POAG stage III). In stage IV (n = 27) no localized nerve fiber bundle defects were detected in eyes with POAG, but in 4 of 7 patients with LTG, defects could be seen. In glaucoma stages II and III, nerve fiber bundle defects can be detected with the HRT. The control group shows, that the appearance of a nerve fiber bundle defect is no artifact caused by the HRT. However, examination of eyes with OH and POAG stage I also shows that this method is not sensitive enough to be useful for early diagnosis of glaucoma. Nevertheless, it may be possible to develop the confocal HRT method further as a nerve-fiber diagnostic method for glaucoma. PMID- 7549342 TI - [Results of cyclodialysis combined goniotomy in treatment of dysgenetic glaucoma]. AB - This study was performed in order to investigate the results of "cyclodialysis ab interno," i.e., a combination of goniotomy and cyclodialysis, in patients suffering from dysgenetic glaucoma. From 1985 to 1993, 69 patients (98 eyes) underwent at least one cyclodialysis ab interno. The development of intraocular pressure was examined 2 days, 4 weeks, 1/2 year, 1 year and 3 years postoperatively after the first and last cyclodialysis ab interno. Additionally, we examined intraoperative bleeding, preoperative pressure level, previous operations, reoperations, age at manifestation, and monocular or binocular manifestation for their influence on the results. The effect the operation had on visual acuity was also examined. One year after the last cyclodialysis ab interno, the IOP was regulated in 77% of the eyes. The prognosis was better for eyes with preoperative pressure values of less than 30 mmHg than for eyes with pressure values of more than 30 mmHg. It was also clearly better when the glaucoma had become manifest at the age of more than one year. Previous operations, intraoperative bleeding, and monocular or binocular manifestation had no influence on the results. In comparison with the data available for goniotomy and trabeculotomy, cyclodialysis ab interno shows a tendency for better results. PMID- 7549343 TI - [Progress in laser sclerotomy ab externo] Enlarging the sclerostomy channel and local mitomycin administration]. AB - In a previous study we presented our first results after sclerostomy using a pulsed erbium/holmium: YAG laser. At a fiber diameter of 300 microns a success rate of 20-30% was found after a 1-year follow-up. This study concentrates on the improvement of the parameters for filter survival. METHODS. The fiber diameter and thus the size of the fistula increased to 400 microns. Patients who had undergone fibrosis for a previous fistula were treated intraoperatively with topical mitomycin administration. RESULTS. After a follow-up of 6 months, filter function in the 300 microns group was maintained in 26% of the treated eyes (n = 23), while 400 microns fistulas were successful in 48% (n = 26). In the mitomycin treated group (n = 6), sclerostomy achieved an IOP regulation in four patients. Postoperative hypotony was more frequent, but did not exceed 2 weeks. CONCLUSION. An increase in fistula diameter improves the long-term results of laser sclerostomy. Mitomycin is useful in maintaining filter function in patients with an unfavorable prognosis. PMID- 7549344 TI - [Effect of mitomycin C on aqueous humor flow after trabeculectomy]. AB - The intraoperative application of the antimetabolite mitomycin C has been recommended extensively in the literature for trabeculectomy in eyes with poor prognosis glaucoma. The modulation of wound healing with the antimetabolite mitomycin C has increased the success and predictability of filtration surgery. However, eyes in which mitomycin C is applied topically via a sponge during surgery bear the risk of developing postoperative hypotony and a decrease in visual acuity. Anterior chamber fluorophotometry (Fluorotron Master II, Coherent, Palo Alton) was used to study the effect of trabeculectomy with and without mitomycin C on the aqueous humour dynamics in glaucomatous eyes 6 months postoperatively. A 0.5-mg/ml solution of mitomycin C was applied topically via a sponge under the conjunctival flap. In a second group the same type of limbus based flap was performed without the use of mitomycin C. The subsequent procedure was identical in both groups. Six months after surgery the eyes without mitomycin C revealed normal aqueous humour flow during the afternoon hours, comparable with the unoperated fellow eyes (mean 2.1 microliters/min). Eleven eyes treated with mitomycin C showed a reduction of aqueous humour flow of about 56% (mean 0.9 microliter/min) when compared with the unoperated contralateral eye and an age-, race- and sex-matched group. The fluorophotometric data suggest that the significant reduction of aqueous humour flow is the reason for the hypotony seen in glaucomatous human eyes following trabeculectomy with mitomycin C. PMID- 7549345 TI - [Multicenter double-blind study with 0.25% timolol in Gelrite (TG) once daily vs. 0.25% timolol solution (TS) twice daily. German Study Group]. AB - In a 12-week double-masked trial we compared the ocular hypotensive effect of 0.25% timolol in Gelrite administered once daily (TG) to that of 0.25% timolol solution administered twice daily (TS). A second objective was to compare the tolerability and the safety of these treatments. Timolol in Gelrite is a new topical formulation of timolol in an anionic heteropolysaccharide gellan gum. A total of 156 patients entered the study after an appropriate wash-out. The medication schedule included one drop of test drug in each eye at 9 a.m. (active drug for both groups) and 9 p.m. (placebo for the TG group, active drug for the TS group). At trough, the mean decrease from baseline intraocular pressure (after appropriate wash-out) ranged from 5.7 to 6.3 mmHg for the TG group and from 5.9 to 6.2 mmHg for the TS group. The difference between the treatment group means ranged from -0.4 to 0.4 mmHg. At peak, the mean decrease from baseline IOP ranged from 5.3 to 6.2 mmHg for TG group and from 5.1 to 6.1 mmHg for the TS group. The difference between the treatment group means ranged from -0.7 to 0.4 mmHg. The results of this study support the hypothesis of a comparable hypotensive effect at peak and trough of 0.25% timolol in Gelrite q.d. to 0.25% timolol solution b.i.d. Furthermore, timolol in Gelrite has an acceptable tolerability profile. The incidence of blurred vision was higher in the Gelrite group, but this different was not statistically significant. The incidence of foreign body sensation was significantly higher in the Gelrite group (P < 0.022).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549346 TI - [Trabecular aspiration. A new surgical method for improving chamber angle facility in pseudoexfoliation glaucoma]. AB - BACKGROUND: The main reason for elevation of intraocular pressure (IOP) in glaucoma capsulare is secondary plugging of the intertrabecular spaces by pigment and fibrillous material. The goal of this study was to evaluate the clinical results of trabecular aspiration, a new surgical concept to improve trabecular facility in glaucoma capsulare. METHODS: Trabecular aspiration was performed under the operating microscope prior to extracapsular cataract extraction (ECCE) in half of the chamber angle circumference, using a specially designed irrigation aspiration device in 27 glaucomatous eyes. The hand-held instrument has three outlets, one for aspiration (400 microns wide and 45 degrees horizontally angulated to meet the slope of the meshwork) and two (650 microns) for irrigation to maintain a deep anterior chamber and to keep the iris away from suction. Trabecular debris and pigment was cleared with a suction force of 100-200 mmHg. RESULTS: The mean preoperative IOP (with and without glaucoma therapy) was 31.6 +/- 7.2 mmHg; At different times after operation, IOP values were: day 5, 15.1 +/ 4.2 mmHg. 1 month, 17.5 +/- 3.2 mmHg; 3 months, 17.2 +/- 2.5 mmHg; 6 months, 17.5 +/- 2.6 mmHg; 12 months, 21 +/- 4.5 mmHg. The mean antiglaucomatous medication score decreased from 3.81 medications/day preoperatively to 0.52 medications/day 12 months postoperatively. CONCLUSIONS: In a limited number of patients suffering from pseudoexfoliative glaucoma and cataract, the IOP-lowering effect of trabecular aspiration in combination with ECCE could be substantiated. Removal of inter- and pretrabecular debris prior to ECCE decreased IOP by 44% from baseline (12-month results), which indicates clinical relevance. PMID- 7549347 TI - [Cyclocryocoagulation in treatment of special types of glaucoma]. AB - In this retrospective study, we examine the efficiency of cyclocryocoagulation in eyes with secondary and congenital glaucoma. We studied 137 eyes with advanced, inadequately controlled glaucoma, which were operated from 1984 to 1993. Besides the intraocular pressure (IOP), we assessed the effect on the visual acuity and the optic nerve. We also compared the efficiency of two different cryomethods (CO2/N2 cyclocryocoagulation). Fifty eyes were cyclocryocoagulated with CO2 and 87 with N2. After a period of 6 months, 90 eyes were regulated (IOP between 7 and 21 mm Hg). At this time the mean IOP was 16 mmHg, whereas it had been 40 mm Hg before the cyclocryocoagulation. Serious complications like a phthisis bulbi were seen in only 18 eyes. Five eyes became blind despite therapy because of an absolute glaucoma. Concerning the different types of glaucoma, the neovascular glaucoma was the most difficult type to regulate. We obtained better and more lasting results with N2 cyclocryocoagulation (68% regulation) than with CO2 cyclocryocoagulation (62% regulation). Thus, cyclocryocoagulation represents a good method for the treatment of advanced, inadequately controlled glaucoma eyes when other methods have failed. PMID- 7549348 TI - [Lowering intraocular pressure by phacoemulsification and posterior chamber lens implantation]. AB - It is suspected that PE+PCL will often lower IOP. IOP was measured by Goldmann applanation tonometry in 50 consecutive patients (12 males and 38 females) who were unliterally pseudophacic (28 right and 22 left eyes n.s.). They had an intact posterior capsule and were not suffering from glaucoma. Mean age was 76 +/ 10 years. Time intervals since cataract surgery ranged from 2 to 68 months (mean 15 +/- 17 months). Seventy-four percent (99% confidence interval: 61% < P < 87%) of the patients showed lower IOP (less than 90% of reference value) in the pseudophacic eye. Median IOP in pseudophacic eye was 12 mmHg vs 14 mmHg in phacic control eyes (P < 0.01). These results are in accordance with the hypothesis that PE+PCL usually leads to a long-term reduction in IOP. PMID- 7549349 TI - [Perimetry with a glaucoma-specific test grid. A clinical study with the GG program]. AB - The GG program pattern of the Humphrey Field Analyzer includes the 76 test points of the 30-2 program and an additional 52 test points in a glaucoma-specific distribution. In 18 test points in the nasal step area beyond 30 degrees the threshold is also determined. The findings obtained with the GG program and the 30-2 program were investigated by means of three questions: (1) Do the additional test points increase the sensitivity in ocular hypertension and glaucoma in stage I? For this purpose 41 eyes of 41 patients were examined. (2) Is the new grid useful for the determination of form and size of scotomas? For this purpose 46 eyes of 46 patients were examined. (3) What information is gained from a new asymmetry index, the glaucoma hemifield index (GHI), evaluated on the retinal threshold sensitivity of the GG program grid? To evaluate the information gained, the GHI of the test points in program 30-2 was compared to the GHI of the GG program in 11 eyes of 11 healthy persons, 21 eyes of 21 patients with ocular hypertension, and 32 eyes of 32 patients with primary open-angle glaucoma, stage I and II. RESULTS: (1) In ocular hypertension and glaucoma stage I, the higher grid density of the GG program leads to a higher sensitivity in comparison to program 30-2. (2) In the 46 eyes with scotomas in stages II-IV, the condensed grid allowed better assessment of form and size of them. Ninety-one percent of the eyes with glaucoma stage II-IV showed nasal scotomas out of 30 degrees excentricity, which were now detected with the GG program. (3) The GHI of program 30-2 and the GG program showed no significant difference in retinal threshold sensitivity in glaucoma stage I. The asymmetry of the visual field loss in glaucoma stage II with localized scotomas can be detected significantly better with the GHI of the GG program. At the beginning of visual field loss with diffuse sensitivity loss, the grid density of program 30-2 seems to be sufficient. In stage II with localized scotomas, the higher grid density of the GG program yield more information for the detection of hemifield asymmetries. After examination with the 30-2 program it seems to be useful to examine the 52 additional test points and combine these two with the print-out from the GG program. PMID- 7549350 TI - [ProTon--a new portable tonometer. Initial experimental and clinical results]. AB - To determine the accuracy of a new portable tonometer using the Mackay Marg principle (ProTon, Tomey) we measured three cadaver eyes in vitro and 196 eyes of 98 patients in vivo. The 325 manometric determined IOP values were compared to ProTon measurements in three enucleated, canulated human eyes. The correlation was r = 0.99. In a clinical study 196 eyes were measured with both the Goldmann and the ProTon tonometer. The results showed good correspondence with a correlation of r = 0.95. The statistical significance (paired t-test) was P < 0.001. PMID- 7549351 TI - [Etiology of secondary glaucoma at university clinics in Munich and Nairobi]. AB - Glaucoma is one of the most common blinding diseases worldwide. For early detection and treatment of this insidious ailment investigations of risk factors are necessary, most of which require extensive technically demanding and time consuming procedures. Basic information on risk factors for the development of glaucomatous damage is essential for diagnosis of glaucoma, for the identification of groups at risk, and for the planning of prevention and intervention programs. Epidemiological data on secondary glaucomas is especially scarce, while at the same time the strict distinction between primary and secondary glaucomas has been abandoned with changing pathophysiological rationale. We compared the distribution of the main causes of secondary glaucomas in the university eye clinics in Munich and Nairobi, with the aim of finding possible risk factors and obtaining basic information that might be used in preventive programs. We examined 150 patients in Munich and 132 in Nairobi in 1991 and 1992. In addition to tests of visual acuity and intraocular pressure, gonioscopy, ophthalmoscopic assessment of the optic disc and perimetry were performed. Eyes with intraocular pressures above 22 mmHg and a detectable causative condition were selected for the study. The average age of the patients in Munich was 65 years, and most of them presented with glaucoma caused by iris neovascularization (42%), followed by exfoliation syndrome (14%), intraocular inflammation (11%), trauma (10%) and preceding ocular surgery (9%). In Kenya patients were significantly younger, with an average age of 41 years. Here most glaucomas were posttraumatic (40%); 70% of these patients were aged under 30. Other causes were earlier operations (17%), inflammation (16%), lentogenous changes (10%) and neovascularizations (10%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549352 TI - [Devitalization of lens epithelium cells by dye-enhanced therapy]. AB - Hitherto secondary cataract has been usually treated by Nd:YAG laser, which results in a dissection of the capsule with possible complications e.g. retinal detachment. Selective removal of the epithelial cells from the posterior capsule has not been possible. By use of dyes in connection with laser light of a suitable wavelength, lens epithelial cells could potentially be selectively destroyed. We investigated the dye indocyanine green, which has a peak absorbtion at the emission wavelength of commercially available diode lasers. METHODS: Human lens epithelial cells and 3T3 cells (mouse embryonal fibroblasts) were grown in MEM with 10% FBS. Confluent and preconfluent layers were incubated with indocyanine green and treated with a diode laser attached to a slit lamp (810 nm, Zeiss VISULAS, Zeiss Oberkochen, Germany). RESULTS: Indocyanine green alone was toxic in concentrations above 5 mg/ml (60 min). Combined with laser treatment (200 mW, 100 ms, 160 microns), indocyanine green led to cell damage in preconfluent cultures at concentrations above 0.01 mg/ml (10 min). After about 2 days irradiated cells became necrotic and began to desquamate from the culture disc. Cells in confluent cultures could be devitalised at concentrations above 0.05 mg/ml (10 min) using the same power. In contrast, neither indocyanine green alone nor diode laser alone could interfere with epithelial proliferation. DISCUSSION: The experiments show the possibility of dye-enhanced therapy of lens epithelial cells with indocyanine green and diode laser. In contrast to other photosensitizers, indocyanine green is widely used in human function tests. Therefore its use for the treatment of secondary cataracts may merit discussion. PMID- 7549353 TI - [Neuroradiology in ophthalmology. 1]. PMID- 7549354 TI - A new method for studying plaque morphology. AB - A histologic method was developed for three-dimensional (3-D) analysis of atherosclerotic plaques removed from the carotid bifurcation during endarterectomy. By sectioning the plaque at frequent intervals (0.5 to 1.0 mm), it is possible to obtain important information on plaque constituents with regard to their volume and distribution within the lesion. These data from each section are combined with those from other sections and displayed in a 3-D format for the entire length of the lesion. The tissues making up each of the 10 carotid plaques were outlined and digitized for each histologic section by position along the lesion. From the areas outlined a 3-D model was created by a computer-aided design program. Quantitative information on tissue distribution within the plaque was measured. Fibrous tissue constituted between 35% and 70% of plaque volume; loose necrosis from 0.5% to 30% of the plaque and thrombus occupied, at a maximum, 10% even though if was present in six of the 10 plaques. To investigate the distribution of constituents about the long axis, measurements were also made from each of the four quadrants of each section. The reproducibility of the measurements of three sets of sections at 10-mm separation showed that estimates of the amount of some constituents were very reproducible whereas others had considerable variation related to the small volume they occupied within the lesion. By generating a complete 3-D reproduction of the contents of atherosclerotic plaques, it may be possible to identify those features of the plaque that are most responsible for the development of ischemic events. PMID- 7549355 TI - Nitrate versus rest myocardial scintigraphy with technetium 99m-sestamibi: relationship of tracer uptake to regional left ventricular function and its significance in the detection of viable hibernating myocardium. AB - Rest technetium 99m-sestamibi uptake may underestimate myocardial viability in asynergic territories. Because nitrate administration was reported to improve thallium 201 uptake in perfusion defects, this study aimed to test the influence of nitrates on 99mTc-sestamibi uptake and on the tracer capability to recognize viable tissue in asynergic segments. In 23 patients with prior infarction and left ventricular dysfunction, regional wall motion was assessed by echocardiography before and after revascularization (13 segments/patient). Group 1 included 97 normokinetic; group 2, 97 hypokinetic; and group 3, 105 akynetic or dyskinetic segments; group 3 was divided into group 3A (72 segments unchanged after revascularization) and group 3B (33 segments with functional recovery). 99mTc-sestamibi uptake was graded using a scoring scheme in the same 13 segments both at rest and, on a separate day, injecting the tracer during isosorbide dinitrate infusion (ISDN). At rest, the mean 99mTc-sestamibi uptake decreased significantly from group 1 through group 3. With ISDN, the mean 99mTc-sestamibi uptake increased in all groups compared with rest, but the increase was significant only in groups 2 and 3, and within the latter, only in group 3B. Thus, with ISDN group 3B was no longer different from group 2. Only 6% of group 3A segments showed an improved uptake with ISDN, versus 33% of group 3B (P < .00005). At rest only 14 of 33 segments of group 3B showed a normal or slightly reduced uptake, whereas these were 25 of 33 with ISDN (P < .02). In conclusion, the acute administration of ISDN increases the uptake of 99mTc-sestamibi mainly in those asynergic territories that show postrevascularization functional recovery. Therefore, ISDN 99mTc-sestamibi imaging might improve the tracer capability to detect viable hibernating myocardium. PMID- 7549356 TI - Accurate coronary calcium phosphate mass measurements from electron beam computed tomograms. AB - Precipitates of calcium phosphate in coronary arteries always indicate the presence of atherosclerosis. The mass of these precipitates is related to the severity of atherosclerosis. To determine the accuracy of electron beam computed tomographic (CT) mass estimates, we imaged 21 human hearts inside an anthropomorphic chest phantom using an Imatron C-100 electron beam CT scanner (Imatron, San Francisco, CA). We then incinerated the 63 imaged coronary arteries and weighed the mineral ash. We calculated the mass estimates from the images using an algorithm derived from a model that assumes simple radiographic properties of the coronary arteries. We also calculated the currently used coronary calcium score for each artery. Although both the mass estimates and the scores correlated with the actual mass of the incinerated specimens (r = .97 and r = .93), the correlation with the mass estimates was better (P = .02; William's test). The regression equation relating the actual mass to the mass estimates was y = 1.37 x + 14, indicating that the CT mass estimates consistently underestimate actual coronary calcium phosphate mass. We conclude that relative mass estimates using electron beam CT scanning are accurate and that both these and the currently employed calcium scores reflect the actual mass of precipitated calcium phosphate in diseased coronary arteries. PMID- 7549357 TI - Echocardiography in infective endocarditis: reassessment of the diagnostic criteria of vegetation as evaluated from the precordial and transesophageal approach. AB - The echocardiographic characteristics of vegetation used by precordial echocardiography (PE) have been transferred unchanged to transesophageal echocardiography (TEE), which has different image definition and structural resolution. Twelve diagnostic criteria of vegetation were tested for their accuracy in 52 patients evaluated by PE and TEE for suspected endocarditis (36 men, 16 women; mean age, 62 +/- 18 years; 42 with proven endocarditis). Results of PE and TEE were validated against gross anatomic and histologic findings. Significant differences (P < .05) included the fact that TEE disclosed more vegetations not prolapsing in the subvalvular region and in absence of valvular regurgitation. At TEE vegetations presented motion distinct from the endocardial surface, irregular conformation, and uneven margins. Only chaotic motion was significantly associated with vegetations at PE; size < 0.5 cm and increased echogenicity characterized pseudovegetations at PE. Other features such as shaggy echoes or location out of the annular zone (previously indicated as typical of vegetations) were not significantly associated with infective lesions. Discriminant analysis of TEE characteristics of vegetations disclosed that chaotic motion was the variable most significantly (P = .008) associated with vegetation. Coexistence of this sign with size < 0.5 cm and uneven margins was associated with 93.3% sensitivity and 83.7% specificity. In conclusion, the echocardiographic aspect of vegetations is rather different when examined from the precordial and the transesophageal approach. Learning about pitfalls and normal variants should improve TEE specificity in the assessment of infective lesions. PMID- 7549358 TI - Initial experience with a steerable intravascular ultrasound catheter in the aorta and pulmonary artery. AB - The aim of this protocol was to test the feasibility and safety of a prototype steerable intravascular ultrasound (IVUS) catheter (Boston Scientific, Waterton, MA) in comparison with standard IVUS catheters. A 3.5F, 20-MHz mechanical echo transducer was incorporated into a bendable sheath with a blunt tip. The flexible IVUS catheter was compared with a standard IVUS catheter in 13 patients. Seven patients underwent catheterization of the left side of the heart, and six patients had catheterization of the right side of the heart for suspected recurrent pulmonary embolism. In the aorta, three lumen area measurements were made: (1) midway between the aortic arch and the aortic root, (2) at the most cranial part of the aorta, and (3) in the descending aorta at the level of the diaphragm. Evaluation of the accuracy of luminal dimension measurements by both types of catheters in perpendicular positions to the vessel wall was evaluated in a hollow rubber cast of an human aorta and its side branches, representing luminal diameters from 3 to 26 mm. We performed 20 measurements with each type of catheter. The results were compared with ruler measurements, after the cast had been cut in slices. The equation for the standard 3.5F IVUS catheter was: y = 0.89x + 0.15; SE = 0.17; r = .97; for the 4.8F 20-MHz standard IVUS catheter: y = 0.97x + 0.05; SE = 0.18; r = .98; and for the steerable catheter, y = 0.94x + 0.09; SE = 0.12; r = 0.97.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549359 TI - In vivo assessment of nonlinear myocardial deformation using finite element analysis and three-dimensional echocardiographic reconstruction. AB - In vitro data have shown that the myocardium exhibits nonlinear passive stress strain relationship and a non-linear pressure-volume relationship. A finite element (FE) analysis and optimization algorithm was used on three-dimensional reconstructed left ventricular (LV) geometry using echocardiographic images, along with hemodynamic measurements, in seven closed-chest dogs to show a nonlinear stress-strain relationship in vivo. Our analysis included the computation of Poisson's ratio from the measured volumetric strain of the myocardium and a simulated pericardial pressure load ("equivalent pericardial pressure") applied to the epicardial surface of the reconstructed LV. LV geometry was reconstructed in three or four incremental time steps in diastasis and the myocardium was assumed to be homogeneous, isotropic, and linearly elastic during these short intervals in this initial study. Simultaneous LV chamber pressure and equivalent pericardial pressure were incorporated into the algorithm to predict actual LV expansion. Computations were performed iteratively at each interval to compute the optimized elastic modulus. By performing the FE analysis and optimization at each interval (a step-wise linear analysis approach), a linear relationship between the myocardial elastic modulus and LV chamber pressure was derived (r = .87 to .98). Such a linear relationship is equivalent to an exponential myocardial stress-strain relationship in vivo. Detailed measurement of nonhomogeneous regional deformation are becoming possible with the advent of sophisticated imaging techniques. The methodology described in this study, with appropriate modifications in the FE analysis and optimization algorithms, can be applied to assess the complex three-dimensional pressure-deformation characteristics in vivo. PMID- 7549360 TI - Color flow Doppler assessment of valvular regurgitation: qualitative limitations and quantitative promise. PMID- 7549361 TI - The clinical utility of transesophageal echocardiography in patients with left sided infective endocarditis. PMID- 7549362 TI - Two-dimensional echocardiography is preferable for measuring left ventricular mass: all that glitters is not a reference standard. PMID- 7549363 TI - Ultrafast computed tomography for detection of coronary artery calcification. AB - This review summarizes coronary artery calcification as a marker of coronary atherosclerosis as historically noted and detected with radiographic imaging modalities. This review concentrates on the use of ultrafast computed tomography (CT) for coronary artery calcification imaging. Ultrafast CT is uniquely qualified for imaging of the coronary arteries and calcific deposits in particular because of the x-ray attenuation characteristics of calcium. Current validation studies are summarized. In particular, the sensitivity of ultrafast CT coronary calcification in angiographically proven obstructive disease has been shown by many investigators to be above 90%, but with specificity on the order of 40% to 50%. These numbers may be different for younger men and women, where the detection of smaller calcific deposits is more difficult. The positive predictive value has been calculated as between 72% by angiographic validation and 40% in a population with a lower pretest bias. PMID- 7549364 TI - The role of scintigraphic perfusion imaging for predicting ischemic cardiac events in noncardiac surgery. AB - The prediction of perioperative cardiac events remains a challenging issue in clinical cardiology. The most important cause of perioperative cardiac morbidity and death continues to be myocardial infarction, which is associated with a high prevalence of occult coronary artery disease. A large amount of data substantiates the validity of scintigraphic perfusion imaging for accurate diagnosis of coronary artery disease, as well as the prediction of prognosis in stable coronary disease, post-myocardial infarction, and in the preoperative setting. A normal stress perfusion scan predicts an extremely low rate of future cardiac events. The best predictor of future adverse cardiac events is the quantitative extent, severity, and reversibility of jeopardized myocardium, parameters that are easily assessed with perfusion scintigraphy. Although the sensitivity of scintigraphy for detecting coronary disease is high, the reported specificity of abnormal perfusion scans for predicting cardiac events is variable. Indiscriminant use of this technique with subsequent referral for angiography based on the presence of any perfusion abnormality will result in excessive, costly, and potentially dangerous screening procedures. However, thoughtful consideration of both the extent and severity of perfusion abnormality can appropriately stratify the majority of patients into low-, intermediate-, and high-risk groups. Further efforts should be directed at prospectively evaluating the use of quantitative perfusion defect and reversibility scores in various subgroups of patients to optimize the specificity of these techniques and determine the optimal degree of defect extent and severity that warrants invasive evaluation. PMID- 7549365 TI - The clinical utility of transesophageal echocardiography in ischemic papillary muscle rupture. AB - Ischemic papillary muscle rupture is a potentially lethal complication of myocardial infarction. Acute mitral regurgitation, shock with pulmonary edema, may ensue as its result. Transthoracic echocardiography is the initial noninvasive tool used to confirm the diagnosis. Transesophageal echocardiography has overcome some of the limitations of transthoracic imaging, permitting timely surgical management of these critically ill patients. PMID- 7549366 TI - Effect of clonidine on ICP and on the hemodynamic responses to nociceptive stimuli in patients with brain tumors. AB - The effect of clonidine on intracranial pressure (ICP), mean arterial pressure (MAP), cerebral perfusion pressure (CPP), heart rate (HR), and drug requirements was studied in 24 patients scheduled for elective cerebral tumor resection (ICP < or = 20 mm Hg). The patients were randomly assigned to one of two groups: Group P (placebo), 12 patients; Group C (clonidine 3 micrograms/kg 10 min before induction), 12 patients. In all patients, anesthesia was induced with a propofol infusion (500 micrograms/kg/min) combined with fentanyl 2 micrograms/kg, lidocaine 1.5 mg/kg, and vecuronium 0.1 mg/kg. Propofol was also used for maintenance. During the preinduction period, clonidine had no effect on ICP or HR, but in clonidine-treated patients, MAP and CPP decreased significantly in comparison to those of the placebo group. During induction, ICP and HR were stable and similar in both groups. MAP and CPP remained significantly lower in Group C. At intubation and Mayfield clamp application, ICP increased in both groups, with similar values at all times. MAP increased in both groups at intubation, Mayfield clamp application, and incision, staying lower, however, in Group C. CPP followed a pattern similar to that of MAP. Propofol requirements up to the 20th min were lower in Group C than in Group P (2.08 +/- 0.83 vs. 3.3 +/- 0.7 mg/kg, p < 0.05). Finally, throughout the study, eight patients in Group C versus two in Group P had a CPP value < 60 mm Hg for > or = 1 min (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549367 TI - Electrophysiological monitoring for selective shunting during carotid endarterectomy. AB - Selective shunt during carotid endarterectomy is more and more widespread, but it requires a monitoring system able to identify severe brain ischemia correctly. In 255 endarterectomies for severe carotid stenosis, we evaluated cerebral activity by means of sequential use of computerized two-channel electroencephalogram (EEG) and somatosensory evoked potentials (SSEPs). In 1.96% of cases, we observed changes referable to severe cerebral ischemia: in one case, in spite of shunting, EEG asymmetry persisted till the end of the operation, and the patient awoke with irreversible aphasia. In two other cases, a progressive disappearance of the cortical wave (N20) occurred in spite of a normal EEG pattern. None of the unshunted patients had postoperative deficit. Computerized EEG is an easily interpretable method of monitoring and reveals rapidly developing cerebral ischemia, but severe SSEP changes can occur in spite of a normal EEG pattern when cerebral ischemia has a slow onset. Although SSEP monitoring is a slower method of recording, it can give a finer distinction of less severe cerebral ischemia. PMID- 7549368 TI - Accuracy of continuous jugular bulb venous oximetry during intracranial surgery. AB - We compared readings obtained from the Baxter-Edwards continuous jugular bulb venous oximetry catheter with those obtained from blood gas analysis of simultaneously drawn samples from the catheter in 12 patients undergoing neurosurgical procedures. Within the range studied (SjvO2, 42-95%), the 111 (median, nine samples per patient; range five to 17) oximetric catheter readings correlated well with hemoglobin oxygen saturation values obtained from in vitro analysis of simultaneously drawn blood samples from the catheter (y = 0.93x + 3.4, r = 0.94, p < 0.001). Fiberoptic light signal was suboptimal (signal quality index = 3 or 4) on fewer than five occasions per patient during an average surgical procedure duration of seven h, and these occurrences were generally corrected by flushing the catheter. We conclude that the Baxter-Edwards jugular bulb oximetric catheter provides an accurate measure of SjvO2 during neurosurgical procedures. PMID- 7549369 TI - Upper motor neuron dysfunction reduces the EMG evidence of myasthenia gravis. AB - Hyposensitivity to the action of nondepolarizing muscle relaxants has been frequently observed in patients with upper motor neuron dysfunction and in patients treated with antiepileptic drugs. To ascertain whether the same conditions are also characterized by a reduced sensitivity to the effects of anti acetylcholine-receptor antibodies occurring in myasthenia gravis, bilateral investigations of neuromuscular transmission were conducted on proximal and distal muscles of a myasthenic patients with a long history of hemiparesis and epilepsy. It was observed that electromyographic evidence of myasthenia gravis was absent in distal muscles (and low in proximal ones of the paretic side. Even though the influence of antiepileptic treatment could not be assessed with certainty in this patient, indirect evidence suggests that hyposensitivity to anti-acetylcholine-receptor antibodies is an unlikely consequence of neuromuscular changes induced by anticonvulsants. PMID- 7549370 TI - Do recently developed techniques for skull base surgery increase the risk of difficult airway management? Assessment of pseudoankylosis of the mandible following surgical manipulation of the temporalis muscle. AB - We report our experience with anesthetic care for six patients with pseudoankylosis of the mandible following neurosurgical procedures, four of whom required fiberoptically guided intubation for anesthesia. We suggest that the development of operative approaches and reconstruction techniques in skull base surgery may increase the risk of difficult airway due to limitation of mouth opening. PMID- 7549371 TI - The role of nitric oxide in cerebrocortical laser Doppler flow response to halothane in the rat. AB - Laser Doppler flowmetry was utilized to investigate whether nitric oxide (NO) plays a role in the cerebrocortical hyperemic effect of halothane in rats. A particular objective was to elucidate whether the increased vascular tone or the removal of basal NO secondary to NO synthase inhibition influenced the response to halothane. The animals were anesthetized with i.p. pentobarbital for surgery and 90 min later were ventilated with 1.0 minimum alveolar concentration (MAC) halothane for 1 h to achieve a steady-state baseline. The control group was infused with either 1 ml of saline or 20 mg/kg of D-NAME, and the treatment group received 20 mg/kg of L-NAME intravenously. In a subset of the treatment group, we restored baseline flow and vascular tone using i.v. sodium nitroprusside (SNP). Mean arterial pressure (MAP) was maintained constant with an infusion of phenylephrine (0.5-5 micrograms/kg/min). Then, 30 to 45 min later, inspired halothane was raised to 1.7 MAC in each group, and the increase in laser Doppler flow (LDF) was measured. On increasing halothane MAC in the control group, LDF increased by 28 +/- 4%. L-NAME increased MAP by 21 +/- 4% and reduced baseline LDF by 26 +/- 2%. In the L-NAME-only treated group, 1.7 MAC halothane increased LDF by 12 +/- 3%, significantly less than control. The decrease in cerebrovascular resistance induced by increasing inspired halothane MAC was similar in the control group and in the L-NAME treated group at 23 +/- 6% and 22% +/- 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549372 TI - Normal perfusion pressure breakthrough syndrome: entity or excuse? PMID- 7549373 TI - Occlusive hyperemia remains the most logical explanation for the hemodynamic complications of resected intracerebral arteriovenous malformations. PMID- 7549374 TI - Predicting perioperative stroke. AB - The purpose of this review is to examine the incidence and mechanisms of perioperative stroke and to identify possible associated risk factors. The perioperative period can be subdivided into preoperative, intraoperative, and postoperative periods; we focus here on assessment in the preoperative period, as this is the time when risk prediction usually occurs. Surgical technique itself will not be considered, nor will intraoperative monitoring to detect cerebral ischemia be considered. This review will examine perioperative stroke in relation to general surgical procedures, including peripheral vascular reconstruction, orthopedic, gynecologic, and head and neck surgery. PMID- 7549375 TI - Cerebral metabolic rate and hypothermia: their relationship with ischemic neurologic injury. AB - Hypothermia results in a progressive depression of cerebral electrical activity and metabolism. In the setting of cerebral ischemia, large reductions in temperature are associated with a better preservation of high-energy phosphates, a reduced accumulation of toxic metabolites, and an improvement in post-ischemic outcome. With temperature reductions of < or = 6 degrees C, the brain is also partially protected from ischemic neurologic injury; however, this protection does not correlate with measurable alterations in high-energy phosphate depletion or lactate accumulation. Thus, cerebral protection by hypothermia may be due to a variety of factors, including alterations in basal metabolism, ion homeostasis, agonist-specific receptor activity, and cellular structure. Further, the relative influence of these factors may change with progressive reductions in temperature. PMID- 7549376 TI - Electrophysiological monitoring and mild hypothermia. AB - Hypothermia affects cerebral metabolism including a variety of neurotransmitter systems. Neurophysiologic changes during hypothermia are characterized by decreases in the membrane resting potential and amplitude. The duration of the action potential is prolonged and nerve conduction velocity is decreased. Nonsynaptic responses are reduced to a lesser extent than synaptic responses. Mild hypothermia to 33.5 degrees C produces only minimal changes in the EEG with small shifts in frequencies to theta- and beta-activity. Electro-cerebral silence occurs at a temperature below 22-25 degrees C. Evoked potentials (EP) of all modalities are affected by hypothermia to a similar degrees. Amplitudes are decreased and latencies are prolonged. Cortical EP components are more profoundly affected than early, subcortical potentials. EP may be reliably recorded at temperatures > 25 degrees C. Changes in EEG and EP are not specific for hypothermia. In addition, a hysteresis between cooling and rewarming has to be taken into account for the interpretation of temperature effects on brain electrical activity. The interactive effects of anesthetics and temperature may preclude EEG and EP measure for a graded quantification of hypothermia. PMID- 7549377 TI - [Characteristics and key point of the study of meridians]. PMID- 7549378 TI - [Effects of red ginseng on the congestive heart failure and its mechanism]. AB - Forty-five patients with class IV cardiac function were divided into three groups: group I (digoxin group), group II (Red Ginseng group) and group III (Red Ginseng plus digoxin group). Each group consisted of 15 cases. After treatment, the improvement of the hemodynamical and biochemical indexes of group II and group III were greater than those of group I, and group III was the most significant amongst all. The results suggested that Red Ginseng and digoxin had synergism for treatment of congestive heart failure, and Red Ginseng was an effective and safe adjuvant without any side effects. PMID- 7549379 TI - [Effect of Astragalus membranaceus on T-lymphocyte subsets in patients with viral myocarditis]. AB - The efficacy of Astragalus membranaceus (AM) oral liquor combined with routine therapy and routine therapy alone on T-lymphocyte subsets of peripheral blood in viral myocarditis patients have been studied. The results showed that the T lymphocyte subsets profile and OKT4/OKT8 ratio of peripheral blood were significantly lower in viral myocarditis patients than that in healthy control (P < 0.05, 0.01). Routine therapy combined with AM could significantly enhance OKT3, OKT4 and OKT4/OKT8 ratio in the above-mentioned patients (P < 0.05, 0.01). The possible pharmacodynamic mechanism of improved cell immunity in viral myocarditis patients by combining routine therapy with AM was discussed. PMID- 7549380 TI - [Relationship between tongue colour and cardio-vascular function]. AB - The cardiac diastolic and systolic function, arterial compatibility and total arterial resistance of 116 patients was investigated. Using RM-6000 polygraph recorder, five kind of tongue pictures were described, and their impedance cardiogram, carotid arteriogram and their differential spectrum, cardiophonogram and electrocardiogram were examined. The results showed that the PEP index in presphygmic period of purplish tongue group was larger than that of other groups, while the PEP/LVET, HI, SV, SVI and CI indexs of purplish tongue group was less than that of other groups. The cardiac function index of the pale and dark-red tongue group were lower than that of pale-red tongue group. The cardiac function index of red tongue was similar to pale-red tongue. According to cardiac function index, the cardiac function displayed that: pale red tongue > red tongue > dark red tongue > pale tongue > purplish tongue. PMID- 7549381 TI - [Therapy of cantharides extract for perennial allergic rhinitis and its effect on total IgE in serum]. AB - The therapy of 10% Cantharides extract in treating 50 cases of perennial allergic rhinitis (PAR) was studied. The extract was plastered and blistered on Dazhui, Neiguan point. It was observed by nasal mucosa provocative test, cells in nasal secretion test and serum total IgE test. The results showed that its effective rate was 88%, the allergic nasal mucosa provocative test of treated group alleviated obviously after the treatment (P < 0.01), the number of eosinophil and basophil in nasal secretion decreased (P < 0.01, P < 0.05); and the serum total IgE also reduced significantly (P < 0.01). PMID- 7549382 TI - [Clinical and experimental studies on tiaoshen liquor for infantile hyperkinetic syndrome]. AB - One hundred children with hyperkinetic syndrome were treated by using Tiaoshen Liquor (TL) consisted of Chinese herbal drugs. After the treatment, the behavior grading lowered greatly, their attention was improved, and their academic records raised. The total effective rate reached 94%. The results of the animal experiments showed that TL could reduce the spontaneous activities of the healthy mice, thus lowering remarkably the spontaneous activities in the mice with hyperkinetic behavior caused by taking scopolamine; reinforce the learning memory in the healthy mice; and improve in different degrees the learning memory of the mice with dysmnesia caused by administering scopolamine, sodium nitrite and alcohol respectively, which indicate that the therapeutic mechanism of TL for this syndrome was probably related to the improvement of information transfer function of the cholinergic neuron synapses of central nervous system and to the enhancement of hypoxia tolerence of the cerebral tissues. PMID- 7549383 TI - [Localization value of intra-esophageal atrial pacing by modified digitus medius proportional unit]. AB - The length of lead electrode which was eleven times the length of modified digitus medius proportional unit used for intra-esophageal atrial pacing on 86 patients was compared with the length lcm more or less than the lead length itself showed very significant difference in pacing threshold, which was low, adverse reaction was mild, the intra-esophageal P wave display was satisfactory. It could alleviate the patient's pain, and was easy to manipulate, practical, rapid and inexpensive in diagnosis and treatment of arrhythmia, rescuing indoor or outdoor emergency cases as well as monitoring operation. PMID- 7549384 TI - [Effects of zhengda zhenhua 851 on improvement of memory and fluency of language of kidney deficiency patients]. AB - A study was conducted on the effects of Zhengda Zhenhua 851-R oral liquor on the improvement of memory and fluency of language of patients suffering from Kidney Deficiency and control group. It was found that the treated group did better in memory tests and the index of mental quotient (MQ) was higher than that of control group. They had markedly higher scores in the tests of directional memory which requires one's initiative and the free recollection of pictures. The scores achieved by the treated group were significantly higher than that of control on the tests of the fluency of language and expressions, particularly with words of different categories. The results showed that Zhengda Zhenhua 851-R oral liquor obviously had the function of improving the memory and fluency of language for Kidney Deficiency patients. PMID- 7549385 TI - [Experimental and clinical study of shuanghuanglian aerosol in treating acute respiratory tract infection]. AB - Two hundrde and two cases of acute respiratory tract infection (ARI) were treated with Shuanghuanglian (SHL) aerosol. In these cases, the majority of cases were virus infection and 64% of them was caused by the respiratory syncytial virus (RSV). The virostatic and bacteriostatic test were done in vitro by the cell culture method and it was shown that SHL could inhibit the RSV, para-influenza I IV and 23 kinds of pathogenic bacteria such as Staphylococcus aureus etc. The bacteriostatic effect was positively correlated to the SHL concentration. Experimental study showed that SHL could enhance the NK cell activity, promote the production of alpha-interferon and raise the rate of lymphocyte transformation. The controlled observation on SHL preparation with various dosage forms revealed that the SHL aerosol in treating early ARI showed better results than that of injections and oral liquor symptomatologically (P < 0.01). The effective rate was 96%. PMID- 7549386 TI - [Activity of cholinesterase of erythrocyte membrane in patients with hot or cold syndrome]. PMID- 7549387 TI - [Mechanism of tonifying kidney and removing blood stasis recipe in modulation of immune senescence]. AB - This experiment was from 3 aspects to study the relationship between modulating immune senescence and the improvement of free radical metabolism by the recipe of Baweidankun decoction (BWDKD). The results showed that the production of IL-2 decreased, the content of LPO increased and the activity of catalase (CAT) declined in old mice. After medication of BWDKD the above-mentioned indices were modulated. The ozone inhaled mice revealed similar changes of aging in the above indices. After BWDKD administration, the content of LPO decreased, meanwhile, the activity of CAT and IL-2 were strengthened and showed linear correlation. It was discovered that the production of IL-2 decreased significantly after adding H2O2 into the culture of splenic lymphocytes, but when BWDKD was added simultaneously, the IL-2 production was restored to the level of control group, in which no H2O2 was added. The results suggested that the modulating effect of BWDKD on immune senescence was closely related with the improvement of free radical metabolism, and it provided a partial evidence for the viewpoint of "Vitality deficiency with blood stasis is the principal mechanism of senescence". PMID- 7549388 TI - [Effects of reducing injection on plasma TNF-alpha and IL-6 levels in rabbits with endotoxin-induced DIC]. AB - Plasma TNF-a and IL-6 levels of endotoxin-induced DIC in rabbits were measured by ELISA methods. Simultaneously, the effects of Reduqing (RDQ) on TNF-alpha and IL 6 were also studied. The results showed that TNF-alpha and IL-6 levels increased markedly in model group, which were confirmed with obvious damage of tissue or organ. Co-administration of RDQ could reduce plasma TNF-alpha and IL-6 levels, together with mild tissue or organ damage. These results indicated TNF-alpha and IL-6 might be involved in pathogenesis of endotoxin-induced DIC, RDQ might be used in preventing or treating DIC through mechanism of reducing TNF-alpha and IL 6 levels. PMID- 7549389 TI - [Transmission electron microscopic observation on the liver and cerebral cortex in aging mice treated with sijunzi decoction]. AB - By means of Transmission Electron Microscope (TEM), it was observed that Sijunzi Decoction (SD) affected the liver and ultrastructure of cerebral cortex in aging mice. The results showed that SD could improve the liver cells and the ultrastructure of cerebral cortex in aging mice, nourish the Viscera with Qi and blood by replenishing Spleen and Qi, adjust the Deficienoy of Viscera and promote the health of body and longevity. PMID- 7549390 TI - [Diagnosis of blood consumptive disease instead of "xuelao" in hematologia diseases--observation on immune function of red blood cells]. PMID- 7549391 TI - [Recent developments of traditional Chinese medical therapy in treating aplastic anemia]. PMID- 7549392 TI - [A survey on the treatment of acne]. PMID- 7549393 TI - Feeding and treatment: where to draw the line. PMID- 7549394 TI - Project launches major boost for learning disability nurses. PMID- 7549395 TI - Preoperative visiting from the perspective of the theatre nurse. AB - Many issues surround the concept of preoperative visiting. This article explores these issues in the light of recent research findings which question the benefits of using 'psychological' techniques to reduce preoperative anxiety, and examines the strategies in current use. PMID- 7549396 TI - Handwashing: the fundamental infection control principle. AB - This article examines the evidence linking handwashing practices with hospital acquired infections, the basis for the selection of an appropriate handwashing agent, and ways in which health-care workers may be encouraged to comply with handwashing. PMID- 7549397 TI - Behavioural treatments in post-traumatic stress disorder. AB - Post-traumatic stress disorder (PTSD) is increasingly becoming recognised as a serious mental health problem (Department of Health, 1991). Psychological treatments for PTSD remain in their infancy, though limited research has demonstrated the efficacy of behavioural and cognitive-behavioural interventions. PMID- 7549398 TI - Will the nurse practitioner be a mini doctor or a maxi nurse? PMID- 7549399 TI - The UKCC's criteria for specialist and advanced nursing practice. AB - This article examines the development of the UKCC's position on specialist and advanced practice. It considers the evolution of the work within the overall context of postregistration, education and practice. PMID- 7549400 TI - The politics of specialist/advanced practice: conflict or confusion? AB - This article explores the discrepancies between bench marks and the ad hoc development of specialisms. It suggests an agenda for remedying the invisibility of nursing in the shaping of health-care policy. Particular reference is made to the need for nurses to shape future policy on how the nursing workforce should be developed and utilised. PMID- 7549401 TI - Setting up nurse practitioner roles: issues in practice. AB - This article examines the process of developing a nurse practitioner role. Areas which must be considered before and during the implementation of such a role are general and may be applied to any area of practice, although examples here are from the author's own practice. PMID- 7549402 TI - Nebulised therapy and nursing knowledge. AB - Authoritative training from a multidisciplinary resource provides nurses with an excellent breadth and depth of knowledge. This study examines the impact of such training on nurses' knowledge of nebulised therapy and shows an encouraging improvement. Follow-up measures are also described. PMID- 7549404 TI - Student nurses must be supported. PMID- 7549403 TI - Common problems in wound care: malodorous wounds. AB - Malodour is a problem for patients with various types of wounds. The problem has a physical and psychological component. Accurate assessment of the cause will facilitate treatment with wound management products that are designed to alleviate the odour. PMID- 7549405 TI - Is marketing an obligatory skill in the nursing role? AB - The introduction of an internal market to the NHS is altering the culture to one that is more customer-oriented. This article argues that nurses, whether in the public or private sector, need to develop marketing skills in order to function effectively. PMID- 7549406 TI - Law series: 12. Children: suspected child abuse. PMID- 7549407 TI - Images of the Jewish body: three medical views from the Jewish enlightenment. PMID- 7549408 TI - The campaign for medical microscopy in antebellum America. PMID- 7549409 TI - Trains, brains, and sprains: railway spine and the origins of psychoneuroses. PMID- 7549410 TI - "Knocking out the cholera": cholera, class, and quarantines in New York City, 1892. PMID- 7549411 TI - Systemic and regional haemodynamic effects of caffeine and alcohol in fasting subjects [corrected]. AB - It is well established that caffeine and alcohol can have acute effects on heart rate and blood pressure, but it is not known whether cardiac output and peripheral blood flow are also affected. Such information is needed before any possible effects of caffeine or alcohol on the haemodynamic responses to food can be assessed. Thus, the present study determined the haemodynamic effects of caffeine or alcohol. Eight healthy young subjects were studied in the fasting state before and after ingestion of water (200 ml), coffee containing caffeine (3.5 mg/kg in 200 ml) or alcohol (0.5 g/kg made up to 200 ml), in random order on three separate occasions. All of the subjects had abstained from both caffeine and alcohol for at least 12 h. Measurements of blood pressure (BP), heart rate (HR), cardiac output (CO), superior mesenteric artery blood flow (SMABF) and calf blood flow (CBF) were made at baseline and for 2 h after each drink. With the alcohol drink, blood alcohol levels estimated using breath measurement peaked at 18.8 mmol/l. HR fell slightly after water but increased after alcohol (+ 7 beats/min) and after caffeine (+ 16 beats/min, group x time interaction analysis of variance, p < 0.03), with peak responses at 40-60 min and a return to baseline by 120 min. Systolic BP increased significantly with time after all three drinks, with no significant differences between the drinks. CBF rose slightly after the drink of water, more so after alcohol but fell slightly after caffeine (group x time interaction analysis of variance, p < 0.01). PMID- 7549412 TI - Daily variability in resting levels of cardiovascular variables in normal subjects and those with homozygous sickle cell disease. AB - Measurements were made of cardiovascular variables and oral temperature in 16 male subjects with homozygous sickle cell disease (SS) and in 17 matched controls (AA) at 10.00 a.m., 1.00 p.m. and 4.00 p.m. All subjects were in a rested state throughout. At 10.00 a.m., mean arterial pressure was lower, while heart rate, total forearm blood flow and cutaneous red cell flux in the forearm were higher in SS than AA. Vascular resistance in total forearm and forearm skin, calculated by dividing arterial pressure by blood flow or red cell flux, were lower in SS but hand cutaneous red cell flux and vascular resistance were not significantly different in SS and AA. In both SS and AA, there were parallel increases over the three sessions, in mean arterial pressure (by approximately 12 and 10%, respectively) forearm vascular resistance (by approximately 17 and 27%) and hand cutaneous vascular resistance and hand cutaneous resistance (by approximately 2240 and 350%) whereas forearm blood flow and hand cutaneous red cell flux fell. By contrast, forearm cutaneous resistance showed no change during the day in SS, but increased progressively in AA (by approximately 75%). These results indicate that, during the day, there is progressive vasoconstriction in forearm muscle and hand skin in SS and AA and also in forearm skin of AA that contributes to a progressive rise in the resting level of mean arterial pressure. We suggest this daily variability should be considered in studies of cardiovascular function: within a given study they should be performed at the same time of day. PMID- 7549413 TI - Changes in the sweatspot test with ageing and relation to cardiovascular autonomic function. AB - The sweatspot test assesses the local sweat response to an intradermal injection of acetylcholine. It has been reported as a more sensitive indicator of autonomic dysfunction in important diabetic men than either pupillary or cardiovascular tests, and has been used to establish the presence of autonomic dysfunction in patients with idiopathic chronic constipation. However, the usefulness of this test as a simple and quick method of diagnosing autonomic dysfunction in an elderly population has not been established. This is important given the high prevalence of reduced autonomic function with ageing and hypertension. We compared the age-associated responses in the sweatspot test and its relation to cardiovascular autonomic function in elderly normotensive and hypertensive subjects. We studied eleven normotensive and 24 untreated hypertensive elderly subjects (mean age 75.7 years, range 63-85) and compared the results of the sweatspot test to a young control group (n = 11, mean age 32.0 years, range 25 41), and to a standard battery of cardiovascular autonomic function tests. The median sweatspot score was significantly lower in the elderly compared with young subjects (1.9 vs. 12.0, p < 0.0001) although there was no difference between elderly normotensive and hypertensive subjects (1.6 vs. 2.4, p = 0.8). No correlation was demonstrated between the median sweatspot score and the number of abnormal cardiovascular tests. The sweatspot test was grossly abnormal in all elderly subjects and was not correlated to changes in cardiovascular autonomic function. Its diagnostic use in the elderly is therefore of very limited value. PMID- 7549415 TI - Cardiovascular reactivity to a new mental stress test: the maze test. AB - The response to the maze test was studied in 31 subjects on no medication, including seven subjects with mild essential hypertension. Beat-by-beat finger blood pressure (BP) was measured with a Finapres device. Pulse interval from which heart rate (HR) was derived was obtained from the BP signal. A period of 5 min baseline was used to define resting levels of systolic BP and HR using 20-s averaged values for the calculation of the 95% confidence limit of the two variables. The average test duration was 10 min. During this period a series of video-displayed mazes had to be solved with easy mazes preceding difficult ones. No subject had been exposed to the test before. The maximal BP elevation associated with the test was 27 mmHg. The HR response averaged 11 beats/min. Significant elevations were observed for half the test duration. No significant relationship between these two responses was observed and some subjects exhibited isolated systolic BP or HR responses. When indices obtained during this active mental stress were compared to resting values, positive correlations were observed. When the resting standard deviation (SD) of systolic BP was higher the systolic response was greater. A similar relationship was observed for HR. In addition, when the systolic BP level at rest was higher, the systolic BP reactivity index was greater. However, systolic BP or HR at baseline were not predictive of the systolic BP or HR responses. This study demonstrates the applicability of a new test to assess cardiovascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549414 TI - Vagal tone is reduced during paced breathing in patients with the chronic fatigue syndrome. AB - Patients with chronic fatigue syndrome (CFS) often complain of an inability to maintain activity levels and a variety of autonomic-like symptoms that make everyday activity intolerable at times. The purpose of the study was to determine if there were differences in vagal activity at fixed breathing rates in women with CFS. Twelve women with the diagnosis of CFS between the ages of 32 and 59 years volunteered for the study. Healthy women, who were between the ages of 30 and 49, served as controls. Full signal electrocardiograph and respiratory signals were collected during a paced breathing protocol of three fixed breathing rates (8, 12 and 18 breaths/min) performed in the sitting and standing postures. Vagal activity was analyzed by means of heart rate spectral analysis to determine the subject's response to specific breathing rates and postures. Heart rate variability was used as a non-invasive method of measuring the parasympathetic component of the autonomic nervous system. Using this method, although there was significantly less vagal power in the sitting versus the standing postures for both groups, the overall vagal power was significantly lower (p < 0.034) in the CFS group versus healthy controls. Vagal power was also significantly lower (p < 0.01 to p < 0.05) at all breathing rates in both postures except while standing and breathing at 18 breaths/min. Knowledge of the differences in vagal activity for CFS patients may allow stratification for the analysis of other research variables. PMID- 7549416 TI - Comparison of autonomic function using Valsalva ratio and 30:15 postural ratio prior to and after haemodialysis treatment. AB - In published studies of chronic haemodialysis patients, the frequency of autonomic dysfunction varies widely. One reason for the variation may be the time of testing with respect to time of dialysis. The current study tests the hypothesis that autonomic function--as measured by heart rate responses to the Valsalva manoeuvre (Valsalva ratio) and 30:15 electrocardiogram (ECG) R-R interval to upright posture (postural ratio)--is different when patients are above 'dry weight' (predialysis) than when they are at or below dry weight (postdialysis). The study also reviews available literature to analyze other factors that may affect the results of autonomic testing in this population. A total of 25 chronic haemodialysis patients underwent standard Valsalva and 30:15 R-R interval postural autonomic testing prior to and after haemodialysis. In addition, pre- and postdialysis orthostatic responses were measured and compared with a control population. The 30:15 ratio increased after dialysis (p = 0.001). The Valsalva ratio did not change with dialysis. Out of 25 subjects, seven had an abnormal 30:15 ratio prior to dialysis decreasing to two out of 25 patients postdialysis (p < 0.03). Orthostatic responses predialysis did not differ from those in the control group. Review of the literature shows great variability in definition of normal Valsalva and postural (30:15 R-R interval) ratios. Diabetic patients in the current and prior studies were more likely to have abnormal responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549418 TI - The effects of vasoactive intestinal polypeptide and substance P on methacholine induced sweating and vascular flare in diabetic neuropathy. AB - Vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity are reduced in the cutaneous nerves of diabetic patients with peripheral neuropathy. The functional significance of this finding was studied by measuring the forearm sweat response to intradermal methacholine and the effect of coadministration of VIP and SP in six normal subjects, and in six diabetic patients with neuropathy and eight without. Flare responses to the two peptides were also measured. Methacholine-induced sweat output was significantly greater in neuropathic patients compared with the other groups (p < 0.05), suggesting upper limb denervation supersensitivity. VIP and SP alone did not evoke sweating in any subject. Injection of VIP or SP reduced methacholine-induced sweating to a similar degree in all groups, except that the reduction was smaller in the non neuropathic group than in the others (p = 0.028 versus normal subjects, p = 0.014 versus neuropathic diabetic patients). Flare responses to the peptides were markedly reduced in the neuropathic patients compared with the other groups (p < 0.01). In neuropathic patients, increased sweat responses and decreased flare coexist with diminished neurophysiological measurements; cutaneous sweating and flare responses provide valuable additional information to conventional methods of neurological assessment in diabetic neuropathy. PMID- 7549417 TI - Predicting cardiac autonomic neuropathy in type I (insulin-dependent) diabetes mellitus. AB - A total of 24 subjects with type I insulin-dependent diabetes mellitus were studied. Cardiac parasympathetic function was measured by supine heart rate variability (HRV) in the respiratory frequency 0.10-0.50 Hz and the sympathetic index was measured as the ratio of HRV between 0.055 and 0.098 Hz to that between 0.004 and 0.5 Hz. Factors assessing diabetic control and complications, and factors unrelated to diabetes but possibly influencing HRV, were recorded. Association with depressed HRV was assessed with correlation, and prediction of depressed HRV was determined with multiple regression. Factors associated with depressed HRV but not independently predictive were renal dysfunction and elevated thyroid stimulating hormone. Elevated glycosylated haemoglobin was not significantly correlated with depressed HRV. Four factors (presence of diabetic retinopathy, male gender, duration of diabetes and increasing age) were significant in the regression and sufficed to predict 81% of the sample variance. The relative weights (beta) were -0.65, 0.40, -0.40 and 0.26, respectively. Supine sympathetic index was not sufficient to demonstrate sympathetic dysfunction. It is proposed that the regression model may be used to identify patients likely to have cardiac parasympathetic autonomic dysfunction. PMID- 7549419 TI - Hyposensitivity of peripheral alpha-adrenoceptors in respirator-dependent amyotrophic lateral sclerosis assessed by intravenous norepinephrine infusion. AB - Intravenous norepinephrine infusion test was performed in eight patients with amyotrophic lateral sclerosis (ALS) supported by respirators and nine control subjects, to examine alpha-adrenoceptor function of peripheral resistant blood vessels. Baseline plasma norepinephrine concentrations in ALS patients were significantly higher than those in control subjects, indicating basal sympathetic hyperactivity (normal 218.2 +/- 59.7 pg/ml; ALS 450.0 +/- 288.4 pg/ml). The stimulus-response curves in the patients were similar to those in control subjects, and there were no significant differences between mean gains of the stimulus-response curves in both groups (normal 18.7 +/- 5.5; ALS 15.2 +/- 11.2). However, three ALS patients, two of whom had circulatory fluctuation and sympathetic hyperactivity, revealed lower gain levels than the mean minus 2 SD in control subjects (4.7, 1.1 and 3.7). This indicates hyposensitivity or down regulation of the alpha-adrenoceptor function of peripheral blood vessels in these ALS patients. For early detection of sympathetic hyperactivity and prediction of circulatory collapse, it would be useful to measure the plasma norepinephrine concentration and the gain of the norepinephrine infusion curve in respirator-dependent ALS patients. PMID- 7549421 TI - Energy expenditure and anaemia of autonomic failure. PMID- 7549420 TI - The effects of clonidine on cardiovascular responses to standing in healthy volunteers. AB - This study assessed the effects of clonidine on blood pressure (BP) and heart rate responses to active standing, recorded continuously using a Finapres monitor. Ten subjects were given a placebo infusion over 1 h, followed by clonidine hydrochloride 1.5 micrograms/kg over 2 h. During placebo and at 1, 3 and 19 h following the clonidine infusion, heart rate and blood pressure were recorded during the second half of supine rest for 10 min, active standing and quiet standing for 7 min. Clonidine did not alter the size of immediate drop in BP on standing, although the nadir was lower. BP recovery was impaired, with a loss of the usual BP overshoot in most subjects and with delays in reaching supine levels of diastolic BP (6.1 versus 9.6 s; p < 0.01) and systolic BP (8.1 versus 12.3 s; p < 0.05). The compensatory initial heart rate rise was significantly increased from 47 to 53 beats/min (p < 0.05), although the peak rate reached was reduced from 114 to 104 beats/min (p < 0.05). These results demonstrate that impairment of central sympathetic vasomotor drive leads to a delay in BP recovery and loss of initial BP overshoot immediately after standing, together with impaired maintenance of early steady-state BP. PMID- 7549422 TI - Positional cloning reaches maturity. AB - The identification of genes involved in human genetic disease is no longer the province of those who would make a career of 'not finding' a gene. Developments from the human genome initiative have vastly facilitated the process of localizing genetic intervals segregating mutations, as well as that of obtaining the physical regents necessary for characterizing the region. In a few years' time, efforts aimed at the assignment of genes to the physical map, coupled with increasing quantities of sequence data from both cDNA and genomic sources, will provide numerous candidate genes for analysis, with consequences for the approaches used to define the gene and mutations(s) involved in the disease of interest. PMID- 7549423 TI - FISH in genome research and molecular diagnostics. AB - Fluorescence in situ hybridization (FISH) has profoundly altered the aspect of genome research and molecular diagnostics. Deletions of only a few kilobases can be detected by hybridizing probes to naked DNA fibers. Loss or gain of chromosomal material in tumor cells can be visualized using comparative genome hybridization. Further diversification of FISH application will result from new ultrasensitive detection techniques. PMID- 7549424 TI - CpG islands and genes. AB - Of the estimated 45,000 CpG islands in the human genome, the overwhelming majority are found at the 5' ends of genes and their identification and cloning are proving very useful for finding and isolating genes. Recent work has shed light on the chromosomal distribution and origin of CpG islands. It has been shown unequivocally that CpG islands are concentrated in the R band chromosomal regions and that intact transcription factor binding sites and required for their maintenance. Cases of methylation of CpG islands and inactivation of the associated genes have been reported which may be important in ageing, tumorigenesis and imprinting. PMID- 7549425 TI - Human genome organization. AB - Recent advances have been made in addressing three intriguing aspects of human genome organization: the organization of protein-coding sequences within chromosomes, the structural basis of the metaphase chromosomal banding pattern, and the function of non protein coding DNA. At the cytogenetic level, R band heterogeneity has been examined by fluorescence in situ hybridization using complex fractions of genomic DNA as probes. DNA fractionated according to GC content and CpG is island density both generated patterns related G and R bands and directly demonstrated regional variations in gene densities. A structural basis for metaphase bands has been proposed that is based on the differential size and packing of DNA loops and matrix attachment sites in G versus R bands. The model presents interesting opportunities for structure/function and organization investigations. At the molecular level, the human genome initiative has resulted in extensive genomic clone coverage for many large chromosomal regions, permitting detailed documentation of CpG islands, base composition and repeat sequence context, as well as fueling comprehensive gene searches. Sequence and functional characteristics are being examined at the kilobase level and are prompting new suggestions of roles for 'junk' DNA. Because of these developments, opportunities are now emerging for direct assessment of the molecular characteristics of individual metaphase bands. PMID- 7549426 TI - The molecular basis of fragile sites in human chromosomes. AB - Fragile sites on chromosomes have been classified into a number of groups according to their frequency and the conditions required to induce them. A number of the rare folate-sensitive fragile sites have been characterized and shown to be amplified methylated CCG trinucleotide repeats. One common fragile site has been partly characterized and appears to be a region of fragility, rather than a single site. PMID- 7549428 TI - Novel approaches to linkage mapping. AB - The recent progress seen in the development of high-quality genetic markers and high-density human genetic maps is facilitating new strategies for mapping disease-causing and disease-susceptibility genes. These strategies, which include homozygosity mapping, DNA pooling, and the utilization of linkage disequilibrium, have been successfully applied to the mapping and fine mapping of several loci causing diseases segregating in genetically isolated populations. PMID- 7549427 TI - Mapping human chromosomes. AB - The construction of integrated maps at all levels of resolution will facilitate determination of the DNA sequence and, ultimately, the entire gene content of the human genome. In the past two years, the need for extensive frameworks on which to anchor the maps of the human chromosomes has been emphasized. The first framework has been provided following construction of the genetic map using microsatellite markers. This is now being united with extensive collections of expressed sequence tags and other landmarks using radiation hybrid mapping into higher resolution map. Rapid progress is also being made towards integrating these reagents through the construction of physical clone maps using yeast artificial chromosomes which provide near-complete cloned coverage of human chromosomes. PMID- 7549429 TI - YAC transgenics and the study of genetics and human disease. AB - Advances in yeast artificial chromosome (YAC) technologies over the past decade have enabled the precise identification and manipulation of large genomic regions (>100 kb) of DNA. Introduction of YACs into the mouse germline has now been accomplished through transfection of mouse embryonic stem cells as well as through pronuclear microinjection, allowing the efficient transfer defined genomic loci into mice. YAC transgenics will have a profound impact on the development of transgenic mice as bioreactors and as models of human disease, and on the functional analysis of higher order genomic structure. PMID- 7549430 TI - An update and lessons from whole-genome sequencing projects. AB - A number of prokaryotic and eukaryotic genomes are currently being sequenced. Already, the nucleotide sequences of four yeast chromosomes and of 2.2 Mb from Caenorhabditis elegans have been reported. Human genomic sequences have also been used in comparative studies with both mouse and Fugu rubripes. PMID- 7549431 TI - Finding genes involved in human developmental disorders. AB - Recent advances in the human genome initiative have accelerated positional cloning efforts toward identification of a number of genes responsible for human developmental anomalies, particularly those involving the skeletal system. Genotype/phenotype comparison and functional analysis of these genes will further elucidate pathways of normal and abnormal human development of the skeletal and other organ systems. PMID- 7549432 TI - The genetics of hypertension. AB - In the past few years, a number of key insights have been made concerning the genetic basis of hypertension and blood pressure regulation. The genes responsible for two Mendelian forms of hypertension, glucocorticoid-remediable aldosteronism and Liddle's syndrome, were identified. In addition, research into the role of the renin-angiotensin system in blood pressure regulation has further implicated the angiotensinogen and angiotensin-converting enzyme loci in hypertension and its complications, such as myocardial infarction. Finally, several new candidate genes for hypertension have been identified through the use of genome scanning and contemporary gene expression assays in model organisms. PMID- 7549433 TI - Mapping and cloning hereditary deafness genes. AB - In the past two years, considerable progress has been made in the mapping and cloning of human deafness genes. Highlights are the chromosomal localization of at least five genes for autosomal forms of non-syndromic deafness and, more recently, the cloning of an X-linked deafness gene, DFN3, and the Usher syndrome type IB gene. This last gene encodes a myosin-like protein and was identified as the human homolog of the mouse shaker-1 gene. The DFN3 gene Brain 4 encodes a POU domain containing transcription factor that is involved in the development of the inner ear. PMID- 7549434 TI - Mapping genes for psychiatric disorders and behavioral traits. AB - In the past year, some of the most exciting findings in the genetic investigation of mammalian behavior have been obtained through mapping and through gene manipulation studies in the mouse system. These include the localization of a gene for circadian periodicity in the mouse, gene knockouts of serotonin receptors, and the development of a transgenic model of Alzheimer's disease. The recent development of genetic maps covering the entire human genome and the implementation of new approaches to genetic analysis may now facilitate elucidation of complex behaviors in humans, particularly psychiatric disorders. PMID- 7549435 TI - Identification of mismatch repair genes and their role in the development of cancer. AB - Mismatched base pairs are generated by damage to DNA, by damage to nucleotide precursors, by errors that occur during DNA replication, and during the formation of intermediates in genetic recombination. Enzyme systems that faithfully repair these DNA aberrations have been identified in a wide variety of organisms. At lease some of the components of these repair systems have been conserved, both structurally and functionally, throughout evolutionary time. In humans, defective mismatch repair genes have been linked to hereditary nonpolyposis colon cancer as well as to sporadic cancers that exhibit length polmorphisms in simple repeat (microsatellite) DNA sequences. The involvement of mismatch repair defects in microsatellite instability and tumorigenesis suggests that a generalized mutator phenotype is responsible for the large number of genetic alterations observed in tumors. PMID- 7549436 TI - Gene therapy: a long and winding road. PMID- 7549437 TI - Genetics of disease. PMID- 7549438 TI - Age-dependent phenotypic expression of a pattern dystrophy of the retina. AB - We studied a family with pattern dystrophy of the retina (PDR) in order to elucidate the clinical course of the disorder, relations between the different forms, and the mode of inheritance. Thirty-nine family members, representing three generations, underwent a thorough ophthalmological examination, with fluorescein angiography whenever a macular abnormality was suspected. Of family members over the age of 32 years, 46.7% showed signs of PDR. We classified the clinical forms of PDR in this family into four types: minimal lesion, pseudovitelliform type, butterfly-spider type, and late-stage lesion. These forms were predominant in the order cited in age groups 31-40 years, 41-50, 51-60, and > 60 years. Significant visual loss occurred only after the age of 50 years, when 8 of 14 eyes had visual acuity of less than 20/25. Inheritance was autosomal dominant. PDR presented different clinical forms in members of this family, and in successive age classes patterns of increasing severity prevailed. Thus, the different phenotypic forms apparently represent transient stages related to the age of the patient. PMID- 7549439 TI - Management of posterior segment foreign bodies and long-term results. AB - The management of 158 posterior segment intraocular foreign bodies (IOFB) was retrospectively analyzed: transscleral magnet extraction via the pars plana was used for 40 magnetic IOFB, transscleral extraction via the IOFB bed for 35 magnetic and 4 non-magnetic IOFB, pars plana vitrectomy (PPV) for 44 magnetic and 32 non-magnetic IOFB, and open-sky vitrectomy for 3 non-magnetic IOFB. Final visual acuity of 0.02 and better was achieved in 104 out of 119 magnetic IOFB (87%) and 24 out of 39 non-magnetic IOFB (62%), and final visual acuity 0.05 and better in 79 magnetic IOFB (66%) and 17 non-magnetic IOFB (44%). Transscleral extraction via the IOFB bed under ophthalmoscopic control and IOFB removal by PPV proved to be the operations of choice for an increasing number of IOFB. For magnetic IOFB, these techniques yielded better final functional results than transscleral magnet extraction via the pars plana. Final visual acuity did not depend on the interval between injury and IOFB removal, and with regard to the risk of endophthalmitis, IOFB need not be considered an absolute indication for immediate intervention. IOFB size up to 5 mm2 and initial visual acuity of 0.5 and better were significant positive factors for both magnetic and non-magnetic IOFB. PMID- 7549440 TI - Lp(a) is not related to retinopathy in diabetic subjects. AB - PURPOSE: To examine the association between Lp(a) concentrations and the severity of retinopathy in 22 younger-onset and 48 older-onset diabetic subjects from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), a population-based study of diabetic retinopathy. METHODS: We used a subset of the WESDR population with standardized protocols and stereoscopic color fundus photography to determine the severity of diabetic retinopathy in relation to Lp(a) concentrations. Lp(a) concentrations were measured by a monoclonal anti-Lp(a) antibody. RESULTS: Lp(a) levels were not significantly different between younger onset or older-onset subjects with and without retinopathy. CONCLUSION: Our results do not support a link between higher levels of Lp(a) and severe retinopathy in either younger-onset or older-onset diabetic subjects but this needs confirmation in larger prospective studies. PMID- 7549441 TI - The behavior of two cell lines obtained from murine (B16-F10) and human (G-361) melanomas implanted into the eyes of New Zealand white rabbits, and their microscopic, immunohistochemical and ultrastructural appearances. AB - A transcleral inoculation of 15x10(6) melanocytes of the B16-F10 and G-361 cell lines was carried out in the anterior chamber of one eye in New Zealand white rabbits. Tumor growth occurred in 24 eyes (77%) of the B16-F10 group and in 22 (73%) of the G-361 group. The tumors of the latter group were mostly amelanic and showed local aggression with ocular perforation and extrascleral growth one month post-implant, while the tumors of the B16-F10 group were intensely pigmented and grew over the iris although they did not perforate the eyeball. Microscopically, the tumors of both groups were of the mixed type, made up of epithelioid and fusiform melanocytes. S-100 protein and Nki C3 monoclonal immunohistochemical techniques showed a positive immunoreaction in all cases of tumor growth. Ultrastructurally, the G-361 melanocytes showed a few melanosomes corresponding to maturity state II and, occasionally, state III, while totally melanized state IV cells predominated in the B16-F10 group. Aberrant melanosomes were common in both groups. The inoculation of 15x10(6) melanocytes of either cell line was useful to produce ocular melanomas. PMID- 7549442 TI - Penetration of [3H] tiazofurin into guinea pig eye by a saturable mechanism. AB - This study investigated the transport of tiazofurin (2-beta-D-ribofuranosyl thiazole-4-carboxamide) across the blood-aqueous humor barrier, using the vascular perfusion method in the guinea pig. Volume of distribution (Vd) of [3H] tiazofurin increased almost linearly in time, from 4% of its plasma concentration at 3 min to 10% after 12 min of perfusion. Unidirectional transport constant, K(in) was 7.01 +/- 1.06 x 10(-3) ml/min/g. These results indicate that tiazofurin penetrates the aqueous humor to a considerable extent. Addition of unlabelled tiazofurin to the perfusing medium caused a significant decrease in the uptake of [3H] labelled tiazofurin (K(in) = 2.60 +/- 0.91 x 10(-3) ml/min/g). Therefore, penetration of tiazofurin from blood into aqueous humor seems to be a saturable process with a diffusional component that cannot be disregarded. Such findings could be of considerable importance since this molecule is known to affect tissue metabolism. PMID- 7549443 TI - Drug induced myopia associated with treatment for gynecological problems. AB - We present three cases of women who developed acute transient myopia caused by drugs for gynecological problems. One patient was treated with disothiazide for premenstrual edema. The second had acute cystitis and was treated by sulphonamide and the third developed myopia coincident with metronidazole treatment for trichomonas vaginalis. We followed these patients by A scan ultrasonographic ocular measurements, documenting reduction of the anterior chamber depth combined with lens thickening. The ocular and myopic changes cleared up completely on discontinuation of the causative agent. The possible mechanism is thought to be an allergic reaction to the drug. PMID- 7549444 TI - Retinal pigment epithelium tear following toxemia of pregnancy. AB - A 28-year-old woman with toxemia during the 34th week of pregnancy complained of visual loss in one eye. Two weeks after abruptio placentae and delivery a retinal pigment epithelium (RPE) tear which presumably followed a RPE detachment (PED) was found in the macular region of the right eye. Visual acuity was 0.8 at the time of diagnosis but was 1.0 in both eyes one year after the delivery. Some authors have described peculiar choroidal lesions associated with central serous chorioretinopathy (CSC) in pregnancy and ischemic exudative choroidal lesions during severe toxemia. However, the findings we describe are highly unusual, indicating that the mechanisms of choroidal and RPE pathology are still poorly understood. We also demonstrate that a pathological tear following toxemia of pregnancy is compatible with good acuity in spite of the anatomical damage. PMID- 7549445 TI - Morphological and biochemical assessment of the cornea in a Gaucher disease carrier with keratoconus. AB - Ocular abnormalities such as corneal opacities and some specific alterations in ocular movements have been described in the neuropathic forms of Gaucher disease. This study was designed to correlate the clinical, morphological and biochemical findings in the corneal button obtained after keratoplasty in a Gaucher disease carrier with keratoconus. Morphologically, the cornea showed keratocytes with marked dilatations of the rough endoplasmic reticulum and intracytoplasmic "dark inclusions"; the acidic lipid profiles presented alterations in the cornea of the Gaucher disease carrier when compared with healthy controls and a clear deficiency in beta-glucosidase activity was detected as well. Our data suggest that the cornea may serve as a good marker of an early target organ in lipid metabolism disorders such as Gaucher's disease. PMID- 7549446 TI - Ocular pathology in light chain deposition disease. AB - Light-chain deposition disease (LCDD), a rare form of monoclonal gammopathy, is characterized by deposits of amorphous light-chain material, mainly in the kidneys but also in various other organs. Here we present the first report of a light-, electron microscopic and immunohistochemical study of the globes of a patient suffering from LCDD secondary to multiple myeloma. Massive deposits of kappa light chains similar to those typically present in the kidneys were found beneath the basement membrane of the ciliary pigment epithelium, on vessels of the ciliary body, within the collagenous zones of Bruch's membrane, and in the innermost part of the choroid. The choriocapillaris in the macular area was partly obstructed by these deposits, and an exudative retinal detachment was present. Whether this detachment was the consequence of disturbed circulation of the choriocapillaris remains speculative. PMID- 7549447 TI - Ciprofloxacin ophthalmic solution versus rifamycin ophthalmic solution for the treatment of conjunctivitis and blepharitis. AB - The efficacy and safety of ciprofloxacin (0.3%) ophthalmic solution and rifamycin (1%) ophthalmic solution for the treatment of bacterial conjunctivitis and blepharitis was compared in this randomized, double-masked, parallel-group study. Forty-one patients, 19 on ciprofloxacin and 22 on rifamycin, were culture positive on admission and evaluated for efficacy. There was clinical improvement in more than 90% of patients in each group at the end of the seven-day treatment period. However, clinical cure rates on day 7 appeared to be higher with ciprofloxacin (53%) than rifamycin (23%; p = 0.061, Mann-Whitney test). Bacteriological eradication rates were comparable: the infecting organisms were eradicated in 68% of patients on ciprofloxacin and 77% with rifamycin. There were no serious adverse reactions to either treatment. One patient in each group was withdrawn on account of a mild allergy. In conclusion, topical ciprofloxacin was effective and well tolerated and would be a particularly useful agent for the treatment of bacterial conjunctivitis and blepharitis. PMID- 7549448 TI - Systemic blood pressure and capillary blood-cell velocity in glaucoma patients: a preliminary study. AB - Systemic hypotension and vasospasm have both been suggested as risk factors for glaucomatous damage. Furthermore, a relationship between the incidence of vasospastic disorders and systemic hypotension has been reported. This preliminary study investigated the relationship between these two risk factors. In 20 glaucoma patients suspected to have vascular risk factors, time of blood flow standstill was measured under cold provocation in nailfold capillaries and 24-hour blood pressure was monitored. Nine were high-tension glaucoma patients but progressing despite medically well-controlled intraocular pressure, and 11 had normal-tension glaucoma. Thirteen patients were vasospastic, and seven were not. Patients without vasospasm had a significantly lower mean systolic blood pressure during the daytime (113.4 +/- 7.1 mmHg) than vasospastic patients (122.6 +/- 12.8 mmHg), p = 0.026. The time of blood-flow standstill in vasospastic patients, however, correlated with the lowest individual systolic blood pressure reading (r = -0.56; p = 0.049). These low readings occurred mostly during the night ("deepers"). PMID- 7549449 TI - PERG and P-VEP after surgical trabeculectomy in primary open-angle glaucoma. AB - Pattern-reversal electroretinograms (PERG) and visual evoked potentials (P-VEP) were measured for 10 eyes from 10 rigorously selected patients with bilateral advanced primary open angle glaucoma (POAG), before and after surgical trabeculectomy. The aim was to establish whether electrofunctional examinations improved after major IOP reduction. Only one eye at random was operated, and the fellow eye was used as control. The aim of this study, using electrofunctional examinations, was to clarify whether ganglion cell damage was reversible after marked reduction of IOP by surgery. The results indicate that glaucomatous damage seems to be irreversible. PMID- 7549450 TI - Correlation of microcirculation architecture with ultrasound backscatter parameters of uveal melanoma. AB - Ultrasound tissue characterization parameters of size and acoustic concentration can be used to sub-classify uveal melanoma and estimate the patient's risk of death. Histologically determined microcirculation patterns have also been found to sub-classify uveal melanoma and predict patient's risk of death. This study examines the spatial relationship between tumor features detected by the two techniques. Three dimensional ultrasound images that depict the size and relative concentration of scattering elements in uveal melanoma within a range of approximately 50-120 microns were compared with PAS (without hematoxylin) stained histological sections graded and localized according to tumor microvascular patterns. Both ultrasound parameter imaging and histopathology were accomplished by workers masked to the other procedure. In three of five patients vascular networks were identified histopathologically. The predominant ultrasound feature seen in the regions of the histologically identified networks were clusters of scatterers in the range of approximately 50-80 microns. In the two cases without a network vascular pattern, lower range scatterers dominated the tumor volume. All the cases could be distinguished by the size and distribution of contiguous spatial clusters of acoustic scatterers. Scatterer size features detected in three dimensional ultrasound parameter images can identify tumor regions containing a specific microvascular pattern. Ultrasound tissue characterization features used to sub-classify uveal melanoma may have a biophysical basis related to patterns of tumor microcirculation. PMID- 7549451 TI - Effects of metamfetamine on auditory P3 component of event-related potentials in rats. AB - Three rats were trained with an oddball paradigm, in which 5 kHz frequent and 10 kHz target rare tones were presented with a probability of 0.8 and 0.2. Pressing the lever within 2 s following the onset of the target tones was rewarded with a drop of food paste. Long latency event-related potentials (ERPs) corresponding to those in humans could be recorded in rats. After the administration of metamfetamine (0.2 mg/kg), the amplitude of surface and intracortical N1 increased, indicating an augmentation of the primary cortical response. Surface P3 showed a decrease of amplitude, accompanied by an increase of intracortical negativity. These alterations of ERPs may be caused by the dysregulation of central noradrenergic system. PMID- 7549452 TI - Decreased methionine adenosyltransferase activity in erythrocytes of patients with dementia disorders. AB - ATP:1-methionine S-adenosyltransferase (EC 2.5.1.6, MAT) activity was analyzed in erythrocytes from nine patients with a clinical diagnosis of probable Alzheimer's disease (Pro.AD), four with possible Alzheimer's disease (Pos.AD), three with mild cognitive dysfunction (MCD) and two with dementia of vascular origin (VD), and 10 age-matched control subjects. Significantly lower kinetic parameters (Vmax and Km towards methionine) for MAT were observed in all the dementia cases. In the subgroup of Pro.AD patients who also had low plasma levels of vitamin B12 (B12), the reduction in MAT Km was significantly correlated with an increase in the serum levels of homocysteine, while no such correlation was observed in all the other dementia groups. Treatment for 6 months of this subgroup of Pro.AD patients with B12 (1 mg x 7 days + 1 mg/week, i.m.), S-adenosylmethionine (SAM, 200 mg twice daily, p.o.) and folate (2.5 mg every 2 days, p.o.) caused a significant decrease in homocysteine in parallel with a significant increase in Km for MAT. These findings support the hypothesis that aberrations in the B12 dependent transmethylation reactions might be involved in the pathogenesis of dementia, and suggest that the evaluation of erythrocyte MAT activity may be a useful marker for the detection of such an aberration. PMID- 7549453 TI - Adrenalectomy does not prevent the ability of 8-OH-DPAT to decrease the ejaculatory threshold in male rats. AB - The present experiments demonstrate that 8-OH-DPAT (0.25 mg/kg SC, - 15 min) produced a decrease in the ejaculatory threshold to the same extent in adrenalectomized male rats as in sham operated controls. Both groups of animals displayed a marked and statistically significant decrease in number of mounts and penile intromissions preceding ejaculation and in the ejaculation latency, as a result of treatment with 8-OH-DPAT. Adrenalectomy per se did not affect any aspect of the male rat's sexual behavior (latency to first intromission, number of mounts or penile intromissions, ejaculation latency or the postejaculatory interval). The surgical removal of the adrenals was verified by measurements of plasma corticosterone levels. It is concluded that well documented effects of 8 OH-DPAT on adrenal secretions do not contribute to its ability to decrease the ejaculatory threshold in male rats. PMID- 7549454 TI - Social isolation and social interaction alter regional brain opioid receptor binding in rats. AB - Endogenous opioid systems have been implicated in the consequences of social isolation and in the regulation of social behavior, although their precise role is not clear. There is not much information on a possible locus in the brain at which opioids exert their effects on social behavior. In an effort to address this issue we analyzed regional opioidergic activity upon social isolation induced social interaction using in vivo autoradiography. Animals were either socially isolated for 7 days or group housed, and tested singly or in a dyadic encounter. Subsequently, a tracer dose of [3H]diprenorphine was administered and in vivo autoradiographic analysis was performed. Seven days of social isolation caused changes in both social behavior (dyadic encounters) and non-social behavior (singly tested animals). Opioid receptor binding was increased in the medial prefrontal cortex and the parafascicular area in isolates, suggesting that social isolation may evoke an upregulation of opioid receptors in these areas. Social interaction increased opioid binding in the parafascicular area of non isolated rats. In substantia nigra para compacta and ventral tegmental area binding was increased upon social isolation, and social interaction decreased opioid binding in isolates, but these changes failed to reach significance. These observed local changes in opioid receptor binding suggest a role for opioid systems in discrete areas in the consequences of social isolation and the regulation of social behavior in rats. PMID- 7549455 TI - Relationship between the Newcastle scale and sleep polysomnographic variables in major depression: a controlled study. AB - In order to investigate the reliability of the endogenous concept of depressive illness with some sleep EEG parameters, we studied 39 male inpatients suffering from a nonbipolar major depressive episode (15 endogenous (MDDE) and 24 nonendogenous (MDDNE)) and 20 age and sex matched normal controls (C). All patients were diagnosed according to the Research Diagnostic Criteria (RDC) and the endogenous character of the episode was assessed with the Newcastle Endogenous Depression Diagnostic Index. We found significant differences for the following variables between the three groups (MDDE, MDDNE and C): sleep period time (SPT), REM latency, stage II, slow wave sleep (SWS), REM latency expressed as a continuous variable and REM latency expressed as a dichotomizing variable with a threshold of 50 min. These variables were used to compare the endogenous and the nonendogenous depressed patients and also the major depressed patients and the normal controls. Significant differences were observed between all depressed patients and control subjects for amount of SWS and REM latency which were both reduced in endogenous and nonendogenous depressed patients. No significant difference was observed between endogenous and nonendogenous depressed patients, except for the REM latency expressed with a threshold of 50 min (more frequently observed in endogenous depressed patients). Our data support the observation that SWS and REM latency are decreased in major depressive patients. However, in this age and sex controlled study, subtyping nonbipolar major depressive disorder for an endogenous character by the Newcastle Endogenous Depression Diagnostic Index (NEDDI) did not reveal further significant differences for sleep EEG variables, except for the shortening of the REM latency expressed as a dichotomizing variable. PMID- 7549456 TI - Dimensional classification and behavioral pharmacology of personality disorders; a review and hypothesis. AB - Nosological orientation in psychiatry has severely hampered the progress of research in biological psychiatry, especially in the case of personality disorders. Dimensional approaches have redefined the characteristics of these disorders and their possible pathogenetic factors. The significance of arousal and stress, so far relatively neglected in clinical research, and its important function in adaptive and coping strategies has to be included in the study of the behavioral pharmacology of personality disorders. Some preliminary clinical data are available suggesting the potential therapeutic use of serotonin modulating agents in the key symptomatology of certain personality disorders such as disturbed impulsive regulation and increased stress reactivity. PMID- 7549457 TI - Delirium associated with mianserin. AB - Delirium may develop in vulnerable subjects under normal doses of psychotropic drugs. The occurrence of delirium in patients treated with tricyclic antidepressants is attributed to the anticholinergic activity of these drugs. Mianserin, characterized by low anticholinergic activity, has been recommended for treatment of depression in elderly patients. We report five cases of delirium in elderly patients treated with mianserin. The incidence of organo-mental side effects associated with this drug may be higher than previously reported. PMID- 7549458 TI - Guidelines on psychotropic drugs for the EC. Clinical investigation of medicinal products in the treatment of generalized anxiety disorder, panic disorder and obsessive-compulsive disorder. PMID- 7549459 TI - Role of D1/D2 dopamine and N-methyl-D-aspartate (NMDA) receptors in morphine tolerance and dependence in mice. AB - Chronic treatment with the dopamine (DA) agonist B-HT 920 (0.25-1 mg/kg) or bromocriptine (1 mg/kg) followed by morphine (10 mg/kg) on days 1-9 prevented the development of tolerance to the antinociceptive effect of morphine as measured by the tail-flick test in mice, but failed to suppress the development of morphine dependence as assessed by naloxone (2 mg/kg)-precipitated withdrawal jumps on day 10 of testing. Repeated administration of SKF 38393 (5 mg/kg) followed by morphine for 9 days significantly reduced naloxone-precipitated jumps on day 10 but failed to produce any significant change in tail-flick latency from the saline-pretreated group of mice on days 9 and 10 of testing. Repeated administration of B-HT 920 or bromocriptine enhanced the ability of MK-801 to attenuate the development of morphine tolerance and dependence while SKF 38393 failed to do so. The above data suggest a preferential role of D2 DA receptors in morphine tolerance and D1 receptors in the development of morphine dependence. D2 DA receptor stimulation may also play an important role in enhancing the effectiveness of MK-801 in the treatment of opiate tolerance and dependence. PMID- 7549461 TI - Effects of 6-hydroxydopamine lesions of the medial prefrontal cortex on local cerebral blood flow and D1 and D2 dopamine receptor binding in rats: a quantitative autoradiographic study. AB - The effects of lesions of the dopamine (DA) nerve terminals in the medial prefrontal cortex (MPFC) on local cerebral blood flow (LCBF) and DA receptor binding in rats were investigated. 4 micrograms of 6-hydroxydopamine (6-OHDA) was infused stereotaxically into the area of the bilateral MPFC of rats pretreated with desmethylimipramine, and control rats received a vehicle solution. Twenty four days after the operation LCBFs of 23 brain regions were measured using the quantitative autoradiographic N-isopropyl-p-[125I]iodoamphetamine technique D1 and D2 DA receptor binding in various brain regions was also quantified autoradiographically using [3H]SCH 23390 and [3H]YM 09151-2 as the respective ligands. 6-OHDA lesions of MPFC produced significant increases in LCBF of the nucleus accumbens, the dorsolateral portion of the caudate-putamen and the anterior cingulate cortex. The lesioned animals did not show decreased LCBF in MPFC per se. D1 and D2 DA receptor binding was not affected in any brain region examined. These results suggest that lesions of the DA nerve terminals in MPFC induce an enhancement of functional activity in the terminal regions of the subcortical DA systems, and that hypofunction of the mesocortical dopamine system does not elicit reduced metabolic activity in the prefrontal cortex. PMID- 7549460 TI - Subchronic morphine increases amphetamine-induced potentiation of brain stimulation reward: reversal by DNQX. AB - The ability of morphine and amphetamine to potentiate brain stimulation reward was studied in rats with monopolar electrodes in the medial forebrain bundle. Animals received seven daily injections of morphine (3 mg/kg, s.c.) or saline followed 10 min later by either DNQX (100 mg/kg, i.p.) or its vehicle. On the 8th day the self-stimulation (SS) response was examined 60 min or 30 min after the administration of either morphine (3 mg/kg, s.c.) or amphetamine (1.5 mg/kg, s.c.), respectively. It was found that the subchronic administration of morphine sensitizes animals to subsequent amphetamine-, but not morphine-induced activation of SS, which may be prevented by the coadministration of DNQX, an antagonist of the non-NMDA subtype of glutamate receptors. PMID- 7549462 TI - Inhibition of HSV-1 proliferation by decoy phosphodiester oligonucleotides containing ICP4 recognition sequences. AB - Transcriptional control in eukaryotes results from the interplay between DNA sequences in promoters, enhancers, or silencers and transcription factors. Selective control of gene expression can thus be achieved by inhibiting specific transcription factor/DNA interactions. Transcriptional activity of DNA binding transcription factors can be inhibited by competition with double-stranded oligonucleotides (decoys) that contain their specific recognition sequences. The immediate early protein ICP4 of herpes simplex virus type 1 (HSV-1) is a sequence specific DNA binding protein that is essential for viral replication. We synthesized double-stranded hairpin phosphodiester oligonucleotides carrying ICP4 sites and demonstrated their ability to specifically titrate ICP4. Upon addition to Vero cells, ICP4 hairpin decoys significantly reduced HSV-1 titers (IC50 = 0.3 microM), whereas a control hairpin oligonucleotide had no activity. Antiviral activity of ICP4 hairpin decoys was correlated to their relative binding affinities. These results show that phosphodiester oligonucleotides can compete for binding of specific transcription factors within cells, thus providing a potential therapeutic tool to control disease-causing genes. PMID- 7549463 TI - Structural constraints for DNA recognition by Myc and other b-HLH-ZIP proteins: design of oncoprotein analogues. AB - DNA recognition is a critical property of many transcription factors, some of which play important roles in human disease. Disruption of this recognition may profoundly influence the biology of these factors. One such factor, the Myc oncoprotein, utilizes a basic/helix-loop-helix/leucine zipper motif to recognize the DNA target CACGTG. As discussed here, this recognition appears to occur through recognition by one face of a basic region alpha helix utilizing amino acid side chains highly conserved among CACGTG binding proteins. This basic domain alpha helix, however, requires DNA binding for stabilization. To circumvent this energetic requirement, analogues were produced that introduce multiple alanines, displaying substantially increased spontaneous alpha helicity and significantly enhanced DNA affinity. These studies simplify our understanding of the structural constraints for DNA recognition by this family and may serve as a template for the design of small molecule transcription-targeted therapeutics. PMID- 7549465 TI - Gene expression as a target for new drug discovery. AB - Over the last 7 years we have carried out a major research effort focused on gene transcription as a novel approach to drug discovery. The goal is to identify small molecular weight compounds that modulate the expression of a target gene in a specific manner, thereby either increasing or decreasing the concentration of the corresponding protein product. Transcriptional modulation not only provides a potential means to replace recombinant proteins as drugs, but also provides a novel approach to manipulate key gene targets in many therapeutic areas. This article describes some of the features and advantages of transcription-based pharmaceuticals and illustrates how this approach can be applied to drug discovery with a program we are pursuing to identify new treatments for sickle cell disease and beta-thalassemia. PMID- 7549464 TI - Transcription factors as drug targets: opportunities for therapeutic selectivity. AB - Many traditional drugs target cell surface receptors. Medicinal chemists and pharmacologists have not ventured into the field of transcription regulation due to the fear that drugs that interfere with transcription regulation may not be selective or efficacious. The past 5 years have seen some exciting developments in the field of signal transduction in general, and transcription regulation in particular. Our understanding of mechanisms of regulated and basal transcription is advanced to a degree that it should be possible to selectively modulate a target gene directly. In this review we have argued that sufficient diversity exists in the combinatorial interplay of the transcription factors to offer opportunities for selective therapeutic intervention. We have focused our attention on transcriptional factors that play a role in three different therapeutic areas: osteoporosis, immune modulation, and cardiovascular diseases. Human estrogen receptor is considered as a model transcription factor. The role of estrogen in bone remodeling is discussed. Opportunities for tissue-specific modulation of estrogen receptors are described. For selective immune modulation, we have discussed the role of NF-AT (nuclear factors for activated T cells) transcription factors in interleukin-2 gene regulation. The last section focuses on the transcriptional mechanisms conferring tissue specificity in regulated expression of the apoAI gene, a major component of HDL, in liver. We have highlighted opportunities for rational development of transcription-based drugs useful for raising HDL plasma levels and atherosclerosis prevention. PMID- 7549468 TI - XIIIth International Symposium on Glycoconjugates. Seattle, USA, August 20-26, 1995. Abstracts. PMID- 7549469 TI - Environmental sampling: a brief review. AB - Proper application of statistical principles at the onset of an environmental study can make the difference between an effective, efficient study and wasted resources. This review distills some of the thoughts current among environmental scientists from a variety of backgrounds and organizes them according to statistical principles. Collection and review of preliminary information is important for guiding sampling design. Problems must be carefully defined before rational sample selection is possible. Many potential sources of variability exist, and as many of these sources as possible should be identified, assessed, and, ideally, minimized. Sample selection itself will depend on the precise questions that are to be addressed by the study and on what assumptions can be reasonably made about structures and patterns of contaminants in the field. Careful specification of protocols for sample procurement are needed. Choices of statistical analyses will depend on how data are collected, and intended analyses should be predetermined as part of the sampling plan. Proper attention to these principles will substantially increase the quality and efficiency of an environmental sampling program. PMID- 7549467 TI - Translation-targeted therapeutics for viral diseases. AB - Viruses utilize the protein synthetic machinery of their host. Nonetheless, certain features of the synthesis of viral proteins are distinct from those of host-cell translation. Examples include internal ribosome entry sites in some viral mRNAs and ribosomal frameshifting during production of retroviral proteins. Viruses often inhibit host translation and/or possess mechanisms leading to preferential synthesis of viral proteins. In addition, a participant in the cellular antiviral response is the enzyme PKR (protein kinase, RNA activated), which is involved in the control of cellular translation. Thus, viruses and host cells wage war on the battlefield of translation. The distinctive features of protein synthesis in virally infected cells provide potential targets for therapeutic intervention. Translation-targeted therapeutics have precedence in antibiotics like tetracycline and erythromycin. Means for discovery of translation-targeted therapeutics for viral disease are discussed. PMID- 7549466 TI - Signal transduction by Ras-like GTPases: a potential target for anticancer drugs. AB - Members of the ras family of GTPases are involved in a wide variety of cellular processes including cell proliferation, differentiation, apoptosis, and transformation. The ras oncogene is one of the most frequently mutated genes in human cancer. In addition, other oncogene and tumor suppressor gene products are components of the signal transduction pathways in which Ras or other Ras-like GTPases play key regulatory functions. Current progress in the elucidation of these signal transduction pathways will be reviewed and the potential use of these insights for the development of novel therapeutic agents for the treatment of cancer will be discussed. PMID- 7549471 TI - Assessing historical exposures of children to power-frequency magnetic fields. AB - One risk factor for human cancer currently being studied is residential exposure to power-frequency magnetic fields. A key problem in such research is how best to use contemporaneous measurements to assess past magnetic-field exposures. The main goal of the research presented in this paper was to examine the effectiveness of various surrogate measures in predicting historical exposures and to determine if residential power consumptions and the loads served by neighborhood electric networks could be used to improve the accuracy of such predictions. Residential magnetic-field data were collected during 24-h periods in the spring of 1990 and, again, in the winter of 1990-1991 for 35 children living in Western Massachusetts and Northern California. Measurements included spot magnetic fields in rooms occupied by subjects for an average of one or more hours per day, 24-h recordings at locations selected to emphasize ground-current and power-line fields, personal exposures, wire codes, residential power consumptions, and loads served by neighborhood electric networks. The geometric means of time-weighted-averaged (TWA) room spot magnetic fields measured during earlier and later visits to each home were 0.052 microT and 0.060 microT, respectively. Geometric-mean personal exposures for these visits were 0.084 microT and 0.111 microT and were significantly larger. Wertheimer-Leeper wire codes were associated with exposure. These codes, TWA spot fields, and the 24-h averages of the magnetic-field recordings taken to emphasize power-line contributions were about equally effective in explaining between-home variability in personal exposures measured eight months in the past or future. In contrast, personal exposure measurements were ineffective surrogates for past or future exposure. The study yielded little evidence suggesting that residential power consumption or neighborhood electric power flow are helpful in explaining temporal changes in personal exposure. PMID- 7549470 TI - Time-activity patterns and diurnal variation of respiratory status in a panel of asthmatics: implications for short-term air pollution effects. AB - To understand the short-term health risks to people from air pollution exposure, we investigated time-activity patterns and temporal variation of the respiratory status in 49 asthmatic Los Angeles area residents 18-50 years old. During the summer (May-September) and winter (November-March), subjects measured their lung function two to four times daily at home for one week periods, and every hour recorded their symptoms, medication, and activity hourly in diaries. Almost all subjects recorded heart rates (HR), which were converted to ventilation rate (VR) estimates using individual laboratory exercise data. Most subjects' lung function and symptoms varied diurnally, and were worst in early morning. For subjects with clinically mild asthma, diurnal forced expired volume in 1 sec (FEV1) changes averaged 7%, versus 12% in those with moderate symptoms, and 18% in severely asthmatic subjects. Lung function was similar in summer and winter, but symptoms and medication use decreased in winter. In the aggregate, subjects reported spending 75% of waking hours indoors at self-rated slow activity and 11% in vehicles. HR records usually corroborated reports of medium or fast activity. Mean estimated VR at slow, medium, and fast activity was 19, 37, and 61 L/min for men, and 16, 24, and 32 L/min for women. Outdoor fast activity, representing the greatest vulnerability to outdoor pollution, occupied approximately 0.2% of waking hours (2 min/day on average); outdoor medium activity occupied about 2% of waking hours (19 min/day on average). Estimated cumulative ventilation was higher than that of previous healthy panels because of asthmatics' higher VR at slow activity. If these activity patterns are typical, asthmatics may be especially vulnerable to pollutants with effects dependent on cumulative inhaled dose. Effects dependent on high inhaled dose rates over a short period, e.g., sulfur dioxide effects, would be unlikely, except perhaps for uncommonly active individuals in uncommonly polluted areas. PMID- 7549472 TI - The extractability of Cr(VI) from contaminated soil in synthetic sweat. AB - Hexavalent chromium is a common skin irritant that can cause contact dermatitis in sensitized individuals. Soil samples from two chromium waste sites having different Cr(VI) concentrations were extracted in synthetic sweat solutions over a pH range of 4 to 8 and analyzed for Cr(VI). These extractions were performed to determine the potential availability of Cr(VI) from contaminated soils upon contact with human sweat. The samples were also extracted using the EPA Method 3060 alkaline digestion. Overall, hexavalent chromium was readily extractable from both samples in the solutions tested. One of the samples contained Cr(VI) crystals, known as blooms, and had a Cr(VI) concentration approximately 40 times greater than the sample without blooms. The pH of the synthetic sweat solution had little effect on the Cr(VI) concentration determined for the sample containing blooms, whereas the Cr(VI) concentration determined for the sample without blooms increased with increasing pH. The Cr(VI) extracted in the synthetic sweat solutions was consistently lower than the amount extracted by the EPA 3060 alkaline extraction. PMID- 7549473 TI - Gasoline-contaminated ground water as a source of residential benzene exposure: a case study. AB - In a private residence using gasoline-contaminated ground water (approximately 300 micrograms/l benzene), a series of experiments were performed to assess the potential benzene exposures that may occur in the shower stall, bathroom, master bedroom, and living room as a result of a single 20-min, shower. Integrated fixed site SUMMA TM-polished canister and Tenax GC air samples were collected in the target microenvironments over 20, 60, and 240 min. periods. These results were compared with the long-term personal Tenax GC samples (6 h) and grab samples collected with glass, gas-tight syringes at 0, 10, 18, 20, 25, 25.5, and 30 min. Maximum benzene concentrations occurred in the shower stall (758-1670 micrograms/m3) and bathroom (366-498 micrograms/m3) during and immediately after the shower. Inhalation exposures in the shower stall during the 20-min. shower were 2.1-4.9 times higher than corresponding 20-min, bathroom exposures. The total benzene dose resulting from the shower was estimated to be approximately 281 micrograms, with 40% via inhalation and 60% via the dermal pathway. This total is 2 to 3.5 times higher than the mean inhalation dose received during a concurrent 6 h occupation of the house. These results indicate that domestic use of gasoline-contaminated water can produce relatively high benzene exposures that vary significantly according to an individual's proximity to the water use zone. The information in this document has been funded wholly or in part by the U.S. Environmental Protection Agency. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The entire experiment was reviewed and approved by the Research Triangle Institute Human Subjects Review Committee. PMID- 7549474 TI - PCB and dioxin re-entry criteria for building surfaces and air. AB - A number of fires involving polychlorinated biphenyl (PCB)-containing transformers and capacitors have occurred in the United States. PCB fires generate by-products such as polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzodioxins (PCDDs) and, when the transformer is in a building, contaminate the interior. Considerable concern exists over the potential human health effects associated with exposure by inhabitants to residual levels of PCBs, PCDFs and PCDDs. Office workers, for example, may be exposed to these chlorinated compounds via inhalation of contaminated particulates and vapors, dermal contact with contaminated surfaces, and incidental ingestion of dusts. A wide range of re-entry or cleanup levels have been developed for PCDDs and PCBs to protect workers who re-occupy a building following a PCB fire. Re-entry criteria have been used by property owners and regulatory agencies to determine whether the building is safe to re-occupy or to determine the extent of needed remediation. This paper presents a mass balance approach to deriving risk-based re-entry surface and air criteria for PCBs and PCDD/PCDFs. These criteria were based on a lifetime risk level of 10(-5), recent toxicological data on PCDDs and PCBs, and plausible exposure scenarios. Our analysis suggests that 125 ng/m2 2,3,7,8-TCDD TEQ for surfaces and 10 pg/m3 for air are acceptable. Based on Aroclor 1260, risk-based re-entry criteria for PCBs on surfaces and in air were 750 micrograms/m2 and 0.1 microgram/m3, respectively. In comparison to most previous guidelines, these risk-based criteria are less stringent, but can still be considered conservative. The surface criteria are 5 to up to 125 fold higher than previous guidelines. Air criteria range up to 5 times higher than criteria used at past PCB fire sites. Air concentrations associated with these were modeled and were negligible. For PCBs in air, the NIOSH guideline of 1 microgram/m3 is also appropriate for occupational settings. PMID- 7549475 TI - Inter-individual variability in blood/air partitioning of volatile organic compounds and correlation with blood chemistry. AB - In vitro blood/air partition coefficients (KB/A) for acetone, 1,1,1 trichloroethane, toluene, and styrene were measured in blood samples from 73 human subjects and correlated with blood chemistry parameters (hematocrit, total cholesterol, serum triglycerides, serum albumin, total plasma proteins, Na+, K+, Cl-, and HCO3-). Statistically significant inter-individual variation existed in KB/A between some subjects. Substitution of group or generic in vitro KB/A values for values determined in some individuals could introduce errors of up to 50%. However, most subjects could be well represented by group averages (mean +/- SD; acetone, 301 +/- 22; 1,1,1-trichloroethane, 6.0 +/- 0.8; toluene, 19 +/- 3; styrene, 62 +/- 10). The KB/A values for acetone, 1,1,1-trichloroethane and toluene were normally distributed. The data for styrene appeared to deviate from a normal distribution and may have been bimodal. The KB/A values for the two structurally related compounds, toluene and styrene, were strongly correlated within individuals, while the KB/A values for compounds with less structural similarity, such as acetone and styrene, were poorly correlated. At most, 15% of the variation in KB/A among individuals could be explained by variation in the measured blood chemistry parameters. When the entire sample group was considered, blood chemistry parameters were not significantly correlated with KB/A for any compound. The KB/A of 1,1,1-trichloroethane was significantly correlated with the concentration of cholesterol and triglycerides in females. Sex was a significant grouping variable for the correlation of albumin concentration with the KB/A of styrene. Age was not a significant correlation variable. Blood chemistry parameters which previously have been correlated with KB/A in small sample groups do not appear to be significantly correlated in our larger sample group. PMID- 7549476 TI - A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. AB - Seawater-strength salt stress of the ice plant (Mesembryanthemum crystallinum) initially results in wilting, but full turgor is restored within approximately 2 days. We are interested in a mechanistic explanation for this behavior and, as a requisite for in-depth biochemical studies, have begun to analyze gene expression changes in roots coincident with the onset of stress. cDNAs that suggested changes in mRNA amount under stress were found; their deduced amino acid sequences share homologies with proteins of the Mip (major intrinsic protein) gene family and potentially encode aquaporins. One transcript, MipB, was found only in root RNA, whereas two other transcripts, MipA and MipC, were detected in roots and leaves. Transcript levels of MipB were of low abundance. All transcripts declined initially during salt stress but later recovered to at least prestress level. The most drastic decline was in MipA and MipC transcripts. MipA mRNA distribution in roots detected by in situ hybridization indicated that the transcript was present in all cells in the root tip. In the expansion zone of the root where vascular bundles differentiate, MipA transcript amounts were most abundant in the endodermis. In older roots, which had undergone secondary growth, MipA was highly expressed in cell layers surrounding individual xylem strands. MipA was also localized in leaf vascular tissue and, in lower amounts, in mesophyll cells. Transcripts for MipB seemed to be present exclusively in the tip of the root, in a zone before and possibly coincident with the development of a vascular system. MipA- and MipB-encoded proteins expressed in Xenopus oocytes led to increased water permeability. mRNA fluctuations of the most highly expressed MipA and MipC coincided with turgor changes in leaves under stress. As the leaves regained turgor, transcript levels of these water channel proteins increased. PMID- 7549477 TI - Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle. AB - Protein phosphorylation by the complexes of cyclin and cyclin-dependent kinase plays a key role in cell cycle progression in all eukaryotes. The amplification by polymerase chain reaction of a cyclin box from developing root nodules and root apices of soybean showed the expression of a number of different molecular species of mitotic cyclins in plant meristems, and they were classified into five distinct groups based on their sequence similarities. The complete soybean cyclin cDNAs, cyc1Gm to cyc5Gm, corresponding to each group were isolated, and their predicted amino acid sequences showed clear similarities to mitotic cyclins identified from various organisms. These genes are expressed predominantly in such meristematic tissues as root and shoot apices and young developing nodules. Double-target in situ hybridization involving histone H4 as an S-phase marker allowed us to estimate the phases during which these cyclin genes are abundantly expressed. The results indicated that cyc5Gm is expressed in G2-to-M phases and cyc3Gm is expressed from late S-to-G2 phases. These expression patterns, together with the sequence criteria, strongly suggest that cyc3Gm and cyc5Gm encode the plant cognates for A- and B-type cyclins, respectively. In addition, the expression of cyc1Gm was restricted during a short period in S phase, suggesting that it belongs to a novel class of plant cyclins. Sequence comparison of 18 plant mitotic cyclins cloned thus far showed that they can be divided into four distinct structural groups with different functions in cell cycle progression. PMID- 7549478 TI - Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector. AB - In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins. PMID- 7549479 TI - The L6 gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N. AB - The L6 rust resistance gene from flax was cloned after tagging with the maize transposable element Activator. The gene is predicted to encode two products of 1294 and 705 amino acids that result from alternatively spliced transcripts. The longer product is similar to the products of two other plant disease resistance genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis. The similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half. The truncated product of L6, which lacks most of the leucine rich C-terminal region, is similar to the truncated product that is predicted from an alternative transcript of the N gene. The L6, N, and RPS2 genes, which control resistance to three widely different pathogen types, are the foundation of a class of plant disease resistance genes that can be referred to as nucleotide binding site/leucine-rich repeat resistance genes. PMID- 7549480 TI - Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. AB - Genetic analysis of host specificity in the rice blast fungus (Magnaporthe grisea) identified a single gene, PWL2 (for Pathogenicity toward Weeping Lovegrass), that exerts a major effect on the ability of this fungus to infect weeping lovegrass (Eragrostis curvula). The allele of the PWL2 gene conferring nonpathogenicity was genetically unstable, with the frequent appearance of spontaneous pathogenic mutants. PWL2 was cloned based on its map position. Large deletions detected in pathogenic mutants guided the gene cloning efforts. Transformants harboring the cloned PWL2 gene lost pathogenicity toward weeping lovegrass but remained fully pathogenic toward other host plants. Thus, the PWL2 host species specificity gene has properties analogous to classical avirulence genes, which function to prevent infection of certain cultivars of a particular host species. The PWL2 gene encodes a glycine-rich, hydrophilic protein (16 kD) with a putative secretion signal sequence. The pathogenic allele segregating in the mapping population, pwl2-2, differed from PWL2 by a single base pair substitution that resulted in a loss of function. The PWL2 locus is highly polymorphic among rice pathogens from diverse geographic locations. PMID- 7549482 TI - Diverse roles for MADS box genes in Arabidopsis development. AB - Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development. PMID- 7549481 TI - Genetic separation of third and fourth whorl functions of AGAMOUS. AB - AGAMOUS (AG) is an Arabidopsis MADS box gene required for normal development of the third and fourth whorls of the flower. In previously described ag mutants, the third whorl stamens are replaced by petals, and the fourth whorl is replaced by another (mutant) flower. We describe two new ag alleles, ag-4 and AG-Met205, retaining partial AG activity. Both produce flowers with stamens in the third whorl and indeterminate floral meristems; however, ag-4 flowers contain sepals in the fourth whorl, and AG-Met205 produces carpels. The ag-4 mutation results in partial loss of the C terminus of the K domain, a putative coiled coil, and AG Met205 contains a site-directed mutation that causes a single amino acid change in this same region of the K box. Two models that might explain how these changes in AG result in the separation of different AG activities are discussed. PMID- 7549483 TI - AGL15, a MADS domain protein expressed in developing embryos. AB - To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID- 7549484 TI - An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts. AB - The self-incompatibility locus of Brassica consists of a coadapted gene complex that contains at least two genes required for the recognition and inhibition of pollen by the stigma when self-pollinated. Here, we report the identification of a third S locus-linked gene from the S2 haplotype of Brassica oleracea. This gene, which we designated SLA (for S Locus Anther), is a novel gene with an unusual structure. SLA is transcribed from two promoters to produce two complementary anther-specific transcripts, one spliced and the other unspliced, that accumulate in an antiparallel manner in developing microspores and anthers. The sequence of the spliced transcript showed the presence of two open reading frames that predict proteins of 10 and 7.5 kD. Neither transcript was produced in a self-compatible B. napus strain carrying an S2-like haplotype, indicating that the SLA gene in this strain is nonfunctional. Interestingly, sequences related to SLA were not detected in DNA or RNA from plants carrying S haplotypes other than S2. The haplotype specificity of SLA, its anther-specific expression, and its physical linkage to the S locus are properties expected for a gene that encodes a determinant of S2 specificity in pollen. PMID- 7549485 TI - The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts. AB - To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon. PMID- 7549486 TI - The maize Dwarf3 gene encodes a cytochrome P450-mediated early step in Gibberellin biosynthesis. AB - Gibberellins (GAs) are phytohormones required for normal growth and development in higher plants. The Dwarf3 (D3) gene of maize encodes an early step in the GA biosynthesis pathway. We transposon-tagged the D3 gene using Robertson's Mutator (Mu) and showed that the mutant allele d3.2::Mu8 is linked to a Mu8 element. The DNA flanking the Mu8 element was cloned and shown to be linked to the d3 locus by mapping in a high-resolution population developed by selecting for recombination between d3 and linked genetic markers. To establish unambiguously the identity of the cloned gene as D3, a second mutant allele of D3 (d3.4) was also cloned and characterized using the d3.2::Mu8 sequences as a probe. d3.4 was found to have a novel insertion element, named Sleepy, inserted into an exon. A third mutant allele, d3.1, which has the same size 3' restriction fragments as d3.4 but different 5' restriction fragments, was found to contain a Sleepy insertion at the same position as d3.4. On the basis of the pedigree, Sleepy insertion, and restriction map, d3.1 appears to represent a recombinational derivative of d3.4. The D3 gene encodes a predicted protein with significant sequence similarity to cytochrome P450 enzymes. Analysis of D3 mRNA showed that the D3 transcript is expressed in roots, developing leaves, the vegetative meristem, and suspension culture cells. We detected reduced D3 mRNA levels in the mutant allele d3.5. PMID- 7549488 TI - The National Survey of Women's Health Centers: current models of women-centered care. PMID- 7549489 TI - Breast cancer incidence and screening practices among university faculty women. PMID- 7549490 TI - Breast and cervical cancer among lesbians. PMID- 7549487 TI - Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low. AB - The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves. PMID- 7549492 TI - Does universal health care coverage mean universal accessibility? Examining the Canadian experience of poor, prenatal women. PMID- 7549493 TI - Can what counts be counted? Reflections on the content, costs, benefits, and impacts of prenatal care. PMID- 7549491 TI - Listening and learning from women about mifepristone: implications for counseling and health education. AB - The careful, reflective, and honest way in which the women in the study analyzed, questioned, and explored the benefits and disadvantages of a mifepristone abortion compared with vacuum aspiration yielded an extensive list of information needed by women to make informed choices as well as an understanding of the diverse social contexts in which choices are made. Needed information identified by this study included technical information about the drugs themselves and their mechanisms of action, roles and responsibilities of health personnel, and descriptions of other women's experiences with mifepristone. A multiplicity of factors entered the decision-making process, demonstrating at the same time a complexity and flexibility of thought. In their hypothetical evaluation of mifepristone, women weighed such factors as experience with childbirth, spontaneous abortion and vacuum aspiration, specific issues for teenagers, lack of a support system, experience with herbal emenagogues and nonprescription drugs intended as abortifacients, and the relative dependence on health care providers. Social, personal, and cultural factors entered into women's interpretation of the different options. These socio-cultural contexts can profoundly influence decisions and potentially affect clinical outcomes. If health care professionals are not proactive, do not fully provide answers to questions (even if unasked), and fail to probe for specific life circumstances, then poor choices and poor outcomes may follow with long term negative consequences for clients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549494 TI - Armed by fear: self-defense handguns and women's health. PMID- 7549495 TI - Women's health and the pharmacy profession--an alliance whose time has come. PMID- 7549496 TI - Interleukin-12. AB - Interleukin (IL)-12 was originally identified as a factor produced by human Epstein-Barr virus-transformed B cell lines. It was detected by one group as cytotoxic lymphocyte maturation factor, a cytokine that synergized with IL-2 in the induction of lymphokine-activated killer cells and cytotoxic T lymphocytes. A second group characterized it as a natural killer (NK) cell stimulatory factor, due to the enhancement of cytotoxicity and IFN-gamma synthesis by NK cells. Human IL-12 was purified to homogeneity and cloned by both groups. We had identified a murine factor, provisionally termed T cell-stimulating factor (TSF), which was involved in the proliferation, synthesis of IFN-gamma and cell adhesion of CD4+ Th1 cells. TSF was produced in the antigen-specific interaction between Th1 cells and macrophages as antigen-presenting cells, partially purified from supernatants of such cultures, and shown to be identical to IL-12. Monocytes/macrophages appear to be the major source of IL-12. It is rapidly produced by phagocytic cells after stimulation with several bacteria/bacterial products and other microorganisms. In the light of its effects on NK cells as well as CD4+ and CD8+ T cells, IL-12 can be regarded as a cytokine that connects the innate immune system with the acquired immunity. IL-12 has a broad range of activities already reviewed in three papers. These include the regulation of cytokine synthesis and proliferation of T and NK cells, the promotion of Th1 cell development, the differentiation of CD8+ T cells and effects on hematopoiesis. When applied in vivo, IL-12 was shown to enhance the resistance to bacterial and parasitic infections, to promote antitumor immunity, and to influence antiviral responses including HIV in vivo or in vitro. This review will briefly summarize these effects, but mainly focus on recent results concerning the regulation of the production and the activity of IL-12, its role in the differentiation of Th cells and the implications for delayed- and immediate-type hypersensitivity reactions, its importance for organ-specific autoimmune diseases, and the possible role of the IL-12p40 homodimer as a specific inhibitor of the IL-12 heterodimer. PMID- 7549497 TI - Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA. AB - This paper demonstrates that human colostrum can inhibit the invasion of HEp-2 cells by enteroinvasive Escherichia coli (EIEC) of serotypes O28:H- and O29:H-, and that IgA antibodies mediate the inhibitory process. Seventy three of 77 (95.9%) colostrum samples prevented invasion of HEp-2 cells by E. coli O28:H-. Most of these samples contained high levels of IgA reactive to EIEC in immunoenzymatic assays. IgA eluted from an affinity chromatography column strongly inhibited HEp-2 invasion by EIEC, whereas IgA-depleted colostrum had no inhibitory effect on bacterial invasion. Immunoblots of colostrum samples with high (> 60%) invasion-inhibiting levels were performed with water extracts of invasive and noninvasive strains. Bacterial antigens from the invasive strain were recognized and the size of some was consistent with the invasion plasmid antigens (Ipas) A, B, C, and D, with stronger reactions with Ipas A and C. Colostrum samples with high inhibitory levels showed a strong reaction in Western blot assays, in contrast to the faint bands observed with poor-inhibitory samples. The results obtained in the present study suggest that colostrum IgA may protect infants against invasive E. coli infections. PMID- 7549498 TI - Purification and characterization of the major allergen from apple and its allergenic cross-reactivity with Bet v 1. AB - The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1. PMID- 7549500 TI - Progesterone modulation of anaphylactically induced ion transport in uterus and intestine. AB - The objective of this study was to investigate the role of progesterone in modulating electrophysiological responses to antigenic challenge that are reflective of immune functions in uterine and jejunal mucosae. In the rat Trichinella spiralis, host-parasite system, an enteric infection with a microscopic nematode sensitizes both the jejunum and uterus. Electrophysiological correlates of local anaphylaxis or type I hypersensitivity were monitored in vitro throughout the estrus cycle and during pregnancy. The magnitude of the anaphylactic response was correlated with serum levels of progesterone. Functional immunity was measured directly in the intestine by assessing the development of acquired resistance to reinfection with T. spiralis. Results support the conclusion that high progesterone output, as occurs during metestrus and pregnancy, and exogenously administered hormone suppress uterine responsiveness. However, the reduced uterine sensitivity to challenge could be attributed to nonspecific physiological effects of progesterone rather than direct effects on immunological components involved in the transduction of the antigenic signal. The electrophysiological response to antigen in the intestine was relatively refractory to effects of progesterone. These observations add to our understanding of endocrine control of mucosal responses to antigenic challenge and underscore site selectively of hormonal action. PMID- 7549499 TI - Mucosal histamine content and histamine secretion in Crohn's disease, ulcerative colitis and allergic enteropathy. AB - Histamine exhibits various biological effects in inflammatory and immunological reactions. To further define its potential role in allergic enteropathy and inflammatory bowel disease, both gut mucosal histamine levels and histamine release from endoscopic biopsy samples were measured. Tissue histamine content resulted from addition of the released amount of histamine and the remaining part of tissue histamine. The results demonstrate highly elevated mucosal histamine levels of the large intestine in allergic enteropathy. In inflammatory bowel disease histamine content and secretion were found to be significantly increased particularly in affected mucosa of Crohn's disease and ulcerative colitis than in unaffected tissue or in healthy controls. These findings give strong evidence that mast cell mediators like histamine play a role in the pathogenesis of these diseases. Mucosal histamine is thus concluded to contribute to the immuno inflammatory reactions of the intestine found in these disease states and to reflect the degree of colonic inflammation in Crohn's disease and ulcerative colitis. PMID- 7549501 TI - Effects of cyclosporine and dexamethasone on IgE antibody response in mice, and on passive cutaneous anaphylaxis in the rat. AB - The suppressive effects of cyclosporine A (CsA) and dexamethasone (Dxt) on antigen-specific IgE responses to ovalbumin (OA) were studied in BALB/c mice. The effects upon other isotypes were also analyzed. The antiovalbumin IgE response did not change when low doses of CsA [8 mg/kg intraperitoneally (i.p.)] were administered; IgA also remained unchanged, while IgG and IgG1 decreased significantly. At higher CsA doses (16 mg/kg i.p. or orally), a decrease was noted for all the ispotypes assayed. Dxt administered orally at 0.3 mg/kg selectively inhibited IgE and IgA but did not influence IgG or IgG1 levels. Delayed hypersensitivity reactions to OA were not modified by CsA, but were depressed by Dxt. Although CsA had not effect on passive cutaneous anaphylaxis in the rat, Dxt significantly reduced this reaction when it was administered 6 h before challenge. These results suggest that Dxt has more specific antiallergic activity than CsA. PMID- 7549502 TI - Characterization of leukocyte-derived neutrophil chemotactic factor-2 and its possible roles in neutrophil infiltration in allergic inflammation in rats. AB - This study sought to clarify the responsible chemotactic factor for neutrophils in allergic inflammation in rats. When the leukocytes collected from the pouch fluid 4 h after injection of the antigen solution into the air pouch were incubated, the neutrophil chemotactic activity in the conditioned medium increased time-dependently with higher levels for the leukocytes from the immunized rats than the nonimmunized ones. The chemotactic activity did not result from cytokine-induced neutrophil chemoattractant (CINC) because CINC concentrations in the conditioned medium were low. Neutrophil chemotactic factors in the conditioned medium were separated by isoelectric focusing into two factors, leukocyte-derived neutrophil chemotactic factor (LDNCF)-1 and LDNCF-2. The activity of LDNCF-2 was more than 75% of the total chemotactic activity in the conditioned medium. LDNCF-2 was purified by gel chromatography and reverse phase HPLC. The N-terminal amino acid sequence (1-20) of the purified LDNCF-2 was identical to the 32-51 amino acid sequence of the pro-form of rat macrophage inflammatory protein (MIP)-2. Higher levels of MIP-2 mRNA in the leukocytes from the immunized rats than that from the non-immunized rats were proved by the reverse transcription-polymerase chain reaction. In vivo, concentrations of CINC in the pouch fluid were low, and did not represent the chemotactic activity in the pouch fluid. These results suggest that LDNCF-2 (MIP-2) is an important chemotactic factor for neutrophils in the allergic inflammation in rats. PMID- 7549503 TI - Cysteinyl leukotriene production in anaphylactic reactions. AB - Anaphylactic reactions are systemic, potentially life-threatening allergic reactions. In several animal models, evidence has been presented that leukotrienes may be of major pathophysiological significance. The aim of the present study was to obtain information on cysteinyl leukotriene production in anaphylactic reactions in humans in vivo. Urinary leukotriene E4 plus N-acetyl leukotriene E4 were determined in nine patients during clinically apparent anaphylaxis and 2-11 days later following recovery. The concentrations of these established parameters of endogenous leukotriene production were strongly enhanced in urine sampled during or shortly after the anaphylactic reaction; they declined to normal or were slightly elevated subsequently. In one patient suffering from exercise-induced anaphylaxis, leukotriene production was provoked together with clinical symptoms by moderate exercise on a bicycle ergometer. Our data provide the first direct evidence that leukotrienes may be involved in anaphylactic reactions in humans in vivo. PMID- 7549505 TI - Allergy to natural honeys and camomile tea. AB - Precipitation of food allergy reactions is well known in some patients with pollinosis when they consume natural food, such as honey or camomile tea. We present 9 patients with hay fever, with or without asthma, who experienced systemic allergic reactions after ingestion of natural honeys from two local areas (Andujar and Granada) and/or camomile tea. Pollen analysis showed a high level in sunflower pollen (23.6% of pollen grains) in the honey from Andujar but not in that from Granada. The diagnosis of food and respiratory allergy was based on history, skin prick tests and specific IgE activity against pollen from Compositae. Conjunctival challenge with camomile extract also gave positive results. The above allergological tests and the inhibition studies carried out, suggest that pollen of Compositae may be responsible for allergic reactions to certain natural foods and that the reactions are mediated by an IgE-related mechanism. PMID- 7549504 TI - IgE antibodies reactive with silverfish, cockroach and chironomid are frequently found in mite-positive allergic patients. AB - Approximately 30% of the house dust mite allergic patients in The Netherlands have IgE antibodies reactive with silverfish, cockroach and/or chironomid. In allergic patients without IgE antibodies against Dermatophagoides pteronyssinus less than 5% have IgE antibodies reactive with these insects. By means of RAST inhibition studies it is shown that cross-reactivity exists between D. pteronyssinus and silverfish, cockroach or chironomid. This means that a positive RAST for silverfish, cockroach, chironomid or D. pteronyssinus cannot be taken as evidence for exposure. PMID- 7549506 TI - Soluble interleukin-4 receptor in atopic children. AB - The levels of natural soluble interleukin-4 receptor (shuIL-4R) were determined in the peripheral blood of 29 children with stable asthma, 10 children with asthma and acute respiratory infection, 11 healthy children with acute airway infection and 31 healthy controls. Healthy controls revealed the highest levels (median 1,082 pg/ml, range 524-1,900 pg/ml); which differed significantly from levels obtained with the blood of stable asthmatics (p < 0.01, median 658 pg/ml, range 329-1288 pg/ml), patients with asthma and acute respiratory infection (p < 0.01, median 663 pg/ml, range 0-1,250 pg/ml) and patients with respiratory infection alone (p < 0.01, median 674 pg/ml, range 466-1,110 pg/ml). In contrast, there was no significant difference in shuIL-4R content of cord blood obtained from newborns with a high or low risk of atopy. Additional analysis of interleukin-4 receptor (huIL-4R) on cultured lymphocytes from 13 stable asthmatic children and 14 healthy children indicated higher expression on CD4 cells (p < 0.05, median 2.2%, range 0.8-7.8%) compared to healthy controls (median 1.3%, range 0.7-3.3%). Therefore, diminished shuIL-4R concentrations in plasma may be related to inflammatory states but not specifically to atopy. The results support the notion that huIL-4R expressed on the cell surface may be regulated differently from the soluble form. PMID- 7549507 TI - The modulatory effect of tetrandrine on the CD23, CD25 and HLA-DR expression and cytokine production in different groups of asthmatic patients. AB - The therapeutic effect and mechanism of action of tetrandrine on asthma are not defined. Recently, it has been proposed that mononuclear cell (MNC) infiltration in the airway plays a role in the pathogenesis of asthma. In this study, we evaluated the effect of tetrandrine on the cell receptor expression and cytokine production of MNC from two groups (young atopic and old non-atopic) of stable asthmatic patients. MNC separated from peripheral blood of both asthmatic patients and normal individual were cultured in serum free RPMI-1640, with or without phytohemagglutinin (5 micrograms/ml) and tetrandrine (2 micrograms/ml). After culture, MNCs were harvested and stained with monoclonal antibodies for HLA DR, CD23, CD25 and CD3. MNC supernatants were collected for the measurement of IL 2, IL-4 and interferon-gamma (IFN-gamma). The results show that tetrandrine may inhibit (1) MNC proliferation, (2) the production of IL-2, IL-4 and IFN-gamma, and (3) the expression of HLA-DR, CD23 and CD25 on CD3 positive T cells. They were inhibited to a similar extent in both groups of asthmatic patients. These results suggest that tetrandrine might have some therapeutic role in relation to the suppression of lymphocyte function in asthmatics. PMID- 7549508 TI - Effect of some immunosuppressors on allergic bronchial inflammation and airway hyperresponsiveness in mice. AB - The effects of two new immunosuppressors, FK-506 and mizoribine, on antigen induced bronchial inflammation and reactivity to acetylcholine in mice were studied in comparison with those of cyclosporin A and cyclophosphamide. Three inhalations of an antigen by actively sensitized BALB/c mice resulted in an increase in airway reactivity to acetylcholine. Twenty-four hours after the final inhalation, the number of leukocytes (mononuclear cells and eosinophils) and the amount of interleukin 5 (IL-5) increased significantly. In BALB/c nu/nu mice (athymic mice), three inhalations of antigen caused no significant change in either airway inflammation or hyperresponsiveness. The administration of each of the four immunosuppressors clearly inhibited antigen-induced airway eosinophilia. Moreover, FK-506, mizoribine and cyclophosphamide clearly inhibited the antigen induced IL-5 production and cyclosporin A showed the tendency to inhibit IL-5 production. Whereas FK-506, mizoribine and cyclosporin A clearly inhibited the antigen-induced airway hyperreactivity in BALB/c mice, cyclophosphamide did not show a significant effect on this airway hyperreactivity. These results indicate that FK-506, mizoribine and cyclosporin A, but not cyclophosphamide, inhibit antigen-induced airway hyperreactivity in mice. The mechanism which inhibits antigen-induced airway eosinophilia and IL-5 production is not involved in the inhibitory mechanism of airway hyperreactivity by FK-506 and mizoribine. PMID- 7549510 TI - Successful pregnancy in a Churg-Strauss syndrome patient with a history of intrauterine fetal death. AB - Allergic granulomatosis and angiitis was first reported as a disease entity separate from polyarteritis nodosa in 1951 by Churg and Strauss. It is characterized by bronchial asthma, eosinophilia, and vasculitis and is especially rare in women of reproductive age, though, when present, may be associated with fetal mortality in pregnancy. We report a successful pregnancy in a patient who previously experienced intrauterine fetal death at 30 weeks of gestation. PMID- 7549509 TI - Mechanism of allergic cross-reactions. V. High incidence of unanticipated cross stimulation by natural allergens of rat basophilic leukemia cells sensitized with monoclonal IgE antibodies. AB - The incidence of cross-stimulations by natural allergens was investigated using RBL-2H3 cells sensitized with five different mouse monoclonal anti-DNP IgEs and four mercury-induced rat monoclonal IgEs. Cells sensitized with 3 of the 5 monoclonal anti-DNP IgEs (clones SPE-7, SRT-1, LB4) responded by serotonin release upon stimulation by natural allergens such as Dermatophagoides pteronyssinus, horse dander and mugwort extracts. Serotonin release could be inhibited by monovalent DNP-lysine, indicating the involvement of DNP-binding sites of IgEs. Two of the clones (LO-DNP-30 and LA2) were negative on all tests with allergens. All but one (Hg32) of the mercury-induced rat IgE monoclonal antibodies tested positive with DNP-BSA, and with at least one of the six allergen extracts. IgE clone Hg12 mediated serotonin release with 5 of the 6 allergens tested. PMID- 7549511 TI - Reduced level of soluble ICAM-1 in the serum of patients with atopic dermatitis. PMID- 7549512 TI - Induction of eosinophil cytokine generation by chemoattractants. AB - Recent studies have shown that eosinophils are capable of generating and releasing cytokines, illustrating a novel biologic aspect of eosinophils in regulating allergic inflammation by either autocrine or paracrine mechanisms. The effect of chemotactic agonists on eosinophil cytokine generation was examined by determination of interleukin-8 (IL-8) as a main parameter. Both complement C5a and N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated eosinophils to release IL-8, but platelet activating factor (PAF) did not exert any significant effects. The generation of IL-8 by chemoattractants was absolutely dependent on the presence of cytochalasin B. Pertussis toxin completely attenuated C5a- and FMLP-induced IL-8 production, indicating the involvement of pertussis toxin sensitive G proteins in the signal transduction process. In situ hybridization showed that both C5a and FMLP promoted eosinophil IL-8 production via transcriptional gene activation. Furthermore, C5a and FMLP, but not PAF, induced significant secretion of granulocyte-macrophage colony-stimulating factor from eosinophils. These results indicate that C5a and FMLP stimulate eosinophils to elaborate cytokines, which could be an important mechanism in the regulation of allergic inflammation. PMID- 7549513 TI - Serum concentrations of interleukin-5 and the production of lymphocytes in reactive eosinophilia. AB - The concentrations of interleukin-5 (IL-5) in serum and in IL-2-stimulated lymphocyte culture medium (L-IL2-CM) prepared from patients with reactive eosinophilia were measured by enzyme-linked immunosorbent assay. The serum IL-5 concentration was increased in 16 of 34 cases. The concentrations of IL-5 in L IL2-CM were increased in 8 of 21 patients. PMID- 7549514 TI - Role of platelet-activating factor in the release of neutrophil and eosinophil chemotactic attractants from cultured guinea-pig tracheal epithelial cells. AB - Platelet-activating factor (PAF) has been implicated in the pathogenesis of allergic disease, particularly bronchial asthma, by recruitment and activation of inflammatory cells. In an effort to further elucidate this function of PAF, guinea-pig tracheal mucosa was cultured in the presence of PAF in vitro, and culture supernatants were injected intradermally into normal guinea-pigs. After 6 h, recruitment of the inflammatory cells in the tissue was evaluated as a marker of chemotactic activity. Neutrophil and eosinophil recruitment increased significantly 1 h after PAF stimulation, the latter more than the former. After fractionation of the culture supernatant using molecular sieve filters, the fraction of 10-30 kDa showed greater chemotactic activity than the fractions of below 10 kDA or greater than 30 kDA. This activity was inhibited in a dose dependent manner (over about 1-100 mg/ml) by treatment with anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibody. These results suggest that PAF induces the release of chemotactic factors for neutrophils and eosinophils from guinea-pig tracheal epithelial cells, and one of these factors may be GM-CSF. PMID- 7549515 TI - Analysis of bronchoalveolar lavage cells in chronic eosinophilic pneumonia before and during corticosteroid therapy. AB - Bronchoalveolar lavage was performed in patients with chronic eosinophilic pneumonia of unknown origin diagnosed histopathologically, and the alveolar fluid cells were investigated before and during corticosteroid therapy. The counts of eosinophils in bronchoalveolar lavage fluid decreased markedly during steroid therapy compared with before therapy, and the ratios of interleukin 2 receptor positive T cells and HLA DR-positive T cells to all T lymphocytes in the lavage fluid also decreased. Both before and during steroid therapy, many macrophages in bronchoalveolar lavage fluid phagocytosed EG2-positive granules and stained immunohistochemically as CD71-positive 'responsive macrophages'. The decrease of activated T lymphocytes and the active phagocytosis of eosinophil granules by macrophages might play an important role in the mechanism of healing in chronic eosinophilic pneumonia. PMID- 7549516 TI - Chemotactic heterogeneity of eosinophils in idiopathic pulmonary eosinophilia. AB - Heterogeneity in the chemotactic response of eosinophils to 5 T cell line eosinophilic chemotactic factors (ECFs) was assessed in 5 patients with idiopathic pulmonary eosinophilia. Eosinophils from 2 patients responded to all 5 ECFs (group 1), whereas eosinophils from the other 3 patients responded to ECF-PI 5, PI 6, PI 7 and PI 8 but failed to respond to ECF-PI 9 (group 2). It was further found that group 1 showed an elevated level of lactate dehydrogenase and a positive tuberculin reaction, whereas group 2 showed neither. The effects of steroid therapy on the chemotactic responses of eosinophils were also examined. In group 1, the chemotactic response of eosinophils to ECF-PI 9 was significantly diminished after therapy; in contrast it was elevated in group 2. This change was accompanied by resolution of both clinical symptoms and pulmonary infiltration of eosinophils. These findings suggest that pulmonary eosinophilia can be divided into two types on the basis of eosinophil chemotactic response and laboratory findings. The heterogeneous responses of eosinophils to ECFs may provide a useful marker for classification of pulmonary eosinophilia and evaluation of therapy. PMID- 7549517 TI - Evaluation of airway hyperreactivity in interleukin-5 transgenic mice. AB - Interleukin-5 (IL-5) transgenic mice constitutively produce IL-5 and show massive eosinophilia in peripheral blood as well as infiltration of eosinophils into various tissues. This study investigated whether airway hyperreactivity occurs in the airways of IL-5 transgenic mice. These mice showed marked eosinophilia in bronchoalveolar lavage fluid, but no increase in reactivity to acetylcholine. IL 5 transgenic mice actively sensitized with ovalbumin displayed more profound airway hyperreactivity in response to acetylcholine 24 h after inhalation challenge with ovalbumin. Wild-type mice treated the same way showed little, if any, airway hyperreactivity. These findings indicate that this mouse model of airway hyperreactivity is useful in understanding the mechanism underlying pulmonary eosinophilia and the consequent inflammation. PMID- 7549518 TI - Inhibitory effect of transforming growth factor beta 1 on cytokine-enhanced eosinophil survival and degranulation. AB - The effects of transforming growth factor (TGF) beta 1 on cytokine-enhanced eosinophil survival and degranulation were investigated in vitro to determine whether it is an inhibitory regulator of allergic inflammation. Peripheral blood eosinophils purified by Percoll density gradient centrifugation and the CD16 negative selection technique were incubated in the presence of eosinophil activating cytokines (interleukin-5 (IL-5), IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon (IFN)-gamma) with and without TFG-beta 1 for 1-3 days. On day 1, eosinophil protein X release was measured by radioimmunoassay. Eosinophil viability on day 3 was determined by staining the cells with fluorescein diacetate and propidium iodide, and on the same day DNA was extracted and subjected to gel electrophoresis to test for fragmentation. TGF beta 1 significantly inhibited eosinophil survival enhanced by IL-5, IL-3, GM-CSF and IFN-gamma in a dose-dependent manner. The inhibitory effects of TGF-beta 1 on IL-5-enhanced survival was partially reversed by high concentrations of IL-5 and was completely neutralized with anti-TGF-beta antibody. IL-5 inhibited DNA fragmentation of eosinophils in vitro. TGF-beta reversed the effect of IL-5, indicating that TGF-beta 1 activates the pathway of apoptosis. TGF-beta 1 significantly suppressed eosinophil protein X release induced by IL-5. These results suggest that TGF-beta 1 may play a role in the modulation of allergic inflammation. PMID- 7549519 TI - Effects of physiologic soluble agonists on leukotriene C4 production and degranulation by human eosinophils. AB - The capacities of physiologic soluble agonists to induce leukotriene C4 (LTC4) formation and eosinophil cationic protein release from normal human eosinophils were studied. The most effective stimulus for LTC4 production by eosinophils was N-formyl-methionyl-leucyl-phenylalanine, while that for eosinophil cationic protein release was complement factor 5a. Interleukin-3 (IL-3) and IL-5 modulated both LTC4 formation and eosinophil cationic protein release induced by soluble agonists in a similar fashion, whereas tumor necrosis factor and nerve growth factor affected only LTC4 production. The optimal preincubation period for priming of LTC4 production by IL-3 or IL-5 was 90 min, while that for eosinophil cationic protein release was 10 min. These results indicate that degranulation and the generation of lipid mediators are separately regulated cellular responses, and priming by cytokines may qualitatively change the pathophysiologic consequences of eosinophil activation by soluble agonists. PMID- 7549520 TI - Ultrastructural observation of adherence of eosinophils to their surroundings. AB - This study concerned the way in which eosinophils adhere to their surroundings. Many fibrils were seen to adhere to the cell membrane of eosinophils migrating into the submucosa. Those eosinophils migrating into the epithelial layer adhered to the epithelial cells in 2 different ways: through the thin filaments bridging 2 cells and at the top of the cell projections. Eosinophils were found to move chemotactically through this eosinophil adhesion to the surrounding filaments or epithelial cells. It is possible that eosinophil granular protein directly affected the epithelial cells as a regulatory agent of the expression of adhesion molecules on individual epithelial cells, because eosinophil peroxidase was detected in the filaments bridging 2 cells. Accordingly, the expression sites of adhesion molecules must momentarily change under the direct cross-talk between 2 cells. Adherence between the eosinophils as well as large particles stretching between 2 eosinophils were also visualized. PMID- 7549521 TI - Effect of RANTES on intracellular expression of EG2 antigen in eosinophils. AB - RANTES, which is released from thrombin-stimulated platelets, is a member of the 8-kDa cytokine family that has been shown to possess chemotactic activity for eosinophils. The effect of RANTES on the intracellular expression of EG2 antigen in eosinophils was examined in this study. RANTES augmented the intracellular expression of EG2 antigen in eosinophils. These findings suggest that RANTES not only modulates the chemotactic activity of eosinophils but also intensifies the function of eosinophil activation. PMID- 7549522 TI - Increased eosinophil oxidative metabolism by treatment with soluble intercellular adhesion molecule-1. AB - Adhesion molecules may play an important role not only in adherence of inflammatory cells (particularly eosinophils) to an inflamed focus but also in activation of these cells. It is therefore of interest to evaluate eosinophil activation via intercellular adhesion molecule-1 (ICAM-1) and the beta 2-integrin family, namely CR3 (Mac-1), lymphocyte function-associated antigen (LFA)-1 alpha and LFA-1 beta, which are ligands for ICAM-1. Reactive oxygen species generated by eosinophils have also been considered capable of causing airway injury at the inflamed focus. This study examined the effect of recombinant soluble ICAM-1 and its ligands on eosinophil-induced radical oxygen products in terms of luminol dependent chemiluminescence. Recombinant soluble ICAM-1 augmented eosinophil oxidative metabolism. It was concluded that signaling via adhesion molecules might play an important role in the pathogenesis of allergic inflammation through activation of eosinophils, e.g. an increase in oxidative metabolism. PMID- 7549523 TI - Adhesion to fibronectin augments eosinophil radical oxygen products. AB - Fibronectin, an extracellular matrix component, is a ligand for very late activation antigen (VLA)-4, which is one of the beta 1-integrin family of molecules expressed by eosinophils. This study examined the effect of adherence to fibronectin on radical oxygen products from eosinophils. Adhesion of eosinophils to fibronectin resulted in enhancement of eosinophil production of radical oxygen species, as determined by luminol-dependent chemiluminescence of eosinophils stimulated with calcium ionophore. It was concluded that eosinophil adhesion to extracellular matrix via adhesion molecules may be important in the pathogenesis of allergic inflammation through eosinophil activation. PMID- 7549524 TI - Effect of fibronectin on the production of leukotriene C4 by eosinophils. AB - The effect of fibronectin on leukotriene C4 (LTC4) production by eosinophils was examined. Eosinophils obtained from patients with bronchial asthma were cultured for 3 h with or without fibronectin and culture supernatants were assayed for LTC4 by specific enzyme immunoassay. LTC4 was produced in a time- and dose dependent manner in the presence of fibronectin, and maximum production of LTC4 was observed 3 h after addition of 100 micrograms/ml of fibronectin. These results suggest that eosinophils may be activated by interaction with extracellular matrix proteins, including fibronectin, and play a role in the pathogenesis of bronchial asthma. PMID- 7549525 TI - Possible release of eosinophil granule proteins in response to signaling from intercellular adhesion molecule-1 and its ligands. AB - The presence of a large variety of membrane receptors and the identification of cytotoxic molecules (mainly granule basic proteins) have indicated that eosinophils should br considered as effector cells. It has recently been suggested that adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), play an important role in allergic inflammation, for example in bronchial asthma. This study therefore investigated the possible release of granule protein in response to signaling from ICAM-1 and its ligands. The concentrations of eosinophil cationic protein and eosinophil-derived neurotoxin in supernatants of eosinophils were significantly greater (p < 0.05) in the presence of recombinant soluble ICAM-1 than without it. These results suggest that signaling from ICAM-1 and its ligands might induce eosinophil activation and might be involved in degranulation of eosinophil granule proteins, e.g. eosinophil cationic protein and eosinophil-derived neurotoxin. PMID- 7549526 TI - Surface antigens on eosinophils in bronchial asthma and eosinophilic pneumonia. AB - Surface antigens on peripheral blood eosinophils from 23 patients with bronchial asthma, 6 with eosinophilic pneumonia and 8 controls were examined using a new direct method. Peripheral blood eosinophils in bronchial asthma and eosinophilic pneumonia showed higher complexity and/or granularity than those from controls. The percentage expression of HLA DR, CD4 and CD45RO on peripheral blood eosinophils from patients with bronchial asthma were increased compared with those from patients with eosinophilic pneumonia and from the controls. These results suggest that peripheral blood eosinophils in bronchial asthma may play a role in immunoregulation via the expression of human leukocyte antigens, such as HLA-DR, CD4 and CD45RO, that interact with lymphocytes, and may function as antigen-presenting cells. Furthermore this study suggests that there may be different phenotypes of eosinophils with differing surface antigens and intercellular reactions between eosinophils and lymphocytes. PMID- 7549527 TI - Effect of RANTES on eosinophil adhesion to plates coated with recombinant soluble intercellular adhesion molecule-1 and expression of beta 2-integrin adhesion molecules on eosinophils. AB - RANTES, a member of the 8-kDa cytokine family, has been shown to possess chemotactic activity for eosinophils. In this study, RANTES augmented isolated human eosinophil adhesion to plates coated with recombinant soluble intercellular adhesion molecule 1. In addition, RANTES did not augment the expression of beta 2 integrin adhesion molecules (lymphocyte function associated antigen-1 alpha (LFA 1 alpha), LFA-1 beta, complement receptor type 3 (CR3)) on human eosinophils. These findings suggest that RANTES not only modulates the chemotactic activity of eosinophils but also intensifies the function of eosinophil adhesion without augmenting the expression of beta 2-integrin adhesion molecules. PMID- 7549528 TI - Planar chromatography for screening of ivermectin residues in swine and cattle tissues. AB - A method was developed for qualitative detection of ivermectin residues. It involves extraction of 1 g tissue sample with 5 mL acetonitrile and C18 solid phase extraction. After derivatization with trifluoroacetic anhydride, the extract is purified again on the same but previously reconditioned C18 column. The eluate is evaporated, and the residue is diluted in methanol. The method allows detection of residues in swine and cattle meat, liver, and fat at levels as low as 5 micrograms/kg. PMID- 7549529 TI - Comparison of four commercially available rapid test kits with liquid chromatography for detecting penicillin G residues in bovine plasma. AB - Four commercially available rapid tests (Brilliant Black reduction test, LacTek test, Charm Farm test, and Charm Test II receptor assay) were compared with a liquid chromatographic (LC) method (lowest quantitatable level of 5 ng/mL) in their efficiency, reliability, and sensitivity to detect penicillin G in bovine plasma. Samples were obtained from 16 steers treated with procaine penicillin G alone or in combination with its long-acting form, benzathine penicillin G. The steers were injected intramuscularly with penicillin G doses ranging from label dose to about 9 times label dose. When results of the Brilliant Black reduction, LacTek, Charm Test II, and Charm Farm tests for penicillin G in plasma (with detection sensitivities of 5, 10, 20, and 30 ng/mL, respectively) were compared with results of LC, none of the rapid tests gave false-positive results. Each rapid test elicited a positive response when used to test bovine plasma containing penicillin G residues at concentrations above the test's detection sensitivity. The simplicity, selectivity, and sensitivity of the rapid tests, coupled with rapidity with which results are obtained, make them suitable for use in large-volume preslaughter screening of penicillin-treated cattle. PMID- 7549530 TI - Confirmation of chloramphenicol residues in egg by gas chromatography/high resolution mass spectrometry and comparison of quantitation with gas chromatography-electron capture detection. AB - Determination of chloramphenicol (CAP) residues in egg by gas chromatography/high resolution mass spectrometry (GC/HRMS) with negative chemical ionization and gas chromatography with electron capture detection (GC-ECD) is described. A cleanup based on acetonitrile extraction followed by solid-phase extraction with silica gel and gel filtration columns was developed for extraction of CAP residues from whole egg. For quantitation, the internal standards used were the meta isomer of CAP (m-CAP) for GC-ECD and both m-CAP and deuterium-labeled CAP (D5-CAP) for GC/HRMS. For GC/HRMS, evaluation was performed by selecting characteristic ions at m/z 466 for CAP and m-CAP and at m/z 471 for D5-CAP. Both methods were validated with egg samples fortified at 0.4-2.0 micrograms/kg and lyophilized egg samples from animals treated at the 1 microgram/kg concentration level. The coefficient of variation was below 10%. Limits of detection and quantitation of both methods were about 0.3 and 0.5 microgram/kg, respectively. For confirmation of CAP residues, the relative ion abundance (m/z 466 and 468) was calculated. Further confidence was obtained by comparison of accurate monoisotopic masses from a spectrum library. PMID- 7549531 TI - Liquid chromatographic determination of sulfadiazine in salmon by postcolumn derivatization and fluorescence detection. AB - A reversed-phase (ODS-2) liquid chromatographic method was developed to determine low nanogram-per-gram levels of sulfadiazine (SDZ) in salmon muscle tissue. SDZ was extracted with acetonitrile-aqueous 2% acetic acid (pH 3.0), partitioned into methylene chloride, and cleaned up by using a strong-cation-exchange, solid-phase extraction cartridge. SDZ was derivatized postcolumn with fluorescamine and detected by fluorescence. The limit of detection was 0.2 ng SDZ/g tissue. Recoveries from coho salmon tissue fortified with 1, 5, 10, and 20 ng SDZ/g tissue averaged 84.5, 85.0, 83.6, and 83.9%, respectively; recoveries from Atlantic salmon tissue fortified with 10 ng SDZ/g tissue averaged 82.6%. PMID- 7549532 TI - Food and Drug Administration Pesticide Program: residue monitoring 1994. PMID- 7549534 TI - Gas chromatographic determination of yohimbine in commercial yohimbe products. AB - The bark of Pausinystalia yohimbe [K. Schumann] Pierre (Rubiaceae), long valued as an aphrodisiac in West Africa, recently has been promoted in the United States as a dietary supplement alternative to anabolic steroids for enhancement of athletic performance. As the number of yohimbe products on the retail market increases, concerns about their safety are raised because of the reported toxicity of yohimbine (the major alkaloid of the plant). Although plant materials are usually identified microscopically, we were unable to identify them in many of the products, because as their labels indicated, the products were mixtures of various botanicals or were bark extracts and contained little or no plant material. A method for extraction and capillary gas chromatographic (GC) separation of the alkaloids of P. yohimbe was, therefore, developed and used to analyze a number of commercial yohimbe products. The method involved solvent extraction and partitioning in chloroform-water followed by separation on a methyl silicone capillary GC column (N-P detection). Comparisons of chromatograms of extracts of authentic bark with those of commercial products indicated that, although many products contained measurable quantities of the alkaloid yohimbine, they were largely devoid of the other alkaloids previously reported in this species. Concentrations of yohimbine in the commercial products ranged from < 0.1 to 489 ppm, compared with 7089 ppm in the authentic material. Authentic bark has been reported to contain up to 6% total alkaloids, 10-15% of which are yohimbine. The possible presence of undeclared diluents in the products was indicated by peaks in product chromatograms but not in those of authentic bark. PMID- 7549533 TI - Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis. AB - We have developed an improved method, using 96-well microtiter plates, for the microbiological assay of folic acid. With this method, the tedium of conventional microbiological analysis is substantially decreased. Culture volumes have been reduced 33-fold, and pipetting procedures have been simplified. Assay time has been reduced to 14 h, and sensitivity has increased 10-fold (0.1 ng/mL). Analytical recoveries range from 98 to 104%. Intra-assay and interassay variabilities are less than 11%. The assay does not require extensive manipulation of inoculum. Day-to-day variability has been minimized by using saline aliquots of the bacterial suspension stored at 4 degrees C. The procedure is accurate, selective, and useful for direct measurement of folic acid in multivitamin formulations. PMID- 7549535 TI - Comparison of on-column and precolumn derivatization for liquid chromatographic determination of molybdenum in seawater and bovine liver. AB - A rapid, accurate, and sensitive method was developed for the determination of molybdenum in seawater and bovine liver samples by reversed-phase liquid chromatography (LC) using a C18 column. The method was based on chelation of the metal with 8-hydroxyquinoline by on-column or precolumn derivatization followed by LC elution with acetonitrile-0.02M acetate buffer (3 + 2; pH 4.1) and 1 x 10( 3)M of the ligand as the mobile phase. The spectrophotometric detection was made at 390 nm. Data obtained from the 2 derivatizations (on- and precolumn chelation) were compared in terms of detection limit, precision, accuracy, and ease of LC determination of molybdenum. No significant difference exists in the detection limit and the sensitivity of the 2 methods. However, the on-column chelation method, that is, the direct injection of aqueous samples onto the column, showed relatively less interference from manganese and greater simplicity in operation compared with precolumn chelation; therefore, on-column chelation is preferable for routine analyses. The on-column method produced mean levels of 9.6 +/- 0.4 micrograms/L for the seawater sample and 3.6 +/- 0.2 microgram/g (certified value, 3.5 +/- 0.5 microgram/g) for bovine liver. PMID- 7549536 TI - Recovery of Salmonella spp. from refrigerated preenrichment cultures of dry food composites. AB - Refrigerated preenrichment 72 h and selective enrichment cultures arising from 25 g analytical units of dry foods can be used to increase the analytical flexibility and productivity of laboratories for the detection of foodborne Salmonella spp. by AOAC method 994.04. Results of this intralaboratory study using artificially contaminated dry foods validate the extended application of the refrigerated preenrichment approach to dry food composites (375 g). All samples found to be contaminated by AOAC/Bacteriological Analytical Manual methods were identified readily from the homologous, refrigerated preenrichment broth cultures. This extended application of the refrigeration approach was recently adopted First Action by AOAC and was included as a modification to method 994.04. In addition, ancillary work on the diagnostic value of prolonged (48 h) incubation of lysine iron (LI) agar as described in the AOAC Official Method 967.26 led to a recommendation that the 48 h period of incubation be revoked in favor of a 24 h incubation of inoculated LI medium. PMID- 7549537 TI - Comparison of IR and Raman forms of vibrational optical activity. AB - Natural vibrational optical activity consists of two principal forms. The IR form is known as vibrational circular dichroism (VCD) and is simply the extension of electronic circular dichroism into the IR vibrational region of the spectrum. The Raman form, known as Raman optical activity (ROA), is a new form of optical activity that has no counterpart in the classical forms of optical activity. In this paper, the similarities and differences of the IR and Raman forms of vibrational optical activity will be examined. Although both VCD and ROA were discovered and confirmed in the period from 1973 to 1975, each field has evolved independently with key advances in theoretical description, instrumentation and application coming at different times over the past 20 years. The current relative strengths and weaknesses of VCD and ROA will be discussed, and specific examples of VOA spectra of (-)-alpha-pinene and the amino acid L-alanine, for which overlapping VCD and ROA data are available, will be presented. PMID- 7549538 TI - Vibrational circular dichroism in ephedra molecules. Experimental measurement and ab initio calculation. AB - Vibrational circular dichroism (VCD) spectra in the OH- and NH-stretching regions have been measured for six pharmaceutical molecules in the ephedra class, (1S,2R) norephedrine, (1), (1S,2S)-norpseudoephedrine (2), (1S,2R)-ephedrine (3), (1S,2S) pseudoephedrine (4), (1S,2R)-N-methylephedrine (5) and (1S,2S)-N methylpseudoephedrine (6), all in dilute C2Cl4 solution. Ab initio calculations of geometries and vibrational frequencies for a number of conformers of 1 to 6 have been carried out. The recently developed locally distributed origin gauge model for VCD was used to calculate VCD spectra of the conformers for each drug. This ground-state ab initio model, which does not require sum-over-states, magnetic field perturbation or localized molecular orbitals, has been found to give good agreement with experiment in this frequency region. Composite spectra obtained from weighted averages of the calculated conformer spectra agree within a factor of two with observed IR and VCD intensities for five of the ephedra drugs. For (1S,2S)-pseudoephedrine, the discrepancy between experiment and calculation has been interpreted in terms of a coupled-oscillator effect absent in the other molecules. PMID- 7549539 TI - IR circular dichroism of turns in small peptides. AB - The observation of circular dichroism (CD) in vibrational transitions in the infrared spectral region offers new possibilities for the determination of peptide solution conformation. Infrared or vibrational CD (VCD) is a particularly sensitive probe for the conformation of various peptide turns. Results of small linear and cyclic peptides containing beta- or gamma-turns are reported. PMID- 7549540 TI - Contributions of tryptophan side chains to the circular dichroism of globular proteins: exciton couplets and coupled oscillators. AB - We have applied exciton theory to estimate the circular dichroism (CD) contribution of the Trp Bb transition to the far-UV circular dichroism of globular proteins. Strong exciton couplets are predicted for a number of proteins, including dihydrofolate reductase (DHFR), chymotrypsin and chymotrypsinogen. These predicted CD spectra are dominated by the contributions of the closest pair, W47-W74 in DHFR and W174-W215 in chymotrypsinogen. The sign and magnitude of the predicted couplets are consistent with experimental data for DHFR and its W74L mutant, and with the previously unexplained CD changes upon chymotrypsin activation. More extensive coupled-oscillator interactions among all aromatic and peptide chromophores are described for DHFR and barnase. The total far-UV CD spectra predicted for these proteins agree poorly with experiment, primarily owing to difficulties in the calculation of the peptide CD. Nevertheless, difference spectra calculated between the wild-type spectra and those of mutants in which individual aromatic residues are replaced with non chromophoric side chains show satisfactory agreement with experiment in most cases. PMID- 7549542 TI - Quantitative analysis of vibrational circular dichroism spectra of proteins. Problems and perspectives. AB - Experimental and computational aspects of the quantitative analysis of vibrational circular dichroism (VCD) of proteins are discussed. Experimentally, the effect of spectral resolution, sample concentration, cell selection and spectral normalization effects are considered. The influence of random intensity variations on the results of quantitative analysis of amide I' VCD are shown to be minor up to a 15% variation in spectral intensity. A computational algorithm, based on factor analysis of the spectra and multiple linear regression calculation of fractions of secondary structures (FC), was designed to analyse quantitatively the details of the VCD spectra-structure relationship. It also enabled the results of VCD measured independently for the amide I' and amide II regions to be combined. Our study is based primarily on the optimization of the calculation to predict FC values for proteins not included in the reference data set used for regression. The best prediction is obtained with the function using only part of the observable independent VCD spectral components. Inclusion of all components actually reduces the prediction accuracy of the analysis. Spectroscopic reasons for such behaviour and the consequences of the interdependence of the crystallographic FC values on the spectra-structure analysis are discussed. Finally, the possibility of utilizing VCD spectra to obtain quantitative structural information about the protein beyond the conventional secondary structure composition is explored. A matrix descriptor of super-secondary structure features for proteins is designed, and preliminary results for prediction of this descriptor from amide I' VCD spectra are presented. These latter calculations use a novel design of the back-propagation neural network. PMID- 7549541 TI - Empirical studies of protein secondary structure by vibrational circular dichroism and related techniques. Alpha-lactalbumin and lysozyme as examples. AB - Vibrational circular dichroism (VCD) has been shown to be sensitive to secondary structure in proteins and peptides and has been used as the basis for quantitative secondary-structure-prediction algorithms. However, the accuracy of these algorithms is not matched by the apparent qualitative sensitivity of the VCD spectra. This report provides examples of the use of VCD to follow structural change spectrally and to clarify the qualitative nature of the structural changes underlying the spectral variation. The VCD spectra and the complementary UV electronic CD (ECD) and FTIR spectra of alpha-lactalbumin (LA) have been studied as a function of pH, denaturation, Ca2+ ion and solvent conditions for several species. Spectral data for lysozyme were compared with those of LA because of their very similar crystal structures. In fact, these proteins in D2O-based pH 7 solution have quite different spectra using these optical techniques. Even for the LA proteins, the human differs from the bovine and goat species. Furthermore, under low pH conditions, where the LAs are in a reversibly denatured, molten globule form, the spectra are more similar, species variation is minimal and the spectral differences from lysozyme are in fact smaller. Our data are consistent with native, pH 7, alpha-lactalbumin having a less well organized structure than lysozyme, possibly in a dynamic sense. Conversely, in the low-pH, molten globule form of LA, tertiary structure is lost which could relax constraints that might distort the helical segments in the native form. The differences between the interpretation of our results and those from X-ray and NMR data may be due to motional sampling of various geometries in LA which all contribute to the spectral signatures seen in optical spectra but whose contributions are washed out in NMR or frozen out in the crystal structure. Part of this flexibility may relate to the rather large 3(10)-helical content in the LA protein structure. Fluctionality may have specific functional effects, perhaps allowing LA to bind better to beta-galactosyl transferase and form the biologically active lactose synthetase complex. PMID- 7549543 TI - Vibrational circular dichroism measurements of ligand vibrations in haem and non haem metalloenzymes. AB - We have measured the vibrational circular dichroism (VCD) spectra of the stretching vibrations of azide and cyanide ligated to the Fe3+ atoms of haemoglobin (Hb) and myoglobin (Mb). The antisymmetric azide-stretch of the low spin haems have an anomalously large g-value of ca. -1 x 10(-3). In contrast, CN- has a g-value of ca. +2.4 x 10(-3). We also show, for the first time, that a significant VCD occurs for the azide ligand antisymmetric stretches of non-haem proteins; we measure a g-value of ca. -1 x 10(-4) for azide bound to haemerythrin. We have examined the mechanism of the VCD phenomenon by: (1) reconstituting Mb with haems substituted such that they insert differently in the haem pocket; (2) replacing the Fe3+ with Mn3+; (3) examining proteins where replacements occur for E-7 His and E-11 Val distal amino acids close to the haem and (4) examining an Mb mutant where the proximal F-8 His is replaced by Gly, and where an imidazole ligand inserts into the resulting crevice and binds to the haem in a way similar to that of the proximal histidine in the native protein. The VCD anisotropy appears insensitive to the haem substituent replacements used in this study. Exchange of the E-7 distal His or the E-11 Val has a dramatic effect on the g-value. Exchange of the F-8 proximal His reverses the sign of the g-value for the azide complex, but not for the cyanide complex. The work to date indicates that VCD has the potential to become a sensitive technique for examining the structure of metalloenzymes. Work is needed to determine the mechanism giving rise to the large g-values and to correlate the VCD spectrum with the metalloenzyme structure at the active site. PMID- 7549544 TI - Consequences of chirality in second-order non-linear spectroscopy at surfaces. AB - Surface second-harmonic generation (SHG) is recognized as a second-order non linear laser-based spectroscopy offering good surface sensitivity and selectivity. With the goal of developing optical methods to study chiral surfaces, a way to detect dichroic behaviour in SHG in analogy to circular dichroism (CD) spectroscopy has been elucidated. The SHG efficiency from a monolayer of oriented chiral molecules [(R)- or (S)-2,2'-dihydroxy-1,1' binaphthyl (BN)] has a strong dependence upon whether the excitation beam has left- or right-handed circular polarization. The relative differential SHG signal is much larger than in ordinary CD spectroscopy. The large chiroptical effects in SHG are attributed not to R, the rotational strength responsible for traditional CD (R = Im mu.m), but to the electric dipole-allowed second-order non-linear tensor, chi (2). There is a chiral element of chi (2), chi (xyz), that must be included in descriptions of the SHG response from chiral surfaces. The ability to measure chiral properties of an adsorbed molecule separately, with no background from its achiral properties, provides another form of chiral spectroscopy, different from CD and having no analogue in linear spectroscopy, but related in a well understood manner. PMID- 7549546 TI - Universal health care and high-tech medicine. AB - The purpose of this article is to review the Canadian universal health care system, focusing on the benefits and consequences of high-tech medicine. Such knowledge could be valuable in guiding the evolving universal health care system in Taiwan. The Canadian Health Care System was developed under the ideals of universality, accessibility and portability. It has successfully provided health care to all citizens regardless of their financial resources and is supported by the great majority of Canadians. However, in spite of various efforts to control health care costs, the governmental financial burden continues to escalate, seriously affecting the development and availability of high-tech medicine for patients who require such services. Delay in the acquisition of high-tech equipment, scarcity of high-tech resources relative to the population size and long waiting periods for certain services have been experienced in Canada. An aging population and advances in medical technology will force the continued escalation of health care costs in spite of optimization of the health care delivery system, and the most serious impact will be felt in high-tech medicine. A number of possible solutions such as user fees or rationing of health care services in high-tech medicine are so far unacceptable in Canada, both for ideologic and ethical reasons. However, rationing and user fees, which the author considers to be inevitable, can be designed to diminish undesirable effects and address the problem of deteriorating high-tech medical care in a universal health care system. Approaches such as a graded user fee based on income, and technologic rationing based on a cost/utility ratio may have to be explored.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549545 TI - [Directory of the Society of Exotic Pathology. 1995]. PMID- 7549547 TI - Notes on the epidemiology of multiple sclerosis. AB - The single most important feature that determines the reliability of epidemiologic studies of multiple sclerosis (MS) is the use of well-defined, generally recognized diagnostic criteria. The long-accepted direct relationship between prevalence rates (PR) and latitude is no longer valid and has been replaced by the realization that genetic factors play an important role in the acquisition of the disease. The nature of environmental factors, which may be of more importance in influencing the clinical manifestations of the disease, remains obscure. Most potential risk factors that have been studied lack biologic plausibility. Biostatistical interpretation of epidemiologic data can only indicate possible causal relationships but cannot conclusively rule out their existence. Differences in PR among different ethnic groups in similar locations suggest that, in addition to genetic factors, there may be enhancing and/or protective influences which are probably environmental in nature. Migration studies have been interpreted as suggesting that MS is acquired before puberty and that age at migration is important in determining the risk of having clinical disease. Epidemics by age of onset vanish when recalculated on the basis of age of acquisition, and probably mean that more early and mild cases are being diagnosed. A new definition of PR is proposed, ie, onset-adjusted PR, which includes patients who had symptoms at the time of the survey, but had not yet been diagnosed as MS. However, individuals who had symptoms at the time of migration to a study area must not be included in PR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549548 TI - Treatment of pyogenic splenic abscess: nonsurgical procedures. AB - This paper reviews 10 cases of splenic abscess seen from January 1984 to December 1993. Predisposing conditions included preceding pyogenic infections, contiguous infection, trauma, and diabetes. Fever, chills and pain over the left upper quadrant of the abdomen were the most common symptoms. Routine laboratory tests uncovered common abnormalities which included marked leukocytosis and abnormal chest film with left pleural effusion. All 10 patients had a solitary abscess. Enterobacteriaceae and anaerobes were the most common offending organisms and one patient had polymicrobial infections. Nine of the 10 patients were successfully treated with percutaneous sonographically-guided drainage without significant complications. Only one patient underwent splenectomy because of rupture of the splenic abscess into the peritoneal cavity. All 10 patients survived. This review indicates that percutaneous drainage may replace splenectomy as the initial approach in cases of a solitary splenic abscess. PMID- 7549550 TI - Population genetic study of selected tetranucleotide repeat DNA polymorphisms on chromosomes Y and 12. AB - Microsatellite polymorphisms of 75 unrelated Taiwanese males were analyzed for (GATA) repeats in DYS19 on chromosome Y and tetranucleotide simple repeats in D12S67 on chromosome 12. A simple nonisotopic technique based on polymerase chain reaction electrophoresis on native polyacrylamide gels followed by silver staining was used. In the 75 unrelated males, four alleles on DYS19 and five alleles on D12S67 were detected. Different allele frequencies, heterozygosity rates and information on the nature of the repeat loci were determined in these unrelated individuals. The resulting polymorphisms are of great assistance in ethnologic studies, forensic investigation and deficiency cases in paternity testing. PMID- 7549549 TI - Varicella zoster virus infection after allogeneic or autologous hemopoietic stem cell transplantation. AB - A retrospective study was carried out in 161 patients who underwent allogeneic or autologous hemopoietic stem cell transplants. The aim was to determine the frequency, outcome and risk-factors associated with varicella zoster virus (VZV) infection. Post-transplant VZV infection occurred in 29 patients. The median onset of infection was 6.5 months post-transplant, with 82% of cases occurring within the first year. Localized herpes zoster was seen in 27 patients, one patient had varicella, and one patient had simultaneous presentation of both herpes zoster and varicella. No cutaneous or visceral dissemination was noted in the series. Each patient was treated with intravenous acyclovir. Mild complications with postherpetic neuralgia were reported by three patients. There were no deaths from VZV infection. Two risk factors noted to be associated with VZV infection were the presence of graft-versus-host disease in allogeneic transplants and leukemia as the underlying disease in autologous transplants. The overall incidence of post-transplant VZV infection in the present series was relatively low compared with that of other reports involving either allogeneic or autologous bone marrow transplantation. PMID- 7549551 TI - Detubularized folded O-shaped ileal bladder. AB - Using a submucosal needle tunneling technique for ureteral implantation, detubularized folded O-shaped ileal bladders were created for seven patients in need of bladder replacement. A segment of ileum of only 300 to 400 mm in length was required. Anastomosis of the reservoir to the urethra was simply performed by tailoring the ileum to reduce tension at the anastomosis site. Follow-up periods ranged from 24 to 46 months. Excellent upper tract function and good evacuation function with minimal residual urine were demonstrated. Urodynamic studies revealed a mean bladder capacity of 367 mL, with low intra-ileobladder pressure. All patients are continent during the day. Mild enuresis was noted in three patients. No reflux was noted on follow-up voiding cystourethrography. This type of ileal bladder is simple to create and provides reliable results. The presence of a short mesentery is not a serious limiting factor. PMID- 7549552 TI - Risk factors for subsequent epilepsy after febrile convulsions. AB - To evaluate the risk of subsequent epilepsy after febrile convulsions and the long-term prognosis of such patients, the risk factors for afebrile seizures following initial febrile convulsions were studied in 154 hospitalized children: 122 with simple febrile convulsions and 32 with complex features of febrile convulsions. The mean follow-up period was 7 years 2 months (range, 4 yr - 11 yr 2 mo). Nineteen patients (12.3%) developed subsequent epilepsy in the follow-up period. The seizure types in patients with subsequent epilepsy following febrile convulsions included generalized seizures in seven patients, complex partial seizures in five patients, partial seizures evolving to secondary generalized seizures in six patients and benign childhood epilepsy with centrotemporal spike in one patient. The occurrence of epilepsy was strongly associated with complex features of febrile convulsions, pre-existing neurodevelopmental abnormalities, family history of epilepsy, and abnormal electroencephalographic findings. However, the number of recurrences of febrile convulsions, sex, family history of febrile convulsions, age of onset and long-term prophylactic use of anticonvulsants for febrile convulsions were not significant factors for subsequent epilepsy. This study demonstrates the importance of identifying the risk factors for subsequent afebrile seizures after febrile convulsions. PMID- 7549553 TI - Effect of prostaglandins and nitric oxide on basal blood flow and acetylcholine induced vasodilation in rat diaphragmatic microcirculation. AB - To assess the effect of prostaglandins and endothelium-derived relaxing factor (EDRF) on diaphragmatic microcirculation under basal conditions and after acetylcholine (ACh) stimulation, we studied a diaphragmatic preparation in anesthetized rats. With bicarbonate-buffered Ringer's solution suffusing the abdominal surface of the left costal diaphragm, laser-Doppler flowmetry was used to record microvascular blood flow (QLDF). Microvascular conductance (CLDF) was derived from QLDF by dividing by the systemic blood pressure. Drugs were applied to the surface of the diaphragm. Four series of experiments were performed. In Series 1 (n = 9), ACh (3 x 10(-5)-10(-3) mol/L) elicited a concentration dependent increase in QLDF and CLDF. In Series 2 (n = 11), ACh-induced QLDF and CLDF changes were significantly attenuated after 30 minutes suffusion of indomethacin (10(-5) mol/L), although baseline QLDF and CLDF were little affected. In Series 3 (n = 7), following suffusion of N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) mol/L) for 30 minutes, there was no change in baseline QLDF and CLDF. The ACh-induced QLDF change was abolished, while there was still a slight increase in CLDF (172 +/- 26%) at high concentrations of ACh (10(-3) mol/L). In Series 4 (n = 5), co-administration of indomethacin (10(-5) mol/L) and L-NAME (10(-4) mol/L) for 30 minutes did not completely prevent the increase in CLDF (143 +/- 13%) induced by high concentrations of ACh (10(-3) mol/L). The data suggest low basal activities of both vasodilatory prostaglandins and EDRF in diaphragmatic microvascular beds of the anesthetized rat, while both mediators independently modulate microvascular responses to ACh.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549554 TI - Aggressive temporal lobectomy for uncal herniation in traumatic subdural hematoma. AB - Over a period of 28 months, 29 patients who sustained uncal herniation originating from fronto-temporal acute subdural hematoma with major contusion and swelling of the temporal lobe were treated surgically. These 29 patients were divided into two groups according to whether or not they underwent aggressive temporal resection in two study periods. During the initial 16 months, from February 1991 to June 1992, 16 of the 29 (group A) underwent classic surgery (subtemporal decompression for evacuation of the hematoma and debridement of the contused brain). In the 12 months from July 1992 to June 1993, the remaining 13 patients (group B) underwent an aggressive temporal lobectomy in addition to the afore-mentioned procedures, including 10 complete temporal lobectomies and three anterior temporal lobectomies. In group A, there were nine operative deaths, with a mortality of 56%, and the mean Glasgow Outcome Scale (GOS) score was 2.2 +/- 0.4 (mean +/- SEM) after 24 to 36 months of follow-up. In group B, one patient died after operation, and the mean GOS score was 4.0 +/- 0.4 in a follow-up period of 12 to 24 months. Compared with group A, group B showed a better survival rate and more favorable functional outcome. Favorable recovery in group B was noted only after a complete temporal lobectomy (good in seven and moderate disability in three). The three patients who underwent an anterior temporal lobectomy had unfavorable recoveries (two vegetative states and one death).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549555 TI - Fabry's disease: report of a case. AB - Fabry's disease is a rare hereditary disorder of glycosphingolipid metabolism. Its clinical features have not been adequately described in Taiwan. This paper reports on a 32-year-old man who had painful acroparesthesia, disseminated skin angiokeratomas, whorled corneal opacity, mitral valve prolapse and renal insufficiency. There was also involvement of the central motor pathways and the autonomic nervous system. A sural nerve biopsy showed loss of small myelinated and unmyelinated fibers. A reduced serum activity of alpha-galactosidase A and a large amount of urinary globotriaosylceramide confirmed the diagnosis of Fabry's disease. PMID- 7549556 TI - Serotypes, biotypes and antibiotic susceptibility of 126 clinical isolates of Haemophilus influenzae. AB - Serotypes, biotypes, and antibiotic susceptibility of 126 Haemophilus influenzae isolates were determined. Five of the 126 isolates were from blood and were encapsulated type b strains; those taken from other sites were not typable. There were 13% biotype I, 36% biotype II, 38% biotype III, 5% biotype IV, 4% biotype V, and 4% biotype VI isolates. Antibiotic susceptibility tests using the standard disk diffusion method showed the following resistance: ampicillin 51%, cefamandole 10%, cefuroxime 3%, chloramphenicol 28%, tetracycline 37% and sulfamethoxazole-trimethoprim 49%. None of the five type b isolates were resistant to cefotaxime, a third generation cephalosporin. The second generation cephalosporins, cefamandole and cefuroxime, showed a superior activity against H. influenzae isolates, compared to other antibiotics. Multiple drug resistance was found in 64 (51%) isolates. Four of the five type b isolates were resistant to multiple drugs. The multiple-resistance pattern most frequently observed was to ampicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethoprim. Most clinical isolates did not contain plasmids; therefore, the antibiotic resistance of these H. influenzae strains was probably chromosome-mediated. PMID- 7549557 TI - Umbilical venous line related pericardial effusion in a premature neonate: report of a case. AB - Cardiac tamponade occurs very rarely, but is life-threatening in the newborn. This paper reports a premature newborn who developed profound shock 25 hours after undergoing umbilical venous catheterization. Echocardiography taken later, showed marked pericardial effusion. An umbilical venous catheter was located in the left atrium. Immediate pericardiocentesis was performed, 11 mL of a clear straw-colored fluid was removed and the umbilical venous catheter was withdrawn into the inferior vena cava. The heart rate and blood pressure recovered immediately. Analysis of the pericardial fluid showed a high glucose level of 2,451 mg/dL. There was no pericardial effusion reaccumulation thereafter. Rapid diagnosis and treatment of pericardial effusion are mandatory to prevent subsequent morbidity and mortality when disastrous episodes, such as in the present case, occur. PMID- 7549558 TI - Ultrasonic detection of persistent small unilateral hematocolpos in two girls. AB - Two girls with unilateral hematocolpos are reported. In both cases, a small amount of blood which had accumulated in the partially obstructed hemivagina was detected by real-time high resolution ultrasonography, and was confirmed by magnetic resonance imaging. Both patients were asymptomatic, and were regularly followed up at an outpatient clinic. To date, the hematocolpos persists but continues to be small. PMID- 7549559 TI - Correlation between tumor angiogenesis and metastasis in breast cancer. AB - New vessel formation (angiogenesis) plays an important role in the metastasis of cancer cells. To investigate the correlation between tumor angiogenesis and metastatic potential in breast cancer, the microvessel counts of tumor specimens from 81 women with primary infiltrating ductal carcinomas were examined. Histologic parameters (nodal status, tumor size and tumor grade) and hormone receptor status were also analyzed. We found that axillary node metastasis correlated significantly with the microvessel counts per 200x field and with tumor size, but not with age, tumor grade or hormone receptor status. Tumors without axillary node involvement had a lower microvessel count, irrespective of age, tumor size, tumor grade or hormone receptor status. Logistic regression demonstrated that the microvessel count provided the most important estimate of the relative risk of metastasis. These results suggest that, in invasive breast carcinoma, angiogenesis is closely related to metastatic potential. PMID- 7549562 TI - Enterococcal meningitis: analysis of twelve cases. AB - From 1986 to 1993, 12 cases of enterococcal meningitis were identified at the Veterans General Hospital, Taipei. Most of the cases were caused by Enterococcus faecalis and three cases were mixed bacterial meningitis. There were six adult and six pediatric patients. Among the adult patients, four had undergone neurosurgical procedures before onset of meningitis and the other two had severe chronic underlying diseases. In contrast, all six pediatric patients had underlying central nervous system (CNS) pathology such as meningomyelocele or hydrocephalus. Placement of CNS devices was the most important predisposing factor of enterococcal meningitis in this study series. Direct microbial invasion via CNS devices or neural tube defects was considered the most likely portal of entry in most patients. Only one patient had primary meningitis, and one other had an enterococcal infection outside the CNS. The overall mortality rate was 25%. None of the pediatric patients died. Underlying conditions were considered the most important factor influencing the prognosis of these patients. PMID- 7549560 TI - Anaerobic metabolism in patients undergoing intra-aortic balloon counterpulsation for cardiogenic shock. AB - To find the best predictors of outcome for patients undergoing intra-aortic balloon counterpulsation (IABP) for cardiogenic shock, we prospectively studied 30 consecutive patients by examining hemodynamic parameters and cardiac lactic acid extraction ratios before the procedure. 1 hour after and then every 6 hours for 3 days. Complete hemodynamic data were obtained from the recordings of Swan Ganz catheterization. Blood samples were drawn from the peripheral artery, central vein, pulmonary artery wedge position (PAW) and right atrium (RA) to quantify lactic acid levels. The simplified lactic acid extraction ratio was defined as the concentration of lactic acid in RA--the concentration of lactic acid in PAW/the concentration of lactic acid in PAW. The traditional systemic lactic acid production ratio was defined as the concentration of lactic acid in the central vein--the concentration of lactic acid in the peripheral artery/the concentration of lactic acid in the peripheral artery. Of the 30 patients studied, 19 died of cardiogenic shock, (group 1), while the surviving 11 patients formed group 2. The lactic acid extraction ratios at each of the time periods after the procedure were all significantly different, while the differences in the systemic lactic acid production ratio and hemodynamic parameters, including the systemic arterial pressure, cardiac output, cardiac index and PAW pressure, became significant at varying times after IABP. In group 2, the lactic acid extraction ratios 1 hour after IABP and later were all significantly higher than at baseline. This trend was not seen in group 1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549561 TI - Protective effects of propranolol on experimentally head-injured mouse brains. AB - Catecholamines have a deleterious effect on the traumatized brain. Propranolol, a beta-adrenergic blocker, can act on the vasomotor centers within the hypothalamus and brain stem to reduce the activity of the sympathetic nerves and the cerebral metabolic need for oxygen and glucose. This study investigated the effects of racemic (d,l) propranolol on the neurologic recovery of mice with head-injuries. Male BALB-C mice (n = 110) received a 1,100 g-cm head injury from a 50 g weight dropped from a height of 22 cm resulting in immediate loss of consciousness in all animals and some deaths. The survivors were divided into treatment (n = 46) and placebo (n = 45) groups. The treatment group received an intraperitoneal injection of propranolol 2.5mg/kg immediately after the head injury. The placebo group received the same amount of 0.9% normal saline by injection in the same time interval. Neurologic status was evaluated 1 hour after injury. A group of 50 mice without head injury was also tested to evaluate the performance of normal mice. All three groups of mice were then decapitated. Mice with depressed fractures of the skull (n = 1) or intracranial hematomas (n = 1) after head injury were excluded from the study. Small pieces of brain parenchyma were used to check the water content by gravimetric method. The treatment group showed better neurologic recovery than the placebo group by both the string test scoring system and grip test time. The brain edema was significantly reduced at the temporal lobes in the treatment group as compared to the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549563 TI - Rapid detection and biovar differentiation of Ureaplasma urealyticum in clinical specimens by PCR. AB - On the basis of the nucleotide sequence of the multiple-banded (MB) antigen genes of Ureaplasma urealyticum, a polymerase chain reaction (PCR) technique was developed for rapid detection and biovar differentiation of U. urealyticum in a total of 100 urogenital specimens from 50 female patients. Positive PCR UM-1 amplification was found in 28 cervical swabs and 31 urine samples. Overall agreement between PCR and culture was 95%. Members of the two biovars of U. urealyticum could be distinguished by the size of the PCR UM-1 amplification products. Biovar differentiation was also demonstrated by two additional sets of PCRs: PCR UM-2 and UM-3. The PCR UM-2 was used to amplify biovar 1, while PCR UM 3 amplified biovar 2 specifically. The results indicated that use of the MB antigen gene as a target for PCR amplification could provide rapid and specific detection and biotyping of ureaplasma DNA in urogenital samples. PMID- 7549565 TI - Guided tissue regeneration demineralized freeze-dried bone allograft: treatment of furcation defects in mandibular molars. AB - The present study evaluated the effects of combined guided tissue regeneration (GTR) and demineralized freeze-dried bone allograft (DFDBA) therapy on the healing of grade III furcation lesions in mandibular molars of seven periodontitis patients. De novo surgical debridement of furcation roofs by fine diamond bur was introduced. Routine presurgical preparation of teeth and a strict plaque control program were performed for at least six weeks before surgery. A papillary conserved full thickness mucoperiosteal flap was used in all cases. In addition to conventional debridement, odontoplasty was performed on the furcation areas with a diamond bur to eradicate inaccessible fissures or grooves and ensure calculus-free root surfaces. Following debridement, the bony defects were filled with DFDBA and covered with polytetrafluoroethylene (ePTFE) membranes. The flaps were then closed by interproximal sutures coronally positioned through the contact point. The ePTFE membranes were removed 6 to 7 weeks after operation. Clinical parameters such as probing depth (PD), gingival recession (GR), probing attachment level (PAL), tooth mobility (TM), and periapical x-ray were recorded at the baseline and 0, 3, 6, 9, and 12 months after removal of the ePTFE membrane. The results showed a significant increase in the probing attachment level and radiographic evidence of bone fill at the furcation sites. Thus, the addition of fine diamond bur debridement on the furcation in the GTR procedure with DFDBA grafting may be effective in the treatment of grade III furcation involvement. PMID- 7549564 TI - Modulation of periodontal ligament fibroblasts by cementum and alveolar bone extracts. AB - This study investigated the effect of acetic acid extracted proteins of bone and cementum on periodontal ligament fibroblasts. Alveolar bone and cementum obtained from clinically healthy subjects were subjected to 0.5 M acetic acid extraction, dialysis and lyophilization. Effects of both extracts on cell attachment and stimulation of DNA synthesis were studied in fibroblast-like cells cultured from human periodontal ligament tissue. A wide spectrum of proteins, such as 11-14, 17, 19, 22, 24, 27, 30, 34-44, 47 and 55 kDa, were detected in 10% SDS polyacrylamide gel electrophoresis of both extracts. A few tissue-specific bands (19, 22, 47 kDa in cementum extract; 34-44, 55 kDa in bone extract) were noted. These extracts were found to promote cell attachment and stimulate DNA synthesis of periodontal fibroblasts. Cementum extract appeared to have a higher activity in stimulating DNA synthesis than bone extract, while promotion of cell attachment was similar with both extracts. Addition of serum evoked a synergistic effect on the promotion of DNA synthesis of both extracts. These preliminary observations suggest that acetic acid extracts from bone and cementum contain distinct components capable of modulating cellular activities of periodontal fibroblasts. PMID- 7549566 TI - Enamel pitting in childhood tuberous sclerosis. AB - Dental enamel pitting was studied as a diagnostic sign of pediatric tuberous sclerosis (TSC). Thirteen patients aged 2.5 to 18 years with varying degrees of TSC were evaluated. They were checked for the presence of enamel pitting by the use of two to three drops of dental plaque disclosing stain which was applied to the labial surfaces of dry teeth. This technique provides a remarkable color contrast allowing for the detection of many small and subtle enamel pits. A control group of 39 unrelated patients without TSC were also examined. A total of 77% of TSC patients (10/13) revealed enamel pitting, compared with 13% of controls. The distribution of enamel pitting among TSC patients and normal controls of each sex was statistically significant. The total number of enamel pits in each patient varied from 1 to 26 and increased with age; 90% of the teeth with enamel pitting displayed one to two pits per tooth. The youngest patient with enamel pitting was 5 years old. The simplicity of this test and the high probability of pitting in TSC make the examination useful in the assessment of patients in whom the diagnosis of this serious genetic disease is being considered. PMID- 7549567 TI - Fat in the prediction of bone strength of porcine lumbar vertebrate by quantitative computed tomography. AB - The influence of fat content on quantitative computed tomography (QCT) values was studied using porcine lumbar vertebrae as a model. A total of 72 cancellous bone columns, prepared from 18 vertebrae, underwent QCT examination before and after defatting. Mechanical testing and ashing were performed to obtain the ultimate strength and bone mineral content. The ultimate strength was calculated as a power function of apparent density using linear regression on a log-log plot and the power coefficient was 1.88. QCT values were positively correlated with bone density. QCT values of defatted specimens correlated much better with ultimate strength than those of fresh specimens. The average fat content was 27.2%. The QCT values decreased linearly with increasing fat content with a slope of 7.9 mg/cm3/% fat and a correlation coefficient of 0.65. The results provided basic data for the calibration of the machine and the principles for the interpretation of QCT values excluding the fat influence. PMID- 7549568 TI - Factors affecting thyroid function after thyroidectomy for Graves' disease. AB - A series of 108 patients with surgically treated Graves' disease were analyzed retrospectively in an attempt to determine factors that could affect postoperative thyroid function. Univariate analysis showed that remnant size, resected thyroid weight, antithyroid microsomal antibody and antithyroglobulin antibody titers were all significantly related to the postoperative outcome. Remnant size outweighed the other factors in prediction of thyroid status following subtotal thyroidectomy. When all the potential prognostic factors were individually adjusted by remnant size, only the degree of lymphocytic infiltration and antithyroid microsomal antibody titer were significantly associated with the postoperative outcome. Within the range of optimal remnant size, the higher the antithyroid microsomal antibody titer and the degree of lymphocytic infiltration, the greater the likelihood of postoperative hypothyroidism and the lower the chance of recurrence. Further stepwise discriminant analysis revealed that remnant size can be planned according to the known preoperative antithyroid microsomal antibody titer to achieve the greatest likelihood of a postoperative euthyroid state. The model suggested that when the antibody titer is < or = 640, the remnant size should be 3 to 5 cm3; if the antibody titer is 1,280 to 2,560, then the remnant size should be 4 to 5 cm3; and if the antibody titer is > or = 5,120, then the remnant size should be around 5 cm3. PMID- 7549569 TI - Atypical skeletal tuberculosis mimicking tumor metastases: report of a case. AB - Skeletal tuberculosis (TB) generally involves the spine and large joints. Involvement of most other bones has been reported, but tuberculosis of the pubic symphysis is relatively unusual. This paper reports an unusual case of multiple bone lesions in the right symphysis, left sacroiliac joint and left elbow. The radiologic appearance simulated widespread metastatic disease or chondrosarcoma, but the diagnosis of tuberculosis was proven by biopsy and culture. Eight months after starting antituberculous treatment, the patient experienced an improvement in pain and limping gait, and felt well. Radiography of the pelvis also showed improvement. It is proposed that antituberculous therapy in such cases should be maintained for at least 1 year. PMID- 7549570 TI - Amphotericin B-induced leukoencephalopathy in a patient with cryptococcal meningitis. AB - A 43-year-old male was diagnosed as having cryptococcal meningitis based on his clinical condition, cerebrospinal fluid (CSF) profile and high titer CSF cryptococcal antigen. The patient received intravenous amphotericin B at a total dose of 91 mg over 5 days. The therapy was then abandoned due to acute hepatorenal failure. The patient's neurologic status remained stable for 2 months, after which he began experiencing somnolence, confusion and a personality change. Cranial computed tomography revealed multifocal white matter lesions, and a brain biopsy showed a demyelination change with concurrent IgM and C1q deposition. A clinical diagnosis of amphotericin B-induced leukoencephalopathy was made. The patient's clinical condition was partially reversed after low-dose steroid therapy. PMID- 7549571 TI - Pseudocyst formation after trapezius myocutaneous flap reconstruction: management with chemical obliteration. AB - A trapezius myocutaneous flap is frequently used in head and neck reconstruction surgery. Pseudocyst formation over the flap donor area after its dissection is a rare occurrence. When a cyst occurs after flap application in reconstructive surgery for head and neck malignancies, or is noted concomitantly with a local recurrence of a head and neck malignancy, differentiation between malignant effusion and pseudocyst is important. Differentiation is made by cytologic and biochemical analyses of the cystic contents. Instillation of a chemical irritant can successfully obliterate such pseudocysts. PMID- 7549572 TI - Gestational trophoblastic diseases: current trends and perspectives. AB - Gestational trophoblastic diseases (GTD) include a spectrum of diseases from the potentially premalignant hydatidiform mole to the highly aggressive choriocarcinoma. Most complete moles have diploid chromosomes, nearly always of pure paternal origin, whereas most partial moles have triploid chromosomes, containing one haploid maternal set and two paternal sets. The first-line treatment of molar pregnancies is suction evacuation. In patients with persistent trophoblastic diseases or choriocarcinoma, single agent or multiagent chemotherapy is indicated, depending on the prognostic score of the individual patient. With careful follow-up and appropriate treatment, nearly all patients with gestational trophoblastic diseases can be cured. Although many advances have been made in the cytogenetics, molecular biology and immunobiology of GTD, the reasons for its unique curability remain unclear. Studies comparing induction of apoptosis and multidrug resistance gene expression, in normal trophoblasts and GTD, may elucidate the mechanism behind the good response of GTD to chemotherapy. This may give some innovative insight into chemoresistance. PMID- 7549573 TI - Blood-brain barrier damage in children with central nervous system infections. AB - The blood-brain barrier (BBB) function in childhood central nervous system (CNS) infections was evaluated, using cerebrospinal fluid (CSF) and paired serum samples from 30 controls and 74 patients aged from 1 to 15 years with various CNS infections. The 74 patients included 34 cases of aseptic meningitis, 24 of encephalitis and 16 of purulent meningitis. The degree of BBB damage was graded by CSF/serum albumin ratio, and IgG production by IgG index (IgG ratio/albumin ratio of CSF to serum). In the acute stage, patients with purulent meningitis had greater elevation of the albumin ratio than other study groups. The patients with encephalitis, especially chronic encephalitis, had a selectively elevated IgG index compared to other groups. Most patients with aseptic meningitis showed little or no elevation of albumin ratio and IgG index. This study demonstrated the nature of various degrees of BBB damage and intra-BBB IgG production in different infectious CNS diseases in children. BBB damage is most severe in cases of purulent meningitis, while the intrathecal IgG production is greatest in chronic encephalitis. These abnormalities disappear as the inflammatory processes subside. PMID- 7549574 TI - Effect of assisted hatching by partial zona pellucida dissection on mouse embryos in vitro. AB - The zonae pellucida of mouse embryos were dissected to examine the effect of partial zona dissection (PZD) at the four-cell stage on embryonic development and the hatching process in vitro. Micropipettes were used to create a gap of one sixth the zona circumference, to avoid damage to the blastomere. A total of 484 embryos at the four-cell stage (defined as day 2 embryos) were randomly divided into two groups, the PZD group (n = 238) and the control group (n = 246). Embryos of both groups were cultured in vitro under identical conditions and examined on the following day. The hatching rates were as follows: day 3, 11/238 (4.6%) in the PZD group and 0/246 (0%) in the control group; day 4, 105/238 (44.1%) in the PZD group and 54/246 (22.0%) in the control group; day 5, 71/238 (29.8%) in the PZD group and 36/245 (14.5%) in the control group. On day 4 the rates of complete hatching were 7/238 (2.9%) in the PZD group and 2/246 (0.8%) in the control group. On day 6 they were 88/238 (37.0%) in the PZD group and 66/246 (26.8%) in the control group. A significantly higher percentage of PZD embryos than controls initiated and completed hatching in similar culture conditions. The present study indicates that the creation of an opening in the zona pellucida is not harmful to embryonic development and may facilitate hatching. Whether the enhanced hatching ability of embryos in vitro caused by PZD can improve the implantation rate requires further study. PMID- 7549575 TI - Left ventricular thrombi in children with dilated cardiomyopathy. AB - To define the nature and course of left ventricular (LV) thrombi in pediatric patients, the charts and echocardiographic records of 32 pediatric patients with dilated cardiomyopathy from 1986 to 1991 were retrospectively reviewed. Five patients who were identified according to echocardiographic evidence of LV thrombi fulfilled the criteria of dilated cardiomyopathy. Systemic embolization occurred in three patients with LV thrombi who invariably died despite anticoagulant treatment. Although it was not statistically significant, patients with LV thrombi tended to have a lower ejection fraction compared to patients without LV thrombi. The morphology of LV thrombi in patients with systemic embolization showed nonprotruding features. Thus, children with dilated cardiomyopathy are at risk of developing LV thrombi and systemic embolization. Although it was not possible to identify the risk factors, the risk of developing LV thrombi and systemic embolization may be higher in patients with lower ejection fraction of the left ventricle. PMID- 7549577 TI - Body composition and its relationship with physical activity and anthropometric factors in Chinese adults. AB - To evaluate the relationship of body composition with physical activity and other anthropometric factors, a group of 437 healthy Chinese men and women were recruited. Body composition was measured using the bioelectric impedance technique. Levels of physical activity (LPA) were classified as light, moderate and heavy. Anthropometric factors evaluated included body mass index (BMI), ponderal index (PI), waist-to-hip ratio (WHR), triceps skinfold thickness (TSF), mid-arm circumference, mid-forearm circumference, arm muscle circumference (AMC) and arm muscle area (AMA). Sex was found to be highly associated with body composition, with females having a higher percentage of body fat and males having a higher percentage of body lean mass. This gender difference was independent of age, physical activity or other anthropometric factors. An increased level of physical activity was associated with higher body lean mass and lower body fat. Sex, LPA, TSF, PI and WHR were all entered into the regression equations to estimate body composition. Sex and physical activity have an independent effect on body composition and the commonly used estimators for adiposity and fat distribution such as BMI, PI, TSF and WHR are closely correlated with body composition. PMID- 7549576 TI - Detection of Y-chromosomal DNA with marker chromosomes in Turner's syndrome. AB - Patients with clinical features of Turner's syndrome may have a 45,X/46,X + mar or 46,X + mar karyotype. It is estimated that phenotypic females or intersexuals with a Y chromosome and gonadal dysgenesis have a 20% risk of developing gonadoblastoma, so it is crucial to know whether Turner's syndrome patients have a Y chromosome. We studied the chromosomal make-up of four patients with Turner's syndrome using the polymerase chain reaction (PCR). Nine Y-chromosomal loci including four loci (PABY, SRY, ZFY, DYS14) on the short arm, one loci (DYZ3) on the centromere, and four loci (DYS132, DYS1, DYZ1, DYZ2) on the long arm were amplified to determine the origin of marker chromosomes. Three patients were identified as having Y chromosome DNA. Patient 1 contained the presumed gonadoblastoma locus (DYS132) and a prophylactic gonadectomy was carried out. DNA extracted from dysgenetic gonads did not show Y chromosome DNA. A rapid, highly sensitive and isotope-free method for detection of abnormal Y chromosomes in Turner's syndrome patients has been developed. Chromosome in situ hybridization analysis is required to confirm the PCR results, to provide further evidence for molecular organization of these marker chromosomes. PMID- 7549578 TI - Magnetic resonance imaging of acoustic schwannomas. AB - The magnetic resonance imaging (MRI) findings of 27 histologically proven acoustic schwannomas in 24 patients (13 men, 11 women, age 20-79 yr) are described in detail. Three patients had bilateral tumors. Twenty-two tumors (82%) had intra- and extracanalicular components, three tumors (11%) were limited to the internal auditory canal (IAC) and two tumors (7%) were limited to the cerebellopontine angle (CPA). The diameters of extracanalicular lesions in the CPA ranged from 12 to 50 mm, and most of them were round in shape. All IAC portions of CPA tumors had a funnel-shaped appearance on the axial images and short-club-shaped configuration on the coronal images. There was strong homogeneous contrast enhancement of the solid components in 12 tumors (44%) and heterogeneous enhancement in 15 tumors (56%). The cystic components of the tumors correlated well with the histologic features. All tumors could be demonstrated in their enterity by MRI. The "short-club sign", first described in this study, helped to confirm the intracanalicular component of acoustic schwannomas, which were usually found in the cerebellar cistern. The results of this study show that MRI is a sensitive imaging modality for the assessment of acoustic schwannomas located at the CPA or IAC, or in both regions. MRI is non-invasive and does not involve ionizing radiation. It should be considered the imaging examination of choice to evaluate patients with suspected acoustic schwannomas. PMID- 7549579 TI - Long-term effects of gingival fibroblast-coated hydroxylapatite graft on periodontal osseous defects. AB - This study was designed to examine the long-term effects of gingival fibroblast coated hydroxylapatite (GF-HA) grafting on the healing of osseous defects caused by inflammatory periodontitis. Gingival fibroblasts were cultured from biopsy specimens of a healthy portion of gingiva in a periodontal therapy patient. Growth of cells on hydroxylapatite (HA) particles was established in vitro and the GF-HA complex was transplanted into the periodontal osseous defects. Clinical data of the gingival and plaque index, probing depth and periapical X-ray were monitored at baseline and at regular intervals up to 28 months after surgery. Postoperative plaque control and regular maintenance were meticulously performed. In addition to disappearance of gingival inflammation, results showed that the GF HA treated sites had a marked pocket reduction and probing attachment gain at reentry and later recalls. Clinical bone fills of osseous defects were also demonstrated in periapical radiographs. These observations suggest that GF-HA grafting treatment may lead to regeneration of periodontal tissue in periodontitis-affected osseous defects. PMID- 7549580 TI - Mitral valve aneurysm and infective endocarditis: report of four cases. AB - From March 1992 to March 1994, four cases of mitral valve aneurysm were diagnosed at National Taiwan University Hospital. Mitral valve infective endocarditis was documented in three patients, while aortic valve infective endocarditis was found in the other. The diagnosis of mitral valve aneurysm was based on characteristic echocardiographic findings. The mitral valve aneurysms in these four cases were all visualized by transesophageal but not transthoracic echocardiography. At the time of diagnosis, three patients with a history of mitral valve endocarditis had perforated mitral valve aneurysms and severe mitral regurgitation. Although not found before surgery, the remaining patient with a history of aortic valve endocarditis was noted to have an unperforated mitral valve aneurysm one month after aortic valve replacement. All three patients with severe mitral regurgitation underwent mitral valve replacement and the patient with an unperforated mitral valve aneurysm was managed conservatively and obtained a stable clinical condition. In conclusion, mitral valve aneurysm usually appears to be associated with infective endocarditis and transesophageal echocardiography is more helpful in the diagnosis of mitral valve aneurysm than transthoracic echocardiography. Furthermore, unperforated mitral valve aneurysms may be managed conservatively with careful follow-up. PMID- 7549581 TI - Peduncular hallucinosis following microvascular decompression for trigeminal neuralgia: report of a case. AB - Peduncular hallucinosis usually presents as a visual disorder and is often a genuine hallucination associated with mesencephalic lesions. A 62-year-old woman with trigeminal neuralgia underwent microvascular decompression for facial pain through a lateral suboccipital approach. The pain completely disappeared after surgery, but a visual hallucination developed on the second postoperative day and continued for 5 days. Magnetic resonance imaging confirmed the lesions around the mesencephalon. PMID- 7549582 TI - Pulmonary nocardiosis in a child with systemic lupus erythematosus: report of a case. AB - Nocardiosis is an uncommon complication in children with systemic lupus erythematosus (SLE). This is a case report of pulmonary nocardiosis in a 13 year old girl with SLE, who presented with fever, chest pain and tachypnea. She had been treated with steroids and cytotoxic agents for five months. Nocardia asteroides was isolated from a surgical specimen culture. The pulmonary nocardiosis was treated by surgical excision in combination with trimethoprim sulfamethoxazole. Nocardia sp should be considered as a possible agent of infection in immunocompromised patients, especially those treated with corticosteroids at the time of infection. PMID- 7549583 TI - Insulin edema in a diabetic child. AB - A 10-year-old boy had poorly-controlled diabetes for one year. He experienced weight gain and pitting edema in both lower legs and the scrotum 5 days after initiation of insulin therapy for diabetic ketoacidosis. The diagnostic work-up for this patient revealed no evidence of cardiovascular, renal, or hepatic disease. Thiazide was prescribed, but the patient did not take it. Because of family problems, he discontinued insulin therapy and the edema subsided within 2 to 4 days. This case illustrates that the possibility of localized or generalized edema should be considered during the introduction of insulin therapy in poorly controlled diabetes. PMID- 7549584 TI - [Drug therapy in patients with chronic type B hepatitis]. AB - Chronic hepatitis B virus (HBV) infection is a serious problem because of its world wide distribution and possible adverse chronic sequalae such as cirrhosis and hepatocellular carcinoma. Over the past 20 years, many antiviral or immunomodulatory agents, or both, have been used in patients with chronic HBV infection. Among immunomodulatory agents, levamisole, BCG, picibanil and interleukin-2 have been shown to be ineffective. Corticosteroid therapy is also ineffective and can cause deleterious effects in chronic HBV infection. Thymosin alpha 1 therapy is currently in phase III clinical trial. Among antiviral agents, acyclovir, dideoxynucleosides, suramin, zidovudine and ganciclovir have been shown to be ineffective and have intolerable side effects. While adenine arabinoside (Ara-A) and its monophosphate derivative (Ara-AMP) are effective agents if the treatment course is long enough, they have been withdrawn from investigative use because of their substantial neuromuscular toxicity. Interferon alpha may directly inhibit HBV replication and enhance hepatocyte HLA class I antigen expression with subsequent increase of T-cell mediated cytotoxicity. Randomized, controlled clinical trials have shown that 25% to 50% of adult patients with elevated alanine transaminase (ALT) levels lost HBeAg and HBV-DNA when treated with IFN-alpha at a dose of 5MU daily or 10 MU three times a week for 3 to 6 months. In view of the fact that the response rate is far from satisfactory, particularly in Asian patients, combination therapies including interferon alpha with Ara-AMP, acyclovir, didoxynucleoside or interferon-gamma have been studied. Most forms of combination therapy have been shown to be of limited effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549585 TI - [Head injuries in children and adolescents: treatment and outcome]. AB - We conducted a retrospective study to investigate the treatment and outcome of head injuries in children and adolescents in Taiwan. The study group consisted of 820 children and adolescents with head injuries admitted to Chang Gung Memorial Hospital from January 1985 to December 1989. The data were obtained from chart review and questionnaire. The cases were divided into four age-groups: group A (0 4 years old), group B (5-9 years old), group C (10-14 years old) and group D (15 17 years old). Computed tomogram of the brain was performed in 92% of the cases and most had abnormal findings. The frontal, temporal and parietal regions in that order accounted for the most focal sites of injuries on computed tomogram. Brain and other major surgery were necessary in 23.7% and 11.3% of the cases, respectively. Of the 15.3% of cases with seizures, most had early-onset seizures. A questionnaire of 398 survivors, showed that most patients achieved a good recovery level (94.2%) on the Glasgow outcome scale, a normal recovery (83.8%) of motor function and independent walking (96.0%) in locomotion. Vision and hearing were impaired in only 9.3% and 6.0% of cases, respectively. Approximately 97% of the patients aged 5 years or over recovered to be independent in their daily living activities and in normal communication. There were no significant differences in their above outcome-analyses among the different age-groups. PMID- 7549587 TI - [Psychosocial responses of infertile couples attending an assisted reproduction program]. AB - The purpose of this study was to evaluate gender differences in the psychosocial responses of 85 couples who attended the assisted reproduction program at Chung Shan Medical & Dental College Hospital from September 1991 to February 1992. The infertile couples were investigated by a self-administered structured questionnaire about demographic data, infertility history, and psychosocial responses and 75 couples completed the questionnaire. The average age of husbands was 35.0 years/and wives, 31.6 years. The average duration of infertility and treatment was 52.7 and 34.6 months, respectively. Parental expectation was the leading source of stress for men, while women counted being unable to meet childbearing demands as the main source of stress. Infertile women showed a higher psychosocial distress than their partners on the global measures and all the subscales of the infertility questionnaire and psychiatric symptoms test. Except for hostility, the differences between couples in subscales of self esteem/body image, guilty, and all psychiatric symptoms reached statistical significance (p < 0.05). Among varied psychosocial responses of infertile couples, concordant response was demonstrated only in response to sexuality and physical condition. We consistently conclude with previous reports that infertile women showed a higher level of distress than their spouses, and that men and women displayed different psychosocial responses to infertility. PMID- 7549586 TI - [Morphometric analysis of corneal epithelium in normal subjects and soft contact lens wearers]. AB - It is difficult to perform specular microscopic observation of the corneal epithelium in vivo due to the interference of light scattering by the tear film layer. In this study, we clearly observed the morphology of superficial corneal epithelial cells with a specular microscope contact lens that has the same refractive index as the corneal tissue. A total of 61 corneas from normal subjects and 21 corneas from soft contact lens wearers who had worn their lenses on a regular daily basis were studied using a specular microscope to take pictures of the corneal epithelium. The pictures were then analyzed with a computer-assisted morphometric analysis system. In normal subjects, the mean epithelial cell area and corresponding mean coefficient of variation (CV) were 618 +/- 97 microns 2 and 30.7 +/- 5.0% respectively. There was a significant correlation between the mean epithelial cell area and age in male group (p < 0.05) but not in the female group; whereas a significant correlation existed between CV and age in the female group only (p < 0.05). In the soft contact lens group, the mean epithelial cell area was 675 +/- 143 micron 2 and the CV was 29.6 +/- 6.0%. Compared to the corneal epithelium of normal subjects, the epithelial cell area and CV in the soft contact lens group was not significantly different; however, abnormal morphologic patterns in the corneal epithelium appeared more frequently. Our study showed that modified specular microscopy is a noninvasive and clinically valuable method by which to assess subtle alterations in the corneal epithelium that are barely detectable with routine slit lamp biomicroscopy. PMID- 7549588 TI - [Compliance with tuberculosis treatment in Hualien aborigines]. AB - We studied the treatment outcome and compliance in 93 Hualien aborigines patients with pulmonary tuberculosis registered from November 1990 to December 1993. There were 60 men and 33 women with a mean age of 42 years (range, 14-91 Years). There was no difference in age distribution between men and women. Antituberculosis (anti-TB) chemotherapy consisted of isoniazid, rifampin, pyrazinamide and ethambutol for 2 months followed by isoniazid, rifampin, and ethambutol for 4 months. Anti-TB treatment was completed by 68 (73.1%) patients within 9 months while the other 25 Patients (26.9%) failed to complete their medication. It was found that single patients (unmarried, divorced, widowed), those with a low tuberculosis recognition score, and alcohol consumers had a higher chance of non compliance and hence lower chance of completing treatment. Other factors such as sex, age, race, education, occupation, family Apgar score, associated diseases, side effects of anti-TB drugs, complicity of taking drugs, and treatment in general hospital/clinic or in Public health TB clinic did not significantly contribute to treatment failure. PMID- 7549589 TI - [Appropriation of time for professional staff and appropriation of costs for services, education and research at a major teaching hospital]. AB - Cost analysis of hospital graduate medical education (GME) cannot follow standard accounting procedure because most of the services, teaching and research activities involved are performed simultaneously. This paper surveys work time allocation and analyzes the budgetary appropriation of a major teaching hospital in Taiwan, ROC based on answers given in a specially designed questionnaire. Percentages of working time used for services, teaching, research by the 2206 professionals at the hospital were 84.0%, 10.3% and 5.7% respectively; for the 776 physicians the figures were 64.1%, 22.7% and 13.2% respectively; GME represented a 12.22% share of the operation budget. In view of the fact that the IRB (Intern-Resident/Bed) ratio of the hospital reaches a high of 45%, GME investment is apparently insufficient. To obtain a reasonable ratio of teaching activities, hospital physicians expressed the opinion that the GME expenditure rate should be at least 18.95%. The Labor Insurance Plan, currently the biggest of its kind in this country, pays 3% of GME expenses to all hospitals. This payment neglects the different teaching load in different hospitals. PMID- 7549591 TI - The quest for quality in mental health services. PMID- 7549590 TI - [Angioplasty for subclavian artery stenosis: report of two cases]. AB - We present our experience of percutaneous transluminal angioplasty (PTA) in the treatment of subclavian artery stenosis in two patients. One patient was a 66 year-old asymptomatic man. His left arm systolic blood pressure was 40 mmHg lower than that of his right arm. He had irregular segmental stenotic lesions (50% to 80%) at the proximal portion of the left subclavian artery. PTA was attempted via the left brachial artery. After the procedure, the stenotic lesion seemed totally dilated and his left arm systolic blood pressure was 8 mmHg lower than that of his right arm. Restenosis of the left subclavian artery was noted four months after the procedure. The second patient was a 51-year-old man with symptoms of intermittent dizziness, nausea and vertigo. His left arm systolic blood pressure was 30 mmHg lower than that of his right arm. He had 43% diameter stenotic lesion at the proximal portion of the left subclavian artery. PTA was attempted via right femoral artery. After the procedure, the stenotic lesion seemed markedly dilated and his left arm systolic blood pressure was 14 mmHg lower than that of his right arm. He is well three years post-procedure without evidence of restenosis. PMID- 7549592 TI - Contemporary quality management in mental health. AB - Contemporary approaches to quality management in mental health utilize methods derived from traditional quality assurance and industrial quality improvement. Quality assurance involves the use of measurable indicators to monitor and evaluate care. Sentinel event indicators refer to high risk events that require review each time they occur. Rate indicators refer to events that are reviewed in the context of trends and patterns. The process of choosing and developing indicators should involve identification of the indicator topic, the monitoring objectives, data collection mechanisms, and responsible staff. As well, indicator data must be evaluated, actions taken, and communication of results must occur. Positive results, like positive diagnostic findings, point to a probability of the presence of a quality related issue. Quality improvement methods have led to a further evolution of efforts to promote quality care. A major goal of quality management is to utilize data to improve clinical decisions. Reaching this important goal is difficult in mental health due to the uncertain relationship between interventions and outcomes of care. In the effort described,, interventions were initiated to improve decisions relating to clinical risk management and resource utilization. Administrative restructuring enabled real time review procedures and retrospective data analysis focused on decisions related to clinical instability and on permissive and restrictive clinical decisions. Permissive decisions were defined as decisions to allow off-ward privileges for patients whose level of functioning scores suggested the need for greater restriction. Restrictive decisions involved extending hospitalization of low risk patients. Inappropriate permissive or restrictive decisions are undesirable from both risk management and quality-of-care perspectives.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549593 TI - The imperative of outcome assessment in psychiatry. AB - This report describes the current conceptualization of outcome assessment in psychiatry and focuses on how assessment instruments can be built into psychiatric facility-based practice. First, the domains of clinical assessment are outlined, with an emphasis on three elements: level of psychiatric symptoms, clinical functioning, and patient satisfaction. Examples of outcome instruments then are provided as well as the elements of their successful implementation. Finally, the value of linking outcome assessment to data on patient characteristics and service utilization are discussed in order to gain insight into the relationship between treatment and outcome. The clinical, fiscal, and regulatory imperatives emerging for outcome assessment call for its demystification and widespread application. PMID- 7549594 TI - An effective computer-based tardive dyskinesia monitoring system. AB - To promote early recognition and treatment of neuroleptic-induced tardive dyskinesia we used our facility's pharmacy and appointment data bases to develop an automated reminder system that significantly improved physician monitoring of patients receiving antipsychotic drug therapy. The system prompts staff to perform regular examinations for abnormal involuntary movements and to review patients' consent to therapy with antipsychotic medication. The average prevalences in the 15 months after automated reminders began, in a population of over 800 patients, increased from 53% to 85% for an annually completed abnormal involuntary movement scale in medical records and from 38 to 74% for a statement of informed consent. Now, 45 months later, prevalences of both measures approaches 100%. The integrated design of the Department of Veterans Affairs computer system allowed linking pharmacy and appointment scheduling data and facilitated the project. The reminder system effectively promoted rapid, marked, and sustained change in physicians' documentation of antipsychotic drug therapy. PMID- 7549595 TI - The effect of utilization management on the treatment of schizophrenic inpatients. PMID- 7549596 TI - A review of quality evaluation systems for mental health services. AB - Mental health quality of care evaluation systems found in an extensive search are reviewed. The quality measures are differentiated from other seemingly similar types of scales and instruments. The quality systems are reviewed in reference to 1) reliability and validity of data, type, and source of data, allowance for patient variables, standards of quality, and other parameters and 2) range and comprehensiveness, particularly with regard to coverage of the range of disorders and conditions, of age groups, care needs, care settings, treatment modalities, and of types of provider performance. Most measures were reliable and valid. Some gave consideration to patient variables and case mix, to timing, and to treatment intensity. There were no measures of providers' interpersonal performance. With regard to range and comprehensiveness, there was a substantial imbalance toward the medical model and pharmacotherapy and away from systems orientation, from psychotherapy, and from other modalities, interventions, and types of programs. PMID- 7549597 TI - Patients' perceived control over their health care: an outcome assessment of their psychological adjustment to renal failure. AB - Previous research has sometimes produced conflicting results regarding patients' psychological adjustment to end-stage renal disease (ESRD). It is generally accepted that treatment outcomes vary across treatment modality, with successful transplant patients reporting a higher subjective quality of life than patients on center hemodialysis. Hemodialysis and transplantation therapies vary in many ways, including the intrusiveness of treatment on all aspects of the patient's life. In the present study, 65 individuals being treated for ESRD with center hemodialysis or a renal transplant were assessed for health locus of control, for generalized control over 11 life dimensions, and for beliefs about control over specific aspects of treatment for ESRD. The study also included indicators of psychological adjustment. The results indicated that control over life dimensions, which includes control over illness, is a significant factor in psychological adjustment. Beliefs that one can comply specifically with diet and fluid restrictions were related to treatment outcome. These data suggest that interventions designed to increase patients' perception of control are likely to have a positive impact on the qualitative aspect of treatment. PMID- 7549598 TI - Commentary: balancing quality and cost in the health care market. PMID- 7549599 TI - Connective tissue fibre production in keratoconus. AB - In a histochemical and electron microscopic study of 45 cases of keratoconus, oxytalan fibres, a member of the elastic fibre family, were found in the extracellular tissues of the cornea. Oxytalan fibres were identified beneath the epithelium over the thinned central region of the conical cornea as well as in areas of stromal scarring. In a number of instances the apparently thickened Descemet's membrane was shown to include an oxytalan-containing retro-corneal layer. Oxytalan fibres were found to be immunostained by antibodies raised against collagen type VI. PMID- 7549600 TI - Storage and repeated recovery of Neisseria gonorrhoeae using cryovials. AB - One hundred gonococcal isolates, representing eight protein IA serovars and 14 protein IB serovars, were stored at -70 degrees C using the Pro-Lab Microbank cryovial storage system. At monthly intervals a bead from each cryovial was removed and cultured on modified New York City medium. The overall recovery rate was 98.6% (2365/2400), the 35 failures representing 13 separate isolates. There was a small but significant decrease in recovery in the last 12 months of the trial (97.8%) compared with the first 12 months (99.8%), which may have been due to a sampling problem rather than a temporal phenomenon. Failures were significantly associated with minor serovars, suggesting that the transmissibility/viability of minor serovars may be lower than that of common serovars and could be a significant factor in the epidemiology of gonococcal infection. We conclude that Pro-Lab Microbank cryovials provide a highly effective and convenient system for storage of Neisseria gonorrhoeae, particularly when multiple retrieval is required, and the system offers many advantages over conventional lyophilisation. PMID- 7549601 TI - Detection of reverse transcriptase in culture medium for mammary tumour cell lines: a comparison of an established radio-labelling technique and a contemporary non-isotopic technique. AB - Classically, radio-label techniques have been employed to analyse biological samples for reverse transcriptase (RT) activity. More recently, however, non isotopic kits have been developed for retroviral quantification. Nevertheless, until the present investigation it has not been known if these contemporary methods are more sensitive at detecting reverse transcriptase activity. In our study, a non-isotopic ELISA method was shown to be considerably more sensitive than the radio-label technique at detecting reverse transcriptase in growth medium used to culture the murine breast cancer cell line GR/A. Using the ELISA, less reverse transcriptase activity was demonstrated in growth medium from human mammary adenocarcinoma MCF-7 cells than the murine source. This ELISA did not detect reverse transcriptase activity from a pure source of Moloney murine leukaemia virus. In light of this, the broad applicability of this ELISA for reverse transcriptase from different viral sources must be investigated before it can be used to monitor biological supernatants for the presence of retroviruses. PMID- 7549602 TI - Immunohistochemical techniques: the effect of melanin bleaching. AB - This study addresses two questions: i) which antigens can withstand bleaching by 2.5 g/L of potassium permanganate followed by 10 g/L of oxalate, before immunohistochemical staining; and ii) are any other steps in the immunohistochemical staining technique resistant to bleaching? A panel of 10 antigens was stained immunohistochemically and the results compared with staining performed with a bleaching step interpolated at different steps in the procedures. Four antigens (HMB-45, S-100, factor VIII-related antigen and collagen type IV) were unaffected by bleaching; two antigens (CD-20 and CD-45) had their staining enhanced by bleaching; one had the staining reduced (hsp27); and in three it was abolished (CD-45Ro, CD-31 and Ulex/anti-ulex antibody) by bleaching. Two antibodies (UCHL-1 and L-26) showed evidence for altered specificity following bleaching. None of the steps after application of the primary antibody was resistant to bleaching. Three chromagens used for peroxidase demonstration-amino ethyl-carbazole, diaminobenzidine and chloro-naphthol-were also found to be sensitive to bleaching. While some antigens were resistant to the effects of bleaching, some were not, and no other step in the immunohistochemical procedure could withstand bleaching. PMID- 7549603 TI - Uses and misuses of serum hCG--a clinical audit. AB - A 24-hour laboratory-based service was established in the City Hospital, Birmingham, England, in 1987 for semi-quantitative serum human chorionic gonadotrophin (hCG) assay and specific clinical criteria for requests were agreed. However, a 309% increase in workload between 1988 and 1992 prompted a detailed clinical audit of this service. During a representative month only 35% of 142 requests conformed to the agreed criteria. Those for routine diagnosis of pregnancy, threatened miscarriage, and 'abdominal pain screen' comprised 49% of total requests and seemed inappropriate. No pregnancy was diagnosed by 'abdominal pain screen'. Adhering to agreed clinical criteria, therefore, would not jeopardise patient care and could halve the cost of the service. As a result of this audit the established criteria for serum hCG estimation were reinforced and re-issued to all relevant clinical teams. Additionally, to improve delivery of service and reduce costs a semi-quantitative urine hCG assay was introduced in the Accident and Emergency Department for use by approved staff. Records in this department showed no increase in hCG requests during the six months after the service change, compared with those in the preceding half year. However, theoretical cost savings of 5173 pounds per annum were eroded by an estimated 10% because 208 (26%) tests were unaccounted for. The reasons for this apparent 'wastage' are unclear, and highlight the difficulty in auditing a biochemical test performed outside the laboratory. PMID- 7549604 TI - Atherosclerosis risk factors: variation in healthy hospital workers and members of local communities asymptomatic for vascular disease. Implications for normal controls. AB - The major risk factors for atherosclerosis were measured in 100 middle-aged members of the local community aged between 40 and 66 attending hospital for minor operations, hernia repair, varicose veins or endoscopy, and their healthy accompanying spouses. Levels in this group were compared with those measured in 75 age- and sex-matched hospital workers. Ten of the 12 risk factors measured were more unfavourable in the local population (P = 0.039). Levels of total cholesterol (P = 0.0013), low-density lipoprotein (LDL) cholesterol (P = 0.0002) and body mass index (P = 0.0074) were higher in members of the local community. There was no difference in levels of triglycerides, high-density lipoprotein cholesterol, von Willebrand factor (vWf, an index of damage to the endothelial cell), fibrinogen, glucose, systolic and diastolic blood pressure, waist-to-hip ratio or the proportion of smokers. We also found systolic blood pressure (P = 0.014) and vWf (P = 0.021) to be higher, while high-density lipoprotein (P = 0.022) was lower in the 35 smokers, but we could not identify any factor that correlated with age. However, systolic (P = 0.028) and diastolic (P = 0.0072) blood pressures, triglycerides (P = 0.029) and waist-to-hip ratio (P < 0.0001) were all lower, while high-density lipoprotein was higher (P < 0.0001) in the 80 women compared to the men. We conclude that precise definition of the identity of the control group is necessary in studies of risk factors for atherosclerosis, or in frank disease, if mis-interpretation is to be avoided. PMID- 7549605 TI - Immunocytochemistry in the diagnosis of Kaposi's sarcoma and angiosarcoma. AB - Immunocytochemistry in the assessment of Kaposi's sarcoma and angiosarcoma is reviewed, with emphasis on the technical appraisal of the appropriate panel of markers. Problems incurred in the interpretation of the pathological spectrum of both tumours are discussed. PMID- 7549606 TI - von Willebrand factor: its function and its measurement in the laboratory. AB - von Willebrand factor (vWF) is a complex protein essential to normal haemostasis. It is vital to platelet adhesion in the high shear stress conditions encountered in the small blood vessels. Deficient overall levels of, or the presence of abnormally functioning variants of, vWF lead to the common bleeding disorder termed von Willebrand's disease. Laboratory investigation is aimed at both quantitative and qualitative investigation with a view to classification of the abnormality for each individual patient into a particular sub-type. PMID- 7549607 TI - Assessment of food intakes: are we measuring what people eat? AB - Accurate assessments of dietary intake are a pre-requisite for assessing the relationships between diet and health. However, the quantification of errors in dietary data has remained largely undetected because of the absence of techniques to verify dietary survey methodology. Recent studies using doubly-labelled water estimates of total energy expenditure to validate self-reported energy intakes have demonstrated that the majority of these are systematically biased towards under-estimation of usual requirements. Bias is unlikely to be consistent in population studies. Subjects who report the highest energy intake tend to estimate their requirements more accurately than those who report low intakes. Mechanisms for cross-checking energy intake data against estimated energy requirements have been developed and provide limited guidance for checking the physiological plausibility of reported intakes, with special emphasis on under reporting. In the absence of independent validation the evaluation of dietary survey data should be approached with caution. PMID- 7549608 TI - Sweat sodium and chloride analysis using BM/Hitachi 911 ion-selective electrodes. AB - The suitability of Hitachi 911 ion-selective electrodes to analyse neat sweat samples for sodium and chloride was investigated. Sweat samples obtained using the Gibson and Cooke method were analysed by flame photometry for sodium and coulometry for chloride. Specimens obtained by the Wescor Macroduct collection procedure were analysed by ion-specific electrodes. No significant differences between sodium and chloride results were found between the two different collection methods and analysis procedures. Analysis of neat sweat can reduce errors in sweat analysis by eliminating extraction and dilution stages. Errors could occur with use of a serum compensator in the calibration of ion-selective electrodes. PMID- 7549609 TI - Turbidimetric urine screening. AB - Several studies have shown that urine clarity can be used as an infection screening method. Previous publications have involved patients from specific clinics or of specific age groups, and turbidimetric assessment has often been subjective and unscientific in nature. In this study all specimen types and sources with the exception of catheterised samples were included. Turbidimetric readings were performed using a double-beam turbidimeter, followed by quantitative microscopy and culture. Clinical significance was assessed using a combination of culture results, microscopy and clinical details. Performance varied in relation to the chosen turbidity threshold level, but at a sensitivity of 94.1%, 55.7% of samples could be discarded as 'screen-negative'. The rapid nature of this screening method, combined with the ability subsequently to concentrate resources on the remaining 'screen-positive' samples, can result in an improved service at reduced cost. PMID- 7549610 TI - Autofluorescence of Cyclospora. PMID- 7549611 TI - Direct sample polymerase chain reaction for the detection of Mycoplasma pneumoniae: a simple system for clinical application. AB - A semi-nested polymerase chain reaction system with primers derived from the P1 adhesin gene of Mycoplasma pneumoniae was evaluated for sensitivity and specificity for detection of M. pneumoniae. The method used target DNA within samples of M. pneumoniae broth cultures and clinical material, without a formal extraction process. The sensitivity for detection of DNA was found to be to a level of one copy per sample and was specific to M. pneumoniae only, discriminating from the other human mycoplasma and ureaplasma species tested. The system offers the opportunity for a simple and highly specific laboratory method for diagnosis which may be compared to currently available serological methods, and may provide another method of studying mycoplasma-induced pathology. PMID- 7549613 TI - One nurse practitioner's journey through the litigation process. PMID- 7549612 TI - Marie Osborn: rural health care nurse practitioner. Interview by Maryann T Hardesty. PMID- 7549614 TI - Treating obsessive-compulsive disorder. PMID- 7549615 TI - Rehabilitation management of chronic pain syndromes. AB - Chronic pain is an often misdiagnosed and poorly understood problem. Pain complaints are the most frequently reported symptom to physicians and nurse practitioners. Unremitting pain can have an adverse effect physically, psychologically, and socially. When chronic pain is not relieved, anger, frustration, and depression can lead to maladaptive behaviors that interfere with the person's ability to pursue goals and activities. Chronic pain is best treated through a multidisciplinary approach, with best results achieved by programs specialized in pain management. The goal of rehabilitation in chronic pain management is not total pain relief because this is often impossible; restoration of the ability to function productively and satisfyingly is the target goal. PMID- 7549616 TI - Recognizing, treating, and preventing mild traumatic brain injury. PMID- 7549617 TI - Evaluating and treating common upper extremity nerve compression and tendonitis syndromes ... without becoming cumulatively traumatized. AB - Cumulative trauma disorder, repetitive motion injury, and overuse syndrome are all umbrella descriptions of many specific injuries that require careful evaluation, diagnosis, and treatment to restore function, decrease pain, and minimize time spent in a patient role. This article describes anatomic sites of common nerve compressions and tendonitis in the upper extremity (UE). Evaluation techniques are described with emphasis on clinical, environmental, and psychological mitigating factors essential in identifying microtrauma of nerves and tendons to achieve the correct differential diagnosis. Conservative treatment recommendations, including splinting, ergonomics, corticosteroid injection, and hand surgery, are outlined to assist the practitioner in discerning the most appropriate course of action. Surgical intervention and postoperative care and follow-up for median, ulnar, radial, and thoracic outlet nerve compression syndromes are briefly described. PMID- 7549618 TI - Pediatric injury control: strategies for the nurse practitioner. AB - This year, 8,000 children under age 15 years will die of unintentional injuries and 50,000 will become permanently disabled. The financial cost of pediatric trauma to society will reach approximately $14 billion. The nurse practitioner (NP) is in a key position to reduce the number and extent of injuries by participating in comprehensive injury control measures including engineering devices, education, enactment, enforcement, and evaluation. The most convenient and productive strategy for the NP is parent anticipatory guidance. This article will alert the practitioner to specific predispositions children have towards injuries. Age-related injury prevention information will be discussed for integration into primary care visits. PMID- 7549619 TI - Geriatric rehabilitation. AB - The United States population is aging, and the number of people over age 85 is expected to quadruple early in the next century. Advancing age increases the likelihood of acquiring a disabling condition. By the next century, every other visit to a health provider will be made by an elderly person. Nurse practitioners will see increasing numbers of older patients. They will be called on to differentiate normal aging states from pathological conditions. A focus on promoting vigorous aging, minimizing disabling conditions, and restoring or preserving function and independence will assist the practitioner in appropriate evaluation and treatment of the elderly. PMID- 7549620 TI - Local research ethics committees. PMID- 7549621 TI - Which diabetic patients should be taking aspirin? PMID- 7549622 TI - Training of overseas qualified doctors in Britain. PMID- 7549623 TI - Preventing scalds to children. PMID- 7549624 TI - Long-term care in later life. PMID- 7549625 TI - HIV virus does cause AIDS, research shows. PMID- 7549626 TI - South Africa steps up fight against HIV and AIDS. PMID- 7549627 TI - Newspaper discloses US Medicare "millionaires". PMID- 7549628 TI - Employers in UK are wary of mental illness. PMID- 7549629 TI - Family asks to end life of woman in coma. PMID- 7549630 TI - Incidence of cancer among Finnish airline cabin attendants, 1967-92. AB - OBJECTIVE: To assess whether occupational exposure among commercial airline cabin attendants are associated with risk of cancer. DESIGN: Record linkage study. SETTING: Finland. SUBJECTS-1577 female and 187 male cabin attendants who had worked for the Finnish airline companies. MAIN OUTCOME MEASURE: Standardised incidence ratio; expected number of cases based on national cancer incidences. RESULTS: A significant excess of breast cancer (standardised incidence ratio 1.87 (95% confidence interval 1.15 to 2.23)) and bone cancer (15.10 (1.82 to 54.40)) was found among female workers. The risk of breast cancer was most prominent 15 years after recruitment. Risks of leukaemia (3.57 (0.43 to 12.9)) and skin melanoma (2.11 (0.43 to 6.15) were not significantly raised. Among men, one lymphoma and one Kaposi's sarcoma were found (expected number of cases 1.6). CONCLUSIONS: Although the lifestyle of cabin attendants is different from that of the reference population--for example, in terms of social status and parity- concentration of the excess risks to primary sites sensitive to radiation suggests that ionising radiation during flights may add to the cancer risk of all flight personnel. Otherwise the lifestyle of cabin attendants did not seem to affect their risks of cancer. Estimates of the effect of reproductive risk factors only partly explained the increased risk of breast cancer. If present estimates of health hazards due to radiation are also valid for cosmic radiation, then the radiation doses of cabin attendants seem too small to account entirely for the observed excess risk. PMID- 7549631 TI - Who is nutritionally vulnerable in Bosnia-Hercegovina? AB - OBJECTIVE: To monitor nutritional status and food security in order to identify nutritionally vulnerable groups. DESIGN: Members of five different household groups (urban and rural residents, displaced people in collective centres and private accommodation, elderly people living without younger family) and all residents of two old people's homes were prospectively followed. Households were selected from 20 local communities and nine collective centres. SETTING: Monitoring carried out in three besieged areas of Bosnia-Hercegovina (Sarajevo, Tuzla, and Zenica). SUBJECTS: 1739 individuals sampled. INTERVENTIONS: Data collected every month from December 1993 to May 1994. Information on household food security was collected through structured questionnaires. All subjects were weighed and their heights measured. Weight for age Z scores were calculated for children; body mass index was calculated for adults and elderly people. RESULTS: From December 1993 to February 1994, before a temporary cease fire, access to food was reduced. In February 1994 no significant signs of undernutrition were detected among children or adults, but elderly people had higher than expected levels of undernutrition (15.5% with body mass index < 18.5), a higher rate of weight loss than adults (1.2 kg over two months), and a higher prevalence of self reported illness. CONCLUSIONS: Elderly people in Bosnia-Hercegovina are at greater risk of undernutrition than other age groups. Undernutrition may be precipitated in elderly people by sickness, cold, stress, and problems related to food preparation. The health and welfare of elderly people during the emergency in Bosnia-Hercegovina require special attention, and integrated age care programmes are needed. PMID- 7549632 TI - Glucose tolerance in patients with cystic fibrosis: five year prospective study. AB - OBJECTIVES: To study prevalence and incidence of diabetes mellitus in patients with cystic fibrosis. DESIGN: Five year prospective study with annual oral glucose tolerance tests. SETTING: CF Center Copenhagen, Denmark. SUBJECTS: 191 patients with cystic fibrosis aged above 2 years. MAIN OUTCOME MEASURES: Glucose tolerance, plasma glucose concentrations after fasting and after glucose loading, and haemoglobin A1c levels. RESULTS: Prevalence of diabetes increased from 11% (n = 21) to 24% (n = 46) during study, with annual age dependent incidence of 4-9%. Diabetes was diagnosed at median age of 21 (range 3-40). At diagnosis of diabetes, symptoms of hyperglycaemia were present in 33% of patients, fasting hyperglycaemia (> or = 7.8 mmol/l) was seen in 16%, and increased haemoglobin A1c levels (> 6.4%) were seen in 16%. Impaired glucose tolerance implied higher risk for development of diabetes than normal glucose tolerance (odds ratio 5.6). In 58% of cases with impaired glucose tolerance, however, glucose tolerance was normal at next annual test. Normal glucose tolerance was found in only 37% of patients at all five tests. Within this group of patients, median plasma glucose concentrations after fasting and after glucose loading and haemoglobin A1c levels increased by 6-8% during study. CONCLUSIONS: Prevalence and incidence of diabetes in cystic fibrosis patients was high and increased with age. Since hyperglycaemic symptoms, fasting hyperglycaemia, and increased levels of glycated haemoglobin did not reliably identify diabetes mellitus, we recommend annual oral glucose tolerance tests in all cystic fibrosis patients aged over 10 years. PMID- 7549633 TI - Ethics approval for a national postal survey: recent experience. PMID- 7549634 TI - Cross district comparison of applications to research ethics committees. PMID- 7549635 TI - Ethics committees: impediments to research or guardians of ethical standards? PMID- 7549636 TI - Randomised clinical trial of medical evacuation and surgical curettage for incomplete miscarriage. PMID- 7549637 TI - Review of prescribed treatment for children with asthma in 1990. AB - OBJECTIVE: To review treatment prescribed to asthmatic children in Great Britain during the 12 months after publication of the first guidelines and to assess effectiveness of prophylactic treatment. DESIGN: Review of prescribing information from January 1990 to June 1991 in a representative sample of general practices in Great Britain with a Compufile/AAH Meditel computer. SUBJECTS: 17,846 children with asthma aged 4-17 years. MAIN OUTCOME MEASURES: Numbers of children prescribed different asthma treatments; estimated use of inhaled beta agonists in those receiving prophylactic treatment. RESULTS: From January to December 1990, 9,362 (52.5%) children were prescribed preventive treatments. 16,211 (90.8%) children were prescribed bronchodilators of some kind. 3,055 (17.1%) were prescribed sodium cromoglycate, and the proportion decreased significantly during the study (from 19.5% (95% confidence interval 18.6% to 20.4%) to 17.2% (16.4% to 18.1%), P < 0.001, in children aged 4-11 years and from 14.9% (14.0% to 15.9%) to 11.3% (10.4% to 12.2%), P < 0.001, in those aged 12-17 during January-July 1991). 6,952 (39.0%) were prescribed inhaled steroids, and the proportion increased during the study (from 35.1% (34.0% to 36.2%) to 44.1% (43.0% to 45.2%), P < 0.001, in children aged 4-11 years and from 38.7% (37.4% to 40.0%) to 44.1% (42.7% to 45.5%), P < 0.001, in those aged 12-17 during January July 1991). Only 1,358 of the 9,362 children (14.5%) received sufficient repeat prescriptions to suggest that they might be taking the prophylactic treatment regularly. Among these children short acting inhaled beta agonists were being used on average four to eight times a day. CONCLUSIONS: These results are useful baseline data for audit of the impact of published clinical guidelines, particularly in terms of reducing the need for short acting inhaled beta agonists with prophylactic treatment. PMID- 7549638 TI - Clinical ethics committee. AB - An informal clinical ethics committee was set up to advise on ethical problems in prenatal diagnosis in Leeds. It was used twice in six months but was not called on again in the subsequent year, and we describe this experience. In North America similar committees are often used to advise on clinical moral dilemmas, and we review the published evidence from there and discuss some of the advantages and problems. Our committee's advice may have altered clinicians' actions considerably, but perhaps doctors in Britain are not yet ready to surrender this aspect of clinical autonomy. PMID- 7549639 TI - Second opinions: a right or a concession? PMID- 7549640 TI - ABC of medical computing. CD ROMS, multimedia, and optical storage systems. PMID- 7549641 TI - Acquired haemophilia: an important but often unrecognised cause of bleeding in elderly people. PMID- 7549642 TI - Write an obituary for the BMJ. PMID- 7549644 TI - Drug donations must be strictly regulated. Georgia has tight guidelines. PMID- 7549643 TI - Drug donations must be strickly regulated. Assortment of drugs causes more problems than it solves. PMID- 7549645 TI - Hospital acquired infections could still be prevented. PMID- 7549646 TI - Increasing the number of organ transplants. PMID- 7549647 TI - Oral versus intravenous antibiotics. Exclusion of sickest patients invalidates study. PMID- 7549649 TI - Health care rationing. Implicit rationing is not open to public scrutiny. PMID- 7549648 TI - Provision of services for cleft lip and palate. PMID- 7549650 TI - Health care rationing. Implicit rationing should take place within a framework. PMID- 7549651 TI - Health care rationing. Muddling through leaves matters to chance. PMID- 7549652 TI - Bone densitometry in clinical practice. Clinical uses of densitometry are not yet proved. PMID- 7549654 TI - Publishers' copyright policy. PMID- 7549653 TI - Bone densitometry in clinical practice. Is indicated after long term treatment with heparin. PMID- 7549655 TI - Ethical imperative to publish extends to academics too. PMID- 7549656 TI - Reasons for increased incidence of tuberculosis. PMID- 7549657 TI - 100 years of x rays. PMID- 7549659 TI - Should older women be offered in vitro fertilisation? Authors' premise is faulty. PMID- 7549658 TI - Anti-therapeutic community mental health law. PMID- 7549660 TI - Telephony remains useful in medical informatics. PMID- 7549661 TI - Should older women be offered in vitro fertilisation? Counselling may be a better use of resources. PMID- 7549662 TI - Lowering patients' cholesterol. Excluding patients from trials increases uncertainty. PMID- 7549663 TI - Lowering patients' cholesterol. Few eligible patients currently receive treatment. PMID- 7549664 TI - Should older women be offered in vitro fertilisation? Existing children are treated differently from embryos. PMID- 7549665 TI - Lowering patients' cholesterol. Extrapolating results of trial of simvastatin gives room for doubt. PMID- 7549666 TI - Occupational health: undefined, under reported, and uncompensated. Definitions of disease categories are important. PMID- 7549667 TI - Occupational health: undefined, under reported, and uncompensated. Occupational health pilot study finds unmet need. PMID- 7549668 TI - Occupational health: undefined, under reported, and uncompensated. Prescribed diseases yield substantial compensation. PMID- 7549669 TI - Incidence of toxoplasma retinochoroiditis. PMID- 7549670 TI - Coping with a diagnosis of breast cancer. PMID- 7549671 TI - Informed consent to postmarketing research. British rules outlaw disguised promotion of drugs. PMID- 7549672 TI - Informed consent to postmarketing research. Patients may feel pressured to participate. PMID- 7549673 TI - Breast cancer and hormonal supplements in postmenopausal women. PMID- 7549674 TI - After a first episode of venous thromboembolism. PMID- 7549675 TI - To beta block or better block? PMID- 7549676 TI - Magnesium sulphate: the drug of choice in eclampsia. PMID- 7549677 TI - Short termism in the NHS. PMID- 7549678 TI - Drug combination adds fuel to US abortion debate. PMID- 7549679 TI - India's pneumonic plague outbreak continues to baffle. PMID- 7549680 TI - Canada considers capitation fee for GPs. PMID- 7549681 TI - Dutch doctors tighten euthanasia guidelines. PMID- 7549682 TI - WMA rethinks patients' rights. PMID- 7549683 TI - Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors. AB - OBJECTIVE: To investigate the relation between seropositivity to chronic infections with Helicobacter pylori and Chlamydia pneumoniae and both coronary heart disease and cardiovascular risk factors. DESIGN: Cross sectional study of a population based random sample of men. Coronary heart disease was assessed by electrocardiography, Rose angina questionnaire, and a history of myocardial infarction; serum antibody levels to H pylori and C pneumoniae were measured, risk factor levels determined, and a questionnaire administered. SETTING: General practices in Merton, Sutton, and Wandsworth, south London. SUBJECTS: 388 white south London men aged 50-69. MAIN OUTCOME MEASURES: Evidence of coronary risk factors and infection with H pylori or C pneumoniae. RESULTS: 47 men (12.1%) had electrocardiographic evidence of ischaemia or infarction. 36 (76.6%) and 18 (38.3%) were seropositive for H pylori and C pneumoniae, respectively, compared with 155 (45.5%) and 62 (18.2%) men with normal electrocardiograms. Odds ratios for abnormal electrocardiograms were 3.82 (95% confidence interval 1.60 to 9.10) and 3.06 (1.33 to 7.01) in men seropositive for H pylori and C pneumoniae, respectively, after adjustment for a range of socioeconomic indicators and risk factors for coronary heart disease. Cardiovascular risk factors that were independently associated with seropositivity to H pylori included fibrinogen concentration and total leucocyte count. Seropositivity to C pneumoniae was independently associated with raised fibrinogen and malondialdehyde concentrations. CONCLUSIONS: Both H pylori and C pneumoniae infectins are associated with coronary heart disease. These relations are not explained by a wide range of confounding factors. Possible mechanisms include an increase in risk factor levels due to a low grade chronic inflammatory response. PMID- 7549684 TI - Long term effects of smoking on physical fitness and lung function: a longitudinal study of 1393 middle aged Norwegian men for seven years. AB - OBJECTIVE: To study association between smoking habits and long term decline in physical fitness and lung function in middle aged men who remained healthy. DESIGN: Baseline and follow up measurements performed during 1972-5 and 1980-2 respectively. SETTING: National University Hospital of Oslo, Norway. SUBJECTS: 1393 men aged 40-59 at baseline who were all healthy at baseline and at follow up. MAIN OUTCOME MEASURES: Forced expiratory volume in one second and physical fitness (defined as total work done during a symptom limited bicycle ergometer test divided by body weight. RESULTS: Initial fitness was substantially lower among 347 persistent smokers than among 791 persistent non-smokers (1349 J/kg v 1618 J/kg), as was initial forced expiratory volume (3341 ml v 3638 ml). Mean (95% confidence interval) decline in fitness over 7.2 years was 217 (185 to 249) J/kg among smokers compared with 86 (59 to 113) J/kg among non-smokers (P < 0.001). Corresponding declines in forced expiratory volume were 271 (226 to 316) ml in smokers and 116 (85 to 147) ml in non-smokers (P < 0.001). Differences between smokers and non-smokers remained practically unchanged after adjustment for age and level of physical activity. Changes in fitness and forced expiratory volume among 199 men who had stopped smoking mimicked the findings for persistent non-smokers, and 56 men who started smoking presented findings close to those of persistent smokers. CONCLUSION: Decline in physical fitness and lung function among healthy middle aged men was considerably greater among smokers than among non-smokers and could not be explained by differences in age and physical activity. PMID- 7549685 TI - Social cost of land mines in four countries: Afghanistan, Bosnia, Cambodia, and Mozambique. AB - OBJECTIVES: To document the effects of land mines on the health and social conditions of communities in four affected countries. DESIGN: A cross design of cluster survey and rapid appraisal methods including a household questionnaire and qualitative data from key informants, institutional reviews, and focus groups of survivors of land mines from the same communities. SETTING: 206 communities, 37 in Afghanistan, 66 in Bosnia, 38 in Cambodia, and 65 in Mozambique. SUBJECTS: 174,489 people living in 32,904 households in the selected communities. MAIN OUTCOME MEASURES: Effects of land mines on food security, residence, livestock, and land use; risk factors: extent of individual land mine injuries; physical, psychological, social, and economic costs of injuries during medical care and rehabilitation. RESULTS: Between 25% and 87% of households had daily activities affected by land mines. Based on expected production without the mines, agricultural production could increase by 88-200% in different regions of Afghanistan, 11% in Bosnia, 135% in Cambodia, and 3.6% in Mozambique. A total of 54,554 animals was lost because of land mines, with a minimum cash value of $6.5m, or nearly $200 per household. Overall, 6% of households (1964) reported a land mine victim; a third of victims died in the blast. One in 10 of the victims was a child. The most frequent activities associated with land mine incidents were agricultural or pastoral, except in Bosnia where more than half resulted from military activities, usually during patrols. Incidences have more than doubled between 1980-3 and 1990-3, excluding the incidents in Bosnia. Some 22% of victims (455/2100) were from households reporting attempts to remove land mines; in these households there was a greatly increased risk of injury (odds ratio 4.2 and risk difference 19% across the four countries). Lethality of the mines varied; in Bosnia each blast killed an average of 0.54 people and injured 1.4, whereas in Mozambique each blast killed 1.45 people and wounded 1.27. Households with a land mine victim were 40% more likely to experience difficulty in providing food for the family. Family relationships were affected for around one in every four victims and relationships with colleagues in 40%. CONCLUSIONS: Land mines seriously undermine the economy and food security in affected countries; they kill and maim civilians at an increasing rate. The expense of medical care and rehabilitation add economic disability to the physical burden. Awareness of land mines can be targeted at high risk attitudes, such as those associated with tampering with mines. PMID- 7549686 TI - Interpreting self reported limiting long term illness. AB - OBJECTIVE: To examine the association between self reported limiting long term illness and other dimensions of self reported health. DESIGN: Stratified random sample of general population. SETTING: Lothian region, Scotland, in 1993. SUBJECTS: 6212 men and women aged 16 and over. MAIN OUTCOME MEASURES: Limiting long term illness was assessed by the same question as used in the 1991 United Kingdom census. The short form 36 health survey was used to assess other dimensions of health. RESULTS: Rates of limiting long term illness were much higher than reported in the census. Scores on general and physical health scales had strong associations with limiting long term illness, but after adjustment for these associations psychosocial health measures had little influence on limiting long term illness. Being at the lower rather than the upper quartile on the physical functioning scale more than doubled the odds of having limiting long term illness. Reported prevalence of many common illnesses was between two and three times higher among those with limiting long term illness. CONCLUSIONS: A positive response to the question used by the census to define limiting long term illness was strongly associated with physical limitations on activity and less strongly influenced by scores on scales of mental and social wellbeing. Socioeconomic effects on limiting long term illness seem largely mediated through measures of general health and physical limitations on health. PMID- 7549687 TI - Urban hypothermia in the west of Scotland. West of Scotland Accident and Emergency Trainees Research Group. PMID- 7549688 TI - Survey of scope of neonatal screening in the United Kingdom. PMID- 7549689 TI - What do we known about fundholding in general practice? AB - The general practice fundholding scheme was introduced four years ago. So far its impact has not been formally evaluated nationally, but review of published research shows some trends. Fundholding has curbed prescribing costs and given general practitioners greater power to lever improvements in hospital services- for example, reducing waiting times for hospital treatment--but fundholding practices may have received more money than non-fundholding practices. The impact of fundholding on transactions costs, equity, and quality of care (particularly for patients of non-fundholding general practitioners) is unknown. Research into costly reforms such as fundholding needs to be coordinated. PMID- 7549690 TI - Treatment of acute anaphylaxis. PMID- 7549691 TI - How to do it. Get patients' consent to enter clinical trials. AB - Gaining patients' consent to enter clinical trials is essential, but not easy. Giving careful thought to the design of the study itself, information which patients receive, and the use of a signed consent form may all help. To be properly informed, patients need to know something about their condition, the proposed study, and alternative options. The type and amount of information will vary and investigators need to judge the level appropriate for each person. Patients should understand that taking part in a clinical trial is voluntary and that their decision will not affect the quality of care they receive. The process of obtaining consent requires time and good communication. Working with young, elderly, or mentally impaired patients, or those particularly vulnerable to coercion, requires special sensitivity to the potential dangers. PMID- 7549692 TI - Lumbar physiological striae in adolescence suspected to be non-accidental injury. PMID- 7549693 TI - ABC of medical computing. Using computers in clinical audit. PMID- 7549694 TI - Advice on lifestyle dilutes important smoking message. PMID- 7549695 TI - Asking patients to write lists. Clinic audit supports it. PMID- 7549696 TI - Asking patients to write lists. Randomised controlled trials support it. PMID- 7549697 TI - Faxed electronic summaries are valued by general practitioners. PMID- 7549698 TI - Analgesic effect of sucrose. Heel pricks were unnecessarily painful. PMID- 7549699 TI - Babies' deaths linked to suboptimal care. PMID- 7549700 TI - Late diagnosis of HIV infection in children is common and devastating. PMID- 7549701 TI - Analgesic effect of sucrose. Cuddle deprivation may have confounded experiment. PMID- 7549702 TI - Adult survivors of child sex abuse. Group therapies are also effective. PMID- 7549703 TI - Protecting adolescent girls against tetanus. Targeting both sexes may avoid misunderstandings. PMID- 7549704 TI - Protecting adolescent girls against tetanus. Induced abortion can predispose to tetanus. PMID- 7549705 TI - Nestle obeys Indian label laws. PMID- 7549706 TI - Non-Hodgkin's lymphoma and skin cancer. Ultraviolet light is unlikely explanation for association. PMID- 7549707 TI - Non-Hodgkin's lymphoma and skin cancer. Pathogenesis is multifactorial. PMID- 7549708 TI - Non-Hodgkin's lymphoma and skin cancer. Association may be iatrogenic. PMID- 7549709 TI - Non-Hodgkin's lymphoma and skin cancer. American data refute ultraviolet hypothesis. PMID- 7549710 TI - Meta-analysis of antidepressant prescribing. Acceptability of side effects as reason for stopping may bias results. PMID- 7549711 TI - Meta-analysis of antidepressant prescribing. Drop out rates presented in a misleading manner. PMID- 7549712 TI - Meta-analysis of antidepressant prescribing. Important differences exist between second generation antidepressants. PMID- 7549713 TI - Postgraduate deans will deal with accusations of fraud. PMID- 7549714 TI - Head lice resistant to pyrethroid insecticides in Britain. PMID- 7549715 TI - Flaws in agist arguments. Doctors need pragmatic strategies. PMID- 7549716 TI - Flaws in agist arguments. Philosopher acquiesces to prevailing social mores. PMID- 7549717 TI - Healthy eating in Wales. PMID- 7549718 TI - Integrating pharmacy into the primary care team. "One stop clinic" has advantages for patients. PMID- 7549719 TI - Integrating pharmacy into the primary care team. Freedom from the dispensary is essential. PMID- 7549720 TI - Misleading meta-analysis. PMID- 7549721 TI - Definitions in palliative care. PMID- 7549722 TI - Emergency delays. PMID- 7549723 TI - NHS computer network will breach venereal disease regulations. PMID- 7549724 TI - Hepatitis C and haemophilia. PMID- 7549725 TI - Careers advice for medical undergraduates. PMID- 7549726 TI - Israeli Medical Association shirks "political aspects" of torture. PMID- 7549727 TI - Private prescribing of controlled drugs. PMID- 7549728 TI - Barrett's esophagus and esophageal adenocarcinoma: the scope of the problem. PMID- 7549729 TI - Simultaneous detection of esophageal and gastric carcinomas. AB - The case histories of six patients are presented where the diagnosis of carcinoma of the esophagus and the stomach was made at the same time by barium studies. The clinical history was elusive in two patients. In four of the six patients, histological proof of squamous carcinoma of the esophagus and adenocarcinoma of the stomach was obtained, but in the other two biopsy of the gastric lesion was not possible because of the extensive esophageal cancer. PMID- 7549730 TI - Percutaneous feeding gastrostomy in patients with a partial gastrectomy: transhepatic approach with CT guidance. AB - BACKGROUND: Percutaneous gastrostomy in patients with a partial gastrectomy is rarely performed because the gastric remnants are generally small, positioned high subcostally, and overlaid by the transverse colon and the left lobe of the liver. METHODS: We performed percutaneous transhepatic feeding gastrostomy in four patients with a partial gastrectomy whose conditions precluded oral feeding. The gastric remnant was punctured with a 22-gauge percutaneous transhepatic cholangiography needle through the left lobe of the liver with computed tomography guidance and following tract dilation over the guidewire 8 or 9F. Cope loop catheters were fluoroscopically placed. RESULTS: No obvious complications were encountered during the procedures. The catheter feedings were continued for 2 to 7 months without any serious problems. CONCLUSIONS: We conclude this technique can be performed easily and may be safe because of the presence of the adhesion between the remnant and the liver, which prevents a massive hemorrhage or the displacement of the catheters. PMID- 7549732 TI - Calcified splenic metastasis from gastric carcinoma. AB - Isolated splenic metastases are uncommon. We report CT and MRI features of an isolated, large, calcified splenic metastasis from a primary adenocarcinoma of the stomach. Presence of a calcified splenic mass with a known primary malignancy of mucinous nature should suggest the diagnosis of splenic metastasis even if it occurs in isolation. PMID- 7549731 TI - Obstructive jaundice in gastric carcinoma: cause, site, and relationship to the primary lesion. AB - OBJECTIVE: Obstructive jaundice is frequently present in patients with advanced gastric carcinoma. The purpose of this study was to assess the cause and preferential site of bile duct obstruction in patients with gastric carcinoma and to evaluate correlativity of biliary obstruction with the nature of the primary gastric lesion. METHODS: Cholangiographic findings of 54 patients with metastatic gastric carcinoma presenting with obstructive jaundice were reviewed retrospectively. The level of the bile duct obstruction was divided into four segments: segment 1, from an individual intrahepatic duct to the biliary hilum; segment 2, common hepatic duct (CHD) involvement from the biliary hilum to the level of the cystic duct; segment 3, the proximal half of the common bile duct (CBD); segment 4, the distal half of the CBD. To evaluate the characteristics of the primary gastric lesion, operative records and pathologic findings were reviewed. RESULTS: Obstruction sites were segment 1 in eight patients (15%), segment 2 in 25 (46%), segment 3 in 17 (32%), and segment 4 in four (7%). The causes of obstruction were metastatic lymphadenopathy in the hepatoduodenal ligament (50 of 54) and direct invasion of the primary or recurrent tumor (four of 54). The location of the primary gastric lesions was the antrum, antrum and body, and body in 36 (67%), 17 (31%), and 1 (2%), respectively. Borrmann type 3 lesions were present in 72% of cases, and type 2 lesions in the remaining 24%. Histologic type was undifferentiated adenocarcinoma in 91% of patients, and differentiated adenocarcinoma in the remaining. Serosal invasion was shown in 96% of the patients. CONCLUSION: Our results show that the cause of bile duct obstruction in advanced gastric carcinoma is predominantly metastatic lymphadenopathy in the hepatoduodenal ligament, and its preferential site is around the level of the cystic duct. Obstructing lesions showed characteristic cholangiographic findings. PMID- 7549733 TI - CT diagnosis of intestinal ascariasis. PMID- 7549734 TI - CT identification of ascaris in the biliary tract. AB - Ascariasis is one of the most common helminthic diseases worldwide. The presence of this worm in the biliary tree causes biliary colic, recurrent pyogenic cholangitis, pancreatitis, hepatic abscesses, and septicemia. The diagnosis of biliary ascariasis is usually made by ultrasound (US). We report the computerized tomography (CT) aspects that allowed the identification of ascaris in the biliary tract in two patients. PMID- 7549735 TI - Ultrasound in the diagnosis of gallbladder ascariasis. AB - Ascariasis is the most common helminthic infection, but the invasion of worms into the gallbladder is quite rare. This report illustrates the ultrasonographic findings in gallbladder ascariasis of a typical echogenic structure which exhibits nondirectional movements and contains a central anechoic tube, along the long axis of the gallbladder. The role of ultrasound as a diagnostic tool and in the follow-up treatment is stressed. PMID- 7549736 TI - Ultrasound examination of the small bowel: comparison with enteroclysis in patients with Crohn disease. AB - BACKGROUND: Screening for inflammatory small bowel disease has hereto relied on barium examination, usually performed after duodenal intubation. A noninvasive technique for imaging of the small bowel in such patients would be preferable. METHODS: A total of 59 patients were included in the study. A small bowel barium examination (SBE) was performed after duodenal intubation using a barium and air double-contrast technique. Ultrasound (US) of the right lower quadrant was performed with a 3.5- or 5-MHz transducer. The patients fasted overnight. RESULTS: In 37 of 39 patients with a normal SBE, US was also normal. In 20 patients, SBE showed lesions compatible with Crohn disease and in 18 of these the US study showed thickening of the bowel wall. One of these patients later tested positive for Yersinia enterocolitica. There were two false-positive and two false negative US examinations. For detection of inflammatory disease of the small bowel, US was calculated to have a sensitivity of 0.95, specificity of 0.93, accuracy of 0.93, predictive value of a positive test was 0.90, and a predictive value of a negative test was 0.95. CONCLUSIONS: US, therefore, seems to be a reliable method in the workup of patients suspected of having inflammatory small bowel disease. Thereby, US probably can select patients for SBF. PMID- 7549738 TI - Drug smuggling by ingested cocaine-filled packages: conventional x-ray and ultrasound. AB - PURPOSE: To evaluate the imaging characteristics and the diagnostic value of both abdominal ultrasound and conventional abdominal x-ray in identifying ingested drug packages in "body packers." MATERIALS AND METHODS: Twelve individuals were studied prospectively by abdominal ultrasound and by abdominal x-ray to rule out incorporated drug containers. RESULTS: Both abdominal ultrasound and abdominal x ray correctly identified the 7 of 12 individuals who had ingested multiple drug packages. In 5 of the 12 individuals, ultrasound as well as abdominal x-ray were correctly unremarkable with regard to abdominal foreign bodies. CONCLUSIONS: Abdominal ultrasound and abdominal x-ray both represent valuable diagnostic tools in the assessment of ingested drug packages. Abdominal x-ray is superior to ultrasound in differentiating ingested drug-filled condoms from "pseudocondoms." PMID- 7549737 TI - Spiral CT demonstration of hypervascularity in Crohn disease: "vascular jejunization of the ileum" or the "comb sign". AB - OBJECTIVE: To report spiral CT imaging features of the vascular alterations observed in active, early, or recurrent Crohn disease of the small bowel and colon. METHODS: Spiral CT examinations of the abdomen and pelvis following oral and intravenous contrast were performed in two cases of active Crohn disease. RESULTS: Spiral CT features of hypervascularity involving the mesenteric vessels of affected segments of bowel include vascular dilatation, tortuosity, and conspicuous prominence and wide spacing of the vasa recta (the "comb" sign). This is attributed to the factors of increased flow and the fibrofatty proliferation in the mesentery and serosa of the affected bowel. CONCLUSION: Clinical application of these observations may enhance the diagnosis of Crohn disease presenting initially or as an acute recurrence in the differential diagnosis from other conditions. PMID- 7549739 TI - The inside dope: cocaine, condoms, and computed tomography. PMID- 7549740 TI - Duct ectasia due to mucus-producing cancers with intraductal extension: histopathologic correlation with radiologic imagings. AB - BACKGROUND: Although duct ectasia due to mucus-producing pancreatic cancer has been well known, its occurrence in other organs has not been reported. We have studied the pathologic basis of the radiologic features in cases of mucus producing cancer with intraductal extension. METHODS: We conducted a comparative study of detailed various radiographic images and pathological findings in 10 cases of mucus-producing cancers (four of mucus-producing pancreatic cancer, two of mucinous gastric cancer with diffuse lymphangitic liver metastasis, and four cases of bronchoalveolar cell carcinoma with cyst or cavity). RESULTS: When mucus producing cancer occurs in a secretory duct or extends into lymphatic ducts, the normal duct is dilated due to the mucus and increased internal pressure, leading to the formation of a cyst and cavity. Because of having the liquid property of mucus floating cancer cells, the lesion can easily progress to continuous ducts as well as regionally. CONCLUSIONS: In cases of mucus-producing cancer with extension into the ductal structure not only in the pancreas but also in the lung and liver, continuous duct ectasis and cysts filled with mucus or cavities of all sizes made by the nature of the mucus may be detected by CT and US. PMID- 7549741 TI - Imaging of the liver: a survey update of prevailing techniques for conventional CT scanning. AB - BACKGROUND: A survey of the Society of Computed Body Tomography/Magnetic Resonance (SCBT/MR) was performed to assess current techniques in liver CT scanning. METHODS: The study was designed as an update to a study performed in 1987. The survey was distributed to 67 members of the SCBT/MR at 35 institutions. RESULTS: Twenty-six institutions responded. As in 1987, none relied solely on noncontrast scans. In 1987, only 54% (12/22) of institutions performed contrast enhanced scans as their primary technique compared with 73% (19/26) in 1993. Ionic contrast was used exclusively in the earlier study, whereas in the present study 58% used nonionic contrast in the majority of cases and 38% used nonionic contrast routinely. In 1987, 41% performed scans with a power injector compared with 85% in the present study. Enhanced scans were performed during the contrast bolus in 36% of institutions in 1987 compared with 76% in this study. No institution relied on noncontrast scans alone. In the previous study the delay between injection and scanning was variable (0-60 s), whereas in the present study 83% specified a delay of 21-45 s. CONCLUSION: Significant refinements in CT technique, wider use of power injectors, utilization of nonionic contrast, and a more critical approach to optimize liver imaging have created a significant impact on the practice of liver CT. PMID- 7549742 TI - Magnetic resonance cholangiography. AB - Magnetic resonance cholangiography (MRC) can be performed with data from a routine imaging protocol, without the need for additional pulse sequences or special equipment. We studied three patients with obstructive jaundice who underwent magnetic resonance imaging (MRI) of the liver. T2-weighted fat suppressed fast spin-echo sequences were processed with a maximum intensity projection algorithm to create three-dimensional images of the dilated portions of the biliary tree. Results were correlated with endoscopic retrograde cholangiopancreatography and computed tomography. These images compared favorably with those acquired on scanners in which special breath-holding gradient echo protocols are used. PMID- 7549743 TI - Peripheral cholangiocarcinoma: comparison of MRI with CT. AB - To evaluate the clinical utility of computed tomography (CT) and magnetic resonance imaging (MRI) in the diagnosis of peripheral cholangiocarcinoma of the liver, 11 patients with pathologically proven peripheral cholangiocarcinoma were examined with both CT and MRI. On CT scans in 10 cases, the tumors appeared as irregular, low-attenuation masses with a wide variation in heterogeneity. Contrast enhancement of the tumors was mild in nine cases and moderate in one case, at the periphery. Tumor was not identified in one case. On T1-weighted MRIs, the tumors showed low intensity in eight cases and isointensity in three cases. On T2-weighted images, the tumors showed high intensity in all 11 cases. Focal dilatation of the intrahepatic bile ducts around the tumor was seen in one case on MRIs and in four cases on CT scans. Portal vein invasion of the tumors was seen in one case, and lymphadenopathy was seen in four cases on both MRIs and CT scans. MRI was slightly superior to CT in detecting the tumors, was inferior to CT in delineating focal ductal dilatation around the tumors, and was equal to CT in assessing extent of the tumors. PMID- 7549745 TI - Transitional cell carcinoma of the upper urinary tract: staging by MRI. AB - BACKGROUND: This study evaluates the ability of MRI to stage transitional cell carcinoma of the upper urinary tract. METHODS: Nine patients who had transitional cell carcinoma of the upper urinary tract detected by other imaging modalities underwent MRI examination at 1.5 T. Imaging included pre- and postgadolinium-DTPA T1-weighted images (9 patients) pre- and postgadolinium chelate T1-weighted fat suppressed spin echo (7 patients). Postcontrast images were acquired prior to the presence of gadolinium within the collecting system (< 2 min postcontrast), intermediate (2.5-8 min), and late (> 10 min) postcontrast. Images were prospectively interpreted and lesion staging was determined. Correlation with histopathology was obtained in all cases. RESULTS: Transitional cell cancers were demonstrated in 9/9 patients, and tumors ranged in size from 2 to 8 cm (mean = 3.8 cm) in one dimension. Correct tumor staging was performed in 8/9 patients. The staging error in one case occurred because direct tumor extension into the renal parenchyma was not detected. CONCLUSIONS: The results of this preliminary study show that MRI stages transitional cell cancers relatively well; however, MRI is not able to detect superficial invasion of renal parenchyma. PMID- 7549744 TI - MRI findings in Caroli's disease and intrahepatic pigmented calculi. AB - This paper reports a case of Caroli's disease confined to the left lobe of the liver that mimicked left portal vein thrombosis on MRI studies because of the very high signal intensity on T1-weighted images of intrahepatic pigmented calculi. The preoperative diagnosis was a cholangiocarcinoma infiltrating the left hepatic bile duct and portal branch. The final macroscopic and histological diagnosis was Caroli's disease of the left liver lobe with wide enlarged left bile duct containing multiple pigmented calculi. PMID- 7549746 TI - Interureteric ridge edema: incidence and etiology. AB - BACKGROUND: Interureteric ridge edema may be seen at intravenous urography (IVU) and is related to acute lower ureteral obstruction, trauma, or calculi. The purpose of this study was to explore the relationship between interureteric ridge edema and acute distal ureteral obstruction caused by ureteral calculi. METHODS: A total of 338 patients who had IVU for various indications during a 6-month period were reviewed for the presence of interureteric ridge edema. RESULTS: Interureteric ridge edema was seen in 12 (4%) of 338 patients, all with acute lower ureteral obstruction from stones. Interureteric ridge edema was best demonstrated with the partially filled bladder film or post-void bladder film in all cases. CONCLUSION: Interureteric ridge edema is most commonly caused by stone induced distal ureteral obstruction and is less commonly seen with recent passage of a stone or other etiologies. Interureteric ridge edema was present in 26% of patients with acute lower ureteral obstruction. PMID- 7549747 TI - Pelvocalyceal thickening in HIV-associated nephropathy. AB - BACKGROUND: Previously reported causes of renal pelvocalyceal thickening (PCT) include infection, acute tubular necrosis and obstruction. This study was performed to evaluate the significance of PCT noted sonographically in patients with hyperechoic native kidneys. METHODS: We evaluated sonograms of 178 consecutive patients with hyperechoic native (excluding small and hydronephrotic) kidneys for the presence of PCT, and reviewed medical charts of patients with this finding. We also reviewed sonograms of two control groups: 20 consecutive patients with nephrosis unrelated to human immunodeficiency virus (HIV) infection who underwent renal biopsy, and 30 consecutive patients with severe hypoalbuminemia unrelated to kidney disease. RESULTS: PCT was observed in 20 study patients with hyperechoic kidneys. HIV-associated nephropathy (HIVAN) was diagnosed in 15 patients (14 with bilateral PCT) in whom previously reported causes of this appearance were excluded. PCT was not observed in control patients. CONCLUSION: Renal PCT can occur in patients with HIVAN in the absence of HIV-related complications. PCT in HIVAN does not merely reflect nonspecific nephrosis or hypoalbuminemia. HIVAN should be considered if PCT is noted sonographically in hyperechoic kidneys, even in patients not clinically suspected of harboring HIV infection. PMID- 7549748 TI - Castleman disease with renal amyloidosis: imaging findings and clinical significance. AB - We report two cases of renal amyloidosis associated with mesenteric and mediastinal Castleman disease of plasma cell type. Computed tomographic (CT) scan revealed a mesenteric mass with multiple nodal enlargement in one patient and a right paratracheal mass with irregular central calcifications in the second patient. Renal ultrasonography (US) showed increased echogenicity of the renal parenchyma with prominent medullas. Doppler waveform obtained in the kidney associated with mesenteric disease showed complete absence of diastolic flow signal, although it was normal in the patient with mediastinal disease. Recognition of Castleman disease as the cause of renal amyloidosis and the understanding of CT and US findings of this entity are important for the management, as well as its diagnosis. PMID- 7549749 TI - Fibrous pseudotumor of the tunica vaginalis testis: imaging appearance. AB - Fibrous pseudotumor is a benign paratesticular tumor that typically presents as a painless mass of the hemiscrotum. Because this tumor can mimic a malignant process, it is usually not diagnosed preoperatively. We describe a case of fibrous pseudotumor of the tunica vaginalis, demonstrating the ultrasound and magnetic resonance image (MRI) appearance with pathologic correlation. PMID- 7549750 TI - Screening for colorectal cancer and suspected lower gastrointestinal bleeding. PMID- 7549751 TI - Complications of barium enema study following colonoscopic biopsy. PMID- 7549752 TI - Colonoscopic biopsies in colitis. PMID- 7549753 TI - Micronuclei and carcinogen DNA adducts as intermediate end points in nutrient intervention trial of precancerous lesions in the oral cavity. AB - In cancer chemoprevention trials, biomarkers as intermediate end points have gained importance. A variety of biomarkers have been proposed as intermediate end points for upper aerodigestive tract cancers. This study was aimed at studying the frequency of micronucleated cells and carcinogen DNA adducts as indicators of DNA damage and intervention end points in chemoprevention trials. Reverse smokers of chutta (rolled tobacco) from four villages numbering 298 in total were selected. Out of these, 150 were supplemented with four nutrients (vitamin A, riboflavin, zinc and selenium) and 148 controls received placebo, one capsule twice a week for 1 year. Slides of buccal smears were prepared and stained with Fuelgen reaction and counterstained with Fast Green and examined microscopically for the presence of micronucleated cells. Oral cell washings were collected and centrifuged. The DNA adducts were evaluated by the 32P post-labelling assay method. Protein and RNA free DNA (adducted) isolated from the cells was digested with MN/SPD and the DNA adducts isolated by the butanol enrichment procedure. The DNA adducts were identified and quantitated by multidimensional chromatography on PEI-TLC sheets by screen enhanced autoradiography and presented as RAL (relative adduct labelling) values. Both the micronuclei and DNA adducts were significantly elevated in subjects with lesions. At the end of 1 year the frequency of micronuclei decreased significantly (P < 0.001) in the supplemented subjects with or without lesions. The DNA adducts in the supplement group at the end of 1 year also reduced significantly. The adducts decreased by 95% in subjects with all categories of lesions and by 72% in subjects without lesions. No such effects were noted in the placebo group. The two biomarkers investigated in the case study appear to be modifiable by the administration of micronutrient supplements.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549754 TI - Salivary immunoglobulins in cancer patients with chemotherapy-related oral mucosa damage. AB - In 40 patients with neoplastic disorders treated with chemotherapy containing anthracyclines or 5-fluorouracil, objective changes of the oral mucosa were registered in 22 patients (55%). Serum IgG and IgA levels and the mean serum IgG/IgA ratio were normal. On the contrary, the mean IgG/IgA salivary ratio was 1.27 (normally below 1.0) due to an increased salivary concentration of IgG (mean 0.095 g/l), but also due to a decreased IgA concentration (mean 0.075 g/l); the IgG/IgA ratio in saliva was higher in patients with objective changes of the oral mucosa (1.53). Values of the periodontal indices were compatible with the diagnosis of a manifest periodontal disease, which tended to be more severe than in control groups. A positive correlation between the gingival index and concentration of IgG in saliva, a non-linear correlation between the gingival index and salivary IgA and a positive linear correlation between serum IgA concentration and intensity of periodontal attachment level recession, indicate local and systemic immune responses to periodontal tissue alterations and dental plaque components. The IgA related local humoral immune response is, however, operating at a lower concentration level than in healthy individuals. PMID- 7549755 TI - Referral delay in diagnosis of oro/oropharyngeal cancer in Israel. AB - The delay, stage at diagnosis and referral pattern of 543 oro/oropharyngeal cancer patients in Israel were investigated. About two-thirds of the malignancies were diagnosed at early stages. In more than two-thirds there was a delay of at least 2 months from the onset of signs and symptoms up to diagnosis, with a non significant relationship between stage and delay. A significant correlation (P = 0.002) was found between delay and malignancy site. A delay of more than 4 months was observed in 71% of the patients with lip cancer. Cancers of the lip, palate and buccal mucosa were more frequently diagnosed at early stages. A significant correlation (P = 0.001) was found between stage at diagnosis and referral source. Although 86% of the cancers were diagnosed by physicians, nearly half were already at an advanced stage; when the cancers were diagnosed by dentists, 81% of the remainder were at early stages. No significant relationship was found between the referral medical profession and site of diagnosis. No nasopharyngeal, oropharyngeal or laryngeal cancers were diagnosed by dentists. PMID- 7549756 TI - High prevalence of expression of p53 oncoprotein in oral carcinomas from India associated with betel and tobacco chewing. AB - A recent study reported a low prevalence of p53 expression (11%) in oral squamous cell carcinomas (SCCs) from South Asia, in contrast to a high prevalence (averaging 52%) in other studies. It was proposed that the different aetiologies for oral SCCs in the South Asia population, i.e. betel and tobacco chewing in combination with smoking and alcohol consumption as compared to smoking and alcohol consumption alone in other populations, may account for the low prevalence of p53 expression. To confirm this hypothesis, we examined p53 expression immunohistochemically in 23 cases of oral SCC from patients in Southern India. Thirteen of the 23 SCCs (56.5%) demonstrated nuclear p53 staining. The expression of p53 was strongly correlated with the number of tobacco-containing quids chewed per day (r = 0.8). These data support the hypothesis that carcinogens derived from tobacco and betel chewing may induce p53 mutations, which in turn are involved in the development of oral cancer. PMID- 7549757 TI - Prognostic evaluation of HPV-associated precancerous and microinvasive carcinoma of the oral cavity: combined use of nucleolar organiser regions (AgNOR) and proliferating cell nuclear antigen (PCNA). AB - Nucleolar Organiser Regions (NORs) and Proliferating Cell Nuclear Antigen (PCNA) were investigated on routine paraffin embedded histologic sections of 30 oral biopsy specimens (six cases of leukoplakia with low-degree of dysplasia, nine cases of leukoplakia with moderate-degree of dysplasia, nine cases of leukoplakia with severe-degree of dysplasia, six cases of squamous microinvasive carcinomas), tested for HPV-DNA by in situ hybridisation (ISH). The absolute number of NORs per nucleus and the percentage of nuclear positivity for PCNA were found to be different in each group of pathology, with further diversity due to the presence or absence of HPV-DNA. In the major part of HPV-positive lesions, the AgNOR number and percentage of cells positive for PCNA were found to be generally lower than in corresponding negative forms. Conversely, a few cases of HPV+ lesions showed significantly higher values both of AgNOR and PCNA, if compared to the other cases of HPV+ and HPV- lesions. These data suggested that high values of AgNOR and PCNA, in moderate and high grade oral dysplasia, could represent an "alarm signal" of a worse prognosis, and then a possible indication for a strict clinical management and/or a stronger treatment of some HPV-associated preneoplastic lesions. PMID- 7549758 TI - Alcohol, tobacco, diet and the risk of oral cancer: a pooled analysis of three case-control studies. AB - This combined analysis of data from three large case-control studies of oral cancer confirms the important effect of tobacco in the aetiology of the disease. The studies have been conducted in the United States, Italy and China and results for risks associated with tobacco smoking were generally consistent across centres, while those for alcohol were not; increased risks amongst alcohol drinkers were evident in two centres but not in the study conducted in Turin, Italy. In addition, the combined analysis had large enough numbers to analyse the risk of tobacco consumption in non-drinkers. In females these showed increased risks while in males the effect of tobacco alone was weaker. Given the popularity of tobacco smoking, and its consequent high attributable risk in terms of oral cancer it is reassuring, in terms of public health, that cessation will result in a substantial reduction in risk; a 30% reduction in risk for those stopping smoking between 1 and 9 years, and a 50% reduction for those stopping more than 9 years. Although encouraging smokers to stop should be the principal aim, decreases in risk for everyone could be achieved by encouraging high fruit and vegetable consumption. PMID- 7549759 TI - Expression of tenascin in hamster buccal pouch mucosa during experimental carcinogenesis. AB - Experimental carcinogenesis by topical application of 7,12 dimethylbenz(a)anthracene (DMBA) in hamster buccal pouch mucosa was evaluated for expression of tenascin, an extracellular matrix glycoprotein expressed at the epithelial-mesenchymal interface during embryonic and fetal development, wound healing and in the stroma of various neoplastic lesions, by using immunohistochemical methods. The buccal pouch mucosa in normal hamsters showed immunoreactive tenascin either as a linear delicate band or without reactivity at the immediate vicinity of the basement membrane. During carcinogenesis, in the second to fourth week of application of DMBA, the hyalinous changes in the submucosal connective tissue had a weak but diffuse immunoreactivity for tenascin. The hyperkeratinised and hyperplastic mucosa following 5 weeks of application of DMBA showed focal areas of enhanced expression in the vicinity of the basement membrane. Subsequently, specimens showing hyperplasia, dysplasia, carcinoma in situ and invasive carcinomas had comparatively more widespread stromal immunoreactivity where the extent of enhanced reactivity positively correlated with the advancing lesion. These results compared with the results of expression in human normal mucosa, leukoplakia and squamous cell carcinoma of the oral cavity (Shrestha et al., Oral Oncol, Eur J Cancer 1994, 30, 132-137) suggest that the expression of tenascin in experimental carcinogenesis of hamster buccal pouch mucosa, as a model, faithfully mimics the same in human oral mucosa. PMID- 7549760 TI - Evaluation of utilisation of health workers for secondary prevention of oral cancer in Kerala, India. AB - The utilisation of primary health workers (HWs) for cancer control in developing countries has often been suggested, based on the experience of feasibility studies in India and Sri Lanka. We initiated a project in 1988 to evaluate the long-term feasibility of using trained HWs in secondary prevention of oral cancer, to bring about earlier detection of oral cancer in the communities served by them. Two hundred and eighty-two HWs attached to 14 primary health centres (PHCs), serving approximately 0.92 million rural population in the northern half of Trivandrum district in Kerala, India, were trained in oral visual inspection to detect precancerous, malignant and other suspicious lesions of the oral cavity and refer them for confirmation and treatment. They were asked to examine subjects aged 35 years and above and to give person to person health education on tobacco in their target population. The HWs belonging to the PHCs serving approximately 1 million rural population in the southern half of Trivandrum district were not trained, and this region served as the control area. In addition to several process measures, stage distribution of oral cancer in subjects reporting from the intervention and control areas, as well as oral cancers referred by the HWs, as a proportion of total oral cancers from the intervention area, were the outcome measures evaluated. Only 9/282 (3.2%) trained HWs were motivated and they examined 17,812 eligible subjects in 3 years and referred 408 subjects with lesions; 258/408 (63.2%) referred subjects reported for further examination and ten oral cancers were detected among them.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549761 TI - Minor salivary gland tumours. A retrospective study of 164 cases in a Brazilian population. AB - One hundred and sixty-four cases of intraoral salivary gland tumours retrieved from the files of the Surgical Oral Pathology laboratory of the University of Sao Paulo (Brazil), between 1970 and 1993, were studied. Of these, 164 tumours, 62% were classified as benign and 38% malignant. The palate was the main site of occurrence of the tumours followed by the buccal mucosa and upper lip. There was a slight predominance for female patients, with a female to male ratio of 1.3:1. The mean age for benign tumours was 39.9 years (40.8 for females, and 39.7 for males). For malignant tumours the mean age was 43.5 years (42.6 for females and 44.7 for males). Pleomorphic adenoma was the most common of the benign tumours, whereas mucoepidermoid carcinoma and adenoid cystic carcinoma were the most common malignant tumours. In general, benign tumours presented as an asymptomatic nodule. On the other hand, pain, ulceration and radiographic changes were more frequently associated with malignant lesions. PMID- 7549762 TI - Attendance and compliance at an oral cancer screening programme in a general medical practice. AB - The purpose of this study was to measure the attendance and compliance rates in a demonstration invitational screening programme for oral cancer. 4348 subjects aged 40 years or over registered at an inner city medical practice in north London were invited for screening by post. The socioeconomic profile of the group was determined by analysis of residential areas. Screening was conducted by one of several dentists and a referral pathway was established for patients requiring follow-up. Attendance rates for screening and referral for follow-up were measured. The response rate was 985/3826 (25.7%) after removing 522 subjects whose invitations could not be delivered or who refused appointments. No reply was obtained for 2841 patients. Attendance for referral of lesions considered to have malignant potential was 67% (8/12), compared to 92% (11/12) for patients requiring referral for incidental benign lesions. The low compliance suggests that oral cancer screening may not be able to achieve the desired benefits of reducing morbidity and mortality, and establishment of such a programme may not, therefore, be cost-effective. Further research is required into how to identify people in high risk groups and motivate them to present themselves for screening. PMID- 7549763 TI - Oral fasciitis. PMID- 7549764 TI - Effects of live Saccharomyces cerevisiae cells on zoospore germination, growth, and cellulolytic activity of the rumen anaerobic fungus, Neocallimastix frontalis MCH3. AB - The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements. PMID- 7549766 TI - Detection of Bacteroides fragilis in clinical specimens by polymerase chain reaction amplification of the neuraminidase gene. AB - The polymerase chain reaction (PCR) was used in an attempt to detect Bacteroides fragilis by amplifying a segment of the gene encoding B. fragilis neuraminidase. Forty-five reference strains representing 45 species and 113 clinical isolates were tested. Only B. fragilis was PCR positive, except for Bacteroides merdae ATCC 43184, which gave a band by ethidium bromide staining that showed no signal by Southern hybridization. Using a protocol that employed DNA extraction by Sepa Gene kit and a highly sensitive digoxigenin-chemiluminescence detection system, detection of B. fragilis by PCR was in complete agreement with culture results for 44 clinical specimens from which a wide range of aerobic and anaerobic organisms and fungi were recovered. PMID- 7549765 TI - Chitinolytic activity of the anaerobic rumen fungus Piromyces communis OTS1. AB - The anaerobic rumen fungus, Piromyces communis OTS1, was isolated from a fistulated goat. Its chitinolytic activity in the supernatant of media containing different types of chitin was studied. The fungus grew well in our basal medium, with and without colloidal chitin and chitin powder. N-Acetyl-beta glucosaminidase activity was not detected in any of the culture media. Chitinase activity, however, was detected in the basal medium with and without colloidal chitin and chitin powder. The extracellular chitinase concentrated from the fungal culture's supernatant by ammonium sulfate (80% saturation) showed highest activity at 40 degrees C and at pH 5.5. In the other cell fractions of P. communis OTS1, N-acetyl-beta-glucosaminidase was not detected, but chitinase activity was detected by 4-methylumbelliferyl reagents. Thus, it was found that the rumen fungus P. communis OTS1 has chitinase activity. Chitinases from the extracellular, cytosolic, and the microsomal fractions were mainly of the endo type of chitinase activity. PMID- 7549767 TI - Studies on siderophore production and effect of iron deprivation on the outer membrane proteins of Madurella mycetomatis. AB - The purpose of this investigation was to determine whether Madurella mycetomatis, the most frequent agent of eumycotic mycetomas, produces siderophores and synthesizes new outer membrane proteins under iron-starvation conditions. Siderophore production, only of the hydroxamate type, was demonstrated in all nine strains tested. It was regulated by extracellular iron concentrations. Under iron-restricted conditions, M. mycetomatis expressed various outer membrane iron regulated proteins, particularly of 24-kilodalton, that may participate in iron metabolism. PMID- 7549768 TI - Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata. AB - beta-D-Glucosidase, beta-D-fucosidase, beta-D-xylosidase, and beta-D cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. beta-D-Galactosidase and alpha-L-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi. PMID- 7549769 TI - ssp genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus. AB - It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae. PMID- 7549770 TI - Catabolite repression of induction of aldose reductase activity and utilization of mixed hemicellulosic sugars in Candida guilliermondii. AB - NADPH-dependent aldose reductase activity induced by D-xylose or L-arabinose was detected in cell-free extracts of Candida guilliermondii, but only negligible activities were observed if D-glucose served as carbon source. The induction of aldose reductase activity on mixed sugars was investigated under resting cell conditions. D-Glucose repressed enzyme induction by D-xylose or L-arabinose to varying degrees, and L-arabinose inhibited enzyme induction by D-xylose. During incubation in a mixture of D-xylose-D-glucose, glucose consumption by cells was fast and simultaneous with D-xylose utilization. Repression of D-xylose consumption by D-glucose was dependent on hexose initial concentration. L arabinose consumption was poor when it was present as the only sugar and in a mixture with D-glucose; this pentose depletion occurred only when all hexose was consumed. When D-xylose and L-arabinose were present in a mixture, the consumption of both pentoses was reduced by the presence of the second sugar, although both sugars were consumed simultaneously by cells. The results show that induction of aldose reductase activity and D-xylose utilization by cells of Candida guilliermondii are under control of glucose repression. PMID- 7549771 TI - Identification, cloning, and sequencing of DNA essential for encapsulation of Streptococcus pneumoniae. AB - This paper reports the cloning and sequencing of a region of DNA from Streptococcus pneumoniae serotype 3 surrounding transposon Tn916, insertion of which was previously shown to result in lack of expression of the extracellular capsule. Sequence analysis revealed that the transposon inserted into a consensus insertion site 71 bp from the 5' end of the cloned fragment. Within the clone, 3' downstream regions from two different pneumococcal lytA genes were identified, as well as a putative 194 AA open reading frame (ORF1). Moreover, two copies of the repeat element BOX, oriented in opposite directions, were located immediately 3' of orf1. Within the region bounded by the first pair of internal sequencing primers, analysis revealed that the fragment amplified by PCR was always of the same size. Moreover, Southern blotting showed that for all serotypes examined to date, homology exists with the cloned fragment. These results indicate that this region of the chromosome is highly conserved and, taken together with other independently derived data, suggest that interruptions or deletions within this DNA lead to unencapsulation. PMID- 7549772 TI - Alcohol, other drugs, and what we gain from our patients. PMID- 7549773 TI - Loss of control and inability to abstain: the measurement of and the relationship between two constructs in male alcoholics. AB - The constructs "loss of control" and "inability to abstain" have been used extensively in alcohol research. Examination of the literature, however, reveals inconsistencies in the ways researchers have operationalized and measured these constructs and a dearth of empirical investigation of the validity of the constructs. The current study examines a number of methods of operationalizing loss of control and inability to abstain and investigates the relationship of the two constructs with each other. Ninety-seven male alcoholics who participated in an outpatient conjoint alcoholism treatment study provided data on pre-treatment alcohol consumption and self-report measures of lifetime drinking behavior. Nine interview or questionnaire items that best approximated items used in the literature to measure inability to abstain and loss of control were chosen for analysis. Items measuring both constructs were highly intercorrelated and were associated more strongly with quantity rather than frequency of alcohol consumption, suggesting significant overlap between the constructs. None of the items were associated with a particular drinking pattern. The results provide support for a unitary dimension of impaired control, as used in the alcohol dependence syndrome, and suggest that the value of loss of control and inability to abstain as distinct and meaningful constructs should be reconsidered. PMID- 7549774 TI - Alcohol and suicide--the Portuguese experience. AB - An analysis of regional (18 regions) and temporal (1931-89) covariation of suicide rates and indicators of alcohol use and abuse in Portugal is reported. In the time series analysis, a positive relationship was found. An increase in per capita alcohol consumption of one litre is accompanied by a simultaneous increase in the male suicide rate of 1.9 per cent. This is comparable to what has been reported for France and Denmark, but considerably less than that found in Norway, Sweden and Hungary. In the regional data, there was a substantial negative correlation between the variables. However, after controlling for religious and family integration, the latter correlation became small and insignificant but still negative. A possible explanation is suggested for the different outcomes of the two analyses. PMID- 7549775 TI - Suicide rates and alcohol consumption in the United States, 1970-89. AB - This study investigates the aggregate relationships between suicide rates and beverage specific measures of alcohol consumption for states in the United States over periods of from 14 to 20 years. Time series cross-sectional analyses of these aggregate state level data are presented which control for exogenous differences between states, time trends and covariations over time in nine measures; age composition, male population, non-white population, per capita land area, metropolitanism, income, unemployment, measures of religious preferences and divorce. After correcting for substantial autocorrelations in measurement error, the analyses revealed that suicide rates were specifically associated with spirits sales, age composition of state populations, per capita land area, unemployment and religious preferences over time. While suicide rates increased significantly as a function of increased spirits sales, beer and wine sales were not associated with suicide rates. These findings suggest that it is not the consumption of ethanol per se but rather the consumption of ethanol in the form of spirits that is related to suicides. Rather, it would appear that a population based preference for the consumption of spirits is associated with suicide events. PMID- 7549776 TI - Prevalence of female alcohol dependence and abuse in Sweden. AB - Data on prevalence of female alcoholism are scarce, especially outside North America. It is even more rare that prevalence is estimated using clinical interviews, as well as by utilizing multiple sources of information. Our sample consisted of five cohorts of adult women (n = 3130) in a mainly suburban area. In a first phase we screened for alcohol-related problems. In a second phase a strategic sample was interviewed (n = 399). Prevalence was calculated for clinical and CIDI-SAM diagnoses, both according to DSM-III-R, also taking medical record data into consideration. It was found that life-time prevalence of alcohol dependence and abuse was 3.27% and 12-month prevalence was 1.49%. Agreement over alcohol dependence was very high for the different diagnostic methods, but lower for alcohol abuse. Prevalence of dependency/abuse was not higher in the attrition group. The alcohol abuse concept was found to be relevant, both in epidemiological research and for clinical purposes. PMID- 7549777 TI - Conditioned responses to drug-related stimuli: is context crucial? AB - Research into the elicitation of conditioned responses (CRs) by drug-related stimuli in drug and alcohol addicts has produced apparently conflicting results in relation both to the form of the CR (drug-like or drug-opposite) and to its importance in influencing relapse rates after detoxification. Most studies looking at cue-elicited responses directly have taken place with recently detoxified addicts in a laboratory setting. The present study examined cue elicited reactions in eight currently dependent opiate users (receiving prescribed physeptone) during their drug preparation rituals. Seven were seen in their own homes and one in hospital. Subjects rated their mood and physical state before getting out the drug and paraphernalia, during preparation to inject, and after injection. By contrast with the withdrawal-like reactions, which are the most common finding in laboratory exposure studies where drugs are not subsequently accessible for use, these subjects showed a significant reduction in subjectively rated withdrawal symptoms during the pre-injection drug preparation ritual. The results are discussed in relation to the impact of the context of exposure in influencing subjective expectancies, with perception of drug availability seen as a critical part of the conditioned stimulus complex. PMID- 7549778 TI - Self-reports of HIV risk behavior by injecting drug users: are they reliable? AB - While most studies of AIDS risk behavior rely on self-reports, few studies have assessed the reliability of these reports. The present study examines self reports of drug-related and sexual risk behavior among pairs of injecting drug users (IDUs) recruited from the streets in New York City. Since both members of the pair were interviewed, it was possible to compare their responses in order to assess reliability. Subjects reported on their contacts' demographic data (age, gender, race/ethnicity) and on shared risk behaviors, including syringe sharing. Despite the private and/or illegal nature of AIDS risk behaviors, IDU subjects were generally reliable in their reports of both demographic and AIDS risk behaviors. PMID- 7549779 TI - Prospective effects of perceived risk of developing HIV/AIDS on risk behaviors among injection drug users in Puerto Rico. AB - The relationship between perceived risk of developing AIDS and subsequent behavioral risk status is estimated for 1740 Puerto Rican injection drug users (IDUs). Prospective behavioral effects were examined comparing data collected at two intervals approximately 6 months apart. We estimated the association between perceived risk at baseline and risky behaviors at follow-up with unadjusted odds ratios. We confirmed the results with adjusted odds ratios using logistic regressions which included baseline risk status as well as socio-demographic and health status covariates. The analyses showed that having a high HIV/AIDS risk perception was related to subsequent sharing of needles, injection of drugs in shooting galleries and sharing of cookers. None of the tests between risk perception and sex risk behaviors showed a significance association. Increasing IDUs' perceived vulnerability to HIV/AIDS might not be effective in helping reduce HIV risk behaviors. IDUs perceiving themselves to be at high risk of AIDS might believe there is little they can do to reverse the consequences of risky behavior. PMID- 7549780 TI - The harmful effects of drugs as perceived by the Spanish public. AB - A regional survey on peoples' attitudes towards drugs was conducted in the fall of 1992 on 2500 individuals aged 14-70 years. The great majority of those surveyed considered the taking of heroin (98%), cocaine (95.9%) and amphetamines (94.7%) to be "very risky" for one's health. These percentages were considerably lower in the case of tobacco (41.3%) and alcohol (26.7%). Those surveyed pointed to 'heroin' (35.6%), 'all illegal drugs' (27%), 'legal drugs' (17.2%) and 'cocaine' (14.2%) as the most dangerous drugs for society. The study shows how alcohol and tobacco are perceived by Spaniards as less dangerous than illegal drugs. PMID- 7549781 TI - Injecting drug use and body mass index. PMID- 7549782 TI - Comments on brief intervention of alcohol problems: a review of a review. PMID- 7549783 TI - Interaction of concanavalin A with a fraction of dermatan sulfate containing a very high anticoagulant activity. AB - A dermatan sulfate (DS) fraction with high anticoagulant activity was isolated from the precipitate of the interaction with Concanavalin A (Con A). Under the optimum conditions found, the interaction depends absolutely on the presence of Ca++ or Mn++ ions. The pH curve of the activity showed an acute maximum at pH 4.4. The active fraction showed, in average 5.4 folds higher specific activity than the fraction that remained in the supernatant. Native DS was desulfated by acid hydrolysis: a drastic reduction in the interaction with Con A was observed during the first ten min. at 100 degrees C and a parallel decrease in the anticoagulant activity was also detected. Sulfate liberation followed the same pattern and after 30 min. at 100 degrees C no sulfate groups remain bound. PMID- 7549784 TI - Induction of protein disulfide isomerase during proliferation arrest and differentiation of SH5Y neuroblastoma cells. AB - The relationship of genes associated with contact inhibition of cell growth and the commitment for differentiation was studied in the human neuroblastoma cell line SH5Y. These cells could be induced to differentiate in vitro into neuronal like cells upon incubation with retinoic acid, an event that was accompanied by an enhancement in levels of neuron-specific acetylcholinesterase. The kinetics of differentiation, based on morphology and acetylcholinesterase levels, showed that proliferation arrest always preceded differentiation and may be a prerequisite for differentiation. To determine if this growth arrest is mediated by the same pathway underlying contact inhibition of proliferation, the expression of a gene associated with the induction of contact inhibition, protein disulfide isomerase (PDI), was quantified by Northern blot analysis and enzymatic activity after retinoic acid treatment. Retinoic acid caused a significant elevation of PDI-mRNA within 24 hrs. after treatment with a corresponding increase in enzyme activity which immediately preceded proliferation arrest and differentiation. Bacitracin, a specific inhibitor of PDI, abrogated the ability of retinoic acid to induce differentiation. However, treatment with interferon also increased PDI activity and caused proliferation arrest and SH5Y differentiation but into a fibroblastoid cell without neurite outgrowth. These results suggest that the commitment for differentiation of SH5Y cells involves a form of proliferation arrest in which activation of PDI activity is a required and early event but one that does not determine the final differentiation pathway. PMID- 7549785 TI - Alterations in cell-cell communication in human papillomavirus type 16 (HPV16) transformed rat myoblasts. AB - A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts. PMID- 7549786 TI - Immunocytochemical study of NA+ K(+)-ATPase alpha 1 and beta 1 subunits in human and rat normal hepatocytes using confocal microscopy. AB - Hepatic Na+ K(+)-ATPase was recently shown to be composed of two alpha 1 and beta 1 subunits similar to that of kidney. Its localization on hepatocyte plasma membranes was not clearly established. We have studied the localization of alpha 1 and beta 1 subunits using immunocytochemical method and confocal microscopy. In accordance with previous cytochemical findings, the catalytic alpha 1 subunit was distributed in basolateral and bile canalicular membranes of hepatocytes. The beta 1 subunit was not demonstrated, due to its very low amount in the liver. The controversy about the bile canalicular localization was discussed. PMID- 7549787 TI - Studies of radioimmunoassay for measurement of insulin-like growth factor-I (IGF I) by using anti-IGF-I monoclonal antibody. AB - Radioimmunoassay system for measurement of insulin-like growth factor-I (IGF-I) was established by using anti-recombinant human IGF-I monoclonal antibody (MAb). This MAb is capable of recognizing not only human but also rat IGF-I. It was thus suggested that this RIA system can quantify IGF-I in human and rat sera. MAb used in this paper was clarified to be an antibody against the common epitope of C region of human and rat IGF-I. Gly32-Ser33-Ser34 sequence of C-region of IGF-I is discussed to be an antigenic determinant to which this antibody might specifically bind. MAb does not cross-react with proinsulin and insulin as well as anti-IGF-I polyclonal antibody (PAb). And it is general that PAb has almost 2% cross-reactivity with IGF-II. But this MAb did not cross-react with IGF-II. Actually, the value of IGF-I measured by this system was lower than that measured by RIA using PAb. PMID- 7549788 TI - Synthetic octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn promotes differentiation in promyelocytic HL-60 cell line. AB - The activity of the octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn in the control of cell growth and differentiation of human myeloblastic leukemia cells HL-60 is reported. Treatment with peptide slightly slows down the rate of cellular proliferation and this effect becomes more evident in cells grown for several weeks in the presence of the effector. An enhanced effect (40-50% inhibition respect to the control) is found in reversibly permeabilized cells and after 1% DMSO is added to the medium. Moreover the presence of peptide markedly increases the percentage of cells differentiated by DMSO and RA. The effect in DMSO-induced cells is more evident than that observed in RA-induced cells. This in agreement with our hypothesis that DMSO facilitates the peptide entry and its effect is due to an intracellular action. PMID- 7549789 TI - Neutron activation study of the natural elementary composition of edible sea urchins (Paracentrotus lividus Lamarck) in the National Park of Port-Cros (Mediterranean, France). AB - Neutron activation study of elementary composition was performed on edible sea urchins Paracentrotus lividus from the National Park of Port-Cros. The analysis allows the identification and quantification of 22 elements: antimony, arsenic, baryum, bromine, cerium, chromium, cesium, cobalt, gold, iron, lanthane, potassium, sodium, rubidium, samarium, scandium, selenium, silver, strontium, thorium, uranium and zinc. The concentration levels were higher in the soft organic parts (alimentary canals and gonads) for all the elements, except for strontium, which developed a strong affinity with calcareous hard parts (tests, spines, masticating apparatus). We also found high rates of baryum, arsenic, zinc, bromine and iron. The hypothesis on the origin of these elements is discussed. The data obtained on this referential zone will soon be used to appreciate the perturbation of the elementary composition of urchins by pollution in various parts of the French seashore. PMID- 7549790 TI - Neutron activation study of the elementary composition of edible sea urchins (Paracentrotus lividus Lamarck) in marine creek area polluted by city sewages of Marseille (France). AB - Neutron activation study of elementary composition (Ag, As, Au, Ba, Br, Ce, Co, Cr, Cs, Fe, K, La, Na, Rb, Sb, Sc, Se, Sm, Sr, Th, U and Zn) was performed on edible sea urchins (Paracentrotus lividus Lamarck) from marine creek area polluted by city sewages from Marseille (France) after a purifying plant was brought into operation. Concentration levels were higher in soft organic parts (alimentary canals and gonads) for all the elements except for Sr which showed a strong affinity for calcareous parts (tests, spines and masticating apparatus). Certain elements present in the soft organic parts are rarely found in the hard parts. More than half the elements studied show higher concentrations in Marseille samples than in the referential region of Port-Cros National Park (France). That must be due to sewage rejection for a large number of them. This hypothesis is confirmed with regard to elements the values of which decrease significantly with the distance from the sewage emission point, e.g. Cr, Fe and Zn, and a little less for Ag, Ba, Ce, Rb, Sb, Sc, Se, Sm and Th. These results raise a question: has that artificial change on the elementary composition any toxicological effects on the urchin populations? This question is discussed. PMID- 7549791 TI - Expression of yeast O6-methylguanine-DNA methyltransferase (MGMT) gene. AB - We cloned the full genomic DNA of yeast (Saccharomyces cerevisiae) O6 methylguanine-DNA methyltransferase (MGMT) gene and examined its expression. The expression of yeast MGMT gene is decreased when cells reach stationary phase and cannot be induced by the pretreatment with alkylating agents, methylmethanesulfonate (MMS) or N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG). The transcription initiation site was determined by primer extension analysis. This analysis showed that the authentic start codon is the ATG at position +32 from transcription initiation site. PMID- 7549792 TI - Synthesis and secretion of proteins by the human proliferative, secretory, decidual and hyperplastic endometrium. AB - Human endometria were analysed for the synthesis and secretion of proteins following short term culture of human endometrial tissue in the presence of 35S methionine. During the menstrual cycle secretory proteins of molecular weight (MW) 30, 35, 45, 50, 59, 74, 97 and 135 kDa showed increased synthesis during the proliferative phase. The synthesis of these proteins decreased in the secretory phase but the induction of a 26 kDa protein in the early secretory phase and a 28 kDa protein in the late secretory phase was observed. The synthesis of the above secretory proteins of the human endometrium was also confirmed by two dimensional gel electrophoresis. Further, the results demonstrated that the secretory protein profile of human decidual endometria and endometria exhibiting irregular ripening was identical to that of normal secretory phase endometria. But, endometria exhibiting hyperplasia, cystic glandular hyperplasia and adenomatous glandular hyperplasia presented similar secretory protein profiles which were identical with the secretory protein profile of normal proliferative phase endometrium. The present study confirms that a number of proteins are synthesised by the human endometrium during the normal menstrual cycle and during pregnancy. It also provides data for the first time on the proteins secreted by the endometria exhibiting irregular ripening, hyperplasia, cystic glandular hyperplasia and adenomatous glandular hyperplasia. PMID- 7549793 TI - Fibroblast growth factors and their specific binding sites in normal urothelium and transitional cell carcinoma (TCC) of the human bladder. AB - In order to further investigate the involvement of FGF1 and FGF2 in the transitional cell carcinoma of the bladder (TCC), we compared the expression of FGF1, FGF2 and their membrane binding sites by biochemical and immunological methods in both human normal urothelium and transitional cell carcinoma of the bladder. Using a specific enzyme immunoassay (EIA), we observed that neoplastic tissue exhibited a lower FGF2 content than normal tissue. FGF1 and FGF2 Western blot analysis revealed in both types of tissues a major FGF1 form with a low molecular weight (14 kDa) and a single FGF2 form (21 kDa). Immunohistological studies showed that FGF1 was specifically located in normal or transformed urothelial cells, while FGF2 was associated with bladder stroma. Binding analysis in tissue membrane preparations revealed a dramatic drop in both high and low affinity binding sites for FGF2 in TCC bladder in comparison to normal bladder. FGF2 cross-linking experiments illustrated a qualitative modification of FGF2 binding complexes in tumors. These data suggest that invasive bladder carcinoma is associated with modifications of FGF1 and FGF2 tissue levels and alterations in the characteristics of the FGF2 membrane binding sites. PMID- 7549795 TI - Study on the aging changes of DNA and protein synthesis of bipolar and photoreceptor cells of mouse retina by light and electron microscopic radioautography. AB - The DNA and protein synthesis of the bipolar cells and photoreceptor cells of aging mouse retina from fetal to postnatal 1 year were studied by light and electron microscopic radioautography. Meanwhile the ultrastructure of these cells was observed by electron microscopy. The bipolar and photoreceptor cells incorporated 3H-thymidine from embryonic day 19 until 1 week after birth, while 3H-leucine from embryonic day 19 to postnatal 1 year. The 3H-thymidine labelled cells had undeveloped cell organelles in their cytoplasm. On the contrary, the unlabelled cells had well developed cell organelles, mainly mitochondria and endoplasmic reticulum in their cytoplasm. The mitochondria became elongated when the outer segments were well developed. The protein synthesis of the bipolar cells and photoreceptor cells as demonstrated with 3H-leucine labeling was vigorous at embryonic day 19 and early postnatal days when the inner and outer segments were formed. The peak of this phenomenon was observed at 1 day after birth. The activity of protein synthesis was also detected at the adult stage. However there was no significant fluctuation in any stages from 14 days after birth to 1 year. PMID- 7549794 TI - The glycan keratan sulfate in inner ear crystals. AB - The otoconial matrix (OM) of chicks (Gallus domesticus) inner ear was analyzed. Histochemically the OM was reacted with phosphotungstic acid (PTA) and immunohistochemically with the monoclonal antibody antikeratan sulfate (antiKS). The OM was digested with the enzyme endo-beta-galactosidase (E beta Galase) or separated by 1D and 2D gel electrophoresis. PTA which reacts with glycoproteins precipitated the OM, suggesting that the OM contains glycoproteins. A central core in each crystal had no PTA staining, suggesting that the core lacked glycoproteins. Anti KS antibody stained the OM with increased density in older embryos as determined by color thresholding. E beta Galase, which cleaves the lactosamine repeating units in KS, decreased the immunostain by 30% in the OM and by 20% in the cartilage. The OM from the utricle, saccule and macula lagena contained similar molecular weight bands. Five dense bands in the OM were less dense in tissue and blood controls, suggesting that such bands are enriched in the OM. Isoelectric focusing of the OM showed a negatively charged high molecular weight smear not present in blood and faint in tissue controls. The high affinity of the OM for the cationic PTA stain, the strong immunohistochemical reaction of the OM with anti KS antibody and high molecular weight negative smear in 2D gels taken together suggest that: a) the OM contains large amounts of glycoproteins and glycans, one of which is keratan sulfate, because its immuno stain with antiKS antibody was decreased by the enzyme E beta Galase, b) the utricle, saccule and macula lagena may have similar composition, and c) the concentration of KS may increase gradually until complete mineralization of the OM is reached. PMID- 7549796 TI - Treatment with human chorionic gonadotropin and risk of breast cancer. AB - Studies of the induction of mammary tumors by 7,12-dimethylbenz(a)anthracene in a rat model show that human chorionic gonadotropin (hCG) administration reduces tumor incidence in a manner comparable to that of a completed pregnancy. On the basis of their studies, Russo and Russo (Cancer Epidemiol., Biomarkers & Prev., 3: 353-364, 1994) have proposed that hCG treatment of young nulliparous women would reduce their breast cancer risk in a manner similar to that of a term pregnancy. As part of a population-based, case-control study of breast cancer among women ages 40 years or younger, we asked women whether they had received hCG injection as part of a weight loss regimen or as a component of infertility treatment. Participants in this study were 744 women newly diagnosed with breast cancer between July 1983 and December 1988 and 744 controls individually matched on birthdate (within 36 months), race (white), parity (nulliparous/parous), and neighborhood of residence. Forty-five cases and 65 controls reported exposure to hCG (multivariate odds ratio = 0.77, 95% confidence interval = 0.50-1.19). Risk was reduced significantly among women whose maximum nonpregnant body mass index was less than 27.5 kg/m2 but no reduction in risk was observed among more obese women. Although the odds ratios were reduced substantially for both nulliparous and parous women with maximum nonpregnant body mass indices less than 27.5, only the result for nulliparous women was statistically significant. These results are consistent with the effects proposed by Russo and Russo based on their animal model. Although not definitive, these results suggest that hCG may be a means for reducing breast cancer risk. PMID- 7549797 TI - Prenatal and neonatal risk factors for childhood myeloid leukemia. AB - Information about the etiology of childhood myeloid leukemia is limited. A population-based nested case-control study of prenatal and neonatal risk factors for childhood myeloid leukemia was performed with the use of the Swedish National Cancer Register and the Swedish Birth Register. A total of 98 cases of myeloid leukemia were identified in successive birth cohorts from 1973 through 1989. From the Birth Register, five controls were matched to each case. Fourteen of the 98 cases with myeloid leukemia and none of the controls had Down syndrome [odds ratio (OR) = infinity; 95% confidence interval (CI) = 21.0-infinity]. The risk for myeloid leukemia also increased among children who had physiological jaundice (OR = 2.5; 95% CI = 1.2-5.0; children who had been treated with phototherapy (OR = 7.5; 95% CI = 1.8-31.9); or who had been treated in an incubator (OR = 3.5; 95% CI = 1.2-10.2). Excluding cases with Down syndrome, however, decreased these risks, so that their 95% lower confidence interval included the no-effect value. Maternal age < 20 years old (OR = 2.5; 95% CI = 1.1-6.0), hypertension (OR = 2.4; 95% CI = 1.2-5.0), Cesarean section (OR = 2.5; 95% CI = 1.3-4.9), maternal smoking (OR = 2.4; 95% CI = 0.9-6.5), and being one of a multiple birth (OR = 3.6; 95% CI = 1.1-11.3) increased the risk for myeloid leukemia among those without Down syndrome. When the analyses were repeated, by restricting the cases to those with acute myeloid leukemia, the risk associated with young maternal age declined and became nonsignificant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549798 TI - Pelvic inflammatory disease and the risk of epithelial ovarian cancer. AB - Infertility is a common complication of pelvic inflammatory disease (PID) and may result in decreased parity. Low parity and possibly infertility are risk factors for ovarian cancer. We therefore examined the association between ovarian cancer and history of PID in a case-control study conducted during 1989-1992 in metropolitan Toronto and nearby areas of Southern Ontario, Canada. In total, 450 histologically verified new primary epithelial ovarian cancer cases ages 35-79 years were interviewed concerning their reproduction history. Over the same period, 564 randomly selected population controls, frequently matched to the cases according to three 15-year age groups, were interviewed similarly. Continuous unconditional logistic regression methods were used for analysis. It was found that cases were more likely than controls to report having had one or more episodes of PID; adjusted for age, parity, duration of oral contraceptive use, and other factors the odds ratio (OR) was 1.53 [95% confidence interval (CI), 1.10-2.13; P = 0.012]. Higher risk was present for women with recurrent PID (OR, 1.88; 95% CI, 1.13-3.12; P = 0.014). The elevated risk associated with PID was seen particularly among women < 60 years of age at interview (OR, 1.60; 95% CI, 1.09-2.35; P = 0.016), for women of parity 0 or 1 (OR, 2.40; 95% CI, 1.39 4.15; P = 0.0017), among women who had never ever had infertility (OR, 3.74; 95% CI, 1.28-10.9; P = 0.016), and for the small number of women who reported having PID before age 20 (OR, 3.08; 95% CI, 1.17-8.13; P = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549799 TI - Ovarian cancer mortality among immigrants in Australia and Canada. AB - This study examined the impact of changing environments on ovarian cancer by comparing age-standardized mortality rates of numerous immigrants groups in Australia and Canada to those in the origin countries for the period 1984-1988. Mortality rates by length of residence in Australia (0-29 and 30+ years) were also calculated. In Australia, the mortality rates for all four immigrant groups from low-risk countries and 53.8% from high-risk countries (n = 13) shifted toward the rate of the native-born Australians. In Canada, rates for 88.9% of immigrant groups from low-risk countries (n = 9) and 30.0% from high-risk countries (n = 10) converged to the rate of native-born Canadians. Among individual immigrant groups there was not a consistent pattern of convergence with length of residence in Australia. There was evidence of convergence among the long-term residents of some of the groups and in the aggregate analysis. The increased mortality among the majority of immigrant groups is consistent with the reported inverse relationship between parity and ovarian cancer and the generally lower parity of immigrant women compared to those in their home country. The period of residence and analyses suggests that long-term environmental and lifestyle factors in the new place of residence may also influence ovarian cancer mortality. PMID- 7549800 TI - Risk factors for progression of cervical intraepithelial neoplasm grade III to invasive cervical cancer. AB - Data from four case-control studies on invasive cervical cancer and on cervical intraepithelial neoplasia grade III (CIN III) that were concurrently conducted in Spain and Colombia were used to look for factors that might favor the progression from CIN III to the invasive stage. These studies were compared in two ways; a case-case comparison and an estimation of the ratio of odds ratios with the use of of special logistic regression model that took into account the different design of each study and possible confounding factors. Variables studied were human papillomavirus status, viral load, viral types, sexual behavior, sexually transmitted diseases, reproductive patterns, oral contraceptives, and smoking. Both CIN III and invasive cervical cancer have a very similar profile of risk factors and none of them was different in a consistent way to suggest a role in the progression from CIN III to invasive cervical cancer. Some methodological problems such as cohort-specific differences and some selection biases could be adjusted for with a careful statistical analysis. Other problems derived from the cross-sectional nature of the design are unavoidable and should be considered in the interpretation of the results. PMID- 7549801 TI - A cohort study of twins and cancer. AB - Given the current explosion of knowledge of the genetics and molecular biology of cancer, the possibility of widespread testing for inherited predisposition to cancer has been raised. The main objective of this study was to assess the effect of inherited predisposition on cancer mortality among the National Academy of Sciences-National Research Council Twin Registry. The twins were white male United States veterans of World War II, who were born during the period 1917 1927. The follow-up period was from 1946 to 1990, and some cause of death was determined with the use of death certificates. We compared concordance for death from cancer among 5690 monozygotic twin pairs to that among 7248 dizygotic pairs. A possible effect of inherited predisposition to death from cancer was considered present if concordance for cancer mortality among monozygotic twin pairs was greater than it was among dizygotic twin pairs. Among monozygotic and dizygotic twins, a total of 1918 cancer deaths was observed. Concordance for death from cancer at all sites among monozygotic twins was higher than it was among dizygotic twins (overall rate ratio, 1.4; 95% confidence interval, 1.0-2.0). For each zygosity group, two or fewer pairs were observed to be concordant for death from cancer of a specific site, with the exception of lung cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549803 TI - Dietary modulation of serum platelet-derived growth factor-AB levels. AB - Epidemiological studies have demonstrated that diets high in vegetables and fruit are associated with a decreased risk of cancer and, possibly cardiovascular disease. Certain constituents of vegetables and fruit inhibit the in vitro activity of platelet-derived growth factor (PDGF), a potent mitogen implicated in both cancer and cardiovascular disease. Few studies have measured PDGF in relationship to diet in vivo. Specifically, there are no data regarding the changes in PDGF levels of mitogenic activity after a dietary intervention. In this study, 19 young, healthy individuals consumed four (9-day) experimental diets in random order: (a) control diet alone; (b) control diet plus carotenoid rich vegetables; (c) control diet plus cruciferous vegetables; and (d) control diet plus soy foods. Compared to the control diet, there was a significant elevation in PDGF-AB serum levels when the individuals were consuming the soy diet (P = 0.016). Increased PDGF-AB levels were also noted for the carotenoid diet. There was no change from baseline levels when individuals were consuming the cruciferous diet. Overall, mitogenic activity did not change on any of the experimental diets. This study suggests that high soy and carotenoid diets increase serum levels of PDGF-AB. This may represent an additional mechanism by which diet influences individual risk of cancer; further investigation into the role of diet and growth factors is warranted. PMID- 7549802 TI - Cancer incidence in Thailand, 1988-1991. AB - Results from three cancer registries (Chiang Mai, Khon Kaen, and Songkhla) in different regions of Thailand and from a cancer survey in the population of Bangkok during the years 1988-1991 are presented, together with an estimate of the incidence of cancer for the country as a whole. Overall, liver cancer is the most frequent malignancy, but there are large regional differences in incidence and in histological type, with very high rates of cholangiocarcinoma in the northeast (associated with endemic opisthorchiasis) but a more even distribution of hepatocellular carcinoma. Lung cancer is second in frequency, with the highest rates in northern Thailand, where the incidence in women (Age Standardized Rate, 37.4 per 100,000) is among the highest in the world. A link with tobacco smoking is suggested by similarly raised rates, especially in women, for cancers of the larynx and pancreas. Cervical cancer is the most common malignancy in women, with relatively little regional variation in risk, while the incidence of breast cancer is low. Other cancer sites showing moderately increased rates include the lip and oral cavity, particularly in females from the north and northeast, where the chewing of betel nut remains common among older generations, nasopharyngeal cancer, carcinoma of the esophagus in the southern region, and penile cancer, especially in the north and northeast. Previous studies which have investigated the etiological factors underlying these patterns are reviewed, and the implications for future research and for national cancer control policies are discussed. PMID- 7549804 TI - Plasma carotenoids as biomarkers of vegetable intake: the University of Minnesota Cancer Prevention Research Unit Feeding Studies. AB - High vegetable intake has been associated with a decreased risk for various human cancers in epidemiological studies. Carotenoids are plant compounds that may both possess chemopreventive activity and be useful biomarkers of vegetable and fruit intake. Nineteen men and women were randomized into a controlled cross-over feeding study to measure the effect of vegetable intake on plasma carotenoid concentrations. Participants consumed each of 4 experimental diets for 9 days. The control diet consisted of commonly consumed foods and was essentially carotenoid free. High vegetable diets (carotenoid, cruciferous, and soy) consisted of the control diet plus carrots and spinach (carotenoid), broccoli and cauliflower (cruciferous), and tofu and FriChik (soy). Plasma carotenoid concentrations were highest on the carotenoid and cruciferous diets. When compared to the control, mean plasma alpha-carotene, beta-carotene, and lutein concentrations were 5.2, 3.3 and 2.2 times higher on the carotenoid diet, respectively (P < 0.001). Mean plasma lutein concentrations were 2.1 times higher on the cruciferous versus the control diet (P < 0.001). There were no differences between diets in plasma beta-cryptoxanthin and lycopene concentrations. These data indicate that plasma alpha-carotene, beta-carotene, and lutein may be useful biomarkers of carotenoid-rich food intake and that lutein may act as an intake biomarker of commonly consumed vegetables in the Cruciferae family. These findings should prove useful in undertaking dietary intervention trials because they suggest the feasibility of monitoring intake of some plant foods and of distinguishing among plant food groups. PMID- 7549805 TI - A simplified method to quantify isoflavones in commercial soybean diets and human urine after legume consumption. AB - Reliable and economical quantification of micronutrients in diets and human is a critical component of successful epidemiological studies to establish relationships between dietary constituents and chronic disease. Legumes are one of the major dietary components consumed by populations worldwide. Consumption of legumes is thought to play a major role in lowering breast and prostate cancer risk. In this study, a simplified method that uses solid-phase extraction and gas chromatography was developed to measure isoflavones at levels down to 10 micrograms/5ml. With the use of this method, 12.5 g miso (a soybean paste), 12 ounces Isomil, and 12 ounces soymilk had daidzin/daidzein levels of 2, 5, and 12.4 mg, respectively, and genistin/genistein levels of 3, 6.5, and 13.7 mg, respectively. In these products, most of the isoflavones were present as glucosides. With the same method, urinary levels of isoflavones in six 15-17-year old subjects were determined after soymilk ingestion. Each subject was placed on unrestricted nonsoya diets, and three 12-ounce portions of soymilk were given at 12-h intervals. Males excreted 15.02 +/- 2.74 (SD) mg of daidzein glucuronides/sulfates [mean recovery, 40.4 +/- 7.4% (SD)] by 24 h after the third soymilk ingestion, whereas females excreted 25.56 +/- 5.10 mg (68.7 +/- 13.7%) of daidzein conjugates, which was more than males (P = 0.02). Males and females excreted 7.73 +/- 1.95 mg and 9.11 +/- 0.84 mg of genistein glucuronides/sulfates (20% recovery of genistin intake), respectively, in the urine. Most of the isoflavones were excreted within 24 h after ingestion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549806 TI - Stability of ascorbic acid, carotenoids, retinol, and tocopherols in plasma stored at -70 degrees C for 4 years. AB - Aliquots from 40 plasma pools preserved with metaphosphoric acid were assayed for their ascorbic acid values after 12, 24, and 42 months of storage at -70 degrees C. Similarly, aliquots from 16 plasma pools were assayed for values of retinol, several carotenoids, and two tocopherols at 15.5, 27.5, and 51.5 months of storage at -70 degrees C. There were no indications of important losses of these antioxidant micronutrients during storage from the first to the last assay. PMID- 7549807 TI - Validity for epidemiological studies of long-term cryoconservation of steroid and protein hormones in serum and plasma. AB - Prospective studies based on the storage of biological samples at low temperature have opened new perspectives in etiological research on cancer. In planning these studies a crucial question is to evaluate whether the long-term preservation of samples is able to affect the categorization of the subjects involved. In the frame of the ORDET project, a prospective study of hormones and diet in the etiology of breast cancer provided with a -80 degrees C biological bank, we have evaluated the stability of estradiol, free and total testosterone, and prolactin in serum and plasma samples over 3 years of cryoconservation. Study results showed that the subjects maintained almost the same rank by hormonal concentration throughout the 3-year period for all hormones. Looking at the stability over time, estradiol, prolactin, and total testosterone had fairly good performance for both serum and plasma. Serum-free testosterone increased in time up to 30%, whereas progesterone decreased by about 40% of the initial concentration. However, the reliability of the individual categorization by hormonal level suggests the validity of low temperature storage for epidemiological purposes, at least for hormonal parameters. PMID- 7549808 TI - Energy restriction and oxidative DNA damage in humans. AB - The cancer-preventive effect of energy restriction in rodents has been related to a decrease in oxidative damage to DNA. We have investigated the effect of energy restriction on the rate of oxidative DNA modification estimated from the urinary excretion of the repair product, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in healthy, normal weight men. Before and after 10 weeks on a diet containing 80 (n = 16) or 100% (n = 8) of the estimated weight-maintaining energy, resting metabolic rate (RMR) was measured and 24-h urine was collected for 8-oxodG determination by HPLC. During the study, the weight loss was 10 and 2.5% of the initial weight, mostly in terms of fat, and the RMR decreased by 13 and 8% in the energy-restricted and control groups, respectively. With the use of t tests there was no significant difference within or between groups with respect to 8-oxodG excretion. However, if RMR was included as a covariate in multifactorial ANOVA, an average relative 17% (2-31%; 95% confidence interval) increase in 8-oxodG excretion in the energy-restricted group was significantly different from the corresponding value of the control group (P < 0.02). In the energy-restricted group the change in 8-oxodG excretion was correlated closely with the decrease in RMR (r = 0.63; P = 0.013). In the present study, 20% energy restriction for 10 weeks did not reduce oxidative DNA damage; we question a beneficial effect on cancer risk in normal weight humans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549809 TI - Levels of proteolytic activities as intermediate marker endpoints in oral carcinogenesis. AB - It is essential to identify intermediate marker endpoints of carcinogenesis for the evaluation of the effectiveness of cancer-chemopreventive agents. We have observed that levels of proteolytic activities (as detected by 4 different substrates) are increased 2-3-fold (P < 0.003) in oral buccal mucosa cells of smokers and patients with oral leukoplakia or erythroplakia as compared to a nonsmoking comparison group. In addition, proteolytic activity levels in the buccal cells were increased nearly 3-fold in patients with oral trauma (P < 0.01) or diabetes (P < 0.02), as well as pregnant women (P < 0.04). Excluding these subgroups of patients in epidemiological studies increase the differences in levels of proteolytic activities between both the nonsmoking comparison group and smokers and between the comparison group and patients with oral leukoplakia or erythroplakia. Evaluation of prerandomization levels of proteolytic activities of patients in cancer chemoprevention trials will increase the statistical power by allowing stratified randomization based on levels of proteolytic activities. The observed increases in levels of proteolytic activities in tissues at higher than normal risk of cancer development suggest that levels of proteolytic activities should be used as immediate marker endpoints in human cancer prevention trials using protease inhibitors as potential anticarcinogenic agents. PMID- 7549810 TI - A study of gender-based cytochrome P4501A2 variability: a possible mechanism for the male excess of bladder cancer. AB - One hypothesis for the well known gender difference in bladder cancer risk is that males and females metabolize carcinogens differently. The caffeine breath test (CBT) was performed on a group of healthy men and women to determine whether there was a gender difference in P4501A2 activity. Results consistent with previous data suggest an elevation of CBT in men were observed, although this increase was not statistically significant. Among women, however, there was a significant difference between nulliparous and parous women(P = 0.03). Parous women had CBT values similar to men, whereas the results of women who had never given birth were lower. Confirming earlier studies, women taking oral contraceptives had low CBT values. Our data suggest an effect of recent caffeine consumption, with heavy coffee drinkers having higher rates of caffeine clearance. Adjustment for other weak effects, such as age, exposure to environment cigarette smoke, history of smoking, recent meat and cruciferous vegetable consumption, and use of alcohol or other medications, did not alter these findings. The finding of a difference between parous and nulliparous women requires further study. PMID- 7549811 TI - Glutathione S-transferase mu genotype, diet, and smoking as determinants of sister chromatid exchange frequency in lymphocytes. AB - Polymorphisms in inherited metabolic traits and intake of dietary antioxidants have been reported to be associated with risk for the development of lung cancer in smokers. This increased risk of lung cancer is presumably attributable to the accumulation of DNA damage. We conducted a study to investigate whether genetic metabolic variants and antioxidant consumption affected the sister chromatid exchange (SCE) level in lymphocytes. Study subjects were 78 friends and spouses of cases from a case-control study of lung cancer designed to investigate the association of metabolic polymorphisms with lung cancer. The metabolic traits studied included glutathione S-transferase class mu and variants of P-450 isoenzymes CYP1A1 and CYP2D6. Intake of antioxidants including vitamins A, C, and E and selenium was determined through the administration of a validated, semiquantitative food frequency questionnaire. Detailed information on smoking, family history of cancer, medical history, and environmental and occupational exposures was also obtained in an interviewer-administered questionnaire. Smoking status was found to be significantly associated with SCE frequency. In addition SCE frequency decreased with the period of time since quitting smoking. The presence of one or more glutathione S-transferase class mu alleles was associated with significantly lower SCE. Higher intake of vitamin A and selenium was also inversely associated with SCE level. Thus, the results suggest that glutathione S transferase class mu and the intake of vitamin A and selenium may modulate the accumulation of chromosomal damage in lymphocytes. PMID- 7549812 TI - Mutations in the p53 gene in lung cancer are associated with cigarette smoking and asbestos exposure. AB - It has been proposed that the patterns of mutations in the p53 tumor suppressor gene will provide clues to the mechanisms of cancer occurrence. Cigarette smoking is known to be the greatest risk factor for lung cancer. Epidemiological evidence has also implicated radon and asbestos as exposures that significantly increase this disease risk; asbestos exposure synergistically enhances the lung cancer risk of smokers. Previous studies of the mutational spectra of the p53 gene in lung cancer have shown cigarette smoke and radon exposure to be associated with the induction of particular lesions or classes of lesions. We have investigated the p53 gene in surgically resectable lung cancers in 85 patients from the Massachusetts General Hospital. We found 25 (29%) patients to have somatic p53 mutations in their tumors. The patients with p53 mutations who were current smokers were significantly older (75.1 versus 59.8 years; P < 0.01 and had smoked for significantly more years (56.8 versus 41.2 years; P < 0.01) than had those without p53 changes. Consistent with other reports, we observed a large number (40%) of G:C to T:A transversion mutations, noting that their occurrence increased with increasing cumulative exposure to cigarette smoke. Interestingly, we also found that p53 mutations occurred significantly more frequently in patients with a history of occupational exposure to asbestos [3 of 60 (5%) for patients without p53 mutations versus 5 of 25 (20%) of those with p53 mutations; P < 0.05].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549813 TI - High levels of transforming growth factor beta 1 correlate with disease progression in human colon cancer. AB - Several genes have identified that play a role in colon cancer development. However, less is known about factors that increase the rate of progression of colon cancers to metastasis. One candidate is transforming growth factor beta 1 (TGF beta 1), which can enhance the aggressiveness of human colorectal cell lines in vitro and in vivo. The amount of TGF beta 1, TGF beta 2, and TGF beta 3 protein isoforms expressed in primary site colorectal cancers were measured to determine whether any correlation existed between protein levels and disease recurrence in a series of Memorial Sloan-Kettering Hospital patients who underwent potentially curative resections. Intense staining for TGF beta 1 correlated significantly (P < 0.0013; odds ratio, 18) with disease progression to metastasis and was independent of nodal status and the degree of differentiation of the primary tumor. Therefore, in this study, patients with high TGF beta 1 protein levels in their primary site colorectal cancer were 18 times more likely to experience recurrence of their disease than were patients whose tumors exhibited low levels of TGF beta 1. In this case-control study, patients whose cancer recurred and those remaining cancer free were age and sex matched. The disease recurred at a mean of 26.8 +/- 4.3 (SE) months, whereas the mean follow up time in patients whose disease did not recur was over twice as long, 57.3 +/- 6.6 months. Ninety-four % of the patients in each group were node positive at the time of resection, with equal mean numbers of positive nodes per patient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549814 TI - Effects of dietary fat intervention on mental health in women. AB - Several studies have identified potential detrimental sequelae of cholesterol and fat-lowering interventions in randomized trial. Little research has been published to document changes in mental health in women as a result of fat and cholesterol lowering interventions to prevent chronic disease. This paper examines the relationships among changes in dietary fat consumption and mental health in the Women's Health Trial, a randomized, controlled trial to determine whether lowering fat consumption to 20% of daily calories could reduce the incidence of breast cancer in women ages 45-69 years. Assessments were made at baseline and at the 12-month follow-up of several aspects of quality of life, including negative and positive affect and past, present, and future perceptions of health. Mental health variables were measured by the Mental Health Inventory, a standardized scale used in the Medical Outcomes study. Dietary intake was assessed for all subjects with the use of semiquantitative food frequency questionnaires. The change in mental health values (follow-up minus baseline) was significantly different between intervention and control groups for three of the four psychological variables: (a) anxiety; (b) depression; and (c) vigor. In all three cases, the direction of the change for intervention women was positive. Neither randomization assignment nor percent of calories from fat at the follow up visit were significant predictors of mental health at the 1-year follow-up. Cholesterol changes were not related to levels of mental health variables in a sample of the women. These data indicate that lowering fat in the diets of healthy women does not produce overall lowering of any mental health variables.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549815 TI - Awareness of heightened breast cancer risk among first-degree relatives of recently diagnosed breast cancer patients. The High Risk Breast Cancer Consortium. AB - This investigation had two goals: (a) to determine the proportion of first-degree relatives of recently diagnosed breast cancer patients who are unaware of their elevated risk for breast cancer; and (b) to identify demographic medical, and lifestyle factors that characterize these women. The ultimate objective was to identify women at increased risk who could benefit from breast cancer risk education. Three hundred ninety-five female first-degree relatives, ages 30-75 years, completed a structured telephone interview. Twenty-five % of these women believed that their risk for breast cancer was the "same as or lower than" women who do not have a family history of breast cancer, despite the fact that they had an objectively increased risk. Bivariate analyses revealed that women who were unmarried (chi2 = 14.8; P = 0.001) and had less than or equal to a high school education (chi2 = 9.2; P = 0.002) were significantly less likely to perceive themselves as being at increased risk for breast cancer. In addition, almost one half of African-American women were unaware of their increased risk compared to only 19% of white women (chi2 = 29.9; P < 0.001). More smokers were unaware of their elevated risk compared to nonsmokers (43 versus 21%; chi2 = 15.1; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549817 TI - Human papillomavirus integration is not associated with advanced epithelial ovarian cancer in German patients. PMID- 7549816 TI - Models of breast cancer show that risk is set by events of early life: prevention efforts must shift focus. AB - We have recently published a mathematical model of the etiology of breast cancer based on the data from the Nurses Health Study that extends the Pike model of breast cancer (see Appendix). The most salient feature of the model is that it identifies the years before the first birth of a child as the most crucial in establishing future risk of breast cancer. The extended model includes several additional details of reproductive risk factors, allowing us to quantify the relative importance of each of the reproductive risk factors and to estimate the effect of changes in key determinants of breast cancer. In this review, we present the evidence from both animal studies and epidemiological research that corroborate the critical importance of the exposures that occur before first birth. We argue that research and preventive interventions should now focus on youth. Population-wide prevention strategies are necessary because the inherited genetic risk for breast cancer accounts for no more than 10-15% of all breast cancer cases, leaving 85% of cases diagnosed among women who are not in this high risk subgroup of the population. An example of a population-based intervention would be the promotion of increased physical activity among young girls that could result in the delay of menarche. An example of additional research that focuses on the importance of early life exposures would be an analysis of the relation between diet and other lifestyle factors during adolescence and the subsequent risk of breast cancer and studies of precursor lesions (atypical hyperplasia). Shifting the focus of breast cancer prevention to this age group is urgently needed. PMID- 7549818 TI - Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) PMID- 7549819 TI - Trends in smoking in Europe. PMID- 7549820 TI - Quantitative evaluation of mammographic densities: a comparison of methods of assessment. AB - Differences in the proportion of the breast occupied by mammographic densities have been shown to be associated with differences in breast cancer risk. However, estimation of these densities by radiologists may be subject to error, and it is likely that quantitative measurement will reduce misclassification of densities and strengthen their association with risk of breast cancer. The objective of this study was to compare the extent of mammographic densities estimated subjectively by an experienced radiologist with the measured extent of densities using a digital planimeter. A total of 225 sets of mammograms from women aged 40 49 years and enrolled in the Canadian National Breast Screening Study (NBSS) were selected. The extent of the radiological densities was estimated visually by one radiologist. Independently, the total area of the breast and the areas of density were traced and measured using a digital planimeter. Visual estimations and measurements of mammographic densities were then compared to determine the extent of agreement between the two methods. In general, the two methods showed good agreement (kappa = 0.78). The measured area of mammographic densities tended to be slightly greater than the radiologist's estimations. Both methods were highly reproducible (radiologist-dependent method, kappa = 0.89; quantitative method, r = 0.95, P = 0.0001). Our results indicate that measurement of the area of mammographic density using a quantitative method is reliable, and correlates well with assessment by an experienced radiologist. The method may be useful for identifying women at increased risk of breast cancer. PMID- 7549821 TI - Parenchymal patterns of the breast defined by real time ultrasound. AB - The association of mammographic parenchymal patterns of the breast with breast cancer risk has been studied extensively but there is little information about the distribution of different patterns in populations at different risks for breast cancer. Such information could be obtained if a risk-free method of breast examination were available that could be applied to the general population. We have evaluated real time ultrasound for this application by comparing the parenchymal pattern as assessed by mammography with the extent of echogenicity in the breast on ultrasound examination in 102 subjects. Subjects were examined by both methods, the mammographic and ultrasound images independently classified, and the proportion of the breast occupied by radiological density or ductal prominence compared with the extent of echogenic areas on ultrasound. These two methods of classifying mammographic parenchymal patterns were found to be strongly correlated. Real time ultrasound may therefore be useful in the epidemiological study of mammographic pattern and breast cancer risk. PMID- 7549822 TI - Herault breast screening programme: results after 30 months of a mobile French schedule. AB - Breast screening in the Herault Department (France) has been carried out since July 1990. As with other French programmes, it is a population-based screen, whose main features are related to its organization in that: (a) it is performed by a specific unit (the 'Institut Montpellierain d'Imagerie Medico-Biologique'); and (b) the group of women targeted is those aged 40-70 years. The two-view mammography is performed every 2 years in mobile units. This paper presents the results after 30 months: 26,026 participants were screened from a target population of 52,617 women, giving a participation rate of 49%. The recall rate and the biopsy rate observed were 7% and 1.5%, respectively. The predictive positive value was 8.4% for a positive test while it was 35% for the biopsy; the values increased with age. 137 cancers were observed giving a detection rate of 5.3%, of which 82% were in situ. Small (< or = to 10 mm) tumours represented 41.5%, and 71.5% of the cancers were node negative. The surgical treatment was conservative for 66% of them. PMID- 7549823 TI - Influence of anethole treatment on the tumour induced by Ehrlich ascites carcinoma cells in paw of Swiss albino mice. AB - The anticarcinogenic potential of anethole was studied in Ehrlich ascites tumour (EAT) in the paw of Swiss albino mice. The antitumour activity was evaluated from the cytotoxicity of EAT-cells in the paw and their biochemical changes were determined from nucleic acids, protein, malondialdehyde (MDA) and glutathione (NP SH) concentrations. Furthermore, the observations on survival rate, tumour weight, its volume and body weight of EAT-bearing mice were made. The EAT-bearing paws were also evaluated for histopathological changes. Additional studies were undertaken on the cytological effects of anethole in order to establish its clastogenic and mitodepressive activity in normal mice. The results obtained in the present study revealed anethole to increase the survival time, reduce the tumour weight and volume and body weight of the EAT-bearing mice. It caused a significant cytotoxic effect in EAT cells in the paw, reduced the levels of nucleic acids and MDA, and increased NP-SH concentrations. The histopathological changes observed after treatment with anethole were comparable to the standard cytotoxic drug cyclophosphamide. The results on the frequency of micronuclei and the ratio of polychromatic erythrocytes to normochromatic erythrocytes showed anethole to be mitodepressive and non-clastogenic in the femoral cells of mice. Our results indicate the anticarcinogenic, cytotoxic and non=clastogenic nature of anethole. Further studies are warranted to explore the mode of action and safety of anethole for its possible use in cancer therapy. PMID- 7549824 TI - Influence of some covariates on the reproducibility of an Italian semi quantitative food frequency questionnaire. AB - We studied the influence of age, sex, education and time between interviews on the reproducibility of a semi-quantitative food frequency questionnaire (FFQ) developed in Italy for a case-control study on cancers of the breast and digestive tract. The questionnaire had been administered twice to 452 Italian men and women and included the weekly consumption of 77 food items or groups of foods, seven summary questions and three questions on some general dietary habits. Spearman correlation coefficients for the 77 dietary items plus the seven summary questions did not differ between males (median 0.61) and females (median 0.58), volunteers younger than 50 years (median 0.58) and aged 50 or more (median 0.59), volunteers with fewer than 10 years of education (median 0.58) and with 10 or more (median 0.59). A slightly higher Spearman correlation coefficient was found when the two interviews were conducted 5-6 months apart (median 0.60) or more than 6 (median 0.59) than when less than 5 (median 0.55). Similarly, Pearson correlation coefficients for the intake of 27 nutrients or micronutrients, plus caloric intake, computed from the FFQ showed no differences between males (median 0.65) and females (median 0.64), two age groups (median 0.67 for subjects aged < 50 years and 0.65 for those aged > or = 50), and two educational levels (median 0.68 for < 10 years and 0.65 for > or = 10 years). The median Pearson correlation coefficient for nutrients was slightly higher for those subjects who were interviewed with the planned interval (5-6 months, median 0.71) than when the interval was shorter (median 0.57) or longer (median 0.64).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549825 TI - Fish, n-3 fatty acids and human colorectal and breast cancer mortality. AB - Although there is a strong positive association between total fat consumption and colorectal and breast cancer risk, there is evidence that n-3 fatty acids, mainly found in fish oil, are protective. If this is so, we would expect to be able to detect an inverse correlation between fish consumption and colorectal cancer and breast cancer mortality. Mortality data for breast and colorectal cancer in 24 European countries were correlated with current fish and fish oil consumption and with consumption 10 and 23 years previously. In males there was an inverse correlation between colorectal cancer mortality and current intake of fish (P = 0.036), a weaker correlation with fish consumption 10 years earlier (P = 0.042) and none with consumption 23 years earlier (P = 0.12). The data were not statistically significant in females. There was no correlation at all between breast cancer mortality and fish or fish oil consumption at any time. It is concluded that fish consumption is associated with protection against the later promotional stages of colorectal carcinogenesis, but not with the early initiation stages. The results are discussed in terms of the role of prostaglandins in colorectal carcinogenesis. PMID- 7549826 TI - Molecular analysis of the 5q- chromosome. AB - Acquired interstitial deletions of the long arm of chromosome 5, are seen in anomalies of the myeloid cells. The refractory anemia (RA) or 5q- syndrome, in which the erythroid and megakaryocytic lineages are predominantly affected, is a relatively indolent clinical entity distinguishable, from the constellation of preneoplastic myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) with trilineage involvement. Recent molecular evidence suggests that the critical region of 5q deletion in MDS/AML resides in the D5S89 locus, which is proximal (centromeric) to the minimal region of loss in the 5q- syndrome RA. The invariable loss of the D5S89 locus in MDS/AML qualifies it for the MDS/AML tumor suppressor locus. The telomeric 5q31 gene governs erythroid and megakaryocytic differentiation and can be termed the RA locus. Isolation and characterization of these genes will lead to an understanding of molecular mechanisms underlying normal hematopoiesis and leukemic transformation. PMID- 7549827 TI - Intrinsic expression of the multidrug transporter, P-glycoprotein 170, in multiple myeloma: implications for treatment. AB - Multidrug resistance, mediated by the P-glycoprotein 170 transport pump, is a serious problem in multiple myeloma. In this review we discuss the expression of P-gp as a differentiation antigen on normal T and B lymphocytes. In myeloma, circulating presumptively malignant B cells express P-gp prior to chemotherapy. A variety of evidence characterizes these circulating B cells as members of the malignant clone in myeloma, including the demonstration that they share immunoglobulin heavy chain (IgH) rearrangements with bone marrow plasma cells, and their extensive DNA aneuploidy. In some patients the only components of the clonal populations that express P-gp are the circulating B cells suggesting that they represent a reservoir of multidrug resistant cells that maintain malignant growth and spread in myeloma. We speculate that exposure to chemotherapy alters clonal homeostasis and exerts positive selection pressure on generative components of the myeloma clone. Thus the possibility exists that chemotherapy perpetuates rather than eradicates myeloma stem cells. P-gp is detectable on bone marrow plasma cells in myeloma but appears to be in an inactive form that is unable to mediate efflux of marker dyes. A similar phenomenon is seen for normal human monocytes which have surface P-gp but lack any functional export of P-gp substrates. P-gp appears to vary depending in a cell-type specific manner suggesting that it may be feasible to design inhibitors of P-gp which selectively block P-gp export by malignant cells and spare the function of P-gp on normal tissue, including lymphocytes and normal hematopoietic stem cells. PMID- 7549828 TI - Regulation of human B cell lymphopoiesis by adhesion molecules and cytokines. AB - Recent advances in the ability to culture normal human B cell precursors have emphasized the supportive relationship between these cells and stromal cells in the bone marrow microenvironment. It is now possible to examine the role of adhesion molecules and cytokines in the regulation of different stages of human lymphopoiesis using these culture systems. Direct cell-cell adhesion mediated by the integrin adhesion molecule VLA-4 plays a critical role in supporting stromal dependent proliferation of human B cell precursors. In addition, human B precursor cell lines migrate underneath the stromal layer. This transmigration is VLA-4 dependent but not inhibitable by antibody to known VLA-4 ligands. IL-7 is secreted by the stromal layer, and is necessary for stromal-dependent proliferation of early human B cell precursors. Proliferation of early human B cell precursors or mature B cells. Since the bone marrow stroma is a source of cytokines with B cell precursor growth stimulatory activity, it is possible that adhesion interactions may play a co-stimulatory role with respect to cytokine secretion or response. As the cytokine requirements for human B cell lymphopoiesis become more completely defined, it will be important to uncover the cell-cell signals that regulate lymphopoiesis either directly or through modulation of cytokine secretion by supporting cells in the bone marrow microenvironment. The dependent relationship between human B cell precursors and the bone marrow microenvironment provides a model system for these cell-cell interactions which may be applicable to progenitor development in other lineages. PMID- 7549830 TI - Autocrine and paracrine regulation of neoplastic cell growth in hairy cell leukemia. AB - Hairy cell leukemia (HCL), a rare haematological disorder of B-cell origin, mainly presents with bone marrow infiltration, haematopoietic insufficiency, and splenomegaly. In some cases, osteolytic lesions can be observed. Many of these clinical features, especially haematopoietic insufficiency and osteolytic lesions are likely to be caused by soluble factors, such as cytokines. There is evidence that these factors are produced by the malignant hairy cells themselves, suggesting a paracrine pathway. The importance of autocrine as well as paracrine growth loops in growth regulation of HCL-cells is supported by a series of excellent studies, performed within the last few years. It could be clearly shown that cytokines are involved in this autocrine and paracrine regulatory process. The most important cytokines which should be mentioned in this respect are tumor necrosis factor alpha, (TNF alpha). Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6) and B-cell-growth factor (BCGF). The role of other factors such as viruses and oncogenes remains rather unclear. Nevertheless, recent data suggest that the c-fms, which encodes for the macrophage colony stimulating factor (M-CSF) may be involved in the pathophysiological control of HCL growth. In this review, we summarise the important data and studies performed recently which shed light on the complex network of autocrine and paracrine growth regulation of HCL. PMID- 7549829 TI - HRX gene rearrangement in secondary acute lymphoblastic leukemia. AB - The human tri-thorax gene (HRX) also called ALL-1 (Acute Lymphocytic Leukemia-1) as well as MLL (Myeloid-lymphoid or Mixed-lineage Leukemia) gene, is disrupted in the majority of leukemias with chromosomal abnormalities involving 11q23. The alteration of the gene is related to leukemogenesis of various types such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and acute mixed lineage leukemia. The gene is also rearranged in cases of secondary AML developing after exposure to chemotherapeutic agents, especially topoisomerase II inhibitors. In at least one report, genomic analysis of this recombination site showed the breakpoint to be a topoisomerase II binding site and that exposure to the inhibitor could induce the rearrangement. If exposure induces the rearrangement of the gene, secondary ALL as well as secondary AML could occur after exposure to these agents, because the type of leukemias with rearranged HRX gene is not limited to AML. We present here such a case of secondary ALL with this gene rearrangement which occurred during adjuvant chemotherapy for breast cancer. Although less cases of secondary ALL are reported in comparison with those of secondary AML, such case reports have been accumulating. The incidence of this type of leukemia should be clarified in the future. PMID- 7549831 TI - Interleukin-2 therapy for myelodysplastic syndrome: does it work? AB - Recent clinical studies suggested that interleukin-2 (IL-2) has therapeutic potential for some hematologic malignancies, but the therapeutic role of IL-2 for myelodysplastic syndrome (MDS) is still unclear. MDS is a clonal malignant disorder which often involves a variety of immunologic abnormalities. Examination of the effects of IL-2 on MDS in vitro yielded the following results: (1) IL-2 did not induce the proliferation of blasts in most MDS cases. (2) The cytotoxicity of IL-2-induced lymphokine-activated killer (LAK) cells for cell lines and MDS blasts was reduced in the high-risk MDS group (refractory anemia with excess blasts (RAEB), RAEB in transformation and MDS transformed to acute leukemia), but it was still preserved in the low-risk MDS group (refractory anemia (RA) and RA with ringed sideroblasts). However, considerable variation in LAK cell cytotoxicity was noted in each group. (3) The reduced LAK cell cytotoxicity observed in MDS was explained, at least in part, by the presence of a reduced of number of natural killer (NK) cells amongst the LAK cells. (4) MDS patients who have a high blood soluble IL-2 receptor (sIL-2R) level often had defects in NK and CD8+ T cells. These in vitro findings suggest that the response to IL-2 is heterogeneous in MDS patients, and those who have a low-risk MDS subtype and/or a low blood sIL-2R level, may be prone to respond to IL-2 therapy. Clinical trials are mandatory in order to elucidate the efficacy of IL-2 therapy in the treatment of MDS. PMID- 7549832 TI - Clinical, morphologic, cytogenetic and prognostic implications of CD34 expression in childhood and adult de novo AML. AB - Expression of CD34 by leukemic blasts was analyzed in 230 pediatric and 251 adult patients with de novo AML enrolled in two large multicenter trials (AML-BFM-87 and AMLCG respectively). The association between CD34 positivity and morphological classification according to FAB criteria, cytogenetic aberrations, immunophenotypic features and clinical characteristics was investigated. CD34 was expressed (> or = 20%) by leukemic cells from 45% of childhood and 43% of adult AML patients. CD34+ AML was often associated with M1/M2 morphology as well as the coexpression of CD7 and TdT. Translocation t(8;21), inv(16) and chromosome 5 and 7 aberrations were more frequently observed in CD34+ AML. There was a low frequency of CD34 expression in infant AML but no age dependency was evident in adult patients. CD34 expression exerted no influence on the rate of complete remissions (CR) after intensive multidrug induction therapy. In adults, 56% of the CD34-positive and 64% of CD34-negative cases achieved CR (P = 0.29), and the childhood trial even revealed a slight advantage for CD34+ AML with a CR rate of 80% vs. 71% for CD34-negative cases (P = 0.068). Long-term follow-up disclosed no significant differences in remission duration or event-free survival between the CD34-positive and CD34-negative groups. In conclusion, CD34+ AML patients comprise a heterogeneous group with good as well as poor risk factors. Though characterized by some distinct features, CD34 lacks prognostic significance in de novo AML patients submitted to intensive polychemotherapy. PMID- 7549833 TI - A phase I-II study of high-dose thiotepa, busulfan and cyclophosphamide as a preparative regimen for autologous transplantation for malignant lymphoma. AB - Thirty-four adults with malignant lymphoma at high-risk for relapse were treated on a Phase I-II study of high-dose thiotepa (THIO), busulfan (BU) and cyclophosphamide (CYC) with autologous marrow or peripheral blood stem cell support. Four patients were in untreated relapse, 7 had stable or progressive disease, 12 were in partial remission, and 11 were in complete remission after reinduction chemotherapy before proceeding to the preparative regimen. Median follow-up time is 22 months. Grades 3-4 regimen-related toxicity occurred in 3 (14%) of 22 patients treated with THIO 250 mg/m2 x 3, BU 1 mg/kg x 10 and CYC 50 mg/kg x 3, and this was considered the maximal tolerated dose-schedule. Of the 23 patients with active disease at the time of transplantation, 52% (95% CI 31-73%) achieved a complete response, and 26% (95% CI 10-48%) achieved a partial response. For all patients, median time to progression was 13 months, median survival was 16 months, and disease-free survival at 18 months was 34% (95% CI 18 51%). The combination of THIO, BU and CYC is tolerable as a preparative regimen for lymphoma and has a high response rate, but further measures are needed to reduce the relapse rate for patients with advanced disease. PMID- 7549834 TI - Skewed rearrangement of the VH4-21 gene during pre-B acute lymphoblastic leukemia. AB - Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71-4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire. PMID- 7549835 TI - Survival time and season-of-treatment initiation in children with acute lymphoblastic leukemia. AB - We examined survival in childhood acute lymphoblastic leukemia (ALL) as a function of season-of-treatment initiation. Survival for 1797 children aged 15 and under from the Surveillance, Epidemiology, and End Results (SEER) cancer registry, diagnosed and treated from 1973-1986 was obtained. There was no significant seasonal pattern for survival by season-of-treatment initiation. Neither age, gender, nor treatment type served as effect modifiers or confounders in this relationship. Female gender, age between one and ten years, and certain broad categories of treatment type were significant predictors of survival in separate univariate analyses, however. The nonsignificance of the results do not support the concept that season-of-treatment initiation is a prognostic factor in survival from ALL in children. PMID- 7549836 TI - In vitro sensitivity of B-CLL cells to fludarabine and interferons. AB - In this study we evaluated the cytotoxicity of Fludarabine (FAMP) both alone and in combination with alpha and beta interferon (IFN) against B-cells from patients affected by chronic lymphocytic leukemia (CLL). We used an in vitro colorimetric assay based on the bioreduction of the tetrazolium salt XTT by viable cells. Fludarabine concentrations ranging from 0.03 to 30 microM were tested on cells collected from 22 B-CLL patients. For each fludarabine concentration, 800 I.U. of either alpha or beta IFN were added. Interferon alone did not exert any cytotoxic effect, while Fludarabine showed a strong cytotoxicity against B-CLL cells. The concentration of Fludarabine required to induce a 50% cytotoxicity (IC50) was below 3 microM (the achievable serum level after standard dose in vivo administration) for 19 out of 22 patients. After IFNs supplementation to Fludarabine, it was possible to identify three groups of samples. The first in which IFNs addition did not produce almost any significant change in Fludarabine cytotoxicity (13/22), the second in which there was an improvement in FAMP IC50 (6/22), and finally the third group in which IFNs worsened it (3/22). Stage of disease was the only identified factor accounting for these different results. The second group included samples from 5 patients at stage A and one at stage B, while in the third group all three samples were from patients at stage C. Interferon-alpha and -beta induced the same degree of FAMP IC50 variation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549837 TI - Trisomy 14: a recurring cytogenetic abnormality associated with myeloid disorders. AB - Trisomy 14 as single karyotype aberration was detected in three patients, two with acute myeloblastic leukemia, AML-M2 type, and one with aplastic anemia. These new observations and the 28 previously reported cases confirm that trisomy 14 is a primary non random change, mostly confined to myeloid disorders. PMID- 7549838 TI - HTLV-I positive progressive spastic paraparesis (TSP) associated with a lymphoid disorder in three Chilean patients. AB - We describe the clinical and laboratory features in three Caucasian Chilean patients with tropical spastic paraparesis (TSP) associated with/or preceded by a lymphoproliferative disorder involving cutaneous lesions and localised lymphadenopathy. The neurological symptoms and signs were characteristic of TSP and CSF examination revealed the presence of oligoclonal bands. All three patients had a moderate leucocytosis (10-14 x 10(9)/l) with eosinophilia and a minority (2-4%) of circulating atypical polylobed or ATLL-like lymphocytes. Lymph node histology showed a diffuse pattern of infiltration (1 case) and marked expansion of the paracortical zone with convoluted lymphocytes and immunoblasts (2 cases). Skin biopsy demonstrated a dermal lymphoid infiltration with epidermotropism. Antibodies to HTLV-I were detected in the serum and CSF in the three patients and Southern blot analysis of peripheral blood mononuclear cells showed a monoclonal integration of HTLV-I proviral DNA in one case whereas in the two others the pattern was indicative of low level polyclonal integration. All three patients were treated with prednisolone and one with PUVA with transient partial response on the skin and neurological manifestations. Two patients died months to 5 years from presentation and the other is alive 12 years from diagnosis with active neurological and skin disease. The simultaneous occurrence of HTLV-I associated TSP with smouldering ATLL and a cutaneous ATLL or pre leukaemic form is discussed. PMID- 7549839 TI - A randomized study of MOD versus VAD in the treatment of relapsed and resistant multiple myeloma. AB - 67 patients with relapsed or resistant multiple myeloma were randomized to receive either VAD (vincristine, doxorubicin, dexamethasone) or MOD (mitozantrone, vincristine, dexamethasone). 12/30 (40%) patients receiving VAD and 15/37 (41%) patients receiving MOD achieved plateaux. The median duration of plateaux was significantly longer on VAD (15 months) than on MOD (8 months). No significant difference in overall survival was seen between the two treatment arms. The only toxicity which was severe in more than 5% of treatment cycles on either treatment arm was myelosuppression. No toxicity was significantly more severe on MOD than VAD. However, hair loss was significantly more severe on VAD than MOD. The frequencies of thrombocytopenia, haematuria and cutaneous toxicity were significantly greater on VAD than on MOD. Raised serum direct bilirubin levels were seen significantly more often on MOD than VAD. MOD and VAD have similar efficacy in relapsed/resistant multiple myeloma. MOD is the less toxic of the two regimens. PMID- 7549840 TI - Rising serum lactate dehydrogenase often caused by granulocyte-or Granulocyte macrophage colony stimulating factor and not tumor progression in patients with lymphoma or myeloma. AB - Since rising serum lactate dehydrogenase (LDH) heralds progression in patients with lymphoma or myeloma we investigated the significance of its elevations during chemotherapy supported by granulocyte (G-) or Granulocyte-Macrophage (GM-) colony stimulating factors (CSF). To Exclude effects of resistant disease we analyzed 52 courses of therapy in 36 responding patients. During hematologic recovery LDH increased above normal in 53% and 85% of patients with leukocyte counts of 10,000/microL and 15,000/microL, respectively. After CSF discontinuation LDH fell to or towards normal during 20 courses with adequate follow-up. Therefore rising serum LDH in patients with lymphoma or myeloma may be caused the CSF administration during chemotherapy and not by progressive disease. Proper identification of this effect can prevent unnecessary tests or treatment delays. PMID- 7549841 TI - Leukocyte alkaline phosphatase score in plasma cell dyscrasias: correlation with disease severity and circulating levels of granulocyte-colony stimulating factor. AB - In this study the leukocyte alkaline phosphatase (LAP) score in 106 patients with multiple myeloma (MM) in various phases of the disease (66 at diagnosis, 18 in plateau phase, 22 in relapse) was examined and compared with the score of 68 patients with monoclonal gammopathy of undetermined significance (MGUS) and 53 normal volunteers. In addition, the circulating levels of granulocyte-colony stimulating factor (G-CSF) were measured to explore the possible involvement of this cytokine in the pathogenetic mechanisms that lead to increased LAP activity. The results showed that the mean LAP score in patients with MGUS was comparable to normals and significantly lower than in MM (p < 0.001). Also, it increased with increasing tumor mass, and was lower in myelomas with stable disease than in those with active disease. G-CSF concentrations closely mirrored the behaviour of LAP score (r = 0.850, p < 0.001), significantly differing between each group of individuals. Its mean levels in MGUS were comparable to those of controls, whereas they were significantly increased in MM (p < 0.001), again with escalating values from cases with low tumor mass to advanced stages, and with lower concentrations in patients in plateau phase than in those in relapse. A significant correlation was found between G-CSF and neopterin levels (r = 0.578, p < 0.001), thus indicating an origin of the cytokine from monocytes and macrophages. These findings suggest that LAP scoring may assist in distinguishing benign from malignant paraproteinemias and may be used to follow the progression of plasma cell neoplasias.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549842 TI - Dexamethasone and suramin inhibit cell proliferation and interleukin-6-mediated immunoglobulin secretion in human lymphoid and multiple myeloma cell lines. AB - Uncontrolled growth of neoplastic cells and unregulated production of immunoglobulin are major components of the morbidity and mortality of multiple myeloma. Suramin, a polysulfonated napthylurea, has antitumor activity in a number of malignancies, but also significant dose-related toxicity. Suramin has been reported to have major antiproliferative effects in a variety of lymphoid cell lines. Glucocorticoids have long been recognized to have activity in lymphoid malignancies and multiple myeloma while IL-6 has been reported to be an autocrine growth factor for multiple myeloma. This study examines growth inhibition and inhibition of IL-6-mediated secretion of immunoglobulin in human lymphoid and myeloma cell lines by dexamethasone and suramin. Dexamethasone and suramin show synergistic inhibition of cell proliferation at their IC10 concentrations. IL-6-mediated immunoglobulin secretion is also inhibited by both dexamethasone and suramin in an additive fashion. Both dexamethasone and suramin induce apoptosis of lymphoid cell lines, and suramin inhibits the binding of IL-6 to its receptor in a multiple myeloma cell line. These findings suggest that the synergistic growth inhibitory activities of dexamethasone and suramin may be related to induction of apoptosis by both agents and inhibition of IL-6-mediated autocrine growth stimulation and immunoglobulin production. These results indicate that the combination of low-dose suramin (in concentrations not associated with significant clinical toxicity) and dexamethasone merit further study in the treatment of myeloma or lymphoid malignancies. PMID- 7549843 TI - A practical procedure for the cryopreservation of marrow cells intended for autotransplantation. AB - A simple and practical method of unfractionated bone marrow processing and cryopreservation was studied. The date shows that RBCs can be rapidly sedimented by methylcellulose or sodium carboxymethyl starch within 15-45 min. The cells can be cryopreserved in a mixture consisting of 5% DMSO, 6% HES and 4% Albumin prepared in a Lactated Ringer solution which is widely used, and can be simply immersed into a -80 degrees C freezer and stored in liquid nitrogen until infusion. Recovery percentages of nucleated cell, cell viability and CFU-G were similar to those cryopreserved with the conventional method. Clinical toxicity was mild in 12 infused patients. Of them 8 patients had received high dose chemotherapy +/- TBI and their peripheral WBC recovery was rapid. The recovery of WBC or Platelet (PLT) in the study group was similar to that of the control group whose marrow cells were cryopreserved in 10% DMSO. Therefore, cells cryopreserved with this method can also accelerate the hematopoietic recovery in myeloablatively treated patients. PMID- 7549844 TI - Immunocytochemical studies of an antigen in a human T-cell lymphoma line (Jurkat) recognized by certain monoclonal antibodies to CD-13 (aminopeptidase-N). AB - The immunofluorescent staining of Jurkat cells by a panel of eight CD-13 monoclonal antibodies has been investigated. With unfixed cells all antibodies were negative, in contrast to the CD-13-positive cell line, HL60, which gave staining in each case. These results were in line with our previous enzymological studies in which we showed that the aminopeptidase activity observed with intact Jurkat cells was located in the cytoplasm and was distinct in properties from aminopeptidase-N (CD-13), which is expressed by HL60 cells. After fixation of Jurkat cells, four antibodies, 22A5; MCS-2, 72a and WM-15, gave consistent positive staining, while the other four, WM-47, RMAG6, MY7 and CLB-mon-gran/2 were consistently negative. Prior fixation was an absolute requirement for staining, but was observed over a range of concentrations of paraformaldehyde from 0.4% to 4%, as well as with acetone or methanol/acetic acid. Confocal fluorescence microscopy confirmed that the positive fluorescence given by 22A5 was located in the cytoplasm, contrasting with that given by an antibody to a cell-surface marker, HLA-DR, which was associated with the plasma membrane. The fixatives thus appeared to have rendered the cells permeable to the antibodies, but the molecular size of the antibodies could not explain the different behaviour of the two classes of antibodies, since IgG1 isotypes were present in both positive and negative groups. We exclude the possibility that Jurkat cells express an intracellular form of aminopeptidase-N; the identity of the protein(s) bearing the epitope(s) to which four of the antibodies bound has yet to be determined. PMID- 7549845 TI - Peripheral neuroectodermal tumor of the chest wall in a patient treated for Hodgkin's disease. AB - The occurrence of second malignancies after treatment for Hodgkin's disease is a well recognized phenomenon. In this report we describe a case of a 22-year-old male who developed a peripheral neuroectodermal tumor of the chest wall four years after completing successful treatment with combined chemotherapy and radiotherapy for Hodgkin's disease. PMID- 7549846 TI - Juvenile chronic lymphocytic leukemia with unusual intracisternal inclusions: an ultrastructural study. AB - The electron microscopic analysis of intracisternal inclusions in lymphocytes of the bone marrow and peripheral blood in a case of juvenile chronic lymphocytic leukemia is described. These inclusions consist of well-ordered microtubules attached to a central axis. The contribution of electron microscopic analysis in establishing the substructural pattern of these inclusions is discussed. PMID- 7549847 TI - Occupational respiratory disease. PMID- 7549848 TI - Pre-eclampsia. PMID- 7549850 TI - Adverse reactions after measles vaccination in India. AB - BACKGROUND: One hundred and sixty million doses of measles vaccine have been used since the Expanded Programme of Immunization began in 1985. Mild-to-moderate vaccine reactions are not infrequent and are accepted by parents but occasionally severe reactions and even death occur. We studied these severe reactions. METHODS: From 1986 to 1994, 1762 batches of measles vaccine were tested and found to be satisfactory by the World Health Organization criteria. These were released for mass immunization. RESULTS: After 40 reported incidents of severe reactions or deaths in the field, 59 intact samples of vaccine produced by different manufacturers were tested and found to be safe, i.e. they were not toxic and were sterile. However, on testing reconstituted or used vials a few were found to be toxic and many were unsterile. Reactions occurred in 115 vaccinees resulting in death of 79 children. These reactions were characterized by high fever, vomiting and profuse watery diarrhoea resulting in death within 24 hours. CONCLUSIONS: Reactions to the vaccines were more likely to be related to the toxic shock syndrome due to the use of unsterile syringes and needles and perhaps the use of reconstituted vaccines beyond their specified time for administration resulting in contamination. PMID- 7549849 TI - Byssinosis in a Bombay textile mill. AB - BACKGROUND: Till a national campaign against dust-related lung diseases was launched by a voluntary agency in Ahmedabad in 1992, government records for the 150-year-old textile industry showed no cases of byssinosis--the disabling occupational disease caused by cotton dust. The worldwide incidence of byssinosis among workers in the dusty sections of textile mills is nearly 40%. We assessed the prevalence of byssinosis in a Bombay mill so that the Employees State Insurance Scheme would start conducting medical checks in all the 55 textile mills in Bombay and officially recognize the disease. METHODS: The study was conducted under the auspices of the Occupational Health and Safety Centre, a voluntary organization. Textile workers were called to a camp conducted over 3 nights and 3 days. We asked them to answer a questionnaire and tested their lung function using a Wright's ventilometer. The diagnosis of byssinosis was made if there was a feeling of chest tightness on exposure to cotton dust, and if the FEV1 was less than 60% of the expected result or the FEV1/FVC was less than 75%. RESULTS: Of the total 1075 workers in the mill only 273 came to the camp; 54 (30%) of the 179 individuals working in the dusty sections of the mill had byssinosis. In the non-dusty departments, 16 (17%) out of the 94 workers were affected. Among those working for less than 10 years in textile mills, 24% had byssinosis and among those working for more than 30 years, 45% had the disease. CONCLUSION: We found a prevalence of byssinosis among textile workers which is similar to that reported worldwide. The disease affected those who worked in both the dusty and non-dusty sections of the mill. There are an estimated 40,000 affected workers in Bombay and we suggest that the disease be recognized by the Employees State Insurance Scheme, and that the textile mill workers be compensated if they are affected by byssinosis. PMID- 7549851 TI - Primary drug resistance to Mycobacterium tuberculosis in renal transplant recipients. AB - BACKGROUND: After renal transplantation, patients have an up to 5% chance of being infected with Mycobacterium tuberculosis and there are reports from western countries of a 24% mortality if the infection is drug resistant. We investigated primary drug resistance in renal transplant recipients in Vellore, Tamil Nadu. METHODS: Between January 1987 and December 1993 we studied 695 patients (who had received 717 renal allografts) for evidence of tuberculosis, and performed drug sensitivity tests. RESULTS: Forty-three patients had culture-proven infection with Mycobacterium tuberculosis of whom 40 had drug sensitivity tests done. Initial drug resistance was seen from 1991. Rifampicin resistance was seen in 2, 1 and 4 patients and isoniazid resistance in 1, 2 and 2 patients in 1991, 1992 and 1993, respectively of the 23 isolates tested for drug susceptibility. Multi drug resistance was seen in 1 and 2 patients in 1992 and 1993. CONCLUSIONS: This is probably the first report in India of primary drug resistance of Mycobacterium tuberculosis in renal allograft recipients. It is a cause for concern as it may indicate a large reservoir of drug-resistant patients in the community. PMID- 7549852 TI - Laryngeal changes following long-term stenting with polyvinyl chloride tubes. AB - BACKGROUND: To treat tracheal stenosis and permit wound healing to occur an inert stent is required which resists the contraction process and prevents re-stenosis. The newer stents of polyvinyl chloride are not readily available in India and endotracheal tubes are used in many centres. In our centre polyvinyl chloride tubes are used nasotracheally for prolonged periods. We prospectively studied the pattern of laryngeal changes that occur because of prolonged nasotracheal stenting with polyvinyl chloride tubes. METHODS: The incidence and pattern of laryngeal changes due to stents was prospectively studied at the time of removal of stents, using an operating microscope. Twenty-nine patients underwent tracheal reconstructive surgery for tracheal stenosis (post-intubation or post tracheostomy) and tracheal injuries. RESULTS: Twelve different lesions were noted and their pattern was related to the period of stenting. Fibrosis, fibrous band formation and webs were noted after four weeks. A high incidence of laryngeal changes was seen in all age groups with an overall incidence of 79% (males 79%; females 88%; adults 82%, children 75%). The mean (SD) duration of stenting was 50 (33) days. Patients with laryngeal changes had a significantly (p < 0.05) longer duration of stenting [56 (34) days] compared to those without any lesion [29 (15) days]. CONCLUSIONS: After four weeks of stenting the severity and incidence of laryngeal changes increase. We recommend restriction of the use of polyvinyl chloride stents to less than four weeks to prevent permanent laryngeal damage. PMID- 7549854 TI - Propranolol or sclerotherapy to prevent variceal rebleeding. PMID- 7549855 TI - Double reading of magnetic resonance imaging films. PMID- 7549853 TI - Storage of haemopoietic stem cells for autologous bone marrow transplantation. AB - For reinfusing autologous bone marrow cells after high-dose chemotherapy and/or radiotherapy it is necessary that an effective technique for their storage is available. The traditional method uses 10% dimethyl sulphoxide as cryoprotectant, a rate-controlled computerized freezer programmed to cool the cells at a constant rate of 1 degrees C per minute and liquid nitrogen as the storage system. The method is time-consuming, expensive and requires technical expertise. Moreover, it is often associated with varying levels of clinical toxicity following infusion of the preserved cells. Processing the harvest to reduce the initial volume and the mature cells has been shown to be beneficial in reducing the volume of the cryoprotectant and the incidence of toxicity. An alternative, cost effective method using a cryoprotectant mixture of 5% dimethyl sulphoxide, 6% hydroxyethyl starch and 4% albumin has been found to be effective even when the cells are stored at -80 degrees C without rate-controlled freezing. However, its efficacy needs to be evaluated for extended periods. The current use of purging and cell sorting methods seems to be promising. PMID- 7549856 TI - Carcinoma of the gallbladder: can radical resection prolong survival? PMID- 7549857 TI - Epilepsy. PMID- 7549858 TI - A young Kenyan male with fever, hepatomegaly and hydropneumothorax. Postgraduate Institute of Medical Education and Research Chandigarh. PMID- 7549859 TI - Archie Cochrane and clinical outcomes. PMID- 7549860 TI - Mushrooming nursing homes. PMID- 7549861 TI - Malignant mesothelioma of the pleura. PMID- 7549862 TI - Poisoning by antidiarrhoeals. PMID- 7549863 TI - Renal transplantation--a fundamental right? PMID- 7549864 TI - Detection of H. pylori infection. PMID- 7549865 TI - On keyhole surgery. PMID- 7549866 TI - Poison information services. PMID- 7549868 TI - Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding. AB - Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k. PMID- 7549867 TI - Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme. AB - The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6 P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1 kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms. PMID- 7549869 TI - Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant. AB - Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038 1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation. PMID- 7549870 TI - Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I. AB - Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly. PMID- 7549871 TI - A three-dimensional model of aromatase cytochrome P450. AB - P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate. PMID- 7549872 TI - Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase. AB - The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase. PMID- 7549873 TI - Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry. AB - A simple biochemical method that combines enzymatic proteolysis and matrix assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA-binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max--a member of the basic/helix-loop-helix/zipper family of DNA-binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max-specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100-fold reduction in cleavage rates. Over a 2-day period, both complete and partial proteolysis of the Max-DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N-terminal and helix-loop-helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N-terminals. Less protection is seen at the C-terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix-loop-helix protein Max, the present method appears well suited for a range of other structural biological applications. PMID- 7549874 TI - Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain. AB - The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains. PMID- 7549875 TI - A phage display system for studying the sequence determinants of protein folding. AB - We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein. PMID- 7549876 TI - Interaction of SecB with intermediates along the folding pathway of maltose binding protein. AB - SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state. PMID- 7549877 TI - On the role of Glu-68 in alcohol dehydrogenase. AB - Theoretical computations (molecular dynamics and combined quantum chemical and molecular mechanical geometry optimizations) have been performed on horse liver alcohol dehydrogenase. The results provide evidence that Glu-68, a highly conserved residue located 0.47 nm from the catalytic zinc ion, may intermittently coordinate to the zinc ion. Structures with Glu-68 coordinated to the zinc ion are almost as stable as structures with Glu-68 at the crystal position and the barrier between the two configurations of Glu-68 is so low that it can readily be bypassed at room temperature. There is a cavity behind the zinc ion that seems to be tailored to allow such coordination of Glu-68 to the zinc ion. It is suggested that Glu-68 may facilitate the exchange of ligands in the substrate site by coordinating to the zinc ion when the old ligand dissociates. PMID- 7549879 TI - Comparison of methods for searching protein sequence databases. AB - We have compared commonly used sequence comparison algorithms, scoring matrices, and gap penalties using a method that identifies statistically significant differences in performance. Search sensitivity with either the Smith-Waterman algorithm or FASTA is significantly improved by using modern scoring matrices, such as BLOSUM45-55, and optimized gap penalties instead of the conventional PAM250 matrix. More dramatic improvement can be obtained by scaling similarity scores by the logarithm of the length of the library sequence (In()-scaling). With the best modern scoring matrix (BLOSUM55 or JO93) and optimal gap penalties (-12 for the first residue in the gap and -2 for additional residues), Smith Waterman and FASTA performed significantly better than BLASTP. With In()-scaling and optimal scoring matrices (BLOSUM45 or Gonnet92) and gap penalties (-12, -1), the rigorous Smith-Waterman algorithm performs better than either BLASTP and FASTA, although with the Gonnet92 matrix the difference with FASTA was not significant. Ln()-scaling performed better than normalization based on other simple functions of library sequence length. Ln()-scaling also performed better than scores based on normalized variance, but the differences were not statistically significant for the BLOSUM50 and Gonnet92 matrices. Optimal scoring matrices and gap penalties are reported for Smith-Waterman and FASTA, using conventional or In()-scaled similarity scores. Searches with no penalty for gap extension, or no penalty for gap opening, or an infinite penalty for gaps performed significantly worse than the best methods. Differences in performance between FASTA and Smith-Waterman were not significant when partial query sequences were used. However, the best performance with complete query sequences was obtained with the Smith-Waterman algorithm and In()-scaling. PMID- 7549878 TI - pH dependence of the stability of barstar to chemical and thermal denaturation. AB - Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7549880 TI - Incorporation of pairwise interactions into the Lifson-Roig model for helix prediction. AB - The helix/coil equilibrium of a peptide in solution can be modulated by a variety of side-chain interactions that are not incorporated into the standard statistical mechanical models for prediction of peptide helical content. In this report, we describe a recursive formulation of the Lifson-Roig model that facilitates incorporation of specific pairwise side-chain interactions as well as nonspecific individual side-chain capping interactions. Application of this extended model to a series of host/guest peptides indicates that the apparent delta G value for a pairwise apolar interaction is dependent upon the spacing and orientation but not the sequential location of the participating residues. The apparent delta G values for such interactions are about 40% greater than the corresponding apparent delta delta G values obtained from difference measurements. PMID- 7549881 TI - Domains in folding of model proteins. AB - By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36-mer) proteins, the all-or-none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48-mer, a simple all-or-none transition was observed in only three cases. For the other nine sequences, three states of behavior-the native, denatured, and intermediate states-were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other. PMID- 7549882 TI - Fuzzy cluster analysis of simple physicochemical properties of amino acids for recognizing secondary structure in proteins. AB - Fuzzy cluster analysis has been applied to the 20 amino acids by using 65 physicochemical properties as a basis for classification. The clustering products, the fuzzy sets (i.e., classical sets with associated membership functions), have provided a new measure of amino acid similarities for use in protein folding studies. This work demonstrates that fuzzy sets of simple molecular attributes, when assigned to amino acid residues in a protein's sequence, can predict the secondary structure of the sequence with reasonable accuracy. An approach is presented for discriminating standard folding states, using near-optimum information splitting in half-overlapping segments of the sequence of assigned membership functions. The method is applied to a nonredundant set of 252 proteins and yields approximately 73% matching for correctly predicted and correctly rejected residues with approximately 60% overall success rate for the correctly recognized ones in three folding states: alpha-helix, beta-strand, and coil. The most useful attributes for discriminating these states appear to be related to size, polarity, and thermodynamic factors. Van der Waals volume, apparent average thickness of surrounding molecular free volume, and a measure of dimensionless surface electron density can explain approximately 95% of prediction results. hydrogen bonding and hydrophobicity induces do not yet enable clear clustering and prediction. PMID- 7549883 TI - Automatic recognition of hydrophobic clusters and their correlation with protein folding units. AB - A method is described to objectively identify hydrophobic clusters in proteins of known structure. Clusters are found by examining a protein for compact groupings of side chains. Compact clusters contain seven or more residues, have an average of 65% hydrophobic residues, and usually occur in protein interiors. Although smaller clusters contain only side-chain moieties, larger clusters enclose significant portions of the peptide backbone in regular secondary structure. These clusters agree well with hydrophobic regions assigned by more intuitive methods and many larger clusters correlate with protein domains. These results are in striking contrast with the clustering algorithm of J. Heringa and P. Argos (1991, J Mol Biol 220:151-171). That method finds that clusters located on a protein's surface are not especially hydrophobic and average only 3-4 residues in size. Hydrophobic clusters can be correlated with experimental evidence on early folding intermediates. This correlation is optimized when clusters with less than nine hydrophobic residues are removed from the data set. This suggests that hydrophobic clusters are important in the folding process only if they have enough hydrophobic residues. PMID- 7549884 TI - De novo prediction of polypeptide conformations using dihedral probability grid Monte Carlo methodology. AB - We tested the dihedral probability grid Monte Carlo (DPG-MC) methodology to determine optimal conformations of polypeptides by applying it to predict the low energy ensemble for two peptides whose solution NMR structures are known: integrin receptor peptide (YGRGDSP, Type II beta-turn) and S3 alpha-helical peptide (YMSEDEL KAAEAAFKRHGPT). DPG-MC involves importance sampling, local random stepping in the vicinity of a current local minima, and Metropolis sampling criteria for acceptance or rejection of new structures. Internal coordinate values are based on side-chain-specific dihedral angle probability distributions (from analysis of high-resolution protein crystal structures). Important features of DPG-MC are: (1) Each DPG-MC step selects the torsion angles (phi, psi, chi) from a discrete grid that are then applied directly to the structure. The torsion angle increments can be taken as S = 60, 30, 15, 10, or 5 degrees, depending on the application. (2) DPG-MC utilizes a temperature dependent probability function (P) in conjunction with Metropolis sampling to accept or reject new structures. For each peptide, we found close agreement with the known structure for the low energy conformational ensemble located with DPG MC. This suggests that DPG-MC will be useful for predicting conformations of other polypeptides. PMID- 7549885 TI - Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method. AB - Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better. PMID- 7549886 TI - Crystallization and preliminary X-ray investigation of lipoxygenase-3 from soybeans. AB - Soybean lipoxygenase-3 has been crystallized by the vapor diffusion method in 16 20% polyethylene glycol (average M(r), 3,400), 0.2 M sodium acetate buffer, pH 5.7, at 21 degrees C, at a protein concentration of 8-15 mg/mL. The crystals, which diffract to 3-A spacings, belong to the monoclinic space group C2. Cell constants are a = 111.9, b = 136.4, and c = 61.6 A and beta = 95.7 degrees. The calculated value of Matthews's constant, Vm = 2.48 A3/kDa, is consistent with the presence of one molecule of lipoxygenase per crystallographic asymmetric unit (Z = 4). PMID- 7549887 TI - Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein. AB - Type III antifreeze protein, more specifically the recombinant QAE-Sephadex binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative. PMID- 7549889 TI - A putative FAD-binding domain in a distinct group of oxidases including a protein involved in plant development. AB - Using methods for database screening with individual protein sequences and alignment blocks, a conserved domain is delineated in a group of proteins including several FAD-dependent oxidases. Two motifs within this domain resemble phosphate-binding loops and may be directly involved in FAD binding. These motifs can be readily distinguished from previously described nucleotide-binding sites using a method for database screening with position-dependent weight matrices derived from alignment blocks. Unexpectedly, this group of known and predicted FAD-dependent oxidases includes the product of the DIMINUTO gene, which is involved in Arabidopsis development, and its homologues from man and Mycobacterium leprae. PMID- 7549888 TI - Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology? AB - Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2 strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed. PMID- 7549890 TI - Regulation of granulocyte-macrophage colony-stimulating factor and interleukin 3 expression. AB - Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL 3) are multilineage acting hematopoietic growth factors which have overlapping but distinct biological properties. Cellular sources of IL-3 are confined to activated T cells, natural killer (NK) cells, mast cells and possibly megakaryocytes, while these cells and activated macrophages, fibroblasts and endothelial cells are important sources of GM-CSF. In vitro studies have implicated both cytokines in the autocrine growth of human myeloid or murine mast cell leukemias. The human GM-CSF and IL-3 genes map to the long arm of chromosome 5, show similar genomic structures, and share several conserved elements in their 5' and 3' flanking regions. The promoters of these genes contain a variety of positive and negative regulatory regions, and the level of expression of these genes is controlled by both transcriptional and post-transcriptional mechanisms. PMID- 7549891 TI - Selective elimination of malignant stem cells using photosensitizers followed by light treatment. AB - The pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors. PMID- 7549892 TI - Allogeneic marrow transplantation and the use of hematopoietic growth factors. AB - Three hematopoietic growth factors, recombinant human granulocyte-macrophage colony-stimulating factor, recombinant human granulocyte colony-stimulating factor and erythropoietin, which are commonly used in other clinical situations are being increasingly studied in the setting of allogeneic marrow transplantation. Major questions being addressed are: 1) can administration of growth factors post-transplant accelerate hematopoietic recovery, 2) are growth factors of use in the treatment of patients with poor graft function or graft failure, 3) can growth factor mobilized peripheral blood stem cells be used as a substitute for allogeneic marrow and 4) is there a role for the use of growth factors in the treatment of patients with specific types of infection post transplant? This review will discuss where we are in answering these four questions. PMID- 7549893 TI - Do autologous peripheral blood cell transplants provide more than hematopoietic recovery? AB - Bone marrow damage caused by myeloablative radiation therapy and/or chemotherapy can be repaired by intravenously infusing viable stem/progenitor cells collected from either blood or bone marrow. The hematopoietic graft product contains both stem/progenitor cells and populations of hematopoietic and nonhematopoietic (accessory) cells. The frequency of accessory cell types varies with the source of the graft product; marrow or blood. Reinfusion of these accessory cells causes effects other than the hematopoietic restoration provided by the stem/progenitor cells such as graft versus host disease and graft versus leukemia effect after allogeneic transplants. Effects of infused accessory cells in the autologous setting are less well studied and could provide ancillary advantages and/or disadvantages to the patient. Do these additional effects actually occur, and, if they do, are they more likely to appear following peripheral blood cell transplants (PBCT) or after autologous bone marrow transplants (AMBT)? Preliminary data are beginning to accumulate which suggest that reinfusion of occult tumor cells is less likely with PBCT, that immune reconstitution is different depending on the source of the autograft and that, for certain diseases, patient event-free survival following PBCT rather than ABMT may be better. However, infusion of occult tumor cells may result in re-establishment of the malignancy. If the accessory cells (including potential occult tumor cells) are eliminated from the product before transplant, will the patient have a better clinical outcome, or would benefits provided by infused accessory cells outweigh the risks of infused occult tumor cells? These controversial issues are in the very early stages of investigation. PMID- 7549895 TI - Signal transduction through the IL-4 and insulin receptor families. AB - Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549894 TI - Primary thrombocythemia: a current perspective. AB - The relationships among thrombocytosis, abnormal platelet aggregation and altered hemostasis in primary thrombocythemia remain poorly understood. Consequently, the appropriate management of asymptomatic patients is controversial and needs to be individualized. For symptomatic patients, conventional therapy, usually hydroxyurea, is directed primarily at lowering the platelet count by suppression of megakaryocyte activity. Recombinant interferon alpha can selectively lower platelet counts and may offer a reasonable alternative. Recent experience with anagrelide is also most promising in both symptomatic and asymptomatic patients. Current thoughts on the pathogenesis and management guidelines are presented here. PMID- 7549896 TI - Mechanism of action of DNA topoisomerase inhibitors. AB - DNA topoisomerases are enzymes that regulate DNA topology and are essential for the integrity of the genetic material during transcription, replication and recombination processes. Inhibitors of the mammalian enzymes are widely used antitumor drugs. They stabilize topoisomerase-DNA cleavable complexes by hindering the DNA relegating step of the catalytic reaction, thus resulting in DNA cleavage stimulation. Investigations on the sequence selectivity of DNA cleavage stimulated by chemically unrelated compounds established that specific nucleotides flanking strand cuts are required for drug action. Moreover, structure-activity relationship studies have identified structural determinants of drug sequence specificities, thus eventually allowing the design of new agents targeted at selected genomic regions. The initial cellular lesion, i.e., the drug stabilized cleavable complex, is a reversible molecular event; however, how it may lead to cell death remains to be fully clarified. Several laboratories focused in past years on molecular and genetic aspects of drug-activated apoptosis. Irreversible double-stranded DNA breaks, generated from collisions between cleavable complexes and advancing replication forks, were suggested to increase p53 protein levels, thus triggering the cell death program. Other genes were also shown to cooperate in modulating the cell response to drug treatments. Recently, several groups have evaluated the possible prognostic value of topoisomerase II levels in solid tumors and hematopoietic neoplasms. Topoisomerase II inhibitors may also have genotoxic effects. Secondary leukemias, characterized by a translocation between chromosomes 11 and 9, have been reported in disease-free patients after treatments with drug regimens that included anti topoisomerase II agents. It has been proposed that an impairment of topoisomerase activity may be involved in the molecular pathogenesis of secondary leukemias. PMID- 7549897 TI - Tyrosine kinases in megakaryocytopoiesis. AB - Protein-tyrosine kinases (PTKs) are of vital importance in a variety of cell functions. Recent studies have provided considerable insight into the binding of growth factors to tyrosine kinase receptors and the consequent induction of signal pathways that lead to a biologic response. Future studies will further delineate the signals that result in a proliferative response and those that induce a differentiation response. Current studies, reviewed here, indicate an important biologic role for PTKs in the regulation of megakaryocyte development and maturation. Whether PTKs function in megakaryocytes in signaling pathways that are similar to pathways in other cells will need to be examined in future studies. PMID- 7549898 TI - Antigens recognized on human tumors by cytolytic T lymphocytes: towards vaccination? AB - It has been known for many years that cytolytic T lymphocytes that specifically recognize the tumor cells of the same patient can be derived from the blood of melanoma patients. Several of the antigens recognized by these antitumor T lymphocytes have now been completely identified. Some of them are sufficiently tumor-specific to envision their use as antitumor vaccines in selected cancer patients. PMID- 7549899 TI - Synergistic effects of stem cell factor and interleukin 6 or interleukin 11 on the expansion of murine hematopoietic progenitors in liquid suspension culture. AB - The synergistic effects of stem cell factor (SCF) in combination with other growth factors including interleukin (IL)-6, IL-11, IL-3, GM-CSF, G-CSF, IL-1 alpha and interferon-gamma (IFN-gamma) on the expansion of murine hematopoietic progenitors were studied in a short-term liquid suspension culture system. Bone marrow (BM) cells obtained 2 days after 5-fluorouracil (5-FU) injection were cultured for up to 18 days in serum-containing and serum-free cultures in the presence of combinations of various cytokines. The numbers of nucleated cells, total colony-forming cells (CFC), mixed-colony forming units (CFU-Mix) and high proliferative potential colony-forming cells (HPP-CFC) before and after liquid suspension cultures were measured in the presence of different combinations of cytokines. Combinations of SCF with IL-11, IL-6 or IL-1 alpha markedly increased the numbers of total CFC, CFU-Mix and HPP-CFC. A combination of SCF and IL-3 also expanded the number of total CFC; however, the fold increase was smaller than those of SCF plus IL-11, IL-6 or IL-1 alpha. Three or four factor combinations including SCF with IL-3, IL-6 and IL-11 did not yield increased numbers of total CFC over that supported by SCF plus either IL-6 or IL-11. The addition of IFN gamma to the culture containing SCF plus IL-11 resulted in a decrease of the expansion efficiency. However, this difference is not statistically significant. In contrast, the addition of IFN-gamma to the cultures containing SCF plus IL-6 did not affect the expansion efficiency. Interestingly, the addition of IL-1 alpha in the culture containing SCF plus IL-3 significantly increased the number of HPP-CFC over that supported by SCF plus IL-3 (p < 0.01). In contrast, IL-1 alpha did not significantly affect the expansion efficiency in the presence of SCF plus IL-6 or IL-11. These results suggest that combinations of SCF plus either IL-6 or IL-11 or a combination of SCF, IL-3 and IL-1 alpha can most effectively expand murine hematopoietic progenitors derived from day-2 post-5-FU BM cells in vitro. PMID- 7549901 TI - The levels of granulocyte colony-stimulating factor in the plasma of the bone marrow aspirate in various hematological disorders. AB - We developed a sensitive method of measurement of granulocyte colony-stimulating factor (G-CSF) by an enzyme-linked immunosorbent assay, which we applied in the plasma of the bone marrow aspirate in 70 patients with various hematological disorders. The lowest limit of detection by this method is 2 pg/ml. G-CSF was detected in all but two of the patients. Compared to the G-CSF level in normal healthy controls, those in non-Hodgkin's malignant lymphoma, aplastic anemia, agranulocytosis and multiple myeloma were significantly higher, while the level in refractory anemia was not different. The G-CSF level in acute myelogenous leukemia patients was either elevated or decreased regardless of the French American-British subgroup. The level in acute lymphoblastic leukemia was not different from the normal value, as was that in refractory anemia with an excess of blasts, and that in chronic lymphocytic leukemia. A patient with chronic myelomonocytic leukemia showed initial elevation of G-CSF with normalization after entering complete remission. The G-CSF level in chronic myelogenous leukemia was significantly decreased, although one patient in hematological remission who was under alpha-interferon therapy showed normal levels. The level in polycythemia vera was not significantly different from the normal value. The G CSF level for the entire group showed an inverse, although not statistically significant, correlation with the percentages of myeloid cells of the bone marrow (r = -0.174, p = 0.1703, n = 80). These results are thought to reflect the regulatory mechanism of granulopoiesis in the bone marrow in various hematological disorders, and it is concluded that this method may be of clinical use in the treatment of patients with these disorders and in the selection of candidates likely to benefit from G-CSF administration. PMID- 7549900 TI - Granulocyte-macrophage colony-stimulating factor as adjunct therapy in relapsed lymphoid malignancy: implications for economic analyses of phase III clinical trials. AB - With the increasing concern over the high cost of health care, policy makers have incorporated economic analyses into phase III clinical trials as the randomized clinical trials can provide important information on the efficacy and potential cost-effectiveness of new pharmaceutical agents. Economic analyses of single hospital experience during phase III trials of granulocyte-macrophage colony stimulating factor (GM-CSF) as adjunct therapy for high dose chemotherapy with autologous stem cell support found significant shortening of neutropenia with GM CSF at each hospital, but shortened hospitalization (and lower costs) at only two of three hospitals. In this study, we added data from three additional hospitals and found that the 103 patients who received GM-CSF had, on average, 5.7 days shorter durations of severe neutropenia than the 95 patients who received placebo (p < 0.0001) and 3.4 days shorter in hospitalization (p = 0.06). However, the duration of hospitalization, the primary determinant of health care costs, was shorter for GM-CSF patients in only four of the six centers and the duration of hospitalization of placebo patients was shorter at the other two centers. Careful analyses must be carried out when phase III clinical trial results are used to derive estimates of cost-effectiveness of new pharmaceutical agents. The interpretation of economic analyses of phase III clinical trials raises issues related to the perspective of the investigators, study design, collection of data on resource utilization, learning curve effects and generalizability of the results to other settings. PMID- 7549903 TI - Effect of lymphocytes on the production of granulomonopoietic enhancing factor by fully mature macrophages. AB - The granulomonopoietic enhancing factor (GM-EF) is a novel myelopoietic regulator produced by human monocyte-derived lipid-containing macrophages (MDLMs). In the present study, we examined the effect of lymphocytes on GM-EF production by preincubation of MDLMs with various preparations of lymphocyte subpopulations in cell-mixed and in double agar layer cultures. Our results showed that a cell concentration-dependent suppression of GM-EF production was noted in cultures with mitogen-activated T cells, and mitogen-activated/resting B cells, while those containing resting T cells had no such effect. Thus, GM-EF production in the presence of 1 x 10(5)/ml activated T cells or activated/resting B cells was greatly reduced to 5% or 20%, respectively. The lymphocyte-induced suppression was evident in both cell-mixed and double layer cultures, implying that the effector cells might exert their influences via mediators. Assay for cytokine activity revealed that a high level (648.2-685.2 pg/ml) of tumor necrosis factor alpha (TNF-alpha) was found in MDLM cultures with resting/activated B cells, and in those with activated T cells high levels of both TNF-alpha (510.5 pg/ml) and interferon-gamma (IFN-gamma) (321.3 pg/ml) could be detected, whereas in cultures with MDLMs and/or resting T cells, these cytokines were not measurable. Treatment of MDLMs with either recombinant (r) TNF-alpha or rIFN-gamma invariably resulted in a dose-dependent decrease in GM-EF production with intense suppression at doses between 400-800 U/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549902 TI - The presence of lymphoid-associated antigens in adult acute myeloid leukemia is devoid of prognostic relevance. AB - The immunophenotype of 110 adult patients with diagnosis of acute myeloblastic leukemia (AML) was analyzed using a wide panel of monoclonal antibodies (mAbs). Leukemic blasts were tested by applying direct immunofluorescence analysis and dual-fluorescence staining, and two groups of patients were identified: 56/110 (51%) expressing only myeloid antigens (My/AML) and 54/110 (49%) expressing both myeloid and lymphoid antigens (Ly/AML). CD13 and CD33 were expressed in almost all FAB subtypes, whereas CD14, frequently expressed in M4 and M5 subtypes (70%), was rarely expressed in M0 + M1 cases (9%). On the contrary, CD34, expressed in 77% of M0 + M1 cases, was practically absent in M3 and M5 subtypes (6% and 7%, respectively). CD2 and CD7 antigens were found in 34% and 42% of patients respectively, whereas B cell-associated antigens, such as CD10 and CD19, were found in 31% and 18% of patients. Cytogenetic abnormalities characteristically present in AML patients were also analyzed and, except for t(8;21) which was found in both groups of patients, the other abnormalities were frequently found in cases coexpressing lymphoid-associated antigens. Finally, the complete remission (CR) rate, survival and event-free survival were analyzed according to the presence of lymphoid markers and also of some specific antigens such as CD7 and CD34. The only prognostic difference was represented by CD34+ patients who showed a reduction in the CR rate compared with CD34- patients (65% versus 82%) (p = 0.05) which became more evident when the mean intensity of fluorescence was considered.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549904 TI - Role of p53 in leukemogenesis of chronic myeloid leukemia. AB - This review attempts to provide current information on the role played by the p53 gene in normal and leukemic hematopoiesis with particular emphasis on chronic myeloid leukemia. On the basis of the currently available data we can argue that p53 acts as a negative regulator of proliferation of myeloid mature cells and CD34+ progenitors, and its action is mediated through changes in cell cycle kinetics, mainly before the S phase. The p53-dependent pathway is also regulated by several proteins, including p16, p21, p27 (cyclin-dependent kinase [CDK] inhibitors), and a few oncogenes (bcl-2, bax, MDM-2). Although there is some information about the changes in the p53 gene seen in various types of leukemia, the functions and biological importance of these changes in the pathogenesis of leukemia are still largely elusive. During the past several years, accumulated evidence suggests that changes in the p53 gene are commonly associated with blast crisis of chronic myeloid leukemia (CML) but rarely with chronic phase, and they are represented by rearrangements, deletions and point mutations. As for most of the tumors, the majority of point mutations occur between exons 4 and 8 (hot regions). In patients with CML in blastic crisis the most frequent mechanism of p53 inactivation is complete deletion of one allele in association with a point mutation in the remaining allele.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549905 TI - Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases. AB - Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively. PMID- 7549907 TI - Effects of nicotinamide on hepatocyte viability and secretion of albumin and alpha 1-acid glycoprotein by adult rat hepatocytes in primoculture. Comparison with dexamethasone and recombinant human interleukin-6. AB - The effects of nicotinamide on hepatocyte viability and secretion of albumin and alpha 1-acid glycoprotein were studied in the absence or presence of dexamethasone and/or recombinant human interleukin-6 either after cell attachment (2 h) or after 24, 48, and 72 h of culture. The evolution of hepatocyte survival during the culture was appreciated by measurement of total DNA content. The secretion of albumin and alpha 1-acid glycoprotein was measured after a 4-h period following cell attachment or after 24, 48 and 72 h of culture. The important decrease of DNA content, mRNA levels and secretion of albumin and alpha 1-acid glycoprotein in control cultures after 2-3 days was not prevented by the addition of nicotinamide. In contrast, dexamethasone alone or with recombinant human interleukin-6 improved DNA content and albumin secretion with no additional effect of nicotinamide. The secretion of alpha 1-acid glycoprotein was largely induced by dexamethasone alone or dexamethasone and recombinant human interleukin 6. The increase of alpha 1-acid glycoprotein secretion was not modified by the addition of nicotinamide and averaged respectively 27- and 60-fold for dexamethasone alone and dexamethasone and recombinant human interleukin-6 after 48 h. These observations suggested that nicotinamide, at least in the conditions tested here, is unable to prevent alterations of hepatocyte viability and gene expression of cultured hepatocytes. PMID- 7549906 TI - Behaviour of the small GTP-binding protein rab6 in the liver of normal rats and rats presenting an acute inflammatory reaction. AB - While it is known that the small GTP-binding protein rab6 is localized in vitro to the Golgi apparatus of several mammalian cells, its behaviour in vivo has not yet been investigated. The aim of this work was to compare by immunocytochemistry and immunoblotting the distribution of rab6 in hepatocytes from normal rats and from rats with an acute inflammatory reaction, a circumstance where the synthesis and secretion of plasma proteins by the hepatocytes is increased and which is accompanied by several changes in the Golgi apparatus. Our results show that in normal rats, rab6 was present in all hepatocytes irrespective of the location of the cell in the hepatic lobule. At the ultrastructural level, rab6 was mainly visible on the three Golgi saccules, but in some cells it appeared to be absent in saccules corresponding to the cis or the trans saccule. The inflammatory reaction was accompanied by an increase of the immunocytochemical labelling at the light and electron microscopy levels. However, by immunoblotting, no differences in the total amount of rab6, nor in its subcellular distribution were found in liver cells after acute inflammatory reaction. These results demonstrate that rab6 is restricted in vivo to the Golgi apparatus and that no significant redistribution occurs during an acute inflammatory reaction. PMID- 7549908 TI - Functional and structural recovery of myotubes from mice with muscular dysgenesis after co-culture with normal, non-myoblastic cells. AB - Muscular dysgenesis is a mutation which is characterized by paralysis of skeletal muscle cells. Excitation-contraction coupling is deficient and muscle cells display atypical ultrastructure. In vitro, mutant myotubes recover a normal phenotype when co-cultured with spinal cord cells from normal animals or with normal fibroblasts. We have shown that other types of cells, eg certain glial cells present in the spinal cord or in other tissues, have this capacity. In contrast, intervention of neurons in the recovery does not appear likely. Very different types of non-myoblastic cells, then, are capable of restoring contractile activity of dysgenic myotubes in vitro, suggesting that a non specific mechanism is involved in the phenotypic reversion of affected muscle cells. The restoration process seems to imply a close relationship between myotubes and normal glial cells. PMID- 7549909 TI - Relationship between the acid phosphatases of the Kurloff body and the major 30 35 kDa glycoproteins of the Kurloff cell. AB - This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 microns diameter) periodic acid-Schiff-positive lysosomal inclusion body present in Kurloff cells (KC). KC AcPase were extracted, with similar yields, either with non-ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE-cellulose chromatography of such crude Dounce extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I)1). The main protein content consisted of, as testified by SDS PAGE analysis, major KC glycoproteins of 30-35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N glycosylproteins among which the presence of alpha (2,6) sialoglycoproteins was previously established. Following electrophoresis on a 4-15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I. After Clostridium-derived sialidase digestion of peak I, the highly active bands observed at pH 3.5-5.2 disappeared. These zymographic patterns were similar to those obtained with crude extracts. After Concanavalin A (ConA)-Sepharose chromatography of peak I, the single ConA-bound glucosamine labelled peak, eluted at 200 methyl-alpha-D-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activity. After SDS-PAGE analysis, the ConA-bound fraction appeared to correspond only to a single broad protein band in the 30-35 kDa zone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549910 TI - Cytochalasin D induces changes in cell shape and promotes in vitro chondrogenesis: a morphological study. AB - One of the initial events required for the expression of cartilage-specific macromolecules in monolayer cultures is the reversion to the initial round shape of chondrocytes. Thus, considerable research efforts have focused on developing reliable procedures to maintain a round morphology of cultured chondrocytes. Our study focuses on evaluating the response of dedifferentiated fetal rat chondrocytes to cytochalasin D, an actin-disrupting agent, with special emphasis on the morphological events. Immediately after exposure to the drug, cells round up but flatten again after removing the agent. However, immunocytochemical procedures revealed a disorganization of microfilaments and intermediate filaments. Phase-contrast and scanning electron microscopic observations revealed that on day 6 of culture, cells located at the top of the cell layer adopted a spherical morphology. Prominent differences were noted in control cultures where cells had to aggregate prior to overt chondrogenesis. Transmission electron microscopy confirmed the round morphology of the cells situated at the top layer but also revealed the presence of cell contacts between the cells. In addition, cells located at the central part of the cell layer displayed a typical morphology of mature chondrocytes, separated by an extensive extracellular matrix. These morphological changes occurred parallel to the expression of type II collagen and chondroitin sulfate, both hallmarks of the chondrocyte phenotype strong in experimental cultures, relatively weak in control cultures, and only restricted on areas of polygonal cellular aggregates. Furthermore, [35S]-sulfate incorporation into sulfated glycosaminoglycans increased rapidly with the period of culture to a maximum after 7 days and was then two-fold in treated cultures. Taken together, these findings indicated that cytochalasin D stimulates chondrogenesis in response to modification of cytoskeleton architecture and the subsequent rounding up of the cells. PMID- 7549911 TI - Interferon alpha/beta-induced abnormalities in adipocytes of suckling mice. AB - The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN-treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non-homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN-treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 +/- 1.5 vs 20.1 +/- 1.9, P < 0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN treated than in control animals (1.9 +/- 0.7 vs 0.9 +/- 0.7 for mitochondrial section, P < 0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN-treated mice. The present work demonstrates that, in the BAT of suckling mice, IFN-treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BAT of aged animals. PMID- 7549912 TI - RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro. AB - Porcine granulosa cells cultured in serum-free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite-2000, an integrin-binding synthetic peptide containing RGD (Arg-Gly-Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin beta 1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F-actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin beta 1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite-2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature commitment of cells towards luteinization rather than completion of follicular preovulatory differentiation. PMID- 7549913 TI - Heterogeneous localization of epitopes along axonemes of mammalian cilia. AB - The specificity of four monoclonal antibodies, raised against mammalian ciliary axonemes, was determined by both immunofluorescence and immunoblot experiments. Three antibodies reacted with epitopes which are differentially located along axonemal length. Among these, antibody 3.12 recognized an epitope common to different dynein heavy chains, reacted only with tracheal cilia and specifically stained the proximal portion of the ciliary axoneme. PMID- 7549914 TI - Immunocytochemical study of lampbrush chromosomes of the urodele Pleurodeles waltl: axial granules are recognized by the mitosis-specific monoclonal antibody MPM-2. AB - The lampbrush chromosomes of the urodele Pleurodeles waltl have been studied using the mitosis-specific monoclonal antibody MPM-2. Immunofluorescence studies revealed that MPM-2 stains structures associated with axial granules, numerous other chromomeres, telomeres and certain chiasmata. These structures showed a negative reaction with the anti-DNA monoclonal antibody AC-30-10. In course of meiotic condensation of the chromosomes, in growing and maturating oocytes, the number of such structures associated with the chromosome axis was found to diminish progressively. These granular structures have been found to be formed by fine fibrils about 5 nm in diameter. Immunogold labeling confirmed the results of immunofluorescence studies. MPM-2 was also found to stain two other types of structures observed in association with the lampbrush chromosome axis in P waltl, viz the sphere organelle (only in later stages of oogenesis) and the structure known as 'M' which is singular to this material. PMID- 7549915 TI - Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C beta 1 in cells exposed to mitogenic or differentiating agonists. AB - The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta 1 isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF I), a rapid and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC beta 1 isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC beta 1 activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP2 in the nucleus, accompanied by a decrease of the intranuclear amount of PLC beta 1 isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling. PMID- 7549916 TI - Ultrastructural study of intestinal lactase gene expression. AB - The distribution of the mRNA encoding rat intestinal lactase-phlorizin hydrolase (LPH) was analyzed by ultrastructural in situ hybridization, and compared to the distribution of the cognate protein. Different pictures were obtained depending on the epithelial cell position along the crypt-villus axis: i) cells localized at the crypt base were devoid of LPH mRNA and protein; ii) cells of the crypt villus junction and of the villus base showed a high level of mRNA but a low amount of enzyme; and iii) enterocytes of the middle and upper part of the villi exhibited an intense protein immunolabeling, but a low content of LPH mRNA. The analysis of the intracellular distribution of the LPH mRNA revealed a gradient of concentration along the cellular axis, as the transcripts were the most abundant in the apical and subapical domains of the enterocytes. In addition, LPH mRNA labeling was often paired or arranged in a circular shape, suggesting that the transcripts may be located in closed vicinity to rounded structures within the cytoplasm. The results indicate that distinct patterns of mRNA and protein occur along the villus height during the continuous process of cell differentiation of the intestinal epithelium. In addition, the emergence of the functional polarity of the enterocytes comes together with the establishment of an intracellular gradient of concentration of the mRNA encoding a brush border enzyme. A link between the intracellular distribution of LPH mRNA molecules and the membranous flow is proposed. PMID- 7549917 TI - Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: a combined freeze-etch electron microscopy and biochemical study. AB - The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimensions as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer. PMID- 7549918 TI - The important role of PKC in controlling polyploidy formation in cultured fibrosarcoma cell line. AB - Induction of polyploidization by colcemid in cultured fibrosarcoma cells (Meth-A cells) was examined. Activators of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) and ATP, inhibited colcemid-induced polyploidization, but not colcemid-induced cell proliferation cessation. These findings suggest that a down regulation of PKC activity results in checkpoint "dysfunction" which induces polyploidization and that inhibition of polyploidization induction by PMA and ATP is not a result of the inhibition of colcemid-induced depolymerization of tubulin. PMID- 7549919 TI - Molecular species of phospholipids of interleukin-2-dependent murine cytotoxic T lymphocytes. AB - We reported that phospholipids of interleukin-2 (IL-2)-dependent murine cytotoxic T lymphocytes (CTL) contained high amounts of ether lipids (Biochem. Mol. Biol. Int., 33, 137-146, 1994). In this study, the molecular species of fatty acids at the sn-1 and sn-2 positions were determined. First, phosphatidylcholine mainly consisted of the 1,2-diacyl subclass (78%) in which the major molecular species was 16:0/18:1 (24%). It should be noted that arachidonic acid was abundant in both 1-O-alkyl-2-acyl subclasses in the form of 18:0/20:4 (21%) even though the content of the subclass was low (8%). Second, phosphatidylethanolamine mainly consisted of ether subclasses (93%) and contained a large amount of arachidonic acid in the forms of either 18:0/20:4 (19%) or 16:0/20:4 (10%). Third, phosphatidylinositol, a putative phospholipid involved in transmembrane signal transduction, had a significant amount of arachidonic acid at the sn-2 positions of 1,2-diacyl and 1-O-alkyl-2-acyl subclasses. Fourth, phosphatidylserine, consisting of mostly the 1-O-alkyl-2-acyl subclass (86%), contained arachidonic acid dominantly in the form of 18:0/20:4 (33%). Considering these results, it appears that CTL have the potential to mediate its signal transduction mediated by the bioactive lipids, i.e. arachidonic acid metabolites and the ether lipids. PMID- 7549920 TI - The myristoylated alanine-rich C-kinase substrate (MARCKS) is not required for mitogenic signalling via protein kinase C in cultured rat intestinal epithelial (RIE-1) cells. AB - Activation of protein kinase C (PKC) by angiotensin II or 12-O tetradecanoylphorbol-13-acetate (TPA) was associated with a mitogenic response in RIE-1 rat intestinal epithelial cells. However, whereas in control experiments using Swiss 3T3 cells TPA stimulated phosphorylation of the major PKC substrate, MARCKS, the agent did not induce the phosphorylation of any protein with the electrophoretic mobility pattern of MARCKS in RIE-1 cells. However, TPA was able to activate PKC in RIE-1 cells since the agent reduced ('transmodulated') 125I EGF binding to the cells. The failure of TPA to induce phosphorylation of MARCKS in RIE-1 cells was due to the lack of expression of MARCKS protein and mRNA by these cells. MARCKS is not therefore required for mitogenic signalling via PKC in RIE-1 cells. PMID- 7549921 TI - 2-Methyl-L-tryptophan is a substrate of tryptophanase. AB - Tryptophanase was generally considered to be inactive towards tryptophan derivatives substituted at 2-position of the indole ring. We have shown that cells containing tryptophanase catalyze the formation of 2-methyl-L-tryptophan from 2-methylindole and L-serine, and from 2-methylindole, pyruvate and ammonium ion. The kinetics of pyruvate formation from 2-methyl-L-tryptophan and its alpha deuterated analogue catalyzed by homogeneous tryptophanase were examined. The primary deuterium isotope effect (kH/kD = 4.0) as well as the absorption spectrum of tryptophanase complex with 2-methyl-L-tryptophan indicate that the rate of enzymatic reaction of 2-methyl-L-tryptophan is in a considerable degree determined by the stage of removal of alpha-proton. PMID- 7549922 TI - Purine arabinonucleoside 5'-triphosphates with substituents at 2' position as substrates for DNA polymerases. AB - Analogues of araNTPs carrying an azido or aminogroup instead of the 2' hydroxyl exhibited substrate properties towards several mammalian and viral DNA polymerases. At the same time, introduction of a bulky hydrophobic DNP group into the 2' position inactivated the compounds as substrates. HSV-1 and CMV DNA polymerases were an interesting exception: they effectively incorporated the modified nucleotide residues with DNP group into the 3'-termini of the DNA chain. This is a reliable distinction of these enzymes from cellular DNA polymerases. PMID- 7549923 TI - Maturation of immunoglobulin-A binding proteins in the seeds of jackfruit. AB - The accumulation of proteins in the jackfruit seeds was monitored by extracting seeds proteins at various stages of growth. The early developing seed had several protein bands distributed over a molecular weight range of 10-67 kDa. But the mature seeds contained fewer bands with two of them being prominent at 12 and 15.4 kDa. Upon isoelectric focusing, the mature seed proteins were resolved into several bands distributed over a pH range of 4-9. While the mature seed proteins contained powerful hemagglutinating activities, these were not detected in the early developing seed. In contrast, the early developing seed had immunoglobulin A (IgA) binding activity as determined by enzyme-linked immunosorbent assay (ELISA). By Western blotting it was found that the early developing seed contained a novel 10 kDa IgA binding protein that was not present in the mature seeds. PMID- 7549924 TI - Cloning and characterization of an alpha-amylase gene from Streptomyces sp WL6. AB - An alpha-amylase gene from Streptomyces sp WL6 was cloned on a 3.1kb DNA fragment, which was completely sequenced. The 3088 nucleotide sequence obtained contains three putative coding regions in the same orientation. The one corresponding to the structural region of the alpha-amylase gene has a deduced amino acid sequence of 459 residues, showing up to 71% identity to other alpha amylases. An incomplete ORF was identified upstream the alpha-amylase gene, and the deduced product presents some homology to proteins involved in catabolic regulation. PMID- 7549925 TI - Biophysical factors affecting the expression of isopenicillin N synthase isozymes in Escherichia coli. AB - The influence of biophysical factors over the production of isozymes of isopenicillin N synthase (IPNS) from fungal and bacterial sources in Escherichia coli was examined. The effect of these parameters on the expression of Cephalosporium acremonium IPNS was determined by using two expression vectors containing the lac- or the T7-promoter. Under the lac-promoter, IPNS expressed in E. coli reached 5-15% of total soluble proteins and under the T7 promoter, the level reached approximately 50% of total soluble proteins. Furthermore the high level of expression did not affect aggregation of IPNS proteins. Analysis of the hydrophobicity of the C. acremonium IPNS and site-specific mutant enzymes relative to other IPNS isozymes suggested that proteins of low gravy scores (< or = -0.515) tend to be soluble whereas higher gravy scores (> or = -0.512) form aggregates. Neither the GC content of the gene nor the relative percentage proline content of the protein affected the expression of the IPNS isozymes in E. coli. PMID- 7549926 TI - Expression of pS2 gene in rat brain. AB - We have previously shown that the expression of pS2 mRNA, which encodes a secreted 60-amino acid protein of unknown function, is widely distributed throughout the entire body of the mouse including the brain. We report herein that pS2 mRNA is also expressed in the brain and in peripheral tissues of rats. In adult rat brain, pS2 mRNA was predominantly expressed in hippocampus, followed by the cerebral cortex and cerebellum. The developmental expression of pS2 mRNA in hippocampus, which region is known to mature after birth, showed a clear peak in 1- or 3-day-old rats, then gradually decreased by 7 weeks after birth. In midbrain, the maturation of which occurs at an early developmental stage, pS2 mRNA level was retained at a low level from postnatal 1 day to week 7. These results suggest that pS2 protein plays an important role in the development of central nervous system. PMID- 7549927 TI - Biphasic effect of staurosporine on thymocyte apoptosis. AB - When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), apoptosis was induced dose- and time-dependently. Staurosporine-induced apoptosis was dependent on macromolecular synthesis, and it was also dependent on protein phosphorylation sensitive to 1-(5-isoquinolinesulfonyl-2-methylpiperazine dihydrochloride (H-7). Whereas, staurosporine at high doses (above 500 nM) did not induce significant DNA fragmentation, rather it inhibited the DNA fragmentation induced by 12-O-tetradecanoyl-13-acetate, A23187, and dibutyrylcyclic AMP, as H-7 did. K252a, a derivative of staurosporine, induced apoptosis, which was inhibited by H-7, even at high doses. These results indicate that staurosporine had a biphasic effect on thymocyte apoptosis, a stimulatory effect at low concentration, and an inhibitory effect at high concentration. K252a had only the former action. PMID- 7549928 TI - Lipid peroxidation products mediate damage of superoxide dismutase. AB - Membrane lipid peroxidation processes yield products that may react with superoxide dismutase (SOD), one of the key antioxidant enzymes against oxidative stress, resulting in oxidative modifications. We have investigated this possibility and have found that three types of SOD--CuZnSOD, MnSOD, and FeSOD- exposed to autoxidized linolenate in the presence of Fe3+ caused the loss of dismutase activity, fragmentation of peptides, and an increase in protein oxidation. Lysates from E. coli oxyR strains treated with tert-butyl hydroperoxide, which presumably induces lipid peroxidation, resulted in the activation of SOD. The lipid peroxidation-mediated damage to SOD may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. PMID- 7549929 TI - Inhibition of protein kinase C by snake venom toxins: comparison of enzyme inhibition, lethality and hemolysis among different cardiotoxin isoforms. AB - Cardiotoxins, neurotoxins and phospholipase A2 (PLA2) are three major classes of toxic components present in the Taiwan cobra, Naja naja atra of the Elapidae family. Cardiotoxins (or called cytotoxins), a group of major polypeptides of around 60 amino-acid residues present abundantly in the elapid family of snakes, comprise about 45-55% of the crude venom of Taiwan cobra. In contrast to another prominent group of structurally similar neurotoxins with well-established acetylcholine receptors and modes of action, cardiotoxins showed no defined cellular targets and very diverse pharmacological functions. A systematic structure/function comparison of these toxins was made by their relative inhibitory effects on protein kinase C (PKC) isolated from mouse brains. Lethality and hemolysis of various cardiotoxin isoforms were also compared in order to shed some insight on the biological targets and mechanisms of these surface-active amphiphilic polypeptides. A structure comparison of these cardiotoxins based on computer model-building revealed that some defined and subtle differences can be detected upon the superposition of these three dimensional polypeptide chains, which may reflect the intrinsic differences in the hydrophobic peptide segments present on the surface loops of toxin molecules. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by cobrotoxin and PLA2 on PKC. PMID- 7549930 TI - Effect of cyclosporin A on Ca2+ fluxes and the rate of respiration in Ehrlich ascites tumour cells. AB - Cyclosporin A at micromolar concentration decreases the respiration of Ehrlich ascites tumour cells (with pyruvate as substrate) but prevents the inhibition of oxygen uptake produced by glucose or deoxyglucose (the Crabtree effect). Cyclosporin A also diminishes the increase of cytoplasmic free Ca2+ concentration elicited by deoxyglucose and almost completely abolishes this increase induced by extracellular ATP and thapsigargin but does not decrease the size of endoplasmic reticulum Ca2+ stores as revealed after addition of ionomycin. It is concluded that cyclosporin A inhibits the inositol trisphosphate-sensitive Ca2+ channel and the passive permeability of the endoplasmic reticulum to Ca2+ in Ehrlich ascites tumour cells. PMID- 7549931 TI - A filter paper assay for hyaluronic acid synthetase: application to the enzyme from Swiss 3T3 fibroblasts. AB - An improved assay for hyaluronic acid (HA) synthetase is described that is suitable for rapid processing of large numbers of samples. High background levels of unincorporated radioactivity are removed by passage of the reaction through a Sephadex G-50 spin column. The labeled HA product is then precipitated onto glass fiber filters with cetylpyridinium chloride. Apparent Km values for HA synthetase from Swiss 3T3 fibroblasts are 10.8 and 58.4 microM for UDP-glucuronic acid and UDP-N-acetylglucosamine, respectively. HA synthetase activity of quiescent cells is 4.5% of that found in actively growing cells and is stimulated in response to 10% calf serum. There is a greater than 10-fold increase in HA synthetase activity when cells are harvested with hyaluronidase as compared with trypsin. PMID- 7549932 TI - Mitochondria-bound hexokinase from rabbit reticulocytes is resistant to the inactivation induced by Fe(II)/ascorbate. AB - Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms. PMID- 7549933 TI - Modelling cortical cataractogenesis. 16. Leakage of lactate dehydrogenase: a new method for following cataract development in cultured lenses. AB - The in vitro effect of glucose on cultured rat lenses was correlated with (i) the time-dependent leakage of lactate dehydrogenase (LDH) into defined medium; (ii) the appearance of the lens under the dissection microscope; and (iii) the leakage of lens cytosolic proteins. A protective effect of 1 mM Vitamin C (VC) was also found: the extent of opacification, LDH and gamma-crystallin release were reduced if 1 mM VC was included in the medium. Using the above parameters provides a new and more rapid technique to follow lens opacification in vitro. PMID- 7549934 TI - Expression of human plasminogen activator inhibitor-2 in the baculovirus expression system. AB - A cDNA coding for human plasminogen activator inhibitor-2 (PAI-2) was transferred into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus synthesized a 43 kDa PAI-2 protein, the majority of which was secreted. The recombinant PAI-2 was identical to native PAI-2 purified from human placenta with respect to interaction with polyclonal antibody and inhibition of urokinase-type plasminogen activator. PMID- 7549935 TI - Common promoter features in human and mouse liver type phosphofructokinase gene. AB - The liver-type phosphofructokinase is a key glycolytic enzyme encoded by genes residing on human and mouse chromosomes 21 and 10 respectively. Genomic DNA regions upstream of the initiator ATG spanning 2.6Kb and 3.4Kb of human and mouse liver-type phosphofructokinase gene were sequenced and analyzed. The proximal 0.4Kb region of both genes featured a CpG island containing 60%-73% GC residues. The first 120 nucleotides preceding the ATG are highly conserved displaying 73% of sequence similarity between human and mouse genes. While this region lacks TATA and CAAT boxes it contains four Sp1 binding sites and was capable of promoting a non regulated expression of the reporter gene chloramphenicol acetyl transferase, in transfection assays. Additional conserved elements were found further upstream at the 5'-region of both the human and mouse genes. They consisted of two Alu repeats and several sequence motifs known to serve as transcription factors binding sites. PMID- 7549936 TI - The effect of temperature and concentration of LDL and HDL cholesteryl esters and lipoprotein deficient serum on the cholesteryl ester transfer reaction in vitro. AB - We studied the effect of temperature, concentration of low-density (LDL) and high density lipoprotein (HDL) cholesteryl esters, and the concentration of human lipoprotein deficient serum (LPDS, a source of cholesteryl ester transfer activity) on the fluxes of cholesteryl esters between human LDL and HDL in vitro. Incubation temperatures above 37 degrees C resulted in an increase of the fluxes, and a six-fold increase was observed at 55 degrees C. Decreasing the temperature had the opposite effect, and no transfer activity was measured below 10 degrees C. Increasing the concentration of LPDS without changing the concentration of LDL and HDL resulted in a linear increase of the fluxes. Increasing the concentration of LDL and HDL cholesteryl esters without changing the concentration of LPDS resulted in a nonlinear increase of the fluxes, and the results could be described by a Lineweaver-Burk plot. When both the concentration of LPDS and HDL and LDL cholesteryl esters were increased in the same proportions, the fluxes increased more than proportionally. PMID- 7549937 TI - Purification and characterization of an acid phosphatase from Arachis hypogaea. AB - An acid phosphatase from Arachis hypogaea (peanuts) has been purified. The electrophoretically homogeneous enzyme preparation is free of any phophodiesterase activity. The enzyme has a molecular weight of 120,000. Among the various phosphomonoesters tested, p-nitrophenylphosphate was found to be its most effective substrate. The Km for p-nitrophenylphosphate was 1.21 mM at pH 5.0 and 25 degrees C. The enzyme was thermostable and did not loose activity after 1 hr at 50 degrees C. PMID- 7549938 TI - Production of functional human bone morphogenetic protein-2 using a baculovirus/Sf-9 insect cell system. AB - A clone of a human Bone Morphogenetic Protein-2 (hBMP-2) cDNA was obtained from a cDNA library established from human dental pulp cells. After subcloning hBMP-2 cDNA into Autographa californica nuclear polyhedrosis virus, the recombinant baculovirus was transfected to Sf-9 cells. Immuno-reactive recombinant hBMP-2 (rhBMP-2) was detected by a polyclonal antibody against Xenopus BMP-2 in the transfected insect cells but not in the culture media. Three days after treatment with the lysate of the transfected Sf-9 cells, increase in alkaline phosphatase activity of a murine stromal cell line, ST2, was detected. Subcutaneous implantation of rhBMP-2 produced in the insect cells induced formation of cartilage, bone and bone marrow in the rats. The present data indicated that the rhBMP-2 preparation produced in the insect Sf-9 cells had a comparable activity to that produced in mammalian cells. PMID- 7549939 TI - Ultrastructural in situ hybridization and autoradiographic detection of foreign DNA in zebrafish spermatozoa. AB - Conditions have been established which permit specific hybridization of foreign DNA to complimentary DNA in ultra thin sections of zebrafish sperm prepared for electron microscopy (EM)1 after fixation in 2% paraformaldehyde, 2.5% glutaraldehyde and embedded in spurs resin. Zebrafish sperm incubated with foreign DNA were subjected to ultrastructural in situ hybridization and autoradiographic detection to determine if the sperm could retain and internalize the foreign DNA sequences. Specific and intense signals were detected from the sperm head indicating that the zebrafish sperm are capable of internalizing foreign DNA. PMID- 7549940 TI - Protein phosphatase type-1 mRNA levels in response to starvation-refeeding and streptozotocin-diabetes. AB - Levels of the mRNA encoding the catalytic subunit of protein phosphatase type-1 (PP-1cat) were reduced in skeletal muscle but not liver in response to short-term (2h) chow refeeding after prolonged (40h) starvation in the rat. This reduction did not appear to be mediated by insulin per se since streptozotocin-induced diabetes was associated with a reduction in PP-1cat levels in skeletal muscle. It is suggested that glucose levels may be one factor that modulates skeletal muscle PP-1cat mRNA levels. Despite the changes in PP-1cat mRNA levels in skeletal muscle, total protein phosphatase-1 catalytic activity was not altered by either chow refeeding or streptozotocin-diabetes. By contrast, although total hepatic PP 1cat mRNA levels were not altered in response to chow refeeding, there was a marked reduction in glycogen phosphorylase phosphatase activity in the cytosol but not in the glycogen/microsomal fraction. PMID- 7549941 TI - Scavenging effects of baicalin on free radicals and its protection on erythrocyte membrane from free radical injury. AB - Using electron spin resonance (ESR) spin trapping technique, we found that baicalin (B) could scavenge hydroxyl radicals generated from Fenton reaction. It also could scavenge superoxide radicals generated from the reaction system containing xanthine (X) and xanthine oxidase (XO), as was found by using chemiluminescence (CL) method. Kinetic studies on the competition between baicalin and a spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) in trapping OH showed that baicalin had a kinetic reactive rate constant of the order of 7.7 x 10(11) M-1 s-1 in its reaction with OH, and the studies on the competition between baicalin and ferricytochrome c (f.c.) in trapping O2- gave a kinetic reactive rate constant of 3.2 x 10(6) M-1 s-1 for baicalin in its reaction with O2-. Furthermore, we have investigated the protective effects of baicalin on erythrocyte membranes from hydroxyl free radical injuries. The results showed that baicalin could reduce hydrogen peroxide-induced hemolysis, protect the conformation of sulfhydryl groups (-SH) on membrane proteins and the membrane fluidity of erythrocytes incubated with hydrogen peroxide. The results indicated that baicalin could protect the membranes of erythrocytes from free radical injuries, and it was even more effective than alpha-tocopherol. PMID- 7549942 TI - Antioxidant effects of "beta catechin". AB - The free radical scavenging effect of "beta catechin", an antioxidant preparation containing green tea extract, ascorbic acid, sunflower seed extract, dunaliella carotene and natural vitamin E, was evaluated. Two techniques were used: electron spin resonance (ESR) spectrometry to measure radical-scavenging activity, and measurement of its effect on iron-induced lipid peroxidation in brain. A 0.05% solution of "beta catechin" completely scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals (6.1 x 10(15)spins/ml). A 10% solution of "beta catechin" completely scavenged superoxide (4.2 x 10(15) spins/ml) generated by the hypoxanthine-xanthine oxidase system. An undiluted solution of "beta catechin" scavenged about 90% of hydroxyl radicals (3.5 x 10(15) spins/ml) generated by the Fenton reaction. "beta catechin"s effect on the accumulation of thiobarbituric acid-reactive substances (TBARS) was evaluated from tissue obtained from the ipsilateral cortex of FeCl3-induced epileptic rats. Oral administration of "beta catechin" (1 or 2ml/kg body weight) both inhibited TBARS formation and increased the activity of superoxide dismutase (SOD) in the ipsilateral cortex 30 min after iron-salt injection into the left sensory motor cortex. These data suggest that "beta catechin" has an antioxidant effect and may have a prophylactic effect against aging and other neurological diseases related to free radical mechanisms. PMID- 7549943 TI - Hepatocyte growth factor-induced signal transduction in two normal mouse epithelial cell lines. AB - We have investigated HGF-induced signal transduction in two normal mouse epithelial cell lines (M23 and MM55). Both cell lines display HGF-induced mitogenesis and high level HGF-induced autophosphorylation of MET/HGFR. In both M23 and MM55 cells, HGF induces association with MET/HGFR and increased tyrosine phosphorylation of the SH2-domain containing proteins PI3K, GAP and NCK. PLC gamma exhibited neither HGF-induced increases in tyrosine phosphorylation nor an association with MET/HGFR in these cell lines. Additionally, HGF induced increased transcription of c-fos, c-jun, junB, junD, and c-myc early response genes in both cell lines. We therefore suggest that the second messenger proteins PI3K, GAP and NCK, and possibly the protein products of the c-fos, c-jun, junB, junD and c-myc genes, are important elements in the HGF-induced mitogenic pathway in the normal mouse epithelial cell lines M23 and MM55. PMID- 7549944 TI - A new point of view on the problem of G-protein aggregation. Assembly-disassembly of tubulin as an oscillation process. AB - Basic principles of nonlinear thermodynamics are used to develop a theory of tubulin assembly. The presence of GTP/GDP switch provides different ways for tubulin aggregation. The GTP-tubulin assembly results in determining the regime of limit cycle favoring growth of tubulin spiral with the formation of microtubules. The GDP-tubulin assembly, on the contrary, results in forming double rings. The difference between the GTP- and GDP-tubulin assemblies is that the former is a dynamical oscillation process while the latter is no more than a transition from a weakly nonequilibrium point to the equilibrium state along a thermodynamical path. Corresponding equations for the GTP- and GDP-tubulin assemblies are proposed. PMID- 7549945 TI - Unexpected nuclear localization of a chimeric beta-galactosidase lacZ reporter gene product in mammalian cells. AB - A lacZ cassette was designed to include a synthetic amino terminus optimized for translation in eukaryotic cells, as well as multiple restriction sites for the insertion of heterologous regulatory elements both 5' and 3' of the reporter. The cassette was placed under the control of the metallothionein promoter in combination with the SV40 enhancer and this plasmid was introduced into mammalian cells. High levels of beta-galactosidase were observed in several cell types, demonstrating efficient expression of the reporter. Unexpectedly, most of the chromogenic reaction product appeared to be intra- or peri-nuclear, indicating that the enzyme is similarly localized. The synthetic amino terminus does not resemble known nuclear localization signals and thus may constitute a novel signal. PMID- 7549946 TI - Helicobacter pylori lipopolysaccharide inhibition of gastric mucosal somatostatin receptor. AB - A gastric mucosal somatostatin receptor was isolated from the solubilized epithelial cell membrane by affinity chromatography on a column consisting of covalently coupled [D-Tryp8]SRIF-14 to Affi-Gel 10. The receptor protein displayed a molecular weight of 61kDa and exhibited specific affinity towards 125I-labeled [Tyr11]SRIF-14 with the optimum range of 4-8 micrograms/ml. The binding of somatostatin to its mucosal receptor was inhibited by lipopolysaccharide from H. pylori. The inhibitory effect was proportional to the concentration of lipopolysaccharide up to 50 micrograms/ml and reached a maximum of 94.1% inhibition. The results suggest that H. pylori, through its lipopolysaccharide, is capable of interfering with somatostatin regulatory effect on gastric mucosal G-cell function. PMID- 7549947 TI - Evaluation of bilirubin displacing effect of indomethacin by determination of erythrocyte-bound bilirubin. AB - The effect of indomethacin on bilirubin binding to human erythrocytes at different bilirubin/albumin molar ratios varying from 0.5-3.0 was studied. Progressive addition of indomethacin produced a linear increase in erythrocyte bound bilirubin. This increase was more pronounced up to 280 microM drug concentration at all molar ratios being maximum at higher bilirubin/albumin molar ratio. On the other hand, percent increase in erythrocyte-bound bilirubin produced by the drug was found to be maximum at lowest bilirubin/albumin molar ratio and decreased on increasing bilirubin/albumin molar ratio. From these results we conclude that indomethacin induces displacement of bilirubin from albumin, thus increases bilirubin binding to human erythrocytes to a significant degree even at lower concentrations. PMID- 7549948 TI - Isolation of cDNA encoding the human liver phosphorylase kinase alpha subunit (PHKA2) and identification of a missense mutation of the PHKA2 gene in a family with liver phosphorylase kinase deficiency. AB - X-linked liver glycogenosis (XLG) due to liver phosphorylase kinase (PHK) deficiency is the most frequent liver glycogen storage disease. The affected patients present in early childhood with hepatomegaly and growth retardation. We isolated and determined the structure of human liver alpha subunit of PHK (PHKA2) cDNA. The 3705 base pair open reading frame encodes a polypeptide of 1235 amino acid residues, and the deduced amino acid sequence shows 93 and 68% homology to that of rabbit liver alpha subunit of PHK and human muscle alpha subunit of PHK, respectively. We identified a missense mutation, a valine substitution for glycine at amino acid 193, in the PHKA2 gene of a family with XLG. PMID- 7549949 TI - A cytosolic endothelin converting activity in guinea-pig lung: purification of a novel metalloprotease. AB - A proteinous fraction that produces endothelin-1 (ET-1) from big ET-1 in guinea pig lung cytosol is described. An active protein has been successfully purified to homogeneity by combinations of sequential column chromatographies. The purified enzyme was a metalloenzyme based upon its sensitivity to chelating agent, and a molecular mass of the enzyme was 38 kDa estimated by gel filtration and SDS-PAGE. Further investigations revealed that the enzyme activity was abolished by sulfhydryl modifier such as N-ethylmaleimide, but inhibited neither by phosphoramidon, by thiorphan nor by captopril. The enzyme actually produced ET 1 with the Km value of 14.5 microM for big ET-1. These results indicate that this enzyme seems to be a novel metalloprotease that converts big ET-1 to ET-1. PMID- 7549950 TI - Laser high performance liquid chromatography determination of prostaglandins in pleural effusions. AB - We measured prostaglandins (PGs) in human pleural effusions of 43 patients with various diseases using microcolumn high performance liquid chromatography with a He/Cd laser induced fluorescence detection system. PGD2 was not detected in the transdate effusions. In contrast, PGD2 was detected in pleural exudates (malignant effusions: 0.22 +/- 0.07 nmol/ml, tuberculous effusions: 0.28 +/- 0.08 nmol/ml). PGE2 and 6-keto-PGF1 alpha concentrations in malignant effusions (1.62 +/- 0.17 nmol/ml, 14.40 +/- 1.33 nmol/ml, respectively) were significantly increased compared with tuberculous effusions (0.98 +/- 0.13 nmol/ml, 10.36 +/- 0.92 nmol/ml) and transdates (0.60 +/- 0.06 nmol/ml, 6.91 +/- 0.61 nmol/ml). On the contrary, there was no significant difference in PGF2 alpha concentrations between malignant effusions and tuberculous effusions. From these results, not only PGE2 but also PGD2 and PGI2 might be implicated in the pleural fluid accumulation. PMID- 7549951 TI - Identification and immunolocalization of ecto-ATPDase in chicken stomach. AB - The distribution of the extracellular adenosine tri- and di-phosphatase (ecto ATPDase) in adult chicken tissues was investigated using a monoclonal antibody (MC18) generated previously from chicken oviduct. Ecto-ATPDase was determined to be most abundant in stomach by Western blot analysis of crude tissue homogenates. The ecto-ATPDase activity from solubilized stomach microsomes and a purified oviduct control was depleted 64% and 72%, respectively, by immunoprecipitation with MC18. Both oviduct and stomach ecto-ATPDases had an M(r) of approximately 80 kDa based on SDS-PAGE analysis. In addition, the enzymology of the ecto-ATPDase from both tissues was very similar. It is concluded that the same ecto-ATPDase is present in stomach and oviduct. Furthermore, immunolocalization of the stomach ecto-ATPDase with MC18 showed the enzyme to be localized in the apical membranes of the oxyntico-peptic cells, suggesting a role for the ecto-ATPDase in secretion. PMID- 7549952 TI - Vitamin D affects Krebs cycle NAD-linked oxidoreductases from chick intestinal mucosa. AB - Vitamin D3 administration affects the NAD-linked oxidoreductase activities of Krebs cycle from intestinal mucosa of vitamin D-deficient chicks. Vmax values were increased in all of them, while K0.5 for substrate remained unchanged except for 2-oxoglutarate dehydrogenase, which showed lower affinity for oxoglutarate. Addition of Ca2+ to the incubation medium increased the affinity of 2 oxoglutarate dehydrogenase and NAD-isocitrate dehydrogenase for their substrates either in the vitamin D3 treated group or in the control one. The activity of succinate dehydrogenase, a FMN-dependent oxidoreductase, was not modified by vitamin D3 administration. The oxygen consumption of the intestinal mitochondria was not altered by cholecalciferol treatment to vitamin D-deficient chicks. The reason why vitamin D3 selectively affects the NAD-linked oxidoreductase activities of the Krebs cycle remains unknown. The vitamin D hormone, 1,25(OH)2D3, appears to be the mediator of the response. PMID- 7549953 TI - Quercetin, a bioflavonoid, inhibits the DNA synthesis of human leukemia cells. AB - Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. It has been proposed that flavonoids may have potential as anticancer agents. To test an aspect of this hypothesis, we examined the effects of the flavonoid, quercetin, on the DNA synthesis of the human leukemia cell, HL-60. Quercetin induced a dose-dependent inhibition of DNA synthesis in the test range of 1 microM to 1 mM. The inhibitory effect on DNA synthesis was evident as early as 24 h after the addition of quercetin. At the concentrations of 10 microM, 100 microM and 1 mM, 50, 82 and 85% of DNA synthesis, respectively, was inhibited by quercetin as compared to the control. After 48 and 72 h incubation of the cells with 100 microM and 1 mM quercetin, DNA synthesis was almost completely abolished. These results suggest that the inhibitory effects of quercetin on HL 60 cell DNA synthesis is not due to a non-specific cytotoxic effect, since following removal of quercetin, the treated cells regrew normally. PMID- 7549954 TI - Human placental 17 beta-hydroxysteroid dehydrogenase: secondary structure and circular dichroism demonstration of conformational changes upon NADP binding. AB - The secondary structure of human placental 17 beta-hydroxysteroid dehydrogenase in the absence and presence of NADP has been studied by circular dichroism spectroscopy. The conformational analysis of the NADP-containing enzyme shows that is an alpha/beta protein with 60% of regular secondary structure (38% of alpha helix, 22% of beta-strand structures), 20% of beta-turn and 20% of non repetitive structure. These results were in good agreement with the information obtained using statistical and homology methods based on amino-acid sequence. On the other hand, 25% alpha-helix, 55% beta-strand, and 20% non-repetitive structure were estimated by circular dichroism for the cofactor-free enzyme. Addition of varying concentrations of NADP to the cofactor free enzyme is accompanied by circular dichroism spectral changes. From the variation in the magnitude of the positive band at 193 nm with increasing NADP concentration, a dissociation constant of 34 nM was obtained. PMID- 7549956 TI - Rabbit brain endoplasmic reticulum membranes as target for free radicals. Changes in Ca(2+)-transport and protection by stobadine. AB - Incubation of rabbit brain endoplasmic reticulum membranes with either ferrous sulfate/EDTA or ferrous sulfate/EDTA and hydrogen peroxide led to the loss of efficiency of membranes to sequester Ca2+, which did not correlate with changes in conjugated diene formation. The production of practically non-detectable amount of conjugated dienes that occurs during the period of incubation of microsomes with lipid peroxidation initiators represents lipid peroxidation that is enough to produce changes in membrane permeability towards Ca2+. Addition of stobadine was able to prevent Ca2+ transport damage in a dose-dependent manner and drug concentrations higher than 200 microM were able in our model system to confer the defense against free radical and heavy metal initiated lipid peroxidation. The EC50 values for microsomes treated with Fe2+ and Fe2+/H2O2 were 12 microM and 25 microM, respectively. In our model system stobadine seems to be at least as effective as butylated hydroxytoluene, which is considered to be a good chain-breaking antioxidant. In contrast to stobadine alpha-tocopherole acetate was less potent; the effect of 1 mM alpha-tocopherole acetate being comparable to the effect of 20 microM stobadine. PMID- 7549955 TI - Changes in beta 1- and beta 2-adrenoceptor mRNA levels in alveolar type II cells during cultivation. AB - Alveolar type II cells, the progenitor of type I epithelial cells, synthesize and secrete pulmonary surfactant. In this study, we determined the levels of mRNA for beta 1- and beta 2-adrenoceptor in cultured type II cells using a quantitative RNase protection assay. The mRNA levels for beta 1- and beta 2-adrenoceptors in the freshly isolated cells were 0.51 +/- 0.07 and 1.92 +/- 0.19 amol/microgram total cellular RNA, respectively. In the cells cultured for over 2 days, only beta 1-adrenoceptor mRNA level was remarkably reduced. These results suggest that each mRNA expression may be separately regulated in cultured alveolar type II cells. PMID- 7549957 TI - Involvement of a lysine residue in the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde. AB - Leuconostoc mesenteroides NRRL B-512F dextransucrase was rapidly and irreversibly inactivated by o-phthalaldehyde. The dextransucrase-o-phthalaldehyde adduct showed a characteristic fluorescence maxima at 417 nm when excited at 337 nm. These results were consistent with the isoindole derivative formation in which the sulfhydryl group of cysteine and epsilon-amino group of lysine participate in the reaction. The stoichiometric determinations gave one isoindole derivative per enzyme molecule upon complete inactivation by o-phthalaldehyde. The enzyme showed no inhibition on treatment with thiol specific reagents. This indicated that cysteine is present in close proximity of the lysine and is involved in the isoindole derivative formation but is not participating in the catalysis. These results established for the first time that one lysine residue present at the active site is required for the activity of dextransucrase. PMID- 7549958 TI - Relationship between protein damage and ferrylmyoglobin. AB - The effect of ferrylmyoglobin (ferrylMb) on proteins and its inducement of protein damage is shown, using bovine serum albumin (BSA) as the protein. Oxidation of sulfhydryl (SH) groups of BSA, cross-linking of BSA with myoglobin and inactivation of SH enzymes by ferrylMb were investigated. Our results have shown that ferrylMb rapidly oxidates BSA but failed to oxidate N-ethylmaleimide treated BSA. During the reaction ferrylMb was reduced to metmyoglobin (metMb) by BSA, myoglobin-binding BSA was formed, and there was a rapid loss of SH enzyme inactivity. PMID- 7549959 TI - Chicken acidic ribosomal phosphoprotein PO: isolation and molecular characterization of cDNA clones. AB - Differential screening of a cDNA library prepared from chicken mitochondrial DNA less cells led to the isolation of the acidic phosphoprotein PO, as inferred from similar sequences from human, rat and mouse deposited in databases. The chicken PO protein is highly conserved among vertebrates. Nucleotide identities between the chicken and mammalian cDNA sequences are greater than 94%. Amino acid sequence identities greater than 92% are observed between the chicken protein and its mammalian counterparts, and when changes to conservative amino acids are considered, similarities range from 99.4 to 100%. The gene evolved mainly by silent transitions occurring at the third codon position. The highly antigenic carboxy-terminal sequences of mammalian PO proteins are identical to that of the chicken. Southern blot analysis of restricted DNA from chicken embryo fibroblasts suggests that only one gene encoding PO exists in the chicken genome. By Northern analysis, the PO probe detects a major RNA species 1.1-kilobases long, and a minor species 4.0-kilobases long which has no equivalent thus far in mammals. PMID- 7549960 TI - Use of glucose transport mutants to examine the intrinsic properties of glucose transport processes in rat myoblasts. AB - Glucose transport mutants were used to examine the intrinsic properties of glucose transport processes in rat myoblasts. Studies with mutants devoid of any functional glucose transporter revealed that substantial amount of sugar analogues was internalized via simple diffusion; however, equilibration of these analogues across the plasma membrane was not achieved after 1 min of incubation at 23 degrees C. The rates of internalization were substantially higher with sugar analogues that were phosphorylated by intracellular kinases. Mutants harbouring only one functional GLUT transporter were also used to examine the intrinsic properties of specific GLUT transporters. The preferred substrate for the GLUT 1 transporter was 2-deoxy-D-glucose (dGlc); the transport affinity for this substrate was reduced by energy uncouplers. Studies with mutants possessing only the GLUT 4 transporter revealed that this transporter existed in a high and a low affinity form. The former was responsible for dGlc uptake; whereas the latter was for the uptake of both 3-O-methyl-D-glucose (MeGlc) and dGlc; only the former was affected by energy uncouplers. These studies illustrated the usefulness of mutants in characterizing glucose transport processes. PMID- 7549961 TI - Cisplatin-modified DNA-binding proteins in a nuclear extract of rat-liver cells. AB - A mobility shift DNA-binding assay showed that cisplatin-modified DNA-binding proteins are present in the nuclear extract of rat-liver cells (Ac2F cells). The major binding proteins of 22.5, 23, 96.5 and 112 kDa were identified in vitro. The binding activity of the 23 kDa protein was markedly enhanced in the cells that were cultured in the presence of cisplatin, and was inhibited by addition of cycloheximide to the culture. Our previous work [Hibino et al. (1994) Biochem. Biophys. Res. Commun. 202, 749-756] has shown that an Mg(2+)-dependent endo exonuclease activity endogenous to Ac2F-cell nuclei is enhanced in the presence of cisplatin. Thus, the results of the present study imply that specific binding of the nuclear proteins to cisplatin-DNA lesions in rat-liver cells is involved in the mechanism of enhancement of this nuclease activity. PMID- 7549962 TI - Plasmid mediated organophosphate pesticide degradation by Flavobacterium balustinum. AB - A bacterium capable of degrading methyl parathion, an organophosphorus insecticide into paranitrophenol (as evidenced by TLC) and other metabolites, was isolated from the agricultural soils of Anantapur district, Andhra Pradesh, India. This bacterium, identified as Flavobacterium balustinum was found to harbour an indigenous plasmid of approximately 86 kb in size. The degradative enzyme, parathion hydrolase, was found to be encoded by this plasmid. No enzyme activity was observed in plasmid cured strain. PMID- 7549963 TI - Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose-glucose oxidase. AB - A new spectrophotometric assay of superoxide dismutase (SOD) is described. The assay is based on the SOD-mediated inhibition in the rate of nitroblue tetrazolium reduction to the blue formazan at alkaline pH. The optimized assay of SOD is performed in 50 mM glycine-NaOH buffer, pH 9.5, at 25 degrees C. The SOD concentration is determined from the V/v ratio of rates measured in the absence (V) or the presence (v) of SOD. One unit of SOD has been defined as the concentration that decrease the rate to 50% (V/v = 2). The assay is simple, sensitive, uses commercially available reagents, rapid and easy to perform and could be used routinely for monitoring superoxide dismutase levels in purified protein fractions. PMID- 7549964 TI - 2,4,6-Trinitrobenzenesulphonic acid as a probe for lysine at the active site of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. AB - Modification of Leuconostoc mesenteroides NRRL B-512F dextransucrase with 2,4,6 trinitrobenzenesulphonic acid (TNBS) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity. The kinetic profiles of inactivation showed that the reaction followed pseudo-first order reaction. Absorption spectra of TNBS modified enzyme gave characteristic maxima at 367 nm. The inactivation could not be reversed by dilution or dialysis. The substrate sucrose, provided protection to the enzyme against inactivation by TNBS, indicating that the essential residues are present at or near the active site. The stoichiometric results indicated that four mol of lysine are modified per mol of dextransucrase upon complete inactivation. However, more than 50% of the activity loss was accompanied by modification of 1 lysine residue. All these approaches suggested that one lysine residue present near or at the active site is essential for the enzymatic activity of dextransucrase. PMID- 7549965 TI - Purification and characterization of homoserine dehydrogenase from spinach leaves. AB - Homoserine dehydrogenase (HSDH) has been purified to homogeneity from spinach leaves using ammonium sulphate fractionation followed by ion exchange chromatography, gel filtration and FPLC techniques. The purified enzyme has a relative molecular mass of 220,000 and subunit molecular mass of 55,000 and probably occurs as a tetramer. The enzyme was found to be sensitive to threonine and also exhibited aspartate kinase (AK) activity, which was also sensitive to threonine suggesting that it is a bifunctional protein. The enzyme protein also gave a positive cross reaction with antibodies raised against purified AK isoenzymes. Both HSDH and AK activities were stimulated by calcium and calmodulin. PMID- 7549966 TI - Phosphatidylserine synthesis in phorbol ester treated glioma C6 cells. AB - Phosphatidylserine (PS) synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of the phorbol ester, 12-O-tetradecanoylphorbol 13 acetate (TPA). It was found that incubation of the cells with 10 nM or 100 nM TPA for 1 h inhibited PS formation by 30% and 60%, respectively. Long-term (18 h) treatment of the cells with 100 nM TPA diminished PS formation and further addition of TPA to down-regulated cells did not affect PS synthesis. The data show that the changes in PS synthesis can be associated with alterations in morphology of cell and the actin cytoskeleton organization. The role of protein kinase C in this process is discussed. PMID- 7549967 TI - Covalent immobilization of invertase and horseradish peroxidase on concanavalin A Seralose via carbohydrate moieties. AB - Invertase from Baker's yeast (Saccharomyces cerevisiae) and Horseradish peroxidase (HRP) were covalently immobilized on Concanavalin A precoupled to Seralose via carbohydrate moieties. Covalent coupling of glycoenzymes was achieved by periodate induced aldehydic groups of glycosyls with amino groups of Concanavalin A, at different pH values. A bifunctional reagent such as glutaraldehyde crosslinks the glycoenzymes with lectin both intra and intermolecularly. Therefore, attempts were made to introduce covalent linkages between glycoenzymes and Concanavalin A-Seralose without intramolecular crosslinking in either. The immobilized preparations of glycoenzymes exhibited high yield of immobilization and eta value. About 90 and 85% covalent coupling could be observed in invertase and HRP at pH 7.0 respectively, as determined by treatment with 0.5 M methyl alpha-D-mannopyranoside. All immobilized glycoenzyme preparations exhibited marked stabilization towards thermal inactivation. PMID- 7549969 TI - Differential responses of hormone-sensitive lipase gene to nutritional transition in adipose tissue, liver, and skeletal muscle of pigs. AB - To study the regulation of hormone-sensitive lipase gene in pigs, we amplified and sequenced partial porcine hormone-sensitive lipase cDNA. Nucleotide analysis indicated that porcine hormone-sensitive lipase cDNA was 86% homologous with the rat. In agreement with the rat, a 3.3 kb mRNA transcript was detected in adipose tissue but not in skeletal muscle of pigs by Northern hybridization. With more sensitive PCR method, hormone-sensitive lipase mRNA was found in adipose tissue, liver, heart, skeletal muscle, testis, and spleen. The gene expression in adipose tissue and liver was elevated after 2 days of fasting, and refeeding for another 2 days decreased the mRNA abundance. In contrast, the levels in skeletal muscle were not altered during identical nutritional transition. Combined evidences suggest that differential control may occur in porcine hormone-sensitive lipase gene in a tissue-specific fashion. PMID- 7549968 TI - Plasma antioxidant enzymes and oxidized lipoproteins in hypercholesterolemic rabbits. AB - The activities of superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPx, EC 1.11.1.9) and the levels of alpha-tocopherol and oxidized lipoproteins were investigated in the plasma of New Zealand rabbits either before or after cholesterol-diet induced hypercholesterolemia. Plasma SOD activity increased while GPx activity decreased after 60 days of cholesterol feeding. However, in the cholesterol-fed rabbits the release of superoxide dismutase fraction C from vasculature by heparin was lower than that in control rabbits. The levels of triglyceride hydroperoxides increased in low density and high density lipoproteins after feeding rabbits with the cholesterol-rich diet during 60 days. Also, a trend for increasing cholesteryl ester hydroperoxides was observed in beta-very low density and high density lipoproteins. An increase in alpha tocopherol concentration (microM) was observed in very low density and low density lipoprotein fractions, but after normalization of these results to the cholesterol content of lipoprotein particles only the alpha-tocopherol content of low density lipoprotein remained higher after 60 days of cholesterol feeding. The data suggest that low glutathione peroxidase and superoxide dismutase fraction C activities may facilitate intravascular lipoprotein oxidation by oxidant species generated by the endothelium or blood cells. PMID- 7549970 TI - Carbohydrate-lipid interactions during gestation and their control by insulin. AB - 1. During the first two thirds of gestation, coinciding with a minimal accretion by the conceptus, the mother is in an anabolic state which is supported by her hyperphagia and the more efficient conservation of exogenous nutrients when she eats. During this phase maternal fat deposits are accumulated thanks to the enhancement in adipose tissue lipogenic and glycerologenic activity. In contrast, in the latter part of gestation, the rapid fetal growth is sustained by the intense transfer of nutrients from maternal circulation. 2. Glucose is quantitatively the most abundant of the several substrates that cross the placenta and despite increased maternal gluconeogenesis this transfer is responsible for the maternal tendency to hypoglycemia. This causes a switch to a net catabolic state which is especially evident in the net breakdown of fat depots. 3. Enhanced release of adipose tissue lipolytic products, free fatty acids (FFA) and glycerol, facilitates the liver synthesis of triglycerides and their later release into circulation associated to very low-density lipoprotein (VLDL). Glycerol is also used as an important gluconeogenic substrate and FFAs are broken down through beta-oxidation for ketone body synthesis. Flow through these pathways becomes increased when food is withheld and this actively contributes to the availability of fuels to the fetus which becomes partially preserved from maternal metabolic insult. Increased liver production of VLDL triglycerides and decreased extrahepatic lipoprotein lipase contribute to exaggerated maternal hypertriglyceridemia which, besides being a floating metabolic reserve for emergency conditions such as starvation, constitutes an essential substrate for milk synthesis around parturition in preparation for lactation. 4. While the maternal anabolic tendencies found during the first two thirds of gestation seem to be facilitated by hyperinsulinemia in the presence of a normal responsiveness to the hormone, it is proposed that most of the metabolic changes taking place during the last third of gestation seem to be caused by the insulin-resistant state which is consistently present at this stage, since its reversion caused by sustained exaggerated hyperinsulinemia also reverts several of these metabolic adaptations. PMID- 7549971 TI - Early events in virus-plant interactions. AB - 1. Plant viruses can only enter their host through a wounded plant cell. Once in the cytoplasm, the virion must be disassembled, and for certain viruses with a "+" RNA genome, cotranslational disassembly of virus particles has been described. 2. Subsequent to viral protein synthesis which requires the host translational machinery, the "+" RNA genome is replicated in the cytoplasm. Viral genome amplification requires at least one viral-coded non-structural protein in conjunction with one or more host factors. 3. Early events in virus infection can be studied in systems that hinder these events. This is the case of natural hosts that are resistant to viruses: mutant viruses which overcome such resistance have been described. It is also the case of genetically engineered plants that are protected from virus infection. Both types of systems should help in determining the mode of interaction involved, and possibly also the host factor(s) involved in the various steps of virus infection. PMID- 7549972 TI - Molecular signals for the production of lymphocytes in bone marrow. AB - Bone marrow culture and molecular cloning techniques have permitted rapid progress to be made in identifying molecules that stimulate or inhibit particular steps in blood cell formation. Two types of factors that influence B lymphocyte precursors are considered in this brief review. Cell adhesion molecules are probably needed to correctly position immature precursors within the marrow and some of the most important of these have been identified. Recent studies suggest that local or systemically derived hormones must also be added to the list of agents which can markedly influence the production of lymphocytes. PMID- 7549974 TI - Partial purification of an endoglucanase from Biomphalaria glabrata. AB - Previous studies have shown that Biomphalaria glabrata contains a complete cellulolytic system which includes an endoglucanase, an exoglucanase and a beta glucosidase. In the present report, a scheme for the purification of the endoglucanase from this invertebrate is proposed. Two major problems were encountered during the study: 1) the presence of a green-brownish pigment, which could not be eliminated by thermal shock or ammonium sulfate precipitation and 2) relative instability of enzymatic activity. Various alternatives were tested and the best sequence of steps was: 1) a sample of the crude extract, obtained by homogenization of the digestive glands in 50 mM Tris-HCl buffer, pH 8.4, and ultracentrifugation, was applied to a Q-Sepharose FPLC column (50 mM Tris-HCl buffer, pH 8.4; 10 mm x 22.2 cm column; flow rate 1.5 ml/min; 0.1 to 0.5 M NaCl gradient); 2) the eluate peak containing activity was dialyzed, lyophilized and eluted from a Superdex-75 gel filtration FPLC column (50 mM ammonium acetate buffer, pH 4.8; 16 mm x 60 cm column; flow rate 1.0 ml/min). A low degree of purification (about 36-fold) and recovery (about 12%) were observed, probably due to enzyme instability. SDS-electrophoresis of the active fraction showed a major peak of 30 kDa. In order to improve the purification scheme, further studies are required to stabilize this enzyme during purification and storage. PMID- 7549973 TI - Metabolism of glucose, glutamine and pyruvate in lymphocytes from Walker 256 tumor-bearing rats. AB - This study examined the effect of Walker 256 tumor growth in vivo on the metabolism of glucose, glutamine and pyruvate in lymphocytes. A comparison between the metabolism of Walker 256 tumor cells obtained in vivo with that of lymphocytes was also carried out. Lymphocytes and tumor cells were isolated and incubated for 1 h for the following measurements: lactate production from glucose (5.6 mM) and pyruvate (3 mM), glutamate and aspartate formation from glutamine (3 mM) and decarboxylation of [U-14C]-glucose, [U-14C]-glutamine, [1-14C]-pyruvate and [3-14C]-pyruvate. The presence of the tumor increased lactate production (2.7 fold from glucose and 2-fold from pyruvate), decarboxylation of [U-14C]-glucose (3.7-fold) and [1-14C]-pyruvate (4.4-fold) and the formation of aspartate (6.3 fold) and glutamate (4.6-fold) from glutamine. The conversion of glucose to lactate and CO2 was higher in tumor cells as compared to lymphocytes. Tumor cells also showed a higher production of glutamate and an 8-fold increased decarboxylation rate of [U-14C]-glutamine in tumor cells, which was more active than that of lymphocytes even from tumor-bearing rats. Tumor growth stimulated glucose and glutamine metabolism in lymphocytes; however, the importance of this fact for the function of these cells remains to be elucidated. PMID- 7549975 TI - Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli. AB - The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture medium. If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times. It is known that the Fe(2+)-o-phenanthroline(I) and Fe(2+)-o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe(2+)-o-phenanthroline(III) complex is formed slowly. Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2. Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+. PMID- 7549976 TI - Assessment of glomerular filtration rate utilizing subcutaneously injected 51Cr EDTA. AB - 1. 51Cr-EDTA injected with lidocaine and epinephrine, as a subcutaneous button, is slowly absorbed, and a plasma level that is relatively stable can be maintained for a time sufficient to permit measurement of the renal clearance of EDTA, which is a measure of glomerular filtration rate (GFR). We studied this procedure in 32 normal volunteers and 24 patients with different glomerulopathies, comparing EDTA and creatinine clearances. In 20 patients these measurements were also compared with inulin clearance. 2. Creatinine clearance overestimates GFR due to tubular secretion of creatinine. This secretion is present even in patients with significantly reduced glomerular filtration rates. As a consequence, the lower the GFR the higher the overestimation will be. 3. A good correlation was obtained between the 51Cr-EDTA and inulin clearance: y(EDTA) = 4.21 + 0.88 x (inulin), r = 0.98. The procedure is simple to perform, and the radiotracer utilized is significantly less expensive than iothalamate. PMID- 7549977 TI - Blood compatibility of tubular polymeric materials studied by biological surface interactions. AB - Tubular polymeric materials modified by radiation techniques can be used as vascular prosthesis and components of prosthetic devices. The biological interaction between these materials and blood was studied by in vitro and ex vivo methods. Silicone rubber tubes were copolymerized with acrylamide and N vinylpyrrolidone by radiation-grafting techniques. The irradiation was performed with gamma-rays from a 60Co source at a constant dose rate (0.2 kGy/h) for various time intervals (4-15 h). To evaluate the antithrombogenicity of the grafted tubes, the surface adsorption of 125I-albumin and 125I-fibrinogen was studied. All graft copolymers show a preference for albumin, and the degree of preference appears to correlate with antithrombogenic tendency. In the ex vivo experiment with animals, tubes were implanted in the carotid artery of dogs and the blood flow in the graft copolymers was detected with an ultrasonic flow meter. The blood flow rate in the ungrafted implants decreased more rapidly (stopped completely after 15 to 210 min) compared to the flow rate in the grafted ones (decreased slowly from 38 to 35 ml/min and 70 to 60 ml/min). There was a direct relationship between both methods in the study of blood compatibility of the materials. The results suggest that the graft copolymers can be used as biomaterials for long-term use in cardiovascular systems. PMID- 7549978 TI - The interaction of blood proteins with alpha-alumina. AB - The use of alumina (alpha-Al2O3) as a material for cardiovascular applications was investigated on the basis of protein adsorption and thrombus formation on the material. The adsorption of 125I-labelled albumin and fibrinogen from phosphate buffered saline (pH 7.35, 0.100 M NaCl, 8.66 mM KH2PO4 and 41 mM Na2HPO4) solution on ceramic discs of alumina was studied. Both albumin and fibrinogen presented affinity for ceramic surfaces, with adsorptions of 1.47 +/- 0.06 ng/cm2 and 0.198 +/- 0.01 ng/cm2, respectively. Scanning electron micrographs of the alpha-Al2O3 surfaces after contact of the discs with whole human blood showed a thrombogenic behavior of alumina alpha. These results indicate a hemoincompatible property. Although critical surface tension (gamma C: 21.8 dynes/cm) of the disc surfaces determined by contact angle technique of sessile drops indicates that alumina alpha is a biocompatible material, by this criterion, the data reported here indicate that alpha-Al2O3 cannot be used for cardiovascular applications. PMID- 7549979 TI - Donated organs as a source of cytomegalovirus (CMV) in renal transplant patients. AB - Two patients receiving the same cadaver kidney graft were investigated prospectively for cytomegalovirus (CMV) infection using the polymerase chain reaction (PCR) and serologic tests (ELISA and IFI). The data indicate that a strain of CMV was probably transmitted from the same donor to both kidney recipients including one who was seropositive for CMV. PMID- 7549980 TI - Cells involved in the regulation of the autoimmune response to rat male accessory glands. AB - 1. The immunization of Wistar rats with 5 mg of chemically modified rat male accessory glands saline extract (MRAG) incorporated into complete Freund's adjuvant (CFA) induced a delayed type hypersensitivity (DTH) response to rat male accessory glands (RAG). Pretreatment with peritoneal cells (PC) obtained from rats 2 h after an intraperitoneal (ip) injection of a partially purified fraction (FI) of RAG (FI-PC2h) suppressed the DTH response to MRAG after immunization with MRAG-CFA, while pretreatment with PC obtained 24 h after an ip injection of FI RAG (FI-PC24h) induced potentiation of the DTH response to MRAG. 2. The injection of spleen mononuclear cells (SpM), obtained from rats rendered unresponsive to MRAG by pretreatment with FI-PC2h, into normal syngeneic recipients markedly prevented the DTH reaction to MRAG. The transfer of SpM cells from animals injected with FI-PC24h (potentiated animals) to suppressed recipients (recipients of FI-PC2h on days -10 and -3, prior to immunization with MRAG-CFA) showed that SpM cells did not eliminate the suppression state in these recipients, but when they were transferred to normal recipients, they were able to induce a positive response to RAG (P < 0.005). 3. The study of the phenotypic characteristics of the SpM cells prior to transfer showed similar numbers of CD4 and IL-2R SpM cells in both potentiated and normal animals. However, the number of CD8 cells was significantly reduced in SpM cells from potentiated animals compared to that observed in SpM cells from normal animals (P < 0.05). PMID- 7549981 TI - Isolation of Babesia bigemina and babesia bovis merozoites by ammonium chloride lysis of infected erythrocytes. AB - 1. We describe the isolation of viable merozoites from erythrocytes infected with Babesia bovis or Babesia bigemina organisms by ammonium chloride lysis. 2. Parasite morphology was examined by both light and transmission electron microscopy. Erythrocyte-free parasites maintain their viability and infectivity, retain their antigenicity and are suitable for use in the indirect fluorescent antibody assay. PMID- 7549983 TI - A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction. AB - A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69 kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM3 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HCl, pH 7.4; 50 mM Tris-HCl, pH 7.4/0.75 M MgCl2; 50 mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 micrograms PT, 8.14 micrograms FHA, 6.3 micrograms AC, 3.37 micrograms 69-kDa, 9.54 micrograms FIM2 and 2.23 micrograms FIM3) and from P21 (with 0.175 micrograms PT, 0.28 micrograms FHA, 0.002 micrograms 69-kDa, 0.005 micrograms FIM2 and 0.122 micrograms FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA. PMID- 7549982 TI - A rapid and efficient purification method for horse IgG(T) using a rat monoclonal antibody. AB - 1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened by ELISA using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti horse IgG(T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 10(10) M-1. 3. Ascites was induced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T). PMID- 7549984 TI - Detection and neutralization of B. jararaca venom in mice. AB - 1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom i.m. or i.d. 2. The results showed that even 10 min after i.m. injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected i.d., the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the i.d. or i.v. routes only if the antivenom was given a short time after venom injection, even when antivenom is administered in high concentration. 4. In contrast, experiments performed in mice receiving venom i.d. and treated by i.d. or i.v. routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection. 5. We suggest that a feasible amount of antivenom administered i.d. could be given as a first aid measure after a snake bite accident. However, further experimental studies using the i.d. route for antivenom administration are essential to confirm this possibility. PMID- 7549985 TI - Acute effect of intracerebroventricular administration of zinc on the drinking behavior of rats induced by dehydration or central cholinergic and angiotensinergic stimulation. AB - 1. Zinc is a metal important for several biological functions including neuromodulation and neurotransmission in the central nervous system. 2. In the present paper we studied the acute effects of third ventricle injections (2 microliters) of minute amounts of zinc acetate on the water intake of male, adult, Wistar rats (N = 7-14) under three conditions: water deprivation (14 h, overnight) and after third ventricle injections of carbachol (11 nmoles/rat in 2 microliters) or angiotensin II (AII, 9.6 pmoles/rat in 2 microliters). 3. Central injections of zinc acetate in different doses (0.3 and 3.0 nmoles/rat) induced a partial blockade of water intake of rats under all three conditions studied. Water intake after 120 min in control dehydrated rats (those receiving NaAc instead of Zn(Ac)2) was 7.89 +/- 0.47 ml while dehydrated animals receiving Zn(Ac)2 in the highest dose employed (3.0 nmoles/rat) was 2.90 +/- 0.75 ml. Carbachol-induced water intake after 45 min was 5.49 +/- 0.83 ml. This value was significantly reduced in zinc-treated (3.0 nmoles/rat) animals receiving carbachol (2.41 +/- 0.84 ml). Angiotensin-treated animals exhibit a water intake of 3.85 +/- 0.48 ml after 45 min, a value reduced to 1.13 +/- 0.6 ml in those animals receiving angiotensin II plus zinc (3.0 nmoles/rat). 4. It is suggested that zinc alters the functional integrity of cholinergic and angiotensinergic systems in the central nervous system mediating water-intake behavior in rats. PMID- 7549986 TI - Inhibition of bovine brain acetylcholinesterase by aluminum. AB - We report the in vitro inhibitory effect of very low concentrations of aluminum salts (IC50 = 4.1 x 10(-12 M) on bovine brain acetylcholinesterase (AChE). The enzymatic assays were performed using acetylcholine bromide in a buffered pH 7.4 solution at 37 degrees C. The relevant enzyme interacting species is the Al3+ ion, whose concentrations were fixed at pM levels by a citrate metal ion buffer system. The IC50 demonstrates that Al3+ is a potent inhibitor of AChE. PMID- 7549987 TI - Myosin-V is present in synaptosomes from rat cerebral cortex. AB - The subcellular localization in brain of an unconventional, calmodulin-binding myosin (myosin-V) found in neurons, astrocytes and other secretory cells of vertebrates has been investigated by probing Western blots of synaptic fractions from rat cerebral cortex with affinity-purified polyclonal antibodies against myosin-V. Myosin-V was detected in intact synaptosomes and in lysed synaptosomes associated with a particulate fraction. Our data suggest a role for brain myosin V in membrane-cytoskeleton function in the synaptic region. PMID- 7549988 TI - ONSET and OFFSET inhibitions: effect of increase and decrease of cue luminance on manual reaction time to a visual target. AB - Simple reaction time (RT) to a peripheral visual target is shortened when a non informative cue is flashed at the target location 100-150 ms before target onset (early facilitation). With longer intervals, RT to targets appearing at cue hemifield is lengthened (inhibition of return). In the present study, we investigated these effects inverting the stimulus contrast in relation to background to see how these effects are related to the onset and/or to the offset of a cue darker or brighter than background. Ten subjects were asked not to respond to a non-informative cue (S1) appearing on a computer screen 6 degrees to the right or to the left of the center of a fixation cross (FP), but to respond, by pressing a microswitch, to a target (S2) occurring at 4 degrees from the FP in the same hemifield as S1 or in the opposite hemifield. There were two different types of sessions. In one, S1 and S2 were bright against a dark background and in the other, S1 and S2 were dark against a bright background. In each session there were two types of trials. In OFF trials, each trial began with the presentation of FP. Five hundred ms later, S1 appeared and remained on for 700 ms. S2 appeared 100 or 800 ms after the offset of S1. In ON trials, S1 onset occurred 1200 ms after the beginning of the trial and remained on until the end of trial. S2 appeared 100 or 800 ms after S1 onset.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7549989 TI - Partial purification and pharmacological characterization of a neurotoxic fraction isolated from the venom of the spider Lycosa erythrognatha. AB - The effect of the venom of the spider Lycosa erythrognatha on the frog sciatic nerve was investigated with the single sucrose-gap method. Solutions containing the crude venom (40 micrograms protein/ml) markedly increased the duration of compound action potentials and caused the appearance of long-lasting depolarizing post-potentials. These effects were only partially (20%) reversed by extensive washing with control solution. The active material was sensitive to proteolytic treatments with pronase or trypsin and was separated with 20% acetonitrile and 0.1% trifluoroacetic acid by reverse phase chromatography. The effect of this fraction (LycIV) on the post-potential amplitude was concentration-dependent, and was fitted with a quadratic hyperbola having a half maximal effect of 0.9 microgram protein/ml. SDS-polyacrylamide gel electrophoresis of LycIV showed an enriched polypeptide band with apparent molecular weight of approximately 8 kDa. The observed effects were similar to those of toxins that inhibit sodium channel inactivation and different from the effects of potassium channel blockers. Pore formation or membrane disruption could be ruled out. It was concluded that the venom contains a neurotoxic polypeptide that alters the repolarization of action potentials, probably by inhibiting sodium channel inactivation. PMID- 7549990 TI - A comparison of the effects of UTP and ATP on the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum. AB - Heavy sarcoplasmic reticulum (SR) membrane fractions from rabbit and porcine skeletal muscle were incorporated into planar lipid bilayers and the activity of the Ca2+ release channels was recorded under voltage clamp, using Cs+ as the current carrier. The effects of the nucleotides adenosine triphosphate (ATP) and uridine triphosphate (UTP) on channel activity were studied at a holding potential of +30 mV. UTP (0.1-1.0 mM) had no effect per se on the conductance or the gating properties of the Ca2+ release channels. In contrast, ATP (> 0.1 mM) increased P(o), the open channel probability, and both tau o1 and tau o2, the open time constants, and decreased tau c1 and tau c2, the closed time constants. The Ca2+ channel conductance, however, was not affected by ATP. Ruthenium red (1 2 microM), a well-known inhibitor of the SR Ca2+ release channel, abolished the ATP-induced channel activation. These electrophysiological data provide support for our contention (G. Suarez-Kurtz et al. (1993). Anais da Academia Brasileira de Ciencias, 65:330) that the UTP-induced tension in mammalian "skinned" muscle fibers is not due to stimulated release of SR-stored Ca2+ via the release channel. PMID- 7549991 TI - Analysis of right and left ventricular performance of the rat heart with chronic myocardial infarction. AB - 1. The rat heart develops a compensatory hypertrophy after infarction which is secondary to volume overload in the left ventricle (LV) and to pressure overload in the right ventricle (RV). This study was undertaken to determine whether the reduced inotropic response to Ca2+ presented by the LV of infarcted rats extends to the RV and whether this hemodynamic profile in vivo affects the contractile performance of the ventricles assessed in vitro. 2. Male adult rats were submitted to left coronary artery ligation to produce infarction (Inf) or to a sham surgery (SO) and used 4 to 5 weeks later. The hemodynamic data were recorded in the anesthetized animals and the systolic performance of both ventricles was evaluated in vitro in the hearts perfused by the Langendorff technique. The isovolumic systolic pressure (ISP) developed by both ventricles was measured at various diastolic pressures (0 to 30 mmHg) and Ca2+ concentrations (0.62, 1.25, and 2.50 mM). 3. The RV systolic pressure was higher in Inf (N = 12) than in SO (N = 12) rats (42 +/- 5 vs 26 +/- 1 mmHg, P < 0.05). A positive and linear correlation (r = 0.86, P < 0.01) between RV systolic pressure and the RV weight to body weight ratio in Inf rats was observed. 4. Length-dependent activation, evaluated by using normalized ventricular function curves, was unchanged in the RV of Inf hearts. In the Inf LV, however, a slight improvement of the normalized systolic performance was observed in relation to SO hearts for diastolic pressures higher than 15 mmHg. 5. A similar depression of the inotropic response to Ca2+ was observed in both ventricles of Inf hearts. Moreover, for Inf hearts the increase of the ISP to Ca2+ flattened at lower Ca2+ concentrations in relation to the SO group. 6. The present results demonstrate that LV infarction in rats depresses the contractile performance of both ventricles. The reduced inotropic response to Ca2+ presented by both ventricles may contribute to the reduced capacity of the Inf heart to generate external work under conditions of higher metabolic demand. PMID- 7549992 TI - Different effects on rat arterial pressure and heart rate when losartan is injected into the third or fourth ventricle. AB - Cardiovascular responses to central losartan (LOS), a non-peptide angiotensin II (ANG II) receptor antagonist, were investigated by comparing the effects of LOS injection into the 3rd and 4th cerebral ventricles (3rdV, 4thV) on mean arterial pressure (MAP) and heart rate (HR). Adult male Holtzman rats were used (N = 6 animals per group). Average basal MAP and HR were 114 +/- 3 mmHg and 343 +/- 9 bpm (N = 23), respectively. LOS (50, 100 or 200 nmol/2 microliters) injected into the 3rdV induced pressor (peak of 25 +/- 3 mmHg) and tachycardic (peak of 60 +/- 25 bpm) responses. LOS injected into the 4thV had no effect on MAP, but it induced bradycardia (peak of -35 +/- 15 bpm). KCl (200 nmol/2 microliters) injected into the 3rdV or into the 4thV had no effect on either MAP or HR compared to 0.9% saline injection. The results indicate that LOS injected into the third ventricle acts on forebrain structures to induce its pressor and tachycardic effects and that bradycardia, likely dependent on hindbrain structures, is obtained when LOS is injected into the fourth ventricle. PMID- 7549994 TI - Some questions concerning science funding in Brazil: immunology as an example. PMID- 7549993 TI - Ethical guidelines for FAPESP-sponsored research on human populations. Group on Bioethics and Population Research. Fundacao de Amparoa Pesquisa do Estado de Sao Paulo. PMID- 7549995 TI - Productivity versus promised results: one of the dilemmas of biotechnology in Brazil. AB - In 1983 a new funding program--PADCT ("Programa de Apoio ao Desenvolvimento Cientifico e Tecnologico")--was created in Brazil to stimulate the development of research projects related to what was established as technological priorities. In this paper 3 features of the Biotechnology/Health subprogram of PADCT were studied: a) to what extent productivity of the leaders of the research projects affected the way they were evaluated by the award panel, b) the conflict of interests that might result from the composition of the award panel, and c) the final impact of the program on science and technology. In our sample of 210 submitted projects, 62 were funded. The data suggest that the selection of projects did not ensure a better funding for the more productive research leaders. The presence on the award panel of at least one member from the same institution of a given project increased the chance of its approval but, after approval, it had no influence on the amount of funds granted. In a subsample of 21 scientists, support to 24 projects did not increase the productivity level and 4 products in a preliminary phase of development were reported. The issue of trying to solve problems in areas where there is no established scientific competence is discussed. PMID- 7549996 TI - Pathogenicity of Candida albicans: quest for a molecular switch. AB - Candida albicans is an opportunistic pathogen of human beings and other mammals. Two other features, besides its pathogenicity, have made it a popular organism for study. It exists in different cellular forms and can change from one form to another, depending on growth conditions. Thus, it is being used as a model system to study cellular differentiation. It can also heritably and reversibly switch its cellular and colony morphologies. PMID- 7549997 TI - New disulfide bond-forming reactions for peptide and protein synthesis. AB - 1. In order to solve the difficult problems for the synthesis of peptides or proteins containing several disulfide bonds, three new disulfide bond-forming reactions were developed. 2. Thallium(III) trifluoroacetate oxidation. This reagent, a mild oxidant with a soft acid character, cleaved various S-protecting groups of cysteine in trifluoroacetic acid, with spontaneous formation of cystine. Thus, some model peptides containing a disulfide bond were prepared by direct oxidative conversion of the respective S-substituted cysteine peptides. 3. Sulfoxide-directed disulfide bond-forming reaction. The disulfide bond was formed at the sulfur atom of Cys(R)(O) (sulfoxide) intermolecularly as well as intramolecularly following liberation of the SH group from the coreactant, Cys(R'), by a suitable acid. Thus, formation of the disulfide bond was found to be feasible at the position of Cys(R)(O). 4. Silylchloride-sulfoxide procedure. In the presence of diphenylsulfoxide, methyltrichlorosilane in trifluoroacetic acid cleaved various S-protecting groups to form cystine within 10 to 30 min at 4 degrees C. By combination of this new disulfide bond-forming reaction and others, some peptides containing two or three disulfide bonds were synthesized successfully. PMID- 7549998 TI - Molecular genetics of hereditary thyroid diseases due to a defect in the thyroglobulin or thyroperoxidase synthesis. AB - 1. Hereditary goiter and the various degrees of thyroid hypofunction are the result of structural changes in the thyroglobulin (Tg) or thyroperoxidase (TPO) proteins, the inability to couple iodotyrosines or defective iodination, impairing or substantially altering the synthesis of T4 and T3. 2. The first mutations in the Tg and TPO genes responsible for human cases of dyshormonogenesis have been described. The mutation in two siblings with hereditary goiter and marked impairment of Tg synthesis was a cytosine to thymine transition creating a stop codon at position 1510. The point mutation is removed by the preferential accumulation of a 171-nt deleted Tg mRNA. In another subject, molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at position minus 3 in the acceptor splice site of intron 3. 3. Genomic DNA studies identified a duplication of a 4 base sequence in the eighth exon of the TPO gene. Interestingly, besides abolishing the enzymatic activity by disrupting the reading frame of the messenger RNA and introducing stop codons, the GGCC duplication also unmasks a cryptic acceptor splice site in exon 9. 4. In conclusion, the identification of different molecular defects provided evidence that hereditary goiter associated with abnormal Tg or TPO synthesis is caused by heterogeneous genetic alterations. PMID- 7549999 TI - Neuroanatomical and neurochemical mechanisms underlying amygdaloid control of defensive rage behavior in the cat. AB - 1. It is well established that the hypothalamus and midbrain periaqueductal gray (PAG) play important roles in the expression of defensive rage behavior. While defensive rage is not elicited from the amygdala, this region of the limbic system nevertheless serves an important role in the modulation of defensive rage behavior. The present paper attempts to address the question of how the amygdala modulates defensive rage behavior in the cat. The studies were conducted using brain stimulation, pharmacological, neuroanatomical and immunocytochemical methods to identify the likely neural pathways and their associated neurotransmitters by which different regions of the amygdala modulate defensive rage behavior in the cat. 2. The experimental evidence provided thus far establishes that three regions of the amygdala have been identified as powerful modulators of defensive rage behavior. These include the medial nucleus, basal complex and central nucleus of the amygdala. Experiments involving dual stimulation of an amygdaloid nucleus and sites within the medial hypothalamus or PAG from which defensive rage behavior was elicited demonstrated that two of the regions facilitated defensive rage --the medial nucleus and basal complex--and a third region--the central nucleus--suppressed defensive rage. The mechanisms and substrates underlying modulation for each of these regions are different. Medial amygdaloid facilitation of defensive rage involves a pathway (i.e., the stria terminalis) that projects directly to the medial hypothalamus and utilizes substance P as a neurotransmitter. Basal amygdaloid facilitation of defensive rage behavior makes use of a pathway to the PAG in which excitatory amino acids acting on NMDA receptors are utilized as a neurotransmitter. The central nucleus also projects to the PAG. However, it is strongly inhibitory and utilizes enkephalins that act upon mu receptors within the PAG. PMID- 7550000 TI - Molecular pore structure of voltage-gated sodium and calcium channels. AB - 1. Voltage-gated ion channels have a high homology in their primary structure but yet show quite distinct ion selection properties. Therefore, ion selection was studied in voltage-gated wild-type and mutant sodium (Na RBII) and calcium (Ca BI) channels, which were heterologously expressed in Xenopus oocytes, using two electrode voltage clamp and patch clamp methods. 2. In order to facilitate the electrophysiological experiments, fast sodium channel inactivation was abolished by introducing the point mutation F1489Q into the linker between the homologous repeats III and IV. 3. The ion pore in rat brain sodium channel II was located by testing single-point mutants for their sensitivity to externally applied tetrodotoxin (TTX), leading to a model in which part of the linker S5-S6 folds into the membrane to form at least part of the ion pore; two amino acids from each domain are thereby of major importance for TTX association and ion permeation, presumably forming two rings of predominantly charged residues in three-dimensional space (outer ring: E387, E945, M1425, D1717; inner ring: D384, E942, K1422, A1714). 4. When the residue F385 in repeat I of Na RBII channel is mutated to a cysteine, the residue being at the homologous position in cardiac sodium channels, the channel lost its high sensitivity for TTX and gained a high sensitivity for external Cd2+ and Zn2+, properties found in cardiac sodium channels. Mutation of the other cysteine in the pore region, C940, had no effect on channel block by TTX or by external divalent cations. 5. The inner ring in sodium channels constitutes a selectivity filter: after mutation of residues K1422 and A1714 into glutamates, the homologous residues in calcium channels, the sodium channels show ion selection properties similar to calcium channels: a) no selectivity among monovalent cations, b) strong block of monovalent current by divalent cations, c) permeation of divalent cations. 6. Reverse mutations in the BI calcium channel, where glutamate residues were mutated into neutral glutamines, showed that also in this channel these amino acids form a selectivity filter. The individual residues interact differently with the permeating ions suggesting an asymmetric spatial arrangement of the four pore-forming regions of the ion channel pore. PMID- 7550001 TI - Detection of infectious disease agents in tissue by immunocytochemistry. AB - 1. Immunocytochemical procedures have played an increasingly larger role in the identification of infectious disease agents in tissue sections owing to the increased availability and specificity of antibody reagents, the great sensitivity of the methods, and the relative facility with which the studies are performed. 2. Immunocytochemical methods can be applied to routine formalin-fixed tissue for the detection of infectious agents such as viruses, bacteria, fungi, and protozoa among other microorganisms for diagnostic and research purposes. PMID- 7550002 TI - Heart prolyl endopeptidase activity in one-kidney, one clip hypertensive rats. AB - 1. Heart mass, prolyl endopeptidase activity and fractionated proteins from heart tissue were studied in one-kidney, one clip hypertensive rats (N = 6) and compared to sham-operated rats (N = 6). 2. Body weight, arterial pressure and tissue mass were measured 4 weeks after artery clipping. Z-Gly-Pro-p-nitroaniline hydrolysis was used to measure tissue prolyl endopeptidase activity in the homogenate. Protein was fractionated into the soluble and myofibrillar fractions. 3. In the normotensive rats, prolyl endopeptidase activity expressed in terms of protein specific activity (microM substrate hydrolyzed h-1 mg supernatant protein 1) occurred in atria and was 2.5-fold higher than in the ventricles (3.79 +/- 0.20 vs 1.44 +/- 0.02, P < 0.05). In the one-kidney, one clip hypertensive rats, the left ventricle tissue increased 1.7-fold (2.27 +/- 0.11 vs 3.72 +/- 0.11 mg wet weight tissue/g body weight, P < 0.001), the soluble protein fraction (54.86 +/- 3.60 vs 57.38 +/- 6.64 mg/g wet weight tissue) was unchanged, while the myofibrillar fraction increased 1.9-fold (118.9 +/- 9.09 vs 229.8 +/- 8.47 mg/g wet weight tissue, P < 0.001). 4. The specific activity of the atrial and ventricular prolyl endopeptidase decreased in atria and increased in ventricles as the result of hypertension (3.79 +/- 0.2 vs 2.84 +/- 0.13 and 1.44 +/- 0.02 vs 1.87 +/- 0.13; respectively). These regional differences in prolyl endopeptidase enzyme content caused by one-kidney, one clip hypertension in neurosecretory and non-neurosecretory heart areas suggest that this enzyme plays a local role in the turnover of specific polypeptides. PMID- 7550003 TI - A biochemical comparison between latex from Carica candamarcensis and C. papaya. AB - 1. A group of plant proteinases is present mainly in the unripe fruit of the papaya tree (Carica papaya), which is a member of the genus Carica. C. candamarcensis is another species that belongs to this group. Its latex contains several proteinases displaying high proteolytic activity. 2. We used several electrophoretic techniques to compare the protein composition of the latex from the two species. Acid electrophoresis followed by staining or Western blot revealed a total of 17 proteins in C. candamarcensis and 7 proteins in C. papaya. Some of the proteins observed in C. papaya have been previously reported in the literature. 3. Electrophoresis on denaturing gels, followed by staining or Western blot revealed the presence of 14 proteins in C. candamarcensis and 6 proteins in C. papaya. Non-equilibrium isoelectrofocusing of the latex from both species showed a larger array of proteins in C. candamarcensis. The analysis of esterase and proteolytic activities on gel fractions after electrophoresis revealed the presence of distinct areas presenting enzyme activity. Some proteins detected in C. candamarcensis have different mobilities when compared with proteins from C. papaya. 4. These results support the view that latex from C. candamarcensis contains a wider diversity of proteins compared to C. papaya, and that some of the proteins not in C. papaya present esterase and proteolytic activity. PMID- 7550004 TI - Cell wall deficiency in "slime" strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and beta-D-glucan synthase activity. AB - 1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form of an fz, sg, os-1 ("slime"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (Pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkali soluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of beta-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10 fold the formation of cell wall relative to total biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose- and galactose containing polymers, and also showed progressively lower amounts of beta-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-beta-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wild type enzyme (i.e., Km app., Vmax, stability at 30 degrees C, activation by GTP gamma S, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and a GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15% by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0 M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-beta-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-beta-D-glucan synthase activity which was exaggerated in the presence of high osmolyte concentrations. PMID- 7550005 TI - Kinetic studies on the membrane form of intestinal alkaline phosphatase. AB - We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2 monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1 monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate. PMID- 7550006 TI - Isolation and properties of a multicatalytic proteinase complex from Xenopus laevis skin secretion. AB - A multicatalytic proteinase complex present in the skin secretion of Xenopus laevis was purified and its enzymatic activity towards natural and synthetic peptides was investigated. We identified three activities: i) a C-terminal deamidation enzyme activity which exhibited selectivity for the Asp-Phe-NH2 and Phe-Leu-NH2 motifs of cerulein, minigastrin Leu-enkephalinamide, (des-Tyr1)Leu enkephalinamide and diaminobenzylthiocyanate-DVDERDVRGFASFLNH2 (DABTC-DR8kermit); ii) an endopeptidase activity that cleaves peptide bonds on the carboxyl side of hydrophobic amino acid residues such as Tyr-Gly of LHRH, Ile-Ala of PGLa and Leu Ala of buccalin; iii) an enzyme activity that cleaves peptide bonds at the dibasic sites of peptides of the dynorphin family. The molecular weight determined by Sephacryl S-400 molecular sieve filtration indicated an M(r) about 600 kDa. The activities characterized here exhibit an optimal pH of about 7.4. The activities of the multicatalytic complex were differentially inhibited by the classical inhibitors of proteases. PMID- 7550007 TI - Arrhythmogenic effects of combined orally administered theophylline and albuterol in patients with chronic obstructive pulmonary disease. AB - 1. Studies in asthmatic subjects have reported conflicting results about the arrhythmogenic effects of beta agonist and theophylline. The purpose of the present study was to evaluate the effects of the combination of these drugs in patients with chronic obstructive pulmonary disease (COPD). 2. Twelve COPD patients (FEV1 = 1.2 +/- 0.3 L; PaO2 = 65.7 +/- 9.0 mmHg) were evaluated by 24-h Holter monitoring on three different days. The first evaluation was done after the patient had been without any treatment for at least 24 h, the second after sustained-release theophylline for one week and the third after oral beta agonist (albuterol) and theophylline for one week. 3. Mean serum level of theophylline was 1.9, 15.6 and 11.7 micrograms/ml, and mean heart rate was 78.3, 82.0 and 84.5 beats/min for the first, second and third period, respectively. Four patients showed more than 10 premature atrial contractions/h in the baseline Holter, and this rate did not increase after either treatment. Three patients had more than 10 premature ventricular contractions/h (PVC) at baseline, with no increase while receiving theophylline or the combination of theophylline and albuterol. However, one patient did have worsening of the arrhythmia while taking both drugs. There were 5 single PVCs/h at baseline and 150 single and 9 coupled PVCs/h plus 1 episode of non-sustained ventricular tachycardia during combined therapy. 4. We conclude that the combination of theophylline and a beta agonist (albuterol) may increase the premature ventricular contraction rate and the complexity of ectopic activity in COPD patients. PMID- 7550008 TI - Gill diffusion as a physiological mechanism for hydrogen peroxide elimination by fish. AB - Hydrogen peroxide is metabolized by the specific enzymatic action of catalase and glutathione peroxidase in animal tissues. The relatively low catalase and glutathione peroxidase activities found in the blood of fish may be related to the ability of gills to eliminate hydrogen peroxide into the aquatic environment. Poecilia vellifera releases hydrogen peroxide apparently by gill diffusion into the environment, resulting in a steady-state H2O2 concentration of about 0.6 microM in the surrounding water. This physiological mechanism resembles ammonia excretion by teleost fish. PMID- 7550010 TI - The use of filter paper monoclonal antibodies in a Dot-blot test for typing Neisseria meningitidis B. AB - A simple method for the collection, preservation, shipment, and testing of minute amounts of dried monoclonal antibodies for typing Neisseria meningitidis B is described. The monoclonal antibodies collected on filter paper were extracted in PBS and evaluated by Dot-blot employing whole cells of N. meningitidis B as antigen. The dried filter paper with monoclonal antibodies could be stored at room temperature for as long as 30 days without detectable changes in antibody response when used for typing outer membrane antigens of N. meningitidis B. PMID- 7550009 TI - Effect of combined administration of counterregulatory hormones during insulin induced hypoglycemia in rats: lipolysis mediated by a beta-adrenergic mechanism contributes to hyperglycemia. AB - The synergistic effect of combined injection of glucagon (G), cortisol (C) and phenylephrine+isoproterenol (E) during hypoglycemia in male adult Wistar rats was investigated. For this purpose we injected insulin (1 mg/kg) and 30 min later saline (controls), C (20 mg/kg), G (0.02 mg/kg), or E (1 mg/kg), individually or combined (G+C, G+E, C+E and C+G+E). All drugs were injected i.p. and all rats were killed 60 min after insulin injection. The rise in glycemia with C+G+E was greater (delta = 107 mg/dl) than the sum of the responses to injection of C, G and E individually, or in double combination plus any single hormone injection. This synergistic effect was reproduced by G + C + isoproterenol (Iso) but not by G + C + phenylephrine (delta = 0 mg/dl). The results also showed a clear relationship between hyperglycemia and lipolysis. Thus, lipolysis mediated by a beta-adrenergic mechanism played a significant role in promoting hyperglycemia when Iso was combined with G and C. PMID- 7550011 TI - Anxiety induced by simulated public speaking and stroop color word test in healthy subjects: effects of different trait-anxiety levels. AB - 1. The objective of the present study was to compare the effects of two anxiety inducing tests, simulated public speaking (SPS) and the stroop color word test (SCWT), in healthy subjects with different trait-anxiety levels. 2. The mean (+/- SD) trait-anxiety score of 524 university students, measured by Spielberger's state-trait anxiety inventory (STAI), was 40.8 +/- 8.9. Based on these scores, 26 students from our sample were divided into low (score less than 1 SD from the mean, N = 10), medium (score between -1 and +1 SD from the mean, N = 7) and high (score more than 1 SD from the mean, N = 9) trait anxiety. 3. Each subject was submitted to the SPS and SCWT tests in the same experimental session. The sequence of test presentation was randomized between subjects. No effect of test order presentation was found. 4. SPS induced significant increases in the anxiety factor of Norris' visual analogue mood scale (VAMS) in all groups, without difference between them. The SCWT, on the other hand, did not induce an increase in subjective anxiety in any group. In the high trait group, however, there was a general increase in anxiety feelings that was evident even before the test, and might have been caused by the presence of the experimenter. 5. The results suggest that SPS is a more effective anxiety-inducing test, and is not dependent on previous trait-anxiety levels. PMID- 7550012 TI - Effect of electrolytic and chemical lesion by ibotenic acid of the amygdala on salt intake. AB - Sodium chloride intake was studied in male Holtzman rats weighing 250-300 g submitted to electrolytic and chemical lesion of the cell bodies, not fibers of the amygdaloid complex. Sodium chloride (1.5%) intake increased in animals with electrolytic lesion of the corticomedial nucleus of the amygdala. Sodium chloride (1.5%) intake increased after ibotenic acid injection into the corticomedial nucleus of the amygdala to a larger extent (26.6 +/- 9.2 to 147.6 +/- 34.6 ml/5 days). The results indicate that sodium intake response can be induced by lesions, which involved only cell bodies. The fibers of passage of the corticomedial nucleus of the amygdala produce a water intake less consistent than that induced by ibotenic acid, which is more acute. The results show that cell bodies of this region of the amygdala are involved in the control of sodium chloride intake. PMID- 7550013 TI - Injection of ramipril into the lateral ventricle interferes with the drinking response induced by pharmacological and natural thirst stimuli. AB - We investigated the effects of ramipril, an angiotensin I-converting enzyme (ACE) inhibitor, on water intake by male Holtzman rats (250-300 g) with cannulae implanted into the lateral ventricle. Intracerebroventricular (i.c.v.) injection of ramipril (1 microgram/microliter) significantly reduced drinking in response to subcutaneous (s.c.) injection of isoprenaline (100 micrograms/kg) from 8.49 +/ 0.69 to 2.96 +/- 0.36 ml/2 h, polyethyleneglycol (PEG) (30% w/v, 10 ml/kg) from 9.51 +/- 2.20 to 1.6 +/- 0.34 ml/2 h or water deprivation for 24 h from 12.61 +/- 0.83 to 5.10 +/- 1.37 ml/2 h. Ramipril had no effect on water intake induced by cellular dehydration produced by sc injection of hypertonic saline (2 M NaCI). These results are consistent with the hypothesis that ramipril acts as an ACE blocking agent in the brain. The possibility that ramipril is transformed to ramiprilat, the active drug, by the brain is suggested. PMID- 7550014 TI - Embryotoxicity of methanol in well-nourished and malnourished rats. AB - 1. The objective of the present study was to investigate whether maternal protein energy malnutrition alters methanol-induced embryotoxic effects in rats. 2. On day 0 of pregnancy, dams were assigned at random to one of the following treatment groups: well-nourished methanol (WNM), well-nourished control (WNC), malnourished methanol (MNM) and malnourished control (MNC). Malnourished animals received half of the well-nourished food intake (ca 12 g/day) throughout pregnancy. Methanol was administered by gavage (2.5 g/kg body weight) from gestation day 6 to 15. 3. Rats were weighed on days 0, 6 to 15, and 21 of pregnancy. On day 21 rats were submitted to cesarean section. The number of implantations, living and dead fetuses, resorptions and corpora lutea was recorded. All fetuses were weighed, examined for externally visible malformations, fixed, and examined for skeletal anomalies after clearing and staining with Alizarin Red S. 4. An increased proportion of fetuses with skeletal malformations, particularly cervical extra ribs, was found in the methanol treated groups (fetuses with skeletal malformations: WNC = 5.6%, WNM = 45.4%, MNC = 3.8%, and MNM = 38.8%). Malnutrition produced fetal growth retardation, but did not cause any increase in the occurrence of gross structural malformations. The methanol-induced increase in the proportion of fetuses with extra ribs was not altered by malnutrition, but methanol potentiated the malnutrition-induced increase in the proportion of fetuses with signs of delayed ossification (WNC = 18.6%, WNM = 25.4%, MNC = 39.7%, and MNM = 78.4%). 5. These findings suggest that methanol-induced gross structural malformations are not affected by maternal malnutrition, but the delay in ossification caused by malnutrition is aggravated by treatment with methanol. PMID- 7550015 TI - Proceedings of the 1994 European Stem Cell Club meeting and 1st Symposium on Bone Marrow Transplantation in Stem Cell Disorders. Barcelona, Spain, October 6-8, 1994. Abstracts. PMID- 7550016 TI - Pharmacokinetics and biodistribution of radiolabelled idoxifene: prospects for the use of PET in the evaluation of a novel antioestrogen for cancer therapy. AB - With a view to evaluating the role of PET imaging in the development of new anticancer drugs, we are investigating the novel antioestrogen pyrrolidino-4 iodotamoxifen (idoxifene). [125I]idoxifene and [131I]idoxifene have been produced in no-carrier-added form using a tributyl stannylated precursor, and the bio distribution and dynamic behaviour of the compound investigated using syngeneic transplantable mammary tumours in the rat. Our findings support the use of PET imaging with 124I to study the clinical pharmacology of idoxifene. Factors other than hormone receptor levels appear to influence tumour uptake and therefore, possibly the biological effects of this compound. PMID- 7550017 TI - Biodistribution and tumor localization of 111In-labeled unmodified and modified F(ab')2 fragments of human monoclonal IgM (16.88) in a nude mouse model. AB - Unmodified F(ab')2 and modified Fab'-BMH-Fab' fragments of human monoclonal IgM (16.88) were compared for biodistribution and tumor localization in nude mice bearing LS-174T human colon carcinoma xenografts. Although both unmodified and modified fragments of IgM cleared rapidly from the blood, the radioactivity retentions for each fragment in liver and kidney were significantly different. Kidney uptake of the modified fragment was about 4-fold lower than kidney uptake of the unmodified fragment. Radioactivity uptake in liver was 2-4-fold higher for the modified fragment. Lower liver and higher kidney uptake of unmodified fragments reflected the labile disulfide linkage of F(ab')2 in their hinge region and the subsequent behavior of the Fab' fragments resulting from the reduction of the disulfide linkage. Higher liver and lower kidney retention of modified fragments, on the other hand, resulted from the different cleavage mechanism of the stable thioether linkage. Tumor targeting was similar for unmodified and modified fragments at approx. 4% of injected dose per gram. These results indicate that the changes in fragment linkage chemistry may provide different pharmacokinetic patterns in vivo and improve the therapeutic application of radiolabeled fragments in human patients. PMID- 7550018 TI - Tumor targeting and pharmacokinetics of unmodified and modified F(ab)2 fragments of an anti-CEA murine monoclonal antibody (Immu-14). AB - Pharmacokinetic studies were performed with two different 111In-labeled F(ab)2 fragments of an anti-CEA murine monoclonal immunoglobulin G (Immun-14). Unmodified F(ab)2 and modified Fab-BMH-Fab fragments were compared in nude mice bearing a LS-174T human colon carcinoma tumor xenograft. Tumor accumulation is significantly higher for modified fragments than for unmodified fragments at all time points. At 24 h post injection, tumor uptake for modified and unmodified fragments reached 40 and 25% ID g, respectively. The retention of radioactivity in the liver was approx. 2-fold higher for modified fragments. Kidney uptake of modified fragments was at least 2-fold lower than that of unmodified fragments. Although blood radioactivity decreased rapidly for both fragments, the cumulative tumor activity was 40% higher for Fab-BMH-Fab fragment. Modified F(ab)2 fragments can deliver higher radiation doses to the tumor. PMID- 7550019 TI - Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands. AB - Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4 hydroxypiperidin-3-yl]-spirol[1H-i nde ne-1,4'- piperidine] (5), 1'-[1-(3 iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4- dihydrospiro[indene-1,4'-piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4- dihydrospiro[naphthalene-1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550020 TI - [125I]iomazenil binding in the brains of spontaneously epileptic rats: an ex vivo quantitative autoradiographic study. AB - Central benzodiazepine receptor binding was studied in spontaneously epileptic rats (SER) and in their parent strain. Kyoto-Wistar (KW) rats, by ex vivo quantitative autoradiography with [125I]iomazenil. Thirteen-week-old SER developed tonic and absence-like seizures following mild stimulation by tapping at 5 min before injection of the radioligand. When compared with age-matched KW rats, a significant 76% elevation of radioactivity was observed in the SER hippocampus, while there was no difference in hippocampal blood flow obtained using [125I]N-isopropyl-p-iodoamphetamine. Since benzodiazepine receptors act to enhance inhibitory GABA transmission, this finding suggests a possible mechanism for seizure-induced enhancement of inhibition. PMID- 7550021 TI - Labelling small quantities of monoclonal antibodies and their F(ab')2 fragments with technetium-99m. AB - Addition of sulfhydryl groups with 2-iminothiolane (2-IT) is an important new method for labelling monoclonal antibodies (mAb) and fragments with 99mTc. F(ab')2 fragments were prepared by digestion of 1B7.11 and BCD-F9 with pepsin. Optimal conditions for labelling 20-100 micrograms mAb or F(ab')2 involved a 2000:1 molar ratio of 2-IT:protein in phosphate buffer pH 7.4 for 30 min followed by addition of 99mTc-pertechnetate and stannous glucoheptonate. Recovered yields were > 70% and radiochemical purities were > 90% with a total preparation time of < 90 min. PMID- 7550022 TI - Studies with [11C]alprazolam: an agonist for the benzodiazepine receptor. AB - We have built a system for the synthesis of high specific activity carbon-11 alprazolam (Xanax), a high affinity agonist for the benzodiazepine receptor. The system produces 30-40 mCi of the compound with a specific activity of > 12,000 Ci per millimole. Using this compound we have performed PET studies on 6 normal subjects and studied the cerebral influx and efflux of the compound. The uptake in the brain was low, approx. 1% of the administered dose. However, the levels of the compound in the circulation at early time points are heavily affected by the specific activity of the tracer, i.e. when pharmacologically active doses are used as blocking doses the concentration of radioactive material is higher in the circulation and more material enters the brain. We attribute this to a depot effect where the compound is trapped in saturatable sites in an organ, probably the lungs, and is slowly released over time. In the presence of blocking doses of agonist, the compound washes out of the brain more quickly suggesting that some blockade of the receptors is occurring. However, the pharmacological activity of the compound does not permit the administration of enough material to ensure complete receptor blockade. The compound shows definite signs of acting as a receptor binding ligand but the unusual pharmacokinetics complicate the interpretation of the data. PMID- 7550024 TI - Evaluation of radioselenium labeled selenomethionine, a potential tracer for brain protein synthesis by PET. AB - The blood-brain transfer, protein incorporation and metabolism of L [75Se]selenomethionine (SeMet) of relatively high specific activity (> 400 GBq mmol) were studied in male Wistar rats. The highest uptake was found in the pancreas, followed by the tumor, kidney, liver, brain and muscle. In addition, plasma and brain samples of rats were analyzed for labeled fractions of free SeMet, metabolites, and SeMet bound to t-RNA and proteins. For example, free SeMet represented more than 80% of brain radioactivity at 1.5 min while it was less than 15% at 360 min. A concomitant increase was observed for protein bound SeMet in brain. A three-compartment model was applied to calculate the blood brain transfer constant (K1 (0.15 +/- 0.070 mL g-1 min-1) and the rate constant of SeMet incorporation into proteins (k3 = 0.026 +/- 0.008 min-1). The apparent incorporation of methionine into proteins was estimated to be about 0.73 nmol g-1 min-1. From the studies it is concluded that the use of L-[75Se]selenomethionine may be appropriate to measure brain protein incorporation in humans with PET. PMID- 7550023 TI - Preparation and preliminary evaluation of 4-[211At]astato-N-piperidinoethyl benzamide. AB - The potential therapeutic agent, 4-[211At]astato-N-piperidinoethyl benzamide (4 APAB) was synthesized via a halodestannylation reaction. Radiochemical yields were 69% for a 5 min reaction and reached 74% by 25 min, whereas 82% radiochemical yields were obtained under similar reaction conditions for radioiodination. A simplified procedure was adopted for the purification of the target compound. In vitro binding of 4-APAB to SK-MEL 28 melanoma and D247 glioma cell lines was 20.7 +/- 1.3% and 12.2 +/- 1.3%, respectively. In comparison, binding of 4-[131I]iodo-N-piperidinoethyl benzamide (4-IPAB) to SK-Mel 28 cells was 13.9 +/- 1.9%. Paired label biodistribution studies were performed in normal Balb/c mice using 4-IPAB and 4-APAB. Thyroid uptake at 1, 2, and 6 h was significantly higher for 4-APAB. Differences in liver accumulation between the two compounds were small but statistically significant at most time points. A higher accumulation of 211At compared with 131I was observed in lungs and spleen at all time points studied. These results indicate that 4-APAB is not stable in vivo, suggesting the need for a better sigma receptor ligand for use in 211At. PMID- 7550025 TI - Pharmacokinetics and metabolism of 2-[18F]fluoro-2-deoxy-D-glucose (FDG) in mammary tumors of antiestrogen-treated rats. AB - Attempts are being made to evaluate 2-[18F]fluoro-2-deoxy-D-glucose (FDG) as a noninvasive marker of therapy response in malignant tumors. We studied rats with 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinomas by measuring the differential absorption ratio (DAR) and the metabolites of FDG in tumor homogenates. Half the rats were treated with the antiestrogen toremifene for 14 days and half were untreated. The histology of the tumors was studied by morphometry. The animals were killed 15, 45 or 240 min after injection. Regardless of whether the rats received toremifene or not, the fractional change in tumor volume correlated better with the DAR at 15 min [r = 0.284 (untreated) and r = 0.721 (treated)] and at 240 min [r = 0.932 (untreated)], than at 45 min [r = -0.137 (untreated) and r = 0.265 (treated)]. Inverse relations were found for the fraction of unmetabolized FDG and change in tumor volume [r = 0.070 (45 min) and r = -0.872 (240 min) for untreated tumors and r = -0.963 (15 min) and r = -0.715 (45 min) for treated tumors]. The DAR and the fraction of unmetabolized FDG correlated also [r = -0.420 (15 min), r = -0.647 (45 min) and r = -0.976 (240 min) for untreated tumors, and r = -0.963 (15 min) and r = -0.213 (45 min) for treated tumors]. No significant therapy-induced morphometrical changes were observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550026 TI - Synthesis and biological evaluation in mice of (2-[11C]methoxy)-6',7' dihydrorotenol, a second generation rotenoid for marking mitochondrial complex I activity. AB - Evidence has accumulated suggesting that impairment of the function of the complexes of the mitochondrial respiratory chain might be involved in the pathology of neurological diseases including Parkinson's and Huntington's diseases. Recently we reported the synthesis of (2-[11C]methoxy)rotenone ([11C]ROT) as a tool for in vivo studies of complex I. In an effort to develop a complex I imaging radiotracer which might be easier to synthesize and less likely to be metabolized, we prepared (2-[11C]methoxy)-6',7'-dihydrorotenol ([11C]DHROT). The radiotracer was synthesized by [11C]methylation of 2-O desmethyl-6',7'-dihydrorotenol under basic [11C]alkylation conditions. (2 [11C]Methoxy)-6',7'-dihydrorotenol was produced in 30-35% radiochemical yields (decay corrected), with synthesis times shorter than 35 min. Radiochemical purities were over 95% and specific activities averaged 1000 Ci/mmol. The brain distributions of [11C]ROT and [11C]DHROT were investigated in mice after intravenous injections. For both radiotracers, distribution of radioactivity was similar in all brain regions examined. However, significantly higher uptake was observed with [11C]DHROT than with [11C]ROT, indicating that the alterations introduced in the structure of rotenone during the design of [11C]DHROT resulted in a tracer with greater brain barrier permeability. PMID- 7550027 TI - In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with [125I]MRK-16 monoclonal antibody. AB - Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with 125I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb c mice were each xenografted with colchicine-resistant or sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [125I]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targetting of [125I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g +/- SD) (18.76 +/- 2.94 vs 10.93 +/- 0.96; p < 0.05). Quantitative autoradiography verified these findings (19.13 +/- 0.622 vs 12.08 +/- 0.38, p < 0.05). Specific binding of [125I]MRK-16 was confirmed by comparison to [131I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [125I]MRK 16.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550028 TI - Synthesis and biological evaluation of radioiodinated phospholipid ether analogs. AB - Previous work has shown that radioiodinated phospholipid ether analogs with the iodine-125 substituted on the meta position of the aromatic ring readily localized in a variety of animal tumors. In an effort to ascertain the importance of such meta substitution, three phospholipid ether analogs with the iodine-125 in the para position were synthesized for evaluation as potential tumor localizing imaging agents. 12-(p-Iodophenyl)dodecyl phosphocholine, 1-O-[12-(p iodophenyl)dodecyl]-1,3-propanediol-3-phosphocholine, and 1-O-[12-(p iodophenyl)dodecyl]-2-O-methyl-3-rac-glycerophosphocholine were synthesized and labeled with iodine-125 via an isotope exchange procedure. Similar to previous results with the meta substituted analogs, tissue distribution studies with the three para analogs demonstrated tumor localization and retention of radioactivity at 24 h after i.v. injection. In all three cases, the para isomers showed greater tumor avidity than the meta isomers and clearance of the radiotracer from the tumor was much slower than the clearance from nontarget tissue. 12-(p Iodophenyl)dodecyl phosphocholine afforded the greatest tumor-to-nontarget tissue ratio. For example, the tumor-to-blood and tumor-to-liver ratios at 24 h were 10.96 and 1.85, respectively. As a result of such selective tumor retention, it was possible to clearly delineate the tumor using gamma-camera scintigraphy. PMID- 7550029 TI - Detection of acute infection/inflammation with Tc-99m labeled intact polyvalent human IgG. AB - Intact polyvalent human immune globulin (IgG) labeled with Tc-99m by a mild chemical method was investigated with animals infected with either S. aureus or E. coli in the thigh muscle. Focal infection was clearly visualized by Tc-99m IgG scintigraphy within 1 h postinjection. Tc-99m IgG appeared to be concentrated in the liver, spleen, kidneys and urinary bladder. It cleared rapidly via the kidneys resulting minimal of tissue background activity and high infection-to normal organ ratios. At 24 h postinjection, the ratios of infectious lesion to blood, normal muscle and bone averaged 10:1, 23:1, and 24:1 for S. aureus infection vs 4:1, 9:1 and 9:1 for E. coli infection, respectively. Tc-99m labeled IgG also concentrated in terpentine-induced aseptic inflammatory lesion with a target-to-blood ratio of 4:1, bone 6:1 and normal muscle about 10:1. These findings suggest potential value of Tc-99m IgG as an imaging agent for the early detection of focal infection and inflammation. PMID- 7550030 TI - The chemical nature, purity and stability of 99mTc-N-(2,6-Diethyl-3-iodo phenylcarbamoylmethyl)-iminodiacetic acid. AB - The chemical purity and stability of a radio pharmaceutical 99mTc-N-(2,6-diethyl 3-iodo-phenylcarbamoylmethyl)-iminodiacetic acid (IODIDA) has been studied using HPLC. The HPLC results indicate that IODIDA decomposed into at least three species after 99mTc labelling, and the amount of these varied with time. Mass spectra obtained by negative ion FABMS of no-carrier-added (NCA) Tc-labelled IODIDA showed, in addition to the molecular ion and the sodium adduct of N-(2,6 diethyl-3-iodo-phenylcarbamoylmethyl)-iminodiacetic acid, peaks corresponding to sodium containing dimers, trimers and tetramers. Several peaks were also found that may be assigned to 99Tc(I) containing dimers, trimers and tetramers. PMID- 7550031 TI - Amino- and amido-tetrabenazine derivatives: synthesis and evaluation as potential ligands for the vesicular monoamine transporter. AB - Tetrabenazine (TBZ) and dihydrotetrabenazine are well known inhibitors of the CNS vesicular monoamine transporter (VMAT), which is responsible for the packaging of monoamine neurotransmitters in presynaptic vesicles. Amino and amido derivatives of tetrabenazine were prepared as potential ligands for the vesicular monoamine transporter. Ultimately, organotin derivatives of promising ligands were prepared for radiolabeling with 125I. The compounds were evaluated for their ability to inhibit the specific binding of a selective radioligand to the transporter in rat striatal homogenates. Of the compounds evaluated, three amine derivatives of TBZ (primary, secondary and tertiary) were found to have modest to high affinity for the transporter, while two amides exhibited low to undectable affinity. The secondary propargyl amine was found to possess the highest affinity (Ki = 7.6 nM) and was chosen for further evaluation. The organotin derivative of this compound was synthesized in order to prepare the corresponding radioiodinated ligand. However, our inability to synthesize and characterize the iodinated amine precluded its evaluation as a potential radioiodinated ligand for the transporter. Alternative approaches for decreasing the lipophilicity of TBZ analogs while maintaining high binding affinity are currently being explored. PMID- 7550032 TI - Pingyangmycin as a 99mTc carrier in tumor localization. AB - A simple method of preparation of 99mTc-pingyangmycin (PYM) for clinical use has been established using super-micro-amounts of SnCl2 as a reductant under dark conditions. The labeling efficiency was higher than 96%, and further purification was not necessary. The effect of ascorbic acid on the distribution of 99mTc-PYM had been investigated. Ascorbic acid increased uptake of 99mTc-PYM in the tumor. Tumor uptake increased with increasing concentration of ascorbic acid. Tumor blood, tumor-liver, tumor-lung ratios at 1.5 h after 99mTc-PYM administration were 5.19 +/- 1.64, 2.71 +/- 0.51 and 4.15 +/- 0.57, respectively. Preliminary clinical trials in nine patients showed that 99mTc-PYM is a potentially useful tracer for tumor detection with good sensitivity and specificity (true positive 7/7, true negative 1/1, and false positive 1/1). PMID- 7550033 TI - An improved method for the synthesis of radiolabeled McN5652 via thioester precursors. AB - An improved procedure that facilitates routine production and increases the radiochemical yield of [11C]McN5652 (trans-1,2,3,5,6,10b-hexahydro-6-[4 ([11C]methylthio)-phenyl]pyrrolo- [2,1-alpha]-isoquinoline) is presented. Specifically, thiol acetate, butyrate, and benzoate derivatives of McN5652 were prepared as the precursors for the [11C]McN5652 synthesis. These thioesters offer greater stability than the previously used thiol precursor (desmethyl McN5652) and enable a single batch of material to be used for multiple radiolabelings. Hydrolysis of the thioester functionality (tetrabutylammonium hydroxide, 10 min) unmasked the free thiol which, without purification, was reacted with [11C]iodomethane in DMF at 40-45 degrees C for 1 min. The average decay-corrected radiochemical yield for [11C]McN5652 was 26% with an average specific activity of 2290 mCi/mumol (end of synthesis). This facile radiolabeling method, utilizing the butyrate thioester of McN5652, was also employed in the preparation of [3H](+)- and (-)McN5652 [trans-1,2,3,5,6S (or 6R),10bR, (or 10bS)-hexahydro-6-[4 ([3H]methylthio)phenyl]pyrrolo-[2,1,alpha]- isoquinoline] from [3H]iodomethane. PMID- 7550034 TI - [Aspects of pathological physiology of the dog in extracorporeal circulation]. AB - During ECC, dogs ventilate spontaneously according to classical physiology. Further, given the CO2 clearance insured by the ECC oxygenator, many hours of reversible apnea are tolerated without sequellae. Surgical removal of the lungs shows that an emptied chest still responds to chemical drives, but the absent lung-recoil causes hyperinflation with lethal failure. Finally, liquid ventilation induces metabolic acidemia through phasic gut ischemia and respiratory muscle strain that destroys the delicate lung structure. PMID- 7550035 TI - [Absorbable osteosynthesis: experimental and clinical approach in surgery of the hand]. AB - Given the advances in the field of high molecular-weight polylactides, it is now possible to obtain biomaterials of great strength with a resorption rate that ranges from a few months to several years. Experimental evaluation of the biomaterials was done in rabbits with implantation of intramedullary rods after fracture of the knee. The results of histological studies and assessment, using MR imaging, indicated good compatibility of the intramedullary rods. Applications for human clinical practice centered on metacarpophalangeal arthrodesis of the thumb performed for rheumatoid polyarthritis and intractable sprains of the metacarpophalangeal. Operations were performed on 52 patients with a rate of consolidation of 96%. No synovitis-like reactions were observed concurrent with massive release of the biomaterial. As a first step, this type of osteosynthesis could be applied to the upper extremity where stresses are relatively minimal. Eventually, screws of reduced diameter could be developed for the treatment of articular and unstable fractures of the metacarpophalangeals. There are already 3.5 mm-screws that can be used for malleolar fixation at the level of the lower extremity. Because of the wide variety of biomaterials that are currently available, it is important to be familiar with their composition, so that patients will be spared certain complications linked to resorption, especially inflammatory reactions. The biomaterials presented here belong to a class of biomaterials that have demonstrated good compatibility. At the present time, the cost of biodegradable materials remains high but it is important to emphasize the social and economic consequences of their use, since there is no need for a secondary procedure to remove them. PMID- 7550036 TI - [Current therapeutic approach to diabetes using esters of dicarboxylic mitochondrial metabolites]. AB - In non-insulin-dependent diabetes, site-specific defects of glucose metabolism in the pancreatic B-cell may account for a preferential alteration of its secretory response to the hexose. A novel therapeutic approach could thus be based on the use of non-glucidic nutrients that would bypass the metabolic defect in the islets of diabetic subjects. In this respect, the esters of mitochondrial dicarboxylic acids, such as succinic or glutamic acid, offer the advantage of being well transported into the islet cells and, hence, of stimulating efficiently both proinsulin biosynthesis and insulin release. PMID- 7550037 TI - [Molecules of the family of aspartic proteinases in the placenta of ruminants: hormones or proteins?]. AB - The ruminant placenta contains binucleate trophoblastic cells synthesizing proteins, migrating cross the barrier and fusing with endothelial cells of the endometrium. Recently described were two glycoproteins from the family of aspartic proteases, apparently lacking the enzymatic activity: the pregnancy associated glycoproteins I and II (PAGI and PAGII). The first (PAGI) is largely secreted in maternal blood, this characteristic copes with the lack of proteolytic activity. The second (PAGII) is not completely characterized. However, it binds to lutropin (LH) receptors with high affinity. This binding allows to assume that PAGII is likely the same as the bovine chorionic gonadotropin identified earlier (bCG). A better characterization of these glycoproteins (PAGI and PAGII) and other members of the family (PAGIII...) will answer these questions together with the unexplained invasive process of the placenta. PMID- 7550038 TI - [Academic eulogy of Professor Rene Truhaut, foreign honorary member]. PMID- 7550039 TI - [In memoriam Professor Henry Baylon, foreign honorary member]. PMID- 7550041 TI - [Malaria in 1994: a challenge for the clinician]. AB - Physiopathology of malaria, and the immune reactions to this infection are poorly understood. Infection by P. falciparum is potentially fatal, and acquires an important position, both in the pathology of tropical countries as in imported pathology. Outside of endemic areas, diagnosis of the infection is important but sometimes difficult. Problems linked with malaria are compounded by the growing resistance of the infecting parasite to the classical drugs. A worldwide effort to identify new drugs has only led to partially satisfying results. It is possible that derivatives of a chinese medication, "Quing-Hao-Shu" open new perspectives for the treatment of malaria. PMID- 7550040 TI - [Observations on the implantable automatic defibrillator]. AB - Thanks to the fantastic job made by Michel Mirowski, the automatic implantable cardioverter-defibrillator is now an effective treatment for patients with malignant, life-threatening arrhythmias. And in the last ten years, many technical improvements have been made (weight, size, reconfirmation, Holter function, endocardial implantation). But if sudden death has been significantly reduced in implanted patients, the long term global cardiac mortality remains very high in these people. This explains why the indications to implant such an expensive device are difficult to define. Apart from special cases in which the indications (or contraindications) are obvious, many factors must be taken into consideration, including the presentation of the arrhythmia, the results of the programmed ventricular stimulation, and the underlying cardiac disease. The European society of Cardiology has published guidelines for the use of A.I.C.D., but from our own experience in Hospital Lariboisiere (Paris, France) we have learned that each case must be discussed; and, if it is possible to suggest some therapeutic orientations, it is yet illusory to believe that decisional algorithms will solve all the problems. PMID- 7550042 TI - FDA Office of Women's Health. PMID- 7550043 TI - Work redesign: what it means for nurses. PMID- 7550044 TI - What are the current recommendations for routine cancer screening in women? PMID- 7550045 TI - Many women return home. PMID- 7550046 TI - Lesbian health invisible to mainstream. PMID- 7550047 TI - Helping new mothers with the stay-at-home choice. PMID- 7550048 TI - When antepartum women go home on strict bed rest, what suggestions can be provided to help them cope with this unexpected situation? PMID- 7550049 TI - Neisseria gonorrhoeae with decreased susceptibility to ciprofloxacin in British Columbia: an imported phenomenon. PMID- 7550050 TI - Isolates of Neisseria gonorrhoeae in Ontario showing chromosomally-mediated resistance to penicillin combined with plasmid-mediated resistance to tetracycline. PMID- 7550051 TI - An overview of pharmacoepidemiology. AB - Pharmacoepidemiology is the application of epidemiological principles and methods to the study of drug effects in human populations. The goal of this discipline is to characterize, control and predict the effects and uses of pharmacological treatment modalities. Pharmacoepidemiology is also concerned with the economic impact and health benefits of unintended drug effects. The increasing importance of pharmacoepidemiology has been created by the need to develop a more accurate portrait of how drugs are used in the general population. Sophisticated and potent drug therapies require surveillance beyond the scope of the carefully controlled clinical trials of Phases I, II and III. Case-control and cohort studies, which allow scientists to evaluate the effects of patient variables on clinical outcomes, provide a wealth of information regarding the study of unexpected drug effects, drug utilization, treatment costs and the individualization of therapy. PMID- 7550054 TI - Analysis of pharmacotherapy of hypertension in out-patients. AB - In the present pilot study an attempt was made to evaluate the usefulness of results obtained about the treatment of patients suffering from high blood pressure for a pharmacoepidemiological study of the therapeutic value of antihypertensive agents. Data from 90 hypertensive patients were used in the present retrospective-prospective study. These patients all attended an out patient clinic located in one of the East-Bohemian districts that are participating in the MONICA programme. Instead of the analysis of the population data, regular blood pressure measurements, plasma glucose levels, and total cholesterol concentrations were measured and analysed. The consumption of various antihypertensive drugs in this study was similar to that found in larger studies performed in other health districts of the Czech Republic. Diuretics were the drugs most frequently prescribed for hypertensive patients. General practitioners rarely prescribed calcium channel blockers and angiotensin-converting enzyme inhibitors in 1990 and 1991. The metabolic effects of antihypertensive drugs, mostly diuretics, were not significantly evident. The most frequent occurrence of non-compensated blood pressure was recorded for the treatments with Trimecryton and methyldopa. However, the validity of these findings should be tested in a larger group of hypertensic patients. PMID- 7550052 TI - Pharmacotherapy of insomnia: practice and prospects. AB - Insomnia is a complex complaint which is often multifactorial in origin. Pharmacotherapy can only be an adjunct in the treatment of insomnia and hypnotics should be given on an intermittent basis for short periods of time. An overview is presented of the currently available hypnotics, of which benzodiazepines are still the most widely prescribed. New drugs which bind to specific receptor subtypes or which are partial benzodiazepine receptor agonists might overcome the disadvantages associated with chronic benzodiazepine use, but more long-term investigations are needed. PMID- 7550053 TI - Scavenging of reactive oxygen species by letosteine, a molecule with two blocked SH groups. Comparison with free-SH drugs. AB - Acute production of reactive oxygen species by polymorphonuclear neutrophils during the respiratory burst may induce tissue injuries. In this in vitro study, it was demonstrated that letosteine, a mucolytic agent containing two blocked thiol groups, had antioxidant activity, but only when it was first submitted to alkaline hydrolysis. In a cell-free system, hydrogen peroxide, hypochlorous acid and hydroxyl radical concentrations were reduced by half by letosteine concentrations of 200, 15 and 350 mumol/l, respectively. The mechanism of letosteine action may be related to the -SH group liberated in vitro by hydrolysis, which seemed to react by scavenging the reactive oxygen species in the same way as acetylcysteine and MESNA, free-thiol drugs known for their antioxidant properties. So, letosteine, a compound with blocked -SH groups which in vivo can metabolically become free, may have a therapeutic application in preventing oxidative tissue injury damage induced by the respiratory burst. PMID- 7550055 TI - Stability of bupivacaine hydrochloride with diamorphine hydrochloride in an epidural infusion. AB - The stability of diamorphine (0.02 mg/ml as the hydrochloride) in 250 ml bupivacaine hydrochloride (0.15% wt/vol infusion) was studied by high pressure liquid chromatography at temperatures in the range 7 to 45 degrees C. Diamorphine hydrochloride was degraded by approximately 0.13% per day at 7 degrees C. No bupivacaine hydrochloride degradation was detectable during the study. The storage life of the combination at 7 degrees C, based on the lower 95% confidence limit of the time to 5% diamorphine hydrochloride degradation, was 14 days. The stability at 25 degrees C was adequate to allow transport and administration over 24 h at ambient temperature. Stability was also maintained for at least 24 hr at 32 and 45 degrees C. Infusion of the mixture with an ambulatory infusion pump which uses a standard polyvinyl infusion bag is therefore possible. A study of its compatibility with different infusion pump medication reservoirs was not undertaken. The drugs were also stable on frozen storage at -18 degrees C for up to 6 months. PMID- 7550056 TI - Orphans of the 'Real': I. Some modern and postmodern perspectives on the neurobiological and psychosocial dimensions of psychosis and other primitive mental disorders. AB - To understand and treat patients suffering from psychoses (especially schizophrenia) and from other primitive mental disorders, it has become increasingly important to integrate the "soft science" of psychoanalysis, including its derivate, psychotherapy, with the "hard science" findings of neurobiology, infant development research, and newly emerging trauma research. The author presents such an integrated perspective, and also discusses a paradigmatic change within psychoanalysis itself, one that can be described as a transformation from the one-person ("modern") model to the intersubjective ("postmodern") model. This change is also characterized by a shift from drive theory to that of semiotic meaning and hermeneutics. In light of this transformative change and integration, the author formulates the concept of a generic type of patient, "orphans of the 'Real,'" whose illness can be described as the result of a premature awakening from the protective blanket of innocence because of hypersensitivity and/or traumatic abruption. These patients experience the "Real" without the protective blanket of imagination or symbolization. PMID- 7550057 TI - Orphans of the 'Real': II. The future of object relations theory in the treatment of the psychoses and other primitive mental disorders. AB - Objective relations theory is a broad, multifaceted concept that covers a number of differing paradigms. The author highlights the historical development of this concept. Originally developed by Freud and Abraham, it gradually became a shibboleth that identified both the Kleinian and the Independent schools in Great Britain and the object relations school within the American school of ego psychology. The two British schools differed between each other and from the American school. Among these schools of thought, two major paradigms have emerged: (1) the one-person psychoanalytic model, and (2) the two-person model. More recently, derivatives of the two-person model have emerged as the schools of relationism, self psychology, and intersubjectivity. In addition, the contributions of the Lacanian school have altered concepts about the fundamental nature of object relations. What is consequently at issue in terms of the psychoanalytic conception and treatment of psychoses and primitive mental disorders (with patients suffering from being "orphans of the 'Real'") is (1) the differing conceptions of what is meant by internal objects, part-objects, selfobjects, and object-representations, and (2) the differing conceptions of unconscious mental life. PMID- 7550058 TI - Schizophrenia and oxymorons. AB - From the viewpoint of character structure, patients with schizophrenia can be seen as the diametric opposite of persons with a nonpsychotic organization. They construct adaptations and defenses to deal with an externalized primitive mental orientation, whereas nonpsychotic persons tend to develop defensive mechanisms to render these primitive parts inactive. Patients with schizophrenia do not have a continuum between primary process id levels and secondary process reality operations. Thus polarities are not differentiated, leading to a series of paradoxes and oxymorons. These patients may view themselves as omnipotent and grandiose, yet at the same time lead a miserable, drab existence. Primitive parts of the personality may feel alive and powerful, whereas the ego is viewed as dead -the fundamental oxymoron of the living dead. The author presents several illustrative case vignettes of these features. PMID- 7550059 TI - A clinician with a family member with schizophrenia: a case report. AB - Clinicians who treat persons with schizophrenia often disagree with their patient's family about the etiology, treatment, and prognosis of the illness. Traditionally, such discrepancies have been a source of major tension and misunderstanding. The author reports a case that illustrates how a clinician, who herself had a family member with schizophrenia, became better able to bridge the gap in conceptual and empathic understanding that is often apparent between clinicians and families of schizophrenic patients. This clinician's personal views regarding the etiology, treatment, and prognosis of her schizophrenic family member are examined. Parallels are also drawn between the views of this clinician-family member and the current research on family acceptance patterns of the illness. PMID- 7550060 TI - A comprehensive cognitive-behavioral/educational program for schizophrenic patients. AB - Modern treatment of schizophrenia emphasizes pharmacology and nonspecific rehabilitative strategies, but there is renewed interest in psychologically oriented treatments as well. Borrowing from dynamic, cognitive-behavioral, and psychoeducational traditions, the authors describe a program aimed at teaching patients with schizophrenia about their illness and at addressing key cognitive, social, and emotional areas of difficulty from which they generally suffer. The authors document the enthusiasm of the patients for such an approach and provide a case example. PMID- 7550061 TI - Psychodynamic psychiatry and the treatment of psychosis in the French community. AB - The author reviews the development of psychodynamic psychiatry in France, comparing and contrasting it with trends in the United States. In particular, he describes the role of dynamic psychiatry in the treatment of psychotic patients. He traces the movement from institutional treatment toward deinstitutionalization and treatment in the community, culminating in the official adoption in France of catchment area psychiatry. He also notes the emergence and influence of antipsychiatry. He concludes that although psychodynamic psychiatry has experienced a decline resulting from both external (economic) and internal (theoretical) influences, this perspective continues to improve the understanding and treatment of psychotic patients, whether they are treated in the institution or in the community. PMID- 7550062 TI - Treatment of psychotic disorders in the United States and France: three comparisons. PMID- 7550063 TI - A gamma-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas. AB - gamma-Tubulin is a putative component of microtubule initiating material. To further explore its subcellular distribution in plant and animal cells, we have raised a polyclonal antibody, Rb27, directed towards a conserved region (EEFATEGTDRKDVFFY) of the gamma-tubulin molecule. Immunoblotting of cell protein extracts with Rb27 reveals a polypeptide band of M(r) 49 kD in HeLa and a 58 kD band in Chlamydomonas. Although these polypeptides are comparable in size to forms of gamma-tubulin detected previously in mammalian and plant protein extracts by other antibodies to gamma-tubulin, by immunofluorescence microscopy Rb27 gives localization patterns not previously seen. It localizes specifically with the centrioles in HeLa cells and with the basal body complex in Chlamydomonas. Other gamma-tubulin antibodies label pericentriolar material. Because of the similarities in the size of the polypeptides recognized by our and other gamma-tubulin antibodies, and a restricted co-localization with known microtubule-organizing centres in evolutionarily distant organisms, we propose that Rb27 recognizes a novel conserved gamma-tubulin isotype. PMID- 7550064 TI - Germ line chimera produced by transfer of cultured chick primordial germ cells. AB - Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads. PMID- 7550065 TI - Leucocyte involvement in lectin-induced deciduomata formation. AB - Intra-uterine injection of the lectin Concanavalin A (ConA) on day 5 of PSP induced a rapid and persistent infiltration of leucocytes into the rat uterine stroma. Although the infiltration of leucocytes was witnessed along the entire length of the uterine horn, areas of stromal oedema, indicative of decidualisation (as indicated by the positive Pontamine Sky Blue reaction), were only associated with regions in which the movement of leucocytes across the uterine epithelium was evident. Epithelial disruption and trauma was frequently noted within these regions. We believe that ConA may initiate decidualisation through indirectly causing epithelial trauma. PMID- 7550066 TI - F-actin in mitotic spindles of synchronized suspension culture cells of tobacco visualized by confocal laser scanning microscopy. AB - TRITC-labelled phalloidin was used to visualize F-actin distribution during mitosis in Nicotiana tabacum BY-2 suspension cells. Aphidicolin was used to synchronize cell suspensions, which enabled sufficient numbers of mitotic cells to be obtained. F-actin was present in the spindle, and its orientation seemed to correlate with the known microtubular arrays. The use of confocal microscopy greatly reduced background fluorescence, and therefore fine actin filaments could be observed in spindles previously thought to be devoid of actin. PMID- 7550067 TI - Gene expression and protein distribution of inter-alpha-trypsin inhibitor in three human hepatoma cell lines. AB - In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously. PMID- 7550068 TI - Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells. AB - Dimethoxy-2-nitrobenzyl adenosine-3',5' cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1-10 microM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either 3H-thymidine incorporation into DNA or as percent S-phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP. PMID- 7550069 TI - Dye-coupling between term pregnant human myometrial cells before labor: carboxyfluorescein versus lucifer yellow. AB - Term pregnant human myometrial cells in whole mounts were microinjected by pressure with the fluorescent probes Lucifer Yellow and carboxyfluorescein. Tissues obtained from acute and elective sections displayed weak dye-coupling when injected with Lucifer Yellow. Injection of carboxyfluorescein into cells from the elective sections resulted in a more extensive dye-coupling than that observed with Lucifer Yellow. These results indicate that term pregnant human myometrial cells are metabolically coupled before labor and carboxyfluorescein is superior to Lucifer Yellow in detecting the coupling. PMID- 7550070 TI - Studies on the permeability of the blood-testis barrier in stainless steel administered mice. AB - Previous trials performed in our laboratory have demonstrated that stainless steel corrosion products induced morphological changes in mice seminiferous cells. In this study the functional nature of the blood-testis barrier was investigated, in view of the extensive use of this metallic alloy in orthopaedic surgery. The uptake of horseradish peroxidase by the seminiferous tubules was used as a tracer method being exposed in vitro for different periods of time and observed at the ultrastructural level. Both boundary cells as well as Sertoli cells of experimental group absorbed the tracer, as manifested by vesicles and vacuoles filled with the reaction product, evidencing its role within the testis. Germ cells were also able in the uptake of this macromolecule. However, the tracer did not spread into the junctional complexes between neighboring Sertoli cells, at any interval of time used in this study, demonstrating the functional integrity of this barrier. PMID- 7550071 TI - Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas cells. AB - It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardtii contain polypeptides cross-reacting with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either alpha- or beta-spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent. PMID- 7550072 TI - Effects of bradykinin on Ca2+ mobilization and prostaglandin E2 release in human periodontal ligament cells. AB - In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca2+ ([Ca2+]i). When external Ca2+ was chelated by EGTA, the transient peak of [Ca2+]i was reduced and the sustained level was abolished, implying the Ca2+ mobilization consists of intracellular Ca2+ release and Ca2+ influx. Thapsigargin, a specific Ca(2+)-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-IP3)-sensitive Ca2+ pool, induced an increased in [Ca2+]i in the absence of external Ca2+. After depletion of the intracellular Ca2+ pool by thapsigargin, the increase in [Ca2+]i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP3. These results suggest that bradykinin stimulates intracellular Ca2+ release from the 1,4,5-IP3-sensitive Ca2+ pool. Bradykinin stimulated prostaglandin E2 (PGE2) release in the presence of external Ca2+, but not in the absence of external Ca2+. Ca2+ ionophore A23187 and thapsigargin evoked the release of PGE2 in the presence of external Ca2+ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca2+ mobilization via activation of phospholipase C and PGE2 release caused by the Ca2+ influx in HPDL cells. PMID- 7550073 TI - Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors. AB - Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with 125I-ET 1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with Kd values of 1 x 10(-10) M and 5 x 10(-9) M. The numbers of high- and low-affinity receptors were 1 x 10(4) and 2 x 10(5) per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-beta 1, IL-1 beta, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells. PMID- 7550074 TI - The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin. AB - In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion. PMID- 7550075 TI - Effects of retinoic acid and dimethylsulfoxide on the morphogenesis of the sea urchin embryo. AB - Sea urchin embryos of the species Paracentrotus lividus were treated continuously with different concentrations of all-trans retinoic acid (RA) or dimethylsulfoxide (DMSO) at different developmental stages. A delay in embryonic development was observed when embryos were cultured in the presence of 2 x 10(-5) M RA, between 1 and 12 hours of development. Hence, at 48 hours of development, while control embryos had reached the pluteus stage, RA-treated embryos were at the prism stage. At 72 hours of development RA-treated embryos recovered and continued normal development reaching the pluteus stage. No effect was observed when treatment was performed before 1 hour or after 12 hours of development. DMSO treatment had no effect on normal sea urchin embryo development, although we observed that pigment cells, clearly visible at the pluteus stage, become visible earlier with respect to control embryos. This report confirms the advantages that the sea urchin embryo offers for the study of problems in cellular and developmental biology. PMID- 7550076 TI - N-acetyl-cysteine enhances cell adhesion properties of epithelial and lymphoid cells. AB - In this paper, we show that the antioxidant N-acetyl-cysteine (NAC) is capable of enhancing the adhesion properties of the epithelial cell line A431 and of the lymphocytic cells with cytotoxic activity from human peripheral blood: the natural killer (NK) cells. This effect leads to an increased efficiency of A431 cells to form a monolayer and of NK cells to kill their targets. In both cases a specific effect of NAC was found in the distribution of those molecules of the cytoskeleton which are generally involved in cell substrate and cell-to-cell contact region formation, e.g., the actin microfilaments. NAC could thus behave as a drug influencing certain cytoskeleton-dependent cell processes in a non histotype dependent manner. PMID- 7550077 TI - Involvement of tubulin in MPP+ neurotoxicity on NGF-differentiated PC12 cells. AB - In vivo, the neurotoxin MPTP is oxidated to MPP+, which is toxic to dopaminergic neurons. In this paper, we have used MPP+ as a tool to evoke neurotoxicity in the PC12 cell line and investigate the intracellular events that are involved. A cytotoxicity test, performed on undifferentiated and NGF-differentiated PC12 cells, showed that MPP+ is much more toxic on differentiated cells and indicated the suitable range of concentrations for studying the starting events evoked by the neurotoxin. By indirect immunofluorescence we have shown that the localisation of alpha- and beta -tubulin in NGF-differentiated cells was modified by a 24 h treatment with 15 mumol/l MPP+. A biochemical analysis was performed on cell extracts and the results showed that MPP+ treatment induced an increase in alpha -tubulin levels and a decrease in beta -tubulin levels. These results suggest the involvement of the two microtubule proteins in MPP+ neurotoxicity on NGF-differentiated PC12 cells. PMID- 7550078 TI - TGF beta 1 and 8701-BC breast cancer cells: effect on growth and invasion in vitro. PMID- 7550079 TI - Periodontal training and implant dentistry. PMID- 7550080 TI - Radiographic evaluation of alveolar crest obscured by dental implants. AB - This study evaluated the diagnostic accuracy of periapical, tomographic, and cross-sectional occlusal radiographic techniques in the assessment of facial and lingual bone loss at implant obscured sites. An edentulous dry human mandible was prepared with implants in the incisor, premolar, and molar regions. Successive round bur sizes (No. 1 to 6) were used to create circumferential infrabony periodontal defects in the alveolar crest adjacent to the implants. Periapical radiographs were made with custom stents and imaged each site in a conventional parasagittal orientation. Linear tomograms imaged sites in a cross-sectional coronal orientation. Cross-sectional occlusal radiographs imaged anatomy in the axial plane. Radiographic anatomy mesial and distal to each implant was masked with opaque tape before evaluation by a panel of six observers. Images were displayed in pairs consisting of an image at baseline and a second image having an equal chance of displaying an increment of bone removal or no bone removal. Image pairs were displayed in random order. A five-point scale was used to evaluate accuracy of detection of the presence or absence of changes in bone density as well as observer confidence in that assessment. ANOVA demonstrated significant variation due to image modality and increment of bone removal. Cross sectional occlusal views provided significantly greater mean observer confidence scores than either periapical images or tomograms (P < .01). However, tomograms may provide greater utility in actual clinical practice. PMID- 7550081 TI - The use of high-density polytetrafluoroethylene membrane to treat osseous defects: clinical reports. AB - Alveolar bone resorption can result from tooth loss, periodontal disease, or trauma. Guided tissue regeneration is used in an attempt to exclude tissues devoid of osteogenic potential from a bone defect or cavity and promote new bone growth to replace missing osseous structure. Many types of barrier membranes have been used, but none have been found to be ideal for every clinical situation. Macroporous membranes, such as expanded polytetrafluoroethylene, require primary closure and a second surgical procedure for their removal. Macroporous membranes can incorporate bacteria and may become infected if exposed in the oral cavity. Membranes manufactured of resorbable polymers require primary closure of the augmentation site and exhibit variable patterns of resorption, introducing a degree of unpredictability into the procedure. The use of high-density polytetrafluoroethylene membrane to promote deposition of bone for ridge augmentation in the oral cavity is described. Two clinical reports are presented. PMID- 7550082 TI - The effect of cementing agent and technique on the retention of a CeraOne gold cylinder: a pilot study. AB - Endosseous dental implants are routinely used for the replacement of missing teeth. The CeraOne system uses a gold cylinder that is luted to a metal abutment. This study evaluated two luting agents and two luting techniques on the retention of a CeraOne gold cylinder to a CeraOne titanium abutment. A mechanical testing instrument was used to provide an uniaxial tensile force on the gold cylinder, which was cemented with zinc phosphate or Tempbond NE. The access opening to the gold abutment screw was filled or unfilled. Filling the access opening resulted in significantly higher retentive values (P < 0.01) compared to leaving the access opening unfilled. The gold cylinder was significantly more retentive when cemented with zinc phosphate as compared with Tempbond NE (P < 0.01). A Tukey post hoc test demonstrated that all means were significantly different at the P = 0.01 level. The results suggest that the choice of luting agent and luting technique may affect retention of a CeraOne gold cylinder. PMID- 7550083 TI - Undergraduate education in implant dentistry. AB - A special interest group meeting devoted to clinical educational programs in implant dentistry for undergraduate students was held in March 1994 at the annual session of the American Association of Dental Schools in Seattle, Washington. Summaries of the presentations of representatives from three United States dental schools and the moderator's introductory remarks are included in this article. PMID- 7550084 TI - Biologic response to an intraoral extraosseous implant system: a pilot study. AB - Extraoral force systems in orthodontics are dependent on patient cooperation. Endosseous implants that offer intraoral anchorage are being investigated, but the implant cannot be loaded for 4 months, is limited in placement by skeletal anatomy, and must be surgically removed. The purpose of this pilot study was to investigate an intraoral extraosseous implant used as an anchorage source. Titanium ramus implants were placed on the lateral surface of the left rami of 12 sheep. Clinical evaluations were made of implant stability and tissue condition. Bone changes were assessed at the time of killing, which ranged from 43 to 84 days. Infection was present in most animals at the time of killing. The implants were mobile in all animals, and the rami experienced considerable changes due to bone resorption and apposition. The results of this animal pilot study suggest that an intraoral extraosseous implant system such as that used in this investigation may result in bone resorption, implant mobility, and infection. PMID- 7550085 TI - Angulation assessment of anterior single tooth root form implants: technical note. AB - A technique is presented to evaluate the angulation and position of an anterior single tooth root form implant by means of a surgical template, a pilot osteotomy, and an abutment guide. The space available for the restorative materials can be assessed and the angulation modified, if necessary, to improve the esthetic potential. If the angulation is satisfactory and sufficient room is available for the prosthesis, the surgeon can continue with implant placement at the initial site. PMID- 7550086 TI - Dual-tray impression technique for implant-retained overdentures. AB - Accurately recording implant positions and the edentulous areas during the fabrication of implant-retained overdentures can be a difficult task. A modification of the dual or split-tray technique is suggested for implant retained overdentures. This procedure permits the construction of the prosthesis on one master cast and the use of two impression materials with different properties incorporated into a single impression to correspond with the needs of specific anatomic areas of the mouth. PMID- 7550087 TI - Structural analysis of alkaline-soluble polysaccharide, P-1, from the kernels of Prunus mume Sieb. et Zucc. AB - A polysaccharide fraction extracted with cold 0.5 M NaOH from the kernels of Prunus mume exhibited some biological activities. A polysaccharide, P-1, was purified from the 0.5M NaOH extract by ion-exchange chromatography and gel filtration. The results of the structural analysis of P-1 to determine the relationship between the activities and the structure are described in this paper. In the mild acid hydrolysis of P-1, the nondialyzable hydrolysate (I-3) believed to be its core portion was obtained. The yield of I-3 was 26.0% and contained 59.8% uronic acid as galacturonic acid (GalA). The neutral sugars of I 3 were composed of rhamnose, xylose and galactose in a molar ratio of 1.0:3.4:0.3 following analysis by gas-liquid chromatography. The molecular weight of I-3 was estimated to be ca. 14000 by gel-filtration on Toyopearl HW55F. I-3 exhibited the mitogenic activity toward spleen cells as well as P-1. These facts appeared to confirm that I-3 was the core part of P-1 and important for its biological activity. I-3 was successfully reduced by the Taylor and Conrad method to avoid so much repetition. Methylation analysis of the reduced hydrolysate by gas-liquid chromatography and gas chromatography-mass spectroscopy showed that the ratio of 1,4-linked galactopyranosyl and 1,3,4-linked galactopyranosyl residues were significantly increased in comparison with native I-3. These results suggested that I-3 was composed of 1,4- and 1,3,4-linked galacturonic acid residues in the main chain. PMID- 7550088 TI - Radioimmunoassay method for the determination of 22-oxacalcitriol, a novel analog of 1 alpha,25-dihydroxyvitamin D3 having valuable clinical potency, in rat and human plasma. AB - 22-Oxacalcitriol is a synthetic analog of 1 alpha,25-dihydroxyvitamin D3, which is expected to be a novel agent for the treatment of patients with secondary hyperparathyroidism, psoriasis, and/or breast cancer. A sensitive and practical RIA for 22-oxacalcitriol in rat and human plasma has been developed. A rabbit antiserum raised against 22-oxacalcitriol 3-hemiglutarate conjugated with bovine serum albumin was used in combination with a tritium-labeled 22-oxacalcitriol having high specific radioactivity. A plasma sample was extracted with ethyl ether and the extract was successively purified with Bond Elut C18 and a normal phase HPLC prior to the RIA to remove interfering substances. Accuracy of the RIA was assessed by various studies including serial dilution and recovery tests. Intra- and inter-assay coefficients of variation were lower than 10%, and the quantification limit was low enough for practical use (33 pg/ml plasma). The present RIA will be useful for the pharmacokinetic studies of 22-oxacalcitriol in both preclinical and clinical stages. PMID- 7550089 TI - Identification of organic cation transporter in rat renal brush-border membrane by photoaffinity labeling. AB - As an approach to identification of the organic cation transport system in brush border membranes, we designed a photoaffinity probe, 1-cyano-2-(4-azido[3,5 3H]benzoylethyl)-3-[2-[[(5-methyl-4-imidazo lyl ) methyl]thio]ethyl]-guanidine ([3H]AMC) based on the molecular structure of cimetidine, which is taken up by the organic cation transport system in brush-border membrane vesicles. The effect of nonradioactive 1-cyano-2-(4-azidobenzoylethyl)-3-[2-[[(5-methyl-4- imidazolyl)methyl]thio]ethyl]guanidine (AMC) on tetraethylammonium uptake was investigated in rat renal brush-border membrane vesicles. We examined the photolysis of AMC in which the azido group was converted to an active nitrene group using UV light at a wavelength of 254 nm and established a half-life of 7 s. This half-life duration did not significantly impair brush-border membrane vesicles during the exposure to light for photo-labeling. Photoaffinity labeling of brush-border membrane vesicles from the rat renal cortex with [3H]AMC resulted in the covalent incorporation of radioactivity into membrane polypeptides; an apparent 36 kDa polypeptide was predominantly labeled. Photolabeling specificity was shown by a reduction in the labeling of the 36 kDa polypeptide in the presence of organic cations, cimetidine, tetraethylammonium and N methylnicotinamide whereas the organic anion, fur osemide, had no effect on labeling patterns. These data demonstrate that AMC, as well as organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide, interact with a common 36 kDa membrane polypeptide, which may be the transport system or one of its brush-border membrane components. PMID- 7550090 TI - Nonenzymatic glycation of transferrin: decrease of iron-binding capacity and increase of oxygen radical production. AB - The total iron-binding capacity (TIBC) and iron contents of diabetic rat serum, as well as the iron-binding capacity of glycated transferrin and oxygen radical production by the glycated proteins were examined. The TIBC and iron content of diabetic rat sera were found to be much lower than those of control rat sera. Incubation of human serum with glucose in vitro resulted in a significant fall of its unsaturated iron-binding capacity (UIBC) with time. When apotransferrin was incubated with glucose, its UIBC significantly decreased. The iron content of holotransferrin was markedly reduced by incubation with bathophenanthroline sulphonic acid (BPSA) in the presence of glucose, although the content was not altered by incubation with BPSA alone. The generation of superoxide radical (O2-) and hydroxyl radical (OH.) by the glycated holotransferrin was much greater than that by glycated apotransferrin. Glycated holotransferrin showed significantly accelerated hydroxyl radical production by the hypoxanthine-xanthine oxidase system, while intact holotransferrin did not. Treatment of holotransferrin with glucose caused the fragmentation of the protein, while the same treatment of apotransferrin did not. These results suggest that iron ions in the glycated transferrin molecule are bound loosely to the protein and are redox-active and the glycated holotransferrin produces oxygen radicals including O2- and OH. efficiently, and that the glycated transferrin does not function as an iron binding protein. PMID- 7550091 TI - Effects of phosphoramidon on endothelin-1 and big endothelin-1 production in human aortic endothelial cells. AB - Using cultured human aortic endothelial cells, we examined the effects of phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, on the release of endogenous endothelin-1 (ET-1) and big endothelin-1 (big ET-1), and on the generation of ET-1 from exogenously applied big ET-1. Phosphoramidon, at concentrations of 10(-6) to 2 x 10(-4) M, caused a biphasic alteration of the ET 1 release, i.e., at lower concentrations of the drug, there were slight but unexpected increases of the release, whereas higher concentrations led to a decrease which is due to the drug-induced inhibition of ECE. The former effect appears to be based on the inhibition of ET-1 degradation by neutral endopeptidase 24.11 (NEP), since kelatorphan, a specific NEP inhibitor, produced a similar increasing effect on ET-1 release. Phosphoramidon enhanced the big ET-1 release from the cells in a concentration-dependent manner. When high concentrations of phosphoramidon were added, there was a dramatic increase in the release of big ET-1, which cannot be explained only by the drug-induced inhibition of ECE. This increase in big ET-1 release appeared to be partly due to a transient stimulation of the expression of prepro ET-1 mRNA. The amount of ET-1 generated from exogenously applied big ET-1 was markedly decreased by phosphoramidon in a concentration-dependent manner. In a similar fashion, phosphoramidon markedly inhibited ECE activity of the membrane fraction of cultured cells. Thus, ET-1 generation from exogenously applied big ET-1 reflects the functional phosphoramidon-sensitive ECE activities in human aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550092 TI - Potentiating effects of beta-eudesmol-related cyclohexylidene derivatives on succinylcholine-induced neuromuscular block in isolated phrenic nerve-diaphragm muscles of normal and alloxan-diabetic mice. AB - beta-Eudesmol, a sesquiterpenoid alcohol contained in Atractylodes lancea, potentiates succinylcholine (SuCh)-induced neuromuscular blockade. The potentiating effect is greater in diabetic muscles than in normal ones. As a ligand for affinity chromatography to study the potentiating mechanism, we designed and synthesized newly beta-eudesmol-related cyclohexylidene derivatives (2-(3-hydroxy-3-methylbutyl)cyclohexylidene; KTE-13, 2-(3-hydroxy-3-methylbutyl) 4-cyclohexylidene carboxylic acid; KTE-32 and 4-tert-butoxycarbonyl-2-(3-hydroxy 3-methylbutyl) cyclohexylidene; KTE-33). We examined the potentiating effects of those compounds in phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. KTE-33 (100 microM) potentiated more greatly SuCh-induced neuromuscular blockade in diabetic muscles than in normal ones (the potentiating ratios in normal and diabetic muscles were 6.7 and 10.6, respectively), while KTE 13 (100 microM) and -32 (200 microM) potentiated weakly. These results suggest that the ester group in KTE-33 rather than a carboxyl group in KTE-32 is important in inducing the potentiation of SuCh-induced neuromuscular blockade in diabetic state. PMID- 7550093 TI - Activation of the renin-angiotensin system in anti-glomerular basement membrane antibody-induced glomerulonephritis. AB - Activity of the renin-angiotensin system in the nephrotic syndrome was investigated in rats with acute nephritis induced by anti-glomerular basement membrane (GBM) antibody. Injection of anti-GBM antibody resulted in a transient 2 fold elevation of both plasma renin and angiotensinogen with a peak at 12 h. Angiotensinogen mRNA levels in the liver also rapidly and transiently increased 4 fold at 3 h. The manifestation of acute nephritis, indicated by proteinuria, hypoalbuminemia, hypercholesterolemia and an increase in serum creatinine, following injection of anti-GBM antibody, was inhibited by a single administration of the selective angiotensin II type 1 receptor antagonist TCV-116 (1 mg/kg, p.o.) 2 h before an injection with the antibody, but not by successive administration of this drug for 1 week from 3 d after the injection of antibody. These results suggested that the enhanced generation of angiotensin II by elevated levels of both renin and its substrate in the early phase of anti-GBM nephritis promotes the evolution of acute nephritis via angiotensin II type 1 receptor. PMID- 7550095 TI - Cytotoxic effects of several hopanoids on mouse leukemia L1210 and P388 cells. AB - The cytotoxic effects of hopanoids, including bacteriohopane-32, 33, 34, 35 tetrol (Tetrol), bacteriohopane-32-ol (Monol), diploptene, diplopterol and acetylated Monol (AcO-Monol) isolated from Acetobacter aceti, were tested against two leukemia cell lines. Tetrol and Monol have been shown to be toxic to mouse L1210 and P388 compared to the other hopanoids. By measuring the ESR spectra of the spin labeled membranes of these cells, it was shown that the incorporation of Monol resulted in a decrease in the fluidity of the membranes. The decrease in membrane fluidity may correlate, in part, with the cytotoxicity of hopanoids against the two cell lines. PMID- 7550094 TI - Regional characteristics of [125I]cholecystokinin octapeptide specific binding to rat brain membranes following 6-hydroxydopamine treatment. AB - The relative changes in the amount of specific [125I]cholecystokinin octapeptide (CCK8) bound to regional brain membrane preparations after 6-hydroxydopamine (6 OHDA) treatment were examined. The specific binding in the frontal cortex and striatum decreased and reached a minimum on the 3rd day after 6-OHDA treatment. Thereafter, the specific binding recovered to 60% and 65% of control values in the frontal cortex and striatum respectively on the 28th day. On the other hand, in the nucleus accumbens, where CCK8 co-exists in the dopamine neuron, the specific binding decreased gradually, and its recovery was delayed compared with that of the frontal cortex and striatum. In the hippocampus, 6-OHDA treatment had no effect on the specific binding throughout the experimental period. The decrease of [125I]CCK8-specific binding could be caused by enhanced release of CCK8 in the frontal cortex, striatum, and nucleus accumbens, and the recovery of specific binding could be induced by depletion of CCK8 in nerve terminals. Particularly in the nucleus accumbens, the delayed recovery of specific binding suggests the loss of the pre-synaptic binding site, which exists in the CCK8/DA co-existing neuron, together with a change in CCK8 release. PMID- 7550096 TI - Potentiation of the cytotoxicity of chloroethylnitrosourea by O6 arylmethylguanines. AB - It was reported recently that monomeric O6-benzylguanine (1) acts as an alternative substrate for a DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), and that therefore pretreatment of cells with 1 induces depletion of AGT resulting in an enhanced cytotoxic response to alkylating antitumor agents. In order to study the interaction of O6-benzylguanine derivatives with AGT and to obtain greater AGT depletion, we synthesized the following O6-arylmethylguanine derivatives and related compounds: O6-(4-, 3- and 2-fluorobenzyl)guanines (2, 3, 4), O6-(4-, 3- and 2 trifluoromethylbenzyl)guanines (5, 6, 7), O6-(4-, 3- and 2-pyridylmethyl)guanines (8, 9, 10), O6-(2- and 1-naphthylmethyl)guanines (11, 12), O6 biphenylmethylguanine (13), S and Se analogues of O6-benzylguanine (14, 15) and O6-phenylguanine (16). Ten of these are new compounds. All these compounds were tested for their potentiation of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2 chloroethyl)-N-nitrosou rea (ACNU) cytotoxicity using HeLa S3 and C6-1 cells. Compounds 2, 3, 5, 8, 9, 11 and 13 were active, as was 1. Compounds 7 and 12, with a substituent at the alpha position of the benzyl group, and compound 10, the alpha-nitrogen analogue of 1, were almost completely devoid of potentiating activity. These results suggest that the alpha-position of the O6-benzyl group plays an important role in the interaction of O6-benzylguanines with AGT. Of the other compounds, 4 and 6 exhibited very weak activity and 14, 15 and 16 were inactive. Possible reasons for these differences in activity are discussed in relation to the biomimetic dealkylation rates of O6-benzylguanine derivatives and the chemical characteristics of their substituents. PMID- 7550097 TI - Effects of alkyl substituents of xanthine on phosphodiesterase isoenzymes. AB - The structure-activity relationships of a series of alkylxanthine derivatives were investigated. The partition coefficient of alkylxanthines enlarged with an elongation of the alkyl chain at the 1-, 3-, or 7-position of xanthine. There was a mild correlation between the apparent partition coefficient and the tracheal relaxant activity or the inhibitory activity on phosphodiesterase (PDE) IV isoenzyme, while the tracheal relaxant activity closely correlated with the PDE IV inhibitory activity. Regarding substituents at different positions, the alkylation at the 3-position increased the inhibitory activity on every PDE isoenzyme. The alkylation at the 1-position potentiated the inhibitory activity on PDE IV with the alkyl chain length, but decreased the activities on other PDE isoenzymes. The alkylation at the 7-position was characteristic in its decrease in inhibitory activity on PDE III. These results suggested that the potency of the inhibitory activity of xanthine derivatives on PDE isoenzymes is not dependent simply upon their hydrophobicity but upon change in the affinity for the active sites on PDE isoenzymes by the introduction of the alkyl group at particular positions of the xanthine skeleton. PMID- 7550099 TI - Combined effect of d-limonene pretreatment and temperature on the rat skin permeation of lipophilic and hydrophilic drugs. AB - The combined effect of d-limonene and temperature on the skin permeation of lipophilic and hydrophilic penetrants has been investigated in rats in vitro. Prednisolone was used as a lipophilic penetrant, and glucose and isoniazid were used as hydrophilic ones, respectively. When the skin was pretreated with 30% ethanol without d-limonene, the steady state permeability coefficient (P) of every penetrant through the skin was difficult to calculate because of very low permeability. On the other hand, the cumulative amount of each penetrant increased with an increase in temperature when the skin was pretreated with 1.5% d-limonene in 30% ethanol. The Arrhenius plots of P values for glucose and isoniazid showed a linear relationship, and the activation energies of skin permeation were estimated to be 87.6 and 66.5 kJ/mol, respectively. When prednisolone was used as penetrant, however, the Arrhenius plot of P values exhibited a convex curvature. This may suggest that the combined use of d limonene and temperature effectively changes the barrier structure of the non polar pathway in the stratum corneum, while no synergistic effect was observed on the polar pathway. PMID- 7550098 TI - Anti-tumor-promoting activities of euglobals from Eucalyptus plants. AB - To search for possible anti-tumor-promoters (chemopreventive agents), we carried out a primary screening of 21 euglobals (acylphloroglucinol-monoterpene or sesquiterpene structures) isolated from the juvenile leaves of five species of Eucalyptus plants using an in vitro synergistic assay system. Of these compounds, euglobal-G1--G5 (1-5), -Am-2 (15) and -III (16) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12 O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the effects of compounds 1 and 16 on the cell cycle of Raji cells were also examined by a flow cytometer, and both compounds 1 and 16 exhibited strong inhibition on the effect of the cell cycle induced by TPA. These two euglobals (1 and 16) exhibited remarkable anti tumor-promoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. PMID- 7550101 TI - Biliary excretion of furosemide glucuronide in rabbits. AB - Furosemide (F) was administered to rabbits intravenously and intraduodenaly and the biliary excretion was studied. The major metabolite excreted in bile was furosemide glucuronide (FG). F and acyl migration isomers of FG (FG-iso) were also excreted in bile. The biliary excretion rates of total F (F+FG+FG-iso) following intraduodenal administration of F were much smaller than those following intravenous administration. The fraction of (F+FG-iso) in bile following intraduodenal administration of F were larger than those following intravenous administration. Stability of FG or FG-iso in bile and supernatant solution of the duodenum homogenate of rabbits was studied. FG was unstable in both media and its degradation followed apparent first-order kinetics in both media. In bile, FG degraded to produce several FG-iso and F, while in the supernatant solution of the duodenum homogenate, it hydrolyzed immediately to F. FG-iso were hardly detected in the supernatant solution. These results indicated that FG excreted in bile degraded easily to FG-iso and F. FG might easily hydrolyze to F enzymatically in the duodenum, and the resultant F might be reabsorbed from the intestinal tract. Unabsorbed FG-iso and F might be excreted in the feces. PMID- 7550102 TI - Occurrence and antigenic heterogeneity of L-2,4-diaminobutyrate decarboxylase in Acinetobacter species. AB - We have previously reported that a novel enzyme, L-2,4-diaminobutyrate decarboxylase (DABA DC), which is responsible for the formation of 1,3 diaminopropane, occurs in two Acinetobacter species. The present study extends this observation to additional Acinetobacter species and strains (6 reference strains and 30 clinical isolates). Furthermore, the DABA DC protein was detected in every strain by Western blot analysis with the antiserum against the enzyme purified from A. baumannii ATCC 19606. However, only the DABA DCs in the A. calcoaceticus and Acinetobacter genospecies 3 in addition to A. baumannii strains strongly cross-reacted with the antiserum, suggesting antigenic heterogeneity among the DABA DC proteins in Acinetobacter species. Therefore, immunological testing of the DABA DC protein may provide an additional method for differentiating and identifying Acinetobacter strains. PMID- 7550100 TI - Uptake of low molecular weight fractionated [3H]heparin by rat hepatocytes in the primary culture. AB - The uptake of low molecular weight fractionated [3H]heparin (LMWFH: 7000 Da) was examined, for comparison with that of high molecular weight fractionated [3H]heparin (HMWFH:20000 Da), in a primary culture of rat hepatocytes. The uptake of LMWFH increased almost linearly with time up to 60 min (extended uptake), although a faster uptake was observed in the initial 2 min (initial uptake). Both the initial and extended uptake were saturable, and the maximum uptake velocity (Vmax) and the Michaelis constant (Km) were estimated to be 10.7 pmol/min/mg protein and 398 nM, respectively, for the initial uptake and 0.34 pmol/min/mg protein and 116 nM, respectively, for the extended uptake. The Km for the extended uptake was 5 times larger than that of 21 nM for HMWFH, but the other parameters were comparable with those for HMWFH. Thus, an increase in Km, or a decrease in the apparent affinity, with a decrease in molecular weight in the extended uptake may be responsible for the reported lower hepatic uptake of low molecular weight heparin, compared with unfractionated heparin. It was also shown that both the initial and the extended uptake of LMWFH were inhibited by several analogs of heparin, including HMWFH, and anionic compounds such as 4,4' diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suggesting that LMWFH and HMWFH, in spite of a large difference in the molecular weight, share the same specialized uptake mechanism, in which an anionic moiety and/or heparin-like structure plays an important role. PMID- 7550104 TI - Suppressive effect of molybdenum on hepatotoxicity of N-nitrosodiethylamine in rats. AB - In order to elucidate the mechanism by which molybdenum prevents the carcinogenesis of N-nitroso compounds, the effects of Na2MoO4-pretreatment on N nitrosodiethylamine (NDEA)-induced DNA strand breaks, fluctuation in cation contents and lipid peroxidation levels in rat liver were examined. Male Wistar rats weighing 170-190 g were pretreated with Na2MoO4 (1.24 mmol/kg body weight, i.p., once a day) for 3 d and on day 4, they were exposed to NDEA (50 mg/kg body weight, once, i.p.). Three days after exposure to NDEA, the nitroso compound caused DNA strand breaks and disrupted potassium (K) and calcium (Ca) metabolism in the liver but did not affect lipid peroxidation levels. Na2MoO4-pretreatment prevented both NDEA-induced DNA damage and disruption of the metabolism of those cations but rather enhanced lipid peroxidation. These results suggest that Mo prevented NDEA-induced DNA damage by preventing disruption of intracellular Ca metabolism while stimulating the metabolism of the nitroso compound via a nontoxic pathway. PMID- 7550103 TI - A sapecin homologue of Holotrichia diomphalia: purification, sequencing and determination of disulfide pairs. AB - We purified and characterized a sapecin homologue, named holotricin 1, from the hemolymph of immunized larvae of a coleopteran insect, Holotrichia diomphalia. We determined its complete amino acid sequence and three disulfide pairs. Holotricin 1 consisted of 43 amino acid residues and showed potent antibacterial activity against gram-positive bacteria, but antibacterial activity against gram-negative bacteria was not obvious. PMID- 7550105 TI - Study on anti-cataract drugs from natural sources. II. Effects of buddlejae flos on in vitro aldose reductase activity. AB - The inhibitory effects of nine crude drugs were tested on unpurified rat lens aldose reductase, an enzyme involved in the complications of diabetes. Among the crude drugs, a 70% methanolic extract of Buddlejae Flos (flower of Buddleja officinalis) exhibited the highest inhibition. Luteolin, luteolin-7-O-beta-D glucopyranoside, apigenin and acacetin-7-O-alpha-L-rhamnopyranosyl-(6-1)-beta-D glucopyranoside isolated from Buddlejae Flos showed the inhibitory activity, the IC50 (concentration of 50% inhibitory percentage) values of which were 0.21, 0.28, 0.58 and 0.75 microM, respectively. It is suggested that the inhibitory effect of Buddlejae Flos on aldose reductase is partially attributable to these flavonols or their glycosides. PMID- 7550106 TI - Inhibitory effects of steroidal saponins on 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced 32P-incorporation into phospholipids of HeLa cells and proliferation of human malignant tumor cells. AB - Certain Lilium plants contain (25S)-spirost-5-ene-3 beta,27-diol glycosides embracing 3-hydroxy-3-methylglutaric acid at the C-27 hydroxy position. One of their derivatives, methyl ester of (25R)-27-O-[(S)-3-hydroxy-3-methylglutaryl] spirost-5-ene-3 beta,27-diol 3-O-(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D glucopyranosyl-(1-->4)] - beta-D-glucopyranoside) was found to inhibit 12-O tetradecanoylphorbol-13-acetate (TPA)-stimulated 32P-incorporation into the phospholipids of human cervical cancer (HeLa) cells and also to inhibit the proliferation of various kinds of human malignant tumor cells, pancreatic cancer (PANC-1), osteosarcoma (OST), human gastric cancer (HGC-27), pheochromocytoma (PC 12) and HeLa cells, in vitro. PMID- 7550107 TI - Pharmacokinetics of theophylline: effects of hepatic fibrosis in rats induced bile duct ligation. AB - This experiment was performed to evaluate the usefulness of an experimental fibrosis model by bile duct ligation as a pharmacokinetic model of a disease state. First, experimental liver fibrosis was produced by bile duct ligation. At 4 weeks postoperation, a fibrotic condition was characterized by measurement of the aminoterminal procollagen type III peptide (PIIINP) level in serum, total collagen content in liver and light microscopic histology. Four weeks after bile duct ligation there was an increase in total collagen content of the liver to 430% of the initial values, accompanied by an increase of serum-PIIINP (385%). Secondly, we examined the pharmacokinetics of theophylline in the fibrotic rat induced by bile duct ligation. An i.v. dose of 8 mg of theophylline per kg of body weight was administered, and the levels of theophylline in serum were assayed by high performance liquid chromatography. The area under the serum concentration-time curve of theophylline was increased significantly in fibrotic rats compared with that of the control, and the total clearance of drug in fibrotic rats was low, averaging 22.6 mg/kg/h vs. 36.1 and 60.9 ml/kg/h in the control and the normal rat, respectively. However, the value of distribution during the beta-phase was not significantly affected by experimental liver fibrosis. PMID- 7550108 TI - Stability, specific binding activity, and plasma concentration in mice of an oligodeoxynucleotide modified at 5'-terminal with poly(ethylene glycol). AB - Oligodeoxynucleotide (ODN) composed of 15 nucleotides was modified at 5'-terminal phosphate with hexylamine linker and chemically activated poly(ethylene glycol). This derivative showed improved characteristics in terms of enzymatic stability, binding activity, and in vivo retention in mouse. The data are discussed in comparison with those of corresponding unmodified and phosphorothioate ODNs. PMID- 7550109 TI - Interactions between bacteriohopane-32,33,34,35-tetrol and liposomal membranes composed of dipalmitoylphosphatidylcholine. AB - The effects of bacteriohopane-32,33,34,35-tetrol (Tetrol), on liposomal membranes composed of dipalmitoyl-phosphatidylcholine (DPPC) were examined and compared with those of bacteriohopane-32-ol (Monol) and cholesterol (Chol) by means of ESR and NMR techniques. 1H-NMR spectra of Tetrol-incorporated DPPC membranes showed splitting of the signals of the choline N-methyl resonance, whereas Monol- and Chol-incorporated membranes showed not splitting of signals above the transition temperature of DPPC. It was suggested that the incorporation of Tetrol affected only the fluidity near the polar head groups of the DPPC membranes. The characteristics of the interactions between Tetrol and membranes are due to the fact that in DPPC bilayers Tetrol and Monol have an inverted orientation contrary to Chol, and that the hydroxy groups of Tetrol suppress the hydrophobic interaction between DPPC molecules whereas the methyl groups of the hopanoid ring promote this. PMID- 7550110 TI - Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67. AB - Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH. PMID- 7550111 TI - Binding and uptake of oxidized low density lipoprotein (LDL) by macrophage scavenger receptors are enhanced by substrate-bound fibronectin. AB - The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages and the resulting accumulation of low density lipoprotein (LDL) lipids within the cells has been implicated in the pathogenesis of atherosclerosis. The effect of fibronectin (FN) on the binding and uptake of ox-LDL by macrophages was investigated using thioglycollate-induced mouse peritoneal macrophages. The ability of the macrophages to bind ox-LDL was assessed by the binding of mouse red blood cells (RBC) pre-coated with ox-LDL (ox-LDL-RBC) prepared in vitro to macrophages at 37 degrees C. The binding of ox-LDL-RBC to macrophages was significantly enhanced when the macrophages were plated on a FN-coated substrate. Similar enhancement was observed when the macrophages were plated on a substrate pre-coated with Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide, an adhesive sequence of FN involved in binding to the cells, but not with the control Gly-Arg-Gly-Glu-Ser Pro (GRGESP) peptide. The effect of FN was inhibited when GRGDSP, but not GRGESP, was present during the macrophage attachment to the FN-coated substrate, suggesting that the specific interaction of this sequence and the FN-receptor is responsible for the effect of FN. The addition of FN or GRGDSP in solution to the macrophage layers on an uncoated substrate was ineffective. Thus, attachment to a substrate is necessary for FN to be effective on the macrophages.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550112 TI - The occurrence of nicked human chorionic gonadotropin (hCG) by a thermolytic endoprotease. AB - A nicked form of human chorionic gonadotropin (nicked hCG), in which only one peptide bond between residues 47 and 48 (-Gly-Val-) of beta-subunit is cleaved, has been found in the urine and blood of pregnant women. In this study, we investigated the occurrence of nicked hCG and the localization of the nicking enzyme for hCG. First, to determine what type of protease nicks hCG, an in vitro proteolytic study using various proteases was performed. Amino-terminal amino acid sequence analysis of the beta-subunit purified from protease-treated hCG indicated that thermolysin actively nicks hCG. Secondly, to determine which tissues are related to the formation of nicked hCG, the distribution of radioactivity in various tissues after i.v. administration of radiolabeled hCG to female rats was examined. The radioactivity accumulated predominantly in the kidney (17%), liver (9.3%) and ovary (0.9%) after 30 min of injection. Analysis of molecular species of beta-radiolabeled hCG in various tissues and body fluids, using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography, indicated that a nicked hCG-like molecule was found in the kidney, predominantly, as well as in the serum and urine. To examine the role of the kidney in producing nicked hCG, hCG was incubated with rat kidney particulate fraction (KPF). Immunoblot analysis of KPF-treated hCG indicated that KPF produced a nicked hCG-like molecule. Furthermore, the possibility that placental trophoblast cells produce nicked hCG was also examined using the choriocarcinoma cell BeWo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550113 TI - Relationship between cytotoxic and microtubule disruptive activities of (+)-, (-) and (+/-)-indenestrol A and B monomethyl ethers in Chinese hamster V79 cells in culture. AB - We report the effects of indenestrol A, a metabolite of diethylstilbestrol, and its analogue, indenestrol B, on the relative plating efficiency and cellular microtubular architecture of Chinese hamster V79 cells. In this study, the effects of the monomethyl ethers of indenestrols A and B on these biological activities and their affinity for estrogen receptors in cytosol from mouse uteri were investigated. The results indicate that among eight optically active and four racemic methyl ethers, the 4'-methyl ether of [(-)-3S]indenestrol B exhibits both the strongest cytotoxicity in, and greatest disruption of, the cellular microtubular architecture of Chinese hamster V79 cells in culture. For the 6- and 4'-monomethyl ethers of optically active indenestrols A and B, some correlation was found between cytotoxicity and the effect on the distribution of cellular microtubular networks in Chinese hamster V79 cells. Estrogen receptor competitive binding studies revealed that stereochemistry and the position (6 or 4') of the methyl ether group contributed greatly to their binding affinity. However, no correlation was observed between the affinity for estrogen receptors and the cytotoxicity of the monomethyl ester tested. This suggests that the important causes of cytotoxicity in this series of compounds involve the inhibitory activity on cellular microtubule networks and not the affinity for estrogen receptors. PMID- 7550114 TI - Biosynthesis and processing of lysosomal cathepsin D in primary cultures of rat hepatocytes. AB - To investigate the intracellular transport and maturation of lysosomal cathepsin D, we carried out an in vivo pulse-chase analysis with [35S]methionine in the primary cultures of rat hepatocytes. Cathepsin D was initially synthesized as a proenzyme of 45 kDa. The proenzyme was subsequently processed, becoming a mature enzyme of 43 kDa. The proenzyme and mature enzyme showed complete susceptibility to endoglycosidase H treatment, suggesting the presence of high-mannose type oligosaccharide chains. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, the 42.5-kDa unglycosylated precursor polypeptide appeared in the cell, and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results support the notion that the oligosaccharide moieties are of importance in addressing the lysosomal hydrolases to the lysosomes. However, in the presence of chloroquine, proteolytic processing of the proenzyme was prevented, and the enhanced release of proenzyme from the cells was observed. These results indicate that the processing of proenzyme to mature enzyme would take place in the lysosomes. PMID- 7550115 TI - Biochemical properties and intracellular processing of lysosomal cathepsins B and H. AB - Lysosomal cysteine proteinases of cathepsins B and H were isolated to a homogeneous state from rat liver by employing Sephadex G-75, DEAE-Sephacel, CM Sephadex, and Mono S column chromatography. Each of the purified cathepsins B and H was demonstrated to be composed of a mixture of a single-chain form and the processed two-chain form upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To investigate the proteolytic maturation of lysosomal cathepsins B and H, turnover kinetics of these enzymes were studied by comparing the specific radioactivities of the incorporated [3H]leucine into either the single-chain form or two-chain form in vivo. The specific radioactivity derived from each protein band of lysosomal cathepsin H in SDS-PAGE at 1, 3, 6, 12, 24 and 48 h after the injection of a radiolabel showed that the peak of specific radioactivity of the single-chain form of cathepsin H appeared at 6 h and that after 6 h, the radiolabel was sequentially incorporated into the two-chain form, while the radiolabel in the single-chain form started to gradually decrease, suggesting that the single-chain form was processed to generate the mature enzyme after the enzyme was incorporated into the lysosomes. In contrast, in the case of cathepsin B, the appearance of a radiolabel in the single-chain form or in the two-chain form was observed almost concomitantly without time lag, indicating that the processing of cathepsin B occurred very rapidly in the lysosomes. PMID- 7550116 TI - Purification and characterization of PK-120, a novel substrate for plasma kallikrein, from guinea pig plasma. AB - PK-120 is a novel substrate of plasma kallikrein isolated from human plasma. Western blotting using anti-human PK-120 polyclonal antibodies demonstrated the presence of a PK-120-like protein in guinea pig, rat and mouse plasma. We purified the immunoreactive-120 kDa protein from guinea pig plasma by polyethylene glycol fractionation followed by ion-exchange chromatography using Q Sepharose, heparin-Sepharose, Mono-Q and gel filtration with TSK G3000. The 120 kDa protein thus isolated was similar to human PK-120 with respect to limited proteolysis by kallikrein, amino acid composition, and the N-terminal amino acid sequence, indicating that the immunoreactive 120 kDa protein was guinea pig PK 120. PMID- 7550117 TI - The effect of ethanol and diazepam on the fatty acid composition of plasma and liver phospholipids in the rat. AB - The main interest of the present study was to determine possible alterations in fatty acid composition in rat plasma and liver phospholipids (PL) caused by chronic ethanol consumption, diazepam treatment and chronic consumption of alcohol and diazepam together. Chronic ethanol consumption (11 g/kg/d) elevated the proportion of plasma saturated (SFA) and mono-unsaturated fatty acids (MUFA), and decreased the most important polyunsaturated fatty acids (PUFA) in plasma and liver PL. The alterations in fatty acid composition in liver PL indicate that ethanol can change the composition of cell membrane lipids. Diazepam treatment (10 mg/kg/d) elevated the contents of SFA and MUFA in plasma PL. On the other hand, diazepam produced a drastic decrease in 22:6n-3 docosahexaenoic acid (DHA) in plasma and liver PL. These changes in plasma PL fatty acid composition indicated a disturbance of fatty acid metabolism. The changes in fatty acid contents of plasma and liver PL were the greatest in rats treated with a combination of ethanol and diazepam, in which there was a summation of the effects of ethanol or diazepam alone, and the effects were intensified by an ethanol-diazepam interaction. PMID- 7550118 TI - A polysaccharide fraction of shosaiko-to active in augmentation of natural killer activity by oral administration. AB - Shosaiko-to (Xiao-chai-hu-tang, SHO), which is a Kampo medicine prepared by decocting a prescription of 7 kinds of medical plants, has been used mainly to treat chronic hepatitis in Japan. Previously, we reported that an oral administration of SHO augmented natural killer (NK) activity in the peripheral blood. To characterize its active substance, SHO was fractionated. The high molecular weight fraction showed the ability to augment NK activity by oral administration, but the low molecular weight fraction did not. Furthermore, we obtained an active acidic polysaccharide from the high molecular weight fraction. This polysaccharide fraction, with a molecular weight of approximately 1.2 x 10(5), is probably responsible for the effect of the original Shosaiko-to. It contained no protein. The sugar moiety was composed of rhamnose, arabinose, mannose, galactose, glucose and galacturonic acid in molar ratios of 1:17:3:21:100:87. PMID- 7550119 TI - Mechanism of anti-urease action by the anti-ulcer drug ecabet sodium. AB - To investigate the mechanism of the anti-urease action of ecabet sodium (ecabet) observed in Helicobacter pylori in vitro, the effects of ecabet on purified urease from jack bean were studied in comparison with the effects of the specific urease inhibitor benzohydroxamic acid (BHA). After incubation of the enzyme with the test drug for a period of time, urease activity was measured. Ecabet depressed the activity below pH 5, and the lower the pH, the greater the degree of depression. The degree of depression by ecabet increased gradually during incubation and reached a plateau in 20 min, whereas that by BHA attained a maximum rapidly. The IC50 values of ecabet and BHA were 2.1 mg/ml and 0.5 microgram/ml, respectively. When the incubation mixture of urease with an inhibitor was diluted and further incubated, the depressed activity by BHA reverted gradually, but that by ecabet did not. When the incubation mixture of urease with ecabet was centrifuged, the urease activity of the mixture decreased in parallel with the reduction in protein concentration of the supernatant. When the incubation mixture of urease and 14C-ecabet was ultrafiltered to remove the drug, the radioactivity in the retentate remained in parallel with the degree of reduction of urease activity in the retentate. These results indicate that ecabet irreversibly depresses the urease activity of jack bean, and suggest that the depression is caused by irreversible binding of ecabet to urease followed by denaturation of the enzyme protein. PMID- 7550120 TI - Oral tolerance to ovalbumin in mice as a model for detecting modulators of the immunologic tolerance to a specific antigen. AB - Oral tolerance is thought to have a role in preventing allergic responses and immune-mediated diseases. Modulation of this tolerance by drugs and chemicals can cause or suppress them. An improved model of oral tolerance to ovalbumin (OVA) in mice was developed to detect modulators of the tolerance and to apply it to selected immunomodulating substances, cyclophosphamide (CP), Escherichia coli lipopolysaccharide (LPS) and cadmium chloride (Cd). Male C3H/HeN mice given an oral administration of 20 mg OVA were immunized 7 d later with an i.p. injection of 0.1 mg OVA in complete Freund's adjuvant. Effects of oral OVA and agents on systemic immunity were assessed by enzyme-linked immunosorbent assay (ELISA) of immunoglobulin (Ig) levels in serum collected 7 or 14 d after immunization. Oral tolerance was adequately induced on day 7 after immunization and was more effective in C3H/HeN mice than in BALB/c mice. It was primarily associated with the decreased serum levels of anti-OVA IgG (including both IgG1 and IgG2a subclasses regulated differently by T-helper subpopulations, Th2 and Th1 cells, respectively). The C3H model of oral tolerance was further examined to detect modulators of the tolerance. An i.p. injection of CP prior to oral OVA, or 5 consecutive daily oral administrations of LPS after oral OVA elevated or reduced serum levels of anti-OVA IgG in C3H mice hyposensitized by the oral OVA, respectively. Concerning IgG subclasses, CP restored anti-OVA IgG2a but not IgG1 levels, while LPS caused greater suppression of both anti-OVA IgG1 and IgG2a levels. Oral administrations of Cd for 5 d after oral OVA also suppressed anti OVA IgG1 levels further.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550121 TI - Interaction among secretagogues on pepsinogen secretion from rat gastric chief cells. AB - We examined the interaction among secretagogues that stimulate pepsinogen secretion through different pathways in vivo and in vitro. In in vitro study, a combined administration of secretin and carbachol or cholecystokinin octapeptide (CCK-8) to the culture medium of chief cells potentiated pepsinogen secretion. Moreover, the response induced by carbachol or CCK-8 with forskolin was greater than that with secretin. We examined the interaction among receptor-related second mediators, and found that carbachol- or CCK-8-induced intracellular Ca2+ concentration ([Ca2+]i) increase was not affected by secretin or forskolin. Both these substances, however, significantly reduced secretin-induced cAMP production. On the contrary, CCK-8 significantly increased forskolin-induced cAMP production, while carbachol increased it slightly. Calcium ionophore, A23187, or protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), did not alter secretin- or forskolin-induced cellular cAMP production; and the reductive effect of carbachol or CCK-8 on secretin-induced cAMP production was restored by their competitive antagonists, atropine or lorglumide. EC50 of those antagonists was almost the same value as IC50 on pepsinogen secretion and [Ca2+]i increase. These results indicate that secretin-induced cAMP production is interfered with by receptor related agonists like CCK-8 and carbachol. It may be suggested that there is a kind of "cross-talk," between the adenylate cyclase system, that is, the secretin receptor, and carbachol or CCK-8 receptor. The interactions between secretin and other secretagogues (carbachol, CCK-8, tetragastrin and histamine) were examined using the perfused rat stomach.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550123 TI - Metabolism of 4-aminobiphenyl and 4-acetylaminobiphenyl in perfused guinea pig liver. AB - To obtain further knowledge of the metabolic fate of carcinogenic 4-aminobiphenyl (ABP) and 4-acetyl-aminobiphenyl (AABP) in intact guinea pig liver, we performed in situ liver perfusion using a recirculation method. Following a biexponential disappearance of ABP from the perfusate, not only AABP but also its secondary metabolites 4'-hydroxy AABP (4'-OH AABP) and 4-glycolylaminobiphenyl (GABP) appeared as the major metabolites. 4'-Hydroxy ABP (4'-OH ABP) was also detected in the perfusate as another predominant metabolite, while N-hydroxy ABP (N-OH ABP) as a minor one. Most of these metabolites, except for AABP and GABP, found in both perfusate and bile were conjugated with glucuronic acid. In addition, considerable amounts of all the metabolites were also detected in either unconjugated or conjugated form in a homogenate of the liver tissue after perfusion. When AABP was infused, an almost similar metabolic profile to that of ABP was obtained, but the amount of ABP was quite small and N-OH AABP rather than N-OH ABP was found. These results demonstrate that both ABP and AABP are rapidly metabolized by a combination of N-acetylation, C- and N-hydroxylations as well as glucuronidation in guinea pig liver. PMID- 7550124 TI - Effects of roxithromycin on proliferation of peripheral blood mononuclear cells and production of lipopolysaccharide-induced cytokines. AB - Roxithromycin (RXM), a new macrolide antibiotic, has a 14-member macrocycline ring structure which is similar to that of erythromycin. We investigated the effects of RXM on the proliferation of peripheral blood mononuclear cells (PBMCs) and the production of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) by PBMCs stimulated with lipopolysaccharide (LPS). At concentrations greater than 25.0 micrograms/ml, RXM suppressed the proliferation of PBMCs stimulated with phytohemagglutinin, probably due to cytotoxicity. When the PBMCs were incubated with RXM for 7 d, the number of adherent cells (monocyte/macrophages) increased. Incubation with RXM at a concentration of 25.0 micrograms/ml induced the greatest increase (p < 0.05). IL-1 beta and TNF-alpha were present 3 h after LPS-stimulation, and IL-1 beta production reached a peak at 12 h and TNF-alpha production at between 6 and 12 h, and then their production declined. RXM (25 micrograms/ml) suppressed the production of IL-1 beta and TNF alpha slightly during the entire course of the incubation. This suppression was dose-dependent. Anti-human granulocyte-macrophage colony-stimulating factor and anti-human macrophage colony stimulating factor antibodies had no effect on the RXM-induced proliferation of adherent cells. Suppression of the production of IL 1 beta and TNF-alpha by RXM suggested that this drug might have anti-inflammatory and immunosuppressive effects. PMID- 7550125 TI - A new pharmacokinetic model including in vivo dissolution and gastrointestinal transit parameters. AB - When dissolution is the rate-limiting step in absorption of a drug, both it and gastrointestinal transit can play dominant roles in determining absorption. Therefore, a pharmacokinetic model including the process of dissolution, gastrointestinal transit rate and gastrointestinal pH profile was proposed to explain the behavior of drug formulations in vivo. Clarithromycin (CAM), which exhibits pH-dependent solubility and has high partition coefficients, was used as a drug to test the model, and the effect of a meal on serum concentrations of CAM after a single oral administration to healthy volunteers was evaluated. The results of pharmacokinetic analyses of serum concentration-time data for a single oral administration of CAM to humans in the fasting and postprandial states were well fitted by the new pharmacokinetic model. By substituting the pharmacokinetic parameters obtained into the model equations, the in vivo rate of dissolution could be determined. Pharmacokinetic parameters affecting drug bioavailability were also examined using the model. Factors influencing the behavior of a drug formulation in the gastrointestinal tract were determined. These findings indicate that the model we have proposed can be used to evaluate the behavior of drug formulations in vivo. PMID- 7550122 TI - Oxidative modification of low density lipoprotein by diesel exhaust particles. AB - Oxidized low density lipoprotein (LDL) has a variety of hazardous influences on biological systems. Oxidative modification of LDL by diesel exhaust particles (DEP) was studied in vitro to assess its in vivo health effects. DEP suspensions (1, 10 and 100 micrograms/ml) were incubated for 1 h with LDL (1 mg protein/ml) at 37 degrees C. Conjugated diene formation and negative charges in LDL were increased by DEP-treatment in a dose-dependent manner. When native LDL and DEP treated LDL (DEP-LDL) were incubated for 18 h with macrophage, J774A.1 cell at 37 degrees C, significantly more DEP-LDL was taken up into cells than native LDL. Accumulation of cholesterol ester in cells incubated with DEP-LDL was 4 to 8 times higher than that with native LDL while there was no significant difference between them in free cholesterol content. Incubation (18 h) of J774A.1 with DEP LDL caused an increase in leakage of lactate dehydrogenase from cells in a DEP concentration dependent manner, but the incubation with native LDL or DEP per se did not increase the leakage except at the highest concentration of DEP. These results suggest that DEP oxidatively modified LDL giving it cytotoxic, inflammatory and atherogenic properties characteristic of so-called oxidized LDL; these initial modifications of LDL may be one of the underlying mechanisms for diseases associated with DEP. PMID- 7550126 TI - Morphologic examination of rabbit nasal mucosa after the nasal administration of insulin peanut oil suspension and a powder dosage form with soybean-derived sterylglucoside. AB - Soybean-derived sterylglucoside mixture (SG) is a potentially effective absorption enhancer for the nasal absorption of insulin. Insulin is a peanut oil suspension dosage form and in a powder dosage form with SG were administered into the rabbit nasal cavity. After 0.5 or 1 h, nasal mucosa was taken from the nasal cavity and side effects were examined by an optiphoto light microscope. The insulin in the peanut oil suspension produced a histological change in the nasal epithelium mucosae, as well as mucodermal phlogistic cellular infiltration. The peanut oil suspension with SG showed side effects slightly stronger than without SG. SG alone and the insulin powder dosage form with SG produced no signs of inflammation, erosion or squamous metaplasia. The results from this study indicate that SG can be considered safe. PMID- 7550127 TI - Susceptibility of insulin to proteolysis in rat lung homogenate and its protection from proteolysis by various protease inhibitors. AB - The objective of this study was to determine the stability of insulin in rat lung homogenate and to confirm the effectiveness of various protease inhibitors for insulin delivery from the lung. The stability of insulin in rat lung homogenate was compared with that in the small intestine. Insulin was rapidly degraded in lung homogenate similarly to degradation in the small intestinal homogenate. The effects of various protease inhibitors on the degradation of insulin were studied in the lung. Protease inhibitors such as Na-glycocholate (Na-GC; 10 mM), aprotinin (10 mg/ml), soybean trypsin inhibitor (STI; 10 mg/ml) and bacitracin (20 mM) effectively reduced insulin degradation in lung homogenate. The rank order of effectiveness for decreasing the insulin hydrolysis in lung homogenate was bacitracin > aprotinin > STI > Na-GC. In addition, a slight correlation was observed between the in situ pulmonary absorption of insulin and its stability in vitro in the lung homogenate in the presence of various protease inhibitors. These findings suggest that the coadministration of protease inhibitors would be useful for improving the stability of insulin in the lung. PMID- 7550129 TI - Enzyme immunoassay of elcatonin in human plasma. AB - A sensitive and specific double-antibody enzyme immunoassay (EIA) for detecting an elcatonin-like immunoreactive substance (ECT-IS) in human plasma has been developed. In competitive reactions, the ECT antibody was incubated with a plasma sample (or ECT standard) and beta-D-galactosidase-linked synthetic ECT. Free and antibody-bound enzymes were separated using an anti-rabbit IgG-coated immunoplate. Enzyme activity on the plate was determined by fluorescence analysis. This immunoassay allows the detection of 20 to 300 fmol/ml (67 to 1000 pg/ml) of ECT. The EIA was applied to determine the pharmacokinetic behavior of ECT after a single intramuscular administration (20 IU). The maximum level was achieved 30 min after administration, at approximately 30 pg ECT/ml of plasma. PMID- 7550128 TI - Studies on the antiviral activity of protein kinase inhibitors against the replication of vesicular stomatitis virus. AB - Several derivatives of K-252a, a protein kinase inhibitor isolated from Nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (VSV) in BHK-21 cell cultures. Among those we tested, KT5926, which preferentially inhibits the myosin light chain kinase (MLCK), suppressed the viral replication by 95-99% at 15 microM. K-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. Other derivatives, KT5720 and KT5823, that are known to inhibit the cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, did not suppress VSV replication even at a high concentration as 15 microM. None of these inhibitors affected the Sindbis virus replication in BHK-21 cells under similar assay conditions as used for VSV. KT5926 and K-252a seemed to affect the VSV replication at the step(s) after the viral invasion, resulting in decreased viral RNA synthesis. Neither substance inhibited cellular casein kinase (CK) II which is known to be involved in phosphorylation of the nonstructural (NS) protein, a non-catalytic subunit of the viral RNA polymerase. These results suggest that the inhibition of VSV replication by KT5926 and K-252a is not a secondary effect due to generalized suppression of host cell activities, and that the VSV replication requires the KT5926-sensitive function(s) in the cell which would be performed by an enzyme(s) other than CK II. PMID- 7550130 TI - Possible involvement of peripheral-type benzodiazepine receptors in erythroid differentiation of human leukemia cell line, K562. AB - Possible involvement of the peripheral-type benzodiazepine receptor (PBR) in hemin/protoporphyrin-induced erythroid differentiation of human leukemia K562 cells was investigated by the use of the ligands, diazepam and PK11195. Diazepam itself exhibited differentiation-inducing activity on K562 cells. The PBR specific antagonist, PK11195, dose-dependently inhibited both diazepam-induced and hemin/protoporphyrin-induced K562 cell differentiation. The results imply that PBR is involved in the erythroid differentiation of K562 cells. PMID- 7550131 TI - Na,K-ATPase receptor subunits alpha 1, alpha 2 and alpha 3 mRNA in dilated cardiomyopathy. AB - Na,K-ATPase receptor density has been shown to be down-regulated with decreasing ejection fraction in patients with chronic heart failure. It was the aim of the present study to determine whether down-regulation is detected also at the mRNA level. Six donor hearts and six explanted hearts due to dilated cardiomyopathy (ejection fraction 23 +/- 5%) were analyzed. RNA was extracted. Quantitative Na,K ATPase receptor catalytic subunit alpha 1, alpha 2 and alpha 3 mRNA expression was determined by solution hybridization. No cross-reactivity occurred between the three probes. alpha 1 mRNA was expressed at about 5 and 10 times higher (p < 0.001) concentrations than alpha 2 and alpha 3 mRNA, respectively, and alpha 2 mRNA higher (p < 0.001) than alpha 3. There were no differences between right and left ventricles and between donor hearts and patients with dilated cardiomyopathy. In conclusion, Na,K-ATPase alpha 1 mRNA is the predominant subunit expressed in human myocardium. Depressed ejection fraction in dilated cardiomyopathy is not associated with changed mRNA subunit expression. Documented downregulation of Na,K-ATPase activity, therefore, may be associated with the structural and membrane-related beta subunit or posttranscriptional modification of the catalytic subunits. PMID- 7550132 TI - Inhibitory effect of alkaloids extracted from the stem bark of Hunteria zeylanica on 5-lipoxygenase activity in vitro. AB - The effects of alkaloid extract from the stem bark of Hunteria zeylanica Gard. (H. zeylanica) on the activities of cyclooxygenase and 5-lipoxygenase in A23187 stimulated rat mast cells were investigated. H. zeylanica alkaloid extract (0.3 300 micrograms/ml) inhibited leukotriene C4 (LTC4) production by 5-lipoxygenase in a concentration-dependent manner and it blocked the production by 50% at 300 micrograms/ml. On the other hand, the extract had no effect on prostaglandin D2 (PGD2) production by cyclooxygenase. Neither (-)-eburnamine nor pleiomutinine, major constituents of H. zeylanica alkaloid extract, inhibited the production of PGD2 and LTC4 in the A23187-stimulated mast cells. The inhibition of arachidonic acid metabolism via 5-lipoxygenase pathway may be due to minute amounts of other components as stated in the Discussion. PMID- 7550134 TI - Increase of plasma sialic acid upon UV-B irradiation in mouse. AB - In many inflammatory conditions including burns, the plasma sialic acid level rises as an acute responder. Sunburn is a kind of burn. In this study, sunburn was experimentally caused in mice by UV-B irradiation and their plasma sialic acid levels were measured. The levels increased, and reached the maximum 3 d after irradiation. This level was maintained for about 2 d, then it returned to normal within about one week. The increase in the level correlated with the UV dose and the severity of inflammation resulting in sunburn. This assay system was applied to assess the virtue of UV-cut cream in experimental sunburn. PMID- 7550133 TI - Resistance of hepatic lysosomes, mitochondria and microsomes of protoporphyrin administered rats to peroxidative damage. AB - Antioxidative inhibition by protoporphyrin (PP) of peroxidative damage in lysosomes, mitochondria and microsomes of rat liver was investigated at 24 h after an intravenous administration of PP. Using a lysosome-containing (3500 x g) fraction, the release of lysosomal marker enzymes, acid phosphatase and aryl sulfatase, from lysosome which had been stimulated by L-ascorbic acid (AsA), was decreased dose-dependently, as was the inhibition of lipid peroxidation by PP in the fraction. Swelling of mitochondria induced by Fe2+ and AsA was also inhibited in the PP-injected rat. In microsomes, lipid peroxidation stimulated by AsA caused a decrease in activity of a microsomal marker enzyme, glucose 6 phosphatase, and in P450 content. The extent of the decrease by AsA, both in activity and content, was diminished in PP-administered rat liver microsomes. These results indicate that PP protects those subcellular fractions from deterioration by lipid peroxidation. PMID- 7550135 TI - Novel assay system favorable for the study of cell-to-cell transmission of HIV-1 and its application to the evaluation of anti-HIV drugs. AB - The cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was studied using MOLT-4 cells chronically infected with a variant strain of HIV-1SF 2 (MOLT-4/HIV-1SF-2H) and CD4+ human lymphoid MT-4 cells. MOLT-4/HIV-1SF-2H cells produced less than 1 TCID50 infectious particles per day as determined by the cytopathogenicity in MT-4 cells. However, the expression of envelope glycoproteins gp120 and gp41 on the MOLT-4/HIV-1SF-2H cell membrane was satisfactory for syncytium formation with the uninfected MOLT-4 cells. When MOLT 4/HIV-1SF-2H and MT-4 cells were co-cultured, severe cytopathogenicity was observed in MT-4 cells without being accompanied by the formation of multi nucleated cells. Thus, the system consisting of MOLT-4/HIV-1SF-2H and MT-4 cells is convenient for exclusive study of the mechanism of cell-to-cell transmission of HIV-1. Using various compounds, it was confirmed that cell-to-cell transmission required both gp120/gp41-CD4 binding and de novo DNA synthesis. PMID- 7550138 TI - Metabolism of peptide drugs by the microorganisms in rat cecal contents. AB - The metabolism of insulin and calcitonin by microorganisms was examined in rat cecal contents. Both insulin and calcitonin were markedly degraded. Calcitonin was more susceptible to proteolysis in rat cecal contents than insulin. Calcitonin was also rapidly degraded in supernatant, while we found few degradation products of insulin. These findings suggest that care should be taken to metabolize the peptide drugs by microorganisms when they are administered to the large intestine for colon-specific drug delivery. PMID- 7550140 TI - [The biological effects of heavy charged particles. The main results and prospective research in the context of interplanetary flights]. AB - In experiments with biological objects varying in levels of their organization, comprising protozoa and cells and total bodies of large laboratory animals, the relative biological effectiveness (RBE) of accelerated charged particles in a wide range of doses and LET, i.e. high energy protons, heavy ions with energies up to 10 MeV/nucleon, charged particles of high and relativistic energies simulating individual components of GCR and SCR has been estimated. Early and delayed radiation effects were also studied. RBEs of protons in the energy interval of 50-660 MeV and standard radiation were shown to be equal, whereas RBE factors of heavier particles are a function of LET and many other physical and biological agents. Discussed are the specific properties of biological agents of heavy charged particles and the prospects for investigations in view of long-term interplanetary missions. PMID- 7550139 TI - [Long-term G loads (+Gz) and the prediction of their tolerance]. AB - The impacts of comparatively low (1.5-3 G) though prolonged (up to 40 min) "head pelvis" (+Gz) accelerations at the final and most challenging stage of space flight, i.e. the approach to landing site, are numbered among the extreme factors discomforting the flights on board advanced spacecraft. Specificity of the effects of prolonged g-loads and the ways to predict their tolerance are still little known. 52 flyers aged 24-42 were centrifuged at 3.0 g for 15 min with rise gradient 0.2 g/s. 23.3% of test-subjects developed a set of deadaptive disorders including a critical decrease of arterial pressure in the ear lobe leading to precollapsing state (9.6%), stable relative bradycardia with concurrent manifestations of autonomous nervous reactions (5.8%), apparent (up to 170 beats/min) sine tachycardia (7.9%). This syndrome was generally exhibited by subjects with autonomous-vascular instability. All members of the group of healthy subjects showed good tolerance of prolonged (15 min) accelerations. In addition to centrifuge runs, 52 subjects were exposed to the lower body negative pressure test (-50 mm Hg, up to 15 min) in the sitting position. Similar to g load test, the worst decompression tolerance was observed in flyers with autonomous-vascular instability. The mean tolerance time in these subjects made 7.9 +/- 0.7 min, while in healthy subject it amounted to 14.0 +/- 0.6 min (p < 0.01). The most convincing correlation between decompression tolerance and prolonged g-loads was established at 10 min of decompression (Pearson factor Ra = 0.51).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550136 TI - Bioactivity of nickel(II) complex containing N-glycosides derived from D glucosamine and ethylenediamine against pathogenic yeast, Candida albicans. AB - A nickel(II) complex containing N-glycosides derived from the reaction of D glucosamine (D-GlcN) and ethylenediamine (en) [Ni(D-GlcN-en)2]Cl2.H20 showed effective antifungal activity against pathogenic yeast, Candida albicans, where the MIC (minimal concentration of inhibition) value of the complex is 0.25 mM. PMID- 7550137 TI - 9-Hydroxymethyl-10-carbamoylacridan in human serum is one of the major metabolites of carbamazepine. AB - In sera of 29 patients under the chronic administration of carbamazepine (CBZ, dose: 10.9 +/- 4.3 mg/kg/day), a ring-contracted metabolite of CBZ was detected at a relatively higher concentration of 0.57 +/- 0.30 mu g/ml (mean +/- S.D.) by high-performance liquid chromatogaphic assay. The structure of the metabolite was identified as 9-hydroxymethyl-10-carbamoylacridan (HMCA) by the liquid chromatography-mass spectral examination, and also by comparison with the authentic sample. Although HMCA was already reported in a few papers as a minor metabolite of CBZ in human urine, any reliable report for the determination of HMCA in human blood has never been published. Our results show that HMCA is one of the major metabolites of CBZ. PMID- 7550141 TI - [The concentration of methane and trace organic admixtures in the air expired by submariners during a dive simulation in a hyperbaric chamber]. AB - The article describes the procedure of determining methane and some organic trace elements in expired air during simulated "dive" in a hyperbaric chamber. Partial pressures of methane breathed out by divers in 29 experiments are reported. Concentrations of 8 trace organic components have been measured in the expired air of two divers. The results obtained show that expired methane increases by 3 10 times during simulated descent down to 80-115 m. PMID- 7550142 TI - [The effect of hyperbarism on the electrical stability and aggregation ability of liposomal membranes]. AB - Helium pressure (100 bar) increases the electric breakdown potential of lipid bilayer by 4-5 mV and inhibits liposomal aggregation. These effects may result from the gain in surface tension of lipid/water interface, and the change in effective shape of lipid molecules. The studied effects may extenuate pressure effects on the synaptic junction. PMID- 7550143 TI - [New methodological approaches in assessing adaptive body reactions in the system of flight medicine expertise]. AB - The "cost" of adjustable systemic and cellular reactions in the human body in extreme environments have been assessed from the standpoint of their potentialities for upgrading special methods of evaluating and predicting functional status practiced in flight certification examination. As was stated, the functional reserves of blood circulation should be assessed by chronotropic activity of cardiac regulation in response to hypoxic exposure. The adequacy of adaptive processes on the cellular level can be drawn from the dynamics of malon dialdehide (the by-product of lipid peroxidation) and plasma monoaminoxidase. The biochemical investigations of peripheral blood and saliva also show promise. PMID- 7550144 TI - [The organizational and methodological aspects of flight medicine expertise in cardiovascular diseases]. AB - Among diseases causing pilot disqualification ahead of time the cardiovascular disorders amount to 60 percent. In this connection there has been generalized the experience of examining the Armed Forces Aviation pilots with cardiovascular disorders beginning from 1979 when by the order of the Ministry of Defence N 220 the Regulations of Aeromedical Examination of pilots have been come into force. The features of examining the pilots with cardiovascular disorders are presented. The present-day methods for the instrumental examination which have been tested in cardiology are systematized in an effort to use them during aeromedical examinations of the pilots. The algorithm of the examined pilots with cardiovascular disorders is set. For the first time the three main groups of approaches to the appraisal of partial or complete loss of the work capacity with known result of diseases. The proposed organization-and-methodologic approach to medical examination of the flying personnel with consideration for the features of stage diagnostics of cardiovascular disorders, individual assessment of their manifestations enables one to handle more qualitatively the aeromedical examination issues with the aim of maintaining the professional working capacity. PMID- 7550145 TI - [The current approaches to flight medicine expertise on persons with dissociated atrioventricular conduction]. AB - The expert criteria with regard to the pilots with dissociation of atrioventricular conduction in the context of modern cardiological advancements are subject to the considerable improvement. It is demonstrated that the disqualification decision taken with regard to the individuals with the anomalies of conduction system depends not on the type of flying activities but on the paroxysmal supraventricular tachyarrhythmia. The required volume of examination which must include the modern cardiological methods of the noninvasive cardiologic study (ECG monitoring, echocardiography, transesophageal electrophysiological examination). The appropriate changes and improvements included in the Pilot Examination Regulation would take the valid expert decisions in intent to increase the professional longevity of the pilots. PMID- 7550147 TI - [The 60th anniversary of the Institute of Aviation and Space Medicine]. PMID- 7550146 TI - [The nature of the changes in the indices of intracerebral blood circulation in flight personnel performing functional tests]. AB - The state of intracerebral hemocirculation in the pilots during expert examinations to determine the tolerance for special exercise tests was studied. The cerebral circulation reactions were evaluated with the use of ultrasonic transcranial Doppler vasography. It is found that exercise tests depending on an intensity of exposure and directionality result in different degree of changes in the cerebral circulation parameters. In an effect of the flight-surgeon's appraisal the use of transcranial Doppler vasography during LBNP test to identify the persons susceptible to the syncopal states is advisable. PMID- 7550148 TI - [The work experience of the Central Military Aviation Research Hospital in the occupational rehabilitation of flight personnel after lithotripsy]. AB - The evolution of the domestic-certified lithotriptors has made a complete professional rehabilitation of all pilots (100%) suffered from urolithiasis without operative intervention possible. Of 156 pilots who have been exposed to remote shock-wave lithotripsy (RSWL) in recent three years 120 individuals (93%) had the urate stones in the renal calyces. Before introducing the RSWL in clinical practice such concrement were recognized as inoperable and the urolithic pilots have no prospects for continuation of their flying activities. After the RSWL application there was a decrease in the secretory-excretory renal function due to the disturbed urodynamics by the migratory fragments of crushed stones in the renal system. The recovery of renal function occurred after complete passage of the crushed stone fragments. Depending on the composition of a crushed concrement, severity of inflammatory process taking place in the kidneys and urinary tracts, the special program of countermeasures to prevent relapsed lithogenesis was recommended for use by the patients. The relapse of renal stone formation after RSWL application was noted in 4 (3.2%) pilots. In these pilots, the concrements were recrushed by the mentioned method and professional qualification of above individuals was retained. Until recently, the treatment of cholelithic disease was recognized as a purely surgical problem--with the introduction of RSWL of renal stone into clinical practice there appeared real possibility to apply the similar approach to crushing the gallstones. To attain these ends, the experimental studies with the use of lithotriptor "URAT-P" have been performed. 140 gallstones of 0.5 to 2.0 cm in size were crushed. The parameters of shock waves are determined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550149 TI - [The current aspects of the computed tomographic and clinical diagnosis of degenerative changes in the spine of flight personnel]. AB - The paper deals with the modern aspects of the computed tomographic and clinical diagnostics of the degenerative changes in the vertebral column of the pilots. There has been elaborated the classification of degenerative changes with consideration of the present-day requirements of clinical diagnostics and medical examination. The more frequently seen damages of intervertebral disks in the pilots in the form of protrusions and prolapses are indicated, the computed tomographic and clinical characteristics are compared. PMID- 7550150 TI - [Current problems in the diagnosis, treatment and prevention of cholelithiasis in flight personnel]. AB - In 50% of afflicted pilots, cholelithiasis is asymptotic and develops with the minimum anatomicomorphological changes of the biliary system. An early diagnostics of cholelithiasis in the pilots will enable one to treat them timely with the aim of returning the pilots to flying activity in the future. The introduction of nonsurgical treatment methods into the clinical practice with an application of distance shock-wave cholelithotripsy will elevate the percentage of pilots returning to flying activities. A need for developing domestic special devices for cholelithotripsy is ripe. PMID- 7550151 TI - [The characteristics or urolithiasis in flight personnel and the treatment results using a method of extracorporeal shockwave lithotripsy on the Russian lithotripter Urat-P2]. AB - Urolithiasis occurs more frequently in the pilots than in the ground-service personnel. To decrease a damaging effect of the focused shock waves on the renal tissue while using remote lithotripsy technique, a new domestic-certified lithotriptor "URAT-P2" which shows a significantly low energy of shock wave with sufficient effectiveness of crushing the renal stones, is devised. Urolithiasis in the pilots is apparent at the early stages of its development and its complicated forms are less common than in the subjects of other professions. The stones are predominantly located in the renal calyces (73,3% of cases), in the left kidney and urethra in 63,3% of cases and there were urate stones in 40% of cases. The clinical use of the URAT-P2 complex in 30 pilots indicated that it holds advantages over the production domestic-certified lithotriptor. The stones are crushed in all the patients but the complications associated with a negative effect of the focused shock waves on the renal tissue are absent. The use of the new lithotriptor for treating urolithiasis in the pilots will allow one to improve the treatment response and to maintain professional worthiness of the pilots for a long time. PMID- 7550152 TI - [The effect of chronic diseases on the future duration of flight operations for pilots and navigators]. AB - The analysis of the physical fitness disqualification of 1433 pilots and navigators indicates that the coming time of flying activities (CTFA) is a convenient index for predicting the flight longevity. In some cases the CTFA differs mainly due to a different disqualification rate during a year after identification of a disease. In individuals pronounced fit for flying activity the CTFA depends in the main on the age but differs little in some diseases. PMID- 7550153 TI - [The Jupiter-2 slow-rotation system]. AB - The experience of space missions shows that functional disorders in crewmembers on the type of space motion sickness (SMS) may develop on the initial stage of flight. Longer exposure in micro-g causes a wide range of debilitative changes in the vital body systems. Artificial gravity produced by spacecraft rotation might be a universal tool to counteract the impacts of prolonged microgravity on the human body. However, the significance of SMS does not become less high because of a new factor, i.e. the rotating environment. Research system Jupiter 2 is a stand alone slow-rotating ground facility for simulating motion sickness equivalent to its space form. The merits of this facility are the possibilities to control the intensity of exposure, perform long-term investigations of two active subjects simultaneously, and study the stages of body adaptation to this agent, and assess physical and operator's performance. The facility carries large expectations to occupational selection. PMID- 7550155 TI - [The reactions to weightlessness exposure: adaptation or disease?]. AB - The arguments characterizing the poor theoretical validity and low profitability of the concept of "weightlessness disease" developed during recent few years by its authors are advanced. The morphofunctional rearrangements occurred in the body during space flights are considered as the result of adaptation disadaptation rearrangements directed towards supporting the equilibrium with a set of reduced environmental requirements. Understanding the mechanisms of adaptation rearrangements creates the real but not forced basis for developing the measures for cosmonauts health stabilization and spaceflight safety. PMID- 7550154 TI - [The perception of direction during counterrotation of the head and trunk]. AB - Investigations of perception of the "straight forward" direction and a visual target have been carried out with the use of a unique test facility providing negative feedback between horizontal rotations of the head and the chair. Administration of tests with visible or screened hand demonstrated a deviation of subjective axis "straight forward" from the body median by 13 degrees during synchronous but diversely directed rotations of the head and the chair. Under the same test conditions hand movements towards the target were highly precise. The results show a weak interrelation of egocentric head and body directions. PMID- 7550156 TI - [Aerospace radiobiology: the problems and the colleagues (on the 60th anniversary of the Institute of Aviation and Space Medicine)]. PMID- 7550157 TI - [The adaptive-compensatory reactions in adjusting to space flights]. AB - In this paper an evolutionary approach is used to substantiate the steps of life evolution on Earth towards overcoming the gravitational forces with the formation of metabolic cycles controlling the energetic of anti-G processes. The step of an adaptation to hypogravity was similar to return of surface animals to an aquatic environment. The impossibility of coming back to land was the "price" of adaptation to the aquatic environment. This phenomenon was used by the author as a logical model of human adaptation to a weightless environment. The concept of adaptation is examined from two points of view: adaptation and compensation. The scheme contains 4 steps of adaptation to space mission environments: Step I preadaptation (phase of primary reactions); Step II-compensation of body structures not being in line with the conditions of a novel environment; Step III the formation of parameters of an organism corresponding to the norm of adaptation to weightlessness; Step IV-the return of cosmonauts to Earth under hypergravity conditions. In compliance with these steps we consider the tasks of supporting space missions to facilitate body readaptation after return to Earth, i.e., to decrease the "price" of adaptation. PMID- 7550160 TI - [The problem of the effect of cosmogeophysical factors on the formation of the properties of developing organisms during early ontogeny]. AB - The paper is dedicated to a poorly understood but highly perspective direction of modern biology and medicine, namely, to the elucidation of bioinformative role of the natural physical cosmoplanetary fields. The problem associated with the phenomenon of heliomagnetic imprinting, the effect of heliogeophysical factors on developing organism has been touched on. There present the literature data pointing to existing the dependence of a number of morphofunctional properties as well as some peculiarities of human body pathology on when the pregnancy and labor occurred which in particular can be related to the changes in the field structure of the biosphere depending upon the space processes dynamics. PMID- 7550158 TI - [The Tsiolkovskii Lectures over 30 years (1966-1995):the problems of space biomedicine]. PMID- 7550161 TI - [The contribution of space biology and medicine to the solution of ecological problems]. AB - Space biology and medicine solving the problems of space exploration by a man is thereby contributed to the solution of the ecological problems. Theoretically, it is an enrichment of the existing ideas of the biosphere and biological consequences of an anthropogenic effect on the biosphere. Practically, these are: the novel means and methods of the environmental monitoring; the databases of the ecology of a healthy man; the countermeasures and methods to prevent and protect a man from the impacts of unfavourable environmental factors. PMID- 7550159 TI - [Ecology, adaptation and biorhythms]. AB - The interfusion of environmentology and chronobiology makes it possible to explore various physiological mechanisms which arose in the process of evolution as a response to the external challenges. Therefore, a living organism adapts to a changing situation on the environmental and evolutional time scale. Since the temporal structure of environment is rhythmic by nature, body parameters may be strongly dependent on the environmental factors during adaptation. Results of the in-depth environmental and physiological studies of representatives of different climatic-geographical regions pointed to a series of common and individual properties of the human body reaction to an environment. The combination of hypoxia and hypercapnia (rebreathing test) brought into light a relationship of the reciprocal reaction of respiration and circulation to the body mass index and the average annual temperature in a region. The higher the temperature, the lower the body mass index, the weaker the hemodynamic reaction and the more expressed the ventilatory reaction. PMID- 7550162 TI - [The Ecology Department of the Russian University of the Friendship of Peoples--a scientific, pedagogical and social concept]. AB - The paper is about the foundation of the Environmental Department in the Russian University of Friendship of Peoples with a scientific-educational center of environmental biology and advanced technologies, the faculties of system and industrial environmental biology, human and radiation environmentology with the purpose to boost environmentalism and eco-culture. PMID- 7550163 TI - [Biotechnology manufacture as a possible source of environmental pollution and a risk factor for human health]. AB - The biotechnology-oriented quantitative criteria and a plan of primary sanitary hygienic evaluation of pathogenicity of microorganisms-producers were first established. New evidence of the negative effects of strains-producers on microbiocenosis and self-purification of soils and reservoirs were obtained. The requirement of an all-round hygienic assessment of microorganisms-producers consisting of two mandatory steps, i.e. the primary sanitary hygienic evaluation of pathogenicity of strains and the investigation of effects of these microorganisms on microbiocenosis and soil and reservoir self-purification has been substantiated. PMID- 7550164 TI - [The morphological state of the adenohypophyseal somatotrophs during body development under conditions of protein-energy insufficiency]. AB - The quantitative morphometric analysis of the somatotrophs of adenohypophysis of the mice developing in prenatal and early postnatal ontogenesis at the protein energetic insufficiency (PEI) was conducted at the light and electron-microscopic levels. The PEI was produced by feeding the female mice starting from the 1st day of their pregnancy with the diet containing half as many, as compared to control, nutrients (5% of protein included) at the cost of incorporating the cellulose. Adenohypophyses of 20-day old badly nourished experimental mice had a changed relationship among the types of cells: the proportion of chromatophobic cells was 39% which was significantly higher than control values (25%). The decrease in the portion of differentiated cells has occurred in the main at the expense of somatotrophs: their portion was 49% (in control--61%). The volume of cytoplasm of somatotrophs in the hypophyses of test animals is less than control value by 30%. Quantitative electron-microscopic analysis demonstrated that volumetric fractions of somatotrophs cytoplasm occupied by the endoplasmatic reticulum, Golgi complex, mitochondria, lysosomes in the malnutrition test do not significantly differ from the appropriate control values. Under protein insufficiency, a decrease of the medium diameter of the secretory granules (by 1.9 times as compared to control) and their content (by 1.7 times) in the somatotrophs has been noted. Thus in the adenohypophysis of mice developed under conditions of prenatal and early postnatal protein insufficiency there prevails the type of somatotrophs with the small volume of cytoplasm and small-sized secretory granules. This type of somatotrophs is classified with the actively functioning cells. PMID- 7550165 TI - [The ecological-morphological aspects of adaptation and the role of the macrophages in body dehydration]. AB - The morphological aspects of rearrangement of macrophages in white rats' subcutaneous areolar tissue during dehydration and their impact on the recovery of osmotic homeostasis were studied. A response of macrophages to dehydration with preliminary injection of antioxidant dibunol was revealed. Macrophages respond to dehydration by a rise in number, elevated cytoplasmic activity of lipolytic enzymes (beta-oxibutyrate dehydrogenase), and a peculiar ultrastructural rearrangement, i.e. a decrease of vacuoles, their sizes, an increase of the lipid volumetric fraction. However, the higher activity of macrophages in terms of production of metabolic water is concurrent to increased malonic dialdehyde production and inhibited activity of the cell antioxidant defense (superoxide dismutase). To correct the observed changes, dehydration was performed on the background of antioxidant dibunol injection. The antioxidant and subsequent dehydration did not annul the response of macrophages to increased intrabody osmolality although slightly masked the effect of dehydration. Survivability in this group of animals was by 22% higher than in the water deprived group. Hence, the areolar macrophages can be considered the controllers of adaptive reactions of the body during dehydration, whereas antioxidants are the correction factor for these reactions. PMID- 7550166 TI - [The ecological conditions in the Aral Sea area and the functional reserves of the human body]. AB - The authors give their judgment of the cardiorespiratory status of the population in the ecologically disaster area to the south of the Aral. The data from examination of various age groups of the native population inhabiting eco-varying regions are published. The authors make a conclusion about an important role of environment in formation of the bronchopulmonary system. PMID- 7550167 TI - [Changes in the infradian rhythm of the dehydrogenases in the blood lymphocytes of rats due to epiphysectomy and the action of weak alternating magnetic fields]. AB - In experiments with 110 white inbred male rats, the infradian rhythm changes in succinate- and alpha-glycerophosphate dehydrogenases in rat's lymphocytes due to epiphysectomy, exposure to transitory magnetic field (TrMF) at 8 Hz and 5 microTl, and their combined effect were studied. According to spectral analysis, the infradian rhythms of lymphocyte succinate dehydrogenase, alpha glycerophosphate dehydrogenase mean activities in intact animals amounted to 26.5 +/- 2.5; 19.1 +/- 1.1; 13.5 +/- 1.1; 9.1 +/- 0.8; 6.9 +/- 0.7; 3.6 +/- 0.2 days. Epiphysectomy decreases the observed rhythm amplitudes, shifts phases, disrupts temporal organization of the redox processes, i.e., induces desynchronosis. TrPM at 8 Hz also causes desyncronosis in intact animals though less pronounced than in epiphysectomized rats. TrMP partially normalizes temporal organization of the dehydrogenase status in epiphysectomized rats. Our findings point to the possibility to apply TrMF for "functional epiphysectomy" of intact animals and, in case of desynchronoses, as biorhythm "time sensors". PMID- 7550168 TI - [The effect of geomagnetic disturbances on the functions of attention and memory]. AB - The effect of geomagnetic disturbances on the basic types of voluntary attention and short-term memory was studied. The three-hour K-indices and diurnal AK indices as parameters of geomagnetic disturbances and psychological tests of attention and memory were used for IBM PC/AT processing. As a result, the test subjects were distributed into two groups according as the attention and memory parameters exhibited a decline or a rise. Decrement of memory was more apparent than that of attention. The short-term memory displayed a decline more frequently than mechanical or image-bearing, whereas the intensity of attention increased more often than memory span or distribution. These data suggest a non-specific impact of geomagnetic disturbances on the processes of attention and memory in humans. PMID- 7550171 TI - [Human ecology and sustainable development under current conditions]. PMID- 7550170 TI - Morphological changes in rat's brain choroid plexus after exposure to low doses of high energy oxygen ions, fast neutrons, and gamma radiation. AB - Ultrastructural and morphometrical changes in choroid plexus cells of the rat's brain in the delay period after irradiation with low doses of oxygen ions [= 300 MeV/nucleon], and fast neutrons [1.5 MeV], and gamma rays (Co60) were described. The applied irradiations provoked similar ultrastructural changes in choroid plexus cells; however, the obtained morphometrical data showed differing effects of these radiations, due to, probably, different mechanisms of their effect on the cells. PMID- 7550169 TI - [Hematopoietic dynamics in mammals under combined radiation exposures (mathematical modelling)]. AB - The mathematical models describing the dynamics of hemopoiesis in the mammals exposed to a combination of chronic irradiation are devised and studied. The models reproduce the increased radiosensitivity of the systems of thrombocytopoiesis, erythropoiesis and lymphopoiesis in the animals resulting from prolonged radiation exposure. Succeeding acute radiation exposure causes a more severe damage of the above-mentioned systems than it occurs for the species not being previously exposed to radiation. The model of granulocytopoiesis simulates both the decrease and the increase in radiosensitivity of this system due to the effect of chronic exposure using low and somewhat higher radiation doses, respectively. The postchronic acute radiation exposure has respectively the decreased or increased damaging effect. Within the limits of the models an interpretation of these effects is suggested. The results of modelling have an important theoretical significance in studies of the mechanisms of an effect of small doses of radiation on the mammalian organism as well as point to the perspectives of simulation experiments when evaluating the real radiation risks during long-term space missions. PMID- 7550172 TI - [Decreased oxygen content in the atmosphere--an ecological disaster imperceptibly sneaking up?]. AB - With the use of gas chromatography there have been done 112 analyses of an oxygen content in the air of St. Petersburg. The results were computer-processed with an application of the standard programs of statistical analysis and program of analysis of the extreme values which permitted to provide the stability of the studied process. In an autumn-winter period (November-January) the values of oxygen content in the air in 37 measurements have been different from the norm in that they declined more than 0.5% and in 12 measurements-approximately by 1%. The mean values of December and January differed from the norm by 0.652; 0.639; 0.834%, respectively. In February, the mentioned changes were smoothed over. As a result of a decrease of oxygen in the period when the green plants stop air purification, the air composition ceases to content, which is the indication of a partial disorder of regulating activity of the biota. PMID- 7550173 TI - [Changes in the structures of the kidney medullary layer in rats during adaptation to high altitude]. PMID- 7550174 TI - [The All-Russian Congress on the Rational Use of Natural Resources in Russia in the Socioeconomic Transitional Period (the current status and developmental outlook)]. PMID- 7550175 TI - [The HUBES experiment]. PMID- 7550176 TI - [The general concepts of development and the principle of sustainable development]. AB - A concept of the sustainable development principle proposed by the United Nations/International Committee on Environment and Development (ICED, 1987) and gained acceptance at the United Nations Conference on Environment and Development (Rio-de-Janeiro, 1992) lies not in the notion of a sustained character of the development but in an understanding of the development as a steadily and long existing and maintained process that is in advocating the stability of the development. It is known, that the state instability of open self-organizing and self-developing systems including the social ones is the the source of the development having a probabilistic, bifurcate and not quite predictable character. Such an understanding of the development should be well represented in the action initiated in the frame work of national and international state ecological policies. PMID- 7550177 TI - [The international cooperation of the Institute of Biomedical Problems in 1994]. PMID- 7550178 TI - Individual-patient monitoring in clinical practice: are available health status surveys adequate? AB - Interest has increased in recent years in incorporating health status measures into clinical practice for use at the individual-patient level. We propose six measurement standards for individual-patient applications: (1) practical features, (2) breadth of health measured, (3) depth of health measured, (4) precision for cross-sectional assessment, (5) precision for longitudinal monitoring and (6) validity. We evaluate five health status surveys (Functional Status Questionnaire, Dartmouth COOP Poster Charts, Nottingham Health Profile, Duke Health Profile, and SF-36 Health Survey) that have been proposed for use in clinical practice. We conducted an analytical literature review to evaluate the six measurement standards for individual-patient applications across the five surveys. The most problematic feature of the five surveys was their lack of precision for individual-patient applications. Across all scales, reliability standards for individual assessment and monitoring were not satisfied, and the 95% CIs were very wide. There was little evidence of the validity of the five surveys for screening, diagnosing, or monitoring individual patients. The health status surveys examined in this paper may not be suitable for monitoring the health and treatment status of individual patients. Clinical usefulness of existing measures might be demonstrated as clinical experience is broadened. At this time, however, it seems that new instruments, or adaptation of existing measures and scaling methods, are needed for individual-patient assessment and monitoring. PMID- 7550179 TI - Evaluating the quality of life associated with rifabutin prophylaxis for Mycobacterium avium complex in persons with AIDS: combining Q-TWiST and multiattribute utility techniques. AB - Our objective was to evaluate the effect of rifabutin prophylaxis in patients with AIDS and CD4 counts of less than 200 per cubic millimetre using a combination of Q-TWiST (quality-adjusted time without symptoms and toxicity) and multiattribute health utility assessment. The design consisted of a secondary analysis of two previously reported multicentre, randomized, placebo-controlled clinical trials conducted in 78 academic, community and Department of Veterans Affairs HIV centres and private practices. 542 patients with AIDS and CD4 counts of less than 200 per cubic millimetre were assigned to rifabutin 300 mg/day and 562 were assigned to a placebo. A modified Q-TWiST approach was used for comparing treatments based on the occurrence and duration of time with and without severe symptoms and clinical endpoints. Health states were constructed to represent combinations of clinical events experienced by study patients. Five physicians assigned utilities for health states using a six-attribute health classification system. These utilities were used to adjust survival for QOL. The rifabutin and placebo groups were compared using estimated quality-of-life adjusted days. The incidence of MAC was 9% for the rifabutin group and 18% for the placebo group (p < 0.001). Differences, although not statistically significant, were observed for rates of survival and hospitalization. The rifabutin group experienced less anaemia (p < 0.02), and fever and night sweats (p < 0.02) than the placebo group. Average Q-TWiST days were 325 for the rifabutin group and 309 for the placebo group (p < 0.05). Q-TWiST days were significantly lower for patients with MAC bacteraemia (p < 0.04) and hospitalizations (p < (0.003). Rifabutin prophylaxis resulted in fewer MAC infections and greater quality-of-life-adjusted days of survival compared to no rifabutin. Quality-of-life-adjusted survival, based on a combination of the Q TWiST and multiattribute health utility index, is a feasible approach for evaluating the outcomes of medical treatment. Future studies should, however, use patient-assigned utility weights to compute Q-TWiST scores, since physician generated utilities may differ significantly from those of patients. PMID- 7550180 TI - The relationship between visual acuity and functioning and well-being among diabetics. AB - Given the enormous recent interest in functional capabilities related to vision, the goal of this study was to examine the relationship of standard clinical measures of vision (e.g. Snellen acuity) to functioning and well-being. The association between Snellen visual acuity, Amsler grid distortion and presence of diabetic retinopathy with self-reported functioning and well-being (SF-36) were examined in a sample of 327 diabetics from the Medical Outcomes Study (MOS). There was little or no correlation between Snellen visual acuity, Amsler grid distortion or diabetic retinopathy and functioning and well-being (i.e. SF-36 scales). Maximum product-moment correlation was 0.15 with worst eye visual acuity, 0.13 with best eye visual acuity, 0.08 with presence of retinopathy, and 0.10 with Amsler grid distortion. Analysis of variance revealed that visual acuity (both best and worst eye) was statistically related only to the physical function scale; no other exam measure was related to any other SF-36 scale score. Snellen visual acuity, Amsler distortion and diabetic retinopathy correlate weakly with patient self-reported functioning and well-being. Thus, the information provided by functioning and well-being measures is complementary to that of standard clinical measures of visual ability. PMID- 7550182 TI - The impact of lower urinary tract symptoms on general health status and on the use of prostatectomy. AB - Our objective was to determine the extent to which lower urinary tract symptoms affect the general health status of men and contribute to the decision to undergo surgery. A cross-sectional population survey using postal questionnaires was conducted in the North West Thames health region, followed by a prospective cohort study of men undergoing prostatectomy (North West Thames and Oxford regions). The subjects in the first survey were 221 men aged 55 and over with previously reported mild, moderate or severe urinary symptoms; subjects in the second study were 388 men undergoing prostatectomy. Main outcome measures were self-reported symptom severity, bothersomeness and general health status (Nottingham Health Profile, Part 1). The response rate among eligible responders in the population survey was 85.7%. Increasing symptom severity was associated with worsening NHP scores for energy, emotional reactions, sleep and physical mobility (p < 0.01). Increasing bothersomeness of symptoms was associated with emotional reactions, sleep and pain (p < 0.05). Men undergoing surgery reported worse health status than men in the population with the same severity of symptoms as regards emotional reactions, energy and pain. For a given level of symptom severity, the impact of those symptoms on aspects of a man's general health status may be the determinant of seeking and undergoing surgery. Greater understanding of the factors that affect a man's response to his symptoms is needed in interpreting the decision to seek and accept treatment. PMID- 7550181 TI - Measuring changes in quality of life following magnetic resonance imaging of the knee: SF-36, EuroQol or Rosser index? AB - Evidence suggests that magnetic resonance imaging (MRI) allows accurate diagnosis of meniscal and ligamentous injuries of the knee. However the link between improved diagnosis through MRI and improved patient quality of life (QOL) has yet to be shown. Previous studies aimed at establishing this link have found no significant improvements in health related quality of life (HRQOL) as measured by the Rosser classification and index. This paper presents the results of three HRQOL questionnaires (SF-36, Rosser and EuroQol) used to measure health change in 332 patients referred for MRI of the knee. Before imaging, patients reported poorer HRQOL than the general population on two of the three questionnaires (SF 36 and EuroQol). The same two questionnaires recorded significant improvements in patient health at six months, although patients' health had not yet reached that experienced in the general population. There was evidence to suggest that the index values attached to the Rosser classification made it unresponsive in this group of patients, which may have predisposed the null results of previous studies of the influence of MRI on HRQOL. Some evidence is provided to suggest that the EurolQol may be less responsive in assessing change in health status than the SF-36. PMID- 7550183 TI - MRC quality of life studies using a daily diary card--practical lessons learned from cancer trials. AB - The UK Medical Research Council (MRC) Daily Diary Card for quality of life (QOL) assessment was first designed over ten years ago, and since then has been in continuous use in cancer clinical trials organized by the MRC Cancer Trials Office. However, clinical trials using this technique have only reached maturity since 1989 and later. Thus it is timely to review the experiences gained by use of this method of assessing QOL. Results from a series of clinical trials confirm that the Daily Diary Card appears to be a valid, reliable and sensitive instrument which is able to detect the transient changes in health and QOL which occur on a day to day basis during therapy for cancer. However, in early studies we also encountered a number of problems which were addressed during later MRC trials; we believe that many of these are of general importance to any investigation of QOL in patients, many of the issues raised remain controversial, and the practical lessons learned from applying the Diary Card will be of relevance to those planning future studies of QOL assessment. PMID- 7550184 TI - Comparison of the sensitivity to change of long and short form pain measures. AB - Our objective was to assess the sensitivity to change over time of three methods of assessing pain. We conducted a comparison of the sensitivity to change of three pain assessment measures in a double blind, double dummy parallel group study evaluating the efficacy of one of two doses of oral bromfenac and one of two doses of sublingual buprenorphine. Our subject pool consisted of 75 patients following general surgical and orthopaedic procedures. Pain intensity was measured by the affective, sensory and evaluative dimensions of the McGill Pain Questionnaire together with total score and word score, a 10 cm Visual Analogue Scale and by a four word scale. Effect size calculations indicated considerable variation in the sensitivity of the instruments to change. However, despite variation in the size of effect indicated by different measures, there was consistent agreement as to the most effective treatment regimen. This study suggests that simple measures of pain are sensitive to change and are worthwhile indicators of the impact of an intervention upon acute pain, although some care is needed in interpreting results from such instruments. More complex forms of assessment, such as the calculation of the MPQ sub-scales may not add anything to such data. PMID- 7550185 TI - Health-related quality of life in endstage renal failure. AB - The objective of this study was to describe the health-related quality of life (HRQOL) of patients on different forms of treatment for endstage renal failure in such a way that the data could be used in a cost-utility analysis of renal failure treatment in Britain. Twenty-four British renal units participated in this study. 997 adult dialysis and transplant patients were randomly selected from these units using the European Dialysis and Transplant (EDTA) Registry Database. The Health Measurement Questionnaire was completed by 705 of the 900 patients who were alive at the time of the survey (response rate of 78%). The HRQOL data were linked with comorbidity data and with clinical data from the EDTA Registry. Compared to the general population, patients with endstage renal failure experienced a lower quality of life. Many factors contributed to this, but uncertainty about the future and lack of energy emerged as key components. Transplant recipients reported better HRQOL than dialysis patients, they reported fewer problems with physical mobility, self-care, social and personal relationships and usual activities. They also experienced significantly less distress, while dialysis patients reported problems with depression, anxiety, pain and uncertainty about the future. These differences remained after controlling for age and comorbidity. PMID- 7550186 TI - Surgical hemostasis by pneumatic ankle tourniquet during 3027 podiatric operations. AB - A retrospective study was performed at the Denver Doctors Hospitals in which 3818 surgical cases on the foot and/or ankle were reviewed over a 4-year period from July 1986 through May 1990. From the 3027 ankle tourniquet cases reviewed, it was determined that pneumatic ankle tourniquets are safe and effective in providing hemostasis during foot surgery. There were five postoperative complications noted with ankle cuffs, with post-tourniquet syndrome being the most common (three cases). Over the 4-year period, ankle tourniquets failed a total of 50 times, a 1.8% failure rate (0.25% failure rate in the last 17 months). The most common pressure setting used for ankle cuffs was 325 mm. Hg (400 mm. Hg for thigh cuffs). Tourniquet ischemia lasted from 4 to 139 min.; the most common duration of ischemia noted for ankle tourniquets was 30 to 60 min. (60 to 90 min. for thigh tourniquets). A review of the potential complications associated with tourniquets, as well as safeguards, recommendations, and contraindications are presented. PMID- 7550187 TI - Exploiting the viscoelastic properties of pedal skin with the Sure Closure skin stretching device. AB - This manuscript introduces a device for harnessing the inherent viscoelastic properties of skin, to mechanically assist primary and delayed primary wound closure. The current indications, contraindications, and general use of the device are discussed. Also included is an initial series of device applications, with two case reports reviewed in detail. PMID- 7550188 TI - Double calcaneal osteotomy in the treatment of posterior tibial tendon dysfunction. AB - This article presents an alternative treatment for tibialis posterior tendon rupture in a select group of patients with recent rupture and planovalgus foot structure. Double calcaneal osteotomy is used to realign weightbearing forces in situations where soft-tissue repair alone is not sufficient, and arthrodesis is premature. A single case is presented followed by a discussion, with encouraging results. PMID- 7550189 TI - A severe acute Achilles rupture and repair. AB - An unusual type IV rupture is described, whereby the Achilles tendon was ruptured in two places. This required several innovative techniques to repair this injury, which are described herein. The patient recovered uneventfully and returned to his exercise activities in 3 months. PMID- 7550190 TI - Chevron bunionectomy fixation: in vitro stability assessment of plate-and-screw system compared with Kirschner wire. AB - Many methods for internal fixation of the Chevron bunionectomy have been described. Currently available fixation devices stabilize the osteotomy sufficiently to ensure healing. Each has unique advantages and disadvantages. A low-profile plate buried beneath capsular tissue eliminates some complications of other forms of fixation. The authors compared the structural characteristics of a low-profile plate-and-screw system and of a single Kirschner wire. Testing was performed on dissected human cadaveric specimens by using a Telos strain gauge. Loads were applied both from the medial to the lateral aspect and from the plantar to the dorsal aspect at the capital fragment. Results indicate that both modes of fixation can resist Chevron osteotomy displacement in vitro. PMID- 7550191 TI - The Romano drill: a new surgical curved drill system. AB - A new surgical curved drill system that drills curved holes into bone is presented, along with several examples of its use in foot and ankle surgery. The technical information involving soft tissue fixation, wiring, or suturing around a bone bridge, by drilling a curved tunnel for tendon, capsule, and ligament reconstruction is also reviewed. PMID- 7550192 TI - Biofix fixation techniques and results in foot surgery. AB - Biofix absorbable rods have been used for internal fixation for 10 years, and one of its most utilized applications has been osteotomies of the first metatarsal. This study evaluates 59 procedures on 49 patients completed over a 3-year period, using the 2.0-mm. Biofix rods. The transarticular technique for the fixation of osteotomies in the 1st and 5th metatarsals and the intramedullary technique used for digital fusions are described. All of the patients were followed for a minimum of 3 months, but most for > 1 year, and all of the procedures were evaluated for failure of fixation, osteolytic bone changes, sterile sinus development, and infection. There were no failures of fixation and no infections in this series. Patients (22.4%) developed radiographic osteolytic changes, and 4.1% of the patients developed sterile sinus discharge. However, these had no effect on the procedures' success or outcomes. An introduction to future application of the technology using Biofix screws and plates is made with a pantalar fusion case as an example. PMID- 7550193 TI - Tibiofibular arthrodesis for malunion of the talocrural joint. AB - Reconstruction of malunited or an incongruous talocrural articulation is relatively unrecognized within the literature and few procedures have been described. This article reviews a case of a 36-year-old male who presented with chronic ankle pain, secondary to a malunion of the ankle joint. The case presentation, including surgical treatment and follow-up care, is presented. A review of the literature is also provided. PMID- 7550194 TI - An unusual cause of tarsal tunnel syndrome. AB - An unusual cause of tarsal tunnel syndrome is discussed, along with many of the other etiologies of this painful condition. The diagnosis was difficult because the patient's symptoms only occurred during activity. Because of burning paresthesias, it was felt that reflex sympathetic dystrophy was the etiology. Only through persistence of the treating podiatrists and the patient's agreeing to an exploration was this entity discovered. PMID- 7550195 TI - Surgery for chronic Achilles tendon problems. AB - The author introduces a new procedure for surgical treatment of Achilles tendocalcinosis and other techniques for treatment of various chronic Achilles pathologies. Results of 21 procedures on 19 patients are presented. PMID- 7550196 TI - Extraosseous Ewing's sarcoma in the foot. AB - Extraosseous Ewing's sarcoma is an extremely rare neoplasm that may occur in the lower extremity. The neoplasm is highly aggressive and is usually fatal, however, early diagnosis and treatment greatly increase the chances of survival. A case report and review of the literature is presented. PMID- 7550197 TI - A retrospective comparison of endoscopic plantar fasciotomy to open plantar fasciotomy with heel spur resection for chronic plantar fasciitis/heel spur syndrome. AB - The authors review the etiologies and treatments of plantar fasciitis or heel spur syndrome. They offer results of a retrospective study. Comparison of the return to work time after surgery for this condition, examinations of the effects of patient age at the time of surgery, gender, duration of pain prior to surgery, and type of surgical procedure, either endoscopic plantar fasciotomy or open plantar fasciotomy with heel spur resection, is provided. PMID- 7550198 TI - Cheilectomy, chondroplasty, and sagittal "Z" osteotomy: a preliminary report on an alternative joint preservation approach to hallux limitus. AB - The surgical correction of hallux limitus can be most rewarding for both patient and surgeon when marked degenerative changes exist in older individuals. Many foot and ankle surgeons would perform an implant arthroplasty in a patient over 60 years of age with hallux limitus or rigidus. In a patient without gross first metatarsophalangeal joint arthrosis, but with limitation in range of motion, the literature is replete with biomechanically sound surgical options. However, surgical repair of the moderate-to-severe arthritic first metatarsophalangeal joint in younger individuals remains quite a challenge. The authors present the use of a sagittal plane "Z" osteotomy of the proximal phalanx along with cheilectomy and chondroplasty for the treatment of hallux limitus and rigidus in the young, active patient. PMID- 7550200 TI - Bronchogenic carcinoma with metastases to the foot: a report of two cases. AB - Pedal metastases from a primary sarcoma or carcinoma are rare occurrences. The hallux appears to be one area with which these have been identified. Two case reports of metastasis from the lung to the foot are described. PMID- 7550199 TI - Review of Elmslie's triple arthrodesis for post-polio pes calcaneovalgus deformity. AB - Twelve patients with residual talipes calcaneovalgus deformity due to poliomyelitis were reviewed. A two-stage Elmslie's arthrodesis was performed on nine patients with rigid deformity. Tendon transfer (peroneus brevis in one, peroneus brevis and longus in two) to the tendo Achillis insertion is used to correct the calcaneus deformity, after reduction of the calcaneus in the subtalar joint, using a Steinmann pin passed through the calcaneus. Good results were obtained in patients treated by arthrodesis. However, the deformity recurred in patients treated only with tendon transfer. PMID- 7550201 TI - Whither pulmonary rehabilitation? PMID- 7550202 TI - Asthma death. PMID- 7550203 TI - Spirometric reference values in school children from Dalmatia. AB - Data on spirometric reference values in children are based on small, selected populations. We performed spirometric testing (forced vital capacity (FVC); forced expiratory volume in one second (FEV1); forced mid-expiratory flow rate (FEF25-75%)) in 2,500 healthy nonsmoking school children (1,250 boys and 1,250 girls) in the region of Dalmatia, Croatia. Significantly higher FVC and FEV1 values were noted in boys. Standing and sitting heights correlated significantly with FVC and FEV1 in boys and girls. Regression equations were derived, utilizing the multiple linear regression analysis, for FVC, FEV1 and FEF25-75% related to five (sex, age, weight, standing and sitting heights) or three (sex, age and standing height) variables. The reference spirometric values in the Dalmatian school boys were in close agreement with Knudson et al., while the values in girls were closest to those of Knudson et al. and Cotes et al. It is suggested that for calculation of ventilatory reference values in boys and girls the age and the standing height should be used. PMID- 7550204 TI - Cardiopulmonary adaptation to exercise after acute weight loss in severely obese subjects. AB - The aim of our study was to investigate the effects on baseline and exercise cardiopulmonary function and metabolic parameters of an acute (3-4 weeks) loss of at least 10% of initial weight in severely obese patients. Eight obese patients, 3 males and 5 females, mean age +/- SEM 42 +/- 6 yrs, body mass index (BMI) > 35 kg.m-2, underwent two cardiopulmonary function tests separated by 3 weeks of very low calorie protein-sparing diet (589 kcal.day-1) and a weight loss of 10% of initial weight (mean 8.43 +/- 0.72 kg). Eight normal subjects, matched for sex and age with the obese patients, served as controls. In the obese subjects, maximal workload (+15 +/- 3.6 W) and maximal oxygen consumption (V' O2) (+188 +/- 40.5 mL.min-1) were significantly increased after weight loss. Interestingly, exercise capacity at anaerobic threshold was not significantly different in obese subjects after weight loss with respect to normal subjects. Our results show that an acute but significant weight loss obtained with a very low calorie diet in obese patients is able to significantly improve maximal V' O2 and V' CO2 at anaerobic threshold. Moreover, this acute weight loss is able to partially reverse all of the cardiopulmonary alterations seen in obese patients both at baseline and during exercise. PMID- 7550206 TI - Asthma death induced by ibuprofen. AB - We describe the case of a 40 year old woman, who had suffered from asthma since infancy, with a negative case history of asthma induced by aspirin or nonsteroidal anti-inflammatory (NSAID) agents, who died after ingesting 400 mg of ibuprofen. The autopsy specimens collected 3 months after death had all the characteristic pathological features of fatal asthma. The medical records showed that the patient was not informed of the potential fatal outcome of an asthma attack, and that she was not properly treated. PMID- 7550205 TI - Clinical evaluation of oscillating positive expiratory pressure for enhancing expectoration in diseases other than cystic fibrosis. AB - The benefit of chest physiotherapy in patients with cystic fibrosis has been well documented. However, the benefit of similar rehabilitation in patients with large amounts of sputum who have diagnoses other than cystic fibrosis has not been clearly demonstrated. The aim of this study was to evaluate the acute effectiveness of a device advised for home chest physiotherapy in comparison to postural drainage combined with chest percussion in removing secretions in patients with high sputum production due to diseases other than cystic fibrosis. Fourteen in-patients, known to spontaneously produce more than 25 mL sputum.day 1, underwent two sessions each of two treatment modalities in random order. Treatment A consisted of postural drainage and manual chest percussion. Treatment B included breathing through a commercial device claimed to combine oscillating positive expiratory pressure with oscillations of the airflow. Expiratory flows and oxygen saturation were monitored and recorded throughout the study. The amount of sputum produced in the 30 min preceding, during, and in the 60 min after completing each treatment session was recorded, together with the sensation of "chest unpleasantness due to secretions" as assessed by means of a modified visual analogue scale. The mean time that the patients tolerated treatment was not different for A and B (18.7 +/- 5 and 19.3 +/- 5 min, respectively). Sputum significantly increased during both treatment periods and in the same amount for the two modalities (2.9 +/- 2.9 to 10.9 +/- 7.1 and 2.8 +/- 3.1 to 10.1 +/- 10.8 mL for A and B, respectively). Visual analogue scale score significantly decreased at the end of each treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550208 TI - Pulmonary input impedance or pulmonary vascular resistance? AB - Pulmonary artery input impedance (PAII) expresses the opposition of pulmonary vessels to pulsatile blood flow, in the same way that vascular resistance expresses opposition to steady flow. PAII spectrum includes information concerning not only the mean blood flow, pressure and resistance, but also proximal vessels stiffness, pulse wave velocity and arterial wave reflection. It is an adequate expression of the right ventricular afterload, and it is quite suitable for the description of the right-ventricular-pulmonary arterial interaction. Pulsatile properties must be taken into account, for instance, to understand the effects of pulmonary vasodilation on cardiac output. Characteristic PAII is increased in pulmonary hypertension due to stenosis, pulmonary thromboembolic disease, and in unexplained pulmonary hypertension. PMID- 7550207 TI - Long-term treatment of pulmonary tuberculosis with ofloxacin in a subject with liver cirrhosis. AB - We report on the clinical case of a patient suffering from active pulmonary tuberculosis associated with liver cirrhosis, which limited the use of the major antituberculosis drugs. The patient was treated with a 12 month ofloxacin and ethambutol therapy. At completion of the therapy, the lung cavity disappeared with a considerable reduction of the lung infiltration, as seen in the chest radiograph. At the same time, a positive to negative conversion of the sputum was noted. The only significant side-effect was hyperuricaemia. PMID- 7550209 TI - Immunomodulatory actions of xanthines and isoenzyme selective phosphodiesterase inhibitors. AB - Theophylline and related xanthines have been used in the treatment of airways diseases such as bronchial asthma for over 50 years. The therapeutic effectiveness of this class of drugs was traditionally thought to be derived from their ability to elicit bronchodilation, however evidence is accumulating to suggest that theophylline in particular possesses immunomodulatory and anti inflammatory activity. The molecular mechanisms of action of theophylline have not yet been clarified, although several putative mechanisms of action have been proposed. One of these suggests that theophylline via non-selective inhibition of the phosphodiesterase (cAMP) and cyclic guanosine monophosphate (cGMP) levels resulting in relaxation of airway smooth muscle and inhibition of inflammatory cell activation. To date seven different families of the PDE enzyme have been defined according to a variety of criteria including substrate specificity, sensitivity to selective inhibitors and the effect of allosteric modulators. The type IV isoenzyme is the predominant isoenzyme in most inflammatory cells. This article reviews some of the in vitro, in vivo and clinical studies which have demonstrated that theophylline and selective PDE inhibitors possess anti inflammatory and immunomodulatory activity. PMID- 7550213 TI - Respiratory muscle function and COPD. PMID- 7550212 TI - How does chronic heart failure cause dyspnoea? PMID- 7550210 TI - Standards for rehabilitative strategies in respiratory diseases. PMID- 7550215 TI - The Head Injury Severity Scale (HISS): a practical classification of closed-head injury. AB - The authors introduce a two-dimensional scale for rating closed-head injury, the Head Injury Severity Scale (HISS). This system is based on a five-interval severity classification (minimal through critical), determined primarily by the initial post-resuscitation Glasgow Coma Scale score. The second dimension is predicated on the presence or absence of complications, appropriate for each severity interval. The outcomes of almost 25,000 patients with head injury encountered at our institution over a 7-year period were evaluated. We discovered that adding a complication dimension to each severity category resulted in significant outcome differences and effectively divided patients into groups with very different risks, prognosis and treatment requirements. The HISS is proposed as a framework on which further research can be done to guide care to predict outcome and to perform audits on head-injured patients. PMID- 7550214 TI - Neurobehavioural dysfunction following mild traumatic brain injury in childhood: a case report with positive findings on positron emission tomography (PET). AB - The present case study describes the neurobehavioural, neurodiagnostic, and positron emission tomography (PET) scan findings in a child who sustained a whiplash-type injury in a motor vehicle accident. Although neck and back pain were reported immediately, neurobehavioural symptoms, such as staring spells, gradually increased in frequency over a 2-year period following the accident. At 4 years after the accident the patient's symptoms persisted, as reported by teachers and parents, and more extensive diagnostic work-up was initiated. Standard EEG was normal while two ambulatory EEGs were abnormal and interpreted as epileptiform. A PET scan showed evidence of marked hypometabolism in both temporal lobes. Neuropsychological findings were consistent with PET findings and reflected verbal and visual memory deficits in the context of high average intelligence. Treatment with carbamazepine, verapamil, and fluoxetine greatly improved the patient's symptoms. The present case illustrates an example of a poor outcome in a paediatric case of mild traumatic brain injury, the importance of PET in demonstrating definitive evidence of brain dysfunction, and the child's positive response to anticonvulsant medication. PMID- 7550211 TI - Patients' interest in educational programmes on asthma. AB - The compliance of patients in educational programmes on asthma is sometimes lacking and this may be due in part to patients' scarce interest or refusal to expand their knowledge of the disease. In the present study, we aimed to evaluate the interest of a group of asthmatic patients in education on asthma and to point out the asthma-related topics considered particularly worth discussing. The study was conducted before starting the educational programme. Sixty adult patients with mild-to-moderate asthma were asked to complete a questionnaire, including a group of 14 questions aimed at evaluating the level of patients' interest in various asthma-related topics, one question regarding patients' willingness to attend a small-group educational course on asthma, and a further question asking patients to suggest topics of particular interest for discussion. For each of the first 14 questions, patients indicated their level of interest according to a score ranging 1-3. The sum of the scores obtained for each topic (partial score) and the sum of partial scores (total score) were calculated and classified into three classes of interest (I: low, II: medium; III: high), and the distribution of patients into the three classes was analysed (chi-squared test).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550216 TI - Balance abilities in individuals with moderate and severe traumatic brain injury. AB - Balance characteristics, latency, amplitude, and symmetry were measured in 23 moderate and 23 severe traumatic brain-injured (TBI) patients. Patients received two graded forward and two graded backward linear perturbations. Although within normal limits, latency was high. Patients were able to appropriately grade force in relation to the size of the perturbation. Standing posture prior to and during perturbation was recorded by the amount of force generation through each lower extremity. Some individuals used an asymmetrical balance response following perturbations, whereas others shifted to a more symmetrical weight-bearing balance response. TBI patients may be prone to instability due to a combination of long latency of onset of the balance response coupled with asymmetrical stance patterns during recovery from an unexpected linear perturbation. PMID- 7550217 TI - A multi-factor account of disability after brain injury: implications for neuropsychological counselling. AB - Persons with brain injuries demonstrate an inconsistency in functioning over time and circumstances that requires a consideration of factors in addition to the size and location of the acquired brain lesions. A multi-factor account is presented in which neuropsychological disability on any occasion is attributed to some combination of neuropsychological deficits plus one or more of four personal factors (negative thinking, tension-arousal, fatigue, physical symptoms) and three situational factors (demands for complex attention, demands for rapid processing, external distractions). Citing pertinent research the paper discusses the development of, and possible mechanisms of influence by, these seven factors. It then broadly prescribes clinical interventions that might assist clients to reduce their adverse effects. It is suggested that the scope of brain injury rehabilitation be extended to include training clients to identify and manage non organic conditions that may combine with direct brain injury effects to compromise productive work, complicate social relationships, or render the affected person less able to cope with stress. PMID- 7550218 TI - Impairments of discourse abilities and executive functions in traumatically brain injured adults. AB - Preliminary findings from an ongoing investigation of the potential relationship between narrative discourse performance and executive functions in adults with traumatic brain injuries (TBI) are reported. Narrative stories were elicited from 32 adults with TBI. Stories were analysed at three levels: sentence production, intersentential cohesive adequacy, and story episode structure. These measures were then correlated with scores from the Wisconsin Card Sorting Test (WCST), the primary measure of executive function. A significant correlation was noted between a factor score from the WCST and the measure of story structure, but not sentence production or cohesive adequacy. These results suggest that executive functions may be a promising avenue to pursue in the search for underlying causal factors of narrative discourse dysfunction and, therefore to better delineate the nature of communicative deficits secondary to TBI. PMID- 7550219 TI - Dimensions of neurobehavioural dysfunction: cross-validation using a head-injured sample. AB - Principal-components analysis of 44 items from the Minnesota Multiphasic Personality Inventory (MMPI) identified as neurologically related was conducted on profiles obtained from 196 individuals with head injury. Four principal factors, that accounted for 36.5% of the total variance, were extracted. The first factor was composed of items tapping attention and memory complaints. The second factor consisted of items focusing on somatic complaints. Items primarily tapping behavioural disturbance made up the third factor, and the fourth factor was composed of items reflecting specific somatic or neurological complaints. These findings partially confirmed those obtained in previous research, and further illuminate the need for developing a specific and brief measure to characterize neurobehavioural dysfunction for neurological samples. PMID- 7550221 TI - Team development and memory training in traumatic brain injury rehabilitation: two birds with one stone. AB - The need for developing team cooperation procedures when treating patients with traumatic brain injury (TBI) is stated. One approach in promoting team cooperation is to combine team development with a specific training programme. A memory training programme used in a subacute TBI rehabilitation unit is described. A combination of a team development procedure and memory training programme was performed in the unit. A questionnaire to assess team members' attitudes to team cooperation was administered before and after team development, and memory training procedures were implemented. The post-training questionnaire administration indicated a more positive perception among team members of how the team functioned. The efficacy of memory training showed variable results. The programme described may illustrate the advantages of combining a specific treatment programme with efforts to promote team development. PMID- 7550220 TI - Sinemet and brain injury: functional versus statistical change and suggestions for future research designs. AB - During the past decade the usage of dopaminergic agonists for the brain-injured population has become a more common treatment option during both the acute and subacute phases of recovery. We attempted to use Sinemet to address the functional limitations of a 74-year-old woman who was 7 months status post traumatic brain injury secondary to a motor vehicle accident. The patient was administered a 3-month trial of Sinemet while continuing to receive structured sensory stimulation. Her baseline performance using Rappaport's Coma/Near-Coma (CNC) scale yielded a score in the moderate coma range. Weekly evaluations were conducted throughout the drug trial period using the CNC scale. Although the patient displayed modest improvement in her total score, functional change was negligible. Long-term follow-up suggested an absence of retention of earlier gains obtained on the CNC scale. The authors suggest that flawed research methodologies, heterogeneous patient populations, and potential difficulties obtaining reliable dependent measures makes the interpretation of brain injury research findings equivocal. However, given the many limitations, the current research design suggested that the long-term practical utility of Sinemet was unremarkable. Suggestions for future pharmacological research designs with the brain-injured population are discussed. PMID- 7550222 TI - Verbal learning and memory impairment in adult civilians following penetrating missile wounds. AB - Verbal memory and learning patterns, as measured by the California Verbal Learning Test (CVLT), following penetrating head injury (PHI) from gunshot wound were studied in 10 acutely injured patients (mean age 25.3 years) at a mean of 2.1 months post-injury. Primary impairment was found on measures of free recall of new verbal information which appeared to be related to deficits in organisational and retrieval functions: (1) the group's learning characteristics were marked by disorganization and an underutilization of active learning strategies; (2) rate of acquisition also appeared to be mildly decreased; (3) nevertheless, the PHI group did not show severe disruption in all aspects of learning and memory. In fact, the group showed a relatively intact capacity to store new information in memory. PMID- 7550224 TI - The psychological, political, and economic realities of brain injury rehabilitation in the 1990s. AB - Gans, in 1983, detailed the prevalence, causes, and implications of hate in the rehabilitation setting, and offered suggestions for therapeutic responses to it. Mullins, in 1989, noted that during the 1980s rehabilitation became a rapidly expanding, increasingly privatized, big business, with seemingly limitless opportunities for advancement and profit. He asserted that during those years hate had been joined by power, envy and greed in the rehabilitation setting. The present article builds upon the two earlier ones and reviews recent events and their effects, including: the continued development of rehabilitation as a business, the national economic slow down, the health-care crisis, the rise of managed care, the fear that rehabilitation workers feel for their jobs, and the loss of control that people feel throughout rehabilitation. Suggestions for improving the current situation are offered to clinicians, administrators, insurers, and educators, and all are urged to do what they know is right. PMID- 7550223 TI - Traumatic brain injury: factors predicting return to work or school. AB - Prediction of outcome for brain-injured individuals will facilitate effective rehabilitation programme planning which will ultimately improve the patient's quality of life. A sample of traumatic brain-injured subjects who had completed their rehabilitation were contacted post-discharge to identify predictive factors for return to work/school. Subjects were given a telephone interview and their charts were examined retrospectively along five subsets of variables in the predictive matrix: sociodemographics, chronicity, indices of severity, physical impairment, and cognitive functioning. Forty-five subjects were used as the study sample to investigate their vocational and educational outcome, and to generate the best predictive model for return to work/school. Twenty subjects made up the test sample used to evaluate the generalizability of the predictive model. Performance IQ score of the Wechsler Adult Intelligence Scale-Revised emerged as the most significant predictor of return to work/school. Implications of these findings for treatment and rehabilitation are discussed. PMID- 7550225 TI - Mutations in muscle phosphofructokinase gene. AB - Mutations in the muscle phosphofructokinase gene (PFK-M) result in a metabolic myopathy characterized by exercise intolerance and compensated hemolysis. PFK deficiency, glycogenosis type VII (Tarui disease) is a rare, autosomal, recessively inherited disorder. Multiple mutations, including splicing defects, frameshifts, and missense mutations, have recently been identified in patients from six different ethnic backgrounds establishing genetic heterogeneity of the disease. There is no obvious correlation between the genotype and phenotypic expression of the disease. PFK-M deficiency appears to be prevalent among people of Ashkenazi Jewish descent. Molecular diagnosis is now feasible for Ashkenazi patients who share two common mutations in the gene; the more frequent is an exon 5 splicing defect, which accounts for approximately 68% of mutant alleles in this population. PMID- 7550226 TI - Homozygosity for a null allele of the insulin receptor gene in a patient with leprechaunism. AB - Mutations in the insulin receptor gene can cause genetic syndromes associated with extreme insulin resistance. We have investigated a patient with leprechaunism (leprechaun/Qatar-1) born of a consanguineous marriage. Postnatally, the proband had episodes of severe hypoglycemia and hyperinsulinernia, with blood glucose levels ranging from 0.9 to 9.9 mmol/L. The C peptide concentration with 1880 nmol/L, and the total insulin concentration was 1409 mU/L. The patient died outside the hospital at the age of four months. All 22 exons of the patient's insulin receptor gene were screened for mutations using denaturing gradient gel electrophoresis. Thereafter, the nucleotide sequences of selected exons were determined directly. The patient was homozygous for a mutation in exon 13; thirteen base pairs were deleted and replaced by a 5 b.p. sequence. This mutation shifts the reading frame and introduces a premature chain termination codon downstream in exon 13. Thus, the mutant allele is predicted to be a null allele that encodes a truncated receptor lacking both transmembrane and tyrosine kinase domains. PMID- 7550227 TI - Mutation heterogeneity of cystic fibrosis in France: screening by denaturing gradient gel electrophoresis using psoralen-modified oligonucleotide. AB - In order to establish counseling guidelines and aid carrier risk assessment, we sought to establish the frequencies of cystic fibrosis (CF) mutations that are present in CF families living in the Ile de France region, a region notable for its ethnic heterogeneity. We studied 470 CF chromosomes in which we identified more than 90% of the CF mutations. We systematically screened 21 exons and the adjacent sequences of the CF transmembrane conductance regulator (CFTR) gene by denaturing gradient gel electrophoresis using chemical clamps. We detected 41 different CF mutations located in 14 exons. One of these mutations had not been previously described. Given the heterogeneity of these mutations, population screening does not appear to be readily feasible in our population. PMID- 7550228 TI - Detection and genetic analysis of beta-thalassemia mutations by competitive oligopriming. AB - A new approach for the detection of beta-thalassemia mutations has been applied, based on competitive oligonucleotide priming (COP) of in vitro DNA amplification at the mutation site. This method allows genotyping of the template DNA, through differential labeling of the allele-specific competitive oligoprimers and biotinylation of the common reverse primer. The system provides a basis for rapid, simple, and reliable detection of the numerous known beta-thalassemia mutations, revealing the precise nature of the mismatch in each case, and thereby facilitating the molecular genetic analysis of the disease. PMID- 7550229 TI - Molecular characterization of galactosemia (type 1) mutations in Japanese. AB - We characterized two novel mutations of the galactose-1-phosphate uridyltransferase (GALT) gene in two Japanese patients with GALT deficiency and identified N314D and R333W mutations, previously found in Caucasians. One novel missense mutation was an G-to-A transition in exon 8, resulting in the substitution of arginine by histidine at the codon 231 (R231H). GALT activity of the R231H mutant construct was reduced to 15% of normal controls in a COS cell expression system. The other was a splicing mutation, an A-to-G transition at the 38th nucleotide in exon 3 (318A-->G), resulting in a 38-bp deletion in the GALT cDNA by activating a cryptic splice acceptor site. In seven Japanese families (14 alleles for classic form and one allele for Duarte variant) with GALT deficiency, the R231H and 318A-->G mutations were found only on both alleles of the proband. The N314D and R333W mutations were found on one allele each. The Q188R was prevalent in the United States but not in Japanese patients. The N314D mutation was associated with the Duarte variant in Japanese persons, as well as in the United States. We speculate that classic galactosemia mutations appear to differ between Japanese and Caucasian patients. Our limited data set on galactosemia mutations in Japanese suggests that the N314D GALT mutation encoding the Duarte variant arose before Asian and Caucasian people diverged and that classic galactosemia mutations arose and/or accumulated after the divergence of Asian and Caucasian populations. PMID- 7550230 TI - Three novel aniridia mutations in the human PAX6 gene. AB - Aniridia (iris hypoplasia) is an autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the study reported here we describe PAX6 mutations in one sporadic and five familial cases with aniridia. Of the four different mutations identified, one was identical to a previously reported mutation (C-->T transition at codon 240), and three were novel: two in the glycine-rich region and one in the proline/serine/threonine-rich (PST) region. One PAX6 mutation found in the PST region was associated with cataracts in an aniridia family. Another splice mutation in the PST domain occurred in an aniridia patient with anosmia (inability to smell). The six new aniridia cases reported here have mutations predicted to generate incomplete PAX6 proteins. These results support the theory that human aniridia is caused by haploinsufficiency of PAX6. PMID- 7550231 TI - Mutations in the myelin protein zero gene associated with Charcot-Marie-Tooth disease type 1B. AB - Charcot-Marie-Tooth type 1 (CMT1) disease is an autosomal dominant neuropathy of the peripheral nerve. The majority of CMT 1 cases are due to a duplication of an 1.5-Mb DNA fragment on chromosome 17p11.2 (CMT 1a). Micromutations were found in the gene for peripheral myelin protein 22 (PMP22) located in the duplicated region of CMT 1a, and in the peripheral myelin protein zero (PO) located on chromosome 1q21-q23 (CMT 1b). We have characterized two new mutations in the PO gene in two french families presenting CMT disease. Both mutations occur in the extracellular domain of the PO protein. One mutation is a de novo mutation and is from paternal origin. PMID- 7550232 TI - Four novel mutations underlying mild or intermediate forms of alpha-L-iduronidase deficiency (MPS IS and MPS IH/S). AB - The alpha-L-iduronidase deficiency diseases (Mucopolysaccharidosis I) cover a spectrum of clinical severity ranging from the very severe (Hurler syndrome, MPS IH) through an intermediate (Hurler/Scheie syndrome, MPS IH/S) to a relatively mild form (Scheie syndrome, MPS IS). Numerous mutations of the gene encoding alpha-L-iduronidase (IDUA) are known in Hurler syndrome, but only three in the other disorders. We report on novel mutations of the IDUA gene in one patient with the Scheie syndrome and in three patients with the Hurler/Scheie syndrome. The novel mutations, all single base changes, encoded the substitutions R492P (Scheie), and X654G, P496L, and L490P (Hurler/Scheie). The L490P mutation was apparently homozygous, whereas each of the others was found in compound heterozygosity with a Hurler mutation. The deleterious nature of the mutations was confirmed by absence of enzyme activity upon transfection of the corresponding mutagenized cDNAs into Cos-1 cells. These results provide additional information for genotype-phenotype correlations. PMID- 7550233 TI - Haplotype analysis in gelsolin-related amyloidosis reveals independent origin of identical mutation (G654A) of gelsolin in Finland and Japan. AB - Familial amyloidosis, Finnish type (FAF) (gelsolin-related amyloidosis) is an autosomal dominant form of systemic amyloidosis characterized by corneal lattice dystrophy and peripheral polyneuropathy. The accumulating protein in FAF consists of fragments of gelsolin, an actin-modulating protein. The gelsolin mutation G654A has been found in both Finnish and Japanese patients. To study the origin of the gelsolin mutation in these patients we performed haplotype analysis in 10 Finnish and 2 Japanese FAF families. Poymorphic DNA markers GSN, D9S103, AFMa061xd9, and AFMa139xb9 revealed a uniform disease haplotype in all the disease-associated chromosomes of the Finnish FAF families, which was different from the one observed in the Japanese families. The present results and the previously detected gelsolin mutation G654T in Czech and Danish FAF patients suggest that nucleotide 654 may represent a mutation hot spot in the gelsolin gene. The DNA markers studied here will be useful in future genealogical analyses of FAF. PMID- 7550234 TI - Heterozygosity for an exon 12 splicing mutation and a W234G missense mutation in an American child with chronic tyrosinemia type 1. AB - Hereditary tyrosinemia type 1, an autosomal recessive disorder caused by deficiency of fumarylace-toacetate hydrolase (FAH), manifests in either an acute or a chronic form. We used reverse transcription and the polymerase chain reaction to amplify the FAH cDNA of a 12-year-old American boy with chronic tyrosinemia type 1. The patient is a compound heterozygote for mutations in the FAH gene. One allele contains a missense mutation in codon 234 changing a tryptophan to a glycine; this allele was of maternal origin. Mutagenesis and transfection into COS cells demonstrated that the W234G mutation abolishes FAH activity. The patient's paternally derived allele is a splicing mutation in the +5 position of intron 12, causing either insertion of a 105 bp fragment due to a cryptic splice site, or skipping of exon 12, or skipping of both exons 12 and 13. The chronic phenotype of tyrosinemia type 1 in this patient may be due to some residual, correct splicing by the allele with the splicing mutation. PMID- 7550235 TI - Where phenotype does not match genotype. PMID- 7550236 TI - Using information content and base frequencies to distinguish mutations from genetic polymorphisms in splice junction recognition sites. AB - Predicting the effects of nucleotide substitutions in human splice sites has been based on analysis of consensus sequences. We used a graphic representation of sequence conservation and base frequency, the sequence logo, to demonstrate that a change in a splice acceptor of hMSH2 (a gene associated with familial nonpolyposis colon cancer) probably does not reduce splicing efficiency. This confirms a population genetic study that suggested that this substitution is a genetic polymorphism. The information theory-based sequence logo is quantitative and more sensitive than the corresponding splice acceptor consensus sequence for detection of true mutations. Information analysis may potentially be used to distinguish polymorphisms from mutations in other types of transcriptional, translational, or protein-coding motifs. PMID- 7550237 TI - Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3). AB - Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire beta 3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene. PMID- 7550238 TI - Identification of a one-basepair deletion in exon 6 of the dystrophin gene. PMID- 7550239 TI - Identification of a mutation, N543H, in exon 11 of the low-density lipoprotein receptor gene in a French family with familial hypercholesterolemia. PMID- 7550240 TI - An Alu1- polymorphism in the HEXA gene is common in Ashkenazi and Sephardic Jews, Israeli Arabs, and French Canadians of Quebec and northern New England. PMID- 7550241 TI - Identification of five novel inactivating mutations in the human thyroid peroxidase gene by denaturing gradient gel electrophoresis. AB - Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones. Defects in the TPO gene are reported to be the cause of congenital hypothyroidism due to a Total Iodide Organification Defect (TIOD). This type of defect, where iodide taken up by the thyroid gland cannot be oxidized and bound to protein, is the most common hereditary inborn error causing congenital hypothyroidism in the Netherlands. Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene of TIOD patients from nine apparently unrelated families, and seven different mutations were detected. Three frameshift mutations were found: a 20 bp duplication in exon 2, a 4 bp duplication in exon 8, and an insertion of a single nucleotide (C) at pos. 2505 in exon 14. In addition, four single nucleotide substitutions were identified: one single-base mutation resulted in a premature termination codon (C ->T at pos. 1708 in exon 10), two single-base substitutions changed an amino acid in highly conserved regions of the gene (Tyr-->Asp in exon 9 and Glu-->Lys in exon 14). The fourth single-base mutation located at the exon 10/intron 10 border altered a conserved Gly into Ser and could also affect splicing. Nine TIOD patients from five families were compound heterozygotes and six patients from four families were homozygous for one of the mentioned mutations in the TPO gene. PMID- 7550242 TI - Mucopolysaccharidosis type I: identification of 13 novel mutations of the alpha-L iduronidase gene. PMID- 7550243 TI - Relatively high prevalence of the CFTR mutations, G85E and 1154insTC. PMID- 7550244 TI - Molecular cloning of a putative Na(+)-K(+)-2Cl- cotransporter from the Malpighian tubules of the tobacco hornworm, Manduca sexta. AB - Fluid secretion in insects is controlled, in part, by diuretic hormones which bind to receptors on the surface of Malpighian tubule cells, leading to an increase in intracellular cAMP. Recent evidence suggests that Na(+)-K(+)-2Cl- cotransporters may be involved, upon stimulus by diuretic hormones, in transport of ions from the hemolymph to the lumen of the Malpighian tubules. In this report, I describe the cloning of a putative bumetanide sensitive Na(+)-K(+)-2Cl- cotransporter from the Malpighian tubules of the tobacco hornworm, Manduca sexta. This Na(+)-K(+)-2Cl- cotransporter is a Malpighian tubule specific isoform by Northern blot analysis. The protein consists of 1060 amino acids, contains 12 putative membrane spanning regions, and is related to other Na(+)-(K+)-Cl- cotransporters that have been cloned from shark, flounder, rat, rabbit and mouse. This protein is the first Na(+)-K(+)-2Cl- cotransporter that has been identified in an invertebrate. PMID- 7550246 TI - Genomic organization and expression of a trypsin gene from the spruce budworm, Choristoneura fumiferana. AB - A 7 kb (kilobase) genomic fragment containing a trypsin gene from the spruce budworm Choristoneura fumiferana was isolated and sequenced. The coding sequence is interrupted by two introns; these occur at the same positions as the first two introns of mammalian trypsin genes. The three exons encode 256 amino acids. The deduced protein sequence displays all of the structural features that characterize trypsin enzymes in other eukaryotic organisms. Genomic Southern hybridization showed that there is only one copy of the trypsin gene in the Choristoneura genome. This gene is expressed in the insect midgut, where the pH is extremely high. The complete lack of Lysine residues may be an adaptation to the high pH conditions. Extensive sequencing of the flanking regions did not reveal the presence of any linked trypsin-encoding genes. Instead, several short repetitive sequences and a sequence homologous to a Drosophila reverse transcriptase gene was identified in this genomic region. PMID- 7550245 TI - Insect nuclear receptors: a developmental and comparative perspective. AB - The appearance of puffs on the polytene chromosomes of insect salivary glands incubated with 20-hydroxyecdysone provided the first demonstration that steroids act directly at the gene transcriptional level to bring about subsequent cellular changes (Becker, 1959; Clever and Karlson, 1960). Despite that auspicious beginning, learning about the molecular mechanisms that underlie the hormonal regulation of insect development was impeded for many years by the difficulty associated with isolating and identifying rare regulatory factors from limited tissue sources. The advent of recombinant DNA methodology and powerful techniques such as the polymerase chain reaction (PCR) along with the recognition that many important endocrine factors are structurally conserved across a wide range of species has, however, all but eliminated the technical obstacles once facing the insect endocrinologist trying to isolate and study these regulatory molecules. This review will discuss recent progress and recall some earlier experiments concerning the molecular basis of hormonal action in insects focusing primarily on the members of the nuclear hormone receptor superfamily in Drosophila melanogaster. Two members of this family comprise the functional ecdysteroid receptor and at least a dozen other "orphans" have been identified in Drosophila for which no cognate ligand has yet been found. Many of these orphans are regulated by ecdysteroids. A discussion of juvenile hormone binding proteins that are not family members has been included because of their potential impact on nuclear receptor function. As receptor homologues have been identified in other insects, several general ideas concerning insect hormonal regulation have begun to emerge and these will be examined from a comparative point of view. PMID- 7550247 TI - Highly polymorphic repetitive sequences in Rhynchosciara americana genome. AB - Rhynchosciara americana genomic DNA when digested with EcoR1 or BamH1 presents visible fragments suggestive of repetitive sequences after fractionation on EtBr stained agarose gels. The cloning and molecular analysis of some of these fragments showed a highly polymorphic family of repetitive sequences. These were mapped by in situ hybridization to telomeres and some heterochromatic regions on polytene chromosomes. PMID- 7550248 TI - Primary structure of a novel neuropeptide isolated from the corpora cardiaca of periodical cicadas having adipokinetic and hypertrehalosemic activities. AB - A new neuropeptide hormone was isolated from the corpora cardiaca of the periodical cicadas, Magicicada species. Primary structure of the peptide as determined by a combination of automated Edman degradation after enzymatic deblocking with pyroglutamate aminopeptidase and mass spectrometry is: pGlu-Val Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH2. Synthetic peptide assayed in the green stink bug Nezara viridula caused a 112% increase in hemolymph lipids at a dose of 0.625 pmol, and a 67% increase in hemolymph carbohydrates at a dose of 2.5 pmol. Based on these results we designate this peptide, a first from order Homoptera, as Magicicada species-adipokinetic hormone (Mcsp-AKH). PMID- 7550249 TI - Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms. AB - A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced including 2015 bp of 5' untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3' untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis. PMID- 7550250 TI - Proceedings of the Gatorade Sports Science Institute Conference on Nutrition Ergogenic aids. November 1994. PMID- 7550251 TI - Fat loading: the next magic bullet? AB - The depletion or reduction of bodily carbohydrate reserves is associated with fatigue during endurance exercise. Various carbohydrate supplementation and exercise regimens have been used experimentally to increase carbohydrate reserves before exercise or to maintain the availability of carbohydrate for oxidation during exercise. On the other hand, the improved endurance capability observed after aerobic training has been attributed to an increased oxidation of fat relative to carbohydrate; this carbohydrate sparing presumably delays the point at which reduced carbohydrate reserves cause fatigue. This effect has led to the suggestion that a greater availability of fat during exercise can improve performance via the carbohydrate-sparing effect of "fat loading." Although this is a plausible hypothesis, it is not supported by a sufficient number of valid, credible, and replicated studies. Thus, it appears prudent to advise endurance athletes to consume a diet that is largely carbohydrate to optimize training and competitive performance and, more importantly, to promote optimal health. PMID- 7550252 TI - Creatine and its application as an ergogenic aid. AB - Phosphocreatine (PCr) availability is likely to limit performance in brief, high power exercise because the depletion of PCr results in an inability to maintain adenosine triphosphate (ATP) resynthesis at the rate required. It is now known that the daily ingestion of four 5-g doses of creatine for 5 days will significantly increase intramuscular creatine and PCr concentrations prior to exercise and will facilitate PCr resynthesis during recovery from exercise, particularly in those individuals with relatively low creatine concentrations prior to feeding. As a consequence of creatine ingestion, work output during repeated bouts of high-power exercise has been increased under a variety of experimental conditions. The reduced accumulation of ammonia and hypoxanthine in plasma and the attenuation of muscle ATP degradation after creatine feeding suggest that the ergogenic effect of creatine is achieved by better maintaining ATP turnover during contraction. PMID- 7550253 TI - Effects of bicarbonate, citrate, and phosphate loading on performance. AB - Since the 1930s, scientists have attempted to determine if increasing the body's ability to buffer metabolic acids will enhance physical performance. The buffer of major interest has been bicarbonate; to a lesser degree, citrate and phosphate salts have been investigated. In theory, the buffers facilitate performance by decreasing the accumulation of hydrogen ions that would otherwise presumably inhibit glycolysis and interfere with energy production or impair cross-bridge formation between myofilaments and thereby reduce force production. Literature findings indicate variable results, but overall it appears that bicarbonate salts taken at dosages of 0.3 g.kg-1 may improve performance during repeated sprints or at the end of a progressively more intense exercise test. Athletes are advised of potential ill effects of bicarbonate ingestion, such as gastrointestinal distress. Prior to applying the agents in a competitive setting, athletes should test the effects of buffers on performance during training sessions and consider the sport governing body's stand on buffer usage. PMID- 7550254 TI - Antioxidants, carnitine, and choline as putative ergogenic aids. AB - Three nutritional products that have very different mechanisms of action are antioxidant vitamins, carnitine, and choline. Antioxidant vitamins do not appear to have a direct effect on physical performance in well-fed people but have been touted for their ability to detoxify potentially damaging free radicals produced during exercise. Carnitine purportedly enhances lipid oxidation, increases VO2max, and decreases plasma lactate accumulation during exercise. However, studies of carnitine do not generally support its use for ergogenic purposes. Choline supplements have been advocated as a means of preventing the decline in acetylcholine production purported to occur during exercise; this decline may reduce the transmission of contraction-generating impulses across the skeletal muscle, an effect that could impair one's ability to perform muscular work. However, there are no definitive studies in humans that justify choline supplementation. Much of the scientific data regarding the aforementioned nutrients are equivocal and contradictory. Their potential efficacy for improving physical performance remains largely theoretical. PMID- 7550255 TI - Dietary carbohydrate as an ergogenic aid for prolonged and brief competitions in sport. AB - Reduction of body stores of carbohydrate and blood glucose is related to the perception of fatigue and the inability to maintain high-quality performance. This has been clearly shown with aerobic, endurance events of moderate intensity of over 90 min duration. Carbohydrate intake may also have relevance for athletes involved in short, high-intensity events, especially if body weight control is an issue. Prevention of carbohydrate depletion begins with a high-carbohydrate training diet of about 60-70% carbohydrate. If possible, carbohydrate beverages should be consumed during the event at the rate of 30-70 g/hr to reduce the chance of body carbohydrate depletion. Finally, replacement of body carbohydrate stores can be achieved most rapidly if 40-60 g of carbohydrate is consumed as soon as possible after the exercise and at repeating 1-hr intervals for at least 5 hr after the event. PMID- 7550256 TI - Carbohydrates, branched-chain amino acids, and endurance: the central fatigue hypothesis. AB - The mechanisms of central fatigue are largely unexplored, but the central fatigue hypothesis suggests that increased brain serotonin (5-HT) can cause a deterioration in sport and exercise performance. There is now convincing evidence that exercise-induced increases in the plasma free tryptophan (f-TRP)/branched chain amino acids (BCCA) ratio are associated with increased brain 5-HT and the onset of fatigue during prolonged exercise. Furthermore, when drugs are administered to alter brain 5-HT, they have the predicted effects on exercise performance. The influence of nutritional manipulations of f-TRP/BCCA on performance is less well established. The effects of BCCA supplementation on exercise performance are mixed, and the published studies often suffer from methodological flaws. Alternatively, dramatic reductions in f-TRP/BCCA and enhanced performance accompany carbohydrate feedings during prolonged exercise. However, it is difficult to distinguish between the effects of carbohydrate feedings on mechanisms that reside in the brain versus the muscles themselves. PMID- 7550257 TI - Do athletes need more dietary protein and amino acids? AB - The current recommended daily allowance (RDA) for protein is based primarily on data derived from subjects whose lifestyles were essentially sedentary. More recent well-designed studies that have employed either the classic nitrogen balance approach or the more technically difficult metabolic tracer technique indicate that overall protein needs (as well as needs for some specific individual amino acids) are probably increased for those who exercise regularly. Although the roles of the additionally required dietary protein and amino acids are likely to be quite different for those who engage in endurance exercise (protein required as an auxiliary fuel source) as opposed to strength exercise (amino acids required as building blocks for muscle development), it appears that both groups likely will benefit from diets containing more protein than the current RDA of 0.8 g.kg-1.day-1. Strength athletes probably need about 1.4-1.8 g.kg-1.day-1 and endurance athletes about 1.2-1.4 g.kg-1.day-1. PMID- 7550258 TI - Fluid needs in hot and cold environments. AB - Of all the physiological perturbations that can cause early fatigue during exercise, dehydration is arguably the most important, if only because the consequences of dehydration are potentially life threatening. The rise in body temperature that normally accompanies exercise stimulates an increase in blood flow to the skin and the onset of sweating. Normal hydration is protective of these thermoregulatory responses, whereas even a slight amount of dehydration results in measurable declines in cardiovascular and thermoregulatory function. Mild to severe dehydration commonly occurs among athletes, even when fluid is readily available. This voluntary dehydration compromises physiological function, impairs exercise performance, and increases the risk of heat illness. Recent research illustrates that maintaining normal hydration (or close to it) during exercise maintains cardiovascular and thermoregulatory responses and improves exercise performance. Consequently, it is in the athlete's best interest to adopt fluid-replacement practices that promote fluid intake in proportion to sweat loss. PMID- 7550259 TI - Micronutrients (magnesium, zinc, and copper): are mineral supplements needed for athletes? AB - Mineral elements, including magnesium, zinc, and copper, are required by the body in modest amounts for the maintenance of health and for the development of optimal physiological function. For athletes, adequate amounts of these minerals are required for physical training and performance. Studies of athletes during training, as compared to nontraining control subjects, indicate the potential for increased losses of minerals in sweat and urine. Some studies report suboptimal intakes of minerals, particularly among athletes who are actively attempting to lose weight to meet standards for competition. However, most athletes consume diets that provide adequate amounts of minerals to meet population standards. Athletes should be counseled to consume foods with high nutrient density rather than to rely on mineral supplements. General use of mineral supplements can alter physiological function and impair health. PMID- 7550260 TI - Caffeine and performance. AB - Caffeine ingestion (3-9 mg/kg body weight) prior to exercise increases performance during prolonged endurance exercise and short-term intense exercise lasting approximately 5 min in the laboratory. These results are generally reported in well-trained elite or recreational subjects. However, there is a lack of well-controlled field studies to determine the applicability of laboratory results to the athletic world. Caffeine does not appear to enhance performance during incremental exercise tests lasting 8-20 min and during sprinting lasting less than 90 s, although research examining sprinting is rare. In addition, the mechanisms responsible for any improvement in endurance and short-term exercise have not been clearly established. The ergogenic effects of caffeine are present with urinary caffeine levels that are below the limit of 12 micrograms/ml allowed by the International Olympic Committee, which raises serious ethical issues regarding the use of caffeine to improve athletic performance. One solution would be to add caffeine to the list of banned substances, thereby requiring athletes to abstain from caffeine ingestion 48-72 hr prior to competition. PMID- 7550261 TI - Experience with 500 extracorporeal shockwave lithotripsy patients using a low cost unit: the "Econolith". AB - The Econolith (Medispec Ltd.) is a modular extracorporeal shockwave (SWL) system that uses the spark gap as its source for lithotripsy. In this study, the immediate and late results and complications in 500 patients (640 treatments) treated with this machine during the last 2 years were summarized. An average fragmentation rate of 86% was achieved after the first treatment. A stone-free situation at 3 months was achieved in 75% of the cases; 18% had small (< 5mm) fragments, and 7% had larger fragments. Auxiliary procedures were required in 8% of the patients. Minor complications such as hematuria, colic, and urinary tract infection were seen in 6% of the patients. The Econolith system is safe and effective. Its mobility and low cost enable any urologist to use this technology even with a low monthly turnover of patients. PMID- 7550262 TI - Early experience with extracorporeal shockwave Dornier lithotriptor "compact". AB - One of the latest developments in extracorporeal shockwave lithotripsy (SWL) is a combination of an electromagnetic energy source with an upgraded parallel online ultrasound imaging for localization. The device is compact, requiring no significant installation or site preparation. Furthermore, it increases the margin of safety of SWL by virtue of the continuous ultrasound monitoring. A hundred sessions of SWL were performed on 88 patients using the Dornier Compact machine. Ninety three renal units having an average of 1.7 stones were treated (two had upper ureteric stones). Of the 135 stones, 2 (1.5%) were radiolucent and 4 (3%) were of faint opacity. Intravenous sedation was used in all patients except one 13-year-old patient, who required general anesthesia. Patients received an average of 2409 shocks per session, and the maximum power setting ranged from 1 to 6 (average 4.7). A plain film was obtained immediately before and after treatment. Early adverse effects were uncommon, and all were mild. Patients were followed by plain films at 2 weeks and 3 months. In 35 sessions (25 solitary and 10 multiple stones), the stones were considered completely fragmented; in 63 sessions, the stones (43 solitary and 20 multiple) were judged to be partial fragmented; and 2 sessions (solitary stones) resulted in poor fragmentation. The mean (+/- SD) stone dimension in the group with complete fragmentation was 9 +/- 4.8 (range 2-24) mm, compared with 11 +/- 6.3 (range 2 38) mm in those with no or partial fragmentation (P = 0.0095). After 2 weeks, 23 of 69 systems (33%) were stone free, while 46 showed residual stones.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550263 TI - Safety and effectiveness of Lithostar shock tube C in the treatment of urinary calculi. AB - Over 14 years of clinical use of extracorporeal shockwave lithotripsy (SWL), great technical modifications resulted in the development of many second generation lipthotripters. The Siemens Lithostar machine, with its standard shockwave tube, was introduced in 1986. The objective of this study was to assess the safety and effectiveness of the newly proposed Lithostar shock tube C in the treatment of urinary calculi. Between July 1992 and August 1993, 319 patients (214 males and 105 females, average 49.7 years) with 433 renal or ureteral stones or both were treated at five centers in Canada and the United States. Most of the stones (72%) were located in the kidneys, while 28% were located in the ureters. Most (81%) of the treated sides (side = kidney and ureter) presented with single stones, 11% presented with two stones, and 8% presented with three or more stones. The average stone burden was 13.6 mm. The average duration of treatment for the whole population of patients was 39.3 minutes using an average number of shockwaves of 3633 in a minimum and maximum energy setting of 0.11 and 3.82, respectively. The majority of treatments (92%) were performed without anesthesia. Fragmentation was achieved in 93.5% of treatments, with a 3-month stone-free rate of 62.5% and a success rate (stone free or fragment < 5 mm) of 72%. Auxiliary procedures were necessary in conjunction with 108 treatments, and most of them were in form of catheter/stent placement. Treatment applied on a separate occasion to different stones but in the same collecting system (either a kidney or a ureter) were considered retreatments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550264 TI - New Lithostar treatment technique for difficult upper ureteral stones. AB - In situ treatment of ureteral stones with the Siemens Lithostar is highly successful. In a rare patient, however, despite various positioning maneuvers, an upper ureteral or lower pole renal stone overlies vertebral bone when the shock head is raised. Unless the patient chooses to delay treatments until the stone has moved distally, such patients would ordinarily be forced to undergo percutaneous stone manipulation or ureteroscopy. We describe a technique to facilitate in situ Lithostar treatment of these uncommon but vexing upper ureteral stones using the contralateral shock head with stone side-posterior oblique positioning. Nine of ten patients were stone free at 3 months without further treatment. PMID- 7550265 TI - Modification of Dornier HM-4 lithotriptor for pediatric extracorporeal shockwave lithotripsy. AB - Extracorporeal shockwave lithotripsy has enjoyed increasing acceptance and utility in pediatric urology. The Dornier HM-4 lithotriptor has had good results in adults, but the gantry design does not allow smaller children to be treated. We have employed a specially designed yet simple 'spacer' to allow smaller children to be treated safely. The device may also have application in other patients with deformities who could not be treated in the past. PMID- 7550266 TI - Severe perinephric hemorrhage after shockwave lithotripsy. AB - We report a case of a 69-year-old man who, after a second session of shockwave lithotripsy for multiple stones in the right kidney, showed symptoms of severe hemorrhage and flank pain unresponsive to analgesics, with the gradual development of extensive and serious perinephric hematoma. The bleeding necessitated nephrectomy. Unrecognized chronic pyelonephritis may have been a predisposing factor. PMID- 7550267 TI - Cost-effectiveness v patient preference in the choice of treatment for distal ureteral calculi: a literature-based decision analysis. AB - Ureteroscopy (URS) and extracorporeal shockwave lithotripsy (SWL) battle for supremacy in the management distal ureteral calculi. In order to clarify issues surrounding this controversy, we created a decision tree modeling URS or SWL with literature-based probabilities and used as endpoints both cost and patient preferences. Ureteroscopy was more successful than single-session or multiple session SWL, 92.1% v 74.3% or 84.5%, and had a lower retreatment/complication rate. Although initial SWL was only slightly more expensive than URS, $4,420 v $4,337, the difference increased when the additional costs of complications and retreatment were calculated, $6,745 v $5,555. Using values for an "average" patient, SWL was preferred to URS in terms of patient satisfaction. The most important factors distinguishing between URS and SWL were the success of treatment, the cost of initial therapy, and patient attitudes toward unplanned ancillary procedures and retreatment. Although no alteration of success rates and cost figures within reasonable ranges made URS less cost-effective than SWL, individual differences in patients' aversion for complications allowed URS to be preferred to SWL in some situations. Therefore, SWL is less cost-effective than URS and is not necessarily preferred by patients. The physician should be aware of the principal determinants of the choice between URS and SWL treatment of distal ureteral calculi. PMID- 7550269 TI - Preliminary experience with holmium: YAG laser lithotripsy. AB - Lithotripsy with the pulsed-dye laser has been shown to be a safe and effective method of intracorporeal urinary stone fragmentation. Recently, a new device, the holmium: YAG laser, has been developed with both soft tissue and lithotripsy applications. We present our preliminary experience with the Ho:YAG laser for lithotripsy of urinary calculi. Ureteroscopy and lasertripsy resulted in successful fragmentation of ureteral stones in 92% of 21 patients when the laser was used alone or in combination with electrohydraulic lithotripsy. One complication directly related to the use of the laser occurred, a ureteral perforation, when the device was utilized under fluoroscopic control. In four patients undergoing percutaneous flexible nephrolithotripsy, the laser demonstrated utility in fragmenting caliceal stones remote from the nephrostomy tract. In summary, the Ho:YAG laser has demonstrated its efficacy as a method of intracorporeal lithotripsy. The tissue effects of this device, as well as the optimal energy/pulse and frequency settings, need further evaluation in order to minimize the risks of tissue injury. PMID- 7550268 TI - Special considerations in the endourologic management of stones in continent urinary reservoirs. AB - To various degrees, all continent pouch designs are subject to stones, which often are infected. We report on the endourologic management of large stone burdens in three types of continent reservoirs. Stone in a UCLA and a Kock pouch were managed endoscopically, and stones in an augmented pouch with a Mitrofanoff valve were managed percutaneously. Recommendations are made with regard to the optimal endourologic management of significant stone burdens in each of the common continent urinary reservoirs. PMID- 7550270 TI - Sixteen-month experience with video-assisted extraperitoneal laparoscopic bladder neck suspension. AB - After an extensive favorable experience with the Lapides-Ball open retropubic bladder neck suspension, we chose to perform this operation laparoscopically. We describe video-assisted extraperitoneal laparoscopic bladder neck suspension (VELBNS), in which we employ a single laparoscopic cannula and a laparoscopic bladder suspension set for suturing and simultaneously use video-assisted cystoscopy and laparoscopy for precise suture placement. Over 16 months, we operated on 70 patients with stress urinary incontinence secondary to hypermobility of the urethra. We describe the patient evaluation and selection and the surgical principles and techniques. Five procedures were converted to open operations because of bladder lacerations (two cases) or inability to dissect the space of Retzius because of scar tissue (three cases). At 3 months, 4 patients had unresponsive de novo urgency incontinence, and 61 patients were dry and without protection. One patient had a recurrence at 6 months that was corrected by open surgery. All 12 patients seen at 1 year were dry. We intend to survey our patients annually to obtain long-term results. PMID- 7550271 TI - Laparoscopic adrenalectomy: clinical results in 25 patients. AB - Adrenal tumors would be eminently suitable for laparoscopic surgery because of their size and location. We have performed 25 cases of laparoscopic adrenalectomy, and the purpose of this study was to evaluate the clinical results. Since November 1992, 12 men and 13 women with a mean age of 47.3 (range 29-71) years underwent laparoscopic adrenalectomy. Fifteen lesions were on the left side, and ten lesions were on the right side. The clinical diagnosis was primary aldosteronism in eight patients, Cushing's syndrome in seven, and a nonfunctioning adrenal tumor in ten. The mean operating time was 254 +/- 72 minutes (range 155-412 minutes), but the time on the last 10 cases was 216 +/- 40 minutes, which was equal to the mean operating time of the 15 open adrenalectomy cases (190 +/- 66 minutes). There was no difference in the operating time by clinical diagnosis. The time to first oral intake after the operation was shorter in the laparoscopic group, the need for analgesics was less, and the hospital stay and the time until return to preoperative activity were shorter than after open surgery. The laparoscopic cases had no significant complications, and every operation was performed completely. Although there was the learning curve for the performance of laparoscopic adrenalectomy, its operating time was equal to that of open surgery, and the postoperative recovery was significantly faster. Therefore, laparoscopic adrenalectomy would be useful compared with open surgery on the basis of invasiveness and cost. PMID- 7550272 TI - Laparoscopy-assisted cystoplasty. AB - A new clinical endoscopic cystoplasty technique is described. The patient presented with a microbladder and a markedly dilated left ureter. One month earlier, he had had a right-side nephrectomy for tuberculosis. Five trocars were introduced: one of 10 mm via the umbilicus, one of 5 mm in each iliac fossa, and one of 11 mm in each flank. We opened the peritoneum and freed the bladder walls to the pelvic floor, dissected free and sectioned the ureter as low as possible, and withdrew it with a loop of intestine through a minilaparotomy. We isolated a segment of intestine and restored continuity. The ureter was anastomosed to the isolated segment and reintroduced into the abdomen. The intestinal segment was taken around the bladder and fixed on each side. One jaw of the EndoGIA was introduced into a small incision in the bladder dome and the other into the intestinal segment, and the instrument was triggered. The operation was concluded by introducing an appropriately oriented conventional Roticulator stapler via the minilaparotomy to grip the bladder-intestinal breach and triggering. The patient's bladder capacity was effectively increased, and 20 months later, he is asymptomatic and the intervals of diurnal micturition are more than 3 hours. PMID- 7550273 TI - Ethanol sclerotherapy for symptomatic simple renal cysts. AB - Twenty-eight patients with 30 symptomatic simple renal cysts were treated by percutaneous aspiration of the cyst and injection of 95% ethanol. All patients were available for follow-up during mean period of 19 (range 14-40) months. In all patients, successful resolution of the cyst with disappearance of the symptoms was observed within 1 month after treatment. During the whole period of follow-up, no patient experienced recurrence of symptoms or required repeat therapy. Of all renal cysts treated, resolution was complete in 25 (83%) and partial (recurrence of < 50% of original cyst volume with no symptoms) in 5 (17%). The degree of response to sclerotherapy (whether complete or partial) correlated significantly with cyst size (P > 0.0008). Major complications were not encountered, while microscopic hematuria was seen in two patients, and low grade fever was observed in another two. Ethanol sclerotherapy is simple, noninvasive, and highly cost-effective and should be recommended for the treatment of symptomatic simple renal cysts. PMID- 7550274 TI - Adhesion formation after transperitoneal nephrectomy: laparoscopic v open approach. AB - The risk of postoperative adhesion formation is a significant concern with transperitoneal laparoscopic surgery. To evaluate the incidence of adhesions after major interventional urologic laparoscopy, 16 pigs underwent either laparoscopic (N = 8) or transperitoneal open (N = 8) nephrectomy. Adhesions occurred at 12.5% of the operative sites in the laparoscopic group compared with 75% of the operative sites of the open group. Adhesions were present along the incision site in all the pigs in the open group compared with 5 of 40 (12.5%) trocar sites in the laparoscopic group. Adhesion number, grade, and extent were significantly greater in the open group than the laparoscopic group for both nephrectomy and access (incision/trocar) sites (p < 0.05). Transperitoneal laparoscopic urologic surgery in the porcine model results in a marked decrease in adhesion formation compared with incisional transabdominal surgery. PMID- 7550275 TI - Laparoscopic cyst decompression in polycystic kidney disease. AB - Three cases of cyst decompression accomplished laparoscopically in patients had autosomal dominant polycystic kidney disease are reported. The patients experienced a sustained resolution of symptoms after cyst decompression. PMID- 7550276 TI - Balloon dilation of the prostate: correlation with magnetic resonance imaging and transrectal ultrasound findings. AB - Alternative treatments for benign prostatic hyperplasia are under intense scrutiny. Initial reports on balloon dilation therapy showed success rates of 60% to 90%, although follow-up was brief. We present a prospective non-blinded study assessing the efficacy of an investigational balloon dilatation catheter (105 Fr at 3 atm) as well as the MRI findings preoperatively and postoperatively. Twenty seven men underwent balloon dilation and have been followed for at least 1 year. Twelve patients (44%) ultimately required definitive transurethral prostatectomy during follow-up. A mild improvement was noted in the symptom score and flow rate in the responder group. Fracture of the anterior commissure was accomplished in only 5 patients (18%) despite diligent efforts. The MRI scans showed no change in the prostate in any patients. Intraoperative transrectal ultrasound scanning suggested that proximal balloon migration can occur. Our experience with this balloon system leads us to recommend that it remain an investigational procedure. PMID- 7550277 TI - Urolithoscintigraphy: preliminary report of a new imaging modality for urolithiasis. PMID- 7550278 TI - Circadian rhythms: peering into the molecular clockwork. AB - Recent developments in our understanding of the molecular basis of circadian rhythms look set to provide one of the most elegant demonstrations of the relationship between the activity of individual genes and the execution of biologically relevant and complex patterns of behaviour. At the same time the lid is being opened on one of the classic 'black boxes' of biology, the circadian clock. PMID- 7550280 TI - Localization in the nervous system of Drosophila melanogaster of a C-terminus anti-peptide antibody to a cloned Drosophila muscarinic acetylcholine receptor. AB - Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly. The staining of a discrete group of neurosecretory cells in the pars intercerebralis of the brain indicates a possible new role for this mAChR in the regulation of neurosecretion. Very little staining is detected in the thoracic nervous system. PMID- 7550279 TI - Effect of lactation on hypothalamic preproenkephalin gene expression. AB - Enkephalin appears to modulate several aspects of reproductive function in female rats. The purpose of this study was to determine if lactation influences preproenkephalin gene expression in one or more hypothalamic nuclei known to be involved in maternal or reproductive behavior and prolactin secretion. Lactating rats were killed on day 3 (LAC 3) or day 10 (LAC 10) of lactation. Controls consisted of regular 4-day cycling rats that were killed on diestrous day 1, with 9 to 12 females per group. We used in situ hybridization histochemistry to assess preproenkephalin gene expression in individual cells in the medial preoptic nucleus, anterior, medial and posterior arcuate nucleus, magnocellular and parvocellular aspects of paraventricular nucleus, and ventromedial nucleus. Preproenkephalin mRNA in the anterior arcuate nucleus increased to reach significance (P < 0.05) at day 10 of lactation. Levels in the medial arcuate nucleus increased significantly (P < 0.001) by day 3 of lactation (LAC 3) and remained elevated on day 10 (LAC 10). No significant differences between lactating and control rats were detected in preproenkephalin mRNA levels in the posterior arcuate nucleus, medial preoptic nucleus or in the ventromedial nucleus. Substantial levels of preproenkephalin mRNA were found in the paraventricular nucleus, particularly in a limited region of the magnocellular portion. However, these levels did not change with lactation. These data provide evidence for differential regulation of the preproenkephalin gene during lactation. This change may contribute to lactational hyperprolactinemia and suppressed GnRH secretion, leading to reproductive acyclicity. PMID- 7550281 TI - Three dimensional culture of pineal cell aggregates: a model of cell-cell co operation. AB - Three dimensional (3-D) cultures of pineal cell aggregates were obtained by constant gyratory shaking the heterogenous cell populations, obtained from the rat pineals, in the DMEM (Dulbecco's modified Eagle's medium). Within 4 days, the pineal cells became organized into a tissue like configuration appearing as a compact ball, evidenced by the scanning electron microscopy. The 3-D aggregates seemed to be mainly composed of pinealocytes (round-oval cells), glial (elongated cells) and other unknown cells. The heterogenous cells were separated by intercellular spaces. The ultrastructural characteristics revealed by transmission electron microscopy exhibited the presence of granular lysosomes, typical of pinealocytes actively involved in the secretion. These pineal cell aggregates secreted melatonin and other indole amines i.e. 5-methoxytryptamine (5 MT), indole acetic acid (IAA), 5-methoxy-3-indole acetic acid (5-MIAA), tryptophol (TOL) and 5-methoxytryptophol (5-MTL) in the culture medium, indicating the functional aspect of pinealocytes. The 3-D aggregates cultures had advantages over the pineal monolayer cultures as, after 4 days of culture, the amounts of indole amines secreted by 3-D aggregates were higher than those secreted by monolayer cultures. Besides, the 3-D aggregates remained functional till 24 days in the gyratory culture conditions. In the continuous perifusion system, the 3-D aggregates secreted melatonin while challanged with isoproterenol. This 3-D model of pineal cell aggregates might be useful, in future, to perform other kinetic studies of the release of indole amines in perifusion experiments as this system allows the maintenance of pineal cells for a long period of time. PMID- 7550283 TI - Resistance of growth hormone secretion to hypoglycemia in the mouse. AB - Although previous studies have demonstrated that acute hypoglycemia inhibits growth hormone (GH) secretion due to stimulation of hypothalamic somatostatin (SS) neurones in the rat, the effect of hypoglycemia on GH secretion has not yet been elucidated in the mouse. In this study, the effects of insulin-induced hypoglycemia on mouse GH secretion, hypothalamic c-fos expression, GH-releasing hormone (GRH) and SS mRNA levels were investigated in conscious male mice. Seven days after implantation of chronic atrial catheters, blood samples were taken every 20 min from 1200-1600 h under unrestrained conditions. Insulin was administered iv every 20 min from 1200-1240 h to induce moderate hypoglycemia (MH) and severe hypoglycemia (SH), respectively. Expression of hypothalamic c-fos protein was examined 30 min and 60 min after induction of hypoglycemia by immunohistochemistry. Hypothalamic GRH and SS mRNA levels were examined 1 h and 3 h after induction of hypoglycemia by Northern blot analysis. The lowest mean plasma glucose levels after insulin injections were 49.1 +/- 4.1 mg/dl and 34.2 +/- 5.6 mg/dl in conscious mice, respectively. However, pulsatile GH secretion was not significantly altered in either group. Although both MH and SH markedly stimulated c-fos expression in specific hypothalamic nuclei including the paraventricular nucleus, they did not induce c-fos protein in the periventricular nucleus. Neither MH nor SH altered hypothalamic GRH or SS mRNA levels. These results suggest that hypoglycemia does not activate SS neurons which inhibit GH secretion in the mouse. PMID- 7550282 TI - Melatonin receptors couple through a cholera toxin-sensitive mechanism to inhibit cyclic AMP in the ovine pituitary. AB - The nature of melatonin receptor-G-protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins. ADP-ribosylation of ovine PT membrane proteins using 32P-NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins. Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX. Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for 125I-MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%). A CTX-sensitive component also mediates the inhibition of forskolin-stimulated cyclic AMP accumulation as pre-treatment of PT cells with CTX (5 micrograms/ml) for 16 h blocked this response. Gs alpha is a major substrate for ADP-ribosylation by CTX, and 16 h pre-treatment of PT cells with CTX (5 micrograms/ml) caused a down regulation of Gs alpha. Northern analysis showed only one major transcript of Gs alpha of about 2 kb, which would encompass all of the known splice variants of the Gs gene. Screening of a cDNA library from ovine PT for Gs-related genes and sequencing of clones, combined with RT-PCR of PT mRNA, revealed no novel products. On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the Gs class of G protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550284 TI - Increased fos expression in preoptic calcitonin gene-related peptide (CGRP) neurones following mating but not the luteinizing hormone surge in female rats. AB - The functional relationship between sexually dimorphic neural populations and sex differences in reproductive functioning is unclear. The present study has investigated the function of the sexually dimorphic, estrogen-receptive, calcitonin gene-related peptide (CGRP) neurones in the female preoptic area by examining patterns of Fos immunoreactivity within these cells in relation to the luteinizing hormone surge and lordosis behaviour. In the first experiment, ovariectomized rats were treated with estradiol alone or estradiol plus progesterone to induce the luteinizing hormone surge. The percentage of CGRP neurones with Fos-positive nuclei was not different in estradiol alone (18 +/- 4%) and estradiol/progesterone-treated (24 +/- 3%) rats although the number of Fos-immunoreactive cells in the medial preoptic nucleus was increased 2-fold (P < 0.01) in estrogen/progesterone-treated rats and 40 +/- 5% of luteinizing hormone releasing hormone neurones were found to express Fos in this group. In the second experiment, ovariectomized rats were treated with estradiol and progesterone and either, mated with a single male or placed in an empty cage, for 30 min. The number of Fos-immunoreactive cells in the medial preoptic nucleus was increased 4 fold in mated rats (P < 0.01) and the percentage of CGRP neurones with Fos positive nuclei increased from 24 +/- 3% to 38 +/- 2% (P < 0.01) in mated animals. No differences were detected in the number of luteinizing hormone releasing hormone neurones with Fos-positive nuclei in mated and non-mated animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550285 TI - A 5-day estradiol therapy, in amounts reproducing concentrations of the early-mid follicular phase, prevents the activation of the hypothalamo-pituitary-adrenal axis by interleukin-1 alpha in the ovariectomized rhesus monkey. AB - In a previous report, we have shown that intracerebroventricular (icv) administration of the cytokine interleukin-1 alpha (IL-1 alpha) in the ovariectomized (OVX) rhesus monkey results in the acute activation of the hypothalamo-pituitary-adrenal (HPA) axis and the inhibition of LH and FSH secretion. Here, we compare the cortisol response to IL-1 alpha administration in OVX monkeys and in OVX animals replaced with estradiol (E) to reproduce E concentrations typical of the early-mid follicular phase. Cortisol, LH and FSH were measured after an icv infusion of physiological saline or IL-1 alpha (2.1 or 4.2 micrograms/30 min) in both groups. E-containing capsules were implanted sc 5 days prior to the experiment. In OVX, E concentrations were < 5 pg/ml. Cortisol concentrations decreased throughout the afternoon after saline infusion (to 49.7 +/- 5.1% of baseline at 5 h; n = 7), but increased significantly after IL-1 alpha to 158.3 +/- 13.8% (n = 7). In OVXE, cortisol also declined after saline (to 76.4 +/- 16.2%; n = 5). There were 2 types of response to IL-1 alpha: in grp 1 (mean E: 18.0 +/- 0.7 pg/ml), the cortisol response was similar to that in OVX (160.8 +/- 17.0%; n = 5), while in grp 2 (E: 30.7 +/- 3.1 pg/ml), the cortisol response was absent (66.6 +/- 7.2% of baseline at 5 h; NS vs saline in OVXE; n = 7). The cortisol response to IL-1 alpha was restored in 2 monkeys when E was increased to > 100 pg/ml, confirming our previous observations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550286 TI - Induction of cellular follicle-stimulating hormone in the hamster adenohypophysis requires intermittent stimulation by luteinizing hormone releasing hormone. AB - We investigated the effectiveness of continuous vs intermittent LHRH stimulation of the neonatal female anterior pituitary gland on inducing cellular FSH immunoreactivity in the Golden Syrian hamster. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hosts with a catheter implanted in the external jugular vein. In experiment 1, vehicle or LHRH (6 ng/h) was infused continuously or LHRH was pulsed at 1 h (6 ng) or 12 h (72 ng) intervals through the catheters for 8 days. Hamsters were decapitated for collection of trunk blood shortly after the end of treatment, and grafts were prepared for immunocytochemical staining for LH and FSH. Anterior pituitary glands removed from neonatal (day 1) and day 9 female pups also were stained for LH and FSH. The mean percentage of adenohypophysial cells staining for LH increased from 11% in neonatal pups to mean percentages (24-28%) that were similar in day 9 pups and in all groups with grafts. The mean percentage of adenohypophysial cells staining for FSH increased from 1% in neonatal pups to percentages (16-21%) that were similar in day 9 pups and in grafts in hosts administered 6 or 72 ng LHRH pulses. By contrast, the mean percentage of FSH cells did not increase in grafts in hosts administered vehicle or LHRH by continuous infusion. Serum LH concentration was low in hosts given vehicle or LHRH by continuous infusion but elevated in hosts given 72 ng LHRH pulses and in all but one host given 6 ng LHRH pulses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550287 TI - Sex-steroid control of galanin in the rat hypothalamic-pituitary axis. AB - To further investigate how sex steroids regulate galanin (GAL) in the rat pituitary and hypothalamus, we examined the effects of prepubertal gonadectomy (Gx) and long-term (9 weeks) replacement with estradiol (E2) or testosterone (T) on pituitary and hypothalamic GAL concentrations in Wistar rats (5-6/group). Sham operated animals served as controls (CTR). Pituitary GAL concentration was markedly higher in random-cycling CTR-females than in CTR-males (1391 +/- 247 vs 39 +/- 5 pg/mg protein, P < 0.01) and decreased after Gx only in females (20 +/- 3 pg/mg protein, P < 0.01). E2 strongly increased pituitary GAL in Gx-females and Gx-males (4470 +/- 365 and 3853 +/- 347 pg/mg protein, P < 0.01), whereas T had no effect. Inversely, hypothalamic GAL was higher in CTR males than in CTR females (5.4 +/- 0.3 vs 4.0 +/- 0.5 ng/mg protein, P < 0.05), and decreased significantly after gonadectomy in males (3.7 +/- 0.2 ng/mg protein, P < 0.01). The only steroid treatment that significantly modified hypothalamic GAL in Gx animals was administration of E2 to females (5.7 +/- 0.4 ng/mg protein, P < 0.01 vs non-treated Gx). We also studied in hypophysectomized (Hx) rats (8/group) the effects of sex steroids on hypothalamic GAL concentration and distribution. The low hypothalamic GAL concentration observed in male and female Hx rats (1.0 +/- 0.1 ng/mg protein) was significantly increased by T in males and in females (respectively, by 40% and by 50%, P < 0.02) and by E2 in males (by 60%, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550288 TI - The emerging concept of relaxin as a centrally acting peptide hormone with hemodynamic actions. AB - The novel finding that relaxin has an action on the brain was first published in 1984. Since then, it has been shown that exogenous relaxin affects the release of a number of hypothalamo-pituitary hormones and has a robust pressor action. In this paper, we review the accumulating evidence that relaxin affects the release of oxytocin and vasopressin by an action at the level of the brain. The potential mechanisms of this central action are discussed and the evidence presented for the interaction between relaxin and the forebrain angiotensin-II system. Furthermore, we articulate the possible physiological influences of relaxin on the changes in cardiovascular function that occur during pregnancy. PMID- 7550289 TI - Antisense vasopressin oligonucleotides: uptake, turnover, distribution, toxicity and behavioral effects. AB - The uptake, turnover, distribution, toxicity and behavioral effects of antisense vasopressin oligonucleotides were investigated to define how these compounds interact with neural tissue to inhibit translation of a target mRNA. Both phosphorothioate modified and unmodified oligonucleotides are rapidly taken up by mammalian neural tissue. Turnover of the unmodified oligonucleotide was found to be fast (t1/2 < 1 h) relative to the phosphorothioate modified oligonucleotide (t1/2 = 12 h). The phosphorothioate vasopressin antisense oligonucleotide suppressed vasopressin synthesis in vivo at concentrations below the toxic threshold of approximately 5 microM. Intracranial injections of phosphorothioate antisense oligonucleotide into the region of the SON in vivo, resulted in a small decrease in vasopressin mRNA and a compensatory drinking response within the first 24 h, consistent with a deficit in vasopressin translation with kinetics similar to those observed in vitro. Water intake returned to normal by the second day indicating relatively rapid clearance of the oligonucleotide and minimal side effects. Although the mechanisms of accumulation and details of the molecular interactions are still unknown, our observation of preferential uptake and/or retention of oligonucleotide within a subset of neurons in vitro suggests some process of selective targeting. Thus, low concentrations of oligonucleotides targeted to the untranslated 5' end of vasopressin mRNA can be effective for the acute and reversible control of vasopressin synthesis in mammalian CNS with relatively rapid onset of behavioral effects and minimal side effects. PMID- 7550290 TI - Disruption of reproductive rhythms and patterns of melatonin and prolactin secretion following bilateral lesions of the suprachiasmatic nuclei in the ewe. AB - To determine whether the photoperiodic responses of reproductive and prolactin (PRL) rhythms in the ewe requires an intact suprachiasmatic nucleus (SCN) driving the pineal rhythm of melatonin secretion, four groups of ovary-intact ewes over a 6-year period were subjected to bilateral (n = 40) or sham lesions (n = 15) of the SCN. Animals were exposed to an alternating 90-120 day photoregimen of 9L:15D and 16L:8D photoperiods. Blood samples taken twice weekly were assayed for prolactin and for progesterone to monitor oestrous cycles. On several occasions blood samples also were taken at hourly intervals for 24 h and analyzed for melatonin. Melatonin concentrations in sham lesioned ewes were basal during the lights-on period and rose robustly during darkness. Those sheep bearing unilateral lesions of the SCN (n = 13) or where the lesion spared the SCN entirely (n = 8) had patterns of melatonin secretion similar to sham ewes. The remaining ewes, having complete (n = 9) or incomplete bilateral (n = 8) destruction of the SCN, with one exception, had disrupted patterns of melatonin secretion. The nature of this disruption varied from complete suppression to continuously elevated levels. In lesioned ewes where melatonin secretion was not affected the onset and cessation of ovarian cycles were similar to sham ewes; stimulation of oestrous cycles under 9L:15D and cessation of oestrous cycles under 16L:8D. In contrast, 13 of 17 ewes with disrupted melatonin secretion also exhibited disrupted patterns of ovarian activity. In these animals oestrous cycles were no longer entrained by photoperiod but still occurred in distinct clusters, that is, groups of cycles began and ended spontaneously. Sheep with normal melatonin patterns showed low levels of PRL secretion during short days and elevated PRL levels during long days. However, 8 of 13 ewes with disrupted melatonin showed patterns of PRL secretion that were no longer entrained by photoperiod. A minority of ewes with disrupted melatonin patterns still showed reproductive (n = 4) and PRL (n = 5) responses similar to those of sham-lesioned ewes. These results show that bilateral destruction of the SCN in the ewe disrupts the circadian pattern of melatonin secretion and that this disruption usually, but not always, is associated with altered photoperiodic responses. These results strongly suggest that the SCN are important neural elements within the photoperiod time-keeping system in this species. A role for the SCN in the generation of endogenous transitions in reproductive activity (refractoriness) and prolactin secretion is not supported. PMID- 7550291 TI - Glutaminyl-cyclase expression in the bovine/porcine hypothalamus and pituitary. AB - The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing. PMID- 7550292 TI - Central oxytocin mediates stress-induced tachycardia. AB - To address the role of oxytocin in the control of cardiovascular reactivity, we examined the effect of central injection of oxytocin, vasopressin and mixed base antisense oligodeoxynucleotides on stress-induced cardiovascular and endocrine changes. Antisense oligomers were injected into the paraventricular nucleus (PVN), 4 h prior to the stress test. The oxytocin antisense abolished the tachycardia produced by 5 min of shaker stress. The blood pressure and plasma oxytocin responses were not different between the groups. PVN levels of OT were reduced in the oxytocin antisense-treated group while brain stem levels were increased. These results demonstrate the importance of a specific peptide system, the PVN/oxytocin axis, in stress-induced tachycardia. Further, the data illustrate the effectiveness of short-term treatment with antisense oligomers on physiological responses. PMID- 7550293 TI - Regulation of corticotropin-releasing factor secretion and synthesis in the human neuroblastoma clones- BE(2)-M17 and BE(2)-C. AB - The BE(2)-M17 and BE(2)-C human neuroblastoma cell lines have been shown to synthesize and secrete corticotropin-releasing factor (CRF) following retinoic acid treatment. It has been demonstrated that CRF secretion and intracellular synthesis increases in response to forskolin treatment. In this report, we have further characterized these cells in response to protein kinase C activators, dexamethasone, interleukin-1 alpha, as well as various neurotransmitters and peptides. Nanomolar concentrations of the phorbol ester--phorbol 12 myristate 13- acetate (TPA), increased intracellular CRF content in both cell lines while increasing secretion only in the BE(2)-M17 cell. Nanomolar concentrations of dexamethasone were not able to alter basal levels of secretion and content in either cell type. However, in the BE(2)-M17 cell but not the BE(2)-C cell, the same concentrations of dexamethasone added to 30 microM forskolin augmented levels of CRF secretion and content. Likewise, the same augmented response in CRF secretion and content was seen only in the BE(2)-M17 cell line when nanomolar concentrations of dexamethasone were added to 20 nM TPA. Furthermore, only in the BE(2)-M17 cell line were micromolar levels of the biogenic amine serotonin able to increase levels of CRF secretion and content. No effects on CRF in both cell lines were demonstrable with picomolar levels of interleukin-1 alpha as well as micromolar levels of acetylcholine, norepinephrine, arginine-vasopressin, oxytocin, and angiotensin-II. The potential usefulness of these cells as models of central nervous system or placental CRF-containing neurons is discussed. PMID- 7550294 TI - S-100 antigen-positive folliculostellate cells are not the source of IL-6 gene expression in human pituitary adenomas. AB - We have investigated the expression of IL-6 in a random selection of 27 human pituitary adenomas, comprising 8 somatotroph, 5 corticotroph, 3 mammotroph and 11 endocrinologically inactive adenomas, using a 35S-labelled 1.1kb riboprobe complementary to human IL-6. Positive and negative IL-6 transcript controls were generated from the IL-6-secreting human bladder carcinoma cell line T24/83. Tissue from a malignant melanoma was used as a positive S-100 immunocytochemical control tissue. Of the 27 human pituitary adenomas examined by in situ hybridization, 7 (26%) contained IL-6 transcripts: these were 3 of 5 corticotroph adenomas, 2 of 8 somatotrophinomas and 2 of 11 endocrinologically inactive adenomas. In each case, IL-6 transcript-positive cells constituted less than 1% of the total pituitary tissue mass examined. Alternate wax embedded 3 microns thick sections from 5 of the 7 IL-6 transcript positive tumours were examined immunocytochemically for S-100 antigen, or by in situ hybridization for IL-6 transcripts. Immunocytochemistry for S-100 antigen was completely negative in 3 of the 5 tumours and in the remaining 2, there was no evidence of IL-6 transcripts and S-100 antigen co-localization in any of the sections examined. This suggests that in pituitary adenomas, cells other than classical folliculostellate cells are responsible for IL-6 production. PMID- 7550296 TI - Caloric intake stimulates growth hormone secretion in food-deprived rats with anterolateral deafferentation of the medial basal hypothalamus or administered antiserum to somatostatin. AB - In rats, food deprivation inhibits episodic growth hormone (GH) secretion. On the basis of previous studies, we hypothesized that during a recovery from prolonged fasting, caloric intake stimulates the release of GH-releasing factor (GRF) and this process does not depend on the specific macronutrients in the meal, while protein in the meal acts to restore characteristic ultradian rhythmicity of GH secretion. To test this hypothesis, the effect of caloric intake on GH secretion was examined in fasted adult male Wistar rats devoid of somatostatin (SS) influence on GH secretion either by anterolateral deafferentation (ALC) of the medial basal hypothalamus (MBH) or administration of anti-SS goat serum (ASS). Rats were provided with an indwelling right atrial cannula and were deprived of food for 72 h. ALC was performed 2 weeks prior to the study. ASS was given i.v. 8 h and 7 h prior to refeeding, respectively. Serial blood specimens were collected every 10 min. In rats with ALC (ALC rats) or rats given ASS (ASS rats), the blood GH level revealed irregularly occurring small fluctuations, instead of the usual high bursts and low trough level. The baseline GH level and the mean GH level of fasted ALC rats or fasted ASS rats were significantly lower than those of fed ALC rats or fed ASS rats. Feeding the isocaloric mixed meal, the protein meal or the protein-deficient meal increased the GH pulse frequency, the pulse amplitude, the baseline GH level and the mean GH level in 72-h fasted ALC rats. These changes in GH secretory pattern persisted during the period of observation and were independent of the type of meal ingested. Following feeding the mixed meal, similar changes in the GH secretory pattern demonstrated in 72-h fasted ALC rats were also observed in 72-h fasted ASS rats, suggesting that the stimulation of GH secretion following caloric intake is not limited to ALC rats. Since the influence of SS on GH secretion has been largely eliminated in ALC or ASS rats, it is highly unlikely that the augmentation of GH secretion following feeding after prolonged food deprivation was the consequence of inhibition of SS secretion. Although GRF measurement was not performed, it is conceivable that the signal of caloric intake is conveyed to the MBH and acts to stimulate GRF release. PMID- 7550295 TI - Contribution of the ventral subiculum to inhibitory regulation of the hypothalamo pituitary-adrenocortical axis. AB - Anatomical studies indicate that the ventral subiculum is in a prime position to mediate hippocampal inhibition of the hypothalamo-pituitary-adrenocortical (HPA) axis. The present study evaluated this hypothesis by assessing HPA function following ibotenic acid lesion of the ventral subiculum region. Rats with lesions of the ventral subiculum (vSUB) or ventral hippocampus (vHIPPO) did not show changes in basal corticosterone (CORT) secretion at either circadian peak or nadir time points when compared to sham-lesion rats (SHAM) or unoperated controls. However, rats with vSUB lesions exhibited a prolonged glucocorticoid stress response relative to all other groups. Baseline CRH mRNA levels were significantly increased in the medial parvocellular paraventricular nucleus (PVN) of the vSUB group relative to controls. CRH mRNA differences were particularly pronounced at caudal levels of the nucleus, suggesting topographic organization of vSUB interactions with PVN neurons. Notably, the vHIPPO group, which received large lesions of ventral CA1, CA3 and dentate gyrus without significant subicular damage, showed no change in stress-induced CORT secretion, suggesting that the ventral subiculum proper is principally responsible for ventral hippocampal actions on the HPA stress response. No differences in medial parvocellular PVN AVP mRNA expression were seen in either the vSUB or vHIPPO groups. The results indicate a specific inhibitory action of the ventral subiculum on HPA activation. The increase in CRH biosynthesis and stress-induced CORT secretion in the absence of changes in baseline CORT secretion or AVP mRNA expression suggests that the inhibitory actions of ventral subicular neurons affect the response capacity of the HPA axis. PMID- 7550297 TI - Knee muscle strength in elite male gymnasts. AB - This study was launched to establish the profile of knee dynamic concentric strength in elite male gymnasts after it was found that three of the 10-member Canadian men's gymnastics team had incurred anterior cruciate ligament (ACL) rupture. The dynamic concentric force characteristics of the quadriceps and hamstring muscles of 84 male gymnasts were studied at the Canadian National Championships using a Kin-Com isokinetic dynamometer. These tests were performed at 90 degrees/sec and 230 degrees/sec and revealed that the hamstrings to quadriceps peak torque ratio was not only unusually low (0.5) when compared with data collected in previous research, but that this ratio was consistent across all ages, from 12 to 27 years. The torque ratios were also reported at 30 degrees, 45 degrees, and 60 degrees and it was found that the ratios decreased as the joint angle increased and again was consistent across the four age groups. It was also found that the hamstrings to quadriceps peak torque ratio did not increase (hamstrings becoming stronger relative to quadriceps) as velocity of movement increased as has been reported in other studies. It was hypothesized that the large shear forces that are generated about the knee in gymnastics (extrinsically from backward landing and intrinsically from the quadriceps eccentrically contracting), combined with the relatively weak hamstrings, could be one cause for the increasing incidence of ACL injuries in that sport. The results of this study indicate that it would be prudent for clinicians involved with gymnasts to test for knee strength imbalance and to prescribe exercises to correct it when necessary. PMID- 7550298 TI - Patellofemoral pain in female ballet dancers: correlation with iliotibial band tightness and tibial external rotation. AB - Review of the literature reveals that ballet dancers have a high incidence of idiopathic patellofemoral pain. Twenty-four female ballet dancers were subjects in a study of the relationship between: 1) iliotibial band (ITB) tightness and patellofemoral pain, and 2) ITB tightness and degrees of tibial external rotation used in the dance demi-plie. Dancers were initially assessed by questionnaire to determine if any had knee pain. Twelve subjects met the study criteria for patellofemoral pain, and 12 dancers without knee pain served as controls for the study, Iliotibial band tightness was measured (Ober test), and degrees of tibial external rotation used during knee flexion (demi-plie) in standing were measured in both legs of all 24 subjects (48 legs). Chi-square analysis of the collected data revealed that there was an association between ITB tightness and patellofemoral pain in the dancers. Data analysis using the Wilcoxon Rank Sum test revealed that the degree of tibial external rotation used by dancers with iliotibial band tightness was significantly greater than those without ITB tightness. This study confirms the assumption that ITB tightness in dancers may be a contributing factor to patellofemoral pain. Follow-up study is indicated to determine if the preservation or restoration of functional ITB length is effective in the prevention and/or treatment of patellofemoral pain in ballet dancers. PMID- 7550299 TI - The effect of knee and foot position on the electromyographical activity of the superficial quadriceps. AB - Isometric quadriceps exercises are used early in knee rehabilitation. Varying knee or foot position is hypothesized to selectively activate specific quadriceps muscles. This study examined the activities of the vastus medialis oblique, vastus lateralis, and rectus femoris during isometric contractions at 90 degrees, 150 degrees, and 175 degrees knee angles with internally rotated, neutral, and externally rotated foot positions. Subjects performed three perceived maximal isometric contractions at each knee angle/foot position while electromyographic activity (EMG) was collected. Statistical analysis consisted of a three-way repeated measures multiple analysis of variance, with post hoc analysis as was appropriate. Although no significant differences were detected among foot positions for the rectus femoris at 90 degrees, this knee angle produced significantly greater EMG activity for the neutral position compared with 150 degrees or 175 degrees. The 90 degrees angle was also superior to 175 degrees for the externally rotated position. The results for both the vastus medialis and vastus lateralis were similar, with the 90 degrees angle producing greater EMG activity than 175 degrees in the externally rotated position. In addition, the neutral position produced significantly more activity than the internally rotated position at 90 degrees. At 150 degrees, the neutral position was superior to the externally rotated position. Finally, at the 175 degrees knee angle, the highest level of EMG activity was with the foot internally rotated. Considering the combination of effects for EMG activity across all muscles tested, the 90 degrees knee angle with a neutral foot position may provide the most effective condition for rehabilitation of all muscles tested.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550300 TI - Case study: the painful os trigonum syndrome. AB - The painful os trigonum syndrome is one cause of posterolateral ankle pain. This syndrome is most prevalent in athletes who perform frequent and/or forceful plantar flexion. The painful os trigonum may be misdiagnosed as Achilles and/or peroneal tendinitis. In this case, the patient was misdiagnosed for 15 months and treated for tendinitis. The appropriate clinical tests to evaluate the os trigonum as a source of posterolateral ankle pain are outlined. The surgical and postoperative management for the patient are discussed. Clinicians should be aware of the painful os trigonum syndrome as a possible source of posterolateral ankle pain. PMID- 7550302 TI - Video views. PMID- 7550301 TI - Factors associated with menstrual irregularities and decreased bone mineral density in female athletes. AB - Menstrual irregularities occur in some female athletes. The most extreme form of menstrual irregularity is amenorrhea, which has been linked to significant decreases in vertebral bone density and increases in injury prevalence. Many authors have sought to determine the causal factors of athletic amenorrhea, some of which include hormonal status, training and physical parameters, nutritional balance, and psychological stress. The purpose of this paper was to compare studies that have examined the relationship of these variables to menstrual irregularities and bone density. Controversy exists regarding the relative contribution of these variables. The etiology is likely multifactorial and should be evaluated as such. Clinicians treating female athletes must be knowledgeable about the negative consequences associated with menstrual irregularities. Furthermore, it is critical that clinicians provide thorough patient education in order to prevent injuries and the long-term loss of bone density. Appropriate medical and/or psychological referral of the athlete with menstrual irregularities may be necessary. PMID- 7550303 TI - The obsession with obesity. PMID- 7550304 TI - Ceruloplasmin gene defect associated with epilepsy in EL mice. PMID- 7550306 TI - ITD in Ashkenazi Jews--genetic drift or selection? PMID- 7550305 TI - Reeler gene discrepancies. PMID- 7550307 TI - A major founder Y-chromosome haplotype in Amerindians. PMID- 7550308 TI - The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues. AB - We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17 beta-estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones. PMID- 7550309 TI - Night blindness in Sorsby's fundus dystrophy reversed by vitamin A. AB - Sorsby's fundus dystrophy (SFD) is an autosomal dominant retinal degeneration caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. Mechanisms of the visual loss in SFD, however, remain unknown. In a SFD family with a novel TIMP3 point mutation, we tested a hypothesis that their night blindness is due to a chronic deprivation of vitamin A at the level of the photoreceptors caused by a thickened membrane barrier between the photoreceptor layer and its blood supply. Vitamin A at 50,000 IU/d was administered orally. Within a week, the night blindness disappeared in patients at early stages of disease. Nutritional night blindness is thus part of the pathophysiology of this genetic disease and vitamin A supplementation can lead to dramatic restoration of photoreceptor function. PMID- 7550310 TI - Human embryo use in developmental research. PMID- 7550311 TI - Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue. AB - Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease. PMID- 7550312 TI - Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. AB - GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis. PMID- 7550313 TI - Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. AB - In humans, deficiency of the tissue non-specific alkaline phosphatase (TNAP) gene is associated with defective skeletal mineralization. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem (ES) cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal. Defective metabolism of pyridoxal 5'-phosphate (PLP), characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma aminobutyric acid (GABA) in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet. Rescued animals subsequently develop defective dentition. This study reveals essential physiological functions of TNAP in the mouse. PMID- 7550314 TI - Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization. AB - Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only. PMID- 7550315 TI - Heterogeneous hypertension. PMID- 7550316 TI - Interaction between undulated and Patch leads to an extreme form of spina bifida in double-mutant mice. AB - The aetiology of spina bifida involves genetic and environmental factors, which may be why major genes contributing to pathogenesis have not been identified. Here we report that undulated-Patch double-mutant mice have a phenotype reminiscent of an extreme form of spina bifida occulta in humans. This unexpected phenotype in double-mutant but not single-mutant mice shows that novel congenital anomalies such as spina bifida can result from interaction between products of independently segregating loci. This example of digenic inheritance may explain the often sporadic nature of spina bifida in humans. PMID- 7550317 TI - MSH2 deficient mice are viable and susceptible to lymphoid tumours. AB - Alterations of the human MSH2 gene, a homologue of the bacterial MutS mismatch repair gene, co-segregate with the majority of hereditary non-polyposis colon cancer (HNPCC) cases. We have generated homozygous MSH2-/- mice. Surprisingly, these mice were found to be viable, produced offspring in a mendelian ratio and bred through at least two generations. Starting at two months of age homozygous-/ mice began, with high frequency, to develop lymphoid tumours that contained microsatellite instabilities. These data establish a direct link between MSH2 deficiency and the pathogenesis of cancer. These mutant mice should be good models to study the progression of tumours and also to screen carcinogenic and anti-cancer agents. PMID- 7550318 TI - Epileptic seizures caused by inactivation of a novel gene, jerky, related to centromere binding protein-B in transgenic mice. AB - Epidemiological data and genetic studies indicate that certain forms of human epilepsy are inherited. Based on the similarity between the human and mouse genomes, mouse models of epilepsy could facilitate the discovery of genes associated with epilepsy syndromes. Here, we report an insertional murine mutation that inactivates a novel gene and results in whole body jerks, generalized clonic seizures, and epileptic brain activity in transgenic mice. The gene, named jerky, encodes a putative 41.7 kD protein displaying homology to a number of nuclear regulatory proteins, suggesting that perhaps the jerky protein is able to bind DNA. PMID- 7550319 TI - Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. AB - Sensitivity of blood pressure to dietary salt is a common feature in subjects with hypertension. These features are exemplified by the mendelian disorder, Liddle's syndrome, previously shown to arise from constitutive activation of the renal epithelial sodium channel due to mutation in the beta subunit of this channel. We now demonstrate that this disease can also result from a mutation truncating the carboxy terminus of the gamma subunit of this channel; this truncated subunit also activates channel activity. These findings demonstrate genetic heterogeneity of Liddle's syndrome, indicate independent roles of beta and gamma subunits in the negative regulation of channel activity, and identify a new gene in which mutation causes a salt-sensitive form of human hypertension. PMID- 7550320 TI - Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa. AB - Junctional epidermolysis bullosa (JEB) is a heterogeneous autosomal recessively inherited blistering skin disorder associated with fragility at the dermal epidermal junction. Characteristic ultrastructural findings in JEB are abnormalities in the hemidesmosome-anchoring filament complexes. These focal attachment structures, which extend from the intracellular compartment of the basal keratinocytes to the underlying basement membrane, have been shown to be hypoplastic or rudimentary in different forms of JEB. Previously, in different JEB phenotypes, mutations have been found in the three genes for the anchoring filament component laminin 5 (LAMA3, LAMB3, and LAMC2) and in the gene for the hemidesmosome-associated integrin beta 4 subunit. Here, we describe the first mutations in the gene encoding the 180-kD bullous pemphigoid antigen (BPAG2), a transmembranous hemidesmosomal collagen, also known as type XVII collagen (COL17A1). The patient is affected with generalized atrophic benign epidermolysis bullosa (GABEB), a rare variant of JEB, and is a compound heterozygote for premature termination codons on both alleles. These novel findings emphasize the molecular heterogeneity of this group of genodermatoses, and attest to the importance of BPAG2 in maintaining adhesion between the epidermis and the dermis. PMID- 7550321 TI - Dominant mutations in the type II collagen gene, COL2A1, produce spondyloepimetaphyseal dysplasia, Strudwick type. AB - The chondrodysplasias are a heterogeneous group of disorders characterized by abnormal growth or development of cartilage. Current classification is based on mode of inheritance as well as clinical, histologic, and/or radiographic features. A clinical spectrum of chondrodysplasia phenotypes, ranging from mild to perinatal lethal, is due to defects in the gene for type II collagen, COL2A1. This spectrum includes Stickler syndrome, Kniest dysplasia, spondyloepiphyseal dysplasia congenita (SEDC), achondrogenesis type II, and hypochondrogenesis. Individuals affected with these disorders exhibit abnormalities of the growth plate, nucleus pulposus, and vitreous humor, which are tissues that contain type II collagen. The Strudwick type of spondyloepimetaphyseal dysplasia (SEMD) is characterized by disproportionate short stature, pectus carinatum, and scoliosis, as well as dappled metaphyses (which are not seen in SEDC). The phenotype was first described by Murdoch and Walker in 1969, and a series of 14 patients was later reported by Anderson et al. The observation of two affected sibs born to unaffected parents led to the classification of SEMD Strudwick as an autosomal recessive disorder. We now describe the biochemical characterization of defects in alpha 1(II) collagen in three unrelated individuals with SEMD Strudwick, each of which is due to heterozygosity for a unique mutation in COL2A1. Our data support the hypothesis that some cases, if not all cases, of this distinctive chondrodysplasia result from dominant mutations in COL2A1, thus expanding the clinical spectrum of phenotypes associated with this gene. PMID- 7550322 TI - Gotta have GATA. PMID- 7550323 TI - Benign neurofibromas in type 1 neurofibromatosis (NF1) show somatic deletions of the NF1 gene. AB - Neurofibromatosis type 1 (NF1) is one of the most common human autosomal dominant diseases. NF1 is characterized by cafe-au-lait spots (CLS), axillary freckles and Lisch nodules of the iris. Another hallmark of NF1 is the development of neurofibromas, benign tumours that arise from peripheral nerve sheaths. NF1 patients also have an increased incidence of certain malignant tumours. Malignancies in NF1 are believed to follow the 'two-hit' hypothesis, in which one allele is constitutionally inactivated while the other allele is subsequently inactivated ('second hit') at the somatic level. This hypothesis has not, however, been fully tested in the aetiology of benign neurofibromas. This is a crucial issue since it addresses not only the basic mechanism behind the genesis of neurofibromas, but may also indicate a mechanism common to many or all NF1 features. Using both NF1 intragenic polymorphisms as well as markers from flanking and more distal regions of chromosome 17, we have investigated loss of heterozygosity (LOH) in 22 neurofibromas from five unrelated NF1 patients. Eight of these tumours revealed somatic deletions involving NF1, indicating that inactivation of NF1 is associated with at least some neurofibromas. PMID- 7550324 TI - A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36. AB - Sacral agenesis is a rare disorder of uncertain incidence that has been reported in diverse populations. Although usually sporadic and most commonly associated with maternal diabetes, there is a hereditary form which may occur in isolation or with a presacral mass (anterior meningocele and/or presacral teratoma) and anorectal abnormalities, which constitute the Currarino triad (MIM 176450). The radiological hallmark of hereditary sacral agenesis is a hemi-sacrum (sickle shaped sacrum) with intact first sacral vertebra. Bowel obstruction is the usual neonatal presentation, but, unlike other neural tube defects, adult presentation is not uncommon. The major pathology is confined to the pelvic cavity and may present as a space-occupying lesion or meningitis due to ascending infection. All recurrences in families have been compatible with autosomal dominant inheritance except for those associated with the isomerism gene at Xq24-q27.1 (ref. 3). Several associated cytogenetic defects have been reported, including 7q deletions. Previous studies failed to detect linkage to HLA markers, but we now present evidence for a location on 7q36. The same region also contains a gene for holoprosencephaly, an early malformation of the extreme rostral end of the neural tube. PMID- 7550325 TI - Mutational hotspot in the human biotinidase gene causes profound biotinidase deficiency. AB - Biotinidase deficiency is an autosomal recessive inherited disorder that is characterized by neurological and cutaneous symptoms. Biotinidase-deficient children cannot recycle endogenous biotin, an essential water-soluble B vitamin. Biotin is covalently attached to epsilon-amino groups of lysyl residues of four carboxylases. These carboxylases are subsequently degraded to biocytin (biotin epsilon-lysine). Biotinidase cleaves biocytin to biotin and lysine, thereby completing the biotin cycle. The symptoms of biotinidase deficiency can be resolved or prevented by treatment with biotin. Therefore, it is important that biotinidase deficiency is diagnosed early so that permanent neurological damage can be prevented. Many states and countries currently perform newborn screening for biotinidase deficiency. We have recently isolated and characterized the cDNA for normal human biotinidase and localized the gene to chromosome 3p25 (ref. 9). We have now identified the first mutation that causes profound biotinidase deficiency. It occurs in a distinct region of the gene that encodes the putative signal peptide. Fifty percent of symptomatic children studied have a 7-bp deletion coupled with a 3-bp insertion in at least one of their alleles of the biotinidase gene. This mutation appears to be a common cause of biotinidase deficiency in symptomatic children. PMID- 7550326 TI - Monoallelic mutation analysis (MAMA) for identifying germline mutations. AB - Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC). PMID- 7550327 TI - Risk assessment and religion. PMID- 7550329 TI - Mental modelling. PMID- 7550328 TI - Tune into the weaver channel. PMID- 7550330 TI - Parthenogenesis in man. PMID- 7550331 TI - A common BRCA1 mutation in the Ashkenazim. PMID- 7550332 TI - HEAT repeats in the Huntington's disease protein. PMID- 7550333 TI - Absence of Sry in species of the vole Ellobius. PMID- 7550334 TI - Gender equality in Machado-Joseph disease. PMID- 7550335 TI - Gender equality in Machado-Joseph disease. PMID- 7550336 TI - Settling the myelin protein zero question in CMT1B. PMID- 7550337 TI - Pressing ahead with human genome sequencing. AB - While waiting for revolutionary techniques to emerge, conventional approaches have been used to generate more than 40 million bases of DNA sequence from large scale projects involving different organisms. About eight megabases are from the human genome. Does this progress signal the time to move ahead for a massive human genomic sequencing effort, or should we bide our time, waiting for more technical developments? The answer is to move ahead now, choosing carefully those strategies that can be easily adapted to future methodologies. PMID- 7550338 TI - A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. AB - Early events in neuronal differentiation are generally considered to be regulated by factors independent of alterations in membrane permeability. Weaver mice harbour a mutation that blocks neuronal differentiation just after cessation of cell division, prior to cell migration and synaptogenesis. Cerebellar granule cells in homozygous weaver mice fail to differentiate, either because intrinsic cues are absent or because the granule cells are unable to respond to those cues. We now report that weaver mice have a missense mutation in a gene encoding a G protein coupled inward rectifier potassium channel. The mutation alters the putative ion-permeable, pore-forming domain of the protein, suggesting that granule cell differentiation is regulated by changes in membrane permeability. PMID- 7550339 TI - A gene (PEX) with homologies to endopeptidases is mutated in patients with X linked hypophosphatemic rickets. The HYP Consortium. AB - X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterised by impaired phosphate uptake in the kidney, which is likely to be caused by abnormal regulation of sodium phosphate cotransport in the proximal tubules. By positional cloning, we have isolated a candidate gene from the HYP region in Xp22.1. This gene exhibits homology to a family of endopeptidase genes, members of which are involved in the degradation or activation of a variety of peptide hormones. This gene (which we have called PEX) is composed of multiple exons which span at least five cosmids. Intragenic non-overlapping deletions from four different families and three mutations (two splice sites and one frameshift) have been detected in HYP patients, which suggest that the PEX gene is involved in the HYP disorder. PMID- 7550340 TI - Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1). AB - Hereditary multiple exostoses is an autosomal dominant disorder that is characterized by short stature and multiple, benign bone tumours. In a majority of families, the genetic defect (EXT1) is linked to the Langer-Giedion syndrome chromosomal region in 8q24.1. From this region we have cloned and characterized a cDNA which spans chromosomal breakpoints previously identified in two multiple exostoses patients. Furthermore, the gene harbours frameshift mutations in affected members of two EXT1 families. The cDNA has a coding region of 2,238 bp with no apparent homology to other known gene sequences and thus its function remains elusive. However, recent studies in sporadic and exostosis-derived chondrosarcomas suggest that the 8q24.1-encoded EXT1 gene may have tumour suppressor function. PMID- 7550341 TI - Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. AB - We now report a mutation in the nuclear-encoded flavoprotein (Fp) subunit gene of the succinate dehydrogenase (SDH) in two siblings with complex II deficiency presenting as Leigh syndrome. Both patients were homozygous for an Arg554Trp substitution in the Fp subunit. Their parents (first cousins) were heterozygous for the mutation that occurred in a conserved domain of the protein and was absent from 120 controls. The deleterious effect of the Arg to Trp substitution on the catalytic activity of SDH was observed in a SDH- yeast strain transformed with mutant Fp cDNA. The Fp subunit gene is duplicated in the human genome (3q29; 5p15), with only the gene on chromosome 5 expressed in human-hamster somatic cell hybrids. This is the first report of a nuclear gene mutation causing a mitochondrial respiratory chain deficiency in humans. PMID- 7550342 TI - Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice. AB - Airway hyperresponsiveness is a key characteristic of human asthma and a marker for asthma-like conditions in animals. F1 mice derived from A/J and C57BL/6J display a phenotype which resembles the asthma-like phenotype of the A/J mice. Since airway responsiveness failed to segregate as a mendelian trait, we show significant linkage at two loci, Bhr1 (lod = 3.0) and Bhr2 (lod = 3.7) on chromosomes 2 and 15. A third locus, Bhr3 (lod = 2.83), maps to chromosome 17. Each of these loci maps near candidate loci implicated in the pathobiology of asthma. Our study represents the first linkages established through a genome-wide survey of airway hyperresponsiveness in any mammal. PMID- 7550343 TI - Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. AB - The expansion of CAG triplet repeats in the translated region of the human HD gene, encoding a protein (huntingtin) of unknown function, is a dominant mutation leading to manifestation of Huntington's disease. Targeted disruption of the homologous mouse gene (Hdh), to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, we propose that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway. PMID- 7550344 TI - A human parthenogenetic chimaera. AB - In mice, parthenogenetic embryos die at the early postimplantation stage as a result of developmental requirements for paternally imprinted genes, particularly for formation of extraembryonic tissues. Chimaeric parthenogenetic<==>normal mice are viable, however, due to non-random differences in distribution of their two cell types. Species differences in imprinting patterns in embryo and extra embryonic tissues mean that there are uncertainties in extrapolating these experimental studies to humans. Here, however, we demonstrate that parthenogenetic chimaerism can indeed result in viable human offspring, and suggest possible mechanisms of origin for this presumably rare event. PMID- 7550345 TI - Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. AB - Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of beta-hexosaminidase A resulting in defective GM2 ganglioside degradation. Through disruption of the Hexa and Hexb genes in embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models show very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model was severely affected. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans. PMID- 7550346 TI - A mouse model for Down syndrome exhibits learning and behaviour deficits. AB - Trisomy 21 or Down syndrome (DS) is the most frequent genetic cause of mental retardation, affecting one in 800 live born human beings. Mice with segmental trisomy 16 (Ts65Dn mice) are at dosage imbalance for genes corresponding to those on human chromosome 21q21-22.3--which includes the so-called DS 'critical region'. They do not show early-onset of Alzheimer disease pathology; however, Ts65Dn mice do demonstrate impaired performance in a complex learning task requiring the integration of visual and spatial information. The reproducibility of this phenotype among Ts65Dn mice indicates that dosage imbalance for a gene or genes in this region contributes to this impairment. The corresponding dosage imbalance for the human homologues of these genes may contribute to cognitive deficits in DS. PMID- 7550347 TI - Mice lacking ornithine-delta-aminotransferase have paradoxical neonatal hypoornithinaemia and retinal degeneration. AB - Deficiency of ornithine-delta-aminotransferase (OAT) in humans causes hyperornithinaemia and gyrate atrophy (GA), a blinding chorioretinal degeneration. Surprisingly, OAT-deficient mice produced by gene targeting exhibit neonatal hypoornithinaemia and lethality, rescuable by short-term arginine supplementation. Post-weaning, these mice develop hyperornithinaemia similar to human GA patients. Subsequent studies in one human GA infant also showed transient hypoornithinaemia. Thus, the OAT reaction plays opposite roles in neonatal and adult mammals. Over several months, OAT-deficient mice develop a retinal degeneration with involvement of photoreceptors and pigment epithelium. OAT-deficient mice appear to be an excellent model of human GA. PMID- 7550349 TI - The carrier frequency of the BRCA1 185delAG mutation is approximately 1 percent in Ashkenazi Jewish individuals. AB - Since BRCA1, the first major gene responsible for inherited breast cancer, was cloned, more than 50 unique mutations have been detected in the germline of individuals with breast and ovarian cancer. In high-risk pedigrees, female carriers of BRCA1 mutations have an 80-90% lifetime risk of breast cancer, and a 40-50% risk of ovarian cancer. However, the mutation stats of individuals unselected for breast or ovarian cancer has not been determined, and it is not known whether mutations in such individuals confer the same risk of cancer as in individuals from the high-risk families studied so far. Following the finding of a 185delAG frameshift mutation in several Ashkenazi Jewish breast/ovarian families, we have determined the frequency of this mutation in 858 Ashkenazim seeking genetic testing for conditions unrelated to cancer, and in 815 reference individuals not selected for ethnic origin. We observed the 185delAG mutation in 0.9% of Ashkenazim (95% confidence limit, 0.4-1.8%) and in none of the reference samples. Our results suggest that one in a hundred women of Ashkenazi descent may be at especially high risk of developing breast and/or ovarian cancer. PMID- 7550348 TI - Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. AB - Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy. PMID- 7550350 TI - A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. AB - Epilepsy affects at least 2% of the population at some time in their lives. The epilepsies are a heterogeneous group of disorders, many with an inherited component. Although specific genes have been identified in a few rare diseases causing seizures as part of a more diffuse brain disorder, the molecular pathology of the common idiopathic epilepsies is still unknown. Linkage has been reported for some generalised epilepsy syndromes, but only very recently for familial partial epilepsy syndromes. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial epilepsy causing frequent, violent, brief seizures at night, usually beginning in childhood. The gene for ADNFLE maps to chromosome 20q13.2-q13.3 in one large Australian kindred. The neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) maps to the same region of 20q (ref. 12) and the gene is expressed in all layers of the frontal cortex. We screened affected family members for mutations within CHRNA4 and found a missense mutation that replaces serine with phenylalanine at codon 248, a strongly conserved amino acid residue in the second transmembrane domain. The mutation is present in all 21 available affected family members and in four obligate carriers, but not in 333 healthy control subjects. PMID- 7550351 TI - Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse. AB - p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/Cdk complexes, and is a negative regulator of cell proliferation. The gene encoding human p57KIP is located on chromosome 11p15.5 (ref. 2), a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it a tumour suppressor candidate. Several types of childhood tumours including Wilm's tumour, adrenocortical carcinoma and rhabdomyosarcoma display a specific loss of maternal 11p15 alleles, suggesting that genomic imprinting plays an important part. Genetic analysis of the Beckwith-Wiedemann syndrome has indicated maternal carriers as well as suggested a role in genomic imprinting. Here, as a first step towards elucidating the genesis of human cancers in this region, we showed that a mouse homologue of p57KIP2 is genomically imprinted. The paternally inherited allele is transcriptionally repressed and methylated. This murine gene maps to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2, H19 and Mash2 (refs 14-18). PMID- 7550352 TI - Genetic association of the very low density lipoprotein (VLDL) receptor gene with sporadic Alzheimer's disease. AB - A specific isoform of apolipoprotein E has been associated with the accelerated rate of disease expression of sporadic Alzheimer's disease (AD) and late-onset familial AD (FAD). An earlier age at onset has also been demonstrated in familial AD patients with mutations in the amyloid precursor protein (APP) gene (APP717 and APP670/671)13 carrying the APOE epsilon-4 allele compared to those who do not, but not in familial AD patients with APP692 or 693 mutations, or in chromosome 14-linked familial AD patients. Hypothesizing that receptors for apoE containing lipoproteins act as a potential risk factor for AD, we performed an association study using a polymorphic triplet (CGG) repeat in the gene for the VLDL receptor (VLDL-R), a receptor for apoE-containing lipoproteins. The frequency of the 5-repeat allele was significantly higher in all of the Japanese sporadic AD patients (P < 0.02) than in the Japanese controls. Moreover, the odds ratio was significantly increased in the AD patients homozygous for the 5-repeat allele (OR = 2.1, 95% CI = [1.1-4.2]). Multiple logistic regression analysis reveals that the relative risk conferred by the presence of two copies of the 5 repeat allele and at least one copy of the APOE epsilon-4 allele is 8.7 (95% CI = [2.9-25.8]). Our results suggest that the VLDL-R gene is a susceptibility gene for AD. PMID- 7550354 TI - The locus for Meckel syndrome with multiple congenital anomalies maps to chromosome 17q21-q24. AB - Autosomal recessive Meckel syndrome (OMIM 249000) (MES), first described in 1822 by Johann F. Meckel, is a major monogenic malformation syndrome with a neural tube defect leading to death of the fetus in utero or shortly after birth. The hallmarks of the syndrome are occipital meningoencephalocele, very large kidneys with multicystic dysplasia, cystic and fibrotic changes of the liver and polydactyly (Fig. 1). Other typical malformations for MES are cleft lip and palate, urinary tract anomalies, ambiguous genitals in the males and club feet. Although MES has been reported worldwide, reports on the true birth prevalence of MES in different populations are scarce. In Finland MES is effectively screened and relatively frequent with a birth prevalence of 1:9,000 and a disease gene frequency of 0.01 (ref.4) which is of the same order of magnitude as that of the most common recessive diseases belonging to the 'Finnish disease heritage', that is genetic disorders enriched or only encountered in Finland. However, in MES, comparable or even higher incidences are also reported from other populations. Here, we report the assignment of the MES locus to chromosome 17q21-q24 in the 13 cM region, and exclude some of the potential candidate genes located in this critical chromosomal region. PMID- 7550353 TI - Frequency of homozygous deletion at p16/CDKN2 in primary human tumours. AB - Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15. PMID- 7550355 TI - Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. AB - Congenital muscular dystrophies (CMDs), are heterogeneous autosomal recessive disorders. Their severe manifestations consist of early hypotonia and weakness, markedly delayed motor milestones and contractures, often associated with joint deformities. Histological changes seen in muscle biopsies consist of large variations in muscle fibre size, a few necrotic and regenerating fibres and a marked increase in endomysial collagen tissue. Diagnosis is based on clinical features and on morphological changes. In several CMD cases, we have demonstrated an absence of one of the components of the extracellular matrix around muscle fibres, the merosin M chain, now referred to as the alpha 2 chain of laminin-2 (ref.3). We localized this CMD locus to chromosome 6q2 by homozygosity mapping and linkage analysis. The laminin alpha 2 chain gene (LAMA2) maps to the same region on chromosome 6q22-23 (ref. 5). We therefore investigated LAMA2 for the presence of disease-causing mutations in laminin alpha 2 chain-deficient CMD families and now report splice site and nonsense mutations in two families leading presumably to a truncated laminin alpha 2 protein. PMID- 7550358 TI - Clinical neuropsychology and brain injury rehabilitation in Israel: a twenty-year perspective. AB - The development of the field of neuropsychology in Israel is primarily the result of the development of rehabilitation services for traumatic brain-injured patients. The responsibility to care for and help disabled veterans has always been an important motivation for the establishment of rehabilitation services. Israel is probably one of the first countries in the world to develop community based services specifically designed to address the needs of young patients with severe traumatic brain injury. The fairly extensive therapeutic and community services available today for both military and civilian brain-injured persons in Israel ae the result of initiatives and funding by the Israel Ministry of Defense's Department of Rehabilitation. There are two principles that characterize most of the programs in Israel: (1) multidimensional remedial intervention and (2) life-time commitment to provide support. The accessibility of patients in a small country enables professionals to conduct follow-up studies in order to evaluate the long-term effects of brain injury. Current developments in neuropsychology are in three directions. First, formal training programs in neuropsychology are being set up. Second, the involvement of neuropsychologists is being extended beyond the treatment of young patients suffering from traumatic brain injury to include the treatment of different brain pathologies in children and the elderly. Third, sophisticated neuroimaging techniques are being applied to studies in cognitive neuropsychology. PMID- 7550357 TI - Neuropsychology in Denmark. AB - The origins and development of neuropsychology in Denmark are briefly overviewed, as are the education and training opportunities for Danish neuropsychologists. Areas of application and research are presented, and the role of the rehabilitation of brain injury is emphasized, with specific reference being made to the Center for the Rehabilitation of Brain Injury at Copenhagen University, wherein the rehabilitation program and pertinent research ensuing from the center is presented. Future trends in Danish neuropsychology are also considered. PMID- 7550360 TI - One hundred years of neuropsychology in Japan: retrospect and prospect. AB - The development of neuropsychology in Japan since the end of the 19th century will be outlined, including a number of pioneering works that gave a scientific basis to contemporary Japanese neuropsychology. The activities of societies/associations of related disciplines as well as the educational systems of clinical and experimental neuropsychology, which represents in Japan an interdisciplinary field, will be described. After national and international activities are mentioned, a review of significant Japanese contributions to main topics in neuropsychology will be given in some detail: dementia, memory disorders, frontal lobe syndromes, aphasia-alexia-agraphia, callosal syndromes, and specific right hemisphere syndromes. The issues of these interdisciplinary scientific investigations have found application in language and cognitive disabilities of brain-damaged patients, as well as evaluation of medical treatments and comedical rehabilitational effects, care, and counseling. Finally, some comments will be presented on possible and desirable developments of Japanese neuropsychology in the future. PMID- 7550359 TI - Neuropsychology in France. AB - The main steps in the development of neuropsychology in France are briefly reviewed, from the early contribution of Paul Broca (1861) up until the early 1990s. The current epistemological and institutional crises is analyzed with respect to some general factors inherent to the domain (e.g., disputes between the proponents of the psychological and biological dimensions) and to particular factors related to the French situation (e.g., retirement of prominent neuropsychologists). As regards clinical practice and research activities, the future will probably show some fractionation of goals and techniques prior to new synthesis. PMID- 7550356 TI - The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families. AB - Genetic linkage studies place a gene causing early onset familial Alzheimer's disease (FAD) on chromosome 14q24.3 (refs 1-4). Five mutations within the S182 (Presenilin 1: PS-1) gene, which maps to this region, have recently been reported in several early onset FAD kindreds. We have localized the PS-1 gene to a 75 kb region and present the structure of this gene, evidence for alternative splicing and describe six novel mutations in early onset FAD pedigrees all of which alter residues conserved in the STM2 (Presenilin 2: PS-2) gene. PMID- 7550361 TI - Microglia: intrinsic immuneffector cell of the brain. AB - Microglia form a regularly spaced network of resident glial cells throughout the central nervous system (CNS). They are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage. In the ultimate vicinity of the blood-brain barrier two specialized subsets of macrophages/microglia can be distinguished: firstly, perivascular cells which are enclosed within the basal lamina and secondly juxtavascular microglia which make direct contact with the parenchymal side of the CNS vascular basal lamina but represent true intraparenchymal resident microglia. Bone marrow chimera experiments indicates that a high percentage of the perivascular cells undergoes replacement with bone marrow-derived cells. In contrast, juxtavascular microglia like other resident microglia form a highly stable pool of CNS cells with extremely little turnover with the bone marrow compartment. Both the perivascular cells and the juxtavascular microglia play an important role in initiating and maintaining CNS autoimmune injury due to their strategic localization at a site close to the blood-brain barrier, their rapid inducibility for MHC class II antigens and their potential scavenger role as phagocytic cells. The constantly replaced pool of perivascular cells probably represents an entry route by which HIV gets access to the brain. Microglia are the first cell type to respond to several types of CNS injury. Microglial activation involves a stereotypic pattern of cellular responses, such as proliferation, increased or de-novo expression of immunomolecules, recruitment to the site of injury and functional changes, e.g., the release of cytotoxic and/or inflammatory mediators. In addition, microglia have a strong antigen presenting function and a pronounced cytotoxic function. Microglial activation is a graded response, i.e., microglia only transform into intrinsic brain phagocytes under conditions of neuronal and or synaptic/terminal degeneration. In T-cell-mediated autoimmune injury of the nervous system, microglial activation follows these lines and occurs at an early stage of disease development. In experimental autoimmune encephalomyelitis (EAE), microglia proliferate vigorously, show a strong expression of MHC class I and II antigens, cell adhesion molecules, release of reactive oxygen intermediates and inflammatory cytokines and transform into phagocytic cells. Due to their pronounced antigen presenting function in vitro, activated microglia rather than astrocytes or endothelial cells are the candidates as intrinsic antigen presenting cel of the brain. In contrast to microglia, astrocytes react with a delay, appear to encase morphologically the inflammatory lesion and may be instrumental in downregulating the T-cell-mediated immune injury by inducing T cell apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7550362 TI - Large-scale cortical networks and cognition. AB - The well-known parcellation of the mammalian cerebral cortex into a large number of functionally distinct cytoarchitectonic areas presents a problem for understanding the complex cortical integrative functions that underlie cognition. How do cortical areas having unique individual functional properties cooperate to accomplish these complex operations? Do neurons distributed throughout the cerebral cortex act together in large-scale functional assemblages? This review examines the substantial body of evidence supporting the view that complex integrative functions are carried out by large-scale networks of cortical areas. Pathway tracing studies in non-human primates have revealed widely distributed networks of interconnected cortical areas, providing an anatomical substrate for large-scale parallel processing of information in the cerebral cortex. Functional coactivation of multiple cortical areas has been demonstrated by neurophysiological studies in non-human primates and several different cognitive functions have been shown to depend on multiple distributed areas by human neuropsychological studies. Electrophysiological studies on interareal synchronization have provided evidence that active neurons in different cortical areas may become not only coactive, but also functionally interdependent. The computational advantages of synchronization between cortical areas in large-scale networks have been elucidated by studies using artificial neural network models. Recent observations of time-varying multi-areal cortical synchronization suggest that the functional topology of a large-scale cortical network is dynamically reorganized during visuomotor behavior. PMID- 7550363 TI - Contingent tolerance to the anticonvulsant effects of carbamazepine: relationship to loss of endogenous adaptive mechanisms. AB - Contingent tolerance to the anticonvulsant effects of carbamazepine on amygdala kindled seizures develops when the drug is repeatedly given prior to but not after the electrical stimulation. Such tolerance can be reversed by kindling the rats for several days without drug or even by continuing to give the drug but after each seizure has occurred. Contingent tolerance can be slowed by reducing the electrical stimulus intensity and by chronic continuous (as opposed to repeated paired) drug administration. Contingent cross-tolerance has been demonstrated from carbamazepine to PK11195 (a drug active at peripheral-type benzodiazepine receptors) and valproate, but not to clonazepam and diazepam (two drugs active at central-type benzodiazepine receptors) or phenytoin. Endogenous physiological changes occur in conjunction with contingent tolerance, exemplified by the decrease in seizure threshold that returns to normal upon reversal of tolerance. We suggest that contingent tolerance is associated with a loss of seizure-induced adaptations, since many biochemical changes that occur following seizures (or in non-tolerant animals given drug after seizures) are not observed in tolerant animals. These include a loss of seizure-induced up-regulation of GABAA receptors and a loss of increases in mRNA expression for corticotropin releasing-factor (CRF), thyrotropin-releasing-hormone (TRH), neuropeptide Y (NPY), glucocorticoid receptors and brain-derived neurotrophic factor (BDNF). Thus, several putative seizure-induced anticonvulsant adaptations, such as increases in GABAA receptors and TRH and NPY mRNA fail to occur in tolerant animals. These findings are consistent with the novel observations that, paradoxically, seizures themselves appear to facilitate the anticonvulsant effects of carbamazepine or diazepam on amygdala kindled seizures. That is, animals given a 'vacation' from seizures show a decreased response to these agents, a phenomenon we have called the 'time-off seizure' effect. Thus, seizures are postulated to induce adaptive changes that influence seizure thresholds and potentiate the anticonvulsant effects of exogenously administered drugs such as carbamazepine and diazepam. Taken together, these data suggest that seizures are associated with endogenous adaptations lasting days to weeks and that a selective failure of some of these to occur during contingent drug administration may underlie the development of contingent tolerance. These observations suggest tht endogenous illness-related mechanisms may participate both in the therapeutic responses of some agents and that their failure to occur could relate to loss of drug efficacy via tolerance; these processes may reveal new potential targets for therapeutic intervention. PMID- 7550364 TI - Dopamine D2 receptor gene variants: association and linkage studies in impulsive addictive-compulsive behaviour. AB - Drug and alcohol seeking behaviour has become a great global problem affecting millions of inhabitants with a cost to society in the billions. Dopaminergic reward pathways have frequently been implicated in the etiology of addictive behaviour. While other neurotransmitters have also been implicated, to date the only molecular genetic defect which has been found to associate with alcoholism, drug dependency, obesity, smoking, pathological gambling, attention-deficit hyperactivity disorder (ADHD), Tourette syndrome, as well as other related compulsive behaviours, are the variants of the dopamine D2 receptor gene (DRD2). In this review of the available data on the subject, we report a number of independent meta-analyses that confirm an association of DRD2 polymorphisms and impulsive-additive-compulsive behaviour (IACB), which we have termed "Reward Deficiency Syndrome". While we agree that Meta-analyses of all exant studies support an association of variants of DRD2 and IACB, correct negative findings with alcoholism may be due to differences in assessing controls and inclusion/exclusion criteria for selection of diseased probands. PMID- 7550365 TI - Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. AB - Chlorzoxazone, a muscle-relaxing drug, is metabolized by carbon-hydroxylation at position 6. Chlorzoxazone has been suggested as an in vivo probe for CYP2E1. We studied the specificity of such a substrate using vaccinia virus expressed human P450 forms and the effect of inhibitors for chlorzoxazone metabolism by human liver microsomes. The 6-hydroxylation of chlorzoxazone was mediated by CYP1A2 as well as by CYP2E1. The Km value of CYP1A2 and CYP2E1 for the reaction was 5.69 microM and 232 microM, respectively. However, the Vmax value of CYP2E1 for the reaction was approximately 8.5-fold higher than that of CYP1A2. The CYP1A inhibitor, alpha-naphthoflavone, as well as the CYP2E1 inhibitor, diethyldithiocarbamate, decreased chlorzoxazone 6-hydroxylation at a low substrate concentration by human liver microsomes. Our results raise questions about the suitability of chlorzoxazone as an in vivo probe for hepatic CYP2E1 activity. In human liver microsomal samples, the Km = 40 microM was different from either the Km of CYP1A2 or CYP2E1. We think that this discrepancy is due to the co-expression of similar levels of CYP1A2 and CYP2E1 in human liver. Furthermore, it is suggested that the role of CYP2E1 in 6-hydroxychlorzoxazone formation at the physiological chlorzoxazone concentration of 30-60 microM is almost the same when compared to that of CYP1A2. PMID- 7550366 TI - Polymorphisms of human Ah receptor gene are not involved in lung cancer. AB - The Ah receptor (Ahr) is a ligand-dependent transcription factor that positively regulates inducible expression of the CYP1A1 gene. Based on the sequence information of the human Ahr and the intron-exon junctions of the mouse counterpart, an analysis of single-strand conformational polymorphism (SSCP) was carried out to detect subtle base differences in the coding region of the gene among individuals. We found that the Ahr protein has at least two forms of variants in a Japanese gene pool, and that these variants can be ascribed to one amino acid replacement of Arg by Lys at codon 554. The frequencies of Arg-coded and Lys-coded alleles were 0.57 and 0.43, respectively. We found, however, that this germ line polymorphism of the Ahr gene did not show a significant association with aryl hydrocarbon hydroxylase (AHH) inducibility nor with lung cancer incidence. PMID- 7550367 TI - Genetic analysis of the CYP2D locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects. AB - The CYP2D6 genotype and the debrisoquine and mephenytoin hydroxylation phenotypes were studied in 63 Oriental subjects including 21 Chinese, 21 Japanese and 21 Koreans. All subjects were extensive metabolizers of debrisoquine. The incidence of the S-mephenytoin poor metabolizer phenotype was 14% in the Chinese, 24% in the Japanese and 24% in the Korean population, respectively, which is similar to previous reports. The CYP2D6 genotype was analysed by Xba I and Eco RI RFLP, and by allele-specific PCR analysis for the presence of several allelic variants of the CYP2D locus. No CYP2D6A or CYP2D6B alleles, two of the most common defect alleles among Caucasians, were found among the Oriental subjects. The frequency of the CYP2D6D allele was similar to that in Caucasian populations and consistent with the low incidence of the poor metabolizer phenotype in all three Oriental populations. The CYP2D6L2-allele with duplication of an active CYP2D6L gene was identified in one Korean and one Chinese allele in association with high CYP2D6 activity. The CYP2D6Ch alleles CYP2D6Ch1 and Ch2, identified by RFLP and PCR for the -1338C-->T and 188C-->T mutations, were the most frequent allelic variants in all three populations studied, and were related to a decreased CYP2D6 activity as previously shown in Chinese. In conclusion, the present pilot study revealed major similarities in the polymorphic CYP2D locus between Korean, Japanese and Chinese populations. PMID- 7550368 TI - Susceptibility mutations in the mitochondrial small ribosomal RNA gene in aminoglycoside induced deafness. AB - Aminoglycoside induced deafness has been linked recently to a predisposing homoplasmic mutation in the 3' end of the small ribosomal RNA (rRNA) gene of the human mitochondria (1555 A-->G) that makes the mitochondrial rRNA structurally more similar to its bacterial counterpart. This mitochondrial DNA mutation was consistently found in families in which the susceptibility to develop ototoxic deafness was inherited through the maternal lineage. However, the 1555 A-->G mutation was rarely found in sporadic patients in China, where a significant proportion of the population has been exposed to aminoglycosides. To further characterize the mutations predisposing to aminoglycoside ototoxicity, we analysed the 12S rRNA gene in 35 Chinese sporadic patients without the 1555 A-->G mutation. Using single stranded conformational polymorphism (SSCP) analysis, heteroduplex (HD) analysis, sequencing, and allele specific oligonucleotide hybridization, we found three out of 35 sporadic patients with unique sequence changes in the 12S rRNA gene. Two of the patients had homoplasmic mutations. One patient displayed localized heteroplasmy around nt 961, with an absence of the thymidine at this position and different populations of mitochondrial DNA with varying numbers of inserted cytosines. The description of these putative susceptibility mutations, in particular the heteroplasmic mutation around nt 961, provides further support for the important role of the mitochondrial 12S rRNA in genetic predisposition to aminoglycoside induced ototoxic deafness. PMID- 7550369 TI - Codeine metabolism in three Oriental populations: a pilot study in Chinese, Japanese and Koreans. PMID- 7550370 TI - Availability of furafylline. PMID- 7550371 TI - The formation of mRNA 3'-ends in plants. PMID- 7550372 TI - PvAlf, an embryo-specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters. AB - Mutations in Vp1 and ABl3 genes of maize and Arabidopsis lead to drastic reductions in the synthesis of a subset of maturation-specific products including seed storage proteins. Gene Phaseolus vulgaris ABl3-like factor (PvAlf), whose protein product is similar to the ABl3 and Vp1 proteins, has been cloned. Here, it is shown that PvAlf positively regulates phaseolin and phytohemagglutinin (PHA L) promoters in particle bombardment assays. PvAlf mRNA expression is embryo specific and temporally complex. PvAlf mRNA abundance is highest during two periods (9-14 and 22-35 days after flowering) that precede the onsets of seed maturation and seed abscission, respectively. Protein fusions with the DNA binding domain of the yeast transcriptional activator GAL4 demonstrated that the N-terminal 243 amino acids of PvAlf function as a strong transcriptional activation domain in yeast (Saccharomyces cerevisiae) and plant cells. This domain consists of a central cluster rich in serine, threonine and proline (STP cluster) flanked by two negatively charged regions containing bulky hydrophobic residues similar to acidic activation domains of Vp1, the herpes simplex virus virion protein VP16 and transcription factors GCN4 and HAP4 from yeast. Together with the Vp1 proteins of maize and rice and ABl3, PvAlf constitutes a class (Vp1/ABl3-like factors or VAlfs) of regulatory factors that are pivotal for the promotion of seed maturation and dormancy in angiosperms. PMID- 7550373 TI - Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation. AB - Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation. PMID- 7550376 TI - Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean. AB - Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian delta-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian delta-type PI-PLCs and yeast PI-PLC1, the putative Ca(2+)-binding site of the soybean PI PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane. PMID- 7550375 TI - Isolation of Arabidopsis mutants altered in the light-regulation of chalcone synthase gene expression using a transgenic screening approach. AB - Transgenic Arabidopsis expressing beta-glucuronidase (GUS) driven by a chalcone synthase gene (CHS) promoter were produced. GUS activity in the leaves increased with increasing fluence rates of white light in parallel with endogenous CHS transcript levels. An isogenic line homozygous for the transgene was obtained and mutagenized seedlings of this line were screened for altered light-induction of the transgene. Putative mutants with low GUS activity were not altered in the light-induction of endogenous CHS transcripts and are therefore not regulatory mutants. Two mutant lines (A12 and C10) with elevated levels of GUS activity in the light show a corresponding increase in CHS transcript levels. The A12 mutant was focussed upon and designated icx1 (increased chalcone synthase expression). This mutant has enhanced light-stimulation of CHS expression since CHS transcript levels in darkness in icx1 are very low, as in the wild-type. The transcript levels of two other genes involved in flavonoid biosynthesis are elevated in the light in icx1 as is anthocyanin formation. However, there is no alteration in LHCII chlorophyll a/b-binding protein gene (CAB) transcript levels under the same conditions. The altered gene expression phenotype of icx1 co-segregates with several other phenotypic characteristics, including fewer leaf trichomes and alterations to the seed coat. On the basis of these data and comparison with the Arabidopsis ttg (transparent testa glabra) mutant, it is suggested that the ICX1 gene product may be concerned both with the light-regulation of gene expression and with developmental processes occurring in the epidermis. PMID- 7550377 TI - Expression of an amino acid biosynthesis gene in tomato flowers: developmental upregulation and MeJa response are parenchyma-specific and mutually compatible. AB - The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the 'conditioning' of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue specific response to MeJa, in conjunction with developmental control, is discussed. PMID- 7550374 TI - Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize. AB - The 18 kDa high-methionine delta-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of delta-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine delta-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown. PMID- 7550378 TI - An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a delta 7-sterol-C-5-desaturase from yeast. AB - The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS) mutagenized population of Arabidopsis thaliana which consisted of 22,000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples. STE 1 was characterized by the accumulation of three delta 7-sterols concomitantly with the decrease of the three corresponding delta 5-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these delta 7-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC MS) and proton nuclear magnetic resonance spectrometry (1H-NMR). The accumulation of delta 7-sterols suggested that the mutant is deficient in the activity of the delta 7-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of delta 7-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the delta 7-sterols in STE 1 are good surrogates of physiologically active delta 5-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the delta 7-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in delta 5-sterols and a concomitant decrease in delta 7 sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of delta 7-sterols in the STE 1 mutant is due to a deficiency of the delta 7-sterol-C-5 desaturation step in the plant sterol biosynthesis pathway. PMID- 7550380 TI - Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia. AB - Alternative splicing is a well-known post-transcriptional regulatory mechanism in eukaryotic organisms but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase multi-enzyme complex from the C4 species Flaveria trinervia revealed the presence of two transcript populations that differ in the length of their coding regions by six nucleotides. Otherwise, including their 3' nontranslated region, they are identical. From a genomic Southern analysis and from the sequencing of several independent cDNA clones it is evident that both types of transcript are derived from a single-copy gene. This gene, FTgdcsH, has been cloned and sequenced. It comprises four short exons. The two alternative splice sites are located at the end of intron 1. The shorter transcript closely corresponds to published H protein mRNA sequences from other organisms. The longer transcript encodes two additional alanine residues very close to the N-terminus of the mature H-protein. A quantification of the relative amounts of both transcripts in different organs revealed that, with 80-90% of the total H-protein mRNA, the alternative mRNA dominates in leaves whereas roots contain more of the 'correctly' spliced transcript. It is concluded that, in F. trinervia and with a distinct organ preference, alternative splicing leads to the synthesis of two different H proteins of the glycine cleavage system. PMID- 7550379 TI - Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts. AB - The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to beta-carotene when expressed in Escherichia coli. The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene beta-cyclase activity when expressed in E. coli. A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re channeling of linear carotenoids into beta-cyclic carotenoids in C. annuum ripening fruits. PMID- 7550381 TI - Organ-specific differences in the ratio of alternatively spliced chorismate synthase (LeCS2) transcripts in tomato. AB - The primary transcript of one of the two chorismate synthase genes (LeCS2) of tomato is differentially processed due to an alternative splicing of the third intron. A novel observation was made when the abundances of the two LeCS2 specific transcripts in different organs were analysed. The ratio of these two transcripts differs in RNA populations from different organs. Possible explanations for this finding and its potential physiological impact for plant metabolism are discussed. PMID- 7550382 TI - Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. AB - Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described. Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction experiments indicate that the technique can recover single copy sequences from genomes as complex as common wheat (1.5 x 10(10) bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci. PMID- 7550383 TI - Molecular cytogenetics: unraveling of the genetic composition of individual cells by fluorescence in situ hybridization and digital imaging microscopy. AB - Molecular biology techniques allow the unraveling of the genetic alterations that cause or accompany malignant disease. Since tumors are often heterogeneous, biochemical analysis of tissue homogenates is of limited diagnostic value. This paper gives examples of methods that are presently operational to analyze the genetic composition of individual cells. They are based on fluorescence in situ hybridization (FISH) and digital imaging microscopy. First, the current status of indirect and direct FISH staining methods with respect to probe labeling, detection sensitivity, multiplicity, and DNA resolution is summarized. Microscope hardware as well as charge-coupled device (CCD) cameras required for FISH analysis are then described. Applications potentially important for the analysis of urological malignancies, such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells) and high-resolution DNA mapping on highly extended chromatin, are described in detail. Finally, the limitations of the present methodology and its future prospects are discussed. PMID- 7550384 TI - Diagnosis and prognosis of renal-cell tumors: a molecular approach. AB - This review discusses the impact of molecular genetics on the diagnosis and prediction of the prognosis for renal-cell tumors. Through the use of molecular techniques, it has become possible to divide renal-cell tumors into genetically and biologically well-defined entities. The new classification has many advantages over morphological ones: (1) in contrary to tumor phenotype, genetic markers are constant during tumor progression and allow a precise diagnosis; (2) genetic analysis discriminates between papillary (multiple origin) and nonpapillary renal-cell carcinomas (solitary tumors); (3) genetic analysis allows a precise diagnosis of renal adenoma; (4) the new classification system offers a genetic prognostic system; and (5) the new diagnostic-prognostic system could easily be adapted to modern detection systems such as automated sequence and fragment-length analysis, which allow a quick and correct diagnosis with a high throughput. PMID- 7550385 TI - Localized renal-cell carcinoma: detection of abnormal cells in peritumoral tissue. A cytophotometry and immunocytochemistry study. AB - To spare organ function, partial resection of early diagnosed renal-cell carcinoma (RCC) is applied for well-localized and small-volume RCC with increasing frequency, although recurrence of the tumor in the same kidney is occasionally observed. The aim of the present study was to establish objective prognostic parameters that would allow the selection of tumors suitable for an organsaving procedure. Of the 160 patients undergoing a radical nephrectomy, 67 were included in this study. In 7/45 patients with lymph-node dissection (15.6%), clinical staging revealed a false-negative lymph-node status. By means of conventional histopathology, multifocality could be demonstrated in 2/67 patients (3%); in 1/67 patients (1.5%), the ipsilateral adrenal gland was unexpectantly tumor-involved. Both tumor tissue and normal peritumoral tissue were examined for the presence of premalignant and tumor cells on the basis of DNA ploidy and of the expression of the tumor-associated G250 antigen, which is specifically expressed at the surface of renal cancer cells. In 40/67 (59.1%) peritumoral tissue specimens, cells with an abnormal DNA content could be observed using automated image analysis. In 12/67 cases (18%), cells obtained from peritumoral tissue also showed an aneuploid DNA histogram; 4/67 (6%) had a tumor-correlated DNA ploidy. Additionally, 38/67 (56.9%) of these tissues, histopathologically classified as normal, contained cells expressing the G250 antigen. These observations were independent of the stage or histological grade of the tumor. These data indicate that classic pathological parameters for tumor staging are insufficient for the detection of multifocality, occurring in more than 15% of cases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550386 TI - Morphological classification of renal cancer. AB - The current classification of renal-cell adenomas (RCAs) and carcinomas (RCCs) is based on eight basic cell and tumor types (entities) with characteristic morphologic features: (1) RCCs of clear-cell type, (2) RCAs/RCCs of chromophilic cell type, (3) RCAs/RCCs of chromophobic-cell type, (4) RCCs of duct Bellini type, (5) RCCs of transitional-cell type, (6) RCCs of neuroendocrine type, (7) RCAs of oncocytic type, and (8) RCAs of metanephroid type. Tumors with a proposed histogenesis from the proximal tubule (clear-cell and chromophilic tumors) amount to 85% of renal cancers, whereas tumors with a proposed histogenesis from the connecting tubule/collecting duct (chromophobic-, oncocytic-, and duct Bellini type tumors) amount to only 11%. The remaining tumor types represent rare entities (less than 1% each). Tumor cytogenetics data confirm the proposed eight morphologic subtypes and give further indications for type-specific tumor development and progression. PMID- 7550387 TI - Radical surgery for renal-cell carcinoma: caval neoplastic excision, adrenalectomy, lymphadenectomy, adjacent organ resection. AB - A radical nephrectomy typically includes early ligation of the renal vessels, excision of the kidney and perinephric tissue, a regional lymphatenectomy, and an adrenalectomy. More controversial may be excision of supradiaphragmatic caval neoplastic extension and adjacent organ resection. Although survival is low in these unfavorable groups of patients, some patients may benefit from the extensive local resection of tumor, including adrenalectomy, lymphatenectomy, caval resection, and resection of adjacent organs. PMID- 7550389 TI - Renal-cell carcinoma with intracaval neoplastic extension: stratification and surgical technique. AB - Surgical removal continues to be the mainstay in the treatment of renal-cell carcinoma with neoplastic venous extension. The steady improvement of surgical and anesthesiological techniques and the introduction of complete circulatory arrest has dramatically improved the morbidity even of patients with extensive thrombi. If ultrasound or computerized tomography (CT) scanning suggests the presence of a venous extension in a patient with renal-cell carcinoma, cavography, magnetic resonance imaging (MRI), transesophageal color-coded ultrasound, and echocardiography may be needed to resolve the questions of cranial extension and vascular wall infiltration. Surgical stratification and, thus, classification of the venous extension depend on the potential need for complete circulatory arrest. Surgical removal is done en bloc for smaller venous extensions and in a two-step procedure (radical nephrectomy followed by thrombectomy) for more extensive thrombi. In patients with infiltration of the suprahepatic inferior vena cava, the hepatic veins or atrium, pending thrombotic embolism, or large masses of suprahepatic thrombotic material, the use of cardiopulmonary bypass and complete circulatory arrest is recommended. PMID- 7550388 TI - Current controversies in nephron-sparing surgery for renal-cell carcinoma. AB - The role of nephron-sparing surgery for renal cell carcinoma is well established in patients with an anatomical or functional solitary kidney (imperative indication) in which a radical nephrectomy would render the patient anephric with subsequent need for hemodialysis. This also encompasses patients with a unilateral renal cell carcinoma and a functioning contralateral kidney when the opposite renal unit is affected by a disease that might threaten its future function, such as renal artery stenosis, chronic pyelonephritis, stone disease or systemic conditions such as diabetes. A functioning renal remant of at least 20% of normal renal parenchyma seems to be necessary to avoid end-stage renal failure in these patients [16]. There have been several reports in the literature of excellent 5-year cancer-specific survival rates of over 80% in such circumstances [12, 15]. These results were confirmed in our institution, with a 5-year cancer specific survival rate of 83% in over 70 patients with an imperative indication for nephron-sparing surgery. Thereby the prognosis was significantly influenced by the local tumor stage and the grade of malignancy. These data support the efficacy of nephron-sparing surgery in this clinical situation. PMID- 7550390 TI - Adjuvant treatment of locally advanced renal cancer with autologous virus modified tumor vaccines. AB - We report on 208 patients with locally advanced renal-cell carcinoma who received a surgical adjuvant vaccination with autologous, Newcastle disease virus (NDV) modified, and lethally irradiated tumor cells in combination with low-dose recombinant interleukin-2 and interferon-alpha. The pathological stage was defined as pT2-3a, N1-2, MO (n = 107); pT3b-4 NO, MO (n = 68); and pT3b-4, N1-2, MO (n = 23). The follow-up of 203 evaluable patients showed a median disease-free survival of 21+ months (range, 2-64+ months). In all, 18 relapses (9%) occurred in spite of initial vaccination therapy. Those patients presented with local relapse (n = 3), lymph node metastases (n = 10), and/or distant organ metastases (n = 9). All patients relapsing during the first 6 months after the onset of treatment had primary lymph node involvement of the disease. An analysis of the patient subgroup with a follow-up of more than 22 months showed 10 relapses among 56 patients (18%) along with a median follow-up of 39 months (range, 23-64 months). Toxicity was very mild, manifesting as flu-like symptoms and fevers of up to 38 degrees C. At 8 and 24 weeks after the start of vaccination, anti-NDV serum antibodies were detectable in 70% and 100% of the patients tested, respectively. In comparison with historical data based on the natural course of patients with locally advanced renal-cell cancer, our results demonstrate an improvement of the disease-free survival after surgical adjuvant treatment with autologous, NDV-modified tumor vaccines in combination with low-dose cytokines. PMID- 7550391 TI - Biochemotherapy of advanced metastatic renal-cell carcinoma: results of the combination of interleukin-2, alpha-interferon, 5-fluorouracil, vinblastine, and 13-cis-retinoic acid. AB - We conducted a phase I/II clinical trial evaluating the sequential outpatient combination of S.C. recombinant human interleukin-2 (rIL-2; given at 10 MIU/m2 b.i.d. on days 3-5 weeks 1 and 4 and at 5 MIU/m2 on days 1, 3, and 5 of weeks 2 and 3), s.c. recombinant human alpha-interferon (rIFN-alpha; given at 6 MIU/m2 on day 1 of weeks 1 and 4 and on days 1, 3, and 5 of weeks 2 and 3 and at 9 MIU/m2 on days 1, 3, and 5 of weeks 5-8), i.v. bolus 5-fluorouracil (5-FU; given at 1,000 mg/m2 once weekly during weeks 5-8), and i.v. bolus vinblastine (given at 6 mg/m2 once weekly during weeks 5 and 8) in conjunction with p.o. 13-cis-retinoic acid (13-C-RA; given at 35 mg/m2 daily during weeks 1-8). Therapy was always given in the outpatient setting. Grade 3 constitutional symptoms (malaise, chills, fevers, anorexia) were observed in 4%-8% of treatment cycles and required a 50% reduction in the doses of rIL-2 and rIFN-alpha. None of the patients experienced major 5-FU-related toxicities such as severe diarrhea and/or stomatitis; up to 20% of patients developed vinblastine-associated peripheral polyneuropathy, which was reversible after the cessation of therapy. 13-cis Retinoic acid produced no significant side effect; no toxic death occurred. Among 24 patients with progressive metastatic disease, there were 4 complete remissions (lung, lymph nodes) and 6 partial remissions (lung, pleura, liver, lymph nodes, and peritoneal carcinosis), for an overall objective response rate of 42% (95% confidence interval, 22%-63%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550394 TI - Elastin morphology in normal and obstructed guinea-pig bladders. Localization of elastin to the trigone. AB - Elastin is the major component of elastic fibers in aortic tissue and has been suggested to be important for the stretch and recoil of the bladder. We have mapped the histologic distribution of elastin throughout the entire guinea-pig bladder as well as changes in the distribution of elastin during bladder-outlet obstruction. We used an animal model in which a jeweler's ring is placed around the urethra of the young guinea pig. As the animal grows, outlet obstruction is gradually induced, and the bladder gradually increases in total weight to as much as 8-fold that of controls. We localized the elastin histologically in normal and obstructed bladders by serially sectioning entire bladders and staining the sections with an elastin-specific stain, Verhoff-van Gieson stain (VVG). Surprisingly, we found that the elastin was predominantly localized only to the trigone of the bladder and that the elastin in this area was increased during obstruction. These results are consistent with the predominantly mesodermal embryologic origin of the trigone, since mesodermal tissues are more often associated with elastin expression. PMID- 7550392 TI - Gene therapy on renal-cell carcinoma: magic bullet or tragic insanity? AB - Correction of the aberrant genetic code as a means of rational therapy has been a challenge since the first discoveries of an abnormal genetic link to expression of certain disorders. Our growing understanding of the molecular basis of cancer has also led us into a new era in cancer therapy. The possibility of gene therapy represents one of the biggest potential returns on the investment in molecular biology research over the past several years. As a massive gene therapy attack mounts against many forms of malignancy employing various techniques, strategies, and concepts, there appears to be reason to be optimistic, with expectations thus far decidedly outweighing results. Scientists and clinicians have joined together to target directly the molecular basis of tumorigenesis through the restoration of tumor-suppressor gene function or inhibition of oncogene expression. In addition, scientists mapping the human genome have supplied us with a number of genes that can be used to destroy cancer cells selectively [e.g., the herpes simplex-thymidine kinase (HS-tk) gene], induce a potent antitumor immune response (e.g., interleukin 2), and afford protection to normal tissues from the toxic effects of standard chemotherapy [e.g., multidrug resistance gene type 1 (mdr 1)]. These new anticancer tools provide new opportunities for more specific tumor cell destruction in vivo without the common regional and systemic side effects related to conventional forms of chemotherapy, immunotherapy, radiation, and surgery. Hence, over the next 5-10 years, gene therapy is likely to become a realistic treatment option for certain cancers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550393 TI - Radiolabeled monoclonal antibody G250 in renal-cell carcinoma. AB - The current status of iodine 131-radiolabeled monoclonal antibody G250 (mAb G250) in renal-cell carcinoma (RCC) is described. This mAb recognizes a tumor associated antigen that is expressed on the cell surface of almost all RCC but is not expressed on normal tissues, with the exception of gastric mucosa and larger bile ducts. On the basis of these favorable characteristics, this mAb seemed a prime candidate for clinical investigations. Preclinical animal studies and ex vivo perfusion experiments in tumor-bearing kidneys showed excellent targeting of mAb G250 to RCC tumors. Supported by these investigations, a phase I study was initiated to define the imaging and biodistribution characteristics of 131I labeled mAb G250 in RCC patients. Specific localized of [131I]-mAb G250 to G250 antigen-positive primary and metastatic RCC was observed. In several patients, [131I]-mAb G250 imaging revealed thus far unrecognized, i.e., occult, disease. Values obtained for [131I]-mAb G250 uptake, relative as well as absolute, were among the highest reported for tumor biopsies obtained 8 days after intravenous mAb administration. The specific localization and high accumulation encouraged us to begin a phase I/II radiotherapy trial with [131I]-mAb G250. The maximal tolerable dose was reached at 90 mCi/m2 [131I]-mAb G250. In the subsequent phase I/II radiotherapy study, we observed stable disease in a great number of patients as well as minor responses in a small number of patients. Multiple treatments seemed necessary to achieve better response rates. However, anti-mouse responses prevented multiple dosing with the murine mAb G250.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550396 TI - Evaluation of nitroxynil and closantel activity using ELISA and egg counts against Fasciola hepatica in experimentally and naturally infected cattle. AB - The responses of cattle infected with Fasciola hepatica to treatment with nitroxynil or closantel were monitored by faecal egg counts and by ELISA assay of anti-fluke antibodies. A first trial with experimentally infected heifers showed an increase in anti-fluke antibody titre as early as 2 weeks post-infection. Eggs were first detected in the faeces 10 weeks after infection. Egg output increased steadily over the next 8 weeks and then rapidly decreased. Treatment of a 20-week infection with nitroxynil was followed by a slow decrease in antibody titre 4 weeks later. This decrease continued over the next 40 weeks, but returned to pre infection levels in only 2 out of 4 animals. The faecal egg count fell to zero 2 weeks after treatment and remained so for the following 30 weeks, although 1 animal produced a few eggs 32 and 34 weeks post-treatment. Within this period, neither diagnostic technique discriminated between this persistently infected animal and the others. In a second trial, 45 cattle from a naturally infected herd were treated with nitroxynil or closantel. The faecal egg counts of the treated cattle were zero within the following 2 months, whereas there were eggs in the faeces of the control (untreated) group. Nevertheless, the treated cattle showed a small, non-significant drop in anti-fluke antibody titre. These results demonstrate the need for new tools to monitor and evaluate accurately the efficacy of anthelmintic treatment. PMID- 7550395 TI - Nucleolar organizer regions in low- and high-grade carcinomas of the prostate. AB - The number of silver-stained nucleolar organizer regions (AgNORs) and the status of nucleoli were studied in biopsies from 10 cases of normoglandular prostatic tissue and the findings were compared with the results obtained for 10 cases of hyperplastic prostatic tissue (BPH) and 74 cases of prostatic carcinoma of various grades of malignancy. There was an increase in the number of AgNORs detected between the grade of malignancy found in highly versus poorly differentiated carcinomas of the prostate. Furthermore AgNOR values differed significantly among the groups compared (P < 0.001). The nucleolar status confirmed these differences. In addition, the significantly different survival of patients suffering from prostatic carcinomas support our idea of subdividing GII carcinomas into group IIa (low risk) and group IIb (high risk). This subgrading implies the possibility of preoperatively identifying patients with a prostate carcinoma of high malignancy within the GII carcinoma group. PMID- 7550397 TI - [Epidemiology of infectious hematopoietic necrosis (IHN) of salmonid fish in France: study of the course of natural infection by combined use of viral and seroneutralization test and eradication attempts]. AB - Infectious haematopoietic necrosis (IHN), a rhabdoviral infection of salmonid fish, was considered to be an exotic disease in Europe until it was recognized in France and Italy in 1987. In France, the existence of this new condition led the authorities in charge of animal health to order epidemiological studies to be undertaken. These studies were based upon virological, serological and experimental diagnostic methods and also encompassed disease eradication attempts. Studies were conducted at 7 fish farming sites, involved 1,545 salmonid fish, of which 848 were sacrificed, and represented 262 virological examinations and 1,782 serum neutralization tests. The presence of the IHN virus was detected in the 7 trout farm fish populations that were located in 5 regions, one of which was situated 600 km from the place where the first isolation of IHN virus was made. Moreover, 6 out of 7 rainbow trout populations reared in these farms also harboured viral haemorrhagic septicaemia virus (VHSV) often resulting in overt disease. Rainbow trout was the only salmonid fish species found infected with IHN. Overt infection, which was observed in fish ageing less than 2,200 degrees days, always occurred at water temperatures below 14 degrees C, and the younger fish were more susceptible (mortality rate > or = 80%). Although the IHN virus is easily isolable from fish undergoing overt infection, it was hardly detectable in survivors until they were adults, at which stage the virus was shedded via sexual products which constituted suitable materials for virological examination and disease transmission assays. Survivors of overt and dormant IHN infection developed consistent immune response and special attention was paid to neutralizing antibodies (NAb) to IHN virus. The detection of such NAb in fish from infected farming sites or other NAb from presumably IHN-free sites, correlated fairly well with the presence and further detection of IHN virus among such fish populations. Our data provide arguments for considering the serological technique as a suitable means of completing fish health surveillance programmes for IHN. Although our results are in agreement with part of the existing knowledge on IHN, they differed in several points: rainbow trout was the only susceptible fish species; overt IHN was always recorded in juveniles and at water temperatures below 14 degrees C; IHN virus could not be recovered from the mucus of infected broodfish; IHN infection usually coexisted with VHS infection in same fish population; and serology was widely and successfully used for the diagnosis of IHN. PMID- 7550398 TI - Ability of bronchodilators to prevent bovine experimental respiratory distress. AB - This cross-over trial involving 6 Blue Belgian double-muscled calves aimed to investigate whether ipratropium bromide inhaled alone or in combination with clenbuterol hydrochloride could prevent the dramatic clinical and pulmonary disturbances that are observed during an experimentally induced bronchoconstriction. Inhaled bronchodilators significantly influenced the clinical and functional responses of bovines subjected to a 5-hydroxytryptamine perfusion. However, despite a mean (standard error) wash-out period of 11.2 (3.1) and 7.5 (0.9) d after the 1st and the 2nd one-day challenges, respectively, first order carry-over effects (ie those remaining from the previous treatment), effects of the period during which the treatment was allocated and interaction effects did not allow a definitive interpretation of overall treatment differences. PMID- 7550400 TI - Frequency of interferon-alpha-secreting blood leukocytes in irradiated and bone marrow-grafted pigs. AB - The effects of irradiation were studied on porcine interferon-alpha (IFN-alpha) secreting cells (IFN-alpha SC). IFN-alpha SC were characterized by an ELISPOT assay on non-adherent PBMC following incubation with the transmissible gastroenteritis coronavirus. In vitro irradiation of PBMC was followed by a decrease in the number of IFN-alpha SC while IFN-gamma production and cell viability were not affected. These data indicate that porcine IFN-alpha SC are relatively radiosensitive. Indeed, the frequency of blood IFN-alpha SC decreased markedly and rapidly after in vivo whole body or partial lymphoid irradiation. In addition, within several days of compatible bone-marrow engraftment in the irradiated animals, the number of blood IFN-alpha SC returned to normal values. These data demonstrate that circulating porcine IFN-alpha SC are derived from bone-marrow progenitors. PMID- 7550399 TI - Effect of oxytocin on estrogen and progesterone receptors in the rat uterus. AB - The effect of oxytocin on uterine estrogen and progesterone receptors (ER and PgR) was investigated in vivo in groups of immature female rats that were treated subcutaneously with oxytocin, 0.5 or 5 IU (1 and 10 micrograms) for 5 and 3 d, respectively. Receptor concentrations and affinities were estimated by Scatchard analysis, using radioactive hormones as ligands. Statistical analysis was performed by Student's t test and by ANOVA. Oxytocin did not alter the receptor affinity for either steroid hormone, but the lower dose significantly decreased the concentrations of receptors: ER = 486 +/- 76 fmol/mg vs 346 +/- 105 fmol/mg (P < 0.001) and PgR = 686 +/- 237 fmol/mg vs 433 +/- 236 fmol/fmol/mg (P < 0.01) (mean +/- SD values for control and treated animals, respectively). There were no significant effects on the plasma 17 beta-E and Pg concentrations. In in vitro studies with mature rats, uterine specific binding of estradiol and progesterone in the presence or the absence of oxytocin showed no modification. Oxytocin could be a negative modulator of ER and PgR in the uterus, even though the mechanism of its action remains unknown. It could have potential implications on reproductive capacity and fertilization. PMID- 7550401 TI - Partial sequencing of hog cholera virus Alfort strain genome and its comparison with other pestivirus strains. AB - After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined. This sequence was compared with homologous parts of previously published pestivirus genomes. The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67%. Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively). Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC. These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage. Genomic relationships among the pestiviruses are discussed. PMID- 7550402 TI - Plasma progesterone, oestradiol-17 beta, oestrone sulphate, corticosteroids and a metabolite of PGF2 alpha: evolution throughout pregnancy, before, during and after parturition in buffalo cows. AB - The concentration of plasma progesterone, oestradiol-17 beta, oestrone sulphate, corticosteroids and 13,14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) was measured in 12 buffalo cows during the whole period of gestation, around parturition and for 15 d postpartum. The concentration of progesterone and oestradiol-17 beta increased slightly during the first 2 months (3.5 +/- ng/ml) and 4 months (14.8 +/- 2.1 pg/ml) of pregnancy, respectively. Their values remained consistently below these levels until near the end of the pregnancy period when progesterone concentrations decreased significantly (P < 0.05) at d 7 prepartum (0.9 +/- 0.1 ng/ml) and oestradiol-17 beta increased markedly (P < 0.01) at d 10 prepartum (26.3 +/- 2.6 pg/ml). Progesterone showed basal values (< 0.5 ng/ml) from d 4 prepartum to d 15 postpartum. Oestradiol-17 beta concentrations were maximal (82.8 +/- 3.6 pg/ml) during labour and returned to their basal values (< 12 pg/ml) at d 5 postpartum. The concentrations of oestrone sulphate remained low (< 140 pg/ml) during the first half of gestation period. It increased sharply (P < 0.01) thereafter to 5,620 +/- 116.5 pg/ml by 30 d prepartum and afterwards declined to about 50% of this value before calving reaching basal level (< 80 pg/ml) at d 2 postpartum. The concentration of corticosteroids fluctuated narrowly (1.7 +/- 0.3 ng/ml) throughout gestation, increasing significantly (P < 0.05) at d 12 prepartum (5.3 +/- 1.8 ng/ml) and peaking to 16.8 +/- 3.2 ng/ml at the moment of delivery. Its value declined below 3 ng/ml from d 3 onwards postpartum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550403 TI - A novel BoLA class II molecule with a tissue distribution different from BoLA-DR or BoLA-DQ molecules. AB - This study was designed to investigate the lymphoid tissue distribution of a new BoLA class II molecule defined by a unique monoclonal antibody H42A. We immunostained various lymphoid organs of 4-month-old Holstein calves with this monoclonal antibody and compared its tissue distribution to the BoLA-DR and BoLA DQ expressions. Our results demonstrate a unique tissue distribution of the H42A defined molecules, restricted to epithelial cells of the thymic medulla but extending in the periphery to the different cells involved in antigen presentation (B-cells, macrophages and dendritic cells). The peculiar distribution of this new BoLA class II molecule suggests that it has a specific function. PMID- 7550405 TI - Trials of olfactory attractants to enhance trap catches of Glossina fuscipes fuscipes (Diptera: Glossinidae) in the Central African Republic. AB - Host odours increased the trap catches of Glossina fuscipes fuscipes in cattle breeding areas of the Central African Republic. The increase was significant with zebu urine (x 1.4) and the principal reptilian host, the monitor lizard (x 1.7). The greatest effect (x 4.2) was obtained for male G f fuscipes with zebu urine when the densities of flies were low (less than 5 males per trap per day). It seems that olfactory baits in urine could improve the control of G f fuscipes by trapping. Reptile odour contains attractants that should be identified. PMID- 7550404 TI - Activity of a monoclonal antibody against Besnoitia besnoiti endozoites. AB - Besnoitia besnoiti multiplication in vitro was inhibited by a specific organelle complex-directed monoclonal antibody (MoAb) raised against the endozoite stage. Multiplication rates in antibody-treated cultures were lower at parasite/cell ratios of 1:1, 1:2 and 1:10, than in untreated cultures, after 4 or 8 d of incubation. At 1:100 ratio, there was no difference between test and control cultures irrespective of the incubation period. In in vivo experiments, the anti B besnoiti MoAb had no neutralizing effect on the infectivity of endozoites. Inoculation of gerbils with antibody-preincubated endozoites, followed by treatment with these MoAb through 5 successive days ultimately failed to prevent death in any of them. In Western blots the specific anti-B besnoiti MoAb of the IgG1 subclass recognized a 70 kDa endozoite protein in the cytosolic and the insoluble membrane fraction of endozoites. PMID- 7550406 TI - [Treatment with dialysis of chronic renal patients]. PMID- 7550408 TI - [Quantitative evaluation of the duodenogastric reflux after cholecystectomy and transduodenal sphincteroplasty: experimental study in dogs]. AB - The role of cholecystectomy and transduodenal sphincteroplasty on duodenogastric reflux (DGR) is still controversial. PURPOSE--To assess the effect of cholecystectomy alone, transduodenal sphincteroplasty alone and cholecystectomy combined with transduodenal sphincteroplasty on bile reflux into the stomach. METHODS--40 dogs were divided into four groups: control group (C) (n = 12), cholecystectomy group (Co) (n = 10), transduodenal sphincteroplasty group (Es) (n = 10) and cholecystectomy plus transduodenal sphincteroplasty group (Co+Es) (n = 8). A quantitative index of DGR was obtained in each case by determining the percentage of the intravenously injected 99m Technetium-DISIDA that was recovered in the gastric juice. RESULTS--The DGR was significantly higher in Co+Es group (mean: 2.26 +/- 2.69%) than in the C group (mean: 0.23 +/- 0.26%), and the Co group (mean: 1.52 +/- 1.77%) and the Es group (mean: 1.23 +/- 1.23%) (p < 0.05). The DGR did not differ significantly between C, Co and Es groups (p > 0.05). CONCLUSION--The results shows that only cholecystectomy plus sphincteroplasty increases duodenogastric reflux. PMID- 7550407 TI - [Control of the rejection process treated with cyclosporine and RS-61443, in allogeneic microsurgery transplantation of a limb, in rats]. AB - Composite tissue allotransplantations would contribute to the reconstructions of the congenital, traumatic and tumors deformities. PURPOSE--The aim of this study is to prevent or inhibit the rejection in limb vascularized allotransplantation; decrease the toxicity of available immunosuppressive agents; determine whether combination subtherapeutic doses of CsA +RS-61443 will have an additive immunosuppressive effect. METHODS--Five groups were studied, using Brown-Norway limb donors and Fischer 344 recipient rats: group A Untreated autograft controls; group B Untreated allograft controls; group C Allografts: CsA 1.5 mg/kg/d SQ; group D Allograft: RS-61443 15 mg/kg/d; group E Allograft: combination CsA + RS 61443. RESULTS--The results were the following concerning the rejection: Group A animals displayed no rejection clinically (0%). All the skin biopsies obtained were devoid of rejection (grade 0). Group B animal developed rejection at 10-13 days post-transplantation. Skin biopsies confirmed the epidermal necrosis (grade 4). Group C animals developed rejection in 55%. Group D animals developed rejection in 94%. In contrast, Group E animals had 96% rejection-free survival up to POD 172 thus far. CONCLUSION--Combination subtherapeutic doses of CsA + RS 61443 was effective in preventing acute rejection of limb allografts and had an additive immunosuppressive effect because of the agents immunosuppressive synergistic effect. PMID- 7550409 TI - [Hospital infection--ethical aspects]. AB - The author focuses the question of hospital infection, analysing the background on which the control committees were created. The hospital infection is discussed under bioethical principles and the Medical Ethics Code, examining the aspects related to the government, the Hospital Directorship, the Committee and the Control Service of Hospital Infection, and the assisting physician. A closer integration between the activities of the Program of Control of Hospital Infections and those of the Medical Ethics Committee is proposed, aiming at the patient and at the community, "targets of total medical attention". PMID- 7550410 TI - [New indications for the clinical use of human growth hormone: in search of a balance]. PMID- 7550411 TI - [Osteopenia and treatment with L-thyroxine]. AB - Thyroid hormones exert potent effects in bone as evidenced by bone loss which accompany untreated thyrotoxicosis. Conventional thyroxine replacement therapy may be associated with some degree of biochemical or tissue hyperthyroidism, increasing the risk for late onset osteoporosis. This has led to recommendations for reductions in L-T4 dosages. More recently, however, other reports have indicated that L-T4 therapy does not have adverse effects on bone mass. The overall analysis of both past and recent published reports has indicated that the evidence that conventional thyroxine therapy has adverse effects on bone mass is hard to ignore, although the magnitude of these effects is likely to be relatively small and closely related to estrogen deficiency. PMID- 7550413 TI - [Prevalence of intraabdominal aneurysm in elderly patients]. AB - BACKGROUND: A study in old patients on the incidence of aneurysms in abdominal arteries (AAA) and on the maximum diameter of the aorta below the renal arteries in those patients without arterial dilatation. MATERIAL AND METHODS: The study comprised 411 individuals, 218 women and 153 men with an average age of 74.4 years. Physical examination of the abdomen (EF) and abdominal ultrasonography (US) were done, and the latter was considered the "golden standard" of reference. In relation to the aorta, it was considered as an aneurysm the maximum artery diameter larger than 30 mm and for the iliac arteries, the maximum diameter larger than 15 mm. RESULTS: The US showed the presence of aneurysm in the aortic iliac territory in nine patients, one woman and eight men, corresponding to a prevalence of 2.1%, 4.1% in men and 0.4% in women. Two such aneurysms were in the iliac arteries (one aneurysm in a common iliac artery) and the other seven in the aorta below the renal arteries. The bearers of iliac aneurysm are men. The prevalence of the AAA was of 1.7% (7/411), 3.1% in men and 0.4% in women. The EF showed suspicion of the presence of aneurysm in 3 of these patients. The other 6 patients had no aneurysm. Considering all the aneurysms of the aortic-iliac territory, the EF had a sensitivity of 33.3%, a specificity of 99% and a positive prediction value of 33.3%. Considering only AAA, the sensitivity of the EF was 42.8%, the specificity 98.5%, and the positive prediction value, 33%. In the 402 patients without arterial aneurysm the maximum diameter of the aorta varied from 11 to 29 mm, with an average value of 16-21 mm. CONCLUSIONS: US is a non-invasive diagnostic procedure that should be used for the old age population. PMID- 7550412 TI - [Diabetic ketoacidosis induced by immunologic insulin resistance]. AB - Although rare, ketoacidosis may be induced by the occurrence of antibody mediated insulin resistance. Cases of 3 patients with ketoacidosis precipitated by immunologic insulin resistance (IIR) are reported. CASE REPORT--Three patients were admitted to the primary care unit of Hospital Sao Paulo in Diabetic Ketoacidosis. Demographic data of the patients (HML, DRJ and DIS) included: age (46.39 and 54 y.o.); sex (2F, 1M); diabetes mellitus (2 DM II and 1 pancreatic); duration of diabetes (6, 11 and 9 years) and BMI (17.5; 25.5 and 24.3 kg/m2. Admission laboratory data were: glucose (40, 38 and 22 mmol/L); pH (7.2; 6.9 and 7.2) and all had ketonuria. Insulin requirements for metabolic control were: HML: 1494U; DRJ: 1496U; DIS: 450U in a period of: 212, 206 and 72h. The plasmatic leves of Anti insulin antibodies (IA) measured by RIA (nU/mL) and ELISA (EI), where: HML: 7186, 3.26; DRJ: 7879, 3.42 and DIS: 8377, 2.88. HI was associated with marked decrease of both, insulin requirements and IA (HML: 3393, 1.39 after 10 months and DRJ: 4673, 2.34; DIS: 1510, after 18 months) at follow-up. DISCUSSION--The High Insulin requirements and time necessary to achieve the metabolic control guided us to the diagnosis of IIR. It was confirmed by high levels of AI and by the improvement in the metabolic control after the introduction of HI. CONCLUSION--The physician must be alert to severe IIR if there is no response after standard therapy to ketoacidosis. HI can be considered a valid alternative of treatment for IIR. PMID- 7550414 TI - [Staphylococcus aureus septicemia in children: bacterial tolerance to vancomycin and serum bactericidal activity]. AB - PURPOSE: To evaluate the susceptibility and of strains of Staphylococcus aureus isolated from children with septicemia, and to evaluate the importance of the serum bactericidal test. METHODS: Seventeen children with Staphylococcus aureus septicemia admitted to the Semi Intensive Care Unit of the Instituto da Crianca do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo were studied. Twenty nine tests in the pico and 23 in the nadir of the antibiotics were made. RESULTS: Strains of Staphylococcus aureus from hospital origin were resistant to all the antibiotics but vancomycin and pefloxacin. The phenomenon of tolerance was seen in 5 (50%) of the strains that were tested for vancomycin, and 4 of the children had a bad evolution. The serum bactericidal tests showed titles in the pico > or = 1/8 in 55.5% of the observations; in this group the evolution was better. CONCLUSION: Strain of Staphylococcus aureus from hospital origin are multiresistant. The phenomenon of antimicrobial tolerance, as well as the serum bactericidal test may be related to a bad therapeutic evolution. The increasing value of the serum bactericidal test as a way to evaluate the therapeutic evolution in severe infections, and the role of the tolerance of the Staphylococcus aureus to vancomycin more studies. PMID- 7550415 TI - [The family physician or the family's physician]. PMID- 7550416 TI - [Traumatic injuries of the gallbladder and of the extrahepatic biliary tract: analysis of 45 cases]. AB - Injury of the extra-hepatic biliary tract is infrequent, occurring in approximately 3.5% of all patients with blunt and penetrating abdominal trauma. The incidence of this injury caused by blunt abdominal trauma is rare. PURPOSE- Retrospective analysis of 5069 patients with abdominal trauma treated at the Department of Surgery University of Sao Paulo School of Medicine over a six-year period from 1986 through 1991. METHODS--Forty five patients with gallbladder and extra-hepatic ducts injury were identified (0.89%) and divided in two groups according to the nature of trauma: 12 caused by non-penetrating injuries and 33 to penetrating injuries. Records, including operative and pathology reports, were reviewed to study the site of injury, associated intra-abdominal injuries, incidence, trauma scores, treatment, morbidity, mortality rates and correlated with the nature of the trauma. RESULTS--Overall mortality was 24.4%. The incidence was greater in patients sustaining penetrating abdominal trauma (p < 0.05). Forty of the 45 patients (88.9%) had liver lacerations, the most commonly seen injuries. The patients with blunt abdominal trauma had significant different trauma scores (p < 0.05) than those with penetrating trauma, indicating greater severity in this group of patients. CONCLUSION--There is a relation between severity of trauma and incidence of extra-hepatic biliary tract injury. However, in the penetrating trauma, the incidence of trauma is correlated with the direction of the wound and there is no relation with the severity of trauma. The greater mortality seen in the patients sustaining non-penetrating injury (p < 0.05) supports this observation. PMID- 7550417 TI - [Comparison of the subjective global nutrition assessment x objective nutrition evaluation]. AB - OBJECTIVE: Nutritional Assessment methods (ONA) are traditionally employed in hospitalized patients (anthropometry, laboratorial exams and immunological tests). The Subjective Global Assessment (SGA) estimates weight loss and changes in dietetic intake as being allegedly efficient for protein-caloric malnutrition diagnosis of the hospitalized adult. PURPOSE: Compare the efficacy of SGA against the traditional ONA. METHODS: The prospective study was done with 100 hospitalized patients from 3/90-5/90 at the Hospital Beneficencia Portuguesa de Sao Paulo. The Detsky et al. questionnaire was applied besides anthropometric measurements (body weight, triceps skinfold thickness-TSF, mirdam circumference MCA) and laboratorial examinations (serun albumin, hemoglobin, peripheral blood lymphocytes) all of them in the first 3 days of admission. SGA always preceded ONA, and was always done by the same observer. RESULTS: There was a significant reduction on the average values of anthropometric and laboratorial measurements with the progressive worsening of nutritional status assessed by SGA. There were significant associations (p < 0.05) between hypoalbuminemia (< 3.5 g/dL) loss of TSF (< 10 mm) and MAC (< 23.3 cm) in those patients classified as moderately and severely malnourished by SGA (14%). Hypoalbuminemia, low TSF and SGA malnutrition were all significantly associated to mortality (p < 0.05). CONCLUSION: SGA in our hospital is a reliable diagnostic method for the diagnosis of malnutrition of hospitalized adult patients and malnutrition of hospitalized adult patients and keeps prognostic association with mortality. PMID- 7550418 TI - Transitory complete atrioventricular block associated to ingestion of Nerium oleander. AB - BACKGROUND: Self-medication with plants can lead to severe poisoning. Oleander (Nerium oleander) is an ornamental plant whose toxicity to man is due to a mixture of nondigitalis cardiac glycosides. The clinical manifestations of oleander poisoning combine cardiac and gastrointestinal symptoms, and are similar to those of a digitalis overdose. CASE REPORT: Following the ingestion of tea made of the leaves of oleander, a 33-year-old woman developed dizziness, vomiting and abdominal cramps as main symptoms, and complete atrioventricular block that reverted within 24 hours. She remained with bradycardia, dizziness and diarrhea for about 6 days. On admission she had a serum potassium of 6.7 mEq/L and a serum creatinine of 2.3 mg%, that progressively returned to normal levels. CONCLUSION: Clinicians must include oleander poisoning in the differential diagnosis of bradyarrhythmias, particularly in children and young people without known cardiovascular disease, in areas where this plant either is used as a herbal medicine or is known as poisonous. PMID- 7550419 TI - [Indications for surgical resection of pulmonary metastases]. PMID- 7550420 TI - [Answers to human organ procurement: donations, research, and prevention]. PMID- 7550422 TI - [The effect of chemical sympathectomy with 6-hydroxydopamine on the activity of the sodium-potassium pump and on the contractile responses of the mesenteric artery and vein]. AB - After chemical sympathectomy of mesenteric arteries and veins, different increase of responsiveness to noradrenaline, potassium chloride and ouabain was found. Stronger changes occurred in the vein which seems to be due to different adrenergic innervation of arteries and veins. PMID- 7550423 TI - [The perception of muscle loading and the role of locomotor and respiratory sensation]. PMID- 7550421 TI - [Multiple neoplasms after a well succeeded treatment of Hodgkin's disease]. AB - The authors refer to a 21-year-old Caucasian (white) woman, who in 1977 presented fever and cervical and axillary adenopathy, whose biopsy showed nodular sclerosis Hodgkin's Disease, stage IIIB. The patient received six chemotherapy cycles associated with immunotherapy and supplemented with radiation therapy with good response. RESULTS--In 1985, after routine gynaecological examination and a hysterectomy, cervical intraepithelial neoplasia grade 3 (CIN 3) and atypic leiomyoma of the uterine body were diagnosed. Five years later, biopsies diagnosed invasive duct carcinoma in the right breast and homolateral axillary and cervical nodes. The patient was submitted to chemo and radiation therapy and died nine months later. CONCLUSION--The possibility of later occurrence of a second or multiple new malignancies in patients successfully treated for Hodgkin's Disease points out the need for a more complete long-term follow-up, including periodic mammography. PMID- 7550424 TI - [The chronic action of an alternating magnetic field in murine ontogeny]. AB - Alternating magnetic field induced changes in the blood system both on the level of functioning cell group and on the level of proliferative elements in white mice. The field inhibits the differentiation processes in seminal glands. The field seems to be a biologically active factor exerting its effect in crucial periods of the ontogeny. PMID- 7550425 TI - [The effect of diazepam on the activity of carboxypeptidase N and the angiotensin converting enzyme in sections of the brain of rats with differing normal emotionality and in emotional stress]. AB - Diazepam increased the carboxypeptidase H activity and decreased the angiotensin converting enzyme activity in the rat brain. Joint action of diazepam and emotional stress changed the carboxypeptidase H activity to the least extent and the angiotensin converting enzyme activity to the utmost extent as compared to the agents' separate effects. The role of both enzymes in the diazepam effect is discussed. PMID- 7550426 TI - [The dynamics of the ion currents in the membrane of an isolated mollusk neuron under the action of high pressure]. AB - The effect of high ambient pressure on the inward and outward currents in the membrane of the snail isolated neuron was studied. A significant inhibition of the inward (sodium) current occurred at 101 ata. No significant modification of the outward (potassium) current was found. The data obtained corroborates the hypothesis of inhibitory effect of high pressure on some membrane structures involved in generation of neuronal activity. PMID- 7550427 TI - [A comparison of the measurements of local cerebral blood flow by hydrogen clearance with hydrogen inhalation and by its electrochemical generation in brain tissue]. AB - The goal of the present investigation was to compare data received by modifications of H2-clearance method with inhalation (H2-Inh) and electrochemical generation in brain tissue (H2-Gen) of H2 from the same recording electrodes in acute experiments (urethane 1g/kgip) with Wistar rats (n = 39). Block of three or four Pt electrochemically sharpened electrodes to tip diameters of 20-40 mu, 0.3 0.5 mm apart and 0.8-1.0 mm in length were inserted through the dura into sensorimotor cortex. One electrode was used for H2 generation (DC current 3-5 microA) and the others for PH2 recordings by polarographical method. Durations of H2 inhalation and H2 electrochemical generation were 3 or 10s with similar amplitudes of PH2. Data was recorded with a MacLab analog-digital system and Macintosh 11si computer. Recordings of resting LCBF were made with H2-Gen and H2 Inh the beginning of the experiment and after 40-60 min. LCBF was stimulated with inhalation of 7.5% CO2 for 60s. In 6 preparations LCBF was measured 10-20 min. after occlusions of two branches of the middle cerebral artery. After experiments the brain was perfused with India ink and cut in frozen sections for morphological analyses. The mean values of resting LCBF measured by H2-Inh was 1.67 +/- 0.54 ml/g/min. (+/- SD, N = 149), and by H2-Gen 3.17 +/- 0.91 (N = 147). The diffusional component was estimated as 1.2-2.5 (equivalent units, ml/g/min.) in dead cortex. The ratio of clearances in H2-Gen to H2-Inh varied in different experiments from 1.0 up to 4.0.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550428 TI - [A universal method for performing microelectrode and neurochemical studies of the subcortical brain structures in waking cats]. PMID- 7550429 TI - [The synchronous recording of the neuronal activity and of the dynamic content of intracellular bound calcium in the cat cerebral cortex]. PMID- 7550430 TI - [The sex dimorphism in the dynamics of interhemispheric asymmetry during the processing of a transcallosal signal]. AB - Two main patterns of interhemispheric domineering were distinguished: the right left-right shift type and the left-right-left one. The domineering of the right hemisphere at the beginning and the end of processing of a transcallosal signal, was stronger in female rats in some areas, whereas in the majority of the areas under study domineering of the left hemisphere occurred more frequently in male rats. The data obtained suggests a greater participation of the right hemisphere cortex in females and the left hemisphere in males. PMID- 7550431 TI - [The sensory and motor components of different forms of learned movement during a change in the activity of the neostriatal cholinergic system]. AB - The role of the neostriatal cholinergic system in the regulation of sensory controlled movements were studied using two behavioral models: 1) maintenance acquired extension of forelimb during proper time (rats) and 2) prolonged conditional flexion of hindlimb (dogs). It was found that injections of carbacholine (Car, 0.03 mkg) into the dorsolateral part of nucleus caudatus (NCd) of rats did not considerable change the movement connected with maintenance of forelimb tonus, while injections of Car (0.05-0.1 mkg) into the same area of the NCd of dogs were accompanied by improved performance of movement connected with maintenance of hindlimb tonus. It is possible to suggest the follows: 1. The neostriatal mechanisms of regulation of fore- and hindlimb tonus maintenance are different. 2. The neostriatal mediator of mechanisms of tonus regulation and regulation of somesthetic control of maintenance of this tonus are probably different too. Bilateral Car microinjections (0.03 mkg) into NCd of rats improved the discriminative active avoidance learning in T-maze. The discrimination of sound signals in dogs were also significantly improved after Car injections (0.05 0.1 mkg) into the NCd. Thus, data obtained at the different behavioral models and different animals suggest that cholinergic system of Neostriatum takes part in the regulation of both motor and sensory components of learned movements realization. The possible mechanisms of this regulation are discussed. PMID- 7550432 TI - [The participation of the glutaminergic input of the nucleus accumbens in regulating the synaptic release of dopamine during associative learning]. AB - The effect of MK-801 on basal and conditioned dopamine release in the rat n. accumbens, was studied. Exposure of the rats to the box where they had been given electrical shock, caused an immediate long lasting increase in the dopamine release. Administration of the MK-801 into the n. accumbens abolished the delayed increase in the dopamine release while exerting no effect on the immediate increase. PMID- 7550433 TI - [The electrical activity of symmetrical areas of the rat cerebral cortex during the use of a low-intensity UHF field]. AB - Electrical asymmetry of electrograms of the rat symmetrical brain areas was decreased under the UHF irradiation of the animals. Non-modulated UHF field increased the 1.5 and 6 Hz rhythms in the left hemisphere, whereas modulated UHF field decreased the 1 Hz rhythm in the right hemisphere. The effects were only occurring at the initial period of the UHF fields use. PMID- 7550434 TI - [The dynamic impulse activity of the neocortical neurons in monkeys with the task of visual recognition after short-term oxygen starvation]. AB - During a 2.5-min. hypoxia in monkeys, a rearrangement of the unit activity occurred in the visual and prefrontal cortex. Following this, the motor response in delayed visual spatial reaction tack did not restore during several hours. The rearrangement involved appearing of sequential phases of hyperactivation and inhibition. Duration of the posthypoxic inhibition and ratio of posthypoxic hyperactivation frequencies to the background one were significantly greater in the prefrontal cortex than in the visual one. PMID- 7550435 TI - [The molecular cellular mechanisms of the protective effect of short-term anoxia]. AB - Recovery of unit activity was studied after a 5-min anoxia following a short-term anoxia in the cat brain cortex. The short-term anoxia's protective effect was only revealed in case when it had induced the phenomenon of asphyxic hyperactivity. The protective effect seems to be due to the short-term anoxia induced changes in the calcium, polyphosphoinositide and cAMP regulatory systems' activity. PMID- 7550436 TI - [The effect of the composition of an artificial gas environment under pressure up to 5.1 MPa on the oxidation-reduction reactions in the human body]. AB - In 500-m dives, lipid and carbohydrate metabolism was found to depend on the composition of hyperbaric gas mixture. Reduction of the oxygen partial pressure for over 2.0 kPa from the optimal one depressed aerobic energy-producing biochemical reactions in the human blood cells. 5 per cent of nitrogen in the hyperbaric gas mixture at 5.1 MPa suppressed the aerobic metabolism. PMID- 7550437 TI - [The consequences of physical loading in puberty for lactation]. AB - Consequences of physical loads in pre- and puberty periods told both on the rats and their litter. Adaptation to physical load occurring with participation of the androgen told negatively on the mammo- and lactopoiesis. PMID- 7550438 TI - [The relationship of systemic pressor reactions to the initial (spontaneous) blood pressure level]. AB - Relative responses of the systolic and diastolic pressure to polyglukine and mesatone administration were found to be more obvious in anesthetised rats with initial blood pressure less than 100 mm Hg. A reverse linear correlation was found between the above parameters at the high level of connexion for both agents. Exclusion of sinocarotid or cardioaortic areas did not affect the dependence of the responses to measatone on the initial blood pressure, whereas such a dependence was found for polyglukine at intact baroreceptors. PMID- 7550439 TI - Human bilharzial ureters: I. Fine structure of eggs deposited in the submucosa and muscularis. AB - The internal fine structure of the partially calcified Schistosoma haematobium eggs in the lower ureteral segments of Saudi patients with chronic urinary schistosomiasis is described. The egg shell is penetrated by cribriform pores and consists of the three previously described layers: outer microspinous, middle intermediately dense, and inner dense layers. The space between the egg shell and the developing embryo is partitioned by three layers. An outer acellular Reynolds' layer of unknown origin and function consists of a fibrillar material mixed with a finely granular matrix extending to the egg-shell pores via racimose channels. The middle von Lichtenberg's envelope consists of a single layer of flattened epithelial cells containing several mitochondria in other previously described Schistosoma eggs suggesting an active, and perhaps selective, transport in or out of the egg shell. The inner fluid filled cavity, or Lehman's lacuna, between the von Lichtenberg's envelope and the embryo contains numerous lipoid bodies suggesting a relation to vitelline cells. - Four systems (out of eight previously described for the free, mature Schistosoma miracidium) have been recognized for the first time in the developing miracidium within S. haematobium eggs and include: 1) ciliated epidermal plates representing the epithelial system, 2) an outer circular and an inner longitudinal muscle layers forming the musculatures, 3) lateral penetration glands, and 4) ciliated flame cells representing the excretory system. PMID- 7550440 TI - A study on the prevention of prenatal and galactogenic Toxocara canis infections in pups by treatment of infected bitches with ivermectin or doramectin. AB - Investigations were carried out to test the efficacy of a two dose treatment with ivermectin or doramectin on reactivated larvae of Toxocara canis in bitches to prevent prenatal and galactogenic infections of their pups. Thirty pregnant bitches were treated by subcutaneous injection of ivermectin or doramectin on day 40 and 50 post conception (p.c.) each with a dose of 1 mg/kg body weight. The efficacy of the treatments was determined by faecal examinations and by comparing the number of infected pups and the onset of patency in each group of pups. 1. Patient infections with T. canis were seen in pups from untreated bitches from day 21 after birth and all pups were coproscopically positive one week later. To prevent severe clinical symptoms all pups in this group were treated with anthelmintics on day 42 after birth. 2. Pups from ivermectin treated bitches occasionally exhibited patent infections from day 28 after birth onwards; pups from doramectin group exhibited infections from day 56 after birth. The number of patently infected pups in both groups increased till 70 days after birth, probably because of postnatal infections. 3. 28 days after parturition, T. canis infections became patent in all untreated bitches. Single bitches in the ivermectin and doramectin groups developed patent infections after day 49 after parturition. All coproscopically positive bitches had patently infected pups in their litters. 4. The contamination of the environment with eggs of T. canis was calculated from the results of the faecal examinations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550441 TI - Morphology and ultrastructure of the redia and pre-emergent cercaria of Opisthorchis viverrini (Trematoda: Digenea) in the intermediate host Bithynia siamensis goniomphalus (Prosobranchia: Bithyniidae). AB - The intramolluscan stages of the human liver fluke Opisthorchis viverrini were investigated by light, scanning, and transmission electron microscopy. The morphology of the tegument, and the excretory system of the redia and pre emergent cercaria as well as glandular cells, tail structure, and the eye spots of the cercarial stage are described. Possible environmental adaptations are discussed. The redia is characterized by a tegument which is folded concentrically and covered with densely arranged microvilli. This tegument is composed of a typical syncytium with submerged tegumental cells. The pre-emergent cercaria is characterized by a long tail with prominent fin folds and obliquely striated muscles. The oral sucker is well developed and protrusible, whereas the ventral sucker is faintly developed. Proteinaceous tegumental spines which are arranged transversally and different kinds of presumed sensory structures cover the cercarial body surface. Two well pigmented eye spots with rhabdomeric photoreceptors are located in the anterior part of the body. An extended protonephridial system is connected to a voluminous excretory bladder. The excretory tube opens at the end of the body and does not lead into the tail. Three types of glandular cells could be differentiated according to their vesicles, which are shed into the syncytial layer. PMID- 7550443 TI - On the diagnostics and nomenclature of Sarcocystis species (Sporozoa) in roe deer (Capreolus capreolus). AB - Three species of sarcocysts from European roe deer (Capreolus capreolus) are characterised using light and electron microscopy. The hitherto existing descriptions of four species are attributed to the actual state of nomenclature. PMID- 7550444 TI - Epidemiological studies of coccidiosis in the domesticated fowl (Gallus gallus): I. The fate of ingested oocysts of Eimeria tenella during the prepatent period in susceptible chicks. AB - A small proportion of sporulated and unsporulated coccidial oocysts (Eimeria tenella) inoculated per os into domesticated fowl were recovered intact within about one day, well within the usual 5-day ?prepatent period.? Recovery rates of sporulated oocysts were ca. 2-9% from chicks inoculated at all ages tested from 2 to 28 days. From chicks within the same age range, the recovery rates for unsporulated oocysts were ca. 15-21%. The higher recovery rates of unsporulated oocysts indicated that they were more resistant to disruption in the gizzard than sporulated oocysts. Up to 83% of unsporulated oocysts that survived passage through the host's gut were able subsequently to sporulate. The first oocysts recovered, either sporulated or unsporulated, appeared at virtually the same time in all the chicks tested, despite the oldest birds having guts twice as long as the youngest. Moreover, the lengths of oocyst recovery periods in the oldest birds were 10-25% of those in the youngest. PMID- 7550442 TI - The in vitro culture of Onchocerca spp.: II. The excretory/secretory products of the first and third stage larvae of Onchocerca lienalis and O. ochengi. AB - The developing first and third larval stages of two bovine Onchocerca species were maintained in vitro in the presence of 35S-methionine, following their initial development in the vector species Simulium ornatum s.1. to characterise the expression and secretion of their metabolites. The first larval stages of O. lienalis and O. ochengi did not release any E/S products. In contrast the supernatants of the third stage larvae of both species contained a double band of 21 and 20 kDa for O. ochengi and 23 and 22 kDa bands for O. lienalis when kept at room temperature. A temperature shift to 37 degrees C led to the increased expression of the 23 kDa protein with the infective larvae of O. lienalis. The possible role of these molecules is discussed. PMID- 7550446 TI - Prevalence of ticks in relation to their role as vector of Borrelia burgdorferi under autochthone conditions. AB - A total of 59 (78.7%) of 75 areas were found to be inhabitated by ticks; all the 4335 collected ticks were identified as Ixodes ricinus. Both nymphs and adults were found to be more active during the late spring and early summer months. Ticks of 29 recreation areas were found to be carrying spirochaetes. Considering the total number of 75 recreation areas examined and the total number of 59 areas inhabitated by ticks the percentage was 38.7 and 49.2 respectively. The positivity rate by dark field microscopy was 16.7% in 653 tick samples (single nymphs 8.8%, pooled nymph samples 21.5%, single females 10.9% and single males 10.8%). Overall positivity rate by culture examination of 245 samples was 22.8%. It was maximal in nymphs (24.7%) followed by males (21.1%) and females (19.7%). 10.2% of the 177 samples, found negative by DFM, were positive by this method. A positivity rate of 23.9% in 96 tick samples was found by IFAT. A total of 56 spirochaete-strains were obtained of which 51 could be maintained further; 50 strains were identified as Borrelia burgdorferi. PMID- 7550445 TI - Epidemiological studies of coccidiosis in the domesticated fowl (Gallus gallus): II. Physical condition and survival of Eimeria acervulina oocysts in poultry house litter. AB - The survival of coccidial oocysts in poultry-house litter has been examined. The experimental design ensured that viable oocysts were not continuously replenished by multiplication in chicks. Hence, reliable oocyst survival data were obtained, which is not possible in a commercial house where there is a continuously changing population of coccidia. Litter was seeded directly from infected chicks with unsporulated oocysts of Eimeria acervulina. Whilst sporulation proceeded rapidly, oocysts simultaneously began to deteriorate after only 24 h. After five days, most oocysts were disintegrating. Viable oocysts were, however, detected (using sentinel birds) up to day 23, although subsequently the viability of the oocyst population began to wane. Few viable oocysts were detectable after 38 days, despite some intact sporulated oocysts being evident up to 54 days. The physical condition of oocysts is therefore not a reliable indication of their viability. The surface temperature of the litter was 25-28 degrees C throughout and the moisture content varied from 31.0 to 62.1%; these conditions were not limiting factors for oocyst sporulation. PMID- 7550447 TI - Immunophysiology: the immune system as a multifunctional physiological unit. AB - In this review, the immune system is described as a physiological system in its functions and relations with other organs. Hence, communications are mediated by common receptors and cytokines. The immunophysiology is a bidirectional system where the immune system is influenced by endocrine apparatus and vice versa. At the cellular level, we have attempted to define this "cross-talk" by proposing one- or two-signal models, where the first signal is mediated via binding of hormones or neurohormones to the lymphocytes and the second signal is generated by binding of the antigen to the recognition sites or of cytokines to their lymphocyte receptors. The physiological reality of these interrelationships is discussed. PMID- 7550449 TI - Differential effects of tumor necrosis factor-alpha on the expression of fibronectin and collagen genes in cultured bovine endothelial cells. AB - The effects of recombinant human tumor necrosis factor-alpha (TNF-alpha) on the expression of fibronectin and types (IV), (III), and (I) procollagen genes in cultured bovine pulmonary artery endothelial cells were examined in this study. Findings indicate that TNF-alpha increases steady-state levels of alpha l (IV) and alpha l (III) procollagen mRNAs while it decreases levels of fibronectin and alpha 2 (1) procollagen mRNA and leaves cytoskeletal actin mRNA levels unchanged. Both dose and exposure time moderated these effects. Treatment with TNF-alpha increased the stability of alpha l (IV) procollagen mRNA. The half-life of this mRNA, previously unreported in the literature, was increased by 118%, while the stability of fibronectin mRNA decreased by 44%. The stability of mRNAs for procollagens alpha l (III) and alpha 2 (I) were unchanged. Except in the case of procollagen alpha l (III), these effects were blocked by cycloheximide. Protein production induced by TNF-alpha was evaluated by immunoprecipitating proteins from the media and cell lysates. Our data indicate that TNF-alpha has a strong pretranslational regulatory role in cultured endothelial cell's expression of extracellular matrix protein genes. PMID- 7550448 TI - Tissue-specific, temporally regulated expression mediated by the proximal ovine beta-lactoglobulin promoter in transgenic mice. AB - The ovine beta-lactoglobulin gene is expressed abundantly in the mammary gland. This study determines whether the ovine beta-lactoglobulin promoter is sufficient for targeting tissue-specific expression in transgenic mice. To address this, the expression profile of an ovine beta-lactoglobulin promoter driven bacterial chloramphenicol acetyltransferase reporter construct was analysed. Comparison of the expression frequency of this hybrid transgene to that of a genomic beta lactoglobulin transgene indicates that additional sequences, downstream of the promoter, are required for position-independent expression in transgenic mice. Nevertheless, the hybrid transgene was expressed specifically in the mammary gland. Furthermore, the hybrid transgene was expressed in the appropriate temporal pattern during pregnancy and lactation. Thus, the proximal promoter of the ovine beta-lactoglobulin gene contains sufficient sequence information to target expression to the mammary. This construct constitutes the basis for a compact mammary expression vector. PMID- 7550450 TI - Functional consequences of substitutions of the carboxyl residue glutamate 779 of the Na,K-ATPase. AB - Carboxyl-containing amino acids in the transmembrane segments appear to be important for sodium- and potassium-activated adenosinetriphosphatase (Na,K ATPase) activity. Substitution of Glu779 with Leu in a ouabain-resistant isoform inactivates the overall enzyme activity (Jewell-Motz & Lingrel, 1993). Chemical modification of this residue results in inactivation of Na,K-ATPase in a Na+ and K+ protectable manner (Arguello and Kaplan, 1991, 1994). These experiments suggest that this residue is important in cation binding. To further understand the role of Glu779 in Na,K-ATPase function, we have substituted this with four amino acids (Gln, Asp, Ala, and Leu) using site-directed mutagenesis coupled with expression and characterized the expressed enzyme. The amino acid substitutions were introduced into a modified sheep RD alpha 1 isoform that is relatively resistant to this drug. Enzyme carrying the E779Q and E779A replacements conferred ouabain resistance to the sensitive HeLa cells, while expression of enzyme carrying the E779D and E779L substitutions did not. Further analysis of isolated plasma membranes containing altered enzymes E779Q and E779A confirmed that they retain Na,K-ATPase activity. Analysis of cation stimulation of Na,K ATPase activity revealed that the E779Q substituted enzyme exhibited a similar apparent affinity for K+ and a 2.6-fold decrease in affinity for Na+ compared with control enzyme. The E779A replacement caused a 6.6-fold and 5-fold decrease in apparent affinity for K+ and Na+, respectively. There is no difference in apparent affinity for ATP at the low affinity site for either E779Q or E779A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550451 TI - Demonstration of differentiation in hepatocyte progenitor cells using dipeptidyl peptidase IV deficient mutant rats. AB - The presence of progenitor or stem cells in the adult liver and their potential roles in oncogenesis are unresolved issues. The study of hepatocyte progenitor cells has been limited by a lack of convenient in vivo systems allowing unequivocal cell localization and demonstration of differentiation into hepatocytes. To develop an in vivo progenitor bioassay, early (E14) fetal Fischer 344 rat hepatoblasts were transplanted into the spleen of syngeneic, weaning rats deficient in dipeptidyl peptidase IV (DPPIV) activity. The donor status of transplanted hepatoblasts was demonstrated by DPPIV expression. Localization of hepatoblasts was facilitated by the use of an ectopic site, as well as weanling recipients, which readily allowed identification of very small numbers of transplanted cells. Fetal rat hepatoblasts were demonstrated to undergo cellular differentiation along the hepatocyte lineage by acquiring glucose-6-phosphatase activity within 5 d of transplantation. A critical review of previous transplantation studies of hepatocyte progenitor cells and the role of the local microenvironment at inducing differentiation indicates that this novel bioassay should facilitate analysis of progenitor cells. PMID- 7550452 TI - The tyrosine phosphorylation of a p72syk-like protein in activated murine resident peritoneal macrophages. AB - As a marker of macrophage activation, IL-1 alpha was measured after stimulation of murine resident peritoneal macrophages (RPM) with endotoxin-associated protein (EP). Significant IL-1 alpha was produced by EP-stimulated RPM from both C3H/OuJ and C3H/HeJ mouse strains. This EP-mediated IL-1 alpha production was blocked by tyrosine kinase inhibitors including genistein and tyrphostin, suggesting the involvement of a protein tyrosine kinase in the activation of RPM by EP. Immunoblot analysis using antiphosphotyrosine antibody showed that EP induces the tyrosine phosphorylation of a 71 kD protein (p71). The p71 and the spleen tyrosine kinase p72syk found in other cell types share common features including: similar molecular weight, PKC independent tyrosine phosphorylation, and inhibition of phosphorylation by piceatannol. Furthermore, immunoblot analysis using anti-p72syk antibody detected the p72syk kinase in EP-activated RPM. These results suggest that the activation of RPM involves an early tyrosine phosphorylation of p72syk or a p72syk-like protein. PMID- 7550453 TI - Inhibition of cleavage by restriction endonucleases due to modifications induced in SV40 DNA by methyl methanesulfonate. AB - Methyl methanesulfonate (MMS), a direct mutagen, methylates DNA bases and causes distortions in DNA structure. Supercoiled SV40 DNA was treated in vitro with varying concentrations of MMS from 0.001 mM to 10 mM MMS either for 30 min or 3 h and analysed by electrophoresis in 1% neutral and alkaline agarose gels. The electrophoretic mobility (EPM) of native DNA did not change after treatment with the mutagen, while alkaline gels revealed low MW DNA fragments due to single strand breaks at alkali-sensitive sites generated by the action of MMS. By two dimensional electrophoresis, we find that all three native DNA forms contain alkali-sensitive sites after treatment with MMS. To examine the effect of base modification by MMS on DNA-protein interactions, we have used as probes, restriction endonucleases. These cleave DNA in a sequence-specific manner, and their activity is dependent upon the methylation status of the substrate DNA. We find that cleavage by these restriction endonucleases is inhibited due to methylation by MMS. PMID- 7550454 TI - Fast inducible repair of microinjected UV-irradiated SV40 DNA in monkey kidney cells. AB - In monkey kidney cells (TC-7), microinjected with UV-irradiated (103-362 J/m2) SV40 DNA, the expression of viral antigens decreases in a UV-dose-dependent manner and the viral genes are not repaired constitutively. When the viral DNA is microinjected 4 h after UV-irradiation (40 J/m2) of host cells, the expression of viral antigens is restored in all cells. The time course of restoration of viral gene expression function shows that in UV-irradiated cells the repair is induced rapidly and fully within 2 h and the induced state is maintained for 24 h. Intact viral DNA molecules, microinjected during the period of induction of cellular UV repair, are expressed less efficiently than UV-irradiated viral genomes. PMID- 7550455 TI - NADPH-diaphorase activity in piglet intestinal mucosa. AB - The distribution of the enzyme synthesizing nitric oxide (NO) has been characterized in several mammalian enteric nervous systems. Two methods, immunohistochemical staining, employing anti-nitric oxide synthase antibodies, and histochemical localization of NADPH-diaphorase (NADPH-D), have given the same results. On the other hand, few studies have investigated nitric oxide synthase (NOS) in the gastrointestinal mucosa. Our study demonstrated the presence and distribution of the enzyme, NADPH-D, throughout all layers of the neonatal piglet intestinal tract. In the neonatal piglet, NADPH-D activity was found in nerve fibers parallel to the circular and to the longitudinal muscles and in the ganglion cells of Auerbach's plexus. However, the majority of NADPH-D activity was localized to the mucosa. Furthermore, the most intense activity in the mucosa was observed in villous epithelial cells. Other mucosal cells which were NADPH-D positive included the glandular epithelium and crypt cells. In addition, glandular epithelium in the deeper submucosa had very strong NADPH-D activity. Our results support the hypothesis that locally produced NO mediates physiological functions in the intestinal mucosa and submucosa. PMID- 7550456 TI - How Medalia made primary care its cornerstone. Interview by Christopher Serb. PMID- 7550457 TI - Leadership. PMID- 7550458 TI - Consumers--paid to pump up. PMID- 7550459 TI - Leadership ... conducting a survey. PMID- 7550460 TI - Payment--putting the tab on plastic. PMID- 7550461 TI - Doctor pay hikes slow in 1994 for some specialties. PMID- 7550462 TI - Employers ... "In Good Company," a training program for employee assistance professionals. PMID- 7550463 TI - Mean streets. Five lessons from the front lines of reengineering. AB - Two-thirds of all reengineering attempts fail. Why? CEOs cede responsibility to consultants, push through ?slash-and-burn? layoffs thinly disguised as redesign, and often just lack the guts to follow through. Worse, still, is that they forget the process is about people. But you can avoid these potholes along the way. Here are five lessons from the front lines. PMID- 7550465 TI - Hispanic Americans. Who are they, where are they, and how do we talk to them? PMID- 7550464 TI - Combine & conquer. Lessons learned from a Minnesota merger. AB - Despite the fact that the doctors at Park Nicollet Clinic brought in most of the patients at Methodist Health System's hospital, their alliance was tentative. So when the medical group decided to build a facility in order to compete with Methodist, anything could have happened. Health care organizations poised to merge can learn from their tale. PMID- 7550466 TI - Seeing green. The Rural Wisconsin Health Cooperative helps keep the state's rural hospitals from being put out to pasture. Here are three of their stories. PMID- 7550468 TI - Home health--the nursing circuit. PMID- 7550467 TI - Wellness--taking health to heart. PMID- 7550469 TI - Employers--prepping for patients. PMID- 7550470 TI - Managed care--saying no to hospital-controlled PHOs. PMID- 7550471 TI - Protocols--going down the clinical path. PMID- 7550472 TI - Facilities--a new hospital takes wing. PMID- 7550473 TI - Online--grand rounds in cyberspace. PMID- 7550474 TI - Public health--catching breast cancer early. PMID- 7550475 TI - Targeting health care consumers. PMID- 7550477 TI - Waterbirth: a 10-year retrospective. PMID- 7550476 TI - Opportunities to learn. PMID- 7550478 TI - "My mouth tastes like sand". PMID- 7550479 TI - Searching for underlying causes. PMID- 7550480 TI - How can we persuade women to choose a midwife for their births? PMID- 7550481 TI - Burnout control. PMID- 7550482 TI - Turning toxemia around. PMID- 7550485 TI - Highlights from Eugene: something for everyone at the Fourth Annual Midwifery Today West Coast Conference. PMID- 7550486 TI - Birthing our message. PMID- 7550483 TI - Educating the public. PMID- 7550484 TI - Changing viewpoints. PMID- 7550487 TI - Opening doors to the next generation. PMID- 7550488 TI - Walking our true path. PMID- 7550489 TI - From which we come. PMID- 7550490 TI - Soothing. PMID- 7550491 TI - A change of mind. PMID- 7550492 TI - Choices with no regrets. PMID- 7550494 TI - Noninvasive glucose monitoring in diabetic patients. PMID- 7550493 TI - Calcification and allergy. PMID- 7550495 TI - Designing medical devices for conformance with harmonized standards. The European Community's Medical Devices Directives and their effect on the product development process. AB - The European Community's Medical Devices Directives represent an ambitious effort to streamline the regulation of medical devices within the European Economic Area, and area comprising more than 380 million people. In this, the first of two special reports, Richard C. Fries and Mark D. Graber describe the Medical Devices Directives and their effect on the product development process. In the second report, appearing on page 294 Jean A. Goggins uses a case study format to demonstrate the process that would be used to gain European approval for a hypothetical medical device. PMID- 7550496 TI - Designing medical devices for conformance with harmonized standards: a case study of non-active implants. AB - The European Community's Medical Devices Directives represent an ambitious effort to streamline the regulation of medical devices within the European Economic Area, an area comprising more than 380 million people. In this, the second of two special reports, Jean A. Goggins uses a case study format to demonstrate the process that would be used to gain European approval for a hypothetical medical device. In the first report, appearing on page 284, Richard C. Fries and Mark D. Graber describe the Medical Devices Directives and their effect on the product development process. PMID- 7550497 TI - The battery enigma: are third-party rechargeable batteries as good as OEMs'? PMID- 7550498 TI - Benchmark indicators for medical equipment repair and maintenance. PMID- 7550499 TI - Productivity improvement in the community hospital. PMID- 7550500 TI - A hydrostatic method assessing accuracy and reliability while revealing asymmetry in blood pressure measurements. AB - This study describes a method for noninvasive hydrostatic validation of blood pressure devices. It is based on the hydrostatic pressure exerted by a column of blood. The mid-position of an upward-held wrist blood-pressure cuff marks the top of the column, and a cuff on the opposite wrist pointed downward similarly determines the bottom of the column. Twenty-one healthy subjects comprising 12 right-handed and nine left-handed men and women entered this study. Each repeatedly alternated the arm positions ten times. Linear regression analysis yielded correlation coefficients for comparison of the Dinamap devices against the hydrostatic differences of 0.57, 0.70, and 0.70 for systolic pressure (SP), diastolic pressure (DP), and mean pressure (MP), respectively. These coefficients combined with the results of the graphic method of Altman and Bland demonstrated the best results for DP and matched least for SP. Overall results presented mean errors (ME: Dinamap-hydrostatic) +/- standard deviations (SD) of 9.3 +/- 10.1, 1.0 +/- 5.6, and 6.3 +/- 6.7 mmHg for SP, DP, and MP, respectively. These results are numerically comparable to some of the prior reports of ME +/- SD analysis of Dinamap devices by conventional validation studies. Numerically, DP is within and MP is close to the AAMI standards. The results of ME +/- SD, however, revealed a remarkable asymmetry for SP. The results of all the left-arm-up/right-arm-down position had ME +/- SD 12.3 +/- 11.1, -0.6 +/- 5.8, and 7.2 +/- 7.1 mmHg for SP, DP, and MP, respectively. The results of the opposite positioning were 6.3 +/- 7.9, -1.3 +/- 5.5, and 5.4 +/- 6.2 mmHg, respectively. This asymmetry of the SP differences seems independent of handedness (left or right) and gender, and may be related to the reflectance caused by intrathoracic vasculature asymmetry, which suggests that for validation purposes, the left-arm-down/right-arm-up position should be used. The alternative position may be used to detect and/or study this asymmetry. Such detection should have important clinical consequences for patient blood pressure monitoring. During surgery, if both arms must be placed shoulder-high, for instance, the right arm should provide more accurate and reliable blood pressure measurement. PMID- 7550501 TI - A warning detector for urinary incontinence for home health care. AB - A telemetry system for monitoring urinary incontinence has been developed using two principles, temperature and impedance changes of a diaper. The system is composed of a pair of sensors, a transmitter, and a receiver. Temperature changes are monitored using thermistors, one in the center of the diaper and the other attached to the abdomen, and the temperature differences between them after urinary incontinence is detected. For the impedance method, two electroconductive cloths as electrodes placed in the diaper are used as sensors. Urine acts as a conductor to produce a current between the sensors. Clinical evaluation showed that both methods operate well; 13 of 17 incontinence episodes were detected using the temperature method and 32 of 35 with the impedance method. The misdetections were caused by faulty sensor arrangement for temperature measurement and by detection of exudates by the conductive sensors. These monitors may be used for the care of elderly people who use diapers for home health care, to save care time and help maintain hygiene. PMID- 7550502 TI - "Silent" malfunction of a critical-care device caused by electromagnetic interference. AB - Although there have been many previous reports of serious medical device malfunctions caused by electromagnetic interference (EMI), it is not widely recognized that many such malfunctions were "silent," having occurred without triggering a device alarm. The authors describe one such malfunction, and its implications. An 8-year-old radiant heater, which appeared to be operating normally in a neonatal intensive care unit, was observed, by mere chance, to intermittently register an increased, or decreased, neonatal skin temperature of about 1 degree C, depending on personnel movements and on its location in the room. The possibility that EMI had been the cause of this malfunction was investigated. The malfunction stopped in an adjacent windowless room, but again occurred when a 146-MHz walkie-talkie (100 mW) was used within a 1-2-m radius. More complete (0.1-1,000 MHz, 4.5-10 V/m maximum) test-chamber assessment of EMI susceptibility showed that the heater malfunctioned at electric field strengths above 0.3-1 V/m over four roughly 50-MHz bands between about 10 and 600 MHz. A previous electromagnetic environment survey had documented fairly high fields (0.01-0.3 V/m, 30-1,000 MHz) in the original malfunction area. Combined results suggested that the malfunction had been due to EMI from nearby fixed-source FM transmission antennas, which could be seen from the room's windows. The device had probably been malfunctioning for months, even though it had been regularly checked during preventive maintenance. Other identical, but newer, units functioned normally, or malfunctioned negligibly. Although operation of the older device deviated only "slightly" from normal, the consequences of this malfunction could have been serious.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550503 TI - Nitrogen and CO2 monitoring. PMID- 7550504 TI - Profession in crisis? Redefining clinical engineering. PMID- 7550506 TI - The new medical-surgical nurse. PMID- 7550508 TI - Cost considerations of implementing OSHA tuberculosis regulations. AB - The resurgence of tuberculosis and the enforcement of the Occupational Safety and Health Administration (OSHA) regulations has health care organizations struggling to budget for the cost of implementing The Enforcement Guidelines for Occupational Exposure to Tuberculosis in Health Care Facilities. One acute care hospital spent approximately $102,487 implementing the OSHA standards. Nurses and other health care providers need to become more aware of the regulation requirements to assist the institution in maintaining compliance, minimizing occupational exposure, and containing health care spending. PMID- 7550507 TI - Congestive heart failure: meet the challenge. AB - Congestive heart failure is an increasingly common, complex disease process with far reaching consequences for patients and caregivers. Thorough knowledge of the pathophysiology, assessment, and management of congestive heart failure affords nurses the opportunity to improve patient functional status and quality of life. PMID- 7550509 TI - Increasing compliance with breast self-examination. AB - The number of women who actually perform breast self-examination (BSE) monthly is less than desired. This is unfortunate since BSE is critical to the early detection of breast cancers. Common motivators and barriers to performance, and nursing strategies to increase the effectiveness of BSE, are explored in this review. PMID- 7550505 TI - Mastering the magic of the future. PMID- 7550513 TI - Physiology of healing: the basis for the principles of wound management. PMID- 7550511 TI - The nurse's role in assisted suicide. AB - Assisted suicide is creating intense discussion and controversy within the health care system and society in general. As a result, nurses may be placed in situations where they are asked to participate in the suicide of an individual. Very little has been written about the role and responsibility of the nurse in assisted suicide. The purpose of this article is to provide insight into the history of suicide, address legal and ethical issues in assisted suicide, and provide information on the nurse's role in assisted suicide. PMID- 7550510 TI - Pain control in hospitalized postsurgical patients. AB - The purpose of this study was to describe the pain experience of 100 randomly selected postsurgical patients, identify discrepancies between physician, nurse, and patient reports of pain intensity, and characterize the documentation of patients' pain experiences within the hospital record. PMID- 7550512 TI - Removing communication barriers for the hearing-impaired elderly. AB - Hearing impairment is a significant disability affecting many older adults. A number of technologies and services can provide the means to improve communication with these individuals. Nurses play an important role in identifying patients experiencing hearing loss and providing information and assistance that can reduce the consequences of this serious problem. PMID- 7550514 TI - The restructuring debate: rhetoric versus reality. PMID- 7550515 TI - Disregarding a physician's order: insurrection or compassion? PMID- 7550516 TI - Cognitive dysfunction associated with cancer and cancer therapy. PMID- 7550517 TI - Subacute care: a new opportunity for nurses. PMID- 7550518 TI - Sulfonylurea overdose. PMID- 7550519 TI - Techniques for the experimental measurement of human eating behaviour and food intake: a practical guide. AB - OBJECTIVE: Increases in the prevalence of overweight and obesity strengthen the importance of understanding human eating behaviour. A variety of methodologies and strategies have been devised during the last three decades with the intention of placing human food consumption under experimental scrutiny. The purpose of this review is to examine their diversity, their usage, and their strengths and limitations. CONTENT OF REVIEW: It is argued that meaningful research should be conducted within an explicit theoretical framework and with an awareness of how the methodological context influences instrument use and data interpretation. Techniques describing and quantifying the act of eating, the food consumed and the disposition to eat are reviewed. CONCLUSION: A strong and comprehensive methodology devised to measure the various components of eating behaviour is essential to achieve an understanding of the underlying causal mechanisms. A practical guide such as this should encourage further research and the construction of novel and more powerful procedures to elucidate appetite control and its failure. PMID- 7550520 TI - Attitudes about overweight and dating among secondary school students. AB - OBJECTIVE: To understand social preferences for thinness and the stigmatization of obesity, this study examined attitudes of adolescents towards dating (courtship) of overweight people. DESIGN: A cross-sectional survey was performed in upper class high school students at a public and private school. MEASUREMENTS: A written questionnaire that included questions assessing attitudes about dating overweight individuals, including a Guttman Dating Overweight Partners Scale (DOPS), was completed by 786 students. RESULTS: Women were more concerned than men about their own body weights, while men emphasized thinness in partners more than did women. The DOPS scale revealed low comfort in engaging, in dating activities with very overweight partners, moreso by men than by women. CONCLUSION: Stigmatization of overweight students in dating activities may be an important influence on desires of adolescents to be thin to be attractive. PMID- 7550523 TI - Anthropometry and lipoproteins-related characteristics of young adult males in Taiwan. AB - OBJECTIVES: To investigate the prevalence of obesity and the lipoprotein-related characteristics among young male adults in the Taiwan area. DESIGN AND SUBJECTS: After cluster sampling, a cross-sectional survey with a total of 936 males (mean age 20, 18-24) were enrolled. MAIN OUTCOME MEASURES: The distribution of anthropometric and lipoprotein-related variables and their correlations in young male adults were measured. The prevalence of obesity by different criterion and the lipoprotein characteristics of obese and non-obese were analyzed separately. RESULTS: The prevalence of obesity was 9.6% by the criterion of body weight greater than 20% of ideal body weight, or 12.6% by the criterion of body mass index (BMI) greater than 25. The obese subjects had significantly higher serum total cholesterol (CHOL), triglyceride (TG) and apo-lipoprotein B (apo B) and lower higher density lipoprotein-cholesterol (HDL-C) levels than the non-obese. The apo A1 levels were 141.3 and 141.9 mg/dl and the lipoprotein [a] (Lp[a]) were 17.4 and 17.1 mg/dl in obese and non-obese respectively, the difference being not statistically significant. Pearson correlation coefficients of body weight, body height, BMI, waist circumference, hip circumference and waist-hip ratio (WHR) to lipoprotein variables showed that both BMI and WHR are positively correlated with CHOL, TG and apo B, but negatively correlated with HDL-C. Furthermore the lipoprotein variables were better correlated with BMI than WHR in lean subjects (BMI < 25). However, this phenomenon was quite different in obese (BMI > 25) subjects, where the WHR was more highly correlated with lipoprotein variables than with BMI. CONCLUSION: The prevalence of obesity is slightly higher than reported in previous studies in Taiwan. The obese subjects had various abnormal lipoprotein metabolic characteristics, such as higher CHOL, TG, and apo B and lower HDL-C levels than non-obese subjects. The BMI was more highly correlated with lipoprotein variables than was WHR in lean subjects, but the WHR was more highly correlated with lipoprotein variables than was BMI in obese subjects. PMID- 7550524 TI - Is the intra-uterine period really a critical period for the development of adiposity? AB - OBJECTIVE: The intra-uterine environment may be a critical period for the development of adiposity. Alternatively, most of the co-variance of relative weight from birth to adulthood may be genetic in origin. This study tested whether birth weight 'tracks' into adulthood, independent of genetic factors. DESIGN: Observational twin study. Birth weights and gestational ages from birth records of 8040 twins from the Minnesota Twin Registry including 699 monozygotic (MZ) male pairs, 609 dizygotic (DZ) male pairs, 939 MZ female pairs, 880 DZ female pairs, and 893 opposite sex DZ pairs ages (yrs) 28 to 52 (Mean = 40.3; s.d. = 6.2) were compared to self-reported adult height and weight. RESULTS: The correlation of birth weight with adult height was 0.236, with adult weight 0.188, and with adult body mass index 0.078 (all P-values < 0.0005). To test if this tracking was independent of genetic influences, we analyzed intra-pair differences for MZ twins. If differences in birth weight between members of a MZ twin pair correlate with adult relative weight, this association cannot be attributed to genetic influences. The correlation of intra-pair differences in birth weight with intra-pair differences in adult height was 0.316 (P < 0.0005), with adult weight 0.136 (P < 0.0005), and with adult BMI 0.026 (P = 0.331). Results were unchanged when multivariate regression modeling was employed. CONCLUSION: These data suggest that the intra-uterine environmental influences on birth weight have an enduring impact on adult height but not on adult relative weight. This suggests that the intra-uterine period is a critical period for the development of height but not for adiposity. PMID- 7550521 TI - Prescription of exercise intensity for the obese patient: the relationship between heart rate, VO2 and perceived exertion. AB - OBJECTIVE: It has been suggested that relative heart rate reserve (% HRR), relative oxygen uptake (% VO2peak), and rating of perceived exertion (RPE) can be used interchangeably to prescribe exercise intensity. The purpose of this study was to examine the relationship between % HRR, % VO2peak, and RPE in obese females prior to and following substantial weight loss. SUBJECTS: 122 obese females (% Body fat = 45.9 +/- 5.0%) who participated in a weight loss intervention program. METHOD: Functional aerobic capacity was measured at baseline and following 12 weeks of exercise training and weight loss using a multi-stage Modified Balke treadmill protocol. The VO2, heart rate (HR), and RPE were measured at each stage, with VO2 and HR data converted to the percentages of the peak levels attained. These multi-stage data were then analyzed using mixed model regression procedures to examine the relationship between % HRR, % VO2, and RPE. RESULTS: With RPE as the dependent variable, results indicated that % HRR and % VO2, corresponded to RPE values consistent with existing guidelines (70% = 13-14 RPE), and this was true for analyses performed at baseline and following weight loss. Further, baseline results indicated that % HRR and % VO2 corresponded to similar levels of exercise intensity (40-70% HRR = 40-70% VO2peak). However, following weight loss, % HRR represented a higher level of intensity than its corresponding % VO2peak. CONCLUSIONS: The results of this study suggest that RPE can be used as subjective marker of exercise intensity in an obese female population. However, despite adhering to existing guidelines prior to weight loss, there may be a discrepancy in the relationship between % HRR and VO2max following severe weight loss, possibly due to the decrease in resting HR following exercise training and weight loss. These findings directly impact the prescription and monitoring of exercise intensity for obese patients. PMID- 7550522 TI - Cyclic AMP and glycerol concentrations in freeze-clamped human omental and subcutaneous adipose tissues. AB - OBJECTIVE: To compare intracellular concentrations of cyclic AMP, GTP, ATP, AMP and glycerol in omental and abdominal subcutaneous adipose tissues. DESIGN: Samples of omental and/or abdominal subcutaneous adipose tissues of 14 women undergoing elective surgery were freeze-clamped, deproteinized with perchloric acid and neutralized. All subjects were obese since it was not possible to freeze clamp lean individuals. MEASUREMENTS: The concentrations of ATP and GTP were determined by ion-paired high performance liquid chromatography and AMP and glycerol enzymatically. Cyclic AMP was determined by radioimmunoassay. Intracellular nucleotide concentrations were calculated from tissue weights, water contents and relative amounts of sodium and potassium. RESULTS: The intracellular concentration of cyclic AMP in omental adipose tissue (31 mumol/l) was twice as high as that in subcutaneous fat while those of ATP, GTP and AMP were similar at the two sites. Glycerol concentrations were higher in subcutaneous (472 mumol/l) than in omental (325 mumol/l) adipose tissue. CONCLUSION: The results suggest higher relative stimulation of lipolysis by cyclic AMP in omental adipose tissue than in the subcutaneous region in situ. However, probably because of differences in blood flow and possibly in the lipolytic machinery, this is not reflected as a higher glycerol concentration. PMID- 7550525 TI - Central obesity and hypertension: pathophysiologic role of renal haemodynamics and function. AB - OBJECTIVE: To investigate the role of alterations in renal haemodynamics and function and in plasma renin activity on obesity-induced hypertension. DESIGN: Renal haemodynamics and function, salt-regulating hormones and structural cardiac parameters were evaluated in 20 lean normotensives and in 64 obese subjects with central or peripheral fat distribution, 43 of them were normotensives and 21 of them were hypertensives. Obesity and central fat distribution were defined according to sex-specific 85th percentile respectively of Body Mass Index (BMI) and Waist to Hip Ratio (WHR). MEASUREMENTS: Serum immunoreactive insulin (IRI), plasma renin activity (PRA), plasma aldosterone (PA), microalbuminuria (UAE) and 24h urinary excretion of sodium (NaU) were evaluated by current methods. Renal haemodynamics was evaluated by radionuclide study according to Schlegel's and Gate's methods. By radionuclide study, effective renal plasma flow (ERPF), effective renal blood flow (ERBF), glomerular filtration rate (GFR), filtration fraction (FF) and renal vascular resistances (RVR) were measured. Left ventricular mass (LVM) and indexed for body height (LVM/H), cardiac output (CO) and total peripheral resistances (TPR) by ecocardiography were also calculated. RESULTS: CO, LVM and LVM/H were significantly (P < 0.05) higher in all the obese groups than lean controls. In addition, LVM and LVM/H were significantly (P < 0.05) higher in obese hypertensives than obese normotensives either with central fat distribution. TPR values were significantly (P < 0.05) higher in central obese hypertensives than peripheral obese hypertensives and than central obese normotensives. Moreover, IRI levels were significantly (P < 0.05) higher in central normotensive and hypertensive obese subjects than lean subjects. ERBF and ERPF were significantly (P < 0.05) lower and PRA levels were significantly higher only in central obese than lean subjects. On the contrary RVR were significantly (P < 0.05) higher in both obese hypertensive groups and in central obese normotensives than lean subjects. Comparisons between peripheral and central obese groups indicated that PRA, RVR and UAE were significantly (P < 0.05) higher and ERBF and ERPF values were significantly (P < 0.05) lower in both central obese groups than comparable subjects with peripheral obesity. Multiple regression analysis indicated that RVR increased significantly (P < 0.05) with WHR and PRA but not with CO and IRI. CONCLUSIONS: Our results indicate that obesity with body fat distribution of central type, more than obesity of peripheral type, is associated to abnormalities in renal haemodynamics and function. These data are consistent with the indication that change in renal haemodynamics take place at an early stage in the obesity-induced hypertension. PMID- 7550526 TI - Impact of obstructive sleep apnea and sleepiness on metabolic and cardiovascular risk factors in the Swedish Obese Subjects (SOS) Study. AB - OBJECTIVE: To determine if obstructive sleep apnea (OSA) is independently associated with cardiovascular risk factors and health status in subjects with severe obesity. DESIGN: Cross-sectional analysis of epidemiological data. SUBJECTS: 3034 participants in the Swedish Obese Subjects (SOS) Cohort. Two sub groups with a high and low likelihood for OSA based on questionnaire data were analysed in detail. MEASUREMENTS: General health questionnaires, anthropometric data including CT calibrated values for body fat distribution and lean body mass, blood pressure, fasting insulin, triglycerides, cholesterol, uric acid, glucose. RESULTS: Self-reported loud snoring and observed breathing pauses (high likelihood of OSA) was associated with increased frequency of WHO Grade 4 dyspnea, admissions to hospital with chest pain, myocardial infarction, blood pressure, fasting insulin, fasting triglyceride (women only), uric acid (women only) after adjustment for body fat distribution and other potential confounders. CONCLUSION: OSA may be another medical disorder which contributes to morbidity in severe obesity and is associated with some of the components of the metabolic syndrome observed in the centrally obese. PMID- 7550528 TI - Fat, friendless and unhealthy: 9-year old children's perception of body shape stereotypes. AB - OBJECTIVE: Society has a very negative view of overweight, leading to prejudice and discrimination. In an examination of the scope of these views, 9-year old children's attributions of the social functioning and health of thin and overweight body shapes were investigated. METHOD: One hundred and eighty eight girls and boys completed a series of ratings of four silhouette figures depicting a thin and heavy boy and girl. The children's own body shape preference, dietary restraint, height and weight were also measured. RESULTS: The ratings of the four silhouette variations were dominated by the size of the figure being judged. The overweight body shape was associated with poor social functioning, impaired academic success, and low perceived health, healthy eating and fitness. Gender and the raters own body weight had only limited impact on these stereotypical judgements. Children's judgements of themselves on the same attributes showed a greater perceived relevance of weight for girls and a confusion of healthy eating with dieting. CONCLUSION: Pre-adolescent children's perception of thinness and overweight echo the prejudices against overweight voiced by society. Health promotion at all ages will require a more considered presentation of the health implications of overweight. PMID- 7550527 TI - Metabolic effects of an increase of sympathetic activity in healthy humans. AB - OBJECTIVE: To determine the metabolic effects of catecholamines released at sympathetic nerves ending in the postabsorptive and postprandial states. DESIGN: Sympathetic activity was acutely increased by lower body negative pressure (LBNP; -15 mmHg) on two occasions in a group of eight healthy volunteers: (1) in the postabsorptive state; and (2) after glucose ingestion. MEASUREMENTS: Plasma norepinephrine concentrations were determined by HPLC and energy expenditure and substrate oxidation rates were assessed with indirect calorimetry. RESULTS: After glucose, LBNP increased plasma norepinephrine by 27% and lipid oxidation by 72% and decreased glucose oxidation by 14%. Energy expenditure was not altered. In the postabsorptive state, LBNP increased plasma norepinephrine by 34%, but had no significant effect on energy or substrate metabolism. CONCLUSIONS: In healthy humans, LBNP-induced activation of the sympathetic nervous system decreases glucose oxidation and increases lipid oxidation after oral glucose, but does not quantitatively affect energy expenditure. PMID- 7550529 TI - Prevalence of obesity among adult Kuwaitis: a cross-sectional study. AB - OBJECTIVE: The association between obesity and different diseases is well documented, especially with non-insulin dependent diabetes, hypertension and heart disease. To prevent proliferation of these and other obesity-related diseases, and to plan adequate health education programmes for the control of obesity, base-line data are required on the prevalence of this condition. The present study is concerned with this need and its purpose is the determination of the prevalence of obesity among adult Kuwaitis. METHOD: A cross-section of 3435 Kuwaiti adults forms the study sample. Corpulence was assessed from body mass index (BMI kg/m2) and the mean BMI (+/- standard deviation) was 28.3 (+/- 5.3). RESULTS: According to the WHO BMI criteria, the prevalence of obesity was found to be 70.2% (Grade 1) and 36.4% (Grade 2) with BMI as > 25 and > 30, respectively, and was significantly higher (P < 0.001) among women. Obesity among Kuwaitis was found to be higher than reported elsewhere in the world. PMID- 7550530 TI - Proceedings of an International Symposium on Insulin Resistance as a Risk Factor for Cardiovascular Disease. Sainte Adele, Quebec, 22-24 June 1993. PMID- 7550531 TI - Hypofibrinolysis and the insulin resistance syndrome. AB - Atherothrombosis seems now to be the most appropriate term to describe the pathogenic events leading to the development of cardiovascular disease. An impaired fibrinolysis may not only contribute to and aggravate the tendency to thrombosis but fibrin deposit may also play a role in the development of the atherosclerotic plaque. Hypofibrinolysis is observed among obese subjects and it has been shown that an excess of plasminogen activator inhibitor 1 (PAI 1) the main regulator of the fibrinolytic system, is closely associated to other components of the insulin resistance syndrome, namely, excessive body weight, high waist to hip ratio, elevated blood pressure, hyperinsulinemia and hypertriglyceridemia. PAI 1 levels decrease with measures attempting at decreasing insulin resistance. However, the mechanisms leading to increased PAI 1 levels are still unknown. On the basis of epidemiological studies, and in vitro studies with PAI secreting cells such as hepatocytes or endothelial cells, insulin, insulin precursors, and lipoproteins, mainly VLDL, appear candidates for triggering this excessive secretion, but no definite answer has yet been found. PMID- 7550532 TI - Alterations in lipoprotein lipase in insulin resistance. PMID- 7550533 TI - Are insulin resistance and/or compensatory hyperinsulinemia involved in the etiology and clinical course of patients with hypertension? PMID- 7550535 TI - Metabolic disruptions in the adipocyte-hepatocyte fatty acid axis as causes of HyperapoB. AB - HyperapoB is the atherogenic dyslipoproteinemia characterized by increased numbers of LDL particles in plasma due to increased secretion of B100 lipoprotein particles by the liver. The lipid phenotype in affected patients is variable but an increased plasma apoB points to the increased LDL particle number. The adipocyte-hepatocyte fatty acid axis refers to the traffic in fatty acids from adipocytes to hepatocytes and back again. Our central thesis is that a reduced rate of adipocyte triglyceride synthesis leads to increased traffic to and fro along this axis. This article outlines the ways in which impaired adipose tissue function leads to increased flux of fatty acids to the liver which leads, in turn, to increased secretion of hepatic B100 particles. The Adipsin-ASP pathway is a newly described biological pathway which appears to play a critical role in regulating adipose tissue triglyceride synthesis. Impaired function of this pathway appears to be the commonest reason for the increased fatty acid traffic in the adipocyte-hepatocyte axis leading to HyperapoB. PMID- 7550534 TI - LDL subclass phenotypes and the risk factors of the insulin resistance syndrome. AB - The insulin resistance syndrome is characterized by a constellation of risk factors including obesity, central body fat distribution, hypertension, glucose intolerance, elevated plasma insulin levels, increased triglyceride and decreased high-density lipoprotein (HDL) cholesterol. Similarly, low-density lipoprotein (LDL) subclass phenotype B, characterized by a predominance of small, dense LDL particles, is associated with increased triglyceride and lower HDL cholesterol levels. Both the insulin resistance syndrome and phenotype B have also been related to increased risk of coronary heart disease. Using a sample of nearly 700 women who participated in the Kaiser Permanente Women Twins Study, we investigated the associations of LDL subclass phenotype B with the risk factors that characterize the insulin resistance syndrome. The results demonstrate that LDL subclass phenotype B was strongly associated with both age and diabetes status. Among nondiabetic women, phenotype B was more prevalent in those with higher body mass index and in those with higher waist-hip ratio. In addition to the expected associations with triglyceride and HDL cholesterol, mean values of both systolic and diastolic blood pressure were higher in women with phenotype B. Finally, fasting and post-load insulin levels and post-load glucose levels were higher in those with phenotype B. In general, these relationships persisted in multivariate statistical analyses. Therefore, a predominance of small, dense LDL particles appears to be an integral feature of the risk factors that characterize the insulin resistance syndrome. PMID- 7550536 TI - The acylation stimulating protein-adipsin system. AB - Considerable evidence indicates that obesity, and in particular abdominal obesity, is a risk factor for both heart disease and non-insulin dependent diabetes mellitus. In spite of this, little is known of the regulation of triacylglycerol synthesis in adipose tissue other than by insulin. Acylation stimulating protein (ASP), a human plasma protein, stimulates triacylglycerol synthesis in adipose tissue and is also produced by human adipocytes. ASP may play a physiological role in the regulation of efficiency of adipose tissue fat storage and affect clearance of triglycerides from plasma. PMID- 7550538 TI - Genetics and the metabolic syndrome. PMID- 7550539 TI - Insulin resistance: the consequence of a neuroendocrine disturbance? AB - Visceral obesity in man is followed by several abnormalities in endocrine secretions, including an elevated cortisol secretion as well as hyperandrogenicity in women, and a relative hypogonadism in men, suggesting a central neuroendocrine background. A pronounced insulin resistance is a main symptom of this condition and may be at least partially following these endocrine abnormalities. For example, mimicking hyperandrogenicity in women by administration of testosterone (T) in moderate doses to female rats is followed by severe muscular insulin resistance, as well as a low type I/type II muscle fiber ratio, and a low capillary density in muscle. These findings are identical to those in visceral obese women with hyperandrogenicity and insulin resistance. Studies of details of function and morphology in the muscles in this rat model have revealed that insulin stimulation of glucose transport, glycogen synthesis and the insulin sensitive part of the glycogen synthase is diminished at submaximal insulin concentrations. However, insulin receptor number and function, as well as the total number of glucose transporter 4 appear to be normal, supported by observations that the insulin resistance is overcome by elevated insulin concentrations. These observations suggest that normal cellular mechanisms for glucose metabolism in muscle in this model are under-utilized, and that the explanation to apparent insulin resistance may be found proximal to the machinery of the muscle cell. The histochemical changes of the muscles may provide a clue to the understanding of the mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550537 TI - Hyperinsulinemia and smooth muscle cell proliferation. AB - Diabetic and obese subjects run a greater risk of developing atherosclerosis than the rest of the population. Several epidemiological studies suggest that hyperinsulinemia, which characterizes both obese and insulin resistant diabetic subjects, may be involved in atherosclerosis. Because insulin stimulates proliferation of arterial smooth muscle cells (SMC) in culture, it was supposed that insulin may exert an atherogenic role by promoting proliferation of SMC in the intima which is considered as one of the most important initial steps in atherogenesis. However, direct evidence of the growth-promoting effect of insulin on SMC in vivo is still lacking. Using an animal model of arterial injury with a balloon catheter (deendothelialization), we have investigated the short and long term effect of high circulating insulin, associated or not with plasma glucose variations on aortic SMC proliferative response to endothelial denudation. SMC proliferation was appreciated by measuring the incorporation of 3H-thymidine in the DNA of intima-media layers. In a first series of experiments, we induced concomitant variations of plasma glucose and insulin concentrations by submitting rats with experimental aortic injury to fasting and refeeding. In control rats, the proliferative response to endothelial denudation was dramatically increased on day 2 after injury, remained high on day 4, and progressively declined thereafter. In fasted-refed (FR) rats, the increase on day 2 was less pronounced than in controls; concomitantly a sharp decrease in plasma glucose and insulin concentrations was observed. Refeeding was associated with a significantly less marked decline in SMC mitotic activity since day 4 after injury; at the same time, plasma glucose and insulin concentrations were sharply increased. These results suggested an involvement of variations of both glucose and insulin in SMC proliferation. However, when the surge of glucose and insulin after refeeding was prevented by adding acarbose to the diet, the SMC mitotic activity remained higher in FR rats than in controls, casting doubt as to whether hyperglycemia and hyperinsulinemia could be of importance in this process. Moreover, when deendothelialized rats were rendered either euglycemic-hyperinsulinemic or hyperglycemic-hyperinsulinemic by simultaneous infusion of glucose and insulin, no difference with controls, in the SMC proliferation, was observed. Taken together these data indicate that the acute mitotic response of SMC to endothelial denudation is not influenced by insulin associated or not with high levels of glucose. The long-term of long-lasting hyperinsulinemia was investigated using the obese Zucker rat model which is characterized especially by early spontaneous development of hyperinsulinemia and insulin resistance. Compared to controls, the SMC mitotic activity was not significantly increased from day 2 to day 7 after injury, but, from day 14 to day 30 after endothelial denudation, SMC proliferation was significantly less decreased in obese than in lean rats. Moreover, on day 30, aortic thickness, as measured by histomorphometric technique, was much higher in obese than in lean rats. This difference was entirely due to an increase in SMC number within the intima.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7550540 TI - Nutritional determinants of the insulin resistance syndrome. AB - Recent data show that fat balance is the only component of macronutrient balance which is not precisely regulated. The adherence to a lifestyle characterized by high fat food habits and sedentariness thus favor a positive fat balance which may persist for a long period of time. The resulting body fat gain contributes to the restoration of fat and energy balance because of the enhancing effect of fat gain on fat oxidation. Individuals predisposed to obesity seem to be particularly vulnerable to a positive lipid balance because they are characterized by a reduced fat oxidation when their body weight is normal and by a reduced capacity to increase fat oxidation when exposed to a high fat diet. Body fat gain, particularly in the deep abdominal area, is also associated with an increase in insulinemia which is known to acutely inhibit fat mobilization and oxidation. Beyond these peripheral metabolic effects, hyperinsulinemia with euglycemia can influence neuronal regulation of energy balance which ultimately results in an increase in sympathetic nervous system activity. An increased sympathetic tonus exerts an enhancing effect on fat oxidation and energy expenditure and a reducing effect on food intake. As discussed in this report, hyperinsulinemia thus appears to be an adaptation which is part of a regulatory system aimed at restoring fat and energy balance under conditions favoring body fat accumulation. PMID- 7550541 TI - Role of glucocorticoids and fatty acids in the impairment of lipid metabolism observed in the metabolic syndrome. AB - The metabolic syndrome is discussed in terms of insulin resistance linked to an increased regulation of metabolism by cortisol and fatty acids. This change in hormonal balance is associated with diabetes, android (visceral) obesity, hypertension, hypertriglyceridemia, hyperapobetalipoproteinemia and low concentrations of HDL; a cluster of risk-factors that predisposes to the development of premature atherosclerosis. It is proposed that the metabolic syndrome is accompanied by a derangement in the hypothalamic-pituitary-adrenal axis such that the effects of cortisol are exaggerated relative to those of CRF. Excessive action of fatty acids and cortisol causes insulin resistance and increase the hepatic secretion of glucose and VLDL. Furthermore, cortisol can decrease the uptake of LDL by the liver. Cortisol in the presence of relatively high insulin concentrations can promote the deposition of energy and lead to obesity. Chronic treatment of rats with D-fenfluramine has been shown to decrease the release of cortisol and fatty acids in response to stress, and to improve insulin sensitivity. The effects of D-fenfluramine were also tested in male JCR:LA corpulent rats which are prone to develop atherosclerosis and myocardial lesions. D-fenfluramine improved insulin sensitivity, decreased the hypertriglyceridemia, and prevented the development of necrotic myocardial lesions caused by ischemia. The data presented demonstrates a link between excessive action of cortisol and fatty acids in predisposing to insulin resistance and the pathologies that are associated with the metabolic syndrome. PMID- 7550542 TI - The insulin resistance-dyslipidemic syndrome: contribution of visceral obesity and therapeutic implications. PMID- 7550543 TI - 2-amino-3-phosphonopropionic acid (AP3) and its N-methyl analog improve learning and memory in rats. AB - The influence of 2-amino-3-phosphonopropionic acid (AP3), an antagonist of metabotropic glutamate receptors and its N-methyl analog (N-methyl AP3) on acquisition, consolidation and recall of information in a passive avoidance situation was tested. Both compounds improved three examined parameters. The significant effect of AP3 appeared at the icv dose of 12.5 micrograms per rat when its influence on acquisition and consolidation was tested, and at the icv dose of 100 micrograms when its influence on retrieval process was evaluated. The significant improvement of acquisition of information was observed at the icv dose of 50 micrograms per rat of N-methyl AP3, but significant facilitation of consolidation and retrieval processes appeared at the higher dose of 100 micrograms per rat. Significant improvement of performance in a passive avoidance situation persisted in all experiments to the highest used dose of both compounds, 250 micrograms. PMID- 7550544 TI - Attenuation of the fentanyl-induced muscle rigidity by the selective 5HT1A agonist 8-OH-DPAT. AB - Fentanyl given in a low dose of 25 microgram/kg (ip) evoked a marked muscle rigidity measured directly by a mechanographic method in non-anesthetized rats. The selective 5HT1A receptor agonist 8-OH-DPAT (0.1, 0.3 and 1.0 mg/kg ip) showed only a tendency to attenuate the natural muscle tone. However, when that compound was given 40 min before (0.3 and 1.0 mg/kg ip) or 20 min after (1.0 mg/kg ip) fentanyl, it abolished the muscle rigidity. It is concluded that the serotonergic transmission, possibly via 5HT1A receptors, may participate in elucidation of the mechanism(s) of the fentanyl-induced muscle rigidity. These results seem to be clinically important in case other 5HT1A agonists, buspirone or gepirone (potent anxiolytics), also prevented fentanyl-induced muscle rigidity in humans. PMID- 7550545 TI - The effect of imipramine on lipid peroxidation in the rat cerebral cortex. AB - The effect of imipramine (IMI) on lipid peroxidation in the rat cerebral cortex was investigated in ex vivo and in vitro study. It was found that IMI when given to rats chronically (14 x 10 mg/kg ip) but not acutely (10 mg/kg ip), inhibited lipid peroxidation in the cortical membranes of rat brain. When added in different concentrations (0.125-10.0 nmoles/sample) to the cerebral cortical membranes of naive rats in vitro, IMI inhibited lipid peroxidation in concentration-dependent fashion. Using [3H]-IMI it was found that employed procedure for membrane preparation did not removed IMI from cortical membranes of rats treated chronically with the drug and this might explain the lack of antioxidant effect of IMI in animals treated with a single dose of IMI in ex vivo study. A comparative study of IMI and chlorpromazine (CPZ)-whose inhibitory effect on the free radicals formation has been found before-indicated the higher potency of IMI than that of CPZ as radical scavenger in ex vivo and in vitro studies. The results imply that IMI might affect the brain function also through its effect on lipid peroxidation. PMID- 7550546 TI - Concanavalin A increases N-methyl-D-aspartate-induced Ca2+ influx in cerebellar granule cells in culture. AB - Desensitization of glutamatergic receptors can be inhibited by various endogenous and/or exogenous agents e.g. by lectins. Effect of lectin, concanavalin A (con A) on N-methyl-D-aspartate (NMDA)-induced Ca2+ influx was studied in cultured cerebellar granule cells. Additionally the effect of con A on 3H-(+)-5-methyl 10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (3H-(+)MK-801) binding to NMDA receptors was investigated. Preincubation with con A enhanced NMDA efficacy (but not potency) to stimulate Ca2+ influx. The potency of MK-801 to inhibit NMDA effect was similar in both control and con A pretreated cells. Preincubation of cortical membranes with con A resulted in a robust decrease of Bmax 3H-MK-801 binding and modest decrease in affinity. This contradiction between Ca2+ influx and binding experiments may result from the difference in the subtype of NMDA receptors or different expression of con A action in both assays. PMID- 7550548 TI - Dopaminergic neuronal systems modulate the central cardiovascular effects of TRH in rats. AB - Thyrotropin releasing hormone (pGlu-His-Pro-NH2-TRH) is a hypothalamic peptide that exerts pharmacological actions on the mammalian central nervous and circulatory systems independent of hormonal activity. In rats TRH produces an increase in blood pressure, heart rate and respiratory rate, following its intracerebroventricular (icv) administration. Because of a suspected role of the central dopamine (DA) system in these effects, we examined the influence of assorted DA receptor agonists and antagonists on the central cardiovascular and respiratory effects of TRH. Adult male Wistar rats were anesthetized with urethane (1.5 g/kg ip), implanted with an intracranial cannula for icv injections, and continuously monitored for (a) mean arterial blood pressure via an indwelling catheter in the right carotid artery, (b) heart rate via an ECG lead II signal and (c) respiratory rate via a photoelectric transducer. TRH (100 nmol) produced a sustained increase in blood pressure (ca. 20 mm Hg), heart rate (ca. 60 beats per min-bpm) and respiratory rate (< or = 200 breaths per min) during a 30 min observation period. The DA D1 receptor agonist SKF 38393 HCl (0.3 or 3.0 mg/kg ip) attenuated the effects of TRH on blood pressure and heart rate by > 50%, while the DA D2 receptor agonist quinpirole HCl (1.0 mg/kg ip) potentiated the chronotropic response to TRH by > 50%. The predominant DA D2 receptor antagonist haloperidol lactate (1.0 mg/kg ip) potentiated the effects of TRH on blood pressure and heart rate by > 50%, with the latter effect on heart rate being mimicked by the D2 receptor antagonist spiperone HCl (1.0 mg/kg ip). Chlorpromazine HCl (5.0 mg/kg ip), a low potency non-selective D2 receptor antagonist, had effects opposite to that of haloperidol. The effect of TRH on respiration was potentiated by quinpirole but not modified by other DA receptor agonists or antagonists. These findings indicate that the DA system is involved in a complex manner in the central cardiovascular actions of TRH, while DA D2 receptors alone appear to be involved in modulating respiratory effects of TRH. PMID- 7550547 TI - Influence of halothane on the level of enkephalins in discrete brain areas of rabbits. AB - With regard to the fact, that theory of anesthesia, based on the endogenous opioid peptides requires further investigations the aim of this paper was to measure the concentration of leucine- (LENK) or methionine-enkephalin (MENK) in discrete brain areas of rabbits during and after 1 h halothane (2% v/v) anesthesia. The level of LENK and MENK was measured in discrete brain areas using the radioimmunoassay. Animals were divided into the following groups, depending upon the duration of anesthesia: I-15 min; II-60 min; III-1 h after anesthesia termination; IV-control group of non-anesthetized animals. After halothane anesthesia evident decrease in the concentration of leu-enkephalins in hypothalamus (HT) and the opposite effect in hippocampus (H) were observed. The MENK level significantly increased after halothane (for 60 min) in hypothalamus, hippocampus and mesencephalon (M). The change in the level of LENK in the thalamus (Th), hippocampus and mesencephalon and in the level of MENK in the hypothalamus and mesencephalon persisted after withdrawal of anesthesia. It was found that the alterations in the level of enkephalins in discrete areas of rabbit's brain is a feature of halothane anesthesia. The explanation of this phenomenon is possibly important for the understanding of the mechanism of halothane anesthesia and requires further investigations. PMID- 7550549 TI - Evaluation of the effect of oral enalapril on neutrophil functions: comparison with the in vitro effect of enalapril and enalaprilat. AB - Polymorphonuclear neutrophils (PMN) participate in the development of myocardial injury by releasing free oxygen radicals and by involvement in the no-reflow phenomenon. Neutrophil-mediated myocardial injury, therefore contributes to the pathogenesis of heart failure. We investigated the effect of oral treatment with enalapril on neutrophil free oxygen radical production, aggregation and adherence in patients with moderate heart failure (New York Heart Association-NYHA II and III degrees). Samples were taken before and 48 h after a single 10 mg oral dose. Oral enalapril inhibited hydrogen peroxide released by unstimulated PMN, but did not affect stimulated H2O2 release, superoxide anion production, adhesion or aggregation of PMN. Enalaprilat in vitro stimulated PMN to release H2O2 and superoxide anions. Furthermore, in the in vitro conditions both enalaprilat and enalapril inhibited hydrogen peroxide release by stimulated cells. We conclude that, despite certain modifications of neutrophil function in vitro, oral administration of enalapril seems to exert a limited biological effect on circulating neutrophils. PMID- 7550551 TI - In vitro generation and decomposition of S-nitrosothiols from direct and indirect nitric oxide donors. AB - Nitric oxide which was released in aqueous solutions (> or = 10 microM) of direct NO-donors such as 3-morpholinesydnonimine (SIN-1) and S-nitroso-N-acetyl penicillamine (SNAP) consumed avidly sulfhydryl groups of N-acetylcysteine > cysteine > glutathione. In case of SIN-1 generation of nitrites run in parallel to disappearance of sulfhydryl groups of N-acetylcysteine and glutathione, however, for a pair of SIN-1 and cysteine the rate of formation of nitrites was much slower than the rate of consumption of sulfhydryl groups. We infer that kinetics of formation and breakdown of S-nitrosothiols varies depending on the type of a thiol which reacts with a NO-donor. Indirect NO-donors such as glyceryl trinitrate (GTN), molsidomine (MSD) or sodium nitroprusside (NaNP) at concentrations < 100 microM did not consume sulfhydryl groups of cysteine unless pretreated with the xanthine/xanthine oxidase system. We suppose that in this last case superoxide anions react with nitric oxide to form peroxynitrites with a higher potency than nitric oxide itself to destroy sulfhydryl groups. We conclude that out of three studied thiols N-acetylcysteine is the best substrate for the formation of S-nitrosothiols, while S-nitrosocysteine is the slowest releaser of nitric oxide. Moreover, unlike SIN-1 and SNAP, NaNP is not a direct NO-donor but behaves rather like GTN. Minute amounts of nitric oxide released either from NaNP or GTN gain from superoxide anions an amplification as SH-scavengers. PMID- 7550550 TI - Interactions between reactive oxygen species and sulfhydryl groups of cysteine, acetylcysteine and glutathione. AB - Oxidative potencies of hydrogen peroxide, superoxide anions or hydroxyl radicals towards sulfhydryl groups of cysteine, acetylcysteine or glutathione were studied. Acetylcysteine was the least susceptible to oxidation by reactive oxygen species. The most vigorous oxidant of sulfhydryl groups was hydrogen peroxide. PMID- 7550552 TI - Minaprine, but not oxiracetam, prevents desipramine-induced impairment of avoidance learning in mice. AB - The tricyclic antidepressant desipramine impaired shuttle-box avoidance acquisition in mice of the CD-1 strain. The nootropic drug oxiracetam was unable to prevent the desipramine-induced learning impairment, while a protective action was exerted by minaprine, a psychotropic agent regarded as an atypical antidepressant drug, possessing dopaminergic and related memory-enhancing properties. It seems likely that the dopaminergic action of minaprine played a determinant role in its avoidance improving effects in desipramine treated mice, because similar effects were produced by amphetamine. However, in contrast to amphetamine, minaprine did not enhance locomotor activity and did not show signs of general behavioral stimulation. PMID- 7550553 TI - The effect of interaction between (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD and noradrenaline on cyclic AMP accumulation: different actions in brain slices, primary glial and neuronal cell cultures. AB - The existence of multiple metabotropic receptors for excitatory amino acids is firmly established by recent cloning experiments. Several subtypes of those receptors are coupled to adenylate cyclase. (1S,3R)-1-aminocyclopentane-1,3 dicarboxylic acid (1S,3R)-ACPD) is a selective agonist for metabotropic glutamate receptors, it influences cyclic AMP accumulation in brain and is able to enhance the effect of other substances which stimulate cyclic AMP accumulation. Here we present data showing that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid enhanced the action of noradrenaline on cyclic AMP accumulation in rat brain cortical slices (EC50 40 microM). On the contrary, it inhibited the action of noradrenaline in primary glial cell cultures (EC50 50 microM), being without any effect on noradrenaline-induced cyclic AMP accumulation in primary neuronal cell cultures. The differences may reflect stimulation of different types of metabotropic glutamate receptors in various preparations from rat brain. The nature of interaction between noradrenaline and (1S,3R)-ACPD remains to be studied. PMID- 7550554 TI - Anxiolytic-like action of intra-hippocampally administered NMDA antagonists in rats. AB - The effects of intra-hippocampally administered NMDA receptor antagonists MK-801, and AP-7 on rat behavior in the Vogel's and open field tests of anxiety were examined. MK-801 and AP-7 given locally stimulated at the same time rat exploratory and motor activities, in the open field. In the Vogel's test, both compounds attenuated the suppressive effect of shock on drinking, in a dose-range not affecting rat motility. It is concluded that hippocampal NMDA innervation may play a role in the control of motor activity. Furthermore, the Vogel's test data indicate more selective involvement of hippocampal NMDA innervation in processing of fear-related signals. PMID- 7550555 TI - Central activity of 2-amino-4-phosphonobutyric acid (AP4). AB - The influence of intracerebroventricular (icv) injections of 2-amino-4 phosphonobutyric acid (AP4) on the activity of central nervous system was examined in rats. The compound (100 and 200 micrograms icv) significantly enhanced apomorphine stereotypy, in doses of 50, 75, and 100 micrograms decreased haloperidol-induced catalepsy and did not change motility of rats estimated in the "open field" test. AP4 significantly improved learning and memory processes in passive and learning in active avoidance situation. The highest significant effect was observed after icv dose of 100 micrograms per rat, when acquisition, consolidation and recall of information was evaluated in the passive avoidance behavior. AP4 significantly improved only acquisition of information in active avoidance situation at the icv dose of 50 micrograms per rat. The mechanism of the positive effect of AP4 on learning and memory is at present unclear, and its explanation needs future investigations. PMID- 7550557 TI - [Progestagen contraceptives]. PMID- 7550556 TI - [Lowering the steroid dose in oral contraception and risk of functional ovarian cysts]. PMID- 7550558 TI - [Detection of fetal cells in maternal blood: towards a noninvasive prenatal diagnosis]. AB - Fetal cells exist in maternal blood and can be utilized for prenatal genetic diagnosis. These cells are present during the first and second trimesters, with frequency increasing as gestation advances. Enrichment of erythroblasts by various density gradient techniques and either magnetic activated or flow sorting techniques can be followed by FISH with chromosome-specific DNA probes. This approach has allowed detection of trisomy 21, trisomy 18, Klinefelter syndrome 47,XXY and 47,XYY. Polymerase chain reaction (PCR) analysis of maternal blood has enabled the detection of fetal sex, certain Mendelian disorders (e.g. beta-globin mutations), HLA polymorphisms, and fetal Rhesus (D) blood type. The fetal cell types receiving the most attention has been nucleated erythrocyte (erythroblast) and the trophoblast. Lymphocytes and granulocytes are also present in maternal blood; however, lymphocytes are considered the cell type most likely to persist after pregnancy. A large scale collaborative is now underway in the U.S. that will allow determine whether sensitivity and specificity of this technique provide a noninvasive alternative to conventional methods of prenatal cytogenetic diagnosis. PMID- 7550560 TI - [Oocyte maturity and quality: value of intracytoplasmic sperm injection. Fertility of microinjected oocytes after in vitro maturation]. AB - Many studies in IVF practice, have tried to assess the maturity and quality of oocytes prior to insemination and relate it to IVF efficiency and to the pattern of ovarian stimulation. They were based on the indirect evaluation of the aspect of the cumulus-corona-cell complex (CCC) which was rapidly shown to be poorly correlated to the oocyte nuclear and cytoplasmic maturity in stimulated cycles. For sperm microinjection procedures, oocytes need to be peeled off from any follicular cell through hyaluronidase action in order to gain an easy access to the zona pellucida and the ooplasm. If therefore becomes possible to precisely known at recovery the nuclear status of the oocyte cohort as well as the rate of degenerative gametes. Immature oocytes can be further matured in vitro. Moreover, the ICSI procedure (intracytoplasmic sperm injection) allows a direct assessment of the cytoplasmic maturation, whatever the maturity of the ZP and its receptors and of the plasma membrane. On a preliminary evaluation of 70 ICSI cycles performed in our collaborative group from september to november 1994 and leading to a 24 % pregnancy rate per oocyte pick-up we focused on the true maturation of the oocyte cohort, its outcome and correlation with ICSI efficiency and stimulation protocol. On the 760 oocytes, 13.9% were atretic, 9% at the germinal vesicle stage (GV), 3.4% in metaphase 1 and 73.7% in metaphase 2 at recovery.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550561 TI - [In vitro fertilization failures related to a sperm hyperactivation defect: indication for microinjection?]. AB - Development of hyperactivated motility is considered as necessary for penetration through zona pellucida. In a study concerning 114 IVF attempts, we could not establish a significant correlation between fertilization rate and hyperactivation rate (HA). On the contrary, most fertilization failures (16/21) we found for HA values < 10%. 10 of such couples could benefit of ICSI for the next attempt: fertilization rates were strictly similar to the general rate observed for ICSI in our group (48%). PMID- 7550563 TI - [Intracytoplasmic injection and embryo quality: comparison with conventional in vitro fertilization]. AB - We report here the results of a study comparing the quality of the embryos obtained after conventional IVF and after ICSI (respectively 872 and 459 embryos for 184 and 108 cycles). In the ICSI group, the female age was lower than in the IVF group, the oestradiol level on the day of hCG injection was higher, so that the number of retrieved oocytes and the number of mature oocytes. The policy of transfer being the same in the two groups, the mean number of transferred embryos was identical. The percentage of embryos without anucleate fragments, the percentage of embryos without irregularities, the percentage of 1, 2, 3, 4 or 5 cell embryos and the distribution of embryos in the 5 embryo scores were similar. In both IVF and ICSI groups, the transfer score (sum of the embryo scores of each transferred embryo) was higher for the patients who achieved pregnancy. PMID- 7550559 TI - [Study of a new lactic acid and pH 5.2 lactoserum emulsion for feminine hygiene. Results of a clinical study]. AB - A new solution intended for use in feminine hygiene, and composed of a lactic acid base and of lactoserum at pH 5.2, was tested for a period of 8 weeks on 40 women. This test focused on its influence on the pH and the flora, as well as on the tolerance of the mucous membrane. Successive measurements of the vulvar and vaginal pH revealed no statistically significant variation between the beginning and the end of the trial. At the study inclusion thirty patients had a normal flora; of these, twenty-eight showed no change during the eight weeks of the trial. A candidiasis appeared in two patients in the middle of the trial; one of these patients received no treatment, while the other was given a five-day treatment due to similar occurrences in her past medical history. The product under study was continued throughout the trial, at the conclusion of which the flora was found to have returned to normal. At the study inclusion ten patients had either a candidiasis or a vaginitis. Their lack of symptoms and the absence of any previous problems resulted in no treatment being made in five of the ten cases, while in four cases the presence of such prior problems led to the immediate carrying out of short-length treatments. In every case, the product under study was used until the end of the trial. Bacteriological studies showed that in all the patients--whether or not they had been treated--the flora rapidly returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550564 TI - [Cytogenetic analysis of human oocytes after sperm microinjection]. AB - After standard in vitro fertilization (IVF) chromosome analysis of uncleaved oocytes has shown that in about 80% of the cases, there was no fertilization at all while in 10% of the cases there was a premature chromosome condensation and a development arrest. After micro-injection our results suggest that both the technique itself and the pathologies which require micro-injection can influence the results. On the other hand about 20-30% of the metaphase II oocytes are cytogenetically abnormal after IVF attempt while ICSI might increase chromosome breakage. Our results suggest that both the technique used and the gamete pathologies can influence the different steps of development arrest. PMID- 7550565 TI - [Microinjection in France: report of the BLEFCO multicentric study]. AB - In order to evaluate the assisted fertilizations in France, BLEFCO association has analysed 436 and 1,781 attempts for SUZI and ICSI respectively. For SUZI, the transfer rate was 62% and the pregnancy per attempt rate was 10%. For ICSI, whatever indications, the transfer rate was 86% and the pregnancy per attempt rate was 21%. ICSI in cases of altered sperm without previous IVF represented 46% of ICSI attempts. No relationship between the transfer rate and the indication of ICSI has been observed. However, in case of unexplained fertilization failures, the pregnancy per attempt rate is significantly lower when compared to the other indications. PMID- 7550562 TI - [Selection and characterization of human acrosome-reacted sperm: application to subzonal insemination]. AB - Acrosome reacted spermatozoa were selected with immunobeads coated with an antibody directed against the inner acrosomal membrane (GB24) and then detached immunologically using an anti-Fab'2 antibody. Using this method, we have shown that acrosome reaction occurs in the most morphologically normal spermatozoa and is followed by a loss in motility and a decrease in longevity. Moreover, when injected in the perivitelline space of hamster oocytes, the selected spermatozoa had a higher fertilization rate than unselected spermatozoa, confirming that acrosome reaction is necessary for the fusion with the oocyte plasma membrane and that the selection method does not alter the fertilizing ability of the spermatozoa. Therefore, this method represents a new way for understanding the sperm functions. PMID- 7550567 TI - [1994 evaluation of reproduction with gametes donation. French Federation of CECOS]. AB - Those results record the referred gametes donation practiced by CECOS French Federation (22 centers) and IFREARES-Toulouse (1 center). In 1994, the activity was quite the same than the previous year, the AID data are most satisfying. Men's sterility is the greater indication of the 2 2337 requests for a first conception and we tried to detail the sperm parameters. The issue of the 2,030 pregnancies obtained in 1993 and the new-born children health are studied. Sperm preservation is still growing and it seems that the increase of sperm storage is connected to the hope given by ICSI. PMID- 7550568 TI - [Cocultures in France: situation in 1995. Geff-BLEFCO]. AB - Co-cultures of human embryos, in particular those concerning Vero cells, are used by French teams mainly in case of repeated failures of implantation. Most of them continue the co-cultures until obtaining one blastocyst, expanded in possible. 1,603 co-cultures have been reported by 11 groups during 2 years (1993-1994). 41,8% of cleaved eggs (day 2) reached the blastocyst stage. The pregnancy rate per transfer was 32,9%; the implantation rate of blastocysts was 24,8% which represents a significant improvement when compared to the implantation rate of 2 days old embryos. The rate of multiple pregnancies remained high (29,1%), which would incite to transfer not more than 2 blastocysts. The rate of anomalies at birth or detected in utero, was not different of the rate observed in the general population, taking account of the maternal age. PMID- 7550566 TI - [Indications for intracytoplasmic sperm injection: possible influence of oocyte quality on the results]. AB - Intracytoplasmic sperm injection is theoretically the first line treatment of many forms of severe male infertility, but the prognostic factors are to be determined according to the indications. Thus the results of two clinical teams working with the same biological Unit show statistically different pregnancy rates per oocyte pick-up (15.9% versus 35.7% - p < 0.02) and per transfer (16.9% versus 34.9% - p < 0.03). They could by explained by female factors such as age and ovarian response to stimulation, which probably have influence on embryonic quality. PMID- 7550572 TI - Ultrastructure of Psammobatis extenta (Rajidae) electrolytes and cytochemical localization of acetylcholinesterase, acetylcholine receptor and F-actin. AB - 1. The electrocytes from the electric organ of the Patagonian ray P. extenta are very unusual cells: semicircular in shape, multinucleated and highly polarized. They have an anterior, concave, innervated face, which exhibits positive histochemical reactions for acetylcholinesterase and for the nicotinic acetylcholine receptor. Multiple nerve-endings are covered with Schwann cell projections, similar to those present in skeletal muscle. Their posterior face is convex, non-innervated and is in contact with collagen fibres. 2. The cytoplasm of these electrocytes possesses abundant filamentous actin (F-actin), orderly distributed in the cell and exhibiting intense fluorescence with NBD-phallacidin. The F-actin is in contact with Z-lines as in muscle, and in contrast with Torpedo (Kordeli et al., 1986, 1987) and Discopyge (Vidal et al., 1986, 1989a) electrocytes, where it is confined to the non-innervated face. 3. Electrocytes of this Rajidae are an ideal model for the study of F-actin because of the similar embryological origin with skeletal muscle tissue and also because of the peculiar characteristics of their cytoplasm, packed full with F-actin. In addition, electric organs could constitute an alternative biological source for the study of the cholinergic synapse. PMID- 7550569 TI - [French in vitro fertilization registry. 1994 report]. AB - FIVNAT registry collected information on 25,806 individual assisted reproductive technology (ART) cycles in 1994 and the present analysis concerns 20,473 cycles arrived on time to be incorporated, 93.5% being in vitro fertilization (IVF) cycles, and 5.6% microinsemination cycles. The clinical pregnancy rates decreased from 20.4% to 18.5% per oocyte recovery cycle and from 25.5% to 23.6% per transfer, compared with 1993. The mean oocyte number (8.7 +/- 5.8) increased, but the mean embryo number (3.83 +/- 3.87) and the fertilization rate were slightly decreasing, and the mean number of transferred embryos decreased from 2.73 +/- 1.09 to 2.61 +/- 1.05. In 1994 the proportion of infertilities of pure tubal origin was still decreased to 33.7%, and that of male infertility increased. The pregnancy rate decreased only for infertilities involving a male factor, the semen characteristics becoming worse. Most of the stimulation regimen used the GnRH analogues protocols with a long blocking period (74.3%): they obtained the highest pregnancy rate per transferred embryo (24.7%). An increased proportion of transfers involved 2 embryos, and they were associated with a higher clinical pregnancy rate compared with 1993 (23.0% vs 21.8%). Those two facts indicate that the transfer policies involved a lower number of transferred embryos. The analysis of 13,446 clinical pregnancies resulting from recoveries realised between 1986 and 1993 showed a preterm birth rate of 9.3% for singleton. For those singleton pregnancies, in utero mortality concerned 6.2% of the children, and the neonatal mortality rate (< 7 days) was 5.2%. The overall malformation rate (major and minor) was 2.5%. PMID- 7550575 TI - Mitosis and genome partition in trypanosomes. PMID- 7550570 TI - [Multiple pregnancies. FIVNAT]. AB - FIVNAT register has collected 10,510 deliveries after IVF attempts from 1986 to 1993. The multiple pregnancy rate is 27.2%, 23.5% for twin pregnancies, 3.6% for triplets pregnancies and 0.15% for multiples pregnancies over 3. Triplet pregnancy proportion, before embryo reduction, reached 8% in 1989 then decreased to 6.1% in 1993. In 1994 the proportion of 3 and 4 embryos transfers has decreased and that of 2 embryos transfers has increased, but global pregnancy rate per transfer is 1.6% lower than pregnancy rate in 1993. This point shows how difficult it is to decrease multiple pregnancy rate without decreasing global pregnancy rate. PMID- 7550571 TI - [The results of classical in vitro fertilization in male infertility. FIVNAT]. AB - Between 1988 and 1993, FIVNAT register collected data on 1,218 IVF cycles, with less than 500,000 motile and morphologically normal ejaculated spermatozoa per ml on the day of IVF. 700 embryo transfers were performed (57.5% per oocyte retrieval) and the mean number of transferred embryos was 2.4 +/- 1.0. 175 clinical pregnancies were obtained: consequently the clinical pregnancy rate was 14.4% per puncture and 25.0% per transfer. The pregnancy outcome is known for 102 clinical pregnancies (58.3%) and is similar to that of clinical pregnancies obtained from normal sperm. When sperm characteristics display such alterations, numeration lower than 5 millions per ml and total motility lower than 10% impair cleavage rate. The effect of teratozoospermia is not clear, because in this study severe teratozoospermia (> 90%) were associated with high numeration and motility. PMID- 7550574 TI - Alteration of morphogenetic cell interactions by 1-beta-D arabinofuranosylcytosine in Bufo arenarum embryo. AB - Amphibian gastrulation was used as a model system to study the action of the nucleoside 1-beta-D-arabinofuranosylcytosine (Ara-C) on the early events of amphibian morphogenesis. Ara-C inhibits both glycoprotein and glycolipid synthesis and interferes with DNA synthesis. Thus, it is useful to investigate the importance of the cell surface and the nucleous during Bufo arenarum morphogenesis. Living embryos were incubated with Ara-C at blastula and gastrula stages. Treated-embryos undergo abnormal gastrulation, most of the embryos exogastrulate, although some do not gastrulate at all. This antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. We have proven that Area-C disturbs the typical intercellular organization and inhibits the radial intercalation of the blastocoelic roof. The mesodermal migration is the most affected morphogenetic process. The results described in this paper demonstrate that the timing of gastrulation movements strongly involves the participation of surface and extracellular molecules in cell recognition and cell interaction but does not involve a significant increase in cell division rate and can also occur in the absence of the cell division. PMID- 7550573 TI - Serotonin reverses the inhibitory effect of a brain soluble fraction on synaptosomal membrane Na+, K(+)-ATPase activity. AB - Normal operation of the Na+ pump (Na+, K(+)-ATPase) is essential for the maintenance of neurotransmission. Filtration through Sephadex G-50 of a brain soluble fraction allowed the separation of peaks I and II fractions, respectively stimulating and inhibiting synaptosomal membrane Na+, K(+)-ATPase activity. Peaks I and II were isolated from rat cerebral cortex and their effect together with serotonin (5-HT) was studied on ATPase activity by estimating K(+)-p nitrophenylphosphatase activity in brain cortex synaptosomal membranes. It was observed that 10(-5) or 10(-4) M 5-HT failed to modify either control membrane enzyme activity or peak I activatory effect; on the other hand, such 5-HT concentrations significantly suppressed peak II inhibitory effect. This ability of 5-HT to reverse the inhibitory effect of endogenous factors on Na+, K(+) ATPase activity could well be a new 5-HT modulatory action within the brain. PMID- 7550576 TI - New and peculiar cytoplasmic membranous bodies in the acinar cells of the harderian gland of the armadillo Chaetophractus villosus. AB - A new and peculiar morphological feature in acinar cells of the Harderian gland of the South American armadillo Chaetophractus villosus (Mammalia, Dasipodidae) is reported. The gland of adult males and females was studied at macroscopic, microscopic and electron microscopic levels. The gland is the largest structure in the bony orbit. It is located in its medial (nasal) and basal side. It shows a tubuloalveolar structure characterized by large alveoli with a single layer of columnar or cuboidal cells. Myoepithelial cells are located between the secretory ones and the basement membrane. The peculiar morphological feature consists of large intracellular membranous bodies located in the supranuclear cytoplasmic region. They are seen in every acinar cell of males and females. Their size is prominent being almost as large as the nucleus. Only one body is observed in each cell. The structure of the bodies displays an outstanding geometrical pattern which differs completely from other membranous structures described in other species. PMID- 7550577 TI - Galanin-like immunoreactive expression in the central nervous system of the toad. AB - The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested. PMID- 7550578 TI - Evidence that molecules on the surface of one cell can adhere to the oligosaccharide portion of gangliosides on the surface of another cell. AB - While there have been numerous reports concerning the possible role(s) of gangliosides in neuronal development and their efficacy, or lack thereof, as possible therapeutic agents for the treatment of neuronal injury, the molecular mechanisms by which they induce specific cellular effects are not well understood. This review presents evidence for the existence of cell surface molecules able to adhere to the oligosaccharide portion of specific gangliosides and describes methods employed for their study. The identification of such cell surface molecules permits the hypothesis that the binding of the oligosaccharide portion of the ganglioside by its cell surface receptor is responsible for initiating the intracellular reactions that lead to modified cell behavior. PMID- 7550579 TI - Neutrophil chemotaxis, phagocytosis, and generation of reaction oxygen species show a hierarchy of responsiveness to increasing concentrations of N-formyl-Met Leu-Phe. AB - We assessed the effect of varying concentrations of N-formyl-methionyl-leucyl phenylalanine (fMLP) on neutrophil chemotaxis, phagocytosis, and reactive oxygen species generation. Boyden chamber chemotaxis was first elicited at an fMLP concentration of 10(-11) M, reached a peak at 10(-10) M, and declined at higher concentrations. Phagocytosis was first activated at 10(-10) M, reached its highest level at 10(-9) M, and declined at higher concentrations. O2-, H2O2 and OH. generation were elicited to a significant degree only at a fMLP concentration of 10(-8) M, or higher, reaching a peak at 10(-6) M. Thus, a distinct hierarchy was observed in the order of activation of these three neutrophil functions to varying concentrations of a soluble agonist. A teleologic model of neutrophil function that accounts for these observations is proposed. PMID- 7550580 TI - Parathyroid hormone-activated phosphoinositide degradation and calcium channels in human dermal fibroblasts. AB - Previous studies have shown that there are two types of parathyroid hormone (PTH) receptors on human dermal fibroblasts. We evaluate the effects of PTH on intracellular cyclic AMP, calcium and phosphoinositides in order to define the effector systems of the PTH receptors. The results show that PTH stimulates cyclic AMP accumulation in the human dermal fibroblasts with half-maximal effect at 1-5 nM. PTH also increases intracellular calcium as measured by the technique using Fura-2 fluorescent dye and stimulates phosphoinositide degradation as evaluated by column chromatography. The half-maximal effect on AMP occurred at 5 nM, while those effect on calcium and phosphoinositide turnover occurred at 500 nM of PTH. The results show that cyclic AMP, intracellular calcium and phosphoinositides are second messengers for PTH in human dermal fibroblasts. These results provide further evidence to support the concept of heterogeneity of the PTH receptor and its effector system. PMID- 7550582 TI - 5-Methoxypsoralen inhibits 6-hydroxylation of melatonin in the rat. AB - Pharmacokinetic studies of exogenous melatonin (100 or 1 micrograms) administered to 5-methoxypsoralen (MOP)-treated rats, showed that MOP administration induced an increase in the half-life of melatonin from 22 to 67 min. This delayed degradation of melatonin was the consequence of 5-MOP-induced inhibition of the 6 hydroxylation of melatonin. Under in vitro experimental conditions, 5-MOP did not affect melatonin synthesis and release. These results demonstrate that the elevated plasma melatonin concentrations observed in vivo following 5-MOP administration is due to inhibition of the hydroxylation of endogenous melatonin. PMID- 7550581 TI - 2-[125I]iodomelatonin binding sites in the quail liver: characterization and the effect of guanosine 5'-O-(3-thiotriphosphate). AB - Melatonin receptors were studied in quail livers using the melatonin agonist 2 [125I]iodomelatonin ([125I]MEL) as the radioligand. The specific binding of [125I]MEL to membrane preparations of liver was rapid, stable, saturable, reversible and of high affinity. Scatchard analysis of the specific binding data indicated an equilibrium dissociation constant (Kd) of 19.4 +/- 1.01 pmol/l (n = 7) and a maximum number of binding sites (Bmax) of 1.16 +/- 0.19 fmol/mg protein (n = 7) in the quail liver collected at mid-light. The Hill coefficient approached 1.0, suggesting a single class of [125I]MEL binding sites in the quail liver. The diurnal variation study showed that the value of Kd was 64.4% higher (p < 0.05) at mid-dark compared to mid-light, with no significant change in Bmax. The kinetic analysis showed that the Kd value was 25.0 +/- 3.94 pmol/l at mid light, which was comparable with values determined from saturation studies. Aside from 2-iodomelatonin, melatonin and 6-chloromelatonin, all indole analogs and neurotransmitters tested in inhibition studies had slight or no displacement of [125I]MEL binding. These studies demonstrated that [125I]MEL binding sites were highly specific for melatonin. The presence of 10 and 50 mumol/l guanosine 5'-O (3-thiotriphosphate) significantly increased (p < 0.05) the Kd values and depressed the Bmax values, proposing that [125I]MEL binding sites in quail livers were coupled to a G-protein. Our results indicate that melatonin may exert a direct action on liver functions. PMID- 7550583 TI - Expression of daily torpor in the Djungarian hamster (Phodopus sungorus) is correlated with the testosterone-driven vasopressin content in the lateral septum. AB - In the Djungarian hamster (Phodopus sungorus), torpor bouts appear after exposure to short photoperiods (SP) for 11 weeks. Castration accelerates the occurrence of daily torpor when performed between the 1st week before and the 4th week after transfer to SP. In animals castrated 8 weeks before transfer to SP, daily torpor was observed nearly at the same time as in the control group. Vasopressin immunoreactivity (VP-ir) in the lateral septum (LS) of the Djungarian hamster was examined. Dense VP-ir was seen in sexually active and in sexually inactive hamsters implanted with testosterone (T) capsules. No VP-ir could be detected in sexually inactive animals. Thus, the presence of VP-ir in the LS depends on circulating T levels. In control animals exposed to SP, complete disappearance of VP-ir in the LS was observed after 8 weeks. Castration on the day of transfer to SP reduced this period to 6 weeks. In these animals, the early occurrence of daily torpor is related to the quick disappearance of VP-ir in the LS. When castrated animals were maintained under long photoperiods the disappearance of VP ir was not complete. However, VP-ir disappeared completely after transfer to SP. The results suggest that the SP message acts on daily torpor indirectly via testicular regression but also 'directly' via VP septal innervation. PMID- 7550584 TI - Effects of 5-HT denervation of the suprachiasmatic nuclei or lesions of the median raphe nucleus on daily torpor in the Djungarian hamster, Phodopus sungorus. AB - In the Djungarian hamster (Phodopus sungorus), using 5,7-dihydroxytryptamine (a specific neurotoxin), depletion of the serotonin (5-HT) fibers innervating the suprachiasmatic nuclei (SCN) was performed in animals displaying torpor bouts. The characteristics of postoperative torpor bouts in lesioned hamsters were similar to those observed before the denervation. 5-HT innervation of the SCN thus seems unnecessary for the expression and temporal organization of daily torpor. After chemical or thermic lesions of the 5-HT neurons of the anterior median raphe nucleus (MR) in hamsters displaying torpor bouts, the phenomenon of torpor was not prevented. In addition, hamsters lesioned on the day of transfer to short photoperiod (SP) displayed their first torpor bouts after the same duration of SP exposure as intact hamsters. These results suggest that, contrary to what is observed in hibernation, 5-HT neurons of the anterior part of the MR are not involved in the mechanisms governing the occurrence and seasonal timing of daily torpor. PMID- 7550586 TI - Outcome of referrals to a district immunisation advisory clinic. AB - We have studied referrals to and outcomes of consultation at a district immunisation advisory clinic, aiming to examine and confirm the rarity of true contraindications, the safety of immunising children with possible contraindications, and the influence on parents of counselling by well informed professionals. We examined medical records and postal questionnaires to parents, representing 30 months' work. Three hundred and fifty-eight children were referred to the clinic, 248 of whom were seen and 110 (31%) did not attend. Fourteen children who attended (6%) had a true contraindication to the vaccine(s) in question and 202 of the remaining 234 (86%) proceeded to vaccination with no adverse effect. Forty of the 110 (36%) who were referred but did not attend were vaccinated, and no adverse effects were reported, but the immunisation rate of this group was significantly lower than that seen in the clinic (chi 2 test, p < 0.001). True contraindications to childhood immunisation were rare, and children with difficult problems were safely vaccinated. The immunisation advisory clinic is effective in counselling parents who are uncertain about childhood immunisation. PMID- 7550587 TI - Outbreaks of infectious intestinal disease associated with person to person spread in hotels and restaurants. AB - Twenty-eight outbreaks of infectious intestinal disease, reported as being transmitted mainly by the person to person route, were identified in association with retail catering premises, such as hotels, restaurants, and public houses, in England and Wales between 1992 and 1994. Five thousand and forty-eight people were at risk in these outbreaks and 1234 were affected. Most of the outbreaks (over 90%) occurred in hotels. Small round structured viruses were the most commonly detected pathogens. Diarrhoea and vomiting were common symptoms and most of the outbreaks occurred in the summer months. Control measures to contain infectious individuals and improved hygiene measures are necessary to contain such outbreaks. PMID- 7550585 TI - Virological surveillance of influenza in England and Wales: results of a two year pilot study 1993/94 and 1994/95. PHLS Influenza Subcommittee. AB - Ten public health laboratories and a group of collaborating general practitioners were recruited to participate in a two year pilot study of virological surveillance of influenza in England and Wales, which was carried out during the winters of 1993/94 (first year) and 1994/95 (second year). The study was designed to facilitate rapid diagnosis and reporting of the results from the specimens submitted each week. Nose and throat swabs were obtained from 240 patients seen by general practitioners in the first year, 14 of which (6%) were positive for influenza virus; in the second year 66 specimens (14%) from a total of 477 nose and throat swabs were positive. The highest proportion of positive results were in children in both years. Fewer specimens were submitted to the study than expected, but the study obtained more timely data on the contribution of influenza virus infection to the overall level of influenza-like illness in the community than are normally available through routine surveillance. PMID- 7550588 TI - Gastroenteritis associated with oysters. AB - An outbreak of gastroenteritis occurred in catering students attending three classes at a Yorkshire college in February 1994. The three classes were held on the Monday, Tuesday, and Thursday of the same week and had identical menus. Thirty-seven of the 90 students were affected. A cohort study, with a 94% response rate, showed a highly significant association of illness with the consumption of raw oysters grown in English coastal waters. PMID- 7550589 TI - Outbreak of hospital acquired multidrug resistant tuberculosis. PMID- 7550590 TI - Epidemic methicillin resistant Staphylococcus aureus. PMID- 7550591 TI - A case of anthrax septicaemia in a London teaching hospital. PMID- 7550592 TI - Cluster of cases of Legionnaires' disease associated with travel to Turkey. PMID- 7550593 TI - Outbreak of Escherichia coli O157 in North Wales. PMID- 7550598 TI - Dietary changes, compulsions and sexual behavior in frontotemporal degeneration. AB - The occurrence of weight gain, sweet and carbohydrate craving, hyposexuality, and compulsions in frontal lobe dementia (FLD) compared to Alzheimer's disease (AD) was evaluated. FLD is a progressive dementia with a high rate of misdiagnosis and therefore better diagnostic criteria for FLD are needed. Fourteen patients meeting research criteria for AD were compared to 14 with suspected FLD. All had cerebral perfusion measured with xenon-133 and imaged with HMPAO using brain dedicated SPECT. The FLD group showed frontotemporal and AD posterior temporoparietal hypoperfusion. The primary caregivers were queried regarding weight gain, sweet/carbohydrate preference, sexual drive, and compulsions. Differences were compared with Fisher's exact test. The following was found in FLD versus AD: Weight gain in FLD patients amounted to 64% (AD 7%), carbohydrate craving was 79% (vs. 0%) and compulsive behavior 64% (vs. 14%). The differences for these symptoms were statistically significant, whereas for the symptoms increased sexual drive (8 vs. 8%) and reduced sexual drive (54 vs. 23%) no significant difference could be found. In FLD the first symptoms were often dietary changes or hyposexuality. Compulsions were more bizarre and severely disabling in FLD than in AD. Dietary changes, hyposexuality, and disabling compulsions are prominent early symptoms in FLD but not AD. The cause of these symptoms may be due to both frontal and subcortical serotonin loss and dysfunction of the anterior temporal lobes. PMID- 7550595 TI - Transient enhancement of cholinergic neurochemical markers induced by NGF in aged F344 rats. AB - Two experiments were conducted to assess the durability of nerve growth factor (NGF) effects on cholinergic neurochemical markers. Artificial CSF or NGF was infused via osmotic pumps for 2 weeks into the lateral ventricles of young adult (3- to 6-month-old) and aged (22- to 26-month-old) Fischer 344 rats. Assessment of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) within the cortex, striatum, and hippocampus was conducted either approximately 3 (experiment 1) or 12 (experiment 2) weeks after termination of treatment. A variety of age-related deficiencies were found in the two experiments with decreased marker levels within the dorsal hippocampus and striatum being most consistent. NGF increased cholinergic marker enzyme activity in experiment 1 only. Specifically, NGF (a) attenuated age-related CAT and AChE deficits within the dorsal hippocampus and striatum, (b) enhanced CAT activity within the frontal cortex and ventral hippocampus in aged animals, and (c) increased CAT activity within the dorsal hippocampus in young subjects. It is concluded that NGF may be beneficial in enhancing cholinergic neurochemical parameters, especially in aged animals, but such effects are most likely transient. PMID- 7550596 TI - A decrease in neural sialyltransferase activity in Alzheimer's disease. AB - The activities of both a particulate and soluble form of the sialyltransferase enzyme have been examined in post-mortem brain samples from Alzheimer's disease patients and age-matched controls. There was a considerable decrease in the activity of both the soluble and membrane-bound forms of the enzyme in the frontal and temporal cortical lobes, although no change was observed in the hippocampus. There was, however, no change in activity of the Golgi marker enzyme thiamine pyrophosphatase. Therefore, it is suggested that the decrease in sialyltransferase enzyme activity may be a specific biochemical event associated with the AD-like neurodegeneration. PMID- 7550597 TI - Familial Alzheimer's disease and cortical Lewy bodies: is there a genetic susceptibility factor? AB - The reason for the occurrence of Lewy body disease (LBD) in Alzheimer's disease (AD) patients is unknown. If brains from etiologically different AD groups differ in their tendency to develop cortical Lewy bodies, the concurrence of LBD in AD patients may be a manifestation of the AD process in specific AD subsets. To address this issue, we counted cortical Lewy bodies in AD patients with genetic abnormalities on chromosome 14 (n = 19), and chromosome 21 (n = 3), sporadic AD (n = 27), Down's syndrome (n = 1) and control (n = 26) patients. Cortical Lewy bodies were occasionally present in AD patients with long-duration disease in most of the above AD subgroups, but were not present in any of our age-matched control cases. We suggest that cortical Lewy body formation may be an intrinsic part of the late pathologic changes of AD regardless of etiology and that it is not specific to any of the AD subtypes studied. PMID- 7550594 TI - Influenza A: a school outbreak to start the term. PMID- 7550599 TI - Leukocyte hexokinase activity in aging and Alzheimer disease. AB - We determined the effects of clinical variables on hexokinase (HK) activity in leukocytes from Alzheimer's disease (AD) patients and controls. Age accounted for 51% of the variance in HK activity in the young and 23% in the old. Duration of illness in both familial and sporadic AD accounted for HK levels by 32 and 38%, respectively. Hexokinase activity increases with age and does not discriminate between familial and sporadic AD. PMID- 7550600 TI - Atrophy of hippocampal formation subdivisions correlates with stage and duration of Alzheimer disease. AB - The hippocampal formations of 13 subjects with severe Alzheimer disease [AD; Global Deterioration Scale (GDS) stage 7] and of 5 age-matched subjects without symptoms of dementia were reconstructed from serial sections. Functional assessment staging (FAST) was used at the time of demise to assess 9 patients at stages 7a-c (incipient averbal and nonambulatory) and 4 patients at stages 7e-f (immobile). The duration of the disease from FAST stage 5 until demise ranged from 2 to 8 years in the first of these subgroups, and from 10 to 13 years in the second. The volumes of the entire hippocampal formation and of the cornu ammonis, its sectors and layers, the dentate gyrus, the subicular complex, and the entorhinal cortex were calculated. Hippocampal formation volume decreased by 36% in the incipient averbal and nonambulatory patients and by 60% in the severely functionally impaired immobile patients, in comparison with controls. In the final substages of AD, immobile patients exhibited significant atrophy, in comparison with controls, in the cornu ammonis and all of its sectors and layers except CA4, the subicular complex and all of its parts, and the entorhinal cortex (p < 0.05). Within the AD patient group, significant correlations were noted between both the magnitude of functional severity and the duration of AD and the volumes of most hippocampal formation subdivisions studied. For the cornu ammonis, subicular complex, and entorhinal cortex, volumetric loss correlations with FAST stage 7 ordinally enumerated substages were r = -0.71, -0.79, and 0.62, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550601 TI - Synaptophysin immunoreactivity is stable 36 h postmortem. AB - The effects of postmortem time, age and tissue storage time on synaptic density were investigated in the prefrontal cortex of human brains. Thirty normal cases and 10 patients with a variety of neurologic diseases were studied, using quantitative immunohistochemistry with a monoclonal antibody to synaptophysin. Synaptophysin immunoreactivity was stable during the first 36 h after death, showing no significant change at 1, 8, 12, 24 and 36 h postmortem in normals. However, at 48 and 72 h postmortem, synaptophysin immunoreactivity decreased significantly. A similar pattern of postmortem changes was found in the disease group. In 14 normal cases with a wide range of ages, a significant reduction in synaptic density with age was observed while the time of tissue storage as paraffin-embedded blocks had no significant effect on the synaptophysin immunoreactivity. These results support the validity of using synaptophysin immunohistochemistry as a measure of synaptic density in human brain autopsied within 36 h of death and stored as paraffin-embedded blocks for a long time. PMID- 7550602 TI - The cortical neuritic dystrophy of Alzheimer's disease: nature, significance, and possible pathogenesis. AB - It has been increasingly appreciated in the last decade that the neocortex of the Alzheimer's disease brain is characterized by a widespread neuritic dystrophy, spatially distinct from that observed surrounding neuritic amyloid plaques. The nature and significance of this cortical neuritic dystrophy and its possible pathogenesis are the topics of this review. It may represent a regenerative sprouting response of neurons to partial deafferentation following cell loss; alternatively it may reflect a primary aberrant growth phenomenon leading to cell loss. Such changes could result from derangement of neurite growth-promoting and growth-inhibitory factors in the Alzheimer's disease brain. PMID- 7550603 TI - THA--historical aspects, review of pharmacological properties and therapeutic effects. AB - The authors reviewed historical data in the development of THA, as well as recent data on its pharmacological properties and therapeutic effects on cognitive dysfunction. A medline search was conducted to identify the trials conducted with THA in Alzheimer's disease (AD) patients. The findings seem to lend support to some palliative action of THA, especially at higher doses, but in these doses about 2/3 of the patients experience significant adverse reactions. The significance of these findings is discussed, with emphasis on the their relevance for the management of AD patients. PMID- 7550604 TI - Striatonigral degeneration combined with olivopontocerebellar atrophy with subcortical dementia and hallucinatory state. AB - We present an autopsied case of striatonigral degeneration (SND) combined with olivopontocerebellar atrophy (OPCA) with subcortical dementia and hallucinatory state. A Japanese woman without a remarkable family history showed hand tremor at the age of 35 years, followed by bradykinesia, muscle rigidity, orthostatic hypotension, neurogenic bladder and pyramidal signs. No obvious cerebellar symptoms were found. Various antiparkinsonian drugs were administered, but were not markedly effective for the parkinsonism. She developed a mild dementia characterized by mild memory disturbance with preservation of orientation, slowing of thought processes, emotional lability toward sadness, impaired ability to manipulate acquired knowledge and poor calculating, and by the absence of aphasia, apraxia and agnosia. The features in this patient were consistent with those seen in subcortical dementia. She also had auditory hallucinations. MRI revealed hypointense T2 signals in the putamina and substantia nigra. T1-weighted MRI demonstrated atrophy of both the pons and cerebellum in addition to atrophy of the putamina and substantia nigra. EEG showed slowing of background activity. She died of cardiac failure at the age of 47. Autopsy disclosed brain stem tegmental atrophy, SND, OPCA and many glial cytoplasmic inclusions in the central nervous system, but well-preserved cerebrum. We discuss the relationship between the psychiatric symptoms and pathologic findings of brain stem tegmentum. PMID- 7550605 TI - Cell migration in the small and large bowel shows a strong circadian rhythm. AB - Migration velocity estimates have been determined at each position along the crypt length for both the small and large intestine of the mouse at 6 different times of the day. Measurements also have been made of crypt circumference and length. Dramatic, and significant (P < 0.001), changes in migration velocity as a function of time of day were observed in the small intestine with a maximum 0.84 cell positions (cp) per hour at 0900 h and a minimum of -0.46 cp/h at 1700 h, although the negative velocity was probably artefactual. The 24-h mean velocity rose smoothly as a function of cell position to a peak of 0.45 cp/h at cell position 17 (around the top of the proliferative zone). Much more modest changes were seen in the percent of 3HTdR labelled cells (minimum 30.8%, maximum 38.3%, P < 0.001) and crypt circumference (minimum 16.9 cells, maximum 17.9 cells, P = 0.003). The migration velocity was rather less well determined in the large intestine with a peak in the 24-h mean velocity (0.26 cp/h) occurring at cell position 10. At this position significant circadian variation was detected (minimum -0.39 cp/h, maximum 0.75 cp/h, P = 0.006). Changes were seen in the percent of labelled cells (minimum 9.4%, maximum 22.3%, P < 0.001) and crypt circumference (minimum 18.3 cells, maximum 19.2 cells, P < 0.001). In both tissues it is suggested that the combination of the modest changes in cell proliferation rates in conjunction with the changes in crypt cell number can account for the large amplitude in variation of crypt output, and that the reservoir effects of changes in crypt geometry are an essential part of the process governing the maintenance of intestinal cell numbers. PMID- 7550606 TI - Murine dendritic epidermal T cells (DETC) express the homophilic adhesion molecule E-cadherin. AB - Cadherins mediate homotypic intercellular adhesion in epidermis and other epithelia. E-cadherin is also involved in interactions between murine epidermal Langerhans cells (LC) and keratinocytes (KC). Dendritic epidermal T cells (DETC) comprise another subpopulation of epidermal leukocytes. Using flow cytometry, we determined that DETC expressed levels of E-cadherin similar to those expressed by LC and KC. DETC also adhered congruent to three-fold better to KC and E-cadherin transfected fibroblasts than to normal fibroblasts. Treatment of DETC with trypsin in the absence of calcium caused a loss of E-cadherin and resulted in an congruent to 80% decrease in DETC-KC adhesion whereas treatment of DETC with trypsin in the presence of calcium did not significantly affect E-cadherin expression or DETC-KC binding. Thus, E-cadherin may be involved in adhesion of DETC to KC. DETC are derived from TCR V gamma 3+ thymocytes that transiently populate embryonic murine thymus. We determined that TCR V gamma 3+ thymocytes as well as other early (fetal day 16) TCR gamma/delta+ thymocytes expressed E cadherin; TCR gamma/delta+ (TCR V gamma 3-) thymocytes that developed later did not. These results indicate that cells of the T cell lineage can express E cadherin, and suggest that E-cadherin may play a role in adhesion of DETC (and/or DETC precursors) to KC. PMID- 7550608 TI - Stimulatory guanine nucleotide binding protein in pig epidermis: transient increase of the 45KDA cholera toxin substrate (Gs alpha) in the tape stripping induced hyperproliferative state. AB - Cholera toxin catalyzed the transfer of ADP-ribose from [alpha-32P] NAD to 45kDa protein in pig epidermis. Western blot analysis using anti-Gs alpha antibody identified the 45kDa protein to be Gs alpha. In contrast to pertussis toxin catalyzed ADP-ribosylation of Gi alpha, the cholera toxin-catalyzed ADP ribosylation was enhanced by the presence of Mg2+ in the reaction mixture. The cholera toxin-catalyzed ADP-ribosylation of the epidermal 45kDa membrane protein was significantly decreased, when samples were prepared from the cholera toxin pretreated epidermis. The results, coupled with our previous report (Tsutsui and Iizuka 1990), indicate that pig epidermis contains functional G proteins (Gs and Gi), that affect the epidermal adenylate cyclase activity. Tape stripping-induced hyperproliferative epidermis showed an increased cholera toxin-catalyzed ADP ribosylation of the 45kDa protein (Gs alpha) at 12-24 h following the tape stripping. Immunoblot analysis, however, showed no remarkable change in the level of Gs alpha compared with non-stripping controls. There was no significant difference in the level of the pertussis toxin-induced ADP-ribosylation of 40kDa protein (Gi alpha) in the tape-stripped epidermis. Immunoblot analysis showed no change in Gi content, either. Forskolin-induced cyclic AMP accumulation was markedly increased in the tape stripping-induced hyperproliferative epidermis. Cholera toxin-induced cyclic AMP accumulation was slightly increased, but this was not statistically significant. These results indicate that the alteration of Gs that is documented by cholera toxin-catalyzed ADP-ribosylation, is among the functional derangements of adenylate cyclase of tape stripping-induced hyperproliferative epidermis. PMID- 7550607 TI - The kinetic organization of the ulcer-associated cell lineage (UACL): delineation of a novel putative stem-cell region. AB - We have examined the proliferative organization of the ulcer-associated cell lineage (UACL), in a series of human ileal tissues involved by Crohn's disease, using antibodies to trefoil peptides and Ki-67. The UACL arises in conjunction with endodermal damage and is likely to play a role in subsequent tissue repair. Our study supports the view that the UACL initially forms through extrusion from the base of a parent intestinal crypt without indigenous mitotic activity. Eventually a duct forms extending to the gut surface and within this duct a proliferative zone develops. We also confirm that the trefoil peptides pS2 and hSP, which play a major role in the repair process, are spatially segregated within the UACL with a zone of overlap in the duct. Using triple antibody labelling techniques we have demonstrated that the zone of overlap of these peptides coincides with the proliferative region and provides evidence for a novel stem cell zone. PMID- 7550609 TI - Association of transglutaminase with the reconstituted keratin filaments isolated from rat vaginal epithelial cells. AB - We report a novel association of the calcium dependent cross-linking enzyme, transglutaminase (TGase) with the urea soluble reconstituted keratin filaments (RKF) isolated from the rat vaginal epithelial cells (VEC). This was ascertained by measuring the activity using 14C-spermidine incorporation and also by an increase in keratin filament aggregation by the addition of only TGase cofactor calcium. These events were specifically inhibited by the treatment of calcium chelator, EDTA at a concentration > 2 mM as well as by pretreating the RKF with histamine, a TGase substrate inhibitor. The association was also exemplified by immunoblotting analysis where a specific and preferential polypeptide of molecular weight 58 kDa cross-reacted with TGase antibody amongst the other keratins. This phenomenon was not seen in the keratins isolated from skin, a non targeting tissue for estradiol action. PMID- 7550610 TI - Rates of clearance of the epithelial surfaces of mouse oral mucosa and skin. AB - Cell desquamation, by removing material adherent to the epithelial surface, appears to play an important role in limiting bacterial colonization of epithelia. Data are available concerning rates of epidermal surface clearance but similar data for oral mucosal epithelia, which exist in an environment more conducive to colonization, have not been reported. The rates of surface clearance of six regions of murine oral mucosa and skin were assessed by two different methods: (a) EM autoradiography to examine the rate of passage of a 3H-histidine label through the stratum corneum, and (b) measurement of rates of cell proliferation and of the size of the superficial cells of the stratum corneum. The autoradiographic (direct) method indicated regionally-varying epidermal clearance rates (6.3-15.9 h) which compared well with published data. This method indicated considerably faster (2.3-3.4 h) clearance rates for the three oral mucosal regions. The rates of surface clearance that were calculated indirectly, from rates of cell proliferation and of cell size, similarly showed faster clearance rates for mucosa (2.0-3.9 h) than for skin (6.0-17.9 h) but differed in the relative rates calculated for the individual regions of mucosa and skin. PMID- 7550612 TI - Hippocampal long-term potentiation does not affect either discrimination learning or reversal learning of the rabbit nictitating membrane response. AB - The theoretical premise that the acquisition and storage of information occurs through the strengthening of synaptic connections has contributed to the popularity of long-term potentiation (LTP) as a candidate neural mechanism for associative learning. However, whether experimentally induced LTP facilitates, disrupts, or has no effect on subsequent learning is a controversial issue. The present study examined the reported facilitative effect of LTP within hippocampal perforant path-dentate gyrus synapses on subsequent discriminative conditioning of the rabbit nictitating membrane response. In addition, the effect of LTP on subsequent reversal learning of the initial discrimination was examined. LTP did not significantly affect acquisition of the initial discriminative response or subsequent reversal learning. Furthermore, the magnitude of LTP could not be used to predict the rate of acquisition of either task. The failure to find an effect of LTP on classical conditioning of the rabbit nictitating membrane response mirrors the recent failures to replicate the disruptive effect of LTP on spatial learning in the rat. Thus, the potential contribution of an LTP-like mechanism to associative learning remains equivocal. PMID- 7550611 TI - Spontaneous and synaptic input from granule cells and the perforant path to dentate basket cells in the rat hippocampus. AB - To characterize excitatory inputs to dentate basket cells from dentate granule cells and the perforant path, the whole-cell recording technique was used in neonatal rat hippocampal slices. Spontaneous excitatory input to basket cells was also examined and compared to that of other interneurons in the dentate gyrus. Basket cells were separable from other neurons in the dentate gyrus based on morphology and location, as determined by biocytin staining following recording, and by resting membrane potential, propensity to fire action potentials spontaneously, and miniature excitatory postsynaptic current (EPSC) characteristics. Minimal electrical stimulation of the granule cell layer evoked in basket cells short latency EPSCs that were composed of both N-methyl-D aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) components as judged by their time course, voltage dependence, and blockade by selective antagonists. Perforant path EPSCs exhibited slower kinetics than EPSCs evoked by granule cell stimulation. Like granule cell evoked EPSCs, however, perforant path EPSCs were composed of both NMDA and AMPA components. Minimal electrical stimulation of the granule cell layer and perforant path evoked monosynaptic EPSCs in only 67% and 62% of the trials, respectively, suggesting that these inputs are as unreliable as previously determined inputs from CA3 pyramidal cells (48%). Tetrodotoxin-insensitive spontaneous miniature EPSCs were frequent in basket cells and non-basket interneurons residing either at the border between the granule cell layer and the hilus or deep within the hilus. Miniature EPSCs recorded from all cells held at -70 mV were blocked completely by 3 microM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Though a component of the miniature EPSCs recorded from border and deep hilar interneurons at +40 mV was blocked by the NMDA receptor antagonist D-2-amino-phosphonovaleric acid (D-APV), miniature EPSCs in basket cells were insensitive to D-APV. We conclude that input from granule cells and the perforant path results in activation of basket cells via glutamatergic synapses that employ both NMDA and AMPA receptors. These inputs to basket cells likely contribute to feedback and feedforward inhibition of granule cells. The absence of an NMDA receptor component in spontaneous miniature EPSCs of dentate basket cells implies a difference in organization of excitatory synapses made onto basket cells compared with other hilar interneurons. PMID- 7550613 TI - Simulation of spatial learning in the Morris water maze by a neural network model of the hippocampal formation and nucleus accumbens. AB - Cells in the hippocampal formation show spatial firing correlates thought to be critical to the role played by this structure in spatial learning. Place cells in the hippocampus proper show location-specific activity, whereas cells in the postsubiculum fire as a function of momentary directional heading. One question which has received little attention is how these spatial signals are used by motor structures to actually guide spatial behavior. Here we present a model of how one kind of spatial behavior, instrumental learning in the Morris water maze, could be guided by the spatial information in the hippocampal formation. For this, we concentrate on the hippocampal projection to the nucleus accumbens, which is strongly implicated in instrumental learning. In the model, simulated firing patterns of place cells and head direction cells activate "motor" cells in the "accumbens." Each motor cell causes a particular locomotor movement in a simulated rat. In this way, the "rat" locomotes through the simulated environment. Each step places the animal in a slightly different location and directional orientation, which, in turn, activates a different set of place and head direction cells, thus causing the next locomotor response, and so on. Connection strengths between cells are initially set randomly. When the animal encounters the reward location, however, connections are altered, so that recently active synapses are strengthened. Thus, successful moves in a particular locational and directional context are "stamped in." Simulated rats show rapid learning, similar in many ways to that of actual rats. In particular, they generate efficient routes to the goal after minimal experience, and can do so from somewhat novel starting positions. Consideration of the model architecture shows that 1) combined use of directional and place information is an example of a linearly inseparable problem and that 2) some types of novel route generation, often thought to require a "cognitive mapping" strategy, can be generated from the S-R type model used here. PMID- 7550614 TI - Information acquired by the hippocampus interferes with acquisition of the amygdala-based conditioned-cue preference in the rat. AB - White and McDonald (1993, Behav Brain Res 55:269-281) previously reported that animals with amygdala lesions failed to acquire a conditioned-cue preference (CCP) based on spatial cues, but that animals with fornix lesions exhibited larger CCPs of this type than normal animals. The present experiments focused on the hippocampal interference with amygdala-based CCP learning inferred from this finding. In experiment 1 we tested the hypothesis that this interference was due to the acquisition of information about the maze and its environment during a 10 min period of free exploration of the maze before the start of CCP training, hitherto given to all animals in these experiments. Normal animals that were not preexposed to the maze and animals that were preexposed to a similar maze in a different room both exhibited larger CCPs than animals that were preexposed to the same maze in the same room as CCP training and testing. This suggests that normal animals acquire context-specific information during the preexposure period and that this may be the cause of the hippocampus-based interference with the amygdala-mediated CCP. In experiment 2 we attempted to determine if the information thought to be acquired by the hippocampal memory system interferes with acquisition or expression of the CCP. As previously demonstrated, animals that received fornix lesions before preexposure (i.e., before the start of the experiment) exhibited larger than normal CCPs. Animals that received fornix lesions after preexposure but before CCP training and animals that received fornix lesions after CCP training but before testing both exhibited normal CCPs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550615 TI - Changes in exploratory activity following stimulation of hippocampal 5-HT1A and 5 HT1B receptors in the rat. AB - The object exploration task allows the measure of changes in locomotor and exploratory activities, habituation, and reaction to a spatial change and to novelty. The effects of intrahippocampal (dorsal CA1 field) microinjections of serotonin 1 receptor (5-HT1) agonists on these behavioral components were evaluated in the rat. 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 5 micrograms/microliters) was used as a 5-HT1A agonist, 3-(1,2,5,6-tetrahydropyrid 4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93,129,16 micrograms/microliters) as a 5-HT1B agonist, and scopolamine (10 micrograms/microliters) as a muscarinic cholinergic antagonist. Scopolamine induced a long-lasting increase in locomotor activity and a lack of reaction to spatial change; both these results are in agreement with the known crucial influence of the septo-hippocampal cholinergic system in hippocampal functioning. Stimulation of 5-HT1A and 5-HT1B receptors induced a decrease in object exploration and habituation without affecting the retrieval of spatial information. But stimulation of hippocampal 5-HT1B receptors induced a selective change in the animal's emotional state, i.e., an initial decrease in locomotor activity and a neophobic reaction in response to a new object; such effects did not occur following stimulation of 5HT1A receptors. These results have to be considered in the light of the anxiogenic property of 5-HT1B agonists. On the whole, they support the hypothesis of the involvement of the serotonergic system, via 5HT1A and 5-HT1B receptors, in the modulation of hippocampal functions. PMID- 7550616 TI - Effect of adenosine on bicuculline-resistant paired-pulse inhibition in the rat hippocampal slice. AB - This study extends previous investigations into the effect of adenosine on bicuculline-resistant paired-pulse inhibition between field potentials evoked 300 ms apart in the CA1 area of the rat hippocampal slice. A direct assessment of the effect of adenosine on paired-pulse inhibition is complicated by the facts that adenosine directly depresses evoked potentials and bicuculline-resistant paired pulse inhibition is greater between pairs of small potentials than between pairs of larger potentials. Adenosine increased bicuculline-resistant paired-pulse inhibition when stimulus strength was constant between adenosine and control but paired-pulse inhibition of responses in adenosine was markedly less than paired pulse inhibition of control responses of the same size. Furthermore, adenosine decreased the size of conditioned potentials to a significantly lesser extent than unpaired potentials of the same initial size. Taken together the results indicate that adenosine can decrease bicuculline-resistant paired-pulse inhibition in the hippocampus. A possible mechanism for this effect is that adenosine is suppressing transmission at excitatory terminals onto interneurones which would suggest that these receptors are more sensitive to adenosine than those on the Schaffer collateral/CA1 pyramidal cell synapses. In this case adenosine should reduce paired-pulse inhibition at lower concentrations than are required for depression of single evoked potentials. A comparison of the concentration-response relationships for the effects of adenosine on paired-pulse inhibition and on single evoked potentials ruled out greater sensitivity of adenosine receptors at excitatory terminals onto interneurones as an explanation for adenosine's action on bicuculline-resistant paired-pulse inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550617 TI - Postnatal development of CA3 pyramidal neurons and their afferents in the Ammon's horn of rhesus monkeys. AB - Previous studies described the postnatal development of CA3 pyramidal neurons and their afferents in the rat. However, the postnatal development of the primate hippocampus was not previously studied. Thus, pyramidal neurons of the CA3 area of the monkey hippocampus were analyzed postnatally in the present study. At birth, a few thorny excrescences, the complex spines postsynaptic to mossy fibers, were found on the proximal segments of both apical and basal dendrites, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number until the third month. A continuous increase in the number of spines per unit length along the distal dendrites was observed during the first 12 months. The ultrastructural features of somata and dendrites of pyramidal cells in newborn monkeys were similar to those of adults. The analysis of the afferents to the CA3 pyramidal neurons was limited to the development of mossy fibers, the axons of granule cells, and myelinated axons in the alveus, stratum oriens, and stratum lacunosum-moleculare. At birth, most mossy fiber terminals were densely packed with synaptic vesicles and formed mainly axospinous synapses with CA3 pyramidal cells. By 1 month of age, the number of mitochondria and embedded spines increased to mature amounts. In the first postnatal month, degenerating axons and axon terminals were frequently observed in the mossy fiber bundles in stratum lucidum. The proportion of myelinated axons increased simultaneously in all three examined layers. At birth most axons were unmyelinated, whereas at 7 months of age the proportion of myelinated axons was similar to that found in adults. The present study indicates that most pyramidal neurons of the CA3 region in monkeys are in an advanced stage of development at the time of birth. Thus, mossy fibers from granule cells in the dentate gyrus have established mature-looking synapses, and the thorny excrescences of pyramidal cells that are postsynaptic to mossy fibers are also adult-like. Nevertheless, several of the adult features, such as the spine density of distal dendrites of pyramidal neurons and the myelination of afferent axons, develop during an extended period of time in the first year. The significance of this early anatomical maturation in a brain region involved in memory function is consistent with recent behavioral data that show a rapid postnatal maturation of limbic-dependent recognition memory in rhesus monkeys. PMID- 7550618 TI - The role of the hippocampus in declarative memory: a comment on Zola-Morgan, Squire, and Ramus (1994) PMID- 7550619 TI - Fast rhythms in the hippocampus are a part of the diffuse gamma-response system. PMID- 7550620 TI - Thumb movements, motions, and moments. AB - An understanding of the movements of the thumb is essential for understanding thumb function and pathology and for planning therapeutic interventions. The thumb has three joints, which move about axes of rotation. The carpometacarpal and metacarpophalangeal joints have two offset axes of rotation and the interphalangeal joint has one. The thumb muscles act about these axes, and imbalances resulting from paralysis are explained by this model. The effects of tendon transfers and splints can be predicted by analyzing their effects at each joint and axis. PMID- 7550622 TI - Splints: mechanics versus convention. AB - Splints are specialized engineering machines that are created to solve specific upper-extremity problems. Astute manipulation of mechanical concepts increases splint efficiency, enhances patient comfort and function, and improves splint durability, while diminishing cost and frustration. Creation of splint designs should be based on mechanical fact rather than on mode-of-the-day bias. Those who are responsible for the treatment of upper-extremity dysfunction and use splinting as a treatment modality must have a thorough working understanding of the engineering concepts involved. Splinting is both science and art but the ultimate criterium is does it work mechanically? If a splint does not work mechanically, then there is no reason for its application. The insight provided by understanding engineering concepts opens new horizons in patient treatment for those who take the time to learn. PMID- 7550621 TI - Application of biomechanics to tendon transfers. AB - This article has focused on considerations important in the application of biomechanics to tendon transfers and has used an example protocol. Different surgeries require different protocols. What is most important is that specific protocols are used, and that they are both safe and effective. The communication among the therapist, surgeon, and patient is essential with the use of any protocol. As Brand has stated, "A hand is a very personal thing. It is the interface between the patient and his or her world. It is an emblem of strength, beauty, skill, sexuality, and sensibility. When it is damaged it becomes a symbol of the vulnerability of the whole person." For the patient who has damage from nerve palsy, paralysis, or injury resulting in a dysfunctional hand, a tendon transfer procedure may prove to be a viable option to restore balance and function, especially if the biomechanics of deformity and correction are considered. PMID- 7550623 TI - Biomechanics of soft-tissue growth and remodeling with plaster casting. PMID- 7550624 TI - Objective and subjective observations of low-temperature thermoplastic materials. AB - There is much more to a therapist's selection of materials for splints than the qualities of conformability and rigidity, although these are important in a busy clinic. As new materials become available, they may replace existing materials in popularity as more therapists become open to trying them. PMID- 7550625 TI - Guidelines for evaluating assessment instruments. PMID- 7550627 TI - Threshold detection and Semmes-Weinstein monofilaments. AB - Semmes-Weinstein monofilaments provide a repeatable instrument stimulus with a small standard deviation in contrast to other handheld test instruments, making them an optimum choice for objective sensory testing in a variety of clinics. Normal sensory detection thresholds for the entire body, and the stimulus force for each filament, were determined by Weinstein. He found a nylon filament of 0.005 in wide and 38 mm long (mean force, 68 mg) to be a good predictor of "normal" light touch-deep pressure threshold for the hands and most of the body. However, manufacturers of the nylon used in making the filaments allow an 8-10% tolerance in diameter. This small change in diameter can result in small variations in mean force among filaments of a given size. It has not been previously determined what effect this small variance in force has on the accuracy of the 2.83 (marking number) 0.005-in wide filament most often used for normal threshold detection. This study compared the 2.83 filaments available at the Gillis W. Long Hansen's Disease Center, which have a mean force of 62 mg, with those from North Coast Medical, Inc., which have a mean force of 95 mg. The filaments were used by 6 examiners in a standard testing protocol for the hands, arms, faces, legs, and feet of 130 subjects. Heavier and lighter filaments of measured force were also included. Results showed a high correlation in responses for two values for the 2.83 filaments in the range specified. On detailed analysis between kits there were some differences for site and age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550628 TI - Objective measures of joint stiffness. AB - Objective measures of joint stiffness allow for the evaluation of the effectiveness of treatment modalities. Without this, the effectiveness of therapy is not quantifiable. Presently, joint stiffness can be quantified by either passive range of motion (PROM) measurement or torque range of motion (TqROM) measurement. PROM measurement does not control the force applied, nor does it require that the other joints in the kinematic chain be held fixed. Also, it demonstrates poor interrater reliability. An idealized device melding existing technologies of constant passive motion devices and computerized workstations is proposed to allow for easier measurement of TqROM angles for analysis data for the determination of the effectiveness of treatment modalities. PMID- 7550626 TI - Clinical assessment, objectivity, and the ubiquitous laws of instrumentation. AB - With the Carville approach to interdisciplinary rehabilitation, special emphasis is placed on improved objective measurement of human function. This article defines essential elements for objective measurement, based on the experiences of a biomedical engineer on the rehabilitation team. Applied to manmade system, these elements are straightforward. Applied to living systems, these elements present difficulties, and special consideration must be given to the methods of measurement applied in the clinical setting. The rules for successful instrumentation and measurement are discussed with respect to the hand therapist's continuing challenge to measure the threshold to tactile sensation. PMID- 7550630 TI - Biomechanics: the forces of change and the basis for all that we do. PMID- 7550629 TI - Computer simulation of the upper extremities. AB - The development of real-time, interactive, three-dimensional, computer graphic simulation of the musculoskeletal system with emphasis on the upper extremities is described. Developments in image analysis, scientific visualization, and interactive computer methods and the continuous improvement in knowledge of musculoskeletal function have combined to provide an exciting new tool for musculoskeletal research. Interactive simulation also has promising applications in medical education and clinical rehabilitation. Only those developments that fit the specific criteria of realism, real-time response, intuitive interaction, three-dimensional structures, and modeling flexibility are discussed. It is hoped that an improved understanding of this emerging tool and an appreciation for its potential applications will be gained. PMID- 7550632 TI - Basic biomechanics. PMID- 7550631 TI - A walk through the anatomy of the hand and forearm. PMID- 7550634 TI - Mechanical factors in joint stiffness and tissue growth. PMID- 7550633 TI - Dynamic properties of soft tissues and their interface with materials. AB - Treatment for the human hand involves application of forces and torques to the skeletal system. These are applied through the soft tissues, frequently using protective materials such as splints and other static and dynamic therapeutic orthoses. A view of soft tissues as polymeric materials provides a more universal approach. The role of shear in soft tissues is discussed, as well as the response of the tissues of the hand to the mechanical challenges of treatment. A connection between the underlying concepts presented is provided and how this influences clinical treatment is discussed. PMID- 7550636 TI - Alternative method of assessing toxicity. PMID- 7550635 TI - Biomechanics of the wrist. AB - This article summarizes six years of studies with an experimental model, which uses a static positioning frame, pressure-sensitive film, and a microcomputer based videodigitizing system, to characterize the biomechanics of the human cadaver wrist by measuring the contact areas and pressures in a variety of normal, simulated traumatic, and surgically treated conditions. The results of these studies have provided clinically relevant information about the normal anatomy and functional mechanics of the wrist, as well as guidelines for the treatment for a number of different fractures and ligament injuries. The results of studies of radial carpal instabilities and fractures have demonstrated an increased load in the areas where degenerative arthritis is seen in these clinical conditions, and have furnished a means by which the biomechanical efficacy of certain surgical treatments can be measured. PMID- 7550637 TI - The FRAME cytotoxicity test. PMID- 7550638 TI - LLC-RK1 cell screening test for nephrotoxicity. PMID- 7550639 TI - Allium test. PMID- 7550640 TI - Measurement of cell membrane toxicity by means of 2-deoxy-D-glucose. PMID- 7550641 TI - MTT assays. PMID- 7550642 TI - V79 cytotoxicity test for membrane damage. PMID- 7550643 TI - SIRC cytotoxicity test. PMID- 7550644 TI - Rabbit articular chondrocyte functional toxicity test. PMID- 7550645 TI - Preparation and use of cultured astrocytes for assay of gliotoxicity. PMID- 7550646 TI - Balb/c 3T3 cytotoxicity test. PMID- 7550647 TI - The pollen tube growth test. PMID- 7550648 TI - HET-CAM test. PMID- 7550649 TI - The use of the bovine isolated cornea as a possible in vitro test for ocular irritancy. PMID- 7550650 TI - Cytotoxicity in an anchorage-independent fibroblast cell line measured by a combination of fluorescent dyes. PMID- 7550652 TI - The murine local lymph node assay. PMID- 7550651 TI - Cell culture phototoxicity test. PMID- 7550653 TI - Polymorphonuclear leukocyte locomotion. PMID- 7550654 TI - Immune function assays. PMID- 7550655 TI - Human thyroid culture. PMID- 7550656 TI - The DNA alkaline unwinding genotoxicity test. PMID- 7550658 TI - Gene mutation assays in mammalian cells. PMID- 7550657 TI - Measurement of unscheduled DNA synthesis in vitro using primary rat hepatocyte cultures. PMID- 7550659 TI - Measurement of chromosome aberrations in vitro using human peripheral blood lymphocytes. PMID- 7550660 TI - Mutation assays in bacteria. PMID- 7550661 TI - Chick embryotoxicity screening test (CHEST I and II). PMID- 7550662 TI - Frog embryo teratogenesis assay. Xenopus (FETAX). PMID- 7550664 TI - The Hydra attenuata assay. PMID- 7550663 TI - The Drosophila melanogaster assay. PMID- 7550665 TI - Thyroid follicular cells in monolayer culture. In vitro models for thyroid toxicity testing. PMID- 7550666 TI - Dust toxicity in rat alveolar macrophage cultures. PMID- 7550667 TI - Hepatoma cell cultures as in vitro models for hepatotoxicity. PMID- 7550668 TI - Cytotoxicity, DNA fragmentation, and DNA repair synthesis in primary human hepatocytes. PMID- 7550669 TI - Model cavity method. INVITTOX. PMID- 7550670 TI - Human esophageal culture. PMID- 7550671 TI - The application of in vitro models of anterior pituitary function in toxicity testing. PMID- 7550672 TI - The application of in vitro models of hypothalamic function in toxicity testing. PMID- 7550673 TI - Methods of immunization to enhance the immune response to specific antigens in vivo in preparation for fusions yielding monoclonal antibodies. PMID- 7550674 TI - Use of monoclonal antibodies in immunoelectron microscopy. PMID- 7550675 TI - Use of monoclonal antibodies for western blotting with enhanced chemiluminescent detection. PMID- 7550676 TI - The use of directly and indirectly labeled monoclonal antibodies in flow cytometry. PMID- 7550677 TI - Use of monoclonal antibodies and flow cytometry to cluster and analyze leukocyte differentiation molecules. PMID- 7550679 TI - Culture conditions that optimize outgrowth of hybridomas. PMID- 7550678 TI - In vivo and in vitro production of monoclonal antibodies. Bioreactors vs immune ascites. PMID- 7550680 TI - Separation of monoclonal antibodies from cell-culture supernatants and ascites fluid using thiophilic agarose. PMID- 7550681 TI - Detection, purification, and utilization of murine monoclonal IgM antibodies. PMID- 7550682 TI - Preparation and use of immunoaffinity columns with monoclonal antibodies without purification from ascites and tissue-culture medium. PMID- 7550683 TI - Antigen purification by monoclonal antibody immunoaffinity chromatography. PMID- 7550685 TI - Coupling of monoclonal antibodies with biotin. PMID- 7550684 TI - Coupling of monoclonal antibodies with fluorophores. PMID- 7550687 TI - Use of monoclonal antibodies with magnetic particles to separate cell subpopulations by negative selection. PMID- 7550688 TI - Use of monoclonal antibodies with magnetic particles to separate cell subpopulations by positive selection. PMID- 7550690 TI - Production of stable bovine-murine interspecies hybrids. PMID- 7550689 TI - Production of stable heterohybridomas producing human monoclonal antibodies. PMID- 7550686 TI - Coupling of monoclonal antibodies with enzymes. PMID- 7550692 TI - Production of monoclonal antibodies in horses. PMID- 7550691 TI - Production of monoclonal antibodies in swine. PMID- 7550693 TI - Use of electric-field-mediated cell fusion to produce hybridomas secreting monoclonal antibodies. PMID- 7550695 TI - Methods of measuring antibodies in sera/plasma, ascites, and tissue-culture media. PMID- 7550694 TI - Rapid isotyping of mouse monoclonal antibodies. PMID- 7550697 TI - Use of monoclonal antibodies in immunohistochemistry. PMID- 7550696 TI - Measurement of immunoglobulin synthesis using the ELISPOT assay. PMID- 7550699 TI - Antibiotic resistance profiling. PMID- 7550701 TI - The direct epifluorescent filter technique (DEFT). PMID- 7550702 TI - Use of commercially available ELISA kits for detection of foodborne pathogens. PMID- 7550700 TI - The direct immunofluorescent filter technique (DIFT). PMID- 7550698 TI - Methods of immunization to enhance the immune response to specific antigens in vitro. PMID- 7550703 TI - Detection of foodborne pathogens using DNA probes and a dipstick format. PMID- 7550704 TI - Preparation of bacterial genomic DNA. PMID- 7550705 TI - Pulsed field gel electrophoresis. PMID- 7550706 TI - Bacteriophage typing. PMID- 7550707 TI - Application of total DNA restriction pattern analysis to identification and differentiation of bacterial strains. PMID- 7550708 TI - Determination of rRNA gene restriction patterns. PMID- 7550709 TI - Preparation of nonradioactive DNA probes. PMID- 7550710 TI - Preparation of bacterial plasmid DNA. PMID- 7550711 TI - Plasmid profile typing and plasmid fingerprinting. PMID- 7550712 TI - Polymerase chain reaction for the detection of Listeria species and Listeria monocytogenes. PMID- 7550713 TI - Development of bacterial species-specific DNA probes based on ribosomal RNA genes using PCR. PMID- 7550714 TI - Identification of microorganisms using random primed PCR. PMID- 7550715 TI - Ligase chain reaction. PMID- 7550716 TI - The analysis of bacterial proteins by SDS polyacrylamide gel electrophoresis. PMID- 7550718 TI - Analysis of bacterial outer membrane proteins. PMID- 7550717 TI - Lipopolysaccharide chemotyping. PMID- 7550719 TI - Bacteriocin typing. PMID- 7550722 TI - Pyrolysis mass spectrometry. PMID- 7550721 TI - Fast atom bombardment-mass spectrometry. PMID- 7550720 TI - Multilocus enzyme electrophoresis. PMID- 7550723 TI - Electroporation theory. Concepts and mechanisms. AB - The basic features of electrical and mechanical behavior of electroporated cell membranes are reasonably well established experimentally. Overall, the electrical and mechanical features of electroporation are consistent with a transient aqueous pore hypothesis, and several features, such as membrane rupture and reversible electrical breakdown, are reasonably well described quantitatively. This gives confidence that "electroporation" is an attractive hypothesis, and that the appearance of temporary pores owing to the simultaneous contributions of thermal fluctuations ("kT energy") and an elevated transmembrane voltage ("electric field energy") is the microscopic basis of electroporation. PMID- 7550724 TI - Escherichia coli electrotransformation. PMID- 7550725 TI - Electrotransformation in Salmonella. PMID- 7550726 TI - Electrotransformation of Pseudomonas. PMID- 7550727 TI - Electroporation of Xanthomonas. PMID- 7550728 TI - Transformation of Brucella species with suicide and broad host-range plasmids. PMID- 7550729 TI - Electroporation of Francisella tularensis. PMID- 7550730 TI - A simple and rapid method for transformation of Vibrio species by electroporation. PMID- 7550732 TI - Electrotransformation of Agrobacterium. PMID- 7550731 TI - Genetic transformation of Bacteroides spp. using electroporation. PMID- 7550733 TI - Electroporation of Helicobacter pylori. PMID- 7550735 TI - Transformation of Lactococcus by electroporation. PMID- 7550734 TI - Electrotransformation of Streptococci. PMID- 7550736 TI - Transformation of Lactobacillus by electroporation. PMID- 7550737 TI - Electrotransformation of Staphylococci. PMID- 7550738 TI - Electroporation and efficient transformation of Enterococcus faecalis grown in high concentrations of glycine. PMID- 7550739 TI - Introduction of recombinant DNA into Clostridium spp. PMID- 7550740 TI - Electroporation of mycobacteria. PMID- 7550741 TI - Electrotransformation of the spirochete Borrelia burgdorferi. PMID- 7550742 TI - Yeast transformation and the preparation of frozen spheroplasts for electroporation. PMID- 7550744 TI - Instrumentation. PMID- 7550743 TI - Ten-minute electrotransformation of Saccharomyces cerevisiae. PMID- 7550745 TI - Electroporation of Schizosaccharomyces pombe. PMID- 7550746 TI - Gene transfer by electroporation of filamentous fungi. PMID- 7550747 TI - Transformation of Candida maltosa by electroporation. PMID- 7550749 TI - Electroporation of Dictyostelium discoideum. PMID- 7550748 TI - Electroporation of Physarum polycephalum. PMID- 7550751 TI - Transfection of the African and American trypanosomes. PMID- 7550752 TI - Electroporation in Giardia lamblia. PMID- 7550750 TI - Gene transfer by electroporation in Tetrahymena. PMID- 7550753 TI - Direct plasmid transfer between bacterial species and electrocuring. PMID- 7550754 TI - Transfer of episomal and integrated plasmids from Saccharomyces cerevisiae to Escherichia coli by electroporation. PMID- 7550756 TI - Electroporation of RNA into Saccharomyces cerevisiae. PMID- 7550755 TI - Production of cDNA libraries by electroporation. PMID- 7550757 TI - Electrofusion of yeast protoplasts. PMID- 7550758 TI - Environmental factors and primary T-cell sensitisation to inhalant allergens in infancy: reappraisal of the role of infections and air pollution. PMID- 7550759 TI - Ontogeny of IL4 production. AB - There is evidence to suggest that the production of some cytokines in childhood is different to that in adults. The production of IL4 in PHA-stimulated PBMC cultures was examined in healthy neonates, children and adults to determine the ontogeny of IL4 production throughout childhood. In vitro IL4 production was found to be significantly reduced in neonates and children under 10 years of age as compared to adults, and to increase progressively with age. The mechanisms leading to reduced IL4 production in neonates were shown to be different to those in children, with a defect in signal transduction demonstrated for lymphocytes from neonates but not children < 10 years. The presence of an inhibitory factor in cord blood plasma was also noted. These age-dependent variations in IL4 production and response to stimulation with PMA/Ca may reflect differences in naive and memory T cell populations. PMID- 7550760 TI - Ontogeny of human mannan-binding protein, a lectin of the innate immune system. AB - Mannan-binding protein (MBP) is a member of the collectins. These lectins are composed of polypeptide chains which contains a collagen-like region and a calcium dependent carbohydrate recognition domain (named a C-type CRD). MBP binds via the CRD to carbohydrate structures on microorganisms. MBP can activate the complement system when bound to carbohydrate. MBP is thus thought to play a role in the defence against microorganisms. The present report describes the ontogeny of MBP. The level of MBP increase during the first 3 months of life, at which time a stable level is reached, comparable to that seen in adults. On average, the level of MBP at term was 37% of the post 3-month level. Measurements on infants born prematurely showed a 3-fold increase in MBP concentration from the time of birth at around 30 weeks from conception to the 10th week of life. PMID- 7550761 TI - Age patterns of immunoglobulins G, A & M in healthy children and the influence of breast feeding and vaccination status. AB - Serum levels of immunoglobulins G, A and M were determined by nephelometry in 414 infants and children aged 1 month to 14 years. The children were admitted to "Aghia Sophia" Teaching Hospital for Children for surgical corrections of minor anatomical abnormalities, but they were otherwise healthy. Statistical analysis was performed through multiple regression after logarithmic transformation of the immunoglobulin values. Immunoglobulin G (IgG) levels increased significantly by 18% per year until the age of 5 years and by 2% per year thereafter. Among children less than 5 years old, IgG levels tended to increase significantly with the number of doses of either DTP or Sabin vaccine. Immunoglobulin A (IgA) levels increased significantly by 27% until the age of 7 years and by 4% thereafter. Among children less than 7 years old, history of breast feeding was significantly associated with higher levels of IgA. Vaccination with either DTP or Sabin was associated with elevation of IgA levels among younger and, to a lesser extent, older children, but the elevations did not reach statistical significance. Immunoglobulin M (IgM) levels increased rapidly during the first 12 months of life and very slowly thereafter and they were significantly higher among girls. Among children less than one year old, there was evidence that vaccination with either DTP or Sabin was associated with elevated IgM levels, although the differences were not statistically significant possibly because of small numbers of infants in the study sample.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550762 TI - Mucosal IgA, mucosal cow's milk antibodies, serum cow's milk antibodies and gastrointestinal permeability in infants. AB - We have studied development of the levels of IgA cow's milk (CM) antibodies in the saliva, faeces and serum of 20 term and 20 preterm infants from birth to 8 months. All infants already had IgA in their saliva during the first week of life. The levels peaked at the age of one month, thereafter decreasing in both groups; from the age of three months levels remained stable. Term infants had higher levels than preterm infants, but no differences were found between breastfed and CM-fed infants. Breast-fed infants had higher levels of IgA in their faeces than did CM-fed infants; the IgA levels were similar in breast-fed term and preterm infants, being highest at birth, and decreasing thereafter. We also showed rising titers of serum IgA CM antibodies, with higher levels in infants regularly exposed to CM than in breast-fed infants. We sought associations between the magnitude of intestinal permeability to human alpha lactalbumin (ALA) measured at the ages of 4-7 days and one month and the levels of IgG antibodies to CM, but no such relation was found. PMID- 7550763 TI - Latex allergy in children with no known risk factor for latex sensitization. AB - We describe latex allergy in 11 atopic children, aged 0.7-11.1 years, without any known risk factor. A skin prick-test (SPT) for latex was positive in 8/11, and latex specific IgE was found in all. Latex glove challenge was positive in 9 assessed. These patients demonstrate that latex allergy should be looked for not only in children who have had several operations or those children reporting symptoms from rubber, but also in children with severe atopic eczema, banana allergy, or urticaria or anaphylaxis for which the cause is unknown. PMID- 7550764 TI - Frequency of food allergy in a pediatric population from Spain. AB - We evaluated the prevalence and characteristics of the principal foods implicated in 355 children diagnosed with IgE-mediated food allergy. Diagnosis was established on the basis of positive clinical history for the offending food, positive specific IgE by skin prick test and RAST, and open food challenge. Our results showed the principal foods involved in allergic reactions are: eggs, fish, and cow's milk. These are followed in frequency by fruits (peaches, hazelnuts and walnuts), legumes (lentils, peanuts and chick peas) and other vegetables (mainly sunflower seeds). The legumes demonstrated the highest degree of clinical cross-reactivity. Most patients with food allergy reacted to one or two foods (86.7%). Only 13.3% of patients reacted to 3 or more foods, mostly to legumes and fruits. We found that food allergy begins most frequently in the first (48.8%) and second (20.4%) years of life. Allergy to proteins of cow's milk, egg, and fish begins predominantly before the second year, demonstrating a clear relationship with the introduction of these foods into the child's diet. Allergy to foods of vegetable origin (fruits, legumes and other vegetables) begins predominantly after the second year. PMID- 7550765 TI - Systemic reactions to specific immunotherapy in children with respiratory allergy: a prospective study. AB - The aim of this multi-centre prospective study was to evaluate the prevalence of systemic reactions to specific immunotherapy in children with allergic asthma and or rhinitis. One thousand and fifty-six children (653 boys and 403 girls), median age 8.5 years, were enrolled in this three-year prospective study. All the children were treated with injections of the following allergenic extracts: 689 of house dust mite, 291 of grass, 109 of Parietaria, 13 of Alternaria, 6 of Artemisia and 11 of Olea. Among 1056 treated children, 41 (3.7%) had systemic reactions: 40 children (3.7%) experienced mild symptoms such as asthma and/or urticaria, and only one shock (0.08%). A total of 47,247 injections were administered, and the rate of systemic reactions, according to the number of total injections was only 0.08%. According to the allergenic extract, systemic reactions occurred in 29/689 children (4.2%) treated with house dust mites extract (0.09% of the injections), in 9/291 children (3.1%) treated with grass extract (0.07% of the injections) and in 3/109 children (2.8%) treated with Parietaria extract (0.06% of the injections). The prevalence of systemic reactions was significantly higher (p < 0.0001) in the children treated with house dust mite extract in comparison with those treated with pollen extracts. All the systemic reactions appeared within 30 minutes following the administration of the extract and occurred in 37/41 cases (90.2%) with the same dose, previously tolerated. Most reactions were mild, and were readily controlled by immediate emergency treatment. There was no need for hospitalization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550766 TI - Changes in the concentration and distribution of immunoglobulin-producing cells in SIDS palatine tonsils. AB - Seventeen sudden infant death syndrome (SIDS) cases and 9 controls, were examined immunohistochemically with regard to the presence of IgA-, IgM-, IgD, and IgG, as well as for the subtypes IgG1-, IgG2-, IgG3-, and IgG4-immunocytes. Differences in compartmentalization were also investigated. Differences were demonstrated between SIDS and controls in total number of IgG cells per 0.1 mm2 tissue area (median: 18.3, range: 12.3-30.2 versus median: 6.3, range: 2.0-14.6) (p < 0.01), and for IgA immunocytes (median: 3.9, range: 2.4-5.0 versus median: 1.5, range: 1.1-3.7) (p < 0.05), while no differences were demonstrated for IgM cells (median: 1.8, range: 1.2-3.3 versus median: 1.8, range: 0.7-5.6) or IgD cells (median: 1.9, range: 0.8-2.9 versus median: 1.6, range: 0.7-2.4). Differences were demonstrated between SIDS and control IgG plasma cells in all the four palatine tonsillar compartments; germinal centre (p < 0.01), mantle zone (p < 0.05), interfollicular area (p < 0.01) and reticular epithelium (p < 0.01). Furthermore, the number of IgA cells was higher in SIDS vs. controls in both the germinal centre (median: 1.4, range: 0.6-2.1 versus median: 0.6, range: 0.3-1.3) (p < 0.05) and in the interfollicular area (median: 2.2, range: 1.1-3.1 versus median: 0.5, range: 0.4-2.0) (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7550767 TI - Infant mortality time series are random walks with drift: are they cointegrated with socioeconomic variables? AB - Previous time series analyses of infant mortality have failed to provide evidence to support their implicit assumption that infant mortality data used behaved as a stationary time series. The present study applies the augmented Dickey Fuller Test to infant mortality time series for Sweden (1800-1989), United Kingdom (1839 1989) and United States (1915-1989). The null hypothesis that each of these series is non-stationary is accepted at standard levels of significance. A conceptual framework of infant mortality which uses a combination of physical and social overhead capital as factors in a production function is developed to explain the finding of non-stationarity as derivative from the non-stationarity of a stock of health-enhancing capital. Estimation of econometric models of the socioeconomic determinants of infant mortality using differenced data with ARIMA estimation is inconclusive. Estimation of a bivariate cointegration model supports the hypothesis that infant survival and GNP/Capita are cointegrated for 19th century Sweden but not for 19th century UK. Bivariate analysis of 20th century Sweden, UK, and US data demonstrated no cointegration. This may be due to the onset of disequilibrium in the economic determination of infant mortality in the present era as technological advances and demographic shifts began to play a larger role. Supplementing the bivariate analysis with measures of unemployment, and crude birth rate in the 20th century permitted the detection of cointegration in US and UK. The multivariate results may suggest that improvements in 20th century UK GNP/capita have had greater impact on infant survival relative to US GNP/capita. PMID- 7550768 TI - Health, health care, and the environment. Econometric evidence from German micro data. AB - The paper develops and applies a Grossman-style health production model set up in discrete time to explain the impact of environmental pollution on the demand for both health and health care. In order to introduce the environment, our analysis takes changes in environmental conditions to influence the rate at which an individual's stock of health depreciates. While the theoretical part of our paper also contains a discussion of the full model, we restrict our empirical analysis to a submodel which is known as the pure investment model. This is because the other submodel, the pure consumption model, implies a rather implausible case of satiation with respect to the individual's preferences. Our empirical findings are based on data taken from the German Socio-economic Panel. The stock of health capital and environmental pollution are treated as latent variables and estimated using a Linear Covariance Structures model. The quality of the environment turns out to be an important determinant of health capital. From the point of view of health economics, improvements in environmental conditions can be interpreted as preventive measures. In terms of prevention, public policies designed to protect the environment also yield significant health effects. As regards health care demand the influence is not clearcut, i.e., one cannot necessarily expect a reduction in resource use. PMID- 7550769 TI - A comparison of alternative models of prescription drug utilization. AB - The two-part estimation technique has been advocated for estimating models using individual level health care utilization data characterised by a large proportion of non-consumption and small proportions of heavy users. This paper compares the two-part model to several other estimators, including the Poisson, negative binomial and 'zero altered' negative binomial models on the basis of within sample forecasting accuracy and non-nested model selection tests. The empirical model estimates the differential effect of the removal of copayments for prescription medicines on the prescription drug utilization by older adults with differing levels of health status. The two-part estimator of this model is found to dominate the competitors. Results from this model indicate that utilization increases appear to be higher among individuals with lower levels of health status. PMID- 7550770 TI - Determinants of expenditure variation in health care and care of the elderly among Finnish municipalities. AB - In Finland, municipal health care expenditure varies from FIM 3 800 per capita to FIM 7 800 per capita. The objective of this study was to estimate the impact of different economic, structural and demographic factors on the per capita costs of health services and care of the elderly. Using regression analysis we attempted to explain observed differences in expenditure by determining separately the effects of allocative and productive inefficiency and the effects of factors influencing the demand for services. We found income level of local population, generosity of central government matching grant, allocative efficiency (the mix of care between institutional and non-institutional care), productive efficiency of service providers, and factors associated with the need of services (age structure, morbidity) to be the most important determinants of health care expenditure. Our results reveal that municipalities have the means at their disposal (by shifting resources to outpatient care and increasing productivity) to significantly reduce expenditure on health services and care of the elderly. PMID- 7550771 TI - Patient charges and the utilisation of NHS prescription medicines: some estimates using a cointegration procedure. AB - Since the inception of the NHS, user charges have been present for prescription medication. However since 1968 there has been a steady increase in this charge, particularly notable during the 1980s. The main justification for user charges is their revenue raising potential, and a recent government report has backed the use of user charges for prescription drugs. Whilst there is extensive evidence of the impact of user charges on utilisation of health care in the US, few studies exist in the UK. An accurate estimation of the price elasticity of utilisation is necessary if the full consequences of user charges are to be examined. This paper uses a cointegration estimation technique to estimate the price elasticity for prescription drug utilisation in the UK. PMID- 7550772 TI - Subjective health measures and state dependent reporting errors. AB - The use of subjective health measures in empirical models of labour supply and retirement decisions has frequently been criticized. Responses to questions concerning health may be biased due to financial incentives and the willingness to conform to social rules. The eligibility conditions for some social security allowances, notably Disability Insurance benefits, are contingent upon bad health. Even if the decision to apply for a disability allowance is to some extent motivated by financial considerations or a relatively strong preference for leisure, respondents will be inclined to play down these motives and emphasize the importance of their health condition. As a consequence, reporting errors may depend on the labour market status of the respondent and self-reported health variables will be endogenous in labour supply and retirement models. The objective of this paper is to assess the importance of state dependent reporting errors in survey responses and to propose and estimate a model that can be used to account for this kind of systematic mis-reporting. The estimation results indicate that among respondents receiving Disability Allowance, reporting errors are large and systematic. Using such subjective health measures in retirement models may therefore seriously bias the parameter estimates and the conclusions drawn from these. PMID- 7550774 TI - Scientific truths: weighing the evidence. PMID- 7550773 TI - A grouped data regression approach to estimating economic and social influences on individual drinking behaviour. AB - General Household Survey (GHS) data sets, covering the period 1978-1990, are pooled to investigate the relationship between the riskiness of individuals' self reported drinking behaviour and a wide range of personal characteristics and economic factors. A grouped data regression approach is used to reduce problems with the inaccuracy of self-reports of alcohol consumption and clustering of observations in the consumption data. Results for males aged 18 to 24 years are presented, and possible methods for interpreting the results of grouped data regression are illustrated. Controlling for other factors, current smokers are estimated to be at a 75% higher risk of drinking over recommended levels than non smokers. Particular attention is paid to the interactions between the price of alcohol, income and heavy drinking. At average levels of income, a 5% increase in the real price of alcohol is predicted to reduce the probability of 'at-risk' drinking by 1.5%. At lower initial levels of income, drinking patterns are found to be more responsive to both price and income changes. Grouped data regression is proposed as a way of focusing policy analysis on individual risks of alcohol related health and social problems. PMID- 7550775 TI - Wound C.A.R.E.: an acronym for action. PMID- 7550776 TI - Pressure ulcer pain: assessment and quantification. AB - A cross-sectional study design was used to document perception of pressure ulcer pain in 132 patients in an acute care setting. Subjects were evaluated by means of the Folstein Mini-Mental State Examination, Beck's Depression Inventory, the Faces Pain Rating Scale, and the Visual Analog Scale for pain intensity. Charts were reviewed for demographic data and related medical treatments. The group comprised 44 subjects (33.3%) who were able to respond to the evaluation instruments and 88 subjects (66.7%) who were unable to respond to the evaluation tools. Forty-one percent of the respondents denied pressure ulcer pain and 59% reported pain of some type. According to the Faces Rating Scale, 32% of this group reported no pain and 68% reported some degree of pain. The respondents included 48% who scored below 24 on the Folstein Mini-Mental State Examination, indicative of cognitive impairment, and 52% who were found to be cognitively intact, with scores of 24 or above. Only 2% (n = 3) were given analgesics for pressure ulcer pain within 4 hours of the interview. PMID- 7550777 TI - Necrotizing fasciitis: early recognition and aggressive treatment remain important. AB - Necrotizing fasciitis is an infection caused by aerobic or anaerobic microorganisms that rapidly spread along the fascial plane, causing necrosis of the fascia, overlying skin, and vasculature. Septicemia and systemic toxic effects may lead to death within as short a time as 24 to 96 hours. Successful treatment, comprising radical surgical debridement, relies on early recognition. Despite the importance of early recognition, however, a review of nursing literature during the last 10 years revealed only one article dealing specifically with this condition. The purpose of this article is to review the causes and treatment of cellulitis complicated by necrotizing fasciitis. PMID- 7550778 TI - Kaposi's sarcoma with elephantiasis associated with AIDS. AB - Nurses in many parts of the United States have limited experience in caring for patients with Kaposi's sarcoma. The severity of the disease can range from a few lesions to extensive cases with fungating, encrusted wounds. This case study describes the wound management of a 43-year-old patient with an advanced case of Kaposi's sarcoma with elephantiasis of the lower extremities. Use of a collaborative care approach resulted in dramatic improvement of this patient's wounds. Although the patient's serum albumin level remained low, no secondary skin breakdown or infection occurred. PMID- 7550779 TI - Multidisciplinary management of altered body image in the patient with an ostomy. AB - Many clinical studies have found patients with ostomies to be a group facing multiple adjustment demands. One of these demands is coping with a significant change in body image. At the Medical College of Wisconsin, a team approach has been initiated; the ET nurse, the psychologist, and the surgeon deal with body image concerns together. Problems requiring counseling have included difficulty with personal acceptance, personal and social body-image disruption, sexual concerns, reduced self-care skills, and the management of surgical complications. This article represents a study employing a methology of selected case presentations. Cases were chosen to outline the types of problems encountered and were selected from referrals made for psychologic intervention by the surgeon and ET nurse. The patients included four women and three men, ranging in age from 22 to 79 years. Data were compiled by examining the records of the surgeon, ET nurse, and psychologist. The primary needs revolved around personal or social acceptance of altered body image. By addressing these needs in a straightforward, time-limited manner, postsurgical counseling was delivered effectively for these patients. In conclusion, we have demonstrated the multidisciplinary approach to be successful in facilitating adaptation to an altered body image. PMID- 7550780 TI - Caring for the patient with peristomal pyoderma gangrenosum. AB - Pyoderma gangrenosum is an ulcerating skin condition associated with inflammatory bowel disease and other diseases. During and 18-month period beginning in the fall of 1991, seven patients were followed up for pyoderma gangrenosum. Three of these cases, with assessment and treatment plans, are presented. A discussion of treatment principles for managing pyoderma gangrenosum follows. PMID- 7550781 TI - Problem solving and troubleshooting: the indwelling catheter. AB - Long-term use of an indwelling catheter is seldom free of problems, and the complications associated with indwelling catheters cause significant morbidity and mortality. Often there are alternatives to long-term catheterization; it is assumed in this article that the patient will have been appropriately assessed for other methods of continence control. In this article, leakage around the catheter (bypassing) is discussed. This problem is multifactorial: irritation caused by the catheter balloon, improper sizing of catheter, confusion of the patient, bacteriuria, constipation or fecal impaction, blocked catheters, problems related to materials used in catheter construction, and improper positioning of the catheter are all potential problems contributing to leakage in the patient with an indwelling catheter. Emphasis is placed on etiology because understanding the underlying problem is crucial to implementing effective treatment. PMID- 7550782 TI - Patient with Roux-en-Y jejunostomy for enteral feedings. PMID- 7550783 TI - Continence management and the ET nurse: how far should we go? PMID- 7550784 TI - How a three-campus heart service line improves clinical processes and outcomes. AB - BACKGROUND: In 1993, Intermountain Health Care's three Salt Lake Valley Hospitals formed service lines in four clinical areas, one of which was heart services. After experimenting with various organizational structures, the Salt Lake Valley Hospitals formed a cardiac executive council and three specialty work teams--the clinical process and outcome, satisfaction, and resource teams--to allow for unified planning and greater teamwork. CASE STUDY--OPEN HEART TEAM: The team mapped out the current process and identified areas for potential improvement in the care of patients undergoing coronary artery bypass graft (CABG) surgery. One of the key processes selected for study was extubation. Patients were extubated for an average of 20.41 hours (range, 6 to 120 hours). Analysis of practice patterns demonstrated that extubation was related to staffing patterns, not the patient's readiness. The team created a weaning path, which reduced extubation time to an average of 8.89 hours. LESSONS LEARNED: A common vision and an organized structure to support integrated services is essential. Cross-training of staff helps ensure that the same standards of care apply across the three campuses. Even when the medical staff and hospital departments each have their own structures for dealing with quality issues, cohesiveness among physicians treating a certain group of patients, such as cardiac patients, can be promoted. In conclusion, a "cardiac culture" that is evident throughout the three hospitals promotes performance improvement. PMID- 7550785 TI - Reporting on QI efforts for internal and external customers. AB - BACKGROUND: In April 1993 the Methodist Hospital of St Louis Park, Minnesota, released its first internal quality report on outcomes and quality improvement (QI) initiatives. When a local television news reporter mentioned the report in a segment on health care quality, public interest led the hospital to launch an annual series of external quality reports in addition to its internal quality reports. When the eight-page external report was first released in 1994, consumer response was weak, but the report generated a strong response from the mass media, trade publications, the business community, and other health care organizations nationwide. DATA COLLECTION AND USE: Data on sentinel events and outcomes analysis of a variety of clinical and administrative functions have assisted in identifying opportunities for improvement. For example, the hospital monitors the five-year survival rate for patients with myocardial infarction. With the adoption of treatment with streptokinase, data indicated frequent hypotension. Increase of infusion from 30 to 60 minutes led to a decrease in hypotension. THE REPORTS: The external report included, in shorter and simpler form, almost all the sections in the internal report, such as QI activities (teams, training, critical paths), clinical outcome measures, community health, patient satisfaction, value, and accreditation. The indicators included in the external report were selected to minimize potential misinterpretation by public audiences. CONCLUSIONS: By increasing the visibility of QI within the hospital, the internal quality reports have helped generate further QI activity, and the external report augmented further positive publicity among the local health care press. The reports are proven effective tools for communicating the hospital's ability to sustain and improve the quality of its services over time. PMID- 7550786 TI - Quality with a human face? The Samuels Planetree model hospital unit. AB - BACKGROUND: Hospitals across the country are seeking to restructure the delivery of care. Planetree, an international consumer health care organization, works with hospitals to cultivate educated health care consumers and to create caring inpatient environments. This article gives an overview of Planetree's philosophy, examines staff and patient satisfaction, and contrasts Planetree with total quality management (TQM)/continuous quality improvement (CQI) as practiced at one hospital. PLANETREE'S PHILOSOPHY: Planetree's model differs from other patient centered or patient-focused care models because of its emphasis on educating patients and making them active partners in the care process. To help make the hospital environment less forbidding, more homelike, and more conducive to social interaction, Planetree physically redesigns the hospital space. STUDY RESULTS: Planetree hopes to achieve many goals with its restructuring of patient care, two of which are staff and patient satisfaction. Preliminary surveys indicate that nurses and nursing assistants on Planetree are more satisfied than are staff on comparable units. The results of patient surveys, however, are more ambiguous. Planetree patients are no more, or no less, satisfied with their care than patients on comparable units. DISCUSSION: Both Planetree and TQM/CQI have goals of improving the delivery of patient care, and there are times when these approaches work in concert. There are times, however, when the different vantage points of TQM/CQI and Planetree may raise different questions and foster different solutions. Questions are also raised regarding whether Planetree benefits all patients in the same way. Furthermore, since it is not clear if Planetree's vision of humanizing patient care brings the results it hopes for, a long-term multifaceted research program is called for. PMID- 7550787 TI - Using clinical practice improvement: an interview with Susan Horn. Interview by Steven Berman. PMID- 7550788 TI - Prevention and management of glucocorticoid-induced osteoporosis. PMID- 7550789 TI - MRS and NIRS for muscle disease evaluation. PMID- 7550792 TI - Performing arts rheumatology. PMID- 7550790 TI - Case management study: osteoarthritis of the knee. PMID- 7550791 TI - Scleroderma: diagnosis and treatment, Circa 1995. PMID- 7550793 TI - Assessment of exposure to environmental pollutants. PMID- 7550796 TI - Oilseed rape and bronchial reactivity. AB - OBJECTIVES: To investigate atopy and changes in symptoms, peak flow rate, and bronchial reactivity in people complaining of symptoms during the oilseed rape flowering season. METHODS: 37 people who had given positive answers to questions about the presence of symptoms in relation to the flowering season of oilseed rape and 24 controls with no such symptoms were studied, although not all took part in all parts of the study. All had been previously identified in a cross sectional survey of a random sample of a rural population. Atopy was assessed by means of skin prick tests, total immunoglobulin E (IgE), and radioallergosorbent test (RAST) assays. Bronchial reactivity (PC20) was measured by histamine challenge. Total IgE and bronchial reactivity were measured both before and during the oilseed rape flowering season. Day to day changes were monitored by subjects who kept a record of their symptoms and peak flow morning and evening, starting before the flowering season and continuing during it and into the grass pollen season. RESULTS: Only two cases, of 23 tested, showed evidence of allergy to oilseed rape and only 10 of 23 tested, including these two, were atopic. Eye, nasal, and headache symptoms increased in the season in cases, which validated the questionnaire used in the previous cross sectional survey. 12 of 16 cases tested and seven of 15 controls showed a seasonal fall in PC20; the fall in the cases was significantly greater than in the controls. Peak flow charts showed no evidence of fall or of increased variability during the season. CONCLUSIONS: People who complained of symptoms in relation to the flowering of oilseed rape were rarely allergic to the plant and fewer than half were atopic. Nevertheless, they usually showed increased bronchial reactivity during the season, which may have been due in some cases to other allergens but in others to non-specific irritant effects of the air. Whether these are due to chemicals released by the crop, to rising summer ozone levels, or to other factors remains unclear. PMID- 7550794 TI - How have Zambian businesses reacted to the HIV epidemic? AB - OBJECTIVES: To evaluate the impact of HIV on businesses in Zambia and to assess attitudes towards HIV and HIV education in the workplace. METHODS: The personnel managers of 33 companies with a total workforce of 10,204 in Lusaka and in towns in the Copperbelt were visited by two members of the study team. The study was discussed and a questionnaire about the impact of HIV on their company was explained and left for completion from company records. RESULTS: All 33 questionnaires were returned. HIV was recognised to be a problem by 30 companies questioned. Seven said that it had affected recruitment and 11 production. 23 companies carried out pre-employment medicals. 17 companies demanded that some or all of their employees had an HIV test before employment. Nine companies were sure that a positive HIV test would prevent employment, 15 were unsure saying that there was no particular company policy. Two companies had recently changed their policy and had stopped discriminating against those with HIV. 12 companies had some HIV educational material available for their employees and five had someone (or an organisation that they used) to whom they could refer employees for HIV information and advice. Condoms were provided free to staff by five of the companies. All thought that HIV education in the workplace was an appropriate intervention. Mortality data showed a sevenfold increase in the crude mortality from 0.25-1.8 per 100 person-years from 1987-93, and an increasing trend in reported deaths from AIDS and HIV related conditions. CONCLUSIONS: HIV is having an important impact in the workplace in urban Zambia. Although many companies insist on pre-employment medicals, often including HIV testing, few have developed policies relating to test results. Some companies have instituted HIV education but there is a demand for this service to be available more widely. There has been a striking increase in mortalities in this working population, which seems likely to be related to HIV, although the cause of most deaths was not recorded. PMID- 7550795 TI - Occupational hypersensitivity pneumonitis in Japan: data on a nationwide epidemiological study. AB - OBJECTIVES: Diagnostic criteria were prepared for hypersensitivity pneumonitis (HP) and a nationwide survey was conducted to investigate epidemiological and clinical characteristics of HP in Japan. The results are presented with special focus on occupational HP and on the key to the diagnosis of HP. METHODS: A questionnaire was completed by 185 doctors from 185 hospitals (response rate 89.5%). All cases were verified according to diagnostic criteria; 835 cases were classified as HP (653 definite and 182 probable). These 835 cases (total HP) and 99 possible cases of HP diagnosed during the 1980s were analysed and presented as a case series study. RESULTS: Occupational HP was noted in 115 cases (13.8%). 21 occupations, and 20 aetiological antigens were listed. Farmer's lung: 68 cases (59% of occupational HP) was the most prevalent diagnosis followed by 19 industrial workers who handled chemicals (for example, isocyanate) and 10 office workers. Unique cases of mushroom, greenhouse, and silkworm farmers, and a new type of bagassosis are also described. Adverse environmental conditions, immunological findings on examination, antigen challenge, and pathological findings were all significantly lower for possible than for total HP. This was not true for clinical findings. The differences in antibody analysis (6% positive of possible HP v 59% of total HP) and environmental challenge (3% v 74%) were notable. CONCLUSION: These data suggest that a careful interview about the environment and an antigen panel matched to variations in exposure are the key to the diagnosis. PMID- 7550797 TI - Work related distal airway obstruction in an agricultural population. AB - OBJECTIVE: To assess the prevalence of distal airway obstruction and its risk factors in agricultural areas. METHODS: A cross sectional study of respiratory symptoms and lung function was performed among French farmers and their spouses (1122 subjects) who came for preventive medicine examinations. They answered a respiratory questionnaire and performed pulmonary function tests on a portable spirometer. Diagnoses of chronic bronchitis were made on the basis of reported chronic respiratory symptoms. Airway obstruction was determined from predicted values. Odds ratio (OR) and linear regression coefficients were calculated after stratification by smoking and history of cardiac and other respiratory diseases. RESULTS: Of respiratory symptoms prevalence of chronic cough was 8.47%, and chronic bronchitis 7.66%. Prevalence of distal airway obstruction was 11.4%, and overall airflow obstruction 3.2%. Smokers were 20.2% men, and 5.7% women. Linear regressions showed high association between pack-years in smokers or exsmokers and forced expiratory volume in one second/forced vital capacity (FEV1/VC) and forced expiratory flow between 25% and 75% of vital capacity (FEF25-75). In non smokers without any history of cardiac or respiratory diseases, age and the size of farms had the highest correlations with these variables. OR for distal airway obstruction was 2.1 in subjects > 50 years old v the younger ones and 3.02 in the smaller farms v the larger ones. CONCLUSION: After stratification by smoking and history of cardiac and respiratory diseases, distal airway obstruction is present in agricultural areas. The age, and the size of farm are the highest respiratory risk factors in non-smokers. PMID- 7550798 TI - Bladder cancer among French farmers: does exposure to pesticides in vineyards play a part? AB - OBJECTIVE: To appraise the potential contribution of pesticides sprayed on vineyards to the genesis of bladder cancer among agricultural workers. METHODS: A pesticide exposure index (PEI), based on labour time and the proportion of agricultural land used as vineyards, was constructed for 89 French geographical units (departements). The standardised mortality ratios (SMRs) for bladder cancer, as well as tobacco consumption and economic status of male farmers and farm labourers aged 35-74 in the same areas were estimated for the period 1984-6. Models were fitted to the geographical data with Poisson regressions and extra Poisson models with geographically structured and unstructured random effects. RESULTS: Mortality from bladder cancer among farmers was lower (but not significantly so) than within the overall population (SMR 0.96, 95% confidence interval (95% CI) 0.85-1.08), but there was a significant link with exposure to pesticides in vineyards by univariate analysis (relative risk (RR) 1.17, 95% CI 1.10-1.24) and by multivariate analysis (RR 1.14, 95% CI 1.07-1.22). CONCLUSION: These results add some evidence to the view that pesticides in vineyards cause mortality from bladder cancer among farmers, and could explain the French south north gradient in bladder cancer, as vineyards are mainly located in Southern France. PMID- 7550799 TI - Association of exposure to polycyclic aromatic hydrocarbons (estimated from job category) with concentration of 1-hydroxypyrene glucuronide in urine from workers at a steel plant. AB - OBJECTIVES: Increased risk of lung cancer has been associated with employment in the steel industry. This association is thought to be due in part to increased concentrations of polycyclic aromatic hydrocarbons (PAHs) in air found in this work environment. Measurement of PAH metabolites in human urine provides a means of assessing individual internal dose of PAHs. This study examined the relative contribution of occupation and smoking to urinary concentration of 1 hydroxypyrene glucuronide (1-OHPG) among a group of workers at a steel plant. METHODS: Concentrations of 1-OHPG in urine from 44 workers with jobs associated with increased air concentrations of PAHs and 40 workers with jobs with low or no exposure to PAHs were measured. 20 workers in each group were not current smokers. Urinary 1-OHPG was measured by synchronous fluorescence spectroscopy after immunoaffinity chromatography specific for PAH metabolites. RESULTS: Mean (SEM) urinary 1-OHPG concentration was 2.16 (0.42) pmol/ml urine among the 44 occupationally exposed workers compared with 0.38 (0.05) among the 40 workers with no or low exposure (P < 0.0001). Mean urinary 1-OHPG concentration was 1.82 (0.41) pmol/ml urine among the 44 current smokers compared with 0.75 (0.20) among the 40 non-smokers (P < 0.005). Mean 1-OHPG concentrations in non-smokers were 0.26 (n = 20), 0.70 (n = 15), and 2.84 pmol/ml urine (n = 5) for strata of exposure to PAHs (no or low, mid, and high) based on job category; the corresponding values in smokers were 0.55 (n = 20), 0.94 (n = 12), and 4.91 pmol/ml (n = 12), respectively. Multiple linear regression showed significant differences between subjects in different PAH exposure with increased concentrations of 1-OHPG in urine. Amounts of foods containing PAHs ingested by this group of workers were relatively low and did not contribute significantly to urinary 1-OHPG concentrations. CONCLUSIONS: These results indicate that 1-OHPG is a common urinary metabolite in people with recent occupational exposure to PAHs and is associated with both job category and estimated stratum of PAH exposure. PMID- 7550800 TI - Exposure to polycyclic aromatic hydrocarbons in coal liquefaction workers: impact of a workwear policy on excretion of urinary 1-hydroxypyrene. AB - OBJECTIVE: This study was undertaken to assess whether contaminated personal clothing worn beneath a coverall (normal workwear) is a source of potentially significant dermal exposure to polycyclic aromatic hydrocarbons (PAHs) in coal liquefaction workers. METHODS: An intervention study was conducted over a two week period involving 10 workers that reflected the range of activities performed at the factory. A cross over design was used to examine the influence of normal workwear (personal clothing worn beneath a coverall) and intervention workwear (new coverall, shirt, trousers, underwear, socks, and boots) upon excretion of urinary 1-hydroxypyrene (1-OHP) and skin pad deposition of pyrene. RESULTS: The impact of intervention was noted in three ways: (1) A notable reduction (55%) in the mass of 1-OHP excreted on the first day of the intervention phase was found. The median reduction in mass excreted (22.7 nmol) was significant from zero at the 5% level; (95% confidence interval (95% CI) 9.5-40.8 nmol). (2) A notable reduction (82%) in skin pad deposition of pyrene on the first day of the intervention phase was found. The median reduction of 13.20 ng.cm-2 was significant from zero at the 5% level; (95% CI 7.3-26.4 ng.cm-2). (3) About a 50% reduction in 1-OHP concentration over the working week occurred during the intervention phase; an increase of 2.07 mumol/mol creatinine was found from the start to the end of the work period during the intervention phase compared with an increase of 4.06 mumol/mol creatinine during the normal phase. This reduction was not significant at the 5% level. CONCLUSION: The results indicate that on the first day of the working week investigated, significant reductions in absorbtion (as measured by excretion of urinary 1-OHP) and deposition of PAHs (as measured by skin pad deposition of pyrene) can be effected by improvements in workwear policy. The impact of the improved workwear regimen was also detected by reduction in spot urinary 1-OHP concentrations, although this effect was less pronounced. One implication of the findings is that exposure to PAHs may arise from workers' own contaminated personal clothing. As a consequence of this study an improved workwear policy has been implemented at the factory. PMID- 7550801 TI - Risk of gastric cancer in pneumoconiotic coal miners and the effect of respiratory impairment. AB - OBJECTIVES: This study was carried out to investigate the mortality patterns in a group of 3790 coal miners. The study population had abnormal chest x ray films at a routine medical examination that was performed in the 1950s. METHODS: The total group of 3790 coal miners were followed up for mortality up to 1 January 1992. Causes of death, determined and coded at time of death, were traced with help from the Central Bureau of Statistics. RESULTS: Total mortality in this group of coal miners with abnormal chest x ray films was significantly higher than expected (SMR 127.1, 95% CI 122.5-131.6), mainly a reflection of the increase in mortality from non-malignant respiratory disease (SMR 411.0, 95% CI 382.3-441.3). Mortality from gastric cancer was also significantly increased (SMR 147.5, 95% CI 122.3-176.3). This risk of mortality from gastric cancer was confined to workers with no pneumoconiosis or only a mild form. Despite the strong relation to duration of employment and pneumoconiosis the group of workers with more severe manifestations of pneumoconiosis did not experience an excess in mortality from gastric cancer. CONCLUSION: This study confirms the earlier reported risk of gastric cancer in coal miners. Also it confirms the hypothesis that this risk of gastric cancer is limited to workers with a mild degree of pneumoconiosis or none. In workers with severe forms of pneumoconiosis the pulmonary clearance system is impaired in such a way that the inhaled coal dust does not reach the digestive tract. PMID- 7550802 TI - Biological monitoring of exposure to benzene: a comparison between S phenylmercapturic acid, trans,trans-muconic acid, and phenol. AB - OBJECTIVES: Comparison of the suitability of two minor urinary metabolites of benzene, trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (S-PMA), as biomarkers for low levels of benzene exposure. METHODS: The sensitivity of analytical methods of measuring tt-MA and S-PMA were improved and applied to 434 urine samples collected from 188 workers in 12 studies in different petrochemical industries and from 52 control workers with no occupational exposure to benzene. In nine studies airborne benzene concentrations were assessed by personal air monitoring. RESULTS: Strong correlations were found between tt-MA and S-PMA concentrations in samples from the end of the shift and between either of these variables and airborne benzene concentrations. It was calculated that exposure to 1 ppm (8 hour time weighted average (TWA)) benzene leads to an average concentration of 1.7 mg tt-MA and 47 micrograms S-PMA/g creatinine in samples from the end of the shift. It was estimated that, on average, 3.9% (range 1.9% 7.3%) of an inhaled dose of benzene was excreted as tt-MA with an apparent elimination half life of 5.0 (SD 2.3) hours and 0.11% (range 0.05%-0.26%) as S PMA with a half life of 9.1 (SD 3.7) hours. The mean urinary S-PMA in 14 moderate smokers and 38 non-smokers was 3.61 and 1.99 micrograms/g creatinine, respectively and the mean urinary tt-MA was 0.058 and 0.037 mg/g creatinine, respectively. S-PMA proved to be more specific and more sensitive (P = 0.030, Fisher's exact test) than tt-MA. S-PMA, but not tt-MA, was always detectable in the urine of smokers who were not occupationally exposed. S-PMA was also detectable in 20 of the 38 non-smokers from the control group whereas tt-MA was detectable in only nine of these samples. The inferior specificity of tt-MA is due to relatively high background values (up to 0.71 mg/g creatinine in this study) that may be found in non-occupationally exposed people. CONCLUSIONS: Although both tt-MA and S-PMA are sensitive biomarkers, only S-PMA allows reliable determination of benzene exposures down to 0.3 ppm (8 h TWA) due to its superior specificity. Because it has a longer elimination half life S-PMA is also a more reliable biomarker than tt-MA for benzene exposures during 12 hour shifts. For biological monitoring of exposure to benzene concentrations higher than 1 ppm (8 h TWA) tt-MA is also suitable and may even be preferred due to its greater ease of measurement. PMID- 7550803 TI - Fibrogenic effect of wollastonite compared with asbestos dust and dusts containing quartz. PMID- 7550804 TI - Methods in cohort studies. PMID- 7550805 TI - Offspring sex ratios as an index of pollution hazard in residential environments. PMID- 7550806 TI - The three faces of welfare. Public opinion and the debate over reform. PMID- 7550807 TI - The public and the welfare reform debate. AB - OBJECTIVES: To identify the core beliefs and policy preferences of the American public toward changing the welfare system and providing support for low-income families. DESIGN: Results are presented from 19 telephone and in-person surveys of adults nationwide between 1937 and February 1995. SETTING: At-home interviews with adults. PARTICIPANTS: Seventeen surveys; each survey involved 1000 to 2000 adults nationwide. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The results showed that the public supports strong welfare reform measures (eg, time limits and work requirements), but it is reluctant simply to cut off welfare benefits to people and leave them without some means of basic support. The surveys identified five underlying beliefs that shape the public's policy preferences: (1) welfare causes more harm than good because it discourages work and causes families to break up; (2) welfare should be a temporary transition to work, not a long-term subsidy for low-income families; (3) the country spends too much on welfare programs; (4) lack of economic opportunity as well as personal responsibility is the reason people need welfare; and (5) both government and people themselves have a shared responsibility for ensuring that people have a minimum standard of living. CONCLUSIONS: The outcome of the welfare reform debate will have a substantial impact on the 21% of the nation's children who now live in poverty. The jury is still out on what the public will support in the welfare reform debate. The Medicaid program is caught in the middle of the welfare reform debate, and its ultimate fate may rely on state rather than federal decision making. PMID- 7550808 TI - What mothers say about why poor children fall behind on immunizations. A summary of focus groups in North Carolina. AB - OBJECTIVES: To develop a more thorough understanding of the factors that impede poor parents' utilization of health care services for their children and to refine interventions to improve immunization rates. METHODS: We conducted focus group sessions with mothers whose children received care at the health departments in five North Carolina counties. Mothers were uninsured or were receiving Medicaid. A total of 50 women participated; group size varied from three to seven mothers. RESULTS: Socially disadvantaged mothers faced barriers at multiple points in the process of obtaining preventive care for their children. Organizational barriers, such as a lack of flexibility in scheduling and long waiting times, were exacerbated by personal barriers, such as a lack of reliable transportation, chaotic home environments, and employment conflicts. Lack of knowledge regarding the timing of childhood immunizations and misperceptions about the safety of immunizations were also important obstacles. Mothers made several suggestions, such as changes in scheduling, greater assistance with transportation, improved waiting facilities, and increased health education. CONCLUSIONS: Our study suggests that even with improved financing of well-child care, many important barriers to adequate immunization will remain. Many of the changes that mothers in our focus groups advocated are not related to insurance coverage and would be simple and inexpensive to implement. To help with these changes, we developed a checklist for use by health departments to determine which organizational barriers exist at their facility and suggest strategies to overcome the problems. Organizational, personal, and attitudinal barriers pose serious problems for socioeconomically disadvantaged families. To improve vaccination rates for children, new personnel and programs are probably less important than careful strategies to maximize existing resources. PMID- 7550809 TI - Housing subsidies and pediatric undernutrition. AB - OBJECTIVE: To test the hypothesis that receipt of housing subsidies by poor families is associated with improved nutritional status of their children. DESIGN: Cross-sectional study. SETTING: Pediatric emergency department of an urban municipal hospital. PATIENTS: Convenience sample of 203 children younger than 3 years and their families who were being seen during one of twenty-seven 24 hour periods. MAIN OUTCOME MEASURES: Anthropometric indicators (z scores of weight for age, weight-for-height, and height-for-age), and the proportion of children with low growth indicator (weight-for-height below the 10th percentile or height-for-age below the fifth percentile, or both, of the reference population). RESULTS: Multivariate analysis controlling for demographics and program participation showed that receipt of housing assistance contributed significantly to z scores for weight-for-age (P = .03) and weight-for-height (P = .04). The risk of a child's having low growth indicators was 21.6% for children whose families were on the waiting list for housing assistance compared with 3.3% for those whose families received subsidies (adjusted odds ratio = 8.2, 95% confidence interval = 2.2 to 30.4, P = .002) CONCLUSION: Receiving a housing subsidy is associated with increased growth in children from low-income families, an effect that is consistent with a protective effect of housing subsidies against childhood undernutrition. PMID- 7550810 TI - Overweight prevalence and trends for children and adolescents. The National Health and Nutrition Examination Surveys, 1963 to 1991. AB - OBJECTIVE: To examine prevalence of overweight and trends in overweight for children and adolescents in the US population. DESIGN: Nationally representative cross-sectional surveys with an in-person interview and a medical examination, including measurement of height and weight. PARTICIPANTS: Between 3000 and 14,000 youths aged 6 through 17 years examined in each of five separate national surveys during 1963 to 1965, 1966 to 1970, 1971 to 1974, 1976 to 1980, and 1988 to 1991 (Cycles II and III of the National Health Examination Survey, and the first, second, and third National Health and Nutrition Examination Surveys, respectively). MAIN OUTCOME MEASURES: Prevalence of overweight based on body mass index and 85th or 95th percentile cutoff points from Cycles II and III of the National Health Examination Survey. RESULTS: From 1988 to 1991, the prevalence of overweight was 10.9% based on the 95th percentile and 22% based on the 85th percentile. Overweight prevalence increased during the period examined among all sex and age groups. The increase was greatest since 1976 to 1980, similar to findings previously reported for adults in the United States. CONCLUSIONS: Increasing overweight among youths implies a need to focus on primary prevention. Attempts to increase physical activity may provide a means to address this important public health problem. PMID- 7550811 TI - Survival trends in adolescents with human immunodeficiency virus infection. AB - OBJECTIVE: To examine trends in survival and death in nonhemophiliac adolescents with human immunodeficiency virus (HIV) infection. DESIGN: Case series. SUBJECTS: All 117 HIV-seropositive adolescents (age range, 13 to 21 years) without hemophilia whose cases were reported through December 1992. SETTING: A state with mandatory, name-linked reporting of cases of HIV. METHODS: Review of death certificates and public records, and interviews with subjects and key informants. ANALYSES: Kaplan-Meier and Cox proportional hazards regression analyses of time from the diagnosis of HIV infection to death. RESULTS: Documentation of death or survival was available for 92% (108/117) of the sample. Fourteen percent (15/108) of the subjects were known to have died, mainly of well-recognized complications of acquired immunodeficiency syndrome. Among deceased subjects, the median duration of survival after the diagnosis of HIV was 3 years. Cumulative survival at 8 years after the diagnosis was 52%, with 4 years being the median period of observation (range, < 1 year to 8 years). Stratification of survival functions by gender, race, and mode of transmission revealed no significant (P < .05) differences between groups. However, advancing age at the time of the diagnosis was inversely associated with survival. CONCLUSIONS: Despite an apparent advantage to young age, overall survival in this adolescent cohort was shorter than expected. Rather than an inherently rapid progression of disease during adolescence, delays in diagnosis and treatment might better explain the results. PMID- 7550813 TI - Consistent but not the same. Effect of method on chronic condition rates. AB - OBJECTIVE: To determine rates of chronic physical conditions for children and youths. DESIGN: Secondary analysis of the Health Examination Surveys, cycles 2 and 3, National Center for Health Statistics. SETTING: National household survey of noninstitutionalized civilians in the United States. PARTICIPANTS: Multistage probability samples of children aged 6 to 11 years (cycle 2, 1963 to 1965) and youths aged 12 to 17 years (cycle 3, 1966 to 1970), and a longitudinal subsample assessed at both times. Excludes persons with an IQ lower than 80. RESULTS: Selected items from developmental and medical histories and screening physical examinations were used to classify those with chronic physical conditions. Rates of persons with chronic conditions are 25.2% for the children and 35.3% for the youths. Parental histories produce rates equal to or greater than 11%, and physical examinations produce rate of 15.7% for children and 22% for youths. About 4% in each cycle are identified by both screening physical examination and parental history. Little overlap occurs in identification by the two sources, accounting for the higher rates for the combined indicators. Combining data sources identifies 13% of the longitudinal subsample as having a chronic physical condition at both times. CONCLUSIONS: Rates of chronic physical conditions seem to be altered by combining different indicators to categorize children. For either source used alone (eg. parental report or physical screening physical examination) the estimate is similar in magnitude, but the composition of the affected group differs. These findings have major implications for research, service, and policy. PMID- 7550812 TI - Malnutrition in hospitalized children with congenital heart disease. AB - OBJECTIVE: To determine the prevalence of malnutrition among hospitalized children with congenital heart disease by age, disease process, and clinical status. DESIGN: Cross-sectional, retrospective chart review. SETTING: Pediatric cardiology units at a 150-bed tertiary care teaching hospital in Ann Arbor, Mich. PATIENTS: Patients (n = 160) were randomly selected from consecutive admissions to the Pediatric Cardiology and Thoracic Surgery Services during a 1-year period. INTERVENTION: None. MAIN OUTCOME MEASURES: Acute and chronic malnutrition, assessed by comparing the patients' weight and height with established means. RESULTS: Acute and chronic malnutrition occurred in 33% and 64% of the patients, respectively. Age, diagnostic category, and symptoms were associated with malnutrition. Eighty percent of infants presented with acute malnutrition compared with 18% of patients of other ages (P < .001). Malnutrition affected 60% of patients with left-to-right shunts, 53% of patients with complex heart disease, and no patients with primary rhythm disturbances. Acute malnutrition affected 11% and chronic malnutrition affected 50% of patients with left-sided heart obstruction. Acute or chronic malnutrition occurred in 70% or more of patients with cyanosis and/or congestive heart failure but in only 30% of patients with neither (P < .001). CONCLUSION: Malnutrition in hospitalized children with congenital heart disease remains common, highlighting the importance of nutritional screening and intervention. PMID- 7550816 TI - The epidemiology of infantile hypertrophic pyloric stenosis in New York State, 1983 to 1990. AB - OBJECTIVES: To investigate an apparent decline in the rate of infantile hypertrophic pyloric stenosis (IHPS) and to examine the characteristics of children with IHPS and any associated malformations. DESIGN: Cohort study in which children with IHPS were compared with the population of live births. Trends of IHPS were compared in two data sets: a population-based birth defects registry and hospital discharge data. PARTICIPANTS: Children with IHPS identified from a birth defects registry and the population of live-born infants born to residents of New York State from 1983 to 1990. MAIN OUTCOME MEASURE: Trends in the incidence of IHPS in the two data sets, and demographic characteristics and malformations associated with IHPS. RESULTS: The rate of IHPS declined from 2.4 per 1000 live births in 1984 to 1.7 in 1990. White race and male gender were associated with a higher occurrence of IHPS; high birth order, older maternal age, higher maternal education, and low birth weight were associated with lower occurrence. Seven percent of children with IHPS had a major malformation compared with 3.7% of the general population. Three major malformations occurred more frequently in children with IHPS: intestinal malrotation, obstructive defects of the urinary tract, and esophageal atresia. Fewer cases were found in the birth defects registry than in the hospital discharge data. CONCLUSIONS: Underreporting of IHPS to the birth defects registry accounts for some of the decline. Children with IHPS have more major malformations than the general population, although some of the excess could be attributed to increased detection. Further investigation is needed into the environmental factors, especially socioeconomic, associated with IHPS. PMID- 7550814 TI - Influenza A vaccine decreases the incidence of otitis media in 6- to 30-month-old children in day care. AB - OBJECTIVE: To determine if the use of influenza vaccine in children in day care decreases the incidence of otitis media during the influenza season. DESIGN: Prospective cohort study. SETTING: Eight day-care centers in North Carolina. PARTICIPANTS: One hundred eighty-six children aged 6 to 30 months. INTERVENTION: Half the participants received trivalent subvirion influenza virus vaccine. MEASUREMENTS: Acute otitis media (AOM) and serous otitis media (SOM) were assessed biweekly from mid-November 1993 to mid-March 1994 by visual and tympanometric examinations performed by "blinded" observers. The winter season was divided into three periods-before, during, and after influenza season--and the number of children with AOM or SOM during each period was determined. Unadjusted and adjusted odds ratios (ORs) were computed, while controlling for race and sex using logistic regression methods. RESULTS: Influenza vaccine was protective against AOM (OR = 0.69, 95% CI, 0.49-0.98) during the influenza season. Although there may have been some protection against SOM (OR = 0.75, 95% CI, 0.54-1.02) statistical significance was not achieved. Myringotomy tubes were also significantly protective against AOM and SOM during all three time periods, with ORs between 0.34 and 0.52, but the greatest protection was seen during the influenza period. CONCLUSIONS: Influenza vaccination of 6- to 30-month-old children in day care was associated with a decreased incidence of otitis media during the influenza season. Myringotomy tubes protected against AOM and SOM during all 16 weeks monitored. PMID- 7550815 TI - Malnutrition in hospitalized pediatric patients. Current prevalence. AB - OBJECTIVE: To document the current prevalence of protein-energy malnutrition compared with that reported from the same institution in 1976. DESIGN: All inpatients of this tertiary-care facility were assessed by anthropometric, laboratory, and clinical nutrition assessment methods in a 1-day cross-sectional survey. The comparison study from 1976 was also a 1-day cross-sectional survey. SETTING: A tertiary-care facility in Boston, Mass. PATIENTS: The entire inpatient population was assessed on a single weekday in September 1992. MAIN OUTCOME MEASURES: Prevalence of acute and chronic malnutrition as judged by anthropometric and laboratory data. Data on demographics, admission classification, underlying disease, route of nutrition, and global nutritional status were also assessed. RESULTS: The prevalence of acute protein-energy malnutrition (weight for height) based on the Waterlow criteria was as follows: severe, 1.3%; moderate, 5.8%; mild, 17.4%; and none, 75.5%. The prevalence of chronic protein-energy malnutrition (height for age) was as follows: severe, 5.1%; moderate, 7.7%; mild, 14.5%; and none, 72.8%. Although the prevalence of acute and chronic protein-energy malnutrition was significantly less in 1992 than in 1976 (P = .03 and P < .001, respectively), the numbers are still alarmingly high. Children younger than 2 years and older than 18 years and those with chronic medical conditions had a higher prevalence of protein-energy malnutrition. Twenty-four percent of patients had a serum albumin level less than 30 g/L, 34.8% had a total lymphocyte count less than 1.5 x 10(9)/L, and 24.9% had a hemoglobin concentration less than 105 g/L. One fourth of all patients were obese (> 120% weight for height), with the greatest prevalence in children aged 2 to 18 years. CONCLUSIONS: Acute and chronic protein-energy malnutrition remains common in hospitalized pediatric patients in the United States. Important risk factors may be underlying chronic disease, periods of normally rapid growth, and recognized need for nutrition intervention. PMID- 7550818 TI - Precision and accuracy of clinical and radiological signs in premature infants at risk of patent ductus arteriosus. AB - OBJECTIVE: To determine the precision (interobserver agreement) and accuracy (agreement with criterion standard) of clinical and radiological signs in premature infants at risk of patent ductus arteriosus (PDA) with left-to-right shunting. DESIGN: Masked comparison of clinical and radiological examination with Doppler flow echocardiography (criterion standard). SETTING: Neonatal intensive care unit. PATIENTS: One hundred infants with birth weights less than 1750 g were studied once between days 3 and 7 of life. A third of the cohort was intubated at the time of study. INTERVENTION: Five independent observers noted the presence or absence of an increased pulse volume, an active precordium, a heart murmur, a cardiothoracic ratio greater than 60%, increased pulmonary vascular markings on a concurrent chest x-ray film, and a relative increase of the cardiothoracic ratio compared with that from the previous chest x-ray film. Pulsed and color flow Doppler echocardiography was performed within 4 hours. All 100 tapes were reviewed by a second pediatric cardiologist. RESULTS: Twenty-three infants had a PDA with left-to-right shunting. The precision of clinical signs was modest, with average kappa values of 0.15 for pulse volume, 0.32 for precordium, and 0.41 for murmur. Pulse quality (43%) and murmur (42%) had the highest mean sensitivities. Corresponding specificities were 74% for pulse volume and 87% for murmur. The combination of a cardiac murmur with an abnormal pulse volume had the highest positive predictive value (77%). The radiological examination did not improve the observers' ability to distinguish between patients with and without PDA. CONCLUSIONS: The precision and accuracy of clinical and radiological signs of a PDA with left-to-right shunting are unsatisfactory. Therefore, Doppler flow echocardiography is required to diagnose PDA confidently in preterm infants between days 3 and 7 of life. PMID- 7550819 TI - Initial lesion size as a predictive factor in determining the response of port wine stains in children treated with the pulsed dye laser. AB - OBJECTIVE: To evaluate the response of facial port-wine stains in children to pulsed (450 microseconds) dye (577- or 585-nm) laser treatment based on the age of the patient and the size of the port-wine stain at the initiation of treatments. DESIGN: Case series. SETTING: Outpatient dermatology clinic at the University of Colorado School of Medicine, Denver. PATIENTS: A consecutive sample of 83 children who had facial port-wine stains that were treated in their entirety at each visit. INTERVENTION: Patients were treated with a pulsed (450 microseconds) dye (577- and 585-nm) laser. MAIN OUTCOME MEASURE: Decrease in size of the port-wine stain. RESULTS: Overall 18 (22%) of the 85 patients responded with complete clearing of their port-wine stains. The port-wine stains in 32% (7/22) of the patients who began treatment before 1 year of age responded with complete clearing of their port-wine stains compared with 18% (11/61) in older patients (P < .01). Fifteen (32%) of 47 patients with port-wine stains smaller than 20 cm2 at initial evaluation were totally cleared vs three (8%) of the 36 patients with port-wine stains larger than 20 cm2 (P < .05). This size effect is apparent at all ages. CONCLUSION: When considering treatment for facial port-wine stains with pulsed dye lasers, it is important to consider not only the age of the child at the beginning of treatments, but also the initial size of the lesion. PMID- 7550817 TI - A 'barrier-free' health care system does not ensure adequate vaccination of 2 year-old children. AB - OBJECTIVES: To assess vaccination status in a cohort of 2 year olds with access to health care at no cost and to delineate factors associated with failure to be fully vaccinated. DESIGN: Retrospective cross-sectional study. Children not up-to date on vaccinations by age 2 years compared with children up-to-date by medical record review. A telephone survey was conducted for those without medical records or whose records lacked complete documentation. SETTING: Large military tertiary care hospital. PARTICIPANTS: A cohort of 844 children born between August 31, 1988, and September 1, 1989. MAIN OUTCOME MEASURES: Timeliness of vaccination, factors associated with undervaccination, number and nature of missed opportunities. RESULTS: Best estimate of coverage with entire primary vaccination series was 72% by age 2 years. Attendance at military day care was associated with full vaccination (odds ratio [OR] = 1.80, confidence interval [CI] = 1.12 to 3.24) as was completion of well-baby visits. Increasing number of other visits did not predict full vaccination. Children followed up by family practice were more likely to be up-to-date than children followed up by pediatrics (OR = 3.67, CI = 1.47 to 9.73). Seventy-two percent of children who were not up-to-date had at least one missed opportunity for vaccination. If vaccinations had been offered at all visits, 93% of children could have been fully vaccinated by age 2 years. CONCLUSIONS: Offering free vaccinations in a "barrier-free" system will not ensure vaccination levels of 90%. Health care providers should offer vaccinations during acute, non-well visits to improve coverage. PMID- 7550820 TI - Wasted health care dollars. Routine cord blood type and Coombs' testing. AB - OBJECTIVE: To determine if selective newborn cord blood testing (NCBT) could contain costs without increasing morbidity of hemolytic disease of the newborn (HDN). DESIGN: A national telephone survey confirmed the common practice of routine blood type and Coombs' NCBT. Two 12-month study arms, retrospective and prospective, were conducted. Hemolytic disease of the newborn was studied retrospectively under an unrestricted NCBT policy. Then, HDN was studied after a policy change that restricted NCBT to patients in newborn intensive care units and normal newborns with clinical jaundice or Rh-negative mothers, and/or positive maternal antibody screenings, or unavailable maternal blood testing. PARTICIPANTS: All newborns (N = 8501) at the Metro-Health Medical Center, Cleveland, Ohio, were studied (retrospective arm, all 1989 admissions; prospective arm, all July 1990 to June 1991 admissions). OUTCOME MEASURES: Blood type and Coombs' NCBT, maternal blood type and antibody screening, Hobel risk scores for clinical severity of newborn hospitalization, duration of hospitalizations, and peak serum bilirubin levels. RESULTS: No quantitative or qualitative increases in morbidity from jaundice were detected by retrospective analysis with unrestricted NCBT, or prospectively after selective testing on 4498 newborns. Each study arm resulted in 15 readmissions for jaundice; these included two patients with ABO HDN. Furthermore, selective testing resulted in performance of NCBTs on only 390 infants in the "normal" nursery (24% of the original sample). Estimates projected on 1991 US births (4,111,000) showed that selective NCBT offers potential yearly savings above $30.8 million of patient charges, savings above $11.3 million of hospital costs, and the reassignment of more than 112 personnel full-time equivalents. CONCLUSION: Selective NCBT decreases the use of resources and costs without apparent additional patient morbidity from HDN. PMID- 7550821 TI - Pediatric clinical clerkships are associated with an excess risk of acute infection. AB - OBJECTIVE: To determine the incidence of acute respiratory and gastrointestinal tract illness and associated absence by third-year medical students during pediatric and nonpediatric (control) clinical clerkships. DESIGN: A self administered questionnaire was completed by students after the first 4 weeks of two pediatric clerkships (inpatient and outpatient) and two non-pediatric clerkships (obstetrics-gynecology and psychiatry). Information was obtained on the symptoms of acute respiratory and gastrointestinal tract illness and related absences. ANALYSIS: Results from each student's pediatric clerkship were compared with the results from the same student's control clerkship by means of matched pair analysis. SUBJECTS: Students who made up the junior class at the University of Texas Southwestern Medical School at Dallas, July 1, 1990, to June 30, 1991. RESULTS: Of 177 students (77%) who completed questionnaires after one pediatric and one control clerkship, 108 students (61%) had had an acute illness while on pediatric clerkships in contrast to 69 students (39%) on control clerkships (odds ratio, 3.3; 95% confidence interval, 2.0 to 5.4; P < .001). More students were absent for illness during pediatric than control clerkships (23 [13%] vs nine [5%], respectively; odds ratio, 2.7; 95% confidence interval, 1.2 to 6.2; P = .02). The higher risk of illness during pediatric clerkships was not related to the order of the pediatric or control clerkship, the order of inpatient and outpatient pediatrics, or the season of the year. CONCLUSION: Pediatric clinical clerkships in the third year of medical school were associated with excess morbidity from acute infectious illness. Studies are needed to determine whether emphasizing infection control practices decrease this morbidity and any resulting nosocomial spread to patients. PMID- 7550823 TI - On-line resources for pediatricians. AB - BACKGROUND: Medical information is increasingly available on computer networks. OBJECTIVES: To familiarize the pediatrician with some of the terms associated with these on-line resources, and to describe what is available via the Internet and dial-up computer bulletin boards. DATA SOURCES: Information for this article was taken from publicly available on-line services and Internet servers. DATA EXTRACTION: Only resources relevant to pediatric practice were included. DATA SYNTHESIS: Medical information on the Internet is in a state of evolution. Useful resources for pediatricians are sparse but expanding rapidly. CONCLUSIONS: Pediatricians may use on-line computer networks to communicate. Familiarity with computer networks will become more important to pediatricians as electronic medical records, on-line medical literature, and telemedicine (the practice of medicine over long distances) become more common. Medical libraries and commercial on-line services are good places to begin to investigate the availability of medical information over computer networks. PMID- 7550824 TI - Radiological case of the month. Eosinophilic gastroenteropathy. PMID- 7550825 TI - Picture of the month. Harlequin color change. PMID- 7550822 TI - What is left of futility? The convergence of anencephaly and the Emergency Medical Treatment and Active Labor Act. AB - In 1986, Congress passed legislation intended to prevent hospitals from "dumping" patients who were unable to pay for their care. This legislation was appended to the Consolidated Omnibus Budget Reconciliation Act as the Emergency Medical Treatment and Active Labor Act (EMTALA). The EMTALA imposes two sets of duties on all hospitals that receive Medicare funds. Since 98% of hospitals in the United States participate in the Medicare program, EMTALA applies to nearly all hospitals in the United States. In short, hospitals with an emergency department must provide an appropriate screening examination to any patient who requests treatment. The purpose of the medical screening examination is to determine whether an "emergency medical condition" exists. If an emergency medical condition is discovered, the hospital must either provide treatment sufficient to stabilize the patient's condition or transfer the patient to another medical facility in an acceptable fashion. PMID- 7550826 TI - Pathological case of the month. Globoid cell leukodystrophy (Krabbe's disease). PMID- 7550827 TI - Just say no ... welfare. PMID- 7550828 TI - Just say no ... welfare. PMID- 7550829 TI - Just say no ... welfare. PMID- 7550830 TI - Just say no ... welfare. PMID- 7550831 TI - Neurobiology of arrhythmias in children prenatally exposed to cocaine. PMID- 7550832 TI - Meningitis in the child with a smile. PMID- 7550833 TI - [Reflexions apropos of recent progresses in molecular genetics of myopathies]. PMID- 7550834 TI - [What method of sampling is best for prenatal diagnosis of genetic diseases?]. PMID- 7550835 TI - [Pharmacokinetics of clofibrate in jaundiced newborn infants at term]. AB - BACKGROUND: Clofibrate (CFB) has been proposed to increase elimination of bilirubin in neonates with hyperbilirubinemia. Nevertheless, its disposition, at this age, remains unknown. The aim of this work was to characterize pharmacokinetics of an oil formulation of CFB in neonates at term with jaundice. PATIENTS AND METHODS: Two groups (G1 and G2) of eight neonates, presenting with jaundice, entered an open, non randomized and comparative study. Five blood samples were collected over 50 hours following a single oral administration of 100 mg/kg or 50 mg/kg CFB, respectively, in G1 and G2. Serum concentrations of both CFB and clofibric acid (CFA) were measured by HPLC and the pharmacokinetic analysis was made by a non-compartmental method. Data were compared to those obtained in adults receiving 2 g dose of CFB. RESULTS: Tolerance to the treatment was excellent. Pharmacokinetic profiles were similar in both groups of infants. There was a slow and prolonged formation of CFA whose serum concentrations remained high 50 hours after drug administration. Non-hydrolyzed CFB was found in the blood of three neonates. Elimination of CFA was prolonged corresponding to a terminal half-life (t1/2m) often above 100 hours and sometimes incalculable. MRTo ->50 (h) was similar in both groups (ie 26.2 +/- 2.0 vs 25.5 +/- 1.3, respectively). The decrease of t1/2m was related to the decrease of the clearance of CFA. CONCLUSIONS: The decrease in CFB's metabolism in newborns is probably the result of at least two concurrent phenomenons: partial hydrolysis of CFA, especially at high doses, and decrease in the hepatic capacity to conjugate the active metabolite. A single oral administration of 50 mg/kg CFB seems to be a suitable schedule. PMID- 7550836 TI - [Precocious puberty and polycystic ovarian syndrome: apropos of 13 cases]. AB - BACKGROUND: Precocious puberty and polycystic ovarian syndrome are two different entities which appear at different stages of ovarian development. Their association is uncommon. POPULATION: Thirteen girls presented idiopathic central precocious puberty with sexual development before the age of 8 years; menstruations were seen at the age of 9.5 years in one patient. Nine of them were given medroxyprogesterone or cyproterone acetate and two patients LHRH analog. Menarche occurred at a mean age of 11.8 +/- 1.5 years. After a mean free interval of 22 months, these thirteen patients developed hirsutism with irregular menstruations (eight patients) and weight gain (seven patients). The diagnosis of polycystic ovarian syndrome was confirmed by increased plasma testosterone (mean 91.1 +/- 14 ng/dl) and LH levels during LHRH test and by ultrasonography or coelioscopy. The treatment included cyproterone acetate plus 17 beta oestradiol; it was discontinued in eleven cases after 2 years of treatment. Plasma testosterone levels were normal 6 months later in association with regular menstruations. But three patients presented clinical and hormonal recurrence one year later, requiring repeated treatment. CONCLUSION: This association seems to be related to the same gonadotropin dysfunction. It is necessary to regularly follow patients treated for precocious puberty. PMID- 7550837 TI - [Modality and causes of 259 deaths in a pediatric intensive care unit]. AB - BACKGROUND: There are few data available from European pediatric intensive care units (PICU) regarding the modes of death and their causes. POPULATION AND METHODS: Two hundred and fifty nine children, not including neonates, died in the PICU over a 7-year period (1987-1993). Data were obtained from a computerized data base and the retrospective review of medical records by two intensivists. Deaths were classified into three groups according to the terminal event: brain death (BD), unsuccessful resuscitation (UR), do-not-resuscitate order and limitation and/or withdrawal of therapy (LWT). RESULTS: BD was the most common mode of death (38%); UR accounted for 34% and LWT for 28% of deaths. There was no significant annual variation in the proportion of BD, UR and LWT. Age and sex were similar in the three groups. The predominant organ system failure involved upon admission was the central nervous system (52%) in the LWT group, and the cardiovascular system (54%) in the UR group. Severe chronic disease (37%) and immunosuppression (19%) were more prevalent in the LWT group than in the BD group. Time from admission to death was longer in the LWT group (median = 119 hours) as compared to the UR group (10 hours) and the BD group (54 hours). Ten percent of the BD patients became organ transplant donors. Sixty-seven per cent of BD patients had medical contraindication for organ donation: parents did not accept organ donation in 61% of potential cases. Thirty deaths (12%) seemed to be avoidable; dehydration from acute infectious gastroenteritis (n = 7) was the most common cause of avoidable death. CONCLUSIONS: The modes of death in our PICU were statistically not different from those seen in two of four North-American PICUs; LWT was less prevalent than in the two other PICUs, but the patient populations were very different (presence of neonates and many cardiovascular surgery patients). Assessment of the severity of illness at admission and of functional outcome in the survivors are mandatory in future studies. PMID- 7550838 TI - [Frequency of gastroesophageal reflux in infants and in young children with cystic fibrosis]. AB - BACKGROUND: Gastro-esophageal reflux (GER) is common in chronic bronchopulmonary diseases, and may aggravate them. The aim of this study was to study frequency and characteristics of GER in infants and toddlers with cystic fibrosis (CF). POPULATION AND METHODS: Twenty-five CF patients, 45 to 954 days-old (mean 219 days) without clinical evidence of bronchopulmonary infection, were studied by prolonged pHmetry (19.45 +/- 1.89 hours). Their pulmonary state was evaluated by the number of previous courses of antibiotic treatment and analysis of the X rays. RESULTS: Nineteen children had GER (76%); four of them had no clinical sign of GER. All 19 patients had mild or moderate signs of pulmonary impairment. The reflux index (percentage of time with pH < 4) was 12.82%. The GER episodes were more frequent during walking time (p < 0.001), but longer during sleeping time (p < 0.001). CONCLUSIONS: GER is frequent in CF patients, even the youngest. It seems primary, and not related to pulmonary impairment. Its frequency and its potential severity lead to suggesting a pHmetry to every new CF patient. PMID- 7550839 TI - [Value of extracorporeal shockwave lithotripsy in primary hyperoxaluria type I]. AB - BACKGROUND: Application of extracoporal shock wave lithotripsy (ESWL) has revolutionized the management of many types of urolithiasis, including that observed in primary hyperoxaluria where surgical attempts to remove calculi sometimes result in worsening of renal function. CASE REPORTS: Three unrelated patients aged 8, 10 and 11 years, respectively, with type I primary hyperoxaluria, suffered from recurrent bouts of abdominal pain (two patients) or anemia (one patient). Two patients had chronic renal failure. Plain abdominal films and ultrasonography showed several large bilateral stones. ESWL was applied to the three patients permitting complete removal of stones following three sessions in one patient and partial removal following four sessions in the second patient who developed infection after the first session with transitory increase in creatinemia. Several sessions of ESWL failed to fragment stones in the third patient. CONCLUSION: ESWL may represent a safe procedure for attempting stone removal in patients with primary hyperoxaluria. PMID- 7550841 TI - [Congenital erythropoietic porphyria. Apropos of a fatal case in the neonatal period due to acute hemolysis with hepatic failure]. AB - BACKGROUND: Congenital erythropoietic porphyria, an autosomal recessive disease, is characterized by deficiency of uroporphyrinogen III synthase. Clinical variability of the disease is related to the different mutations found in the patients. CASE REPORT: A newborn suffered one hour after birth from jaundice and polypnea with acute hemolysis. Severe cutaneous photosensitivity occurred after phototherapy. Congenital erythropoietic porphyria was suspected because of reddish-colored urine and confirmed by porphyrin analyses. The baby died one month later due to severe hemolytic anemia with hepatic failure. Uroporphyrinogen III synthase activity was decreased by 99% in bone marrow cells and established lymphoblastoid cells from the patient. Molecular biology studies demonstrated the presence of the Cys 73-->Arg substitution at the homozygous state in the patient. CONCLUSION: This mutation, the most frequently found in this disease, is responsible for a severe phenotype. Molecular characterization provides genotype/phenotype correlations in this porphyria and allows to clarify unusual cases of porphyrias. PMID- 7550840 TI - [Darier-Ferrand dermatofibrosarcoma in children]. AB - BACKGROUND: Dermatofibrosarcoma is a tumor relatively ignored in childhood. CASE REPORT: A 11 year-old boy was referred because he suffered from a tumor of the neck, which had progressively developed after a cervical traumatism occurring 4 years earlier. Examination showed a tumor moderately infiltrated, attached to the overlying erythematous skin. Biopsy showed ill-limited dermal and hypodermal proliferation of fusiform cells with a storiform arrangement. Two successive excisions were necessary and the patient is well 2 years later. CONCLUSION: This case confirms responsibility of previous traumatism, progressiveness of course and necessity of large excision. PMID- 7550842 TI - [Adenocarcinoma on villous tumor of the rectum in an adolescent]. AB - CASE REPORT--A 15-year 6-month-old boy suffered from isolated recurrent rectal bleeding. Rectoscopy and colonoscopy allowed to identify and to excise one large size villous polyp, which was the site of dysplasia and liberkuhn adenocarcinoma. Transrectal ultrasonography showed thickened mucosa and submucosa and suspect adenopathy. Proctectomy, ganglionic curage and coloanal anastomosis were then performed; one of the excised adenopathies was metastatic. The patient died twelve hours after surgery from an unexplained cardiovascular collapse. CONCLUSIONS--Unspecific symptoms of colorectal cancer explain that its diagnosis is often delayed. Prognosis is poor related to the advanced stages at diagnosis and mucinous adenocarcinomas. Better knowledge of the clinical presentations and of high risk situations as polyposis, ulcerative colitis, hereditary non polyposis colorectal cancer could improve the prognosis. PMID- 7550843 TI - [Scorpion stings in children. Saudi Arabian experience]. AB - Scorpion stings are a major public health problem in Saudi Arabia. The main victims are children who accidentally tread on a scorpion, about 900 children being admitted to hospitals each summer for such stings. Many cases are fatal. The pathophysiology of scorpion stings is complex and its management is difficult. Almost all body systems are affected but circulatory shock and pulmonary oedema are the usual cause of death. The treatment is based on scorpion antivenom and symptomatic treatment of the envenoming manifestations in an intensive care unit. The currently used antivenom is a polyvalent horse serum preparation given intravenously. PMID- 7550844 TI - [Perioperative fasting in children: current data]. AB - Perioperative fasting aims at decreasing the incidence of gastric content inhalation during anesthesia. Current knowledge concerning gastric emptying and the epidemiology of pulmonary aspiration authorizes new perioperative fasting guidelines. If prolonged fasting does not guarantee gastric emptiness at the induction of anesthesia, shortening preoperative fasting by allowing clear fluids two hours before surgery does not modify gastric content and does not increase the risk of gastric content aspiration, while enhancing the patient comfort by reduction of the fasting period. On the other hand, after surgery, the mandatory intake of water significantly increases the incidence of postoperative vomiting. Therefore new guidelines may be applied for children operated in ambulatory surgery settings: 1) clear fluids may be allowed until 2-3 hours before operation, 10 ml.kg-1, or even ad libitum for some authors (by clear fluids one means water, tea, coffee, apple juice, syrup with water); 2) drinking is not absolutely necessary before discharge from day care surgery unit and should be left to the child's own assessment. PMID- 7550846 TI - [Radiological case of the month. Cystic lymphangioma of the omentum]. PMID- 7550845 TI - [Magnetic resonance spectroscopy: a new technique for the exploration of brain metabolism in pediatrics]. AB - In vivo nuclear magnetic resonance spectroscopy (MRS) has been developed recently in order to analyze brain metabolism in adults and children. After a short presentation of the methods and of the metabolic signals detected by phosphorus and proton MRS of the brain, the impact of MRS in pediatrics is examined with two main indications: the study of cerebral maturation in normal or pathological neonates, and the study of inborn metabolic encephalopathies. In the near future, brain MRS will be performed routinely after conventional MRI, and will become a valuable metabolic complement to the anatomical evaluation of cerebral pathologies. PMID- 7550847 TI - [Treatment of febrile convulsions in infants]. AB - Guidelines for the management of febrile seizures are presented. Intrarectal administration of diazepam (0.5 mg/kg) is the first line therapy for complex or prolonged seizures. Preventive treatment of recurrence is mainly indicated in case of complex seizures using either prolonged daily monotherapy, preferentially sodium valproate, or intermittent administration of diazepam during febrile illnesses. PMID- 7550849 TI - [Early hematological consequences in infants born to HIV seropositive mothers treated with zidovudine during pregnancy]. PMID- 7550850 TI - [Metabolic control in young diabetics by insulin therapy: follow up after two years]. PMID- 7550848 TI - [Resistant pneumococcal meningitis revealing sickle cell anemia: efficacy of cefotaxime-vancomycin combination]. PMID- 7550851 TI - [Death during sickle cell anemia crisis: possible role of morphine?]. PMID- 7550852 TI - Effects of Helicobacter pylori infection on gastric mucosal defense factors in Japanese monkeys. AB - The pathogenic role played by Helicobacter pylori in gastric mucosal defense was investigated in Japanese monkeys infected with H. pylori. Serum gastrin levels and ammonia concentrations in gastric juice were compared in H. pylori-infected (n = 6) and control (n = 7) groups. The gastritis score, the intracellular content of periodic acid-Schiff (PAS)-positive substance and hexosamine, and the bromodeoxyuridine (BrdU) labeling index in the gastric mucosa were compared in the two groups in the antrum and the corpus. The ammonia concentration in the gastric juice was significantly higher in the infected group (P < 0.01). The gastritis scores were significantly higher in the antrum and corpus in the infected group (P < 0.01, and P < 0.05, respectively). The content of PAS positive substance and hexosamine was significantly decreased in the antrum of the infected group compared with that in the controls (P < 0.01, and P < 0.05, respectively), but there was no significant difference between the two groups in the corpus. The BrdU labeling indices were significantly higher in the antrum and corpus of the infected group (P < 0.01, and P < 0.01, respectively). Colonization by H. pylori injures the gastric mucosa by depressing the gastric mucosal defense factors, and, consequently, the cell kinetics are accelerated. PMID- 7550853 TI - Cholecystokinin is not a physiological regulator of gastric pepsin secretion in rats. AB - The physiological relevance of cholecystokinin (CCK) in gastric pepsin secretion is unclear, although CCK has been reported to stimulate pepsin secretion in intact animals and in dispersed chief cell. To clarify the physiological role played by this peptide in pepsin secretion, we determined the effects of intravenous infusions of CCK on gastric pepsin release, and investigated the effect of endogenous CCK released by small amounts of trypsin inhibitor on pepsin secretion in conscious rats. The infusion of CCK-8 at 1 nmol/kg per h resulted in a plasma CCK concentration of 204 pM and a 2.5-fold increase in pepsin secretion compared to the baseline rate. The infusion of CCK-8 at 0.3 nmol/kg per h resulted in a plasma CCK concentration of 41.8 pM and also caused a significant increase in pepsin secretion compared to the baseline rate. However, the infusion of CCK-8 at 0.1 nmol/kg per h (plasma CCK level, 19.9 pM), which is still far beyond the physiological plasma levels of CCK, did not significantly affect pepsin secretion. In addition, the intraduodenal infusion of soybean trypsin inhibitor increased the plasma CCK concentration to 4.4 pM, a value comparable to that observed after feeding (3.3 pM), but again, this had no effect on gastric pepsin secretion. We conclude that CCK is not a physiological regulator of gastric pepsin secretion in rats. PMID- 7550854 TI - Serum pepsinogen in screening for gastric cancer. AB - To establish a sensitive and efficient screening method for gastric cancer using serum pepsinogen, we investigated the characteristics of serum pepsinogen I and II levels and the I/II ratio and their cut-off points. We found that the pepsinogen I level and the I/II ratio were significantly lower in patients with gastric cancer than in control subjects, especially in patients with cancers of the differentiated type, the elevated type, and the depressed type without ulceration. However, sex, depth of invasion, and location of tumor did not correlate with the pepsinogen levels. A suitable cut-off point in screening for gastric cancer was a pepsinogen I level of less than 50 ng/ml and a I/II ratio of less than 3.0, as determined by receiver operator characteristics curves. The sensitivity, the specificity, and the accuracy of detection for all types of gastric cancer were approximately 55%, 75%, and 72%, respectively. If restricted to cancers of the elevated and the depressed type without ulceration, the sensitivity was approximately 85%, and the specificity and accuracy were approximately 76% and 77%, respectively. These results suggest that, in screening for gastric cancer when using pepsinogen levels and morphological examinations, the suitable cut-off point in regard to specificity is as stated above. However, regarding sensitivity, when the pepsinogen method is used alone, a pepsinogen I level of less than 70 ng/ml and a I/II ratio of less than 3.0 is acceptable. PMID- 7550855 TI - A proton pump inhibitor, E3810, has antibacterial activity through binding to Helicobacter pylori. AB - Helicobacter pylori infection is causally related to atrophic gastritis, and it may also be associated with peptic ulcer and gastric carcinoma. Eradication of H.pylori is recommended in patients with such diseases, especially in those with peptic ulcer. A new potent proton pump inhibitor, E3810, had an antibacterial effect on H. pylori, as has been reported for omeprazole and lansoprazole, two other proton pump inhibitors. The minimum inhibitory concentration of E3810 was 1.57-3.13 micrograms/ml, lower than that of omeprazole or lansoprazole. To clarify the mechanism of the antibacterial effect of E3810, we analyzed the characteristics of E3810 binding to H. pylori. Scatchard plot analysis of this binding showed a curvilinear profile, indicating the presence of several molecules with different affinities to E3810 on H. pylori. The binding capacity of E3810 to H. pylori was calculated to be about 2 x 10(6) sites/cell. These results suggested that E3810 has an antibacterial effect against H. pylori and that the effect may be mediated through direct binding to H. pylori. PMID- 7550856 TI - Fulminant second-set allograft rejection and endoscopic findings following small bowel transplantation in the rat. AB - In the presensitized recipient who has been exposed to donor antigens, second-set rejection takes the form of severe hyperacute graft rejection. Second-set allograft rejection was studied following small bowel transplantation in the rat. Heterotopic intestinal grafting was performed from DA (RT1a) donors to PVG (RT1c) recipients 4 weeks after DA skin sensitization. The endoscopic images and histological specimens were compared with those of syngeneic and first-set rejected grafts. Endoscopically, diffuse erosions of the graft were detected from day 1. Mucosal necrosis progressed rapidly, and was accompanied by massive bleeding on days 3-5. These findings were similar to the course of severe necrotizing hemorrhagic enteritis. Histologically, interstitial edema and hemorrhage with massive infiltrations of neutrophils were manifested from day 1. Mesenteric vessels were completely occluded by thrombi on days 3-5. The grafted intestine had became totally necrotic by day 5. Microscopic findings strongly suggested that destructive graft necrosis was due to vascular damage caused by humoral factors. All the presensitized rats (n = 11) died showing systemic septic signs by day 11 after small bowel transplantation. We conclude that lethal hyperacute rejection occurred in presensitized recipients, even when the graft was transplanted heterotopically. Endoscopic evaluation is beneficial for the early diagnosis of graft rejection. Immediate graft removal should be mandatory as a rescue treatment in second-set rejection of the small intestine. PMID- 7550857 TI - Rabbit granulomatous enterocolitis induced by injection of muramyl dipeptide emulsified with Freund's incomplete adjuvant. AB - We induced granulomatous enterocolitis in rabbits by injecting them with muramyl dipeptide (MDP), a subunit of the peptidoglycan polymers that endow the bacterial cell wall with structural rigidity, emulsified with Freund's incomplete adjuvant (FIA). Injections of 0.1 ml of a water-in-oil emulsion of MDP and FIA were given submucosally at six sites in the rectum and colon, 10 cm proximal to the anus, using a flexible endoscope. Four rabbits each were sacrificed 1, 2, and 4 weeks after a single injection of the emulsion. Another 4 rabbits each were injected six times at 1- and 2-week intervals, and were sacrificed 1 and 2 weeks after the last injection of the emulsion, respectively. In all 20 rabbits, injected with the MDP emulsion, histological findings of the colon consisted of cellular infiltrations of plasma cells and lymphocytes, granulomatous lesions, and granulomas, although the findings differed in degree. Cellular infiltration in hyperplastic villi and denuded epithelia of the small intestine were seen in 2 of 8 rabbits repeated that received MDP emulsion injections. The histological changes in this animal model may be useful for studying the pathogenesis of inflammatory bowel disease in humans. PMID- 7550859 TI - Efficacy of combination therapy with interferon and azidothymidine in chronic type C hepatitis: a pilot study. AB - The effects of interferon are seen in only a limited number of patients with hepatitis C virus (HCV) of the K1 type, indicating that a combination therapy with other antiviral drugs may be essential to obtain better results. In the present pilot study, the effects of combination therapy with interferon (IFN) and an antiviral drug azidothymidine (AZT) were analyzed. The combination therapy was conducted in 22 patients with chronic hepatitis C after obtaining their informed consent (combination group). Three or six million units of natural IFN alpha was administered daily for 3 weeks and then three times a week for 21 weeks. Combination therapy was initiated at the beginning of the 8th week of IFN treatment, 500 mg of AZT per day being given for 8 weeks. As a control, changes in HCV-RNA were also analyzed in patients treated with interferon alone (IFN alone group). At the end of the treatment, blood was negative for HCV in 32.5% of the IFN-alone group and in 50.0% of the combination group, the difference not being significant. However, in patients with HCV-K1, HCV-negative rates were 14.2% in the IFN-alone group and 45.5% in the combination group, showing a significant difference. In patients with other HCV genotypes, HCV-negative rates did not different between the two groups. These results suggest that combination therapy with IFN and AZT may be an effective treatment for chronic type C hepatitis caused by the K1 type virus, although further studies on larger number of patients will be needed to obtain definite conclusions. PMID- 7550858 TI - Clinical significance of the trimethadione tolerance test in chronic hepatitis: a useful indicator of hepatic drug metabolizing capacity. AB - Trimethadione (TMO) was chosen as an indicator of quantitative hepatic microsomal function, and its pharmacokinetics were studied in 52 patients with chronic hepatitis. Findings in these patients were compared with those for 26 healthy subjects and 13 patients with renal failure. Patients with chronic hepatitis, but not those with renal failure, showed significant reduction in clearance (CL) and prolongation of half-life (t1/2), and the extent of abnormalities was found to reflect the severity of histologic changes in liver tissue. The serum dimethadione (DMO)/TMO ratio 4 h after the administration of TMO altered in parallel with the CL and t1/2 of TMO, and abnormalities in this simple ratio were also related to the histologic severity of changes in the liver tissue. A low DMO/TMO ratio (< 0.4) was associated with advanced histologic changes (chronic active hepatitis with bridging or chronic active hepatitis with cirrhosis), whereas a high DMO/TMO ratio (> 0.4) was associated with mild histologic changes (chronic persistent hepatitis or chronic active hepatitis) (sensitivity, 0.81; specificity, 0.86). These results indicate that the DMO/TMO ratio, which can be obtained from a single blood sampling, reflects the histologic severity of changes in tissue liver, and that the TMO tolerance test is a useful indicator of quantitative liver function. PMID- 7550860 TI - Effects of short-term administration of the CCK receptor antagonist, KSG-504, on regeneration of pancreatic acinar cells in acute pancreatitis in rats. AB - Cholecystokinin (CCK) receptor antagonists have been reported on have an inhibitory effect on acute experimental pancreatitis, but their long-term administration is also reported to block pancreatic regeneration. We examined whether the short-term administration of KSG-504 (KSG), a synthetic CCK-A receptor antagonist, inhibited the regeneration of pancreatic acinar cells after ethionine-induced acute pancreatitis in rats. KSG (50 mg/kg), given 12 times by subcutaneous injection at 6-h intervals, prevented the reduction of protein, amylase, and trypsinogen levels, and the DNA content of the pancreas and facilitated the recovery of these values. Ornithine decarboxylase activity in pancreatic tissue and a 5-bromo-2'-deoxyuridine labeling study indicated that DNA synthesis was accelerated in rats treated with KSG. These findings suggest that the short-term administration of KSG inhibits the development of ethionine induced acute pancreatitis and facilitates the regeneration of acinar cells. PMID- 7550862 TI - Esophageal stricture secondary to candidiasis without underlying disease. AB - Candidiasis of the esophagus progressing to stricture formation in a 74-year-old male is reported. Esophageal candidiasis develops in the presence of various predisposing conditions such as long-standing administration of antibiotics or corticosteroids, and malignancy. The first endoscopic examination of this patient revealed unusual multiple black plaques in the esophagus. Despite intensive exploration, no predisposing factors were found. The stricture was progressive, despite the administration of adequate antifungal therapy, and its presence necessitated several attempts at dilatation. Case reports of esophageal candidiasis without underlying disease are very rare. PMID- 7550861 TI - Comparative effects on biliary concanavalin A-bound glycoproteins and calcium ion on cholesterol crystal nucleation and growth in model bile. AB - The concanavalin A-bound glycoproteins in human gallbladder bile have recently been demonstrated to be strong promoters of cholesterol crystal nucleation. In the present study, we investigated the mechanism(s) whereby such promoters affect cholesterol crystal nucleation and/or growth, and compared these mechanisms with those of another promoter, calcium ion. Concanavalin A-bound glycoproteins were isolated from the Helix pomatia-unbound fraction of gallbladder bile from stone free patients, and determined by electrohoresis to consist of six subclasses (MW 143, 98, 80, 58, 50, and 40 kDa). A cholesterol crystal growth assay showed that concanavalin A-bound glycoproteins both accelerated nucleation time and increased growth rate, whereas calcium ion affected nucleation time only. In the presence of both concanavalin A-bound glycoproteins and calcium ion, both cholesterol nucleation and growth were markedly enhanced. A gel permeation chromatographic study revealed that concanavalin A-bound glycoproteins shifted a considerable amount of cholesterol from micelles to vesicles, whereas calcium ion did not. These results suggest that concanavalin A-bound glycoproteins promote cholesterol crystal nucleation and growth, partly by shifting cholesterol from stable micelles to metastable nonmicellar fractions in bile. In contrast, calcium ion promotes these processes by other mechanisms and, therefore, enhances the effect of concanavalin A-bound glycoproteins. PMID- 7550864 TI - Undifferentiated carcinoma of the duodenal ampulla. AB - This report demonstrates a case of undifferentiated carcinoma of the duodenal ampulla. A 74-year male experienced jaundice lasting for 3 weeks. An upper gastrointestinal series demonstrated a polypoid, ovoid filling defect in the second portion of the duodenum, and duodenoscopy disclosed a protruding mass involving the orifice of the papilla of Vater. Cholangiography demonstrated obstruction due to compression in the terminal common bile duct. Pylorus preserving pancreatoduodenectomy was performed on the diagnosis of ampullary carcinoma. The gross specimen showed a polypoid mass, measuring 3.5 cm in diameter, in the ampulla, located mainly in the duodenal submucosal layer and invading the terminal common bile duct. Histologically, the tumor was small cell type, undifferentiated carcinoma, arising from the duodenal epithelium adjacent to the ampulla. PMID- 7550863 TI - Pericardiac metastasis from advanced gastric cancer. AB - A 64-year-old man complaining of anterior chest pain, weight loss, and neck tumors was found to have advanced gastric cancer with pleuritis carcinomatosa and multiple lymph node and bone metastases. The patient was treated with combination chemotherapy consisting of mitomycin C (MMC), tegafur (UFT), and lentinan, and then with MMC and 5-fluorouracil (5FU) instillation into the pleural spaces after pleural drainage. With these treatments, the primary tumors and cancerous ulcers of the stomach improved markedly, and the lymph node enlargement and pleural effusion disappeared completely. Afterwards pericardiac metastasis complicated by cardiac tamponade occurred, but repeated pericardiocentesis and administration of MMC into the pericardiac cavity effectively eliminated the effusion. These treatments appeared potentially useful for advanced gastric cancer with generalized metastases including pericardiac involvement. However, the patient died of cardiac tamponade with massive pericardiac bleeding, probably due to the repeated pericardiocentesis and/or the administration of anticancer drugs. PMID- 7550865 TI - Colonic and peritoneal tuberculosis associated with coloduodenal fistula. AB - We report a very rare case of tuberculous colitis that showed relatively long segment involvement of the colon near the hepatic flexure with coloduodenal fistula that caused severe malnutrition. The formation of fistula in abdominal tuberculosis is very rare. This is the eighth reported case of abdominal tuberculosis with fistula and the first reported case with a coloduodenal fistula. PMID- 7550867 TI - Venous tumor thrombosis and cavernous transformation of the portal vein in a patient with gastric carcinoma. AB - A case of extensive extra- and intrahepatic portal tumor thrombosis, with no metastatic foci in liver parenchyma, secondary to advanced gastric carcinoma in a 69-year-old man is reported. The portal tumor thrombosis was characterized by enlargement of the thrombosed segment of the vein, decreased density mass without intraluminal enhancement of the involved vein, nonvisualization of the portal venous branch in the involved lobe, and the so-called cavernous transformation of the portal vein. The surgically resected gastric specimen showed Borrmann type 3 advanced papillary adenocarcinoma. The portal tumor thrombus is presumed to have arisen from vascular invasion in the primary foci of gastric carcinoma, and then to have permeated the portal vein without invasion of liver parenchyma. PMID- 7550866 TI - Primary sclerosing cholangitis with marked eosinophilic infiltration in the liver. AB - A 16-year-old boy was diagnosed as having primary sclerosing cholangitis (PSC), based on retrograde cholangiography showing mixed features of narrowing and dilatation of the common hepatic and intrahepatic bile ducts. However, periductal fibrosis was not observed in the needle biopsy liver specimen. The liver biopsy specimen obtained 11 years previously, at the onset of the disease had disclosed a marked infiltration of eosinophils in the portal tract with eosinophilic catinonic protein immunostaining, with marked eosinophilia (54%) being noted. In Japanese reports, eosinophilia of more than 7% was reported in 13 of 32 (40.6%) PSC patients. However, the early stage of PSC, with marked eosinophilia and eosinophilic infiltration in the liver, such as in the present case, has rarely been reported. The findings in this case suggest that eosinocytes are related to the pathogenesis of PSC. PMID- 7550868 TI - Hepatitis E probably contracted via a Chinese herbal medicine, demonstrated by nucleotide sequencing. AB - Hepatitis E is endemic in developing countries and may occur as imported hepatitis in industrialized countries. A 46-year-old Japanese man developed immunoserologically diagnosed acute hepatitis E in Japan 4 months after he had made a trip to China. He had bought a Chinese herbal medicine there, taking it occasionally until approximately 6 weeks prior to the onset of acute hepatitis. Nucleotide sequencing of the 3' terminal region of the viral cDNA amplified from the patient's serum by polymerase chain reaction revealed a high degree of homology (99.8% of 752 nucleotides) with the Chinese strain. Thus, the results of sequencing suggest that his hepatitis E was caused by infection with the Chinese strain, via the Chinese herbal medicine. PMID- 7550869 TI - Acute hepatitis in an adult with acquired rubella infection. AB - We report a case of acute hepatitis in a 28-year-old male with acquired rubella infection. Serological tests revealed acute rubella virus infection and ruled out infection by other common viruses, including type A and type B hepatitis viruses. The patient showed not only marked increase of lactate dehydrogenase (LDH) activity, with only slight liver dysfunction, but also platelet and kidney injury, suggesting systemic rubella virus infection. Because the liver dysfunction was slight, liver biopsy was not performed. When a patient has mild, transient hepatitis accompanied by high LDH activity in comparison with both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, we should take a common viral infection such as rubella into consideration when making a diagnosis. PMID- 7550870 TI - Heterotopic gastric mucosa of the gallbladder. AB - A case of heterotopic gastric mucosa in the fundus of the gallbladder is reported. A 23-year-old man, who had been healthy and asymptomatic, visited our hospital because of abnormal findings in a liver enzyme test given during a routine health screening. Ultrasonography demonstrated a highly echogenic polypoid mass in the fundus of the gallbladder. The gallbladder mass was confirmed by both computed tomography and intravenous cholangiogram. After a 10 month follow up, laparoscopic cholecystectomy was performed. Intraoperative touch smear cytology of this lesion revealed class II cells. The surgical specimen revealed a 15 x 10 x 5 mm polypoid lesion in the fundus, with no gallstones in the gallbladder. Histologically, the polypoid lesion consisted of both fundic type and pyloric type gastric glands located in the mucosa of the gallbladder. In the literature, 42 cases of heterotopic gastric mucosa of the gallbladder have been reported, only 3 of which, including this present case, were found incidentally, with no apparent symptoms. PMID- 7550871 TI - Adenomyoma of the common hepatic duct. AB - A very rare case of adenomyoma of the common hepatic duct is described. A 54-year old woman was admitted with impending obstructive jaundice secondary to adenomyoma of the common hepatic duct. Our impression, formulated from her clinical presentation, endoscopic investigations, and biochemical and radiological findings, was a cancer of the proximal common hepatic duct. The patient was treated successfully by combination surgical resection and hepaticojejunostomy. Despite our obtaining an intraoperative frozen section, final histological examination was required to confirm the diagnosis. The patient remains well 16 months postoperatively. A survey of the world literature revealed that this is the second report of adenomyoma occurring in the common hepatic duct. PMID- 7550872 TI - High nitric oxide synthase activity in endothelial cells in ulcerative colitis. AB - Endothelial nitric oxide (NO) synthase, a unique NO synthase (NOS) isoform that is expressed constitutively by the vascular endothelium both in vivo and in vitro, is believed to be essential to systemic and/or local vascular integrity. NOS expression by endothelial cells may indicate vascular activation. We successfully established a simple method for the culture of microvascular endothelial cells from a small amount of tissue and investigated ulcerative colitis (UC), in which condition vascular factors have not been studied extensively. We cultured endothelial cells from the mesenteries of surgical patients with UC and assayed NOS activity by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Strong NOS activity was demonstrated in the cells from all UC patients (5/5), whereas no activity was detected in the cells from human umbilical veins and the mesenteries of colon cancer patients (0/10 and 0/5, respectively). This strong NOS activity was not diminished by incubation with a high concentration of glucocorticoid, suggesting that it was constitutive. These results indicate a close relationship of vascular activation (high NOS activity) with the pathogenesis of UC. PMID- 7550873 TI - Porcelain onlays. AB - For years porcelain-fused-to-metal crowns have been the only restoration for posterior teeth in which strength and esthetics were important. Predictable results can now be achieved with a much more conservative approach--the bonded porcelain onlay. This article reviews the preparation designs as well as several types of materials currently available for the cosmetic onlay. An application using porcelain onlays to restore vertical dimension is explored. With this technique, moreover, the porcelain onlay is combined with porcelain veneer to achieve a functional and esthetic restoration. PMID- 7550874 TI - Marketing the cosmetic practice via television. AB - Everyone is marketing their dental practice whether they know it or not. It is far better to design the desired effect than to send mixed messages to your clients. All marketing must be carried out with a plan or mission that is internalized by the doctor and by each member of the staff. The mission coupled with creative talent becomes the proprietary personality of your office and the focus of your marketing efforts. Internal marketing is perhaps the most economical and effective--and most used--method of creating positive results for the practice. External marketing takes your office out to the public most often in print via newsletters, direct mailings, and the yellow pages. Getting the message out through newspapers, radio, and television has and will become increasingly important. This article takes you through the steps we used to identify and market to our cosmetic clients. The results of our demographic and psychographic studies afford unique insight into the type of person attracted by our services. Marketing to this group has to do with diversity in appeal, "the boomers," and experiential motivation. PMID- 7550875 TI - Differing porcelain systems. AB - Current research in ceramics has centered on improving the strength, fit, and bondability of porcelain. Several different approaches to accomplishing these ends have led to the development of stronger cores, castable glass, computer generated restorations, and chemically altered basic ceramics. Improvements in these basic parameters of clinical success have led to the use of all-ceramic systems such as inlays, onlays, conservative veneers, anterior and posterior crowns, and even fixed partial dentures. Porcelain veneers and full-coverage ceramic crowns have held up well clinically. Porcelain inlays and fixed partial dentures have presented problems with fit and strength, respectively, that may need correcting before they are clinically predictable in all situations. Nevertheless, it is apparent that modern dental porcelain technology has come a long way since it was originally introduced as an esthetic replacement for less cosmetic, metal-based restorations. PMID- 7550876 TI - Esthetic periodontics. AB - Periodontal therapy has developed beyond the scope of the treatment of periodontal pathoses. Periodontal plastic surgery has evolved beyond the techniques of mucogingival surgery and now consists of the regenerative and reconstructive procedures designed to enhance esthetics. This article briefly reviews the historical development of root coverage, ridge augmentation, and papilla preservation and provides an update of the current pertinent literature. PMID- 7550880 TI - Restoring the aging dentition. AB - The aging of the population has put new demands on the range and capabilities of the dentist. Patients are no longer satisfied with the straightforward restoration of their mouths. They are demanding a more youthful appearance as an essential element of therapy. To provide this element, the dentist must have a thorough understanding of the physiology of tooth and facial aging, the treatments available, and the problems and limitations that might be incurred. PMID- 7550879 TI - Color management of cosmetic restorations. AB - Ideally, a cosmetic restoration should be indistinguishable from the surrounding unrestored dentition. The cosmetic dentist faces the challenging task of creating the restoration using materials that do not possess the light-transmitting properties of natural tooth structures. A thorough knowledge of the physical and physiologic properties of light, color production and perception, and skill in the art of color matching can lead to rewarding success. PMID- 7550878 TI - Treating dental disharmony with mixed media. AB - The art of designing a new smile often involves more than one cosmetic dental procedure. It is not uncommon to combine media such as tooth whitening with porcelain veneers and crowns to create an entirely new smile. To maintain dental harmony, it is paramount that media are indistinguishable from each other and are consistent with the existing dentition. PMID- 7550881 TI - Methods and materials for porcelain veneers. AB - Porcelain veneers are an esthetic, relatively conservative restoration used to cover the facial surface of the teeth. Following proper guidelines, the practitioner can achieve successful results as a matter of routine. This article reviews the most current literature published on this subject to bring practitioners up to date on clinical methods and materials used for porcelain veneers. PMID- 7550877 TI - Anterior esthetics and the visual arts: beauty, elements of composition, and their clinical application to dentistry. AB - The challenge of developing a pleasing smile is an artistic venture. A study of how the visual arts have explored the nature of beauty and the elements of artistic composition will enhance our artistic abilities in cosmetic dentistry. This review discusses the perception of beauty and important features of that which we call beautiful. The discussion uses important works of art to demonstrate elements of composition, which are then made relevant in a dental application. PMID- 7550882 TI - Indirect composite restorations. AB - The evolution of composite and adhesive technology has benefited dentistry significantly. The 1990s have brought us, for the first time in the history of our profession, the ability to restore tooth structure in a conservative and esthetic fashion. The ability to give consistently high-bond strengths to both enamel and dentin and the excellent physical properties of these restorations have made them a permanent part of restorative dentistry. This paper summarizes the authors' current opinion about indirect composite restorations, outlines indications for their use, and describes a clinical technique for their preparation and cementation. PMID- 7550883 TI - Porcelain veneer adhesion systems. AB - The outstanding bonds achieved by composite resin luting systems to etched enamel and to etched and silanated porcelain are well documented. These bond strengths, in concert with the excellent and ever-improving dentin bonding systems, encourage the use of porcelain veneers in a continually expanding range of clinical situations and ensure predictable results. Clinicians desiring to offer their patients ultimate cosmetics in conjunction with optimal, conservative restorative techniques will need to monitor scientific and clinical results obtained by leaders in the field of adhesive dentistry and continually update their technique. This review brings the clinician up to date on current research and gives the clinician an understanding of the components of today's adhesive systems technology. PMID- 7550884 TI - Advances in adhesive technology. AB - Advances in adhesive technology have occurred at a remarkable pace over the past few years. It seems as though every month brings a "new" and "better" bonding system onto the market. Clinical protocol is constantly changing. Just when clinicians have mastered one technique, they find it has been supplanted by another. This often creates confusion and suspicion on the part of the dental practitioner. There are, however, some fundamental concepts applicable to almost all adhesive systems. This paper attempts to present a lucid and comprehensible review of some of these concepts. Many of the latest innovations, such as total etch and wet bonding, are discussed. This paper is not intended as a comprehensive review of all aspects of adhesive dentistry. (For example, adhesion to metals is not covered.) It is my hope that readers will come away with a basic understanding of some current concepts and beliefs in the constantly evolving field of adhesive dentistry. PMID- 7550885 TI - The high-tech dental office. AB - The 1990s ushered in technologic advances affecting the forefront of dentistry. This technology was the harbinger of a change in dentistry from a need-driven to a want-driven profession. As the twenty-first century approaches, dentistry is embracing these novel advances. Concurrently, the multifarious high-technology equipment brings with it the responsibility for the dentist to educate patients about potential dental benefits that can further enhance their overall health. PMID- 7550886 TI - A new esthetic impression system. AB - Communication between the restorative dentist and the laboratory technician is essential to establish a predictable esthetic result. Errors in the laboratory can create the need for excessive clinical try-ins and modifications that waste valuable time. This paper reviews the use and potential benefits of a new facial impression system to minimize such esthetic errors. This system provides the laboratory technician with a three-dimensional reference of the local soft tissue frame during the fabrication of restorations. The application of this system to orthognathic surgical template fabrication is also discussed. The objective is to obtain the essential esthetic qualities of dentofacial harmony in the laboratory and not at the chairside. This system is compared with other methods of communication. PMID- 7550887 TI - Repairing porcelain and ceramic materials. AB - Technologic breakthroughs in bonding and ceramics have given us materials and techniques that are more reliable in the long term. Nevertheless, we may still be confronted with fractured composites or porcelain. For reasons of finance, technology, psychology, or time, we may find it difficult to abandon the restoration and redo the job. We are then confronted with the problem of repairing the esthetic material, either temporarily or permanently. This paper reviews the recently published articles on techniques and materials used in the repair of dental restorations. PMID- 7550888 TI - Esthetic diagnosis. AB - To ensure esthetic success, dental practitioners must respond to patient inquiries with a codiagnostic approach, allowing the clear identification of esthetic problems and the visualization of solutions. This structured approach involves a therapeutic mind set whereby a common esthetic language is established, and a communication triangle is formed between the patient, the dentist, and the technicians. The problem is clearly identified through the use of the esthetic evaluation form, diagnostic casts, and computer imaging. The solutions are visualized and treatment methods established through a diagnostic wax-up and computer imaging. This approach works by building a high-energy, high trust relationship between the doctor and the patient, which serves to increase the probability of successful and consistent treatment and improves the dentist's general understanding of esthetic concepts. PMID- 7550889 TI - A case study of lasers in cosmetic dentistry. AB - The dental laser offers revolutionary advantages over traditional cosmetic dental treatment for our patients. These advantages include precision, hemostasis, sterility, and minimal postoperative pain and swelling. The laser interacts with tissue to vaporize it in a predictable manner that produces predictable results. It is used in periodontal plastic surgery, gingivectomy, soft-tissue crown lengthening, crown and bridge gingival retraction, composite curing, biopsy, frenectomy, stage II implant recovery, and more. Fixed prosthetic impressions can be taken immediately following gingivoplasty and sutures and postoperative dressings are eliminated. This paper presents a case study in cosmetic dentistry to demonstrate many of these applications and summarizes current opinion regarding them. It also briefly describes what a laser is and how it works. PMID- 7550890 TI - [Assessment of factors determining the dynamics of pollution by Cs-137 of agricultural production after the Chernobyl AES accident]. AB - Analysis of the dynamics of 137Cs content in the main kinds of plant and animal products obtained on the basis of a separate farm located at 40-50 km to the north from Chernobyl NPP has shown that in 1987-1991 the decrease of 137Cs transfer from soil to agricultural crops was on the average 2--4-fold, and for milk--6--7-fold during the pasture period and 10-fold--during stabling one. The half-life periods of decrease of 137Cs constant in the main kinds of plant production vary from 2.1 to 5.9 years; for milk this parameter equals to 1.4 years in the pasture period, and in the stabling one--2.8 years. The main contribution (67-80%) to the decrease of 137Cs transfer to plant products is made by biogeochemical processes causing decrease of mobility of 137Cs in soil; as for agrochemical countermeasures, these contribute 20--33%. Decrease of contamination of milk, on the contrary, is mainly due to realization of countermeasures- contribution of the latter reaches 60%. PMID- 7550891 TI - [Regularities of changes in Cs-137 contents in animal products in the territory of the Russian Federation polluted as a consequence of the Chernobyl AES accident]. AB - Dynamics of 137Cs content in main types of animal production obtained in region of Russia contaminated as a result of the Chernobyl NPP accident, 1986, is described. Decrease of contamination of agricultural production during 1987--1992 was of not regular character. The most rapid decrease of 137Cs content in production was registered in the first post-accidental period which was connected with intensive realization of countermeasures in agriculture at that time. Half life periods of 137Cs content (major dose-forming radionuclide) decrease in production for areas of Russia subjected to the most intensive contamination amounted of 1.6 to 4.8 years, as dependent on the scale of countermeasures carried out. PMID- 7550892 TI - [Radiobiological constant in irradiation stimulation of cells for the subsequent adaptive response]. AB - It was shown that the maximum adaptive response to damaging irradiation dose would be achieved after preirradiation when for latter the product of the dose by the dose rate (i. e. D*D/t) is equal to constant value. The radiobiological constant is the reciprocal of this product, i. e. k = t/D2 = tau/delta 2, where delta is an average dose received by the cell for single pass of ionizing particle through the cell sensitive volume with a given density. The triggering signal for the adaptive response program initiated by preirradiation is a certain reaction, which is completed for a time interval tau between two consistent acts of absorption. The radiobiological constant is determined by this time interval tau and by a dose delta. In human lymphocytes in the case of signal initiated by X-rays the value of tau is within the range of 1--5 s. PMID- 7550893 TI - [DNA analysis of retroposon-like genetic LINE elements in blood plasma of rats exposed to radio-diapason electromagnetic waves]. AB - The elevation of LINE-elements' DNA level was revealed in blood plasma of rats exposed to electromagnetic waves. The amount of full-size 5'-containing LINE elements copies was increased especially. Connection of this effect with retrotransposon activation and genetic instability condition of organism development is supposed. PMID- 7550894 TI - [Changes in structural and functional properties of the erythrocyte membrane under the action of ionizing radiation]. AB - The effects of lethal and sublethal doses of ionizing radiation on structural and functional characteristics of erythrocytes membranes was studied. Statistically reliable increase in K+ escape from cell and decrease in membrane potential. Model membranes made of phospholipids extracted from irradiated membranes exhibit increased ion permeability. Radiation induced changes in some membrane phospholipids content were revealed. PMID- 7550895 TI - [Features of ontogenesis of the progeny of both irradiated parents]. AB - Acute gamma-irradiation with a dose of 4 Gy of germinal cells of both parents in postmeiotic stages of gametogenesis results in increase of ineffective copulation rate. Antenatal and postnatal ontogenesis of first generation progeny shows worse consequences as compared with only one parent germinal cell irradiation. PMID- 7550896 TI - [Radiation effects in ontogenesis of the progeny of one or both irradiated parents]. AB - The purpose of the investigation is the study and comparative analysis of radiation effects observed in antenatal and early postnatal ontogenesis of first generation progeny when one or both parents have been irradiated in doses 2, 3, 4 Gy. It was shown that the effects for the posterity of both exposed parents were attributed mainly to a female if mature oocytes were exposed to radiation, or to a male if maturing oocytes were exposed. If both parents have been irradiated at the spermatid, spermatozoid or mature oocyte stages and got 4 Gy aggravated effects in progeny are observed. It is may be related to synergistic mechanism of realization of this phenomenon. PMID- 7550897 TI - [Regularities of radiation effects in ontogenesis of progeny of the 1st generation of Wistar rats after irradiation of germ cells of both parents at different stages of gametogenesis]. AB - The data on antenatal and postnatal periods of ontogenesis of the posterity of the first generation of Wistar rats exposed by doses of 2-4 Gy at various terms before conception showed that the realization of radiation effects depended on the stage of gametogenesis of sex cells of both parents at radiation exposure. Stimulating radiation effect (hyperovulation) is accompanied by the higher rate of antenatal and early postnatal death of the posterity. In the rest cases it died mainly at embryogenesis. The most unfavourable factor for ontogenesis of the posterity appeared to be a participation in the fertilization of sex cells exposed at postmeiotic stages of gametogenesis, with the degree of manifestation of radiation effects depending on radiation dose. PMID- 7550898 TI - [Effects of sorbents of different chemical structure in bacterial toxemia after radiation and burn injuries]. PMID- 7550899 TI - [Endocrine function of the thymus and ionizing radiation]. AB - It is shown that external as well as incorporated radiation inhibit the endocrine function of the thymus in the result of the direct and indirect effects. Postradiation deficiency of thymic hormones plays a significant role in pathogenesis of functional disorders in the immunity system. Immunocorrection with thymic agents is the most effective method when the mechanism of inhibition of thymic hormonal function are taken into account. PMID- 7550901 TI - [In vivo study of the functional activity of peripheral blood leukocytes of intact and irradiated dogs with an inactivation test of endogenous hemopoietic stem cells of sublethally irradiated mice]. AB - Peripheral blood leukocytes of intact dogs, when transplanted to sublethally irradiated mice, have been found to suppress endogenous colony formation. The inactivation effect in hemopoietic stem cells of mice is enhanced with an increase in the number of effector cells in the graft. Radiation exposure results in failure of the effector function of leukocytic cells at the peak of acute radiation sickness. Nine months after irradiation, the inactivating capacity of dog leukocytes is restored and exceeds that of the control. Administered to dogs 9 months after irradiation, proteus vaccine stimulated the effector function of leukocytes at days 7 and 28 and suppressed it at days 14 and 62 post administration. PMID- 7550903 TI - [Radiation-protective effectiveness of MIGI-K preparation in irradiation of animals with medium-lethal and sublethal doses of x-ray and gamma irradiation]. PMID- 7550902 TI - [Effects of MIGI-K on general resistance of the body]. AB - The data on the effect of MIGI-K preparation, acidic hydrolyzate from mussel meat, on organism resistance are presented. The effect is based on the ability of MIGI-K to influence the level of endogenous compounds responsible for radioresistance, such as biogenic amines and lipid hydroperoxides, as well as certain parameters determining immune status of organism. PMID- 7550905 TI - [Spectral and energy parameters of the biological effect of UV-irradiation and approaches to its standardization]. AB - Human and animal health effects of UV-radiation are considered. Action spectra and biologically effective doses for UV-induced erythema, keratoconjunctivitis, cataract, skin cancer and immunosuppression are presented. Admissible doses of UV irradiation with regard to damage to skin and eye, recommended in some foreign countries, are discussed. PMID- 7550904 TI - [Radiation sensitivity of the seeds of winter wheat from chronically exposed agrocenosis]. AB - The morphophysiological properties of two generations of seeds from chronically exposed agrocenoses of the Chernobyl NPP accident zone were investigated. The difference in reactions between exposed and control seeds to acute stimulating irradiation and to treatment with biologically active substances was shown. The relationship between the degree of radiation depression of the seed morphological development indexes and biometric characteristics of plants on the plots with different radioactive contamination was revealed. PMID- 7550906 TI - [The age-related characteristics of the muscular layer of the common bile duct in man]. AB - Walls of 22 common bile ducts of corpses of both sexes people aged from 21 to 73 were studied. Longitudinal and cross sections of the common bile ducts walls obtained by the improved histotopographical method were stained with hematoxylin eosin after Jenner-Giemsa, picrofuchsine after Van-Gieson and orsein impregnated after Gordon-Svit and Kuprianov in modification corresponding with biliary tracts tissues. Specific volumes of myocyte bands and connective tissue forming the demonstrated smooth myocytes bands were determined and their correlation was defined in 4 age groups with research material divided in correspondence with the age classification adopted. Changes of capillary-tissue coefficient in ontogenesis were also determined. It was demonstrated that in proximal middle regions the common bile duct muscular coat was represented with longitudinal smooth myocyte bands, isolated from the surrounding loose connective tissue by reticulo-elastic framework. Regular order of these bands forms the crimpet construction of the common bile ducts in longitudinal direction of the tube. By the end of the 2nd maturity period and in elderly age separate location of the myocytes and zigzag course of the bands, their fragmentation with connective tissue layers were observed. Substitution of smooth myocytes with connective tissue fibres occurs. Analysis of the numeral values of the specific volumes obtained mathematically confirms all the morphological transformations listed above. Capillary-tissue coefficient increases in elderly and senile and age indicates disturbance in the tissue metabolism resulting from significant worsening of blood supply. PMID- 7550900 TI - [State of cellular immunity effectors of the lung in inhalation exposure to low transportable Pu-239]. AB - The reduction of populations of lung cell immunity effectors as well as changes in their cytotoxic and phagocytic activity was observed in rats after inhalation exposure to polymeric 239Pu. Dose-response curves described as a function of absorbed dose of alpha-irradiation in lung. PMID- 7550908 TI - [Changes in the adrenergic innervation of the large intestine in the postnatal ontogeny of the white rat]. AB - Age-related changes of adrenergic structures in intramural (myenteric and submucosal) plexuses of the albino rats large intestine were studied in different stages of postnatal ontogenesis (5 days, 1,5,13 and 24-26 months). It was established that adrenergic link of innervation develops most rapidly during the first month of postnatal life. This results in decrease of adrenergic nerve fibres number and catecholamine content in them with the simultaneous hypertrophy of part of the structures. Speed of the destructive processes development in the intestinal nerve plexuses grows with age, which leads to greater reduction of the adrenergic structures number. PMID- 7550907 TI - [The structure of the motor cortex in the rat in hypokinesia]. AB - Ultrastructure of neurons, synapses and glial elements was studied in the rat motor cortex under exposure to hypokinesia lasting for 40 and 90 days. Changes prevail in all cortical layers in 90-days hypokinesia and in upper ones in 40 days hypokinesia. These are basically reactive or not deep destructive changes. Cell elements and synapses with structural manifestations of low functional activity prevail with the increase of the experimental terms. Astrocyte number including satellite forms significantly grows in 40-days hypokinesia. In 90-days changes occur in all cortical layers with the increase of both astrocyte and oligodendrocyte number. PMID- 7550910 TI - [The deformation-strength properties of the vertebral and common carotid arteries in fetuses and newborn infants]. AB - General carotid and vertebral arteries of 51 corpses of foetuses and newborns with 28-40 weeks gestation were studied. The entire material was divided into 3 groups: 28-32 weeks, 33-36 weeks and 37-40 weeks. The thickness of the wall and its certain layers and connective tissue fibres architectonics were studied. General tensile strength, its limit, maximum relative deformation and the arterial wall Junge module were studied at longitudinal tension on ZM-40 tension machine. General carotid artery wall of foetuses and newborns is more tensile and has greater strength and less rigidity than that of the vertebral artery. The wall of the arteries studied has tensility reserve, safe for its structure in account of its fibroarchitectonics peculiarities. All the tensile strength parameters of the vessels investigated, with the exception of general carotid artery relative deformation, significantly change with the gestation increase. Tensile strength properties change depending on gestation term and body weight of foetuses and newborns may be approximated with linear function Y = A + B x X + C x Z (X--gestation term, Z--body weight). PMID- 7550909 TI - [The toxic effect of serotonin in direct and mediated actions on rat embryos]. AB - Embryotoxic effect of serotonin in direct (intra-amniotic administration) and indirect (subcutaneous administration) treatment was studied. In both cases serotonin causes embryo death and different abnormalities in development in small number of embryos. Mechanism of serotonin embryotoxic effect is under discussion. PMID- 7550911 TI - [The function of the immune, reproductive and endocrine body systems during irradiation under exposure to stress factors of a non-radiation nature]. AB - Diverse indexes of the state of immune, reproductive and endocrine systems were studied by morphological, biomechanical and biological methods under the effect of non-radiation stress factors, accompanying irradiation of the animals and their changes under effect of the ionizing irradiation 7 and 360 sGy in doze. It was demonstrated that short-time extreme effect lead to hormonal homeostasis destabilization, compensatory-adaptive transformations in immune system and also influence the indexes of copulation efficiency and intrauterine losses. Ionizing irradiation modifies the response to stress-factors effect with modification depending on the irradiation doze. PMID- 7550912 TI - [The indices of the physiological maturity of the progeny under conditions of chronic cholestatic lesions of the liver in the mother]. AB - Chronic cholestatic lesions of the liver in female rats causes the birth of physiologically immature progeny, which is reflected in more prolonged reservation of immaturity manifestation, decrease of everyday body weight accretion index, high perinatal mortality. Besides, depression of cell immunity and increase of humoral one was demonstrated in most stages in the animal group. PMID- 7550914 TI - [The types of macrophages in the central lymph of rabbits during the use of radon baths]. AB - Three types of macrophages circulating in central lymph were revealed by original method of lymph getting from rabbits. Type I has common morphological properties, type II and III are described in central lymph for the first time. Processes and protrusion are present in these types. Macrophages with processes are called by the author dendritic macrophages. They get into contact with lymphocytes. Type II and III macrophages number increases after radon balneotreatment in proportion with radon content, which is connected with radon effect on the skin receptor area, where intraepithelial macrophages are located. The latter are the precursors of dendritic cells of the other types and are the main antigen presenting cells in the initial phase of the immune response. PMID- 7550915 TI - [The tissue basophils of the rat mesentery under the influence of a permanent magnetic field]. AB - Effect of constant magnetic fields (CMF) of low energies (B = 35 mT, E = 350 Oe) on the system of mesenterial mast cells (MT) was studied by methods of light and electron microscopy in 140 outbred albino rats of both sexes for 1 day. The results obtained indicate active reaction of mesenterial MC on the CMF effect. Their activity is cyclical. High lability of the cellular system was demonstrated. Sexual peculiarities of the reactivity on different stages of CMF action also exist. Mesenterial MC reaction forms the complex of reactions of nonspecific adaptation to light irritants. Mesenterial MC response occurs stagely: orientation, training and relative training. The latter provides change of the tissue homeostasis on a qualitatively new level. Increase of passive and active resistance occurs simultaneously in training phase. This confirms the possibility of use of the CMF of low energies in complex of therapeutic treatment. PMID- 7550913 TI - [The histological, histochemical and ultrastructural characteristics of induced ectopic chondrogenesis]. AB - Implantation of the chondrogenesis inductor (demineralized osseous powder) into the subcutaneous fat in the area of the rat lower extremity muscles caused clear histological, histochemical and ultrastructural manifestations of ectopic chondrogenesis in certain zones of close contact with the inductor. Fibrogenic mesenchyme cells and fibroblasts proliferating by the glottis between the 6 th postoperative day and forming the bands, invading the glottis between the inductor granules were probably the object of chondrogenic transformation. Stages of these cells chondrogenic differentiation, hyaline cartilage forming and manifestations of its following ossification were followed up. The process of the induced ectopic chondrogenesis after fibrogenic cells transformation into chondroblasts passes the stages similar to that of the cartilage embryogenesis. PMID- 7550916 TI - [The localization of protein gene product 9.5 in the lungs of children with bronchitis and bronchiectasis]. AB - Human respiratory tract has a rich innervation with nerve fibers. Presence and distribution of nerve elements labeled with protein gene product 9.5 (PGP) were studied in the lungs of children with bronchitis and bronchiectasis. Smooth myocytes, blood vessels and mixed glands of the walls of bronchi principales were surrounded with single PGP-positive nerve fibers in perichondrium grows smaller with age. Nerve fibers also surround lymph nodules of the bronchi principales mucosa. Small or medium number of them in nerve plexuses and surround basically blood vessels. As neuropeptide function in respiratory tract vary, they may be of certain importance in pathogenesis of chronic lung diseases. Morphological base of this suggestion was given. PMID- 7550917 TI - [The surface status of the gastric mucosa in retinol deficiency]. AB - Changes of the stomach mucosa in retinol deficiency of the organism were studied by methods of scanning and transmission electron microscopy. Observation of stomach mucosa state of the animals was performed in dynamics (in proportion with increase of the vitamin deficiency). The disturbance of integrity of the apical part of the external cell membrane of the superficial epithelium and development of apical erosions in cells were established. Disturbance of integrity of the external membrane apical part in epitheliocytes provides erosions and ulcers development in retinol deficiency. PMID- 7550918 TI - [Isolated enterocytes as an object of functional morphology]. AB - Morphological examination of isolated enterocytes obtained-from the rat jejunum initial region by mechanical method after the previous treatment with ethylendiamintetracetic acid disodium salt included staining with hematoxylin, carmin and janus green. Histochemical reaction to alcaline phosphatase, succinate dehydrogenase, lactate dehydrogenase, ATP-ase and glycosaminoglycans were performed. Significant resemblance between the main cytological and cytochemical characteristics of the isolated enterocytes and those of the intestinal epithelium was demonstrated. Morphophysiological examination of isolated enterocytes, incubated in media, containing different substrates (glucose, maltose, glycine, trioleine) and under effect of oxygenated and not-oxygenated media demonstrated that mitochondria intracellular topography and succinate dehydrogenase activity significantly depend on their functional state. PMID- 7550920 TI - [The morphofunctional changes in the EC cells of the duodenum in the white rat during starvation]. AB - EC-cells morphofunctional changes were studied in the albino rats duodenal mucosa in different periods of starvation (1, 2, 3, 6 days). Dynamics of the endocrinocyte number increase and change of argentaffine reaction extent indicate active participation of duodenal EC cells in the development of compensatory adaptive reactions induced by starvation. PMID- 7550919 TI - [The cytochemical properties of the protective barrier of the duodenum in carnivorous animals in ontogeny]. AB - Studying the duodenal ontogenesis by means of morphometric and histochemical methods demonstrated that protective barrier of the organ is formed in the embryonic period and may be characterized by chemical composition of its secretion as a resistant barrier, formed by neutral and acid glycoproteins. PMID- 7550921 TI - [The role of anatomical factors in predicting the probability of the development of an abdominal ischemic syndrome]. AB - Anatomo-topographical studies, performed in 100 human corpses established that predispositions to development of the extravasal compressions (EC) of the coeliac trunk (CT) may be observed at high location of the aortal canal mouth, which is usually observed in case of dolichomorphic constitutional type. Complications of not diagnosed coeliac trunk extravasal compressions were the possible reason in 3% of sections. Acute substernal angle (< or = 70 degrees) is an easily determined and significant indicator of dolichomorphic type. This should be taken into account by clinicians in cases of unclear abdominal syndrome. PMID- 7550922 TI - [Changes in the tissues of a revascularized transplant of small intestine in the dog under different operating conditions]. AB - Histomorphological changes of revascularized autotransplant were studied in 34 dogs in different conditions of the operation. It was established that revascularization is necessary for transplantation. Organ disintegrates without vascular peduncle. Prolonged operative ischemia (up to 3 hrs) of the transplant causes its essential alternative and destructive changes up to the complete disintegration of part of the villi. Autotransplant of the small intestine preserves tissue and cell composition on the neck for a long period (more than a year) and may be used in replacing operations on the esophagus. However, complete organotypic and tissue restoration of the structure does not occur. Villi disappear, crypt epithelium forms structures resembling terminal regions of cardial glands, simple intestinal epithelium is replaced with the stratified one. PMID- 7550924 TI - [The intravital noninvasive assessment of the density of bone substantia spongiosa by using magnetic resonance tomography]. PMID- 7550923 TI - [The histotopography of the endocrinocytes in the rectal epithelium of hens in ontogeny]. AB - Histotopography and content of argyrophilic and argentaffin endocrinocytes per 1 mm2 of section of the hen rectal mucosa in ontogenesis were studied by method of light microscopy. Endocrinocytes were demonstrated in a part of epithelial layer from 12 day of embryogenesis. By 21 day their number increased 23 times. By the 3rd day of postnatal development endocrinocyte number reaches definitive values. Endocrinocytes are evenly distributed in the rectal epithelium, their shape varies. Forming of the endocrine apparatus structural organization in the hen rectum deals with the processes of the early endocrinocyte differentiation in a part of the epithelial layer during embryogenesis. PMID- 7550925 TI - [The structure of the arteries and the hemodynamic characteristics in the area of the ostia of efferent vessels]. PMID- 7550926 TI - Alexia without either agraphia or hemianopia in temporal lobe lesion due to herpes simplex encephalitis. AB - We report a case of alexia without either agraphia or hemianopia following herpes simplex encephalitis. The patient had a temporal lobe lesion with involvement of the occipitotemporal gyrus. This is an unusual cause of alexia without agraphia. The location of the lesion supports the view that transcallosal fibers from the right hemisphere to the left angular gyrus course inferior to the posterior horn of the left lateral ventricle and pass close to the left occipitotemporal gyrus. PMID- 7550928 TI - Neuro-ophthalmology and systemic disease--Part I. An annual review (1994). PMID- 7550927 TI - Isolated third-nerve palsy associated with frontal sinus mucocele. AB - Isolated third-nerve palsies are seen most commonly with aneurysms, vascular disease, trauma, or neoplasms (1-5). Sinus mucoceles have been known to cause orbital symptoms including proptosis and cranial nerve palsies. Rarely, an oculomotor nerve abnormality may be seen in the setting of mucoceles of the paranasal sinuses. We describe an unusual case of an isolated pupil-sparing third nerve palsy as the presenting sign of a frontal sinus mucocele. Emphasis is placed on the discussion of sinus mucoceles and their relation to orbital symptoms. PMID- 7550929 TI - Magnetic resonance visualization of the swollen optic disc in papilledema. PMID- 7550930 TI - Optic nerve sheath fenestration for anterior ischemic optic neuropathy? The answer is in. PMID- 7550931 TI - Uhthoff and his symptom. AB - At the turn of the last century, Wilhelm Uhthoff was a renowned clinical neuro ophthalmologist, and probably the first clinician whose entire career was devoted to this discipline. His achievements are among those that mark the commencement of contemporary neuro-ophthalmology. Uhthoff's symptom of visual loss with exercise is most frequently associated with optic neuritis. The symptom carries a major risk for recurrence of optic neuritis and development of multiple sclerosis. This study and its companion in the publication show that, independently, a metabolic byproduct of exercise or increases in body temperature cause a reversible conduction block in demyelinated optic nerves and result in temporary loss of vision [corrected]. PMID- 7550933 TI - Spontaneous intracranial hypotension. A review. PMID- 7550932 TI - Visual evoked potentials during hyperthermia. AB - OBJECTIVES: We sought to evaluate the effect of hyperthermia (HT) on central conduction pathways by alterations in pattern visual evoked potentials (PVEPs) in normal and demyelinated optic nerves. MATERIALS AND METHODS: We studied PVEP peak latency and amplitudes in 10 normal subjects and six patients with demyelinating optic neuropathy before and during HT. RESULTS: In normal subjects, a mean rise in temperature of 2.5 degrees C resulted in a decrease in the second positive peak (P2) latency of 6.1 ms (p < 0.0001) and a slight decline in P2 amplitude of 1.16 muV (p < 0.009). These results were compared to those obtained from six patients with multiple sclerosis. These patients had a history of monocular optic neuritis; two patients had had bilateral optic neuritis, and one patient had not had involvement of the optic nerve. Average temperature elevations during PVEPs were 1.60 degrees C. PVEPs among these patients showed decrease in mean P2 latencies, except in patients with multiple sclerosis, who showed an increase in latency with 60 min check size in the left eyes. There was a consistent decline in P2 amplitudes. Loss of amplitude was greater among the six optic nerves of those patients having transient, mild losses in visual acuity during HT. Reductions in P2 amplitude were best explained by partial or complete conduction block. CONCLUSIONS: These changes in conduction time and amplitude during HT provide a neurophysiologic correlation to the well-known sensitivity of demyelinated optic nerves to elevated temperatures. They are also relevant to the monitoring of central pathways in the operative or intensive care setting. The demonstrated reversible loss of amplitudes also gives promise to therapeutic manipulation of impaired pathways by impeding the loss of current from denuded nerve fibers. PMID- 7550934 TI - Extreme eyelid swelling as an unusual presentation of dysthyroid orbitopathy. AB - We describe seven cases of dysthyroid orbitopathy initially seen as severe periocular lid swelling. Imaging techniques typically demonstrate normal extraocular muscles, although moderate muscle enlargement may occur. Patients do not respond to steroids, radiation therapy, antihistamines, or diuretics. We believe this clinical picture represents a unique population of dysthyroid orbitopathy patients who are best managed by long-term observation followed by cosmetic surgery. PMID- 7550936 TI - Restrictive ophthalmopathy associated with linear scleroderma. AB - A patient with a coup de sabre lesion of the forehead developed progressive ipsilateral limitation of ocular motility, primarily involving adduction and depression. Investigation disclosed no other explanation for the ocular motility disturbance, which we suspect represents restrictive myopathy maximally involving ocular muscles immediately subjacent to the area of linear scleroderma. PMID- 7550935 TI - A controlled trial of regional sympatholysis in the treatment of photo-oculodynia syndrome. AB - INTRODUCTION: The mechanism(s) underlying the eye pain syndrome characterized by photo-oculodynia and decreased tears (herein referred to as PODS) is unknown. Postulating a sympathetically maintained pain mechanism, cervical sympathetic ganglion blocks (CSB) were performed in an open-label trial in two patients as a pilot test of our hypothesis. Because these patients experienced clinically dramatic reductions in signs and symptoms, a double-masked controlled trial was initiated. METHODS: With Institutional Review Board approval and written informed consent, four other subjects with PODS who had experienced temporary reduction (> 50%) of symptoms with a single lidocaine CSB were enrolled. A randomized, double masked series of three CSBs (saline, bupivacaine, lidocaine) was scheduled for each subject 2-7 days apart. RESULTS: CSB with lidocaine and bupivicaine, but not saline, reduced spontaneous pain and light sensitivity and increased production of tears. Symptom reduction lasted for hours to days, extending beyond clinical signs of local anesthetic-induced sympatholysis. It was also observed that sympatholysis extinguished associated blepharospasm in those subjects with this dystonic condition (one subject in the open-label pilot trial and two of three in the controlled trial). CONCLUSIONS: Results of these preliminary data suggest that the sympathetic nervous system is involved in mediating symptoms associated with PODS in certain individuals. PMID- 7550937 TI - Painful ophthalmoplegia caused by hemangiopericytoma of the cavernous sinus. AB - Progressive, painful ophthalmoplegia developed in a 34-year-old man. MRI scan revealed an enhancing mass in the left cavernous sinus. Histologic examination of resected tumor revealed reticulin staining and cytologic features of hemangiopericytoma. Characteristics of intracranial hemangiopericytoma are reviewed. PMID- 7550938 TI - Surgical management for intractable metatarsalgia. AB - We reviewed the results of arthrodesis of the first metatarsophalangeal joint and excisional arthroplasty of the lesser metatarsophalangeal joints performed on patients who presented intractable metatarsalgia and forefoot deformities. The study included 18 feet (15 patients) in 12 women and 3 men. The follow-up averaged 5.2 years. A good to excellent result was achieved in 15 (83%) feet; pain, which had been moderate to severe before surgery, was reduced to none to mild, and function, which had been severely limited before surgery, was improved to virtually unlimited. The fusion rate of the first metatarsophalangeal joint was 94% (17 of 18 feet). Thirteen of the 15 patients (87%) stated they would have the procedure again. This repair has been demonstrated to be a useful salvage procedure in nonrheumatoid patients with severe metatarsalgia secondary to failed forefoot surgery. PMID- 7550939 TI - Update on women's footwear. AB - Shoes have been implicated as being responsible for the majority of foot deformities and problems that physicians encounter in women. In our original study of 356 women, the majority of women studied wore shoes that were too small for their feet, had foot pain and deformity, and had increased in shoe size since the age of 20. The women without foot pain or deformities also wore shoes that were smaller than their feet but to a lesser degree. In the present study, data on 255 of the original 356 women are evaluated. Tracings were made of the standing foot and the shoe. Measurements were made of forefoot and the heel width. An index of forefoot width to heel width was developed. The indices do not vary much among women. Based on linear measurements, as forefoot width increases, so does heel width. As foot length increases, forefoot width increases to a greater extent than heel width. PMID- 7550940 TI - Triple arthrodesis for diabetic peritalar neuroarthropathy. AB - From 1963 to 1990, the senior authors (R.E.M. and K.G.H.) performed eight triple arthrodeses in seven patients with diabetes mellitus with sensory loss in the lower extremities. By clinical and roentgenographic examination, all patients were diagnosed with peritalar neuroarthropathy before surgery. All patients underwent a two-incision triple arthrodesis with internal fixation. Patient follow-up averaged 44 months and included repeat physical examinations and radiographs. All patients went on to clinical union and were satisfied with the procedure. One patient had prolonged wound drainage that resolved with antibiotic therapy; another had a residual rocker-bottom deformity and plantar ulceration that resolved after modification of custom shoe wear. We believe comprehensive management of diabetic peritalar neuroarthropathy can include surgical arthrodesis of the involved joints. The disease process and surgical indications are discussed. PMID- 7550941 TI - Treatment outcome of major fractures of the talus. AB - Twenty-six patients with major fractures of the talus were studied to assess the long-term outcome. The patients were admitted to a university teaching hospital and major trauma center from 1983 to 1991. The study excluded isolated fractures of the talar dome and posterior tubercle. Fifteen patients were treated using internal fixation and 11 patients were treated using nonsurgical methods. Avascular necrosis was detected in only four of the 26 patients. Subtalar osteoarthritis was a significant problem in 61%. Seven of these patients have come to secondary fusion procedures, with another three contemplating fusion procedures at the time of review. Only one patient developed significant avascular necrosis requiring a fusion procedure. Only three of 26 patients had not returned to work at a mean 6 years after their injury. Eleven of the 26 (42%) had not returned to their premorbid activity level. The majority of these patients (25/26) had sustained multiple injuries, which compromised the functional recovery from the talar injury. Early accurate diagnosis and anatomical reduction gave the best results. The low incidence of avascular necrosis in this study has been attributed to early anatomical stabilization of the fracture. We believe an early CT scan can more accurately assess the severity of the talar fracture and offers the best information for an appropriate treatment plan. PMID- 7550942 TI - Fixation of the Chevron osteotomy utilizing absorbable polydioxanon pins. AB - The Chevron osteotomy has become popular for the treatment of hallux valgus deformity. Displacement of the osteotomy is a known complication. Many methods of internal fixation have previously been reported to prevent the displacement and each has inherent advantages and disadvantages. An alternative method of fixation, utilizing absorbable polydioxanon pins, is presented in this article. Between April 1989 and April 1990, 71 procedures were performed on 50 patients. At 1-year follow-up, there were no complications or infections. Review of the osteotomy after surgery and at 1 year revealed no fractures or displacements, and no pins required removal. Internal fixation utilizing absorbable polydioxanon pins appears to have significant benefits when compared with metallic fixation. PMID- 7550943 TI - Potential for recurrence of hallux valgus after a modified Hohmann osteotomy: a biomechanical analysis. AB - On the basis of a biomechanical model the present study investigates whether a foot-widening effect, which may result in recurrence of bunions, can be demonstrated in operated patients. Therefore, the medial deviation of the first metatarsal head as a result of flexion forces on the hallux was measured in eight patients with clinical idiopathic hallux valgus, who underwent a modified Hohmann osteotomy, and in eight "normal" persons. We found that (1) before osteotomy, all forefeet broadened while pressing the big toe downward, and (2) after surgery, widening of the forefeet on average had significantly diminished, but still existed, which might explain the development of recurrences. (3) After surgery, the patients showed a slight, but statistically nonsignificant, decrease (from 37 N to 25 N) of the isometrically determined average maximal applicable flexion force. These abnormal hallux loads may cause, or result from, a deviation in gait pattern. PMID- 7550944 TI - Integrity of the first metatarsophalangeal joint: a biomechanical analysis. AB - Five fresh-frozen cadaver feet obtained from traumatic amputations were tested during hyperdorsiflexion stress of the first metatarsophalangeal joint. Three different types of injury were observed: (1) rupture of the capsule proximal to the sesamoids, (2) rupture of the plantar plate distal to the sesamoids, and (3) rupture of the capsular structures medially, allowing a lateral swing of the sesamoids around the metatarsal head. Incomplete dislocation can be associated with significant damage to the plantar plate and other soft tissues of the foot. PMID- 7550945 TI - Traumatic focal posterior tibialis muscle denervation. AB - The purpose of this case presentation is to demonstrate posterior tibialis muscle (PTM) denervation as a cause of traumatic plantarflexion inversion weakness. In a 42-year-old woman, severe pain, swelling, and ecchymosis over the medial aspect of her left ankle developed after she twisted it while playing tennis. Plantarflexion inversion weakness developed (grade 3/5). The strength of all other muscle groups of the lower extremity was normal. Her pin and light touch sensation were normal in the left lower extremity. Deep tendon reflexes were equal and active at both knees and ankles. A magnetic resonance image of the left leg, ankle, and foot performed 1 month after injury demonstrated an intact posterior tibialis tendon behind the medial malleolus and edema-like increased signal intensity in the PTM on the T1-weighted image consistent with denervation. On electromyographic testing, there were continuous fibrillation and positive sharp wave potentials in every site tested in the PTM without any voluntary motor unit activity. The left extensor hallucis, left gastrocnemius, and lumbar paraspinal muscles were normal. In conclusion, combined magnetic resonance imaging and electromyographic studies supported denervation of the PTM as the cause of plantarflexion inversion weakness, rather than posterior tibialis tendon rupture in this patient. PMID- 7550946 TI - Volkmann's ischemic contracture of the foot and ankle: evaluation and treatment of established deformity. AB - Fibrotic contracture of skeletal muscle can follow weeks or months after the severe ischemic insult of compartment syndrome. Commonly known as Volkmann's ischemic contracture, the affected limb often becomes dysfunctional and painful, and may lose sensibility. The pathogenesis of the muscle contracture includes prolonged ischemia, myonecrosis, fibroblastic proliferation, contraction of the cicatrix, and myotendinous adhesion formation. Resultant shortening or overpull of involved muscles leads to stiffness and deformity. Simultaneously, nerve injury from initial ischemia or subsequent soft tissue fibrotic compression leads to muscle paresis or paralysis of the involved compartment and of those muscles more distally innervated. The resultant deformity is thus a combination of varying degrees of contracture and weakness depending on which muscles and nerves are affected. Deformity and functional impairment in the foot and ankle secondary to ischemia are determined by many factors, including: (1) which leg compartment, if any, has been affected and to what degree extrinsic flexor or extensor overpull is exhibited, (2) degree of nerve injury sustained causing weakness or paralysis of extrinsic or intrinsic foot and ankle muscles (3) which foot compartment, if any, has been affected and to what degree intrinsic overpull is exhibited, and (4) degree of sensory nerve injury leading to anesthesia, hypoesthesia, or hyperesthesia of the foot. Therefore, a variety of clinical presentations can be encountered following compartment syndrome of the leg and foot. Treatment is based on an appreciation of the pathoanatomy of the deformity. Nonoperative therapy is aimed at obtaining or preserving joint mobility, increasing strength, and providing corrective bracing and accommodative footwear. Operative management is usually reserved for treatment of residual nerve compression or severe and problematic deformities. Established surgical protocols are performed in a stepwise fashion, to include: (1) release of residual or secondary nerve compression, (2) release of fixed contractures, using infarct excision, myotendinous lengthening, muscle recession, or tenotomy, (3) tendon transfers or arthrodesis to increase function, and (4) ostectomy or amputation for severe, refractory deformities. PMID- 7550947 TI - The orthopaedic/podiatric dilemma. PMID- 7550948 TI - Elective foot surgery: relative roles of doctors of podiatric medicine and orthopaedic surgeons. PMID- 7550949 TI - Elective foot surgery: relative roles of doctors of podiatric medicine and orthopaedic surgeons. PMID- 7550950 TI - Long-term costs for foot ulcers in diabetic patients in a multidisciplinary setting. AB - The purpose of this study was to analyze long-term costs for foot ulcers in diabetic patients. Patients were treated and followed prospectively by a foot care team. A retrospective economic analysis was performed of costs for 274 patients during 3 years from healing of an initial foot ulcer, with or without amputation. Costs were estimated for inpatient care, outpatient care, home care, and social service. The cost calculations include costs due to complications and disability related to the initial ulcer, costs related to recurrence of ulcer, and costs for prevention of new ulcers. Expected total present value cost per patient during 3 years of observation was $26,700 (U.S. dollars) for primary healed patients with critical ischemia and $16,100 for primary healed patients without critical ischemia. For patients who healed with an amputation, the corresponding costs were $43,100 after a minor amputation and $63,100 after a major amputation. When estimating the costs for diabetic foot ulcers, it is not sufficient to calculate short-term costs. Long-term costs are high, mainly due to the need for increased home care and social service, but also due to costs for recurrent ulcers and new amputations. PMID- 7550951 TI - Triple arthrodesis with lateral column lengthening for treatment of severe planovalgus deformity. AB - Triple arthrodesis with lateral column lengthening through the calcaneocuboid joint was performed on 22 feet in 14 patients. The primary indication for surgery was severe symptomatic planovalgus deformity unresponsive to conservative measures. All patients achieved solid fusion within 12 weeks. No patient had no decline in ambulatory status. Excellent correction of deformity was achieved and maintained with an average correction of the talus first metatarsal angle of 25 degrees in both the AP and lateral planes. Triple arthrodesis with lateral column lengthening provides for reliable arthrodesis and allows correction of severe planovalgus deformity while maintaining foot length. PMID- 7550952 TI - Tibiocalcaneal arthrodesis. AB - A review of eight patients who underwent nine tibiocalcaneal arthrodeses was performed. The surgical indications were failed previous surgery, posttraumatic talar avascular necrosis, and rheumatoid arthritis. The average age at the time of operation was 54 years. The average time of follow-up was 40 months after the operation. Fusion was achieved in all nine feet, with an average time of fusion of 5 months. Results were excellent in three feet, good in four feet, fair in one foot, and poor in one foot. The average leg length discrepancy was 2 cm. The average position of fusion was 6 degrees of valgus and 2 degrees of plantarflexion. All patients stated that they had improvement of pain and function. Seven of the eight patients stated that they would have the procedure done again. Complications were local infection in two patients, malunion in two patients, wound slough in one patient, and a prominent fibula in one patient. Tibiocalcaneal arthrodesis should be reserved as a salvage procedure because it is technically difficult and has a significant risk of complications. PMID- 7550953 TI - Anterior tibial tendon rupture. AB - Twelve patients with rupture of the anterior tibial tendon are presented. Nine patients were aware of an acute event prior to their symptom onset and three were not aware of any acute event. Complete rupture of the tendon was noted in 10 patients and incomplete rupture was seen in two patients. Treatment was individualized based on age, etiology, preinjury function, patient health, and personal considerations. Five patients were treated without surgery. Three preferred no orthotic devices, and two believed their function was improved with an ankle-foot orthosis. Seven patients were treated operatively using a variety of individualized reconstructive techniques. All operatively treated patients demonstrated increased function and strength. Based on our findings, operative reconstruction is recommended in appropriate patients. PMID- 7550954 TI - Selective hindfoot arthrodesis for the treatment of adult acquired flatfoot deformity: an in vitro study. AB - An acquired flatfoot deformity with significant laxity at the transverse tarsal joint was created experimentally and the amount of correction that was obtained with selective hindfoot fusions was measured radiographically. Results showed that the talonavicular, double (talonavicular and calcaneocuboid), and triple arthrodeses were able to fully correct the deformity, including correction of hindfoot valgus with just a talonavicular fusion. Subtalar and calcaneocuboid fusions failed to completely correct the deformity. This study provides experimental evidence that although the triple joints are interconnected, they differ with respect to their ability to malalignment. We conclude that talonavicular or double arthrodesis will correct deformity in a flatfoot with considerable laxity through the transverse tarsal joint, but that a subtalar fusion will not provide consistent correction. PMID- 7550955 TI - Anatomy of the Achilles tendon and plantar fascia in relation to the calcaneus in various age groups. AB - Ten adult cadaver feet, three neonatal feet, and the feet of two fetuses were dissected to investigate whether an anatomical continuity exists between the fibers of the Achilles tendon and the plantar fascia. Histologic sections of the feet were done in three age groups: neonate, persons in their mid-20s, and the elderly. As the foot ages, there appears to be continued diminution of the number of fibers connecting the Achilles tendon and plantar fascia. The neonate has a thick continuation of fibers, while the middle-aged foot has only superficial periosteal fibers that continue from tendon to fascia. The elderly feet show simply an insertion of fibers of both structures into the calcaneus with periosteum in between. PMID- 7550957 TI - Arthroscopy of the subtalar joint: establishing a medial portal. AB - One of the recently introduced procedures for studying the posterior subtalar joint is subtalar arthroscopy. There is no reference in the literature to the possibility of a medial portal that might be used either for arthroscopic insertion, probing, or instrumental manipulation. The two portals mentioned in the literature are the anterolateral and the posterolateral portals. For evaluating the possibility of establishing a medial portal, six embalmed adult cadaver feet were used to study the anatomical relations to the proposed medial portal. The subtalar joints of another six fresh adult cadaver feet were then arthroscoped, after distraction of the joint, using the anterolateral, posterolateral, and medial portals. Findings indicated that the medial portal gives good visualization of the posterior subtalar joint. Clinical application has not yet been assessed. PMID- 7550956 TI - Dynamic orthopaedic brace in the treatment of ankle sprains. AB - Ankle sprains may lead to disabling sequelae such as joint instability and persistent pain. Immobilization with plaster cast may give rise to joint stiffness and muscle atrophy. Twenty patients with acute inversion sprains of the ankle were treated with a "dynamic" orthopaedic brace after a 10-day plaster immobilization. A control group, consisting of 10 subjects, received a weight bearing short-leg plaster cast for 25 days. A clinical evaluation and an instrumental isokinetic investigation (Cybex) were performed as scheduled. The clinical findings suggest an earlier and more comprehensive functional recovery in the group receiving the "dynamic" brace compared to the casted group. The isokinetic test revealed a statistically significantly better performance for most parameters in the brace group especially regarding the ankle joint invertors. PMID- 7550958 TI - Retrograde intramedullary nailing for ankle arthrodesis. AB - This is a retrospective study of retrograde intramedullary rodding for ankle arthrodesis in 19 ankles in 16 patients. The preoperative diagnosis of 16 patients was diabetic neuropathic arthropathy in seven patients, rheumatoid arthritis in three patients, post traumatic arthrosis in three patients, paraplegia with fixed equinovarus of the foot in two patients, and avascular necrosis of the talus in one patient. Retrograde intramedullary rodding for ankle arthrodesis was done as a salvage procedure in each patient. Fourteen of the 19 ankles had radiographic evidence of solid arthrodesis. In the four patients with five ankles with pseudarthrosis, no case was clinically significant. There was one deep infection and one broken rod. Thirteen of the 16 patients are ambulatory, and nine required either an ankle-foot orthosis or shoe modification. The standard method of ankle fusion using crossed cancellous screws is the procedure of choice because it preserves the subtalar joint. Retrograde intramedullary rodding for ankle arthrodesis should be considered for patients with significant posttraumatic arthrosis and bone loss following distal tibial plafond fractures, concomitant subtalar arthrosis, severe osteopenia, such as in patients with rheumatoid arthritis, or neuropathic arthropathy. PMID- 7550959 TI - Comparison of two fixation methods of oblique lesser metatarsal osteotomies: a biomechanical study. AB - Two methods of internal fixation of oblique lesser metatarsal osteotomies were compared biomechanically using fresh-frozen human cadaver bones. Osteotomies were made obliquely through the metatarsal shafts and fixed with either crossed Kirschner wires or a single AO screw using the lag technique. The specimens were then fixed at their proximal end and loaded to failure using an axial torsion material testing system (MTS, Minneapolis, MN). Load displacement curves were obtained and the stiffness of the constructs were determined. Single-screw fixation was found to be significantly stiffer than the crossed wire configuration (P < .01). Single-screw fixation resulted in a stiffness of 211.2 +/- 111.7 N/cm (mean +/- SD), while stiffness of the crossed wire configuration averaged 56.9 +/- 25.1 N/cm. PMID- 7550960 TI - Ipsilateral peroneus brevis tendon grafting in a complicated case of traumatic rupture of tibialis anterior tendon. AB - Ruptures of tibialis anterior tendon can be caused by open, closed, direct, or indirect trauma, as well as spontaneously. Sixty-three cases of tibialis anterior tendon ruptures have been reported in the international literature. The treatment of choice is the surgical end-to-end or side-to-side anastomosis after previous Z lengthening. The case of a 28-year-old world-class female triathlete who sustained an open laceration of the tibialis anterior tendon from the bicycle chain guard is reported. The primarily applied tendon suture became infected and a wound revision with wide resection of the tendon stumps was necessary. This lead to an extensive defect of the tendon combined with a deep-seated keloidal scar reaction of the skin. The surgical closure was performed using free ipsilateral peroneus brevis tendon grafting. Four months after the operation the patient was completely rehabilitated. Eight months later she became the second European triathlon champion. PMID- 7550961 TI - Stress fracture of the tibia following extensive hindfoot and ankle arthrodesis: a report of three cases. PMID- 7550963 TI - Feeling lumps and bumps in foot surgery. PMID- 7550962 TI - Correction of fixed hammertoe deformity with resection of the head of the proximal phalanx and extensor tendon tenodesis. AB - A method of surgical correction of a fixed hammertoe deformity is presented. It incorporates the resection of the head of the proximal phalanx with an extensor tendon tenodesis to the dorsum of the proximal phalanx. The controlled tension in the extensor tendon provides the necessary stability. It alleviates the use of K wire fixation. PMID- 7550964 TI - Systematic study of field and concentration effects in capillary electrophoresis of DNA in polymer solutions. AB - A systematic study of the separation of double-stranded DNA in hydroxypropylcellulose (HPC) with a molecular mass of 10(6) was undertaken, using a variety of concentrations (from 0.1 to 1%) and different electric fields (from 6 to 540 V/cm). The data show that at high polymer concentrations ( > or = 0.4%) and low fields, the separation mechanism is similar to that occurring in gels. The results are in good agreement with theoretical models, and in particular with a recently proposed theory for gels with a pore size smaller than the persistence length of DNA. For more dilute solutions and high fields, however, the separation pattern cannot be explained by existing theories. The existence of an original mechanism was confirmed by the direct observation of the conformation of double stranded DNA molecules in the polymer solution by fluorescence videomicroscopy. Practical conclusions for the capillary electrophoretic separation of duplex DNA are drawn. PMID- 7550965 TI - Further identification of human plasma glycoproteins interacting with the galactose-specific lectin Jacalin. AB - In this report we show that Jacalin binds the heme-binding protein hemopexin and the C4b-binding protein sgp120 in human plasma. The interaction of Jacalin with hemopexin confirms that a single O-linked oligosaccharide is sufficient to mediate binding of a protein to this lectin. Retention of sgp120 by immobilized Jacalin demonstrated that this protein was O-glycosylated and, therefore, clearly different from another C4b-binding protein, the complement protein C2 which is physicochemically similar but exclusively N-glycosylated. In addition, Jacalin was also shown to bind several proteolytic enzymes which remain to be identified. PMID- 7550966 TI - High-performance liquid chromatographic determination of the novel antitumour drug topotecan and topotecan as the total of the lactone plus carboxylate forms, in human plasma. AB - A sensitive high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantitation of the novel anticancer agent topotecan and topotecan as the total of its lactone and carboxylate forms in human plasma. Linear response in analyte standard peak area were observed over the concentration range 0.05-10 ng/ml using 100-microliters plasma samples. The instability of the drug in the biological matrix necessitated that the plasma fraction was obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). Stability studies have indicated that topotecan is stable in these methanolic extracts for at least 4.5 months at -30 degrees C and 2 months at -70 degrees C. For the total determination of the lactone plus lactone ring-opened forms of the drug as topotecan, plasma samples were deproteinated with methanol and, subsequently, acidified with 7% (v/v) perchloric acid. Plasma samples for the measurement of total levels of the lactone and the ring-opened forms of topotecan were stable for at least 4.5 months when stored at -30 degrees C. After centrifugation, the supernatants were analysed by HPLC using a Zorbax SB-C18 Stable Bond column and methanol-0.1 M hexane-1-sulfonic acid in methanol-0.01 M N,N,N',N'-tetramethylethylenediamine (TEMED) in distilled water pH 6.0 (25:10:65, v/v) as the mobile phase. Detection was performed fluorimetrically. Within-run and between-run precision was always less than 12.1% in the concentration range of interest (0.05-10.0 ng/ml). The limit of quantitation is 0.05 ng/ml. Accuracy measurements ranged between 87.6 and 113.5%. PMID- 7550967 TI - Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction. AB - A new method is described for the determination of cimetidine in human plasma. The drug and internal standard (ranitidine) were separated on a Nucleosil C18 5 microns (25 x 4.6 mm I.D.) column using a mobile phase of acetonitrile-phosphate buffer, pH 6.2 (25:75, v/v) containing 2.5 g/l heptane sulphonic acid. The mobile phase was delivered at a flow-rate of 0.9 ml/min, detection was by ultraviolet absorption at 228 nm and concentrations were calculated on the basis of peak areas. The drugs were extracted from alkaline plasma into ethyl acetate using a salting out procedure which involved the addition of 100 ml of a saturated solution of K2CO3 to each 250-microliters plasma aliquot. The method was validated over the concentration ranges 50-3000 ng/ml and 100-7000 ng/ml for two separate studies. Mean coefficients of variation were less than 6% for both intra and inter-assay in both studies and recoveries varied between 71 and 81%. The method was successfully applied to the determination of cimetidine in plasma for a pharmacokinetic study. PMID- 7550968 TI - High-performance liquid chromatographic determination of urinary 4' hydroxymephenytoin, a metabolic marker for the hepatic enzyme CYP2C19, in humans. AB - The preferential hydroxylation of (S)-mephenytoin to 4'-hydroxymephenytoin (4'-OH M) displays a genetic polymorphism of drug metabolism in humans. Thus the excreted 4'-OH-M is considered to be an important marker for the hepatic (S) mephenytoin 4'-hydroxylase. Accordingly, a mixture of urine containing total 4' OH-M after enzymatic deconjugation and phenobarbital as internal standard (I.S.) was extracted with absolute diethyl ether. The residue remaining after evaporation was dissolved in 50 microliters of eluate and 20 microliters were injected into the chromatographic system. All components were separated isocratically on a reversed-phase column using acetonitrile-water (24:76, v/v) as the mobile phase at a flow-rate of 1.2 ml/min. The effluent was monitored at 204 nm. The retention times for 4'-OH-M and the I.S. were within 6 min. The absolute recovery was in the range 84-89% for 4'-OH-M and that of the I.S. was 75.9 +/- 4.2%. Quantification was performed by measuring the peak-height ratio compared with the ratio of the amount of 4'-OH-M divided by that of the I.S. The intra- and inter-day variations were less than 8% and 10%, respectively. The proposed method is simpler and more convenient than those reported previously. Its practical applicability was assessed by phenotyping the efficient and deficient hydroxylators among the Chinese minorities and Han Chinese populations. PMID- 7550969 TI - Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G. AB - Protein A immobilized polyhydroxyethylmethacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma. PMID- 7550970 TI - Development and validation of a high-sensitivity assay for an antipsychotic agent, CP-88,059, with solid-phase extraction and narrow-bore high-performance liquid chromatography. AB - An analytical method has been developed and validated for the quantitation of CP 88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C18 reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within +/- 10% and +/- 15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase. PMID- 7550971 TI - High-performance liquid chromatographic assay for CI-980, a novel 1-deaza-7,8 dihydropteridine anticancer agent, in human plasma and urine. AB - CI-980, a 1-deaza-7,8-dihydropteridine, is a novel anticancer agent that is a potent mitotic inhibitor acting as a tubulin binder similar to the vinca alkaloids. CI-980 has shown equivalent or superior anticancer activity in vitro compared to vincristine and retains full activity against vincristine resistant tumors in vitro. A high-performance liquid chromatographic (HPLC) assay was developed and validated for human plasma and urine to support Phase 1 clinical trials. CI-980 and PD 080658, internal standard, were isolated from 2-ml samples of human plasma and urine by solid-phase extraction with Bond-Elut C18 cartridges. Urine samples must be pretreated with bovine serum albumin (BSA) to minimize the binding of CI-980 to glass and some plastics. The eluate from the cartridges for both matrices was evaporated to dryness and taken up in mobile phase. Zorbax RX C18 columns, mobile phase buffer of 10 mM ammonium dihydrogen phosphate at pH 7.5 and a flow-rate of 0.75 ml/min were used for both matrices. Column dimensions, column temperature and mobile phase acetonitrile-buffer ratio were 300 mm x 4.6 mm I.D., 30 degrees C and 38:62 (v/v), respectively, for the plasma assay and 250 mm x 4.6 mm I.D., 35 degrees C and 40:60 (v/v), respectively, for the urine assay. Column effluent was monitored fluorometrically for the plasma method using excitation and emission wavelengths of 388 nm and 473 nm, respectively. Ultraviolet detection at 380 nm was used for the urine method. Peak-area ratios were proportional to CI-980 concentrations from 0.2 to 25 ng/ml and 1 to 100 ng/ml for plasma and urine, respectively. CI-980 in water will bind to glass and plastics but not PTFE or stainless steel. Urine calibration standards were frozen prior to use in order to compensate for loss of CI-980 due to freezing in this matrix. The accuracy of the assay was within 4.7%, with a precision of 5.6% for both matrices. Recoveries ranged from 93.8 to 102% and 90.7 to 92.3% for plasma and urine, respectively. CI-980 was stable in plasma and urine for at least 275 and 217 days, respectively, when stored at -70 degrees C. The assay is suitable for studying the clinical pharmacokinetics of CI-980. PMID- 7550972 TI - Determination of a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse, rat, dog, and monkey plasma using high-performance liquid chromatography with ultraviolet detection. AB - A method for the analysis of the AMPA (alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C18 Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 microgram/ml to 50 micrograms/ml with an intra-assay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 microgram/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 microgram/ml in dog and monkey plasma and 0.1 microgram/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at -70 degrees C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies. PMID- 7550973 TI - Exploration of the metabolism of dihydrocodeine via determination of its metabolites in human urine using micellar electrokinetic capillary chromatography. AB - After single-dose administration of 40 or 60 mg of dihydrocodeine (DHC, in a slow release tablet) to four healthy individuals known to be extensive metabolizers of debrisoquine, the urinary excretion of DHC and its four major metabolites, dihydrocodeine-6-glucuronide, nordihydrocodeine, dihydromorphine and nordihydromorphine, was assessed using micellar electrokinetic capillary chromatography (MECC). DHC and two of its metabolites (dihydrocodeine-6 glucuronide and nordihydrocodeine) could be analyzed by direct urine injection, whereas the metabolic pattern was obtained by copolymeric bonded-phase extraction of the solutes from both plain and hydrolyzed urine specimens prior to analysis. The total DHC equivalents excreted within 8 and 24 h were determined to be 30.4 +/- 7.7% (n = 5) and 63.8 +/- 6.1% (n = 2), respectively, and only about 4% of the excreted DHC equivalents were identified as morphinoids. Furthermore, almost no morphinoid metabolites of DHC could be found after administration of quinidine (200 mg of quinidine sulfate) 2 h prior to DHC intake. PMID- 7550974 TI - Thermal degradation of clozapine-N-oxide to clozapine during gas chromatographic analysis. AB - Studies were undertaken to determine if clozapine-N-oxide, the principal urinary metabolite of the antipsychotic agent clozapine, may interfere with the gas chromatographic-mass spectrometric bioanalysis of clozapine. Following injection of clozapine-N-oxide onto a (5% phenyl)methylpolysiloxane capillary column operated at 250 degrees C, significant on-column reduction of clozapine-N-oxide to the parent drug occurred. Accordingly, preparation of biological samples for clozapine determination by gas chromatography should avoid conditions which reportedly co-extract the N-oxide to assure no artifactual contribution of this metabolite in the detection of clozapine. PMID- 7550975 TI - Stereospecific high-performance liquid chromatographic assay of ketoprofen in human plasma and urine. AB - A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of ketoprofen (KT), a 2-arylpropionic acid (2-APA) non steroidal antiinflammatory drug (NSAID), in plasma and urine was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), plasma containing the KT enantiomers and I.S. was extracted by liquid-liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in mobile phase and injected into the HPLC system. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (Chiralpak AD) column with hexane isopropyl alcohol-trifluoroacetic acid (80:19.9:0.1, v/v/v) as the mobile phase pumped at 1.0 ml/min. The enantiomers of KT were quantified by ultraviolet detection with the wavelength set at 254 nm. The assay described allows for the direct quantitation of KT enantiomers without pre-column derivatization, and is suitable for clinical studies of KT enantiomers in human plasma and urine after administration of therapeutic doses. PMID- 7550976 TI - Determination of lansoprazole and five metabolites in plasma by high-performance liquid chromatography. AB - A high-performance liquid chromatographic method for the determination of lansoprazole, a new proton-pump inhibitor, and five of its metabolites in human plasma is described. Lansoprazole, its metabolites, and internal standard (omeprazole) were extracted into diethyl ether-methylene chloride and separation was obtained using a reversed-phase column under isocratic conditions. The method features monochromatic ultraviolet detection at 285 nm, and single extraction, single evaporation sample handling. The lower limit of quantitation, based on standards with acceptable coefficients of variation, was 10 ng/ml for all compounds. No endogenous compounds were found to interfere. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. PMID- 7550977 TI - Solid-phase extraction of nicotine and its metabolites for high-performance liquid chromatographic determination in urine. AB - A solid-phase extraction, using Extrelut-1 glass columns, has been applied to urine samples of both passive and active smokers for high-performance liquid chromatographic determination of nicotine and its metabolites cotinine and trans 3'-hydroxycotinine. Chromatography was performed using a reversed-phase LC8DB column and a mobile phase consisting of water-acetonitrile (80:9, v/v) containing 5 ml triethylamine, 670 mg/l sodium heptanesulphonate, and 0.034 M each of K2HPO4 and citric acid (pH 4.4), at a flow-rate of 1.6 ml/min. The results obtained indicate that solid-phase extraction is a reliable and quick procedure which can be applied also to other nicotine metabolites. PMID- 7550979 TI - Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma. AB - A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z) stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 microliters solvent. After evaporation to dryness, the extract is reconstituted in 20 microliters solvent and evaporated to approximative 10 microliters. A 4-microliter aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m x 0.32 mm I.D., 0.52 micron capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients. PMID- 7550978 TI - Identification of methylated metabolites of inorganic arsenic by thin-layer chromatography. AB - TLC on cellulose plates was used to identify methylated products of inorganic arsenic metabolism (monomethylarsonate and dimethylarsinate) in biological samples. Two solvent systems were tested: methanol-ammonium hydroxide (8:2) and isopropanol-acetic acid-water (10:1:2.5). The latter solvent system produced the most satisfactory separation of radiolabelled methylated arsenic compounds in aqueous solution, in rat liver cytosol incubated with carrier-free or 1 microM [73As]arsenite and in urine of mice given carrier-free [73As]arsenate or 5 mg of [73As]arsenate/kg per os. Oxidation of samples by hydrogen peroxide improved the separation and quantitation of monomethylarsonate in both biological matrices. PMID- 7550980 TI - Analysis of sevoflurane degradation products in vapor phase samples. AB - Sevoflurane degradation products were measured by GC-flame ionization detection in vapor phase samples using manual and automated injection methods. Sample handling techniques allowed the transfer and storage of samples for up to 72 h. Compound A, fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, was the major vapor phase degradation product formed in simulated clinical conditions. Recoveries of 4-32 ppm (v/v) compound A concentrations using the manual method were in the range of 88-117% (n = 12, mean = 102%, R.S.D. = 9%). PMID- 7550982 TI - Automated high-performance liquid chromatographic method for the determination of nedocromil sodium in human urine using bimodal column switching. AB - An automated HPLC method is described for the determination of nedocromil sodium in human urine. An HPLC autosampler is used to inject urine samples onto a short reversed-phase column. This column acts as a concentration column and performs a preliminary extraction. The concentration column is automatically back-flushed onto an ion-exchange column where final separation of nedocromil sodium from urine constituents occurs. Recovery, accuracy, precision, sensitivity and specificity were investigated. The method has been applied to urine samples from clinical studies, and the results were compared to those obtained using a radioimmunoassay developed previously. PMID- 7550983 TI - Determination of the enantiomers of metoprolol and its major acidic metabolite in human urine by high-performance liquid chromatography with fluorescence detection. AB - Enantiomers of metoprolol and its acidic metabolite H 117/04 were determined in human urine by high-performance liquid chromatography (HPLC) with fluorometric detection after chiral derivatization. The carboxyl functional group of the major metabolite was blocked by esterification after solid-phase extraction, which helped to quantitate this compound from interfering substances. The assay method was validated. The recovery of (-)- and (+)-metoprolol from urine was 86.3-90.5%; and the recovery of the (-)- and (+)-acidic metabolite H 117/04 from urine was 74.4-83.9% at different concentrations. PMID- 7550981 TI - Determination of chloramphenicol in animal tissue using high-performance liquid chromatography with a column-switching system and ultraviolet detection. AB - A rapid and universally applicable method for determination of chloramphenicol (CAP) residues in animal tissues using high-performance liquid chromatography with a column-switching system is presented. The clean-up procedure as well as the chromatographic conditions and detection are described. The linearity and repeatability of the data obtained by this method as well as the recovery rates of CAP in several farm animals are presented. PMID- 7550984 TI - Development and preliminary application of high-performance liquid chromatographic assay of urinary metabolites of diazepam in humans. AB - A simple high-performance liquid chromatographic method for the measurement of diazepam (DZP) and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP) and oxazepam (OZP) in urine was developed. Preliminary studies of DZP metabolism were also undertaken in four healthy volunteers after administration of a single oral dose (4 mg) of DZP. The assay allowed the simultaneous determination of all analytes in 1 ml of urine and the detection limit was 2 ng/ml with a signal-to-noise ratio of 3. None of 22 drugs and 17 metabolites, except for mianserin, maprotiline and imipramine N-oxide, interfered with the detection of DZP metabolites. Recoveries of the analytes and the internal standard (prazepam) were > 82%. Intra- and inter-assay coefficients of variation for all analytes were < 5.5 and 4.1%, respectively. The mean (+/- S.D.) cumulative urinary excretions of DMDZP, TZP and OZP over 96 h after a single oral administration of DZP were 3.9 +/- 0.4, 6.6 +/- 1.4 and 2.8 +/- 0.6% of the dose, respectively. The urinary excretion of DZP was under the detection limit. PMID- 7550986 TI - Determination of free concentration of ropivacaine and bupivacaine in blood plasma by ultrafiltration and coupled-column liquid chromatography. AB - A coupled-column liquid chromatographic method for determining the free concentration of ropivacaine and bupivacaine in blood plasma was developed. Following adjustment of the temperature and pH, the plasma samples were ultrafiltrated. Ropivacaine or bupivacaine in the ultrafiltrate was determined by direct injection into a coupled-column liquid chromatographic system, consisting of one reversed-phase and one ion-exchange column. The system was highly selective. Ropivacaine and bupivacaine were detected by UV at 210 nm. The limit of determination was 10 nM and the inter-assay precision at a concentration level of about 100 nM was 6% (R.S.D., n = 30) for ropivacaine and 7% (R.S.D., n = 30) for bupivacaine. PMID- 7550985 TI - Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection. AB - A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (% C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma. PMID- 7550988 TI - Handling staff with chemical dependence. PMID- 7550987 TI - Simple, rapid and sensitive high-performance liquid chromatographic determination of delavirdine and its N-desisopropyl metabolite in human plasma. AB - A method for the determination of a bisheteroarylpiperazine, non-nucleoside HIV-1 reverse transcriptase inhibitor, delavirdine, and its N-desisopropyl metabolite in human plasma, is described. Samples were deproteinized by addition of two parts of a solution of internal standard in acetonitrile (1 microgram/ml) to one part plasma. The supernatant was diluted with 10 mM phosphate buffer, pH 6.0, and injected onto the HPLC system. Fluorescence of the eluent was monitored with excitation at 302 nm and emission at 425 nm. Quantitation of delavirdine and its metabolite was achieved by comparing the peak-height ratio of each component relative to the internal standard to a through-the-origin linear regression curve determined from fortified plasma calibration standards. The assay was linear over the concentration range 0.02-17 microM for both delavirdine and its metabolite. The precision of the method, as expressed by the mean C.V. of the back calculated, non-zero, standard concentrations, was +/- 4.4% for delavirdine and +/- 4.3% for the metabolite. The assay has been validated and utilized to analyze samples from human and animal pharmacokinetic studies. PMID- 7550989 TI - Euthanasia, nursing and care of the dying: rethinking Kuhse and Singer. PMID- 7550991 TI - Pain remembered with a dash of humour. PMID- 7550990 TI - A good many died. PMID- 7550992 TI - Nursing and the law. A bitter lesson. PMID- 7550993 TI - Understanding rostering. Part 6. Changing rosters--managing roster change. PMID- 7550994 TI - Linkage of the athymic nude locus with the myeloperoxidase locus in the rat. AB - In rats of the BUF/Mna strain epithelial thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. In pre-thymoma stage, BUF/Mna rats have extremely large thymuses, when compared with those of other strains of rats. The large thymus size of this strain is contributed by a thymus-enlargement gene, Ten 1. On the other hand, reduced thymus size and suppression of thymoma development were found in heterozygous BUF/Mna-rnu/+ rats. Linkage studies between RNU and microsatellite and restriction fragment length polymorphism markers in ([BUF/Mna rnu/rnu x WKY/NCrj] F1 x WKY/NCrj)- and (WKY/NCrj x [BUF/Mna-rnu/rnu x WKY/NCrj] F1)- backcross rats have led to the localization of RNU on chromosome 10. The rat homolog of mouse Mpo (myeloperoxidase) was also assigned to the chromosome 10. The gene order on the chromosome was MYHSE (myosin heavy chain of embryonic skeletal muscle)--(1.0 centimorgan [cM])--SHBG (sex hormone-binding globulin)- (4.0 cM)--RNU (Rowett rat nude)--(10.0 cM)--MPO--(13.0 cM)--AEP (anion exchange protein). Conserved linkage of homologous loci mapped to rat chromosome 10 and mouse chromosome 11 supports the hypothesis that the RNU and MPO loci are rat homologs of the mouse nu and Mpo loci. PMID- 7550996 TI - Immunohistochemical analysis of giant cell glioblastoma. AB - Giant cell glioblastoma (GCG) is one of a group of rare tumors in which the cell population is abnormally large and includes multinucleated cells of gigantic sizes. Immunohistochemical studies were performed on four GCG cases and found that all giant cells and/or tumor cells were positive for glial fibrillary acidic protein (GFAP), S-100 protein, and vimentin, thus verifying the tumor's glial origin. The nuclei of multinucleated giant cells of three adult cases were frequently immunostained for proteins expressed during the cell cycle (proliferating cell nuclear antigen (PCNA) and Ki-67), thereby demonstrating the proliferative capacity of these cells. By contrast, those of a 12 year old girl expressed these cell cycle markers rather infrequently. Alpha I-antitrypsin was detected with relatively high frequency in the giant cells, and its presence may explain their bizarre sizes and pericellular reticulin fiber formation. A literature review of 32 cases revealed that the GCG that occurs preferentially in young girls is a type of pleomorphic xanthoastrocytoma. By contrast, GCG in adult males has the same age incidence as ordinary glioblastomas and, as these, expresses high levels of cell cycle-related proteins. Thus, GCG, which is subclassified morphologically as ordinary glioblastoma, has distinct biological and clinical characteristics, with that in children requiring re-evaluation because of its similarities to pleomorphic xanthoastrocytoma. PMID- 7550997 TI - Diffuse laminar endocervical glandular hyperplasia. AB - A case is reported of diffuse laminar endocervical glandular hyperplasia, which is very rare among pseudoneoplastic endocervical glandular lesions. The patient was a 54 year old woman who had been suffering from watery vaginal discharge for about 7 years, and the lesion was found in the cervix of the hysterectomy specimen. Microscopically, the lesion showed a diffuse, laminar proliferation of hyperplastic endocervical glands, sharply demarcated from the underlying stroma. The significant nuclear atypia was absent. Inflammatory response was mild. Immunohistochemically, carcinoembryonic antigen was negative. Differential diagnosis from other non-neoplastic conditions and well differentiated adenocarcinoma is discussed. PMID- 7550995 TI - A new monoclonal antibody (IE8) reactive with dendritically shaped cells in the human tonsil. AB - A new monoclonal antibody (mAb), 1E8 (IgG1, kappa), was obtained from a hybridoma prepared by fusion of mouse myeloma cells (NS-1) with splenic cells of mice immunized with a human B blastic malignant lymphoma cell line, HPE-Ret-3 (Ret-3). The mAb showed a reactivity unrestricted to a specific cell lineage on flow cytometrical analysis of the reactivity with human lympho-hematopoietic cell lines. In peripheral blood, 1E8 reacted with the cells of all lineage, that is, lymphocytes, monocytes, granulocytes and platelets, even though its intensity was very low by immunohistochemistry. Immunohistochemical examination of human tonsil with 1E8 showed a characteristic staining pattern. Positive cells scattered in follicular (mantle zone and germinal center), parafollicular (T-dependent area), subepithelial and interstitial connective tissue areas. These positive cells seemed to be categorized into dendritically shaped cells (DSC), including dendritic cells (DC) and a subpopulation of macrophages in follicles, interdigitating cells (IDC) and irregularly shaped mononuclear cells. The localization of 1E8 antigen staining was similar to that of integrin CD11c, although its distribution on hematopoietic cell lines did not coincide with that of 1E8 antigen. Immunobiochemical studies showed that 1E8 bound two cell surface proteins with molecular size of 70,000-90,000 and 35,000 Da each. Consequently, 1E8 antigen might be a novel marker of DSC. PMID- 7550998 TI - Increased serum levels of type IV collagen and laminin in a patient with a giant retroperitoneal schwannoma. AB - A 63 year old woman presented with a giant abdominal tumor. Ultrasonography, computerized tomography and drop-infused pyelography revealed a suspected retroperitoneal tumor. The tumor was removed surgically and weighted 2800 g. The pathological diagnosis of the tumor was schwannoma. Strong immunohistochemical staining specific for type IV collagen and laminin was observed in the tumor, and these components were localized in the pericellular region of Schwann cells. The serum levels of these antigens, as determined by radio-immunoassay, were very high before the operation but decreased rapidly thereafter. PMID- 7550999 TI - Mucinous cholangiocarcinoma featuring a unique microcystic appearance. AB - An autopsy case is presented of a peculiar type of intrahepatic mucinous adenocarcinoma with microcyst formation arising in a 78 year old Japanese man who died of hepatic coma and renal failure 4 months after onset. Macroscopically, the cut surface of the lesion revealed a characteristic honeycomb-like appearance, consisting purely of microcysts, 0.2-0.4 cm in diameter, lined by prolific mucin producing adenocarcinoma cells. The lesion did not have large cystic space, fibrous capsule, or benign cystadenomatous component other than neoplastic microcyst formation. The carcinoma cells showed various proliferating patterns, such as irregularly shaped nest-like, trabecular, papillary and tubular ones, directly invaded the hepatic parenchyma and portal tract with loose or thick fibrosis, and infiltrated extensively into both intrahepatic and extrahepatic stroma along the vascular structures. From these clinicopathological findings, we consider the present tumor to be a variant of mucinous cholangiocarcinoma with characteristic microcyst formation rather than a type of cystadenocarcinoma. PMID- 7551001 TI - Carcinosarcoma of the uterine body of mesonephric origin. AB - A case of carcinosarcoma of mesonephric origin in a 58 year old woman is reported. A cystic tumor with a solid area, measuring 14 cm in greatest diameter, was detected in the pelvic cavity by computerized tomography and ultrasound. Although it was diagnosed as an ovarian cancer for surgical removal, it was found to be entirely located in the myometrium of the left lateral wall of the uterine body and neither ovary was remarkable. Histologically the tumor was composed of epithelial and sarcomatous components. The former showed low papillary pattern, crowded solid nests and cords of cells, and focal tubular structures. The latter showed a solid growth pattern with differentiation to leiomyosarcoma. In the uterine cervix, a 1.2 cm mesonephric (Gartner's) cyst was found. Neither neoplastic lesions nor endometriosis were identified in the cervix, endometrium, fallopian tubes or ovaries. Based on the histologic features and the specific location of the tumor, the coexistence of Gartner's cyst, and the normal appearance of the endocervical mucosa as well as the endometrium, it was diagnosed as a mesonephric carcinosarcoma. The serum levels of carcinoembryonic antigen, CA125, CA19-9, and CA72-4 were within normal ranges in the clinical course. The patient died of disease 8 months after surgery. PMID- 7551000 TI - Carcinosarcomas of the esophagus. AB - Two cases of carcinosarcoma of the esophagus are reported. Both were polypoid tumors occurring in the middle of the intrathoracic esophagus. The tumors were predominantly composed of spindle-shaped sarcoma cells with some squamous cell carcinomas (SCC). One tumor showed many bizarre giant cells with filamentous materials in the cytoplasm. Microscopical examination of both tumors revealed transition from SCC to sarcoma cells. Immunohistochemically, the spindle-shaped sarcoma cells in both tumors displayed a strongly positive immunoreaction to alpha-smooth muscle actin, as did the bizarre giant cells of one tumor to sarcomeric actin. SCC and a few spindle-shaped sarcoma cells near the SCC showed a positive immunoreaction to cytokeratin. Electron microscopy revealed that the spindle-shaped cells had many myofilaments with dense bodies and that the bizarre giant cells had sarcomere structures with Z-bands in their cytoplasm. These findings indicate that both tumors were carcinosarcomas of SCC and myogenic sarcoma. We considered that sarcoma cells might originate in SCC, representing its metaplastic differentiation, or that both SCC and sarcoma might originate in a pluripotent stem cell. PMID- 7551002 TI - Anerythremic form of acute erythremic myelosis (Di Guglielmo's syndrome) causing hepatosplenomegaly due to the infiltration of hemoglobin-bearing blast cells: an autopsy case. AB - An autopsy case of a 42 year old man with the anerythremic form of acute erythremic myelosis (Di Guglielmo's syndrome) is reported. The patient was admitted because of a 1 month history of fatigue and fever. Physical examination showed hepatosplenomegaly. Laboratory data showed leukopenia, mild normocytic anemia, and high levels of serum lactate dehydrogenase and vitamin B12. Bone marrow aspirate revealed an elevated number of erythroblasts, with dyserythropoiesis (E/M = 3.7). After admission, thrombocytopenia progressed rapidly, but blast cells were not seen in the peripheral blood throughout the clinical course. On the 56th hospital day, the patient died of pneumonia. At autopsy, the spleen weighed 550 g and the liver 1800 g. Histologically, the white and red pulps of the spleen and the portal region and sinusoid of the liver were diffusely infiltrated by blast cells that were positive for anti-hemoglobin (Hb) antibody on immunoperoxidase staining. The bone marrow, the lymph nodes, the adrenal glands, the pancreas, and the heart were also infiltrated by the blast cells. This was thus considered to be a rare case of the anerythremic form of acute erythremic myelosis (Di Guglielmo's syndrome), the findings showing that Hb immunoperoxidase staining is useful for the diagnosis of this condition. PMID- 7551003 TI - Malignant fibrous histiocytoma of the right ventricle of the heart. AB - Right-sided cardiac malignant fibrous histiocytoma (MFH) is extremely rare, and to the authors' knowledge only three cases have been reported. In this study, a case of MFH in the right ventricle, the septum, and the pulmonary valves and artery in a 47 year old male is described. The tumor showed typical pathological features of MFH, such as cellular pleomorphism, storiform pattern and abundant mitoses. Immunohistochemical and electron microscopical findings were compatible with MFH, and excluded the possibility of leiomyosarcoma and angiosarcoma. Whole body examination, including Gallium scintigram, localized the primary site to the heart. The details of this case are presented with a review of the reported cases of cardiac MFH. PMID- 7551004 TI - Primary osteosarcoma arising from cirrhotic liver. AB - An autopsy case of a 67 year old man with primary osteosarcoma arising in cirrhotic liver is reported. His son had von Recklinghausen disease and he had had a history of hepatitis C virus infection for 10 years. A large tumor, about 10 cm in diameter, was found in the right liver lobe. This tumor showed marked central necrosis and hemorrhage, and histologically diffuse sarcomatous cell proliferation associated with extensive osteoid formation and calcification of the periphery. Examination of the whole tumor and the cirrhotic liver (155 tissue blocks) showed that the tumor consisted of sarcoma cells mixed with osteoid with no region resembling hepatocellular carcinoma or hepatoblastoma. Minute hepatocellular carcinomas were found in the cirrhotic liver distant from the sarcomatous area. On immunohistochemical examination, the main tumor gave a distinct positive reaction for vimentin, but not for keratin or other epithelial markers. These findings indicate that the tumor was a true primary osteosarcoma, not an osteoid metaplasia of hepatocellular carcinoma. PMID- 7551005 TI - Podocytic degeneration and regeneration in puromycin aminonucleoside nephropathy in the rat. AB - In various types of chronic glomerulonephritis, proliferation of mesangial and endothelial cells is often seen together with extracellular matrix accumulation. In contrast, the proliferation of visceral glomerular epithelial cells (podocytes) has not been detected in any disease in which these cells are the principal target of injury. To examine whether proliferation of podocytes occurs, puromycin aminonucleoside nephropathy was induced in Wistar rats by intraperitoneal injection of puromycin aminonucleoside (15 mg/100 g of bodyweight). In this nephropathy model of nephrotic syndrome with minimal change, the main lesions consisted of vacuolation and flattened foot processes of podocytes, which are the primary target of puromycin aminonucleoside. Urine protein excretion and proliferating cell nuclear antigen (PCNA) positive cells were examined. The PCNA+ podocytic count reached its maximum on day 3, indicating that the most marked podocytic proliferation occurs just after the injection of PAN. Twenty-four hour urinary excretion in this nephropathy model supported the hypothesis that mitosis of podocytes occurs in association with recovery on day 13. PMID- 7551006 TI - Alveolar basement membrane breaks down in diffuse alveolar damage: an immunohistochemical study. AB - Sequential structural changes of the alveoli in diffuse alveolar damage (DAD) were examined by immunohistochemical methods. Lung specimens obtained at autopsy from 52 patients with DAD were stained with antibodies to laminin, 7S collagen (7S) and type IV collagen (type IV) for alveolar basement membrane, to von Willebrand factor, CD-31 and thrombomodulin (TM) for the alveolar capillary endothelial cell, and to epithelial membrane antigen and surfactant apo-protein (PE-10) for the alveolar epithelium. Forty-two of the patients had the exudative form of DAD; 10 of the patients had the proliferative form of DAD. The results were summarized as follows: (i) laminin was most easily impaired both in the epithelial and capillary basement membrane in the early exudative stage; (ii) following laminin, 7S and type IV in the capillary basement membrane were also injured in the early exudative stage, and recovered in the proliferative stage; (iii) subsequently, 7S and type IV in the epithelial basement membrane were also impaired in the late exudative stage, and remained impaired even in the proliferative stage; and (iv) alveolar epithelium regenerated almost completely in the late exudative stage, but staining for TM in the alveolar capillary recovered in the proliferative stage. Because the alveolar basement membrane must govern the homeostasis of alveolar tissue architecture, it was concluded that its preservation is necessary to avoid the abnormal remodeling of the alveoli in the reparative stage of DAD, if the patient survives the acute episodes of the disease. PMID- 7551007 TI - Quantitative analysis of stromal fat content of human parathyroid glands associated with thyroid diseases using computer image analysis. AB - The stromal fat content in the parathyroid glands and its significance are still controversial. Several methods have also been introduced to evaluate the content in the literature. In an attempt to better understand the significance of stromal fat content, a computer image analyzing system was applied and 62 glands were investigated using routinely processed tissue. Our study revealed that the average stromal fat content was 34.8 +/- 18.7% which was lower than that of other studies. The stromal fat content was correlated with the age, grade of obesity, area of the gland and serum parathormone level; however, no consistent patterns were obtained with other clinical parameters. These results indicate that the change of stromal fat content may be related to the general condition of the patient similar to the change of fat in other organs. However, the decrease of the content may indicate the early change of parathormone secretory activity even if the serum examination shows no evidence of hyperfunction of the glands. PMID- 7551008 TI - Angiomyofibroblastoma of the vulva: a clinicopathologic study of seven cases. AB - A clinicopathologic and immunohistochemical review was made of seven cases of angiomyofibroblastoma. The patients were middle-aged women who had a slowly growing mass, measuring 1.5-6 cm in maximum dimension, located subcutaneously in the vulva. The tumors were well-demarcated and characterized by well vascularized, alternating hypercellular and hypocellular edematous areas composed of bland, plump spindle- or oval-shaped stromal cells frequently aggregated around small blood vessels. An epithelioid appearance of the stromal cells was seen in two cases. Immunohistochemically, the stromal cells were consistently positive for vimentin and desmin, but negative for muscle specific actin, alpha smooth muscle actin, myosin, cytokeratins, S-100 protein or von Willebrand factor. Ultrastructurally, the plump stromal cells had a small amount of peripherally located rough endoplasmic reticulum, numerous pinocytotic vesicles and abundant intermediate filaments, on which immunogold probes for desmin were localized, whereas fine filaments were few and there were no electron dense plaques. Thus, while the proliferating stromal cells expressed an immunohistochemical profile of peculiar myoid differentiation, ultrastructural findings differed from those of smooth muscle cells or those seen in typical myofibroblasts. At 1-4 years after surgery, there was no evidence of recurrence. PMID- 7551009 TI - Integrin distribution in gastric carcinoma: association of beta 3 and beta 5 integrins with tumor invasiveness. AB - Immunolocalization of a variety of integrins using monoclonal antibodies against beta 3, beta 5, alpha 3 beta 1 and alpha 6, and polyclonal antibodies against vitronectin receptor (alpha v beta 3) and beta 1 were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The beta 5 integrin, which pairs only with the alpha v subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). beta 5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. beta 3, which is another subunit pairing with alpha v, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.01). beta 3 was also located chiefly in the cytoplasm. Five of the seven beta 5 positive cases showed coexpression of beta 3. Polyclonal antibodies to alpha v beta 3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-beta 1 antibodies showed clear positivity in many cases (89%). Of the beta 1 integrins, alpha 3 beta 1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit alpha 6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma These distinctive patterns of integrin positivity indicate a close association of beta 5 and beta 3 expression with scirrhous invasion in gastric carcinoma. PMID- 7551010 TI - Flow cytometric evaluation of nuclear DNA content in hepatoblastoma: further evidence for the inhomogeneity of the different subtypes. AB - The DNA ploidy pattern of nine hepatoblastomas and three normal liver specimens was investigated by flow cytometry. Areas of unlike differentiation in the same tumor were investigated separately. Normal liver tissue and the fetal subtype were always diploid. Aneuploidy was found in the embryonal subtype. The one case of small cell tumor was also diploid. When the fetal and embryonal areas of the same tumor were analyzed together, there was always an aneuploid peak in the histogram. There are obvious differences in DNA ploidy among the various hepatoblastoma subtypes. Differences in methods used, including the failure in some studies to evaluate areas of unlike differentiation in the same tumor separately, probably account to some extent for the conflicting results of previously reported studies. When flow cytometric analysis of the DNA content of a hepatoblastoma is performed, it is important to ensure that the various types of differentiation in the tumor are represented adequately in the material investigated, especially when conclusions about the prognosis are to be based on these findings. PMID- 7551011 TI - Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization. AB - The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as well as in situ hybridization (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-embedded urinary bladder cancers, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16- or 18 positive signals in all except one of the tumor samples. However, the PCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffin-embedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers. PMID- 7551012 TI - Immunohistochemical and flow cytometric study of neuroendocrine carcinoma of the skin. AB - Immunohistochemical and flow cytometric analysis using formalin-fixed, paraffin embedded sections was performed on 10 neuroendocrine carcinomas of the skin (NCS). Grimelius staining was positive in seven tumors. All tumors showed coexpression of CAM 5.2 and neuron-specific enolase with paranuclear dot-like or diffuse cytoplasmic reactivity. Neurofilament was positive in five cases, chromogranin in six, calcitonin and carcinoembryonic antigen in two each, and somatostatin and S-100 protein in one each. Eight primary lesions were diploid and the remaining two were aneuploid; however, two diploid NCS presented as aneuploid metastatic tumors. The follow-up periods ranged from 3 to 66 months (mean 13.6). Six patients died of metastatic diseases between 3 and 33 months (mean 9.2) after the diagnosis. There were no significant correlations among histologic features, DNA ploidy, S-phase fraction, and clinical outcome of the patients with NCS. These results indicate that a panel of antibodies may be required for immunohistochemical confirmation of neuroendocrine differentiation and that a flow cytometric analysis is not a good tool to predict the biologic behavior of NCS. PMID- 7551013 TI - A collapsing form of glomerulopathy. AB - An unusual presentation of glomerular disease occurring in a 30 year old black female is described. The case was characterized by a rapid deterioration of renal function. Associated glomerular manifestations were diffuse collapse of the glomerular capillary lumina, extracapillary hypercellularity without obliteration of the urinary space and without proliferation of parietal epithelial cells, electron-dense deposits, and deposition of IgG and C3. There was little loss of tubules and minor interstitial fibrosis. This case demonstrated a collapsing form of glomerulopathy with immunoglobulin and electron-dense deposits. PMID- 7551014 TI - A sporadic case of Ehlers-Danlos syndrome type IV: diagnosed by a morphometric study of collagen content. AB - A sporadic case of a young woman with Ehlers-Danlos syndrome (EDS) type IV is described. Multiple aneurysms of medium-sized arteries were noted. Histological study revealed deposition of acid mucopolysaccharides in the media of major arteries and in the intima of smaller arteries with intimal thickening. Systemic changes related to stenosis were observed in arteries more than 100 microns in diameter. Histologic study revealed hyperplasia of adipocytes in the submucosal layer of the intestines and the trachea, and fibrosis of Langerhans' islets of the pancreas. Typical signs for EDS such as skin hyperelasticity and joint hypermobility, and positive family history were not present. However, type III collagen was not detected on frozen sections from either the skin or the anterior cerebral artery by immunohistochemical quantification. Thus, it was concluded that the present case is a variant of EDS. PMID- 7551015 TI - Regional Proteus syndrome: report of an autopsy case. AB - An uncommon autopsy case involving Proteus syndrome with multiple hamartomatous lesions in a 52 year old woman is reported. At birth, hemihypertrophy was noted on the right side of the face. Leiomyoma of the urinary bladder was extirpated at 37 years of age. She died of sepsis due to a brain abscess. At autopsy, diffuse asymmetrical lipomatosis was noted on the right side of the face, scalp, lip, oral mucosa, tongue and on the left side of the cerebellar peduncle. Multiple meningiomas, cavernous hemangiomas and osseous hypertrophy overlapped in the intracranial regions. The present case is considered as a regional type of Proteus syndrome displaying a somatic mosaicism. PMID- 7551016 TI - Fibro-osseous pseudotumor of the digits arising in the subungual region: a rare benign lesion simulating extraskeletal osteosarcoma. AB - A rare case of subungual fibro-osseous pseudotumor of the digits in a 59 year old woman is reported. A painful polypoid mass with ulcerative changes was noted in the subungual portion of the right first toe. Macroscopically, the tumor arose chiefly in the subcutaneous region and was unrelated to the underlying bone tissue. Histologically, the lesion contained fibroblast-like tumor cells producing an extensive osteoid substance in a granulation-like background. Although it was necessary to distinguish the lesion from extraskeletal osteosarcoma, the tumor cells lacked prominent atypical features. It is considered that pathologists and clinicians should add fibro-osseous pseudotumor of the digits to the differential diagnosis of subungual tumor. PMID- 7551017 TI - Sequence motifs in a flagellin of Pseudomonas putida. PMID- 7551018 TI - The planctomycetes: emerging models for microbial ecology, evolution and cell biology. PMID- 7551019 TI - Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. AB - Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed. PMID- 7551020 TI - Adherence of Candida albicans to epithelial cells: studies using fluorescently labelled yeasts and flow cytometry. AB - Candida albicans adherence to epithelial cells is the first step in the infectious process, but in spite of its importance, current methods for the quantitative measurement of adherence of C. albicans to epithelial cells in vitro have some serious limitations. They are based on filtration assays and either microscopic or radiometric analysis. The adherence reaction is usually carried out with a large excess of yeasts (100-fold) over epithelial cells in order to perform the microscopic analysis, which is slow, subjective and limited to 100 200 cells and thus lacks statistical power. The radiometric analysis fails to measure individual cells. A method for measuring yeast adherence that overcomes these problems has been developed. It is based on labelling the yeasts with the fluorogenic marker 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF) prior to the adherence reaction, and analysing 10(4) epithelial cells by flow cytometry, while nonbound yeasts are excluded by gating. Two subpopulations of buccal epithelial cells (BECs) which differ in their mean fluorescence intensities per cell (MFIs) were observed: one with MFI which did not exceed nonspecific fluorescence, and the other with MFI as high or higher than the MFI of labelled yeasts. The two subpopulations represent yeast-free and yeast-binding epithelial cells, respectively, and the MFI increment of the BECs is a quantitative measure of the extent of yeast adherence. Control experiments confirming previously described basic features of adherence, such as enhanced adherence at increasing yeast excess, diminished adherence of trypsin-treated or heat-inactivated yeasts, and the differential adherence of various Candida species, supported the validity of the assay. The possibility of studying adherence reliably at low yeast:epithelial cell ratios, which better mimic adhesion as it occurs in vivo, is an important advantage of the assay. New findings, using this method, included the observation that exfoliated BECs from diabetic patients exhibited the same capacity for C. albicans adherence as cells from healthy controls, and that epithelial cells from early human ontogenic stages had a significantly lower adherence level than those from later stages. PMID- 7551021 TI - Variability in expression of antigens responsible for serotype specificity in Candida albicans. AB - The monoclonal antibody (mAb) B9E, which reacts with a cell wall surface determinant of Candida albicans serotype A, and a polyclonal monospecific antiserum against the antigen 6 (IF6) were used to investigate the expression of the antigens responsible for the serotype specificity in C. albicans under different growth conditions. By indirect immunofluorescence, both antibodies reacted with the cell wall surface of serotype A yeast cells and germ tubes grown in vitro but no reactivity was observed with serotype B yeast cells. In some cases, only a weak reactivity restricted to a zone close to the parent yeast cell was observed in serotype B germ tubes stained with mAb B9E. Both antibodies reacted strongly with yeast cells and germ tubes present in kidney abscesses from rabbits infected with both serotypes, but only serotype A yeast cells and germ tubes present in smears from patients with vulvovaginal candidiasis reacted with B9E and IF6 antibodies. The expression of antigens reactive with both antibodies was modulated by the pH of the environment in which the fungus was grown. Both antibodies showed a similar pattern of reactivity when studied with a spectrofluorometer. Serotype A yeast cells showed maximum reactivity when cells were grown on Sabouraud dextrose broth supplemented with yeast extract at pH 4.6. The lowest reactivity was observed in cells grown at pH 2.0. Conversely, the reactivity of serotype B yeast cells increased at alkaline pH values, the highest being in cells grown at pH values of 7.2 and 9.5.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551022 TI - Identification of glucan-mannoprotein complexes in the cell wall of Candida albicans using a monoclonal antibody that reacts with a (1,6)-beta-glucan epitope. AB - The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied. PMID- 7551023 TI - Basis of cerulenin resistance of two strains of Candida albicans. AB - The basis of cerulenin resistance of Candida albicans strains 4918-2 and 4918-10 has been investigated. Parasexual genetic analyses established that cerulenin resistance to concentrations of at least 5 micrograms ml-1 is dominant in both strains. The results also showed that strain 4918-2 is heterozygous for resistance, while the change from resistance to sensitivity of strain 4918-10 is reversible. Experiments to define the mechanism(s) responsible for resistance focused on cerulenin uptake and fatty-acid synthase activity. Cerulenin uptake by strains 4918-2 and 4918-10 was 24% of that of the wild-type (strain 4918). Uptake was restored in UV-induced cerulenin-sensitive segregants of strains 4918-2 and 4918-10, and varied from 63% to 200% of parental values. Fatty-acid synthase from strains 4918-2 and 4918-10 was resistant to cerulenin as judged by differences in the inactivation of the enzyme by the agent. However, inactivation kinetics of fatty-acid synthase of cerulenin-sensitive segregants did not revert to the parental inactivation profile. Further investigation showed that nine out of ten segregants were resistant to cerulenin at concentrations between 1 and 4 micrograms ml-1 while strain 4918 was sensitive to cerulenin at all concentrations tested. Thus, the results suggest that alteration of fatty-acid synthase and changes in permeability contribute to total cerulenin resistance of each strain. PMID- 7551024 TI - Constitutive glucose-induced activation of the Ras-cAMP pathway and aberrant stationary-phase entry on a glucose-containing medium in the Saccharomyces cerevisiae glucose-repression mutant hex2. AB - Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a nonfermentable carbon source triggers a rapid, transient increase in the cAMP level. The occurrence of this cAMP spike appears to be correlated inversely with the glucose-repression state of the cells. This was also observed for the hex2 mutant, which is deficient in glucose repression and which displayed the cAMP signal constitutively. When cells of the hex2 mutant were starved for nitrogen on a glucose-containing medium, they rapidly lost viability, similarly to mutants with overactivation of the Ras-adenylate cyclase pathway. Flow cytometry measurements showed that G1 arrest of the hex2 mutant under such conditions was incomplete. Trehalose accumulation, a typical feature of cells entering the stationary phase G0, was very short-lived in the hex2 mutant under the same conditions. These results are in agreement with the presence of continuous glucose-triggered activation of cAMP synthesis in hex2 cells on a glucose containing nitrogen-starvation medium. In the course of these experiments a spontaneous suppressor mutant, shx (for suppressor of hex2), was isolated which survived nitrogen starvation on a glucose-containing medium much better than the hex2 strain. It also showed normal G1 arrest and much longer accumulation of trehalose. The suppressor mutation also caused inability to grow on nonfermentable carbon sources and absence of invertase depression, and it was epistatic to hex2 for these characteristics also. The isolation of this epistatic depression mutation supports the idea that the defect in glucose repression of the hex2 mutant is the cause of its rapid loss of viability during nitrogen starvation on a glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551026 TI - Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice. AB - We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N terminal region of Mycobacterium fortuitum beta-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice. PMID- 7551025 TI - Coordination of sucrose uptake and respiration in the yeast Debaryomyces yamadae. AB - Screening in batch cultures identified Debaryomyces yamadae as a yeast that exhibits the Kluyver effect for sucrose: this disaccharide can be respired but, even under oxygen-limited conditions, alcoholic fermentation of sucrose does not occur. Ethanol, glycerol and arabitol were the main fermentation products during oxygen-limited growth on glucose in chemostat cultures. None of these fermentation products were produced in oxygen-limited chemostat cultures grown on sucrose and the fraction of the sucrose that could not be respired remained unused in the culture medium. This absence of alcoholic fermentation was not due to repression of the key fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase. In contrast to some other yeasts that exhibit a Kluyver effect, D. yamadae did not exhibit a preference for ethanol in batch cultures grown on mixtures of ethanol and sucrose. Sucrose metabolism in D. yamadae involves intracellular hydrolysis by an alpha-glucosidase. Incubation of weakly buffered cell suspensions with sucrose led to a rapid transient alkalinization, indicating the presence of a sucrose-proton symport system. The apparent substrate saturation constant of the sucrose-uptake system was 0.2 mmol l-1. Sucrose dependent alkalinization rates were much lower in samples from oxygen-limited cultures than in samples from aerobic cultures. Transient responses of D. yamadae to oxygen limitation were investigated by applying a sudden decrease in the oxygen feed to aerobic sugar-limited chemostat cultures. In glucose-grown cultures, this led to alcoholic fermentation and no significant accumulation of sugar occurred after the switch. In sucrose-limited cultures, sugar accumulation occurred instantaneously after the switch, and ethanol formation was virtually absent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551030 TI - Production of Escherichia coli STb enterotoxin is subject to catabolite repression. AB - Enterotoxigenic Escherichia coli are known to secrete several types of toxins including STb, a heat-stable enterotoxin. STb enterotoxin production was studied in wild-type E. coli strains. Using a quantitative STb-specific inhibition ELISA, the amount of toxin present in the culture supernatant fractions of various E. coli strains was determined. Variation in the production of STb toxin was observed for the wild-type strains. For E. coli strain 82-4247 grown in trypticase soy broth, the toxin was produced after 4 h of growth and was maximal after about 57 h of growth. The amount of toxin in the culture supernatant fraction increased concomitantly with bacterial growth. Using the rat loop assay, the biological activity of STb was retained even after the logarithmic phase of growth when STb production levelled off (i.e. from 24 to 74 h). STb production by E. coli strain 82-4247 varied with the culture medium used. In particular, addition of 1.0% (w/v) compared to 0.1% glucose to Davis minimal medium decreased STb production, whereas addition of 1.0% (w/v) glycerol did not affect STb production. Addition of exogenous cAMP reversed the repressive effect of glucose. Using mutant strains, STb production was shown to be subject to catabolite repression. PMID- 7551028 TI - Differential transcription of the MPB70 genes in two major groups of Mycobacterium bovis BCG substrains. AB - Substrains of Mycobacterium bovis BCG (BCG) have been divided into two major groups, high and low producers, on the basis of the amount of secretion of the MPB70 protein. The antigen is produced in high concentration by BCG Tokyo, Moreau, Russia and Sweden (high-producer substrains), whereas in BCG Pasteur, Copenhagen and Tice (low-producer substrains) it is detected at 1% (w/w) or less of the concentration of BCG Tokyo. To investigate why this protein is secreted differently, the MPB70 genes of BCG Tokyo and Pasteur were cloned, sequenced and compared. The MPB70 genes in two substrains showed exactly the same sequence. Even the upstream and downstream regions of the MPB70 gene were identical. MPB70 gene expression was assessed by means of Northern hybridization analysis and reverse transcriptase polymerase chain reaction. The mRNA was clearly detected in BCG Tokyo, but a very low level in BCG Pasteur. On the basis of these results, the difference in the secretion of the MPB70 protein between BCG Tokyo and Pasteur was attributed to differential transcription efficiencies. PMID- 7551027 TI - A linear B-cell epitope on the class 3 outer-membrane protein of Neisseria meningitidis recognized after vaccination with the Norwegian group B outer membrane vesicle vaccine. AB - The class 3 outer-membrane protein (OMP) of Neisseria meningitidis is a potential target for bactericidal and opsonic antibodies in humans. Synthetic peptides spanning the class 3 OMP from the vaccine strain 44/76 (B:15:P1.7,16:L3,7) were synthesized on pins and screened with serum obtained from Norwegian adolescents immunized with a meningococcal serogroup B outer-membrane vesicle (OMV) vaccine. A strong IgG response to a single peptide (19FHQNGQVTEVTT30) located within loop 1 (VR1) was stimulated after three doses of OMV vaccine in three vaccinees selected on the basis of their antibody response to class 3 OMP. No clear linear B-cell epitopes were recognized by four different murine serotype 15-specific mAbs. A 23mer peptide (D63b2) containing loop 1 of the class 3 OMP was synthesized, and the IgG responses were measured in pre- and post-vaccination serum from 27 vaccinees. Specific IgG rose significantly in 37% of vaccinees 6 weeks after the second dose and in 74% of the vaccinees 6 weeks after the third dose of the OMV vaccine. Most immune sera reacted distinctly on immunoblots with denatured class 3 OMP, and the immunoblotting reactivity correlated strongly with concentration of the IgG antibodies specific for peptide D63b2. When added to a post-vaccination serum from one vaccinee, peptide D63b2 competed efficiently with the class 3 OMP for specific antibody binding on immunoblots and in pin ELISA. The results show that the significant part of the humoral response to the meningococcal class 3 OMP elicited by vaccination with the Norwegian OMV vaccine was directed against a single continuous epitope. PMID- 7551029 TI - Molecular composition of the outermost capsular material of the tubercle bacillus. AB - To gain insight into the pathogenesis of tuberculosis, a molecular definition of the tubercle bacillus cell envelope, which is involved in the early stages of the infection, is required. The cell-surface-exposed material of the pathogen was isolated by mechanical means and chemically analysed. It was shown by scanning electron microscopy that the method used for extracting the surface-covering material preserves the integrity of the bacilli. Surprisingly, in view of the current opinion, only small amounts of lipids (1-6%) were present. Polysaccharides and proteins were the main components of the material. The polysaccharides were neutral and lipid-free D-glucan, D-arabino-D-mannan and D mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 120, 13 and 4 kDa, respectively. Based on NMR spectroscopy and conventional chemical analyses, the major structural motifs of the purified polysaccharides were established as being identical to those of the polysaccharides we previously isolated from the culture filtrate of the tubercle bacillus. Immunocytochemical studies showed that these compounds were not only surface-located but were also present in the inner capsular compartment. The major protein constituents exhibited the same mobilities on SDS-PAGE as those of the culture filtrate on the tubercle bacillus and readily reacted with the monoclonal antibodies directed against these molecules. These proteins included the 19 and 38 kDa lipoproteins, the 30/31 kDa fibronectin-binding proteins and the 40 kDa L-alanine dehydrogenase. These findings suggest that the culture filtrate material represents part of the capsule which, in an in vivo context, could contribute to the electron transparent zone surrounding the tubercle bacillus. The 24 kDa (MPB/T64) protein was found to be a secreted protein, as it was detected almost exclusively in the culture filtrate. Taken together, the data give a new insight into the surface-exposed compounds of the tubercle bacillus and may explain part of the nature and limitation of the host immunity towards the pathogen. PMID- 7551032 TI - Mesentericin Y105 gene clusters in Leuconostoc mesenteroides Y105. AB - Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb DraII fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities. PMID- 7551031 TI - Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus. AB - The genes encoding the production of acidocin B, a bacteriocin produced by Lactobacillus acidophilus strain M46 which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus, but inactive against most other Lactobacillus species, were previously localized on a 4 kb XbaI HindIII fragment of plasmid pCV461. In the present work, DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially code for hydrophobic peptides composed of 60, 91 and 114 amino acids, respectively, and a fourth ORF of opposite polarity which could potentially encode a peptide of 59 amino acids. The middle ORF (ORF-2; acdB) was identified as the gene encoding acidocin B by comparing the amino acid composition of highly purified acidocin B with the deduced amino acid sequence of ORF-2. Our results suggest that acidocin B is synthesized as a precursor which is processed at a site which conforms to the ' -3, -1' rules of von Heijne to yield active acidocin B (59 amino acids). The presence of an immunity-protein-encoding gene on the 4 kb XbaI-BamHI fragment was deduced from the capacity of a plasmid vector harbouring this fragment to confer immunity upon transformation of L. fermentum NCK127. One of the three non assigned ORFs may encode this immunity protein. PMID- 7551034 TI - A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic upshock in Staphylococcus aureus. AB - Four major proteins are induced in Staphylococcus aureus in response to hyperosmotic shock caused by the presence of two different osmolytes, sucrose and NaCl. The gene encoding one of these proteins was isolated using a novel PCR procedure. The derived protein sequence shows extensive similarity to a subunit of alkyl hydroperoxide reductase (AhpC) from both Escherichia coli and Salmonella typhimurium. Exposure of S. aureus to varying concentrations of H202 did not result in the detectable induction of AhpC. PMID- 7551033 TI - Escherichia coli periplasmic protein FepB binds ferrienterobactin. AB - Most high-affinity systems for iron uptake in Gram-negative bacteria are thought to employ periplasmic-binding-protein-dependent transport. In Escherichia coli, FepB is a periplasmic protein required for uptake of iron complexed to its endogenously-synthesized siderophore enterobactin (Ent). Direct evidence that ferrienterobactin (FeEnt) binds to FepB is lacking because high background binding by FeEnt prevents use of the usual binding protein assays. Here the membrane localization vehicle LppOmpA [Francisco, J.A., Earhart, C.F. & Georgiou, G. (1992). Proc Natl Acad Sci USA 89, 2713-2717] was employed to place FepB in the E. coli outer membrane. Plasmid pTX700 was constructed and shown to encode, under lac operator control, the 'tribrid' protein LppOmpAFepB; the carboxy terminal FepB portion lacks at most two amino acids of mature FepB. After short induction periods, most of the tribrid was in the outer membrane. A number of LppOmpAFepB species could be detected; some were degradation products and some may be related to the multiplicity of FepB forms previously observed in minicells and maxicells. Outer membrane harbouring the tribrid and lacking FepA, the normal outer membrane receptor for FeEnt, bound approximately four times more FeEnt than outer membrane from uninduced cells, from cells lacking pTX700 and from cells expressing only an LppOmpA 'dibrid'. Similarly, whole UT5600(fepA)/pTX700 cells induced for tribrid synthesis bound FeEnt and this binding was not affected by energy poisons. The results demonstrated that FepB can bind FeEnt, thereby definitely placing FeEnt transport in the periplasmic permease category of transport systems, and that the LppOmpA localization vehicle can be used with periplasmic binding proteins. PMID- 7551035 TI - The mob locus of Escherichia coli K12 required for molybdenum cofactor biosynthesis is expressed at very low levels. AB - The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using lambda placMu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA. Both of the promoter regions show a poor match to the -35 and -10 consensus sequences for sigma 70 promoters. A 2.2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene. PMID- 7551036 TI - Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine. AB - The Rhizobium meliloti Rm220-3 mos locus producing the rhizopine scyllo-inosamine (SI) in nodules is shown to consist of three ORFs (ORF1, mosB and mosC) arranged in an operon structure. This differs from the R. meliloti L5-30 mos locus, which produces 3-O-methyl-scyllo-inosamine (3-O-MSI), by the complete absence of mosA. The deletion covers a region of 1235 nt and includes the entire mosA gene as well as the sequence both upstream and downstream. As a result, Rm220-3 mos ORF1 shares a 5' region common with L5-30 ORF1 but includes an additional 10 bp insertion and a section in the L5-30 mosA and mosB intercistronic region. Antibodies against L5-30 Mos proteins detected MosB and MosC proteins in Rm220-3 induced nodules but no translation product for either ORF1 or mosA was detected. A construct prepared from the L5-30 mos locus which has a truncated mosA gene produces SI rather than 3-O-MSI, confirming this gene is involved in a methylation step in the production of 3-O-MSI. Further, changes made to this ORF result in reduced levels of the rhizopine. PMID- 7551037 TI - beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria. AB - A series of transposons are described which contain the gusA gene, encoding beta glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out. PMID- 7551038 TI - Functional analysis of the flagellar genes in the fliD operon of Salmonella typhimurium. AB - The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments. Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT. Based on the gene-disruption experiment in E. coli, both the fliS and fliT genes have been postulated to be necessary for flagellation. In the present study, we constructed S. typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted. Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S. typhimurium. However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain. This indicates that the fliS mutation affects the elongation step of filament assembly. The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media. We found that the amount of flagellin excreted was much reduced by the fliS mutation. Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway. PMID- 7551040 TI - Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase. AB - The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor. PMID- 7551039 TI - A gene encoding a thermophilic alkaline serine proteinase from Thermus sp. strain Rt41A and its expression in Escherichia coli. AB - The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present. PMID- 7551041 TI - Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure. AB - High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42 degrees C, but not at 30 degrees C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non permissive temperature. The secretion defect was not linked to the secA/div locus. PMID- 7551043 TI - Activity of mycobacterial promoters during intracellular and extracellular growth. AB - pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains. PMID- 7551042 TI - An operon encoding a novel ABC-type transport system in Bacillus subtilis. AB - Downstream from the surfactin synthetase operon in Bacillus subtilis a new operon type structure has been localized which, on the basis of sequence determination, potentially encodes an ABC-type transport system. The 268 amino acid protein, the product of orf1, represents the solute-binding component of the system whereas the orf2 product, a 234 amino acid protein, is the transmembrane component. Finally orf3 potentially encodes a typical 241 amino acid ATP-binding protein involved in energy supply. Comparison of the three proteins with the subunits of other ABC-type systems suggests that this new system is involved in amino acid transport. PMID- 7551044 TI - A new Myxococcus xanthus gene cluster for the biosynthesis of the antibiotic saframycin Mx1 encoding a peptide synthetase. AB - The gene cluster for the biosynthesis of the heterocyclic quinone antibiotic saframycin Mx1 of Myxococcus xanthus DM504/15 was inactivated and tagged by Tn5 insertions. The tagged genes were cloned in Escherichia coli and used to select overlapping cosmid clones spanning 58 kb of the M. xanthus genome. Gene disruption experiments defined a > or = 18 kb contiguous DNA region involved in saframycin biosynthesis. Sequencing of part of this region revealed a large ORF containing two 600-amino-acid domains with similarity to peptide synthetase amino acid-activating sequences, suggesting that saframycin Mx1 is synthesized by a nonribosomal multienzyme complex, similar to other bioactive peptides. PMID- 7551045 TI - Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase. AB - The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup- strain. PMID- 7551046 TI - A pre-induction sporulation gene from Aspergillus nidulans. AB - Asexual sporulation in Aspergillus nidulans is an inducible developmental process controlled by genes that act before and after the inductive stimulus is applied. Genes that act before induction (pre-induction genes) potentially represent functions required for response to induction. This report describes the isolation and characterization of the acoB pre-induction gene which was cloned by complementation of a thermosensitive aconidial mutant followed by gene rescue. Genetic analysis and gene disruption confirmed the identity of the cloned gene. The mRNA of the acoB gene was present in uninduced vegetative hyphae, induced conidiating cultures and in both conidiospores and ascospores. An ORF in the nucleotide sequence of the acoB cDNA specifies a 327-residue protein unrelated to any known peptide sequence. Sequence analysis of the thermosensitive mutant allele, acoB202, revealed that the mutant phenotype is due to a frame-shift mutation that severely truncates the putative ACOB protein. Disruption of the acoB gene also produced a strain that was thermosensitive for conidiation. These properties suggest that acoB may be a gene that is required for sporulation only at elevated temperatures. Hybridization of acoB DNA with DNA from a variety of Aspergillus species showed that homology to this gene is largely restricted to sexually sporulating species that belong to the nidulans group. PMID- 7551048 TI - Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield. AB - Isogenic strains of Escherichia coli W3110 containing pyruvate dehydrogenase complexes with three (wild-type), two or one lipoyl domains per lipoate acetyltransferase (E2p) chain, were constructed. The maximum growth rates (mumax) for batch cultures growing in minimal medium containing different carbon sources showed that reducing the number of lipoyl domains adversely mumax value of the mutant containing one lipoyl domain per E2p chain was restored by the presence of compatible multicopy plasmids encoding PDH complexes with either one or three lipoyl domains per E2p chain. In glucose-limited chemostat cultures the protein contents of all strains were similar and substrate carbon was totally accounted for in the biomass and CO2 produced. However, the carbon efficiencies (percentage carbon conversion to biomass) were significantly lower when the lipoyl domain content of the E2p subunit was reduced from three to one. Similarly, the cellular maintenance energy (m(e)) and the maximum growth yield (Ymax) were lower in bacteria containing PDH complexes with fewer than three lipoyl domains per E2p chain. Wild-type values were restored by supplementing the medium with either casamino acids (0.01%) or acetate (up to 0.1 mM). The lower growth efficiencies of the mutants were further confirmed in competition experiments where equal numbers of genetically marked (NalR) mutant and wild-type bacteria were used to inoculate glucose-limited chemostat cultures (dilution rate 0.075 h-1). The mutants with one or two lipoyl domains per E2p chain were washed out, whereas in controls, the initial ratio of wild-type (Nals) to reconstructed wild-type (NalR) bacteria was maintained over 50 generations. PMID- 7551047 TI - Unusual effect of myo-inositol on phospholipid biosynthesis in Cryptococcus neoformans. AB - Cryptococcus neoformans is an opportunistic fungal pathogen which preferentially localizes to the inositol-rich environment of the central nervous system. One of its distinguishing traits is its capacity to catabolize inositol. Inositol is a precursor for the synthesis of phosphatidylinositol (PI). This study demonstrated that C. neoformans synthesizes inositol. Three inositol-containing sphingolipids were identified in C. neoformans: ceramide-(P-inositol)2mannose, mannose, ceramide-P-inositol-mannose, and ceramide-P-inositol. These inositol-containing sphingolipids are typical of fungi but not higher eukaryotes. The effect of inositol on the membrane lipid composition of C. neoformans was also examined. In contrast to the nonpathogenic yeast Saccharomyces cerevisiae, neither the PI composition nor the synthesis of methylated phospholipids was altered by exogenous inositol. Hence, C. neoformans appears to have a metabolic mechanism for maintaining a steady lipid composition regardless of the inositol in its environment. PMID- 7551050 TI - Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme. AB - A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0.11 h-1 (td = 6.3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M(r)) of the native GDH was 110,000. SDS-PAGE resulted in one single band that represented a polypeptide with a M(r) of 28,000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4.2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551049 TI - Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase. AB - A sorbitol dehydrogenase (SDH; L-iditol:NAD+ 2-oxidoreductase; EC 1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R. sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene. SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass (M(r)) of the native SDH was 61000 as calculated from its Stokes' radius (rs = 3.5 nm) and sedimentation coefficient (S20,w = 4.23S). SDS-PAGE resulted in one single band representing a polypeptide with a M(r) of 29,000, indicating that the native protein is a dimer. The isoelectric point of SDH was determined to be pH 4.8. The enzyme was specific for NAD+ and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D tagatose and of L-iditol. The apparent Km values were NAD+, 0.06 mM; D-glucitol, 6.2 mM; galactitol, 1.5 mM; NADH, 0.13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11.0 and that of substrate reduction 6.0-7.2. It was demonstrated that SDH is expressed in the wild-type strain R. sphaeroides Si4 together with MDH during growth on D-glucitol. Forty four amino acids of the SDH N terminus were sequenced. This sequence exhibited 45 55% identity to the N-terminal sequence of 10 enzymes belonging to the short chain alcohol dehydrogenase family. PMID- 7551053 TI - Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli. AB - Spontaneous multidrug-resistant (Mdr) mutants of Klebsiella pneumoniae strain ECL8 arose at a frequency of 2.2 x 10(-8) and showed increased resistance to a range of unrelated antibiotics, including chloramphenicol, tetracycline, nalidixic acid, ampicillin, norfloxacin, trimethoprim and puromycin. A chromosomal fragment from one such mutant was cloned, and found to confer an Mdr phenotype on Escherichia coli K12 cells that was essentially identical to that of the K. pneumoniae mutant. Almost complete loss of the OmpF porin in the E. coli transformant, and of the corresponding porin in the K. pneumoniae mutant, was observed. The presence of the Mdr mutation in K. pneumoniae or the cloned K. pneumoniae ramA (resistance antibiotic multiple) locus in E. coli also resulted in active efflux of tetracycline, and increased active efflux of chloramphenicol. After transformation of a ramA plasmid into E. coli, expression of chloramphenicol resistance occurred later than expression of resistance to tetracycline, puromycin, trimethoprim and nalidixic acid. The ramA gene was localized and sequenced. It encodes a putative positive transcriptional activator that is weakly related to the E. coli MarA and SoxS proteins. A ramA gene was also found to be present in an Enterobacter cloacae fragment that has previously been shown to confer an Mdr phenotype, and it appears that ramA, rather than the romA gene identified in that study, is responsible for multidrug resistance. The ramA gene from the wild-type K. pneumoniae was identical to that of the mutant strain and also conferred an Mdr phenotype on E. coli, indicating that the mutation responsible for Mdr in K. pneumoniae had not been cloned. PMID- 7551054 TI - The bldA-encoded tRNA is poorly expressed in the bldI mutant of Streptomyces coelicolor A3(2). AB - The bldA gene, encoding a leucyl tRNA recognizing the UUA codon, is expressed at significantly lower levels in the bldI mutant, Streptomyces coelicolor J703, than in the parent S. coelicolor A3(2). Expression of a TTA-containing reporter gene was reduced in the bldI mutant, as was the mature, 87 nucleotide, form of the bldA-encoded tRNA. This reduced level of the tRNA was also seen when the bldA gene was introduced on a high-copy-number plasmid into the bldI mutant, suggesting that maximal bldA expression may require a bldI-dependent promoter. PMID- 7551052 TI - cAMP-cAMP receptor protein complex: five binding sites in the control region of the Escherichia coli mannitol operon. AB - The control region of the mannitol (mtl) operon of Escherichia coli has been shown to contain five cAMP receptor protein (CRP) binding sequences, the most yet reported for any operon. A DNA fragment encompassing the entire mtl operon regulatory region was generated by PCR, and the binding of the cAMP-CRP complex was studied. Using restrictional analysis to separate, delineate and destroy the various putative CRP binding sites, all five sites were shown to be functional for CRP binding in vitro. Four of these sites bound the cAMP-CRP complex with high affinity while the fifth site (the most distal relative to the transcriptional start site) bound the complex with lower affinity. Simultaneous binding of cAMP-CRP complexes to several of these sites was demonstrated. The results serve to identify and define five dissimilar CRP binding sites in a single operon of E. coli. A model for mtl operon transcriptional initiation and repression complexes is presented. PMID- 7551051 TI - Hydrophobic properties of the cell surface of Candida albicans: a role in aggregation. AB - The ability of Candida albicans to aggregate and adhere to biological surfaces is a topic of major biological and medical importance. One factor which has been implicated in such properties is the hydrophobic nature of the cell surface. Two simple spectroscopic techniques are described which permit the rapid determination of this property. The first involves the use of arylnaphthalenesulfonate, the fluorescence emission maximum of which was shown to be a sensitive indicator of dielectric polarity. This was used to identify the hydrophobic characteristics of the cell surface of C. albicans. The second technique involves the use of 90 degrees Rayleigh-Debye light scattering as an indicator of the aggregation state of a fungal suspension. These techniques were then used to compare the surface properties of three different strains of C. albicans and the effects of culture conditions: the hydrophobicity of the strains varied, and galactose-based culture media promoted the greatest degree of cell surface hydrophobicity. PMID- 7551055 TI - Topological analysis of the lysine-specific permease of Escherichia coli. AB - Escherichia coli accumulates lysine via two systems, one specific for lysine (LysP) and a second inhibited by arginine or ornithine (LAO). The lysP gene encodes a polypeptide of 489 residues. A topological analysis of the LysP protein was performed using gene fusions. Random in-frame fusions of the lysP gene with the lacZ or blaM genes were generated. Site-directed mutagenesis was also used to generate additional blaM fusions at specific locations in the lysP gene. Two methods were used to alleviate the problem of lethal expression of some lysP::blaM fusions. First, ternary fusions were constructed in which the arsD gene was fused at the 5' end of the lysP gene and the blaM gene fused at specific sites within the lysP gene. In these plasmids lysP expression was controlled by the ars promoter. Secondly, an E. coli strain with a pcnB mutation was used with some fusions to maintain the plasmids at a reduced copy number. From analysis of 30 gene fusions, a topological model of the LysP protein is proposed in which the protein has 12 membrane-spanning regions, with the N- and C-terminal in the cytosol. PMID- 7551057 TI - Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment. AB - A major limitation of rRNA-targeted group-specific probes is that they may cross react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes. PMID- 7551056 TI - I-CeuI recognition sites in the rrn operons of the Bacillus subtilis 168 chromosome: inherent landmarks for genome analysis. AB - The Bacillus subtilis 168 circular chromosome yielded ten fragments on I-CeuI endonuclease digestion. I-CeuI recognizes a 26 bp sequence that is located within the gene encoding the 23S subunit of the rRNA in Chlamydomonas eugametos, Escherichia coli and Salmonella typhimurium. The precise locations of the I-CeuI sites of the B. subtilis chromosome were determined on a NotI-SfiI physical map by (i) double digestion analyses with I-CeuI and SfiI, (ii) comparison of mutant strains lacking a specific rrn operon, (iii) using an I-CeuI linking clone and (iv) analysis of nucleotide sequence data of some rrn operons. In conclusion, all the I-CeuI sites were located within the B. subtilis rrn operons and the I-CeuI sites were conserved in all the B. subtilis 168 derivatives tested. Thus, variations in size of the I-CeuI fragments must be due to genome alterations. A B. subtilis 168 strain was investigated with I-CeuI. We demonstrated that the aberrant structure was the outcome of the inversion of an approximately 1700 kb DNA segment. PMID- 7551059 TI - Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection. AB - The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551058 TI - A 46 kDa integral membrane protein from Mycobacterium leprae resembles a number of bacterial and mammalian membrane transport proteins. AB - In this paper we describe the nucleotide sequence of a 3.4 kbp region of the Mycobacterium leprae genome. This region contains an open reading frame of 1290 bp with a coding capacity for a protein of 46,179 Da, designated the 38L protein. Using antibodies against part of the 38L protein, we were able to demonstrate that the 38L protein is present in the membrane protein fraction of M. leprae. The 38L protein showed significant matches with a number of integral membrane proteins involved in the transport of small molecules through the cellular membrane. Among these are a human and a murine protein involved in melanin biosynthesis. The 38L protein might play a role in the hypopigmentation observed in leprosy patients. PMID- 7551060 TI - Cholera toxin (CTX) genetic element in Vibrio cholerae O139. AB - PFGE analysis of the NotI- and SfiI-digested genome of Vibrio cholerae O139 strains isolated from different epidemic regions of India showed that all the strains are of clonal origin and the genome size is about 2.2 Mb. An analysis of the electrophoretic profiles of the genome of O139 strains, the RFLP of the cholera toxin (ctx) gene and Southern blot hybridization of NotI-digested genomes of classical, El Tor and O139 with a NotI-linking clone of classical strain 569B, suggest that these strains closely resemble V. cholerae O1 biotype El Tor, but are widely different from the classical O1 vibrios. Using restriction enzymes which cleave a single site in either the core region or in the direct repeat sequence (RS) of the CTX genetic element, it has been shown that the genome of most of the O139 strains has two copies of the ctx gene in tandem connected by two RSs. The chromosomal location of the CTX genetic element in the O139 strain is the same as that reported for El Tor vibrios. The organization of the virulence gene cassettes in different O139 strains shows genetic heterogeneity in the population. Whilst most of the epidemic strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a single copy of the element has been deleted. PMID- 7551061 TI - Reduction of exogenous ferric iron by a surface-associated ferric reductase of Listeria spp. AB - The reduction of exogenous ferric iron by Listeria monocytogenes, a Gram-positive food-borne pathogen, was investigated. Using an assay incorporating the ferrous iron chelator ferrozine, we showed that intact cells of L. monocytogenes, when exposed to ferric iron, were able to rapidly reduce and solubilize the iron to the ferrous form. Reduction occurred only after direct contact between the bacteria and the iron source. A number of different ferric iron chelates, including transferrin and lactoferrin-bound iron, haemoglobin, ferritin, and iron complexed to siderophores, could be reduced. The ferric reductase activity was expressed by both reference strains and clinical isolates of L. monocytogenes and by all other species of Listeria, although significant quantitative differences were observed. In L. monocytogenes, the expression of ferric reductase was not affected by the growth phase of the bacteria nor by the presence or absence of iron in the growth medium. However, expression was greatly reduced in bacteria grown anaerobically and when cultured in media of reduced pH. In addition, bacteria grown at a cold temperature displayed greater ferric reductase activity than cells grown at higher temperatures. A surface-associated ferric reductase system may be one component of a general iron scavenging mechanism which can be used by Listeria growing in a variety of environments. PMID- 7551062 TI - Attenuated typhoid vaccine Salmonella typhi Ty21a: fingerprinting and quality control. AB - Live attenuated vaccines, developed with molecular genetical techniques, require new approaches for their quality control. To develop novel quality control tests that enhanced and extended existing procedures, the attenuated vaccine strain Salmonella typhi Ty21a and its parent strain Ty2 were characterized by pulsed field gel electrophoresis (PFGE) and direct nucleotide sequence analysis. Mutant and parent strains were distinguished using fingerprints generated by the resolution on PFGE of chromosomal DNA digested with each of the enzymes SfiI, SpeI or XbaI. These fingerprints were stable through multiple in vitro passages of the vaccine strain and were identical from one batch of vaccine to another. It was also possible to distinguish between the mutant and parent strains by direct nucleotide sequence analysis of the galE gene. This analysis identified two base changes in the gene from strain Ty21a: a single base deletion causing a frameshift that would result in a truncated gene product, accounting for the galE phenotype; and a transition that eliminated an AluI restriction site. The consequent change in the AluI fingerprint of the galE gene in strain Ty21a provided a rapid, PCR-based alternative to the use of differential media or biochemical assays for the identification of the vaccine strain. PMID- 7551063 TI - Anti-Candida activity of a novel killer toxin from the yeast Williopsis mrakii. AB - A screening of putative killer yeast strains showed that spore-forming ascomycetous yeasts of the genera Pichia and Williopsis displayed the broadest range of activity against sensitive strains of Candida spp. and Saccharomyces cerevisiae. Williopsis mrakii (NCYC 500) showed extensive anti-Candida activity against strains isolated from clinical specimens. W. mrakii killer factor was produced in minimal media as a function of growth and its activity reached constant levels as cells entered stationary phase. The proteinaceous killer toxin was found to be unstable without a specific range of temperature and pH (above 30 degrees C and pH 4.0), and further analysis showed that the active toxin molecule was an acidic polypeptide with a relative molecular mass between 1.8-5.0 kDa. At critical concentrations the killer factor exerted a greater effect on stationary phase cells of Candida than cells from an exponential phase of growth. At low concentrations, the killer toxin produced a fungistatic effect on sensitive yeasts but at higher concentrations there was evidence to suggest that membrane damage accounted for the zymocidal effects of the killer factor. the cidal nature of the toxin was reflected in a rapid decrease in sensitive cell viability. Findings presented suggest that W. mrakii killer toxin has potential as a novel antimycotic agent in combatting medically important strains of Candida. PMID- 7551065 TI - [The scientific method: the indispensable tool for students]. PMID- 7551064 TI - Lipopolysaccharide and porin of Roseobacter denitrificans, confirming its phylogenetic relationship to the alpha-3 subgroup of Proteobacteria. AB - Roseobacter denitrificans has rough (R)-type lipopolysaccharide, containing 2 keto-3-deoxyoctonate but no hepatoses. Its lipid A has a glucosamine-containing, phosphorylated backbone. It contains the rare 3-oxotetradecanoic (3-oxomyristic) acid as the only amide-bound fatty acid and ester-bound 3-hydroxydecanoic acid, this pattern being characteristic for the alpha-3 subgroup of Proteobacteria. Treatment of the major outer-membrane protein (porin, apparent molecular mass 88 kDa) of Roseobacter denitrificans with EDTA (2 mM, 30 degrees C, 20 min) resulted in the dissociation of the oligomers into monomers (apparent molecular mass 35 kDa). EDTA-sensitive dissociation has so far been observed only within the alpha 3 subgroup of Proteobacteria. The 12 N-terminal amino acids of the monomers exhibit sequence homology with the porins of Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas blastica. Renaming of Roseobacter denitrificans as Rhodobacter denitrificans is suggested. PMID- 7551066 TI - [Effects of personalized counseling in the reduction of pressure levels in patients with severe essential hypertension. WHO 1]. AB - Two intervention systems to evaluate the effect of personalized counseling in severe essential hypertensive patients OMS I, oriented to reduce systolic and diastolic tension pressures and to increase self-esteem, were compared. The independent variable as an intervention method took place during 24 weeks. DEPENDENT VARIABLES: tension pressures and self-esteem were measured. The results showed that essential severe tension pressures and increased their self-esteem more than patients of control group. Finding also a direct dependence between weight and tension pressures Personalized Counseling showed to be a successful strategy of nursing for this group of patients. PMID- 7551067 TI - [Epidemiology for nurses]. AB - The subject of this paper emphasizes the uses of Epidemiology to nurses like an essential tool to afford health service assistance and his application in practice. Equally it evidences that the epidemiology offers important support to the planning, administration, execution and evaluation of the health service and in addition an extremely useful and essential investigation instrument to his professional formation. PMID- 7551069 TI - [Nursing and the hypertensive patient. Report of experiences]. AB - The Arterial Hypertension League (LHA), has enable the team to carry out a multiprofessional approach by highly prinzing the actions of the medical team, comprised of: doctors, nurses, nutritionists, physical education teachers and psychologists. In this working structure, nursing represents an important supporting pillar, as it participates in 50% of the actions. The nurse's work consists of attending the patients in preconsultation, consultation and pos consultation procedures; taking part in the team meetings with the patients (The Hypertension Club); drawing up and carrying out research projects and coordinating the team's efforts. The participation of the nursing staff in the work done by the health team has afforded a greater efficiency to the work, and at same time, it is a great training field for students. PMID- 7551068 TI - [AIDS and nursing: attitudes towards personality traits in the context of hospital care]. AB - The objective of the present study was to determine possible relations between attitudes towards some aspects of facing AIDS and personality traits in nursing personnel for later elaboration of a program of guidance and follow-up of professionals who treat the disease. On the basis of the attitude scales constructed by the theoretical model of FISHBEIN and AJZEN (1975), a survey was carried out on 56 professionals of the Nursing Service of the University Hospital of Ribeirao Preto to determine their attitudes towards the six dimensions of AIDS treatment: the disease itself, support treatment, the stigma attached to the patient, the impotence of the professional, the rapport with the patient, and the embarrassment due to the conditions of care provided. The personality traits of these 56 subjects were later evaluated by applying Questionnaire 16 PF of CATTEL and EBER (1954). Correlation studies and contingency analyses revealed interaction between attitudes towards some factors and the results obtained with the 16 PF, with the level of significance set at p = .05. A detailed analysis of the personality/attitudes towards AIDS relationship was then performed from the results obtained, in combination with data obtained in previous studies. PMID- 7551070 TI - [Detection of visual and auditory problems in Ribeirao Preto school children. A comparative study by socio-economic level]. AB - The evaluation of visual and auditive functions of students entering the first degree of elementary school is considered a basic action of the student health. The child, upon entering the elementary school, begins or continues learning the alphabet which is considered one of the most important process of the educational area. The child needs normal vision and hearing (or with adequate correction) to carry on this process in the easiest way. In this paper, we tried to study the visual and auditive problems in elementary school students in the city of Ribeirao Preto, from different social layers concerning early detection of cases and resolution of problems (or not) found by the family and school. PMID- 7551071 TI - [Attitudes of professional nurses towards burn patients: elaboration and reliability tests of a measurement instrument]. AB - In this study it was developed an Instrument for Measuring the Attitudes of Nursing Professionals toward Patients with Burns. 194 items, divided into subscales, expressing favorable and unfavorable attitudes toward patients with burns, were analyzed, concerning their apparent and context validity. Following this psychometric analysis 170 items were selected to compose the definitive scale. The confiability coefficient estimated by the split-half method for the different subscales ranged from 0.61 to 0.82. The confiability coefficient of the total scale was equal to 0.92. PMID- 7551072 TI - [Studies using the methodology of phenomenologic investigation]. AB - The author reports on her experience as an adviser in studies carried out according to the methodology of phenomenologic investigation. This experience involves advising processes at several levels: scientific initiation, scientific training, master's and doctorate. The phases of investigation of this type are discussed, with emphasis on the critical and decisive points along the methodological pathway. Emphasis is placed on the importance of a significant investment in the search for an in-depth understanding of the principles of phenomenology on the part of researchers, so that the study to be performed will be conducted in a serious, rigorous and appropriate manner, thus effectively generating new knowledge. PMID- 7551073 TI - [A program of adult health promotion by health care workers]. AB - The purpose of this work is to evaluate an alternative methodology of education in adult health care undertaken with health care workers. The evaluation was made by the directional question: what did the Adult Health Course mean to you? The analysis consisted of detecting in the responses, the affirmatives that were respective and grouping them in seven categories: 01. Learning new things. 02. Learning to orient people who goes to the basic Health Unit. 03. Interaction with colleagues. 04. Feeling professionally appraned. 05. Worrisome with sons. 06. Improving way of life. 07. Suggestions. The results refers to the presuppositions presented in the introduction of the article, as well as, with the Epidemiologic Model proposed by BLUM. PMID- 7551074 TI - [A common language for nurses: a persistent dilemma]. AB - The author during her discussion tries to explain that the theme needs another approaching perspective. Therefore, she uses a critical model to find out the ideology of the theme; the factors that have contributed to the dilemma inside and outside the profession; presents a general view of the situation in Latin America and finalizes the paper with the indication of some actions aiming at transforming Nursing and enabling the discussion and work with this theme. PMID- 7551076 TI - [Evaluation of theoretical knowledge of diabetics in an interdisciplinary program]. AB - Using an instrument of evaluation of theoretical knowledge, the authors have assessed the capacity of understanding and memorizing often groups of diabetes' carriers, that were being followed by a multiprofessional team from the ambulatory of the HURNP. The evaluation included the discussion of a variety of subjects which were part of the activities of an Educational Program. Only one group of patients showed a satisfactory performance. Individual analysis showed that young diabetes' carriers, that were motivated and had good level of understanding, had a better performance. It was concluded that the evaluation allows the selection of patients that need specific reinforcement and that a plan of activities can be elaborated according to the tested subjects. PMID- 7551075 TI - [Nurses' aides and the practice of nursing research: study of a reality in hospitals]. AB - The present study aims at demonstrating the situation of nurses of a particular hospital institution with respect to nursing research, focusing their previous and actual research, the limited and favourable elements to elaborate scientific works and the meaning of research to nursing, according to this study participant's opinion. The data collection were made through questionnaire and semi-structured interview. PMID- 7551077 TI - [Living the pregnancy: processes and subprocesses of a data-based theory]. AB - This paper shows the results of a qualitative study, that adopted as a methodological line the "Grounded Theory" and as a method to the data collection the participating observation. The goal was to explore and understand the period of a pregnancy, as seen by pregnant women. The data were collected with four sample groups, including 96 women, from january to december 1987. Results of the study are a starting point to a substantive theory in development, "Living the Pregnancy": a dynamic process occurring in the woman's life within a context. Living such a process could be planned or not. Such course of living occurs along four sequential and interconnected stages, which were called Preceding the Beginning, the Beginning, the Middle, and the End of pregnancy. PMID- 7551078 TI - [Study on the incidence of adolescent births in a municipality of the state of Sao Paolo]. AB - The main goal of this study is to find out the rate of childbirth by teenager mothers in Sao Carlos-SP from july, 1993 to july, 1994. The data were collected during one year and showed that in 14.72% of the birth the mothers were 11 to 18 years old. The data were also analysed in terms of, number of pregnancies, occupation, type of birth, and the mother's marital status. The results are discussed in terms of their implication with regard to educational programs to adolescents. PMID- 7551079 TI - [Personal depositions of hospital nurses from the 50's: a help to the understanding of current nursing]. AB - This study is a part of a more extensive project that proposes to rescue significant aspects related to nursing assistance evolution from the 1950s to the 1990s. Aiming at accomplishing this objective, the authors used the technique of oral declaration with active and retired registered nurses, in the context of a school-hospital at the state of Sao Paulo. The present study emphasizes the outcomes referring to the 1950s. The results show that the nurse's efforts are evident in the struggle for profession's recognition and prestige; there are intense and deep transformations related to nurse's new roles and their leadership as a member of the nursing staff and a member of the medical team. PMID- 7551080 TI - [The nursing process in a burn unit: analysis and reformulation of teaching]. AB - The present study aims at elaborating and applying an education plan for nurses in an Unit for Burnt Patients, with the proposal of analysing and reformulating the practice of the nursing process in this service. The framework proposed by Charles MAGUEREZ, was employed as a guideline, which led to the selection and application of the teaching and learning strategies implemented in this plan. We concluded that the framework proposed by MANGUEREZ made possible a substantial improvement in the nursing process in this service, and that this new reality claims for further studies. PMID- 7551081 TI - [Review of the practice of care in a health unit: Experience developed in an integration project between school and service]. AB - The paper shows a program concerning the review of the assistance practice in a Health Unit at the target region of the School-Service Integration Project between the College of Nursing at Ribeirao Preto/University of Sao Paulo and Sao Paulo city. The majority of the health workers took part in the program which was developed in 8 meetings where they analysed their own working practice and elaborated several proposals aiming at changing it. The greatest merit of this program was to allow the establishment of a collective and participative process of discussion and reflection. PMID- 7551082 TI - [Support services for students offered in Brazilian nursing schools]. AB - The authors present an exploratory study realized with 103 brazilian Colleges of Nursing. They aimed at identifying the types of assistance offered to the undergraduate students. The results of 61 courses revealed two categories: 1- offering pedagogic-administrative support services, showing the concern with the formal orientation about nursing education; 2- adding any kind of care delivery to the students needs. Nine courses surpassed the essential needs (mentioned above), concerning with the student's mental health. PMID- 7551083 TI - [Evaluation of a computer system by nursing students]. AB - This study was undertaken to investigate the opinion of nursing undergraduate students of the first year about the discipline "Informatic applied to health". Data were collected using a questionnaire with open and closed questions applied through three consecutive years (1989, 1990, 1991). The results showed that undergraduate students considered this course important. Student's expectations were related to the necessity to get familiar with computers. The mentioned critiques were related to the necessity of more time and equipments to support a better learning process. PMID- 7551084 TI - [Patients with cardiac disorders: diagnostic profile elaborated for nursing students]. AB - The present study aimed at searching patterns of human responses and diagnoses categories identified by 16 undergraduate nursing students in 22 patients with cardiac alterations admitted in a School Hospital. 77 Nursing Diagnoses (ND) were found, 72.7% in the pattern of EXCHANGING, 7.7% in the pattern of MOVING, 6.4% in the pattern of FEELING and 2.5% in the pattern of RELATING. The predominant diagnoses categories were: knowledge deficit (9%), decreased cardiac output (7.7%), fluid volume excess (6.4%), altered nutrition less than body requirements (6.4%) and high risk for infection (5.1%). PMID- 7551085 TI - [Interaction with hospitalized children. Use of therapeutic techniques and measures]. AB - The aim of this study is to describe an experience of a nursing student caring for a child in a pediatric unit. Using therapeutic communication techniques and therapeutic nursing measures, the student developed a help relationship with the child, which enabled the implementation of a thorough nursing process, where the student got fully involved with the patient sharing beneficial experiences for both. PMID- 7551086 TI - Colors of the spectrum. Plastic and reconstructive surgery nursing. PMID- 7551088 TI - Helping patients learn. PMID- 7551087 TI - Baby and me--out 1,2,3. PMID- 7551091 TI - On the other side of the call bell: when the nurse is the patient. PMID- 7551090 TI - Continuing nursing education--a key to staying marketable. PMID- 7551089 TI - Management perspectives. I am coming to the end of my tenure as president of a nursing association. PMID- 7551092 TI - Cancer registrar--oncology resource. PMID- 7551093 TI - Military nurse anesthetists remembered. PMID- 7551094 TI - Becoming a nurse manager. PMID- 7551095 TI - Guide to grad school entrance exams. PMID- 7551098 TI - Management perspectives. I am a nurse manager in a large healthcare organization. PMID- 7551096 TI - Colors of the spectrum. Nurse attorneys. PMID- 7551097 TI - First things first. PMID- 7551099 TI - Pioneering nurse-managed adult day care. PMID- 7551100 TI - Hospital head honchos: RNs at the top. PMID- 7551101 TI - RN singer-songwriter blazes onto children's circuit. PMID- 7551102 TI - Colors of the spectrum. Perioperative trauma nursing. PMID- 7551103 TI - Victor or victim in a critical care war? PMID- 7551104 TI - Management perspectives. On occasion, we get complaints about the care in our unit. PMID- 7551105 TI - Critical care RNs launch program for patients' loved ones. PMID- 7551106 TI - Building a PCU from the ground up. PMID- 7551107 TI - Too sick to move? PMID- 7551108 TI - Glued to the bedside: staff veterans. Interview by Barbara Barzoloski-O'Connor. PMID- 7551110 TI - Demyelinating diseases. PMID- 7551109 TI - Inflammatory diseases. PMID- 7551111 TI - Headache. PMID- 7551112 TI - Emerging pathogens. PMID- 7551113 TI - Neurologic complications of Bartonella henselae infection. AB - Bartonella henselae can cause cat scratch disease and bacillary angiomatosis, a multisystem disorder seen primarily in patients with the acquired immunodeficiency syndrome. Both of these diseases are associated with neurologic complications, particularly encephalopathy. B. henselae may also cause bacteremia and endocarditis, and has been associated with aseptic meningitis and with dementia in patients also infected with the human immunodeficiency virus. Recent advances in identification of this difficult-to-culture organism will lead to recognition of more neurologic complications. PMID- 7551114 TI - Acute viral infections. AB - There have been important recent advances in the diagnosis of acute viral infections of the nervous system. Polymerase chain reaction amplification of nucleic acids in cerebrospinal fluid and tissues is a rapid and accurate tool in the diagnostic evaluation of patients with suspected infections. It has proved to be highly valuable in the noninvasive diagnosis of herpes simplex virus encephalitis and also in establishing the role of herpes simplex virus infection in Mollaret's meningitis. Improved diagnosis of herpesvirus infections should lead to more appropriate antiviral therapy and better outcomes. PMID- 7551115 TI - Neurologic opportunistic infections. AB - Three common opportunistic infections in patients with the human immunodeficiency virus are being more accurately diagnosed and effectively treated as a result of recent advances. Toxoplasmosis may be prevented in some cases, and can be recognized and treated noninvasively in most cases. Cryptococcal therapy has been enhanced by successful development of oral azole therapy. Cytomegalovirus encephalitis is now an entity that can be diagnosed antemortem, and thus efforts to treat it can now be evaluated. PMID- 7551116 TI - Human T lymphotropic virus, type-I myelopathy: an immunopathologically mediated chronic progressive disease of the central nervous system. AB - The human T lymphotropic virus, type-I is associated with a slowly progressive neurologic disorder termed human T lymphotropic virus, type-I associated myelopathy/tropical spastic paraparesis. Considerable information on the pathology, epidemiology, virology, and immunology is known about this disorder and will serve as a framework to define the role that a human retrovirus can play in human disease. In particular, this review will discuss the role of cellular immune responses to the virus in patients with human T lymphotropic virus, type-I associated myelopathy/tropical spastic paraparesis and how these responses may be associated with the pathogenesis of this disorder. By defining virus-specific functional cellular host responses we hope to improve our understanding of the underlying mechanisms that may be involved in the neuropathology of human T lymphotropic virus, type-I associated neurologic disease. PMID- 7551117 TI - Progressive multifocal leukoencephalopathy: clinical and laboratory observations on a viral induced demyelinating disease in the immunodeficient patient. AB - Progressive multifocal leukoencephalopathy is a viral-induced demyelinating disease of the central nervous system usually occurring in the immunocompromised individual. The incidence of progressive multifocal leukoencephalopathy has risen sharply over the past decade because of widespread human immunodeficiency virus infection leading to immunodeficiency. This increased incidence of progressive multifocal leukoencephalopathy may also be due to better recognition of its clinical signs, and more rapid and reliable laboratory diagnosis of JC virus, the etiologic agent. There have also been advances in the molecular detection of the JC virus and the identification of variations in the viral genome sequence that may affect its multiplication cycle in different tissues. Clinical and basic research have resulted in a better understanding of the pathogenesis of progressive multifocal leukoencephalopathy and have provided sufficient information to plan new approaches for treatment. PMID- 7551118 TI - Pathogenesis of inflammatory demyelination: implications for therapy. PMID- 7551119 TI - The treatment of multiple sclerosis: current and future. AB - During the past year observations have been published that might lead to further improvement in the design of future clinical trials. At the same time, results of clinical trials have become available that suggest that a number of treatments could be of benefit in the care of patients in the various phases of multiple sclerosis. Future multiple sclerosis clinical trials should involve a blinded investigator restricted to assessing the clinical outcome variables, and because current evidence suggests that magnetic resonance imaging gives an objective and sensitive reflection of the biological evolution of the disease, such scanning should also be included. The use of a composite outcome variable in a trial of chronic progressive multiple sclerosis should also be considered in order to increase the percentage of patients reaching the clinical endpoint. In 1994 recommendations were published for the selection of relapsing-remitting patients for treatment with interferon beta-1b; furthermore, large and well performed clinical trials demonstrated that interferon beta-1a and copolymer-1 are also partially effective, though not curative, for these patients. Two smaller studies suggested that low-dose methotrexate and cladribine might have a beneficial effect on the course of the disease in patients with secondary chronic progressive multiple sclerosis, the former drug probably being less toxic. Unfortunately, therapeutic perspectives for patients with primary progressive multiple sclerosis are less promising at present. Several studies suggest that 4 aminopyridine and tizanidine have therapeutic potential for symptomatic treatment; the former by improving neurological deficits, the latter by relieving troublesome spasticity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551120 TI - Magnetic resonance imaging and spectroscopy in multiple sclerosis. AB - Stimulated by the impressive effect of interferon beta on magnetic resonance imaging-detected disease activity, the past year or so has seen a particular interest in the use of magnetic resonance imaging to monitor treatment. To be effective in this regard, there needs to be a high sensitivity and accuracy in detecting disease activity, and the imaging findings should be predictive of clinical outcome, especially disability. A number of approaches show promise as being more sensitive than the conventional T2-weighted sequence. Furthermore, conventional magnetic resonance imaging cannot identify demyelination and axonal loss, the pathological substrates of irreversible disability. Newer magnetic resonance imaging techniques may be more pathologically specific, for example magnetization transfer imaging and magnetic resonance imaging spectroscopy. Such techniques provide new insights into the pathophysiology of multiple sclerosis. Improved methods for examining the often disabling spinal cord lesions have also emerged. PMID- 7551121 TI - Neuropsychology of multiple sclerosis. AB - Neuropsychological investigations have demonstrated that cognitive disorders are common (45-65%) in patients with multiple sclerosis. This review summarizes our current understanding of the nature and etiology of these disorders. Specifically, neuropsychological deficits regularly occur on measures of recent memory, attention, information-processing speed, executive functions, and visuospatial perception. In contrast, general intelligence, language, and certain aspects of memory (short-term capacity and implicit) are preserved. The degree and pattern of cognitive dysfunction is highly correlated with the amount and location of white-matter disease within the cerebral hemispheres. Natural history studies indicate that changes in cognitive test performance are correlated with increasing lesion load on magnetic resonance imaging. The development of brief cognitive-testing instruments for clinical screening and measuring treatment effects in clinical trials is underway. A clear weakness in the literature is the dearth of studies evaluating potential treatments for cognitive disorders. PMID- 7551122 TI - Adrenoleukodystrophy. AB - The main advances concerning adrenoleukodystrophy have been in the fields of genetics and therapy. Abnormalities in the 'putative gene' reported in 1993 have been confirmed. Mutations in this gene have been demonstrated in all of the 80 adrenoleukodystrophy families studied so far in various parts of the world. The same unusual dinucleotide deletion was present in approximately 20% of families. The remaining deletions involved nearly all parts of the gene and in most instances were unique in each family. There was no correlation between the phenotype and the nature or location of the mutation. Follow-up of patients treated with Lorenzo's Oil suggests that this therapy does not alter the course of the illness in symptomatic patients. However, dietary therapy started before the development of symptoms may reduce the frequency and severity of subsequent neurological disability. PMID- 7551123 TI - Acute disseminated encephalomyelitis and isolated central nervous system demyelinative syndromes. AB - The clinical and pathological characteristics of acute disseminated encephalomyelitis are briefly outlined. The possible relation between acute disseminated encephalomyelitis and other isolated monophasic inflammatory demyelinating episodes in the central nervous system, such as acute optic neuritis, transverse myelitis or brainstem lesions, is noted and the risk of progression to multiple sclerosis is examined. The surprising results of a clinical trial of corticosteroids in optic neuritis are of particular interest in this regard, and we speculate on an explanation in terms of a spread of antimyelin T cell receptor gene usage over time. Finally, the risk factors for the progression of demyelinating ther than optic neuritis to multiple sclerosis are explored. Further work to elucidate better the relationships between these pathological entities is suggested. PMID- 7551124 TI - Some unsolved problems in the epidemiology of headache. PMID- 7551125 TI - Headache as a symptom of organic diseases. AB - A review of the literature dealing with patients presenting with headache caused by underlying disease reinforces the classic teaching that these headaches are hardly ever completely typical of benign syndromes such as migraine. More often than not they are accompanied by features, such as other symptoms or physical abnormalities, that suggest, to the careful clinician, the suspicion of a lesion and the need for investigation. PMID- 7551129 TI - I treated; now I want to prevent. PMID- 7551127 TI - Treatment of cluster headache and its variants. AB - In cluster headache, subcutaneous administration of sumatriptan has been established as an efficient, safe and well tolerated treatment for acute attacks. For prophylactic treatment, capsaicin (intranasal administration) and leuprolide (a synthetic slow-release gonadotrophin-releasing hormone) seem to be promising. Hyperbaric oxygen therapy seems to be effective in acute attacks, but it is of interest mostly because of its apparent interruption of the cluster period in some patients. In chronic paroxysmal hemicrania, which clinically resembles cluster headache, indomethacin is still necessary for diagnosis, and it is the treatment of choice in most patients. However, because of its potential side effects, alternative treatments should be sought. PMID- 7551128 TI - Association between psychopathology and headache syndromes. AB - During the past year, several important papers have confirmed the association between psychological factors and headache. Recent studies are beginning to identify the specificity of the general association of headache subtypes with subtypes of psychiatric disorders and psychological traits. Studies of this topic have increasingly moved from the clinical arena to the community at large. Finally, much of the research on this topic is now driven by the goal of understanding the possible mechanisms for the concurrence of psychopathology and headache. PMID- 7551126 TI - Acute treatment of migraine attacks. AB - Migraine is a chronic neurological disorder, characterized by attacks of severe, usually unilateral and throbbing headache accompanied by nausea, vomiting, and photophobia and photophobia. Sometimes transient neurological (aura) symptoms may precede or accompany the headaches. Acute drug therapy comprises nonspecific drugs, including simple analgesics and non-steroidal anti-inflammatory drugs, often in combination with antiemetics, and specific antimigraine drugs, such as ergotamine, dihydroergotamine and sumatriptan. Sumatriptan is a potent and selective serotonin1D receptor agonist, which can be administered orally and via the subcutaneous or intranasal route. The drug is well tolerated and is consistently highly effective in most patients. Significant limitations, however, include the occurrence of chest symptoms, suggestive of cardiac ischaemia; recurrence of the headache within 24 h after initial successful treatment; and in a minority of patients, abuse of sumatriptan with daily 'sumatriptan-dependent headaches'. Administration during the aura phase does not affect the aura itself, but is not recommended because the subsequent headache will not be prevented in that case. Preliminary data of new serotonin1D receptor agonists, such as 311C90 and MK-462 are promising in terms of increased efficacy after oral administration, but side-effect profile and incidence of headache recurrence are similar to those observed after the use of sumatriptan. Intranasal administration of dihydroergotamine may also be effective, but data are very limited. PMID- 7551131 TI - Food behaviors of southern rural community-living elderly. AB - OBJECTIVE: To examine how food behaviors and indicators of nutritional status of elderly living in rural communities might relate to age, race, income, medication use, activities of daily living and instrumental activities of daily living, living arrangements, and other lifestyle and health measures. DESIGN: In-home survey administered by trained interviewers. SETTING: Communities in rural North Carolina. PARTICIPANTS: A total of 2178 adults, aged 60 years and older, representing a 2% random stratified example of the region's elderly. MAIN OUTCOME MEASURES: Weight status described by body mass index, functional status as activities of daily living and instrumental activities of daily living, and self reported food and health behaviors. RESULTS: Substantial diversity exists among households by age, race, residential location, and household size. Almost three fourths of respondents had seen a physician in the last 6 months. More than 33% of women and 28% of men were obese, but none reported having been prescribed a weight-loss diet. About 18% of the respondents were underweight. Weight was not related to income, perceptions of health, chronic disease, medication use, feelings of depression, instrumental activities of daily living, or exercise. Most (65%) ate three meals per day, and almost all were able to eat without assistance. Few drank much liquid or participated in structured exercise programs. Research is needed to determine the impact of weight status on the quality of life of elderly persons living in rural areas. CONCLUSIONS: Current literature descriptions of food behaviors and nutritional status of the elderly may not appropriately describe rural elderly. Attention should be given to the identification and treatment of malnutrition in the elderly. PMID- 7551130 TI - Nedocromil in the outpatient management of asthma. AB - OBJECTIVE: To more closely approximate the use of a nonsteroidal inhaled anti inflammatory medication for asthma, nedocromil sodium, under actual ambulatory practice conditions. DESIGN: Large, open-label trial. PATIENTS: One thousand two hundred one patients from 286 primary care and specialty centers. INTERVENTION: Four weeks of treatment with nedocromil sodium (4 mg delivered from the valve and 3.5 mg delivered from the mouthpiece of a metered inhalor [2 puffs, four times daily]). MAIN OUTCOME MEASURES: Asthma symptom scores, peak expiratory flow rate, a lifestyle assessment measures questionnaire, and mean number of days missed per month from work or school. RESULTS: Statistically significant improvements were seen after 1 and 4 weeks of treatment for cough, daytime and nighttime asthma, morning tightness, peak expiratory flow rate, and all four measured lifestyle assessment factors (P < .001). An additional clinically relevant outcome measure, mean number of days missed per month from work or school, was reduced by 75% (P < .001). No serious adverse reactions were reported. CONCLUSION: This study reproduces the high level of efficacy and safety of nedocromil that was previously reported in placebo-controlled clinical studies. PMID- 7551132 TI - Colorectal cancer trends by race and anatomic subsites, 1975 to 1991. AB - OBJECTIVES: To determine whether colorectal cancer rates among black men and women show the abrupt declines seen in whites since the mid-1980s and to determine how the cancer trends vary by anatomic subsites. DATA SOURCES: Mortality data from the National Center for Health Statistics, Hyattsville, Md, and incidence and survival data from the Surveillance, Epidemiology, and End Results program of the National Cancer Institute, Bethesda, Md. MAIN OUTCOME MEASURES: Trends in incidence, survival, and mortality rates. RESULTS: For white men and women, cancer incidence rates declined for the right colon, left colon, and rectum after 1985. Stage-specific incidence rates for white men and women for each subsite had generally similar patterns. Distant-disease incidence rates declined beginning in the late 1970s, whereas regional-disease rates increased until the early to mid-1980s and then declined. An exception is the right colon in men, for which the incidence rates of distant disease did not decline, although the regional-disease pattern was similar to other sites. For blacks, colorectal cancer incidence rates changed little in the 1980s for men or women. In particular, there were no significant declines in the cancer incidence rates of the colorectum or of subsites after 1985. Black colorectal cancer mortality trends showed gender and age differences. Black men had significantly increasing colorectal cancer mortality rates from 1975 through 1992, but the increase after 1985 was observed only in men 65 years of age and older. The colorectal cancer mortality rates did not increase overall in black women in the 1980s, but the mortality rates increased slightly in women 65 years of age and older while declining in women younger than 65 years. CONCLUSIONS: For whites, the trends in colorectal cancer rates by anatomic subsite support the contention that early detection procedures, such as sigmoidoscopy and colonoscopy, are contributing to the declines in incidence and mortality rates since 1985. The absence in blacks of significant declines in colorectal incidence or mortality rates since 1985 suggests the need for a greater emphasis on early-detection programs in the black community, particularly for elderly blacks. PMID- 7551133 TI - Prevalence of mental disorders in primary care. Implications for screening. AB - OBJECTIVES: To determine the prevalence of five mental disorders in primary care and to identify patient groups that have a relatively high prevalence of these disorders. DESIGN: Two-stage case identification design that involves administration of a 16-item screening instrument followed by an independent diagnostic assessment. SETTING: Three family practice offices in Rhode Island. SUBJECTS: A total of 937 primary care patients completed the brief screen, 388 of whom completed the independent diagnostic assessment. PREVALENCE ESTIMATION: A Bayesian procedure was used to estimate prevalence of mental disorder from screening and assessment results. Independent assessments were based on the Structured Clinical Interview for DSM-III-R administered by a mental health professional. RESULTS: The prevalence estimates were alcohol abuse or dependence, 3.2%; generalized anxiety disorder, 2.8%; major depressive disorder, 14.1%; obsessive-compulsive disorder, 2.2%; panic disorder, 6.2%; and any of the five disorders, 22.0%. The prevalence of any of the five disorders was higher in patients returning for follow-up visits (27.9%) than in those either presenting with a new illness (21.7%) or seeking a routine physical examination (11.8%). The combined prevalence was also higher in patients with a chronic medical problem (25.8%) than in those without (16.7%). CONCLUSIONS: Patients returning for follow up care and, to a lesser extent, those with chronic medical problems appear to be at increased risk of having a mental disorder. The practice of selectively screening new patients for mental health problems is questioned. Screening efforts in primary care should include established patients and those with chronic medical illnesses as well as new patients. PMID- 7551134 TI - Familial patterns in patients with infrequent panic attacks. AB - OBJECTIVE: To evaluate the family environment in patients with infrequent panic attacks. DESIGN: Survey. SETTING: Waiting room of a family health center at a university-based family practice residency program. The center primarily serves low-income or underinsured patients, 80% of whom are Hispanic. PATIENTS OR OTHER PARTICIPANTS: Randomly selected patients completed the panic disorder section of the Structured Clinical Interview of the Diagnostic and Statistical Manual of Mental Disorders, Third Edition. Thirty patients with infrequent panic attacks were compared with 30 control patients without panic attacks matched for age, gender, and ethnicity. Both groups completed in-depth interviews. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The in-depth structured interview included family environment instruments--Family Adaptability and Cohesion Evaluation Scales and Duke Social Support and Stress Scale--as well as a genogram. Family violence and sexual abuse were assessed by means of the Conflict Tactic Scales and the Sexual Stress Questionnaire. RESULTS: Although patients with infrequent panic attacks were of lower birth order than patients without panic attacks (Wilcoxon chi 2 = 2.13, P < or = .02), no differences in childhood or current family functioning were found. However, patients with infrequent panic attacks reported higher levels of childhood (paired t = 3.97, P < or = .001) and current (paired t = 3.05, P < or = .005) family stress. Although the prevalence of family violence was similar between groups, the group with infrequent panic attacks reported more violent events in the past year (paired t = 2.60, P < or = .02) than did the group with no panic attacks. Similarly, 60% of patients with infrequent panic attacks reported childhood sexual abuse, as opposed to 13% of patients without panic attacks (McNemar's chi 2 = 10.5, P < or = .005). CONCLUSIONS: Although no association between infrequent panic attacks and family functioning or support were found, the group with infrequent panic attacks reported more frequent violent events currently and higher levels of family stress. The high rate of childhood sexual abuse may have important causative implications for infrequent panic attacks. PMID- 7551135 TI - Management of disorders of cholesterol, triglyceride, and lipoprotein metabolism. Report 6 of the Council on Scientific Affairs. AB - The American Medical Association House of Delegates adopted a set of diagnostic and therapeutic recommendations concerning the management of disorders of cholesterol, triglyceride, and lipoprotein metabolism at its annual meeting in 1994. In recognition of the advances made during the past decade in the prevention and management of coronary heart disease, these recommendations updated those made previously. In addition, recommendations concerning the management of clinical risk factors for coronary heart disease were expanded to include infants, children, and adolescents. PMID- 7551136 TI - Selective causes of fever in adult human immunodeficiency virus-infected patients relative to CD4+ cell counts. AB - Fever is a common finding in the patient who is infected with the human immunodeficiency virus. As immunocompetence wanes, febrile episodes become more common, although the virus itself is seldom the cause of the fever. A thorough evaluation, based on the history and physical findings and directed by the level of immunosuppression relative to the CD4+ cell count, provides the framework upon which an approach to this complex problem is based. Noninfectious causes of fever, for example, drug reactions or adverse effects or neoplasms, should be considered in the differential diagnosis. Finally, health care workers should discuss the diagnostic evaluation with the patient before starting the process, as some individuals may be reluctant to undergo such an investigation. PMID- 7551138 TI - Reproduction and rationality. PMID- 7551137 TI - Solitary kidney and sports participation. AB - A questionnaire was sent to the 1994 membership of the American Medical Society for Sports Medicine to describe attitudes regarding allowance of sports participation for athletes with a single kidney. Using a hypothetical scenario of a high school or collegiate preparticipation examination, the respondents were asked if they would preclude or allow participation in collision and contact sports for an athlete with a single kidney. Of 438 respondents, 237 (54.1%) indicated that they would allow full participation in sports after discussion of the possible risks. The percentage decreased to 41.6% if the athlete was their son or daughter. These results indicate that the decision to allow participation in this situation remains controversial, but they also provide some support for those physicians who allow participation. PMID- 7551139 TI - Ethical and policy issues in human embryo twinning. PMID- 7551140 TI - Clones, harms, and rights. PMID- 7551141 TI - Transparent women, visible genes, and new conceptions of disease. PMID- 7551143 TI - The new eugenics and medicalized reproduction. PMID- 7551142 TI - Informed consent in the human genome enterprise. PMID- 7551144 TI - The day-to-day realities: commentary on the new eugenics and medicalized reproduction. PMID- 7551147 TI - The road that I see: implications of new reproductive technologies. PMID- 7551146 TI - Abortion and the ethics of genetic sexual orientation research. PMID- 7551148 TI - Social and ethical implications of in vitro fertilization in contemporary China. PMID- 7551149 TI - Report from Hong Kong. PMID- 7551145 TI - Germ-line therapy to cure mitochondrial disease: protocol and ethics of in vitro ovum nuclear transplantation. PMID- 7551150 TI - Gene therapy in Japan: current trends. PMID- 7551151 TI - Designs of life: choice, control, and responsibility in genetic manipulation. PMID- 7551152 TI - Immortality through the fertility clinic. PMID- 7551154 TI - Computer systems in dysmorphology. AB - A number of computer systems have been dedicated to some aspect of dysmorphology. These systems have generally been developed in isolation and demonstrate much variation in their design. Some systems are purely database applications designed to take advantage of computer technology in order to provide up to date syndrome compendia. Conversely, a number of research projects have endeavoured to build an intelligent system that can formulate a diagnosis, either through its own knowledge-base, or through interaction with an expert user. This report reviews computer systems in dysmorphology with reference to these two alternative design methodologies. The London Dysmorphology Database and POSSUM provide case studies in a standard database approach. The Skeletal Dysplasia Diagnostician (SDD) is described in order to demonstrate how an expert system might operate in dysmorphology. Other work in the field is reviewed in terms of the common and distinctive aspects of their design with respect to the three aforenamed systems. PMID- 7551153 TI - Case: responding to a request for genetic testing that is still in the lab. PMID- 7551155 TI - Neural tube defects, chromosome abnormalities and multiple closure sites for the human neural tube. AB - The hypothesis that during human embryogenesis there is multi-site closure of the neural tube, with possibly regionally distinct genetic control, is examined in the light of the neural tube defects (NTD) observed in trisomy 13, trisomy 18 and triploidy which survive at least to the mid-trimester. In a series of mid trimester fetuses examined post-mortem, there were two spina bifidas in 25 cases of trisomy 13; eight spina bifidas and one anencephalic among 38 trisomy 18 individuals; and three spina bifidas in 13 triploids. Not only was there a predominance of spina bifida, but all the spina bifidas were similar in location along the neuraxis: regardless of chromosome constitution, they were all sacral or lumbosacral. A survey of the literature confirms this finding. While this regionally distinct type of NTD is not specific to a particular chromosome abnormality and thus by implication to any particular gene, it does seem to be specific to an imbalance caused by an excess of genetic material of a type which permits survival past the first few post-conception weeks, and thus lends some support to the hypothesis. PMID- 7551156 TI - Heterogeneity of SPONASTRIME dysplasia: delineation of a variant form with severe mental retardation. AB - We report a child with short stature, osteopenia with metaphyseal striations and severe mental retardation. This child shows radiological and clinical features of SPONASTRIME dysplasia. Only three sibships with this disorder have been reported. In two families, affected patients were of normal intelligence. In the third one, as well as our case, the dysplasia was complicated by severe mental retardation of unknown origin. The severity of the retardation in our case and a previous report, and some difference in the gestalt and radiological aspects, suggest that SPONASTRIME dysplasia is a heterogeneous disorder. We provisionally propose to split SPONASTRIME dysplasia in two phenotypically distinct subgroups, and to delineate here a 'new' variant with microcephaly and severe mental impairment. PMID- 7551158 TI - Opsismodysplasia: another case and literature review. PMID- 7551157 TI - Patterson-Lowry rhizomelic dysplasia: a possible second example. AB - We present a possible example of Patterson-Lowry rhizomelic dysplasia. If so, this is only the second reported individual with this disorder and the first instance of its recognition in a child. Clinical features in this child are compared with the other described individual and differential diagnosis is discussed. Primary features include rhizomelic foreshortening most markedly affecting the arms, limitation of shoulder abduction, mild generalized foreshortening of the long bones, mild short stature, coxa vara, and specific radiological features. PMID- 7551159 TI - Wolcott-Rallison syndrome. AB - Two sibs with early onset diabetes and epiphysed dysplasia (Wolcott-Rallison syndrome) are described. The epiphyseal changes were radiologically apparent at 6 months of age in one of them, and both developed insulin dependent diabetes in the first few weeks of life. The clinical and radiological features of this syndrome are reviewed. PMID- 7551160 TI - Microcephalic osteodysplastic primordial dwarfism type II. AB - We report a child with osteodysplastic primordial dwarfism type II. The literature is reviewed. PMID- 7551161 TI - The Wiedemann-Rautenstrauch neonatal progeroid syndrome: a case report and review of the literature. AB - We present the case of a post-term newborn with intrauterine growth retardation, pseudohydrocephalus, a tiny face and mouth, thin wrinkled skin, an aged appearance, lipoatrophy and a normal cranial CT scan, suggestive of the Wiedemann Rautenstrauch neonatal progeroid syndrome. He developed hypothyroidism on day 18 due to a partial organification disorder as did a later born sib. His mental development remains normal at age 2 with delayed growth at -2.5 SD. The case is presented and discussed and the literature is reviewed. PMID- 7551162 TI - Ectrodactyly, diaphragmatic hernia, congenital heart defect, and agenesis of the corpus callosum. AB - A unique case of a female born with four major malformations, ectrodactyly, diaphragmatic hernia, ventricular septal defect, and agenesis of the corpus callosum is reported. The patient had a normal birth weight, normal head circumference and a normal karyotype. There was no significant facial dysmorphism. The family history was unremarkable for birth defects, recurrent pregnancy loss, limb anomalies or consanguinity. We propose that this represents a new constellation of multiple malformations. PMID- 7551163 TI - A case of lateral facial cleft, cleft lip and palate, anophthalmia, microtia, clavicular agenesis and asternia. AB - We present a female infant with a complex pattern of congenital malformations including a lateral facial cleft, cleft lip and palate, anophthalmia, microtia, clavicular agenesis and asternia. A differential diagnosis is proposed and discussed. PMID- 7551164 TI - A patient with a previously undescribed overgrowth syndrome. AB - A 34-year-old man is described with a novel and distinctive overgrowth syndrome. His bone age was markedly advanced in early childhood and, unlike some other overgrowth syndromes, his final height is greatly increased. PMID- 7551165 TI - Marden-Walker syndrome in an adult. AB - We report on a 23-year-old male with growth retardation, blepharophimosis, congenital contractures, minor anomalies of the face, and severe mental retardation. This patient is the second adult reported with the Marden-Walker syndrome. Blepharophimosis, contractures, growth retardation, and developmental delay persisted whereas minor anomalies of the face were less noticeable as the patient grew older. Behaviour changed from kindness in childhood to restlessness, hyperactivity and aggressiveness in adolescence. PMID- 7551166 TI - Sonographic diagnosis of epignathus (oral teratoma), prosencephaly, meromelia and oligohydramnios in a fetus with trisomy 13. AB - Epignathus (oral teratoma), an extremely rare congenital tumour is generally known as an isolated anomaly with benign histology. We present a case of epignathus detected prenatally in an oligohydramniotic 24 week fetus in which anophthalmia, a single nostril, holoprosencephaly, meromelia, bilateral renal dysplasia and a club foot deformity were also present. Chromosome analysis revealed trisomy 13. PMID- 7551167 TI - Ophthalmo acromelic syndrome. PMID- 7551168 TI - Characteristic facies in type B brachydactyly? PMID- 7551169 TI - Target preference of embryonic retina cells and retinal cell lines is cell autonomous, position-specific, early determined and heritable. AB - Retinal ganglion cells (RGCs) form the topographic connection between retina and optic tectum in the developing avian embryo. In vitro, neurons with the morphological traits and marker expression of RGCs were found both in single-cell cultures from embryonic day (E) 6 chick retina and in retinal cell lines derived from E3.5 quail retina. Rapid and substantial differentiation of RGC-like cells could be induced in the lines by addition of fibroblast growth factor aFGF or bFGF. RGC-like cells were examined with respect to their target discrimination properties as single cells in the stripe carpet assay. In this assay system, alternating stripes of membrane vesicles prepared from the anterior and posterior tectum are offered to growing axonal processes as a substrate. Temporal RGC-like cells, both primary cells prepared from the temporal retina and immortalized cells of those retinal lines derived from the temporal retina, avoid stripes of membrane vesicles from posterior tectum; they prefer to grow on membrane vesicles from the anterior tectum, which is their in vivo target. Nasal RGC-like cells did not exhibit a target preference, in accordance with previous findings. Together the experiments show that target preference of RGCs is a cell-autonomous and heritable mechanism that is determined early and is position-dependent. PMID- 7551170 TI - NMDA-dependent GABAA-mediated polysynaptic potentials in the neonatal rat hippocampal CA3 region. AB - Evoked inhibitory postsynaptic potentials (IPSPs) were studied in CA3 hippocampal neurons from brain slice preparations of rats ranging from 5 to 18 days of age (P5-18) using intracellular recording techniques. With KMeSO4-filled electrodes the evoked inhibitory response consisted of fast and slow IPSPs mediated by GABAA and GABAB receptors respectively. In recordings obtained with electrodes filled with 2-(triethylamino)-N-(2,6-dimethylphenyl) acetamide and KMeSO4, electrical stimulation evoked monophasic IPSPs in mature slices (P10-18) and biphasic IPSPs with an early and a late phase in neonatal slices (P4-7). In neonates both the early and late phases of the IPSP were mediated by GABAA receptors. Pharmacological investigation revealed that the early phase arose from both direct and feedforward activation of GABAergic interneurons involving non-NMDA receptors, while the late phase resulted from polysynaptic activation of GABAergic interneurons mediated by NMDA receptors. PMID- 7551172 TI - Fasciculation of granule cell neurites is responsible for the perpendicular orientation of small inhibitory interneurons in mouse cerebellar microexplant cultures in vitro. AB - To study the cellular and molecular mechanisms involved in the perpendicular orientation of stellate and/or basket cells to the direction of fasciculating granule cell neurites, we have used cultures of microexplants from early postnatal mouse cerebellum that show this cellular behaviour in vitro. When these cultures were maintained in the presence of antibodies to the neural cell adhesion molecules L1 and N-CAM or immunoglobulin-like domains I, II and IV of N CAM, the resulting decrease or increase in the fasciculation of granule cell neurites changed the perpendicular orientation and morphology of the small inhibitory interneurons. Additives which did not perturb fasciculation did not affect the perpendicular orientation and morphology of stellate and/or basket cells. Furthermore, when perturbation of fasciculation was prevented, neither L1 nor N-CAM antibodies modified the positioning or morphology of interneurons. These observations indicate that ordered fasciculation of granule cell neurites is an important parameter in the perpendicular orientation and elaboration of the typical morphology of the small cerebellar inhibitory interneurons. PMID- 7551171 TI - Behaviour of small inhibitory interneurons in early postnatal mouse cerebellar microexplant cultures: a video time-lapse analysis. AB - The aim of this work was to investigate how the environment of the neuropil determines the positioning and differentiation of neurons that are postsynaptic to them. We investigated how stellate and basket cells, the small inhibitory interneurons of the cerebellar cortex, find their perpendicular orientation to the direction of fasciculated granule cell axons. Cultures of early postnatal mouse cerebellar microexplants showing this cellular behaviour in vitro were analysed by video time-lapse cinematography and evaluated by morphometry. The small interneurons were first detectable when they migrated, intermingled with granule cells, away from the explant along the radial fascicles of granule cell neurites. During migration some cells suddenly changed their orientation by extending neurites in perpendicular orientation to the radial fascicles. These cells were all GABA-immunoreactive and expressed the cytoskeletal markers tau in the thin axon-like process and MAP2 in the thicker dendrite-like arborizations at the opposite pole of the cell body. After having translocated in perpendicular orientation, these neurons were again able to turn back to move along the radial neurite bundles to another position. Furthermore, while in perpendicular orientation, the processes of these cells repelled each other upon contact of their growth cones, leading to equal spacing between the cell bodies with time in culture. PMID- 7551173 TI - Expression of the mRNA of seven metabotropic glutamate receptors (mGluR1 to 7) in the rat retina. An in situ hybridization study on tissue sections and isolated cells. AB - We have studied the expression of mRNAs for seven metabotropic glutamate receptors (mGluR1-7) in the retina of the adult rat by in situ hybridization with tissue sections and isolated cells using [alpha 35S]dATP-labelled oligonucleotide probes. Hybridization revealed the expression of six of the metabotropic receptor mRNAs, mGluR1, 2 and 4-7, in the retina, while mGluR3 was not detected. Each of the expressed receptor mRNAs showed a distinct pattern of expression. In the outer nuclear layer, corresponding to photoreceptor somata, no labelling was detected. In the outer part of the inner nuclear layer, putative horizontal cells were labelled for mGluR5. More proximal in this layer, corresponding to the position of bipolar cell somata, there was strong labelling for mGluR6. A small number of bipolar cells were also labelled for mGluR5 and mGluR7. In situ hybridization with isolated cells showed that mGluR6 was expressed by rod bipolar cells. Subsets of amacrine cells, with cell bodies along the border between the inner nuclear layer and the inner plexiform layer, were positive for mGluR1, 2, 4 and 7, suggesting considerable heterogeneity of these receptors among amacrine cells. None of the seven metabotropic receptor mRNAs was expressed in isolated Muller glial cells. In the ganglion cell layer, virtually every ganglion cell and displaced amacrine cell was labelled for mGluR1 and mGluR4. Some cells in this layer (approximately 20% of the total), most likely both ganglion cells and displaced amacrine cells, were also labelled for mGluR2 and mGluR7. These findings suggest that metabotropic glutamate receptors are considerably more widespread among neurons in the retina than indicated by previous physiological and pharmacological investigations. PMID- 7551175 TI - Expression of GABA receptor rho 1 and rho 2 subunits in the retina and brain of the rat. AB - We have investigated the distribution of GABA receptor rho 1 and rho 2 subunits in the rat central nervous system. Cloning of rat rho 1 and rho 2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (rho 1) and 88% (rho 2) at the protein level. Whereas the human rho 2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse-transcribed total RNA (RT-PCR) from different brain tissues revealed that transcript for the rho 1 subunit was present in the retina only. The rho 2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that rho 1 and rho 2 transcripts are present in the inner nuclear layer. RT-PCR and in situ hybridization of isolated retinal cells showed that both rho subunits are present in rod bipolar cells. Since these cells express bicuculline-insensitive GABA receptors, our results further support the idea that rho subunits are part of the GABAc receptor. PMID- 7551177 TI - Orienting behaviour and superior colliculus sensory representations in mice with the vibrissae bent into the contralateral hemispace. AB - When a tactile stimulus touches the body on one side, animals show an orienting response toward that side with the eyes, the head or the entire body. This movement requires the transformation of sensory information into motor commands. The superior colliculus is supposed to be a fundamental part of the brain where this sensorimotor transformation occurs and where one of the possible mechanisms could be the alignment among sensory and motor topographies. We changed the body shape of mice in order to analyse the development of new orienting responses following tactile stimulation. To do this, we bent the left vibrissae from left to right such that they were located in the right portion of the visual hemifield. If left-right inversion was performed in adults, tactile stimulation of the left vibrissae performed from the right produced wrong orienting movements to the left. Conversely, if left-right inversion was performed in newborns, mice learned to respond correctly to the right. By recording from superior colliculus multisensory neurons of mice whose vibrissae were displaced at birth, we found a shift of visual and auditory receptive fields from left to right in those multisensory neurons receiving tactile input from the displaced vibrissae. These results show the strict relation existing between the neuronal modifications in the superior colliculus and the changes in orienting behaviour. These findings also suggest two important conclusions. First, sensory mapping in the superior colliculus depends on sensory inputs coming from the same portion of space.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551176 TI - Noradrenergic modulation of cholinergic nucleus basalis neurons demonstrated by in vitro pharmacological and immunohistochemical evidence in the guinea-pig brain. AB - The effects of noradrenalin were tested upon electrophysiologically characterized cholinergic nucleus basalis neurons in guinea-pig brain slices. According to their previously established intrinsic membrane properties, the cholinergic cells were distinguished by the presence of low-threshold Ca2+ spikes and transient outward rectification that endowed them with the capacity to fire in low threshold bursts in addition to a slow tonic discharge. A subset of the electrophysiologically identified cholinergic cells that responded to noradrenalin had been filled with biocytin (or biotinamide) and documented in previously published reports as choline acetyltransferase (ChAT)-immunoreactive. The noradrenalin-responsive, biocytin-filled/ChAT+cells were mapped in the present study and shown to be distributed within the substantia innominata amongst a large population of ChAT+ cells. Slices from another subset of noradrenalin-responsive, electrophysiologically identified cholinergic cells were stained for dopamine-beta-hydroxylase to visualize the innervation of the biocytin-filled neurons by noradrenergic fibres. These biocytin-filled neurons were surrounded by a moderate plexus of varicose noradrenergic fibres and were ostensibly contacted by a small to moderate number of noradrenergic boutons abutting their soma and dendrites. Applied in the bath, noradrenalin produced membrane depolarization and a prolonged tonic spike discharge. This excitatory action was associated with an increase in membrane input resistance, suggesting that it occurred through reduction of a K+ conductance. These effects persisted when synaptic transmission was eliminated (by tetrodotoxin or low Ca2+/high Mg2+) and were therefore clearly postsynaptic. The excitatory effect of noradrenalin was blocked by the alpha 1-adrenergic receptor antagonist prazosin and not by the alpha 2-antagonist yohimbine, and it was mimicked by the alpha 1-agonist L phenylephrine but not by the alpha 2-agonists clonidine and UK14.304, indicating mediation by an alpha 1-adrenergic receptor. There was also evidence for a contribution by a beta-adrenergic receptor to the effect, since the beta antagonist propranolol partially attenuated the effect of noradrenalin, and the beta-agonist isoproterenol produced, like noradrenalin, alone or when applied in the presence of the alpha 1-antagonist prazosin, membrane depolarization and an increase in tonic spike discharge. These results indicate that through a predominant action upon alpha 1-adrenergic receptors, but with the additional participation of beta-adrenergic receptors, noradrenalin depolarizes and excites cholinergic neurons. This action would tend to drive the cholinergic cells into a tonic mode of firing and to stimulate or increase the rate of repetitive spike discharge for prolonged periods.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7551174 TI - Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons. AB - Immunocytochemistry has been used to examine the location of trkA, the high affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA-immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 microns in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA-immunoreactive cells express calcitonin gene related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non-peptide-containing cells. Eighteen percent of trkA-immunoreactive cells belong to the 'large light' subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA-expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide-producing population. The receptor is present not only on cell bodies but also on central terminals. Non peptide-containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA-immunoreactive and therefore most probably are functionally independent of nerve growth factor. PMID- 7551178 TI - Involvement of substance P in ultraviolet irradiation-induced inflammation in rat skin. AB - The possible involvement of substance P released from primary afferents in rat skin was investigated in cutaneous inflammation following ultraviolet (UV) irradiation. Recordings from c-fibres innervating the UV-exposed hindpaw skin showed long-lasting low-frequency (0.8-1.25 Hz) spontaneous activity. Spontaneously active c-fibres increased to constitute 35.3% of the total population 72 h after UV exposure. Immunohistochemical analysis of substance P containing nerve fibres in hindpaw skin revealed a significant increase in substance P immunoreactivity 24 h after UV irradiation. Average length of substance P-immunolabelled nerve fibres was about two times higher in UV-exposed compared to control skin. UV-induced oedema was investigated in rat ears using an ear-swelling test. Intradermal injection of either peptide (Spantide) or nonpeptide (CP-96,345) substance P antagonists and epicutaneous application of CP 96,345 reduced UV-induced oedema significantly in the late phase of sunburn (> 12 h after UV exposure). The UV-induced increase in skin blood flow was investigated in hindpaw skin up to 72 h by the laser Doppler technique. Epicutaneous application of CP-96,345 reduced erythema significantly between 12 and 72 h after UV exposure. Thus, our findings suggest the involvement of neurogenic substance P as a proinflammatory mediator in the late phase of UV-induced cutaneous inflammation in rats. PMID- 7551179 TI - BDNF protein measured by a novel enzyme immunoassay in normal brain and after seizure: partial disagreement with mRNA levels. AB - Messenger RNA for brain-derived neurotrophic factor (BDNF) is distributed in many brain regions and regulated by excitatory neuronal activity. Despite numerous studies of BDNF mRNA, the distribution and regulation of BDNF protein are poorly understood because of the difficulty of its quantitative measurement. We have established a two-site enzyme immunoassay that detects trace amounts of BDNF protein (> 1 pg/assay) but not other neurotrophins or growth factors. The highest levels of BDNF in adult rat brain were found in the hippocampus, followed by the hypothalamus, neocortex, cerebellum, thalamus and striatum. This pattern is similar, but not identical, to the distribution of BDNF mRNA. A similar disparity between BDNF protein and mRNA levels was observed in their changes after hilus lesion-induced limbic seizures. In limbic structures, BDNF concentrations remained elevated 4 days after seizure onset, whereas BDNF mRNA has been reported previously to return to basal levels within 46 h. The temporal and spatial differences between the dynamics of protein and mRNA levels suggest the importance of post-translational and/or subcellular processes for BDNF production. The persistence of the increases in BDNF content was also reflected in its biological activity, e.g. peptidergic differentiation activity. After limbic seizures, neuropeptide Y content was most markedly and persistently elevated in the entorhinal/amygdaloid region, where the most sustained up regulation of BDNF protein was observed. These results suggest that the sustained increase of BDNF protein in these limbic structures is involved in prolonged post seizure phenomena, including peptidergic alterations. PMID- 7551180 TI - Human motor planning, motor programming, and use of new task-relevant information with different apraxic syndromes. AB - Patients with ideomotor apraxia (n = 2) or ideational apraxia (n = 2) after left brain stroke and patients with constructional apraxia (n = 2) after cerebrovascular accident of the right hemisphere, as well as 16 non-brain-damaged control subjects, were given a standardized simple motor task: they were asked to make triangular arm movements of specific size, configuration and spatial orientation without visual control. Motion was analysed three-dimensionally in great detail prior to and after kinaesthetic training using a triangular stencil, and 1 day later. The experiment was conceptualized to assess three aspects of motor behaviour: (i) motor planning, operationalized as specification of content parameters of the movement as a whole; (ii) motor programming, the specification of spatiotemporal parameters of movement segments; and (iii) the ability to make use of task-relevant information provided by the training. Patients with ideational apraxia showed signs of impaired motor planning: they had difficulty in selecting the body parts to be moved, and movement concept and configurational aspects were deficient. The kinaesthetic sensorimotor training given seemed not adequate to reduce behavioural deficits. Kinematic peculiarities of patients with ideomotor apraxia can be understood as deficits in programming movement elements. Submovements were more segmented, showed irregularities as well as additional, not-requested elements. Their impairments could be reduced by task-specific sensorimotor training. Patients suffering from visuoconstructive apraxia after right brain damage might have difficulties in making use of new sensorimotor information relevant for spatial-motor aspects, as suggested by training-induced behavioural impairment with a severely constructional apraxic patient. PMID- 7551181 TI - The entorhinal cortex entrains fast CA1 hippocampal oscillations in the anaesthetized guinea-pig: role of the monosynaptic component of the perforant path. AB - Entorhinal inputs reach the hippocampal CA1 field through a trisynaptic circuit involving dentate granule cells and CA3 pyramidal neurons, as well as through a monosynaptic path ending on the distal apical dendrites of CA1 pyramidal cells. The influence of monosynaptic entorhinal inputs onto CA1 operations is poorly understood. In this study, we characterized the involvement of the monosynaptic pathway in the generation of the fast CA1 oscillation bursts (30-60 Hz) that occur in the dorsal hippocampus of anaesthetized guinea-pigs after partial cortex removal. Using multiple-site extracellular and intracellular recording, we found that in this particular preparation, devoid of theta rhythm, fast oscillations are temporally coherent over a large portion of the CA1 region along the hippocampal septotemporal axis. Current source density analysis revealed that fast CA1 oscillations involve two dipoles reflecting synchronous synaptic activities in the stratum lacunosum-moleculare of the hippocampus proper and in the stratum moleculare of the dentate gyrus. These layers constitute the two major termination zones of entorhinal afferents, suggesting that the entorhinal cortex entrains fast CA1 oscillations. This hypothesis was corroborated by the concomitant occurrence of fast oscillation bursts in the entorhinal cortex and CA1 region. Furthermore, fast CA1 oscillations were abolished by lidocaine or tetrodotoxin injections in the entorhinal cortex. Finally, acute interruption of the hippocampal trisynaptic loop did not affect the stratum lacunosum-moleculare dipole recorded extracellularly, but also intracellularly, as high-frequency postsynaptic potentials in CA1 pyramidal cells. These results indicate that the monosynaptic pathway is involved in the genesis of fast CA1 oscillations. PMID- 7551182 TI - Properties of K+ conductances in cat retinal ganglion cells during the period of activity-mediated refinements in retinofugal pathways. AB - During ontogeny retinal ganglion cells manifest pronounced changes in excitable membrane properties. To further our understanding of the ionic conductances underlying such functional changes, the whole-cell voltage-clamp variation of the patch-clamp technique was used to record potassium currents in 220 ganglion cells dissociated from cat retinas ranging in age from embryonic day 31 to postnatal day 10. Potassium currents were isolated by blocking voltage-gated Na+ and Ca2+ currents with tetrodoxin (TTX) and CoCl2 respectively and were characterized by their pharmacology, kinetics and voltage-dependence of activation and inactivation. In all cases, a combination of three currents accounted for the total outward calcium-independent K+ current: (i) a steady linear conductance; (ii) a voltage-gated transient current, IA, and (iii) a voltage-gated sustained current, IK. Both voltage-gated currents were affected by the application of 4 aminopyridine and tetraethylammonia (TEA): IA showed a greater sensitivity to 4 aminopyridine, while IK was more sensitive to TEA. Both voltage-gated currents were present throughout the developmental period examined; however, the percentage of retinal ganglion cells (RGCs) expressing IA showed a marked decline from 82% at E31 to 45% at postnatal ages. During this developmental period there was an increase in the density of the two voltage-gated and the linear conductance. Additionally, with maturation, significantly slower inactivation kinetics were observed for IK. These findings, and our previous results dealing with maturational changes in the TTX-sensitive voltage-gated Na current, are related to the generation of excitability in developing retinal ganglion cells. Furthermore, the presence of cells with and without transient K+ conductance throughout development suggests that the different spiking patterns observed in RGC classes may be partially due to differences in their membrane properties. PMID- 7551184 TI - Corticosteroid regulation of gene expression and binding characteristics of vasopressin receptors in the rat brain. AB - Arginine-vasopressin (AVP) plays significant roles in neuroendocrine and autonomic regulation, and in processing of cognitive information. Its synthesis and secretion are subject to control by circulating glucocorticoids. The lateral septum and subdivisions of the hippocampus are innervated by AVP-ergic fibres and, together with AVP-producing neurons in the hypothalamic paraventricular nucleus, are major neural targets of glucocorticoid negative feedback. In this study, we investigated the effects of chronic adrenalectomy (ADX) and subsequent treatment with supraphysiological doses of corticosterone (B) on the gene expression of AVP receptors of the V1a subtype in the septum, hippocampus and hypothalamic arcuate (ARC) nucleus using semiquantitative in situ hybridization histochemistry. Adrenalectomy did not alter AVP receptor expression in any of the structures studied. Supplementation with B significantly decreased AVP receptor expression in the lateral septum and hippocampus, whereas receptor mRNA levels in the ARC were indistinguishable from those measured in controls. In a complementary study, we investigated the binding characteristics of V1 AVP receptors in membrane preparations from the hippocampus. Adrenalectomy significantly decreased the number of AVP binding sites, and chronic corticosteroid treatment was associated with a further suppression of AVP receptor concentrations in this structure. These results indicate that the gene transcription of V1a AVP receptors in the brain is regulated by circulating glucocorticoids in a site-specific fashion that largely reflects the corticosteroid sensitivity of the corresponding structure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551183 TI - Calcium deposit formation and glial reaction in rat brain after ibotenic acid induced basal forebrain lesion. AB - The mechanisms underlying amino acid neurotoxicity may involve a rise in the intracellular concentration of calcium. Some neurons appear to die as a consequence of increased intracellular calcium levels induced by excitatory amino acids. One month after injection of ibotenic acid in the rat basal forebrain, the induced formation of calcium deposits and concomitant glial reaction were studied. Alizarine Red-positive calcium deposits were observed after ibotenic acid injection in the ventral part of the globus pallidus, but not in the medial septum. These deposits were present in the globus pallidus, ventral pallidum, substantia innominata, zona incerta, lateral hypothalamic area, entopeduncular nucleus, medial amygdaloid nucleus and several thalamic nuclei. Three types of round shaped deposit were identified morphologically. Differential astroglial and microglial reactions, studied autoradiographically with the monoamine oxidase-B marker [3H]Ro19-6327 and the peripheral benzodiazepine receptor marker [3H]Ro5 4864 respectively, were observed after both lesions. Our data suggest that excitotoxic lesions in the globus pallidus and medial septum lead with time to different neurodegenerative consequences and glial reactions. This differential sensitivity is discussed on the basis of the presence of different glutamate receptor subtypes and calcium-binding proteins. PMID- 7551185 TI - The unique characteristics of inflammatory responses in mouse brain are acquired during postnatal development. AB - The kinetics of leukocyte recruitment during acute inflammation in adult mouse brain differ from the stereotyped response occurring in non-CNS tissues; neutrophil recruitment is minimal and monocyte recruitment occurs after a 48 h delay. One aspect of the CNS microenvironment which may contribute to restricted leukocyte recruitment is the highly differentiated nature of resident CNS macrophages, the microglia. Thus we studied the inflammatory response to intracerebral injections of endotoxin in neonates in which microglia are less differentiated and resemble more closely macrophages of non-CNS tissues. Mice injected with endotoxin on the day of birth exhibited both neutrophil and monocyte recruitment to the parenchyma, but the response differed from that occurring in non-CNS tissues such as skin. Leukocyte recruitment was very slow, the mononuclear phagocyte response peaking 14 days after endotoxin injection. This sluggish inflammatory response was reminiscent of that previously described in fetal wounds. However, when endotoxin was injected into brains of 7-day-old neonates the inflammatory response resembled that seen in non-CNS tissues; i.e. prolific neutrophil recruitment and a brisk mononuclear phagocyte response. Thus the unusual inflammatory cell kinetics are a property of the mature CNS microenvironment; all signals necessary to support typical leukocyte recruitment are present in the brain by 7 days of age but the brain becomes able to restrict leukocyte immigration during subsequent postnatal development. Developmental changes in the host response to identical inflammatory challenges suggest a window during which the brain may be particularly vulnerable to inflammatory bystander damage. PMID- 7551186 TI - The rate of Wallerian degeneration in cultured neurons from wild type and C57BL/WldS mice depends on time in culture and may be extended in the presence of elevated K+ levels. AB - Wallerian degeneration of severed axons is delayed in C57BL/WldS mice. We have examined this further in cultured sympathetic, sensory and CNS neurons using superior cervical ganglion (SCG), dorsal root ganglion (DRG) and cerebellar granule neurons respectively from neonatal mice. We found that the time taken for the neurites to degenerate depends upon the length of time in culture before cutting, reaching a maximum by approximately 7 days when C57BL/WldS neurites survive for > 6 days after axotomy. The onset of degeneration could also be extended in SCG and DRG neurites from wild type C57BL/6J mice. After 7 days in culture these neurites normally degenerate within approximately 12-16 h of axotomy, but in the presence of raised K+ (50 mM) degeneration often did not begin until a further 2 days had lapsed. Under similar conditions degeneration of neurites from C57BL/WldS mice was also found to be further delayed, extending survival from approximately 5-6 days to > 7 days. The L-type Ca2+ channel blockers nifedipine (5 microM) and verapamil (10 microM) both blocked the effect of raised [K+], although not completely. Thapsigargin, which raises cytoplasmic [Ca2+], and the cAMP analogue 8-(4-chlorophenyl-thio)cAMP were also able to delay degeneration, but only when added 24 h prior to axotomy. These results show that it is possible to influence the course of Wallerian degeneration and that increases in levels of cytoplasmic Ca2+ can protect neurites from its onset. PMID- 7551187 TI - Inhibition of glutamate-induced neurotoxicity by a tau antisense oligonucleotide in primary culture of rat cerebellar granule cells. AB - Short-term exposure of primary cultures of cerebellar granule cells from neonatal rat brain to high concentrations of glutamate resulted in a significant increase of both immunoreactivity to and mRNA levels of tau protein. Time-course experiments revealed the increases of tau immunoreactivity and mRNA levels to be maximal 2 h after the glutamate pulse. To investigate the relationship between newly synthesized tau protein and glutamate-induced neurotoxicity, neurons were preincubated with a specific tau antisense oligonucleotide. This treatment resulted in (i) inhibition of the glutamate-induced increase of tau immunoreactivity and (ii) a decrease in the sensitivity of the neurons to neurotoxic concentrations of glutamate. These data indicate that induction of the cytoskeleton-associated tau protein participates in the cascade of events promoted by glutamate leading to neurodegeneration. PMID- 7551188 TI - Comparison of frequency-specific c-Fos expression and fluoro-2-deoxyglucose uptake in auditory cortex of gerbils (Meriones unguiculatus). AB - Induction of c-Fos in the auditory cortex of gerbils was investigated immunocytochemically 1 h after single, triple or 1 h continuous stimulation with a series of narrow band frequency-modulated tone bursts. With single stimulation c-Fos immunoreactive neurons were chiefly found in the primary auditory field (AI), where they formed a narrow frequency-specific column across layers II-VI. Side-band-like patterns adjacent to this column appeared characteristically with triple stimulation. Immunoreactive cell density in the anterior auditory field and the caudal fields was sparse and location not frequency specific with single or triple stimulation. Spatial comparisons of c-Fos immunoreactive neuron density with 2-deoxy-2-fluoro-D-glucose (FDG) autoradiography in the same animals after 1 h of stimulation revealed spreading of c-Fos expression in neurons across the tonotopic maps of the AI and in the rostral and caudal fields of the auditory cortex. The pattern of the highest density of c-Fos labelled cells in the AI still matched the peak labelling of FDG autoradiographs. The results show that the postsynaptic marker c-Fos reflects the frequency representation in the AI with single or triple stimulation yet with a higher spatial resolution than the deoxyglucose technique. Longer stimulation causes nontonotopic intracortical spreading of the c-Fos-inducing message, a phenomenon potentially reflecting the effects of cooperativity in the maps. PMID- 7551189 TI - NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain. AB - Injection of N-methyl-D-aspartate (NMDA) or kainate in the striatum of 7-day-old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome-sized fragments, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris-HCl or in animals treated with MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10 imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non-apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post-NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7551190 TI - Persistence of neuromuscular junctions after axotomy in mice with slow Wallerian degeneration (C57BL/WldS). AB - The present study was undertaken to examine the fate of neuromuscular junctions in C57BL/WldS mice (formerly known as OLA mice) after nerve injury. When a peripheral nerve is injured, the distal axons normally degenerate within 1-3 days. For motor axons, an early event is deterioration of motor nerve terminals at neuromuscular junctions. Previously, the vulnerability of motor terminals has been attributed either to a 'signal' originating at the site of nerve injury and transported rapidly to the terminals or to their continual requirement for essential maintenance factors synthesized in the motor neuron cell body and supplied to the terminals by fast axonal transport. Mice of the WldS strain have normal axoplasmic transport but show an abnormally slow rate of axon and myelin degeneration. Structure and function are retained in the axons of distal nerve stumps for several days or even weeks after nerve injury in these mice. The results of the present study show that WldS neuromuscular junctions are also preserved and continue to release neurotransmitter and recycle synaptic vesicle membrane for at least 3 days and in some cases up to 2 weeks after nerve injury. Varying the site of the nerve lesion delayed degeneration by approximately 1-2 days per centimetre of distal nerve remaining. These findings suggest that the mechanisms of nerve terminal degeneration after injury are more complex than can be accounted for simply by the failure of motor neuron cell bodies to supply their terminals with essential maintenance factors. Rather, the data support the view that nerve section normally activates cellular components or processes already present, but latent, in motor nerve endings, and that in WldS mice either the trigger or the cellular response is abnormal. PMID- 7551191 TI - Simultaneous expression of long-term depression of NMDA and long-term potentiation of AMPA receptor-mediated synaptic responses in the CA1 area of the kainic acid-lesioned hippocampus. AB - This study investigates the plasticity of the excitatory synapses in an experimental model of epilepsy, the kainic acid-lesioned rat hippocampus. Stimulation of afferents in the CA1 area of lesioned hippocampi produced an epileptiform burst of action potentials, with an underlying synaptic potential composed of mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 80%) and N-methyl-D-aspartate (NMDA; 20%) receptor-mediated components. Tetanic stimulation yielded a long-term potentiation (LTP) of the mixed AMPA/NMDA receptor-mediated population excitatory postsynaptic potentials. However, the same type of tetanus resulted in a long-term depression (LTD) of pharmacologically isolated NMDA receptor-mediated responses. This LTD occurred independently of the antagonism of AMPA receptors. This suggests that tetanic stimulation produced LTP of AMPA and LTD of NMDA receptor-mediated responses simultaneously. Finally, both LTP and LTD were shown to be NMDA dependent. This property has profound functional implications for the control of excitatory networks in temporal lobe epilepsy. PMID- 7551192 TI - Microsatellite DNA and isozyme variability in a west African population of Anopheles gambiae. AB - Microsatellites are defined as tracts of tandemly repeated short DNA sequences. Polymorphisms in this class of DNA are currently being used to generate a genetic map of the mosquito Anopheles gambiae. In the present study we explore the potential of microsatellites as a tool for studying the genetic structure of natural populations of this malaria vector. Genetic polymorphism at twenty enzyme coding gene loci and eleven microsatellite DNA loci was surveyed in a population of An. gambiae from Mali, West Africa. All of the microsatellite loci surveyed were polymorphic, as compared to 40% of the isozyme loci. The mean heterozygosity for the isozyme loci was only 0.097 (+/- 0.0035), but for the microsatellite loci it was 0.732 (+/- 0.060). The pattern of variability was very different between isozymes and microsatellites. Typically, at an isozyme locus a single allele occurred at a frequency > or = 0.75, whereas at microsatellite loci the most common allele had a frequency < 0.50. We conclude that microsatellites provide a rich source of genetic polymorphisms for the study of the population genetics of An. gambiae and are in many ways superior to isozymes for this purpose. We discuss the potential for utilizing genetically mapped microsatellite loci to explore the effect of chromosomal inversions on the distribution of genetic polymorphisms in An. gambiae. PMID- 7551194 TI - Evaluation of a viral thymidine kinase gene for suicide selection in transfected mosquito cells. AB - An Aedes albopictus cell line previously shown to be deficient in thymidine kinase activity was transfected with a thymidine kinase gene (tk) from Herpes simplex virus. Survival of the transfected lines in a 'TK+ selective medium' indicated that the viral gene was actively expressed at a level sufficient to rescue the TK-deficient phenotype of the parent line. Unlike the parental cells, TK+ transformants (TK6:hsv cells) were sensitive to 5-bromodeoxyuridine, and contained DNA corresponding to the constructs introduced by transfection. This TK selection system will facilitate recovery of other cotransfected, non-selectable mosquito genes in cultured cells. Transformed cells were treated with several antiviral drugs to define conditions for a 'suicide selection' system, in which cells expressing the viral thymidine kinase enzyme (TK) under the control of an inducible promoter would be selectively destroyed, whereas cells expressing the endogenous mosquito enzyme would remain relatively unaffected. The anti-herpetic drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) showed greater cytotoxicity against transformed cells expressing the viral enzyme, and less toxicity to wild type mosquito cells. This cell culture system provides a model for initial evaluation of suicide selection systems that may ultimately be adapted to the mosquito using sex- or tissue-specific promoters to drive expression of heterologous genes. PMID- 7551193 TI - Organization and expression of the hemolin gene, a member of the immunoglobulin superfamily in an insect, Manduca sexta. AB - Hemolin is a protein from the immunoglobulin (Ig) superfamily found so far in the haemolymph of two lepidopteran insect species, Hyalophora cecropia and Manduca sexta. Injection of bacterial into these insects induces the expression of hemolin. We have isolated the hemolin gene from M. sexta and determined its DNA sequence and transcription start site. The hemolin gene is 3127 bp long and contains six exons. The only correspondence between exons and the four Ig domains of hemolin is in domain 4, which is encoded by exon 6. Southern blot analysis indicates that there is one copy of the hemolin gene in the M. sexta genome. Analysis of the 5'-flanking sequence of the hemolin gene resulted in identification of potential regulatory sequences. Hemolin mRNA accumulated in haemocytes, as well as fat body, in response to injection of larvae with bacteria. Hemolin was detected by immunocytochemistry in only one of the five morphological haemocyte types in M. sexta, the granular cells. PMID- 7551195 TI - Primary sequence, copy number, and distribution of mariner transposons in the honey bee. AB - A single honey bee mariner transposon (TnM1a) was sequenced, revealing a transpositionally non-autonomous element of 937 bp delimited by 30 bp perfect inverted terminal repeats. The element is flanked by the TA duplication typical of mariner elements in general. There are approximately 435 copies of TnM1a homologous elements per haploid genome. These elements appear, by Southern blot analysis, to be dispersed throughout the genome. Thirteen individual genomic clones with an average size of 15 kb, were found to contain only a single element each, which also suggests that the elements are not tightly clustered. Finally, mariner elements are neither inactivated by methylation nor sequestered into a methylated fraction of the genome. PMID- 7551196 TI - The Simulium damnosum species complex: phylogenetic analysis and molecular identification based upon mitochondrially encoded gene sequences. AB - The DNA sequence of portions of the 16s rRNA and the NADH dehydrogenase subunit 4 (ND4) genes were used to determine phylogenetic relationships in the Simulium damnosum s.l. species complex. Results suggested that at least two major clades existed in the S. damnosum species complex, and that members of the S. damnosum s.l. species complex were not closely related to North American Simulium species. The sequence variability of the ND4 gene was exploited to develop a method to distinguish the sibling species of the S. damnosum s.l. species complex, based on directed heteroduplex analysis of PCR products derived from the ND4 gene. This method was capable of classifying the six sibling species into at least five groups. PMID- 7551198 TI - Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector. AB - The scorpion, Androctonus australis Hector, produces an insect-specific toxin (AaHIT) encoded by the Scotox gene. To assess the toxicity of AaHIT for mosquitoes, we have taken a novel approach to express the Scotox gene in vivo. We have engineered a double subgenomic Sindbis (dsSIN) virus that contains the Scotox gene in the viral genome and intrathoracically inoculated the virus (TE/3'2J/Scotox) into mosquitoes (Aedes aegypti, Ae. triseriatus and Culex pipiens), houseflies (Musca domestica) and ticks (Dermacentor andersoni). Mosquitoes, which normally show no pathologic effects from Sindbis (SIN) virus infections, died 1-5 days after infection with TE/3'2J/Scotox virus. Neither flies nor ticks were killed. The mosquitocidal action of AaHIT in mosquitoes makes AaHIT a potential candidate for inclusion in molecular-based methods of mosquito control. The expression of an arthropod gene in vivo demonstrates the utility of dsSIN expression vectors for future use to examine and potentially disrupt endogenous gene functions in mosquitoes. PMID- 7551197 TI - Identification of a mariner element from the tsetse fly, Glossina palpalis palpalis. AB - In the present study, the polymerase chain reaction was used initially to demonstrate the presence of mariner sequences in seven species/subspecies of tsetse flies. DNA hybridization experiments show mariner sequences to be dispersed within the tsetse genome and that there are large variations in copy numbers among the various taxa. A genomic library was used to isolate and characterize a full-length mariner element from G. p. palpalis. The results indicate that this element is 1257 bp in length, flanked by two 32 bp inverted repeats differing at only one position, and belongs to the mellifera subfamily. The nucleotide sequence that is translated into a reading frame of 337 amino acids requires the introduction of two frame shifts and one stop codon to maximize sequence homology with a mariner element from Drosophila erecta. Based on this evidence, we conclude that the G. p. palpalis mariner element clearly represents a non-functional transposable element and that the protein product is not an active transposase. PMID- 7551199 TI - Platelets and hypersensitivity. AB - Platelets are anucleated blood cells not exclusively committed to hemostasis. They participate in inflammation, tissue repair, the immune response and hypersensitivity. In this review, the role of platelets in hypersensitivity is summarized. In order to understand the profound implications of these cells, the authors introduce the involvement of platelets in inflammation and immune reactions. Both the pathophysiology and the biology of the interaction of platelets with other cells are then summarized. These interactions justify the actual knowledge about the possibilities for the involvement of platelets in clinical states of hypersensitivity. Furthermore, the participation of platelets in allergy is supported by biological and clinical evidence, including in vitro studies of platelet activity in hypersensitivity states, the demonstration of platelet killing and the response to parasite contact, as well as animal studies in which the response to immune aggression is dependent on platelet reactivity. However, the need for more complete and developed clinical studies is clearly stressed, as several therapeutic approaches can already be proposed for a better control of these processes. PMID- 7551200 TI - Nonspecific changes in immunotherapy with house dust extract. AB - Despite the possible complications and inadequacies of the immunotherapy of allergic patients with house dust extracts (HDE), this is widely applied in Latin America and is commonly considered to be a cost-effective treatment for allergic diseases. Doubts about its efficacy and mechanism of action prompted us to evaluate the immunological changes that occurred in a group of 99 patients selected for their confirmed allergic sensitivity to house dust, and who showed significant clinical improvement after a minimum of 12 months of immunotherapy with HDE. The favorable clinical response of these patients was associated with some of the mechanisms reported for immunotherapy with pollen or venom allergens, such as increased levels of IgG or IgG4 "blocking" antibodies, and the increased affinity of these antibodies for house dust antigens, or diminished basophil sensitivity to HDE. However, the magnitude of these changes was relatively small, and their biological significance questionable. In contrast, particularly evident was a reduction in the cutaneous reactivity not only to the specific allergen, but also to the nonspecific mast cell degranulator codeine and to histamine. These results suggest that as yet unidentified "nonspecific" mechanisms might contribute to the effect of immunotherapy with extracts as heterogeneous and complex as HDE. PMID- 7551201 TI - Sublingual immunotherapy in Parietaria pollen-induced rhinitis: a double-blind study. AB - A double-blind, placebo-controlled study of immunotherapy was conducted in 31 patients with allergic rhinitis due to Parietaria pollen to evaluate the efficacy and safety of high doses of allergen via the sublingual route. The patients were assessed before and after a 10-month period of treatment by clinical (symptom medication scores and specific nasal reactivity) and immunological (total IgE, specific IgE, IgG and IgG4 antibodies) parameters. High doses of Parietaria extract corresponding to a cumulative dose of 105 BU for each patient were administered with negligible side effects. The actively treated patients had significantly lower medication scores than those on placebo (p < 0.05) when the maximum pollen count was recorded, and at the end of the trial they showed a significant decrease in nasal reactivity (p < 0.02) and a significant increase in serum specific IgG4 (p = 0.02). No differences were detected in any of these parameters in the placebo group. Possible explanations for the mechanisms of sublingual immunotherapy are proposed. PMID- 7551202 TI - IgE antibodies against snake venoms. AB - A similar event was detected in the clinical records of a small group of atopic patients living in the northern provinces of Argentina, i.e., they were bitten by a snake of the Bothrops species (or yarara) during their rural activities (woodcutters, cattle-drivers and farmers). Those who were bitten twice suffered an acute episode of hives and angioedema within 15 minutes after the snake bite. The presence of specific antibodies against Bothrops alternata (Ba) extract was detected by means of RAST for IgE and Ouchterlony and Boyden for IgG. The Ouchterlony also demonstrated crossreactivity among the venoms of the Bothrops species and the positivity of the six fractions obtained by DEAE-cellulose column fractionation against the horse anti-Ba serum. The Ba antigen induced a definite inhibition of the RAST. We presume that hives and angioedema in atopic patients immediately after a second snake bite could be attributed to the presence of a specific IgE antibody against the venom, and must not be misinterpreted with the toxic effects that appear later. PMID- 7551203 TI - Erythrocyte antigens as immunogenetic markers of respiratory atopic diseases in Georgians. AB - The aim of this study was to investigate the role of erythrocyte antigens of several systems (ABO, Rh, MNSs, P, Duffy, Kell) in the predisposition to and development of respiratory atopic disease (RAD) in the Georgian population. Two hundred and ninety-three patients with RAD, 83 patients with intrinsic bronchial asthma (IBA) and 215 healthy subjects were included. Associations between blood groups and RAD (atopic bronchial asthma [ABA] and pollinosis), the severity of the disease and the type of sensitization were established, involving the ABO, MNSs and Duffy systems. The groups at higher risk for the development of RAD were carriers of phenotypes O(I), MN, O(I)MN, MNFya(-), O(I)Fya(-) and O(I)MNFya(-). Resistance to RAD was associated with phenotypes A(II), AB(IV), N, Fya(+) and B(III)N. Markers for ABA and pollinosis were also revealed. Allergy to house dust allergen was associated with phenotype MN, whereas pollen sensitization was associated with the M group of MNSs. The risk for the development of severe ABA was higher in patients with B(III), M and B(III)M phenotypes. For the patients with groups O(I) and MN, a mild to moderate degree of severity of ABA was more characteristic. In healthy Georgians, erythrocyte polymorphism was not associated with immune status. In cases of house dust allergy, erythrocyte antigen P1 was associated with high levels of IgE. In patients with RAD higher B-lymphocyte counts were associated with O(I) and lower counts with A(II). PMID- 7551204 TI - Comparative study of terfenadine and cetirizine in hay fever: assessment of efficacy and central nervous system effects. AB - A daily dose of either terfenadine 120 mg or cetirizine 10 mg was compared in two parallel groups of patients suffering from hay fever. According to a double blind, double-dummy, randomized design, 28 patients were treated with one of the two drugs once daily in the morning for 2 weeks during the 1990 grass pollen season. The severity of nasal congestion, rhinorrhea, sneezing, nasopharyngeal itching and itchy, watery, red eyes was evaluated by the investigator after a 1 week run-in period and at the end of the treatment. The patients made a daily record of the severity of symptoms on a diary card. In addition, drug-related central nervous system (CNS) effects were assessed at baseline and at the end of the treatment by neuropsychological tests aimed at investigating selective and sustained attention, visuomotor abilities and anxiety, and by quantitative, bit mapped EEG. Both terfenadine and cetirizine produced a significant improvement in symptoms at endpoint without any significant difference between the two drugs. Drowsiness was referred by one patient in each treatment group. No significant impairment of psychomotor performance occurred with either drug. Quantitative EEG showed a significant power increase in the relative (%) delta band in both groups of treated patients. Although the difference was not statistically significant, a tendency towards greater involvement of the CNS was observed with the use of cetirizine. In conclusion, the results of this study confirm that terfenadine and cetirizine are equally effective in the management of hay fever. Some differentiated untoward EEG changes were also observed in relation to the drugs used, without any variation in neuropsychological performance. PMID- 7551205 TI - Adverse reactions to food in adults. AB - This study was carried out to assess the occurrence of food hypersensitivity (type I) in adults. The diagnosis of food allergy, based on suggestive history and positive skin prick test and/or RAST, was established in 30 (0.98%) of 3034 patients over 14 years of age who were attended between June 1992 and March 1993. The foods implicated were as follows: fruit (46.8%), dried fruits (19.1%), seafood (14.9%), vegetables (12.8%), fish (4.2%) and chicken eggs (2.1%). This study confirmed the low occurrence of food allergy in adulthood. The differences found with regard to the sensitizing foods (cow's milk and eggs were infrequent; fruit, seafood and vegetables were common) may be attributed to dietary habits in our geographical region. PMID- 7551206 TI - Evaluation of theophylline clearance in children with bronchial asthma. AB - The total body clearance of theophylline was examined in 95 children aged 6 months to 15 years with chronic bronchial asthma. Each patient received sustained release theophylline preparations and was evaluated at steady state. Blood samples were obtained 1-8 hours after a morning dose, and individual total body clearance was determined with a Bayesian forecasting computer model. Changes in theophylline clearance were evaluated within age groups, with the age determined at the time of blood sampling. Total body clearance tended to increase with age up to 10 years. In children over 10 years old, total body clearance significantly decreased with age. The interpatient variability of theophylline clearance was most noticeable at 2-4 years of age. Changes in volume of distribution were similar to those in clearance. In conclusion, there are pronounced age-dependent differences in theophylline clearance that require individual adjustment of dosage regimens on the basis of serum concentrations and clinical response in children. Individualization of the oral dose based on frequent serum measurements is necessary to maintain theophylline levels in the therapeutic range. PMID- 7551208 TI - What is asthma? An update on the mechanisms. AB - Asthma is currently best described by the presence of characteristic symptoms and by variable airways narrowing and airways hyperresponsiveness to a variety of inhaled bronchoconstricting stimuli. For more than 100 years, asthma has also been known to be an inflammatory disease of the airways. More recently, the importance of airways inflammation in the pathogenesis of asthma, ranging in severity from mild to severe, or in transient asthma after exposure to an inflammatory stimulus, has been recognized. Airways inflammation can be defined as the presence of activated inflammatory cells in the airways. Many studies have now demonstrated the presence of activated eosinophils and of mast cells in the airways lumen and airways wall of patients with asthma, even those with mild disease. The presence and survival of these inflammatory cells may be promoted by the presence of increased levels of proinflammatory cytokines, such as GM-CSF, in asthmatic airways. These cells have the capacity to release potent bronchoconstricting mediators, such as the cysteinyl leukotrienes, which are responsible, at least in part, for airways narrowing in asthma and for allergen-, exercise- and aspirin-induced asthma. Other cells, such as a subset of T lymphocytes (Th2), may also be important in maintaining the inflammatory cascade. Airways structural changes caused by persisting inflammation, such as airways epithelial damage, or altered smooth muscle function or volume are likely to be important in the pathogenesis of stable, long-standing airways hyperresponsiveness.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551207 TI - Mustard allergy: three cases of systemic reaction to ingestion of mustard sauce. AB - Very few cases of hypersensitivity due to ingestion of mustard have been described in the literature, although this spice enjoys widespread use. We present three cases of anaphylactic reactions to ingestion of a small amount of mustard sauce. In our patients we performed skin prick tests with mustard and with the rest of the vegetables belonging to the Cruciferae family. We also performed skin prick tests with common pneumoallergens. We measured specific serum IgE to mustard using the CAP System (Pharmacia). Oral challenges with the other vegetables of the Cruciferae family were performed in the patients with no evidence of previous tolerance. Skin prick tests and specific serum IgE determinations to mustard were positive in all three patients. The tests with the rest of the vegetables belonging to the same family were negative. In conclusion, we consider that skin prick tests and the measurement of specific serum IgE to mustard are good methods for the diagnosis of mustard hypersensitivity. We did not detect cross-sensitivity with other vegetables of the same family or with pneumoallergens. PMID- 7551209 TI - Recent advances in clinical neurotology. AB - In recent years, owing to significant technological developments and an increased number of investigators entering the field, there have been spectacular advances in our understanding of the basic anatomy and physiology of the vestibular system. Unfortunately, these advances in basic science are slow to impact the clinical management of patients. We have selected a few important advances in clinical neurotology that have impacted the diagnosis and treatment of patients with vestibular disorders. This material was originally presented at the "Mechanisms of Vestibular Function and Dysfunction" symposium of the 1994 Neural Control of Movement meeting in Waikoloa, Hawaii. PMID- 7551210 TI - Vestibularly evoked potentials (VESTEPs) of the horizontal semicircular canals under different body positions in space. AB - We recorded vestibularly evoked potentials (VESTEPs) using natural vestibular stimuli simulating the form of a normal head movement ("raised cosine"). Its smooth course was designed to minimize all possible sources of artifacts of mechanical, electrical, and physiological origin. This motion profile was applied to subjects (Ss) sitting upright, thus stimulating the horizontal semicircular canals alone, as well as when they were tilted in different positions in steps of 30 degrees about the interaural y-axis, which had the effect of modulating the otolithic contribution to the horizontal VESTEP. The transient bell-shaped VESTEP recorded in the upright sitting S was more and more superimposed or replaced by a sustained negativity the more the S was tilted toward a horizontal position. Thus, the VESTEP gradually changes from a velocity response in the upright S (canals only) to a position response in the lying S (canals plus otoliths). Differences in the VESTEPs obtained for clockwise (cw) and counterclockwise (ccw) turns in the tilted S were found. The reason for this is not clear at present and requires further experimentation. However, the VESTEPs obtained under conditions involving backward tilts and cw turns corresponded to those obtained with forward tilts and ccw turns. A similar relationship was found for the VESTEPs obtained with backward tilts and ccw turns and those obtained with forward tilts and cw turns. The physiological basis of this is discussed. PMID- 7551212 TI - Visual and nonvisual contributions to perceived ego-motion studied with a new psychophysical method. AB - The perception of ego-velocity (PEV) during sinusoidal linear acceleration (otolith stimulation) is investigated with a psychophysical method proposed by Wertheim (1990;1). This method is based on the threshold for object motion perception measured during ego-motion. In a first experiment, PEV was measured both with and without illumination of the experimental room. It was shown that, for the particular frequency and amplitude of ego-motion used, PEV in the light approached physical ego-velocity, whereas in darkness PEV was significantly smaller than physical ego-velocity. In a second experiment the PEV was measured again without illumination of the experimental room. It was found that in response to a sinusoidal ego-velocity stimulus, PEV was sinusoidal as well with a gain of 0.8 and a phase-lead of 4 degrees at 0.15 Hz. PMID- 7551213 TI - Normal vestibular function in chicks after partial exposure to microgravity during development. AB - Sixty-four fertilized chicken eggs, half at developmental Day 2 and half at Day 9, were exposed to micro-gravity for 5 days aboard the shuttle. Postflight examination showed that none of the Day 2 flight embryos had survived, whereas the Day 9 flight group and both groups of synchronous ground control embryos appeared viable. One-half of the Day 9 flight and ground control embryos were dissected and the temporal bones preserved in acetone for morphological examination. The other half was allowed to hatch to examine vestibularly related behavioral changes. Morphology of the lagenar otoconia was evaluated by scanning electron microscopy. Behavioral changes were accessed by a battery of reflex tests and recordings of spontaneous and vestibularly driven head movements. The results from both the morphological and behavioral studies showed no consistent difference between the flight and the control animals. Several hypotheses may account for this negative result. Because all the Day 2 embryos failed to survive, the remaining Day 9 chicks may have passed the critical developmental period of the chick's vestibular system. Also, the reexposure of the developing chick embryo to earth's 1-g environment may have masked any adverse behavioral effects that exposure to Microgravity may have caused. PMID- 7551211 TI - Mediolateral differential projection to the nodulus from the inferior olive in the albino rat: a retrograde WGA-HRP study. AB - The mediolateral topographic organization of olivocerebellar projections to the nodulus in rats was investigated by a retrograde WGA-HRP method. The nodulus received olivary afferents from the contralateral dorsal cap, nucleus beta, ventrolateral outgrowth, and dorsomedial cell column. The mediolateral extent of terminal areas of the olivocerebellar fibers arising from these subnuclei differed. The dorsal cap projected to the entire mediolateral area of the contralateral nodulus; the rostral and caudal parts of the dorsal cap projected to the lateral and medial parts of the nodulus, respectively. The nucleus beta projected to the medial and intermediate parts of the nodulus. Differential projections from the nucleus beta were observed in a rostrocaudal direction as those from the dorsal cap; the rostral and medial parts of the nucleus projected to the intermediate and medial parts of the nodulus, respectively. The ventrolateral outgrowth projected only to the intermediate part of the nodulus. The dorsomedial cell column projected to the lateral half of the nodulus, including its lateral tip. These and previous findings are compared with respect to this mediolateral topography. PMID- 7551214 TI - Geometric adjustments to account for eye eccentricity in processing horizontal and vertical eye and head movement data. AB - Neglecting the eccentric position of the eyes in the head can lead to erroneous interpretation of ocular motor data, particularly for near targets. We discuss the geometric effects that eye eccentricity has on the processing of target directed eye and head movement data, and we highlight two approaches to processing and interpreting such data. The first approach involves determining the true position of the target with respect to the location of the eyes in space for evaluating the efficacy of gaze, and it allows calculation of retinal error directly from measured eye, head, and target data. The second approach effectively eliminates eye eccentricity effects by adjusting measured eye movement data to yield equivalent responses relative to a specified reference location (such as the center of head rotation). This latter technique can be used to standardize measured eye movement signals, enabling waveforms collected under different experimental conditions to be directly compared, both with the measured target signals and with each other. Mathematical relationships describing these approaches are presented for horizontal and vertical rotations, for both tangential and circumferential display screens, and efforts are made to describe the sensitivity of parameter variations on the calculated results. PMID- 7551215 TI - Head-only rotational testing: influence of volition and vision. AB - Head-only rotational testing has been suggested as a method of assessing the vestibuloocular reflex in patients with reduced vestibular function as a result of ototoxicity. The purpose of this study was to investigate the influence of vision and volition on horizontal vestibuloocular reflex responses during head only rotation in normal subjects to better define the test parameters for future clinical application. Ten asymptomatic, healthy subjects were tested using various combinations of volitional and visual conditions during head-only rotation. Results indicated that visual fixation enhanced responses slightly and that response dynamics were altered somewhat by volition. These findings provide motivation for future studies to further define head-only rotation test parameters for patients with suspected vestibular abnormalities. PMID- 7551216 TI - Immunological and formulation design considerations for subunit vaccines. PMID- 7551217 TI - Laboratory empiricism, clinical design, and social value. The rough road toward vaccine development. PMID- 7551218 TI - A compendium of vaccine adjuvants and excipients. PMID- 7551219 TI - Adjuvant properties of aluminum and calcium compounds. AB - It is likely that aluminum compounds will continue to be used with human vaccines for many years as a result of their excellent track record of safety and adjuvanticity with a variety of antigens. For infections that can be prevented by induction of serum antibodies, aluminum adjuvants formulated under optimal conditions are the adjuvants of choice. It is important to select carefully the type of aluminum adjuvant and optimize the conditions of adsorption for every antigen since the degree of adsorption of antigens onto aluminum adjuvants markedly affects immunogenicity. The mechanism of adjuvanticity of aluminum compounds includes formation of a depot at the site of injection from which antigen is released slowly; stimulation of immune-competent cells of the body through activation of complement, induction of eosinophilia, and activation of macrophages; and efficient uptake of aluminum-adsorbed antigen particles by antigen-presenting cells because of their particulate nature and optimal size (< 10 microns). Limitations of aluminum adjuvants include local reactions, production of IgE antibodies, ineffectiveness for some antigens, and inability to elicit cell-mediated immune responses especially cytotoxic T-cell responses. Calcium phosphate, which has adjuvant properties similar to aluminum adjuvants, has the potential advantages of being a natural component of the body and of not increasing IgE production. There is a need for alternative adjuvants, particularly for diseases in which cell-mediated immune responses are important for prevention or cure. PMID- 7551220 TI - Structure and properties of aluminum-containing adjuvants. AB - This chapter is concerned with the identification, characterization, and behavior of aluminum-containing adjuvants with proteins and anions similar to those occurring in vaccines and interstitial fluid. Aluminum-containing adjuvants referred to commercially as aluminum hydroxide have been identified as poorly crystalline aluminum oxyhydroxide with the structure of the mineral boehmite. Relevant properties of this material include its high surface area and its high pI, which provide the adjuvant with a high adsorptive capacity for positively charged proteins. Aluminum phosphate and alum-precipitated adjuvants may be classified as amorphous aluminum hydroxyphosphate with little or no specifically adsorbed sulfate. Variations in the molar PO4/A1 ratio of amorphous aluminum hydroxyphosphates result in PI values that range from 5 up to 7; the materials are negatively charged at a physiological pH of 7.4. The amorphous nature of these compounds gives them high surface area and high protein adsorptive capacity for positively charged proteins. Observations on the interactions of anions and charged proteins with charged adjuvant surfaces have provided a framework for predicting behavior of complex systems of vaccines and for designing specific combinations of adjuvants and antigens to optimize the stability and efficacy of vaccines. PMID- 7551222 TI - Development of vaccines based on formulations containing nonionic block copolymers. AB - In summary, data indicate that nonionic block copolymers in several different delivery formats can effectively enhance antibody responses to a variety of viral, parasite, or bacterial antigens. Polymers have historically been evaluated as polymers alone in aqueous buffer, in oil-in-water and water-in-oil emulsions. Several of those formulations can induce protective antibodies in preerythrocytic or erythrocytic malaria vaccine models or in pneumococcal vaccine models. In those models, protective immunity is associated with the development of IgG2a subclass antibodies. These results tend to indicate that copolymer adjuvant can influence isotype development, possibly by stimulating the appropriate T-cell subsets. Although there are some data suggesting that microfluidized vaccines containing the L121 nonionic block copolymer can induce CTL, equivalent experimental results with larger block polymers, which are effective in induction of greater proportions of IgG2a, have not yet been obtained. Several of the basic formulations with an appropriate copolymer may be suitable for clinical evaluation in conjunction with either current or future subunit antigens. Other formulations containing copolymers may also be suitable for mucosal administration. PMID- 7551221 TI - MF59. Design and evaluation of a safe and potent adjuvant for human vaccines. AB - MF59 is a safe, practical, and potent adjuvant for use with human vaccines. The formulation is easily manufactured, may be sterilized by filtration, and is both compatible and efficacious with all antigens tested to date. MF59 has been shown to be a potent stimulator of cellular and humoral responses to subunit antigens in both animal models and clinical studies. Toxicology studies in animal models and Phase I-III studies in humans have demonstrated the safety of MF59 with HSV, HIV, and influenza vaccines. PMID- 7551223 TI - Development of an emulsion-based muramyl dipeptide adjuvant formulation for vaccines. PMID- 7551224 TI - Liposomal presentation of antigens for human vaccines. AB - Liposomes are considered prime candidates to improve the immunogenicity of both antigens with hydrophobic anchor sequences and soluble, nonmembrane proteins or synthetic peptides. During the 20 years since liposomes were first demonstrated to have adjuvant potential, studies have shown that variation in liposomal size, lipid composition, surface charge, membrane fluidity, lipid-protein composition, anchor molecules, and fusogenicity can significantly influence results. In addition, antigen location (e.g., whether it is adsorbed or covalently coupled to the liposome surface or encapsulated in liposomal aqueous compartments) may also be important. Analysis of these variables as well as a comparison of the various techniques used to ensure the efficacy, stability, homogeneity, and safety of liposomal vaccine have been discussed. PMID- 7551225 TI - Liposome design and vaccine development. PMID- 7551226 TI - Lipid matrix-based vaccines for mucosal and systemic immunization. AB - For more than a decade our laboratories have been combining concepts in biochemistry, virology, and immunology in order to develop a conceptual basis for vaccine design. Our long-term goals have been to construct simple and well defined immunogens that would stimulate specific immune responses in vivo. Using this approach, we hypothesized that it should be possible to define the structural and biochemical parameters of an immunogen that are necessary and sufficient to stimulate designated effector arms of the immune system. Through the use of covalently coupled peptide complexes, we have been able to define minimal requirements for the induction of humoral immune responses (Mannino et al., 1992). This represents a significant advance in eliciting an immune response to peptides, because it requires only peptides and phospholipids in the absence of additional adjuvants. It is different from the previous use of peptides and liposomes since here the peptides are covalently linked to a hydrophobic anchor and integrated into the phospholipid complex, rather than passively adsorbed or encapsulated. The presentation of peptide as part of a peptide-phospholipid complex (in contrast to encapsulation or nonspecific absorption) may be more similar to the natural presentation of an epitope in the context of an in vivo antigenic challenge. This technology also allows us to incorporate B and Th epitopes in a number of forms--as a single peptide, as two peptides in the same liposome, or as a peptide with viral glycoproteins in the same liposome. These data also demonstrate that Th epitopes do not have to be covalently linked to the B-cell epitope in order to provide help for that epitope. The implications of the data reported here are significant for both basic science and applied technologies. In basic science, the peptide-phospholipid complexes are potentially useful for analyzing the cooperative effects of B- and T-cell epitopes in the in vivo immune response. Since the peptide-phospholipid complexes are totally synthetic and highly immunogenic, they may be constructed in any formulation required to answer questions on the roles of B and T cells in promoting an immune response. Furthermore, since the number of antigenic sites is limited only by the number of peptides included in the peptide-phospholipid complexes, these constructs may be useful in producing antisera or monoclonal antibodies to weakly antigenic regions of a large protein, since the lack of antigenic competition should enhance the immunogenicity of these regions. Clinically, this technology will expand the potential for subunit vaccines.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7551227 TI - Polymer microspheres for vaccine delivery. PMID- 7551228 TI - Vehicles for oral immunization. PMID- 7551229 TI - Public health implications of emerging vaccine technologies. AB - The field of public health and medicine stands to benefit immensely from the emerging vaccine technologies and improved application of existing technologies. Technological advances may promote: (1) greater flexibility and simplicity in the design and operation of immunization campaigns or ongoing prevention programs, including reduction in number of vaccine doses, cold chain elimination, slow release/prolonged antigenic stimulation, reduced cost and hazard and increased ease of administration through noninvasive, oral delivery systems, greater population levels of immunization and health; (2) the development of documents by FDA, WHO, and other regulatory authorities and groups, to assist the manufacturer in the appropriate manufacturing, preclinical, and clinical development of these new vaccines; (3) a greater array of vaccines to protect the civilian and military populations; (4) increased vaccine potency; (5) vaccines eliciting mucosal immunity, cytotoxic T cells, and/or neutralizing antibody. At the end of the 20th century there remain many unconquered pathogens and noninfectious indications for which medical science suggests that vaccines could be effective. New technologies may provide the best hope to address this wide array of public health needs. PMID- 7551230 TI - Design and production of single-immunization vaccines using polylactide polyglycolide microsphere systems. PMID- 7551231 TI - Nanoparticles as adjuvants for vaccines. AB - PMMA nanoparticle adjuvants can be manufactured in a physicochemically reproducible manner. Their particle size can be controlled within narrow limits. Immunogens may be either incorporated or adsorbed to these nanoparticles. PMMA nanoparticles induced significantly higher and more prolonged antibody responses against a variety of immunogens, including influenza virions and subunit vaccines, BSA, and HIV-1 and HIV-2 split vaccines. In addition, a protective immune response against challenge with live influenza virus was induced and a better stability of the immunogen was observed after incorporation or adsorption of influenza virions or subunits to PMMA nanoparticles. The observation that PMMA did not induce antibodies against gp120 contained in the HIV-2 split vaccine demonstrates that different adjuvants or carriers may be required for different antigens. A combination of two or more different adjuvants or carriers may be necessary to induce the optimal immune response against antigen mixtures as present in most vaccine preparations. PMMA seems to be a safe adjuvant material. It is very slowly biodegradable and has been used in surgery in humans for over 40 years, and now warrants continued investigation as a vaccine adjuvant. PMID- 7551232 TI - Water-soluble phosphazene polymers for parenteral and mucosal vaccine delivery. AB - PCPP can be used in two different ways to potentiate an immune response. The soluble form of the polymer has been found to have immunoadjuvant activity. A single subcutaneous injection of polymer/influenza dramatically increases the ELISA, neutralizing, and HI antibodies to influenza virus compared to CFA. The polymer has also succeeded in dramatically increasing the amount of ELISA antibodies to TT. The antibody response elicited was predominantly of the IgG1 isotype. PCPP has also been used to generate micron-sized hydrogel microspheres through a process of divalent ion cross-linking of the polymer strands. These microspheres can induce significantly higher anti-TT serum IgG titers after a single intranasal immunization than TT alone. PMID- 7551234 TI - Structural and immunological characterization of the vaccine adjuvant QS-21. PMID- 7551233 TI - Monophosphoryl lipid A as an adjuvant. Past experiences and new directions. AB - The current era of vaccinology has resulted in antigens produced by synthetic chemistry and by genetic engineering techniques; these may provide more effective and safe vaccines. Many of these antigens can be produced in large quantities and in relatively pure form. However, inherent in their simplified, defined structures is a lack of immunogenicity and they require an immunostimulant or adjuvant to elicit appropriate immunity. It has been the purpose of this chapter to document the potential use of MPL as a vaccine adjuvant. In this regard, it has been demonstrated that (1) MPL has retained the useful immunostimulating activities of the parent LPS molecule, but has greatly attenuated toxicity; (2) MPL produces diverse effects on the cellular elements of the immune system, including macrophage activation and T and B cell interaction, with concomitant cytokine and lymphokine release; (3) MPL has proven adjuvant activity, in both the cellular and humoral effector arms of immunity; (4) MPL has adjuvant activity when used alone, or in combination with other immunostimulants and delivery vehicles; and (5) MPL has been administered safely to humans in various clinical vaccine trials. Thus, MPL is emerging as a promising new adjuvant for use in human vaccines. PMID- 7551235 TI - A novel generation of viral vaccines based on the ISCOM matrix. PMID- 7551236 TI - Vaccine adjuvants based on gamma inulin. AB - Algammulin and gamma-IN comprise a novel class of vaccine adjuvant. Their use in vaccines is to exploit the humoral defense known as the alternative pathway of complement. They use a "natural" mechanism and the biochemical basis of their action is well understood in general terms. They are fully researched up to the stage of specific commercial application. Inulin itself is registered for human use as a solution and is without physiological effect except for ACP activation as gamma-IN particles. The ACP comprises a relatively harmless part of the inflammatory response. Gamma inulin is nontoxic in several species including humans and is nonpyrogenic. The amount of systemic C3a produced from adjuvant active doses of gamma-IN is expected to be very much less than that routinely tolerated without effect by human renal dialysis patients. Registration of gamma IN should not be difficult. Gamma inulin in vivo is either dissolved and excreted unchanged or metabolized to simple foodstuffs. Its primary chemical structure is completely known, and it is inexpensive, readily available, and easy to handle and manufacture. It is completely stable under normal conditions of use and storage. Patent cover is either fully granted or accepted for granting in most developed countries. Alum is also registered for human use and its combination with gamma-IN known as Algammulin is equally nontoxic especially in the fine formulation, and is equally stable. The partial coating with inulin in Algammulin greatly reduces the undesirable effects of alum such as granuloma formation and IgE generation. Combinations of gamma-IN with immunogen carriers other than alum are feasible, either as hybrid particles or as simple mixtures of particles of similar size. Gamma inulin, and especially Algammulin, are potent enhancers of the Th1 immune response pathway, boosting seroconversion rates and immunological memory in protective Ab classes and enhancing cell-mediated immunity. The responses can equal those of CFA. They are also Th2 pathway enhancers, especially for IgA, and the emphasis on Th2 might be varied by altering the alum-to-inulin ratio in the final formulation. A dual response (balanced Th1 and Th2) may be desirable for several reasons. Their primary targets in vivo are probably lymphocytes rather than macrophages. Gamma inulin-based adjuvants therefore comprise new, safe, potent, and attractive candidates for enhancing responses to human and veterinary vaccines, especially those requiring cell-mediated defenses. PMID- 7551238 TI - Preclinical safety assessment considerations in vaccine development. PMID- 7551237 TI - A new approach to vaccine adjuvants. Immunopotentiation by intracellular T-helper like signals transmitted by loxoribine. AB - Loxoribine is a potent new immunostimulant with a relatively broad spectrum of immunobiological activities. Both loxoribine and its analogues function as agonists of immune responses in a variety of species, including humans. They upregulate the activity of B cells, T cells, NK cells, macrophages, and LAK cells. Induction of enhanced cytokine secretion has been found to involve IFN alpha/beta, IFN-gamma, TNF-alpha, TNF-beta, IL-1, IL-6, and the 40 kDa chain of IL-12. Evaluation of in vivo activity has been undertaken only for antibody production, NK cell-mediated cytotoxicity, induction of certain cytokines, and LAK cell-mediated cytotoxicity; all four types of activity are markedly upregulated by loxoribine in vivo. Augmentation of antibody production has been observed for protein, recombinant protein, and synthetic peptide antigens, among others. Because loxoribine and its analogues transmit a T-helper-like signal to antibody-producing B cells, it is a highly effective adjuvant even for synthetic peptides that lack T-cell epitopes, effectively replacing the function of T helper cells in this milieu. It thus provides an alternative, T-cell-independent vaccination strategy if it becomes desirable to avoid untoward T-cell-mediated effects, or in patients with functional or absolute T-cell deficiency. There are a number of features unique to loxoribine that are highly advantageous under specific circumstances: (1) T cell independence; (2) loxoribine augments antibody responses from an intracellular location (rather than at the surface membrane), independently of protein kinase C involvement; this may be particularly relevant for patients with membrane receptor/signal transduction defects; (3) adjuvanticity of loxoribine is essentially free of cytokine dependency; this may be of particular value for organ transplantation patients whose cytokine dependent immunity is pharmacologically suppressed; (4) loxoribine bypasses functional immunological immaturity, rendering it particularly useful for vaccines in infants. In preclinical safety studies, the drug has exhibited a relatively benign profile. Phase I clinical studies to date have produced no toxicity higher than grade 1. The drug appears to be quite stable, and compares very favorably in direct evaluations with a number of other immunostimulators. A number of clinical trials have been planned for the future. PMID- 7551239 TI - Stearyl tyrosine. An organic equivalent of aluminum-based immunoadjuvants. PMID- 7551240 TI - Cytokines as vaccine adjuvants. Current status and potential applications. PMID- 7551241 TI - Cytokines as immunological adjuvants. PMID- 7551242 TI - Cytokine-containing liposomes as adjuvants for subunit vaccines. PMID- 7551243 TI - Haemophilus influenzae type b conjugate vaccines. AB - In summary, all of the Hib conjugate vaccines are highly immunogenic and efficacious in children older than 12-15 months of age, and HbOC, PRP-OMPC, and PRP-T are highly immunogenic and demonstrated to be efficacious in infants as young as 2 months old. HbOC, PRP-OMPC, and PRP-T have been licensed in numerous countries for infants and are recommended for infant immunization. However, perhaps the greatest tribute one can pay to all four Hib vaccines described in this review is to note the dramatic decrease in the incidence of Hib disease that has occurred since their introduction. In fact, according to the Morbidity and Mortality Weekly Report (March 4, 1994), the incidence of Hib disease in children less than 5 years old has declined by 95% from 41 cases per 100,000 in 1987 to 2 cases per 100,000 in 1993, timing that coincides with the availability and use of the Hib conjugate vaccines (Anderson, 1994). As universal administration is achieved and the apparent vaccine-induced reduction in carriage of Hib by the population continues, Hib vaccines may follow the lead of past vaccines (such as smallpox, measles, mumps, rubella, and polio) toward eradication of disease or at least a high degree of medical control, thereby virtually eliminating the mortality and insidious morbidity associated with invasive Hib diseases. PMID- 7551244 TI - Pneumococcal conjugate vaccines. PMID- 7551245 TI - Lyme vaccine enhancement. N-terminal acylation of a protein antigen and inclusion of a saponin adjuvant. PMID- 7551246 TI - Vaccine research and development for the prevention of filarial nematode infections. AB - The development of vaccines for the prevention of filarial nematode infections is in a state of relative infancy in comparison to vaccines for other parasitic diseases, such as schistosomiasis and malaria. There are many reasons for this slow start. Some of the principal problems are: (1) the lengthy and complex life cycle of these organisms with attendant complex immune responses, (2) the unique characteristics associated with a relatively large number of different pathogens, (3) the lack of suitable model systems for study of medically important infections, (4) the paucity of parasite material for antigen discovery and recombinant library construction, (5) the lack of substantial evidence suggesting the natural occurrence of protective immune responses, and (6) the limited data on mechanisms responsible for protective immunity. As technical hurdles are considered, it is also critical to focus on the characteristics of a vaccine necessary for its eventual utility. In the case of a vaccine for D. immitis a completely successful product will need to approach a 99+% efficacy. This is because of the 99+% efficacy of competitive chemotherapeutic products and the fact that microfilaremia observed on blood examination, resulting from as few as two worms, would present as a vaccine failure. Although very low worm burdens in large dogs could be perceived as success in the context of protection from clinical disease, because of the option of virtually complete chemoprophylactic protection, the typical veterinary practitioner would probably fail to appreciate less than complete vaccine protection. In contrast, a vaccine that produced a reduction in adult worm burdens without complete protection in either lymphatic filariasis or onchocerciasis would be very important. Highly effective chemoprophylactic agents are not widely available for prevention of the human filariases, and dramatically reduced clinical disease provided by less than a completely effective vaccine could occur as the result of fewer adult worms. The importance of developing these vaccines has outweighed the obstacles to this research. There has been a great deal of epidemiological and experimental evidence to suggest a vaccine is feasible and antigen discovery has progressed relatively rapidly within just the past few years. Efforts to generate appropriate larval cDNA libraries are beginning to yield dividends and a variety of fascinating vaccine candidates have been cloned. Additional antigen discovery, research on appropriate modalities for overexpression of genes from these parasites, and the complex tasks associated with vaccinology remain as significant research and development obstacles.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7551247 TI - Retrovirus and retrotransposon particles as antigen presentation and delivery systems. PMID- 7551248 TI - Rationale and approaches to constructing preerythrocytic malaria vaccines. PMID- 7551249 TI - The MAP system. A flexible and unambiguous vaccine design of branched peptides. PMID- 7551250 TI - Regulatory considerations in vaccine design. AB - To summarize, vaccines are regulated in the United States as biologics by CBER and must meet requirements for safety, purity, and potency (efficacy). Although general requirements exist for safety, purity, and potency, specific standards for each vaccine are agreed to by the manufacturer and CBER. The final standards for any vaccine are relevant to the technology used to produce the vaccine. Vaccine efficacy is demonstrated through conducting one or more Phase III trials. A single, definitive, well-controlled, double-blind, placebo-controlled Phase III trial often provides sufficient efficacy data for licensing a vaccine. Pivotal efficacy data may be derived from U.S. or outside the U.S. studies. Bridging studies may be required to link the efficacy data to the intended marketing target population. In the United States, approval for conducting clinical trials is obtained from the FDA through the mechanism of the IND application. Marketing approval is obtained through the mechanism of the PLA and ELA. Postmarketing Phase IV clinical trials are generally requested to develop large-scale field data for safety. Timely communication with the FDA throughout the development and approval process is the most efficient mechanism for meeting all regulatory requirements in the shortest possible time. PMID- 7551252 TI - Design and testing of peptide-based cytotoxic T-cell-mediated immunotherapeutics to treat infectious diseases and cancer. PMID- 7551251 TI - Design of experimental synthetic peptide immunogens for prevention of HIV-1 and HTLV-I retroviral infections. PMID- 7551253 TI - Development of active specific immunotherapeutic agents based on cancer associated mucins. AB - As a result of aberrant glycosylation, cancer-associated mucins expose to the immune system certain carbohydrate, peptide, and possibly glycopeptide epitopes that are not exposed on the normal mucins. This provides the basis for our development of synthetic carbohydrate, peptide, and glycopeptide-based ASI agents corresponding to the cancer-associated mucin epitopes. Our studies on ASI formulations based on carbohydrate structures such as TF and STn have demonstrated their ability to induce immune response relevant to the native epitopes on the cancer cells in animal models and in cancer patients. Further, such immune responses were able to mediate cancer rejection in an animal model. Similar studies on peptide epitopes of a cancer-associated mucin, MUC1, have also shown the ability of the synthetic antigen to induce anticancer immune responses in an animal model. Ongoing studies on the carbohydrate and peptide epitopes would allow us to define the most important target structures on cancer associated mucins that can selectively stimulate cancer-specific immune responses. Our long-term goal is to develop multiepitopic glycopeptide ASI formulations capable of stimulating strong CMI responses against common carcinomas. PMID- 7551255 TI - Synthetic hormone/growth factor subunit vaccine with application to antifertility and cancer. PMID- 7551254 TI - Synthetic peptide vaccines for schistosomiasis. PMID- 7551256 TI - Clinical considerations in vaccine trials with special reference to candidate HIV vaccines. PMID- 7551257 TI - Survival of patients with stage IA malignant melanoma. AB - There is ongoing clinical research on prognostic parameters relevant to stage IA melanoma. The object is to identify those factors associated with an increased risk of recurrence. The charts of 197 patients first treated at our Institute between 1980 and 1992 along with 62 patients referred for follow-up or treatment of recurrent disease, all having been initially diagnosed with stage IA disease, were reviewed. Only one patient (0.5%) of those first treated at our Institute manifested recurrence and this was a local recurrence. No statistically significant differences were found between patients who relapsed and those who did not with regard to lesion thickness, level of invasion, evidence of ulceration, location of the primary lesion, gender, or age. Generally, stage IA melanomas have excellent prognosis. However, there are patients who experience recurrent and metastatic disease. At the present time, there are no reliable indicators available for use in predicting which patients are at risk. PMID- 7551258 TI - Gastrointestinal metastases from malignant melanoma. AB - Between 1980 and 1992, 68 patients with clinical indications of involvement of the gastrointestinal (GI) tract with metastatic melanoma were treated at Roswell Park Cancer Institute. Presenting symptoms were anaemia, abdominal pain, nausea and vomiting. Sites commonly involved were the small bowel (75%), the large intestine (25%), and the stomach (16%). Twenty-one patients were considered unsuitable for surgery; their median survival after diagnosis of GI metastases was 2.9 months. Forty-seven patients underwent abdominal surgery; effective palliation was achieved in most of them. Complete resection of GI metastases was accomplished in 47% of patients. The median survival after operation was 27.6 months for patients with complete resection of GI metastasis and no other disease, 5.1 months for patients with resection of involved GI tract and other metastases present, and 1.9 months for patients who had a by-pass procedure only. The 5-year survival for patients with complete resection of GI metastases and no other evidence of disease was 28.3%. The other groups had only 1-year survivors. Surgical intervention is justified on the basis of these findings, and extended palliation can be achieved in patients with complete resection of metastatic disease. PMID- 7551259 TI - The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer. AB - Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumor cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion. PMID- 7551260 TI - Pre-operative induction chemotherapy for small cell carcinoma of the oesophagus: a case report. AB - Induction chemotherapy was prescribed to a patient who had locally advanced small cell carcinoma of the oesophagus which was considered to be irresectable on presentation. Significant tumour downstaging was observed and the patient remains recurrence free 16 months after a successful Ivor-Lewis oesophagectomy. PMID- 7551263 TI - Proliferating cell nuclear antigen may be superior to argyrophilic nucleolar organizer regions in predicting shortened survival of patients with non-small cell lung cancer. AB - We examined proliferating cell nuclear antigen (PCNA) in 102 patients with surgically treated non-small cell lung cancer (NSCLC). PCNA labelling index (LI) tended to be higher in tumours of higher stages than those of early stages, in squamous cell carcinomas than adenocarcinomas, or in poorly differentiated adenocarcinoma than in well-differentiated. A positive correlation was observed between the PCNA LI and argyrophilic nucleolar organizer regions (Ag-NOR) count which we previously examined (r = 0.31, P = 0.002). In survival analysis of 79 patients who died of lung cancer, only age, stage and PCNA LI were found to be significant prognostic factors on multivariate analysis among seven potential prognostic factors including sex, age, year of operation, histological type, stage, Ag-NOR count, and PCNA LI. We conclude that PCNA may be superior to Ag-NOR in predicting shortened survival of patients with non-small cell lung cancer. PCNA staining can be performed with ease and it may be applied in a clinical laboratory on a routine basis to help predict prognosis of NSCLC. PMID- 7551261 TI - Management of pancreatic carcinoma. AB - Pancreatic cancer is the fifth-leading cause of cancer death in the United States. It is estimated that 27,000 patients die each year with this diagnosis. The overall five-year survival of patients with pancreatic cancer remains less than 5%, but some subsets of patients may have a better prognosis. Advanced imaging and laparoscopy have improved staging to better select patients for potentially curative surgery, while the operative morbidity and mortality of the Whipple resection have decreased in recent years. Non-surgical therapies in current use include chemotherapy, external-beam radiation therapy, and brachytherapy, as well as pain management. More recently, gene insertion therapy has shown promise in animal models. This review discusses current diagnostic and treatment strategies for these patients and documents the need for new strategies in the future. PMID- 7551265 TI - Clinical and epidemiologic predictors of recurrent coronary artery disease. PMID- 7551262 TI - Adenocarcinoma of the gastric cardia. AB - One hundred and ninety-five patients operated on for adenocarcinoma of the gastric cardia during the years 1961-90 were analysed and the present data indicate that the more enthusiastic attitude adopted towards resective surgery led to a significant increase in operative explorations performed and in resectability rate, from 50% (44/88) and 35% (28/88) during the years 1961-75 to 84% (90/107) and 56% (60/107) during the years 1976-90, respectively. The difference between radical resections, 54% (15/28) and 67% (34/60), remained non significant. The overall postoperative mortality and morbidity after resective surgery were 14% and 35% and these rates did not rise with time. The anastomotic leakage rate was 15%. Anastomotic leakage was, in fact, not only the most common postoperative complication but also the most common cause of death. Overall cumulative survivals at 1, 3 and 5 years were 47%, 11% and 5%. Comparison of the cumulative survival rates between the 15-year periods indicated that there were no differences in overall survival or in survival after resective surgery. We regard these results disappointing, because over half of the patients died in 1 year and because the long-term survival remained dismal. PMID- 7551264 TI - Prognostic significance of peritumoural blood and lymphatic vessel invasion by tumour cells in T4 non-small cell lung cancer following induction therapy. AB - We investigated the impact of new biological prognostic factors is in 28 patients receiving a median of two courses of cisplatin-based chemotherapy with (n = 14) or without (n = 14) radiation and operation for stage IIIB (T4) non-small cell lung cancer (NSCLC). After induction therapy, 5 patients had a complete and 21 a partial response; 2 had a stable disease. A complete resection was made in 26 patients (93%). Five patients (18%) had their primary tumour and involved vestiges completely sterilized. In the remaining 23, the majority of the tumours showed abnormalities in the p53 gene expression (56%), harboured proliferating cells (91%) and induced angiogenesis (91%). Peritumoural blood and lymphatic vessel invasion (PBLVI) by tumour emboli was observed in 6 tumours. With a median follow-up of 25 months, overall 3-year survival was 48%; disease-free survival (DFS) has not been reached yet. The only significant factor influencing DFS in multivariate analysis was PBLVI by tumour cells; PBLVI-positive patients had a significantly higher likelihood ratio (P = 0.000001) of developing metastasis than their PBLVI-negative counterparts. This study documents the prognostic implication of PBLVI by tumour cells in T4 NSCLC. PMID- 7551266 TI - Clinical investigations of the arrest and reversal of coronary artery disease. AB - Trials of lipid-lowering therapy with angiographically monitored endpoints have demonstrated that regression of atherosclerosis can be achieved. Additionally, the unexpected clinical benefit seen in some trials has suggested that stabilization of lesions may be even more important clinically than regression, and more readily achievable. However, much additional research is needed to clarify the effects of therapy on the heterogeneous population of lesions and the precise mechanisms by which such therapy influences event rates. It is nonetheless clear that aggressive lowering of LDL cholesterol is indicated in patients with established coronary disease, who are at high risk for future events. Accordingly, the new guidelines of the USA National Cholesterol Education Program (NCEP) have adopted an LDL cholesterol goal of less than 100 mg/dl (2.6 mmol/l) in patients with confirmed atherosclerotic disease. Ongoing and future strategies for investigating lipid-lowering therapy and the progression of atherosclerosis include new imaging modalities, such as ultrasonography of the carotid arteries and PET scanning, and use of more potent lipid-lowering interventions. PMID- 7551267 TI - Risk factor intervention and coronary artery disease: clinical strategies. PMID- 7551271 TI - Concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) correlated with expression of VCAM-1 mRNA in the human atherosclerotic aorta. AB - BACKGROUND: Adherence of circulating monocytes and lymphocytes to arterial endothelium is detectable early in experimental and human atherosclerotic plaque formation. The purpose of the present study was to assess vascular cell adhesion molecule-1 (VCAM-1) mRNA expression and the properties of soluble VCAM-1 in patients with atherosclerotic aortic disease. METHODS: Thirteen patients with aortic disease (mean age 64 years) and 40 healthy volunteers (mean age 32 years) were included in the study. We investigated the expression of VCAM-1 mRNA in eight human aortic specimens obtained during surgery. Of these, two showed no evidence of atherosclerotic plaque formation [aortic dissection (n = 1) and annuloaortic ectasia (n = 1)], whereas six had demonstrable complex atherosclerotic formation [abdominal aneurysm (n = 5) and aortic dissection (n = 1)]. The RNase protection assay was performed using an alpha 32P-labeled 121 base pair VCAM-1 cRNA probe. We also measured concentrations of the soluble VCAM-1 using two-site enzyme immunoassays in 40 healthy volunteers and in 13 patients with abdominal and thoracic aortic aneurysms. RESULTS: In human samples, VCAM-1 mRNA expression was found to be higher in the six patients with complex atherosclerotic formation [optical density (OD), 0.71 +/- 0.14] than in the two patients with no evidence of atherosclerotic plaque formation (OD, 0.53 and 0.49). The concentration of serum soluble VCAM-1 was higher (850 +/- 298 ng/ml) in patients with aortic or thoracic disease than in the healthy volunteers (494 +/- 94 ng/ml). In addition, there was a relationship between VCAM-1 mRNA expression and the concentration of soluble VCAM-1 (y = 2088x-554, r2 = 0.73). CONCLUSION: VCAM-1 expression is higher in the human aorta in patients with atheromatous changes. Furthermore, concentrations of soluble VCAM-1 may provide important information about endothelial activation or in-vivo damage. PMID- 7551270 TI - Assessment of left ventricular myocardial perfusion and diastolic function during acetylcholine-induced diffuse coronary vasoconstriction by Doppler echocardiography and thallium-201 scintigraphy. AB - BACKGROUND: We investigated whether diffuse coronary vasoconstriction induced by acetylcholine caused myocardial ischemia. METHODS: We studied 30 patients (40 coronary arteries) with spontaneous chest pain or equivocal studies on treadmill exercise testing and no significant coronary stenosis or previous myocardial infarction. During the acetylcholine provocation test, Doppler echocardiography was performed, and thallium-201 was injected intravenously for scintigraphy. We used Doppler echocardiography to measure the ratio of early-to-late peak mitral flow (E:A ratio). RESULTS: When acetylcholine was injected, the coronary arteries showed three different responses. Diffuse coronary vasoconstriction without chest pain or ischemic changes on the ECG was induced in 18 (45%) arteries and the E:A ratio decreased from 0.83 +/- 0.13 to 0.77 +/- 0.13 (P = 0.031). In 17 vessels (control arteries), the E:A ratio did not change significantly (from 0.88 +/- 0.15 to 0.88 +/- 0.18; P = 0.95). In five arteries, focal spasm was induced and the E:A ratio decreased from 0.83 +/- 0.18 to 0.66 +/- 0.13 (P = 0.043). Transient defects on thallium-201 scintigraphy were observed in the territory of eight (80%) arteries with diffuse vasoconstriction and in one (20%) control artery (P = 0.047). CONCLUSIONS: Diffuse coronary vasoconstriction induced by intracoronary acetylcholine can decrease the regional myocardial blood flow (as shown by thallium-201 scintigraphy) and can cause global left ventricular diastolic dysfunction (as shown by the results of Doppler echocardiography). PMID- 7551269 TI - Differences in vasodilatory response to dipyridamole between patients with angina and normal coronary arteries and patients with successful coronary angioplasty. AB - BACKGROUND: Previous studies reported a reduced coronary blood flow reserve, assessed by the intravenous administration of dipyridamole, in patients with angina and normal coronary arteries, and early after successful coronary angioplasty, which suggests the presence of small coronary vessel dysfunction. This study aimed to establish whether the mechanisms of small coronary vessel disease in these two groups of patients are similar. METHODS: The effects of the intracoronary infusion of adenosine and dipyridamole (maximum dose 2.7 and 7.5 mg/min, respectively) on coronary blood flow velocity were assessed in 11 patients with angina and normal coronary arteries (group A) and in 12 patients immediately after successful coronary angioplasty (group B) using a 0.018" Doppler wire. RESULTS: Baseline coronary blood flow velocity was significantly higher in group B than group A (34 +/- 14 versus 19 +/- 8 cm/s; P = 0.001). In group A, coronary blood flow velocity was higher during adenosine than dipyridamole infusion (74 +/- 17 versus 58 +/- 21 cm/s; P < 0.001), whereas in group B velocities were similar (85 +/- 30 versus 78 +/- 32 cm/s; NS). CONCLUSIONS: In patients with angina and normal coronary arteries, a maximal dose of adenosine causes a greater coronary dilation than that of dipyridamole. Given that dipyridamole operates mainly through an inhibition of adenosine re-uptake, it can only dilate the arteriolar segments exposed to endogenous adenosine. Therefore, the lower response to dipyridamole than to exogenous adenosine observed in patients with angina and normal coronary arteries suggests an impairment of the pre-arterioles that are not influenced by endogenous adenosine, resulting in a limited flow-mediated dilation in response to arteriolar dilation. Such an impairment is not apparent immediately after successful coronary angioplasty, where the most obvious abnormality is an increase of baseline coronary blood flow velocity. PMID- 7551274 TI - Bibliography of the current world literature. PMID- 7551272 TI - Vasoreactivity and restenosis after coronary angioplasty in the atherosclerotic pig model. AB - BACKGROUND: The effect of coronary angioplasty on coronary spasm remains unknown. We examined the effects of balloon angioplasty, using an oversized balloon, on coronary hypercontraction and eventual restenosis in a pig model of coronary spasm. METHODS: We performed balloon angioplasty, using an oversized balloon at the site of coronary spasm superimposed on atherosclerosis with 6 (group A, n = 14) or 1 (group B, n = 14) atmospheres of inflation pressure in miniature pigs. Using coronary angiography we assessed the coronary basal diameter and diameter change in response to histamine and serotonin before, immediately after, and 4 weeks after angioplasty. Histological examinations were performed immediately after and 4 weeks after the angioplasty. RESULTS: Before angioplasty, histamine- and serotonin-induced hypercontraction was reproducibly noted at the atherosclerotic site. Immediately after angioplasty, the coronary diameter became larger (P < 0.01) than before angioplasty in group A, but there was no significant difference in group B. The hypercontraction in group A was abolished immediately after and 4 weeks after angioplasty, whereas the hypercontraction in group B was unchanged. Histological examination revealed medial necrosis immediately after and medial fibrosis 4 weeks after angioplasty, which were more prominent in group A. Four weeks after angioplasty, the magnitude of restenosis was greater in group A than in group B. CONCLUSIONS: Angioplasty using an oversized balloon with the higher atmosphere of inflation pressure abolished coronary hypercontraction immediately after and 4 weeks after angioplasty, which probably resulted from the irreversible damage to medial smooth muscles; however, this did not prevent restenosis. PMID- 7551275 TI - Laparoscopic cholecystectomy in 200 consecutive patients using an ultrasonically activated scalpel. AB - Ultrasonic energy has not been previously used for surgical cutting and coagulating. Work in our laboratory has led to the development of an ultrasonically activated scalpel that safely and effectively cuts and coagulates tissue in animals. The purpose of this study was to determine if ultrasonic energy can replace monopolar electrosurgery in human laparoscopic surgery. Two hundred consecutive patients underwent laparoscopic cholecystectomy with the ultrasonically activated scalpel. The scalpel was the sole energy form in 98 of the first 100 patients, and in all of the last 100 patients. There were no common duct injuries, reoperations, or mortality. No patient had more than a 3-g drop in hemoglobin or transfusion. The ultrasonically activated scalpel is a safe and effective energy form for cutting and coagulating tissue during laparoscopic cholecystectomy in humans. The absence of need of monopolar electrosurgery combined with hemostatic effectiveness supports the concept that the ultrasonically activated scalpel can replace electrosurgery for laparoscopic cholecystectomy. PMID- 7551268 TI - Cost-effectiveness of accepted measures for intervention in coronary heart disease. PMID- 7551276 TI - Laparoscopic retrieval of disconnected ventriculoperitoneal shunt catheters: report of two cases. AB - Two patients previously treated with ventriculoperitoneal shunt for hydrocephalus had the distal catheters disconnected, and they migrated to the free peritoneal cavity. The signs and symptoms of increased intracranial pressure occurred as a result of mechanical failure of the shunt system. Both patients successfully underwent a laparoscopic retrieval of the catheters using endoscopic grasping forceps, and revision of the ventriculoperitoneal shunt by inserting the new catheters into the peritoneal cavity was performed through a small entrance for the laparoscope. Recently, conventional laparotomy has been often replaced by laparoscopic surgery, and general surgeons are well familiar with the laparoscopic procedures. The significant advantage of this procedure is the ability to explore the entire peritoneal cavity for both diagnosis and treatment, and it is by far a less invasive approach than conventional laparotomy. The present report describes the laparoscopic procedure for removal of the migrated catheters and discusses its advantages over conventional laparotomy. PMID- 7551273 TI - Elective coronary angioplasty with and without surgical standby: clinical and angiographic criteria for the selection of patients. AB - BACKGROUND: The cardiac catheterization laboratory (CCL) of our hospital is the only facility for invasive cardiology in a large district. No cardiac surgery is carried out in our hospital at present, the nearest facility being approximately 50 miles away. METHODS: Over a period of 2 years we recommended percutaneous transluminal coronary angioplasty (PTCA) with surgical standby for 164 cases, who were referred to CCLs with on-site surgical standby, and PTCA without surgical standby for 232 cases, 199 of whom underwent PTCA in our CCL. Criteria used in the selection of patients for PTCA without surgical standby were the following: (a) either limited extent or severely impaired function of the ventricular segment in jeopardy; (b) normal or near-normal function of the uninvolved myocardial segments; (c) absence of lesions of the left main or left anterior descending coronary arteries when the target stenosis was in the left coronary artery; (d) non-applicability and high risk-benefit ratio of emergency surgical revascularization in the individual patient. Clinical and angiographic characteristics of patients assigned to PTCA with and without surgical standby are compared. RESULTS: An initial success was achieved in 186 cases (93%) in our CCL. In 12 cases (6%), PTCA was not successful, and in two cases (1%) it was complicated by myocardial infarction. None of the patients died, or had to undergo further coronary interventions within 1 month. Coronary stents were implanted in 24 cases. CONCLUSION: For many PTCA candidates, emergency coronary surgery is not an option in case of occlusive complications. Our data suggest that PTCA can be performed with minor complications in these patients in the absence of surgical standby, provided strict criteria are used in the selection of cases. PMID- 7551277 TI - Significance of laparoscopic excisional biopsy for polypoid lesions of the gallbladder. AB - Polypoid lesions of the gallbladder taken from 103 patients were examined to correlate clinical indicators with pathological characteristics. By ultrasonographic examination, no carcinoma was found in polypoid lesions with a diameter less than 1 cm. However, the incidence of carcinoma in those with diameters greater than 1 cm was 23%, a lower rate than expected from previous pathological studies. Comparison of ultrasonographic and actual measurements suggested that the size of cholesterol polyps tended to be overestimated by ultrasonography, suggesting that the apparent risk of cancer was decreased by cholesterol polyps. Laparoscopic cholecystectomy was performed in 30 patients with polyps. No complications were encountered, and operative time was significantly shorter (p < 0.01) than in the 116 laparoscopic cholecystectomies for cholelithiasis. This study demonstrated the difficulty in distinguishing large benign polyps from carcinoma by ultrasonography. To complete the diagnosis with less invasive intervention, we propose the use of laparoscopic cholecystectomy for patients with polypoid lesions larger than 1 cm by ultrasonographic estimation. PMID- 7551278 TI - Laparoscopic cholecystectomy in cirrhotic patients. AB - Cholecystectomy in cirrhotic patients is commonly followed by high morbidity and mortality. In our study, laparoscopic cholecystectomy was performed in 12 cirrhotic patients (eight Child A and four Child B) in an effort to obtain lower complication and mortality rates. The mean age of the group was 51.8 years; seven of the 12 patients were men. Four patients had liver disease from virus B, five from virus C, one because of alcohol, and two had cryptogenic liver cirrhosis. The average operative time was 157 min. Intraoperative cholangiography could be performed in eight cases. No mortality or need to perform laparotomy occurred. Only one patient required blood transfusion (8.3%). Postoperative complications occurred in four patients, but these were easily controlled and included renal failure, diabetic impairment, hematoma with ascitic leakage through the wound, and wound abscess, one case each. No postoperative liver failure was observed. All patients walked and were refed in the first 24 h after surgery. They were dismissed in an average period of 2.5 days. Our primary view demonstrated that laparoscopic cholecystectomy was safe and well tolerated by selected cirrhotic patients (Child A and B) with clear indication for surgery. PMID- 7551279 TI - Laparoscopic resection of two liver hemangiomata. AB - New laparoscopic instrumentation coupled with standard surgical techniques allows one to perform procedures previously thought impossible via the laparoscope. This report reviews the natural history, the indications for resection, and the technique of laparoscopic resection of hepatic hemangiomata. Two women, 24 and 62 years of age, were first seen with abdominal pain. A diagnosis of hemangioma was made in each case, and both lesions were removed laparoscopically. Operative blood loss was 200 cc in each case, and neither patient required transfusion. Diets were started on the first postoperative day (POD), and the patients were discharged on the second and fourth PODs without narcotic analgesia. If the size and location of the tumor are favorable, laparoscopic resection of liver hemangiomata can be performed safely. Blood loss comparable to that of open procedures and a quicker recovery support an endosurgical approach to resection of liver hemangiomata in selected cases. PMID- 7551280 TI - Laparoscope-assisted Billroth I gastrectomy. AB - We performed a distal gastrectomy, including regional lymph node dissection, under laparoscopic observation followed by Billroth I. (B-I) anastomosis using autosuture in five patients with early gastric cancer. We had previously evaluated the technique for reconstructive surgery in animal experiments. Our method was determined to be safe without marked deformation caused by applying the autosuture for gastroduodenostomy. The wound was small, and an almost closed operation was possible. Furthermore, unlike endoscopic mucosal resection or laparoscopic partial resection, a major part of the regional lymph nodes can be extirpated; the lesion site may not be limited to the half of the stomach on the distal side. Our method of distal gastrectomy under laparoscopy is a potentially useful limited operation in patients with early gastric cancer. PMID- 7551281 TI - Laparoscopic treatment of biliary and gastric outlet obstruction. AB - To avoid laparotomy in patients with biliary or gastric outlet obstruction in inoperable carcinoma of the pancreas, we evaluated the feasibility of double bypass operation in 20 pigs. In model A (n = 10), biliary bypass was a Roux-en-Y loop for cholecystojejunostomy after choledochal clip occlusion. In model B (n = 10) an additional Roux-en-Y loop was attached to the stomach after transection of the duodenum. Model A showed functioning biliary bypass with patent and leak-free anastomoses at necropsy on day 28. In model B, four pigs were sacrificed earlier, but nine had normal functioning double bypass (two intussusceptions of the ileum, one bleeding ulcer at the gastroenterostomy, one necrotic Roux-en-Y-loop). Based on the presented bypass function, we are encouraged to perform laparoscopic biliary and gastroenteral bypass in humans to treat malignant jaundice and gastric outlet obstruction. PMID- 7551282 TI - The role of endoscopic retrograde cholangiopancreatography in small children and adolescents. AB - Diagnostic and therapeutic endoscopic retrograde cholangiopancreatography (ERCP) was performed on 16 patients aged 2 months to 18 years at Jordan University Hospital in the period between May 1990 to January 1993. The adult side-viewing duodenoscope was used, and visualization of appropriate ductal system was accomplished in 100% of the cases with no complications. Intravenous sedation was used in 69% of the cases with diazepam and pethidine (meperidine). ERCP provided valuable additional information in patients with hepatobiliary and pancreatic diseases. However, it did not demonstrate significant pathology in patients with otherwise unexplained abdominal pain. Endoscopic sphincterotomy (ES) was successfully performed on four infants and small children with common bile duct stones. It is concluded that ERCP and ES are relatively easy and safe techniques when applied to infants and children with pancreatobiliary disorders. PMID- 7551283 TI - Minimally invasive abdominoplasty: surgical technique development and report of three cases. AB - Endoscopically assisted rectus abdominis plication is a safe and effective method of repair in patients with mild to moderate central abdominal wall flaccidity. A procedure was developed in animals and in a human cadaver using a single umbilical incision, gasless dissection of the anterior rectus abdominis fascia, and suture plication under endoscopic guidance. This procedure was performed successfully in three patients. Early postoperative results were comparable to those obtained by standard rectus abdominis plication. Moreover, adjunctive suction lipectomy or a limited abdominal skin resection can be used to expand the contouring capability of this technique. PMID- 7551284 TI - Partial laparoscopic splenectomy for trauma: technique and case report. AB - We describe the course of a patient who underwent partial laparoscopic splenectomy after a fall while skiing. Computed tomography revealed intact liver and kidneys with fragmentation of the upper third of the spleen and a left upper quadrant hematoma. Splenic artery angiogram showed devascularization of the superior pole, and selective cannulation of the superior polar artery demonstrated extravasation of contrast media. The superior polar artery was embolized with three coils to control bleeding. After laparoscopic exploration, the patient underwent partial laparoscopic splenectomy of the superior pole, which consisted of ligating the short gastric vessels and incising the splenic parenchyma 6 to 8 mm above the ischemic demarcation line produced by embolization of the superior polar artery, where a deep transverse laceration was located. The specimen was retrieved in a plastic bag. Intraoperative blood loss was less than 50 ml, and the patient left the hospital on the 3rd postoperative day after an uneventful recovery. Partial laparoscopic splenectomy can be performed safely, and its role needs to be delineated. PMID- 7551286 TI - Injury in laparoscopic surgery. AB - A prospective study was made to evaluate injury caused by laparoscopic surgery, in terms of physiological response. Two groups of patients were established: Group 1 (laparoscopic surgery, n = 26) and Group 2 (open surgery, n = 18). The groups were homogeneous in terms of age, sex, body mass index (BMI), duration of surgery, and anesthetic technique. Both groups exhibited significant postoperative increases in plasma adrenocorticotropic hormone (ACTH), growth hormone (GH), insulin, and cortisol (p < 0.05), with a significant decrease in follicle-stimulating hormone (FSH) and T3 (p < 0.05). Significant increases were noted in 24-h urine cortisol and catecholamine levels in Group 2 (p < 0.05). No correlation was noted between the duration of surgery and the intensity of neuroendocrine response. Acute-phase postoperative metabolic response was greater in Group 2 and was correlated to the duration of surgery. No postoperative hydrosaline or acid-base alterations were recorded in either group. Injury was graded in terms of neuroendocrine and metabolic response and proved highest in Group 2. Complex laparoscopic surgery (e.g., sigmoid colon and esophageal hiatus) exhibited the least neuroendocrine response, whereas laparoscopic inguinal hernioplasty involved the least metabolic response. To conclude, laparoscopic surgery globally involves less neuroendocrine and metabolic response than does open surgery. PMID- 7551285 TI - Laparoscopic electrosurgical injuries: survey results and their implications. AB - This article provides the results of a survey on electrosurgical complications and surgical techniques during laparoscopy from the American College of Surgeons. Of the respondents, 18% stated that they had personally experienced an electrosurgical burn to their patient during laparoscopy. The survey shows that the majority of surgeons, 74%, employ coagulation mode most commonly during surgery. Of the surgeons, more than one third routinely employ high-voltage coagulation and blend mode operation at power settings > 40 W. We discuss the possible complications that may occur from high-voltage-high-power settings, such as direct coupling, insulation failure, and capacitive coupling of unintended current into internal tissue such as bowel. We further describe techniques that may be employed to minimize the likelihood of unintended electrosurgical burns as well as technologies that can eliminate or greatly decrease the likelihood of electrosurgical complications. PMID- 7551288 TI - Pulmonary interstitial edema after probable carbon dioxide embolism during laparoscopy. AB - Carbon dioxide embolism is a well-known complication of laparoscopy that can be lethal. We describe a patient who showed signs of pulmonary interstitial edema revealing a probable gas embolism. This event occurred during a gynecologic laparoscopy performed for uterine perforation after a curettage. PMID- 7551289 TI - Laparoscopic cholecystectomy for gallbladder torsion. AB - Although torsion of the gallbladder is most commonly a disease of the elderly, an 18-year-old woman presented with this condition. This is the first reported case of laparoscopic cholecystectomy for gallbladder torsion. Signs and symptoms of gallbladder volvulus are often subtle. Radiologic evaluation may be normal. A high index of suspicion is necessary to make the preoperative diagnosis. If treated laparoscopically, gallbladder decompression and detorsion prior to cholecystectomy are helpful techniques to avoid bile duct injury. A literature review of this uncommon problem is provided. PMID- 7551287 TI - Thoracoscopic resection of a localized visceropleural mesothelioma. AB - Herein we present a case report of a 46-year-old man with a localized visceropleural mesothelioma detected incidentally during a health examination. The patient underwent a thoracoscopic resection, and his postoperative course was uneventful. A thoracoscopic resection of peripheral localized nodules can be easily performed using thoracoscopic techniques, and this method should not be considered inferior to thoracotomy in terms of curability. PMID- 7551290 TI - Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms. AB - We have developed and tested a rapid and sensitive method of detecting expansion of T-cell clones using the polymerase chain reaction (PCR) and a single set of consensus primers for the V and J regions to amplify rearranged T-cell receptor gamma (TCR-gamma) genes. Monoclonality was continued in all of the 18 cases of T cell neoplasms tested, but not in reactive lymphadenopathy, non-Hodgkin's B lymphomas, and Hodgkin's disease. PCR analysis, using the primer sequence outlined in this study, had an overall specificity of 100% when compared with Southern blot analysis. No false-negative results were observed, certainly owing to the choice of consensus primers and to the control of PCR reactions on agarose gels before testing for clonality by separation of PCR products on polyacrylamide gels. This method for the detection of T-cell monoclonality can be especially useful in cases that are diagnostically problematic with standard histological and immunological analysis and in cases where the material available is limited. PMID- 7551292 TI - Human papillomavirus infection in the malignant and premalignant head and neck epithelium. AB - A prospective study was undertaken to determine the prevalence and histological correlates of human papillomavirus infection in the head and neck epithelium. Oral, pharyngeal, and laryngeal paraffin-embedded samples were analyzed by polymerase chain reaction with use of human papillomavirus E6 consensus sequence primers. Human papillomavirus infection was detected in 20 of 126 (15.9%) patients. Twenty-five of 230 (10.9%) samples contained human papillomavirus DNA. Papillomaviruses were detected in 15 of 131 (11.4%) head and neck squamous cell carcinomas, in 3 of 32 (9.4%) dysplasias, and 2 of 19 (10.5%) keratoses. The most commonly identified human papillomavirus in cancerous, precancerous, and keratotic lesions was type 16 (80.0% of the isolates). Five papillomas were shown to contain human papillomavirus type 6. No other lesion in 42 samples contained human papillomavirus. Our data are consistent with the hypothesis that infection with certain types of human papillomavirus contributes to head and neck cancer although this may be so only in a minority of cases. Human papillomavirus infection may play a role in the earliest stages of tumorigenesis, since papillomaviruses can be found in laryngeal premalignant and keratotic lesions, which are closely linked to tobacco use. PMID- 7551291 TI - Clonality of thyroid nodules in sporadic goiter. AB - Clonality studies have suggested that neoplasms are monoclonal and hyperplasias are polyclonal. To investigate this question in thyroid, we analyzed the clonality of 26 morphologically characterized hyperplastic nodules from 19 patients with sporadic goiters. For comparison we studied six thyroid carcinomas. We used the highly informative M27 beta probe that maps to the X-chromosome DXS255 locus (X cen-p11.22). Material was obtained from 52 nodules; tissue from nine nodules was rejected because of contamination with normal elements, five patients (eight nodules) were homozygous at Pst I sites in nonnodular thyroid tissue, and three nodules were excluded for technical reasons. Methylation patterns after Hpa II digestion confirmed polyclonality in all nontumorous thyroids of informative patients. Seven hyperplastic nodules were polyclonal, and 18 were monoclonal; one showed loss of heterozygosity. One nodule exhibited aberrant methylation. Multiple nodules were obtained from four patients; in three, all were monoclonal with activation of the same allele. Three papillary carcinomas were monoclonal; two exhibited aberrant methylation. One follicular carcinoma showed loss of heterozygosity. Our data indicate that morphologically indistinguishable hyperplastic thyroid nodules may be monoclonal or polyclonal. These findings suggest that variable molecular mechanisms are involved in the pathogenesis of nodules in sporadic goiter. Future studies will need to explore the biological significance of nodules of variable clonal origin. PMID- 7551293 TI - Fluorescence in situ hybridization (FISH) detection of MYCN oncogene amplification in neuroblastoma using paraffin-embedded tissues. AB - The expression and degree of amplification of the MYCN oncogene in neuroblastoma provide an important indicator of disease prognosis. Detection of MYCN amplification has been described using Southern blotting or polymerase chain reaction (PCR) on DNA from fresh or frozen tissue samples, and using in situ hybridization mainly on metaphase spreads or smears of cultured neuroblastoma cells. In this article, we describe fluorescence in situ hybridization (FISH) results on detection of MYCN amplification in formalin-fixed, paraffin-embedded samples of 25 neuroblastoma and 20 nonneuroblastoma pediatric tumors. MYCN amplification was readily detectable by FISH in eight of the neuroblastomas; correlation with results obtained by Southern analysis was perfect. Of the nonneuroblastoma tumors, only one of three retinoblastoma cases showed MYCN amplification. In contrast to the Southern blot technique, FISH demonstrated the state of amplification heterogeneity of the tumor cells as well as the nature of the amplification units: double-minute chromosomes (DMs) or homogeneously staining regions (HSRs). The results indicate that FISH is a rapid and reliable method for detection of MYCN oncogene amplification in routinely processed samples and may be used to supplant the Southern blot technique. PMID- 7551294 TI - Coexpression of TGF alpha, epidermal growth factor receptor, and P-glycoprotein in normal and benign diseased breast tissues. AB - Twenty-four normal or benign breast tissues were examined for the expression of transforming growth factor-alpha (TGF alpha), epidermal growth factor receptor (EGFR), and P-glycoprotein, the product of the mdr-1 gene. Specific staining for all three proteins was observed in the majority of the samples. P-glycoprotein staining was present in most (88%), and confined to the lumenal surface of the ductal epithelium. Membranous EGFR expression was observed in epithelial cells in 92% of the specimens and 42% displayed both myoepithelial and epithelial cell staining. TGF alpha staining was intense and uniformly distributed through the cytoplasm (96%). Coexpression of EGFR, TGF alpha, and P-glycoprotein in normal human breast tissues suggests a role for each of those proteins in normal breast physiology. An interaction may be present in normal breast tissue between the EGF receptor pathway and P-glycoprotein. PMID- 7551295 TI - In situ hybridization analysis of chromogranin A and B mRNAs in neuroendocrine tumors with digoxigenin-labeled oligonucleotide probe cocktails. AB - The chromogranin/secretogranin (Cg/Sg) molecules are a family of acidic proteins present in neuroendocrine cells and tumors with secretory granules. They have been frequently used to characterize neuroendocrine cells and tumors by immunohistochemical analyses. Immunoreactivity for CgA is related to the presence of secretory granules in these tumors, so immunohistochemical staining for CgA may be absent in neuroendocrine tumors with only a few secretory granules. RNA in situ hybridization with a series of oligonucleotide probes for CgA and CgB was used to detect the mRNA transcripts for CgA and CgB with digoxigenin-labeled probes in 31 neuroendocrine tumors. These results were compared to ISH with 35S labeled probes and with immunohistochemical staining for CgA and synaptophysin in the same neoplasms. ISH with 35S-labeled probes for CgA and B detected mRNA transcripts in 31 of 31 tumors, whereas the digoxigenin-labeled probe cocktails for CgA and B were positive in 19 of 31 cases when used separately and in 24 of 31 cases when used together. Immunohistochemical staining for CgA was positive in 22 of 31 cases and for synaptophysin in 23 of 31 cases. The CgA and B oligonucleotide probe cocktails were highly specific, since nonneuroendocrine cells and tumors did not stain and the hybridization signal was abolished by ribonuclease A pretreatment. These results indicate that non-isotopic ISH with digoxigenin-labeled probe cocktails for CgA and B or with 35S-labeled probes can be used in characterizing neuroendocrine cells and tumors in formalin-fixed paraffin-embedded tissue sections even when the CgA protein is not detected by immunohistochemistry. PMID- 7551296 TI - Molecular diagnostics. Reimbursement and other selected financial issues. PMID- 7551298 TI - Nasal messenger RNA expression of interleukins 2, 4, and 5 in patients with allergic rhinitis. AB - In nasal biopsies from 17 adult patients with seasonal allergic rhinitis and from 10 healthy controls, cytokines were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). The time-course study during winter included repeated local allergen provocation with subsequent nasal biopsies as well as biopsies taken during pollen season. The RT-PCR for CD44 yielded positive bands in 65 of 71 cases, in which cases mRNA for interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5) were thus investigated by means of seminested PCR. IL-4 mRNA was found almost exclusively in the allergic patients. During provocation a significant increase in IL-4 was noticed compared with controls (p = 0.043). Equally, during the natural pollen season, IL-4 mRNA expression was significantly higher in patients not using nasal corticosteroids compared with those who did (p = 0.011). No differences in IL-2 or IL-5 were observed between the groups. These findings also indicate, together with earlier observations of T-cell activation, a phenotype switch toward T-helper 2 (Th2) cells, and the accumulation (homing) of these T cells in the nasal mucosa, that T cells constitute the main source for IL-4 in the nasal mucosa. Therefore, allergic patients have an increased synthesis of IL 4 when provoked with the allergen, and during natural pollen season this synthesis can be downregulated by corticosteroids. Furthermore, this study exemplifies the versatility of molecular biology in surgical pathology and that even low-copy-number cytokine mRNA can be examined in routinely snap-frozen surgical specimens. PMID- 7551299 TI - mdm2 gene amplification and overexpression in non-small cell lung carcinomas with accumulation of the p53 protein in the absence of p53 gene mutations. AB - Fifty-three non-small cell lung carcinomas (NSCLC), previously investigated for p53 abnormalities, were studied to evaluate the status of the mdm2 gene by Southern and Northern blot analysis and expression of the mdm2 protein by immunohistochemistry with specific monoclonal antibodies. Amplification and overexpression of the mdm2 gene and nuclear accumulation of its protein product were observed in three (6%) of the NSCLC examined. All of the tumors having mdm2 abnormalities belonged to a subset of NSCLC characterized by a strong accumulation of the p53 protein in the absence of p53 gene mutations. Since mdm2 is capable of forming tight complexes with p53, possibly stabilizing it, our results suggest that this event may take place in a low percentage of NSCLC. Moreover, all of the mdm2-positive tumors were histologically classified as lung adenocarcinomas. This may indicate that the mdm2 gene is preferentially altered in this particular subtype of lung tumors. PMID- 7551297 TI - Introduction to quantitative reverse transcription polymerase chain reaction. PMID- 7551300 TI - Comparison of serologic analysis and in situ localization of PCR-amplified cDNA for the diagnosis of hepatitis C infection. AB - The purpose of this study was to compare the serological analysis for hepatitis C infection using the recombinant immunoblot assay (RIBA) to the direct in situ localization of the hepatitis C viral genome using the reverse transcriptase (RT) in situ PCR technique. Concurrent sera and liver biopsies from 42 patients with clinical and histologic evidence of chronic liver disease were studied. Antibodies against hepatitis C specific antigens were demonstrated by RIBA in the sera of 39/42 (92%), and PCR amplified viral cDNA was detected in the biopsies of 21 (54%) of the 39 seropositive patients. The detection rate using standard in situ hybridization for the tissues known to be viral positive with RT in situ PCR was 9/21 (42%). It is concluded that approximately one-half of patients with chronic hepatitis and serologic evidence of hepatitis C infection will not have virus detectable in their liver biopsy even with a highly sensitive PCR-based technique. PMID- 7551301 TI - Internal defibrillation with smaller capacitors: a prospective randomized cross over comparison of defibrillation efficacy obtained with 90-microF and 125-microF capacitors in humans. AB - INTRODUCTION: The size of current implantable cardioverter defibrillators (ICD) is still large in comparison to pacemakers and thus not convenient for pectoral implantation. One way to reduce ICD size is to defibrillate with smaller capacitors. A trade-off exists, however, since smaller capacitors may generate a lower maximum energy output. METHODS AND RESULTS: In a prospective randomized cross-over study, the step-down defibrillation threshold (DFT) of an experimental 90-microF biphasic waveform was compared to a standard 125-microF biphasic waveform. The 90-microF capacitor delivered the same energy faster and with a higher peak voltage but provided only a maximum energy output of 20 instead of 34 J. DFTs were determined intraoperatively in 30 patients randomized to receive either an endocardial (n = 15) or an endocardial-subcutaneous array (n = 15) defibrillation lead system. Independent of the lead system used, energy requirements did not differ at DFT for the experimental and the standard waveforms (10.3 +/- 4.1 and 9.5 +/- 4.9 J, respectively), but peak voltages were higher for the experimental waveform than for the standard waveform (411 +/- 80 and 325 +/- 81 V, respectively). For the experimental waveform the DFT w as 10 J or less using an endocardial lead-alone system in 10 (67%) of 15 patients and in 12 (80%) of 15 patients using an endocardial-subcutaneous array lead system. CONCLUSIONS: A shorter duration waveform delivered by smaller capacitors does not increase defibrillation energy requirements and might reduce device size. However, the smaller capacitance reduces the maximum energy output. If a 10-J safety margin between DFT and maximum energy output of the ICD is required, only a subgroup of patients will benefit from 90-microF ICDs with DFTs feasible using current defibrillation lead systems. PMID- 7551302 TI - Electrophysiologic determinants of ventricular rate in human atrial fibrillation. AB - INTRODUCTION: The mechanisms of the ventricular response during atrial fibrillation (AF) remain uncertain. The parameters obtained during an electrophysiologic study, including atrial rates during AF, were analyzed to clarify further the determinants of the ventricular rate during AF. METHODS AND RESULTS: Thirty patients without manifest preexcitation in whom AF was induced during electrophysiologic study were divided into two groups. Group 1 consisted of 20 patients (ages 55 +/- 10 years) without a dual AV nodal pathway. Group 2 consisted of 10 patients (ages 53 +/- 13 years) having a dual AV nodal pathway. The correlation coefficient between the mean RR interval during AF (mRR) and the mean intra-atrial potential interval during AF (mff) was positive (0.05 [P < 0.05] in group 1 and 0.37 [P = NS] in group 2). The correlation coefficient of the mRR against the functional refractory periods of the AV node (AVFRP) was 0.73 (P < 0.001) in group 1. The correlation coefficients between mRR and the fast AV nodal pathway functional refractory periods and the slow AV nodal pathway effective refractory periods (SPERP) were 0.58 (P = NS) and 0.7 (P < 0.05) in group 2, respectively. The correlation coefficients between mRR against (mff x AVFRP)1/2 in group 1 and (mff x SPERP)1/2 in group 2 were 0.8 (P < 0.001) and 0.72 (P < 0.05), respectively. CONCLUSIONS: This clinical study did not indicate an inverse relation between the atrial and ventricular rates that had been reported by the previous experimental study. The ventricular rate during AF appeared to be quantitatively related to the atrial rate via AV node function. The importance of the slow pathway in determining the ventricular rate during AF was observed. PMID- 7551303 TI - Assessment of pacing maneuvers used to validate anterograde accessory pathway potentials. AB - Four pacing maneuvers have been proposed to validate an anterograde accessory pathway potential (APP): (1) atrial pacing to induce complete block between the atrial electrogram and the APP; (2) ventricular pacing to advance the APP without altering the timing of the atrial electrogram; (3) atrial pacing to induce complete block between the APP and the ventricular electrogram; and (4) ventricular pacing to advance the ventricular electrogram without altering the timing of the APP. The purpose of this study was to assess these validation techniques by applying them to electrograms that simulated APPs but which were known to be atrial in origin. In 32 patients undergoing an electrophysiology procedure, a split atrial electrogram containing two components separated by at least 30 msec (mean 54 +/- 15 msec) was recorded. Using an atrial extrastimulus technique, complete block between the two components of the atrial electrogram (criterion 1) could never be induced, but complete block between the second component of the atrial electrogram and the ventricular electrogram (criterion 3) consistently was induced. Using a ventricular extrastimulus technique, the second component of the atrial electrogram consistently could be advanced by 10 to 40 msec without altering the timing of the first component (criterion 2). In addition, with ventricular pacing, the ventricular electrogram consistently was advanced without altering the timing of the two components of the atrial electrogram (criterion 4). In conclusion, among the four pacing maneuvers used to validate an anterograde APP, the only one that may be specific for an APP is the ability to induce complete block between the atrial electrogram and the APP. PMID- 7551305 TI - Heart rate variability and mortality and sudden death post infarction. PMID- 7551304 TI - Circadian variation of heart rate variability in postinfarction patients with and without life-threatening ventricular tachyarrhythmias. AB - INTRODUCTION: Determination of heart rate variability (HRV) is widely used for noninvasive assessment of cardiac autonomic tone. A decreased HRV is associated with an increased mortality in patients surviving an acute myocardial infarction. There are, however, only sparse data about the circadian variation of different components of HRV that may be linked to the well-known circadian fluctuations in the occurrence of sudden death. In addition, the potential prognostic impact of circadian variations of HRV has not been examined. METHODS AND RESULTS: The present study compared the circadian variation of HRV from 14 postinfarction patients who had survived at least one episode of out-of-hospital cardiac arrest (cardiac arrest group) with that of 14 age- and sex-matched patients without a history of malignant arrhythmias after their index infarct (control group). Several time- and frequency-domain measures of HRV were assessed from 24-hour Holter recordings. Circadian variations of high- (HF), low- (LF), and total frequency (TF) components were determined by calculating for each parameter the hourly difference from the day's mean. The average of these differences was calculated for every hour as well as for predefined day and night periods. There was no significant difference between the two groups with regard to HRV indices that predominantly reflect vagal tone, such as SDNN (78 +/- 25 vs 96 +/- 24 msec), pNN50 (2.7% +/- 4.6% vs 4.9% +/- 4.2%), or HF (6.3 +/- 3.0 vs 7.8 +/- 3.2 msec; cardiac arrest vs control group). There was also no significant difference in the circadian variation of LF or TF between the two groups during daytime and nighttime. However, a significant difference in circadian variation of HF was found during daytime (0.02 +/- 0.5 vs -0.6 +/- 0.5 msec; P = 0.006) and nighttime (0.19 +/- 0.64 vs 1.5 +/- 0.75 msec; P = 0.0002). In cardiac arrest survivors, there was no difference in the mean deviation of HF between the day- and the nighttime periods. CONCLUSIONS: These results show an almost complete abolition in circadian variation of parasympathetic tone in postinfarction patients surviving an episode of out-of-hospital cardiac arrest, whereas circadian variation of sympathetic tone is comparable to that of postinfarction patients without arrhythmic episodes. These findings indicate that determination of diurnal variation of HRV may add to the prognostic value of HRV with respect to identifying patients at high risk of sudden death. PMID- 7551307 TI - Calculation of transmembrane current from extracellular potential recordings: a model study. AB - INTRODUCTION: A mathematical/computer model of cardiac tissue was used to study the estimation of transmembrane current (EIm) from extracellular potential recordings. METHODS AND RESULTS: The simulated EIm of transmembrane current was compared with the simulated transmembrane current (Im), and both simulated values were compared with experimentally derived EIm obtained during sinus rhythm and ventricular fibrillation in dogs. We found that although EIm measurements slightly overestimate the duration of the Im waveform, they provide a reasonable approximation of Im during normal conduction and during decremental conduction and conduction block. CONCLUSIONS: There is a very clear linear correlation between the time spent at or below 25% of the peak inward transmembrane current (Im25), its corresponding estimate (EIm25), the peak inward Im and EIm, and the peak ionic current, providing some evidence that EIm25 may be a suitable in vivo measure of peak ionic current. PMID- 7551306 TI - The effect of cardiac compression on defibrillation efficacy and the upper limit of vulnerability. AB - INTRODUCTION: We determined the effects of decreasing the ventricular blood volume and altering cardiac geometry on defibrillation, the upper limit of vulnerability (ULV), and the relationship between them. METHODS AND RESULTS: In six pigs, fibrillation/defibrillation trials were performed with a left ventricular apex patch to a superior vena cava catheter electrode configuration and a biphasic waveform. Thirty trials each were performed on a compressed versus noncompressed (normal) heart. Compression was achieved using direct mechanical ventricular actuation. Dose-response curves were constructed, and the 50% probability points (ED50) were compared for leading edge voltage (LEV), leading edge current (LEI), and total energy (TE). In another 12 pigs, triplicate defibrillation thresholds (DFTs) and ULVs were determined for each heart state. The T wave was scanned with shocks in 10-msec steps for determining the ULV. Compression resulted in decreased ED50s for LEV (delta = 138 +/- 77 V, P < 0.05, mean +/- SD), LEI (delta = 1.57 +/- 0.7 A, P < 0.05), and TE (delta = 4.9 +/- 3.6 J, P < 0.05) compared to normal. In the second study, compression significantly reduced DFT (P < 0.02) and ULV (P < 0.02) for LEV, LEI, and TE compared to normal. The ULV tended to be lower than the DFT for the normal heart state (delta = 23 +/- 46 V LEV: P = NS). However, the ULV was significantly greater than the DFT for the compressed heart state (delta = 19 +/- 25 V LEV; P < 0.03). CONCLUSIONS: Shock delivery during cardiac compression improves defibrillation efficacy. Additionally, cardiac compression decreases both DFT and ULV, which supports the ULV hypothesis of defibrillation. Finally, maintaining the heart's geometric and volumetric state during ULV testing in paced rhythm and DFT testing in ventricular fibrillation moves the ULV higher than the DFT-the position predicted by the ULV hypothesis for defibrillation. PMID- 7551308 TI - Tachycardia-induced cardiomyopathy in a cardiac transplant recipient: treatment with radiofrequency catheter ablation. AB - INTRODUCTION: Two years after orthotopic cardiac transplantation, a 60-year-old man presented with unexplained congestive heart failure and an incessant atrial tachycardia. METHODS AND RESULTS: Electrophysiologic evaluation identified the underlying arrhythmia as automatic atrial tachycardia with site of origin at the high anterior lateral right atrial wall. Radiofrequency catheter ablation successfully eliminated the tachycardia, which resulted in prompt improvement of this patient's congestive heart failure. CONCLUSION: This is the first reported case of tachycardia-induced cardiomyopathy in a cardiac transplant patient. Radiofrequency catheter ablation can be used successfully in this patient population. PMID- 7551309 TI - Spontaneous echo contrast following delivery of a shock from a transvenous implantable cardioverter defibrillator. AB - Spontaneous echo contrast has never been described in association with cardiac defibrillation. In this report, we present a patient who developed dense echo contrast as a result of a shock delivered from a transvenous defibrillator system. PMID- 7551310 TI - AV nodal-His-Purkinje reentry: a novel form of tachycardia. AB - INTRODUCTION: Bundle branch reentry (BBR) typically occurs in patients with dilated cardiomyopathy and infra-Hisian conduction system disease. The macroreentrant circuit of BBR is confined to the His-Purkinje system (HPS) and ventricular myocardium. As such, the atrioventricular (AV) node plays no role in the tachycardia circuit. METHODS AND RESULTS: In the present study, we identified a novel form of wide complex tachycardia in a patient with coronary disease and severe aortic regurgitation. The tachycardia morphology was right bundle branch block with a left superior axis. Ventriculoatrial block was present during tachycardia. An unusual feature of this rhythm was two sequential His-bundle deflections (H and H') for each ventricular beat of tachycardia. The H'V interval was identical to the HV interval during supraventricular rhythm. Changes in the ventricular cycle length (VV) preceded changes in the HH interval, consistent with retrograde activation of the first His-bundle deflection. Changes in the H'H' interval preceded changes in the VV interval, consistent with anterograde activation of the second His-bundle deflection. Tachycardia could be terminated with ventricular extrastimuli that did not capture the proximal HPS as well as with ventricular extrastimuli that advanced the His deflection, consistent with block in the HPS and in the AV node, respectively. Reproducible termination of the tachycardia following the first His deflection was demonstrated with adenosine, consistent with an upper pivot in the AV node. CONCLUSIONS: We have identified a new form of reentrant tachycardia in which the AV node, HPS, and ventricular myocardium each obligatorily participates in the tachycardia circuit, with the left posterior fascicle and right bundle functioning as the anterograde and retrograde limbs, respectively. Unlike BBR, however, the His bundle is activated twice as the wavefront pivots in the AV node. This model requires longitudinal dissociation at the levels of the AV node and His bundle. PMID- 7551311 TI - Electrocardiographic and clinical precursors of ventricular fibrillation: chain of events. AB - Ventricular fibrillation is the final event in the majority of cases of sudden death. The ECG and clinical precursors of ventricular fibrillation are discussed in this article. Ventricular fibrillation usually appears as a consequence of a chain of events that leads to the appearance of this lethal arrhythmia. We review the markers of the vulnerable myocardium prone to ventricular fibrillation, the triggers and modulators that act on this vulnerable myocardium, and the event(s) that constitute the final step preceding this arrhythmia. The final step may be as unique as a sudden waterfall or present as a cascade of successive phenomena. PMID- 7551312 TI - Nonlinear analysis of epicardial atrial electrograms of electrically induced atrial fibrillation in man. AB - INTRODUCTION: We applied methods from the theory of nonlinear dynamics to characterize unipolar epicardial right atrial electrograms of electrically induced atrial fibrillation (AF) in man. METHODS AND RESULTS: Electrograms were selected from a high-density mapping study, which confirmed the existence of at least 3 different types of induced AF (types I, II, and III) in patients undergoing open chest surgery. We analyzed sets of 5 electrograms (4 sec, sampling frequency 1 kHz, resolution 8 bits) in 9 patients (AF type I, n = 3; type II, n = 3; type III, n = 3). The Grassberger-Procaccia method was applied to estimate the correlation dimension and correlation entropy from the electrograms. In 2 patients (AF type I) some electrograms (2 of 5 and 3 of 5, respectively) showed scaling at normalized distances ranging from 0.2 to 0.5 in phase space. Correlation dimension D ranged from 1.8 to 3.2 and correlation entropy K from 2.2 to 3.8 nats/sec. The patients were ranked according to increasing coarse-grained correlation dimension Dcg (range 3.7 to 7.9) and coarse-grained correlation entropy Kcg (range 5.6 to 18.6 nats/sec). The method of surrogate data was applied to detect nonlinearity in the electrograms. Using the correlation integral as test statistic, it could be excluded that electrograms of type I AF have been generated by linear stochastic dynamics. Episodes of sinus rhythm (D ranging from 1.0 to 5.1 and K from 2.0 to 8.6 nats/sec) and induced atrial flutter (D ranging from 2.7 to 4.2 and K from 2.2 to 4.2 nats/sec) in 2 different patients showed features of low-dimensional chaos. CONCLUSION: Nonlinear analysis discriminated between electrograms during electrically induced AF in humans. The results are consistent with a classification of AF into 3 types based on the spatiotemporal complexity of right atrial activation patterns. PMID- 7551313 TI - Distinguishing cardiac randomness from chaos. PMID- 7551314 TI - In vivo and in vitro electrophysiologic effects of terodiline on dog myocardium. AB - INTRODUCTION: Terodiline hydrochloride, widely prescribed for urinary incontinence, has been reported to cause bradycardia and torsades de pointes. METHODS AND RESULTS: In this study, we characterized the electrophysiologic effects of terodiline in dog cardiac tissues in vivo and in isolated canine cardiac Purkinje fibers. Terodiline (1 to 10 microM) resulted in dose-dependent reduction of action potential amplitude and maximal upstroke velocity (Vmax). The threshold for these effects was approximately 2 microM (0.6 mg/L), and the changes were cycle-length dependent. Terodiline (> or = 2 microM) also depressed the action potential plateau but did not significantly alter action potential duration at concentrations < or = 10 microM. In vivo studies demonstrated that high doses of terodiline (3 mg/kg) lengthened AH and HV intervals, slowed spontaneous sinus rate, prolonged ventricular refractoriness, and inhibited vagally induced slowing of the sinus node. Sympathetic effects on spontaneous sinus rate were unchanged. In both isolated canine Purkinje fibers and anesthetized dogs, terodiline did not evoke afterdepolarizations, repetitive firing, or ventricular tachyarrhythmias under normal or hypokalemic conditions. CONCLUSION: Our findings suggest that terodiline (> or = 1 to 2 microM) leads to blockade of sodium and calcium channels as well as muscarinic receptors in canine cardiac tissues. PMID- 7551315 TI - Na-Ca exchange tail current indicates voltage dependence of the Cai transient in rabbit ventricular myocytes. AB - INTRODUCTION: In mammalian cardiac myocytes, a rise of intracellular calcium (Cai) is well known to activate Ca extrusion via forward Na-Ca exchange, which generates an inward membrane current. This can be observed as an inward "tail" current (INa-Ca) when the membrane is repolarized after a depolarization activated rise of Cai. If, during a voltage step, the membrane is repolarized at the time of the peak of the Cai transient, the size of the INa-Ca tail might be expected to reflect the magnitude of the Cai transient. Therefore, it might be possible to estimate the amplitude and voltage dependence of the Cai transient without, for instance, using fluorescent indicators that can interfere with Cai regulation. The first aim of this study was to use INa-Ca tails to investigate the voltage dependence of the Cai transient in whole cell patch clamped rabbit ventricular myocytes dialyzed with a "normal" level of internal Na. The second aim was to investigate how the voltage dependence of the INa-Ca tails varied with changes to the dialyzing Na concentration. The third aim was to test the correlation of voltage dependence of INa-Ca tails with the voltage dependence of the Cai transient obtained using a fluorescent Ca indicator. METHODS AND RESULTS: Experiments were performed at 35 degrees to 37 degrees C using whole cell patch clamp, and the holding potential was set at -40 mV. Depolarization elicited a Cai transient that peaked in 40 to 50 msec. We reasoned, therefore, that membrane repolarization after 50 msec would cause the raised level of Cai to activate an inward current on forward Na-Ca exchange. The amplitude of INa-Ca measured shortly (10 msec) after repolarization should reflect the peak amplitude of the Cai transient elicited by the depolarization. In cells dialyzed with 10 mM Na containing solution and depolarized for 50 msec to differing test potentials, the INa-Ca tail on repolarization increased progressively after pulses to between -40 and +20 mV. The INa-Ca tail was maximal after a +20-mV pulse and showed no decline after depolarizations to more positive potentials, up to +100 mV (P > 0.1; n = 8). This implies that the Cai transient has a similar amplitude for depolarizing pulses between +20 and +100 mV. When Na-free solution dialyzed the cell, the voltage dependence of the INa-Ca tail became bell-shaped, with a maximum at +20 mV (n = 4). Voltage dependence of the INa-Ca tail was little affected by raising dialyzing Na from 10 to 20 mM (n = 4); but the amplitude of the INa-Ca tail increased. Inhibition of the Na-K pump with strophanthidin in cells dialyzed with 10 mM Na had qualitatively similar effects to increasing dialyzing Na. In Fura-2 loaded cells dialyzed with 10 mM Na, the Cai transient exhibited a similar voltage dependence to the INa-Ca tail (n = 6). CONCLUSION: The results of this study suggest that in cells dialyzed with 10 mM Na, the voltage dependence of the Cai transient is different from the L-type Ca current, since this current declines at potentials > +20 mV. The results obtained using Fura-2 suggest that the INa-Ca tail current measurement tracked the Cai sufficiently well to reflect the voltage dependence of the Cai transient. The data also confirm that the voltage dependence of the Cai transient in rabbit cells can be modulated by altering dialyzing Na concentration. PMID- 7551316 TI - Alteration of ventricular fibrillation by propranolol and isoproterenol detected by epicardial mapping with 506 electrodes. AB - INTRODUCTION: We hypothesized that drugs which alter ventricular refractoriness or excitability produce quantifiable changes in ventricular fibrillation. METHODS AND RESULTS: We used a 528-channel mapping system to quantify the effects of the beta-antagonist, propranolol, and the beta-agonist, isoproterenol, on activation patterns in ventricular fibrillation. A plaque of 506 (22 x 23) electrodes spaced 1.12 mm apart and covering about 5% of the ventricular epicardium was sewn to the anterior right ventricle in 18 pigs (30 kg). Propranolol (0.25 to 0.4 mg/kg) increased the refractory period at a right ventricular epicardial site while isoproterenol (3 to 5 micrograms/min) shortened it. Ventricular fibrillation was induced by programmed stimulation, and unipolar electrograms were recorded from the 506 plaque electrodes for 2 seconds beginning 1, 15, and 30 seconds after the onset of fibrillation. Active epicardial recording sites were identified from the first derivative of the unipolar potentials (dV/dt) detected at each electrode. Then, neighboring active sites were grouped into activation fronts by computer analysis. In six pigs the effect of repeated inductions of ventricular fibrillation was assessed by comparing ventricular fibrillation after saline with a preceding control episode of fibrillation. Each activation front excited 40% +/ 46% of the mapped region before blocking. No changes were observed with saline and multiple inductions of fibrillation. In another six pigs, ventricular fibrillation after propranolol was compared with a preceding control episode of fibrillation. Ventricular fibrillation after propranolol exhibited a decreased activation rate per epicardial recording site and fewer activation fronts per second. There was no change in the amount of tissue excited by each activation front or the number of reentry cycles per activation front compared with control. In addition, there was no change in the maximum negative dV/dt detected per activation at an epicardial site. In six pigs ventricular fibrillation during isoproterenol was compared with control episodes of ventricular fibrillation before and 45 minutes after washout of the drug. The control episodes of fibrillation were not different from each other. Compared with control, ventricular fibrillation during isoproterenol exhibited an increased activation rate per epicardial site, an increased amount of tissue excited by each activation front, and an increased maximum negative dV/dt for each activation. There was no change in the number of activation fronts per second or the number of reentry cycles per activation front compared with control. CONCLUSION: Quantitative analysis revealed that propranolol and isoproterenol do not have symmetrically opposite effects on ventricular fibrillation. Propranolol decreased the number of activation fronts while isoproterenol increased the amount of tissue excited by each activation front. Thus, drugs that alter ventricular refractoriness or excitability alter ventricular fibrillation. PMID- 7551317 TI - Cure of interfascicular reentrant ventricular tachycardia by ablation of the anterior fascicle of the left bundle branch. AB - INTRODUCTION: Fascicular reentrant ventricular tachycardia (VT) using the anterior fascicle of the left bundle anterogradely is rare and may produce identical QRS morphology during sinus rhythm and VT. Catheter ablation of this type of VT has not been described in detail. METHODS AND RESULTS: In a postinfarct patient with dilated left ventricle and recurrent VT (showing a QRS configuration of right bundle branch, left posterior fascicular block), endocardial recordings from the His-Purkinje system showed that VT was due to interfascicular reentry. Induction of VT occurred after progressive retrograde conduction delay on increasing the prematurity of the extrastimulus. Anterograde conduction occurred exclusively over the left anterior fascicle, which caused identical QRS morphology during sinus rhythm and VT. During VT, the left posterior fascicle was used retrogradely. The usual target for bundle branch reentry ablation, the right bundle, did not participate in the reentrant circuit. While performing left ventricular endocardial mapping, VT was interrupted when positioning the catheter on the left anterior fascicle, and "reversed" nonsustained bundle branch reentry occurred with anterograde conduction over the posterior fascicle and retrograde conduction over the anterior fascicle. Ablation of conduction in the anterior fascicle led to cure of the VT. CONCLUSION: Interfascicular reentrant VT with right bundle branch block, right-axis QRS configuration can be cured by catheter ablation of anterior fascicle conduction. PMID- 7551318 TI - Regular wide QRS complex tachycardia during atrial fibrillation in a patient with preexcitation syndrome: a case report. AB - We report an unusual case of a relatively regular wide QRS complex tachycardia alternating with periods of an irregular narrow QRS complex tachycardia during atrial fibrillation in a patient with Wolff-Parkinson-White syndrome. Both tachycardias resulted from atrial fibrillation, the wide QRS complex tachycardia being due to 2:1 AV conduction of a type I atrial fibrillation across a posteroseptal accessory AV connection. PMID- 7551319 TI - The molecular basis of anisotropy: role of gap junctions. AB - Electrical activation of the heart requires transfer of current from one discrete cardiac myocyte to another, a process that occurs at gap junctions. Recent advances in knowledge have established that, like most differentiated cells, individual cardiac myocytes express multiple gap junction channel proteins that are members of a multigene family of channel proteins called connexins. These proteins form channels with unique biophysical properties. Furthermore, functionally distinct cardiac tissues such as the nodes and bundles of the conduction system and atrial and ventricular muscle express different combinations of connexins. Myocytes in these tissues are interconnected by gap junctions that differ in tissue-specific manner in terms of their number, size, and three-dimensional distribution. These observations suggest that both molecular and structural aspects of gap junctions are critical determinants of the anisotropic conduction properties of different cardiac tissues. Expression of multiple connexins also creates the possibility that "hybrid" channels composed of more than one connexin protein type can form, thus greatly increasing the potential for fine control of intercellular ion flow and communication within the heart. PMID- 7551320 TI - Management of sterile necrosis in instances of severe acute pancreatitis. AB - BACKGROUND: The clinical management of sterile pancreatic necrosis is still a matter of debate. In this study we analyzed the clinical course and outcome of patients with sterile necrotizing pancreatitis treated surgically versus nonsurgically. STUDY DESIGN: Between May 1982 and December 1993, 249 patients with necrotizing pancreatitis (NP) entered this study, of which 172 (69 percent) had intraoperatively or fine needle aspiration-proven sterile NP. One hundred seven of 172 patients underwent surgery (S group) with necrosectomy and continuous postoperative closed lavage and 65 of 172 were treated by nonsurgical means (NS group). RESULTS: Median Ranson and admission APACHE II scores were 4.7 (range, 1 to 10) and 11 (range, 1 to 29) in the S group, significantly higher than those in the NS group with 3.0 (range, 0 to 6) (p = 0.022) and 8 (range, 1 to 23) (p = 0.036). After 48 hours of intensive care treatment, APACHE II scores persisted at 10.5 (range, 1 to 29) in the S group and decreased to 6 (range, 0 to 15) (p = 0.013) in the NS patients. Median C-reactive protein (CRP) levels on admission were 179 mg/L and 68.5 mg/L (p = 0.023), respectively. Within 72 hours, 61 (94 percent) of 65 NS-managed patients responded to intensive care therapy, whereas organ complications persisted or increased and thus led to surgery in the S group. Mortality rates were 13.1 percent in the surgically treated patients and 6.2 percent in the nonsurgically treated patients (p = NS). CONCLUSIONS: Most patients with limited and sterile pancreatic necrosis respond to intensive care treatment. Indication for surgery in sterile NP should be based on persisting or advancing organ complications despite intensive care therapy. APACHE II scores and admission CRP levels represent a helpful tool in decision making for surgical or nonsurgical management of NP. PMID- 7551321 TI - Postburn cardiac contractile function and biochemical markers of postburn cardiac injury. AB - BACKGROUND: In vivo assessment of cardiac injury and contractile deficits after thermal injury remain difficult as neurohumoral compensatory mechanisms maintain cardiac output. While measurement of creatine kinase (CK) and the isoenzyme of creatine kinase (CKMB) have been used as clinical indicators of cardiac injury, these biochemical markers are not completely specific for cardiac muscle. Cardiac protein troponin I (cTnI) is unique to the myocardium but can be detected in the systemic circulation within three to four hours after cardiac injury. The purpose of this study was to examine cardiac contractile function at several postburn intervals and to correlate the appearance of cardiac dysfunction with biochemical measures of cardiac injury (serum concentration of CK, CKMB, and cTnI). STUDY DESIGN: New Zealand white rabbits were deeply anesthetized and a scald burn comprising 34 percent of the total body surface area (n = 36) or sham burn (n = 36) was accomplished using a template device. All burn rabbits were given lactated Ringer's solution (4 mL/kg/percent burn, Parkland formula). Blood samples were collected immediately prior to sacrifice in six animals from both burn and control groups, and animals were sacrificed either two, four, six or 24 hours after burn. Cardiac function was assessed in left ventricular preparations (Langendorff) and serum CK, CKMB, and cTnI levels were determined. RESULTS: Cardiac dysfunction occurred at all times after burn as indicated by a lower peak systolic left ventricular pressure and +/- dP/dt maximum compared with time matched shams and the shift of left ventricular function curves plotted for burn groups downward and to the right of those calculated for shams, p < 0.05. Left ventricular systolic dysfunction after burn correlated with a progressive rise in cTnI and CK but not CKMB. CONCLUSIONS: The cardiospecificity of the cTnI eliminates concerns about tissue source associated with CK and CKMB and provides a biochemical measure of cardiac injury that is consistent with in vitro assessment of cardiac dysfunction. PMID- 7551322 TI - Hemodynamic alterations during massive incisional hernioplasty. AB - BACKGROUND: The operative treatment of a large abdominal incisional hernia increases intra-abdominal pressure (IAP). This study was done to verify if this IAP elevation acts on the cardiocirculatory function. STUDY DESIGN: Hemodynamic measurements were performed in five patients who underwent massive incisional hernioplasty before and after abdominal wall closure. RESULTS: Reduction of a large abdominal hernia increases (+226 percent) IAP, which can produce serious hemodynamic alterations, manifested in two patients by a fall of cardiac output ( 30 percent), without significant variations of heart rate and arterial pressure. Cardiac output is decreased secondary to decreased venous return, despite the increase in measured central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP). CONCLUSIONS: During massive incisional hernioplasty, CVP cannot be used as an indicator of venous return to the heart as it reflects a composite of venous filling pressure, pleural pressure, and transmitted IAP. Transmural CVP and PCWP, and not directly measured CVP and PCWP, should be used as clinical indicators of venous return to the heart in this situation. PMID- 7551323 TI - Phase I-II randomized study on prehepatectomy recombinant interleukin-2 immunotherapy in patients with metastatic carcinoma of the colon and rectum. AB - BACKGROUND: This study, which investigates prehepatectomy immunostimulation with recombinant interleukin-2 (rIL-2), had two goals: to evaluate the tolerance of rIL-2 in association with major hepatectomy, and to verify whether preoperative immunostimulation is effective (neoadjuvant immunotherapy). In animal experiments, the conjunction of shed tumor cells into the circulation during surgery and severe surgery-induced immunodepression can promote the dissemination of the disease. Perioperative immunostimulation by rIL-2 reduces the incidence of metastases in animals. STUDY DESIGN: This prospective phase I-II randomized study included 19 patients with potentially resectable hepatic metastases from adenocarcinomas of the colon and rectum. Two-thirds were randomized to the rIL-2 treated group and one-third to the control group. Preoperative rIL-2 was administered by continuous intravenous infusion over five days and stopped two days before major hepatectomy. The dose of rIL-2 was gradually increased, from 3 x 10(6) IU/m2/day to 12 x 10(6) IU/m2/day. At least three patients were studied at each dose level before increasing the rIL-2 dose. Nineteen patients were eligible for the study (12 in the rIL-2 group and seven in the control group). Toxicity during infusion and intraoperative and postoperative complications were evaluated. Immunological monitoring consisted of repeated determination of lymphocyte counts and phenotypic analysis of lymphocyte subpopulations. Non-major histocompatibility complex-restricted cytotoxicity was assayed against K562 and DAUDI tumor cell lines. RESULTS: Toxicity during rIL-2 infusion was acceptable, similar to that of other phase I studies, and surgery was never delayed. Morbidity and mortality rates after major hepatectomy were not different between the two groups and it appeared that prehepatectomy rIL-2 at a dose of 12 x 10(6) IU/m2/day was well tolerated. During the postoperative course, the mean lymphocyte count was higher in the rIL-2 group (p < 0.05), without qualitative modification of the lymphocyte subsets. Immunological modifications were dose related. High cytotoxicity against K562 and DAUDI cells was constant at the 12 x 10(6) dose level. CONCLUSIONS: Preoperative five-day infusion of rIL-2 before major hepatectomy for colorectal metastases is well tolerated and reverses postoperative immunodepression. PMID- 7551324 TI - A new technique for pancreaticogastrointestinal anastomosis without suturing the pancreatic parenchyma. AB - BACKGROUND: In an attempt to lessen the incidence of pancreatic fistula and the disruption of pancreatic anastomosis after pancreatoduodenectomy, we have developed a new technique for pancreaticogastrointestinal anastomosis that consists of pancreatectomy using the ultrasonic dissector and implantation of the pancreatic duct into the gastrointestinal tract without suturing the pancreatic parenchyma. The purpose of this study is to evaluate the safety and reliability of this new technique in a canine model of pancreaticogastrostomy and pancreaticoduodenostomy using 10 beagle dogs. STUDY DESIGN: Canine pancreas was resected using the ultrasonic dissector. In the distal pancreas, a 1-cm long stump of the main pancreatic duct was freed and the other smaller pancreatic ducts were skeletonized and securely ligated. The main pancreatic duct was implanted into the stomach or the duodenum and fixed to the seromuscular layer with purse-string sutures without suturing the pancreatic parenchyma. RESULTS: There was no anastomotic leakage, signs of peritonitis, or abscess formation, and the pancreas was grossly normal in appearance seven days after operation. Histologic examination of the specimens harvested 30 days after operation revealed good connective tissue union between the pancreas and the gastric or duodenal wall, and good mucosal continuity between the pancreatic duct and the stomach or duodenum. CONCLUSIONS: This new technique is simple, safe, and reliable, and is recommended as an alternative method for restoring pancreaticogastrointestinal continuity after pancreatoduodenectomy. PMID- 7551326 TI - Study of rat liver transplantation from non-heartbeating cadaver donors. AB - BACKGROUND: The aim of this study was to determine if livers could tolerate prolonged warm ischemia and to determine optimum timing of intervention in the cadaver donor to preserve liver viability. Livers harvested from non-heartbeating donors (NHBDs) would greatly expand the donor pool for transplantation. However, sensitivity of the donor liver to warm ischemia is a major obstacle for successful use of livers from NHBDs. The limit of non-heartbeating time in the donor remains unknown. STUDY DESIGN: Hepatic transplantation was performed in ten groups of rats with stepwise increase of cardiac arrest time in the donor from zero to 180 minutes with 20-minute intervals. The cardiac arrest time was counted from ligation of the base of the donor's heart to the beginning of cold flushing of the liver. Bile flow, bile acid outputs, hepatic functions, and rat survival rates in each group were compared. RESULTS: Survival rates from groups 1 to 10 were 83.3, 100, 83.3, 50, 66.7, 50, 16.7, 16.7, zero, and zero percent, respectively. The stepwise increase of non-heartbeating time significantly reduced recipient survival rates after 100 minutes. In addition, the increase caused a stepwise decrease of bile flow rates and bile salt outputs and an increase of serum aspartate aminotransferase and lactate dehydrogenase activities. CONCLUSIONS: The liver is less sensitive to warm ischemia than formerly believed. Rat livers can be used for transplantation after cardiac arrest up to 140 minutes with a chance of survival. PMID- 7551325 TI - Malpractice litigation involving patients with carcinoma of the breast. AB - BACKGROUND: We sought to evaluate recent trends in the United States of America regarding malpractice awards for patients with carcinoma of the breast. STUDY DESIGN: A retrospective review was performed of 118 cases of purported malpractice in the diagnosis and management of patients with carcinoma of the breast and related problems. The information was tabulated from Westlaw Transmission, a computerized database. RESULTS: Gynecologists were the specialists most often sued and accounted for 47 percent of the physicians involved in lawsuits. Radiologists were cited in only 13 percent of the cases. Health maintenance organizations (HMOs) were cited in 5 percent of the cases. The most common complaint was delay in diagnosis, made by a plaintiff who detected her own breast mass (52 percent). In 15 percent of the cases, the plaintiffs complained that a mammogram was not obtained, and 9 percent complained that other diagnostic tests, such as ultrasound or fine-needle aspiration biopsy, were not performed. The average delay in diagnosis was 14 months. The average award to plaintiffs with carcinoma of the breast was $691,449. The average plaintiff's age was 44 years. CONCLUSIONS: Most malpractice complaints related to carcinoma of the breast are instituted by women under the age of 50 years who identified the breast mass by themselves and were assumed by their physicians to have fibrocystic disease of the breast. Complaints can be expected to increase regarding failure to order further diagnostic tests, such as ultrasound or fine needle aspiration biopsy, despite a negative mammogram. Complaints against HMOs are now also being made, citing failure to properly diagnose or treat patients with carcinoma of the breast. PMID- 7551328 TI - Total mesorectal excision in the operative treatment of carcinoma of the rectum. AB - BACKGROUND: Total mesorectal excision (TME) is reported to reduce local recurrence and improve survival rates in patients with carcinoma of the rectum. STUDY DESIGN: Two hundred forty-six consecutive patients with Dukes' B (T3, N0, M0) and C (T(any), N1-2, M0) primary rectal carcinomas underwent operation according to the principle of TME. Kaplan-Meier estimates of survival and pelvic recurrence rates were calculated from a database of patients followed prospectively. RESULTS: The operative mortality rate was 0.8 percent (two of 246). The Kaplan-Meier five-year survival rate for patients with stages B and C was 74.2 percent; for patients with stage T3, N0, M0, 86.7 percent; for patients with stage T(any), N1-2, M0, 64.0 percent; and for patients with substage T3, N1 2, M0, 68.0 percent. Pelvic recurrences were observed in a total of 18 patients (7.3 percent) with or without metastases. In the 246 patients with Dukes' stages B and C, pelvic recurrence rates were 4.0 and 8.1 percent, respectively, in the presence or absence of distant metastases, and 3.0 and 5.8 percent, respectively, in the absence of distant spread. Statistically significant risk factors for pelvic recurrence were N2 disease and perineural invasion. Adjuvant radiation therapy was of no statistical benefit in preventing local recurrences. CONCLUSIONS: Total mesorectal excision cures carcinoma of the rectum and provides excellent local control through resection of the entire unit of regional spread that is excised, intact and with negative circumferential margins. Total mesorectal excision is compatible with autonomic nerve preservation and with sphincter preservation. The current role of combined modality adjuvant therapy, which is standard therapy following conventional surgery, should be reconsidered in patients who have undergone resection in accordance with TME. PMID- 7551327 TI - Role of P-selectin in total hepatic ischemia and reperfusion. AB - BACKGROUND: Ischemia and reperfusion of the liver are associated with changes in the interaction of leukocyte-endothelium cells. The role of an adhesion molecule, P-selectin, is studied in ischemia and reperfusion injury of the liver. STUDY DESIGN: Total hepatic ischemia was produced in the rat for 90 minutes, using a portosystemic shunt. To determine the role of P-selectin in ischemia and reperfusion, a murine IgG1 monoclonal antibody to P-selectin (1 mg/kg) was used at different times (30 minutes before and at reperfusion and five minutes and 24 hours after reperfusion). Rats survived for seven days, and tests showing hepatic injury, myeloperoxidase in hepatic tissue, and histologic studies were analyzed at four hours postreperfusion. RESULTS: Survival improved from 15 percent for the rats in the ischemia control group to 55 percent for those in the group receiving anti-P-selectin antibody given 30 minutes before reperfusion (p < 0.05). We observed an improved statistically significant difference in tests demonstrating hepatic injury, myeloperoxidase in hepatic tissue, and histologic studies in the treated and ischemia control groups. The other groups did not show consistent significant differences. CONCLUSIONS: P-selectin has a significant role in ischemia and reperfusion injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreased neutrophil adhesion and migration and consequently diminished damage to the liver. PMID- 7551329 TI - Laparoscopic control of short gastric vessels. AB - BACKGROUND: The Nissen fundoplication is currently the most commonly performed antireflux surgery whether performed as an open procedure or through the laparoscope. Extensive experience with open Nissen fundoplication has shown that dividing the short gastric vessels to mobilize the fundus ensures that the wrap will be loose and without tension. The standard laparoscopic technique for fundal mobilization is dissecting out the short gastric vessels, applying hemoclips, and then dividing them. For surgeons new to laparoscopic surgery, this can be an intimidating task. Introduction of the ultrasonic coagulating shears, a new energy source technology, offered the possibility of making this process quicker and easier. STUDY DESIGN: We present a randomized prospective study comparing two methods of ligating the short gastric vessels during laparoscopic fundoplication. Thirty-one patients were enrolled and randomized into two groups: those who underwent short gastric ligation by dissection, clipping, and dividing, and those in whom the ultrasonic laparoscopic coagulating shears were used. The results were tabulated and subjected to statistical analysis. RESULTS: Fifteen patients had laparoscopic fundoplication with ligation of the short gastric vessels by using clips and 16 by using the ultrasonic coagulating shears. There was no demographic difference between the two groups and the number of short gastric vessels ligated was the same in both groups. Significance was seen for the median operating time for short gastric control (22 minutes for clipping versus 12 minutes for coagulating shears), median blood loss (70 mL for clipping versus 2.5 mL for the ultrasonic coagulating shears), and technical difficulty (93 percent with clipping versus 19 percent with the coagulating shears). CONCLUSIONS: Based on the results from this randomized prospective study, the ultrasonic coagulating shears are easier to use and less prone to intraoperative "complications" and postoperative morbidity. We believe that this new technology will make it easier and more desirable for surgeons to mobilize the gastric fundus during laparoscopic fundoplication. PMID- 7551330 TI - Eugene Polya: the Billroth of Budapest. PMID- 7551331 TI - Sterile pancreatic necrosis: is operation necessary? PMID- 7551332 TI - A hemi-double stapling method to create the Billroth-I anastomosis using a detachable device. PMID- 7551333 TI - Central venous catheter change over a guide wire: a simple technique. PMID- 7551334 TI - Current views on the pathogenesis of abdominal aortic aneurysms. PMID- 7551335 TI - The optimal evaluation and treatment of feeding disorders in children with neurological disorders. PMID- 7551336 TI - Emergency shunt for variceal bleeding. PMID- 7551338 TI - Ploidy analysis on Wilms' tumour touch imprints using ethidium bromide and automated image analysis integrated confocal laser scanning microscopy. AB - Although image analysis (IA) is increasingly being used to quantitate nuclear DNA, comparative data between fluorescence methods of IA and flow cytometry (FCM) is limited. In this study fluorescence IA was compared with FCM data in a series of Wilms' tumour touch preparations. Airdried touch imprints that had previously been Giemsa stained were restained with ethidium bromide. Confocal fluorescence images were obtained with a confocal laser scanning microscope and assessed by a fully automated IA package. Data was collected from 400 nuclei per imprint. The resulting DNA histograms were analysed and ploidy status and DNA indices determined using standard criteria. Results were compared with those derived from FCM analysis of nuclear suspensions. Ten of twelve tumours were concordant by both techniques. However in two cases assessed as diploid by FCM, IA identified aneuploidy. Excellent correlation between DNA indices as assessed by both techniques was observed (r = 0.987). In the three cases for which both unstained and Giemsa stained touch imprints were available for IA, the histogram configurations did not differ significantly. Fluorescence IA is an accurate and sensitive technique for DNA quantitation, which appears at least comparable to FCM assessment and which has a number of important advantages. PMID- 7551340 TI - A rare case of pigmented paraganglioma. PMID- 7551339 TI - Anaplastic large cell Ki-1 lymphoma with bone involvement: report of two cases. AB - Two cases of anaplastic large cell Ki-1 lymphoma involving bone as the most prominent and initial manifestation are reported. The first patient was a 20-year old male who had back pain and incomplete paraparesis due to vertebral involvement. The second was a 14-year-old girl, whose first clinical signs were fever of unknown origin and sternal bone pain. Radiologically, skeletal lesions were lytic and destructive. Histopathologically, the tumour cells had pleomorphic bizarre nuclei and abundant basophilic cytoplasm. Immunohistochemically, Ki 1(CD30) reactivity was strongly positive in both cases. Tumour cells were also CD3, CD4, epithelial membrane antigen and interleukin-2 receptor positive in the first case, and CD10, HLA-DR positive in the second case. The former tumour was considered to be of T-cell lineage and the latter of lymphoid progenitor cell origin. Radiation and chemotherapy were temporarily effective. However, both patients died 14 and 7 months after diagnosis, respectively, due to systemic lymph node involvement. These observations suggest that the prognosis for Ki-1 lymphoma involving bone is poorer than indicated in previous reports. PMID- 7551342 TI - Correlation of p53 protein expression with apoptotic incidence in colorectal neoplasia. AB - The wild-type p53 gene suppresses cell proliferation and induces apoptosis when it is transfected into human colon cancer cell lines. Therefore, mutation of the p53 gene, which correlates closely with p53 protein overexpression, would be predicted to activate cell proliferation and limit apoptosis. We tested this hypothesis by correlating p53 protein expression with cell proliferation and apoptosis in 70 neoplasms (29 adenomas and 41 carcinomas) using p53 and Ki-67 immunohistochemical staining and DNA nick end labelling. The p53 immunoreactivity was independent of the Ki-67 positivity. The apoptotic incidence was less frequent (P < 0.005) in tumours with diffuse p53 protein overexpression than in those with the sporadic overexpression, defined as p53 staining of isolated or scattered expression. In addition, apoptotic incidence only correlated directly (P < 0.05) with Ki-67 positivity in tumours with sporadic p53-protein expression. These results indicate that p53 protein that is expressed sporadically in colorectal neoplasms is probably wild-type protein and induces apoptosis in response to active cell proliferation. In contrast, diffusely overexpressed p53 protein in colorectal neoplasms is probably mutant and correlates with a reduction in apoptotic cell death independently of cell proliferation. PMID- 7551341 TI - Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours. AB - A series of 26 malignant peripheral nerve sheath tumours (MPNST) and 24 benign peripheral nerve sheath tumours (BPNST) were analysed immunocytochemically for p53 expression and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 (with MIB1). In 23/26 MPNST, 5%-65% of the tumour cell nuclei were immunoreactive for Ki67 with MIB1 while none of the 24 BPNST had nuclear staining exceeding 5%. Greater than 50% nuclear PCNA staining was detected in 25/26 MPNST compared with 8/24 BPNST; 17/26 MPNST showed 5-100% nuclear staining for p53 (13/26 > 20%), whereas none of the BPNST had nuclear staining exceeding 1%. The Ki67, PCNA and p53 immunostaining results correlated significantly with benign versus malignant (P < 0.001, P < 0.001 and P < 0.005, respectively) as well as mitotic rate (P < 0.001, P < 0.05 and P < 0.05). Ki67 immunostaining results correlated significantly with PCNA and p53, as did p53 and Ki67 and PCNA (P < 0.001 in both). Stepwise (logistic regression forward) multivariate analysis of the variable, benign versus malignant, revealed the strongest correlations with PCNA (P = 0.007) and Ki67 (P = 0.021). Direct confirmation of the presence of p53 protein was obtained by western blot analysis of 3 MPNST and 5 BPNST. Two MPNST, showing 90% and 30% immunoreactivity, were positive for p53, while one MPNST with 5% immunoreactivity and all 5 BPNST were negative. Southern blot analysis performed on the two MPNST with high p53 protein levels revealed no amplification of the MDM2 gene, suggesting that high p53 levels in MPNST are likely to be due to mutation. The results also indicate that PCNA and Ki67 are potentially useful in distinguishing BPNST from MPNST, particularly in problematic cases of cellular schwannoma versus MPNST. The detection of p53 in a large percentage of cells of a plexiform neurofibroma giving rise to MPNST and Ki67 in 5% and 25% of cells of two similar cases suggests that malignant transformation may be detected in some cases by p53 and proliferation markers prior to overt histological evidence of malignancy. PMID- 7551345 TI - Proteoglycans in haemodialysis-related amyloidosis. AB - Changes in extracellular matrices of articular tissue, intervertebral discs and systemic organs in patients with haemodialysis-related amyloidosis were investigated by immunohistochemical and biochemical examination of proteoglycans. Increased staining for chondroitin sulfate (CS) was detected in the amyloid deposits of all patients, ranging from early to advanced stages. Degenerative tissue changes around early-stage amyloid deposits in the intervertebral discs also showed positive staining for CS. Heparan sulfate (HS) was detected in amyloid deposits, especially in the synovial membrane. Biochemical analysis of connective tissues containing amyloid supported the immunohistochemical studies; CS was the major glycosaminoglycan species in these tissues, accounting for 55 81% of the total glycosaminoglycans. Although previous studies have stressed the importance of HS in amyloidogenesis, the present study showed that CS, which increased significantly in articular tissues associated with mechanical stress, also has a close relationship with amyloidogenesis. PMID- 7551346 TI - Nodular fibrosis of the lung in diabetes mellitus. AB - The target organs in diabetes mellitus include the kidneys, the eyes and the small vessels. In these organs some specific histopathological changes have been described but there are few reports of histopathoogical changes in the lung in diabetic patients. Several reports describe abnormal pulmonary function in diabetic patients and consider these abnormalities to be due to histopahtological changes found in the pulmonary vessels. We have studied the histopathological changes in the diabetic lung comparing the findings in the autopsies of 61 diabetic and 50 non-diabetic patients. Statistically significant differences in the incidence of chronic obstructive lung disease and pulmonary haemorrhage exist. There are no differences in the incidence of fibrosis of the alveolar walls or intimal and medial thickening of small vessels, changes associated with diabetes according to the literature. We have found a specific type of nodular fibrosis not previously reported which we believe to be typical of diabetes. PMID- 7551347 TI - Intramural mesenteric venulitis. A new cause of intestinal ischaemia. AB - Venous damage is an uncommon cause of intestinal ischaemia. We report on a 44 year-old woman who presented signs and symptoms of acute intestinal ischaemia requiring surgical treatment. Histological examination of the resected right colon showed features of an intramural lymphocytic venulitis with no other demonstrable causes of ischaemic injury of the bowel. Extramural mesenteric veins appeared dilated and congested, without evidence of thrombotic occlusion or of inflammatory involvement. The patient, who was not taking any long-term medication and had no clinical evidence of collagen-vascular disease, promptly recovered after surgery. Follow-up for 7 months with no recurrences suggested a self-limited or indolent process. We propose the name 'intramural mesenteric venulitis' for this condition and believe that it could represent one extreme (the microscopic variant or intramural phase) of the spectrum comprising entero colic phlebitis and mesenteric inflammatory veno-occlusive disease. The immunohistochemical evidence of a marked preponderance of T phenotype in the perivenular lymphocytes suggests lymphocyte-mediated vascular damage as the pathogenesis of the lesion. PMID- 7551344 TI - Inverse modulation of intraepithelial Langerhans' cells and stromal macrophage/dendrocyte populations in human papillomavirus-associated squamous intraepithelial lesions of the cervix. AB - Ninety-four cervical biopsies from normal tissue to high-grade squamous intraepithelial lesion (SILs) were examined for the presence of intraepithelial Langerhans' cells and subpopulations of stromal macrophages/dendrocytes by immunohistochemistry using anti-S100, -L1, -CD68 and -factor XIIIa antibodies. Human papillomavirus (HPV) detection was performed in all cases by using first a mixture of DNA probes for 14 HPV types commonly found in anogenital biopsies at low stringency conditions (Tm -40 degrees C) and by reanalyzing the tissues at high stringency (Tm -10 degrees C) with HPV 6/11, 16/18 and 31/33/35 biotinylated probe cocktails and individual digoxigenin-labelled probes. SILs and metaplastic tissues were significantly associated with a depletion of S100-positive intraepithelial Langerhans' cells when compared with normal epithelium. In contrast, there was a significant increase in L1-positive stromal macrophages in SIL biopsies compared with normal or metaplastic cervix. A significantly higher density of CD68-positive macrophages was also observed in high-grade SILs compared with normal or metaplastic biopsies and with low-grade SILs. The density of factor XIIIa-positive dendrocytes was found to be higher in SILs compared with metaplastic tissues and in high-grade SILs when compared with normal cervical biopsies. No specific relationship was found between the densities of these cells and the HPV type detected in SILs separated into low grade and high grade. The significance of this inverse modulation of intraepithelial Langerhans' cells and stromal macrophages/dendrocytes in normal and SIL biopsies is discussed in relation to HPV infection and malignant transformation. PMID- 7551343 TI - Prognostic evaluation of oestrogen-regulated protein immunoreactivity in ductal invasive (NOS) breast cancer. AB - Determination of steroid receptors and several oestrogen-regulated proteins in mammary carcinomas is useful in the prediction of their evolution and of the likely success of endocrine therapy. Cathepsin D (Cat D), pS2 peptide and heat shock protein 27 (Hsp 27) were detected immunohistochemically in 63 infiltrating ductal (NOS) breast carcinomas, and our results were qualitatively correlated with several clinicopathological indicators and patients' overall survival. Cat D immunostaining of tumour cells was strongly associated with axillary nodal involvement (Pf = 0.0005) and so, it is directly connected with the metastatic capacity of malignant cells. pS2 immunoreactivity was correlated with oestrogen and progesterone receptor positivity (Pf = 0.0009 and Pf = 0.05 respectively) and, nonsignificantly, with good differentiation of the tumours (Pf = 0.06). Neoplastic cells expressing this protein are therefore characterised by a highly organised state of cellular physiology. Hsp 27 was expressed predominantly in tumours with one to four infiltrated lymph nodes (Pt = 0.05), and Hsp 27-positive patients were inclined to rather short survival, possibly due to chemotherapy resistance. In future, prognostic estimation of each one of the examined markers should be performed in specific large subgroups of patients. The findings of this study contribute to the establishment of criteria by which these subgroups should be formed. PMID- 7551337 TI - Hepatic neoplasia: reflections and ruminations. PMID- 7551349 TI - Ultrastructural changes in the lung in Niemann-Pick type C mouse. AB - The biochemical and morphological aspects of BALB/c mice with many features of the Niemann-Pick disease type C in man (NP-C mouse) have been studied extensively. However, the pulmonary pathology has not been studied extensively and we describe here some unique ultrastructural features of the lung in the NP-C mouse. Ultrastructurally, macrophages in younger mice contained osmiophilic dense granules and annulolamellar structures, but larger multilamellar concentric structures increased in the macrophages of older mice. In contrast, endothelial cells and type I pneumocytes showed membrane-bound bodies with dense granules and vesicular or vesiculogranular structures as well as amorphous materials. Type II pneumocytes were unremarkable throughout. Our study suggests that endothelial cells and type I pneumocytes are the major site of metabolic derangement resulting in pronounced morphological changes with granular and round membranous structures in the lungs of NP-C mouse. Alveolar macrophages with multilamellar concentric structures may be a result of disturbed disposal of surfactant material from type II pneumocytes rather than that from storage material of type I pneumocyte. PMID- 7551352 TI - [Nursing research: luxury or a tool for the nursing process?]. PMID- 7551350 TI - Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis. AB - An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of +/- 1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guerin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence labelled PCR products will presumably lead to the discovery of new restriction enzyme sites. PMID- 7551348 TI - The significance of cytological examination on reperfusion in rat small intestinal transplantation. AB - We examined the cytology of the exudate in preserved intestinal grafts on reperfusion and compared it with the histological findings in rat small intestinal transplantation. The jejunal graft was harvested from the Lewis rat and was preserved in University of Wisconsin solution for 6, 12, 24 and 48 h at 4 degrees C (n = 6, in each group) and was then syngeneically transplanted. On reperfusion, the exudate was collected and studied cytologically. Full thickness biopsies were performed at the end of the preservation and at 30 min after reperfusion for histological examination. Histological examination after reperfusion showed that the crypt layer was preserved until 24 h. However, it was destroyed by 48 h preservation. The cytological findings correlated with the depth of tissue injury shown histologically. The degeneration of villus epithelial cells, the decrease in the content of mucin in both the goblet cells as well as villus cells, and the appearance of crypt cells are all considered to be signs of poor graft viability. Cytological examination is therefore recommended as an effective, non-invasive and real-time method for evaluating graft viability just after reperfusion in small intestinal transplantation. PMID- 7551353 TI - [Patient lifter in a practical test. A spine-saving aid or bulk in the storage room?]. PMID- 7551354 TI - [New collection of information. Everyday habits of patients as a basis for the development of quality of care according to section 80 SGB XI]. PMID- 7551351 TI - Renal cysts and associated renal tumours in male ddY mice injected with ferric nitrilotriacetate. AB - In experiments using ferric nitrilotriacetate (Fe-NTA) as a renal carcinogen, multiple renal cysts are often observed in addition to renal tumours. In the present study, we used 3-week-old male ddY mice and examined the relation between renal cysts and cancer development. Four months after the start of Fe-NTA administration, we observed cysts in the renal cortex in all Fe-NTA-treated mice, but not in Fe-free NTA-treated mice. Three types of cysts were observed, but only those which originated from the renal proximal tubules showed multilayered or papillary growth of cyst epithelial cells. Using histochemical staining, we found a cyst formation-tumour induction sequence, and the supposed cystic-papillary tumour induced by Fe-NTA was of proximal tubular cell origin. We also found that the minimum dose of Fe-NTA capable of inducing renal tumours in ddY mice was 10 mg of iron/kg/day, four times in 2 weeks. PMID- 7551355 TI - [Tired of living. Suicide and suicide proneness in the aged]. PMID- 7551356 TI - [War nursing and the Red Cross movement. Henri Dunant and Florence Nightingale]. PMID- 7551357 TI - [Knock-out in the operating room. The right to retraction]. PMID- 7551360 TI - [Dangerous, adequate or optimal? Quality criteria for ambulatory nursing services]. PMID- 7551359 TI - [Discussion: clinical teaching]. PMID- 7551358 TI - [To Slovenia by way of Essen]. PMID- 7551361 TI - [The hospice movement in North-Rhine-Westphalia]. PMID- 7551363 TI - Rethinking exercise testing in children: a challenge. PMID- 7551362 TI - Guidelines for the initial management of community-acquired pneumonia: savory recipe or cookbook for disaster? PMID- 7551364 TI - Serum house-dust-mite antibodies and reduced FEV1 in adults of a Norwegian community. AB - In this cross-sectional study we investigated whether the presence of specific serum IgE antibodies to house dust mite, timothy, birch, cat, and mold was associated with a reduced FEV1 in adults. We performed complete examinations on 82% of a stratified random sample of 18 to 73-yr-old adults (n = 1,239). Subjects with house-dust-mite antibodies had lower (p = 0.002) sex, age, and height standardized residuals of FEV1 (SFEV1) than those without any specific IgE antibody. This relationship did not differ significantly by sex, age, smoking habit, total serum IgE level, or season, and remained significant after excluding subjects with obstructive lung disease. For house-dust-mite antibodies we also observed a dose-response relationship between antibody levels and impaired lung function. In a final multiple linear regression analysis the presence of house dust-mite antibodies was the only significant predictor (regression coefficient: 0.425; SE = 0.189; p = 0.02) of reduced SFEV1 after adjusting for smoking habit and lifetime tobacco consumption, season, total serum IgE level, and respiratory symptom and disease status. Thus, house-dust-mite allergy is an independent predictor of reduced lung function in adults of a wide age range. PMID- 7551365 TI - Effect of diuretics on allergen-induced contractions of passively sensitized human bronchi in vitro. AB - Inhaled furosemide has been shown to protect subjects with asthma from bronchoconstriction induced by a wide variety of stimuli, including allergen, but the mechanism of action is controversial. We have used an in vitro model of allergen-induced bronchoconstriction to examine the effects of furosemide and other ion transport inhibitors. Human bronchial rings were passively sensitized by incubation with serum from an atopic donor and were challenged with Dermatophagoides pteronyssinus. Allergen-challenged bronchial rings developed bronchoconstriction which was effectively inhibited by the cysteinyl-leukotriene antagonist ICI 198,615 (10(-7) M) and to a lesser extent by terfenadine (10(-5) M). Assessed over 60 min furosemide 10(-6), 10(-5), and 10(-4) M inhibited contractions by a mean (95% confidence interval [CI]) 7.9% (-23.5, 39.3%, p > 0.05), 44.2% (12.9, 75.2%, p < 0.01), and 86.9% (55.5, 118.3%, p < 0.001) respectively (n = 5). The same concentrations of bumetanide inhibited contractions by 21.5% (-8.4, 51.4%, p > 0.05), 13.6% (-16.3, 43.4%, p > 0.05) and 51.6% (21.7, 81.4%, p < 0.01) respectively (n = 5). The sodium transport inhibitor amiloride and the anion transport inhibitor 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) were without effect (both 10(-4) M; n = 4). Furosemide increased PGE2 production by the bronchial rings by 134% (95% CI 53, 259%). Indomethacin (3 x 10(-6) M) blocked the furosemide-induced increase in PGE2 production and reduced the protection afforded by 10(-4) M furosemide against allergen-induced contractions from 67.9% to 34.7% (mean difference 33.2%; 95% CI 9.7, 56.6%; p < 0.01; n = 8).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551366 TI - Regular formoterol treatment in mild asthma. Effect on bronchial responsiveness during and after treatment. AB - Regular beta 2-adrenoceptor agonist therapy may lead to a rebound increase in bronchial responsiveness on discontinuation of therapy and a reduction in bronchoprotective effects. Formoterol, a long-acting beta 2-agonist, is effective in single doses in the prevention of methacholine-induced bronchoconstriction. In a double-blind, placebo-controlled cross-over study, we examined the effect of an inhaled long-acting beta 2-adrenoceptor agonist, formoterol (24 micrograms twice a day) for 2 wk on airway function and responsiveness in 17 subjects with mild asthma (mean age, 26.3 +/- 1.4 yr) who were not taking inhaled glucocorticosteroids. FEV1 and the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) were measured at 36, 60, and 108 h and at 2 wk after the last dose of regular treatment. In addition, PC20 was measured 12 h after the first and the last dose of formoterol and placebo. PC20 values at 36, 60, and 108 h and at 2 wk after formoterol treatment cessation were not significantly different from those after placebo. Mean FEV1 was 3.44 +/- 0.18 L after placebo compared with 3.79 +/- 0.20 L after formoterol (p < 0.001) 12 h after the first dose, and mean PC20 was 0.53 (GSEM 1.4) mg/ml after placebo compared with 2.0 (GSEM 1.4) mg/ml after formoterol (p < 0.001). After 2 wk of regular treatment, mean FEV1 at 12 h after the final dose of formoterol fell to 3.51 +/- 0.23 L compared with 3.41 +/- 0.18 L after the final dose of placebo (p = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551367 TI - Ozone exposure in humans: inflammatory, small and peripheral airway responses. AB - We exposed eight normal adults to filtered air (FA) and 0.35 ppm ozone (O3) and compared responses in spirometry, including isovolume (isoV) flows at intermediate-to-low lung volumes, against levels of inflammatory markers in bronchoalveolar lavage fluid (BALF) and peripheral lung resistance (Rp) measured through a wedged bronchoscope. Spirometry was performed at the end, 25 min and 24 h after exposure, bronchoscopy at 24 h after exposure. The percentages of neutrophils, fibrinogen, albumin, PGE2, PGF2 alpha, and kinins were elevated in BALF after O3 compared with FA. The percentage reduction in (isoV) FEF25-75 at 25 min and 24 h after administration of O3 correlated closely with the rise in fibrinogen concentrations in BALF, a marker of altered vascular permeability. Rp, a measurement dominated by very small or peripheral airways, was unaffected in 7 of 8 subjects. The absence of change in Rp might have reflected insufficient penetration of O3 into these airways to produce or sustain an effect for 24 h; alternatively, the bronchoscopic procedure which included atropine and lidocaine pretreatment may have reversed an O3 effect. An unexpected finding was the significant association between baseline Rp (after FA) and the magnitude of the spirometric response to O3. Our results suggest that small airway dysfunction in the immediate post-O3 period is a marker of lung inflammation. PMID- 7551368 TI - Menopause, postmenopausal estrogen preparations, and the risk of adult-onset asthma. A prospective cohort study. AB - We prospectively evaluated the association of hormone replacement therapy and asthma incidence in a cohort of pre- and postmenopausal women 34 to 68 yr of age. During 582, 135 person-years of follow-up between 1980 and 1990, 726 new cases of asthma were documented. Postmenopausal women who were never users of replacement hormones had a significantly lower age-adjusted risk of asthma than premenopausal women (relative risk = 0.65; 95% confidence interval [CI] = 0.46 to 0.92). Among naturally menopausal women, the age-adjusted relative risk of asthma for ever use of postmenopausal hormones was 1.49 (95% CI = 1.10 to 2.00); for current use of hormones (conjugated estrogens with or without progesterone), 1.50 (95% CI = 0.98 to 2.30); and for past use, 1.52 (95% CI = 1.08 to 2.13), compared with never use of hormones. Ever users of 10 or more years' duration had twice the age-adjusted risk of asthma compared with women who never used postmenopausal hormones (95% CI = 1.39 to 2.87). Among current users of conjugated estrogens, there was a positive dose-response demonstrated between daily dose and asthma risk (p for trend = 0.007). While confirmatory studies are warranted, these data suggest that estrogen plays a role in the pathophysiology of asthma and that long-term use and/or high doses of postmenopausal hormone therapy increase subsequent risk of asthma. PMID- 7551369 TI - Pulmonary inflammation after segmental ragweed challenge in allergic asthmatic and nonasthmatic subjects. AB - Inflammatory reactions in the airways are thought to play an important role in asthma pathogenesis. The goal of this work was to test prospectively the hypothesis that the pulmonary inflammatory response to segmental antigen challenge is greater in allergic asthmatic (AA) subjects than in allergic nonasthmatic (ANA) subjects. A total of 46 ragweed-allergic subjects, 27 AA and 19 ANA, took part in these studies. Subjects had normal or nearly normal pulmonary function, were on no chronic medication, and were characterized as to their skin sensitivity to intradermal ragweed injection, their nonspecific responsiveness to methacholine, and the presence (or absence) of a late asthmatic response after whole-lung antigen challenge. Subjects then underwent bronchoscopy, bronchoalveolar lavage (BAL) of a control lung segment, antigen lung challenge of a contralateral lung segment with 5 ml of a concentration of ragweed solution 100-fold higher than that required to produce a positive skin reaction, and finally, BAL of the challenged segment after 24 h. AA did not differ from ANA in any inflammatory parameter measured in BAL fluid (total cells/ml, macrophages/ml, lymphocytes/ml, eosinophils/ml, neutrophils/ml, total protein, albumin, urea, or eosinophil cationic protein) 24 h after challenge. In addition, there was no relationship between nonspecific bronchial responsiveness to methacholine and eosinophils recruited to the lung by segmental antigen challenge. Rather, in both groups a marked inflammatory response was seen only in the subgroup of subjects who demonstrated a late airway reaction after whole-lung antigen challenge, regardless of disease classification.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551370 TI - Effects of a PAF antagonist, Y-24180, on bronchial hyperresponsiveness in patients with asthma. AB - The involvement of platelet-activating factor (PAF) in bronchial hyperresponsiveness (BHR) in bronchial asthma has been controversial. To determine whether PAF is involved in BHR in humans, we carried out a randomized, double-blind, placebo-controlled, two-phase cross-over study on the effects of Y 24180, a potent, specific, orally active PAF receptor antagonist, on BHR to methacholine in patients with asthma. The subjects were 13 patients with extrinsic stable asthma. The provocative concentration of methacholine producing a 20% fall in FEV1 (PC20-FEV1) was measured as an index of BHR. Y-24180 (20 mg twice a day) or a placebo was orally administered for 2 wk, respectively. At the time of cross-over from the first treatment regimen to the second regimen, administration of the test drug was suspended for 2 wk. The methacholine challenge test was performed four times, before and after the first treatment period and before and after the second treatment period. Compared with the placebo, Y-24180 significantly (p = 0.005) improved the PC20-FEV1 value without carryover effect and period effect by analysis of variance. These results suggest that PAF is an important mediator involved in the BHR of bronchial asthma in humans. PMID- 7551371 TI - Effect of chronic 5-lipoxygenase inhibition on airway hyperresponsiveness in asthmatic subjects. AB - The leukotrienes are known bronchoactive agonists with potential proinflammatory effects that may be involved in mediating airway hyperresponsiveness. We investigated the effects of zileuton, an inhibitor of 5-lipoxygenase (5-LO), on airway responsiveness to cold, dry air in patients with moderate asthma. A group of 10 asthmatic patients underwent cold, dry air hyperventilation challenge; challenges were performed before drug treatment and 1 to 10 d after the completion of treatment with study drugs. The cold air minute ventilation required to cause a 15% decrease in FEV1 (PD15 VE) increased by 58% compared with the response before treatment, 1 to 10 d after the completion of 13 wk of treatment with zileuton. The geometric mean (geometric mean/SEM and geometric mean x SEM) PD15 VE increased from 24.5 (20.4, 29.5) L/min to 38.8 (34.7, 43.7) L/min (p = 0.01). Zileuton treatment inhibited 5-LO as measured ex vivo by ionophore-stimulated LTB4 levels in whole blood. In four of seven subjects, LTB4 levels before zileuton ingestion fell from 110.88 +/- 25.42 to 5.40 +/- 1.95 ng/ml 2 h post-zileuton dosing (p = 0.02, pre- versus 2 h postzileuton ingestion). Consistent with the short half-life of zileuton, 6 h postzileuton dosing the ionophore-stimulated, LTB4 levels in whole blood had increased to 89.68 +/- 35.54 ng/ml (p = 0.41, pre- versus 6 h postzileuton ingestion). Based on the first-order kinetics of zileuton, its effect on 5-LO activity should have been dissipated less than 16 h postingestion. Thus, chronic zileuton treatment decreased airway hyperresponsiveness as determined by reactivity to cold, dry air.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551373 TI - Monocyte responsiveness and a T-cell subtype predict the effects induced by cotton dust exposure. AB - The aim of the study was to evaluate whether peripheral cellular parameters could predict susceptibility to decreased lung function and associated symptoms, in response to a single exposure to cotton dust. Previously nonexposed subjects (n = 42) inhaled an aerosol of cotton dust in a model cardroom during a period of 5 h. The subjects were examined before the exposure for FEV1, procoagulant activity (PCA) in blood mononuclear cells (BMNC), and serum IgE antibodies against a pool of inhalant antigens. Blood lymphocytes were typed into the helper/inducer (CD4+) and cytotoxic/suppressor (CD8+) T cells in combination with surface markers subdividing these populations. A questionnaire was used to identify atopic and nonatopic subjects. Immediately after exposure, the subjects were tested for FEV1 and PCA, and symptoms were recorded with a questionnaire. The dust exposure induced a decrease in FEV1 that was larger for the atopic group, but did not change the PCA in BMNC. The decrease in FEV1 was positively related to the preexposure PCA in both atopics and nonatopics. Symptoms from the airways after the exposure were reported to the same extent in the atopic and nonatopic group, and the subject group reporting chest tightness had a larger preexposure PCA. The atopic group had a larger proportion of blood CD8+ T lymphocytes negative for the monoclonal antibody anti-S6F1 (CD8+S6F1-), and in this group the decrease in FEV1 was significantly related to the proportion of this cell type. Also, in the atopic group, the proportion of CD8+S6F1- cells correlated positively with the preexposure PCA, and a negative correlation was found for this cell and serum levels of IgE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551372 TI - Enhanced arachidonic acid metabolism in alveolar macrophages from wheezy infants. Modulation by dexamethasone. AB - To test the hypothesis that alveolar macrophages (AM) from wheezy infants release increased amounts of eicosanoids, as do AM from adults with asthma, we compared eicosanoid release by unstimulated- and ionophore-A23187-stimulated AM from 13 wheezy and six nonwheezy infants and analyzed its regulation by dexamethasone in vitro. Alveolar macrophages from wheezy infants released greater amounts of thromboxane A2 (TxA2) and leukotriene B4 (LTB4) under resting conditions and of TxA2 upon stimulation than did those from control subjects. Dexamethasone induced a dose-dependent inhibition of the spontaneous and A23187-stimulated release of TxA2, but not of the A23187-stimulated release of lipoxygenase products. The inhibition of TxA2 formation was maintained when free arachidonic acid was added during A23187 stimulation, demonstrating that dexamethasone acted mainly at a postphospholipase A2 site. AM exposed to acetylsalicylate and then incubated overnight exhibited de novo cyclooxygenase synthesis, suggesting the presence of the inducible cyclooxygenase as a target for inhibition by dexamethasone. In conclusion, our findings suggest that AM from wheezy infants are activated in vivo to release eicosanoids, as are AM from asthmatic adults, and they support the therapeutic indications of glucocorticoids in severe recurrent wheezing of infancy. PMID- 7551374 TI - Effect of blood flow on the leukocyte-endothelium interaction in pulmonary microvessels. AB - Circulating leukocytes are retained in the microcirculation of the lung. The site of leukocyte retention, however, is still a subject of controversy, and the effects of microvascular blood flow on the leukocyte-endothelium interaction in pulmonary microvessels are unknown. We used in vivo fluorescence microscopy to analyze microhemodynamics and the flow behavior of in vivo-labeled leukocytes in pulmonary arterioles, venules, and alveolar capillaries. Microvascular blood flow was altered by variation of cardiac output. Leukocytes were found to roll and to stick on arteriolar and more pronouncedly on venular endothelium. During their passage through alveolar capillaries, a fraction of passing leukocytes became static for 0.1 to > 5 s. Under control conditions, leukocytes were concentrated approximately 8-fold more in arterioles and 24-fold more in venules than in the blood passing through these vessels. The concentration in capillaries was 1.5 times greater than in venules. The velocity of rolling leukocytes in arterioles and venules correlated significantly with the shear rate in these vessels, whereas the density of sticking cells was negatively correlated with the shear rate. The differences between leukocyte rolling and sticking in arterioles and in venules cannot be explained by respective hemodynamic conditions. In alveolar capillaries, the percentage of temporarily static leukocytes and the time of their stasis were inversely correlated with red-blood-cell (RBC) velocity. We conclude that leukocytes are retained in pulmonary arterioles, venules, and alveolar capillaries according to microvascular blood flow and endothelial factors. PMID- 7551375 TI - Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. AB - Lung liquid clearance, epithelial permeability for Na+, mannitol and albumin, as well as Na,K-ATPase activity in alveolar type 2 (AT2) cells were studied during the acute and the recovery phase of hyperoxic lung injury. Rats exposed to 100% oxygen for 64 h were studied at 0, 7 and 14 d after removal from the hyperoxic chamber and compared with control rats breathing room air. In the isolated perfused, liquid-filled rat lung, the albumin flux from the perfusate into the air spaces increased immediately after the oxygen exposure (220 +/- 56 mg/h) and returned to control values (28 +/- 7 mg/h) after 7 and 14 d of recovery. The small solutes (Na+ and mannitol) flux across the alveolar epithelium normalized only after 14 d of recovery in room air. Active Na+ transport and lung liquid clearance were reduced by approximately 45% immediately after oxygen exposure when compared with control values, increased by approximately 56% above control values after 7 d of recovery, and returned to control values after 14 d of recovery. Paralleling these changes the Na,K-ATPase activity decreased by approximately 41% in AT2 cells isolated from rats after 64 h of breathing 100% O2 and increased by approximately 25% after the rats recovered in room air for 7 d. These results suggest that alveolar epithelial Na,K-ATPase may contribute in the recovery from the hyperoxic lung injury by participating in the clearance of lung edema. PMID- 7551377 TI - Effects of PEEP on acinar gas transfer in healthy and lung-injured dogs. AB - We measured cardiorespiratory variables and 133xenon washout from a nonperfused lung region (XeW) in six anesthetized/paralyzed dogs, mechanically ventilated with 60% O2 at different positive end-expiratory pressures (PEEP). XeW in this technique represents directly measured acinar gas transfer (3). Measurements were repeated after induction of lung injury by lavaging the lungs 11 to 13 times with 600 ml saline. In control dogs, lung compliance (CL), alveolar ventilation (Valv), and XeW all decreased with increasing PEEP from 0 to 25 cm H2O (p < 0.05), while lung resistance (RL) did not change. After lavage, CL, Valv, and XeW below 15 cm H2O PEEP were all less than control values (p < 0.05), while RL was higher than control values. As PEEP increased from 0 to 20 cm H2O, Valv and XeW increased, but CL did not change; RL decreased only from 0 to 5 cm H2O. At 20 cm H2O PEEP, Valv and CL were not different from control values (p > 0.05), and XeW was higher than control values (p < 0.05). At estimated alveolar volumes above 400 ml, values for XeW before and after lavage were similar. We conclude that, during severe lung injury: (1) increasing PEEP to moderate levels will increase acinar gas transfer but, after a certain lung volume is reached, further increases in PEEP will have effects similar to the healthy condition; (2) overall mechanical properties of the lung do not reflect the responses to PEEP of the lung periphery. PMID- 7551376 TI - Clinical risk factors for pulmonary barotrauma: a multivariate analysis. AB - Previous investigations have suggested that elevated airway pressures increase the risk of ventilator-induced pneumothorax. However, risk factor analysis using multivariate techniques has not been done. We investigated the hypothesis that airway pressures would not independently correlate with pneumothorax when underlying disease was considered. All ventilated patients over a 1 yr period in the Hohenburg Critical Care Unit at the University of Alabama were followed until death or discharge from the ICU. Ventilator data were collected daily and the presence of pneumomediastinum and pneumothorax determined by review of chest radiographs. Maximal values of airway pressures, minute ventilation, tidal volume, and respiratory rate, as well as age, sex, and underlying disease, were entered into logistic regression analysis. A total of 168 patients was studied, and 20 experienced pneumothorax. Multivariate analysis of the entire ventilated population revealed that only the presence of ARDS independently correlated with pneumothorax. A similar analysis performed on the ARDS population revealed independent correlation only with male sex. Trends toward elevation in airway pressures were seen that did not reach statistical significance. We conclude that development of pneumothorax is most closely correlated with underlying disease, specifically ARDS, and that the associations previously noted between airway pressures and barotrauma largely relate to the occurrence of high airway pressures in ARDS. PMID- 7551378 TI - The load of inspiratory muscles in patients needing mechanical ventilation. AB - We studied 31 consecutive mechanically ventilated patients with acute respiratory failure in two stages: (1) During spontaneous breathing through the respirator, switching from full mechanical assistance to continuous positive airway pressure mode with 0 cm H2O pressure. We measured maximum inspiratory pressure and continuously monitored the pattern of breathing. After 8 to 25 min, none of the patients were able to sustain spontaneous breathing and mechanical ventilation was required to resume. (2) Subsequently, during mechanical ventilation, by manipulating the variables of the ventilator we simulated the pattern of spontaneous breathing the patients had just before the re-institution of mechanical ventilation. We assessed the respiratory mechanics by the constant flow end-inspiratory and end-expiratory occlusion method. Intrinsic positive end expiratory pressure was present in 29 patients. The ratio of the mean inspiratory pressure per breath over the maximum inspiratory pressure (Pi/pimax), as well as Ppeak/pimax, had excessively high mean values, equal to 0.42 +/- 0.11 and 0.56 +/ 0.10, respectively. Pressure-time index was 0.14 +/- 0.04. When we plotted the Pi/Pimax and Ppeak/Pimax against the dynamic increase in FRC, we found that the Pi/Pimax of 13 patients (42%) and the Ppeak/Pimax of 25 of 31 patients (81%) were placed above a hypothetical critical line, representing the critical inspiratory pressures above which fatigue may occur. In addition, almost all patients were gathered around the critical line. We conclude that during discontinuation from mechanical ventilation (MV) almost all patients breathe against a high inspiratory load and their inspiratory muscles perform work that may lead to fatigue. PMID- 7551379 TI - Postnatal lung function in lambs after fetal hormone treatment. Effects of gestational age. AB - We previously found that a single dose of betamethasone in combination with thyroxine given by intramuscular injection to fetal sheep 48 h before preterm delivery at 128 d gestation improved postnatal lung function. We have now asked how the combination of 0.5 mg/kg betamethasone and 15 micrograms/kg T4 given by a single fetal intramuscular injection changes lung response 48 h after treatment at 121 and 135 d gestation. At 121 d gestation the fetal hormone treatment significantly improved postnatal lung function. Compliance increased by 55%, arterial PO2 increased from 39 to 215 mm Hg, PCO2 decreased from 109 to 79 mm Hg, and maximal lung volumes increased by 112%. The hormone treatment decreased the severity of the respiratory failure, although these very preterm lambs still had severe respiratory failure. At 135 d gestation, the fetal hormone treatment decreased the ventilatory pressure requirements that were needed to normalize PCO2 values from 30 to 21 cm H2O. Compliance increased by 40%, and maximal lung volumes increased by 33%. Alveolar or lung tissue, saturated phosphatidylcholine, or alveolar SP-A pool sizes did not change with hormone treatment at 135 d gestation. We conclude that fetal hormone treatment significantly improved postnatal lung function at both gestational ages, although the characteristics of the responses were different. PMID- 7551381 TI - Physiologic effects of nutritional support and anabolic steroids in patients with chronic obstructive pulmonary disease. A placebo-controlled randomized trial. AB - Nutritional depletion commonly occurs in patients with COPD, causing muscle wasting and impaired physiologic function. Two hundred seventeen patients with COPD participated in a placebo-controlled, randomized trial investigating the physiologic effects of nutritional intervention alone (N) for 8 wk or combined with the anabolic steroid nandrolone decanoate (N + A). Nandrolone decanoate or placebo (P) was injected intramuscularly (women, 25 mg; men, 50 mg) in a double blind fashion on Days 1, 15, 29, and 43. Nutritional intervention consisted of a daily high caloric supplement (420 kcal; 200 ml). Also, all patients participated in an exercise program. In the depleted patients, both treatment regimens induced a similar significant body weight gain (2.6 kg) but different body compositional changes. Particularly in the last 4 wk of treatment, weight gain in the N group was predominantly due to an expansion of fat mass (p < 0.03 versus P and N + A), whereas the relative changes in fat-free mass (FFM) and other measures of muscle mass were more favorable in the N + A group (p < 0.03 versus P). Maximal inspiratory mouth pressure improved within both treatment groups in the first 4 wk of treatment, but after 8 wk only N + A was significantly different from P (p < 0.03). Nutritional supplementation in combination with a short course of anabolic steroids may enhance the gain in FFM and respiratory muscle function in depleted patients with COPD without causing adverse side effects. PMID- 7551380 TI - Effect of smoking cessation on airway inflammation in chronic bronchitis. AB - To investigate the effect of smoking cessation on the airway inflammatory process present in nonatopic subjects with chronic bronchitis, we obtained bronchial biopsies from nine current smokers and seven exsmokers, all with symptoms of chronic bronchitis at the time of the study, and from seven healthy nonsmoking subjects. The exsmokers had stopped smoking on average 13 yr before the study, yet cough and production of sputum had persisted. Bronchial biopsies were assessed using immunohistochemical techniques to investigate the number of inflammatory cells, the markers of mononuclear cell activation, and the expression of endothelial adhesion molecules and cytokines in the subepithelium. Current smokers and exsmokers had an increased number of macrophages, IL-2R positive cells, VLA-1-positive cells, ICAM-1-positive vessels, and E-selectin positive vessels compared with normal nonsmoking subjects, but the number of cells positive for neutrophils, EG-2, CD3, CD4, CD8, TNF-alpha and IL-1 beta were similar among the three groups. No differences were observed between current smokers and exsmokers for any parameter examined. In conclusion, the inflammatory process present in the airway mucosa of current smokers may persist after smoking cessation in subjects who continue to have symptoms of chronic bronchitis. PMID- 7551382 TI - Contraction, relaxation, and economy of force generation in isolated human diaphragm muscle. AB - Contraction, relaxation, and energetics of normal human diaphragm strips (n = 10) were investigated over the whole load continuum in both twitch and tetanus modes. For a given level of isotonic total force and over a large part of the contraction phase, instantaneous velocity was shown to be a unique function of instantaneous length, regardless of time and initial length. In afterloaded tetanic contractions and over a wide range of loads, the peak lengthening velocity (VL) linearly decreased when maximum extent of muscle shortening (delta L) decreased. Stimulation mode modulated the VL versus delta L relationship, the slope of this linear regression being greater in tetanus than in twitch. Conversely, over a wide range of load, an increase in load linearly accelerated the peak rate of force decay, regardless of the stimulation mode. The energetics of human diaphragm muscle was evaluated in terms of both mechanical activity and economy of force generation. Maximum mechanical work (Wmax) differed significantly according to the stimulation mode, and the relative force at which Wmax occurred was higher in tetanus than in twitch (p < 0.05). The G curvature of the P-V hyperbola and maximum mechanical efficiency were significantly higher in tetanus than in twitch. This strongly suggests that the economy of force generation is higher in tetanus contractions than in twitch. PMID- 7551383 TI - Factors limiting exercise performance in long-term survivors of bronchopulmonary dysplasia. AB - The long-term impairment of pulmonary function during exercise was assessed in 12 children, aged 6 to 12 yr, who developed BPD after prematurity (gestational age 30 +/- 2 wk [mean +/- SD] and birth weight 1,400 +/- 335 g) and 16 age-, sex-, and physical activity-matched healthy children born at term, who served as controls. The children performed pulmonary function tests at rest and a maximal stepwise exercise on a treadmill. Oxygen consumption (VO2), carbon dioxide output (VCO2), and minute ventilation (VE) were monitored during the run. Baseline mean spirometric values (% of predicted) were in the normal range for both groups but were lower in BPD children with respect to control children (p < 0.05). At rest, arterial oxygen saturation (SaO2) was > or = 98% in all BPD children, but at peak exercise, 4 of them had a SaO2 fall > or = 4%. The postexercise FEV1 fall, with respect to the baseline, was 8 +/- 6%, in BPD and 2 +/- 1% in control children (p < 0.01). Maximum VO2 and VE were significantly lower in BPD children with respect to the control group (25.2 +/- 10.3 versus 37.1 +/- 10.4 ml/min/kg and 20.8 +/- 9.4 versus 30.7 +/- 7.9 L/min, respectively, both p < 0.01). Also, at submaximal levels of exercise dynamic, VO2 and VE responses were significantly lower in the BPD group (ANOVA, p < 0.001), with a ventilatory pattern characterized by lower tidal volumes. Anaerobic threshold was 20.6 +/- 9 in BPD and 28.8 +/- 8.6 ml O2/min/kg in healthy children (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551384 TI - Nasal lavage cytokines in normal, allergic, and asthmatic school-age children. AB - Inflammation can be demonstrated in the airway mucosa of asthmatics, even in the absence of overt symptoms, but the pathogenesis of this chronic inflammation is incompletely defined. It has been suggested that inflammatory cytokines produced by epithelium may play important roles in this process. Therefore, we measured the cytokines interleukin-8 (IL-8), IL-6, and granulocyte-macrophage colony stimulating factor (GM-CSF) in nasal lavage fluids from school-age children who were (1) "normal" (nonallergic/nonasthmatic), (2) allergic to house-dust mite antigen but nonasthmatic (no history of wheezing), or (3) allergic and asthmatic (history of > or = 10 wheezing episodes). Children underwent a single nasal lavage procedure while asymptomatic and on no anti-inflammatory medications or anti-histamines. In addition to cytokine concentrations, cell counts, differentials, albumin, histamine, and eosinophil cationic protein (ECP) concentrations were determined in nasal lavage fluids. Significant increases in IL-8 and ECP were observed in asthmatics compared with both normals and allergic nonasthmatics. Overall, IL-8 in nasal lavage fluids correlated significantly with ECP. Allergic nonasthmatics did not have significant increases in cytokines or other mediators compared with normal subjects. Concentrations of IL-6 did not differ significantly among the three groups, and GM-CSF was undetectable in all samples tested. We conclude that increased IL-8 production and eosinophil activation are characteristic of the airways of asthmatic children when asymptomatic, and we speculate that IL-8 plays a role in the maintenance of airway inflammation in asthma. PMID- 7551385 TI - Supplemental oxygen during sleep in children with sleep-disordered breathing. AB - Supplemental O2 is sometimes used to treat children with the obstructive sleep apnea syndrome (OSAS). However, its effects have not been studied. We therefore evaluated the use of supplemental O2 during sleep in children with OSAS. Oxygen and room air were delivered via nasal cannula at 1 L/min for 4 h each in a randomized, double-blind fashion. Twenty-three children were studied (mean age, 5 +/- 3 [SD] yr). Patients had a higher mean SaO2 and higher SaO2 nadir when breathing O2. There was no difference in the number (10.9 +/- 20.6/h on O2 versus 13.5 +/- 19.3 on room air) or duration of obstructive apneas. Although there was no overall change in end-tidal PCO2 when patients breathed O2, two children showed a significant increase. We conclude that breathing supplemental O2 during sleep in children with OSAS results in improved oxygenation and in most cases does not exacerbate sleep-disordered breathing. However, end-tidal PCO2 should be monitored in children with OSAS receiving O2 therapy. We speculate that supplemental O2 does not depress the ventilatory drive during sleep in most children with OSAS. PMID- 7551386 TI - Relation of the course of bronchial responsiveness from age 9 to age 15 to allergy. AB - Bronchial responsiveness to methacholine was measured in a birth cohort of New Zealand children at ages 9, 11, 13, and 15. Overall bronchial hyperresponsiveness (BHR) decreased with age. While the response at age 9 was significantly related to responsiveness, symptom severity, and low lung function at age 15, these relationships were much closer when bronchial response levels after age 9 were taken into account. Also, among children who were unresponsive to methacholine at age 15, those with previous BHR had more frequent wheeze and lower lung function than those whose previous tests were all unresponsive. Both the overall tendency to BHR and the tendency to retain BHR were closely related to high serum IgE levels (determined at age 11) and to positive allergy skin tests (determined at age 13). These evidences of allergy, closely related to the severity and course of methacholine response, appeared to be important determinants of the frequency of wheeze and the degree of impairment of lung function at the end of follow-up. In view of the variability in BHR, a single estimate of bronchial responsiveness taken at an arbitrary point in time may not be an accurate index of the overall tendency to BHR. PMID- 7551387 TI - Community-acquired pneumonia: impact of immune status. AB - This cross-sectional and prospective one year study evaluated adults admitted to an inner city hospital with community-acquired pneumonia. The study used extensive diagnostic methods to evaluate the etiologies of community-acquired pneumonia in hospitalized patients with differing immunologic status. Of 385 study patients, concurrent problems associated with immunosuppression were noted in 221 (57%) patients, 180 of whom were human immunodeficiency virus (HIV) infected. The five most common causes of community-acquired pneumonia were: Streptococcus pneumoniae, Pneumocystis carinii, aspiration, Hemophilus influenzae, and gram-negative bacilli. Only 8.3% of patients had either Legionella, Chlamydia pneumoniae or Mycoplasma pneumoniae. Despite use of state of-the-art diagnostic techniques, no diagnosis was made in 46 of 180 (25.6%) HIV infected patients, 56 of 164 (34.1%) immunocompetent patients, and 20 of 41 (48.8%) non-HIV-infected immunosuppressed patients. The diagnostic yield of pre antibiotic sputum culture for conventional bacteria was 99/155 (63.9%) compared to 52 of 169 patients (32.7%) with adequate post-antibiotic sputum culture (p < 0.0001). Although S. pneumonia continues to be the most commonly identified etiologic agent of community-acquired pneumonia, it is surpassed by P. carinii in the HIV-infected patient population. The apparent decline in the frequency of S. pneumoniae in our series presumably reflects administration of antibiotics prior to procurement of sputum culture. The paucity of atypical agents in this study support the current American Thoracic Society guidelines for selective use of macrolide therapy in immunocompetent adults hospitalized with community-acquired pneumonia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551388 TI - Bacterial infection in chronic obstructive pulmonary disease. A study of stable and exacerbated outpatients using the protected specimen brush. AB - The lower airways of asymptomatic chronic obstructive pulmonary disease (COPD) patients can be colonized by bacteria, mainly Haemophilus influenza, Streptococcus pneumoniae, and Moraxella catarrhalis. However, the role of lower airway bacteria in stable and exacerbated COPD has not been well defined. To determine the importance of lower airway bacterial infection in COPD we studied 40 outpatients with stable COPD (Group A: age 61.1 +/- 9.9 yr; [mean +/- SD]; FEV1/FVC 51.7 +/- 12.5) and 29 outpatients with exacerbated COPD (Group B: age 63.4, SD 9.0 yr; FEV1/FVC 52.0, SD 9.6), using the protected specimen brush (PSB) for microbiology sampling. Group A consisted of outpatients with stable COPD having normal or near-normal chest X-rays, with clinical indications for performing fiber-bronchoscopy (pulmonary nodule, remote hemoptysis); Group B consisted of patients with exacerbated COPD who voluntarily accepted lower airway microbiology sampling. To avoid contamination by upper airway flora the PSB was used for bacterial sampling in all the cases and concentrations > or = 1,000 colony-forming units/milliliter (CFU/ml) were considered positive. Results were as follows: Group A: Lung function data in outpatients with stable COPD were lower than the reference values for this population (FVC 2.97 +/- 1.02 L, FVC% 71.4 +/- 22.4, FEV1 1.59 +/- 0.79 L, FEV1% 51.2 +/- 23.0). Positive PSB cultures were obtained in 10 of 40 cases (25%), mainly of H. influenzae and S. pneumoniae. Two of 40 cases had positive cultures at concentrations > or = 10,000 CFU/ml (5.0%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551389 TI - Treatment of presumed and proven acute rejection following six months of lung transplant survival. AB - The gold standard for the diagnosis and subsequent treatment of acute rejection of lung allografts is the demonstration of rejection on transbronchial biopsy specimens. However, treatment may be initiated in the case of a compatible clinical scenario in the absence of definitive histologic documentation. In the Toronto Lung Transplant Program, we have treated patients with a decline in FEV1 and no evidence of infection with augmented systemic steroids for a presumed diagnosis of rejection. We retrospectively reviewed all episodes of acute rejection that occurred beyond 6 mo after transplant where treatment with augmented steroids had been initiated. A total of 72 treatments with augmented steroids were initiated in 45 patients who underwent 47 transplant procedures. FEV1 showed at least a 10% improvement following steroids in 14 of 72 (19%). FEV1 continued to decline by at least 10% in 32 of 72 (44%). Changes in FEV1 between +10 and -10% occurred in 26 of 72 (36%); of those episodes, 19 showed a decline of < 10%. Histologic evidence of at least grade II rejection was documented in only 16 cases. In those cases, FEV1 improved by at least 10% in 7 of 16 (44%), whereas it declined by at least 10% in 4 of 16 (25%). Spirometric evidence of bronchiolitis obliterans syndrome developed within 3 mo of the treated rejection episode in at least 20 of 47 transplants (43%). We conclude that treatment with augmented systemic steroids for presumed and histologically proven acute rejection beyond 6 mo after transplant is often ineffective in improving spirometry. PMID- 7551390 TI - Cyclic adenosine monophosphate-mediated release of nitric oxide from canine cultured tracheal epithelium. AB - Nitric oxide (NO) may play a part in pulmonary vascular regulation and bronchomotor control and has been detected in exhaled air. We report the release of NO from airway epithelial cells and its regulation by cyclic adenosine monophosphate (cAMP). To directly measure NO release, a highly specific amperometric sensor for NO made of Pt/Ir alloy coated with a three-layered membrane consisting of KCI, NO-selective resin, and normal silicon resin was developed. Immersion of this sensor in the medium containing canine cultured tracheal epithelium detected baseline levels of NO at 9.6 +/- 1.6 nM (mean +/- SE), which was reduced by NG-nitro-L-arginine methylester (L-NAME) but not by D NAME. This inhibition was reversed by L-arginine. Addition of isoproterenol, 3 isobutyl-1-methylxanthine, and forskolin caused a rapid increase in NO, an effect that was not altered by Ca(2+)-free medium in the presence of the intracellular Ca2+ chelator BAPTA-AM and the calmodulin antagonist W-7. Bradykinin, ionomycin, and ATP were without effect on NO release. The forskolin-induced NO release was accompanied by intracellular accumulation of cAMP and Ca2+. In contrast, bradykinin increased intracellular Ca2+ but not cAMP levels. Cytochemistry of cultured tracheal epithelium showed a positive staining with NADPH diaphorase activity. These results suggest that airway epithelial cells spontaneously release NO and that the release may be stimulated specifically through cAMP dependent mechanism. PMID- 7551391 TI - Anti-CD18 antibody does not block ileal injury induced by phorbol myristate acetate. AB - Acute lung injury (ALI) is frequently complicated by systemic microvascular injury. In previous studies, ALI from acid aspiration resulted in ileal microvascular injury. This was due to systemic inflammation and was prevented by MoAb 60.3, an antibody against the CD18 antigen that mediates leukocyte adherence. The present study was designed to test the hypothesis that ileal microvascular injury during phorbol myristate acetate (PMA)-induced systemic inflammation is also blocked by MoAB 60.3. We assessed the injury by measuring the concentration ratios of lymph protein to plasma protein (i.e., CL/CP) at steady-state lymph flows in autoperfused cat ileum preparations. As expected, the CL/CP increased in the ilea of animals given PMA (15 micrograms/kg; n = 5) compared with the ilea of control animals (n = 5) (0.202 +/- 0.024 versus 0.106 +/- 0.010; p = 0.006) and was accompanied by widespread morphologic alterations. Intravenously administering MoAb 60.3 (2 mg/kg) to animals before the PMA infusion (n = 5) yielded a CL/CP value indistinguishable from that of the PMA group (0.222 +/- 0.024 versus 0.202 +/- 0.024; p = NS). These results suggest that CD18-mediated leukocyte adherence is not important in the mechanism of PMA induced ileal microvascular injury. PMID- 7551392 TI - Vaccination promotes TH1-like inflammation and survival in chronic Pseudomonas aeruginosa pneumonia in rats. AB - In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF) we studied whether the inflammatory response could be altered by vaccination. Rats were immunized with either a depolymerized alginate toxin A conjugate (D-ALG toxin A), purified alginate, an O-polysaccharide toxin A conjugate, or sterile saline. After challenge none of the rats immunized with D ALG toxin A died, in contrast to the other two vaccine groups combined (p = 0.03). A significant reduction in the severity of the macroscopic lung inflammation was seen in rats immunized with D-ALG toxin A, compared with the other three groups (p = 0.009). The histopathologic response in the control rats was dominated by numerous polymorphonuclear leukocytes (PMN) surrounding the alginate beads. In contrast, the histopathologic response in rats immunized with D-ALG toxin A changed within the first week after challenge from predominantly PMNs (TH2-like) to a chronic-type inflammation dominated by mononuclear leukocytes (TH1-like). In accordance, the antibody titers induced by the D-ALG toxin A vaccine were not different from those of the control rats after challenge. This study identifies a possible new way of modifying the inflammation and thereby preventing the PMN-mediated lung tissue damage during chronic P. aeruginosa lung infection in CF. PMID- 7551393 TI - Increased respiratory system resistance and bronchial smooth muscle hypertrophy in children with acute postoperative pulmonary hypertension. AB - Following surgery for congenital heart disease, there is often an increased reactivity of the pulmonary vasculature to stimuli, resulting in rapid increases in pulmonary artery pressure and a clinical impression of stiff lungs. Lung mechanics were measured in 30 children, mean age 6.7 +/- 4.1 mo, who were ventilated and had pulmonary artery pressure monitoring following surgery for congenital heart disease. A group of 15 patients developed postoperative pulmonary hypertension. In these patients, respiratory system resistance was 43% higher (p = 0.001) and compliance 11% lower (p = 0.004) during acute pulmonary hypertension compared with baseline pulmonary artery pressure. No changes in resistance or compliance were seen in the 15 patients who did not develop pulmonary hypertension. The changes in lung mechanics interfered with mechanical ventilation, resulting in a 9.4% rise in PaCO2 during pulmonary hypertension. The bronchial smooth muscle was found to be increased by 68%, and the vascular smooth muscle was more than twice normal in lung biopsies from 9 pulmonary hypertension patients compared with 6 age-matched postmortem controls patients who had no cardiac or pulmonary disease. The bombesin-immunoreactive pulmonary neuroendocrine cells (PNEC) were also increased in the pulmonary hypertension patients. These findings suggest a coconstriction and cohypertrophy of bronchial and vascular smooth muscle during pulmonary hypertension. Mediators, such as bombesin, endothelin-1, and serotonin, are known to be produced by PNEC and may be involved in the observed vasoconstriction, increased respiratory system resistance, and smooth muscle hypertrophy. PMID- 7551394 TI - Screening Young syndrome patients for CFTR mutations. AB - Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles. PMID- 7551395 TI - Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor alpha and interferon beta. AB - Respiratory syncytial virus (RSV) is the major pathogen causing severe lung disease in children. RSV initially replicates efficiently in the respiratory tract but becomes undetectable by 7 to 21 d after infection in normal children, suggesting that intrinsic cellular mechanisms, as yet undefined, may restrict virus replication. To provide an in vitro model to examine mechanisms that restrict RSV replication, three human lung epithelial cell lines were exposed to RSV in vitro and virus replication proceeded in a dose- and time-dependent manner, although less efficiently than the highly permissive CV-1 cell line (monkey kidney epithelial cell). Tumor necrosis factor alpha (TNF alpha) and/or interferon beta (IFN beta) markedly inhibited RSV replication in a dose- and time dependent manner. TNF alpha combined with IFN beta essentially aborted RSV replication in A549 epithelial cells. TNF alpha and/or IFN beta did not induce cell membrane damage, cause cell lysis, or inhibit cellular protein synthesis. RSV-infected human alveolar macrophages, which produce TNF alpha, failed to productively infect lung epithelial cells in co-culture. Together these studies suggest that endogenous TNF alpha coupled with exogenous IFN beta could restrict RSV replication in lung epithelium. PMID- 7551396 TI - Defective bronchus-associated lymphoid tissue in long-term surviving rat lung allografts. AB - In a previous study we found that a local immune response did not develop in the bronchus-associated lymphoid tissue (BALT) of infected rat allografts. We hypothesized that the BALT in rat lung allografts was damaged after allotransplantation. Therefore, we investigated three prerequisites for a normal function of the BALT, i.e., its structure, the uptake of antigens, and the lymphocyte migration to the BALT in three groups of rats (n = 10 each): (1) Brown Norway(BN)-to-Lewis (LEW) allografts; (2) LEW-to-LEW isografts; and (3) normal LEW rats. All rats were immunosuppressed with CsA (injected on days 2 and 3). Six mo after transplantation the structure of the BALT and the uptake of intrabronchially injected carbon particles in the BALT were determined histologically; the migration of intravenously injected, fluoroscein isothiocyanate labeled lymphocytes to the BALT was determined immunohistochemically. In the allografts the BALT was defective in all three investigated aspects. It was reduced in size and lymphocyte density and was largely replaced by fibrous tissue. Twenty-four h after administration no carbon particles and only a few labeled lymphocytes were found in the BALT. In contrast, in the syngeneically transplanted and nontransplanted lungs the BALT consisted of a large and dense collection of lymphocytes. In these BALTs large numbers of carbon particles and labeled lymphocytes were found. In conclusion, after allogeneic transplantation the BALT in the lung becomes defective in structure and function. The BALT is most likely damaged by rejection, since the BALT is syngeneic lung transplants was perfectly normal. PMID- 7551398 TI - Allergic bronchopulmonary mycosis caused by Fusarium vasinfectum. AB - We present a case of allergic bronchopulmonary fusariosis (ABPF). This is the first case of allergic bronchopulmonary mycosis (ABPM) caused by Fusarium vasinfectum reported in the medical literature. The patient presented with a history and radiographic picture highly suggestive of allergic bronchopulmonary aspergillosis (ABPA), yet with negative ABPA serology results. The demonstration of a positive skin test and precipitins against F. vasinfectum, elevated serum IgG and IgE antibodies against F. vasinfectum, and a decline in total serum IgE with treatment clearly establish the diagnosis of ABPF. The diagnosis of ABPM caused by organisms other than Aspergillus represents an important consideration in patients who appear clinically to have ABPA but whose serology results do not confirm this diagnosis. As in ABPA, early diagnosis and appropriate therapy may be necessary in order to prevent progressive lung disease. PMID- 7551397 TI - Analysis of K-ras gene mutations in malignant and nonmalignant endobronchial tissue obtained by fiberoptic bronchoscopy. AB - Using polymerase chain reaction DNA amplification, we identified K-ras oncogene mutations in bronchial biopsies obtained from patients undergoing bronchoscopy for clinical indications. We hypothesized that these mutations would be found in a field encompassing malignant and nonmalignant tissues in patients with pulmonary carcinomas, and also possibly in tissue from some patients who smoked but did not have pulmonary malignancy. We found K-ras mutations in endobronchial biopsies from nine of 22 patients (41%) with carcinoma of the lung. In five of these patients, mutations were found in both malignant and nonmalignant specimens. In two instances, mutations were found only in the neoplastic tissue, and in two cases mutations were present only in the nonmalignant tissue. Furthermore, we identified two patients with K-ras mutations among seven patients in whom no clinical malignancy was apparent. Our data suggest that some oncogene activations may occur over a broad area in smokers with and without malignancy, and they imply that K-ras mutations may serve as surrogate markers for malignant potential. PMID- 7551399 TI - Clinical features of vocal cord dysfunction. AB - Vocal cord dysfunction (VCD) is a respiratory condition characterized by adduction of the vocal cords with resultant airflow limitation at the level of the larynx. Previously, this condition was described in case reports and in small series. This study reviews all patients hospitalized from 1984 through 1991 in whom VCD was diagnosed. Demographic, historical, physiologic, laboratory, and psychiatric factors were statistically analyzed. Ninety-five patients met the criteria for proved VCD; of these, 53 also had asthma. All patients had laryngoscopic evidence of paradoxical vocal cord motion, with inspiratory and/or early expiratory vocal cord adduction. The patients with VCD without asthma were predominantly young women. In these patients, asthma had been misdiagnosed for an average of 4.8 years. Their medications were identical to those of a control group of patients with severe asthma. Thirty-four of the 42 patients with VCD without asthma were receiving prednisone regularly at an average daily dose of 29.2 mg. Medical utilization was enormous with the VCD group, averaging 9.7 emergency room visits and 5.9 admissions in the year prior to presentation. Also, 28% of the patients with VCD had been intubated. We conclude that VCD can masquerade as asthma and that it often coexists with asthma. This study helps to define the historical and clinical features of VCD. PMID- 7551400 TI - Semirecumbent position protects from pulmonary aspiration but not completely from gastroesophageal reflux in mechanically ventilated patients. AB - The aim of this study was to evaluate the effect of two body positions (supine and semirecumbency) on the dynamics of gastroesophageal reflux (GER) in 15 patients requiring mechanical ventilation and having a nasogastric tube in place. Samples of gastric contents, pharyngeal and bronchial secretions, and blood were obtained at baseline and every hour during a period of 5 h after nasogastric tube isotopic instillation of 37 megabecquerels of Tc99m. Radioactivity counting (RAc) was performed using a gamma counter with correction for decay. Irrespective of the body position, all patients showed at 3, 4, and 5 h after the isotope instillation a significant increase in RAc of the oropharyngeal contents (p < 0.05, each), indicating GER. Although RAc values in the pharynx were higher in supine from 1 through 4 h (p < 0.05), at the end of the study (5 h) the values did not differ between each position. Likewise, the slopes of the regression lines of sequential oropharyngeal RAc values were not different between each position (0.39 +/- 0.09 versus 0.45 +/- 0.11, respectively). In contrast, RAc values in bronchial secretions were higher at 5 h in the supine position compared with baseline (p < 0.05) and to semirecumbency (p < 0.01). These results strongly support that GER in mechanically ventilated patients with a nasogastric tube is a feature occurring irrespective of body position. Semirecumbent position does not protect completely from GER and subsequently from oropharyngeal colonization from gastric origin. PMID- 7551401 TI - Albuterol delivery in a model of mechanical ventilation. Comparison of metered dose inhaler and nebulizer efficiency. AB - Using an in vitro model, we compared efficiencies of jet nebulizers and metered dose inhalers (MDI) with actuator devices to deliver albuterol in various conditions of mechanical ventilation. Factors tested included influence of humidification, MDI actuator device (Aerovent spacer or Marquest 172275 MDI adaptor), and synchronization of MDI to the respiratory cycle. With the nebulizer (AeroTech II) filled with 2.5 mg albuterol sulfate in 3 ml water and run until dry, inhaled mass was 42 +/- 2.6% and mass median aerodynamic diameter (MMAD) was 1.3 microns on a nonhumidified circuit. With the MDI+ Aerovent, the inhaled mass percentage per 90 microgm puff was 15.4 +/- 0.2% with humidification and 25.1 +/- 3.7% without humidification, actuations being synchronized with the beginning of inspiration and separated by a 1-min pause. Failure to synchronize actuations with inspiration or to pause 1 min resulted in significant reductions in inhaled mass (by 35 and 72%, respectively). The Marquest adaptor was less efficient, with an inhaled mass of 7.2 +/- 0.7% under optimal conditions. Metered-dose inhaler actuation during expiration resulted in relatively large particles (MMAD = 2.0 microns). All other MDI actuations led to essentially biphasic distributions, with particles greater than 1 micron following a distribution similar to the nebulizer and the overall MMAD estimated to be 0.22 microns. The AeroTech II delivered a cumulative 1,000 micrograms of drug (2,500 x 0.40) over 40 min. To achieve that amount, the MDI connected to the Aerovent and used in its most efficient sequence would require 45 timed puffs (90 micrograms per puff, 25.1% mean inhaled mass) and take 45 min of an experienced therapist's time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551402 TI - Training programs in adult pulmonary disease and critical care medicine and training programs in pediatric pulmonary disease. 1995 editions. American Thoracic Society/American Lung Association. PMID- 7551403 TI - Pneumocystis carinii pneumonia: a major complication of immunosuppressive therapy in patients with Wegener's granulomatosis. PMID- 7551404 TI - Pneumonia associated with nosocomial sinusitis: a different risk according to the pathogen involved. PMID- 7551405 TI - Influenza viruses and cell membranes. AB - Influenza viruses are spherical, about 1000 A in diameter, and consist of an as yet undefined central structure containing the eight negative-sense RNA molecules of the genome (1) in association with the transcriptase required for mRNA synthesis, an abundant nucleoprotein, and an equally abundant matrix protein. This core is surrounded by a membrane derived from the cell surface in a budding process by which newly formed viruses are released from the infected cell. During infection cell membranes are modified by the incorporation of newly synthesized virus membrane proteins, and the finally released viruses contain exclusively two different types of virus-specified glycoprotein, hemagglutinin and neuraminidase, and a proton channel protein, M2. All three of these molecules have been studied extensively, particularly the glycoproteins, and in this paper information on their structures and functions will be summarized and related to modifications in cellular membranes that occur during virus infection. PMID- 7551406 TI - Influenza viruses, cell enzymes, and pathogenicity. AB - Proteolytic cleavage of the influenza virus hemagglutinin glycoprotein (HA) by cellular proteases is a prerequisite for virus infectivity, spread of the virus in the infected organism, tissue tropism, and viral pathogenicity. Production of infectious virus depends upon the structure at the HA cleavage site as well as the substrate specificity and the distribution of appropriate enzymes. Differences exist in the specificities of the endoproteases that recognize the different sequence motifs at the cleavage site. With avian influenza viruses that cause lethal systemic infections, the cleavage site consists of multibasic amino acids. Furin, which activates this type of HA, is a member of the subtilisin family and represents the prototype of ubiquitously occurring membrane-bound proteases. On the other hand, serine proteases secreted from a restricted number of cell types and some bacterial enzymes recognize a monobasic cleavage signal at HA of the mammalian and the apathogenic avian influenza viruses. The limited occurrence of these proteases results in only localized infection. Implementation of these defined conditions for virus activation may represent a novel type of disease control. PMID- 7551407 TI - Potential roles of apoptosis in viral pathogenesis. AB - Apoptosis is a physiologic mechanism of cell death that is induced during development or reversible tissue expansion or after tissue damage to eliminate unwanted cells. Recently, viral gene products have been shown to either specifically induce or specifically inhibit apoptosis. In this review, our current understanding of the regulation of apoptosis is outlined. The mechanisms by which viruses can inhibit or induce apoptosis are described. The role of apoptosis in modulating the immune response to viral infection is discussed. Finally, the hypothesis is advanced that the balance between induction and inhibition of apoptosis is modulated by viruses to optimize the course of viral infection. PMID- 7551408 TI - Interspecies transmission of influenza viruses. AB - In this report we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species. Two partly overlapping reservoirs of influenza A viruses exist in migrating water-fowl and shorebirds throughout the world. These species harbor influenza viruses of all the known hemagglutinin and neuraminidase subtypes. In contrast to the rapid, progressive changes in both the nucleotide and amino acid sequences of mammalian virus gene lineages, avian virus genes show far less variation and, in most cases, appear to be in evolutionary stasis. There are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. The periodic exchange of influenza viruses between species has been illustrated by the appearance of new pandemic influenza viruses in humans, including the Spanish influenza of 1918, the Asian influenza of 1957, and the Hong Kong influenza of 1968. Transmission of avian influenza viruses to swine in Europe in 1979 has resulted in the appearance of human-avian reassortant influenza viruses in pigs in Italy and in children in the Netherlands. These studies provide evidence supporting the possibility that pigs serve as a mixing vessel for reassortment between influenza viruses in mammalian and avian hosts and raise the question of whether the avian influenza viruses now circulating in European swine are the precursors of the next human pandemic virus. PMID- 7551410 TI - Rhinovirus infection of the normal human airway. AB - Selected events in rhinovirus infection of the normal human airway can be regarded as occurring sequentially. Initial steps in rhinovirus pathogenesis are believed to include viral entry into the nose, mucociliary transport of virus to the posterior pharynx, and initiation of infection in ciliated and non-ciliated epithelial cells of the upper airway. Viral replication peaks on average within 48 h of initiation of infection and persists for up to 3 wk. Infection is followed by activation of several inflammatory mechanisms, which may include release or generation of interleukins, bradykinins, prostaglandins, and possibly histamine and stimulation of parasympathetic reflexes. Pathophysiologic processes are initiated, which include vasodilatation of nasal blood vessels, transudation of plasma, glandular secretion, and stimulation of nerve fibers, causing pain and triggering sneeze and cough reflexes. The resultant clinical illness is a rhinosinusitis, pharyngitis, and bronchitis, which, on average, lasts 1 wk. PMID- 7551409 TI - Rhinoviruses and cells: molecular aspects. AB - Recent developments in our understanding of the molecular structure and functioning of the human rhinoviruses are briefly reviewed. The determination of virus structure at near atomic resolution has provided a wealth of information that can be used to help interpret dynamic properties of the virus. The mechanisms involved in capsid assembly, attachment to cell surfaces, uncoating, and the initiation of infection and antibody-mediated neutralization of infectivity can all be examined afresh in the light of the structural data. In addition, molecular techniques for manipulating and analyzing the biological properties of nucleic acids and proteins are providing a wealth of information on details of the replication machinery of the viruses. Probably one of the least understood aspects of viral replication is the precise nature of the interactions between host and viral components during infection. Unfortunately, despite all of these impressive scientific advances the cure for the common cold is still elusive. PMID- 7551411 TI - The clinical impact of human respiratory virus infections. AB - Acute respiratory infections are the most frequent illnesses of the human host. Most infections are caused by viruses and bacteria; the proportion caused by viruses is much greater. The viruses most frequently involved are adenoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, and rhinoviruses. Acute respiratory infections are more common in young children, have rather specific seasonal occurrences, and some agents are associated with specific respiratory syndromes. Risk factors associated with increased incidence or severity of respiratory infections are occurrence in the very young or the elderly; crowding; being male; inhaled pollutants; anatomic, metabolic, genetic or immunologic disorders; and malnutrition, including vitamin or micronutrient deficiency. Respiratory infections are a much greater problem in developing countries than in developed countries and are the leading causes of death in children under 5 yr of age. The same agents cause infections, and the incidence of total respiratory infections is the same as in the developed countries. The precise causes of increased morbidity and mortality in the developing world are unclear, but crowding, inhaled pollutants, and malnutrition are likely candidates. The interactive role of viruses and bacteria is not clear but may play a role in increased severity of respiratory infections. PMID- 7551413 TI - Natural and experimental rhinovirus infections of the lower respiratory tract. AB - Rhinoviruses are the cause of the majority of common colds, but their role in lower respiratory disorders is less clear. Recent studies using the polymerase chain reaction to detect rhinoviruses have established respiratory viral infections as major factors in the induction of acute exacerbations of asthma in both adults and children, both in mild exacerbations and in more severe exacerbations leading to hospital admission. Rhinoviruses were the major virus type detected in these studies, accounting for two-thirds of viruses detected. It is not known whether rhinoviruses produce their effects by directly invading the lower airway or by indirect means. Previous clinical studies provide some evidence that rhinoviruses are capable of infecting the lower airway. However, the immunologic response, both in the upper and lower airways, remains poorly defined. Recent studies have provided evidence of increased cellular activation in peripheral blood and in bronchial biopsies in atopic subjects compared with normal subjects during experimental rhinovirus infections. The reasons for these different cellular responses are unclear. Rhinoviruses as well as other respiratory viruses have been shown to increase levels of a variety of cytokines from respiratory epithelium, monocytes, or macrophages. Prominent among these cytokines is interleukin (IL)-8. We have detected increased levels of IL-8 in nasal secretions from subjects with wild-type rhinovirus infections. We studied the mechanisms of rhinovirus-induced IL-8 production and and found protein release from both pulmonary epithelial and peripheral blood mononuclear cells. This protein production was accompanied by increased mRNA expression and evidence of infection of both pulmonary epithelial and monocyte cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551414 TI - Psychological stress and susceptibility to upper respiratory infections. AB - The biologic plausibility of a link between psychological states and host resistance is discussed. Although there is substantial evidence for the association between psychological stress and both cellular and humoral immune response, these data do not necessarily suggest increased susceptibility to infectious agents among stressed persons. Epidemiologic and viral-challenge studies suggest that psychological stress is a risk factor for upper respiratory infections with the strongest evidence provided by recent well-controlled, prospective viral-challenge trials. However, there is still little direct evidence for the nature of neuroendocrine, immune, or behavioral pathways through which stress might alter susceptibility. PMID- 7551412 TI - The effects of rhinovirus infections on allergic airway responses. AB - Although it has long been recognized that the common cold is a potent trigger for symptoms of asthma, the mechanisms underlying the association between upper respiratory infection and increased lower airway obstruction remain obscure. The use of experimental infection of volunteers with or without respiratory allergies has enabled direct comparisons of common cold symptoms in these two groups. Furthermore, techniques such as bronchoalveolar lavage and segmental antigen challenge have been used to directly sample lower airway fluids and tissues during acute viral infection. This review summarizes the findings of studies examining the separate and combined effects of rhinovirus infection and allergen exposure on airway physiology and inflammation. PMID- 7551415 TI - Immunity and immunopathology to respiratory syncytial virus. The mouse model. AB - Infection with respiratory syncytial virus (RSV) is a major unsolved challenge for vaccine development. RSV is worldwide in distribution and infects almost all children during the first 2 yr of life. The mouse model of RSV lung disease has been very successful in reproducing many aspects of the human disease. In particular, the role of antiviral T cells in both eliminating virus and causing enhanced disease has been shown dramatically. This immunopathologic paradox is now more clearly understood than for any other common human infection, largely due to insights gained from the mouse model. This review focuses on the unique ability of different RSV proteins to prime for specific functional subsets in the mouse, and the association between sensitization to the major surface glycoprotein G, the induction of T helper 2 cells, and the subsequent appearance of lung eosinophilia during RSV infection. PMID- 7551417 TI - Interferons, Mx genes, and resistance to influenza virus. AB - Human influenza is primarily an infection of the upper respiratory tract and central airways. The interferon (IFN) system appears to have a role in limiting viral spread and initiating recovery before the development of T-cell and B-cell responses in primary infection. All cellular responses to IFNs result from interaction with cell surface receptors that trigger the expression of a number of cellular genes. Among the IFN-inducible gene products, the Mx proteins have attracted much attention because they have potential activity against influenza virus and possibly against other viruses. Mx proteins are guanosine triphosphate (GTP)-binding proteins with intrinsic GTPase activity. They seem to act indirectly against viruses by modifying cellular functions needed along the viral replication pathway. In mice the Mx1 protein has been shown to be necessary and sufficient to protect against influenza virus infection because the resistance does not require a functioning immune system. In humans the MxA protein has antiviral activities against influenza viruses. The MxA protein is encoded on the distal part of the long arm of chromosome 21 together with several other proteins implicated in the IFN system. Patients with Down's syndrome (trisomy 21) have an increased expression of MxA protein, and their cells display an increased sensitivity to IFNs in vitro because of gene dosage effects. These patients, however, are more susceptible to upper respiratory infection than normal individuals. This susceptibility has been related to deficiencies in the immune system. Therefore, induction of MxA in man does not sem sufficient to prevent influenza spreading, and, in contrast to the murine Mx system, a functioning immune system is necessary for protection. PMID- 7551416 TI - Pathogenesis of respiratory syncytial virus vaccine-augmented pathology. AB - Respiratory syncytial virus (RSV) is an important respiratory pathogen for which vaccine development has been thwarted by the legacy of vaccine-enhanced illness. A formalin-inactivated, alum-precipitated, whole virus vaccine did not protect children from infection and was associated with severe illness. Clues from clinical studies of RSV and vaccine-induced atypical measles illness suggest that an aberrant CD4+ lymphocyte response occurred in vaccinees. There is a growing body of evidence from murine models that show vaccine formulations can selectively activate different populations of CD4+ T helper lymphocytes that produce distinct cytokine expression patterns. The cytokine milieu in turn can influence the composition of the immune response and thereby impact the efficiency of virus clearance, type of pathology, and magnitude of illness. Major priorities of current vaccine development are to define the optimal combination of T lymphocyte subsets to safely clear RSV and to learn ways to modulate the composition of the immune response to vaccine antigens. PMID- 7551420 TI - The information and learning needs of the caregiving family of the adult patient with cancer. AB - The need for information by the caregiving family of an adult patient with cancer has been identified by research. This paper is a literature review of the information and learning needs of caregiving relatives. By meeting these needs the health care professional is able to help support the family, and therefore the patient, with the difficult adjustment to the diagnosis and through the subsequent course of the disease. The health care professional facilitates the individual caregivers' coping mechanisms and the benefits can be seen in the caregiving outcome for both the family and the patient. Information needs vary from individual to individual and are dynamic throughout the cancer experience. This paper describes the role of the nurse in this area, and encourages the nurse to act as a resource person for the caregiver by the appropriate delivery of information and by ensuring that the information given to the family by other health care professionals is understood and fulfils their needs. PMID- 7551419 TI - Bob Tiffany annual Nursing Lecture. Expert nursing: a necessary extravagance. PMID- 7551418 TI - A review of attenuation of influenza viruses by genetic manipulation. PMID- 7551421 TI - Appropriate palliative care: when does it begin? AB - The transition between a curative and a palliative approach to the care of a patient with cancer may be filled with uncertainty for patients, their families and health care professionals. A conventional model of treating the patient with cancer through curative, palliative and terminal phases is examined. The boundaries between the phases of care are blurred. A model of care based on respect for patient autonomy ensures that the timing of the switch from curative to palliative care is appropriate. PMID- 7551422 TI - The development of the Bottomley Cancer Social Support Scale. AB - At present, no social support scale exists that is cancer-specific. The objective of the study was to develop a cancer-specific scale that not only had validity in reflecting the experiences of cancer patients, but also one that was quick and easy to use in a busy clinical environment. Sixty patients with a primary diagnosis of cancer were selected from oncology wards and out-patient clinics, and they were administered the Bottomley Social Support Scale and the Hospital Anxiety and Depression Scale. The results indicate a valid and reliable social support scale that could be used in conjunction with other measures in a clinical setting. The clinical implications of the measure are that it will allow medical and support staff to assess the levels of social support and implement any appropriate social support interventions. PMID- 7551425 TI - An analysis of the concept of participation within the context of health care planning. AB - This paper analyses the concept of participation using the model of concept analysis developed by Walker and Avant (1988). The concept was chosen for analysis because of its extensive but often unqualified use in literature associated with the reform of the British National Health Service. The Walker and Avant model was chosen because of its capacity to identify the empirical referents of the concept. The analysis considers the use of the concept in the health and social policy literature from the 1960s to date, identifies the defining attributes prior to creating a model, a borderline and a contrary case, summarizes the antecedents and consequences of participation and offers three empirical referents which could be used in the construction of instruments for nursing research. The paper concludes with an assessment of the limitations of the analysis, an example of the use of the empirical referents in a research review and the identification of issues for those who manage or develop nursing services. PMID- 7551424 TI - Is managed care for nursing too? PMID- 7551423 TI - Acute leukaemia and malignant lymphoma patients' experiences of disease, treatment and nursing care during the active treatment phase: an explorative study. AB - Five acute leukaemia or highly malignant lymphoma patients at a hospital in southern Sweden were interviewed about their daily living problems, their coping strategies and their opinions about the nursing care they received during the active phase of their treatment. In addition the EORTC QLQ-C30, the Global Life Quality and the Sense of Coherence scales were administered. The data were analysed from a hermeneutic phenomenological perspective and interpreted to indicate that the patients sensed a threat to their lives, loss of control, and having to live with uncertainty stemming from the disease and the treatment. They had problems with fatigue, diarrhoea, nausea and vomiting, loss of appetite, sore mouth and high temperature. However, they seemed to minimize the importance of these problems and instead focused on gaining control of the situation, developing their knowledge of the disease and relying on the support of their family. Contradictions appeared in their statements about the quality of care, the information given was said to be good but difficult to understand; although the quality of the nursing care was judged to be high it had to be asked for. That is, help was received on request. The patients' perspective of the family and the nurses should be studied in further research in order to fully understand the patients' coping strategies and how nursing care can support them. PMID- 7551427 TI - A study of nurse managers' constructs of nurse researchers. AB - Recent fundamental changes in health care policy and provision have emphasized the need for a research culture within the National Health Service (NHS). The argument underpinning this revolves around the assumption that increasing the evidence-base of practice may have a significant impact on the quality of patient care. In consequence, members of the paramedical professions have been urged to become more research aware and research active. Despite these exhortations, however, there is mounting concern generally within the professions allied to medicine and, within nursing in particular, that the corpus of essential research evidence is not sufficient and that which exists is only partially effective in informing practice. While numerous explanations have been put forward to account for this, it is conceivable that the way in which the nurse's role is construed both by the nurse and by associated personnel is dysfunctional to the development of the nurse as researcher. To test this hypothesis, two groups of nurse managers were asked to consider how much of each of 15 bipolar dimensions was possessed by a nurse described either as 'a good clinician' or as 'a good researcher'. The results suggested that a nurse described as a good researcher was attributed with fewer of the characteristics assumed to be inherent to a good nurse. In this way, to be a good researcher appears to be fundamentally incompatible with being a good nurse. Moreover, a good researcher was also less likely to be employed. These results implicate the role of traditional attributions of nursing and are discussed in the context of the development of nursing research. PMID- 7551426 TI - The management of quality assurance in nursing. AB - The results of a national survey of hospital patients as they relate to nursing are presented. A brief review of the literature locates the study in the context of developments in nursing quality assurance, examining, in particular, conceptual issues of how the nursing process may be measured, and the role of the nursing profession in quality assurance. Results of the study identify a number of problems with the process of nursing care, including pain management, discharge planning and physical care, with wide variation between hospitals on many issues. The implications of these results for managers of nursing are discussed. PMID- 7551428 TI - The different appraisal profiles of a group of nurses and nursing aides: implications for policy initiatives. AB - This paper focuses upon the extent to which different groups of nurses and nursing aides are operating within appraisal systems and highlights how appraisals are largely offered on an ad hoc basis to a selected few. The implications of these findings are discussed in the light of recent developments within the health service generally and the nursing profession more specifically. PMID- 7551431 TI - 18th annual International Association of Human Caring Research Conference- Charlottesville, Virginia. PMID- 7551429 TI - Excavating health care policy: are league tables likely to dig out the truth about hospital performance or dig it in? AB - This paper considers the extent to which the publication of performance data on aspects of activity within the health service illuminates, or inhibits, judgments as to its effectiveness. In order to set the question of league tables into a wider perspective their use in relation to schools, and the impact of that initiative, is considered first. Research is shown to be sceptical as to the degree to which performance data can act as a proxy for institutional effectiveness. Misgivings centre around the criteria that determine, and the adequacy of the definition of, effectiveness together with concerns that the debate is at heart a political, not a professional one. In considering the question of the publication of performance data in the health service the same three issues, concerning the criteria determining, the definition of, and the politics of, effectiveness are applied. It is concluded that a case exists for the publication of data concerning institutions funded by the taxpayer, provided the information published does concentrate on institutional effectiveness, rather than serving as an attack on professional groups or as a distraction from concern about levels of expenditure. PMID- 7551430 TI - Developing the involvement of nurses in purchasing. AB - This article addresses the fact that nurses appear to be under-represented in National Health Service (NHS) purchasing, a situation which has recently been examined by a working group from the NHS Executive (1994) entitled 'Building a stronger team: the nursing contribution to purchasing'. A number of potential reasons for this situation, which is so important for the future of the nursing profession, will be outlined. A different perspective will then be introduced in the form of a psychological instrument, the Myers Briggs Type Indicator (MBTI) which will be described and related to this specific topic. The final section concerns a piece of current research which has the MBTI as its focus and contains material which is particularly relevant to the management of this problem. PMID- 7551432 TI - Tenacity--a central component in the preparation of clinical nurses. PMID- 7551433 TI - A drama within a crisis--relatives in the resuscitation room. PMID- 7551435 TI - Reflective process in action: the uncovering of the ritual of washing in clinical nursing practice. AB - Reflection can be utilized to reveal aspects of clinical practice which remain shrouded in ritualistic origin. The underlying themes surrounding the action of washing patients can be traced to Victorian legacies and the nurse education process. The nurse-patient relationship serves as a template for the deliverance of nursing interventions. An incomplete awareness of the nurse-patient relationship persists as to its initiation, continuation and usefulness in clinical nursing. Following reflection, there has been an increased awareness of feelings and attitudes surrounding the activity of washing a patient. PMID- 7551434 TI - Quality audit--a review of the literature concerning delivery of continence care. AB - This paper outlines the role of quality audit within the framework of quality assurance, presenting the concurrent and retrospective approaches available. The literature survey provides a review of the limited audit tools available and their application to continence services and care delivery, as well as attempts to produce tools from national and local standard setting. Audit is part of a process; it can involve staff, patients and their relatives and the team of professionals providing care, as well as focusing on organizational and management levels. In an era of market delivery of services there is a need to justify why audit is important to continence advisors and managers. Effectiveness, efficiency and economics may drive the National Health Service, but quality assurance, which includes standards and audit tools, offers the means to ensure the quality of continence services and care to patients and auditing is also required in the purchaser/provider contracts for patient services. An overview and progress to date of published and other a projects in auditing continence care and service is presented. By outlining and highlighting the audit of continence service delivery and care as a basis on which to build quality assurance programmes, it is hoped that this knowledge will be shared through the setting up of a central auditing clearing project. PMID- 7551437 TI - Assessing health needs in people with severe learning disabilities: a qualitative approach. AB - The nursing assessment, and the contribution it makes to the identification of an individual's health needs, is often a complex and multidimensional process. This is always true when the individual concerned has a severe learning disability. The nurse should develop the assessment in partnership with individuals who may experience major communication difficulties, or have unappreciated levels of perceptual or cognitive dysfunction. They may also live with the ongoing possibility that their affective or behavioural responses may be misinterpreted as 'challenging behaviour'. This case study focuses upon one particular young man with a severe learning disability living in a community home. Drawing upon the principles of qualitative research methods, especially those of 'grounded theory', this study aims to demonstrate how these can be used to develop a uniquely person-centered assessment. A systematic process of observation, analysis and reflection produces a detailed picture of the individual and his or her particular health states. The strength of this approach is threefold. Firstly, the person is considered as an active participant and remains the central focus. Secondly, in contrast to many other forms of assessment, the person is viewed as dynamic with ever changing needs. Thirdly, the process of nursing judgement is made explicit and integrated into the assessment process. PMID- 7551438 TI - Standardized Assessment for Elderly People (SAFE)--a feasibility study in district nursing. AB - Current policy requires systematic and comprehensive assessment of elderly people. Few standardized tools exist for assessment. This paper is in two parts. First it reports on the SAFE assessment measures agreed by a multidisciplinary working party initiated by the Royal College of Physicians and the British Geriatrics Society in 1992. The second part reports the findings of a feasibility study of SAFE in district nursing practice. The outcomes examined were the proportion of patients with completed assessments, the time taken to carry out the assessments, acceptability to the patient, carer and district nurse, and the training required. The findings show that SAFE is a practical and acceptable set of instruments in the assessment of elderly people by district nurses, but that further refinement is desirable. PMID- 7551439 TI - The description of levels in nursing degrees: an illustration and analysis of the variations. AB - This study has analysed the level descriptors of Higher Award degree programmes offered by a random sample of 15 colleges of nursing and midwifery in England. The analysis revealed a wide variation in the way each level (1, 2, 3 and M) had been defined. The variation occurred in a range of different ways. The main categories of variation were: the style of the descriptor; the features defined by the descriptor; the use of descriptors in the cognitive domain; the use of dimensions of professional knowledge and practice such as autonomy and reflective practice; and progression between the levels. The context of the study is described and the implications of the study and possible ways forward are discussed. PMID- 7551440 TI - Nursing home residents' perceptions of relocation. AB - The lived experience of relocating to a nursing home is explored from the perspective of 19 residents of Australian nursing homes. The interpretative research methodology is informed by hermeneutic phenomenology. In-depth interviews were conducted with 19 nursing home residents living in nursing homes in NSW. Interviews were audiotaped, transcribed and analysed to identify emergent themes. The themes are described and interpreted to provide an understanding of the relocation experience. PMID- 7551436 TI - An action research project to develop and evaluate the role of an advanced nurse practitioner in dementia. AB - This paper outlines the first phase of a project to generate and evaluate the role of an advanced nurse practitioner inductively from first principles. The role was developed by interviewing a wide range of health-care professionals, careers and patients. The resulting role is responsive to perceived need at grassroots level rather than to the needs of managers with no clinical experience. Although the role might not be generalizable to other settings, the method can be applied to any role in any clinical area. PMID- 7551442 TI - Community psychiatric nurses: their role as trainers in schizophrenia family work. PMID- 7551444 TI - [De Bleerinck in the Netherlands. A special village for 186 patients with dementia]. PMID- 7551443 TI - [Care for cancer patients. A continuing education seminar on the promotion of competence and health for nursing personnel and medical technologists]. PMID- 7551445 TI - [Care and environmental protection. How cellulose is made from wood]. PMID- 7551441 TI - The art of clinical supervision. AB - Helping nurses to become effective and confident as primary nurses involves developing their interpersonal skills, sharpening their decision-making and nurturing their creativity. In our action research study, we found that traditional methods of ward teaching did not help to promote this kind of professional growth. We therefore developed, tested and refined three distinct strategies for clinical supervision which specifically aimed to foster more thoughtful and more sensitive nursing practice. Very importantly, we found that these approaches to supervision were feasible and practical to use in a busy hospital ward. In this paper, we describe the three supervision strategies, using a clinical story to illustrate what each demands from the supervisor and what it offers the nurse under supervision. We also suggest ways in which expert nurses can be helped to become effective clinical supervisors. PMID- 7551446 TI - [Water alone is not enough. Even intact skin needs care]. PMID- 7551447 TI - [FIPS, Inc.--home pediatric nursing. For sick children too there is an alternative to hospital care]. PMID- 7551448 TI - [Counseling in the hospital. Incontinence is not an unalterable fate]. PMID- 7551449 TI - [Working time legislation 1994. Important provisions for hospitals]. PMID- 7551450 TI - [A new concept in teaching First Aid. Questioning shows: education according to the care-centered first aid concept is judged positively]. PMID- 7551451 TI - [Compensation in nursing. A new model of tariffs by the nursing union]. PMID- 7551452 TI - [Home care services impaired]. PMID- 7551453 TI - [Safeguarding the intimate sphere while bathing the genitals. Study of a taboo topic]. PMID- 7551454 TI - ["Not this way, Mr. Blum!"]. PMID- 7551455 TI - [Nursing counseling at the Colon University Hospitals: seven women are beating the theory-practice conflict]. PMID- 7551456 TI - [Nursing and environmental protection. III. Diapers "made in The Netherlands"]. PMID- 7551457 TI - [Temporary dwelling. The bed is the place of security for the hospitalized patient]. PMID- 7551459 TI - [3000 want to experience Naomi Feil]. PMID- 7551458 TI - [Nursing emphasis on pneumonia--prevention and therapy. The second most frequent hospital infections are pneumonias]. PMID- 7551460 TI - [New perspectives for the nursing service--experiences from the St. Josef Hospital in Moers]. PMID- 7551461 TI - [Germ reduction in the hospital. Surface and spray disinfections are no cure alls]. PMID- 7551462 TI - [The problematic right to a dignified death. 1. Limits to assistance to die under criminal law]. PMID- 7551463 TI - [Work time legislation 1994. The hospital needs specific service times]. PMID- 7551464 TI - [Nursing in Namibia. Nurses have extensive responsibilities]. PMID- 7551466 TI - [Scientific activities in nursing. Nursing research has grown up in Germany]. PMID- 7551465 TI - [A topic neglected in education. Sexuality is not a privilege of the young]. PMID- 7551467 TI - [Computers in schools: special software is facilitating school administration]. PMID- 7551468 TI - Pathways to suicide prevention. AB - Society places a responsibility on the medical profession for the prevention of suicide. In this article, current trends in suicide, social prevention strategies and the role and limitations of medical intervention are reviewed. PMID- 7551469 TI - Insufficiency fractures of the sacrum and os pubis. AB - Insufficiency fractures are increasingly seen among postmenopausal women. Sacral fractures are difficult to detect clinically and radiographically, while os pubis fractures may mimic malignancy. Diagnosis is made by bone scintigraphy, supplemented by computed tomography. As patients respond well to conservative management, increased awareness and use of appropriate imaging may avoid unnecessary and invasive investigations. PMID- 7551470 TI - Essential tremor and related disorders. AB - Essential tremor is a common neurological disorder. This article reviews the phenomenology of essential tremor and related disorders (excluding parkinsonian and dystonic tremors), summarising current theories on their pathogenesis. The various treatment options are also discussed. PMID- 7551471 TI - Drug delivery in asthma. PMID- 7551472 TI - Uses and limitations of pulse oximetry. AB - Pulse oximeters are a useful method of monitoring patient welfare in many circumstances. However, the technique of pulse oximetry has limitations, and there is little or no formal training available. Patient safety could be compromised if pulse oximeters were used by staff with inadequate knowledge. PMID- 7551473 TI - The place of research in surgical training. PMID- 7551474 TI - Heart failure: an old problem. AB - Heart failure is an increasingly common problem in the UK, particularly in older patients. This article reviews the available information is available on heart failure in the older population, and provides clinically useful guidelines on the investigation and treatment of patients with this condition. PMID- 7551475 TI - An unusual family with precocious puberty. PMID- 7551476 TI - Herpes zoster ophthalmicus masquerading as giant cell arteritis. PMID- 7551477 TI - Contracting through district health authorities. AB - Contracts are the glue of the new NHS, the vital tool linking the two main structures: health-care commissioning and health-care provision, carried out by GP fund holders and district health authorities (the purchasers) and the NHS trusts and others (the providers) respectively. Like all adhesives, contracts are of great benefit if used carefully, but if applied inappropriately or with an overly heavy hand they have the potential to become an obstructive mess. PMID- 7551478 TI - Anaesthesia during carotid surgery. PMID- 7551479 TI - Anaesthesia during carotid surgery. PMID- 7551480 TI - Anaesthesia during carotid surgery. PMID- 7551481 TI - Airway control in a patient with an unstable cervical fracture and delirium tremens. PMID- 7551484 TI - Advance statements about medical treatment. AB - The BMA recently produced comprehensive professional guidance on advance statements and medical treatment, in the form of a Code of Practice and explanatory notes. However, some of the guidance is problematic. This article reviews various aspects of the Code and recommends its use as a set of essential explanatory notes to health-care professionals. PMID- 7551482 TI - The 6-week postnatal visit: are we doing it right? AB - The 6-week postnatal visit has been practised for a long time as a follow-up visit after childbirth. Although the idea behind it is still as sound as it was half a century ago, its timing and contents need to change in line with recent research findings. PMID- 7551483 TI - Drug therapy of peptic ulcer disease. AB - Healing of peptic ulcers can be achieved by using a variety of anti-ulcer medications. The most commonly used agents include the histamine-2 receptor antagonists (H2RAs) and the proton pump inhibitors. They are also efficacious in preventing ulcer recurrence providing maintenance treatment is given. The ideal treatment for peptic ulcers today is aiming at eradication of Helicobacter pylori infection. Successful elimination of the latter not only heals the ulcers but also provides a cure for the disease, so that the patients will no longer require lifelong maintenance medical therapy. PMID- 7551485 TI - Siderosis bulbi: an unusual cause of a unilaterally dilated pupil. PMID- 7551486 TI - Cholangiocarcinoma of the gall bladder presenting as a gastrohepatic fistula. PMID- 7551489 TI - Angiotensin-converting enzyme inhibitors. PMID- 7551492 TI - The challenge of nuclear medicine. PMID- 7551491 TI - An academy of medicine: a future for British postgraduate medicine? PMID- 7551488 TI - Abdominal anaesthesia. PMID- 7551490 TI - Suppurative thrombophlebitis. PMID- 7551493 TI - Tumour imaging. AB - This review looks at the nuclear medicine scanning techniques that are available for tumour imaging. It aims to promote an understanding of the specific value of this imaging modality, and thereby help to conserve resources, save time, and assist clinicians in providing optimal care. PMID- 7551487 TI - Cultural changes within the NHS. AB - The NHS is in the throes of profound change. How much of it is cultural? Indeed, what do we mean when we talk about the culture of an organisation? And if we succeed in defining culture, how does an understanding of this concept help us to manage the service better? This article sets out to answer these questions and argues that an understanding of organisational culture is a necessary part of good human resource management. PMID- 7551494 TI - Scintigraphic techniques for delineation of infection and inflammation. PMID- 7551495 TI - The clinical challenge of nuclear medicine in gastroenterology. PMID- 7551496 TI - Glomerulonephritis in children. AB - Children with glomerulonephritis may present with acute nephritic or nephrotic features. Glomerulonephritis may be a primary diagnosis or may be secondary to systemic illness. Children with persisting renal impairment, hypertension or proteinuria require careful investigation and assessment by a paediatric nephrologist. PMID- 7551497 TI - Advances in diabetic ketoacidosis and hyperosmolar syndrome pathogenesis and management. PMID- 7551498 TI - [Analysis of the increased frequency of cesarean section in the Czech Republic]. AB - From the results of the nationwide analysis of the quality of perinatal care made every year ensues that in the period from 1986-1993 the mean annual increase of caesarean sections (SC) increased by 0.5%. In this increment more than 90% account for deliveries of neonates with a birth weight of 2,000 g and more without VVV. The effectiveness of the increment of SC from 7 to 10% measured by the decrease of perinatal mortality rate represents a decrease by 0.7% only. This effectiveness is higher in perinatological centres than in hospitals which refer pathological pregnancies to centres, and is lowest in hospitals which do not refer these cases. As to different indications which participated in the increased frequency of SC participated mainly foetal hypoxia, preceding SC, dystocia and disproportion. The higher the frequency of SC, the more frequently the decision taking of the doctor as regards these indications is influenced also by non-medical standpoints. PMID- 7551499 TI - [Actocardiography in obstetrics]. AB - Introduction of ultrasonic techniques made more detailed follow-up of the motor activity of the foetus in its natural environment possible. The objective of the submitted paper is to present actocardiography as a method which makes it possible to visualize differences of motor activity of the foetus, in particular during the third trimester and to map different types of movements. The authors obtained thus objective records in a group of pregnancies which will be suitable for comparison with the motor activity of foetuses with signs of hypoxia, with foetuses with retarded growth or foetuses threatened by other risks. This method can be thus included and used as a method of routine in the spectrum of examinations of imminent foetal stress. PMID- 7551500 TI - [The transport system in the in vitro fertilization program]. AB - The programme of in vitro fertilization helps at present successfully to resolve the problem of sterility in an ever increasing number of married couples. It is, however, a method which is very pretentious from the technical aspect, from the aspect of time and financial demands. Therefore in February 1994 with the Institute for the Care of Mother and Child in Prague-Podoli collaboration in the Transport IVF programme was started. In our clinic the first two stages of the IVF programme are implemented, i.e. stimulation of the ovaries and aspiration of the follicles. The patients of our centre for sterility are included in the programme, if the following indications are present: tubal factor 82%, idiopathic sterility 4%, the remainder being endometriosis, andrological factor and others. The majority of women were in the age group from 30-35 years. Stimulation is most frequently implemented by GnRh analogues and menopausal gonadotropins, aspiration of follicles by the transvaginal route with short analgosedation under so-called semi-ambulatory conditions. The aspirated follicular fluid is immediately stored in a special transport incubator G-812 of K-system Co., Denmark. Within 20 minutes it is taken by the patient's husband to the embryological laboratory of the Institute for the Care of Mother and Child in Prague-Podoli. Between February and December 1994 a total of 69 aspirations in 56 patients were made and 15 pregnancies were achieved. It is 23% pregnancies by transfer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551503 TI - [Successful pregnancy and delivery after assisted fertilization in the IVF and ET program at the Institute for Maternal and Child Care]. PMID- 7551502 TI - [Transportation in utero]. PMID- 7551501 TI - [Vulvar varices]. AB - The authors treated in 1983-1993 72 patients with vulvar varicosities who suffered particularly during pregnancy. Typical symptoms are pruritus, pain caused by pressure in the vulvar area and the sensation of prolapse. According to the authors surgical treatment is unnecessary and involves risk. They recommend compressive sclerotherapy as described by Fegan, using sodium tetradecyl sulphate S.T.D. Hegefort England not only in case of a marked clinical symptomatology but also as prevention of dangerous haemorrhage during delivery. PMID- 7551504 TI - [The polycystic ovary syndrome]. PMID- 7551505 TI - [Prevention, diagnosis and therapy of thromboembolic complications and disseminated intravascular coagulation in obstetrics]. PMID- 7551506 TI - A versatile two-stage hypospadias repair. AB - One-stage repair of hypospadias is currently fashionable and is undoubtedly attractive in concept but the methods that are presently available all have inherent limitations and drawbacks. The author presents a two-stage method which offers a unique combination of versatility, reliability and refinement, and can be used for almost any hypospadias deformity, be it primary repair in a child or salvage surgery in an adult. A personal series of 600 cases is analysed. PMID- 7551507 TI - The extended gracilis muscle flap for reconstruction of the lower leg. AB - The width of the gracilis muscle was measured before and after removal of the epimysium in 10 fresh cadavers. The average extent of muscle widening achieved by epimysium removal was over 100% (mean 112.6%; standard deviation 11.9%). This extended muscle flap enabled us to cover successfully even large soft tissue defects measuring up to 300 cm2. In 27 consecutive patients, soft tissue defects of the lower leg with exposed bone have been repaired by free tissue transfer of a gracilis muscle flap, covered with split skin grafts. The advantages of the gracilis muscle flap were the low donor site morbidity with almost no recognisable functional loss, the easy surgical access, the thin and flat shape of the muscle, and its adequate size after excision of the epimysium. Reconstruction with a gracilis muscle flap resulted in an inconspicuous, stable, and flat contour of the lower leg. The entire length of the vascular pedicle of the gracilis muscle was easily harvested in our patients by routinely dissecting the pedicle on both sides of the adductor longus muscle. PMID- 7551508 TI - Photodynamic therapy of primary skin cancer: a review. AB - The role of photodynamic therapy (PDT) in the treatment of primary non-melanoma skin cancer is examined. Prolonged systemic skin photosensitivity limits the usefulness of PDT using conventional photosensitisers such as Photofrin-II. However in exceptional circumstances, such as multiple or widespread basal cell carcinomas, this therapy provides a useful and seemingly effective alternative mode of treatment. For Bowen's disease, PDT using topical 5-aminolaevulinic acid (ALA) yields high response rates and excellent cosmetic results. For large lesions and those in anatomically difficult sites or in poorly vascularised skin, ALA-based PDT can be considered the treatment of choice. Recent pharmacological and technological developments may further enhance the efficacy and convenience of photodynamic therapy, and make it more generally available in non-specialist centres. PMID- 7551510 TI - Paradoxical clinical consequences of peripheral nerve injury: a review of anatomical, neurophysiological and psychological mechanisms. AB - This paper reviews some of the possible explanations and mechanisms that may be responsible for variation from expected clinical findings soon after nerve injury in certain patients, and for subjective sensations and objective sensibility which can appear to arise from within the autonomous zone of the cutaneous distribution of a divided nerve. A number of features of peripheral innervation, central nervous system physiology and sensory psychology are discussed. These include: (1) the normal extent of overlap- or cross-innervation between the territories of adjacent peripheral nerves; (2) anomalous innervation due to normal anatomical variation; (3) ectopic impulse generation and cross-excitation between neurons in the peripheral nervous system after nerve injury; (4) neurophysiological responses and mechanisms of re-innervation other than axon regeneration across the site of nerve repair; (5) cortical somatotopic reorganisation in response to nerve injury; and (6) phantom sensory phenomena including the psychology of sensory perception. PMID- 7551511 TI - Repair of complete syndactyly by tissue expansion and composite grafts. AB - Repair of complete syndactyly by a combination of tissue expansion and composite grafts from the glabrous non-weight bearing areas of the foot has been performed on three syndactylies in two patients. The commissure and the lateral areas of the proximal and middle phalanges were covered with expanded skin and the separated fingertips were covered with composite grafts. Without using an ordinary skin graft, this method can provide aesthetically excellent results with good skin colour and texture. PMID- 7551509 TI - Paradoxical clinical consequences of peripheral nerve injury: conduction of nerve impulses does not occur across the site of injury immediately following nerve division and repair. AB - The occasional apparent clinical phenomenon of the immediate although transient return of peripheral nerve function after nerve division and primary repair has been previously reported. Electrophysiological findings from the sciatic nerve of the rabbit have been previously presented in support of the concept of the transmission of nerve impulses across a freshly divided and repaired peripheral nerve for a short period until the onset of Wallerian degeneration. This paper presents experimental evidence to show that these findings were misinterpreted and that a compound action potential cannot be transmitted across a surgically repaired division in a peripheral nerve. Observations from previous experimental research in neurophysiology are discussed which confirm these conclusions. The claim that failure to diagnose a peripheral nerve injury at presentation can be explained in some circumstances by "jump transmission" is not based on sound evidence and the concept of "jump transmission" cannot be accepted as a defence for clinicians who fail to diagnose a peripheral nerve injury. PMID- 7551512 TI - A neurovascular island flap including a vein for the treatment of an acquired ring constriction. AB - We present a case of an acquired constriction band caused by a tight ring on the left middle finger of a 45-year-old man. Most of the constriction band was excised and the resulting defect reconstructed with a neurovascular island flap, including a dorsal digital vein, from the adjacent ring finger. The distal end of the artery and the nerve in the flap were anastomosed and coapted to the digital artery and nerve, respectively, at the distal edge of the defect after excision of the band. The vein in the flap was anastomosed to a dorsal vein at both the distal and proximal edges of the flap as a vascularised interpositional vein graft. Four months later, the remaining constriction band was excised and the defect closed by an advancement flap. Swelling of the finger decreased and sensation improved. PMID- 7551513 TI - Disappearance of Langerhans cells and melanocytes after cryopreservation of skin. AB - Cryopreserved skin allografts have been extensively reported to remain viable for longer periods after grafting, both in the laboratory and in the clinic, than skin stored by other methods. We investigated the immunocytochemical and electron microscopic properties of samples of cryopreserved human skin (-196 degrees C) for comparison with fresh samples. In an immunocytochemical study of fresh skin, reagents S-100 and CDIa indicated numerous mesenchymal origin cells in the squamous cell layer, basal layer and dermis; 2B7 identified these cells in the basal layer and PC-10 identified them in the basal and squamous cell layers. In cryopreserved skin, however, few cells reacted to these reagents. An electron microscopic study of the cryopreserved skin showed Langerhans cells (LC); however, these had suffered degeneration, with partial defects of the cell membrane and vacuolation in the cytoplasm. We speculate these effects are responsible for the virtually complete abolition of LC membrane and cytoplasm markers. In summary, we detected few mesenchymal origin cells, melanocytes, Langerhans cells, or S-phase cells, in cryopreserved skin by immunocytochemical methods. Langerhans cells existed but had degenerated. These results indicate that cryopreservation at -196 degrees C causes degeneration of Langerhans cells, and that is the reason for the prolonged viability of cryopreserved allograft. PMID- 7551515 TI - Sialadenosis of the salivary glands. AB - Sialadenosis (sialosis) is an uncommon, non-inflammatory condition which usually causes bilateral, diffuse enlargement of the salivary glands, particularly the parotid. We present a series of 7 patients with sialadenosis. Two had bilateral and 4 unilateral parotid involvement. One patient had unilateral submandibular gland sialadenosis. One patient had bilateral parotid and bilateral submandibular gland sialadenosis. The clinicopathological features and management of the condition are reviewed. PMID- 7551514 TI - What are the priorities in plastic surgery? Kay-Kilner Prize Essay, 1994. PMID- 7551516 TI - Abdominoplasty following vertical banded gastroplasty for morbid obesity. AB - We describe 15 patients who underwent abdominoplasty following vertical banded gastroplasty for morbid obesity between 1991 and 1994. Vertical banded gastroplasty was performed (by CMSR) on patients with a Body Mass Index greater than 39. Following this, the patients lost weight rapidly, leading to excess folds of skin and fat on the abdomen, arms and thighs, and were referred for plastic surgery when their weight had stabilised. After abdominoplasty, the Body Mass Indices of all the patients decreased to an acceptable range. Because vertical banded gastroplasty causes little long-term metabolic or nutritional disturbance, the abdominal skin could be undermined up to the costal margin and the umbilicus re-sited without major necrosis of the abdominal wall or umbilicus. All patients received prophylactic low dose heparin perioperatively until early ambulation. Prophylactic antibiotics were not used but there were no major wound infections. Patients were reviewed up to 12 months after abdominoplasty and were satisfied with the results. Abdominoplasty following vertical banded gastroplasty for morbid obesity safely provides acceptable cosmetic results. PMID- 7551517 TI - Fungal growth within a silicone tissue expander: case report. AB - Fungal contamination of tissue expanders has not previously been reported. There are, however, reports of fungi in association with inflatable breast prostheses. Colonisation of a tissue expander with Aspergillus niger resulting in mechanical obstruction of the device is described. The possible modes of inoculation and survival of the organism within the expander envelope were studied including an investigation of the permeability to gases of the silicone expander envelope. Recommendations are made about prevention of this complication. PMID- 7551519 TI - Human tails. PMID- 7551520 TI - Super-thin flaps. PMID- 7551518 TI - Chest wall reconstruction by free latissimus dorsi flap 25 years after accidental exposure to a source of industrial irradiation. AB - 25 years after successful plastic surgical closure of a large, radiation-induced ulcer of the chest wall the patient developed recurrent ulceration and unstable scars requiring a second procedure. A quarter of a century of progress in plastic surgical procedures is demonstrated. PMID- 7551521 TI - The arterial anatomy of the temporal region and the vascular basis of various temporal flaps. AB - The arterial anatomy of the temporal region was examined macroscopically and radiographically in 10 fresh cadavers which had been injected with lead oxide. The blood supply of the temporal region is derived from the superficial temporal, middle temporal, deep temporal, posterior auricular, transverse facial, zygomatico-orbital, zygomaticotemporal, zygomaticofacial, and middle meningeal arteries. The vascular network formed by these arteries can be divided into four arterial networks corresponding to the different layers of the temporal region. With a new understanding of the arterial networks and their anastomoses, the techniques for elevating various flaps in the temporal region are discussed. PMID- 7551522 TI - A retrospective analysis of the results of 218 consecutive rhinoplasties. AB - In a 7-year period, 218 consecutive rhinoplasties were performed within a joint rhinoplasty service comprising a plastic surgeon and an otorhinolaryngologist. Retrospective analysis of these procedures revealed a minor surgical complication rate of 5%, no major complications and requests for revisional surgery in 10% of patients after primary rhinoplasty by the service. However when patients had not had their first operation by our joint service, the revisional operation rate after our first operation was 19%. These results are discussed in relation to other published series. The data generated should enable more precise preoperative patient counselling and act as a useful baseline for subsequent audit of performance. Specifically, patients should know that after rhinoplasty 1 in 10 patients may request revisional surgery. All trainers and trainees should be aware of such data on their own patients in order that standards be set and enhanced with time. PMID- 7551523 TI - The sensate deep inferior epigastric musculocutaneous flap and the twelfth thoracic nerve. AB - A detailed anatomical study of the terminal branches of the twelfth thoracic nerve (subcostal nerve) based on observations made during dissections of 23 embalmed cadavers is presented. In all 23 cadavers, the twelfth thoracic nerve had an ascending branch which joined the deep inferior epigastric vascular pedicle about half way between the lower border of the umbilicus and the symphysis pubis. This observation, we believe, explains how it is possible to preserve sensation in the deep inferior epigastric musculocutaneous flap. PMID- 7551524 TI - A modern translation and interpretation of the treatise on burns of Fabricius Hildanus (1560-1634). AB - At the beginning of the seventeenth century, some surgeons were as interested in the classification, prognosis and treatment of burns as their successors are at the close of the twentieth century. We present extracts from our translation of the visionary treatise on Burns by Fabricius Hildanus (Wilhelm Fabry), the "Father of German surgery", and show how some of the present day approaches to burns have their origins almost 400 years ago. PMID- 7551525 TI - Facial reconstruction after a burn injury using two circumferential radial forearm flaps, and a dorsalis pedis flap for the nose. AB - A 21-year-old man is reported in whom flap tissue was required to reconstruct the face and nose following burns. The whole of both forearms were used as radial forearm flaps for both cheeks, both lower eyelids, the right upper eyelid, upper and lower lips and chin. A Tagliacozzi arm flap and a dorsalis pedis flap were used for the nose. The ears were resurfaced using grafted skin along the line of the superficial temporal arteries. No thinning of the flaps was required and some facial muscle attachments to the forearm deep fascia led to more recovery of facial movement than might be expected with a flap reconstruction. PMID- 7551526 TI - Amniotic membrane as a cover for microskin grafts. AB - Twenty two granulating wounds were grafted with microskin grafts of 1 mm size and amniotic membrane was used to cover these grafts. In 16 patients, epithelialisation was complete by 10 days and the grafts remained stable. Fifteen fold expansion was achieved in all these cases. The remaining 6 patients needed split thickness skin grafts due to failure of the microskin grafts. Human amniotic membrane as a cover for microskin grafts helped early epithelialisation and rapid wound healing. PMID- 7551527 TI - Evaluation of angiogenesis in delayed skin flaps using a monoclonal antibody for the vascular endothelium. AB - The angiogenic process in delayed and undelayed flaps was evaluated in relation to different periods of delay (6 h, 72 h and 7 days) and at different times after the definitive elevation of the flaps (48 h, 96 h and 7 days). An immunohistochemical method has been tested to stain the endothelial cells of capillaries and venules, providing a high specificity for labelling these vessels. No signs of angiogenesis were found in undelayed and 6-h delayed flaps at 48 h but there was an increase of capillaries from 48 h to 7 days after elevation of the flaps. The 72-h and 7-day delayed flaps showed signs of angiogenesis at 48 h and the capillary development went on increasing to the 7th day after definitive flap elevation (p < 0.05). The surviving lengths of these groups of flaps were higher than the control and 6-h delayed flaps (p < 0.05). These results indicate that an angiogenic process can be implicated in the physiology of the delay phenomenon. PMID- 7551528 TI - Secondary procedures to improve function after toe-to-hand transfers. AB - From August 1990 to December 1992, 139 toe-to-hand transfers were performed on 99 patients. Crush, avulsion, and combined crush and avulsion were the mechanisms of injury in 92.8% of the cases. Average duration of follow-up was 17 months (1 to 40 months). 133 transfers were successful, an overall survival rate of 95.7%. 19 transfers (14.3%) required secondary procedures for functional improvement. The incidence of secondary procedure on tendon, bone, joint and soft tissue was 9.0%, 1.5%, 2.3% and 3.0% respectively. Flexor tenolysis was the single most common secondary procedure (6.8%). The results of secondary procedures were satisfactory in all but one instance. Transient neurapraxia of an ulnar digital nerve after tenolysis was the only complication of the secondary procedures. PMID- 7551529 TI - Different muscle graft denaturing methods and their use for nerve repair. AB - In an attempt to enhance recovery through denatured muscle grafts we have used different methods to denature muscle, either using dry heat in a microwave oven or by warming in sterile distilled water. Axonal regeneration was studied at intervals from day 5 to day 60 after insertion of the muscle grafts and compared to results found in frozen-thawed muscle grafts and in autologous nerve grafts used as controls. Axonal regeneration, Schwann cell behaviour and the degree of inflammation were quantified using immunohistochemical techniques and computerised image analysis. Autologous nerve grafts supported the highest rates and volumes of axonal regeneration until day 30 when the microwaved muscle grafts had the highest values for axonal immunostaining and at 60 days the highest level of Schwann cell immunoreactivity. We conclude that microwave heating is suitable as an alternative denaturing method for successful muscle grafts and has a potential clinical use. PMID- 7551530 TI - Urethral reconstruction with autologous vein graft: an experimental study. AB - There is no universally accepted material for urethral reconstruction. This study presents the results of segmental urethral replacement with a free graft of jugular vein in rabbits. Histological examination showed ingrowth of normal transitional epithelium into the venous endothelium. Retrograde urethrograms revealed an excellent result up to 300 days. Fistulas and infection occurred in 4/44 rabbits; these settled spontaneously. No structures or papillary hypertrophy were noted. Segmental urethral reconstruction with autologous vein graft is a simple technique with few complications and appears suitable for use in clinical cases. PMID- 7551531 TI - Closure of an ischial pressure sore using a free gastrocnemius musculocutaneous flap with a long venous pedicle. AB - The recurrent pressure sore in an active paraplegic can become a frustrating challenge as available simple flap options become depleted. The medial gastrocnemius musculocutaneous free flap is an alternative that offers the benefits of muscle from an expendable donor site and still preserves the remainder of the donor limb for future use. Inclusion of an intact greater saphenous vein could augment venous drainage and potentially might be the sole means of outflow if veins at the recipient site were inadequate. This case report describes the closure of a recurrent ischial pressure sore with a free medial gastrocnemius flap, including a greater saphenous vein pedicle. PMID- 7551532 TI - Reconstruction of aplasia cutis congenita of the scalp by split rib cranioplasty and a free latissimus dorsi muscle flap in a nine month old infant. AB - We present a case of reconstruction of severe aplasia cutis congenita. Initial treatment with silver sulphadiazine dressings failed and local scalp repair of the defect at age 3 months also failed to provide a stable reconstruction. At age 9 months the infant was experiencing increasing apparent hydrocephalus, shunt infection and brain herniation. The definitive reconstruction was performed with a split rib cranioplasty and a free latissimus dorsi muscle flap. The reconstruction was successful and the infant has a normal head shape and no neurological deficit 12 months postoperatively. PMID- 7551533 TI - Hanhart's syndrome with facial asymmetry: case report. AB - An unusual variant of Hanhart's syndrome associated with facial asymmetry in a female patient is presented together with surgical treatment of her facial anomalies. Following reconstruction which included bimaxillary osteotomies and osteopericranial and pericranial flaps, a good aesthetic result was achieved which enabled the psychological and social rehabilitation of the patient. PMID- 7551534 TI - Free combined parascapular flap and latissimus dorsi muscle flap for facial palsy and neck reconstruction. PMID- 7551535 TI - Irreversible muscle contracture after functioning free muscle transfer for facial palsy. PMID- 7551536 TI - Irreversible muscle contracture after functioning free muscle transplantation using ipsilateral facial nerve for reinnervation. PMID- 7551537 TI - Cross-face reanimation of the paralysed face, with single stage microneurovascular gracilis transfer without nerve graft. PMID- 7551538 TI - A pre-expanded island scapular flap for contracture of the neck. PMID- 7551539 TI - Treatment of ischial pressure sores with an inferior gluteus maximus musculocutaneous island flap. PMID- 7551540 TI - Genome reorganization from polyteny to polyploidy in the nurse cells found in onion fly (Delia antiqua) and cabbage root fly (Delia radicum) ovaries (Diptera, Anthomyiidae). AB - The material required to ensure successful embryogenesis in the onion fly (Delia antiqua) and the cabbage root fly (Delia radicum) (Diptera, Anthomyiidae) is supplied by 15 nurse cells, while the oocyte chromosomes enter a quiescent stage during prophase I of meiosis. This level of transcription is achieved by the polyploidization of the nurse cell DNA. Elongate polytene chromosomes form in both species, but lack the banding and conspicuous puffing commonly seen in other dipteran tissues. The polytene chromosomes contract until they finally appear as small, densely staining spheres. These fragment into large numbers of endochromosomes that are much smaller than their mitotic counterparts, which then despiralize, resulting in the flocculate appearance of the nurse cell nucleus. Photodensitometry revealed a gradient of DNA values between nurse cells near the oocytes and those further away. Final DNA values 1000 times the haploid level were recorded in the nurse cell nearest to the oocyte compared with 336 times the C-value in the most distal cell. At lower temperatures (< 10 degrees C), the polytene chromosomes become banded and longer. None of the onion flies kept in these conditions produced viable eggs, though there was some reproductive success among the cabbage root flies. PMID- 7551541 TI - High frequency of mutagen-induced chromatid exchanges at interstitial telomere like DNA sequence blocks of Chinese hamster cells. AB - Interstitial telomere-like DNA sequence arrays of Chinese hamster Don cells were delineated by fluorescence in situ hybridization in quadriradial and triradial chromosome configurations induced by X-rays, mitomycin C and teniposide (VM-26). Around 40% of the scored exchanges involved a telomeric-like block of sequences at a rearrangement site. This frequency was independent of the DNA-damaging agent, and the result suggests a general recombinogenic capacity of interstitial telomere-like DNA sequence repeats that, at least in the case of the agents employed, seems not to be related to the initial mechanism of DNA damage. PMID- 7551542 TI - Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involving Leporinus friderici. AB - Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyotype, whereas others probably have several. In the present study we analyzed the NORs of Leporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods, i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A3, and in situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re evaluated by methods such as chromomycin A3 staining and in situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals. PMID- 7551545 TI - Topographic changes in a heterochromatic chromosome block in humans (15P) during formation of the nucleolus. AB - Fluorescence in situ hybridization and multispectral confocal laser scanning microscopy revealed a highly dynamic nucleolar-associated chromosome 15 satellite III heterochromatin cluster in humans. This nucleolar-associated DNA was highly decondensed at the metaphase plate compared with its topography at interphase and appeared to act as a centre for the post-mitotic reorganization of the nucleolus. Our data showed unexpected trans-mitotic changes in the topography of this nucleolar-associated satellite III DNA that suggest that this locus-specific heterochromatin superstructure may be involved in nucleolar organization. PMID- 7551543 TI - Chromosomal evolution in bovids: a comparison of cattle, sheep and goat G- and R banded chromosomes and cytogenetic divergences among cattle, goat and river buffalo sex chromosomes. AB - A G- and R-banding comparison of cattle (Bos taurus, 2n = 60), goat (Capra hircus, 2n = 60) and sheep (Ovis aries, 2n = 54) chromosomes at the 450 band level was made. The study revealed a large number of banding homologies among the autosomes of the three species and resolved some ambiguities in arranging some of their small disputed acrocentrics by direct and indirect comparisons with some bovid marker chromosomes. A loss of the subcentromeric G-positive band in sheep chromosome 2g was observed when the G-banding patterns of sheep 2q and homologous cattle and goat chromosome 2 were compared. The chromosomal divergences among cattle, goat and river buffalo (Bubalus bubalis, 2n = 50) sex chromosomes are shown to have occurred by pericentric and paracentric inversions with a loss (or acquisition of constitutive heterochromatin. PMID- 7551544 TI - Mouse p87wee1 kinase is regulated by M-phase specific phosphorylation. AB - We have cloned a mouse wee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50wee1 kinase reported previously. In a cell cycle, the mouse wee1 kinase is phosphorylated at M-phase, and an in vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the human wee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated by cdc2-cyclin B kinase. Furthermore, the activity of the wee1 kinase was reduced by phosphorylation with the mitotic extract which contained cdc2-cyclin B kinase. PMID- 7551546 TI - Barley telomeres are associated with two different types of satellite DNA sequences. AB - The genomic organization of two different types of satellite DNA sequences was analysed by means of fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) in barley. Satellite HvT01 was detected at all chromosome ends except the long arms of chromosomes 2 and 7. The unrelated satellite pAS1 was found at all chromosome ends except the long arm of chromosome 7 and at two interstitial sites, both located on the long arm of chromosome 4 on the standard karyotype. Southern and in situ hybridization further indicate that pAS1 also occurs interspersed in the barley genome. For most chromosome ends, the linear order of HvT01 and pAS1 could not be determined by in situ hybridization except at the short arms of chromosomes 2 and 6, where HvT01 is more distal than pAS1. This is confirmed by PFGE analysis, HvT01 being frequently associated with the telomeric repeat but not pAS1. Furthermore, we found that HvT01 occurred in clusters up to 1000 kb in size, whereas the pAS1 cluster had a maximum size of 500 kb. Sequence comparison revealed that both satellites are completely unrelated and differ considerably in their G + C contents. PMID- 7551547 TI - Chromosome elimination in three Baltic, south Pacific and north-east Pacific hagfish species. AB - We have confirmed that chromosome elimination occurs in the cells of Myxine glutinosa, collected from the Baltic Sea off Sweden, Eptatretus cirrhatus from the south Pacific Ocean off the east coast of New Zealand, and E. stoutii from the north-east Pacific Ocean off Canada, similar to cells of four Japanese hagfish species. In M. Glutinosa, E. cirrhatus type A, E. cirrhatus type B and E. stoutii, the differences in chromosome number between spermatogonia (44, 72, 80 and 48) and somatic cells (28, 34, 34 and 34) were 16, 38, 46 and 14 respectively. The amount of DNA eliminated from presumptive somatic cells averaged 43.5%, 48.7%, 54.6% and 52.8% respectively. Euchromatic chromosomes and/or parts of chromosomes in addition to heterochromatic chromosomes were clearly eliminated in E. cirrhatus and E. stoutii. Adding our previous observations of four Japanese hagfish species, chromosome elimination occurs in all seven of the hagfish species. These results suggest that this phenomenon, chromosome elimination, generally occurs in the order Myxinida. In addition, B chromosomes were observed in the germ cells of E. cirrhatus and E. stoutii, similar to the cells of E. okinoseanus, E. burgeri and Paramyxine atami (E. atami). This fact suggests that B-chromosomes might exist generally in the family Eptatretidae. PMID- 7551548 TI - Analysis and chromosomal localization of retrotransposons in sugar beet (Beta vulgaris L.): LINEs and Ty1-copia-like elements as major components of the genome. AB - DNA sequences of the reverse transcriptase gene of long terminal repeat (LTR) and non-LTR (non-viral) retrotransposons have been isolated and cloned from the genome of sugar beet (Beta vulgaris). Both retrotransposon types are highly amplified in sugar beet and may account for 2-5% of the genome. The BNR1 family, representing the first non-viral retrotransposon reported from a dicotyledonous species, shows homology to the mammalian L1 family of long interspersed repeated sequences (LINEs) and to retrotransposable elements from maize and lily. Sequences of the Tbv family are homologous to the Ty1-copia class of LTR retrotransposons. The BNR1 and Tbv retrotransposon families are characterized by sequence heterogeneity and are probably defective. The deduced peptide sequences were used to investigate the relation to other retroelements from plants, insects and mammals. Fluorescence in situ hybridization was used to investigate the physical distribution and revealed that both retrotransposon families are present on all sugar beet chromosomes and largely excluded from chromosomal regions harbouring the 18S-5.8S-25S rRNA genes. The BNR1 family is organized in discrete clusters, while the Tbv family of Ty1-copia-like retrotransposons shows a more uniform distribution along chromosome arms and is absent from some chromosomal regions. These contrasting distributions emphasize the differences in evolutionary amplification and dispersion mechanisms between the two types of retrotransposons. The in situ results of both elements reflect significant features of a higher order structure of the genome, as it is known for both short interspersed repeated sequences (SINEs) and LINEs in human. PMID- 7551551 TI - Molecular analysis of chromosomal polymorphism in the South American cricetid, Graomys griseoflavus. AB - Graomys griseoflavus is a South American phyllotine rodent widespread in Argentina that shows a high frequency of Robertsonian fusions (RFs). DNA restriction with EcoRI produced a 250-bp repeated family (EG250) specific for the genus. Southern hybridization and sequencing analysis indicate that the EG250 family is heterogeneous, comprising at least two subfamilies. In situ hybridized EG250 probe showed a centromere location in almost all chromosomes. In all karyomorphs C-banding was negative, but restriction enzyme banding (Re-banding) with Alul and Mbol showed centromeric blocks in the autosomes that will generate Robertsonian fusions. Thus, we found three groups of chromosomes: (a) EG250 and Re-banding negative; (b) EG250 positive and Re-banding negative; and (c) EG250 and Re-banding positive. We consider that group (b) is more the result of chromatin condensation state than that of the frequency of recognition sites for the enzymes used. Restriction enzyme blocks would appear in regions with heterochromatic EG250 subfamilies, while lack of banding would be due to decondensed EG250 subfamilies becoming an easier target for chromosomal restriction. It is suggested that heterochromatic EG250 DNA provides a favourable molecular environment for Robertsonian fusion occurrence. PMID- 7551550 TI - Mapping functional domains of the polycomb protein of Drosophila melanogaster. AB - In Drosophila the Polycomb group (Pc-G) proteins are responsible for the stable and heritable silencing of genes. The Pc-G apparently uses heterochromatin-like mechanisms to transcriptionally inactivate developmental regulators such as the homeotic genes. The Polycomb (Pc) protein is part of a large multimeric complex composed of other members of the Pc-G. We have identified functionally relevant domains of the Pc protein by sequencing different Pc alleles. Additionally, using a Pc-beta gal fusion protein with deleted internal histidine repeats, we found that this mutant protein cannot bind to four particular target loci, but otherwise does not change the remaining overall binding pattern. We show that, in contrast to the dotted subnuclear localization of the wild-type protein, the nuclear distribution of mutant proteins becomes homogeneous. Surprisingly, in Pc mutants the polyhomeotic protein, another member of the Pc-G, is also redistributed in the nucleus. Our results indicate that the appropriate subnuclear localization of the two proteins is critical for the silencing function of the Pc-G complex. PMID- 7551549 TI - Chromosomal location of endogenous geminivirus-related DNA sequences in Nicotiana tabacum L. AB - The N. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivirus infection, during Nicotiana evolution. Each GRD repeat carries sequences similar to the geminiviral AL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus the cis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescence in situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA from N. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool. PMID- 7551552 TI - Imaging of DNA in human and plant chromosomes by high-resolution scanning electron microscopy. AB - We describe a DNA-specific staining procedure, using a blue platinum organic dye, which allows DNA imaging of chromosomes by detection of back-scattered electrons in the scanning electron microscope. DNA distribution is visualized within chromosomal details of the centromeric and satellite regions, or in interphase chromatin, with high-resolution (10-15 nm) for comparison with the corresponding secondary electron image representing DNA plus protein. PMID- 7551553 TI - Direction of DNA sequences within chromatids determined using strand-specific FISH. AB - The 5' to 3' direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5' to 3' direction of three repetitive DNA sequences, each for a different human repeat family. PMID- 7551554 TI - Restriction endonuclease/nick translation procedure on fixed chromosomes of the Atlantic salmon fish cell line. AB - We have used a restriction endonuclease/nick translation (RE/NT) procedure to study the ability of restriction enzymes to cleave DNA in fixed chromosomes of a fish cell line. This technique has proved to be very useful in revealing the chromatin heterogeneity underlying the chromosome structure that remains cryptic to other techniques also able to induce longitudinal differentiation on fish chromosomes. The differences observed in the banding patterns after nick translation procedure seem to be due, at least in part, to differences in activity among the enzymes assayed. The results obtained also reveal some evidence about the origin and evolution of the marker chromosomes of the Atlantic salmon cell line. PMID- 7551556 TI - Theoretical calculations of heavy-atom isotope effects. AB - An overview of calculations of isotope effects on biochemical and chemical reactions using quantum chemistry methods is presented . Usefulness of different levels of theoretical scrutiny for such calculations is critically discussed. PMID- 7551555 TI - In situ quantification of hsp70 and alpha-beta transcripts at 87A and 87C loci in relation to hsr-omega gene activity in polytene cells of Drosophila melanogaster. AB - The hsp70-coding duplicate loci at the 87A and 87C sites (the 87C site also carries heat-inducible alpha-beta repeats) in polytene nuclei are known to puff to different levels under conditions in which heat shock does not induce the non protein-coding hsr-omega gene at the 93D site. To understand the basis of this unequal puffing, the levels of hsp70 and alpha-beta transcripts at the 87A and 87C heat shock loci in polytene chromosomes of Drosophila melanogaster were quantified in situ by hybridization of antisense RNA probes after treatment with heat shock, benzamide, colchicine, heat shock followed by benzamide or heat shock in the presence of colchicine in salivary glands of late third instar larvae. Heat shock, resulting in equal puffing of the 87A and 87C loci, increased the hsp70 transcripts at both sites in proportion to the numbers of hsp70 gene copies at the two loci; levels of alpha-beta transcripts were also elevated at the 87C site following heat shock. Heat shock followed by benzamide treatment, which results in a larger puff at 87A, caused an increase in hsp70 transcripts per gene copy at 87A and a decrease at 87C without any effect on the alpha-beta transcripts; heat shock in the presence of colchicine, which causes the 87C puff to be larger than 87A, resulted in a decrease in hsp70 RNA at 87A but an increase in the levels of hsp70 as well as alpha-beta transcripts at the 87C site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551557 TI - Computation of low-frequency normal modes in macromolecules: improvements to the method of diagonalization in a mixed basis and application to hemoglobin. AB - The method of diagonalization in a mixed basis (DIMB) that was published previously (Mouawad.), L. and Perahia D., Biopolymers 33, 599, 1993), allows the computation of the low-frequency vibrational modes for large macromolecules. Improvements to this method are presented here, namely the single and double truncation window techniques. The best convergence rate is obtained with the double truncation windows, which couple most efficiently the parts of the macromolecule which are far in sequence but close in space. Both methods were applied to the T-state of hemoglobin, to compare their efficiency. The resulting modes are analyzed in order to study the pathways between T- and R-states of this protein. They show that the quaternary conformational are mainly due to one mode at 2 cm-1. PMID- 7551558 TI - Geometrical analysis of structural changes in immunoglobulin domains' transition from native to molten state. AB - Molecular dynamics simulation (300, 320, 340 K) performed on the Fab (Kol) fragment of immunoglobulin G revealed that the structural changes associated with relaxation of peptides after their release from the stabilized by the tertiary interaction native conformation may be considered characteristic of the transition from native to molten state. The configuration of peptide chains at temperatures close to melting, liberated from the constraints associated with tertiary packing, was found to deviate toward helical rather than extended forms. The direction of the shift is diagonal on the phi-psi map. The torsional angles tend to concentrate in the Cea7 region, and some leak to the alphaR area. The geometrical parameters designed to describe the configuration of the peptide chain in Fab fragment also confirmed that during melting the peptides generally moved toward helical form. PMID- 7551559 TI - Epiligrin is decreased in papulonodular basal cell carcinoma tumor nest basement membranes and the extracellular matrix of transformed human epithelial cells. AB - Patients with anti-epiligrin cicatricial pemphigoid have anti-basement membrane autoantibodies that immunoprecipitate a set of disulfide-linked human keratinocyte polypeptides that co-migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with the same complex identified by monoclonal anti-epiligrin (P1E1) and monoclonal anti-nicein/kalinin (GB3) antibodies. In an attempt to further compare the reactivity of patient autoantibodies, P1E1 and GB3, these reagents were tested against the tumor nest basement membranes of 7 papulonodular basal cell carcinomas. These studies found that all of these reagents showed markedly decreased or no reactivity against this substrate. Though their concordant lack of reactivity failed to distinguish these antibodies, these studies did identify a significant defect in papulonodular basal cell carcinoma tumor nest basement membranes. Similarly, integrin subunits alpha 6, beta 4, alpha 3, and alpha 2 as well as bullous pemphigoid antigens 1 and 2 (all potential receptors for the extracellular matrix ligands epiligrin and nicein/kalinin) were also reduced in these tumor nest basement membranes. These findings signify an extensive impairment in the lamina lucida of this neoplasm's basement membrane. Related comparative studies of normal human keratinocytes and transformed human epithelial cell lines (specifically, A-431 and HaCat cells) showed that epiligrin production is markedly decreased in the latter. Decreased expression of epiligrin and nicein/kalinin in papulonodular basal cell carcinoma tumor nest basement membranes in vivo and transformed epithelial cells in vitro indicate that this complex is a transformation-sensitive cell adhesion ligand. PMID- 7551560 TI - Divalent cations (Mg2+, Ca2+) differentially influence the beta 1 integrin mediated migration of human fibroblasts and keratinocytes to different extracellular matrix proteins. AB - Directed migration of keratinocytes and fibroblasts is a fundamental prerequisite in wound healing. Cation-dependent affinity changes of integrins are responsible for cell adhesion to and deadhesion from extracellular matrix proteins and have been implicated in driving cell migration. The specific requirements for divalent cations in the integrin-dependent migration of human dermal fibroblasts and human epidermal keratinocytes to various extracellular matrix proteins have been studied in vitro using blindwell Boyden chambers. The migration of the tested cells to collagen type I was mediated by the alpha 2 beta 1 integrins, to fibronectin by the combined action of the alpha 3 beta 1 and the alpha 5 beta 1 integrin, and the migration of fibroblasts to laminin dependent both on the alpha 2 beta 1 and the alpha 6 beta 1 integrins. No migration of keratinocytes to laminin was detected. Mg2+ alone induced cell migration with an optimum at 2 mM for fibroblasts and at 10 mM for keratinocytes. Ca2+ alone at 2 mM only marginally enhanced fibroblast and keratinocyte migration. At higher concentrations Ca2+ suppressed the stimulatory Mg2+ effect. 2 mM Ca2+ combined with 2 mM Mg2+ showed an additive stimulatory effect on the migration of fibroblasts to fibronectin. These data suggest that extracellular divalent cations differentially influence the integrin-mediated cell migration. A concentration gradient of Mg2+/Ca2+, as reported in tissue injury, thus may play a regulatory role in cell migration required for tissue remodelling. PMID- 7551561 TI - Interleukin-8 and melanoma growth-stimulating activity (GRO) are induced by ultraviolet B radiation in human keratinocyte cell lines. AB - Ultraviolet radiation can induce the transcription and release of cytokines from keratinocytes (KC's). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2-1.4 kb transcript for the chemokine and melanoma growth-stimulatory protein, GRO-alpha and that ultraviolet B radiation (UVB) could upregulate the expression of GRO-alpha mRNA and protein in the KC line A431. The GRO-alpha gene response to UVB was maximal at 48h post irradiation with 70 J/m2. Reverse transcription-polymerase chain reaction (RT PCR) revealed a 4.5-fold increase in GRO-alpha mRNA over basal levels (p < 0.001). GRO-alpha protein was measured in the culture media by enzyme-linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166 +/- 83 pg/ml GRO-alpha protein. After UVB, a time-dependent increase in GRO-alpha protein was seen in the culture media from 6-48h. At 48h post-irradiation the GRO alpha protein content was 27583 +/- 678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre-incubated with 10 micrograms/ml interleukin-1 receptor antagonist (IL-1RA). We also show that another potent leukocyte chemoattractant, Interleukin-8 (IL-8), was induced in A431 cells by UVB. This induction of IL-8 mRNA began as early as 3h post irradiation, when it reached twice basal levels (p < 0.05) and reached 4.5-fold basal levels at 48h post-irradiation (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551563 TI - Rapid purification of human Langerhans cells using paramagnetic microbeads. AB - Detailed studies on the biology of Langerhans cells (LC), which account for only 1-3% of all epidermal cells, require isolation from their cutaneous symbionts. Several techniques of LC isolation have been reported, including positive enrichment with mAb coupled to immunomagnetic beads. The disadvantage of this technique is the size of the beads (approximately 2-5 microns), which can interfere with subsequent phenotypic and functional analyses. This limitation prompted us to test whether paramagnetic microbeads (15 nm) employed by the MACS system could be used to purify LC from human skin. To isolate fresh LC (fLC), epidermal cell suspensions (EC) were stained with anti-CD1a mAb and with appropriate secondary reagents conjugated to microbeads and to FITC. They were then passed over a separation column and exposed to a strong magnetic field. Thereafter both CD1a-depleted and CD1a-enriched cells were collected. Cultured LC (cLC) were isolated by staining 72-h cultured EC with anti-HLA-DR mAb followed by the same isolation procedure. Using this technique, we could routinely isolate viable EC that were 45-88% CD1a+ or HLA-DR+ as determined by FACS. Two-color FACS analysis demonstrated the majority of MACS-purified cells to be CD1a+/HLA-DR+, indicating that they were indeed LC. By transmission electron microscopy (TEM), the MACS-purified CD1a+/HLA-DR+ cells showed typical ultrastructural characteristics of LC. Furthermore, MACS-purified fLC or cLC were functionally intact, because they stimulated the proliferation of alloreactive T cells in a primary, one-way, mixed epidermal cell leukocyte reaction (MECLR). We conclude that MACS-separation is an efficient and rapid method to isolate human fLC and cLC of high purity and unimpaired function. PMID- 7551562 TI - Ultrastructural effects of topical tretinoin on dermo-epidermal junction and papillary dermis in photodamaged skin. A controlled study. AB - We examined the effects of daily topical application of 0.05% tretinoin cream on photodamaged Caucasian facial skin by electron microscopy. Specimens obtained pretreatment, after 6 and 12 months of tretinoin therapy (20 patients), and after 6 months of vehicle treatment (5 patients) were compared in a blinded fashion, with special attention to the dermoepidermal junction and papillary dermis. Baseline specimens disclosed various degrees of damage including reduplication of basal lamina, smudging and sparsity of collagen fibers, and nodular arrangement of degenerated microfibrils in the papillary dermis. No significant changes were observed at 6 months in the papillary dermis of either tretinoin-treated or vehicle-treated patients. After 12 months of tretinoin treatment, however, disorganized collagen fibers, which were conspicuous in 11 patients at baseline, were replaced by new well-organized collagen fibers in a wavy pattern in 6 patients. In addition, the amount of nodularly degenerated microfibrillar material decreased in 15 of 18 patients with this finding at baseline. In contrast, no significant change was noted in the number of anchoring fibrils per unit length of the lamina densa. These observations provide further evidence that topical treatment with 0.05% tretinoin produces papillary dermal reconstruction, for which more than 6 months of application were required. PMID- 7551565 TI - Embryonic expression of the 5-HT3 receptor subunit, 5-HT3R-A, in the rat: an in situ hybridization study. AB - The role of serotonin as a neurotransmitter in the vertebrate central and peripheral nervous systems has been extensively studied. In addition to its well defined role in neurotransmission, serotonin has also been implicated in development. We have used in situ hybridization to localize 5-HT3 receptor mRNA in embryonic rat sections from Embryonic Day 10 (E10) to E18. Expression was first detected in the cranial sensory ganglia starting on E10. Expression was later detected in many regions within the developing CNS. In addition to the cranial sensory ganglia, expression was detected in many regions of the peripheral nervous system including dorsal root, sympathetic, and parasympathetic ganglia, and in the myenteric plexus of the enteric nervous system. Expression was also detected in many nonneuronal cell populations including the choroid plexus, cochlear duct, and olfactory epithelium. Expression in regions of active epithelial-mesenchymal interaction such as the tooth bud, lung, and submandibular gland may indicate a role for 5-HT3 receptors in the process of secondary induction. PMID- 7551564 TI - Restrictions to floor plate induction by hedgehog and winged-helix genes in the neural tube of frog embryos. AB - Intercellular signaling molecules of the vertebrate hedgehog family and transcription factors of the winged-helix family have been implicated in floor plate development. We have examined the consequences of misexpressing the vertebrate hedgehog gene vhh-1 (sonic hedgehog, shh) and the winged-helix gene HNF-3 beta in the neural plate and neural tube of frog embryos. Misexpression of either of these genes induces floor plate differentiation at ectopic locations. However, ectopic floor plate induction in response to both vhh-1 and HNF-3 beta was temporally and spatially restricted. At neural plate stages, ectopic floor plate differentiation was not detected. After neural tube closure, ectopic floor plate differentiation was detected but was restricted predominantly to the dorsal region of the neural tube. The ability of winged-helix and vertebrate hedgehog genes to induce floor plate differentiation in vivo may, therefore, be constrained by additional signals that specify the time and position of floor plate differentiation. PMID- 7551567 TI - In vivo viability of postmitotic Purkinje neurons requires pRb family member function. AB - The product of the retinoblastoma susceptibility gene, pRb, is known to be an important regulator of cell division. Disrupted central nervous system development in RB null mice suggests a critical function for pRb in the proliferative arrest and initiation of terminal differentiation of certain neurons. Previously, we have shown that SV40 T-ag expression targeted to Purkinje neurons in transgenic mice causes cell-specific death. Here we describe that T-ag expression induces DNA synthesis and results in DNA fragmentation in Purkinje neurons. Characterization of transgenic mouse lines expressing mutant T-ags demonstrate that the pRb binding domain of T-ag is required for induction of Purkinje cell loss. These findings indicate that a pRb function is required well beyond the completion of Purkinje neuron differentiation and provide a link between cell cycle regulation and neurodegeneration in vivo. PMID- 7551569 TI - Ceramide: a potential second messenger in the nervous system. PMID- 7551570 TI - NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains. AB - Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric receptors was constructed using cDNA sequences from rat trkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor. PMID- 7551571 TI - Anatomy of cervical spine. AB - A knowledge of the cervical spine anatomy provides the basis for understanding and interpreting magnetic resonance imaging studies. Because magnetic resonance imaging provides multiplanar images, this article describes anatomic relationships in multiple planes. The anatomic relationships of the spinal nerves and the neural foramina and spinal cord in the central canal are emphasized, and the location of fluid and fat, which have distinctive appearances in magnetic resonance imaging, is noted. PMID- 7551566 TI - Chimeric integrins expressed in retinal ganglion cells impair process outgrowth in vivo. AB - Integrin function in retinal ganglion cell (RGC) development was examined in vivo by transfecting genes encoding various dominant forms of the chicken beta 1 integrin subunit into intact eye primordia of Xenopus embryos. RGCs expressing the chimeric chicken/Xenopus integrin receptors exhibited a marked reduction in process outgrowth with only 27% extending an axon and 41% bearing dendrites compared to control levels of 85-88%. None of the integrin constructs impaired the ability of RGC axons to pathfind appropriately or of retinal precursors to migrate to different laminar positions. Chimeric integrin expression also impaired process outgrowth in cells of the inner nuclear layer, although to a lesser extent than RGCs. Transfected diencephalic neurons, by contrast, showed normal levels of process outgrowth. These findings show that beta 1 integrins play an important role in regulating the outgrowth of axons and dendrites from RGCs in the retina but that chimeric integrins do not impair growth cone steering in general. PMID- 7551568 TI - Serine 133-phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals. AB - A mechanism has been characterized by which the transcription factor CREB regulates neurotrophin-induced gene expression. Whereas CREB can mediate calcium- or cyclic AMP-induced c-fos transcription independently of other promoter-bound transcription factors, CREB mediates NGF induction of c-fos transcription via a novel mechanism that appears to require a cooperative interaction with another transcription factor, the serum response factor. A similar transcriptional mechanism may explain how neurotrophins and growth factors induce distinct subsets of delayed response genes. Neurotrophins induce the phosphorylation of CREB at a key regulatory site, Serine 133, with prolonged kinetics that are distinct from the transient kinetics of CREB phosphorylation elicited by growth factors. These results indicate that CREB is a versatile transcription factor that activates transcription via distinct mechanisms in a stimulus-specific manner. In addition, by selectively activating delayed response genes, CREB may confer specificity to neurotrophin signals that promote the survival and differentiation of neurons. PMID- 7551572 TI - Clinical evaluation of cervical radiculopathy and myelopathy. AB - This article considers the entities of cervical radiculopathy and myelopathy. Beginning with a brief historical note and neuroanatomy review of the cord and nerve roots, the discussion proceeds with analysis of symptoms and signs of cervical root disease and then compressive cervical myelopathy. A discussion of diagnostic testing and a differential diagnosis follows. A brief review of conservative medical therapy concludes the article. PMID- 7551573 TI - Computed tomography and myelography in the evaluation of cervical degenerative disease. AB - Computed tomography and myelography have long been used for diagnosing the effects of degenerative diseases of the cervical spine. Despite the continuous improvement of magnetic resonance scanning for this purpose, computed tomography techniques may provide an excellent screen for disc herniation and spinal stenosis. Computed tomography myelography is still considered by some workers to be the gold standard for demonstrating the soft tissue and bony changes that result in nerve root and spinal cord compression syndromes. PMID- 7551575 TI - Myelopathy. AB - Myelitis and myelopathy may be caused by numerous ideologies, such as neoplastic, inflammatory, metabolic, infectious, and physical agents. Cervical spondylitic myelopathy, a spectrum of degenerative change occurring in the cervical spine, is the most common cause of cord dysfunction in patients more than 50 years of age. A variety of ideologies of myelopathy and imaging features are presented in this article. PMID- 7551576 TI - Cervical spine tumors. AB - Magnetic resonance imaging is the central imaging modality for cervical spine neoplasms. The majority of extradural lesions are metastatic. Nerve sheath tumors are the most common intradural neoplasm, followed by meningioma. Intramedullary tumors are less common. This article reviews tumors according to this classification, with emphasis on clinical considerations and imaging techniques and findings. PMID- 7551574 TI - Cervical radiculopathy. AB - Cervical radiculopathy refers to any disease process that compromises the cervical nerve roots. This article briefly reviews the normal anatomy of the cervical spine and the clinical presentation of patients with radiculopathy. Emphasis is then placed on the magnetic resonance evaluation of these patients, with focus on degenerative disease, the most common cause of cervical radiculopathy. Dramatic improvements have been appreciated in the magnetic resonance pulse sequences because they have evolved from standard two-dimensional spin echo studies to a combination of two-dimensional spin echo and three dimensional gradient echo acquisitions. More recently, the gradient echo study has been replaced by hybrid RARE sequences and may soon be supplanted by a three dimensional version of this sequence with the advent of advanced gradient capabilities of newer magnetic resonance imaging systems. PMID- 7551577 TI - Inflammatory and infectious processes of the cervical spine. AB - This article discusses infectious and inflammatory processes of the cervical spine. Major emphasis is placed on infectious discitis/osteomyelitis and epidural abscess, particularly the epidemiologic, bacteriologic, pathophysiologic, and clinical aspects, as well as the major role played by magnetic resonance imaging and other imaging modalities used in the detection and diagnosis of these processes. PMID- 7551578 TI - Congenital anomalies of the cervical spine. AB - A wide variety of congenital pathology affects the cervical spine and underlying spinal cord. This article discusses commonly encountered conditions under the categories of osseous anomalies, Chiari malformations, dysraphic conditions, and skeletal dysplasias. Osseous anomalies tend to involve the upper cervical spine to a greater degree than the lower cervical spine. Chiari malformations warrant inclusion on the basis that they have many manifestations within the cervical spine. Dysraphic conditions resulting from failure of either primary or secondary neurulation are distinctly uncommon in the cervical spine compared with the lumbar spine and are, therefore, only briefly discussed. Skeletal dysplasias encompass a diverse group of uncommon entities but may produce significant radiographic abnormalities and may be associated with clinical symptomatology. PMID- 7551579 TI - Imaging evaluation of cervical spine trauma. AB - Plain radiography remains the standard imaging modality for initial evaluation of cervical spine trauma. CT and MR imaging play an important role in additional evaluation for patients with known or suspected injuries. The role of conventional tomography and myelography is limited. PMID- 7551580 TI - The anterior approach to the cervical spine. AB - This article focuses on the indications for anterior cervical surgery, relevant surgical anatomy, a brief description of the technique of anterior surgery followed by a discussion of results and complications. It is hoped that the reader will achieve a better understanding for the clinical syndrome of cervical disc degeneration and be better able to put into proper perspective the role of diagnostic imaging in the overall evaluation of the patient. PMID- 7551581 TI - The posterior surgical approach to the cervical spine. AB - This article reviews the indications for, and the technique of, posterior operations on the cervical spine. Cervical hemilaminectomy and laminectomy are discussed, including diagnosis, nonoperative treatment, and results and complications of surgery. Laminectomy for other conditions, cervical trauma, and cervical fusion are reviewed also. PMID- 7551582 TI - Cervical spine stabilization. Surgical techniques. AB - Because of the virtually unlimited combination of injuries and disorders that can affect the cervical spine, no single stabilizing procedure can be advocated for all situations. As our understanding in the areas of spinal biomechanics, bone graft properties, internal fixation devices, and external immobilization has improved, advances have been made in the surgical stabilization of the cervical spine. These innovations are not necessarily intended to replace those proven methods that have been traditionally employed, but rather add to the armamentarium of available stabilizing techniques. With this broader selection of techniques, managing patients with an unstable cervical spine can be even more individualized in order to achieve an optimal outcome. PMID- 7551583 TI - Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells. AB - As they respond to numerous extracellular and intracellular stimuli, plants develop various morphological features and the capacity for a large variety of physiological processes during their growth. If we are to understand the molecular basis of such developments, we must elucidate the way in which signals generated by such stimuli can be transduced into plant cells and transmitted by cellular components to induce the appropriate terminal events. In yeast and animal systems, signal pathways that are known collectively as MAPK (mitogen activated protein kinase) cascades have been shown to play a central role in the transmission of various signals. The components of these pathways include the MAPK family, the activator kinases of the MAPK family (the MAPKK family) and the activator kinases of the MAPKK family (the MAPKKK family). The members of each respective family are structurally conserved and signals are transmitted by similar phosphotransfer reactions at corresponding steps that are mediated by a specific member of each family in turn. Both cDNAs and genes that encode putative homologues of these components have recently been isolated from plant sources. Some of them have been shown to be related not only structurally but also functionally to members of the MAPK cascades of other organisms. These findings suggest that plants have signal pathways that are analogues to the MAPK cascades in yeast and animal cells but it remains to be proven that plant homologues do in fact constitute kinase cascades.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551585 TI - Isolation and characterization of a cDNA that encodes maize glutamate dehydrogenase. AB - A full-length cDNA clone, pMGDH1, encoding maize NADH-glutamate dehydrogenase (NADH-GDH) was isolated from a maize root cDNA library. The identity of the cDNA was established by the coincidence of the structure of the purified protein with that inferred from the nucleotide sequence of the cDNA. pMGDH1 had a cDNA insert of 1,638 bp and the open reading frame encoded 411 amino acid residues. The deduced amino acid sequence was similar to putative partial sequences of GDHs from higher plants and to the sequences of GDHs from organisms as diverse as mammals and bacteria. The NH2-terminal sequence deduced from the open reading frame had a typical structure that is associated with the import of proteins into the mitochondrial matrix. The cDNA hybridized to an RNA of about 1.6 kb. This transcript was more abundant in roots than in leaves and was localized in the bundle sheath cells in leaf tissues. Analysis of genomic DNA by Southern hybridization suggested the existence of gene(s) for another NADH-GDH subunit(s). PMID- 7551584 TI - Genomic organization of 251 kDa acetyl-CoA carboxylase genes in Arabidopsis: tandem gene duplication has made two differentially expressed isozymes. AB - Acetyl-CoA carboxylase (ACCase) catalyzes the carboxylation of acetyl-CoA, forming malonyl-CoA a key intermediate in the biosynthesis of fatty acids and a variety of secondary metabolites. Based upon amino acid sequences conserved among rat, chicken, and E. coli ACCases, PCR-primers were used to amplify a genomic fragment which codes for an ACCase of Arabidopsis. The resulting fragment was used for isolation of genomic and cDNA clones. We have determined the complete cDNA sequence coding for an Arabidopsis ACCase consists of 2,254 amino acids with the molecular mass of 251 kDa. This enzyme contains no recognizable plastid transit-peptide sequence. Therefore, this ACCase is presumably the cytosolic isozyme. Southern analysis indicates that there are two ACCase genes in the Arabidopsis genome. Surprisingly, the results of RFLP analysis and physical mapping of the isolated genomic clones demonstrate that these two genes, acc1 and acc2, are contiguously located within a 25-kbp genomic region near the middle of chromosome 1. Both genes are transcriptionally active, as transcripts from each gene were detected by reverse transcription-PCR analysis using gene-specific primers. The acc1 and acc2 transcripts accumulate in leaves and seedlings but only the acc1 transcript accumulates in developing siliques, unexpectedly. The differences in the expression patterns may be indicative of the differential role of the two genes. PMID- 7551586 TI - Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors (ARFs) and characterization of the binding of nucleotides by its product after expression in E. coli. AB - A cDNA clone, designated DcArf1, was isolated from a cDNA library prepared from embryogenic cell clusters of Daucus carota and characterized. The cDNA (883 bp) contained an open reading frame that encoded a protein of 181 amino acid residues with significant homology to the ADP-ribosylation factors (ARFs) of animals and yeast. The DcArf1-related transcripts were detected at a nearly constant level during somatic embryogenesis. The DcArf1-encoded protein was expressed as a fusion protein in E. coli and was shown to have dithiothreitol-independent [alpha 32P]GTP-binding activity. This binding was completely prevented by a 100-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding. The binding of [35S]GTP gamma S to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl2 and dimyristoylphosphatidylcholine (DMPC). The amino acid sequence and the GTP binding characteristics of the DCARF1 protein suggest that the DcArf1 cDNA encodes an ARF of carrot rather than a protein that only resembles such a factor, ADP-ribosylation factor-like protein (ARL). PMID- 7551587 TI - Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.). AB - Phospholipase D (PLD) was purified to high homogeneity from rice bran (Oryza sativa L.). Two peaks of PLD activity were resolved by Mono Q anion-exchange chromatography. The molecular mass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDa in gel filtration. Antibodies raised against the protein in one of the peaks precipitated the enzyme activities in both peaks. Enzymatic characteristics of PLD in the two peaks were identical except for a difference of 0.1 in the isoelectric points. Sequence analysis covering more than 10% of the amino acids of the proteins and peptide mapping did not detect any difference in the primary structure of the proteins. A cDNA for PLD was isolated from rice and it encoded a protein of 812 residues. The N-terminal sequences of purified PLDs matched the deduced amino acid sequence starting from residue 47. A Northern blot showed this gene was expressed in leaves, roots, developing seeds and cultured cells, and a Southern blot detected a single band of rice genomic DNA hybridizing to the cDNA. A cDNA for PLD was also isolated from maize. The similarity of the deduced amino acid sequences of PLD was 90% between rice and maize, 73% between the cereals and castor bean. PMID- 7551588 TI - Isolation of cDNA for a plasma membrane H(+)-ATPase from guard cells of Vicia faba L. AB - cDNA encoding the plasma membrane H(+)-ATPase of guard cells of Vicia faba L. was isolated. The clone encoded a 105-kDa polypeptide (956 amino acids) that was 79 85% identical in terms of amino acid sequence to other plant H(+)-ATPases. High levels of mRNA explain the high H(+)-ATPase activity of these plasma membranes. PMID- 7551590 TI - Psychological interventions in infection with the human immunodeficiency virus. AB - BACKGROUND: Infection with the human immunodeficiency virus (HIV) is associated with substantial psychiatric morbidity. This paper summarises the main forms of psychological intervention currently used, and reviews research evidence for their efficacy. METHOD: Publications on the mental health aspects of HIV infection and psychological interventions were identified through the main HIV journals and general psychiatric and psychological periodicals, with the assistance of AIDS Abstracts. RESULTS: Publications concerned psychological interventions at the time of HIV testing, interventions for infected people at different stages of disease, and risk-reduction interventions. While many publications were identified describing uncontrolled investigations, only a few studies had involved systematic evaluative research. CONCLUSIONS: There is a need for systematic evaluation of psychological interventions for HIV infection, in terms of both efficacy and cost. PMID- 7551589 TI - Population policy and eugenics in China. PMID- 7551591 TI - Economics of treatment of depression. PMID- 7551592 TI - Economics of treatment of depression. PMID- 7551593 TI - The costs of psychotropic medication. PMID- 7551594 TI - Self-measure of neuroleptic side-effects. PMID- 7551596 TI - Charles Bonnet syndrome. PMID- 7551595 TI - Dystonia and neuroleptic medication. PMID- 7551598 TI - The cultural context of hallucinations. PMID- 7551600 TI - ECT seizure threshold. PMID- 7551599 TI - Mental health tribunals. PMID- 7551597 TI - Charles Bonnet syndrome. PMID- 7551601 TI - The Chinese classification of mental disorders. PMID- 7551602 TI - Rhabdomyolysis due to mania. PMID- 7551603 TI - Cardiovascular and autonomic function in Down syndrome--prescribing implications. PMID- 7551604 TI - A Darwinian approach to the origins of psychosis. AB - BACKGROUND: The onset of psychotic illness in the reproductive phase of life with a decrease in fecundity (and approximately constant incidence across populations) requires an evolutionary explanation. What is the survival value of the predisposing gene or genes? METHOD: Evolutionary theories, including the author's, are reviewed and critically compared. RESULTS: Some theories (e.g. Huxley et al, 1964) postulate an advantage outside the nervous system: such theories fail to explain either the characteristic age distribution or constant incidence. More plausible are theories that relate the advantage to diversity of personality structure or social ability, or even to general intelligence, i.e. to the areas of function in which the phenomena of psychosis arise. CONCLUSIONS: It is argued that psychosis arises as the boundary of a distribution of variation in cerebral structure generated in the course of hominid evolution. Language played a central role, with the critical changes taking place on the basis of a mutation that allowed the two cerebral hemispheres to develop with a degree of independence. Sexual selection (differing criteria in females and males in choosing a mate) acting on this genetic innovation has generated a dimension of competence in social interaction in relation to which there has been a progressive increase in cerebral size by delayed maturation (neoteny). A sexual dimorphism in cerebral asymmetry and the sex difference in age of onset of psychosis can be parsimoniously explained if a gene regulating the relative growth of the two hemispheres is X-Y homologous. PMID- 7551605 TI - An evaluation of community-based psychiatric care for people with treated long term mental illness. AB - BACKGROUND: We evaluated integrated, multidisciplinary, community-based care for a cohort of people with treated long-term mental illness over two years in a field trial set in a semi-rural setting. The aim was to organise local psychiatric services on an extramural basis with general practitioner teams as the key element. METHOD: Trained research workers used a structured interview to collect standardised base-line and three-monthly socio-demographic, clinical, social, family adjustment and burden, and treatment measures from patients, informants, and key-workers. Analysis included descriptive statistics and, for longitudinal data, analysis of best-fitted straight lines. RESULTS: We studied 34 patients (14 with schizophrenia, 11 with major affective disorders, and 9 with minor (non-major) affective disorders and anxiety disorders) who had a mean of 12 years continuous illness. At baseline, they were mainly characterised by research workers as mildly ill with fair social adaptive functioning, and by lay informants as having moderate target symptom severity and disturbance of social performance. Over two years, there were statistically significant, slight improvements in clinical global impressions ratings by research workers, and in informants ratings of target symptoms and social performance. Most patients continued to receive prescriptions for psychotropic drugs throughout follow-up, and they had a mean of 47 therapeutic contacts, mainly from nurses. Two patients were admitted to psychiatric hospital. There were few differences between patients in different diagnostic groups. CONCLUSIONS: Integrated, multidisciplinary, community-based psychiatric care for people with treated long term mental illness is feasible in a semi-rural setting: patients receiving pharmacotherapy and regular psychosocial treatments remained relatively stable on clinical and social measures over two years. The unique way in which the service was monitored, by making regular, systematic assessments of patients and carers, serves as an example for other services. PMID- 7551606 TI - Tardive dyskinesia in schizophrenia. Relationship to minor physical anomalies, frontal lobe dysfunction and cerebral structure on magnetic resonance imaging. AB - BACKGROUND: It was hypothesised that schizophrenic patients with tardive dyskinesia show an excess of neurodevelopmental disturbance, particularly minor physical anomalies, in association with cognitive dysfunction and abnormalities of cerebral structure. METHOD: Forty-seven out-patients with a DSM-III diagnosis of schizophrenia were examined for tardive dyskinesia using the Abnormal Involuntary Movement Scale; they were examined also for minor physical anomalies and neuropsychological test performance. Cortical atrophy, signal hyperintensities and lateral ventricular volume were determined on magnetic resonance imaging. RESULTS: Patients with and without tardive dyskinesia could not be distinguished by age, gender distribution or a number of clinical measures; however, patients with tardive dyskinesia sorted fewer categories on the Wisconsin Card Sorting Test (P = 0.04). Cerebral structure in patients with and without tardive dyskinesia could not be distinguished on magnetic resonance imaging but those with dyskinesia, all of whom showed involvement of the orofacial region, showed more evident minor physical anomalies of the head relative to those of the periphery (P = 0.02). CONCLUSIONS: Tardive orofacial dyskinesia in schizophrenia appears to be associated particularly with poorer frontal lobe function, while predominance of craniofacial dysmorphogenesis may constitute a vulnerability factor that is related to the early origins of the disease process. PMID- 7551607 TI - A pilot study of exposure control of chronic auditory hallucinations in schizophrenia. AB - BACKGROUND: Many patients complain less of their auditory hallucinations per se than of lack of control of the experiences. There is reason to believe that a non distraction (exposure) approach could help patients gain more control over persistent auditory hallucinations and teach them that their experience is a form of thinking and has no external source. This study is a pilot test of that idea. METHOD: Five DSM-III-R schizophrenic outpatients with medication-resistant auditory hallucinations improved with a mean of 31 hour-long sessions over 3 months of therapist-guided exposure to their hallucinations and situations likely to evoke them. RESULTS: Improvement was greatest in patients' anxiety and sense of control over their hallucinations, less in social use of leisure and hallucinating time. CONCLUSIONS: These mildly encouraging pilot results warrant a controlled study of exposure for drug-resistant chronic auditory hallucinations and other psychotic experiences which are associated with anxious avoidance. PMID- 7551608 TI - Young offenders, serious crimes. PMID- 7551609 TI - Neuropsychological function in manic-depressive psychosis. Evidence for persistent deficits in patients with chronic, severe illness. AB - BACKGROUND: While neuropsychological deficits are recognised to occur in manic depressive psychosis during episodes of depression and to reverse with clinical recovery, it is uncertain whether they can ever be seen outside episodes of illness. METHOD: Forty-five patients meeting DSM-III-R criteria for major depression or bipolar disorder were screened using tests of memory, executive function and overall intellectual function. All testing was carried out during remission of affective symptoms. RESULTS: None of 24 young patients and 11 elderly patients scored in the impaired range on any of the tests. However, five of ten patients with chronic, severe affective disorder were impaired on one or more of the measures. On more detailed neuropsychological investigation, these five patients were found to show a variable pattern of impairment, ranging from memory and executive deficits in relative isolation, to widespread poor performance. CONCLUSIONS: Enduring neuropsychological deficits may be a feature of chronic, severe manic-depressive illness. PMID- 7551610 TI - Is there a seasonal pattern of relapse in bipolar affective disorders? A dual northern and southern hemisphere cohort study. AB - BACKGROUND: Admission statistics for mania frequently show an increase in the summer. The present two-centre study was designed to test the hypothesis, in a representative sample of bipolar patients, that manic and depressive relapses show a seasonal pattern. METHOD: Two cohorts of bipolar I patients, one in London, England (n = 86), the other in Dunedin, New Zealand (n = 58), were tracked retrospectively during 1985-88 and prospectively during 1989-91, with the onset of all relapses being carefully dated. RESULTS: In the London cohort there were 221 episodes of mania and 76 of depression; in the Dunedin cohort there were 201 of mania and 61 of depression. No consistent seasonal pattern of mania was detected in either centre. There was an autumn preponderance of depressive episodes in both centres. CONCLUSIONS: Relapse of bipolar depression, but not of mania, appears to be determined in part by seasonal factors. PMID- 7551611 TI - Prevalence and correlates of depression in a population of nonagenarians. AB - BACKGROUND: Clinicians see many more nonagenarian patients now and there is a need for epidemiological data relating to this group. The aim of the present study was to investigate the prevalence of depressive symptoms and syndromes in this age group. METHOD: The DSM-IV and the ICD-10 criteria for depression were used and correlated with physical health, disability in daily life, gender, use of drugs, social circumstances and cognitive dysfunction. Data were derived from 329 persons aged 90 and over, registered in a parish of Stockholm, who had been extensively examined by physicians and nurses. RESULTS/CONCLUSIONS: The prevalence of Major Depressive Episode as defined in DSM-IV was 7.9%; and of mild, moderate and severe Depressive Episode (combined); as defined in ICD-10 9.1%. No gender difference was found. Disability in daily life and the use of psychotropic drugs were found to correlate with depressive symptoms and syndromes. PMID- 7551613 TI - Does imagined exposure to the consequences of not ritualising enhance live exposure for OCD? A controlled study. II. Effect on behavioural v. subjective concordance of improvement. AB - BACKGROUND: This study tested whether adding imagined exposure to live exposure would increase the concordance between behavioural and subjective improvement in obsessive-compulsive disorder (OCD). METHOD: 46 OCD out-patients were randomly allocated to 9 weekly sessions of either combined live+imagined exposure/ritual prevention (Exi, n = 23), or only live exposure/ritual prevention (Ex, n = 23). Patients were asked to do 90 min of daily self-exposure at home (corresponding to Exi or Ex). Measures were: (A) behavioural; (B) subjective; (C) clinical global impression (CGI). RESULTS: After 9 weeks of treatment, improvement was greater on behavioural than subjective measures (similar for the Exi and Ex groups). At 20 weeks (3-month follow-up) each group had improved slightly more on subjective measures and slightly less on behavioural ones. Two subjective measures improved less during Exi than Ex, but this difference disappeared at follow-up. The greater difference between behavioural and subjective improvement scores in Exi than in Ex did not relate to clinical outcome at the end of treatment or follow up. CONCLUSIONS: Compared to live exposure alone, combined imagined plus live exposure did not enhance behavioural/subjective concordance. PMID- 7551612 TI - Does imagined exposure to the consequences of not ritualising enhance live exposure for OCD? A controlled study. I. Main outcome. AB - BACKGROUND: This randomised controlled study tested whether adding imagined to live exposure plus ritual prevention would enhance gains in obsessive-compulsive disorder (OCD). METHOD: Out-patients with OCD were randomly allocated to either have nine sessions of daily live self-exposure and ritual prevention to external cues alone (Ex) (n = 23) or to have, in addition to Ex, daily self-exposure to the imagined internal cues of the consequences of not ritualising (group Exi) (n = 23). All patients had the same sessional exposure time of 1 h 30 min (Exi 1 h live, 30 min imagined; Ex 1 h 30 min live), and had to practise either Exi or Ex daily for the same duration and to keep diaries of that self-exposure homework throughout treatment. Patients were followed up to week 32. Outcome measures were YBOCS for rituals and obsessions, compulsion checklist, target rituals and obsession, general anxiety, depression (Beck, Hamilton), work and social disability, clinical global impression (CGI). RESULTS: At weeks 4, 9, 20 and 32 the two groups improved similarly with no significant difference between them, neither for washers nor for checkers. Imagined exposure was more difficult to do than live exposure and there were more drop-outs. CONCLUSIONS: Daily imaged exposure to internal cues did not enhance exposure to external cues. Perhaps longer imagined exposure would have been more enhancing. PMID- 7551614 TI - Panic disorder and suicide attempt. A reanalysis of data from the Epidemiologic Catchment Area study. AB - BACKGROUND: Analysing data from the Epidemiologic Catchment Area (ECA) study, Weissman and colleagues reported that panic disorder was strongly associated with suicide attempt. However, they did not control optimally for comorbid disorders known to increase suicide risk. METHOD: Reanalysing the ECA data, we controlled for comorbid disorders in the aggregate rather than one at a time when we estimated the association between panic disorder and suicide attempt. RESULTS: Panic disorder was not associated with an increased risk of suicide attempt. CONCLUSIONS: Comorbid conditions strongly influence whether people with panic disorder are at especial risk of suicide attempt. PMID- 7551615 TI - Children in institutions. PMID- 7551616 TI - Sexual abuse in patients with eating disorder, patients with depression, and normal controls. A comparative study. AB - BACKGROUND: A history of sexual abuse has been widely reported in patients with eating disorders. However, the association does not appear to be specific, because a high rate of such abuse has also been found in other psychiatric patients. METHOD: A standardised interview method was used to elicit details of sexual abuse in a psychiatrically normal control group and samples of patients with anorexia nervosa, bulimia nervosa, or depression. RESULTS: An equally high rate of abuse was found in all three clinical samples. Among the patients with anorexia nervosa the presence of bulimic episodes was not found to be associated with reports of abuse; and among the patients with bulimia nervosa there was no relationship between abuse and a history of anorexia nervosa. Among the patients with eating disorders, borderline personality disorder, assessed by means of self report questionnaire, was not found to be related to reports of abuse, although there was an association between abuse and both indices of impulsive behaviour and the overall level of personality disturbance. CONCLUSIONS: Childhood sexual abuse appears to be a vulnerability factor for psychiatric disorder in general and not eating disorders in particular. The way in which abuse interacts with other aetiological factors to produce different psychopathological trajectories remains to be elucidated. PMID- 7551617 TI - Cognitive functioning and magnetic resonance imaging in chronic fatigue. AB - BACKGROUND: This study examines whether cognitive dysfunction in chronic fatigue may be accounted for by depression and anxiety or is due to brain pathology evident on magnetic resonance imaging (MRI). METHOD: Twenty-six subjects with chronic fatigue, with and without coexisting depression, and 18 age-matched normal controls were recruited from primary care following a presumed viral illness six months previously. Comparison was made with 13 psychiatric controls with depressive illness on standardised cognitive tests. MRI determined the presence of cerebral white-matter lesions. RESULTS: No substantial differences in performance were shown between subjects with chronic fatigue, most of whom met the criteria for chronic fatigue syndrome, and controls. Subjective cognitive dysfunction increased with psychopathology. White-matter lesions were found in a minority from all groups. Improvement in fatigue and depression coincided with improved performance on cognitive measures. CONCLUSIONS: Subjective complaints of cognitive impairment are a prominent feature of chronic fatigue, but objective cognitive and MRI abnormalities are not. Such complaints probably reflect psychopathology rather than a post-viral process. PMID- 7551618 TI - The neuropsychiatric sequelae of mercury poisoning. The Mad Hatter's disease revisited. AB - BACKGROUND: The detailed effects of mercury poisoning on cognitive function, brain anatomy and regional brain function are largely unknown. We report the case of a 38-year-old man who was exposed to toxic levels of inorganic mercury. METHOD: Four years after exposure, the patient was assessed using magnetic resonance imaging (MRI), single-photon emission computerised tomography (SPECT) and detailed neuropsychological evaluation. RESULTS: The patient developed a myriad of physical and psychiatric complaints, including stomatitis, muscle spasm, tremor, skin rash and the psychiatric syndrome known as 'erythism' (Mad Hatter's disease). Neuropsychological evaluation revealed marked and significant deficits of attention concentration, particularly when under time pressure. The MRI scan was unremarkable; however, SPECT revealed hypermetabolism of the posterior cingulate CONCLUSIONS: Mercury poisoning appeared to result in a dysregulation of posterior cingulate cortex, which was associated with attention/concentration deficits and marked anxiety/agitation. PMID- 7551620 TI - Flims 95: the united psychotherapeutic interventions of psycho-oncology. PMID- 7551621 TI - Can psychotherapy be used to treat cancer? PMID- 7551619 TI - A scale for the assessment of hedonic tone the Snaith-Hamilton Pleasure Scale. AB - BACKGROUND: Hedonic tone and its absence, anhedonia, are important in psychopathological research, but instruments for their assessment are lengthy and probably culturally biased. METHOD: A new scale was constructed from the responses of a large sample of the general population to a request to list six situations which afforded pleasure. The most frequent items were reviewed and those likely to be affected by cultural setting, age, or sex were removed. A pilot study led to an abbreviated scale of 14 items, covering four domains of pleasure response. This questionnaire was subjected to psychometric evaluation in new samples from the general population and psychiatric patients. RESULTS: The scale was found to have a score range that would distinguish a 'normal' from an 'abnormal' response. Validity and reliability were found to be satisfactory. CONCLUSIONS: The new scale, the Snaith-Hamilton Pleasure Scale (SHAPS), is an instrument which may be recommended for psychopathological research. PMID- 7551622 TI - Proven and unproven therapies in cancer treatment: a controversy among German oncologists. PMID- 7551623 TI - Supportive and palliative care of cancer patients at the Kantonsspital St. Gallen, Switzerland. AB - In 1991, the first unit for oncology palliative care within an oncology centre in the country was opened in St. Gallen, Switzerland. The main admission criterion is symptom distress due to advanced cancer disease. The mean duration of hospitalisation was 23 days in 140 patients in 1994. Approximately one third of the patients die within the unit, while one third can be discharged for home care. The aims and strategies of interdisciplinary palliative care are described. The need for the 11-bed unit is documented by its high occupancy, by patient satisfaction and by its influence on palliative care development in and outside the country. PMID- 7551625 TI - A short-term psychoeducational intervention for patients newly diagnosed with cancer. AB - The psychological and medical problems encountered by cancer patients are numerous and unique. The diagnosis of cancer frequently produces psychological distress. A review of the literature and the authors' clinical and research experience suggest that cancer patients may benefit from a variety of psychological intervention programs. A structured, psychiatric intervention consisting of health education, stress management/behavioral training, coping (including problem-solving techniques), and psychosocial group support offers the greatest potential benefit for patients newly diagnosed or in the early stages of their treatment. Early-stage interventions that encourage active behavioral coping and active cognitive coping rather than avoidance or passive acceptance of the illness can be helpful psychologically. These active behavioral and cognitive coping behaviors, which can be learned, can attenuate the psychological distress caused by stressful illness, decrease the amount of psychosocial adjustment to the illness needed, improve overall quality of life, and may also be associated with longer survival times. PMID- 7551624 TI - On organizing an international workshop on psychotherapeutic interventions in cancer patients. A virtual coffeebreak communication. AB - This paper is an unusual summary of my thoughts on the International Workshop on Psychotherapeutic Interventions in Cancer Patients in Flims, Switzerland, in 1995. Based upon virtual coffeebreak communications, I try to explain to my virtual partner how the idea of the workshop developed, how I came to select participants and experts, and why the workshop was structured in such a specific way. Concerning the workshop itself I shall quote personal feedback statements from participants, drawing upon the impressions they took with them from demonstration workshops prepared by the leading experts in the field. During the workshop the impact of psychosocial interventions upon survival was critically evaluated, whereas it was unanimously agreed that the most important target of psychosocial interventions in cancer patients is the improvement in quality of life. After the workshop I personally decided to enhance my professional engagement in the area of helping clinical oncologists and nurses to improve on the psychosocial skills they need in caring for cancer patients. PMID- 7551626 TI - Psychological intervention with couples after mastectomy. AB - Cancer is a family affair. Clinical work and research studies have shown that cancer does indeed invade the entire family, and that family members, especially spouses, are often highly distressed individuals. The family in general and the spouse in particular cannot, therefore, be looked on as natural supporters for cancer patients, but rather as a system that is itself in need of help and support. PMID- 7551627 TI - Group psychological therapy for cancer patients. A brief discussion of indications for its use, and the range of interventions available. AB - The growing evidence for the efficacy of psychological therapy in improving quality of life in cancer patients suggests that we should now consider it an adjuvant treatment in cancer management, analogous to adjuvant chemotherapy. If this is accepted, it follows that the main indication for advocating such adjuvant psychological therapy to patients should be an expert assessment that the patient is likely to benefit; it should not (as is usual at present) be restricted to either those patients with overt psychopathology, or those few who specifically request it. We describe five main kinds of therapy, arranging them on a hierarchy of increasingly active participation by the recipient, and noting the status of evidence for their efficacy. These five types are: providing information, emotional support, behavioral training in coping skills, psychotherapy (of various kinds), and, more speculatively, spiritual/existential therapy. PMID- 7551630 TI - Some problems and some solutions in research on psychotherapeutic intervention in cancer. AB - Several requirements of psychotherapeutic intervention (PI) research are presented. Some major problems are discussed. First, confidence in experimental results may be diminished by interference of possible confounders. A large number of these exist, both physical, perhaps more easily measurable, and psychological or psychosocial. Second, the value and limitations of randomization are presented, with special reference to small N. Validity and reliability are addressed, with emphasis on things that may dilute the strength of both of these measures. Natural remedies are proposed. Internal consistency and reliability are compared and cautions are given regarding their use and possible invalid substitution of consistency for reliability. Comparison of different PIs is commented on, with some relevant examples. Finally, a matter of special interest is discussed: the inconsistency between Spiegel's control survival curve and that produced from local population normative data. PMID- 7551628 TI - Improving quality of life: adjuvant psychological therapy for patients with cancer. AB - The present paper describes a psychological treatment programme--Adjuvant Psychological Therapy (APT)--that has been developed specifically for patients with cancer-related psychosocial disorders. APT is brief (average six sessions), directed at current problems and conducted with individual patients together with their partners or spouses, if possible. Therapy focuses on the personal meaning of cancer to the patient and his or her coping strategies. An outline of APT together with a clinical illustration is given. A randomised trial has demonstrated that APT produces significant improvement in psychological distress and thus in the quality of life of patients with cancer. PMID- 7551631 TI - History of psychotherapeutic intervention in cancer patients. PMID- 7551629 TI - Essentials of psychotherapeutic intervention for cancer patients. AB - Psychosocial treatments, including group, individual and family psychotherapies, are of proven efficacy, and deserve inclusion as standard components of biomedical treatment for cancer patients. Anxiety and depression are very common (and treatable) problems among cancer patients, most of whom can benefit from intervention. Psychotherapy, both group and individual, employs three fundamental approaches: emotional expression, social support, and cognitive symptom management skills. Psychotherapy has been shown to be effective in improving quality of life. Results of studies of various psychotherapies include reduction in depression, anxiety, and pain, and improved coping skills, and, in some cases, there is evidence of extended survival time. PMID- 7551632 TI - The aim of psychotherapeutic intervention in cancer patients. AB - Two main groups of goals in psychotherapeutic interventions in cancer patients are identified. First, process goals, which include the provision of information and instrumental support, assistance in expressing emotions, dealing with the the meaning of the disease, increasing patients' sense of coherence, enhancing personal resources, improving communication, gaining control and improving the ability to cope. Second, primary goals, which are aimed at decreasing somatic symptoms and psychosocial sequelae of cancer and its treatment, and enhancing adjustment, acceptance, and quality of life. PMID- 7551633 TI - The ethics of mercy killing. PMID- 7551634 TI - Fifty years of the SGM. PMID- 7551635 TI - Cf9 and Avr9, two major players in the gene-for-gene game. PMID- 7551636 TI - Typhoid fever and other salmonellosis: a continuing challenge. PMID- 7551637 TI - Virulence in Actinobacillus pleuropneumoniae and RTX toxins. AB - RTX toxins are pore-forming, cytolytic protein toxins that occur widely among pathogenic Gram-negative bacteria. RTX toxins appear to play a direct role in the virulence of Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia. This discovery has led to the development of new diagnostic and epidemiological tools, as well as vaccines, that are useful for a broad variety of serotypes. PMID- 7551639 TI - Epithelial cytokine responses and mucosal cytokine networks. AB - Localized at the border between the external environment and the internal tissue, epithelial cells are exposed to stimulants from two directions. Microorganisms in the lumen can activate the transcription of cytokine mRNA and cytokine secretion, and cytokines in the mucosal environment can modify endogenous and microbially induced epithelial cytokine responses. Epithelial cells thus actively participate in mucosal immunity and inflammation. PMID- 7551638 TI - Genome organization in the caliciviridae. AB - Caliciviruses cause a wide spectrum of important diseases. These viruses have a positive-sense single-stranded RNA genome; recently, the complete genome sequences of several caliciviruses have been determined. This review outlines the genome organization and phylogenetic relationships of the animal and candidate human caliciviruses. PMID- 7551640 TI - Understanding the mechanism of action of the anti-influenza virus drug amantadine. PMID- 7551641 TI - Immunotherapy of plant viral diseases. AB - The stable expression of antibodies in plants is one recent strategy for the unconventional control of plant viruses that is undergoing development. The advantages of this approach are its wide applicability and intrinsic safety; however, to be successful, the 'genetic immunization' of plants requires careful antibody design, efficient expression and targeting to appropriate cell compartments. PMID- 7551642 TI - Growth-phase regulation of plasmid virulence genes in Salmonella. AB - Virulence genes in the genus Salmonella are regulated by growth phase and by environmental signals, which allows a sequential program of expression during infection. Conditions that promote the expression of loci required in systemic infection, including the plasmid-encoded spv genes, are the opposite of the factors that induce genes involved in the invasion of epithelial cells in the gastrointestinal tract. PMID- 7551643 TI - Swarming and pathogenicity of Proteus mirabilis in the urinary tract. AB - Proteus mirabilis is best known for its pattern of swarming differentiation on agar plates, as well as for its association with the development of renal stones in patients with urinary tract infection. Urease and flagella appear to contribute most significantly to virulence, with fimbriae playing a more subtle role, whereas hemolysin does not appear to contribute significantly to pathogenesis. PMID- 7551645 TI - Does fortune favour the bald? Psychological correlates of hair loss in males. AB - In order to investigate the relationship between male hair loss and psychological distress, 182 men were recruited who had a wide range of ages and hair loss varying from none to severe. Care was taken to ensure that hair loss and age were uncorrelated in the sample. Multiple regression was used to predict possible consequences of baldness, controlling for age, and examining the interaction between baldness and age to see if consequences were especially severe in cases of premature baldness. Increasing degrees of hair loss were associated with loss of self-esteem, depression, introversion, neuroticism and feeling unattractive. These effects were more marked for young men in the case of self-esteem, introversion and feeling unattractive. PMID- 7551644 TI - Cognitive structural models: the perception of risk and prevention in coronary heart disease. AB - This paper examines the relationship between causal models and the assessment of actions in a naturalistic domain. Two studies used a novel network diagram technique to elicit individual causal models of the factors affecting the risk of coronary heart disease. The same individuals also rated the effectiveness of different preventive actions. In their diagrams, individuals depicted the causal interrelationships between various factors and estimated the strength of the connecting paths. Total path strength accounted for two-thirds of the variance in ratings of preventive actions. A number of methodological issues were also explored, which supported the utility of the technique. Consensual models allowed a ready comparison between groups of subjects and showed the importance of distinguishing the presence of paths from the strengths of those paths. The studies support the value of the technique in analysing individual differences in the way people represent complex causal structures; various extensions and applications are also proposed. PMID- 7551646 TI - I recognize your face but I can't remember your name: is it because names are unique? AB - Burton & Bruce (1992), and Cohen (1990a), offer alternative accounts of the differences in 'normal' and neurologically impaired subjects' abilities to recall people's names and other pieces of biographical information. Cohen claims that the principal difference between names and other biographical information is in their relative meaningfulness, while Burton & Bruce argue that uniqueness is the crucial variable. As yet existing empirical evidence cannot be explained within the framework of either of these accounts, although the Burton & Bruce (1992) proposal has the advantage of being implemented as an interactive activation model. This paper describes the case of a patient who, following a stroke, retained the ability to access 'unique' semantic information for familiar people she is no longer able to name. It is argued that this pattern of performance provides difficulties for the Burton & Bruce model. However, it can be accounted for by Cohen's account, and a modified version of Burton & Bruce's model (Bruce, Burton & Walker, 1994). PMID- 7551647 TI - Experiences of meaning in life: a combined qualitative and quantitative approach. AB - The present study investigates the relation of aspects of meaning in life with indices of psychological well-being by means of a combined qualitative and quantitative design. Content analysis of subjects' answers to open questions about personal experiences with meaning in life showed findings that are in line with phenomena that are reported in the literature. Meaningfulness was found to be strongly associated with contact with self, others and the world, whereas meaninglessness was associated with a state of alienation from self, others and the world. The Life Regard Index (LRI), an instrument designed to measure the construct of positive life regard, was found to be strongly associated with the interpersonal dimension of well-being. The exchange of both positive and negative feelings was associated with positive life regard. As predicted, effective coping with stressful life events in the past was associated with a current sense of meaningfulness as measured with the LRI. The findings support the clinical significance of the construct of meaning in life and add to the validity of the LRI. The strength and weakness of a combined qualitative and quantitative research approach are discussed. PMID- 7551648 TI - Classifying infants in the Strange Situation with three-way mixture method of clustering. AB - The quality of the attachment relationship between mother and infant is typically determined in the Strange Situation. The assignments of infants to the A (avoidant), B (secure), and C (resistant) attachment classes are largely but not exclusively based on measurements during the reunion episodes. In this paper, the measurements in the reunion episodes are used to derive a clustering of the infants via three-way mixture method of clustering, a technique especially designed for clustering three-way mixture method of clustering, a technique especially designed for clustering three-way data (here: infants, variables and episodes). The results are compared with the A-B-C classification, and the relevance of the outcomes for attachment research are discussed. At the same time, the paper aims to demonstrate the use and usefulness of the three-way clustering procedure for data from the social and behavioural sciences. PMID- 7551650 TI - Patient education: treatment or nice extra. PMID- 7551651 TI - Disease outcomes in rheumatology. PMID- 7551649 TI - Integral bias in the cognitive processing of emotionally linked pictures. AB - It is well established that human cognitive processes are subject to systematic biases associated with particular emotional states. But how do these cognitive biases arise? This question was addressed by means of a pictorial Stroop task which allowed the development of such a bias to be assessed. In each of two experiments, spider-phobic participants were significantly impaired at naming the colours of phobia-related pictures relative to those of control pictures. Investigation of the magnitude of this effect for participants of different ages allowed two contrasting hypotheses concerning the development of such biases to be compared. The integral bias hypothesis asserts that the effects of emotion on cognition reflect integral links between emotional states and cognitive mechanisms, links that are not arbitrary but instead are fixed constituents of our mental architecture. In contrast, the inferred bias hypothesis asserts that these effects arise as a consequence of the gradual establishment of associations between particular emotional states and particular patterns of cognitive behaviour. It was found that the magnitude of the observed bias remained approximately constant irrespective either of the ages of participants or of the directly estimated durations of their phobias. This fixed relation provides strong support for the integral bias explanation of the cognitive effects of an emotional disturbance. Finally, the evolutionary context in which integral links between emotion and cognition may arise is discussed. PMID- 7551652 TI - Therapeutic monoclonal antibodies. AB - Monoclonal antibodies have been used extensively over the last few years in clinical trials of rheumatoid arthritis (RA). Not only are they potential therapeutic agents, but they are also useful probes into the immunopathogenesis of RA. Anti-tumour necrosis factor alpha (TNF alpha) monoclonal antibodies have been shown to be clinically efficacious. Although they produced rapid disease amelioration, the duration of clinical improvement was limited to 4-6 weeks. Re treatments were again effective but long-term studies are required to assess their therapeutic role in RA. So far, the therapeutic effects of lymphocyte depleting antibodies have been disappointing. From the data, it is clear that synovial lymphocytes are more difficult to eliminate than peripheral blood lymphocytes and it is likely that in order to delete all synovial lymphocytes, high doses of depleting antibodies will be required which could lead to severe immunosuppression. Hence, lymphocyte depletion may not be a feasible therapeutic strategy. However, there are a number of clinical trials currently underway attempting to inhibit CD4 lymphocyte function by non-depleting antibodies. In animal models of RA, such antibodies have been shown to induce long-term disease remission. Another possibility is to combine several monoclonal antibodies in order to induce disease remission in RA. This strategy has been used in murine collagen-induced arthritis in which a combination of anti-CD4 and anti-TNF alpha monoclonal antibodies was shown to be synergistic. PMID- 7551653 TI - Specificity of antiperinuclear factor for rheumatoid arthritis in rheumatoid factor-positive sera. AB - Although antiperinuclear factor (APF) has the same specificity for rheumatoid arthritis (RA) as rheumatoid factor (RF), there is no evidence that this specificity is maintained in patients with positive RF-agglutination tests. Thus, we evaluated the specificity and usefulness of APF for RA diagnosis, regardless of RF titre. APF was tested (1:100 threshold) on 214 sera sent for RF evaluation over a 9-month period. These sera were previously determined to have latex or Rose-Waaler (RW) titres > or = 12 or 4 IU, respectively, but not necessarily above the threshold values of 100 and 32 IU. The APF test was performed blindly, and physicians were not advised of the results. In the patient population (119 RA and 95 non-RA) APF still demonstrated good specificity (0.82) for RA. As expected, APF proved useful for RA diagnosis in 28/33 (85%) RA cases with an RF level below 1:100 for latex and 1:32 for RW, thus reducing the number of 'seronegative' RA from 33/119 to 5/119. However, it also improved the serological positive predictive value for RA, even in cases when RW results were > or = 32 IU. Indeed, the positive predictive value for RA when both tests were positive was 0.94 (68/72), whereas concordant results (either positive or negative) for both APF and RW tests allowed correct classification (RA or non-RA) in 94% of cases. PMID- 7551655 TI - A quantitative study of the phagocytosis of urate crystals in the synovial fluid of asymptomatic joints of patients with gout. AB - The objective of this study was to determine whether monosodium urate (MSU) crystals are phagocytosed in the synovial fluid (SF) of the asymptomatic joints of patients with gout. SF samples were obtained from 20 asymptomatic knees of 19 different patients. Cell and differential counts were done. Intracellular MSU crystals were identified by ordinary and polarizing light microscopy. We found that in 19 out of the 20 SF samples intracellular MSU crystals have been found. A mean of 22.55% [confidence interval (CI) 13.92, 31.18; range 0-62] of all the cells contained intracellular MSU crystals. The majority of the cells which contained intracellular crystals were mononuclear cells (MC), and polymorphonuclear (PMN) leucocytes containing intracellular crystals accounted only for 0.5% (CI 0, 1.05; range 0-5) of the total. The total cell count was 527 cells/mm3 (CI 226, 828, range;: 30-2670). Poor correlation was found between the percentage of cells with intracellular crystals and both the total cell count (r = -0.22) and the percentage of PMN leucocytes (r = -0.26). We conclude that cells containing phagocytosed MSU crystals--generally mononuclear cells--are a regular finding in the SF of asymptomatic joints of patients with gout. This finding indicates that other factors besides intra-articular interaction between crystals and cells are necessary to produce arthritis in gouty patients. PMID- 7551654 TI - Polymorphism of the heat-shock protein gene HSP70-2 in systemic lupus erythematosus. AB - We investigate whether a heat-shock protein gene (HSP70-2) is involved in determining susceptibility to systemic lupus erythematosus (SLE) in a Spanish population. A HSP70-2 PstI polymorphism was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified genomic DNA in 90 SLE patients and 117 controls. The PstI site containing allele (B) was significantly increased in SLE patients compared to healthy controls. This was due to a significant increase in the BB homozygous genotype in patients, particularly in those with diffuse proliferative nephritis. Neither allelic nor genotypic differences were detected when compared by the presence or absence of DR3. The HSP70-2 B allele seems tightly linked to the human leucocyte antigen (HLA) haplotypes carrying susceptibility to SLE in our population. An independent role for this gene cannot be confirmed due to its linkage with HLA DR3. PMID- 7551657 TI - The incidence of juvenile dermatomyositis: results from a nation-wide study. AB - There have been no previous attempts to estimate the incidence of juvenile dermatomyositis (JDM) in the UK and Ireland. Consultant paediatricians from these countries were asked to report all cases of JDM which were diagnosed in 1992 and 1993 to the British Paediatric Surveillance Unit. One-hundred-and-twenty-one reports were received, of which 48 were confirmed cases of JDM and three were confirmed cases of polymyositis. The estimated incidence of JDM was 1.9 per million children aged under 16 yr (95% confidence interval 1.4-2.6). The median age at onset was 6.8 yr and the median delay in diagnosis was 4 months. Girls were affected much more often than boys (ratio 5.0:1). In the cases with an onset in 1992 there appeared to be a cluster of cases with an onset in April and May. PMID- 7551656 TI - Characteristics of ulcers of the small bowel induced by non-steroidal anti inflammatory drugs in the rat: implications for clinical practice. AB - Small bowel ulcers were created in the rat after the oral administration of non steroidal anti-inflammatory drugs (NSAIDs). Of the six NSAIDs tested, indomethacin and diclofenac alone were associated with such damage which did not occur in a simple dose-related fashion. Bacteria were observed by electron microscopy in an active state of division in the base of the ulcers. When grown aerobically these were shown to be strains of Escherichia coli and Proteus mirabilis. Anatomically, NSAID-induced ulcers were found throughout the length of the bowel although more abundant in the proximal half. In vivo and in vitro sensitivity to antibiotics suggested that in addition to the bacteria identified, anaerobic beta-lactamase-producing organisms also have an important role in ulcer production in this model. This rat model of NSAID-induced gut toxicity is discussed in relation to the human situation, particularly for patients who take NSAIDs and who have an iron-deficiency anaemia and blood in their faeces, but no lesions in either the upper or lower bowel. PMID- 7551659 TI - The effect of systemic lupus erythematosus and long-term steroid therapy on bone mass in pre-menopausal women. AB - The aim of our study was to assess bone mineral density (BMD) in pre-menopausal women with systemic lupus erythematosus (SLE) and the influence of disease activity and use of corticosteroids. Lumbar and femoral BMD were measured in 43 patients with SLE (28 on regular steroid therapy, 15 with recent onset, non treated) and compared with 43 healthy women matched for age. In addition, 21 SLE patients treated with corticosteroids were followed-up with a mean of 36.6 +/- 12.7 months. BMD was significantly lower in SLE patients with prednisone doses > or = 7.5 mg/day than those of recent onset (lumbar: 1.07 +/- 0.11 vs 1.15 +/- 0.13, P = 0.039; femur: 0.85 +/- 0.13 vs 0.98 +/- 0.21, P = 0.034) and the control group (lumbar: 1.07 +/- 0.11 vs 1.13 +/- 0.10, P = 0.040; femur: 0.85 +/- 0.13 vs 0.93 +/- 0.10, P = 0.028). In addition, 18% of SLE steroid users had osteoporosis. Lumbar and femoral BMD were inversely correlated with the time of treatment and the cumulative doses of prednisone. There were no significant BMD changes during the 3-yr follow-up period. In conclusion, in SLE pre-menopausal patients lumbar and femoral BMD is decreased and related to long-term corticosteroid therapy. PMID- 7551658 TI - Menstrual disorders in girls with systemic lupus erythematosus treated with cyclophosphamide. AB - To study the ovarian toxicity associated with cyclophosphamide in girls with systemic lupus erythematosus (SLE), we retrospectively reviewed the charts of 30 SLE girls aged 16 yr or younger at diagnosis, followed at three university hospitals. Gynaecological history was extracted from the charts or obtained prospectively. Ten had not received cyclophosphamide therapy, six were treated with daily oral cyclophosphamide, 10 with intravenous pulses and four with daily oral and intravenous pulses. Median oral cyclophosphamide dose was 38 g (inter quartile range 75) and median intravenous dose 12.95 g (inter-quartile range 6.2). Six girls had oligomenorrhoea (20%) and one amenorrhoea (3%). Two treated with oral cyclophosphamide had oligomenorrhoea (33%) and one amenorrhoea (17%), two treated with both oral and intravenous pulses had oligomenorrhoea (50%), and none of those treated with intravenous pulses alone had menstrual disturbances (50% oral vs 0% intravenous pulses; P = 0.016). Girls who had menstrual disturbances had received higher doses of cyclophosphamide than those who did not (medians: 63 vs 15 g; P < 0.05). In summary, menstrual disturbances in SLE girls treated with cyclophosphamide are related to the total dose and perhaps to the administration method. PMID- 7551660 TI - Effect of methotrexate alone or in combination with sulphasalazine on the production and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in patients with rheumatoid arthritis. AB - In a recent study from our group, the combination of methotrexate and sulphasalazine (MTX + SASP) seemed superior to MTX alone in the treatment of rheumatoid arthritis (RA). To assess the impact of these therapies on the cytokine cascade, the in vitro production and circulating concentrations of several cytokines and endogenous cytokine antagonists were measured in 30 healthy controls and longitudinally in a subset of 26 patients enrolled in this study. Compared to controls, RA patients had significantly higher circulating concentrations of interleukin-6 (IL-6), soluble receptors for tumour necrosis factor (sTNFR), soluble receptors for interleukin-2 (sIL-2R) and interleukin-1 receptor antagonists (IL-1RA), and their peripheral blood mononuclear cells (PBMNC) showed a higher spontaneous production of interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and IL-1RA (both secreted and cell associated) and a higher stimulated production of cell-associated TNF alpha, IL 1RA and (to a lesser extent) IL-1 beta. Treatment with MTX alone (n = 12) or combined with SASP (n = 14), resulted in significant reductions of circulating IL 6 and sIL-2R but did not alter IL-1 beta, TNF alpha or IL-1RA concentrations. Decreases in circulating levels of sTNFR and in the in vitro production of cell associated IL-1 beta and IL-1RA after stimulation were only observed in patients treated with MTX + SASP. The concentrations of IL-1RA and sTNFR in the circulation exceeded moderately those of IL-1 beta and TNF alpha but this is probably insufficient to block IL-1 and TNF alpha activity. In conclusion, therapy with MTX alone or with SASP modulates IL-6 and sIL-2R concentrations in RA. Decreased production of IL-1 beta and IL-1RA and circulating sTNFR levels were only observed during therapy with MTX + SASP. Whether this relates to the better clinical effect observed with the combination therapy remains to be investigated. Circulating levels of IL-6, sIL-2R and sTNFR seem useful markers of disease activity in RA. PMID- 7551662 TI - Comparison of the validity and reliability of self-reported articular indices. AB - Our objective was to compare the validity and reliability of three formats for self-administered articular indices assessing pain (PAI) or swelling (SAI). Fifty five patients with rheumatoid arthritis were asked to mark the degree of pain on a list of 16 joints (PAI list), to mark 'painful joints' on a mannequin presenting 42 joints (PAI diagram), and to mark 'swollen or tender joints' on a mannequin presenting 38 joints (SAI diagram). The test--retest reliability (intraclass correlation coefficient) ranged from 0.63 (SAI diagram) to 0.67 (PAI diagram) and 0.85 (PAI list). The correlation with clinical parameters was strongest for the PAI list and the SAI diagram. The association of the SAI diagram with clinical parameters increased with omission of the less reliable toe joints and/or weighting for joint size according to Lansbury. As expected, the short and weighted SAI diagram correlated more strongly with the physician derived swollen joint count (r = 0.49), C-reactive protein (r = 0.49) and erythrocyte sedimentation rate (r = 0.41) than did the PAI list whereas the PAI list correlated more strongly with physician-derived tender joint count (r = 0.43), global pain measured on a numerical rating scale (r = 0.57) and the Health Assessment Questionnaire (r = 0.49) than did the SAI diagram. We concluded that patients' rating of tender and swollen joints on a mannequin diagram and calculation of a 26-joint and weighted articular index produces an excellent estimate of total joint inflammation, which may be useful in clinical, health services and epidemiological research. An articular index calculated from ratings of pain degree of 16 joints or joint groups may provide complementary information. PMID- 7551663 TI - Ankylosing spondylitis: the correlation between a new metrology score and radiology. AB - The objective was to compare the Bath Ankylosing Spondylitis Metrology Index (BASMI) with radiology as a measure of disease outcome. Fifty-three patients, covering the entire spectrum of disease severity, were blindly and independently assessed using the BASMI (total of five standardized measurements, scoring range 0-10) and a radiology score of the four main spinal areas affected by ankylosing spondylitis (AS). BASMI correlates positively with the total radiology score (r = 0.74), while the individual BASMI scores for cervical rotation (r = 0.59), wall to tragus (r = 0.61), lumbar side flexion (r = 0.56), lumbar flexion (r = 0.68) and intermalleolar distance (r = 0.50) correlate positively with their respective radiology scores. BASMI and radiology do not relate well to each other as BASMI takes account of normal physical limitation and soft tissue involvement. In addition, although radiology scores are termed a 'gold standard', they are unreliable. Therefore, BASMI may be judged to be more important in assessing AS and become a 'gold standard' itself. PMID- 7551661 TI - The effects of alcoholic beverages on urate metabolism in gout sufferers. AB - The purine contents of commercial, low-alcohol and alcohol-free beers were determined. Four gout sufferers were studied under controlled conditions before and after ingestion of four different beverages containing alcohol, alcohol and purine, purine and neither alcohol nor purine. The results show a significant increase in purine excretion with a fluid load alone and impairment or reversal of this response with the other three beverages. These results are difficult to interpret on the basis of the alcohol and purine contents of the beverages alone. Isohumulones are present in all beers. Their effect on urate metabolism and excretion is unknown but needs further study as a possible explanation of these results. These results suggest that the three beverages other than a fluid load alone are unsuitable for gout sufferers. PMID- 7551665 TI - Health economics as an aspect of health outcome: basic principles and application in rheumatoid arthritis. AB - Health economic evaluation comprises the systematic appraisal of the costs, the benefits and the relative economic efficiency of different medical interventions. The first section of this paper outlines the techniques of economic analysis and how they relate to the efficient use of health resources. This is followed by a review of the health economics of rheumatoid arthritis as a model for the wider application of health economics in the field of rheumatology. PMID- 7551667 TI - Bush rheumatology or rheumatologist gone bush? PMID- 7551664 TI - The effects of joint washout and steroid injection compared with either joint washout or steroid injection alone in rheumatoid knee effusion. AB - Patients with rheumatoid arthritis attending rheumatology out-patients who had a symptomatic knee effusion were randomly allocated to receive one of three treatments: group I, a steroid injection without washout; group II, a joint washout with normal saline; group III, a joint washout with normal saline and steroid injection. Sixty knees in all were studied. Laboratory parameters for disease activity (erythrocyte sedimentation rate, C-reactive protein) were monitored in all patients prior to the study and at 3 months. Clinical assessment of disease activity (pain, morning stiffness involving the knee, circumference of the knee, walking distance and range of movement) were recorded prior to the study, at 1 month and at 3 months. All three treatments, resulted in a reduction of pain and increased movement. However, patients who had a joint washout alone showed significantly less improvement as compared with the other two groups. Symptomatic improvement was marginally greater in patients following joint washout and injection than in those who had had joint injection alone. The results of the study indicate that the simple procedure of joint aspiration and steroid injection, which can be carried out in the out-patient clinic, provides satisfactory relief of symptoms in rheumatoid patients with knee effusions. Joint washout alone was less beneficial. PMID- 7551666 TI - Methodological problems in comparing the severity of rheumatoid arthritis between populations. PMID- 7551668 TI - Atlanto-occipital subluxation in rheumatoid arthritis demonstrated by magnetic resonance imaging. AB - Atlanto-occipital subluxation is a rare but recognized form of subluxation that occurs in rheumatoid arthritis (RA) at the cranio-cervical junction. Magnetic resonance imaging (MRI) clearly demonstrates the bony and soft tissue changes of RA in the cervical spine. We report a single case of atlanto-occipital subluxation in RA demonstrated by MRI. PMID- 7551669 TI - Should tests for proteinuria be included in the monitoring schedule of sulphasalazine? AB - Two patients who developed proteinuria while taking sulphasalazine are described. It is argued that the prevalence of this side-effect is such that tests for proteinuria should be included in the monitoring protocol for sulphasalazine. PMID- 7551670 TI - Receiver operating characteristic analysis of urinary pyridinium cross-links in arthritis. PMID- 7551671 TI - Bath Ankylosing Spondylitis Functional Index. PMID- 7551672 TI - A new case of polyarthritis as a presenting sign of non-Hodgkin's lymphoma. PMID- 7551674 TI - The growth of scientific interest in rheumatology. PMID- 7551673 TI - Quantification of joint laxity. 'The non-dominant (Spanish) modification'. PMID- 7551675 TI - Advice to car drivers with rheumatoid arthritis. PMID- 7551676 TI - Toxic epidermal necrolysis, pancytopenia and adult respiratory syndrome. PMID- 7551677 TI - Clavulones and related tert-hydroxycyclopentenone fatty acids: occurrence, physiological activity and problem of biogenetic origin. AB - The present review is concerned with a group of prostanoids from soft corals, namely, clavulones and their congeners, as well as with related octadecanoids found in plants of the genus Chromolaena (Compositae). Known physiological properties of these prostaglandin-like fatty acids are discussed. Special attention is paid to published hypotheses on the biogenetic origin of clavulones and related fatty acids. According to a newly proposed biosynthetic route, acyclic alpha-ketols are metabolic precursors of cyclopentenolones. PMID- 7551678 TI - Platelet-activating factor mediated potentiation of stimulation-evoked catecholamine release and the rise in intracellular free Ca2+ concentration in adrenal chromaffin cells. AB - The effects of platelet-activating factor (PAF) on catecholamine (CA) release and intracellular free Ca2+ concentration ([Ca2+]i) were studied in cultured bovine adrenal chromaffin cells. PAF (1 nM-1 micron) alone had no effect on [Ca2+]i and basal CA release, but potentiated the [Ca2+]i rise and CA release evoked by acetylcholine (ACh) and by elevated extracellular K+. PAF did not potentiate the responses to caffeine in Ca(2+)-deficient medium or to Bay K 8644. In chromaffin cells pretreated with either BN 50739, tetrodotoxin and amiloride or in Na(+) deficient medium, PAF failed to potentiate the stimulation-evoked [Ca2+]i rise and CA release. In contrast, neomycin, U 73122, 5-(N-ethyl-N-isopropyl)amiloride or pertussis toxin were ineffective in blocking the potentiating effect of PAF. In a membrane fraction prepared from fresh bovine adrenal medulla, ligand-binding studies using [3H]WEB 2086 identified a PAF-displaceable binding site. These results are consistent with a model in which PAF modulates CA release by activating plasma membrane receptors that can enhance the [Ca2+]i rise via an Na(+)-dependent mechanism. PMID- 7551679 TI - Inhibition of leukotriene biosynthesis improves renal function in experimental glomerulonephritis. AB - The development of renal dysfunction in experimental glomerulonephritis (GN) is mediated in part by enhanced leukotriene (LT) formation. In our studies the pathophysiological role of LTs was investigated through pharmacological inhibition of LT biosynthesis in a rat model of nephrotoxic serum nephritis. MK 0591, an indirect inhibitor of 5-lipoxygenase activity, was co-administered to rats injected with nephrotoxic rabbit serum, followed by assessment of renal function, morphology and microsomal LTC4 synthase activity on day 7. A significant improvement in glomerular function was noted (p < 0.05), together with a 50% reduction in proteinuria (p < 0.01) in animals receiving MK-0591 (60 mg kg-1 day-1). In addition, the fall in renal LTC4 synthase activity which occurred in nephritic rats (to 74% of control values, p < 0.01) was prevented in drug-treated animals. Based on these results, it appears that inhibition of LT biosynthesis protects against both renal impairment and alterations in LTC4 synthase activity during the development of experimental GN, and may provide a useful therapeutic adjunct in the treatment of this disease. PMID- 7551680 TI - Role of rat liver plasma membrane phospholipids in regulation of protein kinase activities. AB - The role of rat liver plasma membrane phospholipids in the regulation of protein kinase A, protein kinase C and tyrosine kinase activities was investigated. Plasma membrane composition was modified by phospholipase A2, phospholipase C and phospholipase D treatment and subsequent incorporation of various phospholipids. Phospholipase A2 deactivated the three types of protein kinases, while phospholipase C and D affected the enzymes in a different manner. Phosphatidylcholine and sphingomyelin were found to be the most effective activators of protein kinase A and tyrosine kinase. Incorporation of sphingomyelin and phosphatidylserine into partially delipidated plasma membranes resulted in a significant stimulation of protein kinase C activity. Since sphingomyelin appeared to be a specific effector of the three types of protein kinases under investigation, one might suggest that its role in cellular signaling could be manifested via regulation of protein kinase C as well as via modulation of protein kinase A and tyrosine kinase activities. PMID- 7551682 TI - Phospholipase A2 activity in vitro cultured cells--influence of ascorbate supplementation. AB - Non-malignant LLCMK cells and malignant B16 cells were supplemented with ascorbate, over the concentration range 0-100 micrograms/ml ascorbate. The effects of ascorbate supplementation on cell growth and phospholipase A2 activity of the membrane fractions of the respective cell lines were determined. Increasing ascorbate supplementation had a significant inhibitory effect on the growth of the B16 cells. Phospholipase A2 activity in the control B16 cells was lower than that detected in the control LLCMK cells. Phospholipase A2 activity decreased significantly in the B16 cells upon increasing ascorbate supplementation, while the supplementation of ascorbate to the LLCMK cells did not have any significant effect on phospholipase A2 activity in these cells. PMID- 7551681 TI - Expression of S14 protein gene in rat liver in response to partial hepatectomy, and its regulation with T3 and sucrose. AB - In rat liver a significant decrease in the expression of the protein S14 gene was observed 4 h after partial hepatectomy compared to sham operation performed after overnight fasting. After 24 h a decrease was recorded after partial hepatectomy or sham operation and this low level persisted for 120 h. A single i.p. dose of 3,5,3,'-L-triiodothyronine (10 micrograms/100 g body wt), a potent inducer of gene S14, reversed the inhibition of gene expression at each time interval studied in both the partially hepatectomized and sham-operated rats. Sucrose administration to rats 24 h after partial hepatectomy or to sham-operated rats restored the induction of hepatic mRNA S14 gene expression in approx. 60% of controls as recorded after 4 and 24 h. These data suggest that the expression of gene S14 was altered in the liver after partial hepatectomy or sham operation by food restriction, and no compensatory effect of the increased lipid metabolism was found in rat remnant liver. Our findings indicate that thyroid hormone and sucrose administration exert similar effects on hepatic expression of the protein S14 gene both in the regenerating liver and in the intact liver of sham-operated animals. In spite of the increased lipid metabolism of the liver after resection, sensitivity of mRNA S14 expression to triiodothyronine and sucrose was unchanged and its regulation was operative in a positive manner. These results also suggest that the treatment of a regenerating liver containing both the parent and the daughter hepatocytes with sucrose and/or triiodothyronine resulted in a fully restored response. PMID- 7551683 TI - Leukocyte-induced endothelial dysfunction in the rabbit basilar artery: modulation by platelet-activating factor. AB - We studied the effects of polymorphonuclear leukocytes (PMNLs) activated by N formyl-methionyl-leucyl-phenylalanine on the endothelium-dependent relaxation of the rabbit basilar artery (BA). In the presence of activated PMNLs the maximal vessel relaxation to acetylcholine (ACh) and bradykinin (endothelium-dependent dilators) was decreased from 62 +/- 7 and 48 +/- 6% to 23 +/- 9 and 19 +/- 7, respectively, (p < 0.05). The endothelium-independent relaxation to nitroprusside was not affected by PMNLs. When PMNLs were activated in the organ chamber in the presence of a low concentration of platelet-activating factor (PAF, 10(-10) mol/l), the depression of ACh- and bradykinin-induced relaxation increased by 27 +/- 9 and 23 +/- 7%, respectively (p < 0.05), though at this concentration PAF alone did not cause PMNLs to induce endothelial dysfunction. In addition, in the presence of PAF, activated PMNLs inhibited endothelium-dependent relaxation at lower cell concentrations and shorter periods of contact with the endothelium. PMNL effects on the endothelium were correlated with the level of cell exocytosis as tested by accumulation of beta-glucuronidase activity. In the presence of PAF, accumulation of this activity increased from 46 +/- 6 to 79 +/- 8 U/ml (p < 0.05). Examination of BA segments by scanning electron microscopy revealed that, after the treatment with activated PMNLs, the endothelium was morphologically preserved, but in the presence of PAF PMNLs caused more apparent microlesions in the endothelial layer. We conclude that small quantities of PAF potentiate the activation of marginated PMNLs. These cells then become more aggressive towards the endothelium, producing significant depression of the endothelium-dependent relaxation. PMID- 7551684 TI - Inhibition of platelet type II phospholipase A2 by an acylamino phospholipid does not alter arachidonate liberation. AB - An acylamino phospholipid analogue (2-(R)-N-palmitoylnorleucinol-1-phosphoglycol or (R)-PNPG) was examined for its inhibitory effects against type II phospholipase A2 (PLA2) acting on membranes from Escherichia coli. Using two enzyme sources (rat platelet membranes or recombinant human type II PLA2), (R) PNPG inhibited phospholipid hydrolysis to a maximal value of 80-85%, half-maximal effect being attained at a substrate/inhibitor molar ratio of 80-250. In contrast, (S)-PNPG was 12-fold less potent and thus provided a control for possible non-specific effects of these polar lipids. However, both analogues exerted only marginal effects on the liberation of [3H]arachidonic acid from rat platelets challenged with calcium ionophore A23187. Since, among various animal species, rat platelets contain by far the highest amounts of this enzyme, our data rule out any possible involvement of secretory PLA2 in arachidonic acid liberation from platelet phospholipids, cytosolic PLA2 appearing in this case as the best candidate able to regulate eicosanoid biosynthesis. PMID- 7551687 TI - IL-1 beta and TGF-beta 1 modulate the sulphation grade of chondro-disaccharides in porcine articular cartilage: a capillary electrophoresis study. AB - This report describes the effect of interleukin-1 beta (IL-1 beta) and transforming growth factor-beta 1 (TGF-beta 1) on proteoglycan release from cartilage explants and modification at the sulphation level. Matrix proteoglycans purified by ion-exchange chromatography were composed of two distinct peaks (1 and 2) each showing a different Kav value when they were subjected to size exclusion chromatography on a Sepharose CL-2B column. Glycosaminoglycans (GAGs) of conditioned medium and extracellular matrix proteoglycans were digested by chondroitin ABC and AC lyase, suggesting that chondroitin sulphate (CS) is the major GAG present (80-90%). Structural analysis of disaccharides, by capillary zone electrophoresis, revealed a different pattern of sulphated glycosaminoglycans when cartilage was treated with either IL-1 beta or TGF-beta 1. Analysis of GAGs released into the medium from TGF-beta 1 treated cartilage showed a reduction in the level of 4-S-disaccharide (delta Di4S) and an increase in non-sulphated disaccharides (delta Di0S), while no significant changes were found in IL-1 beta treated cartilage. In the extracellular matrix, IL-1 beta and TGF-beta 1 induced a more complex rearrangement of the GAGs. The level of non sulphated disaccharides was increased whereas that of total sulphated disaccharides was reduced. Taken together, these results suggest that both cytokines modify the structure of GAGs, probably by interfering with the activity or the synthesis of sulphotransferases involved in GAG turnover. PMID- 7551689 TI - Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury. AB - In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC50 = 2.4 +/- 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAF is not an essential component of the cascade leading to restenosis following vascular injury. PMID- 7551685 TI - Pharmacological evidence for the existence of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria. AB - The negative inotropic effect of carbachol, as well as phosphoinositide hydrolysis, was measured in atria from neonatal and adult rats. Carbachol increased phosphoinositide hydrolysis and decreased dF/dt of both neonatal and adult atria; however, the neonatal atria showed hypereactivity to carbachol as compared with adult atria. Inhibition of phospholipase C reduced the supersensitivity to carbachol upon contractility in neonatal atria producing values similar to those of the adult atria, suggesting that muscarinic acetylcholine receptor (mAchR) stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides. Pharmacological analysis with mAchR antagonists tends to support the idea that m1 and m2 subtypes are the most important mediators of the response to carbachol in neonatal atria. In adult atria, the effect of carbachol is coupled only to mAchR m2 subtypes. PMID- 7551688 TI - PAF and haematopoiesis: V. Platelet-activating factor and acetylhydrolase in human femoral bone marrow. PMID- 7551686 TI - Lipid inhibitors of platelet-activating factor (PAF) in normal human plasma. AB - Endogenous, human plasma-derived lipids that inhibit the platelet stimulating activity of platelet-activating factor (PAF) have been identified. Chromatographic fractionation of neutral lipid PAF inhibitors revealed a majority of PAF inhibitory activity comigrating with cholesterol and a second peak localized with free fatty acids. Plasma phospholipids demonstrated three distinct PAF inhibitory fractions in TLC regions corresponding to those of sphingomyelin, phosphatidylcholine and phosphatidylethanolamine. Three fractions (one neutral lipid and two phospholipid) specifically inhibited PAF-induced platelet activation. Thus, there are both specific and non-specific lipid inhibitors of PAF in normal human plasma. These plasma lipids may be important in the specific regulation of the diverse, potent biological activities of PAF in various physiological states. PMID- 7551690 TI - Phospholipids with a short acyl chain modulate phospholipase and acyltransferase activities. AB - 1-Acyl lysophosphatidylcholine prepared from egg yolk has been chemically reacylated to form decanoyl, dodecanoyl, myristoyl and palmitoyl derivatives of phosphatidylcholine. The liposomes formed by these semi-synthetic phospholipids have been characterized by calorimetry, X-ray diffraction and fluorescence probe methods. Asymmetric phosphatidylcholines tend to promote formation of excimers of a codispersed fluorescent phospholipid (1-palmitoyl-sn-2-(1-pyrenedecanoyl)-L alpha-phosphatidic acid) (2 mol%). Excimer formation is correlated with the rate of hydrolysis of the fluorescent anionic phospholipid by Crotalus venom phospholipase A2. Codispersion with the semi-synthetic phosphatidylcholine of cholesterol or unsaturated fluid lecithin modulated both excimer formation and the susceptibility of the fluorescent probe to hydrolysis by venom phospholipase A2 at 22 degrees C. Similar results were obtained with hydrolysis of a radiolabelled substrate, 1-palmitoyl-sn-2-[1 14C]linoleoylphosphatidylethanolamine, codispersed with the semi-synthetic phosphatidylcholine. Enrichment of rat hepatocyte plasma membranes with semi synthetic asymmetric phosphatidylcholines was mediated by incubation of membranes with phospholipid dispersions in the presence of a phospholipid exchange protein. Enrichment of the membranes with semi-synthetic phosphatidylcholines of between 30 and 60% of the membrane phosphatidylcholine was achieved. The resulting alteration of the biomembrane is associated with a decreased activity of endogenous membrane phospholipase A2 acting on extramembranous radiolabelled substrate vesicles. By contrast, the activity of acyl-CoA:lysophospholipid acyltransferase is increased in membranes enriched with highly asymmetric phospholipids. PMID- 7551691 TI - [Work conditions and health status of workers engaged in nickel production using carbonyl method]. AB - Work conditions in modern production of nickel (carbonyl way) are characterized by high concentrations of sodium tetracarbonyl in the industrial compartments, so the acute and chronic intoxications in the workers could occur. Data on biologic control of the exposure (according to nickel level in the urine and carbon dioxide content of the expired air) prove considerable load of sodium tetracarbonyl in the workers and the inadequate protective means. The workers are characterized by increased prevalence of chronic respiratory, gastrointestinal, cardiovascular, endocrine and dermatologic diseases. PMID- 7551692 TI - [Psychophysiological testing system, Combitest 4, in the studies of stress in workers engaged in mental work involved with computers]. AB - The system "COMBITEST 4" enables to conduct psychophysiologic studies according to the scheme "load--strain" in assessment of psychophysiologic reactions in laboratory and field conditions, in real activities of examinees on their workplaces. The patients with vagotonia vs. those with sympathicotonia demonstrated stably effective performance of tasks suggested during the day. Drastic fluctuations of cardiac reactivity parameters (pulse rate, SAa, spectral distribution) in the examinees with vagotonia prove constant reaction to the new conditions, whereas those with sympathicotonia demonstrate more stable physiologic parameters. PMID- 7551693 TI - [Clinical-toxicological aspects of micro mercury poisoning]. AB - Thirty workers were hospitalised after clinical and laboratory screening of 65 women and 35 men from electrical lamp factory. The mercury concentration in air of the working places varied from normal value (0.01 mg/m3) to 0.04-0.05 mg/m3. Target neurological anamnesis, internal and neurological examination, EEG, renal function tests and several laboratory parameters of large extent were carried out. The analysis of results establishes the signs of micromercurialism in 20% of the examinees. The authors determine the peculiar clinical (neurasthenic syndrome, periodontal diseases ) and biochemical (blood Hg level and urinary mercury) parameters of this early, functional stage of the chronic Hg poisoning. PMID- 7551696 TI - [Component analysis of variability as a method of differentiating simple effect from hazardous effect in toxicological experiments]. AB - The article forms the main requirements for every hazard criterion (valid, definite, statistically correct, differential, invariable, sensitive, integral). The suggested hazard criterion is based on additive components of general dispersion. Those components reflect environmental influence (environmental effect) and influence of substance (effect of substance), then the components are compared according to Fisher criterion (F). The criterion works in two-factor dispersion analysis. PMID- 7551699 TI - [Diagnostic method in occupational vegetative trophic dysfunction of upper limb muscles]. AB - The investigators registered the curves of force and quickness in digital compression of transducer during 30 s at rate of 80 per minute. Force and quickness at the certain moments of the movements were measured, times of contraction and relaxation were estimated. The method enables to assess the parameters of muscular contractility, compare them and evaluate the functional state. Lower quickness of contraction is known as the first sign of muscular fatigue. The method is applicable in workshops, handy and fit for mass screening, enables to assess influence of work conditions on the worker's hands. The method is certified by an Authorship No 1683682. PMID- 7551694 TI - [Clinical interpretation of the effects of leading hazardous factor in multifactorial intensive action]. AB - The article "Clinical manifestations of chronic exposure of metallurgists and blacksmiths to heating microclimate" by T.K. Semennikova et al., published in "Industrial medicine and occupational hygiene" No 5, 1992, was reviewed and compared with the author's data. Extensive clinical materials in the article reviewed are traced only to chronic overheating, however the hygienic data presented prove critical levels of noise, local and general vibration, hard work. Index of occupational diseases was calculated, so vibro-acoustic effects and chronic overheating appeared to be comparable. Analysis of etiology and pathogenesis appeared to play an important role in evaluation of effects caused by multiple critical factors. PMID- 7551697 TI - [Occupational vegetative dystonia or psychogenic disorder?]. PMID- 7551695 TI - [Hygienic assessment of occupational hazards resulting form low-soluble lead compounds]. AB - The MACs for aerosols of low-soluble lead compounds should be 10 times higher than those for inorganic lead compounds. PMID- 7551698 TI - [Evaluation of the degree of radiation safety in the use of radioisotope devices in teaching and research centers]. AB - The authors consider degree of radiation danger in exploitation of devices using radio isotopes in research departments of Moscow and the region. The radiation doses accumulated by the staffers using all types of the devices do not exceed the main dose limit for B category (maximum 0.5 rem annually). The main causes of radiation accidents are violated norms of exploitation and loss of the radiation source, the main result of radiation accident with radio isotope devices is environmental pollution and external irradiation of population. PMID- 7551700 TI - [Means of individual protection in cleaning-up consequences of accidents at chemical industries]. AB - The authors considered protective efficiency and physiologic acceptability to compare various types of individual protective means applied in some industries. The article presents general principles and methodic approaches to use of various individual protective means for eyes and respiratory organs. The important notion is that besides individual protective means also pharmacologic agents, various norms and regulations (MAC) should be used to protect humans from chemical hazards. Some revealed common features for chemical enterprises could serve to forecast occupational diseases and therefore determine individual protective means for respiratory organs and define the application of those means. To make quick and correct choice of the individual protective means for respiratory organs during accidents at chemical enterprises, one should refer to PC data bases. PMID- 7551703 TI - [Teratogenic effects of substances polluting surface waters in the district of intensive development of coal and chemical industries]. PMID- 7551702 TI - [A neuropharmacological study of amnesia in animals induced by ultra high frequency electromagnetic irradiation]. PMID- 7551701 TI - [Substantiation of physiologic and hygienic requirements of heat-proof properties of winter headgear]. AB - Experimental studies conducted in cooling environment assessed the features of heat exchange and thermoregulation of head during physical work varying in hardness and type. The studies determined allowable parameters of heat exchange on head (skin temperature and surface heat outflow), which are necessary for construction and evaluation of headgear. The article presents correlations between main heat-proof parameters of headgear (total heat resistance, thickness of materials and their permeability to air). The data of experimental and industrial trials helped to elaborate physiologic and hygienic requirements for heat-proof headgear in various climate conditions. PMID- 7551704 TI - [Effects on the body of chemicals appearing in double superphosphate production]. PMID- 7551706 TI - [Work conditions in concentration of tungsten-molybdenum ore]. AB - Manifold hygienic studies carried out at Tyrnaouz tungsten-molybdenum enterprise served as example to characterize work conditions, microclimate, chemical load, levels of dust, noise, vibration and radiation at modern concentration enterprises. The factors leading in intensity were dust load in air during preparation of ores processing (grinding, grist, etc.), noise, and microclimate especially during cool season. Hygienic importance of occupational factors is shown by data on morbidity with transitory disablement and outpatient medical examinations. PMID- 7551705 TI - [Toxicity of the Extreme East fir ointment]. PMID- 7551707 TI - Evolutionary aspects, and taxonomic definition of viruses together with mobile extrachromosomal elements of relative autonomy into a special highest rank taxon (a review). PMID- 7551708 TI - Nutrient optimization for production of broad spectrum antibiotic by Streptomyces antibioticus SR15.4. AB - Antibiotic production by Streptomyces antibioticus Sr15.4 was studied under various cultural conditions. During nutrient optimization it was found that the strain utilized glycerol as the best source of carbon at 1.044 molar level, and 0.020 molar arginine as the best source of nitrogen. The strain exhibited significant enhancement in antibiotic production when grown at pH 6.8. PMID- 7551710 TI - Effects of some heavy metals on growth, pigment and antibiotic production by Streptomyces galbus. AB - A chromogenic strain of Streptomyces galbus 5ME-13 producing an antifungal antibiotic was used to study the effects of some heavy metals on growth, melanoid pigment and antibiotic production in culture. Mercury and nickel were highly toxic followed by cadmium while cobalt and chromium appeared to be less toxic. All the metals inhibited growth, pigment and antibiotic production with widely variable IC50 values. Cadmium and chromium at lower concentrations unusually stimulated growth and enhanced pigment production in the strain. PMID- 7551709 TI - In vitro suppression of anti-TSH receptor antibody by autologous anti-idiotypic antibody in patients with Graves' disease. AB - Regulation of anti-TSH receptor antibody (anti-TSH-R antibody) in Graves' patients (n = 11) by anti-idiotypic antibody was studied using patients sera before and after one year of Methimazole treatment. Patients sera with high level of anti-TSH-R antibody (110 + 41.9 U/I) were incubated with pooled control and autologous (with low level or anti-TSH-R antibody negative) sera containing equimolar IgG G. The mixture was centrifuged and the supernatants were tested for anti-TSH-R antibodies (TRAK, Henning). It was found that the autologous sera from patients with remission were able to suppress significantly the titre of anti-TSH R antibodies (p < 0.001), whereas the controls were capable of a less remarkable inhibition. F(ab')2 fragments of autologous IgG from remission could also suppress the levels of TSH-R antibodies. It was concluded that anti-anti-TSH-R antibodies in sera of Graves' patients might be, at least in part, responsible for inducing and maintaining remission and suppression of autoantibodies to TSH R. It is hypothesized that the idiotype system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenetic antibodies. On the basis of this observation the autologous sera could have therapeutical implication in an accelerated remission of hyperthyroidism in Graves' disease. PMID- 7551711 TI - Altered humoral immunity against cytomegalovirus and Epstein-Barr virus without detectable virus antigens and virus-DNA in the glomeruli of patients with IGA nephropathy in remission phase. AB - Herpes viruses, such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), very often occur in the nasopharynx. A pathogenetic role of these viruses in immunoglobulin A nephropathy (IgA NP) is a challenging hypothesis, since upper respiratory tract infections are frequently and closely related in time to the acute episodes of IgA NP. However, conflicting reports have been published in this field. We compared the IgA and IgG antibody (AB) titres against cytomegalovirus (CMV) in sera of 41 IgANP patients with 80 healthy controls. The prevalence rates of CMV-IgA and CMV-IgG AB were significantly higher in patients with IgA NP than in healthy controls (CMV-IgA, titre > = 8, p < 0.001; CMV-IgG, titre > = 16, p < 0.05). There was also a significant difference in the concentration of CMV-IgA AB between IgANP patients and controls (IgA NP: 0.34 +/- 0.66, healthy controls: 0.06 +/- 0.33, mean +/- SD, p = < 0.002), but not in the concentration of CMV-IgG AB between NP patients and controls. While examining 60 patients with IgA NP and 75 healthy controls a significantly elevated prevalence rate of IgA AB against EBV capsid antigen (EBV-VCA) was also detected in the sera of IgA NP patients vs controls (titre > = 16; p < 0.05). There was no significant difference in the prevalence of IgG AB to EBV-VCA between patients with IgA NP and healthy controls, except at titre = 1024 (p < 0.05). At a follow-up, CMV-IgA persisted in 4 of 5, and EBV-VCA-IgA in 8 of 12 seropositive patients with IgA NP. We could not detect virus antigens or virus deoxyribonucleic acid (DNA) in the glomeruli of NP patients either with immunohistology using a monoclonal antibody or with the DNA in situ hybridization technique.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551713 TI - Remarks on the appearance of "violine" on Cantal cheese (a note). AB - A mould, Scopulariopsis brevicaulis was isolated from a Cantal cheese. This mould was responsible for the "violine" phenomenon. It's optimum pH growth was about 7 and the mould was able to grow suitably at 10 degrees C, the temperature of cellar for cheese ripening. It was inhibited by high sodium chloride contents or low water activities. PMID- 7551714 TI - Immunomodulating effect of acridine tautomers on eukaryotic cells. AB - The immunomodulating effect of some new amino- and imino-acridine derivatives, were investigated on antibody dependent cellular cytotoxicity (ADCC) and induced blast transformation of lymphocytes. In different concentrations (2.0 x 10(-6) M, 4.0 x 10(-8) M and 2.0 x 10(-5) M) the drugs produced a suppression of PHA and ConA induced cell proliferative response except in the case of 2b, 2d and 2g amino-acridines. The suppressive effects were dose dependent and exhibited a higher inhibitory level in the case of imino-acridines. Some drugs at low concentration exerted a little enhancing effect on ADCC reaction. PMID- 7551712 TI - Longitudinal immunological follow-up of HIV infected haemophiliacs in Hungary. AB - Twenty-five haemophiliacs who had been infected with HIV in 1982 or 1983 were followed up from 1986 to 1993. The absolute number of the CD4+ and CD8+ cells, neopterin levels and more recently the percentage of activated, DR+ T lymphocytes were determined twice a year. In most patients a permanent decline in the CD4+ cell count was observed whereas in two HIV-infected haemophiliacs the absolute number of CD4+ cells did not change during the observation period. In these long term non-progressor patients no clinical symptoms and no increased neopterin levels were observed. T cells subset and neopterin measurements were found to predict the development of AIDS. AIDS developed only in those patients who exhibited both a CD4+ cell count of < 350/microliter and a serum neopterin concentration of > 20 nmol/l. A negative correlation was observed between the percentage of activated. DR+ T lymphocytes and the CD4+ cell counts. PMID- 7551715 TI - The effect of vasopressin on the phosphoinositides of Tetrahymena pyriformis. Relationship with glycogen content. AB - In Tetrahymena pyriformis a 20% decrease of 32P-PIP2 was produced by 10(-6) M vasopressin treatment within 30 s. A 17% decrease occurred in the content of PAS positive material within 5 min after administration. The effect of vasopressin was similar to that of hepatocytes. PMID- 7551716 TI - A model for testing drug susceptibility of Pseudomonas aeruginosa and Staphylococcus aureus grown in biofilms on medical devices. AB - UV-sterilized polyethylene rings infected with Pseudomonas aeruginosa or with Staphylococcus aureus were implanted into artificial wounds of mice. During 5 days incubation biofilm was formed on the plastic surfaces. To determine the Minimal Bactericidal Dose on sessile bacteria, rings with biofilm were removed and incubated in Luria broth containing serial dilutions of different antibiotics. Parallel, the sensitivities of planktonic phase organisms were also determined using cells grown in broth. The biofilm mode of growth strongly reduced the sensitivity of the strains against most of the antibiotics used, especially against polymyxin B. On the other hand, beta-lactam type antibiotics were equally effective against bacteria both in the sessile and planktonic phase of growth. PMID- 7551719 TI - Detection of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) (a note). AB - Since the majority of post-transfusion hepatitis cases are closely related to the HCV infections, the sensitive and specific detection of HCV-RNA is a central issue both in blood transfusion and in follow-up of hepatitis patients. This leaflet provides the technical details of HCV-RT-PCR. PMID- 7551718 TI - Dehydroepiandrosterone modulates the spontaneous and IL-6 stimulated fibrinogen production of human hepatoma cells. AB - The role of an androgen, dehydroepiandrosterone (DHEA) has been studied on the constitutive and IL-6 induced fibrinogen production of HepG-2 cells. DHEA markedly augments the constitutive fibrinogen production of the hepatoma cells in a dose dependent fashion. Oppositely, for IL-6 induced fibrinogen production, DHEA is strongly inhibitory. The effectiveness of DHEA on the constitutive fibrinogen production is further potentiated if the hepatoma cells are preincubated with a glucocorticosteroid, dexamethasone. These findings demonstrate that the complex interaction between the steroid- and cytokine directed regulation of the production of acute phase proteins is further coloured by the action of androgens on immune and hormonal systems. PMID- 7551720 TI - Nurses lead the pathway. PMID- 7551723 TI - Empowering the E-grades. PMID- 7551717 TI - Biofilms produced by Pseudomonas aeruginosa and by Staphylococcus aureus on model medical devices. AB - Polyethylene, teflon, tygon, polypropylene, silicon rubber, and rubber tubes or rings contaminated with Pseudomonas aeruginosa or Staphylococcus aureus were implanted subcutaneously into mice. After 5 days the colony forming units developing on, and attaching to them were determined. The highest numbers of bacteria were observed on rubber and silicon rubber, polyethylene was next in order, while significantly lower values were obtained on teflon and on tygon and polypropylene. Rubber devices were better colonized after heat than after UV sterilization. The number of bacteria rose further, if the already used rubber implant was resterilized, recontaminated and reimplanted. The model seems suitable to test the development of bacterial biofilms on different materials pretreated in different manners. PMID- 7551721 TI - Expand, contract and win. PMID- 7551722 TI - Investment wins interest. PMID- 7551725 TI - Drug error detectives. PMID- 7551726 TI - Super supervision. PMID- 7551727 TI - Enhance your employability. PMID- 7551728 TI - To attempt to insinuate the nurses' code into the heart of the NHS marketplace is a bold move. PMID- 7551724 TI - Health and efficiency. PMID- 7551729 TI - Power of the transformers. PMID- 7551730 TI - Is there a con in your contract? PMID- 7551731 TI - Mycoflora and Fusarium toxins of three types of corn grains in Egypt with special reference to production of trichothecene-toxins. AB - Sixty three species and 2 varieties which belong to 21 genera of fungi were isolated from corn grains (53 + 2, 36 + 1 and 34 species belonging to 19, 13, and 12 genera from white, yellow and popcorn, respectively). Aspergillus (15 species), Penicillium (17) and Fusarium (4) were the dominant genera isolated from the three types of corn. Of four species of Fusarium, F. moniliforme was the dominant species. F. oxysporum and F. solani were detected in all the three substrates, whereas F. subglutinans was isolated exclusively from yellow corn. A biological assay (brine shrimp larvae) and chemical analysis (TLC, UV spectrophotometery and NMR spectroscopy) of chloroform extracts proved that 7 out of 78 samples were toxic. Diacetoxyscirpenol (4 samples, 98.4-128.4 micrograms/kg corn), T-2 toxin (4 samples, 72.8 to 130.4 micrograms/kg) and zearalenone (7 samples, 22.6 to 80.4 micrograms/kg) were detected, whereas 90% methanol-water re extracts revealed that 3 out of 78 samples were toxic due to presence of fusarin C (1 sample, 76.8 micrograms/kg) in addition to an unidentified toxic factor (3 samples). Of 22 Fusarium isolates (F. moniliforme, F. oxysporum, F. solani and F. subglutinans; 10,5,5 and 2 isolates, respectively grown on corn grains, the chloroform extracts of 6,3,3 and 1 isolates were toxic to brine shrimp larvae. Based on chemical analysis, diacetoxyscirpenol (10/22 isolates; 9.8 - 78.6 micrograms/g dry corn grains), T-2 toxin (5/22; 20.8 - 60.4 micrograms/g), HT-2 toxin (3/22; 12.4 - 18.6 micrograms/g) and zearalenone (13/22; 9.8 - 38.4 micrograms/g) were identified.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551732 TI - Physiological characterization of a cadmium-resistant mutant in the fission yeast Schizosaccharomyces pombe. AB - After treatment of the wild-type strain 975 h+ with N-methyl-N'-nitro-N nitrosoguanidine (MNNG) mutants able to grow on YEP-medium containing 15 mM CdCl2 were isolated. Mutant R17 showed a specific resistance to Cd, but no increased tolerance to Pb(NO3)2, CuSO4 or ZnCl2. No higher content of Cd-binding phytochelatins could be detected, but the mutant accumulated lower levels of Cd than the corresponding wild-type. No change, however, was observed in the accumulation of Cu. Under the influence of Cd, the respiratory activity of mutant R 17 is reduced less than in the wild-type. The mechanism of resistance could be an increased secretion or a decreased uptake of Cd. PMID- 7551734 TI - The impact of interfacial interactions on poliovirus infectivity in detergent treated cell monolayer cultures. AB - Monolayer cultures of Buffalo green monkey (BGM) kidney cells were inoculated with different aqueous solutions of anionic detergents prior to infection with polio-myelitisvirus type 1 (PMV-1). Alteration of inoculated cell culture monolayers was assessed by polarized light microscopy. Based on their visual appearance, altered monolayers were arbitrarily assigned different rating categories (r.c.) ranging from 1 to 8. For each detergent concentration, the rate of monolayer destruction was recorded and graphically integrated into the plots of r.c. against detergent concentration. In addition to a direct detergent mediated alteration of the cellular monolayers, inoculation of detergent conditioned BGM cultures with PMV-1 apparently affected virus-mediated alteration as well. The type of alteration as well as the initial rate of cytopathic destruction of the monolayer depended on several factors including the stereochemical properties of the detergent used, the final detergent concentration as well as the organic content and inoculation volume of the preconditioning solution, and the infectious titer of the viral inoculum. Moreover, infectivity kinetics revealed that cytopathic effects may be reactivated after a period of viral inhibition. In this way, at least two cycles of virus generation successively alternating with cell proliferation could be recorded simply by supplementing the system with energy for growth. From the presented results, it became apparent that modifications of cell surface activity contribute to virus infectivity mechanisms. Since treatment of aqueous PMV-1 suspensions has been previously described to largely affect viral infectious titers, viral uptake seems to be triggered by the phase behavior of viral particles upon contact with the plasma membrane. Therefore, aquatic samples that have been treated with anionic detergents in order to optimize virus isolation rates should be extensively washed prior to inoculation on cell culture. PMID- 7551733 TI - A method for selecting Trichoderma strains antagonistic against Sclerotinia minor. AB - Thirty strains of T. hamatum, T. harzianum, T. koningii, T. longibrachiatum and T. viride were selected for in vitro mycoparasitic activity against Sclerotinia minor. High destruction of sclerotia (up to 70% in two weeks) was caused by antagonists only at 25 degrees C, in autoclaved soil, because the optimum temperature for radial growth of Trichoderma strains (25-30 degrees C) was significantly higher than those of phytophatogenic fungus (20 degrees C). The most active strains, which were selected also for competitive saprophytic ability could enhance the percentage of decayed sclerotia 5-9 fold over the control even after 1 week under non sterile conditions. A silicon rubber fixation technique was developed for recovery of irregularly shaped S. minor sclerotia. PMID- 7551736 TI - Filamentous soil fungi and unidentified bacteria in drinking water from wells and water mains near Bratislava. AB - The occurrence of filamentous fungi in 294 samples of drinking water taken from wells and taps near Bratislava was studied. Fungal contaminants were found in 192 samples (44%). 39 genera and 64 species of soil filamentous fungi were identified by routine isolation and cultivation methods. Most of them belong to the Deuteromycetes. In the course of fungal cultivation in Czapek-Dox agar supplemented with 20 mg.l-1 tetracycline, colonies of unknown yellow coloured bacteria were observed. PMID- 7551735 TI - Mode and extent of degradation of adenosine and guanosine by extracts of Aspergillus terricola. AB - The mode of degradation of adenosine by extracts of Aspergillus terricola was suggested to be affected preliminary by adenosine deaminase to inosine and the resulting ribonucleoside was then degraded hydrolytically to give hypoxanthine and ribose. With regard to guanosine, the same extracts could initially catalyze the hydrolytic cleavage of guanosine to guanine and ribose. The resulting base was then deaminated to give xanthine by guanine deaminase. Addition of arsenate to the reaction mixture or dialyzing the extract did not affect the observed hydrolytic activity indicating the absence of phosphorylase activity or phosphorylase-phosphatase activities in the extracts. PMID- 7551737 TI - Degradation of refinery products and oils from polluted sites by the autochthonous microorganisms of contaminated and pristine soils. AB - The degradation of five refinery products with different boiling ranges and four mineral oils extracted from long-term contaminated soils was monitored by measuring oxygen consumption and the concentration of the total hydrocarbons. Degradation was catalyzed by the isolated communities of the microorganisms from contaminated and pristine soils in an aqueous medium, and partly also directly in the soils. Degradation exceeding 95% was achieved for diesel fuel and spindle oil. The higher-boiling raffinates and the extracts of long-term contaminated soils were degraded by 40-60%. The extracted oils have higher boiling ranges and are therefore more difficult to degrade. Moreover, the progressive enrichment of persistent (including lower-boiling) hydrocarbons decreases the degree of degradation attainable of such aged oils. Whereas in the medium term the autochthonous microorganisms of the contaminated soils showed better degradation efficiency, in the long term the populations of the natural soils caught up with them. This may be due to a sociological adaptation process. PMID- 7551738 TI - A composite skin substitute (graftskin) for surgical wounds. A clinical experience. AB - BACKGROUND: Bioengineered skin substitutes offer tissue replacement without requiring a donor site and might produce better healing. OBJECTIVE: To evaluate the recipient's response to grafting a bioengineered skin equivalent onto acute surgical wounds. METHODS: Graftskin, which is made of: 1) a bovine collagen matrix containing human fibroblasts, and 2) an overlying sheet of stratified human epithelium, was grafted onto the excision sites of 15 patients. RESULTS: Blood and cell studies for toxicity were negative. Graftskin proved easy to handle, and a typical clinical appearance of the skin substitute during "take" was detected. Compared with expectations improved healing occurred. Twelve of 15 patients had initial clinical takes. CONCLUSION: Graftskin was not clinically rejected and was not toxic. It often appeared to take and produced better than expected healing. PMID- 7551739 TI - Follow-up study of microsurgical treatment of linear facial wrinkles. AB - BACKGROUND: A new microsurgical technique for the removal of facial wrinkles, first reported in this journal, has been further researched. METHODS: The technique consists of two steps, both performed under a microscope: 1) microsurgical peeling of tissue surrounding a linear wrinkle, and 2) direct excision and closure of the wrinkle. RESULTS: Wrinkles were effectively diminished in 61 patients and the results, despite occasional occurrence of minor hypopigmentation and/or scarring, were satisfactory and long-lasting. PMID- 7551740 TI - Surgical combination therapy for vitiligo and piebaldism. AB - BACKGROUND: Refractory and stable defects of vitiligo and piebaldism may be unresponsive to medical therapy. Melanocyte transplantation can restore the normal pigmentation in some selected patients. OBJECTIVES: To evaluate the efficacy of additional mini-grafting with 1.0-1.2-mm punch grafts to complete the restoration of achromic defects when performing surgical correction of leukoderma. METHODS: Eight patients with refractory stable leukoderma were treated with melanocyte transplantation; three with segmental vitiligo had epidermal shave, by removing the hyperpigmented macules at the periphery of achromic lesions; two others received suction epidermal grafts; and three subjects were treated by in vitro cultured epidermal autografts. All patients received additional mini-grafts in areas of residual achromia. RESULTS: The depigmented defects were 100% restored in seven patients, and in one subject 80% improvement was observed. CONCLUSION: Surgical methods, followed by additional mini-grafting, may be helpful to restore completely the depigmented defects when residual achromia, after treatment with the methods described above, is still present. PMID- 7551741 TI - Cell surface molecules in basal cell carcinomas. AB - BACKGROUND: The factors determining a basal cell carcinoma's (BCC's) growth pattern and invasive potential are not known. In other tumors it has been shown recently that the expression of cellular adhesion molecules may determine a tumor's invasive and metastatic potential. Integrins, cell surface molecules important in cell stroma interactions, are present on BCCs and may help regulate the tumor's growth pattern. OBJECTIVE: We compared the expression of cellular adhesion molecules alpha 2 integrin, beta 1 integrin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), leukocyte function antigen 1a (LFA-1a), and E-selectin in different histological subtypes of basal cell carcinomas. METHODS: BCCs were obtained from patients undergoing Mohs surgery. The BCCs were classified as nodular, micronodular, mixed, infiltrative, and basosquamous types and stained using an avidin-biotin immunoperoxidase technique with antibodies against alpha 2 integrin, beta 1 integrin, ICAM, LFA-1a, VCAM-1, and E-selectin. RESULTS: BCCs expressed alpha 2 and beta 1 integrin, but no significant differences in the amount or pattern of expression was seen in the different histologic subtypes. CONCLUSION: The expression of integrins by BCCs by binding to the surrounding stroma may limit BCC's growth; however, their expression does not appear to correlate with their histological pattern. PMID- 7551743 TI - Sclerotherapy. Continuous wave Doppler-guided injections. AB - BACKGROUND: The usual method of injection of sclerosing agents is based simply on clinical recognition and visualization of the varicose vein. OBJECTIVE: The authors present an original technique for sclerosing injections, named Doppler sclerotherapy. The purpose is to allow more accurate sclerotherapy in situations where varicose veins (usually greater than 4-5 mm) are not palpable in the supine position, but palpable when standing. METHODS: This technique consists of injecting the sclerosing agent using an uncomplicated continuous wave Doppler, a syringe, and a needle, according to a method consisting of four well-defined steps ensuring avoidance of arterial vessels and constant appreciation of the varicose vein to be treated, even during the injection. It requires no assistants and allows the treating physician's gaze to remain at the injection site while listening for very specific Doppler sounds of aspiration and injection. RESULTS: In our multicenter study of 220 patients (approximately 1,400 injections) all but 18 injections were successful for intravascular localization. No serious complications were noted. CONCLUSIONS: The principal indication is the treatment of varicose veins that are palpable while standing but impalpable in the supine position. In addition varicose veins in the groin region, lower third of the thigh, and along the axis of the small saphenous vein may be treated with this technique. In these situations it is a more accessible, faster, and economical technique, although it does not replace duplex ultrasound-guided injections. PMID- 7551746 TI - Identification of arteriovenous anastomoses by duplex ultrasound. Implications for the treatment of varicose veins. AB - BACKGROUND: It has been suggested that arteriovenous anastomoses (AVA) may contribute to the etiology of varicose vein disease. Duplex ultrasound has recently assumed a major role in the diagnosis of venous disease. This noninvasive method can serve as a research tool to better elucidate the role of AVA, and as a guide to the treatment of varicose veins. OBJECTIVE: To determine the incidence and location of AVA identifiable by duplex ultrasound in a population with clinically apparent varicose veins due to underlying saphenous vein reflux. METHODS: The presence and location of AVA detected by duplex ultrasound were noted in 510 patients with varicose veins due to Doppler-positive saphenous vein reflux from three private practices. RESULTS: Twenty-six AVA were found in 19 patients (incidence 3.7%), mostly in the distal half of the long saphenous system. AVA were represented equally on both right and left sides, and regardless of previous vein stripping. Three AVA were found at the base of venous ulcers. AVA were more easily detected using higher resolution ultrasound. CONCLUSION: AVA occurring in association with saphenous vein disease and identified using duplex ultrasound are usually found below the knee in the long saphenous system, and sometimes at the base of venous ulcers. Knowledge of their presence and location may help to avert complications of sclerotherapy, and guide the approach to treatment. PMID- 7551744 TI - The syndrome of the "coup de fouet". Is it always a benign disease? AB - BACKGROUND: The "coup de fouet" syndrome is an uncommon condition characterized by a spontaneous intramuscular venous hemorrhage of the calf, sometimes accompanied by a deep thrombosis of the leg. OBJECTIVE: The purpose of this work is to verify the real incidence of the thrombotic complications, making use of the most recent noninvasive diagnostic instruments, such as the duplex scanner and color Doppler. METHODS: From January 1992 to December 1993, we examined 24 subjects (average age, 38.5 years) suffering from the so-called "coup de fouet" syndrome, which had appeared during physical activity: walking (nine cases), running (six cases), and sudden changes of position (nine cases). In all cases we performed a clinical and instrumental examination (analogical and tensive arterial and venous Doppler, arterial and venous duplex scanner, and color Doppler). RESULTS: Clinically we found, in 17 subjects, a hematoma in the posterior calf with increased thickness and, in 10 cases, with spontaneous sharp pain. In the other seven cases we found a modest hematoma without subjective or other objective symptoms. The arterial Doppler and scanner findings were normal. In the first group of 17 cases, the average clinostatic Doppler venous pressure, measured using a method developed by us and already described, was 32 +/- 4 mm Hg (normal value, 15 +/- 7 mm Hg); in the other seven cases the value was 19 +/- 4 mm Hg. CONCLUSION: The duplex scanner and color Doppler examination confirmed in all subjects the presence of an intrafascial hematoma in the gemellar zone; in 10 cases (41.7%), with more painful symptomatology, in a zone contiguous to the hemorrhage, we found a gemellar (six cases) or popliteal (four cases) phlebothrombosis. Lastly, we must point out that the syndrome is not always benign and that, therefore, a correct and more vigilant diagnostic and therapeutic approach is necessary. PMID- 7551745 TI - Cutaneous microcirculation in skin lesions associated with chronic venous insufficiency. AB - BACKGROUND: There are various types of skin lesions in chronic venous insufficiency. OBJECTIVE: To determine an association between different skin lesions and different changes in the cutaneous vascular bed. METHODS: Twenty eight patients with chronic venous insufficiency (CVI) without and with various types of skin lesions (ie, hyperpigmentation, lipodermatosclerosis, white atrophy, and venous ulcer) and 12 healthy control subjects were examined by video capillaroscopy, transcutaneous oxygen tension measurement, photoplethysmography, and Doppler ultrasonography to detect disturbances of cutaneous microcirculation and venous macrocirculation. RESULTS: There were no significant differences between the different CVI groups with respect to their venous function disorder. However, important variations were noted in the cutaneous microcirculation. In all the patient groups, an increase in the mean halo diameter could be demonstrated even in clinically healthy skin. In all types of skin lesions, the mean capillary diameter was significantly enlarged. Severe morphological capillary changes and a significant reduction of the mean transcutaneous oxygen tension values were found in conjunction with lipodermatosclerosis and white atrophy and in the areas surrounding venous ulcers. CONCLUSION: The results of this study point to the conclusion that, once they have become established, microvascular disturbances play an independent role in the development of the skin lesions associated with CVI. PMID- 7551750 TI - Bovine collagen hemostatic agent used in removal of a basal cell carcinoma of the nose with a good cosmetic result. PMID- 7551749 TI - Cutaneous malignancy and leprosy. PMID- 7551748 TI - Hereditary multiple glomus tumors involving the glans penis. A case report and review of the literature. AB - BACKGROUND: Multiple glomus tumors are extremely rare and seldom demonstrate malignant change. OBJECTIVE: To describe an exceptionally rare case of familial multiple glomus tumors involving the glans penis. METHODS: Multiple glomus tumors in a father and son were studied by light and electron microscopy. RESULTS: The clinical presentation of the two patients was similar, and atypical glomus cells were identified in both son and father. The histology of the tumor cells suggested that they were malignant, yet the clinical course has been benign. CONCLUSION: Despite their malignant histologic appearance, the behavior of familial glomus tumors is benign. A conservative approach to treatment appears warranted. The diagnosis and management of these lesions should be based on clinical behavior rather than histopathology. PMID- 7551747 TI - Squamous cell carcinoma in situ arising within lichen planus of the vulva. AB - BACKGROUND: Lichen planus is a common dermatologic disorder involving the glabrous skin, hair-bearing skin and scalp, nails, and mucous membranes of the oral cavity, penis, and vulva. There have been multiple case reports of squamous cell carcinoma (SCC) developing within lesions of hypertrophic cutaneous lichen planus and erosive oral lichen planus. Recently, there have been several case reports of SCC arising within lesions of the genital mucosa: one case of penile lichen planus, and two cases of vulvar lichen planus. OBJECTIVE: To report a case of SCC in situ developing within erosive vulvar lichen planus. To heighten the awareness, and facilitate the recognition of vulvar lichen planus. RESULTS: We present SCC in situ developing within a lesion of erosive vulvar lichen planus. The patient was treated with simple total vulvectomy by gynecologic surgery. Her vulva remain disease-free 2 years postoperatively. CONCLUSIONS: Vulvar lesions of lichen planus should be recognized and followed closely. In long-standing erosive lesions of the vulva, periodic sampling biopsies should be examined to rule out malignant degeneration. PMID- 7551742 TI - Posttransplant epidermoid cysts secondary to small graft hair transplantation. AB - BACKGROUND: Epidermoid cysts sometimes result from small-graft hair transplantation. Although hair transplant surgeons are aware of this complication, it has not been reported to date. OBJECTIVE: Our purpose is to report the clinical and histological features of posttransplant epidermoid cysts. METHODS: Four patients with posttransplant cysts, and the histological features of two of these cysts, were reviewed. RESULTS: All cysts occurred at the site of a recent micro- or minigraft, and were small; one had become infected. Serial sectioning of one scalp biopsy showed a micrograft lying in the subcutaneous tissue in close proximity to an epidermoid cyst. The cyst apparently arose from the follicular infundibulum of the graft. CONCLUSION: Posttransplant epidermoid cysts are examples of epidermal inclusion cysts. They are treated by conservative excision, and may require drainage for secondary infection. Careful operative technique should prevent some of these cysts. PMID- 7551751 TI - A redefinition of male pattern baldness and its treatment implications. PMID- 7551752 TI - Five feminist myths about women's employment. AB - Feminist sociology has contributed substantial revisions to theory, especially in the sociology of work and employment. But it is also creating new feminist myths to replace the old patriarchal myths about women's attitudes and behaviour. Five feminist myths about women's employment are discussed whose acceptance as fact is not damaged by being demonstrably untrue. Arguably the most pervasive is the myth of rising female employment. The myth that women's work commitment is the same as that of men is often adduced to resist labour market discrimination. The myth of childcare problems as the main barrier to women's employment is commonplace in advocacy research reports. The myth of poor quality part-time jobs is used to blame employers for the characteristic behaviour of part-time workers, including high labour turnover. The issue of the sex differential in labour turnover and employment stability illustrates clearly how feminist orthodoxy has replaced dispassionate sociological research in certain topics. The concluding section considers the implications of such feminist myths for an academic community that claims to be in the truth business and for theories on the sexual division of labour. PMID- 7551753 TI - Chronic ankle instability: the value of talar tilt under general anaesthesia. AB - Ankle inversion injuries are the most common injury in sport and yet treatment is controversial. One result is chronic ankle instability, an injury for which criteria for surgical intervention are unclear. This prospective study of 14 patients showed that a large proportion of patients had no detectable abnormality on preoperative investigation, but were subsequently shown to have an abnormality under general anaesthesia. This suggests that a good history of chronic instability is more sensitive an indicator than conventional investigations. Stress views under anaesthesia may confirm the problem. PMID- 7551754 TI - Bilateral isokinetic variables of the shoulder: a prediction model for young men. AB - Clinicians frequently want to know the pre-injury strength of an uninjured limb. The purpose of this study was to delineate the differences in bilateral isokinetic peak torque (PT) at 60 degrees and 240 degrees, and torque acceleration energy (TAE), average power (AP) and total work (TW) at 240 degrees during shoulder extension and flexion movements, and to develop a method to determine the expected maximal isokinetic variables of the dominant shoulder based upon isokinetic measurements from the non-dominant shoulder. Shoulder isokinetic measurements were obtained from 30 normal young male adults. While bilateral correlation was significant (P < 0.01), results also suggest significant bilateral differences P < 0.01). Thus, it is inappropriate to use the uninjured extremity to predict the pre-injured strength of the injured side without adjustment. In this investigation models were developed relating the expected maximal isokinetic measurement of the dominant shoulder to the non dominant shoulder measurements. PMID- 7551755 TI - Poor ventilatory response to mild hypoxia may inhibit acclimatization at moderate altitude in elderly patients after carotid surgery. AB - Peripheral chemoreceptors (carotid bodies) are the main sensing organs for hypoxaemia. During carotid surgery, the carotic body in the bifurcation of the common carotid artery is often involved and damaged or destroyed. Animals lose their ability to adapt to high altitude after experimental denervation of the carotid bodies. The objective of our study was to evaluate the ability of human patients to adapt to moderate altitude after single side carotid surgery. Blood gas analysis at rest at 171 m and after car and cable car transport to 1600 m before and after carotid surgery was performed. Mean(s.d.) paO2 decreased insignificantly from 74.8(3.56) at 171 m altitude to 71.6(2.07) at 1600 m (P = n.s.), means(s.d.) paCO2 decreased significantly from 36.2(2.86) to 31.4(2.7) mmHg (P < 0.05) before carotid surgery. Months after surgery, a significant drop in paO2 occurred after identical passive exposure to moderate altitude: mean(s.d.) paCO2 at 171 m 74.4(3.65) mmHg, at 1600 m 65.8(3.70) mmHg (P < 0.01), paCO2 did not change significantly. Mean(s.d.) paCO2 at 171 m: 36.0(2.35), at 1600 m 36.2(2.86) mmHg (P = n.s.). Although the sample investigated was small, after single side carotid surgery patients seem to lose their ability for satisfactory ventilatory response to acute exposure to moderate altitude. This is of possible alpine medical importance. PMID- 7551756 TI - Thermoregulation of exercising men in the morning rise and evening fall phases of internal temperature. AB - The purpose of this study was to compare the thermoregulatory responses during exercise in the morning rise (0900 h) and evening fall phases (2000 h) in circadian variation of body temperature. Five healthy volunteers performed bicycle exercises at 30% and 60% of maximal aerobic power (VO2max) at 26 degrees C with a relative humidity of 50%. Whole-body sweat rate (SR), rectal (Tre), mean skin (Tsk) and mean body (Tb) temperature, pulmonary ventilation (VE), oxygen uptake (VO2), and carbon dioxide output (VCO2) and heart rate (HR) were measured during the experimental period. SR during exercise at 30% VO2max was significantly higher at 2000 h than at 0900 h. However, the circadian variation of SR during exercise was not observed at 60% VO2max. At the two experimental times, there were also no significant differences in VO2, VCO2, VE and Tsk in both workloads. In HR, Tb and Tre circadian effects were demonstrated as well as in workload levels. As Tb was plotted against SR during exercise, positive correlations were observed. The data showed that there was a parallel shift in the SR to Tb relationship during exercise in the morning and evening. This rightward shift indicated that there was an increased Tb threshold for the onset of sweating in the evening. Resting Tb at 2000 h was significantly higher when compared with Tb at 0900 h. The present results suggest that the circadian influence on the thermoregulatory response to exercise may be evident only at low workloads. PMID- 7551757 TI - Surgical treatment of ruptures of the Achilles tendon: a review of long-term results. AB - The rupture of the Achilles tendon is frequently sports-related. In the time from 1 January, 1978 until 31 December, 1988, we treated 358 men and 54 women with such an injury at the Staatliche Orthopaedische Klinik in Munich. The average age of these patients was 43 years. The site of the rupture was generally located between 3-5 cm proximal of the distal insertion of the tendon. In the follow-up examination of 122 patients with surgical treatment of tendo calcaneus ruptures 85% showed 'good' to 'very good' subjective results. Of the operated patients 97% would choose the same treatment under similar circumstances. The isokinetic studies demonstrated a loss of static and dynamic strength in plantar flexion of the ankle joint of 9.1%, and 16.7% respectively, when compared to the healthy contralateral side. The ultrasound examination revealed a thickening of the tendon and of the dorsal paratenon with changes in the internal structure of the injured Achilles tendon. In spite of these favourable results, the high complication rate of 15.1% shows the need for new and extensive studies regarding the various alternative treatment forms, such as functional, non-operative options, to finally resolve the debate about the optimal treatment of Achilles tendon ruptures. PMID- 7551759 TI - The relationship between carbon dioxide sensitivity and sprint or endurance performance in young swimmers. AB - There has been some evidence that extremes of CO2 sensitivity can indicate an individual's potential for sprint or endurance athletic performance. This study examined the responses to CO2 rebreathing, and previously validated sprint and endurance tests by subjects who were involved in regular, but not intensive, swimming training. The aim was to determine whether subjects with low CO2 sensitivity might perform better on endurance tests, whilst those subjects with high CO2 sensitivity would be more successful in the sprint tests. Initially, 168 young (mean mean(s.d.) age = 12.4(2.1) years) swimmers were measured using a modified version of the Read CO2 rebreathing technique. From this sample, 17 high (mean means(s.d.) = 2.24(0.39) 1 min-1 mmHg-1) CO2 responders were matched by gender, age, height, weight and FVC with 17 low (mean means(s.d.) = 0.57(0.19) 1 min-1 mmHg-1) responders. Each of these 17 pairs underwent two sprint tests (10 s Tri-level alactic power, 50 m run) and two endurance tests (PWC170, 1.6 km run) in order to determine whether any differences existed between the two groups and the sprint and endurance parameters. The subjects remained unaware of their results of the rebreathing test throughout testing. A dependent t test was then used to compare the results collected from each group. The low CO2 responders recorded a significantly faster 1.6 km run time, but were not superior on the PWC170 ergometer test, than the high CO2 responders. The high CO2 responding group recorded significantly higher results on the 10 s alactic power test, but not the 50 m sprint run, than the low responders. Hence, further study is required to ascertain underlying causes as to why significant differences occurred with CO2 rebreathing, the 1.6 km run and the 10 s alactic power test, but not with the PWC170 and the 50m sprint run. PMID- 7551760 TI - A new gas dilution method for measuring body volume. AB - This study was designed to examine the validity of a new gas dilution method (GD) for measuring human body volume and to compare its accuracy with the results obtained by the underwater weighing method (UW). We measured the volume of plastic bottles and 16 subjects (including two females), aged 18-42 years with each method. For the bottles, the volume measured by hydrostatic weighing was correlated highly (r = 1.000) with that measured by the new gas dilution method. For the subjects, the body volume determined by the two methods was significantly correlated (r = 0.998). However, the subject's volume measured by the gas dilution method was significantly larger than that by underwater weighing method. There was significant correlation (r = 0.806) between GD volume-UW volume and the body mass index (BMI), so that UW volume could be predicted from GD volume and BMI. It can be concluded that the new gas dilution method offers promising possibilities for future research in the population who cannot submerge underwater. PMID- 7551761 TI - Surgical repair of traumatic medial disruption of the elbow in competitive athletes. AB - Five active athletes with acute medial elbow rupture were treated with muscle ligamentous repair and a spiked washer. All patients regained full strength as well as stability of the elbow, and resumed previous sporting activities within 3 months of surgery. Early surgical repair of the ligament and flexor mass should be considered for active athletes who exhibit gross instability of the elbow on a valgus stress test without anaesthesia. PMID- 7551758 TI - The mathematical relationship between the number of events in which people are injured and the number of people injured. AB - In this paper the mathematical relationship between the number of times people sustained injuries and the number of people injured is discussed. The number of injury-causing events in a population will be larger than the number of people injured as people can be injured on more than one occasion. The Poisson distribution is used as a way to study the relationship between the two different sets of numbers. A practical example is given of injuries sustained while running. From this it seems that injury proneness needs to be considered in studying those who were injured on more than one occasion while running during the past year. PMID- 7551762 TI - Traumatic carotid and vertebral artery dissection in a professional jockey: a cautionary tale. AB - Jockeys accept bony fractures and soft tissue injuries as occupational hazards. An average National Hunt jockey falls once in ten races with an injury rate of 4.25%. Head injury is a common cause of morbidity and the benefit of helmets is well recognized. Neck injuries are also common and usually musculoskeletal. Although rare, trauma to the neck arteries may go unnoticed yet have catastrophic consequences. Internal tears can allow arterial blood to dissect the layers of the arterial wall and obstruct the lumen. Severe obstruction may lead to cerebral ischaemia and infarction. An appreciation of the early clinical signs caused by this process may allow effective intervention. We report a case which illustrates these points. PMID- 7551763 TI - Doping taboos. PMID- 7551764 TI - Bilateral rupture of the quadriceps tendon associated with anabolic steroids. PMID- 7551765 TI - Blood lactate levels of decathletes during competition. AB - There are many detailed physiological profiles of athletes who participate in a wide variety of sporting activities but few data have been obtained on decathletes. This study defines some physiological characteristics of these athletes and measures capillary lactate concentrations [La(b)] during a laboratory test of progressive maximal exhaustion and the different events during competition. The treadmill test is similar to the 100 m, 400 m and 1500 m in terms of [La(b)] accumulation but only similar to the 1500 m in terms of velocity. The 400 m is the most demanding event with the greatest blood lactate accumulation (mean(s.d.) 16.38(2.36) mmol l-1). The [La(b)] at the end of the 110 m hurdles is significantly lower than in any other racing events (mean(s.d.) 6.96(1.32) mmol l-1) compared with mean(s.d.) 12.14(2.87) and mean(s.d.) 11.44(2.16) for the 100 m and 1500 m respectively. The [La(b)] after the long jump, the high jump and the pole vault are not significantly different (mean(s.d.) 5.30(2.23), 4.64(1.39) and 5.36(1.34) mmol l-1) respectively). PMID- 7551766 TI - Effect of exercise training programme on bone mineral density in novice college rowers. AB - Exercise has important effects on skeletal mineralization. Changes in bone mineral density (BMD) and bone mineral content (BMC) as measured by dual energy X ray absorptiometry were investigated in a group of 17 male novice college oarsmen over a 7-month period and were compared with eight age-matched controls. The rowing training programme consisted of approximately 8 h rowing, 1 h weight training, and 1 h running per week. After 7 months training the mean BMD of the lumbar spine (L1-L4) had increased significantly by 2.9% (P < 0.001) and the mean BMC had increased by 4.2% (P < 0.001). There was no significant change in the control group. Neither group showed a significant change in BMD or BMC in the femoral neck, greater trochanter or Ward's triangle. This study provides further evidence that exercise plays an important role in bone mineral formation. PMID- 7551768 TI - Acute intracranial epidural haematoma in a basketball player: a case report. AB - A 35-year-old basketball player suffered a serious double head injury during a match. An acute left temporal epidural haematoma, which necessitated surgical drainage, developed. The exceptional circumstances of the trauma are discussed and the literature concerning basketball-related injuries is reviewed. PMID- 7551769 TI - Anxiety responses to maximal exercise testing. AB - The influence of maximal exercise testing on state anxiety was examined in three separate studies. Highly trained male distance runners (Study 1, n = 12) as well as college students with average (Study 2, n = 16) and below average (Study 3, n = 32) physical fitness levels completed graded maximal exercise tests. This last group was also randomly assigned to either a control or an 8 week training programme in order to determine the effect of increased fitness on the psychological responses to maximal exercise testing. Physical fitness was determined by the measurement of maximal oxygen uptake. State anxiety (State Trait Anxiety Inventory) was assessed before and from 2-15 min following exercise. It was found that the state anxiety responses to maximal exercise testing were not influenced by re-testing or by 8 weeks of endurance training. Across the three study groups, the anxiety response was variable during the first 5 min following exercise testing; increases, decreases and no changes in anxiety were observed when compared to pre-exercise levels. The anxiety response to maximal exercise appeared to be dependent on the pre-exercise anxiety levels as well as the timing of the post-exercise assessments. It is concluded that maximal exercise testing can be associated with negative mood shifts during the first 5 min after exercise; however, this response is transitory and followed by positive mood shifts 10-15 min following such tests. PMID- 7551770 TI - Influence of level of identification with a group and physiological arousal on perceived intergroup complexity. AB - Influence of group identification and physiological arousal on perceived intergroup complexity and intergroup evaluations were investigated. Group identification was examined because persons with differing levels of identification differ in the degree to which they possess stereotypes about groups while physiological arousal was investigated because of its known ability to limit cognitive capacity and potentially encourage the use of stereotypes. Specifically, increases in incidental arousal resulting from exercise were expected to result in increased stereotype use, reflected in differential intergroup complexity. This effect was expected only for those high in identification with the in-group, for whom stereotypes were available. The results supported the predictions. In terms of group evaluations, an in-group favouritism effect was found, although it was only exhibited by those high in identification. Arousal level did not significantly influence evaluation of groups. Thus, incidental arousal can decrease complexity, without simultaneously increasing negativity. Discussion focuses on the differences between the current method and those employed in past research. PMID- 7551767 TI - Eccentric activation and muscle damage: biomechanical and physiological considerations during downhill running. AB - An eccentric muscle activation is the controlled lengthening of the muscle under tension. Functionally, most leg muscles work eccentrically for some part of a normal gait cycle, to support the weight of the body against gravity and to absorb shock. During downhill running the role of eccentric work of the 'anti gravity' muscles--knee extensors, muscles of the anterior and posterior tibial compartments and hip extensors--is accentuated. The purpose of this paper is to review the relationship between eccentric muscle activation and muscle damage, particularly as it relates to running, and specifically, downhill running. PMID- 7551773 TI - Vulnerability to depressive mood reactions: retesting the hopelessness model of depression. AB - A replication of Metalsky, Halberstadt & Abramson's (1987) test of the revised hopelessness model of depression was conducted. Depressive mood response to examination outcome was assessed in undergraduate students across two periods of examination. In phase 1, consistent with Metalsky et al. (1987), examination outcome was the sole predictor of depressive mood response on receipt of exam results. There was no evidence for a delayed mood response five days later. In phase 2, the interaction between attributional style and exam outcome was predictive of depressive mood response on receipt of exam results. Taken together, the results provide support for the diathesis-stress and specific vulnerability components of the model with respect to immediate mood response. No support was found for the causal mediation component. Differences between recently published studies are reported and implications for the model discussed. PMID- 7551771 TI - Determinants of perceived consistency: the relationship between group entitativity and the meaningfulness of categories. AB - The concept of entitativity was developed by Campbell (1958) to refer to the extent to which a group is perceived as a coherent whole or entity. This concept is relevant to research in both social perception (e.g. the categorization effects approach to the study of social stereotyping) and social influence (e.g. the consistency attributed to minority groups in theories of minority influence). On the basis of previous research, four variables were expected to play a role in group entitativity judgements. These were intra-group variability, group size, diversity (or variety) and extremity. In two empirical studies it was found that entitativity decreased as variability and diversity increased and that it increased with group size. These effects and interactions between group size and extremity, size and diversity, and variability and extremity are consistent with the idea that entitativity is a function of how meaningful a stimulus pattern is. This is in turn (in part) a function of how unlikely the pattern is. PMID- 7551774 TI - Manners of expressing negative and sympathetic attitudes towards the unemployed. AB - Social psychologists have examined ways in which indirectness and discursive techniques allow the communication of prejudice while not breaching the tolerance norm. The role these techniques play in the expression of other kinds of attitude, however, is as yet unknown. The present study examined the discursive techniques used by 67 employed individuals to express negative and sympathetic attitudes towards the unemployed. A style of directness was used significantly more frequently to express sympathetic attitudes than negative attitudes. Conversely, credibility techniques were used significantly more frequently to express negative attitudes than sympathetic attitudes. These credibility techniques were used in conjunction with either directness or indirectness. However, against hypothesis, indirectness alone was not used more frequently to express negative than sympathetic attitudes; in fact, one type of indirectness was associated to a greater extent with the expression of sympathy than with negativity. It was predicted that professionally employed individuals would tend to use different discursive techniques than non-professionally employed individuals. This prediction, while supported by a difference on a composite measure of the manners of expression, was not confirmed for any of the styles considered separately. PMID- 7551772 TI - Affective responses to social comparison: a study among disabled individuals. AB - The present study examined the effects of social comparison among 112 individuals receiving payments under the Disablement Insurance Act. A part of a fictitious interview with another disabled person was presented to the subjects. This interview contained upward or downward social comparison information about either the problem severity or coping success of a target. In line with the predictions, the results indicated that upward comparison generated more positive affect than downward comparison only for subjects high in perceived control. Downward comparison generated more negative affect than upward comparison, regardless of perceived control. The nature of the comparison dimension (problem severity versus coping success) did not result in differences in positive or negative affect. Furthermore, subjects high in control identified more strongly with the upward target than with the downward target, especially when the comparison dimension was coping success. Some evidence was found that the effect of perceived control on the positive affective consequences of upward comparison was mediated by identification with the upward target. PMID- 7551775 TI - Emotional expression in upside-down faces: evidence for configurational and componential processing. AB - In two experiments, a total of 126 subjects judged the seven emotional expressions of Ekman & Friesen's (1976) pictures of facial affect presented upright or inverted. Inversion reduced accuracy for sad, fear, anger and disgust, and sad was identified as neutral. However, happy was identified almost perfectly on upright and inverted faces, and both anger and disgust were identified significantly often on inverted faces. In addition, the classic confusions between surprise and fear and between disgust and anger occurred on both upright and inverted faces. It is argued that expressions are difficult to identify on inverted faces when they are based on configural information. However, accurate performance on inverted faces and similar confusions on upright and inverted faces are due to componential processing. PMID- 7551776 TI - Mindless processing of requests? Don't ask twice. AB - This study examines the mindlessness hypothesis and its associated compliance gaining paradigm from the perspective of politeness theory. The main prediction of politeness theory, which is not taken into account in the mindlessness literature, is that the perceived magnitude of an imposition is a function not only of the size of favour asked but also of the framing of the request itself. A confederate asked students studying in the library for either one or 10 sheets of paper using appropriate variations of Langer et al.'s three request types: no reason, placebic reason and real reason. Students then completed a questionnaire to determine: (a) how large the imposition on them had been; (b) their verbatim memories for the requests; and (c) self-reported compliance with the requests. In order to examine the effect of reflective thought on subjects' judgements and recall, this questionnaire was completed either immediately following compliance/non-compliance or three minutes later. The analyses established that the perceived imposition is influenced jointly by the actual imposition, the type of request and the time of judgement. Further, contrary to some previous research, the recall memory data provide support for a strong version of the mindlessness hypothesis, as well as new evidence for the reconstructive character of conversation memory. It is concluded that politeness theory describes a powerful heuristic that people use when processing requests. PMID- 7551777 TI - A sub-standard X-ray dose-meter. 1955. PMID- 7551778 TI - Invited review: intrinsic radiosensitivity as a predictor of patient response to radiotherapy. AB - This article reviews the available literature on intrinsic radiosensitivity as a predictor of patient response to radiotherapy. Results have been summarised for tumour, fibroblast and lymphocyte radiosensitivity where results have been correlated with clinical data. Suggestions are made for the possible way forward in this area of radiobiology. PMID- 7551781 TI - The use of xenon-133 for measurement of blood flow through systemic arteriovenous malformations before and after therapeutic embolization. AB - Embolization is increasingly used to treat systemic arteriovenous (AV) shunts although its success, as judged by either angiographic or clinical means, is difficult to quantify. The aim of the study was to quantify blood flow through AV shunts with 133Xe, which, because of its relatively long transit time through peripheral tissues, behaves like microspheres. Following arterial injection, 133Xe entering an AV shunt rapidly arrives in the lung and can be quantified with a scintillation probe. In 17 patients with systemic AV shunts, the reduction in shunt flow following therapeutic embolization was quantified in the angiography theatre by comparing the initial count rates in the lung, recorded by probe, following injection of identical quantities of 133Xe into a supplying artery before and after embolization. By comparing the lung counts with those given by an intravenous injection of 133Xe, the fraction of flow at the catheter tip entering the shunt was also quantified. Tissue perfusion in the vascular territory distal to the shunt was measured at the same time by recording the clearance of non-shunted 133Xe with a second probe over the extremity. Control injections of 133Xe were given in the contralateral limb in order to assess 133Xe transit in the absence of shunting and to compare tissue perfusion between the two sides. Shunt flow ranged from 40% to 100% (of that at the tip of the catheter) (n = 14), while the reduction in shunt flow following embolization ranged from 15% to 96% (n = 19). Tissue perfusion distal to the shunt and in the contralateral limb was about 5 ml 100 ml-1 min-1. Contrast medium had no consistent effect on tissue perfusion in either limb, or on shunt flow. There was no difference in peripheral perfusion between the abnormal and control sides, nor any significant difference in perfusion in the distal tissue on the abnormal side before and after embolization. There was, however, a consistent increase in the fraction of the injected 133Xe delivered to the distal tissue after embolization (median increase 93%, p < 0.001). The technique is relatively simple and merits further development as a means of continuous quantification of systemic AV shunt flow in the angiography theatre at the time of embolization. PMID- 7551780 TI - Does peppermint oil relieve spasm during barium enema? AB - The effectiveness of topical peppermint oil added to barium sulphate suspension in relieving colonic muscle spasm during double contrast barium enema examination was assessed in a double blind study. 141 patients were randomized either to a control group (71 patients) examined with standard barium suspension or to the treatment group which received peppermint oil mixed with the barium preparation. No residual spasm was evident in a significant proportion of patients in the treated group (60%) compared with the control group (35%) (p < 0.001). The patients' acceptability of the procedure was good and there were no adverse effects on the overall quality of the examination. In conclusion, the addition of peppermint oil to the barium suspension seems to reduce the incidence of colonic spasm during the examination. The technique is simple, safe, cheap and it may lessen the need for intravenous administration of spasmolytic agents. PMID- 7551779 TI - Air versus carbon dioxide insufflation in double contrast barium enemas: the role of active gaseous drainage. AB - Air has been traditionally used as the negative contrast agent in double contrast barium enema (DCBE) examinations, but causes abdominal pain in the 24 h following the procedure. The frequency of post-procedural pain is less when carbon dioxide (CO2) is used as the negative contrast agent. We evaluated patients following DCBE examinations (using either air or CO2) by means of a questionnaire, to determine whether active drainage of gas altered the post-procedural pain. There was no difference in the pain experienced in the groups receiving CO2 with either active or passive drainage, or in the group receiving air with active drainage. Compared with the other groups there was a significantly higher incidence and severity of pain in the group receiving air and passive drainage. We conclude that active drainage of air following a DCBE examination is as effective as using CO2 in reducing post-procedural pain and swelling. PMID- 7551783 TI - Explaining variation in radiologists' reporting times. AB - This paper describes an investigation into the reasons for variation in the time taken by senior radiologists to complete radiological reports. An observational study of the reporting process at one UK hospital was undertaken for a 25 day period. An independent health service researcher observed the radiology reporting process and collected data on a variety of factors including the time taken to produce the report, the number and nature of all images viewed, the experience of the radiologist, and the number of disturbances that occurred. The nature of the variation in reporting time was explored using both simple comparative statistics and more sophisticated multiple regression techniques. Data were collected on 2345 report observations and the median report time was 117 s. This research provides the first empirical evidence for systematic variation in reporting time. The results confirm the importance of certain factors that were expected to explain report time variation. For example, the results indicate that report time tended to be significantly shorter in reporting sessions that were busy, and significantly longer when the radiologist was disturbed during the reporting process or was training juniors during a reporting session. More surprising were the results indicating that there was no significant difference in report time for reports categorized as urgent or "hot" and those categorized as less urgent or "cold", and that report time appeared to vary systematically depending on the day of the week and on the time of day. PMID- 7551782 TI - Relationship between the diuretic effect of radiocontrast media and their ability to increase renal vascular resistance. AB - The relationship between diuresis and natriuresis induced by radiocontrast media (RCM) and their renal haemodynamic effects were investigated. The effects of the iso-osmolar iotrolan and the hyperosmolar diatrizoate on the renal vascular resistance (RVR) were studied in the filtering and non-filtering variants of the isolated perfused rat kidney (IPRK) preparation. In the non-filtering model, no tubular regulatory process can be activated. The effect of diatrizoate on the RVR of the filtering IPRK in the presence of fursemide (0.3 mmol l-1) an inhibitor of the tubuloglomerular feedback (TGF) was also investigated. There was no significant difference (p > 0.05) in the response of the filtering (n = 6) and non-filtering (n = 6) IPRK to iotrolan. The induced reduction in the renal perfusate flow (RPF) by iotrolan was 20.5 +/- 3.05% and 22.9 +/- 3.03%, respectively. The reduction in the RPF which was observed with diatrizoate in the non-filtering IPRK (n = 5, 17.5 +/- 3.04%) was significantly less (p < 0.05) in comparison to that of the filtering IPRK (n = 6, 26.9 +/- 4.28%). In the frusemide experiments, a reduction in the RPF comparable to that of the non filtering kidney was observed (n = 5, 13.7 +/- 4.34%). This study demonstrates that the renal vascular effect of diatrizoate is partially dependent on the TGF response. No tubular regulatory mechanism was accountable for the haemodynamic effect of iotrolan. The activation of the tubular response is osmolarity dependent. PMID- 7551784 TI - Interval cancers in the National Health Service Breast Screening Programme. AB - The National Health Service (NHS) Breast Screening Programme defines objectives with acceptable and, where possible, achievable standards. This paper examines the problem of interval cancers. Slopes due to shrinkage of primary breast cancers in response to primary systemic therapy are used as surrogates for growth slopes. From the distribution of these slopes the proportion of tumours which might reach particular sizes in any time interval may be estimated. For specific differences in size between screen and clinical detection levels the number of interval cancers which could appear in 1, 2 and 3 years after screening is estimated. The justification for these predictions is evaluated. The literature in relation to growth is reviewed; we add three growth slopes from our own measurements. The range of volume doubling times and volume halving times is the same whereas the mean value derived from the literature is about 90 days in contrast to only 26 days for inferred growth rates. Because of the difficulties inherent in the measurement of growth, it is concluded that the literature defines the left half of a distribution; the right hand side is completed by shrinkage data. The observed rates for interval cancers from different sources vary widely; there is an underlying assumption that interval cancers are an index of failure. These calculations suggest that there is an irreducible minimum of interval cancers, which will depend upon the screening interval and the size at which tumours are detected. The interval cancers will contain more poorly differentiated tumours. The anticipated achievable standards appear to be over optimistic. PMID- 7551785 TI - Risk and benefit associated with radiation dose in breast screening programmes- an update. AB - This paper discusses and attempts to estimate the very small numbers of women attending the UK Breast Screening Programme for whom the risk of cancer induction may exceed the probability of cancer detection. It updates a previous paper on the same topic. Variations in breast dose between individuals, due to differences in breast size and in numbers of views and films taken, are considered and revised. New data on cancer induction and its variation with age at exposure have been employed. The overall effect of these changes is generally to improve the balance of benefit against risk compared with the previous paper referred to, the very few exceptions being categories where the numbers of women in question remain of the order of one in a million. The implications for certain alternative screening schedules and for some current trials are also discussed, the conclusions being again reasonably reassuring. PMID- 7551786 TI - Assessment of mean glandular dose in mammography. AB - The routine assessment of patient dose in the National Health Service Breast Screening Programme is performed as part of the quality assurance protocol recommended by the Institute of Physical Sciences in Medicine. The mean glandular dose to a standard breast is deduced from measurement of the air kerma at the entrance surface of a 4 cm Perspex phantom by applying a series of conversion factors. The exposure factors for this measurement are those used clinically. The measured mean glandular dose is then compared with nationally accepted action levels. In some centres the assessment of mean glandular dose using Perspex is supplemented by patient dose surveys. The mean glandular dose to a series of patients attending a breast screening unit may be estimated from a knowledge of the exposure factors and compressed breast thickness, using a knowledge of the X ray tube output. Measurements made on units in the Northern Region of England and in Scotland using both methods are presented. The implication of these measurements with regard to patient dose surveys in mammography and quality assurance programmes are discussed. An analysis of the uncertainties associated with the measurement techniques is presented. PMID- 7551787 TI - The optimization and inherent limitations of 3D conformal radiotherapy treatment plans of the prostate. AB - This paper describes the applications of an inverse planning optimization algorithm to the real clinical problem of prostate cancer. The algorithm has been designed to compute optimized beam-weights taking full account of three dimensional spatial information of dose inside the patient. The algorithm is based on fast simulated annealing, utilizing a cost-function containing both linear and quadratic terms. The linear part of the cost function allows for the implementation of "short-cuts" in the cost-function computation, which reduces the calculation time by a factor of about 30. It has been applied to compute optimized beam-weights for a three-field and a seven-field prostate treatment plan. It is shown for the three-field plan that the optimization algorithm can reproduce, and even slightly improve on, the results of an experienced human planner. For the seven-field plan, the human planner experienced difficulty finding beam-weights that gave an acceptable dose distribution. It is shown that the optimization algorithm can achieve good results in this case. The outcome of the optimization of the seven-field plan prompted an investigation into the best results that could be achieved by an "ideal" conformal radiotherapy technique. The results of this investigation are presented and it is shown that the limiting factor for conformal therapy of the prostate is the size of the overlap volume between the planning target volume (PTV) and the rectum. Finally, the efficiency and accuracy of fast simulated annealing is compared with that of classical simulated annealing. The former was found to be at least 10 times faster for the problem studied. PMID- 7551788 TI - Derivation of the optimum dose per fraction from the linear quadratic model. AB - The linear quadratic equation for fractionated radiotherapy has already been adapted to include a time factor for tumour repopulation: loge cell kill (E) is given as a function of dose per fraction (d), number of fractions (n), overall treatment time (T) and the clonogen doubling time (Tp). By incorporating a normal tissue isoeffect and replacing the relationship between T and n by a function f, the equation for E can be rewritten as a more complex function of d. In this form, E and d are continuous variables so that the dose per fraction (d') required to produce maximum values of E for isoeffective late normal tissue effects can be found by differential calculus. The derived equation takes the form (beta kTp-alpha Tp)d2 + 1.386fd + 0.693fk = 0 and when solved for d provides a direct estimation of the optimum dose per fraction. Where normal tissue sparing is possible and the tumour dose z is related to the normal tissue dose d, the optimum dose per fraction z' can be found by solving the equation (beta kTp-alpha gTp)z2 + 1.386fgz + 0.693fk = 0 The results show that a critical minimum dose per fraction is required to counteract rapid tumour clonogen repopulation in both conventional and accelerated radiotherapy. The calculus method is reasonably accurate for larger fraction numbers, when clonogen doubling times are 3.5 days or longer and for conventional radiotherapy given 5 days per week. The model is even more accurate for accelerated hyperfractionated radiotherapy providing that there is complete repair between successive fractions. Where greater normal tissue sparing is possible, as with focal teletherapy methods and brachytherapy, higher tumour doses per fraction can be used to increase further the tumour cell kill without exceeding normal tissue tolerance. These predicted doses per fraction are consistent with clinical experience when the given constraints in terms of frequency of treatment are considered. The model described can be used for tumours in which repopulation occurs at a constant rate throughout treatment. For tumours in which accelerated repopulation occurs, the optimum dose per fraction can be separately calculated for the initial phase of slow repopulation (for which very small doses per fraction are optimal) and also for the second phase of rapid repopulation (for which either accelerated hyperfractionated treatments or hypofractionated focal methods of treatment would be appropriate). The limitations of the model are fully discussed including the need for accurate radiobiological predictive assays. In the future such assays of pre-treatment doubling times and tumour cell radiosensitivities could be used to determine reasonable ranges for the optimum dose per fraction in experimental tumours and subsequently in clinical trails. This approach could produce major improvements in the therapeutic potential of radiotherapy. PMID- 7551789 TI - Dual energy X-ray absorptiometry normal reference range use within the UK and the effect of different normal ranges on the assessment of bone density. AB - The number of different normal ranges used on dual energy X-ray absorptiometry (DEXA) machines in the United Kingdom was determined by means of a postal questionnaire. Both femoral neck and posteroanterior spine L2-L4 regions were considered. It was clear from this survey that a variety of normal ranges were in use for all manufacturers of DEXA systems. The effects of four normal ranges supplied by Norland for use within the UK on the stratification of over 1000 consecutive patients into different grades of bone mineral density (BMD) and Z score were examined. The main outcomes measure the number of patients with BMD less than 80% of mean age-matched BMD and Z-score less than -1.5 over the femoral neck and spine for each normal range. The percentage of patients for each normal range with BMD less than 80% of mean for the femoral neck were 24.6%, 7.4%, 17.5% and 11.1% and for the spine 12.1%, 7.4%, 16.5% and 14.4%, respectively. For the femoral neck, 21.2%, 5.0%, 12.9% and 12.1% of patients had Z-scores of less than 1.5, for the spine this was 7.9%, 8.1%, 14.2% and 13.6% of patients. These differences between ranges are large enough to influence patient management- patients may be diagnosed as osteoporotic using one range and normal using another. The relationship between hip and spine measurements was also studied as some therapeutic agents do not affect BMD equally at all anatomical sites. The number of patients with a lower hip than spine BMD grade varied from 12.3% to 34.4%, and with a lower hip than spine Z-score from 14.0% to 43.4%. Choice of normal range thus has a critical effect on the categorization of osteoporotic patients using DEXA. The wide variety of normal ranges indicates that there are likely to be differences in patient management throughout the country. This will be an increasing problem due to the ability of NHS purchasers to switch contracts from one provider to another. PMID- 7551791 TI - Technical note: a new device for performing sinograms--the Frank catheter. AB - Usually sinography is a difficult and messy investigation, in that most of the contrast medium seeps past the Foley catheter balloon and out of the orifice. A new sinogram catheter has been devised to overcome this problem. PMID- 7551792 TI - Technical note: an assessment of X-ray protective gloves. AB - An assessment of X-ray protective gloves is reported which demonstrates that a significant degree of protection from X-ray exposure can be offered by these gloves. The need for such protection, together with the method of assessment is discussed. It is recommended that X-ray protective gloves are considered when undertaking image intensifier work. PMID- 7551793 TI - Case report: avascular necrosis of the femoral head as a complication of complex embolization for severe pelvic haemorrhage. AB - We present a case of avascular necrosis of the femoral head following embolization of the right medial femoral circumflex artery with alcohol after a failed prior internal iliac artery ligation to control benign pelvic haemorrhage in a 41-year-old woman. No case of late necrosis of the head of the femur as a complication of iliac artery vessel embolization to control haemorrhage has been documented previously. The problems associated with therapeutic pelvic embolization following ligation of the internal iliac artery and the disruption of the femoral head arterial supply are discussed. PMID- 7551790 TI - Ultrasonographic assessment of bone in normal Italian males and females. AB - Recently ultrasound techniques have been proposed to evaluate skeletal status. Speed of sound and attenuation through the bone are the ultrasound properties currently used to assess bone strength and fragility. The speed of sound in m s-1 (SOS), broadband ultrasound attenuation in dB MHz-1 (BUA) and stiffness (S) in 134 healthy females (age range 10-90 years) and in 100 healthy males (age range 10-93 years) was measured using the Achilles scanner (Lunar Corp., Madison, WI, USA). A polynomial function was applied to the observed data to evaluate a pattern of age-related BUA, SOS and S changes. Peak values of SOS, BUA and S were reached in both sexes at the age of 30 years. Average decreases of 12.9% in BUA, 4.9% in SOS and 28.9% in S were found in men aged between 30 and 90 years. In women average decreases of 17.2% in BUA, 4.2% in SOS and 31.9% in S were discovered in those aged between 30 and 90 years. The analysis of SOS, BUA and S changes between pre- and post-menopausal women revealed a significant decrease of these parameters with years since menopause. These data indicate an age-related decrease of ultrasound signals in both sexes. Furthermore, this technique is able to detect, in females, menopause related changes due to oestrogen failure. In contrast, in males, the age-related loss of ultrasound signals appears to be more linear. PMID- 7551794 TI - Case report: pulmonary calcification after liver transplantation in children. AB - Pulmonary calcification following liver transplantation is a recognized phenomenon in adults but has not been previously described in children. We describe two children who developed pulmonary calcification after liver transplantation. Pulmonary calcification should be considered after liver transplantation when radiographic changes fail to resolve with appropriate treatment. PMID- 7551795 TI - Case report: non-Hodgkin's lymphoma presenting as an encasing pleural mass. AB - We report a case of primary non-Hodgkin's lymphoma of the pleura with marked pleural thickening and encasement, apparent on a chest radiograph. This has not, to our knowledge, been previously reported. PMID- 7551796 TI - Case report: bleeding gastric varices secondary to splenic vein thrombosis successfully treated by splenic artery embolization. AB - Splenic vein thrombosis is a complication of pancreatic carcinoma or pancreatitis. It may lead to gastric varices which are difficult to treat and splenectomy may be required to stop variceal bleeding. A case of bleeding gastric varices secondary to splenic vein thrombosis and successfully treated by splenic artery embolization is reported. Embolization was performed by transcatheter deposition of four Gianturco coils into the splenic artery. This resulted in reduced blood flow through the spleen with partial splenic infarction and cessation of variceal bleeding. There has been no recurrence of bleeding in the 6 months since the procedure. Literature review confirms that experience of using this treatment is very limited and it should therefore be restricted to patients at high risk from surgery. PMID- 7551797 TI - Case of the month: a pain in the neck. PMID- 7551799 TI - Tangential breast irradiation. PMID- 7551798 TI - Short communication: proximal tibial stress lines--appearances on magnetic resonance imaging of the knee. AB - Trabecular lines through the proximal tibia in line with the direction of the anterior cruciate ligament are visible in around two-thirds of standard knee magnetic resonance imaging (MRI) examinations. We describe their appearances and postulate that these lines may develop in response to stresses transmitted via the anterior cruciate ligament (ACL). PMID- 7551800 TI - [Serologic response of normal Korean children to Pneumocystis carinii as observed by immunoblot]. AB - Soluble protein of purified Pneumocystis carinii was prepared from experimentally infected rats. SDS-PAGE of the crude antigen resolved about 20 protein bands from 20 to 200 kDa. Out of them, 116 kDa band strongly reacted and 45-55 and 100 kDa bands reacted weakly to the positive reference human serum from U.S.A. Western blot analysis with sera of 130 normal children and 15 newborns in Korea revealed specific IgG antibody reaction to 40-55 and 116 kDa protein bands. Forty percent (40.0%) of the 145 sera were positive with any of the antigenic protein bands of P. carinii. The positive rate was 56% in 50 males and 33.3% in 48 females. The protein bands 40-55 and 116 kDa from rat P. carinii were confirmed to cross-react with human sera in Korea. PMID- 7551801 TI - Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody. AB - Two hybridoma cell lines against Cryptosporidium parvum oocysts (VRI-CN91) were produced. The isotype of these 2 monoclonal antibodies (mAbs) was IgG2b (1E7.2) and and IgM (C6). Enzyme immuno-transfer blotting analysis showed that 1E7.2 reacted specifically to 36 kDa protein and C6 reacted to 67 and 70 kDa proteins. C. parvum was bound specifically to the surface region of oocysts by these mAbs. No cross-reactivity was observed with tachyzoites of Toxoplasma gondii and oocysts of Eimeria zuernii, E. bovis and E. canadensis of bovine origin. The indirect immunofluorescence assay (IIF) using mAb C6 was successful with counterstain. With the IIF using mAb C6, oocysts appeared as 3 to 5 microns spherical objects fluorescing bright apple green against a reddish dark background. The IIF using mAb C6 was agreed in specificity and sensitivity with those of a commercial diagnostic kit. These results demonstrated that the produced mAbs were specific to C. parvum and that the mAb C6 could be used for diagnosis of cryptosporidiosis. PMID- 7551803 TI - An imported case of hepatic unilocular hydatid disease. AB - A 38-year old man visited a private clinic complaining of epigastric discomfort for 2 months. A huge hepatic cyst was found by sonography and computerized tomography. An exploratory laparotomy was performed under the impression of hydatid disease. The cyst was successfully removed. A lot of living protoscolices of Echinococcus granulosus were found from the cystic fluid under light microscopy. During the operation, however, the cyst was accidentally ruptured and the cystic fluid spilled out. The patient was medicated with albendazole, and had been well without any signs of anaphylaxis or recurrence for 1 year follow-up period. He had been in Saudi Arabia for 3 years. This is the 16th case of hydatid disease reported in Korea and a case without immediate complication in spite of rupture of the cyst. PMID- 7551802 TI - [Evaluation of sonography and skin test in diagnosis of clonorchiasis at the Hyongsan-gang (river) area]. AB - This study was performed to evaluate diagnostic sensitivity and specificity of sonography on clonorchiasis. During the 9 months from March to November 1994, sonography, skin test, stool examination, and medical examination were performed to 609 volunteers of Pohang which is located along the Hyongsan-gang (River) and is one of well-known endemic areas of clonorchiasis. The sensitivity of sonography was either 21% if the intrahepatic ducts dilatation was assumed to be the only positive finding or 52% if the periductal echogenicity was also included as one of positive findings. The sensitivity of skin test was 62%. The sensitivity was 46% if the diagnostic criteria were arbitrarily assumed as being positive in skin test and having either sonographic findings. The sonographic finding of fusiform, non-shadowing, weakly echogenic foci in gallbladder or in biliary tree indicating the flukes or aggregates of flukes was not obvious in this study. The specificity of sonography was either 95% if the intrahepatic ducts dilatation was assumed to be the only positive finding or 82% if the periductal echogenicity was also included as one of positive findings. The specificity of skin test was 52%. The specificity was 90% if the diagnostic criteria was arbitrarily assumed as being positive in skin test and having either sonographic findings. PMID- 7551805 TI - [Incidence of Buxtonella sulcata from cattle in Kyonggi-do]. AB - In order to survey the prevalence of Buxtonella sulcata in Kyonggi-do in 1984 and 1994, the feces of cattle were collected and examined. Of 792 cattle in 1984 and 293 in 1994, cysts of B. sulcata were detected from 266 (33.6%) and 101 (34.5%) cattle respectively. The positive rates were 85.9% and 64.2% in breeder cattle, 44.8% and 9.4% in dairy cattle, 7.5% and 14.9% in Korean cattle and 1.8% in beef cattle. In spring, the cyst positive rates were 62.5% in 1984 and 27.8% in 1994; 20.1% and 66.7% in summer; 21.3% and 0% in autumn; and 34.8% and 64.6% in winter. More cattle, 69.1% (184) and 55.5% (56) in 1984 and 1994, had diarrhea among the cyst positive cattle than those of cyst negative, 37.6% (198) and 32.3% (62). PMID- 7551804 TI - Lymphadenitis in experimental murine toxoplasmosis induced by intramuscular injection of tachyzoites. AB - When tachyzoites (RH strain) of Toxoplasma gondii are injected intramuscularly, experimental mice survive up to 7 days, 1-2 days longer than those infected intraperitoneally. We observed sequential histopathological changes in inguinal lymph nodes after intramuscular injection of tachyzoites to thighs of specific pathogen free (SPF) mice. Initial findings on 1 or 3 days after the injection were reactive germinal centers, distended sinuses and epithelioid cell clusters in cortical and paracortical regions. Later on 5 days after the injection, however, effacement of nodal structure with depletion of cells and focal necrosis were observed. Necrotizing lymphadenitis in the experimental murine toxoplasmosis suggests the causal relation between T. gondii infection and the human disease. PMID- 7551806 TI - Two human cases of Thelazia callipaeda infection in Korea. AB - Thelazia callipaeda were observed from a 7-month old baby who lived in Uijongbu in 1989 and from a 42-year old man who lived in Anyang in 1994. These are the 23rd and 24th records of human thelaziasis in Korea as the literature are concerned. PMID- 7551807 TI - Gorgoderid trematodes (Digenea: Gorgoderidae) from the urinary bladder of frogs in Korea. AB - Two species of the family Gorgoderidae (Trematoda: Digenea), Gorgodera japonica Yamaguti, 1936 and Gorgoderina bombinae Yu & Lee, 1983 were collected from the urinary bladder of frogs captured from various localities in Korea. The morphology of each species is described and illustrated. Gorgodera japonica differs from G. cygnoides by having deeply branched vitellaria. Gorgoderina bombinae is considered a valid species. PMID- 7551808 TI - [Taxonomical approach to scabies mites of human and animals and their prevalence in Korea]. AB - Three types, hitherto commonly known as varieties of Sarcoptes scabiei (Linnaeus, 1758) were collected from scabietic patients, pigs and dogs to confirm their taxonomic status. As a result, we consider that the mites of human and pig scabies are different subspecies [S. scabiei scabiei (Linnaeus, 1758) and S. scabiei suis Megnin, 1880, respectively], and that of dog scabies is a different species, S. canis Gerlach, 1857. Infestation rate of human scabies in Korea peaked in 1981-1982 and showed nearly 10% of prevalence rate among outpatients of the dermatology clinic, but decreased dramatically during the past ten years and now lowered below 1% since 1990. On the other hand, dog scabies seems to be increasing recently. PMID- 7551809 TI - [Infection status of anisakid larvae in anchovies purchased from local fishery market near southern and eastern sea in Korea]. AB - A survey was carried out on the larval anisakis in anchovies (Engrauris japonica) which were caught from April to June in 1993 at the southern and eastern sea in Korea. Anisakids in anchovies were morphological classified into Anisakis type I and II and Contracaecum type B and C, after fixation in 70% alcohol and clearing in lactophenol. A total of 171 larval anisakids were collected from 150 anchovies (6.9%) out of 2,180 examined. The infection rates showed no relation with length of the anchovies. The numbers of anisakids by organs of anchovies were 96 (56.1%) from the muscle, 65 (38.0%) from the omentum, 7 (4.1%) from the intestine, 2 (1.7%) from the stomach and 1 (0.6%) from the testis. Infection rates of anchovies with anisakids by localities were 5.1% at Taebyon, 8.0% at Sokcho and 9.2% at Chungmu. The larval anisakids in anchovies may infect humans who often eat raw anchovies. PMID- 7551810 TI - Proposed role for a combination of citric acid and ascorbic acid in the production of dietary iron overload: a fundamental cause of disease. AB - This paper presents a review of the significant body of literature liking dietary iron overload, not only to heart disease, but also to cancer, diabetes, osteoporosis, arthritis, and possibly other disorders. Following an analysis of our understanding of the mechanistic role iron plays in oxidative damage, an interpretation of the fact that plasma concentrations of several antioxidants are decreased in the presence of disease is offered. Evaluation of (1) age-related dietary trends over time and (2) factors involved in iron absorption leads to the hypothesis that the combination of citric acid and ascorbic acid (a synergistic pair of strong enhancers) is instrumental in causing a deleterious increase in iron load in aging populations. Iron overload may be the most important common etiologic factor in the development of the diseases mentioned; therefore, the synergistic combination of citric and ascorbic acids may play a major role in our worsening disease statistics. Evidence to support this hypothesis and possible experiments to test it are included. This combination needs further study, particularly because the iron overload produced may be correctable. PMID- 7551814 TI - Inhibition kinetics of sheep brain glutathione reductase by cadmium ion. AB - Glutathione reductase participates in preventing lipid peroxidation by oxygen radicals which results in cellular damage. The brain is among the organs most susceptible to cadmium-induced lipid peroxidation. The mechanism of free radical generation by Cd2+ is not well understood, but it is known that Cd2+ is an inhibitor of glutathione reductase. In this study, inhibition kinetics of the brain glutathione reductase by Cd2+ was investigated. Sheep brain enzyme (11,000 fold purified) was used for this purpose. The data were analyzed by a nonlinear curve fitting program. It was found that the inhibition was competitive with respect to oxidized glutathione and uncompetitive with respect to NADPH. Inhibition constants were found as 12.3 and 9.4 muM, respectively. These findings might contribute to the understanding of the mechanism of lipid peroxidation by Cd2+ in brain. PMID- 7551813 TI - The human and mammalian N-acetylmuramyl-L-alanine amidase: distribution, action on different bacterial peptidoglycans, and comparison with the human lysozyme activities. AB - N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) specifically hydrolyzes the bacterial cell wall peptidoglycans (or mureins) and the muropeptides. The enzyme splits these molecules into two parts: the peptide subunits and the glycan strands or moieties. The bacterial peptidoglycans and their derived muropeptides display a number of biological properties. Removal of the glycosidic part of these molecules abolishes their beneficial as well as their detrimental properties. We report the high level of enzymatic activity found in all mammalian (including human) sera tested. The enzyme also occurred in human saliva, milk, cerebrospinal fluid, and synovial liquid. Mucosal tissue from different parts of the mammalian digestive tract exhibited enzymatic activity, but the enzyme was not detectable in the lumen content. The range of substrate specificity of the human enzyme was evaluated by measuring its action on the peptidoglycans extracted from several bacterial strains and representing different chemotypes and structures. Time course of the muramylalanine amidase and of the lysozyme (both of human origin) activities on some of these peptidoglycans are also reported, with the enzymes acting separately or together. From these data, we would speculate that a probable physiological role of the muramylalanine amidase is the maintenance of adequate ratios between the biologically active muropeptides and their inactive derivatives in the organism, the amidase activity antagonizing the production of biologically active molecules by lysozyme. PMID- 7551812 TI - Fatty acid synthesis from glutamate in the adipose tissue of normal subjects and obese patients: an enzyme study. AB - In the adipose tissue, besides fatty acid synthesis (FA-S) from glucose, which includes several mitochondrial steps, FA-S from glutamate has been demonstrated. FA-S from glutamate takes place in the cytosol through the backward pathway of Krebs cycle (BPKC) and is due to the sequential action of (1) alanine aminotransferase (ALT, EC 2.6.1.2), which is presence of pyruvate converts glutamate to oxoglutarate; (2) isocitrate dehydrogenase (NADP) (ICDH, EC 1.1.1.42), which converts oxoglutarate to isocitrate; (3) aconitate hydratase (ACO, EC 4.2.1.3), which transforms isocitrate to citrate: and (4) ATP citrate lyase (ATP-CL, EC 4.1.3.8), which splits citrate to yield the acetyl-CoA needed for FA-S. We studied the enzymes involved in BPKC in homogenates of human adipose tissue. In normal subjects, the cytosolic activity (mumol/min/g protein) was: ALT = 10.3 +/- 1.1, ICDH = 29.5 +/- 2.8, ACO = 2.05 +/- 0.23, and ATP-CL = 1.2 +/- 0.2. Mitochondria contained less or no activity, values being 20, 9, 11, and 0% of total for ATL, ICDH, ACO, and ATP-CL, respectively. BPKC enzymes are more active than the enzymes limiting FA-S from glucose, i.e., phosphofructokinase (EC 2.7.1.11), pyruvate carboxylase (EC 6.4.1.1), and pyruvate dehydrogenase (EC 1.2.4.1). In the obese patients, cytosolic ALT and ATP-CL were increased (12.9 +/ 0.7, P < 0.05, and 2.28 +/- 0.27, P < 0.01, respectively) compared to normal, while ICDH was not changed (ACO could not be studied). Similar changes were obtained by expressing enzyme activity per fat cell number.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551815 TI - Impaired glucose uptake and intact gluconeogenesis in perfused rat liver after carbon tetrachloride injury. AB - The dynamics of glucose movement across perfused livers were assessed in carbon tetrachloride (CCl4)-injured rats. Rats were given CCl4 for 8 weeks and became glucose intolerant and hyperinsulinemic. The fasted rat liver was cyclically perfused with 4 mM lactate and various concentrations (0-20 mM) of glucose for 20 min. In the CCl4-injured liver, net glucose output was less suppressed at high glucose levels than in the normal liver (147 +/- 70 vs 18 +/- 10 mumol at 20 mM glucose, P < 0.05). Deposition of the carbon from [14C] glucose into glycogen was stimulated at high glucose levels and was markedly reduced in the CCl4-injured liver compared to the normal liver (0.58 +/- 0.33 mumol vs 1.44 +/- 0.20 mumol at 20 mM, P < 0.01). Conversion of [14C] lactate to [14C] glucose was not different between the CCl4-injured and the normal liver at each glucose level. Deposition of the carbon from [14C] acetate into glycogen in the CCl4-injured liver was larger than that in the normal liver at 0 mM glucose (0.81 +/- 0.15 mumol vs 0.32 +/- 0.06 mumol, P < 0.01), but was similar to the normal at 20 mM glucose. In the CCl4-injured liver, utilization of exogenous glucose was impaired at high glucose levels, and gluconeogenetic activity was not impaired at low glucose levels. These changes in the hepatic glucose metabolism seem to account for postprandial hyperglycemia without fasting hypoglycemia associated with liver diseases. PMID- 7551811 TI - Superoxide dismutase and catalase enhance autoxidation during one-electron reduction of aminochrome by NADPH-cytochrome P-450 reductase. AB - NADPH-cytochrome P-450 reductase catalyzes one-electron reduction of aminochrome to the corresponding ortho-semiquinone, which was found to be unstable as indicated by the occurrence of NADPH oxidation and oxygen consumption. The addition of superoxide dismutase and catalase, alone or together, to the incubation mixture, during reduction of aminochrome catalyzed by NADPH-cytochrome P-450 reductase, did not prevent the autoxidation of ortho-semiquinone, but instead they increased NADPH oxidation. These results contrast with the almost complete inhibition of autoxidation (NADH oxidation) of ortho-hydroquinone during reduction of aminochrome catalyzed by DT-diaphorase in the presence of both superoxide dismutase and catalase. However, the effect of superoxide dismutase and catalase on oxygen consumption was found to differ from the effect on NADH or NADPH oxidation, since these enzymes, alone or together, inhibited the oxygen consumption during the reduction of aminochrome catalyzed by both NADPH cytochrome P-450 reductase and DT-diaphorase. These results support the proposed role of NADPH-cytochrome P-450 reductase in neurodegeneration as a consequence of activation of aminochrome to reactive oxygen species. In addition, they also support the protective and antioxidant role of DT-diaphorase, together with superoxide dismutase and catalase, by competing with NADPH-cytochrome P-450 reductase to reduce aminochrome to ortho-hydroquinone and prevent the formation of reactive oxygen species. A possible mechanism is proposed. PMID- 7551816 TI - An in vivo 31P magnetic resonance spectroscopy study of uridine excess in rats fed orotic acid. AB - Spatially localized 31P NMR spectroscopy was used to assay in vivo the liver of intact rats fed orotic acid (OA) in a diet which produces hepatic steatosis. Twenty-three sets of multiple volume spectra were obtained from twenty-one 265- to 315-g female rats after 0-9 days of feeding either a 1% OA/64% sucrose diet (12 rats) or a 65% sucrose control diet (9 rats). The intensity of the in vivo diphosphodiester resonance ascribed to UDP-hexos(amin)es increased and the phosphomonoester resonance decreased in intensity prior to fatty infiltration. High resolution NMR spectroscopy of extracts of these livers indicated that the UDP-hexos(amin)e peak included four different UDP-sugars including UDP-N acetylglucosamine (UDP-glcNAc), and that lower phosphocholine (P-Cho) accounted for the lower phosphomonoester resonance in vivo. Increased UDP-glcNAc is thought to reflect impaired lipoprotein glycosylation as a mechanism for hepatic steatosis in orotic acid feeding. P-Cho deficiency has been shown to be due to an increased rate of phosphatidylcholine synthesis. Low P-Cho concentration has been shown to be associated with lipid accumulation in a choline-deficient diet, but was not previously associated with hepatic steatosis in OA feeding. Changes in phosphorus metabolites were observed 2 days prior to development of fatty liver. HPLC assay of uridine nucleotides showed a good correlation between magnetic resonance spectroscopy and HPLC quantitation. In this study there were two biochemical correlates of impaired hepatic lipid secretion detectable by in vivo assay with 31P NMR spectroscopy. This method has application for noninvasive assays in ornithine transcarbamylase-deficient patients. PMID- 7551819 TI - Sequential evaluation of plasma retinol-binding protein response to vitamin A administration in very-low-birth-weight neonates. AB - Vitamin A (retinol) deficiency is associated with impaired healing from lung injury in very-low-birth-weight (VLBW) neonates susceptible to bronchopulmonary dysplasia (BPD). Vitamin A supplementation from birth may ameliorate this adverse outcome. We hypothesized that plasma retinol-binding protein (RBP) response to vitamin A administration, which provides a dynamic measure to vitamin A status, might be useful for early recognition of vitamin A deficiency in VLBW neonates at risk for BPD. We prospectively studied 20 VLBW neonates (inclusion criteria: birth weight < 1300 g, gestational age < 30 weeks, need for supplemental oxygen and mechanical ventilation for > 24 h after birth) who were eligible to receive vitamin A supplementation. In addition to sequential assessment of vitamin A status, we measured plasma RBP just before and 3 and 6 h after an intramuscular injection of vitamin A (2000 IU/kg retinyl palmitate) on Postnatal Days 1, 7, 15, 21, 29, and 43. The percentage increase in plasma RBP (delta-RBP) was calculated. A high plasma delta-RBP value ( > 8%) is indicative of vitamin A deficiency. Based on pulmonary outcome, the infants were divided into two groups: BPD (n = 12) and No BPD (n = 8). Mean vitamin A intake ranged from 1414 to 2114 IU/kg/day and did not differ between infant groups. Mean plasma vitamin A concentration increased from baseline levels on Postnatal Day 1 to levels within the desired range of 1.05-2.10 mumol/liter (30.0-60.0 micrograms/dl) during supplementation period in both infant groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551818 TI - Investigation of beta-oxidation intermediates in normal and MCAD-deficient human fibroblasts using tandem mass spectrometry. AB - Mitochondrial fatty acid beta-oxidation was studied by incubating stable isotope labeled fatty acid probes with human fibroblasts in the presence of L-carnitine. The acylcarnitine intermediates produced were analyzed by tandem mass spectrometry. Oxidation by normal fibroblasts produced specific acylcarnitine intermediates corresponding to acyl-CoA dehydrogenase substrates mainly of 10 or less carbons. These probes demonstrated that the pathway, involving all beta oxidative steps, could be examined. Oxidation of the same precursors by cells with medium chain acyl-CoA dehydrogenase (EC 1.3.99.2) (MCAD) deficiency, which is caused by different DNA mutations, produced acylcarnitine profiles which appear to be specific to this enzyme defect, regardless of the DNA mutation. Increased amounts of octanoyl-, decanoyl-, or decenoylcarnitine were detected. The ratios of octanoylcarnitine to decanoyl- or decenoylcarnitine appear specific for MCAD deficiency. Even though the concentration of labeled decenoylcarnitine (C10:1) was elevated in incubations of MCAD-deficient cells with labeled linoleate or with a fatty acid mixture which included palmitate, oleate, and linoleate, the predominant intermediate was octanoylcarnitines. These results suggest that MCAD-deficient cells readily convert decanoyl-CoA into octanoyl-CoA. This in vitro system could be utilized to study fatty acid oxidation disorders and to study the origins of metabolic intermediates associated with them. PMID- 7551817 TI - Determination of hexokinase isoenzyme I and II composition by RT-PCR: increased hexokinase isoenzyme II in human renal cell carcinoma. AB - Hexokinase isoenzyme composition has been noted to vary in different tissues and with the developmental and metabolic status of the cell. Until now these investigations were performed either by isoenzyme electrophoresis or by column chromatography. In this report we described an RNA-PCR method to evaluate the percentage of HK-1 and HK-2 in different rat tissues. Furthermore we applied the method to determine if a shift in isoform composition is detectable in human renal carcinomas compared to normal kidney tissue. In all of our specimens we were able to detect a shift toward HK-2 in the carcinoma specimens. We discuss a possible role for the detection of the shift in isoenzyme composition as a possible marker to discriminate between normal and malignant specimens. PMID- 7551820 TI - Hexose metabolism in pancreatic islets: regulation of the mitochondrial NADH/NAD+ ratio. AB - In rat pancreatic islets, D-glucose in concentrations exceeding 5.6 mM caused a concentration-related decrease of the mitochondrial NADH/NAD+ ratio, as judged from the changes in the islet content of glutamate, NH4+, and 2-ketoglutarate, and assuming that the glutamate dehydrogenase reaction is near equilibrium with the mitochondrial NAD system. The concentration dependency of the response to D glucose was vastly different in islet and parotid cells, respectively. L-Leucine, 2-ketoisocaproate, BCH (a nonmetabolized but insulinotropic analog of L-leucine) and 3-phenylpyruvate also lowered the mitochondrial NADH/NAD+ ratio. In the presence of D-glucose, the latter ratio was also decreased by NH4+ or the absence of extracellular Ca2+, but dramatically increased by aminooxyacetate. Taking into account prior metabolic findings, the nutrient-induced fall in the mitochondrial redox state is thought to reflect an increased clearance of mitochondrial NADH through both the respiratory chain and malate-aspartate shuttle. The nutrient induced decrease in the mitochondrial NADH/NAD+ ratio might favor both the circulation of metabolites in the Krebs cycle and the exit of Ca2+ from the mitochondria. PMID- 7551822 TI - Differential evolution and expression of murine peroxisomal membrane protein genes. AB - Gene segments encoding the 70 and 22-kDa peroxisomal membrane proteins (PMP) have been characterized in mice and compared with other peroxisomal proteins in terms of evolution and expression. The mouse PMP22 gene sequence predicts A16G and I136V substitutions that agree with those defined by cyanogen bromide cleavage analysis, providing additional evidence that this gene encodes the major 22-kDa membrane protein visualized by SDS-polyacrylamide electrophoresis. Mammalian PMP22 genes exhibit high evolutionary rates (0.17% amino acid substitution per million years) than PMP35 (0.14%), PMP70 (0.07%), or catalase genes (0.13%). PMP70 gene regions are conserved throughout vertebrate phylas based on Southern analysis, while PMP22 sequences were only detected in rodents. Amino acid substitutions are clustered in both PMP22 and PMP70 genes, and their pattern supports membrane topologies derived from hydropathy profiles. Northern blot analysis identifies single mRNAs of 4.6 kb (PMP70), 1.4 kb (PMP22), and 2.3 kb (catalase) in several rodent tissues. Quantitative or competitive RT-PCR assays detected two- to three-fold greater numbers of catalase mRNA molecules relative to PMP mRNA molecules in brain, liver, and kidney; PMP22 and PMP35 mRNAs were two fold more abundant than PMP70 mRNA in these tissues. Steady-state levels of PMP22 mRNA were highest in rodent liver kidney, spinal cord, and duodenum with low levels in colon, adrenal, thymus, and spleen. We conclude that PMP genes exhibit independent evolutionary rates and tissue regulation, suggesting that they have unique roles in peroxisome biogenesis and tissue differentiation. PMID- 7551823 TI - Androgen receptor binding studies on heterozygotes in a family with androgen insensitivity syndrome. AB - A large family with androgen insensitivity syndrome (AIS) has been investigated, in order to detect carrier individuals and to investigate their AR binding. We have previously reported that different affected members of this family have different AR gene deletions, and, testing normal female relatives of the affected individuals, we have identified three heterozygote females all of whom carry a deletion of exon E of the AR gene. Androgen binding capacity was measured in cultured genital skin fibroblasts from normal male and female controls, the affected, and the heterozygote individuals in this family. A significant difference was found between the binding ranges for normal male foreskin and suprapubic skin fibroblasts (P < 0.005), and the three individuals with AIS had very low androgen binding capacity in their genital skin fibroblasts (2.6, 5.0, and 3.4 fmol 3HR1881/mg protein) compared to the normal range. The heterozygote females all had binding within the normal female suprapubic skin fibroblast range (12.5, 6.1, and 6.4 fmol 3HR1881/mg protein, respectively, for the three heterozygotes). Thus, we conclude that the absence of one functional AR gene in heterozygote females has not effect on AR binding capacity in cultured genital skin fibroblasts. In addition, bone mineral density was measured in the affected aunt and found to be significantly lowered at the lumbar spine (Z = -2.81) and hip (Z = -1.54); however, the role of the AR in determination of bone mineral density remains to be elucidated. PMID- 7551821 TI - Long-chain acyl-CoA profiles in cultured fibroblasts from patients with defects in fatty acid oxidation. AB - Negative chemical ionization (NCI) mass spectrometry was used to quantify the acyl-CoA intermediates present in human fibroblasts growing in media containing the long-chain fatty acid, palmitate. The acyl-CoA intermediates were detected as the N-acyl pentafluorobenzyl glycinates. In fibroblasts from normal individuals only saturated acyl-CoA esters were detected, supporting the concept that the acyl-CoA dehydrogenase reaction is the rate-limiting step of intramitochondrial fatty acid oxidation. In patients with inherited enzymatic defects of intramitochondrial long-chain fatty acid oxidation, there was not a significant increase in the amount of long-chain acyl-CoA compounds, with palmitoyl-CoA amounts similar to those found in controls. However, there was a sharp decrease in the relative amount of lauroyl-CoA and a resultant sixfold elevation in the palmitoyl-CoA:lauroyl-CoA ratio. In contrast, fibroblasts with a defect involving the transport of fatty acids across the mitochondrial membrane, carnitine palmitoyl transferase 1 deficiency, had a fourfold increase in palmitoyl-CoA. Our results suggest that acyl-CoA esters in biological tissues are readily detectable using NCI mass spectrometry. This approach is significantly more sensitive than previous methods for the detection of these important metabolic intermediates, and may prove useful in the study of fatty acid oxidation in both normal and enzyme-deficient tissues. PMID- 7551825 TI - Nonenzymatic degradation and salvage of dietary folate: physicochemical factors likely to influence bioavailability. AB - We investigated the oxidative degradation pathway of 5CH3-H4PteGlu, the main extracellular folate and the predominant form of the vitamin found in food and blood. 5CH3-H4PteGlu is oxidized to 5CH3-5,6-H2PteGlu which subsequently undergoes C9-N10 bond cleavage yielding a pteridine residue and P-ABG, the latter step resulting in irreversible loss of vitamin activity. Under moderately acid conditions typical of the postprandial gut (pH 3.5) 5CH3-H4PteGlu is fairly stable (t1/2 = 273.6 min), while 5CH3-5,6-H2PteGlu is rapidly degraded (t1/2 = 16.9 min). In a neutral environment (pH 6.4) stability is reversed; 5CH3-H4PteGlu t1/2 = 12.0 mins, 5CH3-5,6-H2PteGlu t1/2 = 1504.6 min. Ascorbic acid was efficacious in the facile salvage of 5CH3-H4PteGlu from 5CH3-5,6-H2PteGlu which occurred rapidly and with significant efficiency (100% conversion) under acid (pH 3.5) conditions, t1/2 = 1.3 min (1 mmol/liter ascorbate), but was less efficient under neutral (pH 6.4) conditions t1/2 = 273.6 min (36% conversion). The presence of zinc and iron broadly maintains the pattern of effect, but increases all reaction rates. PteGlu was stable under all conditions studied. These results obtained in an artificial environment were supported by findings in human gastric juice: at a gastric pH of 1.47 with low endogenous ascorbate (7.0 mumol/liter), 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu both degrade instantly via C9-N10 bond cleavage to yield an equimolar amount of P-ABG. If the same gastric juice is spiked at 58.0 mumol/liter ascorbate (moderate endogenous concentration), 5CH3 H4PteGlu is stable (t1/2 = 334.7 min), while 5CH3-5,6-H2PteGlu is instantly salvaged to 5CH3-H4PteGlu with 43.3% efficiency, and the remaining 5CH3-5,6 H2PteGlu is degraded to P-ABG. In gastric juice with an elevated pH of 7.0 and no endogenous ascorbate, 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu are both stable, with no C9-N10 bond cleavage. This, for 5CH3-H4PteGlu, is in apparent contrast to findings at pH 6.4 in an artificial environment. The same gastric juice spiked to 50 mumol/liter ascorbate did not result in 5CH3-H4PteGlu salvage from 5CH3-5,6 H2PteGlu.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7551826 TI - Cholesterol ester transfer and high-density lipoprotein conversion in normolipidemic, hypercholesterolemic, and hypertriglyceridemic non-insulin dependent diabetics. AB - Non-insulin-dependent diabetes (NIDD) is a situation at elevated risk for atherosclerosis. The plasma concentration of high-density lipoprotein (HDL) is often lowered. This may be accompanied by an abnormal composition and profile of HDL subfractions. These abnormalities might result in part from a defect in the net cholesterol ester transfer (CET) from HDL to apo B-containing lipoproteins. In the present work, we have studied the net CET and HDL conversion in normolipidemic, hypercholesterolemic, and hypertriglyceridemic NIDD, by comparison with control subjects. HDL conversion was determined by gradient gel electrophoresis after 23 h incubation in plasma with HDL3 labeled with a nontransferable synthetic marker. The net CET in normolipidemic NIDD was similar to that of controls, while it was approximately doubled in hypercholesterolemic or hypertriglyceridemic NIDD. In all groups, HDL conversion was comparable, with the exception of hypertriglyceridemic NIDD. In the latter group, the labeled HDL2/HDL3 ratio was increased, indicating a more complete conversion that was correlated with the triglyceride/cholesterol ester ratio in HDL. In addition, when lecithin:cholesterol acyl transferase was inhibited, a distinct peak of small HDL particles appeared in the density range of HDL2 in contrast with the other groups where only small HDL3 was formed. Recombination experiments showed that these abnormalities were attributable to the plasma in which labeled HDL3 was incubated rather than to the origin (control or hypertriglyceridemic NIDD) of labeled HDL3. These data suggest that in NIDD, hypertriglyceridemia may result in abnormalities of HDL conversion due to alterations in HDL composition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551827 TI - Sandwich enzyme immunoassay for endothelin with monoclonal antibodies and its application. AB - The development of a sensitive enzyme immunoassay for endothelin is described. This assay is based on a sandwich method using two different monoclonal antibodies against endothelin-1. A monoclonal antibody, which reacted to the C terminal region of endothelin, was selected as an immobilized antibody. The Fab' fragment of another monoclonal antibody, which might recognize the N-terminal rigid region of endothelin, was used as a horseradish peroxidase-labeled detector antibody. The assay measures endothelin-1 and endothelin-2 with a sensitivity of 1 fmol/ml. We have determined that cultured endothelial cells actually produced endothelin in significant amounts in a time-dependent manner. The levels of plasma endothelin extracted with Sep-Pak tC18 light cartridges could also be monitored. A basal endothelin level was about 0.3 fmol/ml of plasma, and a transient increase was observed 4 h after starting blood collection under in vivo experimentation in the rat. This enzyme immunoassay will facilitate the investigation of physiological roles of endothelin. PMID- 7551828 TI - Superoxide anion formation and glutathione metabolism of blood in patients with idiopathic pulmonary fibrosis. AB - The oxidant-antioxidant imbalance in the lower respiratory tract plays a major role in the pathogenesis in idiopathic pulmonary fibrosis (IPF). However, the systemic oxidant-antioxidant balance in the patients with IPF has not been extensively evaluated. In this study, the metabolism of glutathione (GSH) and superoxide anion production of whole blood were tested in 14 IPF patients and 12 normal subjects. While the total amount of GSH in the blood of IPF patients was not different from that of normal subjects (IPF; 22.9 +/- 4.9 micrograms/ml, control; 26.1 +/- 3.7 micrograms/ml), the amount of oxidized GSH (GSSG) and the ratio of GSSG to the total GSH in blood significantly increased in patients with IPF (IPF; 14.4 +/- 3.5%, control; 7.0 +/- 1.7%, P < 0.01), indicating that the glutathione redox cycle may be impaired in IPF. The production and generation of superoxide anions by blood were significantly greater in IPF than in normal subjects. The level of superoxide anion production was correlated with the GSSG/GSH ratio. These results indicate that IPF patients exhibit an impaired GSH metabolism with an increased oxidant formation of blood. PMID- 7551829 TI - Photoaffinity labeling of lysosomal membrane proteins with [125I]diiodotyrosine, a system h ligand. AB - Percoll-purified rat thyroid FRTL-5 cell lysosomes were photoaffinity-labeled with [125I]diiodotyrosine to identify proteins which bind diiodotyrosine, a ligand for lysosomal transport system h. SDS-PAGE and autoradiography of these membranes showed specific labeling of a 70-kDa protein and weak labeling of three smaller proteins. [125I]Diiodotyrosine photolabeling of the 70-kDa protein was specifically competed against by ligands of lysosomal transport system h ligands. The 70-kDa protein was photolabeled more strongly in lysosomal membranes isolated from thyrotropin-stimulated cells when compared with those grown in the absence of thyrotropin, consistent with previous demonstrations that thyrotropin stimulates system h transport. The 70-kDa protein may represent some portion of the system h carrier protein. PMID- 7551824 TI - Biliary excretion of copper, metallothionein, and glutathione into Long-Evans Cinnamon rats: a convincing animal model for Wilson disease. AB - Long-Evans Cinnamon (LEC) rats, characterized by a gross accumulation of hepatic Cu and the spontaneous onset of hepatitis, have been established to be an animal model for Wilson disease. They were used to estimate the relationships among copper (Cu), metallothionein (MT), and reduced glutathione (GSH) in biliary excretion in this study. Even though a huge amount of MT existed in the LEC rat liver (5016 micrograms/g liver) compared to that (63 micrograms/g liver) of controls (Fischer rats), the biliary excretion of MT (65 ng/ml bile) did not reflect the accumulated MT level in LEC rats. It seems likely that MT does not excrete intrinsically into the bile. Biliary excretion of Cu (0.17 microgram/ml) in LEC rats was significantly lower than that (0.57 microgram/ml) in Fischer rats. The difference in biliary excretion of GSH between the two groups was significant but slight. The reduced excretion of GSH into bile in LEC rats may be due to increased hepatic gamma-glutamyltransferase but not to hepatic GSH levels. There were no differences in biliary potassium and inorganic phosphorous between the two groups. On the other hand, excretion of lysosomal enzymes such as beta-N acetylglucosaminidase into bile was much lower in LEC rats (15.6 units/liter) than in controls (42.5 units/liter). The defective biliary excretion of Cu may be due to impaired lysosomal exocytosis, rather than canalicular membrane impairment. The LEC rat is very useful for research into the dynamics of metal excretion via the hepatobiliary system. PMID- 7551830 TI - An A-to-G mutation at the +3 position of intron 8 of the HEXA gene is associated with exon 8 skipping and Tay-Sachs disease. AB - Tay-Sachs disease (TSD) results from a deficiency of beta-hexosaminidase A (EC 3.2.1.52) activity. A child with late-infantile TSD was found to have two HEXA mutations, 986 + 3A-->G (A-->G at the +3 position of intron 8) and 533G-->A, associated with the variant B1 form of TSD. We were able to detect exon 8 deleted, but no correctly spliced HEXA mRNA, from the non-533G-->A allele in this patient. This suggests that 986 + 3A-->G results in missplicing and, together with 533G-->A, TSD. PMID- 7551831 TI - Effect of short-term treatment with pivalic acid containing antibiotics on serum carnitine concentration--a risk irrespective of age. AB - Treatment with pivalic acid containing prodrugs has been shown to cause carnitine depletion by loss of pivaloyl carnitine in urine. A 7-day standard pivmecillinam treatment of adults lead to a marked decrease of the free serum carnitine concentration (44.6 to 12.9 mumol/liter), whereas no change was seen in those given norfloxacine (40.0 to 40.5 mumol/liter). In some patients irrespective of age the free serum carnitine concentration was decreased to levels (around 10 mumol/liter) at which an impaired ketone-body production may occur. Therefore, there is reason for cautious use of this type of drug irrespective of the age of the patients. PMID- 7551833 TI - The TNM classification of prostate cancer: a discussion of the 1992 classification. The British Association of Urological Surgeons TNM Subcommittee. PMID- 7551832 TI - Effect of glucose and galactose loading in normal subjects on red and white blood cell uridine diphosphate sugars. AB - In order to test the assumption that red blood cell (RBC) uridine diphosphate galactose content is regulated in part by dietary galactose and to help comprehend factors influencing RBC and white blood cell (WBC) uridine diphosphate hexose concentrations in vivo, oral loading studies were performed with 50 g of galactose or 75 g of glucose in normal adults. While elevations of blood glucose did not influence RBC or WBC UDPhexose levels, increased blood galactose concentrations caused transient increases in both RBC and WBC UDPgalactose. In both RBC and WBC, the UDPgalactose-4-epimerase was rate limiting. In comparison to RBC, WBC had larger changes in UDPgalactose levels, synthesized additional UDPglucose through the action of UDPglucose pyrophosphorylase and began to restore the equilibrium ratio between UDPglucose and UDPgalactose concentrations while the plasma galactose level was still increasing. Thus, galactose ingestion alters the steady-state levels of UDPhexoses in circulating cells. The modulation of UDPglucose and UDPgalactose concentrations, occurring after the presentation of galactose to cells, as a result of the combined actions of GALT and UDPgalactose-4-epimerase, may be important in determining rates of synthesis of complex glycoconjugates. PMID- 7551834 TI - Interferon-alpha and survival in renal cell cancer. AB - OBJECTIVE: To establish whether the use of interferon-alpha might result in improved survival, using two large series of patients with advanced renal cell cancer treated during studies of chemotherapy and biological therapy, respectively. PATIENTS AND METHODS: Patients treated either in the Eastern Cooperative Oncology Group (ECOG) chemotherapy protocols (327 patients) or in protocols employing interferon as part of a European randomized study or phase II studies at the Norwegian Radium Hospital (231 patients) were retrospectively analysed. Groups for comparison were matched by exclusion of those with an ECOG performance status > 2, no prior nephrectomy, brain metastases or prior chemotherapy. Univariate analysis of prognostic factors for survival was performed by the log rank method and multivariate analysis by Cox regression. RESULTS: Univariate analysis of the whole population showed that performance status, time from diagnosis to treatment, sites of metastases and the use of interferon carried the greatest prognostic significance. In multivariate analysis, the use of interferon remained a significant predictor of survival (P < 0.001). Subgroup analysis suggested that the impact of interferon treatment was greatest in those patients with two of the following characteristics: good performance status, an interval of > 2 years from diagnosis to treatment and no more than one site of metastasis. CONCLUSION: Although a prospective randomized trial is needed to establish definite benefit from the use of interferon in advanced renal cell cancer, this analysis supports the rationale for performing such a trial, particularly in patients with relatively good prognostic features. Patients should be entered into the Medical Research Council study comparing interferon with medroxyprogesterone acetate. PMID- 7551835 TI - Effects of denervation on muscarinic receptors in the rat bladder. AB - OBJECTIVE: To demonstrate the specific distribution of muscarinic receptors in the rat urinary bladder and to investigate the effects of afferent and efferent denervation on the density and distribution of muscarinic receptors. MATERIALS AND METHODS: Urinary bladders were obtained from female rats which had been injected with vehicle (control), or neonatally with capsaicin (NC, afferent denervation) or which had their pelvic plexus removed (post-ganglionic denervation, PGD, efferent denervation). Tissue sections were used in radioligand binding studies and for autoradiography with the muscarinic receptor ligand l quinuclidinyl[phenyl-4-3H]benzilate (QNB). RESULTS: Binding of QNB was saturable and specific to a single population of binding sites, with a mean dissociation constant (Kd) of 1.05 +/- 0.14 nM in controls and 0.90 +/- 0.13 nM in rats with PGD. Post-ganglionic denervation caused a 37% increase in maximal binding (Bmax) of QNB from 437.1 +/- 39.1 fmol/mg protein (control group) to 599.1 +/- 4.5 fmol/mg protein (P < 0.02). Autoradiograms revealed muscarinic binding sites over the smooth muscle, but none over the epithelium. Smooth muscle binding sites were doubled after PGD but were unchanged after NC treatment. CONCLUSION: Muscarinic receptors were localized over the smooth muscle of the rat bladder and were increased after post-ganglionic denervation. This increase may be responsible for the increased sensitivity to muscarinic agonists reported to occur after bladder denervation. PMID- 7551836 TI - Characterization and autoradiographic localization of [3H] alpha, beta-methylene adenosine 5'-triphosphate binding sites in human urinary bladder. AB - OBJECTIVES: To characterize [3H] alpha, beta-methylene adenosine 5'-triphosphate ([3H] alpha, beta-MeATP, a radioligand for P2x-purinoceptors) binding sites in the washed homogenates and membrane preparations of human urinary bladder and, using autoradiography, to localize [3H] alpha, beta-MeATP binding sites in human bladder. MATERIALS AND METHODS: Specimens were obtained from the fundus of the urinary bladder of male patients aged 56-79 years. The washed homogenates or membrane preparations of the bladder specimens were incubated with [3H] alpha, beta-MeATP and the bound and free radioligand separated by filtration. For autoradiography, cryostat sections were incubated with 10 nM [3H] alpha, beta MeATP, washed, dried and exposed for 2 weeks to emulsion-coated coverslips. In both experiments, 100 microM beta, gamma-methylene ATP was used to determine non specific binding. RESULTS: Six of 16 specimens in the binding assay and three of seven specimens in the localization study showed specific [3H] alpha, beta-MeATP binding. The binding process was saturable and the specific binding sites were composed of a high- and low-affinity component. The specific binding to membrane preparations was reduced in the presence of Mg2+ in the incubation medium. Competitive displacement experiments showed that the order of potency of the unlabelled ligands to displace the [3H] alpha, beta-MeATP binding was alpha, beta methylene ATP > beta, gamma-methylene ATP > suramin > 2-methylthio ATP > ATP > ADP >> adenosine, which indicates that the binding sites are, or are linked to, P2x-purinoceptors. Autoradiography showed that the specific [3H] alpha, beta MeATP binding sites were located only over the smooth muscle of the bladder. CONCLUSIONS: The results suggest that P2x-purinoceptors exist in human urinary bladder, although at a lower density than reported for rodent urinary bladder. PMID- 7551837 TI - The temporal relationship between the severity of hydroureter and the dynamic changes of obstructed ureters in a rat model. AB - OBJECTIVE: To investigate dynamic changes in the obstructed rat ureter and to evaluate the usefulness of the rat model in the study of obstructive uropathy. MATERIALS AND METHODS: Unilateral ligation of the ureter was performed in 26 rats. After ureteric ligation, four to seven rats were killed and examined after 1, 3, 7, 10, and 14 days. The severity of hydroureter and hydronephrosis was assessed by pyelography and the hydroureter-hydronephrosis index. The middle portion of the ureter was prepared for light and electron microscopic observation. Five rats which received a sham operation were also examined as controls. RESULTS: The severity of hydroureter and hydronephrosis in the ligated ureters progressively increased during the period of obstruction (P < 0.005) and reached a maximum 10-14 days after ligation. Hypertrophy of the smooth muscle layer developed on the third day after ligation and proliferation of submucosal connective tissue occurred on the 10th day after ligation. The changes in smooth muscle and connective tissue layers also progressed consistently during ureteric obstruction (P < 0.001 and < 0.005, respectively). The number and size of granular endoplasmic reticulum and Golgi apparatus in the smooth muscle cells increased after ureteric ligation and then caveolae exocytosis developed. Deposition of collagen fibres between muscle cells became apparent later. CONCLUSION: The rat model of hydrouropathy demonstrated a clear temporal relationship between the severity of hydroureter and the dynamic changes in the obstructed ureter and could be used as a conventional method for the study of obstructive ureter. PMID- 7551839 TI - Exenteration as palliation for patients with advanced pelvic malignancy. AB - OBJECTIVE: To examine the place of pelvic exenteration in the palliation of advanced and recurrent pelvic tumours. PATIENTS AND METHODS: The notes of patients referred for palliative exenteration were reviewed retrospectively. Fourteen patients (three men, mean age 52 years, and 11 women, mean age 61 years) with a variety of pelvic tumours associated with severe symptoms, had a laparotomy with a view to pelvic exenteration and 10 underwent total or anterior exenteration. RESULTS: Eight patients achieved excellent or good palliation based on survival and quality of life assessment, and two received no benefit. Four of eight patients were alive and apparently free of tumour at a mean of 17 months after surgery. Four were alive, symptom-free but with evidence of malignancy at a mean of 19 months. Two patients who received no benefit died at 2 and 7 months after surgery. The planned exenteration was abandoned in four patients; three of these four patients were dead at a mean of 13 months and one was alive at 12 months. CONCLUSION: Our results confirm that with careful selection and appropriate multi-specialty care, aggressive pelvic surgery is of value in the palliation of some tumours. PMID- 7551838 TI - Urinary basic fibroblast growth factor in patients with bladder cancer and benign prostatic hypertrophy. AB - OBJECTIVE: To investigate the role of urinary measurements of an angiogenic factor, basic fibroblast growth factor (bFGF), in the assessment of patients with bladder cancer. PATIENTS AND METHODS: Urine from 83 patients was assayed using a commercially available ELISA for bFGF. Thirty-eight patients had a bladder tumour and 21 had a history of bladder cancer but no disease at the time of testing. Twenty-four patients acted as controls, 16 of whom were about to undergo transurethral resection of the prostate (TURP) for benign prostatic hypertrophy (BPH) and eight who had no urological disease. RESULTS: Median urinary bFGF was higher in patients with active bladder cancer than in those with a clear cystoscopy (5.20 and 2.13 ng/g creatinine, respectively; P < 0.005). Median urinary bFGF was also elevated in patients about to undergo TURP (4.52 ng/g creatinine). Using a threshold value of 6.0 ng/g creatinine, the sensitivity of the test for detecting cancer was 42% and specificity was 88%. At a threshold value of 4.0 ng/g the sensitivity was 62% and the specificity 70%. CONCLUSION: The relationship between urinary basic FGF and the presence of bladder cancer was significant. The test is not sufficiently sensitive or specific to use as a screening test for bladder cancer but may be very useful in monitoring the effectiveness of systemic therapies in bladder cancer. Elevated levels of bFGF in the urine of patients about to undergo TURP suggests a role for bFGF in the pathogenesis of BPH. PMID- 7551841 TI - Tamsulosin, a selective alpha 1c-adrenoceptor antagonist: a randomized, controlled trial in patients with benign prostatic 'obstruction' (symptomatic BPH). The European Tamsulosin Study Group. AB - OBJECTIVE: To evaluate the efficacy and safety of tamsulosin 0.4 mg once daily (as a modified-release formulation) compared with placebo in patients with benign prostatic enlargement, lower urinary tract symptoms and prostatic 'obstruction' (symptomatic benign prostatic hyperplasia [BPH]). PATIENTS AND METHODS: Of 313 patients with symptomatic BPH enrolled in a 2-week placebo run-in period, 296 were subsequently randomized to receive either placebo (98 patients) or tamsulosin 0.4 mg once daily (198 patients) for 12 weeks. The primary variables assessed to determine efficacy were maximum urinary flow rate (Qmax) from free flow measurements and the total Boyarsky symptom score. RESULTS: Tamsulosin produced greater improvements in Qmax (1.4 mL/s, 13.1%) than did placebo (0.4 mL/s, 3.8%) (P = 0.028) and a greater decrease in total symptom score (3.4 points, 35.8% reduction) than did placebo (2.2 points, 23.7% reduction) (P = 0.002). Significantly more tamsulosin-treated patients (67%) than placebo-treated patients (44%) had a > or = 25% decrease in total symptom score after 12 weeks (P < 0.001). Treatment with tamsulosin for 12 weeks also produced significant improvements in average urinary flow rate (P = 0.040), irritative (P = 0.013) and obstructive (P = 0.014) symptom scores and symptoms of nocturia (P = 0.022) and hesitancy (P = 0.004). Tamsulosin was tolerated well by the patients. The incidence of adverse events emerging during treatment was comparable in the tamsulosin- and placebo-treated groups (34% and 24% respectively, P = 0.109), as was the incidence of cardiovascular-related adverse events (5% and 7% respectively; P = 0.596). There were no significant differences in changes in blood pressure or pulse rates between the tamsulosin- and placebo-treated groups. CONCLUSION: Tamsulosin 0.4 mg once daily is safe, well tolerated and clinically effective in improving symptoms and urinary flow rate in patients with symptomatic BPH. PMID- 7551840 TI - Long-term results with M-VAC for advanced urothelial cancer: high relapse rate and low survival in patients with a complete response. AB - OBJECTIVE: To describe the long-term results of treating patients with advanced urothelial cancer using a combination of methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC). PATIENTS AND METHODS: Fifty-one patients (37 men and 14 women, median age 66 years, range 41-82) with inoperable or metastatic carcinoma of the bladder, ureter or renal pelvis were treated with M-VAC and their responses evaluated for up to 7 years. Relative dose intensity (RDI) was calculated by dividing the actual dose intensity by the projected total dose intensity and related to outcome. Overall survival was assessed from the date of initiation of treatment with M-VAC, using the Kaplan-Meier method. RESULTS: Of the 51 patients. 10 (20%) had a complete and 18 (35%) had a partial response, giving an overall response rate of 55% (95% CI, 41-68%). There was no significant difference in the median values of RDIs with response to the M-VAC regimen. The median duration of response was 11.9 months for the 10 patients with a complete response and of these, eight relapsed at a median of 10 months after treatment and died at a median of 8.5 months from the time of disease recurrence. Survival of patients with a complete response differed significantly from those with no response at 1 year after the start of treatment, but not subsequently. CONCLUSION: Long-term follow-up revealed a high relapse rate and poor prognosis in patients with a complete response who received the M-VAC as induction therapy. Therefore, new adjunctive therapies are needed for patients with locally unresectable or metastatic urothelial cancer. PMID- 7551842 TI - Experience with the Memotherm permanently implanted prostatic stent. AB - OBJECTIVE: To evaluate the Memotherm (Angiomed) permanently implantable prostatic stent. PATIENTS AND METHODS: Forty-eight men (mean age 70.3 years, range 57-86) with symptoms and urodynamics suggestive of bladder outflow obstruction had a Memotherm stent inserted into the prostatic urethra. Subjective and objective follow-up was performed at 1, 3 and 6 months. RESULTS: All but one patient was eventually able to void. Ten stents have been removed and three patients are awaiting stent removal. Subjective benefits bore no correlation with the maximum urinary flow rate and the findings on cystoscopy. Only 15 of the 48 patients had a satisfactory result. CONCLUSION: Permanently implanted prostatic stents should be considered experimental and only marketed after intensive investigation. As a result of this study, the Memotherm stent is to undergo significant modifications. PMID- 7551843 TI - Effect of age, comorbidity and type of surgery on perioperative complications and mortality of prostatectomy. AB - OBJECTIVE: To study the effect of age, type of surgery and comorbidities (cardiovascular diseases, chronic lung diseases, diabetes mellitus and renal impairment) on the peri-operative complications and mortality of prostatectomy. PATIENTS AND METHODS: A series of 236 consecutive patients who underwent prostatectomy (171 by transurethral resection, TUR, and 65 by open procedure) were reviewed. Ninety patients at risk with one or more of the comorbidities were grouped together and compared with the remaining 146 fit patients. Also, 148 patients aged < or = 70 years were compared with 88 patients aged > 70 years. The peri-operative morbidity variables compared were haemorrhage, > or = 3 units of blood transfusion, bacteriuria, orchitis, pyrexia, uraemia and bed-stay. RESULTS: Mean post-operative bed-stay for patients at risk was significantly longer than that for fit patients (12 +/- 7 and 9.7 +/- 6 days, respectively). Mortality and the other morbidity variables, apart from post-operative bacteriuria which was more frequent in diabetics, were not significantly different between the groups. Similarly, comparing age groups revealed no significant differences. However, open surgery was associated with more haemorrhage, blood transfusion, post operative pyrexia and a longer bed-stay. CONCLUSION: The comorbidities studied and age did not significantly influence the complications and mortality of prostatectomy. However, comorbidities prolonged post-operative bed-stay. Open surgery was associated with more bleeding and pyrexia and a longer bed-stay than TUR. PMID- 7551844 TI - Derivation and application of upper limits for prostate specific antigen in men aged 50-74 years with no clinical evidence of prostatic carcinoma. AB - OBJECTIVE: To derive age-specific upper limits for prostate specific antigen (PSA) level in men 50-74 years of age with no clinical evidence of prostatic carcinoma, and to test the sensitivity and specificity for cancer detection of these upper limits. SUBJECTS AND METHODS: A total of 6166 men were recruited for a multicentre study of prostate cancer detection and underwent a serum PSA determination and digital rectal examination (DRE). Men considered to be clinically free of prostatic carcinoma were those with a normal DRE and a PSA level < or = 4.0 ng/mL, and men with an abnormality in either parameter who underwent ultrasonography-guided prostate biopsy that revealed no evidence of carcinoma. By these criteria, 5469 men had no evidence of prostatic carcinoma. Dividing the population into 5-year age increments, three statistical methods were assessed to derive upper limits for serum PSA level by age; the mean +2 SD, the 99th percentile, and a 97.5% prediction interval based on linear regression. RESULTS: Newly-derived upper limits calculated by each method in the 50-54 and the 70-74 age group were 3.9 ng/mL and 7.6 ng/mL (mean +2 SD), 5.2 ng/mL and 14.0 ng/mL (99th percentile), and 4.7 ng/mL and 8.2 ng/mL (97.5% prediction interval). The sensitivity of the newly-derived upper limits was tested using receiver operating characteristic curves derived from men with no suspicious findings on DRE and a serum PSA concentration > 4.0 ng/mL. Although the specificity of the test increased with increasing PSA upper limits, no upper limits derived from these three methods yielded adequate sensitivity to detect cancer; sensitivities by age range were from 53 to 94%, using mean +2 SD, from 25 to 50% with the 99th percentile, and from 47 to 64% with the 97.5% prediction interval. CONCLUSION: We do not recommend age-referenced adjustments in upper limits for serum PSA concentration, but recommend that an upper limit of 4.0 ng/mL be used in all men 50-74 years of age. PMID- 7551845 TI - Antimicrobial prophylaxis for transrectal prostatic biopsy: a prospective randomized trial of cefuroxime versus piperacillin/tazobactam. AB - OBJECTIVE: To compare the clinical and microbiological outcome of a single-dose administration of cefuroxime or combined piperacillin/tazobactam (PT) after transrectal prostatic core-biopsy (TPB) in a prospective, randomized, open-label study. PATIENTS AND METHODS: Of 111 eligible men consecutively undergoing ultrasonographically guided TPB, 56 received 1.5 g cefuroxime and 55 received 4.5 g PT intravenously 20 min before the procedure. The anterior rectal wall was cleaned with an antiseptic swab and four biopsies were taken. Urine and blood cultures were evaluated before the procedure and again after 48 h, and oral temperature and symptoms recorded for 72 h after TPB. RESULTS: A clinically successful outcome (no symptoms to indicate urinary or systemic sepsis or pyrexia > or = 37.5 degrees C after TPB) was achieved in 100 of 108 men (92.6%), of whom 51 received cefuroxime and 49 PT. Microbiological success (no bacteriuria > 10(5) organisms/mL, or bacteraemia, after TPB) was observed in 98 of 103 men (95%), of whom 48 received PT and 50 cefuroxime. Bacteriuria was present 48 h after TPB in 5% of the men. One man randomized to receive cefuroxime became bacteraemic (with Escherichia coli) and required hospital admission. Four of the five microbiological failures were caused by organisms sensitive to the administered antimicrobial agent. No anaerobic organisms were cultured. The most common adverse event recorded was transient diarrhoea, reported by 16 men receiving PT and two receiving cefuroxime. CONCLUSION: With this prophylactic regimen, there was no significant difference in outcome between the groups. Most of the organisms isolated were sensitive to the administered antimicrobial agent. Thus, further reductions in sepsis after TPB may be achieved by the administration of additional oral antimicrobial prophylaxis. PMID- 7551846 TI - The significance of prostatic intra-epithelial neoplasia. AB - OBJECTIVE: To investigate the relationship between the detection of prostatic intra-epithelial neoplasia (PIN) on initial prostate biopsy and subsequent invasive prostatic adenocarcinoma. PATIENTS AND METHODS: Thirty-six men (mean age 67 years, range 52-82) with PIN underwent digital rectal examination (DRE), serum prostate-specific antigen (PSA) measurement and transrectal ultrasonography (TRUS) before the initial biopsy and documentation of PIN. They were followed up with serial PSA, TRUS and a repeat biopsy every 6 months until either invasive carcinoma was identified or 2 years had elapsed. RESULTS: The initial biopsy revealed Grade I PIN in 33%, Grade II in 22%, and Grade III in 45% of the men. The repeat biopsy showed evidence of invasive carcinoma in 21 patients (58%; Group I), while 15 (42%) showed persistence of PIN (Group II). In Group I, 19 had had high-grade PIN (Grade II/III) on initial biopsy compared with one in Group II. The findings on DRE, and age, were no significantly different between groups. TRUS revealed a hypoechoic lesion in 15/21 patients in Group I compared with 7/15 patients in Group II. There was an increase in PSA level in 18 patients in Group I (from 8.4 to 11.6 ng/mL). CONCLUSIONS: PIN and invasive adenocarcinoma of the prostate were closely associated, and the likelihood for coexistence was higher in patients with high-grade PIN, increasing PSA level or positive findings on TRUS. We recommend that all patients who show high-grade PIN on prostate biopsy be followed very closely with serial PSA measurements and repeat biopsies from both the area of PIN and other areas of the prostate. PMID- 7551847 TI - Pain relief and quality-of-life assessment following intravenous and oral clodronate in hormone-escaped metastatic prostate cancer. AB - OBJECTIVE: To establish the efficacy of intravenous clodronate followed by maintenance oral clodronate in patients with painful bone metastases resulting from hormone-resistant prostate cancer. PATIENTS AND METHODS: A multicentre open study of 27 patients assessed the efficacy of clodronate treatment by estimating the reduction in World Health Organization (WHO) Pain Score, the increase in WHO Performance Status and by a novel quality-of-life/activity score. RESULTS: Ten of 27 patients achieved significant pain relief after receiving 300 mg/day of intravenous clodronate for 10 days. This was matched by an improvement in the activity score and WHO Performance Status. Three of 27 patients continued to have relief from pain after 3 months of oral clodronate therapy. CONCLUSION: Intravenous clodronate therapy was effective in relieving the pain resulting from prostate cancer bone metastases in 10 of 27 patients but the benefit was shortlived. The use of a personal quality-of-life/activity questionnaire which assesses aspects of everyday life that are important to the patient may be more appropriate for patients with very advanced prostate cancer than are other quality-of-life questionnaires in current use. PMID- 7551848 TI - Immunological infertility among Nigerian men: incidence of circulating antisperm auto-antibodies and some clinical observations: a preliminary report. AB - OBJECTIVES: To (i) establish the incidence of circulating antisperm auto antibodies among infertile men; (ii) relate this incidence to the high prevalence of sexually-transmitted diseases (STDs) in sub-Saharan Africa and; (iii) elucidate the effect of steroid and other therapy on semen quality and subsequent fertility of the patients. PATIENTS AND METHODS: Serum samples from 50 infertile men and 50 age-matched controls were assayed by two agglutination techniques for anti-sperm antibodies. Mean sperm concentrations were determined before and after steroid treatment of patients having antibody titres of 1:64 or above. Serum levels of follicle-stimulating hormone, luteinizing hormone, testosterone and prolactin were also determined by radioimmunoassay in 38 patients. Seminal fluid analysis and culture were performed in 35 patients and testicular histology determined in 21. RESULTS: Agglutination was demonstrated in 22 of 50 sera (44%), whilst non-agglutinating cytotoxic antibodies were detected in two. Only two of the 50 control sera (4%) were positive. After steroid therapy, antibody titres were significantly decreased and there was a sixfold improvement in mean sperm concentration and a threefold improvement in motility and morphological characteristics. Bacterial (46%) and non-bacterial (17%) infection were recorded in 22 of 35 patients, 13 of whom showed the presence of antisperm antibodies in their sera. Staphylococcus aureus was the commonest single bacterial isolate. Overall, 13 of 29 patients (45%) improved, nine accounting for 12 pregnancies. Pregnancies and/or improvements in semen quality were observed only among patients with mild histological changes. Low testosterone and prolactinaemia occurred in 29% and 21% of the patients, respectively. Among these, anti-sperm antibodies were also recorded in 18% and 13%, respectively. CONCLUSION: The incidence of antisperm antibodies among infertile men is high in Nigeria and may be related to high prevalence of STDs. Immunologically infertile men can be treated successfully with steroids. Concomitant antibiotic and hormone therapy may also be essential in appropriate cases. Clinicians are advised to adopt a multimodal approach to the treatment of male infertility in sub-Saharan Africa. The presence of non-agglutinating cytotoxic antibodies calls for further investigation of the role of complement in the pathogenesis of immunological infertility. PMID- 7551849 TI - Temporary reappearance of sperm 12 months after vasectomy clearance. AB - OBJECTIVES: To determine the incidence of positive semen analysis 12 months after vasectomy clearance. SUBJECTS AND METHODS: A prospective study was undertaken, starting in 1990, of men undergoing vasectomy. Azoospermia was confirmed by two successive semen analyses 16 weeks after vasectomy. One year later a further sample was analysed for the presence of sperm. RESULTS: Of 1000 men who provided a sample for analysis, six men (0.6%) have had positive semen analyses 1 year after the initial tests showed azoospermia. In all six the sperm count was <10,000 per mL. Five of the six men produced a repeat sample 1 month later which, in all five cases, showed azoospermia. No pregnancies have been reported to date. CONCLUSION: Transitory reappearance of sperm following successful vasectomy occurs in about 0.6% of men. This incidence is 18 times greater than the reported pregnancy rate following successful vasectomy. PMID- 7551850 TI - Vasectomy by section, luminal fulguration and fascial interposition: results from 6248 cases. AB - OBJECTIVE: To determine the incidence of complications, including recanalization, in a series of 6248 consecutive vasectomies performed with a section-fulguration fascial interposition technique. PATIENTS AND METHODS: Over a 38-year period, 6248 vasectomies were performed by one surgeon (S.S.S.) as a clinic procedure under local anaesthesia with no resection of a vasal segment. The mucosa of the cut ends of the vas was destroyed by cauterization and the fascial sheath of the vas was interposed as a barrier. Semen specimens were examined until two specimens, one month apart, showed no sperm. RESULTS: Complications were minimal, with few cases of haematoma or wound infection. Spermatic granulomas were uncommon. No post-vasectomy pregnancies were reported and no patient showed a persistence of sperm. CONCLUSION: The section-fulguration-fascial interposition technique of vasectomy was uniformly effective, with few post-operative problems. PMID- 7551851 TI - Persistent spermatozoa after vasectomy: a survey of British urologists. AB - OBJECTIVES: To determine the rate of, and main indications for, repeat vasectomy in our department, and to assist in policy-making procedures by determining how urologists in England and Wales manage those men who show small but persistent quantities of motile or non-motile spermatozoa in their ejaculate after vasectomy. SUBJECTS AND METHODS: A retrospective review of all of the vasectomies and repeat vasectomies performed by the Urology Department at Southmead Hospital during a 14-month period was undertaken to determine the rate of and indications for repeat vasectomy. Subsequently, every consultant urologist in England and Wales was canvassed with a questionnaire to determine whether they repeated vasectomy in the presence of persistent motile or non-motile sperms and if so, after what time interval. Any experience of pregnancies arising from these groups was also assessed, and any relevant comments invited. RESULTS: The local review revealed that 5% of all vasectomies were repeated within 6-36 months. Of these, 87% were performed because of persistent sperms in post-vasectomy semen samples, the majority of which showed sperm concentrations of one in 50 to one in 100 high power fields. A response of 56% was obtained to the questionnaire and of those responding, 23% never repeated a vasectomy where there were presistent non-motile sperms, but almost all urologists would eventually repeat vasectomy where motile sperms were present. The median interval between the first and second vasectomies was 6 months and 12 months for motile and non-motile sperm, respectively. Apart from those cases already published, there was little experience of pregnancy arising from men with persistently few motile or non-motile sperms. CONCLUSIONS: The risk of pregnancy occurring in the presence of non-motile sperms was estimated to be less than the established risk of late recanalization, and this survey provides both logical and medico-legal support for issuing a 'special clearance' to men with few persistent non-motile sperm after vasectomy, providing the risks of pregnancy are properly discussed and documented. For motile sperm, however, there appears to be a stronger precedent for repeating the vasectomy. The technique used for post-vasectomy semen analysis was also an important consideration when determining any policy regarding such cases. PMID- 7551852 TI - The first case of epispadias: an unknown disease of the Byzantine Emperor Heraclius (610-641 AD). PMID- 7551853 TI - Decreased immunoreactive androgen receptor levels are not the cause of isolated hypospadias. AB - OBJECTIVE: To elucidate whether diminished levels of androgen receptor (AR) are the underlying cause for the development of hypospadias by determining AR levels in the foreskins of boys with hypospadias. PATIENTS AND METHODS: The content of AR was determined by a sensitive immunoblotting technique in extracts from preputial tissue obtained from 15 patients (mean age 2.5, SD +/- 1.5 years) with hypospadias and from seven controls (mean age 2.5, SD +/- 1.5 years). Immunoreactivity of the protein was measured by densitometry. RESULTS: No significant difference in mean AR content was found between those boys with hypospadias (2.1 +/- 0.9 fmol/mg protein, +/- SD) and the age-matched control group (2.2 +/- 0.3 fmol/mg protein). Stratification by the severity of hypospadias (distal and proximal on the shaft, and penoscrotal hypospadias) showed that the severity was not related to tissue AR levels. CONCLUSION: It is unlikely that hypospadias is caused by a decreased expression of AR. PMID- 7551854 TI - Maternal serum human chorionic gonadotrophin during early pregnancy resulting in boys with hypospadias or cryptorchidism. AB - OBJECTIVE: To compare maternal serum human chorionic gonadotrophin (hCG) samples before 18 weeks gestation in mothers of boys with cryptorchidism or hypospadias with that in mothers of normal boys. The effect of season on maternal hCG was also assessed. SUBJECTS AND METHODS: Stored serum samples from mothers of singleton male fetuses born between January 1988 and December 1992 were assayed for total hCG, using a Delfia assay. There were 153 eligible samples from 96 mothers of normal boys, 31 of those with cryptorchidism, and 26 of those with hypospadias. RESULTS: The data from mothers of normal boys were used to construct a time-specific reference range for hCG. Plotting the hCG levels of the other groups and superimposing the reference range showed no significant difference between them (P = 0.09). Maternal hCG was not significantly different between cryptorchid boys requiring orchidopexy and those experiencing early spontaneous descent. An analysis of covariance allowing for gestational age showed that maternal hCG is significantly higher during the peak summer than peak winter months (P = 0.046). CONCLUSION: While a seasonal effect on maternal hCG was demonstrated our data does not suggest this to be the sole cause for cryptorchidism or hypospadias or to account for the observation that both have a seasonal frequency. PMID- 7551856 TI - Endoscopic management of impacted stones in the intramural or meatal part of the ureter without performing meatotomy. PMID- 7551855 TI - Intraprostatic vasopressin before TURP. PMID- 7551857 TI - The posterior sagittal approach for the excision of a prostatic utricle cyst. PMID- 7551858 TI - Parotid metastasis from renal cell carcinoma. PMID- 7551860 TI - Escherichia coli endophthalmitis: a rare presentation of renal parenchymal malakoplakia. PMID- 7551859 TI - High-turnover osteoporosis and diffuse idiopathic skeletal hyperostosis induced by retinoid therapy in a patient with superficial bladder cancer. PMID- 7551861 TI - Small-cell undifferentiated carcinoma of the renal pelvis 26 years after subdiaphragmatic irradiation for non-Hodgkin's lymphoma. PMID- 7551863 TI - Pseudo-renal failure after traumatic bladder rupture--the common features. PMID- 7551865 TI - Primary non-Hodgkin's lymphoma of the prostate mimicking acute prostatitis. PMID- 7551862 TI - Splenunculus masquerading as an adrenal mass. PMID- 7551866 TI - Diagnosis and treatment of renovascular hypertension in a patient with a functional solitary kidney. PMID- 7551864 TI - Ileoneobladder-enteric fistula: a rare early post-operative complication treated conservatively. PMID- 7551867 TI - Retraction method for implantation of penile prosthesis. PMID- 7551869 TI - Fragment of balloon-catheter as a retrograde renal foreign body. PMID- 7551870 TI - Pseudoureterocele: potential for misdiagnosis of an ectopic ureter as a ureterocele. PMID- 7551868 TI - An unusual double pigtail stent story. PMID- 7551871 TI - Development of a life quality questionnaire in bladder cancer surgery. PMID- 7551873 TI - Prevention of recurrent calcium stones: a rational approach. PMID- 7551874 TI - Is nephrocalcin related to the urinary derivative (bikunin) of inter-alpha trypsin inhibitor? AB - OBJECTIVE: To isolate, purify, sequence and characterize nephrocalcin (NC), a urinary protein that may be an important determinant of calcium oxalate (CaOx) kidney-stone disease. MATERIALS AND METHODS: Proteins were isolated from human urine using cellulose and resin columns and were sequenced using Edman degradation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of CaOx crystal growth by the isolated proteins was assessed by measuring the deposition of 14C-labelled CaOx. RESULTS: A protein assumed to be NC on the basis of SDS-PAGE, inhibitory and gel filtration properties was isolated from healthy human urine. Its molecular weight and the amino acid sequences of two of its peptides suggested it was identical to fragment HI-14 of the light chain (bikunin) of inter-alpha-trypsin inhibitor (ITI). CONCLUSIONS: NC represents a portion of the light chain of ITI, although this conclusion must remain tentative until confirmed using authentic NC. PMID- 7551875 TI - The effect of extracorporeal piezoelectric lithotripsy on the contractility of the human pelvicalyceal system. AB - OBJECTIVE: To assess the effect of extracorporeal piezoelectric lithotripsy (EPL) on the contractility of the human pelvicalyceal system. PATIENTS AND METHODS: Contractions of the pelvicalyceal system were measured in 12 patients (mean age 55 years) before and after EPL. Pelvicalyceal pressure was measured via a Cope nephrostomy tube which had remained in situ following stone debulking procedures days or weeks earlier. All patients were treated using a Wolf Piezolith 2300 lithotripter. RESULTS: The pelvicalyceal systems of two patients were acontractile before and after treatment. Immediately after treatment, contractions were completely abolished in eight of the remaining 10 systems, and reduced in frequency in the other two. All 10 systems regained contractions 24 h after treatment. CONCLUSION: Piezoelectric lithotripsy temporarily abolishes upper urinary tract motility. PMID- 7551872 TI - Management of superficial bladder cancer. PMID- 7551876 TI - In situ piezoelectric extracorporeal shock wave lithotripsy of ureteric stones. AB - OBJECTIVE: To evaluate the efficacy of the EDAP LT 02 lithotripter for the in situ treatment of ureteric calculi. PATIENTS AND METHODS: One hundred consecutive patients presenting with ureteric calculi were treated with in situ piezoelectric extracorporeal shock wave lithotripsy (ESWL) using the EDAP LT 02 lithotripter. There were 49 patients with upper, nine with mid and 42 with lower ureteric stones. The largest diameter of the stones varied from 7 to 21 mm (mean 9.6 mm). Mild or severe hydronephrosis was present in 53 cases. Mid and lower ureteric stones were treated with the patients in the prone position, with no anaesthesia or pre-medication, and upper ureteric stones in the supine position, with intravenous sedation in 44 cases. RESULTS: Localization of the stones was easy in 81 cases and more difficult in 19, but an intravenous pyelogram was only necessary in three cases. The number of sessions per patient varied from 1 to 3 (mean 1.17). Complete success rate was achieved in 75% of patients and partial success (residual stones < or = 3 mm) in 6%. The stone-free rate was statistically affected by stone size but was independent of stone localization or the degree of obstruction. The rate of infective and obstructive complications was 14% and auxiliary treatments were necessary in 5% of patients. CONCLUSION: In situ piezoelectric ESWL with the EDAP LT 02 device is a convenient and efficient method for the treatment of ureteric stones. PMID- 7551877 TI - Urological community nursing: a new concept in the delivery of urological care. AB - OBJECTIVE: To investigate the impact of a urological community nursing service on the mode of practice, efficiency, and quality of care in a urological practice. METHODS: A urological community nurse was appointed to investigate the possibility of performing various urological procedures in the community rather than in hospital, with a prospective audit of the results for a period of 1 year. The setting was a busy urological unit serving a mixed urban and rural catchment area of 300,000 people. The outcome was assessed by the number and type of procedures successfully transferred from hospital to community practice. RESULTS: In 1 year, 464 urological episodes were performed in the patients' homes which otherwise would have required transfer or admission to hospital for treatment or, in some cases, a cancelled operation. These episodes included tuition in clean intermittent self-catheterization and dilatation, changes and insertions of urethral, suprapubic and nephrostomy catheters, bladder instillations to treat cancer and interstitial cystitis, voiding trials with no catheter, urosheath fittings, and pre-operative visits and follow-up visits to avoid attendance at the clinic. CONCLUSIONS: The urological community nursing service transferred a significant number of routine urological procedures from hospital to community with considerable financial savings and improvement in the quality and efficiency of urological care. PMID- 7551878 TI - Cutting the cost of catheterization for acute retention--a hospital or domiciliary procedure? AB - OBJECTIVE: To report the views of General Practitioners (GPs) and patients on the most appropriate place and person to catheterize patients in acute urinary retention. PATIENTS AND METHODS: Two separate questionnaires were sent to 50 local GPs and 100 patients who had recently been referred with acute urinary retention. The questionnaires were designed to ascertain the most appropriate place and person to catheterize these patients. They also established the number of hospital admissions due to acute retention, the use of ambulances and the uptake of the district nurse services. RESULTS: Whilst 54% of GPs advocated domiciliary catheterization, only 20% of emergency catheterizations were carried out in the home. Nearly half the patients catheterized in the Accident and Emergency Department were admitted acutely and 41% of patients had travelled to hospital by ambulance. Patients were equally divided about where they would prefer to be catheterized, but 70% of patients stated that they were happy to be catheterized by a trained nurse; no GP opposed this concept. CONCLUSION: The cost/benefit analysis of these results strongly supports the case for establishing best-practice guidelines to promote an emergency domiciliary service led by GPs and trained district nurses and thus hasten the emergency care of acute retention and cut costs. PMID- 7551879 TI - Nottingham health profile measurement in the assessment of clinical outcome after prostatectomy. Northern Regional Prostate Audit Group. AB - OBJECTIVE: To compare the changes in pre- and post-operative symptom scores with changes in Nottingham health profile (NHP) scoring, and thus determine whether NHP scoring offers a reliable assessment of outcome after transurethral resection of the prostate (TURP), and whether NHP scoring could usefully supplement the more traditional method of symptom scoring in this assessment. PATIENTS AND METHODS: An 8-month audit of 1396 TURPs, involving 12 hospital sites, was performed in the Northern region between April 1 and November 31, 1991. A cohort of 371 of these patients, for whom pre- and post-operative NHPs and irritative and obstructive voiding symptoms had been recorded, were investigated. RESULTS: There was a significant decrease in both obstructive and irritative voiding symptom scores 3 months after TURP and a significant fall in the NHP scores for all stations except social isolation. There were significantly higher irritative symptom scores both before and after operation in men with prostate cancer compared with those with benign prostatic hypertrophy, and significantly higher post-operative obstructive symptom scores in men with prostate cancer. However, there were no significant differences in the pre- and post-operative NHP scores for these two subgroups of patients. In patients who had a good outcome on symptom scoring, there was a significant reduction in NHP scores for all stations except social isolation. However, in those patients who had a poor outcome on symptom score, there was no correlation with changes in the NHP scores, with some NHP stations showing a significant reduction after operation. CONCLUSION: NHP quality-of-life scoring cannot replace symptom scoring in the assessment of outcome after TURP and when used alone, added little further information. However, for a comprehensive assessment of outcome, symptom scores and quality-of life assessments should be used together. PMID- 7551881 TI - An open, in-patient incremental safety and efficacy study of desmopressin in women with multiple sclerosis and nocturia. AB - OBJECTIVES: To examine the safety and efficacy of desmopressin in three doses given to women with multiple sclerosis to treat nocturia with or without enuresis. PATIENTS AND METHODS: Eight women with clinically confirmed multiple sclerosis and nocturia with or without enuresis were entered as in-patients into an open, nonrandomized, placebo-controlled study of incremental doses of 20, 40 and 60 micrograms desmopressin. Urinary and serum sodium, plasma arginine vasopressin and urine osmolality were monitored every 4 h for 24 h. A single dose of placebo or desmopressin was given during each of four 24-h periods. RESULTS: There was a significant decrease in nocturnal urinary volumes and a significant increase in nocturnal urinary osmolalities in patients taking desmopressin when compared with those taking a placebo, but there was no difference among the desmopressin doses. There was no significant difference in serum sodium level between the desmopressin doses. However, at the end of the 24-h period with the 60 micrograms dose, serum sodium was decreased significantly. CONCLUSIONS: Neither a significant decrease in nocturnal urinary volumes nor an increase in urinary osmolality was achieved by doses of desmopressin > 20 micrograms. A dose of 60 g was associated with a decreased serum sodium level at the end of the 24-h period but there was no biochemical hyponatraemia. Because there were no benefits and a possibility of clinical hyponatraemia with higher doses, doses of > 20 micrograms desmopressin cannot be recommended. PMID- 7551882 TI - Treatment of stress incontinence by vaginal cones: short- and long-term results and predictive parameters. AB - OBJECTIVE: To evaluate prospectively the short- and long-term results of treatment with vaginal cones for women with urinary stress incontinence and to assess predictive parameters. PATIENTS AND METHODS: Fifty women (mean age 49 years, range 28-76) with differing severities of stress incontinence (mean parity 2.4 and mean 60-min pad-test 20.7 g) were treated with cones for 15 min twice a day for 8 weeks. Treatment results were evaluated immediately after and at a mean of 3 years after the treatment. Parameters were analysed to determine those with predictive value for a successful outcome. RESULTS: Physiotherapy was assessed as being successful, i.e. a complete cure or a reduction of > 50% of the original severity, in seven patients (14%), while in 43 patients (86%) the treatment failed. After 3 years, 13 (30%) reported that the treatment was successful, 27 (61%) reported failure and four (9%) underwent surgery for stress incontinence. Those with lesser amounts of urinary loss and those with less frequent incontinence would be most likely to benefit from treatment with vaginal cones. CONCLUSION: The treatment had a low success rate and we recommend that the vaginal cones should be used only for those with a slight or moderate degree of stress incontinence. PMID- 7551880 TI - Validation of the self-administered Danish Prostatic Symptom Score (DAN-PSS-1) system for use in benign prostatic hyperplasia. AB - OBJECTIVE: To validate the Danish Prostatic Symptom Score (DAN-PSS-1), a self administered quality-of-life questionnaire comprising 12 questions related to voiding problems and the perceived bother of each individual symptom. METHODS: Using published results from several comparisons of other symptom scoring systems with DAN-PSS-1, the test-retest reliability, internal consistency, construct and content validity, and responsiveness of the DAN-PSS-1 system were assessed. RESULTS: The system was internally consistent (Cronbach's alpha = 0.73), the median test-retest reliability of answers to each question was 83.5% (range 0 99.7%) and the questionnaire was well understood by the patients. The DAN-PSS-1 system demonstrated a high degree of construct validity, correlating with the extensively used Madsen-Iversen score system (Spearman's correlation coefficient, rs = 0.51) and with the patients' answers to questions about how bothersome their symptoms were (rs = 0.71). The DAN-PSS-1 system discriminated clearly between patients with benign prostatic hyperplasia (BPH) and control subjects (an area under the receiver operating characteristic curve of 0.94). Finally, the DAN-PSS 1 was sensitive to changes following intervention, with scores decreasing from a median of 20 to zero 4 months after patients underwent transurethral prostatectomy and from a median of 11.5 to 7.5 (65%) after patients had received 4 months treatment with an alpha-blocker. CONCLUSIONS: The DAN-PSS-1 system is reliable, valid and responsive, and therefore can be recommended for assessing the severity of symptoms among patients presenting with lower urinary tract complaints suggestive of BPH and in the follow-up after intervention. PMID- 7551883 TI - Long-needle bladder neck suspension for genuine stress incontinence--does endoscopy influence results? A structured overview. AB - OBJECTIVE: To assess the hypothesis that the use of an endoscope to aid suture placement in long-needle suspension procedures is more likely to be lead to improved results. MATERIALS AND METHODS: A literature survey of 15 journals yielded 77 evaluable publications which provided data to compare the subjective and objective outcomes following a Pereyra procedure (2259 and 301 patients, respectively) or a Stamey procedure (2029 and 456 patients, respectively). RESULTS: There was no statistically significant difference in the mean percentage continence rates, based on subjective assessment of continence, following either the Pereyra or the Stamey procedure. Approximately 7% of patients subjectively claimed to be continent yet were not so upon some form of objective measurement. CONCLUSION: The analysis detected no improvement in the likelihood of continence after long-needle suspension procedures whether or not they were performed under endoscopic control to aid suture placement. This conclusion applied to primary procedures whether outcome was judged subjectively or objectively, and to subjective outcome after previously failed surgery. This study suggests that those surgeons who report their results are sufficiently skilled in judging suture placement as accurately as is needed without the benefit of a cystoscope. PMID- 7551884 TI - Impact of tumour grade, stage, number and size, and smoking and sex, on survival in patients with transitional cell carcinoma of the bladder. AB - OBJECTIVE: To evaluate the impact of the grade, stage number and size of the tumour and of smoking and sex of patient on survival, and the relationship between smoking and tumour grade, stage, number and size, and sex of patient in patients with transitional cell carcinoma (TCC) of the urinary bladder. PATIENTS AND METHODS: The study comprised 252 consecutive patients with histologically verified TCC of the bladder reporting to Oulu University Central Hospital between 1978 and 1986. The average duration of follow-up was 6.7 years. The association between smoking and other covariates was analysed by cross-tabulation and standard chi-squared analyses. The Kaplan-Meier method was used to obtain estimated survival curves and significant differences determined using log rank statistics. RESULTS: Smoking had no impact on tumour grade, stage, size and number or aggressiveness of metastases. Prognosis was noticeably better if the patient did not smoke, as 27% of the non-smokers and 40% of the smokers had died during the first 10 years after diagnosis. Survival was also better in patients with low grade (I, II), non-invasive (Ta/T1) and small (< or = 3 cm) tumours. CONCLUSION: In addition to the well-known prognostic factors (histological grade and stage of the tumour), smoking is a risk factor for bladder cancer and also has an impact on prognosis, leading to higher mortality from the disease in the longer term. PMID- 7551885 TI - The role of urodynamic studies in the evaluation of patients with augmentation cystoplasties. AB - OBJECTIVE: To assess the role and value of videourodynamic studies (VUD) in the post-operative evaluation of 100 patients who were continent following augmentation (clam) enterocystoplasty. PATIENTS AND METHODS: Fifty patients had non-neuropathic detrusor instability and 50 had neuropathic bladder dysfunction. All underwent standard dual channel medium fill cystometry and synchronous video screening before and at 6 months and 2 years after a clam ileocystoplasty. RESULTS: Pre-operative capacity was variable in non-neuropathic patients but post operative capacity in this group, and both pre- and post-operative capacity in the neuropathic group, were more reproducible. Of non-neuropathic patients, 88% became stable and 83% became normally compliant, compared with only 38 and 50% respectively of neuropathic patients. More surprisingly, two of eight neuropathic patients previously stable became unstable and four of 12 of those previously normally compliant became poorly compliant. In all patients voiding spontaneously there was a tendency for capacity to increase and voiding efficiency to decrease with time. CONCLUSIONS: From these results, standard VUD studies contributed little to the post-operative assessment of patients with clam cystoplasties beyond that which could be obtained by ultrasonography of the bladder before and after voiding. The effect or lack of effect of augmentation cystoplasty on compliance in the neuropathic bladder suggests a neuropathic effect on bowel contractility and warrants further investigation. The meaning and significance of 'urodynamic' observations when bowel is incorporated into the bladder also needs careful investigation. PMID- 7551887 TI - Prostate-specific antigen levels in patients receiving long-term dialysis. AB - OBJECTIVE: To investigate the effect, if any, of renal failure upon prostate specific antigen (PSA) levels and the validity of PSA estimation as a marker of prostatic disease in renal failure. PATIENTS AND METHODS: PSA was measured in 65 men (median age 67 years, range 39-84) on regular haemodialysis and 37 men (median age 70 years, range 42-77) on continuous ambulatory peritoneal dialysis (CAPD). Patients with a PSA level > 4 ng/mL underwent prostatic biopsy guided by transrectal ultrasonography. RESULTS: There was no evidence of an artefactual elevation of PSA attributable solely to renal failure. All eight patients with a PSA level > 4 ng/mL had prostatic disease. CONCLUSION: PSA measurements in patients with end-stage renal failure treated by dialysis remain a useful marker of prostatic disease. PMID- 7551888 TI - Urethral anaesthesia with topical bupivacaine. A role for a longer-acting agent. AB - OBJECTIVE: To assess the efficacy and toxicity of bupivacaine as a topical urethral anaesthetic. PATIENTS AND METHODS: This prospective two-part study comprised a pilot study of 10 men (mean age 73 years, range 39-86), to determine the toxicology, pharmocokinetics and suitable preparation of bupivacaine gel, and a study of 40 men (mean age 76 years, range 59-92) to compare the efficacy of bupivacaine with lignocaine gel. All patients were undergoing treatment for benign prostatic hyperplasia by transurethral radiofrequency heating using the Direx Thermex II system. RESULTS: There were no major adverse events. Bupivacaine provided good topical anaesthesia with a mean duration of 141 min, compared with 29 min for lignocaine. Serum samples taken from patients showed that the drug was absorbed slowly, and with a dose of 50 mg there was a wide margin between serum drug concentrations and toxic levels. CONCLUSION: Bupivacaine is safe and effective as a topical anaesthetic agent in the urethra in circumstances where prolonged duration of action is desirable. For lower urinary tract procedures 20 22 mL of anaesthetic gel is required, giving 2-3 h of analgesia/anaesthesia with no significant toxicity or adverse effect. The application of longer-acting anaesthetic agents need not be only during surgical intervention, but might usefully be extended post-operatively to provide early management of pain. PMID- 7551889 TI - Aspiration sclerotherapy for hydroceles in the tropics. AB - OBJECTIVE: To assess the usefulness of aspiration sclerotherapy in the treatment of hydroceles in the tropics. PATIENTS AND METHODS: This prospective study included 82 patients with 94 primary vaginal hydroceles of which 62 hydroceles were treated by aspiration and tetracycline hydrochloride sclerotherapy as an outpatient procedure (mean age of patients 56.5 years, range 40-82) and 28 were treated surgically and included as controls (mean age 52.4 years, range 40-70). All patients were followed for a year. RESULTS: In the sclerotherapy group, the overall cure rate after a year was 95%. Few complications were encountered in this group: six patients (10%) experienced pain during the procedure, seven patients (12%) had local infection and three patients (5%) developed haematoma. No recurrence was encountered in the surgical group, seven patients (25%) had infection and two patients (7%) developed haematoma. The mean hospital stay for the surgical group was 3.4 +/- 1.3 days (range 1-7). Four patients with thick walled hydroceles had persistent swelling after sclerotherapy, the recurrence of which dissatisfied the patients. CONCLUSIONS: Aspiration sclerotherapy for thin walled hydroceles proved to be a curative, simple, safe and cost-effective out patient procedure. It can be an alternative to surgery in developing countries where resources are limited. PMID- 7551891 TI - The incidence of testicular cancer in Royal Air Force personnel. AB - OBJECTIVES: To review the incidence of testicular cancer within the Royal Air Force (RAF) compared with the national incidence. METHODS: The medical statistics for RAF personnel (supplied by the Central Statistics Office, categorized by occupation) were reviewed for the incidence of testicular cancer and compared to the national rate. RESULTS: A significantly elevated incidence of testicular neoplasm was detected in RAF personnel, which was well above the national rate. The increased incidence appeared most marked in those personnel working directly with aircraft. CONCLUSION: The only cause of the significantly higher incidence of testicular cancer in RAF personnel is probably related to working with aircraft. This concurs with a finding that aircraft technicians in the US Navy had an increased incidence of testicular cancer, and the exposure to hydrocarbon carcinogens in the workplace was implicated. PMID- 7551890 TI - Does delayed diagnosis or scrotal incision affect outcome for men with non seminomatous germ cell tumours? AB - OBJECTIVE: To ascertain whether delayed diagnosis or type of orchidectomy affected outcome for men with non-seminomatous germ cell tumours (NSGCT). PATIENTS AND METHODS: The case notes of 454 men resident in the west of Scotland with a diagnosis of NSGCT between 1975 and 1989 were retrospectively reviewed. The clinical record was available for 442 (97%) and included information on time to diagnosis in 92% and diagnostic surgery in 97%. The study end-point for orchidectomy was loco-regional recurrence and for diagnostic delay was survival. RESULTS: A significant minority of men (10.4%) underwent a scrotal orchidectomy or had a scrotal incision before an inguinal orchidectomy (9.4%). More scrotal incisions were performed on patients under the care of general surgeons (28%) than of urologists (12.1%). Of the men who had scrotal surgery, one of 78 (1.3%; 95% CI, 0-4%) developed loco-regional disease as the initial site of recurrence, compared with none of 318 men undergoing inguinal orchidectomy. The median time to diagnosis was 3 months. There was no relationship between time to diagnosis and tumour extent at presentation. A diagnostic delay of > 3 months was associated with inferior survival in univariate analysis, but delayed diagnosis was not an independent influence on survival after adjustment for the major prognostic factors--tumour extent, year of diagnosis and treatment unit. CONCLUSIONS: These results suggest that scrotal incision is unlikely to affect the risk of loco-regional recurrence and that effective cytotoxic therapy has probably reduced the prognostic importance of delayed diagnosis on survival of men with NSGCT. PMID- 7551886 TI - Neomycin-induced perception deafness following bladder irrigation in patients with end-stage renal disease. AB - OBJECTIVE: To report a serious side-effect of intravesical instillations of neomycin in patients with end-stage renal disease. PATIENTS AND METHODS: Three patients (two men and a woman, aged 51.71 and 54 respectively) with end-stage renal disease who had undergone bladder irrigation with neomycin sulphate solution suffered complete irreversible deafness, which was assessed by audiogram. RESULTS: All three patients developed complete perception deafness after 3, 4 and 10 months, with characteristic changes in the audiogram. The severe progressive hearing loss was maximal in the high-frequency range. A high pitched tinnitus was the first sign of impending hearing difficulty and two patients developed a spontaneous nystagmus. CONCLUSION: In view of the doubtful efficacy and, most importantly, the risk of severe disabling side-effects such as ototoxicity, we recommend the avoidance of intravesical instillation with neomycin whenever possible. PMID- 7551892 TI - Fetal penile and urethral development subsequent to vascular insult in the last trimester of gestation: an experimental study in goat fetuses. AB - OBJECTIVE: To investigate a possible mechanism (the vascular theory) in the development of congenital anomalies of the penis and urethra in the fetus of the goat. MATERIALS AND METHODS: Fifteen male Angora goat fetuses underwent an operation at 110-114 days of gestation whereby branches of the internal pudendal artery within the superficial compartment of the perineum were ligated. Five male fetuses served as controls in which the perineum was explored with no ligation. RESULTS: Five treated and three control live offspring were delivered spontaneously. Although no genitourinary anomalies were observed on gross examination, histopathological examination of the penises of treated animals revealed smaller penises with increased fibrosis. CONCLUSION: A defective vascular supply can partly explain the morphogenesis of some aspects of the congenital anomalies of the penis and urethra. PMID- 7551893 TI - Staging problems in the pre-operative chemotherapy of Wilms' tumour. AB - OBJECTIVE: To determine the effect of the International Society of Paediatric Oncology (SIOP) Wilms' tumour protocols (pre-operative chemotherapy based on clinical and radiological findings, with no prior tumour verification by open or needle biopsy) on subsequent intra-operative tumour diagnosis and staging. PATIENTS AND METHODS: The diagnosis and staging of possible Wilms' tumour by clinical, ultrasonographic and radiological assessment were compared with the intra-operative findings in 14 consecutive children (1-12 years of age) treated between 1989 and 1994. RESULTS: A diagnosis of Wilms' tumour was histologically verified in 11 of 14 children. In two children, verification was not possible due to complete necrosis of the tumour following pre-operative chemotherapy. In a 12 year-old boy with an estimated stage IV disease due to a solitary lung metastasis, a renal cell carcinoma was revealed in the nephrectomy specimen while subsequent thoracotomy revealed dysmorphic but not malignant tissue. The estimated tumour stage was correct with regard to localized or metastatic disease in nine of 11 children with histologically confirmed Wilms' tumour, while in two children with an estimated stage II tumour, liver metastases were found intra operatively and the tumour was upstaged to IV. CONCLUSION: Exact tumour diagnosis and staging was difficult in these patients. Although the accuracy of tumour staging depends on the sensitivity of radiological and ultrasonographic examinations, difficulties in tumour diagnosis may be overcome by biopsies of the primary tumour. The justification of upstaging a low-stage I/II tumour to stage III, provoking a more intense post-operative treatment, should be proven by prospective randomized studies. The decision to perform a primary tumour biopsy would be facilitated if possible subsequent deterioration of outcome could be excluded. PMID- 7551895 TI - Denis Browne ring retractor in hypospadias surgery. PMID- 7551894 TI - Concepts in the surgical technique of one-stage hypospadias correction. AB - OBJECTIVE: To report on a single-stage technique for hypospadias repair based on the combination of several features of established methods and to emphasize the importance of identifying and excising the chordee that is almost always present in the tissues proximal to the urethral opening to ensure the complete correction of this deformity. PATIENTS AND METHODS: Over a 3-year period, 84 boys (age range from 8 months to 14 years) underwent a single-stage repair of proximal hypospadias. Chordee was corrected by excising fibrotic tissue representing the atretic corpus spongiosum proximal to the urethral opening. The neourethra was constructed using the epithelium distal to the urethral opening and part of the dorsal prepuce. RESULTS: Satisfactory results were obtained in 79 patients with a single procedure. Only five patients had fistulae and of these, four were closed successfully during the same hospital stay. CONCLUSION: Proximal hypospadias can be corrected using the single-stage technique described, with satisfactory results in most cases. Fistulae are uncommon and can be closed by a primary procedure performed in the post-operative period. PMID- 7551896 TI - A purpose-designed self-retaining kidney retractor for operative retroperitoneoscopy. PMID- 7551898 TI - An unusual complication of ureteroscopy with general anaesthesia: cauda equina syndrome. PMID- 7551897 TI - Pyelogram persisting 1 month in ureteric obstruction. PMID- 7551899 TI - Critical limb ischaemia--a complication of cystoscopy. PMID- 7551900 TI - Recurrence in the other testicle of a non-Hodgkin's lymphoma with no other manifestations. PMID- 7551901 TI - Renal angiomyolipoma with nodal involvement. A 20 year follow up. PMID- 7551902 TI - Garlic and the risk of TURP bleeding. PMID- 7551903 TI - Testicular cancer presenting as loss of vision. PMID- 7551904 TI - Retroperitoneal malacoplakia: an unusual cause of ureteric obstruction. PMID- 7551905 TI - Angiomyolipoma of kidney--a cause of recurrent retroperitoneal haemorrhage. PMID- 7551906 TI - Zinner's syndrome associated with adrenal gland and vena cava malformation: a very rare case. PMID- 7551907 TI - Laparoscopic varicocelectomy. PMID- 7551908 TI - The Kropp-onlay procedure (Pippe Salle procedure) PMID- 7551909 TI - Staging incidental carcinoma of the prostate. PMID- 7551910 TI - Implementing patient-focused care: success indicators for measuring satisfaction. AB - Using success indicators to regularly survey patients, families, physicians, and staff, you can monitor the acceptance of a new Patient-Focused Care model. Unit specific and hospital-wide data reveals problems and successes, and it offers a concrete scale on which you can judge how successful the transition is at any stage. PMID- 7551911 TI - Preparing for future nursing demand. AB - As patient care shifts from the hospital to community settings, the demand for nurses is also changing. Hospitals, long-term care agencies, and ambulatory settings need to identify their future need for nurses and work with schools of nursing to help prepare or retrain nurses for the jobs of the future. This article describes a Robert Wood Johnson Foundation project, administered by the American Association of Colleges of Nursing, to help nursing schools, hospitals, and other employers meet current and future nursing workforce needs in their regions. Organizations involved in employing or training nurses along with hospital associations, boards of nursing, and health education providers can apply for funding as regional nursing workforce consortia. PMID- 7551912 TI - A prospective randomized trial comparing blood- and marrow-derived stem cells for hematopoietic replacement following high-dose chemotherapy. PMID- 7551913 TI - Culture methods for the detection of minimal tumor contamination of hematopoietic harvests: a review. AB - The evaluation of minimal residual disease in patients and hematopoietic cell grafts is of considerable importance for staging disease, determining the response to treatment, and monitoring the efficiency of ex vivo purging or positive selection procedures. The most widely used techniques are immunocytochemical staining and the polymerase chain reaction; however, these assays do not measure the viability or clonogenic capacity of the detected cells. For this purpose, a culture technique must be used. This paper reviews the status, advantages, and limitations of this approach and the detection of tumor cells in bone marrow and peripheral blood. PMID- 7551914 TI - Natural killer cell proliferation is dependent on human serum and markedly increased utilizing an enriched supplemented basal medium. AB - We studied the effects of basal medium and human serum on the ex vivo expansion of CD56+/CD3- natural killer cells (NK). We demonstrated that sorted NK cultured for 18 days with 10% human AB serum without accessory cells in a 2:1 DMEM/F12 basal medium expanded significantly greater (6.4 +/- 0.9-fold, n = 14) than when cultured in standard RPMI 1640 basal medium (3.7 +/- 0.67-fold, n = 16; p = 0.019). Supplementation of the DMEM/F12 mixture with 2-mercaptoethanol (2ME), ethanolamine, L-ascorbic acid, and sodium selenite significantly augmented NK proliferation (16.3 +/- 2.5-fold, n = 11; p < 0.001) compared with DMEM/F12 without supplements. NK growth kinetics demonstrated that both the growth rate and the duration of exponential growth were increased by medium supplements. Addition of 2ME-containing supplements to cultures of NK with accessory cells augments NK proliferation, recruits NK progenitors, and has a serum-sparing effect. For the large-scale expansion of NK, we demonstrate a greater than 30 fold increase (n = 7) in the number of activated natural killer cells (ANK) derived from CD5/CD8-depleted PBMNC when cultured for 35 days in supplemented DMEM/F12 versus RPMI 1640 basal medium (197.6 +/- 95.0-fold versus 6.3 +/- 2.1 fold, respectively). Serum-free media (AIM-V and X-VIVO 10) were unable to support NK proliferation. These data suggest that utilizing 2:1 DMEM/F12 + 2ME containing supplements instead of standard RPMI 1640 as a basal medium can increase the proliferation of cytotoxic NK while reducing the culture interval and the amount of serum needed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551915 TI - Bioreactor expansion of human bone marrow: comparison of unprocessed, density separated, and CD34-enriched cells. AB - Scale-up of human hematopoietic cultures was previously described in continuously perfused systems with bone marrow mononuclear cells (BMMNC), yielding expansion of both progenitors and long-term culture-initiating cells (LTC-IC). We report here on the use of these systems for expansion of unprocessed whole BM cells (WBMC) and CD34-enriched cells. Density separation recovered 84% of CFU-GM and 65% of LTC-IC from WBMC. Subsequent CD34 selection recovered 17% of CFU-GM and 48% of LTC-IC from the MNC fraction. The unabsorbed (CD34-depleted) fraction contained 37% of CFU-GM and 38% of LTC-IC, accounting for most of the lost cells. WBMC, BMMNC, and CD34-depleted cells were each placed directly in bioreactors, whereas CD34-enriched cells were placed in bioreactors containing preformed irradiated stroma. After 14 days, an average of 3.82 x 10(7) (12.7-fold expansion), 3.54 x 10(7) (11.8-fold), 2.85 x 10(7) (9.5-fold), and 3.65 x 10(7) (1298-fold) total cells were obtained from bioreactors inoculated with WBMC, BMMNC, CD34-depleted, and CD34-enriched cells on stroma, respectively. These cultures yielded 1.64 x 10(5) (27.9-fold expansion), 1.69 x 10(5) (14.3-fold), 8.36 x 10(4) (13.0-fold), and 1.91 x 10(5) (41.4-fold) CFU-GM each, respectively. Cell recovery and expansion data were combined to determine the number of expanded CFU-GM obtained per ml of BM aspirate, allowing direct comparison of performance between the four culture inocula. WBMC generated 3.76 x 10(6) CFU-GM per ml BM aspirate, whereas MNC resulted in 1.42 x 10(6) CFU-GM. CD34-enriched cells (on irradiated stroma) gave 7.00 x 10(5) CFU-GM per ml BM aspirate, whereas CD34-depleted cells generated 4.97 x 10(5) CFU-GM. The high productivity from WBMC cultures was studied further and was found to be reproducible at different inoculum densities. WBMC cultures had elevated levels of endogenous EGF and PDGF production, which may have been responsible for the more extensive stromal development observed. Flow cytometric analysis showed that the final culture composition, with respect to T and B lymphocytes, monocytes, granulocytes, and erythrocytes, was not significantly affected by the inoculum composition and in all cases was comprised of multiple lineages. Therefore, each step in cell purification resulted in the loss of primitive and accessory cells, which in turn resulted in a net decrease in the number of expanded cells obtained per ml BM aspirate. PMID- 7551916 TI - Patient condition affects the collection of peripheral blood progenitors after priming with recombinant granulocyte colony-stimulating factor. AB - A total of 258 aphereses were performed in 79 patients with nonmyeloid malignancies after mobilization of peripheral blood stem cells (PBSC) with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Apheresis products were examined for viable mononuclear cell (VMC), CD34+ cell, and clonogenic cell contents. The number of progenitors in aphereses differs in subgroups of patients with different diagnoses. However, the number of CD34+ or clonogenic cells is dependent on age and amount of chemotherapy delivered to patients before collection rather than on the nature of the disease itself. In addition, the actual dose of rhG-CSF used to mobilize PBSC and the number of VMC in aphereses influenced the clonogenicity of CD34+ cells, although the daily dose of rhG-CSF seems to play little role on the number of clonogenic cells in each individual apheresis product. CD34+ cell and CFU-C (or CFU-GM) numbers are related parameters, and the relation can be described as linear. However, the linear relation varies in different patient groups, and most of the linearity is induced by the highest sets of values. We conclude that mobilization with low doses of rhG-CSF alone is feasible and that the probability of collecting a high number of peripheral blood progenitors is increased in young patients undergoing apheresis early in the course of the disease. Although the relationship between CD34+ cells and CFUs can be described as linear in well-defined situations, its relevance may be limited because it is not a universal finding. PMID- 7551917 TI - Proceedings of the Perugia International Cancer Conference IV. Perugia, 18-19 June 1993. PMID- 7551918 TI - Neoadjuvant chemotherapy of locally advanced non-small cell lung cancer. AB - Neoadjuvant chemotherapy was tested in non-small cell lung cancer in an attempt to increase the resectability of the tumor and to treat the microscopic metastatic disease known to be responsible for the majority of failures in surgically treated patients. This review deals with published trials. Most of them are feasibility studies in Stage III NSCLC. Obviously, the heterogeneity of eligibility criteria from one study to another prevents general conclusions on the usefulness of neoadjuvant chemotherapy. However, it is possible to conclude that neoadjuvant chemotherapy has an antitumor activity; the majority of the studies report a 60% objective response rate including a significant number of complete responses and a 50% complete resection rate. Neoadjuvant chemotherapy does not increase morbidity after surgery except when it is combined with preoperative radiation therapy. At the time of writing, one Phase III randomized study comparing neoadjuvant chemotherapy followed by surgery with surgery alone has been published. This study concludes that the combined modality treatment improves the survival of patients with locally advanced non-small cell lung cancer. Taken as a whole, the literature deserves further studies to determine the place of neoadjuvant chemotherapy in lung cancer. PMID- 7551919 TI - Radiotherapy in non-small cell lung cancer: optimal doses and schedules. AB - The doses and schedules for radiation therapy as applied in Stages IIIB and IIIA non-small cell lung cancer patients show a great variety. Depending on its intent, several purely palliative schemes can be used. When a curative intent scheme is considered, patients can be treated with a more experimental approach as several options are tested to increase selectively the radiation dose in the tumour areas. If improvements in treatment schemes are proven, new standards can be set. PMID- 7551921 TI - Biological prognostic factors in non-small cell lung cancer. AB - The results of conventional treatments for lung cancer remain poor and long-term survival rates have changed little over the last 10 years. In the same period of time there has been an explosion in the knowledge on the processes of cellular transformation, tumour progression, invasion and metastasis. The major categories of biological events implicated in non-small cell lung cancer include growth factor receptors expression (epidermal growth receptor, p185c-neu), autocrine growth factor production (transforming growth factor alpha), dominant oncogenes activation (ras genes) and deletion of tumour suppressor genes (p53 gene, retinoblastoma gene) and these are some of the abnormalities associated with specific histological types and with poor prognosis. Additional prognostic information can be obtained from the evaluation of the ploidy and proliferative activity of the tumours, carbohydrate antigens expression, presence of neuroendocrine differentiation and the evaluation of markers of the sequential steps involved in the process of tumour dissemination. PMID- 7551920 TI - Superiority of three-drug combination chemotherapy versus cisplatin-etoposide in advanced non-small cell lung cancer: a randomized trial by the Italian Oncology Group for Clinical Research. AB - To evaluate the efficacy of a three-drug regimen vs. a two-drug CDDP based combination in the treatment of NSCLC, we conducted a three-arm randomized parallel trial comparing (a) CDDP (120 mg/m2 day 1) + etoposide (100 mg/m2 days 1 3) every 3 weeks (PE--arm A); (b) CDDP (120 mg/m2 every 4 weeks) + mitomycin (8 mg/m2 days 1, 29, 71) + vindesine (3 mg/m2 days 1, 8, 15, 22 every 2 weeks) (MVP- arm B); and (c) CDDP (120 mg/m2 day 1) + mitomycin (6 mg/m2 day 1) + ifosfamide (3 g/m2 day 2) every 3 weeks (MIC--arm C). From May 1989 to April 1992, 393 consecutive previously untreated patients with NSCLC Stage IIIB and IV entered the trial; 373 were evaluable for survival and 360 for response. The response rate was significantly better for both the three-drug regimens compared with PE (Table 3). Logistic regression model showed a significantly better response in patients with a good P.S. and in Stage IIIB. Main toxicity consisted of myelosuppression: neutropenia Grade III-IV was recorded in 14% (arm A), 15% (arm B) and 21% (arm C). Thrombocytopenia Grade III-IV was worst in arm C: 10% vs. 5% (arm A) and 3% (arm B). Nephrotoxicity Grade III-IV was more common in arm C: 3.5%. Toxic deaths were 11 (3%: three in arm A, five in arm B, three in arm C). From our data, the three-drug containing regimens, MVP and MIC, appear more active than the two-drug combination PE in treatment of advanced NSCLC. PMID- 7551922 TI - Therapy of non-small cell lung cancer. AB - A few regimens can give clinical responses and more prolonged survival to the patients with NSCLC who respond. In addition, there is often a suppression of morbid symptoms in those patients. This is the situation that can be obtained in about 30% of the treated patients and, in addition, the treatment corresponds, from the psychological point of view, to what the patient expects. The toxicity of chemotherapy and the resulting morbidity and mortality can, to a large extent, be controlled by a close clinical follow-up and the use of various techniques of supportive care. Such a surveillance of patients probably explains why administration of planned therapy can be less expensive than palliative care. PMID- 7551923 TI - Meta-analysis of randomised trials of systemic chemotherapy versus supportive treatment in non-resectable non-small cell lung cancer. AB - We have conducted a meta-analysis of all published and unpublished 'polychemotherapy' versus 'best supportive care' clinical trials in patients with non-resectable non-small cell lung cancer to assess the usefulness of polychemotherapy in this cancer. Seven studies with more than 700 patients were selected for our meta-analysis. Our meta-analysis showed a reduction in mortality during the first months under polychemotherapy. Although quite small, this increase in survival, coupled with a certain improved 'quality of life' suggests that polychemotherapy should be recommended for patients with non-resectable non small cell lung cancer. PMID- 7551925 TI - Paclitaxel (Taxol) and docetaxel (Taxotere): active chemotherapeutic agents in lung cancer. AB - Paclitaxel (Taxol), the prototype of a new class of plant-derived antineoplastic compounds, is a natural product isolated from the Pacific yew. Docetaxel (Taxotere) is a hemisynthetic product derived from the European yew. These agents share a unique mechanism of cytotoxic action by promoting assembly of microtubules and rendering the microtubules resistant to depolymerization. In vitro studies suggest a possible role for radiation sensitization. In Phase I trials, the dose-limiting toxicity was neutropenia for both agents. Other toxicities include infusion-related hypersensitivity reactions, alopecia, neurotoxicity, mucositis, diarrhoea and myalgias. To prevent infusion-related reactions, routine premedication is recommended. Noncumulative cardiac toxicity has been observed with paclitaxel. Fluid retention and rash have been reported with docetaxel. In Phase II studies of paclitaxel in advanced non-small cell lung cancer, response rates of 21% and 24% were observed. In Phase II studies of docetaxel in similar patients, response rates ranging from 28-38% were reported, including patients previously treated with cisplatin. The most common toxicity in these studies was neutropenia. Combination studies with cisplatin and other agents are in progress. Paclitaxel and docetaxel are among the most active chemotherapeutic agents for non-small cell lung cancer patients. Their testing will dominate trials of new therapies in this disease for years to come. PMID- 7551927 TI - Camptothecin analogues in the treatment of non-small cell lung cancer. AB - Camptothecin is a natural product derived from the Oriental tree Camptotheca acuminata which has shown activity in a number of experimental tumors. Its clinical development was halted in the early-70s owing to its unpredictable and formidable toxicities. Two water-soluble camptothecin analogs have been synthesized recently and are currently in clinical trials: topotecan and CPT-11. Camptothecin and its derivatives are unique in that they represent the only family of topoisomerase I inhibitors. Topoisomerase I is a nuclear enzyme which modulates the topological structure of DNA by making transient single-stranded breaks. Pre-clinical studies have shown that CPT-11 and topotecan possess high and broad antitumor activity against a variety of experimental tumors including both non-small cell lung cancer (NSCLC) and small cell lung cancer. Lack of cross resistance with most classical anticancer agents has been also demonstrated. Phase I studies have identified neutropenia to be the dose-limiting toxicity for topotecan while, for CPT-11, either neutropenia or diarrhoea were dose-limiting. Maximum Tolerated Doses (MTD) of both agents are greatly dependent upon the schedule used. A Phase II Japanese study of CPT-11 in advanced untreated NSCLC has been recently published. Given at the dose of 100 mg/m2 as a 90-min infusion, CPT-11 produced a 32% objective response rate out of 72 assessable untreated patients. Similar studies are in progress with topotecan. The same Japanese group has completed Phase I-II studies on the combination of CPT-11 with cisplatin. The optimal dose of CPT-11, which can be safely combined with cisplatin 80 mg/m2, was found to be 60 mg/m2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551924 TI - New drugs in non-small cell lung cancer. An overview. AB - Chemotherapy is rather ineffective in non-small cell lung cancer. However, in the last few years, a number of new anticancer agents have been developed which have definite activity in this disease. Among them are the taxanes and CPT-11, drugs with novel mechanisms of action, new antimetabolites (edatrexate and gemcitabine), and a new vinca alkaloid (vinorelbine). Furthermore, in the near future, the better understanding of lung cancer biology will help in devising new treatment strategies. PMID- 7551926 TI - Gemcitabine in non-small cell lung cancer. AB - The pyrimidine antimetabolite gemcitabine is an analogue of cytosine arabinosid. Gemcitabine is well tolerated when given in doses of 1000-1250 mg/m2 weekly x 3 followed by 1 weeks rest, with mild myelosuppression as the major toxicity. In five studies, including a total of 250 patients with previously untreated non small cell lung cancer (NSCLC), response rates from 20 to 28% were observed, ranking gemcitabine among the active agents in NSCLC. Gemcitabine should be further explored in combination therapy for NSCLC. PMID- 7551929 TI - Biological response modifiers. AB - Biological response modifiers (BRMs) of current clinical interest may be divided into two categories: immunomodulating agents and colony stimulating factors (CSFs). The first are employed to potentiate the host defences towards the tumor but results have proved disappointing in non-small cell lung cancer (NSCLC). With regard to the second, the real dose increment which may be ascribed to CSFs is not sufficient to overcome chemoresistance of NSCLCs. CSFs can, however, reduce the hematologic toxicity of chemotherapy, which represents the most significant result. PMID- 7551930 TI - Future directions and strategies. PMID- 7551928 TI - Edatrexate studies in non-small cell lung cancer. AB - Edatrexate (10-ethyl-10-deaza-aminopterin, or 10-EDAM) is a water-soluble antifolate which is under study in a variety of malignancies. Edatrexate demonstrated greater antitumor activity than methotrexate in several solid tumor models and xenografts, which may be due to a more extensive formation of polyglutaminates within tumor cells by edatrexate metabolites. Phase I studies have recommended a dose of 80 mg/m2 i.v. weekly for tumor specific trials. When used with leucovorin, edatrexate doses more than 10 times as high have been found to be well-tolerated. Dose-limiting toxicity is mucositis, with leukopenia and thrombocytopenia being less prominent. In three Phase II trials without leucovorin in non-small cell lung cancer, edatrexate has shown an overall objective major response rate of 17% in 66 previously untreated patients (95% C.I.: 9-28%), making it one of the more active single agents in this malignancy. With its relatively low degree of myelosuppression, edatrexate has been an attractive agent for use in combination. To date, trials combining this drug with mitomycin plus vinblastine, cisplatin plus cyclophosphamide, paclitaxel, and carboplatin have been initiated. The encouraging response rates and low degree of toxicity make this agent interesting for further investigation in non-small cell lung cancer. PMID- 7551931 TI - Prognostic factors in NSCLC. Recent experiences. AB - In recent years, a group of new prognostic factors have been added to the list of well-known clinical prognostic factors of non-small cell lung cancer. Among these are mutations in the K-ras oncogene, abnormalities in p53, the presence of N-CAM expression as measured by Mab immunostaining and elevated serum levels of NSE. These factors have provided important clinical insights into the biology of lung cancer and prospective studies using these biomarkers are now warranted to provide further important clues about their potential significance in treatment selection of patients. PMID- 7551933 TI - Adjuvant chemotherapy: results and perspectives. AB - The overall 5-year survival of surgically resected non-small cell lung cancer (NSCLC) remains less than 50% and is unlikely to improve until there are effective systemic adjuvant therapies. Two studies of the Lung Cancer Study Group (LCSG) have shown a modest impact of adjuvant CAP chemotherapy on disease-free and overall survival. In addition, a Finnish study, which randomized patients with T1-T3, N0 disease to CAP chemotherapy or follow-up, has also demonstrated an improvement in recurrence-free survival, as well as overall survival, at 5 and 10 years. On the other hand, an LCSG trial of adjuvant CAP in Stage I NSCLC (T2, N0, T1 N1) showed no benefit, although compliance with treatment was poor. Similarly, adjuvant trials using vindesine and cisplatin have not demonstrated benefit in studies done in Montreal and New York. Clearly, more effective systemic therapy is needed and must be evaluated in randomized trials in which patients have been carefully staged intra-operatively. Biological markers, such as vascular invasiveness, oncogene mutations and other factors may allow identification of specific subsets of patients at high risk of recurrence who can be targeted for aggressive adjuvant approaches in the future. PMID- 7551934 TI - Effectiveness and toxicity of preoperative therapy in stage IIIA non-small cell lung cancer including the Memorial Sloan-Kettering experience with induction MVP in patients with bulky mediastinal lymph node metastases (Clinical N2). AB - The use of preoperative chemotherapy with mitomycin, vinblastine and cisplatin (MVP) has led to improved complete resection rates and survival in Stage IIIA non small cell lung cancer with bulky, ipsilateral, mediastinal lymph node metastases (Clinical N2 disease). The addition of preoperative irradiation has also been explored with results not substantially different from preoperative cisplatin based chemotherapy alone. While preoperative chemotherapy has been shown to be feasible, the toxicity of both the chemotherapy and the subsequent resection is of concern with an overall treatment-related mortality of nearly 8%. The careful selection of patients, swift management of neutropenia, and meticulous perioperative pulmonary care has the potential to reduce the mortality from multimodality therapy. Having shown survival benefit in multiple single institution and randomized trials, induction chemotherapy followed by surgery or irradiation is now the treatment of choice for patients with Stage IIIA non-small cell lung cancer with mediastinal lymph node metastases. PMID- 7551935 TI - Mutated K-ras gene analysis in a randomized trial of preoperative chemotherapy plus surgery versus surgery in stage IIIA non-small cell lung cancer. AB - The observation that the proteins encoded by ras genes play a central role in the signalling pathways used by cells to respond to growth factors and the fact that mutated ras proteins are constantly promoting cell division have led to a PCR based hunt for additional clinical information. In the present study, K-ras analysis draws the following conclusions: (1) K-ras point mutation frequency was higher in the surgery group (10 of 24 patients) than in the chemotherapy-surgery group (3 of 20 patients). (2) Mutated K-ras was predominantly observed at codon 12 but five mutations appeared at codon 61. (3) Mutations were identified in the squamous cell carcinoma histological NSCLC subtype except in four cases corresponding to adenocarcinoma. (4) A multifarious pattern of substitutions, especially at codon 12, were noted with aspartic K 12 substitutions more prone to develop bone metastases. (5) Although a genotypic K-ras classification of NSCLC may not yet be formulated, our accumulated data (unpublished) suggest a trend toward it. (6) Patients with mutated K-ras tumors in the surgery group had no different survival than those with normal K-ras. However our pooled data as well as other authors' results assert that mutated K-ras constitute an additional prognostic datum that deserves to be included together with TNM classification. In the design of new preoperative (neoadjuvant) chemotherapy trials, stratification of tumors by K-ras status deserves to be further investigated in order to correlate with response, relapse and survival. Mutated K-ras genotype merits further research. Finally, the paradigm of uneven histological distribution and mutated K-ras spectra among researchers should serve as a stimulus to search for further contributions in this field. PMID- 7551932 TI - Epidemiology of lung cancer: recent trends in mortality with emphasis on Europe. AB - Recent trends in mortality from lung cancer in Europe are reviewed. During the last decade, overall lung cancer mortality in males showed no systematic pattern in Northern and Central Europe, but a modest decline started at younger ages in several countries. In Southern Europe, lung cancer mortality started from lower values, but is still rising, and only in Italy is some flattening of rates at relatively high levels becoming apparent in middle age (35-64 years). The average change in lung cancer rates in Southern Europe over the last decade for males was + 24% for all ages and + 22% in middle age. The upward trends were even more substantial in Eastern European countries (+ 32% in middle age), which now have the highest lung cancer rates in young and middle-aged males. Over the last few decades, female lung cancer rates have risen in all European countries, but only in Denmark and Britain are overall rates now over 20/100,000. There is therefore still ample scope for urgent intervention aimed at controlling a major tobacco related lung cancer epidemic among European women in the near future. Southern and mainly Eastern Europe are becoming priority areas for campaigns for giving up smoking, since the prevalence of tobacco smoking in the young is higher and high tar dark-tobacco cigarettes are still common. PMID- 7551936 TI - A phase II trial of combined chemotherapy and surgery in stage IIIA non-small cell lung cancer. AB - A poor prognosis for patients with Stage IIIA clinical N2 treated by surgery alone has led clinical researchers to find a new treatment modality to improve the curative potential of surgery. Many Phas II trials have been carried out with induction chemo- or chemo-radiotherapy prior to surgery. From June 1988 to July 1991, 46 patients with non-small cell lung cancer (NSCLC) Stage IIIA clinical N2 entered a Phase II induction-chemotherapy trial. Patients received 2-3 cycles of high-dose cisplatin and etoposide. Forty-five were evaluable for response; the response rate was 82% (37/45: 3 CR, 34 PR). Toxicity was primarily hematologic. Surgical resection was performed in 35 patients; radical resection was possible in 28 patients (62%); three patients were incompletely resected and two patients were only explored. Three deaths were surgery-related. Median survival was 24.5 months with a 2-year survival of 53%. Cisplatin with etoposide is an active and safe induction chemotherapy regimen for NSCLC Stage IIIA N2 with a high response rate. The median survival seems to be prolonged and therefore, randomized trials are needed to compare this approach with other treatment modalities. PMID- 7551938 TI - Multimodality therapy in unresected stage III non-small cell lung cancer: the American Cooperative Groups' Experience. AB - Since the mid-1980s, the American Cooperative Cancer Treatment Groups (Cancer and Leukemia Group B, Eastern Cooperative Oncology Group, North Central Cancer Treatment Group. Radiation Therapy Oncology Group, Southwest Oncology Group) have performed several multimodality trials exploring combinations of chemotherapy and radiation in patients with Stage III non-small cell lung cancer. The initial trials had a sequential design with induction chemotherapy followed by radiation. Later trials have emphasized concurrent chemoradiation with or without induction chemotherapy. These trials have begun to have an impact on what is considered 'standard' therapy for patients with Stage III non-small cell lung cancer (NSCLC). PMID- 7551940 TI - Current Perspectives in the Treatment of Non-Small Cell Lung Cancer. Proceedings. Colorado Springs, Colorado, June 28, 1994. PMID- 7551939 TI - Trials with mitomycin, ifosfamide and cisplatin in non-small cell lung cancer. AB - In 1988, we reported a Phase II study of mitomycin, ifosfamide and cisplatin (MIC) in inoperable non-small cell lung cancer. The overall objective response rate was 56% in 66 evaluable cases. An improvement in performance status was observed in responding patients and toxicity was acceptable. Consequently we then embarked upon two major, multicentre randomised trials to test this regimen in cases with localised disease (MIC 1--chemotherapy plus radical radiotherapy versus radical radiotherapy alone) and advanced disease (MIC 2--chemotherapy plus palliative care versus palliative care alone). These trials are still in progress with targets of 500 and 300 randomised cases, respectively. The present interim analysis is based on 317 cases randomised in MIC 1 and 193 in MIC 2. The overall response rate (CR + PR) in the MIC 1 trial is 52% with 10% CRs and in the MIC 2 trial is 42% with 5% CRs. Formal symptomatic assessments are monitored in both trials and the present interim analysis indicates symptom improvement in cases randomised to chemotherapy, including patients who fail to achieve an objective PR or CR. Other Phase II studies employing MIC are also reviewed. PMID- 7551937 TI - Impact of chemotherapy on survival in locally advanced non-small cell lung cancer: the Gustave-Roussy experience. AB - The high local and distant recurrence rate after thoracic radiation deserves the development of new strategies to improve the outcome of patients with Stage III non-small cell lung cancer (NSCLC). The addition of chemotherapy (CT) to radiotherapy has seen a decrease in the metastasis rate in some controlled studies with a favorable impact on overall survival. However, the optimal schedule of both modalities remains to be defined. The persistent poor local control in these studies underestimates the real impact of chemotherapy on patients' outcome and calls for new investigational approaches. PMID- 7551942 TI - Concurrent paclitaxel and thoracic radiation for advanced non-small cell lung cancer. AB - A phase I trial of paclitaxel therapy, administered as a weekly 3-h infusion for 6 weeks with concurrent daily thoracic radiation to patients with advanced or medically inoperable non-small cell lung cancer, was performed. Paclitaxel was escalated in increments of 10 mg/m2/week in successive cohorts of three new patients as tolerated, starting at 10 mg/m2/week. Radiation was administered to the primary tumor and regional lymph nodes (40 Gy over 4 weeks), followed by a boost to the primary tumor (20 Gy in 2 weeks). Twenty-seven patients entered this study through seven dose levels of paclitaxel ranging from 10-70 mg/m2/week for the primary purpose of evaluating the toxicity of concurrent chemoradiation treatment. Esophagitis was the principal and dose-limiting toxicity. Radiation pneumonitis occurred in two patients. Other toxicities were mild. Although not designed as an efficacy study, 23 patients were available for response, and an overall response rate of 74% (confidence interval, 65-83%) was observed. PMID- 7551944 TI - Clinical features and current treatment of diffuse malignant pleural mesothelioma. AB - Malignant pleural mesothelioma is an uncommon cancer for which the major risk factor is prior asbestos exposure. During the past decade, there has been progress in the diagnosis, staging and biology of mesothelioma. Treatment remains controversial and ranges from supportive care alone to aggressive multimodality therapy. Prospective clinical trials in carefully staged patients are needed to determine what approaches to treatment may confer a survival benefit. PMID- 7551943 TI - Dose-finding study of paclitaxel (Taxol) plus cisplatin in patients with non small cell lung cancer. European Lung Cancer Working Party. AB - We conducted a phase I trial in advanced NSCLC to determine the optimal dosage of the new active drug paclitaxel (Taxol), in combination with high-dose ciplastin (CDDP). Taxol was infused over 3 h, followed by CDDP given over 30 min with hyperhydration. Main criteria of selection were the presence of metastatic or locally relapsing pathologically proven NSCLC, stage IIIB or IV and no prior therapy. Out of 17 treated patients, eight objective responses (47%) were documented. Significant but transient neutropenia was observed in steps III and IV. In seven patients, who received more than three courses, treatment was discontinued for severe polyneuropathy in five, cancer progression in one and unrelated disease in one. In conclusion, paclitaxel plus cisplatin, although active, was associated with severe late neurological toxicity prohibiting the administration of multiples courses. PMID- 7551941 TI - Phase II study with paclitaxel for the treatment of advanced inoperable non-small cell lung cancer. AB - Paclitaxel is a plant product isolated from the bark of the Western yew (Taxus brevifolia) that promotes the formation and stabilization of microtubules. This leads to growth arrest in the G2/M phase of the cell cycle. Paclitaxel has demonstrated significant antineoplastic activity in different tumor types, most notably in ovarian and breast carcinoma. In two Phase II trials (Eastern Cooperative Oncology Group [ECOG]/M.D. Anderson) in patients with previously untreated Stage IIIB-IV non-small cell lung cancer (NSCLC), response rates of 21% and 24% were reported. We are performing a Phase II trial investigating the efficacy of paclitaxel in patients with inoperable Stage IIIB-IV NSCLC. Forty three patients were treated, 31 males and 12 females, with a median age of 59 years (range, 29-75), ECOG performance status 0-2, Stage IIIB 30%, Stage IV 70%. Patients were treated every 3 weeks with 225 mg/m2 as a 3-h infusion with standard premedication. Preliminary efficacy results from 37 patients include partial remissions in eight (21.6%) patients, no change in 22 (59.5%) and disease progression in seven (19%) patients. Eight patients are still receiving therapy. The hematologic toxicities (n = 43) were mild, and no World Health Organization (WHO) Grade 4 neutropenia was observed. Nonhematologic toxicities were Grade 1/2 polyneuropathy in 97.6%, Grade 1-3 myalgia/arthralgia in 76%, and Grade 1-3 nausea/vomiting in 18.6% of the patients. In conclusion, paclitaxel is an active single agent in this patient population. Mild hematologic toxicities were observed in the 3-h infusion setting (compared with 24-h infusion) and therapy was well tolerated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551952 TI - Current perspectives in the treatment of small cell lung cancer: educational symposium of the VII World Congress on Lung Cancer. PMID- 7551948 TI - Neoadjuvant (induction) treatment for non-small cell lung cancer. AB - Preoperative treatment for non-small cell lung cancer is not new, but recently it has gained increasing acceptance in the management of locally advanced disease. Preoperative radiotherapy has been studied extensively but has never been demonstrated to improve survival in locally advanced (N2,T3) disease. However, it is still considered part of the accepted management for superior sulcus tumors. In the past 15 years, many Phase II trials have investigated the use of induction chemotherapy or chemoradiotherapy as part of the treatment plan for Stage IIIA patients, especially those with preoperatively identified N2 disease who have a poor prognosis following primary surgical resection, with or without postoperative adjuvant therapy. Preoperative chemotherapy trials, as exemplified by the Memorial Sloan-Kettering Cancer Center and Toronto MVP (mitomycin, vinblastine, and cisplatin) trials, have demonstrated what appear to be improved median survival time and prolonged 5-year survival when compared to historical controls. More recently, three relatively small randomized trials have demonstrated statistically significant improvement in patients treated preoperatively with induction chemotherapy. More widely investigated in Phase II trials has been the use of chemoradiotherapy as an induction protocol. The chemotherapy has usually been platinum based and has been given prior to, or concurrent with, radiotherapy. The two largest series to date (Rush-Presbyterian and Southwest Oncology Group) have both suggested improved median survival times and prolonged survival using this approach. Three recently completed nonsurgical trials have demonstrated the superiority in median and long-term survival of primary chemoradiotherapy compared to primary radiotherapy alone in treating locally advanced, inoperable Stage IIIA and IIIB lung cancer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551947 TI - Molecular events in lung cancer. AB - The observation that genes contributing to the process of malignant transformation are altered forms of genes normally present in eukaryotic cells initiated many of the advances that have increased our understanding of lung carcinogenesis at the molecular level. The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes. Proto oncogenes (normal homologue of the oncogene) participate in critical cell functions, including signal transduction and transcription. Only a single mutant allele is required for malignant transformation. Primary modifications in the dominant oncogenes that confer gain of transforming function include point mutations, amplification, translocations, and rearrangements. A second recently described gene family is the tumor suppressor genes. Tumor suppressor genes appear to require homozygous loss of function either by mutation, deletion, or a combination of these. Some tumor suppressor genes appear to play a role in the governance of proliferation by regulation of transcription. The identification of specific genes that contribute to the development of the cancer cell presents an opportunity to use these genes and their products as prevention and treatment targets. PMID- 7551946 TI - An historical perspective of multi-modality treatment for resectable non-small cell lung cancer. AB - We examine the origins of surgical therapy, radiotherapy, and chemotherapy as they were applied to lung cancer in the mid-portion of this century. Surgical therapy for lung cancer started in the 1930s with pneumonectomies. The prognostic significance of nodal metastases was soon recognized, and surgical staging procedures became an important part of patient workup. Radical radiotherapy for potential cure of lung cancer began in the 1950s with megavoltage linear accelerators. The first application of chemotherapy for lung cancer was the use of nitrogen mustards in the 1940s. Single modality surgical therapy has become the treatment of choice for Stages I and II non-small cell lung cancer, but 50% of clinical Stage I patients die of recurrent disease, and 70% of those recur outside the chest. Biologic markers may identify high risk subgroups of Stage I and II patients who may benefit from adjuvant chemo- or radiotherapy. Within the last decade, several single and multi-institutional Phase II trials and two single institution Phase III trials have reported improved survival in Stage IIIA patients treated with cisplatin-based neoadjuvant chemotherapy prior to surgical resection. These trials have reported high response and resectability rates, but at a substantial toxicity. A new standard of care for Stage IIIA disease has not been conclusively established. PMID- 7551950 TI - Scheduling of chemotherapy and radiotherapy in locally advanced non-small cell lung cancer. AB - In scheduling chemotherapy and radiotherapy for locally advanced non-small cell lung cancer (NSCLC), chemotherapy can be given pre-radiotherapy or concurrently as a single agent or in combination. Optimal scheduling has yet to be established. Optimal pre-radiotherapy for NSCLC requires further development but cisplatin with vinblastine, vindesine, etoposide or navelbine appear the best currently available. A number of new drugs show potential for enhancing radiation effects. Concurrent chemotherapy and radiotherapy has been tested in a number of experimental tumours in cell culture. In these systems cisplatin, carboplatin, 5 fluorouracil, mitomycin-C and other agents appear to improve cell kill compared to chemotherapy alone. Mouse xenograft models allow the study of various concurrent drug and radiation schedules including the effect of radiation with cisplatin, carboplatin, paclitaxel and gemcitabine. In these systems, cisplatin in divided doses shows optimal enhancement with fractionated radiotherapy. There are a number of drug candidates for concurrent chemotherapy and radiotherapy programs. Clinical studies in head and neck cancer, esophageal cancer, small cell lung cancer and NSCLC show promising results with concurrent chemotherapy and radiotherapy. Cisplatin given daily with radiotherapy improved survival in NSCLC compared to cisplatin given weekly with radiotherapy or to radiotherapy alone. To study the toxicity of radiation and concurrent carboplatin, we have studied 170 patients with unresectable locally advanced NSCLC in a 4-arm randomized trial. An analysis of the first 100 patients entered revealed significantly more neutropenia (P < 0.0001) and thrombocytopenia (P < 0.004) with the combined modality arms. Esophagitis was worse on all three experimental arms but was significantly more prolonged with accelerated radiotherapy arms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551949 TI - Combined modality treatment for stage III non-small cell lung cancer. AB - There are increasing reports of studies in which combined modality treatment is being tested in stage III non-small cell lung cancer. Randomized trials in which sequential chemoradiotherapy has been compared to radiation alone and in which single agent cisplatin and simultaneous thoracic radiation were compared to radiation are reviewed and discussed. The largest and the most mature phase II trials of preoperative chemotherapy are also included in this review. Similarly the results from recently reported small randomized trials evaluating preoperative treatment are described. Potential future directions for clinical trials are suggested, including the incorporation of new agents in combined modality regimens. PMID- 7551951 TI - New chemotherapy agents in the treatment of advanced non-small cell lung cancer: an update including data from the Seventh World Conference on Lung Cancer. AB - Over the past two decades, modest gains have been made in chemotherapy for non small cell lung cancer with the addition of cisplatin-based regimens to the therapeutic armamentarium. Over the last decade, several new agents with significant activity have reached the level of Phase II and III testing. This list of new drugs includes: navelbine, the taxanes--taxol and taxotere, gemcitabine, edatrexate and the camptothecins--irinotecan and topotecan. During this period, oral etoposide and epirubicin were re-investigated and biological agents such as the retinoids, interferons and interleukins were also explored as alternatives to traditional chemotherapy. As these new drug investigations proceeded, basic scientists made important discoveries which are now beginning to be applied to therapy. The future promises to combine these active new drugs with therapies directed against targets unique to non-small cell lung cancer cells. PMID- 7551953 TI - The role of surgery in small cell carcinoma of the lung (SCLC). PMID- 7551945 TI - Future directions in non-small cell lung cancer. A personal view. AB - At the 1991 6th World Conference on Lung Cancer sponsored by IASLC and held in Melbourne, the Future Directions summary described several critical clinical trials that were underway, reviewed the progress in cellular and molecular biology and decried the excessive nihilism and pessimism then rampant. The theme today is identical, but the baseline has advanced significantly. There is now a clear consensus that combined modality therapy is superior to single modality therapy for locally advanced NSCLC. The issues for future clinical trials are related to increasing the effectiveness of regimens known to be active in reducing toxicity. Radiation alone for Stage IIIB NSCLC and surgery alone for Stage IIIA NSCLC need not serve as control arms. The availability of several new, and significantly more active, chemotherapeutic compounds will drive the clinical trials in this arena for several years. Meta-analyses also demonstrate a clear benefit for chemotherapy over supportive care and only in the area of post operative adjuvant therapy for resected NSCLC is the efficacy of therapy still in question. Our comprehension of the number and sequence of genetic mutations leading to clinical cancer is rapidly growing, although it is not likely that attempts to reverse specific mutations will lead to successful therapy for established tumors. On the other hand, early detection and prevention are becoming very real possibilities. Knowledge of specific mutations and the development of pharmacologic approaches to overcome them is the next area for clinical development. Optimism remains an appropriate response to developments in the area of NSCLC. PMID- 7551956 TI - Biology of small cell lung cancer. AB - The bombesin-like peptides can function as autocrine growth factors in lung cancer and candidate tumor suppressor genes on chromosomes 3 and 9 play important roles in lung cancer. Bombesin-like peptides can function as mitogens for normal bronchial epithelial cells and lung cancer cell lines. The monoclonal antibody directed against gastrin releasing peptide and bombesin, 2A11, can inhibit the growth of small cell lung cancer in vitro and in vivo and intravenous administration has induced a clinical remission in a patient with relapsed small cell lung cancer. The loss of a portion of one of the two short arms of chromosome 3 (3p) is identified in nearly 100% of tumor cell lines and tumors from patients with small cell lung cancer. Introduction of chromosome 3 into tumor cell lines suppresses their tumorigenicity in athymic nude mice, one of the characteristics of the cancer phenotype. Both copies of the candidate tumor suppressor gene on chromosome 9, CDKN2, are deleted in approximately one-fourth of lung cancer cell lines examined and the protein product of CDKN2, p16 is undetectable in one-third of the lung cancer cell lines studied. The CDKN2 gene is inactivated more commonly in non-small cell lung cancer than small cell lung cancer while the retinoblastoma gene is inactivated more commonly in small cell lung cancer than non-small cell lung cancer. It appears that a single defect in this cell cycle pathway is necessary for unregulated growth in lung cancer and current evidence suggests these defects differ between small cell and non-small cell lung cancer. PMID- 7551955 TI - Combined platinum etoposide with radiation therapy in limited stage small cell lung cancer: an effective treatment strategy. PMID- 7551959 TI - Future directions in the management of small cell lung cancer. AB - The treatment of small cell lung cancer (SCLC) has continued to evolve over the past 20 years and now consists primarily of combination chemotherapy with or without thoracic radiotherapy depending on stage at presentation. However, despite marked improvement in overall survival, a majority of patients continue to die of their disease. It is not likely that conventional chemotherapy agents will substantially alter this dismal fact but new agents are available with novel mechanisms of action some of which yield excellent response rates. The agents with greatest promise include the taxanes and drugs that target topoisomerase I. Real progress in the management of SCLC will come with the identification of methods for overcoming drug resistance, exploration of new treatment strategies including gene therapy and possibly through the use of biological agents. Chemoprevention trials are of the utmost importance given the high incidence of second primary tumors that arise in the few long term survivors of this disease. All these areas are fertile grounds for future investigation in the management of SCLC. PMID- 7551958 TI - Oral therapy for small cell lung cancer. AB - After a remarkable improvement of the very poor prognosis of small cell lung cancer with very simple therapy such as iv and oral cyclophosphamide the role of oral therapy has become minimal. However, since more than a decade results of combination chemotherapy are at a plateau and it is necessary to reconsider the role of simple therapy in patients without the prospect of cure. Oral therapy might be worthwhile because it is probably less effecting the quality of life of the patient and makes it unnecessary to visit the hospital frequently. All drugs available for oral use with known activity against small cell lung cancer are reviewed. The best example of the success of oral therapy is etoposide, other candidates that need to be tested in a modern way are oral cyclophosphamide and hexamethylmelamine. New concepts of prolonged chemotherapy and dose-intensity are easier evaluated by using oral drugs. The discovery of the activity of prolonged oral etoposide is an excellent example how to test a new concept in a very simple way. PMID- 7551957 TI - New drugs for treating small cell lung cancer. AB - Recently there has been a number of new active drugs which have been identified for treating patients with SCLC. These drugs include paclitaxel, docetaxel, CPT 11, topotecan and gemcitabine. The range of response rates are as follows for each of the drugs given: (1) to previously untreated patients: paclitaxel (34 41%), topotecan (39%) and gemcitabine (30%), and (2) to previously treated patients: docetaxel (28%), CPT-11 (47%) and topotecan (35%). Further studies with these new drugs utilized in combination chemotherapeutic regimens are needed to define the role of these agents in the treatment of patients with SCLC. PMID- 7551954 TI - Prophylactic cranial irradiation in small cell lung cancer--an update. AB - The role of prophylactic cranial irradiation (PCI) in the management of patients with small cell lung cancer is reviewed, with emphasis on 11 randomised trials. Several interpretive and methodological problems related to PCI investigations are identified, and it is argued that given the current data PCI should be considered as an optional treatment component in CR patients, that these authors do not recommend, while other investigators do. PMID- 7551960 TI - Carboplatin/etoposide combination chemotherapy in the treatment of poor prognosis patients with small cell lung cancer. AB - Advances in the treatment of SCLC have been slow but sure over the past decade. It is now recognized that a small subset of patients may be cured of this disease, particularly with combined modality treatment with radiation therapy and systemic chemotherapy. However, perhaps only 25% of all patients will be candidates for this intensive/combined modality approach. Therefore, there is a need to identify effective combination chemotherapy regimens that can be easily administered to the remaining 75% of poor prognosis SCLC patients. Our studies and those of others would suggest that the combination of carboplatin and oral etoposide is a highly effective regimen for the treatment of these patients with results equal to and comparable with more intensive schedules. The ease of administration of this combination with acceptable toxicity would suggest that for such a subset of patients it may be the treatment of choice against which other combination schedules should be compared. PMID- 7551961 TI - Etoposide phosphate or etoposide with cisplatin in the treatment of small cell lung cancer: randomized phase II trial. AB - Etoposide phosphate, a water soluble prodrug of etoposide, has several potential advantages including easier and more rapid administration, avoidance of large fluid loads, and elimination of hypersensitivity reactions and other problems related to the solubilizer. This randomized Phase II study was done to evaluate the efficacy and toxicity of etoposide phosphate and etoposide, when used in combination with cisplatin in the treatment of patients with small cell lung cancer. Previously untreated small cell lung cancer patients were randomized to receive cisplatin in combination with molar equivalent does of either etoposide or etoposide phosphate. The patients were evaluated with respect to response rate, time to progression, survival, and toxicity. Response rates with etoposide phosphate and etoposide were 61% (95% confidence interval 55-67%) and 58% (95% confidence interval 52-64%), respectively (P = 0.85). Median time to progression was 6.9 months for patients who received etoposide phosphate and 7.0 months for those with etoposide (P = 0.50). For extensive stage disease patients, median survival with etoposide phosphate was 9.5 months versus 10 months for etoposide (P = 0.93). The corresponding median survivals for patients with limited stage disease were > 16 months and 17 months, respectively (P = 0.62). Myelosuppression was the most common toxicity; Grade 3 and 4 leukopenia occurred in 63% of patients receiving etoposide phosphate compared with 77% receiving etoposide (P = 0.16). The combination of etoposide phosphate and cisplatin is effective in the treatment of small cell lung cancer, and can be administered with acceptable toxicity. This study was not designed to be a formal Phase III comparative trial, but the efficacy and toxicity observed with this regimen were found to be similar to a standard etoposide/cisplatin regimen, using molar equivalent etoposide doses. Etoposide phosphate is preferable to etoposide because it is easier to use. PMID- 7551962 TI - Autism, mental retardation, multiple exostoses and short stature in a female with 46,X,t(X;8)(p22.13;q22.1). AB - A young adult female with multiple exostoses, short stature, autism, mental retardation and 46,X,t(X;8)(p22.13;q22.1) is described. Although the clinical features and translocation breakpoints raise the possibility of a number of specific conditions, the constellation of problems is not consistent with any previously reported genetic syndrome. It is argued that her clinical disorder is likely due to the chromosomal abnormality and that further detailed molecular genetic investigation may shed light on the genetic basis to various components of her phenotype including the autism. PMID- 7551963 TI - A review and reanalysis of Bruno Schulz's "Erkrankungsalter schizophrener Eltern und Kinder [Age at onset of illness in schizophrenic parents and offspring]:" Zeitschrift fur die gesamte Neurologie und Psychiatrie, 168, 709-721, 1940. AB - Nearly all previous evidence of the familial transmission of age at onset of schizophrenia has been in siblings and twins. In his paper, Bruno Schulz examined the age at onset distribution of schizophrenia in affected parent and offspring pairs, using a systematic series of ascertained cases (n = 106), as well as a second series of chronic in-patients (n = 36). The parent-offspring correlation in age at onset, for cases with a definite diagnosis in the systematically ascertained series, was estimated at 0.346 (95% confidence interval 0.134, 0.528). Schulz did not test for differences between the two series and between males and females, but our reanalysis, using correlational methods and a mixed linear model, did not detect any significant differences. These results are consistent with previous findings that age at onset of schizophrenia is influenced by familial factors which may be genetic. PMID- 7551965 TI - Schizophrenia with karyotype mosaic 47,XXY/48,XXY + 8. AB - A case of severe schizophrenia is described in a patient with chromosomal mosaicism of Klinefelter's syndrome (XXY) and trisomy 8. The literature of both syndromes is reviewed and the aetiological implications discussed. PMID- 7551964 TI - Association study of bipolar disorder using a functional polymorphism (Ser311- >Cys) in the dopamine D2 receptor gene. AB - Several pieces of evidence are consistent with the involvement of dopamine neurotransmission in the aetiology of bipolar disorder. We have tested the hypothesis that the functional mutation Ser311-->Cys of the dopamine D2 receptor gene confers susceptibility to bipolar disorder. There was no increased frequency of the mutation in 82 bipolar probands compared with 72 controls, showing that this mutation is not involved in the pathogenesis of (at least) the vast majority of cases of bipolar disorder. Our findings are consistent with other evidence from linkage and association studies against the involvement of the dopamine D2 receptor in bipolar disorder. PMID- 7551966 TI - A possible locus for manic depressive illness on chromosome 16p13. AB - We have previously reported possible evidence for linkage between manic depressive illness and the locus at 16p13.3 encoding the enzyme phosphoglycolate phosphatase (PGP), in the larger of two Danish families. As PGP was not fully informative, 12 additional DNA markers were tested in these families to clarify if a gene involved in the etiology of manic depressive illness might be located on chromosome 16p13. Though not reaching a lod score level of 3.0, the possible presence of a disease gene for manic depressive illness on chromosome 16p13 was still suggested. The evidence for a dominant locus near PGP was weakened. However, when assuming a recessive mode of inheritance and including both families a two-point lod score of 2.52 was found for marker D16S510, and a three point lod score of 2.65 in both families combined and 2.29 in the large family alone was obtained in the same area. Simulations indicated that lod scores as obtained for several markers in the large family alone, would occur only rarely with an unlinked marker. PMID- 7551967 TI - Association of polymorphic VNTR region in the first intron of the human TH gene with disturbances of the catecholamine pathway in schizophrenia. AB - In the present study, five allelic fragments were typed by a polymerase chain reaction (PCR) process with a pair of primers specific for the tetranucleotide (TCAT) repeat sequence in the first intron of the human tyrosine hydroxylase (TH) gene and their sizes (bp) were 114 (A), 118 (B), 122 (C), 126 (D) and 130 (E), respectively. The AE genotypic frequency was found to be significantly higher in unrelated patients with schizophrenia than in unrelated control subjects (chi 2 = 4.18, p < 0.05). ANOVA revealed a significant difference between the three groups (neuroleptic-free patients possessing or not possessing the AE genotype, and unrelated control subjects) in the concentration of serum noradrenaline (F = 4.96, df = 2.79, p < 0.01), but no significant differences were found between the three groups in the concentrations of serum homovanillic acid, phenylalanine and tyrosine. These results suggest that the polymorphic intron 1 of the human TH gene may be associated with disturbances of the catecholamine pathway in schizophrenia. PMID- 7551969 TI - Portal hypertensive hemorrhage: pathogenesis and risk factors. PMID- 7551968 TI - Searching for complex disease genes: can it be made any easier? AB - The search for complex disease genes is gaining momentum. Recent guidelines for expediting this effort, such as "intelligent" genome scanning and statistical criteria for detecting weak signals indicative of linkage, are discussed, with special reference to psychiatric disorders. PMID- 7551970 TI - Systemic hemodynamic changes in portal hypertension. PMID- 7551971 TI - The role of vasoactive mediators in portal hypertension. PMID- 7551973 TI - Hemodynamic consequences of therapeutic portosystemic shunting: recent experience with transjugular intrahepatic portosystemic shunts. AB - TIPS provides a side-to-side portosystemic shunt without a major abdominal operation. Although TIPS is relatively less invasive, it is associated with similar hemodynamic alterations as surgically created side-to-side shunts. An important difference is that a mild degree of portal hypertension is maintained with TIPS similar to small diameter shunts. TIPS provides effective portal decompression and prevents variceal hemorrhage. However, the risk of encephalopathy is relatively high and stenosis is a long-term concern in many patients. An important advance in the future use of TIPS will be tailoring the diameter of the shunt to optimize regional and systemic hemodynamics in a given individual to minimize the risk of bleeding or to decrease ascites accumulation while limiting the risk of hepatic encephalopathy and liver failure. PMID- 7551972 TI - Pharmacological therapy for portal hypertension: rationale and results. AB - Drug therapy for acute variceal bleeding should be viewed as an adjunct to emergency sclerotherapy. Its role in preventing very early rebleeding (within days) following sclerotherapy needs to be established. The best candidates for such a role are somatostatin and octreotide, but glypressin and vasopressin and nitroglycerin combinations have therapeutic effects in the short-term. Propranolol is the drug for long-term prevention of rebleeding and prevention of the first variceal bleed. For primary prophylaxis it significantly reduces the rate of bleeding, and there is a trend towards reducing mortality. It should be used in cirrhotic patients with large varices. For secondary prophylaxis, propranolol significantly reduces rebleeding but does not improve survival. The reduction in rebleeding is similar to long-term sclerotherapy when compared in randomized studies. There is no value in adding beta-blockers to sclerotherapy compared with sclerotherapy alone, but few studies have evaluated the effects after the eradication of varices. beta-Blockers can be used as the first-line therapy to prevent variceal rebleeding. They also have been shown to reduce the frequency of rebleeding from congestive gastropathy. Many patients do not have a portal pressure reduction with propranolol. The addition of isosorbide mononitrate converts many nonresponders to responders. Current clinical trials are evaluating if therapeutic efficacy is improved by these drug combinations. PMID- 7551974 TI - Evolution of the major histocompatibility complex: a current overview. PMID- 7551975 TI - The emergence of xenotransplantation. AB - The field of transplantation is faced with a growing shortage of human organs as the list of potential recipients continues to increase. Those currently listed can already expect long waits; some die waiting. Xenotransplantation is a potential solution to this widening donor-recipient disparity. Consequently, in recent years, there have been several clinical attempts using organs from nonhuman primates and pigs. The results with nonhuman primates as donors have been encouraging, but it is unlikely that these species will provide a long-term solution to the organ shortage. Most recent xenotransplantation research has therefore shifted to more phylogenetically disparate species, such as pigs, as potential donors. The major barrier to transplantation between members of disparate species combinations has been hyperacute rejection (HAR). The elements of humoral immunity involved in this rejection process include (1) naturally occurring antibodies directed against carbohydrate and other antigens expressed on pig endothelium, and (2) the complement system, which is activated by binding of natural antibodies to their targets. Several elegant strategies to prevent HAR are being developed. The creation of transgenic pigs, whose cells express human regulators of complement activation, is one such strategy. Another promising approach has been to remove antidonor antibodies from the recipient by absorption with some recently characterized carbohydrate epitopes of porcine endothelial xenoantigens. Recent experimental work indicates that HAR can successfully be prevented by inhibition or depletion of complement. A delayed type of xenograft rejection, characterized by endothelial cell antibody deposition and cellular infiltration, occurs over the next three to four days. The likely mechanisms involved in delayed xenograft rejection include antibody-dependent cell-mediated cytotoxicity and the phenomenon of endothelial cell activation. PMID- 7551976 TI - Analysis of donor reactive cells in ALS-treated mice bearing skin allografts after the intrathymic injection of donor specific spleen cells. AB - The intrathymic injection of donor spleen cells into antilymphocyte serum (ALS) treated mice induces significant prolongation of donor skin grafts. The intrathymic route of antigen presentation in this model is superior to the intravenous route in achieving unresponsiveness. To elucidate possible mechanisms involved in the induction of unresponsiveness in ALS-treated mice after intrathymic injection of donor spleen cells, we have analysed the reactivity of lymphoid cells from unresponsive mice in various ways. Deletion of donor reactive cells has been studied using the Mls antigen system. Functional inactivation was analysed by a sequential study of the frequency of donor reactive cells. Suppressor cell activity was studied using an adoptive transfer assay. Deletion of donor reactive cells was partial and occurred largely in the spleen in both early and late stages of unresponsiveness. The frequency of donor reactive cytotoxic cells was suppressed in both spleen and lymph nodes from day +/- 13 until grafts were rejected, with the exception of a rebound period at day +/- 22. In contrast, donor reactive cells were not deleted or inactivated in the thymus. Suppressor cell activity could only be detected in mice bearing long-term grafts. These results suggest that donor reactive cytotoxic cells are functionally inactivated in the spleen and nodes in the early and late stages of unresponsiveness after intrathymic injection of antigen. In contrast, donor reactive cells in the thymus do not appear to be affected. PMID- 7551978 TI - Modulation of interleukin-1 secretion by immunosuppressive drugs, alone and in combination. AB - This study evaluates the ability of the immunosuppressive drugs dexamethasone, cyclosporine, FK506 and rapamycin, alone and in combination to suppress interleukin-1 beta (IL-1 beta) secretion in vitro by THP-1 cells when stimulated by lipopolysaccharide. All four drugs, when added to cell culture medium at therapeutic concentrations, significantly decrease secretion of the monokine to well below control levels. However, only dexamethasone completely suppresses IL-1 beta secretion in a dose-dependent fashion. Cyclosporine, FK506 and rapamycin only partially suppress secretion of IL-1 beta at concentrations within their therapeutic ranges and increasing concentrations of the drugs do not result in further suppression of secretion. Likewise, the combination of any two of these three drugs does not provide any additional suppressive effect. Dexamethasone, however, when added in increasing concentrations in combination with any of the other drugs, results in further suppression of IL-1 secretion in a dose-dependent fashion. These data suggest that cyclosporine, FK506 and rapamycin all share a common effect on the production of IL-1 beta, different from that of dexamethasone. PMID- 7551979 TI - Lack of correlation of soluble E-selectin level with renal transplant rejection. AB - E-Selectin is a 115-kDa cell surface glycoprotein transiently expressed on vascular endothelium in response to interleukin-1 and tumour necrosis factor alpha with a peak in expression at four hours. Its distribution in transplant biopsies has been associated with inflammatory events such as allograft rejection. Recently, a soluble isoform of E-selectin has been detected in the culture medium of cytokine activated endothelial cells by an ELISA method. In this study soluble E-selectin levels in renal allograft recipients were compared with the incidence of rejection, acute tubular necrosis (ATN), cyclosporin A (CyA) toxicity, and use of orthoclone OKT3 (muromonab-CD3) to establish whether early endothelial activation and inflammatory damage could be detected. The mean soluble E-selectin level in normal volunteers was 89 ng/ml serum compared to 120 ng/ml for a group of chronic renal failure patients. Soluble E-selectin levels declined upon transplantation but this was not significant, nor was the difference in samples from patients experiencing rejection, ATN or CyA toxicity. A dramatic and sustained rise in soluble E-selectin levels was found within 24 hours of the first dose of OKT3 treatment. This study shows that soluble E selectin does not provide early unequivocal indication of pathological sequelae in renal transplantation, although extensive endothelial activation can be demonstrated with OKT3 treatment. PMID- 7551977 TI - Failure of intrathymic inoculation of donor-specific splenocytes to prolong cardiac or renal allograft survival in dogs. AB - The intrathymic inoculation (ITI) of donor splenocytes into potential organ transplant recipients has been demonstrated to result in donor-specific unresponsiveness and greatly prolonged survival of subsequent organ allografts in rodents without the need for long-term pharmacological immunosuppressive therapy. We have studied the effect of the ITI of saline (controls) (groups 1 (n = 6) and 3 (n = 6)) or donor splenocytes (groups 2 (n = 10) and 4 (n = 8)) in dogs that received either pharmacological immunosuppression (with cyclosporine and prednisone, +/- azathioprine/cyclophosphamide) (groups 1 and 2) or rabbit anti dog antithymocyte globulin (groups 3 and 4) at the time of ITI. Kidney or heart allografting (from the donor of the splenocytes) was carried out 16-74 days after ITI; all but four transplants were performed within 16-22 days after ITI. Mean kidney allograft survival was 6, 10, 9, and 9 days, respectively, in groups 1-4. Mean cardiac allograft survival was 7, 14, 8, and 7 days, respectively. There was no statistical difference in allograft survival between those dogs that received ITI of saline and those that received donor splenocytes. These results would suggest that the protocols developed to date using ITI in rodent species may not be successful in dogs. PMID- 7551980 TI - Manipulation of skin graft rejection in alloimmune mice by anti-VCAM-1:VLA-4 but not anti-ICAM-1:LFA-1 monoclonal antibodies. AB - C3H mice were immunized by repeated skin grafting with B10.BR tail skin. Ten days after the last immunization mice received 100 micrograms (intravenously) of a variety of different monoclonal antibodies (mAbs: anti-ICAM-1, -LFA-1, -VCAM-1, VLA-4), alone or in combination, followed by further B10.BR skin grafts. Control animals received injections of saline only. Skin graft survival was monitored daily in all groups. Further injections of antibody were given every second day until graft rejection occurred. In separate studies lymphoid cells were harvested from various tissues of the grafted mice at 6 and 20 days post grafting. Aliquots of each sample were analysed by polymerase chain reaction for mRNA for different cytokines (interleukins IL-2, IL-4, IL-10 and IFN gamma (gamma-interferon)) believed to be important in the regulation of graft rejection. In addition, lymphoid cells were restimulated in vitro with irradiated B10.BR or third-party stimulator cells in the presence or absence of monolayers of C3H-derived endothelial cells (EC), in an attempt to mimic the in vivo environment of the interactions of cells engaged in alloreactivity in these mice. Only anti-VCAM-1 caused significant prolongation of graft survival in immune mice, while in contrast only the combination of anti-ICAM-1 and anti-LFA-1 produced enhanced survival in naive animals. In each case increased survival was associated with decreased activation of Th1 cells (diminished IL-2, IFN gamma) and increased activation of Th2 cells (increased IL-4, IL-10).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7551981 TI - Nutrition and allorejection impact of lipids. AB - Polyene n-3 and n-6 fatty acids are claimed to have immunomodulating properties. The impact of nutritional variations on transplant rejection was therefore studied in the heterotopic rat heart allotransplant model with particular focus on lipids. Twenty per cent fat emulsions with differing n-3/n-6 fatty acid ratios were continuously infused (9 g fat per kg bodyweight per day; n = 10 in each group) after transplantation until rejection: safflower oil (n-3/n-6 = 1/370), fish oil (7.6/1), soybean oil (1/6.5) and a 1:1 mixture of safflower and fish oil (1/2.1; oil control group). Graft survival time, subpopulations of infiltrating and circulating immunocompetent cells and interleukin-6 release by circulating mononuclear cells were analysed. In the safflower oil, fish oil and soybean oil groups graft survival was prolonged to 13.3, 12.3 and 10.4 days vs. 6.7 days in the oil control group and 7.8 days in the saline control group (p < 0.01). In the two groups with the highest prolongation of graft survival the number of infiltrating cells was reduced by up to 50% and the peripheral blood mononuclear cell interleukin-6 release by up to 45%. Beyond that, circulating T-cells were reduced in the fish oil group. The n-3/n-6 fatty acid ratio determines the immunomodulating effect of lipids. Both n-6 and n-3 fatty acids, if applied as the main fatty acid source, exert immunosuppressive effects by diminished infiltration, mobilization and cytokine release by immunocompetent cells. A n-3/n 6 fatty acid ratio of 1/2 proved to be immunologically neutral. The recipient's disposition to reject an allotransplant is influenced by the fatty acid composition of exogenously supplied lipids. PMID- 7551982 TI - Pre-transplant anti-epithelial cell antibodies and graft failure after single lung transplantation. AB - Eighty-five patients undergoing single lung transplantation have been studied to determine the presence of anti-epithelial cell antibodies (AECA) prior to transplantation using the human lung carcinoma epithelial cell line A549 in a microcytotoxicity assay. In addition, 29 healthy volunteers were also assayed for the presence of AECA. Twenty-seven of the 85 recipients exhibited AECA prior to transplantation compared to none of the 29 control subjects (p = 0.0001). Actuarial graft survival at 1 year was 78% for the AECA negative group compared to 56% for AECA positive recipients (p = 0.01). No correlation was seen between the presence of AECA and graft rejection as determined by transbronchial biopsy. However, there was an association between AECA and post-transplant infection (p = NS) where 16 (64%) of the AECA positive recipients had postoperative infection episodes compared to 25 (47%) of the negative recipients. Sodium dodecylsulphate polyacrylamide gel electrophoresis and Western blotting was also performed for 68 of the recipients and antibody reactivity was detected in 22 patients compared to 26 patients exhibiting AECA detectable by microcytotoxicity. The presence of AECA demonstrable by Western blotting did not correlate with graft survival, rejection or infection. In conclusion, AECA detectable prior to single lung transplantation are associated with a decrease in graft survival and with postoperative infections. PMID- 7551983 TI - Allogeneic lymphocyte chimerism after clinical lung transplantation. AB - Donor lungs contain large amounts of passenger leukocytes which are transferred to the recipient by organ transplantation. In this study we have analysed the fate of these cells and have studied the populations of donor leucocytes detectable in the blood circulation of ten lung transplanted patients during the first postoperative weeks. To this aim we have applied immunocytological as well as flow cytometric analyses using monoclonal antibodies against polymorphic HLA class I antigens that differed between donor and recipient as well as antibodies against cell differentiation markers. The results demonstrate that donor cells can be detected in the circulation of all lung transplanted patients but there is a considerable interindividual variability between 0.9% and 17.5% (mean 5.1%) on postoperative day 3. Cells were usually detectable for 2-4 weeks and had disappeared in all patients after 1 month. The circulating donor cells consisted exclusively of lymphocytes. T cells were the predominant population, most of which seemed to be CD45R0+, but B and NK (natural killer) cells were also present. Probably due to the small numbers of patients studied no correlation between clinical parameters and the extent of donor lymphocyte persistence; there were no clinical graft-versus-host reactions. The findings demonstrate the regular existence of a transient (macro)chimerism due to passenger lymphocytes in the early phase after lung transplantation. The immunological function and the relation between this phenomenon and the long-term microchimerism which frequently develops after solid organ transplantation remain unclear. PMID- 7551984 TI - Bronchoalveolar lavage fluid endotoxin elevation in human single lung transplant recipients during rejection. AB - Endotoxin has been implicated as an aetiological factor in liver transplant rejection and acute graft-versus-host disease. To investigate the role of endotoxin in human single lung transplant rejection we measured the level of endotoxin in bronchoalveolar lavage (BAL) fluid from six subjects at baseline and during rejection, which was defined histologically from transbronchial biopsy. Differential cell counts of BAL fluid cells, the levels of protein and albumin in BAL fluid, and serum albumin levels were also examined. BAL fluid albumin to serum ratio was calculated to evaluate alveolar-capillary leakage. A significant elevation of BAL fluid endotoxin with rejection compared with baseline was observed. Standardizing endotoxin levels to BAL fluid volume, protein, or albumin were all of similar significance. Examination of BAL fluid cell population revealed a significant elevation in the percentage of lymphocytes with rejection. No significant difference between BAL fluid protein levels, BAL fluid albumin levels. BAL fluid albumin to protein ratio, serum albumin levels, or BAL fluid albumin to serum albumin ratio was seen with rejection. We conclude that BAL fluid endotoxin levels increase during lung rejection, endotoxin levels can be accurately standardized to millilitres of BAL fluid, and abnormal alveolar capillary leakage does not appear to account for the increased BAL fluid endotoxin. PMID- 7551986 TI - Pancreatic trauma. PMID- 7551987 TI - Induction of tolerance after transplantation. PMID- 7551988 TI - Transjugular intrahepatic portasystemic stent-shunt in the treatment of variceal haemorrhage. AB - The transjugular intrahepatic portasystemic stent-shunt (TIPSS) is a side-to-side portocaval shunt, performed by interventional radiological methods, linking the hepatic and portal veins through the liver parenchyma with an expandable metal stent. The technique can be performed successfully in over 90 per cent of patients. The procedure-related mortality rate is about 1 per cent, mainly from intraperitoneal bleeding. The main indications for TIPSS insertion are control of acute variceal bleeding in patients with cirrhosis that is refractory to sclerotherapy and recurrent variceal haemorrhage despite sclerotherapy or band ligation. TIPSS insertion is followed by variceal rebleeding in about 10-20 per cent of cases, encephalopathy in 10-20 per cent, transient deterioration of liver function in 25-35 per cent and subsequent shunt dysfunction over a 6-12-month period in 15-60 per cent. The final place of TIPSS insertion in the management of portal hypertension is being evaluated in controlled studies, but its use in the treatment of uncontrolled variceal haemorrhage seems assured. PMID- 7551989 TI - Local excision of rectal tumours. AB - Local excision of colorectal tumour may be palliative or curative. Recent advances in minimal access techniques have allowed curative excision to be offered to a wider range of patients. Absolute indications for potentially curative local excision include mobile tumours, T1 tumours (assessed by ultrasonography), well or moderately differentiated histology (determined by biopsy) and tumour size less than 3 cm. Relative indications include T2 and T3 tumours (by ultrasonography), poorly differentiated histology (by biopsy) and tumour size greater than 3 cm depending on patient fitness. The rationale for these recommendations is described in detail. PMID- 7551985 TI - Effects of FK 506 on chimerism and the induction of donor-specific unresponsiveness following fully allogeneic bone marrow transplantation in mice. PMID- 7551990 TI - Urge incontinence of faeces is a marker of severe external anal sphincter dysfunction. AB - A proportion of patients with faecal incontinence experience debilitating urgency of defaecation and urge incontinence. This study prospectively assessed 56 faecally incontinent patients by means of standard interview, physical examination and anorectal physiology. Patients with urge incontinence, when compared with those without urge incontinence, were symptomatically worse, had had more vaginal deliveries and had more bowel actions each day. Physiological tests included anal manometry, anal electrosensitivity, pudendal nerve terminal motor latency and a standard proctometrogram. The only physiological differences between the groups were a reduction in voluntary squeeze pressure (median (interquartile range (i.q.r.)) 43 (26-67) versus 67 (45-122) mmHg, P = 0.01) and a smaller percentage change in pressure-volume, an integral of sphincter length and squeeze (median (i.q.r.) 43.5 (0-289) versus 247 (71-455), P = 0.02), in those with urgency. The authors conclude that urge incontinence is associated with impairment of the striated musculature of the anal sphincter complex. PMID- 7551992 TI - Manipulation of the L-arginine-nitric oxide pathway in experimental colitis. AB - The role of the L-arginine-nitric oxide pathway in the pathogenesis of colonic inflammation was assessed using L-arginine and its competitive analogue N omega nitro-L-arginine methyl ester (L-NAME) in a rat model of colitis. In the first study oral L-arginine 2 per cent (control: 3.4 per cent L-glycine) was administered with and without L-NAME 100 mg/l. Orally administered L-arginine increased colonic inflammation (P = 0.004) and decreased thymic weight (P = 0.0007). Addition of L-NAME reduced the colonic inflammation and prevented loss of body-weight (P < 0.04). In the second study L-NAME was administered orally in concentrations of 100, 200 and 500 mg/l (control: no L-NAME). L-NAME 500 mg/l reduced colonic inflammation and increased thymic weight and body-weight (P < 0.01). Thymic weight and body-weight correlated positively with the concentration of L-NAME administered orally (rs > or = 0.3, P = 0.04). L-NAME l g/l was administered topically as an enema (control: suspension agent). Topical L-NAME reduced colonic inflammation and increased thymic weight (P < 0.05). These results suggest that the L-arginine-nitric oxide pathway mediates colonic inflammation in this model. PMID- 7551991 TI - Nitrergic inhibitory innervation of porcine rectal circular smooth muscle. AB - This study was performed to determine whether nitric oxide contributes to inhibitory non-adrenergic non-cholinergic (NANC) neurotransmission in the circular smooth muscle layer of pig rectum. Smooth muscle strips were mounted for isometric tension recording in superfusion organ baths. In the presence of atropine sulphate and guanethidine, transmural electrical field stimulation (EFS) produced frequency-dependent relaxation of precontracted muscle strips. N omega nitro-L-arginine (L-NOArg), a powerful competitive antagonist of nitric oxide synthase, reduced the relaxant response in dose-dependent fashion; this response was maximally reduced by mean(s.e.m.) 86.5(2.6) per cent (P = 0.0003) at a concentration of 3 x 10(-5) mol/l L-NOArg. The response was restored by the addition of L-arginine. Sodium nitroprusside, an exogenous donor of nitric oxide, mimicked the relaxant response. Responses to EFS were abolished by tetrodotoxin. These results suggest that inhibitory NANC neurotransmission in this tissue is mediated, at least in part, by nitric oxide. The failure of L-NOArg to completely abolish the relaxant response suggests that additional neurotransmitters may be involved. PMID- 7551994 TI - Cardiac ischaemia induces vagally mediated lower oesophageal sphincter relaxation. AB - Gastro-oesophageal reflux and coronary artery disease frequently coexist. Direct stimulation of myocardial vagal receptors impairs lower oesophageal sphincter (LOS) function but the effect of cardiac ischaemia has not been examined. Eight adult mongrel dogs were studied under general anaesthesia. Each underwent occlusion of the left circumflex coronary artery before and after bilateral cervical vagotomy. Blood pressure, heart rate and LOS responses were measured. Median (range) LOS tone was significantly reduced by coronary artery occlusion, from 9 (6-14) to 6 (3-8) sphinctometer units (P < 0.01). This was accompanied by a reduction in heart rate (P < 0.05) and blood pressure (P < 0.01). After bilateral cervical vagotomy the same ischaemic insult produced no significant alteration in LOS tone or heart rate. These data suggest a direct vagal reflex to explain the high incidence of gastro-oesophageal reflux in patients with coronary artery disease. PMID- 7551993 TI - In vitro regulation of human breast cancer cell adhesion and invasion via integrin receptors to the extracellular matrix. AB - The extracellular matrix consists of the interstitium and the basement membrane. Cellular interaction with fibronectin, laminin and collagen provides a possible mechanism by which cancer cells adhere, invade and metastasize. The integrins are a major family of adhesion molecules that recognize epitopes on the extracellular matrix as ligands. These include the alpha 2 beta 1, alpha 3 beta 1, alpha v beta 1 and alpha v beta 5 integrins, most of which were found to be expressed on MCF 7, T47D, MDA-MB-231, ZR75-1 and Hs578T breast cancer cell lines. Each cell line adhered to the matrix proteins in a dose-dependent manner and was inhibited by monoclonal antibodies against relevant integrins. Only Hs578T was significantly invasive through fibronectin but both Hs578T and MDA-MB-231 invaded through laminin and type IV collagen in an in vitro assay. The invasive potential of these cell lines could be inhibited by integrin antibodies added to cells before incubation, but the addition of antibodies after cells were allowed to adhere to the matrix failed to inhibit invasion. Inhibition of cellular adhesion to the matrix reduced the invasive potential of breast cancer cell lines. As integrin antibodies inhibit cell invasion in vitro, the integrins may be of potential value as antitumour therapeutic agents. PMID- 7551995 TI - Observer variation in assessment of quality of life in patients with oesophageal cancer. AB - Quality of life (QOL) assessment may improve the evaluation of treatment for oesophageal cancer but patient compliance using self completion questionnaires is often poor. The use of a proxy to estimate QOL might improve data collection from patients who are either unable or unwilling to complete the questionnaire. This study examined whether a doctor or patients' carers could accurately assess QOL using the QLQ-C30 questionnaire developed by the European Organisation for Research and Treatment of Cancer Study Group on Quality of Life. One doctor, 52 patients and 39 carers completed the instrument independently. Proxy and patient ratings were analysed for agreement using weighted kappa scores. Agreement was poor or moderate in most QOL scales and items (kappa < or = 60). This study demonstrates that a carer or doctor is not sufficiently accurate to postulate the patient's responses to the questionnaire. Quality of life data, therefore, should come from the patients themselves. PMID- 7551996 TI - Gastric juice epidermal growth factor concentration and Helicobacter pylori in patients with duodenal ulcer. AB - The level of epidermal growth factor (EGF) was measured in the basal and maximally stimulated gastric juice of 20 control subjects and 20 patients each with duodenal ulcer and non-ulcer dyspepsia. Basal gastric juice was analysed for ammonia and urea concentrations, and the [ammonia]3/[urea] ratio was used to show Helicobacter pylori status, as was the [13C]urea breath test in nine controls. There was complete concordance in the nine controls between the two methods for determining H. pylori status. Twenty-five subjects were H. pylori positive (seven with duodenal ulcer, nine with non-ulcer dyspepsia, nine controls) and 35 H. pylori negative (13, 11 and 11 respectively). In H. pylori-positive subjects, the median EGF concentrations in the stimulated secretion of patients with duodenal ulcer and without (non-ulcer dyspepsia and controls combined) were 46.7 and 18.0 ng/ml (P < 0.001), and in H. pylori-negative subjects were 40.0 and 26.5 ng/ml respectively (P < 0.01). There was no difference in EGF concentration between controls and subjects with non-ulcer dyspepsia irrespective of H. pylori status. Lack of EGF is unlikely to be a cause of duodenal ulcer. The increased EGF concentration in patients with ulcer bore no relationship to the H. pylori status of the individual. If this bacterium causes duodenal ulcer, it is not via a reduction in EGF concentration. PMID- 7551997 TI - Bladder injury during laparoscopic abdominoperineal resection. PMID- 7551998 TI - Primary soft tissue sarcoma of the extremities in adults. AB - The modern literature on adult extremity sarcoma implies that adjuvant therapy (usually irradiation) is mandatory for adequate local control, at least in patients with high-grade tumours undergoing limb-sparing procedures. In 152 primary extremity soft tissue sarcomas, wide or radical resection was employed (116 patients) including amputation in nine patients (6 per cent), or local excision followed by adjuvant postoperative radiation therapy (36). Local recurrence alone occurred in 5 per cent of patients, and in combination with distant metastases in 9 per cent. The total rate of local recurrence was 10 per cent after wide resection (with or without chemotherapy) and 25 per cent after conservative resection and radiotherapy (with or without chemotherapy). Limb sparing was possible in 94 per cent of patients. The majority (76 per cent) had surgical resection alone as local treatment with satisfactory results. Wide resection, when feasible, provides acceptable local control and may be preferable to local excision plus radiation therapy. PMID- 7551999 TI - Abdominoperineal resection and anterior resection in the treatment of rectal cancer: results in relation to adjuvant preoperative radiotherapy. AB - The outcome of patients with rectal cancer treated by abdominoperineal or anterior resection, with or without preoperative radiotherapy, was assessed to detect any differences attributable to the operative method and interactions between radiotherapy and type of surgery. The study was based on 1292 patients included in two consecutive controlled randomized trials of preoperative radiotherapy in operable rectal carcinoma. The outcome was not related to surgical method. Radiotherapy increased postoperative mortality and complications and reduced local and distant recurrence, but had no effect on overall survival. Effects of radiotherapy were similar irrespective of the type of surgery, except that the increase in postoperative mortality in irradiated patients was greater in those treated with abdominoperineal resection. Sphincter-saving procedures appear to have no adverse effects on outcome of rectal cancer, but the optimum use of radiotherapy is still to be defined. PMID- 7552000 TI - Clinical outcome and restenosis following percutaneous transluminal angioplasty for ischaemic rest pain or ulceration. AB - The role of percutaneous transluminal angioplasty in the management of severe leg ischaemia is controversial. To investigate further the efficacy of angioplasty and the clinical consequences of restenosis, a randomly selected cohort of 29 patients with ischaemic rest pain or ulceration was studied for 6 months after a technically successful balloon angioplasty. All patients had digital subtraction arteriography at the end of follow-up. Seven of 15 patients undergoing the procedure for rest pain had sustained relief from the initial dilatation. Partial or complete healing was noted in all 14 patients with ulceration and was maintained at 6 months in 11 despite significant (greater than 30 per cent) restenosis at the angioplasty site in eight. There were no complications or clinical deterioration associated with the procedure. Angioplasty is an effective method for treating the severely ischaemic leg, especially when used to achieve ulcer healing; restenosis is often clinically unimportant. PMID- 7552001 TI - Insonation and impedance analysis in graft surveillance. AB - Seventy consecutive patients with infrainguinal bypass grafts entered a 1-year graft surveillance programme involving colour duplex scanning, direct graft insonation and computer-assisted impedance analysis. Graft patients with a positive duplex scan, high frequencies on graft insonation or an impedance value above 0.50 subsequently underwent arteriography. Sixteen patients were excluded before the initial surveillance visit. The 54 remaining patients with grafts (30 vein, 24 synthetic) underwent a total of 137 surveillance visits, with 21 grafts confirmed to be 'at risk'. The sensitivity of an impedance value above 0.55 in identifying these grafts was 86 per cent, rising to 95 per cent when combined with graft insonation. Duplex scanning did not identify any abnormalities in 11 grafts that were either shown by arteriography to be 'at risk' or occluded before arteriography. Impedance measurement and graft insonation are simple screening techniques with a high sensitivity (when combined), which identify 'at risk' infrainguinal grafts. Positive graft insonation or an impedance value over 0.55 will identify all 'at risk' vein grafts while minimizing the number of unnecessary arteriograms. PMID- 7552002 TI - Postocclusive hyperaemic duplex scan: a new method of aortoiliac assessment. AB - Aortoiliac duplex scanning, while accurate, is time-consuming and technically demanding. This study aimed to develop a fast, non-invasive screening test for aortoiliac disease. Colour duplex scanning was used to record common femoral Doppler ultrasonographic waveforms following 3 min of arterial occlusion using a thigh cuff in 25 patients with normal aortoiliac segments and 25 patients with significant aortoiliac disease. The latter patients had a prolonged period of postocclusive hyperaemic flow compared with the former. End diastolic velocity, 70 s after cuff release, was a significant discriminant between the two groups (sensitivity of 88 per cent, accuracy of 92 per cent). The postocclusive hyperaemic duplex (PHD) test performed well when used prospectively in a further 50 limbs (sensitivity of 86 per cent, accuracy of 84 per cent). The test was more sensitive than femoral pulse palpation and compared favourably with arteriography. The PHD test provides a simple, noninvasive assessment for aortoiliac disease that can be performed on the initial outpatient clinic visit. PMID- 7552003 TI - Treatment of iatrogenic false aneurysm by compression ultrasonography. PMID- 7552004 TI - Percutaneous transluminal angioplasty for lower-limb critical ischaemia. AB - Percutaneous transluminal angioplasty (PTA) alone was used to treat 54 (23 per cent) of 232 lower limbs with critical ischaemia. Technical success was achieved in 49 cases (91 per cent) with an immediate symptomatic improvement in 47 (87 per cent). There was no death or limb loss attributable to PTA and three embolic complications were successfully treated by percutaneous aspiration. Symptomatic improvement and haemodynamic patency rates at 24 months were 77 and 78 per cent respectively; 27 of 34 limbs with ulceration or gangrene had healed with minor surgery by a median of 7.5 (range 3.0-18.0) months. Patient survival and limb salvage rates at the same time interval were 76 and 89 per cent respectively. These results demonstrate that, for a selected group of patients with lower-limb critical ischaemia, PTA can be a highly successful therapeutic option with a low associated morbidity. PMID- 7552006 TI - Distal pancreatectomy for gunshot injuries of the distal pancreas. AB - This study comprised 57 patients with gunshot injury of the distal pancreas. There were 16 grade II, 29 grade III and 12 grade IV pancreatic injuries. The intraoperative mortality rate was 12 per cent. Patients with the most severe grade II injuries and all those with grade III and IV injuries had distal pancreatectomy and splenectomy, with a 14 per cent fistula formation rate and 2.3 per cent postoperative mortality rate directly related to the pancreatic injury. The remaining patients with grade II injuries were managed by debridement and drainage; there were no fistulas or deaths. The method of closure of the pancreatic resection margin is unrelated to fistula formation, and identification of the duct for ligation is unnecessary. Liberal use of distal pancreatectomy with splenectomy for gunshot injuries of the distal pancreas is suggested. PMID- 7552005 TI - Favourable prognosis after surgical drainage of gunshot, stab or blunt trauma of the pancreas. AB - The records of 152 patients with pancreatic injury treated over a 5-year period were reviewed. The diagnosis was made at laparotomy in all patients. Gunshot wounds, stab wounds and blunt trauma occurred in 63, 66 and 23 patients respectively with mean ages of 28, 28 and 30 years. Multiple organ injury was most common after gunshot wounds. Intraoperative management was by drainage of the pancreatic injury site alone in the majority of patients in all aetiological groups. The rate of fistula formation was 14 per cent after gunshot wounds, 9 per cent after stab injury and 13 per cent after blunt trauma. Death occurred after 24 h in 8, 2 and 10 per cent of patients following gunshot wounds, stab wounds and blunt trauma respectively, and was attributable to other organ damage. It is concluded that gunshot injury to the pancreas may be more extensive than other injuries, but conservative management with surgical drainage of pancreatic injury is justified irrespective of the mechanism of injury. PMID- 7552007 TI - Trauma and bacterial translocation. PMID- 7552008 TI - Pain relief after inguinal hernia repair: a randomized double-blind study. AB - A randomized double-blind study was undertaken using 0.5 per cent bupivacaine ilioinguinal field block and oral papaveretum-aspirin tablets to assess pain relief after hernia surgery. A consecutive series of 200 men undergoing repair of a unilateral inguinal hernia underwent random allocation into one of the four groups to receive: bupivacaine and papaveretum-aspirin (group 1), bupivacaine and oral placebo (group 2), saline and papaveretum-aspirin (group 3), or saline and oral placebo (group 4). Patients were prescribed postoperative opiates to be given on demand. Pain levels and mobility were assessed at 6 and 24 h after operation. Patients in group 1 reported significantly less pain, required less additional opiates and had better mobility than those in group 4 (pain score P < 0.001 at 6 h and P = 0.002 at 24 h) and group 3 (P = 0.002 for pain and mobility scores at 6 h). Bupivacaine alone provided good immediate postoperative pain relief (P = 0.002 group 2 versus group 4 at 6 h). The combination of bupivacaine and papaveretum-aspirin provided the best results and is suitable for day-case postoperative analgesia. PMID- 7552009 TI - Preoperative chemotherapy for stage III-IV gastric carcinoma: feasibility, response and outcome after complete resection. AB - Despite extensive resection and systematic lymphadenectomy the prognosis of patients with locally advanced gastric carcinoma remains poor. The effect of preoperative outpatient chemotherapy with etoposide, doxorubicin and cisplatin was evaluated prospectively in 30 patients who had been shown by preoperative staging (including endosonography and surgical laparoscopy) to have gastric carcinoma stages IIIA, IIIB or IV. Haematological side-effects were common and necessitated hospitalization in 13 of 30 patients. Complete clinical response to neoadjuvant therapy was observed in eight of 27 evaluable patients. Resection was performed in 27 of 30 patients, with complete macroscopic and microscopic tumour removal in 24. There were no deaths and no major morbidity following operation. On multivariate analysis complete clinical response (P < 0.01) and complete tumour resection (P < 0.01) were the major independent predictors of long-term survival after neoadjuvant chemotherapy. Actuarial survival after complete tumour removal was superior with neoadjuvant therapy compared with results in an age-, sex- and tumour stage-matched control population who had primary resection (P = 0.07). Recurrence occurred in 17 of 23 evaluable patients who had complete tumour removal, with relapse in the tumour bed or area of lymphatic drainage in 11. These data show that neoadjuvant therapy in patients with locally advanced gastric carcinoma is feasible and appears to increase the rate of complete tumour removal. More powerful and less toxic regimens are, however, required to improve the response rate and to delay or avoid recurrence after neoadjuvant chemotherapy. PMID- 7552011 TI - Macroscopically node-negative but histologically node-positive gastric carcinoma. AB - The features of macroscopically node-negative but histologically node-positive (N(-) n(+)) tumours were examined in 521 patients undergoing curative surgery for gastric carcinoma. Of 240 histologically node-positive tumours, 62 (26 per cent) were N(-) n(+). The 10-year survival rate of patients with N(-) n(+) tumour at 66 per cent was worse than that of those with histologically node-negative tumour (91 per cent) (P < 0.01) but better than that of patients with macroscopically and histologically node-positive tumour (40 per cent) (P < 0.01). N(-) n(+) tumour was characterized by size less than 5 cm (47 per cent), invasion not beyond the muscularis propria (53 per cent), lymph node metastasis limited to group 1 nodes (76 per cent), number of positive nodes fewer than seven (90 per cent) and disease of stage II (52 per cent). The survival of patients with N(-) n(+) tumour was determined by the number of positive nodes and stage of disease. N(-) n(+) tumours are associated with a better outcome, owing to less progressive disease, and the number of positive nodes is a prognostic indicator. PMID- 7552010 TI - Two-layer closure of typhoid ileal perforations: a prospective study of 46 cases. PMID- 7552012 TI - Incidence and management of laser-associated oesophageal perforation. AB - The incidence of oesophageal perforation in 350 patients referred for laser treatment to palliate malignant dysphagia is reported. Perforation occurred in 6 per cent of patients or 2 per cent of treatment episodes. Perforation was usually due to oesophageal dilatation undertaken before laser treatment (23 of 25 perforations). Immediate recognition and the institution of an aggressive non operative management protocol resulted in survival in 20 of 23 patients. PMID- 7552014 TI - Intraoperative risk factors associated with hepatic resection. AB - Risk factors associated with complications following hepatic resection were investigated retrospectively in 121 patients between January 1987 and April 1992. Fifty-seven patients recovered uneventfully, but 64 suffered post-operative complications and 15 died within 3 months. All those who died had suffered from hyperbilirubinaemia or bleeding and/or coagulopathy, which were considered critical complications after hepatic resection. Risk factors following hepatic resection were investigated statistically. Stepwise logistic regression analysis showed that serum levels of cholinesterase, the histology of the remaining liver and the volume of intraoperative blood loss were significantly associated with major complications (odds ratio 0.02, 5.14 and 4.97 respectively). Coexisting liver cirrhosis, deterioration of liver protein synthesis and massive intraoperative blood loss are important risk factors following hepatic resection. PMID- 7552013 TI - Intraoperative radiotherapy for pancreatic carcinoma. AB - Intraoperative radiotherapy (IORT) with an electron beam is a therapeutic modality employed predominantly in tumours with a high risk of local recurrence. In this department 35 patients with proven adenocarcinoma of the pancreas underwent surgery and IORT, while 41 patients underwent surgery only. Irradiated patients suffered more serious postoperative complications (21 per cent versus 7 per cent), spent more time in hospital (32 days versus 26 days) and did not show a survival advantage (median of 326 days versus 366 days for two patients with curative resection). Anastomotic breakdown (twp patients), abscess formation (two patients) and pancreatitis in the pancreatic remnant (one patient) occurred only in this group. It is concluded that IORT may increase the risk of postoperative complications. As, on the basis of experimental data, an additional serious long term risk is to be expected, application of IORT should be limited to well controlled study conditions. PMID- 7552017 TI - Percutaneous cholecystostomy: a valuable technique in high-risk patients with presumed acute cholecystitis. AB - Percutaneous cholecystostomy offers a potentially important therapeutic modality for critically ill patients with acute cholecystitis who represent a high risk for general anaesthesia. The aim of the study was to assess experience with percutaneous cholecystostomy in resolving the acute episode of cholecystitis without operative intervention. Twenty-two consecutive patients with a clinical diagnosis of acute cholecystitis underwent the procedure. All were at high risk for general anaesthesia, and all but one developed cholecystitis while hospitalized for another co-morbid condition; 14 were in an intensive care unit. Twenty-one of the 22 patients proved to have acute cholecystitis (11 acalculous, ten cholelithiasis). There were no acute technical complications. Toxaemia resolved in 17 of the 21 patients with acute cholecystitis. Acute cholecystitis failed to resolve in three patients; all died within 48 h from overwhelming generalized sepsis. One patient required emergency cholecystectomy for bile peritonitis when the cholecystostomy catheter became dislodged 24 h after placement. The 60-day mortality rate for the acalculous and calculous patient groups was 55 and 20 per cent, respectively. Only three interval cholecystectomies have been performed at a mean follow-up of 19 months. In conclusion, percutaneous cholecystostomy may be the procedure of choice for the management of acute cholecystitis in the very high-risk critically ill patient. If symptoms fail to resolve quickly, ongoing sepsis, cholangitis or gallbladder necrosis should be suspected. PMID- 7552016 TI - Randomized controlled multicentre study of the prevention of complications by octreotide in patients undergoing surgery for chronic pancreatitis. AB - A randomized double-blind placebo-controlled multicentre trial was carried out in 247 patients undergoing major elective surgery for chronic pancreatitis to clarify whether the perioperative application of octreotide prevents postoperative complications. Eleven complications were defined, including death, anastomotic leakage, pancreatic fistula, abscess, fluid collection, shock, sepsis, bleeding, pulmonary insufficiency, renal insufficiency and postoperative pancreatitis. A total of 124 patients underwent pancreatic head resection, 55 left resection, 61 pancreaticojejunostomy and seven had other procedures. The overall mortality rate was 1.2 per cent (octreotide group 1.6 per cent, placebo group 0.8 per cent [corrected] (P not significant)). The postoperative complication rate in the octreotide group was 16.4 per cent (20 of 122 patients) and in the placebo group 29.6 per cent (37 of 125) (P < 0.007). The perioperative application of octreotide substantially reduces the risk of postoperative complications in patients undergoing major pancreatic surgery for chronic pancreatitis. PMID- 7552018 TI - Surgical pathology of human immunodeficiency virus infection: lessons from Africa. PMID- 7552019 TI - Use of a feeding jejunostomy after oesophagogastric surgery. PMID- 7552021 TI - Endoscopically guided surface repair of inguinal hernia. PMID- 7552022 TI - Endoscopically guided surface repair of inguinal hernia. PMID- 7552020 TI - Efficacy of glutamine-enriched enteral nutrition in an experimental model of mucosal ulcerative colitis. PMID- 7552024 TI - Recurrent inguinal hernia after laparoscopic repair: possible cause and prevention. PMID- 7552015 TI - Prospective evaluation in 121 consecutive unselected patients undergoing laparoscopic treatment of choledocholithiasis. AB - Laparoscopic common bile duct (CBD) exploration was attempted in 115 of 121 consecutive unselected patients with choledocholithiasis (mean age 69 (range 21 92) years) found during routine intraoperative cholangiography. The CBD was successfully cleared of all stones in 100 patients (87 per cent). Ten of 11 patients referred for surgery after failure of endoscopic sphincterotomy had complete laparoscopic choledocholithiasis. Eleven patients (10 per cent) required conversion to open CBD exploration, and laparoscopic exploration was not attempted in six (5 per cent) because of inflammation or fibrosis. Postoperative endoscopic sphincterotomy was required in four patients (4 per cent) for retained stones after laparoscopic exploration. There were no postoperative deaths (39 per cent of patients were aged 75 years or more). Routine intraoperative cholangiography, and when required laparoscopic CBD exploration, should be compared in randomized trials with preoperative endoscopic retrograde cholangiography in patients with suspected choledocholithiasis. PMID- 7552023 TI - Enhanced intra-anastomotic healing by operative lavage with nutrient solutions in experimental left-sided colonic obstruction. PMID- 7552025 TI - Retroperitoneal endoscopic adrenalectomy. PMID- 7552027 TI - New adjustable knot for securing subcuticular running sutures. PMID- 7552026 TI - Low molecular weight heparin started before surgery as prophylaxis against deep vein thrombosis: 2500 versus 5000 XaI units in 2070 patients. PMID- 7552028 TI - Randomized controlled trial of appendicectomy versus antibiotic therapy for acute appendicitis. PMID- 7552029 TI - Definition of local recurrence after surgery for rectal carcinoma. PMID- 7552030 TI - Gastric carcinoma in patients over 70 years of age. PMID- 7552031 TI - Influence of temporary faecal diversion on long-term survival after curative surgery for colorectal cancer. PMID- 7552032 TI - Outcome of patients with abdominal sepsis treated in an intensive care unit. PMID- 7552033 TI - Prognostic relevance of systemic lymph node dissection in gastric carcinoma. PMID- 7552035 TI - Modulation of the cytokine and acute-phase response to major surgery by recombinant interleukin 2. PMID- 7552036 TI - Regional taste sensitivity to NaCl: relationship to subject age, tongue locus and area of stimulation. AB - Using a signal detection procedure and a microprocessor-controlled gustometer, sensitivity to three concentrations of NaCl (0.01, 0.1 and 1.0 M) was measured on the tongue tip, and on a region 3.0 cm posterior to the tongue tip in 12 young (20-29 years of age) and 12 elderly (70-79 years of age) subjects. Stimulus duration was 2 s; the sizes of the tongue areas stimulated were 12.5, 25 and 50 mm2. On average, the young subjects were more sensitive to NaCl on the tongue tip than on the more posterior stimulation site and exhibited, at both tongue loci, an increase in detection performance as stimulus concentration increased. The elderly subjects, on the other hand, performed at chance level at both tongue loci for all three stimulus sizes and concentrations tested. No sex differences were observed. In the young subjects, the mean R-index increased monotonically as a function of stimulus size for the two highest NaCl concentrations at both tongue loci. A hyperbolic function provided the best fit to these data at the tongue tip, and an exponential function at the more posterior tongue region, implying a different pattern of spatial summation at these two sites. Overall, this study demonstrates that marked age-related changes in regional taste sensitivity to NaCl are present in elderly persons. PMID- 7552034 TI - Laparoscopic caecopexy for caecal volvulus. PMID- 7552038 TI - Taste discrimination using alternative solvents for PTC and NaCl. AB - Better discrimination was possible between threshold PTC solutions and pure solvent when the solvent was a tasteless, low concentration NaCl solution to which the subject had adapted, than when the solvent was purified water. The reverse was true for threshold NaCl stimuli. Subjective reports indicated this to be due to the absence or presence of taste cues from the solvent and stimulus after-effects. Interstimulus rinsing with the appropriate solvent improved discrimination. PMID- 7552037 TI - The genetic basis of preference for sweet substances among inbred strains of mice: preference ratio phenotypes and the alleles of the Sac and dpa loci. AB - Five inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BI/6J and DBA/2J) were examined with two-bottle (48 h) preference ratio testing across concentrations of sodium saccharin (3 x 10(-4) M, 10(-3) M, 3 x 10(-3) M and 10(-2) M), d phenylalanine (10(-3) M, 10(-2) M and 10(-1) M), and l-glutamine (10(-2) M, 3 x 10(-2) M, 10(-1) M and 3 x 10(-1) M). Three consistent groupings of strains were observed across substances and concentrations: (1) C57BI/6J (preference at low and high concentrations); (2) BALB/cByJ and C3HeB/FeJ (preference at high concentrations); (3) 129/J and DBA/2J (preference at high concentration for sodium saccharin and indifference to d-phenylalanine and l-glutamine). If a single locus (presumably dpa or Sac) determines these phenotypes, there are likely to be three alleles. If two independent loci (presumably dpa and Sac) determine these phenotypes, an allelic assignment of Sacb/dpa+s for the C57BI/6J strain, Sacb/dpa-s for the BALB/cByJ and C3HeB/FeJ strains, and either Sacd/dpa+s or Sacd/dpa-s for the 129/J and DBA/2J strains is suggested. PMID- 7552039 TI - A novel psychophysical procedure for bitter taste assessment in rats. AB - A persistent problem with attempts to examine bitter taste mechanisms has been the lack of adequate behavioral methodology providing data which parallels that obtained from physiological investigations. We developed a brief contact procedure to assess the ability of rats to detect the presence of a weak bitter compound dissolved in a strong sucrose solution. Male Fischer 344 rats were trained to drink immediately to multiple 10-s presentations of acetaminophen (2, 8, 32, 128 mM), chlorpheniramine maleate (1, 3, 9, 27 mM) L-tryptophan (13.5, 27, 54, 108 mM), pseudoephedrine hydrochloride (1, 4, 16, 64 mM) and quinine hydrochloride (0.008, 0.04, 0.2, 1.0 mM) dissolved in 0.8 M sucrose. The number of licks to sucrose and water were also measured. A microcomputer controlled stimulus presentations and measured the animal's licks of each solution during each 10-s presentation. The responses to the bitter+sucrose mixture were significantly decreased at most concentrations with increasing levels of the bitter component. This was true for all five bitter-tasting compounds, but over different concentration ranges relatively unique to each compound. The present study is the first to characterize the sensory effects of acetaminophen, pseudoephedrine, and chlorpheniramine maleate, all purported to taste bitter to humans. These results demonstrate rats' acute ability to discriminate by taste not only the presence but the concentration of a dilute bitter compound dissolved in a strong sucrose solution. PMID- 7552041 TI - Higher olfactory threshold and decreased odor identification ability in HIV infected persons. PMID- 7552040 TI - Distinct projections of two populations of olfactory receptor axons in the antennal lobe of the sphinx moth Manduca sexta. AB - The central projections of olfactory receptor cells associated with two distinct types of antennal sensilla in the sphinx moth Manduca sexta were revealed by anterograde staining. In both sexes, receptor axons that arise from sexually isomorphic, type-II trichoid sensilla (and possibly some basiconic sensilla) project to the spheroidal glomeruli in the ipsilateral antennal lobe. Each axon terminates in one glomerulus. Axons from a limited region of the antenna project to glomeruli throughout the lobe, arguing against strict topographic mapping of antennal receptor cells onto the array of glomeruli. Axons of sex-pheromone selective receptor cells in the male-specific type-I trichoid sensilla project exclusively to the sexually dimorphic macroglomerular complex (MGC). Axons from sensilla on the dorsal surface of the antenna are biased toward the medial MGC and those from ventral sensilla, toward the lateral MGC. Some receptor-cell axons branch before reaching the MGC, but their terminals are always confined to one of the two main glomerular divisions of the MGC, the cumulus and toroid. These findings confirm that primary-afferent information about pheromonal and non pheromonal odors is segregated in the antennal lobe and suggest that there is a functional correspondence between particular olfactory receptor cells and specific glomeruli. PMID- 7552042 TI - Biogenic amines in the taste organ. AB - The presence and content of biogenic amines in taste disk-bearing fungiform papillae of the frog, Rana esculenta, the only available model of an isolated taste organ, were verified by means of HPLC. Fungiform papillae were found to contain measurable amounts of serotonin, epinephrine and norepinephrine. The amounts of serotonin and epinephrine were significantly higher in fungiform papillae than in the general mucosa of the tongue. Moreover, the epinephrine content of fungiform papillae was found to differ across the tongue, in accordance with previous physiological studies showing an inhomogeneous response of different tongue regions to taste stimuli. Ultrastructural and histochemical investigations confirmed the presence of catecholamine and serotonin. The latter was found to be contained mainly in the basal cells of the frog taste disk. These results extend previous qualitative data on the presence of biogenic amines in taste chemoreceptors. PMID- 7552043 TI - Responses recorded from the frog olfactory epithelium after stimulation with R(+) and S(-)-nicotine. AB - The aim of this study was to determine whether the olfactory system is responsible for the discriminability of the stereoisomers of nicotine. The EOG was recorded after stimulation with different concentrations of undistilled S(-) , distilled S(-)- and distilled R(-)-nicotine separately in three groups of frogs (Xenopus laevis). The responses to all types of nicotine used in the experiments increased with increasing stimulus concentration. The responses to undistilled S( )-nicotine were significantly lower compared to responses to distilled S(-)- and R(+)-nicotine, whereas no significant differences could be found when the purified stereoisomers of nicotine [distilled S(-)-nicotine, distilled R(+) nicotine] were compared. Control measurements of time course and peak concentration employing a UV-detection method demonstrated that the differences between distilled and undistilled S(-)-nicotine could not be explained by different nicotine concentrations. The fact that no differences between the pure nicotine stereoisomers could be found for all concentrations used in our experiments and that experiments in humans revealed similar detection thresholds for both stereoisomers points to a similar receptor affinity of R(+)- and S(-) nicotine within the olfactory system. At this point, it cannot be determined whether the observed differences in the perception of nicotine enantiomers in humans are due to differences in quality coding by stereospecific receptors on the olfactory sensory cells or by specific receptors on the trigeminal nerve endings. PMID- 7552045 TI - Concentration of 1,8-cineol in human blood during prolonged inhalation. PMID- 7552044 TI - The effect of dietary protein levels on the responses of the taste nerve to sodium chloride in spontaneously hypertensive rats (SHRs). AB - The present study was undertaken to clarify the effect of dietary protein level on the response of the taste nerve to NaCl solutions in spontaneously hypertensive rats (SHRs). The results showed that the taste sensitivity to NaCl in SHRs fed a 5% whole-egg protein diet for 3 weeks was significantly lower than in those fed a 15% protein diet. This observation suggests that chronic feeding of a low-protein diet causes an impairment of salt-taste reception or subsequent transduction. PMID- 7552046 TI - Synthesis and characterization of hydroxylated polychlorinated biphenyls (PCBs) identified in human serum. AB - Hydroxylated polychlorinated biphenyls (PCBs) have been identified in wildlife and human samples. Most of these compounds are highly chlorinated (penta hepatachloro) and contain a single meta- or para-hydroxyl group. Using the Cadogan coupling procedure, the following hydroxy-PCBs congeners were synthesized: 2,3,3',4',5-pentachloro-4-biphenylol, 2,3',4,4',5-pentachloro-3 biphenylol, 2',3,3',4',5-pentachloro-4-biphenylol, 2,2',3',4,4'-pentachloro-3 biphenylol, 2,2',3,3',4',5-pentachloro-4-biphenylol, 2,2',3',4,4',5-hexachloro-3 biphenylol, 2,2',3,4',5,5'-hexachloro-4-biphenylol, 2,2',3,3',4',5,5'-heptachloro 4-biphenylol, 2,2',3',4,4',5,5'-hepatachloro-3-biphenylol, 2,2',3,4',5,5',6 heptachloro-4-biphenylol. Many of these compounds have been detected as residues in human serum and current studies are investigating their activities as agonists and antagonists for several endocrine-mediated responses. PMID- 7552047 TI - Comparative evaluation of anaerobic biodegradability of hydrocarbons and fatty derivatives currently used as drilling fluids. AB - The examination of a number of potential and currently used carrier fluids for invert emulsion drilling fluids in the ECETOC screening test revealed clear differences with respect to their easy anaerobic biodegradability. Fatty acid- and alcohol-based ester oils exhibited excellent anaerobic degradation to the gaseous final end products of the methanogenic degradation pathway, methane and carbon dioxide. Mineral oils, dialkyl ethers, alpha-olefins, polyalphaolefins, linear alkylbenzenes and an acetal-derivative were not or only slowly degraded. Although the poor degradation results obtained in the stringent ECETOC screening test may not be regarded as final proof of anaerobic recalcitrance, nevertheless, these results were found to be in line with the present understanding of the structural requirements for anaerobic biodegradability of chemicals. The validity of the conclusions drawn is corroborated by published results on the anaerobic biodegradation behaviour of ester oils, mineral oils and alkylbenzenes in marine sediments. PMID- 7552048 TI - Patient selection and remission rates with the current practice of electroconvulsive therapy in Germany. AB - The current practice of German psychiatric hospitals restricts electroconvulsive therapy (ECT) to patients with profound disability and failure to respond to pharmacotherapy. We studied clinical features and seizure parameters in 63 patients who received ECT in a 3-year period at a German university hospital. Patients with unipolar and bipolar disorder (depressed) (n = 47) showed a complete or partial recovery as assessed 2 weeks after completion of the ECT course in 31.9 and 38.3%, respectively. Multiple linear regression analysis performed on several clinical and ECT seizure parameters failed to identify strong outcome predictors in our selected sample of patients. Most patients who eventually responded did so early in the course of ECT, while there was little improvement when > 10 ECT treatments were given. Our report highlights the efficacy of ECT in the management of severe psychiatric disorders even in a highly selected sample of patients previously found to be resistant to alternative modes of psychiatric treatment. PMID- 7552049 TI - Seizure activity in combined clozapine and ECT: a retrospective view. AB - We examined the medical records of electroconvulsive therapy (ECT) recipients who concurrently received clozapine (n = 7). The patients tolerated ECT, with standard electrical stimulus parameters, without complications. No patients had tardive seizures. The seizure duration during ECT increased, as recorded by electroencephalogram and cuff technique, nonsignificantly by a mean of +2.4 s (range of duration: 17-54 s) while on clozapine. Patient response as evaluated with Brief Psychiatric Rating Scale scores showed a total improvement of 26.9%, improvement in positive signs of schizophrenia of 25.3%, and improvement in negative signs of 21.3%. Hallucinatory behavior (41.7%) and disorganization (45.5%) were markedly reduced. Combined clozapine and ECT appears as safe as other neuroleptics with ECT, with comparable clinical improvements. PMID- 7552050 TI - Effects of ECS on DNA single-strand breaks in rat brain cells. AB - We conducted a series of experiments to evaluate possible molecular mechanisms by which electroconvulsive therapy, a commonly used treatment for depression, may exert its adverse effects such as amnesia. We assessed the effects of repeated electroconvulsive shocks (ECS) alone and in combination with low-level radiograph irradiation on DNA single-strand breaks in cells in the rat brain, using a sensitive alkaline microgel electrophoresis assay method. Our results show that ECS, when administered alone, had no significant effects on DNA single-strand breaks in cells in either the hippocampus or the rest of the brain. However, repeated ECS when combined with a low-level radiograph irradiation produced a small but significant increase in DNA single-strand breaks in rat brain cells. PMID- 7552053 TI - Delusional depression, hyperparathyroidism, and ECT. AB - A woman with delusional depression and unsuspected primary hyperparathyroid disorders responded completely to electroconvulsive therapy (ECT). Hypercalcemia is a complicating factor in the treatment of delusional depression, but there is little evidence that it is causative and the use of ECT should not be delayed. The case adds to the literature confirming the efficacy and safety of ECT with physically ill patients when it may be a life-saving procedure. PMID- 7552054 TI - Subclinical status epilepticus following ECT. AB - Subclinical status epilepticus is a rare complication of electroconvulsive therapy (ECT). We describe the case of a 70-year-old man with psychotic depression who developed prolonged subclinical status epilepticus following an initial ECT treatment; he subsequently received a course of ECT without complications. It is important to consider status epilepticus in the differential diagnosis of patients who do not regain consciousness following ECT, even in the absence of overt motor seizure activity. The development of this complication does not preclude the future use of ECT; in fact, ECT has been repeated successfully in individuals who developed this complication, including this case. Identification of potential risk factors, appropriate preventive measures, and early intervention are important in the management of this complication. The literature is reviewed and appropriate preventive and treatment measures are discussed. PMID- 7552052 TI - ECT administration to a hyperthyroid patient. AB - There are few reported convincing cases of hyperthyroid depressed patients receiving electroconvulsive therapy (ECT). We describe a depressed 66-year-old woman with catatonic depression diagnosed with new-onset hyperthyroidism due to Grave's disease. After commencing propylthiouracil, her Grave's disease was partially treated, but her depression was no better. She subsequently received a course of seven ECT with resolution of her depression and no adverse sequelae. PMID- 7552055 TI - Electroconvulsive therapy and intracranial aneurysm. AB - Little has been written regarding the safety of electroconvulsive therapy (ECT) administered to patients with intracranial aneurysms. The literature is reviewed and we report two additional cases of ECT safely and effectively employed to treat depressed patients with intracranial aneurysms. We found no reported cases of aneurysm rupture associated with ECT. We do not feel the presence of intracranial aneurysm is a contraindication to ECT, in most patients, with appropriate monitoring and control of arterial blood pressure. PMID- 7552051 TI - Tinnitus and ECT. AB - We report the case of a 69-year-old man with major depressive disorder and occupational tinnitus; both problems were refractory to several different treatment modalities, but were successfully treated with electroconvulsive therapy (ECT). The tinnitus was present for 15-20 years, but had worsened during the 3 years before treatment, causing him significant distress. Extensive organic workup did not identify a treatable etiology for his symptoms. To date, he has been hospitalized four times due to depressive symptoms associated with tinnitus. He has received ECT each time, and has responded well on each occasion. PMID- 7552056 TI - The efficacy of caffeine-modified seizures. PMID- 7552058 TI - A prospective study of lithium continuation therapy in depressed patients who have responded to electroconvulsive therapy. AB - Twenty-eight of 34 patients with major depression who completed a course of electroconvulsive therapy (ECT) and were classified as responders were administered lithium carbonate (Li) continuation therapy in the context of an open, prospective study. Twenty-four patients were followed for 6 months or until relapse; four patients dropped out of follow-up while still in remission. The probability of completing 6 months without relapse (by survival analysis, including the patients who dropped out as censored observations) was 65%. The eight patients who relapsed into depression all did so within 13 weeks. They were characterized by a shorter duration of their index depressive episode, a greater likelihood of having suffered an additional depressive episode in the preceding 12 months, and failure of an adequate trial of antidepressant medication before the ECT course. Novel pharmacological strategies may be needed in the post-ECT continuation therapy of patients who have a prior history of relapse and are demonstrably resistant to antidepressant medication. PMID- 7552059 TI - Electrical dose titration for electroconvulsive therapy: a comparison with dose prediction methods. AB - Recent literature on electroconvulsive therapy (ECT) emphasizes the use of moderately suprathreshold electrical stimuli for clinical ECT. Since there is substantial interpatient variability in seizure thresholds, individualization of the ECT stimulus dose has been advocated. Three methods for selecting an electrical stimulus are compared in the present study. The methods compared include empirical titration and dose predictive methods described by two different ECT equipment instruction manuals. The use of ECT stimulus titration was found to be a more consistent means of selecting an electrical dose than either of the two predictive methods. The implications of these findings for the clinical practice of ECT are discussed. PMID- 7552057 TI - The role of ECT in refractory depression. PMID- 7552061 TI - What is rheumatoid arthritis? PMID- 7552060 TI - A history of oral protection for the ECT patient: past, present, and future. AB - Dental injuries and oral soft tissue trauma are recognized risks of electroconvulsive therapy (ECT), which continue to cause concern for patients and providers. The sudden forceful closure of the jaws that precipitates trauma is not avoidable since the pterygoid, masseter, and temporalis muscles are stimulated during treatment. Therefore, the use of a biteguard and oral protection techniques is recommended. No one biteguard has been found to be effective in protecting against all of the oral conditions that exist in the adult ECT patient population. A review of the objectives of ECT oral protection and biteguard design is detailed. The characteristics of patented, commonly used, and home-made ECT oral devices are considered, and recommended techniques for oral protection are detailed. PMID- 7552062 TI - Genes and arthritis. AB - Familial clustering is a common feature of many different arthritic conditions which can be explained by shared genetic or environmental influences or a combination of these. The aetiology of some of these diseases (e.g. multiple epiphyseal dysplasia, Lesch-Nyan syndrome) is clearly monogenic but the majority of common rheumatic conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA) are complex and multi-factorial with a polygenic component. In the last 10 years there has been spectacular progress in unravelling the underlying genetic mechanisms of many monogenic disorders including many that affect the musculoskeletal system. Among these osteogenesis imperfecta (OI) and the Marfan syndrome are striking examples in which molecular defects of matrix protein components of the mesodermal tissues have been revealed. In particular, the power of modern molecular genetics both to test candidate genes (e.g. collagen in OI) and to identify genes where no prior knowledge of the protein defects exists (e.g. fibrillin in the Marfan syndrome) has proved quite remarkable. In the near future many of the techniques that have been applied successfully to these monogenic diseases can be expected to provide insights into the genetic component of common diseases, such as rheumatoid arthritis. However, it is important to realize before embarking on these studies that even monogenic diseases may provide significant problems of analysis and interpretation. Some of these problems are exemplified below (e.g. phenocopies, mosaicism, imprinting). PMID- 7552063 TI - Genetic epidemiology of rheumatoid disease. AB - The aetiology of rheumatoid arthritis (RA) has both genetic and environmental origins. However, the relationship of these with each other and with RA disease is complicated as both genes and environment may be protective or render individuals susceptible. Diagnostic certainty and disease heterogeneity have posed serious hurdles for investigating RA. Patients with similar disease phenotypes may have different aetiologies and it may now be more appropriate to investigate genetic susceptibility with respect to disease process or characteristic immunopathology. Disease concordance studies have been a classical approach to estimating the upper threshold of the genetic contribution to RA and recent studies have approximately halved the much quoted figure of 32% reported by Lawrence for RA concordance in monozygotic twins. However, the interpretation of twin data has to be treated with caution as MZ twins can differ in terms of somatic rearrangement/mutation of immunoglobulin and T cell receptor genes, X chromosome inactivation in females, genomic imprinting and in utero immune relationship. Calculations based on heritability put the genetic component in RA much higher. HLA is an important genetic factor in RA and risk is thought to be associated with a consensus sequence of amino acids within the third hypervariable region of certain DRB1 alleles. It is now clear that HLA is not particularly associated with onset of synovitis but is more associated with progression and severity of the disease process. The HLA-DRB1*0401/0404 genotype is particularly associated with severe, erosive and seropositive RA. Clear differences in RA susceptibility exist between males and females and this may be attributed to hormonal status. Males have a higher threshold requirement for developing RA and this may explain why RA in males is more associated with HLA risk alleles. We have now reached a critical time for the investigation of RA aetio-pathology. Recent advances in molecular biology and automated microsatellite gene scanning technology are making it possible to map disease susceptibility genes over the whole genome in common disorders such as RA. This will require large numbers of well characterised multiplex affected families. It is anticipated that some of the genes identified will fit in with our current concepts of which inflammatory and immune processes are pathologically important in RA. However others may be a major surprise. PMID- 7552064 TI - The anatomy of the rheumatoid lesion. AB - Rheumatoid arthritis primarily involves the synovial membrane of the joints, together with that of the tendon sheaths and bursae. These structures are targeted in a selective and symmetrical manner. The inflammatory cell infiltrate is usually dominated by mononuclear phagocytes. These are recruited into the synovial lining as type A synoviocytes. A proportion of the mononuclear cell population has proven to be specialized antigen-presenting accessory cells. The T and B lymphocyte infiltrate is quite variable in intensity, but may become highly organized. Polymorphonuclear leukocytes pass rapidly into the joint space. The cellular characteristics of rheumatoid nodules have some similarities with those of the synovial membrane. In a minority of patients, immunoglobulin production and circulating immune complex formation reaches levels which precipitate the appearance of a complement mediated vasculitis. This may lead to tissue damage in remote organs. PMID- 7552067 TI - The role of T-lymphocytes in rheumatoid arthritis. AB - The role of T lymphocytes in the pathology of rheumatoid arthritis (RA) is controversial. To some extent this has resulted from contradictory data, but even where specific points of fact are not in dispute, their interpretation often is. Nevertheless, the basic idea of a pathological T cell response in RA receives significant support from the work of several groups who have removed them by thoracic duct drainage, lymphapheresis or total lymphoid irradiation (TLI). Each of these procedures ameliorates RA; in the case of TLI which induces a profound and sustained decrease in CD4+ cells, the effect on synovitis was prolonged. These observations are in line with the general assumption that the strong association of RA with HLA-DR molecules containing a particular conserved region of amino acids implies an important role for T cells in RA, since the physiological role of HLA-DR molecules is to present antigens to T cells. In addition the association with Dw4 and related alleles is strongest for the most severe and persistent forms of disease, arguing that T cells may be important not just in initiation of RA but also in its perpetuation. However, the infiltrating T cells seem to be remarkably inactive and as a population to lack specificity for any particular antigen, leading some to conclude that their role is either passive or irrelevant, while others contend that it is precisely this inactivity which is responsible for the persistence of RA. PMID- 7552065 TI - Leukocyte adhesion molecules. AB - The last few years have seen an enormous expansion of knowledge regarding the role of cell surface adhesion molecules in biological processes. This article discusses the contribution that adhesion molecules make to the traffic of leukocytes into and within synovium and to the capacity of leukocytes to perform their specialised functions. Because of their importance in cellular interactions, adhesion molecules represent a major potential target for antiinflammatory therapy in rheumatoid and other inflammatory arthritides. PMID- 7552066 TI - Rheumatoid factors, B cells and immunoglobulin genes. AB - The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed. PMID- 7552068 TI - T cell receptor usage in rheumatoid arthritis. AB - The structure of alpha beta T cell receptors (TCR) is restricted in a number of rodent and human antigen responses. In several rodent EAE models of multiple sclerosis a limited range of T cell receptors are expressed by T cells which respond to the inciting antigen and are capable of transferring the disease to naive animals. These observations have raised the question of whether in rheumatoid arthritis (RA), a particular T cell receptor structural signature can be identified among T cells derived from the synovium compared to autologous peripheral blood. The parameters which are usually measured are TCR variable region usage, oligoclonality and/or limited junctional region usage. A large number of studies have been carried out and results are variable with some authors claiming evidence for the effect of uncharacterised superantigens expanding or deleting T cells with particular V beta regions while others have suggested that observations of restricted V region usage and limited junctional regions imply that clones of cells have been expanded by antigen. So far none of these studies have led to the identification of an antigen or superantigen which plays a role in RA pathogenesis. PMID- 7552069 TI - Role of antigen presenting cells in rheumatoid arthritis. AB - Histological evidence indicates that activated antigen presenting cells (APC) are present in large numbers within the synovial compartment in rheumatoid arthritis. Their potent function has been demonstrated in vitro using isolated synovial APC populations, although the full picture of which APC populations are involved is still emerging. The ability of these APC to activate T cells which traffic to the joint is likely to play an important role in the maintenance of the synovial inflammatory response. PMID- 7552070 TI - Cytokine expression in chronic inflammatory disease. AB - In chronic inflammatory diseases, typified by rheumatoid arthritis, we speculated that upregulation and/or disregulation of cytokine production in inflamed tissue might contribute both directly and/or indirectly to the pathology in the synovial joint tissue. This chapter summarises studies performed principally by our own group over the last 9 years or so, but also by others in the field who have investigated the expression of cytokines in RA. From our studies we identified one particular cytokine, tumour necrosis factor alpha (TNF alpha) as an important, 'pivotal', molecule in the disease process. This concept has led to the initiation and completion of the first successful clinical trials in RA patients to verify this hypothesis, using a neutralising antibody to TNF alpha. PMID- 7552071 TI - Proteinases and inhibitors. AB - The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases degrading collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases (MMPs) in many of the processes involved in joint diseases. Conventional treatments do little to affect the underlying disease processes and recently the use of proteinase inhibitors has been suggested as a new therapeutic approach. Inhibitors of proteinases can prevent the destruction of animal cartilage in model systems and future patient trials will test the effectiveness of these compounds in vivo. PMID- 7552073 TI - Hypoxia, oxidative stress and rheumatoid arthritis. AB - The synovial cavity has a negative pressure in health. When the joint is exercised, vascular patency is maintained, allowing for nutrition of the avascular cartilage. In rheumatoid synovitis, the situation is altered. The cavity pressure is raised and upon movement this pressure exceeds the capillary perfusion pressure, causing collapse of the blood vessels. This leads to the production of multiple episodes of 'hypoxic-reperfusion injury' generating reactive oxygen species (ROS). Such ROS oxidise: (a) IgG, inducing rheumatoid factor production (b) Hyaluronan, leading to hyaluronan fragmentation products which may alter immune function (c) Lipids, generating aldehydes which are toxic and may alter T cell/macrophage interactions (d) lipoproteins, leading to the production of monocyte chemotactic peptides Progressive hypoxia alters immune function, predominantly by calcium mediated pathways. PMID- 7552072 TI - Intracellular signalling mechanisms of interleukin 1 and tumour necrosis factor: possible targets for therapy. AB - Interleukin 1 (IL-1) and tumour necrosis factor (TNF) are primary inflammatory cytokines. They have highly similar diverse biological actions and act as local and systemic hormones to cause many features of inflammation. Results of studies in animals and humans indicate that blocking their production or action will have marked anti-inflammatory effects. They combine with distinct receptors at the cell surface and rapidly activate intracellular protein kinase cascades. The protein kinases then phosphorylate a variety of cellular protein substrates, including transcription factors, to produce the biological response. In cell culture IL-1 and TNF have the potential to activate all three MAP (Mitogen Activated Protein) kinase cascades, as well as other less well characterized enzymes. The mechanism(s) by which the cytokine receptors activate the kinases is unknown. There is some evidence that IL-1 and TNF activate sphingomyelinase in the cell membrane to generate ceramide, which may have a signalling function. PMID- 7552074 TI - Immunology of systemic rheumatoid disease. AB - Extra-articular or systemic features occur commonly in seropositive erosive rheumatoid arthritis (RA). Clinical, pathological and immunogenetic evidence suggests that nodule formation, vasculitis and Felty's syndrome can be considered as manifestations of the same fundamental disease process as rheumatoid synovitis. However, while synovitis seems likely to be driven by macrophage activation induced by Th1 cells, systemic involvement in RA is characterised by B cell overactivity, immune complex formation and complement consumption, suggesting that Th2 cells are involved in the pathogenesis of extra-articular rheumatoid disease. PMID- 7552075 TI - The genetic origin of responses to drugs. AB - Individual variation in drug metabolism has been extensively investigated. Population studies have shown that there is a wide range in metabolising ability for all detoxification pathways; the distributions may be unimodal (Gaussian) or polymodal, with subsets of individuals who differ from the majority. These may be poor metabolisers (PM) or extensive metabolisers (EM). In many cases, these phenotypes can be linked with the genotype. Frequently the PM phenotype is more susceptible to drug toxicity, while the EM phenotype requires increased dosage for therapeutic benefit. In some cases, phenotypes or genotypes appear to have increased susceptibility to clinical disease. These ideas are discussed for the cytochrome P-450 isozymes, FMO system, cysteine dioxygenase-linked oxidations, glucuronidation, sulphation, acetylation, glutathione conjugation and methylation pathways. PMID- 7552076 TI - Hormonal control of rheumatoid inflammation. AB - As other contributors to this volume have discussed, the genetic basis of rheumatoid arthritis (RA) is multifactorial. One aim of this chapter is to propose that perhaps an additional, non-MHC genetic factor contributing to the pathogenesis of RA may reside in a defective response of the neuroendocrine system to inflammatory stimuli. This proposal is based on work carried out in patients with RA and on lessons learned from animal models of chronic inflammatory disease. This chapter will not consider the effect of sex hormones on the pathogenesis of RA because the data are often contradictory and have yet to crystallize into a coherent hypothesis with testable predictions. PMID- 7552077 TI - How do second-line agents work? AB - The pathogenesis of rheumatoid arthritis remains unknown. Disease modifying anti rheumatic drugs are commonly used to treat rheumatoid arthritis patients. Their use in rheumatoid arthritis was discovered serendipitously and their modes of action remain unknown. This article reviews current hypotheses on the mechanisms of action of established DMARDs. Most of them have anti-inflammatory properties and some are immunomodulators. We also discuss a number of novel agents which are potential new DMARDs. PMID- 7552078 TI - Monoclonal antibody therapy of inflammatory rheumatic diseases. AB - The recognition that certain monoclonal antibodies have immunosuppressive properties led to the therapeutic application in autoimmune rheumatic diseases, rheumatoid arthritis in particular. The therapeutic potential of monoclonal antibodies directed against cell surface antigens mainly present on T-cells has been suggested by open trials in rheumatoid arthritis but the results of controlled studies are disappointing. Open intervention studies with monoclonal antibodies directed at other antigens relevant for the rheumatoid inflammation such as the intercellular adhesion molecule ICAM-1 or the cytokines IL-6 and TNF alpha provided encouraging clinical improvements. The impressive potential of anti-TNF alpha which was already illustrated by the immediate suppression of the acute phase response in open studies could be confirmed by a recently completed controlled trial. The present overview summarizes the available information on the results of these treatment modalities and discusses the possibilities of monoclonal antibodies as a long term treatment for rheumatic diseases. PMID- 7552079 TI - Gene therapy of rheumatoid arthritis via cytokine regulation: future perspectives. AB - Following many years of research using isolated human tissues and animal models, sufficient knowledge concerning rheumatoid arthritis has accumulated so that novel immunotherapies have been proposed. Biological agents are being tested in clinical trials and include antibodies to T cells and cytokines. Currently the most promising of these is intravenously administered neutralizing anti-TNF antibody. In order to establish disease modification, however, therapy needs to be delivered continuously over the long term. The prospect of delivering cytokine inhibitors as genetic material (naked DNA), viruses or in engineered autologous cells is considered as one option for achieving this goal. We compare two strategies, firstly, using immobile cells such as fibroblasts, myoblasts or keratinocytes, and secondly, the migratory cells of the immune system. The former provides a reservoir of systemic delivery of the therapeutic protein whereas the latter provides targeted delivery determined by the antigen specificity of the immune cells. Early validation has begun in animal models of rheumatoid arthritis. PMID- 7552080 TI - Epidemiology of malignant melanoma. PMID- 7552081 TI - Cutaneous melanoma: pathology, relevant prognostic indicators and progression. AB - Malignant melanoma, one of the most rapidly increasing malignancies in man, has recently received substantial attention in the world literature concerning application of traditional morphologic and newer immunologic and molecular biologic methods of predicting progression and ultimate clinical outcome. Although clinical features of patient sex, age and anatomic site of the lesion are important, classic morphologic variables defining prognosis remain the cornerstone of predicting disease outcome. Extent of radial growth phase and the two microstaging methods of measuring tumor thickness and determining level of invasion remain the critical disease progression predictors. Assessment of mitotic rate, number of tumor infiltrating lymphocytes, and determining the presence of regression, ulceration, and epithelioid cell component, microscopic satellites and vascular invasion are also important. More recently a variety of molecular and biochemical prognostic markers have been cited for prediction of disease recurrence and metastasis. Both overexpression and down regulation of a variety of cell adhesion molecules have been implicated in disease progression as has alterations in the plasminogen activation system. A series of growth factors, growth factor receptors, oncogenes and tumor suppressor genes have also been considered. Structural and numerical genomic DNA abnormalities, cell proliferation markers and DNA ploidy status have also been considered. Recently a variety of serum and blood markers indicating disease persistence or progression have been studied including melanin synthesis precursors and intermediate compounds of melanogenesis. Molecular detection by PCR of melanogenesis specific mRNA may become the most sensitive prognostic marker of the future. At present histopathologic and clinical criteria remain the cornerstones of predicting prognosis in malignant melanoma. PMID- 7552082 TI - Melanoma prevention and early detection. AB - In the absence of significant advances in non-surgical treatment of advanced malignant melanoma, efforts to reduce mortality must rely on earlier diagnosis of thinner lesions more likely to be cured by surgery, and also on primary prevention, early detection activities are in progress in many countries with varying levels of built-in audit of their efficacy. In general, however, it would appear that in most countries melanomas are currently detected and treated when they are thinner than was the case 10 years ago. Primary prevention activities are aimed mainly at encouraging sensible sun exposure. Measuring change in sun exposure habits of the public is difficult but surveys indicate that knowledge and attitude to sunburn and the desirability of a tanned skin has moderated over the past 10 years. There is as yet little evidence however that behaviour with regard to sun exposure has changed significantly. PMID- 7552084 TI - Melanoma: chemotherapy. AB - The overall median survival of patients with systemic metastasis from melanoma is about 6 months. Survival is dependent on the sites of first metastasis, the resectability of the metastases, and the number of metastases. Patients with non visceral metastases at first relapse i.e. in skin, subcutaneous tissues, distant lymph nodes, and lung, have a better survival rate than patients with visceral (liver, bone, brain) metastases. Treatment of patients with systemic melanoma should include careful evaluation for the potential role of surgery, radiotherapy, and systemic therapy. The main use of chemotherapy in metastatic melanoma patients remains palliative. The chapter reviews the chemotherapeutic options available for the treatment of malignant melanoma including recent published works on new agents, multi-agent therapy, high dose chemotherapy with autologous bone marrow rescue, adjuvant chemotherapy and regional perfusion chemotherapy. PMID- 7552083 TI - Surgical management of malignant melanoma. AB - Wide and mutilating surgical excision is contraindicated for primary malignant melanoma. Tumours less than 1 mm thick require only 1 cm excision margins while those 1-4 mm thick need only 2 cm margins. Primary closure without skin grafting should always be attempted. Axillary and inguinal block dissection remain standard treatment for established lymphatic metastases but elective block dissection is still controversial and should only be performed in the context of a clinical trial. Selective lymphadenectomy based on intraoperative lymphatic mapping is being evaluated. Isolated limb perfusion plays an important role in palliation, and perfusion with a combination of cytotoxic agents and cytokines is an exciting therapeutic advance. Laser vapourization under local or general anaesthesia is an alternative way of treating multiple small cutaneous and subcutaneous lesions and is much better tolerated. PMID- 7552085 TI - Active specific immunotherapy of melanoma. AB - Active specific immunotherapy, or the use of tumor 'vaccines', attempts to stimulate the patient to reject his or her tumor. Nowhere has this approach been utilized more than in melanoma, often with encouraging results. The best results have occurred in the setting of minimal residual disease after resection of the primary tumor and involved lymph nodes, but responses have also been obtained in disseminated disease. Prolonged survivals of several years have been achieved in both settings, particularly the former, with little toxicity attributable to the treatment. Genetic and biochemical approaches promise considerably improved preparations of 'vaccines', with defined components and improved activity within the immediate future. PMID- 7552086 TI - Targeted gene therapy. AB - Melanin biosynthesis is limited to melanocytes partly as a consequence of transcriptional regulation of the enzymes involved in this pathway. Promoter sequences of these enzyme genes may be utilised to drive expression of complementary DNA coding for therapeutic genes so as to provide transcriptional targeting. We have used the 5'-flanking sequences of the murine tyrosinase or tyrosinase-related protein 1 (TRP-1) genes to show that such transcriptional targeting can be achieved both in vitro and in vivo. Using IL-2 as an example of an immunostimulatory gene and Herpes Simplex Virus thymidine kinase (HSVtk) as an example of a prodrug-activating gene we have shown, in murine model systems, that substantial anti-tumour effects can be achieved by targeted gene therapy approaches. The stage now is set for initial clinical evaluations in human patients. PMID- 7552088 TI - Pathology of ocular melanomas. AB - Primary ocular melanomas usually arise in the uvea, in the choroid and ciliary body. They metastasize primarily and initially exclusively, to the liver. Metastasis and survival is determined by the maximum tumour dimension, the number of epithelioid cells present within the tumour, vascular patterns within the tumour and nucleolar size and activity. Ganglioside and integrin profiles differ from cutaneous melanomas. Iris melanocytic lesions tend not to metastasize, most being naevi of varying degrees of aggressiveness which may cause glaucoma and corneal decompensation. Conjunctival melanoma is a rare unilateral tumour arising either in primary acquired melanosis or de novo rather than within a naevus. Survival of the patient depends on the location of the tumour and the histological subtype. Tumours not arising in the bulbar or limbal conjunctiva have a much poorer prognosis as do eyelid (cutaneous) melanomas if they involve the lid margin. PMID- 7552087 TI - Biological response modifiers in melanoma. PMID- 7552091 TI - Renal pathophysiology. PMID- 7552092 TI - Mineral metabolism. PMID- 7552090 TI - Prospectives for cutaneous malignant melanoma. Considerations of the precursor state and heritability. AB - Each of the discoveries through the years has begotten a series of Gordian knots. We must patiently inspect our new collection of mysteries and focus on those where our efforts may well change the course of the disease to the benefit of affected patients. The proper management of the precursor state, the beginning of the disease, and the patient exploration of the complexities of heritability would seem to be appropriate beginnings. PMID- 7552089 TI - Management of ocular melanoma. AB - Most melanomas in the orbital region are primary tumours. The uveal tract is the commonest site though the skin of the eyelids, the conjunctiva and, very occasionally, the orbit itself may be involved. Direct spread may cause the tumour to present in an adjacent structure so that a uveal tract melanoma with an extrascleral nodule may appear to arise in the conjunctiva or in the orbit whilst a conjunctival melanoma may first be apparent in the adjacent eyelid skin. Metastatic deposits from cutaneous melanoma may occasionally present in the eye or ocular adnexa. PMID- 7552094 TI - Altered vitamin D metabolism and receptor interaction with the target genes in renal failure: calcitriol receptor interaction with its target gene in renal failure. AB - The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with the vitamin D response elements of the target genes. Although decreased VDR concentration in renal failure could diminish the biological action of calcitriol, recent study indicates that uremic toxins could modify the VDR DNA-binding domain and inhibit the binding of the VDR to the vitamin D response elements. The latter reaction could also account for end-organ resistance in renal failure. The inhibitory action of uremic toxins has been tested in vivo by a method using gene transcription. It was demonstrated that uremic ultrafiltrate blocks calcitriol-induced chloramphenical acetyltransferase reporter constructs containing a synthetic vitamin D response element in JEG-3 cells. Taken together, the findings indicate that uremia could underlie the calcitriol resistance in renal failure. The modification of the VDR may involve Schiff base formation between lysine residues of the VDR DNA-binding domain and reactive aldehydes accumulated in uremia. This suggestion is on the basis of the finding that the VDR and other steroid receptors form Schiff bases with pyridoxal 5'-phosphate and weaken the binding of these receptors to the DNA cellulose. PMID- 7552096 TI - Immunomodulatory effects of 1,25-dihydroxyvitamin D3. AB - The activated form of vitamin D, 1,25-dihydroxyvitamin D3, has not only a central role in bone and calcium metabolism, but also has important general effects on cell proliferation and differentiation. Moreover, 1,25-dihydroxyvitamin D3 behaves as a paracrine factor in the immune system as it can be produced by monocytes and has potent actions on all the cellular components of the immune defence system. In recent years, this new physiological role has been studied intensively both in terms of research and with the purpose of exploiting this action in a (pre)clinical setting. Indeed, through chemical alterations of the parent molecule, new substances have been created, called vitamin D analogues. Some of these molecules share the immunological effects of the mother compound, but have decreased effects on calcium and bone metabolism. This makes them potentially useful in clinical practice as immunomodulatory drugs. In the present review, we summarize the data on the in-vitro immune effects of 1,25 dihydroxyvitamin D3 and its analogues and demonstrate that these compounds have clear in-vivo immune modulating properties in the prevention of spontaneous and allergic autoimmune diseases and in the prevention of graft rejection. PMID- 7552095 TI - Oral versus intravenous calcitriol: is the route of administration really important? AB - Deficiency of 1,25-dihydroxyvitamin D plays an important role in the pathogenesis of secondary hyperparathyroidism. Adequate replacement of this hormone is required to normalize parathyroid gland function and restore bone homeostasis in patients with advanced renal failure. Controversy exists regarding the best method of administering 1,25-dihydroxyvitamin D. Although initial, uncontrolled clinical trials suggested the superiority of intravenous calcitriol treatment, more recent controlled investigations have shown that different routes (oral versus intravenous), frequency (daily versus intermittent) and dosing (physiologic versus pharmacologic) of calcitriol administration are equivalent. Overall, the response to calcitriol treatment depends more on the severity of secondary hyperparathyroidism and the presence of confounding variables, such as hyperphosphatemia and acquired abnormalities of parathyroid cell function, than on the method of calcitriol administration. PMID- 7552097 TI - Oestrogens and calcium regulatory hormones: potential implications for bone. AB - Postmenopausal oestrogen deficiency is associated with the development of osteoporosis. Oestrogen therapy prevents further bone loss but does not have an anabolic effect. The only treatment with an anabolic effect on bone is intermittent parathyroid hormone treatment. Oestrogens have a direct action on the parathyroid to increase parathyroid hormone gene expression and parathyroid hormone secretion. They exert this effect at doses that are too low to cause the uterotrophic effect of oestradiol. Osteoporotic patients have a decreased parathyroid hormone secretory response to changes in serum calcium, supporting the experimental data that oestrogens have a direct effect on the parathyroid. The value of parathyroid hormone treatment is limited by the need for parenteral therapy. The ability of oestrogens to increase parathyroid hormone secretion suggests that the intermittent administration of oestrogen analogues, at doses that exert no effects on breast tissue and the uterus, would be the optimal treatment for osteoporosis. PMID- 7552093 TI - Role of the renin-angiotensin system in essential hypertension. PMID- 7552098 TI - The calcium/parathyroid hormone concept of the parathyroid glands. AB - Plasma ionized calcium is the major determinant of parathyroid hormone (PTH) secretion. The minute-to-minute secretory response of the parathyroids to changes in plasma ionized calcium is described by the calcium/PTH concept, but the detailed mechanism is not yet well understood. The recent cloning of a calcium sensing receptor in the plasma membrane of the parathyroid cells will probably yield important information concerning the mechanisms by which calcium and other ions control the parathyroid function. It is likely that autocrine and paracrine factors also participate in the regulation of PTH secretion. PTH, chromogranin A, chromogranin A-related peptides and endothelin-1 have been suggested as autocrine factors. More documentation is needed on the impact of these factors in the physiology of the parathyroid gland. In-vivo investigations of the parathyroid function are difficult to interpret because of the complexity of the PTH secretory response to hypo- and hypercalcaemia. Rate dependency and the ability of the parathyroids to sense the direction of changes in calcium make the existing models for investigating the calcium/PTH relationship inappropriate. In vitro, the models are compromised by a rapid drop in the expression of the calcium-sensing receptor of the cultured parathyroid cells. We, therefore, recommend caution when using the calcium/PTH concept in clinical or experimental investigations. PMID- 7552099 TI - Noninvasive parameters of bone metabolism. AB - Markers of bone formation determined in serum include alkaline phosphatase, bone specific alkaline phosphatase, osteocalcin [bone gamma-carboxyglutamic acid peptide (BGP) and procollagen type I carboxyterminal propeptide. Recently, advances have been made in the immunoassay of bone-specific alkaline phosphatase. This is a marker for osteoblastic activity; it is very stable and is not primarily dependent on kidney function because it is degraded in the liver. BGP is not specific for bone formation because it increases in serum during bed rest (which involves increased bone resorption), and it is not stable. Furthermore, the elimination of BGP is dependent on glomerular filtration rate. Procollagen type I carboxyterminal propeptide is not as sensitive as bone-specific alkaline phosphatase because it increases less in women after the menopause. Urinary pyridinoline and deoxypyridinoline determined by high-performance liquid chromatography are regarded as the best methods for measuring bone resorption. These might be replaced by type I collagen crosslinked N-telopeptide or CrossLaps in the future in laboratories not equipped with a high-performance liquid chromatography system. Serum markers of bone resorption are currently under investigation. An immunoassay for the tartrate-resistant acid phosphatase in serum should be a very promising tool for the quantification of bone resorption. PMID- 7552100 TI - Abnormal protein traffic through the glomerular barrier induces proximal tubular cell dysfunction and causes renal injury. PMID- 7552101 TI - Mathematical description of transport of water and macromolecules through the glomerular capillary wall. AB - Glomerular diseases are associated with changes in glomerular membrane permeability properties that alter filtration rate of plasma water and barrier function of the capillary wall. To estimate intrinsic permeability properties that regulate transmembrane transport of water and macromolecules, theoretical analysis of renal clearance of tracer molecules can be used. The development of adequate theoretical models is required to achieve sufficient accuracy to simulate complicated biological processes. Current research in this area is aimed at improving and validating the presently available models in order to characterize the nature of permeability changes associated with pathological conditions. This is a key step in understanding the pathophysiological nature of glomerular diseases and in the development of effective treatments. PMID- 7552102 TI - Defining the role of endothelins in renal pathophysiology on the basis of selective and unselective endothelin receptor antagonist studies. AB - The sensitivity of the renal vasculature to endothelin-1 together with the effect of the peptide on the kidney prompted investigators to study the role of endothelin-1 in renal pathophysiology. The kidney expresses both endothelinA and endothelinB receptors, which differ from species to species. Experiments employing specific endothelin receptor antagonists in experimental animals have suggested a role for endothelin-1 in ischaemia-induced acute renal failure and renal disease progression. PMID- 7552104 TI - The nitric oxide pathway in glomerulonephritis. AB - Nitric oxide has fundamental roles in the modulation of many cell functions. In glomerulonephritis, generation of nitric oxide by the acutely inflamed glomerulus has recently been confirmed, with evidence that synthesis occurs through induction of the inducible form of nitric oxide synthase. Recent studies implicate infiltrating macrophages as a major source of this activity, although intrinsic glomerular cells may contribute as nitric oxide synthase has been found in cytokine-stimulated mesangial, endothelial and epithelial cells in culture. Cytokines, which are known to have a central role in glomerulonephritis, are the most probable stimulus of nitric oxide synthase induction in vivo, although this awaits confirmation. A role for nitric oxide in the pathophysiology of glomerulonephritis is not clearly defined at present but is strongly suggested by evidence for participation of nitric oxide in other immune and inflammatory diseases. The evidence evaluated in this review emphasizes that the role is certain to be complex. As yet it is not possible to predict whether the modulatory effects of nitric oxide on glomerular haemodynamics, vascular integrity, leucocyte infiltration and intrinsic glomerular cell responses are predominantly protective or cytotoxic. There are presently only two fully published reports of inhibition of nitric oxide in models of glomerulonephritis, one of which showed an exacerbation of acute heterologous nephrotoxic injury, and the other showed amelioration of chronic autoimmune glomerulonephritis. It is therefore premature to speculate on the effects of nitric oxide inhibition in glomerulonephritis. New insights await further understanding of the regulation of the inducible form of nitric oxide synthase in glomerulonephritis and the availability of specific inhibitors of this enzyme. PMID- 7552103 TI - The role of T-cells in inflammatory kidney disease. AB - The fundamental role of cell-mediated immunity in inflammatory disease of the kidney has been established for some time. This review focusses on the recent advances in our understanding of the role of T-cells in the pathogenesis both of glomerulonephritis and interstitial nephritis. In particular, the increasing experimental evidence for a functional role for T-cells in promoting disease initiation and progression is examined. The ever increasing variety of T-cell effector functions and their relevance are also discussed. PMID- 7552105 TI - Chronic renal disease: do metalloproteinase inhibitors have a demonstrable role in extracellular matrix accumulation? AB - Recent evidence suggests that decreased degradation of the glomerular extracellular matrix may contribute to the matrix accumulation that occurs in the progression of chronic renal disease. The presence of matrix metalloproteinases in cultured glomerular cells and possibly in glomeruli in vivo combined with the ability of these proteinases to degrade extracellular matrix components suggests that these proteinases may play important roles in glomerular extracellular matrix degradation. Decreased activity of these proteinases mediated by upregulation of their inhibitors could theoretically contribute to matrix accumulation. In the limited number of studies that have addressed this issue directly, there is evidence both to support and refute this hypothesis. It is reasonable to suspect, however, that either increased matrix formation, decreased matrix degradation, or both, might contribute to extracellular matrix accumulation in progressive renal disease, depending on the primary etiology. PMID- 7552106 TI - Renal toxicity of albumin and other lipoproteins. AB - Proteinuria may be an independent risk factor for nephrosclerosis. One potential mechanism has emerged with the finding that lipoproteins, including albumin, modulate the biology of mesangial cells and proximal tubular epithelial cells when internalized. Consequent alterations in renal-cell lipid metabolism may underlie some of these effects. PMID- 7552107 TI - Glomerulonephritis: from new knowledge on the mechanisms of tissue damage to novel therapies. AB - Remarkable progress has been made in the past two years in the investigation of immunological events and effector mechanisms which lead to glomerular inflammation and tissue damage, suggesting a number of areas in which therapeutic modulation is possible. We review recent advances in the two major areas of investigation in this field: the nephritogenic response to glomerulonephritis and local inflammatory reaction, and the novel therapeutic perspectives that have emerged from insights into the biology and pathology of the immune and inflammatory response. PMID- 7552108 TI - ENSO and disaster: droughts, floods and El Nino/Southern Oscillation warm events. AB - The connection between El Nino/Southern Oscillation (ENSO) events and precipitation and temperature variability worldwide is increasingly well understood. ENSO has been linked to droughts and flooding in some regions. This paper uses the disaster history database of the U.S. Agency for International Development's Office of U.S. Foreign Disaster Assistance to examine the link between ENSO events and droughts or floods of sufficient magnitude to trigger international disasters. Worldwide, disasters triggered by droughts are twice as frequent during year two of ENSO warm events than during other years. No such relationship is apparent in the case of flood disasters. Drought disasters occur during year two of ENSO warm events significantly more frequently than in other years in Southern Africa and Southeast Asia. No regional pattern emerges from a comparable analysis of flood disasters. Those places likely to be affected by ENSO-triggered droughts can take proactive measures to mitigate the impacts. PMID- 7552109 TI - The association between El Nino/Southern Oscillation events and typhoons in the Marshall Islands. AB - An analysis of the historic record of typhoons in the Marshall Islands has identified a significant association between the occurrence of the El Nino/Southern Oscillation phenomenon (ENSO) and the occurrence of typhoons in the Marshall Islands. Whilst typhoons normally occur further to the east, the warming of the ocean waters around the Marshall Islands, as part of the ENSO phenomenon, generates typhoons further to the west. The results suggest that typhoons are 2.6 times more likely to occur during ENSO years, with a 71 per cent chance of a typhoon striking during an ENSO year, and only a 26 per cent chance of one happening during a non-ENSO year. This has implications for planning and public safety, which the relevant authorities may wish to take note of. PMID- 7552110 TI - Malnutrition and poverty in the early stages of famine: North Darfur, 1988-90. AB - In this article we report findings on the relationship between malnutrition and poverty during a period of acute food insecurity in Darfur, Sudan. Children of rich and poor families were equally likely to be malnourished, which is explained in terms of people's responses to the threat of famine. This finding has important implications for targeting interventions in the early stages of famine. Appropriate interventions at the early stages of famine are livelihood and income support to the most vulnerable. The entitlement theory of famine causation assumes that the poor are most vulnerable, and become malnourished and die during famines. In this article we show that this assumption does not hold. Even though poverty is the root cause of malnutrition, it does not follow that anthropometric status can be used to target individual poor families, or even that targeting the poor is appropriate in famine situations. PMID- 7552111 TI - The impact of a reduced and uncertain food supply in three besieged cities of Bosnia-Hercegovina. AB - Nutritional status and household food security were longitudinally monitored in three besieged cities of Bosnia-Hercegovina (Sarajevo, Zenica and Tuzla) during the winter and spring of 1993 to 1994. The objectives were to provide early warning of a deterioration in the food and nutrition situation and identify particularly vulnerable groups so that action could be taken to prevent potential undernutrition and target resources to the most needy. Before the cease-fire, which came into effect at the end of February 1994, trends in various indicators (weight loss, decline of household food stocks, rising food prices, reduction in food aid distribution, sale of possessions) suggested that the situation was deteriorating. Access to food improved as a result of the cease-fire, however, which was reflected in improvements in indicators of food security and weight gain. The impact of, and response to, the food emergency differed between individuals, households and locations; the elderly were found to be more nutritionally vulnerable than children or adults, households with the least access to resources were the most food insecure, while the city of Zenica appeared to be particularly hard hit. The findings show the effects of an uncertain and reduced food supply on a previously well-fed healthy population in an industrialised country. The value and constraints of an Early Warning System set up to monitor trends in an emergency in a European context are discussed. PMID- 7552114 TI - Flood control projects in Bangladesh: reasons for failure and recommendations for improvement. AB - Flood control and drainage projects in Bangladesh are intended to give protection from main river floods, flash floods in the east and northeast of the country, and saline intrusion in the lower delta and to improve drainage in order to avoid crop damage. While in some cases such projects have had positive results, in many others their benefits have fallen well below expectations. One of the major reasons for the poor performance of projects is embankment failure, brought about by poor planning, design, construction, operation and maintenance. Recommended measures to improve the efficiency of projects include the involvement of local people in the planning and operation of projects, better training of management staff and the allocation of adequate funds for maintenance. PMID- 7552116 TI - The assessment of community vulnerability in hazard prone areas, the Royal Society, London, 31 March 1995. PMID- 7552113 TI - The role of food aid in drought and recovery: Oxfam's North Turkana (Kenya) drought relief programme, 1992-94. AB - Regional droughts carry the seeds of catastrophe: the immediate risk is famine; the long-term risk is destitution. Preventing both situations is an appropriate, if not essential, goal for relief agencies. In the past, responses to hunger in Turkana District (north-west Kenya) have taken the form of traditional feeding programmes. A better understanding of the boom/bust cycles in pastoralist systems has, however, produced new relief strategies. A central tenet of these strategies is the acceptance that relief aid should assume two roles: humanitarian--to overcome food deficits--and economic--to overcome 'income' deficits. Arguments in favour of a broader role for food aid are tested with a case study of a drought relief programme in north Turkana between 1992-1994. Field studies confirmed that when food aid is integrated as an asset into household resources, it can strengthen economic recovery. Ultimately, the extent to which relief operations are able to protect both people's lives and their livelihoods is the key to more sustainable development in drought-prone areas. PMID- 7552115 TI - Primary health care in disasters: the NorAid system. PMID- 7552117 TI - Eighth Annual Hunger Research Briefing and Exchange, 'Hunger, scarcity and policy: strengthening people's capacity for development', Brown University, 5-7 April 1995. PMID- 7552119 TI - Windstorm: coming to terms with mankind's worst natural hazard, Royal Academy of Engineering, London, 4 May 1995. PMID- 7552118 TI - First Harvard Symposium on Complex Disasters, Boston, Massachusetts, 10-11 April, 1995. PMID- 7552120 TI - Further refinements of the polyp snare for interuterine surgery--a new modality for treatment of myomas and polyps. AB - Hysteroscopic treatment of 30 patients suffering from menorrhagia or post-partum complications was accomplished using an electrosurgical polyp snare. Using this method, 18 polyps and 12 myomas were successfully removed in less than twenty minutes without complications. Local anaesthesia was used in 12 patients. Three patients have presented with recurrence of menorrhagia, with a minimum of six months follow-up. Benefits of this technique compared to uterine resectoscopy include shorter operative time, decreased risk of fluid overload, and less risk of uterine perforation. The snare is difficult to use and a learning curve exists. Higher currents than that used for resection are required. PMID- 7552112 TI - Long-term effects of Hurricane Andrew: revisiting mental health indicators. AB - Two population-based surveys of South Dade County, Florida, were conducted after Hurricane Andrew to compare hurricane-related symptoms of mental distress and describe the impact of mental health outreach teams. Households were selected by three-stage cluster sampling and findings from the two surveys, 13 months apart, were compared. Response rates were 75 per cent and 84 per cent. The prevalence of symptoms of mental distress decreased over time. However, in the households contacted by the teams (25 per cent of sample), the prevalence of symptoms (50 per cent) did not differ from households not contacted (43 per cent). Households contacted by teams that reported symptoms were just as likely to have been referred for help by the teams (72 per cent) as those without symptoms (68 per cent). Households reporting symptoms were equally likely to get counselling regardless of whether the teams visited. Mental health teams had no significant impact on mental health symptoms or the use of mental health services. Alternative approaches to mental health outreach teams need to be explored. PMID- 7552121 TI - Subtotal hysterectomy revisited. AB - The advent of laparoscopic surgery has enabled gynaecologists to re-evaluate the traditional approaches to hysterectomy. Until the 1940's hysterectomy involved retaining the cervix because the simpler operation avoided damage to the ureter and prevented ascending infection, which was an important consideration before the advent of antibiotics. In order to reduce the risk of developing cervical carcinoma the cervix was traditionally removed at hysterectomy over the last 50 years. Since it is possible to remove the area where cervical carcinoma develops and with the development of an effective screening programme for cervical carcinoma, this needs no longer to be a consideration. A more logical approach to laparoscopic hysterectomy would be to retain the cervix but remove the transformation zone and in so doing there is less risk to the ureter, less postoperative urinary dysfunction, virtually no disturbance to the lower genital tract, thus resulting in little or no impairment of sexual enjoyment. PMID- 7552122 TI - Nursing aspects of gynaecologic endoscopy. AB - Nurses now play an integral part in gynaecological endoscopic surgery, with the video camera enabling the operating room staff to visualise the entire procedure. Endoscopy teams are being developed in the operating room for specialised procedures to ensure that nurses assisting with such procedures are familiar with the endoscopic surgeon's routine, instrumentation and equipment. The team consists of the surgeon, surgical assistant, scrub technician, circulating nurse and laser nurse. PMID- 7552123 TI - Endoscopic capsulotomy by means of laser--a new therapeutic method in cases of capsular contracture. AB - The laser endoscopic capsular technique is a new method introduced to treat patients who develop capsular contracture after the insertion of a breast prosthesis. A capsuloscope was developed in co-operation with M. Boebel (Richard Wolf) to undertake the procedure. Twenty patients have undergone 25 capsulotomies. The technique relieved pain, corrected the deformity and reduced hardness. PMID- 7552124 TI - Endoscopic retropubic colposuspension: "Retziusscopy" versus laparoscopy--a reasonable enlargement of the operative spectrum in the management of recurrent stress incontinence? AB - The technical feasibility of the laparoscopic and extraperitoneal approach to the Burch colposuspension for treatment of grade II and III (moderate to severe) stress incontinence was examined in a randomised prospective study. Irrespective of the endoscopic access, two suspension techniques, namely conventional suture and stapler fixation of alloplastic materials, were compared. A total of 20 patients entered this preliminary evaluation. Three complications occurred; a bladder perforation (during laparoscopic dissection of the space of Retzius), a postoperative detrusor instability and transient urinary retention. Both the endoscopic approaches and the suspension procedures employed proved feasible and safe, and presented the advantages of a "minimal access" procedure, with short hospitalisation and rapid recovery. Short-term follow-up (6-12 months) showed subjective and objective results comparable to those of the conventional abdominal Burch colposuspension. Detailed evaluation of the subgroups is not yet feasible, as the number of patients in this preliminary evaluation was too small and follow-up too short. Final evaluation of the entire study population and long term follow-up will be necessary before these procedures can be generally offered as a therapeutic alternative. PMID- 7552125 TI - Multifunctional instrument for operative laparoscopy: technical, experimental and clinical results in gynaecology. AB - Whilst endoscopic surgical procedures are getting increasingly more complex, in the various surgical disciplines mono- and bifunctional instruments are only slowly being replaced by multifunctional ones. Therefore a complex, intelligent system was developed, the central part of which is a multifunctional instrument. All basic functions necessary for surgical laparoscopy are integrated and comprise: cutting electrodes (unipolar and bipolar) which can be advanced or retracted pneumatically; coagulation forceps with mechanical control; and irrigation and suction devices. All 5 mm instruments can be used and there is an option for others, such as laser or aqua-dissection. The various functions are controlled via the handle of the multifunctional instrument which is connected to the electronic control unit, the MULTILAP system, which supplies the energy, material, and information flow required. In vivo standardised experiments in pigs were first performed to test the new instrument. Operation time was reduced by more than 20% when compared with the same procedure performed conventionally, during which frequent changing of instruments was necessary. Clinical application, without complications in all 30 patients (uterus preserving procedures or reconstructive tubo-ovarian surgery) confirmed the advantages of a multifunctional device, with optimised cutting and coagulation of vessels more than 1-2 mm in diameter, and reduced duration of operation. Safety and ergonomics were improved. Thus, an electronically controlled instrument with multifunctional working channels for lasers, ultrasound appliances, or mechanical instruments is available for application in all domains of operative laparoscopy. PMID- 7552126 TI - Disposable versus reusable instrumentation. PMID- 7552127 TI - Reusable, re-posable and disposable instrumentation. PMID- 7552128 TI - Economic impact of laparoscopic instrumentation: a company perspective. AB - This report represents findings concerning the economic impact of laparoscopic surgery. Specifically, the study addresses hospital costs, and not the hospital charges often given attention by studies in the literature. Hospital expenditures for the equipment and instrumentation required for laparoscopic surgery are important cost factors in laparoscopic surgery. Data for determining hospital costs was obtained from nine hospitals throughout the United States. At each hospital, a research team spent four to five days interviewing surgeons, OR staff, hospital administrators and other personnel as well as gathering data. Analysis of operating room equipment and supplies indicates that single-use laparoscopic instruments are a cost-effective alternative to reusable instruments. In addition, single-use instruments have many benefits that were not possible to quantify accurately in this study. PMID- 7552129 TI - The evolving design of laparoscopic instruments. AB - Due to the rapid acceptance of new laparoscopic techniques, older instruments have been directly adapted to new purposes. The new laparoscopic applications and the evolution of these techniques have led to specific new designs. In this paper the evolution of laparoscopic instruments towards better safety standards with regard to sterility is examined. It is found that although the problem of sterility can be solved, this comes at an increased cost. PMID- 7552130 TI - Single-patient-use laparoscopic instrumentation: a company perspective. AB - The availability of single-patient-use laparoscopic instrumentation has been important for the adoption of laparoscopic surgery. Many surgeons were first introduced to laparoscopic surgery through the use of single-patient-use instrumentation. While there are reusable alternatives in most cases, many surgeons and operating room nurses still prefer single-patient-use instruments because they are sterile, safe, reliable, innovative in design, and always available. They also offer potential long-term hospital cost savings. PMID- 7552131 TI - Disposable and reusable trocars. AB - A wide variety of disposable and reusable trocars have been designed and marketed for use in laparoscopic surgery. The performance of these instruments varies considerably. Disposable trocars usually contain valve designs which allow the easy passage of instrumentation and safety mechanisms which reduce the likelihood of visceral injury from the trocar point. Whilst most reusable trocars have not provided safety mechanisms, and have been less user-friendly, newer designs are beginning to bridge the gap. The choice of a trocar for laparoscopic surgery will depend on each surgeon's perception of the advantages and disadvantages of competing instrumentation, which may need to be balanced against the budgetary requirements of their practice location. PMID- 7552132 TI - Durability and function of disposable versus reusable laparoscopic instrumentation. AB - The durability and function of reusable and disposable laparoscopic instruments are compared, and the relative advantages and disadvantages of each are illustrated. Familiarity with the relevant issues described should allow surgeons to make informed choices on the most suitable instruments for their specific situation. PMID- 7552134 TI - Disposable and reusable instruments. PMID- 7552136 TI - Vaginal vault suspension. AB - A laparoscopic approach to significant pelvic relaxation and vault prolapse, including technical aspects of the procedures involved and an anatomic rationale for the laparoscopic approach, are described. The results demonstrate good clinical outcomes in addition to low intraoperative and post operative morbidity. It is concluded that the laparoscopic approach to vault support has a superior result when compared to open vaginal alternatives. PMID- 7552135 TI - Laparoscopic bladder neck suspension. AB - The preferred therapy for genuine stress incontinence is surgery. The Burch procedure is considered by many to be the gold standard for surgical treatment of genuine stress incontinence. The Burch procedure requires the elevation of the anterior wall of the vagina to the level of the origin of the paravaginal fascia by suspension from Cooper's ligaments (iliopectineal ligaments). A properly performed Burch procedure cures 93 percent. The laparoscopic performance of the Burch procedure results in a decrease in the length of hospital stay, faster recovery, and less scarring due to the smaller incisions. In a consecutive series of thirty patients the average length of stay in hospital was thirty-six hours and the patients were able to return to non-stressful regular activities within one week of surgery. There were no conversions to the open Burch procedure. Intraoperative and postoperative complications included one electrocautery injury of the bladder requiring laparoscopic suture reinforcement of the bladder and three weeks of bladder drainage, three episodes of transient detrusor instability requiring medical therapy for two months, and two episodes of urinary retention requiring bladder drainage for two weeks. The laparoscopic procedure provides results similar to the open operation if a meticulous technique is used. PMID- 7552133 TI - Disposable versus reusable instruments in laparoscopic cholecystectomy. A prospective, randomised study. AB - We quantified and compared the advantages and disadvantages of disposable and reusable laparoscopic instruments in a prospective, randomised study of 158 cholecystectomies. The patients were randomly divided into two groups: 80 underwent surgery with reusable instruments, 78 with disposable instruments. The following parameters were recorded in both groups: duration of surgery, number of complications, technical problems during surgery, rate of conversion to open surgery, subjective postoperative pain, postoperative hospitalisation time, length of inability to work, and postoperative evaluation by the operating room personnel. No overall differences were found in subjective pain, postoperative complications, postoperative hospitalisation time, or time before returning to work. Surgery with disposable instruments was on the whole faster, with fewer conversions to open surgery, but this was statistically non-significant. Reusable instruments were associated with a statistically significant increase in the rate of intraoperative, instrument-related difficulties. In spite of longer operation times and higher personnel costs with reusable instruments, we found that cholecystectomy with reusable instruments costs an average of 1,015 DM less per procedure. PMID- 7552137 TI - Laparoscopic creation of a neovagina: modified Vecchietti method. AB - A new surgical technique for the treatment of vaginal aplasia is described. It consists of a modification of Vecchietti's method, allowing laparoscopic performance of the operation. 9 patients have been treated in this way since January, 1991 with good results. No complications were seen. Being virtually atraumatic, this method requires only brief hospitalisation, which is of vital importance in view of the psychological and physical problems this particular condition entails. Results are identical, so that this technique should always take precedence over the conventional Vecchietti method. PMID- 7552138 TI - Lymph node dissection in gynaecologic malignancy: the role of laparoscopy. AB - Surgical sampling and resection of the pelvic and para-aortic lymph nodes plays a critical role in the management of women with selected gynaecologic malignancies. Techniques to perform laparoscopic bilateral pelvic and para-aortic lymphadenectomies have been developed. Published reports reviewing the use of these techniques are limited and have critical deficiencies. Well-controlled multicentre trials must be completed to confirm the merit and efficiency of this innovative approach prior to it becoming a new surgical standard. PMID- 7552139 TI - Identification of two new mutations in congenital erythropoietic porphyria. AB - Congenital erythropoietic porphyria (CEP) or Gunther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Gunther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen. PMID- 7552142 TI - DNA forensic science 1995. AB - After digression by a National Research Council committee, forensic use of DNA identification is returning to methods that do not violate population genetics and statistics. Polemic has been replaced by normal science as advances are made in molecular techniques and presentation of evidence. Rigorous quality control over the chain of custody and likelihood calculations still need to be implemented, and evidence extended on the structure of forensic populations and the operating characteristics of alternative tests. PMID- 7552140 TI - DNA-based presymptomatic diagnosis for the von Hippel-Lindau disease by linkage analysis. AB - Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited condition characterized by a predisposition to the development of haemangioblastoma, renal cell carcinoma and phaeochromocytoma. The gene which, when altered, causes the disease was cloned in 1993, and maps within a series of known polymorphic loci in the 3p25-p26 region. To optimize a DNA-based presymptomatic diagnosis, we have selected six highly informative microsatellite loci, closely linked to the VHL gene. Genotyping using a multiplex-PCR approach was performed in 26 affected families including 99 asymptomatic relatives born from an affected parent. Ninety six subjects were informative with one or more markers, 76 being informative with markers on both sides of the gene. Combination of age-related and DNA-based risk information improved the accuracy of risk assessment for 90 at-risk patients (91%) and allowed attribution of risk with a confidence limit higher than 0.98 in 79 cases (88%). PMID- 7552141 TI - Ataxia-telangiectasia: closer to unraveling the mystery. AB - Ataxia-telangiectasia (A-T) is a progressive genetic disorder affecting the central nervous and immune systems, and involving chromosomal instability, cancer predisposition, radiation sensitivity and cell cycle abnormalities. Studies of the cellular phenotype of A-T have pointed to a defect in a putative system that processes a specific type of DNA damage and initiates a signal transduction pathway controlling replication and repair. A-T is genetically heterogeneous, with 4 complementation groups. While functional cloning of the A-T gene(s) using gene transfer has proven problematic, positional cloning attempts are zeroing in on a defined interval on chromosome 11q22-23 that probably harbors the mutations for all 4 complementation groups. PMID- 7552143 TI - The Foundation of the European Society of Human Genetics. PMID- 7552144 TI - Cosmid contigs spanning 9q34 including the candidate region for TSC1. AB - The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34. PMID- 7552145 TI - Cosmid contigs from the tuberous sclerosis candidate region on chromosome 9q34. AB - Tuberous sclerosis (TSC) is a heterogeneous multisystem disorder with loci on 9q34 (TSC1) and 16p13.3 (TSC2). The TSC2 gene has recently been isolated, while the TSC1 gene has been mapped to a 5-cM region between the markers D9S149 and D9S114. In our effort to localise and clone TSC1, we have obtained three adjacent cosmid contigs that cover the core of the candidate region. The three contigs comprise approximately 600 kb and include 80 cosmids, 2 P1 clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites. The ABO blood group locus, the Surfeit gene cluster, the dopamine beta-hydroxylase gene (DBH) and VAV2, a homologue of the vav oncogene, have all been mapped within the contigs. Exon trapping and mutation screening experiments, aimed at identifying the TSC1 gene, are currently in progress. PMID- 7552148 TI - Thinking through potential problems. PMID- 7552146 TI - A provisional transcript map of the spinal muscular atrophy (SMA) critical region. AB - YACs from the region containing the spinal muscular atrophy (SMA) locus at 5q12 have been used as probes in a direct screening of cDNA libraries to isolate 8 cDNAs, mapped to different YAC fragments. Three clones showed complete identity to the genes for cyclin B1 (CCNB1), the p44 subunit of the transcription factor BTF2 (BTF2p44), and cofilin (CFL). Two clones showed partial identity to the beta glucuronidase gene (GLCB) and a rat integral membrane glycoprotein gene (RNINMEGLA). CFL turned out to have been identified by a pseudogene sequence. Related sequences occurred on other chromosomes. CCNB1 and BTF2p44 were given an exact location. The GLCB-like gene and the RNINMEGLA-like gene detected loci on both 5q and 5p. The remaining three cDNA clones were localized to the SMA region only. Their sequences did not show identity to any gene for which a function is already known. Two of them have now turned out to be identical to recently reported candidate genes for SMA. PMID- 7552150 TI - Arthroscopic lateral portals revisited. A cadaveric study of the safe zones. AB - Lateral knee portal placement is defined by and divided into five anatomic zones (A through E) beginning just anterolateral to the patellar ligament and proceeding posteriorly at 1-cm intervals. The arthroscopic anatomy and the open dissected lateral knee anatomy are correlated in this study with a fresh frozen cadaver. Zone A includes the anterolateral capsulosynovial layer and zone B includes the anterolateral capsulosynovial layer, anterior portion of the iliotibial tract, and the patellotibial ligament. The lateral capsulosynovial layer and the middle-to-posterior portion of the iliotibial tract comprise zone C. Zone D includes the lateral collateral ligament, the popliteal tendon, and the anterior border of the long head of the biceps femoris tendon. The mid-portion of the long head of the biceps femoris tendon and the peroneal nerve make up zone E. Structures penetrated are defined as safe in zones A, B, and C, and unsafe in zone E. Structures that are potentially penetrable are defined as relatively at risk in zone D. When placing a portal for arthroscopic visualization of the posterolateral compartment of the knee, the placement should be between zone D and the anterior portion of zone E at 90 degrees of knee flexion. This potential space is between the lateral collateral ligament and the anterior portion of the biceps femoris tendon; it enlarges with increasing knee flexion and allows for easier portal placement. PMID- 7552149 TI - Complications of extensor mechanism surgery for patellar malalignment. AB - This review focuses on the complications of extensor mechanism surgery for symptomatic patellar malalignment and ways to avoid those complications. We combined a review of new and referred patients who had complications resulting from extensor mechanism surgery with a review of selected literature specifically addressing similar complications. By studying the pathologic anatomy at the time of arthrotomy, it was possible in many cases to determine the causes of adverse outcomes with this type of surgery. The complications of extensor mechanism surgery can be avoided by performing a thorough preoperative evaluation, determining each component of the malalignment, tailoring the procedure to correct each patient's unique pathomechanics, and respecting the functional anatomy of the extensor mechanism. PMID- 7552147 TI - Isolation of microsatellites from the spinal muscular atrophy (SMA) candidate region on chromosome 5q and linkage analysis in Spanish SMA families. AB - A locus responsible for autosomal recessive spinal muscular atrophy (SMA) on chromosome 5q11.2-q13.3 has been mapped to a critical interval delimited by markers D5S435 and D5S557. By a modification of the Vectorette-(GT)n method, we have isolated three polymorphic CA repeats from two YACs of the SMA region. Two of them (D5S1417 and D5S1416) map within the SMA critical region, and the other (D5S1415) is centromeric to D5S435. Linkage analysis in Spanish SMA families with eleven markers showed that in our families the disease is linked to this region and confirmed that the novel markers are tightly linked to the SMA locus. The most likely order of markers was 5cen-(D5S63/D5S1356)-(D5S125/D5S465)- (D5S435/D5S1417/D5S1416/D5S557)-D5S610- D5S112-D5S127-5qter, with odds against alternative orders > 1,000:1. Genetic distances are in agreement with those previously published. However, the recombination fraction between D5S610 and D5S112 is remarkably greater than expected from the physical distance, suggesting a hot spot for recombination in this region. Our results from haplotype and multipoint analyses show that the SMA locus must lie between D5S465 and D5S112, and lend further support to the current location of the SMA locus. PMID- 7552153 TI - Passing a guide pin through a displaced tibial nonunion. AB - A method is presented for aligning apposing medullary canals in preparation for closed nailing in cases of displaced tibial nonunions. This method involves manipulating an intramedullary guide pin through the nonunion via a bone hook inserted through a small incision at the fracture site. The minimal disruption to the fracture site may reduce the risk of infection and speed healing. PMID- 7552151 TI - Synovial sarcoma of the suboccipital region of the neck. AB - A synovial sarcoma (SS) is an uncommon malignant soft-tissue tumor, which in spite of its name does not arise from synovial tissue. It is so named because of its histologic similarity to synovium. An SS originates from mesenchyme, not from synoviocytes and usually manifests as a biphasic tumor with both malignant epithelial and spindle-cell components. Monophasic epithelial and spindle-cell presentations may cause a diagnostic dilemma. Diagnosis should include immunocytochemistry using cytokeratin and/or epithelial membrane antigen; vimentin further helps to eliminate any histologic confusion. These tumors are most commonly found in the extremities. When located near a joint, invasion occurs only by secondary extension. Rarely are SSs found in the neck, especially in the posterior aspect, as reported here. PMID- 7552152 TI - Brachial artery disruption after closed posterior dislocation of the elbow. AB - The incidence of neurovascular compromise after closed posterior elbow dislocation is extremely rare. Early diagnosis and treatment is imperative. This case report describes the diagnosis and treatment of a wrestler who sustained a closed posterior elbow dislocation with transection of the brachial artery. PMID- 7552154 TI - A 28-year-old woman with swelling of the thumb. AB - The following case is presented to illustrate the roentgenographic and clinical findings of a condition of interest to the orthopedic surgeon. The initial history, physical findings, and roentgenographic examinations are found on this page. The clinical and roentgenographic diagnoses are presented on the following pages. PMID- 7552155 TI - Mediocrates. PMID- 7552156 TI - Mediocrates. PMID- 7552157 TI - HIV-1 virology. Simply Marvelous nuclear transport. AB - HIV-1 MA protein is a viral membrane protein, but it is also involved in nuclear import of the preinitiation complex; the latter requires transfer of some MA molecules to the maturing core particle during virus assembly. PMID- 7552158 TI - Inductive signals. Revolving vertebrates. AB - An old idea about the relationship between arthropod and vertebrate body plans has been given new life by studies of the signalling genes controlling dorsal and ventral development in Drosophila and Xenopus. PMID- 7552159 TI - Intracellular signalling. PI 3-kinase puts GTP on the Rac. AB - Phosphoinositide 3-kinase, an enzyme that is known to transduce signals received by a variety of receptor types, has been found to mediate agonist-dependent membrane ruffling via the small GTP-binding protein Rac. PMID- 7552160 TI - Morphogenetic signalling. Responses to hedgehog. AB - Protein kinase A activity is required for signalling by the extracellular molecule Hedgehog in developing Drosophila imaginal discs, but does the kinase actually respond to the Hedgehog signal? PMID- 7552161 TI - Colour vision. Dalton's eyes and monkey genes. AB - Recent molecular genetic studies show how changes in the protein component of a visual pigment alters its absorbance; they also explain the abnormal colour vision of one of the great pioneers of visual science. PMID- 7552162 TI - Cytokine-processing enzymes. Stopping the cuts. AB - The recently discovered enzymes that process the precursors of inflammatory cytokines are good targets for the design of new anti-inflammatory therapeutic agents. PMID- 7552163 TI - Microvillus assembly. Not actin alone. AB - Transfection studies provide supporting evidence for the proposed role of villin and fimbrin in bundling the core actin filaments of microvilli. PMID- 7552164 TI - Limb development. The budding role of FGF. AB - Implantation of beads soaked in fibroblast growth factor into the flank of a chick embryo causes extra limbs to be formed, suggesting that FGF is important in initiating limb buds. PMID- 7552165 TI - RNA editing. An I for editing. AB - In vitro editing in mammalian nuclear extracts reveals adenosine-to-inosine conversions in glutamate receptor messenger RNAs. PMID- 7552166 TI - Neural crest development. Do developing enteric neurons need endothelins? AB - Recent experiments have led to the unexpected finding that endothelin-3 and the endothelin B receptor are absolutely necessary for the development of the enteric nervous system in the colon, but it is not yet clear why. PMID- 7552168 TI - Behavioral learning. The illuminated songbird. AB - Recent studies of the neural mechanisms of avian song learning suggest that pathways for adult song production are distinct from those essential to juvenile song development. PMID- 7552167 TI - Lymphopoiesis. Transcription factors controlling B-cell development. AB - Two structurally dissimilar transcriptional regulators, E2A and Pax-5, have been shown to play essential roles in B-cell development; the p50 subunit of NF kappa B, by contrast, is required for normal immune responses. PMID- 7552169 TI - Somite differentiation. Sonic signals somites. AB - Sonic hedgehog, a secreted signalling molecule known to play a role in the patterning of the central nervous system and the limb in vertebrates, also controls differentiation of the somites. PMID- 7552170 TI - Integrin-ligand binding. Do integrins use a 'MIDAS touch' to grasp an Asp? AB - Integrins use divalent cations, held within a novel 'MIDAS' motif, for ligand binding. It is proposed that a missing coordination site for the cation is provided by a critical acidic residue in the ligand. PMID- 7552171 TI - Adipocyte differentiation. When precursors are also regulators. AB - As well as being the precursors of the triacylglycerols deposited as fat in adipose tissue, long-chain fatty acids are one class of agents that induce the differentiation of preadipocytes to adipocytes. PMID- 7552172 TI - Apoptosis. A sticky business. AB - Many proteins that resemble Bcl-2 or bind to it have been found using techniques that reflect interactions in vitro or depend on DNA homology, but we still do not know how this master regulator of apoptosis works. PMID- 7552173 TI - Knockouts. Targeting the mouse genome: a compendium of knockouts (Part I) PMID- 7552175 TI - Secretion of the amino-terminal fragment of the hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila. AB - BACKGROUND: The Drosophila segment polarity gene hedgehog encodes a member of a family of secreted proteins that are involved in a variety of patterning processes, in both vertebrates and invertebrates. Some of these processes depend upon short-range or contact-dependent interactions, whereas others seem to involve long-range signalling. Two different models have been proposed to account for the execution of these contrasting processes by the same proteins: one postulates that Hedgehog acts exclusively over short distances, its long-range influences being effected through regulation of other signalling factors; the second postulates that different aspects of Hedgehog activity are mediated by distinct forms of the protein that are generated by autoproteolysis. RESULTS: We have investigated these models by mutating the hedgehog coding region such that only the amino-terminal or carboxy-terminal half of the protein is secreted. Deletion of the carboxy-terminal portion has little effect on the signalling activity of the protein, whereas abolishing the secretion of the amino-terminal half leads to a complete loss of signalling. In addition, we find that increases in the level of expression within the normal hedgehog transcriptional domain of either the wild-type protein or the carboxy-terminal-deleted form expand the range of activity to a limited extent, but have only minor effects on cell identity. CONCLUSIONS: In Drosophila, all of the signalling activity of Hedgehog resides in the amino-terminal portion of the protein, the secretion of which is essential for its function. The range of Hedgehog is limited by the close association of the amino-terminal peptide with the cell surface but can be extended by elevating the level of its expression. PMID- 7552176 TI - Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation. AB - BACKGROUND: The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation. RESULTS: Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh transfected COS cells. Direct transfection of neural plate cells with an Shh expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation. CONCLUSIONS: The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors. PMID- 7552177 TI - Alpha beta and gamma delta T cells can share a late common precursor. AB - BACKGROUND: The subdivision of T cells into alpha beta and gamma delta subtypes is conserved throughout vertebrate development. The respective alpha beta and gamma delta T-cell receptors (TCRs) are encoded by somatically rearranged genes. There has been broad speculation as to whether an individual thymocyte can become either a gamma delta T cell or an alpha beta T cell as a result of stochastic gene rearrangement processes, or whether the two types of T cell are derived from separate lineages. Many of the experimental findings are apparently conflicting, however, and the issue--a basic one in immunology and development--remains unresolved. RESULTS: To address this issue, we have used the recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, which allows us to examine quantitatively the status of TCR gamma and delta genes in postnatal alpha beta T cells and their progenitors. Interestingly, such cells are depleted of productively rearranged delta and gamma genes, which can encode delta and gamma TCR polypeptide chains. However, in mice that can rearrange TCR delta gene segments, but in which the TCR delta gene is non functional in other respects, no such depletion of productive rearrangements is seen. CONCLUSION: The quantitative data that we have obtained fulfill the predictions of the stochastic hypothesis: that is, a progenitor T cell first attempts to become a gamma delta T cell and, if unsuccessful, then attempts to become an alpha beta T cell. Thus, alpha beta and gamma delta T cells can derive from a common precursor thymocyte. In the simplest case, therefore, lineage determining factors are the successful rearrangement of both gamma and delta genes before TCR alpha gene rearrangements occur, which lead to deletion of the TCR delta locus and thereby preclude further gamma delta T-cell differentiation. In contrast, successful rearrangement of the TCR beta locus remains compatible with cells becoming either gamma delta or alpha beta T cells. PMID- 7552174 TI - Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. AB - BACKGROUND: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. RESULTS: In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1 adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator. CONCLUSION: GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters. PMID- 7552180 TI - [Guideline for the selection of articles]. PMID- 7552179 TI - Size-contrast illusions deceive the eye but not the hand. AB - BACKGROUND: When we reach out to pick up an object, not only do we direct our moving limb towards the location of the object, but the opening between our fingers and thumb is scaled in flight to the object's size. Evidence obtained from patients with neurological disorders has shown that the visual processing underlying the calibration of grip aperture and other movement parameters during grasping is mediated by visual mechanisms located in the cerebral cortex that are quite distinct from those underlying the experiential perception of object size and other object features. Under appropriate conditions, such dissociations can also be observed in individuals with normal vision. Here we present evidence that the calibration of grasp is quite refractory to pictorial illusions that have large effects on perceptual judgements of size. RESULTS: We used a variation of the familiar 'Titchener circles' illusion in which two target circles of equal size, each surrounded by a circular array of either smaller or larger circles, are presented side by side. Subjects typically report that the target circle surrounded by the array of smaller circles appears to be larger than the target surrounded by larger circles. In our test, two thin 'pokerchip' discs were used as the target circles. The relative size of the two discs was randomly varied so that on some trials the discs appeared perceptually different but were physically equivalent in size, and on other trials they were physically different but appeared perceptually equivalent. The perceptual judgements made by the 14 subjects in our experiment were strongly affected by this size-contrast illusion. However, when asked to pick up a disc, the scaling of the subjects grip aperture (measured opto-electronically before contact with the disc) was largely determined by the true size of the target disc and not its illusory size. CONCLUSIONS: It would seem that the automatic and metrically accurate calibrations required for skilled actions are mediated by visual processes that are separate from those mediating our conscious experiential perception. Earlier studies on patients with neurological deficits suggest that these two types of processing may depend on quite separate, but interacting, visual pathways in the cerebral cortex. PMID- 7552181 TI - [Effect on FEV1 induced by the administration of salbutamol with aerochamber and metered dose inhaler]. AB - There are several devices known as spacers and aerochambers designed to help in the administration of drugs in spray into pulmonary airways in asthmatic patients. This study was done to determine the effect on FEV1 of salbutamol given through an aerochamber compared with metered dose inhaler and spacer. Thirteen asthmatic patients who met all selection criteria proposed, were included. Spirometry was operformed before and after salbutamol application by means of one of the aforementioned devices, assigned at random. The effect on FEV1 with metered dose inhaler was 22.76%, with spacer it was of 23.35% and with aerochamber it was of 23.94%. Statistical analysis did not show significant differences among groups. These data suggest that each of the three devices tested have the same effectiveness for administration of salbutamol in spray. This is, however, a preliminary study. PMID- 7552178 TI - Selective modulation of the expression of L-selectin ligands by an immune response. AB - BACKGROUND: The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells. The cDNA encoding Sgp50 has been cloned, and its product, which has been designated GlyCAM-1, is secreted. The third ligand, Sgp200, is both secreted and cell-associated. We have investigated how the expression of these sulphated glycoproteins is regulated during an immune response. RESULTS: Here we demonstrated that, during a primary immune response, the expression and secretion of both GlyCAM-1 and Sgp200 are reduced, recovering to normal levels 7-10 days after antigen stimulation. In contrast, the expression of cell-associated CD34 and Sgp200 is relatively unaffected. These results may account for the modest decreases in the binding of an L-selectin-IgG fusion protein to high endothelial venules of inflamed peripheral lymph nodes that have been observed after antigen exposure. In vivo experiments show that, following the decrease in the levels of secreted GlyCAM-1 and Sgp200, migration of lymphocytes from the blood stream into lymph nodes remains L-selectin-dependent, but more lymphocytes home to antigen-primed than unprimed peripheral lymph nodes. CONCLUSIONS: We suggest that the secreted forms of the L-selectin ligands GlyCAM 1 and Sgp200 act as modulators of cell adhesion, and that cell-associated CD34 and Sgp200 are the ligands that mediate the initial loose binding of lymphocytes to high endothelial venules. PMID- 7552182 TI - [Premedication for the prevention of adverse reactions to ionic iodized contrast media. Comparative study]. AB - A study comparing two pretreatment protocols to prevent adverse reactions to iodinated radiocontrast media (IRCM) was performed in 45 adults patients. They followed inclusion and exclusion criteria. Pretreatment protocol A was within 12 hours before using the IRCM and pretreatment protocol B was for 72 hours before. A corticosteroid and two H2 receptor antagonists were given on both protocols. We managed these protocols by an aleatory way, so 22 patients received protocol A and 23 patients received protocol B. Excellent results were found in 63.6% and good results were found in 36.4% for protocol A. Excellent results were found in 78.2% and good results were found in 21.8% for protocol B. However, no statistically differences were detected for both protocols (p = 0.451). PMID- 7552184 TI - [Immunoglobulin in the treatment of asthma]. PMID- 7552183 TI - [Skin tests for different species of acari in asthmatic children from Mexico City]. AB - House dust mites, Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) are the predominant cause of allergic asthma in our population. To detect the presence of sensitivity to other mites besides Dp and Df in 100 children that live in Mexico City, skin tests were performed in their arms with Multi-test devices and glycerinated extracts. There were 34 female and 66 male, aged 4 to 14 years (x = 9.1y), with history of mild and moderate asthma, 2 months to 12 years of evolution. Dp was positive in 96, Df in 80, Euroglyphus maynei in 41, Chortoglyphus in 22, Blomia tropicalis in 17, Trophagus putrescentiae in 12, Glycphagus in 12, Acarus siro in 7, Lepidoglyphus destructor in 7 and Gophieria in 7. Hypersensitivity to all of th species was common. Even we know about the cross-reactivity between them, there are species-specific allergens that could make some of them important in diagnosis and treatment. PMID- 7552185 TI - [Clinical value of receptors, mediators, and cells in asthma]. PMID- 7552186 TI - [Reference values of T-lymphocyte subsets in umbilical cord blood]. AB - In order to determine reference values of T lymphocyte subpopulations in cord blood in the metropolitan area of Puebla city we randomly selected 80 from a total of 400 term, eutrofic and without pathology newborn. The percentage of CD3, CD4 and CD8 T lymphocyte subpopulations in cord blood were determined by flow cytometry. Reference values were gotten from the 25 and 75 percentile in parameters without gaussian distribution. Reference values are CD3 (45.5 to 91.3%), CD4 (26.2 to 69.4%), CD8 (16.2 to 24.1%) and NK (2.0 to 8.5%). The presented data represent valuable normal ranges of T lymphocyte subsets to compare with values observed in sick or in risk of developing immunoallergic pathology children. PMID- 7552187 TI - [Comparative study of the effectiveness of 2 oral corticoids in the control of severe crisis of bronchial asthma: deflazacort and prednisone]. AB - This article contains the results reached after conducting a study with 30 patients hospitalized or seen at the Emergency Ward of the National Institute of Respiratory Diseases, diagnosed as suffering from a severe bronchial asthma crisis, in whom the crisis was controlled with conventional measures. The purpose of this study was to compare the efficacy of deflazacort and prednisone as anti inflammatory agents capable of controlling severe asthma crisis in addition to analyzing their side effects. Deflazacort showed to be as effective as prednisone. PMID- 7552188 TI - [Frequency of allergens in immediate, late, and dual allergic skin response at the National Institute of Pediatrics]. AB - We studied 126 children with allergic illness in Instituto Nacional de Pediatria. We applied cutaneous test to determine the early, late and dual response. The results were 68% for the early response, 6% late response and 26% dual response. The allergens more frequently found in the early and late response were Dermatophagoides pteronyssinus, Dermatophagoides farinae and house dust. Approximately the third part of the children studied presented late cutaneous response (dual response more isolated late response). It is important to make the evaluation of the late response in patients with allergy disease for a good etiologic diagnosis. PMID- 7552190 TI - Salmonellosis: a hundred years old and still going strong. PMID- 7552189 TI - [The use of steroids in asthmatic children]. AB - During the last years it has been discovered that inflammation is the main substrate in asthma psychopathology. Treatment with steroids, inhaled or systemic is the first step in the antiinflammatory course during acute or chronic stages, and the early introduction is recommended to avoid prolongation of the attack. Actually, the disponibility of inhaled steroids, change the way we face with this illness, due their great local potency, their minimal side effects and the availability of metered dose devices. PMID- 7552191 TI - Lameness, lesions of the claw of the dairy cow and subclinical laminitis. PMID- 7552192 TI - The risks of the game: the confidential enquiry into perioperative equine fatalities. PMID- 7552193 TI - Current perspectives in salmonellosis. AB - Salmonellosis remains an important human and animal problem worldwide and, despite extensive research effort, many of the details of its pathogenesis are not known. While there have been recent advances in some aspects of pathogenesis, other areas are not understood. The host adaptation shown by several serotypes and the recent dramatic changes in the predominance of particular serotypes are examples. Molecular techniques using in vitro model systems have identified several genes involved in adhesion and invasion, though their function and even their relevance to disease remain poorly defined. Similarly, several potential toxins have been identified and the genes cloned, although their significance is far from clear. Some of the essential genes on the large virulence plasmids have been defined, and these are known to be necessary for the establishment of systemic infection. Two of these genes are regulatory, but the function of the other genes is unknown. A general theme has been the identification of gene systems involved in regulation of virulence. New vaccines, based on 'rational attenuation' are being designed, and these have also been used to carry heterologous antigens; such vaccines are currently undergoing trials. The improved understanding of the pathogenesis of salmonellosis may also provide a model of wide applicability to a more general understanding of bacterial pathogenesis. New techniques, including the polymerase chain reaction, are being applied to diagnose salmonellosis. PMID- 7552194 TI - The interference by maternally-derived antibody with active immunization of farm animals against foot-and-mouth disease. AB - Foot-and-mouth disease (FMD) is a highly contagious disease affecting ruminants and pigs. In countries in which control of FMD relies predominantly on vaccination, young stock ingest specific anti-FMD virus antibodies in the colostrum. This maternally-derived antibody (MDA) provides immediate protection against infection with FMD virus, but also interferes with the development of active immunity following vaccination. However, susceptibility to infection precedes the ability to respond to vaccination in the presence of MDA. Currently available vaccines cannot overcome this inhibitory effect of MDA, and protection of young stock can only be provided by their isolation from FMD virus. PMID- 7552195 TI - Lesions associated with subclinical laminitis of the claws of dairy calves in two management systems. AB - Claws of ten 6 to 7-month-old dairy calves in two management systems were examined for lesions associated with subclinical laminitis. Sole haemorrhages were found only in claws of calves maintained out of doors. No significant differences in number and severity of haemorrhages were found between left and right or lateral and medial claws, but haemorrhages were more frequent and more severe in hind claws than in front claws (P < 0.05). More haemorrhages of greater severity were present in the white zone at the toe than in all other areas of the weight-bearing surface combined (P < 0.05). No lesions of interdigital dermatitis and heel horn erosion were found in either indoor-housed or outdoor-housed calves. All calves kept indoors on deep bedding developed overgrown claws. PMID- 7552196 TI - Disposition of penicillin G sodium following intravenous and oral administration to Equidae. AB - The present study was designed to determine and compare the plasma disposition and pharmacokinetics of penicillin G sodium following intravenous (i.v.) administration to horses, ponies and donkeys. The plasma disposition and pharmacokinetics of penicillin G was similar in horses, ponies and donkeys (elimination half-lives--39.0, 27.3 and 31.5 min, respectively) and a dosage interval of 6-8 h would be suitable to treat infections caused by susceptible bacteria. Although penicillin G was absorbed rapidly following nasogastric administration, the systemic availability was low (0.12-0.34%), therefore oral administration would be unsuitable for systemic antimicrobial therapy in the equine. The elimination of penicillin G into the gastrointestinal tract following i.v. administration and the absorption of penicillin G from the gastrointestinal tract following oral administration were studied in two ponies with cannulated caecal fistulas. A low concentration of penicillin G (< or = 0.6 micrograms ml-1) was measured in caecal liquor following i.v. administration, however the risk of development of antimicrobial-associated colitis would be high following oral administration of penicillin G since high concentrations of drug (4.96-157.12 micrograms ml-1) were measured in caecal liquor. PMID- 7552197 TI - Impaired absorption of magnesium in the aetiology of grass tetany. AB - Magnesium is absorbed mainly from the reticulo-rumen and there are a number of factors reducing its absorption. The chief of these is the increased potential difference across the rumen epithelium caused by increased intraruminal potassium concentration. A significant amount of magnesium leaves the extracellular fluid each day as saliva. As only a portion of it is reabsorbed the rest is lost through the endogenous faecal excretion of magnesium. Thus, during impaired magnesium absorption, saliva could play an important role in the aetiology of hypomagnesaemia especially during dietary sodium depletion and the resultant increase in the potassium content of the saliva. PMID- 7552198 TI - Effects of treatment with buserelin on plasma concentrations of oestradiol and progesterone and cycle length in the cow. AB - Treatment of cows with 10 micrograms of the gonadotrophin releasing hormone (GnRH) analogue, Buserelin (Receptal; Hoechst) 12 days after mating results in a 12% improvement in conception rate (Drew & Peters, 1991). We have now investigated the endocrine effects and possible mechanism of action underlying this effect. Treatment of cyclic cows (n = 5) with Buserelin on days 11 and 13 did not affect cycle length or plasma concentration of progesterone, but did significantly (P < 0.05) reduce plasma oestradiol concentration from day 12-16. We conclude that the Buserelin treatment acts by causing a fall in plasma oestradiol which results in a reduction in the strength of the luteolytic drive, improving the chance of an embryo being able to prevent luteal regression. PMID- 7552199 TI - Cytogenetic characterization of a fibroma and three haemangiopericytomas in domestic dogs. AB - Cytogenetic evaluation of tumour cells taken from an 11-year-old mixed breed birth with a fibroma, showed trisomy 1 (2n = 79) and often the presence of a third copy of chromosome 4. In a 13-year-old mixed breed Boxer bitch with a haemangiopericytoma, trisomy 9 (2n = 79) was present. In contrast, another haemangiopericytoma (in a 15-year-old rough-haired Dachshund bitch) showed a deleted chromosome 1, several centric fusions and trisomy 2. Trisomy 2 and trisomy 29 were detected in a third haemangiopericytoma from an 11-year-old rough haired Dachshund bitch. PMID- 7552203 TI - Musculotendinous diseases of the shoulder (from clinical manifestations to anatomic lesions, from lesions to handicap and treatment) PMID- 7552200 TI - Congenital abnormalities of the ovine paramesonephric ducts. AB - A 15 month survey of ovine reproductive tracts was undertaken in slaughterhouses in southwest England. A total of 33506 tracts were examined; 23536 from lambs and 9970 from adults. In total, 3.4% of tracts were pregnant and 3.3% exhibited abnormalities. Twenty cases of uterus unicornis, six of uterus didelphys and 11 of segmental aplasia were encountered, such that partial aplasia of the paramesonephric ducts accounted for 3.3% of all abnormalities. Although developmental abnormalities of the ovine female genital system are relatively uncommon, a substantial proportion of these can be accounted for by development defects of the paramesonephric ducts. PMID- 7552201 TI - Pharmacokinetics of gentamicin following single-dose parenteral administration to goats. AB - The disposition kinetics of parenterally administered gentamicin (5 mg kg-1) has been studied in Gaddi goats. The serum concentration-time profile was described by bi-exponential and mono-exponential equations following intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) administration with elimination half life values of 0.96 +/- 0.09, 2.37 +/- 0.47 and 3.56 +/- 0.39 h, respectively. The apparent volume of distribution following i.v. administration (Vdarea: 0.26 +/- 0.041 kg-1) reflected limited extracellular distribution of the drug. The bioavailability was higher following i.m. administration (96.3%) compared to s.c. (76.9%). In view of the significantly longer biological half-life and larger area under the curve values, the s.c. route may be preferred. It is concluded that a suitable and practical dosage recommendation for gentamicin in goats would be 3.35 mg kg-1 body weight given s.c. at 12 h intervals. PMID- 7552202 TI - Aqueous humour and cerebrospinal fluid collected at necropsy as indicators of ante mortem serum 3-OH butyrate concentration in pregnant sheep. PMID- 7552204 TI - Phospholipase A2 inhibitors: a challenge for the future. PMID- 7552208 TI - Skeletal lesions in early congenital syphilis (a review of 86 cases). AB - Early congenital syphilis is still common in Morocco, where it causes a large number of stillbirths and neonatal deaths. Over the last 16 years, we managed 86 cases of congenital syphilis in infants aged one day to four months. Multiple organs were affected in half the patients. Skeletal lesions were found in 65% of patients, Wimberger's sign in nearly 20% (17/86), and pseudoparalysis in 23%. The significance of the skeletal lesions often seen in congenital syphilis in Morocco is emphasized. PMID- 7552205 TI - Fatty infiltration of disrupted rotator cuff muscles. AB - The extent of fatty infiltration of rotator cuff muscles was evaluated on computed tomography displays using a five-point scoring system in 63 shoulders before surgery for a rotator cuff tear and in 57 of these shoulders after surgical repair of the tear (the quality of cuff repair was evaluated in these 57 shoulders by arthrography, usually coupled with computed tomography, after a mean time interval of 8 months since the procedure). Preoperatively, significant fatty infiltration of the supraspinatus muscle was uncommon. Severe fatty infiltration of the infraspinatus and subscapularis muscles was seen in some patients. In most instances, fat accumulated in those muscles whose tendons were torn; however, tendon cleavage was associated with mild fatty infiltration of the corresponding muscle in some instances, and wide tears of the supraspinatus and subscapularis muscles were sometimes accompanied with severe fatty infiltration of the infraspinatus muscle. In half the cases, fatty degenerescence of the infraspinatus muscle occurred within six months of the tendon rupture. After surgery, in most shoulders without evidence of rotator cuff leakage on the postoperative arthrogram, no further accumulation of fat occurred. However, a decrease in the amount of existing fat was rarely documented and occurred only in the supraspinatus muscle. Before and after surgery, increasing severity of the fatty infiltration of the infraspinatus muscle was associated with increasing functional impairment of the same muscle. Recurrent rotator cuff tears, which involved only the supraspinatus muscle, were considerably more common when there was severe preoperative fatty infiltration of the infraspinatus muscle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552206 TI - Diagnostic value of clinical tests for shoulder impingement syndrome. AB - Results of three clinical tests for detecting shoulder impingement syndrome (Neer's, Hawkins', and Yocum's tests) and four tests for determining the location of the rotator cuff lesion (Jobe's test [supraspinatus], Patte's test [infraspinatus], lift-off test [subscapularis], and palm-up test [long head of the biceps brachii]) were compared to intraoperatively observed anatomic lesions in 55 consecutive patients who had surgery for Neer's syndrome. For Jobe's and Patte's tests, both pain (denoting tendinitis) and functional impairment (denoting tendon rupture) were evaluated. All clinical tests were done by the same examiner and all surgical procedures (acromioplasty with or without rotator cuff repair) by the same surgeon. The location and extent of the lesions (size of the tear in the 34 patients with rotator cuff defects) were determined intraoperatively. The sensitivity, specificity, and positive and negative predictive values of each test were calculated. Sensitivity was satisfactory but specificity was poor, in particular for determining the location and type of rotator cuff lesions. The severity of functional impairment during Jobe's and Patte's maneuvers was not correlated with the size of the tear. PMID- 7552207 TI - Systemic effects of epidural dexamethasone injections. AB - OBJECTIVES: to evaluate potential systemic effects of a single epidural injection of dexamethasone. PATIENTS AND METHODS: each of nine patients (five males and four females, mean age 47 +/- 11.8 years) admitted for sciatica was given a single epidural injection of 15 mg dexamethasone acetate. Before the injection (D0) and two (D2), seven (D7) and 21 (D21) days after the injection, the following laboratory tests were performed: serum cortisol and ACTH in the morning after an overnight fast, free cortisol in a 24-hour urine collection, fasting serum levels of glucose, triglycerides and cholesterol, serum levels of sodium and potassium. Blood pressure was measured on D0, D2, and D7. RESULTS: Serum cortisol, ACTH and urinary cortisol were profoundly decreased on D2 and D7 but normal on D21. There were no changes in fasting serum glucose, triglycerides, cholesterol, sodium or potassium levels. CONCLUSION: a single epidural injection of 15 mg dexamethasone acetate is associated with transient adrenal suppression, denoting passage of the steroid into the systemic bloodstream. However, evidence of hypercorticism is usually lacking. PMID- 7552209 TI - Familial multiple myeloma. Report of a case in a father and daughter. Review of the literature. AB - We report a case of familial multiple myeloma in a father and daughter. The same lambda chain was detectable in both patients. Symptom onset occurred three years earlier in the daughter than in the father. We found 52 previously published cases of familial myeloma, of which only 13 occurred in a parent and child. Given the incidence of myeloma in the general population, the occurrence of several cases in the same family is probably not due to chance. The few available data on karyotypes and oncogenes are discordant. The respective role of genetic factors and environmental factors is discussed. PMID- 7552210 TI - A critical appraisal of classification criteria for systemic lupus erythematosus. PMID- 7552211 TI - Spontaneous fracture of the odontoid process in a patient with ankylosing spondylitis. Nonunion responsible for compression of the upper cervical cord. AB - Tetraparesis due to a spontaneous fracture of the base of the odontoid process occurred in a patient with undiagnosed, advanced ankylosing spondylitis. Few cases of insufficiency fractures of the proximal cervical spine in patients with loss of spinal mobility have been reported in the literature. The pathologic lesions that can cause severe neurologic compromise are reviewed. Because functional impairment and neurologic loss are common even after surgical decompression and stabilization, these lesions should be looked for during a careful physical evaluation followed by appropriate investigations. PMID- 7552213 TI - Hypophosphatemic osteomalacia with plantar neurilemoma. A review of the literature (100 cases). AB - A case of hypophosphatemic osteomalacia with recovery after removal of a plantar neurilemoma is reported. One hundred cases of osteomalacia with a connective tissue tumor were found in the medical literature. Both sexes and all age groups were affected. Patients often had severe osteomalacia with decreased serum 1,25 (OH)2 vitamin D and phosphate levels and renal phosphate wasting. These abnormalities resolved immediately after complete excision of the tumor, which was often a small lesion found after the diagnosis of osteomalacia. Of the 100 tumors, 87 were benign and half were vascular (e.g., hemangiopericytoma, hemangioma, angiofibroma). A large number of tumors could not be readily classified because they contained vascular structures, giant cells, spindle shaped cells, and cartilage. Many other histologic variants were observed, including giant cell tumors, nonossifying fibromas, cartilaginous tumors, and osteosarcomas. The tumor was usually located in a limb, generally a lower limb, and was skeletal in nearly half the cases. The tumors produced one or more substances with a capacity for blocking intracellular phosphate transfer and inhibiting renal tube 1 alpha hydroxylase. The link between these two abnormalities remains unclear. PMID- 7552212 TI - Acute polyarthritis after BCG-therapy for bladder carcinoma in a patient with ankylosing spondylitis. AB - A case of polyarthritis following intravesical administration of bacillus Calmette-Guerin (BCG) for bladder carcinoma is reported in a patient with long standing ankylosing spondylitis. Possible links between BCG-therapy and joint manifestations are discussed in the light of data from the literature. Patients who develop joint manifestations after BCG-therapy are often HLA B27-positive. PMID- 7552214 TI - Polyradiculoneuropathy induced by gold therapy. PMID- 7552215 TI - Amiodarone-induced neuromyopathy mimicking polymyositis. PMID- 7552216 TI - Remitting seronegative symmetrical synovitis with pitting edema in elderly subjects: a plea for a limited definition. PMID- 7552217 TI - Afferent projections to the hypothalamus in ranid frogs. AB - Hypothalamic afferents were examined following applications of wheat germ agglutinin-horseradish peroxidase in the infundibulum of bullfrogs (Rana catesbeiana). Neuronal populations were generally labelled bilaterally, with an ipsilateral predominance. In the telencephalon, most labelled cells were present in the medial pallium, ventral lateral pallium, lateral amygdala, and lateral septal nucleus. A moderate number of cells were found in the medial amygdala and fewer still in the caudal striatum, olfactory tubercle/diagonal band nucleus, medial septal nucleus, and median septum. In the diencephalon, labelled neurons were primarily found in the anterior and central thalamic nuclei, anterior preoptic area, and suprachiasmatic nucleus. Fewer cells were present in the ventral habenular nucleus, anterior division of the lateral thalamic nucleus, posterior thalamic nucleus, ventromedial thalamic nucleus, magnocellular preoptic nucleus, posterior entopeduncular nucleus, and along the medial forebrain bundle. In the midbrain, a few labelled cells were found in the nucleus of the medial longitudinal fasciculus, suprapeduncular nucleus, and posteroventral tegmental nucleus. The secondary isthmal nucleus contained a large number of labelled neurons and moderate numbers of labelled cells were present in the grey medial to nucleus isthmi and in the isthmal raphe. A few lightly labelled cells were also seen in the parabrachial nucleus. Finally, after the largest applications, a few cells were seen in the obex region ventrolateral to the solitary tract. PMID- 7552218 TI - Afferent projections to the ventromedial hypothalamic nucleus in a lizard, Gekko gecko. AB - Afferent projections to the ventromedial hypothalamic nucleus (VMH) of a lizard, Gekko gecko, were studied with wheat germ agglutinin conjugated to horseradish peroxidase. Results indicated that the shell and core areas of the VMH are largely innervated by different telencephalic nuclei but similar brain-stem nuclei. The common brainstem projections include the superior raphe, two isthmal populations (the ventral isthmal nucleus and parvocellular isthmal nucleus), and a dorsal thalamic projection from the posteroventral nucleus. Brainstem projections to the shell but not to the core of the VMH arise from the laterodorsal tegmental nucleus. Telencephalic projections to the VMH core originate from the ventrolateral septum, centromedial DVR, lateral amygdala, medial amygdala, interstitial amygdala, and ventral anterior amygdala. Telencephalic projections to the VMH shell come from the ventral pallidum, the anterior septal nucleus, the dorsal septal nucleus, the striatoamygdalar area, and the ventral posterior amygdala. These results, combined with connectional and topological information from other studies in amphibians, reptiles, and mammals, will be used to suggest homologies between a number of limbic areas, including several amygdalar nuclei. PMID- 7552219 TI - Afferent projections to the lateral and dorsomedial hypothalamus in a lizard, Gekko gecko. AB - Afferent projections to the lateral hypothalamic area and dorsomedial hypothalamic nucleus of the lizard Gekko gecko were studied after applications of wheat germ agglutinin conjugated to horseradish peroxidase. Large applications into the hypothalamus labeled several telencephalic populations not observed after smaller injections. These included the rostrolateral area of the dorsal cortex, a sheet of cells deep to the caudal pole of the lateral cortex, the external amygdala, and part of the dorsal ventricular ridge. Other populations were labeled in the diencephalon, including the supraoptic nucleus and nucleus ovalis; in the medulla the medial reticular area was labeled. Injections into the lateral hypothalamic area labeled neurons in the rostrolateral dorsal cortex, anterior, lateral, and dorsal septal nuclei, the striatoamygdalar area, nucleus accumbens, vertical limb of the diagonal band, nucleus of the accessory olfactory tract, the interstitial, ventral anterior, and ventral posterior amygdalar nuclei, several hypothalamic nuclei, and the posteroventral thalamic nucleus. Labeled brainstem populations included the ventral tegmental area, torus semicircularis, parvocellular and ventral isthmal nuclei, superior raphe, and the solitary nucleus. Injections in the dorsomedial hypothalamic nucleus labeled neurons in the rostral and caudolateral poles of the dorsal cortex, anterior septal nucleus, horizontal limb of the diagonal band, nucleus of the anterior commissure, several hypothalamic areas, the lateral habenula, the posteroventral thalamic nucleus, and cells scattered around the dorsolateral anterior thalamic nuclei. Labeled brainstem populations included the torus semicircularis, ventral tegmental area, superior raphe, parvocellular and ventral isthmal nuclei, and the lateral dorsal tegmental nucleus. The results of these studies are compared with findings in amphibians and mammals. PMID- 7552220 TI - Excitatory and inhibitory transmission from the optic tectum to nucleus isthmi and its vicinity in amphibians. AB - Intracellular recordings were made from nucleus isthmi (NI) and its vicinity in toads (Bufo bufo gargarizans) in response to electrical stimulation of the bilateral optic tract. The results suggest that NI mainly receives its excitatory input from the ipsilateral tectum and its inhibitory input from the contralateral tectum. Tegmental cells in a region medioventral to NI were usually characterized by strong spontaneous activity and an excitatory-inhibitory sequence of postsynaptic potentials in response to stimulation of either side of the bilateral optic tract. Sixty of the recorded cells were stained with Lucifer yellow to show their morphological features and anatomical positions within NI and its vicinity. PMID- 7552221 TI - Thomas Szabo 1924-1993. PMID- 7552222 TI - Connections of the olfactory bulb in the chinook salmon (Oncorhynchus tshawytscha). AB - This report describes the connections of the olfactory bulb in juvenile chinook salmon (Oncorhynchus tshawytscha) as revealed by the retrograde and anterograde transport of biotinylated dextran amine (BDA). Pressure injections of BDA to discrete locations within the bulb reveal that the olfactory bulb projects via medial and lateral tracts to various regions of the dorsal and ventral telencephalon as well as to the diencephalon. Terminal-like boutons are present in the ventral, dorsal, lateral and supracommissural nuclei of the ventral telencephalon and in the lateral-ventral and posterior zones of the dorsal telencephalon. There is also a projection to the nucleus taenia and to the contralateral olfactory bulb. A diencephalic terminal field occurs in the posterior tuberal region of the ventral diencephalon. Olfactory bulb fibers in the preoptic region have axonal varicosities near neurons containing releasing hormones. A synaptic connection is suggested allowing for the possibility that output from the olfactory bulb may directly modulate the activity of these neurons. Telencephalic injections of BDA are used to determine the origins of olfactory bulb efferents. Neurons are retrogradely labeled by BDA in a Golgi-like manner, allowing one to visualize the morphology of olfactory bulb efferents. Three types of neurons in the bulb are filled by injections into the posterior zone of the dorsal telencephalon: mitral cells and ruffed cells in the external cellular layer, and neurons in the internal cellular layer. Although it has been known that both mitral cells and neurons in the internal cellular layer project out of the bulb in teleosts, the use of BDA allowed for an accurate description of the morphology of these extrinsically projecting neurons. This is the first evidence that the ruffed cells in salmon project out of the bulb. The results of this study suggest that the secondary olfactory projections in chinook salmon are consistent with those in other teleosts with minor variations. The data also show that olfactory bulb efferents originate not solely from mitral cells in the external cellular layer but also from ruffed cells in the external cellular layer and neurons in the internal cellular layer. It is proposed that the extrinsically projecting neurons in the internal cellular layer constitute an anterior olfactory nucleus in salmon. PMID- 7552223 TI - Instrumental learning in toads (Bufo arenarum): reinforcer magnitude and the medial pallium. AB - Two experiments studied the effects of shifts in the amount of water reinforcement on the instrumental performance of toads (Bufo arenarum) using one trial per day. In the first experiment, a shift from a large to a small reward magnitude led to a gradual change in performance without evidence of negative contrast. A shift from large or small reward magnitude to extinction led to similar extinction rates and provided no indication of the magnitude of reinforcement extinction effect. In the second experiment, the gradual change in performance (with no contrast) after a shift from a large to a small reward magnitude was completely eliminated by ablation of the medial pallium. These results provide support for the hypothesis that the medial pallium in amphibians is homologous to the hippocampal formation in mammals and also indicate that species differences in these learning phenomena may be related to the differential development and differentiation of the hippocampal formation. PMID- 7552224 TI - A quantitative analysis of agonistic behavior in juvenile American lobsters (Homarus americanus L.). AB - In these studies a quantitative analysis of agonistic (fighting) behavior in lobsters in presented as a first step in our attempt to relate patterns of behavior to underlying neurobiological mechanisms. The agonistic behavior of juvenile American lobsters (Homarus americanus L.) was studied in laboratory tanks at the New England Aquarium. Using video analyses and statistical techniques: (1) an ethogram of agonistic behavior was constructed; and (2) the temporal structure of the behavior was identified. We demonstrated that fighting in juvenile lobsters proceeds according to strict rules of conduct. All animals exhibit six common behavioral patterns in a stereotypical manner. A temporal sequence of these patterns was evident, representing an increase in intensity during confrontations. The typical scenario of an encounter begins with extensive threat displays upon first contact, continues with periods of ritualized aggression and restrained use of the claws, and terminates in a brief session of unrestrained combat. Predictions of game theory (i.e. assessment strategies) provide a useful framework for the understanding of fighting in lobsters. The presence of a highly structured behavioral system may reduce the potential for damage in fights among conspecifics, and may prove useful in attempts to study the neurobiological causes of complex behavioral patterns such as aggression. PMID- 7552225 TI - Studies on the optic chiasm of the leopard frog. I. Selective loss of visually elicited avoidance behavior after optic chiasm hemisection. AB - We hemisected either the posterior or anterior portion of the optic chiasm and found that frogs were unresponsive to large looming stimuli anywhere in the visual field. Nonetheless, the animals responded to prey stimuli throughout the visual field. Responses to looming stimuli returned in 1 to 8 weeks post-surgery. After complete transection of the chiasm animals were unresponsive to both prey and large looming stimuli. Frogs responded normally to prey and looming stimuli if less than half the optic chiasm was cut or if the postoptic commissure was cut. Since responses to looming stimuli returned before cut optic fibers could regenerate, these results suggest that visual information concerning prey and large looming objects are mediated by separate optic nerve fiber systems. PMID- 7552227 TI - Representation, referentiality, and processing in agrammatic comprehension: two case studies. AB - A number of investigators have argued that agrammatic comprehension, the pattern of sentence comprehension often associated with Broca's aphasia, can be characterized in terms of a representational disruption in one or another module of the normal grammar. In this study, these proposals are reviewed and their adequacy is examined in light of two case studies of agrammatic comprehension. In particular, we present data from sentences that have composed the core of the agrammatic comprehension pattern, as well as data from three different classes of sentences including comprehension of the matrix clause of center-embedded relative constructions, pronoun and anaphor dependencies, and Wh-questions. Our conclusion is that none of the existing representational models provides a fully adequate account of the data, and we propose some alternative approaches that distinguish between referential and nonreferential elements and potential processing differences between the two. PMID- 7552226 TI - Studies on the optic chiasm of the leopard frog. II. Organization of retinotectal fibers in the optic chiasm. AB - The organization of retinotectal fibers in the optic chiasm was investigated using horseradish peroxidase (HRP) histochemistry and electrophysiological recording. HRP injection into a small region of the tectum led to retrograde staining of labeled fibers in a circumscribed region of the chiasm and staining of labeled ganglion cells in the contralateral retina. In each instance labeled tissue was spread over a greater proportion of the area of a chiasm section than over the flattened retina. Fibers originating in central (older) retina are located in dorsal chiasm. Fibers originating in peripheral (younger) retina are located in ventral chiasm. Viewed with the electron microscope, labeled unmyelinated fibers are admixed with labeled myelinated fibers. Neuronal activity was monitored with an extracellular microelectrode from points in dorsoventral tracks in the chiasm. Multiple units were recorded at each chiasm location. Using visual stimuli, the receptive fields of the units were mapped. The fields were distributed along an arc across the visual field. At ventral chiasm recording sites the arc was in the peripheral part of the visual field. In succeeding dorsal sites the arcs were concentrically arranged so that the more dorsal the chiasm recording site, the more central was the arc in the visual field. Thus, in the optic chiasm, retinal fibers appear to be organized chronotopically but not retinotopically. Fibers of the same age but from different locations in the retina are mixed together. PMID- 7552228 TI - Functional categories in agrammatism. AB - This paper comments on Hagiwara's (1994b, this volume) proposals to account for the language deficits found in agrammatic aphasia, with special emphasis on Functional Categories (FCs). Some contemporary assumptions about FCs are reviewed focusing on their contentfulness, morphological features, and depiction as a heterogeneous set. This paper also discusses the view that the target of impairment in agrammatism constitutes a natural class. Treatment of FCs in their connection to chains and Lexical-Relatedness is discussed in terms of feature checking and morphological characteristics. PMID- 7552231 TI - The breakdown of functional categories and the economy of derivation. AB - In this paper I argue that in a hierarchical structure of a sentence, the lower the position of the functional head and its projection, the more accessible they are to an agrammatic aphasic. The major empirical basis for this includes spontaneous speech data and an acceptability judgment experiment by Japanese agrammatic patients as well as the available French and Italian data. The previous cross-linguistic agrammatic data from verb second languages are also examined. The unified account for the status of functional categories in agrammatism is presented based on the principles of the Economy of the Minimalist framework (Chomsky 1994). Finally, the empirical consequences of agrammatic data for language acquisition are discussed. PMID- 7552230 TI - Agrammatism as evidence about grammar. AB - A variety of experimental paradigms has yielded surprisingly fine-grained evidence about the kinds of syntactic information to which agrammatic aphasics are sensitive. This paper contrasts three accounts of agrammatism which draw quite different conclusions about the implications of this disorder for normal function: the chain-disruption, trade-off, and mapping hypotheses. Counterarguments to the chain disruption and trade-off hypotheses are presented, and it is argued that agrammatism provides considerable support for the modularity of syntax but provides no evidence more specific than that regarding the psychological reality of government binding theory vis-a-vis other current theories of grammar. PMID- 7552229 TI - A restrictive theory of agrammatic comprehension. AB - In this paper I propose a new, restrictive theory of Trace-Deletion in agrammatism. This theory subsumes the Trace-Deletion Hypothesis (TDH; Grodzinsky, 1984a,b, 1986, 1990), which maintains that traces are deleted from agrammatic representations and that a cognitive strategy augments the patients' performance. This claim accounts for the pattern of loss and sparing observed in these patients' comprehension of a wide variety of syntactic constructions and is thus important for our understanding of the neural representation of syntax. Yet there are reasons for revising the account and making it more precise, stemming from both recent empirical findings and new developments in the theory of syntax. The original TDH was based on observations of agrammatic comprehension of structures containing traces resulting from either NP- or Wh-movement. Nevertheless, heads (as opposed to phrasal projections) also move and leave traces behind. Head movement (of verbs, for instance) has come to play a central role in linguistic theory (which currently postulates a wider variety of empty categories than any previous theoretical framework). Recent findings suggest that verb movement is retained in agrammatism, indicating that a sweeping claim regarding the deletion of all empty categories is too strong. This motivates the first restrictive move, resulting in a theory that picks out a restricted set of traces--only those for which deficient performance is indeed observed. All other empty categories are left intact. Trace-Deletion is tied to theta-positions. The second restrictive move is motivated by two types of surprising asymmetries that have recently been discovered for agrammatic comprehenders: First, agrammatic comprehension on passives of psychological predicates provides an error pattern that distinguishes this construction from agentive passive, indicating that the deficit is tied to the thematic properties of the predicate: Second, asymmetries have been observed in agrammatic comprehension of questions and quantifiers. These findings motivate a modification of the augmentative strategy, whose domain of application is restricted to referential NPs. Thus, the new account amounts to the claim that only traces in theta-positions are deleted, and that the strategy applies to referential NPs alone. This, I argue, not only derives all the data precisely but is also conceptually superior to any previous account of agrammatism. Finally, I discuss the consequences of this account to linguistic theory, and to theories of brain/language relations. PMID- 7552232 TI - PDA comments: SUPAC-IR, ICH Q1C, FDA draft guideline. Parenteral Drug Association. AB - Following are comments prepared by PDA on three draft regulatory documents all relating to stability for post-approval changes, new dosage forms and related issues. The comments on SUPAC-IR and ICH Q1C were submitted to FDA on February 10, 1995. The comments on the December 12, 1994 draft guideline were submitted on March 10, 1995. The documents have different sources and content, however, there is some overlap between the three. Readers may wish to peruse the table attached to the March 10, 1995 PDA comments for a graphic analysis of the contents of the three documents. Readers are cautioned that these comments are based on draft proposals by FDA and/or the International Conference on Harmonization. More recent drafts may be available. Copies of all three documents are available from PDA (SUPAC-IR: Document #FDA 79; ICH Q1C: #FDA 93; December 12, 1994 guideline: #FDA 77). See the PDA Letter or contact the PDA staff for additional information. Further clarifying comments can be found in the January, March and April 1995 issues of the PDA Letter. Staff contact is James Lyda. PDA expresses thanks to the working group which drafted the comments on very short notice: Raymond Shaw, Jr. Ph.D., Merck (now of Wyeth-Ayerst) (Chair); Robert Barraza, Burroughs Wellcome; Fred Gustafson, Abbott; Paulette Kosmoski, and Russel Madsen and James Lyda, PDA. PMID- 7552234 TI - Aggregation of insulin and its prevention by carbohydrate excipients. AB - Aggregation of peptide/protein drugs is of concern as it may lead to reduced bioactivity, immunogenic reactions, blockage of infusion pumps or unacceptable physical appearance. Aggregation of insulin and its prevention by carbohydrate excipients was investigated in this study. Aggregation was induced in solid-state by incubating with moisture at 37 degrees C, or in solution by lyophilization, shaking or multiple passage through a needle. Moisture-induced aggregation in solid state with bovine and human insulin resulted in soluble aggregates which were analyzed by light scattering technique. They were found to be noncovalent by size exclusion chromatography. Three of the four carbohydrate excipients used, i.e., dextrose, Emdex and hydroxypropyl beta-cyclodextrin minimized the moisture induced aggregation of bovine insulin, with dextrose and Emdex being somewhat more effective. In lyophilization studies, bovine insulin was found to aggregate more than human insulin. In syringe/needle study, aggregation increased as a function of the number of times the solution was passed through the needle. Aggregation induced by shaking insulin solutions resulted in insoluble aggregation, which could also be minimized by carbohydrate excipients. PMID- 7552233 TI - Sterility test incubation issue. AB - The article published in the September-October 1993 issue of the Journal of Parenteral Science and Technology, entitled "The Incubation Period in Sterility Testing" is a subject that should stimulate further scientific dialogue. Let's examine the technical ramifications of the publication. PMID- 7552236 TI - Validation: an unconventional review and reinvention. PMID- 7552235 TI - The effect of excipients on glass transition temperatures for FK906 in the frozen and lyophilized states. AB - FK906, a tripeptide, has been used as a model compound to study the effect of excipients on the glass transition temperature for peptides and related compounds in the frozen solution and lyophilized states. Three kinds of excipients were chosen for study, low molecular weight sugars, high molecular weight polymers and salts. Our results indicate that the Gordon-Taylor equation can be used to predict the glass transition temperature of FK906 in the presence of sucrose, lactose, trehalose and maltose in the frozen solutions and in lyophilized products. Dextrans having different molecular weights can have different effects on the glass transition temperature of FK906 in the frozen state. Cooling rate can also have an effect on the glass transition temperature for FK906 frozen solutions in the presence of dextran. Finally, salts have a significant effect on the glass transition temperature of FK906 in the frozen state but not in lyophilized FK906. PMID- 7552237 TI - Application of a spray drying technique in the production of TRH-containing injectable sustained-release microparticles of biodegradable polymers. AB - Copoly (dl-lactic/glycolic acid) microparticles for sustained release of a water soluble drug (Thyrotropin releasing hormone: TRH) were prepared by a spray drying method. A higher entrapment ratio was achieved with the spray drying method with the in-water drying method. In order to avoid agglomeration of the microparticles, a double-nozzle spray drying method was designed using mannitol as an anti-adherent. The surface of the spray-dried microparticles was coated with mannitol, and the extent of agglomeration was decreased. Acetonitrile was the most suitable solvent for microencapsulation using the double-nozzle spray drying method because the initial burst of TRH from the microparticles during the first day was the smallest. When PLGA with a weight-average molecular weight of 14,000 was used, constant release of TRH continued for one month with a small initial burst. In conclusion, the production of biodegradable microparticles by the double-nozzle spray drying method appears to be an attractive alternative to conventional microencapsulation methods. PMID- 7552238 TI - Regulatory science. AB - The mission of regulation is to provide the general population with the most desirable products from among the many new ones currently available. Scientific activities supporting this mission can be regarded as Regulatory Science--the science of estimation and evaluation. Regarding regulatory science, research that can help produce accurate estimates is actually of greater value than the fruits of basic or applied research. Regulatory science exists not merely for the purpose of regulation but to support development as well. Rules that are neutral and open to public scrutiny must be created through cooperative research among industry, academic and regulatory scientists. PMID- 7552239 TI - The significance of sieve-retention to the filter validation process. AB - Current efforts at filter validation involve the need to test under "worst case conditions" the bacterial retentive action of filters utilizing the very drug being processed. These concerns recognize that the adsorptive retentions of organisms vary in their efficiencies, depending upon the filtration conditions and the presence or absence of surfactant. The sieve retention of organisms is independent of such conditions, deriving solely from considerations of organism size, pore-size relationships. Flow-decay studies, can serve to differentiate between filter actions based on sieve-retentions and adsorptive-retentions. A filter's dependence solely upon sieving effects is demonstrable. What is involved is a plotting of flow rates as a function of time in accordance with established mathematical equations. Appropriate plots would indicate the exclusive sieve retention of organisms, a condition of absolute filter reliability deriving from organism-pore size relationships. The clear demonstration by a filter company that its sterilizing filter retains organisms solely by the sieve retention mechanism would suffice to validate that filter for all pharmaceutical filtrations, eliminating the need for individual user validations. The advantage of this mode of filter validation, one based on structurally-inherent filter pore architecture, is that pharmaceutical manufacturers would be free from the need to validate a given filter for each and every pharmaceutical preparation. This would also simplify the FDA's confirmation of filter validation, since a single inspection of the filter manufacturer's records would suffice for all the filter's users. PMID- 7552240 TI - ASEAN GMP and pharmaceutical industries in Indonesia. AB - Indonesia was appointed by the ASEAN Technical Cooperation in Pharmaceutical as a focal point and to coordinate the development of practical guidelines for the implementation of GMP. The ASEAN GMP Guidelines were endorsed by the ASEAN Technical Cooperation in Pharmaceutical in 1988, which among others required separation of Beta-Lactam dedicated facilities and three degrees of cleanliness for production areas. As it was realised that drug manufacturers in developing countries need more detailed guidelines to be able to implement the GMP, an Operational Manual for GMP was also prepared for providing examples of SOPs lay outs, documentation etc. It was agreed by the technical cooperation group to leave the implementation of GMP to each member country. However, the ASEAN Manual for Inspection of GMP was drafted and endorsed by the group and training of ASEAN Drug Inspectors was organized to support the implementation. The ASEAN GMP is being implemented in Indonesia through a five-year, stepwise implementation plan, starting in 1989. PMID- 7552241 TI - Quality concept of parenterals in Europe. AB - Different types of dosage forms are available for parenteral use. They include solutions and suspensions for injection and infusion, sterile powders and lyophilisates which are administered after reconstitution with water for injection or other solutions and finally, controlled-release products (e.g., microparticles or implants). For the quality characterization of the drug products the quality concept of the drug substance has to be considered in addition to the finished product. Several Guidelines on the European basis and others are available, but the harmonisation of the quality concept on an international basis is highly appreciated by pharmaceutical industries. The ICH process is an extremely positive development aiming for a unique chemical and pharmaceutical documentation which can be submitted to all authorities worldwide. Beside the existing standards already internationally harmonised additional aspects will be discussed which include for the quality concept of the drug substance the manufacture and quality control during manufacture and specification of impurities and for the drug products particulate matter tests, specification of content, uniformity of mass and content and the biopharmaceutical characterization. PMID- 7552242 TI - 5-HT1A agonist and dexamethasone reversal of para-chloroamphetamine induced loss of MAP-2 and synaptophysin immunoreactivity in adult rat brain. AB - Serotonin and dexamethasone act as differentiating agents during development. Reducing circulating adrenal steroids or central 5-HT levels via adrenalectomy (ADX) or the tryptophan hydroxylase inhibitor, para-chlorophenylalanine (PCPA), respectively, has been shown to have de-differentiating effects in the adult brain. Morphometric analysis of 5-HT, S100 beta, MAP-2 and synaptophysin immunoreactivity (IR) was used to follow the molecular plasticity of several brain regions after lesioning of 5-HT nerve terminals by para-chloroamphetamine (PCA; 2 x 10 mg/kg s.c.), a serotonin neurotoxin. Two weeks after PCA treatment we observed reductions of 5-HT, S100 beta, and MAP-2 IR in parietal and temporal cortex, temporal pole, hippocampus and hypothalamus. The reductions in MAP-2 and synaptophysin-IR were reversed by 3 days of treatment with dexamethasone (10 mg/l drinking water) or ipsapirone, a 5-HT1A agonist (1 mg/kg s.c.). The loss of S100 IR was reversed only by the 5-HT1A agonist. These results indicate that both dexamethasone and serotonin have effects on adult neuronal plasticity but may work via different mechanisms. The implications of these findings to the loss of synaptophysin and MAP-2 staining in Alzheimer's disease are discussed. PMID- 7552244 TI - Nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester decreases ischemic damage in reversible focal cerebral ischemia in hyperglycemic rats. AB - We tested the hypothesis that the exacerbation of post-ischemic brain tissue injury associated with hyperglycemia in rats is due to toxic metabolism of nitric oxide. We used magnetic resonance imaging (MRI) techniques to measure neuronal and cerebrovascular injury in a 2-h transient focal cerebral ischemia model in normoglycemic and hyperglycemic rats at 3 and 24 h post-ischemia onset. We determined the effect of low dose (3 mg/kg i.p.) treatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Compared to normoglycemia, preexisting hyperglycemia increased the volume of brain tissue exhibiting hyperintensity in diffusion weighted MRI (DWI) by factors of 5.6 and 6.2 at 3 h and 24 h post-ischemia, respectively. A similar increase in tissue volumes exhibiting hyperintense signal in T2-weighted MRI (T2WI) (3.3-fold and 5.6-fold) was observed. Cerebral blood volume MRI indicated a large focal no reflow zone in hyperglycemic rats. Treatment with L-NAME eliminated the no-reflow zone in the hyperglycemic rats, and reduced tissue volumes of DWI hyperintensity by 86% and 93% at 3 h and 24 h, respectively. Similarly, tissue volumes of T2WI hyperintensity were reduced by 80% and 94% at 3 h and 24 h, respectively. Thus, nitric oxide is an important mediator in the exacerbation of post-ischemic brain injury in hyperglycemic rats. Inhibition of nitric oxide synthase limits edema formation, improves perfusion and reduces infarct volume. PMID- 7552243 TI - Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia. AB - Polyclonal antisera were generated against two identical regions of rat and human A1 adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A1 receptors. Immunohistochemistry of CHO cells transfected with the rat or human A1 adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A1 adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A1 receptor expression and support the concept of cellular specificity of A1 adenosine receptor action. PMID- 7552245 TI - Dose-dependent effects of buspirone on behavior and cerebral glucose metabolism in rats. AB - In this study we compared the effects of the anxiolytic buspirone on behavior and regional cerebral metabolic rates for glucose (rCMRglc) with those of the reference serotonin (5-HT)1A agonist 8-hydroxy-2(di-N-propylamino)tetralin (DPAT). Behavioral effects were assessed by scoring the 5-HT syndrome. rCMRglc was measured in 56 brain regions by using the quantitative autoradiographic [14C]2-deoxyglucose technique, at 10 min after i.p. injection of DPAT (1 mg/kg) or buspirone (0.4, 4 and 40 mg/kg) in awake male Fischer-344 rats. Whereas DPAT produced an intense 5-HT syndrome, buspirone had no behavioral effect. A low dose (0.4 mg/kg) of buspirone reduced rCMRglc in 18 brain areas (32%), more markedly in limbic areas and raphe nuclei. These were the only rCMRglc effects buspirone had in common with the potent 5-HT1A agonist DPAT and suggest that low dose buspirone activates preferentially 5-HT1A receptors. Hence, this receptor subtype may mediate buspirone functional effects on the limbic system and, given the role of these brain areas in mood control, possibly buspirone therapeutic actions. High doses (4 and 40 mg/kg) of buspirone produced widespread rCMRglc decreases in 46 (82%) and 44 (79%) of the areas studied and increased rCMRglc in one brain area, the lateral habenula, that was not affected by DPAT or a low dose of buspirone. The topographic distribution and direction of rCMRglc changes by high doses of buspirone differ from those produced by the 5-HT1A agonist DPAT. Instead these changes resemble the rCMRglc effects of dopaminergic D2 antagonists like haloperidol and are consistent with some pharmacological and binding properties of buspirone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552246 TI - Water deprivation induces regional expression of c-fos protein in the brain of inbred polydipsic mice. AB - We studied the effects of water deprivation on the expression of c-fos protein (Fos) in the brain of inbred polydipsic mice, STR/N strain, that show extreme polydipsia without a lack of vasopressin in the body. Non-polydipsic mice, ICR strain, were used as controls. All male animals were deprived of water for 24 and 48 h. Fos-like immunoreactivity (Fos-LI) in the brain was studied by immunohistochemical techniques. In both groups of mice water deprivation induced a remarkable increase in Fos-LI in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the median preoptic nucleus (MnPO), the organum vasculosum laminae terminalis (OVLT) and the subfornical organ (SFO). A far more increase, however, was seen in the MnPO, the SFO and the area postrema (AP) of the polydipsic mice compared to those of the non-polydipsic control mice. In the nucleus of the tractus solitarius (NTS) and in the anteroventral part of the PVN (avPVN), water deprivation caused a clear increase in Fos-LI in the polydipsic mice, while in the non-polydipsic mice the same treatment induced no Fos-LI in the NTS and no change in the avPVN. These results indicate that neurons in the circumventricular organs and the NTS are strongly activated by water deprivation in the polydipsic mice, suggesting that these brain structures play an important role in the polydipsia. PMID- 7552247 TI - Efferent projections from the region of the medial zona incerta containing A13 dopaminergic neurons: a PHA-L anterograde tract-tracing study in the rat. AB - Potential efferent projections of A13 dopaminergic (DA) neurons were identified in the present study by examining the distribution of labelled fibers following iontophoretic injection of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into the medial zona incerta (MZI), the region of the diencephalon containing A13 DA neuronal perikarya. One week after injection, PHA-L labelled fibers were found throughout the brain with the heaviest labelling occurring ipsilateral to the injection site in the anterior hypothalamic area, lateral hypothalamus, lateral preoptic area, horizontal diagonal band of Broca, and parvocellular region of the paraventricular nucleus. Moderate labelling was observed in the ipsilateral median preoptic nucleus, lateral septum, lateral aspect of the bed nucleus of the stria terminalis, and central nucleus of the amygdala. Moderate labelling was also found in the contralateral MZI and parvocellular region of the paraventricular nucleus. Light labelling was detected in the ipsilateral medial preoptic area, supraoptic nucleus, ventromedial nucleus, arcuate nucleus, vertical limb of the diagonal band of Broca, and in the contralateral lateral hypothalamus. Few immunopositive fibers were present in the dorsomedial nucleus of the hypothalamus or the magnocellular region of the paraventricular nucleus. These results reveal that neurons located in the MZI (possibly A13 DA neurons) have ipsilateral efferent axonal projections to a variety of brain regions including the lateral hypothalamus, lateral preoptic area, and the limbic structures at the diencephalic-telencephalic juncture. PMID- 7552248 TI - NMDA receptors mediate expression of one form of functional plasticity induced by olfactory deprivation. AB - Unilateral olfactory deprivation for the first 3 postnatal weeks results in an enhancement of granule cell mediated, feedback inhibition of mitral cells in the rat olfactory bulb. Granule cells are excited by mitral cells by both non-NMDA and NMDA receptors. The present report describes the effect of the NMDA antagonist MK-801 (0.75 mg/kg) on the expression of deprivation enhanced inhibition. The results demonstrate that (1) enhancement of lateral olfactory tract paired-pulse inhibition of evoked potentials in deprived bulbs was stimulus intensity dependent, with the greatest difference expressed at highest stimulus intensities; (2) MK-801 reduced inhibition in both undeprived and deprived bulbs in a stimulus intensity dependent manner, with the greatest reduction occurring at highest stimulus intensities; (3) MK-801 eliminated the stimulus intensity effect on inhibition in both groups; and (4) following MK-801, deprived bulbs showed the same or less inhibition than undeprived bulbs. PMID- 7552249 TI - Stress regulation of mineralocorticoid receptor heteronuclear RNA in rat hippocampus. AB - Localization and regulation of mineralocorticoid receptor (MR) heteronuclear RNA (hnRNA) was assessed in rat hippocampus using an intron-directed in situ hybridization approach. The presence of hnRNA in the cell nucleus reflects recent gene transcriptional events and can be used as an index of neuronal transcriptional activation or inhibition. In the present study, sections incubated with an MR intron probe labeled cells in all regions known to contain MR mRNA. Signal generated by the intron probe was localized specifically to the nuclear compartment, consistent with recognition of hnRNA. Analysis of distribution across hippocampus indicated that MR hnRNA was particularly abundant in dentate gyrus (DG). In contrast, MR mRNA shows similar levels of expression across all hippocampal subfields. Exposure of animals to acute restraint markedly reduced MR hnRNA levels in CA1 and DG 60 min and 120 min following stress exposure, consistent with reduced gene transcription. These data support the hypothesis that stress-induced glucocorticoid secretion can rapidly affect transcription of steroid receptor genes. MR mRNA levels did not decrease over comparable time periods, suggesting either a significant lag-time between transcriptional changes and changes in cytoplasmic mRNA pools or stimulus-driven alterations in post-transcriptional RNA processing. PMID- 7552250 TI - The effect of chronic treatment with a novel aryl-piperazine antipsychotic on monoamine receptors in rat brain. AB - The effects of chronic treatment of rats with RWJ 37796, a novel aryl-piperazine containing antipsychotic drug, on brain monoamine receptors were studied. Rats were treated daily with RWJ 37796 (1.3 mg/kg), the typical antipsychotic haloperidol (1 mg/kg) or vehicle (control) for 21 days, and were sacrificed 3 days after the last injection. Binding of [3H]Sch-23390 and [3H]spiperone to D1 and D2 dopamine receptors, respectively, and [3H]8-hydroxy-2-(di-n-propylamino) tetralin ([3H]8OH-DPAT) to 5-HT1A receptors were measured in various brain regions using quantitative autoradiography. Binding to D2 dopamine receptors was significantly elevated in the caudate-putamen of rats treated with haloperidol or RWJ 37796 as compared to controls. However, the magnitude of the increase in D2 binding was significantly greater in haloperidol-treated (+38%) compared to RWJ 37796-treated (+21%) rats. Haloperidol treatment also increased binding (+35%) to D2 dopamine receptors in the nucleus accumbens, where RWJ 37796 treatment had a considerably smaller effect (+12). No changes in D1 dopamine or 5-HT1A receptor binding were detected following either antipsychotic treatment in any brain regions studied. Thus, at comparable doses, the novel antipsychotic RWJ 37796 produces less up-regulation of D2 dopamine receptor binding in the striatum than does the typical antipsychotic haloperidol. PMID- 7552251 TI - Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine. AB - Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance. PMID- 7552252 TI - Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats. AB - The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5-25 micrograms: 5 microliters) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 micrograms) increased MAP and HR by 32 +/- 3 mmHg and 45 +/- 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 micrograms; i.c.v.) blocked the effects of physostigmine (25 micrograms; i.c.v.). Also, the pre-administration of the M2 muscarinic antagonist, methoctramine (25 micrograms; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M1 muscarinic antagonist, pirenzepine (100 micrograms; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M3 muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 micrograms; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25-30 mg/kg), physostigmine (25 micrograms; i.c.v.) evoked a decrease in MAP and HR of 13 +/- 6 mmHg and 15 +/- 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats are possibly mediated through stimulation of central M2 muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR. PMID- 7552253 TI - The 'glibenclamide-shift' of centrally-acting antinociceptive agents in mice. AB - Morphine-induced antinociception is antagonized by the K(+)-channel blocker glibenclamide (glyburide; Glib), implicating ATP-sensitive (KATP) K+ channels in the analgesic effect of opioids. The present study examined the generality of this conclusion by measuring the effect of Glib on supraspinal (intracerebroventricular; i.c.v.) antinociception produced by representative mu opioids and the non-opioids pilocarpine and two alpha 2-adrenoceptor agonists (clonidine and tizanidine) using the mouse tail-flick test. Concurrent administration of Glib (40 micrograms, i.c.v.) produced a significant rightward shift of the dose-response curve of morphine, levorphanol, methadone, pilocarpine, clonidine and tizanidine; a modest, but not statistically significant, rightward shift of the dose-response curves of the mu-selective peptides DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin) and PL017 ([N-Me-Phe3,D Pro4]-morphiceptin); and no shift of the dose-response curves of alfentanil, carfentanil, fentanyl, sufentanil, or beta-endorphin. Glib produced a leftward shift of the dose-response curve of etorphine. These data support the involvement of KATP-type K+ channels in mediation of supraspinal antinociception, differentiate Glib-sensitive and Glib-insensitive opioid agonists, and reveal fundamental differences among antinociceptive agents in the extent of demonstrable utilization of this transduction pathway. PMID- 7552254 TI - Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro. AB - Many aspects of retinal physiology are controlled by a circadian clock including at least two steps in the melatonin synthetic pathway: the activity of the enzyme, N-acetyltransferase (NAT), and mRNA levels of the rate-limiting enzyme trytophan hydroxylase (TPH). Light and dopamine (through D2-like dopamine receptors) can phase shift the clock, and can also acutely inhibit NAT activity, resulting in supressed melatonin synthesis. In this paper, we show that eyecups cultured in constant darkness maintain a clock-controlled rhythm in TPH mRNA, with low levels in early day, rising to a peak in early night. Both eyecups and isolated retinas, cultured in light during the day, also exhibit a similar increase in TPH mRNA levels, indicating that this expression is not acutely inhibited by light. Treatment with light or quinpirole (D2 dopamine receptor agonist) in early night, at a time and dose that acutely inhibits NAT activity, does not change levels of TPH mRNA. Addition of eticlopride (D2 dopamine receptor antagonist) during the day, also has no effect on the normal daytime increase in TPH message levels. Therefore, TPH mRNA level is controlled by a circadian clock located within the eye, but acute effects of light or dopamine are not detected. PMID- 7552255 TI - Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons. AB - An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95% O2/5% CO2) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 +/- 5.4% (P < 0.001), while the latency increased to 115.6 +/- 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 +/- 0.1 ms to 2.1 +/- 0.1 ms (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive 'fast' K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 +/- 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption. In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of 'fast' K+ channels contributes to posttraumatic axonal dysfunction. PMID- 7552257 TI - Potentiation by neurosteroids of muscimol/adenosine interactions in rat hippocampus. AB - Extracellular recordings were made from the CA1 pyramidal cell layer of hippocampal slices in response to stimulation of Schaffer collateral fibres in stratum radiatum. Alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one potentiated the inhibitory effect of muscimol on the population spike size at low concentrations (0.5 and 1 microM) that had no significant effect on the spike size by themselves. This profile is in agreement with other reports which have described the effect of these neurosteroids as barbiturate-like. Alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one also at low concentrations potentiated the inhibitory effect of adenosine alone and in the presence of 1 mM barium which blocked adenosine activated potassium channels. Alphaxalone failed to potentiate the inhibitory effect of adenosine in the presence of 1 microM bicuculline. It is concluded that these neurosteroids enhanced the potentiative interaction between adenosine and muscimol in the presence of barium. The results indicate that adenosine's effects are normally enhanced by virtue of the potentiative interaction occurring with endogenous GABA. PMID- 7552256 TI - In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina. AB - Divergence of neural retinal and retinal pigment epithelial (RPE) lineages from the optic vesicle neuroepithelium starts at a very early stage of eye development. Partially or even fully differentiated RPEs of some vertebrate species are capable of transforming into neural retina. In the present study, we have shown that mammalian RPE possesses the ability to transdifferentiate into neural retina at early embryonic stages. If cultured in serum-free medium, presumptive rat RPE became pigmented and expressed a molecular marker of mature RPE. In the presence of basic fibroblast growth factor (bFGF), cultured early embryonic rat RPE did not acquire pigment and grew to form retina-like multilayer structure containing neuronal cells and cells that express markers of retinal ganglion, amacrine and rod photoreceptor cells. The effects of bFGF occurred independently of effects on cell division and became irreversible after periods that varied with tissue age. This study has demonstrated that already differentiated embryonic rat RPE still retain the ability to become neural retina up to certain stage. PMID- 7552259 TI - GABAB receptors modulate synaptically-evoked responses in the rat dentate gyrus, in vivo. AB - We assessed the effects of systemically injected baclofen, a GABAB agonist, on single and paired-pulse responses in the dentate gyrus of urethane-anesthetized rats, in vivo. Baclofen (10 mg/kg) significantly increased the duration of single excitatory responses. This increase was blocked by the GABAB receptor antagonist, CGP 35348, indicating that baclofen was acting through GABAB receptors. To determine the mechanism underlying this increase in response duration, the NMDA antagonist, D-2-amino-5-phosphonopentanoic acid (D-APV), was administered intracerebroventricularly (i.c.v.) after baclofen. D-APV by itself had no effect on the duration of the population excitatory post-synaptic potential (EPSP). However, when infused after baclofen, D-APV blocked the baclofen induced increase in EPSP duration. This indicates the prolonged EPSP duration caused by baclofen resulted from an enhancement of an NMDA receptor mediated component of the response. We then examined the effect of baclofen on population responses to paired stimuli. Baclofen attenuated paired-pulse inhibition of population spike amplitudes at a 25 ms interstimulus interval. CGP-35348 reduced the effect of baclofen on paired-pulse inhibition, indicating that baclofen suppressed paired pulse inhibition by acting on GABAB receptors. In contrast to its disinhibitory effect at the 25 ms interval, baclofen had an inhibitory effect on responses evoked at a 150 ms interstimulus interval. Under control conditions, we observed that when stimuli were delivered 150 ms apart, both the EPSP duration and population spike amplitude evoked by the second stimulus were enhanced. Baclofen suppressed this enhancement.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552258 TI - Incorporation of amino acids into the axoplasm is enhanced by electrical stimulation of the fiber. AB - The effect of sustained electrical stimulation upon the incorporation of amino acids into the axoplasm was studied in the goldfish Mauthner (M) axon with light autoradiography. An extracellular pulse of tracers applied between M-axons in the medulla resulted in a local and substantial labeling of the M-axoplasm and a faint labeling of the M-perikaryon 4-5 mm away from the site of injection. After 18 h of direct electrical stimulation of the M-axon at 0.3-0.8 Hz, the local incorporation of amino acids into the M-axoplasm doubled. This enhancement declined to reach the baseline within 24 h. A 4 h electrical stimulation did not enhance the incorporation. Transynaptic activation of the M-neuron through the auditory input at 0.1-0.2 Hz for 18 h did not raise the amino acid incorporation in the M-axoplasm. We conclude that electrical discharge of the axon modulates the local incorporation of amino acids into the axoplasm. PMID- 7552261 TI - Redistribution of GABA immunoreactivity following central retinal artery occlusion. AB - gamma-Aminobutyric acid (GABA) is normally primarily in amacrine cells in the rat retina. Immediately after an ischaemic insult, attained by occlusion of the central retinal artery for 60 min, GABA is then found to be associated with Muller cells. During subsequent reperfusion, the distribution of GABA immunoreactivity gradually reverts from the glial cells back into neuronal elements of the retina. Twenty-four hours after ischaemia, GABA staining is indistinguishable from that seen in control animals. It is suggested that during central retinal artery occlusion, Muller cell energy levels are sufficient to allow the active uptake of released GABA, but insufficient to metabolise it to glutamine. The normal cycle of GABA metabolites from Muller cells to neurones is thus inhibited. Restoration of blood flow and the consequent increase in retinal energy levels, as indicated by a slight recovery of the electroretinogram b-wave, facilitates glutamine shunting between glial cells and amacrine cells, resulting in the synthesis of neuronal GABA. PMID- 7552260 TI - Neuronal mechanism underlying dystonia induced by bicuculline injection into the putamen of the cat. AB - Microinjection of bicuculline (Bic) into the unilateral putamen (Put) of the cat resulted in periodic occurrence of contralateral movement of the head (dystonia). Corresponding to this dystonic movement, repetitive spikes of the field potential occurring in the Put of the injected side and strong inhibition of the extracellularly recorded unit discharges in the neurons of the substantia nigra pars reticulata (SNr) of the same side were observed. PMID- 7552262 TI - Colocalization of fructose-1,6-bisphosphatase and glial fibrillary acidic protein in rat brain. AB - Immunofluorescence studies of rat brain sections demonstrated an exclusive colocalization of the gluconeogenic key enzyme fructose-1,6-bisphosphatase (FBPase) with the astroglial marker glial fibrillary acidic protein, indicating FBPase in brain as an astrocyte-specific enzyme. This conclusion was supported by the presence of FBPase activity in astroglia-rich but not neuron-rich primary cultures derived from rat brain. PMID- 7552263 TI - Methamphetamine-induced serotonin neurotoxicity is mediated by superoxide radicals. AB - Methamphetamine (METH) causes deleterious effects in brain monoaminergic systems. Evidence has accumulated to suggest that these effects may be mediated via the overproduction of the superoxide radicals. We have recently shown that METH induced dopamine (DA) depletion is attenuated in copper-zinc superoxide dismutase (CuZnSOD) transgenic (Tg) mice. In the present study, we have used receptor autoradiographic studies of [125I]RTI-55 labeled serotonin (5-HT) uptake sites to evaluate the effect of a two dosing schedule (5 mg/kg or 10 mg/kg x 4) of METH on striatal 5-HT uptake sites in nontransgenic (Non-Tg), heterozygous (Hetero) and homozygous (Homo) SOD-Tg mice. The low dose caused no significant changes in striatal 5-HT uptake sites in any of the groups. The high dose caused marked decreases (-74%) in striatal 5-HT uptake sites in Non-Tg mice. In contrast, 5-HT uptake sites showed only a 31% decrease in homozygous SOD-Tg mice whereas heterozygous SOD-Tg mice showed 63% depletion. These results show that increased SOD activity can protect against METH-induced neurotoxicity in striatal serotonergic terminals. These data provide further evidence for a role of oxidative stress in the neurotoxic effects of METH. PMID- 7552264 TI - Electron microscopic evidence that axon terminals from the mediodorsal thalamic nucleus make direct synaptic contacts with callosal cells in the prelimbic cortex of the rat. AB - A combined study of anterograde axonal degeneration and HRP retrograde labeling has shown that there exist monosynaptic connections between afferent fibers from the mediodorsal thalamic nucleus (MD) and callosal cells in the prelimbic cortex of the rat. Degenerating axon terminals from MD made asymmetrical synaptic contacts with dendritic spines from apical dendrites of layer III pyramidal cells that were retrogradely labeled with HRP after its injection into the prelimbic cortex contralateral to MD lesions. PMID- 7552266 TI - Predegeneration enhances regeneration into acellular nerve grafts. AB - In the present study, we determined the regeneration rate and the initial delay in rat sciatic nerve grafts first made hypercellular by predegeneration then acellular by freeze-thawing. 7-day predegenerated nerve pieces from the distal nerve stump on the right side were made acellular by repeated freeze-thawing and inserted as grafts into a 10-mm long freshly created defect on the left contralateral side. Freshly made (no predegeneration period) acellular nerve grafts were used as controls. Both types of grafts supported outgrowth of regenerating axons as demonstrated by the sensory pinch test. However, the predegenerated acellular nerve grafts had a significantly shorter initial delay period (2.7 days) as compared with freshly made acellular nerve grafts (9.5 days). The initial delay period for predegenerated acellular nerve grafts was similar to that for fresh cellular nerve grafts but significantly longer than that for predegenerated cellular nerve grafts [24]. The rate of regeneration appeared independent of the type of grafts used. We suggest that modifications of the basal lamina and/or factors produced during the predegeneration period by non neuronal cells survive the freeze-thawing cycle and account for the decrease in the initial delay period. PMID- 7552268 TI - Free D-serine in post-mortem brains and spinal cords of individuals with and without neuropsychiatric diseases. AB - We have measured the concentrations of free D-serine post-mortem in the prefrontal cortex, parietal cortex, cerebellum and spinal cord from individuals with and without (controls) neuropsychiatric diseases using high-performance liquid chromatography with fluorometric detection. The levels of D-serine were found to be high in the prefrontal and parietal cortex (around 100 nmol/g wet weight) and very low in the cerebellum and spinal cord (below 10 nmol/g wet weight). The uneven distribution of the D-amino acid in the human central nervous system (CNS) resembles that observed in rodents, suggesting that, as shown in the rat CNS, the regional variation of D-serine content in the human brain might also be closely correlated with those of the N-methyl-D-aspartate (NMDA) type excitatory amino acid receptor. In the prefrontal cortex, the gray and white matter had a similar concentration of D-serine. These findings, together with the selective action of D-serine at the NMDA-related glycine site and the non neurogenic nature of extracellular D-serine release, add further support to the view that D-serine could be an intrinsic modulator of the NMDA receptor liberated from certain glial cells in the mammalian brain. Despite the anti-psychotogen activity of D-serine in the rat, there were no statistically significant differences between the D-serine contents in the prefrontal or parietal cortex of controls and those of patients with schizophrenia or dementia of the Alzheimer type. PMID- 7552265 TI - The putative dopamine D3 agonist, 7-OH-DPAT, reduces dopamine release in the nucleus accumbens and electrical self-stimulation to the ventral tegmentum. AB - The present experiments were designed to test further the idea that 7-OH-DPAT (7 hydroxy-N,N-di-n-propyl-2-aminotetralin), a putative dopamine (DA) D3 agonist, has effects at DA autoreceptors to reduce intracranial DA levels and to reduce behaviours that are DA-dependent. Rats were trained to respond on a self stimulation protocol for electrical stimulation to the ventral tegmental area (VTA). Each press of a lever delivered a 0.5 s train of square wave, 1.5 ms duration, 100 Hz, 90-120 mA stimulation. Systemic administration of 7-OH-DPAT at 0.01-0.3 mg/kg i.p., quickly dose-dependently reduced responding. Electrical stimulation using similar parameters to those that supported self-stimulation were then applied to the VTA of anaesthetized rats. Fast cyclic voltammetry (FCV) revealed that this stimulation released DA in the nucleus accumbens (NAC). 7-OH DPAT i.p. (0.1-3.0 mg/kg) quickly and potently reduced the size of the DA generated voltammetric signal. This effect of 0.3 mg/kg 7-OH-DPAT was not blocked by sulpiride (60 mg/kg, i.p.) a D2-specific antagonist that may preferentially block D2 autoreceptors. These data are discussed with reference to the possibility that 7-OH-DPAT reduces the release of dopamine in the NAC, at D3, but not at D2, autoreceptors and that this in turn may reduce the rewarding effect of VTA stimulation. PMID- 7552269 TI - Muscimol induces state-dependent learning in Morris water maze task in rats. AB - Effects of muscimol on the place learning in Morris water maze task were investigated in rats. Rats were given 4 training trials per day with the submerged platform at a fixed location in the maze for 4 days. On day 4, rats were required to swim in the pool without the platform after 4 training trials (probe test). Compared to the saline-treated rats, the rats treated with muscimol on day 1-4 showed no modifications of place learning in the training trials and the probe test. However, in the rats treated with muscimol on day 1-3 and treated with saline on day 4, there was increased latency to reach the platform and reduced duration in the quadrant where the platform had been located on day 4. The increased latency in the training trials and reduced duration in the probe test on day 4 was blocked by bicuculline, when bicuculline and muscimol were co administered on day 1-3, and saline was injected on day 4. Moreover, in the rats treated with muscimol on day 1-3, co-administration of bicuculline and muscimol on day 4 blocked place learning: increased latency in the training trials and reduced duration in the probe test was observed. These results suggest that muscimol induces state-dependent learning (SDL) in Morris water maze task, and that muscimol-induced SDL is mediated by GABAA receptors. PMID- 7552267 TI - Dopamine D4 receptors and effects of guanine nucleotides on [3H]raclopride binding in postmortem caudate nucleus of subjects with schizophrenia or major depression. AB - The densities of dopamine-D4 receptors were determined in postmortem samples of caudate nucleus from patients with schizophrenia (n = 9) and age-matched controls (n = 10). D4 receptor binding was defined as the difference between binding sites labeled by [3H]YM-09151-2 (D2 + D3 + D4 receptors) and those by [3H]raclopride, in the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p) (D2 + D3 receptors). D4 receptor binding was measurable in all the subjects with schizophrenia (mean = 3.8 pmol/g tissue) but only in 3/10 controls. To determine the specificity of these findings for schizophrenia, D4 receptor binding was also measured in the caudate nucleus of suicide victims with major depression (n = 6) and age-matched controls (n = 6). A small amount of D4 binding was noted in some of the controls + depressed subjects and there was no significant difference between controls and patients with major depression. The addition of 200 microM Gpp(NH)p to the assay significantly increased the amount of specific binding of [3H]raclopride in control tissues, but not in tissues from subjects with schizophrenia, suggesting an abnormality in the G-protein component coupled to the D2 receptor. [3H]Raclopride binding was also significantly increased by Gpp(NH)p in subjects with major depression. These results confirm a previous report of Seeman et al. (1993) and suggest that measurable D4 receptor binding in the caudate nucleus is more frequent in patients with schizophrenia as compared with normal controls and subjects with major depression and that guanine nucleotides do not enhance [3H]raclopride binding in schizophrenia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552270 TI - Neovascularization of rat fetal neocortical grafts transplanted into a previously prepared cavity in the cerebral cortex: a three-dimensional morphological study using the scanning electron microscope. AB - Neovascularization within syngeneic rat fetal neocortical grafts transplanted into a previously prepared cavity in the cerebral cortex was studied 1 to 3 months after transplantation, utilizing scanning electron microscopy of vascular corrosion casts. The grafts were easily identified and the outer surface of the grafts, especially at the host-graft interface, was surrounded by large regenerated vessels of leptomeninges and connective tissue (e.g. dura). Large vessels originating from the choroid plexus also coated the grafts in animals whose lateral ventricles had been opened at the time of cavitation. These large regenerated vessels were mainly observed on the surface of the grafts, and they ramified markedly to form capillary networks in the vicinity of the host-graft interface. Occasionally several relatively large regenerated vessels were noted to extend into the grafts, and to ramify and connect with graft capillary networks having the same features as that of the host brain. Moreover, direct vascular connections between host capillaries and those within the grafts were observed. In some animals, arteries and arterioles which fed the grafts were identified in the perimeter of the grafts with their characteristic morphology. The interior microvasculature structure of the grafts was largely composed of the capillary network of graft origin, and of several relatively large penetrating vessels originating from the regenerated leptomeningeal vessels or the vessels of the choroid plexus. The present study demonstrated that the blood supply to the solid grafts transplanted into the previously prepared cavities originated primarily from the regenerated host vessels. These host vessels perfused the intrinsic graft vessels via new anastomoses which formed predominantly at the host-graft interface. PMID- 7552271 TI - Monoaminergic and cholinergic modulation of REM-sleep timing in rats. AB - The effects on sleep structure of systemic administration of benchmark cholinergic, serotonergic, and noradrenergic antagonists (QNB, ritanserin, metergoline, and prazosin) were characterized in rats using a new technique for identifying transitions (NRTs) from non-REM (NREM) sleep to REM sleep. In agreement with previous studies, all agents tested reduced REM-sleep expression (by 36-86%). In addition, the serotonergic and noradrenergic antagonists reduced NRT frequency (by 58-81%). The cholinergic antagonist QNB had no effect on NRT frequency. These findings suggest that blockade of serotonergic or noradrenergic receptors increases the interval between REM-sleep episodes, perhaps reducing the rate of accumulation of REM-sleep propensity. Blockade of cholinergic receptors, by contrast, decreases REM-sleep expression by interfering with REM-sleep maintenance, not by modulating REM-sleep timing. These conclusions are contrary to the predictions of a number of published models of REM-sleep timing. PMID- 7552272 TI - Kappa opioid inhibition of morphine and cocaine self-administration in rats. AB - Two kappa agonists, U50,488 and spiradoline, produced dose-related acute decreases in both morphine and cocaine self-administration in rats; higher doses of both agents were required to decrease rates of bar-pressing for water. On the day after kappa agonist administration, both agents produced extinction-like patterns of responding in many rats self-administering morphine or cocaine but not in rats responding for water. Two days after their administration, both U50,488 and spiradoline produced significant decreases in both morphine and cocaine intake; some rats continued to show decreases in drug self-administration for 5-6 days. Although the kappa antagonist nor-binaltorphimine (10 mg/kg s.c.) had no effect itself on either morphine or cocaine self-administration, it fully antagonized the effects of U50,488 (10 m/kg i.p.). The results suggest that although endogenous kappa opioid systems may not tonically modulate mechanisms involved in drug reinforcement, pharmacological activation of kappa pathways may be a novel and effective pharmacological approach to treating both opioid and stimulant addiction. PMID- 7552273 TI - Brain distribution of c-fos expression as a result of prolonged rapid eye movement (REM) sleep period duration. AB - Auditory stimulation (AS) or recovery from sleep deprivation (SD) has been shown to increase REM sleep periods in rats, cats and humans. This increment in REM has been credited to an amplified level of excitability in a widely distributed neuronal network throughout the brain. Fos-like immunostaining (FLI) has been useful in constructing maps of post-synaptic neuronal activity with single cell resolution, and has been proposed to be tightly related with progressing neuronal activation. This study utilized FLI as a marker to determine the number of neurons and structures which express c-fos in broadly distributed areas of the brain in animals with REM periods prolonged by either AS or SD. The results indicated that the brain stem and diencephalon present FLI increases in a variety of structures that possibly share various functional aspects of the REM sleep mechanism. These results are discussed in terms of the possibility that REM maintenance is related to an increase in the recruitment of REM-on neurons. PMID- 7552274 TI - Dopamine D2-like receptors labeled by [3H]YM-09151-2 in the rat hippocampus: characterization and autoradiographic distribution. AB - Dopamine D2-like receptor labeled by [3H]YM-09151-2 in the rat hippocampus proper was examined by in vitro receptor autoradiography. In the dorsal hippocampus, [3H]YM-09151-2 bindings were high in the whole layers of CA1, the stratum pyramidale of CA4 and the stratum molecular of gyrus dentatus, moderate in the stratum oriens of CA3 and hilus of the gyrus dentatus, and low in remaining CA3 and the subiculum. In the ventral hippocampus, the binding densities were high in the stratum oriens and the stratum radiatum of CA1, the stratum pyramidale of CA4, and the stratum moleculare of gyrus dentatus, moderate in the stratum lacnosum moleculare of CA1 and the hilus of the gyrus dentatus. Saturation analysis using hippocampal sections demonstrated that the Kd value was about five times higher than that using striatal sections. The rank order potency of competition on [3H]YM-09151-2 binding by dopaminergic ligands in the hippocampus was YM-09151-2 > (+)-butaclamol > dopamine > sulpiride > SCH-23390; which shows the appropriate dopamine D2-like receptor profile. The hippocampal [3H]YM-09151-2 binding did not represent serotonergic receptors (5-HT1A and 5-HT2) and sigma receptor, since Ki values of ketanserine, serotonin, 8-OH-DPAT and DTG were much lower than D2-like receptor antagonists. These findings suggest tha [3H]YM-09151 2 binds hippocampal D2-like receptor site with different association kinetics of striatal D2-like receptor site, and demonstrates widespread distribution of D2 like receptor in the hippocampus with distinct region-specific profile. PMID- 7552276 TI - Inhibitory effects of dopamine and methylenedioxymethamphetamine (MDMA) on glutamate-evoked firing of nucleus accumbens and caudate/putamen cells are enhanced following cocaine self-administration. AB - Rats were allowed to self-administer cocaine during a 3-h session for 15 days. One to 11 days after the last cocaine exposure, rats were anesthetized with urethane and effects of microiontophoretically-applied dopamine on glutamate evoked firing of neurons in the nucleus accumbens and in the caudate/putamen were tested. Dopamine produced a dose-dependent inhibition of glutamate-evoked firing in both the nucleus accumbens and the caudate/putamen of rats that had been repeatedly exposed to self-administered cocaine and in control rats. However, the DA-induced inhibition was significantly greater in the group that had self administered cocaine. The cocaine self-administration group was significantly sensitized to the inhibitory effects of dopamine in both early (1-3 day) and later (9-11 days) periods of cocaine abstinence. Following cessation of repeated cocaine self-administration sessions, nucleus accumbens cells were also sensitized to the inhibitory effects of methylenedioxymethamphetamine (MDMA), a drug that increases extracellular levels of DA and serotonin in the nucleus accumbens. This sensitization to DA- and MDMA-induced inhibition in the nucleus accumbens and in the striatum indicates that long-term neuroadaptations occur in these regions of the nervous system following repeated exposure to self administered cocaine. PMID- 7552277 TI - Behavioral consequences of neonatal injury of the neocortex. AB - Several strains of autoimmune mice spontaneously develop molecular layer ectopias that are similar in appearance to those seen in humans and are caused by disturbances in neocortical neuronal migration. These mice also exhibit behavioral anomalies, some of which correlate with ectopias, others with the immunological disorder. In this study, we induced neocortical ectopias (via puncture wounds) and microgyria (via freezing lesions) in the neocortex of 1-day old (newborn) mice without immune disorders in an attempt to further disentangle the effects of autoimmunity and of cortical malformation on behavior. In addition, we wished to compare the behavioral effects of small ectopias to larger microgyric lesions. DBA mice were assigned at birth to receive either a puncture wound or freezing lesion of either the left or right hemisphere. An independent group was subjected to sham surgery. In adulthood, these mice were given a battery of tests designed to measure lateralization and learning capacity. Lesioned mice (irrespective of hemisphere or type of damage) performed poorly when compared to sham-operated animals in discrimination learning, in a spatial Morris Maze Match-to-Sample task, and in a Lashley Type III maze. In shuttlebox avoidance conditioning, where immunological disorder has been shown to compromise behavioral performance in autoimmune mice, there was no difference between lesioned and sham animals. These results (1) support the dissociation between the effects of developmental neocortical anomalies and autoimmune disease on behavior (2) reveal similarities between spontaneous and induced neocortical malformations and (3) fail to support a difference in behavioral effects between ectopias and microgyria. PMID- 7552278 TI - Alteration of brain nicotinic receptors induced by immobilization stress and nicotine in rats. AB - Immobilization stress (2 h.day-1, for 2 weeks) caused downregulation of the nicotinic receptors ([3H]cytisine binding sites) in the rat brain (cerebral cortex and midbrain). However, chronic nicotine treatment abrogated stress induced downregulation of the [3H]cytisine binding sites, suggesting that chronic nicotine treatment may block stress activated nicotinic receptor-mediated neurotransmission. PMID- 7552275 TI - Diminished [3H]inositol(1,4,5)P3 but not [3H]inositol(1,3,4,5)P4 binding in Alzheimer's disease brain. AB - Levels of the calcium mobilising receptors for the phosphoinositide hydrolysis derived second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and inositol(1,3,4,5) tetrakis-phosphate [Ins(1,3,4,5)P4] were compared in the cerebellum, superior temporal and superior frontal cortex of a series of Alzheimer's disease and matched control cases. Membrane [3H]Ins(1,4,5)P3 radioligand binding experiments performed under steady state conditions revealed that the number of Ins(1,4,5)P3 recognition sites was significantly decreased in all three brain regions of the Alzheimer's disease cases, compared to controls. In contrast, [3H]Ins(1,3,4,5)P4 binding levels, as assessed in competition analyses, were not significantly different between the groups in any brain region. Moreover, the Hill coefficients for inhibition of [3H]Ins(1,3,4,5)P4 binding by non-radioactive Ins(1,3,4,5)P4 were less than unity in both the control and Alzheimer's disease brains, suggesting that the heterogeneity of these binding sites are also maintained in the disease. It is concluded that disruptions of the phosphoinositide hydrolysis pathway in Alzheimer's disease brain are associated with a selective loss of calcium mobilising Ins(1,4,5)P3, but not Ins(1,3,4,5)P4 receptor sites. These alterations may contribute to an altered calcium homeostasis in Alzheimer's disease, as well as providing one reason for the lack of success of cholinergic replacement therapies aimed at enhancing muscarinic receptor-mediated phosphatidylinositol hydrolysis. PMID- 7552279 TI - Impairment of long-term potentiation in rats fed with vitamin E-deficient diet. AB - Our previous results indicate that alpha-tocopherol induces a slowly developing long-term potentiation (LTP) of excitatory postsynaptic potentials (EPSP) in hippocampal slices. In the present study on hippocampal slices obtained from rats fed with vitamin E-deficient diet for 3 months, a tetanic stimulation of the stratum radiatum or applied alpha-tocopherol phosphate failed to induce LTP of CA1 neuronal EPSPs. However, LTP could be induced in rats fed on a matching control diet. These results indicate that vitamin E-deficiency leads to an impairment in LTP induction. PMID- 7552280 TI - Long-lasting conditioning of the human soleus H reflex following quadriceps tendon tap. AB - Percussion of the quadriceps tendon was used to test the hypothesis that knee extensor muscle spindle discharge initiates down-regulation of the gain of the soleus H reflex. Seven subjects participated. Soleus H reflex magnitude was observed for up to 15 s, following conditioning tendon taps of 60 N or 80 N force and 10 ms duration, with the knee at 60 degrees or 90 degrees of flexion. The tap elicited quadriceps stretch reflexes in four subjects, with a mean latency of 42 ms. The major component of the conditioning of the soleus H reflex was significant attenuation of magnitude by 30-90% of controls, starting as early as 36 ms post-percussion and lasting as long as 3-8 s. The attenuation of reflex magnitude was evident, whichever combination of duration and force of tap was used. Preceding and/or following this inhibition, there was mild facilitation. Static stretch of quadriceps also significantly reduced soleus H reflex magnitude. These results support the spindle receptor origin for the gain attenuation seen during movement. The time course of the gain attenuation suggests a spinal route, by which the spindle discharge of the heteronymous extensor muscles initiates presynaptic inhibition of transmission through the reflex pathway. PMID- 7552281 TI - Sympathectomy does not modify the levels of dopa or dopamine in the rat dorsal root ganglion. AB - In the present study we report on the levels of dihydroxyphenylalanine (DOPA), dopamine and their metabolites, 3-O-methyl-DOPA, homovanillic acid and dihydroxyphenylacetic acid, in the dorsal root ganglion (DRG) where dopaminergic sensory neurons have recently been identified. HPLC with electrochemical detection was used. Chemical sympathectomy, induced by daily injection of 30 mg/kg/sc of guanethidine over 4 days, did not modify these levels. DOPA, dopamine and their metabolites were also detected in the dorsal root in both intact and sympathectomized rats. The present results show that in the DRG, DOPA and dopamine do not derive from sympathetic nerves, and agree with previous reports suggesting that there is a peripheral dopaminergic sensory innervation. In addition the identification of 3-O-methylDOPA, the extraneuronal metabolite of DOPA is not only the precursor of dopamine in the DRG but may be released from DRG cells. The high 3-O-methyl-DOPA/DOPA ratio found in the DRG and the dorsal root suggests that DOPA could play a role as neurotransmitter or modulator in the peripheral sensory innervation, as it has been proposed for the central nervous system and the sympathetic system. PMID- 7552282 TI - Expression of stereotyped behaviour requires stimulation of nigral D1 dopamine receptors. AB - Dopamine receptors in the pars reticulata of the substantia nigra were inactivated following bilateral injections of N-ethoxycarbonyl-2-ethoxy-1,2 dihydroquinoline (EEDQ). Loss of D1, but not D2, receptors was associated with a significant decrease in the incidence of stereotyped head-down sniffing elicited by the mixed D1/D2 receptor agonist, apomorphine. The results support the hypothesis that D1 receptors in the substantia nigra are necessary for the expression of dopamine mediated stereotyped behaviour. PMID- 7552285 TI - Astroglia proliferate in response to oxytocin and vasopressin. AB - The effects of the peptides oxytocin and vasopressin on the proliferation of cultured cortical and hypothalamic astroglia were assessed by two corroborative methods. Both hemocytometer cell counts, and immunocytochemistry for bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic protein (GFAP) expression indicate that oxytocin increases the rate of proliferation of both cortical and hypothalamic astroglia. While vasopressin also had an effect on cortical cells, no conclusive evidence for vasopressin affecting proliferation of hypothalamic astroglia was found. PMID- 7552283 TI - Acute dopamine depletion potentiates independent stimulatory and inhibitory D1 DA receptor-mediated control of striatal acetylcholine release in vitro. AB - Fractional release of [3H]ACh was evaluated under basal and evoked conditions in striatal slices from normal and acutely dopamine-depleted adult rats for the influence of D1- and D2-DA receptor agonists. The D1 ligand had no effect on normal slices but DA depletion unmasked two independent but simultaneous supersensitive responses: augmentation of K(+)-evoked and inhibition of glutamate evoked release. The D2 ligand inhibited evoked release in normal slices and this effect was not potentiated. This is a new cholinergic model of acute D1 receptor supersensitivity. PMID- 7552286 TI - Crowding stress impairs the pituitary-adrenocortical responsiveness to the vasopressin but not corticotropin-releasing hormone stimulation. AB - To evaluate the effect of social crowding stress on the CRH and vasopressin induced hypothalamic-pituitary-adrenocortical (HPA) response, both those neuropeptides were administered intracerebroventricularly and intraperitoneally to rats crowded for 3 days. Crowding stress did not affect the corticosterone response to CRH given by either route (1 micrograms i.c.v. or 2 micrograms/kg i.p.) but totally abolished or considerably diminished the response to vasopressin given i.p. (5 micrograms/kg) or i.c.v. (5 micrograms), respectively. Social crowding stress considerably impairs central vasopressin but does not change the CRH-system involved in the HPA stimulation. PMID- 7552284 TI - Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release. AB - An antibody directed against the lumenal NH2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La3+ or alpha-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin Ii immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface. PMID- 7552287 TI - Immunohistochemical localization of neuropeptides in the human ciliary ganglion. AB - In human ciliary ganglia, 18% of neurons were in contact with substance P (SP) and 12% with calcitonin gene-related peptide (CGRP) like-immunoreactive (LI) varicose axons. CGRP was colocalized with SP. Numerous SP-LI and CGRP-LI non varicose nerve fibers were found between the ganglion cells and in nerve trunks that entered the ganglia. Axons immunoreactive for neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH) never contacted neuronal cell bodies. Perikarya of ciliary neurons neither stained for any of the neuropeptides nor for DBH. 23% of ciliary perikarya were TH-immunoreactive. These observations suggest an innervation of human ciliary ganglion neurons by peptidergic primary afferent collaterals presumably of trigeminal origin. PMID- 7552288 TI - Impairments and compensatory adjustments in spontaneous movement after unilateral dopamine depletion in rats. AB - Rats with unilateral dopamine (DA) depletions (hemi-Parkinson rats) display directional biases in their locomotion in spontaneous and drug induced tests. These biases have been explained as being due either to changed responsiveness to sensory stimulation, changes in motor ability, or to central changes, but as yet their basis is not fully understood. The purpose of the present experiment is to examine the posture of immobility and the posture and strategies of locomotion in rats with unilateral DA depletions. The rats are found to display impairments in their bad limbs (contralateral-to-lesion limbs) in adjusting posture and moving. They compensate by supporting themselves mainly on their good hindlimb, using the bad hindlimb and tail for balance and by disproportionately relying upon their good limbs to turn and to walk. Thus, their center of gravity is shifted to the good side and movement is preferentially directed toward the good side, in part to maintain equilibrium and in part to remove weight from the bad limbs so that they can enter the swing phase of the stepping cycle. It is proposed that the bad limbs may be unable to apply force to adjust posture and produce movement. These results provide a basis for predicting the movements that the animals will use in various situations and they expand the test repertoire this hemi-Parkinson model provides for studying recovery processes after loss of dopamine. PMID- 7552289 TI - Altered GABA distribution in hamster brain is an early molecular consequence of infection by scrapie prions. AB - Antibodies specific for GABA, glutamate and taurine were used to study the distribution of these amino acid neurotransmitters during the progression of scrapie in hamsters. Immunohistochemical distribution of glutamate and taurine were unaffected in scrapie hamsters compared with controls, but the distribution of GABA was altered by 21 days after inoculation. We found both a greater number of neurons showing GABA-like immunoreactivity and more intense staining in those neurons in scrapie-inoculated hamster brains, particularly in the hippocampus, inferior colliculus, frontal cortex and cerebellum. The overall concentrations of aspartate, GABA, glutamate and taurine, measured in seven different brain regions by PITC-amino acid analysis, were not significantly different between normal and scrapie-affected hamsters. The subtle alteration in GABA metabolism detected in this scrapie model suggests that PrPSc interacts directly with a component of the GABA system. PMID- 7552291 TI - A study of the effects of noradrenaline in the rat olfactory bulb using evoked field potential response. AB - In the rat, the main olfactory bulb receives a strong noradrenergic (NA) input from the locus coeruleus which is critical for different types of olfactory learning. However, the resulting effect of NA modulation on on the olfactory bulb electrical activity and its pharmacology are not well understood. In this study, we investigated the action of NA on the bulbar neuronal population using evoked field potentials (EFP) elicited antidromically in the olfactory bulb of anesthetized rats, by stimulation of the lateral olfactory tract (LOT). EFPs in response to single and paired-pulse stimulation of the LOT were collected before, during and until 2 h after a 10 min perfusion of pharmacological agents through a push-pull cannula. Four concentrations of NA were tested ranging from 10(-5) M to 10(-2) M. NA induced a reversible dose-dependent effect. The major effect was observed at 10(-3) M. It consisted of an increase in Component 2 amplitude (depolarization of granules cell dendrites) and a decrease in Component 3 amplitude (depolarization of granule cell bodies). In parallel, paired-pulse inhibition of mitral cells by granule cells was increased. The alpha 1 agonist phenylephrine (10(-3) M) mimicked most of the effects of NA whereas the alpha 1 antagonist prazosin (10(-3) M) blocked its main action. Isoproterenol (beta agonist, 10(-3) M) and clonidine (alpha 2 agonist, 10(-3) M) could not reproduce the effects of NA. Thus mainly through the activation of alpha 1 receptors, NA enhances synaptic activation of granule cells and increases feed-back inhibition of mitral cells. Consequences of such effects in the context of learning and memory are discussed. PMID- 7552290 TI - Specific binding sites for (3-8)angiotensin in C6 glioma cells. AB - A novel putative receptor for (3-8)angiotensin (AngIV) has recently been found in various tissues, including the brain. However, the localization of AngIV binding sites to specific types of brain cells has yet to be established. In this study tissue culture was used to determine the presence and characteristics of AngIV binding in a glioma cell line (C6) and in rat primary glial cells. Using [125I]AngIV as a radioligand, C6 glioma cells were found by radioreceptor assay to bind with a high affinity and in a saturable, reversible manner. The best fit to the data was for a two-binding-site model; a higher-affinity site with a Ka of 2.49 +/- 0.46 nM-1 and a density of 33.71 +/- 7.8 fmol/mg protein, and a second low-affinity site with a Ka of 176 +/- 7 microM-1 and a density of 563 +/- 190 fmol/mg protein. The ligand specificity of the AngIV sites was determined from competitive displacement assays with AngIV, AngIII, (4-8)AngII, [Sar1,Ile8] AngII, losartan (an angiotensin subtype 1 receptor ligand) and PD123319 (an angiotensin subtype 2 receptor ligand). The relative order of binding affinity was AngIV > AngIII >> (4-8)AngII, while losartan, PD123319 and Sar1,Ile8-AngII failed to compete for the AngIV sites, even at 1 microM. The radioreceptor assay data were confirmed by receptor autoradiography on cells grown on glass slides. Moreover, the bound radioligand was shown by HPLC to be [125I]AngIV and not a breakdown product. Preliminary experiments with primary astrocyte cultures showed the presence of AngIV binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552293 TI - Physiological levels of beta-amyloid increase tyrosine phosphorylation and cytosolic calcium. AB - The a beta peptide is a neurotoxic peptide that accumulates in the brains of Alzheimer patients, but is also present in body fluids at subnanomolar levels. The potential effects of these low levels of a beta are unclear. We now show that one such action is to increase tyrosine phosphorylation in PC12 cells and olfactory neuroblasts. Application of a beta 25-35 or a beta 1-40 induces a dose dependent increase in the tyrosine phosphorylation in both whole cells and in vitro. The increase in tyrosine phosphorylation is both rapid and sensitive, being stimulated by picomolar doses of a beta and occurring within 1 min of application. Calcium imaging experiments provide further support for the role of tyrosine phosphorylation in the action of a beta. While a beta does not alter calcium metabolism under basal conditions, the addition of a beta induces a rapid increase in cytoplasmic calcium in olfactory neuroblasts that have been treated with the tyrosine phosphatase inhibitor, sodium orthovanadate or in PC12 cells treated with nerve growth factor. These responses could be blocked by the tyrosine kinase inhibitor, herbimycin. These calcium responses displayed an obligate requirement for the presence of matrix proteins. The identification of a rapid, sensitive assay for the action of a beta may facilitate investigations of its mechanism of action. PMID- 7552294 TI - Multiple types of binding sites for atrial natriuretic peptide in rat olfactory bulb membranes and synaptosomes. AB - The binding of atrial natriuretic peptide (ANP) to rat olfactory bulb membranes and synaptosomes was examined. [125I]ANP (rat, 99-126) bound specifically to a single class of binding site on olfactory bulb membrane preparation with dissociation constant (Kd) of 106 pM and maximum binding capacity (Bmax) of 13.6 fmol/mg protein. Comparable results were obtained when the binding was characterized using displacement and kinetic experiments. The ring deleted analog of ANP, C-ANP (rat, 4-23) displaced [125I]ANP only minimally from its binding site in the membrane preparation. Saturation, displacement and blocking experiments on [125I]ANP binding to rat olfactory bulb synaptosomes revealed the presence of two distinct binding sites. Simultaneous analysis of homogeneous and heterogeneous displacement curves and blocking experiments revealed the quantitative characteristics of these receptors to be: Kd1 = 44 pM, Bmax1 = 42 fmol/mg protein and Kd2 = 1050 pM, Bmax2 = 173 fmol/mg protein, for the high and low affinity binding sites, respectively. Kinetic experiments further confirmed the differences between the receptors present in the membranes and the synaptosomes preparations. The demonstration of multiple ANP binding sites in olfactory bulb synaptosomes but not membrane preparations raises the possibility of a particular function of ANP in nerve terminals. PMID- 7552292 TI - Functionally mature olfactory neurons from two anosmic patients with Kallmann syndrome. AB - Patients with Kallmann syndrome (KS) exhibit hypogonadotropic hypogonadism and anosmia [Kallmann et al., Am. J. Mental Def., 48 (1944) 203-236] secondary to failure of gonadotropin-releasing hormone (GnRH)-producing neurons to migrate from the olfactory placode to the brain, and to agenesis of the olfactory bulbs. It has been hypothesized that olfactory neurons (ON) from individuals with KS are immature partly on the basis of studies in animals showing that lack of synaptic connection of ON with the olfactory bulb results in expression of immature ON [Schwob et al., J. Neurosci., 12 (1979) 880-883]. To test this assumption, we obtained olfactory tissue samples from two males diagnosed with KS on the basis of medical history and MRI studies. Both patients were anosmic. The functioning of cells isolated from biopsies taken from the upper middle turbinate and septum was studied by measuring changes in intracellular Ca2+ concentration ([Cai]) using dual excitation fluorescence microscopy. Biopsies from both patients yielded cells that morphologically appeared to be ON. Seven of 16 cells that morphologically resembled ON responded with a change in [Cai] upon stimulation with an odorant mixture. These studies show that at least some ON in KS individuals are functionally mature and suggest that complete development of the olfactory bulbs is not required for differentiation of mature human ON. PMID- 7552295 TI - Effects of kainic acid induced seizures on immediate early gene expression in mice with a targeted mutation of the CREB gene. AB - The present study examined the response of immediate early genes following kainic acid induced seizures in mice lacking the alpha and delta isoforms of CREB. mRNA levels for c-fos, c-jun, and Krox-24 were measured following limbic seizure activity and were found to be induced in wild type as well as CREB mutant mice. This effect was also seen for these three mRNAs at the protein level as well as for FOS-B. Furthermore the time course of expression of FOS, JUN, KROX-24, and FOS-B proteins were essentially the same in CREB mutant mice as compared to wild type controls. These data suggest that CREB alpha and delta are not required for the induction of immediate early genes following pharmacologically induced seizures. PMID- 7552297 TI - Sexual differences in vasopressin receptor binding within the ventrolateral hypothalamus in golden hamsters. AB - In the following studies, the presence of a sexual difference in arginine vasopressin (AVP) receptor binding was tested within the ventrolateral hypothalamus (VLH), an area rich in gonadal steroid receptors. The density of AVP receptor binding was estimated by in vitro quantitative autoradiography within the entire rostro-caudal extent of the VLH. The density of AVP binding was higher in males than in females at all levels of this area. Furthermore, dependency on testosterone treatment was also compared between gonadectomized males and females. While gonadectomy resulted in a near total disappearance of binding in both males and females, testosterone treatment resulted in equally high levels of binding in both sexes. Indeed, a high density of AVP receptor binding was observed at all levels of the VLH in both testosterone-treated males and females. These results show that adult female golden hamsters are equally capable as males of expressing high levels of AVP receptor binding in the VLH in response to high levels of testosterone. Together, our results suggest that, while AVP receptor binding within the VLH is sexually different in gonadally-intact animals, these differences are not related to differential responsiveness to testosterone, but rather to a differential production and availability of the hormone. PMID- 7552296 TI - Time course of hypothalamic growth hormone-releasing hormone and somatostatin content in streptozocin diabetic rats: evidence for early changes in hypothalamic regulation. AB - Growth hormone secretion is markedly suppressed early in streptozocin induced diabetes mellitus of the rat. Our studies were designed to delineate early changes in hypothalamic regulation by growth hormone-releasing hormone (GHRH) and somatostatin (SS) with the aim of determining the best time period for hypothalamic secretion studies. Although hypothalamic GHRH content (ng/hypothalamus) and SS concentration (ng/mg wet weight) were unchanged at 17 to 20 days in previous studies, we anticipated changes earlier in the time course from transient imbalances in release and synthesis. We examined hypothalamic GHRH content and SS concentration in control, diabetic, and insulin treated diabetic rats (n = 5-13; streptozocin 100 mg/kg i.p.) at 0, 2, 4, 7, 10 and 21 days. In diabetic rats GHRH content was greater at day 2 (142 +/- 9% of control-same day, P < 0.05) and day 4 (139 +/- 17%, P < 0.05), but was less at day 10 (67 +/- 4%, P < 0.01). GHRH content of insulin treated diabetic rats was elevated at day 2 (158 +/- 10%, P < 0.05), but subsequently was unchanged from control. In diabetic rats SS concentration was decreased at day 4 (78 +/- 5%, P < 0.01) and at day 21 (91 +/- 3%, P < 0.05). Our results show earliest changes compared to control in GHRH content at 2 days and in SS concentration at 4 days. These findings support early changes in hypothalamic secretion, define a time period of 1 to 10 days for further studies of release and gene expression, and suggest complex relationships of gene expression, peptide synthesis, and peptide release. PMID- 7552298 TI - Schwann cell responses to forskolin and cyclic AMP analogues: comparative study of mouse and rat Schwann cells. AB - Forskolin and cAMP analogues (8-bromo cAMP and dibutyryl cAMP) induced proliferation or surface galC expression, depending on the concentrations, in rat and mouse Schwann cells in vitro. However, rat Schwann cells required much higher concentrations of these agents than mouse Schwann cells in both proliferation and surface galC expression. The concentrations needed for cellular proliferation were 0.5 microM forskolin or 10 microM cAMP analogues in mice and 2.5 microM forskolin or 50 microM cAMP analogues in rats. Surface galC was expressed in mouse Schwann cells at concentrations of 10 microM forskolin or 100 microM cAMP analogues, while in rat Schwann cells, 50 microM forskolin or 500 microM cAMP analogues was needed for expression of surface galC. Rat Schwann cells transformed from an elongated spindle shape to flattened cells by the addition of these agents. However, mouse Schwann cells remained spindle shaped and their processes were apparently elongated at concentrations of more than 1.0 microM forskolin or 100 microM cAMP analogues. These results may reflect the differences in the cellular metabolism between mouse and rat cells. Moreover, the elongation of mouse Schwann cell processes appeared to be associated with surface galC expression, suggesting that elongation may be an initial signal for differentiation in mouse Schwann cells. PMID- 7552300 TI - Effect of hypothyroidism on vasoactive intestinal polypeptide-immunoreactive neurons in forebrain-neurohypophysial nuclei of the rat brain. AB - We have recently reported that hypothyroidism increases immunoreactive (IR) vasoactive intestinal polypeptide (VIP) and VIP mRNA content in both parvocellular and magnocellular neurons of the rat, hypothalamic paraventricular nucleus (PVN). As VIP can stimulate vasopressin (AVP) secretion, we conducted an anatomical investigation to determine whether VIP-containing neurons in other regions of the brain that are involved with homeostatic mechanisms of water and salt conservation are also affected by hypothyroidism. The distribution and intensity of VIP immunostaining in neurons and fibers of the magnocellular neurohypophysial system, including the hypothalamic PVN, supraoptic nucleus (SON) and accessory magnocellular cell groups, circumventricular subfornical organ (SFO), preoptic and anterior hypothalamus, midline thalamus, subthalamic zona incerta and posterior septal nuclei were studied using a highly sensitive immunocytochemical technique and unbiased neuronal counting methods, based on the optical dissector principle. Hypothyroidism increased the intensity of VIP immunostaining and/or the number/section, percentage and numerical density of IR VIP neurons in the PVN, SON, nucleus circularis, periventricular preoptic nucleus of the hypothalamus and SFO. In addition, IR-VIP perikarya and/or fibers in the hypothalamic medial preoptic area and anterior periventricular nucleus, nucleus reuniens of the thalamus and dorsal fornix-triangular septal nucleus complex were also apparent in the hypothyroid animals while no immunostaining was seen in these areas in control animals. No quantitative and/or qualitative modifications in IR-VIP neurons and fibers were noted in the anterior hypothalamic area, suprachiasmatic nucleus, thalamic paraventricular nucles an subthalamic zona incerta between hypothyroid and control animals. These findings suggest an inverse relationship between thyroid hormone and VIP content and/or distribution of IR-VIP neurons in specific forebrain regions involved in the control of AVP release, extracellular fluid volume, thirst, blood pressure and anterior pituitary secretion. This raises the possibility that changes in fluid homeostasis and cardiovascular function occurring in hypothyroidism may be mediated, at least in part, by VIP-producing neurons in diverse regions of the brain. PMID- 7552299 TI - NF-Y activates mouse tryptophan hydroxylase transcription. AB - Tryptophan hydroxylase catalyses the rate-limiting step in the biosynthesis of serotonin, a neurotransmitter which has been implicated in the etiologies of clinically important psychiatric illnesses. Tryptophan hydroxylase is expressed in a tissue-specific manner, but little is known about its transcriptional regulation. By analysing transcriptional activities of a set 5'-deletion constructs of promoter-reporter plasmids in P815-HTR mastocytoma cells, we found that transcription was activated by sequences between nucleotides -343 and -21. DNase I footprint analysis, using nuclear protein extracts from P815-HTR cells, revealed a protein-DNA interaction between nucleotides -77 and -46. A double stranded oligonucleotide, representing this binding site, specifically bound nuclear protein in a gel shift assay. Methylation interference analysis of this complex revealed that nuclear protein interacted with an inverted GGCCAAT element, which is a high-affinity binding motif for the transcription factor NF-Y (also known as CP1 or CBF). An NF-Y specific antibody abolished protein binding in a gel shift assay. Mutagenesis of specific base pairs abolished protein binding in vitro, and mutagenesis of the same base pairs in a reporter gene construct resulted in a 65% decrease in transcriptional activity. Our results suggest that the transcription factor NF-Y binds to a GGCCAAT motif in the tph proximal promoter and activates transcription. PMID- 7552302 TI - Brain grafts of cerebral ganglia have effectiveness in growth restoration of damaged Helix aspersa mesocerebrum. AB - The microsurgical extirpation of the mesocerebrum from the brain of fast-growing juvenile snails (Helix aspersa aspersa: H.a.a.) stops their growth. This suggests that neurosecretory cells of the mesocerebrum secrete a growth hormone. Neural grafting has been used as a tool to restore the impaired growth function after mesocerebrum removal in juvenile H.a.a snails. The transplantation of desheathed cerebral ganglia (CG) (i.e. CG with their glioconjunctive outer covering removed), into the place where the mesocerebrum had been re-established growth which depended on the age of the donors. For the grafts of H.a.a CG into H.a.a, it was CG from the youngest donors that restored growth best. However, the CG of adult snails still conserved a slight growth-stimulating activity. Transplantation of the CG from the large, fast-growing sub-species H. aspersa maxima (H.a.m), into the brain of H.a.a with mesocerebrum removed induced faster growth than the H.a.a CG probably because of a more abundant secretion of growth hormone. Our results show that intracerebral CG grafts are well tolerated in snails and that labeling of the neurones of the transplanted CG with a vital fluorescent stain (Fast blue), allowed the observation, over several months, of their integration into the lesion zone of the host brain. PMID- 7552301 TI - Rat brain neurotransmitter turnover rates altered during withdrawal from chronic cocaine administration. AB - This experiment utilized neurotransmitter turnover rates to assess the effects of withdrawal from chronic cocaine on the brain. A triad-littermate design was used to evaluate the importance of response dependency on the effects of withdrawal from chronic cocaine administration upon brain biogenic monoamine and amino acid putative neurotransmitter turnover rates. Each member of a triad was exposed to one of three conditions. Cocaine infusions (0.33 mg/inf) were used to engender and maintain lever pressing by one rat under an FR 2 schedule, while the second and third rats received simultaneous infusions of either cocaine or saline, respectively. After a minimum of 15 days exposure to the three treatment conditions and 24 h after the start of the last drug session, the triads were pulse labeled with [14C]glucose, [3H]tyrosine and [3H]tryptophan and killed 60 or 90 min later by total immersion in liquid nitrogen, The frozen brains were removed and dissected at -20 degrees C into 22 areas. The content and specific radioactivities for dopamine (DA), noradrenaline (NA), serotonin (5-HT), aspartate (Asp), glutamate (Glu), glycine (Gly) and gamma-aminobutyric acid (GABA) were determined in each brain region using high pressure liquid chromatography with electrochemical (biogenic monoamines) or fluorescence (amino acids) detection followed by liquid scintillation spectrometry. Turnover rates (TOR) were calculated and compared across treatment conditions. The significant decreases in TOR resulting from chronic cocaine exposure included 5-HT in the frontal cortex, DA in the cingulate cortex, entorhinal-subicular and motor somatosensory cortices and NA in the inferior colliculus. Significant increases in TOR were also observed which included 5-HT in the preoptic-diagonal band region, DA in the hippocampus and NA in the pyriform and temporal-auditory cortices, the dentate gyrus and brainstem. GABA TOR was also increased in the preoptic-diagonal band region, dentate gyrus and brainstem of both groups receiving cocaine as was Glu TOR in the pyriform cortex and cerebellum. In addition, changes were seen in the rats under the ratio schedule of cocaine self administration that were not seen in rats receiving yoked-cocaine infusions that included increased TOR of 5-HT in the pyriform cortex, NA in the caudate-putamen and GABA in the pyriform and motor-somatosensory cortices. Decreased 5-HT TO was seen in the motor-somatosensory cortex and dentate gyrus in the rats that had self-administered cocaine compared to the yoked-cocaine infused group. Perhaps the most interesting changes were those seen in the yoked-cocaine group that were reversed in the rats whose responding was maintained by cocaine.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7552303 TI - Autoradiographic identification of brain angiotensin IV binding sites and differential c-Fos expression following intracerebroventricular injection of angiotensin II and IV in rats. AB - A unique angiotensin binding site specific for the hexapeptide, angiotensin II(3 8) (AngIV), has been previously reported by our laboratory in the guinea pig brain and is presently described in the rat brain. This angiotensin receptor subtype has been termed AT4 and is prominently distributed in cerebral cortex, piriform cortex, hippocampus, habenulae, colliculi, septum, periaqueductal gray, several thalamic nuclei, the arcuate nucleus of the hypothalamus and cerebellum. In the second part of the present investigation, separate groups of rats received i.c.v. injections of angiotensin II (AngII), AngIV or artificial cerebrospinal fluid (aCSF) and were euthanized 2 h later for the purpose of evaluating for brain c-Fos expression. After i.c.v.-injected AngIV, Fos-like immunoreactivity was present in the hippocampus and piriform cortex. This immunoreactivity was unaffected by i.c.v. pretreatment with the AT1 angiotensin receptor antagonist DuP 753 (losartan) or the AT2 receptor ligand PD123177 but was blocked by the AT4 angiotensin receptor antagonist, divalanal-AngIV. I.c.v. injection of AngII resulted in Fos-like immunoreactivity in the dorsal third and lateral ventricles, subfornical organ, lateral hypothalamus and amygdala. Pretreatment with losartan or PD123177 significantly interfered with this AngII-induced immunoreactivity while divalanal-AngIV did not. These results indicate that in both guinea pig and rat brains the AT4 receptor has a distribution different than that previously reported for AT1 and AT2 receptor subtypes. The c-Fos expression results suggest that different brain neuronal pathways are activated by i.c.v. injection of AngII and AngIV. PMID- 7552305 TI - Traumatic neuronal injury in cortical cell culture is attenuated by 21 aminosteroids. AB - The effect of the 21-aminosteroids U74500A and U74389F, alone and in combination with the NMDA receptor antagonist MK-801, on traumatic neuronal injury was quantitatively assessed in murine neocortical cell cultures. Consistent with prior observations, a mechanical insult to the culture monolayer resulted in widespread neuronal death over the following 24 h. Treatment with either U74500A or U74389F provided moderate protection, reducing neuronal death as measured by lactate dehydrogenase release by 25-50%. This effect was most consistent when these agents were preincubated for 2 h prior to injury. Combined treatment with a 21-aminosteroid plus the NMDA receptor antagonist MK-801 reduced injury more than either drug alone. Approximately 40% of the neuronal death occurring in the presence of MK-801 was blocked by concomitant treatment with 10 microM U74500A or U74389F. These results suggest that free radicals may contribute to cell death in this in vitro model of traumatic neuronal injury. PMID- 7552304 TI - Toxic effects of L-DOPA on mesencephalic cell cultures: protection with antioxidants. AB - The toxicity of L-3,4-dihydroxyphenylalanine (L-DOPA) was studied in neuronal cultures from rat mesencephalon. The survival and function of DA neurons were assessed by the number of tyrosine hydroxylase-positive (TH+) cells and 3H-DA uptake and those non-DA neurons by the exclusion of Trypan blue and the high affinity 3H-GABA uptake. L-DOPA was toxic for both DA and non-DA neurons. DA neurons were more severely affected than non-DA neurons after short periods of treatment and with exposure to a low dose of L-DOPA (25 vs. 100 microM) and less selectively affected after 1 or 2 days of treatment. After incubation with L DOPA, a disruption of the neuritic network and an overall deterioration were observed, more evident for TH+ cells in the whole culture. Auto-oxidation to quinones is responsible in part for L-DOPA toxicity in non-DA neurons since the levels of quinones correlated well with the severity of cell death in the cultures. The damage of DA neurons took place before the rising of quinones, suggesting that quinones are not essential in L-DOPA toxicity for DA neurons. Antioxidants, such as ascorbic acid and sodium metabisulfite, completely prevented L-DOPA-induced quinone formation as well as the death of non-DA neurons. In contrast, they could only partially prevent the damage produced by L DOPA in DA neurons. Mazindol, a selective inhibitor of DA uptake, protected TH+ cells from L-DOPA. PMID- 7552306 TI - Inhibition of serotonin-induced Ca2+ mobilization by interleukin-1 beta in rat C6BU-1 glioma cells. AB - To study the potential interaction between cytokine and serotonin (5-HT) signal transduction, we evaluated the effect of interleukin-1 beta (IL-1 beta) on the 5 HT2 receptor-mediated mobilization of intracellular Ca2+ in cultured rat C6BU-1 glioma cells. Pretreatment of cells with IL-1 beta significantly inhibited the 5 HT-induced mobilization of Ca2+ in a dose (30-1000 U/ml)- and time (12-24 h) dependent manner. Inhibition was observed when cells were stimulated with concentrations of 5-HT of > or = 1 microM, which induced the maximal 5-HT response. Lipopolysaccharide (1 microgram/ml) also inhibited 5-HT-induced Ca2+ mobilization, but heat-inactivated IL-1 beta as well as interferon-alpha (1000 U/ml), interferon-gamma (1000 U/ml), and tumor necrosis factor-alpha (2000 U/ml) did not. The inhibitory effects of IL-1 beta and LPS were significantly prevented by genistein, a selective tyrosine kinase antagonist, and by H7, a potent inhibitor of protein kinase C. These results indicate that IL-1 beta and LPS inhibit 5-HT2 receptor-mediated Ca2+ mobilization via pathways that include the activation of a tyrosine kinase and protein kinase C. The interaction between cytokines (IL-1 beta) and monoamines (5-HT) may serve to modulate signal transduction in the central nervous system. PMID- 7552307 TI - Simultaneous single unit recording in the medial nucleus of the medial geniculate nucleus and amygdaloid central nucleus throughout habituation, acquisition, and extinction of the rabbit's classically conditioned heart rate. AB - The present study examined single neuron activity in the medial nucleus of the medial geniculate (mMG) and amygdaloid central nucleus (ACe) simultaneously across several phases of differential heart rate conditioning (habituation, acquisition, and extinction). Within the same animals, the magnitude of mMG and ACe unit responses to two tone conditioned stimuli (CS) exhibited habituation, differential acquisition, and extinction. Neurons in each area developed a differential response latency to the CSs during acquisition, suggesting that mMG and ACe may be involved in changes of synaptic efficacy. Units in both areas rapidly developed a differential response magnitude to the CSs (< 6 acquisition trials), however, mMG units responded to the CSs with a shorter latency than ACe units across all phases of training. This suggests that unlearned and learned CS information may access mMG before ACe. These results are consistent with the notion that conditioning-induced plasticity which occurs in mMG may influence the conditioning-induced plasticity that occurs further downstream in the amygdala. PMID- 7552308 TI - Systemic nitroglycerin induces Fos immunoreactivity in brainstem and forebrain structures of the rat. AB - Nitroglycerin is a vasodilator which induces vascular relaxation by releasing nitric oxide in the wall of blood vessels. It has been suggested that the cardiovascular inhibitory responses which are induced by this drug are mediated by central structures. In this study, we evaluated the distribution and intensity of Fos immunoreactivity in rat brain nuclei following the systemic administration of nitroglycerin. In the medulla, a significant number of Fos-immunoreactive neurons were observed in the nucleus tractus solitarius, ventrolateral medulla, area postrema and spinal trigeminal nucleus caudalis. A robust staining was seen in the parabrachial nucleus, locus coeruleus and ventrolateral periaqueductal grey. In the hypothalamus, Fos-positive cells were densely packed in the paraventricular and supraoptic nuclei. Other areas where significant staining was observed include the central nucleus of the amygdala and the subfornical organ. These findings demonstrate that the systemic administration of nitroglycerin is capable of activating a spectrum of functionally diverse brain regions. This spectrum includes areas involved in reflex adjustments to nitroglycerin-induced hypotension, areas involved in sensory nociceptive perception and areas associated with integrative regulation of autonomic, behavioral and neuroendocrine functions. PMID- 7552309 TI - The temporal profiles of ICAM-1 protein and mRNA expression after transient MCA occlusion in the rat. AB - Leukocytes may contribute to ischemic cell damage. ICAM-1 expression on endothelial cells facilitates the migration of leukocytes into tissue. Therefore, we measured the temporal profiles of ICAM-1 mRNA and protein in rat brain after transient (1 or 2 h) of middle cerebral artery (MCA) occlusion. Male Wistar rats (n = 86) were subjected to 1 or 2 h MCA of occlusion, or 2 h of MCA occlusion followed by reperfusion for a variety of durations ranging from 1 h to 1 week. 10 additional control animals were employed. ICAM-1 mRNA and protein were measured during ischemia and reperfusion, and immunohistochemical methods were used to identify specific cell types expressing ICAM-1. ICAM-1 mRNA was detected 1 h after the onset of ischemia. mRNA maximized at 10 h of reperfusion and persisted out to 1 week of reperfusion. ICAM-1 significantly increased in microvascular endothelial cells at 2 h of reperfusion, maximized at 46 h and persisted out to 1 week of reperfusion (P < 0.05). ICAM-1 mRNA and protein are present in ischemic brain early after the onset of ischemia and reperfusion, respectively. These data provide support for the role of ICAM-1 in mediating leukocyte-endothelial adhesion after transient MCA occlusion in the rat. PMID- 7552313 TI - Neuronal membrane conductance activated by amyloid beta peptide: importance of peptide conformation. AB - Whole-cell voltage-clamp recording and circular dichroism (CD) spectroscopy were used to assess the importance of amyloid beta peptide (A beta) conformation for eliciting homeostatically disruptive membrane conductances in embryonic rat hippocampal neurons. A beta that assumed a random coil conformation when freshly dissolved in water did not alter cell resting ('leak') membrane conductances. In contrast, after several days incubation ('aging'), the same peptide samples became capable of activating a large, rapid onset and potentially toxic increase in leak membrane conductance that coincided temporally with a transition in peptide conformation from random coil to beta-sheet. Interestingly, this membrane activity was not mimicked with chemically equivalent A beta s that immediately adopted a beta-sheet conformation in water ('pre-aged'). These findings suggest that, under conditions that allow for a gradual transition of random coil A beta to beta-sheet structures, peptide conformation may be an important determinant of the toxic consequences of A beta-mediated membrane conductances. PMID- 7552312 TI - Y chromosomal effects on hippocampal mossy fiber distributions in mice selected for aggression. AB - The influence of the non-pseudoautosomal region of the Y chromosome (YNPAR) on the sizes of the hippocampal intra- and infrapyramidal mossy fiber (IIPMF) terminal fields were examined in wild house mice. For this purpose selection lines for short attack latency (SAL), long attack latency (LAL), and their respective congenics for the YNPAR were used. We found an incremental effect of the (non-aggressive) LAL YNPAR, combined with an additive effect of the line background on the sizes of the IIPMF terminal fields. In contrast, only the line background affected attack latency. The implications of this finding for the previously observed correlation between the size of the IIPMF and aggression in male house mice are discussed. PMID- 7552311 TI - Hypoosmotic volume regulation and osmolyte transport in astrocytes is blocked by an anion transport inhibitor, L-644,711. AB - Cell volume, potassium content, and potassium influx were measured in rat cerebral astrocytes grown in primary culture following exposure to hypoosmotic medium containing either 3.2 mM or 50 mM potassium. Some solutions also contained 1 mM L-644,711, an anion transport inhibitor. L-644,711 inhibited volume regulation and taurine efflux induced by hypoosmotic exposure in medium containing either potassium concentration. L-644,711 also inhibited potassium uptake associated and not associated with the sodium/potassium pump. The correlation of reduced taurine efflux and volume decrease produced by L-644,711 exposure indicates the important role for this amino acid in hypoosmotic astrocyte volume regulation. However, the effects of L-644,711 on potassium transport indicate that multiple actions of this drug may be important factors in its effect on astrocyte volume regulatory mechanisms. PMID- 7552314 TI - Immunocytochemical localization of 155 kDa myosin light chain kinase and myosin heavy chain in bovine brain. AB - We immunocytochemically stained the bovine brain with monoclonal antibodies against 155 kDa MLCK and myosin heavy chain (MHC) and a polyclonal antibody against calmodulin. In the bovine brain, a strong immunoreactivity with the anti MLCK antibody was observed in neurons in all layers of the cerebral cortex and basal ganglia, and astrocytes in the white matter. The antibody also stained Purkinje, granular and molecular cells in the cerebellum. An anti MHC antibody stained neurons and astrocytes in the cerebral cortex and Purkinje cells similarly to the anti-MLCK antibody, whereas the MHC immunoreactivity was detected in the cerebellar glomerulus and there was no immunostaining of MHC in the granular and outer stellate cells. These results suggest that the 155 kDa MLCK might be related not only to the Ca-calmodulin-myosin system but also other intracellular system. PMID- 7552315 TI - GABA-containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain. AB - The ventral tegmental area receives a gamma-aminobutyric acid (GABA) innervation from the nucleus accumbens and contains GABA immunoreactive neurons believed to be interneurons. We combined the immunocytochemical detection of retrogradely transported Fluoro-Gold (FG) from the nucleus accumbens (Acb) with the detection of GABA within the same section of tissue in the ventral tegmental area (VTA) of the rat brain to determine whether there might also be reciprocal GABAergic projections in the mesolimbic pathway. Immunoperoxidase labeling for FG and immunogold-silver labeling for GABA were most readily distinguished within perikarya and dendrites in sections examined by electron microscopy. Ultrastructural observations indicated that 36% (n = 110) of the FG-labeled perikarya and dendrites also contained GABA immunoreactivity. The present results provide the first evidence that GABA is contained in a subpopulation of neurons in the mesolimbic pathway from the VTA to the Acb. The reciprocity of this circuitry may provide an important feedback loop thus facilitating inhibition of motor activity. PMID- 7552310 TI - Regional differences in the effects of forced swimming on extracellular levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. AB - The effects of forced swimming for 30 min on extracellular 5-hydroxytryptamine (5 HT) levels were examined in five brain regions in rats using in vivo microdialysis. A single dialysis probe was implanted under surgical anesthesia into either the striatum, ventral hippocampus, frontal cortex, amygdala, or lateral septum on the day before the study. Dialysate content of 5-HT and 5 hydroxyindoleacetic acid (5-HIAA) was measured by HPLC. Forced swimming elevated extracellular levels of 5-HT in the striatum to a maximum of 90% above baseline. In contrast, forced swimming reduced 5-HT levels in the amygdala and lateral septum to 50 and 40% of baseline, respectively. In the hippocampus and frontal cortex, 5-HT levels were not altered significantly by forced swimming. In all five brain regions, forced swimming reduced 5-HIAA levels to 45-60% of baseline. These results suggest that forced swimming modulates 5-HT neurotransmission in a regionally specific manner. Aside from being a significant biological stressor, the forced swimming test is used as an animal behavioral model to detect antidepressant drugs, including drugs that alter 5-HT neurotransmission. It is possible that the alterations of extracellular levels of 5-HT produced by forced swimming in certain brain regions may be associated with the ability of antidepressant drugs to selectively alter behavioral performance during the forced swimming test. PMID- 7552316 TI - Neuromagnetic fields of the brain evoked by voluntary movement and electrical stimulation of the index finger. AB - Neuromagnetic fields from the left cerebral hemisphere of five healthy, right handed subjects were investigated under two different experimental conditions: (1) electrical stimulation of the right index finger (task somatosensory evoked fields, task SEF's), and (2) voluntary movement of the same finger referred to as movement-related fields, (MRFs). The two conditions were, performed in random order every 5-8 s. In addition, the task SEF's were compared to control SEF's recorded at the beginning of the experiment in order to find the optimal dewar position for localizing the central sulcus. The magnetic signals of the sources corresponding to the main components of the somatosensory evoked fields (early ones at 24 ms and at 34 ms, and late ones after 50 ms) and movement-related fields (motor field, MF and movement-evoked field I-MEF I) were mapped and localized by means of a moving dipole model. In four out of five subjects the MEF I dipoles were found to be located deeper than the early task SEF dipoles. In addition, all of the task SEF's components were found to exhibit larger amplitudes than the control SEF's components. The results are discussed in respect to the ability to selectively analyze contributions of mainly proprioceptive (area 3a) and cutaneous (area 3b) areas in the primary somatosensory cortex using magnetoencephalography. An additional finding of the study was that all of the task SEF's components were found to exhibit larger amplitudes than the control SEF's components. PMID- 7552317 TI - Male preference for the odors of estrous female mice is reduced by the neurosteroid pregnenolone sulfate. AB - The effects of the neurosteroid, pregnenolone sulfate (PS), on the responses of male mice to the odors of estrous female mice were examined in an odor preference test. Control untreated mice displayed a significant preference for the odors of an estrous female, spending more time in a Y-maze in the vicinity of the odors of an estrous than a non-estrous female. Administration of PS decreased male preference for the odors of estrous females, causing a significant dose-related (0.01-10 mg/kg) decrease in the amount of time spent in the proximity of the odors of the estrous female, while having significantly less of an effect on the responses to the non-estrous female odors. Neither pregnenolone nor sodium sulfate had any significant effects on the olfactory responses. The effects of PS were significantly reduced by peripheral administrations of the non-competitive N methyl-D-aspartate (NMDA) receptor antagonist, MK-801, but were not significantly affected by either the GABAA antagonists, bicuculline and picrotoxin, or the benzodiazepine antagonist, Ro 15-1788. These results suggest that pregnenolone sulfate has inhibitory effects on olfactory mediated male sexual interest, preference, or 'motivation' that, in part, involve interactions with NMDA receptor mediated mechanisms. PMID- 7552318 TI - Hippocampal dentate granule cell degeneration after adrenalectomy in the rat is not reversed by dexamethasone. AB - Although adrenalectomy has been reported to induce a selective and sometimes nearly complete degeneration of hippocampal dentate granule cells, Azmitia and colleagues recently reported (Mol. Brain Res., 19 (1993) 328-332) that normal hippocampal structure can nonetheless be restored within a matter of days by dexamethasone in the drinking water. We have attempted to confirm this remarkable finding. Four months after adrenalectomy, rats were given vehicle or dexamethasone for 5 days and then sacrificed. Histological analysis revealed that vehicle-treated adrenalectomized rats exhibited a full spectrum of granule cell loss, which spanned mild to nearly complete cell loss. Dexamethasone-treated adrenalectomized rats did not differ from vehicle-treated adrenalectomized rats and, in fact, exhibited a virtually identical spectrum of granule cell loss. These results confirm that adrenalectomy reliably induces hippocampal granule cell degeneration in a majority of animals and indicate that dexamethasone does not restore normal hippocampal structure once granule cell loss has occurred. PMID- 7552319 TI - Possible involvement of atrial natriuretic peptides in olfaction. AB - Atrial natriuretic peptides (ANP) are a family of humoral compounds released from the heart atria and involved in water and salt homeostasis. ANP immunoreactivity and ANP-binding sites were also found in several areas of the central nervous system including the olfactory bulb. In the present study, the possible involvement of ANP in olfaction was tested by measuring the content and distribution of IR-ANP and ANP-binding sites in rat olfactory bulb in control and rats. The results implicate ANP in the processes leading to olfactory perception. PMID- 7552320 TI - Short-latency excitation of phrenic motor output mediated by non-NMDA receptors. AB - This study tested the hypothesis that the short-latency excitation of the phrenic motor output elicited by superior laryngeal nerve (SLN) stimulation requires non NMDA receptor-mediated neurotransmission in the region of the dorsal respiratory group (DRG) of the adult cat. Injection of the non-NMDA receptor antagonist NBQX into the DRG severely attenuated or abolished the short-latency excitation, indicating that the short-latency excitation requires non-NMDA receptor-mediated neurotransmission within the DRG. PMID- 7552322 TI - Increased density of mu-opioid receptors in the postmortem brain of suicide victims. AB - The biochemical status of mu-opioid receptors in suicide was evaluated by [3H]DAGO specific binding in postmortem human brains from 15 suicide victims and 15 controls. The density (Bmax) in frontal cortex and thalamus was directly correlated with age. In the frontal cortex and caudate but not in the thalamus of suicide victims the density of mu-opioid receptors was 36-39% higher than in controls. KD values in suicide victims were similar to those in control group (range 1.4-2.2 nM). The results suggest an increase of mu-opioid receptors in suicide associated to some brain areas showing the mu2-opioid receptor subtype. PMID- 7552321 TI - Regional distribution of the AMPA glutamate receptor subunits GluR2(4) in human hippocampus. AB - In order to characterize the regional and cellular distribution patterns of individual ionotropic excitatory amino acid receptor subunits in the human hippocampus we performed an immunohistochemical analysis using the monoclonal antibody 3A11 to the AMPA GluR2(4) subunit. The study was based on paraffin embedded hippocampal specimens of five human brains obtained at autopsy. GluR2(4) immunoreactivity was consistently higher in hippocampus as compared to the adjacent areas of the mesial temporal lobe. Virtually all neurons showed intracytoplasmic staining of the perikarya and dendritic profiles with well defined laminar patterns. The most intense GluR2(4) immunoreactivity was observed in the target structures of mossy fibers, thus indicating that GluR2(4) AMPA subunits may be involved in NMDA-independent synaptic transmission pathways and long-term potentiation. Glial cells were not immunoreactive. These findings may provide basic information for studies of the GluR2(4) subunit in human hippocampus during various neuropathological conditions, such as temporal lobe epilepsy, ischemia and Alzheimer's disease. PMID- 7552323 TI - Ethanol withdrawal does not induce a reduction in the number of spontaneously active dopaminergic neurons in the mesolimbic system. AB - The effect of ethanol withdrawal, after chronic administration, on the electrophysiological properties of antidromically identified mesoaccumbens dopaminergic neurons was studied in two groups of rats with relative controls (withdrawal from chronic saline). The first group was anesthetized with chloral hydrate whereas the second was immobilized with D-tubocurarine. In chloral hydrate anesthetized rats, a significant reduction in the number of spontaneously active dopamine neurons was observed as compared with chronic saline withdrawn controls. In contrast, in ethanol-withdrawn D-tubocurarine treated rats, the number of spontaneously active dopamine neurons, as measured by the cells/track index, was found not different than chronic saline withdrawn controls. Further, intravenous administration of apomorphine, did not reverse the reduced cells/track index in chloral-hydrate anesthetized rats but consistently inhibited dopaminergic firing. Apomorphine-induced inhibition of firing was significantly more pronounced in ethanol withdrawn chloral-hydrate anesthetized rats. Firing rate and firing pattern were found decreased during ethanol withdrawal irrespective of experimental conditions. The results do not support the possibility that dopaminergic neurons of the mesoaccumbens pathway might be affected by depolarization inactivation during ethanol withdrawal. Rather, they confirm a reduction of neuronal activity already reported by previous studies. The reduced cells/track index observed in chloral hydrate anesthetized rats during ethanol withdrawal awaits an alternative explanation to the depolarization inactivation mechanism. PMID- 7552324 TI - Effects of an every other day rapid kindling procedure in prenatally protein malnourished rats. AB - Prenatally protein (6/25) rats have been reported to require significantly more stimulations to attain a stage 5 seizure than well-nourished controls (25/25) when using either a traditional or rapid every day, kindling procedure. In the present study, a rapid kindling procedure was utilized where both prenatally malnourished and control rats received every other day perforant path kindling (50 Hz, 10 s train) 12 times a day at 5-min intervals. Using this procedure, stage 5 seizures and a fully state were attained in both nutritional groups at approximately the same rate. It is postulated that it is the every other day component of the present procedure which overcomes seizure-induced inhibition in the 6/25 subjects, thereby allowing them to attain stage 5 seizures at the same rate as controls. PMID- 7552326 TI - Amygdala kindling in rats with brainstem bisection. AB - The effect of midsagittal bisection of the brainstem (the midbrain to the pons) on amygdala (AM) kindling at the primary and the secondary sites was examined in rats. It was found that the kindling rate was not affected at either primary or secondary sites regardless of the extent of bisection. However, seizure progression beyond stage 4 bilateral clonus did not occur with extensive dorsal bisection. The findings suggest that the dorsal midline brainstem structure bisected in this study participates in the mechanism of stage 5 seizure but is not involved in the transhemispheric positive transfer mechanism in rodent AM kindling. PMID- 7552325 TI - Modulation of N-methyl-D-aspartate (NMDA) antagonist-induced darting behaviour by the peptidomimetic PAMTA. AB - The N-Methyl-D-Aspartate (NMDA) receptor has attracted much attention in recent years due to its involvement in both the functions and dysfunctions of CNS neurotransmission. The existence of multiple sites by which NMDA receptor channel function can be pharmacologically modified and the interaction between glutamate and other neurotransmitter systems such as dopamine, provide exciting therapeutic avenues for related CNS disorders. In the present study, a novel synthetic analogue of the endogenous brain peptide L-prolyl-L-leucyl glycinamide (PLG) has demonstrated a significant modulatory action on the NMDA receptor. On the basis of radioligand binding studies, the novel synthetic peptide 5-[1(S)-(2(S) pyrrolidinylcarbonyl)amino-3-methylbutyl]-2- tetrazolylacetamide (PAMTA) has been suggested to act at a polyamine site on the NMDA receptor complex. Scatchard analysis of [3H]MK-801 binding revealed that in the presence of 100 microM PAMTA, a single binding site was obtained with the Kd being increased from 2.5 +/- 0.2 nM to 6.2 +/- 0.1 nM. The ability of PAMTA to inhibit the binding of [3H]MK-801 was sensitive to the presence of both spermidine (polyamine agonist) and arcaine (polyamine antagonist). Analyses of the binding profiles of various NMDA receptor antagonists support PAMTA's interaction with the polyamine site on this receptor complex. Furthermore, we have investigated the behavioural profile of the peptidomimetic PAMTA, by studying its effect on stereotypic behaviours induced by the NMDA receptor antagonist, CPP (3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid). Male Sprague-Dawley rats cannulated bilaterally into the medial prefrontal cortex were injected with PAMTA, CPP, a CPP/PAMTA combination, or a saline control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552329 TI - Beta-amyloid(1-40) effects on behavior and memory. AB - Beta amyloid 1-40 is a primary protein in plaques found in the brains of patients with Alzheimer's disease. There is evidence that unaggregated soluble beta amyloid may be neurotoxic and may have behavioral effects on some types of memory. In the current study, the 1-40 fragment of beta-amyloid protein (beta A4), or vehicle, was bilaterally injected into the rostral hippocampus of rats performing under stable food-maintained schedules of reinforcement or under a delayed conditional discrimination procedure. Under the first procedure, rats were trained to stability under a multiple fixed interval 15 s, fixed ratio 30 reinforcement schedule. This reinforcement schedule has proven sensitive to low dose drug effects. Acute bilateral hippocampal beta A4 (1.0, 2.0 and 3.0 microliters of 10(-3) M) administration did not significantly alter responding, compared to vehicle, under either reinforcement condition. Following the acute single-injection regimen, rats were administered chronic daily beta A4 (1 microliter of 10(-3) M), bilaterally, for 15 days. No significant changes in lever-pressing performance were observed during the chronic injection regimen, but performance declined significantly 30 days after termination of the chronic daily regimen. Histological examination revealed three of six rats showed positive reactions under Thioflavin S staining in and around the area of cannulae termination. The second assessment employed a delayed conditional discrimination procedure to evaluate the effects of intrahippocampal injections of beta A4 on short-term working memory. This conditional discrimination procedure assesses appropriate responding, dependent on a previously presented stimulus, after delays of various lengths have been imposed between the stimulus and the response opportunity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552327 TI - Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes alpha and beta. AB - Using a monoclonal antibody the translocation of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes alpha/beta was studied in hippocampal slices after stimulation of glutamate receptors or induction of long-term potentiation. In submerged slices preincubated for 60 min in a medium usually used in electrophysiological studies, cytosolic PKC was not detectable and the amount of membrane-associated enzyme was increased. The treatment of these slices with 10( 6) M phorbol-12,13-dibutyrate induced a time-dependent translocation of alpha/beta PKC from the membrane-associated into the membrane-inserted state. The glutamatergic agonists N-methyl-D-aspartate, quisqualate and trans-ACPD did not cause a membrane insertion of alpha/beta PKC as observed for the phorbol ester when applied alone or in combination. Furthermore, 2 min and 15 min after induction of LTP in the Schaffer collateral-CA1 pathway the distribution of alpha/beta PKC between the two membrane fractions remained unchanged. An increase in the total amount of PKC immunoreactivity was measured immediately after tetanization (142.6% of controls). The data suggest that a membrane insertion of alpha/beta PKC is not a prerequisite for the LTP-induced increased phosphorylation of PKC substrates and that the enzyme might be recruited from a previously inactive pool. PMID- 7552328 TI - Sensitization to apomorphine-induced rotational behavior in 6-OHDA-lesioned rats: effects of NMDA antagonists on drug response. AB - The development of drug-induced behavioral sensitization is thought to underlie many of the motor complications that accompany chronic L-DOPA treatment of patients with Parkinson's disease. As the development of sensitization to some dopaminergic behaviors has been linked to alterations in NMDA neurotransmission in animal models, we sought to determine whether or not NMDA antagonists can block the development of sensitization to rotational effects of dopamine agonists in rodents with unilateral nigrostriatal lesions. Rats with unilateral 6 hydroxydopamine lesions received either a single dose or eight daily doses of apomorphine, each dose preceded by the NMDA antagonists MK-801 or CPP. Three days after the last apomorphine dose, the circling behavior produced by the D1 agonist SKF 38393 was measured. A single dose of MK-801 (0.1 mg/kg) prevented the subsequent response to SKF 38393 but neither repeated treatment with MK-801 (0.1 or 0.3 mg/kg) nor CPP (0.1 mg/kg) preceding apomorphine prevented the subsequent response to SKF 38393 or attenuated the response in comparison to a control group. Each of the chronic treatment groups exhibited an increase in rotational effects of apomorphine despite MK-801 or CPP pretreatment. These data suggest behavioral sensitization in unilateral nigrostriatally lesioned rats chronically treated with apomorphine is not dependent upon stimulation of NMDA receptors. PMID- 7552331 TI - Discrimination training in a GO/NOGO-procedure alters the 2-deoxyglucose pattern in the Starling's forebrain. AB - European starling's (Sturnus vulgaris L.) were used to measure differences in the glucose metabolism in the auditory forebrain between birds performing an auditory discrimination task and birds habituated to the same acoustic stimuli. One group (n = 5) of individuals was trained in an operant GO/NOGO-procedure to report 1 kHz tone signals in a background of 4-kHz stimuli. The other group (n = 5) was habituated to the experimental set-up and to the same sequence of tones presented to the trained birds. [14C]2-deoxyglucose (2DG) uptake was determined in the caudal auditory telencephalon and the nucleus ovoidalis of well trained and habituated birds by autoradiography of brain sections. The tissue areas having grey values above predefined threshold values of labelling were determined in every brain section of each bird and then combined to volumes of labelled tissue. No significant differences of the 2DG uptake in the nucleus ovoidalis were found between the two experimental groups. In the caudal auditory telencephalon, however, significant differences in 2DG-labelling were found. In the trained birds, the labelling in the caudal auditory telencephalon was confined to smaller brain regions than in the habituated birds. These results suggest a differential processing of sounds in the trained and habituated birds which is discussed in the context of sharpening of the frequency representation by GABAergic inhibition and processes of attention. PMID- 7552332 TI - Calretinin-immunoreactivity in mitral cells of the rat olfactory bulb. AB - We addressed the question whether the projection neurons of the olfactory bulb, i.e. the mitral and tufted cells, are immunoreactive for the calcium-binding protein, calretinin. The following approaches were adopted: (1) light and electron microscopic calretinin-immunocytochemistry; (2) neuroanatomical tracing combined with calretinin-immunocytochemistry according to double-peroxidase and double-fluorescence protocols; (3) unilateral lesion of the olfactory bulb combined with calretinin-immunocytochemistry. The experiments were carried out in rats. Immunostaining of brain sections revealed weakly calretinin-immunopositive mitral cell bodies. Tufted cells were immunonegative. In contrast, fibers in the lateral olfactory tract were strongly immunopositive. Dense immunostaining was also present in a superficial band in layer I of the olfactory tubercle, piriform cortex, periamygdaloid cortex, and in the lateral entorhinal cortex. In electron microscopic preparations of these target areas we observed immunoreaction product in axons and axon terminals. The latter invariably formed asymmetrical synapses, mostly with dendritic spines. Injections of the neuroanatomical tracer biotinylated dextran amine (BDA) into the olfactory bulb produced labeled fibers which remained completely restricted to the superficial, calretinin immunopositive band in layer I in the above-mentioned cortical forebrain areas. We noted colocalization of transported BDA and calretinin-immunoreactivity in mitral cells, in fibers in the lateral olfactory tract and in fibers in the piriform cortex. Olfactory bulb lesions produced depletion of calretinin immunoreactivity in the lateral olfactory tract and the superficial band in the olfactory cortex-related areas. Together these data firmly indicate that mitral cells and their axons are calretinin-immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552330 TI - In vivo measurement of a diurnal variation in vasopressin release in the rat suprachiasmatic nucleus. AB - Diurnal changes in the intranuclear release of vasopressin (VP) and oxytocin (OT) in the suprachiasmatic (SCN), paraventricular (PVN) and supraoptic nuclei (SON) of the rat were studied by means of brain microdialysis. A significant diurnal variation in VP release in the SCN was detected, with the highest levels occurring during midday and a trough around midnight. OT release from the SCN was below detection limit. The release of neither of these neurohypophysial peptides showed diurnal variations within the PVN or SON. PMID- 7552333 TI - Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion. AB - The viral transneuronal labeling method was used in combination with immunohistochemical procedures to identify CNS neuropeptide and monoamine neurons that innervate the sympathetic preganglionic neurons (SPNs) which project to the stellate ganglion--the principal source of the sympathetic supply to the heart. Transneuronal labeling was found at three CNS levels: spinal cord, brainstem, and hypothalamus. In the thoracic spinal cord, apart from the pseudorabies virus (PRV)-labeled stellate SPNs, PRV-labeled neurons were localized in laminae I/II, IV, V, VII, and X as well as in the lateral spinal nucleus and lateral funiculus. In the C1-C4 spinal segments, labeled neurons were found in the lateral funiculus as well as laminae V and VII of the spinal gray matter. PRV-labeled cells were identified in lamina V and the dorsolateral funiculus of the lumbar spinal cord. Three medullary areas were consistently labeled: rostral ventromedial medulla (RVMM), rostral ventrolateral medulla (RVLM), and caudal raphe nuclei. The greatest concentration of labeling was found in the RVMM. This projection arose from adrenergic, serotonergic (5-HT), thyrotropin releasing hormone (TRH), substance P, somatostatin, enkephalin, and vasoactive intestinal peptide (VIP) immunoreactive neurons. The RVLM projection originated mainly from C1 adrenergic neurons, some of which contained immunoreactive neuropeptide Y (NPY). C3 adrenergic-NPY neurons lying near the floor of the 4th ventricle were also labeled. Enkephalin-, somatostatin- and VIP-immunoreactive RVLM neurons also contributed to this projection. 5-HT neurons of the caudal raphe nuclei (raphe pallidus, raphe obscurus, and raphe magnus) were labeled; some of these contained substance P or TRH-immunoreactivity with an occasional neuron staining for all three putative neurotransmitters. In the pons, catecholamine neurons in the A5 cell group, subcoeruleus and Kolliker-Fuse nuclei were labeled. The midbrain contained relatively few infected cells, but some were present in the Edinger Westphal and precommissural nuclei. Forebrain labeling was concentrated in the paraventricular hypothalamic nucleus (PVN) with lesser amounts in the lateral hypothalamic area (LHA) and the perifornical region. In the PVN, oxytocin immunoreactive neurons accounted for the greatest chemically-defined projection while corticotrophin releasing factor (CRF), vasopressin-, and angiotensin II immunoreactive neurons provided successively lesser inputs. In the LHA, angiotensin II-immunoreactive neurons were labeled. In summary, this study provides the first detailed map of the chemically-coded CNS neurons involved in the control of the cardiosympathetic outflow. PMID- 7552334 TI - Functional implications of kappa opioid receptor-mediated modulation of glutamate transmission in the output regions of the basal ganglia in rodent and primate models of Parkinson's disease. AB - Parkinson's disease is characterized by an increased excitatory amino acid transmission in the internal segment of the globus pallidus and the substantia nigra pars reticulata. The effects of the kappa receptor agonist enadoline (CI 977) on glutamate transmission were investigated in vitro. Enadoline reduced the K(+)-evoked release of glutamate from slices of substantia nigra in a concentration-dependent manner (maximum effect: 78% inhibition at 200 microM). This effect was blocked by the selective kappa receptor antagonist nor binaltorphimine. The endogenous ligand for kappa receptors is thought to be dynorphin. Dynorphin released from terminals of striato-pallidal and striato nigral pathways might thus act as an endogenous modulatory agent on glutamatergic transmission in the basal ganglia. In vivo experiments were carried out in rodent and primate models of Parkinson's disease to assess the potential of manipulating kappa receptors as a potential treatment for Parkinson's disease. Enadoline reduced reserpine-induced akinesia when injected in the entopeduncular nucleus of the rat. Similarly, injections of CI-977 in the internal segment of globus pallidus (GPi) of the MPTP-treated marmoset alleviated parkinsonian symptoms and allowed the animal to recover its locomotor activity. This suggest that reducing the overactive glutamatergic transmission in the output regions of the basal ganglia by activating kappa receptors might potentially form the basis of a novel anti-parkinsonian therapy. PMID- 7552335 TI - Effects of deprivation of oxygen or glucose on the neural activity in the guinea pig hippocampal slice--intracellular recording study of pyramidal neurons. AB - The block of synaptic transmission and neural activity during deprivation of oxygen or glucose has been simply attributed to the lack of energy due to the disorder of energy production. To clarify the interrelation between neural activity and energy metabolism during hypoxia or glucose deprivation, we studied the changes in ATP levels and electrical events of pyramidal neurons in the CA3 region and [Ca2+]i mobilization of the dendritic and cellular region of CA3 area, using guinea pig hippocampal slices. The studies of field potentials and intracellular recording from the pyramidal cell of CA3 area during hypoxia or glucose deprivation revealed that the cessation of synaptic activity and the depolarization of resting potential occurred earlier than during glucose deprivation while the increase of [Ca2+]i was slow during hypoxia but rapid during glucose deprivation although the ATP level of CA3 area was maintained at its original level for 20 min during both conditions. When glucose was replaced by lactate, ATP concentration was not reduced but the electrical activity decayed and [Ca2+]i increased with the similar time course as observed during lack of glucose, only. These results suggest that different mechanisms underlie the block of synaptic transmission in the CA3 pyramidal neurons during hypoxia and glucose deprivation and that lactate cannot substitute for glucose in the maintenance of neural activity. PMID- 7552337 TI - Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins. AB - Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or -4/5 (NT-3; NT-4/5) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF and NT-4/5. NT-4/5 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent increases in the number of ChAT-positive neurons, compared with either factor alone. NT-4/5 was also found to be additive with NGF, whereas cotreatment with BDNF and NT-4/5 showed no additivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF and NT-4/5), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins. PMID- 7552336 TI - The AMPA antagonist NBQX protects thalamic reticular neurons from degeneration following cardiac arrest in rats. AB - Thalamic reticular (RT) neurons are selectively vulnerable to degeneration following global ischemia. The degenerative mechanism is thought to involve an excitotoxic component, mediated in part by sustained post-ischemic activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) type excitatory amino acid (EAA) receptors. In order to test this hypothesis, the selective competitive AMPA type EAA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxalinedione) was administered at 30 mg/kg to rats 1, 3, and 6 h after resuscitation from 10 min cardiac arrest. NBQX treatment resulted in a 2-fold increase of spared RT neurons, from a mean density of 3.6 +/- 0.8 x 10(3) neurons/mm3 in cardiac arrest cases to 7.4 +/- 1.1 x 10(3) neurons/mm3 in the NBQX treated group, which represents sparing of 41.7% of the normal population of RT neurons, and protection of 26.9% of vulnerable RT neurons. Neurons within the central core of the RT manifest both a higher degree of vulnerability to ischemic degeneration, > 92% loss, and a higher sensitivity to sparing following NBQX administration, 460% increased sparing, than neuronal sub-populations in the medial or lateral 1/3 of the RT. Protection by post-arrest administration of NBQX suggests that sustained post-arrest stimulation of AMPA receptors is an important component in the process of ischemic degeneration of RT neurons. PMID- 7552338 TI - Changes in brainstem calcitonin gene-related peptide after VIIth and VIIIth cranial nerve lesions in guinea pig. AB - The present study investigated the effect of seventh and eight cranial nerve lesions on the prominence of calcitonin gene-related peptide in the hypoglossal (XII), facial (VII), abducens (VI), and oculomotor (III) cranial nerve nuclei. Guinea pigs were anesthetized and subjected to unilateral cochlear removal, vestibular end organ ablation, and seventh nerve transection. After a survival period ranging from 4 h to 5 days, each animal was anesthetized and perfused intracardially. Frozen sections were collected through the brainstem and stained immunohistochemically for calcitonin gene-related peptide using a polyclonal antibody with the Vectastain ABC kit and protocol. Positive cells were counted in each nucleus bilaterally and analyzed for side to side differences. Nuclei XII and III showed no significant difference in the numbers of cells staining positively for calcitonin gene-related peptide between the ipsilateral and the contralateral sides to the lesion. However, nuclei VII and VI showed elevated numbers ipsilateral to the lesion on some days, but not all. For VII, there was no significant difference before 24 h, but there were significant differences 1-5 days after the lesion. Similarly, in VI, there was no difference before 24 h, but differences were significant beginning with day 1 and continuing through day 3, and finally disappearing by day 4. Changes in the numbers of CGRP positive cells in VII measurable 24 h after the lesion and continuing for at least 5 days afterward indicate a central nervous system retrograde response to peripheral motor nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552339 TI - Pressor responses to stimulation of non-NMDA receptors in the superficial laminae of the cat spinal cord. AB - Microinjection of L-glutamate (109 mM; 12-18 nl) or AMPA (150-300 microM; 12 nl) into the superficial laminae of the L7 dorsal horn of decerebrate or chloralose anesthetized cats significantly increased mean arterial pressure. In contrast, microinjection of NMDA (300 microM; 12 nl) had no effect on mean arterial pressure. The pressor response to L-glutamate microinjection was blocked by prior microinjection of CNQX, an antagonist to non-NMDA receptors, but not by AP-5, an antagonist to NMDA receptors. We conclude that stimulation of non-NMDA receptors in the superficial laminae of the lumbar dorsal horn increases arterial blood pressure. PMID- 7552340 TI - In situ transblot and immunocytochemical comparisons of astrocytic connexin-43 responses to NMDA and kainic acid in rat brain. AB - Intracerebral injection of kainic acid (KA) in rat brain was previously found to cause altered immunohistochemical recognition of connexin-43 (Cx43) epitopes (epitope masking) with different sequence-specific antibodies against this gap junction protein. We demonstrate here that similar alterations occur when nitrocellulose membranes containing protein transferred from fresh cryostat sections of KA-injected brain are probed with these antibodies (in situ transblotting), indicating that epitope masking is not a result of epitope alteration due to fixation conditions used in earlier studies. Alterations in immuno-recognition of astrocytic Cx43 subsequent to injections of NMDA were also observed and were similar to those seen with KA in some, but not all respects. The results provide further indications of Cx43 molecular modification in excitotoxin-lesioned tissue and suggest that the sequelae of reactions by astrocytes and their gap junctions in these tissues is dependent on cell-type susceptibility to excitotoxin action. PMID- 7552341 TI - Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal. AB - Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nigra, striatum (not Leu-enkephalinArg6) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg6 levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were affected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats. PMID- 7552342 TI - Cholesterol oxidation reduces Ca(2+)+MG (2+)-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes. AB - These experiments examined effects of cholesterol oxidation on Ca(2+)+Mg(2+) ATPase activity, Na(+)+K(+)-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 from Brevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a 1t1/2 of 1.19 +/ 0.09 min and a second pool with a 2t1/2 of 38.30 +/- 4.16 min. Activity of Ca(2+)+Mg(2+)-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca(2+)+Mg(2+)-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca(2+)+Mg(2+)-ATPase activity was not observed. Activity of Na(+)+K(+)-ATPase was not affected by different levels of cholesterol oxidation (5%-40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relationship observed between SPM interdigitation and Ca(2+)+Mg(2+)-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases. PMID- 7552343 TI - Fos-determined distribution of neurons activated during the Bezold-Jarisch reflex in the medulla oblongata in conscious rabbits and rats. AB - Experiments were performed in unanaesthetized rabbits and rats to investigate the distribution, within the medulla oblongata, of neurons activated during the Bezold-Jarisch reflex. Repeated intravenous injections of phenylbiguanide evoked depressor and bradycardic responses in both rabbits and rats. Fos-positive neurons were present in the nucleus tractus solitarius and in the caudal ventrolateral medulla oblongata. Double-label tyrosine hydroxylase (TH) immunohistochemical studies in the ventrolateral medulla showed that most Fos positive neurons in the caudal ventrolateral medulla were TH-negative neurons scattered between A1 noradrenaline cells, in the rabbit and in the rat. Approximately 20% of neurons in the caudal ventrolateral medulla in rabbits, and 50% in rats, were immunoreactive for both Fos and TH. Some Fos-positive, TH negative neurons in the caudal ventrolateral medulla were retrogradely labelled with cholera toxin B-Gold after injection of this tracer into the sympathoexcitatory region of the rostral ventrolateral medulla. Our data suggests that neurons in the nucleus tractus solitarius, and rostrally projecting TH negative neurons in the caudal ventrolateral medulla, are part of the pathway by which stimulation of cardiopulmonary receptors inhibits sympathetic vasomotor tone to decrease blood pressure during the Bezold-Jarisch reflex. PMID- 7552344 TI - Detection of membrane-bound guanylate cyclase activity in rat C6 glioma cells at different growth states following activation by natriuretic peptides. AB - We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differentially located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes. PMID- 7552345 TI - The site of the inhibitory action of endogenous opioids in the superior cervical ganglion of the cat. AB - A low-frequency stimulus train to the preganglionic input inhibits synaptic transmission in the superior cervical ganglion (SCG) of the cat. The inhibition is blocked by naloxone as well as by selective antagonists at mu and delta opiate receptors, which suggests that the mediator is an endogenous opioid [27,29]. Exogenous opioid peptides, including methionine-enkephalin (Met-Enk), which is present in preganglionic axons of the SCG, inhibit ganglionic transmission by a naloxone-sensitive mechanism. In the present study we test, in the anesthetized cat, whether the naloxone-sensitive synaptic inhibition is mediated by a pre- and/or post-synaptic mechanism. As a test of presynaptic inhibition, we measured the acetylcholine (ACh) released by preganglionic stimulation into the venous effluent of the perfused SCG. As a test of post-synaptic inhibition, we measured the effect of a preganglionic conditioning train on the ganglion cell firing evoked by ganglion-stimulant drugs injected into the arterial supply of the ganglion. In presence of naloxone (3 microM), which blocked the synaptic inhibition, the amount of ACh released by stimulated preganglionic axons did not change. Thus, the endogenous opioid which mediates the naloxone-sensitive inhibition does not act by depressing ACh release. In contrast, the ganglion cell firing evoked by ganglion-stimulant drugs was markedly depressed by a conditioning train, and naloxone blocked the depression, which suggests that the endogenous mediator of the naloxone-sensitive inhibition acts postsynaptically to decrease the excitability of ganglion cells. Exogenous Met-Enk depressed both ACh release by preganglionic stimulation and the firing of ganglion cells evoked by ganglion-stimulant drugs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552346 TI - gamma-Aminobutyric acid type A/benzodiazepine receptors regulate rat retina neurosteroidogenesis. AB - It has been previously shown that retinal ganglion cells have the ability to synthesize steroids including neuroactive steroids such as pregnenolone sulfate. Since ganglion cells possess GABAA/benzodiazepine (BZ) receptors and neurosteroids modulate retinal GABAA receptor function, we investigated the role of these receptors in isolated rat retina neurosteroidogenesis. Ligands for central-type BZ receptors stimulated retinal pregnenolone synthesis. Clonazepam was the most potent ligand examined acting at nanomolar concentrations. Moreover, the effective steroidogenesis stimulatory dose (ED50) for these ligands and the Ki to inhibit [3H]flunitrazepam binding showed a coefficient of correlation of r = 0.87, suggesting the involvement of the central-type BZ receptors in this event. Ro 5-4864, which preferentially binds to peripheral-type BZ receptors, was less efficacious and potent whereas PK 11195 did not affect the basal pregnenolone formation and did not antagonize the Ro 5-4864 stimulated steroid synthesis. The GABAergic agonist muscimol, stimulated neurosteroid synthesis and this effect was reversed by the GABAergic antagonists bicuculline and picrotoxinin. In addition, these antagonists decreased basal pregnenolone formation, suggesting a tonic GABAergic control of the steroidogenic pathway, and reduced clonazepam-stimulated steroidogenesis. These results, together with the reported ability of neurosteroids to modulate GABAA receptor function, suggest a novel regulatory mechanism to control the inhibitory transmission. PMID- 7552348 TI - Effects of acetylcholine and adrenaline on axoplasmic transport at different regions of mouse superior cervical ganglion cells in culture. AB - Adrenaline and acetylcholine (ACh) were applied locally at three different positions in cultured superior cervical ganglion cells, i.e., cell body, neurite, and growth cone and the effects on the axoplasmic transport were measured with a video-enhanced microscope. Local ACh application to the cell body, neurite, and growth cone caused the same decreasing effect, but the effects of local adrenaline application were different from each other. Local adrenaline application to the cell body and growth cone caused an increase of axoplasmic transport, but local application at the neurite caused no effect. These data may indicate that there was a lack of beta 2 adrenergic receptors in the neurite. Desensitization of axoplasmic transport was also examined in the SCG neurons. Repetitive adrenaline application to the cell body caused desensitization to the stimulus of adrenaline application. PMID- 7552347 TI - Distribution of Fos-positive neurons in cortical and subcortical structures after picrotoxin-induced convulsions varies with seizure type. AB - The distribution of Fos protein was mapped in rat brain following a single non focal convulsive seizure. Single seizures were induced with intravenous picrotoxin in unhandled animals housed in isolation. Different convulsive behaviours occurred unpredictably. The least severe seizures were predominantly localised to the face, head and forelimbs, without loss of posture control (restricted seizures). The most extensive seizures affected all limbs and trunk, sometimes with falling (generalised seizures). There was a correlation between seizure behaviour and distribution of Fos induction. After restricted seizures, Fos was induced at highest levels in neocortex and piriform cortex and was prominent in entorhinal cortex, caudal-ventral caudate-putamen and amygdala. Regions of thalamus were consistently and lightly labelled, but Fos induction did not occur in hippocampus. After generalised seizures, there was Fos induction in cortex but less than after restricted seizures and, in three of four animals, also in dentate gyrus, hippocampus and subiculum. There was occasional or variable labelling of thalamus, basolateral amygdala and caudate-putamen. One animal with generalised seizures showed no hippocampal Fos induction. The findings indicate that picrotoxin induces seizures with at least two different patterns of neuronal involvement. The cortex, part of the caudate-putamen, amygdala and thalamus are involved in restricted seizures while the hippocampus, cortex and thalamus are involved in generalised seizures. The results do not support the view that generalised seizures are a progression from restricted forms. Cortical Fos involvement is entirely consistent with the participation of cortex in non-focal epilepsy. In these non-focal seizures, the dentate hippocampus may be a source of excitation to cortex in the generalised group while the cortex appears to be the predominant site of excitation in the restricted group. PMID- 7552349 TI - Sodium channel distribution in a spider mechanosensory organ. AB - A site-directed antibody was used immunocytochemically to measure the distribution of sodium channels in the tissues of a spider mechanoreceptor organ. The VS-3 slit sense organ contains 7-8 pairs of bipolar sensory neurons; these neurons are representative of a wide range of arthropod mechanoreceptors. Sensory transduction is thought to occur at the tips of the dendrites and to cause action potentials that are regeneratively conducted to the cell bodies, although it has not been possible to confirm this by direct intracellular recordings from the dendrites. Wholemount preparations were labelled by immunofluorescence and thin sections were immunogold labelled, using an antibody to the highly conserved SP19 sequence of the voltage-activated sodium channel. Labelling for sodium channels was found in the neurons and in their surrounding glial cells. Both cytoplasm and membranes were labelled, but immunogold particles were clearly aligned along cell membranes, indicating that the majority of labelling represented membrane-bound sodium channels. Channel density in the dendrites was similar to the axons and higher than in the cell bodies, supporting the idea of active conduction in the sensory dendrites. Labelling in glial cell membranes was indistinguishable from the neighboring neurons, suggesting a significant role for sodium channels in the functions of these supporting cells. PMID- 7552350 TI - Time course of reinnervation of the olfactory bulb after transection of the primary olfactory nerve in the pigeon. AB - Horseradish peroxidase (HRP) histochemistry was used to study the time course of reinnervation of the pigeon olfactory bulb after simple transection of the primary olfactory nerve. At selected intervals (9, 13, 29, 61 and 93 days) after transection of the right olfactory nerve, a concentrated solution of HRP was instilled in both nasal cavities. Intracarotid perfusion was performed 3 days after nasal instillation of HRP and 40-microns sections of olfactory bulb processed with the tetramethylbenzidine (TMB)-HRP histochemical protocol to visualize olfactory receptor axon terminals reinnervating the glomerular layer of the bulb. The total area of reinnervation of four representative regions in the bulb of the transected side were compared with that on the control bulb. The area of innervation by newly reconstituted olfactory axons was approximately 17% of control values at the 12-day posttransection time interval. A progressive increase in the area of reinnervation was observed over time. Reinnervation of the right bulb was approximately 70% complete at the 32-day posttransection time interval and indistinguishable from the left control bulb at the 64- and 96-day posttransection time intervals. A uniform pattern of reinnervation of different bulb regions was observed at all time intervals. These results indicate that the peripheral olfactory system of the pigeon is capable of complete reconstitution after nerve transection. Our findings should be useful in guiding functional comparisons of normal and newly reconstituted peripheral olfactory systems in the pigeon. PMID- 7552352 TI - Potent inhibitory effects of glial derived neurotrophic factor against kainic acid mediated seizures in the rat. AB - Recently messenger RNA (mRNA) for glial derived neurotrophic factor (GDNF), a recently discovered member of the TGF-beta superfamily, was shown to increase in the hippocampus after kainic acid-induced seizures. The possibility that exogenous recombinant human (rh) GDNF may have anticonvulsant properties was investigated using a model of temporal lobe epilepsy in the rat. rhGDNF, vehicle or inactive rhGDNF were injected intracerebroventricularly 1 h before peripheral administration of kainic acid. rhGDNF suppressed kainic acid-induced tonic-clonic convulsions when compared to animals treated with vehicle or inactive rhGDNF. The inhibition of kainic acid-induced seizure activity by rhGDNF also prevented the associated neuronal cell loss in hippocampal, thalamic and amygdaloid regions. These results suggest that rhGDNF should be evaluated in other seizure and acute neural disorders that are associated with excitotoxic processes. PMID- 7552351 TI - Transport of [125I]transferrin through the rat blood-brain barrier. AB - Transferrin receptors are present in the plasma membrane of brain endothelial cells but it is unclear if these receptors mediate transport of transferrin across the blood-brain barrier (BBB). In the present study, the transport of rat holo-transferrin (rTf) across the BBB in vivo was evaluated in ketamine anesthetized rats (250-300 g) both by in situ brain perfusion coupled with capillary depletion analysis and by thaw-mount autoradiography. [125I]rTf was infused through the right internal carotid artery at a rate of 3.65 ml/min for 2.5-5 min. After a 5 min perfusion, the volume of distribution (VD) of [125I]rTf in the brain homogenate, the postvascular supernatant, or the vascular pellet was 55.8 +/- 4.5, 43.5 +/- 4.8, and 8.7 +/- 2.3 microliters/g, respectively. Co infusion of [125I]rTf with unlabeled rTf or with a high dose of OX26 monoclonal antibody to the rat transferrin receptor significantly reduced the [125I]rTf transport, and in the presence of 10% rat serum [125I]rTf transport was nearly entirely abolished. The transport of [125I]rTf across the BBB in vivo was demonstrated by thaw-mount autoradiography, which showed silver grains well within brain parenchyma following a 5 min internal carotid artery perfusion. These studies are consistent with the following conclusions: (a) in the absence of competing plasma transferrin, [125I]holo-transferrin is transported through the BBB at a rate comparable to the OX26 monoclonal antibody; and (b) the ability to detect measurable transport of perfused [125I]transferrin is greatly inhibited by a small contamination of the perfusate by rat serum, which contains high concentrations of competing transferrin. PMID- 7552353 TI - Major differences in response to graded hypoxia between hypoglossal and neocortical neurons. AB - Intracellular electrophysiologic recordings were performed in brain slices from adult rats to compare the response of brain stem hypoglossal neurons (XII) and layer II/III neocortical neurons (NCX) to two levels of oxygen deprivation (hypoxia, pO2 = 15-20 Torr; anoxia, pO2 = 0 Torr). These recordings were also used during re-oxygenation after hypoxia or anoxia to study neuronal recovery. Both groups of neurons showed a greater response to anoxia than hypoxia in terms of membrane potential (Vm) and input resistance (Rm). When the two groups were compared at each level of O2, XII depolarized more and in a shorter period of time than NCX. During anoxia, XII neurons responded with anoxic depolarization (AD) of > 20 mV/min by 3 min, along with a large decrease in Rm. NCX neurons, on the other hand, exhibited AD after a mean latency of approximately 9 min and 18% of NCX neurons did not even show AD. Although all neurons (both XII and NCX) recovered when re-oxygenated before or at AD, XII neurons failed to recover from periods of anoxia that were well tolerated by NCX neurons. We conclude that: (1) there are marked differences in the magnitude and trajectory of membrane depolarization between XII and NCX neurons in response to O2 deprivation, with NCX neurons showing a much longer latency to AD during anoxia than XII; and (2), when exposed to periods of anoxia of similar duration and severity, XII neurons are less likely to recover than NCX neurons and XII neurons may, therefore, be inherently more vulnerable to anoxia-induced injury than NCX neurons. PMID- 7552354 TI - Monocular deprivation alters the morphology of glial fibrillary acidic protein immunoreactive astrocytes in the rat visual cortex. AB - Monocular deprivation was used to examine the experience-dependent structural plasticity of astrocytes in Oc1M and Oc1B visual cortex of young and adult rats. Stereological techniques were employed to assess the numerical density (Nv) of cells and surface density (Sv) of processes immunoreactive for glial fibrillary acidic protein in laminae II/III, IV, V and VI in the hemisphere opposite the deprived eye. In one group of pups eyelids were sutured on postnatal day 12 (P12) and maintained until P80 (MD), while a second group had the sutures removed at P75 followed by 5 days of light exposure (MD + L). An unoperated light experienced group was used for comparisons (L). The Sv of astrocytic processes in lamina IV but not laminae II/III, V and VI was significantly decreased in the MD group. The ratio of Sv to the Nv of neurons, an estimate of the amount of astrocytic membrane per neuron, was also significantly decreased in layer IV. The Nv of astrocytes was not significantly different among the three groups. In adults that were monocularly deprived for 5, 10 and 30 days the Nv of astrocytes and Sv of their processes were not significantly altered in layer IV. There was however an increase in the Nv of all types of glial cells combined in layer IV following 10 and 30 days. These results indicate that the structure of astrocytes is influenced by visual experience during development whereas merely altering the level of visually-driven activity in the adult was not sufficient to induce astrocytic structural change. PMID- 7552356 TI - Involvement of cholinergic systems in the deficit of place learning in Morris water maze task induced by baclofen in rats. AB - Effects of oxotremorine on the deficit of place learning in the Morris water maze task induced by baclofen and scopolamine were examined to determine the involvement of brain cholinergic systems in the deficit of learning induced by baclofen. Rats were given 4 training trials per day with the submerged platform at a fixed location in the maze for 4 days. On day 4, rats were required to swim in the pool without the platform after the 4th training trial (probe test). Baclofen as well as scopolamine dose-dependently increased the escape latency in the training trials. In the probe test, baclofen as well as scopolamine dose dependently reduced the duration in the quadrant where the platform had been originally located. Increased latency in the training trials and reduced duration in the probe test induced by scopolamine were dose-dependently attenuated by oxotremorine. Increased latency and reduced duration in the baclofen-treated rats were improved by oxotremorine as well as 2-hydroxysaclofen. Baclofen but not scopolamine induced motor incoordination in the rotarod test. Oxotremorine failed to improve motor incoordination induced by baclofen. These results suggest that cholinergic systems may be involved in the deficit of place learning induced by baclofen, and that the ameliorative effects of oxotremorine may not be due to improvement of motor incoordination. PMID- 7552355 TI - Stereochemistry of the Aplysia neuronal 12-lipoxygenase: specific potentiation of FMRFamide action by 12(S)-HPETE. AB - Nervous tissue of the marine mollusc, Aplysia californica, generates arachidonic acid metabolites in response to neurotransmitters such as histamine or FMRFamide. In addition, identified neurons of Aplysia respond to the pharmacologic application of some of these products, particularly those of the 12-lipoxygenase pathway. We investigated the chirality of the initial Aplysia 12-lipoxygenase product, 12-HPETE, in preparation for more detailed metabolic studies and for the analysis of the physiological activity of the endogenous lipid. Neural homogenates and intact ganglia exclusively generate 12(S)-HPETE as do the better characterized mammalian lipoxygenases. The direct application of 12(S)-HPETE to cultured sensory neurons induced a hyperpolarization which averaged 2.6 mV. We did not find any difference between the response to the naturally-occurring 12(S) HPETE and its diastereomer, 12(R)-HPETE which is not generated in Aplysia. Both isomers were significantly more effective than 15(S)-HPETE. In contrast, 12(S) HPETE, but not 12(R)-HPETE, was a potent modulator of the action of the molluscan neuropeptide, FMRFamide. Prior application of 12(S)-HPETE to cultured sensory neurons increased the subsequent response to a submaximal dose of FMRFamide by 60%. On the other hand, 12(R)-HPETE reduced the subsequent response to the peptide by 30%. The lack of stereospecificity in the direct effect of the lipids differs markedly from their stereospecific effects as modulators of FMRFamide action. This suggests that there may be an important neurophysiologic role for these lipid modulators which is distinct from their direct effects, and also indicates that there are multiple sites and mechanisms by which lipid hydroperoxides act on neurons in Aplysia. PMID- 7552358 TI - Increased sensitivity to cocaine place-preference conditioning by septal lesions in rats. AB - Rats bearing electrolytic lesions of medial septum and sham-operated controls were trained on cocaine place-preference in a 3-compartment apparatus. Cocaine was paired with a white or a black compartment. An unbiased design was used, in which cocaine was paired with the preferred side in half the animals and with the unpreferred side in the other half. Two low doses of cocaine HCl were used: 2.5 and 5.0 mg/kg. Only two pairings of drug with environment were used to minimize the influence of drug sensitization. Rats with septal lesions, but not controls, showed preference conditioning to the black side at 2.5 mg/kg; lesioned and control animals showed similar conditioning to the black side at 5.0 mg/kg. Lesioned animals could not be conditioned to the white side at either dose. This was attributed to a drug-induced enhancement of a previously described increased reactivity to brightness following septal lesions. Controls conditioned to either side at 5.0 mg/kg. It was concluded that septal lesions lowered the cocaine dose required for preference conditioning, consistent with reports that such damage enhances some behavioral effects of psychostimulants. PMID- 7552357 TI - N-type Ca2+ channels mediate transmitter release at the electromotoneuron electrocyte synapses of the weakly electric fish Gymnotus carapo. AB - The effects of omega-conotoxin-GVIA (omega-CgTX) on synaptic transmission were studied in the electromotoneuron-electrocyte synapses of the electric organ (EO) of the weakly electric fish Gymnotus carapo. omega-CgTX selectively and irreversibly blocked excitatory postsynaptic potentials (EPSPs) in a dose dependent-manner. The toxin had no effect on: (a) resting postsynaptic membrane potential and conductance; (b) postsynaptic action potentials elicited by depolarizing transmembrane current pulses; (c) the action potential conduction in the presynaptic fiber; (d) acetylcholine (ACh)-induced postsynaptic responses. Nifedipine - a selective dihydropyridine antagonist of the L-type voltage dependent Ca2+ channels (VDCCs) - did not affect synaptic transmission. Transmission was also undisturbed by the peptide omega-Agatoxin (omega-Aga-IVA), the low molecular weight polyamine, funnel-web toxin (FTX) - both included in the venom of the spider Agelenopsis aperta - and its synthetic analog sFTX, all selective blockers of P-type VDCCs. Since omega-CgTX irreversibly blocks the N type VDCCs, we conclude that presynaptic N-type VDCCs mediate transmitter release at electromotoneuron terminals. The VDCCs involved in fish peripheral electromotoneuron-electrocyte presynaptic transmitter release are therefore similar to those in amphibian, reptilian and avian peripheral synapses, but differ from mammalian and invertebrate motoneuron terminals. PMID- 7552359 TI - Functional network interactions between parallel auditory pathways during Pavlovian conditioned inhibition. AB - Using covariance structural equation modeling and fluorodeoxyglucose (FDG) autoradiography this study examined auditory system interactions when the learned associative effects of a tone were inhibited by a light. Two groups of rats received pairings of a tone (conditioned excitor: T+) with a mild footshock. Group TL- was trained in a Pavlovian conditioned inhibition paradigm (T+/TL-) where the tone-light compound signaled the absence of footshock, making the light the inhibitor (L-). Group TL degree was trained with the tone as the excitor and the light as a 'neutral' stimulus. After FDG injection, all rats were presented with the tone-light compound. Group differences in auditory system FDG uptake were observed only in the ventral division medial geniculate nucleus (MGV), where group TL- had relatively lower incorporation. Structural equation modeling was used with the covariances of FDG activity to determine the functional influences through the auditory system anatomic connections. Differences were noted mainly at the level of the inferior colliculus (IC) and medial geniculate, possibly reflecting the unique anatomic relation of these regions with extraauditory areas. Ascending and descending influences from the IC differed with stronger influences for group TL-. Intracollicular and the ascending influence influences of MGV and medial division of the medial geniculate nucleus (MGM) on the auditory cortex also differed mainly in the sign of the functional interaction. These results demonstrate how interactions among parallel auditory pathways can code the behavioral significance of auditory stimuli and emphasize that a full appreciation of neural operations underlying learning can only be gained through examination of both regional activity and interregional interactions. PMID- 7552360 TI - Neurotensin increases the cationic conductance of rat substantia nigra dopaminergic neurons through the inositol 1,4,5-trisphosphate-calcium pathway. AB - Whole-cell patch-clamp recordings were used to investigate electrophysiological effects of neurotensin on acutely isolated dopaminergic (DA) neurons of the rat substantia nigra pars compacta (SNC). During current-clamp recordings, neurotensin depolarized DA neurons and triggered action potentials. Under voltage clamp recordings, neurotensin evoked an inward current at a holding potential of 50 mV. Neurotensin-induced inward currents reversed the direction at -5 mV and became smaller as the membrane potential was hyperpolarized from -75 mV. With potassium-free recording solutions, neurotensin evoked voltage-insensitive cationic currents. With sodium-free external solution, neurotensin also caused inward currents by reducing the inwardly rectifying potassium conductance. Neurotensin-induced inward currents mainly resulted from an increase in a non selective cationic conductance. Neurotensin-evoked cationic currents were inhibited by the intracellular perfusion of 1 mM guanosine-5'-O-(2 thiodiphosphate). In DA neurons internally perfused with 0.5 mM guanosine-5'-O-(3 thiotriphosphate), the cationic current produced by neurotensin became irreversible. Pretreating DA neurons with 500 ng/ml pertussis toxin (PTX) did not significantly affect the ability of neurotensin to evoke cationic currents. Internal perfusion of heparin (2 mg/ml), an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, and buffering intracellular calcium with the Ca(2+)-chelator BAPTA (10 mM) suppressed neurotensin-induced cationic currents. Dialyzing DA neurons with protein kinase C (PKC) inhibitors, staurosporine and PKC(19-31), failed to prevent neurotensin from evoking cationic currents. It is concluded that PTX-insensitive G-proteins mediate neurotensin-induced enhancement of the cationic conductance of SNC DA neurons.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552362 TI - Is nestin a marker for chemosensory precursor cells? AB - The vomeronasal and olfactory systems are unique in that their chemosensory neurons undergo continuous neurogenesis after development. Immunoreactivity to nestin, a neuronal precursor marker protein, was investigated in the developing rat vomeronasal organ to determine its potential as a cell marker. From postnatal day 1 (P1) to P22, the distribution of nestin positive cells became progressively restricted to the area adjacent to the basement membrane. By P29, the vomeronasal organ reached structural maturity and only a few nestin positive cells were observed. Results suggest that nestin may be a useful marker for neuronal precursor cells in studies of neurogenesis and development of chemosensory systems. PMID- 7552361 TI - Rapid and semiquantitative analysis of HSP72 and HSC73 heat shock mRNAs by mimic RT-PCR. AB - The amounts of mRNAs for 72 kDa (HSP72) and 73 kDa (HSC73) heat shock proteins were measured semiquantitatively with competitive polymerase chain reaction (PCR) techniques after reverse transcription (RT) using a heterologous DNA fragment (PCR mimic) as an internal standard. The changes of signal intensities of PCR products were well correlated with the amount of mRNA in rat brain measured as optical densities of Northern blot. Thus, an analysis with mimic RT-PCR technique provides a rapid, semiquantitative, and safe method to detect changes of HSP72 and HSC73 mRNAs after brain ischemia. PMID- 7552363 TI - Exogenous thrombin inhibits neuritogenesis in cultured neuroblastoma cells but not in rat hippocampal neurons. AB - Thrombin has been reported to inhibit neurite outgrowth from neuroblastoma cells grown in serum-containing medium after switching to serum-free medium. A test of the serum and substrate-dependence of this inhibition became possible with the development of Neurobasal/B27 serum-free medium. Inhibition of sprouting of Nb2a neuroblastoma cells by thrombin occurred from the substrate where it was bound to material adsorbed from serum. Neuritogenesis of primary hippocampal neurons was unaffected by exogenous thrombin on polylysine substrates with or without serum treatment. However, sprouting of hippocampal neurons was stimulated by treating the substrate with hirudin, a highly specific thrombin inhibitor. This suggests that hippocampal neurons are not directly responsive to added thrombin, perhaps because they produce their own thrombin. PMID- 7552364 TI - Cocaine increases extraneuronal levels of aspartate and glutamate in the nucleus accumbens. AB - Intracerebral microdialysis was used to assess the effects of cocaine-HCl on extracellular concentrations of the excitatory amino acids aspartate and glutamate in the nucleus accumbens of awake, freely moving rats. After an initial equilibration period, cocaine (7.5, 15 or 30 mg/kg) or saline was injected i.p., and samples were collected for an additional 2 h. The highest dose of cocaine (30 mg/kg, i.p.) caused a 4-fold increase in glutamate levels and an 18-fold increase in aspartate levels over baseline. To verify that the source of the extracellular aspartate and glutamate was neuronal, additional experiments were conducted using Ca(2+)-free microdialysis buffer, and buffer containing 10 microM tetrodotoxin. Local perfusion with Ca(2+)-free buffer reduced the increase of extracellular aspartate and glutamate in rats injected with 30 mg/kg cocaine. Tetrodotoxin significantly decreased the cocaine-induced increase in excitatory amino acids, but not the behavioral response. PMID- 7552365 TI - Castration increases [125I]MK801 binding in the hippocampus of male rats. AB - This study examines the effect of castration and androgen replacement on [125I]MK801 binding in the hippocampus. In castrated male rats, [125I]MK801 binding was significantly increased in both the stratum oriens and radiatum and the pyramidal cell layer of CA1. In contrast, no increase in [125I]MK801 binding was observed in the stratum oriens and radiatum of CA1 of castrated rats that were treated with dihydrotestosterone. No change in [125I]MK801 binding was observed in the CA3 region or dentate gyrus after castration. The observed increase in [125I]MK801 binding in pyramidal cell neurons within CA1 suggests that androgens may potentially affect hippocampal function by modulating pyramidal cell NMDA receptors. PMID- 7552367 TI - NBQX, a selective antagonist of the AMPA receptor, affects neither field potentials nor long-term potentiation in vivo. AB - NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), a selective antagonist of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors, at an anticonvulsant dose of 40 mg/kg (i.p.), was shown in vivo to have no effects on either the field potentials in the piriform cortex or those in the dentate gyrus of the hippocampus. The same dose of NBQX also exerted no significant effects on long-term potentiation in the hippocampus. These results suggest that the mechanism underlying the anticonvulsant action of NBQX in vivo does not involve a suppressive action on physiological synaptic transmission. The possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs is suggested. PMID- 7552366 TI - Hydrogen peroxide hyperpolarizes rat CA1 pyramidal neurons by inducing an increase in potassium conductance. AB - It has been suggested that hydrogen peroxide is involved in cascades of pathological events affecting neural cells. The aim of this study was therefore to examine whether this molecule is able by itself to modify membrane properties of pyramidal neurons in the CA1 region of the rat hippocampus. Intracellular recordings in the slice preparation showed that 3.3 mM hydrogen peroxide hyperpolarized all neurons tested (n = 41) by 11 +/- 3 mV. This effect persisted in the presence of tetrodotoxin. It developed slowly, was reversible and reproducible. In the presence of tetrodotoxin, the extrapolated reversal potential of this effect was -95 +/- 5 mV in 2.5 mM external potassium. This value was not significantly different from the one obtained with the GABAB agonist baclofen (10 microM) (-98 +/- 5 mV). It shifted when the concentration of external potassium was increased to 10.5 mM (from -96 +/- 5 to -62 +/- 4 mV), in close agreement with the Nernst equation potassium ions. The hyperpolarization was significantly reduced (by 65 +/- 22%) by the potassium channel blocker barium (100 microM). We suggest that hydrogen peroxide is able to induce an increase in potassium conductance in rat CA1 pyramidal neurons. The exact mechanism by which it produces this effect (direct action on channels or indirect effect) remains to be determined. PMID- 7552368 TI - Effects of orochemical stimulation on postnatal development of gustatory recipient zones within the nucleus of the solitary tract. AB - Previous receptor damage studies and artificial rearing (AR) studies in rat have demonstrated that orochemical stimulation between the postnatal ages of P2 and P14 is necessary for development of primary gustatory axons and terminal endings in the rostral nucleus of the solitary tract (NST). Objectives of the present experiment were to evaluate the qualitative nature of orochemical stimulation and amount of orochemical stimulation that is necessary to produce normal axonal and terminal development in gustatory recipient zones of the rostral and intermediate NST. To this end, ultra-pure water, 30, 150, and 500 mM NaCl, 80 and 340 mM lactose, whole rat milk, and rat milk that had been subject to extensive dialysis (12-14 kD MWCO) was delivered to independent groups of rat pups during AR procedures. Unstimulated AR animals and matched mother-reared (MR) animals were used as controls. AR animals received experimental orochemical stimulation between the ages of P4 and P10, and were thereafter returned to lactating dams until the time of weaning; MR animals received experimental orochemical stimulation during the course of normal nursing. Following maturation, anterograde fluorescent dual-labeling experiments were conducted to map the course and distribution of primary gustatory axons within the NST. Results show that experimental stimualtion with water during AR procedures is not sufficient to produce normal development of primary gustatory axons and terminal endings in the gustatory NST. Stimulation with 30, 150, and 500 mM NaCl produced normal axonal development in the NST, as did 80 and 340 mM lactose, whole rat milk, and dialyzed rat milk.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552369 TI - Chemosensitivity of neurons in the dorsal motor nucleus of the vagus that responded to portal infusion of hypertonic saline in rats. AB - Neural responses in the dorsal motor nucleus of the vagus (DMV) to topical administrations of sodium and portal infusions of hypertonic saline were investigated electrophysiologically by using multibarrel electrodes in anesthetized rats. Of 102 neurons that showed antidromic response to electrical stimulation of the ventral gastric vagus or the accessory celiac vagus, 51 neurons increased and 13 neurons decreased their discharge rates in response to the electrophoretic administration of sodium. The other 38 neurons did not respond to this stimulation. The portal infusion of hypertonic saline elicited neural responses of some DMV neurons whose axons are involved into either the ventral gastric or the accessory celiac vagus. Further, effects of the topical administration and the portal infusion of hypertonic saline were examined on 33 neurons. Typical response was characterized by an increase in discharge rate responding to both of the portal infusion and the topical administration. In conclusion, the DMV neurons receiving the afferent inputs from hepatoportal osmoreceptors may have an enteroceptor function detecting the change in osmotic pressure of their environment. PMID- 7552370 TI - Serotonin 1B receptor regulation after dorsal subiculum deafferentation. AB - The subiculum may be the key structure in the transfer of relevant processed information from the hippocampal formation to cortical areas. We investigated the location of the serotonin 1B receptor (5-HT1B) in the hippocampus with the specific ligand serotonin-O-carboxymethyl-glycyl[125I]tyrosinamide in rat brain sections using in vitro autoradiography. A high density of 5-HT1B binding sites was found in the dorsal subiculum (DS), in the lacunosum moleculare, and in the most dorsal layer of the stratum oriens of the CA1 field. CA1 pyramidal neurons that contain 5-HT1B mRNA project primarily to the DS. We interrupted the pyramidal CA1 axons unilaterally by a stereotaxic knife cut. Histological analysis showed that the lesion was restricted to a trial of cells lost between CA1 and DS. Specific 5-HT1B binding site density was decreased in the DS on the ipsilateral side of the lesion compared to the contralateral side. We conclude that 5-HT1B receptors are located on CA1 pyramidal axon terminals in the DS. Serotonin, acting on these receptors, should inhibit CA1 neurotransmitter release and, in this way, modulate subicular functions. PMID- 7552371 TI - Calcium deposits in the thalamus following repeated cerebral ischemia and long term survival in the gerbil. AB - We investigated the long-term changes in the gerbil brain following three episodes of 2-min forebrain ischemia at 1-h intervals in comparison with a 6-min period of ischemia. The animals were sacrificed after 1 month and 6 months. Following either ischemic insult, the hippocampal CA1 region showed a loss of pyramidal neurons together with a diffuse calcium accumulation as shown by alizarin red S staining. Three 2-min ischemic insults additionally produced neuronal damage in the striatum and thalamus. The thalamic damage was accompanied by an accumulation of small calcium granules after 1 month and large calcium concretions after 6 months. Calcium staining in the striatum was weak. Thus, the thalamic neuronal damage was accompanied by an active process of calcification, which has not been described in experimental cerebral ischemia models. The observations show that repeated ischemic insults produce different long-term effects in different brain regions. PMID- 7552373 TI - The possible involvement of the nonstrial pathway of the amygdala in neural control of sexual behavior in male rats. AB - The effect of medial amygdala lesions on male sexual behavior in male rats with stria terminalis cut was examined. First, castrated male rats received bilateral transections of the stria terminals (STC) or sham cut (SC). Most STC males showed no ejaculation, but displayed mount and intromission, although the frequencies were not high compared to those of males with SC. Next, bilateral lesions of the medial amygdala (MAL) or sham lesion (SL) were performed in males with STC or SC. The MAL caused severe loss of all aspects of copulatory behavior in males with STC as well as in males with SC. The suppressive effect of the MAL on copulatory activity was stronger than that of the STC. These results indicate that a neural pathway other than the stria terminalis is involved in the regulation of male sexual behavior by the amydgala in male rats. PMID- 7552374 TI - The parabrachio-subfornical organ projection in the rat. AB - Previous physiological studies have shown that both the parabrachial nucleus and the subfornical organ are involved in drinking behavior and cardiovascular controls. The purpose of the present work was to study the direct connections between these two structures by using anterograde and retrograde transport methods. A mixture of wheat germ agglutinin conjugated with horseradish peroxidase and free horseradish peroxidase or Fluorogold was injected into either the parabrachial nucleius (PBN) or the subfornical organ (SFO). The results indicated that the parabrachial nucleus sends a substantial projection to the entirety of the subfornical organ, and this input appears to be distributed to both the central and peripheral regions of this structure. Neurons that give origin to this projection are mainly located in the outer layer of the lateral division of the parabrachial nucleus, including the superior, internal, dorsal, and external lateral subnuclei. These findings suggest that, besides the already known connections, there is an additional parabrachio-subfornical pathway that may be involved in the central integration of cardiovascular function and drinking behavior. PMID- 7552372 TI - Dipyridamole attenuates the development of iminodipropionitrile-induced dyskinetic abnormalities in rats. AB - The present investigation was undertaken to study the effect of dipyridamole on experimental dyskinesia in rats. The movement disorders were produced by intraperitoneal administration of iminodipropionitrile (IDPN) in the dose of 100 mg/kg per day for 12 days. Dipyridamole was administered orally, daily 30 min before IDPN in the doses of 0.5 g/kg, 1 g/kg, and 1.5 g/kg bodyweight in three different groups of rats. Twenty-four hours after the last dose of IDPN, animals were observed for neurobehavioral changes including vertical and horizontal head weaving, circling, backwalking, grip strength, and righting reflex. Immediately after behavioral studies brain specimens were collected for analysis of vitamin E, conjugated dienes, and lipid hydroperoxides as indices of oxygen-derived free radical (OFR) production. Our results showed that concurrent use of dipyridamole significantly protected rats against IDPN-induced neurobehavioral changes in a dose-dependent manner. Treatment of rats with dipyridamole inhibited IDPN-induced decrease of vitamin E and increase in conjugated dienes and lipid hydroperoxides in brain. These findings suggest the involvement of OFR in dipyridamole induced protection against the development of IDPN dyskinesia. Further studies are warranted to determine the role of dipyridamole as a prophylactic agent against the drug induced dyskinetic abnormalities. PMID- 7552375 TI - Citalopram, a serotonin reuptake inhibitor, and brain ischemia in SHR. AB - The effects of citalopram, a serotonin (5-HT) reuptake inhibitor, on cerebral blood flow (CBF) and concentration of 5-HT and its metabolite were investigated in spontaneously hypertensives rats (SHR) subjected to forebrain ischemia. Cerebral ischemia was induced by bilateral carotid artery occlusion. The concentration of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), increased during cerebral ischemia in most brain regions examined, while that of 5-HT increased only in the frontal cortex and the striatum. Citalopram restored the 5-HIAA concentrations to the preischemic normal levels. Citalopram had no effect on the cortical CBF, before and during ischemia. These results suggest that citalopram attenuates ischemia-induced hypermetabolism of 5-HT in the brain. The effects of citalopram are independent of hemodynamic factors including cerebral blood flow, and are likely to be mediated by a direct inhibition of the neuronal 5-HT reuptake system. PMID- 7552376 TI - Afferent connections of the lateralis medialis thalamic nucleus in the cat. AB - We have used anterograde and retrograde horseradish peroxidase tracing methods in this study. Peroxidase injections in the lateralis medialis thalamic nucleus (LM) of the cat resulted in labeled neurons in cortical and subcortical structures that averaged 71% and 29%, respectively. Every LM sector receives abundant projections from the polymodal sylvian anterior cortical area, the reticular thalamic nucleus, and the stratum opticum and intermediate layer of the superior colliculus. Less abundant but consistent projections were detected in cingular, auditory II, lateral suprasylvian and anterior ectosylvian visual cortices, and cortical area 7. A topographical distribution of afferent connections to different LM sectors arising from other cortical and subcortical structures could be established. The ventromedial sector receives connections mainly from the insular agranular, limic and prefrontal cortical areas, as well as from brain stem structures and the contralateral pretectal region. The dorsolateral sector is mainly related to cortical areas and subcortical structures processing visual information. The existence of overlap among neuronal LM populations receiving and sending connections to and from various cortical areas suggests that this nucleus is an appropriate substrate for effective interaction between different and distant cortical areas. PMID- 7552377 TI - Loss of neonatal hypoxia tolerance after prenatal nicotine exposure: implications for sudden infant death syndrome. AB - Maternal cigarette smoking has a high correlation with sudden Infant Death Syndrome, a condition in which cardiorespiratory failure occurs during an hypoxic episode, as in sleep apnea. Pregnant rats were given nicotine infusions of 2 or 6 mg/kg/day throughout gestation, regimens that produce plasma nicotine levels spanning the range in smokers. The day after birth, animals in the high dose group displayed excessive mortality during hypoxic challenge. These animals were found to be deficient in an essential response component, namely adrenomedullary catecholamine release that is required to maintain neonatal cardiac rhythm during hypoxia; the defect was in adrenal cell function rather than in altered innervation or nicotinic receptor desensitization. We also examined brainstem and forebrain noradrenergic mechanisms that are involved in neonatal respiratory control. The nicotine group showed suppressed spontaneous neuronal activity, but were hyperresponsive to hypoxia. As these projections are inhibitory for respiration, the nicotine-induced sensitization would be expected to contribute to respiratory arrest during hypoxia. Prenatal nicotine exposure may thus provide a useful animal model with which to study the physiological mechanisms that underlie Sudden Infant Death Syndrome, while at the same time providing a biological explanation for the association of the syndrome with smoking. PMID- 7552379 TI - Projection from nucleus reuniens thalami to piriform cortex: a tracing study in the rat. AB - To study the cells of origin and area of termination of the projection from the nucleus reuniens thalami (NRe) to the piriform cortex (PC) we used anterograde and retrograde tracing with the B subunit of the cholera toxin. Tracer injections in the NRe resulted in anterogradely labeled fibers in the dorsolateral part of the PC layers and I and III. Following injections in the PC, retrogradely labeled cells were observed primarily in the dorsal subdivision of the NRe. Moreover, a topographical organization was observed in this subdivision: its anterior part projects to the posterior part of the PC, whereas its middle part projects to the anterior part of the PC. The present findings suggest that the NRe may exert different modulatory influences on the dorsolateral part of both anterior and posterior PC areas. The possible role of the NRe in the olfactory information processing is discussed. PMID- 7552378 TI - A 3D digital map of rat brain. AB - A three dimensional (3D) computerized map of rat brain anatomy created with digital imaging techniques is described. Six male Sprague-Dawley rats, weighing 270-320 g, were used in the generation of this atlas. Their heads were frozen, and closely spaced cryosectional images were digitally captured. Each serial data set was organized into a digital volume, reoriented into a flat skull position, and brought into register with each other. A volume representative of the group following registration was chosen based on its anatomic correspondence with the other specimens as measured by image correlation coefficients and landmark matching. Mean positions of lambda, bregma, and the interaural plane of the group within the common coordinate system were used to transform the representative volume into a 3D map of rat neuroanatomy. images reconstructed from this 3D map are available to the public via Internet with an anonymous file transfer protocol (FTP) and World Wide Web. A complete description of the digital map is provided in a comprehensive set of sagittal planes (up to 0.031 mm spacing) containing stereotaxic reference grids. Sets of coronal and horizontal planes, resampled at the same increment, also are included. Specific anatomic features are identified in a second collection of images. Stylized anatomic boundaries and structural labels were incorporated into selected orthogonal planes. Electronic sharing and interactive use are benefits afforded by a digital format, but the foremost advantage of this 3D map is its whole brain integrated representation of rat in situ neuroanatomy. PMID- 7552382 TI - [The present status of iodine deficiency disorders in Slovakia and perspectives in their prevention]. AB - During 1949-1953 more than 157,000 of inhabitants were examined (i.e. more than 5% of total population) (Podoba, 1962) and the average prevalence of goitre of about 70% was found in women and about 40% in men. In the districts with the highest goitre prevalence also cretinism was found. The average values of urinary iodine in 86 villages were less than 35 micrograms/24 h. In 1951-1953 the production and mandatory consumption of iodized salt has been initiated and 10 years later a considerable decrease in goitre prevalence especially in adolescents was found. Systemic measurements during 1952-1994 showed that in about 25% of salt samples from the distribution network the iodine content was less than the requested amount of 25 +/- 10 mg KI/kg. Further decrease was found in the salt samples from households which apparently resulted in lower intake of iodine than originally expected. After 1987 some institutions joined the actions organized by ICC/IDD (International Council for Control of Iodine Deficiency Disorders), the aim of which was to evaluate the present status of iodine intake and thyroid volume namely in children and adolescents from posttotalitary countries with which it was not possible to establish any cooperation before. The examination of thyroid volume by sonographic volumetry in 4254 children and adolescents showed that 9% of values were higher than 97 percentile level of what is currently being recognized as normal range (Gutekunst and Teichert, 1993).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552380 TI - Neuron transplantation into mice hippocampus alters sensitivity to barbital narcosis. AB - The role of several hippocampal innervations in the sensitivity to barbital induced narcosis was studied in selected mice strains. The outbred and inbred mouse strains HS/lgb, SABRA/HUC, C57BL, CBA/LAC, and BALB/c were tested for barbital-induced sleep (315 mg/kg). The relatively short sleeping HS/lbg (HS) and the longest sleeping BALB/c (BALB) were chosen for further investigation. Cholinergic (ACh), serotonergic (5-HT), and noradrenergic (NE) innervations were studied in HS strain; whereas BALB, which possesses both an unusually high sensitivity to barbital and unique NE innervations in the cortex and hippocampus, was employed in a detailed study of the NE innervations. Transplantation of embryonic NE cells from the mouse embryo into the hippocampus of adult HS mice increased barbital narcosis by 65% (p < 0.05), whereas transplantation of 5-HT cells decreased barbital narcosis by 54% (p < 0.001). Transplantation of ACh cells had no significant effect on barbital-induced narcosis. BALB mice were subjected to NE cell transplantation into the hippocampus and cortex. Similarly to HS, BALB receiving NE transplants into their hippocampus slept 34% longer than control after barbital challenge (p < 0.025). Noradrenergic cell transplantation into frontal cortex had no effect on barbital sleep. The results suggest that (a) enhancement by neural grafting of the NE innervation to the hippocampus accentuates and enhancement of the 5-HT innervations attenuates the sensitivity to barbital narcosis, whereas ACh innervations have no effect on the sensitivity to barbital narcosis, and (b) the unusually high sensitivity of BALB mice to barbital may not be related to its unique NE innervations. PMID- 7552381 TI - Modifications of head turning and circling movement following sulpiride microinjections into nucleus accumbens in the rat. AB - The aim of this study was to determine whether a relationship exists between nucleus accumbens D2 receptors, circling behavior, and its first stage, the head turning. Rats were unilaterally lesioned in the substantia nigra with 6 hydroxydopamine and afterward treated with d-amphetamine IP following bilateral intraaccumbens microinjections (1, 5, 10 micrograms/0.5 microliters) of sulpiride, a D2 receptor antagonist. Computer-assisted video analysis allowed the study of some parameters (number of turns, type of turn, head turning duration, degree and speed) characterizing rotatory activity. Sulpiride microinfusion resulted in a dose-dependent decrease of the number of turns and head rotation speed and in a dose-dependent increase of head-turning duration. Two turn types were observed in relation to the animal's position: a large head-to-tail position with a short-diameter turn type following sulpiride microinjection, and a close head-to-tail position in relation to a wide diameter turn type in the control condition (saline). The results show a relationship between head turning parameters, circling behavior, and D2 receptors in nucleus accumbens, which may be also involved in the regulation of some mechanisms related to sensory-motor integration in the rat. PMID- 7552383 TI - [Latent hypothyroidism--a minor laboratory variation or a disease?]. AB - Latent hypothyreosis is a state which is characteristic by normal values of thyroid hormones (T3, T4) and moderately increased values of thyroid-stimulating hormone (TSH), or extensive response of TSH after stimulation with thyrotrophin releasing hormone (TRH-test). The characteristic symptoms include goitre, symptoms of mammary glands (galactorrhea, benign mammary dysplasia), impairment of menstruation cycle and fertility and psychic disturbances (depressive symptomatology, deterioration of memory). The characteristic laboratory alterations include increased basal or stimulated values of prolactin and impairment of lipoprotein metabolism (significant decrease of HDL, increase of LDL). This aspect often makes the indication of substitution therapy justified in spite of normal concentrations of peripheral hormones. (Ref. 26). PMID- 7552384 TI - [The role of antioxidants in the prevention of tumors]. AB - The potential role of antioxidant vitamins (ascorbic acid, beta-carotene, alpha tocopherol), minerals (selenium) and non-vitamin natural antioxidants (e.g. glutathione) in the prevention of cancer diseases is reviewed. Free oxygen radicals, especially the hydroxyl radical .OH modify nitrogen bases, split DNA, stimulate oncogene activators and probably in many other ways participate in carcinogenesis. In a great number of experimental and epidemiological studies a significant increase of cancer risk in laboratory animals and in humans with low antioxidant status was found. Significant protective effects of ascorbic acid, beta-carotene, alpha-tocopherol and selenium against the incidence of gastrointestinal and lung cancer were achieved in most, but not in all prospective and intervention studies. It is probable that extremely high premature cancer mortality in postcommunist countries of Central and East Europe is caused by high consumption of cigarettes, spirits and saturated fats, by pollution, and by very low consumption of the chief sources of natural antioxidants (fruits, vegetables). (Fig. 13, Ref. 29). PMID- 7552385 TI - [Location of atrial and ventricular insertions of accessory atrioventricular pathways using surface ECG mapping and its importance in catheter ablation therapy]. AB - BACKGROUND: The modern non-pharmacological therapy of the WPW syndrome by means of catheter ablation is based on the interruption of the accessory pathway(-s) by radiofrequency current energy. Destruction of the morphologic substrate of the arrhythmia alters the activation wave spread in the heart. OBJECTIVES: It was the aim of this report to demonstrate the diagnostic potential of BSM in localizing both overt and concealed accessory pathways. Presented study analyzes the alterations of the cardioelectric field by means of body surface ECG mapping in two female patients with accessory pathways before and after their successful curative treatment by radiofrequency catheter ablation. METHODS: Five patients were analyzed prior and after RF catheter ablation by BSM, two of them were selected for this presentation. One patient with WPW syndrome suffered from frequent supraventricular tachycardia due to in one female patient with an overt accessory pathway. The other patient was for several years incessantly in permanent junctional reentry tachycardia due to a concealed accessory pathway. The examination comprised 12-lead ECG, orthogonal vectorcardiogram according to Frank, BSM using a regular 80-electrode-array system and signal-averaged ECG. RESULTS: The RF ablation was successful in both patients and their arrhythmia was abolished. By means of a detailed analysis of the ventricular activation prior RF ablation in the patient with WPW syndrome the precise site of the ventricular insertion of the accessory pathway in the left lateral free wall was predicted. Furthermore, alterations of the terminal QRS complex were observed when comparing pre- versus post-ablation maps. In the second patient the atrial insertion of the accessory pathway with retrograde and decremental conduction was successfully localized to the right septal region by means of pre-ablation BSM. CONCLUSIONS: Both ventricular and atrial activation can be in detail analyzed by means of BSM. Such analysis offers more precise information on the spatial component of the activation wave spread. This case report gives further evidence that BSM is a useful method for precise localization of both ventricular and atrial insertion sites of accessory pathways in patients with paroxysmal tachycardias due to this electrophysiologic abnormality. This information gained recently clinical impact since it can be directly used for faster arrhythmogenic substrate targeting during ablation therapy. (Fig. 5, Ref. 17.) PMID- 7552386 TI - [Adhesion molecules in leukocyte and endothelium interactions]. AB - The subject of this survey are the leukocyte-endothelial cell interactions, which modulate leukocyte migration and vascular permeability. Understanding of the interaction processes play an important role in insights into the mechanisms of vascular injury. Adhesion molecules (ADM) of blood elements and endothelial cells are important in the leukocyte adhesion and transendothelial migration. Adhesion structures are divided into three major categories: selectins, integrins and members of the immunoglobulin superfamily. The bidirectional interactions between leukocytes and endothelial cells are influenced by cytokines. These protein mediators induce changes in the expression of adhesion receptor and may regulate the number and type of leukocytes that migrate from the blood into the inflamed tissues. Von Willebrand factor (vWf) is a suitable marker of endothelial cell injury, as a specific product of the endothelium. The role of vWf in the adhesion process is to form a molecular bridge between the platelet and the subendothelium of an injured vessel wall. ADM that participate in the immune reactions, represent a target of novel therapeutic approaches for the treatment of inflammatory and other diseases. (Fig. 1, Tab. 1, Ref. 43.) PMID- 7552387 TI - [Chemotherapy of chiasmal gliomas in children]. AB - Chiasmal gliomas are rare tumors occurring predominantly in childhood. Their optimal treatment is still controversial. In the past only neurosurgeons (performing partial or subtotal removal of the tumor, biopsy or shunting procedure in hydrocephalus) and radiotherapeutists participated in their treatment. In the middle of the eighties there was only a single article dealing with chemotherapy of these tumors (Rosenstock, 1985). Since that time there was an increased number of articles about harmful effects of radiotherapy on the developing child's brain. Neurosurgeons are aware that they will not solve this problem alone. During the past 7 years we have observed gradual retreat from radiotherapy and an inclination to combined chemotherapy of the chiasmal gliomas in children. The author has been engaged in the research of this clinical entity for more than 10 years and he offers to readers a summary of the contemporary knowledge about chemotherapy of chiasmal gliomas in children. Despite the fact that there is lacking experience with long-term survivors after chemotherapy, which is extremely important especially in this disease, the preliminary short term results of combined chemotherapy of chiasmal gliomas in children are promising. Rapid development of chemistry and pharmacology in the last few years is promising for the discovery of further, more effective anti-tumor drugs. Their new combinations could improve present non-satisfactory results of treatment of chiasmal gliomas in children. (Ref. 25.) PMID- 7552388 TI - [Bacterial virulence factors and their role in the pathogenesis of pyogenic infections of the abdominal cavity and mediastinum]. AB - Purulent peritonitis are caused predominantly by anaerobic bacteria which come from physiological intestinal flora. Mediastinitis is caused amidst other etiologic factors also by bacteria inhabiting the oropharyngeal region, as well as microorganisms causing diseases localized in the proximity of mediastinum. Anaerobic sporulating bacteria both Gram positive and negative cause often miscellaneous infections due to Staphylococcus aureus, Enterobacteriacae, Streptococcus, Enterococcus. It involves a group of bacteria which are able to produce a fibrin network in their proximity, to resist phagocytosis by their structures, to distruct the components of the complement system and immunoglobulins, to impair membranes of cells which leads by means of their factors of virulence to formation of defectuous immunity. Microbiological examination requires the material to be sent for anaerobic cultivation and the antimicrobial therapy must take into account its polymicrobic etiology. (Ref. 23.) PMID- 7552389 TI - [Inflammation, immunity and surgery]. AB - The activation of endothelial and phagocytic cells, with concomitant formation of a range of adhesive molecules and inflammatory mediators, are integral parts of the host response to injurious agents (trauma, infection, altered antigens, toxins, chemicals etc.). The excessive mediator responses are associated with increased morbidity and lethality. Cytokines, soluble mediators secreted by cells, play an integral role in the metabolic and immune responses to the injurious agents. Widespread tissue damage, when associated with fulminant sepsis may induce massive release of cytokines (TNF, IL-1, IL-6), triggering certain steps of reactions involving multiple organs and culminating in the systemic inflammatory response syndrome, sepsis syndrome or multiple organ failure syndrome. (Fig. 2, Ref. 21.) PMID- 7552390 TI - [Pancreatic abscesses]. AB - The paper deals with pancreatic abscess as one of three entities of pancreatic infection. In spite of the common origin with infected pancreatic necrosis and infected pancreatic pseudocyst, the treatment of each kind of pancreatic necrosis has its own requirements. By comparing 30 pancreatic abscesses (mortality 18%), 29 infected pancreatic pseudocysts (mortality 6%) and 35 infected pancreatic necroses (mortality 43.5%) we concluded that the best treatment for pancreatic abscess is surgical evacuation with external drainage, for that of infected pancreatic pseudocyst internal drainage operation, and for infected pancreatic necrosis surgical debridement with external drainage, lavage and open-abdomen procedure. Differences in CT scan, treatment and prognosis require different surgical approach to each type of pancreatic necrosis. (Fig. 3, Ref. 24.) PMID- 7552391 TI - [Suppurative complications in acute pancreatitis]. PMID- 7552392 TI - [Personal experience with management of abscessing pancreatitis]. PMID- 7552393 TI - [Laparostomy in abscessing necrotizing pancreatitis]. PMID- 7552394 TI - [Results of treatment of diffuse peritonitis in data from the surgery department of St. Barbara Hospital in Roznava]. AB - Authors evaluated their own group of 318 patients (5.4% of all patients treated by operation between 1990-1994) 91 patients (28%) had a severe peritonitis. Authors emphasised the need for complete therapy, and an early and appropriate operation. In the conclusion of their study authors emphasised the necessity of evaluation of relevance illness before making a decision in managing peritonitis. (Tab. 2, Fig. 1, Ref. 9.) PMID- 7552395 TI - [Suppurative complications in Crohn's disease]. PMID- 7552396 TI - [Abscessing cholangitis after endoscopic manipulation of the biliary tract]. PMID- 7552397 TI - [Recurrent intra-abdominal abscesses]. PMID- 7552398 TI - [Enteral nutrition by means of puncture jejunostomy in patients with septic complications in organs in the abdominal cavity]. AB - Authors quoted some own results and experience with using pharmacology define medicine of enteral nutrition feeding served by puncheon jejunostoming in case of difficult malnutrition caused especially by septical condition and also after larger operation of throat and stomach. They stated, that enteral feeding apply into the upper part of jejuna with thin cattier is physiological, secure and economical effective method of nutrition support. Combination serving of nutrition with enteral and parenteral feeding they find out hyperalimentary effect at examined group of patients which was confirmed by watching the anthropometrical parameters. PMID- 7552399 TI - [Treatment of patients with acute mediastinitis]. PMID- 7552401 TI - [Esophageal perforation at the thoracoabdominal juncture]. PMID- 7552400 TI - [Mediastinitis--still a dangerous disease]. PMID- 7552402 TI - [Rational therapeutic approach in thoracic empyema]. PMID- 7552403 TI - [Thoracic empyema--still a real diagnosis]. PMID- 7552404 TI - [Post-traumatic pleural empyema]. PMID- 7552405 TI - [We are opening up to the world and the world is opening up to us--the gateway is Internet. Welcome to the 21st century, Mr. Netizen!]. PMID- 7552406 TI - [Non-homogeneity of the average life span in Slovakia]. AB - The life expectancy at birth of males in the Slovak Republic (SR) is one of the shortest in Europe. Exact explanation of this phenomenon is still unknown. The statistical significance of the relation between life expectancy and various factors (infant mortality rate, ethnical structure, educational level, religious belief, economic activity of inhabitants and environmental factors) was analysed by multifactorial methods in all 38 districts of Slovakia. A surprising paradox phenomenon was observed in the areas with the highest pollutant emissions, where the life expectancy was the longest. Infant mortality rate, ethnical structure and particularly the educational level of the districts were significantly correlated with life expectancy. The life expectancy was negatively influenced mostly by the proportion of elementary--educated males. We suppose the style of life presents the background of these relations, but there is a lack of statistically reliable data from the individual districts of SR for more profound analysis. It must be stressed that neither the statistically high significant correlation is evidence of the causal connection between observed parameters. (Tab. 1, Fig. 5, Ref. 16.) PMID- 7552409 TI - [Modern views on pneumococcal infections in infants and toddlers]. AB - Pneumococci colonize the upper respiratory tract predominantly in sucklings and toddlers. By means of their factors of virulence (capsule, ahesines, peptidoglycan, and polysaccharides of the capsule, cytoplasmic membrane enzymes) they can either avoid or impair the immunity mechanisms causing thus severe infections especially in children younger than three years of age and in patients with immunity defects. Regarding the possibility of occurrence of pneumococci with altered susceptibility to penicillin and some other drugs (erythromycin, cotrimaxazol, ceftriaxon, chloramphenicol, tetracycline) it is necessary to treat severe pneumococcal infections on the basis of in vitro detected susceptibility. PMID- 7552407 TI - [Antiphagocytic activity of sera treated with organic solvents (chloroform, ether)]. AB - The opsonization of C. albicans by means of sera treated with chloroform and ethyl ether resulted in significantly decreased values of PMN leucocyte phagocytic activity. In contrast to ethyl ether treated sera (decrease of phagocytic activity about 45%), chloroform processed sera possessed increased antiphagocytic activity (decrease of phagocytic activity about 63%). Significant changes were observed in the candidacidal capacity to C. albicans (decrease about 50%) and in the capacity of NBT (INT) reduction measured spectrophotometrically (decrease about 66% in chloroform treated sera and about 42% in ethyl ether treated sera). (Fig. 1, Ref. 22.) PMID- 7552408 TI - [Hypoglycemia--occurrence, causes and hormonal counterregulatory mechanisms in healthy persons and in patients with IDDM]. AB - The occurrence of hypoglycemia is common in patients with insulin-dependent diabetes mellitus. They experience an average of one to two episodes of symptomatic hypoglycemia per week; 10-20% suffer at least one episode of severe hypoglycemia in a given year. The incidence of severe hypoglycemia in diabetic patients treated with intensive insulin therapy is threefold higher than in patients on conventional insulin therapy and 55% of all episodes occurred in sleep. Prevention of hypoglycemia or the restoring of euglycemia involve the dissipation of metabolic insulin effects and activation of counterregulatory hormone responses (glucagon, catecholamines, growth hormone, cortisol). Among counterregulatory mechanisms there exists hierarchy. The glycemic threshold for release of glucoregulatory hormones in normal people is about 3.8 mmol/l. The glucose counterregulatory system is often impaired in patients with diabetes mellitus with the onset early after diagnosis of diabetes mellitus. Impaired glucagon response is almost universally present after 5 years of diabetes. The defect is selective and the mechanism is not fully explained. Most often it is considered a defect in signal perception of alfa-cells, a rearrangement of islets of Langerhans with predominance of delta-cells or mechanism of central adaptation. After 5-10 years of diabetes glucose counterregulation becomes further impaired with a defect in epinephrine secretion. A possible mechanism is the abnormality at the CNS level. Autonomic neuropathy is a contributory factor. Defective hormonal counterregulation correlates well with duration of diabetes and strict glycemic control. If the glucagon response is intact the hormonal counterregulation is sufficient even in the presence of the impairment of other glucoregulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552411 TI - [Dynamics of hemodynamic parameters in the early neonatal period]. AB - BACKGROUND: The fetal circulation is subdued to alteration after birth. Fetal vascular communications enclose (ductus arteriosus Botalli and foramen ovale) and vascular resistance of the lungs and pressure in the pulmonary artery decrease. Low-resistance placenta is excluded from the systemic circuit, leading thus to abrupt pressure elevation and to its further increase. OBJECTIVES: The aim of the study is to judge the dynamics of alterations during the postnatal period by means of Doppler's echocardiography:--the left ventricular ventricle,--parameters characterizing circulation in the pulmonary bed. METHODS: 23 healthy randomly selected newborn infants born at full term were examined on the 1st and 2nd day of their life. RESULTS: Parameters of the left ventricular function did not change between the 1st and 2nd days of life. Parameters from the outflow tract of the right ventricle on the 5th day of life of infants: the ratio of PEP (preejection period) to AT (acceleration time) significantly decreased, the ratio of PEP to ET(ejection time) did not change. Parameters of the pulmonary artery on the 5th day of life of infants: significant decrease of the ratio PEP/AT, as well as of PEP/ET, the peak and mean velocity of blood flow significantly increased. CONCLUSIONS: The left ventricular function is not subdued to alterations during early postnatal period. The parameters characterizing the pulmonary circulation prove a decrease of pressure in the pulmonary bed on the 5th day of life. MEANING FOR PRACTICE AND THEORY: The results are utilizable in interpretation of echocardiographic examination in postnatal age. (Fig. 8, Ref. 19.). PMID- 7552410 TI - [DNA analysis in the determination of a predisposition for hypertension]. AB - Angiotensinogen gene belongs to the genes designated as hypertension candidate genes. These genes might participate in development of hypertension. The aim of this work was to establish frequency of the mutant allele M235T on angiotensinogen gene in Slovak population and compare this frequency with that obtained from the group of hypertensive patients. We tested DNA from 120 healthy individuals and 20 hypertensive patients. By polymerase chain reaction followed by restriction analysis we determined frequency of mutant allele M235T in healthy population as well as in the group of hypertensive patients. We have found that frequency of the mutant allele in Slovak population was 0.33, while among hypertensive patients 0.45. Percentage of heterozygosity for M235T allele was 44.5%. Frequency of this mutant allele was significantly higher among women compared to men (0.38 vs. 0.27). Increased frequency of M235T allele among hypertensive patients compared to healthy population confirm that M235T mutation is bound to increased blood pressure. This quick and noninvasive method should help in the future to determine the possible risk of hypertension development. (Tab. 1, Fig. 2, Ref. 9.) PMID- 7552412 TI - [Serum selenium levels in children and adolescents in selected regions in Slovakia]. AB - BACKGROUND: There is a limited number of data concerning blood serum selenium levels in children and adolescents in the Slovak Republic. OBJECTIVES: The aim of this study was to access blood serum selenium levels in 102 healthy children (11 14 y) and 122 adolescents (15-18 y) from two regions: The district of Cadca and Modra--Pezinok area. METHODS: Serum selenium was determined by electrothermal atomic absorption spectrophotometric method using Pd as a matrix modifier. RESULTS: The mean (+/- SD) serum selenium concentration was 0.644 +/- 0.154 mumol/l in children and 0.730 +/- 0.175 mumol/l in adolescents. A large proportion of the individuals (28.43% in the children group, vs 14.05% in adolescent group) exhibited serum selenium levels below 0.57 mumol/l (45 micrograms/l). A significant correlation between serum selenium concentration and age (p < 0.001) was found. The correlation between serum selenium concentration and sex was not significant. CONCLUSIONS: In comparison with serum selenium levels in children and adolescents from other countries, the concentration of serum selenium in Slovak children and adolescents is relatively low. This differences could probably be attributed to the selenium content in the soil and to the lower average daily selenium intake via food. (Tab. 5, Fig. 2, Ref. 32). PMID- 7552413 TI - [Grawitz tumor in a female patient with minor manifestations of herpes zoster detected by an active dermatologic examination]. PMID- 7552415 TI - [Monocytoid B-lymphocyte lymphoma: a new type within the spectrum of non Hodgkin's B-cell lymphoma]. AB - BACKGROUND: Clonal proliferation of monocytoid B-lymphocytes (MBLy)--monocytoid B cell lymphoma (MBCL) represents a "new" type of lymphoma within the spectrum of B cell malignancies. OBJECTIVES: The aim of the study was to evaluate the possibilities of a routine histological and immunohistochemical diagnosis of MBCL. METHODS: Three cases of MBCL diagnosed in peripheral lymph nodes (n = 2) and in mammary gland with infiltration of regional lymph node (n = 1) were analyzed both histologically and immunohistochemically using a panel approach (Ig chains, CD30 antigen, markers of B-cells, T-cells and of monocytes/histiocytes). RESULTS: Morphological appearance of neoplastic cells of MBCL is identical to that of MBLy in reactive conditions--kidney-shaped nuclei, bright clear PAS negative cytoplasm, and small inconspicuous nucleoli. CONCLUSIONS: Morphological appearance together with immunophenotypic results (positivity of CD20 and Ki-B5, negativity of CD3, CD43, CD45RO, and of lysozyme, negativity of CD30) are considered to represent sufficient diagnostic criteria of MBCL, including its differential diagnosis of other B-cell low grade malignancies. An increase of large cell type MBLy might represent a feature of a secondary blastic transformation of MBCL. (Tab. 2, Fig. 5, Ref. 27.) PMID- 7552414 TI - Immunohistochemical markers in uterine tumour diagnostics. AB - The recent fast growth of data on new markers and their identification in different cellular structures results in the need of narrow selection with regard to their applicability. The aim of the review is description of application possibilities of immunohistochemical methods in the diagnostics of pathological processes in the cervix and body of uterus. (Fig. 5, Ref. 43.) PMID- 7552416 TI - [Atelocollagen/hydroxylapatite composite material as bone defect filler in an experiment on rats]. AB - Many types of biomaterials are used as skeletal bone fillers in reconstructive surgery. An attention is paid to hydroxyapatite due to its high biocompatibility with the surrounding tissue. The paper deals with the testing of new collagen/hydroxyapatite composite material applied to the bone defect on os parietale of rats. The composite material was prepared from the bovine atelocollagen dispersion and the dispersion of hydroxyapatite. Collagen serves as a matrix in which the particles of hydroxyapatite are anchored. The composite presents the advantage that after the saturation with physiological solution it is compact and can be shaped. The composite material was implanted in the form of plate into six male Wistar rats to the ground bed on the surface of os parietale. The implants were taken out after four moths. The macroscopic finding of soft tissue, bones and implants gave evidence about good healing without any undesirable reaction. This was also confirmed by the histological observations. Collagen was resorbed and the rest of the material strongly adhered to the bone. The marked osteocytes were present in the zone of the newly formed bone and the dividing line between new and old bone was clear. The experimental results give preconditions to the clinical use of this new composite implant material the structural improvement of which is in progress. (Fig. 2, Ref. 12.) PMID- 7552417 TI - [Histochemistry of a new Tritrichomonas mobilensis lectin with sialic acid specificity]. AB - BACKGROUND: Histochemistry with lectins highly specific for one sugar represent a sensitive and accurate method for the localization of glycoproteins in tissues. OBJECTIVES: The paper evaluates the possibilities of histochemical applications of the novel lectin from Tritrichomonas mobilensis (TML) with specificity for sialic acid. METHODS: TML purified by affinity chromatography was used for anti TML monoclonal antibody production and tested with the use of avidin-biotin peroxidase technique. RESULTS: In human biopsy material from different organs and tissues, TML showed strong membrane reactivity with vascular endothelium with the exception of glomerular and pulmonary capillaries and sinuses in the spleen and the bone marrow. Luminal membrane positivity dominated in glandular epithelia, especially in the lining of ducts. In squamous epithelia, the membrane positivity was present in cells of the basal layer, in transitional epithelium also in umbrella cells. Mucus in respiratory tract was positive, in gastrointestinal tract the positivity was irregular. Nerve tissue and endocrine glands were mostly negative. CONCLUSIONS: The lectin from tritrichomonas mobilensis can be considered a perspective reagent for identification of sialylated glycosubstances based on its high specificity for one sugar--the sialic acid and excellent results in histochemistry. (Fig. 7, Ref. 29.) PMID- 7552418 TI - [Pathologic findings in the uterine cervix--diagnostic aspects]. AB - The study presents results of routine bioptic diagnoses. 651 lesions of cervix uteri have been diagnosed during a five-year period. The presented number represented a summary of 26% of the absolute number of 2513 registered neoplastic and precancerous lesions of the female genital. The group of patients yielded dominance of precancerous lesions of exo and endocervix, namely in 445 cases, i.e. 68%. Malign tumours of exo and endocervix uteri constituted a group of 185 cases, i.e. 28%. The study proves the world's trends of the development of pathological changes in cervix uteri where HPV infection represents one of the most significant factors. The two-year lasting evaluation of 27,093 cytologically examined patients revealed HPV infection in 748 female patients, i.e. in 2.8%. Summarization of the findings detected on the basis of cytologic and bioptic examinations proved the histologic correlation of HPV infection in 124 patients out of 467 patients afflicted with dysplastic or malign lesion of exocervix. (Tab. 1, Fig. 3, Ref. 13.) PMID- 7552419 TI - Serum autoantibodies in recurrent parotitis in children. AB - BACKGROUND: Recurrent parotitis in children is a relatively frequent disease, the pathogenesis of which has not been cleared yet. OBJECTIVES: The possibility of autoimmune mechanisms participating in the disease development was evaluated. METHODS: 20 sera samples from patients with recurrent parotitis in children were examined. The presence of autoantibodies was tested on cryostat sections of the parotid gland tissue using direct immunofluorescence. RESULTS: In 17 sera the presence of autoantibodies reacting with the cytoplasm of acinar cells and in 8 antibodies reacting with the cytoplasm of ductal cells were found. Mostly they were of IgM class, less frequently the IgG type. Anti-nuclear antibodies were not identified. CONCLUSIONS: With regard to the clinical finding of isolated and mostly unilateral affliction of the parotid gland, the found autoantibodies cannot be considered pathognomic, causing an autoimmune disease. It is assumed that these autoantibodies are produced in response to the primary damage of the salivary gland, the cause of which has not been fully explained so far. (Tab. 1, Fig. 4, Ref. 14.). PMID- 7552420 TI - [Deep soft-tissue leiomyoma]. AB - Deep soft tissue leiomyomas (DSTL) are very rare. For this reason they are neither histologically, nor clinically reliably characterized, especially regarding their dignity. The authors present two cases of leiomyomas which grew from the deep soft tissue in the region of the head and perineum. Both cases involved women 43 and 63 years of age. Characteristically morphological traits, as e.g. good borderline of lesion, the presence of fibrous pseudocapsule, and classical morphologic appearance of leiomyoma were present in both cases. While in the first case the foci of dystrophic calcification within regressively altered foci of the tumour were found, the second case involved the presence of foci of myxohyalin degeneration of stroma. The first tumour contained also parts with a palisade arrangement of nuclei which gives a hint of resemblance to that of neurilemmoma. Mitoses did not exceed the HPF index of 2/10 in neither of the cases. Both cases were S-100 immunohistochemically negative, actin and human muscle actin were significantly positive, desmin was weakly or mediately positive, and vimentin significantly or mediately positive. The first case involved a clinical recurrence of a histologically verified leiomyoma after 5 years in the same location with MI being of zero value. Regarding the presented characteristics, the authors classify the described leiomyomas as DSTL. These tumours with an uncertain prognosis can metastatize despite their benign morphology. (Tab. 2, Fig. 4, Ref. 19.) PMID- 7552421 TI - [Cervical spondylotic myelopathy]. AB - Cervical spondylotic myelopathy, the most frequent type of spinal cord's function impairment in patients over 55 years of age, is caused by mechanical and dynamical changes in the degenerating lower cervical segment of the spine leading to the reduction of the perimedullar space, disturbing thus the blood supply or spinal cord per se. The clinical picture of neurologic changes and their development is very variable. The suspicion of CSM is supported by the finding of the stenosis of the spinal canal to 11 mm, or the decrease in Pavlov's index below 0.8. The diagnosis is based dominantly on CT myelographic examination and MRI. Operative solution has a favourable prognosis providing that the irreversible changes in the spinal cord are absent. (Tab. 1, Fig. 7, Ref. 12.) PMID- 7552422 TI - Review of forty years of rehabilitation issues in spinal cord injury. PMID- 7552423 TI - Late-life spinal cord injury and aging with a long term injury: characteristics of two emerging populations. AB - Modern care of patients with spinal cord injury is leading to greater numbers of individuals surviving into old age and the emergence of a cohort that has sustained injury at an advanced age. The clinical characteristics of either group of patients has not been well characterized. Analyses from the Aging with a Long Term Disability Research Program database, which is enriched by the presence of a high quality Spinal Cord Injury Service, revealed a population of 510 recently assessed individuals with a mean age of 50 years, ranging from 16 to 84 years. Twenty-three percent of the patients were at least 65 years of age. Spinal cord injuries were usually the result of automobile accidents in individuals injured younger than 50 years of age and falls in individuals injured when older than 50 years of age. Patients surviving late life injury are much more likely to have incomplete injuries predominantly affecting the cervical spine. A number of conditions were found to be more prevalent in older patients. These included carpal tunnel syndrome, chronic obstructive pulmonary disease, myocardial infarction, diabetes, kidney stones, pressure ulcers and hypertension. The development of diabetes, kidney stones and perhaps pressure ulcers was directly related to aging with SCI, but not just to aging alone. The better functional outcomes in late life spinal cord injury may be secondary to selective survival. The excess morbidity associated with late life spinal cord injury has significance for future planning of healthcare needs for the spinal cord injured patient. PMID- 7552424 TI - An unusual location for heterotopic ossification: lumbar anterior longitudinal ligament. AB - Heterotopic ossification (HO) is a well known complication of spinal cord injury. It usually affects the hips and knees, with less common involvement of the shoulders and elbows. We present a patient with incomplete tetraplegia who developed HO in the left hip and in the plane of the lumbar anterior longitudinal ligament from L3 to L5. During evaluation for the patient's complaints of low back pain, a fracture was noted in the HO which was confirmed by three dimensional CT scan. We postulate that this fracture contributed to his symptoms. Review of the literature on HO in spinal cord injured patients indicates this to be an unusual location for HO. Although HO of the lumbar anterior longitudinal ligament is a rare occurrence, this possibility should be considered in the evaluation of back pain in spinal cord injured patients. PMID- 7552425 TI - Diazepam and body weight in myelopathy patients. AB - Because diazepam appeared to affect body weight, spastic myelopathy patients for whom this drug had been prescribed but in whom the dose was altered were compared with similar patients, without changes in antispastic medication. A retrospective survey averaging 10 months was conducted for these two groups to determine weight changes of 10 pounds or more. After reduction or discontinuation of diazepam in seven patients, all lost weight -12 to 35 lbs at rates of 0.9 to 3.5 lb per month. On unchanged medication, only one of twelve patients lost as much as 10 lb, p < 0.001. After partially or fully restoring diazepam in four patients, all gained weight -7 to 26 lbs-at rates of 1.8 to 4.3 lbs per month. Three of the four patients and two of the 12 without medication change gained as much as 10 pounds, p = 0.03. We conclude that body weight in myelopathy patients is affected by the use of diazepam. The effect of other medications used for spasms was not assessed. PMID- 7552426 TI - Pulmonary blastoma presenting as a solitary lip metastasis: case report and review of the literature. AB - Pulmonary blastoma (PB) is an uncommon primary lung malignancy. This neoplasm was first described by Barrett and Barnard in 1945. The tumor is composed of immature epithelial and mesenchymal tissues which may recapitulate early embryological lung development. Under the microscope, the globular component resembles immature bronchus and connective tissue as seen in embryonic lung. More than one hundred cases have been reported in the literature. PB is more frequent in older people and in males and tends to affect blacks at younger ages. Symptomatology varies from asymptomatic to symptoms of a non-specific pulmonary disease. Cough, hemoptysis, dyspnea, chest pain, respiratory distress, fever, anorexia and weight loss are the most common presenting features. The most common roentgenologic pattern is a well-demarcated peripheral lesion, encapsulated by compression or atelectatic lung tissue, although in some cases there is a tendency to lobulation and cavitation. The size of the mass varies from a small peripheral nodule to a mass occupying the entire lobe or hemithorax. The treatment of choice has been surgical excision, radiation and, in selected cases, a combination of chemotherapy with radiation. The prognosis of this malignancy is poor; overall five-year survival is approximately 16 percent. No correlation has been established between histopathologic criteria and survival. The factors that indicate poor prognosis are tumor recurrence, metastasis at initial presentation, tumor size over 5 cm and lymph node metastasis. Liver, central nervous system and bones are the most frequent location of distant metastases. A rare case is presented of a pulmonary blastoma with an upper lip metastasis occurring in a paraplegic male. Diagnosis was confirmed by autopsy findings.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552427 TI - Auditory P3 event related potentials (ERP) and brainstem auditory evoked responses (BAER) after spinal cord injury in humans. AB - Previous studies have observed altered somatotopic sensory fields after experimental deafferentation in animals as well as enhanced somatosensory evoked potentials and altered cortical motor pathways following spinal cord injury (SCI) in humans. These observations indicate that cortical reorganization may occur subsequent to SCI. In earlier work, we have observed attenuated amplitudes for both tactile P3 and auditory N1/P2 orienting event-related potentials (ERP) in spinal cord injured groups. These results suggest that the reorganization process may have functional perceptual and cognitive consequences. In an effort to determine if deafferentation affects the P3 ERP using stimuli other than somatosensory, we measured brain activity from central recording sites during an auditory "oddball" task. Additionally, we obtained brainstem auditory evoked responses (BAER) in order to assess subcortical primary auditory pathways as well. Results show that the SCI groups produced significantly attenuated N1/P2 complexes and P3 when compared to controls. Also, the quadriplegic group exhibited increased latencies of the P3 at frontal and central sites. There were no differences between groups in BAER results. These findings suggest that primary subcortical auditory information processing stages remain intact after SCI although later stages may be significantly altered. PMID- 7552428 TI - Assessing the behaviour of the newborn infant. PMID- 7552429 TI - Parents in recovery: parental and staff attitudes. PMID- 7552430 TI - Conflict in decision making: do nurses have a role? PMID- 7552431 TI - Disability within the family unit. PMID- 7552432 TI - Meningitis in childhood. PMID- 7552433 TI - Who wears the stethoscope? PMID- 7552434 TI - At the cutting edge of care.... PMID- 7552436 TI - No turning back. PMID- 7552435 TI - Fit for the future. PMID- 7552437 TI - Managing multiple births. Part 1: The quest for parenthood. PMID- 7552438 TI - Down's syndrome: a positive approach. PMID- 7552439 TI - Parent education for Asian mothers. PMID- 7552440 TI - Bridging the gap between purchaser and provider. 2: The future of maternity care. PMID- 7552441 TI - Wales shows the way. PMID- 7552442 TI - A new Midwives' Act: who will really benefit? PMID- 7552443 TI - Critically ill babies: life and death issues. PMID- 7552444 TI - Dilemmas in practice: the pregnant drug user. PMID- 7552445 TI - United, knowledgeable, communicating, collaborating. PMID- 7552446 TI - Engaging practicing nurses in the quality-improvement process: a dual approach. PMID- 7552447 TI - Standard cost accounting. PMID- 7552448 TI - Quality is a journey more than a destination. PMID- 7552449 TI - Quality: the role of the nurse manager. PMID- 7552450 TI - Creating an environment to support hardiness and quality patient care. AB - Patient care environments play a significant role in the nurses' ability to provide quality patient care and receive personal satisfaction through caregiving work. Hardiness is defined in terms of the presence of three characteristics: control, or the belief that individuals can control or influence occurrences in their lives; commitment, or an ability to feel involved in the activities of their lives; and challenge, or the anticipation of change as an exciting challenge to further development. These characteristics can be cultivated through the environment. Leadership actions and behaviors influence environmental development to stimulate and cultivate the characteristics of hardiness. PMID- 7552451 TI - Making a transition from quality assurance to quality improvement. AB - Health care leaders now recognize the importance of adding continuous quality improvement activities and the measurement of clinical outcomes to their longstanding quality programs. From other industry leaders, they have learned that there is great promise on quality/cost returns when quality programs focus on measuring performance outcomes rather than just using the longstanding process of measuring variation in staff and system capacity processes. The purpose of this article is to describe how one urban acute care hospital is taking steps to change its quality paradigm and therefore change its quality program, which until recently consisted largely of unrelated quality assurance activities and projects. The new system will be a truly coordinated hospital wide continuous quality improvement program that will align quality activities with the mission of the organization and focus on the measurement of outcomes. PMID- 7552452 TI - The primacy of the patient and family in a quality-improvement environment. AB - The primary customers of health care services are the patient and family. It is important to adopt a mission and philosophy that put the patient and family at the center of all quality improvement programs. The principles put forth by Deming in his 14 points can be applied to patient-focused quality improvement measures. Creating a foundation for the professional practice of nursing and using and expanding tools that are already in use can help care providers meet the needs of their customers and help people to live healthier, better lives. PMID- 7552453 TI - Quality improvement in the emergency department. AB - Health care institutions across the country are recognizing that their success is dependent on satisfied patients. The Emergency Department in this community teaching hospital dramatically improved their patient satisfaction rates with a customer-focused program. The effort was based on defining the patient's perception, developing a multidisciplinary team to address the issue, and implementing a plan in which every employee was a part of the solution. PMID- 7552455 TI - Quality improvement with a culturally diverse staff: implications for nurse managers. AB - Cultural diversity is a noticeable reality in today's work force. The problems resulting from cultural diversity pose special challenges to nurse managers. If nurse managers are to be successful at managing diversity, they need to not only be aware of the specific issues created by diversity, but they must plan effective management and leadership strategies to enable staff to collectively meet the desired goals of the work setting. The authors of this article are employed in one of the most culturally diverse work settings in the world. Their experiences in successfully implementing a quality improvement program, and in using strategies to gain shared vision and commitment by management and staff, will be explored and the implications for nurse managers outlined. PMID- 7552454 TI - High quality and lower cost: they can coexist! AB - The need to control costs must be balanced with consumers' demands for high quality in their health care. An integrated approach that uses a multidisciplinary team, a case manager, and a clinical path to improve the quality of patient care offers one method by which both goals may be achieved. The report details the results of a case study in which these three elements were applied to the care of patients undergoing total hip replacement surgery. Results included a length of patients' stay of 3.92 days, cost reduction of $667 per patient in direct costs, no reported cases of readmission for complications during the 2 year process, reductions in length of stay for patients referred to extended care facilities, and a shortened period in home health services. These results are attributed to extensive use of prior screening for all patients by multiple care disciplines, better patient education processes before admission and surgery, improved communication between team members on patient progress, and coordination of care across the continuum. PMID- 7552456 TI - Creating a culture of quality in home health care. AB - The unique environment of home care requires integrated strategies on both the organizational and staff levels to foster a quality culture. This article lists the many strategies that a home care agency can use effectively to achieve that goal. PMID- 7552457 TI - Creating the environment necessary to ensure quality patient care in the community setting. AB - Quality patient care in the community setting can be challenging to community based practice nurses. Using the combined components of structure, process, and outcome can ensure the delivery of quality patient care. This article presents a framework using the broader environment as well as the unique contributions of nurses to address the issue of quality in the community setting. PMID- 7552458 TI - The patient's perceptions of quality: implications for nurse managers in long term care. AB - Nurse managers are in key positions to promote quality care in their institutions. In long-term care, the patient's perspective of quality has important implications. Knowledge of these perceptions can provide a basis for the development of educational, administrative, and practice modalities that impact positively on the quality of care and the quality of life for these patients. A move from quality assurance to quality improvement programs helps to focus energies, improve processes, and assist nurse managers in setting standards that are realistic, effective, and efficient. PMID- 7552460 TI - Evaluation of current perception threshold testing as a screening procedure for carpal tunnel syndrome. PMID- 7552459 TI - Color vision: replication, rather than nonreplication of findings. PMID- 7552461 TI - Current perception threshold mis-evaluation. PMID- 7552462 TI - Pharmacologic mechanism of antidotes in cyanide and nitrile poisoning. PMID- 7552464 TI - Environmental health in medical school curricula: views of academic deans. AB - Academic deans at 126 US medical schools were surveyed in Spring 1994. Comparisons of means and frequencies, multiple regression, and factor analysis were used. Study results showed only low to moderate expectations for graduate competence in seven environmental health competency areas. Over two-thirds of deans (70%) indicated that there was "minimal" emphasis on environmental health at their schools; 61% thought that ideally there should be "moderate" emphasis. An "already crowded curriculum" and "too few qualified faculty" were frequently cited as barriers to greater emphasis on environmental health. Students were identified most commonly as the group expressing the greatest support for environmental health education. Although there was not a clear consensus, occupational medicine departments were most often selected as best suited to offer environmental medicine education. PMID- 7552463 TI - Cancer incidence among Finnish workers exposed to halogenated hydrocarbons. AB - Epidemiologic studies and long-term carcinogenicity studies in experimental animals suggest that some halogenated hydrocarbons are carcinogenic. To investigate whether exposure to trichloroethylene, tetrachloroethylene, or 1,1,1 trichloroethane increases carcinogenic risk, a cohort of 2050 male and 1924 female workers monitored for occupational exposure to these agents was followed up for cancer incidence in 1967 to 1992. The overall cancer incidence within the cohort was similar to that of the Finnish population. There was an excess of cancers of the cervix uteri and lymphohematopoietic tissues, however. Excess of pancreatic cancer and non-Hodgkin lymphoma was seen after 10 years from the first personal measurement. Among those exposed to trichloroethylene, the overall cancer incidence was increased for a follow-up period of more than 20 years. There was an excess of cancers of the stomach, liver, prostate, and lymphohematopoietic tissues combined. Workers exposed to 1,1,1-trichloroethane had increased risk of multiple myeloma and cancer of the nervous system. The study provides support to the hypothesis that trichloroethylene and other halogenated hydrocarbons are carcinogenic for the liver and lymphohematopoietic tissues, especially for non-Hodgkin lymphoma. The study also documents excess of cancers of the stomach, pancreas, cervix uteri, prostate, and the nervous system among workers exposed to solvents. PMID- 7552465 TI - Mortality following cotton defoliation: San Joaquin Valley, California, 1970 1990. AB - A proportional mortality study comparing the cotton-growing areas of the San Joaquin Valley with the rest of the State of California was performed by the Office of Environmental Health Hazard Assessment as a continuation of earlier studies related to mercaptan-releasing pesticides. This mortality study found a pattern of increased proportion of "respiratory causes" mortality (ICD codes 460 519), statistically significant at less than the .05 probability level, for 15 of 21 years between 1970 and 1990, for the time period during and immediately following cotton defoliation. Defoliants which have the potential to produce acute symptoms include DEF and Folex, both of which release odorous butyl mercaptan gas as a degradation product. This paper tests the hypothesis that exposure to cotton defoliant breakdown products may be associated with a disproportionate increase in mortality. Prediction of the mortality proportions by pounds of DEF and Folex used was not statistically significant in the unadjusted models or in models adjusted for air pollution variables. One air pollution adjustment factor, total suspended particulates, was a statistically significant independent mortality proportion predictor. This finding suggests that total suspended particulates, not defoliants, may be related to mortality differentials during defoliation season. Possible confounding by demographic variation of the counties was not controlled in the analysis. PMID- 7552467 TI - Mortality of iron foundry workers: IV. Analysis of a subcohort exposed to formaldehyde. AB - In the final phase of the mortality study of workers at an automotive iron foundry, a subset (N = 3929) of the original cohort of 8147 men, consisting of those exposed to formaldehyde during the period from January 1960 through May 1987, was analyzed. In addition to the external US population, an internal population (N = 2032), consisting of men who had worked in the same foundry during the same time period but not in formaldehyde-exposed jobs, was also used as a referent. Follow-up continued through December 31, 1989. Smoking status was ascertained for 65.4% of the exposed and for 55.1% of the unexposed cohorts. Detailed work histories and evaluation of occupational exposures by an industrial hygienist enabled us to categorize cumulative formaldehyde and silica exposures. Standardized mortality ratios were used to compare the mortality experience of the exposed cohort with the US population and, because of concerns about the healthy worker effect, with an occupational referent population. Relative risks for race, formaldehyde exposure status, smoking status, and silica exposure level were estimated by fitting a Poisson regression model to four causes of death: cancers of the buccal cavity and pharynx, lung cancer, diseases of the respiratory system, and emphysema. No association between formaldehyde exposure and deaths from malignant or nonmalignant diseases of the respiratory system was found. Cigarette smoking and silica exposure were found to be significantly associated with deaths attributed to lung cancer and disease of the respiratory system. PMID- 7552466 TI - Study of employees with anhydride-induced respiratory disease after removal from exposure. AB - The purpose of this study was to determine clinical and immunologic status of hexahydrophthalic anhydride (HHPA) employees who have had immunologic respiratory disease and who have been removed from exposure for at least 1 year. In a retrospective study, 16 consecutive employees with HHPA-induced immunologic respiratory disease who had been removed from exposure for more than 1 year were evaluated. Eleven had asthma, allergic rhinitis, or both; five had hemorrhagic rhinitis. Respiratory symptoms were obtained by physician-administered questionnaire. Physical examination, spirometry, and chest film were obtained. Antibody against HHPA conjugated to human serum albumin (HHP-HSA) was determined by enzyme-linked immunosorbant assay. Symptoms, signs, and pulmonary functions were normalized in all employees. There was a decline in antibody titers for both IgE and IgG against HHP-HSA. There were no chest film findings attributable to HHPA. In this group, there appeared to be no evidence of permanent anatomic sequelae after removal from exposure for at least 1 year. Specific antibody was still present, but titers were lower at follow-up than at presentation for a substantial proportion of the sample. PMID- 7552468 TI - A nonconventional approach to the treatment of "environmental illness". AB - Twenty patients with symptoms of "environmental illness" were subject to a controlled study of deep versus superficial acupuncture. The patients were evaluated by a detailed questionnaire concerning their occupational, environmental, and medical history. Blood samples were taken as well. Patients were randomized to deep or superficial acupuncture. Both groups improved significantly in key variables during and after treatment. There were no group differences. There were no changes in biological variables apart from a gradual and continuous increase in serum cortisol and a decrease in neuropeptide Y, which was somewhat more accentuated in those receiving deep acupuncture. This rise in cortisol may have contributed to decreased dermal symptoms among the participants. It is hypothesized that the positive treatment results observed are partly due to weakening of the conditioned response, linking bodily symptoms to environmental agents. To date, a number of different methods have been tried in the management of patients with environmental illness. However, only rarely have the treatments been evaluated in controlled studies. PMID- 7552469 TI - Needlesticks in medical students in university hospitals. AB - The incidence of needlesticks and the effect of experience and other personal characteristics on the risk of needlesticks in medical students are unknown. Eighty-nine medical students were given a self-administered questionnaire. The questionnaire was completed by 86 students, and there were 91 sticks in 43 students. The incidence of needlesticks was 5.8/1000 procedures on the first rotation and 0.1/1000 during the second 4-month period (relative risk, 6.5, with 95% confidence interval (CI) of 3.4-125; P < 0.001). Personal characteristics were not associated with needlesticks except for tension felt while drawing blood and accident proneness. Those who were stuck during the first rotation were more likely to be stuck subsequently (odds ratio, 9.0, with 95% CI of 1-422; P < 0.05). We conclude that experience decreases the risk for needlesticks and therefore effective instructional intervention may have the biggest impact during the first medical student ward experience. Emphasis may have to be placed on those students who are accident prone or have been stuck in the past. Further studies are warranted to substantiate our findings and to test the effectiveness of various interventional approaches. PMID- 7552470 TI - Bronchiolitis obliterans from exposure to incinerator fly ash. AB - Inhalation of toxic substances in the workplace can result in a variety of respiratory disorders. One relatively rare sequela of the inhalation of toxic fumes is bronchiolitis obliterans, a condition characterized by fibrosis and narrowing of the small airways. Several substances have been reported to cause bronchiolitis obliterans, including ammonia, chlorine, hydrogen fluoride, hydrogen sulfide, nitrogen dioxide, ozone, phosgene, and other irritant fumes. Little has been reported on the pulmonary effects of fly ash produced by the incineration of coal and oil. We report a case of bronchiolitis obliterans with a component of partially reversible airway obstruction in a 39-year-old male occupationally exposed to incinerator fly ash. PMID- 7552471 TI - High-density lipoprotein-cholesterol: determining hygienic factors for intervention. AB - Current guidelines of the Adult Treatment Panel on High-Density Lipoprotein Cholesterol (HDL-C) emphasize the protective effect of HDL-C in reducing one's risk for coronary heart disease and recommend that individuals with serum HDL-C levels below 35 mg/dL utilize hygienic means to raise them. A cross-sectional study was performed to examine the relationship of the hygienic factors obesity (measured by percent body fat and body mass index), smoking, and aerobic exercise to HDL-C. The sample, consisting of 1701 male employees of a large aerospace hardware assembly plant, were evaluated by health risk appraisal and anthropometric measurement. Regression analysis revealed a significant negative relationship between body mass index, percent body fat, age, smoking and the level of HDL-C in the blood. Alcohol consumption was directly related to HDL-C, and Whites had a lower HDL-C than all other races combined. Aerobic exercise was not found to be significantly related to HDL-C. A model (multiple R2 = .1136) consisting of age, race, alcohol consumption, smoking, and body mass index fit the data well. These findings justify weight management and smoking cessation interventions for raising HDL-C. However, the role of aerobic exercise was not supported in this study as a means of raising HDL-C. Future studies should use maximum oxygen consumption as a measure of aerobic capacity, which may be a better indicator of aerobic exercise level. The role of medication and genetic and dietary factors in HDL-C management should also be explored. Although findings from this study support smoking cessation and weight management interventions, longitudinal research is needed to determine the most effective strategy for HDL-C management. PMID- 7552472 TI - Correlation of measurements of pressure perception using the pressure-specified sensory device with electrodiagnostic testing. AB - Quantitative sensory testing of pressure threshold has been recommended for diagnosis and monitoring of peripheral nerve problems, yet there has been no validation of the results of such testing with electrodiagnostic testing (EDT), the "gold standard." The Pressure-Specified Sensory Device (PSSD) was used to measure the pressure threshold in 72 clinical nerve entrapment syndromes (23 carpal, 23 cubital, and 16 tarsal tunnel syndromes, and 10 common peroneal nerve entrapment at the fibular head), each of which also had EDT. There was diagnostic agreement between both EDT and PSSD in 54 of the 72 nerve entrapments (75%). The sensitivity of the PSSD was 100% for each of the four nerve entrapments. In those patients in whom there was a disagreement, the PSSD was abnormal when the EDT was normal. In conclusion, quantitative sensory testing with the PSSD has a high sensitivity, but a low specificity, when compared with EDT for diagnosis of peripheral nerve entrapment. PMID- 7552473 TI - Increase in percentage of CD45RO+/CD8+ cells is associated with previous severe primary HIV infection. AB - The purpose of the study was to examine how memory (CD45RO) and naive (CD45RA) phenotypes of CD4+ and CD8+ T-cell subpopulations changed with respect to progression and duration of human immunodeficiency virus (HIV) infection. Forty three HIV-seropositive (HIV+) subjects with known time for seroconversion were included in this cross-sectional study. They were divided into the following groups for comparison: persons with and without AIDS, persons who had seroconverted > 72 and < 72 months before entering the study, persons with or without previous severe primary infection, persons who had developed AIDS > 72 and <72 months before entering the study. Furthermore, the HIV+ group was compared with an HIV-seronegative (HIV-) age- and sex-matched group. There was no difference in the proportion of total naive relative to total memory cells between HIV+ and HIV- subjects, showing an equal loss of naive and memory CD4+ cells in this study. Moreover, there was no difference in the proportion of total naive relative to memory CD8+ cells, showing an equal increase in both subgroups of CD8+ cells in HIV+ subjects. However, HIV+ subjects who had experienced severe primary symptoms resembled the AIDS group regarding shift in the CD8 phenotype from naive to memory and by down-regulation of amounts of CD45RA protein. Furthermore, the results showed that during infection with HIV the amounts of both CD45RA and CD45RO markers on CD4+ cells and CD45RA on CD8+ cells were down regulated, although with different kinetics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552474 TI - Positive correlation between the natural killer and gp 120/41-specific antibody dependent cellular cytotoxic effector functions in HIV-infected individuals. AB - Using our recently developed target system model of env gene-transfected cells for gp120/41-specific antibody-dependent cellular cytotoxicity (ADCC) assays, we evaluated the ADCC effector function of the peripheral blood mononuclear cells (PBMC) in 39 human immunodeficiency virus type 1 (HIV-1)-infected individuals. The natural killer (NK) activity of the PBMC from these individuals against K562 was also determined. A significant positive correlation (p < or = 0.05, r = 0.37) was found between these two activities. On the basis of our data, we built a regression equation for the two activities whereby the ADCC effector function of PBMC of an HIV-infected individual can be predicted from their NK activity. We also found that the endpoint titer of a given serum for ADCC-mediating antibodies in the ADCC assay varied depending on the NK activity of the effector cells used: The effectors with high NK activity tended to give higher titers as compared with the effectors with low NK activity. To our knowledge, this is the first formal documentation of the existence of a positive correlation between the ADCC and NK activities of the PBMC. It also shows that the ADCC titer of serum in ADCC assay may vary depending on the NK activity of the effector cells used. This observation underscores an important variable of ADCC assays. Using our statistical model, one can predict the ADCC effector function of the PBMC from HIV-infected individuals from their NK activity and thus help clarify the role of this important immune response in HIV infection and AIDS prognosis. PMID- 7552475 TI - Effect of a retroviral immunodeficiency syndrome on resistance to MCMV: a role for natural killer cells. AB - Mice exhibiting T cell immunodeficiency and lymphadenopathy induced by LP-BM5 MuLV (MAIDS) were assessed for their ability to control murine cytomegalovirus (MCMV) infection. Elevated replication of MCMV was observed in spleens 4-6 days after MCMV infection. Production of interferon alpha/beta in response to MCMV was abrogated by MAIDS and natural killer (NK) cell activity, assessed by lysis of YAC-1 target cells, was correspondingly depressed on Day 1. However, this was elevated at later times. As previous studies attribute early control of MCMV in C57BL mice with splenic NK1.1 cells, we confirmed that YAC-1 lysis declines and MCMV titres are elevated in M- mice given antibodies to NK1.1 or asialo GM1. However, M+ mice were less severely affected by these regimes. Hence M+ mice may acquire cells that do not express NK1.1 or asialo GM1, but are able to lyse YAC-1 target cells and partially control MCMV replication. MCMV was essentially cleared from the spleens and livers of M+ and M- mice by Day 11, but in the salivary gland MCMV replication was enhanced by MAIDS 11-30 days postinfection. The role of CD4 T cells in this phenomenon is being investigated. Since patients with acquired immunodeficiency syndrome exhibit suppressed T and NK cell activity and experience severe cytomegalovirus disease, the model warrants further study. PMID- 7552476 TI - Effects of viral virulence on intrauterine growth in SIV-infected fetal rhesus macaques (Macaca mulatta). AB - Studies with a simian immunodeficiency virus (SIV)-infected fetal monkey model were conducted with a focus on fetal growth and viral pathogenesis. Twenty-six fetuses were inoculated in utero via ultrasound guidance with an uncloned pathogenic strain of SIV or vehicle during the second or third trimesters [gestational day (GD) 65, 110, or 130], sonographically monitored weekly (biometrics, blood flow), then necropsied at incremental time points postinfection. Peripheral blood hematologic (complete blood counts, clinical chemistries), immunologic (immunophenotyping), and endocrine studies [insulin like growth factor (IGF), IGF-binding proteins (IGFBP)] were conducted. Severe intrauterine growth restriction (IUGR), oligohydramnios, and altered lymphocyte counts were noted for fetuses infected on GD 65. Less severe effects were detected for fetuses inoculated at the later time points, with severity dependent upon the length of SIV infection in utero. IGF studies indicated significant reductions in IGF-I and elevated immunoreactive levels of IGFBP-3 in infected fetuses during the third trimester. Parallel studies conducted with four fetuses infected on GD 65 with a nonpathogenic, molecularly cloned virus (SIVmac1A11) resulted in normal fetal growth, with no effects on hematopoiesis or IGF/IGFBP levels, and no evidence of clinical disease. Taken together, these studies show that (1) infection of fetuses during the early second trimester with an uncloned pathogenic strain of SIV results in severe IUGR and a disruption in the molar ratio of IGF:IGFBP-3, and (2) outcome of fetal SIV infection is determined by the timing of infection and the virulence of the viral inoculum. PMID- 7552477 TI - Assessment of antiretroviral therapy by plasma viral load testing: standard and ICD HIV-1 p24 antigen and viral RNA (QC-PCR) assays compared. AB - To assess the utility of quantitative competitive-polymerase chain reaction (QC PCR) measurements of plasma human immunodeficiency virus type 1 (HIV-1) RNA and other viral load markers for assessment of antiretroviral therapy, we used archived cryopreserved specimens from a randomized controlled clinical trial of 135 patients (CD4+ T cell count < or = 500/mm3), comparing zidovudine (500 mg/day) versus the nonnucleoside reverse transcriptase inhibitor L-697, 661 (50, 300, or 1,000 mg daily). We evaluated treatment-associated changes in plasma viral load by standard and immune complex-dissociated (ICD) HIV-1 p24 antigen assays, and, in a representative subset of patients (n = 46), by QC-PCR determination of virion-associated HIV-1 RNA. At baseline, HIV-1 RNA was quantifiable by QC-PCR in all patients tested (100%), whereas standard and ICD HIV-1 p24 antigen tests were positive (> or = 30 pg/ml) in 42% and 56%, respectively. All viral load parameters showed significant decreases from baseline within 1 week of initiation of zidovudine, as measured by standard p24 antigen assay, ICD p24 assay, and QC-PCR. At 1 week, patients treated with either 300 or 1,000 mg/day of L-697,661 showed significant decreases from baseline in plasma standard and ICD p24 antigen and QC-PCR-determined HIV-1 RNA levels. Whereas viral load decreases seen with zidovudine were sustained for the duration of treatment, plasma viral markers often returned to pretreatment levels despite ongoing L-697,661 treatment, with evidence of the emergence of drug-resistant virus. Whereas standard p24, ICD p24, and viral RNA levels changed similarly in response to treatment, the superior sensitivity and available dynamic range of plasma viral RNA assays like QC-PCR analysis provide an advantage for clinical monitoring of plasma viral load, allowing tracking of treatment-related changes even in patients with earlier stage disease and lower levels of viral load. PMID- 7552478 TI - Safety, pharmacokinetics, and antiviral response of CD4-immunoglobulin G by intravenous bolus in AIDS and AIDS-related complex. AB - To assess the safety, pharmacokinetics, and antiviral effects of intravenous recombinant CD4 immunoglobulin G (CD4-IgG), a 12-week Phase One study with an optional maintenance phase was performed. Twenty-two subjects with advanced human immunodeficiency virus (HIV) infection were enrolled; 15 subjects completed the initial 12 weeks. CD4-IgG doses were 30, 100, or 300 micrograms/kg weekly; 1,000 micrograms/kg once, twice, or three times per week; or 3,000 micrograms/kg twice weekly. Serum concentrations of CD4-IgG increased linearly with dose, with average peak serum concentrations of 22 micrograms/ml with 1,000 micrograms/kg. CD4-IgG was well tolerated; one patient had self-limited tachycardia and flushing associated with CD4-IgG therapy. No changes were seen in CD4 cell counts, hematologic or coagulation studies, serum chemistries, HIV p24 antigen titers, or plasma HIV titers. No subject developed anti-CD4 antibodies. HIV isolates from five patients had IC90 values that were higher than the peak concentrations of CD4-IgG achieved in those patients. Additional studies that achieve higher CD4 IgG concentrations are necessary to evaluate the antiviral activity of this compound. PMID- 7552480 TI - Substitution of didanosine sachets by chewable tablets: a pharmacokinetic study in patients with AIDS. AB - We have conducted a pharmacokinetic study of didanosine (ddI), formulated in sachets and in tablets, in patients with acquired immune deficiency syndrome (AIDS). Fifteen subjects received 250 or 167 mg of ddI twice daily as the sachet formulation and used this for at least 1 month. Subsequently, the patients were converted to receive ddI chewable/dispersible tablets (250-mg sachets to 200-mg tablets; 167-mg sachets to 125-mg tablets). Four subjects withdrew because of clinical deterioration or adverse effects. Serial blood samples were collected for pharmacokinetic monitoring during the use of the sachets and after 1 month of use of the tablets. No statistically significant differences were found in the maximum plasma concentration (Cmax), the time to reach Cmax (tmax), the area under the plasma concentration-time curve (AUC), or the terminal elimination half life (t1/2) between the two formulations. Patients who received low-dose ddI (sachets, 167 mg; tablets, 125 mg) displayed lower plasma concentrations than did the patients receiving high-dose ddI (sachets, 250 mg; tablets, 200 mg), despite an equal weight-normalized dose of ddI in these two groups. PMID- 7552479 TI - Efficacy, safety, and tolerance of low-dose, long-term interferon-alpha 2b and zidovudine in early-stage AIDS-associated Kaposi's sarcoma. AB - Seventeen male homosexual outpatients with limited but progressive AIDS associated Kaposi's sarcoma (KS) of skin, lymph nodes, oral cavity, and gastrointestinum were consecutively enrolled in an open, nonrandomized, prospective study. Patients received low-dose IFN-alpha 2b (3 MIU t.i.w.) in combination with 250 mg ZDV b.i.d. After remission of KS, continuation of treatment as maintenance therapy was provided. Eleven (65%) of 17 patients had a complete (CR) or partial remission (PR) of KS lasting more than 24 months in four patients. Response to treatment was strongly dependent on pretreatment values of CD4+ lymphocyte counts, CR and PR occurred only in patients with pretreatment levels higher than 250 CD4+ lymphocytes per microliter. Treatment was generally well tolerated and without severe hematologic side effects attributable to the addition of IFN-alpha 2b to ZDV treatment. This study suggests that a combination of low-dose IFN-alpha 2b and ZDV is therapeutically effective and well tolerated in early stages of progressive KS. PMID- 7552481 TI - Risk factors for dideoxynucleoside-induced toxic neuropathy in patients with the human immunodeficiency virus infection. AB - Dideoxynucleosides induce a dose-related toxic neuropathy; however, there is a paucity of information on whether other risk factors influence the development of neuropathy. We reviewed the records of 103 patients at an AIDS Clinical Trials Unit who were taking didanosine and/or zalcitabine to determine the risk factors for dideoxynucleoside-induced toxic neuropathy. Most were homosexual or bisexual (85%) men with a mean age of 39 years. The median CD4+ lymphocyte count was 59 cells/mm3, and 35% had a previous diagnosis of AIDS. Toxic neuropathy was more common in patients taking zalcitabine compared with those taking didanosine (14 of 51 versus seven of 55, p = 0.08). In the patients who took zalcitabine, those who had a low baseline serum cobalamin level, a history of heavy ethanol consumption, or a history of symptoms of peripheral nerve dysfunction were more likely to develop a toxic neuropathy (10 of 14 versus 12 of 37, p = 0.01). Conversely, there were no factors associated with the development of didanosine induced toxic neuropathy. Dideoxynucleoside-induced toxic neuropathy is a common problem that can be disabling but is usually reversible. A history of symptoms of peripheral nervous system disease, heavy ethanol consumption, or a low serum cobalamin level may be useful in distinguishing patients at higher risk of developing zalcitabine-induced toxic neuropathy. PMID- 7552482 TI - HIV incidence among New Haven needle exchange participants: updated estimates from syringe tracking and testing data. AB - This article provides updated estimates of the rate of new HIV infections among participants in New Haven's legal needle exchange program from syringe tracking and testing data. We previously reported that based on data collected from November 1990 through May 1992, the maximum likelihood incidence rate was zero with a 95% confidence interval of 0-10.2 new infections per 100 drug injectors per year. Expanding this data set through August 1993, the same statistical methods yield a maximum likelihood estimate of 1.63 new infections per 100 drug injectors per year with a 95% confidence interval of 0-7.2. Given these data, the null hypothesis of no new infections cannot be rejected, providing further support for the efficacy of New Haven's needle exchange program. PMID- 7552483 TI - Human immunodeficiency virus type-1 seroconversion trends among young adults serving in the United States Army, 1985-1993. United States Military Medical Consortium for Applied Retroviral Research. AB - The direct measurement of the incidence of new infections with the human immunodeficiency virus type 1 (HIV-1) can be made among soldiers because of the routine and periodic nature of HIV-1 testing in the United States Army. Between November 1985 and October 1993, 978 HIV-1 seroconversions were seen among 1,061,768 soldiers, contributing over 3.6 million person-years of follow-up [seroconversion rate (95% confidence interval) = 0.27/1,000 person-years (0.25 0.29)]. A significant decreasing trend in HIV-1 seroconversion rates was seen over the analysis period. The rate of new infections declined significantly from the first interval, 1985-1987, (0.43/1,000 person-years) to the second interval, 1987-1988, (0.28/1,000 person-years), but stabilized at approximately 0.22/1,000 person-years after 1988, representing new infections in approximately 100-150 soldiers annually. The risk of seroconversion among active duty soldiers was significantly associated with racial/ethnic group, age, gender, and marital status. Surveillance of HIV-1 seroconversion rates in the U.S. Army continues to offer a unique opportunity to assess temporal trends in the evolving HIV-1 infection epidemic. Monitoring the rate of new HIV-1 infections allows for identification of subgroups in need of intervention, refocusing of intervention strategies, and evaluation of their effectiveness. PMID- 7552484 TI - Diagnostic utility of immune-complex-dissociated p24 antigen detection in perinatally acquired HIV-1 infection in Rwanda. AB - We evaluated the diagnostic utility and prognostic value of immune-complex dissociated (ICD) HIV-1 p24 antigen detection in a prospective cohort study of perinatally acquired HIV-1 infection in Butare, Rwanda. HIV-1 p24 antigen was measured after acid dissociation in plasma samples collected at 6 weeks and 3 months of age from 36 HIV-1 infected and 80 uninfected children born to HIV-1 infected women. Among children with persistent HIV-1 antibody after 12 months of age, 26.7% of samples tested ICD HIV-1 p24 antigen positive. Among children who died between 3 and 12 months of age but were classified as HIV-1 infected, 17.6% tested p24 antigen positive at 6 weeks of age and 33.3% at 3 months of age. Female infants were more likely to test p24 antigen positive than male infants (p = 0.046). The specificity of the assay was 96.8%. Cumulative probabilities of child survival to 36 months of age were computed by HIV-1 status of the child and early p24 antigenemia. The mean survival time was 7 months for HIV-1 infected children with a high (> 50 pg/ml) p24 antigen concentration versus 20 months for children with p24 antigen concentrations between 10 and 50 pg/ml (p = 0.02). Cumulative probabilities of survival to 36 months of age were 0% among HIV-1 infected children with at least one positive p24 antigen test, 44% among confirmed HIV-1 infected children who were p24 antigen-negative at both 6 weeks and 3 months of age, and 95% among seroreverting children (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552486 TI - Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians. AB - To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals. PMID- 7552485 TI - Relation between human T-lymphotropic virus type I and neurologic diseases in Panama: 1985-1990. AB - Human T-cell lymphotropic virus type I (HTLV-I) is endemic in the Caribbean basin and in Japan. HTLV-II, a closely related virus, is endemic in several groups of native Americans, including Panamanian Guaymi. In Panama, a nationwide HTLV-I/II seroprevalence of 1-2% has been reported. We evaluated the frequency of HTLV-I/II infection in patients with neurologic diseases admitted to state tertiary hospitals in Panama City between 1985 and 1990. Nineteen of 322 patients with eligible diagnoses had antibodies to HTLV-I/II, 17 with HTLV-I and 2 with HTLV II. HTLV-I was associated with spastic paraparesis (13 of 23, 56.5% versus 4 of 299, 1.3%, p < 0.001) and with cerebellar syndrome (2 of 13, 15.4%) and multiple sclerosis (2 of 54, 3.7%) (p < 0.05 for both diseases compared with subject with none of these diagnoses). The two HTLV-I infected patients with cerebellar syndrome later developed spastic paraparesis. HTLV-II infection was noted in one patient with cerebellar syndrome and one with amyotrophic lateral sclerosis. All patients with other diagnoses were seronegative. Among patients with spastic paraparesis, HTLV-I-infected patients were clinically indistinguishable from seronegative subjects. There is apparently an overlapping clinical spectrum of neurologic diseases associated with HTLV-I and HTLV-II infection. PMID- 7552487 TI - HTLV-II detection in an Amerindian family in Colombian southern Andean region. PMID- 7552488 TI - Interest in HIV vaccines among Italian injection drug users. The Network Interregionale SERT. PMID- 7552489 TI - Simulation of the San Francisco HIV epidemic: AZT a possible epidemiological modulator. PMID- 7552490 TI - 7th International Conference on Human Retrovirology HTLV and Related Viruses. Paris, France, October 17-21, 1995. Abstracts. PMID- 7552491 TI - Heterogeneity of spindle cells in Kaposi's sarcoma: comparison of cells in lesions and in culture. AB - The immunophenotype of spindle cells in epidemic, endemic, and classic (sporadic) Kaposi's sarcoma (KS) lesions was defined by the demonstration of various cell markers and compared with that of KS-derived cell lines. No significant histological or immunophenotypic differences were observed between the three clinical types of KS at comparable stages. The spindle-cell compartment of the different KS types was composed predominantly of a mixture of proliferating CD45+/CD68+ bone-marrow-derived monocytes and TE7+/collagen+ fibroblastic cells with varying expression of EN4/PAL-E/CD31/CD34/CD36 endothelial-associated antigens and/or smooth-muscle-specific alpha-actin (alpha-actin). The latter cells appeared to represent transitional forms of fibroendothelial and fibromyocytic cells. The in vitro cultured KS-derived cell lines (KS-3, KS-6, and KS-8) expressed the fibroblastic antigen TE7 and smooth-muscle-specific alpha actin but not leukocytic or endothelial-associated antigens consistent with the phenotype of fibromyoid spindle cells of primary lesions. Neither HIV antigen nor provirus DNA was demonstrable in the epidemic KS lesions. The observed heterogeneity of the spindle-cell compartment further substantiates the view that Kaposi's sarcoma, irrespective of clinical setting, expresses salient features more compatible with reactive, tumor-like lesion than clonal sarcoma. PMID- 7552493 TI - Sequence specificity in the higher-order interaction of the Rev protein of HIV-1 with its target sequence, the RRE. AB - The Rev protein of human immunodeficiency virus type 1 (HIV-1) multimerizes along RNAs containing the Rev target sequence, the RRE. Although sequence-specific information is recognized in the high affinity or initial interaction, it is not known what role RNA-contained information plays in higher-order binding events. We have quantitatively studied the binding of Rev protein to the primary Rev binding domain (II + III) of wild-type and mutant RREs. RRE mutations that retain the basic secondary structure of wild type can separately and differentially alter the Kds for formation of the first, second, and third Rev/RRE complexes (C1, C2, and C3). The data suggest that Rev recognizes sequence-specific information in the RRE when it forms higher-order complexes. However, the formation of higher-order complexes is not as dependent on sequence-specific information as the first or lowest order binding interaction, which involves recognition of the high-affinity site. PMID- 7552492 TI - An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity. AB - Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation. PMID- 7552495 TI - Natural killer cell immunodeficiency in HIV disease is manifest by profoundly decreased numbers of CD16+CD56+ cells and expansion of a population of CD16dimCD56- cells with low lytic activity. AB - Natural killer (NK) cells were enumerated by three-color immunofluorescence in 255 uninfected and 399 human immunodeficiency virus-infected adults. Several dramatic alterations were observed. First, the median number and percentage of CD16+CD56+ NK cells, the subset that comprises > 90% of the NK cells in healthy adults, were severely decreased (median, 175/mm3 in uninfected controls; 63/mm3 in HIV-infected non-AIDS subjects). Even subjects with > 800 CD4+ cells/mm3 had decreased CD16+CD56+ NK cell levels (97/mm3). Second, the number of CD16+CD56- cells, an NK population that is rare in healthy adults, was elevated (median, 20/mm3 in uninfected controls; 64/mm3 in HIV-seropositive non-AIDS subjects). Third, the expression of CD16 on the NK cells was markedly reduced; some CD56+ cells and virtually all CD56- cells were CD16dim. Fourth, fluorescence-activated cell-sorting studies revealed little NK- or antibody-dependent cellular cytotoxic activity in the CD16dimCD56- cell population. These results indicate that the pathogenesis of HIV disease includes numerical alterations in subpopulations of NK cells. A better understanding of how HIV infection causes this aspect of pathogenesis is needed. PMID- 7552494 TI - Evidence of active cytomegalovirus infection in clinically stable HIV-infected individuals with CD4+ lymphocyte counts below 100/microliters of blood: features and relation to risk of subsequent CMV retinitis. AB - To determine the frequency and significance of cytomegalovirus (CMV) viremia and viruria in HIV-positive subjects with low CD4+ lymphocyte counts but with no clinical indications for culture, we studied 100 consecutive clinically stable subjects with CD4+ cells < or = 100/microliters of blood who agreed to culture of blood and urine. Serum was tested for CMV antibody, p24 antigen, neopterin, and liver enzyme concentrations, and patients were offered funduscopic examination. Subjects' records were reviewed an average of 9.1 months after enrollment for evidence of subsequent CMV retinitis. Three of the original cohort proved ineligible because of CD4+ count > 100/microliters; CMV antibody was present in 96% of the remainder. Isolation of CMV from blood was uncommon (2 of 93 seropositive subjects) whereas viruria occurred in 51.6%; likelihood of having a positive urine culture was significantly related to the subject's absolute CD4+ lymphocyte count: 60% for those with CD4+ < or = 50/microliters, vs. 26.1% for those with CD4+ 51-100/microliters. Neither serum p24 antigen nor neopterin was predictive of CMV in urine or blood. No subjects submitting to ophthalmologic exam had unsuspected CMV retinitis. Subsequent development of retinitis correlated with CMV viruria on entry: 13.5% if urine-positive, 1.9% if negative (p = 0.029; Fisher exact test). PMID- 7552497 TI - Validity of the Karnofsky Performance Status in an HIV-infected sample. AB - The Karnofsky Performance Status (KPS) is the most widely used health status measure in human immunodeficiency virus (HIV) medicine and research. Because there are limited data on KPS metric properties in this setting, we present evidence of the construct validity of the KPS in a sample of HIV-infected persons using data from the AIDS Time-Oriented Health Outcome Study (ATHOS). The sample consisted of 160 primarily white, homosexual/bisexual men with a mean age of 45 years and a mean KPS score of 82 (range, 40-100). Ninety percent were classified in Centers for Disease Control (CDC) clinical category B or C. Pearson's product moment correlations were strong between the KPS and measures of global health status, physical disability, numbers of symptoms, CDC clinical category, social function, days off work, and energy/fatigue (r = 0.39-0.52, p < 0.0001). Correlations with measures of mental health and cognition were less impressive but statistically significant. Analysis of variance followed by Student Newman Keuls test showed significant differences among three KPS groupings for global health status but not for physical disability. Regression analysis indicated three significant variables accounting for KPS variance: visual analogue global health status (27%), days off work (10%), and energy/fatigue (1.7%). We conclude that the upper range of the KPS reflects global health status better than physical performance and much better than psychosocial constructs, in persons with HIV infection. Further research examining the ability of the KPS to detect clinically significant change over time is needed. PMID- 7552496 TI - The consequences of a positive prenatal HIV antibody test for women. AB - As more women of childbearing age are affected by the human immunodeficiency virus (HIV), many providers have demanded routine perinatal HIV screening, arguing that the medical benefits of testing outweigh the socioeconomic, medical, and psychological risks of a positive HIV test for women. In this primarily urban poor population, we used a semistructured interview to evaluate differences in health care discrimination, economic losses, risk behaviors, relationships changes, and psychological status in 20 HIV-positive and 20 HIV-negative mothers matched for HIV risk, race, income, and delivery date. Many (35%) seropositive and no seronegative women cited health care discrimination due to HIV status. Although seropositive women reported greater satisfaction with social support from friends (100%) and family (80%), many women had not disclosed their HIV status to any friends (65%) or family (25%), indicating fear of abandonment. Only 56% of HIV positive and 44% of seronegative women knew their partners' HIV status, and many HIV-positive and HIV-negative women reported having sex without condoms after the HIV test. Mean standardized anxiety (p < 0.05) and depression scores were higher in seropositive women. Despite added social support and medical treatments, HIV-positive women showed higher levels of health care discrimination, personal isolation, and psychological sequelae than their seronegative counterparts. As the medical benefits to prenatal HIV testing increase, we will need to develop focused medical, social, and mental health services addressing the needs of HIV-positive women. PMID- 7552499 TI - Weight loss prior to clinical AIDS as a predictor of survival. Multicenter AIDS Cohort Study Investigators. AB - In this analysis the aim was to determine the independent effect of moderate to severe weight loss prior to an AIDS diagnosis on survival after AIDS. The study was conducted as part of the Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV-1-seropositive gay or bisexual men. Measured weight and self reported weight loss data were collected semiannually from 1984 through 1993. The study population included 962 HIV-1-seropositive men who developed clinical AIDS during the follow-up period. Median survival after AIDS was significantly lower for men with measured weight loss of > or = 4.5 kg 3-9 months and 3-15 months prior to AIDS, or who had lost > 10% of their baseline body weight compared with men with less weight loss or weight gain. Men with self-reported unintentional weight loss of > or = 4.5 kg 3-9 months prior to AIDS had significantly poorer survival (median = 1.05 years vs. 1.48 years; p = 0.0001) compared with men not reporting weight loss. After adjusting for potential confounding factors, men in the high measured weight loss group 3-9 months prior to AIDS still had significantly poorer survival [relative hazard (RH) = 1.36; p = 0.02]. Similar trends were seen for the two longer intervals prior to AIDS (RH = 1.38, p = 0.01; and RH = 1.50, p = 0.02, respectively). Men who self-reported weight loss > or = 4.5 kg 3-9 months prior to AIDS also had significantly poorer survival after AIDS (RH = 1.43; p = 0.002) in multivariate analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552498 TI - The epidemic of HIV/AIDS in Abidjan, Cote d'Ivoire: a review of data collected by Projet RETRO-CI from 1987 to 1993. AB - We present a review of epidemiologic data collected by Projet RETRO-CI between 1987 and 1993 on trends in human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections and on cases of AIDS in Abidjan, Cote d'Ivoire. Overall rates of HIV infection in pregnant women had already reached 10% in 1987, and have increased only modestly since then. In contrast, in 1992-1993, rates in men with sexually transmitted diseases and in female commercial sex workers reached 27 and 86%, respectively. The increases in infection rates have been largely due to transmission of HIV-1, whereas rates of HIV-2 have remained stable or have declined. Among persons with tuberculosis and hospitalized patients, rates of 46 71% have been reached, increases in recent years again being largely attributable to HIV-1. Among the 15,245 AIDS cases reported by Projet RETRO-CI, a steady decline in the male:female sex ratio has occurred over time, from 4.8:1 in 1988 to 1.9:1 in 1993. It is likely that AIDS cases were initially concentrated among a core group of female commercial sex workers and their male clients. A substantial proportion of sex workers and their clients originate from neighboring countries, and migration is likely to have contributed to the spread of HIV infection in West Africa. Including HIV-associated pulmonary tuberculosis as an AIDS-defining illness increased AIDS cases reported by Projet RETRO-CI by 13% in 1993. Despite a need for interventional research, careful description of the evolution of HIV/AIDS in this region remains essential. PMID- 7552500 TI - How important is race/ethnicity as an indicator of risk for specific AIDS defining conditions? AB - In order to examine differences in the prevalence of AIDS-defining conditions by race/ethnicity, we analyzed U.S. surveillance data for 203,470 adolescents and adults diagnosed with AIDS from 1988 through 1992. A number of AIDS-indicator conditions were more common among certain racial/ethnic groups. The prevalence of extrapulmonary tuberculosis was higher among blacks, Hispanics, Asians/Pacific Islanders, and American Indians/Alaskan Natives than among whites. The prevalence of isosporiasis and toxoplasmosis was higher among Hispanics than among blacks or whites. Furthermore, the likelihood of being diagnosed with extrapulmonary tuberculosis (TB), toxoplasmosis, or isosporiasis was generally higher among foreign-born than among U.S.-born persons of all racial/ethnic groups. The prevalence of all malignancies was higher among whites than among blacks or Hispanics. However, the magnitude of prevalence differences by race/ethnicity was reduced when we controlled for other demographic and exposure risk categories. Although race/ethnicity was significantly associated with the prevalence of a number of conditions, the relative frequency and patterns of AIDS-indicator conditions in different populations are probably most influenced by differences in (1) underlying prevalence or exposure to various etiologic agents causing these conditions, (2) diagnosis and reporting of conditions, and (3) access to care and therapy for HIV-related conditions. PMID- 7552501 TI - Concordance of PCR and antibody results from HIV testing of injecting drug users. AB - Standard HIV-1 testing relies on the enzyme immunoassay (EIA) for detecting antibodies specific to HIV-1. This technique may misclassify persons as HIV-1 negative in instances where testing follows infection but precedes development of antibody to HIV-1. To evaluate the occurrence of HIV infection in the absence of positive antibody, polymerase chain reaction (PCR) for viral DNA in the blood has been applied. Research comparing these two testing techniques has generally focused on populations of homosexual and bisexual men. This study compares PCR and antibody testing of 337 injecting drug users recruited from street settings in San Francisco. Of 286 HIV-1 antibody-negative samples, 3 (1.0%) were PCR positive. Of 49 HIV-1 antibody-positive samples, 1 (2.0%) was PCR-negative. Two samples were antibody-indeterminate and PCR-negative. This yielded an overall concordance of 331/335 (98.8%), excluding the indeterminate results. These results suggest that current antibody methodology is adequate. However, misclassification among recently infected individuals may occur, which is of concern in high-incidence groups. PMID- 7552503 TI - Whole-body hyperthermia. PMID- 7552502 TI - Medical eligibility, comprehension of the consent process, and retention of injection drug users recruited for an HIV vaccine trial. AB - Injection drug users (IDUs) at high risk for human immunodeficiency virus (HIV) infection are being identified as a population for HIV vaccine trials. We studied willingness of drug users to enroll and their comprehension of consent procedures in the context of a Phase II trial at one site. Of 175 people screened for enrollment and whose data sets were complete, 119 (68%) were IDUs. Of the 71 who were eligible, 39 (55%) were IDUs. Exclusion of IDUs was usually for medical reasons. Using a 17-item true/false test, comprehension of the informed consent procedure was high (median score, 16 of 17 for IDUs and non-IDUs); only three subjects (all of whom were IDUs) were excluded from enrollment due to lack of comprehension. Follow-up rates were similar for IDUs and non-IDUs. These data suggest that recruitment of IDUs into HIV vaccine trials is feasible, that IDUs can comprehend and complete the informed consent procedures, and that they return for follow-up visits. PMID- 7552504 TI - Serum selenium predicts outcome in HIV infection. PMID- 7552505 TI - Surrogate Markers of HIV: Strategies and Issues for Selection and Use. Proceedings of a symposium. Arlington, Virginia, October 12-14, 1994. PMID- 7552506 TI - Evaluating surrogate markers. AB - We discuss approaches for efficiently evaluating potential surrogate markers; in particular, we focus on case-cohort designs in which marker evaluation is undertaken only for a random sample of subjects within a randomized trial and for all other subjects who develop a major clinical outcome. These designs will be useful in clinical trials in which a highly significant treatment difference on clinical outcome has been obtained. In addition, we describe a method for using data from all available studies using a meta-analysis to explore the association of treatment effects on the potential marker and on clinical outcome. This may be the most effective approach for marker evaluation because it uses data from both large and small trials and incorporates information from trials in which nonsignificant treatment differences on the major clinical outcome are obtained. PMID- 7552507 TI - Clinical research and drug development of antivirals in HIV: an industry perspective. AB - Clinical research can be viewed from three perspectives: research, patient care, and regulatory. Clinical research strategies can be either drug oriented, following the traditional phase I-II-III flow, or disease oriented. The drug oriented strategy has as its goal regulatory approval, which means widespread patient-care usage. The disease-oriented strategy focuses on improving the overall care of the disease in question. At times, a dynamic tension exists between the two strategies. In HIV, as in all diseases, industry mainly is involved in drug-oriented studies emphasizing the regulatory perspective. In HIV a critical issue involves accelerated approval, following expanded access, based on surrogate markers. This initial approval is for end-stage therapy (the expanded access population). Additional claims require a full supplemental New Drug Application utilizing clinical end points. This leads to a situation in which the label, over time, becomes less relevant to how the drug is used in actual practice. This has caused concern among the patient advocates and is becoming a major challenge for the industry. PMID- 7552508 TI - Consensus statement. Scientific Advisory Committee on Surrogate Markers of HIV. PMID- 7552510 TI - Viral phenotype and genotype as markers in clinical trials. AB - Treatment of AIDS and HIV infection is increasingly dependent on the use of surrogate markers to assess the efficacy of drug and biologic therapies in individual patients and in the clinical trials. Recent developments in laboratory techniques have resulted in new assays to measure circulating viral RNA in HIV infected individuals, standardized methods to assess genotypic changes in virus associated with drug resistance and biologic assays for syncytia-inducing phenotype, a viral characteristic associated with rapid clinical progression. Studies from our laboratory have examined the relationship between surrogate markers of drug efficacy, CD4 cell changes, quantitative HIV plasma RNA and cell dilution cultures, genotypic changes associated with drug resistance and the syncytia-inducing phenotype. The results of these studies suggest that drug resistance genotype and syncytia-inducing phenotype are independent factors that contribute to disease progression in patients receiving zidovudine. The design of studies of drugs and biologics for the treatment of HIV should include assessment of genotypic and phenotypic characteristics of HIV in addition to CD4 cell numbers and virus load. PMID- 7552509 TI - Virologic parameters as surrogate markers for clinical outcome in HIV-1 disease: verification, variation, and validation. AB - The criteria used to evaluate which virologic measurements are used to monitor HIV-1 infection should include an assessment of verification, variation, and validation. Rationale for use should first be based on understanding the role of the measurement in the pathogenesis of the disease. Subsequently, the prevalence of the measurement, an understanding of its intrinsic variation, and ease of use will determine the utility of the measure. The usefulness of the measurement will depend on its validation in relation to disease prognosis, antiviral activity, and antiviral efficacy. PMID- 7552511 TI - Performance characteristics for the quantitation of plasma HIV-1 RNA using branched DNA signal amplification technology. AB - Highly sensitive assays that quantitate human immunodeficiency virus type 1 (HIV 1) RNA may be valuable for clinical research and the treatment of HIV-1-infected patients. In this study we evaluated the reproducibility and accuracy of the first-generation branched DNA (bDNA-1.0) signal amplification assay under conditions that are relevant to routine use in a clinical context. We show that the bDNA-1.0 assay was able to discern two- to three-fold changes in plasma HIV-1 RNA levels as significant. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was less reproducible and required a 3.7- to 5.8-fold change in plasma HIV-1 RNA levels to be statistically significant. The accuracy of the bDNA 1.0 assay in RNA quantitation was not affected by HIV-1 genotypic variation or by the presence of hemoglobin, bilirubin, lipemia, or any of a dozen therapeutic drugs. Using the bDNA-1.0 assay, we show that HIV-1 RNA levels in plasma specimens were stable when stored at -80 degrees C and were able to withstand at least three freeze-thaw cycles without significant loss. We also examined the performance of an ultrasensitive bDNA assay with improvements to the signal amplification technology. The ultrasensitive bDNA assay displayed a quantitation limit of approximately 500 RNA Eq/ml, yet maintained a dynamic quantitation range up to 1.6 x 10(6) RNA Eq/ml. Like the bDNA-1.0 assay, the ultrasensitive bDNA assay was not affected by HIV-1 genotype variability. PMID- 7552512 TI - Potential applications of proviral load measurement in clinical retrovirology. AB - We have previously noted an association between proviral load and the severity of immune disease in individuals with a wide range of CD4 cell counts. Using the quantitative DNA polymerase chain reaction technology developed in our laboratory, we sought to extend these observations, with a view to establishing guidelines for the use of proviral load in a clinical context. We studied 199 patients with a range of CD4 cell counts attending an urban tertiary care center. Proviral load/10(6) peripheral blood mononuclear cells (PBMCs) was measured using a microtiter plate assay designed specifically for this purpose. Human immunodeficiency virus proviral DNA was detected in 193 of 199 clinical samples. Levels of proviral load were tabulated for patients and evaluated in seven categories defined by CD4 cell counts. Although a wide range of proviral loads was observed in each category of patients, there was a trend toward increasing proviral load with decreasing CD4 cell count. Statistically significant relationships were observed between proviral load and the CD4 cell count and the CD4 cell percentage (Spearman's correlation coefficient -0.19, p = 0.01 for both absolute CD4 and CD4 percentage). These relationships were quite weak and could not be taken to explain disease progression in isolation. If we defined a cutoff between low and high proviral loads at 100 copies/10(6) PBMCs, we noted that 52% (24 of 46) of patients with CD4 cell counts > 400/microliters had lower loads, as compared with 16% (24 of 143) of those with more advanced disease (p < 0.01). There is a weak, but statistically significant association between proviral load and CD4 cell depletion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552513 TI - Viral load in peripheral blood mononuclear cells as surrogate for clinical progression. AB - During the symposium on surrogate markers of HIV, the Scientific Advisory Committee posed the following question: "Which surrogate markers currently deserve the greatest commitment of investigative resources to validate them in a clinical setting?" The Committee concluded that for antiretroviral drugs measurements of HIV RNA in plasma deserve the greatest priority, and for immune based therapies assessing viral load still had the highest priority. But it was recognized that assessing viral load should not be restricted to plasma RNA because the primary mechanisms of action are different from antiretroviral drugs. Thus, the Committee voted that based on current knowledge, investigating the clinical relevance of changes in HIV-1 DNA and RNA copy number in peripheral blood mononuclear cells (PBMCs) with validated assays should be given equal focus for immune-based therapies. This article reviews the rationale for using HIV-1 DNA and RNA load in PBMCs for the monitoring of clinical trials and presents recent data that indicate that the postseroconversion level and the dissemination of proviral DNA in the blood cells have prognostic value, i.e., high levels correlate with disease progression. In addition, longitudinal studies show that an increase in proviral DNA and/or HIV mRNA load correlates with disease progression. We present evidence that these markers are relevant activity markers for anti-HIV therapies. Changes in both DNA and RNA load can be achieved using either antiretroviral drugs or immune-based therapies. These results suggest that these markers should be evaluated in clinical studies to firmly establish their value as surrogates of clinical progression. PMID- 7552515 TI - Immunologic and virologic markers determining progression to AIDS. AB - Infection with the human immunodeficiency virus type 1 (HIV-1) results in a severe immunodeficiency characterized by a depletion of CD4+ T-helper cells. Furthermore, it is well documented that in asymptomatic persons the number of CD4+ cells is also a good predictor of progression to AIDS. However, persons with similar CD4+ cell counts may differ with regard to clinical progression. For this reason the development of additional markers predictive of disease progression is of major clinical importance. In this review three additional progression markers are discussed: rate of decline of CD4+ cells, T-cell reactivity, and HIV biological phenotype. Besides their usefulness as independent progression markers they also provided insight into immunopathologic mechanisms responsible for the eventual development of AIDS. PMID- 7552514 TI - Sampling lymph node content of human immunodeficiency virus type 1 nucleic acids and p24 antigen by fine-needle aspiration in early-stage patients. AB - The potential of lymph node fine-needle aspiration (LNFNA) for sampling viral load was evaluated in excised, peripheral lymph nodes from five patients with early-stage human immunodeficiency virus type 1 HIV-1 disease (asymptomatic, CD4 cells > 300/mm3). The preponderance (> 80%) of viral RNA was within follicular germinal centers as noted by in situ hybridization on lymph node frozen sections (LNFSs) as well as within cohesive groups of 10-20 lymphoid cells (microfragments) in LNFNA preparations. Quantification of cells expressing HIV-1 RNA by in situ hybridization, quantification of HIV-1 gag RNA and gag DNA per 10(5) cells by polymerase chain reaction, and measurement of p24 antigen per 10(5) cells yielded similar values for LNFNA, lymph node mononuclear cells (LNMCs) from tissue homogenates by Ficoll-Hypaque separation, and LNFS. Sampling of lymph node viral load by LNFNA appears to capture viral components associated with both individual expressing cells and follicular germinal centers. Due to the advantages in terms of patients' morbidity, repeatability, and cost, assessment of lymphoid tissue viral load by LNFNA warrants an in vivo feasibility trial as an alternative to lymph node biopsy. PMID- 7552516 TI - Quantification of the variation due to laboratory and physiologic sources in CD4 lymphocyte counts of clinically stable HIV-infected individuals. AB - We have conducted a study to quantify the amount of variation in the CD4 lymphocyte counts of HIV-infected individuals due to laboratory and physiological factors. Thirty HIV-infected male volunteers had blood drawn on six occasions: three times in each of 2 weeks, 4 weeks apart. Two tubes of blood were drawn at each visit, and duplicate measurements were obtained from one of the tubes of blood. Differences between duplicate measurements from a single tube of blood and between CD4 counts obtained from two tubes of blood drawn on the same day were attributed to laboratory factors. Differences between CD4 counts obtained on different days were attributed to a combination of laboratory factors and physiologic factors, which included the effects of exercise, tobacco, and the consumption of alcohol and caffeine. The mean absolute CD4 count at the first visit was 450 (range 86-1,081). The short-term coefficient of variation of CD4 count was 13.7 (95% CI: 12.9, 14.6). Physiologic and laboratory factors accounted for 85% and 15% of the variation in CD4 counts, respectively. Variation in the absolute white blood cell count, lymphocyte percentage, and CD4 percentage accounted fo 52%, 29%, and 19% of the physiologic variation in CD4 counts, respectively. Our results confirm a high degree of short-term variability of CD4 counts among HIV-infected individuals, which can be largely attributed to physiological factors. This variability can be minimized more effectively by repeating CD4 counts over time than by repeating measurements at a single visit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552517 TI - Safety and immunogenicity of a gp120-depleted, inactivated HIV-1 immunogen: results of a double-blind, adjuvant controlled trial. AB - We report the safety and immunogenicity results of a double-blind adjuvant controlled trial of the human immunodeficiency virus type 1 (HIV-1) immunogen. Healthy, asymptomatic HIV-1-seropositive individuals received either three 100 microgram doses of the inactivated HIV-1 antigen in incomplete Freund's adjuvant (IFA) or three doses of IFA alone. The results of this study show that this HIV-1 immunogen is safe, with mild and transient adverse events. No difference in the number or type of adverse events was noted between the treatment groups. Rises in HIV-1-specific humoral and cell-mediated immune (CMI) responses were also noted, favoring the HIV-1 immunogen-treated group. The results of this study confirm and extend the safety and immunogenicity profile of this HIV-1 immunotherapeutic agent. The observation that this treatment can augment HIV-1-specific CMI is encouraging because this immunological marker may represent a key surrogate end point in HIV-1 infection and disease. PMID- 7552518 TI - In vitro comparison of selected triple-drug combinations for suppression of HIV-1 replication: the Inter-Company Collaboration Protocol. AB - Ten different three-drug combinations have been analyzed for their ability to prevent HIV-induced cytopathic effects (CPEs) in a continuous human T lymphoblastoid cell line. Agents acting at the same as well as at different sites in the HIV-1 replicative cycle were used. Each compound was analyzed at peak and trough plasma levels achieved in monotherapy and in the presence of HIV-1 strains 3B and MN at a viral inoculum varying from 1 x TCID50 (50% tissue culture inhibitory dose) to 1,000 x TCID50. Using a viral inoculum of 10 x TCID50 HIV-1 3B, it was determined that triple-drug combinations had greater antiviral activities than the corresponding double-drug combinations, which had greater antiviral activities than zidovudine (AZT) monotherapy. The most consistent triple-drug combination, demonstrating superior activity at all concentrations of virus, was AZT + dideoxyinosine + lamivudine which reduced the AZT IC95 (95% inhibitory concentration) by 208-, 57-, 133-, and 25-fold at of 1,000, 100, 10, and 1 x TCID50 HIV-1 3B, respectively, as compared with the IC95 for AZT monotherapy. For all antiviral regimens tested, higher viral inoculum resulted in less inhibition of viral replication and a higher IC95 for AZT. This observation argues for therapeutic intervention at an earlier stage in HIV infection, when viral burden is lower. PMID- 7552519 TI - Insights from monitoring the CPCRA didanosine/zalcitabine trial. Terry Beirn Community Programs for Clinical Research on AIDS. AB - The design, conduct, and analysis of clinical trials that evaluate the safety and efficacy of treatment interventions in patients with HIV infection provide many scientific challenges. A recently completed randomized trial of didanosine (ddI) and zalcitabine (ddC), sponsored by the Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA), is an especially valuable resource for illustrating these challenging issues and for providing insights into how they might be properly addressed. Establishing equivalence of treatment effects on clinical efficacy end points is illustrated through the use of the confidence interval approach. The striking changes in treatment efficacy results that occurred during the course of the CPCRA trial provide important insights into how a data and safety monitoring board can reduce the risk of inappropriate early study termination. The trial also provides valuable insights into how treatment effects should be assessed, revealing inconsistencies between effects on the CD4 surrogate end point and effects on primary clinical efficacy end points and showing the incompleteness of the standardly employed definition of AIDS progression. Finally, the results of this ddI/ddC trial are used to examine the role of covariate adjustment. PMID- 7552521 TI - Survival effects of ZDV, ddI, and ddC in patients with CD4 < or = 50 cells/mm3. AB - Seven major clinical trials for the treatment of HIV-infected individuals are investigated. The treatments used in these trials were zidovudine, dideoxyinosine, dideoxycytosine, and one combination for patients with CD4 cell counts < 500 cells/mm3. Patients in each trial are partitioned into two subgroups based on their baseline CD4 cell counts: patients with CD4 < or = 50 cells/mm3 and patients with CD4 > 50 cells/mm3. The difference between treatment effects, using survival as a measure of effect, within each subgroup is calculated separately for each trial; this difference is referred to as "subgroup response." For each trial the difference between the subgroup responses is calculated and standardized. A meta-analysis is conducted over all seven trials for the differences between subgroup responses; the consistency of responses is evaluated across all trials among patients within baseline CD4 subgroups. Based on the result of this meta-analysis we conclude that there is no evidence that the treatment effects in patients with CD4 < or = 50 cells/mm3 are different from those among patients with CD4 > 50 cells/mm3. This result is observed in patients with different antiviral experience and different baseline characteristics. Risk ratios as well as chi 2 statistics are used to quantify the response rates in different subgroups. Kaplan-Meier curves of survival for these trials are depicted for all patients and each subgroup separately. In most of the trials the Kaplan-Meier curves for the patients with CD4 < or = 50 cells/mm3 resemble those for all patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552520 TI - Inter-Company Collaboration Combination Trials. Clinical Trial Subcommittee of the Inter-Company Collaboration for AIDS Drug Development. AB - The Inter-Company Collaboration for AIDS Drug Development (ICC) represents a collaborative effort among member companies to facilitate the conduct of clinical trials on AIDS drugs. One of the goals of the ICC is to expedite the development of combination antiretroviral therapy through data and compound sharing. Recently, the ICC formed a consensus master protocol to evaluate rapidly the safety and efficacy of triple-drug combinations of antiretroviral therapy for treatment of HIV-infected patients. This concept builds upon historical work with combination chemotherapy that resulted in treatments to successfully control chronic immunosuppressive, infectious or malignant diseases, such as tuberculosis, leprosy, childhood acute lymphoblastic leukemia, and Hodgkin's lymphoma. Because of limitations on potency and the continuing emergence of drug resistance seen with use of currently available antiretroviral agents in monotherapy and two-drug combination regimens, triple-combination regimens should represent a more promising approach to maximize antiviral activity, maintain long term efficacy, and reduce the incidence of drug resistance. The ICC master protocol is a randomized, controlled, double-blind study with a treatment duration of 52 weeks. Patients eligible to enroll in this study must have documented HIV infection, with CD4 counts between 200 and 500 cells/mm3, and no history of antiretroviral therapy. The first four triple-drug combinations will be evaluated in two trials. These regimens have been selected based on encouraging data from laboratory and clinical studies. Each ICC trial will consist of three arms, with 75 patients per arm. Protocol ICC 001 will include AZT + zalcitabine (ddC) + saquinavir, AZT + ddC + nevirapine, and AZT + ddC as the control arm.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552522 TI - Carotid endarterectomy: who should perform it? PMID- 7552524 TI - Fibrinogen and markers of fibrinolysis and endothelial damage following resolution of critical limb ischaemia. AB - OBJECTIVES: To assess the effects of resolution of critical limb ischaemia on the elevated plasma fibrinogen, cross-linked fibrin degradation products (FDP), and von Willebrand factor antigen (vWF) levels, reported in peripheral arterial occlusive disease. DESIGN: A prospective study of patients undergoing surgery for chronic critical limb ischaemia. SETTING: Two vascular surgery units providing tertiary referral services for the West of Scotland. MATERIALS: Venous blood samples were assayed for plasma fibrinogen, FDP D-dimer, and vWF levels, prior to surgery, together with fibrinolytic and rheological parameters, in 82 patients. Sampling was repeated 4 months after resolution of critical limb ischaemia. OUTCOME MEASURES: Levels of these parameters following successful resolution of critical limb ischaemia were compared with pre-operative levels, and with an age matched random population sample. MAIN RESULTS: Plasma fibrinogen and vWF levels were significantly lower (both p < 0.005, Wilcoxon matched pairs) following successful resolution of critical limb ischaemia in the 56 patients available for review, although levels remained higher than in population controls (p < 0.01, Mann-Whitney U-test). FDP levels were unchanged following surgery, remaining higher than in age-matched population controls (p < 0.01). CONCLUSIONS: Resolution of critical limb ischaemia fails to reduce plasma fibrinogen, fibrin turnover, and vWF levels to those seen in population controls. This implies that increased fibrinogen and fibrin turnover in peripheral arterial disease is not solely a consequence of tissue ischaemia, while the persisting prothrombotic state following resolution of critical limb ischaemia has potentially important implications for graft and patient survival. PMID- 7552523 TI - Perioperative vascular flushing perfusion in acute mesenteric artery occlusion. AB - AIM: To evaluate in animals and patients a perioperative vascular flushing perfusion with antioxidants in order to improve postischaemic condition of the bowel and hence, to prolong the warm ischaemia tolerance time. MATERIALS: 40 rats and 10 patients with acute mesenteric artery occlusion. METHODS: Intestinal ischaemia was induced in 40 rats by clipping the superior mesenteric artery for 1 h. Thirty animals received a vascular flushing perfusion with oxygen radical scavengers (ascorbate, tocopherol or oxypurinol). Histology, ATP, ADP levels were examined in tissue biopsies and malondialdehyde, lactate dehydrogenase, alkaline phosphatase levels were measured in blood during ischaemia and 60 min after reperfusion. RESULTS: ATP restoration was particularly improved in the oxypurinol group (ATP/ADP-ratio = 2 vs. 1.2 in the control group). Malondialdehyde increase observed after reperfusion as a marker of radical induced lipid destruction was significantly attenuated (control group 160% of base level vs. 127% in the ascorbate group, 133% in the tocopherol group, 121% in the oxypurinol group). Histological alterations during ischaemia/reperfusion were markedly less extensive in the perfusion groups than in control. The patients treated perioperatively with arterial flushing perfusion had an overall mortality of 10%. CONCLUSION: Vascular flushing perfusion with oxygen radical scavengers prevents radical induced ischaemic damage and may prolong the warm ischaemic tolerance time of the intestine. PMID- 7552526 TI - Plasminogen activator and plasminogen activator inhibitor expression by normal and aneurysmal human aortic smooth muscle cells in culture. AB - OBJECTIVES: Matrix metalloproteinases may play a major role in aortic wall degeneration causing abdominal aortic aneurysm (AAA). Available plasmin, required for MMP zymogen activation, is regulated by plasminogen activators (uPA, tPA) and plasminogen activator inhibitors (PAI-1, PAI-2). To appraise the impact of these regulatory proteins on aortic wall degeneration, expression at the level of translation and transcription was determined in human aortic smooth muscle cell (SMC) cultures. METHODS: Explants were derived from aneurysmal aortic tissue (n = 6) and from three male organ donors. Cells were also stimulated with 0.1-1.0 ng/ml IL-1 beta. RESULTS: uPA antigen and tPA antigen in the conditioned medium (CM) of normal SMCs were not detectable but AAA SMCs produced uPA (values are given as median (interquartile range)), [1.97 ng/ml/10(6) cells (1.50-8.21)] and tPA [7.02 ng/ml/10(6) cells (6.20-14.90)]. uPA antigen was absent from normal SMC cell extract (CE) but present in small amounts in AAA SMC CE [0.80 ng/mg protein (0.40-1.24)]. tPA was detectable in the CE of normal SMCs [3.2 ng/mg protein (1.90-3.40)] but at a lower concentration than in AAA SMCs [13.14 ng/mg protein (7.20-27.00)] (p < 0.05). IL-1 beta suppressed production of uPA and tPA in both types of SMCs. The difference in baseline PAI-1 release from normal SMCs and AAA SMCs was not statistically significant. Selective upregulation of PAI-1 was observed for AAA SMC after IL-1 beta stimulation. Northern analysis of AAA SMCs grown in serum containing medium exhibited clear bands for uPA. CONCLUSIONS: Human AAA SMCs are a source of elevated plasminogen activators. In the absence of a reciprocal rise of PAI an imbalance is created favouring proteolysis. PMID- 7552525 TI - Intravascular ultrasound predictors of outcome after peripheral balloon angioplasty. AB - OBJECTIVE: This study investigates the potential role of intravascular ultrasound (IVUS) in the outcome in patients undergoing percutaneous transluminal angioplasty (PTA) of the superficial femoral artery. MATERIALS: Angiographic and the qualitative and quantitative IVUS data obtained at the narrowest site derived from 39 patients before and after PTA were analysed. RESULTS: Angiographically the diameter of the remaining stenosis seen after PTA was classified as < 50% in 31 patients (success); in eight patients a failure was encountered. Evaluating at 6 months the functional and anatomic results of the PTA in 31 patients, the intervention was a success in 14 patients (Group I) and a failure in 17 patients (Group II). The remaining eight patients defined as angiographic failure following PTA comprised Group III. Neither qualitative nor quantitative IVUS data obtained before PTA could predict outcome. Conversely, after PTA, the extent of dissection was significantly more severe in Groups II and III than in Group I. Similarly, significant differences were found between Groups I and II for mean free lumen area (13.2 vs. 9.7 mm2, respectively) and mean free lumen diameter (4.1 vs. 3.5 mm, respectively). Quantitative data obtained in Group II were similar to those in Group III. CONCLUSION: This preliminary study demonstrates that following PTA the extent of dissection, free lumen area and diameter seen with IVUS are predictive factors of patency. Future studies with more patients are mandatory to further highlight the sensitivity of these observations. PMID- 7552527 TI - The Gracz arteriovenous fistula evaluated. Results of the brachiocephalic elbow fistula in haemodialysis angio-access. AB - OBJECTIVES: To assess fistula patency and complication rates in braciocephalic elbow fistulas of both the Gracz and the side-to-side configuration. DESIGN: Retrospective clinical study. METHODS: Life table patency and complications of 50 Gracz fistulas and 23 side-to-side elbow fistulas created between 1988 and 1993 were reviewed. RESULTS: The cumulative patency for the elbow fistulas was 84% after 1 year and 78% after 3 years. No difference was found between the Gracz fistula and the side-to-side elbow fistula. Stenosis was the most frequent indication for revision and thrombosis was the most frequent cause of fistula failure. CONCLUSION: The elbow fistula has a long patency with few complications and performs as well as wrist fistulas and better than the graft fistulas reported in the literature. The Gracz elbow fistula has results as good as the side-to-side elbow fistula. Graft fistulas should be reserved for tertiary procedures only. PMID- 7552528 TI - Simple hyperaemia test as a screening method in the postoperative surveillance of infrainguinal in situ vein bypasses. AB - OBJECTIVES: To develop a simple protocol for ultrasound Duplex surveillance of infrainguinal vein bypasses. DESIGN: The value of three Doppler waveform parameters, obtained from a single point of the bypass, for identification of stenoses was studied in 91 in situ vein bypasses. Midgraft peak systolic velocity (PSV), pulsatility index (PI) and ratio of hyperaemic and resting time-average mean velocities (TAMV), (TAMV ratio = TAMVhyperaemia/TAMVrest) were correlated with the presence and severity of stenoses as assessed by conventional Duplex scanning and ankle-brachial index (ABI) measurements. The optimal value of the waveform parameters for discrimination between bypasses with and without evidence of stenoses was determined by receiver operating characteristics (ROC) analysis. MAIN RESULTS: Complete Duplex scanning of the entire graft revealed an increase in the peak systolic velocity by a factor 2.5 indicative of significant stenoses in 24 (26%) patients. A PSV below 55 cm/s was a poor indicator of stenoses (sensitivity 46%, specificity 76%) and PI < or = 3.8 only allowed suboptimal discrimination between normal and stenotic bypasses (sensitivity 63%, specificity 75%). The hyperaemic response assessed by TAMV ratio proved the best parameter for identification of graft stenoses. A TAMV ratio of 2.0 or less correctly identified 21 of the 24 lesions (sensitivity 88%, specificity 75%) and none of the three bypasses with evidence of stenoses and TAMV ratios exceeding 2.0 failed during follow-up. CONCLUSIONS: Single point waveform analysis of vein bypass velocity profile at rest and during reactive hyperaemia is a simple screening method providing diagnostic and prognostic information which may be of value in the postoperative surveillance of infrainguinal vein bypasses. PMID- 7552529 TI - Late reoperation in vascular surgery. AB - OBJECTIVES: Assessment of late reoperation (after 30 days) following vascular surgery. DESIGN: Analysis of a prospectively collected database of consecutive patients undergoing vascular surgery. SETTING: A single teaching unit's experience between 1986-1993. MATERIALS: Patients undergoing 2501 primary arterial reconstructions. CHIEF OUTCOME MEASURES: Reoperation after 30 days. MAIN RESULTS: One hundred and fifty eight patients (6%) underwent further operations, at more than 1 month after the primary procedure. Primary procedures at highest risk for reoperations were axillobifemoral bypasses and femorodistal bypasses with respective late reoperation rates of 20% and 16%. The majority of patients required late reoperation because of graft occlusion or stenosis. Overall, of the 158 late reoperations performed, 114 were related to the same arterial segment with the same presenting symptoms as the primary operation, and 44 for a different indication. A second or subsequent reoperation was required in 54 patients and the overall operative mortality was 11%. CONCLUSION: Patients undergoing certain vascular procedures, should be informed of the high risk of a subsequent procedure when consent is obtained. PMID- 7552530 TI - Culture of human adult endothelial cells on endarterectomy surfaces. AB - OBJECTIVES: Endothelial cell seeding of prosthetic grafts has not been as successful as initially hoped and the application of seeding technology to alternative reconstructive procedures such as endarterectomy and angioplasty has been increasingly considered. The success of such seeding depends on the ability of the seeded cells to attach to, and form a monolayer on the endarterectomised vessel wall which was the aim of this study. METHODS: Using a seeding chamber model, heterologous human adult endothelial cells were seeded onto fresh human endarterectomy specimens and cultured. Studies of endothelial call adherence to endarterectomy specimens were performed using 111-Indium oxine labelled cells using methodology analogous to graft seeding. RESULTS: Mean endothelial cell adherence of 70% (S.D. 10%) after 1 h incubation was achieved and the successful development of a monolayer of human adult venous endothelial cells on endarterectomised arteries was demonstrated in vitro. CONCLUSIONS: These results indicate that closed endarterectomy appears to offer a surface with cell attachment that is superior to prosthetic grafts. Where femoral endarterectomy is appropriate, endothelial seeding potentially offers a method of reducing thrombogenicity and intimal hyperplasia, improving patency and avoiding a prosthetic graft whilst preserving collateral circulation and autologous vein. PMID- 7552531 TI - Lymph drainage and the development of post-reconstructive leg oedema is not influenced by the type of inguinal incision. A prospective randomised study in patients undergoing femoropopliteal bypass surgery. AB - OBJECTIVES: To see whether the type of groin incision influenced the degree of postoperative leg oedema and the occurrence of lymphatic damage. DESIGN: Prospective randomised clinical study. SETTING: University Department of Surgery. MATERIALS: Twenty-four patients undergoing femoropopliteal bypass reconstruction with either a lateral groin incision (Group A, n = 12) or a direct incision over the femoral vessels (Group B, (n = 12). CHIEF OUTCOME MEASURES: The leg volume increase was measured according to the formula of a truncated cone. Deep venous thrombosis was excluded by air plethysmography and colour-coded Duplex scanning. Lymphatic lesions were detected by lymphoscintigraphy using 99mTc labelled human serum albumin. MAIN RESULTS: One week following vascular reconstruction the median leg volume increase was 24.5% in Group A vs. 23.3% in Group B (NS). Lymphoscintigraphy revealed obstruction of the lymphatics in five patients of Group A vs. three patients of Group B (NS). Neither the occurrence of lymph cysts nor extravasation of lymph differed between the two groups. In seven patients no lymphatic lesion was observed. Patients with interruption of the lymphatics (n = 8) had a higher leg volume increase compared to the remaining patients with no or minor lymphatic lesions, 31.2% vs. 19.6%, respectively (p < 0.05). CONCLUSIONS: Leg oedema and the occurrence of lymphatic damage following femoropopliteal bypass surgery is not reduced by applying a lateral approach to the femoral artery in the groin. However, the higher leg volume increase in patients with lymphatic obstruction indicates that lymphatic damage could play a part in the leg oedema formation. PMID- 7552532 TI - Results of vascular reconstructions for atherosclerotic arterial occlusive disease of the lower limbs in young adults. AB - OBJECTIVE: To evaluate early and long-term results of vascular reconstructions for arterial atherosclerotic occlusive disease (AOD) of the lower limb in young patients under the age of 40 years. DESIGN: Retrospective study. SETTING: University hospital. MATERIALS: Twenty-nine young adults, who underwent vascular reconstruction for histologically proven AOD of the aortoiliac and/or femoropopliteal segments in a 15-year period. CHIEF OUTCOME MEASURES: Early and late mortality, failure of vascular reconstructions, additional procedures, other manifestations of atherosclerosis, recurrence of symptoms. MAIN RESULTS: Nine patients (31%) died, seven related to atherosclerotic disease. In 21 patients (72%) initial vascular reconstruction(s) failed. Twenty-two patients (76%) underwent surgery for failures and/or progression of AOD in other segments of the lower limb. Amputation was performed in five patients (17%). At the end of the follow-up period only 25% of surviving patients were asymptomatic. CONCLUSION: Young patients undergoing vascular reconstructions for AOD of the lower limbs, in particular those who initially have extensive and progressive atherosclerosis, have a poor outcome in terms of a high mortality and a high operative failure rate. A liberal attitude towards reconstructive surgery, particularly in claudication, is not warranted. PMID- 7552533 TI - Management of lower limb ischaemia associated with the use of intra-aortic balloon pumps during cardiac surgery. AB - OBJECTIVE: To audit the lower limb vascular complications associated with the use of an intraaortic balloon pump (IABP) on a cardiothoracic unit over a 12 month period. DESIGN: Retrospective analysis. SETTING: Regional university cardiothoracic unit. PATIENTS: Fifty four IABPs inserted into 51 patients. RESULTS: Seventeen patients (33%) died from cardiogenic shock in the immediate postoperative period. Of the remaining 34 patients (37 IABPs), lower limb vascular complications occurred in nine patients (26%) who underwent 11 IABP insertions (30%). Vascular complications included groin haematomas (n = 2 insertions), compartment syndrome (n = 2 insertions), femoral artery trauma (n = 7 insertions). CONCLUSIONS: Prompt management by peripheral vascular surgeons resulted in limb salvage in 10 legs and only one death from a pulmonary embolus. PMID- 7552534 TI - The impact of renal revascularisation on renal dysfunction. AB - AIM: To determine the value of kidney revascularisation in patients with impaired renal function and correctable renal artery stenosis, the authors reviewed their surgical experience from 1978 to 1990. PATIENTS AND METHODS: The study population included 23 patients with ischaemic nephropathy whose preoperative baseline creatinine level exceeded 20 mg/l (range 21-65 mg/l). This represents 20% of all patients operated on for renal artery disease during the same time interval. Preoperative risk profile, operative mortality, impact on hypertension and on renal function, and late survival were analysed. Renal function response to kidney revascularisation was defined as favourable (20% or more reduction of serum creatinine), moderate (stabilised serum creatinine values) or bad (further deterioration of renal function). All patients had atherosclerotic renal artery disease, involving a solitary kidney in five, both kidneys in 15 and one of the two kidneys in three patients. Hypertension was present in 74%. Revascularisation was unilateral in 10, bilateral in nine and associated with controlateral nephrectomy in four patients. RESULTS: Four patients died postoperatively (three myocardial infarctions, one stroke). Four patients needed postoperative short term dialysis. After operation, renal function improved in 13, stabilised in six and deteriorated in four patients (of whom two died). Follow-up among the survivors averaged 46 months. The mean serum creatinine value at last follow-up visit was 26.2 mg/l, a decrease of 7.7 mg/l compared to preoperative values (p < 0.05). Overall, 69% of azotemic patients submitted to renal revascularisation manifested a favourable response (45% improved and 24% stabilised). Three patients required long-term dialysis. The 5-year survival rate was 48%. CONCLUSION: These data suggest that kidney revascularisation in patients with ischaemic nephropathy can restore or stabilise renal function, preventing evolution and end-stage renal disease and dialysis dependency. PMID- 7552535 TI - The influence of age on operative mortality and long-term relative survival following emergency abdominal aortic aneurysm operations. AB - OBJECTIVE: To study operative mortality and long-term survival following emergency operations for abdominal aortic aneurysm. DESIGN: Retrospective survey in a university hospital. MATERIALS: Two hundred and twenty-seven patients with median age 72 years, (17% women). METHODS: Founded on data from the Norwegian Registrar's Office, operative mortality and long-term survival was estimated using the life-table method. Expected survival for demographically matched subgroups was calculated from death rate tables issued by the Norwegian Central Bureau of Statistics. RESULTS: Operative mortality was 41% for the 175 patients with ruptured aneurysms and 17% for the 52 with imminent rupture. The 6-year survival rate was 61% for all the successfully operated patients, and not different from that of a demographically matched population. For the patients of 72 years or older the 6-year survival rate was 53%. This was equal to that of an age and sex matched population. The younger patients had an observed 6-year survival rate of 64%, which was significantly lower than the expected of 84%. The standard mortality rate for this group was 2.25. No statistically significant difference in long-term survival was detected between the two age groups. CONCLUSIONS: Age at the time of the operation for a symptomatic abdominal aortic aneurysm does not seem to influence long-term survival. Consequently, younger patients experience a higher relative mortality compared to the older. PMID- 7552536 TI - Colour duplex ultrasonographic imaging and provocation of popliteal artery compression. AB - OBJECTIVES: Provoked compression of the popliteal artery by active plantar flexion of the foot is still mainstay in the assessment of the popliteal artery entrapment syndrome. In this study, the effects of several provocation manoeuvres of the foot on the flow patterns of the popliteal arteries of normal healthy volunteers were evaluated. DESIGN: Experimental study among healthy volunteers in a vascular laboratory. MATERIALS AND METHODS: The popliteal artery of 16 healthy volunteers was studied with Duplex scanning in rest and during active and passive plantar and dorsal flexion of the foot. RESULTS: Active plantar flexion was the only movement that influenced popliteal artery flow patterns. Changes were found in 27 arteries (85%), consisting of either a complete occlusion (n = 19, 59%), a significant lumen reduction (n = 4, 13%) or a low flow state (n = 4, 13%). CONCLUSION: Compression of the popliteal artery by active plantar flexion of the foot is a physiologic phenomenon and its value in the assessment of patients suspected of the popliteal artery entrapment syndrome is limited. PMID- 7552538 TI - The long-term outcome of infrainguinal vein graft surveillance. AB - OBJECTIVES: Vein graft surveillance is of proven benefit in improving graft patency at least in the first year after arterial bypass surgery. The aim of this study was to look at the longer term outcome of a vein graft surveillance programme on graft patency. METHODS: One hundred and twelve consecutive vein grafts in 106 patients were prospectively entered into a vein graft surveillance programme. The median (range) follow up was 34 (1-76) months. RESULTS: There were 23 (21%) early (less than 1 month) failures and a further 15 grafts occluded during follow up. The primary, primary assisted and secondary patencies at 4 years were 27%, 59% and 67% respectively. Thirty-eight grafts (34%) developed at least one stenosis at a median interval of 5 months after the operation. Eight (21%) of these stenoses occurred more than 12 months after surgery and would have been missed had surveillance been curtailed after 1 year. CONCLUSIONS: The benefits of graft surveillance extend beyond one year and surveillance should therefore continue indefinitely. PMID- 7552539 TI - Treatment of intermittent claudication: the impact on quality of life. AB - OBJECTIVES: To measure changes in claudicant's quality of life after surgery, angioplasty or unsupervised exercise. To explore the relationship between clinical indicators of limb perfusion and patient's perception of health change. DESIGN: Prospective study. SETTING: University Hospital vascular outpatients. MATERIALS AND METHODS: 202 claudicants referred for Duplex of lower limb arterial disease over a 12 month period. The short form 36 questionnaire was used to determine quality of life. Ankle pressures and walking distances were determined. MAIN RESULTS: The SF-36 was completed by 186 patients (92%) before and after treatment (34 operative patients, 74 angioplasty and 78 treated by exercise alone). Baseline quality of life was worse in surgical patients. Unsupervised exercise produced minimal changes in quality of life. Angioplasty and operation produced similar, significant improvements in physical functioning and pain. Changes in physical function or pain scores were unrelated to changes in ankle pressure. CONCLUSIONS: Unsupervised exercise programs are unlikely to significantly improve patient's quality of life. The benefits of surgery and angioplasty support a relaxation in the indications for investigation and treatment of claudicants. Patients with impaired perceived health should not be denied treatment on the basis of preintervention ankle pressure or walking distance alone. PMID- 7552537 TI - Oral ciprofloxacin versus intravenous cefuroxime as prophylaxis against postoperative infection in vascular surgery: a randomised double-blind, prospective multicentre study. AB - OBJECTIVES: To test the hypothesis that oral ciprofloxacin is equally effective as intravenous cefuroxime in preventing postoperative infectious complications in patients undergoing peripheral arterial surgery involving the groins. DESIGN: Prospective, randomised, double-blind multicentre study. MATERIALS: 580 patients undergoing arterial surgery involving the groins were randomised to ciprofloxacin (Ciproxin, Bayer) 750 mg x 2 p.o. or cefuroxime (Zinacef, Glaxo) 1.5 g x 3 i.v. given only on the day of surgery. The primary endpoint was wound/graft infection within 30 days postoperatively. Wound infection was defined as pus. RESULTS: The wound infection rate in the ciprofloxacin group was 9.2% (27 patients) and in the cefuroxime group 9.1% (26 patients) according to intention to treat. For correct treatment the corresponding numbers were 9.5% (23 patients) and 9.7% (22 patients), respectively. There were three graft infections (0.5%). The infection rate was 7.1% (31/433) in the absence and 14.9% (22/147) in the presence of distal ulcers (p < 0.05). S. allreus was the most common bacteria isolated. Forty percent of the wound infections were localised to the groins. By multivariate analysis presence of distal ulcer was the only factor of prognostic significance. CONCLUSIONS: The infection rate was similar in the two groups. Thus, oral administration of ciprofloxacin is an attractive, cost-effective and safe alternative to prophylaxis in vascular patients capable of taking oral medication on the day of surgery. PMID- 7552540 TI - Arterial complications of the thoracic outlet syndrome. AB - OBJECTIVES: Arterial complications due to compression of the thoracic outlet are uncommon. The objective of this study was to review our fairly extensive experience with this problem with particular reference to its management. METHODS: Patients entered into the Vascular Clinic database were reviewed over an 11 year period. Twenty six records were found. In 24 patients the vasculopathy was caused by a cervical rib (complete in 15) and in two by an anomaly of the first rib. In all patients the basic arteriopathy was a fibrous structure with a post-stenotic aneurysm in 13. Seventeen presented with a fixed pulse deficit; 13 had a palpable aneurysm and 12 had distal embolisation. RESULTS: Two patients refused operation. In 22 with cervical rib, the rib was removed via a supraclavicular incision, an anterior scalenectomy was performed and the arterial pathology repaired on its merit, usually by vein graft replacement or bypass. In two with first rib anomalies these were resected by the transaxillary route. Twenty three patients have been followed for between 3 months and 10 years; 20 are cured and three have residual claudication. CONCLUSIONS: Our results show that simple excision of the cervical rib via the supraclavicular route together with vascular reconstruction is adequate. This is in disagreement with the view of those who advocate routine excision of the first rib in addition to cervical rib excision. PMID- 7552541 TI - Aortic aneurysms in patients with autoimmune disorders treated with corticosteroids. PMID- 7552542 TI - Oesophago-vascular fistulae secondary to benign peptic ulcer disease: a rare cause of fatal haemorrhage. PMID- 7552543 TI - Superior mesenteric artery thrombosis in a patient with the antiphospholipid syndrome. PMID- 7552546 TI - Femoropopliteal bypass. PMID- 7552544 TI - Aortic graft infection with mycoplasma (Ureaplasma urealyticum). PMID- 7552545 TI - Native hepatic artery pseudoaneurysm after liver transplantation: an unusual presentation with biliary leak. PMID- 7552547 TI - Thoracoscopic sympathectomy. PMID- 7552548 TI - Thoracoabdominal aneurysms. PMID- 7552549 TI - Limb threatening ischaemia. PMID- 7552551 TI - Structure: the unspoken word in community treatment. PMID- 7552550 TI - Mesothelial or endothelial? PMID- 7552552 TI - Do hospitalized psychiatric patients have a right to smoke? PMID- 7552553 TI - The American Academy of Child and Adolescent Psychiatry's managed care strategy. PMID- 7552554 TI - Trends in research on temperament. PMID- 7552555 TI - Special section on assertive community treatment: an introduction. PMID- 7552556 TI - Assertive community treatment: an update of randomized trials. AB - OBJECTIVE: Results of randomized clinical trials of assertive community treatment for seriously mentally ill patients published between 1990 and 1994 are reviewed to synthesize the state of knowledge about this research and to clarify continuing research directions. METHODS: Randomized trials of interventions that used treatment principles and practices consistent with the Program for Assertive Community Treatment model or close adaptations whose results were published since 1990 were identified by literature searches using MEDLINE and PsychLit and by contact with investigators of ongoing trials. RESULTS: Controlled clinical trials have been conducted with a wide range of severely mentally ill populations, including patients in Great Britain, patients with recent-onset schizophrenia, veterans, dually diagnosed clients, and homeless persons. Methodological improvements in some studies include increased attention to monitoring the experimental and comparison interventions, as well as larger sample sizes and longer duration of the clinical trials than in earlier efficacy trials. Strong positive effects of assertive community treatment on hospital days and on patient and family satisfaction were found. Gains in functional outcomes, such as employment, may require interventions specifically targeted to these outcomes. CONCLUSIONS: Questions about the role of assertive community treatment as time limited treatment, as an adjunct to other services and treatment, or as a comprehensive and continuous service system for adults with severe mental illness require further research. The growing research base should provide valuable information on costs, outcomes, and indications for assertive community treatment that can be evaluated by policy-makers. PMID- 7552557 TI - Dissemination of assertive community treatment programs. AB - OBJECTIVE: The study sought to estimate the number of programs in the U.S. for severely mentally ill adults that used the assertive community treatment model and to describe variations in characteristics of the intervention across programs. METHODS: Assertive community treatment programs identified by state mental health authorities completed a 12-item survey. The survey included questions on caseload, composition of the treatment team, nature of services, and structure of service provision. RESULTS: A total of 303 of 340 programs (89 percent) identified by states responded to the survey. More than 75 percent provided most of their services in the field, delivered medications, included medical staff on the assertive community treatment team, and had caseload ratios of less than 20 consumers for each provider. CONCLUSIONS: Assertive community treatment programs have disseminated quite unevenly across 33 states, with the highest concentrations of programs in midwestern and eastern states. PMID- 7552558 TI - Implementing assertive community treatment teams. AB - The Connecticut Department of Mental Health began creating assertive community treatment teams in 1987. The authors describe the approach taken by the department in defining the assertive community treatment model, in creating new assertive community treatment teams, and in monitoring the creation and functioning of these teams to ensure that fidelity to the assertive community treatment model is maintained. Assertive community treatment teams can be created even in the absence of funding for new staff by reconfiguring current community based staff and by moving staff from state hospitals to the community. Preliminary data from a randomized trial in Connecticut comparing assertive community treatment with high-quality case management in areas with an array of community services indicated that the intended models were replicated, with variations in practice style across programs. Clients in assertive community treatment were in the hospital about half as often as clients in standard services and were also less likely to be without a permanent residence. Training and ongoing monitoring of assertive community treatment teams are necessary to detect practices that diverge from the intervention model so that corrective action can be taken. PMID- 7552559 TI - Modifying the PACT model to serve homeless persons with severe mental illness. AB - The success of the Program for Assertive Community Treatment (PACT) has led to its replication with different client populations, especially those who are underserved by the traditional treatment system. This paper describes a program in Baltimore that has adapted the PACT model to serve homeless persons with severe mental illness. Although the essential ingredients and philosophy of the original model were maintained, the original team approach has been modified by the use of "miniteams." All staff share knowledge of all program clients through formal mechanisms such as daily meetings; however, each client is assigned to a miniteam composed of a clinical case manager, a psychiatrist, and a consumer advocate. Another deviation from the PACT model is that services can be time limited. The authors describe four phases of treatment and problems, including interventions characteristic of each phase. PMID- 7552560 TI - Evaluating use of continuous treatment teams for persons with mental illness and substance abuse. AB - OBJECTIVE: Continuous treatment teams serving persons with co-occurring severe mental disorders and substance abuse disorders at seven sites in New Hampshire were evaluated to determine their fidelity to a model based on the Program for Assertive Community Treatment. METHODS: Continuous treatment teams and standard case management programs at the seven sites were evaluated on 13 criteria for fidelity to the continuous treatment team model over a 27-month period. Data sources included clinicians' activity logs, agencies' management information systems, interviews, observation of staff activity and practices, and clinical records and other documents. RESULTS: The continuous treatment teams scored significantly higher than the case management programs on ten of the 13 criteria. The teams were more effective than the case management programs in implementing substance abuse treatment. CONCLUSIONS: Evaluation of the programs' fidelity to the model criteria allowed differentiation of successfully implemented continuous treatment teams from standard case management and from an unsuccessfully implemented team. The results confirm the need for careful measurement of model implementation and for investigation of organizational issues such as administrative support and clarity of program mission. PMID- 7552562 TI - Implementation and activities of protection and advocacy programs for persons with mental illness. AB - In 1986, following public hearings, the United States Congress enacted a federal grant program to enable the states and territories to create independent protection and advocacy programs to investigate reports of abuse and neglect of persons with mental illness in residential facilities and to pursue legal, administrative, and other remedies on behalf of those persons. The author discusses implementation of the law and performance of the protection and advocacy agencies in the program's first six years. About 20,000 individuals were served by the program in 1991 and in 1992. The largest category of complaints in those years concerned access to services. Besides representing individuals, protection and advocacy agencies provide referral and information services, public education, outreach, training, and class-action representation. As mental health systems change and fewer persons live in residential facilities, access to protection and advocacy services for persons with mental illness living in the community will become increasingly important. Advocacy will also be important in ensuring enforcement of new laws and policies affecting care and treatment. PMID- 7552561 TI - A multisite study of client outcomes in assertive community treatment. AB - OBJECTIVE: This study examined outcomes of clients admitted to assertive community treatment programs simultaneously implemented at six sites in northeastern Indiana. METHODS: A total of 212 clients at risk for psychiatric rehospitalization were assessed at baseline and at six-month intervals for 18 months after admission to assertive community treatment programs. Data on rehospitalization, quality of life, and level of functioning were compared using t tests. Progressive improvement was also examined by linear trend analysis. RESULTS: Frequency of psychiatric hospitalization was reduced by one-third and the number of inpatient days by 50 percent after admission to the program. Improvements were progressive, with continued reductions over the 18-month period. Progressive improvements also occurred in quality of life as measured by both client and staff ratings. Case managers rated clients as having improved family and social support, increased self-reliance and independence, and improved daily living skills. Clients reported significantly more legal problems, which may have been an artifact of increased monitoring during treatment. A key element of the programs' success was the position of clinical coordinator, important functions of which are described. CONCLUSIONS: Results of this study provide support for wide-scale dissemination of assertive community treatment as an effective form of community care for persons with serious mental illness. PMID- 7552563 TI - Employment supports for people with mental illness. AB - People with psychiatric disabilities often require extensive support to maintain employment. The authors describe five areas that supported employment programs should carefully examine when creating a supportive workplace environment: "natural" co-worker supports, personal network supports, training supports, self management supports, and organizational supports. The different support needs of persons with developmental disabilities and those with psychiatric disabilities are highlighted, particularly in regard to how social stereotypes of these two groups influence decisions about how to provide support. The authors also discuss strategies that can be used by transitional employment programs for creating more effective supports within the limitations of the transitional setting. PMID- 7552564 TI - Factors influencing disposition decisions for patients seen in a psychiatric emergency service. AB - OBJECTIVE: The study examined factors influencing clinicians' decisions about disposition of patients seen in a psychiatric emergency service. METHODS: A stratified random unduplicated retrospective sample of 378 patient records was drawn from the records of 1,823 patients who visited the emergency service of an acute care psychiatric hospital in Israel during a seven-month period. Patients were selected from each of the following dispositions: not admitted (N = 96), discharged after brief observation in the emergency service (N = 90), admitted to a open unit (N = 104), and admitted to a locked unit (N = 88). Data on demographic and clinical characteristics of patients and on some clinician and system variables were analyzed using univariate statistical techniques and stepwise logistic regression. RESULTS: Patients were more likely to be admitted if they were judged by clinicians to be suicidal, had more than three previous hospitalizations, were psychotic, had suicidal behavior as the presenting complaint, and were brought to the hospital involuntarily. Variables favoring assignment to a locked unit were age between 20 and 30, dangerousness to self or others, male gender, and a low Global Assessment of Functioning score. CONCLUSIONS: Patients' level of psychopathology and dangerousness were the primary factors influencing clinicians' decisions about disposition of patients from the emergency service. PMID- 7552565 TI - Therapeutic alliance and outcome in a VA intensive case management program. AB - A total of 143 clients and their case managers in a Veterans Affairs (VA) intensive case management program modeled on the Program for Assertive Community Treatment rated their therapeutic alliance after two years in the program. Strong case-manager-rated alliance was associated with reduced symptom severity and improved global functioning as rated by independent assessors; it was also associated with higher client ratings of community living skills and more positive outcome as perceived by both clients and case managers. Strong client rated alliance was associated only with more positive client-perceived outcome. Alliance ratings were not associated with use of inpatient psychiatric hospitalization. The case manager-client alliance appears to be a significant component of therapeutic effectiveness. PMID- 7552566 TI - Factors predicting patients' satisfaction with managed mental health care. AB - Patients' satisfaction with a managed mental health care program was examined using a mail survey that included questions about patients' perceptions of various elements of service delivery and of their functioning after treatment. Completed surveys were returned by 292 patients who received outpatient psychotherapy, a 30 percent return rate. Patients who reported better psychological functioning and who rated the managed care staff as helpful and their therapist as skillful and conveniently located were more likely to be satisfied with the services they received. PMID- 7552567 TI - Factors contributing to nurses' difficulty in treating patients in short-stay psychiatric settings. AB - Difficulties experienced by nurses in treating a total of 249 patients in four short-stay psychiatric units were examined using a rating scale that assessed such factors as overall extent of treatment difficulty, patients' problem behaviors, adequacy of treatment resources, and staff members' affective responses. Data from each site were analyzed separately using stepwise hierarchical regression. The findings indicated that patient problem behaviors and treatment resources were comparable between settings. Except for patient violence, none of the problems were consistently related to overall treatment difficulty across settings. The results suggest that treatment difficulty is related to the unique combination of patient characteristics, resource deficits, and treatment philosophies in particular treatment settings and that efforts to reduce treatment difficulty should address setting-specific issues. PMID- 7552568 TI - Clinical decision making during assessment for involuntary psychiatric admission. AB - Twenty-four clinicians (five psychiatrists, five registered nurses, and 14 counselors) participated in a psychophenomenological study of the decision-making process in assessing clients for involuntary psychiatric hospital admission. In interviews, the clinicians were asked to describe a situation in which the admission assessment was especially difficult. Analysis of the interviews identified nine essential structural elements of the decision-making process: systematic and individualized process, state-mandated criteria, investigation of alternatives, decision not made alone, intuitive reasoning, connection with the client, caution, and inability to control all contingencies. PMID- 7552569 TI - Treatment of coexisting substance dependence and posttramatic stress disorder. AB - This paper describes the treatment of a physician who developed posttraumatic stress disorder (PTSD) and a polysubstance use disorder after he was shot and held hostage by a patient. Inpatient treatment combined pharmacological and behavioral approaches, including systematic re-exposure via talking about the event in therapy groups. Standard methods for achieving and maintaining abstinence were used, such as asking for and accepting peer support and discussing painful feelings. Cognitive aspects of treatment included education about interactions between the two conditions. After 12 weeks the physician was free of symptoms and had minimal anxiety when exposed to salient cues of the traumatic event. PMID- 7552571 TI - Differences in managed care. PMID- 7552570 TI - Race and psychiatric illness. PMID- 7552572 TI - Consumer vs. patient. PMID- 7552573 TI - Parainfluenza virus type-3 infection attenuates the respiratory effects of antigen challenge in sensitized guinea pigs. AB - Respiratory viral infections not only exacerbate asthma symptoms but may also be important in the pathogenesis of the disease. We therefore explored the effects of respiratory viral infection on the respiratory response of sensitized guinea pigs to antigen challenge. Lung tissue obtained from uninfected guinea pigs sensitized to ovalbumin aerosol released histamine upon incubation with the antigen in vitro. After antigen challenge in vivo, sensitized animals had significantly greater numbers of eosinophils in their bronchoalveolar lavage fluid than did nonsensitized animals and exhibited airway hyperresponsiveness to methacholine aerosol. When ovalbumin sensitization was initiated 7 days after inoculation with parainfluenza virus type-3 (PI-3), antigen challenge elicited little histamine release from infected lung tissue in vitro. Likewise, subsequent to antigen challenge in vivo, animals failed to exhibit airway hyperresponsiveness or an increased eosinophil population in bronchoalveolar lavage fluid. Similar effects were observed when sensitization was begun 19 days after PI-3 virus inoculation. The mechanism(s) responsible for the attenuated responses to antigen in PI-3 infected animals are unknown but may involve virus induced effects on immune cells. PMID- 7552574 TI - Determination of histidine decarboxylase mRNA in various rat tissues by the polymerase chain reaction. AB - Histidine decarboxylase (HDC) mRNA in various rat tissues were quantitated by using a reverse transcription-polymerase chain reaction (RT-PCR) in which a mouse mRNA was used as an internal standard. The stomach HDC mRNA level was the highest followed by the brain, skin, jejunum, spleen and liver. There was no measurable HDC mRNA in the kidney. The stomach HDC activity was also the highest followed by the brain, skin, spleen, jejunum, liver and kidney. A significant correlation (r = 0.940, p < 0.0001) was observed between the HDC mRNA levels and HDC activities in these tissues. We have also examined the HDC mRNA levels in fasting rats and found that HDC mRNA levels in the stomach were reduced after the 48-hr-fasting with the decrease in HDC activities. These observations indicate that there may exist a gene regulation, at least at the basal level, for the HDC activities in the rats. PMID- 7552575 TI - Renal vasodilation to histamine in vitro: roles of nitric oxide, cyclo-oxygenase products and H2 receptors. AB - The aim of this study was to evaluate the roles of nitric oxide (NO) and prostanoids in vasodilation to histamine in the preconstricted isolated perfused rat kidney. Kidneys were excised from Hypnorm/Hypnovel-anaesthetised Wistar rats and perfused at constant flow in vitro. Renal perfusion pressure was elevated similarly with methoxamine (3 microM) or modified Krebs Henseleit solution containing high KCl (30 mM) and vasodilation to histamine (10, 30 nmol) and papaverine (30, 100 nmol) was then examined before and during perfusion with the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) or the cyclo-oxygenase inhibitor, indomethacin (10 microM). Furthermore, the vasodilator response to 30 nmol histamine was examined in the presence of the H2 receptor antagonist, ranitidine (0.1-10 microM). Vasodilation to histamine (10, 30 nmol) was found to be unaffected by L-NAME (0.3 mM) or indomethacin (10 microM), while ranitidine (0.1-10 microM) antagonised vasodilation to 30 nmol histamine with an estimated pA2 of 6.67. Vasodilation to histamine in the isolated perfused rat kidney is therefore probably independent of NO and prostanoids and mediated by H2 receptors. PMID- 7552576 TI - Conversion of endogenous arachidonic acid to 5,15-diHETE and lipoxins by polymorphonuclear cells from patients with rheumatoid arthritis. AB - The conversion of endogenous arachidonic acid (AA) by polymorphonuclear cells (PMN) from patients with rheumatoid arthritis (RA) was studied before (D0) and one day (D1) after antiinflammatory drug therapy. The biosynthesis of 5,15-diHETE and lipoxins (LXS), were investigated ex vivo, after PMN stimulation by ionophore A23187 without exogenous addition of 15-HETE. The eicosanoids were resolved by RP HPLC and simultaneously detected at 246 and 302 nm respectively. Large amounts of 5,15-diHETE (50 to 400 ng/10(7) PMN) and significant levels of LXS (from 2 to 20 ng/10(7) PMN) were produced with individual differences between donors. Metabolite levels varied between patients but this work showed for the first time a linear relationship between the amounts of 5,15-diHETE and LXS. Moreover LXS production after treatment may be related to long-term clinical improvement of patients. PMID- 7552577 TI - Inhibition by actinomycin D of neurogenic mouse ear oedema. AB - We have investigated the effects of actinomycin D on mouse ear oedema induced by capsaicin, neuropeptides, and established inflammatory mediators. Actinomycin D (0.5 mg/kg, i.v.) significantly (P < 0.01) inhibited ear oedema induced by topical application of capsaicin, while adriamycin (6.0 mg/kg, i.v.) and cycloheximide (6.0 mg/kg, i.v.) had no effect on oedema. The ear oedema induced by intradermal injection of neuropeptides such as mammalian tachykinins, calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), was markedly (P < 0.05, P < 0.01 or P < 0.001) suppressed by actinomycin D. The drug was also effective (P < 0.01 or P < 0.001) in inhibiting bradykinin (BK)- and compound 48/80-induced ear oedema, but did not inhibit oedema induced by histamine, 5-HT, leukotriene C4 (LTC4), and platelet activating factor (PAF) at a dose of 1 mg/kg. In mast cell-deficient W/WV mice, actinomycin D (1.0 mg/kg, i.v.) failed to inhibit substance P (SP)-induced ear oedema whereas spantide (0.5 mg/kg, i.v.) was an effective (P < 0.01) inhibitor of oedema formation. Furthermore, actinomycin D (10-100 microM) dose-dependently prevented histamine release from rat peritoneal mast cells evoked by SP, compound 48/80, and the ionophore A23182, respectively. These results strongly suggest that an inhibitory effect of actinomycin D on neurogenic inflammation is due primarily to the prevention of mast cell activation mediated by neuropeptides, rather than an interaction with DNA or receptors of neuropeptides. PMID- 7552578 TI - Inhibition of some aspects of acute inflammation of guinea-pig lung by intraperitoneal dexamethasone and mifepristone: demonstration of agonist activity of mifepristone in the guinea-pig. AB - We have determined the inhibitory activity of dexamethasone as an inhibitor of histamine-induced plasma protein extravasation (PPE) in guinea-pig lung and skin, and of lipopolysaccharide (LPS)-induced neutrophilia and platelet activating factor (PAF)-induced eosinophilia in guinea-pig lungs. Dexamethasone inhibited PAF-induced eosinophilia in guinea-pig lung (ED50 1.4 mg/kg i.p.). Higher doses of dexamethasone were required to inhibit LPS-induced neutrophilia (ED50 10.8 mg/kg i.p.). However, at doses up to 150 mg/kg i.p. dexamethasone did not inhibit histamine-induced plasma protein extravasation (PPE) in guinea-pig lung, but did inhibit PPE in guinea-pig skin. These preparations have previously been shown to be equally sensitive to inhibition by the beta 2-adrenoceptor agonist salmeterol. Dexamethasone inhibited PAF-induced eosinophilia (5 mg/kg) or LPS-induced neutrophilia (50 mg/kg) when given 3 h or 1 h prior to challenge. Inhibitory activity was lost when dexamethasone was administered 23 h prior to LPS or 1 h after PAF. The glucocorticoid antagonist mifepristone (1-100 mg/kg i.p.) caused dose-related inhibition of PAF-induced eosinophilia but not of LPS-induced neutrophilia. The highest dose of mifepristone used (100 mg/kg) did not reverse the inhibitory actions of dexamethasone (50 mg/kg) on LPS-induced neutrophilia. We suggest that the different inhibitory activity of dexamethasone in the preparations studied indicates differences in the sensitivity of the target cells involved to inhibition by dexamethasone. We also suggest that inhibition of PAF induced eosinophilia by mifepristone reflects the partial agonist activity of this agent, demonstrated by others in different experimental systems. PMID- 7552579 TI - Eicosanoid and gastroprotection by copper derivatives and NDGA. AB - We investigated the effect of oral administration of graded doses of: nordihydroguaiaretic acid (NDGA), CuNSN, a bis(2-benzimidazolyl)thioether and CuCl2 on ethanol-induced gastric damage in the rat and the role of leukotrienes and prostaglandins in attenuating this damage. In the experiments we determined ex-vivo eicosanoid release in the rat gastric mucosa pretreated with the above mentioned compounds. The results indicate that the gastric lesion is accompanied by an increase in mucosa-synthesize LTC4, while PGE2 formation remains unchanged. Pretreatment with NDGA, CuNSN and CuCl2, protects the gastric mucosa from damages and reduces the increase in LTC4 mucosal formation. CuNSN and CuCl2 increase the PGE2 release, while NDGA has no effect on this pathway. These results suggest that one of the possible mechanisms of the NDGA protective effect is related to the inhibition of LTC4 formation, while the PGE2 increase in synthesis together with the leukotriene inhibition could contribute to the protective effect of CuNSN and CuCl2. PMID- 7552581 TI - John M. Eisenberg, MD. AB - The complicated interaction between government, academic medical centers, health care payers, and burgeoning market forces has tested the leadership skills of a generation of academicians with little formal training in economics. The emergence of a new breed of physician investigator with solid business credentials has therefore proved attractive to many segments of the medical community. John M. Eisenberg received his MD from Washington University in 1972, his MBA from the Wharton School in 1976, and shortly thereafter headed the division of general internal medicine at the University of Pennsylvania. In addition to championing the role of the generalist in health care delivery, Eisenberg has also played a major part in the reformation of Medicare reimbursement. He has been a commissioner on the Congressional Physician Payment Review Commission since 1986, serving as chairman since 1993. After assuming the chairmanship of the department of medicine at Georgetown University in 1992, Eisenberg served as an advisor to the Clinton administration during its efforts towards national health care reform. Interviewed in his office in Georgetown, Eisenberg reflected on the economic forces twisting post-graduate medical education, the role of non-physician providers in future health care delivery, and the evolving relationship between specialists and generalists. PMID- 7552582 TI - Wilson's disease: a new gene and an animal model for an old disease. AB - Wilson's disease is an autosomal recessive, inherited disorder of copper metabolism. In normal individuals, copper homeostasis is controlled by the balance between intestinal absorption of dietary copper and hepatic excretion of excess copper in bile. In Wilson's disease, hepatic copper is neither excreted in bile nor incorporated into ceruloplasmin and copper accumulates to toxic levels. The Wilson's disease gene (WND) encodes a putative copper-transporting protein that is expressed almost exclusively in the liver. The predicted structure of the protein product is that of a P-type ATPase with striking homology to bacterial copper transporters and the gene product of another inherited disorder of copper metabolism, Menkes' disease. A rat model of Wilson's disease has recently been identified. The Long-Evans Cinnamon (LEC) rat manifests elevated hepatic copper, defective incorporation of copper into ceruloplasmin, and reduced biliary excretion of copper. The rat homologue of the WND is abnormal in LEC rats. Clinical manifestations of Wilson's disease arise directly from copper-induced damage to hepatocytes (hepatic presentation) or indirectly after the release of copper from the liver with subsequent damage to the brain (neuropsychiatric presentation) and other organs. Genetic heterogeneity (different mutations in a single gene) may account for some of the variability in Wilsonian presentations. The diagnosis of Wilson's disease depends on the demonstration of disordered copper metabolism, manifested as elevated urinary and hepatic copper and low ceruloplasmin levels. However, none of the abnormal findings in Wilson's disease is pathognomonic. Genetic diagnosis, in the absence of family studies, is likely to be difficult since many different mutations result in the disease. Management of Wilson's disease involves decreasing excess levels of copper accumulated in the liver, brain, and other organs. Copper chelation therapy, to increase urinary excretion of copper, is the mainstay of treatment. In addition, oral zinc therapy may be useful at decreasing absorption of dietary copper and rendering tissue copper nontoxic, by increasing the formation of complexes with copper-binding proteins. Liver transplantation can be necessary for individuals with acute hepatic failure or complications of cirrhosis. Gene therapy may evolve in the future; however, medical management is effective in most patients. PMID- 7552583 TI - Acute promyelocytic leukemia: retinoic acid response and resistance. PMID- 7552580 TI - The effect of histamine on the oxidative burst of HL60 cells before and after exposure to reactive oxygen species. AB - During an inflammation neutrophils are stimulated to produce reactive oxygen species (ROS). These ROS induce the release of histamine from mast cells, which are also present at the inflammation site. In this study dibutyryl cAMP differentiated HL60 cells are used as a model for human neutrophils. The effect of histamine on formyl-methionyl-leucyl-phenylalanine (fmlp) stimulated cells is examined. Except for histamine also an accumulation of ROS takes place at the inflammation site and we investigated if ROS can influence the response of the stimulated HL60 cells. It is found that 10(-3) M histamine can inhibit the fmlp induced superoxide anion radical production. This occurs partly via an H2 receptor because H2 antagonists like famotidine, mifentidine and ranitidine could partially antagonize this effect of histamine. When HL60 cells are exposed to hydrogen peroxide or hypochlorous acid (20 min), an increased fmlp response is found while the inhibiting effect of histamine remains unchanged. PMID- 7552584 TI - A glimpse of the coronary microcirculation with myocardial contrast echocardiography. PMID- 7552585 TI - The unveiling of calreticulin--a clinically relevant tour of modern cell biology. PMID- 7552586 TI - Pulmonary responses to purified zinc oxide fume. AB - BACKGROUND: Metal fume fever is a flu-like illness caused by zinc oxide fume inhalation and mediated by unknown mechanisms. It is one of a group of work related febrile inhalational syndromes. We studied bronchoalveolar lavage (BAL) obtained from cigarette smoking and nonsmoking human volunteers after controlled exposure to purified zinc oxide fume to explore the possible roles of proinflammatory cytokines in this condition. METHODS: We studied 14 volunteers after inhalation exposure to purified zinc oxide fume and after sham exposure to air. The mean cumulative exposure was 537 +/- 232 mg min per cubic meter elemental zinc. Twenty hours after exposure we performed BAL. We analyzed BAL cells and studied BAL supernatant for cytokines including tumor necrosis factor alpha (TNF alpha), interleukin(IL)-8, and IL-1 by enzyme-linked immunosorbant assay (ELISA). RESULTS: Polymorphonuclear leukocytes (PMNs) were significantly increased in the BAL fluid obtained post-exposure compared to sham (mean difference = 41.3 +/- 16.8 x 10(3) per mL; p < 0.05). Cumulative zinc exposure positively correlated with exposure-sham differences in BAL supernatant concentrations of both TNF (r2 = 0.58; p = .002) and IL-8 (r2 = 0.44, p = 0.01). Exposure-sham concentration differences in BAL supernatant IL-8 and BAL PMNs were also positively correlated (r2 = 0.60; p < 0.001). Cigarette smoking was not associated with exposure-sham differences in BAL TNF or IL-8, but did demonstrate a packs-per-day dependent increase in BAL supernatant IL-1 (t = 2.3, p = 0.04) post-exposure compared to sham, after taking into account the zinc exposure response. CONCLUSIONS: Purified zinc oxide fume inhalation causes an exposure dependent increase in proinflammatory cytokines and PMNs in the lung. This supports a role for cytokine networking in mediating metal fume fever. PMID- 7552587 TI - Transduction of CD34-enriched human peripheral and umbilical cord blood progenitors using a retroviral vector with the Fanconi anemia group C gene. AB - BACKGROUND: Fanconi anemia (FA) is an autosomal recessive inherited form of bone marrow failure. FA cells are characterized by their extreme sensitivity to DNA cross-linking agents that cause DNA instability and cell death. Four genetic complementation groups for FA have been identified and the gene for the complementation C group (FACC) has been cloned. Genetic transfer of the FACC gene should provide a growth advantage in transduced hematopoietic cells. We have previously demonstrated efficient retroviral-mediated gene transduction and correction of FA(C) cell lines and peripheral blood-derived CD34+ progenitors from patients carrying mutant FACC alleles. In this report we sought to define the optimal conditions for transduction of CD34+ progenitors from mobilized peripheral blood and umbilical cord blood. METHODS: Peripheral blood hematopoietic progenitors were obtained by G-CSF mobilization followed by apheresis. Human fetal cord blood cells were obtained from full-term gestation deliveries. Cells were immunoselected for CD34 antigen expression and then incubated with recombinant retroviruses containing a selectable marker gene (neomycin). Recombinant colony stimulating factors were added to facilitate viral transduction. Cells were plated in methylcellulose and resulting hematopoietic colonies were isolated and analyzed by PCR. RESULTS: Transduction efficiency of peripheral blood progenitors (from normal individuals) using a retrovirus encoding the FACC cDNA was comparable to that of the retroviral producer G1Na.40 currently being used in clinical gene therapy marking studies. We extended our standard transduction protocol to analyze CD34+ and CD34+ CD38-subpopulations of progenitors derived from umbilical cord blood (from normal pregnancies). In addition, we tested whether FACC cDNA transduction could be improved by vector infection supported by autologous stroma. For FA(C) hematopoietic cell infection, vector supernatant transduction in the presence of recombinant human IL-3, IL-6, and SCF was found to be superior to transduction supported by autologous FA(C) patient stroma. CONCLUSIONS: We documented efficient retroviral transduction of umbilical cord blood and peripheral blood enriched for hematopoietic progenitor cells. These results suggest the feasibility of a clinical gene therapy protocol utilizing progenitor cells from both peripheral blood and umbilical cord blood. PMID- 7552588 TI - Inflammatory mediators and bronchopulmonary dysplasia. PMID- 7552590 TI - Abundance of endothelial nitric oxide synthase in newborn intrapulmonary arteries. AB - A monoclonal antibody to endothelial NOS (eNOS) was used to demonstrate the distribution and density of eNOS in the developing porcine lung. Lung tissue from large white pigs aged from less than 5 minutes to 3 months was immunostained and, using light microscopy, distribution of eNOS was assessed by a semiquantitative scoring system. At all ages eNOS was located on the endothelial cells of pulmonary and bronchial arteries and veins. Immunoreactivity for eNOS was greater in the larger, more proximal pulmonary arteries than at the periphery. In the lung of newborn pigs immunoreactivity for eNOS was present in arteries of all sizes but some showed no positive staining. At 2-3 days of age almost all arteries showed positive immunoreactivity. By 3 months of age the amount of eNOS had decreased and was less than that seen in the newborn. The highest level of eNOS was seen immediately after birth when the pulmonary arteries are dilating. eNOS may therefore play an important part in adaptation to extra-uterine life. PMID- 7552589 TI - Low birthweight infants and total parenteral nutrition immediately after birth. III. Randomised study of energy substrate utilisation, nitrogen balance, and carbon dioxide production. AB - This study aimed to investigate energy substrate utilisation and nitrogen balance in low birthweight infants receiving total parenteral nutrition during the first days of life, and in particular, to determine the effect of two different glucose intakes on carbon dioxide production. Twenty infants (mean (SE) birthweight 1314 (65) g, mean (SE) gestation 30.9 (0.4) weeks) were recruited to the study. Immediately after birth they were randomised to a carbohydrate intake of 8 g/kg/day (5.5 mg/kg/minute) or 12 g/kg/day (8.3 mg/kg/minute). After 24 hours they were changed to the alternative regimen which was continued for a further 24 hours. Fat and protein intakes were kept constant throughout the study. Indirect calorimetry was performed during each of the regimens, urine was collected for urinary nitrogen, and substrate utilisation calculated for 12 infants. The carbohydrate utilisation rate was increased during the higher carbohydrate intake. Lipid utilisation rates were significantly different, with net lipid synthesis occurring during high carbohydrate intake. Protein utilisation rates were not influenced by the different carbohydrate intakes. The mean plasma glucose concentration was higher during the high carbohydrate intake but the mean highest and lowest values were not significantly different during the two study periods. A plasma glucose below 2.6 mmol/l was recorded more frequently during the low glucose intake (9/20 v 5/20). Capillary PCO2 values measured during high and low glucose intakes were similar (5.9 (0.2) v 6.2 (0.3) kPa. Carbon dioxide production rates were increased during the higher carbohydrate intake but the differences were not significant. Similarly, there was no significant difference in the respiratory quotients (RQ), oxygen consumption, or energy expenditure during the two study periods. PMID- 7552591 TI - Randomised, double blind trial of two loading dose regimens of diamorphine in ventilated newborn infants. AB - AIMS: To compare the safety and efficacy of two loading doses of diamorphine in 27 ventilated newborn infants in a randomised double blind trial. METHODS: Fifty or 200 mcg/kg were infused intravenously over 30 minutes, followed by a 15 mcg/kg/hour continuous infusion. Serial measurements were made of physiology, behaviour, and stress hormones. RESULTS: Both loading doses produced small but significant falls in blood pressure. The 200 mcg/kg dose produced greater respiratory depression, and two infants deteriorated clinically, requiring resuscitation. Loading reduced respiratory effort in most of the infants, but had little effect on behavioural activity. Stress hormone concentrations were reduced at six hours in both dosage groups; differences between loading doses were not significant. Morphine, morphine-3-glucuronide, and morphine-6-glucuronide were detected in the plasma of all patients. No significant differences in concentrations between loading doses were found. CONCLUSIONS: Diamorphine reduces the stress response in ventilated newborn infants. A high loading dose confers no benefit, and may produce undesirable physiological effects. A 50 mcg/kg loading dose seems to be safe and effective. PMID- 7552592 TI - Randomised controlled trial of allopurinol prophylaxis in very preterm infants. AB - Allopurinol, an inhibitor of xanthine oxidase (an enzyme capable of generating superoxide radicals following hypoxiaischaemia), was investigated in preterm infants to determine its ability to prevent the complications of neonatal intensive care which may be associated with oxidative injury. Four hundred infants of between 24 and 32 weeks' gestation were randomly allocated to receive enteral allopurinol (20 mg/ml) or an equivalent dose of placebo for seven daily doses. At admission, plasma hypoxanthine concentrations were significantly higher in infants who subsequently developed periventricular leucomalacia (PVL), bronchopulmonary dysplasia (BPD), or retinopathy of prematurity (ROP), but there was no difference in the primary endpoint (PVL) between the treated and control groups. The failure of allopurinol prophylaxis in this group of infants is probably related to the complex nature of the pathological processes and to the timing of treatment. If oxidant injury is an important mechanism of cellular injury in these preterm infants, an alternative biochemical modulator would be required, or a combination of agents might be effective. PMID- 7552593 TI - Use of the CRIB (clinical risk index for babies) score in prediction of neonatal mortality and morbidity. AB - A prospective study of the outcome of care of a regional cohort of very low birthweight (< 1500 g) and very preterm (< 32 weeks) infants was carried out. Its aims were to assess the ability of the CRIB (clinical risk index for babies) score, rather than gestational age or birthweight, to predict mortality before hospital discharge, neurological morbidity, and length of stay, and to access CRIB score as an indicator of neonatal intensive care performance. 676 live births fulfilled the criteria and complete data were available for 643 (95%). Compared with gestation and birthweight, CRIB was better for the prediction of mortality, was as good for the prediction of morbidity, and was not as good for the prediction of length of stay. CRIB adjusted mortality did not demonstrate better performance in units providing the highest level of care. Either the CRIB score was not sensitive to performance or the level 3 hospitals in this study were performing badly. On the basis of this analysis purchasers and providers of neonatal intensive care cannot yet rely on the CRIB score as a performance indicator. PMID- 7552594 TI - Ureaplasma urealyticum as a cause of pneumonia in preterm infants: analysis of the white cell response. AB - The tracheal isolation of Ureaplasma urealyticum from critically ill infants was investigated to determine if the organism was associated with an inflammatory response. Twenty nine neonates consecutively admitted for acute respiratory disease, with birthweights of < 1301 g and no evidence of viral, chlamydial, or bacterial infections, were identified. Culture results for ureaplasmas were correlated with white cell counts and clinical and radiographic features. Sixteen infants had tracheal aspirates and/or blood specimens positive for U urealyticum. Pneumonia was diagnosed more frequently in the U urealyticum positive infants than in the 13 who were negative for the organism. The mean total white cell count, absolute neutrophil, and band form counts were significantly higher in the U urealyticum positive group than in the negative group. These data suggest that U urealyticum can induce an inflammatory response in selected individuals who present with clinical, radiographic, and, in some instances, histological features of pneumonia. PMID- 7552596 TI - Fetofetal transfusion in triplets. AB - A case of fetofetal transfusion syndrome (FFTS) in a monochorionic triplet pregnancy, in which all three fetuses shared a common circulation, is reported. All babies were born alive, although two died within two days of delivery. This case highlights the problem of FFTS with accompanying high perinatal morbidity and mortality in naturally occurring monochorionic triplet gestations. PMID- 7552597 TI - Sodium glycerophosphate in the treatment of neonatal hypophosphataemia. AB - Nineteen very low birthweight (mean (SD) gestational age 28 (3) weeks) were parenterally fed nutrition solutions containing inorganic calcium and phosphorus salts. All infants had hypophosphataemia. Plasma concentrations were maintained between 1.5 mmol/l and 2.2 mmol/l. Plasma phosphorus concentrations reached 1.5 mmol/l or greater in three patients after 12 hours, in a further nine patients after 36 hours, and in all patients by 60 hours. Changes in plasma calcium concentrations were not significant. PMID- 7552595 TI - Low birthweight infants and total parenteral nutrition immediately after birth. I. Energy expenditure and respiratory quotient of ventilated and non-ventilated infants. AB - The aim of this study was to determine the energy expenditure and respiratory quotient (RQ) of ventilated and non-ventilated low birthweight infants during the first five days of life, in order to determine optimal feeding regimens. Eighty six infants, of birthweight less than 1750 g, were grouped according to whether they were artificially ventilated or breathing air spontaneously, and whether they were parenterally or enterally fed at the time of study. Energy expenditure and respiratory quotient were measured during days 1-5 and the relation of energy expenditure to several explanatory variables was investigated using multiple regression analysis. The energy expenditure of ventilated infants was less than that of spontaneously breathing infants; the differences were significant on days 1-3. The respiratory quotient (mean (SE)) was greater in intravenously fed infants compared with milk-fed--0.99 (0.03) v 0.92 (0.01) (P < 0.05), with 42% of studies of infants receiving total parenteral nutrition (TPN) producing an RQ of > 1.0 compared with 16.6% of milk-fed infants (P < 0.01). There was a significant correlation between glucose intake and RQ (r = 0.39, P < 0.001). The activity scores were measured during 75 studies and scores were significantly higher in spontaneously breathing milk-fed infants compared with ventilated parenterally fed infants. Factors independently related to energy expenditure were: postnatal age (P < 0.01); milk feeds (P < 0.01); and physical activity (P < 0.05). A mix of carbohydrate and fat from day 1 may not only meet energy needs but may also reduce respiratory quotient. PMID- 7552598 TI - Group B streptococcal infection: when does vertical transmission take place? AB - Expression of CD45 isoforms was used to estimate when group B streptococci had infected a child born with low Apgar scores who subsequently died. The measure suggested that infection was present more than 24 hours before delivery, thus distinguishing perinatal infection as the primary event which preceded intrapartum asphyxia in this case. PMID- 7552600 TI - Soranus of Ephesus (circa AD 98-138) and perinatal care in Roman times. PMID- 7552601 TI - Fatal familial surfactant protein B deficiency. PMID- 7552602 TI - Application of measured spontaneous inspiratory and expiratory times to improve infant ventilation. PMID- 7552599 TI - Mechanism of visceral damage in fetofetal transfusion syndrome. PMID- 7552603 TI - Interleukin-8: a potent neutrophil chemotactic factor. PMID- 7552605 TI - Knee joint proprioception in below-knee amputees. AB - This study determined knee joint proprioception in below-knee amputees using two methods of measurement of knee joint proprioception. The threshold for detection of passive motion and the ability to reproduce specific limb positions were recorded in nine below-knee amputees. A significant difference was observed between the prosthetic and sound limbs in the amputees in the threshold of detection test. The large angles (5 degrees to 25 degrees) involved in position reproduction tests may have provided cues through the cuff suspension. Different strategies are used by below-knee amputees to discern large and small movements of the knee. PMID- 7552606 TI - Arthrodesis after infected knee arthroplasty using long arthrodesis nail. A report of five cases. AB - Safe methods to perform secondary arthrodesis in the treatment of infected total knee arthroplasty (TKA) after revision arthroplasty has failed are increasingly needed as TKA becomes more common. Possible treatments include external fixation (with single- or double-framed fixators), internal fixation, or amputation. This article reports five cases of infected TKA treated with arthrodesis using a long intramedullary nail. Arthrodesis was performed in a two-stage manner, with surgical revision and insertion of a long intramedullary nail. All arthrodeses healed on an average of 4.8 months. PMID- 7552604 TI - Low birthweight infants and total parenteral nutrition immediately after birth. II. Randomised study of biochemical tolerance of intravenous glucose, amino acids, and lipid. AB - This randomised study aimed to compare the biochemical tolerance of three parenteral regimens administered during the first 48 hours of life. Twenty nine infants were randomised to either: (a) glucose 10%; (b) glucose 10%/amino acids; (c) glucose 10%/amino acids/lipid. Blood samples for plasma amino acid profiles, cholesterol, and triglyceride concentrations were taken on arrival in the neonatal unit and again between 36 and 48 hours of life. Arterial or capillary blood gas analysis and blood glucose estimates were performed routinely during the first 48 hours of life. There was a sharp decline in plasma amino acid concentrations in the group following (a) compared with the two groups following (b) and (c) regimens. In all groups plasma triglyceride and cholesterol were not significantly different before and after 48 hours of lipid infusion. Peak mean (SE) bilirubin concentrations (203 (12) v 181 (19) v 220 (20) mumol/l) and the need for phototherapy (nine v eight v five infants) were similar for each of the groups. Hypoglycaemia occurred most frequently during the (b) regimen and least commonly in the (c) group. There are potential health gains from giving parenteral nutrition to low birthweight infants immediately after birth, and this study indicates that restriction of nutritional intake immediately after birth in preterm infants may cause significant metabolic disturbance. This can be prevented by starting a regimen of intravenous amino acids and lipid immediately after birth. PMID- 7552607 TI - Arthroscopic treatment of an avulsion fracture of the intercondylar eminence of the tibia. Case report. AB - Multiple methods exist for reduction and fixation of an intercondylar eminence fracture of the tibia. Modes of treatment are based on the amount of displacement of the fracture. This article describes an arthroscopic technique as an alternative treatment method in a younger patient population. The arthroscopic reduction of the tibial eminence and cast fixations provides no trauma to the physeal plate, and no further operative procedures for hardware removal are required. Associated injuries may be diagnosed and treated at the time of arthroscopy without the need for arthrotomy. PMID- 7552608 TI - Isolated lateral ring meniscus. Case report. AB - Reports of ring menisci are rare. This article describes a case of a congenital lateral ring meniscus and includes a literature review. The anatomy of the menisci of various vertebrates is described with an attempt to discuss the possible origin of the anomaly. Although ring menisci are asymptomatic, they are clinically significant in that they need to be differentiated from old bucket handle tears. It is also easy to fail to visualize the intercondylar bridge between the two "horns" with conventional magnetic resonance imaging. PMID- 7552609 TI - Is MRI justified as a routine preoperative study in the evaluation of the meniscus? PMID- 7552610 TI - The relationship between hematocrit, blood lost, and blood transfused in total knee replacement. Implications for postoperative blood salvage and reinfusion. AB - The relationship between patient hematocrit level, red blood cell volume lost, and blood units transfused is important in determining conservation strategies in patients undergoing total knee replacement surgery. In a series of 30 such patients, 3 (10%) received allogeneic blood, despite preoperative autologous blood donation in 28 patients. There was no evidence that the degree of anemia affected rate or volume of postoperative wound blood drainage. The wound drainage volume that could have been salvaged and reinfused in bilateral procedures was substantial. A combination of one or more conservation techniques along with conservative transfusion practice is necessary to achieve minimal allogeneic blood exposure. PMID- 7552611 TI - Does tranexamic acid reduce blood loss in knee arthroplasty? AB - In a retrospective study of 179 total knee arthroplasties, 70 patients received tranexamic acid (Cyklokapron, Kabi Pharmacia, Uppsala, Sweden) before the tourniquet was released to reduce postoperative blood loss. A group of 109 patients who underwent surgery before this treatment was introduced served as controls. Multiple regression analysis showed that the average postoperative blood loss was 340 mL less in treated patients compared with controls. Blood transfusions were reduced from 2 to 0 units (median values). Complications did not differ between the two groups apart from the number of postoperative hematomas. PMID- 7552612 TI - MRI versus arthroscopy in evaluating knee meniscal pathology. AB - The purpose of this project was to evaluate magnetic resonance imaging (MRI) accuracy for diagnosing and defining meniscal pathology. The charts of 45 patients who underwent both MRI and arthroscopy were viewed retrospectively. All cases were seen by one of two orthopedic surgeons. Magnetic resonance imaging scans were reviewed by staff radiologists at the University of Michigan Hospital Medical Center. Arthroscopy was used as the standard for comparing MRI results. Data were analyzed two ways with respect to questionable (code 3) MRI lesions. First, all code 3 lesions were interpreted as either true positives or negatives. Second, all code 3 lesions that corresponded with a negative arthroscopy finding were interpreted as false positives. The MRI accuracy was 87.8% when considering nonmatching code 3 lesions as true positives or negatives. The accuracy declined to 82.2% when considering code 3 lesions as false negatives. Magnetic resonance imaging sensitivity for medial meniscal and lateral meniscal tears was 84.6% and 87.5%, respectively, regardless of code 3 interpretation. The specificity for medial meniscal tears with and without the negative effect was 71.9% and 84.4%, respectively. The specificity with and without considering code 3 lesions as false positives was 89.2%, and 91.9%, respectively. PMID- 7552613 TI - Orthopedic management of knee dislocations. Comparison of surgical reconstruction and immobilization. AB - Thirty-nine patients with 43 complete knee dislocations managed between 1973 and 1990 were reviewed retrospectively to compare the results of surgical reconstruction to nonreconstructive treatment of these injuries. The average patient age was 34 years and the average follow-up was 5 years (range: 1 to 18 years). Patients were evaluated by physical examination and the Lysholm knee scoring scale. Fourteen knees (33%) sustained popliteal vessel injury and five (9%) required amputation. Peroneal and tibial nerve injuries involved 13 knees (30%). Twenty-five of 39 patients (64%) sustained other associated fractures. Three patients sustained associated paraplegia and nine were lost to follow-up, leaving 25 knees available for follow-up examination. Thirteen knees were managed by surgical reconstruction of their ligamentous injuries and 12 were managed by nonreconstructive means. The surgically treated group had an average Lysholm knee score of 80 compared with the nonreconstructive group with an average score of 66. Average range of motion of was 106 degrees for the surgical group and 95 degrees for the nonreconstructive group. Despite the severity of the initial injury and the potential presence of vascular/nerve injuries, surgical reconstruction provides superior results to immobilization alone in the management of these injuries. PMID- 7552614 TI - Doxorubicin for inflammatory breast cancer: a red humor for a red tumor? PMID- 7552615 TI - Benefit of aggressive multimodality treatment for gastric cancer. PMID- 7552616 TI - Therapy for inflammatory breast cancer: impact of doxorubicin-based therapy. AB - BACKGROUND: Inflammatory breast cancer (IBC) carries an ominous prognosis. Before 1988, women with IBC at our institution were treated with neoadjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with or without vincristine and prednisone (CMF/VP). After 1988, women with IBC were treated with cyclophosphamide, doxorubicin, and 5-fluorouracil (FAC). This study compares these two regimens with regard to response and survival. METHODS: The records of all women presenting between January 1973 and December 1991 with a stage IIIB (T4d, any N, MO) breast cancer with proven dermal lymphatic invasion by tumor cells were reviewed retrospectively. RESULTS: The study comprised 38 women; 28 received CMF (22 CMF, 6 CMF/VP), and 10 received FAC. The overall response rate to induction chemotherapy in the CMF/VP group was 57% (40% PR, 17% CR), and 100% (60% PR, 40% CR) in the FAC group. The median overall survival for women receiving CMF/VP was 18 months compared with 30 months for women receiving FAC (p = 0.02). The median disease-free survivals for the CMF/VP and FAC groups were 6 and 24 months, respectively (p < 0.001). When comparing responders and nonresponders with CMF/VP induction therapy, the responders had a significantly longer overall median survival (24 versus 10 months) (p < 0.001) and disease-free median survival (8 versus 2 months) (p < 0.001). All of the five patients remaining alive received FAC with 80% (four of five) having a complete response. These four patients subsequently underwent mastectomy and radiation. CONCLUSION: This study suggests that a doxorubicin-containing chemotherapy regimen improves overall and disease-free median survivals when compared with the previously used CMF combination in the treatment of IBC. A favorable response to induction chemotherapy also appeared to be associated with an improved survival. PMID- 7552617 TI - Treatment of adenocarcinoma of the stomach with resection, intraoperative radiotherapy, and adjuvant external beam radiation: a phase II study from Radiation Therapy Oncology Group 85-04. AB - BACKGROUND: Fewer than 10% of patients presenting with adenocarcinoma of the stomach in the United States can expect to be cured. These discouraging results have led to trials of various adjuvant therapies. Some studies suggest a role for radiation in improving regional control. Radiation doses, however, are limited by the tolerance of abdominal organs. METHODS: Between 1985 and 1989, the Radiation Therapy Oncology Group conducted a phase II study to determine the feasibility of using intraoperative radiotherapy (IORT) in the treatment of adenocarcinoma of the stomach. Forty-three patients were entered into the study. Patients underwent maximal surgical resection (subtotal or total gastrectomy and regional node dissection) and IORT doses of 12.5-16.5 Gy were delivered in 27 patients. Adjuvant external beam radiation was given to 23 of the 27 patients with total doses ranging from 24 to 50 Gy. RESULTS: Two-year actuarial survival in the 27 patients receiving IORT was 47% and median survival was 19.3 months. Disease-free survival was 27%. Fifteen percent failed locally only, 26% with distant metastases only and 22% with both. Acute postoperative complications occurred in 14% with one fatality. Severe late complications occurred in 7% with one fatality. CONCLUSIONS: Intraoperative radiotherapy combined with surgical resection and postoperative radiotherapy appears to be feasible without excessive morbidity in a multiinstitutional study. Its ultimate value requires further study. PMID- 7552618 TI - Effect of reexcision on the success of breast-conserving surgery. AB - BACKGROUND: The success of lumpectomy and radiotherapy is dependent on minimizing the residual tumor burden in the breast. Histologic margin status is one measure of the extent of residual tumor. This study was undertaken to determine the success rate of a single conservative lumpectomy in obtaining negative margins and to evaluate the incidence of residual tumor after biopsies with positive or unknown margins. METHODS: This is a retrospective study covering a 5-year period (June 1988-June 1993). RESULTS: Three hundred sixteen women had lumpectomies. In 239, lumpectomy was the initial operation after a positive fine-needle aspiration or as a diagnostic procedure. Thirteen cases had positive margins. Reexcision was performed in 90 cases. The indication for reexcision was a positive margin in 42 cases (4 with gross tumor) and unknown margin status in 48. Nineteen of the reexcisions for positive margins and 20 of the reexcisions for unknown margins contained residual tumor. Eighty-six (96%) of the 90 reexcised patients underwent breast preserving surgery. Patient age, menopausal status, histologic tumor type, tumor size, and clinical presentation were not predictive of residual tumor. CONCLUSIONS: The need for reexcision does not preclude breast preservation. Because single-stage lumpectomy is successful in achieving negative margins in 95% of patients, diagnostic biopsy without margin evaluation should be abandoned to avoid routine reexcision. PMID- 7552619 TI - Lymph node metastasis as a significant prognostic factor in early gastric cancer: analysis of 1,136 early gastric cancers. AB - BACKGROUND: Gastric cancer is the most frequent cancer and the leading cause of death from cancer in Korea. Early gastric cancer has been defined as a gastric carcinoma confined to mucosa or submucosa, regardless of lymph node status, and has an excellent prognosis with a > 90% 5-year survival rate. From 1974 to 1992, we encountered 7,606 cases of gastric cancer and performed 6,928 gastric resections. Among them, 1,136 cases were early gastric cancer (14.9% of all gastric cancer cases and 16.4% of resected gastric cancer cases). METHODS: A retrospective analysis of 1,136 cases of early gastric cancer was performed to evaluate the prognostic significance of clinicopathologic features (sex, age, tumor location, gross type, histologic type, depth of invasion, status of lymph node metastasis, resection type). Lymph node metastasis was classified into three groups: N(n = 0) for no lymph node metastasis; N(n = 1-3) for one to three lymph node metastases; and N(n > 3) for more than three lymph node metastases. All patients received radical total or subtotal gastrectomy with lymph node dissection. RESULTS: In univariate and multivariate analysis of these nine factors, the only statistically significant prognostic factor was regional lymph node metastasis (p < 0.001). The others had no statistically significant association with prognosis. Lymph node metastasis was present in 178 cases (15.7%). The factors associated with the lymph node metastasis were depth of invasion and gross type [protruding type (e.g., types I, IIa)]. One hundred twenty-five of these patients had one to three lymph node metastases, and 53 cases had more than three lymph node metastases. The difference in 5-year survival rates among these groups was statistically significant: 94.5% for N(n = 0), 88.3% for N(n = 1-3), and 77.3% for N(n > 3). CONCLUSION: We propose that for early gastric cancer, lymph node dissection is necessary in addition to gastric resection, at least in patients with a high risk of lymph node metastasis. PMID- 7552620 TI - Clinically occult breast carcinoma: diagnostic approaches and role of axillary node dissection. AB - BACKGROUND: Nonpalpable breast lesions are being detected with increasing frequency with the advent of improved mammographic techniques. Although only 20 30% of these lesions are malignant, definitive diagnosis has usually required a needle-localization excisional biopsy, which is costly and increases the psychological stress on the patient. The purpose of this retrospective study was to determine the sensitivity of ultrasound-guided fine-needle aspiration (FNA) biopsy of nonpalpable breast masses and the incidence of axillary nodal metastases in these subclinical lesions. METHODS: Seventy-one patients treated for clinically occult malignant breast tumors between 1985 and 1992 were identified. Charts were reviewed to determine the accuracy of breast ultrasonography in detecting occult mass lesions and whether ultrasound guidance improved the accuracy of FNA biopsy. In addition, the incidence of axillary lymph node involvement was noted. RESULTS: Of the 71 malignant tumors, 35 were in situ and 36 were invasive. The median diameter was 0.5 cm for noninvasive lesions and 0.8 cm for invasive tumors. A mass was seen on mammography in 32 (45%) patients, microcalcifications were seen in 36 (51%), and both a mass and microcalcifications were seen in three (4%). Of the 30 patients who underwent an axillary node dissection, 4 (13%) had disease-positive nodes. Ultrasound-guided FNA was performed in 15 patients with a mass lesion, with a sensitivity of 93%. CONCLUSIONS: These results indicate that ultrasound-guided FNA cytologic analysis is an accurate diagnostic technique even in small (< 1 cm), mammographically detected breast masses. In addition, the incidence of axillary nodal metastases indicates that an axillary lymph node dissection should be performed in invasive lesions, even those < 1 cm in diameter. PMID- 7552622 TI - Intraaortic stop-flow infusion: pharmacokinetic feasibility study of regional chemotherapy for unresectable gastrointestinal cancers. AB - BACKGROUND: This study attempted to increase the exposure of gastrointestinal tract tissues to chemotherapy by prolonging the first pass of intraaortically administered drug by temporary occlusion of vascular structures. METHODS: Bolus infusion of 14C-labeled mitomycin C (MMC) mixed with unlabeled MMC was performed in dogs. Distribution of MMC in gastrointestinal tract tissues was studied under different types of major vessel occlusion. Three dogs with intravenous infusion constituted the control group. Vascular flow was controlled in four ways for 30 min: type I--stop-flow infusion (SFI) with clamping of the abdominal aorta above the celiac and below inferior mesenteric artery; type II--with additional clamping of the inferior vena cava above the diaphragm; type III with additional clamping of the portal vein in the hepatoduodenal ligament; and type IV--with surgical exclusion of nongastrointestinal branches of the aorta in addition to type II clamping. RESULTS: Type II and IV produced a 3-10-fold increase in exposure to MMC of major gastrointestinal tissues as compared with intravenous infusion. Area under the curve ratios with type IV were most prominent in the following tissues: stomach, pancreas, liver, and mesenteric lymph node. CONCLUSION: Access of MMC to several gastrointestinal tissues was increased through SFI. Type IV infusion was the most effective. Tissue exposure to MMC was especially advantageous for stomach, pancreas, liver, and mesenteric lymph node. PMID- 7552621 TI - Routine bone scanning in patients with T1 and T2 breast cancer: a waste of money. AB - BACKGROUND: Bone scans are often performed as routine staging procedures for patients with T1 and T2 breast cancers. Bone scanning in this patient population is evaluated with respect to cost and impact on clinical management. METHODS: Three hundred sixteen women with clinical T1 or T2 breast cancer who had bone scans were treated at Fox Chase Cancer Center from January of 1991 to December of 1992. We reviewed clinical and pathologic tumor stage, nodal status, laboratory studies, symptoms, bone scans (frequency, results, and cost), and resultant studies. RESULTS: Sixty-three women (20%) had bone scans that were interpreted as positive or suspicious for metastatic disease on initial presentation, resulting in 105 confirmatory studies, including 80 plain films, 10 computed tomography (CT) scans, 11 magnetic resonance imaging (MRI) scans, and four biopsies. Seven patients (2%) had skeletal metastases, six of whom had clinical stigmata of distant disease. A single patient (0.3%), with no signs or symptoms of distant disease, had bone metastases. The initial bone scans cost $224,676; additional tests cost another $53,122. The initial positive predictive value of bone scans in detecting metastatic disease was 11%. The "baseline" bone scans were followed by 130 "follow-up" scans over 2 years at a cost of $92,400. Seven patients developed metastatic disease, confirmed by 31 additional studies. Again, only one patient was asymptomatic. The availability of initial studies for comparison did not prevent thorough evaluation in women with worrisome follow-up bone scans. CONCLUSIONS: Bone scans of 316 woman at initial presentation with T1 or T2 breast cancer identified one incurable patient whose management was changed. The cost was $277,798. Bone scans contribute little information to the management of asymptomatic patients. PMID- 7552624 TI - Augmentation of carcinoembryonic antigen release from intact, viable tumor cells by a factor in human serum. AB - BACKGROUND: Measurement of carcinoembryonic antigen (CEA) levels in human serum is frequently used to detect tumor recurrence in patients with resected primary colorectal cancers. These levels are highly variable from patient to patient, and the mechanism that determines these levels is still poorly understood. METHODS: Using a 6-h in vitro CEA-release assay, we determined that a factor in human and fetal bovine sera significantly augments the release of CEA from the tumor cell surface into cell culture supernatants. RESULTS: As little as 1% serum admixed with tumor cells results in CEA release up to 200% greater than that of serum free controls. It is not inhibited by 1,10-phenanthroline or heat inactivation (of serum) but is calcium dependent. The electrophoretic mobility and membrane linkage of CEA released by serum appear to be identical to those of CEA released by bacterial phospholipase C. Because bacterial phospholipase C is known specifically to cleave the phosphoinositol (PI) glycan moiety that anchors CEA to the tumor cell surface, a mechanism of action for serum cleaving this anchor is suggested. CONCLUSIONS: The large range of CEA levels observed in patients with colorectal cancers may be related to differential sensitivity of the CEA membrane anchor to the CEA-releasing factor in serum. PMID- 7552623 TI - Altered glutathione metabolism in the tumor-bearing state. AB - BACKGROUND: Because glutathione (GSH) appears to be important for tumor growth and many tumors contain the capacity (gamma-glutamyltranspeptidase) to transport GSH, we examined GSH metabolism in MCA sarcoma-bearing rats (TB). METHODS: Tumor, liver skeletal muscle, kidney, and serum were collected from 47 MCA sarcoma (TB) rats and 26 normal (CTL) rats. Amino acids, GSH, gamma-glutamylcysteine synthetase (GCS), and gamma-glutamyl transpeptidase (GGTP) were determined. RESULTS: Significant activity of GGTP (117.8 +/- 16.0 mU/min/mg protein) was present in tumors. Liver GCS activity (nanomolar per hour per milligram protein) in TB rats (106.6 +/- 37.7) was increased (p < 0.01) compared with CTL rats (57.5 +/- 12.3) and correlated positively with tumor burden (R = 0.77). Muscle GGTP was decreased (p = 0.001) in TB rats (1.7 +/- 1.1) compared with controls (6.8 +/- 1.1). Serum GSH concentrations (microM) were lower (p < 0.05) in TB rats (14.97 +/- 1.72) versus control rats (16.82 +/- 1.54) and correlated negatively with tumor burden (R = -0.83). CONCLUSIONS: In this tumor-bearing model, tumor has significant capacity (GGTP) for the uptake of GSH. Serum GSH is depleted in TB rats and correlates negatively with tumor burden. Liver GCS is increased in TB rats and skeletal muscle GGTP is decreased, which may preferentially benefit the tumor by increasing the bioavailability of glutathione for its own use. PMID- 7552625 TI - Intraoperative radiotherapy in the treatment of neuroblastoma: report of a pilot study. AB - BACKGROUND: External beam radiotherapy in advanced neuroblastoma is limited by the volume of normal radiosensitive tissues included in the radiation field. Limitations to external radiation are the late effects to these tissues. Intraoperative radiotherapy (IORT) delivers a single high-radiation dose to a tumor while displacing normal tissues that would have been included in an external field. Standard external radiotherapy can still be done after "boost" IORT. METHODS: Eight advanced-stage neuroblastoma patients who received IORT as part of their multimodality therapy were reviewed to identify the impact of IORT on operative time, complications, and tumor control in the treatment field. The IORT was accomplished by patient transport from the OR to the radiation therapy suite; these were separated by three floors. RESULTS: IORT added 30-75 min to the operative procedure. Tumors in the resection/IORT fields showed no evidence of disease (one), stable tumor size (six), and tumor recurrence (one). Two complications were identified: a urinary fistula and CO2 retention, which was detected and corrected before the IORT. Neither of these complications was related to the IORT. Two patients who had subsequent tumor resection after IORT demonstrated tumor differentiation to ganglioneuromatous tissue. CONCLUSIONS: IORT usually can be completed in less than an hour. No IORT-associated complications were identified. IORT along with maximal tumor resection, external radiation, and chemotherapy enhances local tumor control. PMID- 7552627 TI - Prospective hospital-based survey of attitudes of Southern women toward surgical treatment of breast cancer. AB - BACKGROUND: Breast-conserving surgery is equivalent to total mastectomy in the treatment of breast cancer. The Southern part of the United States has a low rate of breast conservation. METHODS: We surveyed 300 women: 100 hospital personnel, 100 cancer clinic patients, and 100 non-cancer clinic patients. The women were asked about their attitudes toward breast cancer, surgery preferences, and factors that might influence their decisions. RESULTS: One hundred eighty-nine chose mastectomy as the best operation, 106 women chose lumpectomy, and five women were undecided. There was no difference in mean age, racial distribution, education level, income level, percentage of women who considered themselves Southern women, concerns about breast cancer, recent mammograms, previous breast surgery, previous breast cancer treatment, or acquaintances with breast cancer between the mastectomy and the lumpectomy groups. Women interested in saving the breast were more likely to pick lumpectomy (35 vs. 84%, p = 0.001). A fear of cancer recurrence played a role in the decision (88 vs. 40%, p = 0.001). Fear of radiation therapy (76 vs. 57%, p = 0.002) and of the side effects (80 vs. 63%, p = 0.005) was a significant factor. CONCLUSIONS: The choice of surgery for breast cancer is an individual process between a woman and her surgeon. Attitudes and fears regarding cancer recurrence and radiation therapy may make women select mastectomy over lumpectomy. PMID- 7552626 TI - Treatment of hepatic-metastatic colorectal cancer with a chemotherapeutic emulsion: interim results of a phase I trial. AB - BACKGROUND: Hepatic arterial infusion of 5-fluoro-2-deoxyuridine (FUdR) is associated with a 60% response rate among previously untreated patients who have hepatic-metastatic colorectal cancer. One obstacle to further dose escalation has been concomitant hepatic toxicity. We are evaluating a FUdR-containing chemotherapeutic emulsion to further dose intensify therapy without associated toxicity. METHODS: The in vitro pharmacokinetics of the emulsion were determined using high-pressure liquid chromatography (HPLC). The rate at which FUdR is released from emulsion into an overlying aqueous phase was determined in static and dynamic assays. Fifteen patients with hepatic-metastatic colorectal cancer were treated with intrahepatic arterial infusions of emulsion on a phase I dose escalating clinical protocol. Serum collection determined systemic drug levels using HPLC. RESULTS: In vitro studies demonstrate that FUdR is slowly released from emulsion into overlying aqueous medium. The emulsion serves as a depot for FUdR. Therapy was well tolerated. Emulsion was sequestered in the liver after infusion in all treated patients. CONCLUSIONS: This Ethiodol-based, oil-in-water emulsion serves as a sustained-release preparation of FUdR. An Ethiodol-based oil in-water emulsion is a clinically effective vehicle for delivering FUdR to hepatic-metastatic colorectal tumors. PMID- 7552628 TI - Potential uses of interferon alpha 2 as adjuvant therapy in cancer. AB - BACKGROUND: The purpose of this study was to provide an overview of the potential uses of adjuvant interferon (IFN) therapy for resected solid tumors at high risk for postsurgical relapse. METHODS: A MEDLINE search (1970-1994) of the English language literature for original articles, reviews, and abstracts addressing IFN use in the adjuvant setting together with the authors' collective experience formed the basis for this review. RESULTS: The use of adjuvant IFN-alpha has been studied most extensively in conjunction with the treatment of melanoma. Fewer data are available on IFN-alpha use for the treatment of other solid tumors. In melanoma, there is evidence from Intergroup trials (Eastern Cooperative Oncology Group and World Health Organization) that IFN-alpha 2a given for 1-3 years prolongs the interval to relapse and may have a survival benefit. Trials of adjuvant IFN, with and without chemotherapy, are ongoing in the treatment of renal cell carcinoma and colorectal adenocarcinoma. Its value in the treatment of osteosarcoma and high-grade astrocytoma is unknown. CONCLUSIONS: The use of IFN in the adjuvant setting is an exciting area of medical and surgical oncology and has the potential to prolong the time to relapse and to increase survival of patients with melanoma. Its role in the adjuvant therapy of other solid tumors remains to be defined. PMID- 7552629 TI - Malnutrition: incidence, diagnosis, causes, effects and indications for nutritional support. AB - Various methods for the diagnosis of malnutrition, and some of the physical signs commonly associated with it, are discussed. The incidence and possible causes of malnutrition and its effects are then addressed. Finally, the indications for nutritional support, using either the enteral or the parenteral route, are examined. PMID- 7552630 TI - Enteral nutrition. AB - This review summarizes research studies published in recent years that are useful for clinicians considering the enteral route as a means of nutritional support for their patients. In this period, there has been a move away from emphasis on what nutrients are given to a more considered appraisal of how nutrients are provided. Thus, the technical aspects of enteral feeding have been refined, enabling a greater number of patients to be fed safely and effectively using the enteral route. PMID- 7552631 TI - Techniques for the provision of parenteral nutrition. AB - Parenteral nutrition should be provided by a regularly trained nutrition team. The objectives of nutritional support should be decided by considering both the prognosis of the disease to be treated and the risk-to-benefit ratio of parenteral nutrition. Only when other nutritional therapy has failed should the costly and complicated protocol of parenteral nutrition be implemented. Although patients' consent to artificial nutrition is usually implicit, the goals and modalities of parenteral nutrition should be explicit. This approach avoids misunderstanding and may allow patients to participate in their own care, thus decreasing the likelihood of complications, especially those of a technical type, and possibly increasing the quality and efficacy of parenteral nutrition. PMID- 7552632 TI - The short-bowel syndrome. AB - Patients with a short bowel have usually had a bowel resection for Crohn's disease. Two types of short-bowel patient can be distinguished: those with a jejunostomy and those with their jejunum anastomosed to a functioning colon. Both types of patient have problems with macronutrient absorption, although those with a colon experience fewer problems because some energy from unabsorbed carbohydrate is salvaged in the colon. Patients with a jejunostomy have problems with large stomal losses of water, sodium and magnesium, whereas those with a jejuno-colic anastomosis rarely have problems with water and electrolyte absorption. Patients with a jejunostomy 100-200 cm from the duodeno-jejunal flexure ('absorbers') usually absorb more from the diet than they pass through the stoma and therefore require oral electrolyte or nutrient supplements. Those with a residual jejunal length of less than 100 cm usually secrete more from the stoma than they take in orally ('secretors') and therefore require long-term parenteral fluid or nutrient supplements. A high output resulting from a jejunostomy is treated by reducing the oral intake of hypotonic fluid, administering a sipped glucose-saline solution and, often, by giving drugs that reduce intestinal motility (most effective in absorbers) or gastrointestinal secretions (most effective in secretors). Gallstones are common both in short bowel patients with and in those without a colon (45%), and calcium oxalate renal stones occur in the former (25%). However, it is now possible to provide adequate nutrition and fluid supplements for most patients with a short bowel, and the prospects for the rehabilitation of such patients are good. PMID- 7552633 TI - Inflammatory bowel disease. AB - Since the early 1970s there has been considerable debate over the role of nutritional support in the treatment of patients with acute inflammatory bowel disease. The role of enteral feeding as a primary therapeutic option in patients with acute Crohn's disease remains controversial. This article reviews the role of nutritional support both as an adjunct to standard medical therapy and as a primary therapeutic option. PMID- 7552634 TI - Nutritional support in liver disease. AB - Protein-energy malnutrition is highly prevalent in patients with chronic liver disease, especially in those with advanced cirrhosis, and seems to influence their survival. Enteral and parenteral approaches to the treatment of alcoholic hepatitis have yielded controversial results. The use of polyunsaturated lecithin might be a promising alternative for the future. Enteral nutrition is the preferred way of providing artificial nutrition to patients with advanced cirrhosis. In some trials, it appears to improve the short-term survival of these patients. The long-term use of oral nutritional supplements may widen the scope of enteral nutrition in cirrhotic patients. PMID- 7552635 TI - Helicobacter pylori: who is positive and who is not? PMID- 7552636 TI - Gastrointestinal intra-epithelial lymphocytes in man: the saga continues. PMID- 7552638 TI - Anismus and biofeedback: who benefits? AB - BACKGROUND: Biofeedback is the main treatment for dyschezia in patients with anismus, but retraining may fail because of the frequent association of pelvirectal disorders with anismus. We set out to identify indices of biofeedback failure in the treatment of anismus. PATIENTS AND METHODS: From May 1990 to May 1993, 27 patients (20 women and seven men; median age 46 years) with anismus in which dyschezia was not improved by laxative agents were enrolled in a biofeedback retraining programme. All patients underwent proctologic examination, anal manometry and defecography. Anismus was defined as an increase in anal pressure during attempted defecation in conjunction with an impairment of rectal emptying as assessed using an objective test (barium paste expulsion). Associated disorders were encountered frequently. These included abnormal perineal descent (22 cases), large rectocoele (12 cases), high-grade rectal prolapse (six cases), abnormally high anal canal pressures at rest (seven cases) and abnormal rectal response to inflation (20 cases). Anismus was the sole abnormality in 12 patients when perineal descent, low-grade prolapse and abnormal rectal sensations were not taken into account. RESULTS: Biofeedback retraining did not suppress dyschezia in 13 out of 27 patients. Neither associated disorders (rectocoele, rectal prolapse, abnormal perineal descent, anal pressure and abnormalities of rectal sensation) nor a relevant past history (hysterectomy, laxative abuse, use of antidepressive agents) were encountered more frequently in these 13 patients than in the other 14. The duration of symptoms before treatment was significantly longer in the group unresponsive to biofeedback retraining (81 +/- 61 compared with 33 +/- 34 months for the responsive group, P < 0.01), but the total duration of symptoms and the number of retraining sessions attended did not differ significantly between the two groups. CONCLUSIONS: (1) Extensive examination (defecography and manometry) before biofeedback retraining of anismus is not mandatory because the failure of retraining (48%) is not related to the presence of associated pelvirectal disorders. (2) A long past history of dyschezia seems to provide an index of the failure of biofeedback retraining. PMID- 7552637 TI - Adhesion molecule expression on gastric intra-epithelial lymphocytes of patients with Helicobacter pylori infection. AB - OBJECTIVE: To examine the expression of adhesion molecules on gastric intra epithelial lymphocytes (IELs) from patients infected with Helicobacter pylori. DESIGN: The expression of adhesion molecules and T-cell activation markers by gastric IELs from patients with gastritis or duodenal ulceration was examined using two-colour flow cytometry. Ten of the patients were H. pylori positive and eight were H. pylori negative. RESULTS: Expression of lymphocyte function associated antigen-1 (LFA-1) on IELs was significantly lower (P < 0.05) in patients with H. pylori infection than in patients negative for H. pylori. There were no significant differences in the expression on IELs of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), human mucosal lymphocyte (antigen)-1 (HML-1), very late antigen-4 (VLA-4) or CD43 between H. pylori-positive and H. pylori-negative individuals. In addition, the transferrin receptor, a maker associated with proliferation and activation, was found on a small population of IELs from H. pylori-positive individuals. CONCLUSION: A reduction in the expression of LFA-1 has previously been reported on activation of IELs. The finding of reduced LFA-1 expression and increased transferrin receptor expression on IELs from H. pylori-positive individuals suggests that activation of these cells is associated with H. pylori infection. PMID- 7552640 TI - Endoscopic intubation of oesophago-gastric malignancy. AB - OBJECTIVE: To determine what percentage of inoperable oesophageal and oesophagogastric malignancies could be successfully intubated for palliation, and to compare Atkinson and Celestin tubes. DESIGN: A consecutive series of 210 patients who had been referred for palliation underwent endoscopy and intubation with the Nottingham introducer. PATIENTS: One hundred and nineteen men and 91 women aged 31-91 (mean age 71) years with inoperable malignant obstruction of the oesophagus or gastro-oesophageal junction were studied. One hundred and five tumours were in the lower third of the oesophagus, 78 in the middle third, 25 in the upper third and seven in the gastric fundus. Four tracheo-oesophageal fistulae resulting from bronchial carcinomas were also successfully intubated. MAJOR OUTCOME MEASURE: Eighty-nine per cent of gastro-oesophageal malignancies were successfully intubated using 120 Atkinson and 67 Celestin tubes. RESULTS: Eleven patients (5.2%) suffered oesophageal perforations during intubation, of whom six died. Nine of the perforations occurred in the first 100 patients treated but only two (2.3%) in the subsequent 87. Seven patients (3.7%) developed aspiration pneumonia, of whom five died. The mean survival time after intubation was 4.5 months (range 0.5-20 months), and 74% of patients required no further procedure. During follow-up, five (4.2%) Atkinson tubes displaced upwards, compared with 12 (17.9%) Celestin tubes (P < 0.01). Eighteen (9.6%) patients had recurrent dysphagia as a result of bolus obstruction and 11 (5.9%) had tumour overgrowth of the tube. CONCLUSION: Palliative intubation of malignant dysphagia is possible in approximately 90% of patients. The mortality associated with the procedure is low (5%) and effective relief of symptoms is achieved, with 74% of patients requiring no further treatment. PMID- 7552639 TI - Prevalence of viral markers among refugees from southern Albania: increased incidence of infection with hepatitis A, B and D viruses. AB - BACKGROUND: Since 1991, thousands of refugees from southern Albania have entered north-western Greece, an area with low-to-moderate endemicity for infection with hepatitis viruses. We examined the prevalence of several markers of viral infection in this population in order to ascertain the likely impact of its presence on the epidemiology of hepatitis infections in north-western Greece. DESIGN: Consecutive unselected serum samples were obtained from refugees resident in three different reception camps. SETTING: A university hospital. STUDY POPULATION: One thousand and twenty-five refugees (662 males and 363 females, age range 0-81 years) and 1984 healthy controls (1293 males and 691 females, age range 0-80 years). INTERVENTIONS: None. RESULTS: We found a significantly greater prevalence of markers of infection with hepatitis A virus (prevalence of antibodies to hepatitis A virus 98.2%), hepatitis B virus (HBV; prevalence of HBV s antigen 22.2%, prevalence of HBV c antibody 70.6%, prevalence of HBV s antibody 40.5%, prevalence of HBV e antigen 21.1%, prevalence of HBV e antibody 46.2%), hepatitis C virus (prevalence of antibodies to hepatis C virus 1.75%) and hepatitis D virus (prevalence of antibodies to hepatis D virus 12.7%) among refugees from southern Albania than in healthy Greek controls. These markers were found with significantly greater frequency among younger refugees (< 30 years of age) than in older members of the same population. CONCLUSIONS: We conclude that refugees from southern Albania are a new immigrant population characterized by a high incidence of infection with hepatitis A, B and D viruses. This finding may reflect the low socioeconomic status of the immigrant population and the poor hygienic conditions experienced by its members. The high incidence of HBV and HDV infections in the population from Albania will probably increase the prevalence of infection with these viruses in Ioannina and subsequently in the whole of the Epirus region. We therefore believe that rigorous adherence to general precautions and the initiation of hepatitis B vaccination programmes will be necessary in future, both in our area and in Albania. PMID- 7552642 TI - Colonoscopic diagnosis and treatment of acute appendicitis. AB - An atypical case of acute appendicitis is reported, in which the initial presentation was not suggestive of this diagnosis. A routine work-up revealed elevated levels of inflammatory markers, an ultrasonographically normal abdomen and normal endoscopic findings in the upper gastrointestinal tract. During colonoscopy, performed 3 days after admission, the inflamed appendiceal orifice was localized and intubated using an endoscopic retrograde cholangio pancreatography (ERCP) catheter; pus was aspirated. Elective appendectomy was carried out 5 months later on the asymptomatic patient. Histological analysis revealed fibrous thickening of the appendiceal wall, correlating with an old inflammatory process, along with changes typical for acute ulcerophlegmonous appendicitis. To the best of our knowledge, this is the first case of an endoscopically diagnosed and treated acute ulcero-phlegmonous appendicitis. We draw the following conclusions: (1) Acute appendicitis may present atypically, leading to an extensive clinical work-up including gastroscopy and colonoscopy. (2) This condition may be effectively treated, at least temporarily, by colonoscopic suction of appendiceal pus, provided that clinical urgency for laparotomy is absent. (3) Histological changes typically associated with acute ulcero-phlegmonous appendicitis may be present in asymptomatic patients and in patients without fever and with normal erythrocyte sedimentation rates and blood leucocyte counts. (4) Elective appendectomy should be considered in patients with conservatively healed acute appendicitis or periappendiceal abscess. PMID- 7552641 TI - Low prevalence of anti-neutrophil cytoplasmic antibodies in ulcerative colitis patients with long-term remission. AB - OBJECTIVES: In spite of a strong positive association between ulcerative colitis and the presence of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), the immunogenetic significance of these antibodies remains unclear. We studied patients with quiescent disease to clarify whether ANCAs are present even in the absence of inflammation. DESIGN: The prevalence of ANCAs was estimated blindly in 137 patients with ulcerative colitis, 128 of whom had quiescent disease with a mean duration of complete clinical and biochemical remission of 14 years. For comparison, we studied sera from 110 patients with Crohn's disease, 27 of whom had a low or intermediate grade of inflammatory activity. The mean duration of complete remission in these patients was 8.5 years. METHODS: ANCAs were detected using indirect immunofluorescence and enzyme-linked immunosorbent assays (ELISAs). RESULTS: Only 13 (9%) of 137 patients with ulcerative colitis had ANCAs (5% had p-ANCAs). Three patients had previously undergone colectomy. In patients with Crohn's disease, ANCAs were observed in 17 of 110 patients (15%, 6% had p ANCAs). Fifteen of these patients had colonic disease. CONCLUSION: In patients with ulcerative colitis free from inflammation for prolonged periods of time, ANCAs occurred less frequently than has previously been reported. Patients with Crohn's disease had the expected frequency of ANCA positivity, which for colonic Crohn's disease was comparable to that found in patients with ulcerative colitis. These findings suggest that the titre of ANCAs decreases with time in inactive disease and may be undetectable with conventional assays after several years of complete remission. PMID- 7552643 TI - Mesenteritis secondary to the use of a pneumatic jackhammer. AB - Mesenteritis is an uncommon condition characterized by a variable clinical course, in which spontaneous remission may occur, but in which uncontrollable disease activity leads to death [corrected]. We describe a construction worker with mesenteritis who presented with vague abdominal discomfort and mucoid diarrhoea. A large, irregular mass was found in the abdomen. Upon explorative laparotomy, a large, rubbery, grey and yellow, irresectable tumorous mass was found, which apparently originated from the mesentery of the small bowel. The large bowel was expanded over the tumorous mass. A defunctioning colostomy was made. Histologic examination of biopsy specimens revealed only fatty necrosis of the mesentery and no evidence of malignancy. It is assumed that a pseudotumour of the mesentery had developed after chronic repetitive abdominal trauma caused by the continuous vibration of a pneumatic jackhammer, on which the patient pressed his abdomen during construction work. In this case of mesenteritis, the clinical course was one of spontaneous and complete recovery. PMID- 7552644 TI - Critical issues in the management of gastroesophageal reflux disease. AB - AIM: To discuss some of the critical issues in the management of gastroesophageal reflux disease (GERD). OPINION: GERD is a chronic relapsing disease characterized by pathological exposure of the distal esophagus to gastric acid. Diagnosis of the condition can often be made on the basis of symptomatology alone. Endoscopy can help in assessing the degree of esophageal damage, influencing the choice of therapy, and should be performed at least once during a symptomatic patient's lifetime to exclude a diagnosis of Barrett's esophagus. However, endoscopy is mandatory at diagnosis if alarm symptoms are present. Treatment should aim to provide the lowest degree of acid suppression needed for the control of symptoms. Proton pump inhibitors (PPIs) represent the most cost-effective treatment option for the short- and long-term management of GERD. Compared with standard- and high dose H2-receptor antagonists, PPIs result in superior and faster healing and symptom relief across all grades of esophagitis and are more effective at maintaining patients in symptomatic and endoscopic remission. Treatment with PPIs has also been shown to reduce the rate of recurrent stricture after initial dilatation. PPIs are generally well tolerated, and to date there have been no reports of gastric dysplasia resulting from their long-term use. Anti-reflux surgery should be reserved for patients who are unresponsive to continuous PPI therapy or perhaps for young patients. It will be several years before the impact of laparoscopic fundoplication as a cost-beneficial therapy for GERD can be assessed. CONCLUSION: The superior clinical efficacy of PPIs when compared with any other drug regimen for GERD make them the treatment of choice for the short- and long-term management of this troublesome condition. PMID- 7552645 TI - Helicobacter pylori and gastric cancer. PMID- 7552646 TI - Isthmic lumbar spondylolisthesis with sciatica: the role of the disc. AB - Thirty-one patients with isthmic spondylolisthesis were investigated using MR imaging. Twenty-one of these patients had selectively unilateral sciatica and no abnormalities on adjacent discs. In 18 patients there was a clear correlation between the degree of foraminal stenosis and the symptomatic side. In 20 patients there was evidence of root compression by disc tissue. PMID- 7552647 TI - The relationship between isthmic and degenerative spondylolisthesis and the configuration of the lamina and facet joints. AB - Advanced degenerative change in the facet joints leads to displacement of the vertebral body, and the configuration of facet joints and lamina is closely related to the development of degenerative spondylolisthesis. For this study 103 patients and 25 controls were examined with respect to the configuration of the lamina, anteroposterior diameter of the vertebral canal, interarticular distance, interlaminar distance, disc degeneration, and degree of arthritic changes of facet joint, all as shown of plain radiography, and facet angle, interfacet distance, and contour of the canal side of the inferior articular process and lamina, as shown on computed axial tomography. The results of this study showed that those patients with a narrow facet angle were more likely to have developed degenerative spondylolisthesis. If the sum of both facet angles was less than 77.9 degree, the risk of development of degenerative spondylolisthesis was 2.5 times higher than if the sum was greater than 77.9 degree. Those with type N lamina, detected on plain radiographs, were especially likely to have developed degenerative spondylolisthesis. This suggested that degenerative spondylolisthesis may be due to the less effective check mechanism preventing a vertebra from slipping forward on the vertebra below. We recommend fusion of the degenerated spinal segments when operation is considered in cases of acute facet angle with symptoms. PMID- 7552648 TI - Sudden onset of back pain. AB - Patients attending a back pain clinic completed a structured interview, with particular reference to whether onset of the first episode of back pain was sudden or insidious. They were classified into four diagnostic groups. A significantly higher proportion of patients who had experienced a sudden onset of pain suffered from sciatic pain with positive root tension signs than was the case with patients whose pain had started more insidiously (50.8% vs. 20.6%; P < 0.001). These patients were also more likely to be male, to have been lifting or twisting with a weight, and to have been confined to bed or hospitalised, and have undergone manipulations, than patients with insidious onset of their pain. PMID- 7552649 TI - Active treatment programs for patients with chronic low back pain: a prospective, randomized, observer-blinded study. AB - Several new studies have indicated that an active approach to patients with chronic disabling low back pain (LBP) seems effective. Some of these studies emphasize the importance of dealing with the patient's total situation in comprehensive multidisciplinary programs--the bio-psycho-social model. However, these programs are expensive. The aim of this study was to evaluate the rehabilitation outcome from three different active programs in terms of: (1) return-to-work rate, (2) days of sick leave, (3) health-care contacts, (4) pain and disability scores, and (5) staying physically active. The subjects included 132 patients randomized to the study, of whom 123 started one of the treatment programs. They had all had at least 6 months of chronic LBP. The patients were randomized into one of three programs: group 1--a full-time, intensive 3-week multidisciplinary program, including active physical and ergonomic training and psychological pain management, followed by 1 day weekly for the subsequent 3 weeks; group 2--active physical training, twice a week for 6 weeks, for a total of 24h; group 3--psychological pain management combined with active physical training, twice a week for 6 weeks, also for a total of 24h. The results presented here are based on data collected 4 months following treatment, which shows an 86% response rate. The initial examination and the follow-up evaluation were performed by a blinded observer. The results show that 4 months after treatment, the intensive multidisciplinary program is superior to the less intensive programs in terms of return-to-work rate, health-care contacts, pain and disability scores, and staying physically active.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552650 TI - Loads in the spinal structures during lifting: development of a three-dimensional comprehensive biomechanical model. AB - Epidemiological studies have shown that loads imposed on the human spine during daily living play a significant role in the onset of low back pain. The loads applied to the lumbar spine are shared by a number of structures: muscles; posterior elements, including facets and ligaments; and the disc of a ligamentous motion segment. In vivo, it is not practical to determine forces in these structures using experimental techniques. Biomechanical models, based on an optimization technique of electromyographic activities of the trunk muscles, have been proposed to predict forces in the load transmitting structures. The mathematical models reported in the literature are based on information collected from a wide variety of sources, of which the subject that takes part in the experiment is only one. The present study describes techniques developed in our laboratory to collect from the subjects themselves all the data needed for the formulation of a biomechanical model. The results demonstrated that back lifting with 0 N (no load), 90 N, and 180 N in the hands created maximum external flexion moments respectively of 109.6 Nm, 137.9 Nm, and 161.7 Nm, at the L3-4 disc level. The corresponding external axial compression forces on the disc were 469.5 N, 511.8 N, and 601.5 N. The predicted disc compression varied from 3.4 to 5.0 times the body weight. In comparison to the static lifting mode, the dynamic lifting task caused an increase in the disc compression force ranging from 15.8% to 39.4% depending on the load being lifted (e.g., 3256 N for the dynamic mode vs. 2516 N for the static mode when the subject lifted 90 N). The salient features of the entire protocol developed by the authors and the need for further improvements are also presented. PMID- 7552651 TI - The Graf stabilisation system: early results in 50 patients. AB - The results of the first 50 consecutive patients using the Graf stabilisation system are presented. The average age of the patients was 41 years; there were 32 women and 18 men in the group. All patients suffered from intractable symptomatic degenerative disc disease which could be localised to one or more levels. All patients gave a history of chronic back pain, but the mean period of severe disability was 24 months. The mean preoperative disability score (Oswestry questionnaire) was 59%. The average period of follow-up was 24 months (range 19 36 months). At the latest review, the mean disability score was 31%. The clinical results were classified as "excellent" or "good" in 72% of patients, "fair" in 10%, "the same" in 16% and "worse" in 2%. All but three patients felt that surgery was worthwhile. The results have not deteriorated over the period of follow-up. PMID- 7552653 TI - Motor evoked potentials: appropriate positioning of recording electrodes for diagnosis of spinal disorders. AB - The interpretation of normal and pathological findings of motor evoked potential obtained by the use of transcranial magnetic stimulation depends on adequate examination technique, including the appropriate positioning of the recording electrodes over the muscle. On the basis of knowing the location of the motor end plate zones in muscles, magnetic stimulation of the motor cortex of 30 healthy adults was performed in order to explore the influence of the position of the surface recording electrodes on potential parameters and to establish the standard location of the recording electrodes over the biceps brachii, medial vastus, anterior tibial and abductor hallucis muscles for diagnostic use in spine disorders. The cortical latencies and peak-to-peak amplitudes of the evoked potentials were analysed by varying the location of the recording electrodes and the stimulus intensities. The latencies were significantly shorter when the different electrode lay more proximally over the muscle belly. Reproducible potentials with sharp negative onset and maximum amplitude were recorded with a separation of 5-7.5 cm between the different electrode, located over the motor end plate area, and the different electrode, located over the distal myotendinous junction. This implies that the parameters of evoked potentials depend on the position and separation distance of the recording electrodes over the muscles and that it is possible to record the potentials using a lower stimulus intensity and, above all, on relaxed muscles, which may prove to be applicable for intraoperative monitoring of the spinal cord using magnetic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552654 TI - Surgical management of haemangioendothelioma of the spine. AB - Four patients with haemangioendothelioma of the spine which was treated surgically are presented. Two were male and two female, mean age 30 years (range 5-60). All tumours were sited in the thoracic spine between T5 and T10; three were primary and one metastatic from a hepatic haemangioendothelioma. Each patient had a significant neurological deficit at presentation; three were paraplegic. A diagnosis of vertebral neoplastic disease was suggested on plain radiographs, and in three cases this was supported by computed tomography or magnetic resonance imaging. Two patients underwent anterior decompression and posterior instrumented stabilisation, one anterior decompression alone and one posterior decompression followed by tumour vessel embolisation and then anterior decompression. Intra-operative blood loss was a significant feature despite the use of hypotensive anaesthetic techniques and local haemostatic agents. Three of the tumours were tested for Factor VIII (a tumour for vascular tumours), and all proved positive. In these, sufficient histological material was available to grade the tumours according to the classification of Campanacci et al. [1]. All were grade II. Three patients recovered completely from paraglegia; one had residual mild spasticity which required the use of a walking aid. The mean improvement in Frankel grade was 2.5 (range 1-4). In two the tumour recurred outside the spine within 18 months; one had subsequently died. The presentation, investigation and results of surgery for haemangioendothelioma of the spine are presented. Particular attention is drawn to the neurological status at presentation, the effect of pre-operative tumour embolisation and the dramatic recovery that can be achieved in these patients following surgery. PMID- 7552652 TI - Parameter variation in stable scoliosis. AB - The reliability of scoliosis parameters such as the Cobb angle, the angle of trunk rotation and moir e topography is known from several publications. These studies concern intra- and interobserver variations; they are in general of transversal nature, which means the measurements were performed on the same subject within a short time period. From clinical experience it appears that these variations may be greater when scoliosis patients are followed over several years. In order to determine the parameter variation in the follow-up of children with scoliosis, a retrospective study was undertaken. From among our outpatient population, 41 children with stable scoliosis over a period of at least 2 years were selected. Stable scoliosis was defined as a spinal deformity which, in retrospect, had not deteriorated during at least a 2-year period prior to the end of growth. Hence no indication for treatment was found for this group. The measurements of the angle of external trunk rotation, moire topography and Cobb angle of these children during the follow-up period were submitted to statistical computations using one-way analysis of variance. Variations in the results of Cobb angle measurement and the measurement of the angle of trunk rotation were found to be no different to the variations in intra- and interobserver measurements as reported mainly from transversal studies. The variation found in moire topography, in moire contour lines and in angle moire determination, was, however, considered too great for measurements to be reliable. In conclusion, only Cobb angle determination and external measurement of trunk rotation can be accepted as valid follow-up parameters from a methodological viewpoint. PMID- 7552655 TI - Fracture of the occipital condyle: case report and review of the literature. AB - Fracture of the occipital condyle is a rare injury that can be easily overlooked. We report a patient with an occipital condyle fracture who presented with pyramidal syndrome and normal plain radiographs. The diagnosis was made by high resolution computed tomographic scanning with sagittal and coronal reconstructions and magnetic resonance imaging. Surgical treatment was deferred because of spontaneous recovery. This fracture should be considered whenever a trauma patient presents neck pain and plain cervical spine radiographs show no abnormalities. PMID- 7552656 TI - CDH: preliminary report on a new anterior spinal instrumentation. AB - CDH (Cotrel-Dubousset-Hopf) instrumentation was developed with the aim of improving stability in ventral operation procedure and facilitating treatment of all anterior spinal diseases. The implantation of anterior plates and drawers, the use of a double-rod fixation within the implant in nonparallel directions, which provide an automatic locking mechanism against displacement, the prevention of dislocation of the cancellous bone srews, and the crosslink principle are its main characteristics. The device can be applied to the spine in accordance with its three-dimensional anatomy by any kind of force (distraction, compression, and rotation). Additional posterior instrumentation and postoperative external support are unnecessary in most cases because of improved stability. No reoperation was necessary following the mono- and multisegmental application of this method in 60 patients (28 with scoliosis, 12 with spondylodiscitis, 8 with primary tumors or isolated metastasis, 6 with fractures, 3 with failed back syndrome, 1 with kyphotic deformity, 1 with spondylolisthesis on two levels, and 1 with loss of correction after the dislocation of another posterior spinal instrumentation). Average blood loss was 950 ml; the average operating time was 3 h. In all, 16 monosegmental and 44 multisegmental procedures were carried out. In 25 patients, in particular those with paralytic scoliosis, a double-stage anterior and posterior spondylodesis was done. PMID- 7552657 TI - Amyloidosis: author's response. PMID- 7552658 TI - Resolution of lameness associated with Scottish fold osteodystrophy following bilateral ostectomies and pantarsal arthrodeses: a case report. AB - Bilateral hind-limb lameness, associated with tarsal exostoses in a Scottish fold diagnosed as having Scottish fold osteodystrophy, resolved following staged bilateral ostectomies and pantarsal arthrodeses. Degenerative changes in the phalangeal joints of the hind limbs have progressed radiographically, but lameness has not recurred 48 weeks following the second arthrodesis. Additional skeletal abnormalities were detected radiographically in both carpi and in several caudal vertebrae. A partial, left-sided conduction deafness was diagnosed by evaluating brain stem auditory-evoked responses. PMID- 7552659 TI - Histopathological evaluation of canine stifle joint synovial membrane collected at the time of repair of cranial cruciate ligament rupture. AB - Synovial membranes from 54 dogs were collected at the time of surgery to stabilize the stifle joint following rupture of the cranial cruciate ligament. Histological evaluation revealed lymphoplasmacytic synovitis with the formation of distinct, nodular aggregates in 36 synovial membranes. The remainder had evidence of chronic synovitis with variable numbers of diffuse, mononuclear cells. The presence of a potentially immune-mediated synovitis in the majority of cases suggests the need for further study. PMID- 7552661 TI - Hypoadrenocorticism in a family of leonbergers. AB - Hypoadrenocorticism was diagnosed in four related leonbergers. Two of the four dogs also had low-resting serum thyroxine (T4) levels and signs consistent with hypothyroidism. The familial association of the affected dogs and the presence of coexisting thyroid dysfunction are similar to what is seen in human type II polyglandular autoimmune syndrome. PMID- 7552660 TI - Canine immune-mediated diabetes mellitus: a case report. AB - A four-year-old, spayed female toy fox terrier presented with hyperglycemia and severe anemia. A diagnosis of immune-mediated diabetes mellitus was made based upon the finding of beta-cell specific antibodies. Immune-mediated hemolytic anemia was diagnosed based on findings of a regenerative anemia, spherocytosis, hyperbilirubinemia, hemoglobinuria, and bilirubinuria. The anemia resolved following two months of immunosuppressive therapy. The diabetes was treated with insulin for four months, after which time treatment was no longer necessary. However, the dog remained positive for beta-cell antibodies which may be a predictive marker for the recurrence of diabetes mellitus in the future. PMID- 7552662 TI - Concurrent hypoadrenocorticism and hypoalbuminemia in dogs: a retrospective study. AB - A retrospective study was undertaken to determine the prevalence of hypoalbuminemia in dogs with hypoadrenocorticism. During a 94-month interval, 17 of 44 dogs (38.6%) with hypoadrenocorticism were found to have hypoalbuminemia. When compared to all dogs with hypoalbuminemia (9.8%) observed in the hospital population during this same interval, there appeared to be a significant association between canine adrenocortical insufficiency and hypoalbuminemia. The pathogenesis of the hypoalbuminemia in this study is speculative; possible mechanisms include gastrointestinal blood loss, protein-losing enteropathy, malassimilation, or decreased albumin synthesis. Renal protein loss was not supported by routine urinalysis findings. PMID- 7552663 TI - Recurrent, nonobstructive, idiopathic feline lower urinary tract disease: an illustrative case report. AB - A three-year-old, spayed female domestic shorthair was evaluated because of recurrent hematuria, dysuria, and pollakiuria of one year's duration. With the exception of hematuria and proteinuria, results of other physical, clinicopathological, radiographic, and microbiologic evaluations were normal. Low concentrations of bovine herpesvirus-4 (BHV-4) antibodies (titer 1:40) were detected by an indirect fluorescent antibody test (IFAT). A diagnosis of nonobstructive, idiopathic feline lower urinary tract disease was established by exclusion of other known causes of hematuria and dysuria. Clinical signs resolved in approximately seven days without symptomatic therapy. During the next 69 months, the owners observed five episodes of self-limiting, gross hematuria and pollakiuria. Persistent low titers of BHV-4 antibodies were detected by the IFAT. This case typifies the clinicopathological, radiographic, and microbiologic findings and the natural course characteristics of many cases of nonobstructive, idiopathic feline lower urinary tract disease. PMID- 7552664 TI - Periosteal osteosarcoma in the long head of the triceps in a dog. AB - A 2.5-year-old Labrador retriever was evaluated for forelimb lameness. Fine needle aspirates of a mass in the proximal brachium were suggestive of a mesenchymal tumor. The dog was euthanized and necropsied. Radiographic evaluation and gross and histopathological findings provided a diagnosis of periosteal osteosarcoma in the long head of the triceps. PMID- 7552665 TI - Prognostic factors in dogs with osteosarcomas of the flat or irregular bones. AB - The medical records of 45 dogs with histopathologically confirmed osteosarcomas arising from flat or irregular bones were reviewed. Thirty-five percent of the dogs eventually developed metastases. Telangectatic tumors and tumors arising from the rib and scapula had the highest prevalence of metastases. Survival times were short, with an overall median survival time of 120 days. Anatomic site, body weight, and completeness of surgical excision were found to be prognostic factors. Dogs with mandibular osteosarcomas in this study had shorter survival times than those times recently reported. 21 Local recurrence was the most common cause of death or euthanasia in this population of dogs. PMID- 7552666 TI - The inheritance of osteochondritis dissecans and fragmented coronoid process of the elbow joint in labrador retrievers. AB - Five matings between dogs bilaterally affected with osteochondritis dissecans and fragmented coronoid processes resulted in a distribution of lesions in the elbows of the offspring that indicated the diseases were inherited independently as polygenic traits. PMID- 7552667 TI - Perineal herniorrhaphy: perioperative data from 100 dogs. AB - One hundred dogs (83 intact males, 15 castrated males, and two intact females) underwent 110 perineal herniorrhaphy procedures. Mixed-breed dogs (n = 32), miniature poodles (n = 14), Boston terriers (n = 11), and Pekingese (n = 9) were represented most frequently. Perineal swelling (n = 48) and a perineal defect on rectal palpation (n = 31) were common clinical signs. Twenty dogs had urinary bladder retroflexion and were significantly more likely to have elevated serum urea nitrogen and creatinine concentrations, hyperkalemia, hyperphosphatemia, and neutrophilic leukocytosis. Only five of 43 dogs evaluated radiographically had prostatomegaly. Of 30 dogs receiving oral barium, all had rectal deviation. The most frequent complications during the hospitalization period were incisional (n = 35), followed by rectal prolapse (n = 9), tenesmus (n = 8), and depression (n = 8). Fifty-six of 70 dogs with follow-up had no complications. PMID- 7552669 TI - A retrospective evaluation of 51 cases of peripheral nerve sheath tumors in the dog. AB - Fifty-one cases of canine peripheral nerve sheath tumors were reviewed. Signalment, presenting clinical signs, duration of clinical signs, physical and neurological examination findings, results of diagnostic procedures, type of surgery performed, tumor location, relapse-free intervals and survival times, and causes of death were evaluated. Tumors were divided into three anatomical groups: tumors involving nerves distal to the brachial or lumbosacral plexus (Peripheral Group), tumors involving nerves of the brachial or lumbosacral plexus (Plexus Group), and tumors involving the vertebral canal (Root Group). The most common clinical findings were unilateral forelimb lameness and muscle atrophy. The most useful diagnostic tests were myelography and electromyography. Although there was no significant difference, dogs in the Root Group tended to have shorter relapse free intervals and survival times than dogs in the Plexus Group. The overall prognosis for surgical management of peripheral nerve sheath tumors is guarded to poor. PMID- 7552668 TI - Partial splenectomy in the dog: a comparison of stapling and ligation techniques. AB - Partial splenectomy was performed in 17 adult dogs by using either surgical stapling instrumentation or conventional suturing techniques. Blood loss was minimal as determined by clinical observation and comparison of packed cell volumes and total protein levels before and after surgery. Surgical stapling instrumentation or suturing techniques may be used safely for partial splenectomy in the dog; however, the stapling technique significantly decreases operative time. PMID- 7552670 TI - The distal biceps tendon. Two potential mechanisms involved in its rupture: arterial supply and mechanical impingement. AB - The purpose of this anatomic study was to evaluate potential causes of rupture of the distal biceps tendon, to assess the dynamic relationship of the proximal radioulnar joint during pronation and supination, and to identify potential sites of impingement of the distal biceps tendon. For the anatomic study specimens were evaluated by light microscopy, multiplanar gross dissections, and Spalteholz vascular injection. For the radiographic study computed tomography was used to assess dynamic changes in the radioulnar space in pronation, neutral position, and supination. Three vascular zones were identified in the distal biceps tendon. Vascular contributions were consistently noted from the brachial artery proximally and from the posterior recurrent artery distally. A hypovascular zone averaging 2.14 cm was evident between the proximal and distal zones. On sectioning through the proximal radioulnar joint 85% of the space was occupied by the distal tendon in full pronation. In addition, computed tomography imaging revealed a 50% reduction in the radioulnar joint at the radial tuberosity from full supination to full pronation. Mechanical impingement on the biceps tendon during forearm rotation and hypovascularity within sections of the tendon may contribute to attritional ruptures of the distal biceps tendon. PMID- 7552671 TI - Ultrasonographic measurement of humeral torsion. AB - This article presents a new method for the measurement of humeral torsion with the use of ultrasonography and discusses clinical applications of the method such as the evaluation of cubitus varus deformity after a supracondylar elbow fracture. Torsion angle increased 14 degrees from the time the patients were newborns to when they were adults. No significant difference was found between the right and left sides in healthy subjects. When humeral shaft or supracondylar fractures are treated, or when rotational deformities of the humerus are corrected, this method of measurement is very practical, because the humeral torsion of the unaffected side provides a standard for the individual. With this method of measuring humeral torsion, we also found that cubitus varus deformity after supracondylar fracture in children is caused not only by an increase in varus angulation but also by internal rotation of the distal fragment. PMID- 7552673 TI - Neuroanatomy in elbow arthroscopy. AB - An injury to the median nerve from within the joint during an arthroscopic synovectomy prompted a study of the relationship of the nerves to the capsule and bones of the elbow. Six pairs of cadaveric elbows frozen in 90 degrees of flexion and one pair frozen in extension were sectioned at 5-mm intervals, and the distances from the major nerves to the bones and capsule were recorded. One elbow joint in each pair was filled with saline solution. Saline solution insufflation increased the nerve-to-bone distance with the elbow in flexion. The results were 12 mm for the median nerve and 6 mm for the radial nerve. The capsule-to-nerve distance was affected little by insufflation and was as narrow as 6 mm in three specimens. Extension eliminated the protective effects of insufflation and brought the nerves closer to the bone. These findings confirm (1) the importance of flexion and insufflation in portal placement, (2) that insufflation does not improve the capsule-to-nerve distance, and (3) the potential for "from within out" injury in synovial procedures. PMID- 7552672 TI - Percutaneous reduction of displaced radial neck fractures. AB - Twenty patients with acute, displaced, radial neck fractures were treated by percutaneous reduction with a periosteal elevator. In all cases acceptable radiographic reduction was achieved, and the reduction position was maintained on serial x-ray examinations with a mean observation period of 3 years. Clinical results were satisfactory in all but one case with slightly limited elbow extension. PMID- 7552674 TI - Anterior acromioplasty: effect of litigation and workers' compensation. AB - Seventy-five consecutive anterior acromioplasties were performed in 74 patients with chronic inflammation of the supraspinatus tendon caused by the impingement syndrome. Thirty-six patients (49%) had filed workers' compensation claims (group 1), 21 patients (28%) were involved in accident litigation (group 2), and 17 patients (23%) had no financial gain associated with their shoulder pain (group 3). The patients were followed up for a minimum of 12 months after surgery (range 12 to 48 months). Sixty-one patients (82%) had excellent results, 11 patients (15%) had good results, and 2 patients (3%) failed to improve from surgery. Ninety-one percent of employed patients were able to return to full employment. Although group 1 patients required a significantly longer time to return to work (average 14.2 weeks vs. 4.7 weeks in group 2 and 2.5 weeks in group 3), these patients were all involved in heavy labor demanding a more complete return of shoulder endurance. Satisfactory pain relief and return to preinjury work activities can be achieved by acromioplasty in patients who have the potential for secondary gain. PMID- 7552676 TI - Heterotopic ossification after acromioplasty and distal clavicle resection. AB - A retrospective review of acromioplasty and distal clavicle resections disclosed 40 cases in which postoperative ectopic bone formation caused recurrent shoulder impingement or acromioclavicular joint pain. Symptomatic lesions either encroached on the supraspinatus outlet or were located in the acromioclavicular interval and were large in size. The incidence of symptomatic heterotopic ossification occurring after acromioplasty or distal clavicle excision was 3.2% and was disproportionately seen in patients with chronic pulmonary diseases (p < 0.05). Heterotopic bone formation could not be correlated with the method of bone resection and occurred after both open and arthroscopic procedures. No evidence of bone remnants or calcific deposits was seen in 17 patients in whom postoperative radiographs were taken within 8 weeks of the operation. It thus appeared that the heterotopic bone formed de novo after the procedure. Twenty patients had repeat shoulder surgery to ameliorate symptoms; four of these had a second recurrence of postoperative heterotopic bone formation. Three of the four required a third procedure and had effective prophylaxis against heterotopic ossification. It is suggested that patients at risk (e.g., with a profile of hypertrophic pulmonary osteoarthropathy or active spondylitic arthropathy) be treated with prophylaxis for heterotopic ossification after acromioplasty and distal clavicle resections. PMID- 7552675 TI - Proximal clavicle excision: an analysis of results. AB - Medial clavicle excision has been reported by several authors, but few cases are documented, and long-term follow-up information is lacking. The purpose of this study was to examine the long-term results of medial clavicle excision in regard to function, pain, cosmesis, and complications. Fifteen patients ranging in age from 18 to 64 years (average 43 years) were evaluated an average of 4.6 years (range 1 to 14 years) after proximal clavicle excision. The indications for excision were unstable anterior subluxation/dislocation of the sternoclavicular joint (four cases), unstable posterior dislocation (one case), sternoclavicular osteoarthritis (nine cases), and proximal clavicle osteomyelitis (one case). An average of 2.9 cm of the medial clavicle was excised (range 1 to 4 cm). Fourteen of the 15 patients received significant relief of pain. On a strict grading scale four patients had an excellent result, five a good result, four a fair result, and two a poor result. Regeneration of the clavicle appeared to contribute to a poor result. No operative complications occurred. These findings aid our understanding of surgical options and outcome in the treatment of sternoclavicular joint disease. PMID- 7552677 TI - Trauma-instability-voluntarism classification for glenohumeral instability. AB - Classification of glenohumeral instability is confusing. We think that the existence of trauma, directions of instability, voluntarism, and other factors make classification difficult. The purpose of this article is to create a new classification. One hundred eighty-nine patients with glenohumeral instability involving 207 joints (mean patient age 21.5 years) were subjects of this investigation. Our new classification, which is composed of three main factors (level of trauma, direction of instability, and voluntarism) and some subfactors, simplified it quite well. Abbreviations also make it easier to indicate each joint's condition. About half the subjects had no trauma or mild trauma. Two thirds of the joints with more than one dislocation or subluxation showed instability in other directions in addition to the direction of dislocation or subluxation. This classification is very useful to compare pathogenesis and results of treatment in patients with glenohumeral instability. PMID- 7552679 TI - Dynamic analysis of intraarticular pressure in the glenohumeral joint. AB - Dynamic analysis of intraarticular pressure was performed in 180 glenohumeral joints. The intraarticular pressure demonstrated characteristic changes during shoulder movement. In the healthy group minimum pressure was measured at 40 degrees of elevation in the scapular plane. In patients with contractures the pressure increased in the early phase of elevation and persisted. Pressure changes of the group with incomplete tears of the rotator cuff resembled those of the healthy group, whereas those in the massive tear group showed only slight pressure changes. When patients with rotator cuff tears had no limitation of arm elevation, pressure changes close to the normal pattern were found. However, when active elevation was markedly limited, no significant pressure changes were noted in some cases. In unstable shoulders the rate of pressure increase was slower, and the range of pressure changes was more limited than that in the healthy group. It is speculated that changes in intraarticular pressure reflect the intraarticular volume. Measurement of the intraarticular pressure may be clinically useful in functional assessment. PMID- 7552678 TI - Shoulder muscle forces and tendon excursions during glenohumeral abduction in the scapular plane. AB - Muscle force values and tendon excursions across the glenohumeral joint during unconstrained glenohumeral abduction in the scapular plane were evaluated with a dynamic shoulder testing apparatus. This evaluation was achieved by simulated rotator cuff and middle deltoid activity applied in four plausible muscle force ratios: (1) equal force to each tendon, (2) 2:3 ratio of force applied to the middle deltoid/supraspinatus tendons, (3) 3:2 ratio of force applied to the middle deltoid/supraspinatus tendons, and (4) zero force applied to the supraspinatus tendon to simulate supraspinatus paralysis. The glenohumeral joint was then moved to 5 degrees, 15 degrees, 30 degrees, 45 degrees, 60 degrees, and maximum glenohumeral abduction while muscle forces, tendon excursions, and glenohumeral joint kinematics were monitored. Full glenohumeral abduction was achieved in all four test conditions. When the muscle force combination favored the middle deltoid, the smallest supraspinatus force was required from 30 degrees to maximum glenohumeral abduction; however, when the supraspinatus was favored, the largest supraspinatus force was necessary to achieve maximum glenohumeral abduction. With simulated supraspinatus paralysis the middle deltoid required the greatest increase in force from 15 degrees through 45 degrees of glenohumeral abduction. These results indicate that muscle efficiency during glenohumeral abduction is highly dependent on the ratio of applied force between the middle deltoid and supraspinatus. A larger contribution of force from the supraspinatus was required near the beginning of motion, whereas the middle deltoid was more important near the end of glenohumeral abduction in the scapular plane. Tendon excursion for the middle deltoid (6.4 +/- 0.2 cm) and supraspinatus (3.8 +/- 0.2 cm) were proportionately larger than those for the subscapularis and infraspinatus. Humeral head translations on the glenoid were less than 2 mm in all four conditions evaluated; therefore the glenohumeral joint behaves kinematically as a "ball-and-socket" articulation during glenohumeral abduction. Simulated supraspinatus paralysis does not change normal joint kinematics and does not prevent full glenohumeral abduction. PMID- 7552680 TI - Rotator cuff tearing and inferior subluxation of the humeral head: report of two cases. AB - We encountered two patients with atraumatic rotator cuff tears who exhibited inferior subluxation of the humeral head. In both patients shoulder motion was limited by pain. Repair of the rotator cuff tear relieved the pain and restored glenohumeral joint motion. Although the cause of this unusual complication of rotator cuff damage remains unclear, the inferior subluxation was associated with reflex muscle hypotonia, which would have reduced the pressure in the subacromial space and thus would have decreased the pain caused by the cuff lesion. No similar case seems to have been documented in the literature. PMID- 7552681 TI - Subdeltoid lipoma presenting as subluxation of the glenohumeral joint. AB - This case illustrates that space-occupying lesions beneath the heavily collagenated portion of the deltoid can produce a confusing clinical picture mimicking subluxation. This patient's clinical presentation was quite extraordinary in its resemblance to glenohumeral instability. The case also illustrates the importance of imaging studies in confirming questionable diagnoses. Such studies should be read by the surgeon, and any discrepancies from the radiologist's reading should be addressed. PMID- 7552682 TI - Arthroscopic treatment of rotator cuff disease. AB - Rotator cuff disease represents a spectrum of tendon lesions including inflammation of an intrinsically normal tendon, tendon fibrosis, partial thickness tears, complete tears, and cuff tear arthropathy. An in-depth analysis of the entire subject is beyond the scope of this article. This article will focus on the arthroscopic management of the spectrum of rotator cuff disease in an attempt to provide a timely summary of the present state of knowledge. Five stages of rotator cuff disease will be reviewed: stage 2 impingement, partial thickness cuff tears, complete-thickness rotator cuff tears, irreparable rotator cuff tears, and cuff tear arthropathy. In preparing this article I have assumed that the reader is familiar with the diagnosis of rotator cuff lesions through the use of appropriate patient history, physical examination, and radiologic studies. PMID- 7552683 TI - Hip pain in a patient with an earlier fall. PMID- 7552684 TI - Outcomes in surgical treatment of femoral neck fracture: analysis of failures secondary to osteonecrosis. AB - We analyzed failures of surgical treatment of femoral neck fractures due to osteonecrosis in a consecutive series of 26 patients with an average age of 31 (range, 10 to 49 years). These patients were referred for treatment of osteonecrosis that developed after internal fixation of a fracture of the femoral neck. The average Harris hip score at the time of referral was 59. The length of time from fracture treatment to the development of osteonecrosis averaged 22.5 months (range, 5 months to 13 years). All patients were symptomatic at the time of presentation. Internal fixation used at the time of the initial trauma included a variety of cannulated and noncannulated screws and sliding hip screws. Of the 27 fractures, 7 (26%) were nondisplaced, 20 (74%) were displaced, and 2 (7%) were open and displaced. Fifteen patients had previously had removal of hardware, making magnetic resonance imaging of the femoral head possible. Of those patients, 2 had involvement of 25% or less of the femoral head, 7 had involvement of 25% to 50%, 5 had involvement of 50% to 75%, and 1 patient appeared to have complete involvement of the femoral head. Among the displaced fractures, 30% (6/20) had a radiographically evident malreduction. Radiographs were adequate for evaluation of hardware placement in 78% (21/27) of the fractures. Central placement of the hardware was noted in only 9% (2/21). Superolateral positioning of the hardware within the femoral head was most frequently noted. PMID- 7552685 TI - Posterior fracture-dislocation of the hip in a jogger. AB - Posterior fracture-dislocation of the hip is an uncommon athletic injury, occasionally seen in contact and high-energy sports. The mechanism of injury in this case highlights the high hip joint forces possible during running. The key treatment principle is early reduction of the hip joint, since the incidence of osteonecrosis of the femoral head and degenerative arthrosis increases with delay. Operative treatment will frequently be required to achieve a stable, congruent reduction of articular surfaces, essential for good long-term results. Partial weight-bearing exercise such as swimming and bicycling may aid rehabilitation. Long-term follow-up, with serial radiographs, is important to detect late complications. PMID- 7552686 TI - Imaging of the total hip arthroplasty. AB - More than 75,000 total hip arthroplasties are done yearly in the United States. Although modern cementing techniques have improved the longevity of cemented implants, uncemented prostheses have become popular, especially in younger, more active patients. There is also a trend toward modular components. These mechanical devices will all ultimately fail if subjected to sufficient use and stresses over time, though the specific causes and modes of failure vary widely. Loosening remains the primary cause of implant failure. There is a growing awareness of the role of wear particles in periprosthetic bone resorption with or without loosening. Stress shielding, dislocation, periprosthetic and prosthetic fractures, infection, heterotopic ossification, and stress concentration are also frequently encountered clinical problems. Conventional radiographs are the mainstay in evaluating total hip arthroplasty, with computed tomography and nuclear medicine imaging playing smaller roles. PMID- 7552687 TI - Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen. AB - The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources. PMID- 7552688 TI - Lectin affinity electrophoresis. AB - Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensional lectin affinity electrophoresis experiment is described that affords separation of oligosaccharides. The first-dimension is composed of a weak, polyacrylamide, capillary tube gel containing a lectin. The example described involves a mixture of fluorescently labeled disaccharides. The mobility of only the lectin-binding disaccharide is reduced affording a separation in the first-dimension. The tube gel is then extruded and placed onto the second-dimension gradient polyacrylamide gel and subjected to electrophoresis. Mobility in the second-dimension is dependent on molecular size and visualization si by fluorescence under transillumination. This method is also applicable, with appropriate modifications, for the separation and analysis of glycopeptides and glycoproteins. PMID- 7552690 TI - Methods for construction of adenovirus vectors. AB - Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors. PMID- 7552689 TI - Epstein-Barr virus replication studies and their application to vector design. AB - Vectors containing elements of the Epstein-Barr virus genome are used primarily to maintain cloned DNA inserts as plasmids in mammalian cells. However, Epstein Bar-virus-based vectors have also been valuable tools in the hands of those studying the life cycle of Epstein-Barr virus. In this article, we discuss those characteristics of Epstein-Barr virus and its life cycle that have been used in vector construction and describe methods that are particularly applicable to the use of Epstein-Barr-virus-based vectors. PMID- 7552691 TI - Macintosh sequence analysis software. DNAStar's LaserGene. AB - The analysis of information in nucleotide and amino acid sequence data from an investigator's own laboratory, or from the ever-growing worldwide databases, is critically dependent on well planned and written software. Although the most powerful packages previously have been confined to workstations, there has been a dramatic increase over the last few years in the sophistication of the programs available for personal computers, as the speed and power of these have increased. A wide choice of software is available for the Macintosh, including the LaserGene suite of programs from DNAStar. This review assesses the strengths and weaknesses of LaserGene and concludes that it provides a useful and comprehensive range of sequence analysis tools. PMID- 7552692 TI - The potential exploitation of plant viral translational enhancers in biotechnology for increased gene expression. AB - The regulation of gene expression is extremely important for all organisms, not least for viruses that require a maximum rate of production of viral proteins to allow rapid multiplication and spread. Single-stranded positive-sense RNA viruses contain specific nucleotide sequences that can be used to elevate the expression of vital gene products to required high levels. Among plant viruses, translational enhancement has been documented widely, especially over the past few years. Reported candidates include one of the best known and most intensely researched virus, tobacco mosaic virus, members of the potyvirus group, and even a small satellite RNA of tobacco necrosis virus. Enhancement values range from 2 100-fold with different viruses, different reporter genes, and in different systems. Research indicates that an absence of secondary structure alone does not explain translational enhancement and despite attempts to determine the mechanism by which this enhancement occurs very little conclusions can be made as yet. Whatever the mechanism, the presence of these sequences upstream from an open reading frame results in an elevated level of protein production and may feature as important tools for biotechnology in the future. PMID- 7552693 TI - Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy. AB - The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type ("gene therapy"), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal "break out" capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology. PMID- 7552694 TI - Multiple peptide synthesis. AB - The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required. PMID- 7552695 TI - Nonradioactive probes. AB - This work concentrates on a single procedure, namely hybridizing Southern blots with nonradioactive probes. Stress is placed on features of the procedures where attention to detail is necessary to obtain strong signals and clear background. Some indications are given to the future development of the technology. PMID- 7552696 TI - Detection and identification of Campylobacter coli and Campylobacter jejuni by two-step polymerase chain reaction. AB - Flagellin gene was used as target sequence to detect and distinguish C. coli and C. jejuni by a "nested PCR" technique. The method shows a high level of sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about epidemiological and pathogenetic implications of each of these two microorganisms. PMID- 7552697 TI - Battle for BMRB Biological Magnetic Resonance Data Bank. PMID- 7552698 TI - Breathing life into the folding pathway of cytochrome c. AB - In cytochrome c, the unfolding reaction occurs in four discreet stops, as monitored by the exchange of solvent hydrogen for backbone aminde hydrogens under varying denaturant concentrations. PMID- 7552699 TI - Watching protein folding unfold. AB - Direct NMR observation of a transient folding intermediate provides new evidence for the importance of molten globules as general intermediates in protein folding. PMID- 7552700 TI - Complexity in proteins. PMID- 7552702 TI - Structure of the carboxy-terminal domain of bovine rhodopsin. AB - The biologically active carboxy-terminal peptide of the G-protein receptor, rhodopsin, forms a compact structure, suggesting that it is a structural domain in this integral membrane protein. The disposition of serines explains receptor kinase specificity. PMID- 7552701 TI - Eleven down and nine to go. AB - A series of new crystal structures of aminoacyl-tRNA synthetases sheds light on the evolution of specificity in this ancient family of enzymes. PMID- 7552703 TI - A disulphide-reinforced structural scaffold shared by small proteins with diverse functions. AB - We describe the T-knot scaffold, a structural feature shared by the EGF-like proteins, alpha-toxins and proteinase inhibitors from plants. PMID- 7552704 TI - Different enzymes with similar structures involved in Mg(2+)-mediated polynucleotidyl transfer. AB - Comparison of X-ray structures of restriction endonucleases and polynucleotidyl transferase superfamily enzymes reveals a structural resemblance. PMID- 7552705 TI - Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. AB - Crystallization of membrane proteins, a prerequisite for their X-ray crystallographic analysis, remains difficult. Here, we demonstrate that the crystallization of the cytochrome c oxidase from Paracoccus denitrificans can be mediated by co-crystallization with an antibody Fv fragment. The crystals obtained contain all four subunits of this membrane protein complex and the Fv fragment. The approach of co-crystallizing membrane proteins with antibody fragments should be useful in obtaining well-ordered crystals of membrane proteins in general. PMID- 7552706 TI - Picture story. Starting off right. PMID- 7552707 TI - Looking into the energy landscape of myoglobin. AB - Using the haem group of myoglobin as a probe in optical experiments makes it possible to study its conformational fluctuations in real time. Results of these experiments can be directly interpreted in terms of the structure of the potential energy surface of the protein. The current view is that proteins have rough energy landscapes comprising a large number of minima which represent conformational substates, and that these substates are hierarchically organized. Here, we show that the energy landscape is characterized by a number of discrete distributions of barrier heights each representing a tier within a hierarchy of conformational substates. Furthermore, we provide evidence that the energy surface is self-similar and offer suggestions for a characterization of the protein fluctuations. PMID- 7552708 TI - Solvent isotope effect and protein stability. AB - Here we present a comparative study of the stability of several proteins in H2O and D2O as a function of pH/pH*. We show that the substitution of D2O for H2O leads to an increase in the transition temperature and a decrease in the enthalpy of unfolding. The stability of the proteins, however, appears to be largely unchanged as a result of entropic compensation for the decrease in enthalpy. This enthalpy-entropy compensation is attributed to changes in hydration of proteins in D2O compared to H2O. Analysis of thermodynamic data for the transfer of model compounds from H2O to D2O shows that almost all the changes in the enthalpy of unfolding and in the protein-ligand interactions due to water isotopic substitution can be rationalized by changes in hydration of the buried non-polar groups. PMID- 7552709 TI - Cooperatively folded proteins in random sequence libraries. AB - The structural properties of proteins recovered from random sequence libraries can be used to investigate the relationship between folding and sequence information. Here, we show that helical proteins displaying cooperative thermal denaturation transitions can be easily recovered from a library containing 80 residue proteins predominantly composed of glutamine, leucine, and arginine, with an average hydrophobicity level similar to that of natural proteins. The native structure of one of these proteins has a stability and oligomeric form similar to that of many natural proteins but differs in having no slowly exchanging amide hydrogens. PMID- 7552710 TI - Following protein folding in real time using NMR spectroscopy. AB - The refolding of apo bovine alpha-lactalbumin has been monitored in real time by NMR spectroscopy following rapid in situ dilution of a chemically denatured state. By examining individual resonances in the time-resolved NMR spectra, the native state has been shown to emerge in a cooperative manner from an intermediate formed in the dead-time of the experiments. The kinetics of folding to the native state are closely similar to those observed by stopped-flow fluorescence and near-UV circular dichroism. The NMR spectrum of the transient intermediate resembles closely that of the well characterized stable molten globule state formed at low pH. The results suggest that NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding. PMID- 7552711 TI - Structural basis of the stability of a lysozyme molten globule. AB - Hydrogen exchange measurements on equine lysozyme show that amides in three of the four major helices of the native protein are significantly protected in a molten globule state formed at pH 2. The pattern of protection within the different helices, however, varies significantly. Examination of the pattern in the light of the native structure indicates that the side chains of the protected residues form a compact cluster within the core of the protein. We suggest that such a core is present in the molten globule state, indicating the existence of substantial native-like interactions between hydrophobic residues. The formation of clusters of this type during the early stages of folding could be crucial to directing polypeptide chains to their native structures. PMID- 7552712 TI - Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. AB - The crystal structures of thymidine kinase from herpes simplex virus type-1 complexed with its natural substrate deoxythymidine (dT) and complexed with the guanosine analogue Ganciclovir have been solved. Both structures are in the C222(1) crystal form with two molecules per asymmetric unit related by a non crystallographic two-fold axis. The present models have been refined to 2.8 A and 2.2 A, with crystallographic R factors of 24.1% and 23.3% for the dT and Ganciclovir complexes respectively, without the inclusion of any solvent molecules. The core of the molecule exhibits high structural homology with adenylate kinase and other nucleotide binding proteins. These structural similarities provide an insight into the mechanism of nucleoside phosphorylation by thymidine kinase. PMID- 7552713 TI - The structure of satellite panicum mosaic virus at 1.9 A resolution. AB - The crystal structure of satellite panicum mosaic virus (SPMV) has been solved by multiple isomorphous replacement and molecular replacement and refined at 1.9 A resolution. SPMV, a T = 1 icosahedral virus, is the smallest virus structure determined. The coat protein is an eight-stranded 'jelly roll' beta-barrel with an amino-terminal strand that extends into the interior of the virus, presumably interacting with the RNA. Regions of electron density on the interior of the protein capsid may be RNA, although it is not possible to construct any detailed model of the nucleic acid. Basic amino acid residues in contact with the nucleic acid show a considerable degree of disorder. The carboxy-terminal strand of the virus coat protein interacts with adjacent subunits, forming an additional beta strand. PMID- 7552714 TI - Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1. AB - Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new beta-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into beta-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation. PMID- 7552715 TI - Solution structure of the Kluyveromyces lactis LAC9 Cd2 Cys6 DNA-binding domain. AB - The Zn2Cys6 DNA-binding domain has been identified by sequence homology in approximately forty fungal proteins, including the K. lactis LAC9 transcriptional activator. Using 1H NMR spectroscopy, we have determined the solution structure of a cadmium-substituted form of the LAC9 DNA-binding domain. We have complemented this approach by applying a series of 113Cd-1H NMR experiments, including several novel heteroTOCSY-based techniques. The DNA-binding domain forms a core of two alpha-helix/extended strand segments around the Cd2 binuclear cluster, with a network of amide proton-cysteinyl S gamma hydrogen bonds stabilizing the cluster. Comparison with other Zn2Cys6 domain structures provides insight into the common structural elements used in metal coordination and DNA binding. PMID- 7552716 TI - Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface. AB - Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway. The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY. When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site. A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA. The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis. PMID- 7552717 TI - Aspirin and inflammation. PMID- 7552718 TI - Resolving a resolvase. PMID- 7552719 TI - Hammerhead: Part 2. PMID- 7552720 TI - Reading the minor groove. PMID- 7552721 TI - Christian B. Anfinsen 1916-1995. PMID- 7552723 TI - Structural modelling of a type I DNA methyltransferase. PMID- 7552722 TI - Modelling membrane proteins using structural restraints. AB - Here we present a procedure for modelling membrane proteins which employs molecular dynamics simulations incorporating target restraints derived from low resolution structures alongside distance restraints derived from mutagenesis data. The application of the modelling procedure to the closed conformation of the pore domain of the nicotinic acetylcholine receptor is described. This domain is formed by a parallel bundle of five M2 helices. Each M2 helix is kinked due to cumulative distortions of backbone (phi, psi) values. The central region of M2 may adopt a more distorted conformation. This would enable a ring of conserved leucine residues (one from each M2 helix) to pack together, occluding the central pore and thus preventing ion permeation. Molecular dynamics simulations on isolated helices that kink formation is not an inherent property of M2. PMID- 7552724 TI - Picture story. Cyclin switch. PMID- 7552725 TI - The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase. AB - Aspirin exerts its anti-inflammatory effects through selective acetylation of serine 530 on prostaglandin H2 synthase (PGHS). Here we present the 3.4 A resolution X-ray crystal structure of PGHS isoform-1 inactivated by the potent aspirin analogue 2-bromoacetoxy-benzoic acid. Acetylation by this analogue abolishes cyclooxygenase activity by steric blockage of the active-site channel and not through a large conformational change. We observe two rotameric states of the acetyl-serine side chain which block the channel to different extents, a result which may explain the dissimilar effects of aspirin on the two PGHS isoforms. We also observe the product salicylic acid binding at a site consistent with its antagonistic effect on aspirin activity. PMID- 7552726 TI - An enzyme-substrate complex involved in bacterial cell wall biosynthesis. AB - The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP-N-acetylglucosamine, has been solved to 2.7 A resolution. This enzyme is responsible for the synthesis of UDP-N acetylmuramic acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether. PMID- 7552727 TI - The multisubunit active site of fumarase C from Escherichia coli. AB - The crystal structure of the tetrameric enzyme, fumarase C from Escherichia coli, has been determined to a resolution of 2.0 A. A tungstate derivative used in the X-ray analysis is a competitive inhibitor and places the active site of fumarase in a region which includes atoms from three of the four subunits. The polypeptide conformation is similar to that of delta-crystallin and is comprised of three domains. The central domain, D2, is a unique five-helix bundle. The association of the D2 domains results in a tetramer which has a core of 20 alpha-helices. The other two domains, D1 and D3, cap the helical bundle on opposite ends giving both the single subunit and the tetramer a dumbbell-like appearance. Fumarase C has sequence homology to the eukaryotic fumarases, aspartase, arginosuccinate lyase, adenylosuccinate lyase and delta-crystallin. PMID- 7552728 TI - Extremely rapid protein folding in the absence of intermediates. AB - Here we used the cold-shock protein CspB from Bacillus subtilis to study protein folding at an elementary level. The thermodynamic stability of this small five stranded beta-barrel protein is low, but unfolding and refolding are extremely rapid reactions. In 0.6 M urea the time constant of refolding is about 1.5 ms, and at the transition midpoint (4 M urea) the folded and unfolded forms equilibrate in less than 100 ms. Both the equilibrium unfolding transition and the folding kinetics are perfectly described by a N<-->U two-state model. The validity of this model was confirmed by several kinetic tests. Folding intermediates could neither be detected at equilibrium nor in the folding kinetics. We suggest that the extremely rapid folding of CspB and the absence of folding intermediates are related phenomena. PMID- 7552729 TI - A third native one-disulphide intermediate in the folding of bovine pancreatic trypsin inhibitor. AB - A previously unidentified intermediate has been detected in the early stages of the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). The intermediate contains one disulphide bond between residues 14 and 38 and is denoted [14-38]. The 14-38 disulphide bond is also found in native BPTI. Although the other native one-disulphide intermediates, [30-51] and [5-55], are thermodynamically more stable, [14-38] can be populated substantially at the early stages of BPTI folding. Moreover, initial characterization of the kinetic properties of this intermediate strongly suggest that a substantial fraction of BPTI molecules fold by way of the [14-38] intermediate. Our results emphasize the importance of native-like tendencies in protein folding. PMID- 7552730 TI - Residues defining V beta specificity in staphylococcal enterotoxins. AB - The three-dimensional structure of staphylococcal enterotoxin C2 has been determined at 2.7 A resolution by x-ray diffraction, while the structures of enterotoxins A and E have been modelled based on their sequence homology to other staphylococcal enterotoxins. The T-cell receptor-binding sites of staphylococcal enterotoxin (SE) B and SEC2 are compared and the stereochemical interactions likely to be responsible for their differing V beta specificities are identified. A similar comparison is made between SEA and SEE. PMID- 7552731 TI - Structure of porcine aldehyde reductase holoenzyme. AB - Aldehyde reductase, a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of a variety of aldehydes to their corresponding alcohols. The structure of porcine aldehyde reductase-NADPH binary complex has been determined by x-ray diffraction methods and refined to a crystallographic R factor of 0.20 at 2.4 A resolution. The tertiary structure of aldehyde reductase is similar to that of aldose reductase and consists of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. Unlike aldose reductase, the N epsilon 2 of the imidazole ring of His 113 in aldehyde reductase interacts, through a hydrogen bond, with the amide group of the nicotinamide ring of NADPH. PMID- 7552732 TI - Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction. AB - Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction. PMID- 7552733 TI - The problem with going places. PMID- 7552734 TI - Releasing the calcium trigger. AB - NMR structures of calmodulin, troponin C and related proteins are providing the atomic details of the conformational changes that transduce Ca2+ signals into mechanical or metabolic responses. PMID- 7552735 TI - Dance of the dimers. AB - The structure of the apo form of calcyclin, a member of the S100 family of calcium-binding proteins, reveals a novel dimer fold that may reflect the presence of a new interface for target protein recognition. PMID- 7552736 TI - Scratching the surface with the PH domain. AB - Pleckstrin homology (PH) domains bind to membrane surfaces, and inositol phospholipids appear to form part of the binding sites. Recent structural studies provide a model for PH domain anchoring to inositol phospholipids that will open new avenues for functional investigation. PMID- 7552737 TI - All in the family. AB - Identification of the residues involved in the reaction catalysed by aldehyde reductase should aid in the development of drugs for the treatment of diabetic complications. PMID- 7552738 TI - When one and one are not two. AB - Dimeric proteins can arise from monomers by the simple exchange of secondary structural elements or a wholesale swapping of domains. These results have implications for the construction of novel oligomeric molecules and illuminate how existing structures may have evolved. PMID- 7552739 TI - The CHIP28 water channel visualized in ice by electron crystallography. AB - Electron crystallography of frozen-hydrated two-dimensional crystals of deglycosylated human erythrocyte CHIP28 reveals an aqueous vestibule in each monomer leading to the water-selective channel that is enclosed by multiple transmembrane alpha-helices. PMID- 7552740 TI - Projection map of aquaporin-1 determined by electron crystallography. AB - Using cryo-electron microscopy we have determined a projection map of the structure of the water selective pore aquaporin-1. PMID- 7552741 TI - Crystal structures of B-form DNA-RNA chimers complexed with distamycin. AB - Two DNA-RNA chimers, complexed with DNA minor groove binding drugs, have been observed to adopt the B-form conformation for the first time. Thus, the RNA duplex may assume the B-DNA conformation when interacting with drugs, peptides or proteins. PMID- 7552742 TI - Rational design of synthetic heparin analogues with tailor-made coagulation factor inhibitory activity. AB - Computer modelling of the antithrombin III-heparin-thrombin complex inspired the synthesis of novel glycoconjugates, whose factor Xa and thrombin inhibitory activities can be adjusted in a rational way, leading to anticoagulants with unprecedented characteristics. PMID- 7552743 TI - Proposed structure of the A domains of factor VIII by homology modelling. AB - We have predicted a structure for the three A domains of blood coagulation factor VIII by virtue of their homology to blue copper-binding proteins. This structure, consisting of six beta-barrels, is arranged in a triangular configuration with a single type II copper-binding site linking the A1 and A3 domains. PMID- 7552744 TI - Picture story. An unfolding story on binding. PMID- 7552745 TI - One-step evolution of a dimer from a monomeric protein. AB - Deletion of six amino acids in a surface loop transforms staphylococcal nuclease from a monomeric protein into a very stable dimer (Kd < 1 x 10(-8)M). A 2 A X-ray crystal structure of the dimer (R = 0.176) shows that the carboxy-terminal alpha helix has been stripped from its normal position in one monomer and is now incorporated into the equivalent position on the adjoining monomer. This swapping creates an association interface of 2900 A 2. A second, smaller interface of 460 A 2 is also formed. The spontaneous exchange or swapping of secondary structural elements provides a simple pathway for the formation of large, stable protein/protein interfaces and may play an important role in the evolution of oligomeric proteins. PMID- 7552746 TI - Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex. AB - The Bacillus subtilis bacteriophages PBS-1 and PBS-2 protect their uracil containing DNA by expressing an inhibitor protein (UGI) which inactivates the host uracil-DNA glycosylase (UDGase) base-excision repair enzyme. Also, PBS1/2 UGI efficiently inactivates UDGases from other biological sources, including the enzyme from herpes simplex virus type-1 (HSV-1). The crystal structure of the HSV 1 UDGase-PBS1 UGI complex at 2.7 angstrum reveals an alpha-beta-alpha sandwich structure for UGI which interacts with conserved regions of UDGase involved in DNA binding, and directly mimics protein-DNA interactions observed in the UDGase oligonucleotide complex. The inhibitor completely blocks access to the active site of UDGase, but makes no direct contact with the uracil-binding pocket itself. PMID- 7552747 TI - Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. AB - The solution structure of Ca(2+)-free calmodulin has been determined by NMR spectroscopy, and is compared to the previously reported structure of the Ca(2+) saturated form. The removal of Ca2+ causes the interhelical angles of four EF hand motifs to increase by 36 degrees-44 degrees. This leads to major changes in surface properties, including the closure of the deep hydrophobic cavity essential for target protein recognition. Concerted movements of helices A and D with respect to B and C, and of helices E and H with respect to F and G are likely responsible for the cooperative Ca(2+)-binding property observed between two adjacent EF-hand sites in the amino- and carboxy-terminal domains. PMID- 7552748 TI - Solution structure of calcium-free calmodulin. AB - The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca(2+)-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel beta-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca(2+)-free N-terminal domain of troponin C. PMID- 7552749 TI - Calcium-induced structural changes and domain autonomy in calmodulin. AB - We have determined the solution structures of the apo and (Ca2+)2 forms of the carboxy-terminal domain of calmodulin using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results show that both forms adopt well-defined structures with essentially equal secondary structure. A comparison of the structures of the two forms shows that Ca2+ binding causes major rearrangements of the secondary structure elements with changes in inter-residue distances of up to 15 A and exposure of the hydrophobic interior of the four helix bundle. Comparisons with previously determined high-resolution X-ray structures and models of calmodulin indicate that this domain is structurally autonomous. PMID- 7552750 TI - Structures of the troponin C regulatory domains in the apo and calcium-saturated states. AB - Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction. PMID- 7552751 TI - The structure of calcyclin reveals a novel homodimeric fold for S100 Ca(2+) binding proteins. AB - The S100 calcium-binding proteins are implicated as effectors in calcium-mediated signal transduction pathways. The three-dimensional structure of the S100 protein calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among calcium binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of calcium binding proteins. PMID- 7552752 TI - The structure of rat ADP-ribosylation factor-1 (ARF-1) complexed to GDP determined from two different crystal forms. AB - The ARFs are a family of 21,000 M(r) proteins with biological roles in constitutive secretion and activation of phospholipase D. The structure of ARF-1 complexed to GDP determined from two crystal forms reveals a topology that is similar to that of the protein p21 ras with two differences: an additional amino terminal helix and an extra beta-strand. The Mg2+ ion in ARF-1 displays a five coordination sphere; this feature is not seen in p21 ras, due to a shift in the relative position of the DXXG motif between the two proteins. The occurrence of a dimer in one crystal form suggests that ARF-1 may dimerize during its biological function. The dimer interface involves a region of the ARF-1 molecule that is analogous to the effector domain in p21 ras and may mediate interactions with its effectors. PMID- 7552753 TI - The DNA-binding domain of HIV-1 integrase has an SH3-like fold. AB - We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxyterminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design. PMID- 7552754 TI - Physical performance, physical activity and quality of life in elderly people. PMID- 7552755 TI - Aging of human muscle: structure, function and adaptability. AB - With increasing age, human skeletal muscles gradually decrease in volume, mainly due to a reduced number of motor units and muscle fibers, and a reduced size of type 2 fibers. As a result, progressive weakening and impaired mobility occur. High-resistance strength training is beneficial, even in the very old, and could possibly reverse some of the detrimental effects of age-related weakness. The importance of exercise for older people affords an excellent opportunity for the medicine community as a major source of information and promotion of physical activity for this rapidly growing segment of the population. In this review, we summarize the current knowledge of the effects of aging on the human neuromuscular system, describe some of the major underlying mechanisms of the aging atrophy and focus on the importance of strength training to improve muscle function in older people. PMID- 7552756 TI - A maximal cycle exercise protocol to predict maximal oxygen uptake. AB - Maximal oxygen uptake (VO2max) was predicted from maximal power output (MPO) in a progressive cycle ergometer test. The subjects were 232 men and 303 women 15-28 years of age. The relationship between VO2max and MPO was: VO2max (1.min-1) = 0.16 + (0.0117 x MPO) (w). A correlation coefficient of r = 0.88 was found between MPO and VO2max. Test-retest reliability was evaluated by two procedures. Standard deviations of test-retest differences in MPO and VO2max using the same standardized procedure in 35 subjects, were 10% and 8%, respectively, and Pearson correlations between test and retest values were 0.95 and 0.96, respectively. When MPO of tests conducted at the schools was compared to a standardized test performed by a physiologist in 267 subjects, test-retest Pearson correlation was 0.82. A prediction model only including MPO and explaining 80% of the variability in VO2max, is suggested for use in healthy adolescents and young adults. PMID- 7552757 TI - Cardiovascular changes with age and exercise. A 28-year longitudinal study. AB - Various studies report a 0-22% per decade decline in circulatory function (VO2max) with advancing age. Twelve exercising men (E) were followed for 28 years, mean age 43 and 71 years, initial to final measurement, while 12 dropouts (C) detrained for 21 years, with a mean age from first to last measurement of 48 and 69 years, respectively. VO2 max in E changed from 45.9 to 39.4 ml.min-1.kg-1, 5% per decade, whereas C declined from 36.0 to 21.4 ml.min-1.kg-1, 19% per decade. Resting blood pressure was unchanged in E, 119/75 mmHg, whereas C rose from 128/85 to 149/90 mmHg. The data suggest that regular aerobic exercise forestalls the usual loss of circulatory function with increasing age. PMID- 7552758 TI - The influence of training intensity, airway infections and environmental conditions on seasonal variations in bronchial responsiveness in cross-country skiers. AB - The influence of physical training, airway infections and environmental conditions on changes in bronchial responsiveness to methacholine during a training and competitive season was studied in 19 high-performance male cross country skiers 19-21 years old. The longitudinal changes in the methacholine concentration required for a 10% fall in FEV1 (PC10) were negatively correlated with the changes in the volume of physical activity at an intensity level above 90% of maximal heart rate. The variation in physical activity at this intensity level accounted for 54.8% of the change in PC10. No association was found, however, with regard to physical activity at lower intensity levels. Seasonal variation in PC10 was not associated with the changes in occurrence or duration of airway infections provided that the PC10 measurement was postponed for 3-6 weeks after the onset of a recent infection. Seasonal variation in PC10 seemed not to be associated with variations in ambient levels of air pollutants or aeroallergens. PC10 was lowest at the end of the coldest part of the year. In conclusion, seasonal variation in bronchial responsiveness in high-performance cross-country skiers could to a great extent be explained by changes in the volume of physical activity at a very high intensity level. PMID- 7552759 TI - The plantar flexor muscle attachments on the tibia. A cadaver study. AB - Thirty-six tibiae from 11 female and 7 male cadavers were dissected. Anatomical and histological examinations of the plantar flexor muscle origins at the posteromedial border of the tibia were performed. Many individual variations in the type and size of muscle origin were observed. Muscle fibers and/or connective tissue in different proportions attached the muscle to the periosteum or directly to the cortical bone. The length of the attachments varied greatly and there was a considerable overlap of the muscles in some individuals. The attachments of the flexor digitorum longus overlapped the tibialis posterior and the flexor digitorum longus muscle was overlapped by the soleus muscle. In two cases the soleus muscle did not attach to the tibia at all. Our findings may shed some light on the question as to why some athletes sustain posteromedial tibial stress fractures and others develop shin splits or other posteromedial injuries from similar precipitating activity. PMID- 7552760 TI - Contraction of knee flexors and extensors in skiing related to the backward fall mechanism of injury to the anterior cruciate ligament. AB - The purpose of this study was to examine the motion pattern and muscle activity during jumping and landing in downhill skiing to estimate the muscular involvement during the jump-landing-backward fall-anterior cruciate ligament (ACL) injury. A digitized 2-D video motion analysis and a synchronous 8-channel electromyography registration was performed over a jump on a downhill slope during 3 runs by 2 skiers and during 6 control jumps in the laboratory. The knee flexors (lateral gastrocnemius and biceps femoris) were recruited before touchdown and mean 60 ms earlier than the extensors (vastus lateralis and rectus femoris). Extensors and flexors reached peak activity simultaneously at the instant of landing. The knee flexion angle at the instant of landing was mean 36 degrees. As the landing was stabilized, the extensor activity persisted during eccentric work at increasing knee flexion as a corresponding flexor relaxation took place. The control jump in the laboratory showed a similar order of recruitment and timing of muscle contraction. The gastrocnemius was recruited mean 96 ms earlier and the biceps femoris mean 63 ms earlier than the extensors. This recruitment pattern during the landing movement suggest a learned motor program engaging the kinetic chain of the lower extremity to intercept the landing energy. The results also indicate that the knee flexor activity may protect the ACL at the instant of landing but that it is minimal shortly thereafter and probably not able to protect the ACL when a backward fall occurs. The knee was flexed substantially as the extensors became the dominating muscles. Thus, their ability to apply any anterior drawer force able to rupture the ACL is questioned. PMID- 7552761 TI - Repair of Bankart lesions with a suture anchor in recurrent dislocation of the shoulder. AB - This study describes the 3-year results of Bankart reconstruction in 50 consecutive patients with posttraumatic, recurrent, anterior instability of the shoulder. A modified procedure, using suture anchors was used in all patients, to simplify the soft-tissue attachment to the glenoid rim. Forty-seven of 50 (94%) patients regained normal stability. Two recurrent dislocations and one subluxation occurred. The functional results according to the Rowe scoring system were satisfactory in 43 of 50 (86%) of the patients, and unsatisfactory in 7 of 50 (14%). Of the patients with unsatisfactory results, 3 had recurrent instability and 4 had not regained normal range of shoulder motion. The suture anchors were found to simplify the procedure, and no specific complications as a result of the modified technique were seen. PMID- 7552762 TI - Maximal and submaximal oxygen uptake during running: how should body mass be accounted for? AB - Oxygen uptake during running, i.e., the running economy, is an important factor in determining running performance in endurance events. The relation to performance is particularly strong when the aerobic running capacity is calculated, i.e., when running economy is related to the maximal oxygen uptake. There is considerable interindividual variation in running economy, and the reason for this is only partly understood. To some extent, this may be due to the way in which the oxygen uptake during running is usually expressed. This may especially be true when subjects with different or changing body masses are compared. Several lines of evidence, including earlier animal studies as well as more recent human studies, favor the expression of submaximal and maximal oxygen uptake during running in terms of ml.kg-0.75.min-1 rather than as ml.kg-1.min-1. PMID- 7552763 TI - Overuse injuries of the lower extremity in runners. AB - The purpose of this article is to review the literature on overuse injuries of the lower extremity in runners and to discuss briefly today's knowledge concerning etiology, diagnosis and treatment. Running is a natural entity in many sports and a majority of runners will sustain one or more overuse injuries throughout the career, in most cases affecting the lower extremity. A runner may be regarded as an athlete who regularly runs as the predominant physical activity. From that point, we should subdivide the definition "runner" considering the character of different sports or recreational activities performed. Overuse injuries are often described merely from symptoms, including several different etiological and pathoanatomic correlates covering a variety of ailments. The clinical approach should be focused on a thorough history and physical examination. Analysis of possible injury mechanisms, correction of associated extrinsic and intrinsic factors and advice on alternative training should be given. A knowledge of specific demands from the type of running performed is necessary to evaluate the symptoms presented. Overuse etiology has to be considered multifactorial with a yet unsolved exact pathophysiology needing further research. The definition of a "runner", of "running" and of "overuse injury" should be established and agreed upon. This review attempts to draw attention to the huge multidisciplinary work that has to be done to better understand the mechanisms causing an overuse injury in a runner and to define diagnoses on a scientific base, whether or not excentric or intrinsic factors predispose or trigger. PMID- 7552764 TI - Intrapersonal and interpersonal factors in athletic performance. AB - Psychologically athletic performance is a multiplicative function of intrapersonal (for example, intrinsic motivation) and interpersonal (for example, social support) factors. It is theorized that the layer immediately surrounding an athlete's inner core of psychological functioning consists of 4 intrapersonal factors: self-motivation, cognitive capacity and coping skills, affective orientation and mental training skills. The layer surrounding these factors is comprised of interpersonal factors, such as social support and the athlete-coach relationship. A theoretical model was advanced to show that competition-generated intrinsic motivation predisposition and social support are the two main properties of the athletic competition context that buffer against increased athletic and life stress. If such buffering effects occur, athletes' physical and psychological capacity is maintained or even enhanced. This in turn promotes continued competitive athletic participation and performance. Maximization of psychological conditions for successful performance requires that coaches foster athletes' intrapersonal and interpersonal psychosocial resources by serving as facilitators of their autonomous self-regulation rather than as controllers of their goals and behaviors. PMID- 7552765 TI - Mechanical efficiency in athletes during running. AB - The purpose of this study was to compare the external mechanical efficiency (ME) between power-trained athletes (n = 5) and endurance-trained athletes (n = 5). The relationships between biomechanical variables and metabolic cost were also investigated. The subjects ran at 3 different speeds (2.50 m.s-1, 3.25 m.s-1 and 4.00 m.s-1) both on the treadmill and on the track. The external work of the subjects was determined by a kinematic arm, and energy expenditure was determined by measuring oxygen consumption and respiratory exchange ratio. Biomechanical parameters included ground reaction forces, angular displacements of the knee and ankle joints and electromyography (EMG) of the selected muscles. The mean ME (+/- SD) values during running on treadmill were as follows: 49.6 +/- 8.9%, 60.1 +/- 9.6% and 61.2 +/- 10.4% for the endurance group, and 47.1 +/- 3.7%, 52.0 +/- 4.3% and 57.4 +/- 5.5% for the power group. In running on the track the respective values were 57.5 +/- 11.9%, 51.5 +/- 6.1% and 62.2 +/- 9.2% for the endurance group, and 47.0 +/- 8.3%, 45.3 +/- 10.2% and 60.0 +/- 5.9% for the power group. The subject groups did not differ significantly in ME due to high interindividual variance among both subject groups. The metabolic responses such as heart rate, pulmonary ventilation and oxygen uptake differed clearly between the athletic groups but this was not the case for the most of the biomechanical variables (such as EMG, step length and vertical displacement of the centre of the gravity).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552767 TI - Morphology, enzyme activities and buffer capacity in leg muscles of Kenyan and Scandinavian runners. AB - The study comprises data on 12 Scandinavian runners who had either trained for two weeks in Kenya (n = 6; approximately 2000 meters above sea level (m.a.s.l.)) or in Portugal (n = 6; sea level (s.l.)) and on 13 Kenyan runners (9 junior and 4 senior) living and training at approximately 2000 m.a.s.l. Muscle biopsies were taken before and after the training camps in the Scandinavian runners and once on the Kenyan runners from the vastus lateralis (v.l.) and the gastrocnemius muscles. Muscle fiber size and composition were similar in the various groups (4.6-5.1 X 10(3) microns2; ST approximately 60-70%; FTa 30-40%; FTb < 6.0%) with a tendency for some more (approximately 5%) FTa fibers in the gastrocnemius muscle. Mean number of capillaries in v.l. varies between 405-493 cap.(mm2)-1, 2.0-2.7 cap.fiber-1, and 4.4-6.2 cap around the various fiber types, with the Kenyan seniors having the highest and the Kenyan juniors the lowest values. All runners had 10-20% more capillaries in their gastrocnemius muscle. Similar levels for citrate synthase (CS) activity were found in the v.l. of the Kenyan seniors and Scandinavian runners, whereas the Kenyan juniors were 10-15% lower. The 3- hydroxyacyl-CoA-dehydrogenase (HAD) activity was 20% higher in the Kenyan than in the Scandinavian runners. In the gastrocnemius muscle, both enzyme activities were 20-50% higher in Scandinavian and Kenyan runners. The ratio for lactate dehydrogenase (LDH) isoform1-2 and isoform4-5 was increased when training at altitude due to a lowering of LDH4-5 and became close to what was observed in the Kenyan runners. PMID- 7552766 TI - Aerobic exercise capacity at sea level and at altitude in Kenyan boys, junior and senior runners compared with Scandinavian runners. AB - The aim of this study was to characterize Kenyan runners in regard to their oxygen uptake and blood and ammonia responses when running. Untrained Kenyan boys (14.2 +/- 0.2 years) and Scandinavian runners were included for comparison. The studies were performed at altitude (approximately 2.000 m.a.s.l.) and, for several Kenyan and Scandinavian runners, at sea level as well. At altitude sedentary adolescent Kenyan boys had a mean maximal oxygen uptake (VO2max) of 47 (44-51) ml.kg-1.min-1, whereas similarly aged boys regularly walking or running but not training for competition reached above 62 (58-71) ml.kg-1.min-1 in VO2max. Kenyan runners in active training had 68 +/- 1.4 ml.kg-1.min-1 at altitude and 79.9 +/- 1.4 ml.kg-1.min-1 at sea level, with individuals reaching 85 ml.kg-1.min-1. The best Scandinavian runners were not significantly different from the Kenyan runners in VO2max both at altitude and at sea level, but none of the Scandinavians reached as high individual values as observed for some Kenyan runners. The running efficiency, determined as the oxygen cost at a given running speed, was less in the Kenyan runners, and the difference became more pronounced when body weight was expressed in ml.kg-0.75 min-1. Blood lactate concentration was in general lower in the Kenyan than in the Scandinavian runners, and the Kenyans also had extremely low ammonia accumulation in the blood even at very high exercise intensities. It is concluded that it is the physical activity during childhood, combined with intense training as teenagers that brings about the high VO2max observed in some Kenyan runners. Their high aerobic capacity, as well as their good running economy, makes them such superior runners. In addition, their low blood lactate and ammonia accumulation in blood when running may also be contributing factors. PMID- 7552768 TI - Salbutamol and high-intensity treadmill running in nonasthmatic highly conditioned athletes. AB - Salbutamol is a widely used drug among elite athletes. We wanted to provide more information on the effects of salbutamol in nonasthmatic athletes. Seventeen highly conditioned male athletes (VO2max > 70 ml.kg-1.min-1) participated in a randomized, double-blind and placebo-controlled cross-over study. Nebulized salbutamol (0.05 mg/kg) or placebo was inhaled prior to a high intensity (110% of VO2max) treadmill run to exhaustion. The measured variables included endurance time, oxygen uptake, ventilation, breathing rate, heart rate, and oxygen saturation. Lung function was measured as forced expiratory volume in the first second (FEV1) before and after medication, and during the recovery from the run. The high intensity runs led to total exhaustion after 4-10 min. A close-to significant shortening in endurance time was found when salbutamol was given. During the running (0-4 min) the oxygen uptake was slightly lower and the heart rate was slightly higher when salbutamol was given. No differences were found in peak oxygen uptake, peak heart rate, ventilation, breathing rate or oxygen saturation. FEV1 reflected an increase in airway caliber after the inhalation of salbutamol. These changes were still present in the recovery from the test run with the exception of the measurement immediately after the exercise. Although evidence for beta-adrenergic stimulation was found, it was concluded that a therapeutic dose of nebulized salbutamol does not improve performance in highly trained athletes during a high-intensity run to exhaustion. PMID- 7552769 TI - Patellofemoral pain syndrome in young women. I. A clinical analysis of alignment, pain parameters, common symptoms and functional activity level. AB - Clinical alignment, pain variables, common symptoms and functional activity level associated with patellofemoral pain syndrome (PFPS) were evaluated in 40 women with PFPS and 20 healthy controls. No significant difference could be found neither between the patients' most symptomatic knee and least symptomatic knee, nor between the patients and controls regarding clinical lower extremity alignment, such as Q-angle and leg-heel alignment measurements. There were no radiographic signs of malalignment. The patients were significantly more involved competitively in sports but had a significantly lower pain free activity level than the controls. Pain was associated with increased activity. It is suggested that chronic overloading and temporary overuse of the patellofemoral joint, rather than malalignment, contribute to patellofemoral pain. PMID- 7552770 TI - Patellofemoral pain syndrome in young women. II. Muscle function in patients and healthy controls. AB - Muscle function was evaluated in 40 patients with patellofemoral pain syndrome (PFPS) and 20 healthy controls. Patients with PFPS had a significantly lower knee extensor strength in the most symptomatic knee compared to the least symptomatic knee. Further, the patients had less vertical jumping ability and were weaker in the most symptomatic knee compared to the controls, with the largest differences in eccentric knee extension. There were lower strength and EMG activity, in the patients compared to the controls, in the range closer to full extension and significant differences in muscle activity between the vastus medialis and the rectus femoris muscle. The results can be explained by inhibition selective to knee angles and to the vastus medialis muscle. PMID- 7552771 TI - Antisense gene suppression against human ICAM-1, ELAM-1, and VCAM-1 in cultured human umbilical vein endothelial cells. AB - Antisense gene suppression has been carried out for human ICAM-1, ELAM-1, and VCAM-1 in cultured human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1 beta. A panel of antisense phosphorothioate oligodeoxyribonucleotides (PS-ODN), complementary to mRNA or pre-mRNA of these molecules, were tested for their gene suppression activity monitored by radioimmunoassay of the respective cell surface adhesion molecules. Sequences targeted by effective antisense PS-ODNs were located throughout the mRNA and pre-mRNA. "Hot spots" of gene suppression sites for each region were observed. Shift of the PS-ODN hybridizing site upstream or downstream by a few bases resulted in drastic change of gene suppression efficiency. In addition to translation arrest and RNase H activity, a third mechanism was proposed for antisense gene suppression, involving multiple binding sites for PS ODN and the activities of RNase H and RNases other than RNase H. Suppression of ICAM-1, ELAM-1, or VCAM-1 in HUVEC by their antisense PS-ODNs resulted in the reduction of adhesion of monocytes and U937 to HUVEC. This may suggest cooperativity among the adhesion molecule pairs in endothelial-leukocyte adhesion, since decrease of a single adhesion molecule on EC surface significantly reduced cell-cell adherence. PMID- 7552772 TI - Effects of dietary alpha- and gamma-linolenic acids on liver fatty acids, lipid metabolism, and survival in sepsis. AB - The effects of dietary treatment for 3 weeks with soybean oil, linseed- and safflower oil (high alpha-linolenic acid, ALA), or borage oil (high gamma linolenic acid, GLA) on the liver fatty acid profile and lipid metabolism in fed rats, in normal fasted rats, and septic fasted rats, and on survival from sepsis, were studied. The results were the following: 1) Dietary ALA increased incorporation of alpha-linolenic (18:3w3), eicosapentaenoic (20:5w3), and docosapentaenoic (22:5w3) acids in neutral lipids and phospholipids, and docosahexaenoic (22:6w3), dihomo-gamma-linolenic (20:3w6), arachidonic (20:4w6), stearic (18:0), oleic (18:1w9), and linoleic (18:2w6) acids in phospholipids in the livers of fed, fasted, and septic fasted rats. Dietary GLA increased all w6 fatty acids except 18:2w6, and reduced all w3 fatty acids in neutral lipids and phospholipids. 2) Dietary ALA increased liver phospholipid content in fasted as well as in septic fasted rats and was more potent than GLA in lowering serum cholesterol and liver neutral lipids. 3) Dietary ALA counteracted sepsis-related changes in liver weight, platelet count, body temperature, prekallikrein, serum glucose, beta-hydroxybutyrate, and free fatty acids. 4) Dietary GLA reduced survival from sepsis. The results suggest a role for w3 fatty acids to balance w6 fatty acids in the septic state. PMID- 7552773 TI - Modulation of inflammation-induced changes in insulin-like growth factor (IGF)-I and IGF binding protein-1 by anti-TNF antibody. AB - The aim of the present study was to characterize changes in the insulin-like growth factor (IGF) system produced by the nonbacterial nonendotoxic inflammatory agent zymosan and to determine whether these changes were mediated by enhanced production of tumor necrosis factor (TNF). Rats were injected intraperitoneally with either zymosan or saline and studied 18 h later. Animals were pretreated with either nonimmune IgG or neutralizing anti-TNF antibody 2 h before zymosan injection. Zymosan increased the plasma concentration of TNF alpha, and this was associated with a decrease (approximately 40%) in the IGF-I concentration in plasma, liver, heart, and brain. The IGF-I content was not altered in skeletal muscle and kidney. Zymosan also increased the concentration of IGF binding protein (BP)-1 in plasma (120%), liver (90%), and muscle (470%). Circulating TNF alpha was not detectable in rats injected with anti-TNF antibody before zymosan. The neutralizing antibody prevented the zymosan-induced reduction in IGF-I in plasma and blunted the decreased observed in liver, but did not alter the decrease in heart or brain. Anti-TNF antibody also attenuated (40-60%) the increased IGFBP-1 in plasma, liver, and muscle observed in zymosan-treated rats. We conclude that zymosan-induced inflammation not only decreases IGF-I in plasma and selected tissues, but also increases IGFBP-1 in plasma, liver, and muscle, and that these alterations are due in large part to the enhanced production of TNF alpha. PMID- 7552774 TI - Physiologic state severity classification as an indicator of posttrauma cytokine response. AB - A Physiologic State Severity Classification (PSSC) derived from clustering of 17 cardiorespiratory variables was used to predict cytokine response in critically ill posttrauma patients. The PSSC defined physiologic states: A-State (A), normal stress response; B-State (B), metabolic insufficiency; C2-State (C), respiratory insufficiency. Bayesian analysis of these states defined a probability of death (Pdeath). 416 studies from 60 newly studied multiple trauma patients (70% males, Injury Severity Score = 27.5) were analyzed; 45 (75%) had sepsis (s), 28 (47%) had sepsis-adult respiratory distress syndrome (s-ARDS). Of 35 survivors (66% s, 37% s-ARDS, mean Pdeath = .42) 23% were predominantly A, 66% B, and 11% C. Of 25 deaths (88% s, 60% s-ARDS, mean Pdeath = .64) 0% were A, 44% B, and 56% C. PSSC States were correlated with incidence and mean plasma levels (pl) in picograms/mL of cytokines. 23 samples from recovering nonseptic trauma patients were used as controls. PMID- 7552775 TI - Predicting outcome in injured patients and its relationship to circulating cytokines. PMID- 7552776 TI - Delayed "spontaneous" adhesion of leukocytes to rat mesenteric venules is dependent upon reactive oxidants. AB - We investigated the role of reactive oxygen metabolites (ROMs) as potential mediators of delayed "spontaneous" leukocyte adhesion to rat mesenteric venules after the manipulation for in vivo microscopy. Rats were anesthetized via tail vein and prepared for intravital microscopic viewing of a segment of mesenteric venule. Leukocyte rolling, adhesion, and emigration were quantitated every 30 min for 3 h. Intravital observation immediately after routine gentle manipulation revealed a substantial leukocyte rolling and a small number of adherent and emigrated leukocytes. This rolling leukocyte flux then declined to a minimal level for 60 min. The number of adherent and emigrated leukocytes did not change during the initial 90 min. After 120 min, the flux of rolling leukocytes, and adherent and emigrated leukocyte number began to increase and reached significant levels at 180 min. To determine the possible role of ROMs in this "spontaneous" adhesion, rats were treated continuously with superoxide dismutase (SOD) plus catalase given intravenously over 3 h. Rolling leukocyte flux after 120 min was significantly attenuated by SOD plus catalase. SOD + catalase also significantly inhibited delayed leukocyte adhesion and emigration. Allopurinol substantially inhibited xanthine oxidase activity but had no significant effects on the above parameters of neutrophil dynamics. These findings suggest that ROMs, probably derived from a source other than xanthine oxidase, constitute important mediators of "spontaneous" leukocyte adhesion in rat mesenteric venules. PMID- 7552777 TI - Hypertonic/hyperoncotic fluids reverse prostaglandin E2 (PGE2)-induced T-cell suppression. AB - In recent years, hypertonic, and hyperoncotic fluids have been examined for their potential to replace conventional isotonic fluids. This study describes the effects of commonly used intravenous fluids on immune function. The action of increased concentrations of hypertonic saline (HTS), hypertonic saline-dextran (HSD), dextran (Dx), albumin (ALB), and hydroxyethylstarch (HET) on in vitro proliferation of phytohemagglutinin-stimulated normal and prostaglandin E2 suppressed human peripheral blood mononuclear cells was tested. At clinically relevant levels, HTS, HSD (20-40 mM hypertonicity), and ALB (2.5 mg/mL) enhanced T-cell proliferation by 65, 75, and 70%, respectively. Dx and HET had little effect. HTS also reversed prostaglandin E2-suppressed (10 ng/mL) T-cell proliferation to normal levels, and HSD enhanced T-cell proliferation by 40%, in contrast to Dx, ALB, and HET which had minimal effects. The results suggest that hypertonic/hyperoncotic solutions might improve prostaglandin-mediated suppression of T-cell function in patients and may be a useful adjunct to reduce the risk of infection. PMID- 7552778 TI - Hemorrhagic shock increases gut macromolecular permeability in the rat. AB - The effect of hemorrhagic shock on gut permeability to macromolecules was investigated using fluorescein isothiocyanate dextrans (4,300 or 73,100 daltons). These were given orally, then measured in systemic blood during hemorrhage (mean arterial pressure of 30 mmHg) and reperfusion; sham-shocked animals served as controls. Hemorrhage lasting 2 h resulted in a 5-fold increase in concentration of both tracers relative to that found in control groups. Reperfusion reduced these differences, with values approximately threefold greater than those of controls after 2 h. On a molar basis, the smaller molecule was more permeant than the larger one. This study provides evidence that hemorrhagic shock physically perturbs the normal barrier function of the mucosa, permitting very large substances in the gut lumen access to the circulation. It may further explain, at least in part, previous observations that hypovolemic shock results in bacterial translocation and endotoxemia. PMID- 7552779 TI - Hypertonic sodium acetate treatment of hemorrhagic shock in awake rats. AB - Hypertonic sodium acetate has recently been suggested for treatment of hemorrhagic shock. In the present study, the effectiveness of hypertonic sodium acetate (HA) was studied. In controlled hemorrhagic shock, arterial bleeding was followed by a fall in mean arterial pressure (MAP) to 60 +/- 8 mmHg (p < .001). A similar fall in MAP was observed in all animals. Infusion of hypertonic saline (HTS) led to an increase in MAP to 85 +/- 4 mmHg (p < .005) after 30 min, while infusion of hypertonic sodium acetate (HA) was followed by a rise in MAP to 72 +/ 4 mmHg (p < .05), which was significantly lower than in group HTS-treated animals (p < .05). In uncontrolled hemorrhagic shock, arterial bleeding was followed by a fall in MAP to 86 +/- 7 mmHg (p < .001). A similar response was observed in all animals. Infusion of HTS was followed by a further fall in MAP to 47 +/- 8 mmHg (p < .01) after 60 min. Infusion of HA was followed by a fall in MAP to 51 +/- 4 mmHg (p < .01). In the untreated group, MAP decreased to 63 +/- 5 mmHg, which was significantly higher than the HTS (p < .05)- and HA (p < .05) treated groups. The mortality rate after 4 h was 50% in the HA-treated (p < .05) and 75% in the HTS-treated (p < .01) groups. It is concluded that, in awake rats in CHS, both HTS and HA led to a rise in MAP but the response to HTS is significantly higher. In UCHS both HTS and HA led to increased bleeding from injured blood vessels, a fall in MAP, and increased mortality. PMID- 7552780 TI - Volatile anesthetic modulation of lung injury and outcome in a murine model of multiple organ dysfunction syndrome. AB - General anesthesia has been shown to have a significant impact on the inflammatory response. We hypothesized that lung pathophysiology will be attenuated in a mouse model of secondary multiple organ dysfunction syndrome (MODS) elicited by intraperitoneal zymosan suspension in saline. CD-1 mice were anesthetized for 6 h with either 1% halothane or 1.5% isoflurane in 30% oxygen in N2 carrier gas. Another group of mice was exposed to 30% oxygen in N2 carrier gas only. The inflammatory response to zymosan was quantified by measuring lung myeloperoxidase activity (neutrophil recruitment). Lung injury was estimated by determining the degree of lung permeability to radioactive albumin (permeability index). Unanesthetized injured mice exhibited maximal lung myeloperoxidase activity 2 h after zymosan injection (.671 +/- .07 delta OD.min-1), which was significantly attenuated (p < .01) in injured mice anesthetized with halothane (.369 +/- .054) and isoflurane (.324 +/- .055). The maximum lung permeability index occurred 8 h after injection in the unanesthetized, injured mice (.398 +/- .019), and was attenuated (p < .01) in injured mice anesthetized with halothane (.255 +/- .02) and isoflurane (.224 +/- .019). Histopathological findings corresponded to the quantitative myeloperoxidase and permeability index values. Halothane and isoflurane attenuate lung inflammation and injury in this mouse model of multiple organ dysfunction syndrome. This attenuation may be related to modulation of the inflammatory response by volatile anesthetics. PMID- 7552781 TI - Granulocyte colony-stimulating factor improves myelopoiesis and host defense in fulminant intra-abdominal sepsis in rats. AB - The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) was studied in a model of fulminant sepsis in rats. Polymicrobial peritonitis was induced by a 4 mm cecal perforation and 10 micrograms/kg recombinant human G-CSF was given intravenously every 12 h, with the first dose at sepsis induction or 4 h post-induction. Rats were sacrificed at various intervals throughout sepsis to measure levels of neutrophil progenitors in the bone marrow and neutrophils and bacteria in blood and peritoneal fluid. Sepsis gave a sustained neutropenia and bacteremia, but did not affect numbers of blast- or GM-colonies, and only a delayed and moderate proliferation of G-clones was seen. Treatment with G-CSF at sepsis induction improved myelopoiesis by doubling the numbers of GM- and G progenitors at 12 and 24 h post-induction. Concentrations of neutrophils increased twofold in blood and 5-fold in peritoneal fluid, while bacteria counts in the same compartments declined logarithmically. Mortality was 92% in untreated sepsis and declined to 46% when G-CSF therapy was started at sepsis induction, and to 42% following 4 h delayed therapy. PMID- 7552782 TI - rBPI23 attenuates endotoxin-induced cardiovascular depression in awake rabbits. AB - We determined the effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) on hemodynamic and renal sympathetic responses to lethal endotoxemia in unanesthetized rabbits. Endotoxin was continuously infused intravenously (200 micrograms/kg/h) over 120 min with simultaneous infusion of either rBPI23 (3 mg/kg bolus followed by 6 mg/kg/h over 120 min; n = 6) or thaumatin (the same dose as rBPI23), a control cationic protein with a molecular weight and isoelectric point similar to that of rBPI23 (n = 9). Tissue blood flow was also determined using colored microspheres to the left ventricle, renal cortex, liver, and skeletal muscle. Seven of nine animals treated with endotoxin and thaumatin died between 45 and 120 min after start of the infusion, whereas all animals with rBPI23 treatment were alive throughout the entire 2 h experimental period. A transient increase in renal sympathetic nerve activity was observed in the thaumatin-treated animals followed by sympathoinhibition with concomitant decreases in heart rate, blood pressure, and cardiac output. Tissue blood flow to all measured organs gradually decreased in animals receiving endotoxin and thaumatin. However, rBPI23 abolished all these deleterious responses to endotoxin. In conclusion, rBPI23 attenuates the acute lethal sympathoinhibitory and hemodynamic effects of endotoxemia in awake rabbits. PMID- 7552783 TI - Central venous catheters. An overview of Food and Drug Administration activities. AB - This article presents an overview of recent Food and Drug Administration issues pertaining to central venous catheters (CVCs) with emphasis on the CVC working group. Caveats for practitioners concerning CVC use are offered. PMID- 7552784 TI - Peripheral access options. AB - With the dramatic downsizing of acute care facilities and shifting to alternate care settings, infusion therapy is a major aspect of outpatient and home health services. Health care reform has forced health care practitioners to make cost driven clinical decisions, while at the same time demanding quality outcomes. Technology has advanced our capabilities to provide infusion therapies in all settings. Controversial questions are asked regarding the selection of the appropriate device, catheter tip position, tip position verification, and care and management techniques, which only well-designed research protocols can answer. In the meantime, clinical decisions must be based on current science and physiologic principles, rather than tradition, anecdotal observation, or competitive financial agendas. Peripheral access catheters, if widely used, afford a tremendous opportunity for positive outcomes and cost savings. PMID- 7552785 TI - A review of peripherally inserted central venous catheters in oncology patients. AB - The use of semipermanent vascular access devices has become standard in the management of specific clinical populations. Recently, the use of peripherally inserted catheters has gained popularity owing to the relative ease of insertion and lower complication rates. PMID- 7552786 TI - Central venous access catheters. AB - Indwelling central venous access catheters were first introduced into clinical practice about 20 years ago. Today these catheters are an indispensable part of the treatment provided to adult and pediatric oncology patients. This article discusses the indications and major complications of central venous catheters. PMID- 7552787 TI - Difficult access problems. AB - We have described numerous alternatives for establishing central venous access in challenging patients. Using these techniques has proven successful in our experience in essentially all patients, although occasionally initial efforts fail and repeated attempts by alternate routes are required. The keys to eventual success appear to be good basic surgical skills, a strong working relationship between surgeon and interventional radiologist, and perhaps most important, perseverance in the face of frustration. PMID- 7552788 TI - Venous access device options. AB - Venous access devices are widely used today for chemotherapy, TPN, antibiotic therapies, and blood products. As a result, a large variety of products with varying uses have been developed and made available. Those involved with venous access should match individual patients needs with the access device whose characteristics best meet those needs. PMID- 7552789 TI - Central venous catheter related thrombosis. AB - Central vein thrombosis is increasingly recognized as a long-term problem of chronic venous access. The incidence, causative factors, and diagnosis of central vein thrombosis are discussed, along with preventative measures and treatment strategies. PMID- 7552790 TI - Vascular catheter related infections in cancer patients. AB - Immunocompromise due to malignancy or to cytotoxic chemotherapy with resulting neutropenia creates a high risk of infection among cancer patients. The frequent presence of long-dwelling vascular catheters in these patients adds to this risk. Recent data regarding the epidemiology, diagnosis, management, and prevention of vascular catheter related infections are discussed. PMID- 7552791 TI - Interventional radiology and cross sectional imaging in venous access. AB - The role of radiology and the interventional radiologist in the care of patients requiring long term venous access is expanding. This role includes multimodality imaging for anatomic evaluation, guided catheter placement or repositioning, and diagnosis and treatment of catheter occlusion or related venous thrombosis. Interventional procedures have been developed for relief of venous obstruction, repositioning of catheters, and placement of unconventional access devices. PMID- 7552792 TI - Lessons from vascular access procedures for hemodialysis. AB - Although specific requirements for vascular access in chronic hemodialysis differ from those of the oncology patient, many of the problems encountered are common to the two groups. This article reviews the current state-of-the-art of vascular access for hemodialysis. Particular emphasis is placed on the expanding use of dual-lumen silicone central venous dialysis catheters and their use in vascular access for the oncology patient. PMID- 7552793 TI - Current indications for intravenous nutritional support in oncology patients. AB - No clear role has yet been defined for the role of total parenteral nutrition in the management of the cancer patient, and this subject remains an area of controversy. Peri-operative nutritional support, if given for at least 10 days, may reduce morbidity and mortality in some patients, although this subgroup has not been fully characterized. There is no evidence that parenteral nutritional support benefits patients undergoing chemotherapy or radiotherapy in terms of an increased tumor response rate or prolongation of survival. PMID- 7552794 TI - IRONFAN--a sonographic window into the natural history of fetal anomalies. International Registry of the Onset of Fetal Anomalies. PMID- 7552795 TI - Fetal structural disorders--the large series are emerging. PMID- 7552796 TI - End-result of routine ultrasound screening for congenital anomalies: the Belgian Multicentric Study 1984-92. AB - Five ultrasound laboratories from Obstetrics and Gynecology departments of Belgian university hospitals or affiliated institutions conducted a prospective study from 1984 to 1992 in which the results of prenatal ultrasound examinations were compared to examination reports of the neonates. The results of the period 1984-89 (PI) have been published previously, and those of the period 1990-92 (PII) are presented here. Some very minor congenital anomalies, as listed and defined in the EUROCAT Register, were excluded. Of 26,147 pregnant women at normal risk for congenital anomalies, 25,046 had at least one ultrasound scan (96%). A total of 616 fetuses were structurally abnormal (prevalence 2.42%), and 685 abnormalities were recorded. The sensitivity of the ultrasound test was 40.4% in PI and 51.1% in PII for abnormal fetuses (p < 0.05), and 45% (PI) and 64% (PII) for abnormalities (p < 0.01). The specificity was 99.9% and the positive and negative predictive values were 94.2% and 98.6%, respectively; these values did not differ significantly between the two periods. The sensitivity for the detection of anomalies before 23 weeks increased from 21% in PI to 41% in PII, indicating an improvement in the early detection of fetal abnormalities. PMID- 7552797 TI - Prenatal detection of heart defects at the routine fetal examination at 18 weeks in a non-selected population. AB - Few studies have addressed the prenatal detection rate of congenital heart defects in a non-selected population at 18 weeks of gestation. Our objective was to assess the change in the prenatal detection rate of congenital heart defects in such a population that resulted from incorporating the four-chamber view at the second-trimester routine ultrasound examination. The prenatal detection rate of heart defects was prospectively compared between 4435 fetuses in Phase I who were scanned without special attention to the heart, and 7459 fetuses in Phase II who were scanned incorporating the four-chamber view. Of the 49 heart defects in Phase 1, 17 (35%) were critical and three (18%) of these were detected prenatally. Of the 90 heart defects in Phase II, 23 (26%) were critical, six (26%) of these were detected prenatally at the 18 weeks' routine scan, and three were detected in the third trimester, providing a total prenatal detection rate of 39%. A defect was classified as critical when a surgical repair was likely to be required because of gross structural complexity having a functional significance, e.g. transposition of the great arteries, hypoplastic left heart syndrome, atrioventricular septal defect, coarctation of the aorta, and large ventricular septal defect. No non-critical heart defects were detected prenatally in either of the phases. The incidences in the total population were 11 and 12/1000 in Phases I and II, respectively. Thirty-two per cent of the critical and 16% of the non-critical defects had associated abnormalities and/or abnormal karyotype.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552798 TI - Natural history of trisomy 21 fetuses with increased nuchal translucency thickness. AB - Increased fetal nuchal translucency thickness at 10-14 weeks of gestation can identify 80% of trisomy 21 pregnancies. However, a potential disadvantage of screening in the first trimester of pregnancy is that earlier screening preferentially identifies those chromosomally abnormal pregnancies that are destined to miscarry. In this study, we report on the outcome of six fetuses with increased nuchal translucency thickness and trisomy 21 whose parents chose to continue with the pregnancy. During the second trimester, the nuchal translucency resolved in five of the cases and in one it evolved into nuchal edema. Therefore, resolution of translucency with advancing gestation is not indicative of a normal karyotype. All pregnancies resulted in live births, suggesting that increased nuchal translucency does not necessarily identify those trisomic fetuses that are destined to die in utero. PMID- 7552799 TI - Biometry of the fetal cisterna magna: estimates of the ability to detect trisomy 18. AB - Measurements of the fetal cisterna magna were obtained from a prospective sample of patients undergoing diagnostic obstetric ultrasound examinations. These normal measurements were then compared to a retrospective sample of ultrasound scans from fetuses with the diagnosis of trisomy 18 by amniocentesis. The fetal cisterna magna increases in size throughout pregnancy. The sample from fetuses with trisomy 18 was different and had a higher rate of small and large cisterna magnas. After the two samples were mixed, it was determined that a cut-off of 2.5 standard deviations for the fetal cisterna magna would give a sensitivity of 26.3%, specificity of 99.1%, positive predictive value of 50.0% and negative predictive value of 97.6% for trisomy 18. The probability that a fetal cisterna magna of abnormal size will predict trisomy 18 in an early second-trimester ultrasound examination with the 2.5 standard deviation cut-off was estimated at 0.019. A fetal cisterna magna that is of abnormal size appears to be of diagnostic value in identifying the fetus with trisomy 18, but its sensitivity is low. PMID- 7552800 TI - Fetal pelvic kidney: a challenge in prenatal diagnosis? AB - The objective of this study was to establish ultrasonographic guidelines for the prenatal diagnosis of fetal pelvic kidneys and assess the relationship to clinical outcome. The records of all ultrasonographic diagnoses of a fetal pelvic kidney between 1 January 1991 and 31 December 1993 were reviewed. After review of the sonographic evaluation, the prenatal records were obtained, to assess demographic data as well as the obstetric course and neonatal outcome. If a fetal pelvic kidney was suspected on prenatal ultrasound examination, its precise location and size were recorded and compared with neonatal sonograms. Twenty-six cases of fetal ectopic kidney were diagnosed of which 13 were on the right side and 13 on the left (24/26 cases were diagnosed in the late second trimester). The size of the ectopic kidney did not differ from that of the normal kidney. Except for one case of hydronephrosis, there were no associated structural anomalies. All prenatal diagnoses were confirmed by postnatal sonograms and all neonates had normal renal function. Our conclusions are that prenatal sonographic detection of fetal pelvic kidney is feasible, although in most cases the diagnosis is made beyond 24 weeks' gestation. The importance of prenatal diagnosis is that the parents can be reassured that normal renal function is highly probable and that early neonatal intervention is usually unnecessary. PMID- 7552801 TI - Changes of venous blood flow velocity waveforms in fetuses with supraventricular tachycardia. AB - Studies in the fetal lamb have shown that atrial pacing beyond a rate of 300-320 beats/min may be associated with dramatic changes of venous blood velocity waveforms, an increase of venous pressure by up to 75%, hydrops, polyhydramnios and placental edema. The aim of our study was to determine the 'critical' heart rate frequency in the human fetus. In 11 fetuses (five with and six without hydrops) with supraventricular tachycardia, pulsed wave Doppler analysis of flow velocity waveforms of the inferior vena cava, the ductus venosus and the left hepatic vein were performed before and after drug treatment. In ten cases cardioversion was achieved by in utero antiarrhythmic drug therapy; in one case treated with digoxin and flecainide the supraventricular tachycardia was decreased to 160-190 beats/min with disappearance of hydrops. Before intrauterine treatment of supraventricular tachycardia, pulsatile reversal of blood flow in the inferior vena cava, ductus venosus and left hepatic vein was visible, with monophasic forward flow during systole and reversed flow during diastole in ten of 11 fetuses. One fetus with supraventricular tachycardia of 195 beats/min showed a normal biphasic forward flow pattern. During drug-induced sinus rhythm, a normal biphasic forward venous blood flow pattern was shown in all ten cases. In five cases pulsatile reversal was demonstrated during a drug-induced reduction of the heart rate from 280 to 210 beats/min and a normal biphasic forward flow velocity waveform appeared during supraventricular tachycardia below 210 beats/min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552802 TI - Diagnosis of fetal anemia with Doppler ultrasound in the pregnancy complicated by maternal blood group immunization. AB - We investigated whether Doppler measurement of the fetal middle cerebral artery peak systolic velocity can be used to detect fetal anemia in pregnancies complicated by maternal blood group immunization. We first studied normal values for the middle cerebral artery peak systolic velocity in 135 fetuses (Group A), and also in 23 fetuses at risk for anemia who underwent 56 cordocenteses to assess the fetal hematocrit (Group B). A test to detect fetal anemia, based on the middle cerebral artery peak systolic velocity, was developed by using the data of the fetuses of Group A and Group B. Successively, the middle cerebral artery peak systolic velocity was prospectively determined in 16 fetuses at risk for anemia who underwent 42 cordocenteses (Group C) to assess the test developed, in a multicenter prospective fashion, by using the data of Group A and Group B. In the normal fetuses an exponential model expressed the increase of the middle cerebral artery peak systolic velocity values with advancing gestation. By using the data of the fetuses of Group A and Group B, four zones of anemia risk were identified. In Group C, none of the anemic fetuses had the middle cerebral artery peak velocity below the normal mean value, whereas all of the anemic fetuses had the peak velocity above the normal mean. The middle cerebral artery blood velocity increases with advancing gestation and is a non-invasive method of detecting anemia in pregnancies complicated by maternal blood group immunization. PMID- 7552803 TI - Circadian variation in uterine artery blood flow indices during the follicular phase of the menstrual cycle. AB - We investigated for the presence of circadian variation in uterine artery blood flow indices during the late follicular phase of the menstrual cycle. Twenty women with regular menstrual cycles underwent transvaginal color Doppler assessment of blood flow in the uterine arteries on two occasions precisely 12 h apart on the same day in the follicular phase. Ultrasound examinations were performed between 06.00 and 10.00 and repeated between 18.00 and 22.00. Indices of uterine artery blood flow included the pulsatility index (PI) and the time averaged maximum velocity (TAMX). The changes in PI and TAMX were related to variations in serum concentrations of estradiol and luteinizing hormone (LH). We noted a significantly higher PI and lower TAMX in the evening compared to the morning values, p < 0.001 and p = 0.01, respectively. There was no significant change in estradiol or LH concentrations between morning and evening (p > 0.05). There appears to be a circadian rhythm in uterine artery blood flow occurring independently of these hormonal parameters. The circadian changes in PI and TAMX may be larger than the previously described effects of hormones on these parameters. The accurate interpretation of uterine artery blood flow indices should take these time-related fluctuations into account. PMID- 7552804 TI - Endometrial blood flow response to estrogen replacement therapy and tamoxifen in asymptomatic, postmenopausal women: a transvaginal Doppler study. AB - This study was conducted to evaluate endometrial blood flow characteristics in response to estrogen replacement therapy (ERT) and tamoxifen in asymptomatic postmenopausal women, and to correlate the resistance index (RI) with plasma estradiol levels. Transvaginal color Doppler ultrasound examinations were performed in 30 asymptomatic, postmenopausal women who demonstrated thin endometria (< 0.5 cm). Ten healthy women receiving ERT and ten women with breast cancer receiving tamoxifen comprised the study groups. Ten postmenopausal women free of any hormonal treatment comprised the control group. Blood flow response was assessed by visualization of arterial waveforms in the endometrial region. Resistance indices were calculated for analysis and correlated with peripheral blood levels of estradiol. All postmenopausal women treated with tamoxifen or ERT showed continuous forward end-diastolic flow velocities, while the menopausal women without treatment demonstrated partial forward end-diastolic flow. Resistance index values of non-treated women were invariably higher (mean 0.89, range 0.85-0.91) than in those on tamoxifen (mean 0.79, range 0.54-0.90; p < 0.05), while the lowest RI was obtained in women taking ERT (mean 0.66, range 0.54-0.90; p < 0.05). The plasma concentrations of estradiol in women on ERT were found to be significantly higher than those of the tamoxifen and control groups. The observed data suggest that ERT and tamoxifen modify normal postmenopausal endometrial perfusion. Tamoxifen had a weaker estrogenic effect on the endometrial blood flow resistance, and ERT enhanced endometrial blood perfusion through vasodilatation. PMID- 7552805 TI - In utero sonographic appearance of intestinal duplication cysts. AB - The antenatal appearance of gastrointestinal duplication cysts is described. Two cases are presented: a duplication cyst of the pylorus and a cyst of the terminal ileum. The embryogenesis and the clinical utility of prenatal diagnosis of these malformations of the alimentary tract are discussed. Antenatal detection of these cystic masses allowed close neonatal surveillance and timely surgical intervention prior to neonatal complications. PMID- 7552806 TI - Recurrent uterine rupture diagnosed by ultrasound. AB - A case of recurrent rupture of the uterus is presented. The patient had a history of left cornual uterine rupture, which was repaired, and she was considered to be suitable for a subsequent pregnancy. During the subsequent pregnancy, attempts were made to evaluate the condition of the uterine scar by ultrasound in order to anticipate threatening rupture of the scar. No signs of dehiscence could be detected until the patient presented with clinical signs. Ultrasound examination revealed protrusion of the membranes at the fundus uteri. Uterine rupture is a rare, but hazardous, obstetric complication that can affect both mother and child. The possible role of ultrasound in cases of elevated risk for uterine rupture is discussed. PMID- 7552807 TI - Detection of ovarian malignancy. PMID- 7552808 TI - Husband's head trauma--a risk of obstetric ultrasound. PMID- 7552809 TI - Concomitant chemoradiotherapy, neutron boost, and adjuvant chemotherapy for anaplastic astrocytoma and glioblastoma multiforme. AB - The survival rate for patients with malignant gliomas is poor. We describe the results of a prospective study using concomitant chemoradiotherapy, neutron boost, and adjuvant chemotherapy for patients with malignant gliomas. Forty-two patients with anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) were treated with postoperative photon radiation 45 Gy/25 fraction (fxs) with concomitant continuous intravenous infusion of 5-fluorouracil at 300 mg/m2/day x 5 days and hydroxyurea 0.5 g orally every 12 hr for 6 days for 5 consecutive weeks, followed by a neutron boost of 450 N cGy/6 fxs delivered twice weekly. Adjuvant chemotherapy with procarbazine, CCNU, and vincristine (PCV) was given up to 1 year or until tumor progression. Thirty-four patients (81%) had GBM and 8 patients (19%) had AA. Sixteen patients (38%) were ineligible for the neutron boost because of large tumors or poor performance status and instead received a photon boost with concomitant chemotherapy for a total dose of 60-65 Gy to the tumor. The overall median survival is 68 weeks at a median follow-up of 203 weeks (range 166-302 weeks for the 11 patients remaining alive); 7/8 patients with AA are alive, 2 of these with progressive disease. For AA the median survival is not reached at a median follow-up of 203 weeks (range 166-302 weeks for the 7 patients alive with AA). Time to tumor progression for the 1 dead patient with AA was 35 weeks and the other 2 patients failed at 171 weeks and 179 weeks following treatment. The median survival for the 34 patients with GBM was 62 weeks; 4/34 patients with GBM are alive at 285, 238, 216, and 206 weeks. Multivariate survival analysis in the 34 patients with GBM revealed age and Karnofsky performance status as important prognostic factors. Extent of surgery and neutrons did not affect survival. Concomitant chemoradiotherapy was well tolerated by all patients. The only toxicities observed were mucositis < or = grade II in 3 patients (7%) and mild myelosuppression in 1 patient (2.4%). Adjuvant PCV was well tolerated. Continuous concomitant chemoradiotherapy was well tolerated by all patients with acceptable side effects. The survival rate for the patients with GBM suggests no significant impact on the prognosis for these patients. Patients with AA did well; however, the patient numbers are small. PMID- 7552811 TI - Phase I trial of high-dose infused zidovudine combined with leucovorin plus fluorouracil. AB - This phase I trial evaluated a high-dose, short-term infusion of zidovudine (AZT) following oral leucovorin (LV) and bolus 5-fluorouracil (FUra). Thirteen patients with metastatic cancer received 30 cycles of therapy. Plasma monitoring demonstrated a dose-dependent increase in peak plasma levels of AZT through the range of dose levels, from 104.3 +/- 8.7 microM at the 1.5 g/m2 dose of AZT to 1312.6 +/- 165.9 microM at the 11.0 g/m2 dose. While AZT did not potentiate the usual clinical toxicities of LV plus FUra, an unexpected finding of symptomatic hypotension during the AZT infusion was the dose-limiting toxicity in this trial. One partial response was observed in a previously untreated patient with metastatic colorectal cancer. The maximal tolerated dose of AZT, 7.0 g/m2 over 2 hr, is recommended for future phase II evaluation of this novel combination. PMID- 7552810 TI - 5-Fluorouracil and high-dose calcium leucovorin for hepatocellular carcinoma: a phase II trial. AB - A phase II trial of 5-fluorouracil (5-FU) [250-450 mg/m2/day x 5 days as an intravenous (IV) bolus] combined with calcium leucovorin (500 mg/m2/day x 5 1/2 days by continuous IV infusion) administered on a 28-day schedule was performed in 15 patients with advanced hepatocellular carcinoma. The median age was 58 years; performance status ranged from 50 to 100%. Of 15 evaluable patients, 1 (7%) had a partial response lasting 2.4 months; 8 (53%) had stable disease with a median duration of 5.7 months; and 6 (40%) had progressive disease with a median time to progression of 2.7 months. Median survival was 3.8 months. Treatment with 5-FU and calcium leucovorin was moderately well tolerated; 9% of the treatment courses were complicated by grade 3 or 4 hematological toxicity, and 10% of the courses were complicated by grade 3 or 4 gastrointestinal toxicity. Despite the efficacy of the combination of 5-FU and leucovorin in advanced colorectal cancer, our results document the general resistance of hepatocellular carcinoma to modulated 5-FU. PMID- 7552812 TI - Effects of UFT (mixed compound of tegafur and uracil) on cell kinetics and inhibition of thymidylate synthase in L1210 ascites tumor. AB - Previous work in our laboratory showed that UFT (mixed compound of tegafur and uracil, molar ratio 1:4, respectively) caused the prolonged reduction of dTTP in L1210 leukemia cells in comparison with 5-fluorouracil (5-FU). The purpose of this study was to assess the effect of UFT on cell cycle distribution and thymidylate synthase activity of a leukemia cell line as compared with 5-FU. UFT and 5-FU were orally given to BDF1 mice bearing L1210 ascites tumor on day 3 after the tumor inoculation. Cell cycle distribution patterns at 24 hr after the drug administration showed a higher percentage of S phase in tumor cells treated with UFT than in those treated with 5-FU. Until 6 hr after the oral administration of the drugs, UFT inhibited the incorporation of [3H] deoxyuridine into DNA more long than 5-FU did. These results indicated that UFT has longer and stronger inhibitory effects on DNA replication than 5-FU in vivo under the employed experimental conditions (i.e., low and single doses of these fluorinated pyrimidines). PMID- 7552813 TI - The expression of P-glycoprotein in canine lymphoma and its association with multidrug resistance. AB - Canine lymphoma is a spontaneous, naturally occurring disease that is a model for non-Hodgkin's lymphoma in humans. Chemotherapy with antineoplastics results in a high rate of remission; however, relapse and clinical drug resistance are usually seen within 8-10 months. The P-glycoprotein product of the mdr gene is thought to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. To assess the role of mdr gene products in drug resistance in canine lymphoma, membrane preparations of lymphoma cells from 31 dogs with high- or intermediate-grade lymphoma were subjected to Western blotting for detection of P-glycoprotein. In this study, one of 30 samples taken from dogs prior to receiving chemotherapy expressed detectable levels of P glycoprotein. P-glycoprotein was also detected in biopsy samples from 3 of 8 dogs that had become resistant to chemotherapy. This pattern of expression is similar to that in human non-Hodgkin's lymphoma. These studies suggest that canine lymphoma is a useful model for studying multidrug resistance. PMID- 7552814 TI - Insulin-like growth factor-1 inhibits cell death induced by anticancer drugs in the MCF-7 cells: involvement of growth factors in drug resistance. AB - The involvement of growth factors in cell survival in the presence of anticancer drugs was investigated. Cell death was induced in the human breast cancer cell line MCF-7, by the structurally and mechanistically unrelated chemotherapeutic drugs puromycin, actinomycin D, 5-fluorouracil, cisplatin, and adriamycin. The effect of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and insulin on cell death was evaluated by two different methods: (1) trypan blue dye exclusion test and (2) lactic dehydrogenase release into the culture medium. IGF-1 inhibited cell death induced by each of the diverse drugs in a concentration-dependent manner reaching a maximal effect at 40 ng/ml. Insulin mimicked the effect of IGF-1 only at supraphysiological concentration with an optimal effect at 10,000 ng/ml. EGF had no effect on cell death up to 100 ng/ml. Our finding that IGF-1 specifically enhanced MCF-7 cell survival in the presence of different anticancer drugs suggests the involvement of growth factors in the mechanism of drug resistance. PMID- 7552816 TI - Clostridium septicum bacteremia in a patient with large granular lymphocyte leukemia. AB - This is the first case report of Clostridium septicum septicemia in a patient with large granular lymphocyte leukemia. C. septicum infection is highly associated with malignancy and causes a rapidly fatal enterocolitis among patients who are profoundly neutropenic. The need for early recognition and combination of early antibiotic therapy and necessary surgical intervention may help to alter the fulminating nature of C. septicum infection. PMID- 7552815 TI - Dosing of oral etoposide normalized for body surface area. AB - The objective of this clinical and pharmacological study was to determine whether any pretreatment parameters were associated with pharmacological or toxicity parameters after prolonged oral etoposide. Therefore, the relationships between patient characteristics and etoposide concentrations and hematological toxicity were evaluated. Sixty patients with advanced non-small cell lung cancer were treated with etoposide 50 mg/m2/day p.o. for 21 consecutive days and cisplatin 100 mg/m2 i.v. on day 1. Complete blood counts and etoposide plasma concentrations were obtained weekly. Etoposide was measured by high-performance liquid chromatography. The input variables were age, gender, race, weight, weight0.66, weight0.75, height, body surface area, performance status, albumin concentration; and total etoposide dose. The outcome measures were etoposide concentration; nadir values (white blood cells, neutrophils, hemoglobin, and platelets); the absolute decrease, relative decrease, and survival fraction of blood cells; and graded toxicity. No significant correlations were found in 49 fully evaluable patients between any of the input and outcome variables. Among the outcome variables, significant correlations were found between etoposide concentration and the logarithmic transformation of the nadir blood counts. If any of the input variables were significantly correlated to etoposide concentrations or toxicity variables, it would be possible to suggest another predictor variable besides body surface area. As long as treatment is not modified for etoposide concentrations, dosing of oral etoposide must still rely on estimates of body surface area. PMID- 7552817 TI - The endocrine prevention of breast cancer. AB - A clear explanation for the high incidence of breast cancer in modern women is now possible. The risk of breast cancer rises steeply from menarche until menopause. Associated with the reproductive process, the ovary, including the corpus luteum, produces substantial amounts of estrogen and progesterone, both of which induce growth of the breast epithelium. This sex-steroid-driven breast epithelial cell proliferation increases the risk of carcinogenesis by accelerating the occurrence of somatic genetic errors. Postmenopausally, as there is little cell proliferation, the breast epithelium is more "resistant" to mutagenic effects, and breast cancer risk rises at a low rate. Unfortunately, the genetic errors accumulated during the premenopausal period are not lost following menopause, and breast cancer risk remains high. Sex-steroid antagonists, such as tamoxifen, may reduce breast cancer incidence both by blocking breast epithelial cell proliferation and by direct antitumor effects on clinically occult breast cancers. The rationale for a contraceptive designed to reduce breast cell proliferation by decreasing premenopausal sex-steroid exposure is presented. PMID- 7552818 TI - High-dose chemotherapy of metastatic breast cancer: a review. AB - Very-high-dose chemotherapy produces frequent complete responses in patients with metastatic breast cancer. These responses are transient in patients with disease that had relapsed following, or was refractory to, prior conventionally dosed therapy, but when used as first chemotherapy for metastatic disease or as consolidation for patients with responding cancer, a minority achieve durable complete remissions. Improvements in supportive care, especially in hematopoietic support, have resulted in a considerable reduction in the toxicity of this therapy and may allow further intensification through the use of multiple, rapidly cycled courses. PMID- 7552819 TI - Growth following therapy for childhood cancer. PMID- 7552820 TI - Using a community clinical trials network for treatment, prevention, and control research: assuring access to state-of-the-art cancer care. PMID- 7552821 TI - The role of video-assisted thoracic surgery in thoracic oncological practice. PMID- 7552822 TI - The conquest of cancer. PMID- 7552823 TI - Video-assisted thoracic surgery: applications in oncology. PMID- 7552824 TI - Therapy of malignant astrocytoma. PMID- 7552825 TI - Effects of anesthetics on ormaplatin biotransformations in the Fischer 344 rat. PMID- 7552826 TI - Ultrasonography of the fetal neck in the first and second trimesters. Part 2. Anomalies of the posterior nuchal region. AB - Over a 5-year period, the author examined the posterior nuchal region of the fetus in 7200 first- and second-trimester sonograms. Nuchal fluid collections were analysed to differentiate physiologic collections, which resolve spontaneously, from pathologic ones, which necessitate invasive testing for chromosomal abnormalities. Recommendations for evaluating ultrasonographic findings, based on the results of the study, are presented algorithmically in this pictorial essay. A single echogenic line representing a skin reflection was usually visible in normal fetuses. Thin double echogenic lines, observed in eight cases in this series, were due to fetal rotation or the presence of an amniotic membrane adjacent to the normal dorsal pseudomembrane and were not indicative of abnormality. In three cases the spectral reflection of the fetal neck surface was relatively thick, leading to the appearance of thick double echogenic lines; of these, two cases involved Down's syndrome. Therefore, karyotyping is suggested in all cases of thick echogenic lines. Of the 11 nuchal fluid collections up to 5 mm in diameter observed in this series, 10 resolved spontaneously by 24 weeks gestational age. In the other case the collection enlarged, and karyotyping revealed Turner's syndrome. Of the 16 cases of nuchal fluid collections greater than 5 mm in diameter, 5 were associated with an abnormal karyotype (Down's syndrome in 4); karyotyping is recommended in all such cases. Nuchal edema occurred in 22 cases. It was associated with Down's syndrome in 8 cases and with trisomy 18 in 1 case. Among fetuses with nuchal edema, the frequency of aneuploidy was substantially greater if other abnormalities were also present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552827 TI - Journey through the abdominal underpass: the subperitoneal pathway of disease spread revisited. AB - The subperitoneal space is a large potential space in the abdominal cavity created during embryogenesis. It plays an important, although often underestimated, role in the spread of intra-abdominal disease. The various mesenteric folds and ligaments serve as communicating pathways between compartments but sometimes act as barriers to the spread of disease. The authors describe the anatomic features of the subperitoneal space in detail and illustrate the interconnections between organs and compartments by means of case studies. An appreciation of the role of the subperitoneal pathways allows accurate interpretation of complex images and improved diagnostic accuracy. PMID- 7552828 TI - Implications of unsuspected pulmonary embolism detected by computed tomography. AB - OBJECTIVE: To describe the computed tomography (CT) findings and clinical implications of pulmonary thromboembolism noted incidentally on CT. PATIENTS AND METHODS: The authors reviewed the CT studies and medical records for nine patients in whom CT had shown clinically unsuspected pulmonary thromboembolism. The study group consisted of seven men and two women ranging in age from 51 to 75 years, who were referred for CT over a 5-year period for a variety of indications. The location of the emboli and the presence and location of parenchymal and pleural abnormalities were determined. Subsequent changes in patient care were analysed. RESULTS: The locations of the thromboemboli were described according to pulmonary zone. One case involved zone 1 (main pulmonary artery to a lung), all involved zone 2 (first-order branches), and four involved extension into zone 3 (second-order branches). No emboli were distinguished in zone 4 (beyond the segmental arteries). Four patients had pleural-based opacities characteristic of infarcts, and three had pleural effusions. Eight patients underwent confirmatory testing. A vena cava filter was placed in three patients, one of whom also received anticoagulation therapy. The other six patients were treated by anticoagulation alone. All of the patients survived and were discharged. CONCLUSION: Radiologists should watch carefully for occult pulmonary thromboembolism when interpreting CT studies of the thorax. Establishing this diagnosis can result in immediate changes to treatment and possibly a reduction in the substantial morbidity and mortality associated with untreated pulmonary emboli. PMID- 7552829 TI - Bone island: scintigraphic findings and their clinical application. AB - PURPOSE: To evaluate bone islands that showed increased uptake of radiotracer on skeletal scintigraphy and to present an algorithm for examining such lesions to avoid misdiagnosis in difficult cases. PATIENTS AND METHODS: Over an 8-year period, 20 patients (10 men and 10 women ranging in age from 33 to 82 years) with bone islands that showed activity on skeletal scintigraphy were examined with plain radiography (all patients), computed tomography (CT; 5 patients) and magnetic resonance imaging (MRI; 4 patients). For six of the patients the clinical presentation and the radiologic studies suggested malignancy, which prompted biopsy and histopathologic examination. Histopathologic study was also performed for six other patients in whom the bone islands were found incidentally during evaluation for joint replacement surgery for osteoarthritis. In the last eight patients the lesions exhibited the characteristic radiologic features of enostosis, and these patients were followed for up to 3 years without biopsy. RESULTS: In all cases plain radiography showed the characteristic features of a bone island: a homogeneously dense, sclerotic focus in the cancellous bone with distinctive radiating bony streaks ("thorny radiation") that blended with the trabeculae of the host bone to create a feathered or brush-like border. Histopathologic examination of scintigraphically active bone islands showed increased osteoblastic activity, and the lesions were marked by a mixture of compact and trabecular bone. In the patients who did not undergo biopsy but were followed with radiologic examinations, there was no change in the size or appearance of the lesions. CONCLUSIONS: The key to the correct diagnosis of bone island lies in the distinctive radiographic features of enostosis. An asymptomatic, isolated sclerotic bone lesion showing feathered or brush borders is most likely an enostosis, regardless of its size or its activity on scintigraphy. Therefore, a practical algorithm for examining bone islands should flow from their morphologic features as observed on radiographs and CT and MRI scans, rather than from their activity on scintigraphy. PMID- 7552830 TI - Irregularity of the apophysis of the ischial tuberosity evaluated by magnetic resonance imaging. AB - OBJECTIVE: To report the findings of magnetic resonance imaging (MRI) in two cases of apophyseal irregularity of the ischial tuberosity. PATIENTS AND METHODS: The authors describe the MRI findings for two males who presented with pain of sudden onset in the buttock. Both patients were examined by radiography, MRI and computed tomography, and one underwent bone scanning. RESULTS: On T1-weighted images, signal intensity was decreased in both the apophysis and the body adjacent to it; however, the signal intensity on T2-weighted images was increased. These findings are consistent with avulsion fracture of the apophysis. CONCLUSION: The MRI findings in these cases may support the previously suggested hypothesis that apophyseal irregularity with characteristic radiographic changes over time is caused by avulsion fracture of the apophyseal line. The relation between the characteristic radiographic features and the structural changes associated with fracture in this condition is discussed. PMID- 7552832 TI - Parasellar epidermoid cyst presenting with subarachnoid hemorrhage. AB - The authors describe the clinical, radiologic and surgical findings for parasellar epidermoid cyst, which presented as subarachnoid hemorrhage in a 70 year-old woman. The cyst may have provoked the hemorrhage, an association reported only once before. PMID- 7552831 TI - Adrenoleukodystrophy: magnetic resonance follow-up after Lorenzo's oil therapy. AB - PURPOSE: To determine the therapeutic value of Lorenzo's oil (a mixture of glyceryl trioleate and glyceryl trierucate oils) in treating the childhood form of adrenoleukodystrophy (ALD), as indicated by magnetic resonance imaging (MRI) follow-up. PATIENTS AND METHODS: Between January 1992 and March 1994 eight males with biochemically proven ALD, ranging in age from 1 to 28 years, were referred for cerebral MRI and initiation of Lorenzo's oil dietary therapy; treatment continued for a period of 6 to 24 months. Five of the patients underwent follow up cerebral MRI, and the images were reviewed without knowledge of medical status or chronology of imaging relative to therapy; the other three patients were excluded from the study because follow-up examinations were lacking. Signal abnormalities were evaluated according to a modified Loes scale, which yielded a minimum rating of 0 (for normal findings) and a maximum rating of 35 (for the most severe abnormalities). Because an untreated control group was not available for comparison, the Wilcoxon signed-rank test was used to analyse the results. RESULTS: There was no statistically significant improvement in the ratings of abnormalities after treatment (p = 0.31, alpha = 0.10). However, the correlation between MRI findings and clinical symptoms was good. CONCLUSIONS: The Wilcoxon signed-rank test is a useful statistical tool for evaluating the results of studies in which the sample population is small and there is no control group. The authors suggest that although Lorenzo's oil may not be curative, it may have a role in preventing the progress of ALD in asymptomatic patients. A multicentre control trial of Lorenzo's oil treatment, with emphasis on asymptomatic patients, is now underway. PMID- 7552833 TI - Enterovesical fistula in a patient with B-cell lymphoma. AB - Enterovesical fistula is a known complication of several conditions involving the bowel or the genitourinary system. Its occurrence with primary small-bowel lymphoma is rare. The authors report a case of enterovesical fistula associated with secondary small-bowel non-Hodgkin's lymphoma. They review the criteria for classification of small-bowel lymphoma as primary or secondary and discuss the radiologic findings consistent with enterovesical fistula, with reference to the role of computed tomography as the initial radiologic study. PMID- 7552834 TI - Residents' corner. Answer to case of the month #33. Holt-Oram syndrome. PMID- 7552835 TI - Aspects of childhood physical punishment and family environment correlates in bulimia nervosa. AB - Although histories of child sexual abuse among eating disorder patients have attracted considerable attention in the past decade, relatively little is known about parental physical abuse among these patients. We examined aspects of childhood parental physical punishment and its family environmental correlates among women with a lifetime history of bulimia nervosa (BN group; n = 80) and women with no history of eating disorder (Control group; n = 40), recruited primarily by newspaper advertisement. Women in the BN group reported significantly more physical punishment and perceived their discipline to have been more harsh and capricious than women in the Control group. Nonetheless, the groups did not differ significantly in the extent to which they believed they deserved their punishment or in their belief that they were "physically abused." Further, subjects often failed to assert that they had been physically abused despite meeting conservative criteria, while the reverse tendency was uncommon. Finally, increased levels of physical punishment were associated with greater global family pathology in the BN group, but not in the Control group. Our findings underscore the necessity of explicitly inquiring about physically punitive events in the histories of bulimic women, as well as beliefs regarding these events. PMID- 7552837 TI - Comparative conflict resolution patterns among parent-teen dyads of four ethnic groups in Hawaii. AB - Ninety-six high school students reported their own behavior and the behavior of their parents in the resolution of conflicts during the previous year, using the Conflict Tactics Scale (Straus, 1979). Parent-teen dyadic aggression levels for Americans of European, Japanese, Polynesian, and Filipino ancestry were compared in a series of orthogonal contrasts. The adolescent children of Polynesian American parents reported significantly higher parent aggression levels than did adolescents with parents of other ethnicity. Parent aggression was the best predictor of teen aggression directed toward parents. Subjects reciprocated with counteraggression toward European American parents significantly more often than toward parents of other ethnicity. Aggression by one parent was highly correlated with aggression by the other parent. Aggression by either parent was more highly correlated with teen aggression toward the mother, than with teen aggression toward the father. PMID- 7552836 TI - Validity of a scale measuring beliefs regarding the "positive" effects of punishing children: a study of Mexican mothers. AB - This paper discusses the influence that "beliefs concerning the corrective effects of punishment" have on child punishment and abuse in a Mexican population. One hundred and five mothers responded to a questionnaire measuring these beliefs, and their responses were contrasted with the report those mothers gave regarding the physical punishment they inflict upon their children. A scale consisting of six items registering beliefs was developed and administered. The reliability (internal consistency) of the scale was assessed, and its validity was tested by using a factor analytic structural equations model which produced high factorial loadings from a "beliefs" factor to the scale's items. This was interpreted as a confirmation of construct validity. An indication of predictive validity was found in a high, significant structural correlation between the beliefs factor and a "corrective punishment" factor, measured by a series of related items. Mothers reported as abusing their children produced higher scores on the "beliefs" scale as compared to "control" mothers. The direct, significant effect of parent's beliefs on the punishment of children explains much about the child maltreatment problem in the studied society. PMID- 7552838 TI - Ethnic differences in psychological functioning among black and Latino sexually abused girls. AB - Psychological assessments were conducted for Black and Latino sexually abused girls aged 8 to 13 years. Latino girls received significantly higher scores for depression than the Black girls. These differences in depression appeared to be related to ethnic differences in the circumstances of the abuse. Latino girls were abused at a younger age; more likely to be abused by a relative; and more likely to have had a sibling abused. Latinos were also more likely to report high levels of family conflict and somewhat lower levels of maternal support. Ethnicity was also found to be related to psychological functioning independently of the impact of other factors such as the circumstances of the abuse. Cultural and social factors that may influence psychological functioning subsequent to sexual abuse among Black and Latino girls are identified and discussed. PMID- 7552839 TI - Predicting symptomatology and self-blame among child sex abuse victims. AB - Fifty-six sexually-abused girls and their nonoffending female caretakers from primarily low-income. African American families were comprehensively assessed in order to determine factors related to child symptomatology and self-blame. Girls whose overall relationships with their caretakers were somewhat negative exhibited more behavioral difficulties. Girls with disruptions in their relationship with a primary caretaker and who felt powerless as a result of the abuse were rated as functioning less well overall by clinicians. Child age, general attributional style, and caretaker blame of the child were related to child self-blame. Clinical implications of these findings include identification of clients at high risk for negative sequelae and development of interventions targeted at specific clinical issues. PMID- 7552840 TI - A meta-analysis of the relationship of child sexual abuse to adult psychological adjustment. AB - This paper reports on meta-analyses of the relations of child sexual abuse to adult psychological adjustment. Results indicated statistically significant relationships between the experience of child sexual abuse and subsequent difficulties in psychological adjustment as measured by psychological symptomatology, depression, and self-esteem. Significant heterogeneity occurred across studies using a variety of different subject populations, research designs, and assessment methods. Some explanation of the effect size variance was partially accounted for by certain identified study characteristics, most notable in regard to sample source used in the included studies. Student samples consistently generated smaller, more homogeneous effect size estimates than did community or clinical samples. This indicates that abused subjects drawn from student samples may experience fewer impairments in psychological adjustment, when compared to abused subjects drawn from community or clinical samples. The implications of these findings and suggestions for future research are discussed. PMID- 7552841 TI - Child sexual abuse: the female victim's relationship with her nonoffending mother. AB - The present study is an empirical analysis of 60 female victims of sexual abuse and their perceptions of their relationship with their nonoffending mothers. Contrary to much of the sexual abuse literature, which suggests a hostile and rejecting relationship, this study finds that the victims perceived a warm and accepting relationship with their mothers. The present study examines female victims of sexual abuse ages 7-12, who were interviewed within a few weeks of disclosure. Data collection includes the Parental Acceptance/Rejection Questionnaire and the Child Behavior Checklist by Achenbach. Sexual abuse variables related to the nature of the sexual abuse are also analyzed. Results indicate that sexually abused girls who perceived lower levels of maternal rejection had higher competency ratings and fewer behavior problems as compared with victims who perceived higher levels of maternal rejection. Abuse involving force by the offender is correlated with lower competency in the victims. More Violent Abuse is correlated with externalizing behavior problems and sexual behavior problems. PMID- 7552842 TI - "Is this man your daddy?" suggestibility in children's eyewitness identification of a family member. AB - Studies of natural language development suggest that overextension of family memberships terms, such as daddy or papa, occurs in children in the second year of life, but rarely persists thereafter. However, in British Courts it is common for the testimony of 3- and even 4-year-olds to be dismissed on the grounds that these children may not be reliably identifying their father or stepfather when they claim that daddy was the perpetrator of abuse. This study examined whether 3 year-olds could be persuaded to confirm that a stranger who was labelled as daddy was their own father. A mock interview with experienced disclosure interviewers was conducted. Five of the 17 children--all from blue-collar families- misidentified a photograph of their own father when an identification question was repeated. Rather than undermining the validity of all preschoolers testimony, it is suggested that the responses of some children to apparently mundane questions of fact are influenced by contextual factors, including repetition of the question and the perceived omniscience of the interviewer. PMID- 7552843 TI - Information-processing correlates of reported sexual abuse in eating-disordered and comparison women. AB - A history of sexual abuse during childhood or adulthood is reported by a large number of eating-disordered and nonclinical women. However, the cognitive consequences of such abuse are not fully understood. An adaptation of the Stroop test is described, examining information-processing correlates of reported sexual abuse and of clinicians' judgments of the relevance of that abuse to the formulation of cases. Words related to sexual abuse impaired color-naming in eating-disordered and comparison women who reported a history of such abuse. This Stroop interference effect was greater in those eating-disordered women where the abuse was judged to be relevant to their psychopathology. It was also associated with the characteristics of the abuse (use of force, identity of abuser, time since the abuse). Finally, the Stroop interference effect was associated with the degree of bulimic psychopathology in the eating-disordered women who reported abuse. In particular, the frequency of binging was significantly greater in those eating-disordered women who had reported abuse, but that difference was a product of the correlation of the two variables with the degree of information processing bias. A two-stage model of cognitive reaction to sexual abuse is proposed, integrating these effects with the existing literature. The utility of this measure as a research and clinical tool requires further consideration in other clinical groups. PMID- 7552845 TI - Parental empathy, emotionality, and the potential for child abuse. PMID- 7552844 TI - Childhood sexual abuse and the academic adjustment of college women. PMID- 7552846 TI - Empathic responsiveness and affective reactivity to infant stimuli in high- and low-risk for physical child abuse mothers. AB - Empathic responsiveness and affective reactivity to infant stimuli were examined in matched groups of high- and low-risk for physical child abuse mothers. Hypotheses were generated based on models of aggression and the child abuse literature. Although no between-group differences were found in empathy, within group differences were observed. Compared to baseline, high-risk mothers reported no change (p > .05) in empathy across infant conditions (baseline, smiling, quiet, and crying), while low-risk mothers reported an increase (p < .0005) in empathy following presentation of the crying infant. Although there was no change in empathy, high-risk mothers reported more sadness, distress, hostility, unhappiness, and less quietness following presentation of the crying infant. Low risk mothers reported no changes in sadness, distress, and hostility. The data for high-risk mothers are congruent with reports that physical child abusers are less empathic and more hostile in response to a crying child. The findings support aggression models which suggest the lack of empathy and the presence of negative affectivity precede abusive behavior. Post-hoc analyses also provide support for an emotional contagion perspective, where high-risk parents, compared to low-risk parents, are thought to more frequently reflect the emotional state of the infant. PMID- 7552847 TI - What is "good enough"? PMID- 7552848 TI - Condyle position in the temporomandibular joint. Comparison between asymptomatic volunteers with normal disk position and patients with disk displacement. AB - Though the significance of condyle-fossa relationship in the temporomandibular joint has not yet been clarified and normal condyle position not yet defined, efforts have been made to guide the mandibular condyle into a centric position in the glenoid fossa with the aim of relieving the symptoms in patients with orofacial pain and temporomandibular joint internal derangement. The present study investigated the mandibular condyle position in 34 joints in asymptomatic volunteers with normal disk position as verified by arthrography and compared it with the mandibular condyle position in 85 joints in patients with different stages of internal derangement. The results showed that in the volunteers with normal joints including normal superior disk position, the condyles were almost randomly distributed in anterior, centric, and posterior positions in the glenoid fossa. Of the joints with anterior disk displacement approximately half of the number of joints with reducing disks and two thirds of the joints with nonreducing disks appeared to have posterior condyle position. Posterior condyle position cannot, however, be used to diagnose internal derangement because the condyle was found to be either in anterior or centric position in many joints with a displaced disk. The variety in condyle position in the healthy joints ought to be taken into consideration if treatment is chosen to normalize the mandibular condyle position by bringing it into a centric position in the glenoid fossa. PMID- 7552849 TI - Efficacy of FDA guidelines for prescribing radiographs to detect dental and intraosseous conditions. AB - OBJECTIVES: This study compared the FDA guidelines for ordering dental radiographs to a conventional full-mouth examination for the detection of intraosseous disease and conditions affecting teeth other than caries. STUDY DESIGN: We examined 490 patients and selected posterior bite-wing and periapical views as indicated by the patient's signs or symptoms. We compared the radiographic findings with the use of this selected set of radiographs to those from a full-mouth set of radiographs to determine the rates of missed disease when the FDA guidelines were used. RESULTS: The most commonly missed intraosseous findings were osteosclerosis, unerupted teeth, periapical radiolucencies, and primary root tips. Periapical radiolucencies that were most probably periapical cemental dysplasia were missed in six patients. The most commonly missed dental findings were resorbed roots and pulp stones. Three instances of dens in dente were missed. CONCLUSIONS: When we used the FDA guidelines, the number of missed intraosseous and dental conditions was small and most likely inconsequential given the range of variability in dental diagnosis and treatment. PMID- 7552852 TI - Sens-A-Ray characteristics with variations in beam quality. AB - This study examined the relationship between x-ray beam quality and image characteristics with the use of the Sens-A-Ray (Regam Medical Systems, Sundsvall, Sweden). To evaluate the x-ray response, properties of the image sensor, dark current and statistical noise, sensitivity, and contrast resolution were measured. Tube voltage, tube current, and exposure time were varied. Exposures were determined with the use of an ionization chamber. Pixel values beneath a lead strip were used to calculate dark current effects. Contrast was measured with pixel values from the images of a step-wedge. Dark current noise increased in proportion to exposure time. Although the sensitivity at each tube voltage was linear and proportional to the exposure, the gradient was inversely proportional to increased tube voltage. After subtraction of the dark current and statistical noise, the gradient of the sensitivity at low tube voltage remained steeper than at high tube voltages. Hence, sensitivity decreased with increased tube voltage. Sensitivity gradients at each different tube current were similar. Irrespective of exposure, contrast resolution between adjoining step-wedge steps > or = 9 mm were too small to distinguish at 50 kVp. The gray levels for each step below 12 mm in thickness were distinguishable with 90 kVp. Increased dark current noise consequent to long exposure times narrowed the available gray scale range. Lower tube voltage resulted in higher sensitivity with larger gradients than higher tube voltages. PMID- 7552851 TI - Hyperbaric oxygen treatment of osteoradionecrosis of the mandible. Experience in 29 patients. AB - In recent years hyperbaric oxygen has gained an important role in the treatment of osteoradionecrosis of the mandible. In the treatment of 29 patients with osteoradionecrosis of the mandible, a combination of surgical debridement, antibiotics, and hyperbaric oxygen was used in 27 cases. In 20 of the 29 patients the osteoradionecrosis was considered to be resolved after treatment. In 31% of the patients, the continuity of the mandible was lost. It was concluded that combined treatment of surgical debridement, antibiotics, and hyperbaric oxygen gives acceptable results and may, when used in an early phase of the disease, limit the number of patients who lose continuity of the mandible. PMID- 7552850 TI - Accumulation of technetium-99m methylene diphosphonate. Conditions affecting adsorption to hydroxyapatite. AB - Experiments were carried out to explore the mechanism of technetium-99m methylene diphosphonate (99mTc-MDP) adsorption with various calcium compounds including hydroxyapatite powder. 99mTc-MDP adsorption to hydroxyapatite was markedly inhibited by the addition of either pyrophosphate or methylene diphosphonate (MDP). Moreover, adsorbed 99mTc-MDP was partly removed by rinsing with pyrophosphate solution. Adsorption was pH-dependent and was inhibited by univalent cations, adenosine triphosphate solution, and guanosine triphosphate. Adsorption was most apparent to hydroxyapatite and calcium pyrophosphate and was less marked for the other calcium compounds tested. It is suggested that 99mTc MDP adsorption is affected by the hydroxyapatite crystalline structure and environmental factors such as pH and the presence of phosphates, calcium compounds, and various cations. PMID- 7552853 TI - The potential for HIV transmission through allogeneic bone. A review of risks and safety. AB - Over the past two decades, oral and maxillofacial surgeons have gained a greater appreciation for the biology of allogeneic bone healing, resulting in a dramatic increase in its indications and use. Unfortunately, this time period has also ushered in near epidemic proportions of HIV-infected persons, some of whom might be considered as potential donors of allogeneic bone. As this article will discuss, surgeons and tissue bank teams alike must be aware of the clinical and serologic criteria associated with an acceptable donor. Only in this way can contamination-free specimens be obtained and surgically implanted. PMID- 7552854 TI - Frey's syndrome after fracture of the mandibular condyle. PMID- 7552855 TI - Revival of "median rhomboid glossitis"? PMID- 7552856 TI - Use of pedicled fat pad graft as an adjunct in the reconstruction of palatal cleft defects. AB - The purpose of this article is to provide the rationale for the use of pedicled buccal fat pad grafts as an adjunct to the reconstruction of palatal or dentoalveolar clefts in cases when healing by secondary intention may need to be considered and integrated into the initial treatment plan because of the size of the defect or qualitative or quantitative tissue constraints. Four representative cases are presented in which a pedicled buccal fat graft was adjunctively used in conjunction with pedicled mucosal flaps to gain closure of large oroantral or oronasal cleft deformity. PMID- 7552857 TI - Cephalometric comparison of characteristics in chronically snoring patients with and without sleep apnea syndrome. AB - OBJECTIVE: The purpose of this investigation is to cephalometrically study 50 snoring patients with and without sleep apnea and to determine whether cephalograms can be used as a diagnostic tool to differentiate persons who are chronic snorers from persons with sleep apnea. STUDY DESIGN: A sample of 30 sleep apnea patients was compared with a sample of 20 chronic snorers without sleep apnea as documented by polysomnography. Forty cephalometric measurements were determined to study various skeletal, soft tissue, and airway abnormalities. RESULTS: This study showed that both groups presented multiple cephalometric abnormalities. Only four measurements differed significantly between the two samples. In the sleep apnea group the maxilla was retropositioned and the hypoid bone displaced inferiorly and distally as compared to nonapneic snorers. CONCLUSIONS: Because of the overall presence of abnormal cephalometric findings in both samples and given similar age and weight ranges, a differential diagnosis between chronic snorers with and without sleep apnea cannot be reliably based on standard cephalometric evaluation alone. PMID- 7552858 TI - Proliferating cell nuclear antigen. PMID- 7552859 TI - Intraoperatively fabricated bite block in the management of scar contracture hypomobility. AB - Intraoral surgical procedures produce scar tissue formation that may limit mandibular opening. Many procedures have been developed to release these scar contractures, but most lead to further scar formation and greater limitation in opening. The purpose of this article to describe a technique with the use of an intraoperatively fabricated self-curing acrylic bite block combined with aggressive physical therapy in the management of this difficult clinical problem. PMID- 7552860 TI - A double-blind, randomized, placebo-controlled, crossover trial of pentoxifylline for the prevention of chemotherapy-induced oral mucositis. AB - Oral mucositis is a frequent side effect of cancer therapy. No effective method of prophylaxis is currently available. We conducted a randomized, double-blind, placebo-controlled, crossover trial of pentoxifylline to evaluate its potential in preventing mucositis in cancer patients receiving chemotherapy. Ten cancer patients were randomized for treatment with a 15-day course of 400 mg of pentoxifylline given orally four times daily. Concurrent chemotherapy consisted of bolus cisplatin and infusional 5-fluorouracil. Mucositis was evaluated with the use of the Oral Assessment Guide developed at the University of Nebraska. Patients completing two cycles of chemotherapy--one with pentoxifylline and one with placebo--were evaluated for prophylaxis efficacy. Comparison of the oral assessment scores of the two cycles with a two-sided Student's t test failed to demonstrate a cytoprotective effect for pentoxifylline over placebo. We conclude that pentoxifylline as given in this study is ineffective for preventing mucositis in patients receiving cisplatin and 5-FU. PMID- 7552862 TI - Cost-effectiveness analysis of open reduction/nonrigid fixation and open reduction/rigid fixation to treat mandibular fractures. AB - OBJECTIVES: When open reduction and internal fixation is indicated for the management of mandibular fractures, it is generally agreed that nonrigid fixation and rigid fixation represent acceptable treatment alternative. Opinions vary, however, regarding the cost-effectiveness of the two alternative. The purpose of this study was to compare the costs of these treatments of mandibular fractures that required open reduction and internal fixation. STUDY DESIGN: Cost effectiveness analysis was used to determine the most efficient resource used to treat mandibular fractures requiring open reduction and internal fixation. Cost effectiveness was defined as the treatment charges per successfully treated patient. Data were collected retrospectively from patients with mandibular fractures treated between 1991 and 1994. The patient's medical record and hospital billing record were used as data sources. To estimate treatment charges, the sample was divided into three groups: (1) group 1, patients treated with nonrigid fixation without postoperative complications, (2) group 2, patients treated with rigid fixation without postoperative complications, and (3) group 3, patients treated with either procedure who had postoperative complications. The study variables were grouped into two categories: clinical information and charges. Treatment charges for both treatments were estimated and compared. RESULTS: Data were collected for 12 patients in group 1 and 11 patients in group 2. Costs for rigid fixation averaged $1,468 more per patient than for nonrigid fixation in uncomplicated cases. There were 11 patients in group 3. The average cost to treat a postoperative complication was $11,637. Given the institution specific treatment cost and the probability of complications, rigid fixation was a more cost-effective treatment than nonrigid fixation. CONCLUSION: Cost estimates for treating mandibular fractures may vary widely depending on practice patterns and complication rates. Despite these cost variations, one may determine the most cost-effective treatment alternative by estimating treatment costs of both uncomplicated and complicated cases and the postoperative complication rate. PMID- 7552861 TI - Dental findings in geriatric populations with diverse medical backgrounds. AB - OBJECTIVE: To determine whether there is a difference in the oral/dental health in older persons with different life styles and medical status. STUDY DESIGN: Survey (cross-sectional study) included four groups: (1) subjects (n = 123) living in a residential retirement home or community dwelling; (2) subjects (n = 218) seeking dental treatment at a Veterans Affairs Dental Outpatient Clinic; (3) subjects (n = 132) resident in a VA long-term care facility; and (4) subjects (n = 81) recently admitted to a VA acute care ward with a diagnosis of cerebral vascular accident or other neurologic problem. Each subject answered questions on medical and dental health and dietary preferences in a comprehensive interview. They were given a comprehensive dental examination that included measurements of stimulated salivary flow and minor salivary gland output. RESULTS: The data from groups 2 and 3 confirmed previous reports that independent living subjects have better oral/dental health than dependent living subjects. The data from groups 1 and 4, obtained from geriatric populations on the opposite ends of the medical health/disease continuum provide new information that suggests that good medical health and good oral/dental health are linked. The subjects in group 1 were very healthy as judged by their longevity; 54% were > or = 80 years and they had low reported prevalence of medical disease. Only 6% were edentulous and the dentate persons were missing 4.5 teeth. In contrast, over 50% of the patients in group 4 were < 70 years; they had an edentulous rate of 49% and among the dentate persons had an average 12 missing and 5 decayed teeth. CONCLUSIONS: The medically healthy persons had excellent dental health whereas the sickest persons were either edentulous or had many missing teeth. PMID- 7552863 TI - Intraoral squamous cell carcinoma in human immunodeficiency virus infection. A clinicopathologic study. AB - The purpose of this study was to characterize the clinical and histological features of intraoral squamous cell carcinoma in men who were seropositive for the human immunodeficiency virus and to evaluate viral cofactors (human papillomavirus, herpes simplex virus, Epstein-Barr virus), proliferative index (proliferating cell nuclear antigen), a factor associated with invasion (cathepsin D), and mutated tumor suppressor gene and proto-oncogene products (mutated p53, c-erbB-2). Four men who were seropositive for the human immunodeficiency virus and had acquired immunodeficiency syndrome presented with painful oral lesions of variable duration. Oral cancer risk factors included heavy tobacco use (four of four), heavy alcohol use (three of four), and previous radiotherapy (one of four). The lesions consisted of ulcers (two of four), a fungating mass (one of four), and papillary erythroplakia (one of four). Incisional biopsy specimens were obtained. High-stringency in situ hybridization was performed with DNA probes to the human papillomavirus (types 6/11; 16/18; 31/33/35) and Epstein-Barr virus: Immunocytochemical studies for the herpes simplex virus, proliferating cell nuclear antigen, cathepsin D, mutated p53, and c-erbB-2 were performed. Two lesions were moderately differentiated squamous cell carcinoma, one lesion was a basaloid squamous cell carcinoma, and one was carcinoma in situ. Stage of disease at diagnosis was II (one of four), III (two of four), and IV (one of four). Three cases were positive for the human papillomavirus, one case was positive for Epstein-Barr virus, and three cases were positive for the herpes simplex virus. C-erbB-2 was focally positive in one case, and mutated p53 was positive in a separate case.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552864 TI - Detection of human papilloma virus DNA sequences in oral squamous cell papillomas by the polymerase chain reaction. AB - Nineteen clinically diagnosed, and histologically confirmed oral squamous cell papillomas were analyzed for the presence of human papilloma virus DNA sequence by the highly sensitive polymerase chain reaction technique, followed by dot blot hybridization of the polymerase chain reaction product with digoxigenin-labeled, type-specific oligonucleotide probes for human papilloma virus DNA types 6, 11, 16, and 18. Human papilloma virus DNA types 6 and 11 were identified in 68% of these oral lesions, which raises the possibility of an etiologic role for human papilloma virus in the pathogenesis of oral squamous cell papillomas. PMID- 7552866 TI - Simultaneous multifocal polymorphous low-grade adenocarcinoma. Report of two cases. AB - The polymorphous low-grade adenocarcinoma is a recently described variant of salivary gland adenocarcinoma with a comparatively good prognosis. Polymorphous low-grade adenocarcinoma occurs most frequently in the palate with a peak incidence between 30 and 70 years of age. The female/male ratio is approximately 2:1, and the tumors exhibit slow growth with a low recurrence rate. The simultaneous occurrence of polymorphous low-grade adenocarcinoma has not been previously reported. This article reviews the existing literature and describes the first two reports of simultaneous intraoral polymorphous low-grade adenocarcinoma. PMID- 7552867 TI - Lupus erythematosus-like oral mucosal and skin lesions in a carrier of chronic granulomatous disease. Chronic granulomatous disease carrier genodermatosis. AB - We present a case of a young female patient who for 8 years was believed to have discoid lupus erythematosus of the skin and oral mucosae. Only after her infant son had a near-fatal pulmonary infection was the diagnosis of chronic granulomatous disease made and her lupus erythematosus-like mucocutaneous lesions recognized as manifestations of her carrier status for chronic granulomatous disease. The purpose of this report is to raise awareness of and better characterize the mucocutaneous manifestations of carriers of chronic granulomatous disease. Early identification of carriers permits genetic counseling and prenatal diagnosis and forewarns pediatricians so that they can provide better care for affected infants. PMID- 7552865 TI - Primary anaplastic large cell (KI-1 positive) lymphoma of the mandible as the initial manifestation of acquired immunodeficiency syndrome in a pediatric patient. PMID- 7552869 TI - Search for evidence of three viral agents in radicular (periapical) cysts with immunohistochemistry. AB - Sections from 20 paraffin-embedded radicular cysts and positive-control subjects were subjected to immunostaining procedures after treatment of the sections with polyclonal antibodies was performed to search for evidence of human papilloma virus and herpes viruses 1 and 2 in the lining epithelium. All 20 specimens examined were negative for the presence of all three viruses. PMID- 7552868 TI - Quantitation of catecholamines in uninflamed human dental pulp tissues by high performance liquid chromatography. AB - The quantitation of catecholamines has yet to be reported in the human dental pulp. Various methods of chemical detection have shown the presence of catecholamines in pulpal tissue. Pulpal tissue was obtained from nonrestored, uninflamed teeth that needed to be extracted. The samples were collected, extracted, and analyzed by means of high-performance liquid chromatography with ultraviolet detection for levels of catecholamines (dopamine, epinephrine, and norepinephrine). As mediators of vasoconstriction, catecholamines play an important role in the control of intrapulpal pressure. This research describes a method for quantitation of catecholamines. Future studies investigating inflamed tissue can now be accomplished with the use of this analytic method. The results from this research indicate a baseline level of catecholamines in the uninflamed human dental pulp. PMID- 7552870 TI - Herpes zoster infection presenting as an acute pulpitis. AB - A major reason for referral to an endodontic practice is management of pain. Most cases are diagnosed as being of pulpal or periapical origin. However, some turn out quite differently than their initial appearance. This case report presents a patient referred to the endodontic clinic because of symptoms mimicking an irreversible pulpitis. On examination no obvious cause of the symptoms could be found. The patient was treated conservatively after which a herpes zoster viral infection was diagnosed. This case stresses the importance of a thorough investigation of all signs and symptoms and the delay of definitive treatment until a diagnosis is made. PMID- 7552871 TI - Trigeminal neuralgia mimicking odontogenic pain. A report of two cases. AB - Trigeminal neuralgia or tic douloureux is characterized by paroxysmal episodes of facial pain in the distribution of the trigeminal nerve, although patients may have a variety of symptoms that mimic odontogenic pain. This article presents two cases of trigeminal neuralgia that were misdiagnosed and initially treated endodontically as pain of odontogenic origin. A therapeutic regimen of carbamazepine alleviated the pain in both patients. These two cases demonstrate the importance of having a thorough knowledge of both odontogenic and nonodontogenic causes of orofacial pain, as well as the need for careful diagnosis before undertaking any treatment. PMID- 7552872 TI - Intravenous drug users: a consideration for infective endocarditis in dentistry? PMID- 7552873 TI - Retention of third molar roots to prevent damage to the inferior alveolar nerve. PMID- 7552874 TI - Anthropometric profile evaluation of the midface in patients with cleft lip and palate. AB - This study was done to determine those profile anthropometric measurements that are abnormal in the midface profile in patients with cleft lip and palate. The sample population consisted of 30 randomly selected skeletally mature white patients with cleft lip and palate who had been treated by the same team who were accredited by the American Cleft Palate-Craniofacial Association. Twenty patients had unilateral and 10 had bilateral complete clefts. None of these patients had previously undergone orthognathic surgery or definitive rhinoplasty surgery. Fifteen facial anthropometric features were measured on each person's face. The result from this study showed that in patients with cleft lip and palate right versus left side differences did not exist and only four statistically significant differences existed between the unilateral and bilateral cases. However, in all patients, four of these esthetic facial features were consistently and significantly abnormal: obtuse nasofrontal angle: obtuse nasomental angle; a posteriorly positioned infraorbitale relative to globe; and an obtuse general facial angle. Several other features were abnormal in a high percentage of persons in this study. These were lack of supratip break, flat to concave paranasal contour, increased subnasale-alargroove:subnasale-pronasale ratio, decreased nasal protrusion:nasal length ratio, decreased nasolabial angle ratio, decreased maxillary length ratio, increased nasal bridge projection:nasal protrusion ratio, and deficient cheek contour. This data indicates that the major deformity in persons with adult cleft lip and palate exist in the nose and secondarily in other components of the midface. PMID- 7552875 TI - Cephalometric profile evaluations in patients with cleft lip and palate. AB - This study was done to determine those abnormal cephalometric features found in adult cleft lip and palate patients. The sample population consisted of 30 randomly selected white patients with cleft lip and palate who were treated by the same team that had been accredited by the American Cleft Palate-Craniofacial Association. Twenty patients had unilateral complete clefts, and 10 had bilateral complete clefts. Thirteen different cephalometric parameters were measured and compared with normal. The results from this study showed that there were few statistically significant differences between the unilateral and bilateral cleft palate patient populations. There were only three measurements that had statistically significant differences between the unilateral cleft patients and the bilateral cleft patients: subnasale-stomion, subnasale-stomion: stomion-soft tissue menton, and subnasale-lower lip vermillion: lower lip vermillion-soft tissue menton. However, 10 of the 13 measurements had statistically significant variations from normal. These measurements included subnasale=stomion; stomion=soft tissue menton, subnasale=lower lip vermillion; lower lip vermillion=soft tissue menton, interlabial distance, subnasale-perpendicular to upper lip, subnasale-perpendicular to lower lip, subnasale-perpendicular to chin, angle formed between sella turcica=nasion and nasion=A=point, maxillary depth angle, A-point to nasion-pogonion, and angle formed between A=point=nasion and nasion=B=point. The data indicated that a multiplicity of vertical and horizontal abnormalities exist in the person with cleft lip and palate in addition to the well-known transverse deficiencies, and that cephalometric abnormalities are not limited to anteroposterior maxillary deficiency. PMID- 7552877 TI - Electronic thermography for the assessment of inferior alveolar nerve deficit. AB - Neurosensory deficit is one of the major complications encountered in oral and maxillofacial surgery. OBJECTIVES. To determine the efficacy of electronic thermography in objectively assessing neurosensory deficits of the inferior alveolar nerve. STUDY DESIGN. Three studies were conducted measuring skin temperature over the chin region of the face at 0.1 degree C accuracy. RESULTS. (1) Thermal symmetry of the chin region in normal subjects (delta T = 0.2 degree C, SD = 0.02 degree C); (2) Induction of transient thermal asymmetry by local anesthetic injection (delta T = +0.4 degree C, SD = 0.2 degree C); (3) nine subjects with neurologic alterations of the inferior alveolar nerve (delta T = +0.5 degree C, SD = 0.2 degree C). Statistically significant differences were found between control group and experimental groups at p < 0.001 with the use of the Student's t test. CONCLUSIONS. These studies indicate that electronic thermography is capable of detecting sensory changes caused by inferior alveolar nerve injury or by pharmacologic nerve block. PMID- 7552876 TI - A comparison of cardiac rate-pressure product and pressure-rate quotient in healthy and medically compromised patients. AB - Healthy and medically compromised patients were studied to compare blood pressure and heart rate changes in response to stress of routine dental extractions performed while they were under local anesthesia. Thirty-nine patients divided into American Society of Anesthesiologists (ASA) I and II groups were noninvasively monitored every 5 minutes. Systolic, diastolic, and mean arterial pressures and heart rate were recorded. Rate pressure products (RPP) and pressure rate (PRQ) quotients were calculated and compared in each group. Significant results were measures of RPP greater than 12,000 and PRQ less than one. Of the 24 patients in the ASA I category, 50% demonstrated elevated RPP values, but only two of 24 had coincidental PRQ abnormalities. Of the 15 patients in the ASA II category, 80% demonstrated elevated RPP values, but two of 15 had coincidental PRQ abnormalities. Patients in the ASA II category had a higher incidence of RPP and PRQ abnormalities, as was expected. However, it is not known which of these two measures is a more sensitive indicator of increased risk associated with stimulation of the sympathetic-adrenergic axis during oral surgery performed with patients under local anesthesia. Correlation studies with continuous Holter monitoring for ST-T wave changes on electrocardiography are forthcoming. PMID- 7552878 TI - Effectiveness of a low dose of cyclosporine in the management of patients with oral erosive lichen planus. AB - This study investigated the effectiveness of a low-dose cyclosporine rinse used in the treatment of oral erosive lichen planus. Fourteen patients with oral erosive lichen planus provided seven experimental sites treated with cyclosporine and seven control sites treated with a placebo. Participants rinsed with 5 ml (500 mg) of cyclosporine or 5 ml of a placebo for 5 minutes each day over a period of 4 weeks. Cyclosporine blood levels as well as complete blood cell counts with differential and serial multiple analysis were monitored throughout the study. Weekly quantitative measurements of lesion size and character (ulceration, erythema, and reticulation) were recorded with the use of an intraoral grid. Healing was defined as the transition from ulceration to erythema to reticulation or to complete resolution. Pain assessment with the use of a visual analogue scale and a questionnaire pertaining to any side effects of treatment were completed each week. At 4 weeks, a statistically significant difference was observed in lesion healing between the cyclosporine and placebo groups. All experimental sites demonstrated progressive healing with evidence of reduced erythema and ulceration, increased reticulation, and decreased pain scores. In contrast, control sites exhibited minimal change in lesion size or character, and patients reported unchanged or increased pain scores. No significant side effects were reported. Within the parameters of this investigation, topical cyclosporine proved to an effective alternative therapy to currently available medications used in the treatment of oral lichen planus. PMID- 7552879 TI - Hyalinosis cutis et mucosae. Review with a case report. AB - Hyalinosis cutis et mucosae (lipoid proteinosis) is a rare disorder characterized by hyaline and lipid deposits in the skin and mucous membrane of the oral cavity, upper respiratory tract, and internal organs. It usually appears in infancy with hoarseness. This disorder is particularly interesting because of its rarity and wide range of clinical manifestations. We report a case that demonstrated manifestations of lipoid proteinosis. PMID- 7552880 TI - Primary lingual tuberculosis caused by M. bovis infection. AB - Although tuberculosis of tongue has been previously reported, its infrequent clinical presentation and increased chance of being overlooked during routine examination of the oral cavity, make it worthy of documentation. Lingual tuberculosis is usually associated with tuberculosis of the oropharynx, lungs, lymph nodes, and miliary tuberculosis. Primary tuberculosis of the tongue is extremely rare and is seldom reported. Early diagnosis of tuberculosis elsewhere in body and its effective treatment by antituberculous drugs may be the reason for its uncommon presentation. The relationship between consumption of raw or unboiled milk and tuberculosis of the tongue is ill-understood and has evidently not been reported. We take this opportunity to present the first case of primary lingual tuberculosis caused by Mycobacterium bovis related to consumption of unboiled cow milk. PMID- 7552881 TI - Use of INR to assess degree of anticoagulation in patients who have dental procedures. AB - Dental professionals frequently treat patients who are receiving anticoagulation therapy. Proper treatment may require adjustment of the anticoagulant dose usually on the basis of the patient's current prothrombin time. This test has been shown to be less accurate than previously thought. The international normalized ratio is another method that attempts to standardize the degree of anticoagulation and to improve reproducibility of results. This system is slowly being implemented in laboratories in the United States. Practitioners who treat patients taking anticoagulants need to be aware of this system in order to make appropriate management decisions. PMID- 7552882 TI - Tobacco use and cancer. A reappraisal. AB - With approximately six million users, smokeless tobacco has received considerable scrutiny as a risk factor for oral cancer. We review the relationship between smokeless tobacco use, keratosis, and oral cancer. Several features of smokeless tobacco keratosis, including the natural history, clinical presentation, and biologic behavior, differentiate it from other leukoplakias that exhibit greater malignant potential. Previous research has demonstrated that the relative risk of oral cancer with smokeless tobacco use is 4.2, about half of the risk from smoking (relative risk = 10 to 15). Mortality data from populations with sustained high-frequency smokeless tobacco use do not support the mistaken prediction of an epidemic of oral cancer with increasing smokeless tobacco use. In fact, the risks of smokeless tobacco use compare so favorably with those of smoking that smokers who switch to smokeless tobacco reduce their risks for all tobacco-related illnesses including oral cancer. Although some criticize this proposal as less than an ideal solution for the nation's smokers, full adoption of this strategy would eventually save over 400,000 lives each year. PMID- 7552883 TI - Squamous cell carcinoma of the gingiva. A case series analysis. AB - Recent studies suggest that patients with carcinoma of the gingiva exhibit demographic features that differ from those of persons with squamous carcinoma at other intraoral sites. In this study, we sought to explore this hypothesis in greater detail. Records from the University of Connecticut Oral Biopsy Service from 1975 through 1992 inclusive were surveyed for cases of oral carcinoma. A total of 577 cases were retrieved and analyzed with respect to prevalence, gender distribution, and age at diagnosis. We found that the gingiva (alveolar ridge included) was the third most common site for oral squamous carcinoma after carcinoma of the floor of the mouth and tongue. Further, the relative proportion of gingival cancer versus carcinoma at other intraoral subsites remained essentially constant throughout the study period. Male-to-female ratios were significantly greater for cancer of the floor of the mouth as compared with both cancer of the tongue and cancer of the gingiva (ridge included). Age was not a significant predictor of oral cancer subsite, and there were no apparent differences between carcinoma of the dentate gingiva and that of the edentulous ridge. Results of this study indicate that gender-specific predilections exist for squamous cell carcinoma at different intraoral subsites. These differences suggest the possibility of different etiologic factors and pathogenetic mechanisms involved in carcinoma of the gingiva compared with surface carcinoma at other intraoral sites. PMID- 7552884 TI - Intraexaminer and interexaminer reliability in the diagnosis of oral epithelial dysplasia. AB - OBJECTIVES: Pathologists differ in their definition of "dysplasia." This study was done to test the hypothesis that experienced oral pathologists are consistent in diagnosing epithelial dysplasia. STUDY DESIGN: Six board-certified oral pathologists examined 120 oral biopsies exhibiting simple hyperkeratosis to severe dysplasia. No clinical information was given, and presence of dysplasia was judged by histomorphology. Examiners' diagnoses were compared with sign-out diagnoses for each case. Months later, each examiner viewed 60 relabeled slides from the original 120. Each diagnosis was compared with the diagnosis in the first round. RESULTS: Exact agreement with the sign-out diagnosis averaged 50.5% (within one histologic grade 90.4%). Examiners agreed exactly with their own diagnoses 50.8% of the time (within one histologic grade 92.4%). Agreement distinguishing dysplasia from no dysplasia compared with original sign-out diagnosis was 81.5%. Agreement with themselves distinguishing dysplasia from no dysplasia was 80.3%. CONCLUSIONS: Accurate reproducible agreement among experienced board-certified oral pathologists diagnosing oral epithelial dysplasia is difficult to achieve. PMID- 7552886 TI - Rhabdomyosarcoma arising in the course of long-standing ameloblastoma. AB - A rare case of rhabdomyosarcoma preceded by ameloblastoma is presented. The sarcoma seemingly arose from the soft tissue in the stroma of preexisting ameloblastoma. PMID- 7552887 TI - Effects of ultrasonic root-end cavity preparation on the root apex. AB - This study determined the effect of bur and ultrasonic preparation on the root apex. After cleaning, shaping, and obturation of root canals of 47 single-rooted teeth and resection of their apexes were done, 24 root-end cavities were prepared with a bur, and the rest were prepared with ultrasonic tips attached to two different ultrasonic units. After photographs of the prepared root ends were taken, their resin replicas were prepared and examined with a scanning electron microscope. Photographs and scanning electron microscope photomicrographs were examined for the presence or absence of cracks. The results showed a significantly higher incidence of crack formation in the walls of root-end cavities prepared by ultrasonic tips compared with those made by the bur. PMID- 7552885 TI - Rhabdomyosarcoma of the oral cavity. Report of four cases. AB - Clinical and pathologic findings of four cases of rhabdomyosarcoma of the oral soft tissues are described that include findings from immunohistochemistry and electron microscopy. Three cases occurred in children under 16 years of age and one in a 22-year-old. Included is a brief discussion on reported gene abnormalities that may contribute to neoplastic development. PMID- 7552888 TI - Histopathologic effects of kinetic cavity preparation for the removal of enamel and dentin. An in vivo animal study. AB - Recent developments in technology, direct placement restorative materials, and cavity preparation design have renewed interest in kinetic cavity preparation, a term to describe the use of air-abrasion for removal of tooth structure. This study compared the pulpal response of 120 teeth in mixed-breed dogs treated with four kinetic cavity preparation combinations of pressure (80 psi and 160 psi) and aluminum oxide particle sizes (27 microns and 50 microns) to those treated with high-speed rotary burs. Class V buccal preparations were made and restored with an interim material. Teeth were collected 72 hours after surgery, decalcified, sectioned, stained with hematoxylin and eosin, and blindly evaluated by two examiners at the minimal dentin thickness. Samples were graded for extent of displacement, disruption, inflammation, and necrosis of pulpal structures. Differences between groups were analyzed with the use of Bonferroni-adjusted multiple Mann-Whitney-Wilcoxon tests with p < 0.05 being significant. Higher pressures and smaller particles yielded significantly fewer pulpal effects than the high-speed treated teeth whereas lower pressures and larger particles were not significantly different for most effects evaluated. No adverse soft tissue effects were noted when kinetic cavity preparation was directed at attached gingiva. PMID- 7552889 TI - A comparison of single-photon emission computed tomography and planar imaging for quantitative skeletal scintigraphy of the mandibular condyle. AB - A prospective study was designed to compare two methods of quantifying technetium 99m methylene diphosphonate uptake in the mandibular condyle. The standard technique expresses condylar activity as a ratio of condylar uptake to a reference (often the fourth lumbar vertebra) with planar scans (lateral images of the mandible). The experimental technique quantifies condylar activity as a ratio of condyle to clivus uptake with single-photon emission computed tomography (SPECT). The results of this study indicated that the uptake ratio of condyle/clivus by SPECT scintigram was positively correlated (p = 0.039) with the planar scan technique. The SPECT technique, similar to an axial computed tomography scan, was easier to perform with better reproducibility than the standard planar technique. In addition, activity in the clivus showed less variation than activity in the fourth lumbar vertebrae. With the development of normal uptake standards in the clivus, the SPECT technique may replace the planar image technique in nongrowing patients. PMID- 7552891 TI - Angle of the articular eminence in patients with temporomandibular joint dysfunction and asymptomatic volunteers. AB - OBJECTIVE: The purpose of this study was to compare structural variations of the temporomandibular joint anatomy between symptomatic patients with temporomandibular joint dysfunction and asymptomatic controls. STUDY DESIGN: There were 74 symptomatic patients with temporomandibular joint dysfunction, 29 asymptomatic volunteers with normal joints, and 6 asymptomatic volunteers with disk displacement with reduction included in this study. All subjects had bilateral high resolution magnetic resonance imaging scans performed in the sagittal (closed and opened) and coronal (closed) positions. All subjects had right and left full profile laminagraphs, one in the centric occlusal position, and one with the incisors edge to edge. RESULTS: Student-Newman-Keuls tests demonstrated no significant angular or linear differences for depth of the articular fossa, angle of the articular eminence, horizontal and vertical overlap of the incisor teeth, and linear condylar translation. CONCLUSION: This study concludes that there are no significant differences in angular and linear measurements in the articular fossa of asymptomatic volunteers and symptomatic subjects with temporomandibular joint dysfunction. PMID- 7552890 TI - Niobium filtration of conventional and high-frequency x-ray generator beams for intraoral radiography. Effects on absorbed doses, image density and contrast, and photon spectra. AB - We have studied the effects of niobium beam filtration on absorbed doses, on image density and contrast, and on photon spectra with conventional and high frequency dental x-ray generators. Added niobium reduced entry and superficial absorbed doses in periapical radiography by 9% to 40% with film and digital image receptors, decreased the radiation necessary to produce a given image density on E-speed film and reduced image contrast on D- and E-speed films. As shown by increased half-value layers for aluminum, titanium, and copper and by pulse height analyses of beam spectra, niobium increased average beam energy by 6% to 19%. Despite the benefits of adding niobium on patient dose reduction and on narrowing the beams' energy spectra, the beam can be overhardened. Adding niobium, therefore, strikes the best balance between radiation dose reduction and beam attenuation, with its risks of increased exposure times, motion blur, and diminished image contrast, when it is used at modest thicknesses (30 microns) and at lower kVp (70) settings. PMID- 7552892 TI - Reducing antimicrobial dosing errors in a neonatal intensive care unit. PMID- 7552893 TI - Intravenous antihypertensive agents for patients unable to take oral medications. AB - The treatment of chronic hypertension in patients unable to take oral medications is challenging. Little information on the comparative safety and efficacy of i.v. alternatives is available. Hydralazine, methyldopate, enalaprilat, and nicardipine appear to be the best options for patients temporarily requiring i.v. medications for controlling chronic hypertension. Therapy should be selected on the basis of the individual patient's needs and diseases, the potential for adverse events, the monitoring required, drug costs, and the expected duration of therapy. The choices may be limited, but understanding the proper use of i.v. antihypertensives should enhance blood pressure control and patient care. PMID- 7552894 TI - Tacrolimus: a new immunosuppressive agent. AB - The mechanism of action, pharmacokinetics, drug interactions, clinical efficacy, and adverse effects of tacrolimus, a newly approved immunosuppressant drug for use in the prophylaxis of organ rejection after transplantation, are reviewed. Tacrolimus prevents rejection of the transplanted organ by inhibiting the expression of interleukin-2 in T cells and inhibiting T-cell growth and proliferation. Bioavailability after oral administration is 5-67%, and the half life is 4-41 hours. Tacrolimus is extensively metabolized by cytochrome P-450 3A4 isoenzyme, resulting in several known drug interactions. Most experience with tacrolimus has been at one institution, where clinical trials have been conducted in liver, kidney, heart, lung, and intestinal transplantation. Clinical trials have shown that tacrolimus is an effective alternative to cyclosporine for both primary immunosuppression and rescue therapy in liver transplant patients. Fewer reports have been published regarding tacrolimus use in other types of transplantation, but the results show that tacrolimus may be a useful alternative to cyclosporine. The major adverse effects of tacrolimus therapy are nephrotoxicity and neurotoxicity. Tacrolimus is an effective alternative to cyclosporine as a primary immunosuppressant in the prevention of organ rejection and may reduce the incidence of rejection after organ transplantation. PMID- 7552895 TI - Computer-assisted antimicrobial surveillance in a community teaching hospital. AB - A computer-assisted, pharmacy-managed antimicrobial surveillance service is described. Rising drug costs and inappropriate use of antimicrobial drugs led to the January 1994 implementation of an antimicrobial surveillance program at a 369 bed community teaching hospital. A computer interface between the pharmacy and microbiology laboratory data-bases was established that allows pharmacists quick access to culture and susceptibility test results and provides timely reports identifying patients for whom intervention may be necessary. Pharmacists review the reports, assess the patient's condition, and, when appropriate, recommend changes in therapy to the prescribing physician. Data on interventions, costs, and outcomes are reviewed monthly. From January to July 1994, pharmacists monitored the antimicrobial therapy of 1384 patients. Interventions were recommended for 348 patients (25%). Physicians accepted 289 (83%) of the recommendations. The infection subsided in 280 (97%) of the patients for whom recommendations were accepted. The cost avoidance achieved by the interventions exceeded $32,000, which more than justified the one full-time pharmacist position assigned to the service. A computer-assisted antimicrobial surveillance service managed by pharmacists helped control costs and achieve favorable clinical outcomes. PMID- 7552896 TI - Stability of granisetron hydrochloride in a disposable elastomeric infusion device. AB - The stability and sterility of granisetron hydrochloride in 5% dextrose injection or 0.9% sodium chloride injection when stored in a disposable elastomeric infusion device were studied. Granisetron was diluted to 0.02 mg/mL (as the hydrochloride salt) in 5% dextrose chloride injection. The solution was placed in the drug reservoir of a disposable elastomeric infusion device and refrigerated at 4 degrees C for 14 days. A total of eight pumps were prepared, four containing granisetron 0.02 mg/mL in 5% dextrose injection and four containing granisetron 0.02 mg/mL in 0.9% sodium chloride injection. The solutions were assayed for granisetron concentration by stability-indicating high-performance liquid chromatography at 0 hours, 24 hours, 48 hours, 7 days, and 14 days. Each solution was inspected for clarity, color, and precipitation, and sterility testing was performed. Throughout the study, the mean concentration of granisetron remaining was more than 92% of the initial concentration both in 5% dextrose injection and in 0.9% sodium chloride injection. Individual solutions in 0.9% sodium chloride injection consistently maintained more than 90% of the initial drug concentration for only seven days. No microbial growth was detected. No precipitation, color change, or haziness was seen. Granisetron 0.02 mg/mL (as the hydrochloride salt) was stable and free of microbial growth in 0.9% sodium chloride injection for up to 7 days and stable and free of microbial growth in 5% dextrose injection for up to 14 days when stored at 4 degrees C in a disposable elastomeric infusion device. PMID- 7552897 TI - Pharmacist's impact on antimicrobial drug therapy. PMID- 7552898 TI - HMO pharmacy directors' opinions of acquisition of pharmacy benefit management companies. PMID- 7552899 TI - Influence of pharmaceutical sales representatives on the formulary system. PMID- 7552900 TI - Influence of pharmacy residents on pharmacy students' pursuit of residency training. PMID- 7552901 TI - Compatibility of ondansetron hydrochloride and piperacillin sodium-tazobactam sodium during simulated Y-site administration. PMID- 7552902 TI - Stability of ondansetron hydrochloride in a total parenteral nutrient admixture. PMID- 7552903 TI - Stability of pyrazinamide in two suspensions. PMID- 7552904 TI - Stability of cefotaxime sodium and metronidazole in 0.9% sodium chloride injection or in ready-to-use metronidazole bags. PMID- 7552905 TI - Ethical opinion in a formulary recommendation. PMID- 7552906 TI - Tacrolimus in transplantation. PMID- 7552907 TI - Professionalism in the community pharmacy setting. PMID- 7552908 TI - Clarification of venlafaxine chemistry, pharmacology, and pharmacokinetics. PMID- 7552909 TI - Rethinking hospital pharmaceutical marketing. PMID- 7552910 TI - Applying patient outcomes and pharmacoeconomics in patient care. Proceedings of a conference. Boston, Massachusetts, September 1994. PMID- 7552911 TI - The outcomes of patient care. PMID- 7552912 TI - Pharmacoeconomics and outcomes research. PMID- 7552913 TI - Introduction to pharmacist participation in measuring and monitoring patients' health-related quality of life. PMID- 7552914 TI - Pharmacoeconomics and the quality of decision-making by pharmacy and therapeutics committees. PMID- 7552915 TI - Relationship between medication compliance and medical outcomes. PMID- 7552916 TI - Assessing the health status of adult patients with epilepsy. PMID- 7552917 TI - Assessing health status in a dialysis clinic. PMID- 7552918 TI - Workshop synopses. PMID- 7552919 TI - The outcomes movement and health care reform. PMID- 7552920 TI - Prediction of traffic noise: a screening technique. AB - Traffic noise is ubiquitous in many communities and is an important environmental concern, especially for persons located near major roadways. Several different methods are available to estimate noise levels resulting from roadway traffic. These include computational, graphical, and computer modeling techniques. The prediction methodology presented here is a simplified technique that can be used for estimating noise resulting from traffic and for screening traffic noise impacts. This Traffic Noise Screening (TNS) approach consists of a series of traffic noise level prediction graphs developed for different roadway configurations. The graphs are based on the results from using the Federal Highway Administration (FHWA) STAMINA2.0 computerized noise prediction model for various scenarios. Data inputs to the TNS approach include roadway genometries, traffic volumes, vehicle travel speed, and centerline distance to the receptors. The TNS graphs allow easy estimation of traffic noise levels for use in predicting traffic-related noise impacts. This TNS approach is not intended as a substitute for detailed modeling, such as with STAMINA2.0, but as a screening tool to aid in determining when detailed modeling may be necessary. If screening results indicate that noise estimates are significant, or if the scenario is rather complex, then additional, more detailed modeling can be performed. PMID- 7552921 TI - Salmeterol for the treatment of asthma. PMID- 7552922 TI - Management of ocular allergy. AB - OBJECTIVE: Ocular allergy is a frequent accompaniment of systemic allergy and in some situations ocular signs and symptoms are the most prominent features of allergic disease. This paper will describe an approach to ocular allergy intended to aid in the recognition, differentiation from other ocular conditions, and management of ocular allergic disease. DATA SOURCES: Ophthalmologists and allergists have contributed to the body of knowledge concerning ocular allergy during the last several decades. Recent investigations have provided better and more specific treatments for the safe and effective management of ocular allergy. STUDY SELECTION: The results of several review articles, investigation of certain aspects of ocular allergy, and clinical trials have contributed to the approach that ophthalmologists and allergists take in the diagnosis and management of ocular allergy. RESULTS: In most cases, it is not difficult to recognize ocular allergy because of its association with systemic allergy. On the other hand, numerous inflammatory ocular conditions can be confused with ocular allergy. It is important to recognize infectious ocular disease and severe ocular inflammatory disease, such as uveitis, to ensure appropriate treatment. CONCLUSIONS: Allergists and ophthalmologists should be able to recognize ocular allergy and distinguish it from other ocular inflammatory conditions. Most ocular allergy is mild and easily managed. Currently available medications allow for the safe and effective management of most cases of ocular allergy. Many situations can be managed with avoidance of allergens, supportive treatment such as cold compresses, or no treatment at all. It is rare that ocular allergy requires treatment with topical corticosteroids. When such severe circumstances occur, an ophthalmologist should monitor the intraocular pressure and the overall course of the ocular disease. PMID- 7552923 TI - Dramatic resolution of chronic urticaria. PMID- 7552924 TI - Statement on self-monitoring of peak expiratory flows in the investigation of occupational asthma. Subcommittee on Occupational Allergy of European Academy of Allergy and Clinical Immunology, American Academy of Allergy and Immunology, European Respiratory Society, American College of Allergy, Asthma and Immunology. PMID- 7552925 TI - Multifactorial immunologic lung disease: a case report. AB - BACKGROUND: Allergic asthma, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis are all forms of immunologically mediated lung disease. Recognition that more than one of these diseases may co-exist is necessary for proper patient management. OBJECTIVE: To report a case of extrinsic (cat) asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, and potentially fatal asthma. Immunologic mechanisms and treatment of these diseases are discussed. METHODS: The study is a case report from a university hospital setting. Serum precipitating antibodies against avian antigens were measured using gel-diffusion technique. Serum IgE levels were measured and skin testing was performed to selected antigens. RESULTS: The patient had positive cutaneous reactivity to cat antigen and Aspergillus. Serology for allergic bronchopulmonary aspergillosis and serum precipitating antibodies to finch extract were positive. Symptoms improved and prednisone requirements decreased after removal of finches. CONCLUSION: A case of cat asthma, potentially fatal asthma, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis is presented. Early recognition and treatment of multifactorial immunologic lung disease with avoidance measures and use of corticosteroids can prevent progressive respiratory damage. PMID- 7552926 TI - Safety of salmeterol in the maintenance treatment of asthma. AB - BACKGROUND: Salmeterol is the first long-acting inhaled beta 2-agonist available in the US for the maintenance treatment of asthma. OBJECTIVE: To compare the safety of salmeterol with that of the short-acting beta 2-agonist albuterol. METHODS: Two identically designed, prospective, randomized, double-blind, parallel studies were conducted comparing salmeterol 42 micrograms twice daily, albuterol 180 micrograms four times daily, and placebo over 12 weeks in 556 patients (12 to 73 years old) with mild-to-moderate chronic asthma. Patients in each treatment group could use albuterol as needed to control acute symptoms. RESULTS: The incidence of potentially drug-related adverse events was similar among the treatment groups (range: 22% to 23%), with headache being the most commonly reported (range: 9% to 10%). No deaths occurred during the studies. Concomitant use of > 4 puffs of supplemental albuterol per day in the salmeterol group produced no increase in the incidence of adverse events either in general or of a cardiovascular nature. There were no statistically significant differences among treatment groups or clinically significant changes from pretreatment values in mean pulse rate, systolic/diastolic blood pressure, or clinical laboratory values after 12 weeks. There were no clinically significant differences among groups in heart rates nor were there differences in the frequency of supraventricular or ventricular ectopic beats during 24-hr Holter monitoring. The frequency of asthma exacerbations was lowest among patients receiving salmeterol (and highest among those who received placebo), and this rate did not increase over the 12 weeks. Asthma exacerbations were treated successfully with nebulized albuterol (2.5 mg), with no evidence of any increased risk of cardiovascular events. CONCLUSIONS: Salmeterol 42 micrograms twice daily is well-tolerated in patients with asthma, having a similar safety profile as that of albuterol 180 micrograms inhaled four times daily or placebo (plus as needed albuterol). Concomitant use of albuterol, either by MDI or nebulization, did not affect the safety of salmeterol. Extensive cardiovascular monitoring revealed no significant cardiovascular adverse effects or arrhythmogenic effects associated with salmeterol over 12 weeks. PMID- 7552928 TI - Mountain cedar allergens found in nonpollen tree parts. AB - BACKGROUND: Mountain cedar (Juniperus ashei) pollen is the principal aeroallergen in south central Texas from late December through February. The major mountain cedar allergen is a 40-kD glycoprotein, gp40. OBJECTIVE: To identify allergens in mountain cedar wood, leaves, and berries and to detect mountain cedar allergen in smoke from burning male or female trees. METHODS: SDS-PAGE plus mountain cedar human sIgE and monoclonal antibody immunoblots identified mountain cedar allergens within pollen and nonpollen tree part extracts. RESULTS: IgE immunoblots identified a single wood allergen at 36 kD and three berry allergens at 36, 26-27, and 21 kD, in addition to known pollen allergens. Mountain cedar monoclonal antibody bound an allergen epitope present not only on 40, 33, and 28 kD pollen allergens, but also on 36 and 32-kD wood allergens, and the 26-27-kD berry allergen. Immunoblot studies detected no mountain cedar allergen in leaves and no allergen in smoke from burning male and female trees. Allergens constituted a much smaller percentage of extractable protein in wood and berries than in pollen. CONCLUSIONS: Mountain cedar berry allergen content is too small to give credence to the ingestion of berries as a folk medicine treatment of mountain cedar pollinosis. In addition, while smoke from burning mountain cedar trees may be irritating, it contains no allergens that could cause allergic rhinoconjunctivitis. PMID- 7552927 TI - Eosinophil activity reflects clinical status in patients with asthma before and during a prednisolone course. AB - BACKGROUND: Both overtreating and undertreating asthma can be harmful, either because of side effects or development of uncontrolled asthma. As the eosinophil granulocyte is of importance in asthma, it is logical to study this cell in the search for a marker for the right level of treatment. OBJECTIVE: To study the changes in eosinophil activity and the correlation to clinical status in asthmatic patients who have deteriorated and during prednisolone treatment. METHODS: Nine patients were studied on two occasions: with exacerbation (visit 1) and during prednisolone treatment (visit 2). Clinical evaluation was performed as well as analysis of percentage of eosinophils, eosinophilic cationic protein (S ECP) in serum and expression of intracellular ECP, measured by cell flow cytometry using a monoclonal antibody (EG2) against the activated form of ECP. RESULTS: Visit 1: four patients had eosinophils above normal, mean 6.5%, range 1.5 to 13.8. S-ECP was measured in seven patients of whom five had values above normal (16 micrograms/L), mean 29.5, range 1.5 to 13.8. Intracellular expression of ECP was above normal (mean 26.7 +/- 10.8) in seven out of nine (mean 48.1, range 28.8 to 69.6). Visit 2: all patients improved and all eosinophil parameters decreased. The eosinophil concentration fell to a mean of 3.0%, range 1.0 to 6.6, S-ECP to a mean of 10.5 micrograms/L, range 3.2 to 17 and intracellular expression of ECP to a mean of 33.4, range 19.4 to 40.9. CONCLUSIONS: Reduction in eosinophil activity followed improvement in clinical condition. Measuring intracellular expression of ECP may be of value in addition to eosinophil count and S-ECP in monitoring asthma. PMID- 7552929 TI - Effect of topical levocabastine on nasal response to allergen challenge and nasal hyperreactivity in perennial rhinitis. AB - BACKGROUND: It has been demonstrated that some oral antihistamines reduce nasal nonspecific reactivity and that topical levocabastine reduces cellular influx after nasal allergen challenge. This suggests that antihistamines possess other properties besides classical H1-receptor antagonism. OBJECTIVE: To evaluate the effect of 1 week's treatment with topical levocabastine on the nasal clinical response, inflammatory mediators, and nasal hyperreactivity. METHODS: In a double blind, placebo-controlled, 2-period, 2-treatment, crossover study, 21 rhinitic patients allergic to house dust mite participated. After each treatment period patients were challenged with house dust mite extract. Symptom scores and nasal lavages were collected for nine and one-half hours after challenge. Allergen induced nasal hyperreactivity was determined by nasal methacholine challenge 24 hours after allergen challenge. A nasal histamine challenge was performed as well. RESULTS: Patients showed only an immediate nasal response. Levocabastine significantly reduced the symptom score after 100 (P = .0063), 1000 (P = .0035), and 10,000 biological units (BU)/mL (P = .0013) of house dust mite extract. Albumin influx and tryptase release were not significantly reduced by levocabastine. No release of histamine and eosinophil cationic protein was seen. Levocabastine did not reduce nasal response to methacholine. Active treatment significantly reduced histamine-induced nasal secretion (P = .0009) and the number of sneezes (P = .0001). CONCLUSION: A significant effect of levocabastine was shown on the immediate clinical response to house dust mite and to histamine challenge only. Our findings suggest that levocabastine is an effective H1 receptor antagonist without anti-inflammatory properties. PMID- 7552931 TI - Relationship between airway responsiveness and peak expiratory flow variability in subjects with allergic rhinitis. AB - BACKGROUND: Previous studies have demonstrated an increase in diurnal variation of peak expiratory flow (PEF) in subjects with allergic rhinitis who had decreased methacholine PC20 values. The relationship between PEF variability and some indices of the concentration-response curve to methacholine such as the position and level of the maximal response-plateau has not been studied. OBJECTIVE: The study was carried out to determine differences in PEF variability between subjects with allergic rhinitis who showed plateaus on the concentration response curves to methacholine at mild degrees of airway narrowing, and patients in whom no plateau was detected. METHODS: Forty-three nonasthmatic patients with allergic rhinitis were included in the study. Subjects underwent methacholine challenge tests with doubling concentrations from 0.39 to 200 mg/mL and measured their PEF three times daily for the next seven days, using mini-Wright peak flow meters. The concentration-response curves to methacholine were characterized by the threshold value (PC20 = provocative concentration required to produce a 20% fall in FEV1) and, if possible, by the position (EC50 = concentration of methacholine that produced 50% of the maximal response) and maximal response plateau (FEV1 falls < or = 5% after at least three of the highest concentrations). The variability of PEF was expressed as the amplitude percent mean. RESULTS: No correlations were observed between amplitude percent mean and PC20 (r = -.23, NS), EC50 (r = -.07, NS), or level of plateau (r = .02, NS). The amplitude percent mean was higher in subjects who had decreased methacholine threshold values without plateau (geometric mean = 8.3%; range = 3.8% to 14.2%) than in subjects who had decreased threshold values and plateaus (geometric mean = 4.5%; range = 2.4% to 9.9%; P < .01), or subjects who had normal threshold values and plateaus (geometric mean = 4.4%; range = 1.6% to 10.8%; P < .01). Although subjects who had decreased threshold values and plateaus showed higher methacholine responsiveness than subjects with normal threshold values and plateaus (geometric mean PC20 = 6.2 mg/mL versus 107.1 mg/mL, P < .01; geometric mean EC50 = 3.1 mg/mL versus 9.1 mg/mL, P < .01; mean +/- SEM level of plateau = 17.2 +/- 1.8% versus 28.8 +/- 1.1%, P < .001), the difference in amplitude percent mean was not significant. CONCLUSIONS: The loss of plateau on the concentration-response curves to inhaled methacholine at mild degrees of airway narrowing identifies those subjects with allergic rhinitis who show a greater PEF variability. The PEF variability and airway responsiveness are not interchangeable terms. PMID- 7552930 TI - A clinical study of mortality due to asthma. AB - BACKGROUND: Despite identification of the pathophysiologic mechanisms of asthma and improvement in therapy, asthma mortality has not decreased in recent years. OBJECTIVE: The pathophysiology and asthma-related death preventive measures were investigated with a physician-based questionnaire survey. METHODS: Questionnaires were sent to physicians primarily involved in treating asthma in Nagasaki Prefecture, Japan. The clinical characteristics of 32 patients who died of asthma (fatal cases) from 1984 to 1992 were compared with those of 17 patients with severe asthma who survived as a result of treatment by mechanical ventilation (nearly fatal cases). RESULTS: The number of deaths due to asthma increased in the last 2 years. Fatal cases and nearly fatal cases included patients with severe asthma as well as patients with mild asthma. Analysis of the clinical histories of patients judged to have died suddenly revealed the presence of persistent wheezing in these patients for a few days prior to the fatal episode. Airway obstruction was more marked and bronchial hyperresponsiveness was greater in fatal cases compared with those of a group of 70 patients without a history of nearly fatal acute asthma. CONCLUSIONS: The following measures may help prevent asthma deaths. Both patients and physicians should realize that even mild episodes can lead to severe, even fatal acute asthma. The severity of asthma should be evaluated not only by symptoms and peak expiratory flow rates but also bronchial hyperresponsiveness. Treatment should include reduction of bronchial hyperresponsiveness using oral or inhaled corticosteroids. PMID- 7552932 TI - Skin prick tests and in vitro immunoassays with native spices and spice extracts. AB - BACKGROUND: Skin prick tests of native spices (commercial powdered spices) are common in patients with allergy to birch or mugwort pollen. Clinical symptoms from spices are infrequent but occasionally severe. OBJECTIVE: To compare the skin prick test results with native spices and spice extracts and to determine the clinical relevance of test material. METHODS: Skin prick tests with the native spices coriander, caraway, paprika, cayenne, mustard, and white pepper were made twice at 2-month to 2.9-year intervals in 49 patients. During the latter time, tests were also made with spice extracts and spice-specific serum IgE was measured. RESULTS: The reproducibility of skin test results with native spices was 67% to 100%. Spice extracts, except white pepper, elicited positive skin test reactions in half those with positive reactions to native spices. Higher specific IgE concentrations (> or = 3.5 PRU/mL) were seen in cases where the skin tests were positive to the corresponding spices with 5% extracts of > 8 kD Mw. Three-fourths of the patients with positive skin tests to native spices were positive to birch pollen and one-half to a vegetable. Mild clinical symptoms from spices were reported by one-third. CONCLUSIONS: Spice allergens partly crossreact with those of pollens and vegetables. A minority of spice allergens may give clinical symptoms. The > 8-kD 5% extracts may be relevant skin prick test materials for identifying patients at risk of developing severe symptoms from ingested spices. PMID- 7552933 TI - Lack of effect of loratadine on moderate to severe asthma. AB - BACKGROUND: Antihistamines have been shown to be effective in patients with allergic asthma, but their role in chronic and more severe asthma is uncertain. OBJECTIVES: To evaluate whether loratadine, a selective H1 receptor antagonist, given as an adjunct to standard asthma medication would have any effect in patients with moderate-to-severe asthma. METHODS: Thirty-five patients with moderate-to-severe asthma, most receiving inhaled steroids, were enrolled in this double-blind, crossover study. In addition to their maintenance therapy patients received either loratadine, 20 mg once daily, or placebo for 4 weeks before crossing over to the other preparation for a further 4 weeks. Variables of efficacy were daily and nocturnal respiratory symptoms, lung function (PEF, FEV1, FVC), and bronchodilator use. RESULTS: Three subjects were withdrawn from the study because of deteriorating asthma. There was a trend in favor of loratadine treatment with regard to global assessment of drug efficacy but the difference was not statistically significant. There was no objective improvement in asthma control comparing loratadine with placebo but if each treatment week were compared with the run-in period, PEF was significantly (P < .01) improved during the initial phase of loratadine treatment. This effect gradually decreased with time, suggesting tolerance to any bronchodilatory effect of the antihistamine. CONCLUSION: Loratadine, given as an adjunct to standard asthma therapy, has little if any role to play in the treatment of moderate-to-severe asthma. PMID- 7552935 TI - Noncompliance with immunotherapy secondary to adverse effects. PMID- 7552934 TI - Eczema responsive to treatment for Helicobacter pylori. PMID- 7552936 TI - 9th International Symposium on Radiopharmacology. Ann Arbor, Michigan, June 7-10, 1995. Abstracts. PMID- 7552937 TI - AIDS and nuclear medicine. PMID- 7552938 TI - The AIDS epidemic. AB - AIDS epidemic began about 15 years ago; since then, the number of new cases have increased over time. By the end of 1994, 1,025,073 AIDS cases had been reported to the WHO, with a 20% increase in the number of cases since December 1993. As is now evident that the majority of cases occur in developing countries, under diagnosis, under-reporting and delays in reporting explain the big difference existing between reported and estimated AIDS cases, giving a 4.5 million cumulative AIDS cases worldwide, as of late 1994. In the industrialized countries, the spread of HIV infection began in the late 1970's or early 1980's among homosexual men and IVDU's. Heterosexual transmission was, at first, very limited, though recent data has shown an increase in its diffusion. On the contrary, in Sub-Saharan Africa and the Caribbean, transmission was almost exclusively heterosexual. High rates of infection among young women led to a corresponding increase of perinatal transmission, infecting more than 1.5 million children since the beginning of the pandemic. The estimated number of HIV infections worldwide, as of late 1994, is of 17 millions. Thus, even if some evidence indicates a possible decline of the pandemic in industrialized countries over the next few years, probably because heterosexual transmission was less common, developing countries appear to present an increasing number of HIV infections in young people and, consequently, in children. The real impact of such an epidemic is stressed by the fact that HIV is becoming the leading cause of death among, young people, both in industrialized and developing countries.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552939 TI - AIDS: clinical and scientific issues past, present and future. AB - The time from the recognition that AIDS was an infectious disease, to the discovery of HIV as the causative agent and the identification of the first specific antiviral agent that showed some clinical benefit, was impressively short. Over the last few years it would appear that progress against AIDS has slowed down considerably. Neither new treatments nor vaccines have given much grounds for optimism and back to basics has been the main battle cry. However it is easy to overlook the tremendous improvements that have taken place in the management of HIV disease in the absence of a curative treatment. Many of the opportunistic infections that used to kill patients are not only treatable but are able to be treated prophylactically. This has altered the clinical spectrum of disease with many patients surviving several years with virtually no CD4 lymphocytes only to succumb to other HIV related disease such as non Hodgkins lymphoma. This review identifies the major advances that have occurred in our understanding of AIDS and identifies the major problems to be overcome in the next few years at both the clinical and basic levels. PMID- 7552940 TI - New research hypotheses in the immunopathogenesis of human immunodeficiency virus infection. AB - The acquired immunodeficiency syndrome (AIDS) is a clinically multifaceted disease induced by infection with the human immunodeficiency virus (HIV). HIV infection results in a complex pattern of immunologic alterations that leads to the development of acquired immunodeficiency syndrome (AIDS) in the majority of HIV seropositive (HIV+) individuals. The reduction in CD4 T lymphocyte counts is the hallmark of HIV infection nevertheless, long before the reduction of CD4 counts reaches critical levels, a series of profound and complex defects that impair the function of CD4 T lymphocytes can be observed. Thus, HIV infection is characterised by quantitative and qualitative defects affecting CD4 T lymphocytes. It was recently suggested that the qualitative defects observed in HIV infection preferentially impair the production of type 1 cytokines, thus provoking abnormalities that selectively affect cell mediated immunity (CMI). Because the efficacy of CMI and humoral immunity is in a continuous reciprocal balance, the impairment of type 1 cytokines production and CMI is likely to be associated with an increased production of type 2 cytokines and an exaggerated stimulation of humoral immunity. Corollaries of this hypothesis are that: 1) a strong cell mediated immunity is more protective in preventing the progression of HIV infection to AIDS; and 2) signs of hyper/abnormal activation of humoral response are indicators of poor prognosis. Additionally, it was recently suggested that programmed cell death (PCD) is an important mechanism leading to CD4 depletion in HIV infection, and that susceptibility of peripheral lymphocytes to PCD appears to be differentially regulated by diverse cytokines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552941 TI - Role of nuclear medicine and AIDS: overview and perspective for the future. AB - The Human Immunodeficiency Virus (HIV) is the causative agent for the expanding epidemic of the Acquired Immunodeficiency Syndrome (AIDS). Sixteen million individuals were estimated to be infected worldwide with HIV by the World Health Organization in 1995, with over 10 million in Africa and one million in the USA. As the HIV cost in dollars and lives continues to rise it will become more important to understand AIDS and to foresee the potential role of nuclear medicine in HIV diseases. Nuclear medicine may have a role in the assessment of immune function, including the ability to predict if individuals can avoid progression to HIV+status, if pre-AIDS to AIDS conversion can be prevented, and if an individual's immune status requires addition of prophylaxis. Also it can be used for disease detection at an early stage and determination of the extent of disease. It is especially useful to assist clinicians in optimizing therapy and assessing its efficacy. In the future new radiopharmaceuticals for detecting a specific infections and tumors will be needed. This will require collaborative efforts with basic scientists and clinicians working hand in hand to address specific issues related to AIDS. PMID- 7552942 TI - Radiographic evaluation of AIDS patients. AB - Morphological imaging, based on the use of various techniques including ultrasound, X-ray computed tomography (CT), and magnetic resonance imaging (MRI), plays an important role in the characterization, diagnosis and follow-up of patients with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). While the presence of thoracic infections, the most frequently observed illnesses in AIDS patients, can best be performed by using conventional chest films and CT, the assessment of cerebral involvement in AIDS patients--characterized by the presence of focal masses, demyelination, meningitis, and infarction--is best achieved using MRI. The work-up of patients with gastrointestinal symptoms should include the use of ultrasound for the evaluation of visceral involvement and lymphadenopathy, completed by CT to further characterize pathologic conditions in either the bowel or visceral organs. Ultrasound is the screening exam of choice in AIDS patients with suspected renal disease, but other methods may be necessary for the assessment of the complications due to pharmacological treatment. Musculoskeletal complications may require the combined use of all the above methods, since they may be caused by infections, tumors and rheumatologic illness. The use of the radiographic methods for the detection of the numerous forms of infections and malignancies in AIDS patients is described in detail for the various body districts. PMID- 7552943 TI - Correlative morphologic and functional imaging for diagnosis, staging and follow up in AIDS: an overview. AB - The immunocompromised patient can be affected by different opportunistic infections and tumors, that can involve all organ systems, and particularly, the central nervous system, the respiratory system, the gastrointestinal tract. The extreme variability of AIDS presentations requires a specific preparation and cooperation between the different diagnostic imaging specialists and a close collaboration with the clinicians. With AIDS, different morphologic and functional imaging techniques can be used for detection of disease sites, assessment of the extent of the disease and monitoring of disease changes over time and response to treatment. The complexity of AIDS presentations is such that full integration of the complementary information obtained with different techniques could be most useful. Image registration (coregistration, fusion) indicates approaches where precise spatial cross-references are obtained, in order to combine the information acquired with different imaging modalities, in particular by blending morphologic CT/MRI data with functional SPET/PET data. In this paper, an overview of the developments in the field of "image fusion" follows some comments on the results of morphologic and functional assessment of brain, chest and abdominal diseases in the course of AIDS, as illustrative examples of the potential benefits of multimodality image correlation. PMID- 7552944 TI - Use of polyclonal IgG in HIV infection and AIDS. AB - Nuclear Medicine should have a pivitol role to play in the investigation of patients infected with the human immunodeficiency virus (HIV). Unfortunately the use of scintigraphic techniques to localise infection have not become widely used in Europe. Neither 67Ga citrate or labelled leukocytes are ideal. In a search for new agents which can be used to identify the presence of infection both 99mTc and 111In labelled polyclonal immunoglobulin-C have been investigated. It was found that 99mTc labelled polyclonal immune-globulin-G was not able to localise infection in either the chest or the abdomen. In contrast 111In labelled polyclonal immunoglobulin-G had both high sensitivity and specificity for imaging infection in HIV infected patients. If these preliminary results are confirmed immunoglobulin-G could find an important clinical application in this specialised patient group. PMID- 7552945 TI - The role of gallium and labeled leukocyte scintigraphy in the AIDS patient. AB - Because AIDS patients frequently present with minimal symptomatology, radionuclide imaging with its ability to survey the entire body, is especially valuable. Gallium-67 citrate, the most commonly performed radionuclide study for localizing infection in these patients, is most useful for detecting opportunistic infections, especially in the thorax. A negative gallium scan, particularly when the chest X-ray is unremarkable, rules strongly against pulmonary disease. A negative gallium scan in a patient with an abnormal chest X ray and Kaposi's sarcoma, suggests that the patient's respiratory distress is related to the neoplasm. Diffuse pulmonary parenchymal uptake of gallium in the HIV (+) patient is most often associated with PCP. While there are other causes of diffuse pulmonary uptake, the more intense or heterogeneous the uptake, the more likely the patient is to have PCP. Focal pulmonary uptake is usually associated with bacterial pneumonia although PCP may occasionally present in this fashion. Lymph node uptake of gallium is usually associated with Mycobacterium avium complex, tuberculosis, or lymphoma. When corresponding abnormalities are present on thallium scintigraphy lymphoma is likely. Gallium positive, thallium negative, studies suggest mycobacterial disease. Labeled leukocyte imaging is not useful for detecting opportunistic infections probably because of the inflammatory response incited by these organisms. Leukocyte imaging is, however, more sensitive for detecting bacterial pneumonia. In the abdomen, gallium imaging is most useful for identifying lymphadenopathy, while labeled leukocyte imaging is superior for detecting AIDS-associated colitides. In summary, radionuclide studies are valuable diagnostic modalities in AIDS. Their success can be maximized by tailoring the study to the individual's needs. PMID- 7552946 TI - 99mTc DTPA transfer/permeability in patients with HIV disease. AB - Lung 99mTc DTPA transfer/permeability has been widely used to assess the integrity of the lung alveolar-capillary interface. The use in HIV positive patients to provide a rapid assessment of causes of breathlessness or fever is discussed in this overview. There is I believe a clear role for this technique in this group of patients. It is more sensitive than Gallium-67 scanning in defining lung disease and is capable of distinguishing between alveolitic processes and bacterial infections of Kaposi sarcomatous infiltration. The 99mTc DTPA transfer/permeability can also define the absence of lung involvement in those patients with a fever to allow more suitable investigation to proceed. The 99mTc DTPA transfer/permeability measurement is a rapid, easy method of evaluating patients with HIV disease. Depending on the clinicians philosophy it can be used as a basis to initiate treatment or at the very least point to the next necessary investigation. PMID- 7552947 TI - Radionuclide brain imaging in acquired immunodeficiency syndrome (AIDS). AB - Infection with the human immunodeficiency virus-type 1 (HIV-1) may produce a variety of central nervous system (CNS) symptoms and signs. CNS involvement in patients with the acquired immunodeficiency syndrome (AIDS) includes AIDS dementia complex or HIV-1 associated cognitive/motor complex (widely known as HIV encephalopathy), progressive multifocal leucoencephalopathy (PML), opportunistic infections such as Toxoplasma gondii, TB, Cryptococcus and infiltration by non Hodgkin's B cell lymphoma. High resolution structural imaging investigations, either X-ray Computed Tomography (CT scan) or Magnetic Resonance Imaging (MRI) have contributed to the understanding and definition of cerebral damage caused by HIV encephalopathy. Atrophy and mainly high signal scattered white matter abnormalities are commonly seen with MRI. PML produces focal white matter high signal abnormalities due to multiple foci of demyelination. However, using structural imaging techniques there are no reliable parameters to distinguish focal lesions due to opportunistic infection (Toxoplasma gondii abscess) from neoplasm (lymphoma infiltration). In this manuscript we review the use of radionuclide brain imaging techniques in the investigation of HIV infected patients. Brain perfusion single photon emission tomography (SPET), neuroreceptor and positron emission tomography (PET) studies are reviewed. Greater emphasis is put on the potential of some radiopharmaceuticals, considered to be brain tumor markers, to distinguish intracerebral lymphoma infiltration from Toxoplasma infection. SPET with 201Tl using quantification (tumour to nontumour radioactivity ratios) appears a very promising technique to identify intracerebral lymphoma. PMID- 7552948 TI - Panoramic and restrictive intraoral radiography in comprehensive oral radiographic diagnosis. AB - It has been suggested that information from the panoramic radiograph makes it possible to appropriately select supplementary intraoral radiographs to achieve a comprehensive examination of teeth and surrounding bone with less patient dose but no significant information loss. Number of intraoral radiographs selected, information loss and monetary costs with such a procedure was evaluated in 40 patients. Results show that, on average, 5.1 intraoral radiographs were selected to supplement the panoramic radiograph. Of these, 3.1 contained information different from that in the panoramic radiograph but 2.0 did not. An additional 3.4 ought to have been taken to reach the result of the 'gold standard' achieved from a simultaneous evaluation by two expert observers of panoramic radiographs and full mouth surveys with intraoral radiographs. Sensitivity for the combined use of panoramic and supplementary intraoral radiographs was high (80-96%, depending on type of teeth) as regards periapical lesions and marginal bone loss but low for caries (42-96%). Specificity was high for periapical lesions and caries (95-97%) but low for marginal bone loss (50-92%). In Sweden, patient costs become almost the same for a combination of panoramic radiography and 8.5 intraoral radiographs as for a full mouth survey comprising 20 intraoral radiographs. The radiation dose reduction is 40-50% considering that the dose from a panoramic radiograph approximately corresponds to 2-4 intraoral radiographs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552949 TI - Stereologic study of cyclosporin A-induced gingival overgrowth in renal transplant patients. AB - Gingival biopsies were taken from 13 renal transplant patients (mean age 26.5 yr), 11 of whom exhibited cyclosporin A (CsA)-induced gingival overgrowth. Control material was obtained from seven volunteers (mean age 28 yr). Gingival tissue components were analyzed by quantitative microscopy (stereology) on 5 microns-thick sections of interdental papillae. The volume density (Vv) of different tissue components and the surface density of epithelial ridges were calculated by conventional point and intersection counting. The study showed that the volume density of oral epithelium and the surface density of the epithelial ridges in the CsA-induced gingival overgrowth were significantly increased compared to normal gingival tissue. The connective tissue of the lesion exhibited a significant increase in volume density of cells, blood vessels and non collagenous matrix with a corresponding decrease in the collagenous matrix. These results indicate that CsA-induced gingival overgrowth represents a tissue with an altered composition characterized by increased thickness of oral epithelium and relatively increased amount of cells, vessels, non-collagenous matrix and decreased collagenous matrix in the connective tissue. PMID- 7552950 TI - Expression of HLA class II antigens in marginal periodontitis of patients with Down's syndrome. AB - The expression of HLA Class II antigens on the surfaces of immunocompetent cells and the presence of CD1a+ cells (Langerhans cells) are important components of antigen presentation. Quantitative variations in HLA class II expression on antigen-presenting cells play a role in immune regulation. An indirect immunofluorescent technique was used on cryostat sections to reveal such differences qualitatively or quantitatively between chronic marginal periodontitis (CMP) in patients with Down's syndrome (DS) and in otherwise normal patients (NP). We found increased frequency of HLA Class II (HLA-expression on inflammatory cells and on keratinocytes of the oral gingival epithelium) in CMP of DS patients compared to sections from NP. The expression of HLA-DR was more frequent on the keratinocytes of the pocket epithelium in NP than in DS. There were significantly higher numbers of CD1a+ cells and ratios of HLA-DR+/CD1a+ cells and HLA-DP+/CD1a+ cells in the DS group compared to the NP group. Our conclusion is that there is a more pronounced inflammatory process in the gingival sites with CMP of DS patients compared to CMP in NP. The findings also indicate that there is a highly activated immune response in CMP of DS patients. PMID- 7552951 TI - Determination of temporomandibular joint fluid concentrations using vitamin B12 as an internal standard. AB - The aim of the investigation was to test the reproducibility and accuracy of a new method to measure temporomandibular joint (TMJ) fluid concentrations of various substances by saline washing, using exogenous B12 as a marker. An in vitro test was first performed with glucose as a test substance. The difference between a B12-calculated and known standard concentration of glucose was very small. Saline washing of the TMJ was performed on 13 patients having signs of TMJ arthritis, and the aspirates obtained were analyzed for neuropeptide Y-like immunoreactivity (NPY-LI) and interleukin-1 beta (IL-1 beta). Vitamin B12 was mixed with the saline immediately before injection, and a sample of the aspirate was later compared photometrically with the injection solution. There were positive correlations between saline aspirate and joint fluid concentrations for NPY-LI and IL-1 beta, and the correlations were stronger for saline aspirates with high joint fluid content. This study shows that the method is reliable for measurement of joint fluid concentrations of various substances, such as NPY-LI and IL-1 beta. PMID- 7552952 TI - Effects of essential fatty acid deficiency on rat molar pulp cells. AB - In rats fed an essential fatty acid deficient (EFAD) diet, either during pregnancy (DN) or for 4 wk postnatally (ND), the cell density in the central part of the pulp increased about two- and threefold, respectively, of that in rats who had received a conventional diet containing sunflower oil. Cells were especially numerous around capillaries. The cell density was also increased twofold in the subodontoblastic layer in the outer part of the pulp, cells being smaller in ND compared with DN. In contrast, the odontoblasts were reduced in height, and the Hohl cells formed a thin layer in EFAD rats. This emphasizes some aspects of pulp specificity which reacted differently from odontoblasts. We suggest that the function of killer cells which normally destroy cells at the periphery of the pulp may be impaired by the diet, leading to cell accumulation. PMID- 7552953 TI - Incremental lines in human dental cementum in relation to age. AB - Counting incremental lines in dental cementum is an accepted method for estimating age in many wild mammals. In human teeth such countings have given variable results, and the aim of the present investigation was to find out if one incremental line is formed per year in human teeth as well. Canines and single rooted premolars were demineralized, paraffin wax-embedded, sectioned and stained, and the sections were viewed in a fluorescence microscope. Non fluorescent lines, seen against a fluorescent background, were either counted directly or their number calculated by counting only some of them and computing this on the total width of the cementum. The correlation coefficient between tooth age and the number of lines for the whole material was = 0.84 when counted and = 0.73 when calculated. This coefficient was stronger in mandibular second premolars but lower in teeth extracted because of dental diseases. The coefficient was only significant in teeth from individuals below the age of 50 yr. The regression formula with tooth age as dependent variable indicated that only incremental lines formed in about every other year stained sufficiently to be counted. PMID- 7552954 TI - Bafilomycin A1 in bone resorption and tooth eruption in dogs. AB - Tooth eruption depends on bone resorption to form an eruption pathway. We have previously shown that a 2-wk local infusion of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPases in osteoclasts, into the crypts of erupting mandibular premolars in dogs blocks bone resorption during this period and eruption of these teeth is delayed for 8 wk. Here we report the limits of inhibition of resorption that still permit eruption of these teeth. In 3 dogs 10(-6) M bafilomycin was delivered by osmotic minipumps early (18 wk) in eruption to the fourth premolar for 1, 3 or 4 wk. Radiographs taken at weekly intervals thereafter showed that bafilomycin delivery for 1 wk delayed eruption for 3 wk, delivery for 3 wk delayed eruption 9 wk and delivery for 4 wk prevented eruption. These data show that tooth eruption is delayed in direct proportion to the time resorption is blocked, and that this process for dog premolars cannot be blocked for more than 3 wk with 10(-6) M bafilomycin without blocking eruption itself. PMID- 7552955 TI - Efficacy of bone morphogenetic protein (BMP) with osteopromotive membranes--an experimental study in rat mandibular defects. AB - The effect of bone morphogenetic protein (BMP) on healing of standardized bone defects was studied with and without the placement of osteopromotive membranes. Two different bovine BMP (bBMP) preparations were tested. These contained primarily collagen as a carrier. Standardized transosseous bone defects, 5 mm in diameter, were created in mandibles of rats. If left untreated, such "critical size defects" never heal during the lifetime of the animal, whereas covering with an osteopromotive membrane is known to cause complete healing of the defects in 6 weeks. The bBMP was implanted in defects and were either covered with an expanded polytetrafluoroethylene (e-PTFE) membrane (GORE-TEX) or were left uncovered. Control defects did not receive any bBMP and were either covered with membrane or were left uncovered. Histological evaluation was made after 12 d and 24 d of healing, respectively. Implantation of bBMP alone was associated with formation of voluminous amounts of new bone, resulting in essentially complete defect healing at 24 d. However, the combination of membrane and bBMP was clearly less effective in stimulating bone healing, being only about as efficient as when using membranes alone. It was concluded that whereas both bBMP preparations were strongly osteoinductive, no further improvement of bone healing was when the membrane technique was supplemented with bBMP, compared to membrane alone. An explanation may be that the presence of an e-PTFE membrane prevents the degradation of the carrier material in the preparations, thus strongly reducing the availability of bBMP. PMID- 7552956 TI - Effect of specimen preparation method on relative volume of the microvasculature- a light microscopic study in rat tongue. AB - The study of corrosion casts by scanning electron microscope is an established means of investigating three-dimensional microvascular architecture. However, concern has been raised that the volume of the vasculature is affected by the perfusion procedure. Ten rats (group A) were standard perfusion fixed, while 10 rats (group B) were perfused with acrylic resin after fixation. Vascular volume was assessed by computer-aided analysis of thin sections of tongue lamina propria. The results showed that vascular volume in lamina propria of rat tongue is 13.6 +/- 4.1% (mean +/- SD). No significant differences in vascular volume were found between group A and group B specimens. It can be concluded that plastic-perfused specimens may be as reliable as histologic sections in obtaining a true image of vascular systems. Thus, corrosion casts are suitable for histometric analysis of the microvasculature. PMID- 7552957 TI - Effects of delmopinol on antimicrobial peroxidase systems and lysozyme in vitro and in human whole saliva. AB - Delmopinol is a new surface-active agent which can reduce plaque formation and gingivitis. This study was aimed to analyze whether delmopinol (0.0032-0.65 mM) interferes with the activity of two surface-active oral antimicrobial enzymes, salivary peroxidase and lysozyme. In addition to human whole saliva (pH 5.0 and 6.0), the experiments were done in 0.1 M phosphate buffer (pH 6.0) with purified lactoperoxidase (LPO) and myeloperoxidase (MPO). LPO and MPO were significantly inhibited in buffer by delmopinol concentrations > 6.5 mM and > or = 3.2 mM, respectively. No such inhibition was found for total peroxidase activity in mixed saliva. In vitro, delmopinol was found to desorb surface-bound peroxidases in an active form to the liquid phase. In further analyses, the possible effect of delmopinol on peroxidase-generated hypothiocyanite (HOSCN/OSCN-) was studied in saliva and buffer. No effect was found in buffer, but salivary HOSCN/OSCN- declined significantly with 6.5 mM delmopinol. This was obviously due to an enhanced decay of hypothiocyanite, rather than its reduced rate of formation. No delmopinol-related inhibition of lysozyme occurred in saliva or buffer. The results suggest that high concentration (6.4 mM -0.2%) of delmopinol may lower the concentrations of antimicrobial HOSCN/OSCN- in saliva but has no effect on human lysozyme. PMID- 7552958 TI - Effect of Lactobacillus rhamnosus strain GG (ATCC 53103) on the growth of Streptococcus sobrinus in vitro. AB - Lactobacillus GG, a recently characterized L. rhamnosus GG strain (ATCC 53103), has been shown to exert inhibitory activity against a variety of bacterial species, including streptococci. We isolated and studied the effect of the inhibitory substance of Lactobacillus GG on some oral streptococci. The inhibitory activity of the isolated substance was weak, but some growth inhibition was observed in Streptococcus sobrinus pretreated with the substance in comparison with untreated controls. Zones of growth inhibition on agar plates were apparent only at pH values below 5, indicating that the inhibitory activity was restricted to a low pH range. Growth curve experiments showed a statistically significant inhibition between series with and without the isolated substance (P < 0.05). The ultrastructure of S. sobrinus was not affected when treated with the inhibitory substance. The Lactobacillus GG itself did not ferment sucrose. The results offer interesting perspectives for future research focusing on the protective function of normal flora and in the attempt to replace harmful bacterial species in oral microflora with less harmful ones. PMID- 7552959 TI - Effect of temperature and duration of post-cure on in vitro wear and quantity of remaining double bonds of resins containing carboxylic anhydride. AB - The present study determined the effect of post-cure temperature and duration on in vitro wear resistance and quantity of remaining double bonds of anhydride containing resins. Temperatures were varied between 37 degrees C and 225 degrees C, and durations were varied between 0 and 24 h. The quantity of remaining double bonds could not be established for post-cure temperatures of 200 degrees C or more due to melting of the polymer. A temperature of approximately 120 degrees C had optimal effect on wear resistance. Quantity of remaining double bonds and wear were found to decrease with increasing duration of post-cure. Low quantities of remaining double bonds were generally associated with low in vitro wear. PMID- 7552960 TI - Mutans streptococci and dental caries prevalence in a group of Latvian preschool children. AB - Paraffin-stimulated saliva samples were collected from 140 children 3- and 4-yr old attending nine nursery schools in Latvia. The salivary levels of mutans streptococci were rated from zero to 3 after being cultured on a commercially available strip selective for these microorganisms. Of the children, 29.3% were rated at zero (approximately < 10(4) cfu per ml saliva). This group of children demonstrated the lowest mean caries prevalence dmfstot = 1.5 (SD 1.9). The highest dmfstot was found among children in class 2 (38.6%; approximately > 10(5) 10(6) cfu/ml) and class 3 (12.1%; approximately > 10(6) cfu/ml) with a mean caries prevalence of 6.5 (SD 5.8) and 6.4 (SD 6.0), respectively. The study demonstrates the association between high caries prevalence and high salivary levels of mutans streptococci in the young child. It is suggested that early identi-fication of mutans streptococci-colonized children might be of value in selecting at caries risk children for preventive measures. PMID- 7552961 TI - Dissection of the anterior abdominal wall and the deep inguinal region from a laparoscopic perspective. AB - The usual dissection by medical students of the anterior abdominal wall and the inguinal region proceeds from superficial to deep; special emphasis is placed on the sheath of the rectus abdominis muscle and lateral muscular layers. We suggest an alternate approach to dissection of this region that has the following advantages: (1) sparing of delicate deep structures not often fully appreciated by students; (2) provision of an opportunity to visualize the region from a laparoscopic surgeon's vantage point; (3) considerably reduced time spent dissecting and identifying structures and relationships, especially peritoneal reflections important in laparoscopic procedures. Our dissection begins with bilateral subcostal incisions through the entire thickness of the anterior abdominal wall and peritoneum, which extend laterally and inferiorly to the level of the anterior superior iliac spines, thereby forming a large, inverted, U shaped flap. This flap is reflected inferiorly, allowing abdominal viscera to be dissected, and ultimately removed en bloc. The flap is then drawn cranially and stretched somewhat to approximate its position when the abdomen is inflated with CO2 during laparoscopic procedures. Major landmarks, including the deep inguinal ring, are noted and the flap is again reflected inferiorly for dissection beginning with the peritoneum and transversalis fascia. This method of dissecting the anterior abdominal wall and inguinal region results in more facile and timely identification of both superficial and deep structures of the anterior abdominal wall and inguinal region, and provides a clinically relevant demonstration of anatomy from a laparoscopic perspective. PMID- 7552962 TI - Sonographic assessment of normal spleen volume. AB - Thirty-two morphologically normal spleens from adult corpses were excised and immersed into a graduated water tank and the water volume displaced was considered as the actual spleen volume. After that, the splenic maximal height, width, and breadth were determined by a pachimeter. All the reference points were marked with a metal clip. Utilizing the metal clip references ultrasound maximal height, width, and breadth were determined. It was assumed that the ultrasound spleen volume was the result of the multiplication of the three ultrasonographic measurements previously obtained. There was no significant difference between pachimeter and ultrasound determinations. The mean actual spleen volume was 147.5 cm3 (SD = 81.46). The mean ultrasound spleen volume was 283.8 cm3 (SD = 168.27). A roughly linear correlation between actual spleen volume (y) and ultrasound spleen volume (x) was found, y = 14.23 + 0.469 x (R2 = 0.94, P < 0.01). PMID- 7552963 TI - The anatomy of left bronchus syndrome. AB - This explanation of the previously described left bronchus syndrome (Ashour et al., 1990, Thorax, 45:210-212) is based on a prospective study of 17 additional cases with unilateral lung destruction. It is likely that the anatomic peculiarities of the left main bronchus predispose the left lung to more frequent bronchial obstruction and hemodynamic changes than the right. Broncho-pulmonary shunt formation and retrograde filling of the pulmonary artery most likely lead to increased oxygen tension and impaired lymph flow in the entire lung, thus spreading tuberculosis in the lung and ultimately leading to left lung destruction. PMID- 7552964 TI - The sympathetic innervation of the eyes and face: a clinicoanatomic review. AB - Most of the details regarding the course of the sympathetic fibers to human ocular structures are based on anatomical and physiological studies in lower animals. While studying a clinical problem involving pericarotid sympathetic fibers, it became obvious that these animal observations cannot adequately explain the findings in human diseases affecting these pathways. An attempt was made, therefore, to clarify this situation. We were able to gather enough information from human clinical and experimental studies, from our own clinical observations, and from our cadaver dissections to conclude that these pathways are somewhat different from those which are usually described in the literature. Based on this information, we conclude that 1) the oculosympathetic fibers in man do not course through the tympanic plexus and/or trigeminal ganglion, and 2) the sweat glands of the face receive their innervation from both internal and external carotid sympathetic plexuses. We also have suggestive, but inconclusive, evidence regarding the final mode of distribution of these fibers to the dilator of the pupil and the smooth muscle portion (deep layer) of the levator palpebrae superioris muscle (superior tarsal muscle). PMID- 7552965 TI - Anatomical aspects of postintubational subglottic stenosis. AB - The subglottic regions of 54 human adult male and female larynges were studied with regard to anatomical aspects of postintubational stenosis. Fourteen specimens were impregnated with curable polymers and cut into 600-800 microns sections along different planes. Forty formalin-fixed hemilarynges were dissected. Measurements of the upper cricoid lamina and the thickness of the endocricoid soft tissues were taken for statistical analysis. Immediately beneath the glottis, the upper part of the cricoid lamina consists of two lateral plates with an average angle of 110 degrees. Distally, the cricoid adopts a more and more rounded lumen. At the level of the cricothyroid joint, the definite airway lumen is always laterally narrowed by a prominent thickening of the endocricoid soft tissue. Large amounts of loose connective tissue facilitate the development of edema in case of injury in this region. Dorsally, the submucous stratum is smaller and consists mainly of dense connective tissue. The blood vessels are fixed to the cricoid perichondrium by collagenous fibers. Any pressure applied from the airway lumen will force the vessels against the nonresilient cartilage, resulting in occlusion and ischemia. These pathophysiologic mechanisms are important for the development of early laryngeal damage during endotracheal intubation, possibly resulting in posterior stenosis due to scarring later on. PMID- 7552966 TI - The hand as a concept: digital differences and their importance. AB - Digits of the hand vary from each other with respect to bone and joint architecture, muscles, tendons, and degree of functional independence. The hand may be conceived as having a central rigid framework moved at the wrist by flexors, extensors, abductors, and adductors. But on each side of this central structure lies a mobile (hinged) segment necessary for pinch and to increase the strength of grasp. At the distal end of this triplex framework lie multijointed appendages essential for any type of pinch or grasp more sophisticated than that of a lobster claw. The thumb has a highly mobile carpometacarpal joint but lacks a middle phalanx. Therefore, it has no proximal interphalangeal (PIP) joint and consequently no mechanical need for a lumbrical. Since the thumb has excellent coordination without a lumbrical, the lumbrical's function as a sense organ cannot be simply to coordinate finger movement. Mechanically the lumbrical is a deflexor of the PIP joint. Because lumbricals are richly endowed with muscle spindles, their passive stretch by contraction of the flexor digitorum profundus might both inhibit finger extensors and facilitate wrist extensors. Since each digit is unique, accurate mathematical and computer modelling of the hand must take into consideration this uniqueness. PMID- 7552967 TI - Anomalous origins of colic arteries. AB - The topography of the celiac trunk and superior and inferior mesenteric arteries was studied by dissection in 27 embalmed cadavers. Variant vascular patterns were noted in four subjects. These consisted of: (1) an accessory right hepatic artery from the superior mesenteric artery, (2) an anomalous middle colic artery from the proximal segment of the splenic artery, and (3) two instances of an accessory left colic artery originating from the superior mesenteric artery. The precarious course of the middle colic artery (coming from the splenic artery) and its dominance in the formation of the marginal artery were thought to predispose the ascending and transverse colon to an increased risk of vascular damage. These cases also illustrate two variant patterns of formation of the marginal artery. In the case of the anomalous middle colic artery, the only contribution of the superior mesenteric artery to the marginal artery was through the anastomosis of its ileocolic branch with the right branch of the aberrant middle colic artery. In subjects with accessory left colic arteries, the superior mesenteric artery played a dominant role in the formation of the marginal artery by contributing the accessory left colic artery, which supplied the splenic flexure and the proximal part of the descending colon. These arterial variations underscore the importance of doing vascular studies prior to major abdominal surgery. PMID- 7552968 TI - The future of gross anatomy teaching. AB - A survey of U.S. departments of anatomy, physiology, and biochemistry shows that 39% of the respondent anatomy departments reported declines in the numbers of graduate students taking the human gross anatomy course. Similarly, 42% of the departments reported decreases in the numbers of graduate students teaching human gross anatomy. These decreases were greater in anatomy than in physiology and in biochemistry. The percentages of departments reporting increases in students taking or teaching their courses was 6% for human gross anatomy and 0% to 19% for physiology and biochemistry courses. To reverse this trend the establishment of specific programs for the training of gross anatomy teachers is advocated. These new teachers will be available as the need for them is increasingly recognized in the future. PMID- 7552969 TI - Three years of experience with a dissection table ventilation system. AB - A dissection table ventilation system that draws air across the cadaver and away from the table top was designed to fit the Shandon-Lipshaw AN-52 dissection table. Each U-shaped unit consists of a pair of hollow collection arms that attach to a collecting manifold at one end. During dissection the manifold is coupled to a central ventilation system through a flexible duct. The air from the table ventilation system is exhausted after passing through a heat recovery system. The unit is raised from the table surface during dissection of the body cavities to increase the efficiency of fume/odor removal. Eight hour exposure data for formaldehyde concentrations are presented. Data were collected from detectors positioned at selected levels above the cadaver during dissection, and above a tray on the table top containing a known volume of 4% formaldehyde or the West Virginia School of Osteopathic Medicine embalming fluid under varying airflow conditions. The results demonstrate that the table ventilation system is effective in reducing exposure to formaldehyde in the dissection laboratory. PMID- 7552970 TI - Curriculum for medical students. PMID- 7552971 TI - Metabolic responses of the critically ill patient. AB - The care of the critically ill patient has become the point of convergence for substrate and energy metabolism, vital organ support, and cell biology. This article includes a brief review of normal metabolism and its endocrine regulation. The metabolic response to injury is reviewed with special attention to insulin resistance. The metabolic response to infection is considered in the light of rapidly emerging information on inflammatory mediators. Finally, brief attention is given to microcirculation and pericellular fluid as sites of metabolic regulation that have the potential for contributing to each of the three converging disciplines. PMID- 7552973 TI - Nutritional assessment in the critically ill. AB - Although many of the measurements and techniques outlined in this article may be epidemiologically useful and correlate with morbidity and mortality, no single indicator is of consistent value in the nutritional assessment of critically ill patients. Measurements such as anthropometrics, total body fat estimation, or delayed hypersensitivity skin testing either are liable to non-nutritional influences or lack accuracy and precision in individual patients. Plasma concentrations of hepatic proteins are affected significantly by the patient's underlying disease state and therapeutic interventions and therefore lack specificity. Although the measurement of these proteins is of little value in the initial nutritional assessment of the critically ill, serial measurement, particularly of plasma pre-albumin, may be useful in monitoring the response to nutritional support. Nitrogen balance is a widely used and valuable nutritional indicator in the critically ill. Direct measurement of urine nitrogen is the preferred test, although nitrogen excretion often is derived from 24-hour urine urea measurement, an inexpensive and easy procedure, but one that is less accurate. More accurate techniques of assessing change in nutritional status, such as IVNAA of total body nitrogen or isotopic measurement of exchangeable potassium or sodium, are more expensive, less available, unsuitable for repeated analyses, and less feasible in severely ill patients. Total body nitrogen measured using IVNAA and total-body potassium, however, are the most accurate ways of measuring body composition in the presence of large amounts of edema fluid. The application of body composition measurements to patient care remains poorly defined because of the many problems encountered with the various techniques, including cost, availability, and radiation exposure. Improved, more sensitive and, preferably, bedside methods for the measurement of body composition are needed. It is of paramount importance that these methods are validated extensively in the critically ill as well as in more stable patients, not only in terms of analytical accuracy, but also to define the point at which altered body composition influences clinical outcome. The biochemical measurement of levels of vitamins, minerals, and trace elements is invaluable in demonstrating specific deficiencies associated with disease and assessing whether long-term nutritional support is adequate. Such measurements rarely are necessary to make the initial clinical decision to give nutritional support, however. The most widely used measures of nutritional state are nitrogen balance and secretory protein concentrations, and these indices improve when sick patients recover.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7552972 TI - Theories and assumptions on energy expenditure. Determinations in the clinical setting. AB - Energy metabolism is defined as the sum of complex and integrated chemical reactions by which the body derives energy from the environment and maintains the proper functioning of all biologic processes. The final common pathway for all these processes is the complete oxidation of carbohydrates and fats and partial oxidation of proteins to carbon dioxide and water. These processes occur primarily in the mitochondria and are coupled to the biochemical reactions of the tricarboxylic acid cycle (better known as the Krebs cycle). PMID- 7552975 TI - Micronutrients in critical illness. AB - Micronutrients play a key role in many of the metabolic processes that promote survival from critical illness. For vitamins, these processes include oxidative phosphorylation, which is altered in the patient with systemic inflammation, and protection against mediators, in particular oxidants. Trace elements are essential for direct antioxidant activity as well as functioning as cofactors for a variety of antioxidant enzymes. Wound healing and immune function also depend on adequate levels of vitamins and trace elements (Table 6). Of extreme importance is the ease with which a deficiency state can develop in the critically ill because of decreased nutrient intakes and increased requirements. Daily intakes up to or exceeding many times the RDA usually are required. The enteral route is preferred, although, if not available, most of these agents can be given by the parenteral route. In that case, however, dose recommendations are less clear. Attention to micronutrients is paramount both in optimizing the nutritional management of the critically ill and in the overall management of these patients. It also is essential in promoting positive outcomes and decreasing complications. PMID- 7552974 TI - Protein and amino acids in nutritional support. AB - This article discusses the role of amino acids in nutritional support during critical illness. The basis for assessing the requirements for protein and amino acids is presented, and the case for enhanced requirement of particular amino acids is discussed. Specific requirements for branched-chain amino acids, glutamine, and arginine are evaluated. PMID- 7552976 TI - Nutritional support during liver failure. AB - Critically ill patients in varying degrees of liver failure are catabolic and consequently require expeditious caloric support. Unique problems in this group of patients essentially revolve around the diagnosis and management of hepatic encephalopathy. From the overview provided in this text, it can be concluded that, only in overt hepatic coma, should all nitrogen products be withheld while precipitating causes are evaluated. Protein should be reintroduced as rapidly as possible to avoid the consequences of protein deprivation. Once the acute intercurrent illness has resolved, the cirrhotic patient returns to baseline energy and protein requirements indistinguishable from the population at large. PMID- 7552978 TI - Nutritional support during pulmonary failure. AB - Malnutrition is both a cause and a result of pulmonary failure. A successful outcome to the treatment of pulmonary failure often hinges on appropriate and aggressive nutritional therapy. Metabolic stresses caused by nutritional therapeutic intervention in the patient with pulmonary failure must be anticipated. PMID- 7552979 TI - Nutritional support of the septic patient. AB - Nutrition and metabolic support is just one of many weapons at the clinician's disposal in the battle against infection. Much has been learned in the past several decades concerning the host's response to infection and the competition among substrates during health and disease. Despite these advances, we still are remarkably ineffective at preventing the muscle wasting that accompanies sepsis and other chronic catabolic conditions. This likely is the result of our lack of understanding concerning the molecular mechanisms responsible for changes in protein metabolism. It is anticipated that the optimal or preferred mixture of glucose, fat, and protein will be better defined in the future, and that new supplements (e.g., MCTs, amino acids, growth factors, antioxidants) will prove beneficial, so the protein-catabolic response to sepsis can be ameliorated and patient outcome improved. Until that time, nutritional interventions should be initiated early in the septic episode and be assessed and altered frequently. Specific guidelines for nutritional support are presented in Table 3. PMID- 7552980 TI - Nutritional support of the burn patient. AB - Nutritional support of the burn patient is essential to optimize host immune defenses and to promote prompt wound healing. The interdependent relationship between metabolism, nutrition, and infection is discussed, followed by an extensive description of the various means of determining the appropriate type, form, and amount of nutritional support to provide to patients of various ages and with differing burn sizes. A concise discussion of the role of various growth factors and micronutrients completes this article. PMID- 7552977 TI - Nutritional support in the patient with renal failure. AB - Nutritional support in the setting of acute renal failure has become an accepted approach to the overall care of this patient population. It is important to understand the various metabolic alterations imposed by the acute uremic state in order to develop the most appropriate nutritional plan for each individual patient. PMID- 7552981 TI - Nutritional management and the multisystem organ failure/systemic inflammatory response syndrome in critically ill preterm neonates. AB - Current information and concepts regarding unique features and practical aspects of metabolism and the nutritional management of critically ill, very low birthweight neonates are reviewed in this article. The use of "gut priming" (early hypocaloric minimal enteral feeding) and parenteral nutrition and their application to the treatment of specific disease states is discussed. The concepts of critical oxygen delivery and multiorgan failure and their impact on nutritional management in adults show striking similarities to metabolic observations of progressively deteriorating, sick neonates. PMID- 7552982 TI - Protecting bone marrow by gene therapy. PMID- 7552983 TI - Genetic delivery of enzymes for cancer therapy. AB - For many years, antibodies have been examined as means to deliver cytotoxic proteins to kill target cells (immunotoxins). More recently, there have been studies on enzymes that convert prodrugs to active drugs to kill target cells. The advances in gene therapy strategies now allow one to deliver the gene for the protein or enzyme as an alternative. This technique, although in its infancy, promises to overcome some of the problems associated with antibody-mediated delivery. Thymidine kinase and cytosine deaminase are some of the enzymes currently being exploited in this way, but more are on the horizon. However, more research is still needed to enable full exploitation of the transcriptional differences between tumour and normal cells so that more existing cancers can be treated in this way. PMID- 7552984 TI - Influence of interleukin-3, interleukin-6, and stem cell factor on retroviral transduction of rhesus monkey CD34+ hematopoietic progenitor cells measured in vitro and in vivo. AB - As a preclinical test for bone marrow gene therapy, we transduced Rhesus monkey CD34+CD11b- hematopoietic progenitor cells with recombinant retroviruses. We investigated the effects of the recombinant hematopoietic growth factors interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on the susceptibility of in vitro clonogenic progenitor cells and in vivo repopulating stem cells to retroviral transduction. IL-6 did not contribute to transduction of progenitor cells, whereas IL-3 and SCF supported expansion and transduction of progenitors. The combination of IL-3 and IL-6 was most efficient at promoting transduction of more mature progenitor cell types. Cultures containing IL-6+SCF yielded optimal maintenance of CD34+CD11b- cells without evidence for lineage restricted maturation. Autologous transplantation of transduced grafts cultured in the presence of SCF, with or without IL-3 or IL-6, into lethally irradiated Rhesus monkeys resulted in a severely delayed hematopoietic reconstitution as compared with grafts transduced in the presence of IL-3 alone. After in vivo repopulation, transduced cells were found among peripheral blood mononuclear cells, granulocytes and CD34+CD11b- progenitor cells in the bone marrow of engrafted animals. However, no significant difference in transduction efficiency on in vivo repopulating stem cells could be demonstrated among the tested growth factor conditions. PMID- 7552985 TI - The constitutive expression of the immunomodulatory gp19k protein in E1-, E3- adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. AB - The immune response against cells infected by gene therapy vectors may be a major hindrance for gene therapy, destroying infected cells thus limiting the length of exogene expression and quickly eliminating infected cells on repeat administration. Adenoviruses and many other pathogens have evolved strategies for escape from immune surveillance, including the gp19k gene, found in the adenovirus E3 region, known to down-regulate major histocompatibility complex class 1 expression on the cell surface, and thus reduce lysis of the infected cells by cytotoxic T cells. We have constructed an adenoviral vector expressing the genes for beta-galactosidase and gp19k both under the control of constitutive promoters, and compared the capacity of lymphocytes from DBA/2 mice previously injected with the virus or with Ad-beta gal, a virus expressing beta galactosidase but not gp19k, to lyse target cells infected with various viruses. Lymphocytes raised against Ad-beta gal fail to lyse target cells infected with Ad beta gal-gp19k significantly, whereas Ad-beta gal infected target cells and a beta-galactosidase expressing cell line are strongly lysed. The administration of Ad-beta gal-gp19k fails to stimulate the proliferation of anti-vector lymphocytes, and thus these lymphocytes show poor cytotoxic activity against Ad beta gal or Ad-beta gal-gp19k infected cells. PMID- 7552986 TI - An efficient procedure to select and recover recombinant adenovirus vectors. AB - Adenoviruses are efficient gene transfer vectors for a variety of cell types. To date, the most widely used methods to construct recombinant adenoviruses involve either in vitro ligation or recombination between one-half of the virus genome, previously cloned in a plasmid vector and engineered to contain the desired expression cassette, and the other half of the virus genome prepared from virions. Although quite effective, these approaches produce viral progeny containing a mixture of recombinant and parental background virus. Thus the recovery of the recombinant virus can be difficult, especially when it grows more slowly than the parental virus. To improve selection and recovery of recombinant adenoviruses, we have constructed an adenovirus vector, AdTG6553, in which the E1 region has been replaced by the thymidine kinase (tk) gene of herpes simplex virus type 1. We show that infection of cells with AdTG6553 in the presence of the nucleoside analog ganciclovir (GCV) prevents viral replication. The conditional lethal phenotype introduced in AdTG6553 makes it a valuable tool to counter-select parental background virus in the presence of GCV and isolate replication-deficient recombinant adenoviruses in which the tk expression cassette has been replaced by a new gene. PMID- 7552987 TI - Retroviral vector with a CMV-IE/HIV-TAR hybrid LTR gives high basal expression levels and is up-regulated by HIV-1 Tat. AB - We have constructed a new retroviral vector by making modifications to the commonly used Moloney murine leukemia virus (MoMLV) based vector in the long terminal repeat (LTR). The changes include replacement of a portion of the U3 region of the MoMLV LTR with a hybrid regulatory element consisting of the human cytomegalovirus immediate-early enhancer/promoter (CMV-IE) together with the human immunodeficiency virus transactivation response element (HIV-TAR). Transfection of chloramphenicol acetyl transferase (CAT) reporter constructs into a variety of human cell lines showed that the hybrid LTR with the CMV-IE/HIV-TAR enhancer/promoter exhibited basal expression levels which were 10- to 50-fold higher than those obtained from the wild-type MoMLV-LTR enhancer/promoter. Expression from the recombinant LTR was further increased in the presence of the HIV-Tat protein, and surprisingly, Tat up-regulated transcription from both the HIV and the MoMLV TATA boxes. In contrast, a MoMLV enhancer/promoter containing only the HIV-TAR element in the LTR did not respond to Tat. When stably transfected into an amphotropic packaging cell line, the modified retroviral vector containing the hybrid LTR plus an extended packaging signal consistently gave higher titres of retrovirus than did the parental MoMLV based vector. Higher basal expression levels which can be further upregulated by Tat, together with more efficient virion production, suggests that the modified vector should be superior for anti-HIV gene therapy applications as well as for other more general applications in human gene therapy. PMID- 7552988 TI - Resistance to taxol chemotherapy produced in mouse marrow cells by safety modified retroviruses containing a human MDR-1 transcription unit. AB - We used an animal model system to transplant lethally-irradiated mice with one million marrow cells which had been: (1) collected from 5-fluorouracil (5-FU) treated mice; and (2) transduced with retroviruses containing a multiple drug resistance-1 (MDR-1) gene transcription unit. Following recovery from the transplant, we exposed these mice to doses of taxol ranging from 7 mg/kg to 30 mg/kg, which corresponds to doses of 68 to 268 mg/m2 in man. The median white blood cell count by 5 days after taxol (expressed as the percentage of the white blood cell count before taxol) was 83% (range 46-100%) in 11 courses of taxol in mice transplanted once with MDR-1 transduced marrow immediately after transplant, whereas the median white blood cell count by 5 days after taxol in mice not transplanted with MDR-1 marrow was 41% in nine courses of taxol (range 11-66%). This difference is statistically different at the P < 0.001 level (Wilcoxon test). One million marrow cells from the MDR-1 transplanted mice were harvested and serially transplanted through five additional cohorts of mice, and tested with taxol after each cohort. The white blood cell count (expressed as the percentage of pre-taxol white blood cell count) after each cohort ranged from 94 146% in the 29 mice transplanted with the transduced MDR-1 marrow, which had been through more than one transplant. This is statistically different from the median white blood cell count recovery after taxol in mice which have no human MDR-1 modified marrow (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7552989 TI - Chemotherapy resistance to taxol in clonogenic progenitor cells following transduction of CD34 selected marrow and peripheral blood cells with a retrovirus that contains the MDR-1 chemotherapy resistance gene. AB - A retrovirus containing the multiple drug resistance (MDR-1) cDNA, was used to transduce cultures of CD34 selected human marrow cells, on stromal monolayers in the presence of hematopoietic growth factors IL-3 and IL-6, following collection from patients recently recovered from chemotherapy-induced myelosuppression. In one experiment, these CD34 selected cells were grown in Dexter cultures for 35 days or more following MDR-1 transduction, and then plated in methylcellulose. Polymerase chain reaction (PCR) analysis of colonies picked after 10-14 days of methylcellulose culture, using a set of primers that are specific for the endogenous or the retrovirally transduced MDR-1, showed that the long-term culture initiating cells (LTCICs) were transduced by the MDR-1 virus. Analysis of the colonies from the CD34 selected MDR-1 transduced cells, with a reverse transcription (RT) PCR assay that could distinguish viral MDR-1 mRNA from endogenous MDR-1 mRNA, showed that the viral MDR-1 mRNA levels were much higher than that of the MDR-1 mRNA from the endogenous MDR-1 gene in the transduced CD34 selected cells. Fluorescence activated cell sorting (FACS) analysis of the CD34 selected transduced marrow cells within 48 h after the transduction, using the C219 and UIC2 monoclonal antibodies for p-glycoprotein, showed that the transduction frequency under these conditions varied from 7 to 20%. Rhodamine efflux studies showed that this additional p-glycoprotein was functional and that the frequency of cells with high p-glycoprotein levels was higher in the transduced cells than in the non-transduced cells. The resistance to taxol of the CD34 selected transduced cells, as judged by the plating efficiency of clonogenic progenitor cells derived from these cells by growth in methylcellulose supplemented with taxol was much higher in the transduced cells than in untransduced cells. In order to test the reproducibility of the transduction frequency of the retroviral supernatants from PA317 MDR-1 viral producer cells on CD34 selected cells, the virus produced from 12 different lots of supernatants from the PA317 MDR-1 producer cell line was used to transduce CD34 selected marrow cells from four different patients, and to transduce the peripheral blood cells of two additional patients collected following chemotherapy-induced myelosuppression. The supernatant lots used for these transduction experiments were tested by Microbiological Associates (Rockville, MD, USA), by the Mus dunni co-cultivation and amplification tests in the S+L-assay and found to be negative for replication-competent retrovirus, and later approved for human use by the Food and Drug Administration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7552991 TI - HLA-DM: an in vivo facilitator of MHC class II peptide loading. PMID- 7552990 TI - Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes. AB - The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors. PMID- 7552992 TI - Mechanistic aspects of NF-kappa B regulation: the emerging role of phosphorylation and proteolysis. PMID- 7552993 TI - A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen induced negative selection. AB - The transcription factor Nur77, an orphan member of the nuclear hormone receptor superfamily, is highly expressed during T cell receptor-signaled apoptosis, suggesting a possible role for Nur77 in negative selection. We examined this by generating two sets of transgenic mice. In one set of mice, a dominant-negative Nur77 mutant is constitutively expressed and the other in which wild-type Nur77 protein is constitutively expressed in developing thymocytes. We report that inhibition of endogenous Nur77 by the dominant-negative mutant perturbed T cell development and inhibited antigen-induced negative selection in F5T cell receptor transgenic mice. Constitutive expression of wild-type Nur77 protein induced apoptosis in developing thymocytes, resulting in a decreased number of thymocytes and mature T cells. Together, these data support a role for Nur77 in the downstream signaling events in antigen-induced negative selection. PMID- 7552994 TI - Defective B cell development and function in Btk-deficient mice. AB - Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2 deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development. PMID- 7552995 TI - Impaired expansion of mouse B cell progenitors lacking Btk. AB - Mutations in the gene encoding the protein tyrosine kinase Btk are associated with the human B cell immunodeficiency X-linked agammaglobulinemia (XLA). In the mouse, a point mutation in the Btk pleckstrin homology domain segregates with a milder X-linked immunodeficiency (xid). To assess the importance of Btk function in murine lymphopoiesis, we generated multiple embryonic stem cell clones bearing a targeted disruption of the btk gene and examined their potential to produce lymphocytes in both C57BL/6 and RAG2-/- host chimeric animals. These mice provide a complementary set of in vivo competition assays that formally establish the genetic basis for the xid phenotype. Although the null mutation yields a phenotype quite similar to that of xid, it also compromises expansion of B cell precursors. Our results suggest that the murine and human consequences of Btk deficiency differ only quantitatively, and represent the same disease process. PMID- 7552996 TI - Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. AB - To investigate the role of the receptor-type tyrosine kinase, c-kit and its ligand, stem cell factor (SCF) in T cell development, we analyzed c-kit (W/W) and SCF (SI/SI) deficient mice. We also engrafted wild-type or SCF-deficient fetal thymi onto wild-type recipient mice and analyzed the rate of proliferation by in vivo bromodeoxyuridine labeling. The results show that the most immature thymocyte compartment defined as CD3-CD4-CD8- is significantly reduced in SI/SI grafts and W/W thymi compared with wild-type counterparts. Also, the expansion rate of these immature thymocytes in SI/SI graft is reduced by -50%. These experiments provide direct evidence for an important role for c-kit-SCF interactions in expansion of very early thymocytes. PMID- 7552997 TI - A functionally compromised intermediate in extrathymic CD8+ T cell deletion. AB - We have established a model system for analyzing the induction of self-tolerance among mature peripheral T cells in V beta 5 TCR Tg mice. Both CD4+V beta 5+ and CD8+ V beta 5+ cells undergo a superantigen-driven chronic deletion in the periphery of I-E mice. Prior to their disappearance, CD4+ transgene-expressing cells are activated and then rendered anergic to further stimulation through their TCRs. This scenario differs strikingly in the CD8+ cellular compartment, which is characterized by a distinct population of CD8loV beta 5lo cells localized to the blood and spleen. CD8lo cells are small, express the surface phenotype of memory cells, and rapidly incorporate BrdU in vivo. The kinetics of their appearance and disappearance in adult thymectomized mice, the rapid chasing of BrdU from labeled cells, and their in vivo cortisone sensitivity all suggest CD8lo cells are slated for deletion. Furthermore, their functional incompetence can be documented in vitro in the absence of internucleosomal DNA fragmentation. Thus, we have identified an intermediate population of T cells targeted for peripheral deletion that, although functionally compromised, has not yet undergone programmed cell death. PMID- 7552998 TI - Role of B cell receptor Ig alpha and Ig beta subunits in MHC class II-restricted antigen presentation. AB - The ability of the B cell antigen receptors (BCRs) to enhance MHC class II restricted antigen presentation was ascribed to mig-associated Ig alpha/Ig beta heterodimers. The relative role of Ig alpha and Ig beta subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Ig alpha and Ig beta chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Ig alpha targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Ig beta targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response. PMID- 7553000 TI - Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development. AB - Invariant chain (Ii)-negative mice exhibit defects in MHC class II assembly and transport that results in reduced levels of surface class II, altered antigen presentation, and inefficient positive selection of CD4+ T cells. Many CD4+ T cells that do mature in Ii-negative mice express a cell surface phenotype consistent with aberrant positive selection or peripheral activation. Reconstitution of these mice with low levels of either the p31 or p41 form of Ii does not restore transport of the bulk of class II or class II surface expression, but surprisingly does restore positive selection as measured by numbers and surface phenotype of CD4+ T cells. Thus, an Ii-dependent process, independent of effects on class II surface density, appears to be required for normal positive selection of CD4+ T cells. PMID- 7552999 TI - The cytoplasmic and transmembrane domains of MHC class II beta chains deliver distinct signals required for MHC class II-mediated B cell activation. AB - Class II-mediated signals play potential roles in B cell activation and antigen presentation. The regions of the class II molecule participating in B cell signaling are incompletely defined. Our prior analysis of structural requirements of the cytoplasmic domain of A beta revealed that only the eight membrane proximal residues are required for signaling. Here, we report that the sequence and position of two of these are critical, and present direct evidence that the A beta transmembrane domain is also involved in signaling, via a pathway distinct from the cytoplasmic domain. These results demonstrate that specific regions in both the cytoplasmic and transmembrane domains of the class II molecule have distinct signaling functions. PMID- 7553001 TI - Reconstitution of invariant chain function in transgenic mice in vivo by individual p31 and p41 isoforms. AB - MHC class II molecules associate with invariant chain (li) during biosynthesis. If facilitates folding of class II molecules, interferes with their association with peptides, and is involved in their transport. The murine Ii gene encodes two chains, p31 and p41. The role of these isoforms has been studied in vitro only in inappropriate antigen-presenting cells. To circumvent this problem, we have generated invariant chain-deficient mice (delta Ii), which express exclusively the p31 and p41 isoforms. Low level expression of p31 or p41 is not sufficient for rescuing high levels of cell surface class II expression. However, low levels of the typical compact dimer conformation indicative of tight peptide binding are observed. Thus, both isoforms participate in class II folding and assembly. Furthermore, p31 and p41 retrieve the CD4+ T cell population, which is reduced in the (delta Ii) mice. Moreover, the immune response to protein antigen is restored by both isoforms. PMID- 7553002 TI - Major histocompatibility class II peptide occupancy, antigen presentation, and CD4+ T cell function in mice lacking the p41 isoform of invariant chain. AB - We used a "hit and run" gene targeting strategy to generate mice expressing only the p31 isoform of the conserved invariant (Ii) chain associated with major histocompatibility complex (MHC) class II molecules. Spleen cells from these mice appear indistinguishable from wild type with respect to class II subunit assembly, transport, peptide acquisition, surface expression, and the ability to present intact protein antigens. Moreover, these mutant mice have normal numbers of thymic and peripheral CD4+ T cells, and intact CD4+ T-dependent proliferative responses towards a soluble antigen. In short, MHC class II expression and function are surprisingly unaffected in mice lacking p41 invariant chain, implying that the p31 and p41 isoforms may be functionally redundant in the intact animal. PMID- 7553004 TI - Graft-bone healing of a meniscus autograft anchored in bone tunnels. AB - This experiment was designed to histologically study the attachment process between a meniscus autograft and bone when the meniscus autograft is placed within bone at its anterior and posterior horns. In order to provide a time sequenced histologic picture, a canine model was used. The study group consisted of eleven mature canines that underwent complete medial meniscectomy of the stifle joint followed by immediate replacement using an autogenous tissue meniscal scaffold fashioned from the lateral fascia of the thigh. The graft was fixed in bone tunnels at the anterior and posterior horns. One animal was sacrificed immediately after implantation, one at 10 days, three at 28 days, two at 3 months, two at 6 months, and two at nine months. Photographs were taken and histologic sections through bone tunnels were analyzed. The graft consisted of rolled tensor fascia and exogenous clot. RESULTS: The attachment process begins with the invasion of an interface of well vascularized, primitive mesenchymal cells between the soft-tissue graft and the host bone. Histologic evidence of collagenous attachment to bone is visible at one month after graft implantation. Collagen organization with increasing soft tissue attachment to bone is progressive throughout the nine months time frame of the study. The attachment points become increasingly more continuous from three to six months. By six months, the attachment points are confluent and continuous throughout the depths of the tunnel. PMID- 7553003 TI - Synovial reaction and concentrations of proteoglycan fragments in joint fluid after intraarticular knee injuries. AB - In patients with pathology to cruciate ligaments, menisci and cartilage and in some patients with symptomatic knees without gross intraarticular changes, proteoglycan fragment concentrations in joint fluid and inflammatory reaction of the synovium were related to the cartilage condition. There was a weak positive correlation between increased concentrations of proteoglycan fragments in joint fluid and the degree of cartilage degeneration, indicating higher concentrations in knees with more advanced cartilage destruction, but there was no correlation to the synovial reaction at histological inspection. However, in most knees the synovial tissue was infiltrated by inflammatory cells. Symptomatic knees free from gross intraarticular pathology showed similar synovial changes and concentrations of proteoglycan fragments in joint fluid as knees with intraarticular pathology and obvious cartilage degeneration. The complex process of proteoglycan synthesis, release, degradation, and clearance from joint fluid apparently caused the weak or absent correlations between this 'marker' protein and the degree of synovitis and arthrosis stage. PMID- 7553005 TI - Arthroscopic synovectomy of synovial tuberculosis of the knee joint. AB - Synovial tuberculosis is a rare, but important differential diagnosis of synovial diseases. Three cases of arthroscopic synovectomy of tubercular monarthritis affecting the knee joint are reported. The Lysholm scoring scale modified by Klein and Kloos was excellent in two cases, but poor in one. In all three cases there was an excellent improvement in the range of motion compared to the preoperative period and no deterioration of the Larsen score in the follow-up period (25-56 months postoperatively). Considering these results arthroscopic synovectomy can be recommended as an alternative to the open procedure of synovectomy. PMID- 7553006 TI - The value of one-portal endoscopic carpal tunnel release: a prospective randomized study. AB - A randomized prospective study was carried out to compare one-portal endoscopic carpal tunnel release with an open procedure. There were 47 patients (mean age 52.6 years); 25 underwent an endoscopic and 22 an open release. The aim of the study was to evaluate the risks against the benefits for pain, grip, key-pinch strength and ability to return to work. The distribution of age, occupation, sex, neurographic findings and operated hand was similar in both groups. We detected no serious nerve complications. One "open" patient developed a hypertrophic scar, a second "open" patient a disabling reflex sympathetic dystrophy, one "endo" patient a transient neurapraxia. The remaining patients experienced complete relief of symptoms. Improvement of grip strength is significantly better after endoscopic release (P = 0.0001 at 3 months). In contrast, the key-pinch showed a similar pattern of improvement in both groups. The ability to use the operated hand as effectively as the contralateral one developed after 24 days for the endoscopic group versus 42 for the open approach (P = 0.0000). The carpal arch alteration was less important for the endoscopic group (P = 0.013), but without any correlation with the grip strength. Agee's one-portal technique only allows correct placement of a knife, not an inspection of the structures being operated upon. This is a major limitation, reducing the surgeon to a technician. Further development of this procedure demands a device that will enable a fruitful inspection of the carpal tunnel. PMID- 7553007 TI - The Bankart lesion of the shoulder: a biomechanical analysis following repair. AB - The Bankart lesion of the shoulder has long been associated with anterior instability. Our laboratory has developed a biomechanical model of the human shoulder which was used to determine the effects of creating a Bankart lesion on cadaveric specimens and then to compare the effects of two repair techniques. The model simulates the abducted, externally rotated position of the glenohumeral joint and uses pneumatic cylinders to simulate the rotator cuff forces. Specimens were tested intact following a partial Bankart lesion, following a complete Bankart lesion, and after performing a Bankart repair using three Mitek suture anchors. Finally, both the traditional and Mitek repairs were tested until failure. Strain in the inferior glenohumeral ligament (IGHL) and torque resistance was measured as an indication of instability of the joint. Strain was noted to decrease with increasing depth of lesion of the IGHL. Torsional rigidity of the shoulder decreased with increasing depth of lesion as well. Repairing the shoulder restores the strain and rigidity to control conditions. The mean load until failure was greater with the traditional repair than with the suture anchor technique. This study quantitates the effects of a Bankart lesion of the shoulder, and demonstrates that repairing the lesion with a suture anchor technique restores the biomechanics of the shoulder. PMID- 7553008 TI - An electromyographic study of arm muscles during climbing. AB - Upper extremity muscle injuries from rock climbing are common. Knowledge of the activity of specific muscles during climbing may allow the development of training programs to reduce these injuries. This study evaluated the electrical activity of the first interosseous (IN), brachioradialis (BR), flexor digitorum superficialis (FD), and biceps brachii (BB) muscles in seven climbers by integrated electromyography (IEMG) during finger-tip pull-ups. The climbers, with forearms pronated, performed three consecutive pull-ups. Each pull-up consisted of: (1) hanging using four fingers of each hand, (2) pull-up to maximum elbow flexion, (3) slow return to starting position. The IEMG during maximum voluntary contraction (MVC) was obtained for each muscle separately, and the IEMG was normalized to MVC. During hanging, FD showed the highest normalized IEMG (0.64 +/ 0.20). During pull-up, the highest IEMG was produced by FD (0.69 +/- 0.25) and BR (0.67 +/- 0.19), while BB showed only 0.33 +/- 0.12 and IN 0.09 +/- 0.06. During lowering, FD again had the highest IEMG (0.74 +/- 0.24), while the EMG from BR was decreased to 0.42 +/- 0.14 and BB to 0.15 +/- 0.15. BR and BB showed an abrupt peak in EMG during pull-up and lowering, as opposed to FD which remained constantly highly activated, which suggests that FD does not contribute to elbow flexion even though it crosses the elbow joint. The high activation of FD and BR may explain their elevated incidence of injury during climbing. Thus, a reduction in climbing-related muscle injuries may be achieved by a training program that emphasizes conditioning of the BR and FD muscles. PMID- 7553009 TI - Osteoid osteoma in the differential diagnosis of persistent joint pain. AB - We report 20 cases (13 male and 7 female), mean age 21 years) of juxta-articular osteoid osteoma. The distribution of affected joints was as follows: hip joint (7 cases), knee joint (2 cases), ankle joint (2 cases); iliosacral joint (2 cases), lumbar spine (2 cases), carpus (2 cases), shoulder (1 case), second metacarpal (MCP; 1 case) and first metatarsal (MTP; 1 case). The duration between the onset of symptoms and diagnosis varied from 8 months to approximately 4 years. In juxta articular osteoid osteoma, the clinical picture and the radiographic findings are often atypical, and this may lead to misdiagnosis and delayed definitive treatment. In young patients with persistent undiagnosed pain, the possibility of an osteoid osteoma should be considered. When the clinical picture is suggestive but radiological findings are negative, we must proceed to further investigation with bone scintigraphy and computed tomography. These examinations should be repeated 1 year after the onset of symptoms because initially negative findings may become positive at a later date. When the diagnosis of an osteoid osteoma is confirmed, surgical excision leads to complete relief of the symptoms. PMID- 7553010 TI - Untreated acute anterior cruciate ligament tears of the knee: progression and the influence of associated injuries. AB - Arthroscopy was performed on 53 consecutive patients with acutely torn ACL within two weeks after the initial injury. Arthrography was performed on 42 of the 53 patients prior to arthroscopy. Four Segond's fractures, twenty-six meniscus tears (eight medial, eighteen lateral) and nineteen medial collateral ligament (MCL) ruptures were revealed. One medial meniscectomy was performed under arthroscopy, and ten MCL tears were repaired in the acute phase. No surgical procedure was done on the torn ACL, but three excisions of ACL stubs were performed due to a restriction of full extension. The patients were re-examined clinically and radiographically after a period of from 3 years 7 months to 11 years 1 month, averaging 6 years 2 months. Four late meniscectomies were performed on the medial menisci in the chronic phase. Anterior translations of the tibia and knee rating scores of the patients with MCL tears and with Segond's fractures did not differ from those of the others. Anterior translations of the tibia in patients with medial meniscectomies increased (P < 0.05), and their functional scores significantly decreased (P < 0.01) more than those of the other patients. Patients with repaired MCL tears showed significantly higher functional scores than those of the patients with unrepaired MCL ruptures (P < 0.05). PMID- 7553011 TI - Medium-term results of 173 ligamentoplasties of the anterior cruciate ligament using the MacIntosh technique reinforced by the Kennedy ligament augmentation device (LAD). AB - This is a retrospective study of 173 cases of chronic laxity of the knee, surgically treated between May 1985 and December 1988 using the MacIntosh technique (quadriceps-plasty) reinforced with the Kennedy ligament augmentation device (LAD). It involved 171 operations, 113 men and 58 women aged between 15 and 49 years (average 26 years), the majority of whom were active in sports both at competition (51 cases) and at recreational level (119 cases). There were 101 lesions of the medial meniscus (58.4%) and 94 lesions of the lateral meniscus (54.4%); only 41 knees had no meniscal lesion (23.6%). The results at medium term (between 4 and 8 years follow-up) were based on 107 cases (61.8%). The tolerance of the reinforcement was excellent as there were no instances of either acute or chronic synovitis in this series. The anatomical results evaluated using the Lachmann test (maximum manual Lachmann) showed no differential in 24 cases (22.8%), a differential of between 0 and 2 mm in 54 cases (50.5%), of between 2 and 4 mm in 20 cases (19%) and of > 4 mm in 9 cases (8.7%). The functional results evaluated using the Arpege CLAS system showed 83% of results to be satisfactory (score > 23). The radiological results showed that 63 knees (58.9%) had no subclinical or clinical signs of medial or lateral femorotibial osteoarthritis. In total, with an average follow-up of > 5 years (4-8 years), it was found that the MacIntosh quadriceps-plasty reinforced with the Kennedy LAD was not accompanied by any iatrogenic disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553012 TI - Long-term results of the Leeds-Keio anterior cruciate ligament reconstruction. AB - This paper discusses the long-term results of the anterior cruciate ligament (ACL) reconstruction with the Leeds-Keio (LK) prosthetic ligament. For this type of reconstruction we used arthrotomy and an arthroscopy-assisted technique. The fixation was obtained with two bone plugs, and the distal portion was also attached with a staple. A postoperative protocol was used with a progressive range of motion and weight bearing after 50 days. We performed 50 LK operations in professional and amateur athletes aged 17-39 years with an isolated anterior instability. We reviewed at follow-up (5-7 years) 37 patients; 8 were lost, and 5 had a subsequent failure. At the Lysholm score the patients were classified: 19 excellent, 13 good, 3 fair, and 2 poor. At the IKDC grading the patients were classified as follows: 2 class A, 22 B, 8 C, and 5 D. The Lachmann test was 1+ in 15 patients, 2+ in 7, 3+ in 2, and negative in 13; pivot shift was 1+ in 9, 2+ in 7, 3+ in 2, and negative in 25. Results of the KT 1000 test at 30 lb side to side was < 3 mm in 23 patients, 3-5 mm in 6, 6-10 mm in 6, and > 10 mm in 2. In view of the results observed and the progressive deterioration over the years, this procedure should no longer be performed as an ACL substitute. PMID- 7553013 TI - Post-operative problems following anterior cruciate ligament reconstruction. AB - Seventy adult patients were studied during the postoperative rehabilitation period following anterior cruciate ligament reconstruction in order to investigate the role of pre-, intra-, and postoperative factors in range of motion and graft problems. A standard bone-patellar tendon-bone autograft was used for the reconstruction. Pre- and intraoperative factors such as concomitant injuries, time from injury to surgery, age, sex, and tunnel placement were recorded. Tunnel placement was recorded on intraoperative radiographs of the final guide pin placement and compared to pin placement on cadaver knees. The results indicated a significant relation between early reconstruction (< 1 month) following the injury and range of motion problems during the early rehabilitation period (P < 0.001). This relation disappeared by the end of the first postoperative year. Prolonged surgery was also associated with early motion problems (P < 0.05). Graft laxity or failure was correlated with an earlier return of range of motion (P < 0.05). We hypothesized that graft failure can have a biologic cause rather than a mechanical one since intraoperative X-rays indicated a near-anatomic tunnel placement in this patient group when compared to ideal placement in the cadaver knees. PMID- 7553015 TI - An evaluation of knee extensor and knee flexor torques and EMGs in patients with patellofemoral pain syndrome in comparison with matched controls. AB - The relationship between concentric and eccentric isokinetic torques and EMGs of quadriceps and hamstring muscles in patients with unilateral patellofemoral pain was studied in 27 patients (13 males, 14 females). The patients and a group of controls matched for age, gender, and physical activity were tested on a Kin-Com dynamometer at 60 degrees/s and 180 degrees/s angular velocity. EMGs were recorded for eight of the patients and their matched controls. In addition, the reproducibility of isokinetic measurements made under the same conditions but on different occasions in patients with patellofemoral pain was evaluated. Twenty one patients (11 males, 10 females) underwent testing of their quadriceps and hamstring muscles two or three times on a Kin-Com dynamometer. This was performed both concentrically and eccentrically in their painful leg while the patients evaluated their knee pain using Borg's pain scale. The data show that the patients had a significantly lower agonist as well as antagonist EMG activity during knee extension measurements in their painful leg compared with the controls. However, there were no differences in either agonist or antagonist EMG activities during knee flexion measurements between the patients and the controls. The quadriceps muscle torque was considerably weaker in the patients' painful leg compared with both their asymptomatic leg and with the controls. Peak torque for knee extension was reached at a mean of 66 degrees of knee flexion for both patients and controls. However, the patients showed a considerably wider range within which they produced their peak torque in their painful leg than in their asymptomatic and also in comparison with the controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553014 TI - Results of surgical treatment of arthrofibrosis after ACL reconstruction. AB - We prospectively studied 31 knee arthrolyses performed for loss of motion after intra-articular anterior cruciate ligament (ACL) reconstruction. The arthrolysis was performed on average 10.6 months after the reconstruction (range 4-25). Seven knees were localized forms. They were treated with arthroscopic removal of a fibrous nodule and scar tissue anterior to the ACL, which was preserved. Twenty four knees were global forms and treated arthroscopically (14) or in open procedure (10). Suprapatellar, medial, and lateral gutter adhesions were sectioned, and fibrous tissue was removed from the anterior compartment. A posteromedial and/or posterolateral capsulotomy was necessary in 7 knees. The ACL graft was nonfunctional and/or malpositioned in 19 knees. The results were evaluated with the IKDC form with an average follow-up of 3.5 years (range 1.5 7). Preoperatively the localized forms had an average extension loss of 11 degrees and an average flexion loss of 14 degrees compared to the opposite knee. At follow-up all the knees were satisfactory for symptoms. All except one achieved a satisfactory motion (within 5 degrees of extension loss and 15 degrees of flexion loss) and a satisfactory final result. Global forms had a greater preoperative flexion loss (average 34 degrees) and extension loss (average 17 degrees). At follow-up 58% were satisfactory for symptoms and 71% for arc of motion. However, the final result was satisfactory in only 37%. In conclusion, local forms have a good prognosis. In global forms motion may be improved by surgery, but the final result is downgraded by symptoms. Arthrolyses performed within 8 months from index operation had a better outcome. PMID- 7553017 TI - Technical considerations for MR imaging of the hand and wrist. AB - The important technical factors involved in the MR imaging of the hand and wrist are reviewed. The magnet, gradient system, and radiofrequency coils are discussed especially with regard to obtaining small fields of view. The properties of the various pulse sequences including spin echo, fast spin echo, and gradient echo are reviewed, and their uses and potential problems are described. PMID- 7553016 TI - Patellar tendonitis: clinical and literature review. PMID- 7553020 TI - MR imaging of normal and injured wrist ligaments. AB - MR imaging is a rapidly evolving modality for examining the ligaments of the wrist. Recent improvements in magnet design and receiver coil technology have allowed consistent visualization of the wrist ligaments. Image quality can be optimized by selection of the proper receiver coil, careful attention to patient comfort and positioning, and selection of a dedicated imaging protocol. The carpal ligaments can be classified as capsular or interosseous ligaments, with the most important interosseous ligaments being the SLL and LTL as well as the triangular fibrocartilage. It is important for radiologists to understand the anatomy of the carpal ligaments in addition to the normal range of MR appearances as basis for comparison with torn or degenerated carpal ligaments. PMID- 7553018 TI - Normal anatomy of the hand and wrist. AB - An understanding of the anatomy is invaluable when such small structures as described here are to be evaluated. Because MR imaging reveals greater detail, radiologists are obliged to use the information available to benefit the clinician and the patient. A working knowledge of the anatomic elements and their nomenclature also engenders confidence in the imager on the part of the hand surgeon. This discussion serves as the basis for meaningful comprehension of the pathologic entities that are presented in the articles that follow. PMID- 7553019 TI - MR imaging of the triangular fibrocartilage complex. AB - This article describes in detail the anatomic components of normal triangular fibrocartilage complex (TFCC) and its MR appearance on high resolution MR images. It also describes pathologic processes of TFCC and its MR appearance. PMID- 7553022 TI - Synovial lesions of the hand and wrist. AB - Synovial tissue in the hand and wrist is frequently involved by inflammatory, degenerative, infectious, traumatic, metabolic, or neoplastic lesions. Although normal synovium is not shown on MR images, the morphology and signal characteristics of abnormal synovium is generally well demonstrated. Special MR techniques for accurate quantification of pannus are under investigation. PMID- 7553021 TI - Carpal tunnel syndrome. MR imaging diagnosis. AB - Carpal tunnel syndrome is a common condition that is often diagnosed by careful history and physical examination. Symptoms from cervical disc disease, thoracic outlet syndrome, and more proximal entrapment syndromes of the median nerve may be confused clinically with carpal tunnel syndrome. Incision of the flexor retinaculum in these patients will not relieve the symptoms, because the locus of the entrapment is not in the carpal tunnel. Electrophysiologic studies are invasive, painful, and may be equivocal on occasion. Furthermore, they provide little information into the cause of carpal tunnel syndrome. MR imaging is the best modality to image the carpal tunnel. It can define the locus of entrapment to the carpal tunnel. Findings includes swelling of the median nerve just proximal to the carpal tunnel, flattening of the nerve within the carpal tunnel, bowing of the flexor retinaculum, and increased signal intensity of the median nerve. Etiologic findings can differentiate space occupying lesions from diffuse inflammatory causes, and this may aid in management. Also, the signal characteristics of soft-tissue masses may be diagnostic. Knowledge of the course of the median nerve may be helpful when planning corticosteroid injection or surgery, especially with the endoscopic technique. MR imaging also may serve a role in postoperative evaluation of patients with recurrent symptoms by demonstrating an incomplete release of the flexor retinaculum or healing of an incised retinaculum. These unique abilities of MR imaging makes it a useful diagnostic tool not only for the initial evaluation and management but also in the postoperative evaluation of patients with carpal tunnel syndrome. PMID- 7553023 TI - Avascular necrosis of carpal bones. AB - MR imaging is extremely well suited to detection of early phases of avascular necrosis, permitting diagnosis before collapse of the carpal bones has occurred. The sensitivity of this imaging modality allows differentiation of subtle changes in the bone marrow signal. This is used as criteria for a new stage classification of Kienbock's disease based on MR imaging appearance. The prognosis of scaphoid fractures and estimation of likelihood of avascular necrosis of the proximal fragment can be inferred by using gadolinium enhancement to evaluate the bone marrow vascularity. PMID- 7553024 TI - MR diagnosis of tenosynovitis about the wrist. AB - Syndromes of tenosynovitis at the wrist possess a typical appearance at MR imaging. MR provides a useful adjunct to clinical evaluation with regard to differentiation from joint or bone-related entities, allows anatomic localization and determination of disease extent, and provides a means of assessing the effectiveness of conservative treatment. PMID- 7553025 TI - MR imaging of the metacarpophalangeal joints. AB - The metacarpophalangeal joint complex represents an articulation not only between the metacarpal and proximal phalanx but in the case of the thumb also between the metacarpal and sesamoids. The collateral ligaments and volar plate contribute significantly to the stability of the metacarpophalangeal joints. Detailed knowledge of the anatomy of the articulations and soft-tissue supports of this complex is necessary for MR imaging detection of subtle injuries to these structures. The role of MR imaging in both acute and chronic trauma to the collateral ligaments and in the often difficult diagnosis of chronic thumb sesamoiditis is presented, with illustrative examples of the normal and pathologic conditions of metacarpophalangeal joints. PMID- 7553026 TI - MR imaging of musculoskeletal tumors of the hand and wrist. AB - Musculoskeletal tumors of the hand and wrist are relatively uncommon. Yet, despite their overall rarity, certain bone and soft-tissue lesions have a predilection for this location. This article reviews the common musculoskeletal tumors of the hand and wrist. It is not intended as a comprehensive review but as an overview, emphasizing the use of MR imaging in the evaluation of these lesions and highlighting those diagnoses that may be suggested by MR imaging. PMID- 7553027 TI - MR angiography of the hand and wrist. AB - MR angiographic techniques that visualize the arterial anatomy of the hand and wrist currently are available. Further refinements are necessary to ensure that diagnostic studies are routine. In my experience, the most detailed anatomy is obtained with gadolinium-enhanced 3-D sequences. The specific role of MR angiography of the hand and wrist has yet to be defined. Currently, it is used in my practice as a screen for certain vascular disorders and in those patients in whom the risk/benefit considerations of conventional arteriography are deemed unacceptable to the referring surgeon. Future applications may include a quantitative component. This information could prove beneficial in the serial follow up of patients with vasospastic disorders that require intermittent vasodilator therapy. It is hoped that quantitative information might provide a means to direct therapy prior to the onset of debilitating symptoms. Clinical efficacy must be established with comparative studies to the reference standard of conventional angiography. If reliable MR angiography can be obtained with sufficient information for clinical decision making, it is likely that it will have a cost and safety advantage over conventional angiography. PMID- 7553029 TI - [Role of antigen specific T-cell in collagen diseases]. PMID- 7553028 TI - Clinical applications of MR imaging in hand and wrist surgery. AB - MR imaging is emerging as an invaluable tool in the diagnosis of hand and wrist disorders. It is extremely accurate in the diagnosis of stage 1 Kienbock's disease when plain radiographs are equivocal and in evaluating bone and soft tissue tumors of the hand and wrist. MR imaging is replacing arthrography as the imaging modality of choice for disruptions of the TFCC. With refined technology and improved resolution, abnormalities involving the intercarpal ligaments ultimately will be able to be detected with a high degree of precision. PMID- 7553030 TI - [Occurrence of malignancy in patients with biopsy-proven temporal arteritis]. AB - A woman of 79 years was admitted to our hospital because of headache and high erythrocyte sedimentation rate (ESR). Temporal artery biopsy demonstrated giant cell arteritis and nonsteroidal antiinflammatory drugs were effective. Another woman of 69 years was admitted because of headache, high ESR, and polymyalgia. Temporal arteritis (TA) with polymyalgia rheumatica (PMR) was established in biopsy of the temporal artery, and steroid therapy was effective. In our hospital, a third patient of 81 years was also suffering from TA with PMR, but temporal artery biopsy was not performed. Those three cases were followed for several years after the diagnosis of TA. Two years later, cancer in cervix of uterus was diagnosed in first case, and acute myelogenous leukemia in second case. However, such symptoms were not observed in third case. These findings and recently described reports suggest that the patients with biopsy-proven TA may have an increased risk of developing malignancy. PMID- 7553031 TI - [Clinical course of a HIV-seropositive patient with thrombocytopenia and hypergammaglobulinemia who was initially screened by annual complete physical examination]. AB - A 57-year-old man was admitted because of hypergammaglobulinemia which was initially pointed out by annual complete physical examination. No significant abnormal findings were observed except polyclonal hypergammaglobulinemia at that time (IgG; 2,662 mg/dl, IgA; 422 mg/dl). Seven months later, he has progressed to show thrombocytopenia. The laboratory data showing the reduction of peripheral CD 4-positive T cells (CD 4; 0.2 x 10(9)/l, CD 4/8 ratio; 0.19) and positive serum HIV antibody revealed that his hypergammaglobulinemia and thrombocytopenia were resulted from HIV infection. The peripheral platelet count decreased to 28 x 10(9)/l at minimal point, however, it recovered to 160 x 10(9)/l within 7 months without treatment. His good performance status has been still maintained over 4 years. His whole clinical course suggests that there is no significant correlation between peripheral platelet counts, serum gammaglobulin levels or the amounts of PAIgG in this case. This case proposes that we should also consider a possibility of HIV infection for the patients showing hypergammaglobulinemia. PMID- 7553032 TI - [A case of rheumatoid arthritis complicated with salazosulfapyridine-induced aplastic anemia]. AB - A 64 year-old woman with rheumatoid arthritis was admitted to our hospital because of general fatigue. She noticed polyarthralgia 8 months prior to the admission and diagnosis of rheumatoid arthritis was made. Administration of salazosulfapyridine (1.0 g/day) was started 6 months before the admission. Complete remission of rheumatoid arthritis was obtained in 2 months and blood cell counts showed normal values at that time. The results of laboratory tests included hemoglobin; 8.6 g/dl, white blood cell count; 1,900/mm3 with a differential of 19% neutrophils, 77% lymphocytes, and 4% monocytes; platelets were 21,000/mm3. A bone marrow aspiration and biopsy were performed. There were reduced numbers of myelocytes, erythroblasts, and megakaryocytes indicating aplastic anemia. Salazosulfapyridine was discontinued. Prednisolone, anabolic steroid and granulocyte-colony stimulating factor (G-CSF) were administrated. In spite of these therapy, recovery of peripheral blood cell counts have not been observed and supporting therapy including red cell and platelets transfusion have been continued. To our knowledge, this is the first case report which describes occurrence of aplastic anemia in patients with rheumatoid arthritis treated by salazosulfapyridine. PMID- 7553033 TI - [A male case of primary Sjogren's syndrome with interstitial pneumonitis and interstitial tubulo-nephritis in the absence of dry eye and dry mouth: parotid gland MRI is a useful diagnostic method for Sjogren's syndrome]. AB - Here we report a case of primary Sjogren's syndrome with hilar lymphadenopathy, interstitial pneumonitis and interstitial tubulo-nephritis. A 60-year old man was admitted to our hospital in May 1993 because of general fatigue and fever. He was noted to have hypergammaglobulinemia and had positive antibodies to nuclear antigens since 1990 in the absence of clinical manifestations. Since 9 months before admission, he presented with general fatigue, low grade fever and uveitis. On admission, chest X-ray and CT scan showed bilateral hilar lymphadenopathy and interstitial pneumonitis. The negative results for both serum angiotensin converting enzyme and histological findings of the cervical lymph node and the lung excluded the diagnosis of sarcoidosis. Serological examination exhibited marked elevation of polyclonal IgG level and anti-nuclear antibody, but neither anti-SS-A (Ro) nor anti-SS-B (La) antibody was detected. He did not have symptoms of xerophthalmia and xerostomia. Keratoconjunctivitis sicca was diagnosed by positive Schirmer's and rose bengal tests. His labial gland biopsy demonstrated severe mononuclear cell infiltration around the ducts. MRI findings of the parotid glands revealed heterogeneous and dotted high signal intensity similar to those in fat tissues in the T1- and T2-weighted images. These findings depicted that bilateral parotid gland was extensively destructed and was replaced by lipid tissue. Renal biopsy showed interstitial tubulo-nephritis. On the basis of the above findings, he was diagnosed to have primary Sjogren's syndrome and uveitis. Therefore, MR image of the parotid gland is considered to be a noninvasive and useful method for diagnosis of Sjogren's syndrome. PMID- 7553035 TI - [Anti-platelet antibodies in sera from patients with systemic lupus erythematosus by immunoblot analysis]. AB - It has not been clarified that the anti-platelet antibodies are related to thrombocytopenia in systemic lupus erythematosus (SLE). We evaluated the presence of anti-platelet antibodies in sera from patients with SLE using immunoblot analysis. In this study, 19 of 65 cases (29.2%) in SLE had anti-platelet antibodies in their sera, whereas no anti-platelet antibodies were detected in 16 cases of rheumatoid arthritis, 16 cases of progressive systemic sclerosis, 2 cases of polymyositis-dermatomyositis and 10 healthy controls. Six bands of 200 kDa, 140 kDa, 120 kDa, 95 kDa, 80 kDa and 65 kDa molecular weight were detected using immunoblot analysis. The platelet antibodies to 95 kDa and 80 kDa proteins were frequently detected in 12.3% and 16.9% respectively of SLE. These 2 antibodies were absorbed with the platelet pellet derived from healthy person. Therefore, it was suggested that the antibodies to 90 kDa and 80 kDa platelet antigens were specific to platelet membrane protein. Although we could not find any correlation between clinical and laboratory manifestations of SLE and these platelet antibodies, these antiplatelet antibodies were specifically detected in sera from patients with SLE by immunoblot analysis. PMID- 7553034 TI - [Detection of soluble interleukin-2 receptor in idiopathic thrombocytopenic purpura]. AB - We measured soluble interleukin-2 receptor (sIL-2 R) in serum samples from 57 patients with idiopathic thrombocytopenic purpura (ITP). The sIL-2 R level was significantly increased in the ITP patients (481.3 +/- 378.5 U/ml) compared with controls (176.2 +/- 66.9 U/ml) (p < 0.001), and was significantly higher in 8 patients positive for hepatitis C virus (HCV) antibody positive (1,140.7 +/- 194.3 U/ml) than in 49 HCV-antibody negative patients (378.9 +/- 278.6 U/ml) (p < 0.0001). There was also a significant difference between the HCV-antibody negative ITP patients and the controls (p < 0.01). Elevated sIL-2 R levels correlated with the CD 4/8 ratio (p < 0.05), but not with the platelet count or the level of platelet-associated IgG. The increase of sIL-2 R in ITP may be related to the immunological abnormalities underlying this disease. PMID- 7553036 TI - [Efficacy of beraprost sodium on Raynaud's phenomenon in patients with systemic sclerosis]. AB - Raynaud's phenomenon is an important clinical manifestation in patients with systemic sclerosis (SSc). No effective therapy, however, has been established for this phenomenon. Beraprost sodium, a stable prostacycline (PGI2) analogue, has been reported to improve hemorrheological impairment in patients with rheumatic diseases. In this study, we, therefore, examined the efficacy of beraprost sodium on Raynaud's phenomenon in 30 patients with SSc. Sixty micrograms per day of beraprost sodium was found to be effective in 14 patients (47%) in the period of 15.0 +/- 12.5 weeks. Raynaud's phenomenon in patients who responded to beraprost sodium was characterized by infrequent nail fold thrombosis and narrower hand areas affected by Raynaud's phenomenon, with mild secondary symptoms such as pain. These results indicate that beraprost sodium is effective for mild forms of Raynaud's phenomenon in patients with SSc. PMID- 7553037 TI - [Functional abnormalities of natural killer cells in childhood hemophagocytic syndrome]. AB - To analyze the abnormality of natural killer (NK) cells in childhood hemophagocytic syndrome (HPS), we investigated the numbers, cytolytic functions of circulating NK cells, and responsiveness to cytokines by flow cytometry, 51Cr releasing and single-cell assay in 13 patients. In the active phase, the relative number of CD 16+ cells was decreased in 3 patients. NK activity was depressed in 6 patients but normal in the remaining 7 in the active phase. Lymphokine activated killer (LAK) activity showed the similar tendency. The responsiveness of NK cells to IFN-gamma was absent, but that to IL-2 was normal in the active phase. Single-cell assay showed depressed maximal recycling capacity (MRC) and killing capacity values in the active phase and in remission phase, respectively. From these results, NK cells have such functional abnormalities as the absence of responsiveness to IFN-gamma and depressed recycling capacity in the active phase of childhood HPS. Further, abnormality of killing capacity of NK cells may exist even in the remission phase. PMID- 7553038 TI - [Clinical studies on 14 cases of adult-onset Still's disease]. AB - We here report fourteen patients diagnosed as adult-onset Still's disease (AOSD) in our hospital. Seven patients were males (mean age at onset was 26.6 years), and seven were females (30.6 years). All of the cases had spiking fever ( > 39 degrees C) and joint symptoms. Hepatomegaly, splenomegaly, and lymphadenopathy were noted in 50% of the patients, respectively. Skin eruption was seen in twelve patients (85.7%). Among them, nine patients (64.3%) exhibited typical rash. Pleuritis or pericarditis was seen in one case each. Only one patient revealed neurological disorder. Abdominal pain was present in 50% of the cases. The ratio of occurrence of secondary amyloidosis was 14.3%. Four patients (28.6%) were diagnosed to have the apophyseal narrowing at the cervical spine (C2-C3). Two patients (14.3%) accompanied by Sjogren's syndrome were women over 40 years of age. The levels of soluble interleukin-2 receptor were significantly elevated in the sera obtained from seven patients with AOSD and four patients with juvenile onset Still's disease, compared with normal controls. It seems to support the notion that immunopathologic processes via T cell activation play an important role in the pathogenesis of AOSD. PMID- 7553039 TI - [Liver disease in systemic lupus erythematosus]. AB - Liver disease in 193 patients (17 male and 176 female) with systemic lupus erythematosus (SLE) at Kawasaki Municipal Hospital were analyzed. Abnormal transaminase levels were found in 78 case (40.4%). Among them, there were 35 patients whose liver disease were identified. There were 12 patients whom no cause could be found other than SLE. Other liver disease were as follows: fatty liver in 9 cases, virus infection in 5 cases, gall stone and/or cholecystitis in 3 cases, drug allergy in 2 cases, autoimmune hepatitis 2 cases, primary biliary cirrhoses in 1 case. Liver disease with systemic lupus erythematosus was frequent, but there was no severe case. PMID- 7553040 TI - [Preferential proliferation of natural killer cells with bone marrow mononuclear cells in multiple myeloma]. AB - We examined proliferation of natural killer (NK) cells in 10 day mixed culture of peripheral blood mononuclear cells (PBMC) from healthy subjects with irradiated bone marrow mononuclear cells (BMMC) with multiple myeloma. In culture, NK cells increased with 9.5-fold. However, no increase was observed in T cells. The NK cell proliferating activity of PBMC stimulated with BMMC was higher than that of IL-2. NK cells at a purity of 90% or higher purity were collected from 10 day culture. Proliferation of these NK cells was stimulated by the addition of IL-2 but was suppressed by the addition of antibody coated erythrocyte (EA). IFN-gamma production was negligible in cultures of these NK cells alone but was marked in cultures with EA stimulate IFN-gamma production. Next, the NK cell obtained as above showed marked NK activity against K 562 cells, and this activity was further enhanced by the addition of IL-2. Also, while NK cells, these NK cells had some activity against Daudi cells and it was enhanced by the addition of IL 2. These results also suggest the presence of unknown cytokines with NK cell proliferating activities in the bone marrow of patients with multiple myeloma. PMID- 7553041 TI - [Case report: rheumatoid arthritis complicated by chronic myelogenous leukemia treated with interferon]. AB - A fifty-six-year old man complained of arthralgia and swelling of both feet, morning stiffness in both hands and finger joints in March 1987, and was treated with non-steroidal anti-inflammatory agents at another hospital. He has been treated for chronic myelogenous leukemia (CML) since May 1990. He was admitted to our hospital in March 1991 because of worsening of his multiple arthralgias, and a diagnosis of rheumatoid arthritis (RA) (Stage I, Class 2) was made on the basis of gait disturbance, arthralgia persisting for more than 6 weeks, the presence of subcutaneous nodules and X-ray findings. CML was confirmed by peripheral blood and bone marrow findings and the presence of the Philadelphia chromosome and bcr gene rearrangement. High fever and dyspnea developed suddenly 3 days after administration of interferon in May 1991. In addition to pneumonia, a leucostasis was suspected and he was treated with high dose steroids and antibiotics. After improvement, the steroids were tapered and he was discharged from hospital in July 1991. PMID- 7553042 TI - [A case of Sjogren's syndrome with rheumatoid arthritis manifesting transverse myelitis with antineuronal antibody]. AB - We report a sixty-year-old woman with transverse myelitis who had suffered from rheumatoid arthritis since age of 52. She was admitted to our department because of muscle weakness and painful tonic spasm in the bilateral lower extremities, sensory disturbance below the mamillary level and bladder disturbance. She had sicca symptoms. As a result of sialography, Sjogren's syndrome was diagnosed. Antineuronal antibody was found in the sera of the patient. She had no symptom of systemic vasculitis. Lupus anticoagulant and anticaldiolipin antibody were negative. The pathogenesis of transverse myelitis in rheumatic disease is still uncertain. Vasculitis and the immunological reaction of antineuronal antibody have been suggested as possible causes. This report suggests the influence of direct immunological reaction on the central nervous system. PMID- 7553043 TI - [A young woman with rheumatoid arthritis who rapidly developed secondary amyloidosis]. AB - A 22-year-old woman, who had been diagnosed as having rheumatoid arthritis (RA) 2 years before, was admitted to our hospital complaining of watery diarrhea (several times/day). She had been treated with low dose prednisolone (PSL) and auranofin in out-patient clinic. On admission, laboratory data showed moderate proteinuria (0.3 g/day) and positive CRP (2.7 mg/dl). Although the activity of RA was controlled by the administration of low dose methotrexate (7.5 mg/week) in addition to PSL, watery diarrhea and proteinuria did not improve. The biopsy of the stomach, rectum and kidney revealed the deposition of AA type amyloid protein, resulting in the diagnosis of secondary amyloidosis. Secondary amyloidosis has been reported as one of the common complications in RA patients, especially in old patients with a long history of RA. To our knowledge, however, there have been few reported cases who developed secondary amyloidosis so early during the course of RA as our case. We should be careful for the development of secondary amyloidosis even in young RA patients with short history of RA, when the disease is active. PMID- 7553044 TI - [A case of SLE associated with antiphospholipid syndrome and mitral stenosis]. AB - This case involves a 41-year-old woman with SLE. The patient began having symptoms of arthralgia in 1978 and developed fever, pleuritis and lupus psychosis in 1986. Laboratory exams showed positive antinuclear-antibody, LE-cell phenomenon, hypocomplementemia and lupus anticoagulant. Echo cardiography demonstrated mitral regurgitation and stenosis. She was treated with 50 mg of prednisolone and these manifestations subsided. In 1989, she developed dyspnea on exertion and echo cardiography revealed severe mitral stenosis. Pulmonary infarction was detected by MAA lung scintigraphy. At this time, she was diagnosed as SLE associated with antiphospholipid syndrome (APS). A mitral valvular replacement operation was performed in 1991. Pathological studies of mitral valve demonstrated Libman Sacks endocarditis. APS is known occasionally to complicate with left-sided valvular diseases, mitral stenosis is quite rare in both SLE and APS. This patient reveals a rare case of SLE associated with APS and mitral stenosis. It is suggested that this patient developed mitral stenosis with Libman Sacks endocarditis, associated with the presence of antibody against phospholipids. PMID- 7553045 TI - [Pure red-cell aplasia accompanied by neutropenia with increased granular lymphocytes--different effects of prednisolone and cyclophosphamide on improving anemia and neutropenia]. AB - Our report concerns a 53 year-old woman with pure red-cell aplasia (PRCA) accompanied by neutropenia. In July 1992, she experienced palpitations and came to our hospital. A peripheral blood examination demonstrated severe anemia and neutropenia, and an analysis of bone marrow cells revealed a marked decrease in the erythroid series. She was therefore diagnosed as PRCA. Sixteen percent of her peripheral lymphocytes had azur granules, which were considered to be a T-cell lineage based on surface marker analysis. However, gene analysis of T-cell receptors indicated no clonality. Administration of prednisolone (PSL) improved the subject's neutropenia but not her anemia. However, administration of cyclophosphamide (CPM) was effective in reducing the granular lymphocyte count, and finally improved the anemia. In this case, the granular lymphocytes presumably participated in the pathogenesis of anemia and neutropenia. While PSL did not yield sufficient effects on anemia and neutropenia, CPM was effective, suggesting that each drug may have different effects on these disorders. PMID- 7553046 TI - [Primary immunodeficiency syndrome as gene experiment of nature]. PMID- 7553047 TI - [Activation of complement during hemodialysis]. AB - In hemodialysis using 3 types of dialysis membrane materials [regenerated cellulose (RC), cellulose triacetate (CTA), and polysulfone (PS)], activation of the complement, reduction of white blood cells, and variation of vitronectin (VN) were observed. RC membrane caused a significant reduction of white blood cells and elevations of Bb and soluble membrane attack complex (S-MAC), indicating a strong activation of the alternative complement pathway. Especially, the increase of S-MAC persisted for a long time during hemodialysis. Because reduction of VN was transient, it was assumed that the S-MAC escaping removal by VN receptors might have persisted in the circulation. These findings suggested that S-MAC would become useful as an index for evaluating biocompatibility of various artificial organs including dialysis membranes. PMID- 7553048 TI - [Role of autoantibody in preeclampsia]. AB - The role of anti-ssDNA and dsDNA antibody in preeclampsia were studied. Serum samples were taken from 38 pregnants with preeclampsia including 19 cases of severe type. Also, 26 normal pregnant women were studied as the control. Anti ssDNA and dsDNA antibody were measured by enzyme-linked immunosorbent assay (ELISA). Anti-phospholipid antibody (APA), was measured by ELISA. Anti-dsDNA antibody was positive in 1 of the 38 preeclampsia. Anti-ssDNA antibody was positive in 15 out of the 38 cases (39.4%) in preeclampsia. Incidence of anti ssDNA positive preeclampsia is related to it's severity of symptom as following: Six of 19 mild preeclampsia (26.3%) were positive. In severe cases, 10 of 19 (52.6%). However, the relationship between anti-ssDNA antibody and IUGR in preeclampsia, was not confirmed, although the antiphospholipid antibody was related to the IUGR. From these results, we concluded that the autoantibody, such as anti-DNA antibody, might be produced in preeclampsia and the B cell activation might play a role of exaggeration of preeclampsia. PMID- 7553049 TI - [Inhibitory effects of interleukin (IL) -10 and viral IL-10 (vIL-10) on the functions of monocytes/macrophages]. AB - In this study, we examined the effects of IL-10 and vIL-10 on the production of superoxide anion (O2-) and nitric oxide (NO) by human monocytes and mouse macrophages. At an optimal concentration, human IL-10 (hIL-10) and vIL-10 significantly inhibited the production of interferon (IFN)-gamma by stimulated human peripheral blood mononuclear cells (PBMNCs). They also efficiently inhibited the production of O2- by both unstimulated and IFN-gamma-activated human monocytes. Mouse IL-10 (mIL-10) also significantly inhibited the production of NO by lipopolysaccharide (LPS) and IFN-gamma-stimulated mouse peritoneal macrophages. Moreover, the production of O2- and NO was effectively suppressed whether the IL-10 was added before or together with the stimulus, indicating that this cytokine acts primarily at an early stage of monocyte/macrophage activation by IFN-gamma and LPS. We also examined the effects of IL-4 and transforming growth factor (TGF)-beta on the production of O2- and NO by human monocytes and mouse macrophages, and found that they significantly inhibited both the production of O2- by human monocytes and the production of NO by mouse macrophages. Moreover, a combination of any two of IL-10, IL-4 and TGF-beta caused an additive effect on the inhibition of O2- production by human monocytes. These results indicated that IL-10 suppresses monocyte/macrophage activation either indirectly via an inhibition of the synthesis of IFN-gamma, a potent monocyte/macrophage activator, by PBMNCs, or directly via the deactivation of monocytes/macrophages. Moreover IL-10 may act in concert with IL-4 and TGF-beta to suppress monocyte/macrophage activation in vivo. PMID- 7553050 TI - [Establishment of superoxide production assay system using Epstein-Barr virus transformed cell line with chemiluminescence]. AB - We established the system to detect superoxide produced by Epstein Barr virus lymphoblastoid cell line (EB-LCL). Superoxide production of EB-LCL was evaluated by measuring chemiluminescence (CL) enhanced with addition of horseradish peroxidase (HRP). Using this system, we measured CL of EB-LCL established from 13 patients with chronic granulomatous disease (CGD) and 8 normal individuals. Significant elevation of CL was observed in all control EB-LCLs, however, no remarkable CL was seen in any patients' EB-LCLs. We examined the effect of recombinant human interferon gamma (rh-IFN-gamma) and granulocyte colony stimulating factor (G-CSF) on CL of EB-LCL in vitro. With addition of rh-IFN gamma, CL of normal control EB-LCL was significantly enhanced (p < 0.05), on the other hand, G-CSF was shown to have no effect. No significant CL was observed in any CGD patients' EB-LCLs even with addition of rh-IFN-gamma or G-CSF. It was suggested that superoxide produced by EB-LCL detected in this system was dependent on the same NADPH oxidase system which presents in phagocyte. PMID- 7553051 TI - [Immunohistochemical study of collagen in gastric cancer tissue]. AB - The distribution and localization of collagen types were studied immunohistochemically in resected tissues obtained from gastric cancer patients. The expression of transforming growth factor (TGF) -alpha, TGF-beta 1 and TGF beta 2 on cancer cells as well as the aggregation of T lymphocytes in the cancer tissue were also studied, in order to determine the differences between differentiated and undifferentiated type cancer. The interstitial tissues of differentiated type cancer showed intense staining for types I and III collagen, while those of undifferentiated type cancer showed intense staining for types I and III collagen, in addition to the stronger staining for types IV, V and VI collagen. Characteristically, type IV collagen was intensely stained in the interstitium in 18 of 20 undifferentiated type cancer (90%), but was stained in only one of 15 differentiated type cancer (6%). CD 3+ T lymphocytes were aggregated in the interstitial tissue of both the tumors, where the density of CD 4+ cells and the ratio of CD 4 to CD 8 were significantly higher in undifferentiated type cancer than in differentiated type cancer. TGF-alpha was detected in cancer cells in 80% of the differentiated cases and in 45% of the undifferentiated cases. The staining of TGF-beta 1 was also detected in 80% of the undifferentiated cases, which was significantly higher than 47% in differentiated cases. There were no differences in the incidences of staining for TGF-beta 2 between differentiated (33%) and undifferentiated type cancer (40%). These results suggest that there exist different mechanisms in the regulation of collagen production between differentiated and undifferentiated types of gastric cancer. PMID- 7553053 TI - [Sensitivity and specificity for detection of islet cell cytoplasmic antibodies using rat pancreatic sections]. AB - We determined islet cell cytoplasmic antibodies (ICA) using rat pancreatic sections as a test substrate substitutive for human pancreatic sections by indirect immunofluorescent technique. ICA were measured in sera from 58 patients with insulin-dependent diabetes mellitus (IDDM), 456 with non-insulin-dependent diabetes mellitus (NIDDM), 50 patients with autoimmune diseases, and 110 healthy controls. Seventeen of 58 patients with IDDM showed recent-onset (within 3 months). ICA were also measured in some samples using blood group O human pancreatic sections, and the ICA titers were compared with those measured using rat pancreatic sections. The prevalence of ICA was 55.2% (32/58) in patients with IDDM, 1.5% (7/456) in those with NIDDM, 0% (0/50) in those with autoimmune diseases, and 0.9% (1/110) in the healthy controls. Of the 17 recent-onset IDDM ICA were positive in 14 (82.3%). In comparative study of titers for ICA using rat pancreatic sections or human pancreatic sections, rat pancreatic sections yielded ICA titers as high as human pancreatic sections did. These results demonstrate that ICA assay using rat pancreatic sections was disease-specific, and that antigenicity of the substrate was favorable to ICA. Rat pancreas presents the advantage of greater availability, while providing an identical substrate for ICA. In conclusion, rat pancreatic sections are useful substrate for detecting ICA. PMID- 7553052 TI - [Cultured human monocytes activated by cytokines or lipopolysaccharide induce fibronectin]. AB - We studied the effects of cytokines such as IL-1 alpha, IL-6 and TNF alpha, and lipopolysaccharide (LPS) on cultured human monocytes. Increased fibronectin (FN) production, as a indicator of the activation of monocytes, was observed with any cytokines or LPS stimulation those were added in the culture medium. These increased FN production by cytokines showed a dose dependent fashion, and 4 hours culture with these cytokines was enough to obtain the amount of FN measured with radioimmunoassay. The combination of sub-optimal dose of cytokines (IL-1 alpha + IL-6, IL-1 alpha, IL-6 + TNF alpha), that could not induce substantial amount of FN with any single cytokine, also could induce FN by cultured monocytes. The specificity of cytokines for the FN production by cultured monocytes was confirmed by the neutralization using monoclonal antibodies specific for each monokines. Northern blot analysis with cDNA specific for FN confirmed the expression of FN mRNA in cultured monocytes stimulated with cytokines or LPS. Cytokine network plays an essential role for immune and inflammatory reaction, and FN has been shown to induce monokines through VLA-5 receptor on cultured monocytes. Our data suggests that monocytes may not always require high concentration of cytokines for the activation of monocytes in vitro, and that the synergistic action of low concentration of cytokines for the activation was enough for the progression of immune or inflammatory reaction. PMID- 7553054 TI - [Natural killer (NK) and lymphokine-activated killer (LAK) activities of a patient recovered from bladder cancer, and the characteristics of LAK cells generated from lymphocyte subsets]. AB - NK and LAK activities of a patient who recovered from bladder transitional cell carcinoma were determined. He was diagnosed in 1980 and operations, radiations and chemotherapies were repeated until 1986. Since 1987, a relapse has not been recognized, and he is now in a tumor free state. Recently, his very high NK and LAK activities were found. The percentage of CD 16+ in his peripheral blood lymphocytes (PBL) was 52.9%, the CD 4- CD 8- CD 16+ was 26.6% (17.5% and 12.1% in control, respectively). It seems likely that his tumor free condition may be related to these high killer activities. As a high number of CD 4- CD 8- cells were included in his PBL, it was possible to generate a CD 4- CD 8(-)-LAK and compare it with the CD 8(+)-LAK. The CD 4- CD 8(-)-LAK exhibited higher killer activity which was maintained for a long period of the culture, and a strong adherence property against the cultured tumor cells. Whereas, the CD 8(+)-LAK did not show such a high level of killer activity which decreased after two weeks into the culture. However, the CD 8(+)-LAK in this patient demonstrated higher killer activity against the auto-tumor cell line compared to what occurred with the control. A type of specific killing against auto-tumor cells may be contained in his CD 8(+)-LAK, although the MHC-restrictive killing was not confirmed. It remains unclear why the killer activities increased in this patient. PMID- 7553055 TI - [A case of adult onset Still's disease complicated with adult respiratory distress syndrome and disseminated intravascular coagulation]. AB - A 71 year-old man with adult onset Still's disease was admitted to our hospital because of fever, sore throat, myalgia and macular nonpruritic salmon pink eruption. He was treated with prednisolone, 40 mg daily and these symptoms disappeared. When the dose of prednisolone was reduced to 30 mg daily, he began to notice fever. 5 days later he developed adult respiratory distress syndrome (ARDS). The dose of prednisolone was increased to 50 mg daily and oxygen administration was started. All symptoms began to improve immediately and the dose of prednisolone was decreased to 40 mg daily. 10 days later he noticed fever and skin rash. Laboratory investigation showed platelet counts of 69,000/mm3, a ferritin of 37,000 ng/ml, and increased fibrinogen degradation product, indicating increased activity of adult onset Still's disease associated with disseminated intravascular coagulation (DIC). The dose of prednisolone was again increased to 60 mg daily, and 100 mg of nafamostat mesilate was administrated intravenously. All above symptoms associated with adult onset Still's disease and DIC disappeared. The dose of prednisolone was gradually decreased and the clinical course was uneventful with daily administration of 10 mg of prednisolone. Although there are a couple of case report which described the association of adult onset Still's disease with either ARDS or DIC, the association of adult onset Still's disease with both ARDS and DIC have not been reported yet. PMID- 7553056 TI - [The first case of Sjogren's syndrome with anti-Wa antibody]. AB - A 42-year-old woman presented in 1992 with Raynaud's phenomenon, polyarthralgia and morning stiffness and next year developed xerostomia and xerophthalmia. Investigations revealed 20 mm/hr of ESR, positive test for RAPA (1 : 320), ANF (1 : 1280), anti-SS-A antibody and anti-Wa antibody, and positive Schirmer tear test. A biopsied specimen of minor salivary gland of lip showed lymphocytic infiltration around the ducts and fibrotic changes and a sialogram demonstrated diffuse dilatations of the peripheral ducts. Resulting from the data shown above, the diagnosis of Sjogren's syndrome was made. Anti-Wa antibody, which recognizes a 48 kD tRNA associated protein was identified in serum from a patient with systemic sclerosis by Yamagata in 1985. So far the antibody has been specific for systemic sclerosis, because all of six patients with anti-Wa antibody previously reported were diagnosed as having systemic sclerosis. However, we here described the first case of Sjogren's syndrome with anti-Wa antibody. PMID- 7553057 TI - [A case of Sjogren's syndrome complicated with cryoglobulinemia, nephrogenic diabetes insipidus, and renal tubular acidosis]. AB - A 68-year-old woman had been complained of xerostomia since she was 30 years old. Further symptoms of polyuria, polyposia and insomnia had been developed since she was 35 years old. The biopsy material from a minor salivary gland demonstrated the infiltration of lymphocytes into mesenchyme which was compatible with Sjogren's syndrome. She admitted to our hospital because of myalgia in bilateral gastrocnemius and petechiae in both lower extremities in addition to the complaints described above. Complete blood cell counts on admission revealed hemoglobin 9.7 g/dl, platelet count 12.5 x 10(4)/microliters, and white blood cell count 3,300/microliters. Marked polyuria, polyposia (more than 5,000 ml/day, respectively) and low urine gravity (1.005) were observed, although the serum creatinine level showed normal value. Serologic examination showed that the elevation of total serum protein concentration (9.5 g/dl) with marked elevation of serum IgG level (6,190 mg/dl). Her immunoglobulins contained cryoglobulin (cryocrit 20%), and immunoelectrophoresis demonstrated the existence of IgG-kappa monoclonal protein. A positive anti-nuclear antibody at 1:320 dilution, a positive rheumatoid factor and a positive antibody to SS-A (Ro) were also observed. The serial studies of blood gas analysis could not demonstrated the presence of metabolic acidosis. Together with the result of elevated plasma antidiuretic hormone level and results of vasopressin test, Fishberg's concentrating test and the tests of the overload of NH4Cl or bicarbonate, she was diagnosed Sjogren's syndrome with both diabetes insipidus and subclinical renal tubular acidosis. She was initially medicated with prednisolone (40 mg/day, orally), then she was given six courses of intravenous cyclophosphamide (750 mg/body/month).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553058 TI - [A case of Felty's syndrome with marked thrombocytopenia and severe hypocomplementemia]. AB - Felty's syndrome is diagnosed when a patient shows both splenomegaly and leukocytopenia of various degree during the course of rheumatoid arthritis (RA). The accompanying immunologic abnormalities (e.g., antinuclear antibody, antiplatelet antibody, and hypocomplementemia) also characterize Felty's syndrome, but some authors may regard these abnormalities as a transitional form into overlap syndrome [RA + systemic lupus erythematosus (SLE)]. Here we reported a female case of Felty's syndrome who showed marked thrombocytopenia and severe hypocomplementemia. Thrombocytopenia had been refractory against several forms of therapies including high-dose methylprednisolone. Simultaneously, she had various autoantibodies (i.e., antiplatelet antibody, positive Coombs' test, antithyroglobulin antibody, antimicrosome antibody and anti-RNP antibody). Although she did not fulfill the ARA diagnostic criteria for SLE, the degree of thrombocytopenia as well as that of hypocomplementemia argued in favor of the overlap of SLE in this patient. Low-dose cyclosporin A (CsA) combined with small dose of prednisolone could increase both platelet count and level of complement. Notably, the titers of several autoantibodies dropped after CsA was started. These findings might suggest that CsA could normalize the underlying immunologic abnormalities in this patient. However, the disease activity of RA could not be decreased without a help of low-dose methotrexate. PMID- 7553059 TI - [IgD-lambda type multiple myeloma associated with IgG-kappa type benign monoclonal gammopathy]. AB - A 76-year-old man was admitted to Kisen hospital because of lumbago and chest pain. Laboratory examinations revealed a chronic renal failure with marked elevation of the serum BUN (48.8 mg/dl) and creatinine levels (8.2 mg/dl). The serum electrophoresis demonstrated a hypergammaglobulinemia with M peaks. An immunoelectrophoresis demonstrated monoclonal IgD-lambda and IgG-kappa proteins in the serum, and lambda-type Bence Jones protein in the urine (0.4 g/day). Bone marrow smears revealed an abnormal proliferation of atypical plasma cells (43%). A systemic X-ray examination of the skeletal system showed systemic osteoporosis without punched out lesion. The patient was diagnosed as having IgD-lambda type multiple myeloma and IgG-kappa type benign monoclonal gammopathy by quantifying concentration of two M proteins (1,160 mg/dl in IgD, 1,179 mg/dl in IgG, respectively). A combination chemotherapy with melphalan and prednisolone was administered monthly for multiple myeloma, and hemodialysis for the renal failure was performed 3 times a week. A marked improvement of his laboratory findings including a diminution of the serum IgD-lambda M-protein was obtained. On the other hand, IgG-kappa M-protein level was unchanged. Two M-protein levels showed a different behavior after the combination chemotherapy. Although the patient died of congestive heart failure, the partial remission of multiple myeloma has been maintained for 16 months with chemotherapy. PMID- 7553060 TI - [Antibodies of IgM type against small cell lung carcinoma and 29 kD neuronal antigen of rat in a patient with paraneoplastic sensory neuropathy]. AB - 62-year old man who had small cell lung carcinoma (SCLC) with subacute sensory neuropathy had an antibody (IgM) recognizing the antigen on peripheral nerves. The antibody in the serum recognized cell surface antigen on small cell carcinoma cell lines and also reacted with peripheral nerves simultaneously, but not with central nervous system. Two months before admission, he felt difficulty in walking because of paresthesias in the extremities. He was found to have a tumor shadow in chest X-ray, and was diagnosed as SCLC by biopsy of right supraclavicular lymph node. Western blot analysis demonstrated that auto-antibody (IgM) in the serum recognizing the antigen on the neuronal axon with single band at 29 kD. Pathogenesis of carcinomatous neuropathy is still unexplained, but the findings presented here have given rise to the possibility that anti-SCLC antibody may cross-react with neuronal antigen, primarily resulting in neuronal disorder. PMID- 7553061 TI - [Chronic cytomegalovirus infection that present specific clinical course--a case of a boy with common variable immunodeficiency]. AB - We describe an 8-year-old boy with CVID and chronic CMV infection. Although at onset he was diagnosed as IgA deficiency, 4 years after his clinical manifestations because compatible to CVID. During his clinical course he had suffered from various disorders as follows; AIHA, interstitial pneumonia, hemophagocytic syndrome, chronic gastroenterocolitis and so forth. At the age of 8 the PCR of CMV-DNA of biopsy specimen from colon, lung and bone marrow were confirmed to be positive. Hematological examinations revealed abnormal cellular immunity such as decreased CD 4/8 ratio with increased HLA- DR+ CD 8+ T cell, decrease of absolute blood lymphocytes count and reduced response of lymphocytes to blastogenetic agents. These findings brought us to diagnose him as having CVID complicated with chronic CMV infection. This case gives us some impact to speculate what role CMV infection plays in CVID, Whose etiology is unknown. PMID- 7553063 TI - [The 23rd Annual meeting of the Japanese Society of Clinical Immunology. Okayama City, Japan. September 6-8, 1995. Abstracts]. PMID- 7553062 TI - [A case of hemophagocytic syndrome manifesting adult Still's disease and acute hepatitis]. AB - We report a 20 year-old woman with hemophagocytic syndrome. In February 1993, she developed high fever, arthralgia, salmon-like pink eruption, leukocytosis and splenomegaly. She was diagnosed as adult Still's disease and successfully treated with intravenous immunoglobulin and oral prednisolone. In September 1993, she was re-admitted to our hospital complaining of general fatigue and low grade fever and treated with oral prednisolone at a daily dose of 15 mg. On October 2, 1993, she suddenly developed high fever and salmon-like pink eruption on her leg followed by the marked increase of serum transaminase and LDH levels (GOT 3,270 IU/l, GPT 1,880 IU/l, LDH 5,480 IU/l) on October 7. Since hepatic failure progressed, we started methylprednisolone pulse therapy and plasmapheresis. However, because of the progression of pancytopenia caused by hemophagocytosis, the treatment with VP-16 was initiated. However, she died of DIC on November 2, 1993. Autopsy revealed submassive necrosis of the hepatocytes with moderate infiltration of histiocytes. She was retrospectively diagnosed as hemophagocytic syndrome whose manifestations are very similar to those in adult Still's disease and acute viral hepatitis. PMID- 7553064 TI - Calcium-binding proteins 1: EF-hands. PMID- 7553065 TI - Transcription factors 2: helix-loop-helix. PMID- 7553066 TI - HLA-DQ1 is associated with clinical response and survival of patients with melanoma who are treated with interleukin-2. AB - AIM: The risk of developing melanoma, the natural history of this disease, and the response to therapy with biological reagents may be determined, in part, by a patient's human leukocyte antigen (HLA) phenotype. In order to study this, the relationship between HLA type and clinical response to therapy with interleukin-2 (IL-2) was evaluated. METHODS: We retrospectively determined the HLA phenotype of 82 patients with metastatic melanoma who were treated with IL-2-based therapy. Fresh or frozen lymphocytes were serologically typed by standard lymphocytotoxicity techniques (NIH or Amos modified). The treatment regimens included IL-2 alone or with tumour infiltrating lymphocytes, lymphokine activated killer cells, cyclophosphamide, interferon-alpha, and tumour necrosis factor alpha. Initially, the relationship between clinical response and each HLA antigen was evaluated by performing a two-tailed Fisher exact test. Associations with a P value less than or equal to 0.10 without adjustment for multiple comparisons were considered worthy of further study. Independent confirmation of these apparent associations was obtained by studying the relationship between HLA phenotype and patient survival using Cox proportional hazards models. RESULTS: In the initial screening, a statistically significant association between clinical response and the expression of HLA-DQ1 was observed (unadjusted P2 = 0.0017). HLA-DQ1 was also independently associated with prolonged survival (P2 = 0.026). This positive association with survival was evident both for patients who responded to therapy and those who did not respond, as defined by > 50% tumour regression. CONCLUSIONS: Among patients with metastatic melanoma, HLA-DQ1 appears to be associated with clinical response to therapy using IL-2. This apparent association is confirmed by the observation that HLA-DQ1 is independently associated with prolonged survival in this group of patients. PMID- 7553067 TI - In vivo priming effects of interferon-beta ser on NK activity of peripheral blood mononuclear cells in cancer patients. AB - Natural killer (NK) activity was assayed in fresh peripheral blood mononuclear cells (PBMs) from cancer patients receiving interferon (IFN)-beta ser. Patients received a single intravenous injection of IFN-beta ser (90 x 10(6) IU m-2) on alternate days for 2 weeks, followed by a higher dose (180 x 10(6) IU m-2) on the same schedule. PBM NK lysis of K562 target cells was significantly increased in PBMs sampled 24 h after the initial injection (P < 0.05). At the end of the first 2 weeks of the protocol, NK cytotoxic activity of PBMs had fallen below the original baseline levels; the higher IFN dose subsequently given was without effect. However, significant increases in the proportion of CD16+ cells were seen following each injection. A positive correlation was also seen between the increased lytic activity of CD16+ NK cells and the proportion of CD38+ NK cells, but not the proportion of CD56+ NK cells. In vitro IFN-treatment of these in vivo treated PBMs resulted in a further increase in NK activity. Pre-exposure in vivo to IFN-beta ser seems to prime the PBMs to respond to in vitro stimulation by IFN gamma, which otherwise had no effect. Phenotypic analysis of PBMs after in vitro exposure to IFN-beta ser showed that the levels of CD16+, CD38+ and CD56+ cells did not change. All the NK activity responding to IFN-beta ser was found in the CD16+ enriched population of PBM, suggesting that it is unlikely that in vivo redistribution of CD16+ subsets representative of NK cells has occurred in the peripheral blood. PMID- 7553068 TI - Low immunogenicity of therapeutic rat CD45 antibodies when used for pre-treatment of donor organs for transplantation. AB - Immunoassays for the measurement of human anti-immunoglobulin responses against the CD45 specific rat monoclonals, YTH 24.5 and YTH 54.12, have been developed. The assays are based on a 'double antigen' ELISA system and are reproducible with coefficients of variation of less than 15%. The assays were used to measure anti immunoglobulin responses in sera from 40 patients who had received kidneys pre treated with the pair of anti-CD45 monoclonals YTH 24.5 and YTH 54.12. Only two patients elicited a weak human antimurine antibody (HAMA) response. PMID- 7553069 TI - A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity. AB - Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases. PMID- 7553070 TI - Specific down-regulation of anti-allergen IgE and IgG antibodies in humans associated with injections of allergen-specific antibody complexes. AB - Several approaches have recently been put forward describing attempts to suppress the IgE immune response towards allergens, which is thought to be the key event in allergic diseases. In a series of clinical trials we have shown that injections of complexes made up from allergen and specific antibodies are an effective treatment for allergic bronchial asthma and atopic dermatitis. In the work presented here we have examined the humoral immunity changes associated with the use of such complexes in a group of 19 adult patients suffering from atopic dermatitis and hypersensitive to Dermatophagoides pteronyssinus (Dp), and in whom a significant clinical improvement was observed. By comparing serum samples taken prior to and after 4 months of therapy, we show that the administration of immune complexes is associated with: (i) a significant and selective reduction of IgG and IgE antibodies specific for Dp allergens; (ii) a down-regulation that affects only the antibodies present in the complexes; (iii) the induction of corresponding anti-idiotypic antibodies. To our knowledge, this is the first demonstration in humans that an anti-allergen antibody response can be down regulated in a highly selective manner and that this is accompanied by significant clinical improvement. Moreover, the selective reduction of IgG antibodies could be of value in the treatment of some forms of auto-immune diseases. PMID- 7553071 TI - Mucosal tolerance and the immunotherapy of autoimmune disease: a commentary. PMID- 7553072 TI - Induction of a CD8+ cytotoxic T lymphocyte response to soluble antigen given together with a novel muramyl dipeptide adjuvant, N-acetyl-D-glucosaminyl-(beta 1 4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP). AB - We have investigated the ability of the novel muramyl dipeptide, GMDP, to act as an adjuvant for the induction of ovalbumin (OVA)-specific, CD8+ cytotoxic T lymphocyte (CTL) responses. C57Bl/6 mice were twice immunized s.c. with 50 micrograms OVA emulsified with a squalane, L121 pluronic containing Tween-80 vehicle either with (STP-GMDP) or without (STP) GMDP. Splenic precursor CD8+ CTL activity against E.G7-OVA, but not against EL-4 parental targets was detected in STP-GMDP immunized mice after 5 days of in vitro re-stimulation with irradiated E.G7-OVA cells, while mice immunized with OVA in STP alone or OVA alone failed to demonstrate CTL activity. OVA emulsified in a microfluidized STP vehicle formulation without GMDP also elicited the E.G7-OVA precursor CTL. The ability of GMDP to induce a class I-restricted, CD8+ CTL response to a soluble protein antigen may have implications for the development of useful vaccines against viral pathogens or tumours against which CTL responses are desirable. PMID- 7553073 TI - Distribution of rat hepatic steroid 5 alpha-reductase 1 as shown by immunohistochemistry. AB - Peptide fragments of rat liver 5 alpha-reductase were synthesized according to the published primary sequence data. The peptide were used (i) in free form and (ii) linked to keyhole limpet hemocyanin (KLH), respectively, for immunization of rabbits. Resulting antisera showed positive reaction with the respective peptide antigen in an ELISA. In Western blot experiments the antisera specifically recognized a 26,000 mol wt protein in extracts from female and male rat liver. All antisera, as well as isolated immunoglobulins, showed inhibition of 5 alpha reductase activity in microsomes prepared from rat liver. Positive immunohistochemical reactions were observed in the perinuclear cytoplasm of hepatocytes. No reaction was seen in Kupffer- and connective tissue cells. Acinar heterogeneity of the immunoreaction was seen in the male rat liver with a gradient from the portal canal to the central vein. After androgen-deprivation a considerable increase in immunoreactivity was seen in male rat liver cells, indicating androgen-dependent suppression of the enzyme in male liver. PMID- 7553074 TI - Validation of a mechanical method for measuring skin thickness: relation to age, body mass index, skin thickness determined by ultrasound, and bone mineral density. AB - In a number of endocrine disorders, typical changes in skin thickness can be observed which make measurement of skin thickness interesting in this field. A newly developed mechanical method for measuring skin thickness is presented. Using a digital measuring screw on the dorsum of the hand with a defined measuring force of 10 newton and a resulting tissue compression of 1500 mm Hg, highly reproducible results were obtained (mean coefficient of variation 2.56%). In 129 women, 37 to 78 years old, body mass index < 30 kg/m2, there was no significant relation between body mass index and skin fold thickness. A negative correlation between skin fold thickness and age (r = 0.37, p < 0.001) was detected. This has been shown for skin by other methods previously and is well known to occur in bone, another tissue whose matrix as well as dermis consists mainly of collagen type I. In 30 subjects, half hypopituitary patients, half healthy subjects (17 women, 13 men; 43.3 +/- 10.5 years old), skin fold thickness measured mechanically and sonographically determined skin thickness correlated with r = 0.46 (p < 0.01). A significant correlation between bone mineral density measured by single photon-absorptiometry at the ultradistal forearm and skin fold thickness measured mechanically was found and skin fold thickness measured mechanically was found (r = 0.36, p < 0.05), whereas this was not the case for sonographically determined skin thickness and bone mineral density (r = 0.13, n.s.). This newly developed method might be useful in clinical studies on endocrine disorders affecting skin (and bone) metabolism and the regulation of collagen type I metabolism in general. PMID- 7553075 TI - Probucol prevents the progression of fatty liver in MSG obese mice. AB - We investigated the effect of Probucol in preventing fatty liver in monosodium-L glutamate (MSG) treated obese mice and control mice fed a high fat diet. MSG mice became significantly obese 9 weeks after birth with higher levels of serum blood glucose, total cholesterol, HDL-cholesterol, GPT, and cholinesterase, and had greater triglyceride contents in their livers relative to control mice. Morphologically, MSG obese mice also had a marked fatty liver. Administration of Probucol mixed with the high fat diet for 2 weeks significantly decreased the serum levels of total cholesterol and HDL-cholesterol, and liver triglyceride contents in both MSG and control mice. Morphologically, the livers were less fatty after Probucol treatment. These results suggest that Probucol prevents the development of fatty liver, and in addition reduces hypercholesterolemia. PMID- 7553076 TI - Determination of anti GAD65 autoantibodies with an ELISA before and after standardization with the new international reference serum. AB - The serum of a stiff-man syndrome patient was declared international GAD reference standard at the "1st GAD Antibody Workshop" held at the "12th International Immunology and Diabetes Workshop" in Orlando, Florida, USA 1993. A comparative study was performed with 123 diabetic and non-diabetic patients to evaluate whether standardization of this reference serum had changed the properties of a commercially available ELISA assay. All samples classified positive with the old test were confirmed with the new assay. Four additional samples with high "normal" values became positive with the new test. One of them was a control person having a family history of diabetes and genetic loci DR4/DR11. These findings might implicate a higher risk for the development of IDDM. The new standardization and adaptation of the ELISA seems to have influenced the sensitivity of the test positively. PMID- 7553078 TI - Hormone delivery systems to the brain-transthyretin. AB - This paper reviews knowledge on the structure and function and evolution of the thyroid hormone binding protein transthyretin (TTR), with particular reference to factors affecting thyroid hormone distribution and delivery to the brain. The pool of thyroid hormones critical for the biological actions of the hormones is the pool of free thyroid hormone. The size of this pool is determined for short time periods by uptake/release of thyroid hormones into/from cell and binding/release of thyroid hormones by thyroid hormone-binding proteins. Both proportions and absolute concentrations of these proteins differ in blood plasma and cerebrospinal fluid (CSF). The most pronounced difference is found for TTR which is the only thyroid hormone-binding plasma protein synthesized in the brain. TTR is also distinct from the other two thyroid hormone-binding plasma proteins in humans by the absence of genetic deficiencies. TTR gene expression was initiated during evolution much earlier in the brain than in the liver. The structure of the domains of TTR involved in thyroxine (TR) T4 binding has been completely conserved for 350 million years. These observations point to a special functional significance of TTR in the brain. It is proposed that this is the determination of the level of free T4 in the extracellular compartment of the brain. T4 can then be converted in the brain to triiodothyronine T3 by specific deiodinases. This T3 can interact with receptors in the cell nuclei, regulating gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553077 TI - Glucose metabolism and liver cirrhosis. AB - Chronic liver disease is characterized by numerous metabolic alterations, predominantly catabolic, resulting in the clinical picture of malnutrition and even cachexia in some patients. The following review focuses on disturbances of glucose metabolism and of hormonal interactions that could contribute to the clinical picture of malnutrition seen in chronic liver disease. Body composition is altered in a characteristic manner with an increase in fat mass and a significant loss of muscle tissue. Furthermore, defective glucose storage due to reduced insulin sensitivity predominantly of muscle tissue has been observed. The pathogenesis of insulin resistance leading to an impaired glucose tolerance or a manifest diabetes mellitus is as yet unknown. A receptor/postreceptor dysfunction probably exists in chronic liver disease that might be explained by the following factors: 1. Altered membrane lipid composition and increased levels of free fatty acids; 2. long-lasting hyperinsulinemia; 3. increased plasma levels of insulin counteracting hormones such as growth hormone, glucagon, catecholamines and possibly cytokines; 4. a lack of liver-derived humoral factors with insulin-like activity, i.e. insulin-like growth factors I and II. PMID- 7553079 TI - Enhanced cAMP production mediates the stimulatory action of pituitary adenylate cyclase activating polypeptide (PACAP) on in vitro catecholamine secretion from bovine adrenal chromaffin cells. AB - The 38 amino acid peptide pituitary adenylate cyclase activating polypeptide (PACAP) induced a dose dependent increase of catecholamine secretion in cultures of bovine chromaffin cells. This secretagogue activity of PACAP was strictly dependent on the presence of calcium in the culture medium. If calcium was omitted from the medium no effect of PACAP on catecholamine secretion could be detected during an incubation of 20 min. Preincubation of cells with 1 nM PACAP for 5 min facilitated the subsequent nicotine stimulated catecholamine secretion during a 20 min incubation without addition of the peptide. PACAP induced catecholamine secretion was clearly accompanied by a dose dependent increase of intracellular cAMP concentrations. The percentage of cells responding to PACAP with increased catecholamine secretion was assessed by immunocytochemistry of the transient appearance of dopamine-beta-hydroxylase, associated with the membranes of the chromaffin granules on the cell surface during the secretory process. About 70% of adrenal medullary cells responded to 100 nM PACAP with enhanced secretory activity. Though PACAP stimulated catecholamine secretion, we did not observed major effects on intracellular free calcium concentrations ([Ca2+]i) as determined with fura-2 by single cell fluorescence microscopy. In maximally 20% of the cells a rise in [Ca2+]i in response to a challenge with 500 nM PACAP was observed. Lower concentrations of PACAP were without an effect on [Ca2+]i. These data indicate that the stimulatory action of PACAP on in vitro catecholamine secretion from bovine chromaffin cells is linked to a rise of intracellular cAMP. PMID- 7553080 TI - Effect of aphidicolin on DNA synthesis in cultured human thyroid cells. AB - To investigate whether partial inhibition of DNA synthesis affects the growth of thyroid cells, human thyroid cells were cultured in the presence of aphidicolin for 24h. After removal of aphidicolin, the growth of thyroid cells was evaluated by [3H] thymidine uptake and analysis of cell cycle by flow cytometry. In Graves' thyroid cells, [3H] thymidine uptake and the percentage of cells in S+G2/M phase increased during 24 hours after washout of aphidicolin, whereas those were not augmented in normal thyroid cells. Thus, the acceleration of DNA synthesis occurred after the partial inhibition of it by aphidicolin in Graves' thyroid cells, but not in the normal thyroid cells. This phenomenon might, at least in part, explain the difference of growth regulation between Graves' and normal thyroid cells. PMID- 7553082 TI - IGF-I and insulin receptors in bovine skeletal muscle: comparisons of different developmental ages, two different genotypes and various individual muscles. AB - To investigate the regulation of the IGF-I and the insulin receptor in bovine skeletal muscle, we determined their concentrations and their affinity constants in animals of different age, muscle type and breed. Receptors were solubilized and measured by an enzyme-immunoassay. During ontogenesis the concentration of IGF-I receptors decreased from 1650 /+- 600 fmol/mg protein in 3-5-month old fetuses to 105 /+- 20 fmol/mg protein in 16-month old heifers (p < or = 0.01). The insulin receptor concentrations also decreased with age from 136 /+- 28 fmol/mg protein in 3-5-month old fetuses to 50 /+- 11 fmol/mg protein (p < or = 0.05) in 16-month old heifers. In order to compare different muscle types, seven muscles, which represent large differences in fibre type composition and growth impetus, were selected from 6 month old female Jersey calves and were assayed for IGF-I and insulin receptors. We observed significant differences of the IGF-I as well as the insulin receptor concentrations between distinct muscles. However, no relationship could be established between receptor concentration and fibre type composition or growth impetus. In muscle of two cattle breeds, differing markedly with regard to muscle growth intensity, the Jersey and the German Fleckvieh breed, we observed no divergence in IGF-I nor insulin receptor concentrations. We found no differences in IGF-I and in insulin receptor affinities in any of the adult animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553081 TI - Association between diabetic-autonomic-C-fibre-neuropathy and medial wall calcification and the significance in the outcome of trophic foot lesions. AB - The role of peripheral autonomic nerve failure in the development of medial arterial calcification is discussed controversially in the literature. In addition, the influence of medial wall calcification in the development of trophic foot lesions is still not fully understood. We have compared 28 diabetic patients with cardiac autonomic failure and/or decreased acetylcholine-induced sweat response with 22 diabetic patients without evidence of diabetic neuropathy and 27 healthy control subjects. A strong association was found between medial wall calcification and diminished heart rate variation (p.001) or diminished peripheral sweat response (p < 0.001). Only one subject in the control group exhibited a calcification of the tunica media in the lower limb. Medial calcinosis was accompanied by a significant rise of systolic ankle blood pressure (p < 0.05). In addition, there was a strong relationship between medial wall calcification and trophic foot lesions (p < 0.0001). Our results demonstrate a strong association between sympathetic C-fibre neuropathy and the development of medial wall calcification, as well as between calcification of the tunica media and trophic foot ulceration. Further investigation is necessary to determine whether there is a causal connection between medial arterial sclerosis and diabetic foot ulceration, or whether both are independent consequences of diabetic neuropathy. PMID- 7553083 TI - Intense competition shapes PRK delivery market. PMID- 7553085 TI - Have you ever seen a pachoderm? PMID- 7553084 TI - ISL receives IDE for glaucoma procedure. PMID- 7553086 TI - Excimer laser photorefractive keratectomy after radial keratotomy. AB - BACKGROUND: Correction of residual myopia after radial keratotomy may be attempted with repeated keratotomy surgery, but predictability can be less than satisfactory. Excimer laser photorefractive keratectomy (PRK) provides an alternative approach to improving the refractive result in these patients. METHODS: Twenty-five eyes of 20 patients at five clinical locations underwent PRK for residual myopia after radial keratotomy. The number of incisions ranged from 4 to more than 16. Clear zones ranged from 3 mm to 4 mm. Best corrected visual acuity was 20/20 or better in 16 of the 25 eyes, with a range from 20/12 to 20/80. Uncorrected visual acuity was 20/200 or worse in 15 of the 25 eyes, with a range from 20/25 to finger counting. The interval between radial keratotomy and PRK averaged 33.5 months, with a range from 5 to 96 months. Nineteen eyes had 6 months or more of follow up; 15 had 12 months or more. RESULTS: Corneal haze was maximal 1 month after surgery (mean +/- SE, 0.65 +/- 0.09), and declined to 0.35 +/- 0.16 at 12 months. Twelve months after PRK, mean keratometric readings were 40.19 +/- 0.81 diopters (D) and mean spherical equivalent refraction was -1.42 +/ 0.47 D. Nine (60%) of the 15 eyes with 12 months follow up were within 1 D of emmetropia and 12 (80%) were within 2 D. Eight (53%) of the 15 eyes had uncorrected visual acuities of 20/40 or better. Spectacle-corrected visual acuity in the eyes with 12 months follow up improved in 4, did not change in 4, and worsened in 6. CONCLUSIONS: The results of PRK are less predictable in eyes that have previously undergone radial keratotomy, and these eyes respond with more haze after PRK than normal eyes. PMID- 7553088 TI - Histological, immunohistochemical, and ultrastructural findings in human corneas after photorefractive keratectomy. AB - BACKGROUND: Four human corneas that had undergone photorefractive keratectomy (PRK) and subsequent penetrating keratoplasty were examined by means of light and electron microscopy in an attempt to detect possible causes for complications after PRK. METHODS: Four eyes with a central corneal leukoma resulting from a previous PRK treatment underwent penetrating keratoplasty respectively 3 days, 3 months, 5 months, and 13 months after the refractive procedure. Different excimer laser instruments (Meditec MEL 50, Summit UV200, and VISX 20/20) had been used. The corneal buttons removed were submitted for light microscopy, electron microscopy and immunohistochemistry. RESULTS: An immature (one to two layers, basement membrane anomalies) but continuous epithelium was present even in the 3 day specimen; between six and eight epithelial layers could be seen in the 13 month specimen, which still presented an undulated aspect of the otherwise normal basement membrane. A continuous acellular collagen layer underlying the epithelium of the ablated area was detected in the superficial stroma of all examined corneas. No Descemet's membrane or endothelial alterations could be seen. CONCLUSIONS: Despite recovery of a continuous epithelial layer as early as 3 days after PRK, abnormalities of both epithelium and superficial stroma could be detected in all specimens, including the one obtained 13 months after the refractive procedure. PMID- 7553087 TI - Photorefractive keratectomy for myopia using a 4.5-millimeter ablation zone. AB - BACKGROUND: Argon fluoride (193 nm) excimer laser photorefractive keratectomy for myopia is under evaluation by the United States Food and Drug Administration. METHODS: We report a consecutive prospective series of 100 patients (one eye per patient) treated as part of the Phase IIB FDA-approved protocol, with 80 patients followed for 1 year. Patients' ages ranged from 21 to 62 years (mean, 35 years). The Summit Technology, Inc ExciMed UV200LA with a 4.5-mm diameter ablation was used. RESULTS: Baseline spherical equivalent refraction ranged from -2.00 to 6.90 diopters (D) (mean -4.60 D). Ninety-five percent of eyes reepithelialized by 72 hours. At 1 year, the difference between attempted and achieved correction was +/- 0.50 D for 42 eyes (53%) and +/- 1.00 D for 60 eyes (75%). During the first 6 months, there was a trend toward overcorrection and the majority of eyes showed some loss of initial refractive correction; 10 eyes (14%) changed by 1.00 D or more between 6 and 12 months. An uncorrected visual acuity of 20/25 or better was achieved by 50 eyes (63%) and 20/40 or better by 61 eyes (77%). Of the 10 eyes (12%) that lost two or more Snellen lines of spectacle-corrected or glare visual acuity, two had visual acuity of worse than 20/25. Central subepithelial corneal haze was absent to mild in 77 (96%) eyes at 12 months. CONCLUSIONS: Excimer laser photorefractive keratectomy as performed in this study was generally effective and safe in reducing simple spherical myopia. Further studies of the effect of a larger diameter ablation zone, smoother transitional corneal contours, and the effect of postoperative topical corticosteroids may lead to further improvements in outcome. PMID- 7553089 TI - Three-dimensional digital subtraction modeling of corneal topography. AB - BACKGROUND: The introduction of a system of classification of color-coded computerized topography has proved of great clinical value but may be misinterpreted by some who assume that the color plots represent actual corneal contour. METHODS: A three-dimensional system of contour modeling is used to plot equivalents of each category of corneal shape. The round, oval, symmetric bowtie, and asymmetric bowtie of the widely used classification schema are each translated into contour equivalents. Digital subtraction of an appropriate sphere from the contour data is central to the transformation. RESULTS: While round- and oval-type color-coded refractive power plots translate into fairly intuitive contour images, symmetric and asymmetric bowties produce surprising results. We demonstrated that the bowtie appearance is a result of the changing curvatures present between the two principal meridia of the toroidal surface. CONCLUSION: The shape characteristics of the corneal surface dictate the familiar color-coded dioptric plots. PMID- 7553090 TI - Keratoconus detected by videokeratography in candidates for photorefractive keratectomy. AB - BACKGROUND: Patients with corneal shape abnormalities should be identified prior to photorefractive keratectomy (PRK). We used videokeratography screening to detect subclinical corneal abnormalities, including keratoconus, which might have been missed by conventional clinical evaluation. METHODS: One hundred forty-six apparently normal myopic eyes (-1.00 to -7.00 diopters [D] with less than 1.50 D of cylinder) of 91 consecutive patients who were candidates for PRK were screened by videokeratography. RESULTS: In 6 of 91 patients (7 of 146 eyes), unsuspected corneal shape abnormalities were detected by videokeratography. Two patients had definite keratoconus and three were classified as keratoconus suspects by inferior corneal steepening (Rabinowitz I-S index ranging from 1.62 to 6.20 D). One patient had early pellucid marginal degeneration. CONCLUSIONS: Keratoconus suspects and contact lens-induced changes resembling keratoconus are present in the "normal" myopic population that presents for refractive surgery. Videokeratographic screening is the only effective means of identifying these and other corneal shape abnormalities. PMID- 7553091 TI - Refractive keratotomy in keratoconus suspects. AB - BACKGROUND: We utilized videokeratography to determine the frequency of keratoconus suspects in a refractive surgery population, and compared the results of refractive keratotomy in these patients to those of patients with normal corneal topography. METHODS: The records of 67 consecutive patients (120 eyes) undergoing refractive keratotomy were reviewed retrospectively. Quantitative measurements of relative inferior corneal steepening (inferior-superior value, I S value) were obtained from preoperative videokeratography. RESULTS: Three of 120 eyes (3 of 67 patients, 4.5%) were identified as keratoconus suspects based on I S (inferior-superior) values greater than +1.74 (range, +1.76 to +2.00), which represented two standard deviations (+/- 0.66) above the mean (+0.42). All three of these eyes achieved postoperative uncorrected visual acuity of 20/30 or better. Of those eyes with normal preoperative topography (I-S value +1.74 or less), 90 of 112 (80%) achieved postoperative uncorrected visual acuity of 20/30 or better, and 105 of 112 (94%) achieved 20/40 or better. CONCLUSIONS: Although long-term follow up is needed to confirm stability of refractive surgery outcome, it appears that keratoconus suspects can obtain acceptable results with refractive keratotomy. Studies on larger patient populations using videokeratography are needed to better define keratoconus, keratoconus suspects, and normal variants of corneal topography and to determine how eyes with abnormal topography respond to refractive keratotomy. PMID- 7553092 TI - Survival of streptococcus in optisol-GS medium. AB - BACKGROUND: Donor cornea contamination as a cause of endophthalmitis is one of the most serious complications of penetrating keratoplasty. Optisol-GS corneal storage medium with the combination of gentamicin and streptomycin was designed to provide wider antibiotic coverage, most notably against streptococcal species. However, many enterococci are resistant to streptomycin and genta micin. METHODS/RESULTS: We report a case in which Enterococcus faecium was isolated from cultures of the donor limbus prior to corneal excision and again from preoperative cultures of the donor corneal rim despite 5 days of preservation in Optisol-GS. The isolate was found to be resistant to both gentamicin and streptomycin. CONCLUSIONS: This case illustrates the need to raise awareness that streptococcus can remain a viable contaminant of donor corneas despite storage in Optisol-GS medium. PMID- 7553093 TI - Nocardial keratitis following myopic keratomileusis. AB - PURPOSE/METHODS: Interface opacities were detected in a patient who underwent uncomplicated myopic keratomileusis. RESULTS AND DISCUSSION: Nocardia asteroides keratitis was confirmed by microbiologic work-up and guided the correct treatment. The eye recovered 20/60 spectacle-corrected visual acuity and had a residual stromal scar. PMID- 7553094 TI - Laser Corneal Surgery. Proceedings of the 4th Annual Congress of the Summit International Laser User Group. Palm Beach, Florida, December 9-11, 1994. PMID- 7553096 TI - Excimer laser in situ keratomileusis for myopia. AB - Excimer laser in situ keratomileusis was done in 97 eyes of 59 patients to correct moderate and high myopia. Mean follow-up was 150 days. Mean preoperative myopia was -10.75 diopters (D). Mean reduction in myopia was -11.281 D (106.98%) at 1 month and -10.37D (98.417%) at 3 months. At 1-month, 41 eyes (42.27%) had a refraction within -1.00 D and 70 teeyes (72.16%) had a refraction within -2.00 D. At 3 months, 41 eyes (46.59% of 88 eyes) had a refraction within -1.00 D and 66 eyes (75% of 88 eyes) had a refraction within -2.00 D. Mean preoperative keratometric power was 44.00 D; mean postoperative keratometric power was 36.39 D. The keratometric change in power was 7.61 D. Preoperatively, 73 eyes (75%) had a spectacle corrected visual acuity of 20/40 or better; postoperatively, 64 eyes (65.63%) at 1 month and 75 eyes (77.01%) at 3 months reached 20/40 or better. The uncorrected visual acuity was 20/40 or better in 33 eyes (33.72%) at 1 month and in 49 eyes (49.96%) at 3 months. Mean ablation zone diameter was 4.92 mm. Astigmatism was treated separately in 10 eyes. Complications included optical zone decentration (3 eyes with more than 1 mm, epithelial implantation (1 eye), incomplete resection of the disc (2 eyes), severe hypotension, and irregular astigmatism (1 eye). PMID- 7553097 TI - Excimer laser in situ keratomileusis in 124 myopic eyes. AB - The development of corneal haze in some patients treated with excimer laser photorefractive keratectomy (PRK) and the variable predictability of the refractive cut in microkeratome keratomileusis in situ prompted us to evaluate excimer laser in situ keratomileusis (LASIK) for myopia. The Automated Corneal Shaper (Steinway-Chiron) was used to create a hinged corneal flap of 130 to 160 microns thick. PRK was performed on the lamellar bed with a Summit OmniMed laser. Minimum follow up was 3 months. Postoperative results and complications in the first 124 eyes are presented. LASIK is an effective method to correct myopia. PMID- 7553095 TI - Changing concepts in excimer laser corneal surgery. PMID- 7553098 TI - Laser in situ keratomileusis for myopia using manual dissection. AB - Forty-two eyes underwent interlamellar photoablation, a simple method of exposing the stroma to excimer laser treatment using common surgical instruments. A 8 mm diameter line up to 200 microns thick is raised manually from its bed. The stromal bed is reshaped with the excimer laser, and restoring the disc results in correction of high myopia. Our own nomogram was used. This method allows the use of less corticosteroids and final visual acuity recovers between 20 and 90 days postoperatively, time required for interface adherence and epithelial remodeling. Two treatment zones were used when 80% of the patient's myopia was greater than 10.00 diopters (D). One-year follow-up data allowed us to conclude that this method achieved results similar to excimer laser in situ keratomileusis (LASIK). PMID- 7553100 TI - Three-year results of photorefractive keratectomy for myopia. AB - We evaluated the results of single-step photorefractive keratectomy (PRK) on 35 consecutive myopic eyes (19 patients) with follow up of 3 years or more. The range of preoperative myopia was from -2.00 diopters (D) to -6.00 D with astigmatism of less than 1.50 D. The excimer laser used in this study was the ExciMed UV 200LA, (Summit Technology), which was set to a maximum correction of 6.00 D at a 5.0-mm diameter ablation zone. Uncorrected visual acuity better than 20/25 was achieved in 82.9% of eyes. Spectacle corrected visual acuity was equal to or better than that of preoperative levels in 94.3%. The difference between attempted and achieved correction was within 1.00 D in 60.0% of eyes. The myopic regression curve was Y = 3.679-0.6876 log (X). If the regression remains constant, we expect that refraction at 5 years after PRK would be -1.48 D. Trace corneal haze was present in 12 eyes (34.3%). At three years the most common subjective complaint was mild decreased night vision with asthenopia. The findings suggest that myopic regression can occur as long as 3 years after PRK. PMID- 7553101 TI - Excimer laser photorefractive keratectomy for myopia: two-year follow up. AB - This study, comprising 60 eyes with a follow up of at least 2 years, was designed to evaluate the efficacy and safety of excimer laser photorefractive keratectomy (PRK) for myopia in Japanese people. Eyes were divided into three groups: low myopia group (-2.00 diopters (D) to -3.00 D), 11 eyes; medium myopia group (-3.10 D to -6.00 D), 28 eyes; and high myopia group (-6.10 D to -14.00 D), 21 eyes. In 60 eyes, uncorrected visual acuity improved and spectacle corrected visual acuity remained unchanged. At 2 years, 100%, 75%, and 52%, respectively, of the low, medium, and high myopia groups, were within +/- 1.00 D of the attempted correction. Twenty-two eyes (37%) developed corneal haze of grade 2 or more. These data show that in low or medium myopia the predictability is good. But we think we have to pay attention to the strong haze, which seems to have developed more frequently in Japanese people than in previous reports of PRK in Caucasians. PMID- 7553099 TI - Excimer laser in situ keratomileusis. AB - We have been using a 193-nm excimer laser under protocol to ablate the corneal stromal bed after creating a hinged corneal flap with a microkeratome in order to reduce or eliminate refractive error. Thirty-one consecutive eyes are reviewed with a preoperative mean spherical equivalent refraction of -6.25 diopters (D) (range -3.50 to -11.75 D). Preoperative mean astigmatism was +0.87 D (range +0.25 to +2.75 D). At 6 months postoperatively, the mean spherical equivalent refraction was -0.50 D (range -3.50 to +2.00 D). Mean postoperative astigmatism was 0.64 D (range 0.25 to 3.50 D). A result within 1.00 D of attempted correction was achieved in 74.2% of eyes. Uncorrected visual acuity after a single procedure was 20/40 or better in 81% of eyes. Spectacle corrected visual acuity was unchanged in 26 (84%), decreased 1/2 line in 2 (6%), and increased 2 lines in 1 (3%). The incidence of postoperative complications was minimal. Excimer laser ablation in the stromal bed in combination with a hinged corneal flap seems to offer many advantages over excimer laser surface ablation with fewer complications. PMID- 7553102 TI - Simultaneous and sequential photorefractive keratectomy. AB - The purpose of this study was to show the safety, effectiveness, and predictability of simultaneous bilateral as compared to sequential unilateral photorefractive keratectomy in 273 normally sighted myopic eyes. An excimer laser with a fluence of 180 mj/cm2 at a frequency of 10 Hz was used at three ablation zones (5 mm, 6 mm, and 6.5 mm). There were no significant differences between the two groups' results. Mean refraction at six months was 0.34-0.96 D in group 1 simultaneous group and 0.32-0.95 D in group 2 sequential group. At 9 months after surgery, 84% of eyes had an uncorrected visual acuity of at least 20/40. The average pain score on the first postoperative day was 2.4 for group 1 simultaneous group and 2.3 for group 2 sequential group. No serious complications occurred. Despite the short follow up, simultaneous bilateral photorefractive keratectomy appears to be an effective and safe procedure for the correction of myopia. PMID- 7553103 TI - The effect of spherical photorefractive keratectomy on myopic astigmatism. AB - The aim of this report is to present the effects of spherical photorefractive keratectomy (PRK) by excimer laser on myopic astigmatism. Spherical PRK was performed in 96 eyes with myopia associated with astigmatism of between 0.75 and 2.00 diopters (D) using the Summit excimer laser. The reduction both in sphere and in cylinder was found to be highly statistically significant (P < 0.0001 for both). The mean reduction of cylinder was 46%. Astigmatic error of less than 2.00 D can be corrected significantly by spherical PRK. PMID- 7553104 TI - Photorefractive keratectomy for 1.5 to 18 diopters of myopia. AB - The purpose of this study was to evaluate the refractive outcome and the stability of refraction after excimer laser photorefractive keratectomy (PRK). Forty-five eyes were treated in three different refractive groups: low myopes, 23 eyes, -1.50 diopters (D) to -6.00 D; intermediate myopes, 14 eyes, -6.10 D to 10.00 D; and high myopes, 8 eyes, -10.10 D to -18.00 D. At 9 months after surgery the low myopes had a mean refraction of -0.14 D (range -1.00 D to + 1.50 D) and were easy to refract. Intermediate myopes mean refraction was +0.14 D (range 1.50 D to +2.50 D). In the high myope group the mean refraction was -0.47 D (range -10.00 D to +2.50 D) and four eyes (50%) lost more than two lines of spectacle corrected visual acuity. Refraction the high myopes was more difficult compared to the lower myopes. PRK produced good results for myopes from -1.50 D to -10.00 D. Myopes greater than -10.00D are not stable at 9 months after PRK and are difficult to refract. PMID- 7553106 TI - Excimer laser photorefractive keratectomy in Lebanon. AB - We report on 50 eyes of 50 patients treated by photorefractive keratectomy (PRK) using the Summit Technology OmniMed excimer laser. Follow up ranged from 3 to 9 months, and the eyes were divided into three groups on the basis of the initial myopia (< 6.00 diopters (D), 6.00 D to 10.00 D, and > 10.00 D). Mean preoperative spherical equivalent refraction in each group was -4.15 D, -7.88 D, and -12.00 D respectively, and -0.24 D, -1.41 D, and -1.70 D postoperatively. Ninety percent in group 1, 56.25% in group 2 and 35.71% in group 3 had a final refraction within 1.00 D of the attempted correction. Complications consisted of one case of infectious keratitis, medically treated with no sequelae; one case of significant loss of spectacle corrected visual acuity related to corneal haze; and one case of corticosteroid-induced elevated eye pressure controlled with topical treatment. In this series, PRK appeared to be effective and safe for the correction of myopia of less than -6.00 D. For higher myopia, other methods of treatment should be used. PMID- 7553105 TI - Symmetry of outcome after photorefractive keratectomy for myopia. AB - The aim of this study was to find out whether the outcome of excimer laser photorefractive keratectomy (PRK) in the second eye can be predicted from the results of the first, and to determine whether we should modify our therapeutic approach to the second eye according to the results of the first. Bilateral PRK was performed in 73 patients using the Summit Technology excimer laser. All patients included in this study had a follow-up of at least 12 months in both eyes. The change of refraction and the haze of the two eyes were compared. Sixty percent of patients had a difference in refraction between the two eyes smaller than 0.50 diopters (D), and 75% had a difference smaller than 1.00 D. The degree of symmetry in response to treatment was greater in patients with a preoperative spherical equivalent refraction of less than -6.00 D. Most patients had a similar degree of haze in both eyes. The results of our study demonstrate that a considerable degree of symmetry in the outcome of surgery exists between the two eyes of the same patient undergoing PRK, especially in low to moderate myopia. PMID- 7553107 TI - Photorefractive keratectomy for undercorrected myopia after radial keratotomy: two-year follow up. AB - We evaluated the clinical results of excimer laser photorefractive keratectomy (PRK) on 18 myopic eyes of 13 patients with residual myopia after radial keratotomy. Follow up was 24 months or more and the time interval between radial keratotomy and PRK ranged from 12 to 94 months. Patients were divided into two groups for analysis based on their preoperative myopia: group 1 (-6.00 diopters (D) or less), group 2 (more than -6.00 D). Uncorrected visual acuity at 2 years follow up was 20/40 or better in 86% (group 1) and 50% (group 2). Mean manifest refractive change was from -3.55 D to -0.91 D in group 1 and from -7.44 D to 2.50 D in group 2. Predictability within 1.00 D between attempted and achieved correction was 71.4% (group 1) and 80% (group 2). Most complications were transient except decreased spectacle corrected visual acuity in one eye--a complication also reported by others. Glare (61%) and decreased vision at night (50%) were the most troublesome subjective symptoms. These results show that previous radial keratotomy may not influence the myopic correction of PRK. Planned two-stage myopic correction (PRK after radial keratotomy) in high myopia can be considered a useful technique. PMID- 7553108 TI - Excimer photorefractive keratectomy after undercorrected radial keratotomy. AB - Radial keratotomy has been used for the treatment of myopia since 1979. Until recently, patients with undercorrected myopia had recourse only to repeated keratotomy. Now patients undercorrected after two or more radial keratotomy procedures can be treated with the excimer laser to reduce the residual myopia. Nineteen eyes of 17 patients with undercorrected myopia after repeated radial had excimer photoradial keratectomy performed. The mean residual spherical equivalent refractive error after radial keratotomy was -2.74 +/- 1.06 diopters (D). This was further reduced after PRK. Final uncorrected visual acuity ranged from 20/20 to 20/70. Excimer laser PRK offers a safe and more controlled method of treating residual after radial keratotomy. PMID- 7553110 TI - Multizone photorefractive keratectomy for myopia of 9 to 14 diopters. AB - We performed photorefractive keratectomy (PRK) using new multizone software of the Summit Omnimed excimer laser (Summit Technology, Inc., Waltham, Mass.) on thirteen highly myopic eyes. Average preoperative myopia was -10.94 diopters (D) (range -8.60 D to -13.50 D). Mean postoperative spherical equivalent refraction was -0.15 D (range +1.50 D to -4.00 D), at 3 months postoperatively. Uncorrected visual acuity of all the eyes improved markedly. There was no serious complication except one eye that lost two lines of spectacle corrected visual acuity possibly due to decentration. Though we need longer follow-up data and a larger sample size, the new multizone software seems to be effective for correcting high myopia. PMID- 7553111 TI - Multizone photorefractive keratectomy for myopia of 8 to 23 diopters. AB - Multizone photorefractive keratectomy (PRK) was performed on 124 eyes of 69 patients for the treatment of high myopia using 4.5 mm, 5.0 mm, and 6.0 mm diameter ablation zones. Preoperative spherical equivalent refractions ranged from -8.00 diopters (D) to -22.5 D (mean +/- SD, -12.3 +/- 3.9 D). Mean attempted ablation depth was 109.5 +/- 37.8. Follow up ranged from 6 to 9 months. An uncorrected visual acuity of 20/60 or better was achieved in 63% of eyes and 20/40 or better in 48% of eyes. Sixty-seven percent of eyes achieved their pretreatment spectacle corrected visual acuity or were within two Snellen lines of it at 6 months postop. Subepithelial haze was mild in 23% of eyes and moderate in another 34% of eyes. This cleared without a trace in almost all of these eyes within 6 months. Twenty-five percent of the eyes had dense scars and required reablation. PMID- 7553109 TI - Photorefractive keratectomy for myopia of 6 to 12 diopters. AB - To conduct a prospective, nonrandomized clinical trial to assess the efficacy, stability and safety of the use of excimer laser photorefractive keratectomy (PRK) to correct myopia of greater than -6.00 diopters (D) in Singapore. Two hundred thirty-three eyes of 176 patients with a mean preoperative spherical equivalent refraction of -8.70 +/- 2.3 D (range, -5.75 D to -18.13 D) underwent PRK with a 193nm ArF excimer laser (ExciMed, Summit) for myopic corrections of between -6.20 D and -11.90 D. The first 66 of 233 eyes that reached at least 1 year of postoperative follow up are reported. One year postoperatively, the mean manifest spherical equivalent refraction was -1.50 D +/- 1.90 D (range -6.25 D to +3.25 D); 34% of eyes were within +/- 1.00 D of attempted correction; uncorrected visual acuity was 20/40 or better in 75% of eyes. No eyes lost two or more Snellen lines of spectacle corrected visual acuity. Central corneal haze was mild in 20% of eyes and moderate in 3% of eyes. Five eyes developed ocular hypertension that subsequently resolved with treatment. Excimer laser PRK is reasonably effective and safe in the treatment of -6.00 D to -12.00 D of myopia. However, it is less accurate than PRK in eyes with low to moderate myopia and is more likely to result in significant corneal haze. PMID- 7553112 TI - Corticosteroids for reversal of myopic regression after photorefractive keratectomy. AB - With the aim of reversing myopic regression laser photorefractive keratectomy (PRK) after excimer, the effect of high-dose topical corticosteroids in modulating changes in refraction and corneal transparency was assessed prospectively. Twenty-four eyes of 23 patients (mean preoperative spherical equivalent refraction -7.36 diopters (D); range, -4.12 D to -19.25 D), demonstrating myopic regression ranging between 1.00 D and 6.88 D (mean: 2.64 D), were treated according to the same therapeutic protocol. Changes in corneal haze, corneal topography, and refraction were evaluated over 6 months. Eighteen eyes (78.26%) showed 1.00 D or more reversal of myopic regression. Sixteen eyes (69.56%) regained the attempted correction present when corticosteroids were first stopped (+/- 1.00 D). The difference between mean refraction before reintroducing corticosteroids (-2.64 D; range, -1.00 D to -6.88 D) and when stabilization occurred (-0.60; range, +1.50 D to -4.50 D) was statistically significant (P < 0.01). The improvements in corneal haze and spectacle corrected visual acuity were also significant (both P < 0.01). Stabilization occurred between 1 to 4 months after surgery (mean, 2.22). In five eyes (21.74%) increased intraocular pressure of more than 20 mm Hg were reduced with beta-blockers. Topical corticosteroid therapy can modulate refractive changes after PRK, appearing to reduce myopic regression. However, a longer follow up will be necessary to determine the final refractive outcome of these eyes. PMID- 7553113 TI - Management of late corneal haze following photorefractive keratectomy. AB - Subepithelial corneal haze and myopic regression are potential complications following excimer photorefractive keratectomy (PRK). There are many medical and surgical methods of managing this haze. We present a 37-year-old male myope who underwent PRK and subsequently developed central corneal haze late in the postoperative course. The haze was managed initially with topical medications with limited success. Mechanical superficial keratectomy was done to remove the superficial scar tissue but the haze returned necessitating repeat excimer laser PRK, using a transepithelial technique. The haze did not recur. Both mechanical superficial keratectomy and repeat excimer laser ablation may ameliorate haze. Success of these procedures may depend on the morphology of the haze and the patient's individual wound healing response. PMID- 7553114 TI - An empirical model of hyperopic shift with corticosteroid modulation and refractive power prediction after photorefractive keratectomy. AB - Data from a selected sample of 158 patients who received excimer laser photorefractive keratectomy (PRK) in our center and obtained a desirable refractive outcome at 6-months were analyzed retrospectively to establish an empirical model of refractive power after PRK. This model, generated by a computer-assisted regression program, is presented as a multivariate non-linear quadratic equation. Since the coefficients of each variable (attempted correction and postoperative days) are statistically significant, the estimated influence of the variables on the postoperative refraction from this regression model is reliable and consistent. Therefore, this model has been used to direct the use of corticosteroids for modulating post-PRK refraction. The predictability of this model has also been corroborated by separate data from 1-year follow up of PRK recipients. The recipients whose refraction at 6 months agree with the calculation from this model have obtained an excellent outcome; while those whose refractive outcome deviates from this model have gotten undesirable results. PMID- 7553115 TI - Increasing the number of photorefractive keratectomy procedures from a single excimer laser gas fill. AB - Excimer laser ablative procedures are an expensive proposition for both the patient and the user. The high capital expenditure and the cost of consumables, notably the argon-fluoride gas, makes it imperative to achieve maximum utilization in terms of number of cases treated per fill of gas. This is particularly true in countries like India. The maximum number of cases can be treated by understanding not only the limitations of each fill of gas, but by proper patient scheduling and modifications to the treatment protocol. This paper outlines our modified protocol and the patient scheduling software that we developed to meet these criteria. PMID- 7553116 TI - The use of corticosteroid/beta-blocker combinations in the management of regression after PRK for high myopia. AB - After nearly 8 years of clinical experience, and with conflicting evidence from trials with and without corticosteroids, there is still no single, universally agreed postoperative management regimen for photorefractive keratectomy (PRK). All agree that corticosteroids will reverse sudden regressions, and so most still use varying corticosteroid regimens. In Hong Kong we are faced with many patients whose myopia is greater than -6 diopters (D), and we agree with the general observation that the regression/haze rate is much greater for these higher errors, and that increased intraocular pressure (IOP) seems to be associated with increased haze. We placed our high myopia group (greater than -6.00D) on corticosteroid/beta-blocker combinations and observed a much lower incidence of haze and regression from this group. We use this regimen now on all our patients and are achieving more accurate endpoint refraction and less haze. Our hypothesis is that lowering the IOP to less than 16 mm Hg reduces keratocyte migration by "tightening" the corneal lamellae, thus dampening the healing response. Case studies are presented to illustrate this relationship. PMID- 7553118 TI - Patient satisfaction following photorefractive keratectomy for myopia. AB - This study was conducted to evaluate the subjective response of patients who underwent photorefractive keratectomy (PRK) for myopia with the Summit Technology UV 200 LA excimer laser (Summit Technology, Inc., Waltham, Mass.) (3.5 to 5.0 mm diameter ablation zone) 36 months previously. One-hundred thirteen patients with preoperative myopia between -1.50 diopters (D) and -6.50 D with an ablation diameter of 3.5 mm to 5.0 mm answered a questionnaire at the 36-month examination. Sixty-two percent of patients had both eyes treated. Halo problems were experienced always by 34% of patients in the group, 26% sometimes, and 40% never. Permanent night vision problems were experienced by 40%; 30% reported them sometimes and 30% reported no problems at all. Sixty percent never needed to use glasses after treatment, 30% sometimes did, and 10% always wore glasses. Seventy percent of patients thought the results were very good; the remaining 30% experienced different degrees of halo and night vision problems. PMID- 7553117 TI - Effect of photorefractive keratectomy on visual functioning and quality of life. AB - The authors present the results of the psychosocial outcomes of 45 patients undergoing excimer laser photorefractive keratectomy (PRK). The patients were evaluated by means of self-report questionnaires preoperatively and 6 months after surgery to determine the effect of uncorrected vision change on the patients quality of life. The measurements include the following variables: clinical end points (spherical equivalent refraction); visual function (night driving, day driving, near vision, far vision, glare disability); functional status (physical, social, and role functioning, mental status); general well being (health perceptions, personal well-being, overall quality of life); and satisfaction with surgery (expectations, satisfaction with medical staff, postoperative pain, satisfaction with treatment outcomes). Preliminary results indicate that changes of uncorrected vision after PRK are reflected by improvement in each of the quality-of-life functions. PMID- 7553119 TI - Automatic eye tracker for excimer laser photorefractive keratectomy. AB - Acute overlapping of successive laser pulses onto the cornea during photorefractive keratectomy (PRK) is important to avoid refractive distortions. Most excimer laser systems performing corneal ablation lack control of the patient's eye movements and they cannot track the target corneal zone. We developed an eye-tracker based on television monitoring of the pupil and on automatic electro-mechanical deflection of the laser turning mirror, and we applied it to the ExciMed UV200 ArF work station (Summit Technology, Inc., Waltham, Mass.). Basic components are a black and white CCD camera and two fast stepping motors. The circuitry for target discrimination and tracking, and the optical imaging system are designed specifically. The tracker assembly does not interfere with the laser beam path nor with the operator's observation. Tracking of the pupil has been successfully achieved on different color irides, with an accuracy better than 0.1 mm in a 6 x 6 mm2 tracking field. Response time is less than 100 ms. Recordings of eye movements during PRK are presented. Tracking ablations have been performed on moving test eye-balls with plastic corneas. The proposed automatic system appears to be a reliable and effective method for the compensation of patient eye movements appears to be a reliable and effective method during PRK procedures. PMID- 7553120 TI - Photorefractive keratectomy for myopic astigmatism using the emphasis erodible mask. Spanish User Group. AB - Photorefractive keratectomy (PRK) using the Summit emphasis erodible mask was performed on 229 eyes with myopic astigmatism. To analyze the efficacy and safety of the method, we evaluated the following: spectacle corrected visual acuity and refractive error as parameters of efficacy; corneal clarity, decentration of the ablation zone and complications as parameters of safety. Despite the short follow up (the longest was 12 months) our results show that the emphasis erodible mask appears to be an effective surgical method for correcting myopic astigmatism. However, we found a great number of complications and subjective symptoms that worsened the visual outcome. We conclude that the emphasis erodible mask has satisfactory efficacy but unsatisfactory safety. PMID- 7553121 TI - Holmium: YAG laser to treat astigmatism associated with myopia or hyperopia. AB - The Summit Technology, Inc. (Waltham, Mass.) holmium:YAG laser was used to treat astigmatism in 31 myopic and 8 hyperopic eyes. There was slow but continuing regression of effect even 1 year after treatment. This regression was particularly seen in hyperopic astigmatism, larger amounts of myopic astigmatism, and younger patients. In some cases keratometry readings returned to their pretreatment values. Males and females had similar results. The mean myopic shift in 9 myopic astigmatism eyes at 9-month follow up was 1 D. PMID- 7553123 TI - Laser corneal surgery: 1981-1995. PMID- 7553122 TI - Holmium:YAG laser thermokeratoplasty for correction of astigmatism. AB - The effect of astigmatic Holmium:YAG laser thermokeratoplasty in three eyes with previous surgery (extracapsular cataract extraction, penetrating keratoplasty and failed extracapsular cataract extractionntric photorefractive keratectomy 1 to 7 years previously) was evaluated. In the extracapsular cataract extraction eye the effect of a four-spot laser thermokeratoplasty regressed to almost zero within 1 week. In the penetrating keratoplasty and photorefractive keratectomy eyes an eight-spot laser thermokeratoplasty produced an enormous overshoot in astigmatic power of about 15.00 diopters (D), decreasing within 5 months to almost zero in the eye with penetrating keratoplasty and to 4.00 to 5.00 D corneal astigmatic power in the photorefractive keratectomy eye after 8 months of observation. PMID- 7553124 TI - Caring for mother & baby. Keeping safe in the home. PMID- 7553125 TI - Is midwifery safe in the embrace of the UKCC? PMID- 7553126 TI - 15th Dame Rosalind Paget Memorial Lecture. The social meaning of midwifery. PMID- 7553127 TI - "Voting for change". Report of the RCM annual conference, 1995--Belfast. PMID- 7553128 TI - Peanut allergy. PMID- 7553129 TI - Ethics & midwifery practice. PMID- 7553133 TI - Accessory carotid body within the parathyroid gland III of the chicken. AB - In the chicken, the cranial and caudal parathyroid glands (parathyroid gland III and IV), which are connected to each other, are located adjacent to the carotid body. In the present study, we found that a mass of glomus cells surrounded by a thick layer of connective tissue was frequently distributed within the parathyroid gland III. The glomus cells in the parathyroid III, as well as those of the carotid body, expressed intense immunoreactivity for serotonin, chromogranin A, and tyrosine hydroxylase but no immunoreactivity for neuropeptide Y. The cells possessed long cytoplasmic processes containing dense-cored vesicles of 70-220 nm in diameter, and were in close association with sustentacular cells. In and around the glomus cell clusters of the parathyroid III, dense networks of varicose fibers showed immunostaining with the monoclonal antibody TuJ1 to a neuron-specific class III beta-tubulin isotype, c beta 4. Furthermore, the distribution was also detected of numerous galanin-, vasoactive intestinal peptide (VIP)-, substance P-, and calcitonin gene-related peptide (CGRP) immunoreactive fibers. PMID- 7553131 TI - Comparison of the pattern of expression of Leu-M1 antigen in adenocarcinomas, neutrophils and Hodgkin's disease by immunoelectron microscopy. AB - The mouse monoclonal antibody anti-Leu-M1 (CD15) recognizes the carbohydrate determinant lacto-N-fucopentaose III, an oligosaccharide believed to be involved in cell-cell interactions. Anti-Leu-M1 is used in surgical pathology as an aid in the diagnosis of Hodgkin's disease. Additionally, adenocarcinomas derived from various organs stained positively with anti-Leu-M1 at the light microscopic level. Since mesotheliomas do not display positive reactivity to this antibody, Leu-M1 is clinically useful as part of a panel of antibodies in distinguishing adenocarcinomas from mesotheliomas. Previous work was carried out using post embedding protein A-gold immunocytochemistry on thin sections embedded in Lowicryl K4M from a patient with Hodgkin's disease of the nodular sclerosing type; intense and precise labeling by gold particles was revealed in cytoplasmic granules, which were often clustered in a perinuclear location, in the Golgi apparatus, and focally along the plasma membrane of Reed-Sternberg cells. Moreover, polymorphonuclear leukocytes demonstrated similar labelling along the plasma membrane and over cytoplasmic granules. To define precisely the intracellular localization of Leu-M1 in human adenocarcinomas, we have performed post-embedding immunoelectron microscopy with the protein A-gold technique on sections embedded in Lowicryl K4M from neoplasms of the lung, stomach, colon, and breast. The pattern of labeling by gold particles indicative of Leu-M1 binding varied in adenocarcinomas of the different organs. PMID- 7553132 TI - The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution. AB - We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoy-fixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some beta-oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver. PMID- 7553134 TI - Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining. AB - The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3 M MgCl2 with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3 M MgCl2, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule. PMID- 7553135 TI - Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage. AB - The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4 sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage. PMID- 7553136 TI - Nitric oxide synthase is localized predominantly in the Golgi apparatus and cytoplasmic vesicles of vascular endothelial cells. AB - The localization of nitric oxide synthase (NOS) in vascular endothelial cells of submucosal blood vessels from the guinea-pig ileum was examined using NADPH diaphorase histochemistry at the light microscopic level, and endothelial NOS immunohistochemistry at the light and electron microscopic level. The pattern of staining observed following NADPH diaphorase histochemistry and endothelial NOS immunohistochemistry was identical. Endothelial cells of the arterioles, capillaries and venules showed small patches of intense, perinuclear staining. Under the electron microscope, endothelial NOS immunoreactivity was found predominantly in association with the Golgi apparatus and with the membranes of some vesicles. Small regions of the plasma membrane and the rough endoplasmic reticulum also showed some immunoreactivity. The presence of NOS in the Golgi apparatus and in vesicles raises the possibility that NOS may be exteriorized by endothelial cells, and hence that nitric oxide is synthesized extracellularly. PMID- 7553138 TI - Presence of the intermediate filaments cytokeratins and vimentin in the rat corpus luteum during luteal life-span. AB - The presence of the intermediate filament (IF) proteins cytokeratins and vimentin in corpus luteum (CL) and other parts of the ovary from adult pseudopregnant rats was investigated using immunohistochemistry and immunoblot analysis. To induce pseudopregnancy, female rats were mated with sterile male rats. The mating procedure induces a prolonged luteal life-span of 13 +/- 1 days. Positive staining for cytokeratin could be seen in CL, in the theca layer of follicles, and the ovarian surface epithelium with the broad-spectrum monoclonal antibody cocktail AE1/AE3. Weak staining was also seen in CL with antibodies against cytokeratins 8 and 18. A similar distribution was also seen for vimentin, which furthermore was detected in blood vessels. No changes in staining intensity was seen in CL of different luteal age. The strong staining for vimentin in CL was confirmed by immunoblot analysis, where one main band of 57 kDa was observed from day 1 to day 19 of pseudopregnancy. Expression of the IF proteins investigated seems to start in the newly formed CL and the continuous expression indicates that they are not directly regulated by luteal steroids. PMID- 7553137 TI - Ecto-5'-nucleotidase is expressed by pericytes and fibroblasts in the rat heart. AB - Ecto-5'-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5'-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5' nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5'-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5'-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5'-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5'-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings. PMID- 7553141 TI - [Isolation and expression of recombinant diagnostic antigen gene of Schistosoma japonicum]. AB - The sera of patients with chronic infection of Schistosoma japonicum (Sj) were used for screening the cDNA expression library constructed from Sj adult worm, and an antigen-positive clone Sjgt4 which expressed an 148 KD fusion protein was identified. The inserted Sj cDNA fragment was amplified by PCR and subcloned into the vector pGEX with high level of expression. A positive clone SjpG5 which expressed a fusion protein with a molecular weight of 58 KD was obtained. When the fusion protein was used for detection of Sj patients' sera by Western blot, 24 out of 25 sera were positive, a positive rate of 96%. PMID- 7553139 TI - [Prevention and treatment of tropical diseases]. PMID- 7553143 TI - [Effects of cytokine-activated macrophages on intracellular leishmania]. AB - The effects of activated macrophages on Leishmania donovani amastigotes and nitrite (NO2-) release were determined by in vitro inducement of macrophage activation with recombinant IFN-r, TNF-alpha, and rIL-3. The results indicated that rIFN-r or rTNF-alpha at 40,000U/L was able to induce a significant but modest level of macrophage activity, and synergistic effect of rIFN-r and rTNF alpha was apparent in the induction of macrophage activation. 48 hours after macrophages were induced with both rIFN-r and rTNF-alpha, the percentage of the macrophages infected by Leishmania donovani was 16%, and the mean number of the parasite per infected macrophage was 1.2 +/- 0.04. There was a strikingly significant difference in comparison with the control group, in which the infection rate of macrophages was 62% and the mean number of the parasite per infected macrophage was 6.8 +/- 0.10. However, no significant difference was noted between rIL-3 at 1/100 concentration, alone or combined with rIFN-r, and the control in leishmanicidal activity of macrophages. An increase of NO2- in the culture supernatants was paralleled by the ability of various cytokine-activated macrophages to kill the intracellular parasites. PMID- 7553140 TI - [Preliminary study of a recombinant polyvalent vaccine of Plasmodium falciparum and its immunological activity]. AB - A hybrid gene named HGFC coding three protective antigenic epitopes of Plasmodium falciparum and two exogenous T cell activating epitopes was designed and synthesized. A multicopy hybrid gene named HGF-CAC was also constructed. The two genes were cloned into expression vector pWR450-1 and the hybrid fusion proteins containing forgine antigens and beta-galactosidase were expressed in E. Coli. The molecular weights of the fusion proteins were 65KDa and 77KDa respectively. The expression rate was about 35% of total bacterial proteins. The fusion protein could react specifically with mouse and rabbit antibodies against antigens of Plasmodium falciparum. The rabbit immune serum against the purified fusion protein could specifically recognize the antigens of Plasmodium falciparum and effectively inhibit the in vitro development of the parasites. The inhibitory capacity of the immune sera to parasite invasion was enhanced as the amount of the sera increased and the incubation time of the sera with the parasites was prolonged. After 72h incubation at 20% concentration with the parasites, the serum suppressed the multiplication of parasite to a level of 82% and caused degeneration and death of the parasites. The results indicated that the recombinant hybrid antigen of Plasmodium falciparum has immunological activity and protectivity. It is probably a candidate malaria vaccine. PMID- 7553130 TI - The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view. AB - The NADPH oxidase complex catalyzes the formation of superoxide (O2.-) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed. PMID- 7553145 TI - [Detection of enteroviral RNA in paraffin-embedded myocardial tissue from patients with Keshan by nested PCR]. AB - Enteroviral RNA in paraffin-embedded myocardial specimens from 21 patients with Keshan disease and 21 patients with cerebral trauma was investigated by nested polymerase chain reaction. Of the 21 patients with Keshan Disease, 18 (85.7%) showed positive findings. Their myocardial tissues were infected by enterovirus previously. The results suggest that enterovirus infection is the major cause of Keshan disease in the Chuxiong district, Yunnan province. PMID- 7553142 TI - [Long-term effect of leprosy control in both highly endemic prefectures, Weifang of Shandong and Wenshan of Yunnan]. AB - The long-term effect of leprosy control was studied in two prefectures of different geographical conditions and socio-economic status. During 1955-1985, the incidence rate in Weifang of Shandong decreased by 94.5% (9.1-0.5/10,000) and became evident after the introduction of MDT in 1986 (0.04/10,000, 1993). In Wenshan of Yunnan, however, it remained at about 20/10,000 before 1980. After the introduction of rifampin plus dapsone in 1979 and fixed duration MDT in 1986, it decreased by 94% (20-1.2/10,000), whereas the detection rate remained constant (+5/100,000). In comparison with the features of new patients detected since 1980, we noted a steady increase of MB rate and voluntary reporting, and a decrease of deformity rate, in the last 8 years in Weifang. It is estimated that by the year 2000 Weifang may have the detection rate of 0.001260/10 thousand and the incidence rate of 0/00757/10 thousand in contrast to the detection rate of 0.2980/10 thousand and the incidence rate of 0.316/10 thousand in Wenshan. PMID- 7553144 TI - [Rapid detection of platelet glycoprotein IIb, IIIA gene variety by single strand conformation polymorphism analysis]. AB - Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelet defective function. Most of the molecular defects of GT identified in recent years have been caused by point mutation in the platelet glycoprotein (GP) IIb, IIIa genes. Trying to rapidly detect point mutation (or subtle variety) in GPIIb, IIIa genes, we have used single strand conformation polymorphism analysis (SSCPA) and studied the gene structure of platelet GPIIb. IIIa from normal controls and patients with Glanzmann thrombasthenia. After DNA preparation from peripheral blood, all the exons of GPI-Ib, IIIa were amplified by PCR. Denaturated PCR products were separated on mini polyacrylamide gel and stained by silver staining method in the Phast System. Only the PCR products with different migration on the gel were sequenced. 2 GT patients with point mutations and 1 GT patient with 16 bp deletion were found in our study. At the same time, we determined 3 polymorphisms in GPIIb gene. We conclude that SSCPA is a rapid, sensitive and effective method for detecting platelet IIb, IIIa gene variety. PMID- 7553146 TI - [Role of energy metabolism in nutrition management of critically ill patients]. AB - Indirect calorimetric measurements were made with a MedGraphics Critical Care Monitor (CCM) desktop analysis system in the observation of critically ill and malnourished patient's energy expenditure. In 15 critically ill patients, predicted energy requirements based on 1.75 times BEE calculated by Harris Benedict formula or corrected Harris-Benedict formula averaged 32.7% and 27.8% greater than metabolic expenditure measured by indirect calorimetry respectively. In the 20 unstressed malnourished patients, predicted energy requirements based on the Harris-Benedict (BEE) formula averaged 15% to 20% higher than metabolic expenditure measured by indirect calorimetry. When the critically ill patients' total energy intakes were 1.2 times resting energy expenditure, their nutritional state could be maintained in normal conditions. While the malnourished patients were provided with 1.5 x REE in energy intake, the malnourished state could be reversed. We believed that the critically ill and malnourished patients' energy expenditures are better measured than predicted and their nutritional regimens should be guided under the computerized indirect calorimetry. PMID- 7553147 TI - [Causes of postoperative recurrent varicose veins of lower extremities]. AB - We analysed venograms of recurrent varicose veins in 180 cases (206 lower limbs). Deep vein valvular insufficiency resulted in varicose veins in 52.9% cases, and post-thrombotic syndrome in 18.9%. The causes of postoperative recurrent varicose veins varied: blindly performed single superficial veins operation, low ligation of the long saphenous trunk, incomplete stripping of varicose veins, missing of the short varicose saphena or incomplete ligation of the perforating veins and failure of deep vein valvular repairs. Venography is of value in making correct diagnosis and choosing appropriate operations. PMID- 7553148 TI - [Changes of proteoglycans in lumbar intervertebral disc of bipedal rats with aging]. AB - Using a computer based image analysis system and histological sections stained with Safranin O, we investigated systematically the variation of proteoglycans (PG) contents in the low lumbar discs of bipedal rats. Topographically, the PG concentration increased from the outer annulus to the inner nucleus pulposus region, which possesses the highest PG concentration. The PG content decreased with age, and a significant loss of PG was observed in the lumbar discs of bipedal rats as compared with controls, especially in the nucleus pulposus. These observations support the view that the higher abnormal mechanical stress accelerates disc degeneration. PMID- 7553149 TI - [Captopril in improving vascular structure and lowering blood pressure]. AB - SHRs were given captopril 20 mg/kg/day (group A, n = 13) and 100 mg/kg/day (group B, n = 17) from intrauterus period to 16 weeks of age, then the treatment was removed. Experiments were carried out at 40 weeks. SBP, left ventricular mass/body weight ratio, wall/lumen ratio of branch III mesenteric and renal artery were determined. The perfusion pressure response to alpha 1 adrenergic agonist (phenylephrine) of resistance blood vessels in a hindquarter model in the presence of Nw-nitro-1-arginine-methyl ester (LNAME) or L-arginine were examined. Untreated SHR (n = 16) and untreated WKY (n = 17) served as controls. Both doses of captopril treatment completely prevented hypertrophy of vascular vessels to some extent comparable to that of the untreated WKY. But their SBP was still significantly higher than that of the untreated WKY. The curves of perfusion pressure responding to incremental doses of phenylephrine shifted rightward in the captopril treatment groups in a dose dependent manner. The curves of high dose group were almost identical to those of WKY, markedly different from that of the untreated SHR. Captopril decreased the over enhanced vasoconstrictor effect of LNAME in SHR. The attenuated vasoconstrictor effect by L-arginine was greatly augmented by captopril, suggesting that captopril improves the function of resistance of vessels by mediating endothelial cells. Captopril-induced alteration in vascular structure and function may be separated from its hypotensive effect. Clinically, continuing administration of captopril may be beneficial to the improvement of the vascular system in those blood pressure unresponsive patients. PMID- 7553150 TI - [An experimental study of middle ear implantable hearing device]. AB - The partially middle ear implantable hearing device was designed and tested in 7 cats with conductive hearing loss and 5 cats with mixed hearing loss. It was composed of sound processor, connector and ossicular vibrator. The ossicular vibrator was made from piezoelectric ceramic bimorph which was constructed in the form of flat chip 6mm in length, 1mm in width and 0.5mm in thickness. The incus of cat was removed and the lip attachment of the vibrator was attached to the head of the stapes. The base of the vibrator was fixed to the bone wall of bulla with medical glue. The average values of the response threshold of ABR evoked by click via this vibrator got reduction of 37dB and 39dB respectively in conductive hearing loss group and mixed hearing loss group. The gain curve of hearing was flat in the range from 0.5 to 8kHz frequencies. The ABRs evoked by click via ossicular vibrator was similar to that recorded preoperatively in the wave shape. This result indicates that this device is a highly efficient sound conducting divice to inner ear. PMID- 7553151 TI - [Coriaria lactone on gamma-aminobutyric acid secretion and glutamic acid decarboxylase and its receptor binding in rat]. AB - The effects of coriaria lactone (CL) on gamma-aminobutyric acid (GABA) secretion, glutamic acid decarboxylase (GAD) activity and glutamate (Glu) receptor binding were studied by using cultured neurons, P2 component and synaptic membranes from rat cerebral cortex. It was found that GABA secretion was depressed by CL, the depression rate was 8.3%, 10.6%, 14.5% at 12h 24h 48h respectively. GAD activity was depressed by CL in concentration of 0.15-150 mumol/L. The depression rate was 1.32%, 5.96%, 13.24%, 18.76% respectively. The Glu receptor binding capability was decreased by CL with obvious dose-effect relation in the extent of 2.8-350 mumol/L; the decrease rate was 4.4%, 12.7%, 15.2%, 19.5% respectively. The difference was significant as compared to control (P < 0.01). PMID- 7553153 TI - [Gene therapy in combating hepatitis B virus infection]. PMID- 7553154 TI - [Human interleukin-2 gene transfer and expression inhibits hepatitis B virus surface antigen expression and enhances LAK activity of peripheral blood mononuclear cells of normal adults]. AB - Using molecular cloning techniques, we constructed and transfected human interleukin-2 gene expressive retroviral vector into a packaging cell line PA317. Geneticin resistant cell clones were identified and pseudoviral particles titred as 3.75 x 10(6)CFU/ml in their culture supernatants. Human interleukin-2 expression level was 5.71IU/ml for NIH3T3, 6IU/ml for both 2. 2. 15 and peripheral blood mononuclear cells after infection with pseudoviral particles with 8 of multiplicity of infection (M. O. I). Low level interleukin-2 expression potentially inhibited hepatitis B virus surface antigens expression of 2. 2. 15 cell line and enhanced cytotoxicity of peripheral blood mononuclear cells against K562 and Raji as target cells, but neither pseudoviral particles without interleukin-2 cDNA nor reccombinant interleukin-2 at 6 IU/ml. These results indicated that retroviral vector-packaging cell line can efficiently transfer and express interleukin-2 cDNA in human somatic cells and low level expression of interleukin-2 potentially inhibits hepatitis B virus surface antigens expression and enhances LAK activity of peripheral blood mononuclear cells of normal persons. PMID- 7553155 TI - [Inhibition of hepatitis B viral gene expression and replication in vitro by targeted antisense oligonucleotides]. AB - We chose the 2.2. 15 cells as an in vitro cell culture assay system, and identified the surface antigen subtype of hepatitis B virus (HBV) DNA in the cells, by using the amplification of polymerase chain reaction and directed sequencing of amplified products. According to the result of the sequencing, a 16 mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASON) directed against the HBV U5-unique region was synthesized and then linked with two liver targeting ligands. The galactosylated human serum albumin coupled poly-L-lysine and the galactosylated poly-L-lysine. The effect of different modifications of ASONs on the expression of HBV gene was compared by using the 2.2. 15 cells. In the same experimental conditions, the inhibitary effects of surface antigen (HBsAg) and antigen (HBeAg) by PS-ASON were 70% and 58% respectively, and those of HBsAg and HBeAg by ligand-PS-ASONs were 90%-96% and 78%-82%. In the same time, the amount of HBV NDA in culture supernatant and cells was depressed significantly. Thus, the ligands targeted ASON to the hepatocytes were more effective inhibitors of HBV gene expression. ASONs were effective and specific inhibitors of HBV replication and expression, caused the dose-dependent inhibition of viral proteins and had no effect on cellular alpha-fetoprotein synthesis and no cytotoxicity. PMID- 7553156 TI - [Expression of 12 antibody escape mutants of hepatitis B virus surface antigen gene in mammalian cell by using an Epstein-Barr based vector]. AB - A 900bp DNA fragment of hepatitis B virus surface antigen gene by digesting BamHI and HpaI sites on the open reading frame of HBV DNA on plasmid pEcob6 containing double copes of HBV DNA was cloned into SmaI site on the phagmide vector pBluescribt SK +. From this recombinant vector, 12 hepatitis B virus surface antigen gene mutants were obtained by oligonucleotide-mediated site directed mutagenesis. The expression vector-pMEP4HBS mutants are constructed by sub cloning all of these mutant fragments of hepatitis B virus surface antigen gene on pBluescritSK+HBSM into BsmHI and KpnI sites on an Epstein-Barr virus based on eukaryotic expression vector-pMEp4. The vector pMEp4HBSMs were transfected to human hepatocellular carcinoma cell line-HepG2 by calcium phosphate mediated transfection, and resistant cell clones were obtained 3 weeks after selecting by hygromycine B. The results of detection of HBsAg excreted by resistant cell clones with monoclonal antibody to HBsAg showed that all antibody escape mutants of HBsAg except mutant 145R, a substitution of arginine for glycine at amino acid 145 position in HBsAg, were positive. PMID- 7553157 TI - [Rearrangements and fusion gene of AML1 and MTG8 in acute myeloid leukemia M2b]. AB - The t(8;21) reciprocal chromosomal translocation is frequently associated with M2b type of acute myeloid leukemia (AML). Recently, two genes, MTG8 on chromosome 8 and AML1 on chromosome 21, were found. The t(8;21) translocation resulted in rearrangements of the two genes and formation of AML1/MTG8 fusion gene. To clarify the molecular characteristics of AML-M2b, we studied 41 patients with AML M2b. By Southern blot and hybridization, the rearrangements of AML1 and MTG8 genes were detected in 24 of 30 and 22 of 28 patients, respectively. By means of reverse transcription and polymerase chain reaction (RT-PCR), the AML1/MTG8 chimeric transcript was found in all 37 patients. In 4 patients with AML-M2b of normal karyotype, AML1/MTG8 fusion mRNA and/or rearrangements of AML1 and MTG8 genes were detected. However, among 31 patients with other types of AML, these abnormalities were found in only one patient with AML-M6. These results suggest that rearrangements of AML1, MTG8 genes and/or AML1/MTG8 fusion gene could be regarded as gene marker of AML-M2b that can be applied in the diagnosis and monitoring of therapy and minimal residual disease. PMID- 7553152 TI - [Prospects of clinical uses of peptide growth factors]. PMID- 7553158 TI - [Portal vein supply and embolization therapy for hepatocellular carcinoma]. AB - To prove the necessity and feasibility of portal vein embolization for the treatment of hepatocellular carcinoma (HCC), We observed the tumor vascularity and portal vein supply by vascular casting, Doppler pulse sonography and iodized oil embolization in 178 HCC noduls. Portal vascularity was found in 75.6% of noduls (34/45). In few noduls, which were smaller than 3cm in diameter, portal branches existed only without hepatic artery branches (7/35). Tumor portal blood supply was measured in 54.1% of noduls (59/109) by using Dopplar sonography. Portal tumor branch embolization was performed in 24 of patients with unreseatable HCC, all tumor noduls being filled with iodized oil. The combined portal vein and hepatic artery embolization was more effective than hepatic artery embolization in the control of the disease (P < 0.01). PMID- 7553159 TI - [Predictive values for slow pathway modification of atrioventricular nodal reentrant tachycardia]. AB - The predictive values of various ECG parameters for slow pathway modification were studied in 42 slow-fast AVNRT patients. The negative predictive values of a stable ECG, junctional rhythm, and slow pathway potential were as high as 94.8%, 94.9% and 84.9% respectively. But, their positive values were 29.4%, 15.1% and 23.9%. The possibility of success (PS) raised with increasing of width and peak number of A wave. When the width of A wave was > or = 80ms, PS was 50%. When the peak number of A wave was > 7, PS was 80%. A/V ratio varied from 0.14 to 1 in 93.2% of successful sites. When A/V ratio was 0.25, PS reached its peak value of 33.3%. PMID- 7553160 TI - [Epidemiology of adult diabetes mellitus in a population of Capital Iron and Steel Company in Beijing]. AB - To prevent the development of DM in susceptible individuals, 29859 subjects, aged from 30 to 64 years, from 32 units of the Company were studied by using WHO protocal and diagnostic criteria. The results showed that the average prevalence of DM and IGT was 3.63% and 4.19% respectively, standardized by census 1990 in Beijing, and it was increased with age. No sex difference in the prevalence of DM was found, but the prevalence of IGT was higher in females (5.55%) than in males. The obesity, overweight, positive DM family history and giant baby history, as well as high daily intake of protein were closely related to the prevalence of the disease. Smoking was in effective. PMID- 7553161 TI - [Influence of stress-relaxation plate on bone geometrics and mechanical property: an experimental study]. AB - In order to maintain the effective fixation effect of rigid bone plate in the early stage of fracture and to prevent osteoporosis induced by stress shielding effect of rigid plate in later stage of fixation, a washer made of viscoelastic material was placed between the screw and the screw hole of plate to form a stress-relaxation plate system. Both in vitro and in vivo experiments demonstrated that there was no statistical difference between the stress shielding effect of padded and unpadded plate groups. The washer showed significant deformation and damaged 8 weeks after implantation, and 10 weeks after implantation the stress shielding rate of the padded plate group was significantly lower than that of the unpadded group (P < 0.05). As a consequence, the degree of osteoporosis of the tibia in the padded group was significantly milder than the unpadded group, and 20 weeks after implantation, the diameter of the tibia medullary cavity of the padded group was significantly smaller than that of the unpadded group. The thickness of cortical bone and the bending strength of the tibia of padded group were significantly larger than those of the unpadded group (P < 0.05-0.01). These results showed that the stress-relaxation plate system can effectively maintain the stability of fracture fixation at the similar level of rigid plate during the early stage of fracture and can gradually lower down the stress shielding rate so as to prevent severe osteoporosis and reduction of bone mechanical property in the later stage of fixation. PMID- 7553162 TI - [Lugol's solution in endoscopic diagnosis of early esophageal cancer]. AB - About 1500 high-risk subjects of esophageal cancer were found during screening by balloon cytology and all of them were examined endoscopically. Among them, 120 were considered as having early esophageal cancer and precancerous lesions. During the examination, Lugol's solution staining was used and guiding biopsy was taken. 98 subjects with unstained lesions were found, and biopsy showed early esophageal cancer in 60 (61.2%) and moderate and severe dysplasia in 38 (38.8%). It is usually extremely difficult to detect and localize the very early esophageal mucosal and submucosal carcinoma. But endoscopic examination and using Lugol's solution staining with multiple spots biopsy from unstained area are of great assistance. Minute malignant lesions may not be overlooked. PMID- 7553163 TI - [Hepatocyte culture in the study of liver diseases]. PMID- 7553164 TI - [Advances in the pathology of human cardiac transplantation]. PMID- 7553165 TI - Keeping staff quiet. PMID- 7553166 TI - Building a career structure. PMID- 7553168 TI - Elderly need expert nurses. PMID- 7553169 TI - Professional development: what's the best system? PMID- 7553170 TI - Professional development: reaching the peak. PMID- 7553171 TI - Statistics show sick system. PMID- 7553172 TI - A revealing snapshot. PMID- 7553167 TI - Cuts will reduce services. PMID- 7553174 TI - Understanding cultural safety. PMID- 7553175 TI - Students support cultural safety. PMID- 7553177 TI - A lifetime of service. Interview by Anne Manchester. PMID- 7553178 TI - Position paper: peer assistance. The National Nurses Society on Addictions. PMID- 7553173 TI - Who cares for our elderly? PMID- 7553176 TI - Bringing health to families. PMID- 7553179 TI - National Nurses Society on Addictions. Code of ethics for addictions nurses. PMID- 7553180 TI - A comparative study of alcohol education. PMID- 7553182 TI - Environmental factors in wound healing. PMID- 7553181 TI - Outpatient detoxification: guidelines for nurses. National Nurses Society on Addictions. PMID- 7553184 TI - Maintaining quality of life for elderly patients in residential care. PMID- 7553185 TI - The cost of wound care in the community. AB - Prescriptions issued between July 1993 and June 1994 for wound management materials cost the NHS in Wales a total of 5.5 million pounds. A detailed analysis of Welsh PACT (prescribing analysis and cost) data suggests that this expenditure could be reduced by up to 140,000 pounds by the introduction of minor changes in prescribing patterns. These savings would be achieved with no reduction in the quality of care provided to patients, and in some instances the changes proposed would lead to the use of products which are superior in performance terms to those items that are currently being prescribed. More fundamental changes, involving the exclusion of some products of questionable efficacy, would result in additional savings up to a maximum of 527,000 pounds. As expenditure in Wales is generally considered to represent approximately 10% that of the UK as a whole, this figure could approach 5 million pounds on a national basis. PMID- 7553183 TI - Local administration of insulin. PMID- 7553186 TI - The management and repair of wounds of the face. PMID- 7553189 TI - The technique of micrographic surgery for excising skin tumours. PMID- 7553188 TI - The levels of reliability and validity of the Waterlow pressure sore risk calculator. AB - The Waterlow pressure sore risk calculator is widely used in the UK but has not been found to display high levels of reliability and validity. Few evaluative studies have been carried out in the community. This study aimed to evaluate the Waterlow score when used by district nurses to assess elderly patients. The score was not found to display high levels of reliability, and suffered from a lack of operational definitions within risk categories. In line with other studies, it was found to over-predict pressure sore formation. The findings of this small study suggest that the Waterlow score should not be used as the sole basis for decisions concerning the use of pressure sore prevention resources. High Waterlow scores may be a predictor of ill health in elderly people and this hypothesis requires further examination. The findings also indicate that clarification of the role of risk calculators in general is needed, as many mediating factors appear to affect the ability of a tool to predict pressure sore formation. PMID- 7553190 TI - Anchoring the present moment: meditation in the inner city. PMID- 7553187 TI - Formation of the scab and the rate of epithelisation of superficial wounds in the skin of the young domestic pig. 1962. PMID- 7553192 TI - Providing a pleasant birth experience: CNM-staffed birthing center offers alternative to hospitals. Interview by Adam Ezra Segal. PMID- 7553193 TI - Improving compliance with oral contraceptive regimens. Interview by Adam Ezra Segal. PMID- 7553191 TI - Vietnam Women's Memorial provides late healing for advanced practice nursing. PMID- 7553197 TI - The nurse practitioner as entrepreneur. PMID- 7553195 TI - Reaching out to the elderly: the "touch" of the live performance. Interview by Kathleen Savage. PMID- 7553194 TI - Teaching at a distance. PMID- 7553196 TI - Bringing health care to the desert: Nevada's Wendover Clinic. PMID- 7553198 TI - Managed health care plans pose employment risk for NPs. PMID- 7553199 TI - NP leads health trip to El Salvador. PMID- 7553200 TI - Healing patients: therapeutic touch in nursing practice. PMID- 7553202 TI - NP poet/educator shares her life-altering story and journey. PMID- 7553201 TI - Managed urinary incontinence: an independent NP model. PMID- 7553204 TI - Oklahoma City: when disaster struck, NPs were on the scene. PMID- 7553206 TI - C.A.R.E. for the elderly makes a difference. PMID- 7553203 TI - Community outreach: an immunization program that's working. PMID- 7553207 TI - New hope for Rwanda: NP leads the way. Interview by Laura J Ninger. PMID- 7553205 TI - Nurse practitioners in college health services. PMID- 7553208 TI - FNP program for rural and underserved North Carolina. PMID- 7553209 TI - Computer software developed for nurse practitioners. PMID- 7553210 TI - Interview with co-founder of NP movement. PMID- 7553212 TI - Managed care: NPs work to weather the changes. PMID- 7553214 TI - NP turns author, actress, producer to teach about breastfeeding. Interview by Lisa Brusman Zaidel. PMID- 7553213 TI - Independent NP shut out by managed care. PMID- 7553215 TI - Meeting the menopause challenge: nurses take charge. Interview by Kathleen Savage. PMID- 7553211 TI - Columbia faculty: first with hospital privileges. PMID- 7553216 TI - AMA adopts resolutions that attempt to restrict NP practice. PMID- 7553217 TI - Family practice NPs gain hospital admitting privileges. PMID- 7553218 TI - Interferon-alpha elicits downregulation of human papillomavirus 18 mRNA in HeLa cells by selective repression of endogenous viral transcription. AB - We have reported that interferon-alpha inhibits HPV-18 mRNA in HeLa cells. Here we examine mechanisms by which IFN could modulate HPV expression. In northern blot experiments, we observed that interferon-alpha 2b treatment reduced HPV-18 mRNA levels in a time- and dose-dependent manner, with a maximal effect achieved at 48 h. Simultaneously, induction of 2-5A synthetase mRNA was verified as indicative of IFN action. The IFN regulatory effect on HPV-18 mRNA at 48 h required de novo protein synthesis. We performed run-on experiments to determine whether the IFN regulatory effect was at the transcriptional level. HPV-18 endogenous transcription was repressed using 200 and 1000 IU/ml. Interferon treatment did not affect HPV-18 mRNA stability, at least under our experimental conditions. To verify whether HPV-18 enhancer sequences were involved in the interferon effect, we transfected a construct containing the chloramphenicol acetyltransferase driven by the HPV-18 upstream regulatory region. The enzyme activity was unmodified on human keratinocytes and HeLa cells by interferon exposition. Our data demonstrate that interferon-alpha downregulates HPV-18 mRNA levels on HeLa cells by repressing nascent viral transcripts, possibly through regulatory cellular flanking regions. PMID- 7553219 TI - Protective effect of interferon-alpha against cell-mediated human immunodeficiency virus transmission resulting from coculture of infected lymphocytes with fetal trophoblasts. AB - The hypothesis that the low transmission rate of HIV in utero may be due, in part, to the protective effect of IFN-producing placental trophoblasts was explored in vitro. The model consisted of H9 lymphocytes, as surrogates of maternal HIV-infected T cells, incubated for 3 h with JEG-3 trophoblasts in the presence of 10-fold dilutions of leukocyte-derived IFN-alpha (from 1000 to 0.1 IU/ml). The dose effect was monitored either directly, by measuring the levels of proviral DNA by PCR after a single round of infection, or indirectly, by coculturing infected JEG-3 with cord blood-derived MT-4 lymphocytes and determining the levels of p24 production by ELISA. Both assays revealed a dose dependent blocking effect of IFN-alpha on cell-mediated HIV transmission. The complete inhibition of HIV infection was observed in the presence of 100 IU IFN alpha. The efficacy of such a low dose could not be attributed to insufficient viral load because up to 10(8) infectious particles could be transmitted during cell-cell contact. An adhesion assay ruled out the possibility that IFN-alpha acts through prevention of lymphocyte-trophoblast contact. The results suggest that physiologic levels of IFN-alpha, present in the placental environment, may contribute to the protection of the fetus against HIV infection. PMID- 7553220 TI - Induction of apoptosis in B lineage cells by activin A derived from macrophages. AB - A factor produced by P388D1 cell line murine macrophages showed a profound suppressive effect on the in vitro proliferation of B lineage cells. It was purified to homogeneity from conditioned media of P388D1 cells stimulated with phorbol 12-myristate 13-acetate for 48 h by a three-step procedure. The purified factor gave a single band of protein with a molecular mass of 16 kD on SDS polyacrylamide gel electrophoresis. We show here that exposure of B lineage cells to this factor results in the induction of a cytotoxic effect and a significant increase in the proportion of fragmented DNA. DNA fragmentation was detected in B lineage cells after 3 h culture with the factor in the quantitative colorimetric determination. The mechanism of cell death was characterized by a ladder-like electrophoretic pattern of degraded chromosomal DNA, indicating that the factor induces apoptosis. The NH2-terminal amino acid sequence of this factor was identical with that of activin A over the 26 amino acid residues identified. We sought to determine whether apoptosis could be modulated by two kinds of inhibitor of protein kinases, H7 and HA1004, in concentrations that are below their toxicity limits. Apoptosis induced by the factor was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals by protein kinases may regulate apoptotic B cell death by the factor activin A, derived from macrophages. PMID- 7553221 TI - Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene. AB - Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553222 TI - Cord blood natural killer cells are functionally and phenotypically immature but readily respond to interleukin-2 and interleukin-12. AB - Human umbilical cord blood (CB) is being increasingly used both as an alternative to bone marrow to transplant children and for experimental insight into the ontogenic and maturational characteristics of blood cells. We studied the functional and phenotypic characteristics of CB natural killer (NK) cells because of the possibly important role such cells may play in a transplant setting and to gain insight into the little known ontogenic differences and maturational pathways of NK cells. It was found that CB NK lytic activity is usually deficient and that this deficiency cannot be fully explained by the presence of insufficient percentages of CD56+ cells. Although CD16+CD56+ and CD16-CD56+ NK cell subsets typical of adult peripheral blood (PB) are present, a significant population of CD16+CD56- cells also exists in CB. CB CD16+CD56- cells have little or no lytic capabilities; CB CD16+CD56+ cells vary in their lytic capabilities. Although a decreased ability to bind target cells may contribute to the deficient lytic activity of these CB NK cell subsets, studies suggest that other factors must also play a role. Short-term incubation with interleukin-2 (IL-2) or interleukin-12 (IL-12) substantially increases the lytic capabilities of CB NK cells, and long-term incubations induce lymphokine-activated killer (LAK) cell generation. Cell depletion experiments show that activated CD56+ NK cells are responsible for the lytic activity of CB LAK cells. Flow cytometric analysis reveals that during LAK cell generation, CB undergoes phenotypic changes similar to those of PB except that CD16+CD56- cells are still present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553223 TI - Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. AB - Imiquimod (R-837) and its analog, S-27609, belong to a class of imidazoquinolinamines that have potent antitumor and antiviral effects in animals. Much of their biologic activity is a result of the induction of cytokines, including interferon-alpha (IFN-alpha), tumor necrosis factor alpha (TNF), and others. In this study, the cells responsible for S-27609- and imiquimod-induced cytokine production were characterized. E rosette+ T cells were not the major cell population responsible for IFN-alpha and TNF in response to S 27609 or imiquimod. In contrast, E rosette- cells and unseparated PBMC produced similar concentrations of IFN-alpha and TNF in response to S-27609 and imiquimod. Elimination of monocytes by treatment with the lysosomotropic agent L-leucine methyl ester (LME) or depletion using antibody to CD14 and immunomagnetic beads abrogated IFN-alpha and TNF production induced by S-27609, imiquimod, or LPS but not poly(I)/(C). LME treatment also abolished interleukin (IL)-1 alpha, IL-beta, IL-6, and IL-8 production stimulated by S-27609 and imiquimod. Removal of HLA-DR+ or CD36+ monocytes also caused a significant reduction in S-27609- and imiquimod induced IFN-alpha and TNF. Elimination of B cells, NK cells, and dendritic cells did not significantly reduce cytokine induction in response to S-27609. Thus, the cell population responsible for the majority of cytokine release in human PBMC in response to S-27609 and imiquimod is a E rosette-, CD14+, CD36+, HLA-DR+ monocyte. PMID- 7553224 TI - Amino acid sequence analysis, gene construction, cloning, and expression of gelonin, a toxin derived from Gelonium multiflorum. AB - The plant toxin gelonin is an extremely potent inhibitor of protein synthesis, similar in action to ricin. The mature protein primary sequence was obtained using conventional sequencing techniques. Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues. This toxin shares approximately 33% sequence homology with trichosanthin and ricin A chain. A 774 bp synthetic gene encoding gelonin was synthesized and expressed in E. coli. Recombinant gelonin (approximately 28 kD) expression was monitored and demonstrated by western analysis. Purification and functional activity studies demonstrated that this protein behaves identically to that of the natural product. Recombinant gelonin (RG) thus joins a growing list of recombinant toxins currently available for use in the construction of recombinant immunotoxins composed of gelonin fused to binding domains of antibodies, growth factors, or other cytokines. PMID- 7553226 TI - Leukemia inhibitory factor induces leukocyte infiltration and cartilage proteoglycan degradation in goat joints. AB - Recent studies have implicated leukemia inhibitory factor (LIF) in human joint disease. LIF is produced by cultured synovial cells and articular chondrocytes, stimulates cartilage and bone resorption, and has been detected in inflammatory exudates from arthritic joints. The aim of this study was to evaluate the effect of intraarticular injections of human recombinant LIF in the goat. Endotoxin free, sterile normal saline containing 1 micrograms recombinant human LIF (rhLIF) was injected into the right radiocarpal joints (RCJs) of eight angora goats. The left RCJs were injected with an equivalent volume of vehicle alone (n = 6) or vehicle containing 1 micrograms human albumin (n = 2). Goat joints were examined for clinical features of inflammation, and synovial fluid (SF) was aspirated on days 0, 2, and 6 postinjection. Leukocyte counts and concentrations of keratan sulfate, IL-1 beta, and TNF-alpha were determined in the SF. Proteoglycan synthesis was determined ex vivo in cartilage explants obtained on day 6 postinjection. A statistically significant increase in joint swelling and effusion volume was observed in LIF-injected joints but not in control joints. In the LIF-injected RCJs, the leukocyte count increased from 82 +/- 9 cells/microliters before injection to 10,300 +/- 3357 cells/microliters at day 2 postinjection (p < 0.005) and declined to 678 +/- 113 cells/microliters at day 6 postinjection. Polymorphonuclear leukocytes and monocyte/macrophages predominated in the infiltrate. No appreciable change in leukocyte counts was observed in control joints.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553225 TI - Mechanism by which U937 promonocytic cells inactivate human interferon-gamma. AB - Mononuclear phagocytes produce proteinases that are thought to play a role in regulating the activity of cytokines. Activated macrophages secrete urokinase type plasminogen activator (uPA), which mediates the formation of the serine proteinase plasmin from the ubiquitous zymogen plasminogen. We previously observed a correlation between in vitro plasminogen activation by the promonocytic cell line U937 and the apparent ability of these cells to inactivate recombinant interferon-gamma (rIFN-gamma) by proteolysis. The present study was designed to test the hypothesis that plasmin, generated in U937 cell cultures, is both necessary and sufficient to inactivate rIFN-gamma by limited proteolysis. The following observations are consistent with this hypothesis: (1) inactivation of rIFN-gamma was prevented by inhibitors of serine proteinases or an antibody that specifically immunodepleted plasmin activity; (2) purified plasmin inactivated rIFN-gamma as efficiently as U937 culture supernatants and was similarly sensitive to serine proteinase inhibitors; and (3) plasmin removed an 11 amino acid carboxyl-terminal peptide from rIFN-gamma, which included a region known to be required for bioactivity. Overall, these data indicate that plasminogen activation by U937 promonocytic cells leads to the proteolytic inactivation of IFN-gamma by a process that only requires the production of plasmin. PMID- 7553227 TI - Interleukin-10 enhances human immunodeficiency virus type 1 expression in a chronically infected promonocytic cell line (U1) by a tumor necrosis factor alpha independent mechanism. AB - TNF-alpha enhances HIV-1 replication in acutely and chronically infected cells and likely contributes to the wasting associated with the acquired immunodeficiency syndrome. Agents that inhibit TNF-alpha activity should theoretically delay the progression of disease, and several are currently in clinical trials. We hypothesized that IL-10, a cytokine that suppresses the gene expression and synthesis of TNF-alpha in monocytic cells, might inhibit HIV-1 replication. As expected, IL-10 suppressed PMA-induced TNF-alpha production in U1 cells; however, when U1 cells were cultured in the presence of PMA and increasing doses of IL-10, a dose-dependent increase in HIV-1 expression was observed. IL-10 also enhanced IL-1 beta-, TNF-alpha-, and GM-CSF-induced HIV-1 expression in U1 cells, and this occurred, at least in part, at the level of transcription. We next stimulated cells under conditions of TNF-alpha blockade. When PMA-induced TNF-alpha activity and HIV-1 replication were blocked by the presence of soluble TNF receptors, IL-10 independently enhanced HIV-1 replication. In contrast, other agents that are capable of blocking TNF-alpha synthesis or TNF-alpha activity either had no effect (IL-13 and IL-4) or inhibited HIV-1 expression (soluble TNF receptors and pentoxifylline) in U1 cells. These data suggest that IL-10, while inhibiting TNF-alpha synthesis, has an independent mechanism of action that enhances HIV-1 replication. Therefore, IL-10 may have undesirable effects in HIV 1-infected patients. PMID- 7553229 TI - Interferon-gamma inhibits endocytosis of soluble animated beta-1,3-D-glucan and neutral red in mouse peritoneal macrophages. AB - We previously demonstrated that soluble animated beta-1,3-D-glucan (AG) is internalized after binding to a specific beta-glucan receptor on macrophages. Internalization, but not binding, of AG is reduced when the macrophages are treated with IFN-gamma. Because our data indicated that AG is taken up by macrophages through beta-glucan receptor-mediated endocytosis, we wanted to characterize further the inhibitory effect of IFN-gamma on endocytosis. We compared the internalization of AG and neutral red (NR). NR is internalized by macrophages through fluid-phase endocytosis. AG and NR showed a similar influx/efflux pattern. The initial rate of accumulation was much larger for AG than for NR, however, probably because of the involvement of the beta-glucan receptor in the uptake of AG. Internalized AG was associated with membranes of the endocytic vesicles and formed characteristic rings on confocal laser scanning microscopy (CLSM) images. Both the influx and efflux of AG and NR was inhibited by treatment of macrophages with IFN-gamma. Phorbol myristate acetate (PMA) added to the cell cultures increased the accumulation of AG and NR and reversed the inhibitory effect of IFN-gamma. The effect of PMA was dependent on functionally intact microfilaments and microtubules. CLSM showed that the accumulated AG was localized mostly in small vesicles (size < 2 microns) in IFN-gamma-treated cells, in large and small vesicles in untreated cells, and mostly in large vesicles (size > 2 microns) in PMA-treated cells. In conclusion, IFN-gamma inhibits both the beta-glucan receptor-mediated endocytosis of AG and the fluid-phase endocytosis of NR, probably by inhibiting the formation of large vesicles. PMID- 7553230 TI - Interferon-beta, retinoids, and tamoxifen in the treatment of metastatic breast cancer: a phase II study. AB - Based on the additive or synergistic antiproliferative effect of interferon and tamoxifen on breast cancer cell lines and on preclinical and clinical data on retinoids alone and in combination with antiestrogen or interferon, we designed a pilot phase II study to test the toxicity of simultaneous administration of interferon-beta (IFN-beta), retinoids (R), and tamoxifen (TAM) and the efficacy of this combination as salvage therapy in a group of patients with metastatic breast cancer (MBC). A total of 49 stage IV breast cancer patients, 11 pretreated with hormones, 26 with chemotherapy, and 12 with both, received 30 mg TAM and two dose levels of IFN-beta and retinyl palmitate. Among 49 evaluable patients, 27 achieved a clinical response (55%; 95% CI 41-69%), 10 had stable disease (20%), and in 12 (25%) the disease progressed. Toxicity with both dose levels was moderate and mainly hepatic. Median response duration, not statistically different in estrogen receptor-positive and negative patients, was 31.4 months (range 4.9-67). Median overall survival was 19.2 months (range 2-69). We have shown that long-term administration of TAM, IFN-beta, and retinyl palmitate is feasible with moderate toxicity. We have also demonstrated that this regimen is active in pretreated MBC patients and that responses are not influenced by receptor status. PMID- 7553232 TI - Interleukin-1 enhances indoleamine 2,3-dioxygenase activity by increasing specific mRNA expression in human mononuclear phagocytes. AB - The objective of this study was to determine the utility of the THP-1 monocytic leukemia cell line as a model for analyzing molecular mechanisms involved in enhancement of interferon (IFN)-gamma-induced indoleamine dioxygenase (IDO) activity by interleukin-1 (IL-1). Following treatment of THP-1 cells with combinations of IFN-gamma and IL-1, IDO activity and IDO mRNA were quantified by HPLC and radioanalytic imaging of RT-PCR products, respectively. IL-1 increased the amount of IDO activity and the expression of IDO mRNA in IFN-treated cells; IL-1 alone had no effect on untreated THP-1 cells. Because IDO gene regulation might differ between immature THP-1 cells and mature macrophages, experiments were repeated using primary macrophage cultures. IFN-gamma induced IDO activity, and IDO mRNA was expressed in a dose-dependent manner. In the presence of IL-1, 10 times less IFN was required to obtain the same amount of IDO mRNA and IDO activity. Furthermore, IL-1 alone increased IDO mRNA expression. It appears that unlike what was observed in THP-1 cells, IL-1 transcriptionally activates the IDO gene in primary macrophages. However, increases in IDO activity were not observed following treatment with IL-1 alone. Although the THP-1 cell may be used to model cytokine potentiation of IFN-induced IDO activity, some differences in regulation between THP-1 cells and primary macrophage cultures may exist. PMID- 7553231 TI - Randomized controlled trial of recombinant interferon-alpha 2b in the treatment of Chinese patients with chronic hepatitis C. AB - To evaluate the efficacy of recombinant interferon (IFN) alpha 2b in the treatment of Chinese patients with chronic hepatitis C, a randomized controlled trial was conducted in 50 chronic hepatitis C patients: 25 patients received 3 million units of subcutaneously injected recombinant IFN-alpha 2b three times per week for 6 months, and 25 patients received no specific treatment were used as controls. At the end of the IFN treatment, 19 patients (76%) in the IFN-treated group normalized serum ALT compared with only 6 patients (24%) in the control group (p < 0.01). Relapse within 6 months after the completion of treatment occurred in 13 IFN-treated patients (68%). Normalized serum ALT was seen in 6 patients (24%) in the IFN-treated group and 1 patient (4%) in the control group 6 months after discontinuation of IFN therapy (p = 0.10). The presence of serum hepatitis C virus (HCV) RNA measured by reverse transcription-polymerase chain reaction was detected at the end of the IFN treatment in all 13 patients who relapsed after cessation of therapy. In only 3 of 25 IFN-treated patients (12%) was the presence of serum HCV RNA not detectable at the end of the IFN treatment or 6 months after cessation of therapy. No patient in the control group had undetectable serum HCV RNA during the study period. Using multivariate logistic regression analysis, the low pretreatment levels of HCV RNA, measured by a quantitative branched DNA amplification assay, was the only independent predictor of a sustained response to IFN therapy (p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553228 TI - Intravesical liposomal muramyl tripeptide phosphatidylethanolamine treatment of human bladder carcinoma growing in nude mice. AB - The present study evaluated the in vivo activity of synthetic lipophilic muramyl tripeptide phosphatidylethanolamine (MTP-PE) when encapsulated into liposomes (phosphatidylcholine-phosphatidylserine, 7:3 molar ratio) and administered intravesically to athymic nude mice with human transitional cell carcinoma 253J-V cells growing in their bladder. Intravesical liposome-MTP-PE was effective in eradicating the human tumors implanted orthotopically in nude mice. Following therapy, activated macrophages were found in the bladders of liposome-MTP-PE treated mice but not in control mice. In vitro activation of murine macrophages with liposome-MTP-PE increased their cytotoxicity against the 253J-V cell line used in these experiments. This effect was enhanced by cotreatment with interferon-gamma (IFN-gamma). Furthermore, cotreatment of macrophages with both liposome-MTP-PE and IFN-gamma resulted in the secretion of both tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Liposome-encapsulated MTP-PE shows promise as an effective therapeutic agent for bladder carcinoma. PMID- 7553233 TI - Effect of topical interferon-beta on recurrence rates in genital herpes: a double blind, placebo-controlled, randomized study. AB - The aim of this randomized, double-blind placebo-controlled trial was to evaluate the effect of IFN-beta cream applied at the time of recurrent eruptions of genital herpes during 6 months on the overall rate of recurrence. Therapy was initiated at the clinic for the first treated recurrence, and thereafter by the patient for early treatment of eventual subsequent eruptions. Each recurrence was ascertained at the clinic in all 35 evaluable patients. The mean recurrence rate was significantly lower in the group using IFN-beta cream than in the placebo group (p = 0.03). Complete responders without recurrence for the duration of the trial were 36.4% of all patients and 46% among women versus 15.4 and 16.6% in the placebo groups, respectively. A total of 77.3% of all patients were defined as complete or partial responders, their average recurrences/year decreasing from 11 to 2.2 (p < 0.0001). The topical episodic IFN-beta treatment was well tolerated by patients and without side effects. It is concluded that IFN-beta cream application reduces the overall rate of recurrence of genital herpes. PMID- 7553234 TI - Interferon-gamma detection in cultures of newborn cells exposed to Listeria monocytogenes. AB - Clearance of Listeria monocytogenes in experimental models of infection has underscored the importance of interferon-gamma (IFN-gamma) in host resistance to intracellular pathogens. Because L. monocytogenes infections are more severe in newborns than adults, we compared IFN-gamma accumulation in the supernatants of mononuclear cells infected in vitro from newborns with those from adults. Supernatants were assayed for IFN-gamma using an enzyme-linked immunosorbent assay. Uninfected newborn and adult mononuclear cells had less than 50 pg/ml of IFN-gamma at all times tested. IFN-gamma levels in supernatants from infected adult mononuclear cells at 24 h of culture (1.15 x 10(3) +/- 0.92 pg/ml) were greater than supernatants from infected newborn mononuclear cells (0.19 x 10(3) +/- 0.33 pg/ml). IFN-gamma concentrations in newborn cell cultures plateaued on day 3 of culture (1.6 x 10(3) +/- 1.1 pg/ml) and were not significantly less than concentrations from adult cells. However, adult cell IFN-gamma was further increased by day 5 (18.7 x 10(3) +/- 21.8 pg/ml). Because IFN-gamma plays a critical role in the host defense against L. monocytogenes, this delay in the release of IFN-gamma may be a factor in the increased susceptibility and severity of infection in the neonate. PMID- 7553235 TI - Priming with interferon-alpha 1 or interferon-alpha 2 enhances the production of both subtypes simultaneously. AB - A short period of incubation with interferon-alpha ("priming") increases the amounts of IFN-alpha formed by human peripheral blood leukocytes when subsequently induced with a virus. We investigated specifically the effect of priming on the production of two individual subtypes, IFN-alpha 1 and IFN-alpha 2. The rate of interferon synthesis and the amounts formed were equally potentiated in leukocytes primed with either IFN-alpha 1 or IFN-alpha 2. Whichever of these was used for priming had no selective effect on the relative increased production of IFN-alpha 1 or IFN-alpha 2. PMID- 7553237 TI - Correlation of in vitro and in vivo effects of interferon-gamma with the spontaneous regression of a rat histiocytoma. AB - Cytokines trigger activation of different lymphocyte populations, resulting in augmentation of humoral and cell-mediated immunity. We have examined the role of IFN-gamma in mediating AK-5 tumor regression. High levels of circulating IFN gamma and IL-2 marked the process of regression. Moreover, interaction of immune NK cells with antibody-tagged AK-5 resulted in IFN-gamma secretion, providing in vitro evidence for the involvement of this cytokine. IFN-gamma and IL-2 potentiated the cytolytic activity of naive NK cells, suggesting their role in antitumor activity. Furthermore, pretreatment of immune NK cells with protein tyrosine kinase inhibitor downregulated the IFN-gamma release, suggesting that the secretion of IFN-gamma is phosphorylation dependent. Nonimmune cells could be induced to secrete IFN-gamma when exposed to rIL-12, demonstrating IL-12 dependence in inducing IFN-gamma release. In vivo administration of anti-IFN gamma inhibited the cytotoxic activity and the process of tumor regression, further substantiating the role of IFN-gamma in regulating the rejection of AK-5 tumor. These observations suggest a definitive role for IFN-gamma in AK-5 regression. This cytokine in concert with IL-2 and IL-12 might aid in designing effective anticancer therapy. PMID- 7553236 TI - Reevaluation of the superiority of polyethylene glycol-modified interleukin-2 over regular recombinant interleukin-2. AB - Other groups have reported a superior antitumor efficacy of polyethylene glycol modified interleukin-2 (PEG-IL-2) compared with regular recombinant interleukin-2 (rIL-2). However, detailed comparison of the antitumor efficacies of locally applied PEG-IL-2 and rIL-2 in the well-established DBA/2-SL2 model shows a higher antitumor efficacy of PEG-IL-2 only at doses < 800 micrograms IL-2 protein/kg body weight. At doses > 800 micrograms IL-2 protein/kg body weight, rIL-2 has better therapeutic efficacy. The superiority of rIL-2 at doses > 800 micrograms IL-2 protein/kg body weight is a result of the toxicity of PEG-IL-2 at these doses. With either IL-2 preparation, cure rates of approximately 90% can be obtained at nontoxic doses. We conclude that PEG-IL-2 does not have superior antitumor efficacy to rIL-2. The main advantage of PEG-IL-2 is that for optimal therapeutic efficacy a daily injection schedule is not required as seems to be the case for rIL-2. PMID- 7553238 TI - Suppression of tumor growth and metastasis of murine renal adenocarcinoma by syngeneic fibroblasts genetically engineered to secrete the JE/MCP-1 cytokine. AB - The murine monocyte chemoattractant protein 1, JE/MCP-1, like its human counterpart monocyte chemotactic and activating factor (MCAF), attracts monocytes macrophages to tumor tissues. In previous studies we reported that expression of the JE/MCP-1 gene in murine colon carcinoma cells reduced their tumorigenicity and suppressed their metastatic potential. We now demonstrate that the growth and metastasis of the renal adenocarcinoma cell line RENCA are reduced when it was admixed with syngeneic fibroblasts engineered to secrete the JE/MCP-1 cytokine before injection. Culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide (LPS) synergistically activated tumoricidal properties in syngeneic macrophages against RENCA cells. This activity was blocked by anti JE/MCP-1 antibody, indicating that JE/MCP-1 was involved in priming the macrophages to respond to LPS. Moreover, alveolar macrophages isolated shortly after iv injections of JE/MCP-1 transfected cells were cytotoxic to RENCA cells in vitro. Collectively, these data suggest that in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages, thus providing a rationale for the use of the JE/MCP-1 protein as a modality for treatment of metastasis. PMID- 7553239 TI - Are you TH-1 or TH-2? PMID- 7553241 TI - Benzalkonium chloride in a decongestant nasal spray aggravates rhinitis medicamentosa in healthy volunteers. AB - A randomized double-blind parallel study with 20 healthy volunteers was performed to research the effect of a preservative in a decongestant nasal spray on the development of rhinitis medicamentosa. Ten subjects received oxymetazoline nasal spray with benzalkonium chloride and the others used oxymetazoline nasal spray without the preservative three times daily for 30 days. Before starting the course of treatment and after its conclusion, recordings of the mucosal surface positions were made with rhinostereometry followed by histamine challenge tests. Symptoms of nasal stuffiness were estimated on visual analogue scales (0-100) in the morning and the evening just before using the nasal spray. After 30 days, rebound swelling and nasal stuffiness were found in both groups. In the group receiving oxymetazoline nasal spray with benzalkonium chloride the mean rebound swelling was 1.1 mm and the estimated mean evening symptom score for nasal stuffiness was 43. In the group without benzalkonium chloride the corresponding variables were significantly less marked, with a mean rebound swelling of 0.5 mm (P < 0.05) and a mean evening symptom score of 25 (P < 0.05). The increase in histamine sensitivity in both groups was interpreted as a sign of nasal hyperreactivity. A new type of nasal spray bottle was used that has been shown to prevent bacterial contamination. In conclusion, the long-term use of benzalkonium chloride in oxymetazoline nasal spray accentuates the severity of rhinitis medicamentosa in healthy volunteers. PMID- 7553240 TI - Rhinitis medicamentosa. PMID- 7553242 TI - Benzalkonium chloride in nasal decongestive sprays has a long-lasting adverse effect on the nasal mucosa of healthy volunteers. AB - Twenty healthy volunteers participated in the present study on the long-term effects of a nasal decongestive spray composed of either a combination of oxymetazoline nasal spray and benzalkonium chloride or of oxymetazoline nasal spray alone. Three months before the present study the participants had undergone treatment with nasal decongestants for 4 weeks. Ten of the subjects had been treated with oxymetazoline nasal spray without benzalkonium chloride and 10 of them had been treated with oxymetazoline nasal spray with benzalkonium chloride. In a double-blind study the subjects who had been treated with oxymetazoline nasal spray and benzalkonium chloride were again treated with the same combination of substances as before, and the subjects who had been treated with oxymetazoline nasal spray alone were also treated again with oxymetazoline nasal spray alone, but on this occasion only for 10 days. Three variables were studied before and after the 10 days of treatment, i.e. nasal mucosa congestion, nasal reactivity and symptom scores. It was found that only the subjects who were treated with the combination of oxymetazoline nasal spray and benzalkonium chloride had increased nasal stuffiness, estimated by symptom scores and measurements of nasal mucosa swelling after 10 days of treatment. It is concluded that a nasal decongestant spray composed of a combination of vasoactive substance and benzalkonium chloride has a long-term adverse effect on the nasal mucosa. PMID- 7553245 TI - Asthmatic symptoms and indoor levels of micro-organisms and house dust mites. AB - As a part of a worldwide investigation on the prevalence of respiratory symptoms, we have performed a study on the relationship between the indoor environment and asthma-like symptoms in the population of a central Swedish municipality. The study comprised 88 individuals, aged 20-45 years who underwent a structured interview, spirometry, a methacholine provocation test, skin-prick tests and blood samples for measurements of serum concentrations of eosinophil cationic protein (S-ECP), blood eosinophil count and total immunoglobulin E (S-IgE). In the homes, the room temperature, air humidity, respirable dust, house dust mites (HDM) and airborne micro-organisms were measured. The relative humidity in all the homes was found to be above 33%. HDM were found in 13% of homes. In the homes of the 47 subjects with asthma related symptoms, significantly higher total levels of bacteria and mould (P < 0.05) and a higher proportion of detected HDM (OR = 5.3) was found than in subjects with no asthma related symptoms, after adjustment for age, sex, smoking, indoor temperature and air humidity. HDM were found to be an independent risk factor for asthma related symptoms (OR = 7.9) and nocturnal breathlessness (OR = 6.2) (P < 0.05), while the total level of bacteria was a risk factor for asthma related symptoms and wheezing (P < 0.05). We conclude that although HDM is relatively infrequently found in the homes of central-Sweden, the presence of HDM is related to asthmatic symptoms. A relation between levels of airborne bacteria and asthma related symptoms was also found. PMID- 7553243 TI - TNF alpha is localized to nasal mucosal mast cells and is released in acute allergic rhinitis. AB - Allergic mucosal inflammation is characterized by tissue infiltration with eosinophils, and associated activation of mast cells and T lymphocytes. Tumour necrosis factor (TNF) alpha/cachectin is a candidate cytokine relevant to the pathogenesis of these events through its capacity to upregulate the expression of endothelial cell adhesion molecules, mediate granulocyte chemoattraction, and activate eosinophils, mast cells and T cells. To investigate the presence and localization of TNF alpha in the nasal mucosa in allergic rhinitis, nasal biopsies from perennial rhinitic (n = 13) and non-rhinitic volunteers (n = 11) were embedded in glycol methacrylate and immunostained with a monoclonal antibody directed against TNF alpha, and adjacent 2 microns sections stained for tryptase, CD3 and eosinophil cationic protein. This identified positive immunostaining for TNF alpha in the submucosa of both the rhinitic and normal subjects (median cell counts 13 and 23 cells/mm2 respectively, P = 0.24) with cellular localization to mast cells but not to T-lymphocytes or eosinophils. In a subsequent study of seven atopic subjects, nasal allergen challenge produced increases in lavage levels of histamine and albumin, which was associated with significant release of TNF alpha as early as 2 min post-allergen when compared with the saline control day (P = 0.05). This difference was also apparent when studying the area under the curve both at 30 and 60 min post-challenge t-test (P = 0.015 and 0.02 respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553244 TI - Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies. AB - The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26 kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts. PMID- 7553246 TI - Occupational respiratory and skin sensitization caused by polyfunctional aziridine hardener. AB - Polyfunctional aziridine (PFA) is increasingly used as a water-based crosslinker in two-component paints, paint primers, lacquers, topcoats and other protective coatings. The crosslinker is made by reacting multifunctional acrylic monomer with a highly reactive aziridine compound. Respiratory allergy or hypersensitivity from PFA has not been reported previously. During 1978-1991 we came across nine cases with hypersensitivity from PFA: two had allergic contact dermatitis (ACD), four had occupational asthma and three had both of them. Five of the patients were parquet layers, two were fibreboard painters, one was a spray painter and one was a salesman of PFA products. ACD was diagnosed by positive allergic patch test reactions with PFA in a dilution series in petrolatum: 0.32%-0.5% gave a 2(+)-3+ allergic reaction in the five cases with ACD but 0.1% gave only a weak reaction in one case, whereas the methacrylate patch test series was negative. The diagnosis of seven cases of occupational asthma due to PFA hardener was based on symptoms related to exposure to PFA hardener at work, and on positive provocation tests with PFA hardener. One had an immediate type reaction, one a had a dual reaction, and the others had late reactions. The positive reactions with the PFA hardener and the negative reactions with the acrylate compounds indicate that PFA caused ACD which is different from the previous reports in which acrylates present as impurities in the PFA hardener caused the sensitization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553247 TI - Sensitization to Ganoderma lucidum in patients with respiratory allergy in India. AB - Although human sensitization to basidiomycete Ganoderma has been reported in New Zealand, North America and Europe, hypersensitivity due to this fungi is not known in India, in spite of its prevalence in the atmosphere. We have studied the atmospheric concentration of Ganoderma in different localities within Delhi and the sensitization level to this fungi amongst the Indian population. Aerobiological sampling, using a Burkard personal slide sampler, was carried out in Delhi for 2 consecutive years (October 1989-September 1991). The sampler was operated at 10 day regular intervals for 10 min to trap the spores. The peak season for Ganoderma is recorded from July to September with highest average monthly catch of 336 spores/m3 in September 1991 from south Delhi. Antigenic extracts were prepared from both, the spores and whole body of Ganoderma lucidum. The results of intradermal skin tests conducted on 172 patients revealed that 28.48% and 17.44% of patients showed marked skin reactivity to spore and whole body extracts, respectively. A significant correlation (r = 0.963, P < 0.01) was found between intradermal and skin-prick tests. More than 80% of the intradermal test positive patients had significantly (P < 0.01) elevated IgE antibodies to the fungi in question. Thus, sensitization to Ganoderma lucidum has been reported for the first time in the atopic population of India. PMID- 7553248 TI - Experimental intestinal hypersensitivity. Effect of four anti-allergic drugs on protein uptake, permeability to sugars and mucosal mast-cell activation. AB - The ability of four drugs with anti-allergic action to modulate the uptake of bystander protein, lactulose/rhamnose permeability ratios and mast cell activation was studied in rats presensitized with egg albumin in alum and challenged intraduodenally with the same antigen. Beclomethasone dipropionate (BDP) and nedocromil both significantly reduced the uptake of the bystander protein, bovine serum albumin (P < 0.002 and P > 0.02 respectively). BDP also significantly reduced sugar permeability (P < 0.01). In animals with elevated lactulose/rhamnose permeability ratios we confirmed our earlier observation of a significant correlation between levels of the specific mucosal mast cell protease Rat Chymase II (RChyII-previously known as RMCPII) and the sugar ratios. None of the drugs had any influence on the levels of mast cell protease II released following challenge and there was no correlation between the histological light microscopic appearance of the mast cells and the experimental treatment administered. Our results suggest that in the gut the pharmacological effect of anti-allergic drugs may be complex. Some, such as nedocromil, appear to act only on the mechanisms underlying increased protein uptake whereas others, such as BDP, appear to abrogate both increased protein uptake and increased sugar permeability. PMID- 7553249 TI - Podocarpus gracilior and Callitris verrucosa--newly identified allergens that crossreact with Cupressus sempervirens. AB - Thirty-six symptomatic patients, with positive skin reactions to Cupressus sempervirens pollen extract were skin-tested with pollen extracts of Podocarpus gracilior and Callitris verrucosa, of these 17 (47%) had positive responses to P. gracilior, nine (25%) to C. verrucosa, and six (17%) to both. None of the non atopic healthy controls had positive reactions to either of the extracts. Radioallergosorbent test (RAST)-inhibition studies were performed with pooled sera from three patients. Fifty per cent inhibition was obtained with 11 micrograms protein of C. sempervirens, 54 micrograms of P. gracilior, and 71 micrograms of C. verrucosa; however, when pollen extract of Olea europaea, an unrelated allergen, was tested, 265 micrograms protein were needed to obtain 50% inhibition. One-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pollen extracts from the three species revealed that they had several very similar protein bands. Using Western blot analysis, several closely related IgE binding proteins were identified in the three species. It was concluded that the pollen grains of P. gracilior and of C. verrucosa are potentially allergenic. The presence of common allergenic proteins indicate partial crossreactivity with C. sempervirens. PMID- 7553251 TI - Validation and application of a new simple strategy for measurements of urinary leukotriene E4 in humans. AB - To monitor endogenous production of cysteinyl-containing leukotrienes, the end metabolite leukotriene E4 (LTE4) was analysed in urine. Results obtained with a sensitive enzyme immunoassay (EIA), performed on crude urine samples correlated well with data obtained from a previously reported radioimmunoassay. Enzyme immunoassay analysis of unextracted urine was justified by an excellent agreement between analyses in crude samples and measurements achieved after purification on solid phase extraction followed by separation on reversed-phase high performance liquid chromatography. Moreover, LTE4 was stable in urine samples stored at -20 degrees C, for months without the addition of preservatives. The stability of LTE4 in urine was not improved by addition of the antioxidant 4-hydroxy-TEMPO and pH adjustment to 9. As assessed by EIA analysis in crude urine samples, baseline values for urinary leukotriene E4 were not significantly different between atopic asthmatic subjects and non-asthmatic individuals, and there was no diurnal variation in urinary excretion of LTE4 in healthy subjects. However, we confirmed earlier data on significantly higher basal levels of urinary LTE4 in aspirin intolerant asthmatics. In addition, a post-challenge increase in urinary LTE4 levels was detected in association with allergen-induced airway obstruction in atopic asthmatics. The per cent increase in urinary LTE4 was similar, irrespective of whether the samples were purified or not prior to EIA. Thus, combined with random validation by high performance liquid chromatography, the strategy of direct EIA of serially diluted urine samples was found to be a good index of in vivo production of leukotrienes. This was further reinforced by the demonstration that pretreatment with the leukotriene biosynthesis inhibitor Bay x 1005 inhibited the post allergen-challenge increase in urinary LTE4, as shown both with unpurified and purified samples. PMID- 7553250 TI - Influence of the menstrual cycle on skin-prick test reactions to histamine, morphine and allergen. AB - The purpose of this study was to examine the possible influence of the phases of the menstrual cycle on dermal reactivity to skin-prick testing. We studied 15 atopic, menstruating women with seasonal rhinoconjunctivitis and/or asthma, with known sensitivity to olive and parietaria (mean age 25.2 years) and 15 non atopic, healthy, female controls (mean age 24.7 years). Skin-prick tests with histamine, morphine, and in the atopic group with parietaria/and/or olive, were repeated three times during the same menstrual cycle, corresponding to bleeding (day 1-4), midcycle (day 12-16) and the late progesterone phase (day 24-28). None of the patients had received oral antihistamines or exogenous hormones for at least 1 month prior to testing. Results indicate a significant increase in weal and-flare size to histamine, morphine, and parietaria on days 12-16 of the cycle, corresponding to ovulation and peak oestrogen levels. This was observed in both atopic and non-atopic women. Differences in skin reactivity to histamine and morphine between the groups were not significant. Therefore, in women, the phase of the menstrual cycle is another factor that may influence skin-test results. PMID- 7553253 TI - [Treatment of hyperemesis gravidarum by electrostimulation of the vestibular apparatus]. AB - 26 patients hospitalized with Hyperemesis Gravidarum were treated with electrical stimulation of the vestibular system, as the symptoms of Hyperemesis Gravidarum resemble the symptoms of motion sickness, where the electrical stimulation has been used successfully. The patients were treated for one hour daily, two hours before the standard infusion therapy. 89% reported a decrease in vomiting and nausea during the first application, 85% a lasting improvement. Theoretical considerations concerning the mechanism of the effect are discussed. PMID- 7553252 TI - [Administration of pethidine in labor with meconium contaminated amniotic fluid: effect on fetal outcome and respiratory disorders]. AB - OBJECTIVE: The aim of the study was to evaluate the effects of maternal pethidine administration on fetal outcome and incidence of neonatal respiratory distress in deliveries with meconium stained amniotic fluid (MAF). STUDY DESIGN: Between 1989 and 1992, 849 live infants had MAF. For pain relief during labor 214 women received pethidine 50-150 mg i.m. The control group included 401 infants with MAF, who were not exposed to sedatives, analgesics, and anesthetics during labor. Fetal cord pH, Apgar 1 min (A1), Apgar 5 min (A5), and incidence of respiratory distress were compared retrospectively between the groups. Statistics and calculations were done by means of uni- and multivariate analyses. RESULTS: The presence of thickened MAF increased significantly the risk of low A1, low A5, low arterial cord pH, and the incidence of respiratory distress (p = 0.003, = 0.0003, = 0.016, = 0.000). Infants with thickened MAF, low A1, and low fetal cord pH proved to have an exceedingly high risk of respiratory distress. The application of pethidine (0 mg, 50 mg, 100-150 mg) does not discriminate the variables as arterial cord pH, A1, A5, and the incidence of respiratory distress significantly. However the medium cord pH values of infants without pethidine (7.27, s = 0.075) and with 100-150 mg pethidine (7.25, s = 0.078) were significantly different (p = 0.0006). The incidence of arterial pH, 7.10 did not differ significantly in cases with different pethidine doses. CONCLUSIONS: Thickened MAF is an obstetric hazard with small but significantly increased risks for poor fetal outcome and neonatal respiratory distress. This study failed to identify additional neonatal risks engendered by use of pethidine in cases of MAF. PMID- 7553255 TI - [Pregnancy in Takayasu arteritis]. AB - We report the course of pregnancy in a patient with Takayasu's arteritis type I and describe the diagnostical and therapeutical management with repeated Doppler sonography. Immunosuppressive therapy was reduced in pregnancy and could be maintained on a low level even after delivery. In the third trimester arterial blood pressure raised up to 190/60 mm Hg and was sufficiently lowered by prescription of verapamil. Repeated Doppler-sonographic investigation beginning with 20 weeks of gestation revealed indices in the normal range during the whole pregnancy. After 37 weeks of gestation a healthy infant was delivered vaginally. The post partum course was uneventful. PMID- 7553254 TI - [Puerperal inhibition of lactation with metergoline or bromocriptine]. AB - In a controlled, randomised, prospective, clinical study, the effect of prolactin suppression and clinical course of the lactation suppressors Bromocriptine and Metergoline were investigated. During 7 months 150 patients were studied. 81 of those patients, who did not nurse, were treated by Bromocriptine (primary lactation suppression: n = 62, secondary suppression: n = 19) and 69 of the patients were treated by Metergoline (primary suppression: n = 54, secondary suppression: n = 15). The drugs were administrated orally to all subjects, dosed 2 x 2.5 mg/d of Bromocriptine for 14 days and 3 x 4 mg/d of Metergoline for 10 days, starting in average after 13 hours. Puerperal suppression of prolactine were compared with randomised breast feeding subjects (n = 30). In Bromocriptine treated women the average plasma prolactin level decreased from 78.4 +/- 22 ng/ml to 17.0 +/- 3.3 ng/ml during five days of treatment. In Metergoline treated women the plasma prolactin level decreased from 129.7 +/- 15.1 ng/ml to 56.9 +/- 10.0 ng/ml during the first days of treatment. Prolactin level of breast feeding subjects decreased from 233.6 +/- 21.4 ng/ml to 185.8 +/- 23.7 ng/ml during the same period (p < 0.05). There is no statistical significancy of clinical difference of both drugs, but a statistical trend was seen. With Bromocriptine treated women were suppressed efficiently in 71 of 81 cases, 10 refused. Refusals were divided in two quality levels, level I with subjects with moderate complaints and little puerperal lactation, level II with subjects with considerable complaints including strong puerperal lactation. With Metergoline suppressed women, treatment was efficiently in 51 of 69 cases, but refusals of level I were observed in 11 cases and refusals of level II were observed in 7 cases. The results show that Bromocriptine and Metergoline are effective on suppression of lactation. Under the current drug dose of Metergoline an advantage of Bromocriptine were observed. Only further studies could investigate, whether an adaptation of drug dose would improve the clinical efficiency of Metergoline. PMID- 7553256 TI - [Fetal heart rate and pulse oximetry]. PMID- 7553257 TI - [The perinatal concept for improving treatment of congenital diaphragmatic defects]. PMID- 7553258 TI - [Legal problems in transportation of pregnant patients in premature labor from the viewpoint of basic and defined management]. PMID- 7553259 TI - [Comment on the contribution by Benz et al.: Serum screening for Down syndrome in women under 35 years of age with an age independent index]. PMID- 7553260 TI - [Triple test: problem or enhancement of prenatal diagnosis]. PMID- 7553261 TI - [Thoughts and observations on the "E-E-time" in emergency cesarean section]. PMID- 7553263 TI - [The user facing the hospital: an Algerian perspective]. AB - Based on interviews conducted in an Algerian hospital, the study aims at identifying the feelings and reactions of patients. In general, they suffer from lack of humanity and respect from the health workers. However, more importantly, the patients in hospitals suffer from lack of information and communication. PMID- 7553264 TI - [The role of general practitioners in post-occupational health care in an industrial area]. PMID- 7553262 TI - [Para-spinal analgesia in obstetrics]. AB - The use of regional analgesia and anesthesia in obstetrics has been established since the end of the 19th century. According to an agreement between German societies of anesthesiologists and obstetricians, regional anesthesia may be performed by both anesthesiologists and obstetricians under certain preconditions. At the RWTH Aachen hospital, the responsibility for different anesthetic techniques is divided between anesthesiologists and obstetricians in order to reduce legal risks. Caudal anesthesia is done by obstetricians, whereas anesthesiologists perform lumbar epidural anesthesia. A retrospective study over three years has shown comparable results. Lumbar epidural catheters were preferred in primiparous women, while caudal anesthesia was used more often in multiparous women. One-minute Apgar values as well as postpartum fetal arterial blood gas analyses did not differ significantly in both groups. The failure rate of caudal anesthesia was higher than that of lumbar epidural anesthesia. Blood pressure falls exceeding 20 percent of baseline were only seen in the epidural group (incidence 0.9 to 1.5 percent). The incidence of fetal bradycardia and the overall complication rate were similar in both groups. PMID- 7553265 TI - [The mental health of ethnic minorities in the United Kingdom]. AB - The major recent migrations into the United Kingdom took place between 1950 and 1970 from the Caribbean and the Indian sub-continent. According to the 1991 Census, 6% of the population were non-Europeans. Afro-Caribbeans have shown high psychiatric admission rates; there is no clear pattern for "British Asians" (i.e. those from the Indian sub-continent). High rates for compulsory admissions have been reported for Afro-Caribbeans but not Asians. This appears related to similar patterns for the diagnosis of schizophrenia. The excess of schizophrenia among Afro-Caribbeans has been the subject of controversy in terms both of aetiology and service provision. In this article, the mental health of smaller ethnic minority groups is discussed briefly. PMID- 7553267 TI - Society for Academic Emergency Medicine (SAEM) 1995 annual meeting. San Antonio, Texas, May 21-24, 1995. Abstracts. PMID- 7553268 TI - Differential extraction of N-acetylglucosaminidase and trehalase from the cell envelope of Candida albicans. AB - Dithiothreitol (DTT) extraction of N-acetylglucosaminidase and trehalase from intact Candida albicans ATCC 10261 cells was monitored as an index of cell envelope porosity during N-acetylglucosamine-induced morphogenesis. Trehalase, which is secreted into the cell envelope during starvation and bud-formation, displayed similar extraction kinetics in starved, germ tube-forming, and bud forming cells, indicating that the mother cell wall remains largely unchanged during morphogenic outgrowth and that the porosity of bud and mother cell walls is similar. N-acetylglucosaminidase, which is secreted specifically during morphogenesis, was released eightfold more rapidly from germ tube-forming than bud-forming cells, reflecting major differences in porosity between bud and germ tube. In addition, by assaying DTT extracts and extracted cell residues, it was found that the total extracellular N-acetylglucosaminidase activity increased 2- to 2.5-fold during DTT treatment. Thus, DTT unmasks a cryptic form of N acetylglucosaminidase. The cryptic activity was associated with the cell wall fraction. PMID- 7553266 TI - Female and male leaderships: lesson from Norwegian doctors. PMID- 7553269 TI - Phylogenetic ordinal placement based on rDNA sequences of the freshwater genera Ophioceras and Pseudohalonectria. AB - The ordinal placement of two closely related freshwater genera, Ophioceras and Pseudohalonectria, was assessed by using phylogenetic analysis of morphological characters, partial sequences of the large subunit ribosomal DNA and restriction site variations in the internal transcribed spacer (ITS). The two genera have some morphological features that are used to define taxa in both the Sordariales and Diaporthales, and, hence, their phylogenetic relationships are unclear. Equally weighted analyses of thirty-eight morphological characters produced unresolved phylogenetic trees and unequivocal conclusions could not be drawn based on the morphological data. The polymerase chain reaction-amplified ITS region was variable in length between the two genera and restriction sites in the ITS region were determined. Analysis of variation in restriction sites in the ITS region placed Ophioceras and Pseudohalonectria in one clade with taxa sampled from Sordariales. About 350 basepairs of DNA sequence from the 5' end of the large subunit rDNA were also determined. In phylogenetic analysis of the sequence data with Hypocrea lutea and Nectria cinnabarina as outgroups, Ophioceras and Pseudohalonectria showed a closer relationship to Neurospora crassa, Schizothecium sp., and Sordaria fimicola of the Sordariales than to Cryphonectria parasitica and Endothia gyrosa of the Diaporthales. PMID- 7553270 TI - Phylogenetic resolution of Morchella, Verpa, and Disciotis [Pezizales: Morchellaceae] based on restriction enzyme analysis of the 28S ribosomal RNA gene. AB - The large subunit (28S) of the ribosomal DNA repeat of Morchella, Verpa, and Disciotis and a closely related genus (Gyromitra) was enzymatically amplified via the polymerase chain reaction. Restriction fragment length polymorphisms were found among the lines investigated and used to infer phylogenetic relationships. More variability was observed toward the 5' end than toward the 3' end of the 28S rRNA gene. The RFLP data were used to assemble a phylogenetic tree for the taxonomic group. Based on the RFLP data three black Morchella species isolates differed by approximately 0.5, 1.0, and 1.5%, respectively, from all other isolates in the Morchellaceae examined in this study. Gyromitra gigas, used as an outgroup, had approximately 6.2% difference from all members of the Morchellaceae. In some cases more genetic variation was observed intraspecifically than between putative species. Additionally, the hypothesis that Morchella is composed of only a few (possibly three) polymorphic species was supported by our findings. PMID- 7553271 TI - [Reliability of bioelectric impedance analysis as a method of nutritional monitoring in cirrhosis with ascites]. AB - Bioelectric impedance analysis (BIA) was carried out in 55 cirrhotic patients (32 with and 23 without ascites) and 20 healthy controls with the aim of evaluating its usefulness in cirrhosis. Furthermore, in 13 of the ascitic patients BIA was performed immediately before and 24 hours after paracentesis. No differences were observed among the three groups in either resistance or the values of total body water (TBW), lean body mass (LBM) and fatty body mass (FBM) estimated by BIA. On the other hand, reactance and the phase angle were lower in cirrhotic patients. Likewise, the percentage of body cell mass (BCM) was lower in ascitic patients than in the non ascitic patients and the controls (37.7 +/- 1.2 vs 42.3 +/- 1.2 vs 45.5 +/- 1.6%; p = 0.0003). Mid-arm muscle circumference was correlated with TBW and LBM in all the groups and with only BCM in the controls and those without ascites. In general, the precision of the regression lines was lower in the ascitic patients than in the non ascitic patients and controls. TBW decreased following paracentesis (28.3 +/- 1.7 vs 30.3 +/- 2.0 kg; p = 0.008) but this decrease did not correspond with the volume of ascitic fluid obtained in any case. The mean BCM did not change with paracentesis although individual differences ranged between -8.0 and +11.2% (CI 95%). It is concluded that BIA is not a reliable method for nutrition evaluation in cirrhotic patients with ascites in clinical practice and that the anthropometric parameters are still preferable in these cases. PMID- 7553272 TI - [Treatment of persistent or recurrent hemorrhage caused by gastropathy lesions of portal hypertension]. AB - The different therapeutic methods for persistent or recurrent hemorrhage due to portal hypertension gastric lesions are discussed from the clinical experience with 5 cases. In 2 cases endoscopic sclerosis was carried out and in one antrectomy was performed with stabilization of the bleeding and improvement in the clinical situation being achieved in long term follow up. Two patients underwent liver transplantation which led to disappearance of the lesions. PMID- 7553273 TI - [Male with sudden-onset dementia associated with long lasting diarrhea as manifestations of Whipple's disease]. AB - A case of a 55-year old male with Whipple's disease who consulted for dementia (aggressivity, memory loss of recent events, sleep rhythm disturbances) is presented. Furthermore, the patient had referred changes in intestinal movement four years beforehand in addition to erratic arthralgias. Response to treatment (trimethoprim-sulfamethoxazole for one year) was favorable with resolution of both the neurologic and gastrointestinal pictures. The radiologic alterations of this entity are reviewed and suggest that this disease should be considered as a cause of dementia. PMID- 7553274 TI - [Gastric emphysema associated with gastric volvulus]. AB - The gastric volvulus is an infrequent entity requiring surgical treatment in both the acute and chronic cases. The case of an 81-year old female patient attended for an episode of gastric obstruction caused by a volvulus and whose simple stomach radiography showed an image of gastric emphysema is reported. The possible causes of gastric emphysema and its differential diagnosis with emphysematous gastritis are discussed. PMID- 7553275 TI - [Chronic asymptomatic intrahepatic cholestasis associated with Turner's syndrome]. AB - Turner syndrome or the gonadal dysgenesis syndrome which is monosomic because of the lack of an X chromosome (45 X) is associated to a greater incidence of autoimmune, particularly thyroidal, disorders and inflammatory intestinal disease, but is rarely associated to hepatic disorders. A female patient with chronic asymptomatic intrahepatic cholestasis which, to our knowledge, is the first reported in Spain, is herein presented. The 40-year old patient with a 45 X karyotype, feminine phenotype was accidently found to have a chronic alteration in the hepatic profile. Hepatic biochemical tests revealed AST 59 U/L, ALT 90 U/L, GGT 201 U/L and alkaline phosphatase 320 U/L. Hepatic echography was normal. Percutaneous liver biopsy was performed demonstrating minimum changes consisting of sinusoidal dilatation and pigment accumulation in the hepatocyte biliary pole. Treatment with ursodeoxycholic acid 15 mg/kg/day was administered showing a marked decrease in the laboratory parameters during follow up. Different hypothesis which may explain the association between chronic asymptomatic intrahepatic cholestasis and Turner syndrome are discussed. PMID- 7553276 TI - [Focal nodular hyperplasia of the liver: benign lesion with often surgical treatment. Magnetic resonance and laparoscopic findings]. AB - The diagnosis of benign hepatic tumor lesions is every day more frequent. Three cases of focal nodular hyperplasia (FNH) accidently detected during the performance of echography and/or CAT for other reasons are presented. The difficulty or diagnostic doubts which the different imaging techniques may present before these findings, together with the relatively young age of these patients may lead to the adoption of a surgical attitude in lesions of a clearly benign character. The role of magnetic resonance (MR) as a non invasive diagnostic technique in this type of disease is of note. PMID- 7553277 TI - [Ligation or endoscopic sclerotherapy of esophageal varices]. PMID- 7553278 TI - [Predictive factors of response to interferon in chronic hepatitis]. PMID- 7553279 TI - [Scanning electron microscopy in gastric pathology]. PMID- 7553280 TI - Is there a role for recombinant tumor necrosis factor alpha in the intravesical treatment of superficial bladder tumors?--a phase II study. AB - Clinical use of recombinant tumor necrosis factor-alpha is strongly limited by its severe toxicity, mainly cardiovascular, when systemically administered. Recent studies suggest that topical (intrapleural, intraperitoneal, intratumoral) administration is free of significant toxicity. Human recombinant tumor necrosis factor-alpha was administered intravesically, at a dose of 500 mg dissolved in 30 ml of phosphate buffer (pH 7.6-7.8) plus 0.25% human albumin, weekly for two months to 18 patients with papillary transitional cell carcinoma of the bladder. Of the 15 evaluable patients, four (26%) achieved a complete response. Systemic and local tolerability were excellent. PMID- 7553281 TI - Interstitial laser coagulation for benign prostatic hyperplasia: preliminary clinical results. AB - We report our early experience in the clinical application of interstitial laser coagulation of the prostate (ILCP) in the treatment of benign prostatic hyperplasia (BPH). Neodymium: YAG laser energy is transmitted via a specially designed interstitial thermotherapy light-guide. The light guides were inserted transurethrally into each lobe of the prostate by direct puncture, under direct visualization. The prostatic urethra is preserved during the procedure. From December 1993 to March 1994, 37 patients with symptomatic BPH were treated with ILCP. Treatment outcome was evaluated by the International Prostatic Symptom Score (I-PSS), flow rate, postvoided residual urine volume and quality of life score. Significant improvement in I-PSS and peak flow rate was observed at 3 months: I-PSS decreased from a mean of 19.3 to 10.4, and the peak flow rate increased from a mean of 7.2 to 9.4 ml/sec (p < 0.0001). The mean postvoided residual urine volume significantly decreased from 91 to 47 ml (p < 0.01). Two days after ILCP the serum prostate-specific antigen had increased by 860%, evidence of the significant tissue damage produced by laser irradiation. The quality of life score significantly decreased from a mean of 4.8 to 2.1 (p < 0.0001). No serious side-effects were observed. The early clinical results suggest that ILCP is safe and effective as a treatment of BPH and is less invasive than some other methods. PMID- 7553282 TI - Assessment of sextant biopsy of the prostate for detecting cancer prior to therapy in patients clinically diagnosed as benign prostatic hyperplasia. AB - Pathological results and perioperative morbidity were compared in 199 patients who had undergone prostatectomy and/or biopsy in order to determine the extent to which systematic biopsy is effective for detecting prostate cancer prior to therapy in patients clinically diagnosed as having benign prostatic hyperplasia. Seventeen (8.5%) cancers were detected in 199 patients following surgery and/or biopsy. Digitally-guided biopsy as a means of detecting prostate cancer was found to be just as effective as ultrasound-guided biopsy. Seven (12.5%) cancers were detected in 56 patients who had undergone biopsy and transurethral resection. Preprostatectomy biopsy detected only two of three patients with stage T1b disease. All four stage T1a and one stage T1b failed to be diagnosed. Of 90 patients who had biopsy prior to surgery other than TURP, seven (7.8%) cancers were found. Four of these were advanced. The incidence of postoperative fever > 38.0 degrees C and duration of postoperative pyuria did not differ significantly between groups with or without biopsy. Preoperative biopsy did not contribute to perioperative morbidity. Tumors detected by systematic biopsy are usually large and clinically significant. Positive biopsy results are often diagnostic, but the pathological features of a tumor together with clinical parameters should be considered to reduce the chance of overdiagnosing an insignificant tumor. Sextant biopsy would be most applicable to patients scheduled for any type of therapy other than TURP especially in those with markedly elevated serum PSA levels. This procedure may be beneficial particularly for younger patients with long life expectancy who will benefit from definitive therapy. PMID- 7553283 TI - Usefulness of digital rectal examination, serum prostate-specific antigen, transrectal ultrasonography and systematic prostate biopsy for the detection of organ-confined prostate cancer. AB - The detection rate of organ-confined prostate cancer by digital rectal examination (DRE), serum prostate-specific antigen (PSA), and transrectal ultrasound (TRUS) of the prostate, as well as the value of a directed, guided transrectal core biopsy for the prostate (TRUS-guided biopsy) combined with systematic biopsy, were evaluated. The subjects were 171 patients with urinary symptoms suggestive of prostatic disease excluding those with clinical stage C and D prostate cancer. Twenty-five patients (14.6%) had prostate cancer, 127 (74.2%) had benign prostate hypertrophy, four (2.3%) had prostatic intraepithelial neoplasia, eleven (6.4%) had inflammation, and four (2.3%) had normal prostate tissue. The incidence of detection of hypoechoic findings by TRUS in the patients in whom nodules were detected by DRE or who had elevated serum PSA was higher than that in patients with negative diagnostic findings. In 22 of the 25 patients with prostate cancer, the cancer was detected by recognition of a hypoechoic area on TRUS. In 10 of these 22 patients, prostate cancer was also detected by systematic biopsy in isoechoic areas. Prostate cancer was detected by systematic biopsy in three patients without hypoechoic findings. The positive predictive value for patients with abnormal findings on all three tests was 64.3%, which is significantly higher than that for patients with any other combination of findings (p < 0.05). Our results indicate that the combination of DRE, serum PSA and TRUS is useful for the detection of organ-confined prostate cancer, and that TRUS and TRUS-guided prostate biopsy combined with systematic biopsy should be performed in patients with abnormal findings for both DRE and PSA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553284 TI - Mini-laparotomy staging pelvic lymph node dissection for localized prostate cancer. AB - We report our early experience with mini-laparotomy staging pelvic lymph node dissection (PLND) for clinically localized prostate cancer. We have used virtually the same original technique described by Steiner and Marshall. A 5 cm lower midline abdominal incision provides excellent exposure, allowing complete PLND under direct visualization. If radical retropubic prostatectomy is indicated by the state of the pelvic lymph nodes, this can be performed only by extending the same incision. Nine patients with histologically proven prostate cancer underwent mini-laparotomy staging PLND. The average intraoperative time for mini laparotomy PLND was 33 minutes (range, 25-50 minutes). The intraoperative blood loss was 44 ml (range, 20-90 ml). The mean number of pelvic lymph nodes removed was 6.9 (range, 5-10 nodes) on the right and 10.8 (range, 8-21 nodes) on the left. Eight patients underwent immediate radical retropubic prostatectomy and one had radiation therapy. There were no complications directly related to the mini laparotomy staging PLND. Mini-laparotomy staging PLND is an attractive alternative to laparoscopic PLND, especially for patients at low risk of lymph node metastasis. PMID- 7553285 TI - Laparoscopy-assisted radical nephrectomy in combination with minilaparotomy: report of initial 7 cases. AB - We recently developed a new procedure for laparoscopy-assisted radical nephrectomy in combination with minilaparotomy to remove kidneys with renal cell carcinoma. A pararectal incision approximately 7 cm in length was performed from the subcostal region. A 12-mm trocar was placed at the mid-clavicular line at the level of the umbilicus. An 11-mm trocar was placed at the tip of the 12th rib. Under laparoscopic and trans-minilaparotomic observation, intra-abdominal manipulation was begun. The contents of Gerota's fascia were freed from the surrounding tissues and removed through the abdominal incision. Seven patients have been successfully treated with this procedure. The operating time for this procedure was shorter than the time of laparoscopic nephrectomy. There were none of the adverse hemodynamic or ventilatory effects associated with pneumoperitoneum in this procedure. This procedure also resulted in less postoperative pain and a shorter convalescence period when compared with open nephrectomy. PMID- 7553287 TI - Sling operation for male urinary incontinence after ileal neobladder reconstruction: a case report. AB - In July 1992, sling surgery was performed on a 62-year-old male patient with urinary incontinence following ileal neobladder reconstruction. Incontinence ceased immediately after this procedure which was conducted using five nylon sutures and two dacron tubes to protect the posterior urethra. PMID- 7553286 TI - Ectopic ureter opening into the seminal vesicle in an infant: a case report and review of the Japanese literature. AB - A case is presented of an ectopic ureter opening into the seminal vesicle associated with hypodysplastic kidney in an infant. We reviewed 135 cases (139 ureteral units) of male ectopic ureter from the Japanese literature and, of the 139 ectopic ureters, 109 were single-system ectopic ureters, and 26 ureters were associated with the ureteral duplication. Sixty-three and 73 ureters opened into the urinary tract and seminal tract, respectively. In patients 15 years or older, 65 cases of ectopic ureter opened into the seminal tract and 33 cases opened into the urinary tract, whereas in children under 15 years, the ectopic orifice was located more often in the urinary tract (26 cases) than in the seminal tract (8 cases). Presenting symptoms differed according to the location of the ectopic orifice. Ectopic ureters opening into the urinary tract most often presented with urinary tract infection and abdominal or lumbar pain. On the other hand, voiding and ejaculatory symptoms as well as perineal or genital pain were characteristic in ectopic ureters opening into the seminal tract. Of the 83 associated renal segments that were surgically removed, dysplasia, hypoplasia and aplasia were found in 22, 24, 14 cases, respectively. It was noteworthy that 48 of the 53 single ectopic ureters opening into the seminal vesicle were associated with ipsilateral renal dysgenesis. PMID- 7553288 TI - A case of metastatic yolk sac tumor of testis in a child. AB - We report a case of testicular yolk sac tumor in a child aged 3 years and 6 months with multiple bulky metastases to lung and retroperitoneum (stage IIIB2). After three courses of chemotherapy with a PVB regimen (cisplatin, vinblastine and bleomycin), complete and partial responses were obtained for lung and retroperitoneal lymph node metastases, respectively. The patient was followed-up closely. However, on the basis of a re-elevated alpha-fetoprotein (AFP) after 4 months' follow-up, he was treated with three courses of salvage chemotherapy with a modified VAB-6 regimen (cyclophosphamide, etoposide, actinomycin D, bleomycin and cisplatin), followed by retroperitoneal lymph node dissection. Histologically, only necrotic tissue was found. There is no evidence of recurrence 24 months after lymphadenectomy. There is very little information in the literature on the appropriate management of postchemotherapy residual mass in pediatric testicular tumors. PMID- 7553289 TI - Combined intraarterial cisplatin infusion and radiation therapy for invasive bladder cancer. AB - Combined intraarterial cisplatin infusion and radiation therapy were performed as the initial treatment for 23 patients (mean age: 70 years) with invasive bladder cancers (T2 in 17, T3 in 6) who were suitable for total cystectomy. Of these patients, five who had multiple invasive cancers without laterality had their intrapelvic hemodynamics altered by embolizing a contralateral internal iliac artery. Cisplatin (50 mg) was infused into the internal iliac artery through a subcutaneous reservoir twice a week over three weeks while concurrent radiation therapy with 30 Gy, delivered in 15 fractions, was performed. Additional cisplatin infusions were given in six patients. After this combined therapy, total cystectomy and ileal conduit was performed in six patients and transurethral resection of bladder tumor (TURBT) in 17. Two of the patients who underwent total cystectomy were found to exhibit a complete response. Therefore, the overall response rate was 87%, including 13 complete responses and seven partial responses. The complete response rates in patients with clinical stage T2 and T3 disease were 53 and 67%, respectively. The complete response rate was slightly higher in patients with a non-papillary cancer than in those with a papillary one. Toxic reactions included a decrease in bladder capacity in two patients and severe diarrhea due to methicillin-resistant Staphylococcus aureus colitis in one. Other forms of toxicity, including nausea, vomiting, neurotoxicity in the gluteal region, nephrotoxicity and myelosuppression, were tolerable. All but one of the patients are alive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553290 TI - Familial renal cell carcinoma: review of recent molecular genetics. PMID- 7553291 TI - Observations on the growth rate of renal cell carcinoma. AB - We retrospectively reviewed the records of 18 patients to investigate the growth rate of renal cell carcinoma (RCC). Growth rates were calculated from two or more gross measurements of neoplastic foci in the kidney (6 cases) and lung (12 cases). RCCs in primary sites grew slowly and the tumor volume doubling time (DT) raged from 372 to 579 days (468 +/- 84.6). Pulmonary metastases present in 12 cases grew rapidly, with a DT ranging from 20 to 154 days (89.4 +/- 43.0). Tumors in both the kidney and lung were composed of cancer cells with equal proliferative activity, as determined by immunohistochemical analysis of argyrophilic nucleolar organizer regions and proliferating cell nuclear antigen activity. Thus, our results suggest that, in addition to the proliferative activity of cancer cells, the microenvironment of the specific region is an important determinant of the growth rate of cancer cells. PMID- 7553292 TI - Increased incidental detection and reduced mortality in renal cancer--recent retrospective analysis at eight institutions. AB - A retrospective survey of renal cell carcinoma between 1975 and 1993 at eight collaborating institutions was conducted with special reference to the incidental detection and mortality of renal cancer. The analysis demonstrated a recent dramatic increase in the frequency of incidental renal cancer, which now comprises two-thirds of all renal cancers, and a simultaneous recession in non incidental or suspected renal cancer. Incidental renal cancer has remained unchanged during the last decade as far as patient demographics, occasion and method of detection, and the degree of tumor extension are concerned. On the other hand, the annual number of deaths from renal cancer has significantly decreased, and kidney-sparing surgery has been more frequently performed. These results indicate that incidental renal cancers are now in the majority, and earlier detection may contribute to improving the mortality and morbidity from the disease as a whole. PMID- 7553293 TI - The inhibitory effect of kampou extracts on in vitro calcium oxalate crystallization and in vivo stone formation in an animal model. AB - Kampou medicine is a traditional Japanese therapeutic system which originated in China and was used to treat various diseases for hundreds of years until it was superseded by Western medicine. In recent years, there has been a resurgence of interest in Kampou medicine among many physicians. Unfortunately, however, little evaluation has been performed using objective scientific methods until now, and the pharmacodynamics of Kampou medicine are still unclear. Generally speaking, Kampou medicine has been shown to have fewer side-effects than Western medicine based on the experience gained from its long usage. We first selected 16 Kampou extracts for screening as possible calcium oxalate stone prophylactic agents in vitro. This resulted in the selection of two kinds of Kampou extracts, Takusya and Kagosou, as potential Kampou extracts for stone prophylaxis. Next, these two Kampou extracts were tested in vivo for their effects on stone formation in an animal model. Takusya showed significant stone prophylaxis, while Kagosou did not. Lastly, Chorei-to, which contains Takusya and has been approved for prescription as a Kampou medicine for urolithiasis patients in Japan, was examined in vivo at two different concentrations. As a result, a low dose of Chorei-to which corresponded to the human daily dose per unit of body weight exhibited apparent stone prophylaxis, despite the disadvantage of decreasing citrate excretion. In contrast, high doses of Chorei-to did not exhibit stone prophylaxis in vivo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553294 TI - Backscattered electron imaging of crystal matrix protein on the surface of calcium oxalate crystals using colloidal gold. AB - In order to clarify the presence and localization of crystal matrix protein (CMP) upon calcium oxalate crystals, scanning electron microscopy (SEM) and backscattered electron imaging (BEI) techniques were used. This protein exhibits a remarkable affinity with calcium oxalate crystals and may be important in stone pathogenesis. In this paper, rabbit anti-human CMP polyclonal antibody was used as first antibody, and for the second antibody, goat anti-rabbit IgG conjugated with 20 nm immunogold was used. Freshly prepared crystals from male urine were fixed in SEM fixative, then blocked and washed with phosphate-buffered saline and bovine serum albumin (PBS/BSA). First and second antibodies were reacted in PBS/BSA. Crystals were then dehydrated and finally coated for SEM study. The SEM technique showed bipyramidal shaped dihydrate calcium oxalate crystals in every sample and even at high magnification, colloidal gold could barely be seen. BEI clearly demonstrated the presence and localization of the gold on the surface of the crystals as well as on the macromolecules eluted from the crystals by dissolving them in ethylenediamminetetraacetic acid solution. PMID- 7553295 TI - Changes in rat bladder function following exposure to pain and water stimuli. AB - Changes in rat bladder function following the exposure to physical stress were studied in vitro. Rats were divided into two stress groups and two control groups and maintained for 6 months under specific conditions. The stress groups, consisting of animals subjected to water or pain stress and control groups were kept in cages floored with wooden chips or wire net. The body weight of the stressed groups was significantly lower than that of the controls. Gastric ulcer occurred in none of the groups. The detrusor response of the water group to acetylcholine was significantly greater than that of the two control groups. In the pain group, the contractile response was induced by norepinephrine. In the other three groups, however, norepinephrine evoked a relaxation of muscle strips. The contractile response of the bladder to both serotonin (5-hydroxytryptamine) and ATP (adenosine 5'-triphosphate) did not differ between the four groups. PMID- 7553296 TI - Low-count organisms concealed by dominant uropathogenic organisms in urine of patients with asymptomatic bacteriuria. AB - We compared organisms isolated by selective culture media with those isolated by routine bacteriological methods using the same urine specimens. Urine was obtained by suprapubic bladder aspiration from 84 patients suspected of having asymptomatic bacteriuria, including 31 catheterized patients. The selective media included mannitol salt agar, Enterococcus faecalis agar, nalidixic acid cetrimide agar, and desoxycholate hydrogen sulfide lactose agar with polymyxin B or cefsulodin. Colony counts on the selective media approximated to those on heart infusion agar, except for desoxycholate hydrogen sulfide agar with polymyxin B, where the number of organisms was underestimated. In 24 patients the selective media detected a total of 32 strains not reported by the hospital laboratory. The unreported strains included coagulase-negative staphylococci (41%), other cocci (38%), enterobacteriaceae (9%) and glucose-nonfermenting gram-negative bacilli (9%) and fungi (3%), with the number of organisms being mostly < 10(5)/ml. The majority of the unreported strains coexisted with dominant gram-negative bacilli or enterococci. These results indicate that low-count organisms coexisting with dominant organisms tend to be missed by the routine bacteriological methods. PMID- 7553297 TI - Proceedings of the 4th International Bladder Consensus Meeting. Antwerp, Belgium, March 24-26, 1993. PMID- 7553298 TI - Optimal staging procedures, including imaging, to define prognosis of bladder cancer. PMID- 7553299 TI - Which patients are suitable for continent diversion or bladder substitution following cystectomy or other definitive local treatment? PMID- 7553300 TI - The design of clinical trials for new therapies in bladder cancer. Is it time for new strategies? PMID- 7553301 TI - Basic research. AB - Basic research is currently investigating the molecular cascade associated with bladder cancer development. Many new findings are potential leads towards the improvement of the diagnosis and prognosis of this disease. Special care, however, should be taken in the design of protocols for clinical evaluation of the value of these markers. Some initial guidelines have been put forward in this report. PMID- 7553302 TI - Intravesical chemotherapy and immunotherapy: how do we assess their effectiveness and what are their limitations and uses? PMID- 7553303 TI - The current TNM-classification of bladder carcinoma--is it as good as we need it to be? PMID- 7553304 TI - The place of radiation therapy as definitive treatment of bladder cancer. PMID- 7553305 TI - The status of bladder-preserving therapeutic strategies in the management of patients with muscle-invasive bladder cancer. AB - The recommended treatment for medically fit patients with muscle-invading bladder cancer is usually radical cystectomy. However, transurethral resection of the tumor, partial cystectomy, irradiation and systemic chemotherapy are each effective in some patients. These latter treatments allow bladder preservation and cure as an alternative to radical cystectomy although when used unselectively the survival rates are inferior to those of radical cystectomy. The updated results of conservative surgery, radiation therapy and systemic chemotherapy as monotherapy, as well as strategies of combined modality treatment were reviewed. Based on this review many areas of consensus were reached which include: 1. The primary goal of any treatment for a patient with muscle-invading bladder cancer is survival; bladder preservation in the interest of quality of life is a secondary objective. 2. Only a small proportion of carefully selected patients may be cured by transurethral surgery alone, or by partial cystectomy alone. 3. Radiation therapy is currently the standard bladder-preserving therapy against which all other bladder-preserving methods must be compared. 4. Systemic chemotherapy as monotherapy is inadequate and cannot be recommended. 5. The addition of cisplatin-containing systemic chemotherapy to radiation therapy or conservative surgery appears to improve local control. While no multi-modality therapeutic regimen has yet been shown to be clearly optimal with regard to local efficacy and minimizing toxicity, monotherapy for bladder preservation is probably not desirable as a routine approach. 6. Deferring the patient from immediate cystectomy does not appear to compromise survival, nor does the addition of primary systemic chemotherapy appear to significantly increase the morbidity of cystectomy or radiotherapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553306 TI - What is the biology of invasion and metastasis in bladder cancer? PMID- 7553308 TI - Neoadjuvant and adjuvant chemotherapy in advanced disease--what are the effects on survival and prognosis? PMID- 7553309 TI - Current methods of assessing and treating carcinoma in situ of the bladder with or without involvement of the prostatic urethra. PMID- 7553310 TI - What are the risks of cystectomy and the advances in perioperative care? PMID- 7553307 TI - The etiology of bladder cancer--are there any new clues or predictors of behavior? AB - It is well documented that the etiology of bladder cancer involves environmental factors. Most chemical carcinogens probably affect the urothelial cells via their presence in the urine. As an important cofactor, cell proliferative activity may be increased by urinary bladder infection, irritation by bladder stones or through the action of a variety of endogenously produced direct acting chemicals or agents. Among the known causative factors, avoidable major ones are occupational exposure to certain chemicals such as benzidine and 4-aminobiphenyl, cigarette smoking and bilharzial infection, which could be eradicated by a combination of praziquantel, antihelminth therapy, education and improvements in social welfare. An anticarcinogenic drug, cyclophosphamide, used as an immunosuppressive agent also seems to be associated with a high risk of idiopathic induction of bladder cancer and physicians should therefore pay particular attention to its diverse effects when considering its prescription. In contrast to the above, the consumption of coffee and tea including artificial sweeteners is now thought unlikely to be major risk. So far there is no good biochemical tool to predict individual exposure to bladder carcinogens/or relative risk of bladder cancer development. However, acetylation capacity can be applied to assess susceptibility to carcinogenic amines in people exposed in their working environment. Progress in molecular biological analysis will hopefully bring to light etiology-specific DNA damage in the bladder tumors and prove useful for prediction of tumor behavior in the near future. PMID- 7553311 TI - Reclaiming the healthcare agenda--full disclosure, patient advocacy and community participation. PMID- 7553312 TI - Destabilized workforce--restructuring's bequest. PMID- 7553313 TI - The compact with patients--a sacred trust. PMID- 7553314 TI - Managed care--the corporatization of healthcare. PMID- 7553315 TI - How to advocate for patients: laws, regulations, and the consumer. PMID- 7553316 TI - Nurses punished for healthcare warnings. PMID- 7553317 TI - Patient-focused care--as seen from the inside. PMID- 7553318 TI - The unseen hand cutting care--the healthcare consultants. PMID- 7553320 TI - RNs and caregivers confront restructuring. Pandering to patients. PMID- 7553319 TI - ADO forms--the patient/license/job you save may be your own. PMID- 7553321 TI - Confronting common concerns--healthcare workers establish network. PMID- 7553322 TI - CNA in coalition to save community hospital. PMID- 7553324 TI - Patient Watch program builds partnership between RNs and patients. PMID- 7553323 TI - Conferences, coalitions and the future of patient care. PMID- 7553325 TI - 1995 top ten myths on hospital and healthcare restructuring. PMID- 7553326 TI - Role of endogenous CCK in regulation of interdigestive pancreatic exocrine secretion in sheep (Ovis aries). AB - The role of endogenous cholecystokinin (CCK) in the regulation of pancreatic exocrine secretion during the interdigestive period was studied in conscious sheep. Intravenous infusion of proglumide (15, 30 and 60 mumol/kg/min) for 70 min significantly decreased flow rate, protein and amylase output in pancreatic juice by 80% at the maximum dose, as well as atropine (10 micrograms/kg/min), without a significant decrease in plasma concentration of immunoreactive CCK. Intravenous injection of CCK (114 pmol/kg) increased the pancreatic secretion during atropine infusion, but not during proglumide infusion. These results suggest that the regulation of interdigestive pancreatic exocrine secretion in sheep depends greatly on endogenous CCK as well as on the cholinergic mechanism. PMID- 7553327 TI - Effects of carbonic anhydrase inhibitors on oxygen consumption and lactate accumulation in skeletal muscle. AB - In isolated rat soleus and extensor digitorum longus (EDL) muscles, the effects of carbonic anhydrase inhibitors were studied on oxygen consumption as well as lactate release and accumulation after incubation in inhibitors lasting long enough to produce marked changes in contractile parameters and in the concentrations of energy-rich phosphates. The inhibitors used were chlorzolamide (10(-3) M) and NaCNO (10(-2) M). Compared with control muscles, muscles treated with either of the two inhibitors showed a decrease in force, and an increase in time-to-peak as well as in relaxation time. Lactate content and release in soleus and in EDL were increased by factors of 2-3 with both inhibitors. With both inhibitors, oxygen consumption in the red soleus increased by approximately 27%, whereas in EDL, no significant change could be observed. The increase in aerobic metabolic rate in the red soleus only might indicate that the isozyme CA III, which is present only in this type of muscle, is in some way involved in keeping the oxygen consumption low. The increase in anaerobic metabolic rate occurring in both muscles can possibly be explained by increases in Pi and ADP. PMID- 7553329 TI - Dietary nitroprusside alleviates atherosclerosis in hypercholesterolemic Japanese quail (Coturnix coturnix japonica). AB - Male Japanese quail (Coturnix coturnix japonica), susceptible to cholesterol induced atherosclerosis, were rendered hypercholesterolemic by feeding a diet containing 0.5% cholesterol in two experiments. Half the animals also received a dietary supplement of sodium nitroprusside ranging in concentration from 0.005% to 0.015%. After 10 weeks on the diets, serum was obtained for cholesterol analysis, the animals were killed, and the aortae removed and examined for the presence of atherosclerotic lesions. The number of animals having lesions and the severity of the lesions was reduced in a dose dependent manner among those animals receiving nitroprusside. Serum cholesterol was also reduced in response to increasing levels of dietary nitroprusside. These findings indicate that, in this model, dietary nitroprusside, a source of nitric oxide, can reduce the appearance and severity of atherosclerotic lesions in the aorta. PMID- 7553330 TI - Effect of a corn diet during development on [3H]-spiperone binding in the brain of rats at the perinatal stage. AB - A model of undernutrition based on feeding with a corn-diet (tryptophan deficient) was used to study maturation and physiology of the rat brain 5-HTergic system. Previously, using that model, we observed a decrease in 5-HT uptake in rat brain slices, assuming that some compensatory mechanisms could be implicated at the synaptic level on those conditions. Body, brain weight, and total [3H] spiperone binding were recorded at two fetal ages and newborn pups from mothers were fed with a corn-diet and with a corn-lysine diet. Significant decreases in body weight and in [3H]-spiperone binding were observed at all ages studied in the corn-based diet groups with respect to controls at the first postnatal day of age, whereas at the 18th gestation day, the [3H]-spiperone binding was higher in the protein restricted and corn-lysine supplemented group than in the control group. Results suggested that expression of 5-HT receptors and possibly their synthesis may be limited at the synaptic level on undernutrition conditions. PMID- 7553328 TI - Laying hen responses to acute heat stress and carbon dioxide supplementation: II. Changes in plasma enzymes, metabolites and electrolytes. AB - Exposure of laying hens to an acute heat stress period (38 degrees C) produced a decrease (P < or = 0.05) in blood plasma magnesium compared with pre-heat stress (23 degrees C) levels. Blood plasma glucose, alkaline phosphatase, total protein, uric acid and creatinine were not changed (P > or = 0.05) by exposure to 38 degrees C compared with the first 23 degrees C exposure. Inorganic phosphorus, calcium, potassium and sodium levels were not affected by acute heat-stress exposure or carbon dioxide addition. These results suggest that acute heat stress had no dramatic effect on plasma enzymes, metabolites and electrolytes of laying hens. PMID- 7553331 TI - Tityus bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal root ganglion neurons. AB - A novel toxin was isolated from the venom of the Brazilian scorpion Tityus (T.) bahiensis. The N-terminal amino acid sequence of this toxin was shown to be 80% identical to the corresponding segment of T. serrulatus toxin IV-5. The new toxin was thus named toxin IV-5b. Toxin IV-5b was found to markedly slow inactivation of Na channel in dorsal root ganglion neurons from chick embryo. By contrast, Na channel activation was only negligibly delayed, and deactivation completely unaffected. Similarly unaffected by the toxin were K and Ca currents. The slowing effect of the toxin starts to appear at concentrations of c. 80 nM, and shows a KD of 143 nM. With a toxin concentration of 2.4 microM, the Na channel inactivation time constant was increased c. 3-fold with respect to the control. The slowing of inactivation was voltage dependent, and increased with depolarization. PMID- 7553332 TI - Oxygen transport proteins: I. Structure and organization of hemocyanin from scorpion (Buthus sindicus). AB - Scorpions are regarded as the oldest terrestrial arthropods. Scorpion Buthus sindicus (Buthidae) is commonly found in Pakistan and the Mediterranean region. The hemolymph of most arthropods contains large multisubunit, extracellular metalloprotein commonly known as hemocyanin which performs the important function of oxygen transport. The literature available to date shows that no attempt has been made to study hemocyanins or hemolymph proteins from this species. This communication presents the isolation, characterization and partial structural studies on hemocyanin from scorpion Buthus sindicus. (1) The hemolymph was collected by cardiac puncture, centrifuged and subjected to polyacrylamide gel electrophoresis and isoelectric focussing. (2) Crude hemolymph was subjected to gel filtration and high performance ion-exchange chromatography. (3) Purified hemocyanin subunits Bsin 1, 2 and 3 have been analysed for their amino acid composition and N-terminal sequence. The sequence homology was determined by comparison with other arthropod hemocyanin. The results are discussed. PMID- 7553333 TI - Opioid peptides content in the rat brain during the ictal phase and after pentylenetetrazol-kindled rats. AB - We determine the opioid peptide content in the rat brain during the ictal phase and postictal depression after pentylenetetrazol kindling rats. Radioimmunoassays with highly specific antisera risen for Met-enkephalin, Leu-enkephalin and octapeptide, were carried out during the ictal phase, and 15, 30 and 60 min after seizures. We always found an initial IR-Met-enkephalin decrease during the postictal depression content, followed by a reduction in IR-Leu-enkephalin and IR octapeptide tissular concentration. We suggest a functional and differential release of the opioid peptides, during the postictal depression time-course. PMID- 7553334 TI - Effect of cholinergic secretagogue substances on the morphology of the harderian gland in the frog, Rana esculenta. AB - The present study using secretagogue substances was undertaken to investigate Harderian gland secretion in the frog, Rana esculenta. Carbamylcholine chloride and bethanechol injections caused enhancement of the secretory activity and hyperemia, while nicotine did not. Morphological examinations showed reduced cellular height and dilated alveolar lumina, containing secretory granule discharge, nuclei and cytoplasmic fragments, indicating an apocrine and holocrine secretion type. The administration of atropine prevented the enhancement of the secretion. Our data suggest that cholinergic stimulation provokes enhancement of the secretory activity on the frog HG, and this mechanism appears to be mediated by the activation of the muscarinic receptors. PMID- 7553335 TI - Metabolic and ventilatory adjustments and tolerance of the bat Pteropus poliocephalus to acute hypoxic stress. AB - We have investigated the maximum tolerance and the ventilatory responses of a bat, P. poliocephalus (PP), to normobaric hypoxic stress. PP can tolerate inspired PO2s (PiO2) down to 30 torr. This bat is one of the most hypoxia tolerant non-hibernating species of mammals known, and has a tolerance which lies within the range of PiO2s reported for different birds. Unlike most mammals in its size range, PP maintains its normoxic oxygen consumption rate even in deep hypoxia. The maximum hypoxic ventilatory response (HVR), the air convection requirement (Vi/MO2), and the lung oxygen extraction (EL) ability of PP in deep hypoxia are all greater than those of other mammals. These and other data indicate that PP has a superior mammalian tolerance for hypocapnia. The magnitudes of both the V1/MO2 and the EL value of PP fall between those reported for Pekin ducks at corresponding PiO2s, and are inferior to the maximum capabilities of bar-headed geese. Thus, the tolerance and ventilatory adjustments of PP to deep hypoxia are intermediate between those of typical non-flying mammals and the most tolerant avian species, and suggest that at least some of this bat's respiratory adaptations for flight may serve as preadaptations for withstanding acute hypoxic stress. PMID- 7553336 TI - Distribution of digestive enzyme activities along intestine in blue fox, mink, ferret and rat. AB - The activities of amylase, total proteases, monoglyceride lipase, glycyl-leucine dipeptidase and sucrase were investigated in mucosa from five consecutive parts of small intestine in blue fox, mink, ferret and rat. In comparison with rats, the activity gradient of carbohydrates and TPA in mucosa of predatory animals was shifted in the distal direction. The distribution of dipeptidase and monoglyceride lipase along the intestine was similar enough in all animals: the first was exemplarily the same all along the gut, while the second slightly decreased in a distal direction. PMID- 7553337 TI - Influence of torpor on daily energy expenditure of the dasyurid marsupial Sminthopsis crassicaudata. AB - Daily torpor is an energy-conserving mechanism, used by many small marsupials to reduce energy expenditure during adverse environmental conditions. Since little is known about how much energy is actually saved by torpor in marsupials and how this is related to the duration of torpor bouts, we investigated the effect of ambient temperature (Ta) on different metabolic states and the effect of torpor bout duration in Sminthopsis crassicaudata (16 g) on average daily metabolic rate (ADMR). Ta had a significant effect on the active and resting metabolic rates (MR), both being higher at Ta 12 degrees C than Ta 18 degrees C (P < 0.001, t test). In contrast, the ADMR at Ta 12 degrees C and Ta 18 degrees C did not differ significantly, and it appears that the increased cost for thermoregulation in normothermic active and resting animals at Ta 12 degrees C was compensated by a decrease in MR during torpor. Torpor bout duration was negatively correlated with ADMR. Torpor bouts of 5 hr reduced ADMR by 12-16%. Torpor bouts of more than 10 hr reduced ADMRs by 30-50%. Our study shows that torpor can significantly reduce daily energy expenditure in S. crassicaudata, and this may be important for the survival of this species in the wild. PMID- 7553338 TI - Metabolic profiles and risks of diseases in camels in temperature conditions. AB - A survey of 65 female camels has been conducted over a 1-year period in France to determine their metabolic profiles and to study the correlations between this profile and the feeding and health status in temperate conditions. The following parameters were measured: protein (albumin, globulin, total protein), urea, glucose, free fatty acid, liver enzymes (GLDH, GGT, GOT), minerals (Ca, Mg, Cu, Zn) and ceruloplasmin. The values obtained were similar to those reported in desert areas throughout the world, but the standard deviation was generally higher. This important variation might be due to the large variability of feeding conditions: albumin (36.4 +/- 4.7 g/l), total globulin (32.7 +/- 5.1 g/l), total protein (69.2 +/- 6.1 g/l), urea (30.0 +/- 14.8 mg/100 ml), glucose (111.0 +/- 12.2 mg/100 ml), FFA (0.15 +/- 0.15 mmol/l), GLDH (5.8 +/- 10.8 IU/l), GGT (10.1 +/- 5.8 IU/l), GOT (48.1 +/- 14.3 IU/l), calcium (10.2 +/- 6.5 mg/100 ml), magnesium (2.6 +/- 0.3 mg/100 ml), copper (65.4 +/- 20.2 micrograms/100 ml), zinc (34.6 +/- 7.8 micrograms/100 ml), ceruplasmin (41.4 +/- 2.6 UO). The season, the mineral supplementation and the health status had a significant effect on the metabolic profile of the she-camels. PMID- 7553339 TI - Biochemical principles of metabolic depression. PMID- 7553340 TI - kdr-Type resistance in insects with special reference to the German cockroach, Blattella germanica. AB - The phenomenon of knockdown resistance (kdr) was first noted in the housefly (Musca domestica), and has subsequently be found (i.e. kdr-type resistance) in several other insect pests including the German cockroach (Blattella germanica). This type of resistance causes insensitivity of the nervous system to pyrethroids, DDT and a limited number of sodium channel neurotoxins. In the German cockroach, kdr-type resistance is incompletely recessive, monogenic and not sex linked or due to cytoplasmic factors. Additionally, kdr-type resistance is not associated with a change in sodium channel density. kdr or kdr-type loci are tightly linked or identical to the para-homologous sodium channel locus in German cockroach, housefly and tobacco budworm (Heliothis virescens), suggesting that kdr and kdr-type resistance are due to mutations in the para-homologous sodium channel gene. kdr-Type resistance in the German cockroach appears similar, although not necessarily identical, to kdr in houseflies. PMID- 7553341 TI - Fatty acid binding protein in locust and mammalian muscle. Comparison of structure, function and regulation. AB - The flight muscle of adult desert locusts, Schistocerca gregaria, contains a fatty acid binding protein (FABP) that is homologous to mammalian M-FABP (cardiac FABP. In spite of the evolutionary distance between invertebrates and vertebrates, locust muscle FABP is similar to cardiac FABP in its amino acid sequence, structure, and binding behavior. While cardiac FABP is present already in the prenatal period, locust FABP is an adult specific protein; its expression is directly linked to metamorphosis. A correlation seems to exist between fatty acid oxidative capacity and FABP content in both locust and mammals. To accomplish the higher metabolic rate encountered during migratory flight, locust flight muscle cytosol contains more than three times as much FABP as that in mammalian heart. Increased fatty acid utilization by exercise or endurance training apparently induces FABP expression. Similarities and differences between vertebrate and invertebrate M-FABP are discussed in light of the proposed functions of muscle FABP as fatty acid transporter and cytoprotectant. PMID- 7553342 TI - Amino acid sequence determination of a protein purified from the shell of the shrimp, Pandalus borealis. AB - One of the urea-extractable proteins in the shell of the shrimp Pandalus borealis has been purified, and the complete amino acid sequence has been determined by the combined use of mass spectrometry and Edman degradation of the intact protein as well as of enzymatically derived peptides. Characteristic features of the sequence are discussed and compared to sequences from insect cuticular proteins and other structural proteins. PMID- 7553343 TI - A carboxylesterase from the parasitic nematode Ascaris suum homologous to the intestinal-specific ges-1 esterase of Caenorhabditis elegans. AB - We have identified a carboxylesterase in A. suum that appears to be the homolog of the gut-specific C. elegans ges-1 enzyme. The A. suum esterase was purified and its N-terminal sequence found to be 50% identical to the C. elegans ges-1 protein. We have used isoelectric focusing analysis to demonstrate that, unlike the C. elegans ges-1 esterase, the A. suum enzyme is not restricted to the gut but is expressed in a wide range of tissues. PMID- 7553344 TI - The primary structure of a basic (pI 9.0) fatty acid-binding protein from liver of Gallus domesticus. AB - The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226. PMID- 7553345 TI - Rates of beta-oxidation of fatty acids of various chain lengths and degrees of unsaturation in highly purified peroxisomes isolated from rat liver. AB - Highly purified peroxisomes were obtained from the liver of untreated rats, and rates of peroxisomal beta-oxidation were measured using fatty acyl-CoAs differing in chain length and degree of unsaturation. A 20-24-fold purification of peroxisomes, indicated by the specific activities of the marker enzymes catalase and urate oxidase, respectively, was obtained from crude liver homogenate using differential centrifugation techniques followed by a 30% Nycodenz gradient separation. The use of a 30% Nycodenz gradient in the final step of purification was extremely effective (e.g. 5.5-fold reduction) in removing lysosomal contamination. The rate of peroxisomal beta-oxidation with lauroyl-CoA (C12:0) as substrate was the highest of all fatty acyl-CoAs tested. Butyryl-CoA (C4:0) was not oxidized by purified peroxisomes. In general, as chain length of the fatty acyl-CoAs increased above 12 carbons, the rates of beta-oxidation decreased. PMID- 7553346 TI - Peroxisomal beta-oxidation of fatty acids in bovine and rat liver. AB - Hepatic peroxisomal beta-oxidation rates were compared in liver homogenates from cows and rats during different nutritional and physiological states. Peroxisomal oxidation in liver homogenates from cows represented 50% and 77% of the total capacity for the initial cycle of beta-oxidation of palmitate and octanoate, respectively, but only 26% and 65% for rats. Lactation or food deprivation did not alter rates of hepatic peroxisomal beta-oxidation of palmitate or octanoate in cows. Fasting and clofibrate treatment increased rates of total and peroxisomal beta-oxidation of palmitate and octanoate in rat liver. PMID- 7553347 TI - Glutathione regeneration in calcium-loaded erythrocytes: a possible relationship among calcium accumulation, ATP decrement and oxidative damage. AB - Glutathione (GSH) regeneration was studied in rabbit erythrocytes which were loaded with calcium using ionophore A23187. Calcium-loading induced by A23187 and various concentrations of CaCl2 caused a dose-dependent depression in red cell GSH regeneration. The lowered GSH regeneration was mainly due to reduction of ATP level. In an experiment using haemolysate, the effect of calcium per se was negligible, while magnesium strongly affected GSH regeneration by controlling the rate of hexokinase reaction. These results indicate a possibility that cation perturbation, metabolic decay and oxidative damage are all interrelated in the erythrocyte aging process. PMID- 7553349 TI - The effect of GTP on the aluminum fluoride- and forskolin-activated adenylyl cyclase from human embryonic kidney 293 cells. AB - GTP has been shown to inhibit AlF4(-)-stimulated, and to activate forskolin stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4(-) stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4(-)- or forskolin activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4(-)-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4(-)-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4- or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4-. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4(-)-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4-. PMID- 7553348 TI - Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina. AB - The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a Kd for racemic JH III of 33 +/- 6 nM. The density of the binding sites is 212 +/- 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [3H]JH III with JH III > JH II > JH I > JH III acid > JH III diol > JHB3 = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells. PMID- 7553351 TI - Glucose dehydrogenase in beef (Bos taurus) and rainbow trout (Oncorhynchus mykiss) liver: a comparative study. AB - The monomer molecular mass of glucose dehydrogenase (GDH, EC 1.1.1.47) from rainbow trout liver and beef liver were estimated to be 90 kDa for both enzymes, by electrophoresis in the presence of Na-dodecyl-SO4 (SDS). The 90-kDa proteins were partially degraded to about 60 kDa when purified with a delayed procedure without protease inhibitors. Tryptic cleavage of the 90-kDa proteins gave fragments of about 60 kDa and 30 kDa, being similar for trout and beef GDH. Isoelectric points, kinetic and thermodynamic properties of the two enzymes are markedly different. Triton X-100 stimulated and stabilized the reactions catalysed by the purified enzymes. PMID- 7553350 TI - Insulin-like growth factor mRNA expression in tissues of lean and obese male SHFF/Mcc-fa(cp) rats. AB - No differences were detected in serum IGF-I levels between lean and obese male SHFF/Mcc-fa(cp) rats expressing non-insulin-dependent diabetes mellitus (NIDDM). In contrast, serum insulin levels, and blood glucose levels were significantly elevated in obese as compared to lean littermates (P < 0.05), indicating that the obese animals were diabetic. Northern blot analysis of total tissue RNA using labeled cDNAs for IGF-I and IGF-II revealed a decrease in liver and adipose IGF-I mRNA expression in the obese littermates while IGF-II mRNA expression was decreased only in adipose tissue of obese animals as compared to lean littermates. PMID- 7553353 TI - Kinetic properties of ovine adipose tissue fructose-1,6-bisphosphatase. AB - The presence of FBPase was confirmed in both human and ovine white adipose tissue in metabolically significant amounts. The partially purified enzyme from ovine adipose tissue exhibited kinetic properties very similar to other mammalian FBPases (pH optimum of 7.5, absolute requirement for divalent metal ions and strong inhibition by both AMP and F-2,6-P2). The micromolar S0.5 value obtained suggests that the enzyme may be of physiological significance. PMID- 7553354 TI - Isoenzymatic diagnosis of filariae: a method for separation of lactate dehydrogenase isoenzymes from Molinema dessetae (Nematoda: Filarioidea). AB - Lactate dehydrogenase (LDH) is highly active in filariae and could be a valuable tool for phyllogeny studies. Unfortunately, the isoenzymatic diagnosis of filariae is often difficult for LDH because of a poor mobility of the enzymes in starch gels which are the most commonly used in such studies. We propose here a method to separate filarial LDH isoenzymes using disc electrophoresis. The experiments were carried out on male and female Molinema dessetae in order to compare their respective isoenzymes. The study of several parameters such as buffer systems, percentage of bisacrylamide and progression time led to optimize the enzyme separation. LDH from male and female filariae were compared to mammal LDH-H4 and LDH-M4. Five and four LDH isoenzymes were found, respectively, in male and female worms. Relative concentration of each isoenzyme diverged between male and female worms. Mammal muscle LDH-M4 type moved between LDH2 and LDH3 from female worms, and between LDH1 and LDH2 from male worms. Mammal heart H4 type enzyme was very different in electrophoretic mobility. The ratio of each isoenzyme was determined by densitometry. The major isoenzymes from female worms will be studied as a biochemical target for chemotherapeutic attack. PMID- 7553352 TI - The effect of inositol hexaphosphate in the high-affinity hemoglobin of the Andean chicken (Gallus gallus). AB - A strain of high-altitude chickens (Gallus gallus) with a high hemoglobin-oxygen affinity has been found in the Peruvian Andes. The purpose in this work was to investigate the relation of this high affinity with the allosteric regulation of the inositol hexaphosphate effector (IHP). The hemoglobin-oxygen affinity has been determined in phosphate-free hemoglobin solutions before and after the addition of IHP. The results showed that the addition of IHP to the phosphate free hemoglobin solutions increased the affinity significantly less in the high altitude chicken (26.3 Torr) than in the sea-level chicken (42.2 Torr) (P < 0.001). It was concluded that the high-affinity hemoglobin is probably dependent on a molecular change affecting the allosteric regulation, although a change in the intrinsic properties of the hemoglobin cannot be discarded. PMID- 7553355 TI - Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum). AB - Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum, Kinosternidae) were analyzed by gas chromatography mass spectrometry (GC-MS) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Analysis by GC-MS indicates 2,3-dihydroxypropanal and C3-C24 free or esterified fatty acids. Analysis by SDS-PAGE indicates a major protein component with an approximate molecular mass of 60 kDa and minor components ranging from ca. 23 to 34 kDa. The major component of K. subrubrum glandular secretions exhibits a mobility that matches that of the Kemp's ridley sea turtle (Lepidochelys kempi, Cheloniidae), suggesting that these proteins are evolutionarily conserved. PMID- 7553357 TI - Aminotransferase activities in mouse, Mus domesticus, erythrocytes separated according to age. AB - Erythrocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities are often used as indices of vitamin B-6 nutritional status; however, results using a mixed population of erythrocytes can be quite variable. Erythrocytes from two strains of mice (Mus domesticus), A/Ibg and DBA/Ibg, were separated according to age by centrifugation through discontinuous Percoll density gradients into three fractions: top (least dense, youngest), middle and bottom (most dense, oldest). A sufficient yield of age-fractionated erythrocytes was obtained from a single mouse for all of the enzyme measurements. The activities of AST, ALT and three age-marker enzymes, pyruvate kinase, acetylcholinesterase and hexokinase, were found to be significantly higher in the youngest cell fractions, and declined in the older, more dense fractions. A mice had significantly lower AST and ALT activities in the age separated fractions than did DBA mice. The measurement of enzyme activities in low density, young cells may be especially useful in studies involving conditions in which the proportion of young erythrocytes may be elevated with respect to the entire erythrocyte mass. PMID- 7553356 TI - Relationship between plasmin-trypsin-inhibitory and sialyltransferase activities. AB - Previously we have shown that the measurable soluble sialyltransferase (STase) activity released into the medium during the incubation of rat jejunal slices was dependent upon the presence of a heparin-binding fraction (HBF) from heat inactivated serum or a trypsin-binding protein (TBP) isolated from HBF. Both HBF and TBP were able to inhibit trypsin and plasmin. The measurement of galactosyltransferase (GTase) activity which was also released in incubations was not dependent on HBF or TBP. The present study is directed towards further exploring the relationship between STase activity and protease inhibitory activity. Heat-inactivated serum from turpentine-treated rats (HTS), had higher plasmin-trypsin-inhibitory (HTS) activities compared to heat-inactivated serum from control rats (HCS). When HTS was used to supplement jejunal incubations, there was a 25-40% increase in the measurable STase activity in the incubation medium compared to similar incubations carried out in buffer alone. In contrast, with HCS the increase was 10-15%. During incubations with hepatocytes, STase activity detected in the incubation medium was increased with the incubation buffer was supplemented with HTS compared to incubations supplemented with HCS. Serum antiproteolytic activity was higher in turpentine rats compared to controls. Incubation of serum at 37 degrees C led to a progressive decrease in plasmin-trypsin-inhibitory and STase activities. TBP a plasmin and trypsin inhibitor was able to prevent the decrease in STase activity. Overall, serum STase activity was higher in the turpentine treated rats. In contrast, GTPase activity in serum as well as that detected in the medium during jejunal and hepatocyte incubations was not dependent on protease inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553358 TI - Examination of variables affecting syncytium formation by, and serum neutralization of, feline immunodeficiency virus on CrFK cells. AB - The feline immunodeficiency virus (FIV) induces syncytia in Crandell feline kidney (CrFK) cells grown in low fetal bovine serum-containing medium. This finding has allowed the development of sensitive FIV titration and neutralization assays using syncytium formation as an indicator of infection. In this report we examine several variables that can influence number and size of syncytia. In addition, by performing assays under rigidly controlled culture conditions, we confirm that serum neutralization assays based on FIV-induced syncytium formation in CrFK cells detect broadly reactive neutralizing antibodies. PMID- 7553359 TI - Purification of tomato yellow leaf curl geminivirus. AB - Attempts were made to find a good purification procedure for tomato yellow leaf curl virus (TYLCV), a dangerous and continuously spreading whitefly-transmitted germinivirus, up to now only partially purified. Electron microscopy, serology and spectrophotometry were used to evaluate different procedures. The scheme finally adopted was the following: collect leaves and stems from Nicotiana benthamiana graft-infected 45-60 days previously (5-10 g/plant); homogenize with 0.5 M phosphate buffer pH 6 containing 2.5 mM NaEDTA, 10 mM Na2SO3, 0.1% 2 mercaptoethanol, 1% Triton X-100 and 0.1% Driselase (3-4 ml of buffer for each g of material); incubate overnight on ice with gentle agitation; filter; emulsify with 15% cold chloroform; centrifuge at low speed; ultracentrifuge supernatant; resuspend pellets in 0.5 M phosphate buffer pH 7 containing 2.5 mM NaEDTA; centrifuge at low speed; repeat resuspension of the pellets and low-speed centrifugation; ultracentrifuge the pooled supernatant on a Cs2SO4 gradient (e.g. for 5 h at 41,000 rpm); collect the virus band and dialyse or ultracentrifuge the virus. The virus yield was 5-10 mg per kg of tissue. PMID- 7553360 TI - Toxigenicity of culture filtrates of Salmonella enteritidis isolates on three mammalian cell lines. AB - Culture filtrates of 28 Salmonella enteritidis isolates were tested for toxicity on Vero-, CHO-, and human foreskin fibroblast (HFF) cells. Cytopathic effects on HFF cells were extensive, and were observed even with some filtrates diluted 1:256. Vero cells showed effects with filtrates diluted up to 1:16, and CHO cells gave weak or no reaction. All isolates produced iron-binding siderophores as determined by reactions on chrome-azurol-S medium. PMID- 7553361 TI - In vivo and in vitro effects of lactoferrin on Yersinia pseudotuberculosis. AB - Iron is a growth requirement for virtually all microbes. In the human body, extracellular iron is sequestered from microbes by binding to proteins such as lactoferrin. In this study the effect of lactoferrin and human milk on Yersinia pseudotuberculosis was investigated. Its growth in vitro was inhibited by iron free, but not iron saturated, pure lactoferrin or human milk. Iron-free human milk and to a less extent normal human milk were bactericidal for Y. pseudotuberculosis cells that were suspended in deionized water. The in vivo studies also show that iron-saturated lactoferrin enhanced growth, whereas, the viable count was reduced by iron-free pure lactoferrin and EDDA. Nine envelope proteins were decreased or disappeared upon growth in iron-deficient medium, whereas one new high molecular weight protein appeared under the same conditions. PMID- 7553362 TI - Effect of some physical and chemical factors on the bactericidal activity of human lactoferrin and transferrin against Yersinia pseudotuberculosis. AB - The iron-chelating proteins lactoferrin and transferrin have been shown to be bactericidal for a variety of organisms. In this study, the effect of pH, temperature, their concentration, and magnesium and calcium on the bactericidal activity against Yersinia pseudotuberculosis was investigated. The bactericidal activity of lactoferrin was higher at acid pH, whereas, the bactericidal activity of transferrin was higher at alkaline pH. Both were not efficient at 4 degrees, 15 degrees, and 25 degrees C, but they were efficient at 37 degrees C. Lactoferrin, but not transferrin, was very efficient at 42 degrees C. The activity of both were time and concentration dependent. Calcium did not effect their activity up to 60 mM, whereas, magnesium reduced the activity of lactoferrin only. PMID- 7553363 TI - Influence of amoxicillin, erythromycin and metronidazole on adherence of Helicobacter pylori. PMID- 7553364 TI - First data on the distribution and ecology of Vibrio spp. of the Straits of Magellan (South America). AB - During the austral summer of 1991 a study was carried out on the presence and distribution of the genus Vibrio in the Straits of Magellan. Vibrios strains were isolated using membrane filters and Marine Agar 2216 in anaerobiosis. Variations of the populations of total heterotrophic bacteria and vibrios were observed both on the surface and along the column of water. All vibrios are psychrotrophic and were grouped in 4 cluster among which cluster 1, identified as presumed V. anguillarum, seems the most important including 73% of strains. A certain habitat segregation of clusters was noted. Cluster 4 was found only in a deep and permanently colder water mass. The relations between 20 environmental parameters and the bacterial population were also studied. Significant positive correlations were observed between the vibrios population and various fractions of suspended particulate matter. PMID- 7553365 TI - HIV-plasma viral load detection by branched DNA signal amplification. AB - To detect HIV-1 plasma viral load in seropositive patients, we applied a new molecular quantitation technique: branched DNA signal amplification (bDNA). We performed bDNA with 99 sera from HIV-1 seropositive patients undergoing antiretroviral therapy. We compared the results obtained with p24 antigenemia and CD4+ cell count. The bDNA proved quite sensitive and available for routine use in laboratories. PMID- 7553367 TI - The use of surface immunofluorescence assay (SIFA) in the microbiological diagnosis of Lyme borreliosis: a case report. AB - In this report we describe the use of a newly developed immunofluorescence technique performed with living spirochetes to detect serum antibody to B. burgdorferi s.l. in a case of early Lyme borreliosis. The immunofluorescence method used (surface immunofluorescence assay: SIFA) proved useful in the serological evaluation of suspected cases of Lyme disease. PMID- 7553368 TI - An immunofluorescence assay in microwell plate (IFA) for detecting serum antibodies to Chlamydia pneumoniae. AB - An indirect immunofluorescence assay using a 96 microwell cell culture plate (IFA) was developed for the detection of antibodies to C. pneumoniae. The results obtained by IFA on 230 sera of healthy subjects were compared with those obtained with the microimmunofluorescence test. The correlation coefficient for IgG detection with the two methods was 0.96 showing good agreement. PMID- 7553366 TI - Use of a rapid and simple method to extract proviral DNA in the identification of HIV-1 by PCR. AB - DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV 1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity. PMID- 7553370 TI - Resistance to aminoglycosides and other antibiotics among clinical isolates of Enterococcus spp. AB - We conducted a study on 81 clinical isolates of enterococci (65 Enterococcus faecalis and 16 Enterococcus faecium) collected consecutively over a one year period at our University Hospital. Of them, 10 (15%) Enterococcus faecalis and 6 (38%) Enterococcus faecium showed high level resistance to aminoglycosides, were all acquired in surgical units and in five cases they were responsible for invasive infections. Ampicillin and imipenem were active only against Enterococcus faecalis. Vancomycin and teicoplanin were bacteriostatic; however, teicoplanin MICs were lower and in 45% of cases its MBC values were within clinically achievable serum concentrations. Ciprofloxacin and cotrimoxazole were active in vitro. PMID- 7553369 TI - Influence of centrifugation on the infectivity of Chlamydia pneumoniae IOL-207. AB - The influence of centrifugation on the infectivity of Chlamydia pneumoniae IOL 207 was compared with seven serovars of C. trachomatis biovar trachoma, the three serovars of C. trachomatis biovar lymphogranuloma and four C. psittaci strains. Prolonged centrifugation from 1 to 4 hours resulted in a 3-fold increase in the infectivity of C. pneumoniae and variable or no increase in the infectivity of the other Chlamydia strains studied. These findings indicate that a 4 hour centrifugation of C. pneumoniae is recommended to improve the isolation procedures of this organism in cell cultures. PMID- 7553371 TI - In vitro effects of novel glycopeptide antibiotics on the reactivity of the lipopolysaccharide (LPS) of S. minnesota R595. AB - Four novel glycopeptide antibiotics, namely MDL 62708, MDL 63155, MDL 62827, MDL 62873 (mideplanin), plus teicoplanin, which differ in their chemical structure, were used to examine the relationship between the structure of glycopeptides and their LPS neutralization activity. Compound MDL 62708 (100 micrograms/ml) significantly reduced (P < 0.01 vs. antibiotic-free LPS, by Fisher's PLSD Test) metachromatic reactivity of S. minnesota R595 LPS (500 micrograms/ml) as evaluated by the DMB test. The remaining glycopeptides showed a significant reduction of the metachromatic reactivity, although at concentrations (333 and 1000 micrograms/ml) higher than MDL 62708. Data obtained with LAL test appeared in accordance with those of the other techniques: all the glycopeptides used (100 and 1000 micrograms/ml) significantly (P < 0.05) reduced the reactivity of S. minnesota R595 LPS (50 pg/ml), and the lowest concentration of MDL 62708 (10 pg/ml) used produced a substantial, although not significant, reduction of the LPS reactivity with LAL. The antibiotic/LPS ratios associated to a significant reduction of LPS reactivity were 3.3/5 (wt/wt) and 2/1 (wt/wt) for DMB and LAL tests respectively. Such ratio appeared to be even lower for MDL 62708. In conclusion, the four new glycopeptides, when tested at an antibiotic/LPS ratio about 1000 times lower than that which can be found in vivo, were able to reduce the reactivity of LPS in the in vitro models used. Teicoplanin aglycone MDL 62708, which also lacks the teicoplanin fatty acid, seems to have the same anti LPS activity as the parental antibiotic, thus suggesting an important role for the glycopeptide backbone and NH2 groups in LPS-neutralizing effect. PMID- 7553372 TI - Effect of LPS and porins on lymphocytes evaluated by calorimetry. PMID- 7553373 TI - Genetic variability in different Pneumocystis isolates from man and rats. AB - To gain further insight into the genetic variability between different isolates of Pneumocystis carinii of rat and human origin, we sequenced three DNA regions from the mitochondrial rRNA gene, the 5S rRNA gene and the dihydrofolate reductase gene (DHFR) of microorganisms isolated from three different immunosuppressed rat sources and from one HIV + patient with P. carinii pneumonia. Some point mutations and deletions were found among rat isolates in two regions (mitochondrial rRNA gene and 5S rRNA gene). Nucleotide sequence variations in these conserved regions support the hypothesis of the existence of several Pneumocystis strains infecting the same host species. PMID- 7553375 TI - Amelogenesis imperfecta--towards a new classification. AB - This editorial reviews the history of the classification of amelogenesis imperfecta (AI). The limitations of the existing classification systems are discussed. An alternative classification is proposed based upon the molecular defect, biochemical result, mode of inheritance and phenotype in the family involved. While not all of the criteria for the proposed classification can yet be addressed, this scheme is proposed for future classification of AI cases and families. PMID- 7553374 TI - Peripheral giant cell granuloma: a clinical study of 77 cases from 62 patients, and literature review. AB - OBJECTIVES: The aim of this study was to identify the principle clinical features of the peripheral giant cell granuloma (PGCG), and to recognise clinical features of PGCG that are poorly defined. DESIGN: We reviewed retrospectively 77 cases of PGCG from 62 patients, from our files with respect to incidence, sex, patient age, race, clinical symptoms and signs, radiographic features and recurrence following excision. RESULTS AND CONCLUSIONS: Our results were largely in agreement with previous reports, although there is wide variation in the results published between series. In addition, some clinical features of PGCG are poorly defined. Little is known about the relative incidences of PGCG and central giant cell granuloma. An association between PGCG and tooth loss may exist, but is poorly defined, and not all PGCG that involve edentulous areas follow recent tooth loss. Information about PGCG recurrence after excision is limited, and does not necessarily follow incomplete excision. Despite the large number of reported cases of PGCG, clarification of some clinical features is required, and may help formulation and interpretation of future laboratory-based research into this poorly understood lesion. PMID- 7553376 TI - Peripheral giant cell granuloma. An immunohistochemical and ultrastructural study. AB - OBJECTIVE: To study the nature of multinucleated and mononuclear cells from peripheral giant cell granuloma (PGCG). MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded sections of 40 cases of PGCG were immunohistochemically stained for vimentin, alpha I-antichymotrypsin, CD68, S-100 protein, lysozyme, leucocyte common antigen (LCA), factor VIII-related antigen and muscle cell actin. Six cases of PGCG were also studied by transmission electron microscopy. RESULTS: Vimentin, alpha I-antichymotrypsin and CD68 were expressed in both the mononuclear and multinucleated giant cells. Dendritic mononuclear cells, positive for S-100 protein, were noted in 67.5% of the lesions, whereas lysozyme and leucocyte common antigen were detected in occasional mononuclear cells. Ultrastructural examination showed mononuclear cells with signs of phagocytosis and sometimes interdigitations with similar cells. Others presented non-specific characteristics and the third type exhibited cytoplasmic processes and occasional Birbeck granules. Some multinucleated giant cells showed oval nuclei, abundant mitochondria and granular endoplasmic reticulum whereas others presented with irregular nuclei and a great number of cytoplasmic vacuoles. CONCLUSIONS: Immunohistochemical and ultrastructural results suggest that PGCGs of the jaws are composed mainly of cells of the mononuclear phagocyte system and that Langerhans cells are present in two thirds of the lesions. PMID- 7553377 TI - Comparison of the osteolytic activity of surface-associated proteins of bacteria implicated in periodontal disease. AB - OBJECTIVES: To compare the osteolytic activity of surface-associated material (SAM) and lipid A-associated proteins (LAPs) from periodontopathogenic bacteria. MATERIALS AND METHODS: Surface-associated material was extracted from the surface and LAPs from the cell walls of a range of periodontopathic bacteria including Actinobacillus actinomycetemcomitans and Eikenella corrodens. These bacterial fractions were assayed to determine their composition and their capacity to induce bone resorption was determined by use of the neonatal murine calvarial bone resorption assay. RESULTS: The SAMs from E. corrodens and A. actinomycetemcomitans demonstrated bone-resorbing capacity at concentrations as low as 1 ng ml-1 which, given the molecular weights of the active components, is in the picomolar range of activity. In contrast, the SAMs from the other three bacteria were significantly less potent and showed a lower efficacy. The LAPs all showed significant, and similar, capacities to induce bone breakdown. CONCLUSIONS: This is the first demonstration that LAP from periodontopathic bacteria can stimulate bone degradation. The LAPs from diverse bacteria all produced similar levels of bone-resorbing activity. In contrast, the SAM showed significant differences in potency and in efficacy (maximal stimulation). This may mean that in vivo certain periodontopathic bacteria have significantly more bone-resorbing capacity than others and should be therapeutic targets. PMID- 7553378 TI - Oligonucleotide probes to the 16S ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotella intermedia and Prevotella nigrescens. AB - Eight oligonucleotides based upon regions of the small subunit 16S ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two-dimensional structure to rationalise their use in recognising Prevotella intermedia and Prevotella nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA-DNA hybridisation and multilocus enzyme electrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi-2 to I Bi-6 (for P. intermedia) and 2Bi-2 (for P. nigrescens) with the 16S rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52-mer oligonucleotide (designated Bi) reliably detected both species. Because of the high degree of concordance between the 16S rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi-I recognised P. intermedia while I Bi-I detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi-I. These probes were highly specific and did not hybridise with DNA from the closely related P. corporis, nor other periodontal pathogens such as Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola and several pigmented species such as Prevotella melaninogenica, P. denticola, P. loescheii, Porphyromonas asaccharolytica, Py. endodontalis, Py. gingivalis, Py. levii, and Py. macacae. PMID- 7553379 TI - Association between carriage of oral yeasts and malnutrition among Tanzanian infants aged 6-24 months. AB - OBJECTIVE: To determine if there is an association between carriage of oral yeasts and malnutrition in infants. DESIGN: A case-control study within a cross sectional study. The dependent variable was carriage of oral yeasts. The exposure variable was malnutrition and confounders to be adjusted for were age, sex, and breast-feeding. SETTING: A maternal and child health clinic in Dar-es-Salaam, Tanzania that offers routine medical check-ups to all expectant mothers and children aged between 0 and 5 years in its catchment areas. SUBJECTS AND METHODS: 972 infants aged 6-24 months participated. Smears from the tongue and cheek mucosa were examined for candidal hyphae and blastospores. Malnutrition was categorized according to Tanzanian standards (weight-for-age) and World Health Organization (WHO) standards (weight-for-height and height-for-age). MAIN OUTCOME MEASURE: Carriage of oral yeasts (hyphae and blastospores). RESULTS: Carriage of oral yeasts was significantly higher in the 227 malnourished compared with the 745 well nourished adjusted for confounders. Odds ratio for presence of hyphae in smears from the severely malnourished (weight-for-age) was 4.5 (90% CI: 2.0 10.0). Odds ratio for presence of hyphae was 2.3 (90% CI: 1.1-4.8) when weight for-height were used to categorize for malnutrition. CONCLUSION: The study tends to confirm the generally held view that malnutrition may predispose to carriage of oral yeasts and subsequent oral candidiasis. PMID- 7553380 TI - Clinical, microbiological and ultrastructural features of angular cheilitis lesions in Southern Chinese. AB - OBJECTIVE: To obtain baseline data on angular cheilitis in Southern Chinese. DESIGN: A cross-sectional investigation of the clinical, microbiological and ultrastructural features of the condition. SUBJECTS AND METHOD: Thirty six Chinese adults with angular cheilitis; 28 controls matched for age and sex, with no inflammation. Clinical examination, swabs of lesions for microbiology, impressions of lesions for ultrastructure, using replica technique. MAIN OUTCOME MEASURES: Severity of lesions, associated signs and symptoms, incidence and type of microorganisms, ultrastructural features. RESULTS: Of a total 68 lesions 32 were bilateral and four unilateral. Forty four (65%) were mild (Type I) and the remaining 24 (35%) moderate (Type II). Infective agents were isolated from 37 (54%) lesions; pure growth of Candida spp and Staph. aureus was noted in nine lesions each; a mixed growth of the two in II, beta-haemolytic streptococci in three and a mixed flora including coliforms in the other five. Candida spp were present in one control, beta-haemolytic streptococci in two and coliforms in four others. Scanning electron microscopy revealed natural topography of the angular skin with sparse colonisation by bacteria and yeasts. CONCLUSIONS: Angular cheilitis in Southern Chinese seems to be characterised by a milder clinical presentation and classic infective agents of the disease: Candida spp and Staph. aureus. PMID- 7553381 TI - Limitations of flow cytometry in the analysis of CD1a/HLADR+ human oral mucosal Langerhans cells. AB - Suspensions of human oral epithelial cells were stained with antibodies to CD1a and HLADR conjugated with fluorochromes and analysed by flow cytometry with the aim of purifying double-labelled Langerhans cells, a population comprising approximately 2% of the cell total. Whole suspensions had high levels of autofluorescence and a wide range of forward and right angle scatter properties. The mean percentage of CD1a/HLADR+ cells was 2.1%, though the double-labelled cells did not form a discrete group and the percentages of positive cells using control antibodies were similar. Density gradient centrifugation prior to flow cytometry did not facilitate Langerhans cell identification within the suspension. The results indicate flow cytometric analysis of minority cell populations (such as Langerhans cells) within oral epithelium is limited by the autofluorescence of physically heterogeneous keratinocytes, and emphasize the importance of controls in studies of oral epithelium which use this method. PMID- 7553382 TI - Effect of cessation of tobacco use on the incidence of oral mucosal lesions in a 10-yr follow-up study of 12,212 users. AB - OBJECTIVE: To study the effect of cessation of tobacco use on the incidence of lichen planus, leukoplakia and other oral mucosal lesions. DESIGN: A 10-yr cohort study in a rural population of Ernakulam district, Kerala, India. MATERIAL AND METHODS: Some 12,212 tobacco users were interviewed and examined in a baseline survey and re-examined annually for 10 years. At each examination they were exposed to health educational programs to encourage them to quit their tobacco use. The incidence rates were calculated using person-years method among those who stopped their tobacco use and all others. RESULTS: A total of 77,681 person years of observation accrued among men and 32,544 among women. Among men 6.5% of these and among women 14.4% were in the stopped category. The incidence of oral lichen planus did not show any consistent association with cessation of tobacco habits (incidence ratio 1.35) but for leukoplakia there was a substantial drop in the incidence after cessation (incidence ratio 0.31). Several other tobacco associated oral mucosal lesions such as oral lichen planus-like lesion, smoker's palate, preleukoplakia, central papillary atrophy of the tongue and leukoedema showed either zero, or very small incidence, after cessation. CONCLUSION: The reported association between tobacco use and lichen planus appears to be indirect but for all other lesions it is direct. The cessation of tobacco use led to a substantial fall in the incidence of leukoplakia and other lesions implying a reduced risk for oral cancer after cessation of tobacco use. PMID- 7553383 TI - Giant cell lesions of the jaws. PMID- 7553384 TI - Nance-Horan syndrome: a contiguous gene syndrome involving deletion of the amelogenin gene? A case report and molecular analysis. AB - DESIGN: A case of Nance-Horan syndrome in a male is presented, with some features of the condition in his carrier mother and her mother. It is proposed that Nance Horan syndrome might be a contiguous gene syndrome mapping to chromosome Xp21.2 p22.3. SETTING: The proband had congenital cataract microphthalmia and dental abnormalities including screwdriver shaped incisors and evidence of enamel pitting hypoplasia. The region Xp21.2-p22.3 also contains the tooth enamel protein gene, amelogenin (AMGX). RESULTS: Using molecular genetic techniques, we have shown that there is no evidence that the AMGX gene is deleted in this case of the Nance-Horan syndrome. PMID- 7553385 TI - When the immune system puts its hands in a pocket. PMID- 7553386 TI - Orofacial involvement by systemic mycoses in HIV infection. PMID- 7553387 TI - Antibody responses of Porphyromonas gingivalis infected gingivitis and periodontitis subjects. AB - Porphyromonas gingivalis demonstrates a strong association with adult periodontitis although some individuals with the infection do not experience attachment loss. Therefore differences in the immune response to this organism may be of importance to the outcome of the disease. OBJECTIVE: The aim of this study was to determine whether P. gingivalis positive subjects with and without periodontal breakdown, reacted differently to P. gingivalis antigens as assessed by the pattern of serum antibody reactivity. MATERIALS AND METHODS: Two highly defined groups of subjects were chosen for this study. Both demonstrated P. gingivalis in their plaque and both had responded to P. gingivalis as shown by the presence of serum antibodies. The two groups differed only in their apparent clinical susceptibility to periodontal breakdown. Western blots of P. gingivalis membrane antigens were probed with sera from the two groups to determine their reactivity to specific antigens. RESULTS: Analysis of the immunoblots showed that there were no differences in either the total numbers of bands, or bands recognized by the majority of subjects in the gingivitis and adult periodontitis groups. There were however, four bands recognized by the majority of the gingivitis group and not by the majority of the adult periodontitis group, there being a significant difference (P = 0.03) in the recognition of the 91.4-kDa antigen band. A further five antigens of lower molecular weight were seen by the majority of the adult periodontitis group and not by the majority of the gingivitis group. When sera were tested against purified P. gingivalis LPS, the results indicated that the five antigens seen by the majority of the adult periodontitis group had molecular weights which were in the range exhibited by the LPS antigens. CONCLUSION: These results suggest that gingivitis and adult periodontitis subjects with P. gingivalis infection, may recognize different P. gingivalis antigens. PMID- 7553388 TI - Analysis of cultivable Porphyromonas gingivalis with trypsin-like protease enzyme activity and serum antibodies in chronic adult periodontitis. AB - OBJECTIVE: Trypsin-like protease (TLPase) enzyme produced by Porphyromonas gingivalis has been implicated as a virulence factor in the pathogenesis of periodontal disease. The aims of this study were to investigate the relationship between cultivable P. gingivalis, TLPase enzyme activity (BANA hydrolysis) and serum antibody levels against cell sonicate and a purified TLPase antigen from P. gingivalis W50. MATERIALS AND METHODS: Sub-gingival plaque samples were cultured for levels of P. gingivalis together with a chairside analysis of TLPase enzyme activity (Perioscan) from periodontitis and gingivitis sites of adult periodontitis patients. A TLPase from P. gingivalis was purified by gel filtration and ion exchange chromatography from the vesicle fraction for use as a test antigen. RESULTS: Elevated levels of P. gingivalis were found at periodontitis sites, however, there was no correlation with sub-gingival plaque TLPase enzyme activity. Adult periodontitis patients had higher levels of IgG and IgA against cell sonicate and TLPase antigens than did controls. Those patients who were P. gingivalis culture-positive demonstrated an elevated immune response against both cell sonicate and TLPase when compared to P. gingivalis culture negative patients. Treatment resulted in an improvement of clinical indices and no cultivable P. gingivalis could be recovered from the treated sites and there was a concomitant decrease in IgG levels against the TLPase. There was no significant difference in BANA hydrolysis at gingivitis sites or periodontitis sites after treatment. CONCLUSIONS: Further longitudinal studies are suggested to investigate the role of the TLPase in the response to treatment of chronic adult periodontitis patients. PMID- 7553390 TI - Effect of aluminum concentration on dental caries formation in the rat. AB - OBJECTIVE: Topically applied aluminum (AI) reduced fissure caries in rats with indigenous microflora, but neither therapeutic activity nor concentration effects have been studied in animals infected with caries-inducing bacteria. This study investigated the effects of solutions containing various AI concentrations on formation of smooth surface and sulcal caries in cariogenically challenged rats infected with Streptococcus sobrinus. METHODS: Weanling rats, inoculated with S. sobrinus, were randomly assigned to six groups which were topically treated twice daily with water (treatment control), a NaF solution of 100 parts/10(6) F (positive control), or AIK(SO4)2 solutions containing 100, 1000, 2000, or 4000 parts/10(6) AI. Rats were fed a high-sucrose diet and distilled water ad libitum for 10 weeks, then the molars were scored for enamel and dentin caries. RESULTS: Relative to the water control, NaF and all AI solutions significantly reduced prevalence of both smooth-surface and fissure caries. Protection by AI solutions was greater on smooth surfaces than sulcal areas. AI treatments reduced smooth surface scores by 49-71% and sulcal scores by 27-53%. For all comparisons the protective effect of the 100 parts/10(6) AI solution was less than solutions containing 1000 parts/10(6) AI or more. NaF and all AI solutions also significantly reduced the extent of lesions penetration into dentin. CONCLUSIONS: Topically applied AI reduced the formation and progression of both smooth surface and sulcal caries and showed evidence of a dose response in a rat model infected with S. sobrinus. PMID- 7553389 TI - Oral cryptococcosis in a patient with AIDS. A case report. AB - A 59-year-old Caucasian male patient with AIDS is described who presented with an oral lesion of cryptococcosis. The gingival ulceration was the only detectable lesion of cryptococcosis. Diagnosis was established by histopathologic findings from biopsy and detection of serum cryptococcal antigen. The patient was treated with amphotericin B and flucytosine. After four weeks of therapy cryptococcal antigen turned negative. The oral ulceration diminished in size, but an inflammatory tissue reaction persisted. PMID- 7553391 TI - Newer saliva collection methods and saliva composition: a study of two Salivette kits. AB - Saliva is frequently used as a diagnostic fluid and several collection devices have been developed. OBJECTIVE: The aim of the present study was to investigate the validity and reliability of two types of Salivette collection kits (non covered cotton roll and polypropylene covered polyether roll) relative to conventional collection of saliva using paraffin wax chewing stimulation. MATERIALS AND METHODS: Whole saliva samples were collected from 16 healthy volunteers. Following a cross-over design saliva was collected in a standardized way. The flow rate was determined and saliva samples were analyzed for pH, buffer capacity, electrolytes and protein/glycoprotein content. RESULTS: We find that Salivette methods do not allow evaluation of flow rate. pH was unaffected but buffer capacity was lower in Salivette collected than in paraffin wax-stimulated saliva. The non-covered cotton rolls reduced the content of Na+, K+, Cl-, as well as glycoprotein markers (hexosamines, fucose, sialic acid), lysozyme, lactoferrin, salivary- and myeloperoxidase but increased the concentrations of Ca2+, PO4(3)- and SCN-. Polypropylene covered polyether rolls affected saliva composition less than the non-covered cotton rolls. Thus, SCN- and sIgA concentrations were higher and lysozyme activity lower in the former (covered roll) saliva than in paraffin wax saliva. The reliability of the Salivette kits was good. CONCLUSION: We conclude that the Salivette method generates data significantly different from conventional paraffin wax-stimulated saliva such as buffer capacity and several electrolytes and organic components. Care should be taken in interpreting the results when such methods are employed. PMID- 7553392 TI - Transforming growth factor-beta 1 coated beta-tricalcium phosphate pellets stimulate healing of experimental bone defects of rat calvariae. AB - OBJECTIVE: TGF-beta 1-coated beta-TCP pellets were grafted in experimental defects of rat calvariae to study the effects on new bone formation. MATERIALS AND METHODS: The grafted sites were evaluated by light microscopy using hematoxylin-eosin (H-E) staining for histology and detection of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRACPase) activities to demonstrate osteoblastic and osteoclastic cells. Confocal laser scanning microscopy (CLSM) was performed for morphometry of newly formed bone. RESULTS: H-E sections showed more new bone formation in sites grafted with TGF beta 1-coated beta-TCP pellets (experimental sites) than those with beta-TCP pellets only (control sites). TRACPase-positive and ALPase-positive cells at experimental sites were more frequent than at control sites. The bone formation rate calculated by computerized CLSM pixel image analysis showed more new bone formation at the experimental sites than at control sites (3.4 +/- 0.8% vs 9.3 +/ 1.7% on week 2 and 11.8 +/- 2.1% vs 39.8 +/- 10.9% on week 4). CONCLUSION: TGF beta 1-coated beta-TCP pellets promote new bone formation and may be a useful modality in synthetic bone grafting. PMID- 7553393 TI - Oral health in Asia. PMID- 7553395 TI - CDA = can't do-it alone. PMID- 7553394 TI - Dental amalgam. PMID- 7553396 TI - HIV leading cause of death in U.S. PMID- 7553397 TI - Managing emergency patients. AB - Whether it's a new or existing patient who contacts your office about a dental emergency, you and your team have to handle the call with the utmost sensitivity. These patients may be in pain. They need to be treated at a practice that knows how to manage the situation, allowing them to feel relaxed and confident about the impending care. Many new patients find that visiting a dental practice is often an unfamiliar, anxiety-provoking experience. But if you've developed a strong, effective protocol for dealing with dental emergencies, these new patients may ultimately overcome their hesitancy and become enthusiastic patients. It's all about reading and attending to their unique needs. PMID- 7553398 TI - Dental amalgam: toxicological evaluation and health risk assessment. AB - Dental amalgam releases small amounts of mercury, which is absorbed by the body. Available data are not sufficient to indicate whether this poses a health hazard to the general population. There is an urgent need for research into the potential health effects of mercury absorption from this source. However, considering the multiple benefits of dental amalgam, unless new scientific research dictates otherwise there is currently no justification for discontinuing the use of this material. Placing a priority on the preservation of healthy tooth structure through preventive strategies and the judicious use of all restorative materials could lessen current concerns over toxicity and unnecessary treatments. PMID- 7553399 TI - Ceramic inlays and onlays: update 1995. AB - Tooth-colored inlays and onlays are increasingly sought by the public because of a growing interest in restorations that appear natural, and because of concerns about mercury in amalgam fillings. Following the introduction of bonded porcelain inlays in 1985, many of these restorations were placed. These inlays were a natural sequel to the porcelain laminate that reached North America in 1984, yet they did not enjoy the same rate of success. The advent of industrially fabricated porcelain blanks and the ability to copy-mill a positive as an inlay or onlay has resulted in a reliable and predictable dental restoration. Pressed ceramics, which are modelled by wax pattern, are also effective tooth-colored restorations. PMID- 7553401 TI - Factors to consider for predictable post and core build-ups of endodontically treated teeth. Part II: Clinical application of basic concepts. AB - Post and core build-ups represent an important pre-prosthetic procedure prior to the restoration of an endodontically treated tooth. The dental practitioner is presented with the dilemma of selecting from an ever increasing variety of materials, techniques and designs related to this procedure, many of which are harmful and mired in controversy. Part II of this paper will review some basic theoretical concepts related to post and core build-ups with the objective of applying these concepts to address a number of pertinent clinical questions related to the above selection process. This review should provide the dentist with a reference to produce a more predictable post and core build-up. PMID- 7553400 TI - Factors to consider for predictable post and core build-ups of endodontically treated teeth. Part I: Basic theoretical concepts. AB - Post and core build-ups represent an important pre-prosthetic procedure prior to the restoration of an endodontically treated tooth. The dental practitioner is presented with the dilemma of selecting from an ever increasing variety of materials, techniques and designs related to this procedure, many of which are harmful and mired in controversy. Part I of this paper reviews some basic theoretical concepts for the dentist to consider when producing a predictable post and core build-up. An understanding of these basic concepts will assist the dentist in gathering data and establishing a risk/benefit equation, and will also provide an additional aid during the decision-making process for the restoration of an endodontically treated tooth. PMID- 7553402 TI - Reducing marginal flash in the fabrication of direct provisional restorations: a new technique using light-cured resin and transparent silicone. AB - The optimum placement of bonded indirect restorations depends to a large extent on establishing and maintaining the patient's gingival health during the provisional treatment phase, as inflamed sulcular tissues may compromise subsequent isolation efforts. Gingival health is best achieved by providing provisional restorations of correct emergence profile and accurate margination. This paper introduces a fully-cured direct provisionalization technique that minimizes marginal flash and improves the accuracy of the restoration in a fraction of the production time needed for traditional direct techniques requiring remargination. The technique, which relies on the use of a unique transparent over-impression registration material and visible light-curing provisional composite resin, is described in a case report. PMID- 7553403 TI - [Patients with heart valve prostheses: dental care, dental procedures and prevention]. AB - Patients with birth or rheumatic heart defects, or wearing prosthetic cardiac valves, are at high risk of developing bacterial endocarditis following dental treatment. Two hundred patients with cardiac valves were surveyed with respect to both their oral hygiene habits and their last visit to the dentist. More than half of the respondents (112) were fully edentulous, and most of them had not seen a dentist for many years. Of the 88 respondents who had natural teeth, 23 had not been to a dentist since surgery. For the purpose of this study, focus was placed primarily on the answers given by the 65 patients who reported having been to a dentist after surgery. Only 41 of them (63 per cent) reported having received a prophylactic antibiotic therapy while being treated with a risk of bacteremia. Of the 24 others who did not receive prophylactic antibiotic therapy, 20 were given dental treatments with a risk of bacteremia. In light of this study, there should be recommendations to improve the health care given to patients predisposed to bacterial endocarditis. PMID- 7553404 TI - Induction of megakaryocytic differentiation in a newly established human leukemic cell line by 12-o-tetradecanoylphorbol-13-acetate. AB - BACKGROUND: Biological studies on megakaryopoiesis have been hampered by the scarcity of megakaryocytes in normal bone marrow and difficulty in long term culture. Alternatively, leukemic cell lines with megakaryocytic differentiation potential may provide good models to counter these problems. METHODS: Leukemic cells from a patient with acute megakaryocytic leukemia were put into long-term culture and established into a cell line which was designated as VGH-MK1. The VGH MK1 cells were challenged with differentiation agents and/or cytokines, and the differentiation of these cells was examined using morphological, immunocytochemical and surface-marker studies. RESULTS: Morphologically, VGH-MK1 cells had prominent nucleoli and basophilic cytoplasm with some protrusions, but large cells were occasionally seen. Under regular culture condition, the cells had a doubling time of 36-48 hours. The cloned cell line exhibited markers characteristic of megakaryoblasts after differentiation induction. Specifically, when stimulated with 12-o-tetradecanoylphorbol-13-acetate (TPA), cells became larger and had large or multinuclei. They were induced to express platelet glycoproteins GPIb (CD42b), GPIIb/IIIa (CD41), and GPIIIa (CD61) antigens, but not erythroid nor lymphoid markers. Platelet peroxidase (PPO) activity was also induced. Retinoic acid did not exhibit similar differentiation-inducing effects. In contrast, it counteracted the effects induced by TPA. CONCLUSIONS: An unique human leukemic cell line, VGH-MK1, has been established here. It could be induced to exhibit some characteristics of megakaryocytic lineage, and may be an useful model for the biological studies of megakaryopoiesis. PMID- 7553405 TI - Short-term and long-term effects of benazepril in mild to moderate hypertensives. AB - BACKGROUND: Benazepril hydrochloride is a non-sulfhydryl-containing, angiotensin converting enzyme (ACE) inhibitor. The short-term and long-term antihypertensive effects of benazepril remain to be established in Chinese. METHODS: Hypertensive subjects with diastolic blood pressure 95-110 mmHg, after two week placebo run-in first, entered a four-week double-blind phase with treatment of benazepril 10 mg once daily or captopril 25 mg three times daily, then received one-year open treatment of benazepril 10 mg daily with or without diuretics. Ambulatory blood pressure monitoring was performed at the end of placebo run-in, after four-week double-blind phase, and after one-year open treatment. RESULTS: Of the 75 subjects (41 male, 34 female, mean age 57 +/- 12 years, range 34-88 years) who completed the double-blind phase, 42 subjects finished the one-year extension phase. Reasons for withdrawal from the study included irritable cough (16, 21%), hypotension (1, 1%), and poor compliance (16, 21%). During the short-term double blind phase, benazepril reduced clinic and mean 24-h ambulatory blood pressure by -21/-10 mmHg and by -17/-10 mmHg respectively, and captopril by -21/-13 mmHg and by -17/-10 mmHg respectively. After one-year open treatment by benazepril for the 42 subjects, the one-year average clinic blood pressure was 134/88 mmHg (155/104 mmHg at entry and 135/93 mmHg at the end of the double-blind phase), and the mean 24-h ambulatory blood pressure was 137/87 mmHg (149/95 mmHg at entry and 132/84 mmHg at the end of the double-blind phase). CONCLUSIONS: The antihypertensive effect of benazepril 10 mg daily with or without diuretics is not significantly different from that of captopril 75 mg daily in the short-term and can reasonably be maintained for one year. PMID- 7553406 TI - Comparison of intravenous granisetron with metoclopramide in the treatment of chemotherapy-induced emesis. AB - BACKGROUND: It is well known that chemotherapy-induced nausea and vomiting not only produce undesirable physical effects but also reduce patients' willingness to continue chemotherapy. The effect of a 5HT3 receptor antagonist, granisetron, on antiemesis was studied in patients who were receiving cisplatin-based chemotherapy. METHODS: Forty patients with malignant diseases, who were scheduled to receive cisplatin-based chemotherapy, were randomly assigned to receive either a single intravenous dose of granisetron (3 mg) (n = 20) or multiple doses of metoclopramide (2 mg/kg) (n = 20) as trial. The efficacy of antiemetic therapy was assessed based on the patient's subjective assessment of nausea and vomiting, and the physician's global evaluation of nausea and vomiting in the first 24 hours and during the following 6 days. RESULTS: During the first 24 hours, there was a statistically significant advantage for the granisetron group in terms of prevention of both nausea and vomiting. The number of complete responders in the first 24 hours for the granisetron and metoclopramide groups was 16 (80%) and 9 (45%), respectively (p < 0.05). The control rate of anti-nausea over 6 days was 45% and 5% in the granisetron and metoclopramide groups, respectively (p < 0.001). The frequency of adverse events in the granisetron and metoclopramide groups was 10% and 45%, respectively. Analysis of laboratory data showed no significant difference between these two groups prior to and after administration of chemotherapy. CONCLUSIONS: A single 5-minute infusion of 3 mg granisetron was more effective and produced a less adverse effect than multiple doses of metoclopramide in controlling chemotherapy-induced emesis, both in the first 24 hours and over a 6-day period. PMID- 7553408 TI - Jejunoileal atresia in neonates. AB - BACKGROUND: Jejunoileal atresia is the most common intestinal atresia and the most frequent cause of neonatal intestinal obstruction. The prognosis depends on several important factors. The patients reported were collected for evaluation of the factors contributing to their prognoses. METHODS: In the ten-year period from October 1982 to October 1992, all jejunoileal atresia patients who received treatment in this hospital were entered in this study. Possible factors contributing to prognosis, such as prematurity, low birth weight, apple peel atresia, anastomotic leakage, sepsis, short bowel syndrome were evaluated using Mann-Whitney U-test. RESULTS: Twenty-four patients of jejunoileal atresia had been managed in our hospital in 10 years period. Their ages ranged from one to thirteen days old (mean 3.9 days). The numbers and types of atresia were as follow: type I (n = 4), type II (n = 4), type IIIa (n = 10), type IIIb (n = 5), type IV (n = 1). All patients received surgical correction. Nine complications were found in eight patients, which included four anastomotic leakage, three wound infections and two intestinal obstructions. There were three mortalities from sepsis. Total parenteral nutrition was applied to 14 patients (58.3%). The mean hospital stay was 45.1 days, and the survival rate was 87.5%. Low birth weight and anastomotic leakage contributed to prolonged hospital stays (p < 0.05). CONCLUSIONS: Jejunoileal atresia is a common neonatal surgical condition. The prognosis is generally good, except when a patient presented with factors, such as, low birth weight or anastomotic leakage, which prolonged hospital stays. Moreover, sepsis is the only important factor contributing to mortality. PMID- 7553407 TI - Magnetic resonance spectroscopic findings in patients with subcortical ischemic stroke. AB - BACKGROUND: Biochemical changes inside the brain during acute ictus have been extensively studied by means of magnetic resonance spectroscopy (MRS). Previous reports revealed that cerebral cortical infarction causes reduction of N acetylaspartate (NAA) and elevation of lactate. This study was designed to evaluate the biochemical changes in subcortical infarction. METHODS: Nine normal controls and four patients, including two thalamic infarct, one basal ganglia infarct, and one putamen infarct cases were examined using MRS in addition to magnetic resonance imaging (MRI). Evaluation experiments were performed on a Siemens Wagnetom 63SP machine. A spin echo sequence with TE 270 msec was applied for the measurement. The voxel was located at basal ganglia in the controls and at the lesion site, as well as at the contralateral corresponding region in the patients. RESULTS: Three resonance signals were present in the spectrum-NAA, choline-containing compounds (Cho) and creatine-phosphocreatine (Cr). The NAA/Cho and NAA/Cr ratios were reduced significantly on the lesion side compared with the controls. NAA/Cho also decreased on the lesion side compared with the healthy side. CONCLUSIONS: There is a remarkable reduction of NAA concentration even in subcortical infarction. Further study is needed for the detection of changes in lactate in patients with subcortical lesion. PMID- 7553409 TI - Emergency surgical intervention for severe corrosive injuries of the upper digestive tract. AB - BACKGROUND: In treating severe corrosive injury of the esophagus and stomach, prompt diagnosis, adequate fluid resuscitation and warranted surgical intervention are the most important factors in rescue of critically ill patients. The purpose of this study was to evaluate the need for, and the advantages of, a surgical approach to treatment of such corrosive injuries to the upper gastrointestinal (UGI) tract, as well as to select the most suitable technique to achieve a good survival rate. METHODS: From January 1983 to December 1991, 220 patients were treated for caustic ingestion injury to the UGI tract. A retrospective review of their records allowed targeting of 27 patients with severe corrosive injury that surgical intervention was required. In this study, peritoneal sign was taken as the key indicator for early emergency operation. The age, sex, elapsed time from injury to operation, the sort and quantity of caustic agent used, injury mechanism, clinical manifestations, alternative surgical treatment methods and causes of death were also reviewed and analyzed in this study. RESULTS: The patients included 13 men and 14 women, of whom the majority were adults (96.3%) who had attempted suicide (85.2%). All of them had taken liquid corrosive agents, usually hydrochloric acid (63%). Eighteen underwent emergency operations; the other nine received only supportive treatment, given their terminal status. The mortality rates for patients with surgery and supportive treatment were 66.7% and 100%, respectively. Four patients died after undergoing esophagectomy with resection of the stomach using the thoracoabdominal method. Only three of the eight patients who received esophageal stripping combined with resection of the stomach through the abdomen died (37.5%). CONCLUSIONS: The time elapsed between injury and development of peritoneal sign is a good indicator of the severity and extent of the injury. When peritoneal sign manifests at a very early stage, it is an indicator that the corrosive injury is very advanced in its progress and that, no matter what procedures were performed, the outcome would be the same. Yet if there were a six-hour gap then aggressive surgical management can rescue some patients. It is recommended based on experience here, that when using the surgical approach, resection of the stomach with stripping of the esophagus is superior to the thoracoabdominal method. PMID- 7553410 TI - A modified Bristow procedure for recurrent anterior shoulder dislocation. AB - BACKGROUND: Dislocation of the shoulder is one of our earliest frequent injuries. Many procedures have been evaluated for treatment of the condition since the mid 1960s. The modified Bristow procedure was initially described in 1970 by May. Technically easy, the procedure is very effective in prevention of redislocation, and has gained popularity over two decades of use. METHODS: A retrospective study was done of 32 patients with recurrent anterior shoulder dislocation, who had received modified Bristow procedure during the years 1987 through 1991 in this Hospital. Average followup period was 26 months. RESULTS: At postoperative evaluation, average loss of external rotation and abduction as compared to the nonoperated side were 15 and 10 degrees, respectively. There was no limitation on daily activity, but some little restriction in extreme throwing sports was found. Postoperative complications included three patients who had bone block resorption and one patient who had superficial wound infection. There was no redislocation nor implant loosening in the study. CONCLUSIONS: This procedure is technically easy, had a low rate of complication, a high degree of patient satisfaction and very effectively prevents redislocation. PMID- 7553411 TI - Elective interval laparoscopic management for periappendiceal abscess. AB - BACKGROUND: In Far Eastern countries, the right-sided colonic diverticular diseases are more prevalent than the left-sided ones. Accurate differential diagnosis between appendiceal abscess and right-sided diverticulitis with abscess formation is difficult to make preoperatively. Conservative treatment followed by elective interval surgery remains the mainstay of management for patients with periappendiceal abscess. Laparoscopic diagnosis and treatment have been advocated in managing patients with abdominal pain of uncertain diagnosis. The purpose of this study was to evaluate the beneficial effect of interval barium enema and mini-invasive procedures for patients with periappendiceal abscess. METHODS: Patients with periappendiceal abscess (n = 8) were enrolled in this study. Conservative treatment was instituted by administration of antibiotics, and interval barium enema and laparoscopic intervention were scheduled later. Clinical manifestations, results of barium enema, outcomes of laparoscopic intervention and pathological diagnoses were reviewed. RESULTS: The frequency of periappendiceal abscess was 18/263 (6.8%). There were no operative complications. The correlation between barium enema, laparoscopic findings, and pathological diagnosis was quite good. Patients gained the advantages of laparoscopic surgery. CONCLUSIONS: Interval barium enema study and laparoscopic diagnosis and treatment are worthy of trying for patients with periappendiceal abscess, especially in those areas with high prevalence of right-sided diverticular diseases. PMID- 7553412 TI - Intramedullary spinal cord metastasis: a case report. AB - Intramedullary spinal cord metastasis is a rare condition of systemic malignancy. We report a case of breast cancer with thoracic spinal cord intramedullary metastasis. The patient suffered from neurological deficit in both lower extremities. Magnetic resonance imaging (MRI) examination showed two soft tissue lesions located in the medullary region of thoracic spinal cord at T3 to T5 vertebral level. She was treated with local radiotherapy and systemic corticosteroid treatment with much improvement of the symptoms. PMID- 7553413 TI - Hepatic actinomycosis: a case report. AB - A 64-year-old man with hepatic actinomycosis presented with several months of weight loss and poor appetite. However, no fever was noted before admission. Findings on abdominal sonography and computed tomography scan were suggestive of hepatocellular carcinoma. A sono-guided percutaneous liver biopsy specimen demonstrated only chronic fibrosing inflammation. Therefore, laparotomy was performed and the diagnosis of hepatic actinomycosis was established after surgical resection. The patient was then successfully treated. The fact that hepatic actinomycosis may be very similar to hepatocellular carcinoma should be highly suspected. The hospital course of this patient concerning this condition and a brief review of the literature are presented to illustrate the diagnostic difficulties which may be encountered in such cases. PMID- 7553414 TI - Intravenous immunoglobulin infusion in multifocal demyelinating motor neuropathy: a case report. AB - The following report is a case of multifocal demyelinating motor neuropathy (MMN) presenting as a gradual development of asymmetric motor weakness without sensory involvement. Electrophysiological studies showed mainly a conduction block with normal or slightly slow nerve conduction velocity. Cerebrospinal fluid (CSF) protein and serum protein electrophoresis were normal, but serum IgM anti-GM1 ganglioside antibody was elevated. The patient had a poor response to steroid, plasmapheresis and chemotherapy with cyclophosphamide, but significant improvement was noted after intravenous immunoglobulin (IVIG) infusion. MMN is a potentially treatable condition which clinically mimics a motor neuron disease; if treatment with steroid, plasmapheresis and cyclophosphamide have failed, IVIG may be effective. PMID- 7553415 TI - Villous adenoma of ampulla of Vater: a case report. AB - A 56-year-old female patient presented with pain and tenderness over the epigastric region. Endoscopy showed a polypoid mass at the ampulla of Vater. Endoscopic biopsy showed villous adenoma with moderate cellular atypia. Wide excision of the tumor was performed. After resection, histological examination confirmed the tumor to be a villous adenoma of ampulla of Vater. This is a rare neoplasm with an incidence of 0.04-0.12% in postmortem series. A few cases have been reported in the literature, but only two documented in the Chinese literature to date. Wide excision has had good results and should be regarded as the treatment of choice for benign lesions. PMID- 7553416 TI - Piperacillin/tazobactam in comparison with clindamycin plus gentamicin in the treatment of intra-abdominal infections. AB - BACKGROUND: Concerned about the inactivation of piperacillin by beta-lactamase and the risk of aminoglycoside-induced nephrotoxicity and clindamycin-induced enterocolitis, we conducted the following phase III clinical trial. METHODS: Between November 1991 and March 1993, 77 surgical patients with intraabdominal infections were enrolled and randomly assigned in a 3:2 ratio to receive either piperacillin/tazobactam or clindamycin plus gentamicin to compare safety, tolerance and efficacy between both two treatment groups. RESULTS: There were 76 clinically and 50 bacteriologically evaluable patients with 80 isolated pathogens. The demographic data were comparable in both groups. There was no statistically significant difference of clinical response at any time-point of treatment, with 97.8% favorable clinical response rate in piperacillin tazobactam group and 96.6% in clindamycin plus gentamicin group at endpoint. The bacteriological eradication rates were similar, with 97.7% in piperacillin/tazobactam group and 94.4% in clindamycin plus gentamicin group at pathogen level, and 96.7% in piperacillin/tazobactam group and 95.0% in clindamycin plus gentamicin group at patient level. By susceptibility tests, only 3 (4%) isolated pathogens were resistant to piperacillin/tazobactam, which was much superior to the use of piperacillin, clindamycin or gentamicin alone in antimicrobial activity. The piperacillin tazobactam-related adverse experiences included 1 (2.1%) urticaria and 2 (4.3%) diarrhea. However, there were no significant differences in the adverse experiences between these two groups. CONCLUSIONS: This study has demonstrated that piperacillin/tazobactam is comparable with clindamycin plus gentamicin in efficacy, safety and tolerance in the treatment of surgical patients with intra-abdominal infections. The combination of piperacillin/tazobactam could potentially be the treatment of choice in adjunt to surgical management in intra-abdominal infection. PMID- 7553417 TI - Total body bone mineral content in small-for-gestational -age, appropriate-for gestational -age, large-for-gestational -age term infants and appropriate-for gestational -age preterm infants. AB - BACKGROUND: Metabolic bone disease is a recognized complication in very low birth weight infants. Inadequate postnatal intake of calcium and phosphorus is probably important in the pathogenesis of bone disease in the newborn. A few studies have shown lower bone mineral content at birth in small for gestational age (SGA) than in appropriate for gestational age (AGA) infants. The present study was designed to compare total body bone mineral (TBBM) content in AGA, SGA, and large for gestational age (LGA) term infants. Also, it was designed to evaluate extrauterine changes in TBBM in preterm infants. METHODS: Ten SGA [mean +/- S.D. birth weight (B.W.) was 1.7 +/- 0.2 Kg, gestational age (G.A.), 39.0 +/- 0.8 weeks], ten AGA (B.W.; 3.3 +/- 0.4 Kg; G.A.: 39.3 +/- 1.4 weeks), ten LGA (B.W.: 4.4 +/- 0.3 Kg; G.A.: 40.4 +/- 0.9 weeks) term infants and ten AGA preterm infants (B.W.: 1.6 +/- 0.3 Kg; G.A.: 31.9 +/- 1.9 weeks) were enrolled in this study. TBBM content was measured using dual-photon-absorptiometry at 1 week postnatally in SGA, AGA, LGA term infants and in preterm infants at 1, 6, 12 weeks postnatally. Serum total calcium, phosphorus, magnesium, alkaline phosphatase activity (alk-p), parathyroid hormone (PTH), 25-hydroxyvitamin D (25 OHD) and urinary calcium, phosphorus, creatinine were measured at 1 week postnatally in all studied infants and 6, 12 weeks, postnatally in preterm infants. Preterm and SGA term infants received premature formula enriched with calcium, phosphorus and vitamin D. RESULTS: There was no significant difference (p > 0.05) in serum calcium, phosphorus, magnesium, alk-p, PTH, 25-OHD and urinary calcium, phosphorus, creatinine values among SGA, AGA and LGA term infants at one week of age. Also, there was no significant difference in serum biochemical values in preterm infants at 1, 6, 12 weeks postnatally. Significantly lower (p < 0.05) urinary phosphorus values were found in preterm than in term infants. TBBM content was lower (p < 0.05) in SGA term infants than in AGA and LGA term infants. Premature infants had lower (p < 0.01) TBBM values than term AGA infants; however, TBBM values increase with postnatal age in preterm infants. CONCLUSIONS: Biochemical or marked radiological evidence of metabolic bone disease did not develop in any of the studied preterm infants. It appears that feeding permature infants with formula enriched with phosphorus, calcium and vitamin D may provide sufficient mineral for bone mineralization. PMID- 7553418 TI - Evaluation of the hemostatic clot formation of newborns by Sonoclot coagulation analyzer. AB - BACKGROUND: Sonoclot Analyzer has been widely used for more than five years in The United States to evaluate the platelet function and coagulation in adult. No clinical trial with the Analyzer has been reported in the field of pediatrics. The present study was aimed to evaluate the efficacy of the Sonoclot Analyzer for urgent determination of platelet function and coagulation in newborns. METHODS: Venous blood samples were drawn from the umbilical vein in 70 healthy newborns, and from the median cubital vein in 70 healthy adult volunteers, between 24 to 40 years of age, for control. DP2951 Sonoclot II Surgical Analyzer with graphic printer was used to evaluate the hemostatic clot formation. RESULTS: Results demonstrated that Sono ACT (activated coagulation time), peak time and retraction time increased by 36%, 66% and 70% respectively in newborns, while clot rate decreased by 69% in comparison with that of these adults. CONCLUSIONS: The results of using the Sonoclot Analyzer in this study confirmed other previous investigation that platelet function and coagulation are altered in newborns when compared with that of adults. The Sonoclot Analyzer is a simple device, easy to perform; It requires only small amounts of blood sampled (0.4 ml), and provides a rapid coagulation function for newborns. PMID- 7553419 TI - A multivariate analysis of prognostic factors in thymoma. AB - BACKGROUND: Considering the prognostic factors of thymoma, the clinical stage is the most important, while controversies exist about other clinicopathologic factors. A multifactorial study was carried out to clarify the relationship between prognosis and sex, age, tumor size, presence of myasthenia gravis, staging, epithelial subtyping, lymphocyte/epithelial cell ratio (L/E ratio) and other adjuvant pathological features. METHODS: One hundred and thirty-seven cases were collected in Veterans General Hospital-Taipei from 1961 to 1993. The aforementioned features were studied. Kaplan-Meier's actuarial survival analysis was adopted, with Logrank test, to determine the significance of overall heterogeneity and trend. Cox proportional hazard model was also used for multivariate analysis. RESULTS: Among all tested prognostic factors, staging had the highest significance (relative risk = 3.00, p < 0.001) and age had only a moderate one (relative risk = 1.04, p = 0.011). No other clinicopathological factor showed any statistical significance for prognosis. CONCLUSIONS: Staging remains of greatest usefulness in predicting the biological behavior of thymoma. The effect of age requires more investigation. Other clinicopathological features have no definite influence in survival expectancy. PMID- 7553421 TI - Zygomycotic lung abscess: a case report. AB - Zygomycosis is an uncommon, but frequently fatal, fungal infection caused by members of the class Zygomycetes. The risk factors include diabetes mellitus, uremia, leukemia and use of deferoxamine as an iron-chelating agent; healthy persons also are occasionally infected. Those fungi, spread by their ubiquitous spores, most frequently involve the respiratory system. Rhinocerebral zygomycosis occurs predominantly in patients with uncontrolled diabetic ketoacidosis. Pulmonary zygomycosis most frequently is observed in granulocytopenic and corticosteroid-treated patients. Other clinical manifestations are gastrointestinal, cutaneous, disseminated and miscellaneous. This report concerns a previously robust farmer who suffered from left upper lung abscess caused by Rhizopus spp.-one member of the order Mucorales. Initially, it was intended to administer amphotericin B to a total dose of 2,000 mg; however, the patient could not tolerate such side effects as nausea, vomiting and refused further management when the cumulative dose was 948 mg. However, he did recover without further fever and cough. Chest X-ray, followed every three months, disclosed satisfactory improvement. PMID- 7553420 TI - Acute relapsing hepatitis A in Taiwan--a rare event not related to multiple viral infection: a report of two cases. AB - From 1981 to 1993, 40 cases of acute type A viral hepatitis were reviewed and 2 cases (5%) of relapsing hepatitis were reported here. One case relapsed two weeks after remission and the other relapsed three weeks after remission, both had benign courses and recovered within four months. They were negative for serum hepatitis B surface antigen and hepatitis B core IgM antibodies. The serum autoantibodies, hepatitis C antibodies and hepatitis E antibodies were all negative during relapse. In summary, relapsing hepatitis A is rare in Taiwan and it is not related to multiple viral infections. PMID- 7553422 TI - A young adult with tuberculous meningitis diagnosed by polymerase chain reaction on serial CSF samples: a case report. AB - A polymerase chain reaction (PCR) assay was used to diagnose a young adult with tuberculous meningitis who presented with unusual clinical symptoms of an acute illness. He had repeated cerebrospinal fluid (CSF) samples taken, and the PCR assay for detecting mycobacterial deoxyribonucleic acid was negative for the first two CSF specimens, but was positive in the third. He was then treated with antituberculous drugs and had complete restoration three months later. A follow up PCR of the CSF was still positive after one month of therapy using antituberculous drugs, but converted to a negative result one month later. It is concluded that it is valuable to repeatedly sample CSF specimens for PCR study in patients with clinically suspected tuberculous meningitis. PMID- 7553423 TI - Calcifying aponeurotic fibroma: a report of three cases. AB - Calcifying (juvenile) aponeurotic fibroma (CAF) is a rare disease entity characterized by a tendency to develop in the palms and soles of children and young adults. It has definite histologic features of an ill-circumscribed fibroblastic proliferation, with foci of calcification and chondroid differentiation, as well as a recurring or locally aggressive growth pattern. The commonly accepted mode of treatment is wide excision of the tumor; extensive surgery is usually not necessary even for recurrences. Three cases are reported, all of when were female with the age ranging from 8 to 27 years. All lesions were in the hand or foot, and one of them was recurrent. Immunohistochemical study was performed and the literature reviewed. PMID- 7553424 TI - The prognostic value of serum cytokine levels in patients with acute infections. AB - BACKGROUND: Elevation of serum cytokine level, especially tumor necrosis factor alpha (TNF-alpha) and granulocyte-colony stimulating factor (G-CSF), has been reported in acute bacterial infection. Elevation of serum TNF-alpha level upon patients' admission has also been reported to correlate with the fatal outcome. METHODS: The present study is to evaluate, in addition to TNF-alpha and G-CSF, whether serum stem cell factor (SCF), interleukin-3 (IL-3) and granulocyte macrophage-CSF (GM-CSF) levels to see if they were also elevated in patients with acute bacterial infection. It also tries to evaluate whether different degree of elevation of cytokine levels had any relationship to patients' clinical parameter, including fatality. Correlation between different kinds of cytokines was also studied. RESULTS: Patients with bacterial growth in blood culture had higher level of serum G-CSF. Higher level of serum TNF-alpha was significantly associated with the occurrence of septic shock, but not of fatal outcome. Low serum G-CSF and high serum SCF level were significantly associated with fatal outcome; however, all the patients still had serum SCF levels within normal range. GM-CSF and IL-3 play no obvious role as systemic effector molecule in acute bacterial infection. CONCLUSIONS: Higher levels of serum TNF-alpha are associated with the occurrence of septic shock. Low serum G-CSF levels are significantly associated with fatal outcome. Routine monitoring of serum G-CSF level in patients with severe infection in order to supplement recombinant G-CSF can possibly help patients to overcome the disaster. PMID- 7553425 TI - Prevalence of antibodies to hepatitis C virus in patients with nonalcoholic fatty liver. AB - BACKGROUND: Nonalcoholic fatty liver can be caused by obesity, diabetes mellitus, hyperlipidemia or nonalcoholic steatohepatitis. Hepatitis C viral infection can also cause macrovesicular steatosis and such inflammation of the liver as nonalcoholic steatohepatitis. To determine whether the prevalence of anti-HCV is higher in patients with nonalcoholic fatty liver, this clinical study was undertaken; we also tried to detect which factors predict the presence of anti HCV in these patients. METHODS: The study included 205 nonalcoholic fatty liver patients (122 males and 83 females) with a mean age of 46.5 years, diagnosed by ultrasonic examination, on whom anti-HCV antibody tests were then done. RESULTS: Eleven cases (5.4%) were anti-HCV positive with a higher mean age of 56.4 +/- 11.1 years (p < 0.05). Anti-HCV positive patients had higher serum AST and ALT levels than negative patients (95 +/- 59 U/L, 166 +/- 116 U/L vs. 34 +/- 26 U/L, 51 +/- 43 U/L; p < 0.05), while anti-HCV negative patients had higher mean body weight (124 +/- 15% vs. 114 +/- 11% of IBW) and serum cholesterol levels (202 +/- 41 mg/dl vs. 159 +/- 45 mg/dl). There was no statistical difference between anti HCV positive and negative groups in clinical parameters including gender, presence of HBsAg, history of DM, operation, blood transfusion, oral contraceptive usage, and cancer with or without chemotherapy. The prevalence of anti-HCV increased with age from 1.8% in the fourth and fifth decades to 25% in the eighth decade. The prevalence of anti-HCV was 1.4% (1 case) in 72 fatty liver with normal biochemical tests and 7.5% (10 cases) in 133 cases with abnormal biochemical tests, a difference which was statistically marginally significant (p = 0.055). Those with ALT levels greater than three-fold the normal limit had a significantly higher anti-HCV prevalence rate (25.9%) than those with normal (1.4%) or mildly (2.8%) elevated ALT levels (p < 0.001). Logistic regression analysis revealed that only age and serum ALT levels were independent predictive factors for the presence of HCV infection. CONCLUSIONS: Results revealed that the prevalence of anti-HCV in patients with nonalcoholic fatty liver was 5.4%, slightly higher than in the general population. Age and serum ALT level were the predictive factors for the presence of HCV infection. PMID- 7553426 TI - Factors influencing the success rates of endoscopic nasobiliary drainage in treatment of obstructive jaundice. AB - BACKGROUND: Endoscopic nasobiliary drainage (ENBD) is a safe and effective modality which has been well documented for obstructive jaundice. However, factors predicting success rate of ENBD remain inconclusively. This study analyses those factors and discusses the outcome of patients with obstructive jaundice. METHODS: One hundred and sixteen patients (male 99, female 17; mean age 68.2 years) with obstructive jaundice received ENBD after endoscopic retrograde cholangiogram (ERC) by Olympus JF-lT20 endoscope and 7F Wilson-Cook nasobiliary catheter from Sep. 1990 to Oct. 1993. Bile output (QD), serum bilirubin (BIW), liver biochemistry (QW), bile culture (next day), blood culture (if BT > 38.5 degrees C) were checked until definite treatment or death. Adequate drainage was defined as a daily output of bile more than 200cc, a gradual drop in serum bilirubin and no signs of cholangitis. Factors such as causes of jaundice, obstruction level, serum bilirubin, albumin, juxtapapillary diverticulum (JPD), bacteremia, fever before ERCP and ascites were analyzed. RESULTS: The success rate was 86.2% (100/116) in ERC and 78% (78/100) in ENBD. Adequate biliary drainage was 82.1% (64/78), and serum bilirubin was reduced from 14.3 +/- 8.5 mg% to 7.5 +/- 5.6 mg% within one week. In patients with non-cancerous causes, higher success rate and adequate drainage rate were obtained compared with those with cancerous causes (94.3% vs. 69.2%, p < 0.01 and 88.6% vs. 50.8%, p < 0.01, respectively). In all patients, a higher success rate was achieved at the obstruction level at the common bile duct (CBD) compared with periampullary and hilar levels (90.7% vs 69.2%, p < 0.05 and 90.7% vs 28.6%, p < 0.001, respectively). In patients with cancer as a cause, higher success rate was achieved at CBD level than at hilar level (85% vs 28.6%, p < 0.05). Those patients with cancer and serum bilirubin above 15 mg/dl had a higher failure rate in ENBD. Success rate of ENBD was not related to the presence of JPD, bacteremia or fever before ERCP and serum albumin level. CONCLUSIONS: Factors decreasing the success rates of ENBD were underlying cancerous causes, obstruction level at hilum or periampullary region and serum bilirubin level more than 15 mg%. PMID- 7553427 TI - Use of noninvasive positive pressure ventilation via nasal mask in patients with respiratory distress after extubation. AB - BACKGROUND: Noninvasive positive pressure ventilation (NIPPV) has been successfully applied to provide ventilatory support in patients with chronic respiratory insufficiency as well as in selected patients with acute respiratory failure of various etiologies. To investigate the effectiveness of NIPPV via nasal mask in patients who develop respiratory distress after extubation, we prospectively studied 19 such patients (14 elective extubations and 5 self extubations) who required no immediate reintubation. METHODS: The BiPAP ventilatory support system (BiPAP S/T-D, Respironics Inc., Murrysville, PA) via nasal mask was used to provide NIPPV in each patient. Inspiratory pressure support was adjusted to achieve comfort as well as adequate oxygenation and ventilation. RESULTS: Eleven patients were successfully transferred to regular oxygen supplement after an average trial of 38 hours. Persistent oral air leak despite the application of chin strap was observed in four of the eight failure cases and appeared to be the leading cause of NIPPV failure. When these patients were excluded, a success rate of 73% was achieved. Other factors contributing to failure included persistent hypoxemia, inadequate ventilation, worsening of underlying diseases and bronchial secretions. No major complication related to the use of NIPPV was observed. In the success group, respiratory rate significantly decreased by an average of 10 breaths/min at one hour after the commencement of NIPPV (p < 0.01). No significant changes of heart rate, blood pressure, Paco2 and pH were noted in either group. CONCLUSIONS: NIPPV via nasal mask may be considered as an alternative to endotracheal reintubation in selected extubated patients with respiratory distress who require no immediate reintubation. PMID- 7553428 TI - Cerebral venous thrombosis. Demonstration with magnetic resonance angiography. AB - Three patients with cerebral venous thrombosis (CVT) were imaged with magnetic resonance angiography (MRA). Initial spin-echo magnetic resonance (MR) demonstrated acute or subacute thrombosis in all patients. The presence of thrombosis was confirmed with MRA. Repeat MRA in the three patients revealed partial recanalization in one and almost complete recanalization in two patients. The etiology of CVT is reviewed, and the advantages of MRA are compared with conventional MR imaging and computed tomography in the evaluation of CVT. PMID- 7553429 TI - A rare case of cerebellar glioblastoma multiforme in childhood: MR imaging. AB - Glioblastoma multiforme is a highly malignant brain neoplasm that is very rarely discovered in childhood but accounts for approximately 17% of intracranial tumors in the adult. Only approximately 25 children with glioblastoma multiforme in the cerebellum have been described in the literature. We report on a 4 1/2-year-old girl with this tumor in the cerebellar hemisphere and discuss the magnetic resonance imaging findings. PMID- 7553430 TI - Breast tumors associated with nipple discharge. Correlation of findings on galactography and sonography. AB - To compare the specific information provided by ultrasound (US) and galactography and to appreciate the clinical applicability of US as a primary diagnostic method for breast tumors associated with nipple discharge, a comparative study of each method in 15 patients with ductal tumors of the breast was performed. To evaluate the smallest anatomical unit of the lactiferous system demonstrable by US, 30 patients with normal lactating breasts were examined prospectively. The smallest anatomical ductal unit that could be evaluated with US in the normal lactating breast was the terminal duct lobular unit (TDLU). US is superior to galactography in detecting masses smaller than 0.5 cm or lesions involving multiple ducts. Galactography is better for visualizing the duct system and detecting calcification. In conclusion, US serves as a primary noninvasive diagnostic tool in patients with clinically suspicious breast tumor associated with bloody nipple discharge, but galactography also should be done in specific circumstances. PMID- 7553431 TI - Right atrial tumor arising on an atrial septal aneurysm. Assessment by MR imaging. AB - An elderly women presenting with transient ischemic events underwent transesophageal echocardiography, which detected an aneurysm of the interatrial septum. A tumor protruding from the right atrial aspect of the aneurysm also was found incidentally. Not only was magnetic resonance (MRI) imaging helpful in better characterizing the aneurysm, but also the use of gadolinium diethylaminetriamine pentaacetic acid permitted differentiation between the tumor and adherent thrombus. To the best of our knowledge, this represents the first report of a tumor arising from an atrial septal aneurysm. PMID- 7553432 TI - Pseudothrombosis of the right ovarian vein. Pitfall of abdominal spiral CT. AB - We present the computed tomography (CT) findings of pseudothrombosis of the right ovarian vein. In two young multiparous women, pseudothrombosis was detected on early-phase spiral CT scans by the presence of retrograde flow of contrast medium down the left ovarian veins. Asymmetry of density seen in the ovarian veins in the early phase of spiral CT scanning related to reflux in the left ovarian vein should not be mistaken for right ovarian vein thrombosis. PMID- 7553433 TI - Tuboovarian abscess following transvaginal oocyte retrieval for in vitro fertilization: imaging appearance. AB - All physicians participating in the workup of an infertile patient should be aware of the nonspecificity of the computed tomographic or sonographic finding of a complex adnexal cystic lesion occurring following transvaginal oocyte retrieval for in vitro fertilization. Persistent fever and leukocytosis in conjunction with such lesions should lead to an early presumptive diagnosis of tuboovarian abscess with prompt administration of antibiotic therapy. PMID- 7553434 TI - CT assessment of postirradiation changes in the rectum and perirectal region. AB - Postirradiation changes in the rectum and perirectal tissue were assessed by computed tomography (CT) in 55 patients (total of 60 examinations) and correlated with radiation dose and time elapsed. Thickening of the perirectal fascia, increased density of the perirectal fat, and swelling of the rectal wall occurred in 48, 60, and 8% of patients, respectively, regardless of radiation dose within 30 Gy. However, widening of the presacral space was seen in 8% of patients receiving a rectal dose of less than 45 Gy, a percentage that increased to 16% for patients receiving 45 Gy or more. Thickening of the perirectal fascia, increased density of the perirectal fat, widening of the presacral space, and muscular changes were seen regardless of the time from the start of therapy. CT does not appear to be reliable enough in detecting residual or recurrent tumor in the rectum. However, knowledge of the CT features of postirradiation changes is helpful for diagnosis in patients with pelvic malignancies. PMID- 7553435 TI - Sudeck's dystrophy of the hand. MR imaging. AB - We report on Sudeck's dystrophy in a 69-year-old woman who complained of recurrent pain and local inflammation in the right hand. Magnetic resonance (MR) using T1- and T2-weighted images before and after intravenous administration of gadolinium diethylene-triamine pentaacetic acid demonstrated soft-tissue swelling, bone signal intensity changes in some carpals and metacarpals, and small effusions in the adjacent joints. MR imaging provides simultaneous assessment of bone and soft-tissue involvement, proving its value in the diagnosis of this entity. PMID- 7553437 TI - A possible way to solve problems in CT densitometry. AB - The common computed tomography (CT) densitometric scale expressed in Hounsfield units does not possess sufficient representativity. A standard scale is needed to solve problems in CT densitometry. A new scale based on a special phantom with different solutions of calcium chloride permits the elimination of shortcomings of the common scale. Indications for testing CT scanners with the help of the phantom are offered. PMID- 7553436 TI - Periprosthetic mycobacterial infection. CT and mammographic findings. AB - Organisms of the Mycobacterium fortuitum complex are an uncommon but important cause of periprosthetic infection following augmentation mammoplasty or other breast surgery. This etiological agent must be considered in the particular case of periprosthetic infection, because special handling of the fluid is crucial to enhance recovery of the organism. We describe the computed tomography (CT) and mammographic findings in such an abscess with respect to the clinical context and subsequent management. To our knowledge, CT findings associated with any periprosthetic breast infection have not been described. PMID- 7553438 TI - Changes in radiological routines following the introduction of computed tomography. A retrospective study from two Norwegian hospitals. AB - Diagnostic imaging routines have changed rapidly during the last two decades. The real revolution started with the introduction of computed tomography into routine clinical work in the middle of the 1970s. Simultaneously, a tremendous sophistication of ultrasonography took place, and shortly later, magnetic resonance imaging started its "career." The present report explores how the introduction of computed tomography changed imaging routines in two major Norwegian hospitals during the last 10 to 15 years. PMID- 7553439 TI - Transthoracic needle biopsy. What size syringe? AB - Using a vacuum gauge we demonstrated that with less effort, the identical vacuum can be obtained using a 10-cc syringe as opposed to a 50-cc syringe. We recommend using a 20 cc syringe during transthoracic needle aspiration since the syringe is easier to handle and still allows sufficient vacuum to be developed, even if a small amount of air enters the syringe. PMID- 7553440 TI - Pelvic pathology after hysterectomy. A pictorial essay. AB - Hysterectomy, one of the most commonly performed surgical procedures, leads to characteristic changes in the appearance of the pelvis on imaging studies. Women who have had a hysterectomy are often at risk for several complications, both benign and malignant, that are frequently evaluated with cross-sectional imaging. This pictorial essay explores the appearance of the normal pelvis after hysterectomy as well as the many complications that can occur, stressing the appropriate utilization of such studies and correlation with clinical history to aid in patient management. PMID- 7553441 TI - A 41-year-old Guyanese woman is admitted to a hospital with severe hemoptysis. PMID- 7553443 TI - On the humanities in nursing. PMID- 7553442 TI - Moving forward in our philosophizing and theorizing in nursing. PMID- 7553444 TI - Clinical epistemology: a dialectic of nursing assessment. AB - Clinical assessment in nursing combines general knowledge from theory and research with particular knowledge about a client. A philosophical account of this synthesis is required to elucidate the paradox of knowledge that is both general and particular. The approach developed here is a dialectical model of clinical knowledge that culminates in existential safety rather than epistemic certainty. In the model, nursing assessment is the progression from subjective vulnerability through levels of objectivity (disengagement, reduction, holism) to an intersubjectivity in which nurse and client express their combined understanding in a relational narrative. The discussion concludes with reflection on the role of theory in a dialectically complete clinical narrative. PMID- 7553445 TI - Family nursing in tertiary care: history or the promise of things to come? PMID- 7553446 TI - Explanation in nursing science. AB - Is it possible to explain that which we do in nursing and in most human sciences? Is explanation/prediction our goal? Instead, should the goal be understanding/interpretation? Might the goal be both understanding and explanation? Is nursing, as a human science, focused on individuality or generalizability? This paper explores the current debate in relation to explanation and understanding. The traditional view of explanation is addressed as is the dichotomous view of understanding. A third view on explanation and understanding, designed by Miller (1983), is offered as a possible middle ground between the two diverse views as an approach for nursing as a human science. PMID- 7553448 TI - Identifying the psychosocial needs of individuals with cancer. AB - The diagnosis and treatment of cancer creates psychosocial needs that patients often find difficult to resolve. Because most need assessments do not reach beyond enumerating needs to examine barriers to needs resolution, existing social supports or patients' service preferences, we set out to develop a needs assessment inventory to meet these objectives. The first step was to identify need categories using a qualitative methodology. The aims of the research presented in this paper are to: (a) compare patients' and professional caregivers' identification of patients' psychosocial needs, (b) establish categories of psychosocial needs, and (c) verify the categories resulting from the analysis of the data. Twenty-seven need categories were identified. There was a lack of congruency between patients and hospital caregivers and between caregivers at the two cancer centres in the frequencies with which the need categories were cited. The incongruence underscores the importance of gathering information from more than one perspective. PMID- 7553447 TI - The importance of critical theory to nursing: a description using research concerning discharge decision-making. AB - Critical theory has emerged as an important research orientation for nursing. It provides for new and broader research questions and offers the potential to extend the knowledge base of nursing. In this paper I describe some applications of Jurgen Habermas's critical theory (1984, 1987) to nursing, using the example of my recently completed doctoral research (Wells, 1994). The theory was employed as a broad perspective for the study in which I investigated the process of decision-making concerning the discharge of elderly patients from the hospital. When data from 31 patient cases were analyzed, the process was found to be determined largely by systemic forces. Habermas's theory was key in understanding the structure of the process as a means-ends, or instrumental one, and in generating ideas for change in the conceptualization of the process of discharge decision making. Critical theory can advance nursing's understanding of the social organization of everyday practice situations and whether and how they might be reorganized. PMID- 7553450 TI - Molecular techniques and their potential application in monitoring the microbiological quality of indoor air. AB - Health effects associated with poor indoor air quality have created a need for accurate, reproducible methods of monitoring the microbiological content of indoor air. Improved methods of detection may allow researchers to clarify the effect of individual species present in the indoor environment on human health. This review discusses the shortcomings of current methods of identification and detection and focuses on the potential for molecular techniques in this emerging field. Probe techniques, restriction endonuclease analysis, karyotyping, and DNA and polymerase chain reaction fingerprinting methods available to detect and identify bacteria and fungi significant in the indoor air environment are discussed. Problems that may be encountered using these techniques are also considered. The authors have included a brief discussion on current air sampling techniques as well as adapting these techniques for use with molecular detection methods. PMID- 7553449 TI - Nurses' reactions to physical assault by their patients. AB - Using the Assault Response Questionnaire (Lanza, 1988a), the emotional, biophysiological, and social reactions of 35 female non-psychiatric nurses who had been assaulted by their patients were investigated. These nurses were also asked to describe their assault experience, and to identify causes, their coping strategies, and the barriers preventing them from reporting assault. Results showed that as nurses reported more assaults, they experienced more intense emotional, biophysiological, and social reactions. They coped by learning to change their behaviours and they most often cited patient variables as causes for the assault--a finding that runs contrary to the pattern of self-blame reported in earlier studies. PMID- 7553451 TI - The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated. AB - A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product. PMID- 7553452 TI - Interaction of sulfhydryl reactive reagents with components involved in (1,3) beta-glucan synthesis from Candida albicans. AB - Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC50 3 45 microM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 degrees C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 microM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 microM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTP gamma S. PMID- 7553453 TI - Bacillus oleronius sp.nov., a member of the hindgut flora of the termite Reticulitermes santonensis (Feytaud). AB - A new rod-shaped endospore-forming bacterium is described, which was isolated from the hindgut of the termite Reticulitermes santonensis (Feytaud). The isolate stains Gram negative and its DNA has a guanine-plus-cytosine content of 35 mol%. Despite the Gram-staining reaction, both biochemical and physiological features place the isolate in the genus Bacillus and indicate a phenotypic resemblance to the Bacillus firmus-lentus group of species. On the basis of comparative 16S rRNA analysis and some phenotypic features the isolate clearly represents a new species for which the name Bacillus oleronius is proposed. The type strain is Bacillus oleronius Rt 10 (DSM 9356). PMID- 7553456 TI - Metronidazole uptake in Helicobacter pylori. AB - Currently, the mechanism of metronidazole resistance is not understood in Helicobacter pylori. We have looked at uptake of metronidazole into a sensitive and a resistant strain of H. pylori. Both strains displayed rapid uptake of [14C]metronidazole, although the resistant strain accumulated the drug at a slower rate and to a lesser amount than the sensitive strain. Uptake was inhibited by KCN and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) but not by sodium arsenate. Thin-layer chromatography analysis of lysed cell supernatants showed that metronidazole was metabolized in both strains. A variety of related imidazole compounds inhibited metronidazole uptake, consistent with a common transport system for this group of antibiotics. Our data do not support an absence of uptake or metabolism as a cause of resistance in the strain examined. PMID- 7553454 TI - Cell surface properties of Aspergillus fumigatus conidia: correlation between adherence, agglutination, and rearrangements of the cell wall. AB - Culture conditions that lead to swelling and germination dramatically influence cell surface characteristics and properties of Aspergillus fumigatus conidia. Conidial adherence to polystyrene and agglutination markedly increased during swelling, in a time-dependent manner. Agglutination appeared to be sensitive to cycloheximide and calcium. Removal of cell wall polysaccharides by lyticase or sodium metaperiodate suppressed agglutination of conidia. Proteinase K weakly decreased it whereas dithiothreitol strongly dispersed the cells. These observations suggest that both cell surface carbohydrates and proteins are involved in the agglutination process. Electron microscopic observations demonstrated that the cell wall of conidia was subject to some rearrangements during swelling, involving degradation and loss of the external convoluted layer, and subsequent exposure of underlying ligands. This was confirmed using lectins labelled with gold or fluorescein isothiocyanate, which showed that some carbohydrates, particularly those acting as ligands for peanut agglutinin, are largely exposed during the process. Finally, SDS-PAGE revealed major protein changes between resting and swollen conidia. We conclude that the ability of A. fumigatus conidia to aggregate correlates with an increase in adherence and biochemical reorganization of the cell wall. PMID- 7553457 TI - A novel method for cloning DNA of plant-pathogenic mycoplasmalike organisms. AB - A novel method was developed for cloning the DNA from a representative of plant pathogenic mycoplasmalike organisms (MLOs). This procedure utilized random amplified polymorphic DNA (RAPD) and basic recombinant DNA techniques. It consisted of amplification of total DNA from diseased plants using one oligonucleotide primer with arbitrary sequence and separation of RAPD products in agarose gels. Unique RAPD band(s) of MLO origin was (were) then recovered from the gel and cloned into the specifically designed vector pCR II. With this method, a DNA fragment of the SA2 isolate of grapevine yellows MLO was cloned. Southern blot hybridizations revealed that most of the DNA in the unique RAPD band was derived from MLO. Results from dot-blot hybridizations used for screening showed that approximately 60% of transformants harbored MLO-specific recombinant plasmids. Our approach is relatively simple, quite efficient, and not limited by the amount of diseased material available. It does not depend on DNA sequence information for primer design and does not rely on restriction endonucleases for cloning. In addition, it can be used directly for disease diagnosis and for differentiation of closely related MLOs. Our system may serve as a model for cloning DNAs of other fastidious plant pathogens. PMID- 7553455 TI - [Effect of desertomycin on the synthesis of cell wall polymers in Saccharomyces uvarum]. AB - The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of beta(1,3)-glucans linked among themselves by beta(1,6) bonds. Mannan formation was not inhibited such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action. PMID- 7553459 TI - Essential surgery--respectable again. PMID- 7553460 TI - Atherosclerotic disease and stroke. PMID- 7553458 TI - The estrogen debate: to be (old) or not to be (old) PMID- 7553461 TI - Case 5. Presentation. Penetrating injury with pseudoaneurysm of lateral femoral circumflex artery. PMID- 7553462 TI - 1994 Roussel Lecture: a biologic basis for changes in breast cancer management. AB - Cancer treatment in the past has been based on contemporary biologic understanding of the disease process. For most of this century cancer was viewed as an anatomic problem, and the treatment was surgical, with radical and extended radical operations viewed as appropriate. With improved understanding of the true biologic nature of cancer, the extent of surgery has been modified, and clinical trials have confirmed the suitability of breast-conserving surgery. At the same time, adjuvant chemotherapies have extended disease-free survival and survival for many categories of patient. Unfortunately, these gains have been of limited clinical dimensions. More recently, the genetic model of cancer has led to attempts at cancer prevention. In the biologic sense we have entered an era in which surgery is the adjuvant therapy, and systemic treatments have increased breast cancer cure rates. We must now move from a strategy that tries to kill cancer cells to one that seeks to control their growth, which may, if successful provide an approach for true cancer prevention. PMID- 7553463 TI - Hormone replacement therapy: boon or bane? AB - The steadily increasing frequency of breast cancer, especially in elderly women, may be owing to the following three factors, among others: natural aging, the increased use of mammography leading to earlier diagnosis of in-situ carcinoma and occult carcinoma, and the indiscriminate use of hormone replacement therapy (HRT). That there is a correlation of estrogen with breast cancer cannot be refuted. Early menarche, late menopause, late first pregnancy, obesity and dietary factors are directly or indirectly connected with an increased likelihood of breast cancer. The recent flurry of interest in attempting to prevent osteoporosis-induced bony fractures and coronary artery disease among elderly women has not been fully tested as to its efficacy by a scientific, prospectively randomized clinical trial. Therefore, it seems timely to indicate clearly that the use of HRT should be made on an individual basis, with the tacit understanding and approval of the patients, some of whom are symptomatic but the majority of whom have no signs or symptoms of the diseases for which prevention is being advocated. PMID- 7553464 TI - The risks and benefits of hormone replacement therapy. AB - The short-term benefits of hormone replacement therapy (HRT) for the relief of acute menopausal symptoms are beyond dispute. It is equally clear that premenopausal women who have undergone surgical menopause need HRT to prevent the otherwise rapid development of coronary heart disease and osteoporosis. There is epidemiologic evidence that women who have undergone a natural menopause receive similar relief with HRT. Yet patient compliance with long-term HRT is poor. This is due to unacceptable uterine bleeding and the largely unfounded fear that HRT promotes breast cancer. PMID- 7553465 TI - Experience of total hip replacement and its effect on recovery after second hip replacement. AB - OBJECTIVE: To determine whether the knowledge gained through total hip replacement (THR) influences the time to recovery after a second THR. DESIGN: A retrospective study of data gathered prospectively on the recovery course of patients from their preoperative visit to 1 year after surgery. SETTING: A university teaching hospital. PATIENTS: Sixty-nine patients underwent bilateral hip replacements at the Royal Victoria Hospital, Montreal, between May 1985 and May 1992. The inclusion criteria were: (a) similar diagnoses and prostheses bilaterally, (b) time between the two THRs of less than 3 years, (c) both replacements carried out by the same surgeon and (d) attendance at the arthroplasty clinic. Forty-one patients did not fulfil the criteria for inclusion, leaving 28 patients having 56 THRs for the study. An additional 28 patients who had unilateral hip arthroplasty were randomly chosen on the basis of sex and age to act as the control group. INTERVENTION: Bilateral cementless THR. MAIN OUTCOME MEASURES: Harris hip score at one preoperative visit and postoperatively at 6 weeks, 3 months, 6 months and 1 year. RESULTS: There were no significant differences in measures of recovery between the initial and the subsequent hip replacement at any of the time intervals. Significant differences were found only between the various time periods. CONCLUSIONS: Time and not familiarity dictated progress toward recovery for patients who underwent bilateral THR with cementless prostheses. Randomized clinical trials, assessing whether time to recovery, ultimate outcome and cost-effectiveness are affected by preoperative teaching programs, are necessary to further elucidate this important issue. PMID- 7553466 TI - A continuing medical education strategy for care of the elderly by the surgical specialties. AB - As the absolute numbers and percentage of older people rises in Canada, surgeons are required to treat an increasing number of elderly patients. Recognizing the need to enhance the quality of health care for these people by continuing medical education of health care professionals, the Canadian Medical Association sponsored an invitational workshop in May 1992. The workshop group addressing surgery and its specialties identified three essential underprinnings: knowledge of the aging process (altered physiology and response to illness); decision analysis for interventions (risks, benefits and ethical dimensions); and communication skills. Three priorities for continuing education were recommended: improving knowledge of the physiologic changes that impact on the recovery of elderly patients from surgery; management of postoperative care; and improved knowledge and skills in prescribing medications for older surgical patients. Strategies to implement these priorities are outlined. PMID- 7553468 TI - Is there a difference between the epidemiologic characteristics of hip dislocation diagnosed early and late? AB - OBJECTIVE: To determine if there are epidemiologic differences between patients with early and late developmental dislocation of the hip (DDH). DESIGN: A chart review. SETTING: A university-affiliated children's hospital. PATIENTS: Two hundred and forty-three children, 191 with early DDH and 52 with late DDH. MAIN OUTCOME MEASURES: Sex, side involved and prevalence of bilaterality. RESULTS: There were significant differences between the two groups with respect to side involved (p < 0.0002) and bilaterality (p = 0.006) but not in relation to sex. Left-sided dislocations were predominant in the early group and right-sided dislocations in the late group. Bilaterality was more common in the late group. CONCLUSION: There is circumstantial epidemiologic evidence that late DDH may be a different entity from early DDH. PMID- 7553467 TI - Preliminary experience with percutaneous laser disc decompression in the treatment of sciatica. AB - OBJECTIVE: To examine the usefulness of percutaneous laser decompression of a herniated lumbar intervertebral disc. DESIGN: A case series. SETTING: A university-affiliated hospital. PATIENTS: Sixteen patients with clinical and radiologic evidence of herniated lumbar intervertebral discs. INTERVENTIONS: Percutaneous introduction of a fine optical fibre into a herniated lumbar disc and delivery of short pulses of laser energy. MAIN OUTCOME MEASURE: The relief of intractable leg pain. RESULTS: Nine of 14 patients with intractable leg pain experienced total relief after percutaneous laser disc decompression. Four patients required subsequent microsurgical discectomy, and one required a decompressive laminectomy. Of the two patients who had back pain as their major complaint, one required microsurgical discectomy after laser disc decompression. CONCLUSIONS: Percutaneous laser disc decompression can relieve sciatica caused by a herniated intervertebral disc. The technique requires limited use of health resources. Preliminary results suggest that an early return to work can be expected in patients successfully relieved of their leg pain. PMID- 7553469 TI - Comparison of initial laparoscopic cholecystectomy at a community hospital versus a teaching hospital. AB - OBJECTIVE: To compare the initiation of laparoscopic cholecystectomy at a community hospital versus a tertiary-care teaching hospital. DESIGN: Retrospective chart review. SETTINGS: A general community hospital in Prince George, BC, and a tertiary-care teaching hospital in Vancouver. PATIENTS: One hundred and eighty-two patients in the community hospital and 318 patients in the tertiary-care centre. INTERVENTION: Laparoscopic cholecystectomy for symptomatic gallbladder disease. MAIN OUTCOME MEASURES: Preparation of surgeons for the new technique, complication rates, operating time, conversion rates to open cholecystectomy and duration of hospitalization. RESULTS: All community surgeons took didactic and laboratory courses in preparation for the new procedure and assisted each other for their first 10 cases, but surgeons at the teaching hospital had more varied preparation that included additional extensive laboratory work and preceptorships with surgeons experienced with the procedure. The rates of major complications of laparoscopic cholecystectomy were 6.5% at the community hospital compared with 5% at the tertiary-care centre. The rates of minor complications were 5.5% at community hospital and 5.3% at the tertiary-care centre. The rates of conversion to open cholecystectomy were 6.6% for the community hospital versus 4.7% for teaching hospital. The mean (and standard deviation) operating time was shorter at the community hospital than at the teaching hospital: 72.3 (30) minutes versus 106 (32) minutes (p < 0.0001). The mean (SD) length of stay was 2.5 (1.8) days at the community hospital and 3.4 (1.9) days at the teaching hospital. CONCLUSIONS: The introduction of laparoscopic cholecystectomy during a 2-year period was achieved safely at both hospitals. The complication rates were similar. The length of stay and operating times were shorter in the community hospital. PMID- 7553470 TI - Laparoscopic cardiomyotomy with a Dor patch for achalasia. AB - OBJECTIVE: To evaluate early experience with laparoscopic Heller's cardiomyotomy with placement of a Dor patch for achalasia. DESIGN: A prospective case series. SETTING: A university teaching hospital. PATIENTS: Fourteen patients (5 men, 9 women, median age 47 years) with esophageal achalasia, treated between July 1992 and July 1994. INTERVENTIONS: Laparoscopic Heller's cardiomyotomy with a Dor patch. MAIN OUTCOME MEASURES: Clinical relief of symptoms, confirmed by esophageal manometry, 24-hour ambulatory pH monitoring and barium-contrast radiography. RESULTS: Three of the 14 patients required conversion to an open procedure, and 1 underwent early laparotomy for postoperative bleeding. The median operating time was 120 minutes (range from 75 to 210 minutes), and the median duration of hospitalization was 4 days (range from 3 to 18 days). Normal physical activity was resumed after a median of 2 weeks (range from 0.5 to 6 weeks). Symptomatic dysphagia was completely relieved in 12 patients and improved in 2. Only one patient experienced symptoms of reflux postoperatively. Postoperative esophageal manometry (seven patients), 24-hour pH monitoring (five patients) and barium-meal radiography (seven patients) confirmed the clinical results. CONCLUSION: Laparoscopic Heller's cardiomyotomy with a Dor patch provides a viable alternative to open cardiomyotomy and forceful endoscopic dilatation. PMID- 7553471 TI - Laparoscopic appendectomy: a review of 95 consecutive suspected cases of appendicitis. AB - OBJECTIVE: To assess the feasibility and efficacy of laparoscopic appendectomy. DESIGN: A review of a case series. SETTING: A single teaching and referral centre in Vancouver. PATIENTS: Ninety-five consecutive patients referred to the Division of General Surgery with suspected appendicitis requiring surgical intervention. The patients ranged in age from 15 to 85 years. INTERVENTIONS: Laparoscopic appendectomy or open appendectomy. MAIN OUTCOME MEASURES: Feasibility of laparoscopic appendectomy, duration of operation, intraoperative and postoperative complications and duration of postoperative hospital stay. RESULTS: Of the 95 patients, 9 had contraindications to laparoscopic appendectomy and underwent an open operation. Of the 86 patients who underwent laparoscopy, the diagnosis of appendicitis was confirmed in 69 (80%). Following this, 61 (88%) patients underwent laparoscopic appendectomy, and in 8 the procedure was converted to open appendectomy because of periappendiceal abscess or an immobile or perforated appendix. The mean duration of operation was 78.5 minutes. There were no intraoperative complications. The mean duration of postoperative hospital stay was 2.8 days. Postoperative complications consisted of ileus in two patients and diarrhea in two patients. There were no wound infections. CONCLUSIONS: Laparoscopic appendectomy can be performed safely, with minimal complications on most patients with appendicitis; hospital stay is reduced. Laparoscopy allows accurate diagnosis. PMID- 7553473 TI - Spontaneous rupture of a pancreatic pseudocyst into the portal vein. AB - An unusual complication of pancreatic pseudocyst is reported. A 47-year-old woman with chronic liver disease and a history of recurrent pancreatitis died of fulminant, gram-negative septic shock. Acute, bilateral thigh cellulitis with tissue culture positive for Escherichia coli was the only potential infectious source identified. Autopsy revealed a pseudocyst in the head of the pancreas that had eroded into the portal vein, with embolization of mucoid cyst material into intrahepatic portal vein branches. Fibrous organization and recanalization of some of the occluded portal vein branches indicated that this process had been present for weeks to months and was therefore not the direct cause of death. The literature on this unusual complication of pancreatic pseudocyst is also reviewed. PMID- 7553474 TI - Splenic avulsion in a pregnant patient with vomiting. AB - Splenic avulsion in pregnancy is a rare condition that carries a high risk of death to both mother and fetus. The authors describe a 30-year-old woman with hyperemesis who had spontaneous avulsion of the spleen at 30 weeks' gestation. She underwent a splenectomy and cesarean section, resulting in a stillborn fetus. The authors theorize that violent vomiting resulted in splenic avulsion. Prompt surgical intervention by a general surgeon and obstetrician is required in such cases. PMID- 7553472 TI - Benefit of palliative surgery for bowel obstruction in advanced ovarian cancer. AB - OBJECTIVES: To determine the benefit of palliative surgery for patients with advanced ovarian cancer and bowel obstruction and to identify criteria for selecting patients who are most likely to benefit from palliation. DESIGN: A retrospective study of patients treated between 1982 and 1992. SETTING: A university-affiliated hospital. PATIENTS: Fifty-three patients with complete and unresolved bowel obstruction caused by ovarian cancer. INTERVENTION: Surgery for relief of bowel obstruction. MAIN OUTCOME MEASURES: Postoperative survival longer than 60 days, return home and relief of bowel obstruction for longer than 60 days, factors associated with failure of palliative surgery. RESULTS: Successful palliation was achieved in 27 (51%) patients and was associated with the absence of four prognostic factors: palpable abdominal and pelvic masses, ascites exceeding 3 L, multiple obstructive sites and preoperative weight loss greater than 9 kg. Age, time interval between diagnosis of ovarian cancer and bowel obstruction, stage of disease at initial diagnosis, tumour type and grade, site and degree of obstruction, presence of gross residual tumour after initial operation and preoperative use of chemotherapy or radiotherapy did not indicate the success or failure of palliative surgery. CONCLUSIONS: Palliative surgery for bowel obstruction in advanced ovarian cancer can be worthwhile, and there are four prognostic factors that indicate the likely failure of palliation. PMID- 7553475 TI - Stoma closure and wound infection. PMID- 7553477 TI - Gun control. PMID- 7553476 TI - Quebec hopes to overcome province's surgical-delay problem. AB - The Quebec government has released an action plan for eliminating delays in surgery in the province. One of the main goals is to increase the amount of day surgery, thus reducing the demands on hospitals. While most physician groups appear to support the plan, the Quebec Hospital Association wonders whether the follow-up community care needed to support the day surgery will be available. PMID- 7553478 TI - Gun control. PMID- 7553479 TI - Gun control. PMID- 7553480 TI - Forum's insistence on single-tier health care criticized. PMID- 7553481 TI - Hazards of camphorated oil. PMID- 7553483 TI - It's a horse's life. PMID- 7553482 TI - Hearing loss among children who have undergone ECMO. PMID- 7553484 TI - The continuing debate over conjugated estrogens. PMID- 7553485 TI - Physician-assisted suicide and euthanasia: let me count the ways. PMID- 7553486 TI - Anti-circumcision groups criticized. PMID- 7553489 TI - Nothing new on appendicitis. PMID- 7553488 TI - Depiction of coroners more pulp fiction than fact. PMID- 7553487 TI - Anti-circumcision groups criticized. PMID- 7553490 TI - Hereditary aspects of prostate cancer. AB - OBJECTIVE: To review current literature on the hereditary aspects of prostate cancer and to evaluate the importance of family history in history taking and screening for prostate cancer. DATA SOURCES: MEDLINE was searched for articles in English or French published between Jan. 1, 1956, and Oct. 31, 1994, with the use of MeSH headings "prostatic neoplasms," "genetics" and "chromosomes." Additional references were selected from the bibliographies of articles found during the search. STUDY SELECTION: Case-control studies involving the incidence of prostate cancer and relative risk (RR) of such cancer in the families of men with this disease, compared with a control group, were included. Only studies in which prostate cancer was diagnosed on the basis of histologic tests were included. Animal investigations were excluded. DATA EXTRACTION: Ten case-control studies were evaluated critically in terms of design, case and control groups, the size of the samples and statistical results. The incidence of prostate cancer in the families of cases, compared with that in the families of controls, and differences in RR were reviewed. DATA SYNTHESIS: The lifetime risk of prostate cancer is 9.5% and of death from prostate cancer is 2.9% for a man 50 years of age. For first-degree male relatives of men with prostate cancer, the calculated RR ranges from 1.7 to 8.73. "Hereditary" prostate cancer is a term applied to a specific subset of patients with prostate cancer. This form of prostate cancer is transmitted by a rare, autosomal, dominant allele with high penetrance; it accounts for an estimated 43% of early-onset disease (affecting men less than 55 years of age) but only 9% of all prostate cancer in men up to 85 years of age. A greater number of affected family members and early onset among family members are the most significant predictors of risk. CONCLUSIONS: Recent confirmation of the familial clustering and Mendelian inheritance patterns of some prostate cancer has important implications. It increases the potential for directed research into the causes of prostate cancer and for refinements in the current screening practices to detect this common disease. Manoeuvres to detect prostate cancer should be started earlier among men with one or more first-degree relatives with the disease than among other men. PMID- 7553491 TI - Report on activities and attitudes of organizations active in the clinical practice guidelines field. AB - The organizing committee of a workshop on clinical practice guidelines (CPGs) surveyed invited organizations on their attitudes and activities related to five topics to be covered during the workshop sessions: organizational roles, priority setting, guidelines implementation, guidelines evaluation and development of a network of those active in the CPG field. Organizational roles: The national specialty societies were felt to have the largest role to play; the smallest roles were assigned to consumers, who were seen to have a role mainly in priority setting, and to industry and government, both of which were seen to have primarily a funding role. Many barriers to collaboration were identified, the solutions to all of which appeared to be better communication, establishment of common principles and clear role definitions. Priority setting: There was considerable agreement on the criteria that should be used to set priorities for CPG activities: the burden of disease on population health, the state of scientific knowledge, the cost of treatment and the economic burden of disease on society were seen as important factors, whereas the costs of guidelines development and practitioner interest in guidelines development were seen as less important. Organizations were unable to give much information on how they set priorities. Guidelines implementation: Most of the organizations surveyed did not actively try to ensure the implementation of guidelines, although a considerable minority devoted resources to implementation. The 38% of organizations that implemented guidelines actively listed a wide variety of activities, including training, use of local opinion leaders, information technology, local consensus processes and counter detailing. Guidelines evaluation: Formal evaluation of guidelines was undertaken by fewer than 13% of the responding organizations. All the evaluations incorporated assessments before and after guideline implementation, and some used primary patient data. Barriers to evaluation included lack of money, time, data or expertise. CPG Network: Most of the respondents felt that all organizations and individuals interested or involved in guidelines should form the membership of the network. The three most important functions of such a network were deemed to be (a) to facilitate collaboration among those involved in the CPG process, (b) to maintain an information centre on CPGs and (c) to provide expertise to the CPG process. It was felt that the network should have some formal structure and communicate through e-mail and print media. PMID- 7553493 TI - Preferences of program directors for evaluation of candidates for postgraduate training. AB - OBJECTIVE: To determine the preferences of program directors for various grading systems and other criteria in selecting students for residency training positions through the Canadian Resident Matching Service (CaRMS). DESIGN: Questionnaire survey. PARTICIPANTS: All 110 directors of residency training programs in Ontario. SETTING: Ontario medical schools. OUTCOME MEASURES: Weighting of importance of different screening tools (e.g., grading systems, personal interview, dean's letter) used during undergraduate training. RESULTS: Of the 110 directors 96 (87%) responded. Of the 92 who rated the various grading practices 35 (38%) preferred a numeric grading system, 26 (28%) a letter grading system, 23 (25%) an honours/pass/fail system and 8 (9%) a pass/fail system. Most of the respondents from each school favoured a grading system that was more discriminating than the one used at their location. The personal interview was regarded as the most important screening tool by 80 (83%) of the respondents; the dean's letter was considered to be very useful by only 16 (17%). CONCLUSIONS: More value was placed by program directors on a numeric or other more discriminating grading system than on the pass/fail system. Although the grading system provides only one type of screening mechanism it raises the question of whether there should be a policy for uniform grading practices for all Canadian students. PMID- 7553492 TI - Declaring pediatric brain death: current practice in a Canadian pediatric critical care unit. AB - OBJECTIVE: To document the criteria used to declare brain death in a pediatric critical care unit (PCCU). DESIGN: Retrospective chart review. SETTING: Regional PCCU in southwestern Ontario. PATIENTS: Sixty patients 16 years of age or less declared brain dead from January 1987 through December 1992. OUTCOME MEASURES: Presence or absence of documentation of irreversible deep coma, nonresponsive cranial nerves, absent brain-stem reflexes, persistent apnea after removal from ventilator, presence or absence of blood flow detected by radioisotope scanning, presence or absence of electroencephalographic evidence of electrocerebral activity. RESULTS: The 60 patients accounted for 1.5% of all PCCU admissions; 17 were under 1 year of age. In 39 cases brain death was diagnosed using clinical criteria ("certified brain death"), which could not be fully applied in the remaining 21 cases ("uncertifiable but suspected brain death"). Electroencephalography and cerebral blood-flow studies with technetium-99m hexamethyl-propyleneamine oxime were used as ancillary tests in 16 patients with certified brain death and in 17 with uncertifiable but suspected brain death who survived long enough to be tested. Electrocerebral silence was demonstrated in all nine patients who underwent electroencephalography. Cerebral blood flow was undetectable in 26 of the 30 patients tested, and an abnormal pattern of blood flow was seen in the remaining 4, all of whom received a diagnosis of certified brain death. CONCLUSIONS: Pediatricians in this large tertiary care referral centre are using clinical criteria based on the 1987 guidelines of the CMA to diagnose brain death in pediatric patients, including neonates. When clinical criteria cannot be fully applied, ancillary methods of investigation are consistently used. Although the soundness of this pattern of practice is established for adults and older children, its applicability to neonates and infants still needs to be validated. PMID- 7553494 TI - Evaluation of a tuberculosis screening program for high-risk students in Toronto schools. AB - OBJECTIVE: To evaluate the effectiveness of a tuberculosis (TB) screening program for high-risk students in elementary and secondary schools. DESIGN: Cross sectional study of the 1992-93 school screening program conducted by the Department of Public Health of the City of Toronto. Program costs were calculated with the use of 1993 wages, and costs for medical care were based on the 1993 fee schedule of the Ontario Medical Association. SETTING: Elementary and secondary schools in the City of Toronto. PARTICIPANTS: Students enrolled for the first time in any grade who were born in a country where TB is endemic or who were aboriginal Canadians were eligible. Of 44,179 students in Toronto schools 1775 met the eligibility criteria. INTERVENTION: Students were administered a Mantoux skin test, and those with a significant reaction (an induration of 10 mm or more in diameter) were advised to consult a physician for follow-up. OUTCOME MEASURES: Participation rate, number of participants with a significant reaction, percentage of these who were prescribed isoniazid and who completed chemoprophylaxis, potential number of preventable cases and costs associated with preventing each case. RESULTS: Of 1775 eligible students 42.9% (761) agreed to participate, and 40.6% (720) were screened. Significant skin-test reactions were detected in 22.5% (162/720) of the participants screened. Of these, 87.7% (142/162) saw a physician; subsequently, 2 cases of TB (1 active and 1 inactive) were detected. Of the remaining 140 students 44.3% (62) were prescribed isoniazid, of whom 9.7% (6/62) refused chemoprophylaxis. Of the remaining 56 students 82.1% (46) completed at least 6 months of chemoprophylaxis and 10.7% (6) were completing treatment at the end of the study. An estimated 3.2 cases were prevented, at a cost of $13,493.15 per case, whereas the undiscounted cost of treatment for an uncomplicated active case of TB in a patient under 19 years of age was $4503.82. CONCLUSIONS: The effectiveness of this screening program was significantly reduced by poor participation and poor rates of prescription of isoniazid by physicians. Appropriate strategies are needed to reduce barriers to the implementation of these programs. PMID- 7553495 TI - Ocular alkali burn associated with automobile air-bag activation. AB - Alkali burns of the eye can result in permanent visual impairment and are therefore potentially devastating. Immedicate diagnosis and treatment are essential to a good prognosis. The authors report the case of a 52-year-old woman who suffered alkali keratitis as the result of the activation of an automobile air bag. This type of injury will be seen more frequently as more cars are equipped with air bags and should be suspected in drivers and passengers involved in accidents in which air bags have been activated. PMID- 7553500 TI - Montreal Museum exhibit provides clear picture of advances in medical imaging. AB - An exhibit at the McCord Museum of Canadian History, which runs until November, marks the centennial of the discovery of the x-ray. Among the items on display are a portable field x-ray machine used during World War I and one of the machines commonly found in shoe stores between 1930 and 1950 to x-ray customers' feet. The exhibit also features one of the first scanners used in North America. Dr. Denis Melancon, a radiologist at the Montreal Neurological Institute, was the driving force behind the McCord exhibit. "I wanted to show [not only] the historical and scientific aspects of the discovery, but also its medical and social applications," he says. PMID- 7553501 TI - The strange case of the misplaced suppository. AB - In a controversial decision, the General Medical Council has found a consultant anesthetist guilty of serious professional misconduct for giving a painkilling suppository without forewarning the patient and obtaining consent. Even though the suppository was misplaced in the patient's vagina, this was accepted as a mistake, at issue was whether a specific separate consent was required for insertion of the suppository. PMID- 7553498 TI - Medicolegal hell in Texas. AB - In the "war zones" of Texas, lawyers use billboards, television commercials and Yellow Page advertisements to announce their availability to help the "unjustly injured," and medicolegal lawsuits are as common as the rain that sweeps in from the nearby Gulf of Mexico. Almost 75% of the suits are dismissed without award or settlement, since many are plainly frivolous. However, even these can mean torment for physicians, who have to hire lawyers, answer charges, collect paperwork, take time off work for depositions and consultations, and then worry about how insurers will react the next time premiums are due--even if they are cleared. Texas estimates that defensive medicine practised because of legal fears costs the state at least $702 million annually, spending that is bound to continue as long as one lawsuit is filed annually for every 5.3 doctors in the state. PMID- 7553499 TI - The politics of despair: AIDS and the failure of treatment. AB - The recent AIDS-related death of a friend forced Dr. Iain Mackie to think about the disease and the lack of positive news about it. Despite a flood of recent studies that brought pessimistic news about the treatments now in use, Mackie tries to remain optimistic. "I may be treating only myself as I feed off the politics of optimism," he says, "but at least I will not be feeding off the politics of despair." The article appears as Canada marks another National AIDS Week Oct. 2-8. PMID- 7553496 TI - The dilemma of hereditary prostate cancer. AB - The observation that a family history of prostate cancer significantly increases a man's risk of the disease (see pages 895 to 900 of this issue) highlights many of the ethical, emotional and pragmatic controversies in medical circles concerning the management of this common form of cancer. This editorial describes a patient's personal dilemma when he learns that his brother is dying of prostate cancer, a dilemma that illustrates the potential harms and benefits of early detection and treatment. Current evidence does not justify screening asymptomatic men for prostate cancer, but many Canadian physicians do practise early case detection for some patients with a high risk of the disease. Men with a family history of prostate cancer must be informed by their physicians about the known and unknown risks and benefits of early detection and treatment, before they undergo the cascade of diagnostic and therapeutic procedures associated with prostate cancer. PMID- 7553504 TI - Resources available to help family physicians provide advice to travellers. AB - Because many Canadians are travelling to exotic destinations, family physicians may be asked for advice on immunization and health tips to prevent illnesses such as malaria, altitude disease, meningitis and schistosomiasis. A Toronto family physician who is on staff at a travel clinic says a few guiding principles and good resources can help family physicians ensure that their patients are healthy when they return from a trip. PMID- 7553502 TI - University of Calgary marks 25 years of medical training. AB - The University of Calgary Faculty of Medicine celebrates 25 years of innovative medical training in October. Its 3-year degree program has produced more than 1450 physicians since the first students entered the medical school in 1970. A reunion for former staff and students is planned for Oct. 12-14. PMID- 7553503 TI - Ontario's health-information service: a province-wide resource for patients. AB - An innovative service based in Toronto provides consumers with specialized yet understandable information about health care and health promotion. The Consumer Health Information Service gathers articles from medical journals, periodicals, health agencies, support groups, books and medical databases to provide people with better access to readily understandable health information. Much health related information is available, says the chief librarian, but often people have trouble determining where it is. PMID- 7553505 TI - Graduate university course another sign of continuing interest in medical history. AB - Whether the interest is shown through a new graduate course in medical history at the University of Toronto or membership in history-of-medicine clubs, physicians continue to show a keen curiosity about their profession's past. "Other countries have well-established medical-history museums and large permanent collections," explains one museum curator, "and Canada should not neglect her own medical past." Unfortunately, difficult economic times mean universities and hospitals often have a hard time giving priority to medical history. PMID- 7553497 TI - Acknowledging the weather-health link. AB - The impact of weather on health is generally overlooked by physicians. Possible reasons for this include lack of training and insufficient awareness of the significant body of research on human biometeorology. The authors argue that, in the absence of clearly demonstrable causal connections, statistical associations between weather phenomena and health problems should be enough to influence clinical practice. Physicians in Germany make use of daily bulletins from the national weather service to advise patients on the management of common health problems that seem to be exacerbated by certain weather conditions. The authors urge Canadian doctors to follow the lead of their European colleagues by increasing their awareness of the relation between weather and health. PMID- 7553506 TI - Sleep apnea a risk factor for poor driving. PMID- 7553507 TI - Courageous action needed to end smoking. PMID- 7553508 TI - Communications research. PMID- 7553509 TI - Rationing services: another prescription. PMID- 7553510 TI - Lessons from Lithuania. PMID- 7553511 TI - Public health nurses do visit breast-feeding mothers. PMID- 7553512 TI - Public health nurses do visit breast-feeding mothers. PMID- 7553514 TI - Paradox, process and perception: the role of organizations in clinical practice guidelines development. AB - The role of organizations in the development of clinical practice guidelines (CPGs) has received virtually no analytic attention. In a strictly rational and disinterested world, CPGs would be assessed on the basis of the supporting evidence and applicability to practice. However, factors that have more to do with medical sociology play a key role in CPG acceptance and, in some cases, development. The entire concept of CPGs entails troubling paradoxes, many of which turn on the distinction between scientific evidence and the sociologic determinants of validation and implementation. At the root of the question of organizational roles is the issue of values: Whose values should be at the table? What values are legitimate? From what perspectives should the utility of a procedure or technology be addressed? The Canadian health care system is a largely public creature, and CPG development is part of the public policy process. In this context, decisions about organizational roles must be sensitive to conflict of interest and a diversity of values. A provisional model for participation in CPG processes would minimize the role of organizations per se, although individual participants would no doubt reflect the legitimate interests of their affiliations without representing them formally. PMID- 7553515 TI - A new Canadian health care initiative in Tanzania. AB - One of the main conclusions of the World Bank's World Development Report 1993- Investing in Health was that equitable access to a package of essential clinical and public health services could significantly reduce the overall burden of disease in low-income countries. The report argued that more rational and effective decisions with regard to the allocation of limited resources could be made on the basis of burden-of-disease and cost-effectiveness analyses. In collaboration with the Canadian International Development Agency and several other organizations, the International Development Research Centre has developed the Essential Health Interventions Project to test the feasibility of this approach in a few districts in Tanzania. Outcome assessment wil focus on improved planning at the district level and on changes to the health status of the study population. PMID- 7553513 TI - Misuse of asthma-medication inhalers. PMID- 7553516 TI - Comparison of sexual assaults by strangers and known assailants in an urban population of women. AB - OBJECTIVE: To examine the characteristics of sexual assaults by strangers and those by people known to the victims in an urban community-based population of women. DESIGN: Descriptive study. SETTING: Sexual Assault Care Centre, Women's College Hospital, Toronto. PARTICIPANTS: All 677 women who presented to the centre between June 1, 1991, and Sept. 30, 1993, and for whom the victim assailant relationship was known. OUTCOME MEASURES: Assailant's relationship to victim, sex of assailant, number of assailants, number, type and location of assaults, use of weapons, type of coercion and extent of physical trauma or injury. RESULTS: Sexual assault by a person known to the victim accounted for 456 (67.4%) of the assaults reported. In 344 cases the person was known more than 24 hours; 99 (28.8%) were current or previous boyfriends or spouses. Assailants who were strangers were more likely to assault the victim more than once (t = -2.42, 355 degrees of freedom [df], p < 0.05), force the victim to perform fellatio (chi 2 = 8.63, 1 df, p < 0.005), use weapons (chi 2 = 12.01, 1 df, p < 0.001) and use physical coercion (chi 2 = 4.42, 1 df, p < 0.05), whereas assailants who were known to the victims were more likely to assault a woman who was sleeping or drugged (chi 2 = 10.38, 1 df, p < 0.005). Sexual assault by a known assailant was more likely to occur in the home of the victim (chi 2 = 36.27, 1 df, p < 0.001) or the assailant (chi 2 = 8.46, 1 df, p < 0.005), whereas sexual assault by a stranger was more likely to occur outdoors (chi 2 = 89.80, 1 df, p < 0.001) or in a vehicle (chi 2 = 32.81, 1 df, p < 0.001). Overall, the mean number of trauma sites was greater among victims assaulted by strangers than among those assaulted by people they knew (t = -4.29, 180 df, p < 0.001). CONCLUSION: Two thirds of the sexual assaults in this urban population were committed by people known to the victims, and over two thirds of these assaults were associated with physical trauma. Improved victim services and prevention strategies should be built on this knowledge. PMID- 7553518 TI - Bellagio statement on tobacco and sustainable development. AB - Representatives of international, national and scientific organizations met in Bellagio, Italy, in June 1995 to examine the implications of global trends in tobacco production and consumption. The 22 participants agreed that a growing pandemic of tobacco use poses a major threat to sustainable and equitable development in low-income countries. The international Development Research Centre was invited to lead a roundtable consultative process to devise a broad based funding strategy to promote action on tobacco control. PMID- 7553520 TI - Ecosystem and population health: the role of Canadian physicians at home and abroad. AB - Seemingly intractable problems of overpopulation, ecologic degradation, diminishing resources and regional warfare are having a profound effect on global population health. Canadian physicians can assist in ameliorating these problems by helping to modify the overconsumption of natural resources at home and by participating in international health projects focused at the community level, where the health of individuals and that of their environment intersect. The author describes the work of the Canadian Hunger Foundation in Vietnam and Sri Lanka, where a team of professionals worked with local farmers to improve the local water supply, decrease soil erosion and increase food production. The team observed changes in the physical health of communities that resulted in part from interventions that empowered them to address their own problems. PMID- 7553517 TI - Comparison of activity level and service intensity of male and female physicians in five fields of medicine in Ontario. AB - OBJECTIVE: To examine the extent to which physician's sex explains variation in the activity level and service intensity of a cohort of physicians in each of five medical fields after other sources of variation are taken into account. DESIGN: Data from the Ontario Ministry of Health (MOH) and the CMA were analysed by means of multivariate regression techniques for panel data. SETTING: Ontario. PARTICIPANTS: A total of 137 dermatologists, 974 general internists, 330 pediatricians and 941 psychiatrists and a random sample of 2771 family physicians and general practitioners who met the eligibility criteria. Physicians were eligible if they billed the MOH for at least three quarters in 1983, did not bill as a medical laboratory director, provided direct patient care, did not have an alternative funding arrangement with the MOH, remained in the same specialty throughout the study period (1983-90) and billed from an Ontario address. OUTCOME MEASURES: Three measures of total activity level (annual number of services provided, annual fee-for-service billings and annual mean number of patients seen per quarter) and one measure of service intensity (annual mean number of services per patient per quarter). RESULTS: Although several variables (e.g., full-time work status, age, type of practice and recent practice move) influenced the four measures examined, physician's sex contributed significantly to explaining variation in activity in 70% of the regression equations. The women provided 33.0% fewere services per year than the men in family and general practice (p < 0.001), 25.0% fewer services in general internal medicine (p < 0.01), 22.1% fewer services in pediatrics (p < 0.05) and 22.3% fewer services in psychiatry (p < 0.001). Total billings by the women in these fields were also significantly less than those of their male colleagues, the difference being greatest among the family physicians and general practitioners (28.0%) and the general internists (27.0%) (p < 0.001). The women in these four fields saw significantly fewer patients per quarter than their male colleagues, the difference being greatest in psychiatry (33.0%) (p < 0.001). Sex affected service intensity in three fields. The female psychiatrists (14.8%) (p < 0.001) and general intenists (5.5%) (p < 0.10) provided more services per quarter than their male colleagues, whereas the female family physicians and general practitioners delivered 2.2% fewer services per patient per quarter than their male colleagues (p < 0.01). In two specialties differences between women aged 40 years or less and those over 40 years were observed. In general internal medicine the younger women had higher activity levels than the older women (p < 0.01). Conversely, in dermatology the younger women had lower activity levels (p < 0.05) and provided fewer services per patient per quarter (p < 0.001) than the older women. CONCLUSIONS: Although physician's sex explained much of the variation in activity level and service intensity, even after other important correlates were controlled for, the type and extent of differences observed between female and male physicians depended on the particular medical field examined. To understand the effect of the large increase in the number of women on the physician workforce, more detailed analyses by medical field are needed of the volume, mix and intensity of services provided by men and women, with adjustment for any possible differences in the patients seen in their practices. PMID- 7553521 TI - Mouse models and breast cancer. AB - With his colleagues at McMaster University in Hamilton, Ont., molecular biologist Dr. William J. Muller has developed strains of transgenic mice to study the roles of certain genes in the development of mammary epithelial cancer. Genes of particular interest include neu, which codes for a growth factor receptor, and c src, one of the first oncogenes ever described. The outcome of this work is a better understanding of how breast cancer starts and of the prognosis for patients with certain forms of the disease. It is expected that murine models will also be used to test the efficacy of new therapies for breast cancer. PMID- 7553519 TI - As Canadians butt out, the developing world lights up. AB - Although rates of tobacco smoking in Canada have dropped dramatically over the last 30 years this is not a global trend. For every tonne of tobacco that Canadian adults gave up between 1970 and 1990, aggressive marketing by multinational tobacco companies has ensured that an additional 20 tonnes is now consumed in developing countries. The authors describe the dilemma faced by policymakers in their efforts to control the epidemic of tobacco smoking in the developing world: although tobacco consumption leads to increased rates of mortality and morbidity and lost productivity, its production creates employment, generates tax revenue and earns foreign exchange. Canadian experience has proved that trends in tobacco consumption can be reversed through policies that address not only health issues but also economic social and agricultural concerns. The authors propose a framework for harnessing expertise in the service of worldwide tobacco control. PMID- 7553522 TI - Is medicine moving to the right? AB - During the CMA's recent annual meeting in Winnipeg, General Council delegates agreed that as far as Canada's health care system is concerned, maintenance of the status quo is impossible. Some delegates were motivated by the principle of professional autonomy, while others approached the issue from a public-policy perspective. Still others were driven by outrage at what is happening to physicians' incomes. But delegates barely resisted the pull of a vocal group of physicians who favour giving Canadians the right to choose regulated private insurance for all medical services. The compromise position, for now at least, is that delegates want the CMA to lead a public debate on the future of health care. PMID- 7553523 TI - Alberta hospital tries to boost revenue by attracting gastroplasty patients from US. AB - The fate of a surgical weight-reduction program at the 25-bed hospital in Cardston, Alta., is in limbo as Alberta's Conservative government decides whether it will allow the hospital to keep profits made by performing gastroplasty on American patients last year. Cardston Municipal Hospital, which has had more than 1200 Canadian banded-gastroplasty patients since 1979, began recruiting American patients from Montana and Idaho last year. However, rumours that the Klein government will reduce the regional health authority's operating budget by an amount equal to the hospital's profit have jeopardized the program's future. The hospital first marketed the program in the US to cushion short-falls in government funding. PMID- 7553526 TI - Physicians form association to address country's environmental issues. AB - A group of doctors concerned about the environment and health has applied for letters patent and charitable status registration for a new organization, the Canadian Association of Physicians for the Environment. The goal of the new group, says founder Dr. Tee Guidotti, is to create a network for physicians who are interested in the relationship between health and the environment, and to educate and support physicians who take an active interest in environmental issues in their communities. PMID- 7553524 TI - Facts about the fax: MDs advised to be cautious. AB - Physicians' now routine use of fax machines has drawn attention to the need for office policies and procedures to protect the confidentiality of medical records. The growing popularity of faxed prescriptions has also raised questions about prescribing violations. Lawyer Karen Capen outlines some of the legal concerns about the faxing of medical information, and suggests some precautions physicians can take to avoid problems. PMID- 7553525 TI - Some physicians remain sceptical as metamorphosis of health care continues in PEI. AB - Prince Edward Island has restructured its health care system by dismantling former decision-making bodies and establishing provincial and regional health boards and a policy council. The province hopes this move will make people think about health care in a more comprehensive way and strengthen community involvement in it. However, some sceptical physicians think the impetus behind the reforms is the desire to save money, not improve health care. "Health reform is strictly fiscal reform," says Dr. Robert Colborne, immediate past president of the PEI Medical Society. PMID- 7553527 TI - Growing number of female physicians changing the face of Canadian medicine. AB - The growing number of female physicians is changing the way medicine is practised. One recent Canadian study found that "significant differences in practice characteristics and service mix and pattern between men and women." Another change involves differences in the way men and women communicate. One lawyer noted that most medical lawsuits involve a breakdown in communication between doctor and patient, and very few female physicians have been the target of malpractice suits--even in high-risk specialties such as obstetrics and anesthesiology. PMID- 7553528 TI - Canadian physicians considering a move to US can face complicated immigration process. AB - American recruiters are busily trying to coax Canadian physicians to move south of the border to practise, but Canadians must remember that the immigration process can be both lengthy and complicated. In this article, immigration lawyer Henry Chang looks at the requirements that must be met before permanent-resident status is granted by the US. PMID- 7553530 TI - Mechanical testing of materials and the material-tissue interface. AB - This article discusses various aspects of mechanical testing for podiatric applications. Included is a brief overview of the terminology used to describe a variety of mechanical tests, suggestions for the preparation and storage of specimens, and an overview of various mechanical testing techniques that have been used to collect data on internal fixation and osteotomy stability. PMID- 7553529 TI - Mechanical properties of implantable biomaterials. AB - This chapter was intended to review many of the terms used to describe the behavior of biomaterials including linear elastic, plastic, and viscoelastic behavior. Common testing methods for evaluating the behavior of biomaterials were also reviewed. Uniaxial loading, perhaps the most common testing procedure, can characterize both time-independent (i.e., linear elastic and plastic) and time dependent (viscoelastic) materials with a controlled loading condition. Bending tests, because of their ease, are also popular and can describe linear elastic and plastic behavior fairly well. Vibratory methods to measure viscoelastic behavior, on the other hand, are less popular but are essential if the expected loading condition of the biomaterial will be fairly rapid. Taken together, these tests form the foundation for understanding the applicability of a specific material for use as an implant, and can be used to predict not only the failure thresholds for the implant but also the expected remodeling response of the bone once the implant has been placed in situ. For this reason, characterizing the mechanical properties of implant materials has been and will continue to be an important step in implant design. PMID- 7553531 TI - Clinical experience with two-component first metatarsal phalangeal joint implants. AB - The development of the two-component implant systems has generated considerable interest over the past 5 years. It is important to critically document and record the clinical experiences with these devices as they occur. The authors present an objective evaluation of their early findings and comment on possible recommendations for the future. PMID- 7553532 TI - The microscopic interaction between silicone and the surrounding tissues. AB - Biocompatibility is a pivotal issue in the use of any implantable biomaterial. This article describes some of the issues surrounding the use of silicone implants for first metatarsophalangeal joint prostheses. The focus is on the interactions between the implant and the surrounding tissues. Common histologic findings are also discussed. PMID- 7553533 TI - Economic implications of implant selection. AB - Numerous types of implantable biomaterials are available for a variety of applications. Although much has been written about the physical properties or biocompatibility issues, very few papers have focused on the economic feasibility of these materials. This article assesses financial factors associated with first metatarsophalangeal total joint prostheses. PMID- 7553534 TI - Implantable bone substitute materials. AB - This article focuses on materials used as bone substitutes. The materials may be used as substitutes for autografts or, in some cases, along with autografts. Each material has unique properties that may be beneficial for specific applications. Some future developments in bone substitute materials are also discussed. PMID- 7553535 TI - Metallic implants used in foot surgery. AB - Metallic materials have provided a tremendous advantage in the current treatment of many surgical specialties. Further studies on the long-term effects at both local and distant sites of implantation are essential. As this field continues to expand, the podiatric applications of these new devices will grow exponentially. Because all metallic implants in the human body will corrode, several recommendations can be made: 1. Beware of placing reactive components in the younger population. These should be followed accordingly because of latent periods of reported tumor findings in these metals. 2. Consideration should be made toward removal of implant devices when they are no longer effective. 3. The least reactive materials should be considered for implantation. At the present time, titanium alloys have been shown to be more inert than their metallic counterparts. As the search for the ideal human implant material continues, a few important lessons have been learned. First, mixtures of elements such as stainless steel have a higher incidence of antigenicity because they contain a number of different elements, each of which may be an allergen. The smaller the number of elements that are combined to form a metal, the more biocompatible the material will be. Second, certain elements have been found to have increased tissue reactivity, namely nickel, cobalt-chrome, and even vanadium. The less a metal contains these antigenic elements, the less likely the metal will be incompatible. Third, newer "pure" elements with a greater degree of chemical inertness, such as commercially pure titanium and titanium-alloy niobium, are being studied. This ongoing research may lead to a universally biocompatible human implant material that will be used exclusively for all types of implants. PMID- 7553537 TI - Musculoskeletal applications of hyaluronan and hylan. Potential uses in the foot and ankle. AB - Hyaluronan is unusual because it can be used as a highly biocompatible and noninflammatory implant material with mechanical properties that can be customized by altering the polymer configuration. This article suggests that there may be numerous applications in the foot and ankle, including treatments for arthritis, soft-tissue augmentation, and ulcerations. PMID- 7553536 TI - Applications of biodegradable lactides and glycolides in podiatry. AB - A comprehensive review of the clinical applications and the properties of biodegradable lactide and glycolides is presented. Specific podiatric and other orthopaedic applications of polylactic acid, polyglycolic acid, and their copolymers are reviewed also. The authors then discuss numerous biocompatibility studies, basic manufacturing techniques, sterilizations/storage of the polymers, and the development of biodegradable polymers. PMID- 7553538 TI - Future materials for foot surgery. AB - Important advances have been made in the development of biomaterials science and engineering for foot surgery over the past four decades. In this paper, implant materials have been separated into two general categories: temporary implants for bone fixation and permanent implants for joint replacement. As presented, however, currently available temporary implants for bone fixation are often left in place permanently whereas, in the long run, permanent implants for joint replacement cannot realistically be expected to last the lifetime of the average aged patient, and thus are actually only temporary. The benefits and problems of each of these two implant classes were first presented to set the stage for a discussion of possible future directions in the development of new biomaterials that offer the promise of providing improvements for patient care. For bone fixation in foot surgery, the most promising future biomaterials are presented as fully bioabsorbable polymer matrix composites. These implant materials have the potential for development to provide the initial strength and stiffness of currently used metal alloys without concern regarding implant removal. With the development of these materials, clinicians and patients will no longer be forced to choose between the risks of implant retrieval and the risks of leaving the implant behind. Current obstacles that must be overcome before these future materials can be introduced for general clinical use are related to improvements in mechanical property durability and degradation product biocompatibility. For joint replacement, tissue engineered viable biomaterials for permanent articular cartilage replacement are presented as the most important of the future biomaterials. If truly permanent joint replacement materials are to be developed, the implants must be able to regenerate and sustain themselves to permanently retain their properties. Living and sustainable tissues are therefore essential if implant properties are to be permanently maintained, because all nonviable materials are subject to eventual irreversible structural breakdown, degradation, and fatigue. Again, many problems remain to be solved before these envisioned future materials can be brought to accepted clinical use. However, substantial advances have already been achieved and have demonstrated the feasibility of the development of these materials. Biomaterials science and engineering remains a very challenging and exciting field of research and development. As technology advances, the problems that are faced become more complex and, more than ever, now require interdisciplinary cooperation from molecular and cell biologists, biomaterials scientists and engineers, and clinicians. This is especially true in the relatively new field of tissue engineering.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7553539 TI - Mentally disordered offenders and the law. PMID- 7553540 TI - The effects of changes in the law concerning mentally disordered offenders: the Alberta experience with Bill C-30. AB - OBJECTIVE: Until recently, the Criminal Code of Canada, enacted in 1892, stood stalwart to social, political and technological changes, particularly with respect to the regulations pertaining to the management of the mentally ill offender. This became more definitely so with the enactment of the Canadian Charter of Rights and Freedoms (1984) as many regulations in the Code about mentally ill offenders contravened the mandates contained in the Charter. The Supreme Court of Canada's decision on Regina v. Swain spurred the Federal Government to bring the regulations on the mentally ill offender into line with the Charter. The result was the enactment of Bill C-30 which was intended to dramatically change the way in which forensic psychiatry was practised in Canada. This paper presents the Alberta findings from a multi-site evaluation commissioned by the Federal Department of Justice to judge the effects of Bill C 30 on forensic health care practices. METHODS: Health records data were used to compare utilization patterns from the year prior to the enactment of Bill C-30 with the year following. In addition, qualitative data were obtained from key clinical and legal informants outlining implementation difficulties that they had experienced. RESULTS: Results support the judgement that Bill C-30 has not achieved its desired effects with respect to the length of the remand, and has resulted in an increased burden on hospitals and health care providers. In addition, an unanticipated finding was the increased use of the Mental Health Act which was considered to place forensic patients in a position of double jeopardy. PMID- 7553541 TI - Attachment organization and adaptation in sexually-abused women. AB - OBJECTIVE: To explore the possible associations among attachment organization, current functioning and Axis II personality disorder. METHOD: Attachment organization was assessed using the Adult Attachment Interview in a clinical sample of 40 women with a history of childhood sexual abuse. The Global Assessment Scale yielded measures of current psychosocial functioning and the Structured Clinical Interview for Diagnosis on DSM-III-R was used to assess presence of personality disorder. RESULTS: Preoccupation with attachment issues was evident in 68% of the subjects; 60% of the subjects were unresolved in respect to loss and/or trauma; and 88% of the subjects met criteria for one or more Axis II disorders. A relationship between Borderline Personality Disorder and the Unresolved attachment classification is suggested. CONCLUSIONS: Women who have experienced childhood sexual abuse require intervention that focuses on resolution of trauma and loss and responds to individual differences in attitudes toward attachment issues. PMID- 7553543 TI - The role of research in systems reform. AB - Knowledge concerning the care of people with severe and persistent mental illness has grown dramatically, but that knowledge is seldom translated into improvements in care. The authors describe their involvement in three service delivery projects in Ontario and discuss how, by assuming multiple roles, they were able to ensure that planning and policy development were informed by current knowledge. In addition to being an investigator, such roles may include being an administrator, planner, consultant and advocate. The redevelopment of Whitby Psychiatric Hospital resulted in an innovative plan for a comprehensive, integrated service delivery system based upon community support services research. Implementation of the Graham Report in Ontario provided an array of district mental health plans which incorporate current knowledge with regard to service delivery. Ontario's mental health reform strategy represents a close connection between public policy and scientific information. The authors recommend that researchers be prepared to assume multiple roles in the service delivery field so as to reduce the gap between science and practice. PMID- 7553544 TI - Immigrant child and adolescent psychiatric referrals: a five-year retrospective study of Asian and Caucasian families. AB - OBJECTIVE: Referrals to the Bradford (England) Child and Family Psychiatric Clinic were studied over a five-year period with the purpose of comparing native Caucasian and immigrant groups. Punjabi Moslems of Pakistani descent formed a majority of immigrant referrals. METHOD: This immigrant sample was compared with native Caucasians matched for age and sex. RESULTS: Differences between these groups were found in the rates and sources of referral, together with family composition, diagnosis and adherence to treatment. CONCLUSION: These results are discussed in terms of the influence of cultural background and gender socialization. This report makes suggestions with respect to service provision and the assessment of individuals from linguistically unassimilated ethnic minorities. PMID- 7553542 TI - Extended survival of patients on long-term lithium treatment. AB - OBJECTIVE: Findings from a recent international multi-centre trial are compatible with the idea that long-term lithium treatment extends the survival of patients suffering from affective disorders to match the general population. A similar reduction of mortality was found in Canadian patients, although important questions remained to be answered about cardiovascular and suicide mortality, and patient selection. METHOD: Based on data collected in a study (1) from lithium clinics in Canada, Denmark, Germany and Austria, an analysis was carried out of suicide and cardiovascular mortality in patients who received prophylactic lithium treatment. RESULTS: In patients given lithium for two years or longer (n = 641), both suicide and cardiovascular mortality were the same as, or only slightly higher than, in the general population; in patients given lithium for less than two years (n = 186), both mortalities remained high. The reduced mortality is not likely to be the result of selection because the patients who were treated briefly and those treated for a longer time did not differ in important mortality variables. CONCLUSIONS: In addition to its ability to prevent recurrences, prophylactic lithium treatment appears capable of reducing both the excess suicide risk and excess cardiovascular mortality of affective illness. PMID- 7553548 TI - Cultural variations in symptom attribution. PMID- 7553546 TI - Self-reported depressive symptoms in association with medication exposures among medical inpatients: a cross-sectional study. AB - OBJECTIVE: To evaluate associations between exposure to three classes of medications (angiotensin converting enzyme inhibitors, calcium channel blockers, and corticosteroids) and self-reported depressive symptoms. METHOD: The study utilised a cross-sectional study design in a sample of medical inpatients. RESULTS: Associations between self-reported depressive symptoms and exposure to angiotensin converting enzyme inhibitors or calcium channel blockers were not observed. However, an association between self-reported depressive symptoms and corticosteroid exposure was identified. The association was strongest in subjects reporting a past history of depression or a family history of depression. The strength of the corticosteroid-depressive symptom association was comparable with that of associations observed for age, poverty and psychosocial stress. CONCLUSIONS: Depressive symptoms among medical inpatients have a biopsychosocial etiology. Corticosteroid exposure may be a biological risk factor for depressive symptoms in this population. PMID- 7553547 TI - A comparison of comorbid patterns in treatment-resistant unipolar and bipolar depression. AB - OBJECTIVE: To examine the occurrence of concomitant psychiatric disorders in patients with treatment-resistant unipolar and bipolar depression. METHOD: Forty nine patients participated as subjects. Twenty-four (49%) had unipolar depression and 25 (51%) had bipolar depression using DSM-III-R criteria. Structured clinical interviews were conducted with all patients. Chart reviews and interviews with family members were also carried out. Information relating to both current and lifetime diagnoses was obtained. RESULTS: Of the entire sample, 75.5% were found to have at least one other Axis I diagnosis and 46.9% had at least two additional Axis I diagnoses. The unipolar group had significantly more current comorbid diagnoses. When type of diagnoses was examined, unipolar patients had significantly more anxiety diagnoses at the time of the index episode, and over their entire lifetime. Bipolar patients had significantly more lifetime substance abuse diagnoses. CONCLUSIONS: Axis I comorbidity appears to be differentially associated with treatment resistance in unipolar and bipolar depression. PMID- 7553545 TI - The neurology of alcoholic denial: implications for assessment and treatment. AB - Alcohol has neurotoxic effects that frequently result in significant sensorimotor and cognitive deficits. These cognitive deficits may have profound implications for the behaviour and treatment of patients who abuse alcohol. In particular, the deficits in executive cognition that are typical of alcoholic dementia result in difficulties with planning, insight and impulse control. These deficits are frequently misinterpreted as alcoholic denial and are therefore assumed to have a psychodynamic basis. This paper reviews the neurological substrates for insight and self-monitoring and discusses a possible pathophysiology for a subgroup of alcoholic patients who exhibit alcoholic denial. Implications of this model for the evaluation and treatment of alcoholic patients are discussed. PMID- 7553549 TI - A case of inanimate doubles syndrome. PMID- 7553550 TI - New classes of antidepressants that may be lethal in combination. PMID- 7553551 TI - Re: To the memory of Dr. Bruno M. Cormier. PMID- 7553552 TI - [Education of nurse practitioners in Ontario]. PMID- 7553553 TI - Nurse replacement epidemic. Interview by Paula Hickey Guerette. PMID- 7553554 TI - [Heart transplantation]. PMID- 7553555 TI - Teaching i.m. administration by visualization. PMID- 7553556 TI - Heparin has its risks. PMID- 7553558 TI - Valuing life and death. PMID- 7553559 TI - Euthanasia: mercy, morals and medicine. AB - The case of Sue Rodriguez has made it painfully clear that it is the ethical responsibility of every nurse to become involved in the debate on euthanasia. If advocacy is the essence of nursing,as has been suggested, then that advocacy must extend to clients such as Rodriguez. It is our responsibility to help our clients reach decisions that are truly their own, to help them determine the unique meaning that health, illness, suffering and dying have for them as individuals. It is also our responsibility to assist our clients to exercise their freedom of self-determination. To participate fully in this debate it is essential that we examine our own beliefs, determine how they concur or conflict with our role as client advocates, and how they apply to this issue. PMID- 7553560 TI - Euthanasia and assisted suicide: what does the law say? PMID- 7553557 TI - When is it right to die? AB - Advances in technology and the increased awareness and activism of health care consumers have brought a heightened focus to the ethical questions associated with end-of-life decision-making in health care. This attention is manifested in increased discussion, both in the literature and informally, of end-of-life issues; in increased numbers of advanced directives; and, recently in Canada, in the formation of the Senate Committee on Euthanasia and Assisted Suicide whose charge was to develop policy on these issues. Nurses have valuable perspectives to add to this discourse. PMID- 7553561 TI - Testicular self-examination (TSE). AB - The most common form of cancer and the leading cause of death in North American males between the ages of 15 and 35 is testicular cancer. And while the number of cases has increased dramatically in the past 25 years, the reason for the increase remains unclear. What is clear is that the majority of men are not aware of the risk factors and symptoms and do not know how to perform a testicular examination. Of those who are aware of the disease, few examine themselves on a regular basis. Health professionals, including nurses, have done much to promote breast self examination (BSE) in women. Yet the education of young men in the desirability of monthly testicular self examination (TSE) has been largely neglected. PMID- 7553563 TI - Trauma survivor. PMID- 7553562 TI - [University and community health center collaboration]. AB - Nurses from a CLSC in Hull, Quebec are defining a positive role for community health nurses in a multidisciplinary environment. They base their professional day-to-day practice on Orem's conceptual nursing framework. Many of these home care nurses studied in a post-RN baccalaureate nursing program at the University of Quebec in Hull, where they learned different aspects of conceptual frameworks and the nursing process. Two nurse clinicians and a nurse administrator submitted a project based on Orem's model to the administrators of their community health centre. Once it was approved, a nursing professor from the university agreed to act as a consultant and principal researcher to implement the process. The authors describe this three-year collaborative project between home care nurses and the researcher. This includes its objectives, the participants' roles, the tools developed, the process, and the repercussions on nursing care, nurses, and the multidisciplinary team. PMID- 7553564 TI - Comfort levels with the dying. AB - Most people who know they are near death would prefer to die at home or in a home environment. Currently, hospice and community programs that allow such options are being established in some provinces. However, these programs are relatively new and in short supply. The reality is that palliative care in acute care settings will continue, and most people will die in hospitals and institutions. PMID- 7553566 TI - The dynamics of long term care. AB - The relationship between patient and nurse in long term care is unique because of its longevity. Maintaining such symbiotic relationships, both personally and professionally, requires a sustained effort. PMID- 7553565 TI - Not a business matter. AB - Some Canadians believe that turning Canada's public health care system into a privately based operation is the panacea for the health care debt. The Alberta government is at the forefront of this movement toward a privately managed, business-oriented health system. PMID- 7553567 TI - The move to regionalization. AB - Who should decide how health care services are funded and delivered in our communities? Increasingly, provincial governments are placing that responsibility on the shoulders of Regional Health Authorities (RHAs). But what are regional health authorities and why are they being embraced so enthusiastically across the country and around the world? PMID- 7553570 TI - Chancroid and Haemophilus ducreyi: an update. AB - Haemophilus ducreyi is a fastidious gram-negative bacillus that causes the sexually transmitted infection chancroid. Chancroid is a major genital ulcerative disease in Africa, Southeast Asia, the Caribbean, and Latin America and is of increasing concern in the United States. Genital ulcerative disease and chancroid in particular have been associated with facilitating the transmission of human immunodeficiency virus. The diagnosis of chancroid based on the clinical appearance of the genital lesion or on the isolation of H. ducreyi on selective medium is relatively insensitive. However, recent advances in nonculture diagnostic tests have enhanced our ability to diagnose chancroid. There has been renewed interest in understanding the pathogenesis of H. ducreyi. In vitro and in vivo models have been developed to help identify important virulence determinants. Through the use of biochemical and molecular techniques, macromolecular components that may be important in virulence have been identified. PMID- 7553568 TI - Cutaneous defenses against dermatophytes and yeasts. AB - Predispositions to the superficial mycoses include warmth and moisture, natural or iatrogenic immunosuppression, and perhaps some degree of inherited susceptibility. Some of these infections elicit a greater inflammatory response than others, and the noninflammatory ones are generally more chronic. The immune system is involved in the defense against these infections, and cell-mediated immunity appears to be particularly important. The mechanisms involved in generating immunologic reactions in the skin are complex, with epidermal Langerhans cells, other dendritic cells, lymphocytes, microvascular endothelial cells, and the keratinocytes themselves all participating in one way or another. A variety of defects in the immunologic response to the superficial mycoses have been described. In some cases the defect may be preexistent, whereas in others the infection itself may interfere with protective cell-mediated immune responses against the organisms. A number of different mechanisms may underlie these immunologic defects and lead to the development of chronic superficial fungal infection in individual patients. Although the immunologic defects appear to be involved in the chronicity of certain types of cutaneous fungal infections, treatment of these defects remains experimental at the present time. PMID- 7553572 TI - Laboratory-associated infections and biosafety. AB - An estimated 500,000 laboratory workers in the United States are at risk of exposure to infectious agents that cause disease ranging from inapparent to life threatening infections, but the precise risk to a given worker unknown. The emergence of human immunodeficiency virus and hantavirus, the continuing problem of hepatitis B virus, and the reemergence of Mycobacterium tuberculosis have renewed interest in biosafety for the employees of laboratories and health care facilities. This review examines the history, the causes, and the methods for prevention of laboratory-associated infections. The initial step in a biosafety program is the assessment of risk to the employee. Risk assessment guidelines include the pathogenicity of the infectious agent, the method of transmission, worker-related risk factors, the source and route of infection, and the design of the laboratory facility. Strategies for the prevention and management of laboratory-associated infections are based on the containment of the infectious agent by physical separation from the laboratory worker and the environment, employee education about the occupational risks, and availability of an employee health program. Adherence to the biosafety guidelines mandated or proposed by various governmental and accrediting agencies reduces the risk of an occupational exposure to infectious agents handled in the workplace. PMID- 7553569 TI - Recovery of uncommon bacteria from blood: association with neoplastic disease. AB - Table 6 is a summary of the organisms discussed with a listing of the environmental source, the endogenous source, the predisposing factors including neoplasms, and the postulated mechanisms by which the organism can gain access to the circulation. The evidence considered indicates that the entrance of one of these microorganisms into the bloodstream of a human being depends on the presence of multiplicity of predisposing factors. In the majority of cases of bacteremia due to one of these unusual organisms, two or more predisposing factors are present. Certain predisposing factors, such as cancer chemotherapy or intravenous catheterization, often provide a barrier break, while others, such as liver disease, may render the host immune system less capable of clearing organisms from the circulation. For organisms such as Campy-lobacter, Listeria, and Salmonella spp., attributes that allow the invasion of a healthy host are present and seem to be enhanced by the simultaneous presence of a predisposing condition, such as liver disease, in the host. Although somewhat fragmentary, a number of individual case reports describe bacteremia due to one of these organisms occurring weeks to years after surgery and after other therapeutic measures had effected a supposed cure of a cancer. It may be speculated that cancer patients, even after a cure, are still susceptible to bloodstream invasion by one of the aforementioned organisms by virtue of the presence of one or more predisposing metabolic, physiologic, or immunologic factors, even though these factors may be cryptic. The predominance of hematologic malignancies among cases of bacteremia due to these unusual organisms is also apparent. Although, as pointed out by Keusch (169), the reduction in the performance of immune function in hematologic malignancies compared with solid tumors is likely to be responsible, other associations of certain organisms with specific neoplasms warrant further examination. The frequency of bloodstream infections of Salmonella typhimurium and Capno-cytophaga canimorsus in Hodgkin's disease patients seems likely due to a particular mechanism which infection by these species is favored. The specific nature of these mechanisms remains to be determined. The recovery of any unusual bacterium from blood should warrant a careful consideration of the possibility of underlying disease, especially cancer. Microbiologists should advise clinicians of the unusual nature of the identified organism and provide the counsel that certain neoplastic processes, often accompanied by neutropenia, render the human host susceptible to invasion by almost any bacterium. The recovery of such organisms as C. septicum or S. bovis should prompt the clinician to aggressively seek to identify an occult neoplasm if one has not yet been diagnosed. PMID- 7553573 TI - Nucleic acid vaccines. AB - The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines. Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native conformation, glycosylation profiles, and other posttranslational modifications that mimic antigen produced during natural viral infection. Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocyte responses to diverse protein antigens. Advantages of nucleic acid-based vaccines include the simplicity of the vector, the ease of delivery, the duration of expression, and, to date, the lack of evidence of integration. Further studies are needed to assess the feasibility, safety, and efficacy of this new and promising technology. PMID- 7553571 TI - Interaction of pathogenic neisseriae with nonphagocytic cells. AB - The ability to interact with nonphagocytic cells is a crucial virulence attribute of the meningococcus and the genococcus. Like most bacterial pathogens, Neisseria meningitidis and Neisseria gonorrhoeae initiate infections by colonizing the mucosal epithelium, which serves as the site of entry. After this step, both bacteria cross the intact mucosal barrier. While N. gonorrhoeae is likely to remain in the subepithelial matrix, where it initiates an intense inflammatory reaction, N. meningitidis enters the bloodstream, and eventually the cerebrospinal fluid to cause meningitis. Both pathogens have evolved very similar mechanisms for interacting with host cells. Surface structures that influence bacterium-host interactions include pili, the meningococcal class 5 outer membrane proteins or the gonococcal opacity proteins, lipooligosaccharide, and the meningococcal capsule. This review examines what is known about the roles these structures play in bacterial adhesion and invasion, with special emphasis, on pilus-mediated adhesion. Finally, the importance of these structures in neisserial pathogenesis is discussed. PMID- 7553577 TI - The characterization of chromosomal rearrangements by a combined micro-FISH approach in a patient with myelodysplastic syndrome. AB - We report the application of a combined strategy: chromosome microdissection, degenerate oligonucleotide primed-PCR, and reverse chromosome painting (micro FISH), as well as forward chromosome painting, for the characterization of chromosomal rearrangements in a MDS patient with the karyotype 46,XX, -11, +r analyzed by G-banding. The karyotype was refined as 46,XX,der(2)t(2;11) (q35;?p13),der(11)dic r(11)(:p13-->q13::p13-->q13:). Our study demonstrated that the chromosome composition of a neoplasia can be identified more systematically and accurately using these combined molecular cytogenetic approaches. The DOP-PCR methodology modified is suitable for the practical application of micro-FISH on specimens prepared for routine banding analysis. PMID- 7553576 TI - Cutaneous manifestations of opportunistic infections in patients infected with human immunodeficiency virus. AB - Bacillary angiomatosis (BA) presents most commonly as a cutaneous disease and is caused by two organisms. Bartonella (Rochalimaea) henselae and Bartonella (Rochalimaea) quintana. Biopsy confirmation of cutaneous BA is essential because lesions can mimic nodular Kaposi's sarcoma in appearance. Although the vast majority of human immunodeficiency virus (HIV)-infected patients with BA have CD4 lymphocyte counts of less than 100 cells per mm3, the disease responds well to antimicrobial therapy. Staphylococcus aureus is the most common bacterial skin pathogen affecting HIV-infected patients. The prevalence of skin disease due to S. aureus may be explained by high nasal carriage rates for the organism ( > or = 50%) and altered immune function in conjunction with an impaired cutaneous barrier. Herpes simplex virus causes mucocutaneous disease early in the course HIV infection and ulcerative lesions at any site in advanced HIV infection. Herpes zoster is common early in the course of HIV infection; recurrent and disseminated herpes zoster infections are characteristic of patients with advanced HIV disease. Acyclovir resistance is usually seen in patients with large, untreated, ulcerative lesions of herpes simplex virus and in patients with chronic, verrucous lesions of varicella-zoster virus. Cutaneous cryptococcosis, histoplasmosis, and coccidiomycosis are markers of disseminated disease and require biopsy confirmation. Scabies is easily diagnosed but may be atypical in presentation and difficult to eradicate in advanced HIV disease. PMID- 7553578 TI - Additional copies of 1q in sequential samples from a phyllodes tumor of the breast. AB - Cytogenetic and fluorescent in situ hybridization (FISH) analysis has been performed on consecutive samples, taken 4 weeks apart, from a phyllodes breast tumor. This revealed the presence of two different chromosome 1 derivatives, namely a dic(1;10)(q10;q24) in the first sample and an i(1) (q10) in the second. In one cell out of 25 from the second sample both derivative chromosomes were seen. A chromosome 21 was lost in both samples. These results are consistent with phyllodes tumors having a clonal origin. PMID- 7553580 TI - A recurrent translocation, t(3;11)(q21;q13), found in two distinct cases of acute myeloid leukemia. AB - We report two cases of acute myeloid leukemia (M1 and M5B subtypes) with a similar translocation, t(3;11)(q21;q13). We discuss the involvement of these breakpoints in acute leukemia and their putative clinical implications. PMID- 7553579 TI - Fluorescence in situ hybridization (FISH) for retrospective detection of trisomies 3 and 7 in multiple myeloma. AB - The malignant plasma cells of multiple myeloma (MM) have a low proliferative activity and therefore cytogenetic studies of the disease have been severely limited. We evaluated the role of fluorescence in situ hybridization (FISH) in the detection of numerical chromosomal abnormalities in early stages of myeloma and the applicability of the method to stored archival slides. Old air-dried bone marrow smears from 15 myeloma patients obtained at presentation were probed with alpha satellite DNA sequences to chromosomes 3 and 7. Numerical chromosome aberrations were found in eight (53%) of the patients, including six (of 12) with trisomy 7, and two (of eight) with trisomy 3. This study demonstrates that FISH is a sensitive method for the detection of numerical aberrations in myeloma and for the study of old slides for retrospective analysis. PMID- 7553581 TI - Chromosome rearrangements at telomeric level in hematologic disorders. AB - Following retrospective screening of our karyotype data from 414 consecutive non childhood, neoplastic, and preneoplastic hematologic diseases, we have isolated 11 cases with alterations involving one or two chromosome termini, including: a) nonclonal telomeric telomeric associations (tas), b) subclonal terminal rearrangements consisting of additional (add) material of unknown origin fused at the end of the chromosome, c) clonal telomere-centromere fusion (t telcen) with pseudodicentric structure. Most of these abnormalities were present in karyotypes with multiple alterations and associated to an evolutive stage of the disease (9 of 94 cases studied in progression, including three of 22 CML studied in blast crisis). The immunophenotype of the cell populations was lymphoid in eight cases, six of which were NHL, and myeloid, erythroid, and undifferentiated in the other three. More data on telomeric abnormalities may clarify whether there is ubiquitous genomic instability of neoplastic cells or an inborn cell lineage predisposition favoring rearrangements involving telomeres. PMID- 7553574 TI - Bacterial pyrogenic exotoxins as superantigens. AB - The recent discovery of the mode of interaction between a group of microbial proteins known as superantigens and the immune system has opened a wide area of investigation into the possible role of these molecules in human diseases. Superantigens produced by certain viruses and bacteria, including Mycoplasma species, are either secreted or membrane-bound proteins. A unique feature of these proteins is that they can interact simultaneously with distinct receptors on different types of cells, resulting in enhanced cell-cell interaction and triggering a series of biochemical reactions that can lead to excessive cell proliferation and the release of inflammatory cytokines. However, although superantigens share many features, they can have very different biological effects that are potentiated by host genetic and environmental factors. This review focuses on a group of secreted pyrogenic toxins that belong to the superantigen family and highlights some of their structural-functional features and their roles in diseases such as toxic shock and autoimmunity. Deciphering the biological activities of the various superantigens and understanding their role in the pathogenesis of microbial infections and their sequelae will enable us to devise means by which we can intervene with their activity and/or manipulate them to our advantage. PMID- 7553583 TI - An eight-way variant t(15;17) in acute promyelocytic leukemia elucidated using fluorescence in situ hybridization. AB - A complex eight-way translocation was identified, with the aid of fluorescence in situ hybridization (FISH), in a patient diagnosed as having acute promyelocytic leukemia (APL). The balanced translocation was defined as 46,XY,t(1;6;7;6;17;15;12;3) (p22;q27;p15;q13;q21;q22;q13;p13), which includes a der(15) chromosome consistent with the der(15) chromosome of the t(15;17)(q22;q21) typically found in APL. The patient was treated with all-trans retinoic acid (ATRA) and had a clinical course typical of the disease, which is currently in remission after an autologous bone marrow transplant. The other structural rearrangements appeared to have little effect on the biology of the neoplasia. PMID- 7553575 TI - Human antiprotozoal therapy: past, present, and future. AB - Human protozoal infections are ubiquitous and occur worldwide. In many cases, antiprotozoal agents currently in use predate the modern antibiotic era. Despite the relative lag in development of new antiprotozoal agents, the 1990s have witnessed an increasing level of interest in these infections, inspired by international travel and immigration, a growing awareness of antiprotozoal drug resistance, and the significance of acute and recrudescent protozoal infections in immunosuppressed hosts. This review summarizes for nonclinician readers the past, present, and future therapies for common human protozoal infections, as well as pharmacologic mechanisms of action and resistance and common toxicities associated with these agents. PMID- 7553582 TI - Heterogeneity, polyploidy, aneusomy, and 9p deletion in human glioblastoma multiforme. AB - The short arm of chromosome 9 is frequently deleted in malignant gliomas. We used locus-specific probes for interferon-A (IFNA) and D9S3 in combination with a chromosome 9 centromeric probe to detect genetic aberrations on a cell-by-cell basis in touch preparations of 30 glioblastomas by fluorescence in situ hybridization. Seven (23%) of 30 tumors had deletions in > 70% of cells; the IFNA locus was deleted in all seven, but the D9S3 locus was deleted in only five of the seven. The latter data confirm that a tumor suppressor gene on 9p relevant to glioblastoma multiforme lies between D9S3 and IFNA. Eleven tumors had deletions in 20-40% of cells, more than three standard deviations above the level in control tissues. The remaining tumors had deletions in < 20% of cells. The seven tumors with the lowest percentage of deleted cells each had more than one genetically abnormal population of cells. In total, 10 cases were of this type (i.e., aneusomic for chromosome 9). Three of these 10 tumors had hybridization patterns consistent with polyploidy. PMID- 7553584 TI - Translocation (2;14)(p13;q32) in CD10+ ;CD13+ acute lymphatic leukemia. AB - The rare t(2;14)(p13;q32) was previously described in the three pediatric patients with acute lymphatic leukemia. In these cases this abnormality was found at diagnosis, manifested the sole chromosomal abnormality, and was associated with a favorable prognosis. We here describe three cases of leukemia where such translocations were found at relapse, were associated in two of the cases with additional known characteristic chromosomal aberration, and were associated with a grave prognosis. Interestingly enough, the malignant cells of all three patients shared the same surface antigens: CD34, HLA DR, CD10, CD20, and the myeloid marker CD13. The leukemic clone exhibiting t(2;14) probably evolved from a t(1;19)6q- pre-B acute lymphatic leukemia in one of the cases, and from a chronic phase Ph1 chromosome in another. The significance of the translocation and the coexistence of CD10 and CD13 on the same cell are discussed. PMID- 7553585 TI - der(1;15)(q10;10): a nonrandom chromosomal abnormality of myeloid neoplasia. AB - The occurrence of an unusual karyotypic abnormality der(1;15)(q10;q10) is reported in three patients, one with acute megakaryoblastic leukemia and two with myelodysplastic syndrome. A literature review shows that this cytogenetic abnormality is a rare but nonrandom change in myeloid neoplasia/neoplasia. PMID- 7553586 TI - Expression of the ROS1 oncogene for tyrosine receptor kinase in adult human meningiomas. AB - Oncogenes have been implicated in the promotion and progression of cancer in humans. Expression of the ROS1 oncogene, a member of the receptor tyrosine kinase superfamily, was examined in human meningiomas by coupled reverse transcription and polymerase chain reaction (RT-PCR) assays. Two sets of region-specific oligonucleotides, specific for different regions of the ROS1 messenger ribonucleic acid (mRNA), were used in RT-PCR assays to independently examine ROS1 transcripts from primary human meningiomas. ROS1 was expressed at high levels in approximately 55% (17 of 31) of the meningiomas examined, but not expressed in non-neoplastic brain samples. The commonplace expression of the ROS1 oncogene in meningiomas suggests a role for this oncogene in the etiology of these tumors. PMID- 7553587 TI - UV-induced melanoma. A karyotype with a single translocation is stable after allografting and metastasis. AB - Metastatic melanoma cell lines were derived from a lymph node of a laboratory opossum, Monodelphis domestica, which had been exposed to mid-wavelength ultraviolet radiation (UVB) initially as a suckling young, and subsequently as a shaved juvenile and adult. The melanoma cell lines were dendritic and pigmented in vitro and contained a t(6;8)(p13;q13) as the only cytogenetic abnormality. The translocation was detected in 15% of primary cultures (passage 2) from the affected lymph node and in 100% of two ring-clone-derived lines, L1 and L2. The breakpoint or resulting partial trisomy of chromosomes 8 may have played a functional role in the tumorigenesis or metastasis of the tumor. The t(6;8) served as a convenient cytogenetic marker for allogeneic grafting studies in Monodelphis. The L2 cells were allografted subcutaneously (s.c.) into genetically diverse suckling young at 3 weeks of age and resulted in the growth of invasive, pigmented, primary and metastatic lesions affecting lymph nodes, lung, and other tissues. Metastatic variant cell lines, M1 and M3, were derived from the affected lungs of two animals and both lines demonstrated the same t(6;8), without additional numerical or structural chromosomal abnormalities. The maintenance of karyotypic stability with a single translocation during in vivo tumor growth and dissemination in this new allografting model is quiet remarkable, as most human metastatic melanomas exhibit multiple structural and numerical cytogenetic abnormalities. PMID- 7553589 TI - Another case of trisomy 4 with double minute chromosomes in acute non-lymphocytic leukemia. AB - A further case of trisomy 4 with double minute chromosomes in acute non lymphocytic leukemia is reported. The non-random association between these two cytogenetic abnormalities is reinforced. A possible relation with environmental exposure is discussed. PMID- 7553590 TI - Low-grade astrocytoma with a complex four-breakpoint inversion of chromosome 8 as the sole cytogenetic aberration. AB - We report a case of a low-grade astrocytoma in a 10-year-old boy in which the sole cytogenetic aberration was a complex four-breakpoint inversion of chromosome 8 with the karyotype designation 46,XY,der(8)inv(8)(p23q24)inv(8)(q11q21). Two protooncogenes on chromosome 8, MOS at 8q11 and MYC at 8q24, are located at or near the bands which correspond to two of the breakpoints in this inversion. The localization of the structural aberrations to four breakpoints on chromosome 8 provides a rare example of a solid tumor with structural aberrations limited to a single chromosome. PMID- 7553588 TI - Allelic status of chromosome 1 in neoplasms of the nervous system. AB - By using five highly polymorphic markers, the allelic status of chromosome 1 was established in a series of 236 tumors of the nervous system, including all major histologic subtypes: gliomas, meningiomas, neurinomas, neuroblastomas, medulloblastomas, etc. Loss of alleles at 1p was observed at significant frequencies in neuroblastomas (26% of cases), meningiomas (32%), and malignant gliomas (37%) (primarily oligodendrogliomas [94%]). This anomaly was also detected in two of 23 neurinomas, two of three neurofibrosarcomas, one primary lymphoma, and two metastatic tumors of the brain. The analysis of tumors displaying partial 1p deletions suggests the existence of two distinct regions, 1p36 and 1p35-p32, in which loci nonrandomly involved in the development of neurogenic neoplasms might be located. PMID- 7553591 TI - Mouse chromosome 14 is altered in different metastatic murine neoplasias. PMID- 7553593 TI - The pathogenetic significance of acquired trisomy 8 is not reducible to amplification of a single chromosome band. PMID- 7553592 TI - Is i(1)(q10) a chromosome marker in phyllodes tumor of the breast? PMID- 7553594 TI - An XYY male with acute lymphoblastic leukemia. PMID- 7553595 TI - Cytogenetics of 158 patients with regional or disseminated melanoma. Subset analysis of near-diploid and simple karyotypes. AB - We report on the cytogenetic analyses of 158 cases of metastatic malignant melanoma, comprised of 63 cases with regional disease (RD) and 95 cases with distant (metastatic) disease (DD). Clonal structural abnormalities were identified in 126 (80%) cases and were significantly increased ( < 0.01 after adjusting for multiple comparisons) on chromosomes (in order of frequency of involvement) 1, 6, 7, 11, 9, and 3. Clustering of breakpoints occurred at 1p36, 1p22-q21, 6p11-q21, 9p, 11q23-qter, 13p (especially for cases with DD), and 19q13. The most common clonal numerical abnormalities, in a subset of 49 near diploid cases were -10, -22, -9, +7, -19, and -Y. Analysis of chromosome segment gains and losses (CSRP) showed frequent loss of chromosomes 6 and 10, followed by equal rates of involvement of chromosomes 1, 7, and 9. Whole or segmental losses of chromosome 9 (especially 9p) correlate well with recent molecular genetic studies identifying putative suppressor genes, and are also likely important genetic abnormalities. However, based on the frequency of abnormalities in this large series of metastatic melanomas, it is likely that structural abnormalities of 1 and 6, and 10 are important in the pathogenesis of sporadic advanced melanoma. PMID- 7553597 TI - Gene therapy of hepatoma: bystander effects and non-apoptotic cell death induced by thymidine kinase and ganciclovir. AB - A retroviral vector carrying herpes simplex virus thymidine kinase gene was constructed, and transfected into the psi 2 packaging cells. The replication defective retrovirus produced by this cell line (psi 2 tkn cells) was transduced into XC rat hepatoma cells, from which a cell line (XCtkn2) highly sensitive to ganciclovir was cloned. Ganciclovir suppressed the growth of XCtkn2 hepatoma and psi 2tkn cells. Both of these HSV-tk-carrying cells treated with ganciclovir showed potent 'bystander effect' on co-culturing with genetically unmodified XC hepatoma cells. In addition, intratumoral injection of XCtkn2 and psi 2tkn cells into the XC hepatomas transplanted in nude mice and subsequent ganciclovir administration suppressed in vivo growth of the hepatomas. Flow cytometry disclosed that the ganciclovir-treatment increased the relative number of XCtkn2 hepatoma and psi 2tkn cells at the G2 phase of the cell cycle. However, the nuclear fragmentation and internucleosomal DNA cleavage were not observed, indicating that the death of XCtkn2 hepatoma and psi 2tkn cells treated with ganciclovir was not apoptotic. PMID- 7553596 TI - A labile hyperphosphorylated c-Fos protein is induced in mouse fibroblast cells treated with a combination of phorbol ester and anti-tumor promoter curcumin. AB - Curcumin is a potent inhibitor of tumor promotion, and was shown previously to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity. The c Fos protein is inducible by TPA and thus is associated with c-Jun to result in an increased AP-1 activity in mouse fibroblast cells. We therefore hypothesized that c-Fos may be one of the targets of curcumin action. In the present study, the effects of curcumin on TPA-induced c-fos mRNA and protein levels were determined by RNA hybridization and western blot analysis, respectively. Curcumin decreases the TPA-induced nuclear abundance of c-Fos protein in spite of the slight super induction of c-fos mRNA. Upon TPA stimulation, the amount of c-Fos in the quiescent cells increases and reaches maximum at 30 min, and then progressively disappears over a period of 60 min. However, the c-Fos protein seems susceptible to rapid degradation by 45 min if NIH 3T3 cells were treated with TPA in the presence of curcumin. The curcumin-induced hyperphosphorylated forms of c-Fos proteins are significantly more unstable; they entirely disappeared within 40 min after incubation at 37 degrees C. These findings prompted us to suggest that the decrease of c-Fos protein could account for the repressed in vitro DNA binding probably by reducing the Jun/Fos complex formation. PMID- 7553598 TI - Antibodies against murine double minute-2 (mdm2) in sera of patients with various gynaecological diseases. AB - We performed a serologic analysis for anti-murine double minute-2 (anti-mdm2) antibodies in sera of patients with different gynaecological diseases using immunoblotting technique with recombinant mdm2 as antigen. In addition, for large scale screening we established an anti-mdm2 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with breast cancer, ovarian carcinoma, cervix carcinoma and benign gynaecological tissue alterations were tested. We detected antibodies specific for mdm2 in sera derived from cancer patients, as well as from patients with non-malignant diseases. Some of the sera with antibodies against mdm2 also contained antibodies against the growth suppressor gene product p53. PMID- 7553599 TI - Expression of NOR-1 and its closely related members of the steroid/thyroid hormone receptor superfamily in human neuroblastoma cell lines. AB - Previously, we isolated a cDNA of neuron derived orphan receptor (NOR-1), a putative transcription factor with strong homologies to the orphan nuclear receptors NGFI-B and NURR1. In the present study, we examined the gene expression of NOR-1 as well as NGFI-B and NURR1 in human neuroblastoma cell lines by reverse transcription-polymerase chain reaction and nucleotide sequencing. Although the mRNAs of these orphan receptors were detected in all six neuroblastoma cell lines examined, basal expression levels of these genes varied among cell lines. Treatment with forskolin and 12-O-tetradecanoylphorbol-13-acetate rapidly increased the expression of all these genes in neuroblastoma NB-OK-1 cells. This induction did not require de novo protein synthesis, indicating that the NOR-1 gene as well as NGFI-B and NURR1 genes is an immediate-early gene. This is the first demonstration of NOR-1 gene expression in tumor cell lines. PMID- 7553600 TI - Interaction of tamoxifen with cytosolic and nuclear type II estrogen binding sites (type II EBS). AB - The aim of this study was to investigate the interaction of tamoxifen (TAM) with the so-called Type II estrogen binding sites (Type II EBS) in both the cytosolic and the nuclear fraction of the ER-negative A 2780 human ovarian cancer cell line and in an ER-negative ovarian cancer tissue. Although cytosolic and nuclear Type II EBS in A 2780 cells showed substantially similar binding characteristics in terms of ligand affinity and specificity, TAM, while exhibiting the ability to displace [3H]estradiol from cytosolic Type II EBS failed to interact with nuclear Type II EBS. The ability of TAM to interact only with cytosolic Type II EBS seems also to be a characteristic of ovarian cancer tissue and to be shared by several TAM metabolites. The hypothesis that the interaction of TAM with cytosolic Type II EBS could mobilize the true endogenous ligand of Type II EBS which would become available for binding to nuclear Type II EBS was tested by incubating the nuclear fraction with the cytosolic fraction. In the presence of cytosol, TAM acquires the ability to displace the tracer from nuclear Type II EBS but when the cytosolic fraction was DCC, stripped in order to remove the endogenous ligand, the competing activity of TAM for nuclear Type II EBS was abolished. Our results suggest that TAM does not interact with nuclear Type II EBS, but can favor the nuclear binding of endogenous ligand by displacing it from cytosolic Type II EBS. PMID- 7553601 TI - Changes in lipoxygenase activities in human erythroleukemia (HEL) cells during diosgenin-induced differentiation. AB - A human erythroleukemia (HEL) cell line was used as a model to study dynamic changes in human 12-, 15-, 5-lipoxygenases, five lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase gene expression during megakaryocytic differentiation induced by diosgenin (Beneytout, J.L., Nappez, C., Leboutet, M.J. and Malinvaud, G., Biochem. Biophys. Res. Commun., 207 (1995) 398-404). The study was performed at the transcriptional level: 12- and 5-lipoxygenase mRNAs, FLAP mRNA and LTA4 hydrolase mRNA were detected before and after diosgenin treatment in HEL cells while 15-lipoxygenase mRNA was undetected. When HEL cells were incubated with arachidonic acid, 5-, 12-, 15-hydroxyeicosatetraenoic acids (HETE) and LTC4 were synthesized. In contrast, the diosgenin treatment induced the suppression of 12-lipoxygenase activity and only 5-, 15-HETEs and LTC4 were synthesized. PMID- 7553603 TI - Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol. AB - Perillyl alcohol has antitumor activity against rat mammary and liver cancer. Here, we report the chemotherapeutic effects of perillyl alcohol on pancreatic cancer. Perillyl alcohol reduced the growth of hamster pancreatic tumors to less than half that of controls (P < 0.025). Moreover, 16% of perillyl alcohol-treated pancreatic tumors completely regressed whereas no control tumors regressed (P < 0.05). Perillyl alcohol induced contact inhibition in cultured human pancreatic carcinoma cells and inhibited their anchorage-independent growth (P < 0.001). Thus, perillyl alcohol has antitumor activity against pancreatic carcinomas at non-toxic doses, and may be an effective chemotherapeutic agent for human pancreatic cancer. PMID- 7553602 TI - Tamoxifen and somatostatin affect tumours by inducing apoptosis. AB - Tamoxifen is a commonly used chemotherapeutic agent in human breast cancer, although some tumours develop resistance. Somatostatin is also being introduced as an anti-tumour agent. Here we show that the action of these drugs is, at least partly, due to their induction of apoptosis. Both 50 nM somatostatin, and 60 nM tamoxifen significantly enhanced the percentage of cells undergoing apoptosis, when compared to untreated or oestrogen treated control cells. This effect was observed in SK-N-BE(2) human neuroblastoma cells and in MCF-7G human breast cancer cells but not in their drug-resistant counterpart MCF-7A which showed a very low rate of spontaneous programmed cell death. Finally, we propose a simple test of the sensitivity and resistance of individual tumours to these agents by assessing their ability to induce apoptosis in vitro as measured by flow cytometry. PMID- 7553605 TI - Growth inhibition and apoptosis in human neuroblastoma SK-N-SH cells induced by hypericin, a potent inhibitor of protein kinase C. AB - The effect of hypericin, an antiviral agent and inhibitor of protein kinase C (PKC), on cell proliferation and programmed death was investigated in the human neuroblastoma cell line SK-N-SH. Hypericin induced significant growth inhibition in a dose-dependent manner demonstrated by a microculture tetrazolium (MTT) assay. DNA isolated from cells treated with hypericin at concentrations over 1 microM exhibited a 'ladder' pattern of oligonucleosome-sized fragments characteristic of apoptosis. Similarly, treatment of the cells with the PKC inhibitors staurosporine, tamoxifen or phorbol ester PMA for 72 h also resulted in apoptosis, suggesting that hypericin may be triggering an apoptotic signal in neuroblastoma cells, which at least in part may be mediated by the inhibition of PKC. PMID- 7553606 TI - Selective tumorigenesis in non-parenchymal liver epithelial cell lines by hepatocyte growth factor transfection. AB - Hepatocyte growth factor (HGF) has been recently suggested to contribute to tumorigenesis by an autocrine mechanism in fibroblast cells overexpressing its receptor, the MET/HGFR protein. Since epithelial cells represent the primary physiologic target of HGF, we investigated whether inappropriate expression of HGF by epithelial cells which normally express MET/HGFR may also contribute to tumorigenesis. We have transfected a full length rat HGF gene into three mouse epithelial cell lines, one derived from breast (MM55) and two (BNL CL.2 and NMuLi) representing liver non-parenchymal epithelial cells (NPEC). We confirmed the presence of the transfected gene by Southern blot analysis, the production of HGF protein by immunofluorescence, and the preservation of HGF biologic activity by bio-assay. In comparison to untransfected cells, all three HGF-transfected cell lines displayed high level MET/HGFR autophosphorylation and increased ability to proliferate in media containing low serum. The two HGF-transfected liver NPEC lines, but not the HGF-transfected mammary cell line, displayed loss of cell contact growth-inhibition and acquired a markedly increased ability to form colonies in soft agar. Furthermore, the NPEC HGF-transfected cell lines formed much larger tumors in nude mice than the untransfected counterparts, with extensive invasion and sporadic lung metastases. These results demonstrate that overexpression of HGF in at least some epithelial cells contribute to tumorigenesis, and furthermore suggest a possible role for the HGF-MET/HGFR system in the progression of certain epithelial tumors. PMID- 7553604 TI - Effects of three dietary phytochemicals from tea, rosemary and turmeric on inflammation-induced nitrite production. AB - In chronic inflammation, cytokines induce the production of nitric oxide (NO.) that is converted to DNA damaging and carcinogenic peroxynitrite and nitrite. The compounds epigallocatechin gallate (EGCG), carnosol, and curcumin are non-vitamin phytochemicals contained in commonly consumed dietary plants. They are known to be anti-inflammatory and cancer preventive. Therefore, we studied their effect on the generation of peroxynitrite radicals and nitrite. They inhibited lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) induced nitrite production by mouse peritoneal cells by more than 50% at 2.5-10 microM. Cell viability assays verified that the inhibition was not due to general cellular toxicity. PMID- 7553607 TI - Evolving neural networks for detecting breast cancer. AB - Artificial neural networks are applied to the problem of detecting breast cancer from histologic data. Evolutionary programming is used to train the networks. This stochastic optimization method reduces the chance of becoming trapped in locally optimal weight sets. Preliminary results indicate that very parsimonious neural nets can outperform other methods reported in the literature on the same data. The results are statistically significant. PMID- 7553608 TI - Tumor cell growth inhibition by several Annonaceous acetogenins in an in vitro disk diffusion assay. AB - The cell inhibition activities of several Annonaceous acetogenins, covering the three major structural classes of bis-adjacent, bis-non-adjacent, and single tetrahydrofuran (THF) ring compounds and their respective ketolactone rearrangement products, were tested in an in vitro disk diffusion assay against three murine (P388, PO3, and M17/Adr) and two human (H8 and H125) cancerous cell lines as well as a non-cancerous immortalized rat GI epithelial cell line (I18). The results demonstrate a dose-dependent inhibition of cancerous cell growth, while non-cancerous cell growth is not inhibited by the same dosages. All of the acetogenins, irrespective of their various structural types, inhibit the growth of adriamycin resistant tumor cells and non-resistant tumor cells at the same levels of potency. These results show that the Annonaceous acetogenins are an extremely potent class of compounds, and their inhibition of cell growth can be selective for cancerous cells and also effective for drug resistant cancer cells, while exhibiting only minimal toxicity to 'normal' non-cancerous cells. PMID- 7553609 TI - Absence of p53 gene mutations in rat colon carcinomas induced by azoxymethane. AB - The K-ras and p53 genes are two of the most frequently mutated genes found in the human colonic tumors. Since azoxymethane (AOM) induced rat colonic neoplasms are similar to human colonic tumors in their histological features and proliferation characteristics, the rat has been used as an experimental model to study the pathogenesis of colon cancer in humans. Although the presence of K-ras point mutations has been reported in AOM induced rat colonic tumors, there are no reports describing the frequency for mutation of the p53 gene in these tumors. In this study, colon adenocarcinomas induced in rats by AOM were examined for the presence of point mutations in exons 5-8 of the p53 gene, using a combination of single strand conformation (SSCP) analysis, immunohistochemistry and direct DNA sequencing. SSCP analysis showed no differences in banding patterns between the normal mucosa and any of the 20 adenocarcinomas analyzed. Nuclear p53 immunoreactivity was absent in all tumors examined. Since p53 point mutations predominate in malignant colonic tumors, five adenocarcinomas with the greatest local invasiveness were analyzed by direct DNA sequencing of exons 5-8 of the p53 gene. Direct DNA sequencing did not reveal mutations in any of the adenocarcinomas analyzed, within the coding region of p53 gene that were sequenced. The results from the present study indicate that point mutations in the p53 gene, at least in the coding region (exons 5-8) are not involved in the development of colon cancer induced by AOM in the rat. PMID- 7553610 TI - Effects of curcumin on the formation of benzo[a]pyrene derived DNA adducts in vitro. AB - The effects of turmeric (T), curcumins (Cs), aqueous turmeric extract (ATE) and curcumin-free aqueous turmeric extract (CFATE) on the formation of [3H]benzo[a]pyrene ([3H]B(a)P)-derived DNA adducts was studied in vitro employing mouse liver S9. A dose-dependent decrease in binding of [3H]B(a)P metabolites to calf thymus DNA was observed in the presence of T, Cs and ATE but not in the presence of CFATE, suggesting curcumins to be active principles. Further studies employing mouse liver microsomes and individual components of curcumins, i.e. curcumin (C), demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC) showed that all the three components brought about dose-dependent; inhibition of [3H]B(a)P DNA adduct formation and inhibitory activity was in the order C > dmC > bdmC. Investigations on the inhibitory effect of curcumin showed a dose-dependent decrease in cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity resulting in relatively larger amounts of unmetabolized B(a)P in the presence of curcumin. Comparison of structures of curcumins with their activity profile suggested the importance of both parahydroxy (p-OH) and methoxy groups (-OCH3) in the structure activity relationship. Experiments to study the mechanism of action of curcumin indicated that the presence of curcumin was essential for the inhibitory effect, as removal of curcumin resulted in restoration of cytochrome P450 activity and the levels of [3H]-B(a)P-DNA adducts to control values. The present studies demonstrate that curcumin is effective in inhibiting [3H]B(a)P derived DNA adducts by interfering with the metabolic enzymes and its physical presence is essential for this effect. PMID- 7553611 TI - Opposite effect of a cAMP analogue on tumoral growth related to hormone dependence of a murine mammary tumor. AB - Interactions among transcription factors are one of the mechanisms that regulate gene expression. Through protein-protein associations different signaling pathways become connected and the message triggered by each of the molecules involved can modify other related routes. In a murine mammary tumor induced by medroxyprogesterone acetate (MPA), further treatment with this agent showed a different response on tumor growth. In one group of tumors, growth rate was increased (hormone dependent, HD), whereas in the other group the progestin agent failed to modify the rate of tumor development (hormone autonomous, HA). Progesterone receptors (PgR) and estrogen receptors (ER) were expressed in both groups. Administration of 8-CI-cAMP, a cAMP analogue, stimulated tumor growth in the HD subline and inhibited growth in the HA subline. Simultaneous administration of 8-CI-cAMP and MPA resulted in suppression of inhibitory 8-CI cAMP action in the HA tumor subline attributable to changes in molecular configuration of protagonic members of each signaling pathway, whereas in the HD subline growth was additive as if each of the pathways were acting separately. MPA induced down regulation of PgR in both tumor sublines and up regulation of ER in the C4-HD subline. The effect of 8-CI-cAMP alone or associated with MPA was more complex and variations in PgR and ER content by themselves are insufficient to explain changes in tumoral growth. A model consistent with our experimental findings is presented. PMID- 7553612 TI - Postnatal modulation of hepatic biotransformation system enzymes via translactational exposure of F1 mouse pups to turmeric and curcumin. AB - The potential for the transfer of active principle(s) of turmeric (Curcuma longa L.) and curcumin (major pigment in turmeric) via translactational route and its modulatory influence on the hepatic biotransformation system enzymes in the lactating dams and their suckling offspring was assessed. Turmeric (4 g/kg b.w. per day) and curcumin (0.4 g/kg b.w. per day) induced significant (P < 0.01) increase in the hepatic levels of glutathione S-transferase (GST) and acid soluble sulfhydryl (-SH) after 14 or 21 days treatment in lactating dams and translactationally exposed F1 pups. However the lower dose of curcumin (0.2 g/kg b.w. per day) could modulate hepatic GST activity (P < 0.05) and -SH (P < 0.01) only after 21 days of treatment in dams and pups. Cytochrome b5 and cytochrome P450 levels were significantly elevated (P < 0.05) in the dams as well as their suckling pups of both 14 and 21 days age groups at the selected dose levels of turmeric (4 g/kg b.w.) and curcumin (0.4 g/kg b.w.). The induction in hepatic biotransformation system enzymes in lactating dams and F1 progeny suggests the passage of active constituents and/or metabolites of turmeric and curcumin via the translactational route. PMID- 7553613 TI - Increased DT-diaphorase activity in transformed and tumorigenic pancreatic acinar cells. AB - Pancreatic acinar cells from rats treated in vitro with 5-azacytidine and/or transfected with an activated c-H-ras demonstrated transformation and tumorigenic phenotypes. DT-diaphorase (NAD(P)H:quinone oxidoreductase) activity was determined in these non-tumorigenic (3AP) and tumorigenic cells (T3AP and T5AM). T5AM cells were those treated with 5-azacytidine and further treated with N' methyl-N'-nitro-nitrosoguanidine. Higher levels of enzyme activity were found in transformed cells when compared to that in control cells (> 15-fold, 3AP cells; > 40-fold, T3AP cells; > 20-fold T5AM cells). In contrast, NADPH-cytochrome c reductase activity was decreased in transformed cells (> 10-fold, 3AP cells; > 20 fold, T3AP cells; > 10-fold, T5AM cells). These studies demonstrated that pancreatic acinar cells are capable of undergoing alterations in enzyme activity patterns when transformed and that DT-diaphorase may be a good marker for malignant transformation. PMID- 7553614 TI - 2-Methoxy-4-nitroaniline is a selective inducer of cytochrome P450IA2 (CYP1A2) in rat liver. AB - Male F344 rats were treated with a chemical (aniline, nitrobenzene, 2-methoxy-p phenylenediamine, 2-methoxy-4-nitroaniline or 2-methoxy-4-nitroazobenzene) produced by the azo-reduction and/or N-oxidation of 2-methoxy-4-amino-azo benzene, a selective inducer of cytochrome P450IA2 (CYP1A2), and their effects on the induction of CYP1A enzymes in the liver were examined in terms of the protein and mRNA amounts. 2-Methoxy-4-nitroaniline and 2-methoxy-4-nitroazo-benzene, but not other compounds tested, induced CYP1A enzymes, especially CYP1A2, as assessed by Western blotting and Northern blotting. It is noteworthy that 2-methoxy-4 nitroaniline was more selective than 2-methoxy-4-aminoazobenzene for induction of CYP1A2, and it has the smallest molecular size among the known CYP1A2 inducers. PMID- 7553615 TI - Promotion by substance P of gastric carcinogenesis induced by N-methyl-N'-nitro-N nitrosoguanidine in Wistar rats. AB - The effects of prolonged administration of neuropeptide substance P (SP) on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on the labeling index of gastric mucosa were investigated in Wistar rats. Rats received subcutaneous injections of 12 micrograms/kg body weight of SP every other day after 25 weeks of oral treatment with MNNG. Long-term administration of SP significantly increased the incidence of gastric cancers in week 52. However, it did not affect the histological type and depth of involvement of gastric cancers. SP also caused a significant increase in the labeling index of the antral and fundic epithelial cells in week 52. These findings indicate that SP promotes gastric carcinogenesis and suggest that this effect may be related to its stimulation of antral epithelial cell proliferation. PMID- 7553616 TI - Altered regulation of message stability and tumor promoter-responsive cis-trans interactions of ribonucleotide reductase R1 and R2 messenger RNAs in hydroxyurea resistant cells. AB - Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The activity and message levels of the enzyme are elevated in cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13 acetate, and this appears to be mediated through specific cis elements in the 3' untranslated region of the R1 and R2 mRNAs that interact with R1 and R2 binding proteins called R1BP and R2BP, respectively. Hydroxyurea-resistant cells with increased R1 and R2 message levels were observed to have increased R1 and R2 message half-lives. This was accompanied by alterations in R1 and R2 3' untranslated region cis-trans interactions, as judged by band shift and UV cross linking assays, in which R1BP and R2BP binding was markedly reduced. This first description of mutant mammalian cells altered in message stability regulatory determinants indicates another mechanism for acquiring resistance to an antitumor agent. Furthermore, the present study strongly supports the concept that R1BP and R2BP are important general regulators of ribonucleotide reductase message stability and act as message destabilizing factors. PMID- 7553617 TI - Time-dependent pharmacodynamic models in cancer chemotherapy: population pharmacodynamic model for glutathione depletion following modulation by buthionine sulfoximine (BSO) in a Phase I trial of melphalan and BSO. AB - The development of time-dependent pharmacodynamic models in cancer chemotherapy has been extremely limited. A population approach was used to develop such a model to describe the effect of buthionine sulfoximine (BSO), via its active S isomer (S-BSO), on glutathione (GSH) depletion in peripheral mononuclear cells. The Phase I trial utilized escalating doses of BSO, from 5 to 17 gm/m2, as a multiple infusion regimen. The population model consisted of a linear 2 compartment pharmacokinetic model coupled to an indirect response model. The indirect response model consisted of a GSH compartment with input and output rate processes that are modulated as a function of S-BSO and GSH concentrations. The model predicted the observed gradual depletion of GSH, a nadir at approximately 30 h after the last dose of BSO, and a return to baseline GSH levels. On the basis of an IC50 estimate of about 1.6 microM for inhibition of gamma glutamylcysteine synthetase, the target enzyme of BSO, the population model predicted near identical GSH concentration time profiles over the dose range studied. Time-dependent pharmacodynamic models are seen as a powerful means to design dosing regimens and to provide a mathematical platform for mechanistic based models. PMID- 7553618 TI - Transforming growth factor beta 2 (TGF-beta 2) levels in plasma of patients with metastatic breast cancer treated with tamoxifen. AB - Blood levels of transforming growth factor beta 2 (TGF-beta 2) were measured in 20 patients with metastatic breast cancer before and during treatment with the antiestrogen tamoxifen, and in a control group of 7 patients with primary breast cancer before and during adjuvant tamoxifen treatment. The results of this study reveal typical time patterns for TGF-beta 2 in relation to the clinical outcome. Patients in remission showed a significant increase of TGF-beta 2 in the first 4 6 weeks of therapy, followed by a subsequent decrease. Patients who did not respond showed unchanged or diminished TGF-beta 2 values after start of therapy, followed by a later increase preceding the clinical manifestation of tumor progression. Thus, TGF-beta 2 blood levels after 4 weeks of tamoxifen treatment can be used as an early marker for prediction of response. PMID- 7553619 TI - High concentrations of the carcinogen 2-amino-1-methyl-6-phenylimidazo- [4,5 b]pyridine (PhIP) occur in chicken but are dependent on the cooking method. AB - Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds found in meats cooked at high temperatures. Although chicken is consumed in large quantities in the United States, there is little information on its HAA content. The objective of this study was to measure the five predominant HAAs (IQ, MeIQ, MeIQx, DiMeIQx, and PhIP) in chicken cooked by various methods to different degrees of doneness. Chicken breasts were panfried, oven-broiled, or grilled/barbecued. Whole chickens were roasted or stewed. Skinless, boneless chicken breasts were cooked to three degrees of doneness: just until done, well done, or very well done. High levels of PhIP (ranging from 12 to 480 ng/g cooked meat) were found in chicken breasts when panfried, oven-broiled, and grilled/barbecued but not in while roasted or stewed chicken. PhIP concentration increased in skinless, boneless chicken breast with longer cooking time, higher internal temperature, and greater degree of surface browning. PhIP concentration was also high in chicken breasts cooked with skin and bones. MeIQx and DiMeIQx levels increased with the degree of doneness, whereas IQ and MeIQ were not detectable in any of these chicken samples. Certain cooking methods produce PhIP, a known colon and breast carcinogen in rodents and possibly a human carcinogen, at substantially higher levels in chicken than has been reported previously in red meat. PMID- 7553620 TI - bcl-2 suppresses expression of p21WAF1/CIP1 in breast epithelial cells. AB - The tumor suppressor gene p53 regulates G1 checkpoint prior to the initiation of DNA synthesis, which can either induce G1 arrest or signal apoptosis. The involvement of p53 in apoptosis may also be related to its ability to down regulate transcription of the bcl-2 gene. The bcl-2 gene product prevents most types of apoptotic cell death, suggesting that bcl-2 interferes with an essential signaling molecule involved in the apoptotic cell death pathway. Although the bcl 2 protein is shown to be overexpressed in many types of human tumor including breast cancer, its biochemical or pathological consequences are poorly understood. To determine the effects of bcl-2 overexpression on apoptosis and transformation of breast epithelial cells and to investigate whether bcl-2 interferes with the p53 pathway, we introduced the bcl-2 expression vector into MCF10A cells, which were derived from diploid human breast epithelial cells containing the wild-type p53 gene. Overexpression of bcl-2 prevented free radical induced apoptosis and induced a partially transformed phenotype in MCF10A cells. Although overexpression of bcl-2 did not affect the expression of the p53 gene, p53-dependent gene transcription such as p21WAF1/CIP1 was suppressed. These results suggest that bcl-2 may inhibit p53 functional activity and is involved in the regulation of an early commitment step either to proliferate or suicide. PMID- 7553622 TI - Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. AB - Loss of heterozygosity on 9p21, where the p16/CDKN2 tumor suppressor and the p15INK4B cell cycle regulator genes are located, is a common genetic alteration in bladder cancer. However, it has been difficult to demonstrate homozygous deletions and intragenic mutations in either of these two genes in primary transitional cell carcinomas (TCC) of the bladder. Similarly, colon cancer derived cell lines have shown no homozygous deletions of the p16/CDKN2 locus in contrast to a wide variety of tumor-derived cell lines. We have investigated abnormal methylation of the 5' CpG islands of the p16/CDKN2 and p15INK4B genes as an alternative mechanism of inactivation of these genes in bladder and colon cancers. De novo methylation of the 5' CpG island of p16/CDKN2 was observed in 12 of 18 (67%) uncultured bladder TCCs and in 2 of 3 (67%) bladder cell lines. In contrast, only 1 of 10 (10%) colon carcinomas showed methylation of the 5' CpG island of p16/CDKN2. It was striking to find that this region was extensively methylated and the gene not expressed in the normal colonic mucosa of 6 of 10 (60%) patients with colon cancer, whereas 5 of the corresponding colon tumors showed no methylation and high levels of p16/CDKN2 expression. Our data show a significant correlation (P = 0.00001, two-sided) between the absence of p16/CDKN2 expression and methylation of its 5' CpG island in bladder tumors, cell lines, and normal colon mucosa. In contrast, no association was observed between expression and methylation status of the 5' CpG island of p15INK4B. Our results suggest that the p16/CDKN2 tumor suppressor gene may be inactivated by methylation of its 5' CpG island in TCCs of the bladder. We also present evidence of methylation of the 5' CpG island in this autosomal gene in normal colonic tissue. PMID- 7553623 TI - Tumor suppression by p21WAF1. AB - The p21WAF1 gene encodes a cyclin-dependent kinase inhibitor and mediates tumor suppressor gene p53-induced cell cycle arrest. To directly test whether p21WAF1 can act as a tumor suppressor, we have expressed the p21WAF1 cDNA in several human tumor cell lines using a tetracycline-inducible system. Overexpression of p21WAF1 suppresses proliferation and soft agar growth of tumor cells in vitro, as well as tumorigenicity in vivo. Our data provide direct evidence for the tumor suppressive activity of p21WAF1. PMID- 7553621 TI - Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. AB - The tumor suppressor gene CDKN2/p16/MTS1, located on chromosome 9p21, is frequently inactivated in many human cancers through homozygous deletion. Recently, we have reported another pathway of inactivation that involves loss of transcription associated with de novo methylation of a 5' CpG island of CDKN2/p16 in lung cancers, gliomas, and head and neck squamous cell carcinomas. We now show that this aberrant CpG island methylation also occurs frequently in cell lines of breast cancer (33%), prostate cancer (60%), renal cancer (23%), and colon cancer (92%) and is associated with loss of transcription. Primary tumors of the breast (31%) and colon (40%) also displayed de novo methylation of this CpG island. This alteration of p16 in colon cancer was particularly striking, since inactivation does not occur through homozygous deletion in this tumor type. Our data show that in tumors, de novo methylation of the 5' CpG island is a frequent mode of inactivation of CDKN2/p16 and also firmly demonstrate that CDKN2/p16 is one of the most frequently altered genes in human neoplasia. PMID- 7553624 TI - WT1 induces expression of insulin-like growth factor 2 in Wilms' tumor cells. AB - The Wilms' tumor suppressor gene WT1 encodes a zinc finger transcription factor, whose expression inhibits the growth of the RM1 Wilms' tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected promoters containing the early growth response gene 1 consensus sequence. We now show that WT1 has properties of a transcriptional activator in RM1 cells, an effect that may be associated with the presence of a mutated p53 gene in these cells. Stable transfection of wild-type WT1 into RM1 cells results in induction of endogenous insulin-like growth factor 2 (IGF2) but not of other previously postulated WT1-target genes. The induction of IGF2 is dramatically enhanced by WT1 mutants encoding an altered transactivation domain. We conclude that IGF2 is a potentially physiological target gene for WT1 and that its induction may contribute to the growth stimulating effects of WT1 variants. PMID- 7553625 TI - Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations. AB - The von Hippel-Lindau disease (VHL) gene is a novel tumor suppressor gene that plays a role in the pathogenesis of renal cell carcinomas and hemangioblastomas of the central nervous system. To begin an evaluation of the biological functions of the VHL gene product (pVHL), we prepared bacterial fusion protein between glutathione S-transferase and wild-type or mutant pVHLs. The fusion proteins were used to identify cellular proteins that bind to pVHL in vitro. Monkey kidney cells transfected with wild-type or mutant VHL cDNAs were used to identify cellular proteins that bind to pVHL in vivo. Wild-type pVHL consistently bound two cellular proteins with apparent molecular masses of 10 and 14 kilodaltons that were designated p10 and p14, respectively. Mapping studies with a panel of VHL deletion mutant proteins demonstrated that p10 and p14 bound to a 32-amino acid peptide located in the carboxy terminal portion of pVHL. Missense mutation located within this 32-amino acid peptide abrogated the ability of the VHL protein to bind p10 and p14. Of 67 VHL families with identified germline mutations, 42 families had mutations predicted to affect the p10/p14-binding region. Maintenance of the integrity of the p10/p14-binding region appears to be essential for cellular growth regulation by pVHL. PMID- 7553626 TI - Genomic aberrations in human hepatoblastomas detected by 2-dimensional gel analysis. AB - Hepatoblastoma is a frequent embryonic tumor found in children. The mechanism of its formation and development remain unknown. We analyzed genomic DNA aberrations associated with three hepatoblastomas by means of restriction landmark genomic scanning (I. Hatada et al., Proc. Natl. Acad. Sci. USA, 88:9523-9527, 1991). About 2000 NotI restriction landmarks were monitored in each of the hepatoblastomas, which revealed that the intensity of eight spots decreased in the tumor genome, suggesting allelic loss. Three of these spots were identical to those that decreased in human hepatocellular carcinomas. These results suggested that common, early events are involved in the genesis of the two types of liver tumor. PMID- 7553627 TI - The retinoblastoma gene product in acute myeloid leukemia: a possible involvement in promyelocytic leukemia. AB - The retinoblastoma susceptibility gene in leukemia and lymphoma has been investigated using different approaches involving either gene or protein analysis. In this study, a novel method, which evaluates the functional status of the retinoblastoma gene product by a binding assay to an in vitro-translated viral oncoprotein, has been applied to leukemic cells from acute myeloid leukemia patients. One hundred twenty-two cases were considered, and 42 of them were also analyzed by Western blot. Results obtained with the two methods were comparable, with the exception of few cases, where the retinoblastoma protein appeared detectable but unable to bind to the viral oncoprotein. The retinoblastoma protein has been found defective mostly in the M3 promyelocytic subtype. PMID- 7553628 TI - Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro. AB - The invasion and migration occurring in primary neoplastic tissue explants were studied by using a three-dimensional collagen matrix model, subsequent time-lapse videomicroscopy, and computer-assisted cell tracking. We show that not only single cells but groups of clustered cells comprising 5 to more than 100 cells detach from the primary tumor lesion and migrate within the adjacent extracellular matrix. These clusters were highly polarized, resulting in a high directional persistence of migration. Locomoting cell clusters were observed in primary cultures from invasive oral squamous cell carcinomas (6 of 9), ductal breast carcinomas (2 of 3), and rhabdomyosarcoma (1 of 1), whereas normal oral mucosa (0 of 4) was cell cluster negative. Thus, locomoting cell clusters could be a novel and potentially important mechanism of cancer cell invasion and metastasis. PMID- 7553629 TI - Hormone-dependent regulation of BRCA1 in human breast cancer cells. AB - BRCA1 mRNA and protein levels are regulated by the steroid hormones estrogen and progesterone in human breast cancer cells. BRCA1 mRNA and protein levels were significantly decreased in estrogen-depleted MCF-7 and BT20T cells and increased again after stimulation with beta-estradiol. The increase in BRCA1 expression upon stimulation with estrogen was not coordinated with the early induction of the estrogen-dependent pS2 gene but closely paralleled the delayed increase in the S-phase dependent marker cyclin A. T47-D cells deprived of steroid hormones and subsequently stimulated with progesterone also showed a delayed increase in BRCA1 mRNA expression. However, no change in BRCA1 protein was detected in these cells. When considered together, the data suggest that steroid hormones may affect BRCA1 expression indirectly by altering the proliferative status of the cells rather than acting directly on DNA sequences in the BRCA1 gene itself. PMID- 7553630 TI - Increased telomerase activity in mouse skin premalignant progression. AB - It has been postulated that the expression of the ribonucleoprotein telomerase is necessary to overcome cellular senescence and that malignant tumors must express telomerase to maintain their immortality. In most human adult tissues, telomerase activity is not detected. In contrast, several murine tissues express various levels of telomerase. Mouse skin however, does not show telomerase activity. Using the mouse skin chemical carcinogenesis system, a well-characterized model for studying premalignant and malignant progression, we assayed telomerase activity at various stages of premalignant papilloma progression by means of the recently developed telomeric repeat amplification protocol. We observed that at 10 weeks of promotion, only one mouse skin papilloma of 11 analyzed showed high levels of telomerase activity. The number of papillomas showing higher levels of telomerase activity increased at 20 weeks, and at 30 weeks of promotion, 100% of papillomas expressed significantly higher levels of telomerase. We learned from previous studies that early papillomas are diploid, well-differentiated lesions, whereas late papillomas are aneuploid and very dysplastic. It appears that the progressive increase in telomerase activity is associated with the increased level of genomic instability and the phenotypic progression of these premalignant tumors. It is also possible, however, that the increase in telomerase activity could be in part a consequence of an increase in the proportion of proliferating cells. Nevertheless, the mouse skin system may be a very useful in vivo model for the study and development of anti-telomerase therapeutic strategies. PMID- 7553631 TI - An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12. AB - We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region. PMID- 7553632 TI - Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. AB - The growth of solid tumors in vivo beyond 1-2 mm in diameter requires induction and maintenance of an angiogenic response. This can occur through the release of various angiogenic growth factors from tumor cells. One such factor is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a secreted and specific mitogen for vascular endothelial cells. We show that one of the most commonly encountered genetic changes detected in human cancer, i.e., expression of mutant ras oncogenes, is associated with marked up-regulation of VEGF/VPF in transformed epithelial cells. Thus, elevation of the levels of both VEGF/VPF mRNA and secreted functional protein were detected in human and rodent tumor cell lines expressing mutant K-ras or H-ras oncogenes, respectively. Genetic disruption of the mutant K-ras allele in human colon carcinoma cells was associated with a reduction in VEGF/VPF activity. Furthermore, pharmacological disruption of mutant RAS protein function in H-ras transformed rat intestinal epithelial cells by treatment with L-739,749 (a protein farnesyltransferase inhibitor) caused a significant suppression of VEGF/VPF. The results suggest that dominantly acting ras oncogenes may contribute to the growth of solid tumors in vivo not only by a direct effect on tumor cell proliferation but also indirectly, i.e., by facilitating tumor angiogenesis. Hence, pharmacologically targeting mutant ras oncogenes could conceivably suppress solid tumor growth in vivo, in part, by inhibiting tumor-induced angiogenesis. PMID- 7553634 TI - Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII. AB - 2B1 is a bispecific murine monoclonal antibody (BsMAb) with specificity for the c erbB-2 and Fc gamma RIII extracellular domains. This BsMAb promotes the targeted lysis of malignant cells overexpressing the c-erbB-2 gene product of the HER2/neu proto-oncogene by human natural killer cells and mononuclear phagocytes expressing the Fc gamma RIII A isoform. In a Phase I clinical trial of 2B1, 15 patients with c-erbB-2-overexpressing tumors were treated with 1 h i.v. infusions of 2B1 on days 1, 4, 5, 6, 7, and 8 of a single course of treatment. Three patients were treated with daily doses of 1.0 mg/m2, while six patients each were treated with 2.5 mg/m2 and 5.0 mg/m2, respectively. The principal non-dose limiting transient toxicities were fevers, rigors, nausea, vomiting, and leukopenia. Thrombocytopenia was dose limiting at the 5.0 mg/m2 dose level in two patients who had received extensive prior myelosuppressive chemotherapy. Murine antibody was detectable in serum following 2B1 administration, and its bispecific binding properties were retained. The pharmacokinetics of this murine antibody were variable and best described by nonlinear kinetics with an average t 1/2 of 20 h. Murine antibody bound extensively to all neutrophils and to a proportion of monocytes and lymphocytes. The initial 2B1 treatment induced more than 100-fold increases in circulating levels of tumor necrosis factor-alpha, interleukin 6, and interleukin 8 and lesser rises in granulocyte-monocyte colony-stimulating factor and IFN-gamma. Brisk human anti-mouse antibody responses were induced in 14 of 15 patients. Several minor clinical responses were observed, with reductions in the thickness of chest wall disease in one patient with disseminated breast cancer. Resolution of pleural effusions and ascites, respectively, were noted in two patients with metastatic colon cancer, and one of two liver metastases resolved in a patient with metastatic colon cancer. Treatment with 2B1 BsMAb has potent immunological consequences. The maximum tolerated dose and Phase II daily dose for patients with extensive prior myelosuppressive chemotherapy was 2.5 mg/m2. Continued dose escalation is required to identify the maximally tolerated dose for patients who have been less heavily pretreated. PMID- 7553633 TI - Enhancement of transformation in vitro of a nontumorigenic rat urothelial cell line by interleukin 6. AB - Chronic inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. We have shown that acute and chronic inflammation induced by intravesical instillations of killed Escherichia coli strikingly enhances N-methyl-N-nitrosourea (MNU)-initiated rat bladder carcinogenesis. To test the hypothesis that cytokines released during inflammation may be involved in the enhancement of bladder carcinogenesis, we conducted an in vitro experiment. Using soft agar growth as an index of transformation, we examined the effect of inflammation-associated cytokines on the enhancement of MNU-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. In the first experiment, after 1-h exposure to MNU (50 micrograms/ml), cells (5 x 10(4)) were grown in soft agar in the presence of interleukin (IL)-1 alpha, IL-6, IL-8, or tumor necrosis factor-alpha (10 to 100 ng/ml). Colonies consisting of more than 20 cells were counted 4 weeks later. Among the cytokines tested, IL-6 (100 ng/ml) significantly increased colony counts over those for the untreated controls (P < 0.001). In the second experiment, the cells treated with MNU similarly as in the first experiment were cultured with or without IL-6 (100 ng/ml) for 1 week before the cells (5 x 10(4)) were grown in soft agar in the presence or absence of IL-6. IL-6 pretreatment increased colony counts irrespective of subsequent IL-6 treatment (P < 0.05). Moreover, IL-6-stimulated anchorage-dependent growth of MNU transformants far exceeded that of the parental MYP3. However, among the transformants, there was no parallel relationship in response to IL-6 between anchorage-dependent and independent growth. Our results suggest that IL-6 may provide a selective growth advantage to MNU-initiated bladder epithelial cells in vitro and that it may be a factor accounting for the marked enhancement of inflammation-associated rat bladder carcinogenesis. PMID- 7553635 TI - Deregulated alternative splicing of CD44 messenger RNA transcripts in neoplastic and nonneoplastic lesions of the human thyroid. AB - CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, lymphocyte activation and homing, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms, each generated by alternative splicing of up to 10 variant exons (termed v1-v10) encoding parts of the extracellular domain. Using semiquantitative reverse transcriptase-PCR and Southern hybridization, alternative CD44 mRNA splicing was examined in 10 papillary thyroid carcinomas, 8 nodular goiters, 9 adenomas, 2 cases of thyroiditis, and 3 histologically normal thyroid controls. The amount of input cDNA for the CD44 PCRs was standardized against an internal control gene (glyceraldehyde phosphate dehydrogenase). Four papillary carcinomas showed significant overexpression of CD44 transcripts migrating between 750 and 1000 bp. These cases demonstrated reduced levels of the 482-bp standard isoform transcript. In six papillary cancers, we found a prominent v6-containing isoform at 750 bp that was present in only trace amounts in normal thyroid tissue. It is of interest that similar findings were seen in the majority of the goiters and adenomas but not in the cases of thyroiditis. These results show that deregulation of alternative CD44 splicing is a common feature of disordered thyroid follicular cell growth, both in neoplastic and nonneoplastic lesions. The data imply an important role for CD44, including CD44v6, in the pathogenesis of various thyroid lesions. PMID- 7553637 TI - Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and time course of peripheral blood mononuclear cell O6-methylguanine-DNA methyltransferase inhibition in the nonhuman primate. AB - O6-Benzylguanine (O6BG) enhances the cytotoxicity of the nitrosoureas by irreversibly binding and inhibiting the DNA repair enzyme O6-methyl-guanine-DNA methyltransferase (MGMT). The plasma and cerebrospinal fluid (CSF) pharmacokinetics of O6BG and its active metabolite, O6-benzyl-8-oxoguanine, were studied in a nonhuman primate model after 200 mg/m2 had been injected i.v. The parent drug and the metabolite were measured with a reverse-phase HPLC assay. A pharmacokinetic model incorporating separate compartments for O6BG and the O6 benzyl-8-oxoguanine metabolite, first-order conversion of O6BG to the metabolite, and additional first-order elimination rate constants for each compound, was simultaneously fitted to the parent drug and metabolite plasma concentration time data. Elimination of O6BG from plasma was rapid; it had a half-life of 1.6 h and a clearance of 68 ml/min/m2. On the basis of the pharmacokinetic model, essentially all of the O6BG was converted to O6-benzyl-8-oxoguanine. The plasma pharmacokinetic profile of the metabolite differed considerably from that the parent drug. The half-life (14 h) was 10-fold longer and the area under the curve (2420 microM/h) was 11-fold higher than that of O6BG (212 microM/h). The clearance rate of O6-benzyl-8-oxoguanine was 6.4 ml/min/m2. The CSF:plasma ratio was 4.3% for O6BG and 36% for O6-benzyl-8-oxoguanine, and the metabolite area under the curve was 90-fold higher than that of O6BG in CSF. The excellent CSF penetration of the active metabolite provides a rationale for the use of O6BG as a chemosensitizing agent for brain tumors. We also studied the duration of MGMT inhibition in peripheral blood mononuclear cells. By 2 h after a 200 mg/m2 dose of O6BG, > 98% of MGMT activity was suppressed, and > 95% suppression of enzyme activity persisted at 18 and 48 h after the dose. By 2 weeks after the treatment, MGMT levels had returned to baseline. Persistent high concentrations of the active metabolite appear to provide a pharmacological explanation for the prolonged suppression of MGMT activity. PMID- 7553638 TI - Biodistribution of monoclonal antibodies: scale-up from mouse to human using a physiologically based pharmacokinetic model. AB - The efficacy of a novel diagnostic or therapeutic agent depends on its selective localization in a target tissue. Biodistribution studies are expensive and difficult to carry out in humans, but such data can be obtained easily in rodents. We have developed a physiologically based pharmacokinetic model for scaling up data from mice to humans, the first such model for genetically engineered macromolecules that bind to their targets in vivo, such as mAbs. The mathematical model uses physiological parameters including organ volumes, blood flow rates, and vascular permeabilities; the compartments (organs) are connected anatomically. This allows the use of scale-up techniques to predict antibody distribution in humans. The model was tested with data obtained in human patients for the biodistribution of a mAb against carcinoembryonic antigen. The model was further tested for a two-step protocol: bifunctional antibodies and radiolabeled hapten, which compared favorably with data in both mice and humans. The model was useful for optimization of treatment parameters, such as dose and time interval of injections, binding affinities, and choice of molecular carrier. This framework may be applicable to other genetically engineered molecules (e.g., growth factors, antisense oligonucleotides, and gene-carrying vectors). PMID- 7553636 TI - In vitro and in vivo inhibition of human papillomavirus type 16 E6 and E7 genes. AB - Human cervical cancers are often associated with human papillomavirus (HPV). In HPV-positive cervical cancers, the oncoproteins E6 and E7 are consistently expressed. In this study, the effects of antisense inhibition of both proteins were examined. Phosphorothioate oligonucleotides (ODNs) AE6 and AE7 complementary to regions flanking the start codons of HPV16 E6 and E7 genes, respectively, were synthesized. These anti-HPV ODNs inhibited the growth of cervical cell lines CaSki and SiHa, which harbor HPV16 but had little effect on cells that do not. Both ODNs also affected the ability of CaSki cells to form colonies in soft agar. In nude mice, treatment with either AE6, AE7, or a mixture of both led to substantially smaller tumors. AE7 was observed to inhibit E7 synthesis. The AE6 ODN probably exerts its effect by suppressing the expression of E6 as well as E7. Cell cultures and tumors treated with AE6 showed a decrease in E7 expression. In addition, an antisense ODN targeted at the retinoblastoma gene was able to reverse some of the inhibitory effect of AE6 on CaSki cells, indicating that AE6 inhibited E7 synthesis. This study further demonstrates that anti-HPV ODNs may be useful therapeutically. PMID- 7553639 TI - Taxol induction of p21WAF1 and p53 requires c-raf-1. AB - Taxol stabilizes microtubules, prevents tubulin depolymerization, and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized, the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein, we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells, although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells, wild-type p53 protein was also induced after taxol treatment, and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells, p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects, taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol, as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity, but that it is not strictly dependent on wild-type p53. Furthermore, the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression. PMID- 7553640 TI - Microvessel origin and distribution in pulmonary metastases of B16 melanoma: implication for adoptive immunotherapy. AB - To elucidate the role of tumor vascularization on the localization of adoptively transferred, interleukin 2-activated natural killer (A-NK) cells, pulmonary B16 melanoma metastases were analyzed with respect to location, morphological appearance, origin and density of microvessels, and infiltration by A-NK cells. The B16 melanoma metastases could be divided into four subtypes according to their location (superficial or deep in the lung parenchyma) and morphological appearance (compact or loose). Localization of adoptively transferred A-NK cells into the four subtypes of B16 pulmonary metastases differed significantly. More than 800 A-NK cells/mm2 were found in metastases of the deep-loose type, compared to approximately 400/mm2 A-NK cells in the superficial-loose metastases, and less than 200 A-NK cells/mm2 in the compact subtype, regardless of its location (deep or superficial). Although the origin (pulmonary or bronchial) of the blood supply to the metastatic subtypes (as revealed by electron microscopic analyses of lungs perfused with a lanthanum solution) did not account for this difference, the density of microvessels in the metastatic subtypes correlated with the number of A-NK cells that localized into these metastases. The resistance of metastases of the compact type to infiltration of adoptively transferred effector cells might explain, in part, why adoptive immunotherapy seldom results in complete eradication of disseminated cancer. PMID- 7553642 TI - Microdissection based cloning of a translocation breakpoint in a human malignant melanoma. AB - Chromosome translocations in human malignancies have identified the genomic location of several important growth-regulatory sequences (e.g., cellular oncogenes and suppressor genes). Melanomas are characterized by recurring chromosome alterations, including deletion or translocations of the long arm of chromosome 6 (6q). This report details our efforts to clone the t(1;6)(q21;q14) breakpoint in a malignant melanoma to further our understanding of the biology of these tumors. The strategy utilized combined microdissection of the translocation chromosome, development and characterization of a DNA microclone library, isolation of cosmids and YACs from the breakpoint region, ordering of clones by two-color metaphase/interphase fluorescence in situ hybridization, and finally, identification of a YAC spanning the translocation breakpoint. By analogy to other tumor systems, molecular examination of the chromosome 6 breakpoint may provide insight into the pathobiology of this important neoplasm. PMID- 7553641 TI - Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of human prostate carcinoma cell lines. AB - Hormonal treatment of advanced prostatic cancer patients generally results in an initially beneficial response, but the treated patients develop hormonally resistant disease in which no curative therapy is currently available. Recent studies have revealed that interleukin 6 (IL-6) is a growth factor for myeloma, renal cell carcinoma, and certain T-cell lymphomas. Further, IL-6 has been shown to block apoptosis induced by p53, transforming growth factor beta, and certain cancer chemotherapeutic compounds. The objective of the present study was to determine whether IL-6 is a growth factor for two human prostate cancer lines and whether it protects the tumor cells from drug-induced cell death. Two hormone independent prostate cell lines were used in this study, namely PC-3 and DU145, and these have been shown to be relatively resistant to cis diamminedichloroplatinum (CDDP), etoposide (VP-16), and adriamycin (ADR). Both cell lines express IL-6 mRNA and secrete IL-6 constitutively. The addition of anti-IL-6 antiserum to the cell lines resulted in a significant inhibition of cell growth up to day 2, and when additional antibody was added at day 2 the inhibition persisted for 4 days. The coaddition of anti-IL-6 antiserum and CDDP or VP-16 resulted in synergy in cytotoxicity in both cell lines, whereas the combination of antibody and ADR or suramin resulted only in additive effects. Sequential treatment revealed that anti-IL-6 antibody was required to achieve synergy, whereas either sequence of pretreatment resulted in synergy with anti-IL 6 and CDDP but not with VP-16. CDDP treatment of tumor cells down-regulated IL-6 mRNA expression and IL-6 secretion. The present findings demonstrate that IL-6 is an autocrine/paracrine growth factor for DU145 and PC-3 prostate lines. Additionally, the secretion of this cytokine protects the tumor cells against the cytotoxic effect of CDDP and VP-16 and its neutralization sensitizes the cells to cytotoxicity. Overall, the studies suggest that agents that can down-regulate or inhibit protective factors in tumors may overcome drug resistance. PMID- 7553643 TI - Protection against daunorubicin cytotoxicity by expression of a cloned human carbonyl reductase cDNA in K562 leukemia cells. AB - Carbonyl reductase (CBR) catalyzes the reduction of daunorubicin (DN) to its corresponding alcohol, daunorubicinol (DNOL), and changes the pharmacological properties of this cancer chemotherapeutic drug. The DN reductase associated with CBR reduces the C13 methyl ketone group and does not metabolize the quinone ring of DN. Reports comparing DN and DNOL toxicity have resulted in various conclusions depending on the cells tested. Differences in toxicity could be due to variations in several enzymes involved in DN metabolism. In this report, the effects of CBR expression on DN metabolism and cell toxicity were determined by cloning and expressing a human CBR cDNA in DN reductase-deficient myeloid erythroleukemia K562 cells. CBR activity increased 83-fold in the K562 transfected cells and was associated with a 2-3-fold reduction in DN toxicity. Maximum protection occurred at 30 nM DN where 94% of the intracellular DN was converted to DNOL within 2 h. The reduced toxicity was specific for DN. Other CBR substrates such as menadione, phenanthrenequinone, and doxorubicin were equally toxic to both the CBR expresser cells and the control cells under the conditions tested. Our results suggest that high levels of CBR in tumor cells could contribute to drug resistance. The results also suggest that reduction of DN to DNOL protects against DN toxicity by altering interaction of the drug at one or more of the many target sites. PMID- 7553644 TI - Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Waf1. AB - Protein complexes containing cyclins and cyclin-dependent protein kinases (cdks) have been shown to be rearranged in both spontaneous and viral tumor antigen transformed cells. We have examined G1- and S-phase cyclin/cdk complexes as a function of the neoplastic progression of human diploid fibroblasts transfected with the SV40 large T antigen. We find that the expression of cyclin D1 and its association with proliferating cell nuclear antigen (PCNA) and Waf1 remain unchanged in precrisis human fibroblasts transfected with SV40 large T antigen. However, in these same cells the association of cdk4 with cyclin D1, PCNA, and Waf1 is disrupted. Upon immortalization, cyclin D1 protein expression is decreased, and binding of both PCNA and Waf1 with the remaining cyclin D1 is reduced. In contrast, large T antigen increased the expression of cyclin A and cyclin E proteins in both precrisis and immortal cells and did not reduce the binding of PCNA or Waf1 to either cdk2 or cyclin A proteins. These results show that large T-antigen expression in human fibroblasts selectively uncouples cyclin D1 from cdk4, and subsequent immortalization of these cells results in additional changes to the cyclin D1-dependent cell cycle regulatory pathways. PMID- 7553645 TI - Mutational fingerprint induced by the antineoplastic drug chloroethyl-cyclohexyl nitrosourea in mammalian cells. AB - Using the pZ189 shuttle vector approach, we determined two chloroethyl-cyclohexyl nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P < 0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P < 0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5'-GGG-3') or hot spots exclusive for each CCNU concentration (3 mM: position 159, 5'-AGG-3'; 6 mM: position 109, 5'-GGG-3'); (e) the occurrence of GC-->AT transitions was significantly different as a function of CCNU concentration (P < 0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5'Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5' (or 3 bp steps 3') to the mutated G. Our results are consistent with the hypothesis that O6-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O4-alkylthymine and/or N3-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O6-alkylguanine is influenced by the DNA sequence context. All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy related leukemias.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7553646 TI - Significance of chromosome 1p loss of heterozygosity in neuroblastoma. AB - We analyzed 156 primary neuroblastoma tumor samples for loss of heterozygosity at the distal short arm of chromosome 1 (1p LOH). We also compared 1p LOH with known clinical and genetic prognostic variables as well as patient outcome. 1p LOH was detected in 30 of 156 tumors (19%) and was strongly associated with adverse clinical and biological features. 1p LOH was also strongly predictive of a poor outcome in univariate analyses (estimated 4-year survival, 32 +/- 10% SE versus 76 +/- 5% SE; P < 0.001). However, the prognostic value of 1p LOH was equivocal when stratified for amplification of the MYCN oncogene (P = 0.16). We conclude that 1p LOH is an important component of a pattern of genetic abnormalities in neuroblastoma associated with an aggressive clinical course. PMID- 7553648 TI - Elevated levels of syndecan-1 expression confer potent serum-dependent growth in human 293T cells. AB - Syndecan-1 is the best studied integral membrane proteoglycan and functions to modulate epithelial cell attachment and physiology. Extracellularly, syndecan-1 binds both growth factors and extracellular matrix components, and intracellularly, its cytoplasmic portion interacts with cytoskeletal components. To investigate the possible role of syndecan-1 in epithelial cell transformation that is characterized by alteration in extracellular matrix interactions and cytoskeleton architecture, we established stable transfectants of syndecan-1 in a highly transformed human renal epithelial line expressing two viral oncogenes, adenovirus E1a and SV40 large T antigen (293T cell line). Expression of syndecan 1 core protein and appropriate posttranslational attachment of glycosaminoglycan chains was confirmed by enzymatic digestion and Western blot analysis. Overexpresser cells grew at a significantly faster rate than the vector transfected control cells in serum-rich media but showed a proliferative disadvantage in serum-reduced media. In addition to this serum dependency, syndecan-1 overexpression caused a partial reversal of the transformed phenotype with the expressing clones becoming more anchorage dependent and less motile than the vector-transfected counterparts. Surprisingly, the overexpressers were more tumorigenic when injected s.c. into nude mice. These results indicate that syndecan-1 expression plays a role in the control of cell proliferation and suggest that serum-dependent growth may be the more reflective of tumorigenicity in nude mice. PMID- 7553647 TI - Allelotype of pancreatic adenocarcinoma using xenograft enrichment. AB - p53 and MTS1 are known to be mutationally inactivated in pancreatic adenocarcinoma. Other tumor suppressor genes are likely also to play a role. To define chromosomal arms which may harbor additional tumor suppressor genes, we performed an extensive allelotype on pancreatic cancer utilizing a xenograft enrichment technique. Eighty-eight percent (28/32) of primary tumors gave rise to xenografts. Eighteen cases were used in a PCR-based allelotype using 283 polymorphic markers, over 2800 informative assays, and an average coverage of 4.1 informative markers per chromosomal arm per case. Highly frequent allelic loss (> 60%) was seen at chromosomes 1p, 9p, 17p, and 18q. Moderately frequent allelic loss (40-60%) was seen at 3p, 6p, 6q, 8p, 10q, 12q, 13q, 18p, 21q, and 22q. The average fractional allelic loss was 0.36. Allelic and sequence stability was demonstrated among 64 parallel and second-passage xenografts derived from 12 cases of pancreatic adenocarcinoma with the ascertainment of over 3000 single alleles. The findings were confirmed in primary tumors. In only two instances were discrepancies revealed between the allelic loss data obtained from corresponding parallel xenografts, probably due to the xenografting of minor subpopulations, reflecting genetic heterogeneity of the primary tumor. PMID- 7553649 TI - Expression of plasminogen activators and plasminogen activator inhibitors in cutaneous melanomas of transgenic melanoma-susceptible mice. AB - The Tyr-SV40E transgenic mouse model of malignant skin melanoma has been used here to generate melanomas in genetically identical (C57BL/6) mice for analysis of the plasminogen activator (PA) system during tumor development and progression. Twenty-two melanocytic lesions were examined by in situ zymography for PA activity and by immunohistochemistry for concomitant visualization of PA proteins; these lesions encompassed 3 nevi and 19 primary melanomas ranging from melanotic through mixed tumors to amelanotic tumors. Although urokinase-type plasminogen activator (u-Pa) activity was not detected at premalignant stages, it began to appear early in tumorigenesis and became more prominent in later stages of a majority of the tumors. The activity was largely attributable to the endothelium of sprouting capillaries and to a lesser degree to granulocytes, fibroblastic cells, and occasional melanoma cells within tumors. Tissue-type plasminogen activator (t-PA) was undetectable or low in all cases. Of the inhibitors (PAI), PAI-1 was seen in endothelial and fibroblastic cells and in the extracellular matrix, whereas PAI-2 occurred in only one case and was melanoma cell associated. Eleven additional melanomas were analyzed by reverse transcription-PCR for PA expression in RNA extracts from relatively large tumor samples. These were obtained from eight primary melanomas and three metastases, again spanning melanotic, mixed, and amelanotic cases. From four of the mixed primary tumors with distinct melanotic and amelanotic zones, the respective components were propagated separately in transgenic hosts as s.c. transplants to obtain data for clearly identifiable melanotic versus amelanotic parts. u-PA and PAI-1 mRNAs were expressed in all. t-PA expression varied greatly and was notably high in several amelanotic tumors or tumor components, possibly as a result of large blood vessels, as such vessels were seen to be t-PA positive in normal tissue. The u-PA activity in sprouting capillaries may indicate a role in neoangiogenesis. Therefore, according to these mouse models, u-PA may indirectly be a potential therapeutic target against melanoma progression. PMID- 7553650 TI - Plasmin-catalyzed proteolysis in colorectal neoplasia. AB - The expression of different components of the plasminogen activator (PA)/plasmin system was explored in a series of colorectal neoplasia. We have found that urokinase (uPA) and urokinase receptor (uPA-R) gene expression is upregulated in adenomas and carcinomas, and that uPA/uPA-R production is confined to stromal cells in the proximity of epithelial proliferations. In addition, in adenomas, the focal increase in uPA mRNA is not systematically coupled to detectable enzymatic activity, whereas in carcinomas, uPA mRNA accumulation is consistently associated with detectable but variable levels of enzymatic activity. In contrast, in the tumor vasculature, tissue-type plasminogen activator-mediated proteolysis is considerably reduced when compared to normal mucosal and submucosal vessels; this reduction in plasmin formation appears to result from the highly increased production of plasminogen activator inhibitor type 1 by endothelial cells. Our observations demonstrate that colorectal neoplasia are associated with marked alterations in the extracellular proteolytic balance controlled by the PA/plasmin system. They show that contrasting disturbances in plasmin formation take place in distinct stromal compartments but not in epithelial cells, and that these disturbances are maximal during invasive neoplasia. Altogether, our results raise the possibility that alterations in plasmin formation should not be exclusively regarded as promoters of cancer cell invasiveness. PMID- 7553651 TI - Allelic losses on chromosomes 14, 10, and 1 in atypical and malignant meningiomas: a genetic model of meningioma progression. AB - To investigate chromosomal events that underlie formation and progression of meningiomas, we have examined a set of 18 benign (WHO grade I), 15 atypical (grade II), and 13 anaplastic/malignant (grade III) meningiomas for loss of heterozygosity (LOH) on chromosomes 1p, 6p, 9q, 10q, and 14q. Frequent loss of loci on these chromosomes was seen in grade II and grade III tumors, specifically, 14q (II and III, 47 and 55%), 1p (40 and 70%), and 10q (27 and 40%). In contrast, LOH for these loci was infrequent in benign meningiomas, specifically, 14q (0%), 1p (11%), and 10q (12%). The smallest common regions of deletion that could be defined were 14q24-q32, 1p32-pter, and 10q24-qter. These observations indicate the likely presence of tumor suppressor genes in these regions that are involved in the development of WHO grade II and grade III meningiomas. Because LOH for loci on chromosomes 1p and 10q was found in tumors of all grades and because the frequency of LOH in all three regions increased with tumor grade, these results would support a model for the formation of aggressive meningiomas through tumor progression. PMID- 7553652 TI - Mitogenic activity of laminin on human melanoma and melanocytes: different signal requirements and role of beta 1 integrins. AB - The possible mitogenic activity of laminin (LN) on normal and neoplastic cells of the melanocyte lineage was tested by culturing growth-arrested human melanoma cells and neonatal foreskin melanocytes on LN. Serum-deprived, quiescent melanoma cells proliferated, in serum-free medium, in a dose-dependent fashion to immobilized LN as determined by [3H]thymidine incorporation, cell cycle analysis, and change in cell number. The mitogenic activity of LN on melanoma cells was not mediated through autocrine release of growth factors and was observed with primary or metastatic melanoma cells and with clones isolated from the same metastasis but only on cells expressing very late antigen (VLA)-3 and VLA-6 laminin receptors. Proliferation of melanoma cells to LN was significantly inhibited by a mAb to the beta 1 subunit of VLA integrins and by a combination of mAbs to the alpha subunits of VLA-3 and VLA-6. By contrast, LN did not act asa mitogen on human melanocytes expressing VLA-3 and VLA-6 and cultured in serum free medium. However, a costimulatory activity of immobilized LN for proliferation of melanocytes was observed in the presence of a second signal provided by a set of different growth factors. The costimulatory activity of LN on melanocytes could be significantly inhibited by mAbs directed to the alpha and beta chain of VLA-6 but not to VLA-3. These data suggest that LN itself, and not growth factors possibly associated with it, can exert a mitogenic activity on quiescent human melanoma cells and that a change in the signal requirements for response to LN occurs upon neoplastic transformation in the melanocyte lineage. Furthermore, beta 1 integrins are differentially involved in the response of the normal and the neoplastic cells to LN, since VLA-3 and VLA-6 cooperate in the proliferation of neoplastic cells, while VLA-6 is relevant for the response of melanocytes. PMID- 7553653 TI - Differentiation of neuroblastoma enhances Bcl-2 expression and induces alterations of apoptosis and drug resistance. AB - Recent evidence suggests that resistance to antineoplastic therapy may result from mutations in genes mediating the apoptotic response to DNA damage. To determine the effects of epigenetic changes on tumor responsiveness to cytotoxic agents inducing DNA damage, we examined the chemosensitivity of neuroblastoma (NB) after differentiation by retinoic acid (RA). Differentiation of the cell lines SH-SY5Y and SMS-KCNR by RA abolished the cytotoxic effects of adriamycin (Adr) and cisplatin. Chemoresistance was not the result of decreased proliferation induced by RA because: (a) growth arrest by nutrient deprivation did not affect sensitivity; (b) growth arrested NB cell lines, which did not differentiate, remained chemosensitive; and (c) RA concentrations which promoted differentiation without affecting growth, induced resistance. Apoptosis characterized NB cells responding to Adr, although differentiated SH-SY5Y did not apoptose and were resistant to Adr and cisplatin. Marked induction of bcl-2 in NB cells followed RA-induced differentiation, whereas in cell lines failing to differentiate, bcl-2 was not detected. Our data indicate that NB differentiation induces drug resistance after a loss of the apoptotic response to antineoplastic drugs and suggest that bcl-2 overexpression is an important mechanism of resistance in differentiated tumor cells. PMID- 7553655 TI - Transition from the noninvasive to the invasive phenotype and loss of alpha catenin in human colon cancer cells. AB - Loss of epithelioid organization in carcinoma cell lines has been related to invasiveness and poor differentiation of tumors. We investigated the invasion in vitro of various human colon cancer cell lines. Most cell lines were noninvasive into chick heart fragments, and this correlated with an epithelioid morphotype. Only cell lines COLO320DM, SW620, and variants of HCT-8 and DLD-1 were invasive and nonepithelioid. We examined in these cell lines whether invasiveness was related to changes in the structure and function of the E-cadherin/catenin complex. E-cadherin functions as an invasion suppressor and as a cell-cell adhesion molecule when linked to the cytoskeleton via alpha-catenin plus beta- or gamma-catenin. All noninvasive cell lines showed E-cadherin linked to these catenins. The E-cadherin-dependent cell-cell adhesion function in these cell lines was demonstrated by two assays in vitro. It was interesting that all invasive cell lines showed a dysfunctional E-cadherin/catenin complex. COLO320DM, SW480, and SW620 cells were defective in E-cadherin expression, whereas the invasive variants of HCT-8 and DLD-1 lacked the alpha-catenin protein. From clonal epithelioid HCT-8 cultures with functional E-cadherin/catenin complexes, we subcloned, repeatedly, round cell variants that were again invasive and expressed no alpha-catenin protein. Our data suggest that reproducible transformations toward a more invasive phenotype in HCT-8 cells are associated with down-regulation of alpha-catenin. The mechanisms of this transformation and the level of alpha-catenin down-regulation are currently investigated. PMID- 7553654 TI - Cloning and expression of CD24 gene in human hepatocellular carcinoma: a potential early tumor marker gene correlates with p53 mutation and tumor differentiation. AB - To search for genes related to hepatocarcinogenesis, the differential display technique for eukaryotic mRNA was conducted. We have cloned a gene that encodes the CD24 protein from the cDNA library of human hepatocellular carcinoma (HCC). A single 2.1-kb mRNA was identified in HCC specimens and the HuH-7 HCC cell line but only rarely in small amounts in nontumor livers. In 79 unicentric HCC, CD24 mRNA was overexpressed in 52 cases (66%), found in trace amounts in 11, and not detectable in 16 (20%). In 12 cases of multicentric HCC, CD24 mRNA was overexpressed in 21 (68%) of 31 tumor nodules and was helpful for the determination of tumor clonal origin. There was an increased frequency of CD24 mRNA overexpression in patients younger than 50 years with HCC (86% versus 59%, P < 0.025), in serum hepatitis B surface antigen-positive individuals (74% versus 48%, P < 0.023), in those with an elevated serum alpha-fetoprotein level (82%, versus 56%, P < 0.04), and in HCC with alpha-fetoprotein mRNA expression (82% versus 56%, P < 0.04). There was a strong correlation of CD24 mRNA overexpression with p53 gene mutation in HCC (91% versus 46%, P < 0.0005) and poorly differentiated HCC (82% versus 53%, P < 0.0008). Despite its correlation with p53 mutation and the unfavorable outcome of HCC with p53 mutation, the CD24 mRNA expression did not correlate with tumor size, tumor invasiveness, or patient's prognosis. Thus, the CD24 gene expression appears to be a common event in HCC and may serve as an early but not prognostic biomarker for malignant transformation of hepatocytes. PMID- 7553656 TI - Growth advantage and vascularization induced by basic fibroblast growth factor overexpression in endometrial HEC-1-B cells: an export-dependent mechanism of action. AB - The human endometrial adenocarcinoma HEC-1-B cell line was transfected with an expression vector harboring the human basic fibroblast growth factor (bFGF) cDNA under the control of the human beta-actin gene promoter. Stable transfectants were obtained in which a constitutive, limited overexpression of M(r) 24,000, 22,000, and 18,000 bFGF isoforms was observed. When transfectants were screened for the capacity to release the growth factor, significant amounts of bFGF were present in the conditioned medium and extracellular matrix of the bFGF-B9 clone but not of the bFGF-A8 clone, even though both cell lines produced similar levels of intracellular bFGF. When compared to parental cells, bFGF-B9 cells showed down regulation of tyrosine kinase fibroblast growth factor receptors along with up regulation of urokinase-type plasminogen activator expression which was abolished by incubation of the cell cultures with neutralizing anti-bFGF antibody. In vivo, bFGF-B9 cells formed highly vascularized tumors growing faster than parental cells when injected s.c. in nude mice. Also, they were more potent than nontransfected cells in inducing an angiogenic response in the rabbit cornea assay. In contrast, the bFGF-A8 cell phenotype was indistinguishable from parental cells both in vitro and in vivo. In conclusion, clonal differences exist within the HEC-1-B cell line in the capacity to release bFGF. bFGF export by human endometrial adenocarcinoma cells results in autocrine and paracrine effects that confer a growth advantage in vivo associated with increased neovascularization. PMID- 7553657 TI - Integrins in tumour development and spread. AB - The study of integrin receptor expression and function in carcinomas will undoubtedly increase our understanding of the malignant process and clarify the relative importance of the numerous alterations that are identified. Whether this will lead to direct clinical applications is not clear at present, but it will certainly alter our approach to the evaluation of cancer treatment. The more immediate search for prognostic indicators and metastatic site preference in tumours expressing a particular integrin profile is partly hampered by the small numbers of cases studied in most series, where observed trends in tumour subsets barely achieve significance. Perhaps the next phase should be to evaluate promising approaches in large multicentre studies to establish the relative importance of these trends in tumour prognosis and response to treatment, preferably by developing reagents that will permit the demonstration of integrins in routinely processed archival material from human tumour specimens. Future developments in therapy may depend on the knowledge that may emerge from such work. PMID- 7553658 TI - Adherens junction proteins in tumour progression. AB - The loss of epithelial differentiation in carcinomas, which is accompanied by higher mobility and invasiveness of the tumour cells, is often a consequence of reduced intercellular adhesion. The primary cause of the "scattering" of cells in invasive carcinomas appears to be a disturbance of the integrity of intercellular junctions, often involving the cell adhesion molecule E-cadherin. Permanent and transient molecular mechanisms can lead to the impairment of junction integrity of epithelial cells and thus to the progression of carcinomas towards a more invasive state. These include downregulation of E-cadherin expression and interaction between the adherens junction protein beta-catenin and the tumour suppressor gene product APC. PMID- 7553659 TI - The role of ICAM expression in immunity and disease. AB - The ICAM family consists of three members. All are ligands for LFA1, and ICAM1 can also bind Mac1. However, the complete repertoire of other ligands has not yet been defined. The ICAMs are not functionally redundant, since they have quite distinct expression profiles and can bind LFA1 differentially. ICAM3 is constitutively and abundantly expressed by all leucocytes, whereas ICAM1 and ICAM2 are expressed at very low levels. ICAM1 is rapidly upregulated by inflammatory cytokines. A current working model for the interplay of the ICAMs is that ICAM3 is the major ligand for LFA1 in the initiation of immune responses and that ICAM1 takes over after these early stages. ICAM3 not only functions as an adhesion molecule, but also is clearly emerging as a potent signalling molecule. PMID- 7553660 TI - Are CD44 variant isoforms involved in human tumour progression? AB - The transmembrane glycoprotein CD44 exists in a variety of isoforms generated by alternative splicing of the pre-mRNA. In a rat metastasis model, certain variant isoforms (containing exon 6v) are causally involved in lung metastasis formation. We have summarized the data obtained to date on the expression of CD44 variant isoforms in human tumour progression. In non-Hodgkin lymphomas, expression of exon 6v containing isoforms is an independent prognostic factor indicating an adverse prognosis. Upregulation of exon 9v containing isoforms in gastric and renal cell carcinomas relates to a poor prognosis of patients. In colorectal carcinomas, CD44-9v isoforms are strongly expressed already in early adenomas; CD44-6v isoforms are upregulated in late adenomas along with ras and TP53 mutations. No expression of variant isoforms has been detectable in neuroblastomas, but significant downregulation of CD44s correlates inversely with tumour progression and N-myc amplification. Only in breast carcinoma has no correlation of CD44 expression with survival or any other prognostic marker been established. Evaluation of CD44 isoform expression by immunohistochemistry in cases of non-Hodgkin lymphoma, gastric, colon and renal cell carcinomas, as well as neuroblastomas, may be a useful diagnostic parameter indicating invasive processes. PMID- 7553661 TI - The deleted in colorectal cancer (DCC) gene: a candidate tumour suppressor gene encoding a cell surface protein with similarity to neural cell adhesion molecules. AB - Chromosome 18q is among the regions thought to harbour a tumour suppressor gene(s) that is frequently inactivated by LOH during the development of several cancer types, including those of the gastrointestinal tract. In addition, colorectal cancers with 18q LOH have been shown to have a more aggressive clinical behaviour than those without 18q LOH. A candidate tumour suppressor gene from 18q, called DCC, has been identified. The DCC gene is contained within the common region of LOH on 18q, its expression is markedly decreased or absent in the majority of colorectal cancers and cell lines and somatic mutations within the DCC gene have been identified in a subset of cases. Thus, DCC represents the strongest candidate tumour suppressor gene on 18q. At present, however, many questions remain regarding the mechanisms underlying the inactivation of DCC and its decreased expression in cancers. The predicted structural similarity of DCC to the NCAMs suggests that it may function through cell-cell and/or cell extracellular matrix interactions; however, little is known regarding the specific cellular function(s) of DCC. Many reports have detailed the alterations in phenotype observed in cancer cells, including changes in cell morphology and tissue architecture, loss of differentiated phenotype, decreased cell adhesion and aggregation, increased motility and invasive behaviour. These altered properties are likely to account in part for the invasive and metastatic properties of cancer cells in the patient. It is hoped that further studies will identify the means by which DCC inactivation may contribute to the altered growth properties of advanced cancer cells. PMID- 7553662 TI - Role of integrins as signal transducing cell adhesion molecules in human cutaneous melanoma. AB - Tumour progression involves a series of sequential steps leading to metastasis. For several of these steps, tumour cells must be equipped with the appropriate adhesive phenotype. Contact with adjacent cells in the primary tumour must be reduced, and invasion and metastasis require adhesive interactions with ECM components. A group of adhesion molecules called integrins is involved in many of these interactions. Integrins are heterodimeric transmembrane molecules that link the cell to the cytoskeleton. They mediate adhesion to ECM components and to other cells. They may be present in an active or inactive conformation, and in addition to adhesive events, they transfer signals into the cell inducing changes in gene expression. Both functions implicate integrins in tumour progression, and their role in cancer has been the subject of many studies over the past 5 years. Several studies of human cutaneous melanoma have demonstrated that the expression of integrins correlates with tumour progression in vivo. Furthermore, integrin expression and function in melanoma cell lines have been found to correlate with invasive or metastatic potential. Finally, evidence from experimental studies in vitro and in vivo shows that integrins have a role in melanoma tumorigenesis, invasion, angiogenesis and metastasis. Integrins might be used as prognostic markers for clinical outcome and they may be useful therapeutic targets in melanoma. PMID- 7553663 TI - Adhesion mechanisms in liver metastasis formation. AB - Our results strongly indicate that integrin mediated adhesion between metastasizing tumour cells and hepatocytes has a decisive role in the formation of liver metastases. However, it is also clear that tumour cells may use different adhesion pathways and furthermore that the adhesion may be modulated by several factors. The role of adhesion has been demonstrated most clearly for LFA1 on T cell hybridomas, which interacts with ICAM1 present in the liver. Alternative pathways must exist, however, given the high invasive capacity of ESb cells, which is apparently LFA1 independent. A possible alternative is adhesion to fibronectin, which is present in abundance on the hepatocyte surface, both in vivo and in vitro. As there is no basement membrane under the endothelium of liver microvessels, so that tumour cells cannot adhere to laminin and collagen type IV as in other organs, adhesion to this fibronectin may be particularly important for metastasis to the liver. Many tumour types can use this pathway, and many different fibronectin receptors may be involved, including VLA-4, VLA-5 and several alpha V integrins. For carcinomas there is another possibility: Fibronectin receptor deficient cells may still adhere using the integrin alpha 6 beta 4, which binds to an unknown ligand present on the hepatocyte surface. Modulation of adhesion can occur in several ways. One example is steric hindrance by mucins that may strongly affect and even abrogate adhesion, despite high levels of appropriate integrins. Another is the activation required for integrins on lymphoma cells, the best known example of which is the activation of LFA1 on T cell hybridomas. It will be evident, therefore, that the role of adhesion in the formation of liver metastases can only be fully understood if the complete set of adhesion molecules on the tumour cells is known as well as their functional status and the possible effects of both cellular and extracellular modulating factors. PMID- 7553664 TI - Convergent signalling in the action of integrins, neuropeptides, growth factors and oncogenes. AB - These findings have important implications for signal transduction and cell regulation. Most obviously, they suggest that tyrosine phosphorylation of a novel type of tyrosine kinase p125FAK is a point of convergence in the action of integrins, oncogenic forms of pp60src, mitogenic neuropeptides and growth factors (Fig. 3). One inference is that the signal transduction pathways initiated by these diverse groups of molecules have, at least in part, similar consequences for cellular function. The notion of convergence is reinforced by the striking similarity in the overall pattern of tyrosine phosphorylation produced through these different pathways. It is tempting to speculate that p125FAK, paxillin and p130 are components in a common programme of phosphorylation events stimulated by integrins, mitogenic neuropeptides and growth factors. The localization of p125FAK to focal adhesions is clearly consistent with a role for this protein as a junction point in the transduction of signals that regulate cell substrate adhesion and ultimately cell motility and cell shape, as suggested in Fig. 3. The existence of distinct pathways leading to p125FAK phosphorylation raises the possibility of synergistic interactions between integrins and G protein coupled receptors. In fact, integrin mediated p125FAK tyrosine phosphorylation appears to be mediated by a PKC dependent pathway (Vuori and Ruoslathi, 1993). By contrast, bombesin and LPA induce tyrosine phosphorylation of p125FAK and paxillin through a PKC independent pathway (Sinnett-Smith et al, 1993; Zachary et al, 1993; Seufferlein and Rozengurt, 1994). It is possible that tyrosine phosphorylation of p125FAK by bombesin, LPA and pp60v-src bypasses and perhaps mimics the phosphorylation caused by integrin activation. Further experimental work will be required to elucidate whether integrins and neuropeptides increase the autophosphorylation of Tyr-397 in p125FAK, as has been recently demonstrated in src-transformed cells (Schaller et al, 1994). Thus, molecular and cellular aspects of the role of p125FAK in signal transduction remain unclear. Specifically, the molecular steps by which different receptors (integrins, seven transmembrane domain receptors and tyrosine kinase receptors) can transduce signals leading to p125FAK tyrosine phosphorylation and the precise role of p125FAK in cell regulation are important areas for further research. Identification of the substrates of p125FAK, which given its localization are likely to reside in or be associated with focal adhesion, will be crucial for elucidating its role in cell regulation. PMID- 7553665 TI - Desmosomes and cancer. AB - Desmosome expression is downregulated in poorly differentiated transitional cell carcinomas and squamous cell carcinomas of the head and neck. In these tissues, desmosomes may have an invasion and metastasis suppressor function. In colorectal carcinoma, no downregulation is found. This raises the question of how tumours possessing intact desmosomes can invade and metastasize. Temporary modulation of adhesive affinity may be a possible mechanism. Desmosomes are widely distributed in epithelia and other tissues and desmoplakins are expressed in all. Thus, antibodies to desmoplakins are very reliable markers for immunohistochemical diagnosis of carcinomas. Intracranial tumours can also be distinguished because meningiomas possess desmosomes and vimentin intermediate filaments, but metastatic carcinomas possess desmosomes and keratin. PMID- 7553666 TI - Inhibitory effect of diglucosylamines on two beta-glucosidases. PMID- 7553667 TI - A reinvestigation towards the conformation of methyl alpha-D-mannopyranosyl-(1- >6)-alpha-D-mannopyranoside by a combined ROE and molecular dynamics analysis. AB - Conformational analysis of alpha-D-Man p-(1-->6)-alpha-D-Man p1-OMe, by a combination of extensive molecular dynamics calculations in water and ROE buildup series, afforded two main minima, namely, phi/psi = 95/-178 and phi/psi = 140/ 185. Transitions between these minima are observed, which have not previously been demonstrated using other approaches. In contrast to literature data for the glycosidic linkage, describing equal populations of both the gg and the gt rotamers, it was found that the gg conformer is present to ca. 96%. The non reducing mannosyl unit showed approximately a 1:1 ratio for the gg:gt equilibrium, in accordance with earlier reports. PMID- 7553669 TI - Structural analysis of mono- and bis-sulfated glycosphingolipids by negative liquid secondary ion mass spectrometry with high- and low-energy collision induced dissociation. AB - Several underivatized mono- and bis-sulfated glycosphingolipids having gangliotriaose or gangliotetraose core structure were analyzed by negative liquid secondary ion mass spectrometry (LSIMS) with high- and low-energy collision induced dissociation (CID). In the normal negative LSIMS spectra, each mono sulfated glycolipid gave abundant [M - H]- ions and each bis-sulfated glycolipid gave abundant [M + Na - 2H]- ions as well as the hydrogen sulfate anion [OSO3H]-. In high-energy CID spectra of the deprotonated molecule, only ions containing a sulfate ester were clearly observed. When a sulfate was present on the non reducing terminal saccharide residue, a series of ions corresponding to sulfated mono- to tetra-saccharides, resulting from sequential cleavage of glycosidic bonds, were observed. If the sulfate was attached to an internal hexose of the sugar chain, the product ions corresponding to the non-sulfated, non-reducing terminal residue were absent. In contrast, the low-energy CID resulted in extremely simple spectra that contained only one or two major product ions characteristic of each sulfated glycolipid. These results provided clear information on the overall sugar and ceramide compositions, and allowed saccharide structures differing in location and number of sulfate esters to be distinguished. PMID- 7553670 TI - Structural studies of the capsular polysaccharide from Klebsiella type 7. AB - The structure of the capsular polysaccharide elaborated by Klebsiella type 7 has been investigated. NMR spectroscopy together with sugar and methylation analysis have been the main methods used. A uronic acid degradation was also employed. The polysaccharide consists of hexasaccharide repeating units having the following structure. [formula: see text] PMID- 7553668 TI - Structural elucidation of underivatized gangliosides by electrospray-ionization tandem mass spectrometry (ESIMS/MS). AB - The structural characterization of intact polysialogangliosides by electrospray ionization mass spectrometry (ESIMS) and tandem mass spectrometry (MS/MS) is described. The negative-ion ESI mass spectra of gangliosides, such as a tetrasialoganglioside GQ1b, showed molecularly related ions with high sensitivity and accuracy. Collision-induced dissociation (CID) MS/MS provided adequate information to characterize the number and positions of sialic acids. ESIMS and CIDMS/MS techniques were applied successfully also in the positive-ion mode for characterization of intact gangliosides. Two isomeric disialogangliosides, GD1a and GD1b, are distinguishable from one another by CIDMS/MS in both negative- and positive-ion modes. The technique is readily applicable to structural analyses such as determining the positional isomers of glycoconjugates. PMID- 7553672 TI - Varying effect of dietary lipids and azoxymethane on early stages of colon carcinogenesis: enumeration of aberrant crypt foci and proliferative indices. AB - Animal models have been used extensively to study the role of diet in the etiology and prevention of colon cancer. It is recognized that several experimental variables affect disease modulation and outcome. Our objective was to determine whether an interaction between the dose of carcinogen used and dietary factors exist, using aberrant crypt foci (ACF) as a biological end point. Sprague-Dawley male rats were injected with a low or a high dose of azoxymethane (AOM, 5 mg or 20 mg per kg s.c.) or saline (0.2 ml/animal s.c.), and randomly allocated to four diet groups (N = 8/group) 1 week later. Diets varied with respect to type of fat (corn or olive oil) and levels of fat: normal (5 g/100 g) corn or olive oil (CO or OO), or high (23.5 g/100g) corn or olive oil (HCO or HOO). After 8 weeks of dietary treatment animals were injected with colchicine (1 mg/kg). Two and a half hours later they were killed and their colons assessed for number of ACF, number of crypt/focus (crypt multiplicity), and the size of ACF as well as the number of cells in metaphase and their location in the crypt section. The low dose of AOM induced fewer ACF and ACF with higher crypt multiplicity than did the high dose of AOM. Dietary fats exerted a variable effect on ACF, depending on the dose of AOM. In low-dose AOM groups, both CO diets exerted a growth-enhancing effect on ACF compared with OO diets.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553671 TI - Gender-related differences in the distribution of thoracic versus abdominal malignant mesothelioma. AB - The relationship between malignant mesothelioma (MM) and asbestos is well established, but the determinants of host factor susceptibility of MM are not. This study probes susceptibility issues by examining gender-related differences in the distribution of 417 thoracic and 42 abdominal cases of MM from 1988-1989 California Cancer Registry databases. The age-adjusted incidence rate ratio (IRR) for male/female thoracic MM was 6.9 (95% confidence interval [CI]; 5.0-9.6) consistent with greater occupational exposure among men. However, the IRR for male/female abdominal MM was 1.5 (95% CI: 0.6-3.6). Also, average age of onset for thoracic MM was greater than for abdominal MM. Thus, some abdominal MMs may be due to nonoccupational asbestos exposure, occurring over a lifetime, interacting with host factor susceptibility. This study gives impetus to research regarding the importance of host factors and nonoccupational asbestos exposure in the etiology of malignant mesothelioma. PMID- 7553673 TI - Detection of human cytomegalovirus in female and male anogenital lesions with papillomavirus-associated dysplastic changes. AB - The aim of the study was to determine the presence of human papillomavirus (HPV) and cytomegalovirus (CMV) DNA in lesions from mucosal and keratinized cutaneous epithelium of the anogenital tract. One hundred ninety-eight women and 55 men with anogenital lesions were enrolled for the study. Biopsies were prepared for histological analysis and in situ hybridization using biotinylated probes. Potential oncogenic HPV were found in 15% of samples showing no outward signs of HPV infection or inflammation. They were detected in 40% of samples showing low grade squamous intra-epithelial lesions (SIL) and in 95 to 100% of lesions showing high-grade SIL. Cytomegalovirus DNA was detected twice as frequently in women as in men, and was preferentially associated with high-grade SIL. A significant relationship between high-risk HPVs and the presence of CMV was observed in the low-grade lesions. Our data suggest that the CMV genome in the host epithelial cells might act as a permissive factor upon which oncogenic HPV could interact over time to increase the frequency and severity of cervical, penile, and anal dysplasia. PMID- 7553674 TI - A DNA probe study on the origin of the cancer recognition, immunedefense suppression and serine protease protection peptide. AB - A quantitative "two DNA probe" dot blot hybridization assay, using chemiluminescence detection, was used to distinguish between the mRNA and DNA coding for the cancer recognition, immunedefense suppression, and serine protease protection (CRISPP) peptide, and the partially homologous C-terminal end sequence of the alpha1-PI. In cancer cells, there is up to 2.4 times more mRNA and up to 6.3 times more DNA coding for the CRISPP peptide than for the homologous alpha1 PI in their normal cell counterparts. This corroborates results of the "two antibody" immunoassay, which showed that cancer cells produce the CRISPP, peptide in addition to alpha1-PI molecules. The amplified transcription of the CRISPP peptide DNA could be the result of derepression and/or translocation of an inherited, or acquired, heritable damage to the DNA in the exon V of the alpha1 PI gene. Expression of the CRISPP peptide mRNA and DNA is SV40 transformed cells could indicate that a viral transduction causes the amplified transcription of a mutated exon V of the alpha1-PI gene and/or loss of a specific suppressor gene. The CRISPP peptide with its multiple, cancer-promoting biological effects may qualify as a product of a novel, as yet unidentified, oncogene. The detection of the CRISPP peptide DNA regions, using the two DNA probe, or some other suitable methods, might be a useful adjunct in cancer diagnosis of biopsies and/or in predictive diagnosis of familial cancers. PMID- 7553675 TI - Usefulness of coagulation markers in staging of gastric cancer. AB - Thirty-six patients with nonmucinous adenocarcinoma of the stomach, candidates for surgical laparotomy, were studied to evaluate the presence and extent of coagulation disorders in gastric cancer. They were staged according to TNM cancer staging (T: extent of primary tumor; N: lymph node involvement; M: presence of metastases), and a blood sample was collected before surgery. Platelets, platelet factor four (PF4), beta-thromboglobulin (BTG), activated partial thromboplastine time (APTT), prothrombin time (PT), factors five (V) and seven (VII), fibrinogen, cross-linked fibrin degradation products (XDP), fibrinopeptide-A (Fp-A), and antithrombin three (AT III) were assayed. Only fibrinogen, Fp-A, PF4, and factors V and VII were increased in more than 50% of patients. Fibrinogen and Fp-A were positively correlated with T(r = 0.29, p < p < 0.05; and r = 0.35, p < 0.05; respectively), whereas the other parameters did not show any statistically significant relationship with T, N, and M. Considering the subgroups including only the patients with pathological values, Fp-A (31 patients) was positively correlated with N (r = 0.4, p < 0.05), PF4 (25 patients) showed a positive correlation with T and N (r = 0.42, p < 0.05; r = 0.46, p < 0.05; respectively), and a significantly higher median in the presence than in the absence of metastases (median in the M+ subgroup: 42.7 ng/ml, range 38.6 to 102.8; median in the M- subgroup: 33.7, range 20.3 to 85; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553677 TI - Influence of different combinations of tamoxifen and toremifene on estrogen receptor-positive breast cancer cell lines. AB - Acquired tamoxifen (TAM) resistance is supposed to be the major cause of hormone therapy failure in estrogen receptor (ER)-positive breast cancer patients. Toremifene (TOR), a chlorinated TAM-related compound, has been found to be more effective and less toxic than TAM. Moreover 4-hydroxy-toremifene (OH-TOR), like 4 hydroxy-tamoxifen (OH-TAM), is the most effective metabolite in human. To better understand the relative role of TAM, TOR, OH-TAM, and OH-TOR, singly or in combination, we studied their effect on MCF7, ZR-75.1, and T47D cell lines, which, despite their positive receptor status, have a different responsiveness to estradiol and antiestrogenic compounds. The results may be summarized as follows; in MCF7 cells, all compounds, singly or in association, showed an inhibitory effect; ZR75.1 cells were resistance to TAM and OH-TAM, but partially sensitive to TOR and OH-TOR; in T47D cells, all compounds displayed their estrogenic activity and induced cell growth. These result suggest the inefficacy of these triphenylethylene derivatives as a hormone treatment even when given in a simultaneous or sequential combination. PMID- 7553676 TI - Flexible sigmoidoscopy or colonoscopy for colorectal screening: a randomized trial of performance and acceptability. AB - In a randomized clinical trail to assess acceptability, yields, costs, and unwanted effects of screening procedures, 232 subjects (137 with family history of colorectal carcinoma or adenoma, 95 without) were offered either flexible sigmoidoscopy or colonoscopy. Subjects with polyps found on sigmoidoscopy were followed up by colonoscopy. The two procedures were similar in compliance (65%) and yield (19% adenoma, 15% hyperplastic polyps). Polyps of either type were more common in those with a family history (prevalence: 41% compared with 24% without family history, p = 0.04). Costs per procedure were 60% lower for sigmoidoscopy, but follow-up colonoscopy reduced this cost advantage to 20% per subject. The subjects found the preparation for sigmoidoscopy easier, but the procedure more uncomfortable and embarrassing, as colonoscopy was performed under sedation. In this hospital-based study, colonoscopy was as acceptable to subjects, and only slightly more costly than sigmoidoscopy. Advantages of sigmoidoscopy would be greater for use outside hospitals and with less intensive follow up. PMID- 7553679 TI - New thermoluminescent BaSO4:Eu sheet for in vivo measurement of spatial dose distribution in radiation therapy. AB - A new highly sensitive thermoluminescent (TL) sheet has been studied as a means of in vivo measurement of spatial dose distribution of radiation therapy. This TL sheet (40 cm x 50 cm x 0.2 mm), which is composed of teflon mixed with BaSO4:Eu doped powder, is very flexible and can be cut to the desired size. The TL sheet was found to have a linear response with a very wide dynamic range of at least 0.002 cGy to 5000 cGy absorbed dose. In addition, this sheet does not need be shielded, because of its insensitivity to room light, and therefore the sheet can detect low-energy electrons decreased by a few millimeter thick air for 3H(maximum beta-ray energy: 18 KeV). The spatial dose distribution was printed out with a newly developed digital readout system. In addition, another high resolution dosimetry system, which exposes X-ray film with TL photons emitted from the irradiated TL sheet at constant room temperature, is reported. Clinically, the in vivo dose distribution on the surface of the rectal cancer for intracavitary radiation therapy was determined. The applicability of TL sheet for in vivo measurement of dose distribution is discussed. PMID- 7553678 TI - Differentiation and tumor response to retinobenzoic acid RE-80 in a malignant conversion model. AB - The synthetic retinobenzoic acid RE-80 was evaluated for its potential as an inductor of tumor cell differentiation and as a chemopreventive agent. A minimally toxic dose of RE-80 in vitro produced morphologic changes typical differentiation in epidermal tumor cell colonies. Indirect immunofluorescence indicated induction of a differentiation-associated keratin of internal stratified epithelia, K13, and inhibition of the differentiation-associated epidermal keratin K1. Cultured cells were skin-grafted to athymic nu/nu mice to evaluate RE-80 effects on early stage malignant progression in vivo. When tumors had grown to 3 to 4 mm in diameter, mice were treated by intraperitoneal injection with RE-80 (67 micrograms, 170 mmol, i.p., two times per week) or vehicle (100 microliters 20% ethanol). Papillomas (benign) and moderately differentiated squamous cell carcinomas were reduced in volume about 4-fold by RE 80 treatment. Larger, poorly differentiated squamous cell carcinomas were unaffected. RE-80 resulted in a lower proportion of proliferative cells (detectable by bromodeoxyuridine incorporation) and a higher proportion of moderately to well differentiated tumors after 40 days of treatment compared with control tumors, which were mainly poorly differentiated squamous cell carcinomas. K13 induction in vitro was correlated with response to retinoid in vivo. Induction of differentiation may be mechanism of the response to RE-80 in vivo since carcinoma cells expressing K13 did not incorporate bromodeoxyuridine and were on a terminal pathway. PMID- 7553680 TI - T/Tn antigen vaccine is effective and safe in preventing recurrence of advanced breast carcinoma. AB - Since 1974, and as of March, 1993, we have used T/Tn antigen vaccine in safe, specific, effective, long-term intradermal vaccination against recurrence of advanced breast carcinoma (CA). Staging is by the pathologic TNM system. Treatment is ad infinitum. Of 19 consecutive breast carcinoma patients vaccinated, six Stage IV, six Stage III, and seven Stage II all survived > 5 years postoperatively. Three Stage III, three Stage IV, and five Stage II patients (i.e., 11) survived > 10 to > 18 years. Five others are alive but have not reached 10 years; three of them have no evidence of disease (NED). Three patients died of CA before reaching 10 years. An additional three breast CA patients are being treated for > 2 years, but, < 5 years postoperatively, they are NED. The vaccination are presented as a delayed-type hypersensitivity reaction with significant inflammation with increase of helper T lymphocytes and decrease of T suppressor/cytotoxic cell ratio. PMID- 7553682 TI - p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer. AB - The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P = 0.003) at a level of 8900 molecules, 240 (P = 0.005) at a level of 2900 molecules and 1801 (P = 0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay. PMID- 7553681 TI - The relationship between flow-cytometric and immunohistochemically detected c erbB-2 expression, grade and DNA ploidy in breast cancer. AB - Quantification of c-erbB-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erbB-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erbB-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erbB-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erbB-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P = 0.06). Immunohistochemistry revealed a significant relationship between c-erbB-2 expression and aneuploidy (P = 0.04). Cytokeratin-positive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erbB-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erbB-2 expression at a level of more than 3200 molecules (P < or = 0.03). At the level of 3600 molecules significance was increased (P = 0.004). These levels equated with between 15% and 19% of the samples being classified as c-erbB-2-positive. Application of these cut-off points showed no correlation between c-erbB-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P = 0.0015), increased grade correlating with aneuploidy. PMID- 7553683 TI - Expression of cytokine mRNA in human melanoma tissues. AB - We have reported that patients with metastatic melanoma treated with an autologous, dinitrophenol-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with tumor cell destruction. We tested biopsy specimens of eight subcutaneous metastases that had developed inflammation following vaccine treatment for expression of mRNA for interferon gamma (IFN gamma), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF alpha), and IL-10. Post-vaccine, inflamed biopsies contained mRNA for IFN gamma (5/8), IL 4 (4/8) or both (3/8), and for TNF alpha (4/7). In contrast, IFN gamma mRNA was detected in only 1/17 and TNF alpha mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL-10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ the reverse transcriptase/polymerase chain reaction confirmed that melanoma cells were the major source. These findings may provide a new parameter by which to measure the effects of cancer immunotherapy. PMID- 7553684 TI - Immunoscintigraphy of colorectal cancer using 111In-labeled monoclonal antibody to mucin. AB - A murine monoclonal antibody MLS102 recognizes sialosyl-Tn antigen in mucin and immunohistochemically reacts with more than 80% of colorectal cancer tissues. The purpose of this study was to assess the usefulness of this monoclonal antibody for the immunoscintigraphy of colorectal cancer. Planar and SPECT images were obtained on day 2 or day 3 after injection of 2 mg and 74 MBq 111In-labeled MLS102 antibody into 17 patients with colorectal cancer. Nine of 11 primary tumors and 4 of 6 locally recurrent tumors were detected. Positive images were obtained in all tumors larger than 4.5 x 2.7 cm. Three tumors of less than 2.5 cm and 1 recurrent tumor, which was missed by other imaging modalities, were negative. There were no adverse reactions. Human anti-(mouse Ig) antibody developed in 4 patients. Although improvement of detectability for smaller tumors needs to be pursued, the antibody MLS102 is potentially promising for use in immunoscintigraphy of colorectal cancer. PMID- 7553686 TI - Radioimmunotherapy of small-cell lung cancer xenografts using 131I-labelled anti NCAM monoclonal antibody 123C3. AB - We have studied the therapeutic efficacy of 131I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation doses were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2 x 500 microCi, resulted in complete remission of tumours smaller than 200 mm3 and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3 x 600 microCi). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 microCi radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity. PMID- 7553685 TI - Immunotherapy of human colon cancer by antibody-targeted superantigens. AB - T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma. PMID- 7553689 TI - The immunomodulatory effect of levamisole is influenced by postoperative changes and type of lymphocyte stimulant. AB - The results of both laboratory and clinical research into the immunomodulatory activity of levamisole have shown a considerable degree of inconsistency and sometimes contradiction. This is probably a reflection of the lack of understanding of the mechanism(s) of action of levamisole and it is therefore necessary to base conclusions about its immunomodulatory efficacy in the treatment of disease on experimental assays that take into consideration the in vivo conditions. This investigation was designed to compare the immunomodulatory activity of levamisole under clinically achievable and non-achievable conditions as judged by changes in the perioperative proliferative response of lymphocytes from 30 patients with colorectal cancer. The results obtained showed that proliferation in antigen (purified protein derivative, PPD)-stimulated, but not phytohaemagglutinin(PHA)- or staphylococcal-enterotoxin-B(SEB)-stimulated, lymphocyte cultures was consistently and significantly augmented by levamisole in concentrations of 25 ng-25 micrograms/ml. High concentrations of levamisole (25 micrograms/ml and 100 micrograms/ml) were inhibitory to PHA- and SEB-stimulated, but not PPD-stimulated, lymphocyte cultures, especially in the postoperative period. Of particular interest was the observation that, although levamisole temporarily lost its stimulatory activity in the postoperative period (third postoperative day), it did enhance antigen-stimulated lymphocytes at the time of the nadir of the postoperative suppression of lymphocyte proliferation (first postoperative day). Clinically achievable concentrations of levamisole are therefore effective both before and after operation in enhancing the response of lymphocytes to antigens. PMID- 7553690 TI - Human tumor vaccines go molecular. Summary of American Association for Cancer Research Symposium, March 20,1995. PMID- 7553691 TI - [Psychological stress as precipitating factor of the acute manifestations of ischemic cardiopathy]. AB - Psychological stress may play an important role as a risk factor for coronary heart disease (CHD). Psychological stress consists of different components whose diverse role in the onset of the acute manifestations of CHD is still not clear. We studied 80 patients with acute CHD (56 with unheralded myocardial infarction, 24 with unstable angina, mean age 55 +/- 9 years, 55 men) vs 80 controls (mean age 50 +/- 9 years, 52 men) admitted for an acute traumatic event. We proposed to both groups these questionnaires: a retrospective self and etero-evaluation of the degree of psychological stress; the modified Maastricht questionnaire (MMQ) that evaluates psychic and physical manifestations of psychological stress in the period preceding admission; the social support questionnaire (SSQ); the life event assessment that evaluates frequency and importance of life events in the year preceding admission. Psychological stress index was greater in patients (self-evaluation = 7.1 +/- 2.4; etero-evaluation = 7.4 +/- 2.3; MMQ = 91 +/- 30.8) than controls (self-evaluation = 4.3 +/- 2.3; etero-evaluation = 4.3 +/- 2.4; MMQ = 58.6 +/- 32.5; p < 0.001 for all questionnaires). The perception of strong social isolation was greater in patients (SSQ = 29.8 +/- 11.8) than controls (SSQ = 23.8 +/- 9.6; p < 0.001). Painful life events were more numerous in patients (10.7 +/- 2.3) than controls (8.2 +/- 3.4; p < 0.05) and perceived in a more negative way (patients = 12.7 +/- 4.1 vs controls = 10.2 +/- 3.8; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553687 TI - Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-Hodgkin's lymphomas. AB - T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon gamma (IFN gamma), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3. PMID- 7553688 TI - Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients. AB - Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), dot-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins. PMID- 7553693 TI - [The reduction of respiratory function is an independent risk factor in the elderly]. AB - The predictive value of the impairment of lung function and cigarette smoking has been evaluated in 2080 elderly subjects aged > or = 65 years included in the CASTEL (Cardiovascular Study in the Elderly). Some common risk factors, as well as forced expiratory volume, vital lung capacity, and Tiffenau index have been examined with Cox analysis. The 10-year survival equation was as follows: [formula: see text] In disagreement with other studies, in our survey smoking was not a predictor of mortality, although it was able to reduce survival by reducing respiratory function. PMID- 7553692 TI - [ECG-dipyridamole and ECG-exercise test in the assessment of ischemic cardiopathy: effects of the acute administration of nitroglycerin]. AB - It is known that intravenous administration of dipyridamole can induce chest pain and ECG signs of ischemia in patients with coronary artery disease. In the present study we evaluated ECG and hemodynamic changes in response to dipyridamole (0.56 mg/kg in 10 min) under basal conditions and 3 hours after administration of nitroglycerin (10 mg/24 h patch) in 14 patients with coronary artery disease. The effects of nitroglycerin were also compared to those induced by the same drug on a bicycle stress test in the same patients. Exercise stress test induced specific ST changes in all patients when performed off-drug. Nitroglycerin administration completely prevented exercise-induced ischemia in 2 patients, and significantly prolonged exercise time in the remaining patients (p < 0.01). This effect was accompanied by a significant increase in heart rate (HR) and rate-pressure product at the threshold of ischemia (HRBP, p < 0.01); furthermore we observed a significant increase in HR at the maximal work load (p < 0.05). In the absence of treatment, dipyridamole infusion induced ST segment changes and/or typical chest pain in 12/14 patients. Moreover we observed a significant increase (p < 0.05) in HR, BP and HRBP during the test with respect to basal conditions. Following nitroglycerin administration, dipyridamole infusion failed to induce ischemia in 4 patients, and the time to ST depression in the remaining 8 patients (459 +/- 69 vs 610 +/- 127 s; p < 0.05) was significantly prolonged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553694 TI - [Assessment of dispersion++ of ventricular recovery in patients with chronic obstructive pulmonary disease]. AB - Cardiac arrhythmias are common in patients with chronic obstructive pulmonary disease. Several factors may contribute to the development of arrhythmias in these patients including hypoxemia, hypercapnia, acid-base disturbances. The aim of the study was to assess QTc dispersion in patients with chronic obstructive pulmonary disease, who are at high risk of arrhythmias. Interlead QT variability as measured on the 12-lead electrocardiogram is not a technical artifact but probably reflects regional differences in ventricular repolarization. Increased dispersion of ventricular recovery time is believed to provide a substrate which supports serious ventricular arrhythmias. We showed that in patients with chronic obstructive pulmonary disease, free of underlying structural heart disease, the QTc dispersion is significant greater (p = 0.036) than in a control group, despite a similar value in mean QTc value. The data of the present study suggest that the determination of dispersion of repolarization times may be affected by metabolic changes occurring in patients with chronic obstructive pulmonary disease. PMID- 7553696 TI - [Conservative surgical treatment of rheumatic mitral stenosis]. AB - We prospectively evaluated 50 patients with mitral stenosis (43 women and 7 men; mean age 45 years) to assess the results of surgical reconstruction of the mitral valve. All patients underwent a complete echocardiographic examination before and after operation. Surgical reconstruction was extensive, and included commissurotomy, thinning of the valvular leaflets, calcification removal, splitting of subvalvular apparatus, and posterior annuloplasty. Surgical reconstruction resulted in increasing mitral functional area from 0.89 +/- 0.23 to 2.07 +/- 0.42 cm2. NYHA functional class decreased from 2.76 +/- 0.55 to 1.52 +/- 0.71. Before discharging, 10% of patients had moderate mitral insufficiency. All patients were followed at 6-month intervals in our clinic. Mean follow-up was 37 +/- 18 months. During follow-up 5 patients (10%) developed severe mitral incompetence, which required mitral valve replacement. Chi-square and Student t test were used to analyze the correlation between variables and outcome. The occurrence of severe mitral incompetence was correlated with: the degree of enlarged left atrium; chronic atrial fibrillation; postoperative more than mild mitral regurgitation. No correlation was found with anatomical parameters detected by echocardiography, or intraoperative anatomy. In conclusion, surgical reconstruction of mitral stenosis provides satisfactory short-term results. We believe that the low mortality rate and the low incidence of complications justify an effort to save the native mitral valve before considering prosthetic replacement. More attention to the development of residual mitral incompetence with intraoperative control may improve long-term results. PMID- 7553697 TI - [Pericardial effusion in heart surgery interventions]. PMID- 7553695 TI - [Cardiodepressive effects of muscular exercise in spontaneously hypertensive rats]. AB - Conflicting results have been reported on the differences in the cardiovascular reactivity to muscular exercise between normotensive and hypertensive subjects and normotensive subjects with family history of hypertension and normotensive subjects without familial hypertensive history. Also studies performed in spontaneously hypertensive in comparison with normotensive rats have not led to final conclusions. Therefore, the aim of the present study was twofold: to evaluate cardiorespiratory reactivity to different types of muscular exercise in normotensive (WKY) compared to spontaneously hypertensive rats (SHR) at pre hypertensive and hypertensive stages; to verify the role played by the "muscular reflex drive" in mediating the cardiorespiratory responses in rats. We utilized four groups of anesthetized rats: 8 young WKY, 8 SHR in pre-hypertensive stage, 6 adult WKY and 6 SHR in hypertensive stage. We evaluated the cardiorespiratory responses to (rhythmic) dynamic and (static) isometric contractions of gastrocnemius muscles induced by electrical stimulation of the tibial nerve. Cardiorespiratory responses during the initial phase of dynamic and static contractions of hindlimb muscles were studied. Muscle contractions were elicited by stimulating the tibial nerves at 3 and 100 Hz. We measured: mean arterial pressure (MAP), heart rate (HR), and pulmonary ventilation (VE). Both types of exercise caused a significant decrease in MAP and a significant increase in VE while HR did not change significantly. The four groups of rats did not show significant differences in the pattern of cardiorespiratory responses to muscular exercise. The cardiorespiratory reflexes initiated by activation of muscle receptors, verified by interrupting the afferents from the contracting muscles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553700 TI - Coronary artery disease in women: a population-based perspective. AB - Coronary artery disease (CAD) remains a disease of major public health importance among women. In light of this fact, attention has focused recently on the differential expression of the disease in women as compared to men. This review will attempt to describe the characteristics of CAD among women from a population based perspective. PMID- 7553699 TI - Cardiovascular disease in women: facts and statistics. PMID- 7553698 TI - Diagnosis of persistent left superior vena cava by multiplane transesophageal echocardiography. AB - Persistent left superior vena cava occurs in 2-4% of all congenital cardiac defects. The diagnosis is often missed if the lesion is not accurately looked for. Drainage to the coronary sinus is well tolerated, whereas drainage to the left atrium produces right to left shunt and may be associated with brain abscesses and/or embolization secondary to intravenous therapy administered through the left arm. This paper describes for the first time the diagnosis of persistent left superior vena cava by transesophageal contrast echocardiography. Echocardiography is the method of choice in the diagnosis of persistent left superior vena cava, because it can be used at the patient's bedside and allows easy detection of the drainage site. PMID- 7553703 TI - Primary prevention of coronary heart disease in women. AB - Despite the fact that coronary heart (CHD) disease is the leading cause of death among postmenopausal women, research on primary prevention of CHD in women has been relatively sparse. Prevalence of CHD risk factors such as cigarette smoking and hypertension remain high among US women. Moreover, common factors unique to women including pregnancy, oophorectomy, menopause, and use of steroid hormones, appear to have an impact on CHD risk that is often poorly understood. While postmenopausal hormone therapy is increasingly recommended for the prevention of CHD, the potential impact of nonpharmacologic measures such as changes in diet, exercise, and cigarette smoking have been relatively unexplored. PMID- 7553702 TI - Myocardial infarction in women. AB - The leading cause of death in the United States is cardiovascular disease, regardless of gender. Women will more often have angina preceding their first myocardial infarct, but have more unrecognized infarctions than men. Women will be older, have more concomitant disease and present later in the course of their acute myocardial infarction. Although myocardial infarction may have similar clinical presentations in men and women, there are some important differences such as an increased incidence of non Q-wave myocardial infarctions in women, and the higher rate of complication and immortality from a myocardial infarct. Women develop less collateral cardiac vessels, which may explain the increased rate of hemodynamic complications after a myocardial infarct. Both early and late survival rates are also affected by the advanced mean age of women when they do present with a myocardial infarct. There is less data regarding treatment modalities based upon gender differences but in general, it appears that women benefit from established treatment modalities in the acute setting and subsequent to the event. It has been determined that there are epidemiologic differences, but pathophysiologic and clinical factors specific to women in this setting have yet to be determined. PMID- 7553704 TI - Coronary thrombolysis in the treatment of women with acute myocardial infarction. AB - Coronary thrombolysis is a mainstay in the treatment of acute myocardial infarction occurring in men and women. Despite a higher in-hospital mortality that cannot be explained completely by age and comorbid illnesses, women clearly benefit. The relatively high rate of hemorrhagic complications requires further thought and investigation. PMID- 7553701 TI - Education and clinical research issues in women's health. AB - The evolution of women's health both in the United States and abroad is a direct result of an intense commitment to scientific investigation and education. Society serves to benefit greatly from this global effort. PMID- 7553705 TI - Heart failure in women. AB - Observational and other studies suggest gender-related differences in the incidence and prognosis of heart failure. Women appear to live longer after the diagnosis of heart failure when compared with men. After myocardial infarction, women seem more likely than men to exhibit clinical heart failure. Diabetes appears to promote heart failure to a greater extent in women than in men. Review of data from clinical and epidemiologic studies suggests that men and women may differ in their myocardial adaptation to a variety of cardiac insults. Future investigation is necessary to better define gender-related differences and possible sex-specific therapies for those diseases resulting in heart failure. PMID- 7553706 TI - Gender differences in older patients with pressure-overload hypertrophy of the left ventricle. AB - Left ventricular hypertrophy is an adaptive response to increased intraventricular pressure, and is consistently encountered in patients with hypertension or aortic stenosis. While it has long been appreciated that the extent of hypertrophy correlates positively with the level of pressure, the age of the individual, and with indices of body size, recent evidence suggests that left ventricular structure and function also varies with gender. Elderly women with aortic stenosis, in several recent studies, have been shown to develop more marked concentric hypertrophy, lower levels of wall stress, and higher indices of systolic function. Similarly, in recent studies of extreme hypertensive hypertrophy, there is a strong female preponderance. The explanation for these findings is not clear at present, though it is possible that men with extensive hypertrophy have succumbed to complications of vascular disease at an earlier age. The syndrome of extreme hypertrophy complicating hypertension and aortic stenosis, more commonly seen in women, requires a carefully tailored clinical approach. PMID- 7553707 TI - Diagnostic evaluation of women with suspected coronary artery disease. AB - Women are less likely than men to be referred for coronary angiography. It is unclear whether this reflects good clinical judgment or agenda bias. The decision to refer a patient for an invasive evaluation depends upon the prevalence of disease and the population in question, the prognosis of patients with coronary artery disease and the response of these patients of the available therapies. This paper reviews the statistical basis for clinical decision making and the available epidemiologic and risk factor data in women with suspected coronary artery disease. The results of noninvasive testing in women are also reviewed. Finally, guidelines are suggested to help determine when a woman with suspected coronary artery disease should be referred for coronary angiography. PMID- 7553708 TI - Coronary angioplasty and surgical revascularization: emerging concepts. AB - Early reports from the NHLBI PTCA Registry showed that women undergoing percutaneous transluminal coronary angioplasty (PTCA) had lower acute success rates and higher procedural complication rates than men. Similarly, most larger scale studies that have evaluated the differences in outcome between men and women undergoing surgical revascularization have noted a higher operative mortality rate in women. Some, but not all, of these differences are secondary to the higher baseline cardiovascular risk profile and smaller coronary artery diameter found in women. This article reviews the evolution of coronary angioplasty and bypass graft surgery as viable revascularization strategies in women with a focus on gender differences in baseline characteristics, acute outcome and long-term efficacy. PMID- 7553709 TI - Cerebrovascular disease in women. AB - Cerebrovascular disease is one of the leading causes of death in women in the United States. Pregnancy and hormonal therapy have been associated with an increased risk of stroke in young women. Other conditions, more common in women, such as migraine headaches, rheumatologic disorders and antiphospholipid antibodies predispose women to cerebral ischemia. Women may also respond differently to antiplatelet therapy. The identification and treatment of such conditions will improve outcome in women with stroke and may prevent recurrent cerebrovascular events. PMID- 7553711 TI - Activation of ATP-sensitive potassium channels: a novel pharmacological approach to myocardial protection? PMID- 7553710 TI - Peripheral arterial-vascular disease in women: prevalence, prognosis, and treatment. AB - Lower extremity atherosclerosis results in significant morbidity in women, particularly in women following the menopause. Up to 25% of women aged 55 to 74 years are affected by this disease. When noninvasive testing is used to determine the prevalence of lower extremity atherosclerosis, and men in this age group are equally represented. Cigarette smoking, diabetes mellitus, and menopause are risk factors for atherosclerosis of particular interest in women. The prevalence of cigarette smoking is rising rapidly among women, and diabetes appears to be a greater risk factor for atherosclerosis in women than in men. Risk factor reduction, in addition to an exercise program, are important parts of the treatment program for stable claudication. In both men and women with more severe symptoms, an ankle/branchial index (ABI) of less than 0.3 is associated with more severe symptoms, an ankle/brachial index (ABI) of less than 0.3 is associated with a poor prognosis. Men and women fare equally well following revascularization for severe peripheral atherosclerosis. However, there are some data to suggest that women may be offered peripheral revascularization at a lower rate. PMID- 7553713 TI - Cardiac hypertrophy impairs recovery from ischaemia because there is a reduced reactive hyperaemic response. AB - OBJECTIVE: The aim was to investigate whether impaired coronary flow reserve associated with cardiac hypertrophy could significantly limit the flow debt repayment following short periods of coronary occlusion and exacerbate or prolong episodes of myocardial ischaemia. METHODS: Left ventricular hypertrophy was induced in guinea pigs by aortic constriction and the hearts were isolated six weeks later for Langendorff perfusion. Sham operated animals served as controls. The reactive hyperaemic response was studied following various lengths of occlusion of flow and the extent of aerobic and anaerobic metabolism was assessed in each group. RESULTS: Heart weight/body weight ratio was increased by approximately 25% (P < 0.001) with aortic constriction. The reactive hyperaemic flow response in isolated hearts was impaired by hypertrophy in both magnitude (P < 0.05) and duration. The repayment of flow debt was also significantly reduced, suggesting an inadequate recovery of the myocardium following the occlusion. The total amount of O2 consumed by the heart throughout the duration of hyperaemia was less in hypertrophy than in sham operated controls, suggesting a decrease in aerobic metabolism. Total lactate discharge expressed as a ratio of O2 consumed, which provides an estimation of the degree of anaerobic in relation to aerobic metabolism, was greater in hypertrophy than in sham operated controls. CONCLUSIONS: The hypertrophied heart is more vulnerable to brief periods of ischaemia because of an impaired reactive hyperaemic response which results in delayed metabolic recovery. These abnormalities may contribute to the increased morbidity associated with cardiac hypertrophy. PMID- 7553712 TI - Estimation of splanchnic blood flow by the Fick principle in man and problems in the use of indocyanine green. AB - OBJECTIVES: Measurement of splanchnic blood flow is necessary to evaluate the effect of therapeutic interventions on splanchnic tissue perfusion. Systemic indocyanine green (ICG) clearance has been used to estimate splanchnic blood flow, but the results may be compromised by altered hepatic dye extraction. We evaluated the applicability of simultaneous estimation of splanchnic and femoral blood flow by dye dilution and regional blood sampling in intensive care patients. METHODS: 240 simultaneous determinations of regional blood flow were conducted in different patient groups (cardiac surgery, ARDS, pancreatitis, septic shock, preoperative controls). The measurement protocol consists of catheterizations of hepatic vein, femoral artery and vein and primed constant infusion of two different ICG preparations. RESULTS: The method was used successfully in a wide variety of patients. Steady-state dye concentration and sufficient dye extraction was achieved in each group of patients. The coefficient of variation of splanchnic blood flow estimation was 7 +/- 1% and of femoral blood flow estimation 6 +/- 0%. There was a great intra- and interindividual variation of ICG extraction. Use of dobutamine modified the extraction in most patients but did not lessen the performance of the method. ICG extraction was markedly lower and the coefficient of variation of both femoral and splanchnic blood flow markedly higher with propylene glycol-dissolved ICG preparation as compared with the freeze-dried. CONCLUSIONS: The prerequisites for the method of primed, constant infusion of indocyanine green with hepatic vein catheterization are achieved in intensive care patients. The results of splanchnic blood flow estimations based on techniques with peripheral blood sampling should be interpreted with caution, and the use of ICG clearance as a flow-related indicator without the measurement of ICG extraction cannot be justified because of the great variability of dye extraction. Certain indocyanine green preparations may greatly modify the results of the regional blood flow determinations. PMID- 7553714 TI - Vascular acetylcholine response during chronic NO synthase inhibition: in vivo versus in vitro. AB - OBJECTIVE: The aim of this study was to compare the response to NO-mediated vasodilators in vivo and in vitro during chronic NO synthase inhibition. METHODS: NG-Nitro-L-arginine-methyl ester (L-NAME, 0.4 g/l) or vehicle was administered in the drinking water for 6 weeks to male Wistar rats weighing 220-240 g. The effect of acetylcholine and sodium nitroprusside was examined in vivo, on systemic blood pressure and heart rate and in vitro, on the precontracted isolated mesenteric artery. The in vivo response to both vasodilators was examined in awake rats monitored by an indwelling catheter in the femoral artery. Isolated segments of the third-generation mesenteric artery were examined in vitro with a Mulvany dual myograph after precontraction with noradrenaline. RESULTS: In isolated mesenteric arteries obtained from rats chronically treated with L-NAME, the initial relaxant response to acetylcholine was significantly decreased whereas that to sodium nitroprusside was enhanced. A late acetylcholine-induced contractile response was present and abolished by indomethacin. In vivo, the hypotensive action of sodium nitroprusside was also enhanced in the L-NAME-treated rats. Acetylcholine reduced blood pressure in the L-NAME-treated hypertensive animals more than in normotensive controls, but less than in control rats infused intravenously with noradrenaline at a dose increasing their blood pressure to hypertensive levels. CONCLUSIONS: The NO-mediated vasodilation induced by acetylcholine is attenuated during chronic NO synthase inhibition, both in vivo and in vitro. The blunted hypotensive response to acetylcholine can be demonstrated only if blood pressure of control rats is acutely increased to hypertensive levels. PMID- 7553715 TI - Evidence for sodium-dependent hypoxanthine uptake in isolated guinea pig ventricular myocytes: stimulation by extracellular Ni2+. AB - OBJECTIVES: Concentrative transport systems for nucleobases have been identified in renal epithelial cells and jejunal tissue rings. However, the existence and role of nucleobase transport has yet to be described in the heart. The aims of this study were, therefore, to use isolated guinea pig myocytes to: (i) identify whether such a mechanism is present in the heart and (ii) characterise and compare its activity to that already described in other tissues. METHODS: The Na(+)-dependent and Na(+)-independent uptake of [3H]hypoxanthine was quantified in ventricular myocytes. These were isolated from the hearts of adult guinea pigs using a collagenase-based enzymatic digestion technique. The specific incorporation of cellular radioactivity, in the presence and absence of extracellular sodium and other pharmacological agents, was determined by liquid scintillation counting. RESULTS: A system that accumulates hypoxanthine against a concentration gradient in the presence of extracellular sodium was identified. Furthermore, a sodium-independent component of hypoxanthine uptake was also characterised, a proportion (approximately 50%) of which was inhibited by adenine (3 mmol/l). In the absence of sodium and the presence of adenine, the remaining fraction of hypoxanthine uptake was assumed to occur via diffusion. The sodium dependent uptake of hypoxanthine was saturable at 25 degrees C with a Km of 8.1 +/- 2.1 mumol/l and a Vmax of 50.1 +/- 5.2 pmol/mg protein/min. Dipyridamole inhibited sodium-dependent uptake but had no effect on sodium-independent flux. The Na(+)-independent system was saturable with a Km of 157.3 +/- 6.2 mumol/l and a Vmax of 233.6 +/- 5.6 pmol/mg protein/min. Extracellular Ni2+ stimulated sodium dependent uptake in a concentration-dependent manner (0-100 mEq/l). Another divalent cation (Ba2+, 100 mEq/l) had no effect on the either Na(+)-dependent or the Na(+)-independent component. CONCLUSIONS: These data provide the first evidence for sodium-dependent nucleobase co-transport in guinea pig myocytes. The sensitivity of this transporter to Ni2+ has not been described previously and may provide a pharmacological tool to study this uptake mechanism further. PMID- 7553716 TI - Non-uniform recovery of segment shortening during reperfusion following regional myocardial ischaemia despite uniform recovery of ATP. AB - OBJECTIVE: This study focused on transmural postischaemic recovery of ATP and regional contractile function related to reversible and irreversible tissue injury. METHODS: Fifty anaesthetised open-chest cats were randomised into two groups. Groups I: 10 min of LAD occlusion (n = 10) and 10 min of LAD occlusion followed by 180 min of reperfusion (n = 15). Group II: 40 min of LAD occlusion (n = 10) and 40 min of LAD occlusion followed by 180 min of reperfusion (n = 15). Histochemical staining (TTC) was performed in hearts from 5 additional cats subjected to 40 min of LAD occlusion and 180 min of reperfusion. Regional function was measured by sonomicrometry in the circumferential (CIRC) and longitudinal (LONG) axis of the anterior left ventricular midwall. Myocardial blood flow was measured with radiolabelled microspheres. Adenine nucleotides in the subepi- and subendocardium were measured with high-pressure liquid chromatography after LAD occlusion and after reperfusion. RESULTS: Ten minutes of ischaemia induced a transmurally uniform ATP depletion. Repletion of ATP following reperfusion was transmurally uniform. Recovery of regional shortening was non-uniform with better recovery in CIRC (76 +/- 8% vs. LONG; 46 +/- 10%, P < 0.05). Forty minutes of ischaemia induced a more severe ATP depletion in the subendocardium compared to the subepicardium. A slight recovery of ATP following reperfusion was transmurally uniform. Recovery of function was present only in CIRC (48 +/- 6%). Tissue blood flow showed a transmurally homogenous flow restriction during ischaemia and uniform recovery following reperfusion. TTC staining demonstrated predominantly subendocardial infarctions following 40 min of regional ischaemia. CONCLUSIONS: Postischaemic recovery of regional function is non-uniform and independent of ATP repletion and collateral blood flow during ischaemia. Absence of functional recovery in LONG is associated with development of infarction. PMID- 7553717 TI - The effects of antihypertensive therapy on carotid vascular structure in man. AB - OBJECTIVE: An increased carotid intima-media thickness (IMT) has been found to be associated with a number of cardiovascular risk factors such as age, hypertension, cigarette smoking, hypercholesterolaemia and left ventricular hypertrophy. Our objective was to assess whether carotid intima-media thickness in hypertensive patients could be reduced by antihypertensive therapy. METHODS: 13 hypertensive patients, 10 previously untreated, were examined using carotid ultrasonography and echocardiography at baseline and then at 8 weeks and 39 weeks after commencement of antihypertensive therapy with ramipril and the second-line addition of felodipine. RESULTS: By the end of the study significant regression of IMT (0.1(0.05-0.16) mm, F-value 10.2, P < 0.01) and left ventricular mass index had occurred (25(10.7-39.3) g/m2, F-value 9.7, P < 0.01). The reduction in IMT was significantly related to the reduction in mean arterial pressure, r = 0.55, P = 0.05). CONCLUSION: Antihypertensive therapy with ramipril and felodipine causes regression of IMT in hypertensive patients, probably chiefly through blood pressure reduction. Large prospective studies are required to assess whether a reduction in IMT results in a reduction in morbidity and mortality. PMID- 7553718 TI - Vitamin E can protect myocardium against oxidative damage. PMID- 7553719 TI - The discovery of captopril: from large animals to small molecules. PMID- 7553720 TI - 'Til death do us part: a case of failed affection. PMID- 7553721 TI - Outward currents underlying repolarization in human atrial myocytes. AB - OBJECTIVE: The goals of this study were to identify the types of outward potassium (K+) currents that are activated at membrane potentials corresponding to the plateau of the action potential in human atrial myocytes, and to compare their properties with published data describing the K+ channels which have been cloned from a human cDNA library. METHODS: Specimens of right atrial appendages were obtained from patients undergoing cardiac surgery. Single myocytes were isolated enzymatically and whole cell voltage- and current-clamp recording techniques were applied. RESULTS: The outward K+ current in most cells consisted of transient and sustained (non-inactivating) components. 4-Aminopyridine (4-AP, 50 microM) broadened the action potential and increased the plateau height by blocking a Ca(2+)-independent transient outward K+ current (I(t)). The transient and the pedestal components could also be separated by using two pulse voltage clamp protocols to inactivate them: the transient component was inactivated completely by 400 ms depolarizing pre-pulses (-80 to 0 mV). In contrast, the inactivation of the pedestal component was not complete even when very long (2500 ms) pre-pulses were applied. The time-course of inactivation of the K+ currents in most cells could be described mathematically by the sum of two exponential functions. The faster of the two processes underlying inactivation was voltage independent for membrane voltages between +10 and +40 mV. The dependence of the recovery kinetics (reactivation) of I(t) on [K+]o was also studied. When [K+]o was reduced from 5.4 to 1.0 mM, reactivation slowed significantly. In a small fraction of atrial cells, a slowly activating delayed rectifier current was also identified. CONCLUSIONS: These results provide additional information concerning the ionic mechanism(s) for early and late repolarization, and they allow findings from electrophysiologically viable human atrial cells to be related to recent information regarding the molecular biology of potassium currents in human heart. PMID- 7553722 TI - Use of an indwelling catheter for examining cardiovascular responses to pericardial administration of bradykinin in rat. AB - OBJECTIVE AND METHODS: Epicardial application of pharmacologic agonists has been used to study nociceptive and reflex responses to agents such as bradykinin. We utilized a model where intrapericardial bradykinin was administered in a closed chest rat. The procedure allows for reproducible administration of microliter doses of pharmacologic agonists in both conscious and anesthetized animals. RESULTS: Bradykinin (BK) has been shown to produce sympathoexcitatory reflexes when applied to the heart. BK typically produced a dose-dependent (0.001-10 micrograms) decrease in arterial blood pressure and tachycardia in pentobarbital anesthetized rats. In contrast, in alpha-chloralose-anesthetized or awake rats, pericardial administration of BK produced a dose-dependent (0.001-10 micrograms) increase in arterial blood pressure and tachycardia. Maximal cardiovascular changes were produced by 1 microgram BK. The maximum change in arterial pressure was +33.6 +/- 9% in awake, +38.9 +/- 6% in chloralose-anesthetized, and -20 +/- 7% in pentobarbital-anesthetized rats. In alpha-chloralose-anesthetized rats, tachyphylaxis to pericardial administration of 1 microgram BK occurred at 5 and 15, but not at 30 min dosing intervals. Administration of the receptor selective B2-antagonist D-Arg,[Hyp3,Thi5,8 D-Phe7]-BK (200 micrograms) or the mixed B2/B1 antagonist [Thi5,8,D-Phe7]-BK (200 micrograms), produced similar attenuation of the pressor and tachycardia responses to BK. Bilateral transection of the cervical vagus nerve, bilateral removal of the stellate ganglion or ganglion blockade (hexamethonium), but not administration of indomethacin, reduced the magnitude of the tachycardia to BK. Only ganglionic blockade significantly reduced the pressor response to BK. CONCLUSIONS: These results demonstrate that pericardial administration of BK produces a tachycardia and pressor effect in awake and alpha-chloralose-anesthetized rats and a tachycardia and depressor effect in pentobarbital-anesthetized rats. These responses appear to be mediated through activation of BK (presumably B2) receptors on cardiac vagal and sympathetic afferents, and may include a direct action of BK on the heart. This model of pericardial administration of pharmacologic agonists may be useful in studies of cardiac pain and reflex responses. PMID- 7553723 TI - Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1. AB - OBJECTIVE: A blocking monoclonal antibody to intercellular adhesion molecule-1 (ICAM-1), CL18/6, previously has been demonstrated to inhibit neutrophil attachment to isolated vascular endothelium and cardiomyocytes. Due to the well known participation of ICAM-1 in the inflammatory responses associated with myocardial ischemia/reperfusion injury, we investigated if CL18/6 could attenuate myocardial ischemia/reperfusion injury in vivo. METHODS: Saline (3-5 ml, i.v., n = 6), non-blocking control MAb CL18/1D8 or CL18/6 (both 0.5 mg kg-1, i.v., n = 4) were administered prior to coronary occlusion (1 h) and subsequent reperfusion (5 h) produced by inflation of a coronary balloon angioplasty catheter in isoflurane anesthetized, closed-chest dogs. Heart rate and arterial pressure were measured, and regional myocardial blood flow (rMBF), and myeloperoxidase activity (MPO) to index local neutrophil sequestration, were determined. Myocardial infarct size (IS) was evaluated using the tetrazolium staining technique and expressed as a percent of area at risk (AR). RESULTS: Changes in heart rate and arterial pressure were insignificant throughout the experiment. rMBF (mean +/- s.e.m.) in the ischemic subendocardium for each treatment group was: Saline (0.07 +/- 0.02 ml min-1 g-1); CL18/1D8 (0.04 +/- 0.02); CL18/6 (0.06 +/- 0.02). IS/AR% was: saline (37 +/- 3%); CL18/1D8 (39 +/- 9%); CL18/6 (15 +/- 4%*); * = significantly different from CL18/1D8 and saline, P < 0.05. MPO assayed from AR immediately adjacent to the infarct was significantly reduced below infarct MPO only in the CL18/6 treated group-36%). CONCLUSIONS: The results indicate that CL18/6 antagonism of ICAM-1 provided cardioprotection associated with reduced neutrophil activity in vulnerable myocardium, and suggest that ICAM-1 mediated neutrophil sequestration in endangered cardiac tissue is an important mechanism of myocardial ischemia/reperfusion injury. PMID- 7553724 TI - Neuropeptide depletion impairs postischemic recovery of the isolated rat heart: role of substance P. AB - OBJECTIVES: Ischemia of the myocardium stimulates cardiac sensory nerve endings resulting in the local release of neuropeptides. The significance of the release of neuropeptides, such as substance P (SP), for the function of the heart during ischemia and reperfusion is not known. We examined the effects of both chronic and acute neuropeptide depletion and of SP administration on contractile function and rhythmicity of the isolated rat heart during global ischemia and subsequent reperfusion. METHODS: Experiments were conducted on the isolated perfused heart from vehicle and capsaicin-pretreated rats (100 mg/kg) to deplete neuropeptides from peripheral nerve terminals. The hearts were perfused with Krebs-Henseleit solution (95%O2 + 5% CO2, 37 degrees C, at constant pressure of 90 cmH2O). Left ventricular developed and diastolic pressures (LVDevP and LVEDP), heart rate (HR) and coronary flow (CF) were measured. Hearts were subjected to 20 min global no flow ischemia and 30 min reperfusion. RESULTS: Prior to interrupting coronary flow, LV pressures, HR and CF did not differ between vehicle and capsaicin pretreated rats. However, throughout the reperfusion period, the recovery of LVDevP, HR and CF in hearts from capsaicin-pretreated rats was consistently less than in control hearts (P < 0.05), and the incidence of fibrillation during reperfusion was higher (P < 0.05). In other experiments, acute perfusion of isolated hearts with capsaicin (10(-6) M) for 5 min before ischemia had a similar limiting effect during reperfusion. Administration of SP (10(-6)-10(-9) M) to capsaicin-pretreated hearts before ischemia restored their ability to recover contractile function and CF during reperfusion. Administration of SP to untreated hearts before ischemia also improved their recovery above normal during reperfusion and decreased the incidence of fibrillation without affecting postischemic CF. The beneficial effects of SP were abolished by an NK-1 receptor antagonist, CP-96,345 (10(-6) M). CONCLUSIONS: These data indicate that sensory neuropeptides play a role in protection of the isolated heart against ischemic damage and suggest a role of SP in the resistance of the myocardium to ischemia and reperfusion injury. PMID- 7553725 TI - Effect of osmotic stress on sarcolemmal integrity of isolated cardiomyocytes following transient metabolic inhibition. AB - OBJECTIVE: Exposure to hypotonic medium induces sarcolemmal rupture in metabolically inhibited cardiomyocytes. This study investigated the effect of osmotic stress applied during reoxygenation and the possible cooperation between cell swelling and hypercontracture to produce sarcolemmal disruption. METHODS: Freshly isolated adult rat myocytes were submitted to 60 min of metabolic inhibition (NaCN 2 mM). Reoxygenation was simulated by changing to one of 3 inhibitor free buffers: (1) normo-osmotic (312 mOsm); (2) hypo-osmotic (80 mOsm); (3) low Na+ normo-osmotic (312 mOsm). The contribution of hypercontracture induced reoxygenation on sarcolemmal rupture was investigated in myocytes submitted to hypo-osmotic reoxygenation in presence of 2,3-butanedione monoxime 30 mM, a blocker of contractility. Recovery from mechanical fragility was studied by exposing cells to hypotonic buffer 20 or 40 min after restoration of metabolic activity, in either presence or absence of 2,3-butanedione monoxime. Two control groups without metabolic inhibition were used. One was exposed to osmotic stress after 60 min incubation in control conditions, the other was induced to hypercontract by exposure to hypo-osmotic, high-calcium buffer. Cell viability was assessed by the Trypan blue test. RESULTS: Before any intervention 81.9(1.2)% of cells were rod-shaped. After 60 min of metabolic inhibition most cells developed rigor contracture and only 16.4(1.8)% remained rod-shaped. Restoration of metabolic activity induced hypercontracture of most cells with rigor independently of buffer osmolality. Cell viability, however, significantly differed among groups: only 25.9(4.4)% of cells reoxygenated with hypo-osmotic buffer were viable vs. 74.1(7.6)% in the normo-osmotic reoxygenation group, and 82.9(2.9)% in the control group. Addition of 2,3-butanedione monoxime 30 mM during hypo-osmotic reoxygenation prevented hypercontracture and preserved cell viability. Delaying osmotic stress 20 or 40 min after the onset of reoxygenation did not improve viability [19.3(3.9) and 34.9(1.3)%, respectively]. Contractile blockade with 2,3-butanedione monoxime during the first 20 or 40 min of reoxygenation was associated with a reduction in the number of hypercontracted cells after the removal of the inhibitor but did not increase the proportion of hypercontracted viable cells (25% and 27%, respectively). CONCLUSIONS: (1) Osmotic stress following transient metabolic inhibition produces sarcolemmal disruption, and this effect is not related to the low Na+ concentration present in the hypo-osmotic buffer; (2) reoxygenation-induced hypercontracture cooperates with cell swelling to produce sarcolemmal disruption; and (3) osmotic fragility persists for at least 40 min after restoration of metabolic activity. PMID- 7553727 TI - Endogenous nitric oxide (NO) protects against ischaemia-reperfusion injury in the rabbit. AB - OBJECTIVES: Recent studies suggest that nitric oxide (NO) is deleterious in models of shock and hypoxia-reoxygenation However, the role of endogenous NO in ischaemia-reperfusion injury in vivo remains controversial. We tested the hypothesis that blockade of endogenous NO produced during myocardial ischaemia reperfusion or during reperfusion alone in vivo increases infarct size after coronary occlusion in the rabbit, and conversely, supplementation with L-arginine would reduce infarct size. METHODS: Ketamine-xylazine anaesthetised New Zealand white rabbits were subjected to left coronary artery occlusion for 30 min and reperfusion for 120 min. The rabbits were divided into five groups: (1) saline (VEH); (2) L-nitro arginine (L-NA), a NO-synthase inhibitor, was infused intravenously (15 mg/kg bolus followed by 7.5 mg/kg h-1) before coronary occlusion to block NO synthase activity during ischaemia and reperfusion (IR); (3) L-NA was administered during reperfusion only (R) at the same dose as in the IR group; (4) D-arginine (D-ARG) (25 mg/kg bolus followed by 4 mg/kg min-1), the non-metabolised enantiomer of L-arginine was infused intravenously during reperfusion only; (5) L-arginine (L-ARG) (25 mg/kg bolus followed by 4 mg/kg min 1), the physiological precursor of NO, was infused intravenously during reperfusion only. RESULTS: L-NA infusion in the IR and R groups caused an increase in mean arterial pressure and a decrease in heart rate; however, no significant change in pressure rate product (PRP) occurred immediately after drug infusion. PRP did not change significantly during the experiment across groups except at the end of reperfusion. The area at risk was comparable in all groups, averaging 29(1)%. The infarct size (triphenyltetrazolium chloride) expressed as a percent of area at risk was 27(2)% for the untreated vehicle group. In contrast, L-NA significantly (P < 0.05) increased infarct size in the IR group, 51(2)%; this augmented infarct size persisted when NO synthase activity was blocked during reperfusion only in the R group, 50(2)%. There was no significant (P < 0.05) difference in infarct size between the IR and the R groups. D-ARG-treated group showed a comparable increase in infarct size 48(2)% versus the IR and R groups. However, supplementation of NO with L-arginine (L-ARG) showed no reduction in infarct size, 24(3)%, over vehicle group (VEH). CONCLUSIONS: We conclude that (1) blockade of NO synthase activity with L-NA increases infarct size, (2) this effect was expressed primarily during reperfusion, (3) D-arginine mimicked the infarct augmentation of L-NA, while (4) L-arginine supplementation did not reduce infarct size. These data imply that endogenous NO production exerts a tonic cardioprotective effect on myocardial infarct following coronary reperfusion. PMID- 7553726 TI - Combined therapy with dimethylthiourea, diltiazem and amiloride/dimethylamiloride in the ischemic/reperfused heart. AB - OBJECTIVES: The relative contribution of oxygen free radicals, disturbances in calcium homeostasis and Na+/H+ exchange in the development of injury in the ischemic/reperfused heart was investigated. The study was designed to assess whether these factors initiate independent mechanisms of injury or, alternatively, they share a common mechanism of toxicity. METHODS: Isolated working rat hearts were subjected to different periods (30-55 min) of global ischemia and then were reperfused for 30 min. We compared the effects of oxygen radical scavengers (10 mM dimethylthiourea, DMTU and 0.6 mM desferrioxamine), inhibitors of Na+/H+ exchange (0.15 mM amiloride and 15 microM dimethylamiloride, DMA) and of 0.1 microM diltiazem, which was used to limit calcium overload, given alone or in combination, on the rate of myocardial injury development (recovery of hemodynamic function, LDH release, incidence of severe arrhythmias and structural integrity of cardiomyocytes were estimated at reperfusion following different periods of ischemia). RESULTS: All interventions studied, when given alone, provided nearly equivalent cardioprotection. DMTU or desferrioxamine when applied in combination with diltiazem provided additive cardioprotection, relatively limited, however, as compared to the remarkable cardioprotection achieved by DMTU or desferrioxamine in combination with amiloride. CONCLUSIONS: All mechanisms studied may contribute in an equal manner to the rate of injury development in the ischemic/reperfused heart. The oxygen free radicals-induced myocardial injury may be partially attributed to some disturbance in intracellular calcium homeostasis, possibly calcium overload, whereas the damaging effect of the Na+/H+ exchange activated upon reperfusion is probably largely related to some other mechanism. PMID- 7553728 TI - The effects of exogenous nitric oxide on smooth muscle cell proliferation following porcine carotid angioplasty. AB - OBJECTIVES: Nitric oxide reduces platelet adhesion and platelet-thrombus formation following angioplasty and inhibits smooth muscle cell (SMC) proliferation in vitro. In this study we investigated the effects of the nitric oxide donor molsidomine on SMC proliferation and intimal growth following experimental angioplasty. METHODS: Bilateral carotid angioplasty was performed in 24 anesthetized pigs. Animals were randomized to receive oral molsidomine (whose active metabolite is SIN-1; 0.3 mg/kg every 8 h; n = 12) or placebo (n = 12) for 48 h before angioplasty and until the arteries were removed either 7 or 21 days (n = 12 each group) later. SMC proliferation was quantified by immunocytochemical staining with an antibody to the proliferating cell nuclear antigen (PCNA) and morphometric changes by computerized planimetry. SMC's were identified by alpha actin staining. RESULTS: After 3 weeks treatment with molsidomine there was a significant prolongation in bleeding time [mean +/- SEM] (151 +/- 6 to 187 +/- 7 s. P < 0.01) and a sustained increase in arterial wall cyclic GMP (6.57 +/- 1.29 to 13.24 +/- 1.02 pmol/mg protein, P < 0.05). Molsidomine significantly reduced intimal proliferation when compared with placebo in arteries with an intact internal elastic lamina at 7 days (4.3 +/- 0.7 vs. 9.6 +/- 1.9 PCNA index, P < 0.005) and medial proliferation at 7 days (2.4 +/- 0.2 vs. 4.2 +/- 0.7 PCNA index, P < 0.05) and at 21 days (1.3 +/- 0.1 vs. 1.9 +/- 0.2 PCNA index, P < 0.05) after angioplasty. In arteries with rupture of the internal elastic lamina, intimal and medial SMC proliferation were similar in molsidomine- and placebo treated animals. Intimal cell number and intimal area were uninfluenced by treatment with molsidomine in either the presence or absence of rupture of the internal elastic lamina. CONCLUSIONS: These results show for the first time that exogenous nitric oxide inhibits SMC proliferation following balloon angioplasty in vivo. The antiproliferative effects of nitric oxide are overwhelmed when injury is severe and are not associated with a reduction in intimal thickening. The inhibitory effects of nitric oxide on platelet adhesion and SMC proliferation identify a possible role for high local concentrations of nitric oxide to modify the vascular response to balloon angioplasty. PMID- 7553729 TI - Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus. AB - OBJECTIVE: The aim was to examine the feasibility, efficiency and safety of adenovirus-mediated in vivo gene transfer into the canine myocardium by a percutaneous transluminal method using a needle-catheter. METHODS: Either a replication-defective adenovirus (Adex1SRLacZL) or a plasmid (pSRLacZ), both expressing E. coli lacZ coding beta-galactosidase (beta-gal), was directly injected into the left ventricle of dogs through a needle-catheter inserted via a femoral artery. Expression of lacZ was examined by histochemical staining and quantified by measuring beta-gal activity. RESULTS: Injections with Adex1SRLacZL induced lacZ expression as a result of 40 out of 41 injections; the expression level was 10 times higher than that obtained with pSRLacZ. Induced beta-gal activity was detected within 24 h, peaked at 7 days and retained for 2 weeks after gene transfer. A repetitive administration of the same adenovirus at 14 days after the first injection also evoked a reduced but significant level of expression despite neutralizing antibodies to adenovirus in serum. Although injection induced an inflammatory response that peaked at 3 days after injection and gradually subsided without a second peak, the temporal change and the extent of inflammation induced by adenovirus injection was not significantly different from those induced by injection with either saline or plasmid. Neither leakage of enzymes such as CPK or LDH nor alteration in the ECG was detected in the 30 days following gene transfer. CONCLUSIONS: Our findings demonstrate that a catheter mediated direct injection with an adenovirus can induce gene expression in the ventricle more efficiently without additional myocardial damage and inflammation compared with injection with a plasmid. A repeat dose of the same adenovirus elicited gene expression at an attenuated but significant level. This method may potentially have clinical applications: in modifying myocardial phenotype and/or improving general circulation under certain circumstances. PMID- 7553730 TI - Differential suppression of axoplasmic transport: effects of light irradiation to the growth cone of cultured dorsal root ganglion neurons. AB - 1. Growth cones of cultured dorsal root ganglion neurons from mice were irradiated using a mercury lamp. 2. The flux of particles of fast retrograde axoplasmic transport decreased promptly after light irradiation without a change in velocity. 3. That of anterograde transport decreased as well, but with a significant latency. The decrease in anterograde flux was attributed to decreased velocity of particles. 4. Video-enhanced contrast microscopy of growth cones revealed transient swelling of growth cones and transient stagnation of particles in growth cones. 5. The longer the neurite, the larger the latency of the change of the anterograde transport; peripheral information was calculated to be conveyed to the cell body at a speed of 6 microns/min. 6. The mechanism of this information conveyance and the export of materials from the cell body are discussed. PMID- 7553732 TI - Endothelin receptor in microvessels isolated from human meningiomas: quantification with radioluminography. AB - 1. We characterized specific 125I-endothelin-1 (125I-ET-1) binding sites in microvessels isolated from human meningiomas, using an in vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system. 2. This newly developed and highly sensitive method revealed high affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 +/- 0.3 nM; maximum binding capacity, 185 +/- 56 fmol/mg; means +/- SE for nine tumors). 3. In five cases of meningiomas, ET-3 competed for 125I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC50) of 1.6 +/- 0.4 x 10(-6) M], and a selective ETB receptor agonist, sarafotoxin S6c, up to 10(-6) M, did not displace ET binding from the sections. 4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC50 of 1.1 +/- 0.2 x 10(-9) M for the high-affinity component and 1.6 +/- 0.3 x 10(-6) M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC50 = 2.3 +/- 0.2 x 10(-10) M) and 10(-6) M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3. 5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ETA (non-ETB) receptors with a small proportion of ETB receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium. PMID- 7553733 TI - Time course of nitric oxide synthase activity in neuronal, glial, and endothelial cells of rat striatum following focal cerebral ischemia. AB - 1. The time course of nitric oxide synthase (NOS) activity in neuronal, endothelial, and glial cells in the rat striatum after middle cerebral artery (MCA) occlusion and reperfusion was examined using a histochemical NADPH diaphorase staining method. 2. In sham-operated rats, neuronal cells of the striatum exhibited strong NADPH-diaphorase activities. When rats were subjected to MCA occlusion for 1 hr, neuronal damage, including neurons with positive NADPH diaphorase activities, appeared in the striatum at 3 hr after and extended to all areas of the striatum 3-4 days after reperfusion. 3. NADPH-diaphorase activities in the endothelial cells increased in the damaged part of striatum from 3 hr after, peaked at 1-2 days after MCA occlusion/reperfusion, then gradually decreased. 4. In parallel with the development of neuronal damage, some astrocytes and a high proportion of microglia/macrophages located in the perisite and in the center of the damaged striatum, respectively, exhibited a moderate to high level of NADPH-diaphorase activities. Most of these activities disappeared at 4 days after MCA occlusion. 5. These findings provided evidence that an inappropriate activation of NOS in endothelial cells and microglia/macrophages, in response to MCA occlusion/reperfusion, is closely associated with initiation and progression of ischemic neuronal injury in the striatum. PMID- 7553731 TI - Transcriptional regulation of the neuronal L-type calcium channel alpha 1D subunit gene. AB - 1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence. PMID- 7553734 TI - Mechanism of high K+ and Tl+ uptake in cultured human glioma cells. AB - 1. The aim of this study was to elucidate if the K+ uptake was higher in cultured human glioma cells than in cells from other malignant tumors and to analyze the importance of membrane potential and K+ channels for the uptake. 2. K+ transport properties were studied with the isotopes 42K and the K-analogue 201Tl. 3. Comparison with cultured cells from other malignant tumors showed that the specific steady-state accumulation of Tl+ was significantly higher in glioma cells (U-251MG and Tp-378MG). 4. In Ringer's solution at 37 degrees C the rates of K+ and Tl+ uptake were both inhibited by about 55% in ouabain and 60% in furosemide, bumetanide, or Na(+)- or Cl(-)-free medium. This indicated that the routes for K+ and Tl+ uptake were similar and due to Na,K-ATPase-dependent transport and to Na-K-Cl cotransport. 5. About 10% of the uptake was neither ouabain nor bumetanide sensitive. Ba2+, which is known to block inward-rectifying K+ channels and to depolarize glial cells, and other K+ channel blockers (Cs+ and bupivacaine), had no effect on Tl+ uptake. 6. Metabolic inhibition with dinitrophenol reduced the uptake rate to 17%. 7. The washout of Tl+ was unaffected by bumetanide and K+ channel blockers, but dinitrophenol caused a transient increase of 75%, an effect which persisted in the presence of K+ channel blockers. 8. It was concluded that the high specific K+ and Tl+ accumulation in cultured human glioma cells was due not to the presence of inwardly rectifying K+ channels or other identified K+ channels, but to Na,K ATPase dependent transport and Na-K-Cl cotransport. PMID- 7553737 TI - [Laparoscopy in urology]. AB - In the introduction the author analyzes the history of laparoscopy and explains the ways and means how the originally purely diagnostic method developed gradually into a modern, minimally invasive endoscopic surgical technique which with increasing frequency is used by gynaecologists, surgeons as well as urologists. Next the author discusses contemporary indications of laparoscopic operations in urology, he makes the reader familiar with instruments required for surgery and technical principles of its implementation. The author mentions the causes of possible complications and draws attention to contraindications of the operation. In the conclusion he evaluates the perspectives of further development of laparoscopic operations in urology. PMID- 7553738 TI - [Current trends in medical genetics]. AB - Medical genetics made during the last decades immense advances and new findings from the sphere of fundamental research are increasingly penetrating into different clinical disciplines. The greatest attention was concentrated on investigations devoted to the molecular etiology of malignant diseases and the molecular mechanism of embryonic development. Mapping of the human genome is the prerequisite for identification of serious diseases at the gene level. Control of these problems opens possibilities of aimed diagnostics at the molecular level during the postnatal period and as well as the prenatal one. So far we possess knowledge required for genetic counselling and possible prevention. With the extension of our knowledge on gene mechanisms we find that originally apparently clear mechanisms of heredity are much more complicated. In the last three years also the possibility arose of gene therapy of some malignomas and other pathological conditions of the organism. Hitherto assembled experience is relatively optimistic although a major development of these techniques is foreseen only at the end of this millenium. Medical genetics advanced greatly in the prenatal diagnosis of inborn developmental and hereditary foetal diseases. Gradually different screening programmes were introduced to evaluate the status of the foetus in utero. At present also predictive tests are available for some serious diseases. All these methods and technologies are associated with many ethical, moral and psychosocial problems which must be gradually resolved. PMID- 7553739 TI - [Stimulation of endogenous secretion of the growth hormone. Today in diagnosis, tomorrow in therapy?]. AB - BACKGROUND: Treatment with growth hormone (GH) restores the natural growth rate in children with growth hormone deficiency (GHD). This is, however, achieved only after daily injections extending over many years and therefore daily short-term hypersomatotropinaemia. Stimulation of endogenous secretion of GH e.g. by oral administration of growth hormone-releasing peptide (GHRP) may help in future to eliminate these adverse aspects. This treatment could be beneficial in patients with a stimulable endogenous GH secretion. METHODS AND RESULTS: In order to find potential candidates for spontaneous secretion of GH the authors examined, using a test with sermoreline (GHRH1-29NH2), 31 children (21 boys) aged 5.8-16.5 years suffering from idiopathic (GHD), previously treated by daily GH injections. GH rose after stimulation with sermoreline to more than 14 mIU/l in 18/31 children (responders). The ratio of "responders" was higher in the sub-group of children with isolated GHD, as compared with multiple pituitary deficiency (p = 0.05) and insignificantly higher in the sub-group of children born by breech delivery (p = 0.13). CONCLUSIONS: More than half the children treated nowadays with GH could profit in future from the method of spontaneous GH secretions. The success of this procedure is more likely in children with isolated GHD and in breech delivered children. PMID- 7553736 TI - Distribution of catecholamines, indoleamines, and their precursors and metabolites in the scallop, Placopecten magellanicus (Bivalvia, Pectinidae). AB - 1. Although monoamines are well-known to play important roles in molluscan physiology, we are far from fully understanding the synthetic and degradative pathways of these substances, particularly in commercially important bivalve species. In the present study endogenous catecholamines, indoleamines, and their possible precursors and metabolites were detected in the scallop, Placopecten magellanicus, by high-performance liquid chromatography coupled to electro chemical detection. 2. Chromatographic analysis of CNS (cerebral, pedal, and parietovisceral combined), gill, gonad, kidney, mantle, liver, heart, fast adductor muscle, and foot disclosed the presence of the catecholamines 3,4 dihydroxyphenylalanine, dopamine, norepinephrine, and epinephrine and their metabolites normetanephrine, metanephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid. 3. Dopamine was detected most frequently and most consistently among all catecholamines. The concentrations of dopamine (1400 pg/mg wet weight) and its major metabolite 3,4-dihydroxyphenylacetic acid (300 pg/mg wet weight) were highest in the CNS. Following the CNS, dopamine was also abundant in other tissues such as heart, foot, and gill. The concentration of norepinephrine (1000 pg/mg wet weight) was highest in the CNS followed by the heart (700 pg/mg wet weight) and gill (600 pg/mg wet weight). 4. The indoleamine, 5-hydroxytryptamine, was present in considerable amounts in all tissues, but its content was highest in the foot (2700 pg/mg wet weight) followed by the CNS (1150 pg/mg wet weight) and gonad (1000 pg/mg wet weight). The precursor 5-hydroxytryptophan was also abundant in the foot followed by the gonad, CNS, and heart. 5. The oxidative metabolite 5-hydroxy-3-indole acetic acid was detected in the largest amount in CNS (200 pg/mg wet weight), whereas N-acetyl-5-hydroxytryptamine was detected in trace amounts in CNS, gonad and foot. This study also presents evidence for gamma glutamyl dopamine and gamma-glutamyl 5-hydroxytryptamine as the possible alternate catabolic products of dopamine and 5-hydroxytryptamine, respectively, as previously described in gastropods. 6. Thus, the detection of monoamines and their precursors and metabolites in scallop strongly suggests the presence of mammalian-type enzymic action of hydroxylation, oxidation, and methylation pathways leading to synthesis and degradation of detected compounds. Furthermore, this is the first study to disclose the evidence of nonconventional metabolic pathways for dopamine (gamma-glutamyl<--dopamine-->dihydroxyphenylacetic acid- >homovanillic acid) and 5-hydroxytryptamine (gamma-glutamyl 5-hydroxytryptamine<- 5- hydroxytryptamine-->5-hydroxy-3-indoleacetic acid) inactivation in a bivalve species. PMID- 7553740 TI - [Surgical therapy of carcinoma of the thyroid gland]. AB - BACKGROUND: Thyroid carcinoma comprises tumours with a wide spectrum of aggressiveness. The method of first choice is surgery, however, views on its extent and technique are frequently controversial. The objective of the present work was to demonstrate and justify the procedure used at the author's department in treatment of this tumour. METHODS AND RESULTS: During 1988-1992 a total of 919 patients with thyroid disease were operated. The female:male ratio was 4.13:1; a benign finding was confirmed in 76.4% and a malignant one in 23.6%. The female:male ratio in patients with a malignant findings was 3.24:1. Most frequently a papillary carcinoma was confirmed--in 59.9%, followed by follicular carcinoma in 23.11%, medullary carcinoma in 7.0%, Hurtle's carcinoma in 4.25%, anaplastic carcinoma in 1.42% and epidermoid carcinoma in 0.47%. Metastases in the thyroid were present in 0.94% and a myeloma or sarcoma both in 0.47%. In retrosternal goitres papillary carcinoma was most frequent (19 patients) followed by follicular (7 patients). As to surgical operations, total thyroidectomy was performed in 47.6%, total lobectomy in 20.3%, subtotal thyroidectomy in 15.1% and subtotal lobectomy in 2.4% of the operated patients. A palliative resection was made in 11.8% and lymphadenectomy in 2.8% patients with a malignant finding on the thyroid gland. Permanent complications (paresis of the recurrent nerve, hypoparathyroidism) occurred in 0.95% of the patients, temporary complications (abscess in the scar, transient hypoparathyroidism, oedema of the vocal cords) in 8.66% of the operations. None of the patients died. CONCLUSIONS: Surgical results and experience of the clinic for nuclear medicine support a radical approach to all types of thyroid carcinoma in adults as well as in adolescents. PMID- 7553741 TI - [Comparison of experimental models of renal ischemia. Morphologic study]. AB - The authors investigated in rats after unilateral nephrectomy morphological changes in the remaining kidney where the blood flow was arrested for 20, 30, 45 or 60 min. The investigation period was 24 hours, 3, 7, 14, 21 and 28 days after ischaemization. After short-term ischaemia monocellular necroses of the cortex epithelium in the canals occurs and rapid regeneration. In the initial stages after 20 min. ischaemia and in all periods after 30 min. ischaemia the authors observed slight hyperplasia of the juxtaglomerular apparatus manifested by fine granulations. After ischaemia persisting for 45 min. in addition to necroses of the tubules regressive arterial changes are observed with subsequent regeneration from the deep cortex to the surface. After discontinuation of the blood flow for 60 min. the animals die after 7 days with diffuse damage of the cortical parenchyma and regression of smooth muscle cells of the arterial walls with subsequent insudates of the media. PMID- 7553743 TI - [Advances in the diagnosis of phenylketonuria with the introduction of direct detection of PAH gene mutation]. AB - BACKGROUND: Phenylketonuria is as regards the genotype a very heterogenous disease. Successful prenatal and postnatal DNA diagnosis calls for knowledge of different mutations in a given population. The objective of the investigation was to introduce direct detection of 21 mutations in the gene for phenylalanine hydroxylase and to find the distribution and frequency of these mutations in the population of northern and southern Moravia. METHODS AND RESULTS: The authors analyzed a group of 95 patients where according to phenotypic classification classical phenylketonuria was involved which comprised 190 mutant alleles. The presence of mutations was assessed by means of a polymerase chain reaction of a Perkin Elmer DNA Thermal Cycler 480. From the total number of 21 mutations which were sought, 11 were identified in our population, which accounts for 80% of all mutations. It was revealed that mutation R408W is found in 55.3% of our patients. Twenty per cent of the mutations are still unknown. CONCLUSIONS: This investigation laid the foundations for direct DNA diagnosis of phenylketonuria in the Czech Republic. The results assembled in the Moravian region suggest that our population is as regards genotypes relatively homogenous. This gives great hope of successful prenatal diagnosis and postnatal genotype classification. PMID- 7553742 TI - [The human genome--chromosome 12]. AB - One of the most rewarding examples for teaching hereditary metabolic disorders is classical phenylketonuria (PKU) caused by the deficient function of phenylalanine hydroxylase, the locus of which (PAH) is on the long arm of the twelfth chromosome. The twelfth chromosome has also the locus (VWF, F8VWF) the pathogenic alleles of which cause impaired blood clotting--Willebrand's disease and it is at the same time also the site of the family of keratin genes (KRT) responsible for epidermolysis bullosa simplex and other diseases. The question of the relationship between membrane glucose transmitters--GLUT and diabetes (NIDDM) is the subject of many investigations concerned with these loci. PMID- 7553735 TI - Chronic administration of the antidepressants phenelzine, desipramine, clomipramine, or maprotiline decreases binding to 5-hydroxytryptamine2A receptors without affecting benzodiazepine binding sites in rat brain. AB - 1. The effects of chronic administration of five antidepressant drugs on the benzodiazepine and 5-HT2A binding sites in the same rat brain were assessed. 2. Clomipramine, desipramine, maprotiline, fluoxetine, and phenelzine (all 10 mg/kg/day) were administered s.c. for 21 days by Alzet osmotic minipumps. 3. Results showed that none of the drugs changed the density or affinity of benzodiazepine binding sites, yet at the same dose all the drugs with the exception of fluoxetine decreased binding to 5-HT2A receptors in the same animals. PMID- 7553744 TI - [Selegiline in the treatment of Alzheimer's disease]. AB - Alzheimer's disease accounts for 60-70% of all dementias. Although its etiology has not been elucidated so far, its multiple transmitter effect has been proved reliably. Treatment with selegiline, 10 mg per day, which blocks irreversibly monoaminooxidase type B, when administered on a long-term basis, revealed improvement of cognitive functions and of general functional fitness during objective examinations by means of a battery of psychometric tests. The danger of a hypertensive crisis due to excessive supplies of exogenous monoamines ("cheese effect") can be ruled out when this dose is used. Selegiline has moreover a detoxicating potential and the ability to block the transformation of some protoxins into an effective toxin. The reduced formation of free radicals is of particular importance. The latter participate in a significant way in the destruction of cellular membranes, proteins and nucleic acids. In experiments on rats selegiline improves sexual functions, learning and its use significantly prolongs survival. As a symptomatic drug selegiline is the drug of choice in AD. Only future studies will show the justification of extensive preventive medication in advanced age groups. PMID- 7553745 TI - [Legal and ethical problems in drug advertising and their resolution in practice]. AB - Advertising of drugs and other preparations with an impact on human health is an integral part of the present time. The presented paper deals with different types of advertisements and legal and ethical problems associated with them. The authors in more detail discuss principles laid down in directive 92/28/EEC, which are mandatory from January 1993 for countries of the EEC. PMID- 7553746 TI - [Chronic hepatitis--diagnosis and differential diagnosis]. AB - Since the first classification of chronic hepatitis (CHH) more than 25 years have elapsed. Since then conditions for assessment of the etiology, pathogenesis and therapy of the disease have improved. These findings must be applied in the contemporary diagnosis of CHH. The first task is to assess the correct etiology of the disease because it is decisive for treatment, development and prognosis of the disease. The most important cause of CHH is viral hepatitis. Hepatitis B and C are frequently occurring diseases and may develop into CHH and cirrhosis of the liver. New therapeutic procedures, using interferons, can prevent this. Idiopathic autoimmune hepatitis is a serious form where interferon therapy is contraindicated, while the condition usually responds very well to treatment with corticoids and immunosuppressive substances. It is important to take into consideration also possible liver damage by drugs and the fact that the clinical picture is associated with a certain stage of metabolically conditioned changes, in particular Wilson's disease. It is important to examine carefully the histological finding and laboratory results, as it is possible to evaluate from them the severity of the disease. We should also attempt to evaluate these changes quantitatively. PMID- 7553749 TI - [Endothelin in patients with chronic kidney failure]. AB - BACKGROUND: Hypertension, anaemia and arteriovenous shunts represent very important pathogenetic factors in the occurrence of cardiovascular morbidity and mortality in patients with chronic renal failure. It can be expected that endothelin (ET), the most potent vasoconstrictor known at present, can react in a significant way to the haemodynamic changes, caused by the construction of a vascular shunt or anaemia. METHODS AND RESULTS: In 14 patients (7 men and 7 women, mean age 47 years, ranging from 19 to 70 years) the plasma ET concentration was examined before, 24 hours and 7 days after the construction of arteriovenous fistula. In 27 patients (19 men and 8 women, mean age 44 years ranging from 25 to 77 years), undergoing chronic haemodialysis treatment, ET was examined before the erythropoietin (EPO) therapy and after 2 months of EPO therapy, when partial correction of anaemia has been achieved (p < 0.01). CONCLUSIONS: The construction of arteriovenous fistula by itself had no significant influence on the plasma ET concentration. Subcutaneous application of EPO in such doses, which led to gradual correction of anaemia, was not accompanied by the rise of plasma ET. The average plasma concentration of ET was significantly higher in haemodialyzed patients, when compared to healthy controls as well as to patients with chronic renal failure before haemodialysis treatment was started. PMID- 7553747 TI - [Indications for liver resection in childhood]. AB - The authors give an account of indications for resection of the liver in childhood which comprise extensive injuries, cysts, vascular malformations and primary (benign and malignant) and secondary tumours. During the period between 1986 and 1993 34 major resections of the liver were performed and examined. Indications for resection were in 17 cases hepatoblastomas, 4x metastasis, 3x hepatocellular carcinoma, 2x haemangioendothelioma, adenoma, hamartoma and in one case each necrosis after injury, a cyst, cavernous haemangioma and sarcoma. The mean age of the operated children was 4 years and 5 months, the youngest patient was operated on the age of 4 days and the oldest was 17 years old. During the period between 1986 and 1989 the survival rate was 33%. During the period between 1990 and 1993 the survival rate was 86.5%. The authors emphasize the radical character of operations of malignant tumours which has a marked impact on the survival of patients with these disease. PMID- 7553748 TI - [Endoscopic sclerotherapy of esophageal varices in children with prehepatic portal hypertension]. AB - BACKGROUND: Endoscopic sclerotization of oesophageal varices in adult patients with prehepatic portal hypertension has become the method of choice in haemorrhage from these varicosities. The objective of the present work was to prove the effectiveness of this treatment in children. METHODS AND RESULTS: Between November 1987 and May 1993 in the authors' departments endoscopic sclerotization was used to treat 20 children (age 2.5-17 years) with bleeding oesophageal varices (o.v.) associated with prehepatic portal hypertension (PPH), caused by thrombosis of the portal vein. Half the children were treated unsuccessfully before sclerotherapy by surgery, some repeatedly. Complete eradication of o.v. was achieved in 19 children (95%). In the course of sclerotherapy before completed obliteration of all varices 5 children (25%) had another spell of haemorrhage. In three instances this early relapse of haemorrhage was controlled by another sclerotization. In one patient continuing haemorrhage from an oesophageal varix was treated by establishment of a splenorenal anastomosis. In another patient the source of haemorrhage were gastric varices which were ligatured and after the operation the authors proceeded with sclerotherapy. A relapse of o.v. during the average 3-year follow up period was recorded in 7 children (35%), while a relapse of haemorrhage from these neovarices occurred only in one child (5%). CONCLUSIONS: Successful eradication of oesophageal varices by sclerotherapy in 95%, and 95% successful prevention of relapsing haemorrhage from neovarices for a period of three years after surgery justify a change of tactics of treatment. The method of first choice should be always sclerotization of bleeding varices. PMID- 7553751 TI - [The human genome--chromosome 16]. AB - Haemoglobinopathies and thalassaemia caused by qualitative and quantitative disorders of the function of the alpha-chain of haemoglobin are associated with chromosome 16 on the short arms of which two of its loci are placed localized mapped. The majority of the human population has four active alpha-chain genes. Reduction of their number to two or less leads to increasing symptoms of their insufficiency manifested as alpha-thalassaemia--complete loss being lethal. On the 16th chromosome is the cause of the dominantly hereditary disease- polycytosis of the kidney so far inaccurately defined at the gene level. The author mentions also the loci of the cholesterylester transfer protein--which participates in the responsibility for hyperlipoproteinaemia and sclerotic changes and loci of haptoglobin chains--one of the serum proteins oldest as regards knowledge of its electrophoretic polymorphism. PMID- 7553750 TI - [Silent myocardial ischemia in outpatients being treated for hyperlipoproteinemia]. AB - BACKGROUND: Hyperlipoproteinaemias, in particular those associated with hypercholesterolaemia, are in a causal relationship with the development and acceleration of atherogenesis. One of the serious forms of coronary heart disease is silent myocardial ischaemia--an asymptomatic objectively confirmed ischaemic episode. The objective of the present study was to 1. assess the prevalence of this disease in subjects with hyperlipoproteinaemia and 2. to assess the optimal diagnostic procedure to detect it. METHODS AND RESULTS: The group comprises 57 subjects selected at random (23 men and 34 women) from the out-patient department for genetics and treatment of hyperlipoproteinaemias. In all subjects an ergometric loading test was made and 24-hour ambulatory ECG monitoring. Suspected silent myocardial ischaemia (i.e. positive results of the two examinations) was confirmed by load scintigraphy of the heart muscle. Silent myocardial ischaemia was proved in 3 of 23 examined men (13%) and in 4 of 34 women (11.8%). CONCLUSIONS: Prevalence of silent myocardial ischaemia is significantly higher in high risk subjects--with hyperlipoproteinaemia than in the general asymptomatic population. The best screening test for its detection is a loading test and ambulatory ECG monitoring, supplemented by loading scintigraphy of the heart muscle. PMID- 7553753 TI - [Unethical administration of placebos]. PMID- 7553752 TI - [Autosomal dominant hereditary polycystic kidney disease]. AB - BACKGROUND: Molecular genetics are a fundamental turning point in the approach to autosomal dominant hereditary renal polycystosis of adults (ADPKD). DNA analysis makes diagnosis possible at in any ontogenic period, i.e. also during the prenatal period. The objective of the present study was to test the presymptomatic DNA diagnosis in a major group of patients with ADPKD and the possibility to detect the disease in the initial stage and influence its development by early treatment and advise the patients on parenthood. METHODS AND RESULTS: In 1990-1994 the authors contacted 157 patients with polycystic kidney disease, adult type (ADPKD). 87 families were examined by Southern's RFLP method (gene PKD1, 16p13.3, standard probe 3'HVR and restrictase Pvu II). Of 493 members of these families only 25 (5.1%) refused to be examined. So far 378 examinations were completed, 90 proceed. In 40 of 132 examined subjects with the risk of ADPKD transmission of the gene was proved. Of four examinations of the foetus with the risk of ADPKD twice transmission of the gene was proved and the pregnancy was terminated. In three families with three or more members suffering from ADPKD in some there was not agreement between the result of linkage analysis of DNA and the clinical finding. The authors analyzed whether the cause is recombination or ADKPD conditioned by mutation of gene PKD2 (4p13-23). Linkage analysis remains in ADPKD the basic examination as the sequence of gene PKD1 is not yet completed. Discovery of gene PKD2 the mutations of which condition as many as 15% of all cases of ADPKD has an impact on the evaluation of linkage analysis. The reliability of prediction rises with the number of examined subjects in the family. The examination revealed that patients are willing to have DNA examinations (as many as 98%); despite this the number of examined subjects is only a small fraction of the anticipated 10,000 people in the Czech Republic suffering from ADPKD: CONCLUSIONS: DNA analysis in patients with autosomal dominant polycystic kidney disease is the most important examination for its diagnosis. It makes the diagnosis possible in all stages of ontogenesis, incl. prenatal diagnosis. It is a highly valid parameter when these patients decide on parenthood. It makes early treatment possible which can influence the development of the disease and its complications. PMID- 7553754 TI - [The Leksell gamma knife--a radiosurgery instrument]. AB - Radiosurgery means delivery of a single dose of precisely directed ionizing radiation to a small volume of intracranial tissue without opening of the skull after the stereotactic localisation of the preselected target. Leksell gamma knife is a device enabling to deliver focused radiation toward the intracranial volume with the gamma rays emitted by the 201 sources of the isotope Co 60. Suitable indications for radiosurgery are cerebral arteriovenous malformations, benign and malignant intracranial tumors and functional diseases of the brain. PMID- 7553757 TI - [Initial experience of an endocrinologist with the treatment of hypophyseal adenomas with the Leksell gamma knife]. AB - BACKGROUND: Surgery of pituitary adenomas is not quite satisfactory so far and in some patients it is associated with a high risk. Conventional radiotherapy is only partly successful. Only few hospitals have experience with treatment of pituitary adenomas with Leksell's gamma knife. The objective of the presented paper is to give an account of the authors' own results of treatment of pituitary adenomas by irradiation with Leksell's gamma knife. METHODS AND RESULTS: The treated group comprised 13 patients (6 women, 7 men, 25-72 years old, median 44 years) with a pituitary adenoma. Hormonally active acromegaly was recorded in 9 patients, a prolactinoma in one female patient, afunctional adenoma in 3 patients. Twelve patients had been operated already previously, 3 of them twice. For visualization of the pituitary magnetic resonance (NMR) was used on a Magnetom apparatus 1.5 Tesla, Siemens Co. before surgery and one year after surgery. As hormonal indicators the following were examined: growth hormone (GH) profile in the course of the day and during the thyroliberin test (TRH), insulin like growth factor I (IGF I), the prolactin level, the thyrotropin level (TSH) during the thyrotropin releasing hormone test (TRH) test the thyroxine level (T4) and the triiodothyronine level (T3), the plasma concentration of adrenocorticotropin (ACTH), the cortisol level, plasma testosterone level and 17 beta estradiol level. Complete recovery was achieved only in one female patient with acromegaly 18 months after irradiation, and in one patient with a prolactinoma a partial decline of hormone levels was recorded without detectable changes in the size of the adenoma. Only one female patient developed hypopituitarism. No other complications were recorded. CONCLUSIONS: Stereotactic irradiation with Leksell's gamma knife is valuable for treatment of pituitary adenomas and it is well tolerated therapy. Its effect is manifested only after several months. Complications in the sense of hypopituitarism may occur. PMID- 7553755 TI - [Radiosurgical treatment of meningioma with the Leksell gamma knife]. AB - BACKGROUND: Up to the present time radical microsurgery of the tumour was considered the most satisfactory treatment. Now an ever more important part in the treatment of these tumours is played by radiosurgery. It involves minimal surgical stress, a minimal rate of complications and minimal change of lifestyle during the postoperative period. The objective of the submitted work was to evaluate the authors' results with the use of Leksell's gamma knife. METHODS AND RESULTS: In 1992-1994 48 patients with meningiomas were indicated for treatment with a gamma knife. The group comprised 12 men (25%) and 36 women (75%) aged 18 73 years (mean age 52.8 years which did not differ significantly in the two groups). In 19 patients (39.6%) one or more neurosurgical operations were carried out previously, in 4 patients (8.3%) fractionated radiotherapy. Karnofski's score, as an indicator of the general condition, varied between 30-90%, the mean value for the whole group being 71%. The calculated volume of the meningioma was between 0.7 and 25.8 cm3, the average being 7.4 cm3. Collateral oedema in the adjacent area of the tumour was found in 6 patients (12.5%). In 19 patients (39.6%) the tumour was at the base of the skull, in 29 patients (60.4%) at other sites. The radiosurgically administered dose was within the range of 6.5-24 Gy, on average 14,2 Gy, on the isodose on average 50.7% (range 40-75%). The dose below the maximum was within the range of 13-45 Gy), on average 28.3 Gy. Only in two patients fractionated administration of the dose was used. After a 6-month interval following operation 30 patients were examined. In 7 patients (23.3%) partial wrinkling of the meningioma occurred (range 6-18 months), Karnofski's score improved by 10% in 8 (26.6%) and deteriorated in 3 patients (10%). The neurodeficit improved in 7 (23.3%) and deteriorated in 4 patients (13.3%). Collateral oedema developed in 5 patients (16.6%) 5-7 months after operation, in about half the patients it was symptom free. During the follow up period 2 patients died (8 and 5 months following surgery): in one the cause of death was not associated with the basic disease, in the second patient (where radiotherapy was the third neurosurgical operation) the radiosurgical operation could not reverse tha fatal course of the disease. CONCLUSIONS: Radiosurgery of meningiomas is a safe therapeutic method with zero mortality, minimal surgical stress, a minimal rate of complications and a minimal morbidity. It is indicated in meningiomas and possibly their residues after subtotal neurosurgery up to a maximal volume of 30 cm3. It is primary treatment suitable in particular in old.patients, patients with a high per- and postoperative risk and in patients refusing open neurosurgery. PMID- 7553756 TI - [Use of the radiosurgery knife in the treatment of hypophyseal adenomas]. AB - BACKGROUND. Surgery of pituitary adenomas did not produce quite satisfactory results. Therefore radiosurgery using Leksell's gamma knife has become the most widely used method which-with the contribution of MRI-meets the most important demands of aimed irradiation: a sufficiently high dose of radiation of the tumour and minimal radiation load of functionally important surrounding structures. The objective of the investigation was to assemble experience with this therapeutic method. METHODS AND RESULTS. During the period between October 1992 and September 1994 a total of 41 patients were treated: 16 men, 12-66 years old, mean age 40.8 years; 25 women age 16-76 years, mean age 50.2 years. Female:male ratio 1.5 : 1. In 30 patients (73.2%) a microsurgical operation had preceded, in 5 (12.2%) conventional fractionated radiotherapy and in 11 patients (26.9%) primary radiosurgery. As far as the type of pituitary adenoma is concerned, it conditioned acromegaly in 30, Cushing's syndrome in 3 or Nelson's syndrome in 1, or a prolactinoma was involved (in 2 patients). In five instances the adenoma was hormonally inactive. Its localization was most frequently intrasellar (36), less frequently parasellar (5). The range of administered doses varied as regards the maximum between 12.5 and 80 Gy, the average being 46.8 Gy, to the periphery of the adenoma a dose of 10-49 Gy was administered, on average 24.7 Gy using a 50 80% isodose. The time interval after treatment is relatively short for detailed analysis or evaluation. During current evaluation the authors did not observe in any of the patients progression of the disease, and in several patients diminution of the tumour was found. Karnofski's score seemed to improve. CONCLUSIONS. Radiosurgery, using Leksell's gamma knife, is after failure of conservative and microsurgical therapeutic possibilities suitable further treatment of pituitary adenoma. In indicated cases it may be the first choice. Postirradiation follow up indicates promising effects, for more detailed evaluation a several years' interval is necessary. PMID- 7553759 TI - [The human genome--chromosome 17]. AB - The submitted account on genes of the 17th chromosome pays attention to autosomal dominant hereditary neurodegenerative diseases which have some characteristics in common-they are relatively frequent, a considerable proportion of the cases is conditioned by new mutations, contributed mainly by male gametes, and they affect mostly the periphery of the nervous system. In addition to the cause of this group of diseases which at present is not yet quite clear, the 17th chromosome is the carrier of the locus the product of which--p53 protein--interferes with oncogenesis. Its effect twofold--the normal product under normal conditions (natural regulation) exerts an antioncogenic action, its shortage or altered quality-(mutations) exert an oncogenic action. Another important locus which is involved in oncogenic processes is locus RARA--the receptor of retinoic acid which participates in the formation of promyelocytic acute leukaemia and locus BRCA1 the pathogenic alleles of which are a dominant predisposition for breast cancer. PMID- 7553758 TI - [Fractionated stereotaxic radiotherapy with the Leksell gamma knife]. AB - BACKGROUND: Stereotactic radiotherapy is treatment of a small or medium-sized intracranial focus, using a fractionated regime and the stereotactic method. It is carried out usually on linear accelerators. The objective of the present investigation was to test the possibility of stereotactic radiotherapy using Leksell's gamma knife and Leksell's stereotactic frame. METHODS AND RESULTS: During the period between April 1993 and November 1994 38 patients (16 women and 22 men aged 15-72 years) were indicated for stereotactic radiotherapy. The patients had the following diagnoses: adenoma of the epipharynx (1), low-grade gliomas (10), ependymoma (1), germinoma (1), meningioma (2), solitary metastases into the CNS (19), pinealomas (3) and chordoma (1). The volume of the tumour varied from 740 mm3 to 37,000 mm3. Stereotactic fractionated radiotherapy was carried out using a Leksell Gamma Knife, Electa Instrument AB, Sweden. For assessment of the total fractionated dose a linear quadratic model was used. Thus assessed value was further modified in relation to the volume and radiobiology of the tumour, critical structures surrounding the treated lesion and other factors. The total fractionated dose to the maximum varied from 20 Gy to 60 Gy and the total fractionated dose on the periphery of the tumour from 12 Gy to 30 Gy. With regard to the short time interval which has elapsed since treatment it is not yet possible to evaluate completely the achieved results; it will be possible to do so after a time interval of 1-5 years following the operation. The patients tolerated the 2-5 day fixation with a stereotactic Leksell frame very well and none of the patients developed complications. CONCLUSIONS: Fractionated stereotactic radiotherapy with Leksell's gamma knife, using Leksell's stereotactic frame, is a very accurate therapeutic method with a minimal radiation load of surrounding brain tissue. It requires a short hospitalization period and is associated with no or only minimal complications and improves the quality of patient's lives. PMID- 7553760 TI - [The effectiveness of so-called potentiated penicillins (augmentin and tazobactam) in vitro]. AB - The resistance of bacterial strains to beta-lactam antibiotics (penicillins and cephalosporins) is due to transfer of genes coding the production of enzymes-beta lactamases. These enzymes--penicillinases and cephalosporinases--can hydrolyse beta-lactam antibiotics. The using of beta-lactamase inhibitors in combination with some beta-lactam antibiotics is a suitable alternative in the present unfavourable situation in chemotherapy (increase of occurrence of gram-negative and gram-positive penicillin-cephalosporin-resistant bacterial strains). In this communication the authors present the review of efficiency of three, in clinical practice mostly used potentiated penicillins: Augmentin (amoxicillin + clavulanate), Unasyn (ampicillin + sulbactam) and Tazobac (piperacillin + tazobactam). Tazobactam seems to be the most promising beta-lactamase inhibitor which has, unlike clavulanate and sulbactam, its own antibiotic activity. PMID- 7553761 TI - [Report on the activity of the Central Commission on Ethics of the Ministry of Health 1990-1994]. AB - The authors summarize the most important activities of the Central ethical commission at home and abroad during four years of its existence. They mention documents elaborated for the needs of the broad public and local ethical commissions incl. the Statute of the central ethical commission. Experience with evaluation of ethical problems pertaining to relations between health professionals and the lay public indicate above all impaired mutual communication. The authors feel that wide publicity of ethical problems in the professional and lay press is essential and should be permanently stimulated. PMID- 7553762 TI - The neuroendocrine system in hibernating mammals: present knowledge and open questions. AB - The present review describes the distribution and the function-dependent reactivity pattern of those peptidergic and aminergic components of the neuroendocrine system of hibernating mammals that have been studied by histological, pharmacological and physiological techniques. Particular attention has been paid to the intrinsic connectivity of the peptidergic apparatus and its input systems. Since the reactivity patterns of the neuroendocrine system show remarkable fluctuations in relation to the various stages of hibernation and euthermia, these fluctuations have been analyzed with respect to (1) their causative role in the regulation of hibernation and (2) their secondary response to physiological changes during hibernation. The author's investigations described in this review have mainly been performed in European hedgehogs (Erinaceus europaeus), European and golden hamsters (Cricetus cricetus, Mesocricetus auratus), dormice (Glis glis), and in Richardson's and Columbian ground squirrels (Spermophilus richardsonii, Spermophilus columbianus), by the use of light- and electron-microscopic immunocytochemistry and histochemistry, in situ hybridization, radioimmunoassays and stereotaxically guided application techniques. These experiments were also performed in hypothermic animals. The (partially published) results obtained by the author and his associates are reviewed with reference to the body of evidence found in the recent literature. With respect to their reactivity patterns, several neuropeptide and transmitter systems can be regarded as candidates for control systems of hibernation. Neuronal complexes immunoreactive for endogenous opiates, in particular enkephalin, and also for vasopressin, somatostatin, substance P, corticotropin releasing factor and serotonin are probably involved in the neuroendocrine control of hibernation. PMID- 7553763 TI - Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick end DNA labelling. AB - Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland. PMID- 7553764 TI - Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron microscopic and stereological study. AB - The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process. PMID- 7553768 TI - Do vasoactive intestinal peptide (VIP)- and nitric oxide synthase-immunoreactive terminals synapse exclusively with VIP cell bodies in the submucous plexus of the guinea-pig ileum? AB - In the submucous plexus of the guinea-pig ileum, previous light-microscopic studies have revealed that vasoactive intestinal peptide (VIP)-immunoreactive and nitric oxide synthase (NOS)-immunoreactive terminals are found predominantly in association with VIP-immunoreactive nerve cell bodies. In this study, double label immunohistochemistry at the light-microscopic level demonstrated co localization of NOS-immunoreactivity and VIP-immunoreactivity in axon terminals in submucous ganglia. About 90% of nerve fibres with NOS-immunoreactivity or VIP immunoreactivity were immunoreactive for both antigens; only about 10% of labelled varicosities contained only NOS-immunoreactivity or VIP immunoreactivity. The VIP/NOS varicosities were more often seen in the central parts of the ganglia, close to the VIP-immunoreactive cell bodies. Ultrastructural immunocytochemistry with antibodies to VIP was used to determine if NOS/VIP terminals synapse exclusively with VIP-immunoreactive nerve cell bodies. We examined the targets of VIP-immunoreactive boutons in two submucous ganglia from different animals. Serial ultrathin sections were taken through the ganglia after they had been processed for VIP immunocytochemistry. For each cell body, the number of VIP inputs (synapses and close contacts) was determined. The number of VIP-immunoreactive synapses received by the cell bodies of submucous neurons varied from 0-4 and the number of VIP-immunoreactive close contacts varied from 3-10. There was no significant difference between VIP-immunoreactive nerve cell bodies and non-VIP nerve cell bodies in the number of VIP immunoreactive synapses and close contacts they received. Thus, the implication from light microscopy that NOS/VIP terminals end predominantly on VIP nerve cells was not vindicated by electron microscopy. PMID- 7553765 TI - Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies. AB - The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers. PMID- 7553767 TI - Glycosaminoglycans in porcine lung: an ultrastructural study using cupromeronic blue. AB - Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl2 according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium. PMID- 7553769 TI - Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers. AB - The expression and distribution of nitric oxide synthase (NOS) was studied by use of the newly designed specific histochemical NADPH diaphorase staining method and the indirect immunofluorescence technique employing an antiserum to brain NOS in visceral and somatic striated muscles of several mammalian species. Histochemical activity and immunoreactivity were located in the sarcolemma region of type I and II fibers of all muscles investigated. Visceral muscles were more strongly stained than somatic muscles. Furthermore, type II fibers, identified by staining of myosin adenosine triphosphatase activity after pre-incubation at alkaline pH, were more intensely labeled than type I fibers. In addition, NOS activity was detected in the area of the sarcolemma of intrafusal fibers. No obvious differences between species were observed. It was concluded that NOS of striated muscles probably makes up the richest and most important nitric oxide source in mammals. PMID- 7553766 TI - The distribution and co-localization of immunoreactivity to nitric oxide synthase, vasoactive intestinal polypeptide and substance P within nerve fibres supplying bovine and porcine female genital organs. AB - The distribution of nitric oxide synthase-immunoreactive (NOS-IR) axons and their relationship to structures immunoreactive to vasoactive intestinal polypeptide (VIP), substance P (SP) and tyrosine hydroxylase (TH) were studied by means of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) technique or double-labelling immunofluorescence in the genital organs of cow and pig. Relevant neurons were also investigated in the pig. NOS-containing neural structures were TH-immunonegative in bovine or porcine genital organs, or in the studied ganglia. In the bovine ovary, NOS-IR nerves were neither VIP-IR nor SP IR, whereas in the pig, most NOS-containing axons were also VIP-IR. The oviduct was supplied by single NOS/VIP- or NOS/SP-containing nerves, whereas in the uterus, NOS-IR axons were moderate in number, often being immunoreactive for VIP or SP. Numerous NOS/VIP-IR and NOS/SP-IR nerves were found in the vagina of both species. In all tissues studied, NOS-IR axons were mainly related to vascular smooth muscle. Most of the neurons of the paracervical ganglia and some neurons in dorsal root ganglia exhibited strong NOS activity. Only single neurons in sympathetic ganglia were NADPH-d-positive. Most nitrergic neurons in the autonomic ganglia were VIP-IR but SP-immunonegative. The sensory neurons were mostly NOS/SP-IR, whereas only single neurons co-expressed NOS and VIP immunoreactivity. PMID- 7553770 TI - Expression of alpha SM actin in terrestrial ectothermic vertebrates. AB - alpha-Smooth muscle (alpha SM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-alpha SM-1. Immunoreactivity to this antibody has been shown to be localized in the NH2-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian (Triturus vulgaris, Rana esculenta) and three reptilian species (Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti alpha SM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-alpha SM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine alpha SM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac EEED, but not Ac-DEED, as occurs in mammals. alpha SM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-alpha SM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle (P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution alpha SM actin arose earlier than previously proposed. PMID- 7553771 TI - Quantitation of peptide hormone in single cultured secretory neurons of the crab, Cardisoma carnifex. AB - The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1-50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of approximately 150 peptidergic neurons that form the X-organ--sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having "high" (> 33 pg; mean 86 +/- 5, S.E., n = 47) or "low" (< or = 33 pg; mean 22 +/- 2.5; n = 17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2 +/- 2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing. PMID- 7553772 TI - Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland. AB - Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were nonadrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dye-labelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate projecting neurons, which contain VIP. PMID- 7553774 TI - Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat. AB - The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non noradrenergic sympathetic neurons of the pelvic ganglia. PMID- 7553773 TI - Thymic vascular system of the European common frog, Rana temporaria: a scanning electron-microscopic study of vascular casts. AB - Vascular corrosion casts of the thymus of adult individuals of the European common frog, Rana temporaria, were analysed by scanning electron microscopy. The main arterial vessel, which is derived either from the temporal artery or from the auricular ramus, approaches the central territory of the gland and branches into "twigs" that, on penetrating the parenchyma, give rise to capillaries. Most of these capillaries run vertically towards the surface of the gland; they either join the superficial capillary plexus or follow this plexus for a variable distance and then run back towards the medulla, forming capillary loops. The former capillaries link with the extensive venous plexus composed of irregular meshes, whereas the latter capillaries join the venules at the cortico-medullary boundary and finally escape into collecting veins on the gland surface. The venous twigs, which join together near the gland, form the main thymic vein, which empties into the external jugular vein. The details of the thymic vasculature of the anuran amphibian, R. temporaria, are compared with those described in mammalian species, viz. the mouse, rat and guinea pig. PMID- 7553776 TI - Evidence for inositol tetrakisphosphate-activated Ca2+ influx pathway refilling inositol trisphosphate-sensitive Ca2+ stores in hamster eggs. AB - To identify the Ca2+ influx pathway responsible for maintaining Ca2+ oscillations in hamster eggs, changes in intracellular Ca2+ concentration ([Ca2+]i) were recorded using the Fura-2 fluorescent imaging technique during iontophoretic injection of inositol phosphates under voltage clamp. Both inositol 1,4,5 trisphosphate (InsP3) and 1,3,4,5-tetrakisphosphate (InsP4) caused repetitive Ca2+ transients when injected continuously into eggs, although the latter was much less effective. These Ca2+ transients were inhibited by the monoclonal antibody 18A10 to the InsP3 receptor/Ca2+ channel. In Ca(2+)-free medium, InsP4 induced Ca2+ transients were absent or much less frequent than in normal medium. A small but persistent increase in [Ca2+]i during InsP4 injection was revealed when Ca2+ uptake into InsP3-sensitive Ca2+ stores was suppressed by thapsigargin. This Ca2+ rise is due to Ca2+ entry, but not Ca2+ release, because it was: (i) increased by raising the extracellular Ca2+ concentration and abolished in Ca(2+) free medium; (ii) larger at more negative membrane potentials which provide greater electrical driving force for Ca2+ entry; and (iii) not affected by 18A10. A moderate dose of InsP3 did not cause substantial Ca2+ entry, as tested in thapsigargin- and 18A10-treated eggs. InsP4 facilitated the restoration of Ca2+ stores after Ca2+ releases induced by pulsatile InsP3 injections. Thus, we obtained evidence for a Ca2+ influx pathway activated by InsP4 which provides Ca2+ to refill InsP3-sensitive Ca2+ stores in intact cells. PMID- 7553775 TI - Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep. AB - Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohistochemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibres were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep. PMID- 7553778 TI - Interplay of glucose-stimulated Ca2+ sequestration and acetylcholine-induced Ca2+ release at the endoplasmic reticulum in rat pancreatic beta-cells. AB - It is known that the stimulation with high glucose initially decreases as well as subsequently increases the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. In the present study, we aimed at exploring the ionic mechanism and physiological role of the glucose-induced decrease in [Ca2+]i by measuring [Ca2+]i in single pancreatic beta-cells from normal rats. The glucose induced decrease in [Ca2+]i in beta-cells was completely inhibited by thapsigargin (Tg), a specific inhibitor of the endoplasmic reticulum (ER) Ca2+ pump (Ca(2+)-ATPase). On the other hand, neither a Ca(2+)-free nor a low-Na+ condition significantly altered the glucose-induced decrease in [Ca2+]i. At basal glucose concentrations (1-4.5 mM), an insulin secretagogue acetylcholine (ACh) evoked a rather transient increase in [Ca2+]i in the presence and absence of extracellular Ca2+. A rise in glucose concentration from 1 to 4.5 mM produced a sustained decrease in [Ca2+]i and concomitantly augmented the ACh-evoked increase in [Ca2+]i. The resting [Ca2+]i level determined by glucose was tightly and reciprocally correlated with the peak of the [Ca2+]i response to ACh. Successive ACh pulses elicited repeated [Ca2+]i responses, which were progressively inhibited by Tg, suggesting that Ca2+ released by ACh was taken up by the ER Ca2+ pump and thus cycled. The results demonstrate that glucose decreases [Ca2+]i in pancreatic beta-cells mainly by activating the Ca2+ pump in ER from which ACh mobilizes Ca2+. Furthermore, the glucose-stimulated sequestration of Ca2+ by ER results in an augmented [Ca2+]i response to ACh, providing a mechanistic basis for the glucose-dependent action of ACh to initiate insulin secretion. PMID- 7553777 TI - Thapsigargin protects human erythrocyte Ca(2+)-ATPase from proteolysis. AB - The effect of thapsigargin on the activation by partial proteolysis of the plasma membrane Ca(2+)-ATPase was studied in intact human erythrocyte membranes and in the purified enzyme. The enzyme was maximally activated in the absence of thapsigargin within 1 min of exposure to trypsin. However, in the presence of thapsigargin maximal activation was achieved only after 5 min trypsin digestion. Thapsigargin did not alter the pattern of proteolysis as revealed by SDS-PAGE of the tryptic fragments, although it slowed down the rate of appearance of the fragments. Thapsigargin also enhanced the activation of the enzyme by calmodulin. These findings suggest that, although thapsigargin at low concentrations has no effect on the catalytic activity of the Ca(2+)-ATPase in vitro in the absence of calmodulin, it could interfere with its regulation in vivo. PMID- 7553781 TI - A mathematical model of agonist-induced propagation of calcium waves in astrocytes. AB - In astrocytes, calcium signals evoked by neurotransmitters appear as waves within single cells, which spread to other cells in the network. Recent analysis has shown that waves are initiated at a single invariant site in the cell and propagated within the cell in a nonlinear and saltatory manner by regenerative amplification at specific predestined cellular sites. In order to gain insight into local cellular waves and wave collisions we have developed a mathematical model of cellular wave amplification loci. This model is in good agreement with experimental data which includes: ambient calcium gradients in resting cells, wave origination and local amplification and generation of local waves. As observed in experiments, the model also predicts that different locations in the cell can have different frequencies of oscillation. The amplification loci are thought to be specialized areas of the endoplasmic reticulum membrane containing a higher density or higher sensitivity of IP3 receptors. Our analysis suggests that the cellular loci act as weakly coupled oscillators each with its intrinsic latency and frequency of oscillation. Thus the appearance of the propagated calcium wave may be a reflection of these differences rather than an actual diffusional wave propagation. PMID- 7553779 TI - Staurosporine enhances Ca2+ entry induced by depletion of intracellular Ca2+ stores in rat parotid acinar cells. AB - The effect of staurosporine on the Ca2+ signalling induced by the muscarinic receptor agonist carbachol (CCh) was studied in Fura-2-loaded rat parotid acinar cells. At concentrations > 1 nM, staurosporine dose-dependently enhanced the sustained increase in cytosolic free Ca2+ concentration ([Ca2+]i), but did not affect the peak [Ca2+]i seen just after stimulation. The enhancement of the sustained increase in [Ca2+]i was not attenuated by the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate, and not mimicked by another inhibitor of protein kinase C, K-252a, suggesting that the effect of staurosporine on the CCh-induced Ca2+ signalling may be due to a mechanism independent of the inhibitory action on protein kinase C. Staurosporine also enhanced the increases in [Ca2+]i induced by the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG) and the Ca2+ ionophore ionomycin (Iono). When the cells were stimulated by CCh, TG, or Iono in the absence of extracellular Ca2+, a transient increase in [Ca2+]i due to Ca2+ release from intracellular stores was observed. This increase in [Ca2+]i was unaffected by preincubation with staurosporine. However, when Ca2+ was added to the extracellular medium after [Ca2+]i had returned to the resting level, the increase in [Ca2+]i was significantly enhanced by staurosporine. In addition, staurosporine accelerated the Mn2+ influx following the addition of CCh, TG, or Iono. These results suggest that staurosporine modulates the Ca2+ entry system activated by depletion of intracellular Ca2+ stores in rat parotid acinar cells. PMID- 7553782 TI - Distribution of plasma membrane Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor in human platelet membranes. AB - Human platelet plasma membranes were prepared by the glycerol lysis method of Harmon et al. [Harmon JT. Greco NJ. Jamieson GA. (1992) Isolation of human platelet plasma membranes by glycerol lysis. Meth. Enzymol., 215, 32-36]. The membranes were observed to contain a Ca(2+)-ATPase with different properties than those of internal membranes. The specific activity of Ca(2+)-ATPase was lower in plasma membranes (10-40 nmol ATP hydrolyzed/min/mg), but the ATPase was less sensitive to thapsigargin (41% inhibition at 500 nM) and more sensitive to vanadate (50% inhibition at 4 microM) than the Ca(2+)-ATPase in internal platelet membranes. The plasma membranes contained a Ca(2+)-ATPase detectable by monoclonal and polyclonal antibodies against erythrocyte Ca(2+)-ATPase that had a molecular mass of 144 kD. However, an anti-peptide antibody against an N-terminal sequence of the inositol 1,4,5-trisphosphate receptor recognized this protein in internal membranes, but not plasma membranes. PMID- 7553784 TI - Spatial distribution and temporal change in cytosolic pH and [Ca2+] in resting and activated single human platelets. AB - In human platelets, a rapid rise in cytoplasmic Ca2+ and slower rise in cytoplasmic pH follow stimulation by thrombin. With the Ca2+ probe Fura-2 and the pH probe SNARF-1 for digitized fluorescence microscopy, we studied simultaneously the distribution and changes with time of [pH]i and [Ca2+]i in individual human platelets. In platelets coloaded with both probes, the probes had no detectable fluorescence at each other's excitation wavelength. The monovalent cation ionophore, nigericin (2 microM), produced a homogeneous rise in pH but no change in [Ca2+]. Platelets, in contact with glass, spread and developed an irregular, apparently mutually independent rise in both [Ca2+] and pH. Stimulation of platelets by thrombin 1.0 U/ml elevated [Ca2+]i and produced slow alkalinization without initial acidification. Replacement of extracellular Na+ by choline abolished thrombin-induced alkalinization, but had no effect on thrombin-induced [Ca2+]i elevation. ADP 10 microM caused a rapid rise of [Ca2+]i and transient alkalinization. Most stimulated platelets developed a gradient in pH, that was highest in the center. ADP and thrombin caused oscillation of [Ca2+]i but not of [pH]i. We conclude that alkalinization in stimulated platelets, presumably involving Na+/H+ antiport, is not essential for the rise of [Ca2+]i that may accompany it. PMID- 7553783 TI - Immunological stimulation of single rat basophilic leukemia RBL-2H3 cells co activates Ca(2+)-entry and K(+)-channels. AB - The relationship between type 1 Fc epsilon-receptor (Fc epsilon RI) mediated cell stimulation, Ca(2+)-signals and membrane currents was studied in rat mucosal mast cells, subline RBL-2H3 by combining patch-clamp, Fura-2, 45Ca(2+)-uptake and secretory response measurements. Cells were stimulated by Fc epsilon RI clustering either with IgE and antigen or by an IgE specific monoclonal antibody. Both stimuli induced a biphasic increase in the free intracellular Ca(2+) concentration ([Ca2+]i). Fc epsilon RI clustering in Ca(2+)-free solution induces a transient increase in [Ca2+]i reflecting Ca2+ release from the Ins(1,4,5)P3 sensitive stores. Mn2+ applied to a nominally Ca(2+)-free solution, causes quenching of the Fura-2 emission during Fc epsilon RI clustering, indicating activation of a transmembrane pathway for the entry of extracellular calcium ions. Whole-cell current-voltage relationship of resting cells showed strong inward rectification. The inward current component at a potential of -100 mV is increased by 23 +/- 11% (n = 14) upon Fc epsilon RI clustering, whereas the outward component at +50 mV was enhanced by 45 +/- 6%. The Fc epsilon RI activated current was identified as due to K+ ions, because it reversed close to the K(+)-equilibrium potential, was blocked by Ba2+ or Cs+ containing or K(+) free bath solutions. It was also inhibited by TEA and quinidine, while DIDS had no effect. Moreover, an inwardly rectifying K(+)-channel with a conductance of 28 pS was recorded in single channel measurements. The open probability of this channel increased by 39 +/- 16% (n = 8) upon Fc epsilon RI clustering. Superfusion of the cells with nominally K(+)-free solution also significantly inhibited both the Fc epsilon RI mediated 45Ca2+ uptake and the secretory response of the cells. We conclude that activation of K(+)-channels upon Fc epsilon RI clustering is functionally involved in the control and the maintenance of the secretory response of RBL-2H3 mast cells. PMID- 7553780 TI - Multiple intracellular Ca2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca2+ pools. AB - Previously, we have used the classical approach to examine intracellular calcium stores in human foreskin fibroblasts (HSWP) cells. In this classical protocol cells are first permeabilized and then allowed to fill their Ca2+ reservoirs with 45Ca2+ in the presence of ATP. In this paper we present an alternative method to examine intracellular calcium pools. In this alternate protocol, whole cells are loaded to isotopic steady-state with 45Ca2+ and then permeabilized using digitonin. Comparison of the Ca2+ content of cells treated with these two methodologies reveals that cells treated with the alternate protocol have a Ca2+ content 3 orders of magnitude higher than those treated with the classical protocol. Using this alternative technique we demonstrate that there are 3 intracellular calcium pools in HSWP cells. These pools are: (i) an IP3-sensitive, thapsigargin-sensitive Ca2+ pool; (ii) an IP3-insensitive, thapsigargin-sensitive Ca2+ pool; and (iii) an ionomycin sensitive Ca2+ pool. The relationship between the Ca2+ pool mobilized by BK treatment and by IP3 treatment is also explored. Microinjection data shown here suggest that IP3 can mobilize all of the intracellular Ca2+ mobilized by BK. However, in the permeabilized system BK pretreatment followed by IP3 treatment can release more Ca2+ than can be release by IP3 treatment alone. We suggest one plausible explanation for this observation: when cells are treated using the alternative permeabilization protocol, communication occurs between an IP3-sensitive and an IP3-insensitive calcium pool. Thus BK pretreatment would empty the IP3-sensitive Ca2+ pool. This pool would subsequently be refilled with Ca2+ from a previously untapped, IP3 insensitive, Ca2+ reservoir and more Ca2+ would be available for subsequent release by IP3 treatment. PMID- 7553785 TI - Transient increase in cytosolic free calcium evoked by repolarization in type I vestibular hair cells of rats. AB - Simultaneous whole-cell patch clamp and Fura-2 microfluorimetric recordings of membrane currents and intracellular free calcium concentration ([Ca2+]i) were made from type I vestibular hair cells isolated from cristae ampullares of adult rats. Cells held between -110 or -70 mV and depolarized up to -20 mV did not evoke any [Ca2+]i changes for any duration of the membrane depolarization (up to 3 s). Returning the membrane to repolarizing potential induced a transient [Ca2+]i increase. At the pulse break, an inward current was evoked. The [Ca2+]i increase and inward current amplitude were dependent on the duration and the amplitude of the previous depolarization. A liminar value of membrane depolarization of -55 +/- 3 mV (mean resting potential -62 +/- 7 mv) had to be applied to induce [Ca2+]i increase upon subsequent repolarization. [Ca2+]i response and inward current could not be evoked in calcium-free solution. Both responses were restored when calcium was added to the medium. PMID- 7553786 TI - Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum. AB - Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP. PMID- 7553787 TI - Increased capacity for store regulated calcium influx in U937 cells differentiated by treatment with dibutyryl cAMP. AB - We have investigated the mechanisms underlying calcium signalling evoked by cross linking of the high affinity IgG receptor (Fc gamma RI) in populations of the human monocyte-like cell line, U937, following activation of the cells by cytokine treatment, or differentiation to a more macrophage-like state by treatment with dibutyryl cAMP (Bt2cAMP). We have shown previously that a larger and more prolonged entry of external calcium occurs in Bt2cAMP-differentiated cells, although there is a smaller initial release from internal stores in these cells than in those activated by IFN-gamma treatment. In this paper we demonstrate, by use of the endomembrane Ca(2+)-ATPase inhibitor, thapsigargin, that this effect is explained (at least in part) by an enhanced capacity for store regulated entry of calcium in Bt2cAMP-differentiated cells. PMID- 7553788 TI - Quantitative determination of free calcium in subcellular compartments, as probed by Indo-1 and confocal microspectrofluorometry. AB - Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+]i). To perform in situ calibrations of [Ca2+]i in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+]i, intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+]i, intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+]i. The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+]i determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+]i in the cytoplasm than in the nucleus (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553791 TI - Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants. AB - Kinetics of 45Ca outflow and human placental lactogen (hPL) release were characterized in human placental explants. Measurements of the rate of 45Ca and [3H]-sucrose (extracellular space marker) outflow from preloaded explants showed that, after a 40 min washout period, the 45Ca effluent radioactivity presumably originated from an intracellular compartment. This view was further supported by the La3+ and temperature sensitivity of the 45Ca outflow. Moreover, the addition of ionomycin as well as an increase in the extracellular Ca2+ or Ba2+ concentration provoked a dose-dependent rise in both the 45Ca outflow and hPL release. There were no systematic temporal analogies between the pattern of 45Ca outflow and hPL release. Taken together, these observations suggest that the stimulation of 45Ca outflow reflects an increased rate of 40Ca entry. The present data also extend previous observations indicating that hPL release can be stimulated by Ca2+ entry. Lastly, the 'in vitro' method described herein allows one to compare rapid changes in hPL release with associated ionic events and can be used to further document the relationships between placental secretory responses and cell calcium metabolism. PMID- 7553789 TI - [Ca2+]i oscillations in rat chromaffin cells: frequency and amplitude modulation by Ca2+ and InsP3. AB - Rat chromaffin cells in primary culture exhibit oscillations of cytosolic Ca2+ concentration, sustained by the rhythmic discharge of Ca2+ from specialized intracellular stores. Each Ca2+ spike starts from a discrete region of the cell (pacemaker), and then propagates across the entire cytosol. Spike initiation and propagation, governing the oscillation frequency and amplitude respectively, appeared to be controlled by different mechanisms. The pacemaker was found to be directly activated by increases of cytosolic Ca2+ concentration obtained by either K+ depolarization or nicotinic stimulation. On the other hand, muscarinic or B2 stimulation was required for an efficient spreading to occur, thus suggesting a key role of InsP3 in the signal propagation. The pacemaker displayed an autonomous activity, as documented by the presence of local Ca2+ discharges, which were not necessarily accompanied by spreading to the rest of the cell. This uncoupling could be stimulated by the selective increase of the pacemaker firing rate, due to the rise of the intracellular Ca2+ concentration. Modulation of Ca2+ spike amplitude by treatments affecting either the pacemaker or the spreading phase might be related to quantal Ca2+ release from functionally discrete stores. PMID- 7553790 TI - Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines. AB - The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U purinoceptor stimulation which is consistent with the opening of a Ca(2+) sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined. PMID- 7553792 TI - Serum albumin concentrations in rural Zimbabweans. AB - Primary infections, protein malnutrition or simply poor food intake which decrease albumin synthesis often prevail in rural Zimbabwe. Therefore, we postulated that serum albumin levels in rural folk may not compare with published values. To investigate this, blood samples were collected from 1,502 consenting participants aged between 16 and 90 years from randomly selected rural areas in Mashonaland West, Midlands, Matebeleland South and Matebeleland North provinces in Zimbabwe. The effects of smoking, alcohol consumption and pregnancy on serum albumin levels were also investigated. The mean age in years did not differ significantly between females and males (32.5 +/- 0.44, n = 943 vs 33.84 +/- 0.62, n = 559). Serum preserved with sodium azide at -4 degrees C was analyzed for albumin using a Quick Lab 2 Analyzer (Ames Quick Lab) by the bromocresol green dye binding method. Only small differences of albumin concentrations (means +/- S.E.M.) were noted for each studied area and results were pooled. The normal serum albumin ranges were 27 to 52 g/L for females and 26 to 52 g/L for males and were different from those reported for developed countries. The albumin levels in females were significantly (p < 0.01) lower than in males (39.29 +/- 0.20 g/L, n = 943 vs 40.09 +/- 0.25 g/L, n = 559). However, the concentrations of the protein in males who smoked (38.66 +/- 0.38 g/L, n = 174) were significantly low (p < 0.01) by comparison with non smokers. Smoking did not alter albumin levels in females (38.27 +/- 0.53 g/L, n = 87) perhaps because they smoked fewer cigarettes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553794 TI - Indomethacin and alpha-tocopheral enhanced survival in endotoxic rats. AB - Induced endotoxaemia was studied in healthy male albino rats injected intravenously with lipopolysaccharide from Escherichia coli at 10, 20 and 30 mg/kg dose levels. The endotoxic rats were closely observed for mortality within 48 hours and subsequently for seven days. Within five hours post administration over 50 pc mortality was observed at dose levels of 20 to 30 mg/kg. Pre-treatment of rats with Indomethacin (250 mg/kg) alone and alpha-tocopheral (100 mg/kg) produced significant protective effects with the mortality rate reduced to 50 pc at the highest endotoxin dose level. Interestingly a combination of both drugs significantly improved survival with an observed mortality of 20 pc. Prednisolone (500 mg/kg) either alone or in combination with indomethacin or alpha-tocopherol did not offer any advantage. Instead the mortality rate was significantly high. A combination of Indomethacin and alpha-tocopheral significantly improved survival in endotoxic rats. PMID- 7553793 TI - Kufungisisa (thinking too much): a Shona idiom for non-psychotic mental illness. AB - Non-psychotic mental disorders are very common in primary care settings in Zimbabwe. Sociocultural factors play a profound role in the manifestation of such illness, and local idioms and concepts of illness need to be understood and related to biomedical psychiatric concepts originating in EuroAmerican cultures. One such Shona concept is kufungisisa or thinking too much. This article describes some clinical correlates and the contextual meaning of this term summarising the findings of two studies and the details of a third. Kufungisisa is used to mean both a cause and a symptom of illness. Both patients and care providers view this term as being related to mental, social and spiritual distress. The term is strongly related to biomedical constructs of non-psychotic mental illness, but is not specifically related either to depression or anxiety. We suggest that the conceptual equivalent of this term is "feeling stressed" or, in psychiatric terms as a non-specific "neurotic mental illness". Using the term kufungisisa may increase awareness and recognition of non-psychotic mental illness by the community and primary health care providers. PMID- 7553795 TI - Relationship of casual blood pressure to smoking, education and occupation in a high density town near Harare, Zimbabwe. AB - An analysis is presented on associations of smoking, education and occupation with blood pressure among residents of a high density town near Harare, Zimbabwe. A total of 973 persons aged five years and above were surveyed in 1993. Obesity was significantly (p < 0.001) correlated with blood pressure (systolic or diastolic). A significant linear relationship between blood pressure and age for each sex was observed (p < 0.001). Smoking was not associated with blood pressure in both sexes. There was little evidence to suggest that education was associated with systolic blood pressure in both sexes. Meanwhile, there was strong evidence to suggest that education was associated with diastolic blood pressure in males and not in females. Mean differences in diastolic blood pressure levels between unemployed females and females in formal employment (mean difference (MD) = 7.57; standard error (SE) = 3.15; p < 0.05), in self employment (MD = -10.28; SE = 3.27; p < 0.01) and in school (MD = -6.57; SE = 3.06; p < 0.05) were statistically significant. We suggest that further studies utilizing longitudinal data on the risk factors for hypertension be conducted. PMID- 7553796 TI - Laparoscopic and cystopic findings in patients with chronic pelvic pain in Eshowe, South Africa. AB - Forty two Zulu women who reported pelvic pain of more than six months duration were evaluated using laparoscopy and cystoscopy. Chronic pelvic inflammatory disease (57 pc) and pelvic adhesions (12 pc) involving the reproductive organs were the most common pathologies found. An interesting associated finding was a very high incidence (21.5 pc) of schistosomiasis. We are not able to find any pelvic pathology in only 14.4 pc of patients. This study confirms the opinion that chronic pelvic pain is usually associated with organic pathology. PMID- 7553798 TI - Pneumatosis cystoides intestinalis--in Tanzania. AB - Pneumatosis cystoides intestinalis (PCI) is a rare benign condition affecting portions of the intestinal tract, characterised by: multiple subserosa and submucosal gas cysts, unknown aetiology, and non specific clinical presentation. Its diagnosis is often coincidental. Management of PCI entails that of the associated condition. PMID- 7553797 TI - Meralgia paresthetica: a report on two cases treated surgically. AB - Meralgia paresthetica (Bernhart-Roth Syndrome) is characterised by pain, burning, or tingling paresthesias, and decreased touch and pain sensation on the anterolateral aspect of the thigh. It is due to neuropathy of the lateral femoral cutaneous nerve (LFCN). Conservative treatment is usually successful in relieving the symptoms in most of the patients. We describe two patients who required surgical treatment for intractable symptoms. Although neurolysis with transposition is the most common procedure, we preferred neurectomy with excision of a portion of the LFCN for its very low recurrence rate as opposed to neurolysis. The area of anesthesia generated by this procedure in the distribution of the LFCN tends to shrink with time. Moreover, this pathology, to our knowledge, has not been reported much in the African literature. PMID- 7553799 TI - Some considerations for the prophylaxis of malaria in the non-immune traveller. PMID- 7553800 TI - Recent Medical Defence Union news. PMID- 7553801 TI - Association study in migraine. PMID- 7553803 TI - Guidelines for trials of drug treatments in tension-type headache. First edition: International Headache Society Committee on Clinical Trials. PMID- 7553802 TI - Long-term subcutaneous sumatriptan in cluster headache. PMID- 7553805 TI - Migraine with aura and migraine without aura: one entity, two, or more? PMID- 7553804 TI - Headache classification and the Bible: Was St Paul's thorn in the flesh migraine? AB - The conversion of Saul to Paul was a major event in the history of Western culture. Compared with its impact, any medical comments may seem redundant, but they have kept their place in the literature for many centuries. The flashing light that caused Saul to fall is often explained as solar retinopathy or keratitis, a seizure, or even a hysterical fit. These interpretations propose either a trivial injury or disease that would interfere with mental health. Neither version is quite compatible with the dramatic dimension of the event and with Paul's later achievements and sufferings. In later years, Paul became a great manager, preacher and writer who was able to carry on under any kind of duress, though not without very painful reactions. He was suffering from bouts of unilateral headache, and also from a chronic eye condition which gave great trouble to his followers but did not cause lasting damage; the descriptions fulfil the criteria for migraine without aura of the 1988 Headache Classification. If the flashing light that caused Paul to fall down is interpreted as a visual migraine aura, with the additional symptoms of "not seeing" or photophobia and anorexia, it falls into place with his later history of migraine. PMID- 7553806 TI - Migraine with aura and migraine without aura are two different entities. PMID- 7553807 TI - Migraine with aura and migraine without aura are not different entities. PMID- 7553808 TI - Convergence of afferents from superior sagittal sinus and tooth pulp on cells in the thalamus of the cat. AB - We have previously shown convergence of craniovascular and tooth pulp afferents in the cervical spinal cord of cats. This study looked for similar convergence in the thalamus. Fifty-four thalamic cells with input from tooth pulp, superior sagittal sinus, or both, were identified. Twenty-nine cells with tooth pulp and superior sagittal sinus input were located in the ventrobasal complex of the intralaminar nuclei. Most of these 29 cells were also excited by cooling the contralateral tooth pulp, and 21 had receptive fields on the contralateral face or forelimb. Twenty cells excited by stimulation of superior sagittal sinus, and not tooth pulp, were found in several nuclei. The 5 cells excited by stimulation of tooth pulp, but not sagittal sinus, were restricted to the ventrobasal complex. The data confirm convergence from sagittal sinus, tooth pulp, and skin in the thalamus of anaesthetized cats. PMID- 7553810 TI - Pressure-controlled palpation: a new technique which increases the reliability of manual palpation. AB - The objective of the present study was to investigate whether the reliability of tenderness evaluation can be increased by using a new technique called "pressure controlled palpation" (pcp). The technique has been made possible by a newly invented piece of equipment called a palpometer, with which a pressure-sensitive plastic film attached to the index finger records the pressure exerted. In 15 patients with chronic tension-type headache and in 15 healthy volunteers, 2 investigators studied myofascial tenderness using conventional palpation and pressure-controlled palpation. Tenderness was scored on a 4-point scale in each of the examined pericranial regions. The sum of tenderness scores recorded by two observers using conventional palpation differed significantly (p = 0.0003), while results did not differ between observers using pressure-controlled palpation (p = 0.89). During palpation with seven different pressure intensities a positive and linear relation between pressure and pain intensity was found (p = 0.00006). Pain intensity reported by the subjects correlated highly with tenderness scored by the observer (rs = 0.95, p < 0.0001). These results demonstrate for the first time that tenderness scores can be compared between observers if palpation pressure is controlled. Pressure-controlled palpation represents a major improvement on current palpation techniques and should be standard in future research on myofascial pain disorders. PMID- 7553809 TI - Genetic markers: association study in migraine. AB - Eleven genetic markers were typed in 112 unrelated patients with migraine (50 with aura, 62 without aura) and compared with a random sample of healthy individuals. No significant differences were found for the ABO and Rh systems, acid phosphatase 1, phosphoglucomutase 1, adenosine deaminase, haptoglobin, transferrin, alpha-1-antitrypsin, and D1S80. Strong associations between the group of patients with migraine and group-specific component GC 1F-1F and esterase-D ESD 2-2 phenotypes were observed. These associations raise the possibility that a molecular genetic factor for migraine may exist in or near the Group Component (chromosome 4) and Esterase D (chromosome 13) loci, and represent a first comprehensive step in the eventual localization and isolation of the migraine genes. PMID- 7553811 TI - Increased cerebrovascular pCO2 reactivity in migraine with aura--a transcranial Doppler study during hyperventilation. AB - Cerebrovascular reactivity during hypocapnia was tested in 20 migraineurs (8 with aura, 12 without aura) and 30 sex- and age-matched healthy subjects, and during nitroglycerin-induced headache in 12 healthy subjects. Before and during hyperventilation, mean blood-flow velocity (Vmean) in the middle cerebral artery was measured with transcranial Doppler. In each subject a pCO2 reactivity index (RI) was calculated as (delta Vmean/baseline Vmean)/delta pCO2. Interictally, patients with migraine with aura showed higher RI (p < 0.05 ANOVA and multiple range test) than controls, whereas migraineurs without aura did not differ from healthy subjects. Ictal and interictal RIs were similar in 9 patients suffering from migraine without aura. No side-to-side differences were detected in RI. During nitroglycerin-induced headache, the RIs were no different from those recorded during migraine attacks and in non-nitroglycerin-provoked healthy controls (p > 0.05, ANOVA and multiple range test). The exaggerated response in migraine with aura might predispose for the characteristic changes in rCBF seen during attacks. PMID- 7553812 TI - Psychosocial and behavioral characteristics in chronic headache patients: support for a continuum and dual-diagnostic approach. AB - The present study attempted to identify psychological differences among different headache diagnoses defined by IHS criteria as well as psychological differences by headache intensity and frequency. Differences between diagnostic categories reflected characteristics used to assign diagnoses, namely the constancy of pain and distracting behaviors of significant others due to isolating behavior from photophobia and phonophobia. A rating of headache intensity and frequency was a more powerful predictor of psychological ratings than diagnosis. Diagnosis was related to headache frequency but not intensity. The results suggest that a continuum diagnosis based on severity can be useful in conceptualizing headaches, and a dual-diagnostic system integrating headache characteristics with perceptions and coping ability would be helpful in determining treatment options. PMID- 7553813 TI - Twenty-four-hour melatonin and cortisol plasma levels in relation to timing of cluster headache. AB - The cyclic recurrence of cluster periods and the regular timing of headache occurrence in cluster headache (CH) induced us to study the circadian secretion of melatonin and cortisol in 12 patients with episodic CH, during a cluster period, and compare them with 7 age- and sex-matched healthy controls. Blood was sampled every 2 h for 24 h. All subjects were confined to a dark room from 22.00 to 08.00. Plasma melatonin levels were significantly reduced in CH patients (repeated measures ANOVA p < 0.03; mesor p < 0.02), and the cortisol mesor was significantly increased (p < 0.03). Amplitudes and acrophases did not differ between the groups. Individual cosinor analysis showed that 4/12 (33.3%) CH patients had no significant melatonin rhythm, and that 5/11 (45.5%) had no cortisol rhythm. Group analysis of cosinor revealed significantly rhythmicity of melatonin and cortisol secretion in both groups. In controls, the timing of melatonin and cortisol acrophase significantly correlated with each other, indicating that the biorhythm controllers for the secretion of these hormones were synchronized. Such correlation was not found in the CH patients; mesor, amplitude and acrophase of melatonin and cortisol did not correlate with duration of illness, duration of headache in course, or time since last headache attack. PMID- 7553814 TI - Cluster headache attacks treated for up to three months with subcutaneous sumatriptan (6 mg). Sumatriptan Cluster Headache Long-term Study Group. AB - In the first three months of a 24-month open study to assess the safety and efficacy of subcutaneous sumatriptan 6 mg in the long-term acute treatment of cluster headache, 138 patients treated a maximum of two attacks daily each with a single 6 mg injection. A total of 6353 attacks were treated. Adverse events, reported in 28% of sumatriptan-treated attacks, were qualitatively similar to those seen in migraine long-term trials. Their incidence did not increase with frequent use of sumatriptan. There were no clinically significant treatment effects on vital signs, ECG recordings or laboratory parameters. Headache relief (a reduction from very severe, severe or moderate pain to mild or no pain) at 15 min was obtained for a median of 96% of attacks treated. There was no indication of tachyphylaxis, decrease in the speed of response, or increased frequency of attacks with long-term treatment. This study demonstrated that, in long-term use, subcutaneous sumatriptan 6 mg is a well-tolerated and effective acute treatment for cluster headache. PMID- 7553815 TI - Initially migraine-like manifestation of a ruptured spinal arteriovenous malformation. PMID- 7553816 TI - Syncope and seizure-like activity secondary to acute herpes zoster infection of the trigeminal nerve. AB - Syncope may occur with glossopharyngeal neuralgia. We describe a patient with acute herpetic infection of the first branch of the trigeminal nerve associated with episodes of shooting pain, cardiac arrest and tonic-clonic movements. Resemblances with the so-called "cardiovascular" form of glossopharyngeal neuralgia, as well as putative mechanisms of the syncope, are discussed. PMID- 7553818 TI - Early discharge after direct angioplasty for acute myocardial infarction. PMID- 7553817 TI - Early hospital discharge after direct angioplasty for acute myocardial infarction. AB - To determine the feasibility and safety of early hospital discharge after myocardial infarction, we reviewed a 3-yr experience with direct angioplasty: 204 patients had direct angioplasty with in-hospital mortality of 3.4%. Of these patients, 125 were discharged < 5 days after infarction and 98% of these were available for 30-day follow-up. There was one early death (0.8% mortality), two early readmissions without complications, and three late readmissions. Thus early hospital discharge a mean of 3.4 days after infarction can be achieved in > 60% of patients undergoing direct angioplasty with no significant early complications and excellent 30-day survival (99.2%). PMID- 7553819 TI - Timing, mode, and predictors of death after direct angioplasty for acute myocardial infarction. AB - The timing and mechanisms of early (30 day) mortality in 330 consecutive patients treated with direct angioplasty less than 12 hr after onset of myocardial infarction without antecedent thrombolysis were studied. There were 38 deaths (11.5% of pts), with a majority being due to cardiogenic shock (76%). Other causes included acute closure (11%), death after emergency bypass surgery (5%), ventricular arrhythmias (5%), and respiratory failure (3%). No deaths from stroke or cardiac rupture were seen, in contrast to trials of thrombolytic agents. Most deaths were seen early, with 47% occurring within 1 day, 35% from days 2-7, and 18% from days 8-30. Death from cardiogenic shock was the most common cause of death throughout this period: 83% of deaths in days 0-3, 88% of deaths in days 4 6, and 43% of deaths in days 8-30. Significant predictors of early death included older age (P < .0001), multi-vessel disease (P < .05), direct angioplasty failure (P < .05), reduced ejection fraction (P < .0001), and anterior myocardial infarction (P < .0005). Gender, prior myocardial infarction, and prior bypass surgery did not affect mortality. Cardiogenic shock is the most common cause of early death after direct angioplasty for myocardial infarction. Patients with one or more risk factors for early death may benefit from additional myocardial salvage or revascularization efforts in the early post-infarct period. Certain causes of death after direct angioplasty (cardiac, rupture, stroke) appear to be less common than data reported for lytic therapy for myocardial infarction. PMID- 7553820 TI - Death following direct infarct angioplasty: lessons that we've learned. PMID- 7553821 TI - High balloon dilatation pressures in percutaneous transluminal coronary angioplasty are not associated with higher rate of significant complications. AB - To examine the results of high balloon dilatation pressures during percutaneous transluminal coronary angioplasty (PTCA), we retrospectively reviewed 482 angioplasty cases from our institution and divided them into three groups by the peak inflation pressure used during the procedure. Group one was defined by inflation pressures of 1-6 atmospheres (atm), group two by 7-12 atm, and group three by 13-20 atm. There were 166 patients in group 1 (34.4%), 235 in group 2 (48.8%), and 81 (16.8%) in group 3. The success rates were not statistically different; 90% in group 1, 96% in group 2, and 95% in group 3. Large dissections occurred in 27 patients in group 1 (16.3%), 19 in group 2 (8.1%), and 4 (4.9%) in group 3 (P > 0.006). There were no differences in the rates of death (1.2% vs. 0.9% vs. 1.2%), myocardial infarction (3.0% vs. 1.3% vs 3.7%), or in-hospital CABG (3.0% vs. 1.7% vs. 1.2%) in groups 1, 2, and 3, respectively. Six-mo target vessel revascularization rates also were not different: 19% vs. 13% vs. 18%. In summary, selectively using high balloon pressures during PTCA does not result in increased complications. PMID- 7553822 TI - Millimeters and Minutes--Yes: Kilopascals--? PMID- 7553823 TI - Bailout Palmaz-Schatz coronary stenting in 39 patients with occlusive dissection complicating conventional angioplasty. AB - The purpose of this study was to evaluate feasibility, safety, and efficacy of bailout Palmaz-Schatz stenting in a series of 39 patients with coronary dissection associated with acute or unequivocal threatened closure complicating conventional angioplasty. No anatomical characteristics other than reference vessel diameter < 3 mm were considered as contraindications for bailout coronary stenting. Stringent criteria were adopted in defining optimal results (< 10% residual stenosis, no angiographic evidence of residual dissection), suboptimal results (> 10% residual stenosis or angiographic evidence of residual dissection), deployment failure (failure to deploy the stent because of poor trackability or persistent occlusion despite stent deployment). A total of 49 stents and 7 half-stents were implanted in 36 patients (range 1-5; mean 1.45 +/- 0.84). Successful stenting without in-hospital death, urgent or semielective coronary surgery, stent thrombosis, or Q-wave myocardial infarction was achieved in 33/39 patients (85%). A suboptimal result was associated with an increased risk of in-hospital recurrence of ischemia and other related major adverse events (2/5 patients with suboptimal results vs. 1/31 patients with complete deployment success; P < 0.05). Multiple stents implantation did not carry a significant risk of major cardiac adverse events. The results of this study suggest that bailout Palmaz-Schatz stenting may be considered a stand-alone treatment of coronary dissection if an optimal acute angiographic result is achieved. PMID- 7553824 TI - Improving the results of bail-out stenting. PMID- 7553825 TI - Management of balloon rupture during rigid stent deployment. AB - Balloon rupture may occur during stent placement and will only become apparent when the stent has been maneuvered across the target stenosis and deployment is attempted. Withdrawal may be complicated by displacement of the stent off the balloon. We describe two cases in which a power injector was used to momentarily inflate the balloon and partially expand the stent in place. The punctured balloon could then be withdrawn and the stent fully deployed, using a new balloon. PMID- 7553826 TI - The frustrations of coronary stenting. PMID- 7553827 TI - Retrieval of undeployed intracoronary Palmaz-Schatz stents. AB - Two patients undergoing stent placement with the Palmaz-Schatz coronary stent delivery system experienced intracoronary stent embolization. Using a two-wire technique, the stents were successfully removed from the coronary circulation, and this allowed completion of the percutaneous revascularization procedure without any clinical sequelae. These two cases represent the first report of successful percutaneous retrieval of undeployed, retained intracoronary Palmaz Schatz stents. PMID- 7553828 TI - Guidewire transection during rotational coronary atherectomy due to guide catheter dislodgement and wire kinking. AB - Rotational coronary atherectomy is an effective treatment for calcified ostial lesions. We report a case of guidewire transection during rotational atherectomy of a right coronary artery ostial stenosis. Guide catheter dislodgement appeared to have caused prolapse and kinking of the guidewire. Advancement of the burr over the kinked wire resulted in transection. The wire fragment was retrieved successfully using an inflated fixed-wire balloon catheter. This report illustrates the importance of excellent coaxial guide catheter alignment with rotational atherectomy and suggests that operators be vigilant to possible damage to the radiolucent rotational atherectomy guidewire. PMID- 7553831 TI - Retrieval of directional coronary atherectomy guiding catheter with angioplasty balloon catheter. AB - Directional coronary atherectomy (DCA) has been shown to be a safe and effective treatment for occlusive coronary artery disease. We report a case of an atherectomy guiding catheter severing in two with successful retrieval of the dislodged segment by an integrated angioplasty balloon catheter system. PMID- 7553829 TI - Latex versus iodinated contrast media anaphylaxis in the cardiac cath lab. AB - A 65-year-old man had bypass surgery 10 years previously with pulmonary artery catheter monitoring. Shortness of breath and mitral regurgitation necessitated repeat left and right heart catheterization using a pulmonary artery catheter. Before any iodinated contrast media exposure, the pulmonary artery catheter was inserted and within 2 min the patient developed anaphylaxis associated ventricular fibrillation. It was discovered that the pulmonary artery catheter used in the cath lab had a latex balloon and that the patient had been exposed to latex 10 years ago. Latex induced anaphylaxis is rarely considered in the differential diagnosis of patients with hypersensitivity reactions in the cath lab, intensive care unit, and operating room. The principal reason for failure to recognize the latex balloon as a potential allergen is that most health professionals are not aware that almost all pulmonary artery catheters contain a latex balloon. The risk of an allergic response to latex is 0.8% for the general population. Others at high risk include those who have had multiple surgical procedures and interventions with repeated latex exposure. Five to 10% of all U.S. health professionals and those performing household duties wearing latex gloves have an allergic response to latex. Latex hypersensitivity is an IgE dependent reaction, while iodinating contrast medium reaction is an IgE independent reaction. If latex hypersensitivity is suggested by pre-procedural history or if the patient falls into a high-risk group, pre-procedural skin testing and/or latex IgE radioallergosorbent (RAST) should be performed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553832 TI - Perforation of nontarget artery during directional coronary atherectomy. AB - We report a case of perforation of an intermediate branch during attempted directional atherectomy of an ostial left anterior descending artery lesion, resulting from disruption of the cutter drive cable. Potential contributing factors and means of avoiding this complication are discussed. PMID- 7553830 TI - Acute coronary artery closure following intracoronary ultrasound examination. AB - Two patients undergoing intracoronary ultrasound examination were complicated by acute coronary artery closure. One of the complications was thought to be caused by intimal dissection and thrombus formation and the other was thought to be caused by intimal dissection and subsequent embolization. The complications were successfully managed conservatively in both cases. PMID- 7553834 TI - Separation of a flexible nose cone tip during directional atherectomy. AB - An 82-yr-old white female underwent directional coronary atherectomy of a high grade proximal left anterior artery stenosis. The nose cone tip of a Simpson Atherocath SCA-EX separated during loading of the device onto a 360-cm exchange wire. This was recognized prior to insertion through a rotating hemostatic valve and exchanged for another device. Catheterization laboratory policy regarding defective devices is briefly discussed. PMID- 7553833 TI - Dislocation of the rotating cutter during directional coronary atherectomy: a note of caution. AB - Directional coronary atherectomy (DCA) has received increased attention, especially as a bail-out procedure after failed balloon angioplasty. However, this technique may also be burdened by severe pitfalls. We report a patient with a balloon-resistant left coronary artery lesion subsequently treated with DCA. Despite its over-the-wire guidance, as the rotating cutter was advanced, it deviated from its intra-housing course and intruded into the vascular wall. Dislocation of the rotating blade was due to pressure from hard plaque tissue. After having carefully pulled back the complete catheter system, a severe spasm of the left main stem occurred, which was reversed by intracoronary nitroglycerine. The final angiography showed a left coronary artery without significant, residual stenosis. The case report underscores that DCA passes must be performed under continuous fluoroscopic control, especially for balloon resistant lesions because of the unpredictability of DCA-imminent complication. PMID- 7553835 TI - Renal artery stenosis presenting as crescendo angina pectoris. AB - The coexistence of different clinical syndromes due to atherosclerosis in different organs is not rare and emphasizes the diffuse nature of this vascular process. Although renovascular disease may cause hypertension and/or renal insufficiency, it may also occur in the absence of the usual clinical markers that suggest renovascular hypertension. We report a patient with stable coronary anatomy who presented with crescendo angina pectoris. Diagnosis of renovascular hypertension was made by screening renal angiography at the time of the cardiac catheterization. Renal artery stenting resulted in stabilization of the coronary syndrome and obviated the need for further coronary intervention. To our knowledge, this is the first case of renovascular hypertension precipitating an unstable coronary syndrome in a patient with documented stable coronary anatomy. Review of the literature supports that patients undergoing cardiac catheterization are a high risk population for renovascular disease, particularly in the presence of other predictive factors such as documented coronary artery disease, older age, female gender, congestive heart failure, peripheral vascular disease, renal insufficiency, and smoking. Firm recommendations for routine screening renal angiography in patients undergoing peripheral or coronary angiography will need further studies. PMID- 7553836 TI - Importance of a non-dominant right coronary artery occlusion presenting as sudden cardiac death with prolonged right ventricular dysfunction and malignant arrhythmias. AB - We report a case of a patient who presented with sudden cardiac death secondary to a subtotal occlusion of a small non-dominant right coronary system. Catheterization several weeks following the initial episode revealed persistent severe right ventricular dysfunction with moderate hemodynamic compensation. Continued unstable arrhythmogenic potential at this point led to placement of an AICD device. The case highlights the potential hazard and often complacency involved in dealing with benign appearing lesions as this one. PMID- 7553837 TI - Subintimal wire position during angioplasty of a chronic total coronary occlusion: detection and subsequent procedural guidance by intravascular ultrasound. AB - We report a case of intravascular ultrasound (IVUS) imaging of a chronic total coronary artery occlusion angioplasty initially complicated by subintimal wire penetration. IVUS provided unique images of wire position and a "double-barrel" lumen that complemented angiographic data in initial diagnosis and in subsequent guidance of the procedure. PMID- 7553838 TI - Angiographic diagnosis of true and false LV aneurysms after inferior wall myocardial infarction. AB - False aneurysms of the left ventricle are rare complications of myocardial infarction. In this unusual case, a 57-year-old male patient had a false aneurysm, that took origin from a true aneurysm in the inferior wall, both discovered 7 years postinfarct. The aneurysms were demonstrable by ventriculography and were resected because of ventricular tachycardia. PMID- 7553839 TI - Application of translesional pressure and flow velocity assessment in a severely calcified coronary narrowing in a patient with unstable angina. AB - While the angiographic appearance of coronary stenoses commonly directs interventional decisions, it may correlate imprecisely with hemodynamic or physiologic lesional significance. Previous data would suggest that direct measures of translesional physiology can be helpful in assessing the hemodynamic significance of stable coronary stenoses. In unstable ischemic syndromes, however, the hemodynamic severity of lesions may depend on the presence of variably occlusive intraluminal thrombus superimposed on fluctuating vessel tone. Under these circumstances, physiologic lesional assessment can yield helpful information, which nonetheless must be interpreted with caution in light of the clinical context. Determination of optimal management strategies for such patients remains difficult and must await further investigation of prognosis and outcome. PMID- 7553840 TI - Intracoronary Doppler flow to monitor the results of selective saphenous vein graft thrombolytic therapy. AB - Selective intracoronary thrombolytic therapy has recently been advocated as a technique to recanalize occluded coronary bypass grafts. We report a novel approach for monitoring the results of this treatment utilizing a coronary artery doppler flow guidewire. This approach may optimize the duration of thrombolytic treatment in these patients. PMID- 7553841 TI - Angioscopy guided intracoronary thrombolysis and stent deployment in a patient with postinfarction angina. PMID- 7553842 TI - Cloning and functional analysis of TipE, a novel membrane protein that enhances Drosophila para sodium channel function. AB - Voltage-dependent sodium channels are involved in the initiation and propagation of action potentials in many excitable cells. Here we report that tipE, a gene defined by a temperature-sensitive paralytic mutation in Drosophila, encodes a novel integral membrane protein that dramatically stimulates functional expression in Xenopus oocytes of the Drosophila sodium channel alpha subunit encoded by the paralytic (para) locus. Using a heat shock promoter to control tipE+ gene expression in transgenic flies, we demonstrate that tipE+ gene expression is required during pupal development to rescue adult paralysis. In addition, we demonstrate a role for the tipE gene product in adults. PMID- 7553843 TI - glial cells missing: a genetic switch that controls glial versus neuronal fate. AB - The glial cells missing (gcm) gene in Drosophila encodes a novel nuclear protein that is transiently expressed early in the development of nearly all glia. In loss-of-function gcm mutant alleles, nearly all glia fail to differentiate, and, where we can follow them in the PNS, are transformed into neurons. In gain-if function gcm conditions using transgenic constructs that drive ectopic gcm expression, many presumptive neurons are transformed into glia. Thus, gcm appears to function as a binary genetic switch for glia versus neurons. In the presence of gcm protein, presumptive neurons become glia, while in its absence, presumptive glia become neurons. PMID- 7553845 TI - Enigma variations: protein mediators of membrane fusion. PMID- 7553844 TI - glial cells missing: a binary switch between neuronal and glial determination in Drosophila. AB - In the Drosophila CNS, both neurons and glial are derived from neuroblasts. We have identified a gene, glial cells missing (gcm), that encodes a novel nuclear protein expressed transiently in early glial cells. Its mutation causes presumptive glial cells to differentiate into neurons, whereas its ectopic expression forces virtually all CNS cells to become glial cells. Thus, gcm functions as a binary switch that turns on glial fate while inhibiting default neuronal fate of the neuroblasts and their progeny. Similar results are also obtained in the PNS. Analyses of the mutant revealed that "pioneer neurons" can find correct pathways without glial cells and that neurons and glia have a common molecular basis for individual identity. PMID- 7553846 TI - Natural resistance and nitric oxide. PMID- 7553847 TI - Regulation of chromosomal replication in E. coli: sequestration and beyond. PMID- 7553848 TI - Selective protein degradation: a journey's end within the proteasome. PMID- 7553849 TI - Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. AB - The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events. PMID- 7553850 TI - The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. AB - We have reconstituted the fusion and assembly of vesiculated Golgi membranes (VGMs) into functionally active stacks of cisternae. A kinetic analysis of this assembly process revealed that highly dispersed VGMs of 60-90 nm diameter first fuse to form larger vesicles of 200-300 nm diameter that are clustered together. These vesicles then fuse to form tubular elements and short cisternae, which finally assemble into stacks of cisternae. We now provide evidence that the sequential stack formation from VGMs reflects two distinct fusion processes: the first event is N-ethyl-maleimide (NEM)-sensitive factor (NSF) dependent, and the second fusion event requires an NSF-like NEM-sensitive ATPase called p97. Interestingly, while the earliest steps in stack formation share some similarities with events catalyzing fusion of transport vesicles to its target membrane, neither GTP gamma S nor Rab-GDI, inhibitors of vesicular protein traffic, inhibit stack formation. PMID- 7553851 TI - An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. AB - Golgi cisternae regrew in a cell-free system from mitotic Golgi fragments incubated with buffer alone. Pretreatment with NEM or salt washing inhibited regrowth, but this could be restored either by p97, an NSF-like ATPase, or by NSF together with SNAPs and p115, a vesicle docking protein. The morphology of cisternae regrown with p97 and NSF-SNAPs-p115 differed, suggesting that they play distinct roles in rebuilding Golgi cisternae after mitosis. PMID- 7553852 TI - p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. AB - In normal human fibroblasts, cyclin A-CDK2 exists in a quaternary complex that contains p21 and PCNA. In many transformed cells, p21 disappears, and a substantial fraction of cyclin A-CDK2 complexes with p9CKS1/CKS2, p19, and p45. To investigate the significance of these rearrangements, we have isolated cDNAs encoding p19 and p45. In vitro reconstitution demonstrated that binding of p19 to cyclin A-CDK2 requires p45. Addition of these proteins to the kinase had no substantial effect on the kinase activity in vitro. Interference with p45 function in vivo by microinjection of antibodies or antisense oligonucleotides prevented entry into S phase in both normal and transformed cells. Cyclin A-CDK2 has previously been identified as a kinase whose activity is essential for S phase. Our results identify p45 as an essential element of this activity. The abundance of p45 is greatly increased in many transformed cells. This could result in changes in cell cycle control that contribute to the process of cellular transformation. PMID- 7553853 TI - E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. AB - The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration. PMID- 7553854 TI - SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. AB - Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant inherited disorder characterized by degeneration of cerebellar Purkinje cells, spinocerebellar tracts, and selective brainstem neurons owing to the expansion of an unstable CAG trinucleotide repeat. To gain insight into the pathogenesis of the SCA1 mutation and the intergenerational stability of trinucleotide repeats in mice, we have generated transgenic mice expressing the human SCA1 gene with either a normal or an expanded CAG tract. Both transgenes were stable in parent to offspring transmissions. While all six transgenic lines expressing the unexpanded human SCA1 allele had normal Purkinje cells, transgenic animals from five of six lines with the expanded SCA1 allele developed ataxia and Purkinje cell degeneration. These data indicate that expanded CAG repeats expressed in Purkinje cells are sufficient to produce degeneration and ataxia and demonstrate that a mouse model can be established for neurodegeneration caused by CAG repeat expansions. PMID- 7553855 TI - Isolation of a novel gene underlying Batten disease, CLN3. The International Batten Disease Consortium. AB - Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomotor disturbances. The Batten disease gene, CLN3, maps to chromosome 16p12.1. The so-called 56 chromosome haplotype defined by alleles at the D16S299 and D16S298 loci is shared by 73% of Batten disease chromosomes. Exon amplification of a cosmid containing D16S298 has yielded a candidate gene that is disrupted by a 1 kb genomic deletion in all patients carrying the 56 chromosome. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the candidate as the CLN3 gene. The disease gene encodes a novel 438 amino acid protein of unknown function. PMID- 7553856 TI - Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. AB - Hypergonadotropic ovarian dysgenesis (ODG) with normal karyotype is a heterogeneous condition that in some cases displays Mendelian recessive inheritance. By systematically searching for linkage in multiplex affected families, we mapped a locus for ODG to chromosome 2p. As the previously cloned follicle-stimulating hormone receptor (FSHR) gene had been assigned to 2p, we searched it for mutations. A C566T transition in exon 7 of FSHR predicting an Ala to Val substitution at residue 189 in the extracellular ligand-binding domain segregated perfectly with the disease phenotype. Expression of the gene in transfected cells demonstrated a dramatic reduction of binding capacity and signal transduction, but apparently normal ligand-binding affinity of the mutated receptor. We conclude that the mutation causes ODG in these families. PMID- 7553857 TI - Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. AB - The cellular interactions that control the differentiation of dorsal cell types from neural progenitors have been examined in neural plate explants. Certain genes that are expressed in the dorsal neural tube are initially expressed uniformly within the neural plate and appear to achieve their dorsal restriction through a Sonic hedgehog (SHH)-mediated repressive signal from the notochord. The acquisition of definitive dorsal cell fates, however, requires a contact dependent signal from the epidermal ectoderm. BMP4 and BMP7 are expressed in the epidermal ectoderm, and both proteins mimic its inductive activity. BMP4 and a related gene, DSL1, are subsequently expressed by cells in the dorsal neural tube. The differentiation of dorsal cell types, therefore, appears to be initiated at the neural plate stage and to involve the opponent activities of a BMP-mediated dorsalizing signal from the epidermal ectoderm and a SHH-mediated ventralizing signal from the notochord. PMID- 7553858 TI - Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene. AB - Biological function of the BCR-ABL oncogene is dependent on its activated tyrosine kinase. Mutations that inactivate the SRC homology 2 (SH2) domain, the GRB2-binding site in BCR, or the major autophosphorylation site of the kinase domain selectively disrupt downstream signaling but not tyrosine kinase activity. Despite a loss of fibroblast transformation activity, all three mutants retain the ability to render hematopoietic cell lines growth factor independent and transform primary bone marrow cells in vitro. In vivo tests of malignant potential reveal a most critical role for signals dependent on the BCR-ABL SH2 domain. The efficiency of both fibroblast and hematopoietic transformation by BCR ABL is strongly affected by increased dosage of the SHC adapter protein, which can connect tyrosine kinase signals to RAS. The BCR-ABL oncogene activates multiple alternative pathways to RAS for hematopoietic transformation. PMID- 7553859 TI - A central role for microvillous receptor presentation in leukocyte adhesion under flow. AB - Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking. PMID- 7553860 TI - When is a lipid kinase not a lipid kinase? When it is a protein kinase. PMID- 7553861 TI - The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously. AB - Mutations in the C. elegans gene lin-44 lead to reversals in the polarity of certain asymmetric cell divisions. We have discovered that lin-44 is a member of the Wnt family of genes, which encode secretory glycoproteins implicated in intercellular signaling. Both in situ hybridization experiments using lin-44 transcripts and experiments using reporter constructs designed to mimic patterns of lin-44 expression indicate that lin-44 is expressed in hypodermal cells at the tip of the tail and posterior to the cells with polarities affected by lin-44 mutations. Our mosaic analysis indicates that lin-44 acts cell nonautonomously. We propose that LIN-44 protein is secreted by tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail. PMID- 7553862 TI - Complexins: cytosolic proteins that regulate SNAP receptor function. AB - A family of proteins called complexins was discovered that compete with alpha SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis. PMID- 7553863 TI - Degradation of CFTR by the ubiquitin-proteasome pathway. AB - Most cases of cystic fibrosis are caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to the rapid degradation of CFTR molecules that have not matured beyond the endoplasmic reticulum (ER). The mechanism by which integral membrane proteins including CFTR are recognized and targeted for ER degradation and the proteolytic machinery involved in this process are not well understood. We show here that the degradation of both wild-type and mutant CFTR is inhibited by two potent proteasome inhibitors that induce the accumulation of polyubiquitinated forms of immature CFTR. CFTR degradation was also inhibited by coexpression of a dominant negative ubiquitin mutant and in cells bearing a temperature-sensitive mutation in the ubiquitin-activating enzyme, confirming that ubiquitination is required for rapid CFTR degradation. PMID- 7553864 TI - Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. AB - The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER. PMID- 7553865 TI - Alterations in DNA methylation may play a variety of roles in carcinogenesis. PMID- 7553867 TI - Mechanism of corepressor-mediated specific DNA binding by the purine repressor. AB - The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation. PMID- 7553866 TI - A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. AB - The protein kinase MO15/CDK7 has recently been shown to be associated with the general transcription factor TFIIH and to be capable of phosphorylating the RNA polymerase II carboxy-terminal domain. Here, we show that a monoclonal MO15/CDK7 antibody coimmunoprecipitates, from a rat liver nuclear extract, all components of the RNA polymerase II transcription apparatus required for initiation at the albumin and adenovirus major late promoters. The immunoprecipitate includes RNA polymerase II, TFIID, TFIIB, TFIIH, TFIIF, and TFIIE, but is devoid of transcriptional activator proteins, such as HNF1, HNF4, and C/EBP alpha. The finding of an autonomously initiating RNA polymerase II holoenzyme in mammalian cells suggests conceptual similarities between transcription initiation in prokaryotes and eukaryotes. PMID- 7553868 TI - Ancient weapons: NK-lysin, is a mammalian homolog to pore-forming peptides of a protozoan parasite. PMID- 7553870 TI - Displacement of sequence-specific transcription factors from mitotic chromatin. AB - The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs. PMID- 7553871 TI - Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin binding protein EBNA 1. AB - The crystal structure of the DNA-binding and dimerization domains of the Epstein Barr virus nuclear antigen 1 (EBNA1), which binds to and activates DNA replication from the latent origin of replication in Epstein-Barr virus, was solved at 2.5 A resolution. EBNA1 appears to bind DNA via two independent regions termed the core and the flanking DNA-binding domains. The core DNA-binding domain, which comprises both the dimerization domain and a helix predicted to bind the inner portion of the EBNA1 DNA recognition element, was remarkably similar to the structure of the papillomavirus E2 protein, despite a complete lack of sequence conservation. The flanking DNA-binding domain, only a portion of which is contained in the current structure, consists in part of an alpha helix whose N-terminus contacts the outer regions of the EBNA1 DNA recognition element. PMID- 7553869 TI - Overcoming a nucleosomal barrier to transcription. AB - We have studied the kinetics of transcription through a nucleosome core. RNA polymerase transcribes the first approximately 25 bp of nucleosomal DNA rapidly, but then hits a barrier and continues slowly to the nucleosomal dyad region. Here, the barrier disappears and the transcript is completed at a rapid rate, as if on free DNA, indicating that histone octamer transfer is completed as polymerase reaches the dyad. If DNA behind the polymerase is removed during transcription, the barrier does not appear until the polymerase has penetrated up to 15 bp farther into the nucleosome. On a longer template, the barrier is almost eliminated. We have shown previously that the octamer is transferred around the transcribing polymerase via an intermediate containing an intranucleosomal DNA loop. Our results exclude the possibility that polymerase has difficulty breaking histone-DNA contacts and suggest instead that polymerase pauses because it has difficulty transcribing DNA in the loop. PMID- 7553872 TI - Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. AB - We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms. PMID- 7553873 TI - The body language of cells: the intimate connection between cell adhesion and behavior. PMID- 7553874 TI - Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. AB - Erythropoietin (EPO) is the principal growth factor regulating the production of circulating erythrocytes. We introduced null mutations into both Epo and the EPO receptor (EpoR) gene. Both heterozygotes appeared normal. Homozygous animals exhibited reduced primitive erythropoiesis and died around embryonic day 13, owing to failure of definitive fetal liver erythropoiesis. Both types of mutations exhibited identical phenotypes, indicating that EPO and the EPOR are crucial for definitive erythropoiesis in vivo and that no other ligands or receptors can replace them. Committed erythroid BFU-E and CFU-E progenitors were present in both homozygous fetal livers. Thus, neither EPO nor the EPOR is required for erythroid lineage commitment or for the proliferation and differentiation of BFU-E to CFU-E progenitors. EPO and the EPOR are crucial in vivo for the proliferation and survival of CFU-E progenitors and their irreversible terminal differentiation. PMID- 7553875 TI - Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis. AB - The secretion of insulin is controlled by the rate of glucose metabolism in the pancreatic beta cells. As phosphorylation by glucokinase (GLK) appears to be the rate-limiting step for glucose catabolism in beta cells, this enzyme may be the glucose sensor. To test this possibility and to resolve the relative roles of liver and beta cell GLK in maintaining glucose levels, we have generated mice completely deficient in GLK and transgenic mice in which GLK is expressed only in beta cells. In mice with only one GLK allele, blood glucose levels are elevated and insulin secretion is reduced. GLK-deficient mice die perinatally with severe hyperglycemia. Expression of GLK in beta cells in the absence of expression in the liver is sufficient for survival. These mice demonstrate the critical need for beta cell GLK in maintaining normal glucose levels and provide a novel model for one form of noninsulin-dependent diabetes. PMID- 7553876 TI - Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. AB - Transgenic (Tg) mice expressing human (Hu) and chimeric prion protein (PrP) genes were inoculated with brain extracts from humans with inherited or sporadic prion disease to investigate the mechanism by which PrPC is transformed into PrPSc. Although Tg(HuPrP) mice expressed high levels of HuPrPC, they were resistant to human prions. They became susceptible to human prions upon ablation of the mouse (Mo) PrP gene. In contrast, mice expressing low levels of the chimeric transgene were susceptible to human prions and registered only a modest decrease in incubation times upon MoPrP gene disruption. These and other findings argue that a species-specific macromolecule, provisionally designated protein X, participates in prion formation. While the results demonstrate that PrPSc binds to PrPC in a region delimited by codons 96 to 167, they also suggest that PrPC binds protein X through residues near the C-terminus. Protein X might function as a molecular chaperone in the formation of PrPSc. PMID- 7553877 TI - Somatic hypermutation: how many mechanisms diversify V region sequences? PMID- 7553878 TI - Evidence for positional differentiation of prestalk cells and for a morphogenetic gradient in Dictyostelium. AB - We present evidence that Dictyostelium slug tip cells, the pstA cells, may arise by positional differentiation, but at a site remote from that which they will eventually occupy. When first detectable, the pstA cells form a peripheral ring surrounding the other prestalk cell subtype, the pstO cells, but subsequently move above the pstO cells to form the tip. Because pstA cell differentiation requires a 10-fold higher concentration of differentiation-inducing factor, the stalk cell inducer, the initial patterning seems likely to reflect the existence of a morphogenetic gradient. The subsequent redistribution of the two cell types is explicable by their different rates of chemotaxis to cyclic AMP. These results help reconcile the two apparently opposing views of pattern formation in Dictyostelium, that there is positional differentiation and that pattern formation occurs by cell sorting. PMID- 7553879 TI - A triplet of nuclease proteins (NP42-50) is activated in human Jurkat cells undergoing apoptosis. AB - Activation of a triplet of nuclear proteins (NP42-50) was observed in human Jurkat T cell line following treatment with an antibody to CD95 (Fas/Apo-1), a cell surface molecule involved in apoptotic cell death. The nuclease activity, corresponding to a triplet of proteins observed at approximately 42, 45, and 50 kDa in size, was extractable, heat-stable, and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing deoxyribonucleic acids (SDS-PAGE DNA) assay. The NP42-50 activity requires the presence of Mg2+/Ca2+ and is insensitive to inactivation by heating at 80 degrees C for 5 min. Zinc effectively inhibited the enzymatic activity of NP42-50 on SDS-PAGE-DNA and also protected Jurkat cells from the CD95-mediated apoptosis in cell cultures. The nuclease activation, however, was not a unique pathway for the CD95-mediated cell death. The apoptosis induced by arabinofuranosyl cytosine, a chemotherapeutic agent, also activated the NP42-50 nuclease activity in Jurkat cells, suggesting that a similar cascade of subsequent events in apoptosis may occur in most instances although many different signals can initiate apoptotic cell death in various cell types. The nuclease identified by this study appears to be distinguishable from DNase I or DNase II by its molecular characteristics and its enzymatic requirements. The NP42-50, with respect to the nuclease activity closely associated with apoptotic cell death, may serve as a candidate for the endonuclease(s) involved in the cleavage of DNA into fragments during apoptosis. PMID- 7553880 TI - Effects of ultraviolet-B irradiation on human LAK and NK cytotoxic activity. AB - In vitro ultraviolet-B (UVB) irradiation of murine and rodent bone marrow cells prevents GVHD without compromising engraftment while inducing tolerance to donor type allografts. In anticipation of clinical trials of UVB-modified bone marrow grafts, we studied the in vitro effects of UVB irradiation (50-300 J/m2) on human natural killer and lymphokine activated killer cells since both types of cells influence the development of GVHD and graft-versus-tumor effect. Interleukin-2 activated and untreated human lymphocytes were used as effectors in a 51Cr release cytotoxic assay against various tumor cell lines as targets. NK-mediated lysis of K562 targets was decreased by UVB irradiation of the effector cells in a dose-dependent manner. FACS analysis of CD16+ and CD56+ cells 24 hr after UVB exposure showed a UVB-dose-dependent decrease in the number of cells expressing these surface markers. UVB irradiation of lymphocytes prior to activation with high-dose IL-2 resulted in a range of 20- to 89-fold decrease in LAK precursors as measured by limiting dilution analysis using the LAK-sensitive cell line HL60. In contrast, the LAK activity of lymphocytes that had been stimulated in vitro with high-dose IL-2 prior to UVB irradiation was preserved when assayed immediately after UVB modulation; however, there was a significant decrease in lytic activity (with most samples tested) when the assay was performed 24 hr after UVB exposure. It appears that the lymphocyte response to UVB modification is dose dependent, with some cell types displaying higher sensitivity to UVB irradiation than others. These findings suggest that prevention of GVHD by UVB is due, in part, to inhibition of NK activity, and may offer a new strategy to augment the graft versus leukemia effect of UVB-modified bone marrow grafts in clinical transplantation. PMID- 7553881 TI - Administration of a nondepleting anti-CD4 monoclonal antibody (W3/25) prevents adjuvant arthritis, even upon rechallenge: parallel administration of a depleting anti-CD8 monoclonal antibody (OX8) does not modify the effect of W3/25. AB - The aim of this study was to determine the effects of the anti-CD4 monoclonal antibody (mAb) W3/25, found to be nondepleting, on the onset of rat adjuvant arthritis (AA), and, in addition, to ascertain whether depletion of CD8+ cells during the same period could interfere with those effects. Female Wistar rats in which AA had been induced were treated with W3/25 and/or OX8 (anti-rat CD8) mAb during the latency period of arthritis. W3/25 alone or in combination with OX8 prevented the inflammatory process of AA. When the protected groups were rechallenged with a second dose of Mycobacterium butyricum no arthritis was observed. Protected and nonprotected arthritic animals developed the same anti mycobacteria antibody levels as the arthritic control group. This study indicates that a nondepleting anti-CD4 mAb can prevent AA, while CD8+ lymphocytes do not appear relevant for the development of AA and do not seem to have a regulatory role for CD4+ cells. PMID- 7553882 TI - Analysis of the effect of cytokines (interleukins 2, 3, 4, and 6, granulocyte monocyte colony-stimulating factor, and interferon-gamma) on generation of primary cytotoxic T lymphocytes against a weakly immunogenic tumor. AB - We have investigated the role of cytokines (IL-2, IL-3, IL-4, IL-6, IFN-gamma, and GM-CSF) in the generation of primary cytotoxic T lymphocytes (CTL), within a single tumor system. The murine carcinoma line 1 was transfected with expression vectors with cDNA for these cytokines. Line 1 expresses low levels of class I MHC molecules, but can be induced with dimethyl sulfoxide or IFN-gamma to express high levels of class I. Class I low line 1 cells are not susceptible to CTL lysis, while class I high line 1 are lysed by CTL, which allows us to assay for CTL activity. To isolate primary CTL, tumor-infiltrating lymphocytes were isolated from cytokine-expressing or control tumors, growing in vivo. Most cytokines stimulated nonspecific killers, but IL-2 and IL-3 stimulated primary CTL. While IFN-gamma alone did not generate primary CTL, coexpression of IFN gamma with IL-2 resulted in CTL generation. This is the first comparison of the effects of a series of cytokines on primary CTL development, and has important implications for vaccine development and immunotherapy. PMID- 7553883 TI - Administration of monoclonal antibodies against vascular cell adhesion molecule 1/very late antigen-4 abrogates predisposing autoimmune diabetes in NOD mice. AB - The interaction of vascular cell adhesion molecule-1 and very late antigen-4 (alpha 4 beta 1-integrin) has been recently known to be profoundly involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues. To elucidate the role of these molecules in the development of autoimmune diabetes, the expression of these adhesion molecules on inflamed islets and the effects of administration of monoclonal antibodies to these molecules on insulitis and overt diabetes were evaluated in nonobese diabetic (NOD) mice. Immunohistochemical study revealed the overexpression of vascular cell adhesion molecule-1 on vascular endothelium near or within inflamed islets and alpha 4-integrin on islet-infiltrating mononuclear cells. Either anti vascular cell adhesion molecule-1 or anti-alpha 4-integrin monoclonal antibody prevented the transfer of diabetes in irradiated NOD mice which received spleen cells from acutely diabetic NOD mice. When both monoclonal antibodies were administrated to NOD mice during 2-30 weeks of age, neither lymphocytic infiltration to islets nor overt diabetes was observed. Furthermore, administration of these antibodies even from 10 weeks of age could inhibit the development of insulitis and diabetes, whereas administration during 2-5 weeks of age could not. Splenocytes obtained from these treated mice showed no significant change of cytokine production and preserved the ability to transfer diabetes into NOD scid/scid mice. This suggests that treatment with antibodies against these adhesion molecules can inhibit insulitis and diabetes without affecting the Th1/Th2 balance or effector T cells. The blockade of vascular cell adhesion molecule-1/very late antigen-4 interaction would be suitable for therapeutical treatment of the predisposing and latent type I (insulin-dependent) diabetic subjects. PMID- 7553884 TI - Polymorphism in the beta chain of IAq versus IAp influences presentation of protein but not peptide antigens. AB - T cells play a critical role in the development of collagen-induced arthritis (CIA). Immunization with heterologous (chick) type II collagen (cII) results in chronic inflammation with progressive damage to the joints. The expression of specific MHC Class II alpha beta dimers, including IAq, is critical to induction of disease. The alpha chains of IAq and IAp are identical in sequence. The IAq and IAp beta chains differ by only four amino acid residues: 85, 86, 88, and 89. However, mice of the H-2p haplotype are not susceptible to CIA. To examine the impact of these structural differences in IA molecules on T cell Ag recognition, we studied presentation of cII peptides and denatured cII by APCs obtained from H 2q and H-2p mice. We also assessed presentation of ovalbumin, myelin basic protein (MBP), and MBP peptides by these APC populations. H-2q APCs presented both peptides and proteins to our T cell hybrids. In contrast, APCs obtained from H-2p mice presented peptides, but were defective in the processing and/or presentation of protein Ags. We then altered pairs of the residues in IAq to those found in IAp using site-directed mutagenesis and transfected these constructs into M 12.C3 B cells. All transfectants were able to present peptides, but those expressing IAp were unable to present protein Ags. The use of transfectants expressing hybrid molecules (residues 85 and 86 from IAp, 88 and 89 from IAq, or vice versa) allowed us to localize the region responsible for this defect to residues 85 and 86 of the beta chain. The presence of IAp residues (glu and thr versus gly and val in IAq) at these sites severely compromised the capacity for protein presentation. Resistance to CIA in H-2p haplotype mice may be a reflection of the limited repertoire of epitopes to which these mice can respond relative to susceptible H-2q mice. PMID- 7553887 TI - Increased proportion of CD8+ tumor responsive T cells after immunization with tum versus tum+ rat glioma. AB - Previously established immunogenic (tum-) clones of an ENU (ethyl-N-nitrosourea) induced rat glioma, N32, were compared to the original tumor concerning their capacity to induce T lymphocyte responses after in vivo immunization and in vitro restimulation of responder spleen cells in mixed lymphocyte tumor culture (MLTC) assays. Quite unexpectedly, original N32 (tum+) in vivo primed spleen cells proliferated to the same extent in vitro in response to tum+ stimulator cells as did tum- in vivo primed spleen cells. However, flow-cytometric analysis of parallel cultures showed a greatly increased proportion of CD3+CD8+ lymphocytes in the proliferating responder cell population from tum- immunized hosts, contrary to a CD3+CD4+ lymphocyte dominance after tum+ immunization. Although the original tum+ N32 tumor cells are not capable of inducing a clearly demonstrable isograft rejection response, they induce a strong T cell response readily detectable in MLTC assays. We propose that the increased CD8+ lymphocyte proliferation could be an essential feature of the isograft rejection response induced by tum- tumor variants. Possible mechanisms of the augmented CD8+ T cell response are discussed. PMID- 7553885 TI - Phosphorylation of src family lck tyrosine kinase following interleukin-12 activation of human natural killer cells. AB - Interleukin-12 (IL-12) is a heterodimeric cytokine that augments the cytolytic activity of human NK cells and T cells but has little mitogenic activity on resting lymphocytes. The intracellular signaling pathways involved in NK cell activation by IL-12 have not been fully characterized. In the present studies we found that IL-12 induces tyrosine phosphorylation of a 56-kDa protein, identified in Western blot experiments as p56lck, in resting NK cells. IL-12 was active in the range of 0.1 to 1000 U/ml, with maximal activity between 10 and 100 U/ml (30 and 300 pM). The maximal effect was noted 5 min after stimulation and was almost completely inhibited by genistein. IL-12 induced similar effects in resting and IL-2 activated NK cells. Following IL-12 stimulation of resting NK cells, immunoprecipitated lck kinase exhibited increased in vitro autophosphorylation activity 5 and 10 min after activation. Several additional substrates were phosphorylated in vitro following IL-12 stimulation, including proteins of 70 and 110 kDa. These studies indicate that lck tyrosine kinase is involved in the IL-12 signaling pathway in human NK cells. As the primary functional effect of IL-12 on resting NK cells is the enhancement of cytolytic activity and secretory function rather than cell proliferation, these findings suggest that lck tyrosine kinase is involved in these signaling pathways in human NK cells. PMID- 7553886 TI - B7-1 enhances natural killer cell-mediated cytotoxicity and inhibits tumor growth of a poorly immunogenic murine carcinoma. AB - The B7-1 molecule expressed on antigen presenting cells is an important costimulatory molecule for T cell activation. It has been demonstrated that murine B7-1 can enhance host immunity and lead to tumor rejection via its costimulatory function. Here, we investigate how transfection of B7-1 into line 1, a poorly immunogenic murine lung carcinoma, affects the generation and function of different immune effector cells. Line 1 cells expressing B7-1 form tumors that grow at a slower rate than the parental line 1. Our studies have shown that tumor infiltrating lymphocytes present within the B7-1 expressing tumors are primarily composed of nonspecific killer cells with no specific cytotoxic T cells present. To determine if increased nonspecific killer cells could inhibit the tumor growth of line 1 in the presence of B7-1, we examined the cytotoxicity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells on the B7-1-transfected line 1 and the parental line 1. We found that B7-1 augments the NK- but not LAK-mediated killing against line 1 as measured in an in vitro 51Cr-release cytotoxicity assay. This enhancement could be blocked by CTLA 4 Ig. In vivo depletion of NK cells led to growth of the B7-1-transfected line 1 at the same rate as the parental line 1. These results suggest that in addition to its costimulatory role for T cell activation B7-1 could be an accessory molecule that intensifies NK-mediated cytotoxicity. This novel finding may provide a mechanism for the effect of B7-1 on tumors of low immunogenicity. PMID- 7553888 TI - Induction of IL-6 and IL-10 production by recombinant HIV-1 envelope glycoprotein 41 (gp41) in the THP-1 human monocytic cell line. AB - Elevated levels of circulating monokines (IL-6, IL-1, and TNF alpha) have been seen in HIV-1 infection, and the overproduction of these cytokines could contribute to AIDS pathogenesis in various ways. In previous work, we had seen that exposure of human monocytes to HIV-1, including inactivated, noninfectious HIV, led to rapid IL-6 gene expression and secretion. To investigate cytokine production in response to components of HIV by monocytes/macrophages, production of IL-6 and IL-10 were examined in a human monocytic cell line, THP-1, stimulated by HIV proteins. IL-6 production was induced in THP-1 cells by a detergent lysate of HIV, particularly fractions at molecular weight of 25-50 kDa. Recombinant HIV envelope glycoprotein 41 (gp41), but not gp120 or p24, also was seen to induce significant IL-6 production by THP-1 cells. These results suggest that gp41, transmembrane protein, is the primary HIV-encoded protein involved in inducing IL 6 production. IL-10 was also produced with delayed kinetics, following IL-6 production in THP-1 cells stimulated by gp41. To investigate a possible regulatory role for IL-10 in HIV-induced monokine production, recombinant IL-10 was added to gp41-exposed THP-1 cells. IL-10 inhibited gp41-induced IL-6 production and reduced the expression of IL-6 mRNA. When anti-human IL-10 neutralizing antibody was added to THP-1 cells, IL-6 production was enhanced. These results suggest that the IL-6 production may be downregulated by endogenously produced IL-10 and that IL-10 may downregulate cytokine production by HIV-activated monocytes via an autoregulatory mechanism. PMID- 7553890 TI - A revertant TCR gamma delta + cell clone which has lost MHC nonrestricted cytotoxic activity but retains redirected killing upon stimulation of the CD3 receptor. AB - A variant nonkiller (K-) T-cell clone derived from the MHC nonrestricted killer (K+) cell line TALL-103/2 (CD3/TCR gamma delta +) was studied to determine whether its lytic defects were at the tumor-binding or post-binding level. The two TALL-103/2 clones were found to display similar mRNA expression of TCR/CD3 complex epsilon, zeta, gamma, and delta chains, and the same mRNA and protein levels of SRC-like protein tyrosine kinase and kinase activity. However, the K- cells express much less surface CD45 RA and contain smaller intracytoplasmatic cytotoxic granules with a lower expression of the TIA-1 protein. Although the K- cells do not undergo granule exocytosis upon contact with a susceptible (K562) target, they can be triggered to degranulate and display redirected killing by activation of the CD3 receptor. Moreover, OKT3 induces PPI turnover and Ca2+ mobilization in both the K+ and K- cells, whereas K562 cells induces PPI turnover only in the K+ clone. The overall data indicate that, although the K- cells have significant post-binding defects that prevent them from killing MHC nonrestricted targets, they can fully utilize their lytic machinery upon specific activation of the CD3 pathway. PMID- 7553889 TI - Local tumor irradiation augments the response to IL-2 therapy in a murine renal adenocarcinoma. AB - We have previously demonstrated that local tumor irradiation effectively enhanced the therapeutic effect of IL-2 therapy on pulmonary metastases from a murine renal adenocarcinoma, Renca. Irradiation with 300 rad to the left lung only, followed by systemic IL-2 therapy, results in increased tumor reduction in both lungs, suggesting that radiation enhances the systemic effect of immunotherapy. In this study, we show that irradiation of the tumor-bearing organ is essential for the combined effect of both modalities. This effect is radiation dose dependent as increases in the radiation dosage result in greater tumor reduction in the irradiated field as well as systemically in nonirradiated fields when combined with immunotherapy. We find that irradiation has a direct inhibitory effect on Renca cell growth in vitro. Irradiation of Renca cells also causes an upregulation in H-2Kd class I MHC antigen detectable at 300 rad and more pronounced with 800 rad. By in vivo selective depletion of lymphocyte subsets, we demonstrate the involvement of Lyt-2+ and L3T4+ T cell subsets and AsGM1+ cells, including NK cells, in the antitumor effect mediated by tumor irradiation and IL 2 therapy. Immunohistochemistry studies, performed on lung sections, showed a significant infiltration of CD3+ T cells and macrophages in the tumor nodules following treatment with tumor irradiation and IL-2 therapy. Our studies indicate that the mechanism of interaction between tumor irradiation and immunotherapy may include radiation-induced alterations in the tumor growth and antigenicity which may enhance or trigger an anti-tumor response elicited by IL-2 and mediated by T cells, AsGM1+ cells, and macrophages. PMID- 7553892 TI - Expression of ZAP-70 gene in the developing thymus and various nonlymphoid tissues of embryonic and adult mice. AB - The full length of cDNA encoding murine ZAP-70 was obtained from a cDNA library of embryonic mouse thymus. Northern blot analysis indicated that the expression of ZAP-70 mRNA was most pronounced in the thymus and spleen and also slightly in the brain. Analysis by in situ hybridization revealed that the mRNA was expressed not only in T cells in the thymus and spleen, but also in nerve cells of the brain. In the thymus, the distribution of positive cells was different between newborn and young adult mice. In the newborn thymus, positive signal in thymocytes was localized in the subcapsular layer as well as in the medulla, while in the young adult thymus, it was localized exclusively in the medulla. Furthermore, the in situ hybridization revealed that positive signal was also seen in the liver, intestine, and lung of embryonic mice. These findings have suggested that ZAP-70 plays an important role in growth, differentiation, and function of not only T cells but also nerve cells and several embryonic tissues. PMID- 7553891 TI - Signaling by a new anti-Thy 1 monoclonal antibody inhibits T cell proliferation and interferes with T-cell-mediated induction of costimulatory molecule B7-2. AB - Costimulatory activity on antigen-presenting cells is a critical determinant of the fate T cells when the T cell receptors are engaged by MHC:peptide complexes. Therefore, control of the expression of the costimulatory molecules regulates T cell responses. While several types of interactions between T cells and B cells up-regulate costimulatory molecules on antigen-presenting cell, no T cell surface molecules have been implicated in inhibiting the induction of the costimulatory molecules on B cells. Here we characterize a new anti-Thy1 mAb, 21F10, which inhibits T cell proliferation to selective stimuli. T cells stimulated by anti CD3 together with anti-Thy1 mAb are anergic to further stimulation through the CD3, which suggests that the anti-Thy1 mAb interferes with the delivery of the costimulatory activity to T cells. Consistent with this notion, anti-Thy1 mAb 21F10 completely inhibits the induction of B7-2 on B cells. Induction of several T cell surface molecules such as CD69 and CD40 ligand was largely unaffected. As this inhibition requires a bivalent anti-Thy1 mAb and does not require binding of more than 50% of Thy1 molecules on T cell surface, we suggest that Thy1 may mediate a negative signaling pathway which inhibits the T-cell-mediated induction of costimulatory activity, including expression of co-stimulatory molecule B7-2. PMID- 7553893 TI - High-dose cyclophosphamide inhibits anterior chamber-associated immune deviation (ACAID) and the production of extracellular antigen-specific T cell proteins induced by trinitrophenylated (TNP) spleen cells. AB - Injection of antigen into the anterior chamber (AC) of the eye induces the production of IgM and IgG1 antibodies and potentiates the appearance in serum of extracellular antigen-specific T cell proteins (TABM) that are specific for the AC-injected antigen. In contrast, delayed-type hypersensitivity (DTH) to the injected antigen is suppressed. This manifestation of anterior chamber-associated immune deviation (ACAID) is believed to be a key part of the basis of the immune privilege of the eye. Because cyclophosphamide (CY) exerts selective effects on immunoregulatory T cells and macrophages, we sought to determine its effects on the appearance in serum of TNP-specific TABM in mice following intracameral injection with TNP murine spleen cells followed by epicutaneous sensitization and challenge with picrylchloride. Injection of 200 mg/kg CY 2 days before AC injection of TNP spleen cells, sensitization, and challenge prevented the suppression of DTH and the production of TNP-specific TABM. The production of TNP specific immunoglobulins was not affected. Injection of a low dose of 20 mg/kg CY enhanced DTH 100-300% in control animals, but did not prevent either ACAID or the production of TABM. These results provide further evidence that serum TABM may be a serologic indicator of T lymphocyte activity in ACAID and that ACAID is mediated by cells sensitive to high-dose CY. PMID- 7553894 TI - Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2. AB - Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin 15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2. PMID- 7553895 TI - Expression of GPI-anchored complement regulatory proteins CD55 and CD59 differentiates two subpopulations of human CD56+ CD3- lymphocytes (NK cells). AB - CD55 and CD59 are both glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins found on the surface of most hemopoietic cells. Using three color cytofluorographic analysis with antibodies recognizing CD56, CD3, and CD59 or CD55 we determined that CD56+CD3- lymphocytes (NK cells) expressed both CD55 and CD59 but at lower levels than CD3+ lymphocytes. Since the CD56+CD3- lymphocyte population is heterogeneous, we examined expression of CD55 and CD59 on selected CD56+CD3- lymphocyte populations by depleting peripheral blood leukocytes of T cells, B cells, and monocytes. Dual staining of the selected CD56+CD3- cells, which were > 95% CD56+, permitted the distiction of two subpopulations: a major CD16brightCD56dimCD55dimCD59dim subpopulation and a minor CD16dim/negCD56brightCD55brightCD59bright subpopulation. Treatment with phosphatidylinositol-specific phospholipase C released both the CD55 and CD59 antigens from the surface of CD56+CD3- cells, indicating that both are GPI anchored, as they are on other lymphocytes. CD56+CD3- cell subpopulations were individually isolated by anti-CD55 or anti-CD16 negative selection and were functionally compared to the parent CD56+CD3- cell population. The CD16brightCD56dimCD55dimCD59dim cells killed NK targets well but did not proliferate well in response to rIL-2, whereas CD16dim/negCD56brightCD55brightCD59bright cells proliferated well in response to rIL-2 but did not kill NK targets efficiently. We conclude that all CD56+CD3- cells express some levels of the GPI-anchored proteins, CD55 and CD59, and that two CD56+CD3- subpopulations with different functional characteristics can be distinguished by the level of expression of these two antigens. PMID- 7553896 TI - DEC-205, a 205-kDa protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody NLDC-145: purification, characterization, and N-terminal amino acid sequence. AB - The monoclonal antibody NLDC-145 detects an antigen expressed at high levels by mouse dendritic cells (DCs) and thymic epithelial cells. Here we report on the purification and biochemical characterization of this antigen. The antigen detected by the NLDC-145 mAb is an integral membrane glycoprotein, with an apparent molecular mass of 205 kDa by immunoprecipitation and Western blotting. The isoelectric point is pH 7.5, and carbohydrates comprise about 7 kDa of the total mass. Lectin blotting and FACE analysis revealed heterogeneous biantennary N-linked glycans. O-linked glycans were not detected. N-terminal sequence analysis revealed that the antigen is a novel protein. Polyclonal antibodies prepared either to a synthetic N-terminal peptide or to whole purified protein bind the same 205-kDa protein recognized by NLDC-145. We refer to the protein as DEC-205, in view of its abundant expression by dendritic and thymic epithelial cells, and the revised molecular mass. PMID- 7553897 TI - Fas-mediated cytotoxicity remains intact in perforin and granzyme B antisense transfectants of a human NK-like cell line. AB - Cell-mediated cytotoxicity (CMC) has traditionally been thought to involve the release of granule components, including perforin and granzymes, from the effector cell (EC) onto the target cell (TC) membrane. Recently, a granule independent cytolytic mechanism involving the interaction of Fas antigen (CD95) with Fas ligand has been described. We have generated antisense perforin (YT-xP1) and granzyme B (YT-xGrB) transfectants of the human NK-like cell line YT-INDY. These transfectants have greatly reduced cytolytic ability when compared to the vector-transfected control cell line (YT-neo). In this study, however, we demonstrate that the antisense transfectants retain the ability to lyse Fas+ TC. Fas-mediated lysis is Ca(2+)-independent and is inhibited by a monoclonal anti Fas blocking Ab, M3. By RT-PCR, we detect message for FasL in unstimulated YT-xP1 and YT-xGrB transfectants, as well as in unstimulated YT-neo. By flow cytometry, we show that YT-neo, YT-xGrB, and YT-xP1 constitutively express surface FasL. These data indicate that in a human NK-like cell line, similar to the murine system, the granule and Fas pathways of cytotoxicity function independently of one another. At least with the TC tested, our data also indicate that the granule and Fas pathways together account for nearly 100% of the cytolytic ability of YT INDY. PMID- 7553898 TI - "Geometric clutch" hypothesis of axonemal function: key issues and testable predictions. AB - The Geometric Clutch hypothesis integrates a large body of seemingly disconnected analytical measurements and observations into one conceptual framework. It remains to be established whether certain key requirements of the hypothesis are actual attributes of real axonemes. The hypothesis is rich in predictive value, as its fundamental working elements are developed directly from physical properties and structures of the axoneme. Exploration of these predictions will serve to confirm or reject the hypothesis itself, but even more importantly, may contribute to elucidation of the principles underlying ciliary and flagellar functioning. PMID- 7553899 TI - Outer arm dynein from Newt lung respiratory cilia: purification and polypeptide composition. AB - Dyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes > 95% of the outer arm dynein (OAD), and that the beat frequency of OAD-depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199-209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The present study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released approximately 1.4 x 10(7) axonemes during isolation, yielding approximately 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring approximately 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0-8 M urea, 3-5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV-induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (M(r) 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent M(r) of the dynein heavy chains was determined to lie between 430-420 kD. Eight discrete polypeptides (putative intermediate chains, IC1-IC8, M(r), 175-56 kD) copurified with the alpha- and beta-heavy chains by microtubule-alloaffinity. Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two-headed particle represents the entire newt respiratory outer arm dynein. PMID- 7553900 TI - Antibody against phosphorylated proteins (MPM-2) recognizes mitotic microtubules in endosperm cells of higher plant Haemanthus. AB - In diverse cell types, monoclonal antibody MPM-2 recognizes a class of phosphorylated proteins related to microtubule organizing centers and abundant during mitosis. We have used this antibody in an attempt to identify the spatial and temporal localization of putative microtubule organizing centers in endosperm cells of the higher plant Haemanthus. Our results show that MPM-2 recognized epitope is present in interphase cells and enriched in mitotic cells. In interphase the antibody usually stains cytoplasmic granules. During the interphase-prophase transition immunoreactive material appears in the nucleus, at the nuclear envelope, and in association with microtubules. Concomitantly, we observed an increase of immunoreactivity of the cytoplasm. During mitosis the phosphorproteins recognized by MPM-2 are detected in the cytoplasm, in association with microtubules of the spindle, the phragmoplast, and in the newly formed cell plate. After completion of mitosis, only the cell plate and cytoplasmic granules are MPM-2 positive. Extraction of the cells with Triton X 100 prior to fixation removes staining of the cytoplasm by MPM-2. The detergent resistant immunoreactive material remains associated with surrounding the nucleus microtubules of the prophase spindle, the core of kinetochore fibers, and the phragmoplast. In the phragmoplast, however, segments of microtubules which are distal to the cell plate are depleted of MPM-2. These data demonstrate that microtubule arrays of endosperm cells are phosphorylated during mitosis. Thus, similar to animal cells, interphase and mitotic microtubules of higher plants have different properties. Additionally, the localization of detergent resistant MPM-2 antigen points to the difference in microtubule nucleation/organization between higher plant and animal cells. PMID- 7553901 TI - Topographic compensation: guidance and directed locomotion of fibroblasts on grooved micromachined substrata in the absence of microtubules. AB - Fibroblasts cultured on grooved substrata align themselves and migrate in the direction of the grooves, a phenomenon called contact guidance. Microtubules have been deemed important for cell polarization, directed locomotion, and contact guidance. Because microtubules were the first cytoskeletal element to align with the grooves when fibroblasts spread on grooved substrata, we investigated the consequences of eliminating the influence of microtubules by seeding fibroblasts onto smooth and grooved micromachined substrata in the presence of colcemid. Fibroblasts were examined by time-lapse cinematography and epifluorescence or confocal microscopy to determine cell shape and orientation and the distribution of cytoskeletal or associated elements including actin filaments, vinculin, intermediate filaments, microtubules, and kinesin. As expected, cells spreading on smooth surfaces in the presence of colcemid did not polarize or locomote. Surprisingly however, by 24 hours, cells spread on grooves in the presence of colcemid were morphologically indistinguishable from controls spread on grooves. Both groups were aligned and polarized with the direction of the grooves and demonstrated directional locomotion along the grooves. In the absence of microtubules, kinesin localized to some of the aligned stress fibers and to leading edges of cells spreading on grooves. The grooved substratum compensated for the microtubule deficiency by organizing and maintaining an aligned actin filament framework. Thus, microtubules are not required to establish or maintain stable, polarized cell shapes or directed locomotion, provided an alternate oriented cytoskeletal component is available. PMID- 7553902 TI - Flagellar quiescence response in sea urchin sperm induced by electric stimulation. AB - To investigate the mechanism of the flagellar quiescence in sperm, we examined the effect of electric stimulation of individual spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Stimulation with a suction electrode attached to the sperm head elicited a flagellar quiescence response, in which the sperm showed a typical cane-shaped bend in the proximal region of the flagellum when the electrode was used as anode. Cathodic stimulation also induced quiescence, but was much less effective than anodic stimulation. During the quiescence response, which lasted for 1-3 s, no new bend was initiated, and subsequently the flagellum resumed normal beating. The quiescence response required the presence of Ca2+ (> 2 mM) in sea water, and was inhibited by Co2+ and La3+. At low Ca2+ concentrations (2-5 mM), the angle of the cane-shaped bend was smaller than that at 10 mM Ca2+; thus the angle of the cane-shaped bend, characteristic of the quiescence response is dependent on Ca2+ concentration. These results suggest that the quiescence response is triggered by a depolarization of the flagellar membrane, followed by an influx of Ca2+ into the flagellum through Ca2+ channels. The increase in Ca2+ concentration within the flagellum affects the amount of sliding and thus produces a cane-shaped proximal bend of various angles, while inhibiting both the propagation of the proximal bend (principal bend) and the formation of a new reverse bend. PMID- 7553903 TI - Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells. AB - To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE. PMID- 7553904 TI - Polylysine cross-links axoplasmic neurofilaments into tight bundles. AB - We have used axoplasm from the squid giant axon to investigate the effects of anionic and cationic polypeptides on the mobility and organization of axonal neurofilaments (NFs). Intact cylinders of axoplasm were extruded from squid giant axons into an excess volume of artificial axoplasm solution. In a previous study on the mobility of NFs in extruded axoplasm, we showed that these polymers disperse freely and diffusively into the surrounding solution, thereby expanding the axoplasmic cross-sectional area [Brown and Lasek, 1993: Cell Motil. Cytoskeleton 26:313-324]. In the present study, we found that 83nm-long ("long chain") polylysine, a synthetic multivalent cationic protein, inhibited the radial expansion of isolated axoplasm and condensed the axoplasm, thereby reducing the cross-sectional area. Equivalent concentrations of a 7nm-long ("short-chain") polylysine did not inhibit the expansion of axoplasm by long chain polylysine was dependent on the polylysine concentration; condensation of axoplasm was observed at concentrations of 0.01 mg/ml (0.27 microM) or greater. Electron microscopy of the condensed axoplasm showed that the NFs were aligned side-by-side and in parallel in closely-packed bundles. Equivalent concentrations of 91 nm-long ("long-chain") polyglutamate, a synthetic multivalent anionic protein, partially inhibited the expansion of axoplasm but did not cause the NFs to bundle and did not cause the axoplasm to condense. These studies indicate that cationic proteins bind tightly to the highly charged anionic surfaces of NFs and can link them together into compact bundles in a charge-dependent and length dependent manner. The tightly packed organization of these cross-linked NFs differs from the normal loose organization of NFs in healthy axons. However, tightly bundled NFs are sometimes found in certain neuropathologies, such as giant axonal neuropathy. PMID- 7553905 TI - Experimental manipulation of gamma-tubulin distribution in Arabidopsis using anti microtubule drugs. AB - gamma-Tubulin-specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner. During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate. gamma-Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers. At higher drug concentrations, gamma-tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast. During UV induced recovery from colchicine, gamma-tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei. With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles. In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal. As with CIPC, gamma-tubulin is concentrated at focal arrays. Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts. These results support a preferential association between gamma-tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts. Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but gamma-tubulin may also serve another function, such as in structural stabilization. PMID- 7553906 TI - Strikingly different propulsive forces generated by different dynein-deficient mutants in viscous media. AB - The propulsive force generated by Chlamydomonas mutants deficient in flagellar dynein was estimated from their swimming velocities in viscous media. The force produced by wild-type cells increased by 30-40% when viscosity was raised from 0.9 to 2 cP but decreased as viscosity was further raised above 6 cP. The biphasic dependence of force generation on viscosity was also observed in the mutant ida1, which lacks the I1 component of the inner-arm dynein. The mutant ida4, which lacks the inner-arm I2 component, was extremely susceptible to viscosity and stopped swimming at 6 cP, at which other mutants could swim. In contrast, oda1, which lacks the entire dynein outer arm, produced a fairly constant force of about one-third of the wild-type value, over a viscosity range of 0.9-11 cP. In demembranated and reactivated cell models of the wild type, the propulsive force decreased monotonically as viscosity increased. Thus the increase in force generation at about 2 cP observed in live cells may be caused by some unknown mechanism that is lost in cell models. The cell models of oda1, in contrast, did not show a marked change in force generation with the change in viscosity. These results indicate that the force generation by the outer-arm dynein greatly depends on viscosity or the velocity of movement, whereas the complete set of inner-arm dynein present in the oda1 axoneme produces a fairly constant force at different viscosities. These different properties of inner and outer dynein arms should be important in the mechanism that produces flagellar beating. PMID- 7553907 TI - Manipulation of bovine sperm metabolism and motility using anoxia and phosphodiesterase inhibitors. AB - Bovine sperm that were subjected to extended anoxia (2.5 h) in the absence of glycolytic substrates then diluted into oxygenated medium were immotile but metabolically active, producing ATP from lactate via oxidative phosphorylation. In response to anoxia sperm ATP titers dropped from 15-20 mumoles/10(8) cells to 1-2 mumoles/10(8) cells in the first 5 min then remained extremely low until reoxygenation. Cyclic AMP titers declined slowly over the anoxic period, but did not show the same scale of depression as ATP. After dilution and re-oxygenation ATP recovered to pre-anoxia levels within 1 min, and cAMP rose to about the pre anoxia levels. However, motility, which varied quantitatively and qualitatively between ejaculates prior to anoxic treatment, was substantially depressed after extended anoxia in all cases; progressive motility was almost non-existent in post-anoxic sperm. Addition of isobutylmethylxanthine or Cibacron Blue F3GA, both putative phosphodiesterase inhibitors, stimulated a transient peak of cAMP, which was accompanied by motility stimulation. These techniques provide a protocol to manipulate and dissect the biochemical pathways of motility initiation in mammalian sperm. PMID- 7553908 TI - Role of the cytoskeleton in the reaction of fibroblasts to multiple grooved substrata. AB - The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluorescence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 microns width and 0.5, 1, 2, and 5 microns depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 microns. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cells react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colcemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata. PMID- 7553909 TI - cAMP/ATP relationship in the activation of trout sperm motility: their interaction in membrane-deprived models and in live spermatozoa. AB - Live trout spermatozoa initiate flagellar motility for only a short period (30 sec at 18 degrees C) during which their mean beat frequency decreases steadily from 60 to 20 Hz. Motility then stops abruptly. Investigations of the activation of movement in demembranated sperm points to cyclic-AMP being necessary for reactivation (half effect at 0.5 microM) in some conditions. cAMP acts mainly by increasing the percentage of motile cells and not the beat frequency (BF) of the flagellar axoneme. Dibutyryl cAMP does not initiate movement or prolong motility of live sperm. The initiation of movement of demembranated trout sperm was investigated in various incubation conditions relative to previous phases of in vivo movement and to ATP concentration. In the absence of cAMP and in the presence of ATP lower than 25 microM, all sperm cell models were active with BF up to 15-20 Hz whatever their previous physiological conditions. In contrast, at ATP concentrations above 100 microM, the fraction of active spermatozoa decreased proportionally but the BF of the active ones increased so that, at 1 mM ATP, only 5% were active but with a BF of 65 Hz: the addition of cAMP up to 20 microM restored activity to 100% sperm models with a similar BF of 65 Hz. At ATP concentrations higher than 25 microM, cAMP was necessary in a concentration dependent manner in the reactivation, but not in the demembranation medium. This dependence was found to be unrelated to a previous in vivo phase of movement. The antagonistic effects of ATP vs. cAMP were tested at various concentrations of both nucleotides: the apparent affinity for cAMP, measured as the concentration restoring movement of 50% cell models, was decreased from 15 nM at 0.1 mM ATP to 0.5 microM at 1 mM ATP; conversely, the affinity for ATP, measured as the concentration giving rise to the half maximal beat frequency, was not significantly affected when the concentration of cAMP was raised to 0.5 mM. Preincubation with phosphodiesterase (PDE) resulted in motility of 100% of sperm models even at low ATP concentration. This tends to show that cAMP must be constantly present to sustain motility. PMID- 7553910 TI - TEDS rule: a molecular rationale for differential regulation of myosins by phosphorylation of the heavy chain head. PMID- 7553911 TI - Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro. AB - Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide. PMID- 7553912 TI - Identification of two new members of the tubulin family. AB - Analysis of the delta- and epsilon-tubulin sequences indicates that they both consist of two structural domains of which the N-terminal domain can bind to alpha/beta heterodimers while the C-terminal domain probably binds to a non tubulin protein. Both additional tubulins probably bind GTP but lack GTPase activity, while their synthesis requires the TCP1 chaperonine but is not autoregulated. Although these properties resemble those of gamma-tubulin, the low sequence identity (Table I) demonstrates that the gamma-, delta-, and epsilon proteins should be classed as different members of the tubulin family. The identification of these additional members is unexpected. Examination of the cellular expression and distribution of the delta- and epsilon-tubulins, and whether other organisms contain homologous genes, may reveal further features of the eukaryotic cytoskeleton. PMID- 7553913 TI - Overexpression of an epitope-tagged beta-tubulin in Chinese hamster ovary cells causes an increase in endogenous alpha-tubulin synthesis. AB - A Chinese hamster beta-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HA beta 1-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HA beta 1-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HA beta 1-tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HA beta 1-tubulin. The synthesis of both endogenous beta-tubulin and HA beta 1-tubulin was repressed by colchicine. The HA beta 1-tubulin incorporated into microtubules to the same extent as the endogenous beta-tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, transfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HA beta 1-tubulin reduced the synthesis of endogenous wild-type beta-tubulin but increased the synthesis of alpha-tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of alpha-tubulin was found. The results indicate that expression of excess exogenous beta-tubulin perturbs the synthesis of endogenous alpha-tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of alpha- and beta-tubulin for assembly. PMID- 7553914 TI - Suppression of syntheses of high molecular weight nonmuscle tropomyosins in macrophages. AB - In mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two-dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti-TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform-specific cDNA probes, and immunostaining with monoclonal anti-TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti-TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co-localized with F actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm. PMID- 7553915 TI - Porcine brain neurofilament-H tail domain kinase: its identification as cdk5/p26 complex and comparison with cdc2/cyclin B kinase. AB - Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recognizing in vivo phosphorylation sites (SMI31, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract. PMID- 7553916 TI - Cytoskeleton of the Drosophila egg chamber: new observations on microfilament distribution during oocyte growth. AB - The distribution of microfilaments in Drosophila egg chambers stained with rhodamine (Rh)-conjugated phalloidin was studied by laser scanning confocal microscopy and transmission electron microscopy. These techniques revealed new details in the pattern of microfilament localization. We observed in stage 1-3 egg chambers accumulation of filamentous actin in the oocyte cytoplasm between the ring canals connecting the oocyte with adjacent nurse cells. Starting from stages 6-7 short microfilament bundles arranged in basket-like structures were associated with the side of the ring canals facing the nurse cell cytoplasm. We also observed a dramatic decrease in the actin network associated with the cortex of the oocyte in stage 10. During stage 10B the nurse cell cytoplasm was crossed by radial actin bundles that showed a sarcomeric-like cross striation after Rh phalloidin staining. The ring canals also did not uniformly stain but showed a punctate labeling. The implications of the actin cytoskeleton during oocyte growth are discussed. PMID- 7553917 TI - Increasing intracellular concentrations of thymosin beta 4 in PtK2 cells: effects on stress fibers, cytokinesis, and cell spreading. AB - Thymosin beta 4 (T beta 4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of increasing T beta 4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of T beta 4 at a concentration (approximately 28 microM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular T beta 4 concentrations were increased by three different methods: 1) microinjection of an RSV vector containing a cDNA for T beta 4; 2) transfection with the same vector; and 3) microinjection of purified T beta 4 protein. The plasmid coding for T beta 4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure T beta 4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, similar microinjection of pure thymosin beta 4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow regressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these T beta 4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process. PMID- 7553919 TI - Physiology of growth and development. Its relationship to performance in the young athlete. AB - Proper grouping of children for sports is important for injury prevention and for fair competition. We have reviewed several of the classification systems presently used as well as the physiologic underpinnings of pubertal growth and development and the accrual of strength and power. It is during adolescence that the greatest physiologic differences exist mainly because of the wide variations in the timing and tempo of the pubertal growth spurt in normally growing boys and girls. Maturity-based categorization, especially in contact and collision sports, would heighten the competition and lessen rates of injury. PMID- 7553918 TI - Molecular environment of ZO-1 in epithelial and non-epithelial cells. AB - We previously reported the expression of ZO-1 in cell types that do not form tight junctions. Here we compare the molecular environments of ZO-1 in epithelial cells, primary cultures of astrocytes and in the non-epithelial S180 sarcoma cell line. ZO-1 co-localizes with a subset of actin filaments in all cell types. In astrocytes, ZO-1 is found concentrated in discrete bands at points of cell-cell contact. Indirect immunofluorescent microscopy shows that these bands of ZO-1 co localize with the adherens junction proteins vinculin and alpha-actinin, and with the antigen recognized by a pan-cadherin antibody. In contrast, ZO-1 in S180 cells, which exhibit limited cell-cell interactions, is diffusely distributed over the plasma membrane, with concentrations in lamellipodia where actin filaments accumulate. ZO-1 does not co-localize with vinculin at focal adhesions in this cell type. Analysis of ZO-1 immunoprecipitation profiles from different cell types, performed under conditions previously demonstrated to maintain interactions between ZO-1, ZO-2 and p130 from the MDCK epithelial cell line, show that the proteins which co-precipitate with ZO-1 vary with cell type. Precipitation of polypeptides at 165 kDa, potentially ZO-2, and 65 kDa occurs in both a mouse kidney tubule epithelial cell line and the non-epithelial S180 cells. No proteins specifically associate with ZO-1 immunoprecipitated from astrocytes. Spectrin, alpha-actinin, vinculin and cadherin are not detected in immunoblots of ZO-1 immunoprecipitates from any cell type. PMID- 7553920 TI - Overuse injuries in the young athlete. AB - Overuse injuries in the young athlete now are seen more frequently. Using an adult model of these injuries with respect to diagnosis and treatment and applying it to the young athlete does not address many of the unique characteristics that contribute to these injuries. An overview of causative factors that contribute to overuse injuries in young athletes is presented. Examples of specific injuries seen only in this age group and the treatment of these injuries are presented. PMID- 7553921 TI - Head and spine injuries in youth sports. AB - Careful study of the pathomechanics and epidemiology of sports-related spine injuries brings to light many common features. The incidence increases as the sport becomes increasingly violent and aggressive. Improperly conditioned neck muscles and lack of knowledge of the proper techniques of the sport put the athlete who sustains a blow to the head at significant risk for head and/or spine injury. Improper helmet fit and the use of the head as an offensive weapon are also common features. Although recognition of these features has resulted in a dramatic reduction in catastrophic athletic spine injury, the athlete remains at risk for less severe head and spine injury. PMID- 7553922 TI - Upper extremity injuries in young athletes. AB - With the knowledge base of normal anatomy, development, biomechanics, and differential diagnosis, the sports medicine professional can treat injured young athletes with greater efficiency. In addition, microtraumatic injuries may be prevented by emphasizing safe parameters of participation, proper throwing techniques, and careful monitoring of the amount of practice time and intensity. Gymnasts using apparatus should always have spotters. The height of towers and basket tosses by cheerleaders should be limited by age and ability. Proper pitching techniques, not the fastest pitch or youngest curve, should be taught to baseball players. "Play it safe" should be the rule. Finally, by establishing an early and precise diagnosis, potential complications from injuries can be lessened. PMID- 7553923 TI - Back injuries in the young athlete. AB - Back pain in children and young athletes is very different from back pain in adults. Macrotrauma must be carefully evaluated and managed, even in the absence of definitive radiographic findings. Microtrauma must be suspected in at-risk athletes. These athletes require persistent diagnostic evaluation and may require SPECT bone scan to uncover a posterior element stress reaction. Atraumatic back pain requires the elimination of neoplastic, infectious, rheumatologic, or congenital causes. PMID- 7553925 TI - Knee injuries in young athletes. AB - The differential diagnosis of children's knee injuries includes not only the typical adult injuries to bone, ligament, and cartilage, but also growth plate injuries. Bony contours are not as well defined in children due to incomplete ossification, and the practitioner must look for certain types of injuries based on the history and the physical findings because radiographic signs may be difficult or impossible to see. PMID- 7553924 TI - Injuries about the hip and pelvis in the young athlete. AB - Athletic injuries to the hip and pelvis in pediatric and adolescent athletes, although uncommon, may encompass a wide spectrum of entities. A familiarity with this spectrum and a high index of suspicion in the proper clinical setting will ensure timely diagnosis and help to facilitate implementation of a proper treatment plan thereby assuring safe return to play. PMID- 7553926 TI - Lower leg, foot, and ankle injuries in young athletes. AB - Most injuries to the young athlete do have a benign natural course--complete resolution of the difficulty without sequelae. In order to develop a contemporary program of management of the more serious disorders in this active population, the practitioner must be certain to carefully analyze the injury and initiate a rapid course of action. A fracture of the tibia must be reduced, held aligned, and then rehabilitated. A compartment syndrome commonly demands early fasciotomy; the young athlete and his or her parents warrant a sensitive understanding from physicians to quell the anxiety that is paramount to all of these disorders. PMID- 7553928 TI - The young female athlete. AB - It is important that girls and young women participate in sports and develop skills that promote lifelong athletic participation, because of the psychological, sociologic, and physiologic benefits associated with exercise. When an athlete begins intensive, competitive exercise training at a young age, or when the preoccupation with thinness supersedes a desire to be healthy, potential morbidity results. Lack of information and the strong desire to win contribute to this problem. There is relatively little known about the long-term physical and psychological effects of early intensive athletic training and the female athlete triad on the young female athlete. In addition to the need for further research in these areas, there is a need for education of physicians, coaches, trainers, athletes, and parents. The preparticipation physical examination is an excellent opportunity for the physician to screen for the triad disorders and educate athletes and parents on healthy nutrition, normal menstrual function, and the benefits of exercise. PMID- 7553927 TI - Muscle-tendon injuries in young athletes. AB - The pediatric athlete with open physeal plates is more susceptible to growth plate injuries and avulsion fractures rather than ligament and muscle-tendon injuries that most often occur in adults. In general, the pediatric athlete is able to return to full activity quicker than the adult athlete because of a more rapid healing response. There has been a recent interest in the study of the pathophysiology of muscle-tendon injuries and treatment strategies based on these studies have changed. Laboratory studies have several implications regarding the prevention of muscle-tendon injuries. Clinical studies to confirm their efficacy are needed. Complications of re-injury include more serious injury and conditions such as myositis ossificans. Injury prevention has focused on the use of protective equipment and an emphasis on stretching and strengthening programs. PMID- 7553929 TI - The young athlete with chronic disease. AB - Iron deficiency anemia can affect athletic performance. Physicians must be wary of the at risk population, namely vegetarians, female adolescents, and long distance athletes. Particular attention must be paid to nutritional intake and to the use of nonsteroidal anti-inflammatory agents and aspirin. PMID- 7553930 TI - Sports injuries in children and adolescents. Questions and controversies. AB - Certain topics and controversies involving young athletes are addressed, including unclear criteria for volume and intensity of training and acute and overuse injuries of the knee. Osteochondritis dissecans, tibial spine avulsions, peri-pelvic apophyseal avulsions, and back injuries are discussed. PMID- 7553931 TI - Gene PSO5 of Saccharomyces cerevisiae, involved in repair of oxidative DNA damage, is allelic to RAD16. AB - The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H2O2 and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage. PMID- 7553932 TI - Failure to detect an antimutator phenotype following disruption of the Saccharomyces cerevisiae DDR48 gene. AB - The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway. We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator. However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2-1 allele, or forward mutation at the CAN1 locus. Interestingly, the latter gene had been reported previously to be subject to the antimutator effect. DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes. Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype. PMID- 7553933 TI - Molecular analysis of the yeast SER1 gene encoding 3-phosphoserine aminotransferase: regulation by general control and serine repression. AB - Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression. PMID- 7553934 TI - Purification and binding properties of the Mal63p activator of Saccharomyces cerevisiae. AB - Mal63p is a transcriptional activator for maltose fermentation in Saccharomyces cerevisiae. We have purified it to homogeneity from a yeast strain in which the MAL63 gene is under the control of the GAL1-GAL10 promoter. Purification included fractionation of a whole-cell extract by ion-exchange chromatography, chromatography using both non-specific DNA-affinity (calf thymus), and sequence specific DNA-affinity chromatography. Mal63p activity was assayed by its binding to a fragment of the MAL61-MAL62 promoter, using both filter-binding and electrophoretic-mobility shift assays. DNase-I footprinting identified a new binding site (site 3) between the two previously known sites (sites 1 and 2). Mal63p is a dimer, and methylation-protection experiments identify the recognition motif as: c/a GC N9 c/a GC/g. PMID- 7553936 TI - Genetic characterization of two phototropism mutants of Phycomyces with defects in the genes madI and madJ. AB - Two Phycomyces genes, madI and madJ, which are involved in phototropism, were characterized by recombination and complementation analyses. The madI gene was located on linkage group IV of the genetic map of Phycomyces, 27 map units away from the gene carA. Complementation and recombination studies involving the genes madD, madE, madF, and madG, in combination with previous genetic studies, show that the recently isolated mad-407 mutation defines a novel behavioural gene, madJ, of Phycomyces. A regulatory role of the madJ gene product in the light sensory transduction pathway is suggested. PMID- 7553935 TI - Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa. AB - Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents. PMID- 7553937 TI - Genetic variability in Gibberella fujikuroi and some related species of the genus Fusarium based on random amplification of polymorphic DNA (RAPD). AB - One of the most important rice pathogens is Fusarium moniliforme (perfect stage: Gibberella fujikuroi), the causal agent of the super-elongation ("bakanae") disease. Thirty-seven strains of this species from different geographical regions were analyzed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). All GA-producing isolates showed nearly identical RAPD patterns using 51 oligonucleotide nona- and deca mers as arbitrary primers. On the other hand, large differences between GA nonproducing isolates were obtained. Comparison of the RAPD patterns with those of the tester strains of the six known mating populations (A, B, C, D, E, F) of G. fujikuroi showed that all producer strains belong to mating population C and all nonproducer isolates to other mating populations. Evidence for the usefulness of the RAPD technique to distinguish between mating populations was provided by sexual crossings. Consensus phylogenetic trees based on RAPDs were constructed by the Phylogenetic Analysis Using Parsimony (PAUP) system. In combination with morphological analysis, RAPD can distinguish between different species of the genus Fusarium. These investigations may find an application in the diagnosis of unknown Fusarium spp. and in distinguishing isolates of G. fujikuroi within the section Liseola. PMID- 7553938 TI - A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori. AB - A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5' end of the pyrG gene with vectors containing a mutation near the 3' end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus. PMID- 7553940 TI - Inheritance of mitochondrial DNA in sexual crosses and protoplast cell fusions in Lentinula edodes. AB - By using mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) as genetic markers, the modes of mitochondrial inheritance in sexual crosses and protoplast cell fusions of the higher basidiomycete Lentinula edodes were examined. All newly established dikaryons from reciprocal crosses between compatible monokaryons carrying different mtDNA RFLP phenotypes retained mtDNA genotypes from one of the monokaryons, suggesting that mitochondrial inheritance is principally uniparental. In contrast, it was shown that recombinant mtDNA genomes arose in some dikaryons obtained after protoplast cell fusion. Based on these results, a possible mechanism for mitochondrial inheritance in L. edodes is discussed. PMID- 7553939 TI - Inheritance of chromosome-length polymorphisms in Ophiostoma ulmi (sensu lato). AB - We have investigated the mitotic and meiotic transmission of chromosome-length polymorphisms in Ophiostoma ulmi s.l., the causal agent of Dutch elm disease. The North-American aggressive (NAN) strain CESS16K has an atypical electrophoretic karyotype, carrying two chromosome-sized DNAs (chDNAs) that have not been observed in other members of the NAN biotype. Independent CESS16K chDNA preparations, even after repeated inoculation and recovery from the elm host, and analysis of 16 progeny strains after a cross between the NAN strains FG245Br-O and CESS16K, demonstrated that these unique chDNAs are integral components of the CESS16K genome. Analysis of the progeny, by electrophoretic karyotyping and hybridizations with probes specific to individual chDNAs, presented evidence that genome rearrangements can occur as a consequence of meiosis. Even though novel electrophoretic karyotypes and a novel-sized chromosome were observed in the karyotypes of the progeny strains, the low level of reassortment between the chromosomes carrying length polymorphisms presented evidence that there are constraints to genome plasticity for this fungus. PMID- 7553941 TI - Analysis of silent RNA editing sites in atp6 transcripts of Sorghum bicolor. AB - We have observed numerous examples of silent or rare non-silent editing sites in the amino-extension and part of the conserved core of mitochondrial atp6 transcripts of Sorghum. In this region of the 1.4-kb atp6-2 mRNA (position 300 to 550) two editing sites, which alter the amino-acid sequence and occur in all cDNAs analysed, were already known, while nine others were found which are silent or occur in a few mRNAs only. Many aspects of RNA editing in the mitochondria of higher plants are still unknown. This includes the influence of genomic background or silent RNA editing. We were interested in the influence of nuclear and mitochondrial backgrounds on RNA editing. Previous preliminary results indicated the possibility of line-specific editing at silent sites. However, a more comprehensive approach gave no consistent evidence for such editing. These results are discussed with respect to their potential impact on the evolution of mitochondrial genes. PMID- 7553942 TI - The gene for ribosomal protein S10 is present in mitochondria of pea and potato but absent from those of Arabidopsis and Oenothera. AB - A novel group II intron has been identified in the pea (Pisum sativum) mitochondrial genome. The gene harbouring this intron is identified as rps10 (encoding protein S10 of the small ribosomal subunit) by similarity to its known homologues in bacteria and in the mitochondrion of the liverwort Marchantia polymorpha. The rps10 gene is transcribed in pea, the intron is removed, and RNA editing in the rps10 reading frame increases similarity to its homologue in the M. polymorpha mitochondrion. Contrary to the situation in bacteria and Marchantia, rps10 is not part of a ribosomal-protein gene cluster in pea. It is flanked upstream by the genes trnF and trnP, encoding phenylalanine- and proline accepting tRNAs, and downstream by cox1, encoding subunit 1 of the cytochrome-c oxidase. Southern hybridization shows that sequences homologous to rps10 exist in potato mitochondria but not in mitochondria of Oenothera berteriana and Arabidopsis thaliana. The pea rps10 intron is homologous to introns in rrn26 and cox3 in the Marchantia mitochondrial genome, while the Marchantia rps10 gene lacks an intron. PMID- 7553943 TI - Splicing and editing of rps10 transcripts in potato mitochondria. AB - The structure and expression of the potato mitochondrial gene rps10, encoding ribosomal protein S10, has been characterized. The RPS10 polypeptide of 129 amino acids is encoded by two exons of 307 bp and 80 bp respectively, which are separated by a 774-bp class-II intron. Editing of the complete rps10 coding region was studied by sequence analysis of spliced cDNAs. Four C residues are edited into U, resulting in the creation of a putative translational initiation codon, a new stop codon which eliminated ten carboxy-terminal residues, and two additional amino-acid alterations. All these changes increase the similarity between the potato and liverwort polypeptides. One additional C-to-U RNA editing event, observed in the intron sequence of unspliced cDNAs, improves the stability of the secondary structure in stem I (i) of domain I and may thus be required for the splicing reaction. All spliced cDNAs, and most unspliced cDNAs, were completely edited, suggesting that editing is an early step of rps10 mRNA processing and precedes splicing. Earlier work on potato rps10 (Zanlungo et al. 1994) is now known to comprise only a partial analysis of the gene, since the short downstream exon was not identified. PMID- 7553944 TI - A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene. AB - We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trn Y-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3' end of trnY and 25 bp of its 3' flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossing-over. PMID- 7553946 TI - [Paroxetine in the treatment of depressive disorders (pilot study)]. AB - In an open study, 42 depressive patients (according to DSM-III-R) were administered paroxetine at mean minimal and maximum doses of 21 and 48 mg once daily in the morning. Treatment resulted in complete remission as defined by Serejsky in 57%, and 55% of patients were rated, according to CGI, as improved. Global HAMD and FKD scores significantly dropped compared to baseline values and responders and non-responders differed significantly as early as seven days of treatment, although the onset of the antidepressive effect was not clinically apparent before 2 weeks of treatment. Significant reductions were seen in all items except paranoidity and weight loss and hypochondria using the FKD scale. A substantial reduction in suicidal ideation and tendencies was also noted in the group of non-responders, a finding supporting a non-specific anti-suicidal effect of paroxetine, which was therapeutically significantly more successful in women than in men. Side effects occurring in 10% and more percent of treated subjects included fatiguability, sweating, tremor, dry mouth, obstipation and nausea. PMID- 7553945 TI - Structural and functional analysis of plastid genomes from parasitic plants: loss of an intron within the genus Cuscuta. AB - The gene cluster rps12/rps7/psi ndhB of the plastome from the holoparasitic plant Cuscuta europaea has been analysed at the nucleotide level. A comparison with the homologous region of the plastome from the closely related parasite Cuscuta reflexa reveals a complete loss of the cis-spliced intron of the rps12 gene in addition to a drastic size reduction of the ndhB pseudogene. It is demonstrated by RT-PCR analysis that the entire gene cluster is transcribed in the form of a multicistronic transcript which also includes the sequences encoded by the ndhB pseudogene. A cDNA containing the correctly transpliced exon 1 of the rps12 transcript can also be amplified. This shows that trans-splicing of the rps12 transcript persists in the plastids of the holoparasite despite the loss of the cis-spliced intron and the loss of many other gene functions. The rps12 and rps7 genes, therefore, still appear to code for functional ribosomal proteins CS12 and CS7, respectively. The conservation of apparently intact ribosomal-protein genes from which correctly processed transcripts are produced is taken as evidence that the translational apparatus of the plastids is still functional and necessary for the expression of the genes remaining in the reduced plastome of a parasitic plant. PMID- 7553947 TI - [Ca(2+)-channel blockers and binding of tricyclic antidepressive agents]. AB - The interactions between Ca(2+)-channel blockers (verapamil and gallopamil) and synaptic plasma membranes (SPM) from bovine brain or human lymphocyte and platelet plasma membranes were studied. Changes in binding parameters of [3H]imipramine, [3H]desmethylimipramine and [3H]gallopamil were determined after addition of unlabelled verapamil or imipramine and after addition of phosphatidylserine (PS) (PS-stimulation). Specific binding of [3H]imipramine to SPM was decreased and [3H]desmethylimipramine binding was increased by 1 microM verapamil. [3H]gallopamil binds specifically to SPM as well as to platelet and lymphocyte membranes. [3H]gallopamil binding to SPM or lymphocyte plasma membranes was PS-stimulated in contrast to platelet plasma membranes without PS effect on binding. Imipramine inhibited both [3H]gallopamil binding and PS stimulated [3H]gallopamil binding to SPM or lymphocyte plasma membranes. Mutual effects of tricyclic antidepressants and Ca(2+)-channel blockers on their binding sites require relatively high drug concentrations. Mechanism of Ca(2+)-channel blockers action in the treatment of depression may be connected rather with changes in signal transduction through serotonin and catecholamine receptor systems than with direct interaction of drugs with binding sites for tricyclic antidepressants. PMID- 7553948 TI - [Enuresis from the viewpoint of the pediatric psychiatrist]. AB - The etiological aspects of enuresis are diverse. Biological factors such as infection, developmental delay and genitourinary tract malformation must be excluded before the child psychiatrist ever undertakes to treat enuresis. In enuresis patients treated by a psychiatrist, an important role is played by the patients' social setting. These children came more often than controls from broken families, unhappy and quarreling parents who tends to give less attention to their children and encourage early separation. Enuresis is also more frequently associated with psychosocial changes (e.g., hospitalization, birth of a sibling, moving to a new home, etc.). The paper examines developmental stages of the child from the point of view of toilet training, circadian rhythms, and sleep stages from the point of view of enuresis incidence. What has a child psychiatrist to offer? Treatment of enuresis should involve a detailed analysis of the lifestyle, psychotherapy and drug therapy, rehabilitation and re educational training. The most effective drugs are reviewed; these include tricyclic antidepressives (with a number of side effects) and Adiuretin as the drug of first choice, therapeutically hard-to-control enuresis, enuresis in adolescence, and the Peter Pan syndrome. PMID- 7553949 TI - [Personality characteristics of men with psychogenic erectile dysfunction]. AB - Based on psychological examination of 47 males with the diagnosis of psychogenic erectile dysfunction, the following personality characteristics were defined by the authors as typical of these patients: 1. a high level of anxiety and neuroticism, 2. reduced masculinity in the partnership, 3. normal heterosexual development and preserved libido associated with markedly substandard sexual function at present. Judging by the results obtained, the authors believe that the confining the diagnostic and therapeutic approach exclusively to the issue of sexual reactivity would be too much of a simplification in these patients, considering their distinct personality and partnership components. PMID- 7553950 TI - [Psychotherapy in psychiatry (and in medicine in general)]. AB - The author subscribes to the view that psychotherapy has an inalienable place in the bio-psycho-social model in psychiatry, and in medicine in general; psychotherapy makes up part of the plurality of therapeutic methods which is adequate to the stratification of being. The plurality of psychotherapeutic methods is, in turn, adequate to the diversity of problems encountered in mental and psychosomatic disease. Emphasis is laid of the psychotherapeutic aspects of psychiatric examination and on the importance of very short interviews in clinical practice. Future developments as regards the integration of psychotherapy into medicine, and professionalism in the field of psychotherapy remain to be open questions. PMID- 7553951 TI - [Neuropsychologic examinations and rehabilitation]. AB - The presented paper is focused on substantial features of neuropsychological examinations. The author emphasizes the application of different tests focused on the relationship between the brain and behaviour, but in particular the neuropsychological battery. He describes the Halstead--Reitan battery and possibilities of its application in children. He emphasizes the importance of neuropsychological rehabilitation and differentiates it from mere recording of the process of spontaneous recovery. He characterizes the main neuropsychological factors which affect recovery after traumatic brain damage. He mentions some of his own examples where he used these factors in clinical practice. The author describes also his own experience with computer rehabilitation using the SERMION- Train the Brain programme. PMID- 7553952 TI - [Incidence of latent tetany in patients with panic disorder]. AB - The symptoms of panic disorder according to the diagnostic criteria DSM-III-R and MKN-10 are, except a few, virtually identical with those of latent tetany, defined in the tetanic questionnaire developed at the Department of Neurology of the University Hospital at Olomouc. The author formed a group of 20 patients treated in an psychiatric outpatient unit for panic disorder. These patients had examinations necessary for diagnosing latent tetany including clinical neurological examination, biochemical investigation with an emphasis on the levels of serum and red blood cell magnesium, and EMG. In a group of 20 patients treated for panic disorder, the author found a concomitant incidence of latent tetany with known etiology--decreased levels of intracellular magnesium--and causal treatment with magnesium salts in 18 (90%). As these nosological entities appear to occur concomitantly, the author recommends, in all patients treated for panic disorder, to examine the iontogram, red blood cell magnesium and EMG and, if the diagnosis of latent tetany is confirmed, to initiate therapy with magnesium salts whose long-term administration leads to remission of problems common to both nosological entities. PMID- 7553953 TI - [Diaphragmatic paralysis]. PMID- 7553954 TI - [The significance of immune island appeared in pleural and peritoneal effusion]. AB - Macrophage surrounded by transformed or untransformed lymphocytes is called immune island. Aimming at this problem, we made a survey of 120 pleural and peritoneal effusion smears with a variety of patients and discovered that this island appeared in 52 (43.3%) cases. The positive results existed in 11 of 14 (79%) cases with tuberculosis and 29 of 60 (48%) cases with malignant diseases. The average number of island on each positive smear (2.0cm x 2.5cm) is 59.6 +/- 12.8 in the former and 39.2 +/- 18.9 in the latter. The mechanism of this phenomenon may be due to the response to foreign stimulus from pathogenic and cancer antigen. PMID- 7553955 TI - [The cytotoxicity of malignant pleural effusion lymphocytes and LAK cells against autologous tumor cells]. AB - In malignant pleural effusion, lymphocytes (MPEL) and autologous tumor cells (ATC) were obtained by centrifugation on discontinuous density gradients from 16 patients with malignant pleural effusion. The cytotoxicity of MPEL against ATC were compared with that of peripheral blood lymphocytes (PBL). It was demonstrated that the results for separation with 100%: 60% Ficoll-Hypaque which formed a discontinuous density gradients were the best in three sets of discontinuous density gradients. The cytotoxicity of PBL was higher than that of MPEL (P < 0.001), but the cytotoxicity and expansion of MPEL-activated by rIL2 was much higher than that of PBL-activated by rIL2 (LAK cells) (P < 0.001). This shows that local immune reaction within pleural cavity of patients with malignant pleural effusion was in the state of suppression, but the state could be improved by using rIL2. Therefore, we consider that MPEL could be the better effector cells than LAK cells in tumor adoptive immunotherapy. PMID- 7553956 TI - [Intrapleural administration of LAK cells combined with rIL2 in the treatment of advanced lung cancer with malignant pleural effusion]. AB - Thirty-three patients with malignant pleural effusions due to advanced lung cancer were treated by intrapleural administration of LAK cells combined with rIL2. The pleural effusions disappeared in 18 patients and significantly decreased in 12. Three patients did not respond to the treatment. No serious side effect was found in 33 patients. The results indicate that transfer of LAK cells combined with rIL2 in the treatment of patients with malignant pleural effusions due to advanced lung cancer is effective and safe. PMID- 7553957 TI - [The changes and clinical significance of interleukin 1 beta in pleural effusion]. AB - An enzyme linked immunosorbent assay was set up by using anti-recombinant human interleukin 1 beta monoclonal antibodies. The interleukin 1 beta, anti interleukin 1 beta autoantibody and interleukin 1 beta containing immune complex were detected in pleural effusion of 28 patients with bacterial infection cases pleurisy and 13 with lung cancer. The results showed that free interleukin 1 beta could be detectable in the early stage of pleurisy, and then transformed to the immune complex. The interleukin 1 beta containing immune complex were detectable in the pleeural effusions of all the 28 patients with pleurisy, but only two patients with lung cancer. The results suggested that this cytokine behaves differently in these two different kinds of diseases. PMID- 7553959 TI - [Adoptive immunotherapy of malignant pleural effusion with TIL/rIL2 (tumor infiltrating lymphocytes/recombinant interleukin 2)]. AB - TIL of 24 patients with malignant pleural effusions were induced and expanded in vitro. The phenotype of lymphocytes IL2R, NK cytotoxicity activity were analysed in fresh and activated TIL. CEA and cytology of malignant pleural effusion were detected before and after treatment with TIL/rIL2. The results indicated: CD4.CD8.IL2R, NK activity increased markedly, CEA level decreased in all of the patients, tumor cells disappeared in some patients. The pleural effusion disappeared in 63%. The total effective rate of the treatment is 79%. No serious toxicity and side effects were found in 1 to 6 months period of following-up after treatment. PMID- 7553958 TI - [Correlated multiple parameters in pleural effusion with pathology of pleurae for differential diagnosis of tuberculous and carcinomatous pleuritis]. AB - The level of ADA, LZM, leukocyte count, lymphocyte in tuberculous effusion is higher than that of carcinomatous effusion (P < 0.05). All results showed that the change of multiple parameters is related to the pathological manifestation of pleurae. The false positive or negative result was influenced by pathological appearance and bloody character of pleural effusion. Using the ADA, LZM cut off value at 35U and 30mg, respectively, and pleural/serum ADA ratio > 1.4, LZM > 1.1, a sensitivity and specificity of 100 percent were achieved. PMID- 7553960 TI - [Measurement of gamma-interferon and its production capability in pleural effusion]. AB - IFN gamma levels and IFN gamma production capability were studied. The results showed that IFN gamma levels, IFN gamma production in the tuberculous pleurisy (n = 30) were significantly higher than those in the malignant pleural effusion (n = 23) and the pleural transudate. However, there was no detectable IFN gamma in the blood, IFN gamma production were lower in the blood than in the pleural effusion. The finding suggests that IFN gamma may play an important role in local anti mycobacterial infection. PMID- 7553961 TI - [Therapeutical mechanism of 3,4 dihydroxyacetophenone in treating chronic obstructive pulmonary disease]. AB - Hemodynamic parameters were measured by ballon tipped catheter in 11 patients with COPD and plasma levels of endothelin1 (ET1), cAMP and cGMP were detected by radioimmunoassay. The results indicated that 3,4-dihydroxyacetophenone (DHAP) gives with an i.v. dose of 640mg could reduce (1) 15% of mean pulmonary arterial pressure, 39% of pulmonary vascular resistance and 21% of systemic vascular resistance; (2) but not effect on systemic arterial pressure and blood gas analysis; (3) decrease the plasma ET1 as well as cGMP level (P < 0.01, P < 0.05 respectively). The authors think that the decrease of pulmonary arterial pressure in COPD patients may be related to the lowering of plasma ET1 level and the change of the cAMP to cGMP ratio. PMID- 7553962 TI - Alangionosides G-M: glycosides of megastigmane derivatives from the leaves of Alangium premnifolium. AB - Phytochemical investigation of the 1-BuOH-soluble fraction of the MeOH extract of Alangium premnifolium resulted in the isolation of ten megastigmane glycosides, of which, two, (6R,9R)-3-oxo-alpha-ionol apiofuranosylglucopyranoside and roseoside with (6S,9R)-blumeol A as an aglycone were known. The structures of the eight new compounds were determined by spectroscopic methods. PMID- 7553963 TI - Studies on the Chinese crude drug "shoma." IX. Three novel cyclolanostanol xylosides, cimicifugosides H-1, H-2 and H-5, from cimicifuga rhizome. AB - Three new cyclolanostanol xylosides were isolated from a batch of commercial Cimicifuga Rhizome, cimicifugoside H-1 (1), C35H52O9, mp 260-262 degrees C, [alpha]D -43.5 degrees, cimicifugoside H-2 (2), C35H54O10, mp 227-229 degrees C, [alpha]D -38.8 degrees, and cimicifugoside H-5 (3), C35H52O10, mp 262-264 degrees C, [alpha]D -22.9 degrees, together with known glycosides, actein and 27 deoxyactein. Their structures were determined on the basis of chemical and spectrometric evidence including an X-ray crystallographic analysis. The structure of cimicifugoside H-1 (1) was established as (20R,24R)-24,25-epoxy-11 beta-hydroxy-3-beta-(beta-D- xylopyranosyloxy)-9,19-cyclolanost-7-ene-16,23 dione. Cimicifugoside H-5 (3) is the 15-hydroxylated derivative of 1. Since 1 changed into cimicifugoside H-2 (2) on treatment with p-toluenesulfonic acid, 2 has a 24R,25-diol structure derived from 1 by opening its epoxy ring. PMID- 7553964 TI - Synthesis of saframycins. X. Transformation of (-)-saframycin A to (-)-saframycin Mx type compound with the structure proposed for saframycin E. AB - Treatment of (-)-saframycin A (1a) with selenium oxide in acetic acid afforded ( )-saframycin G (1g), and a catalytic reduction and regioselective oxidation sequence afforded the saframycin Mx type compound (3). We applied this methodology to the transformation of (+/-)-5-hydroxysaframycin B (11) to the hydroquinone (1e). Acetylation of 1e with acetic anhydride in pyridine gave the triacetate (13), which is identical with the triacetyl derivative of natural saframycin E. PMID- 7553965 TI - Synthesis and pharmacological evaluation of 1,2,3,4-tetrahydro-beta-carboline derivatives. AB - A series of 1,2,3,4-tetrahydro-beta-carbolines has been synthesized and evaluated for cerebral protecting effects against lipid peroxidation and potassium cyanide intoxication in mice. Most of the compounds synthesized had potent effects against lipid peroxidation. Among them, 1-(3,5-dimethoxyphenyl)-2-propyl-1,2,3,4 tetrahydro-beta-carboline (22) was found to have a combination of potent effects against both lipid peroxidation and potassium cyanide intoxication. Structure activity relationships are discussed. PMID- 7553966 TI - Novel 2-amino-1,4-dihydropyridine calcium antagonists. I. Synthesis and antihypertensive effects of 2-amino-1,4-dihydropyridine derivatives having nitroxy-alkoxycarbonyl groups at 3- and/or 5-position. AB - Novel 2-amino-1,4-dihydropyridine derivatives, which contain nitroxy alkoxycarbonyl groups at the 3- and/or 5-position, were synthesized and their pharmaceutical effect was evaluated in spontaneously hypertensive rats. The structure-activity relationships are discussed in terms of potency, onset rapidity, and duration of antihypertensive activity. Remarkably prolonged duration of antihypertensive action was observed when a tertiary amino group was introduced on either side of an ester chain. PMID- 7553967 TI - Novel 2-amino-1,4-dihydropyridine calcium antagonists. II. Synthesis and antihypertensive effects of 2-amino-1,4-dihydropyridine derivatives having N,N dialkylaminoalkoxycarbonyl groups at 3- and/or 5-position. AB - Novel 2-amino-1,4-dihydropyridine derivatives I, which contain N,N, dialkylaminoalkoxycarbonyl groups at the 3- and/or 5-position, were synthesized and their antihypertensive effects were evaluated in spontaneously hypertensive rats. Remarkably prolonged duration of antihypertensive action was observed when a tertiary amino group was introduced into either the 3- and/or 5-ester side chain of the 1,4-dihydropyridine ring. In particular, the compounds containing cyclic amino moieties at the 3-position showed greater potency than those with acyclic amino moieties. Chemical modification studies indicated that the two ester side-chains of 1,4-dihydropyridine at the 3- and 5-position might function in a different manner in relation to the antihypertensive activities. 3-(1 Benzhydrylazetidinited potent and long-lasting antihypertensive effects with gradual onset of action, and is a promising candidate as an antihypertensive drug. PMID- 7553969 TI - Novel 6-5 fused ring heterocycle antifolates with potent antitumor activity: bridge modifications and heterocyclic benzoyl isosters of 2,4-diamino-6,7-dihydro 5H-cyclopenta[d]pyrimidine antifolate. AB - Structural modifications of an extremely potent inhibitor of dihydrofolate reductase (DHFR) activity and tumor cell growth, N-[4-[3-(2,4-diamino-6,7-dihydro 5H-cyclopenta[d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (1), have led to the synthesis of new cyclopenta[d]pyrimidine-based antifolates, including those with low alkyl substituted trimethylene bridges (2a, b) and isosterically modified bridges (ethyleneoxa, 2c; ethyleneamino, 2d; the N-methyl- and N-ethyl derivatives of 2d, 2e, f) and those in which the benzene ring of 1 has been replaced by heterocyclic isosters (indole, 2g; indoline, 2h; thiophene, 2i). These new analogs are highly potent as DHFR and cell growth inhibitors, and most of them are more potent than methotrexate (MTX) and 10-ethyl-10-deazapterin (10 EDAM) in inhibiting tumor cell growth (P388 MTX-sensitive and MTX-resistant, colon 26 and KB) on 72 h drug exposure. Among them, 2a (the 10-methyl derivative of 1) and 2i were most potent, being 2- to 3-fold more potent than 10-EDAM. On 4 h drug exposure, the growth-inhibitory activity of these analogs was radically influenced by even minor structural changes. Compounds 1, 2a--e, g--i were much more cytotoxic in colon 26 cell line than were MTX and 10-EDAM, with 2d and 2i being most potent, followed by 2a. Structure-activity relationships and their possible significance are discussed. PMID- 7553968 TI - N-alkylated 1,4-dihydropyridines: new agents to overcome multidrug resistance. AB - New N-alkylated 1,4-dihydropyridine derivatives were synthesized and their ability to overcome multidrug resistance was examined in vincristine-resistant P388 cells (P388/VCR cells). Compounds that possessed an arylalkyl substituent on the dihydropyridine ring nitrogen were more potent than verapamil in potentiating the cytotoxicity of vincristine against P388/VCR cells. However, neither drug effectively enhanced the antitumor activity of vincristine in tumor-bearing mice. Introduction of basic nitrogen-containing substituents on the side chain of 1,4 dihydropyridines gave improved activity in vitro and in vivo. The piperazine derivative 12c and 12o were more than 10 times as potent as verapamil in vitro. Four compounds selected for in vivo testing showed superior antitumor activity in P388/VCR-bearing mice in combination with vincristine. The structure-activity relationships of the compounds are discussed. PMID- 7553971 TI - Synthesis of protegrin-related peptides and their antibacterial and anti-human immunodeficiency virus activity. AB - All disulfide analogs (types I, II and III) of protegrin (PG)-1, an 18-residue antimicrobial peptide having two intramolecular disulfide bonds, were synthesized using regioselective disulfide bond formation. Random air-oxidation of the fully reduced PG-1 formed the type III PG-1. In addition, a type III analog containing an amidated carboxy-terminal residue was also prepared. Each analog showed significant and different antibacterial and anti-human immunodeficiency virus (HIV) activity. Deletion of two disulfide bridges caused a significant decrease in activity. PMID- 7553970 TI - Synthesis and structure-activity studies of a series of 1-oxa-8 azaspiro[4.5]decanes as M1 muscarinic agonists. AB - 2,8-Dimethyl-1-oxa-8-azaspiro[4,5]decan-3-one (17), designed by incorporating the tetrahydrofuran ring moiety of muscarone into an 8-azaspiro[4,5]decane skeleton, and related 1-oxa-8-azaspiro[4.5]decanes were synthesized and assessed as M1 muscarinic agonists for the symptomatic treatment of dementia of Alzheimer's type. The compounds were tested for central muscarinic M1 and M2 receptor affinity and in vivo muscarinic activities: namely, amelioration of scopolamine induced impairment in rat passive avoidance tasks, and induction of hypothermia, tremor, and salivary secretion. Compound 17 exhibited potent muscarinic activities in vitro and in vivo with no selectivity. Systematic modifications of 17 were conducted, and a number of compounds, including the 2-ethyl analogue (18), 3-methylene analogue (29), 3-dithioketal analogues (26, 28), and 3-oxime analogue (37) were found to display preferential affinity for M1 receptors over M2 receptors and, in addition, to exhibit potent antiamnesic activity sufficiently separated from hypothermia-inducing activity, taken as an index of cholinergic side effects, compared with the reference compound RS86 (1). Structure-activity relationships are discussed in comparison with those for muscarone analogues. Of these compounds only two, 2-ethyl-8-methyl-1-oxa-8 azaspiro[4.5]decan-3-one (18) and 2,8-dimethyl-3-methylene-1-oxa-8 azaspiro[4.5]decane (29), stimulated phosphoinositide hydrolysis in rat hippocampal slices, indicating partial agonistic activity for M1 muscarinic receptors. The optical resolution of 18 and 29 was performed. Eudismic ratios of both compounds in binding affinity were low, but M1 agonist activity resided preferentially in the (-)-isomers. The absolute configuration of (-)-29 was determined by X-ray crystal structure analysis to be S, being the same as that of muscarone. Based on the in vivo selectivity, (-)-29 was selected for clinical studies. PMID- 7553972 TI - The antagonistic effects of khellactones on platelet-activating factor, histamine, and leukotriene D4. AB - Khellactones of Peucedanum praeruptorum DUUN., including praeruptorins A (= Pd Ia, 2) and B (= Pd-II,11), had an antagonistic effect specifically on platelet aggregation induced by platelet activating factor (PAF) among various aggregating agents examined, and represent a new class of PAF antagonists. We examined the effects of twenty compounds on PAF-induced platelet aggregation and on histamine- and leukotriene D4 (LTD4)-induced contractions in isolated guinea pig ileum. Compounds 2, (+/-)-cis-3',4'-diacetylkhellactone (3), (+/-)-cis-4'-acetyl-3' crotonoylkhellactone (5), (+/-)-cis-4'-acetyl-3'-tetrolylkhellactone (6), (+/-) cis-4'-acetyl-3'-tigloylkhellactone (7), (+/-)-cis-4'-acetyl-3'-(2" methylbutyryl)khellactone (8), (+/-)-cis-3',4'-ditigloylkhellactone (10), and 11 all strongly inhibited PAF-induced platelet aggregation. (+/-)-cis-4'-Acetyl-3' (2"-methyl-2"-dodecenoyl)khellactone (9), (+/-)-cis-4'-ethyl-3' tigloylkhellactone (13), (+/-)-cis-4'-ethyl-3'-[N-(2" triethylammonio)ethylcarbamoyl] khellactone iodide (16), (+/-)-trans-3',4' diacetylkhellactone (18), (+/-)-trans-4'-acetyl-3'-crotonoylkhellactone (19), (+/ )-trans-4'-acetyl-3'-valerylkhellactone (20), (+/-)-trans-4'-acetyl-3' isovalerylkhellactone (21), and (+/-)-trans-4'-acetyl-3'-tigloylkhellactone (22) were weakly inhibitory. Most of the compounds exhibited noncompetitive antagonist actions on histamine- and LTD4-induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553973 TI - New oral dosage form for elderly patients. II. Release behavior of benfotiamine from silk fibroin gel. AB - Silk fibroin gel (SFG) containing benfotiamine (BTMP) was prepared. The release behavior of BTMP from SFG was studied as a function of silk fibroin (SF) content and glycerol content, and the influence of the existence of beta-cyclodextrin (beta-CD) on the physicochemical properties of SFG were investigated. The release rate of BTMP from SFG was retarded by an increase in SF concentration. The addition of beta-CD affected both the release properties and rheological properties of the SFG. It was found from the results of the "paddle-bead method" that the release profiles of BTMP from SFG were inversely proportional to the SFG firmness. PMID- 7553974 TI - Novel antioxidants from roasted perilla seed. AB - Novel antioxidants, 5-(3,4-dihydroxyphenylmethyl)oxazolidine-2,4-dione (1) and 3 (3,4-dihydroxyphenyl)lactamide (2), have been isolated from roasted perilla seed. Compound 1 was the first example of non-synthetic oxazolidinedione. PMID- 7553975 TI - A novel hexahydrodibenzofuran derivative with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells from Lindera umbellata bark. AB - A novel cinnamoyl-hexahydrodibenzofuran derivative (1) was isolated from the bark of Lindera umbellata. The structure was determined by extensive spectroscopic analysis to be (5aR*,6R*,9R*,9aS*)-4-cinnamoyl-3,6-dihydroxy-1-methoxy-6-me thyl- 9-(1-methylethyl)-5a,6,7,8,9,9a-hexahydrodibenzofuran. Compound 1 showed potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells without causing any cytotoxicity in the cultured cells or skin irritation in guinea pig. PMID- 7553976 TI - Achyranthosides C and D, novel glucuronide saponins from Achyranthes fauriei root. AB - Two novel saponins, achyranthosides C and D, were isolated from Achyranthes faurei root, and their structures were characterized based on the chemical and spectroscopic evidence. PMID- 7553977 TI - Application of a unique automated synthesis system for solution-phase peptide synthesis. AB - An automated synthesis system, which is suitable for repetitive syntheses using similar reaction procedures, was used to synthesize systematically a library of all possible dipeptides (25) and tripeptides (125) from 5 protected amino acids. The apparatus has also been applied to the automated synthesis of 10 fragment tripeptide derivatives that are constituents of the hormone PACAP-27. The measured molecular optical rotation values of the library of 125 tripeptides were found to correlate well with calculated values obtained by summation of the molecular optical rotation values for the constituent amino acids. PMID- 7553979 TI - New 5-HT3 (serotonin-3) receptor antagonists. III. An efficient synthesis of carbon 14-labeled (+)-8,9-dihydro-10-methyl-7-[(5-methyl-1H-imidazol-4- yl)methyl]pyrido[1,2-a]indol-6(7H)-one hydrochloride (FK 1052). AB - (+)-8,9-Dihydro-10-methyl-7-[(5-methyl-1H-imidazol-4- yl)methyl]pyrido[1,2 a]indol-6(7H)-one hydrochloride (FK 1052, 1) is a highly potent 5-HT3 (serotonin 3) receptor antagonist. For the study of the metabolism and disposition of FK 1052 (1), we synthesized carbon 14-labeled FK 1052 in three steps from 10 demethyl FK 1052 (8). The Mannich reaction and subsequent hydrogenolysis of the dimethylaminomethyl group enabled the efficient introduction of one carbon atom at the 10-position of the pyrido[1,2-a]indol-6,(7H)-one ring. The Mannich reaction of (+)-8,9-dihydro-7-[(5-methyl-1H-imidazol-4-yl)methyl]pyrido[1,2- ]indol-6(7H)-one (8) with [14C]paraformaldehyde and dimethylamine hydrochloride gave the [14C]-10-dimethylaminomethyl compound (20). Subsequent hydrogenolysis of 20 with palladium on carbon and ammonium formate, followed by recrystallization of the salt with (+)-di-p-toluoyl-D-tartaric acid, gave [14C]FK 1052 with a radiochemical purity of 99.4% and an enantiomeric excess of more than 97%. PMID- 7553978 TI - Amino acids and peptides. XL. Synthesis of Ac-Tyr-Val-Ala-Asp-MCA using newly developed acetylating reagent. AB - 2-Acetoxy-3-benzyl-5-methyl-6-isobutylpyrazine was prepared by cyclization of H Phe-Leu-CH2Cl, followed by acetylation with acetic anhydride. This pyrazine derivative can react with amino groups of amino acids or peptides to produce acetyl amino acids or acetyl peptides without acetylation of hydroxy group of Tyr, Ser and Thr. Using this acetylating reagent, Ac-Tyr-Val-Ala-Asp-MCA, which is a specific substrate of the interleukin-I (IL-I) processing enzyme, was prepared. PMID- 7553980 TI - New 5-HT3 (serotonin-3) receptor antagonists. IV. Synthesis and structure activity relationships of azabicycloalkaneacetamide derivatives. AB - The synthesis and structure-activity relationships of a series of new azabicycloalkanes as 5-HT3 (serotonin-3) receptor antagonists are described. Our study on the azabicycloalkaneacetamide derivatives showed that 2,3-dihydroindole as the aromatic ring moiety afforded potent 5-HT3 receptor antagonist activity, as judged by blockade of bradycardia induced by i.v. injection of 2 methylserotonin in anesthetized rats. 7-Azaindole as the aromatic moiety afforded weak 5-HT3 receptor antagonists activity. The best 5-HT3 antagonists in this study were endo-3,3-diethyl- (9k) and 3,3-dimethyl-2,3-dihydro-1-[(8-methyl-8 azabicyclo[3.2.1]oct-3-yl) acetyl-1H-indole (9d), being approximately 10-fold more potent than ondansetron (1). This study shows that the azabicycloalkaneacetyl group is a new pharmacophoric element as a basic nitrogen and a linking carbonyl moiety. PMID- 7553981 TI - New 5-HT3 (serotonin-3) receptor antagonists. V. Synthesis and structure-activity relationships of pyrrolo[2,1-c][1,4]benzoxazine-6-carboxamides. AB - This paper describes the discovery of structurally novel heterocyclic carboxamides which are highly potent 5-HT3 (serotonin-3) receptor antagonists. Pyrrolo[2,1-c][1,4]benzoxazine-6-carboxamides (12 and 20) were found to possess potent 5-HT3 receptor antagonist activity on the von Bezold-Jarisch reflex in anesthetized rats. Structure-activity studies showed that compounds with small and lipophilic substituents such as chloro and methyl at the 8-position of the aromatic ring portion retained high potency, whereas those with bulky substituents showed essentially no activity. A dimethyl group at the 4-position slightly decreased the potency. 1-Azabicyclo[2.2.2]octan-3-amine as the amine part afforded the most potent activity. From this series, 20a was found to be the most potent 5-HT3 receptor antagonist, being 40-fold more potent than ondansetron (1). PMID- 7553983 TI - Search for naturally occurring substances to prevent the complications of diabetes. II. Inhibitory effect of coumarin and flavonoid derivatives on bovine lens aldose reductase and rabbit platelet aggregation. AB - An EtOAc extract of Artemisiae Capillari Spica inhibited both bovine lens aldose reductase (bovine-LAR) and rabbit platelet aggregation. Two simple coumarins, scoparone (1) and scopoletin (2), and three flavonoids, capillarisin (21), cirsimaritin (22) and rhamnocitrin (23), were isolated from this extract. Scoparone (1) and scopoletin (2) exhibit a potent inhibitory effect on rabbit platelet aggregation induced by four types of agent, ADP, PAF, sodium arachidonate and/or collagen. Capillarisin (21) exhibits a potent inhibitory effect on bovine-LAR. In addition, thirteen simple coumarins, five coumarin glycosides and two flavonoids were tested for their inhibitory effect against bovine-LAR and rabbit platelet aggregation. PMID- 7553982 TI - Development of potent serotonin-3 (5-HT3) receptor antagonists. I. Structure activity relationships of 2-alkoxy-4-amino-5-chlorobenzamide derivatives. AB - A new series of 2-alkoxy-4-amino-5-chlorobenzamide derivatives bearing five- to seven-membered heteroalicyclic rings in the amine moiety was synthesized and evaluated for serotonin-3 (5-HT3) receptor antagonistic activity by assaying the ability to antagonize the von Bezold-Jarisch reflex in rats. The five- to seven membered heteroalicycles comprise pyrrolidine, morpholine, 1,4-thiazine, piperidine, piperazine, 1,4-oxazepine, 1,4-thiazepine, azepine, and 1,4-diazepine rings. Among them, some benzamide derivatives having a 1,4-diazepine ring showed a potent 5-HT3 receptor antagonistic activity. In particular, 4-amino-5-chloro-N (1,4-dimethylhexahydro-1H-1,4-diazepin-6- yl)-2- ethoxybenzamide (96) and the 1 benzyl-4-methylhexahydro-1H-1,4-diazepine analogue 103 showed potent 5-HT3 receptor antagonistic activity without 5-HT4 receptor binding affinity. PMID- 7553984 TI - Screening of tissue cultures and thalli of lichens and some of their active constituents for inhibition of tumor promoter-induced Epstein-Barr virus activation. AB - Inhibition of tumor promoter-induced Epstein-Barr virus (EBV) activation was screened using tissue culture and thallus extracts of lichens. Usnea longissima ACH. thallus and Cetraria ornata MULL. ARG. tissue culture showed strong inhibitory activity. We identified (+)-usnic acid (1), barbatic acid (2), diffractaic acid (3), 4-O-demethylbarbatic acid (4), and evernic acid (5) as inhibitors of EBV activation from the U. longissima thallus. Of these compounds, (+)-usnic acid exhibited the highest inhibitory activity (IC50 = 1.0 microM). PMID- 7553985 TI - Naturally occurring 5-lipoxygenase inhibitors. VI. Structures of ardisiaquinones D, E, and F from Ardisia sieboldii. AB - New 1,4-benzoquinone derivatives, ardisiaquinones D(2), E(4), and F(5) along with the known ardisiaquinones A(1) and B(3) have been isolated from the leaves of Ardisia sieboldii (Myrsinaceae) and shown to be 5-lipoxygenase inhibitors. Their structures have been elucidated by spectroscopic analysis and chemical degradation. The degree of inhibition of 5-lipoxygenase activity by the ardisiaquinones and some derivatives of ardisiaquinone A is reported. PMID- 7553987 TI - Studies on dissolution tests for soft gelatin capsules by the rotating dialysis cell (RDC) method. VI. Preparation and evaluation of ibuprofen soft gelatin capsule. AB - We prepared soft gelatin capsules (SC) containing ibuprofen (IB), a widely used phenylpropionic acid-derived antiphlogistic-analgesic drug. To evaluate the SC, in vitro dissolution tests were performed both by the paddle (PD) method described in the Japanese Pharmacopoeia (JPXII) and by the rotating dialysis cell (RDC) method which we previously developed and evaluated for application. In vivo, the blood IB concentration was determined after administration to beagles. Higher bioavailability was observed after administration of the SC containing IB than after administration of the bulk IB powder. A higher correlation was observed between the in vitro dissolution behavior and in vivo results by the RDC method than by the PD method, suggesting the usefulness of the RDC method in the dissolution test of SC. PMID- 7553986 TI - Solution forms of antitumor cyclic pentapeptides with 3,4-dichlorinated proline residues, astins A and C, from Aster tataricus. AB - Conformational analysis of antitumor cyclic pentapeptides, astins A (1) and C (3), was made by a combination of NMR and computational techniques. These results indicated that the backbone conformations of 1 and 3, with lower activity than astin B (2), were different from that of 2. The backbone conformation together with a cis 3,4-dichlorinated proline residue was considered to play an important role in the antitumor activities of astins. PMID- 7553988 TI - Pharmaceutical characterization of commercially available intravenous fat emulsions: estimation of average particle size, size distribution and surface potential using photon correlation spectroscopy. AB - Particle profiles such as the average diameter, size distribution and dispersion, as well as the zeta potential, of commercially available intravenous fat emulsions of high-calorie nutrient fluids (6 products) and drug carriers (4 products) were examined using photon correlation spectroscopy (dynamic light scattering). Wide variations were observed in number-weighted (dn), weight weighted (dw) and z-average diameters, and dw/dn ratios as a measure of polydispersity. Average size is not sufficient for the pharmaceutical characterization of particles and the determination of size distribution or dw/dn value is essential for more precise information. Although measuring the zeta potential of fat emulsions is of considerable value in estimating their stability on long-term storage, a medium which accurately reflects the environment of the droplets in the system of interest should be chosen when diluting the emulsion. PMID- 7553989 TI - Synthesis of 11Z-8,18-propano- and methano-retinals and their interaction with bovine opsin. PMID- 7553990 TI - Heated humidification: a simple question but... PMID- 7553991 TI - The choice of anaesthetic for carotid endarterectomy: does it matter? PMID- 7553992 TI - Body heat transfer during hip surgery using active core warming. AB - The purpose of this study was to evaluate the effect of core warming on heat redistribution from the core to the periphery as manifested by changes in core, mean skin temperature and mean body heat, investigated in a group of 30 patients undergoing total hip replacement. The control group (n = 10) had no active warming. Core warming was achieved in the humidifier group (n = 10) by using humidified and warmed gases at 40 degrees C, whilst in the oesophageal group (n = 10), an oesophageal heat exchanger was used to achieve active warming. Operating room temperature and relative humidity was standardised. Aural canal and skin temperatures (15 sites) were measured before induction of anaesthesia, at the end of surgery and one hour of recovery after anaesthesia. Mean skin temperatures were calculated for a weighted four and unweighted 15 points, and mean body heat were calculated to quantify the distribution of body heat. Core temperature decreased in the control and the oesophageal groups, but not in the humidifier group at the end of surgery; by mean values +/- SD of 1.9 degrees C +/- 0.6, 1.2 degrees C +/- 0.6 and 0.4 degree C +/- 0.2 degree C, respectively (P < 0.01). Mean skin temperature (MST15) decreased in the control group by 1.0 degree C +/- 1.0, but not in the actively warmed groups where the mean increased by 0.1 degree C +/- 1.4 and 0.2 degree C +/- 0.2 in the oesophageal and humidifier groups, respectively (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553993 TI - Haemodynamic instability and myocardial ischaemia during carotid endarterectomy: a comparison of propofol and isoflurane. AB - The purpose of this study was to compare two anaesthetic protocols for haemodynamic instability (heart rate (HR) or mean arterial pressure (MAP) < 80 or > 120% of ward baseline values) measured at one-minute intervals during carotid endarterectomy (CEA). One group received propofol/alfentanil (Group Prop; n = 14) and the other isoflurane/alfentanil (Group Iso; n = 13). Periods of haemodynamic instability were correlated to episodes of myocardial ischaemia as assessed by Holter monitoring (begun the evening before surgery and ceasing the morning of the first postoperative day). In Group Prop, anaesthesia was induced with alfentanil 30 micrograms.kg-1 i.v., propofol up to 1.5 mg.kg-1 and vecuronium 0.15 mg.kg-1, and maintained with infusions of propofol at 3-12 mg.kg-1.hr-1 and alfentanil at 30 micrograms.kg-1.hr-1. In Group Iso, anaesthesia was induced with alfentanil and vecuronium as above, thiopentone up to 4 mg.kg-1 and maintained with isoflurane and alfentanil infusion. Phenylephrine was infused to support MAP at 110 +/- 10% of ward values during cross-clamp of the internal carotid artery (ICA) in both groups. Emergence hypertension and/or tachycardia was treated with labetalol, diazoxide or propranolol. Myocardial ischaemia was defined as ST segment depression of > or = 1 mm (60 msec past the J-point) persisting for > or = one minute. For the entire anaesthetic course (induction to post-emergence), there was no difference between groups for either duration or magnitude outside the < 80 or > 120% range for HR or MAP. However, when the period of emergence from anaesthesia (reversal of neuromuscular blockade to post-extubation) was assessed, more patients were hypertensive (P = 0.004) and required vasodilator therapy in Group Iso (10/13 vs 5/14; P = 0.038 Fisher's Exact Test). The mean dose of labetalol was greater in Group Iso (P = 0.035). No patient demonstrated myocardial ischaemia during ICA cross-clamp. On emergence, 6/13 patients in Group Iso demonstrated myocardial ischaemia compared with 1/14 in Group Prop (P = 0.029). Therefore, supporting the blood pressure with phenylephrine, during the period of ICA cross-clamping, appears to be safe as we did not observe any myocardial ischaemia at this time. During emergence from anaesthesia, haemodynamic instability was associated with myocardial ischaemia. Under these specific experimental conditions, with emergence, hypertension and myocardial ischaemia were more prevalent with more frequent pharmacological interventions in patients receiving isoflurane. PMID- 7553994 TI - Ventilatory depression by halothane in infants and children. AB - The purpose of this study was to extend previous observations of a greater decrease in tidal volume in infants than in children during halothane anaesthesia. We analyzed the inspiratory flow waveform recorded during spontaneous ventilation in: infants, two to six months of age, and children, one to five years of age. In addition we analyzed the CO2 signal and the pressure waveform during an occluded inspiration. The pressure generated during the initial 100 msec of inspiratory occlusion, an index of respiratory drive, was analyzed to give some insight into the aetiology of the age-related differences. In 15 infants and 15 children, Flow (V), pressure (Pao) and PCO2 were recorded at three concentrations of inspired halothane (FIH): 0%, 1% and 2% which correspond to an endtidal halothane concentration of about 0.3%, 0.9% and 1.3% respectively. Data were analyzed for minute ventilation (Vi) and parameters of timing (Total time (Ttot), Inspiratory time (Ti)), the amplitude of the neural output (mean inspiratory flow (VT/Ti), tidal volume (VT)) and the shape of the inspiratory breath profile (the inspiratory centroid flow (Ci/Ti), the inspiratory duty cycle (Ti/Ttot)). In some, the airway was occluded at end expiration and the slope of the initial 100 msec of occlusion (dP/dt) together with the maximal negative pressure (PMAX) were measured. Estimates of respiratory mechanics E'rs (PMAX/VT) and (VT/Ti)/(dP/dt) were obtained. The VT and Ttot decreased with increasing FIH in both infants and children (P < 0.05). The PETCO2 increased in both groups and the % increase was greater in infants.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7553995 TI - Mivacurium infusion requirements following vecuronium: different response between adults and children. AB - The mivacurium infusion requirements following vecuronium were evaluated in 15 adults and 15 children in an open prospective clinical study. This study was undertaken to elucidate whether potentiation of effect occurred when a mivacurium infusion was administered after vecuronium was used for the facilitation of tracheal intubation. The adult patients were anaesthetized with N2O:02, propofol and fentanyl, the children with halothane (1%) N2O:O2. Vecuronium 100 micrograms.kg-1 was administered during stimulation of the ulnar nerve with train of-four stimuli at 0.1 Hz. The force of contraction of adductor pollicis was recorded. Upon recovery of the twitch response from vecuronium, a mivacurium infusion was started at 4 micrograms.kg-1.min-1, thereafter adjustments were made to maintain the first twitch of the train-of-four (T1) at 1-10% of control. The mean (+/- SE) initial infusion requirements in children of mivacurium was 4.3 (0.4) micrograms.kg-1.min-1 which increased linearly (P < 0.001) over the next 90 min to 10 micrograms.kg-1.min-1. In adults the infusion requirement was lower than in children and remained at approximately 3 micrograms.kg-1.min-1 over the next 75 min. At the end of the surgical procedure, the children recovered faster than the adults with no child requiring reversal. Because of prolonged recovery ( > 20 min), seven adults required reversal with 15-70 micrograms.kg-1 neostigmine. Mivacurium infusion requirements following vecuronium are higher in children than adults. Potentiation of the effects of mivacurium were seen when vecuronium preceded mivacurium. This potentiation of effect lasted longer in adults than in children. PMID- 7553997 TI - Detrimental effects of noise on anaesthetists. AB - To study the detrimental effects of operating room noise, noise levels in operating rooms were first measured and the average noise level was calculated in Decibels, which was 77.32 dB(A). An audiocassette of 90 min duration was prepared recording the operating room noise. The same audiocassette was used later to expose the 20 anaesthesia residents to the operating room noise in the acoustically treated rooms of audiology department. The noise level during exposure was maintained at 77.32 dB(A). Two cognitive functions, mental efficiency and short-term memory were studied. The tests used were the Trail Making Test and Digit Symbol Test for mental efficiency and the Benton Visual Retention Test for short-term memory. The mean pre-exposure scores for the Trail Making Test, Digit Symbol Test and Benton Visual Retention Test were 22.9 +/- 1.94, 83 +/- 2.62 and 9.55 +/- 0.51 respectively. The mean during-exposure scores were 16.35 +/- 1.39, 74.05 +/- 3.46 and 5.8 +/- 0.41 respectively (P < 0.05). In conclusion, we observed that operating room noise reduced the mental efficiency and short-term memory of anaesthesia residents. PMID- 7553998 TI - Substitution of mivacurium for succinylcholine for ECT in elderly patients. AB - Three cases are reported where pre-existing medical conditions (severe osteoporosis, amyotrophic lateral sclerosis, cardiac arrhythmias) made the administration of succinylcholine during ECT potentially dangerous. Therefore, mivacurium was substituted as the muscle relaxant necessary for safe therapy. Full reversal of the non-depolarizing muscle relaxant was assured by post reversal use of the peripheral nerve stimulator with full recovery of train-of four response. PMID- 7553999 TI - Clindamycin-induced neuromuscular blockade. AB - The purpose of this article is to report the case of a patient who developed prolonged neuromuscular block after a large dose of clindamycin (2400 mg). A 58 yr-old, 65 kg woman with severe rheumatoid arthritis was admitted for wrist arthrodesis. After d-tubocurarine (3 mg) and fentanyl (1.5 micrograms.kg-1), anaesthesia was induced with thiopentone (4 mg.kg-1) followed by succinylcholine (1.5 mg.kg-1) and was maintained with N2O in O2 and isoflurane (0.75-1.0% end tidal) and ventilation was controlled. No further neuromuscular relaxants were given although full return of neuromuscular activity in response to train-of-four and 100 Hz tetanic stimulation was observed after succinylcholine. An overdose of clindamycin (2400 mg, instead of the intended 600 mg) was given i.v. soon after the start of surgery. At the end of surgery, 75 min later, the patient made no attempt at spontaneous ventilation, was unresponsive to painful stimuli and naloxone (0.2 mg i.v.) was ineffective. Controlled ventilation was continued in the Recovery Room where neuromuscular testing showed a train-of-four ratio of 0.27 which improved to only 0.47 five minutes after calcium chloride (1.5 mg.kg-1 i.v.), and to 0.62 after edrophonium (20 mg) and neostigmine (2 mg). Nine hours later the patient began to cough, the TOF had returned to 1.0 and two hours later the trachea was extubated and spontaneous ventilation was resumed. Large doses of clindamycin can induce profound, long-lasting neuromuscular blockade in the absence of non-depolarizing relaxants and after full recovery from succinylcholine has been demonstrated. PMID- 7553996 TI - Comparison of haemodynamic changes induced by sevoflurane and halothane in paediatric patients. AB - The purpose of this study is to investigate the haemodynamic effects of 1 MAC and 2 MAC of sevoflurane in children in comparison with halothane. Thirty-eight children (aged from one to six years, average age; 3.6 +/- 0.2 yr) were randomly assigned to four groups, depending on the dose and agent (1 and 2 MAC of sevoflurane: S1 and S2; 1 and 2 MAC of halothane: H1 and H2, respectively). After collecting control data during 0.2 MAC of either anaesthetic, end-expired anaesthetics were kept at 1 MAC or 2 MAC for 15 min. Mean blood pressure (mBP) and stroke volume index (SVI), measured by impedance cardiometry, decreased in all groups without differences between groups. Heart rate (HR) increased in groups S1, S2 and H2 but not in group H1. The HR in S2 was higher than that in H2. The cardiac index (CI), a product of SVI and HR, tended to decrease but not significantly in all groups. These results suggested that the haemodynamic depressant effects of sevoflurane in children were similar to those of equipotent halothane concentration except for HR. PMID- 7554000 TI - Anaesthetic management of labour and delivery in a woman taking long-term MAOI. AB - A case is presented of a woman who, for six years, had been treated for depression with 45 mg daily of the monoamine oxidase inhibitor (MAOI), phenelzine, and who continued taking the drug throughout her pregnancy and labour. Well-documented and potentially fatal interactions between MAOIs and opioids, notably meperidine, meant that her labour analgesia needed careful planning. Opioid- and epinephrine-free epidural bupivacaine analgesia was instituted early with small increments of bupivacaine 0.25% to produce a T10 block, after which an infusion of 8 ml.hr-1 bupivacaine 0.125% was used to maintain analgesia. After 14 hr labour, the epidural was extended uneventfully to allow Caesarean section to be performed for failure to progress. Pressor agents were avoided as indirect-acting drugs can produce severe hypertension. The child appeared normal and the mother had an uncomplicated postoperative course. Epidural analgesia contributed to the safe conduct of labour and Caesarean delivery. PMID- 7554001 TI - Epidural blood patch in a seven-year-old child. AB - We present an unusual case of postoperative headache in a child after an epidural block. A seven-year-old girl (ASA 1) was scheduled to undergo a urological procedure under general anaesthesia combined with an epidural technique for intra and postoperative analgesia. Although there was no evidence of dural puncture when the block was performed, the patient developed a headache postoperatively. The headache, which was accompanied by neck pain, appeared in the sitting or standing position and was relieved by decubitus. According to the mother, her child had never complained of those symptoms in the past. After independent evaluation by an anaesthetist and a neurologist, a postdural puncture headache (PDPH) was considered the most likely diagnosis. As oral analgesics failed to provide relief, and epidural blood patch (EBP) using 10 ml autologous blood was performed under light sedation. This was followed by resolution of the symptoms. Epidural injections of opioids and local anaesthetics are becoming increasingly popular for pain management in children. This implies that there may be an increasing number of unintentional dural punctures that may result in more PDPHs in children. Epidural blood patches are effective in treating PDPHs in adults but their use has been reported very rarely in children. Resolution of the patient's symptoms following EBP supported the diagnosis while illustrating that EBP can be useful in the treatment of PDPH in children. PMID- 7554002 TI - Caudal epidural blood patch for the treatment of a paediatric subarachnoid cutaneous fistula. AB - This clinical report describes the performance of an epidural blood patch in a four-year-old child with acute lymphocytic leukaemia, who developed a subarachnoid-cutaneous fistula from repeated lumbar punctures for chemotherapy. The epidural blood patch was performed using an #18-gauge epidural catheter threaded through a #16-gauge intravenous catheter via the caudal approach. This approach was successful in a child whose lumbar epidural anatomy was distorted due to a collection of subcutaneous cerebrospinal fluid making identification of the epidural space by the usual lumbar approach very difficult. This report demonstrates an alternative technique for the performance of an epidural blood patch. PMID- 7554003 TI - Fixed drug eruption associated with anaesthesia. AB - We report the case of a 65-yr-old woman undergoing bronchoscopy for a lobular lesion who received thiopentone to induce anaesthesia that was maintained with sufentanil, vecuronium and isoflurane. She tolerated the procedure well initially, but developed eruptions about her face and fingers within 24 hr of anaesthesia. Treatment with flucinonide cream (0.05%) resolved the eruptions with no lasting sequelae. Literature review supported the suspicion that the offending agent may have been thiopentone, so when a lobectomy was subsequently required and the patient refused investigation to identify the causative agent, propofol was employed as an alternative to induce anaesthesia. The surgery was well tolerated and the patient was discharged after an uneventful postoperative course. This case is reported to heighten awareness of the delayed onset of adverse effects which may be associated with the use of thiopentone. PMID- 7554004 TI - Sepsis reduces isoflurane MAC in a normotensive animal model of sepsis. AB - Patients with sepsis often require anaesthesia for surgical procedures. Anaesthesia can be unpredictable and the most haemodynamically stable agents are used. No data are available for the minimum alveolar concentration (MAC) requirements in such patients or in animal models of sepsis. We have characterized the effect of sepsis on the MAC of isoflurane in a normotensive rodent model of sepsis. The minimum inhibitory concentration (MIC) of isoflurane to an identical stimulus was determined for rodents subjected to caecal ligation and perforation (CLP n = 8), or sham laparotomy (n = 7). The calculated MAC of isoflurane was reduced in the septic animals compared with the sham animals (MAC of isoflurane, CLP = 0.8%, sham = 1.4% P < 0.003). No statistical differences were found in the haemodynamic variables measured in either group. Isoflurane leads to haemodynamic stability during anaesthesia in this animal model of sepsis. However, the MAC requirement for isoflurane is reduced by sepsis. PMID- 7554005 TI - Comparison of fluid warmer performance during simulated clinical conditions. AB - The study evaluated the warming ability and flow rates associated with four fluid warming devices during pressure driven infusion and during wide open gravity driven roller clamp infusion. Warmers tested were the Astotherm, Flotem IIe, Level 1 System 250 and a modified cardioplegia heat exchanger. Fluids tested were crystalloid, red cells diluted with 200 ml, 0.9% saline, and undiluted red cells. The volume of fluid and outlet temperatures (point where i.v. tubing would be attached to the patient) were measured for each fluid and compared among warmers for each flow rate condition. For pressure driven infusion of red cells and crystalloid, the System 250, and modified heat exchanger delivered warmer fluids (33-35 degrees C) at higher flow rates (160-740 ml.min-1) than the Astotherm and Flotem (23-31 degrees C, 44-268 ml.min-1, P < 0.05). For gravity driven infusion, the System 250 delivered the warmest fluids (33-36 degrees C, P < 0.05) compared with the modified heat exchanger (29-35 degrees C), Astotherm (26-32 degrees C) and Flotem (26-27 degrees C). In conclusion, the modified heat exchanger and System 250 were moderately effective (outlet temperature > 32 degrees C) in warming crystalloid and red cells at pressure driven flow rates. Only the System 250 warmed red cells > 35 degrees C at gravity driven flow rates. The Flotem and Astotherm were not effective in warming rapidly infused solutions. None of the warmers tested was able to deliver fluids at normothermia (> 36.5 degrees C). PMID- 7554006 TI - Carbon dioxide analysers: accuracy, alarm limits and effects of interfering gases. AB - Six mainstream and twelve sidestream infrared carbon dioxide (CO2) analysers were tested for accuracy of the CO2 display value, alarm activation and the effects of nitrous oxide (N2O), oxygen (O2) and water vapour according to the ISO Draft International Standard (DIS)#9918. Mainstream analysers (M-type): Novametrix Capnogard 1265; Hewlett Packard HP M1166A (CO2-module HP M1016A); Datascope Passport; Marquette Tramscope 12; Nellcor Ultra Cap N-6000; Hellige Vicom-sm SMU 611/612 ETC. Sidestream analysers: Bruel & Kjaer Type 1304; Datex Capnomac II; Marquette MGA-AS; Datascope Multinex; Ohmeda 4700 OxiCap (all type S1: respiratory cycles not demanded); Biochem BCI 9000; Bruker BCI 9100; Drager Capnodig and PM 8020; Criticare Poet II; Hellige Vicom-sm SMU 611/612 A-GAS (all type S2: respiratory cycles demanded). The investigations were performed with premixed test gases (2.5, 5, 10 vol%, error < or = 1% rel.). Humidification (37 degrees C) of gases were generated by a Drager Aquapor. Respiratory cycles were simulated by manually activated valves. All monitors complied with the tolerated accuracy bias in CO2 reading (< or = 12% or 4 mmHg of actual test gas value) for wet and dry test gases at all concentrations, except that the Marquette MGA-AS exceeded this accuracy limit with wet gases at 5 and 10 vol% CO2. Water condensed in the metal airway adapter of the HP M1166A at 37 degrees C gas temperature but not at 30 degrees C. The Servomex 2500 (nonclinical reference monitor), Passport (M-type), Multinex (S1-type) and Poet II (S2-type) showed the least bias for dry and wet gases. Nitrous oxide and O2 had practically no effect on the Capnodig and the errors in the others were max. 3.4 mmHg, still within the tolerated bias in the DIS (same as above). The difference between the display reading at alarm activation and the set point was in all monitors (except in the Capnodig: bias 1.75 mmHg at 5 vol% CO2) below the tolerated limit of the DIS (difference < or = 0.2 vol%). The authors conclude that the tested monitors are safe for clinical used (except those failing the DIS limits). The accuracy of the CO2-reading (average of mean absolute bias) is better in the M-type than in the S1- or S2 type analysers although no statistical (nor clinical) significant differences could be detected. Most manufacturers work with stricter limits than those proposed by the DIS. PMID- 7554007 TI - Leg pains after spinal anaesthesia. PMID- 7554008 TI - Oxygen tank check. PMID- 7554009 TI - Intravenous dead space and patient safety in patient-controlled analgesia. PMID- 7554010 TI - Propofol and chemotherapy-induced emesis. PMID- 7554011 TI - Nicardipine, verapamil and response to intubation. PMID- 7554012 TI - Economics of hypertension control. World Hypertension League. AB - This paper summarizes the key aspects of the problem of estimating the economic burden of hypertension and hypertension-related disease, the use of economic models, and the opportunities for containing the costs. More information is needed on the population-attributable risk of hypertension in various countries, which is indispensable to estimate the part of hypertension in the burden of stroke and heart disease. The population and high-risk approaches to hypertension control also have economic consequences, which may vary in different societies and must be assessed to ensure proper allocation of resources. Cost-containment can be achieved by more selective diagnostic investigations and by opting for cheaper drugs, though the choice of treatment is difficult owing to uncertainties in the quality-of-life estimates. PMID- 7554013 TI - Cell culture as a substrate for the production of influenza vaccines: memorandum from a WHO meeting. AB - Influenza has been a significant public health problem worldwide, with three pandemics during the past century. Immunization is the most effective measure to control an influenza pandemic. Since rapid production of large amounts of influenza vaccine depends on the availability of fertile hens' eggs to grow the viruses, there is an urgent need for the development of alternative cell culture systems, which would allow rapid scale-up of production in the event of a pandemic. This WHO meeting discussed the results of studies from several laboratories on the cultivation of influenza viruses in stable cell lines, and made recommendations for further work. PMID- 7554014 TI - Compliance with medication among outpatients with uncontrolled hypertension in the Seychelles. AB - Owing to increasing rates of hypertension and cardiovascular-related diseases in developing countries, compliance with antihypertensive medication is major public health importance. Few studies have reported on compliance in developing countries. We determined the compliance of 187 patients with uncontrolled hypertension in the Seychelles (Indian Ocean), by assessing the presence of a biologic marker (riboflavin) in the urine. The urine tested positive in 56% of the cases. Compliance varied from one physician to another (highest 72% versus lowest 33%, P = 0.003), improved with the level of literacy (62% versus 45%, P = 0.024), and depended on the presence absence of diuretics in the medication (respectively, 45% versus 66%, P = 0.005). The ability of patients to report correctly the number of antihypertensive pills to be taken daily was a predictor of compliance (62% of the patients who gave appropriate answers had positive urine for the marker versus 31% for those giving inappropriate answers). PMID- 7554016 TI - Registration of vital data: are live births and stillbirths comparable all over Europe? AB - International comparisons of the perinatal mortality data derived from vital registration statistics can be made in different ways. In this article we examine the legal and administrative definitions of vital events (live births and stillbirths) in the 27 European countries that participated in an in-depth survey conducted in 1991 by the Institute of Demography, University of Louvain, Belgium. The impact of the various definitions in use on the comparability of vital event data over time and in different countries is illustrated by discussing some of the anomalies exhibited by published data (e.g., age at death in different European countries and the trends in infant mortality or stillbirth rate in selected countries). Analysed is the potential for vital registration systems to produce standardized perinatal mortality data that satisfy WHO recommendations for international comparisons, taking into account the contents of the vital registration forms and the data processing (record linkage) methods useful in different countries. PMID- 7554017 TI - Breast-feeding training for health professionals and resultant institutional changes. AB - Assessed is a breast-feeding training course that was attended by health professionals at the Santos Lactation Center (SLC), Santos, Sao Paulo, Brazil, as well as its impact on the implementation of breast-feeding programmes in maternity hospitals. Eight maternity hospitals were studied--four were randomly allocated to the experimental group and sent three health professionals to attend an 18-day course at SLC; the remaining four institutions constituted the control group. The compliance of all eight hospitals with WHO/UNICEF's "Ten steps for successful breast-feeding" was determined using scores obtained before and 6 months after the training course. Institutions in the experimental group had an improved score, but those in the control group did not. The SLC training course was efficient since it enabled the participants to promote breast-feeding practices. However, in order to succeed in implementing breast-feeding programmes, health professionals require also to develop skills to apply the knowledge they acquire in the course, as well as to involve the whole maternity unit team in the activities. PMID- 7554018 TI - Diagnosis of night blindness and serum vitamin A level: a population-based study. AB - In a cross-sectional survey of 5420 children in northern Bangladesh, 124 were reported to have night blindness by their parents. Of these, 105 cases along with controls matched for age, sex, and neighbourhood had their scotopic vision examined under standard condition using a luxometer, underwent an ophthalmological examination, and had their serum vitamin A level determined. The mean serum vitamin A level was lowest among children identified as night blind by both their parents and the investigators (16.3 micrograms/dl; 95% confidence interval (CI), 13.9-18.7) and highest among those identified as not night blind by both their parents and the investigators (23.6 micrograms/dl; 95% CI, 21.3 25.9). The results show that parents' report of their children's night blindness had low sensitivity compared with diagnosis using standard observations of scotopic vision with a luxometer. PMID- 7554015 TI - The effects of malnutrition on child mortality in developing countries. AB - Conventional methods of classifying causes of death suggest that about 70% of the deaths of children (aged 0-4 years) worldwide are due to diarrhoeal illness, acute respiratory infection, malaria, and immunizable diseases. The role of malnutrition in child mortality is not revealed by these conventional methods, despite the long-standing recognition of the synergism between malnutrition and infectious diseases. This paper describes a recently-developed epidemiological method to estimate the percentage of child deaths (aged 6-59 months) which could be attributed to the potentiating effects of malnutrition in infectious disease. The results from 53 developing countries with nationally representative data on child weight-for-age indicate that 56% of child deaths were attributable to malnutrition's potentiating effects, and 83% of these were attributable to mild to-moderate as opposed to severe malnutrition. For individual countries, malnutrition's total potentiating effects on mortality ranged from 13% to 66%, with at least three-quarters of this arising from mild-to-moderate malnutrition in each case. These results show that malnutrition has a far more powerful impact on child mortality than is generally appreciated, and suggest that strategies involving only the screening and treatment of the severely malnourished will do little to address this impact. The methodology provided in this paper makes it possible to estimate the effects of malnutrition on child mortality in any population for which prevalence data exist. PMID- 7554019 TI - Using clinical signs to diagnose anaemia in African children. AB - Anaemia is a serious and common problem among young children in sub-Saharan Africa. As a first step towards developing guidelines for its recognition and treatment, we conducted a study to evaluate the ability of health workers to use clinical findings to identify children with anaemia. Health care workers examined a total of 1104 children under 5 years of age at two hospital-based outpatient clinics in rural Malawi. Blood samples were taken to determine haemoglobin concentrations. Pallor of the conjunctiva, tongue, palm or nail beds was 66% sensitive and 68% specific in distinguishing children with moderate a anaemia (haemoglobin concentration, 5-8 g/dl) and 93% sensitive and 57% specific in distinguishing those with severe anaemia (haemoglobin concentration, < 5 g/dl). Even without laboratory support, which is often unavailable in rural Africa, clinical findings can identify the majority of children with anaemia. PMID- 7554020 TI - Changes in the resistance of Plasmodium falciparum to chloroquine in Hainan, China. AB - In 1979, in view of the widespread resistance of Plasmodium falciparum to chloroquine in the island of Hainan, China, it use as an antimalarial was suspended throughout the island. A longitudinal survey of the chloroquine sensitivity of P. falciparum was carried out over the period 1981-91 to investigate whether its resistance had changed from the 1979 level. In-vitro assays were carried out every 2-3 years, while in-vivo tests were performed annually over the period 1981-83 and also in 1991. Resistance to chloroquine declined progressively after its use had stopped. The in-vitro tests indicated that the rate of chloroquine-resistant P. falciparum was 97.9% in 1981, but dropped to 60.9% in 1991 (P < 0.001). The mean concentration of chloroquine for complete inhibition of schizont formation was 10.4 pmol/microliters in 1981, but decreased to 3.0 pmol/microliters in 1991 (P < 0.001). The proportion of samples taken from malaria cases that required high concentrations ( > 6.4 pmol/microliters) of chloroquine for complete inhibition of schizont formation was 83.3% in 1981, but only 17.4% in 1991 (P < 0.001); at low concentrations ( > 1.6 pmol/microliters), the corresponding proportions increased from 4.2% in 1981 to 60.8% in 1991 (P < 0.001). In the 4-week in-vivo test, the rate of chloroquine resistant P. falciparum decreased from 84.2% in 1981 to 40% in 1991 (P < 0.001). RII + RIII cases represented 59.4% of the total resistant cases in 1981, but decreased to 37.5% in 1991 (0.02 > P > 0.01). PMID- 7554021 TI - Detecting domestic vectors of Chagas disease: a comparative trial of six methods in north-west Argentina. AB - Six methods for detecting domestic infestations by triatomine bugs were compared in the rural community of Amama, north-west Argentina. An average of three pairs (range, 2-5 pairs) of sensor boxes and sheets of pink typing-paper were tacked to the walls of human sleeping areas in 45 houses for 30 days and then inspected by a two-man team. Triatoma infestans bugs were collected in bedrooms by a different two-man team aided by a flushing-out agent both before and after application of sensing devices. Finally, knockdown collections of bugs after application of one insecticide fumigant canister per bedroom were also made. The proportion of houses with evidence of current domestic bug infestations that were detected by the various methods were as follows: sensor boxes (95.3%), reports of householders (88.4%), knockdown (87.8%), paper-sheets (86.0%), and flushing-out (69.8-76.7%). The detectability of infestations, irrespective of the method used, increased with the density of the bugs. At low or intermediate bug densities, individual sensor boxes were more sensitive than their matched paper-sheets, but at any bug density there were no significant differences between the pooled results for all the boxes and for all the paper-sheets in the house. On average, each sensor box recorded 2.25 times more triatomine faecal smears than its matched paper-sheet, and this relation increased with the density of bugs in the house. Both sensing devices were effective at monitoring unsuccessful attempts of peridomestic triatomine populations to colonize houses. PMID- 7554023 TI - Soil-transmitted helminthiases: nationwide survey in China. AB - A total of 2848 study sites, with about 500 people in each, were randomly sampled for this investigation which covered a total population of 1 477 742. By stool examinations using the Kato-Katz thick-smear and larval-culture techniques, overall prevalences of 47.0%, 18.8%, and 17.2% were obtained for Ascaris lumbricoides, Trichuris trichiura, and hookworm infections, respectively. The number of infections due to Ascaris, Trichuris, and hookworm was estimated as 531 million, 212 million, and 194 million, respectively. Egg counts showed that 75 95% of the subjects had light infections. Higher prevalences of ascariasis and trichuriasis were found in the age group of 5-9, 10-14 and 15-19 years, and among adults for hookworm. Students, farmers (including vegetable growers) and fishermen were the occupational groups with high infection rates. The prevalence of helminthiases was found to be closely associated with climatic and geographical factors. In view of the morbidity and mortality due to these helminthiases, their control, particularly in schoolchildren, is very important. PMID- 7554022 TI - Socioeconomic consequences of blinding onchocerciasis in west Africa. AB - Onchocerciasis or river blindness, a major cause of irreversible blindness among adults, has been the focus of international disease control efforts for over 20 years in West Africa. This paper employs the international classification of impairment, disability and handicap (ICIDH) to interpret results from a field study to assess the socioeconomic consequences of onchocerciasis in Guinea in 1987. In a sample of 136 blind, 94 visually impaired and 89 well-sighted persons, decreasing visual acuity is strongly associated with mobility, occupational and marital handicaps. Individual, household and disease correlates were explored. The implications of these findings for the ICIDH concept of handicap are discussed with particular emphasis on the need to extend analysis beyond the individual when assessing the socioeconomic consequences of disabling disease. PMID- 7554026 TI - Reduced doses of hepatitis B vaccines: is it a good idea? AB - Hepatitis B vaccines are not generic products and it cannot be assumed that all such vaccines can be used in reduced doses in order to save costs. Any use of vaccines in an unlicensed manner should only be performed in the context of a study that is approved by the national control authority and appropriate ethical review committees. PMID- 7554025 TI - Long-term immunogenicity and efficacy of a reduced dose of plasma-based hepatitis B vaccine in young adults. AB - A cohort of seronegative preclinical medical and dental students and another cohort of seronegative national service recruits who were immunized intramuscularly with a reduced dose (10 micrograms) of plasma-based hepatitis B vaccine (Merck, Sharp & Dohme) at the start of the study and at 1 month and 6 months thereafter were followed up for 5 years and 6 years, respectively. Among the medical and dental students, antibody to hepatitis B surface antigen (anti HBs) ( > or = 10 mlU/ml) was detected in 81% of the vaccinees at the end of the 5 year follow-up and the geometric mean titre (GMT) had dropped from 412.6 mlU/ml one year after completion of vaccination to 174.9 mlU/ml after 5 years. Antibody to hepatitis B core antigen (anti-HBc) was detected in 0.4-1.0% of the vaccinees but none was positive for hepatitis B surface antigen (HBsAg) during the follow up period. Among the national servicemen, the anti-HBs seroconversion rate and GMT were considerably lower than those of the preclinical medical and dental students. At the end of the 6-year follow-up, 55% of the vaccinees were positive for anti-HBs ( > or = 10 mlU/ml) and the GMT had dropped from 80.7 mlU/ml one year after completion of vaccination to 30.4 mlU/ml after 6 years. Anti-HBc was detected in 8 (2.7%) and transient HBs antigenaemia in 2 (0.7%) of 293 vaccinees after 4 years.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554024 TI - Evaluation of commercial HTLV-1 test kits by a standard HTLV-1 serum panel. AB - To evaluate the performance of currently available test kits for human T-cell lymphotropic virus type 1 (HTLV-1), we examined two particle agglutination (PA) tests and nine enzyme immunoassays (EIA) using a standard serum panel consisting of HTLV-1-positive and HTLV-1-negative sera that had been characterized by immunofluorescence and the polymerase chain reaction (PCR). The PA kits exhibited 94.0-100.0% sensitivity and 99.5-100.0% specificity; the sensitivity range was ascribed to the quality of the HTLV-1 antigens coated on the particles. The EIA kits had 99.5-100% sensitivity and 98.5-100% specificity; the 98.5%-99.5% specificity exhibited by five of the EIA kits could have been due to nonspecific reactions that were detected through use of an inadequate cut-off value and the use of recombinant proteins. It can be concluded that the sensitivity of the currently available PA and EIA kits is sufficient to permit their use for screening purposes; however, the specificity of some EIA kits should be optimized. PMID- 7554027 TI - Reducing the risk of unsafe injections in immunization programmes: financial and operational implications of various injection technologies. AB - The unsafe use and disposal of injection equipment continues to put patients, health care workers, and the general community at risk of infections such as hepatitis B virus and human immunodeficiency virus. Although the potential for unsafe injection practices varies substantially with the type of equipment that is used, technology alone cannot totally eliminate the risk. A knowledge of the cost, practicality and, most importantly, the potential for misuse, is critical for selecting the most appropriate injection equipment for each immunization setting. Four types of injection equipment are currently available for administering vaccines: sterilizable needles and syringes; standard disposable needles and syringes; autodestruct needles and syringes; and jet injectors. In general, the cost per injection is lowest with sterilizable equipment and highest with autodestruct. However, only autodestruct syringes virtually eliminate the risk of unsafe injection practices. Owing to differences in cost and programme factors, in some settings it may be appropriate to use a combination of equipment. For example, autodestruct syringes may be used in areas where it is difficult to ensure adequate supervision, while in medium-sized, fixed-site clinics with safe injection practices, sterilizable equipment will be the most cost-effective. PMID- 7554028 TI - Noma: a neglected scourge of children in sub-Saharan Africa. AB - Poverty is the single most important risk indicator for noma (cancrum oris), a severe gangrene of the soft and hard tissues of the mouth, face, and neighbouring areas. The risk factors associated with an increased probability of noma developing include the following: malnutrition, poor oral hygiene, and a state of debilitation resulting from human immunodeficiency virus (HIV) infection, measles, and other childhood diseases prevalent in the tropics. There are many similarities between noma and necrobacillosis of the body surface of wallabies (Macropus reforgriseus), and it is proposed that noma results from oral contamination by a heavy load of Bacteroidaceae (particularly Fusobacterium necrophorum) and a consortium of other microorganisms. These opportunistic pathogens invade oral tissues whose defences are weakened by malnutrition, acute necrotizing gingivitis, debilitating conditions, trauma, and other oral mucosal ulcers. The current escalation in the incidence of noma in Africa can be attributed to the worsening economic crisis in the region, which has adversely affected the health and well-being of children through deteriorating sanitation, declining nutritional status and the associated immunosuppression, and increased exposure to infectious diseases. Prevention of noma in Africa will require measures that address these problems, and most importantly, eliminate faecal contamination of foods and water supplies. PMID- 7554029 TI - Transgenic mice and squamous multistage skin carcinogenesis. AB - The use of animals models of human cancers has proved useful in the elucidation of molecular events which occur during tumour development. Mouse skin has been used as a model for human squamous cancer for a number of decades, and analysis of this model has identified a number of changes important for the evolution of malignancy. Transgenic mice offer a further avenue of advancement, allowing refinement of the model, and the ability to examine the consequences of individual events in vivo in greater detail. This article reviews the impact of transgenic approaches to our understanding of multistage squamous carcinogenesis in mouse skin. PMID- 7554030 TI - Murine models of neoplasia: functional analysis of the tumour suppressor genes Rb 1 and p53. AB - Loss of function of one or both of the two tumour suppressor genes p53 and RB-1 has been recognised as an important step in the development of a variety of human neoplasias for some time. By virtue of the ability to manipulate the genome of murine embryonic stem cells in culture, it has become possible to generate strains of mice which bear inactivations of the murine counterparts of these genes. This article attempts to bring together some of the many results obtained from these murine strains which are shedding light both on the normal role played by both of these genes and the consequences of their dysfunction. Surprisingly neither gene product is revealed to have an indispensable role at the level of the single cell. Hence, even though the Rb-1 gene product clearly has an important role in cell cycle regulation animals constitutively deficient in this gene develop relatively normally for the first 10 days of embryogenesis. It is only at and beyond this stage of development that a requirement for Rb-1 becomes clear, in the regulation of certain cell populations through control of both proliferation and apoptosis. That loss of function of Rb-1 is associated with tumorigenesis is confirmed by the development of tumours of the pituitary gland within heterozygotes. The retinas of these animals, the target organ for tumorigenesis in human RB-1 heterozygotes, remain unaffected. The majority of mice homozygous for an inactivating p53 mutation survive to birth, but then rapidly succumb to tumorigenesis. Heterozygotes also develop tumours, but with a delayed time course and altered spectrum. Analysis of several tissue types from the mutant animals has shown p53 to be crucial for the normal induction of apoptosis following DNA damage, and it is thought that failure of this process is a key predisposing step towards tumorigenesis within the mutant animals. Finally, studies on these and other transgenic strains have revealed interactions between pathways governed by these two genes. For example, the fate of Rb-1 deficient cells has been shown, in some tissues at least, to be dependent upon the functional status of p53. PMID- 7554033 TI - Assessing new anti-tumour agents and strategies in oncogene transgenic mice. PMID- 7554031 TI - Apoptosis, cancer and the p53 tumour suppressor gene. AB - One of the most commonly detected abnormalities in human cancer is mutation of the p53 tumour suppressor gene. Intrinsic to the function of p53 is its ability to induce apoptotic cell death and to cause cell cycle arrest. Moreover, p53 plays an important role in controlling the cellular response to DNA damaging agents such as ionizing radiation and cancer chemotherapeutic drugs. Loss of p53 function causes increased resistance to radiation and chemotherapeutic agents, and there is increasing evidence that p53 mutational status is an important determinant of clinical outcome in cancer. This review will focus on recent data describing the biochemistry of p53 function, its role in mediating apoptosis and cell cycle arrest and in the control of tumour growth and death. PMID- 7554032 TI - Transgenic and gene knockout mice in cancer research. AB - Transgenic animal technology, and the use of germline manipulation for the creation of targeted gene mutations, has resulted in a plethora of murine models for cancer research. Our understanding of some of the important issues regarding the mechanisms controlling cell division, differentiation and death has dramatically advanced in recent years through exploitation of these techniques to generate transgenic mice. In particular, the generation of mice with targeted mutations in genes encoding proteins of oncological interest has proved to be a useful way of elucidating the function of these gene products in vivo. Transgenic mouse models have provided some insight into the complex oncogenic events contributing to cellular dysregulation and the loss of growth control that can lead to tumorigenesis. These animal studies have highlighted the fact that there are many different stages at which the loss of cell cycle control can occur, as a result of mutations affecting proteins anywhere from the cell surface to the nucleus. Although mutations affecting growth factors, growth factor receptors, signal transduction molecules, cytoplasmic proteins or nuclear proteins might appear to be very distinct, the end result of these changes may be accelerated and unchecked cell growth ultimately leading to cancer. It is beyond the scope of this review to mention every animal model that has been developed for cancer research, especially since many of the early studies have been covered extensively in previous reviews. This article will instead focus on a small selection of transgenic and knockout animal models which exemplify how proteins from distinct localisations along multiple pathways can contribute to loss of cell cycle control and the pathogenesis of cancer. PMID- 7554034 TI - Utilization of transgenic mice in the study of matrix degrading proteinases and their inhibitors. AB - The extracellular matrix (ECM) acts as both a structural scaffold and an informational medium. Its dynamic status is determined by cells that secrete its constituent molecules and, in most cases, also secrete enzymes that catalyze degradation of these molecules. A stasis between ECM degrading enzymes and their inhibitors maintains the integrity of the matrix. While controlled ECM remodelling is fundamental to several normal processes, uncontrolled disruption underlies diverse pathological conditions. Transgenic mice with specific modulations or a total lack of expression of certain metalloproteinases, serine proteinases or their inhibitors have been generated to elucidate endogenous expression patterns, identify regulatory elements of these genes, and study the physiological consequences of their deregulated expression. With these models we enhance our understanding of the role of proteinases and their inhibitors in diverse normal processes and pathologies including mammary gland development, hemostasis, emphysema and cancer. PMID- 7554035 TI - A phase I and pharmacology study of an oral platinum complex, JM216: dose dependent pharmacokinetics with single-dose administration. AB - JM216 [bis-acetato-ammine-dichloro-cyclo-hexylamine-platinum (IV)] is an oral platinum complex with in vivo activity against murine and human tumor models and a lack of nephro- and neurotoxicity in rodents. During a phase I study of a single-dose schedule, JM216 was given in dry-filled hard gelatin capsules by mouth without hydration or diuresis. In all, 37 patients were given a total of 88 courses at doses ranging from 60 to 700 mg/m2. The study was stopped before the MTD was reached because of nonlinear pharmacokinetics. Myelosuppression was manifest by leucopenia or thrombocytopenia and showed marked variability at 420 700 mg/m2. Vomiting was mild and controllable by antiemetics in approximately 50% of courses. The onset of vomiting was delayed to 4 h after during ingestion. There was no nephro-, oto- or neuro-toxicity. A partial response was recorded in a patient with recurrent ovarian cancer, and significant falls in plasma tumour markers (CA125) were seen in two further cases. Plasma pharmacokinetics were linear and showed moderate interpatient variability at dose levels of < or = 120 mg/m2. At dose levels of > or = 200 mg/m2, Cmax and AUC increased less than proportionally to dose. This was associated with greater interpatient pharmacokinetic variability and reduced urinary platinum recovery. A significant sigmoidal relationship existed between ultrafilterable plasma AUC and the percentage of reduction in platelet count (r2 = 0.78). Nonlinear absorption was a limitation to this single-dose schedule of oral NM216; however, little non haematological toxicity was seen at doses associated with myelosuppression and antitumour activity. Clinical studies of divided dose schedules using doses within the range of pharmacokinetic linearity (< or = 120 mg/m2) are now being investigated. PMID- 7554036 TI - Characteristic antitumor activity of cytarabine ocfosfate against human colorectal adenocarcinoma xenografts in nude mice. AB - The antitumor activity of cytarabine ocfosfate (SPAC) was tested against human colorectal, gastric and lung adenocarcinoma xenografts in nude mice in comparison with the activities of various antitumor drugs used clinically. SPAC showed higher therapeutic efficacy against human colorectal adenocarcinoma xenografts than against human gastric and lung adenocarcinoma xenografts. SPAC was effective against three of four human colorectal adenocarcinoma xenografts, with efficacy higher than that of 1-beta-D-arabinofuranosylcytosine, fluorouracil, cisplatin, doxorubicin, pirarubicin and vindesine sulfate, but lower than that of mitomycin C and cyclophosphamide. These results indicate that SPAC may be useful for induction and/or postoperative chemotherapy against colorectal adenocarcinomas. PMID- 7554038 TI - Gender affects doxorubicin pharmacokinetics in patients with normal liver biochemistry. AB - We studied the variability in doxorubicin pharmacokinetics in 27 patients, all of whom had normal liver biochemistry tests. Blood samples were collected after the first cycle of single-agent doxorubicin given as an i.v. bolus and plasma levels were measured by high-performance liquid chromatography (HPLC). The relationship of doxorubicin clearance (dose/AUC) with biochemical tests (AST, bilirubin, alkaline phosphatase, albumin, creatinine) and physical characteristics (age, gender, height, weight, tumour type) was investigated. The 6 men had a significantly higher doxorubicin clearance than did the 21 women (median values, 59 and 27 lh-1 m-2, respectively; P = 0.002). Doxorubicin clearance was significantly lower in patients with breast cancer than in those with other tumours (median values, 26 and 53 lh-1 m-2, respectively; P = 0.0008). The other biochemical and physical parameters did not correlate with doxorubicin clearance. However, in multivariate analysis, gender was the only factor predicting doxorubicin clearance (r2 = 40%). The ratio of the AUCs for doxorubicinol and doxorubicin (R) was higher in the men than in the women (median values, 0.62 and 0.36, respectively; P = 0.03). We conclude that gender may be an important determinant of doxorubicin clearance in patients with normal liver biochemistry. PMID- 7554039 TI - Serotonin metabolism following platinum-based chemotherapy combined with the serotonin type-3 antagonist tropisetron. AB - The administration of platinum-based chemotherapy induces serotonin release from the enterochromaffin cells, causing nausea and vomiting. This study was conducted to evaluate parameters of serotonin metabolism following platinum-based chemotherapy given in combination with the serotonin type-3 antagonist tropisetron as an antiemetic agent. In nine chemotherapy-naive patients with disseminated germ-cell tumors, parameters of serotonin metabolism in both blood and urine were evaluated during two consecutive courses of platinum-based chemotherapy. Serotonin concentrations in platelet-rich plasma and platelet-poor plasma as well as urinary 5-hydroxyindoleacetic acid (5-HIAA) and serotonin levels were measured during the full length of the courses. By means of comparison with the antiemetic agent chlorpromazine, used on day 1 of the first course only, the effect of the serotonin type-3 antagonist tropisetron, the antiemetic agent used during the rest of the courses, on these parameters was studied. Clinical effects were also recorded. No change in the parameters of serotonin metabolism could be demonstrated during either course by the serotonin type-3 antagonist tropisetron. Also in vitro, no effect of tropisetron on the active serotonin uptake by platelets was found. Serotonin levels in platelets showed no correlation with emetic response. However, the platelet serotonin content decreased significantly between the first and the second course (P < 0.01). The significant reduction in platelet serotonin content observed between the first and the second course indicates a depletion of total body serotonin. The role of a serotonin type-3 antagonist might be affected by the altered serotonin equilibrium during later courses of chemotherapy. PMID- 7554037 TI - Limited sampling models for simultaneous estimation of the pharmacokinetics of irinotecan and its active metabolite SN-38. AB - Irinotecan (CPT-11) is a novel topoisomerase I inhibitor with clinical activity in human malignancies. The objective of this study was to develop efficient limited sampling models (LSMs) to estimate simulataneously the area under the plasma concentration versus time curves (AUC) for both CPT-11 and its active metabolite SN-38. A total of 64 pharmacokinetic sets (> or = 24-h sampling) were obtained in phase I studies at doses ranging from 50 to 750 mg/m2 (0.5-h i.v. infusion). The patients were randomly assigned to a training data set (n = 32) and a test set (n = 32). Multiple linear regression analyses were used to determine the optimal LSMs based on the correlation coefficient (r), bias (MPE%, percentage of mean prediction error), and precision (RMSE%, percentage of root mean squared prediction error). Of these LSMs, the ones including maximal concentrations of CPT-11 (0.5 h, the end of the i.v. infusion) and metabolite SN 38 (approximately 1 h) were favored along with predictive precision and clinical constraints. Several bivariate models including a 6-h time point as the last sampling time (or 7 h) were found to be highly predictive of either the CPT-11 AUC or the SN-38 AUC. The chosen sampling time points were the ones that allowed the best compromise between the accurate determination of either compound alone with the same sampling times. The simultaneously best prediction of both CPT-11 and SN-38 AUCs was obtained with sampling time points harvested at 0.5, 1, and 6 h (or 7 h). With these sampling time points a trivariate model was selected for the determination of CPT-11 AUC namely, CPT-11 AUC (ng h ml-1) = 0.820 x C0.5h + 0.402 x C1h + 15.47 x C6h + 928, and a corresponding model was selected for the determination of metabolite AUC, i.e., SN-38 AUC (ng h ml-1) = 4.05 x C0.5h -0.81 x C1h + 23.01 x C6h - 69.78, where C(t) is the concentration in nanograms per milliliter of either compound at a given time t. These models performed well with the test data sets for CPT-11 AUC (r = 0.98, MPE% = -1.4, RMSE% = 13.9) and for SN-38 AUC (r = 0.95, MPE% = -6.5, RMSE% = 37.7). In addition to the determination of AUCs (and hence clearance), these models also allow the determination of the maximal concentrations of both compounds, which might be needed for pharmacodynamics studies.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554041 TI - Phase I study of percutaneous 4-hydroxy-tamoxifen with analyses of 4-hydroxy tamoxifen concentrations in breast cancer and normal breast tissue. AB - 4-OH-tamoxifen is an active metabolite of tamoxifen that is detectable in the serum and tumour tissue of patients treated by oral tamoxifen. As this metabolite penetrates through the skin, it is possible to compare percutaneous 4-OH tamoxifen (4-OH-TAM) and oral tamoxifen treatments. We report herein a randomized study of percutaneous 4-OH-TAM versus oral tamoxifen in women with breast cancer. This pharmacology study was designed to compare the 4-OH-TAM concentration in breast cancer and normal breast tissue according to the route and dose used for administration of tamoxifen after a 3-week period prior to surgery and tissue sampling. Women were randomized into one of the five following groups: group I, oral tamoxifen given at 10 mg twice a day; group II, 4-OH-TAM delivered percutaneously at 0.5 mg day to both breast areas; group III, 4-OH-TAM applied percutaneously at 1 mg/day to both breast areas; group IV, 4-OH-TAM delivered percutaneously at 1 mg/day to a large cutaneous area excluding the breasts; and group V, 4-OH-TAM applied percutaneously at 2 mg/day to a large skin area excluding the breasts. 4-OH-TAM plasma and tissue concentrations were significantly higher in the oral tamoxifen group as compared with either the high or the low-dose percutaneous 4-OH-TAM group. In group II, percutaneous 4-OH-TAM treatment resulted in tissue concentrations of 1,446 and 352 pg/g in tumour tissue and normal breast tissue, respectively. In group I these concentrations were as follows: tumour tissue, 12, 453 pg/g; and normal tissue, 10,214 pg/g. 4 OH-TAM concentrations in tumour tissue and normal breast tissue did not significantly differ in any group. In the oral group we observed classic effects on coagulation and lipid metabolism when pre- and post-treatment values of these biological variables were compared, whereas no difference was observed in the percutaneous group. Although percutaneous administration of 4-OH-TAM led to a low plasmatic concentration of this active metabolite, the breast tissue concentration remained lower than those observed after oral tamoxifen treatment. Therefore, at the doses described in this study, percutaneous 4-OH-TAM cannot be proposed as an alternative tamoxifen treatment. PMID- 7554040 TI - Cellular pharmacology of N4-hexadecyl-1-beta-D-arabinofuranosylcytosine in the human leukemic cell lines K-562 and U-937. AB - The mechanisms of cytotoxicity, cellular drug uptake, intracellular drug distribution, cellular pharmacokinetics, formation of arabinofuranosylcytosine triphosphate (ara-CTP), and DNA incorporation of N4-hexadecyl-1-beta-D arabinofuranosylcytosine (NHAC), a new lipophilic derivative of arabinofuranosylcytosine (ara-C) formulated in small unilamellar liposomes, were determined in vitro in the human leukemic cell lines K-562 and U-937. Furthermore, the induction of erythroid differentiation by NHAC was tested in K 562 cells. The cytotoxicity of NHAC in both cell lines was not influenced by the deoxycytidine (dCyd) concentration or the presence of the nucleoside-transport blocking agent dipyridamole as demonstrated in coincubations with dCyd and/or dipyridamole, whereas in contrast, the cytotoxicity of ara-C was decreased additively by both drugs. As compared with ara-C, the uptake of NHAC displayed up to 16- and 5-fold increases in K-562 and U-937 cells, respectively, depending on the drug concentration. Studies of the drug distribution and pharmacokinetics of NHAC revealed a depot effect for NHAC in the cell membranes, resulting in half lives 2.6 and 1.4 times longer than those of ara-C in the two cell lines. The ara CTP concentrations derived from NHAC were 150- and 75-fold lower at a drug concentration of 1 microM in K-562 and U-937 cells, respectively. The DNA incorporation of the drugs observed after incubation with 2 microM NHAC was 60- and 30-fold lower as compared with that seen at 2 microM ara-C in the two cell lines. Furthermore, NHAC was capable of inducing irreversible erythroid differentiation to a maximum of only 22% of K-562 cells, whereas ara-C induced differentiation at a drug concentration 100-fold lower in 50% of the cells. These results indicate a mechanism of cytotoxicity for NHAC that is independent of the nucleoside transport mechanism and the phosphorylation pathway and suggest that the mechanisms of action of NHAC are significantly different from those of ara-C. Therefore, NHAC might be used for the treatment of ara-C-resistant malignancies. PMID- 7554043 TI - Etoposide protein binding in cancer patients. AB - The protein binding of etoposide was studied in vivo in 36 cancer patients receiving etoposide therapy. Free etoposide was separated from plasma using an ultrafiltration method and the etoposide concentrations (free and total) were measured by high-performance liquid chromatography (HPLC). Considerable interpatient variation in the protein binding of etoposide was observed. The protein binding of etoposide varied from 80% to 97% (mean, 93%). Univariate analysis showed a significant inverse correlation between the free fraction of etoposide and serum albumin (r = 0.74), daily dose (r = 0.37) and age (r = 0.34). Multivariate analysis demonstrated that serum albumin and age were independent predictors of the etoposide free fraction. Serum bilirubin showed no correlation with etoposide protein binding. There is wide variation in etoposide protein binding in cancer patients, which is mostly dependent on serum albumin concentration. PMID- 7554044 TI - Phase I clinical trial of continuous infusion cyclopentenyl cytosine. AB - Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5'-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i.v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i.v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 microM to 3.1 microM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146 +/- 38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2:1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6% +/- 3.1% at 22 h (mean +/- SE, n = 16), and remained inhibited by 67.6% +/- 7.7% (n = 10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24-48 h after completion of the CPE-C infusion during their first (n = 2), third (n = 1) and sixth cycles (n = 1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2 per h). Hypotension was not seen in patients receiving < or = 2.5 mg/m2 per h CPE C.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554042 TI - The activity of deoxyspergualin in multidrug-resistant cells. AB - Deoxyspergualin, a synthetic analogue of the immunosuppressive anti-tumour antibiotic spergualin, has been shown to possess potent in vitro and in vivo anti tumour activity and is currently in the National Cancer Institute (NCI) decision network. Deoxyspergualin shows similarities in properties and mechanisms of action to the natural-product immunosuppressive agents cyclosporin A and FK506, each of which can act as a modifier of multidrug resistance. We therefore decided to examine the comparative activity of deoxyspergualin in parent and multidrug resistant cells. Deoxyspergualin contains the polyamine spermidine within its structure. Bovine serum copper amine oxidase catalyses the oxidative deamination of spermidine to produce an aminoaldehyde, ammonia and hydrogen peroxide. These aminoaldehydes are believed to be responsible for the toxicity of polyamines in vitro in the presence of bovine serum. For this reason, all experiments were carried out in medium containing bovine serum and in medium containing horse serum (which is low in copper amine oxidase content). We used the tetrazolium (MTT) colorimetric assay to determine drug sensitivity and tritiated daunorubicin accumulation together with inhibition of azidopine binding to study specific mechanisms of resistance modulation. The murine cell lines EMT6/P and EMT6/AR1.0 and the human cell lines H69/P and H69/LX4 were, respectively, 32-, 32-, 372- and 483-fold more sensitive to spermidine and 175-, 133-, 321- and 444-fold more sensitive to spermine in the presence of calf serum than in the presence of horse serum. However, these large differential effects were not seen for deoxyspergualin. It appears that in the presence of horse serum, deoxyspergualin may exert its effect by a mechanism other than polyamine oxidation. Deoxyspergualin did not enhance the accumulation of [3H]-daunorubicin in EMT6/AR1.0 cells. Furthermore, deoxyspergualin (1-20 microM) did not restore the sensitivity of EMT6/AR1.0 or H69/LX4 cells to that of the parent lines. P glycoprotein (Pgp) in membranes prepared from H69/LX4 cells was photo-affinity labeled with [3H]-azidopine. Deoxyspergualin did not inhibit this labeling. Although deoxyspergualin appears to exert its immunosuppressive effect via a mechanism similar to that of cyclosporin A and FK506, it does not share their ability to modify Pgp-mediated multidrug resistance. However, its lack of cross resistance and potent in vivo anti-tumour activity make deoxyspergualin a promising candidate for development as an anti-cancer agent. PMID- 7554045 TI - Interspecies differences in the kinetic properties of deoxycytidine kinase elucidate the poor utility of a phase I pharmacologically directed dose escalation concept for 2-chloro-2'-deoxyadenosine. AB - 2-Chloro-2'-deoxyadenosine (CdA, Cladribine), is a purine antimetabolite currently under investigation in phase II clinical trials for the treatment of lymphoid malignancies. Significant differences in CdA toxicity between mice and humans were observed during phase I clinical evaluation. For the elucidation of interspecies differences in drug toxicity the pharmacokinetics of CdA after subcutaneous injection and the kinetic properties of the CdA-phosphorylating enzyme, deoxycytidine kinase (dCK), were compared in mice and humans. The ratio of the dose lethal to 10% of mice (LD10) to the maximum tolerated dose (MTD) in humans was 50 and the ratio of the area under the curve obtained at approximately one-half the LD10 (AUCapprox. one-half the LD10)/AUC(MTD) was 49. A significant interspecies difference was observed in the kinetic properties of dCK, the main CdA-activating enzyme. With CdA as a substrate, the Michaelis constant (Km) of dCK in crude extracts of mouse thymus was 10 times higher than that in human thymus. An approximately 9-fold interspecies difference in maximum velocity (Vmax)/Km indicated a higher efficiency of dCK for CdA in humans than in mice. The peak plasma concentration was 210 times higher and exceeded the Km in mice. Initial and terminal half-lives were approximately 7 times shorter in mice and trough levels were similar in mice and humans. Thus, the differences in AUCs at equitoxic doses are largely explained by differences in the target enzyme properties and the pharmacokinetic pattern. The observed lower tolerance for CdA in humans as compared with mice confirms the view that antimetabolites may not be good candidates for pharmacokinetically guided dose-escalation schemes unless detailed information on interspecies variability in drug bioactivation is available. PMID- 7554047 TI - Interaction of bulky chemical carcinogens with DNA in chromatin. AB - DNA damage is an important effect of treatment of cells and organisms with chemical carcinogens. Although much has been learned from in vitro studies of the reaction of carcinogens with purified DNA, in vivo the DNA is associated with a variety of histone and non-histone proteins in a complex and dynamic structure known as chromatin. The covalent interactions of bulky chemical carcinogens with chromatin are reviewed. Differences from bulk genomic DNA in adduct density are found in replicating, transcribing and nuclear matrix-bound DNA regions, and between DNA in nucleosome cores and linker DNA regions. These differences range from 2- to 3-fold for linker versus core, to approximately 8-fold close to a replication fork. Much remains to be done to determine the influences of non histone proteins and higher order chromatin structure on carcinogen binding. PMID- 7554046 TI - Retardation of rat sciatic nerve regeneration after local application of minute doses of vincristine. AB - The effect of vincristine on regeneration of rat sural and tibial nerves following a crush lesion of the sciatic nerve was studied in the pinch test. Vincristine locally applied through an osmotic minipump at the site of the lesion dose-dependently retarded regeneration of the tibial and sural nerve at a threshold dose of 5 ng/day, whereas regeneration was blocked at a dose of 200 ng/day. Regeneration of the sural nerve was more sensitive to the retarding effects of vincristine than was regeneration of the tibial nerve. Systemic weekly administration (i.p.) of 1 mg/kg of vincristine for 7 weeks had approximately the same effect as local application of 10-20 ng/day for 1 week. The differences in sensitivity between sural and tibial nerves and the large discrepancy between local and systemic administration are discussed. On the basis of the potent effects of vincristine used at low concentrations, the absence of overt effects of local vincristine on animal behavior and the short time course in which the local vincristine effects are observed, it is concluded that this paradigm is an extremely suitable model for studying vincristine-induced defects of nervous system function. This model may be used for evaluating the neuroprotective effects of neurotrophic agents against vincristine-induced neuropathies. PMID- 7554049 TI - Effect of stage of development on frequency and pathogenesis of kidney tumors induced in Noble (Nb) rats exposed prenatally or neonatally to N nitrosoethylurea. AB - Wilms' tumor of kidney, a common human childhood neoplasm, is modeled by nephroblastomas induced by prenatal exposure of some rodents to alkylating agents. Noble (Nb) rats are especially susceptible. We studied the ontogeny of susceptibility by treatment with N-nitrosoethylurea (NEU) on gestation day 10, 12, 14, 16 or 18 or neonatal day 1, 3, 5, 7, or 10. No nephroblastomas were observed in offspring exposed to NEU on day 10 or 12 of gestation. In contrast, nephroblastomas commonly occurred in rats exposed on gestation day 14, 16 or 18 of gestation, with the highest incidence (48%) after treatment on day 18. Nephroblastomas were rare ( < 10%), but renal mesenchymal tumors were common (25 30%) in rats exposed to NEU on day 1 or 3 after birth. In rats exposed to NEU on day 7 or 10 only renal mesenchymal tumors were seen. Thus our results suggest that the stage of differentiation of fetal and neonatal kidneys at the time of NEU administration determines the frequency and type of kidney tumors induced in Nb rats. Since NEU induces both nephroblastomas and mesenchymal tumors in this strain, this experimental model may prove useful for the study of molecular mechanisms involved in the development of these two histogenetically different types of kidney tumors. PMID- 7554050 TI - DNA repair in human cells: quantitative assessment of bulky anti-BPDE-DNA adducts by non-competitive immunoassays. AB - Mutagenicity and carcinogenicity of the ubiquitous environmental pollutant benzo[a]pyrene is mediated via its reactive diol epoxide metabolite, anti-BPDE, with the predominant formation of N(2)-deoxyguanine adducts in genomic DNA. Polyclonal and monoclonal antibodies specific for (+/-)-anti-BPDE DNA adducts were used for the quantitative detection of genotoxic damage in DNA treated in vitro and in vivo with ( +/- )-anti-BPDE. In non-competitive enzyme-linked immunosorbent assay the polyclonal antiserum (BP1) exhibited higher affinity, avidity and sensitivity than the monoclonal antibody (5D2). A linear antibody binding response was observed over a wide carcinogen dose range with a detection limit of < 0.1 fmol adducts in immobilized DNA. Non-competitive immuno-slot blot assay could detect 0.2 adducts/10(6) nucleotides induced by < 1 nM ( +/- )-anti BPDE. The high sensitivity and mono-adduct specificity of non-competitive immunoassays allowed the detailed study of ( +/- )-anti-BPDE-DNA adduct processing human cells exposed to very low levels of the genotoxin. Analysis of polyclonal antiserum binding sites in DNA from repair-proficient human fibroblasts revealed adduct removal rates directly proportional to the initial genotoxic insult. Despite efficient repair, substantial damage persisted in repair-proficient cells exposed to high doses of carcinogen. At low levels of initial damage (0.882 and 3.44 +/- 0.17 adducts/ 10(6) nucleotides) approximately 50% repair was observed after 4 and 8 h respectively. Cells removed approximately 40% of the lesions in 8 h at an intermediate level of damage (20.7 +/- 1.5 adducts/10(6) nucleotides). At higher DNA damage levels (105 +/- 8 and 177 +/- 1 adduct/10(6) nucleotides) 33 and 19% of the lesions respectively were repaired in 24 h. Repair-deficient xeroderma pigmentosum group A fibroblast cells did not show any significant loss of antibody binding sites at high or low initial modification levels. These data suggest that the level of initial DNA damage has a significant impact on the overall efficiency of cellular repair, with potential implications for the biological consequences of deleterious DNA lesions in mammalian cells. PMID- 7554048 TI - Self-regulation of smoking intensity. Smoke yields of the low-nicotine, low-'tar' cigarettes. AB - It has been assumed for some time that the 'tar' and nicotine data for individual cigarette brands, as reported by the Federal Trade Commission (FTC), do not adequately reflect the levels of exposure to toxic and carcinogenic agents in the smoke. The trend of decreasing 'tar' and nicotine yields of the sales-weighted average US cigarettes was not followed by a proportionate decline of lung cancer incidence and mortality rates. Utilizing a 'tobacco smoke inhalation testing system', we determined smoking profiles for four men and four women who smoked low-nicotine cigarettes ( < or = 0.8 mg/cigarette according to FTC), and for two men and two women who smoked cigarettes with medium-nicotine (0.9-1.2 mg) yields. The recorded smoking profiles were programmed into a smoking machine to establish mainstream smoke yields for 'tar', nicotine, benzo[a]pyrene and 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone. The analytical data obtained for each smoker's cigarette were compared with corresponding measurements in the smoke from the same cigarette brand that was generated by machine-smoking under the standardized FTC conditions (1 puff of 2 s duration and 35 ml volume drawn once/min). Significant increases in terms of total volume of smoke inhaled and exposures to 'tar', nicotine, and lung carcinogens were measured (2- to 4-fold) and, because of smokers' compensation for low nicotine delivery, much greater overall exposure resulted from smoking low-nicotine cigarettes. Although these measurements were obtained for a limited number of smokers, they strongly indicate that both low- and medium-nicotine cigarettes are being smoked much more intensely than would be implied from the FTC-data. Therefore, there is an urgent need to accurately quantify the exposure of consumers of the various types of cigarettes to toxic and carcinogenic agents. PMID- 7554051 TI - Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds. AB - Escherichia coli lacZ strains CC107-CC111, which detect specific frameshift mutations, were used to study the mutational specificities of 2-nitro-3 methylimidazo[4,5-f] quinoline (NO2-IQ) and rat hepatic S9-activated 2-amino-3 methylimidazo[4,5-f]quinoline (IQ). New constructs were made in which UvrABC dependent excision repair was eliminated (strains DJ3107-DJ3111), followed by introduction of plasmid pYG219 conferring acetyl CoA:arylamine N acetyltransferase/acetyl CoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity (strains DJ3207-DJ3211). Sensitivity to mutagens was greatly enhanced. The mutational specificity of NO2-IQ was identical to that of the corresponding amine, IQ. The most prominent mutations caused by the two compounds were -2(CGGC) and 1(CG) frameshifts. +1(AT) Frameshifts play a minor role in the pattern of mutational specificity. Induction of all three mutations was similarly influenced by NAT/OAT activation and UvrABC-dependent excision repair. These new tester strains provide an effective tool for the study of aromatic amine mutational specificity and the influences of excision repair and NAT/OAT activation. PMID- 7554052 TI - Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro. AB - Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues. PMID- 7554054 TI - Rosemary components inhibit benzo[a]pyrene-induced genotoxicity in human bronchial cells. AB - The commonly used spice and flavouring agent, rosemary, derived from the leaves of the plant Rosmarinus officinalis L., displays antioxidant properties in foods and in biological systems. Moreover, in animal models rosemary components were found to inhibit the initiation and tumour promotion phases of carcinogenesis. In this work, we studied the mechanisms by which rosemary components block initiation of carcinogenesis by the procarcinogen benzo[a]pyrene (B[a]P) in human bronchial epithelial cells (BEAS-2B). Whole rosemary extract (6 micrograms/ml) or an equivalent concentration of its most potent antioxidant constituents, carnosol or carnosic acid, inhibited DNA adduct formation by 80% after 6 h co-incubation with 1.5 muM B[a]P. Under similar conditions, cytochrome P450 (CYP) 1A1 mRNA expression was 50% lower in the presence of rosemary components, and CYP1A1 activity was inhibited 70-90%. The observed reduction of DNA adduct formation by rosemary components may mostly result from the inhibition of the activation of benzo[a]pyrene to its ultimate metabolites. Carnosol also affected expression of the phase II enzyme glutathione-S-transferase which is known to detoxify the proximate carcinogenic metabolite of B[a]P. Treatment of BEAS-2B cells with carnosol (1 microgram/ml) for 24 h resulted in a 3- to 4-fold induction of GST pi mRNA. Moreover, expression of a second important phase II enzyme, NAD(P)H: quinone reductase, was induced by carnosol in parallel with GST pi. Therefore, rosemary components have the potential to decrease activation and increase detoxification of an important human carcinogen, identifying them as promising candidates for chemopreventive programs. PMID- 7554055 TI - Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor alpha. AB - Tumor necrosis factor alpha (TNF alpha), a pleiotrophic cytokine present in atherosclerotic lesions, caused a dose-dependent and persistent reduction in gap junctional intercellular communication (GJIC) between primary human smooth muscle cells in vitro. A continuous presence of TNF alpha was required for this persistent inhibition. Pretreatment of smooth muscle cells with ascorbic acid, alpha-tocopherol or glutathione prevented this inhibition of GJIC by TNF alpha. The persistent blockage of GJIC by continuous exposure to TNF alpha suggests that TNF alpha may share some mechanistic similarities with exogenous tumor promoters. Furthermore, this reduction in GJIC by TNF alpha may provide an additional link between the processes of atherosclerosis and carcinogenesis. The protection afforded by antioxidant compounds suggests a role for active oxygen species in the promotion stage of atherosclerosis. PMID- 7554056 TI - Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes. AB - Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU. PMID- 7554053 TI - Effects of single doses of irradiation on the expression of resistance-related proteins in murine NIH 3T3 and human lung carcinoma cells. AB - In this report the effects of single doses of ionizing radiation on the mRNA expression of several proteins involved in multiple drug resistance were analyzed. Murine NIH 3T3 cells treated with single doses of 5, 10 and 20 Gy during the time interval from 1.5 to 72 h after irradiation were compared with their corresponding controls at the same points of time. The glutathione S transferase-pi (GST pi) level was elevated in cells treated with 10 or 20 Gy from 24 to 72 h after irradiation compared with the control. Topoisomerase II alpha and thymidylate synthase were decreased in irradiated cells 24-72 h after exposure. These down-regulations were associated with cellular proliferation, determined by mRNA expression of the proliferation marker histone 3. Irradiated cells exhibited no alteration in the P-glycoprotein or glutathione peroxidase mRNA content. The finding that GST pi mRNA was overexpressed after irradiation was validated by investigations on a human lung carcinoma cell line (LXF 289) on the mRNA and protein level. Thus, our results indicate that irradiation alters the expression of proteins involved in multidrug resistance and may, therefore, play a role in clinical drug response. PMID- 7554058 TI - 32P-postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment. AB - Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form polycyclic aromatic hydrocarbon (PAH)- and nitrated PAH-DNA adducts in rodent skin and lung. The aim of this study was to characterize by 32P postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro and in vivo by diesel extracts. The diesel particle extracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes (B[b,j,k]F) and chrysene. DNA adducts were analyzed in calf thymus DNA incubated in vitro with PAHs activated by S9 mix and in skin and lung DNA from topically treated mice. The main diesel-derived DNA adduct formed in vitro and in vivo did not co-migrate on HPLC and large TLC plates with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene (anti BPDE)-, B[b]F-,B[j]F-,B[k]F-or chrysene-DNA adduct standards. By co-chromatography DNA adducts formed by chrysene from both in vitro and in vivo samples were identified. Nissan diesel extract containing higher PAH concentrations than Volkswagen automobile extract formed skin DNA adducts that co migrated with chrysene- and anti BPDE- DNA-derived adducts. We conclude that the use of a highly sensitive 32P-postlabeling method combined with HPLC improves the identification of PAH adducts formed by complex mixtures such as diesel exhaust extracts. PMID- 7554057 TI - Transforming growth factor-alpha and epidermal growth factor expression in the exocrine pancreas of azaserine-treated rats: modulation by cholecystokinin or a low fat, high fiber (caloric restricted) diet. AB - Expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) was studied in normal pancreatic tissue and in (pre)neoplastic pancreatic lesions of azaserine-treated rats. They were given either a low fat, high fiber (low caloric) diet, to inhibit carcinogenesis, or a low fat diet combined with injections of the cholecystokinin analog caerulein to enhance carcinogenesis. The control groups, maintained on a low fat diet, were injected with azaserine or were not treated at all. Autopsy was performed at 6 and 15 months after the last azaserine injection. After both 6 and 15 months immunohistochemistry revealed a weak expression of EGF and TGF-alpha peptides in the acinar cells, and a stronger expression in the ductular and centroacinar cells. TGF-alpha peptide expression was reduced in both putative preneoplastic and neoplastic acinar cell lesions, but no differences in EGF peptide expression were observed between the various stages of exocrine pancreatic carcinogenesis. After 16 months an increase in TGF-alpha mRNA due to treatment with azaserine was detected by semi-quantitative PCR in total pancreatic homogenates, whereas EGF mRNA expression had decreased. TGF-alpha mRNA levels in macroscopically isolated tumors were significantly lower, but EGF mRNA levels were significantly higher, than in total pancreatic homogenates from azaserine-treated rats. Furthermore, EGF and TGF-alpha mRNA levels in isolated tumors did not differ significantly from mRNA levels in non-carcinogen-treated rats. Neither with immunohistochemistry nor with PCR were differences in EGF or TGF-alpha expression observed due to either inhibition or stimulation of carcinogenesis. It is concluded that putative preneoplastic acinar cell lesions induced in rat pancreas by azaserine may develop into acinar adenocarcinomas independently of TGF-alpha and EGF. The results suggest involvement of these growth factors at the early stage of the carcinogenic process, during the initiation of normal acinar cells into putative preneoplastic cells. However, modulation of azaserine-induced pancreatic carcinogenesis by cholecystokinin or a low fat, high fiber (caloric restricted) diet appeared not to be regulated by EGF or TGF-alpha. PMID- 7554059 TI - Inhibition of intestinal tumor development in rat multi-organ carcinogenesis and aberrant crypt foci in rat colon carcinogenesis by 22-oxa-calcitriol, a synthetic analogue of 1 alpha, 25-dihydroxyvitamin D3. AB - The modifying effects of 22-oxa-calcitriol (OCT), a synthetic analog of 1 alpha,25-dihydroxyvitamin D3, were assessed in a multi-organ carcinogenesis model using male F344 rats initially treated with five kinds of carcinogens. In experiment 1 the rats were given OCT intraperitoneally at doses of 30 micrograms/kg (25 rats) or 3 micrograms/kg (25 rats), three times a week for 24 weeks after initial carcinogen exposure over 4 weeks and a 2 week non-treatment period. Twenty-two rats received the five carcinogens and were given the vehicle intraperitoneally as a control. A further group of 10 rats was given the 30 micrograms/kg dose of OCT without prior carcinogen application. At the end of the total observation period of 30 weeks the carcinoma incidence in the small intestine of rats given 30 micrograms/kg OCT after carcinogen treatment was 0%. This incidence was significantly smaller when compared with the control group. The incidence of large intestine carcinomas in the 30 micrograms/kg OCT group showed a tendency to decrease. The numbers of small and large intestinal carcinomas per rat were also significantly lower in the group given 30 micrograms/kg OCT than after 3 micrograms/kg OCT or carcinogens alone. Attention was, therefore, focused on colon carcinogenesis and in experiment 2 30 micrograms/kg OCT administered six times a week to rats for 8 weeks after the last injection of N,N'-dimethylhydrazine (DMH) exposure. OCT significantly reduced the formation of DMH-induced aberrant crypt foci, considered to be putative preneoplastic lesions. In experiment 3 30 micrograms/kg OCT was administered six times a week to rats for 4 weeks without prior carcinogen treatment. The proliferating cell nuclear antigen labeling index for the colonic epithelium of rats given 30 micrograms/kg OCT was decreased. Ornithine decarboxylase and spermidine/spermine N1-acetyltransferase activities in colonic epithelium, assayed as indicators of cell proliferation, were not significantly decreased as compared with control group values. Furthermore, vitamin D receptors in colonic epithelium were not significantly increased. Thus the present study indicates that OCT can exert inhibitory effects on tumor development in the small and large intestines, although the mechanism is unclear. PMID- 7554060 TI - Uptake and distribution of N-acetylcysteine in mice: tissue-specific effects on glutathione concentrations. AB - Modulation of cellular thiols has been used to ameliorate the toxic side effects associated with cancer chemotherapy and is currently being investigated as a novel therapeutic strategy in cancer treatment. One of the most extensively studied modulators of thiol levels is N-acetylcysteine (NAC), a cytoprotective drug with multiple therapeutic applications, including use as an adjunct to cancer chemotherapy. Tissue-specific protective effects have previously been observed when NAC has been used in conjunction with chemotherapeutic alkylating agents, but the basis for this was unknown. In view of the contrasting cytoprotective effects of NAC in bladder and bone marrow we examined the effect of this compound on mouse liver, bladder and bone marrow glutathione (GSH) levels, as well as the disposition of 14C-labelled NAC. Radiolabelled NAC was taken up by the majority of tissues at varying rates and levels, except for the brain and spinal cord. The bladder, bone marrow and liver all took up the drug or its metabolites within 15 min of injection. NAC was not found to alter GSH concentrations in the liver, but increased GSH levels in the bladder approximately 2-fold. In contrast, the GSH content of bone marrow was found to decrease by 70-50% after NAC administration. When separate bone marrow cell populations were examined the decrease in GSH was associated with granulocytes, as opposed to lymphocytes, whose GSH levels remained unchanged. These findings provide a possible explanation for the differential cytoprotective effects of NAC. PMID- 7554062 TI - Malignant transformation of the liver tumour precursor cell line OC/CDE 22 by the four stereoisomeric fjord region 3,4-dihydrodiol 1,2-epoxides of benzo[c]phenanthrene. AB - In previous work we established the rat liver oval cell line OC/CDE 22 in order to study in vitro mechanisms of liver cell transformation. We have now exposed OC/CDE 22 cells to each of the four optically active fjord region dihydrodiol epoxides of benzo[c]phenanthrene to investigate their capacity for malignant transformation of liver cells. All four configurational isomers, which are among the most potent carcinogenic metabolites of polycyclic aromatic hydrocarbons tested in murine tumour models, malignantly transform OC/CDE 22 cells at a 2 microM dose level, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Neither cell growth in soft agar nor induction of tumor formation in newborn rats were achieved if confluent OC/CDE 22 cell cultures were exposed to each of the four stereoisomers and left in the confluent state for 4 weeks. In contrast, if confluent cells were exposed to the four stereoisomers, immediately split and then subcultured as usual, full transformation was accomplished. Our results indicate that the fjord region dihydrodiol epoxides of benzo[c]phenanthrene are highly efficient transforming agents for rat liver cells and that proliferation plays a pivotal role in the liver cell transformation process induced by polycylic aromatic hydrocarbons. PMID- 7554061 TI - Ornithine decarboxylase inhibitor attenuates NaCl enhancement of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine. AB - The effects of combined administration of NaCl and the ornithine decarboxylase (ODC) inhibitor 1,3-diaminopropane (DAP) on the incidence and number of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on the ODC activity of the gastric wall and the labeling index of the gastric mucosa were investigated in inbred Wistar rats. Rats were given drinking water with or without 2.5 g/l DAP and chow pellets with and without 10% NaCl ad libitum after 25 weeks of oral administration of MNNG. At week 52 feeding 10% NaCl resulted in significant increases in the incidence of gastric cancers, in the ODC activity of the antral portion of the gastric wall and in the labeling index of antral epithelial cells. Administration of both NaCl and DAP significantly reduced the enhancements by NaCl of gastric carcinogenesis, ODC activity of the antral wall and the labeling index of antral epithelial cells. These results suggest that inhibition of ODC attenuates NaCl enhancement of gastric carcinogenesis and that enhancement by NaCl of gastric carcinogenesis is mediated by polyamine biosynthesis. PMID- 7554063 TI - p53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines. AB - p53 inhibits cell cycle progression and DNA damaging cytostatics induce p53 protein expression, indicating that p53 responds to DNA damage. We have measured benzo[a]pyrene (BP)-induced DNA damage in association with p53 expression. The most relevant DNA adducts for carcinogenesis, benzo[a]pyrene-7,8-diol-9,10 epoxide-DNA adducts, were measured by synchronous fluorescence spectrophotometry and p53 immunohistochemistry using polyclonal antibody CM1, which detects both wild-type and mutated forms of p53. Activation of BP in A-549 lung carcinoma and MCF-7 breast adenocarcinoma cell lines containing wild-type p53 was followed by an increase in p53 protein expression. alpha-Naphthoflavone, an inhibitor of cytochrome P450 (CYP)1A1, decreased both the formation of diolepoxide metabolites and the p53 response. The cell lines not able to activate BP, A-427 and SK-LU-1 (both human lung carcinomas), SK-MES-1 (human lung squamous carcinoma) and human fibroblasts, did not show any increase in p53 immunohistochemistry. The OVCAR-3 ovarian adenocarcinoma cell line, containing a mutation in exon 7 of p53, and the SK-LU-1 cell line expressed very high levels of p53 protein before BP treatment and no increase in p53 immunohistochemistry was seen. These findings indicate that p53 protein is part of the response of the cells to BP-induced DNA damage. PMID- 7554065 TI - Up-regulation of resistance-related proteins in human lung tumors with poor vascularization. AB - Microvessel density was investigated by immunostaining endothelial cells for factor VIII antigen in 84 non-small cell lung carcinomas and compared with the expression of several resistance-related proteins. Glutathione S-transferase-pi, thymidylate synthase, metallothionein and, with limitations, P-glycoprotein were overexpressed in tumors with poor vascularization. PMID- 7554064 TI - Effects of consumption of Brussels sprouts on intestinal and lymphocytic glutathione S-transferases in humans. AB - A high intake of glucosinolate-containing cruciferous vegetables, such as Brussels sprouts (Brassica oleraceae), has been linked to a decreased cancer risk, but the underlying mechanism is still unclear. The aim of this study was to reveal possible modulating effects of consumption of Brussels sprouts on duodenal, rectal and lymphocytic (i) glutathione S-transferase (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content. Ten healthy non-smoking volunteers were randomly assigned to two groups in a cross over design. Five persons started on a glucosinolate-free diet (control period), while the other five consumed 300 g/day cooked Brussels sprouts, at the expense of 300 g glucosinolate-free vegetables (sprouts period). After 7 days the regimen was changed for a further week. At the end of both periods blood samples and duodenal and rectal biopsies were taken. Mean GST activity showed marked differences between duodenal, rectal and lymphocytic cytosols (737 +/- 54, 321 +/ 29 and 154 +/- 14 nmol/min/mg protein respectively), but was uninfluenced by the dietary regimen. Isozyme distribution varied greatly between the tissues. In duodenum GST-alpha, -pi, and -mu isozymes were expressed in considerable amounts (8441 +/- 1365, 3002 +/- 223 and 536 +/- 248 ng/mg protein respectively). Rectal biopsies also contained above three GST classes, but here GST-pi was the most pronounced expressed isozyme (2849 +/- 246) followed by GST-mu (495 +/- 242), while GST-alpha was only present in minor quantities (149 +/- 31). In lymphocytes only GST-pi (755 +/- 96) and GST-mu (83 +/- 54) could be detected. As a result of the dietary regimen rectal GST-alpha and -pi levels were slightly increased at the end of the sprouts period, by 30 and 15% respectively. GSH contents were uninfluenced by the dietary regimen. In conclusion, consumption of glucosinolate containing Brussels sprouts for 1 week results in increased rectal GST-alpha and pi isozyme levels. We hypothesize that these enhanced detoxification enzyme levels may partly explain the epidemiological association between a high intake of glucosinolates (cruciferous vegetables) and a decreased risk of colorectal cancer. PMID- 7554066 TI - Early formation of DNA adducts compared with tumor formation in a long-term tumor study in rats after administration of 2-nitrofluorene. AB - 2-Nitrofluorene (NF) is a model compound for nitroarenes which has been identified in diesel exhaust and in urban air. The current study was carried out to observe the carcinogenicity of different doses of NF to rats and DNA adduct formation in different organs at an early stage of NF administration. One group of rats was fed basal diet as a control, whereas the other three groups of rats were fed basal diet supplemented with different amounts of NF (0.24, 0.95 and 2.37 mmol NF/kg diet, referred to as low, medium and high dose, respectively). The rats were exposed to NF continuously for 11 months, after which all groups of rats were fed basal diet without NF for another 13 months. In the high dose group hepatocellular carcinomas were found in all rats (20/20), forestomach squamous carcinomas in 11 and cortical kidney carcinomas in 10 rats. Fifteen out of 19 rats fed the medium dose of NF had hepatocellular carcinomas, 16 had forestomach squamous carcinomas and 15 had cortical kidney carcinomas. The major tumors of the rats fed the low dose of NF were forestomach squamous carcinomas (10/18). DNA adducts formed in tumor target organs after 1, 2, 6 and 10 days NF administration were dose- and time-dependent. Ten days after the start of NF administration DNA adduct levels were found to be 54, 11 and 6 DNA adducts/10(8) normal nucleotides in forestomach, liver and kidney respectively. In the non-tumor target organs levels in the range 1.7-4.8 DNA adducts/10(8) normal nucleotides were found. DNA adduct formation in this study showed a good correlation with the localization of tumors, although there is a need for additional factors for tumor formation. The results indicate that DNA adduct formation is an important factor for tumor formation and suggest that DNA adducts could be used as biomarkers for genotoxic risk. PMID- 7554067 TI - Inhibition of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats by green and black tea. AB - In this study, we investigated the effects of green tea and black tea, when given either during or after carcinogen treatment, on esophageal tumorigenesis in male Sprague-Dawley rats. Rats were treated with N-nitrosomethylbenzylamine (NMBzA) (2.5 mg/kg, s.c., twice weekly) for 5 weeks; 39 weeks after the initial dose of NMBzA, 65% of the rats had esophageal tumors with an average of 1.4 +/- 0.3 tumors per rat. In the groups of rats receiving 0.6% of decaffeinated green tea (DGT) or decaffeinated black tea (DBT) (6 mg tea solids/ml) as the sole source of drinking fluid during the NMBzA-treatment period, esophageal tumor incidence and multiplicity were reduced by approximately 70%. When the tea preparations were given after the NMBzA treatment period, the esophageal papilloma incidence and multiplicity were reduced by approximately 50%. The volume per tumor was much smaller in rats that received black tea after the carcinogen treatment period. In a second experiment, NMBzA was given to rats at a dose of 3.5 mg/kg (s.c., twice weekly) for 5 weeks; after 16 weeks, the tumor incidence was 82% and tumor multiplicity was 6.7 +/- 1.2 tumors per rat. In the groups of rats receiving 0.9% regular green tea (RGT) or DGT after the NMBzA treatment period, tumor multiplicity was decreased by > 55%. The volume per tumor was reduced by approximately 60% in the rats receiving 0.9% RGT. Histological analysis indicated that both the incidence and multiplicity of esophageal carcinoma was decreased by either RGT or DGT. The blood and urine levels of green tea polyphenols due to tea administration were determined in rats, and the levels were comparable to those in humans after tea ingestion. The above results indicate that both green tea and black tea can inhibit the tumorigenic action of NMBzA during the period of carcinogen treatment and the subsequent molecular events important for esophageal tumorigenesis. PMID- 7554069 TI - Chemopreventive activity of Oltipraz against N-nitrosobis(2-oxopropyl)amine (BOP) induced ductal pancreatic carcinoma development and effects on survival of Syrian golden hamsters. AB - The synthetic dithiolethione Oltipraz has marked cancer chemopreventive and phase II enzyme inducing activity in various animal carcinogenesis models, but has not been examined in any animal models of ductal pancreatic cancer relevant to the human disease. The chemopreventive potential of Oltipraz on pancreatic tumor incidence and multiplicity was examined in the N-nitrosobis(2-oxopropyl)-amine (BOP)-induced ductal pancreatic adenocarcinoma model in Syrian hamsters. Animals were maintained on control semipurified diets or semipurified diets containing 300 and 600 mg/kg Oltipraz beginning 2 weeks prior to BOP initiation and throughout the 26 week study. Oltipraz at 300 mg/kg had no effect on the incidence or multiplicity of preneoplastic, neoplastic or metastatic lesions, while at 600 mg/kg dietary Oltipraz the incidence of pancreatic adenocarcinomas was reduced significantly (P < or = 0.05) compared to BOP-treated controls. Dietary Oltipraz at both doses had a significant influence on reducing mortality and morbidity in tumor-bearing animals with metastatic disease. At 26 weeks, total hepatic glutathione-S transferase (GST) activity and GST mu activity were elevated significantly in Oltipraz-treated animals, while total pancreatic GST activity was reduced, albeit not significantly. Serum lipase activity, a marker for pancreatic damage, exhibited a progressive decline in BOP-treated animals administered Oltipraz compared to BOP-treated controls at 12 weeks of the study; by week 26, lipase activity was comparable in all groups and reduced compared to activity at week 12. Positive nuclear immunostaining for the p53 tumor suppressor protein, a hallmark of human pancreatic cancer and a transient response to DNA damage, was observed in only a small percentage of BOP-induced pancreatic lesions and was not influenced Oltipraz administration. Further chemoprevention and pharmacologic studies of Oltipraz in relevant animal models of ductal pancreatic cancer could provide a foundation for future studies in human populations at potential risk for pancreatic cancer. PMID- 7554068 TI - 32P-postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA. AB - 32P-Postlabeling analysis of the bisphosphate derivatives was conducted to characterize the DNA adducts generated from the peroxidase-mediated activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP). Autoradiography of the D1 chromatogram of the postlabeled DNA hydrolysate revealed a major adduct (adduct 1) that migrated at Rf 0.15. An adduct with similar chromatographic characteristics was also obtained by postlabeling the products generated by chemical interaction of: (i) 2',6'-dichlorobenzoyloxy-4-acetylaminobiphenyl with the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) with calf thymus DNA. The adduct derived from chemical reaction exhibited the same mobilities on two-dimensional TLC as that obtained from the peroxidase-mediated DNA binding of N-OH-AABP. Moreover, on HPLC analyses, these bisphosphate derivatives exhibited identical retention times, suggesting that structurally they might be the same. Furthermore, adduct 1 was insensitive to digestion with nuclease P1. In addition to adduct 1, another minor adduct (adduct 2) was also detected in the peroxidase-mediated DNA binding of N-OH-AABP. The adduct 2 in D1 exhibited an Rf of 0.66. Adduct 2 was also observed in the DNA sample chemically interacted with N-OAc-AABP. Both these adducts retained the acetyl moiety, which was confirmed by the presence of radioactivity in the hydrolysate of DNA derived by interaction with N-OAc-[14C-acetyl]AABP (labeled at the N-acetyl group). Based on proton NMR and MS analyses of the 5'-phospho analogs of adducts 1 and 2, the structures of these have been identified as 3 (deoxyguanosine-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) and N-(deoxyguanosine-8 yl)-4-acetylaminobiphenyl (dG-C8-AABP). Analyses of the DNA samples obtained from human uroepithelial cells following exposure to N-OH-AABP revealed primarily the non-acetylated derivative N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP) with trace amounts of dG-N2-AABP. These results suggest that in the target cells for 4-aminobiphenyl carcinogenesis, the prevalence of the peroxidase mediated activation reaction of N-OH-AABP is relatively minor compared to the acetyltransferase pathway. PMID- 7554070 TI - Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed in Escherichia coli and by human liver microsomes. AB - Recent studies indicate that cytochrome P450 (P450) 3A4 plays important roles in the activation of procarcinogens such as aflatoxin B1 and sterigmatocystin, as well as in the oxidation of a number of structurally diverse chemicals and endogenous compounds. Since P450 3A5 has been reported to be present at significant levels in liver microsomes in approximately 25% of human adults, we examined and compared the role of P450 3A4 and 3A5 in procarcinogen activation in humans. Immunoblot experiments with liver microsomes from 60 human samples suggested that 4/30 Japanese and 4/30 Caucasians contained considerable levels of P450 3A5, although P450 3A4 could be determined at relatively high levels in all of the human samples examined. Good correlation was observed between P450 3A4, but not P450 3A5, levels versus activation of aflatoxin B1 and stergmatocystin in these human samples. Comparisons of the activation of procarcinogens in reconstituted monooxygenase systems containing modified P450 3A4 and 3A5 enzymes expressed in Escherichia coli were carried out in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strain for genotoxicity assay, and it was found that P450 3A4 had similar activities to or higher rates than P450 3A5 for the 24 procarcinogens tested. PMID- 7554071 TI - Inhibition of 4-nitroquinoline-1-oxide-induced rat oral carcinogenesis by dietary exposure of a new retinoidal butenolide, KYN-54, during the initiation and post initiation phases. AB - The modifying effect of dietary exposure of a newly synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone (KYN-54) during the initiation and post-initiation phases of oral carcinogenesis initiated with 4 nitroquinoline-1-oxide (4-NQO) was investigated in male F344 rats. At 6 weeks of age, rats were divided into experimental and control groups. At 7 weeks of age, all animals except those treated with KYN-54 alone and those in a control group were given 4-NQO (20 p.p.m.) in the drinking water for 8 weeks to induce oral neoplasms. Seven days after stopping 4-NQO exposure, groups of animals fed the diets containing 100 and 200 p.p.m. KYN-54 for 10 weeks starting 1 week before 4 NQO exposure were switched to the basal diet and kept on this diet until the end of the experiment. Starting 1 week after the cessation of 4-NQO administration, the groups given 4-NQO and the basal diet were switched to the diets containing 100 or 200 p.p.m. KYN-54 and maintained on these diets for 22 weeks. The other group consisted of rats given 200 p.p.m. KYN-54 alone or untreated rats. All animals were necropsied at the termination of the study (week 32). The incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell proliferation activity estimated by bromodeoxyuridine (BrdU) labeling index and by morphometric analysis of silver-stained nucleolar organizer regions protein (AgNORs) were compared among the groups. Feeding of KYN-54 during either initiation or post-initiation phase caused a significant reduction in the frequency of tongue carcinoma (48-71% reduction, P < 0.05) and such chemopreventive efficacy was dose-related. The incidences of preneoplastic lesion in rats fed the diets mixed with KYN-54 at both doses were also decreased (P < 0.05). Dietary administration of KYN-54 also significantly decreased the labeling index of BrdUrd and the number of AgNORs per cell nucleus of the tongue squamous epithelium (P < 0.05). In addition, polyamine levels in the oral mucosa were lowered in rats given 4-NQO and test compound when compared to those given 4-NQO alone, but no significant difference was obtained. These results indicate that the novel synthesized retinoidal butenolide KYN-54 inhibits oral carcinogenesis initiated with 4-NQO and such inhibition may be related to suppression of cell proliferation. PMID- 7554072 TI - Development of a 32P-postlabelling method for the analysis of adducts arising through the reaction of acetaldehyde with 2'-deoxyguanosine-3'-monophosphate and DNA. AB - A 32P-postlabelling assay was developed for the analysis of adducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphate with acetaldehyde, the primary oxidative metabolite of ethanol. The 32P-postlabelling reaction was optimized by testing various parameters such as the kinetics of phosphorylation by T4 polynucleotide kinase, substrate-concentration-dependent labelling efficiency and the concentration of the various ingredients of the phosphorylation reaction. The sensitivity to 3'-monophosphate dephosphorylation activity of nuclease P1 was also studied. Three stable adducts were separated by reversed-phase HPLC. The major stable adduct was structurally characterized and identified as N2-ethyl-2' deoxyguanosine and could be detected, after reduction with NaBH4 or a mixture of ascorbic acid and GSH, in calf thymus DNA samples that had been reacted in vitro with acetaldehyde. DNA adducts were isolated after enzymatic digestion to mononucleotides followed by nuclease P1 digestion of normal nucleotides. The average levels of acetaldehyde-DNA adducts detected in these samples were 12.1 +/ 2.3 (n = 17) and 4.9 +/- 0.9 (n = 9) adducts/10(7) nucleotides after reduction with NaBH4, or ascorbic acid and GSH respectively. The 32P-postlabelling method was further validated by the detection of acetaldehyde adducts in liver DNA from mice treated with ethanol. The average concentration of the adducts detected in these animals was 1.5 +/- 0.8 (n = 7) adducts/10(8) nucleotides, as analyzed by reversed-phase HPLC with online detection of radioactivity. PMID- 7554073 TI - 2,3,7,8-Tetrachlorodibenzo-p-dioxin sensitization of cultured human epidermal cells to carcinogenic heterocyclic amine toxicity. AB - Treatment of cultures of spontaneously immortalized human epidermal cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitized them to carcinogen toxicity. While the tryptophan pyrolysis product 3-amino-1,4-dimethyl-5H pyrido[4,3-b]indole (Trp-P-1) and the mycotoxin sterigmatocystin were highly toxic to the cultures at moderate concentration (1 microgram/ml), the potency of each agent was increased > or = 10-fold in the presence of TCDD. A toxicity increase was also evident in the several-fold stimulation by TCDD of protein and DNA adducts formed by Trp-P-1. In contrast, the cells were insensitive to toxicity from 3-amino-1-methyl-5H-pyrido[4,3-b]indole. DNA damage mediated by Trp P-1 was capable of producing inheritable effects, as judged by the induction of hprt mutants in a TCDD-stimulated fashion. Northern blotting showed that TCDD strongly stimulated expression of P4501A1 and 1B1 in the cells, enzymes important for xenobiotic metabolism. These findings demonstrate the potential usefulness of SIK cultures as a model for studying keratinocyte responses to carcinogens activated by TCDD-induced cytochromes P450. PMID- 7554074 TI - Deregulated expression of cyclin D1 and other cell cycle-related genes in carcinogen-induced rat mammary tumors. AB - Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15 fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans. PMID- 7554075 TI - Lack of effect of retinoic acid and fluocinolone acetonide on mirex tumor promotion indicates a novel mirex mechanism. AB - Mirex, a halogenated hydrocarbon, is a potent skin tumor promoter in 7,12 dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. In the present study retinoic acid (RA) and fluocinolone acetonide (FA), classical inhibitors of phorbol ester- and non-phorbol ester-type skin tumor promoters, were examined for their ability to inhibit mirex tumor promotion. Female CD-1 mice were initiated with 200 nmol DMBA and promoted with equipotent promoting doses of either 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) or 200 nmol mirex twice weekly for 25 weeks and RA (2(1 or 5 nmol), FA (0.5 or 2 nmol) or acetone were applied 30 min prior to each TPA or mirex dose. TPA-promoted papilloma formation was strongly inhibited by > 70% with both doses of RA and by > 90% with both doses of FA. In contrast, mirex-promoted papilloma formation was not inhibited by either dose of RA or 0.5 nmol FA and 2 nmol FA weakly inhibited mirex-promoted papillomas by only 32%. TPA- and mirex-promoted papillomas that were refractory to RA and FA demonstrated the same incidence of Ha-ras mutation as TPA- or mirex-promoted papillomas without RA and FA treatment, further indicating that the inhibitory activity of RA and FA is promoter-dependent and not solely dependent on mutant Ha ras. FA (2 nmol) treatment completely abolished TPA-induced epidermal hyperplasia and proliferating cell nuclear antigen (PCNA) S phase-positive cells, however, FA had no inhibitory effect on the weak proliferative response induced by mirex. Collectively, these results indicate that the promotional activity of mirex, as well as its weak proliferative response, result from a distinct promoter mechanism and/or that mirex promotes a unique population of epidermal cells that are insensitive to FA and RA and cannot be distinguished by their mutant Ha-ras genotype. PMID- 7554076 TI - Higher lung cancer risk for younger African-Americans with the Pro/Pro p53 genotype. AB - A restriction fragment length polymorphism in codon 72 of the p53 gene has been implicated in lung cancer risk, although the functional significance of the polymorphism has not been determined. This association was examined in 109 lung cancer cases (67 African-American and 42 Mexican-American) and 114 controls (74 African-American and 40 Mexican-American) identified from a molecular epidemiological study of lung cancer. The susceptible Pro/Pro genotype was associated with a 1.56-fold higher risk of lung cancer in African-Americans and a 1.95-fold in Mexican-Americans, although neither estimate was statistically significant. In fact, the prevalence of the Pro/Pro genotype was only 2.5% in Mexican-American controls, compared with 20.3% for African-American controls. Patients with the susceptible genotype appeared to have earlier age at diagnosis and lower mean cigarette pack-year exposures than did patients with the Arg/Arg or Arg/Pro genotypes. Risk estimates for the susceptible genotype were 11.29 (1.1, 111.3) for patients < 53 years of age and 14.1 (1.5, 130.6) for patients who reported < 30 pack-years of smoking. The Pro/Pro genotype was not associated with elevated risk in older patients, nor with heavier smokers. If Pro/Pro is a susceptible genotype, the lower prevalence evident in Mexican-Americans may partly explain their lower rates of lung cancer. PMID- 7554077 TI - Association of cigarette smoking and CYP1A1 polymorphisms with adenocarcinoma of the lung by grades of differentiation. AB - We have previously reported on an association of genetic susceptibility to squamous cell carcinoma of the lung with two polymorphisms of the CYP1A1 gene, an MspI polymorphism and an isoleucine-valine (Ile-Val) polymorphism. We report here that the two CYP1A1 polymorphisms were associated with current or ex-smokers with adenocarcinoma of the lung. We first compared smoking status of the patients by differentiation grades of adenocarcinoma. Proportion of current or ex-smokers and their cigarette consumption was the highest among the poorly differentiated. Second, the two polymorphisms of CYP1A1 were examined among current or ex-smokers with three differentiation grades, and we found that poorly differentiated adenocarcinoma showed significantly high frequencies of genotypes C and Val/Val, which have been found to be 'susceptible' in squamous cell carcinoma of the lung. Third, a case-control study was carried out to estimate the genetic risk for CYP1A1 genotypes to differentiation grades, selecting only current or ex-smokers in patients and controls. Current or ex-smokers with 'susceptible' genotype C or Val/Val were at significant risk, with an odds ratio of 3.25 or 3.22 (95% confidence interval (CI), 1.40-7.56 or 1.00-10.32) for adenocarcinoma as a whole, respectively. The odds ratio increased to 4.51 or 4.09 (95% CI, 1.73-11.78 or 1.12-14.91) for poorly differentiated, while the odds ratios for other differentiated grades were not significant. Finally, a relation of the genetic risk to cigarette dose levels was evaluated. PMID- 7554078 TI - Liarozole potentiates the cancer chemopreventive activity of and the up regulation of gap junctional communication and connexin43 expression by retinoic acid and beta-carotene in 10T1/2 cells. AB - Liarozole has been reported to inhibit P450 enzymes responsible for the catabolism of retinoic acid. This suggests that it may increase the effectiveness of cancer chemopreventive agents, such as retinoic acid, and pro-vitamin A carotenoids, such as beta-carotene, which may yield retinoids. To test this we have utilized the 10T1/2 cell assay system of neoplastic transformation. Simultaneous treatment with Liarozole (10(-5) M) potentiated by a factor of 1000 the ability of low concentrations of retinoic acid (10(-10) M) to inhibit carcinogen-induced neoplastic transformation, to up-regulate gap junctional communication and to increase connexin43 expression. When tested under conventional culture conditions, Liarozole itself partially suppressed neoplastic transformation and up-regulated gap junctional communication; this ability appears due to the presence of retinol in the serum component of cell culture medium. When assays for junctional communication and of connexin43 expression were performed under defined conditions, in the absence of serum, Liarozole was ineffective alone, yet still augmented the effects of retinoic acid. HPLC analysis of cell culture medium demonstrated that Liarozole (10(-5) M) completely protected retinoic acid (10(-6) M) from catabolism over a 48 h period. Potential effects of Liarozole on the activities of carotenoids were also examined. Inhibition of neoplastic transformation by the pro-vitamin A carotenoid beta carotene, but not by the non-pro-vitamin A carotenoid canthaxanthin, was moderately potentiated by Liarozole. The augmentation of response to beta carotene was more apparent when tested under defined conditions; here Liarozole strongly increased junctional communication and Cx43 expression. In contrast, Liarozole did not potentiate the activity of canthaxanthin under defined conditions, while increasing the activity of 4-keto retinoic acid, a likely metabolite. Liarozole also elevated connexin43 expression in early passage human fibroblasts. These data indicate that rapid catabolism of retinoic acid limits its in vitro activities, that a portion of the action of beta-carotene as a cancer preventive agent is due to its conversion to retinoic acid and that canthaxathin exerts its chemopreventive action largely independent of conversion to 4-keto retinoic acid. PMID- 7554079 TI - Failure of genotoxic carcinogens to produce tumors in human skin xenografts transplanted to SCID mice. AB - Chemical carcinogenesis of human skin was investigated using human skin xenografts (16 full thickness and 48 split thickness skin grafts) transplanted to CB-17-scid (SCID) mice. Topical application of a carcinogen, i.e. 7,12 dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene, methylcholanthrene or N-methyl N'-nitro-N-nitrosoguanidine, to the human skin xenografts once a week for 25-30 weeks failed to produce skin tumors. Both DMBA application plus UV-B irradiation and alternate applications of the above four carcinogens in combination with UV-B irradiation also failed to produce tumors. All of these treatments induced skin papillomas in skins of host SCID mice. DMBA induced skin papillomas in allogenic CD-1 mouse skin grafts transplanted to SCID mice. These results indicate that susceptibility of human skin to these carcinogenic stimuli is much lower than that of mouse skin. PMID- 7554080 TI - Strong anti-mutagenic activity of the novel lipophilic antioxidant 1-O-hexyl 2,3,5-trimethylhydroquinone against heterocyclic amine-induced mutagenesis in the Ames assay and its effect on metabolic activation of 2-amino-6-methyldipyrido[1,2 a:3',2'-d] imidazole (Glu-p-1). AB - Antimutagenic effects of a novel lipophilic antioxidant, 1-O-hexyl-2,3,5 trimethylhydroquinone (HTHQ), and other known antioxidants against heterocyclic amine- or other mutagen-induced mutagenesis were examined in the Ames assay using Salmonella strain TA 98 to access the chemo-preventive effects of antioxidants on heterocyclic amine-induced carcinogenesis. Further the mechanisms of inhibition by HTHQ were accessed. HTHQ was shown to potently inhibit mutagenesis induced by all of 8 different heterocyclic amines at rates between 100% and 63% in the presence of S9 mix. When the protection of HTHQ against 2-amino-6- methyldipyrido[1,2-alpha:3',2'-d]imidazole (Glu-P-1)-induced mutagenesis was compared with known antioxidants t-butylhydroquinone, propyl gallate, BHA, BHT and alpha-tocopherol, HTHQ showed the greatest effect. Among hexyl, butyl, ethyl and methyl derivatives of 1-O-alkyl-2,3,5-trimethylhydroquinone, HTHQ was the most effective in inhibiting Glu-P-1-, 3-amino-1-methyl-5-H-pyrido[4,3-b]indole (Trp-P-2)- or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced mutagenesis. On the other hand, HTHQ did not inhibit mutagenic activity induced by other mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-(2-furyl)-3-(5 nitro-2-furyl)acrylamide (AF-2) and benzo[a]pyrene. HTHQ weakly inhibited that due to direct mutagen 2-nitro derivative of 2-amino-3,8-dimethylimidazo[4,5 f]quinoxaline (MeIQx) only in the presence of S9 mix. No such influence on a 2 nitro derivative of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis, was observed with or without the S9 mix. HTHQ slightly inhibited mutagenesis induced by activated Glu-P-1, a direct acting proximate metabolite of Glu-P-1, in the absence of the S9 mix. HPLC analysis revealed activated Glu-P-1 to be formed by incubating Glu-P-1 with the S9 mix, but this was considerably decreased by the addition of HTHQ. These results indicate that HTHQ is a powerful antimutagenic compound and specifically acts against heterocyclic amines. Its antimutagenic activity appeared to exert by both inhibiting metabolic activation of heterocyclic amines and action on activated N-hydroxy species. PMID- 7554082 TI - In vivo antimutagenic activity of beta-carotene in rat spleen lymphocytes. AB - The anticarcinogenic effects of beta-carotene (BC) have been extensively investigated, but only in vitro assays have examined the ability of BC to modulate gene mutation. In view of the current interest in the provitamin as a cancer chemopreventive agent, and the association between mutagenesis and carcinogenesis, we have dosed Fischer 344 rats with model carcinogen N-ethyl-N nitrosourea (ENU) and investigated the relationships among BC intake, its tissue accumulation, and antimutagen activity. Animals received drinking water supplemented with BC at doses of 0-0.25% ad libitum, using three dosing schedules. In one group BC dosing commenced before, and continued for three alternating weeks after i.p. injection of 100 mg ENU/kg; another group was given BC only after mutagen treatment. Animals from the first two groups were sacrificed 5 weeks post-mutagen treatment, and cells were isolated from the spleen to determine the frequency of 6-thioguanine- resistant (6-TGr) T lymphocytes. The presence of BC caused a reduction in the frequency of 6-TGr T cells produced by ENU, but the inhibition was non-linear within the range of BC doses used. BC intake only after mutagen treatment was more effective than the combination of pre- and post-mutagen intake. In the third group, rats were treated with 100 mg ENU/kg, and BC administration was continued at a fixed dose of 0.15% in the drinking water for 2, 4, 6, or 8 weeks. Measurement of the frequency of 6-TGr T-cells at the end of the specified times showed > 50% reduction in ENU-mediated mutagenicity throughout the experiment. Analysis of BC levels in the liver and in the spleen following BC intake before and during mutagen exposure revealed higher levels than when BC was given only after mutagen treatment. Continuous intake of BC also showed increased tissue levels. There were some correlations observed between BC tissue levels and the antimutagenic effects for the first two groups, but these correlations were not statistically significant, possibly due to the small numbers of animals used. Taken together, the results demonstrate that intact BC is absorbed, stored, and exerted antimutagenic effects against a chemical carcinogen in rats without first being transformed to retinol in the gastrointestinal tract. PMID- 7554084 TI - Simultaneous immunoaffinity purification of O6-methyl, O6-ethyl-, O6-propyl- and O6-butylguanine and their analysis by gas chromatography/mass spectrometry. AB - A sensitive and specific method has been developed for the simultaneous analysis of different O6-alkylguanines. The cross-reactivity of two different antibodies raised against O6-methylguanosine and O6-butylguanosine for a series of O6 alkylguanines was exploited for the immunoaffinity purification of biological samples before quantitative analysis by gas chromatography/mass spectrometry. The method can be applied to the detection of O6-alkylguanines in DNA and appears to be useful for studying chemical carcinogen mechanisms in animals and possibly for the detection of human exposure to alkylating agents. PMID- 7554083 TI - Inhibitory effect of chlorophyllin on PhIP-induced mammary carcinogenesis in female F344 rats. AB - Chlorophyll and chlorophyllin, a water-soluble salt of chlorophyll, have been reported to inhibit carcinogen-DNA binding and exert antimutagenic activity for some carcinogenic heterocyclic amines and aflatoxins. In the present experiment, the possible inhibitory effects of chlorophyllin on 2-amino-1-methyl-6 phenylimidazo[4,5-b] pyridine (PhIP) carcinogenicity were investigated. Female F344 rats were administered both PhIP, 0.02% in the diet, and chlorophyllin, 1%, in the diet (group 1), or either PhIP (group 2) or chlorophyllin (group 3) alone for 54 weeks. The incidence of mammary adenocarcinomas induced by PhIP was reduced by chlorophyllin co-administration from 40% (8/20 rats) to 15% (3/20). While the difference was not statistically significant, the multiplicity of adenocarcinomas was significantly (P < 0.05) reduced by chlorophyllin co administration from 0.50 per animal to 0.15. On the other hand, incidence of colon adenomas was slightly, but not significantly, increased from 10% to 20%. Neither mammary nor colon adenocarcinomas were observed in group 3. Thus, chlorophyllin reduced PhIP mammary carcinogenesis, suggesting that chlorophyllin is an effective chemopreventor when ingested simultaneously with the carcinogen. PMID- 7554081 TI - P53 polymorphisms and haplotypes in lung cancer. AB - An association between the BstU I 1-1 (Pro-Pro) genotype of the p53 codon 72 polymorphism and lung cancer has previously been reported by Kawajiri et al. A reanalysis of the data by Kawajiri et al. revealed no significant difference between patients and controls with respect to allele frequencies, and the increased frequency of BstU I 1-1 homozygotes was mostly ascribable to a deviation from the Hardy-Weinberg equilibrium. In an attempt to replicate the results by Kawajiri et al. we have studied three p53 polymorphisms (BstU I and Msp I RFLPs in exon 4 and intron 6 respectively and a 16 bp duplication in intron 3) and their haplotypes in Swedish lung cancer patients and controls. The results concerning the codon 72 polymorphism were largely negative. Thus there was no significant association between lung cancer and the BstU I 1-1 type, and only a marginal difference (P = 0.044) with respect to the BstU I allele frequency when lung cancer patients were compared with patients with chronic obstructive pulmonary disease (COPD). However, when the analysis was based on haplotype frequencies larger differences appeared and it was found that only BstU I 1 (pro) alleles linked to 16 bp 1 alleles were associated with lung cancer. Pro alleles linked to the 16 bp duplication appeared instead to confer some protection against cancer. Thus the codon 72 alleles need not be functionally involved in lung cancer, but may rather be markers in linkage disequilibrium with other cancer susceptibility sites on p53. PMID- 7554085 TI - Formation of a mutagen, glyoxal, from DNA treated with oxygen free radicals. AB - Oxygen free radicals cause extensive chemical changes in DNA, including base and sugar modifications and strand breaks. In the present study we found that a mutagen, glyoxal, is produced by exposure of DNA to an oxygen radical forming system (5 mM FeSO4-EDTA, 37 degrees C, 60 min). It was produced with 17 times higher efficiency than 8-hydroxydeoxyguanosine. Thus it is possible that the formation of glyoxal is one of the major types of damage in DNA exposed to oxygen free radicals. PMID- 7554086 TI - The role of mRNA stability and transcription in O6-methylguanine DNA methyltransferase (MGMT) expression in Mer+ human tumor cells. AB - We have recently reported that following depletion of O6-methylguanine DNA methyltransferase (MGMT) activity by acute streptozotocin (STZ) treatment to sensitize innately chloroethylnitrosourea (CENU)-resistant HT-29 cells, the eventual repletion of activity occurs with no concommitant alterations in steady state MGMT mRNA levels. This suggestion of a potentially stable transcript prompted studies to define the relative contributions of MGMT mRNA stability and transcription to cellular MGMT expression. Northern analysis of MGMT mRNA in actinomycin D-treated HT-29, MR-1 and A2182 cells, ranging in relative MGMT expression from high to low respectively, demonstrates relatively long MGMT mRNA half-lives of > 10-12 h. Cell lines with low and moderate levels of MGMT mRNA appear to have longer mRNA half-lives than those with high levels. Run-on transcription in nuclei isolated from cells with low to moderate MGMT mRNA levels demonstrates undetectable basal MGMT transcription rates. Collectively these data suggest that a very low transcription rate, coupled with a stable mRNA molecule, might result in the translation of pre-existing mRNA molecules. This translation may be responsible for the gradual recovery of MGMT and CENU resistance over 24 h following MGMT depletion. PMID- 7554088 TI - Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides. AB - Antisera developed against benzo[a]pyrene diolepoxide (BPDE)-DNA adducts are sensitive tools for detection of DNA adducts in human samples. All antisera currently used for biomonitoring studies were produced against DNA or guanosine modified with racemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbent assay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565-572, 1994) recently tested polyclonal and monoclonal (5D2) antisera for cross reactivity against oligonucleotides containing (+)-and (-)-trans-anti-BPDE-N2 guanine or N6-adenine adducts and showed different stereospecificity for the two antisera. Because of the importance of antiserum specificity in human biomonitoring studies, we have tested several monoclonal (Mab 5D11 and 5D2) and polyclonal (Pab #29) antisera developed against racemic anti-BPDE-DNA adducts, and Mab 8E11 developed against anti-BPDE-guanosine adducts. Stereoisomeric anti BPDE-modified oligonucleotide adducts in the sequence 5'-d(CC-AT-CG*CTACC)-3' where G* = anti-BPDE-N2-dG with (+)-trans, (-)-trans, (+)-cis and (-)-cis adduct stereochemistry at the C10 position of anti-BPDE were tested by competitive ELISA. Two structurally related 5-methylchrysene diolepoxide adducts with G* = (+)- and (-)-trans-anti-5-MeCDE-N2-dG in the same oligonucleotide were also tested. While Mab5D2 had the highest affinity for the (-)-trans-anti-BPDE modified oligomer, Mab 5D11 and 8E11 and Pab #29 recognized the (+)-trans-anti BPDE-modified oligomer better than the (-)-trans-anti-BPDE modified oligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modified DNA adducts better than trans-anti-BPDE-modified oligonucleotides; however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNA adducts and (+)- and (-)-trans-anti-BPDE modified oligomers. All antisera exhibited lower reactivities with both 5-MeCDE modified oligomers. Because of their sensitive detection of (+)-trans-anti-BPDE dG adducts, the primary adduct produced in vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurement of most adducts formed in humans. PMID- 7554087 TI - Colon carcinoma cells with inactive nm23 show increased motility and response to motility factors. AB - nm23H1 has properties of a metastasis suppressor gene. Although its mechanism of action is unknown, nm23 has been implicated in transforming growth factor beta 1 (TGF beta 1) signal transduction. In an earlier study we decreased nm23 mRNA levels 2- to 8-fold by antisense phosphorothiolated oligonucleotides in two HT29 colon carcinoma sublines at different stages in tumor progression with different responses to TGF beta 1: the HD3 subline, which shows TGF beta 1-induced growth arrest and differentiation; and the more tumorigenic U9 subline, whose growth and invasion are stimulated by TGF beta 1. Only TGF beta 1-mediated responses in HD3 cells were inhibited by nm23 antisense oligos, suggesting that nm23 functions in only one TGF beta 1 signaling pathway. In the current report we have extended this study to cell motility. HD3 motility was increased by nm23 phosphorothiolated antisense oligos which decrease nm23 mRNA levels, while HD3 cell motility was conversely decreased by TGF beta 1 which increases nm23 mRNA levels. HD3 motility was not increased by basic FGF, TGF beta 1 or TGF alpha, while the 13-fold higher basal motility of U9 cells was stimulated 3-fold by basic FGF, 4-fold by TGF beta 1 and 5-fold by TGF alpha, but not by scatter factor. Differences in motility and response to motility factors could not be ascribed to differences in either basal levels of proteases or modulation of their levels by TGF beta 1. Both HD3 and U9 cells displayed equal levels of urokinase activity and mRNA, equal expression of the metalloproteinase inhibitor TIMP-1, and no detectable collagenases by zymography. No differential response to TGF beta 1 was seen in any of these assays. Thus limited cell motility and lack of response to motility factors in HD3 colon cancer cells could be correlated with expression of nm23 active in signal transduction. PMID- 7554092 TI - Effect of dietary fat or tamoxifen on the expansion of cells harboring Ha-ras oncogenes in mammary glands from methylnitrosourea-treated rats. AB - Diets containing high levels of fat enhance the formation of methylnitrosourea (MNU)-induced mammary gland adenocarcinomas in rats, while administration of the antiestrogen tamoxifen decreases the incidence of these tumors. It is not known, however, at what stage during tumor development the fat or tamoxifen exert their effects. Here we have used a PCR/liquid hybridization and gel retardation assay to determine the effects of dietary fat and tamoxifen on the growth rate of cells harboring an Ha-ras oncogene in the mammary glands of rats at various times following MNU administration. Glands from animals on a high-fat diet had significantly higher mutant cell fractions than those on a low-fat diet at both 30 and 75 days following MNU treatment. In contrast, there was no difference between the mutant cell fractions of tamoxifen-treated animals and controls at either 30 or 70 days. These results suggest that dietary fat promotes tumor formation early in carcinogenesis by stimulating the growth of cells harboring Ha ras mutations, while tamoxifen delays the appearance of tumors either by acting as a tumoristatic or tumoricidal agent, or by acting to eliminate or retard the growth of preneoplastic cells just prior to the emergence of tumors. PMID- 7554091 TI - DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4,5 f]quinoline (IQ) in liver, kidney and colo-rectum of rats. AB - DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) have been measured in the liver, kidney, and colo-rectum of male Fischer-344 rats given a single oral dose of IQ (20 mg/kg). The pattern and distribution of DNA adducts examined by 32P-postlabeling was similar in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3 methylimidazo-[4,5-f]quinoline (dG-C8-IQ) was the principal adduct identified and it accounted for approximately 50-70% of the observed radioactivity, followed by (deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ) which accounted for 15-20% of the radioactivity. Twenty-four hours after IQ treatment, DNA modification was greatest in the liver at a level of 7.64 +/- 1.08 adducts per 10(7) bases, followed by kidney at 2.04 +/- 0.32 adducts per 10(7) bases, and colorectum at 1.08+/-0.22 adducts per 10(7) bases. Liver and colo-rectum are target tissues of tumorigenesis in the rat during chronic feeding studies with IQ; however, tumors are not formed in the kidney. Therefore, factors in addition to IQ-guanine adduct formation, such as adduct persistence, error-prone repair, and tumor promotion must contribute to organ susceptibility of IQ-induced carcinogenesis. PMID- 7554089 TI - Repair of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-induced damage by mammalian cell extracts. AB - The repair of damage induced by the alkylating antitumor drug 1-(2-chloroethyl)-3 cyclohexyl-1-nitrosourea (CCNU) was investigated using an in vitro excision repair system. Hamster cell extracts prepared from the parental CHO-9 cell line and the ERCC1 mutant 43-3B were both proficient in the repair of CCNU-induced damage. The in vitro repair of CCNU damage was faster than the repair of UV damage and plasmid substrates were rapidly and efficiently incised after incubation with either CHO-9 or 43-3B extracts. 7-Methylguanine (7-meG) and 3 methyladenine (3-meA) glycosylases were active to a similar extent in the CHO-9 and 43-3B extracts. The data indicate that most damage induced by CCNU is repaired via the ERCC1-independent base excision repair pathway, initiating with removal of chloroethylated and hydroxyethylated bases by N-glycosylases. Yet, the sensitive phenotype of 43-3B cells suggests that the ERCC1 gene product is required for the removal of a small subset of CCNU-induced lesions that are important for drug cytotoxicity. PMID- 7554090 TI - Transfer and expression of human O6-methylguanine-DNA methyltransferase cDNA confers resistance of Mer- HeLa MR cells to 1-(4-amino-2-methyl-5 pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a. AB - Previous studies have demonstrated that O6-methylguanine-DNA methyltransferase (MGMT) is a major contributor to tumor cellular resistance toward chloroethylnitrosoureas. To further clarify the effect of MGMT gene expression on cellular chemosensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)-3-(2-chloroethyl) 3-nitrosourea (ACNU) in human cells, a repair-deficient human tumor cell line (HeLa MR) was transfected with a human MGMT expression vector (pSV2MGMT-neo). Multiple unique transfectants were isolated which exhibited variable levels of MGMT mRNA by Northern hybridization analysis. Vector-transfected controls were generated simultaneously. Transfectants expressing high levels of MGMT activity showed an increased resistance to ACNU-induced cytotoxicity. Furthermore, the levels of the protective effect against ACNU correlated generally with the levels of introduced MGMT expression. This study further provided direct evidence of MGMT contribution to ACNU resistance in human tumor cells. Based on the results presented here, we also discussed the perspective of the clinical utility of MGMT cDNA transfer and expression. PMID- 7554094 TI - Osmotic effects of water compartments in protein polymers. PMID- 7554095 TI - Lipid films in water conservation of biological systems. PMID- 7554096 TI - Mitochondrial water loss and aging of cells. PMID- 7554093 TI - Micro-osmosis in gels, cells and enzymes. PMID- 7554097 TI - Cellular water content and volume regulation in animal cells. PMID- 7554098 TI - Hydropharmacology. PMID- 7554099 TI - Bivascular liver perfusion in the anterograde and retrograde modes: zonation of the response to inhibitors of oxidative phosphorylation. AB - The action of cyanide (500 microM), 2,4-dinitrophenol (50 microM) and atractyloside (100 microM) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4 dinitrophenol and attractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 microM, had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge. PMID- 7554101 TI - The secretion of urokinase-like plasminogen activator is inhibited by microtubule interacting drugs. AB - The secretion of proteinases into the extracellular matrix is one of the main features of tumour cells, as related to their invasive behaviour. Considering the role of the microtubule cytoskeleton, and particularly the action of microtubule associated protein (MAPs) in mediating protein secretion, the effects of the anti microtubule drugs estramustine and taxol, on the secretion of urokinase-type plasminogen activator (u-PA) and the 72 kDa gelatinase were investigated. Treatment of 5637 bladder carcinoma cells with estramustine and taxol inhibited u PA secretion into the conditioned medium in a drug concentration-dependent fashion. This inhibition was confirmed by determinations of u-PA enzymatic activities and by measurements of the levels of immunoreactive activator. Studies using gelatin zymograms also showed an inhibition of another tumoural proteinase namely the 72 kDa gelatinase. Time-course uptake experiments showed that estramustine was incorporated into the cells, a process which depended on temperature. On the other hand, immunofluorescence studies indicated that the microtubule network was affected by taxol with the formation of bundles of microtubules at different cell domains. Minor effects were visualized after treatment of the cells with estramustine-phosphate, a drug that blocks primarily the action of microtubule-associated proteins. The studies provide a way to analyse the relationships between u-PA secretion and the integrity of the cytoskeletal network. PMID- 7554100 TI - N-3 but not N-6 fatty acids reduce the expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells. AB - CD36, a multifunctional adhesion receptor e.g. for thrombospondin and collagen, as well as a scavenger receptor for oxidized low density lipoprotein, is expressed e.g. on platelets and monocytes. By this dual role it might be involved in early steps of atherosclerosis like the recruitment of monocytes and formation of foam cells. We therefore studied the effects of n-3 fatty acids on CD36 expression in human monocytic cells. Incorporation of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) into cellular phospholipids resulted in a significant reduction of CD36 expression at the mRNA and protein level, whereas arachidonic acid (AA, C20: 4n-6) and linoleic acid (LA, C18:2n-6) tended to increase CD36 expression compared to the control. This specific down regulation of CD36 by n-3 fatty acids in cells involved in the initiation and progression of atherogenesis and inflammation, represents a further mechanism that may contribute to the beneficial effects of n-3 polyunsaturated fatty acids (PUFA) in these disorders. PMID- 7554102 TI - Cation-induced efflux of calcium from the mitochondria of Hymenolepis diminuta. AB - We have previously reported that Ca2+ influx into the mitochondria of Hymenolepis diminuta, a rat intestinal cestode, takes place through an electrophoretic uniport system. Sodium and lithium were found to induce efflux of 45Ca2+ from the mitochondria of H. diminuta. The two cations induced the efflux in a hyperbolic and linear fashion, respectively. The efflux as well as an exchange of external Ca2+ with internal 45Ca2+ was inhibited by lanthanum. The type of Ca2+ transport system in the cestode organelle has been discussed and compared with that of the host (mammalian) counterpart. PMID- 7554103 TI - Pathways to a child mental health service. AB - The mothers of 46 children who had been assessed by staff from a child mental health service were interviewed about the process by which they had come to be referred. The sequence of contacts with agencies was described and children coming via different pathways compared. Contacts with school teaching staff, and general practitioners were commonly part of the pathway, and health visitors were an important group of carers for younger children. When children had had contact with general practitioners and health visitors they were likely to be referred directly to the mental health service, but school staff had mostly sought help from other agencies. PMID- 7554105 TI - Two cases of severe head-banging parasomnias in peripubertal males resulting from otitis media in toddlerhood. AB - The author presents two case reports of peripubertal boys with severe and chronic head-banging parasomnias, or rhythmic movement disorders (RMDs), which started de novo whilst they were suffering from recurrent and severe otitis media in toddlerhood. Treatment consisting of straightforward behaviour modification techniques and advice concerning healthy sleeping habits proved highly successful. It is proposed that these late-onset or 'acquired' forms of RMD might be distinct from those which continue from infancy which have a stronger developmental aetiology. PMID- 7554104 TI - Sleep disruption in young children. The influence of temperament on the sleep patterns of pre-school children. AB - Sleep problems are a common concern of parents with toddlers. Various reasons have been put forward as to the possible causes and maintenance of sleep disruption. These have included neurophysical differences in the child, perinatal differences such as a long labour and depression and anxiety in the parents themselves resulting in adverse effects on the child's sleep patterns. However, recent research has indicated that a child's temperamental style may be a possible causal factor as to whether the child will present its parents with a sleep problem. This study, therefore, sets out to investigate the importance of temperament as a predictor of whether children may or may not have sleep problems. Environmental factors are controlled as far as possible by selecting toddlers with and without sleep problems from very similar environments. Case studies are also introduced to further illuminate other possible associated factors such as parental handling. The results from this study found significant differences in the temperament profiles of children with and without sleep problems. Children with reported sleep problems were more likely to obtain a 'intermediate high to difficult' profile. Various findings from a group of toddlers with and without sleep problems are discussed in this paper together with implications of the findings and recommendations for further research. PMID- 7554106 TI - Children's help-seeking behaviour. AB - A study of children's help-seeking behaviour with respect to bullying and parental arguing is reported. A number of specific hypotheses and open-ended questions were investigated with boys and girls aged 8-17 years. Female helpers were preferred overall, though a relationship existed between the child's and helper's gender. Parents, friends, and teachers were all chosen, and parent and peer helpers were seen as complementary rather than competitive sources of help. Reasons for choosing helpers were notably varied, but largely related to perceived qualities of the helper rather than the child's own needs. Children depicted helping as an active response on the part of the helper. These findings are discussed and implications for practice noted. PMID- 7554107 TI - Controversy ... in the best interests of the child!!!.... PMID- 7554108 TI - Thrombin and proliferation of vascular smooth muscle cells. PMID- 7554109 TI - Ligation of endothelial alpha v beta 3 integrin increases capillary hydraulic conductivity of rat lung. AB - Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin alpha v beta 3. Vitronectin, SC5b-9, and SC5b-9-enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the alpha v beta 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the alpha v beta 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5 dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located alpha v beta 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation. PMID- 7554110 TI - Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells. AB - The present study investigated the effect of adrenomedullin, a novel vasorelaxant peptide, on the migration of cultured rat vascular smooth muscle cells (SMCs) by using the Boyden-chamber method. Fetal calf serum (FCS) and platelet-derived growth factor (PDGF)-BB strongly stimulated SMC migration. Adrenomedullin clearly inhibited SMC migration stimulated with 5% and 10% FCS in a concentration dependent manner. The migration induced by 10 and 25 ng/mL PDGF-BB was also inhibited by adrenomedullin in a concentration-dependent manner. Inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration was paralleled by an increase in the cellular level of cAMP. In fact, the percent increase in cAMP level was strongly correlated with the percent decrease in migration activity of SMCs after treatment with adrenomedullin. 8-Bromo cAMP, a cAMP analogue, reproduced the inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration. An activator of adenylate cyclase, forskolin, also reduced FCS- and PDGF-induced SMC migration. These data indicate that adrenomedullin inhibits the migration of SMCs stimulated with FCS and PDGF, probably through a cAMP-dependent process. On the basis of these results and the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as a local antimigration factor in some pathophysiological states. PMID- 7554111 TI - Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Asp-dependent endothelial migration in vitro. AB - Osteopontin is an Arg-Gly-Asp (RGD)-containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the alpha v beta 3 integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and beta 3-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, beta 3-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell-derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the alpha v beta 3 receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through alpha v beta 3. These data suggest important roles for osteopontin and beta 3 integrin in regenerating endothelium. PMID- 7554112 TI - Inhibitory effects of insulin on cytosolic Ca2+ level and contraction in the rat aorta. Endothelium-dependent and -independent mechanisms. AB - To determine the mechanism of the inhibitory effect of insulin on vascular tone, contraction was measured simultaneously with endothelial and smooth muscle cytosolic Ca2+ level ([Ca2+]i) in the isolated rat aorta. Insulin (200 mU/mL) increased endothelial [Ca2+]i and decreased resting muscle tone. The removal of endothelium abolished the effects of insulin. In the aorta precontracted with norepinephrine, insulin (3 to 120 mU/mL) induced concentration-dependent inhibition of contraction. The relaxant effect followed the increase in endothelial [Ca2+]i and decrease in smooth muscle [Ca2+]i. The relaxant effect was attenuated by removal of endothelium or by the addition of 10(-5) mol/L NG monomethyl-L-arginine but not by 10(-5) mol/L indomethacin. In the absence of endothelium, the relaxant effect of insulin followed the decrease in smooth muscle [Ca2+]i. These results suggest that insulin inhibits vascular contraction by dual mechanisms in the isolated rat aorta: (1) Insulin acts on vascular endothelium by increasing endothelial [Ca2+]i and releasing NO, which decreases smooth muscle [Ca2+]i and the Ca2+ sensitivity of the contractile elements. (2) Insulin also directly acts on smooth muscle and decreases smooth muscle [Ca2+]i. PMID- 7554114 TI - Differential effects of pressure and flow on DNA and protein synthesis and on fibronectin expression by arteries in a novel organ culture system. AB - Structural adaptation of the blood vessel wall occurs in response to mechanical factors related to blood pressure and flow. To elucidate the relative roles of pressure, flow, and medium composition, we have developed a novel organ culture system in which rabbit thoracic aorta, held at in vivo length, can be perfused and pressurized at independently varied flow and pressure for several days. Histology and histomorphometry, as well as scanning electron microscopy, revealed a well-preserved wall structure. In arteries perfused and pressurized at 80 mm Hg, endothelial injury led to a 2-fold increase in [3H]thymidine incorporation in the media, which peaked at 3 to 5 days and returned to baseline level at 6 to 8 days. In intact endothelialized vessels cultured for 3 days under no-flow conditions, pressure per se had no effect on DNA synthesis. In contrast, in the presence of serum, total protein synthesis, as assessed by [35S]methionine incorporation into the media, was enhanced 6-fold at 150 mm Hg compared with vessels pressurized at 0 or 80 mm Hg. In intact vessels perfused at a constant flow of 40 mL/min for 3 days, DNA synthesis was unchanged regardless of the pressure level when vessels were cultured in the presence of serum but increased 8-fold at both 80 and 150 mm Hg in the absence of serum. Unlike DNA synthesis, total protein synthesis was enhanced 12-fold by flow regardless of the presence or absence of serum. Expression of fibronectin was markedly enhanced at high transmural pressure, and serum potentiated its expression in the arterial wall. This novel organ culture system of perfused and pressurized vessels allowed identification of differential effects of pressure, flow, and serum on DNA and total protein synthesis, including cellular fibronectin expression. PMID- 7554113 TI - Antisense to thyrotropin releasing hormone receptor reduces arterial blood pressure in spontaneously hypertensive rats. AB - We report in the present study the effect of intrathecal treatment with antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH) receptor mRNA on the pressor response to intrathecal administration of TRH and on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester antisense or mismatch oligonucleotides to TRH receptor mRNA (100 micrograms per day) were injected intrathecally for 3 days. Twenty-four hours after the last injection, the magnitude of the pressor response to intrathecal TRH (10 micrograms) was significantly smaller in the antisense-treated group (n = 7) compared with mismatch-treated controls (n = 7) (change in mean arterial pressure, +20.3 +/- 3.0 versus +32.6 +/- 2.5 mm Hg, P < .01). No differences were observed in the pressor responses to injection of N-methyl-D-aspartic acid. Resting arterial blood pressure was unaffected by antisense treatment in WKY rats. In separate experiments, 16-week-old male SHR were treated with antisense (n = 7) or mismatch (n = 6) oligonucleotides for 3 days. Mean resting arterial blood pressure was significantly reduced by treatment with antisense oligonucleotides (from 157 +/- 4.8 to 119 +/- 8.8 mm Hg, P < .01), but no significant changes were observed in mismatch-treated animals. Our results suggest that the expression of TRH receptors in spinal sympathetic preganglionic neurons can be selectively reduced by intrathecal treatment with antisense oligonucleotides and that TRH projections to sympathetic preganglionic neurons play an important role in the elevation of arterial blood pressure in SHR. PMID- 7554116 TI - Corticosterone metabolism and effects on angiotensin II receptors in vascular smooth muscle. AB - It has been postulated that mineralocorticoids can bind to corticosteroid receptors in the kidney, because glucocorticoids are metabolized to inactive compounds. The present study was performed to delineate glucocorticoid metabolism by rat vascular tissue and to determine the activity of these metabolites. Vascular segments converted 25% to 30% of corticosterone (compound B), the major glucocorticoid in the rat, to 11-dehydrocorticosterone (compound A) but not to aldosterone or 6 beta-hydroxycorticosterone. In cultured vascular smooth muscle cells, 10% of compound B was converted to compound A, whereas > 60% of compound A was converted to compound B. The 11 beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone (1 mumol/L) completely blocked conversion in both directions. Whereas 6 beta-hydroxycorticosterone did not upregulate angiotensin II receptor binding (a marker for corticosteroid action in vascular smooth muscle), compound A caused concentration-dependent upregulation. Compound A was almost (75%) as effective and as potent as compound B in upregulating angiotensin II binding. Upregulation elicited by exposure to compound A persisted in the presence of 1 mumol/L carbenoxolone, which completely prevented the conversion of compound A to compound B. Compound A, even in the presence of carbenoxolone, effected other glucocorticoid actions by inhibiting cell growth and potentiating angiotensin II stimulated inositol phosphate formation. In summary, compound B and compound A are interconverted in vascular tissue, and the latter displays significant glucocorticoid action. The concentration excess of compound B in the circulation and the activity of its metabolite compound A will make it difficult for mineralocorticoids to gain access to corticosteroid receptors in the vasculature. PMID- 7554115 TI - L-arginine-induced conducted signals alter upstream arteriolar responsivity to L arginine. AB - Our purpose was to determine whether L-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg i.p.) hamsters (N = 53). The upstream branch arterioles dilated significantly to locally applied L-arginine (100 mumol/L pipette concentration) only if the downstream branches (approximately 1400 microns away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, -3.8 +/- 1.5% (of baseline); third, +58.1 +/- 27%; first, +92 +/- 26% (n = 5); with exposure order upstream to downstream: first branch, -0.4 +/- 3%; third, +5 +/- 11%; last, -5.6 +/- 7.5% (n = 4). Thus, downstream preexposure to L arginine altered the responsivity upstream to locally applied L-arginine. Downstream-applied L-arginine also induced a conducted vasodilation (+17.8 +/- 2.8%; n = 14) 1327 +/- 166 microns upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or N omega-nitro-L arginine (L-NNA, 100 mumol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of L-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1 +/- 2.6%, n = 8) was also completely blocked by sucrose, halothane, or L NNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554117 TI - Oxidant stress enhances adenylyl cyclase activation. AB - The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 mumol/L to a maximum of 166% of control activity; n = 4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149 +/- 13%; H2O2 + tyrphostin A9, 100 +/- 9%; H2O2 + tyrphostin A1, 171 +/- 21%; n = 4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166 +/- 17% of control, n = 5) and sodium fluoride (177 +/- 18% of control, n = 5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation. PMID- 7554119 TI - Propionyl L-carnitine improvement of hypertrophied heart function is accompanied by an increase in carbohydrate oxidation. AB - Propionyl L-carnitine (PLC) is a naturally occurring derivative of L-carnitine that can improve hemodynamic function of hypertrophied rat hearts. The mechanism(s) responsible for the beneficial effects of PLC is not known, although improvement of myocardial energy metabolism has been suggested. In this study, we determined the effect of PLC on carbohydrate and fatty acid metabolism in hypertrophied rat hearts. Myocardial hypertrophy was produced by partial occlusion of the suprarenal aorta of juvenile rats. Over a subsequent 8-week period, a mild hypertrophy developed, resulting in a 17% increase in heart weight in these animals compared with the sham-operated control animals. Myocardial carnitine was decreased in hypertrophied hearts compared with hearts from sham operated animals (4155 +/- 383 versus 5924 +/- 570 nmol.g dry wt-1, respectively; P < or = .05). Perfusion of isolated working hearts for 60 minutes with buffer containing 1 mmol/L PLC increased carnitine content in hypertrophied hearts from 4155 +/- 383 to 7081 +/- 729 nmol.g dry wt-1 (P < or = .05). In the presence of 1.2 mmol/L palmitate, fatty acid oxidation rates were not decreased in the hypertrophied hearts compared with control hearts. PLC treatment did not alter rates of fatty acid oxidation in control hearts but did result in a small increase in rates in the hypertrophied hearts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554118 TI - High- and low-affinity sites for [3H]dofetilide binding to guinea pig myocytes. AB - Dofetilide specifically blocks the rapid component of the delayed rectifier current (IKr) at nanomolar concentrations in a saturable manner, suggesting the presence of a receptor. We characterized two [3H]dofetilide binding sites to ventricular myocytes from adult guinea pigs by using a conventional filter assay. Scatchard analysis revealed two binding sites with different affinities: a high affinity site (Kd, 2.8 +/- 0.3 x 10(-8) mol/L; Bmax, 76 +/- 15 fmol/10(6) myocytes) and a low-affinity site (Kd, 1.64 +/- 0.4 x 10(-6) mol/L; Bmax, 1620 +/ 260 fmol/10(6) myocytes) (n = 11). Kinetic studies showed that there were two dissociation rate constants for [3H]dofetilide (0.02 +/- 0.005 min-1 [high affinity site] and 0.22 +/- 0.064 min-1 [low-affinity site], n = 4), although the observed association rate constant is equally well fit to a single- or two-site model. The ability of known IKr blockers to compete with [3H]dofetilide binding to both sites was assessed. E4031, clofilium, quinidine, and sotalol competed for binding at both sites. Disopyramide and NAPA only competed for a single binding site. The mean IC50 values for inhibition of binding to both the high- and low affinity binding sites correlated with their concentrations required to inhibit IKr in electrophysiological studies. However, inhibition of [3H]dofetilide binding to the high-affinity site by class III antiarrhythmic drugs occurred at pharmacological concentrations, whereas suprapharmacological concentrations were required to inhibit binding to the low-affinity site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554120 TI - An isolated cardiac conduction disease maps to chromosome 19q. AB - Isolated cardiac conduction disease is an autosomal dominant defect that includes various combinations of bundle branch or fascicular blocks. These defects can cause sudden death due to a complete heart block. We used a genome-wide screening approach with polymorphic (CA)n repeat markers to determine the chromosomal position of the gene defect implicated in this disorder. The analyses were carried out on a large Lebanese kindred, which included individuals with either a complete or incomplete right bundle branch block (RBBB) with a vertical-axis deviation (< or = -30 or > or = +100). Linkage to the disease locus was detected with the polymorphic marker D19S604 on the q arm of chromosome 19 (19q13.3) with a multipoint lod score of 7.18. Additionally, we were able to exclude the flanking loci D19S606 and D19S571, which are 13 cM apart because of recombination events in three affected individuals. The histidine-rich calcium-binding protein gene is found in this region and is an attractive candidate gene on the basis of its physiological properties and a tight linkage. There is no expansion in two exon 1 regions known for a variable number of triplet repeats. PMID- 7554121 TI - cGMP and atrial natriuretic factor regulate cell volume of rabbit atrial myocytes. AB - Atrial natriuretic factor (ANF) reduces the volume of atrial myocytes by inhibiting Na+/K+/2Cl- cotransport. We determined the role of cGMP and cAMP in ANF-induced shrinkage by using digital video microscopy to measure cell volume; volumes are reported relative to control. ANF (1 mumol/L) reversibly reduced atrial cell volume from 1.0 to 0.915 +/- 0.005 (mean +/- SEM). This effect was mimicked by 10 mumol/L 8-bromo-cGMP (8-Br-cGMP), which decreased myocyte volume to 0.894 +/- 0.007 with an ED50 of 0.99 +/- 0.05 mumol/L. In contrast, 100 mumol/L 8-bromo-cAMP (8-Br-cAMP) did not affect volume, and activating the cAMP pathway with 100 mumol/L 8-Br-cAMP did not alter the volume decrease caused by 8 Br-cGMP or ANF. Inhibition of Na+/K+/2Cl- cotransport with bumetanide (1 mumol/L) also reduced cell volume and prevented further shrinkage on subsequent exposure to 8-Br-cGMP. Similarly, 8-Br-cGMP (10 mumol/L) prevented further shrinkage by ANF. Block of Na(+)-H+ exchange, a participant in volume regulation in other cells, did not alter the response to 8-Br-cGMP. More evidence implicating cGMP was obtained by altering its metabolism. LY83583 (10 mumol/L), a guanylate cyclase inhibitor, blocked ANF-induced cell shrinkage. Zaprinast (100 mumol/L), a cGMP-specific phosphodiesterase inhibitor, markedly potentiated the effect of a threshold concentration of ANF (0.01 mumol/L). The actions of ANF, LY83583, and zaprinast on cGMP levels were verified by radioimmunoassay. These data strongly support the idea that the cGMP cascade is the intracellular signaling pathway responsible for ANF-induced atrial cell shrinkage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554123 TI - Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca(2+) ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. AB - The objective of this study was to elucidate the role of the sarcoplasmic reticulum (SR) in the transition from compensated pressure-overload hypertrophy (increased left ventricular [LV] mass, normal LV function, and no pulmonary congestion) to congestive heart failure (increased LV mass, depressed LV function, and pulmonary congestion). To address this issue, the descending thoracic aorta was banded for 4 and 8 weeks in adult guinea pigs, and the changes in isovolumic LV mechanics, SR Ca2+ transport, and SR protein levels were determined and compared with age-matched sham-operated control animals. A subgroup of the 8-week banded animals manifested the congestive heart failure phenotype with diminished developed LV pressure normalized by LV mass, reduced rates of LV pressure development and relaxation, and markedly increased lung weight-to-body weight ratios. The cardiac mechanical and morphometric changes were associated with depressed protein levels of the SR Ca(2+)-ATPase (85% of the control) and phospholamban (65% of the control) assessed by quantitative immunoblotting. Resultant rates of SR Ca2+ uptake (Vmax) and the affinity of SR Ca(2+)-ATPase for Ca2+ (EC50) were significantly depressed [32 +/- 6 nmol Ca2+.min-1.mg-1 and 0.59 +/- 0.12 (mumol/L)/L, respectively] compared with the 8 week sham-operated control animals [40 +/- 1 nmol Ca2+.min-1.mg-1 and 0.40 +/- 0.05 (mumol/L)/L, respectively]. We conclude that this model of pressure overload induced cardiac failure is associated with (1) diminished LV force development, rates of pressure development, and decay; (2) depressed protein expression of the Ca(2+)-cycling proteins SR Ca(2+)-ATPase and phospholamban; and (3) decreased Vmax and affinity of the SR Ca(2+)-ATPase for Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554122 TI - Gene expression of natriuretic peptide receptors in myocardial cells. AB - Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that serve to unload the heart through their effects on the kidney and vasculature. Whether the heart itself represents a site of action for these peptides is currently the subject of debate. Although functional studies indicate that ANP has some effects on isolated myocytes, several studies have been unable to detect binding of the hormone to these cells. The present study demonstrates that the genes for all three natriuretic peptide receptor (NPR) subtypes, NPR-A, NPR-B, and NPR-C, are expressed in the rat heart. For microlocalization of the receptor mRNAs in myocytes and nonmyocytic cells, a combination of cell isolation and reverse transcription-polymerase chain reaction (RT-PCR) was used. Cardiac myocytes were isolated by enzymatic dissociation of rat ventricular tissue, purified by density gradient centrifugation, and collected as single cells under microscopic control. Analysis by RT-PCR revealed the presence of transcripts for NPR-A as well as NPR-B and NPR-C. However, cGMP generation in purified myocytes was stimulated only by ANP and BNP, which specifically bind to NPR-A, whereas C type natriuretic peptide (CNP, an NPR-B agonist) was ineffective. Therefore, rat ventricular myocytes appear to produce predominantly NPR-A. The expression of NPR B may be low or even absent. The mRNAs for all three NPRs were also found in cultures of fibroblasts from the rat heart. In contrast to the myocytes, large increases in cGMP were observed in response not only to ANP but also to CNP. PMID- 7554124 TI - New insights into the gating mechanisms of cardiac ryanodine receptors revealed by rapid changes in ligand concentration. AB - We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channels into planar phospholipid bilayers in order to investigate changes in [Ca2+] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca2+] has a direct effect on the gating of the cardiac SR Ca(2+)-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca2+], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca2+] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at -40 mV when the channel is activated by Ca2+ alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation. PMID- 7554125 TI - Nuclear magnetic resonance studies of cationic and energetic alterations with oxidant stress in the perfused heart. Modulation with pyruvate and lactate. AB - The postischemic generation of oxygen-derived free radicals may contribute to myocardial reperfusion injury by affecting sarcolemmal ion transport. Recent evidence indicates that exposure to reactive oxygen intermediates induces rapid increases in myocardial cytosolic free Ca2+ (Ca2+i). The mechanism is undetermined but may involve disturbances in Na+ homeostasis. We tested this hypothesis by interleaving 23Na and 31P nuclear magnetic resonance (NMR) measurements of Na+i and high-energy phosphates in glucose-perfused rat hearts exposed to hydroxyl radicals generated from H2O2 and Fe3+. In separate experiments, K+i and Ca2+i were measured with 39K and 19F NMR, respectively. The hearts rapidly exhibited contracture. Threefold Na+i increases and substantial K+i depletion were observed. Glycolytic inhibition was indicated by rapid sugar phosphate accumulation and cellular energy depletion. Notably, however, severe functional and energetic deterioration and substantial elevation of Ca2+i occurred before substantial Na+i accumulation or K+i depletion was observed. Further experiments investigated the ability of pyruvate to scavenge H2O2 and to protect the myocardium from oxidant stress. Pyruvate (1 or 2.5 mmol/L) dramatically attenuated functional and energetic alterations and alterations in Na+i and K+i, whereas acetate (2.5 mmol/L) offered no protection. Unlike pyruvate, lactate (5 mmol/L) has little or no capacity to scavenge H2O2 but has similar protective effects. In conclusion, pyruvate effectively protects against H2O2/Fe3+, largely by direct H2O2 scavenging. Protection with lactate may involve intracellular pyruvate augmentation. Without exogenous pyruvate or lactate, myocardial Na+ homeostasis can be substantially altered by oxidant stress, possibly via cellular energy depletion. Excess Na+i accumulation may, in turn, hasten metabolic and functional deterioration, but a causal link with the initial alterations in function or Ca2+i was not supported. PMID- 7554126 TI - Optical mapping reveals that repolarization spreads anisotropically and is guided by fiber orientation in guinea pig hearts. AB - Guinea pig hearts were stained with a voltage-sensitive dye and imaged on a photodiode array to record fluorescent action potentials (APs) from 124 sites. Activation and repolarization patterns were recorded from the epicardium during stimulation at different loci and correlated with the underlying fiber architecture. Endocardial APs were recorded by inserting a light guide into the ventricular cavity or by dissecting out the ventricular free wall to expose the endocardium. In hearts paced on the right atrium to simulate sinus rhythm, activation emerged synchronously over a large area of the ventricular epicardium and spread laterally in 5 to 7 ms. The apparent longitudinal and transverse velocities were 2.66 +/- 0.11 and 1.65 +/- 0.09 m/s (n = 12). In contrast, repolarization began near the apex on the endocardium and spread transmurally in 6 +/- 1.3 ms (n = 12) and then anisotropically along the epicardium in 25 to 30 ms with apparent maximum (0.53 +/- 0.11 m/s) and minimum (0.31 +/- 0.10 m/s) repolarization velocities that aligned with the longitudinal and transverse axes of epicardial fibers. When paced on the epicardium, activation of intact hearts (n = 12) and perfused sheets (n = 8) was anisotropic, with longitudinal (0.85 +/- 0.05 m/s) and transverse (0.44 +/- 0.04 m/s) conduction velocities that aligned with the epicardial fiber orientation. When activation was initiated at different sites on the epicardium, repolarization always began near the apex and exhibited patterns similar to those obtained under right atrial pacing, but with slower longitudinal (0.41 +/- 0.09 m/s) and transverse (0.23 +/- 0.07 m/s) repolarization velocities (n = 18). In sheets stretched parallel to the longitudinal axis of surface fibers, AP durations (APDs) increased as a function of fiber length, from the length at zero developed tension to 120% of the length at maximum developed tension (Lmax). Spatial distributions of APDs did not change during stretches along the rising phase of the length-tension curve. In sheets stretched to 50% of Lmax, APDs were shorter and more homogeneous on the endocardium (mean APD, 188 ms; delta APD, 195-186 = 9 ms) than on the epicardium (mean APD, 204 ms; delta APD, 212-186 = 26 ms; n = 8). In guinea pig hearts, activation is rapid; therefore, repolarization depends primarily on intrinsic spatial heterogeneities of APDs. Consequently, repolarization begins at endocardial cells with the shortest APDs and spreads transmurally and then anisotropically on the surface according to the epicardial cell orientation. PMID- 7554127 TI - cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes. AB - Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites. PMID- 7554128 TI - Unique conductance, gating, and selective permeability properties of gap junction channels formed by connexin40. AB - Connexin40 is selectively expressed in specialized cardiac conduction (nodal and His-Purkinje) tissues and the atrium, yet the channel properties formed by this gap junction protein have not been investigated. The conductance, gating, and selective permeability of rat connexin40 (Cx40) gap junction channels between pairs of Cx40-transfected mouse neuroblastoma (N2A) cells in culture were studied by using dual whole-cell voltage-clamp techniques. The macroscopic steady state junctional conductance gating was dependent on transjunctional voltage with a Boltzmann half-inactivation voltage of +/- 50 mV, a residual voltage-insensitive normalized junctional conductance of 35% of maximum, and a gating charge valence of 3. In the presence of 120 mmol/L potassium glutamate, the slope conductance of single rat Cx40 gap junction channels measured 158 +/- 2 pS (n = 4). Lower conductance states equal to 21% to 48% of the main open-state conductance were also occasionally observed in two of the four cell pairs. Multichannel open probabilities were found to be heterogeneous. Ion substitution and dye transfer experiments were performed to determine the relative chloride/potassium conductance and dye permeability of anionic fluorescein derivatives in rat Cx40 channels. The rat Cx40 channel had a maximum conductance of 180 +/- 18 pS (n = 3) in 120 mmol/L KCl and a detectable chloride permeability of 0.29 relative to potassium, indicating some selectivity for cations over anions. Cx40 gap junctions were permeable to 2',7'-dichlorofluorescein (diCl-F) and also to the more polar 6-carboxyfluorescein dye; however, diCl-F dye transfer was not observed to increase with increasing junctional conductance. PMID- 7554129 TI - Effect of mechanical forces on growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role of individual cell types. AB - The effect of mechanical stimuli on pulmonary artery growth and matrix tissue synthesis (and how individual cell types in the vessel wall respond to such stimuli) is incompletely characterized. Rabbit pulmonary arteries were placed in tissue culture medium and subjected to varying magnitudes of stretch or hydrostatic pressure (separately) for 4 days. The rate of protein synthesis in smooth muscle cells (by quantitative autoradiography) was positively related to the magnitude of stretch, as were the percentage of procollagen type I-positive cells and the rate of cell replication. In adventitial fibroblasts, stretch increased the rate of replication but not of protein synthesis. Hydrostatic pressure had little or no effect on the variables measured in either smooth muscle cells or fibroblasts. Stretch also increased the rate of elastin and collagen synthesis in the whole pulmonary artery segment, and after 4 days of stretch, the contents of actin and elastin were increased. Removal of the endothelium did not affect stretch-induced protein, collagen, or elastin synthesis but augmented stretch-induced smooth muscle replication. These data suggest that in the intact pulmonary artery, stretch, but not pressure, can stimulate hypertrophy and hyperplasia in smooth muscle cells and hyperplasia in fibroblasts. Matrix protein synthesis and accumulation are also increased by stretch. Neither stretch-mediated growth nor matrix protein synthesis required endothelium in this model. PMID- 7554130 TI - Modulation of cerebral arteriolar diameter by intraluminal flow and pressure. AB - We determined whether cerebral arterioles in vitro adjust their diameters in response to changes in intraluminal flow rate and pressure. Intracerebral arterioles (38- to 55-microns diameter) were isolated from Sprague-Dawley rats and cannulated with a perfusion system that permitted separate control of intraluminal pressure and flow rates. Increasing pressure at 0 flow, in 20 mm Hg steps from 20 to 100 mm Hg, resulted in myogenic constriction, which was greatest at 60 mm Hg (approximately 20%). Increasing flow rate at a constant pressure of 60 mm Hg elicited a biphasic response. At flow rates of up to 10 microL/min, the arterioles dilated by up to 14.5 +/- 2.2% of their control diameter. At higher (> 10 microL/min) flow rates, however, a progressive restoration of resting diameter was observed. Application of the nitric oxide synthase inhibitor NG-mono-methyl-L arginine (L-NMMA, 0.1 mmol/L) caused a 15.4 +/- 1.7% decrease in control diameter (at 60 mm Hg, zero flow). Although L-NMMA did not affect the responses to increases in pressure or to vasodilators (adenosine and pH 6.8 buffer), it abolished the dilator responses to flow rate increases and to acetylcholine. In contrast, inhibition of prostaglandin synthesis by indomethacin (10 mumol/L) had no effect on flow-induced dilation. These results show that changes in intraluminal flow rates and pressure can independently influence cerebral arteriolar tone and suggest that the flow-induced dilator responses of cerebral arterioles are mediated by an arginine metabolite, such as nitric oxide. PMID- 7554131 TI - Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. AB - This study was designed to elucidate whether nitric oxide (NO) controls norepinephrine (NE) release from sympathetic nerves of the rat heart. Hearts were perfused in the Langendorff mode with Tyrode's solution. The right sympathetic nerve was stimulated with trains of 1 or 3 Hz and NE release was measured. The NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) enhanced the evoked NE release in a concentration-dependent manner. This facilitation was independent of the increase in perfusion pressure and was stereospecifically reversed by L arginine but not D-arginine. Another NOS inhibitor, NG-methyl-L-arginine, produced a similar increase in NE release. The NO-donor compound S-nitroso-N acetyl-D,L-penicillamine, added in the presence of L-NNA, restored the suppression of NE release in a concentration-dependent fashion. A similar suppression was achieved with 3-morpholinosydnonimine. These results demonstrated that NE release is under the inhibitory control of endogenous NO. Western blots demonstrated the presence of neuronal NOS I and endothelial NOS III in the hearts. Perfusion of the hearts with a low concentration of the detergent CHAPS produced functional damage of the endothelium, as evidenced by an increase in perfusion pressure and a conversion of the acetylcholine-induced coronary vasodilation to a constriction. However, CHAPS treatment did not produce a facilitation of NE release (as did the NOS inhibitors), and L-NNA still increased NE release in CHAPS-treated hearts. Double-labeling immunofluorescence histochemistry showed NOS I immunoreactivity in stellate ganglion cells and in neurons of the heart, some of which also stained positive for tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554132 TI - Regional capture of fibrillating ventricular myocardium. Evidence of an excitable gap. AB - Previous investigations have suggested that during ventricular fibrillation (VF) pacing stimuli are incapable of evoking propagated ventricular activations. To determine whether regional myocardial capture could be achieved during rapid pacing in VF, extracellular unipolar potentials were sampled (2 kHz) and recorded from 506 Ag-AgCl electrodes arranged in a rectangular grid (22 x 23, 1.12-mm spacing) embedded in a plaque overlying two pacing electrodes in the epicardium of the anterobasal right ventricle in pentobarbital-anesthetized pigs (25 to 30 kg, n = 6). During separate episodes of electrically induced VF, two bursts of 40 monophasic stimuli (10 mA, 2-millisecond duration) were asynchronously applied to the stimulating electrodes in either a bipolar, unipolar anodal, or unipolar cathodal mode. Evidence of regional capture was provided by (1) animating the first temporal derivative of the extracellular potentials, (2) analyzing inter beat interval patterns, and (3) employing the Karhunen-Loeve decomposition method to quantify the repetitiveness of spatio-temporal patterns of activation. Regional capture of ventricular myocardium during VF was observed when pacing stimuli fell late in the local myocardial activation interval and when the pacing cycle length was 80% to 115% of the mean subplaque activation cycle length. When myocardial activations became phase locked to the pacing stimuli, repeatable spatiotemporal patterns of activation followed each stimulus. Poincare sections at the plaque border revealed that during VF prior to pacing, interbeat intervals were irregular but were driven by pacing to stable fixed values at times corresponding to our qualitative declaration of regional capture. A similar correspondence was demonstrated between the time of capture, defined by direct observation of the activation patterns, and a rise in the power contained in the first two spatial modes of a Karhunen-Loeve decomposition. These data demonstrate that appropriately timed stimuli produce regional capture of fibrillating right ventricular myocardium in the pig and support the existence of an excitable gap during VF in this model. PMID- 7554133 TI - The mechanically active domain of titin in cardiac muscle. AB - One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this approximately 1 micron long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the approximately 1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at approximately 50 to 100 nm. In sarcomeres that are slack (length approximately 1.85 microns), the mechanically active domain is folded on top of itself, and the length of the domain reaches an elastic limit of approximately 550 nm in sarcomeres that are approximately 2.9 microns long. PMID- 7554134 TI - Upregulation of cardiac angiotensin II AT1 receptors in congenital cardiomyopathic hamsters. AB - Angiotensin II (Ang II) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in cardiac myocytes. To gain insight into the role of Ang II in cardiac hypertrophy, we examined the expression and subtype distribution of Ang II receptors in the ventricles of embryonic and of 25- to 350 day-old inbred control and cardiomyopathic (CHF 146) hamsters. Studies were also performed with heterozygous (cardiomyopathic x control) animals. Compared with the control hamsters, cardiomyopathic hamsters presented decreased body weights and increased ratios of ventricular weight to body weight in every adult group studied. Typical histological lesions appeared in the left ventricle of cardiomyopathic animals around 70 to 75 days, and their severity increased with time. Radioligand binding studies with cardiac ventricular membranes indicated that iodinated [Sar1,Ile8]Ang II (sarile) binds to a homogeneous population of sites in membranes derived from adult normal and cardiomyopathic animals. Competition curves using specific receptor subtype antagonists revealed that 125I sarile binding sites were exclusively of the AT1 subtype in both groups of animals. Importantly, the density of AT1 receptors was found to be significantly increased (90% augmentation at 70 to 75 days) in the ventricles of cardiomyopathic hamsters. This augmented expression was observed in all adult groups and was already present at 25 days, when no histological lesions were visible. The affinity of the receptor for losartan did not vary significantly between adult normal and cardiomyopathic animals (mean Kd, 19.6 and 16.7 nmol/L, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554135 TI - Direct effects of smooth muscle relaxation and contraction on in vivo human brachial artery elastic properties. AB - The direct effect of smooth muscle relaxation on arterial elastic properties is controversial. Studies in animals show both a decrease and an increase in elastic modulus. In human subjects, the contribution of smooth muscle to arterial elastic mechanics has been limited by difficulty in separating the direct effects of a vasodilator drug on the arterial wall from the indirect effects due to reduced blood pressure. The purpose of the present study was to assess the direct contribution of vascular smooth muscle to brachial artery elastic mechanics in normal human subjects in vivo. We measured brachial artery compliance and incremental elastic modulus (Einc) in eight normal subjects (age, 22 to 51 years) by using intravascular ultrasound. A 3.5F 30-MHz intravascular ultrasound catheter was placed through a sheath into the brachial artery, and intraarterial pressure, cross-sectional area, and wall thickness were measured simultaneously under baseline conditions and after the administration of intra-arterial nitroglycerin (100 micrograms) and norepinephrine (1.2 micrograms). A pressurized cuff surrounding the brachial artery was inflated to reduce transmural brachial artery pressure. Using this technique, we were able to measure the following arterial characteristics for the first time in human subjects in vivo: (1) the effective unstressed arterial radius and (2) the pressure-area, stress-strain, and pressure-Einc relations over a wide pressure range (0 to 100 mm Hg). Intra arterial nitroglycerin increased brachial artery area by 22% and intraarterial norepinephrine decreased brachial artery area by 17% at 100 mm Hg transmural pressure (P < .001 versus baseline).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554136 TI - Design principles of vascular beds. AB - Hemodynamic parameters were determined in each vessel segment of six complete microvascular networks in the rat mesentery by using a combination of experimental measurements and theoretical stimulations. For a total number of 2592 segments, a strong unified dependence of wall shear stress on intravascular pressure for arterioles, capillaries, and venules was obtained. All three types of segments exhibit an essentially identical variation of shear stress from high to low values (from approximately 100 to 10 dyne/cm2) as intravascular pressure falls from 70 to 15 mm Hg. On the basis of these observations, it is proposed that vascular beds grow and adapt so as to maintain the shear stress in each vessel at a level that depends on local transmural pressure. In contrast to Murray's classic 'minimum-cost' hypothesis, which implies uniformity of wall shear rate throughout the vasculature, the proposed design principle provides an explanation for the functionally important arteriovenous asymmetry of wall shear rates and flow resistance in the circulation. PMID- 7554137 TI - Lymphocytic Na(+)-H+ exchange increases after an oral glucose challenge. AB - The effects of oral glucose challenge on plasma glucose concentration, plasma insulin concentration, arterial blood pressure, cytosolic pH (pHi), cytosolic free Na+ concentration ([Na+]i), and cellular Na(+)-H+ exchange activity were investigated in 16 healthy subjects. The pHi, [Na+]i, and Na(+)-H+ exchange activity were measured in intact lymphocytes by using the fluorescent dye technique. The oral glucose challenge significantly increased plasma glucose, plasma insulin, and the lymphocytic Na(+)-H+ exchange activity, measured as change of pHi per second (control [0 hours], 5.20 +/- 0.53 x 10(-3) dpHi/s; 1 hour after glucose administration, 8.28 +/- 1.07 x 10(-3) dpHi/s; 2 hours after glucose administration, 8.15 +/- 1.18 x 10(-3) dpHi/s; P = .002). The lymphocytic Na(+)-H+ exchange was significantly correlated with plasma glucose concentration (r = .357, P = .041). During steady state euglycemic hyperinsulinemic clamp, the Na(+)-H+ exchange activity was not significantly changed compared with baseline values. The study shows that changes of blood glucose levels can induce an acute increase in Na(+)-H+ exchange activity. Systolic blood pressure and Na(+)-H+ exchange activity were significantly (P < .001) but weakly correlated during an oral glucose challenge. PMID- 7554138 TI - Does ischemic preconditioning in the human involve protein kinase C and the ATP dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. AB - Protein kinase C (PKC) and the ATP-dependent K+ channel (KATP channel) have been implicated in the mechanism of ischemic preconditioning in animal models. This study investigated the role of KATP channels and PKC in preconditioning in human myocardium and whether KATP channels are activated via a PKC-dependent pathway. Right atrial trabeculae were superfused with Tyrode's solution and paced at 1 Hz. After stabilization, muscles underwent one of nine different protocols, followed by simulated ischemia (SI) consisting of 90 minutes of hypoxic substrate-free superfusion paced at 3 Hz and then by 120 minutes of reperfusion. Preconditioning consisted of 3 minutes of SI and 7 minutes of reperfusion. The experimental end point was recovery of contractile function after SI, presented here as percentage recovery (%Rec) of baseline function. %Rec was significantly improved by preconditioning by the KATP channel opener cromakalim (CK), and by the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) compared with nonpreconditioned controls when these treatments were given before the SI insult (control group, 29.5 +/- 3.6%; preconditioned group, 63.5 +/- 5.4%, CK-treated group, 52.9 +/- 3.1%; and DOG-treated group, 48.0 +/- 3.5%; P < .01). The effects of CK could be blocked by the KATP channel blocker glibenclamide (%Rec, 17.8 +/- 3.5%). Preconditioning could be blocked by the PKC antagonist chelerythrine (%Rec, 24.1 +/- 5.0%) and the KATP blocker glibenclamide (%Rec, 24.8 +/- 3.1%). The effects of DOG could also be blocked by glibenclamide (%Rec, 23.1 +/- 2.3%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554139 TI - Matrix metalloproteinases and cardiovascular disease. PMID- 7554140 TI - Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. AB - Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca(2+)-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity. PMID- 7554141 TI - Leukotriene C4/D4 induces P-selectin and sialyl Lewis(x)-dependent alterations in leukocyte kinetics in vivo. AB - The objective of this study was to assess the effect of leukotriene C4 (LTC4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC4 (20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti-P-selectin antibody and soluble sialyl Lewis(x) (sLe(x)). LTC4 also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLe(x). This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at > 5000 times the concentration of LTC4. Treatment with an anti-L-selectin antibody had no effect on the LTC4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4 bioconversion to LTD4 by pretreatment with L serine (100 mumol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4 but not histamine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554142 TI - Effects of lysophosphatidic acid, a novel lipid mediator, on cytosolic Ca2+ and contractility in cultured rat mesangial cells. AB - Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, has the potential to modulate cellular signaling in diverse tissues and cell types, including fibroblasts. In the present study, we assessed the effects of LPA on cultured rat glomerular mesangial cells. Quantitative changes of [Ca2+]i in response to LPA were measured in monolayers of mesangial cells loaded with the fluorescent Ca2+ indicator fura 2. LPA (10 nmol/L to 100 mumol/L) increased [Ca2+]i in a dose-dependent manner and evoked inositol trisphosphate formation. LPA (1 mumol/L to 30 mumol/L) also elicited a marked, albeit transient, contractile response in mesangial cells cultured on collagen gel, as assessed by a decrease in cell surface area (CSA). The contraction persisted for 5 minutes (CSA decreased by 31%), whereupon the mesangial cells gradually relaxed with a consequent increase in CSA. Pretreatment of mesangial cells with isradipine (1 mumol/L), a dihydropyridine-sensitive Ca2+ channel blocker, completely blocked LPA-induced contraction. Isradipine also decreased resting [Ca2+]i levels as well as the peak and the subsequently sustained [Ca2+]i levels induced by LPA, suggesting that the contractile effects of LPA are dependent on Ca2+ entry through voltage-gated Ca2+ channels. Finally, LPA stimulated an increase in both prostaglandin E2 synthesis and cAMP accumulation, indicating that these mediators may modulate the contractile effects of LPA. Our study is the first demonstration that exogenous LPA induces mesangial cell contraction and suggests that the contraction is mediated by mobilization of intracellular Ca2+ by activation of the phosphoinositide cascade as well as by promotion of Ca2+ entry across the plasma membrane. PMID- 7554143 TI - Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. AB - Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4 chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554144 TI - Regional expression of natriuretic peptide receptors during the formation of arterial neointima in the rabbit. AB - In vitro evidence suggests that natriuretic peptide receptors (NPR)-B and NPR-C inhibit vascular smooth muscle (VSM) proliferation. NPR-B is guanylate cyclase coupled and selectively activated by C-type natriuretic peptide (CNP)-(1-22). NPR C is not guanylate cyclase-coupled and, unlike NPR-B, avidly binds atrial natriuretic peptide (ANP)-(1-28) as well as CNP-(1-22). Here, we investigate these receptors during the VSM proliferation and neointimal formation found 5, 7, and 20 days after compressing the central ear artery of the rabbit. Receptors were mapped autoradiographically using [125I-Tyr0]CNP-(1-22), which binds NPR-B and NPR-C, and 125I-ANP-(1-28), which binds NPR-C and NPR-A, another guanylate cyclase-coupled receptor. Normal tunica media had NPR-B-like binding sites, and the level of these did not change significantly after compression. Consistent with this, CNP-(1-22) stimulated cGMP production equally with membranes from normal or damaged arteries and was more effective than ANP-(1-28). Neointima, which became evident 5 to 7 days after arterial damage, expressed NPR-C-like sites and no detectable NPR-B-like binding. NPR-C-like sites also appeared on the media for the first time between 5 and 7 days after compression. Immunohistochemistry for proliferating cell nuclear antigen revealed widespread mitosis in VSM at 5 days after compression, but mitosis was virtually restricted to the neointima at and beyond 7 days after compression. Thus, whereas levels of NPR-B did not change significantly after arterial injury and NPR-A was not detected, NPR-C-like receptors were upregulated as mitosis declined in the media and as a prominent neointima formed. PMID- 7554145 TI - Reduction of intimal hyperplasia by naroparcil, a 4-methylumbelliferyl beta-D xyloside analogue, after arterial injury in the hypercholesterolemic rabbit. AB - 4-Methylumbelliferyl beta-D-xylosides (beta-D-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a beta-D-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol enriched diet (group 1, n = 15) or the same diet with added 100 mg.kg-1 naroparcil (group 2, n = 15) or 300 mg.kg-1 naroparcil (group 3, n = 12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75 +/- 0.54 and 0.85 +/- 0.61 mm2 versus 0.32 +/- 0.25 mm2, respectively; P < .05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1; P < .05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury. PMID- 7554146 TI - Endoplasmic reticulum Ca2+ depletion unmasks a caffeine-induced Ca2+ influx in human aortic endothelial cells. AB - Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+]i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+]i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+]i from 86 +/- 10 to 115 +/- 17 nmol/L (mean +/- SEM); this effect was similar to that of 5 mumol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 mumol/L), ryanodine did not affect [Ca2+]i. In contrast, caffeine induced a large rapid increase in [Ca2+]i (176 +/- 19 to 338 +/- 35 nmol/L, P < .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+]i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+]i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554147 TI - Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP dependent mechanism. AB - Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man. It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrate that 17 beta-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving K+ efflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates K+ channel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17 beta-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17 beta-estradiol on the gating of the large conductance, Ca(2+)- and voltage-activated K+ (BKCa) channels, while 17 alpha estradiol had no effect. Furthermore, blocking BKCa channels in intact arteries inhibited estrogen-induced relaxation. The effect of 17 beta-estradiol on BKCa channels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17 beta-estradiol relaxes coronary arteries by opening BKCa channels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease. PMID- 7554148 TI - Cardiac sarcoplasmic reticulum phosphorylation increases Ca2+ release induced by flash photolysis of nitr-5. AB - Effects on Ca(2+)-induced Ca2+ release due to phosphorylation of sarcoplasmic reticulum (SR) proteins were investigated in isoproterenol-treated saponin permeabilized trabeculae from rat ventricles. In these experiments, Ca2+ release from the SR was induced by a rapid change in concentration of free Ca2+ (ie, trigger Ca2+) achieved by flash photolysis of nitr-5, and the amount of Ca2+ released was assessed by measuring isometric tension. Ca2+ uptake by the SR was more rapid, and the amount of Ca2+ released by a given concentration of trigger Ca2+ was greater in isoproterenol-treated trabeculae compared with control trabeculae. However, under the same conditions of Ca2+ loading, the amplitudes of caffeine-elicited tension transients in control trabeculae were similar to those in isoproterenol-treated trabeculae, suggesting that the Ca2+ available for release was similar in the two cases. Control experiments showed that there were no significant differences in Ca2+ sensitivity of tension between isoproterenol treated and control trabeculae. Also, application of alkaline phosphatase to trabeculae that had previously been treated with isoproterenol returned SR Ca2+ release to control levels. We conclude that the greater release of Ca2+ in isoproterenol-treated trabeculae in response to a given concentration of trigger Ca2+ is due to phosphorylation of SR proteins, most likely the Ca2+ release channel. PMID- 7554149 TI - Description of a nonselective cation current in human atrium. AB - Ion currents were examined in isolated human atrial myocytes by using the whole cell patch-clamp technique. When currents were recorded with a K(+)-containing pipette solution, depolarizing voltage pulses elicited a rapidly activating outward current that decayed to an apparent steady state. Exposure of cells to 10 mmol/L 4-aminopyridine markedly reduced current amplitude; however, a rapidly activating current that was approximately 30% of the steady state current amplitude remained. When pipette K+ was replaced with Cs+, a similar rapidly activating current that reversed polarity at approximately 0 mV was recorded. This current was seen in 100% of the cells tested from 17 different hearts (n = 142), and its amplitude was approximately 40% of the amplitude of the steady state current recorded in the presence of pipette K+. The current amplitude was not significantly different in cells isolated from adult (6.31 +/- 1.35 pA/pF, n = 8) and pediatric (5.54 +/- 1.04 pA/pF, n = 9) hearts. Studies designed to determine the charge-carrying species indicated that changes in bath Cl- concentration had no effect on either the amplitude or the reversal potential of this current, whereas removal of pipette Cs+ and bath Na+ dramatically reduced this current. In addition, this current was not modulated by either isoproterenol (1 mumol/L, 22 degrees C) or cell swelling. This study provides the first description of a nonselective cation current in human atrial myocytes, which may play an important role in repolarization in human atria. PMID- 7554150 TI - Relative densities of muscarinic cholinergic and beta-adrenergic receptors in the canine sinoatrial node and their relation to sites of pacemaker activity. AB - The site of earliest extracellular electrical activation in the sinoatrial node (SAN) is known to shift in response to autonomic stimuli, but the mechanisms underlying this phenomenon and the determinants of the location of dominant pacemaker activity have not been elucidated. The present study was designed to characterize the spatial distribution of muscarinic cholinergic and beta adrenergic receptors in the canine SAN and to determine whether a consistent relationship exists between autonomic receptor densities and the site of dominant pacemaker activity. We used quantitative light-microscopic autoradiography of radioligand binding sites to characterize the spatial distribution of muscarinic cholinergic and beta-adrenergic receptor subtypes in tissue sections containing the SAN and adjacent right atrial muscle from 18 canine hearts. Muscarinic receptor density was 5.4 times greater in SAN cells than in atrial myocytes (P < .01). Total beta-adrenergic receptor density was more than 3 times greater in SAN cells than in atrial myocytes (P < .0001), due entirely to the significantly greater number of beta 1-adrenergic receptors in the SAN. The region of dominant pacemaker activity, localized in 4 hearts with in vitro mapping, consistently exhibited greater densities of muscarinic and beta 1-adrenergic receptors than other SAN regions. Muscarinic receptor density in the dominant pacemaker region was 18 +/- 2% and 29 +/- 7% higher than in adjacent superior and inferior regions, respectively. beta 1-Receptor density in the dominant site was 53 +/- 5% and 26 +/- 4% higher than in adjacent superior and inferior SAN regions, respectively. Thus, the SAN is richly endowed with both muscarinic cholinergic and beta 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554151 TI - Isometric and dynamic contractile properties of porcine skinned cardiac myocytes after stunning. AB - The purpose of this study was to investigate myofibrillar mechanisms of depressed contractile function associated with myocardial stunning. We first tested whether the degree of stunning was directly related to changes in myofilament Ca2+ sensitivity. Variable degrees and durations of low-flow ischemia were followed by 30 minutes of reperfusion in an open-chest porcine model of regional myocardial stunning (n = 27). Ca2+ sensitivity of isometric tension was measured in skinned myocytes obtained from endocardial biopsies taken during control aerobic flow and after 30 minutes of reperfusion. The degree of stunning, as assessed by percent systolic wall thickening, ranged from -3% to 75% of control but did not correlate (r = .11) with changes in pCa50, ie, pCa for half-maximal tension. Only in the group (n = 10) with the most severe level of ischemia was there a significant decrease in pCa50 (from 5.97 +/- 0.06 in the control condition to 5.86 +/- 0.07 after ischemia, P < .05). Less severe levels of ischemia (n = 17) resulted in significant stunning (percent systolic wall thickening, 38 +/- 4% of control) but no change in pCa50. To investigate the possibility that alterations in myofibrillar cross-bridge kinetics contribute to depressed function in stunning, maximum velocity of shortening (Vo) was measured in postischemic myocytes. Vo in postischemic myocytes was reduced to 56 +/- 4% of Vo in control myocytes and was independent both of the degree of stunning (r = .26) and changes in Ca2+ sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554152 TI - Differential regulation of circulating Mg2+ in the rat by beta 1- and beta 2 adrenergic receptor stimulation. AB - Extracellular Mg2+ homeostasis was studied in vivo in the anesthetized rat. Animals were infused with isoproterenol (ISO) for 10 minutes, and serum Mg2+ was measured after the infusion and then 10 and 20 minutes later. A dose-dependent increase in circulating Mg2+ was observed in animals infused with ISO at a rate of 0.1 microgram.kg-1.min-1 or higher. The time course of the effect demonstrated that circulating Mg2+ continued to increase 20 minutes after the end of the ISO infusion. A predicted maximal increase in serum Mg2+ concentration of 19.3% was derived with a predicted EC50 of 0.08 microgram.kg-1.min-1. The maximal percent increase corresponded to a net increase of 6.7 mumol/300 g body wt. Because infusion of ISO resulted in changes in hemodynamic parameters, most notably a drop in blood pressure, a group of animals was infused with nitroprusside to mimic the hypotensive response via a nonadrenergic mechanism. Under these conditions, there was a transient increase in circulating Mg2+ that was largely inhibited by propranolol, indicating that hypotension per se was not responsible for the mobilization of Mg2+. Infusion of salbutamol, but not prenalterol, also induced an increase in circulating Mg2+. Pretreatment with butoxamine, ICI 118551, or propranolol prevented the ISO-induced increase in serum Mg2+. Pretreatment with atenolol minimally affected the ISO-induced changes in circulating Mg2+. Pretreatment with CGP-20271A actually enhanced the ISO-induced increase in circulating Mg2+. This evidence demonstrates the existence of a pool of Mg2+ that is mobilized into the circulation in response to selective beta 2 adrenergic stimulation. PMID- 7554153 TI - Effects of NO modulation on cardiac arrhythmias in the rat isolated heart. AB - It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs' solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion. NG-Nitro-L-arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n = 20, P < .05). The profibrillatory effect of L-NAME was prevented in hearts coperfused with 1 or 10 mmol/L L-arginine (an NO precursor) but persisted in hearts coperfused with D-arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264 +/- 10 versus 309 +/- 5 bpm in control groups, P < .05) and reduced coronary flow before ischemia (6.2 +/- 0.6 versus 9.2 +/- 0.6 mL.min-1.g-1 tissue in controls, P < .05). L-NAME reduced coronary effluent NO levels after 60 minutes of ischemia; during the first minute of reperfusion, values were reduced from 1457 +/- 422 to 812 +/- 228 pmol.min-1.g 1 (P < .05). This effect was prevented by coperfusion with L-arginine (10,344 +/- 1730 pmol.min-1.g-1, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554154 TI - Brain 'ouabain' mediates sympathetic hyperactivity in congestive heart failure. AB - In congestive heart failure (CHF), endogenous compounds with ouabainlike activity (OLA) may contribute to the maintenance of the circulatory homeostasis by peripheral as well as central effects. In the present study, we assessed changes in peripheral (plasma and left ventricle) and central (pituitary, hypothalamus, pons, and cortex) OLA in two animal models of CHF and determined whether brain OLA mediates sympathetic hyperactivity in CHF. Cardiomyopathic hamsters with their controls were studied at 9 months of age for tissue OLA. Rats were studied 4 weeks after acute coronary artery ligation for tissue OLA and sympathetic activity. In both models, left ventricular end-diastolic pressure was markedly increased. CHF was associated with significant increases in both plasma and tissue OLA in both models. In the brain, the most marked (twofold to threefold) increases occurred in the hypothalamus. In vitro, all OLA measured could be blocked by antibody Fab fragments (Digibind). Conscious rats with CHF showed elevated plasma catecholamines and enhanced responses of mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) to air stress and to intracerebroventricular (ICV) injection of the alpha 2-adrenergic receptor agonist guanabenz compared with sham-operated rats. ICV administration of the Fab fragments did not change resting RSNA or responses to air stress at 1 hour. However, 18 hours after injection of the Fab fragments, resting RSNA levels had significantly decreased compared with the control values, and plasma catecholamine levels had decreased to control values.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554155 TI - [Observation on the development of Coelomomyces indica in Culex tritaeniorhynchus larvae]. AB - The present paper reports the development of Coelomomyces indica in Culex tritaeniorhynchus larvae. Experimental observations indicated that the first instar of the mosquito larvae can be infected with fungus. The hyphal bodies developed and produced sporangia in the haemocoele of the mosquito larvae. The sporangia were released in water from the dead mosquito larvae. The development of mature sporangia with zoospores was in water at 34 degrees C within 8-12 hours. The zoospores released from the mature sporangia within 1/2-1 hour under optimal conditions were very active in water and their activities decreased after 1 hour. The zoospores act as the infective stage for the Cyclops (host). PMID- 7554156 TI - Effects of mebendazole, albendazole and praziquantel on alanine aminotransferase and aspartate aminotransferase of Echinococcus granulosus cyst wall harbored in mice. AB - Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of Echinococcus granulosus cyst wall harbored in mice for 10-12 months was measured. The activities of the 2 enzymes were 3,430 +/- 1,370-4,160 +/- 1,790 and 1,560 +/ 840-2,890 +/- 1,470, respectively. When infected mice were treated ig with mebendazole (Meb) 25 mg.kg-1.d-1 for 7-14 d, or 50 mg.kg-1.d-1 for 7 d, the ALT activities of cyst walls of both collapsed and full cysts were inhibited significantly with average inhibition rates of about 50%. Another benzimidazole derivative, albendazole (Alb), also inhibited ALT activity of the cyst wall with an inhibition rate of 40.7%, but this was not the case in the treatment with praziquantel 500 mg.kg-1.d-1 of the infected mice for 14 d. When the infected mice were treated ig with the above mentioned drugs at the same regimens, no apparent effect on AST activities of the cyst wall was seen except for the group treated ig with Meb at a higher daily dose, the inhibition rates being 26% (collapsed cyst) and 43.9% (full cyst). The significance of the results has been discussed briefly. PMID- 7554157 TI - [Sensitivity of Plasmodium falciparum to seven antimalarials in China-Laos border]. AB - The sensitivity of P. falciparum to chloroquine (CHL), piperaquine (PIP), artesunate (ATS), dihydroartemisinine (DAT), artemether (ATM), arteether (ATE) and pyronaridine (PRN) were assayed by in vitro microtechnique in China-Laos border in 1992. The resistance rates of isolates from the south of Yunnan of China in the border were 97.0%, 96.4%, 12.5%, 16.0%, 6.3% and 34.5%, respectively; ID50 were 119.0, 320.0, 7.2, 5.4, 295.0, 74.4 and 31.9 nmol/L, ID95 were 625.0, 2426.0, 42.2, 36.6, 767.0, 371.1 and 325.2 nmol/L. The resistance rates of isolates from the north border of Laos were in order 9/10, 8/10, 1/5 to CHL, PIP, PRN, ID50 were 114.0, 166.9, 16.4 nmol/L, ID95 were 570.0, 631.1 and 107.7 nmol/L. All the isolates from Laos were sensitive to ATS, DAT, ATM and ATE. ID50 were 5.0, 4.4, 91.6 and 56.7 nmol/L, ID95 were 23.4, 11.1, 3276.1 and 223.9 nmol/L, respectively. The results indicate that P. falciparum in the border was highly resistant to CHL and PIP, while most of the isolates were susceptible to the other 5 of the above-mentioned drugs. A comparison between the isolates from China and Laos showed that there was no significant difference in resistance degree to CHL but the sensitivity of isolates from China to other 6 drugs was lower than those from Laos. PMID- 7554158 TI - [Molluscicidal activity of seed of Camellia sinensis]. AB - This paper is a preliminary report on the molluscicidal activity of seed of Camellia sinensis (L) O. Kuntze (SCS). Its LD50 and LD90 were 10 and 16 micrograms/ml, respectively. We found also SCS inhibited oxygen consumption 65% and glycogen content of Oncomelania snail by 80%. The activity of hatching eggs to juvenile snails was inhibited by this seed at 8-16 micrograms/ml. Juvenile snails may also be killed by this molluscicide. PMID- 7554159 TI - [A preliminary study on the inhibitory action of target-primaqine on the exoerythrocytic forms of Plasmodium yoelii yoelii]. AB - Target-primaquine (TPQ)--the primaquine entrapped by galactosylneoglycoalbumin (G NGA), was given intravenously (iv) to Wistar rats 2 h after sporozoite inoculation. The sporozoite viability (SPV) of TPQ 20 mg/kg group was revealed to be 1.2% 42 h after inoculation, which showed a decline of about 6% as compared to the control; besides, degeneration or retardation of development was present in 90% of EEF. In TPQ 10 mg/kg and PQ 20 mg/kg groups, the SPV were 2.8% and 5.0%, respectively, showing drops by 15% and 26% as compared with that of the control. Degeneration or retardation in development was also visible in about half of the EEF. In mice which were treated iv with TPQ 10 mg/kg or PQ 20 mg/kg 2 h after sporozoite inoculation, the prepatent periods were delayed markedly as compared with that of the control. Parasitemia did not appear in 2 mice in 10 mg/kg TPQ group further indicated the effectiveness of the drug. The results suggest that the anti-EEF effect of TPQ is apparently more promising than that of PQ. PMID- 7554160 TI - [Application of Plasmodium falciparum DNA extract from dried blood spot of falciparum malaria patient in polymerase chain reaction]. AB - Polymerase chain reaction (PCR) primered with primers 3,4 and 5,6 was performed using DNA extracted from dried blood spot of a falciparum malaria patient from Mengpeng Township, Yunnan using four different methods, including STE-proteinase K-SDS, methanol-proteinase K-SDS, Chelex-100 and Chelex-100-proteinase K to amplify DNA of apical membrane antigen 1 (AMA-1). A 900 bp DNA band could be seen on the ethidium bromide-stained agarose gel electrophoresis of the PCR products when DNA was extracted using all the above-mentioned methods except STE proteinase K-SDS method. DNA extracted with Chelax-100 was recommended. PMID- 7554161 TI - [Histochemical changes in Brugia malayi microfilariae-infected Anopheles sinensis]. AB - Histochemical changes in normal and Brugia malayi microfilariae-infected Anopheles sinensis were observed using histochemical technique. The results showed that the activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) and the content of glycogen and basic protein in Brugia malayi microfilariae infected Anopheles sinensis were less obviously than those of the normal control. Esterase (EST) activity was reduced slightly and acetylcholinesterase (AChE) remained no change in infected mosquitoes compared with the normal control. It is suggested that the development of filaria larvae in vector mosquitoes has selective effect on their biochemical metabolism. PMID- 7554162 TI - [Effect of Cladonia alpestris on Trichomonas vaginalis in vitro]. AB - In this paper, an experimental research is reported on the effect of water extract of Cladonia alpestris and S-(-) usnic acid on Trichomonas vaginalis in vitro. The results showed that both the water extract of Cladonia alpestris and S (-)usnic acid exhibited a strong effect against Trichomonas vaginalis in vitro. As the time of action of the agents was prolonged, the mortality of Trichomonas vaginalis increased. For S-(-) usnic acid, 0.4 mg/ml was the lowest effective concentration against Trichomonas vaginalis in vitro. No remarkable differences were found in the effect of the S-(-)usnic acid and metronidazole at concentrations of 0.4 mg/ml and 0.6 mg/ml. PMID- 7554163 TI - [Experimental and clinical study on pneumocystosis. IV. Use of a 4D7 monoclonal antibody to detect Pneumocystis carinii by immunoperoxidase staining]. AB - This paper reports on the diagnostic utility of the 4D7 monoclonal antibody (McAb) and immunohistochemical (IHC) staining to detect P. carinii. Specimens of 30 pathological sections of lung tissues and 1 smear of aspirated bronchial material from confirmed cases, and 12 sputum smears from high risk patients with pneumonia were examined by immunohistochemical method using the 4D7 McAb. The results showed that both the sensitivity and specificity of the 4D7 McAb-IHC staining were 100% (31/31). A comparison between immunohistochemical staining with GMS and TBO staining was made, no difference in sensitivity was found. However, the IHC method gave a clear back ground that is useful for definite diagnosis. PMID- 7554164 TI - [Relationship between tumour necrosis factor and anemia of malaria]. AB - In this paper, the relation between the level of the reactive oxygen species (ROS) and relevant free radicals in the blood plasma of the BALB/c mice infected with Plasmodium berghei ANKA strain and their erythrocytic deformability (ED), and the relation between the ED and the Hb indices of these mice were studied by chemiluminescence (CL), induced CL (ICL) and laser diffraction method. The results indicated that the ED decreased with the increase of the level of ROS and free radicals in blood plasma when the anemia is developing. The use of antioxidants may restrain the course and raise the Hb index. The authors deem that the excess TNF produced in the later stage of malaria may induce the release of excess ROS and free radicals from phagocytes, which may cause damage on the deformability of erythrocytes and a drop in ED, resulting in the anemia of malaria. PMID- 7554165 TI - [Exploration on experimental conditions of dot-ELISA for Trichomonas vaginalis using intact parasite antigen-monoclonal antibody detecting system]. AB - This paper deals with the effect of different experimental conditions including the number of parasite, blocking fluid, incubation time and different types of antigens on dot-ELISA for Trichomonas vaginalis. Five microlitres of freshly washed intact worms at a concentration of 7.7 x 10(5)-3 x 10(8) cells/ml were added onto nitrocellulose membrane blocked with 1% BSA, 10% bovine serum or milk power (0.1%, 0.25%, 0.5%, 5%). Dot-ELISA using nitrocellulose membrane containing whole cell antigen 2.5 x 10(7) cells/ml, blocked by 0.5% defatted milk power at 37 degrees C for one hour before the incubation in McAb, incubated at 37 degrees C for one hour in McAb and SPA respectively was found to give more promising results. The whole cell antigen could be preserved for four months at 4 degrees C and -20 degrees C. PMID- 7554166 TI - [A ten-year follow-up observation on serum antibody level in schistosomiasis patients treated with praziquantel]. AB - The detection of the serum antibody in 122 schistosomiasis patients, using enzyme linked immunosorbent assay (ELISA), had been carried out from 1980-1989. The results showed that the potency of serum antibody was on an annual progressive decrease. Before treatment, the rate of ELISA reactive titre above 1:800 was 87.7%, while after treatment it was 21.2% in the first year and 1% in the second year and even lower in the following years. The antibody negative conversion rate with ELISA from the first to the fifth year after praziquantel treatment ranged from 53.2%-95.9%, whereas it was 97.0% in the tenth year. No significant difference was found between the ELISA positive rate in patients treated after 4 years and the false positive rate in normol persons (P > 0.05). The data demonstrate that it took four years after therapy for the noticeable reduction of the residual serum antibody level in patients, and that the ELISA positive reaction titre > or = 1:400 in patients 4 years after treatment might be used as complementary diagnostic index for those uncured patients. PMID- 7554167 TI - [Advances in the study of molecular biology in Cryptosporidium]. PMID- 7554168 TI - A great success in lymphatic filariasis control in China. National Technical Steering Group for Filariasis Control and Research, MOPH. AB - Lymphatic filariasis control was first included in national program in 1956. Since then, a large scale anti-filariasis campaign has been carried out extensively throughout the endemic areas. From 1956-1994, a cumulative total of 707,421,736 person-time were comprised in nationwide blood examination, and a total of 260,041,645 person-time DEC chemotherapy were conducted (including selective treatment, mass chemotherapy and DEC-fortified salt). Up to 1994, all of the 864 endemic counties/cities in 15 provinces/autonomous regions/municipalities had achieved effective control of filariasis successively. Parasitological, entomological and serological surveillance during the past few years demonstrated that the transmission of both bancroftian and malayan filariasis have been virtually interrupted in most of the areas reaching the criterion for effective control of filariasis. PMID- 7554169 TI - Demonstration of in vitro cultured exoerythrocytic schizonts and hypnozoites of Plasmodium vivax (southern China isolate) by an immunoperoxidase antibody technique. AB - By indirect immunoperoxidase staining, different forms of exoerythrocytic (EE) stage of Plasmodium vivax (Southern China isolates) are revealed in d8 cultured material. The mature schizonts are elongated in shape measuring 42-48 microns in diameter, immature schizonts 14-28 microns and hypnozoites 4-7 microns. EE schizonts are stained dark-brown only after conjugated by monoclonal antibody (McAb) 4B2 specific against erythrocytic stages of P. vivax while hypnozoites are only stained after conjugated by McAb 2F2 against sporozoite. These results show that the antigenic components of these two forms of EE plasmodia are quite different. The ratio of EE schizont and hypnozoite found within hepatoma cells (HepG2-A16) is 1.5 to 1. Referred to the clinical manifestations of the isolate, among 5 volunteers not radically cured, two had long incubation period (283 d and 304 d, respectively) and three relapsed 235, 260 and 365 days after the primary attack. These data are unanimous with the comparatively large ratio of hypnozoites in the cultured material. PMID- 7554170 TI - [A study on human behavior and socioeconomic factors affecting malaria transmission and control in Qiongzhong, Hainan]. AB - This study was conducted in Heping District of Quiongzhong County, a hyperedemic mountainous area, in August-September 1992. The comparative surveys between the village and state-run farm, Li and Miao nationalities and Han nationality were carried out by using the sociological method together with the epidemiological methods. Gray relational analysis was conducted between the aforementioned 7 socioeconomic human behavioral factors and IFA rates. The result showed that their degrees of relation (r) were in the following order: (1) percentage of persons who had stayed in the mountain overnight (r = 0.8690); (2) percentage of bed net users (r = 0.7990); (3) percentage of households seeking medical service (r = 0.7990); (4) number of mosquito nets per person (r = 0.7867); (5) percentage of householders knowing malaria transmission route (r = 0.7798); (6) percentage of households with tile-roofed houses (r = 0.6767) and (7) income per capita (r = 0.6636). It indicates that staying in the mountain, using bed net and seeking medical service were three discriminating factors affecting local malaria transmission and control. Therefore, it is suggested that carrying out health education, changing the stay-in-mountain behavior, increasing the utilization of mosquito nets and reinforcing the primary health care should be taken as the fundamental measures for malaria control programme. PMID- 7554171 TI - [Expression of chemically synthesized hybrid genes of Plasmodium falciparum and preliminary characterization of expressed products]. AB - The chemically synthesized hybrid genes HGFC and HGFCAC were respectively recombinated with expression vector pWR450-1 and transferred into Escherichia coli. After adding IPTG to the cultural media, the bacteria harboring recombinant plasmid expressed the fusion proteins of exogenous gene products and partial amino acid of beta-galactosidase. The molecular weights of the two fusion proteins were 65 kDa and 77 kDa, respectively. The expressed fusion proteins could be recognized by antibodies of rabbit anti-RESA peptide EENVEHDA by Western blotting, indicating that the fusion proteins harbored the antigenic epitopes of Plasmodium falciparum. PMID- 7554173 TI - The power to regulate tobacco. PMID- 7554172 TI - [Nationwide survey of the distribution of parasites in China--the characteristics and rules of geographic distribution of human helminth infection]. PMID- 7554174 TI - NHLBI cost-management strategies. A success story. PMID- 7554175 TI - New standard for success of thrombolytic therapy. An earnest proposal. PMID- 7554176 TI - A skeleton in the atherosclerosis closet. PMID- 7554177 TI - Angina pectoris and disease progression. PMID- 7554178 TI - Identification and molecular analysis of two apoB gene mutations causing low plasma cholesterol levels. AB - BACKGROUND: Familial hypobetalipoproteinemia (FHB) is an autosomal codominant disorder characterized by abnormally low plasma levels of apoB and LDL cholesterol. Heterozygotes for FHB almost always have plasma LDL cholesterol levels < 70 mg/dL and are asymptomatic. Because the low cholesterol levels may protect FHB heterozygotes from coronary heart disease, the mechanisms for FHB are of considerable interest. METHODS AND RESULTS: The plasma lipoproteins of 29 subjects with LDL cholesterol levels < 70 mg/dL were examined by SDS-PAGE. One subject who had virtually undetectable levels of LDL cholesterol had a truncated apoB, apoB-44.4, in his lipoproteins; a second subject with an LDL cholesterol level of 44 mg/dL had apoB-55 in his lipoproteins. The apoB-44.4 (2014 amino acids in length) resulted from a frameshift caused by an 11-bp insertion in exon 26 of the apoB gene; the apoB-55 (2494 amino acids) was caused by a nonsense mutation in exon 26 of the apoB gene. The apoB-55 mutation occurred at a CpG dinucleotide pair, a mutational hot spot, and was identical to a mutation described previously in a subject with hypobetalipoproteinemia. Our subject with apoB-55, however, had a different haplotype than the subject described previously, suggesting that the two apoB-55 mutations may have arisen independently. Of note, the apoB-55 proband's father, who had very low cholesterol levels and who probably carried the apoB-55 mutation, had significant coronary and aortic atherosclerosis at autopsy. CONCLUSIONS: In a study of adults with low LDL cholesterol levels, we discovered two subjects with truncated apoB proteins and identified the responsible mutations. ApoB gene mutations causing truncated apoB are not particularly rare in subjects with low cholesterol levels. The role of these mutations in preventing atherosclerosis deserves further study. PMID- 7554179 TI - cAMP response element binding protein is expressed and phosphorylated in the human heart. AB - BACKGROUND: In end-stage failing human hearts and in rat hearts after prolonged in vivo beta-adrenergic treatment, several proteins involved in the cAMP dependent signal transduction are altered on the protein, mRNA, or transcriptional level, eg, beta-adrenoceptors, G-proteins, or proteins of Ca2+ homeostasis. In many tissues, cAMP-dependent transcriptional regulation occurs through the cAMP response element binding protein (CREB) and related transcription factors binding as dimers to cAMP response elements (CREs) in the promoter regions of regulated genes. METHODS AND RESULTS: To investigate a possible role of CREB in the human heart, nuclear protein of explanted failing and nonfailing human hearts was used to test for CRE specific binding properties in gel mobility shift assays. CRE specific binding was found in competition studies, and CREB and its phosphorylated form were immunologically identified in supershift experiments. The alternatively spliced CREB isoforms CREB327 and CREB341 were found to be expressed on the mRNA level by the reverse transcriptase polymerase chain reaction. CONCLUSIONS: We conclude that in the failing and nonfailing human heart, CREB is expressed on the protein and mRNA levels and that CREB is phosphorylated and able to bind to CREs, indicating a functional role of CREB in the human heart. PMID- 7554180 TI - A randomized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction. The STAR Trial Group. AB - BACKGROUND: Recombinant staphylokinase (STAR) was shown recently to offer promise for coronary arterial thrombolysis in patients with evolving myocardial infarction. The present multicenter randomized open trial was designed to assess the thrombolytic efficacy, safety, and fibrin specificity of STAR relative to accelerated alteplase (recombinant tissue-type plasminogen activator [RTPA]). METHODS AND RESULTS: One hundred patients with evolving myocardial infarction of < 6 hours' duration and with ST-segment elevation were allocated to accelerated and weight-adjusted RTPA over 90 minutes (52 patients) or to STAR (the first 25 patients to 10 mg and the next 23 patients to 20 mg given intravenously over 30 minutes). All patients received aspirin and intravenous heparin. The main end points were coronary artery patency and plasma fibrinogen levels at 90 minutes. Thrombolysis in Myocardial Infarction (TIMI) perfusion grade 3 at 90 minutes was achieved in 62% of STAR patients versus 58% of RTPA patients (risk ratio, 1.1; 95% CI, 0.76 to 1.5). With 10 mg STAR, TIMI grade 3 patency was 50% (risk ratio, 0.86; 95% CI, 0.54 to 1.4 versus RTPA); with 20 mg STAR, it was 74% (risk ratio, 1.3; 95% CI, 0.90 to 1.8 versus RTPA). Residual fibrinogen levels at 90 minutes were 118 +/- 47% (mean +/- SD) of baseline with STAR and 68 +/- 42% with RTPA (P < .0005). STAR therapy was not associated with an excess mortality or electric, hemorrhagic, mechanical, or allergic complications. However, patients developed antibody-mediated STAR-neutralizing activity from the second week after STAR treatment. As an addendum to the randomized study, 5 patients were given 40 mg STAR over 30 minutes, resulting in TIMI perfusion grade 3 at 90 minutes in 4 patients without fibrinogen breakdown (residual levels at 90 minutes of 105 +/- 8% of baseline). CONCLUSIONS: STAR appears to be at least as effective for early coronary recanalization as and significantly more fibrin-specific than accelerated RTPA in patients with evolving myocardial infarction. PMID- 7554181 TI - Thrombolytic therapy of peripheral arterial occlusion with recombinant staphylokinase. AB - BACKGROUND: Recombinant staphylokinase (STAR) induces fibrin-specific coronary artery recanalization in patients with evolving myocardial infarction. The present pilot study evaluates its thrombolytic efficacy, safety, fibrin specificity, and immunogenicity in patients with peripheral arterial occlusive disease. METHODS AND RESULTS: Thirty patients (37 to 86 years of age) with angiographically documented thromboembolic peripheral arterial occlusion of recent origin (21 +/- 5.5 days, mean +/- SEM) were treated with heparin and intra arterial STAR given as a 1-mg bolus followed by a 0.5-mg/h infusion in 20 patients or as a 2-mg bolus followed by a 1-mg/h infusion in 10 subsequent patients. With 7.0 +/- 0.7 mg STAR infused over 8.7 +/- 1.0 hours, recanalization was complete in 25 patients, partial in 2, and absent in 3. Two major hemorrhagic complications occurred: one fatal hemorrhagic stroke and one hypovolemic shock caused by bleeding at the angiographic puncture site. Administration of STAR did not induce fibrinogen breakdown or a significant prolongation of template bleeding time. STAR-neutralizing activity and anti-STAR IgG were low at baseline, increased markedly from the second week on, and remained elevated for several months. CONCLUSIONS: Intra-arterial administration of STAR restores vessel patency in patients with peripheral arterial occlusion in the absence of fibrinogen degradation. PMID- 7554182 TI - Rapid angiographic progression of coronary artery disease in patients with angina pectoris. The role of complex stenosis morphology. AB - BACKGROUND: Rapid disease progression commonly underlies acute coronary events, and "complex" stenosis morphology may play a role in this phenomenon. METHODS AND RESULTS: We studied the role of complex stenosis morphology in rapid disease progression in 94 consecutive patients awaiting routine coronary angioplasty. Coronary arteriography was repeated at 8 +/- 3 months' follow-up, immediately preceding angioplasty (68 patients) or after an acute coronary event (26 patients). Disease progression of 217 stenoses, of which 79 (36%) were "complex" and 138 (64%) were "smooth," was assessed by computerized angiography. At presentation, 63 patients had stable angina pectoris and 31 had unstable angina that settled rapidly with medical therapy. At follow-up, 23 patients (24%) had progression of preexisting stenoses and 71 (76%) had no progression. Patients with progression were younger (55 +/- 12 years) than those without (58 +/- 9 years) but did not differ with regard to risk factors, previous myocardial infarction, or severity and extent of coronary disease. Twenty-three lesions (11%) progressed, 15 to total occlusion (11 complex and 4 smooth; 65%). Progression occurred in 17 of the 79 complex stenoses (22%) and in 6 of the 138 smooth lesions (4%) (P = .002). Mean stenosis diameter reduction was also significantly greater in complex than in smooth lesions (11.6% versus 3.9% change; P < .001). Acute coronary events occurred in 57% of patients with progression compared with 18% of those without progression (P < .001) and were more frequent in patients who presented with unstable angina (P = .002). CONCLUSIONS: Rapid stenosis progression is not uncommon, and complex stenoses are at risk more than smooth lesions. PMID- 7554183 TI - Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty. AB - BACKGROUND: An insertion/deletion (I/D) polymorphism in the gene for angiotensin converting enzyme (ACE) has been associated with myocardial infarction and other cardiac pathology. There is evidence for a role of the renin-angiotensin system in cell growth and in the repair of damaged arterial walls, so the ACE gene was postulated to be a candidate gene affecting the important clinical problem of restenosis after percutaneous transluminal balloon coronary angioplasty (PTCA). Because restenosis is influenced by the apolipoprotein E (apoE) genotype, the possibility of a relation between ACE and apoE genotypes and restenosis was also sought. METHODS AND RESULTS: Subjects (< 70 years of age) were prospectively followed and had coronary angiography 6 months after PTCA to determine the presence or absence of restenosis. Those who had angiography earlier and did not have restenosis (> or = 50% loss of gain at PTCA plus > or = 50% luminal diameter stenosis) also had angiography at 6 months. The whole group (n = 207) had a higher DD genotype frequency than did 136 population control subjects (38% versus 26%, P < .02); in PTCA patients, the frequency was the same in those with and without prior myocardial infarction. The distribution of ACE genotypes was not different in the 88 patients with and 119 patients without restenosis, while the epsilon 4/4 genotype was more frequent in those with restenosis (8 of 88 versus 3 of 118, P < .05). There was no effect of the ACE genotype in noncarriers of the epsilon 4 allele, but there was a significant effect in epsilon 4 carriers (P < .005). The combined D and epsilon 4 carrier state showed a 16-fold increase in the odds ratio for restenosis (P < .02). Multiple linear regression examining the loss of lumen as a continuous variable showed significant independent effects of the ACE and apoE genotypes. CONCLUSIONS: Overall, the ACE genotype had no clear influence on restenosis, but there was an interaction between ACE and apoE genotypes. The combined carrier state for the D and apoE epsilon 4 alleles substantially increased restenosis. For loss of lumen as a continuous variable, there were significant effects of both ACE and apoE genotypes. While the observations may not affect current management, they no doubt have implications in pathophysiology. PMID- 7554184 TI - Impaired myocardial tissue perfusion early after successful thrombolysis. Impact on myocardial flow, metabolism, and function at late follow-up. AB - BACKGROUND: Impaired tissue reperfusion after successful recanalization of an epicardial coronary artery has been documented both in animals and in patients with acute myocardial infarction. Whether this phenomenon can be demonstrated with positron emission tomography (PET) and whether it has an effect on late recovery of flow, metabolism, and function are unknown. METHODS AND RESULTS: Thirty patients with an acute myocardial infarction and TIMI flow grade 3 of the infarcted vessel at 90 minutes after thrombolytic therapy were studied. Within 24 hours after thrombolysis, at 5 days and at 3 months, myocardial blood flow was measured with 13NH3. 18FDG uptake was measured at 5 days. Radionuclide left ventricular angiograms were acquired at 5 days and at 3 months. In 11 patients (37%), regional myocardial flow was severely impaired (< 50% of normally perfused myocardium) despite successful thrombolysis. No recovery of left ventricular function occurred in any of these patients at 3 months. In 12 patients (40%), intermediate flows (50% to 75% of normal) were found, with functional improvement after angioplasty only in regions with a PET mismatch. Seven patients (23%) had high flow values early after successful thrombolysis (> 75% of normal) and showed preserved regional contractile function at 3 months. CONCLUSIONS: This study is the first demonstration with PET of impaired myocardial tissue perfusion in patients with an acute myocardial infarction after successful thrombolysis. Functional recovery of the reperfused myocardium is observed only when adequate tissue flow is restored. PET may be helpful in selecting patients in whom additional revascularization can improve recovery of left ventricular function. PMID- 7554185 TI - A study of biochemical markers of reperfusion early after thrombolysis for acute myocardial infarction. The PERM Study Group. Prospective Evaluation of Reperfusion Markers. AB - BACKGROUND: In acute myocardial infarction (AMI), early noninvasive identification of patients with occluded infarct-related arteries (IRAs) after thrombolysis has important prognostic and therapeutic implications. The aims of this study were to evaluate biochemical methods for the early diagnosis of patency after thrombolysis prospectively and to establish the optimal diagnostic criteria retrospectively. METHODS AND RESULTS: In 97 patients with AMI treated with thrombolytic agents < or = 6 hours after the onset of symptoms, myoglobin, troponin T, creatine kinase, the MB isoenzyme and MM isoforms of creatine kinase were measured just before thrombolysis began and 90 minutes later. IRA patency was assessed by means of 90-minute coronary angiography. For each marker, compared with the expected sensitivity and specificity based on published thresholds for the diagnosis of patency, the observed values were consistently lower but were markedly improved in a subset of patients treated > 3 hours after the onset of symptoms. With receiver-operator characteristic curve analysis of the slopes of increase and relative increases in each marker over 90 minutes, the best diagnostic performance was achieved by use of the relative increase in myoglobin, troponin T, and MM3/MM1 creatine kinase isoforms in patients treated > 3 hours after onset (areas under the curve of 0.84, 0.83, and 0.85, respectively). CONCLUSIONS: Effective early noninvasive diagnosis of patency after thrombolysis is possible in patients treated > 3 hours after symptom onset by use of criteria derived from the relative increase over 90 minutes in plasma markers, particularly myoglobin, troponin T, and MM3/MM1 creatine kinase isoforms. The diagnostic performance of the relative increase in myoglobin appears to be less susceptible to small changes in the diagnostic threshold value. PMID- 7554186 TI - Quantitative assessment of coronary vasoreactivity in humans in vivo. Importance of baseline vasomotor tone in atherosclerosis. AB - BACKGROUND: Disturbances in vasomotor tone are closely linked to the development of atherosclerosis and play an integral part in the pathophysiology of myocardial ischemia in patients with coronary artery disease. Currently, evaluation of coronary vasomotor tone relies on assessment of luminal changes in response to vasoactive stimuli by quantitative angiography. Assessment of luminal changes, however, may be misleading because of the effects of geometric magnification induced by atherosclerotic wall thickening and differences in basal tone, which may obscure potential effects of vasoactive stimuli. METHODS AND RESULTS: Combining angiographic measurements of luminal changes with measurements of coronary wall architecture determined by intracoronary ultrasound, we calculated absolute coronary arterial vasomotor tone in 34 patients with angiographically normal or "minimally diseased" coronary arteries. Epicardial artery vasodilator capacity in response to intracoronary nitroglycerin decreased significantly with increasing atherosclerotic wall thickening. An inverse relation existed between local atherosclerotic plaque load and nitroglycerin-induced changes in vasomotor tone (r = -.65, P < .0001) regardless of potential systematically operative factors. At the same time, basal vasomotor tone significantly decreased with increasing local atherosclerotic wall thickening (r = -.38, P = .004). The vasomotor effects of acetylcholine, an endothelium-dependent vasodilator, were inversely related to wall thickening only in segments with very minor degrees of atherosclerosis, whereas no such relation was observed for epicardial artery segments with more advanced atherosclerotic wall thickening. In these segments, however, vasoreactivity to acetylcholine was closely correlated with basal vasomotor tone (r = -.62, P = .0002). CONCLUSIONS: Atherosclerosis is associated with impairment of the vasodilator response to both nitroglycerin and acetylcholine in epicardial arteries in vivo. Basal vasomotor tone appears to be the primary determinant of the altered coronary vasoreactivity in response to vasoactive stimuli. PMID- 7554187 TI - Relation between ischemic threshold measured during dobutamine stress echocardiography and known indices of poor prognosis in patients with coronary artery disease. AB - BACKGROUND: Stress echocardiography has become an accepted methodology for the evaluation of coronary artery disease. One potential advantage of dobutamine over other stressors used with echocardiography is the possibility of assessing the ischemic threshold. However, whether this measurement correlates with indices associated with adverse outcome has not been established. METHODS AND RESULTS: One hundred four patients (91 men and 13 women; age, 61 +/- 9 years) with coronary artery disease were studied with transesophageal echocardiography during infusion of dobutamine 2.5 to 40 microgram/kg per minute. When regional dyssnergy developed, the dobutamine ischemic threshold (the dose of dobutamine at which induced regional wall motion abnormalities were first detected) was identified. The dobutamine stress echocardiogram was abnormal in 90 patients (sensitivity, 87%). The dobutamine ischemic threshold was 25.4 +/- 11.2 micrograms/kg per minute in patients with single-vessel disease, 14.4 +/- 7.9 in patients with two vessel disease, and 9.1 +/- 7.9 in patients with three-vessel disease (P < .0001). The dobutamine ischemic threshold correlated with the ejection fraction response to exercise measured by radionuclide angiography: Patients with low ischemic threshold had a mean fall in ejection fraction, and patients with high ischemic threshold or normal tests had a mean increase in ejection fraction. CONCLUSIONS: In patients with coronary artery disease, the ischemic threshold measured during dobutamine stress echocardiography correlates with both the number of stenosed vessels and the left ventricular ejection fraction response to exercise. Because these variables are associated with poor prognosis, these findings provide further support regarding the utility of dobutamine stress echocardiography in the clinical evaluation of patients with chronic coronary artery disease. PMID- 7554188 TI - Mental stress-induced ischemia in the laboratory and ambulatory ischemia during daily life. Association and hemodynamic features. AB - BACKGROUND: The purpose of this study was to determine the correspondence of mental stress-induced ischemia in the laboratory with ambulatory ischemia and to assess the relationship between hemodynamic responses to mental stress and the occurrence of ischemia. Although exercise testing is usually used to elicit myocardial ischemia, ischemia during daily life usually occurs at relatively low heart rates and in the absence of strenuous physical exercise. Mental stress has been shown to trigger ischemic events in the laboratory at lower heart rates but at blood pressures comparable to exercise. We therefore compared the extent to which mental stress and exercise testing identify patients who develop ischemia out of hospital. METHODS AND RESULTS: One hundred thirty-two patients with documented coronary disease and recent evidence of exercise-induced myocardial ischemia underwent 48-hour ambulatory monitoring and radionuclide ventriculography during exercise and mental stress testing. Patients who displayed mental stress-induced ischemia in the laboratory were more likely to exhibit ischemia during daily life (P < .021). Furthermore, patients who exhibited ischemia during ambulatory monitoring displayed larger diastolic blood pressure (P < .006), heart rate (P < .039), and rate-pressure product responses (P < .018) during mental stress. CONCLUSIONS: Among patients with prior positive exercise stress tests, mental stress-induced ischemia, defined by new wall motion abnormalities, predicts daily ischemia independent of exercise-induced ischemia. Exaggerated hemodynamic responses during mental stress testing also identify individuals who are more likely to exhibit myocardial ischemia during daily life and mental stress. PMID- 7554189 TI - Limb vasoconstriction after successful angioplasty of the left anterior descending coronary artery. AB - BACKGROUND: Coronary vasoconstriction has been described after uncomplicated percutaneous transluminal coronary angioplasty (PTCA). However, it is still unknown whether this phenomenon is limited to coronary circulation. The present study was planned to assess the effects of a successful PTCA on forearm blood flow (FBF) and resistance. The role of alpha-adrenoceptors and calcium antagonist agents on PTCA-induced limb blood flow changes was also investigated. METHODS AND RESULTS: We prospectively studied 37 patients scheduled for elective single PTCA of the left anterior descending coronary artery. All patients had evidence of exercise-induced myocardial ischemia. All vasoactive drugs were withdrawn for at least 48 hours before the study. FBF was measured by calibrated venous occlusion plethysmography. A significant reduction of FBF was observed at 1, 5, and 15 minutes after PTCA (from 3.7 +/- 1.2 to 2.7 +/- 1.5, 3.0 +/- 1.6, and 2.9 +/- 1.9 mL/100 mL tissue per minute, respectively; all P < .05 versus baseline). Vascular forearm resistance also increased at 1, 5, and 15 minutes after PTCA (from 27 +/- 8 to 42 +/- 16, 37 +/- 10, and 43 +/- 19 U, respectively; all P < .05 versus baseline). Phentolamine (12 microgram.kg-1.min-1, n = 7) or verapamil (3.5 micrograms.kg-1.min-1, n = 7) also was infused intra-arterially. PTCA-induced forearm vasoconstriction was completely abolished by pretreatment with regional infusion of phentolamine or verapamil. CONCLUSIONS: After an uncomplicated PTCA of the left anterior descending coronary artery, a reduction in FBF and an increase in forearm vascular resistance were observed. This peripheral vasoconstrictive response was probably due to alpha-adrenergic stimulation and was abolished by intra-arterial infusion of calcium antagonist agents. PMID- 7554190 TI - Vasodilatation to arachidonic acid in humans. An insight into endogenous prostanoids and effects of aspirin. AB - BACKGROUND: Human endothelial and vascular smooth muscle cells synthesize prostanoids. Several of these have been implicated in the physiological and pathophysiological regulation of vascular tone; however, there is no direct evidence that human blood vessels synthesize sufficient prostanoid to alter vessel tone. METHODS AND RESULTS: We explored the effects of local infusions of arachidonic acid on the tone of preconstricted superficial hand veins in healthy volunteers. Aspirin was used to assess the contribution of prostanoids to the responses seen. Local infusion of arachidonic acid produced a dose-dependent dilatation of preconstricted veins. This was abolished by local infusion of aspirin. Oral aspirin was also effective: a high (anti-inflammatory) dose of aspirin (1 g) taken 2 hours before the experiment blocked the arachidonic acid induced venodilatation; however, a low (cardioprotective) dose of aspirin (75 mg) did not. Unlike the responses to arachidonic acid, responses to glyceryltrinitrate and bradykinin were unaltered by aspirin (1 g). Ex vivo platelet aggregation was inhibited by aspirin in both high and low doses. Aspirin (1 g) inhibited arachidonic acid-induced venodilatation for up to 5 days. The time course was similar for vascular and platelet effects. CONCLUSIONS: The present findings demonstrate that local generation of prostanoids in a human vessel in vivo alters vascular tone. The predominant prostanoid synthesized is a dilator and its synthesis can be blocked by an anti-inflammatory but not a cardioprotective dose of aspirin. The results suggest that selective inhibition of platelet aggregation by oral aspirin might be a function of dose rather than the interval between doses. PMID- 7554191 TI - Paracrine coronary endothelial control of left ventricular function in humans. AB - BACKGROUND: Similar to endothelial modulation of vascular tone, myocardial contraction may be modulated by cardioactive agents released from the coronary endothelium. To investigate such modulation in humans, we performed invasive assessment of left ventricular (LV) function before, during, and after bicoronary infusion of substance P, which releases nitric oxide from the endothelium. METHODS AND RESULTS: Eight healthy subjects were investigated during diagnostic coronary angiography and eight transplant recipients during annual catheterization. Tip-micromanometer LV pressure was recorded before, during, and after bicoronary (n = 16) and right atrial (n = 14) infusion of substance P (20 pmol/min). LV angiograms (n = 11) were obtained before and at the end of the substance P infusion. At the end of the intracoronary substance P infusion, we observed (1) a fall in LV peak systolic pressure from 147 +/- 16 to 139 +/- 15 mm Hg (P < .01) in healthy subjects and from 147 +/- 25 to 141 +/- 22 mmHg (P < .05) in transplant recipients; (2) a downward and rightward shift of the average LV end-systolic pressure-volume point consistent with depressed systolic performance; and (3) a rise in LV end-diastolic volume at comparable end diastolic pressure, consistent with increased end-diastolic distensibility. Five minutes after the substance P infusion, LV peak systolic pressure was higher than at baseline in healthy subjects (154 +/- 18 mm Hg; P < .05). Right atrial infusion of substance P did not reproduce these changes. CONCLUSIONS: Bicoronary infusion of substance P modulates LV function in humans, probably through paracrine myocardial action of cardioactive agents released from the coronary endothelium. PMID- 7554192 TI - Carotid intima-media thickness is only weakly correlated with the extent and severity of coronary artery disease. AB - BACKGROUND: Intima-media thickness (IMT) of the common carotid artery (CCA), measured with external vascular ultrasound, has been widely used in clinical trials as a surrogate marker for coronary atherosclerosis. Despite this, the degree of correlation between carotid IMT and the extent and severity of coronary artery disease (CAD) is not known. METHODS AND RESULTS: Common carotid IMT was measured by ultrasound in 350 consecutive subjects of age 60 +/- 10 years (range, 30 to 85 years) on the day of coronary angiography. Carotid mean IMT was 0.83 +/- 0.20 mm (range, 0.43 to 1.80 mm), and maximum IMT was 1.04 +/- 0.27 mm (range, 0.49 to 2.19 mm). Coronary angiograms were analyzed by independent observers for disease severity (number of vessels with > or = 70% stenosis), extent score, and a modified Gensini score. Mean carotid IMT was weakly but significantly correlated with CAD severity (r = .26), extent (r = .23), and modified Gensini score (r = .29, P < .0001 for all correlations). Carotid IMT was not clinically useful, however, because it was not specific or sensitive enough to identify patients with or without significant CAD. Increasing age, male sex, and presence of diabetes were all associated with a significantly (P < .01) higher CAD score than the average for any level of carotid IMT, suggesting differential effects of these traditional risk factors on the coronary and common carotid arteries. CONCLUSIONS: Although carotid IMT is significantly correlated with extent and severity of CAD, the relationship is weak. This relatively poor correlation (r2 < .10) should be considered in the interpretation of clinical trials that use carotid IMT as a surrogate end point for coronary atherosclerosis. PMID- 7554193 TI - Endothelial release of nitric oxide contributes to the vasodilator effect of adenosine in humans. AB - BACKGROUND: The endogenous nucleoside adenosine plays an important role in the regulation of vascular tone, especially during ischemia. Experimental data derived from animal models suggest that nitric oxide (NO) contributes to the vasodilator effect of adenosine. The primary purpose of this investigation was to determine whether the endothelial release of NO contributes to adenosine-induced vasodilation in humans. METHODS AND RESULTS: Venous occlusion plethysmography was used to assess the forearm blood flow (FBF) responses to graded intra-arterial infusions of adenosine (1.5 to 500 micrograms/min). Dose-response curves were constructed before and during intra-arterial infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (2 mg/min, n = 6) or vehicle (n = 6). Before infusion of L-NMMA, adenosine caused a dose-dependent increase in FBF from 2.3 to 15.9 mL.min-1.dL-1. During concurrent infusion of L-NMMA, adenosine increased FBF from 1.7 to 10.0 mL.min-1.dL-1, and this change from baseline was significantly reduced compared with that before L-NMMA (P < .05). L-NMMA also attenuated the FBF response to adenosine when the basal constrictor effect of L NMMA was prevented by coinfusion of the NO donor sodium nitroprusside (n = 6, P < .01). In contrast, L-NMMA did not affect the FBF response to intra-arterial infusion of the endothelium-independent vasodilator verapamil (from 2.0 to 13.9 mL.min-1.dL-1 before L-NMMA and from 1.3 to 13.6 mL.min-1.dL-1 during L-NMMA; n = 6, P = NS). The second objective of this study was to determine whether the adenosine-induced release of NO is mediated by activation of endothelial potassium channels, putatively coupled to adenosine receptors. Thus, the FBF response to adenosine was measured before and during infusion of the ATP dependent potassium channel blocker tolbutamide (1 mg/min, n = 6), or the potassium channel blocker quinidine (0.5 mg/min, n = 6). The adenosine-mediated increments in FBF were not attenuated by either potassium channel blocker. CONCLUSIONS: Adenosine-induced vasodilation in humans is mediated, at least in part, by endothelial release of NO. The transducing mechanism of this phenomenon is not known, but it does not appear to involve the activation of either ATP dependent or quinidine-sensitive potassium channels. PMID- 7554194 TI - Dietary antioxidants and carotid artery wall thickness. The ARIC Study. Atherosclerosis Risk in Communities Study. AB - BACKGROUND: Evidence that dietary antioxidants may prevent atherosclerotic disease is growing. The relationship between the intake of dietary and supplemental vitamin C, alpha-tocopherol, and provitamin A carotenoids and average carotid artery wall thickness was studied in 6318 female and 4989 male participants 45 to 64 years old int he Atherosclerosis Risk in Communities Study. METHODS AND RESULTS: Intake was assessed by use of a 66-item semiquantitative food-frequency questionnaire. Carotid artery intima-media wall thickness was measured as an indicator of atherosclerosis at multiple sites with B-mode ultrasound. Among men and women > 55 years old who had not recently begun a special diet, there was a significant inverse relationship between vitamin C intake and average artery wall thickness adjusted for age, body mass index, fasting serum glucose, systolic and diastolic blood pressures, HDL and LDL cholesterol, total caloric intake, cigarette use, race, and education (test for linear trend across quintiles of intake, P = .019 for women and P = .035 for men). An inverse relationship was also seen between wall thickness and alpha tocopherol intake but was significant only in women (test for linear trend, P = .033 for women and P = .13 for men). There was a significant inverse association between carotene intake and wall thickness in older men (test for linear trend, P = .015), but the association weakened after adjustment for potential confounders. No significant relationships were seen in participants < 55 years old. CONCLUSIONS: These data provide limited support for the hypothesis that dietary vitamin C and alpha-tocopherol may protect against atherosclerotic disease, especially in individuals > 55 years old. PMID- 7554195 TI - Hyperinsulinemia inhibits myocardial protein degradation in patients with cardiovascular disease and insulin resistance. AB - BACKGROUND: Insulin resistance, hyperinsulinemia, and myocardial hypertrophy frequently coexist in patients. Whether hyperinsulinemia directly affects myocardial protein metabolism in humans has not been examined, however. To test the hypothesis that hyperinsulinemia is anabolic for human heart protein, we examined the effects of insulin infusion on myocardial protein synthesis, degradation, and net balance in patients with ischemic heart disease. METHODS AND RESULTS: Eleven men (aged 57 +/- 3 years) with coronary artery disease who had fasted for 12 to 16 hours received a constant infusion of insulin (50 mU.m-2.min 1) while plasma concentrations of glucose and amino acids were kept constant. Rates of myocardial protein synthesis, degradation, and net balance were estimated from steady state extraction and isotopic dilution of L-[ring-2,6 3H]phenylalanine across the heart basally and 90 minutes into infusion. Subjects had elevated fasting plasma insulin concentrations (173 +/- 21 pmol/L) and used little exogenous glucose during insulin infusion, suggesting resistance to the effects of insulin on whole-body carbohydrate metabolism. Basally, myocardial protein degradation, as estimated by phenylalanine release (133 +/- 28 nmol/min), exceeded protein synthesis, estimated by phenylalanine uptake (31 +/- 15 nmol/min), resulting in net negative phenylalanine balance (-102 +/- 17 nmol/min). Insulin infusion reduced myocardial protein degradation by 80% but did not affect protein synthesis, returning net phenylalanine balance to neutral. CONCLUSIONS: Acute hyperinsulinemia markedly suppresses myocardial protein degradation in patients with cardiovascular disease who are resistant to its effects on whole-body glucose metabolism. This antiproteolytic action represents a potential mechanism by which hyperinsulinemia could contribute to the development of myocardial hypertrophy in patients with cardiovascular disease. PMID- 7554196 TI - Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area. A histopathologic correlative study. AB - BACKGROUND: Coronary calcium identified by electron-beam computed tomography (EBCT) correlates poorly with luminal atherosclerotic narrowing, but calcium, an intimate part of coronary plaque, may be more directly related to atheromatous plaque area. METHODS AND RESULTS: Thirty-eight coronary arteries from 13 autopsy hearts were dissected, straightened, and scanned with EBCT in 3-mm contiguous increments. Coronary calcium area was defined as one or more pixels with a density > 130 Hounsfield units (0.18 mm2/pixel). Each artery was divided into corresponding 3-mm segments, representative histological sections were stained, and atherosclerotic plaque area per segment (mm2) was quantified. Coronary artery calcium and coronary artery plaque areas were correlated for the hearts as a whole, for individual coronary arteries, and for individual coronary artery segments. The sums of histological plaque areas versus the sums of calcium areas were highly correlated for each heart and for each coronary artery. However, coronary plaque area was on the order of five times greater than calcium area. Furthermore, minimal diffuse segmental coronary plaque could be present despite the absence of coronary calcium detectable by EBCT. CONCLUSIONS: This histopathologic study confirms an intimate relation between whole heart, coronary artery, and segmental coronary atherosclerotic plaque area and EBCT coronary calcium area but suggests that there is a threshold value for plaque area below which coronary calcium is either absent or not detectable by this methodology. PMID- 7554197 TI - Osteopontin is expressed in human aortic valvular lesions. AB - BACKGROUND: Nonrheumatic stenosis of trileaflet aortic valves, in which calcification is a prominent feature, has been termed a "degenerative" condition, but it has been demonstrated recently that chronic inflammation is a characteristic feature of the developing lesion of aortic stenosis. This observation raised the possibility that calcification in the aortic valve might be actively regulated. Thus, the present study investigated whether osteopontin, a protein implicated in the regulation of both normal and dystrophic calcification, could be detected in lesions of valvular aortic stenosis. METHODS AND RESULTS: Morphological and immunohistochemical studies were performed on 14 human aortic valves, representing a range of pathology from normal to clinically stenotic. The extent of calcification and macrophage accumulation and their relation to the presence of osteopontin protein were characterized. Highly statistically significant associations were found between the degree of osteopontin expression and the degrees of both calcification and macrophage accumulation in early through late lesions of aortic stenosis. Further, in situ hybridization localized osteopontin mRNA to a subset of lesion macrophages. CONCLUSIONS: These results suggest that, rather than representing a degenerative and unmodifiable process, calcification in aortic stenosis may be, in part, an actively regulated process with the potential for control either through modification of inflammation or synthesis of proteins such as osteopontin, which may modulate calcification in this tissue. PMID- 7554198 TI - Quantification of mitral regurgitation by the proximal convergence method using transesophageal echocardiography. Clinical validation of a geometric correction for proximal flow constraint. AB - BACKGROUND: Proximal flow convergence is a promising method to quantify mitral regurgitation but may overestimate flow when the flow field is constrained. This has not been investigated clinically, nor has a correction factor been validated. METHODS AND RESULTS: Eighty-five patients were studied intraoperatively with transesophageal echocardiography and divided into two groups: central convergence (no constraining wall) and eccentric convergence (at least one constraining wall). Regurgitant stroke volume (RSV) and orifice area (ROA) were calculated by ROA = 2 pi r2 Va/Vp and RSV = ROA x VTIcw, where r and va are the radius and velocity of the aliasing contour and vp and VTIcw are the peak and integral of regurgitant velocity. In eccentric convergence patients, convergence angle (alpha) was measured from two-dimensional Doppler color flow maps, and ROA and RSV were corrected by multiplying by alpha/180. For reference, RSV was the difference between thermodilution and pulsed Doppler stroke volumes. In central convergence patients (n = 45), RSV (r = .95, delta = 2.5 +/- 10.8 mL) and ROA (r = .96, delta = 0.02 +/- 0.08 cm2) were accurately calculated, but significant overestimation was noted in the eccentric convergence patients (n = 40, delta RSV = 63.9 +/- 38.0 mL, delta ROA = 0.54 +/- 0.31 cm2), 68% of whom had leaflet prolapse or flail. delta RSV was correlated with alpha (r = -.69, P < .001). After correction by alpha/180, overestimation was largely eliminated (delta RSV = 15.5 +/- 19.3 mL and delta ROA = 0.14 +/- 0.14 cm2) with excellent correlation for the whole group (RSV, r = .91; ROA, r = .95). CONCLUSIONS: A simple geometric correction factor largely eliminates overestimation caused by flow constraint with the proximal convergence method and should extend the clinical utility of this technique. PMID- 7554199 TI - Silent cerebral infarction in patients with nonrheumatic atrial fibrillation. The Veterans Affairs Stroke Prevention in Nonrheumatic Atrial Fibrillation Investigators. AB - BACKGROUND: Cerebral infarction in patients with atrial fibrillation may vary from being clinically silent to catastrophic. The prevalence of silent cerebral infarction and its effect as a risk factor for symptomatic stroke are important considerations for the evaluation of patients with atrial fibrillation. METHODS AND RESULTS: This Veterans Affairs cooperative study was a double-blind controlled trial designed primarily to determine the efficacy of warfarin for the prevention of stroke in neurologically normal patients with nonrheumatic atrial fibrillation. It also was designed to evaluate patients with silent cerebral infarction. Computed tomography scans of the head were performed at entry, at the time of any subsequent stroke, and at termination of follow-up on all patients who completed the study without a neurological event. Of 516 evaluable scans performed at entry, 76 (14.7%) had evidence of one or more silent cerebral infarcts. Age (P = .011), a history of hypertension (P = .003), active angina (P = .012), and elevated mean systolic blood pressure (P < .001) were associated with the presence of this finding. Silent cerebral infarction occurred during the study at rates of 1.01% and 1.57% per year for the placebo and warfarin treatment groups, respectively (NS). Silent cerebral infarction at entry was not an independent predictor of later symptomatic stroke, but active angina was a significant predictor; 15% of the placebo-assigned patients with angina developed a stroke compared with 5% of the placebo-assigned patients without angina. CONCLUSIONS: Silent cerebral infarction is frequently seen in asymptomatic patients with atrial fibrillation. Age, history of hypertension, active angina, and elevated mean systolic blood pressure were associated with silent infarction at entry. The sample size was too small to determine whether warfarin had an effect on the incidence of silent infarction during the trial. Active angina at baseline was the only significant independent predictor for the later development of symptomatic stroke. PMID- 7554200 TI - Cardiac beta-adrenergic neuroeffector systems in acute myocardial dysfunction related to brain injury. Evidence for catecholamine-mediated myocardial damage. AB - BACKGROUND: Ten percent to 20% of potential cardiac donors with brain injury and no previous cardiac history have myocardial dysfunction. We assessed components of the beta-receptor-G-protein-adenylyl cyclase complex as well as the contractile response in 10 explanted acutely failing human hearts (donor heart dysfunction [DHD]) and compared the results with 13 age-matched nonfailing (NF) organ donor controls. METHODS AND RESULTS: As measured by echocardiography, all DHD hearts exhibited a decreased shortening fraction (16 +/- 2%, mean +/- SEM). Although total and subpopulation beta-receptor densities measured by [125I]iodocyanopindolol (ICYP) were similar in the DHD and NF groups, DHD hearts exhibited a 30% decrease in maximum isoproterenol-stimulated adenylyl cyclase activity and a 50% decrease in the maximal response to zinterol. DHD hearts also exhibited decreases in adenylyl cyclase maximal stimulation by forskolin (211 +/- 25 [DHD] versus 295 +/- 23 [NF] pmol cAMP.min-1.mg-1, P < .05) and 5' guanylylimidodiphosphate (12.5 +/- 1.8 [DHD] versus 19.6 +/- 3.2 [NF] pmol cAMP.min-1.mg-1, P < .05), but there was no significant decrease in adenylyl cyclase stimulation by Mn2+, a direct activator of adenylyl cyclase. Right ventricular trabeculae removed from DHD hearts exhibited a profound decrease in the contractile response to isoproterenol (8.7 +/- 1 [DHD] versus 22 +/- 2 [NF] mN, P < .001) as well as reduced calcium responses (7.2 +/- 1.6 [DHD] versus 14 +/- 3 [NF] mN, P = .03). Morphological examination of two hearts revealed some ultrastructural evidence suggestive of catecholamine-mediated injury, but there was no difference in tissue creatine kinase activity between the two groups. CONCLUSIONS: Compared with NF hearts, DHD hearts exhibit marked uncoupling of beta 1- and beta 2-adrenergic receptors from adenylyl cyclase and contractile response stimulation as well as decreased intrinsic systolic function. Thus, acute myocardial dysfunction accompanying brain injury is characterized by marked alterations in beta-adrenergic signal transduction as well as changes in the contractile apparatus, and this profile is markedly different from what occurs in the chronically failing human heart. PMID- 7554201 TI - Noninvasive measurement of tissue magnesium and correlation with cardiac levels. AB - BACKGROUND: Intracellular magnesium ([Mg]i) plays an important role in the regulation of myocardial metabolism, contractility, and the maintenance of transsarcolemmal and intracellular ionic gradients. An understanding of the role of magnesium in the clinical setting, however, is hampered by the lack of an assay of intracellular tissue magnesium levels. METHODS AND RESULTS: We used energy-dispersive x-ray analysis to measure [Mg]i in sublingual epithelial cells and to correlate the level with those in atrial biopsy specimens from the same patients during cardiopulmonary bypass. Levels were also measured in acute myocardial infarction (AMI) patients before and after intravenous magnesium sulfate administration and compared with those from intensive care unit (ICU) patients and healthy individuals. A strong correlation between sublingual epithelial cell (mean, 32.1 +/- 0.3 mEq/L) and atrial tissue (mean, 32.1 +/- 0.3 mEq/L) [Mg]i was present in 18 cardiac surgery patients (r = .68, P < .002). Epithelial and atrial [Mg]i levels were lower than in healthy individuals (33.7 +/- 0.5 mEq/L, P < .01) studied at that time and correlated poorly with serum magnesium. Mean [Mg]i in 22 AMI patients was 30.7 +/- 0.4 mEq/L, which was significantly lower than in 21 ICU patients and 15 healthy individuals (35.0 +/- 0.5 mEq/L and 34.5 +/- 0.7 mEq/L, respectively, P < .001). Intravenous magnesium sulfate was administered to most of the AMI patients (mean dose, 36 +/- 6 mmol). [Mg]i rose significantly in the AMI patients over the first 24 hours, and the magnitude of the increase was greater in those who received higher doses of intravenous magnesium sulfate. CONCLUSIONS: Sublingual epithelial cell [Mg]i correlates well with atrial [Mg]i but not with serum magnesium. [Mg]i levels are low in patients undergoing cardiac surgery and those with AMI. Intravenous magnesium sulfate corrects low [Mg]i levels in AMI patients. Energy-dispersive x ray analysis determination of sublingual cell [Mg]i may expedite the investigation of the role of magnesium deficiency in heart disease. PMID- 7554202 TI - Nitric oxide inhibits the positive inotropic response to beta-adrenergic stimulation in humans with left ventricular dysfunction. AB - BACKGROUND: Nitric oxide (NO) attenuates the contractile response to beta adrenergic stimulation in cultured cardiac myocytes in vitro and in myocardium in vivo. We tested the hypothesis that NO synthesized in the heart inhibits the positive inotropic response to beta-adrenergic stimulation in humans with left ventricular (LV) dysfunction. METHODS AND RESULTS: Patients with various degrees of LV dysfunction and free from epicardial coronary artery disease were instrumented with an infusion catheter in the left main coronary artery and a high-fidelity micromanometer-tipped catheter in the LV. Measurements included LV pressure, aortic pressure, heart rate, and LV peak +dP/dt. In eight subjects, dobutamine was infused via the left main coronary artery (25 or 50 micrograms/min) before and concurrent with intracoronary infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 20 mumol/min for 10 minutes). In six other subjects, dobutamine was infused (6, 10, or 15 micrograms.kg-1.min-1) via a peripheral vein. Intracoronary (n = 8) dobutamine infusions increased LV peak +dP/dt by an average of 33 +/- 3%. The intracoronary infusion of L-NMMA had no effect on baseline LV peak +dP/dt, LV systolic or end diastolic pressures, aortic pressure, or heart rate. The intracoronary infusion of L-NMMA, concurrent with a second infusion of dobutamine, potentiated the +dP/dt response to dobutamine by 30 +/- 10% (P < .04 versus dobutamine alone). The intracoronary infusion of L-NMMA likewise potentiated the +dP/dt response to the peripheral infusion of dobutamine by 37 +/- 18%. CONCLUSIONS: Nitric oxide produced in the heart attenuates the positive inotropic response to beta adrenergic stimulation in humans with LV dysfunction. NO may contribute to beta adrenergic hyporesponsiveness in patients with LV dysfunction. PMID- 7554203 TI - Abnormal forearm vascular responses during dynamic leg exercise in patients with vasovagal syncope. AB - BACKGROUND: We have reported previously that in some patients with normal hearts who present with exercise syncope, abnormal forearm vasodilation is seen during leg exercise and tilt table tests are positive. This suggests that exercise syncope may be a variant of vasovagal syncope. In this study we tested the hypothesis that there is loss of the normal forearm vasoconstrictor response during dynamic leg exercise in an unselected population of patients with classic vasovagal syncope. METHODS AND RESULTS: We evaluated forearm vascular responses during maximal semierect cycle exercise in 28 consecutive patients with vasovagal syncope and compared them with 30 age-matched control subjects. We also evaluated blood pressure responses during erect treadmill exercise (Bruce protocol). While forearm vascular resistance at rest was similar in the patients with vasovagal syncope and the control group, forearm vascular resistance was markedly lower in the patients than in control subjects at peak exercise (85 +/- 54 versus 149 +/- 94 units, P = .002). Forearm vascular resistance fell by 3 +/- 48% during exercise in patients versus an increase of 135 +/- 103% in control subjects (P < .0001). Systolic blood pressure during erect exercise was lower in patients versus control subjects (155 +/- 32 versus 188 +/- 17 mm Hg, P < .0001). Six of the vasovagal patients complained of exercise syncope or presyncope on specific inquiry, and 4 of these 6 exhibited exercise hypotension during erect treadmill exercise testing. CONCLUSIONS: Patients with vasovagal syncope exhibit a failure of the normal vasoconstrictor response in the forearm during dynamic leg exercise. Exercise syncope and presyncope are not uncommon in unselected patients with classic vasovagal syncope, as is exercise hypotension. PMID- 7554204 TI - Pressure-diameter relation of the human aorta. A new method of determination by the application of a special ultrasonic dimension catheter. AB - BACKGROUND: Pressure-diameter relation of the aorta provides important information about the elastic properties of the vessel. However, owing to methodological limitations, data regarding this relation are limited in conscious humans. In the present study, we assessed a new method for the direct estimation of the elastic properties of the aorta in conscious humans by simultaneous acquisition of instantaneous aortic pressure and diameter. METHODS AND RESULTS: With this method, instantaneous diameter of the thoracic aorta was acquired by a newly designed intravascular catheter developed in our institution that incorporates an ultrasonic displacement meter at its distal end. Instantaneous aortic pressure was acquired simultaneously at the same aortic level with a catheter-tip micromanometer. Aortic pressure-diameter loops were derived from computer analysis of data. After in vitro and animal testing, elastic properties of the aorta were investigated in coronary artery disease (CAD) patients (n = 15) and compared with those of control subjects (n = 10). Aortic distensibility was less in the CAD group than in the control group (1.73 +/- 0.33 versus 3.95 +/- 1.09 x 10(-6) x cm2 x dyne-1, P < .001). Compared with control subjects, the mean value of the slope of the pressure-diameter loops was significantly greater in the CAD group (38.89 +/- 8.75 versus 19.62 +/- 5.46 mm Hg.mm-1, P < .001), whereas the mean value of the intercept was lower in this latter group of patients (-785.60 +/- 177.55 versus -313.43 +/- 126.41 mm Hg, P < .001). An excellent correlation was found between the slope of pressure-diameter loop and age in the group of control subjects (r = .827). Ninety-three percent of the patients with CAD had values above the upper 95% confidence limits of the control subjects (P < .001). In a third group of patients (n = 16) in whom assessment of pulse wave velocity was also included in the study of the elastic properties of the aorta, pulse wave velocity had a strong inverse correlation with aortic distensibility (r = -.95) and a strong positive correlation with the slope of the pressure-diameter loop (r = .97). CONCLUSIONS: This new method of determination of pressure-diameter of the aorta enables an accurate and reliable evaluation of the elastic properties of the aorta in conscious humans and may be useful for a profound study of human aorta mechanics. PMID- 7554205 TI - Immunoglobulins and left ventricular structure and function in pediatric HIV infection. AB - BACKGROUND: Progressive left ventricular (LV) dilation is common in children infected with HIV-1 and may be a harbinger of congestive heart failure (CHF). In many HIV-infected children, dilation is associated with inadequate LV hypertrophy, elevated afterload, and reduced LV function. Because CHF has been observed empirically to improve after treatment with intravenous immunoglobulin (IVIG) in other conditions and because LV dysfunction in pediatric HIV may be immunologically mediated, we examined retrospectively the relation between immunoglobulins and LV structure and function in 49 HIV-infected infants and children without CHF. METHODS AND RESULTS: A total of 106 echocardiograms were performed in these children within 30 days of serum immunoglobulin (IgG, IgA, and IgM) measurements; this includes 12 children treated with IVIG therapy. All echocardiographic parameters, blood pressures, and immunoglobulins were adjusted for age or body surface area and subjected to repeated-measures regression. Regression models were adjusted simultaneously for endogenous IgA, IgG, IgM, IVIG therapy, zidovudine therapy, age, HIV disease stage, and weight. Higher endogenous serum IgG levels and IVIG treatment were associated with significantly greater wall thickness and lower peak wall stress. Higher endogenous serum IgA levels were associated with more normal LV wall thickness and LV thickness-to dimension ratios. LV contractility, fractional shortening, end-systolic wall stress, and thickness-to-dimension ratio all showed a trend toward more normal values with higher endogenous immunoglobulin values or during IVIG treatment. CONCLUSIONS: LV structure and function appear to be more normal in HIV-infected children who receive IVIG treatment and in those with higher endogenous IgG levels. These results suggest that both the impaired myocardial growth and the LV dysfunction observed may be immunologically mediated and responsive to immunomodulatory therapy. PMID- 7554206 TI - Postoperative course and hemodynamic profile after the arterial switch operation in neonates and infants. A comparison of low-flow cardiopulmonary bypass and circulatory arrest. AB - BACKGROUND: The neurological morbidity associated with prolonged periods of circulatory arrest has led some cardiac surgical teams to promote continuous low flow cardiopulmonary bypass as an alternative strategy. The nonneurological postoperative effects of both techniques have been previously studied only in a limited fashion. METHODS AND RESULTS: We compared the hemodynamic profile (cardiac index and systemic and pulmonary vascular resistances), intraoperative and postoperative fluid balance, and perioperative course after deep hypothermia and support consisting predominantly of total circulatory arrest or low-flow cardiopulmonary bypass in a randomized, single-center trial. Eligibility criteria included a diagnosis of transposition of the great arteries and a planned arterial switch operation before the age of 3 months. Of the 171 patients, 129 (66 assigned to circulatory arrest and 63 to low-flow bypass) had an intact ventricular septum and 42 (21 assigned to circulatory arrest and 21 to low-flow bypass) had an associated ventricular septal defect. There were 3 (1.8%) hospital deaths. Patients assigned to low-flow bypass had significantly greater weight gain and positive fluid balance compared with patients assigned to circulatory arrest. Despite the increased weight gain in the infants assigned to low-flow bypass, the duration of mechanical ventilation, stay in the intensive care unit, and hospital stay were similar in both groups. Hemodynamic measurements were made in 122 patients. During the first postoperative night, the cardiac index decreased (32.1 +/- 15.4%, mean +/- SD), while pulmonary and systemic vascular resistance increased. The measured cardiac index was < 2.0 L.min-1.m-2 in 23.8% of the patients, with the lowest measurement typically occurring 9 to 12 hours after surgery. Perfusion strategy assignment was not associated with postoperative hemodynamics or other nonneurological postoperative events. CONCLUSIONS: After heart surgery in neonates and infants, both low-flow bypass and circulatory arrest perfusion strategies have comparable effects on the nonneurological postoperative course and hemodynamic profile. PMID- 7554207 TI - A multicenter, double-blind, placebo-controlled trial of aprotinin for reducing blood loss and the requirement for donor-blood transfusion in patients undergoing repeat coronary artery bypass grafting. AB - BACKGROUND: Aprotinin is a serine protease inhibitor that reduces blood loss and transfusion requirements when administered prophylactically to cardiac surgical patients. To examine the safety and dose-related efficacy of aprotinin, a prospective, multicenter, placebo-controlled trial was conducted in patients undergoing repeat coronary artery bypass graft (CABG) surgery. METHODS AND RESULTS: Two hundred eighty-seven patients were randomly assigned to receive either high-dose aprotinin, low-dose aprotinin, pump-prime-only aprotinin, or placebo. Drug efficacy was determined by the reduction in donor-blood transfusion up to postoperative day 12 and in postoperative thoracic-drainage volume. The percentage of patients requiring donor-red-blood-cell (RBC) transfusions in the high- and low-dose aprotinin groups was reduced compared with the pump-prime-only and placebo groups (high-dose aprotinin, 54%; low-dose aprotinin, 46%; pump-prime only, 72%; and placebo, 75%; overall P = .001). The number of units of donor RBCs transfused was significantly lower in the aprotinin-treated patients compared with placebo (high-dose aprotinin, 1.6 +/- 0.2 U; low-dose aprotinin, 1.6 +/- 0.3 U; pump-prime-only, 2.5 +/- 0.3 U; and placebo, 3.4 +/- 0.5 U; P = .0001). There was also a significant difference in total blood-product exposures among treatment groups (high-dose aprotinin, 2.2 +/- 0.4 U; low-dose aprotinin, 3.4 +/- 0.9 U; pump-prime-only, 5.1 +/- 0.9 U; placebo, 10.3 +/- 1.4 U). There were no differences among treatment groups for the incidence of perioperative myocardial infarction (MI). CONCLUSIONS: This study demonstrates that high- and low-dose aprotinin significantly reduces the requirement for donor-blood transfusion in repeat CABG patients without increasing the risk for perioperative MI. PMID- 7554208 TI - Mediastinitis after coronary artery bypass graft surgery. Risk factors and long term survival. AB - BACKGROUND: Mediastinitis is a severe complication of coronary artery bypass graft surgery (CABG). The purpose of the present study was to determine preoperative and intraoperative variables that predict mediastinitis and to determine the impact of this complication on long-term survival. METHODS AND RESULTS: Data on 20 preoperative and intraoperative variables were collected prospectively on 6459 consecutive patients who underwent CABG between January 1987 and January 1994. Eighty-three patients (1.3%) developed mediastinitis postoperatively, and a total of 24 patients (29%) died. Multivariate analysis identified 4 of the 20 variables as highly significant independent predictors for the development of mediastinitis: obesity (P = .0002), New York Heart Association congestive heart failure class (P = .002), previous heart surgery (P = .008), and duration of cardiopulmonary bypass (P = .05). A comprehensive review of the literature identified 13 other studies that evaluated 48 factors as predictors of mediastinitis; these data were critically analyzed and compared with the results from this series. In this series, postoperative interval mortality during the first 90 days after surgery for the patients with mediastinitis was 11.8% compared with 5.5% for the patients without mediastinitis. Interval mortality between 1 and 2 years after surgery remained high for the mediastinitis group (8.1%) relative to the nonmediastinitis group (2.3%). These differences were not eliminated by adjusting for important variables that influenced late survival in this population. CONCLUSIONS: The present study and a review of the literature suggest that obesity and duration of surgery are the most important predictors of mediastinitis. Furthermore, although the early increase in mortality has been well described, the present study documents for the first time that mediastinitis has a significant negative influence on long-term survival independent of the patient's preoperative condition. PMID- 7554209 TI - Single lung transplantation for pulmonary hypertension. Single institution experience in 34 patients. AB - BACKGROUND: The present study considered the uniformity and durability of the cardiopulmonary response to single lung transplantation in patients with severe pulmonary hypertension, as well as its effect on length and quality of survival. METHODS AND RESULTS: Thirty-four patients with pulmonary hypertension underwent evaluation, single lung transplantation, and follow-up assessment between November 1, 1989, and June 1, 1994. Operative survival for the entire group of patients was reasonable, with 91% (31 of 34 patients) surviving and being discharged from the hospital following transplantation. The actuarial survival for these 34 patients at 1-, 2-, and 3-year follow-up was 78%, 66%, and 61%, respectively. In the subgroup of 24 patients with primary pulmonary hypertension (PPH), 96% (23 of 24) were successfully discharged from the hospital after transplantation. The actuarial survival for this isolated PPH subgroup at 1-, 2-, and 3-year follow-up was 87%, 76%, and 68%, respectively. The uniform, early posttransplant normalization of pulmonary vascular resistance and right ventricular ejection fraction appears to persist throughout the 4-year follow-up period. Despite a high prevalence of bronchiolitis obliterans, the majority of survivors remain in New York Heart Association functional class I or II and are employed. CONCLUSIONS: Single lung transplantation can be performed in patients with end-stage pulmonary vascular disease with reasonable expectations for a relatively low operative mortality; immediate, complete, and durable amelioration of pulmonary hypertension and right ventricular failure; and optimal use of limited donor organ supply. PMID- 7554210 TI - Mechanisms of ischemic preconditioning in rat hearts. Involvement of alpha 1B adrenoceptors, pertussis toxin-sensitive G proteins, and protein kinase C. AB - BACKGROUND: Ischemic preconditioning attenuates the effects of subsequent sustained ischemia by a mechanism involving adenosine and G proteins in several species. Adenosine is not involved in ischemic preconditioning of rat hearts, the mechanisms of which are poorly understood. METHODS AND RESULTS: Reduction of isometric tension development was used as an index of the effects of ischemia in isolated, Langendorff-perfused rat hearts. Two 5-minute periods of ischemia followed by 10 minutes of reperfusion attenuated the reduction of developed tension caused by 30 minutes of ischemia and 15 minutes of reperfusion. Pretreatment with pertussis toxin (PTX), depletion of norepinephrine stores with reserpine, or blockade of alpha 1-adrenoceptors with prazosin prevented the effects of ischemic preconditioning. Whereas alpha 1B-receptor blockade with chloroethylclonidine blocked ischemic preconditioning, alpha 1A-receptor blockade with 5-methylurapadil had no effect. The alpha-adrenergic agonist phenylephrine mimicked the effects of ischemic preconditioning in a concentration-dependent manner, and pretreatment with PTX prevented the action of maximally effective concentrations of phenylephrine. The protein kinase C activator phorbol 12 myristate 13-acetate mimicked and the protein kinase C inhibitors 1-(5 isoquinolinesulfonyl)-2-methylpiperazine and bisindolylmaleimide prevented ischemic preconditioning. CONCLUSIONS: Ischemic preconditioning in isolated, perfused rat hearts is caused by stimulation of alpha 1B-adrenoceptors by endogenous catecholamines through the activation of protein kinase C via a PTX sensitive G protein. The PTX-sensitive inhibitory protein Gi, which can be activated by adenosine, muscarinic agonists, and alpha 1-adrenoceptor agonists, may play a central role in ischemic preconditioning mediated by protein kinase C across a broad range of species. PMID- 7554211 TI - Pharmacological evidence for the persistent activation of ATP-sensitive K+ channels in early phase of reperfusion and its protective role against myocardial stunning. AB - BACKGROUND: The activation of cardiac ATP-sensitive potassium channels is reported to protect myocardium during ischemia. However, the behavior and role of this channel during reperfusion remain uncertain. METHODS AND RESULTS: Guinea pig right ventricular walls were studied by use of microelectrodes and a force transducer. Each preparation was perfused via the coronary artery at a constant flow rate and was stimulated at 3 Hz. In the first protocol, the preparation was subjected to 10 minutes of no-flow ischemia, which was followed by 60 minutes of reperfusion. Introduction of ischemia shortened the action potential duration (APD) to 58.7 +/- 3.1% of the preischemic values, in association with a decrease in the resting membrane potential (by 12 +/- 0.8 mV) and action potential amplitude (by 34.6 +/- 1.8 mV). On reperfusion, although the APD was restored, it remained shortened for up to approximately 30 minutes of reperfusion. In the presence of glibenclamide (10 mumol/L), the shortening of the APD during ischemia was significantly attenuated and the restoration of APD after reperfusion was significantly facilitated. When glibenclamide was applied from the onset of reperfusion, the persistent APD shortening was significantly suppressed. The developed tension decreased during ischemia and recovered after 60 minutes of reperfusion (up to 92.0 +/- 6.4% of preischemic values) in the untreated preparations. The application of glibenclamide that was started before ischemia or from the onset of reperfusion significantly suppressed the recovery of contractility (P < .05 versus untreated preparations). In the second series of experiments, 20 minutes of no-flow ischemia and 60 minutes of reperfusion were applied. This protocol produced a sustained contractile dysfunction after reperfusion (to 34.0 +/- 3.2% of preischemic values). In the presence of cromakalim (2 mumol/L), the APD shortening was enhanced during both ischemia and the early reperfusion period. Cromakalim significantly improved the contractile recovery (to 79.3 +/- 4.1% of preischemic values, P < .05 versus untreated preparations). The application of cromakalim that was started from the onset of reperfusion also improved the contractile recovery during this phase and this effect was associated with enhanced APD shortening. However, the cromakalim treated preparations demonstrated a higher incidence of ventricular fibrillation during reperfusion. CONCLUSIONS: Cardiac ATP-sensitive potassium channels are activated by ischemia, and a fraction of these channels remains activated during the early reperfusion phase. The resulting shortening of the APD prevents the heart from developing myocardial stunning. PMID- 7554212 TI - Neutrophil sequestration and pulmonary dysfunction in a canine model of open heart surgery with cardiopulmonary bypass. Evidence for a CD18-dependent mechanism. AB - BACKGROUND: Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the beta 2 integrin CD18, a leukocyte adhesion molecule, mediates this response. METHODS AND RESULTS: Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466 +/- 75 versus 516 +/- 43 PMN/mm2 alveolar surface area, mean +/- SEM, P < .001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H2O2 (305 +/- 45 versus 144 +/- 48 amol H2O2 per phagocyte per 20 minutes, P < .05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457 +/- 20 versus 246 +/- 49 mm Hg, P < .05), and they had a significantly increased lung wet-to-dry weight ratio (5.38 +/- 0.14 versus 4.54 +/- 0.15, P = .003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412 +/- 73 PMN/mm2, P < .001) and significantly inhibited PMN production of H2O2 (146 +/- 18 amol H2O2 per phagocyte per 20 minutes, P < .05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445 +/- 28 mm Hg, P < .05) and tended to have less lung water accumulation after bypass (4.99 +/- 0.20). CONCLUSIONS: Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism. PMID- 7554213 TI - Intra-arterial beta irradiation prevents neointimal hyperplasia in a hypercholesterolemic rabbit restenosis model. AB - BACKGROUND: Intra-arterial gamma irradiation has been shown to reduce restenosis after balloon angioplasty. The use of beta emitters should allow similar effects while inducing less undue tissue irradiation radioprotection problems. METHODS AND RESULTS: Flexible 90-yttrium (90Y) coils inside a centering balloon were used to allow homogeneous intraarterial dose delivery. One carotid and one iliac artery of 21 hypercholesterolemic rabbits were deendothelialized and then irradiated. Four dose schedules were studied: (1) control (dilated, nonirradiated); (2) 6 Gy; (3) 12 Gy; and (4) 18 Gy. Arterial specimens were histologically evaluated at 8 days and at 6 weeks. For all radiation doses at 8 days compared with controls, there was a significant decrease in bromodeoxyuridine-labeled cells (245 +/- 93 cells/cm in control, 42 +/- 27 in 6 Gy, 72 +/- 107 in 12 Gy, and 2 +/- 2 in 18 Gy groups; P < .001) and in total neointimal cells (891 +/- 415 cells/cm in control, 79 +/- 43 in 6 Gy, 192 +/- 264 in 12 Gy and 22 +/- 13 in 18 Gy groups; P < .0002). At 6 weeks, computer-derived histological percent area stenosis was reduced from 26 +/- 10% in the control group to 1 +/- 1.3% in the 18 Gy group (P < .0001), but lower doses had no significant effect. CONCLUSIONS: Administration of intra-arterial beta irradiation with a 90Y source is technically feasible and compatible with an ordinary catheterization laboratory environment. A dose of 18 Gy effectively induces long-term inhibition of neointimal hyperplasia. PMID- 7554214 TI - Vagal modulation of epicardial coronary artery size in dogs. A two-dimensional intravascular ultrasound study. AB - BACKGROUND: Because the role of tonic vagus nerve activity in regulating conduit coronary artery size remains undefined, we investigated the response of epicardial coronary artery size to changes in resting vagal tone resulting from vagotomy and muscarinic receptor blockade. METHODS AND RESULTS: Using intravascular ultrasound to measure left circumflex coronary artery cross sectional area continuously, we examined the effects of vagotomy on left circumflex cross-sectional area in nine dogs. Lumen area decreased 20% from 8.70 +/- 2.81 to 6.92 +/- 1.97 mm2 after right vagotomy, 17% to 7.19 +/- 2.80 mm2 after left vagotomy (both P < .05 versus baseline), and 38% to 5.42 +/- 2.00 mm2 after bilateral vagotomy (P < .05 versus unilateral vagotomy). Vasoconstriction occurred despite increases in heart rate and an unchanged rate-pressure product. In six additional dogs, after acetylcholine (100 micrograms/kg i.v.), lumen area increased by 18%, although heart rate, blood pressure, and rate-pressure product were unchanged. Vasodilation was prevented by prior muscarinic blockade with glycopyrrolate. With glycopyrrolate administration and heart rate control by pacing, lumen area decreased by 26% (P = .011). When stellate stimulation was performed in a third group of eight dogs with heart rate, blood pressure, and rate-pressure product controlled by a combination of pacing and exsanguination, there was no change in coronary area, thus precluding reflex sympathetic activation as a contributor to the vasoconstriction produced by vagal withdrawal. CONCLUSIONS: Vagus nerve activity maintains tonic dilation of the left circumflex coronary artery by muscarinic receptor activation. Each vagus nerve contributes approximately equally to the tonically dilated state. Vagotomy-induced vasoconstriction occurs independently of local metabolic factors and coronary distending pressure and is a result of cholinergic withdrawal rather than reflex sympathetic activation. PMID- 7554215 TI - Intramural delivery of agent via a novel drug-delivery sleeve. Histological and functional evaluation. AB - BACKGROUND: The infusion sleeve is a novel drug-delivery catheter system designed to deliver an agent under controlled conditions into the arterial wall at the site of angioplasty. The purpose of the present study was to characterize the delivery agent via the infusion sleeve in ex vivo and in vivo models. METHODS AND RESULTS: The delivery of horseradish peroxidase via the infusion sleeve was studied in a porcine explanted heart model. Under physiological conditions, arteries underwent balloon injury (approximately 10% overstretch), after which horseradish peroxidase (2.5 mL) was delivered at specific pressures. Cross sectional analysis demonstrated greater staining when the agent was delivered at increasing pressures. The infusion sleeve was evaluated in an in vivo canine coronary model. With an infusion sleeve loaded over a standard dilatation catheter through a 9F guide, overstretch balloon injury was performed, after which fluoresceinated heparin was delivered. Animals were killed 2 hours after delivery. Fluoresceinated heparin-treated segments demonstrated high fluorescence signals, localizing with smooth muscle cell nuclei with less activity in the interstitium. The functional significance of intramural heparin delivery was studied in a porcine carotid model. In the presence of 111In-labeled platelets, arteries underwent overstretch injury followed by delivery of heparin (50 or 100 units/kg) or vehicle. Platelet deposition was reduced at 30 minutes (57%, P < .01) and 12 hours (39%, P = .06) compared with saline controls. CONCLUSIONS: Agent delivery via the infusion sleeve is pressure dependent; transmural delivery is possible with minimal disruption of arterial wall architecture; the infusion sleeve is compatible with standard angioplasty equipment; and heparin delivery at the site of balloon injury significantly reduces platelet deposition in a porcine model for a minimum of 12 hours. PMID- 7554216 TI - The cellular basis of pacing-induced dilated cardiomyopathy. Myocyte cell loss and myocyte cellular reactive hypertrophy. AB - BACKGROUND: Rapid ventricular pacing leads to a cardiac myopathy consisting of an increase in chamber dimension, mural thinning, elevation in ventricular wall stress, and congestive heart failure, mimicking dilated cardiomyopathy in humans. However, contrasting results have been obtained concerning the mechanisms of ventricular dilation and the existence of myocardial hypertrophy. Moreover, questions have been raised regarding the occurrence of myocardial damage and cell loss in the development of the experimental myopathy. METHODS AND RESULTS: The functional and structural characteristics of the heart were studied in conscious dogs subjected to left ventricular pacing at 210 beats per minute for 3 weeks and 240 beats per minute for an additional week. At the time the animals were killed, measurements of myocardial structural integrity and myocyte shape, size, and number were determined by morphometric analysis of the myocardium in situ and enzymatically dissociated cells. The experimental protocol used was associated with overt cardiac failure documented by an increase in left ventricular end diastolic pressure and a decrease in left ventricular systolic pressure and +dP/dt in combination with tachycardia, ascites, and pulmonary congestion. Although cardiac weights were not altered, cavitary diameter was increased and wall thickness was decreased from the base to the apex of the heart. Multiple foci of replacement fibrosis, comprising 6% of the myocardium, were detected across the left ventricular wall. Measurements of myocyte size and number documented a 39% loss of cells in the entire ventricle and a 61% increase in volume of the remaining viable myocytes. Myocyte hypertrophy was characterized by a 33% increase in cell length and a 23% increase in transverse area, resulting in a 23% increase in the cell length-to-cell diameter ratio. Pacing did not alter the relative proportion of mononucleated, binucleated, and multinucleated myocytes in the myocardium. CONCLUSIONS: Myocyte cell loss and myocyte reactive hypertrophy are the major components of ventricular remodeling in pacing-induced dilated cardiomyopathy. PMID- 7554217 TI - Logistic time constant of isovolumic relaxation pressure-time curve in the canine left ventricle. Better alternative to exponential time constant. AB - BACKGROUND: The time constant of left ventricular (LV) relaxation derived from a monoexponential model has been widely used as an index of LV relaxation rate or lusitropism, although this model has several well-recognized problems. In the present study, we proposed a logistic model and derived a "logistic" time constant (TL) as a better alternative to the conventional "exponential" time constant (TE). METHODS AND RESULTS: A total of 189 beats (147 isovolumic and 42 ejecting beats) were investigated in seven canine excised cross-circulated heart preparations. We found that the logistic model fitted much more precisely all the observed LV isovolumic relaxation pressure-time [P(t)] curves than the monoexponential model (P < .05). The logistic model also fitted well both the time curve of the first derivative of the observed P(t) (dP/dt) and the dP/dt P(t) phase-plane curve. Like TE, TL indicated that volume loading depressed LV lusitropism and that increasing heart rate and ejection fraction augmented it. TL was independent of the choice of cutoff point defining the end of isovolumic relaxation; TE was dependent on that choice. CONCLUSIONS: We conclude that the logistic model better fits LV isovolumic relaxation P(t) than the monoexponential model in the present heart preparation. We therefore propose TL as a better alternative to TE for evaluating LV lusitropism. PMID- 7554218 TI - Adrenergic control of the force-frequency relation. AB - This article briefly reviews recent experimental studies which show that beta adrenergic receptor stimulation produces an important enhancement of the force frequency relation on myocardial contractility. The basic property of the force frequency effect to progressively enhance myocardial contractility as heart rate increases is augmented at each level of increasing adrenergic stimulation. This newly described intrinsic mechanism for the control of cardiac inotropic state, graded beta-adrenergic amplification of the force-frequency relation, is strongly manifested during normal exercise and infusion of a beta-adrenergic agonist at rest, and it influences both systolic and diastolic ventricular function. Significant impairment of adrenergic amplification of the force-frequency relation is observed in experimental heart failure and could contribute to impaired cardiac function during stress or exercise in this setting. PMID- 7554219 TI - Our preoccupation with coronary luminology. The dissociation between clinical and angiographic findings in ischemic heart disease. AB - Nearly 40 years after its invention, the angiogram is still considered by most physicians to be the "gold standard" for defining coronary anatomy. Careful investigations have revealed many deficiencies inherent in this approach. The purpose of this article is to outline the evidence that our current preoccupation with coronary "luminology" may be misguided and to propose a rational paradigm for future practice and investigation. Angiography depicts coronary anatomy from a planar two-dimensional silhouette of the lumen. Angiography is limited in resolution to four or five line pairs per millimeter. Confounding factors include vessel tortuosity, overlap of structures, and the effects of lumen shape. After intervention, a hazy, broadened silhouette may overestimate the actual gain in lumen size. Studies show marked disparity between the apparent severity of lesions and their physiological effects. After myocardial infarction, cardiologists too often do not make an attempt to demonstrate the physiological significance of the stenosis before performing percutaneous coronary revascularization. Similarly, the allure of a better, more gratifying angiogram with new interventional devices appears to be a dominant factor in their popularity. Interventional cardiologists should be aware that techniques yielding marked angiographic benefit may also generate important but unrecognized hazards. The dissociation between the angiogram and clinical outcome should influence future research efforts. Our review of the literature indicates that we may benefit from shifting the current focus and preoccupation with coronary luminology to achieving the desired clinical end point: promoting survival and long-term freedom from myocardial infarction and the disabling symptoms of coronary heart disease. PMID- 7554220 TI - Distribution, morphology, and neurochemistry of endocardial and epicardial nerve terminal arborizations in the human heart. AB - BACKGROUND: The heart contains a variety of morphologically distinct nerve terminals known to influence cardiac function. Little is known about the distribution, morphology, and neurochemistry of these terminals in the human heart. METHODS AND RESULTS: We examined the entire endocardial and epicardial surfaces of infant and adult hearts obtained postmortem and at transplantation using immunohistochemical and histochemical staining of whole-mount preparations in conjunction with confocal and fluorescence microscopy. Terminals arising from nerve fibers (diameter, 6 to 10 microns) immunoreactive for myelin basic protein were identified in the atrial endocardium, epicardium, and coronary sinus, and four types were distinguished by differences in immunostained nerve area (range, 358 to 797 microns 2) and dispersion (range, 620 to 4684 microns 2). These terminals displayed immunoreactivity for tyrosine hydroxylase, neuropeptide Y, and the general neural marker protein gene product 9.5. Acetylcholinesterase (AChE) activity was detected in < 5% of endocardial terminals and in no epicardial terminals arising from myelinated fibers. The latter were observed in close proximity to mesothelial cells, and nerve fibers supplying these terminals were found to be associated with local ganglia. A distinct population of terminals (mean stained area, 35 microns 2; 18 to 53 microns 2, 95% CI; and mean dispersion, 59 microns 2; 38 to 80 microns 2, 95% CI) was demonstrated to arise from nonmyelinated fibers (mean diameter, 2.5 microns; 2.2 to 2.8 microns, 95% CI) in the endocardial plexus of the atria and left ventricle and were predominantly AChE-positive. CONCLUSIONS: Specialized nerve terminals are distributed more widely in the human heart than has been described in experimental animals. These terminals express either AChE activity or tyrosine hydroxylase and neuropeptide Y immunoreactivity, suggesting that acetylcholine, catecholamines, and neuropeptide Y may be present in sensory and autonomic nerves in the human heart. PMID- 7554221 TI - Images in cardiovascular medicine. Aortic valves. Shamrock, bileaflet, and lucky? PMID- 7554222 TI - Rapid angiographic progression of coronary artery disease in patients with elevated lipoprotein(a) PMID- 7554223 TI - Lipoprotein(a) may have contrasting effects on different aspects of atherosclerotic coronary disease. PMID- 7554224 TI - Intake of mercury from fish, lipid peroxidation, and the risk of myocardial infarction and coronary, cardiovascular, and any death in eastern Finnish men. PMID- 7554225 TI - Coronary atherosclerosis and the angiotensinogen gene. PMID- 7554226 TI - 68th Scientific Sessions of the American Heart Association. Anaheim, California, November 13-16, 1995. Abstracts. PMID- 7554227 TI - Phorbol esters and PKC signaling regulate proliferation, vimentin cytoskeleton assembly and glutamine synthetase activity of chick embryo cerebrum astrocytes in culture. AB - We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes. PMID- 7554228 TI - Differential regulation of phospholipases C and D by phorbol esters and the physiological activators carbachol and glutamate in astrocytes from chicken embryo cerebrum and cerebellum. AB - Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-alpha/beta). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes, than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development. PMID- 7554229 TI - Long-term consequences of prenatal cocaine exposure on biogenic amines in the brains of mice: the role of sex. AB - Prenatal cocaine exposure leads to multiple abnormalities in the mature offspring. We explored the effects of gestational exposure to cocaine on neurotransmitter systems of adult mice. The subjects were the mature offspring of mice (a) prenatally fed cocaine between gestational day (G) 8 and G19, (b) pair fed chow and water, or fed chow and water ad libitum. The forebrains of the mature offspring were assayed for monoamines and amino acids. Cocaine exposure particularly affected the dopaminergic system and in a sex-specific manner. In males dopamine concentrations were decreased and dopamine turnover was increased, whereas in females dopamine concentrations were increased and turnover was decreased. Neither norepinephrine, the serotonergic system, nor neuroactive amino acids (or their precursors) were affected by cocaine. Thus, in utero exposure to cocaine produces long-lasting, specific defects in the dopaminergic system. PMID- 7554230 TI - Heterogeneity of tau protein and mRNA expression during the development of cerebellar granule cell neurons in vitro. AB - Tau microtubule-associated proteins constitute a group of developmentally regulated neuronal proteins which promote microtubule polymerization and stabilization and hence have important implications during neuronal morphogenesis. We have examined the expression of tau mRNA and protein levels during the differentiation of cerebellar granule neurons over a period of 3 weeks in vitro. Oligonucleotide probes directed towards either immature or mature forms of tau mRNA were detected by in situ hybridization. Such experiments demonstrated that the time interval between 1 and 4 days in vitro represents a developmental epoch in the regulation of tau mRNA whereby the dominant immature tau messages were gradually replaced by mature mRNAs. Analysis of the profile of the various tau isoforms showed further developmental regulation with the transient rise in immature tau variants followed by the appearance of mature isoforms in older cultures. The increase in tau heterogeneity during granule neuron differentiation was enhanced by and could be attributed to intensive post-translational phosphorylation. Dephosphorylation of cell cultures demonstrated that the majority of tau was phosphorylated and that such a modification had profound affects on the localization of tau within developing neurons by immunocytochemistry. This study describes the profile of tau protein and mRNA levels expressed by differentiating cerebellar granule neurons in vitro and clearly demonstrates that tau is developmentally regulated and that important changes in tau expression occur at a time when processes are consolidating their first contacts. PMID- 7554231 TI - Developmental changes in the expression of alpha-, beta- and gamma-subspecies of protein kinase C at synapses in the ventral horn of the embryonic and postnatal rat spinal cord. AB - Developmental changes in expression of alpha-, beta- and gamma-subspecies of protein kinase C (PKC) at synapses in the ventral horn of the rat spinal cord were immunocytochemically investigated. On embryonic day 15, a few synapses were found in the ventral horn, and they gradually increased in number until postnatal day 21 or 28. During the embryonic period, immunoreactivity (IR) for all three subspecies was demonstrated in both the pre- and postsynaptic regions. In the former, IR was detected mainly along the outer surface of the synaptic vesicles, and in the latter, along the postsynaptic membranes. At these stages, synapses were morphologically immature, having a faint postsynaptic density and a few round synaptic vesicles. After birth, IR for PKCs at the postsynaptic densities became stronger, but gradually disappeared in most of the presynaptic regions. In adult, IR for PKCs was detected only at the postsynaptic densities. At the later postnatal stages, the synapses were fully mature, having a thick postsynaptic density, a great number of synaptic vesicles and a distinct synaptic cleft as those in adult animals. In addition, the developmental changes in expression of these subspecies of PKC in the presynaptic regions were quite different. These findings suggest that the increase in expression of PKC at postsynaptic densities might be closely related with the development of synaptic functions, and also that each subspecies of PKC may take part in different aspects of synaptogenesis. PMID- 7554232 TI - AMPA receptors in cerebellar granule cells during development in culture. AB - The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK 801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554233 TI - Neonatal capsaicin treatment (NCT) alters the metabolic activity of the rat somatosensory cortex in response to mechanical deflection of the mystacial vibrissae. AB - Capsaicin, a selective neurotoxin of unmyelinated C-fibers, was administered to neonatal rat pups at birth. Following a recovery period of 10 days, pups were injected with 2-deoxy-glucose (2DG) and subjected to repetitive mechanical stimulation to the left whiskerpad. Their brains were then harvested for autoradiography. The observed changes in 2DG uptake in the somatosensory cortex of capsaicin-treated rats were compared to vehicle-treated rats. The cross sectional area and density of 2DG uptake by the primary and the secondary somatosensory cortex (SSI and SSII, respectively) were measured. Capsaicin treated rats significantly exhibited a reduction in area of activation and a decrease of 2DG uptake in both structures. The present data indicates that neonatal capsaicin affects the functional activity of the rat somatosensory cortex. It is suggested that unmyelinated sensory afferents play a role in the development of the rat somatosensory system. PMID- 7554234 TI - Permeability of the developing blood-brain barrier to 14C-mannitol using the rat in situ brain perfusion technique. AB - The brain penetration of 14C-mannitol was investigated using a bilateral in situ brain perfusion technique followed by capillary depletion analysis. This technique measures the uptake of slowly penetrating solutes in the absence of the systemic circulation, and separates accumulation in brain endothelial cells from uptake into brain parenchyma. Penetration of 14C-mannitol was linear up to 30 min in rats aged 1, 2, 3 weeks and in adults. The brain mannitol space was higher in 1-week-old neonatal rats compared with adults (P < 0.05) and was due to a greater initial volume of distribution (Vi) for mannitol in the neonates, and not due to an elevated transfer rate (K(in)). Thirty percent of mannitol in the neonatal brain was associated with the capillary containing fraction, whereas in the adult only 13% was found in this fraction. This suggests that the permeability of the blood-brain barrier to mannitol does not change significantly with development but that more mannitol is associated with endothelial cells in the neonate. An investigation of 14C-glycine uptake was also carried out, and unlike mannitol the K(in) was greater in the neonate compared to the adult suggesting an elevated rate of transfer for this amino acid into the neonatal rat brain. PMID- 7554235 TI - An activated mutant of the alpha subunit of G(o) increases neurite outgrowth via protein kinase C. AB - The GTP-binding protein, G(o), is present at very high concentration in the neuronal growth cone membrane. The expression of activated mutants of the a subunit of G(o) increases neurite outgrowth. To determine the intracellular mechanism for this outgrowth, we have examined activated alpha o-dependent outgrowth in the presence of agents which modulate different signal transduction cascades. Activation of protein kinase C with phorbol esters or with diacylglycerol prevents the alpha o-dependent increase in neurite extension. Inhibition of protein kinase C with staurosporine, with H7, or with long-term, high dose phorbol ester treatment resulted in greater neurite elongation, and no further increase after activated alpha o transfection. The protein phosphatase inhibitor, okadaic acid, also blocked the effect of activated alpha o. In contrast, tyrosine kinase inhibitors and agents which alter cAMP levels did not alter activated alpha o-dependent neurite extension. We tested a number of compounds which alter intracellular calcium levels. TMB-8 and thapsigargin prevented an increase in outgrowth by activated alpha o, but diltiazem, Bay K8644 and dantrolene had no effect on activated alpha o-dependent outgrowth. These studies suggest that activated alpha o increases neurite outgrowth by inhibiting protein kinase C and by modulating intracellular calcium release. PMID- 7554236 TI - Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth. AB - Intracellular delivery of anti-GAP IgG inhibits the elaboration of neurites by NB2a/d1 cells. However, recent studies indicate that the extent of inhibition is minimized when cells were cultured on poly-L-lysine-coated or laminin-coated versus uncoated plates, suggesting that the role of GAP-43 in neuritogenesis may be specifically related to membrane adhesiveness. We therefore examined the influence of inhibition of thrombin, the neuronal surface protease that restricts neurite outgrowth, on GAP-43-dependent neurite outgrowth. Treatment of cells with the specific thrombin inhibitor hirudin in the presence of serum induced a similar percentage of neurite outgrowth as was observed following serum withdrawal. However, while neurite outgrowth induced by serum deprivation of cells was reduced following intracellular delivery of anti-GAP IgG, neurite outgrowth induced by hirudin treatment of cells was not. That inhibition of neuronal surface protease activity overcomes the inhibition of neurite outgrowth following intracellular delivery of anti-GAP IgG provides further evidence that the role that GAP-43 plays in neuritogenesis is related to membrane adhesiveness. PMID- 7554237 TI - Estrogen-induction of dendritic spines in ventromedial hypothalamus and hippocampus: effects of neonatal aromatase blockade and adult GDX. AB - Treatment of male rats at birth with an aromatase inhibitor (Letrazole), followed by adult gonadectomy GDX, led to an increase in dendritic spine density on ventromedial hypothalamic neurons (VMN) when treated with estrogen as compared to a decrease when vehicle animals were given estrogen. In contrast, estrogen treatment increased dendritic spine density on CA1 pyramidal neurons regardless of neonatal treatment. In addition, in CA1 there was a significant difference between the two estrogen groups. These results suggest that estrogen induction of dendritic spines in the VMN and CA1 is dependent on organizational effects of gonadal steroids. PMID- 7554238 TI - Effect of kainate-induced seizure activity on the polyamine interconversion pathway in juvenile rat brain. AB - The activity of the polyamine interconversion pathway was investigated in the hippocampus and piriform cortex after systemic KA administration in juvenile rats. Pretreatment of 7-day-old rats with the polyamine oxidase inhibitor, MDL 72527, induced a similar accumulation of N-acetylspermidine and N-acetylspermine in control and kainate-treated animals. The results indicate that KA-induced seizure activity has no effect on the polyamine interconversion pathway in developing rat brain. PMID- 7554239 TI - Optimizing the use of cyclosporine in renal transplantation. AB - OBJECTIVE: To review the existing data on the use of cyclosporine (CsA) in kidney transplantation, particularly with respect to therapeutic drug monitoring. DATA SOURCES: A literature search was conducted of applicable articles related to therapeutic drug monitoring of cyclosporine in renal transplantation. Previous consensus guidelines were examined. Discussions on issues related to this topic convened in Toronto, ON, on June 15-16, 1994. DATA SYNTHESIS: The literature was analyzed to examine patient factors and drug interactions affecting CsA concentrations, the effect of CsA concentrations on patient outcome, current methods of analysis, pharmacodynamic monitoring, and new immunosuppressants. CONCLUSIONS: CsA has improved the success of kidney transplantation, reducing the incidence and severity of acute rejection and improving short-term patient and graft survival. The rate of graft loss after the first year (primarily due to chronic rejection) has remained largely unchanged. Sandimmune Neoral offers promise due to its better bioavailability and limited dependence on bile flow for absorption. Long-term studies are underway to determine its effectiveness and safety. Indications for therapeutic drug monitoring for CsA are provided. PMID- 7554240 TI - Technological trends in clinical laboratory science. AB - OBJECTIVE: This article will review the advancements and new developments being made in (1) advanced computers, (2) microtechnology, (3) advanced immunodiagnostics, (4) neural networks, and (5) molecular biology. The influence of these technologies and their products on clinical laboratories is also discussed. CONCLUSION: Significant evolutionary and revolutionary technological changes are occurring in a number of scientific and engineering disciplines that impact the medical profession. As a consequence of this era of rapid change, the discipline of Clinical Laboratory Science is undergoing a "technological explosion" which is having a significant and profound effect on how clinical laboratories of today and tomorrow are and will be staffed, equipped, and operated. PMID- 7554241 TI - A guide to the interpretation of serum prostate specific antigen levels. AB - OBJECTIVE: To review the factors that affect the concentration of prostate specific antigen (PSA) in the serum. RESULTS: The discussion includes the structure of PSA; its distribution and metabolism; various analytical aspects of PSA measurements; the effects of clinical manipulations on PSA, including digital rectal examination, transrectal ultrasound, cystoscopy, biopsy and transurethral resection of the prostate; factors affecting PSA levels in health, in benign disease, and in prostate cancer; the effect of various treatments on PSA; and the issue of reference ranges. CONCLUSION: Laboratory staff and physicians must take many factors into consideration when interpreting PSA results. PMID- 7554242 TI - Quantification of creatine kinase BB isoenzyme in tumor cytosols and serum with an ultrasensitive time-resolved immunofluorometric technique. AB - OBJECTIVES: To develop a highly sensitive immunofluorometric procedure for creatine kinase BB isoenzyme and use it to measure CK-BB in tumor cytosolic extracts and serum of cancer patients and healthy volunteers. DESIGN AND METHODS: For assay development, we used two monoclonal antibodies in combination with time resolved fluorometry and the biotin-avidin system. We measured CK-BB in breast tumor cytosols and studied its association with steroid hormone receptors. We also measured CK-BB in the serum of healthy subjects and patients with prostate cancer. We have examined the molecular weight of CK-BB in serum using high performance liquid chromatography. RESULTS: The evaluation of the method revealed good precision and accuracy. Study of 336 breast tumor cytosols and 9 normal breast cytosols has shown that CK-BB is overexpressed by 95% of breast tumors and that CK-BB is present in its 80 kDa form. A close association between CK-BB and estrogen but not progesterone receptors was found, suggesting that CK-BB overexpression is another marker of estrogen sensitivity of these tumors. Previous studies, using CK-BB radioimmunoassay could not detect CK-BB in the serum of about 50% of healthy subjects. We have assessed CK-BB levels in 80 male volunteers, detected CK-BB in all sera and provided a detailed distribution of values. We further demonstrated that 30% of prostate cancer patients in remission (PSA < 0.4 microgram/L) post radical prostatectomy and 50% of patients with active prostate cancer (PSA > 20 microgram/L) have elevated serum CK-BB levels. The patients with highly elevated CK-BB also had highly elevated serum PSA. We have demonstrated that some patients who have elevated serum CK-BB also have macromolecular CK complexes in their serum with molecular weights of 700 and 350 kDa as well as the 80 kDa CK-BB isoenzyme. Only the latter was recognized by the assay developed. CONCLUSIONS: CK-BB is a marker of estrogen sensitivity in breast cancer; Patients with prostate cancer have elevated CK-BB in their serum; The new highly specific and sensitive assay may be further used to study the role of CK BB in various malignancies. PMID- 7554243 TI - Analytical evaluation of a newly developed ELISA mass assay for pancreatic amylase. AB - OBJECTIVE: To evaluate a newly developed ELISA mass assay for Pancreatic Amylase. METHODS: The serum levels of pancreatic amylase were measured by an in-house developed ELISA assay on microtitre plate and compared with two activity assays: immunoinhibition and electrophoresis. RESULTS: The proposed method was accurate and precise, as indicated by a recovery of 102% and coefficient of variation of less than 5% for within-run and less than 10% for between-run assay. The proposed assay showed good correlation with the activity assays (r = 0.98). The mass concentrations were three times higher than the activities. The relative values (mass or activity units/upper reference limits), however, were concordant in controls and in pancreatic and non-pancreatic conditions. CONCLUSION: Our results indicate that the activity measurements and mass concentrations of pancreatic amylase in serum are comparable and interchangeable after adjusting for the reference range. PMID- 7554244 TI - Enzyme immunoassay for quantification of tenascin in biologic samples. AB - An enzyme immunoassay was developed for quantification of tenascin in biologic samples. An enzyme conjugate prepared by coupling peroxidase to a well characterized, affinity-purified monoclonal antibody EB2 to human tenascin was used as principal reagent. The assay comprises 96-well microtitration strip plates with immobilized monoclonal antibody DB7 to human tenascin. By using a novel monoclonal antibody suppressing human-anti-mouse-factor, MAK33, in the sample buffer, the specificity of the test could be improved. The method has a minimum detectable sensitivity of 1.5 ng tenascin and permits determination of tenascin in various biologic samples. The coefficients of variation within run and between run ranged from 0.9% to 5.0%. The average tenascin concentration in normal plasma was 0.97 mg/L (n = 200) and in serum 0.73 mg/L (n = 200). The tenascin concentrations were also determined in samples of urine, bile, amniotic fluid, seminal fluid, cerebrospinal fluid, bronchoalveolar lavage (BAL) fluid, and pleural fluid showing general applicability of the assay. The method permits the determination of tenascin in samples of different body fluids from various diseases, including cancer, showing increased amounts of the protein at the tissue level. PMID- 7554245 TI - Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method. AB - OBJECTIVE: To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. METHODS: Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. RESULTS: Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. CONCLUSION: Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening. PMID- 7554246 TI - New methods for rapid detection of low-density lipoprotein receptor and apolipoprotein B gene mutations causing familial hypercholesterolemia. AB - Due to a genetic founder effect, five mutations in the low-density lipoprotein receptor gene account for approximately 83% of familial hypercholesterolemia (FH) diagnosed in French-Canadians. The most frequent mutation, present in 61% of heterozygotes, is a > 10 kb deletion of the 5' region of the gene that removes the promoter and the first exon, resulting in a null allele. Other less prevalent mutations include a gene deletion of approximately 5 kb, which removes exons 2 and 3 (2% of cases) and three missense mutations: Trp66-->Gly (exon 3) (12%), Glu207-->Lys (exon 4) (3%), and Cys646-->Tyr (exon 14) (6%). The apoB Arg3500- >Gln mutation was absent in 228 French Canadians with the FH phenotype. Taking advantage of the availability of fluorescent DNA detection, we have substantially improved the assays for these mutations. PMID- 7554247 TI - Poor applicability of the Friedewald formula in the assessment of serum LDL cholesterol for clinical purposes. AB - OBJECTIVE: To determine the accuracy of the estimation of serum low-density lipoprotein (LDL) cholesterol concentration by the Friedewald formula. METHODS: Modifications of the calculation formula are presented on the basis of ultracentrifugal separation of serum high-density lipoprotein and LDL cholesterol in the specimens collected (n = 1215) in a nationwide health survey. RESULTS: The formulas obtained from different subject groups differed relatively little from each other. The accuracy of the original Friedewald formula was poor; in about 36% of the subjects the error was more than 5% compared with ultracentrifugally obtained results. By applying the currently recommended coronary heart disease (CHD) risk categorizations, high proportions (5%-28%) of the subjects were classified into wrong CHD risk categories when LDL cholesterol was calculated with any of the formulas. At high serum triglyceride levels, misclassifications were especially common. CONCLUSIONS: We conclude that even the most accurate LDL cholesterol calculation methods should be used with caution while classifying subjects into categories of CHD risk. In hypertriglyceridemic subjects, the calculation formulas probably should not be used at all. PMID- 7554248 TI - Tissue polypeptide specific antigen, neopterin, and CRP for monitoring heart transplant recipients. AB - OBJECTIVES: The aim of our study was to investigate the usefulness of tissue polypeptide specific antigen, an established tumor marker detecting proliferation of cells, for monitoring heart transplant recipients in order to detect infection and rejection early. METHODS: Tissue polypeptide specific antigen serum levels were compared with neopterin and C-reactive protein serum concentrations. RESULTS: When infections occurred, tissue polypeptide specific antigen serum concentrations were increased approximately five times (mean: 214 U/L, SD 145), while in instances of acute rejection crises, they were increased two times (mean: 76 U/L, SD 16 in comparison with the values during uncomplicated postoperative courses (mean: 45 U/L, SD 26). CONCLUSIONS: Tissue polypeptide specific antigen was the only investigated analyte that showed significant diagnostic validities when infections were compared with rejections. The discrimination between an uncomplicated postoperative course and a rejection or infection episode was only possible with a combination of the three analytes. PMID- 7554249 TI - Abnormal trace elements in a patient on total parenteral nutrition with normal renal function. AB - OBJECTIVE: To describe trace metal changes in a 74-year-old male patient with mesenteric fibrosis and a small bowel fistula who was maintained on total parenteral nutrition in the hospital and at home. METHODS: Trace elements which included chromium and selenium were monitored over a 14-month period as part of his nutrient follow-up. RESULTS: Serum chromium reached levels > 21-fold the upper reference range, and serum selenium, in contrast, was < 0.5 the lower reference range. Plasma aluminum was also measured, and found to be nearly twice the upper reference range, although the patient had normal renal function. We measured the aluminum content of the parenteral nutrients and additives, and found that replacement of calcium gluconate by calcium chloride helped to reduce the aluminum content in the final parenteral solution by 34%. Aluminum and chromium contaminants found in parenteral solutions need to be reduced or removed to avoid toxic accumulation. CONCLUSION: This study illustrates the importance of adequately adjusting essential trace elements, and monitoring contaminants in parenteral fluids in an individual on total parenteral nutrition. PMID- 7554250 TI - Single-strand conformational polymorphisms (SSCP): studies of the genetic polymorphisms of exon 4 of apolipoprotein C III. AB - OBJECTIVE: We used single-strand conformational polymorphism (SSCP). To screen for mutations/polymorphisms in exon 4 of the apolipoprotein C III in 45 patients with hypertriglyceridemia and 46 control individuals, single-strand conformational polymorphism was investigated using restriction endonuclease and amplification refractory mutations systems (ARMS). RESULTS: SSCP identified six patterns corresponding to six genotypes. We confirmed that the different genotypes result from the two polymorphic sites at positions 3175 and 3206 of the apo C III gene. Only three of four possible haplotypes were found in the study population. This resulted in the identification of 6 of the 10 possible genotypes. CONCLUSIONS: SSCP is a useful method to screen for both known and unknown mutations/polymorphisms and should have increasing applications in clinical laboratories involved with the study of genetic markers of a wide variety of diseases. PMID- 7554251 TI - Influence of anti-LDL receptor antibody on the transfer of cholesterol from LDL to isolated lymphocytes in normal subjects. PMID- 7554252 TI - Enzymatic determination of lipids in liver extracts. PMID- 7554253 TI - Correlation among ionized calcium, citrate, and total calcium levels during hepatic transplantation. PMID- 7554254 TI - Human plasma preservation for amino acid analysis by immediate low temperature freezing. PMID- 7554256 TI - Current trends in the treatment of ectopic pregnancy. PMID- 7554255 TI - Umbilical veins in dichorionic twins. A morpho-functional assessment. AB - Investigations on singleton and twin pregnancies show different functional behaviour on maternal-fetal relationship. In some ways twin pregnancies may be considered at risk and they may develop associated pathologies such as hypertension. The aim of this work was to evaluate the morpho-functional behaviours of umbilical cord veins in twin and singleton gestations to better understand the role of these extra-embryonic tissues in the regulation of pregnancies. The umbilical cords were studied from singleton pregnancies and from dichorionic twin pregnancies. Biochemical and morphological investigations were carried out. A significant decrease in the anisotropy values was observed in endothelial cells from twins compared with singletons. Our ultrastructural data show immaturity features at the vein vessel wall level in twins. Furthermore, immunohistochemical investigations showed a lower degree of expressivity concerning adhesion molecules such as ICAM-1 and ELAM. Morphogenetic extracellular glycoproteins like fibronectin and tenascin seem over-expressed in twin pregnancies. Our morpho-functional data well testify the lower maturation degree of umbilical cord veins in twins with respect to singletons. PMID- 7554257 TI - Case report: benign neural tumour of small bowel presenting as a pelvic mass. PMID- 7554258 TI - Antenatal ambroxol usage in the prevention of infant respiratory distress syndrome. Beneficial and adverse effects. AB - A bromhexine metabolite ambroxol, is a relatively new promoter of fetal lung maturation. The data on its efficacy and side effects in humans are not yet as many as those of corticosteroids. We found that in 24 premature labor patients ambroxol reduced the incidence of respiratory distress syndrome when compared with the control group, consisting of 58 patients. There was no concomitant disorder in any patient that would have contributed to the fetal lung maturation. We also observed septic morbidity to be less frequent in the ambroxol group. Thyroid hormone levels were within normal range both in maternal and fetal circulation. There were no side effects attributable to the drug. Maternal liver and renal function test results did not differ significantly throughout the treatment. PMID- 7554259 TI - Short-term use of Goserelin depot in the treatment of dysfunctional uterine bleeding. AB - STUDY-PLAN: an open study aimed at evaluating the results of a short term therapy (3 months) with Goserelin depot as a medical treatment of premenopausal dysfunctional uterine bleeding. PATIENTS: 60 premenopausal women (aged 36-50) with dysfunctional uterine bleeding, presenting simple endometrial hyperplasia. RESULTS: after the treatment, spontaneous menstrual bleeding recurred in 57/60 patients, while 3/60 (5%) patients remained amenorrheal during the whole period of follow-up, showing a postmenopausal hormonal pattern. In the first post therapy menstrual cycle all the 57 patients had a bleeding score < 100; patients relapsing during the second, third and fourth cycle were respectively 2/54 (3.7%), 5/48 (10.7%) and 17/38 (44.7%). The fourth post-therapy cycle was delayed 6-9 months after the last injection of Goserelin. Both the mean blood loss and the mean duration of bleeding were significantly reduced in all post-therapy cycles. Eleven patients were anaemic before therapy (Hb < 12 g%); Goserelin treatment resulted in a normalization of the hematological parameters. At the end of treatment a small area of hyperplasia persisted in only 4/60 patients (6.7%). Localised or diffused hyperplasia were found respectively in 5/54 (9.3%) and in 1/54 patients (1.9%) at three months, and in 5/48 (10.4%) and 4/48 (8.3%) at a six-month follow-up. Side effects were infrequent. CONCLUSIONS: the long symptom free period and the low incidence of side effects indicates Goserelin depot as a valuable medical treatment for dysfunctional uterine bleeding. PMID- 7554260 TI - Development of melanoma throughout two close pregnancies. Case report. AB - Primary melanoma is the most malignant and the least frequent of all cutaneous tumors. Its incidence in pregnancy varies from 0.1 to 2.8/1000. Up to today, impact of pregnancy on melanoma's course is still a much debated question in the literature. Some earlier reports suggested a particularly serious prognosis for melanoma associated with pregnancy, while more recent studies show that pregnancy may influence relapses without significantly altering survival. This paper reports the case of a woman affected by melanoma, whose clinical conditions became more and more serious during her second pregnancy and the following puerperal period. The progressive impairment of her clinical condition has suggested a correlation between the two close pregnancies and the unfavourable course of her disease. PMID- 7554261 TI - Prenatal evaluation of congenital heart disease in high-risk pregnancies. AB - The authors report their experience of echocardiography performed on a group of 736 pregnancies with specific risk-factors for congenital heart disease (CHD). The aim of the study was to evaluate the influence of specific risk-factors (established through the genetic counselling) in detecting fetal cardiac anomalies and diagnostic accuracy of extended fetal echocardiographic examination in high-risk pregnancies. Twenty-seven heart defects were observed (3.6%), of these 24 were detected at ultrasound. Specificity and sensitivity were 99% and 90% respectively. These results suggest the importance of an early detection of pregnancies at increased risk for CHD and confirm the good diagnostic accuracy of a multiple cardiac examination. PMID- 7554262 TI - The comparison of 50 grams glucose challenge test, HbA1c and fructosamine levels in diagnosis of gestational diabetes mellitus. AB - We evaluated the sensitivity, specificity, positive and negative predictive values of 50 grams glucose challenge test, serum fructosamine and HbA1c levels as screening tests for gestational diabetes mellitus. Forty-two pregnant patients between the 24th and 28th week of their pregnancies were included in the study. Blood fructosamine and HbA1c levels did not differ significantly from the 50 grams glucose challenge test and were concluded to be alternatives to this test. Any combination of these 3 tests gives better results than a single test, but no one of the combinations is superior to the others. PMID- 7554263 TI - Preterm labour and neonatal parameters. AB - OBJECTIVES: Our objective was to identify those neonatal factors associated with survival in preterm infants. MATERIAL AND METHODS: We examined a population of 457 preterm newborns delivered between 1 January-31 December 1990, with birthweight between < 1000 gr and > 2000 gr, in respect to umbilical pH values, plasmatic glucose values, 5-minute Apgar score and gestational age. Data were abstracted from the maternal intrapartum records and the neonatal records, with specific attention to neonatal parameters. RESULTS: A positive correlation between birthweight and 5-minute Apgar score, between birthweight, pH and glucose values was noted. No such relationship existed between pH values and 5-minute Apgar score. Within birthweight groups the distribution of neonatal mortality rate was 85.18% in ELBW (Extremely Low Birth Weight) and only 5.26% in LBW (Low Birth Weight). CONCLUSION: The importance of the echographic estimate of the fetal weight must be emphasized, since a birthweight of 1500 gr represents the cut-off for the neonatal morbidity and mortality, and also an accurate clinical evaluation of the risk of preterm labour or pathologies in order to improve the estimate of childbirth timing. PMID- 7554264 TI - Broad ligament abscess after operative hysteroscopy. AB - Delayed complications in hysteroscopic surgery are very rare. This report describes an abscess of the left parametrium which occurred in a young woman after an operative hysteroscopy in which uterine perforation occurred. PMID- 7554265 TI - Juvenile laryngeal papillomatosis from an HPV-positive mother. A case report. PMID- 7554266 TI - Mayer-Rokitansky-Kuster-Hauser syndrome and ovarian cancer. Report of a case. AB - A case of ovarian cancer associated with the Mayer-Rokitansky-Kuster-Hauser syndrome is reported. As far as we know, this is the second report of ovarian neoplasm associated with this syndrome. The need for gynecological control of ovarian and other possible pathologies of the genital tract in these women is discussed. PMID- 7554267 TI - Radiochemical assay of minute quantities of galactose-1-phosphate uridyltransferase activity in erythrocytes and leukocytes of galactosemia patients. AB - A sensitive radioisotopic method has been developed which can detect galactose-1 phosphate uridyltransferase (GALT) activity as low as 0.1% of normal control values in both erythrocytes and leukocytes. This assay utilizes carbon-14 labeled galactose-1-phosphate with high specific activity and requires removal of endogenous galactose-1-phosphate (Gal-1-P) and uridine diphosphate glucose (UDPGlc) through dialysis. Optimal exogenous UDPGlc concentration has been determined with a fixed concentration of Gal-1-P in the incubation. The rate of product, uridine diphosphate galactose (UDPGal), formation is monitored at three different times. Among 423 patients with galactosemia studied by this method, 363 patients exhibited no detectable GALT activity in their erythrocytes and 60 patients were found to have detectable erythrocyte GALT activity ranging from 0.02 to 5.0 units normal values: > 20 units). The former group of patients was designated as classic galactosemia (GG) and the latter group as galactosemia variant (GV). Leucocytes from ten patients belonging to the GG group also showed complete absence of GALT activity while leukocytes from two patients belonging to the GV group showed GALT activity at levels comparable with those found in their erythrocytes. Because there is extensive biochemical heterogeneity among galactosemia patients, we recommend that an assay with increase sensitivity be carried out on blood samples from galactosemia patients so that clinical, biochemical and molecular correlations made by different groups of investigators can be compared. PMID- 7554268 TI - A sandwich enzyme immunoassay for the determination of neutrophil lipocalin in body fluids. AB - Human neutrophil lipocalin was purified from human buffycoat. A polyclonal antibody was obtained by immunisation of rabbits. The antibody reacted with the free lipocalin as well as with the PMNL-gelatinase bound protein. This antibody was used to establish a sensitive sandwich-ELISA for the determination of the protein in body fluids using the biotin/streptavidin system. The mean intra-assay C.V. was 2.3% and the mean inter-assay C.V. 6.7%. The recovery in human plasma was determined to be 98.8%. The ELISA allowed the determination of the protein in the concentration range 0.2-25 micrograms/l. Measurement of the neutrophil lipocalin concentration showed that human plasma of healthy donors contained 9.7 +/- 81 micrograms/l (n = 122) and that the concentrations in serum were significantly higher (P < 0.001) with 133 +/- 90 micrograms/l (n = 122). Neutrophil lipocalin was also found in the urine of healthy donors (8.1 micrograms/l; n = 9). Very high concentrations of this lipocalin were found in the synovial fluids of patients suffering from inflammatory rheumatoid arthritis (1.7 +/- 1.4 mg/l; n = 37). PMID- 7554269 TI - Blood antioxidants (vitamin E and beta-carotene) in long-term low density lipoprotein apheresis. AB - We measured vitamin E and beta-carotene in the serum and in circulating lipoproteins in a large population of 15 patients with familial hypercholesterolaemia who were undergoing long-term treatment by low density lipoprotein (LDL) apheresis. The technique used for apheresis was dextran sulphate cellulose adsorption. The results showed that before LDL apheresis, patients had high vitamin E and normal beta-carotene levels in the serum and in the VLDL+LDL fraction. There were no relationships between serum levels of vitamin E and beta-carotene and the duration of LDL-apheresis. Low vitamin E and beta-carotene levels in the HDL fraction could be related to the low HDL concentrations in these patients. Vitamin E/cholesterol ratios were similar to those of the normolipaemic controls whereas beta-carotene/cholesterol ratios were lower. After LDL-apheresis treatment, the ratios in the HDL fraction fell whereas the ratios in the serum and in the VLDL and LDL fraction did not change. This study shows that these patients exhibited no deficiency in either serum of VLDL LDL of vitamin E or beta-carotene after long-term treatment by LDL-apheresis and that the status of these antioxidants in serum was independent of the duration of treatment. PMID- 7554270 TI - Changed transferrin sialylation in Parkinson's disease. AB - Variation in the sialylation pattern of transferrin was studied in serum and cerebrospinal fluid (CSF) of 90 patients with Parkinson's disease (PD), dementing and non-dementing, de novo and treated, and was compared with the variation observed in a group of 21 age-matched healthy controls. In serum and CSF of PD patients the proportional contribution of the different sialo-transferrins was independent of sex or dementia. However, a significant shift was found towards the more sialylated fractions for serum transferrin in both de novo and treated PD patients. This shift was not observed for CSF transferrin. The contribution of the tau-transferrin fraction, reduced in de novo PD patients, returns on treatment to the level observed for healthy controls. These observations may be important, as the degree of sialylation of transferrin in serum and CSF plays a role in the homeostasis of iron, and suggest that alterations in transferrin sialylation may play a role in the pathophysiology of PD. PMID- 7554271 TI - Enzymatic determination of bicarbonate in serum by flow injection analysis. AB - An automated method for the determination of bicarbonate in human serum based on the enzymatic reaction between the analyte and phospho(enol)pyruvate (PEP) in the presence of PEP carboxylase is proposed. The analytical reaction is coupled with a derivatization reaction in which the NADH consumed is fluorimetrically monitored (lambda ex = 340 nm, lambda em = 460 nm). A stopped-flow/flow-injection approach is used in which the enzymes (PEP carboxylase and malate dehydrogenase) are immobilized on controlled-pore glass. The linear determination range is between 25 and 300 mmol/l (r2 = 0.9973). The %C.V. for the within- and between run studies, performed at three concentration levels, ranges between 1.0 and 3.6% and the sampling frequency is 20 per h. PMID- 7554272 TI - Oxidative stress and erythrocyte membrane fluidity in patients undergoing regular dialysis. AB - Oxidative damage due to free radical production is increased in uraemic patients and has been suggested as a possible factor contributing to the anaemia of chronic renal failure (CRF) and the pathogenesis of atherosclerosis. Oxidative stress was assessed in 40 patients with CRF maintained by either haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) and in 18 healthy controls. Lipid peroxidation (assessed as malondialdehyde, MDA), total glutathione (TG), antioxidant enzyme (glutathione reductase (GSHRx), glutathione peroxidase (GSHPx) and superoxide dismutase (SOD)) activity and antioxidant associated trace metal (selenium, copper, zinc) levels were studied. Erythrocyte membrane fluidity was examined using the fluorescent probe 1,6 diphenyl-1,3,5 hexatriene (DPH). The results indicate increased levels of oxidative stress and altered erythrocyte membrane fluidity in patients treated with CAPD compared with controls and patients treated with HD. Only minor changes were observed in patients treated with HD. Altered free radical activity, oxidative stress and altered erythrocyte membrane fluidity observed in patients with CRF may contribute to the increase in vascular disease in such patients and to the anaemia of CRF. PMID- 7554273 TI - Serum lipoprotein profile in Algerian patients with celiac disease. AB - Celiac disease is characterized by a gluten-induced villous atrophy of the upper small intestine which has an active role in the lipoprotein metabolism. In the present study the lipoprotein profiles of different patients were analyzed to determine the effect of impaired enterocyte function in celiac disease. We compared serum lipid parameters in controls and in celiac disease patients. The major differences between celiac disease patients and the control group were a diminution of cholesterol and phospholipids in HDL and LDL in the former. These differences persisted after treatment; in addition, a lower level of cholesterol in VLDL was observed. Plasma LpAI and apo A-I levels were significantly lower ( 17 and -15%) in celiac disease patients than in controls. Both levels remained low after gluten-free diet. In Algerian patients, treatment with gluten-free diet did not give any return towards normal lipids concentrations. PMID- 7554274 TI - A specific immunoradiometric assay of cationic trypsin(ogen) that does not recognize trypsin-alpha-1-proteinase inhibitor complex. AB - An immunoradiometric assay using two monoclonal antibodies directed to human trypsin 1 was developed for measuring trypsin(ogen) in biological fluids. The assay is different from other assays in that it is specific for cationic trypsinogen and does not recognize the alpha-1-proteinase inhibitor-trypsin complex. It can be used as a complement to classical immunoassays to characterize trypsinogen activation in pathological cases. The evaluation and the specificity of the assay are presented. PMID- 7554275 TI - The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. AB - A number of lines of evidence suggest that red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease (CHD) mortality. This protection over and above that due to ethanol itself may be explained by phenolic components with which red wines are richly endowed. We have studied the effects of the trihydroxy stilbene trans-resveratrol on human platelet aggregation and on the synthesis of three eicosanoids from arachidonate by platelets, i.e. thromboxane B2 (TxB2), hydroxyheptadecatrienoate (HHT) and 12 hydroxyeicosatetraenoate (12-HETE). These effects were compared with the actions of other wine phenolics (quercetin, catechin and epicatechin) and antioxidants (alpha-tocopherol, hydroquinone and butylated hydroxytoluene). trans-Resveratrol and quercetin demonstrated a dose-dependent inhibition of both thrombin-induced and ADP-induced platelet aggregation, whereas ethanol inhibited only thrombin induced aggregation. The other compounds tested were inactive. trans-Resveratrol also inhibited the synthesis of TxB2, HHT, and to a lesser extent 12-HETE, from arachidonate in a dose-dependent manner. Quercetin inhibited only 12-HETE synthesis, and hydroquinone caused slight inhibition of TxB2 synthesis, the remaining compounds being ineffective. De-alcoholized red wines inhibited platelet aggregation; their ability to inhibit the synthesis of TxB2 but not that of 12-HETE from labelled arachidonate by washed human platelets was proportional to their trans-resveratrol concentration. These results are consistent with the notion that trans-resveratrol may contribute to the presumed protective role of red wine against atherosclerosis and CHD. PMID- 7554276 TI - Assay of serum pyridinoline: a potential marker for bone resorption. PMID- 7554277 TI - Surfactant associated protein A determination using a chemiluminescence system- application to tracheal aspirates from newborns. PMID- 7554278 TI - The value of polymorphonuclear elastase in adult respiratory distress syndrome. AB - The clinical usefulness of quantitative plasma polymorphonuclear elastase (PMN elastase) determinations as prognostic markers of adult respiratory distress syndrome (ARDS) in polytraumatized patients was analyzed. PMN-elastase and C reactive protein (CRP) levels were determined in 55 polytraumatized patients admitted into the Intensive Care Unit. Eight patients developed ARDS and 47 patients did not. These parameters were also analyzed in a control group (n = 34). PMN-elastase levels in ARDS cases reached significantly higher values than in patients who did not develop this syndrome (P < 0.01). We conclude that the increase in plasma PMN-elastase levels can be useful in predicting the development of ARDS in polytraumatized patients, in instituting prophylactic actions and monitoring the course of the disease in these high risk patients. This test is easily adaptable to the routine of any hospital laboratory. PMID- 7554279 TI - Polymorphonuclear leukocyte elastase levels in patients with Behcet's disease. AB - Behcet's disease is a chronic multisystem disorder characterized by a relapsing inflammatory process of unknown aetiology. The increased activity of polymorphonuclear leukocytes (PMN) in Behcet's disease has been intensively studied. PMN elastase, an acute phase reactant, was investigated to determine whether it may serve as a biochemical marker in Behcet's disease. Erythrocyte sedimentation rate, protein electrophoresis, immunoglobulins (IgA, IgG, IgM), complement components (C3, C4) as well as PMN elastase were evaluated in 42 patients with Behcet's disease and 40 healthy subjects. The mean PMN elastase levels were found to be 244.2 micrograms/l (median 210 micrograms/l, S.D. 126.8) in patients with Behcet's disease and 44.3 micrograms/l (median 45, S.D. 19.2) in healthy subjects (P < 0.001). In addition, the mean PMN elastase levels were found to be 321.5 micrograms/l (median 300, S.D. 117.9) in the acute phase and 159 micrograms/l (median 162, S.D. 59.3) in remission (P < 0.001). It was concluded that PMN elastase may be a good biochemical marker for diagnosis and therapy control in patients with Behcet's disease. PMID- 7554280 TI - Amino acid sequence of a modified beta 2-microglobulin in renal failure patient urine and long-term dialysis patient blood. AB - We isolated and analyzed beta 2-microglobulin (BM) in urine of two renal failure patients and in ultrafiltrate from long-term dialysis patients. In both groups of patients, we found the native BM with pI 5.7 and some modified BM with pI 5.3. The amino acid sequence of the modified BM was determined and compared with that of the native BM; the N-terminal isoleucine residue was missing and the second glutamine residue was cyclized into pyroglutamic acid in the modified BM. PMID- 7554281 TI - Evidence of toxic metabolite stress in black South Africans with chronic pancreatitis. AB - We report the results of a further study to test our hypothesis that toxic metabolite stress is germane to heightened free radical activity and hence to the genesis of chronic pancreatitis. Consecutive black South African patients with clinically quiescent chronic pancreatitis were studied, provided that the diagnosis had been made within the previous 2 years and that they did not have overt liver disease. All of them had been advised to stop drinking alcohol. Analysis of an early morning sample of urine showed a lower ratio of inorganic to ester sulphate (P < 0.001) and a higher ratio of D-glucaric acid to creatinine (P < 0.02) in the group of 14 patients than in 15 local controls, while plasma analysis showed a lower concentration of glutathione (GSH) in the patients (P < 0.001). This evidence of increased utilisation of phase II conjugative pathways of xenobiotic disposal was in keeping with on-going toxic metabolite stress from heightened phase I oxidative metabolism in the group of patients. Parallel studies of theophylline pharmacokinetics showed heightened drug clearance compatible with induced cytochrome P-4501A2 in two patients, whereas increased activity of gamma-glutamyl transferase in serum suggested persisting induction of P-4502E1, as by ethanol, in several others. The contemporaneous increases in free radical activity and utilisation of xenobiotic disposal pathways in Sowetan Africans with chronic pancreatitis is in line with the toxic metabolite concept of disease pathogenesis. PMID- 7554282 TI - Immunoglobulin cleavage by hypochlorous acid treatment. AB - IgA, IgG and IgM were cleaved by hypochlorous acid treatment. The apparent calculated molecular masses of three polypeptides obtained from IgA were 81.1. 25.8 and 13.9 kDa. The amounts of released IgA fragments were proportional to the amount of HOCl employed. At a HOCl:IgA molar ratio above 320:1, a profound degradation of IgA polypeptide chains occurred, resulting in a yellow-coloured product. The HOCl treatment of IgG resulted in similar effects, the liberation of three fragments, one of them being of a size slightly larger than that of the light chain (30.4 kDa). The treatment of IgM with HOCl also produced three fragments: one corresponding to the monomeric IgM molecule, the second to the light chain (26.4 kDa) and the third of a size smaller than the heavy chain. The optimal protein/HOCl ratios for the degradation of IgG and IgM were 375:1 and 808:1, respectively. PMID- 7554283 TI - Decreased branching, increased fucosylation and changed sialylation of alpha-1 proteinase inhibitor in breast and ovarian cancer. AB - Proteolytic enzymes could be very important in spread of cancer, but the role of the body's natural inhibitors of these enzymes in this process is unknown. One such inhibitor is the serum glycoprotein, alpha-1-proteinase inhibitor (API). In previous studies we showed that the fucose-specific lectin, lotus tetragonolobus, extracted high amounts of API in cancer when patients were unresponsive to treatment. The aim of this study was to determine whether the carbohydrate structure of API is altered in cancer. API was isolated from the sera of healthy women and women with breast or ovarian cancer. By means of high-performance anion exchange chromatography, cancer API was shown to contain more fucose and less N acetylglucosamine than healthy API. Further investigation of the purified specimens using a lectin-binding assay suggested that the cancer API was less branched and contained more alpha 2-6 and less alpha 2-3 sialic acid. Observations from both methods were consistent with an increase in bi-antennary chains terminating in alpha 2-6 sialic acid and possibly more alpha 1-6 fucose in the core of the unit. These distinctive changes could have important consequences for the function of API in cancer and may help to develop more precise markers for monitoring pathological progression in this disease. PMID- 7554284 TI - Selenium dependent glutathione peroxidase activity in human follicular fluid. AB - The purpose of this study was to evaluate the follicular fluid selenium status of in vitro fertilization patients in relation to their individual characteristics and treatment outcome. A total of 135 samples of follicular fluid were collected from 112 patients during transvaginal oocyte retrieval. Selenium levels and glutathione peroxidase (GSHPx) activity were measured in follicular fluid (FF) and blood serum. A selenium dependent GSHPx activity in follicular fluid has been demonstrated. Patients with unexplained infertility had significantly decreased follicular selenium levels as compared with those with tubal infertility or male factor. The mean GSHPx activity in follicles yielding oocytes which were subsequently fertilized exceeded that of the follicles with non-fertilized oocytes. Tobacco smoking resulted in significantly diminished follicular GSHPx activity. The Se/GSHPx pattern of the FF seems to be significant for the evaluation of the follicular milieu. PMID- 7554285 TI - The typing of fetal antigen 2 in human amniotic fluid. AB - Fetal antigen 2 (FA-2) has been identified in amniotic fluid and shown to be of fetal origin. In this study we have extended previous observations on FA-2 heterogeneity with respect to both size and charge using gel filtration, ion exchange chromatography, non-dissociating polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the diversity of forms we have been able to define two principal FA-2 types, type A and type B. Type A has a high molecular mass (140 kDa), has subunits of 33 kDa and 29 kDa, and elutes at approximately 0.27 mol/l sodium chloride from diethylaminoethyl (DEAE)-Sephacel. Type B has the same mass and subunits as type A, but elutes at approximately 0.24 mol/l sodium chloride from DEAE-Sephacel. Other low molecular mass forms of FA-2 have also been identified. All FA-2 forms described were shown to be common to all amniotic fluid samples studied and were not attributable to artefacts of collection or storage. It was also demonstrated that the recently described FA-2 RIA is specific for FA-2 types A and B and the conversion of arbitrary units FA-2 into micrograms applies to type A. The typing is discussed with respect to (i) the aminopropeptide of the alpha 1 chain of human procollagen type I, (ii) the 24 kDa phosphoprotein in developing bone and (iii) fetal calf ligament protein 1 (FCL-1), suggesting that they are the same protein. PMID- 7554286 TI - Demonstration of immunogenic keratan sulphate in commercial chondroitin 6 sulphate from shark cartilage. Implications for ELISA assays. AB - The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (> 120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate correlated well (r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity. PMID- 7554287 TI - Maximization of determination of basement membrane collagen complex in the sera from patients with chronic liver diseases by a sandwich ELISA. PMID- 7554288 TI - Erythropoietin secretion in liver disease. PMID- 7554289 TI - Detection of autoantibodies against oxidized low-density lipoproteins and of IgG bound low density lipoproteins in patients with coronary artery disease. AB - The role of oxidized low-density lipoprotein (ox-LDL) in the pathogenesis of atherosclerosis has been the object of intense investigation. It has been proposed that, due to the antigenic properties of ox-LDL, the anti-ox-LDL antibody titre could represent a useful index of in vivo LDL oxidation. On the other hand, LDL immune complexes (LDL-IC) have been demonstrated in patients with coronary disease and could play an atherogenic role. The goal of our study was to investigate anti-malondialdehyde (MDA)-LDL autoantibodies and LDL-IC in a cohort of patients with coronary artery disease. Seventy control subjects and 70 coronary angiographically documented patients were compared; in addition 32 healthy male non-smokers were compared with 32 healthy male smokers (> 10 cigarettes/day). All patients were matched for age and cholesterolemia. Enzyme linked immunosorbent assay was used to measure anti-MDA-LDL autoantibodies and LDL-IC. Titres of anti-MDA-LDL autoantibodies were not larger in patients with documented coronary artery stenosis and in smokers than they were in controls and non-smokers. The titre of LDL-IC was not higher in patients with coronary artery stenosis than in controls. The results thus indicate that in populations matched for age and cholesterolemia the titres of anti-MDA-LDL autoantibodies and the titre of LDL-IC are not increased in patients suffering from coronary artery stenosis. Furthermore, cigarette smoking does not induce higher titres of anti MDA-LDL autoantibodies in healthy patients. PMID- 7554290 TI - Hyaluronic acid and dermatan sulfate in non-lesional pseudoxanthoma elasticum skin. PMID- 7554291 TI - Pedigree of a family with hyper-alkaline-phosphatasemia apparently inherited as an autosomal recessive trait. PMID- 7554292 TI - Whether, to what degree, and why lipoprotein(a) levels change over time. AB - Our specific aim in this study of 6 +/- 2 serial lipoprotein(a) (Lp(a)) measurements over 1.7 +/- 0.5 years in 145 patients (64 men, 81 women, 135 whites, 10 blacks, mean age 63 years) with high Lp(a) (> or = 25 mg/dl) was to assess whether, to what degree, and why Lp(a) levels change over time. A second specific aim was to study the biological coefficient of variation of Lp(a) (C.V.b). No Lp(a) lowering drugs were given. In the 145 patients, mean (S.D.) and median Lp(a) on entry (70 +/- 34 and 59 mg/dl) did not differ from levels on follow-up (72 +/- 34 and 64 mg/dl) (P > 0.4). Mean (S.D.) C.V.b was 18 +/- 9%, median 17%, minimum 1.7% and maximum 58%. Three or four samples for Lp(a) measurement should be very adequate to establish a 'true' Lp(a) value, since C.V.bs based on three to four samples did not differ (P > 0.1) from those based on five to six, seven, eight, or nine samples. For patients with mean +/- S.D. baseline Lp(a) levels of 39 +/- 3, 49 +/- 3 and 59 +/- 2, 95% of the follow-up values fell in the following ranges: 25-78, 24-93 and 34-104 mg/dl. Lp(a) on study entry was highly correlated with Lp(a) on follow-up (r = 0.84, P = 0.0001) and with Lp(a)'s C.V.b on follow-up (r = 0.24, P = 0.003). Entry Lp(a) was inversely correlated with absolute and percent change in Lp(a) on follow-up (r = 0.38, P = 0.0001 for both).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554293 TI - Quantitation of procholecystokinin and its products in plasma by processing independent analysis. AB - A procedure for processing-independent quantitation of procholecystokinin (proCCK) and its products has been applied to plasma. The procedure is based on tryptic cleavage after Lys61 and Arg71 with subsequent monospecific radioimmuno analysis of fragment 62-71 of human proCCK, which again corresponds to fragment 1 10 of CCK-22. The detection limit of the analysis was 0.2 pmol/l. Plasma was extracted with ethanol. In plasma from 13 healthy volunteers the basal concentration with the above-mentioned radioimmunoassay was 1.1 +/- 0.1 pmol/l (mean +/- S.E.M.) before, and 13.7 +/- 0.6 pmol/l after, incubation with trypsin. Two hours after ingestion of a mixed meal, the plasma concentration was 2.0 +/- 0.1 pmol/l before, and 21.7 +/- 1.2 pmol/l after tryptic cleavage. With a conventional CCK radioimmunoassay specific for the C-terminally amidated and O sulfated bioactive epitope, the concentration was 1.0 +/- 0.1 pmol/l in the basal state and 4.2 +/- 0.4 pmol/l 2 h after a meal. Tryptic cleavage did not increase the concentrations of amidated, bioactive CCK peptides. In plasma from 37 patients with the carcinoid syndrome, the basal concentration of proCCK and its products was 14.1 (2.8-150.4) pmol/l (median (range)), compared with 0.3 (0-18.8) pmol/l for carboxyamidated CCK. Only two patients had significantly elevated CCK concentrations. We conclude that processing-independent analysis is useful for quantitation of proCCK and its products in plasma, since it quantitates CCK cell secretion more accurately than conventional CCK assays. PMID- 7554294 TI - Glycaemia and body mass as determinants of plasma lecithin: cholesterol acyltransferase activity in Nigerian patients with non-insulin dependent diabetes mellitus. AB - There is an inverse correlation between plasma high density lipoprotein (HDL) concentrations and atherogenic vascular morbidity risk. An important pathway for modulating circulating HDL levels is the reaction catalysed by the plasma enzyme, lecithin:cholesterol acyltransferase (LCAT). Thus, determinants of LCAT activity should influence plasma HDL levels and be accessible targets for intervention. We therefore assessed such determinants in Nigerian patients with non-insulin dependent diabetes mellitus (NIDDM), classified into age- and sex-matched groups of obese (body mass index, BMI, > 25 kg/m2) and non-obese (BMI < 25 kg/m2). Our results indicated that 10 obese diabetic patients had significantly reduced plasma LCAT activity and HDL levels and increased plasma triglyceride (TG) levels, in comparison with the observations in 9 non-obese diabetic patients (all P < 0.05). Furthermore, worsening of glycaemic control, when oral hypoglycaemic treatment was discontinued for 1 week in 10 diabetic (5 obese) patients, further reduced fasting levels of HDL and plasma LCAT activity (both P < 0.05). Although plasma HDL concentrations and LCAT activity levels did not correlate significantly, the latter nonetheless had a significant inverse relationship with fasting plasma glucose levels in the obese diabetic subjects (r -0.51, P < 0.05). These results indicate that glycaemia and body mass are important determinants of plasma LCAT activity. Both variables are subject to pharmacological and dietary intervention with the objective of increasing circulating HDL levels. PMID- 7554295 TI - Compositions of very low density lipoprotein subfractions from patients with polydisperse low density lipoproteins. AB - In some hyperlipidemic patients, low density lipoprotein (LDL) shows several peaks (polydisperse) on polyacrylamide gel disc electrophoreses, though LDL usually shows a single peak (monodisperse). In order to clarify the relationship between the LDL polydispersion and VLDL heterogeneity, LDL and VLDL were prepared from hyperlipidemic patients sera with mono- and polydisperse LDL by sequential ultracentrifugation and fractionated by gradient ultracentrifugation and their compositions were analyzed. Polydisperse LDL was rich in triacylglycerol (TG) and poor in esterified cholesterol (CE) as compared with monodisperse LDL and consisted of the lowest and the medium density subfractions when the LDL was separated into six subfractions. The monodisperse LDL was composed of a single major subfraction of a medium density. VLDL from the patients with polydisperse LDL was relatively rich in the dense and poor in the buoyant subfractions as compared with that from the patients with monodisperse LDL. The subfractions in the former contained more CE and less TG than the corresponding subfractions in the latter. There were no significant differences in the apolipoprotein compositions between those VLDLs. The results suggest that polydisperse LDL might be originated from VLDL that differs in particle sizes, densities and compositions from ordinary VLDL. PMID- 7554296 TI - Relationships between triglycerides, lipoproteins, glucose and coronary artery disease. AB - We examined the relationship of apolipoprotein B (apo B), glucose, triglycerides and other lipoprotein lipids to coronary artery disease (CAD). Using receiver operating characteristic curves (ROC), we noticed that the triglyceride ROC curve crossed above other lipoprotein curves at a triglycerides level of approximately 1.4 g/l. We examined subgroups of < 1.4 g/l and > 1.4 g/l. ANOVA (F = 18.9, P < 0.0001) and stepwise logistic regression (P = 0.0002) indicated that triglycerides were the best predictor in the < 1.4 g/l group. The best markers in the > 1.4 g/l group were low density lipoprotein cholesterol and apo B. Glucose did not appear to significantly alter the predictive power of triglycerides. These data suggest that triglyceride appears to be an overall significant univariate marker for CAD because of its effect at lower concentrations. The strong relationship between CAD and triglycerides at low triglyceride levels may reflect increased levels of very low density lipoprotein metabolites in some individuals. We conclude that some triglyceride-rich particles are independently atherogenic, that glucose did not alter this relationship and that when the samples were split into those with high and low levels of triglycerides, triglycerides and apo B but not HDLC was a significant predictor of CAD. PMID- 7554297 TI - Study of the chromium binding in plasma of patients on continuous ambulatory peritoneal dialysis. AB - The distribution of chromium (Cr) among the proteins in plasma of three continuous ambulatory peritoneal dialysis (CAPD) patients and in dialysis solutions was studied with 'in vitro' and 'in vivo' 51Cr-labelled plasma. Radiolabelling allows the kinetics of newly added Cr to be investigated. The separation of the proteins was achieved by ion exchange fast protein liquid chromatography, ensuring quantitative recoveries of the 51Cr activity, transferrin and albumin. In all fractions the proteins were qualitatively characterised by iso electric focusing. The concentrations of transferrin and albumin were determined by nephelometry. The 51Cr distribution among the plasma proteins was similar for the three patients. The 51Cr activity was mainly bound to transferrin and, in the beginning of the incubation, in a lesser degree also to albumin. After 1-6 h a shift was observed of the 51Cr from albumin to an unidentified low molar mass complex (+/- 5000 Da). Two 51Cr species showed up in the subsequently drained dialysate, but they could not be identified. PMID- 7554298 TI - Serum concentrations of the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen in chronic liver disease. PMID- 7554299 TI - Low serum alkaline phosphatase activity associated with severe Wilson's disease. Is the breakdown of alkaline phosphatase molecules caused by reactive oxygen species? PMID- 7554300 TI - QEEG and evoked potentials in central nervous system Lyme disease. AB - Quantitative EEG, flash visual evoked potentials, auditory evoked potentials to common and rare tones, and median nerve somatosensory evoked potentials were obtained from 12 patients with active CNS Lyme disease and from 11 patients previously treated for active CNS Lyme disease. Abnormal QEEG and/or EPs were found in 75% of the active Lyme disease patients and in 54% of the post CNS Lyme disease patients. Three different types of neurophysiological abnormality were observed in these patients including QEEG slowing, possible signs of cortical hyperexcitability, and focal patterns indicating disturbed interhemispheric relationships. In patients tested before and after treatment QEEG and EP normalization was associated with clinical improvement. PMID- 7554301 TI - P300 latency: abnormal in sleep apnea with somnolence and idiopathic hypersomnia, but normal in narcolepsy. AB - To evaluate cognitive abnormalities in excessive daytime sleepiness (EDS) using cognitive evoked potentials (P300), and to evaluate if P300 measures differentiate among disorders of EDS, a series of EDS subjects were administered a polysomnogram, auditory and visual P300 testing using 31 scalp electrodes, and a multiple sleep latency test. P300 variables were compared with those of normal subjects. Forty normal subjects ages 16 to 65 years, and 69 EDS patients ages 16 to 65 years were used. Of these, 39 had profound obstructive sleep apnea (OSA, Respiratory Disturbance Index or RDI > 80/h sleep) with severe somnolence (Mean Sleep Latency < 5 min). Twenty-two had idiopathic hypersomnia (IH). Eight had narcolepsy. The normals and the three EDS groups did not differ in age. IH and profound OSA patients had longer visual P300 latency than normals or narcolepsy patients (p < 0.05). (p < 0.05). IH and profound OSA patients had longer auditory P300 latency than normals. They had smaller auditory P300 amplitude than narcolepsy patients. There were visual P300 latency topographic differences between normals and profound OSA patients. In conclusion, IH and profound OSA patients show cognitive evoked potential evidence of cognitive dysfunction. Narcolepsy patients do not show such evidence. Visual P300 latency differentiates among disorders of EDS. PMID- 7554302 TI - Topographic quantitative EEG measures of alpha and theta power changes during caffeine withdrawal: preliminary findings from normal subjects. AB - Neurophysiological consequences of withdrawal from caffeine are poorly understood. In particular, quantitative studies of EEG changes that may occur during the period of caffeine abstinence in caffeine dependent individuals have not been reported. In this pilot study, 13 physically- and psychiatrically-normal caffeine users were asked to abstain from caffeine for a period of 4 days. Quantitative EEGs were obtained prior to stopping caffeine and on Days 1, 2, and 4 of the caffeine abstinence period. Results indicated that significant increases in alpha and theta absolute power accompany the caffeine withdrawal process with return to the pre-abstinent EEG levels when caffeine usage is resumed. The implications of these findings are discussed with special reference to the possible need to control for the variable of caffeine usage in quantitative EEG studies of other phenomena. PMID- 7554303 TI - How long to treat childhood onset absence epilepsy. AB - The relation between the duration of treatment and achievement of seizure-free state after stopping medication in childhood onset absence epilepsy (COAE) has not been established, and children are usually treated for periods of 2-4 years. We hypothesized that a group of patients defined by prompt total clinical and electrographic remission, verified by 24-hour ambulatory EEG, could be withdrawn from treatment without recurrence. Three patients with new onset of COAE were treated. Ethosuximide (ESM) was used as first line drug. Dosages correlated with blood levels were gradually increased until a clinical and electrographic seizure free state was achieved. The three responded to ESM promptly at therapeutic blood levels (75-90 mcg/ml). The effects of a 5- to 6-week period of withdrawing ESM after 6 months of treatment were as follows: a 10-year-old boy remained seizure free with recurrence of inter-ictal 3/second spike-wave bursts; 5- and 10-year old girls remained seizure-free and their 24-hour ambulatory EEG burst-free. Our findings suggest that optimization of treatment in COAE, aimed at achieving an electroclinical and electrographic seizure-free state, may identify patients in whom therapy can be successfully ended much sooner than has been usual. Thus the same good prognosis may be reached in a shorter period of treatment with minimal risk of recurrence of absence seizures. PMID- 7554304 TI - Quantitative EEG characteristics of children exposed in utero to cocaine. AB - Quantitative EEGs (QEEGs) were evaluated in a group of 6 school age children with in utero cocaine exposure. Their QEEGs showed significant deviations from age expected normal values. Further, the QEEG profile of brain dysfunction seen in these children was extremely similar to that previously reported in a large population of crack cocaine dependent adults. These abnormalities were characterized by significant excess of relative power in the alpha frequency band, and deficits of absolute and relative power in the delta and theta bands. Characteristic disturbances in interhemispheric relationships were also present. The similarities between the QEEG profiles of those adults with chronic exposure and children with prenatal exposure suggests that the brain dysfunction reflected in the QEEG is not a result of a transient change in neurotransmission, but a more profound alteration which persists in these children at school age. Further study is required to extend these findings to a larger group of children, and to investigate the potential relationship between these neurophysiological abnormalities and the developmental, behavioral and co-morbid features observed in such children. PMID- 7554305 TI - Frontal intermittent rhythmic delta activity (FIRDA) in pituitary adenoma. AB - We report a case of FIRDA in the EEG of a patient diagnosed as major depression with pituitary adenoma and hyponatremic encephalopathy. The pituitary adenoma appeared to be a major factor responsible for FIRDA in this case. Although other factors associated with this case, i.e., diffuse encephalopathy and administration of antipsychotic drugs, have been reported to be causative, FIRDA remained in the EEG after these other factors diminished. Although size of the pituitary adenoma that might be associated with FIRDA in the EEG recording was not identified in this study, FIRDA may be associated with a small pituitary adenoma less than 10 mm in diameter. We think a diligent search for additional pathology is recommended if FIRDA is seen in the EEG of an otherwise normal patient. PMID- 7554306 TI - The use of felbamate in patients with periodic lateralized epileptiform discharges (PLEDs). AB - This case report refers to a patient with PLEDs whose EEGs did not change while on steady levels of phenytoin and phenobarbital, but showed a complete disappearance of these discharges when felbamate was used. This report may serve to remind EEG'ers and epileptologists that the acute use of this drug may be effective in dealing with PLEDs that are often difficult to eliminate. However, physicians must keep in mind the significant risks of using this medication. PMID- 7554307 TI - Short stature and growth hormone deficiency. PMID- 7554308 TI - Which adults develop side-effects of growth hormone replacement? AB - OBJECTIVE: Although the nature of the side-effects of GH replacement in adults are well described, the factors influencing their development are ill understood. The aim of this study was to determine whether there were any characteristics of adults with GH deficiency that predicted whether or not they developed side effects of GH replacement. DESIGN: A 12-month study (double blind placebo controlled for the first 6 months and open for the second 6 months) of GH replacement (0.125 IU/kg/week for the first month and 0.25 IU/kg/week thereafter) in adults. PATIENTS: Sixty-three adults (27 men, 36 women, aged 34.9 +/- 1.4 (mean +/- SE, range 20.1-59.5 years)) with GH deficiency (peak serum GH response to provocative testing of less than 10 mU/l) who took part in a 12-month study of GH replacement. Twenty-five patients (40%) did not develop side-effects, 19 patients (30%) developed side-effects which did not necessitate a reduction in dose of GH, and 19 patients (30%) required a reduction in dose of GH because of side-effects. MEASUREMENTS: The three groups of patients were compared according to age, height, weight and body mass index (BMI) at entry into the study and to pretreatment peak serum GH response to provocative testing. They were also compared according to serum concentration of insulin-like growth factor (IGF)-I and IGF binding protein-3, and age-adjusted serum IGF-I standard deviation score (SDS), at entry into the study and by change in these measurements after 6 months of GH replacement. The patient's sex, whether GH deficiency was of childhood or adult onset, estimated duration of GH deficiency, presence or absence of additional pituitary hormone deficiencies, underlying pathological disorder and previous therapeutic interventions were also compared in the three groups of patients. RESULTS: Those patients who required a reduction in dose of GH because of side-effects were more likely to have a peak serum GH response of greater than 1 mU/l (P = 0.005) and to have adult onset GH deficiency (P = 0.04) than those who did not develop side-effects or who did not require a reduction in dose of GH because of side-effects. In addition, those who needed a reduction in GH dose were older (P = 0.002), heavier (P = 0.04) and had a greater BMI (P = 0.003) than those who did not develop side-effects. Those who developed side-effects but did not require a reduction in dose of GH had a greater increment in IGF-I SDS after 6 months of GH replacement than those who did not develop side-effects (P = 0.03). CONCLUSION: Side-effects of GH replacement are more likely to occur in older patients, in those with a peak serum GH response to provocative testing of greater then 1 mU/l, in those with a greater increment in serum IGF-I SDS whilst receiving GH replacement, in those with greater weight and BMI, and those with adult onset GH deficiency. PMID- 7554309 TI - Factors influencing the desire for long-term growth hormone replacement in adults. AB - OBJECTIVE: Growth hormone replacement in adults may be considered beneficial by clinicians, but patients may not perceive any benefits. The purpose of this study was to determine whether there were any factors which influenced whether an adult wished to continue on long-term GH replacement after taking part in a study of GH replacement. DESIGN: A 12-month study (double-blind placebo controlled for the first 6 months and open for the second 6 months) of GH replacement (0.125 IU/kg/week for the first month and 0.25 IU/kg/week thereafter) in adults. PATIENTS: Sixty-three adults (27 men, 36 women, aged 34.9 +/- 1.4 (mean +/- SE, range 20.1-59.5) years) with GH deficiency (peak serum GH response to provocative testing less than 10 mU/l) who entered a 12-month study of GH replacement. Thirty patients (48%) wished to continue on GH replacement and 33 patients (52%) did not wish to continue on GH replacement after the study. MEASUREMENTS: Biochemical, anthropometric and demographic characteristics, and well-being, were compared in those patients who wished to continue on long-term GH replacement and in those who did not. In the two groups of patients the age, height, weight, body mass index, serum insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and IGF-I age matched standard deviation score (SDS) were compared at entry into the study, and changes in IGF-I, IGFBP-3 and IGF-I SDS were compared after 6 months of GH replacement. The patients were compared according to pretreatment peak serum GH response to provocative testing, sex, estimated duration of GH deficiency, whether GH deficiency was of childhood or adult onset, presence or absence of additional pituitary hormone deficiencies, underlying pathological disorder, previous therapeutic interventions, employment status, marital status and living arrangement, and according to development of side-effects of GH replacement and the requirement for reduction in dose of GH because of side effects during the study. Scores on two questionnaire measures of well-being or distress, the Nottingham Health Profile (NHP) and the Psychological General Well Being Schedule (PGWBS), were compared at entry into the study in the two groups, as were change in scores on these questionnaires after 6 months of GH replacement. RESULTS: Those who continued on GH replacement tended to have a greater severity of GH deficiency (median peak serum GH concentration 0.7 vs 2.3 mU/l, P = 0.06), tended to have greater distress in terms of energy (NHP, P = 0.06) and vitality (PGWBS, P = 0.06) at entry into the study and showed an improvement in energy during the study compared with no change in those who did not wish to continue on GH replacement (NHP, P = 0.06). CONCLUSION: Those adults who wished to continue on GH replacement tended to have a greater severity of GH deficiency, to experience more distress in terms of energy and vitality at entry into the study and to experience an improvement in energy after 6 months treatment with GH. PMID- 7554310 TI - Diurnal variation of sex hormone binding globulin and insulin-like growth factor binding protein-1 in women with polycystic ovary syndrome. AB - OBJECTIVES: The aim of this study was to examine (1) the diurnal variation in SHBG and (2) the inter-relationships of insulin, IGF-I, SHBG and IGFBP-1 over 24 hours in 10 women with anovulatory PCOS and compare them with weight-matched ovulatory controls. PATIENTS AND METHODS: The two groups comprised 10 anovulatory women with PCOS (as defined by clinical, ultrasound and biochemical criteria) and 10 weight matched controls. Serum samples were taken at two-hourly intervals for 24 hours and stored for measurement of SHBG, IGFBP-1, insulin and IGF-I. Differences between the groups were compared using the Wilcoxon ranked paired tests of the individual peak and trough concentrations in each group. The variation in insulin, IGFBP-1 and SHBG concentrations over 24 hours was tested using two-way analysis of variance with the factors time and subject. Spearman's correlation coefficient was calculated from the subjects' median value over 24 hours. RESULTS: The median (interquartile range) body mass index (BMI) was 25.2 (22.2-29.3) in the PCOS group and 24.3 (23.2-25.7) kg/m2 in the control group. Serum testosterone (T) and LH levels were significantly raised in the PCOS group compared to the control group; T 3.8 (2.9-5.6) vs 1.9 (1.9-2.5) nmol/l (P < 0.007) and LH 12 (10-15) vs 4.1 (3.6-4.5) IU/I (P < 0.005) respectively. There was no diurnal variation in SHBG. The median (interquartile ranges) of the peak SHBG concentrations was lower in the PCOS group: 29.4 (14.9-39.4) vs 52.1 (39.4 61) nmol/l in the control group (P < 0.01). The fasting levels of insulin at 0600 h (median (interquartile ranges)) were not significantly different between the groups; 6.6 (5.4-9.8) and 6.2 (1.9-7.6) mU/l, respectively, although the peak median concentrations were significantly different; PCOS 66.1 (50.9-129.2) vs 40 (36.1-74.2) mU/l (P < 0.05). Two-way analysis of variance showed a diurnal variation in insulin concentrations in the control group (P = 0.001) but not in the PCOS group (P = 0.1). The diurnal variation in IGFBP-1 was similar in the two groups but the peak median levels were lower in the women with PCOS 54.9 (22.3 79.2) vs 71.5 (60.5-99.3) micrograms/l (P < 0.03). The decline in IGFBP-1 concentrations correlated with the increase in insulin concentrations. The IGF-I concentrations were similar in the two groups. There was a significant negative correlation between SHBG and insulin (P < 0.05) and between insulin and IGFBP-1 (P < 0.01). CONCLUSION: This study demonstrates that there is no diurnal variation in SHBG concentrations and confirms the finding of a marked diurnal variation in the concentration of IGFBP-1. Women with PCOS who are anovulatory have an abnormal pattern of insulin secretion with an absence of diurnal variation compared to weight matched controls. This provides further evidence of the relative insulin resistance which is independent of weight found in women with anovulatory PCOS. The inverse correlations of insulin concentrations with SHBG and IGFBP-1 support the role of insulin as a possible regulator of the circulating levels of these binding proteins although the difference in the time course of their response makes it unlikely that they are co-regulated. PMID- 7554311 TI - Effect of haemoglobin and endogenous erythropoietin on hypothalamic-pituitary thyroidal and gonadal secretion: an analysis of anaemic (high EPO) and polycythaemic (low EPO) patients. AB - OBJECTIVE: Correction of anaemia with recombinant human erythropoietin (rHu-EPO) improves the responsiveness of thyroidal and gonadal axes to exogenous TRH and GnRH in chronic haemodialysis patients, but the mechanisms remain to be fully elucidated. In order to assess the influences of endogenous erythropoietin on the hypothalamo-hypophyseal thyroidal and gonadal axes, we studied the response of polycythaemic and anaemic patients, in comparison to normal controls, after the administration of exogenous TRH and GnRH. DESIGN: Exogenous hypothalamic factors, 500 micrograms TRH and 100 micrograms GnRH, were administered as a bolus and blood samples were obtained over a 3-hour period at 30, 60, 90, 120 and 180 minutes. PATIENTS: Five male polycythaemic patients (low EPO), three male anaemic patients (high EPO) and six normal age and sex matched controls were studied. MEASUREMENTS: Blood samples were centrifuged immediately and the serum was stored at -20 degrees C until assayed for total T4, free T4, free T3, TSH, prolactin, growth hormone (TRH test), and FSH, LH, testosterone (GnRH test). Haematological parameters and biochemical profiles were also measured. RESULTS: After TRH administration, both patient groups showed a normal TSH response; however, their free T4 and free T3 secretion was blunted compared to controls. Normal basal PRL levels increased in an exaggerated fashion, whereas, when compared to chronic renal failure patients on chronic haemodialysis, we did not see a paradoxical GH response or a basal GH increase in these 5 patients. GnRH administration in the study groups elicited a normalization in the LH response without an increase in testosterone levels; however, an exaggerated FSH response was found in the polycythaemic patients (low EPO). CONCLUSIONS: Thus by investigating the role of low endogenous EPO levels in non-anaemic and anaemic patients with high EPO levels, our study suggests that the underlying chronic disease state may be the major contributing factor in the regulation of the hypothalamo-hypophyseal thyroid and gonadal axes, rather than the EPO levels. PMID- 7554312 TI - Nocturnal breathing abnormalities in acromegaly after adenomectomy. AB - OBJECTIVE: The incidence of sleep apnoea is increased in acromegaly. The aim of the study was to determine the occurrence of nocturnal breathing abnormalities and upper airway morphology in acromegalic patients some years after adenomectomy. DESIGN: A case-control study. PATIENTS: Eleven patients with treated acromegaly and two control groups: (1) sleep studies: 197 subjects randomly selected from the population, (2) cephalometry: 27 healthy subjects and 17 patients with obstructive sleep apnoea. MEASUREMENTS: Nocturnal breathing was monitored with a static charge-sensitive bed. The upper airway soft tissues and bone morphology were assessed by cephalometric X-ray photography. The upper airway collapsibility was investigated with dynamic nasopharyngoscopy. Endocrinological investigations were also performed. RESULTS: Nocturnal breathing abnormalities were present in all but one acromegalic patient (91%), which was far more frequent than in the general population (29.4%, P < 0.0001). Treated acromegaly was the most powerful predictor of breathing abnormalities, independent of the other significant predictors, age and body mass index. The predominant breathing abnormality was periodic breathing with symmetrically waxing and waning respiratory effort without a major body movement component. Episodes of complete obstruction with repetitive arousals were rare. Except for the longer soft palate, the cephalometric findings were similar to normal. In comparison to obstructive sleep apnoea, the treated acromegalic patients had rather prognathic than retrognathic mandibles. Fibreoptic endoscopy in the acromegalic patients revealed collapsible upper airways at the level of the soft palate, whereas at the base of the tongue little, if any, dynamic narrowing was observed. CONCLUSION: Our study confirms that nocturnal breathing abnormalities are common in treated acromegaly, and may persist years after the removal of the GH secreting tumour. The breathing abnormalities and the upper airway morphology in acromegalic patients after adenomectomy are different from those observed in primary obstructive sleep apnoea, suggesting a different pathophysiology of the airway obstruction. PMID- 7554313 TI - A new deletion of the 5 alpha-reductase type 2 gene in a Turkish family with 5 alpha-reductase deficiency. AB - The molecular basis for male pseudohermaphroditism produced by the 5 alpha reductase deficiency is becoming increasingly understood. OBJECTIVE: We have performed biochemical and molecular analyses of the 5 alpha-reductase type 2 gene in a Turkish family with a 5 alpha-reductase deficiency. PATIENT: A 46,XY prepubertal Turkish patient with female phenotype showing clitoral hypertrophy, high plasma testosterone and dihydrotestosterone, and normally differentiated and developed testosterone-dependent internal genitalia. MEASUREMENTS: 5 alpha Reductase activity, measured by the conversion of 3H-T into 5 alpha-reduced compounds, was determined from cultured genital skin fibroblasts by both intact monolayer assay and cell-free extracts at various pH values. The five exons of the 5 alpha-reductase type 2 gene were sequenced after enzymatic amplification (PCR) of the patient's genomic DNA. Labelled PCR of the consanguineous parents' DNA was submitted to electrophoresis on a sequencing gel. RESULTS: A marked decrease in the transformation of T into 5 alpha-reduced compounds by intact cells and a diminished 5 alpha-reductase activity at acidic pH by sonicated cell extracts strongly suggested a 5 alpha-reductase type 2 deficiency. Molecular analysis of the 5 alpha-reductase type-2 gene showed a trinucleotide deletion straddling codons 156 and 157, responsible for a methionine residue deletion at position 157 of the protein. The parents' DNA contained both normal and deleted alleles. CONCLUSIONS: This is the third deletion described in the 5 alpha reductase type 2 gene. The deleted methionine 157 is conserved in both types 1 and 2 of human and rat 5 alpha-reductase, which suggests its crucial role in the functioning of the enzyme. This gene rearrangement was thus clearly responsible for the reduced 5 alpha-reductase activity and abnormal genital development in this patient. PMID- 7554314 TI - Growth stimulating antibodies in endemic goitre: a reappraisal. AB - OBJECTIVE: Previous studies, using a variety of methods, have reported growth promoting immunoglobulins (IGs) in a large proportion of patients with endemic goitre. We sought to determine whether thyroid growth-promoting immunoglobulins (TGI) are present in the serum of Indian patients with endemic goitre. DESIGN: IgG was prepared by protein G-Sepharose affinity purification and added to FRTL-5 thyroid cells in the presence of suboptimal concentrations of TSH. PATIENTS: We studied 30 sequential patients with endemic goitre and 16 euthyroid controls without a goitre from the same area. MEASUREMENTS: Two assays for thyroid cell growth were used: 3H-thymidine incorporation, and flow cytometric measurement of the proportion of cells in the S phase and G2/M phase of the cell cycle. RESULTS: Both assays were shown to detect growth produced by TSH and by thyroid stimulating antibodies in IgG preparations from 3 patients with Graves' disease. There was no significant increase in either 3H-thymidine incorporation or the distribution of cells in S or G2/M phase with IgGs from endemic goitre patients, and no difference between the effects of these IgGs and those from the normal subjects. CONCLUSIONS: Thyroid growth-promoting immunoglobulins cannot be detected in Indian patients with endemic goitre. PMID- 7554315 TI - Temperature-induced down-regulation of the glucocorticoid receptor in peripheral blood mononuclear leucocyte in patients with sepsis or septic shock. AB - OBJECTIVE: Activation of the hypothalamic-pituitary-adrenal axis is of vital importance during critical illness. We have studied the adaptive mechanisms which occur at the level of the glucocorticoid receptor in glucocorticoid target tissues in patients with sepsis or septic shock. DESIGN: The effects of hypercortisolaemia, hyperthermia and cellular composition on number of glucocorticoid receptors per cell and their affinity were evaluated, both in vitro and in vivo, in peripheral blood mononuclear leucocytes of control subjects and in patients with sepsis or septic shock. SUBJECTS: Fifteen patients (age 25 79) with sepsis or septic shock who were admitted to an intensive care unit were studied. The control group consisted of 24 healthy laboratory employees. MEASUREMENTS: The binding capacity and affinity of the glucocorticoid receptors were measured and compared to clinical data and the plasma cortisol concentrations. RESULTS: Hypercortisolaemia, in vitro, resulted in a decreased affinity and a decreased binding capacity of the glucocorticoid receptor. In vitro, hyperthermia as well as variations in the cellular composition did not influence the glucocorticoid receptor. In vivo, there was no change in the number of receptors per cell in patients with sepsis or septic shock as compared to healthy controls. However, a decreased affinity of the glucocorticoid receptor was observed. There was a weak but significant negative correlation between body temperature and the number of glucocorticoid receptors in the patient group. There was no relation between circulating cortisol concentrations and glucocorticoid receptor affinity and number. CONCLUSIONS: There is no obvious regulation of the number of glucocorticoid receptors by plasma cortisol concentrations in vivo. The decreased affinity of the glucocorticoid receptor together with the negative correlation between hyperthermia and the number of glucocorticoid receptors in patients with sepsis or septic shock suggest that hypothalamic-pituitary-adrenal axis activation during critical illness is accompanied by peripheral adaptation in glucocorticoid receptor number and affinity. PMID- 7554316 TI - Impaired social status of growth hormone deficient adults as compared to controls with short or normal stature. Dutch Growth Hormone Working Group. AB - OBJECTIVES: In adults with growth hormone deficiency (GHD) social problems have been reported, but so far the relative contributions of GHD, additional pituitary deficiencies and short stature have not been distinguished. We therefore compared social data from GHD patients with social data from controls with short or normal stature. Furthermore we investigated whether social problems are caused solely by the deficiency of GH or also by the associated absence of other pituitary hormones. DESIGN: A questionnaire was sent to patients and controls with items on education, profession, income, partner and living situation. PATIENTS: Two hundred and ten GHD patients treated in childhood but not in adulthood with GH (93 isolated GHD (IGHD), 111 patients with multiple pituitary deficiency (MPD)) were compared with 53 short controls (height in childhood < third percentile for population) and 39 normal stature controls. RESULTS: There were no differences between short and normal controls. There were also no differences between IGHD and MPD patients in any of the investigated items. GHD patients did not differ from controls on education level, but scored lower on the profession scale, had a lower income and had a partner less often; if they had a partner they less often had children; also, more of them lived with their parents. CONCLUSION: Since patients with multiple pituitary deficiency did not differ from patients with isolated growth hormone deficiency, this suggests that the lower scores on the social parameters are the result of the growth hormone deficiency itself. Since short stature controls had higher scores than patients with growth hormone deficiency and did not differ from normal stature controls in any of the aspects investigated, it seems unlikely that the problems of the patients with growth hormone deficiency can be attributed to short stature. PMID- 7554317 TI - Glycosylated and non-glycosylated prolactin forms are increased after opioid administration as part of surgical anaesthesia. AB - OBJECTIVE: Previous studies have shown that non-glycosylated prolactin (NG-PRL) increased more markedly than glycosylated hormone (G-PRL) after TRH or metoclopramide stimulation. The aim of the present study was to determine whether such results could be extended to opioid-induced PRL stimulation. DESIGN: Open and prospective study. Using a newly developed IRMA specific for NG-PRL, we determined G-PRL and NG-PRL immunoreactivities after administration of 0.8-1.2 mg of the opioid drug phenoperidine as part of an anaesthesia. PATIENTS: Ten male patients anaesthetized for surgical treatment of a prolapsed lumbar intervertebral disc. MEASUREMENTS: Samples were obtained hourly pre and post operatively, and every 15 minutes during operation for determination of plasma PRL, NG-PRL and G-PRL. Plasma cortisol, ACTH and GH levels were measured in an attempt to differentiate the respective roles of stress and opiate agonists in the variations of PRL levels during surgery. RESULTS: A dramatic increase in PRL levels was observed in all patients from an average of 300 +/- 90 to 1200 +/- 330 mU/l (mean + SEM) 30 minutes after drug administration. The proportion of G-PRL immunoreactivity was not significantly different when basal (25.2%) and stimulated (27%) values were compared (P > 0.05), and when mean increments of NG PRL and G-PRL were compared (345 and 348%, respectively). The opioid drug induced a significant decrease in cortisol levels after injection and during operation (from 585 +/- 63 to 99 +/- 51 nmol/l) with a concomitant decrease in ACTH levels. GH levels were not significantly altered during anaesthesia but were significantly greater (P < 0.05) after than before surgery (5.0 +/- 1.3 vs 0.98 +/- 0.54 mU/l, respectively). CONCLUSIONS: We conclude from the present and from previous data that opioid induced anaesthesia is accompanied by an increase in both glycosylated and non-glycosylated PRL and that different PRL secretagogues may induce distinct responses in terms of PRL molecular forms. PMID- 7554318 TI - Oestrogen effects on calcitriol levels in post-menopausal women: a comparison of oral versus transdermal administration. AB - BACKGROUND AND OBJECTIVES: In some studies oral oestrogen therapy in post menopausal women has been shown to increase both total and free 1,25 dihydroxyvitamin D (calcitriol) levels, suggesting that oestrogen therapy may prevent post-menopausal bone loss, in part, by increasing calcium absorption as a result of raised calcitriol levels. Transdermal oestrogen, however, has not been shown to increase calcitriol levels although it prevents bone loss. These two routes of administration have not previously been directly compared in the same subjects at bioequivalent doses as assessed by FSH and LH suppression. DESIGN AND PATIENTS: In a randomized cross-over study, 15 women at least 12 months post menopausal (mean age 56 years (range 50-66)) were randomized to either oral conjugated equine oestrogen (1.25 mg daily) or transdermal 17 beta-oestradiol (100 micrograms daily) for 12 weeks after which each subject changed over to the alternative medication. For the last 12 days of each medication, 10 mg medroxyprogesterone acetate (MPA) was added to the treatment protocol. A fasting blood sample was taken at baseline and at the end of each treatment period prior to administration of the MPA. MEASUREMENTS: Serum calcium, phosphorus, albumin, bicarbonate, intact parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (calcitriol), 25-hydroxyvitamin D (25OHD), vitamin D-binding protein (DBP) were measured. The free calcitriol index was calculated as the molar ratio of calcitriol to DBP. Free calcitriol was measured by centrifugal ultrafiltration. RESULTS: The degree of suppression of FSH and LH was similar with the two routes of oestrogen administration. Total calcitriol was significantly higher with oral oestrogen treatment compared to transdermal oestrogen and compared to baseline (mean +/- SEM, baseline 80 +/- 5; oral oestrogen 102 +/- 8; transdermal oestrogen 82 +/- 4) as was DBP (mean +/- SEM, baseline 5.2 +/- 0.2; oral oestrogen 6.9 +/- 0.4; transdermal oestrogen 5.8 +/- 0.2) which accounted for the rise in calcitriol. Free calcitriol measured by equilibrium dialysis showed no rise with either oestrogen preparation. Phosphorus was not different between treatment groups and fell with both oestrogen treatments (baseline 1.32 +/- 0.15, oral oestrogen 1.23 +/- 0.10, transdermal oestrogen 1.17 +/- 0.16) and PTH rose with both treatments (baseline 1.33 +/- 0.21, oral oestrogen 1.52 +/- 0.27, transdermal oestrogen, 1.99 +/- 0.32). Calcium was not different between treatment groups and was not different from baseline. CONCLUSIONS: These results show that in this study the total calcitriol rose after oral but not transdermal oestrogen due to a rise in vitamin D-binding protein. Free calcitriol was not affected by oral or transdermal oestrogen treatment despite a fall in plasma phosphorus and a rise in PTH, both of which are considered agonists for calcitriol production. We may therefore conclude that neither oral nor transdermal oestrogen replacement routinely stimulates free calcitriol levels. In the studies where a rise in free calcitriol was noted, the degree of suppression of bone resorption by oestrogen may have been greater, thus producing a larger demand for calcium due to filling of a larger bone remodelling space with consequent stimulation of calcitriol levels. PMID- 7554319 TI - Familial hyperparathyroidism associated with jaw fibroma: case report and literature review. AB - A 53-year-old female suffering from renal stones and hypercalcaemia was diagnosed as having primary hyperparathyroidism caused by hyperplasia of the parathyroid glands. She underwent total parathyroidectomy and implantation of parathyroid tissue. After one year, she underwent surgery for a jaw tumour. The pathological findings indicated it to be a cementifying fibroma. Jackson et al. (1990) reported the familial association of hyperparathyroidism with jaw tumours, and they suggested that this condition represents a new clinical syndrome. We believe that our case belongs to this syndrome. PMID- 7554320 TI - Isolated combined growth hormone and gonadotrophin deficiency due to hypothalamic dysfunction, associated with insulin resistance. AB - A 47-year-old woman was evaluated for congenital dwarfism, primary amenorrhoea due to hypogonadotrophic hypogonadism, severe hyperlipidaemia with pancreatitis, and overt diabetes mellitus associated with severe insulin resistance requiring 2.5-3 units of insulin per kilogram body weight. Chromosomal analysis with trypsin banding was normal and biochemical evaluation revealed low oestrogen levels, inappropriately low gonadotrophins, very low IGF-I concentrations and GH concentrations unresponsive to insulin or L-dopa administration. Prolactin, pituitary-adrenal and pituitary-thyroid axes were normal. Dynamic testing with GnRH and GHRH produced increases in FSH, LH and GH concentrations. A MRI of the brain revealed no discernible hypothalamic abnormalities and a small pituitary. The presence of congenital combined growth hormone and gonadotrophin deficiency on the basis of a suprapituitary defect suggests the existence of common or related pathways regulating GnRH and GHRH synthesis or secretion and may have contributed to the ultimate development of insulin resistance and hyperlipidaemia. PMID- 7554321 TI - MELAS syndrome associated with diabetes mellitus and hyperthyroidism: a case report from Taiwan. AB - MELAS syndrome is a form of mitochondrial myopathy with manifestations of seizure, stroke-like syndrome, lactic acidosis, ragged red muscle fibres and mitochondrial encephalopathy. The syndrome has been reported in association with a variety of endocrine and metabolic disorders including diabetes mellitus (DM), hypothalamo-pituitary hypofunction, hypothalamic growth hormone deficiency and delayed puberty. Mitochondrial DNA (mtDNA) point mutation may be the major pathological defect. However, association of MELAS syndrome with hyperthyroidism has not previously been reported. A case is reported from Taiwan of a 32-year-old woman suffering from MELAS syndrome with associated DM and hyperthyroidism. When the latter was diagnosed in April 1988, the patient underwent subtotal thyroidectomy. There was no family history of thyroid disease. Because of repeated seizures, she had computed tomography (CT) and magnetic resonance imaging (MRI) of the brain which showed focal, low-density lesions over the cerebral hemispheres. Both serum and cerebral spinal fluid lactic acid levels were elevated. Mild elevations of serum T4 and T3 and a high titre of TSH receptor antibody were still present. Hyperglycaemia was noted during hospitalization and DM confirmed by oral glucose tolerance test. Muscle biopsy showed ragged red fibres. DNA analysis showed an A-to-G transition at the 3243rd nucleotide position of the tRNA(Leu(UUR)) gene of the mtDNA from the patient. Quantitative polymerase chain reaction (PCR) and restriction analysis revealed that about 60% of the blood mtDNA was of mutant type. The patient received antithyroid drugs for hyperthyroidism, diet control for DM and anti-epileptic drugs for seizure. PMID- 7554322 TI - Mineralocorticoid hypertension and congenital deficiency of 11 beta hydroxysteroid dehydrogenase in a family with the syndrome of 'apparent' mineralocorticoid excess. AB - The so-called syndrome of 'apparent mineralocorticoid excess' (AME) is a rare cause of endocrine hypertension thought to result from a defect in the peripheral conversion of cortisol to cortisone. Less than 30 cases have been described. From a consanguineous marriage we present a family comprising 2 and probably 3 affected cases of AME. The index case is a 4-year-old boy with mineralocorticoid hypertension, short stature, failure to thrive, hypokalaemic nephropathy and osteopenia. The ratio of the urinary excretion of tetrahydrocortisone/tetrahydrocortisols was reduced at 0.05 (reference range 1.77 2.11), and the plasma half-life of 3H-11 alpha-cortisol elevated at 152 minutes (reference range 30-50) indicative of severe 11 beta-hydroxysteroid dehydrogenase deficiency. Plasma cortisol concentrations were normal and daily secretion rate reduced. Dexamethasone administration induced a natriuresis in keeping with the observation that cortisol itself is the implicated mineralocorticoid. Treatment with amiloride lowered blood pressure, increased potassium levels, and resulted in an increase in growth rate. The boy's twin brother died at the age of 3.5 years following a trivial diarrhoeal illness and was almost certainly affected. AME was also diagnosed in a younger brother (age 17 months), but both parents are normal. Congenital deficiency of 11 beta-hydroxysteroid dehydrogenase should be considered in any child with mineralocorticoid hypertension and failure to thrive. As cortisol is the 'offending' mineralocorticoid in this condition, the term 'apparent' mineralocorticoid excess is perhaps obsolete.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554323 TI - The syndrome of apparent mineralocorticoid excess and deficiency of 11 beta hydroxysteroid dehydrogenase. PMID- 7554324 TI - Angiotensin converting enzyme inhibitors and the otolaryngologist. PMID- 7554325 TI - Tinnitus and otoacoustic emissions. PMID- 7554326 TI - Antimicrobial activity of glycerine-ichthammol in otitis externa. AB - The clinical efficacy of glycerine-ichthammol in otitis externa may be due to an anti-inflammatory action of ichthammol or a dehydrating effect of glycerine on the oedematous ear canal. Its antimicrobial activity, if any, against the common organisms in otitis externa is not well known. A study of the antibacterial property of glycerine-ichthammol as measured by a growth inhibition test and a modified cidal assay, showed inhibition of selected gram positive organisms (Streptococcus pyogenes and Staphylococcus aureus) by ichthammol and glycerine ichthammol combination, but only negligible antibacterial activity against Pseudomonas aeruginosa and Escherichia coli. Candida albicans was also weakly inhibited. As the activity against gram negative organisms is minimal, incorporation of an anti-gram negative antibiotic such as gentamicin in the glycerine-ichthammol compound to enhance its antibacterial spectrum is suggested. PMID- 7554327 TI - The effect of topical fluticasone propionate on intranasal histamine challenge in subjects with perennial allergic rhinitis. AB - The mechanism of action of topical intranasal steroids is obscure. To investigate this, we have studied the effects of a topical intranasal corticosteroid, fluticasone propionate on nasal airflow resistance (Rnaw), secretions, cytological smears and symptoms. Fluticasone propionate aqueous nasal spray was given to 11 patients with perennial allergic rhinitis in a double-blind, placebo controlled study. On each day, patients were challenged with ascending doses of histamine. Rnaw, secretion volume, total protein, mucin, lysozyme and albumin were measured. Nasal smears were taken and sneezes counted. Diary card data were collected for both treatment periods. There was a significant, dose-related increase in Rnaw and sneezing on histamine challenge. A single dose of fluticasone had no effect on any parameter. After 4 weeks of treatment, resistance measurements were reduced (post-challenge g.m.2.8 cmH2O/l/s, Q1-Q3 1.6 4.8; placebo 4.2, 2.9-5.3: P < 0.0001) as were baseline secretion volumes (mean 2.4 ml/5 min, c.i.1.9-3.0; placebo 3.3, 2.8-3.8: P < 0.05). Eosinophil counts were suppressed (fluticasone 5.8%, c.i. 4.0-15.7; placebo 23.3%, 12.4-34.1: P < 0.05) and the composite symptom score reduced (P < 0.05). Fluticasone has long term effects on the nasal response to histamine in perennial allergic rhinitis and part of this effect is likely to be vascular. PMID- 7554328 TI - Hearing loss in the 4-8 kHz range following tympanic membrane perforation from minor trauma. AB - This study looked at the incidence of sensorineural hearing loss in the 4-8 kHz range in patients with unilateral tympanic membrane perforations from minor trauma who presented to the department in the past year. Eleven patients conformed to our inclusion criteria, one was lost to follow-up. Perforation was present in all patients in the pars tensa and no treatment was required in nine patients. The mean sensorineural hearing loss at 4,6 and 8 kHz was 28.2 dB on initial examination and 14.6 dB by Day 30. All 11 patients had normal hearing in the contra-lateral ear (mean 13.5 dB). In this group of patients with a tympanic membrane perforation from minor trauma the conductive hearing loss was accompanied by a significant (P = 0.001) but reversible high frequency sensorineural deafness. PMID- 7554329 TI - Plasma catecholamine levels during functional endoscopic sinus surgery. AB - Plasma adrenaline and noradrenaline concentrations were measured in 10 consecutive patients during functional endoscopic sinus surgery (FESS). Surgery was preceded by nasal packing with 5 ml 2% cocaine solution, followed by infiltration with 1:80,000 adrenaline and 2% lignocaine. All patients showed a marked rise in plasma adrenaline concentration within 4 min of commencing infiltration, which was not related to the patient's sex, age, weight, the total amount injected, nor the amount injected into the nose. Surgeons should be aware of this marked, but unpredictable, systemic absorption of locally infiltrated vasoconstrictors. Pulse and ECG monitoring should be considered mandatory for such procedures, even when surgery is performed under local anaesthesia. PMID- 7554330 TI - The influence of upper respiratory tract inflammation upon tubal function. AB - Eustachian tube function was examined in four patients (four ears) with a traumatic perforation of the tympanic membrane when they suffered from an upper respiratory tract (URT) infection or seasonal nasal allergy and after they had recovered. Although the tubal opening pressure did not show a distinct difference on the two occasions, the active ventilatory function was apparently impaired during the upper respiratory tract inflammation; particularly the negative middle ear pressure equalizing function, and was still impaired 2-3 weeks later after recovery from the upper respiratory tract inflammation. It is concluded that URT inflammation can affect tubal ventilatory function even in normal individuals. PMID- 7554331 TI - The microbiology and antibiotic treatment of peritonsillar abscesses. AB - Pus from 53 peritonsillar abscesses was cultured and associations between the microbiological results and clinical data were investigated with the aim of developing a clinical protocol for treatment. A positive culture grew in 85% of quinsies and of these 16% produced aerobes and 84% anaerobes. Penicillin resistant organisms were grown from 32% of patients and all but one of these organisms (Haemophilus influenzae) was sensitive to metronidazole. There was no association between clinical presentation and cultured organism which could guide treatment, hence we recommend penicillin and metronidazole as the antibiotic regimen of choice in the treatment of peritonsillar abscesses because of its effectiveness in 98% of patients. PMID- 7554332 TI - The pathophysiology and treatment of rhinitis medicamentosa. AB - To evaluate the treatment of rhinitis medicamentosa, 10 consecutive patients discontinued their use of topical vasoconstrictors and were treated with budesonide nasal spray, 400 micrograms, daily for 6 weeks. The thickness of the nasal mucosa, the decongestive effect of oxymetazoline and the histamine sensitivity were measured with rhinostereometry. All patients were able to stop using the vasoconstrictors and objective variables showed that they needed treatment for at least 6 weeks. The results strongly support the theory that the rebound swelling is due to interstitial oedema rather than to vasodilatation. The presence of tachyphylaxis reflected by a reduction in both the decongestive effect of oxymetazoline and a reduction of drug duration was seen. PMID- 7554333 TI - Neutrophil elastase-alpha 1-antitrypsin in middle ear fluid in chronic otitis media with effusion. AB - Neutrophil elastase-alpha 1-antitrypsin was quantified in samples taken from middle ear effusions collected at operation from 17 children attending for elective myringotomy and grommet insertion. At the time of surgery the effusion was classified as serous or mucoid. Children with a recent history of infection or antimicrobial therapy were excluded. The quantification of immunoreactive neutrophil elastase was by means of enzyme-linked immunosorbant assay (ELISA). The mean value of neutrophil elastase-alpha 1-antitrypsin was 50.6 +/- 38.3 (SD) micrograms/ml in mucoid effusions, which was significantly higher (P < 0.05) than that in serous effusions (5.3 +/- 4.8 micrograms/ml). These results indicate that a mucoid effusion may reflect a more severe inflammatory response and that persistence of neutrophil activity in the middle ear mucosa may contribute to the persistence of at least one group of middle ear effusions. PMID- 7554334 TI - The radio-opacity of fishbones: a cadaveric study. AB - Lateral neck radiographs performed to localize impacted fishbones have a low sensitivity. The differing radio-opacities of the bones of various fish species may be a reason for this. A cadaver head and neck and the rib bones of 10 species of fish were used in this study. A laryngoscope was used to introduce a fishbone into the vallecula and then into the hypopharynx of the cadaver. A lateral radiograph was taken in each case. Ten control films were taken. The radiographs were independently reviewed by a consultant radiologist and otolaryngologist. The presence and position of any bone seen was noted. There was no inter-observer variation. All species of fishbone were visible. One bone was not seen by either observer. Visualization of fishbones is more dependent on position than their degree of radio-opacity. Routine radiography is recommended. PMID- 7554335 TI - Haemorrhage as a complication of inferior turbinectomy: a comparison of anterior and radical trimming. AB - A study was conducted to see whether the turbinectomy technique used influenced the incidence of haemorrhage. In a group of 214 patients who underwent bilateral turbinate surgery, haemorrhage occurred in 0.9% of those who had anterior turbinectomy and 5.8% who had radical turbinectomy (P < 0.05). It is concluded that confining excision to the anterior end of the inferior turbinate offers the advantage of a lower incidence of haemorrhage. PMID- 7554336 TI - Hospital epistaxis admission rate and ambient temperature. AB - The incidence of epistaxis admissions to hospital and their relationship to ambient temperature is examined. A retrospective analysis of 1211 patients with epistaxis sufficiently severe to warrant hospital admission was performed over a period of 1836 consecutive days. Daily average temperature data for this time period were examined and compared with admission rates. A marked increase in hospital attendance was apparent during colder days. Patients were admitted at a rate of 0.829 patients per day for temperatures less than 5 degrees C, (95% Confidence Interval: 0.737-0.928), compared with 0.645 patients per day for temperatures between 5.1 and 10 degrees C, (95% 0.586-0.708). On average the population of epistaxis patients attended on days that were 0.6 degree C colder (95% Confidence Interval: 0.2 degree C-0.9 degree C) than the average temperature for the time examined (P < 0.005, student's t-test). PMID- 7554337 TI - Myringoplasty for subtotal perforation. AB - It is often reported that the repair of subtotal perforations is less successful than that of smaller perforations, mainly because it is technically more difficult. The author's surgical technique for subtotal perforations is presented and the results compared with that of myringoplasty for smaller perforations. The study shows that subtotal perforations are associated with a higher incidence of abnormal middle ear mucosa (chi 2 = 11.75, d.f. = 2) and damage to the malleus (chi 2 = 5.60, d.f. = 1) than smaller perforations. However, the success rate of subtotal perforation closure (92.5%) is as good as the closure rate for smaller perforations (94.1%). It is concluded that the size of the perforation is not a major factor in determining whether the graft will be successful in myringoplasty. PMID- 7554338 TI - Intranasal salbutamol has no effect on mucociliary clearance in normal subjects. AB - Normal mucociliary clearance is essential for the healthy function of the upper and lower airways. Restoring normal clearance times in the presence of chronic disease would be a helpful adjunct in treating these patients. This double-blind placebo-controlled trial in 37 normal subjects compared topical salbutamol against placebo on nasal mucociliary clearance. Clearance rates were measured using the sacharine clearance technique and 34 patients completed the study. There was no effect of treatment, sex, order of treatment or period (interval between treatments) on mucociliary times. There was a weak positive correlation with age and there was a trend towards increased clearance times with salbutamol although this did not reach significance (P = 0.226). The results show that topical salbutamol appears to have no effect on nasal mucociliary clearance times in vivo in normal subjects. PMID- 7554339 TI - Carcinoma of the pyriform sinus: a retrospective analysis of treatment results over a 20-year period. AB - One hundred and one patients with pyriform sinus carcinoma treated at the Netherlands Cancer Institute were studied retrospectively. The patients were staged according to the UICC criteria of 1987: there were no stage I, 23 stage II, 30 stage III, and 48 stage IV patients. The treatment consisted of radiotherapy (n = 45), a planned combination of surgery and post-operative radiotherapy (n = 47) or surgery alone (n = 9). The crude 5-year survival was 27%, whereas the 5-year disease-free survival was 37%. The locoregional disease free survival was 52%. Stage according to the UICC 1987 criteria is an important prognostic variable (P = 0.0026). Furthermore, significantly less locoregional recurrences and a better disease-free survival were seen in the combined surgery and radiotherapy group than in the exclusively irradiated group (P < 0.0001). PMID- 7554340 TI - Thyroid dysfunction following combined therapy for laryngeal carcinoma. AB - The thyroid function of 27 patients who previously had carcinoma of the larynx treated by total laryngectomy with thyroid lobectomy was studied by measuring levels of thyroxine (T4) and thyroid stimulating hormone (TSH). Twenty-two of these patients also received external beam radiotherapy. Abnormal results were found in 45% (10 patients) of those who received combined therapy. Clinical hypothyroidism developed in two patients (9%) and subclinical hypothyroidism (elevated TSH) was seen in eight patients (36%). Eighty-eight per cent of those patients with subclinical hypothyroidism had low or low normal T4 levels. All the patients treated with surgery only had normal thyroid function. To prevent hypothyroidism and identify those at risk of developing hypothyroidism, post operative testing of thyroid function should be carried out on a routine basis in patients receiving combined therapy for laryngeal cancer. In addition we recommend that patients with subclinical hypothyroidism who have had combined treatment should be treated with thyroxine to prevent the complications of this condition. PMID- 7554341 TI - Quantifying the Carhart effect in otosclerosis. AB - The Carhart effect consists of a depression in bone conduction thresholds in the presence of a conductive hearing loss. However, the mathematical relationship between the degree of conductive hearing loss and the degree of depression of bone conduction has not before been described. We have reviewed pre- and post operative pure-tone audiograms performed on 102 consecutive patients having stapedectomy in an attempt to identify relationships between changes in bone conduction and air conduction and air-bone gap closure. Significant linear relationships were found between bone conduction and air conduction at 0.5. 1, 2 & kHz. Bone conduction was linearly related to air bone gap closure at 2 kHz. PMID- 7554342 TI - Factors associated with positive bacteriological cultures of chronic middle ear effusions. AB - Pathogenic bacteria have been isolated from middle ear effusions in a number of studies. Our aim was to identify factors which predispose to patients having positive cultures. Over a 1-year period, prospective data were collected on patients admitted for myringotomy. Middle ear effusions were collected at the time of surgery using specially designed traps, and underwent microscopy and culture. Data on local weather parameters were obtained from the Meteorological Office in Edinburgh, UK and compared with the patterns of positive cultures. A higher proportion of cultures were positive in October, November and December and in February and May than in the other months of the years. We were not able to relate these variations to any of the meteorological data, with the possible exception of relative humidity, or to any other variable. PMID- 7554343 TI - Chromosome 18: a possible site for a tumour suppressor gene deletion in squamous cell carcinoma of the head and neck. AB - The present study analyses tumour samples from 41 patients with squamous cell carcinoma of the head and neck (SCCHN) using the polymerase chain reaction (PCR) to detect genetic alterations on chromosome 18. Microsatellite markers were used to examine each sample for loss of heterozygosity (LOH) and microsatellite instability. Genetic alterations were most commonly noted on the long arm of chromosome 18 (18q). LOH/microsatellite instability occurred on 18q in 20/41 (49%) of patients. The highest single incidence of LOH was found at 18q21.1-21.3 using the microsatellite marker D18S35. LOH occurred in 10/30 (33%) informative cases while LOH/microsatellite instability occurred in 13/30 (43%) informative cases using this marker. Interestingly the tumour suppressor gene known as the 'deleted in colonic carcinoma' (DCC) gene is located in this region at 18q21.3 but is not commonly lost. This suggests that the marker D18S35 is mapping close to a second as yet unidentified tumour suppressor gene in this area. PMID- 7554344 TI - Prediction of complications following caustic ingestion in adults. AB - The records of 86 adults admitted to hospital following caustic ingestion were reviewed. Eighteen patients (21%) developed complications; of these, six were fatal. Patients without symptoms or signs did not develop complications. Complications occurred only following lye ingestion, or intentional ingestion of hydrochloric acid or ammonia water. Of patients with such ingestion, and two signs or symptoms or more, 70% developed complications. Oesophagoscopy did not significantly improve the prediction of complications. There was a strong trend, however, for more severe complications with more severe submucosal, circumferential oesophageal injuries (P = 0.003). The study suggests that only adults with symptoms or signs following strong alkali or strong acid ingestion are at risk of complications. In adults who are symptomatic following ingestion of strong acid or alkali, oesophagoscopy is important to identify the potentially fatal transmural injuries. PMID- 7554345 TI - The accuracy of acoustic rhinometry using a pulse train signal. AB - The signal used in the original acoustic rhinometers was an impulse of short duration and wide energy spectrum. A rhinometer utilizing a new signal of a similarly broad energy but different time course has been developed. We investigated the accuracy of area reconstructions computed using this signal by comparing them with the original areas of simple models. This study demonstrates that, at present, acoustic rhinometry using a pulse train signal is subject to significant systematic errors. Some of these are intrinsic to the rhinometer. Others are caused by the geometry of the models. Areas reconstructions beyond narrow constrictions are particularly inaccurate. The findings are significant because the nasal valve can act as such a constriction. The errors can cause the parameters used in the clinical application of acoustic rhinometry to deviate substantially from the true values. PMID- 7554346 TI - Increased and decreased relative risk for non-insulin-dependent diabetes mellitus conferred by HLA class II and by CD4 alleles. AB - Non-insulin-dependent diabetes mellitus has been recognized to be heterogeneous in etiology, with multiple subgroups. Several genes or chromosomal regions have been implicated in the development of the disease. In this study the association of HLA class II alleles and genotypes and the association of CD4 and CD3 polymorphisms were assessed in a large number of Belgian non-insulin-dependent diabetes mellitus patients. Furthermore, the importance of the DQ alpha 1Arg52/DQ alpha 1Arg52 and the DQ beta 1Asp57/DQ beta 1Asp57 genotypes and the combination of both genotypes were examined. Our results show that in the HLA class II genes only the DQ alpha 1Arg52+/DQ alpha 1Arg52+ genotype was significantly associated with non-insulin-dependent diabetes mellitus compared with controls (p = 0.011, RR = 2.02). We also observed that the frequency of the CD4*A4/*A8 genotype and the CD4*A7 allele was significantly increased and decreased respectively in non insulin-dependent diabetes mellitus patients as compared with the controls (p = 0.018, RR = 2.16 and p = 0.0003, RR = 0.49 respectively). These results therefore suggest that HLA class II and CD4 genes might independently contribute to the susceptibility for non-insulin-dependent diabetes mellitus and that these alleles and genotypes might identify subgroups of patients with different susceptibilities. PMID- 7554347 TI - Partial deletion 11q: report of a case with a large terminal deletion 11q21-qter without loss of telomeric sequences, and review of the literature. AB - We describe the cytogenetic findings and the dysmorphic features in a stillborn girl with a large de novo terminal deletion of the long arm of chromosome 11. The karyotype was 46,XX,del(11)(q21qter). By reviewing previous reports of deletion 11q, we found that cleft lip and palate are most frequently seen in proximal 11q deletions involving 11q21. Telomeric staining using the PRINS technique demonstrated normal telomeric sequences in the deleted chromosome 11. PMID- 7554349 TI - Familial intestinal polyatresia syndrome. AB - Familial multiple-level intestinal atresia is a rare syndrome with autosomal recessive inheritance. Only a few well-documented families have been reported in the medical literature. In this article two new cases from a consanguineous couple are presented. These are the first reported cases of Familial Intestinal Polyatresia Syndrome from Turkey. The importance of this report is that the recognition of multiple intestinal atresias as an invariably fatal syndrome will be helpful for genetic counselling and attempts at early prenatal diagnosis for successive pregnancies in these couples. PMID- 7554348 TI - Evidence for genetic influences on smoking in adult women twins. AB - The purpose of the present study was to examine genetic influences on smoking behavior in women twins, and to compare the results before and after adjusting for environmental covariates. Subjects were participants in the Kaiser Permanente Women Twins Study in Oakland, California. Exam 1 (1978-1979) included 434 pairs of women, and exam 2 (1989-1990) included 352 pairs of women. For comparison with a previous study in male twins, pairwise concordance ratios (OCR) for smoking status between monozygotic (MZ) and dizygotic (DZ) twins were calculated. Multivariate logistic regression analysis was also used to assess the association of smoking behaviors between co-twins, adjusting for age, education, and frequency of contact. At exam 1, OCR's were consistent with previous results in male twins, and supported genetic influences on smoking initiation (OCR = 1.6; 95% Confidence Interval (95% CI) = 1.0, 2.3) and maintenance (OCR = 1.8; 95% CI = 1.3, 2.4). In contrast to the males, however, no clear evidence for genetic influences on smoking intensity or never smoking were present. The adjusted logistic regression models appeared to confirm the findings from the pairwise comparisons. The results were similar for exam 2. These results are consistent with modest genetic influences on smoking initiation and maintenance. Differences between these results and those for the male twins suggest possible genetic environmental interactions. PMID- 7554350 TI - Course of autosomal recessive polycystic kidney disease (ARPKD) in siblings: a clinical comparison of 20 sibships. AB - Forty-two children out of 20 sibships with autosomal recessive polycystic kidney disease were observed pro- and retrospectively over a mean period of 3.7 years in a long-term study on cystic kidney diseases in children. The intra- and interfamilial variability in terms of age at diagnosis, administration of antihypertensive therapy, liver affection, and renal function were evaluated. According to the 1971 subclassification of Blyth & Ockenden, defining different grades of severity, 12 patients were assigned to the perinatal, nine to the neonatal, 13 to the infantile, and eight to the juvenile subtype of autosomal recessive polycystic kidney disease. In 11 of the 20 families different subtypes were observed among affected siblings. In seven families, affected sibs belonged to adjacent subtypes, while major intrafamilial differences were observed in only four families. The defined subtypes, therefore, cannot be regarded as appropriate in distinguishing genetic groups of autosomal recessive polycystic kidney disease. With respect to the severity of autosomal recessive polycystic kidney disease, there is a wide spectrum of phenotypic manifestations, ranging from stillbirths to mildly affected of phenotypic manifestations, ranging from stillbirths to mildly affected adults, while intrafamilial variability of the clinical picture is generally small with multiple allelism as the most likely genetic explanation. Age at death, however, showed gross variation in eight sibships. Differences in the clinical course between several siblings cannot be explained by a sex influence in autosomal recessive polycystic kidney disease. PMID- 7554351 TI - 18p monosomy with GH-deficiency and empty sella: good response to GH-treatment. AB - A patient with 18p monosomy and GH deficiency due to pituitary hypoplasia, who showed an excellent response to GH-treatment, is described. Patients with this syndrome should be considered for endocrine evaluation, as they can benefit from hormonal substitution. PMID- 7554352 TI - Clinical overlap of Beckwith-Wiedemann, Perlman and Simpson-Golabi-Behmel syndromes: a diagnostic pitfall. AB - We report on a child who died in the neonatal period. Major external anomalies included foetal overgrowth, macroglossia, and ambiguous genitalia (micropenis and perineoscrotal hypospadias with cryptorchidism). Necropsy showed a large right diaphragmatic hernia, visceromegaly, multicystic kidney dysplasia, Langerhans islet hyperplasia, nephroblastomatosis, multiple adrenal adenomas, and dysplastic testicles. The child illustrates the difficulties of the differential diagnosis of overgrowth syndromes in the neonatal period, and the phenotypic overlap of Beckwith-Wiedemann, Denys-Drash, Simpson-Golabi-Behmel, Perlman and possibly Meacham-Winn syndromes. Simpson-Golabi-Behmel syndrome was felt to be the most likely diagnosis. If this opinion is correct, genital ambiguity, hydramnios and nephroblastomatosis should be added to the clinical spectrum of Simpson-Golabi Behmel syndrome. Differential diagnosis between the above-mentioned syndromes is of major importance for accurate genetic counseling, considering the differences in recurrence risk. The present case underlines the need for long-term survey of patients suspected of having Simpson-Golabi-Behmel syndrome, who could be at risk for embryonic tumours. PMID- 7554353 TI - Haspeslagh syndrome without severe mental retardation and pterygia? AB - An adult female is described with mild developmental delay, typical facies, dental anomalies, arachnodactyly and camptodactyly. In many respects she resembles four other patients described earlier, but differs in not having multiple pterygia, nor severe mental retardation. We suggest that this entity should be named Haspeslagh syndrome. The differential diagnosis is discussed. PMID- 7554354 TI - Gorlin-Chaudhry-Moss or Saethre-Chotzen syndrome? AB - We report a 2-year-old girl with craniosynostosis, an ossification defect of the cranial vault, midface hypoplasia, low frontal hairline, anti-mongoloid slant of the palpebral fissures, ptosis of the lateral upper lids and high-arched narrow palate. There are additional findings fitting the Gorlin-Chaudhry-Moss syndrome, such as hypoplasia of the labia majora, hypoplasia of the distal phalanges of fingers and toes and conductive hearing loss, but hypertrichosis and dental anomalies are missing, which were described in the four females previously reported with the probably autosomal recessive Gorlin-Chaudhry-Moss syndrome. Since the autosomal dominant Saethre-Chotzen syndrome may show similar cranio facial features, short fingers with non-obligatory cutaneous syndactyly, and ossification defects of the cranial vault, the Saethre-Chotzen syndrome should also be considered in our patient. PMID- 7554355 TI - A small supernumerary marker chromosome X identified by in situ hybridization. AB - Cytogenetic analysis of a girl with moderate mental retardation and dysmorphic features revealed a 46,XX/47,XX,+mar karyotype. Fluorescence in situ hybridization using chromosome specific alpha satellite probes showed that the supernumerary marker originated from the X chromosome. To our knowledge, this is the first reported case of a female patient mosaic for a supernumerary small marker chromosome derived from X, and hence mosaic for trisomy of the pericentric region of the X chromosome. PMID- 7554356 TI - A simple method to detect the RYR1 mutation G1021A, a cause of malignant hyperthermia susceptibility. AB - To search for the mutations RYR1 G1021A in families where malignant hyperthermia (MH) episodes have occurred, we have used an amplification-created restriction sites (ACRS) technique to detect the mutation. The previously described single stranded conformation polymorphism (SSCP) technique was laborious and time consuming, but necessary to detect the mutation, whereas the method described here discriminates quickly and efficiently between homozygotes with the mutation, heterozygotes and homozygotes without the mutation. PMID- 7554357 TI - A highly informative BamHI RFLP in the type IV collagen alpha 5 chain gene (COL4A5). PMID- 7554358 TI - A TaqI RFLP in the type IV collagen alpha 5 chain gene (COL4A5) with the rare allele more frequently found in seven Chinese Alport syndrome patients. PMID- 7554359 TI - Screening of neurofibromatosis type 1 gene: identification of a large deletion and of an intronic variant. AB - Neurofibromatosis type 1 of von Recklinghausen is a common autosomal dominant disorder, characterized by peripheral neurofibromas, cafe-au-lait spots and Lisch nodules of the iris. The high mutation rate at the neurofibromatosis type 1 locus results in a wide range of molecular abnormalities. We have screened seven different exons of the neurofibromatosis type 1 gene, including those codifying for the GAP-related domain, using the RNA-Single Strand Conformation Polymorphism (RNA-SSCP) method in a series of 59 neurofibromatosis type 1 patients. We have also analyzed four intragenic repeats and one RFLP to detect hemizygosity and evaluate informativeness in at-risk families. One deletion and a new intronic normal variant have been detected. Thus the majority of Neurofibromatosis type 1 chromosomes have not been characterized, confirming difficulty in providing proper genetic counselling in neurofibromatosis type 1 families, even following extensive DNA analysis. PMID- 7554360 TI - A recombination event in the closely linked plasminogen and apolipoprotein(a) gene loci. AB - Genetic studies as well as in situ hybridisation data have strongly demonstrated that the genes coding for apoprotein(a) and plasminogen are linked and localised to chromosome 6 at band 6q26-27. We describe in this report the presence of a recombination event in a region of approximately 50 kb of DNA separating the two genes. The recombination was found in an Italian family, in which a mutation affecting both plasminogen plasma level and activity of plasminogen activity has been detected. Polymerase chain reaction and sequencing analysis showed the presence of a mutation different from those previously reported in two Japanese families. PMID- 7554361 TI - Genetic analysis of 20 families with autosomal dominant adult polycystic kidney disease from South West Thames Region. AB - Twenty families with autosomal dominant polycystic kidney disease from S. W. Thames Region were analysed using markers for chromosome 16p13.3, the site of the common mutation (PKD1). Six families gave a negative lod-score for 3'HVR, the most informative distal marker. This could be explained in four cases by recombination events. Of the two families where this was not an explanation, one, of Italian origin, was unequivocally unlinked for all markers, and the other was more likely to be non-PKD1 than linked to 16p13.3. The Italian family was ascertained through the Blood Pressure Unit, and the other via the Genetic Clinic. No members of either family had ever attended a renal clinic. The remaining 18 families either came via renal clinics, or had at least one member attending such a centre. PMID- 7554362 TI - Aplasia cutis congenita and associated disorders: an update. AB - We present an update of disorders in which aplasia cutis congenita is a feature. Localization of the lesion, important other features, and possible etiology are tabulated. Disorders are classified as chromosomal, monogenic, teratological/exogenous, and unknown. Points of particular interest in history taking and examination of patients with aplasia cutis congenita are presented. PMID- 7554363 TI - Problems arising in correlating clinical and molecular data in myotonic dystrophy. AB - The number of copies of CTG trinucleotide repeats in the myotonic dystrophy gene correlates to a certain degree with the clinical symptoms in the patient. Routine molecular analysis of myotonic dystrophy is performed on peripheral blood cells detecting the size of the expansion in leukocytes. However, in some cases somatic mosaicism is responsible for the presence of differently sized myotonic dystrophy alleles in different tissues of the same affected individual, complicating diagnosis and prognosis. Here we report two cases in which the correlation between molecular and clinical analysis performed with standard procedures posed some interpretative problems. The first individual was affected by an atypical clinical picture of myotonic dystrophy, the severity of which was not correlated with the low number of triplet repeats detected in his leukocyte DNA. The second case illustrates a prognostic problem in the presence of a low degree expansion in leukocytes. These examples outline the limits of standard molecular and clinical analysis in myotonic dystrophy. PMID- 7554364 TI - Identification of a breast tumor with microsatellite instability in a potential carrier of the hereditary non-polyposis colon cancer trait. AB - Allelic expansion at microsatellite loci in colorectal tumor DNA indicates a genomic instability caused by defects in DNA mismatch repair. This is observed in a high proportion of tumors from individuals affected by hereditary non-polyposis colorectal carcinoma, but to a lesser extent in sporadic colorectal tumors. In this study we screened 46 colorectal tumors for replication errors (RER). Tumors from six patients were found to be RER positive, two of which had a marked family history of colon cancer. In both cases the RER + phenotype was detected in colon tumors from other family members, suggesting a germline mutation in mismatch repair genes. Additionally, RER + phenotype, distinct from that of the colon and sporadic breast tumors, was found in malignant breast tissue from the mother of one proband. PMID- 7554365 TI - Retinoblastoma in association with the chromosome breakage syndromes Fanconi's anaemia and Bloom's syndrome: clinical and cytogenetic findings. AB - Two children presenting with sporadic unilateral retinoblastoma and exhibiting a high degree of chromosome breakage were noted to have unusual facies, microcephaly and abnormal skin pigmentation. In the first child the pattern of both spontaneous and mitomycin-C-induced chromosome breakage was characteristic of Fanconi's anaemia although the degree of breakage was extreme. She also exhibited a striking increase in X-ray-induced chromosomal damage in G0 lymphocytes as measured by dicentric formation and increase in chromatid-type aberrations. She had a number of typical clinical features, including cafe-au lait patches and abnormalities involving the kidney; however, she demonstrated neither the hypoplasia of radius and thumb nor the typical aplastic phase of this disorder. At age 22 months the child became anaemic with trilineage myelodysplasia, which was rapidly followed by the development of acute myeloblastic leukaemia. The early onset (at age 4 months) of retinoblastoma may have been associated with the underlying genomic instability. The second child exhibited a pattern of chromosome breakage characteristic of Bloom's syndrome, in addition to a moderate increase in damage induced by mytomycin-C. She had the typical stunted growth and malar hypoplasia of Bloom's syndrome although she did not demonstrate the frequently described erythematous 'butterfly rash' Although patients with Fanconi's anaemia and Bloom's syndrome are recognised to be at an increased risk of cancer, retinoblastoma has not previously been described in patients with either condition. We suggest that underlying recessive chromosome breakage syndromes may be underdiagnosed in paediatric cancer patients, with important implications for prognosis and genetic counselling. PMID- 7554366 TI - Detection of alpha-thalassemia-1 (Southeast Asian type) and its application for prenatal diagnosis. AB - A simple non-radioactive method based on the polymerase chain reaction was used to detect the Southeast Asian type of alpha-thalassemia 1 (--). Three oligonucleotide primers, one of which was adjacent to the breakpoint of the alpha thalassemia-1 allele, were used to amplify the 570 and 194 bp DNA fragments. The 570 bp product was specific to the alpha-thalassemia-1 determinant and the 194 bp fragment was amplified from either the alpha-thalassemia-2 (-alpha) or normal alpha-globin (alpha alpha) determinants. In Hb Bart's hydrops fetalis (--/--), only the 570 bp fragment was obtained, whereas the 194 bp fragment was amplified in normal individual (alpha alpha/alpha alpha) and alpha-thalassemia-2 trait ( alpha/alpha alpha). Both 570 and 194 bp fragments were detected in alpha thalassemia-1 trait (--/alpha alpha) and Hb H patients (--/-alpha). This procedure is useful for the rapid screening of alpha-thalassemia-1 trait and prenatal diagnosis of Hb Bart's hydrops fetalis in populations with a high frequency of the Southeast Asian Type of alpha-thalassemia-1. PMID- 7554367 TI - Constitutional heteromorphism of 9q13 --> q21 in a patient with chronic myelogenous leukemia. AB - Cytogenetic studies were carried out in a 44-year-old white male because of newly diagnosed chronic myelogenous leukemia. His initial bone marrow study revealed 46,XY, var(9)(q13 -->q21)/46,XY,var(9) (q13-->q21), t(9;22)(q34;q11) karyotypes and later he also acquired a 47,XY,+8,var(9)(q13-->q21), t(9;22)(q34;q11) clone. The var(9)(q13-->q21) heteromorphism was observed in the normal 9 homolog, in 200 GTG-banded bone marrow metaphases in seven cytogenetic studies (1988-90). This heteromorphism was observed in the normal cell line, in the two chronic myelogenous leukemia-related clones, as well as in 100 mitogen-induced peripheral blood lymphocytes, indicating its constitutional nature. This seems to be the first report of var(9)(q13 --> q21) heteromorphism, involving GTG-positive euchromatic band, in a chronic myelogenous leukemia proband. PMID- 7554368 TI - A further report of Brachmann-de Lange syndrome in two sibs with normal parents. AB - We report on a family in which a girl and a boy in the same sibship show variable manifestations of a less severe type of Brachmann-de Lange syndrome without significant prenatal growth deficiency and reduction deformities of the forearms. Both parents are healthy and phenotypically normal, and no other family members are affected. All the affected sibs except one described so far with normal parents presented the severe type of Brachmann-de Lange syndrome (now sometimes classified as type I: "classic" or "full" Brachmann-de Lange syndrome), with major upper limb anomalies, severe growth and mental retardation and, frequently, early death. We discuss the possible role of genomic imprinting in the etiology of this syndrome. PMID- 7554369 TI - Cystic fibrosis mutations and immotile cilia syndrome. AB - The immotile cilia syndrome (ICS) presents with autosomal recessive inheritance and is a chronic respiratory disease supposed to be caused by different genetic determinants. The hypothesis that cystic fibrosis (CF) heterozygotes may have a predisposition to develop bronchial or respiratory diseases other than CF prompted us to look for CF mutations in patients with ICS. Five patients, as well as the parents and two healthy brothers of one patient were tested for 12 CF mutations, for the polymorphic GATT repeat in intron 6a and for the CF gene flanking markers XV-2c, KM19, MP6d-9, J3.11. None of the 12 mutations at the CF locus have been detected in the ICS patients and no linkage was found between ICS and the polymorphic markers. Thus, based on our data, ICS and CF seem to be two different clinical entities. PMID- 7554371 TI - Normal testicular histology in a mid-trimester 49,XXXXY fetus. PMID- 7554370 TI - A TaqI polymorphism in a gene belonging to the human apoprotein(a)-plasminogen gene family on the telomeric region of chromosome 6 (6q26-27). PMID- 7554372 TI - Clinical follow up of a girl with "mental retardation with pterygia, shortness and distinct facial appearance" (Haspeslagh syndrome) PMID- 7554373 TI - Video display terminals: risk of trisomy 18? PMID- 7554374 TI - Anti-inflammatory agents in allergic diseases. PMID- 7554375 TI - Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis. AB - Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti neutrophil cytoplasm antibodies. The association of this antigen with specific disease entities requires that there is a source of pure antigen present in large quantities. Further delineation of the molecular mechanisms involved in the antigen-antibody interaction requires the ability to manipulate the molecule. The expression of recombinant MPO in Chinese hamster ovary cells has produced a source of pure protein, suitable for molecular studies. We have shown that this protein is an antigen recognized by 95% of anti-MPO antibodies from patients with systemic vasculitis. This recombinant molecule will be of use in providing an additional specific solid-phase assay for these antibodies and further forms of this protein which mirror the antigenicity of native MPO more exactly may replace chemically purified antigen. It will also be of great value in studies examining the epitopes recognized by anti-MPO antibodies and in studies of immunoregulation and T cell activation. PMID- 7554376 TI - HCV infection in patients with primary defects of immunoglobulin production. AB - We tested for infection with hepatitis C virus (HCV) in 58 patients affected by humoral immunodeficiencies: 43 common variable immunodeficiency (CVI), two hyper IgM syndrome (HIM), two IgG subclass deficiency, four ataxia-telangiectasia (AT), and seven X-linked agammaglobulinaemia (XLA). While the assessment of serum specific HCV antibodies in some of these patients was not informative because of the impairment in specific antibody production, the reverse transcriptase polymerase chain reaction (RT-PCR) assay used to detect serum HCV RNA was a useful method for diagnosing infection. We found that 38% of late onset hypogammaglobulinaemic patients (CVI, HIM or IgG subclass deficiency) had evidence of HCV infection. HCV infection was not detectable in patients with XLA or AT. The majority of our patients had persistent viraemia, and those who underwent liver biopsy showed histological findings of chronic hepatitis. Moreover, we could demonstrate in vitro that eight of 18 HCV-infected patients were actively producing anti-HCV antibodies, despite their impaired antibody production. The high rate of HCV infection in hypogammaglobulinaemic patients could be related to several nosocomial routes of transmission, including intravenous immune globulin administration. Despite the persistent viremia only two patients had cirrhosis and none had hepatocarcinoma. PMID- 7554377 TI - Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies. AB - Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (beta-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, beta-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, beta-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system. PMID- 7554378 TI - Immunization with anti-neutrophil cytoplasmic antibody (ANCA) induces the production of mouse ANCA and perivascular lymphocyte infiltration. AB - Wegener's granulomatosis (WG) is a granulomatous necrotizing vasculitis associated with the presence of ANCA, predominantly directed against proteinase 3 (PR3). The titres of ANCA correlate with disease activity and titre increases may precede disease exacerbations. Previously, we have shown that it is possible to induce autoimmune disease (systemic lupus erythematosus (SLE) and anti phospholipid syndrome) in naive mice following active immunization with human autoantibodies, namely anti-DNA and anti-cardiolipin, respectively. The mice developed first anti-autoantibodies and, after about 4 months anti-anti autoantibodies (Ab3), simulating auto-antibodies (Ab1) in their binding activities, and their presence was associated with the development of disease manifestations, characteristic of the human disease. So far, there is no good animal model for WG. In the current study we have immunized mice with human ANCA with the aim of inducing experimental WG. In two separate studies 30 mice were immunized in their footpads with autoantigen-purified IgG fraction (ANCA) from the sera of two patients with untreated WG, emulsified in Freund's complete adjuvant, followed 3 weeks later by ANCA injection in PBS. In the first experiment mice immunized with ANCA developed sterile microabscesses in the lungs after 8 months, and died after 8-15 months. In the second experiment, mice immunized with ANCA developed after 4 months mouse ANCA, with specificity both to PR3 and to myeloperoxidase, as well as anti-endothelial autoantibodies (AECA), as shown by radioimmunoprecipitation. Pathologically, the immunized mice developed proteinuria but not haematuria, and histological sections of the lungs demonstrated mononuclear perivascular infiltration, while diffuse granular deposition of immunoglobulins was noted in the kidneys. Our results point to a pathogenic role of ANCA in WG, and confirm the importance of the idiotypic network in the etiopathogenesis of autoimmune conditions. PMID- 7554379 TI - Anti-M4 antibodies measured by a sulphite oxidase ELISA in patients with both anti-centromere and anti-M2 antibodies. AB - In this study the clinical features of patients with a serological overlap between scleroderma and primary biliary cirrhosis (PBC) were analysed. The entity was defined by the presence of both anti-centromere antibody (ACA) and anti mitochondrial antibodies to the M2 antigen, pyruvate dehydrogenase. In addition the sera were assayed for anti-mitochondrial antibodies to the M4 antigen measured by an ELISA to sulphite oxidase (SO). First, anti-M2 was detected, not only in 58 out of 60 patients with PBC but also in eight out of 61 patients with ACA. These sera, together with sera from 53 normals and 99 from patients with various connective tissue diseases were then evaluated for anti-SO, which has been proposed by Klein and Berg to be a marker of progressive liver disease. Again, a high proportion (62%) of sera from patients with PBC were positive for anti-SO, and three of the eight patients who had ACA and anti-M2 also reacted with SO. We subsequently identified and included for study a further 10 patients positive for ACA and anti-M2, making a total of 18 patients with this profile. Features of limited cutaneous scleroderma were present in 94% and evidence of liver disease in 56%. Eight out of the 18 patients had anti-SO, and of these four had PBC, two had abnormal biochemical liver function tests but two had no evidence of liver disease. These data confirm that detection of anti-SO is limited to an anti-M2 subpopulation, and may be a marker for liver involvement with prognostic significance in scleroderma patients with ACA. PMID- 7554380 TI - Rabbit polymorphonuclear granulocyte function during ethanol administration- migration and oxidative responses in a joint with immune complex synovitis. AB - Functional impairments of polymorphonuclear granulocytes (PMN) are believed to contribute to hampered inflammation and host defence in alcoholics. We studied effects of i.v. ethanol administration on PMN responses in rabbits during induction of a knee-joint synovitis. The synovitis conferred systemic effects, since chemiluminescent responses of peripheral blood PMN to opsonized zymosan and phorpbol myristate acetate (PMA) increased 6.4- and 17.9-fold, respectively. Chemiluminescent responses of synovial PMN were further amplified. This up regulation was reduced to 33% in rabbits treated with ethanol when opsonized zymosan was used as the PMN stimulus; in contrast, PMA responses were unaffected. The appearance and migration of PMN to the synovitis joint were normal despite a blood ethanol concentration of 0.5%. Thus, ethanol impaired release of oxygen metabolites from PMN, but not the delivery of cells at an inflammatory site. PMID- 7554381 TI - Chloroquine inhibits T cell proliferation by interfering with IL-2 production and responsiveness. AB - Chloroquine (Chl) is an anti-rheumatic drug that is widely used in the treatment of rheumatoid arthritis (RA). It seems that T cells are important in the pathogenesis of RA, but it is not known whether Chl acts via inhibition of T cell function. We here present evidence that Chl, just like cyclosporine A (CsA), inhibits T cell proliferation as induced with immobilized alpha CD3 MoAb in a concentration-dependent manner, at least partly through interfering with the production of IL-2 protein and the induction of IL-2 mRNA. Furthermore, Chl impedes the responsiveness of T cell clones to IL-2 since (1) the inhibition of alpha CD3 MoAb-induced proliferation by Chl could not be reversed by rIL-2 and (2) Chl directly blocks IL-2-driven proliferation of cloned T cells. Chl appeared to interfere with the internalization (50% inhibition) and degradation (total blockade) of rIL-2. Finally, the combination of Chl and CsA synergistically inhibited T cell proliferation. We conclude that Chl may inhibit functional properties of human T cells, although the drug is 100- to 1000-fold less potent than CsA in inhibiting T cell proliferation and IL-2 production, respectively. It is speculated that the in vitro effects of Chl might be relevant in explaining the anti-rheumatic effect of this drug in patients with RA. PMID- 7554382 TI - HLA-DR-restricted T cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: lack of reactivity to glutamic acid decarboxylase. AB - T cells reacting with pancreatic islet beta cell proteins play a pivotal role in the pathogenesis of type 1 diabetes in experimental animal models and man, although the islet cell autoantigens against which these T cells are directed remain to be characterized. We have previously shown the presence of disease related antigens residing in the transplantable RIN insulinoma membranes which are recognized by T cells from diabetic NOD mice. We now report on the establishment of CD4+, T cell lines reacting with insulinoma membranes from six newly diagnosed type 1 diabetic patients. Detailed examination of T cell lines from two patients revealed that both the lines continued to react with normal islet cell proteins and, interestingly, were also stimulated by antigens present in brain microsomes. The two T cell lines showed reactivity with different molecular weight proteins of the insulinoma membranes and both the lines were histocompatibility-linked antigen (HLA)-DR restricted. Although the insulinoma membrane preparation is known to contain glutamic acid decarboxylase (GAD), none of the six T cell lines proliferates in response to purified GAD. These T cell lines will be valuable in characterizing novel islet beta cell antigens which are likely to be implicated in type 1 diabetes. PMID- 7554383 TI - Origin of CD57+ T cells which increase at tumour sites in patients with colorectal cancer. AB - Human T cells carrying natural killer (NK) markers, CD57 or CD56 antigens, appear to be distinguishable from other T cell subsets in terms of their granular lymphocyte morphology and their numerical increase in patients with AIDS and in recipients of bone marrow transplantation. At the beginning of this study, we observed that CD57+ T cells as well as CD56+ T cells were abundant at tumour sites in many patients with colorectal cancer. Since all these findings for CD57+ T cells are quite similar to those of extrathymic T cells seen in mice, we investigated how CD57+ T cells are distributed to various immune organs in humans. They were found to be present mainly in the bone marrow and liver, but to be completely absent in the thymus. Similar to the case of extrathymic T cells in mice, they were observed to consist of double-negative CD4-8- subsets as well as single-positive subsets (preponderance of CD8+ cells), and to contain a considerable proportion of gamma delta T cells. These features are striking when compared with those of CD57- T cells, which are characterized by an abundance of CD4+ subsets and alpha beta T cells. Not only at tumour sites but also in the peripheral blood, some patients with colorectal cancer displayed elevated levels of CD57+ cells. These results suggest that CD57+ T cells may be of extrathymic origin, possibly originating in the bone marrow and liver, and may be associated with tumour immunity, similar to another extrathymic population of CD56+ T cells in humans. PMID- 7554384 TI - Localization of T cell receptor (TCR)-gamma delta + T cells into human colorectal cancer: flow cytometric analysis of TCR-gamma delta expression in tumour infiltrating lymphocytes. AB - We analysed TCR-gamma delta expression in tumour-infiltrating lymphocytes (TIL) obtained from 13 patients with colorectal cancer and simultaneously isolated the T lymphocytes from normal intestinal tissue (IL) to compare the frequencies of TCR-gamma delta expression in TIL, IL, and peripheral blood lymphocytes (PBL) in the same patient. Flow cytometric analysis showed that the frequency of TCR-gamma delta expression in TIL (2.75 +/- 1.84%) was significantly lower than that in IL (15.28 +/- 9.45%, P < 0.01). However, a larger quantity of TIL was separated than IL per unit weight of specimen, so the total number of gamma delta T cells obtained per unit weight was not different between tumour tissue and normal intestine. In addition, phenotypic analysis revealed that about half of the TCR gamma delta + TIL were CD8+ (CD4+, 3.0 +/- 3.1%; CD8+, 54.7 +/- 19.9%, mean +/- s.d. of five patients), and a very similar result was obtained in TCR-gamma delta + IL (CD4+, 2.7 +/- 2.4%; CD8+, 53.1 +/- 17.4%). In contrast, most TCR-gamma delta + PBL were double-negative (CD4+, 3.2 +/- 3.0%; CD8+, 20.6 +/- 7.4%). These results indicated that TCR-gamma delta + CD8+ T cells selectively and consistently localized in colorectal tumour tissue, similarly to normal intestinal epithelium. PMID- 7554385 TI - B cells from a distinct subset of patients with common variable immunodeficiency (CVID) have increased CD95 (Apo-1/fas), diminished CD38 expression, and undergo enhanced apoptosis. AB - We investigated the role of apoptosis in the differentiation failure of B cells from a selected subpopulation of patients with CVID delineated by B cell surface marker analysis, in vitro IgE response, and molecular markers of B cell VH gene repertoire. These patients had altered display of B cell surface molecules that play a role in apoptosis. The patients' B cells had a 4.5-250-fold increase in CD95 (Apo-1, fas) expression and increased CD95 display on their T cells. CD38, a molecule important in preventing germinal centre B cell apoptosis, was reduced on the patients' B cells. The expression of this molecule was inducible on the CVID lymphocytes with retinoic acid. Increased spontaneous apoptosis in vitro was observed with the patients' B (23%) and T cells (10%) compared with normal cells (13% and 3%, respectively). Stimulation in vitro with IL-4 and CD40 rescued the B cells from apoptosis and allowed for their differentiation. However, IL-4 plus alpha CD40-driven immunoglobulin production was not quantitatively or qualitatively normal. Failure to overcome apoptosis, a normal step in germinal centre B cell development, may be involved in the lack of differentiation seen in this subset of CVID patients. PMID- 7554386 TI - Autoantibodies against oxidized low density lipoproteins (oxLDL): characterization of antibody isotype, subclass, affinity and effect on the macrophage uptake of oxLDL. AB - Circulating antibodies against oxLDL are present in several inflammatory and autoimmune conditions. Such antibodies are also present in patients with atherosclerosis, although the pathogenic significance of the antibodies is still not known. We have characterized the antibodies with regard to isotype, subclass, affinity and effect on macrophage uptake of oxLDL. Antibodies of IgG and IgM isotype were most common and found both in patients with atherosclerosis and in normal individuals. The subclass of IgG antibodies was mainly IgG2 and IgG3. Scatchard analyses of IgG and IgM antibodies showed that IgG antibodies were heterogeneous with regard to affinity, whereas only one population of high affinity antibodies was found in the IgM antibody population. The high-affinity populations had an average equilibrium constant (K0) of 8.84 x 10(9) M-1 for IgG antibodies and of 1.65 x 10(9) M-1 for IgM antibodies. Incubation of 125I-oxLDL with purified IgG and IgM from sera with high amounts of antibodies enhanced the uptake of 125I-oxLDL in the monocyte-like U937 cell line. Antibody preparations from sera containing no anti-oxLDL antibodies and from sera with antibodies against LDL had less effect on this uptake. The increased uptake was competitively decreased by adding unlabelled oxLDL. This study shows that antibodies against oxLDL are mainly IgG2, IgG3 and IgM. Both IgG and IgM antibodies have a high affinity for the antigen and increase the uptake of oxLDL in a monocyte-like cell line. PMID- 7554387 TI - Progressive renal lesions induced by administration of monoclonal antibody 1-22-3 to unilaterally nephrectomized rats. AB - A new animal model of progressive glomerulosclerosis was developed by administering a single i.v., injection of MoAb 1-22-3 to unilaterally nephrectomized rats. Renal morphological analysis revealed that glomerular lesions characterized by mesangial cell proliferation and mesangial matrix expansion were induced in about 95% of the glomeruli. Approximately 20% of the glomeruli of the unilaterally nephrectomized rats showed sclerosis or segmental sclerosis by week 6 after MoAb injection and crescent formation was observed in some glomeruli (ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor-beta (TGF-beta) was observed in the unilaterally nephrectomized rats treated with MoAb 1-22-3, but we could not demonstrate pathological involvement of platelet-derived growth factor (PDGF), even though early-stage mesangial cell proliferation was observed. The mechanism of mesangial cell proliferation in this model remains to be elucidated. The relatively short period of time needed to induce the sclerotic changes in considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis. PMID- 7554388 TI - Beneficial effect of amrinone on murine cardiac allograft survival. AB - Amrinone is a non-glycoside positive inotropic agent with an inhibitory effect on a cyclic adenosine monophosphate (AMP) phosphodiesterase isoenzyme. In the present study, we examined the immunosuppressive action of amrinone, since several other cyclic AMP-elevating agents have been shown to suppress T lymphocyte activation. First, the in vivo effects of amrinone were investigated. Oral amrinone treatment, at 40 mg/kg per day, significantly prolonged median cardiac allograft survival compared with non-treated controls (22.0 days versus 10.5 days, P < 0.01) when DBA/2 mouse hearts (H-2d) were heterotopically transplanted into C57B1/6 mice (H-2b). Histopathological examination showed that there was less prominent cellular infiltration in the amrinone-treated than in the non-treated allografts. Plasma amrinone concentrations of mice after a single oral dose of 40 mg/kg were within the range of clinical relevance. To clarify the mechanism of action, in vitro studies were done. The generation of specific cytotoxic T lymphocytes after mixed lymphocyte culture was significantly suppressed by addition of amrinone to the culture medium at 5 micrograms/ml. The production of IL-2 and the interferon-gamma during mixed lymphocyte culture was also suppressed by amrinone at 5 micrograms/ml. However, the level of intracellular cyclic AMP in mouse splenic lymphocytes was not affected significantly by the same dose of amrinone. In conclusion, amrinone has immunosuppressive actions at the therapeutic doses, and it may be a beneficial agent for therapy against acute cardiac allograft rejection. PMID- 7554389 TI - Signalling via CD28 of human naive neonatal T lymphocytes. AB - Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate. PMID- 7554390 TI - Glycosylphosphatidylinositol (GPI)-anchored surface antigens in the allogeneic activation of T cells. AB - GPI-linked surface molecules have recently been described as structures with an activation potential for human T lymphocytes. To study the role of these molecules in T cell activation we analysed GPI-deficient or normal T cells from patients with paroxysmal nocturnal haemoglobinuria (PNH). On activation with allogeneic Epstein-Barr virus (EBV)-transformed B cell lines GPI-deficient freshly separated T cells or continuously growing T cell lines exhibited a significantly lower proliferation or cytokine production compared with their normal counterparts. In contrast, stimulation via the T cell receptor-associated CD3 structure resulted in a comparable response. There was no difference in activation of normal T lymphocytes when GPI-deficient B cells were used as stimulators compared with normal B cells obtained from the same PNH patient. We conclude from these data that GPI deficiency in PNH leads to a functional deficiency of GPI-deficient T cells. In contrast, no difference in activation of T lymphocytes for GPI-deficient cells on the stimulator cell level was observed. PMID- 7554391 TI - Delineation of producing ability of IgG and IgA subclasses by naive B cells in newborn infants and adult individuals. AB - Neonatal B cells with the naive (sIgD+) phenotype are able to generate IgG- and IgA-producing cells as well as IgM production in the presence of memory CD4+ T cells expressing L-selectin (CD62L) in pokeweed mitogen-stimulated cultures. We used this system to examine comparatively the ability of naive B cells to produce IgG and IgA subclasses in newborn infants and adult individuals. Naive B cells were enriched from both donors on the basis of sIgD positivity, and memory (CD45RO+) CD4+ T cells with CD62L expression were isolated from adults. We here demonstrate some differences in profiles of IgG and IgA subclass production between neonatal and adult naive B cells. In neonatal B cells, IgG1 and IgG3 were predominantly produced, but IgG2 and IgG4 production was virtually absent. Similar to neonatal B cells, adult naive B cells produced mainly IgG1 and IgG3, although memory (sIgD-) B cells from adults secreted all of the IgG subclasses. It should be noted that low but detectable levels of IgG2 and IgG4 were found in adults' naive B cell cultures. Although IgA produced by neonatal B cells was exclusively IgA1, IgA2-secreting cells were identifiable in adult naive B cells. The results suggest that further class switch of naive B cells to IgG2, IgG4 and IgA2 in addition to IgG1 and IgG3 may be controlled by their own age-dependent maturation process. PMID- 7554392 TI - The effect on immunity of long-term intensive training in elite swimmers. AB - The impact of long-term training on systemic and mucosal immunity was assessed prospectively in a cohort of elite swimmers over a 7-month training season in preparation for national championships. The results indicated significant suppression (P < 0.05) of serum IgA, IgG and IgM and salivary IgA concentration in athletes associated with long-term training at an intensive level. There was also a trend towards lower IgG2 subclass levels in serum in athletes compared with controls (P = 0.07). There were no significant changes in numbers or percentages of B or T cell subsets, but there was a significant fall in natural killer (NK) cell numbers and percentages in athletes over the training season (P < 0.05). After individual training sessions there was a significant decrease in salivary IgA levels for athletes compared with controls (P = 0.002). In athletes there was a downward trend in salivary IgA levels over the 7-month training period in both the pre-exercise (P = 0.06) and post-exercise samples (P = 0.04). There were no significant trends in salivary IgG levels over the study period in either athletes or controls. The only significant change in salivary IgM levels was an increase in detection rate in the pre-competition phase in athletes (P = 0.03). The study suggests that training of elite athletes at an intensive level over both short- and long-time frames suppresses both systemic and mucosal immunity. Protracted immune suppression linked with prolonged training may determine susceptibility to infection, particularly at times of major competitions. PMID- 7554393 TI - The acute phase protein response in patients receiving subcutaneous IL-6. AB - IL-6, tumour necrosis factor-alpha (TNF-alpha) and IL-1 are thought to be the key mediators of the acute phase response although much of the evidence is based on in vitro studies. It is not clear to what extent each of the acute phase proteins are regulated in vivo by each of these cytokines. The aim of this study was to examine the effects of IL-6 treatment in eight patients with cancer on the concentrations of an extensive range of positive and negative acute phase proteins. It was part of a larger investigation to assess the value of IL-6 in the management of chemotherapy-induced thrombocytopenia. IL-6 was administered by a daily subcutaneous injection for 7 days at a dose level of 1, 3, or 10 micrograms/kg/day. Increases in the positive acute phase proteins, serum amyloid A, C-reactive protein, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, haptoglobin, alpha 1-antitrypsin, fibrinogen, complement component C3, and caeruloplasmin, were observed, with the greatest incremental changes and fastest responses being seen for C-reactive protein and serum amyloid A protein. The negative acute phase proteins transferrin, transthyretin and retinol binding protein all fell to a nadir within 48-96 h after the first IL-6 injection. Increases in complement component C4 were only found in two patients, which may be related to the increase in circulating TNF-alpha concentrations found only in these patients. This study has therefore shown that IL-6 is capable of causing changes in the majority of acute phase proteins in vivo. Although secondary induction of TNF-alpha was not observed in the majority of patients examined, it is still possible however that other cytokines involved in regulation of the acute phase response, such as IL-1, may have been induced and contributed to the overall response. PMID- 7554394 TI - Genetic variation in neutrophil accumulation in mice is not mediated through immigrant regulatory cells. AB - Genetic variation of induced peritoneal neutrophilia in mice was accompanied by parallel variation in macrophage responses. The timing of the macrophage responses in high responder (C57B1/10) mice indicated a potential role for these cells in mediating the enhanced neutrophil response. However, adoptive transfer of inflammatory macrophages did not induce neutrophilia. Analysis of peritoneal cytokine levels in high and low responder mice further indicated that IL-1, IL-3, GM-CSF, G-CSF and interferon-gamma (IFN-gamma) were not involved in mediating the genetic variation observed. Exogenous tumour necrosis factor-alpha (TNF-alpha) was effective in inducing the high responder phenotype, despite the absence of detectable TNF-alpha in either peritoneal fluid or serum. A role for genetically determined differential expression of endothelial adhesion molecules in high and low responders is suggested. PMID- 7554395 TI - In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection. AB - We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection. PMID- 7554396 TI - Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen stimulated peripheral blood lymphocytes from HIV-seropositive patients. AB - Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis. PMID- 7554397 TI - Demonstration of virus-specific CD8+ memory T cells in measles-seropositive individuals by in vitro peptide stimulation. AB - CD8+ cytotoxic T lymphocytes (CTL) in measles virus infection have been difficult to investigate due to the strong immunosuppressive effects exhibited by infectious measles virus in vitro. In order to circumvent immunosuppression we used predicted peptide epitopes to induce measles virus-specific CTL. This was done by screening the structural proteins of measles virus for HLA-A2.1 peptide binding motifs with valine in position 2 and leucine in position 9. Synthetic peptides np210-218, np226-234, and np340-348 from the nucleoprotein, peptide hp29 37 from the haemagglutinin protein, and peptide pp 519-527 from the polymerase protein were synthesized and used to expand measles virus-specific CD8+ CTL in vitro. Induction of CTL with synthetic peptides was restricted to HLA-A2-positive peripheral blood mononuclear cells (PBMC) from measles-seropositive individuals. We conclude that this method is a useful tool to demonstrate memory CD8+ CTL in measles-seropositive adults and to evaluate the role of structural proteins in CTL responses against measles. PMID- 7554398 TI - Antigen presentation by naive macrophages, dendritic cells and B cells to primed T lymphocytes and their cytokine production following exposure to immunostimulating complexes. AB - Influenza virus envelope proteins incorporated into immunostimulating complexes (iscoms) are taken up and processed by various kinds of antigen-presenting cells (APC), encompassing peritoneal cells (PEC), unfractionated splenocytes, splenic dendritic cells (DC) or B cells. The iscom-pulsed naive APC stimulated primed T cells to proliferate and produce cytokine in vitro. In contrast, only DC and B cells pulsed with the same antigen (Ag) in the micelle form functioned as accessory cells stimulating the primed T cells to proliferate and produce cytokine. In general, iscoms were better inducers of cell proliferation than micelles. Iscoms stimulated more secretion of IL-2 and interferon-gamma (IFN gamma) than the micelles, but both antigenic forms stimulated secretion of IL-4. DC and B cells pulsed with iscoms stimulated most efficiently the secretion of IL 2 and IFN-gamma. DC were superior to the other APC in stimulating primed T cells to secrete IFN-gamma. On the other hand, micelles stimulated more efficiently than iscoms splenic T cells from micelle-primed as well as iscom-primed mice to secrete IL-10. These data indicate that influenza virus envelope proteins incorporated in iscoms stimulate a broad T cell response, possibly emphasizing a Th1 type of response. The same Ag in a micelle form induce a more prominent Th2 type of T cell response. The results indicate that the administration of an Ag in an adjuvant formulation can superimpose a different cytokine profile on the immune response than that induced by the protein Ag alone. PMID- 7554399 TI - Human T cell responses to peptide epitopes of the 16-kD antigen in tuberculosis. AB - The 16-kD protein constituent of the Mycobacterium tuberculosis complex has been known mainly for its prominent serological immunogenicity and species specificity in tuberculous infection. In this study, we evaluated the T cell immune repertoire in 27 sensitized healthy subjects and 46 patients with active tuberculosis using 14 overlapping 20mer peptides spanning the entire sequence of this protein. Four of the tested peptides individually stimulated proliferation of blood mononuclear cells from more than 50% of healthy controls. Tuberculosis patients reacted to a narrower peptide range and with a 17-27% lower rate of responses to the four most immunogenic peptides, but these differences do not distinguish in any simple way between the T cell repertoire of patients and sensitized healthy subjects. The most immunogenic peptide (91-110) was recognized by 67% of healthy subjects and by 50% of tuberculosis patients. Importantly, several non-responders to this peptide were stimulated with the other three most permissive peptides with sequences of 111-130, 71-91 and 21-40, resulting in an overall response rate to at least one of these four peptides of 93% in healthy controls and 74% in tuberculosis patients. In view of this additive effect between the most immunogenic peptides, their combined use may achieve sufficient sensitivity in a test aimed at the specific discrimination between infected and non-infected healthy subjects. The major interest in testing with these peptides rests in their species specificity, which is not achieved using purified protein derivative (PPD). PMID- 7554400 TI - T cell clones from a non-leprosy exposed subject recognize the Mycobacterium leprae 18-kD protein. AB - Although Mycobacterium leprae shares many protein antigens with other mycobacterial species, there is a degree of specificity in the T cell response to the organism. This is evident in the failure of cross-protection between mycobacterial species and the specific unresponsiveness to M. leprae in lepromatous leprosy patients. The antigenic basis of this specificity is unresolved, but the M. leprae 18-kD protein is one candidate because of its restricted distribution and the isolation of M. leprae-specific T cell clones reactive with the protein from M. leprae-vaccinated subjects. In the course of analysing the human T cell repertoire to mycobacteria we have isolated further CD4+ T cell clones reactive with this protein from a subject who had never been exposed to M. leprae. These clones did not respond to other mycobacteria, including M. tuberculosis and M. bovis (BCG). In addition, they were unreactive with the M. tuberculosis 16-kD protein which has recently been shown to have limited amino acid identity with the M. leprae 18-kD protein. Both clones reacted with peptide 38-50 from the M. leprae 18-kD protein, the T cell response to which is restricted by HLA-DR4. Although homologues for the gene encoding the M. leprae 18-kD antigen have been identified in M. avium and M. intracellulare, the clones failed to respond to preparations of M. avium. Both clones secreted interferon gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) and were cytolytic against autologous targets pulsed with peptide 38-50 or the 18-kD protein. The nature of the antigen which stimulates this apparently 'M. leprae-specific' response is unknown. Nevertheless the recognition of the 18-kD protein by individuals not exposed to leprosy indicates that this protein may not be suitable as a reagent to distinguish between infection with M. leprae and other pathogenic mycobacteria. PMID- 7554401 TI - Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID). AB - Cartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-gamma) synthesis and IL-2 receptor alpha-chain (IL-2R alpha) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2R alpha, IL-2 and IFN-gamma were markedly decreased in patient lymphocytes stimulated with PMA+ionomycin as compared to control lymphocytes. The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c-fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes. PMID- 7554402 TI - Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum. AB - The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs. Little is known about the interaction of BAM and Hc, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis. A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-activated BAM. Fungistatic activity of BAM was determined with a 24-h assay. A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used. Activated BAM killed Hc (reduced inoculum CFU by 25 +/- 12%; n = 4). The fungicidal activity of BAM was abrogated by 0.2 mM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase. In fungistatic assays activated BAM inhibited multiplication of Hc by 61 +/- 4% (n = 3) compared with cocultures with control BAM. However, Hc multiplied 100% more in control BAM cocultures than in medium alone. Data indicated that this was due to advantages that Hc has in the intracellular environment. Only NMMA inhibited fungistatic activity of activated BAM. In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained. In conclusion, activated BAM and PM kill yeast cells of Hc by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway. PMID- 7554403 TI - Cysteine protease of the nematode Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1 antibody response in rats. AB - Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pI 8.5, showed maximum enzyme activity at pH 5.5 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64, completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis, a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and IgG1, production of anti cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response. PMID- 7554404 TI - Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium. AB - TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10( 3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions. PMID- 7554406 TI - Soluble intercellular adhesion molecule-1 (ICAM-1) in sera and bronchoalveolar lavage (BAL) fluids of extrinsic allergic alveolitis. AB - ICAM-1 plays an important role in inflammatory diseases. We analysed ICAM-1 expression on BAL fluid cells and measured soluble ICAM-1 (sICAM-1) concentrations in sera and BAL fluids from patients with extrinsic allergic alveolitis (EAA). We found significantly increased cellular ICAM-1 expression on BAL fluid lymphocytes and alveolar macrophages, and significantly increased values of circulating and BAL fluid sICAM-1 in EAA patients compared with controls. Successive measurement showed prompt decrease of both sICAM-1 values in EAA patients during periods when antigen exposure was prevented. In BAL fluids from EAA patients, sICAM-1 values significantly correlated to neutrophil and ICAM 1+ lymphocyte counts. In EAA patients, circulating and BAL fluid sICAM-1 values has significant negative correlations to values of carbon monoxide diffusing capacity and to time intervals between last episode and sample collection. However, these values had significant positive correlation to values of alveolar arterial oxygen pressure difference. In EAA, antigen exposure appears to induce cellular ICAM-1 expression on BAL fluid cells, and also appears to up-regulate shedding of ICAM-1 in the alveolar lining fluid and in the circulation. The sICAM values appear to reflect disease activity of EAA. PMID- 7554405 TI - Potentiation of in vitro synthesis of human IgE by cyclosporin A (CsA). AB - In this study, we investigated the modulatory effects of CsA on in vitro synthesis of IgE, IgG1 and IgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive effect, we have demonstrated that a low dose of CsA (10(-7) M, 120 ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33 +/- 4.5 to 1346 +/- 290 ng/ml). This potentiation was specific for IgE since no such effect was demonstrable with IgG1 and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell depleted, anti-CD40-stimulated culture (four-fold increase from 400 +/- 48 to 1606 +/- 127 ng/ml). Our data therefore suggest that there are at least two mechanisms for CsA-induced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mediated diseases. PMID- 7554408 TI - 3rd Congress of Federation of Asian and Oceanian Physiological Societies. Shanghai, China, 7-10 November 1994. Abstracts. PMID- 7554410 TI - Deleterious effects of salt intake other than effects on blood pressure. AB - 1. Salt intake is not only known to play an important role in determining blood pressure (BP) but has been shown to have other deleterious effects independent of BP. 2. Epidemiological and animal studies have provided evidence that salt intake may have an adverse effect on stroke mortality independent of BP. 3. Significant correlation between sodium excretion (as a measure of salt intake) and left ventricular (LV) hypertrophy has been shown in many clinical studies. Salt restriction has also been found to produce a significant reduction in LV mass. 4. In animal studies, salt restriction in uninephrectomized spontaneously hypertensive rats retarded renal glomerular injury and suppressed compensatory growth independent of hypertension. Moreover, a high sodium diet accelerated cerebral arterial disease even when no increases in BP could be detected. 5. Epidemiological data have shown an association between asthma mortality and regional purchases of table salt. Furthermore, dietary salt restriction in asthmatic patients results in improvement of symptomatology with lower consumption of bronchodilators. 6. Patients with essential hypertension are known to have increased urinary calcium excretion, and hypertension may be one factor that may increase the likelihood of osteoporosis. High salt intake is also associated with increased hydroxyproline excretion indicating increased resorption of bone. Sodium restriction reduces calcium excretion and may reduce bone demineralization and hip fractures in a similar manner to that seen with diuretics. PMID- 7554409 TI - Left ventricular function following thrombolytic therapy for myocardial infarction. AB - 1. The aim of this study was to evaluate the utility of measurement of left ventricular function in assessing the efficacy of thrombolytic agents. 2. All published studies were reviewed. 3. The major effect of the introduction of thrombolytic therapy on mortality after myocardial infarction has been a dramatic decrease in the number of patients dying from cardiac failure. In the thrombolytic era, left ventricular function has remained the most important prognostic factor after recovery from acute myocardial infarction. There are three trials with the statistical power to evaluate left ventricular function, where both left ventricular function and survival were improved compared to placebo or control treatment. The recent Global Utility of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO) Trial supports these findings, with left ventricular function being strongly correlated with mortality reduction. Left ventricular function, measured at 90 min either as ejection fraction, end-systolic volume or infarct zone contractility, closely correlated with 30 day mortality, P < 0.01. 4. Left ventricular function remains an important factor in the evaluation of the efficacy of different thrombolytic and adjuvant regimens. PMID- 7554411 TI - Alcohol and hypertension. AB - 1. Regular alcohol consumption is a significant contributor to the prevalence of hypertension in drinking communities. 2. The effect is additive to that of obesity and is partly reversible over 2-4 weeks with moderation of ethanol intake. 3. In heavy drinkers acute alcohol withdrawal may lead to more blood pressure elevation following an initial depressor response. 4. Heavy drinking is also associated with an increased risk of haemorrhagic stroke and cardiomyopathy. 5. Lighter drinking habits appear to offer significant protection against ischaemic heart deaths and ischaemic strokes. 6. Antihypertensive drug treatment for alcohol related hypertension may mask some of the adverse cardiovascular effects of alcohol. 7. Arguments as to whether alcohol is a cause of essential hypertension are tautological, given the many reversible lifestyle factors now known to contribute to the rise in blood pressure with aging. PMID- 7554407 TI - Endothelial cells and renal epithelial cells do not express the Wegener's autoantigen, proteinase 3. AB - Proteinase 3 (PR3) is the major antigen for autoantibodies (C-ANCA) against cytoplasmic components of neutrophils which are strongly associated with Wegener's granulomatosis (WG). Recent data that PR3 may be expressed by renal tubular epithelial cells and endothelial cells suggest potential for a direct pathogenic effect against these cells by C-ANCA or cytoxic T lymphocytes. Using a semi-quantitative polymerase chain reaction (PCR), ELISA and indirect immunofluorescence staining we studied endothelial and epithelial cell PR3 expression. By PCR, no PR3 expression was found in human umbilical vein endothelial cells (HUVEC) either untreated, or when treated with interferon-gamma (IFN-gamma) (200 U/ml, 6 h, 24 h), IL-1 (20 U/ml, 6 h), tumour necrosis factor alpha, (TNF-alpha) (200 U/ml, 0, 1, 2, 4, 6 h) or IFN-gamma + TNF-alpha (6 h); iliac vein and artery endothelial cells did not express PR3 either. In contrast, PR3 was detected in HL60 cells and neutrophils by PCR, expression being confirmed by sequence analysis. Three PR3 MoAbs showed no binding to unstimulated or TNF alpha-stimulated HUVEC either by ELISA or by indirect immunofluorescence staining. The epithelial cell line A549 expressed PR3 when assayed by PCR. However, three renal epithelial cell lines (two tubular and one glomerular) showed little or no PR3 expression by PCR or ELISA. These studies fail to demonstrate evidence for PR3 expression by endothelial cells, even when using the highly sensitive PCR assay. Whilst PR3 expression by A549 cells is intriguing, the relevance of this in the pathology of WG is doubtful considering the negligible expression by renal epithelial cell lines. PMID- 7554412 TI - Alcohol, hypertension, coronary disease and stroke. AB - 1. The association between alcohol and hypertension has been recognized for several years. 2. However, it remains a paradox that if alcohol does cause hypertension there is little convincing evidence that alcohol is related to the cardiovascular complications of hypertension such as strokes and heart attacks. The relationship between alcohol and strokes remains inconclusive and there is evidence that moderate alcohol consumption may be protective against heart attacks. 3. It is possible, therefore, that alcohol does not so much cause hypertension, but rather a rapidly reversible rise in blood pressure which does not cause cardiovascular damage. When managing hypertensive patients, however, relevant counselling can bring about a useful fall in blood pressure. PMID- 7554414 TI - Can we recommend fish oil for hypertension? AB - 1. The ability of the n-3 fatty acids in fish oil to lower blood pressure has been established. Dietary fish oil supplementation is effective in mild hypertension and, in certain cases, as an adjunct therapy in drug-treated hypertension. Efficacy may be enhanced by restricting sodium intake. 2. The overall benefit of fish oil in hypertension, however, has not yet been fully evaluated. We still need further information on the relative efficacy of individual omega-3 fatty acids and on additional cardiovascular benefits and possible disadvantages of increasing their consumption. PMID- 7554415 TI - Recent insights into dietary fats and cardiovascular disease. AB - 1. Fat deposition in the arterial intima is fundamental to the atheroma process. Circulating lipoproteins are thought to be the source of much of the deposited fat. The interplay of dietary fat has not been fully clarified. 2. Observational studies have furnished evidence of relationships between the different dietary fats and clinical cardiovascular events. In these, total fat and in particular, saturated fat appear culprit. Mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fats have less consistent relationships with cardiovascular disease, though all classes of fatty acid are found in atheroma. 3. Comparing the effects on lipoproteins of saturates, mono-unsaturates and polyunsaturates, they all increase high density lipoproteins (HDL) and reduce triglycerides when substituted isocalorically for carbohydrate. Saturates increase low density lipoproteins (LDL), while PUFA > MUFA reduce LDL. 4. Upon oxidative modification, lipoproteins are more liable to arterial deposition and, in vitro at least, LDL oxidizability is enhanced by enrichment with PUFA. 5. Trans-MUFA have some unique properties in that they somewhat resemble saturates and seem to predispose to coronary disease, quite possibly because of their adverse effects on LDL, HDL and Lp(a) levels. 6. omega-3 fatty acids seem unique among the dietary fats in that they inhibit thrombosis and platelet aggregation, and can lower blood pressure. 7. The net effect of these various potential influences of fatty acids on atherogenesis in vivo is unclear. It may well be that all fats, with the exception of the omega-3 class, are detrimental with response to atherogenesis. Modification of the diet, with particular attention to fat, has been demonstrated to reduce clinical coronary events in several studies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554413 TI - Vegetarian diet components, protein and blood pressure: which nutrients are important? AB - 1. Evidence that vegetarian dietary patterns lower blood pressure (BP) comes from both population studies and randomized controlled trials in normotensive and hypertensive subjects. 2. The effect has been shown most clearly in those who keep to a strict lacto-ovo vegetarian diet characterized by a relatively low intake of saturated fat, a high polyunsaturated/saturated fat ratio, and a high intake of fruit, vegetables and other fibre containing products. Randomized controlled dietary trials suggest the effects are independent of dietary sodium, additive to that of calorie restriction, and not due to the absence of meat protein per se. Indeed, recent population studies suggest an inverse relationship between dietary protein and BP. 4. Dietary fats, fibre, potassium, magnesium and calcium do not independently seem to account for the effects. A possible role for complex carbohydrate in conjunction with the other dietary factors has yet to be fully explored. PMID- 7554416 TI - Mechanisms for reduction of cardiovascular risk by regular exercise. AB - 1. Regular aerobic exercise causes cardiovascular, neural, humoral and metabolic changes. Many of these are likely to influence cardiovascular risk and the changes vary according to the level and duration of increased physical activity. 2. The case for exercise exerting beneficial effects derives from epidemiological data showing that sedentary subjects have, on average, double the risk of cardiovascular disease of active individuals. Post-infarct rehabilitation studies are also consistent with a beneficial effect of exercise. Large randomized controlled intervention studies have not been performed. 3. These benefits may be due to the effects of regular exercise on risk factors, direct effects on atheromatous vessels, or improvement in survivability in established coronary heart disease by reducing arrythmias or increasing tolerance of ischaemia. 4. There are direct effects of regular exercise on blood pressure (BP) and lipid profiles. These occur at different levels of physical activity. Other changes which would be expected to reduce cardiovascular risk include increased insulin sensitivity, reduced sympathetic activity and increased gain of the baroreceptor heart rate reflex and increased arterial compliance. 5. In hypertensives these changes may improve outcomes, irrespective of the reduction in BP reported in some randomized controlled intervention studies. PMID- 7554417 TI - Assessment of nutrition and physical activity education programmes in children. AB - 1. Studies in children relating blood lipids to the extent of atherosclerosis at post-mortem suggest a link between risk factors for cardiovascular disease in childhood and adult life. Tracking of blood pressure (BP) and cholesterol from childhood also supports this association. However, prospective studies have not yet established the outcome in children with increased levels of risk factors. 2. In a controlled trial in Perth, Western Australia, involving over 1000 10-12 year old children, fitness was improved by physical activity programmes which were associated with a greater fall in diastolic BP and triceps skinfolds in girls compared with controls. Sugar intake decreased in boys and fat intake fell in girls, mainly affecting participants in home nutrition programmes. 3. In higher risk children, identified by cluster analysis, major benefits were associated with the fitness and home nutrition programmes. Physical activity combined with involvement of the family in nutrition education is likely to be the most successful approach to modifying lifestyle in children, including those with higher levels of risk. 4. Undernutrition by too rigid restriction of fat intake must be avoided in young children who need calorie-dense foods. Undernutrition, in itself, may predispose to cardiovascular disease in later life. Programmes should aim to establish a prudent diet appropriate to the age of the child combined with physical activity. As regular activity and a healthy diet in adult life will reduce risks of cardiovascular disease it is likely that childhood education will establish lifestyle habits of potential long-term benefit. PMID- 7554418 TI - Economic comparison of drug and lifestyle treatment of cardiovascular risk factors in high-risk patients. AB - 1. Lifestyle interventions are routinely used in the treatment of cardiovascular risk factors in individual patients but their cost-effectiveness relative to drug treatment is largely unknown. 2. Drug treatment is effective at normalizing risk factors and is known to decrease morbidity and mortality though costs may be high. Lifestyle interventions carry a significant potential to reduce expenditure on drugs, but prospective studies on cost-effectiveness of lifestyle interventions favour drug treatment. 3. According to current evidence, lifestyle intervention in high-risk patients is not more cost-effective than drug treatment. PMID- 7554419 TI - New World syndrome in Western Australian aborigines. AB - 1. It has become clear over the past 25 years that Aborigines in Western Australia (WA) now experience very high rates of type 2 diabetes mellitus and its complications as well as hypertension and cardiovascular disease; these disorders are often associated with obesity and abnormalities of plasma lipids. 2. This experience is similar to that of Aboriginal people in other parts of Australia and to other previously traditional societies now in transition to an urbanized, Westernized existence; this is widely attributed to lifestyle factors and genetic susceptibility. 3. This so-called 'New World Syndrome' is responsible for disproportionately high levels of morbidity and mortality in the Aboriginal population of WA; prevention and improved methods of screening, detection and management are needed to reduce this problem. PMID- 7554420 TI - Ambulatory blood pressure monitoring for evaluating the relationships between lifestyle, hypertension and cardiovascular risk. AB - 1. Ambulatory monitoring provides a unique opportunity for studying the temporal relationships between lifestyle factors and blood pressure (BP). These include physical activity, mental activity, environmental stressors, substances ingested for pleasure such as smoking, alcohol and caffeine, and nutrition. 2. Changes in physical activity play a major role in determining the diurnal profile of BP, whereas the influence of mood is small. 3. Environmental stress, in the form of job strain, has been shown to be associated with a sustained increase of BP throughout the day and night in men, and also with an increased left ventricular mass. The effects are most marked in men who drink alcohol regularly. Job strain does not appear to influence BP in women. 4. Although it is recognized that smoking raises BP acutely, its long-term effects have been unclear. Ambulatory monitoring shows that smokers have a larger diurnal swing of BP than non-smokers, because of a higher daytime pressure. Alcohol also increases the diurnal swing of BP. 5. Ambulatory monitoring has been used relatively little for evaluating nutritional factors, but has the possibility of quantifying their effects on BP more reliably than traditional methods, and also elucidating the underlying mechanisms. PMID- 7554421 TI - Endothelin-1 and the regulation of vascular tone. AB - 1. In 1988, Yanagisawa et al. reported the presence of a potent peptide from the supernatant of porcine endothelial cells. This was later named endothelin-1 (ET 1) and was found to belong to a new family of vasoconstrictor peptides. There are at least three isoforms of endothelin: ET-1, endothelin-2 and endothelin-3. 2. ET 1 is produced from a larger precursor molecule by endothelin converting enzyme (ECE); there may be a number of ECE but the most physiologically relevant appears to be a membrane-bound neutral metalloprotease. The endothelin precursor is produced on demand and is regulated at the mRNA level. 3. Two subtypes of mammalian endothelin receptors have been cloned and sequenced: ETA receptors which mediate vasoconstriction and ETB receptors which mediate both vasoconstriction and vasodilatation. However, functional studies have indicated that other subtypes of endothelin receptors may exist. 4. ET-1 has a wide range of biological actions apart from its direct effects on vascular tone, including constriction of non-vascular smooth muscle, cardiac effects, mitogenesis and stimulation of the release of hormones such as atrial natriuretic peptide and prostacyclin. At low concentrations which have no direct vasoconstrictor action, ET-1 potentiates the effect of other vasoconstrictor agonists. 5. The precise role of ET-1 in health and disease is not well defined at present; however, there are indications that it may have a role in the pathogenesis of some cardiovascular disease states, including subarachnoid haemorrhage, renal ischaemia and certain types of hypertension. PMID- 7554422 TI - Effects of glyceryl trinitrate tolerance on vascular responsiveness to constrictor agents in bovine isolated coronary artery. AB - 1. Recent clinical studies have raised the possibility that intermittent nitrate dose regimens, while limiting the extent of nitrate tolerance development, may trigger rebound aggravation of ischaemia after abrupt withdrawal of nitrate. To date, only limited studies have been performed to examine the mechanism of this phenomenon. 2. In a bovine isolated coronary artery model, we tested the hypothesis that induction of glyceryl trinitrate (GTN) tolerance might affect large coronary artery responsiveness to a variety of non-catecholamine constrictor agents. Glyceryl trinitrate tolerance was induced by pre-exposure to GTN (10 mumol/L) for 60 min followed by 15 min washout. In some experiments, the effect of prior endothelial removal on putative interactions was also examined. 3. Our results showed that GTN tolerance did not affect responses to KCl, the thromboxane A2 mimetic U46619, endothelin-1 and 5-HT, irrespective of presence or absence of endothelium. None of the constrictor agents tested induced endothelium dependent constriction. 4. We conclude that in this model of GTN tolerance induction, there is no evidence of 'rebound' vasoconstriction to this group of non-catecholamine vasoconstrictors with GTN withdrawal. PMID- 7554423 TI - Degranulation of rat salivary glands following treatment with receptor-selective agonists. AB - 1. The aim of this study was to find a drug that induces an almost complete degranulation of secretory cells in rat parotid and submandibular glands. 2. Phenylephrine (alpha-adrenergic), isoproterenol (beta-adrenergic) and mecholine (muscarinic cholinergic) were tested. Time and degree of maximal depletion of acinar and granular convoluted tubule cells were determined morphologically. 3. Following phenylephrine-injection (5 mg/kg or 10.2 mg/kg, i.p.), no effect on the acinar granulation level was observed in either of the glands, while about 50-60% granular convoluted tubules were degranulated for at least 120-180 min post injection. 4. With isoproterenol (5, 10, 40, 70 or 100 mg/kg, i.p.), degranulation of 100% of the acinar cells in the parotid and 80% of the acinar cells in the submandibular gland was observed 90 min post-injection. Granular convoluted tubule cells did not respond to this beta-adrenergic drug. 5. Mecholine (3.75 or 7.5 mg/kg, i.p.) induced mainly degranulation of granular convoluted tubule cells (about 50% after 120 min). Numbers of granulated acinar cells decreased only slightly in both glands (about 10%, 90-120 min). 6. From this study it appears that with a relatively low dosage (5 mg/kg, i.p.) of isoproterenol, a high level of degranulation can be induced in acinar cells of rat parotid and submandibular glands without toxic side effects. Concerning granular convoluted tubules, only moderate degranulation was observed with phenylephrine and mecholine, respectively. PMID- 7554424 TI - Electrophysiological effects of changrolin, an anti-arrhythmic agent derived from Dichroa febrifuga, on guinea-pig and rabbit heart cells. AB - 1. The electrophysiological effects of changrolin (CRL), a Chinese anti arrhythmic drug derived from a traditional antimalarial plant, were examined using the whole-cell patch-clamp method on single cells isolated from guinea-pig and rabbit hearts. 2. At a clinically relevant concentration of 50 mumol/L changrolin inhibited ICa by 19.3 +/- 6.0% and 17.3 +/- 2.6% in guinea-pig and rabbit ventricular cells, respectively. The voltage-dependent channel availability curve was not affected. The CRL effect was enhanced to a small extent during a repetitive stimulation at 2 Hz. 3. INa was resistant to CRL and the channel availability curve was also unaffected. A small use-dependent inhibition was observed only when the INa was elicited at 5 Hz in the presence of 300 mumol/L CRL. 4. At 50 mumol/L, CRL did not affect the time-independent inward rectifier and the delayed rectifier K+ currents (IK1 and IK, respectively), but inhibited the transient outward current (ITO) by 17.7 +/- 2.4%. Changrolin significantly shortened the action potential duration in both guinea-pig and rabbit ventricular cells. 5. In conclusion, CRL inhibits ICa and ITO but has little effect on INa. PMID- 7554425 TI - Sex differences and the effect of gonadectomy on morphine-induced antinociception and dependence in rats and mice. AB - 1. Differences between sexes and the effect of bilateral surgical gonadectomy on the response to morphine analgesia and dependence were examined in rats and mice. 2. The analgesic response to morphine (5 mg/kg) was assessed by the hot plate and the abdominal constriction tests. Dependence was induced by injecting morphine at increasing doses (5-160 mg/kg) for 6 consecutive days and withdrawal signs elicited by injecting naloxone (2.5 mg/kg). 3. The base line reaction times in the intact control, sham-operated and gonadectomized animals of either sex were not significantly different from each other. 4. After treatment with morphine, the percentage increase in the reaction time in gonadectomized male and female rats and in gonadectomized male mice was significantly higher than in their respective controls. 5. The increase in the reaction time, after morphine treatment, was significantly higher in gonadectomized female rats than in gonadectomized male rats. 6. Naloxone-induced withdrawal signs in morphine dependent gonadectomized rats and mice were not significantly different from those in sham-operated controls. However, female rats in both groups exhibited a significantly higher number of escape jumping responses than males. PMID- 7554426 TI - Nitric oxide inhibition does not prevent the hypotensive response to increased renal perfusion in rabbits. AB - 1. The involvement of nitric oxide (NO) and platelet activating factor (PAF) in the systemic depressor responses to increased renal perfusion pressure (RPP) were investigated. 2. In anaesthetized rabbits, the left kidney was perfused via an extracorporeal circuit which allowed RPP to be increased from 65 mmHg to 125 mmHg. The response of systemic blood pressure (SBP) to increasing RPP was measured in the same rabbits. 3. One group of rabbits (n = 5) was treated with NG nitro-L-arginine (NOLA) to inhibit NO synthase activity (20 mg/kg i.v. bolus). Another group (n = 5), received 250 mmol/L NaHCO3 (4 mL/kg bolus) as vehicle treatment. 4. Following an increase in RPP to 125 mmHg, SBP fell at a rate of 0.43 +/- 0.06 mmHg/min in the vehicle treated rabbits. After NO synthase inhibition the rate of fall in SBP of 0.34 +/- 0.07 mmHg/min was not significantly different from that in the vehicle group (P = 0.3). 5. Blockade of NO synthesis did not alter the renal blood flow, renal vascular resistance changes and pressure-related natriuresis and diuresis responses to increased RPP to 125 mmHg. 6. PAF receptor blockade, using WEB 2086 (0.5 mg/kg plus 0.5 mg/kg/h), did not alter the systemic, renal haemodynamic or urinary responses to increasing renal perfusion pressure to 125 mmHg. 7. These findings indicate that neither NO nor PAF play an important role in the blood pressure lowering activity, intrarenal haemodynamics and urinary excretory responses observed when RPP was increased to a level within the physiological range. PMID- 7554427 TI - Spectral analysis of systolic blood pressure and heart rate oscillations related to respiration. AB - 1. Non-invasive assessment of short-term systolic blood pressure (SBP) and heart rate (HR) variability was obtained with the plethysmographic finger blood pressure measurement device. Respiration was measured with a respiratory inductive plethysmograph, which was calibrated prior to each study. 2. The effects of breathing pattern on the respiratory (high frequency, HF) component of the SBP or HR spectrum were analysed by Fourier transform. 3. Our quantification of the changes in the HF (respiratory) peak of the HR or SBP spectrum with changes in tidal volume (VT) and breathing frequency (BF) indicates that the modulus of this component may be predicted for any combination of depth and frequency of breathing. 4. The modulus of this HF component for the HR or SBP spectrum was linearly related to the respiratory sinus arrhythmia (RSA) or to the SBP oscillation related to respiration. PMID- 7554428 TI - Involvement of cytoskeletal proteins in the mechanisms of organophosphorus ester induced delayed neurotoxicity. AB - 1. Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by the presence of swellings in the distal parts of large axons in the central and peripheral nervous systems with subsequent axonal degeneration and paralysis. 2. An early change in OPIDN is enhanced activity and autophosphorylation of Ca2+/calmodulin-dependent kinase II. 3. In OPIDN, there is also a dose- and time-dependent increase in Ca2+/calmodulin dependent kinase mediated phosphorylation of the cytoskeletal proteins, alpha- and beta-tubulin, microtubule associated protein-2, neurofilament triplet proteins and myelin basic protein. 4. Anomalous hyperphosphorylation of neurofilaments decreases their transport rate down the axon relative to their rate of entry resulting in their accumulation. 5. Consistent with the neurochemical results is the presence of anomalous aggregations of phosphorylated neurofilaments in early stages of OPIDN. 6. These findings suggest that aberrant hyperphosphorylation of cytoskeletal proteins is a post-translational modification involved in the pathogenesis of OPIDN. PMID- 7554431 TI - Subunit-dependent action of lead on neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. AB - 1. Pb2+ affects neuronal nicotinic acetylcholine receptors (nAChR) in N1E-115 neuroblastoma cells in a dual manner. At nanomolar concentrations a blockade is observed, while at submillimolar concentrations this blocking effect is reversed. 2. The Xenopus oocyte expression system was used to examine whether the dual effect of Pb2+ is related to a differential action on nAChR subtypes. Effects of Pb2+ were investigated in oocytes expressing nAChR after co-injection of alpha 3 and beta 2 or alpha 3 and beta 4 cDNA. 3. At 1-250 mumol/L, Pb2+ causes a 10 1000% increase of the response mediated by the alpha 3 beta 2 nAChR. 4. Pb2+ blocks ACh-induced inward currents mediated by alpha 3 beta 4 nAChR. The inhibitory potency of Pb2+ greatly varies between cells. In 50% of the cells concentrations < or = 1 mumol/L Pb2+ blocked the nicotinic response by 31-93%. In the other cells even at higher concentrations Pb2+ caused only 0-65% inhibition. 5. These results show that Pb2+ may both potentiate and block nAChR, depending on the type of nAChR subunit expressed. PMID- 7554429 TI - Analysis of the steady-state dynamics of organelle motion in cultured neurites: putative indicator of neurotoxic effect. AB - 1. The objective of this study was to develop a physiologically based method to evaluate the neurotoxic potential of drug candidates in vitro. Rat embryo midbrain cells were grown in micromass culture, and the movement of mitochondria labelled with the fluorescent dye rhodamine 123 was quantified in fasciculated neurites, using a laser cytometer. 2. The rhodamine 123 signal in a defined region of fascicle was quantified and photobleached with the laser. A series of post-photobleach scans revealed the movement of fluorescent-labelled mitochondria into the bleached region from adjacent unbleached regions. Recovery of fluorescence is a measure of the size of the mobile pool of mitochondria relative to the total (moving plus stationary) pool. 3. The steady-state levels of fluorescence recovery was dependent on intracellular calcium and magnesium concentrations, energy status (ATP), and microtubule integrity (post taxol or vinblastine treatment). 4. This technique may be a useful indicator of neurotoxic effect. PMID- 7554430 TI - Comparison of neurite outgrowth with neurofilament protein subunit levels in neuroblastoma cells following mercuric oxide exposure. AB - 1. The objectives of the study were to establish that inhibition of neuronal differentiation in culture (assessed by neurite outgrowth) can be used as a broad spectrum in vitro measure of neurotoxicity. 2. To establish whether a rapid measure of neurite outgrowth could be used. Thus the study examined the relationship between the degree of neurite outgrowth assessed directly by image analysis and neurofilament protein subunit levels measured by an ELISA. 3. SKNSH neuroblastoma cells, exposed for up to 6 days to mercuric chloride during initiation and continuation of differentiation, had lower levels of neurofilament proteins than unexposed cells. 4. Preliminary data from parallel examinations of neurite outgrowth assessed by image analysis and neurofilament protein subunit levels assessed by ELISA support a correlation when neurofilament protein levels are decreased by sub-cytotoxic doses of mercuric chloride in SKNSH cells. PMID- 7554433 TI - Use of differentiated cholinergic and second messenger endpoints to evaluate cholinesterase inhibitors. AB - 1. The effects of organophosphorus compounds on the cholinergic properties of a mouse septal neuronal cell line, SN56, were investigated. Treatment of the cells with 10 mumol/L paraoxon or 10 mumol/L diisopropylphosphorofluoridate (DFP) resulted, respectively, in rapid 27- and 11-fold increases of intracellular acetylcholine content. 2. Time-course studies showed that paraoxon maintained its efficacy whereas the effects of DFP decayed quickly, possibly due to rapid hydrolysis and inactivation of DFP in the growth medium. PMID- 7554432 TI - Use of tissue culture models to study environmental-genetic interactions relevant to neurodegenerative diseases. AB - 1. Clonal cell lines, primary cultured neurones and transgenic animals expressing mutant genes linked to familial forms of neurodegenerative diseases provide models in which to examine the interaction between expression of a predisposing gene and exposure to neurotoxic chemicals. Methods of establishing these models are reviewed. 2. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD 1) have been identified in cases of familial amyotrophic lateral sclerosis linked to chromosome 21. We report that in clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting epoxide, styrene oxide, varied with SOD activity in a manner similar to that previously demonstrated for redox cycling chemicals. These preliminary data suggest that either low or high SOD-1 activities may be associated with greater toxicity of a variety of neurotoxic chemicals and their metabolites. PMID- 7554434 TI - Neurotoxicity of beta-amyloid protein: cytochemical changes and apoptotic cell death investigated in organotypic cultures. AB - 1. This study is an attempt to examine in vitro the cytochemical changes in hippocampal neurones induced by beta-amyloid protein (beta-AP). 2. The mechanism of cell death, and the vulnerability of different regions of the hippocampus to b AP toxicity, has also been explored using TUNEL staining to locate fragmented DNA in both dissociated and organotypic cultures. 3. Apoptotic cell profiles and the detection by immunocytochemistry of ubiquitin and tau protein confirmed the acute neurodegenerative effects of b-AP, and organotypic cultures revealed the dentate gyrus to be especially vulnerable. 4. A scrambled sequence of b-AP, a peptide with similar hydrophobic groups to b-AP, and islet pancreatic amyloidogenic peptide also showed neurodegenerative effects, although less severely than b-AP. 5. It is concluded that organotypic cultures provide a valuable in vitro model with which to observe and characterize the neurotoxic effects of b-AP. These effects, however, may be non-specific and related more to the general amyloidogenicity of the b-AP molecule. PMID- 7554436 TI - Examination of developmental neurotoxicity by the use of tissue culture model systems. AB - 1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity. PMID- 7554435 TI - Developmental neurotoxicity to methamphetamines. AB - 1. To investigate the long-term changes caused by amphetamines in the developing brain, we used both an in vivo and in vitro model of chronic fetal exposure to methamphetamine and related drugs. 2. Offspring of rats, treated with either saline, 2 mg/kg twice a day (b.i.d.) or 10 mg/kg b.i.d. methamphetamine throughout gestation, were examined at 30 days of age for changes in the monoamine system of their brains. 3. At the lower dose methamphetamine was neurotoxic to specific neuronal populations, mostly serotonergic. At the higher dose, methamphetamine retained its neurotoxic properties, but also stimulated the growth of axonal terminals in specific regions as evidenced by an increase in monoamine uptake sites. The neurochemical changes at the higher dose were correlated with deficits in adult behavioural measures. 4. Corresponding in vitro drug treatments of rat neuroblastomas cells also produced a dose-related effect on cellular growth and differentiation patterns. Neurotoxic as well as stimulatory effects of methamphetamine and some related compounds were seen in culture. 5. Our in vivo and in vitro observations demonstrate neurotoxic effects of amphetamines and the remodelling of synaptic morphology in response. PMID- 7554437 TI - Alteration of the morphology and neurochemistry of the developing mammalian nervous system by hydrogen sulphide. AB - 1. Hydrogen sulphide (H2S) is a broad spectrum toxicant that occurs widely in nature and is also released by a variety of industrial activities and processes. 2. The central nervous system (CNS) appears to be the major target organ. 3. There is great potential for insult or injury to the developing or immature CNS. 4. The risk of chronic or repeated exposures to low concentrations have not been well defined. 5. Exposure to low concentrations of H2S to time-pregnant rats from day 5 postcoitus until day 21 postnatal results in architectural modification of cerebellar Purkinje cells, alteration of putative amino acid neurotransmitters and changes in monoamine levels in the developing rat brain up to day 21 postnatal. 6. H2S-induced alterations in monoamine tissue levels observed in the developing rat brain return to control values if exposure is discontinued during development, that is, at day 21 postnatal. PMID- 7554439 TI - Validation and in vitro neurotoxicity. AB - 1. Validation of in vitro systems for studying neurotoxicants generally has not been accomplished, although in vitro tests have been used as screens to identify potential neurotoxic hazards and to study mechanisms. 2. A number of factors need to be taken into account when a test is validated: (i) a rationale for developing the test; (ii) clear biological or pathophysiological relevance of the endpoint to the effect detected in vivo; (iii) a standardized protocol and evidence of intra- and interlaboratory reproducibility; (iv) testing of chemicals representative of the categories of interest, including very toxic, moderately toxic and relatively non-toxic substances; and (v) a method to statistically evaluate the data. 3. Proper validation should lead to methods which can be used by regulatory agencies to make decisions regarding hazard/risk. PMID- 7554438 TI - Involvement of oxidative and L-arginine-NO pathways in the neurotoxicity of drugs of abuse in vitro. AB - 1. Inhibitors of nitric oxide (NO) formation or ADP-ribosylation attenuate methamphetamine (METH)- and methylenedioxymetamphetamine (MDMA)-induced neurotoxicity on dopaminergic and serotonergic cells in primary cultures. 2. They also prevent METH-induced reactive gliosis in dopaminergic cultures. 3. Overexpression of superoxide dismutase (SOD) in cells obtained from SOD transgenic mice also attenuates drug-induced toxicity. 4. These data indicate a role for oxygen-based and NO free radicals in the mechanisms of cell death associated with drugs of abuse in vitro. PMID- 7554441 TI - Cell injury mediated by autoantibodies to intracellular antigens. PMID- 7554442 TI - Differential regulation of human T lymphocyte protein kinase C activity by unsaturated fatty acids. AB - Administration of gamma-linolenic acid suppresses active synovitis in patients with rheumatoid arthritis. We therefore examined the effects of gamma-linolenic acid and its first metabolite, dihomo-gamma-linolenic acid, on protein kinase C, a key element in transduction of signals from cell surface to nucleus. We report here that gamma-linolenic acid and dihomo-gamma-linolenic acid suppress total protein kinase C activity, but facilitate translocation of protein kinase C activity from cytosol to membrane in human peripheral blood T lymphocytes stimulated with phorbol 12-myristate 13-acetate. Arachidonic acid and eicosapentaenoic acid do not influence total protein kinase C activity and have only modest effects on enzyme translocation. These findings in whole cells are in contrast to results of experiments performed with isolated protein kinase C, in which unsaturated fatty acids uniformly enhance protein kinase C activity. The differential effects of unsaturated fatty acids underscore the complexity of protein kinase C regulation and indicate that gamma-linolenic and dihomo-gamma linolenic acids influence T lymphocyte protein kinase C metabolism in a manner that is unique among unsaturated fatty acid precursors of eicosanoids. PMID- 7554440 TI - Esterase comparison in neuroblastoma cells of human and rodent origin. AB - 1. Activities of acetylcholinesterase (AChE), neuropathy target esterase (NTE), and carboxylesterase (CbxE) were compared in neuroblastoma cells of human origin (SH-SY5Y) and murine origin (NB41A3). 2. Mouse neuroblastoma cells had lower specific activities of NTE and CbxE than did human neuroblastoma cells; specific activities in the murine cells correlated with specific activities in mouse brain. 3. AChE activities in mouse and human neuroblastoma cells were considerably lower than AChE activities in mouse or hen brain. 4. Inhibition of esterases did not demonstrate interspecies differences for 12 of the 17 anti esterase compounds tested with human and mouse neuroblastoma cells. PMID- 7554446 TI - Divergence of human and nonhuman primate lymphocyte responses to bacterial superantigens. AB - We compared T cell responses of human, rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) to four bacterial superantigens. When lymphocytes were cultured in media supplemented with species-specific sera, chimpanzee T cells were stimulated by lower doses of staphylococcal enterotoxin (SE) A and toxic shock syndrome toxin 1 (TSST1) than were human T cells, while chimpanzee responses to SEB and SEC1 were nearly equivalent to the human response. Interestingly, rhesus lymphocytes responded to 10,000 times lower amounts of SEA, SEB, and SEC1 and to 100 times lower concentrations of TSST1 than human cells. The greater sensitivity of rhesus T cells to these toxins was not a result of differences in class II binding affinities and was only partly attributable to the presence of anti-SE and TSST1 antibodies in human serum. These results suggest that rhesus T lymphocytes are more sensitive toward these bacterial superantigens than human T cells. PMID- 7554444 TI - Cytotoxicity to endothelial cells by sera from aged MRL/lpr/lpr mice is associated with autoimmunity to cell surface heparan sulfate. AB - Vasculitis is an common clinical feature of systemic lupus erythematosus (SLE) in humans and in animal models of this disease. Humoral autoimmunity against endothelial cells has been previously demonstrated in SLE and other autoimmune disorders, but the precise cell surface antigenic targets involved in the initiation and progression of vascular injury are still essentially unknown. In the current studies, we demonstrate the presence of autoantibodies in the sera of MRL/lpr/lpr mice which bind endothelial cell surface antigens by ELISA and also cause complement-dependent cytotoxicity of these cells. These MRL/lpr/lpr sera induced complement-dependent cleavage and release of 35SO4-labeled material containing primarily cell surface heparan sulfate proteoglycans from these cells, and react with heparin (a glycosaminoglycan related to heparan sulfate) by ELISA and liquid-phase competitive inhibition ELISA. These data indicate that antiendothelial cell autoantibodies present in autoimmune MRL/lpr/lpr mice are directed at least in part against cell surface heparan sulfate proteoglycans. Autoantibodies to cell surface heparan sulfate proteoglycan may play a role in vascular endothelial cell injury in these animals through complement-dependent, autoimmune mechanisms. PMID- 7554445 TI - Activation of human neutrophils by surface-associated IgA is associated with the release of activated collagenase. AB - Neutrophils contain on their surface a receptor for the Fc portion of IgA. Cross linking of this receptor in the fluid phase induces superoxide production and release of granule constituents, but the response to surface associated IgA has not been previously studied. Neutrophils incubated with surface-associated IgA (SAIgA) release significant amounts of activated collagenase in addition to the granule proteins myeloperoxidase and lactoferrin. This activation is associated with release of superoxide as well as hydrogen peroxide and hypochlorous acid. Although neutrophils incubated with soluble aggregates of IgA also release granule proteins and produce superoxide, soluble aggregates of IgA do not trigger the release of activated collagenase and do not generate hydrogen peroxide or hypochlorous acid. In summary, neutrophils activated by surface associated IgA respond differently than when cells are activated by soluble aggregates of IgA. These differences may be important in understanding the mechanisms of tissue injury in patients with inflammatory disorders. PMID- 7554443 TI - Thrombin receptor-mediated synovial proliferation in patients with rheumatoid arthritis. AB - Synovial cell proliferation is one of the pathological bases of rheumatoid arthritis (RA). Several cytokines including IL-1 and IL-6 and growth factors have been shown to be involved in the synovial cell proliferation in RA. Thrombin is a multifunctional protease and acts as a mitogen for several cell types through its specific receptor. To assess whether thrombin is involved in overproliferation of rheumatoid synovial cells, we measured the concentration of thrombin-anti thrombin III (ATIII) complex (TAT) in synovial fluid obtained from patients with RA or osteoarthritis (OA). We also examined the effect of thrombin or thrombin receptor agonist peptide (TRAP) on cell growth of synovial cell clones (SCCs) established from an RA patient. The concentrations of TAT in the synovial fluid from patients with RA were significantly higher than in those with OA. Moreover, both thrombin and TRAP enhanced proliferation of synovial cells in vitro. We also characterized the expression of thrombin receptor mRNA by reverse transcription PCR. The expression of mRNA for thrombin receptor was up-regulated by thrombin or TRAP stimulation. Thrombin receptor antigen was also detected on both SCCs and synovial tissue from RA patients by immunostaining using a monoclonal antibody against thrombin receptor. These findings indicate that thrombin may act as a mitogen for synovial cells through thrombin receptor and may play some role in synovial overproliferation and remodeling in RA. PMID- 7554447 TI - The HIV glycoprotein gp 160 has superantigen-like properties. AB - HIV infection is characterized by paralysis of the immune system and a depletion of CD4+ cells. Recent studies demonstrating modulation of the V beta T cell receptor (TCR) repertoire in HIV patients have suggested that some of these effects may be the result of action by one or more superantigens encoded by the virus. In order to determine whether the HIV envelope glycoprotein, gp160, displays properties reminiscent of a superantigen, the T cell receptor V beta repertoire of T cells from healthy, seronegative individuals activated in vitro with gp160 was determined. In five individuals of disparate HLA type, activation by gp160 resulted in a marked skewing in the relative expression of a common set of V beta gene segments. This activation was HLA class II-dependent and did not require antigen processing. Surprisingly, the V beta segments affected by gp160 bore a striking similarity to those affected by the staphylococcal superantigen SEB. These observations suggest that exposure to superantigens produced by opportunistic infection might play an important role in disease progression. PMID- 7554448 TI - Detection of anti-DNA topoisomerase I antibody by an enzyme-linked immunosorbent assay using overlapping recombinant polypeptides. AB - Five recombinant fusion proteins with overlapping amino acid sequences encompassing the entire DNA topoisomerase I (topo I) molecule were generated, purified, and used as antigens for an enzyme-linked immunosorbent assay (ELISA). IgG, IgA, IgM, and "total" (total of IgG, IgA, and IgM isotypes) anti-topo I antibodies were measured using a mixture of these five fusion proteins in 73 systemic sclerosis (SSc) sera positive for anti-topo I antibody by double immunodiffusion (DID) and 184 control sera negative for anti-topo I antibody by DID. Fragment-specific anti-topo I antibodies were also measured by ELISA using each topo I recombinant protein as antigen. IgG, IgA, IgM, and total anti-topo I antibodies were detected in 67 (92%), 56 (77%), 16 (22%), and 70 (96%) of 73 SSc sera positive for anti-topo I antibody by DID, respectively. The specificity of the total anti-topo I ELISA was 99% when compared with DID. The total anti-topo I ELISA levels were strongly correlated with DID titers (r = 0.907, P < 0.0001). Three sera which recognized a conformational epitope on native topo I or had predominantly IgM anti-topo I antibody showed a false-negative result with the total anti-topo I ELISA. Three SSc sera negative for anti-topo I antibody by DID were positive by the total anti-topo I ELISA, and two were confirmed to recognize the N-terminus of topo I. IgG and IgA antibody levels to the N-terminal and central portion of topo I were correlated with each other, but those to the C terminus were not. These findings indicate that the ELISA using recombinant fusion proteins is a highly sensitive and specific alternative to conventional DID for the detection of anti-topo I antibody. PMID- 7554450 TI - Antimicrobial activity of calprotectin isolated from human empyema fluid supernatants. AB - Abscess and empyema fluid supernatants have zinc-reversible antimicrobial activity that is thought to be due to calprotectin, a calcium- and zinc-binding protein present within neutrophil cytoplasm. The present studies were undertaken to determine if calprotectin isolated from human empyema fluid supernatants demonstrated similar antimicrobial activity to that of the original specimens. The characteristics of the calprotectin complex on SDS-PAGE and Western blotting with specific antisera were similar in neutrophil lysates and in empyema fluid supernatants. Ion-exchange and size-exclusion chromatography were used to obtain highly purified preparations of calprotectin from empyema fluids, and these preparations demonstrated zinc-reversible anti-Candida albicans activity which was similar to that observed in the original specimens. These findings suggest that calprotectin is responsible for most of the growth-inhibitory activity of empyema fluid supernatants against this organism. PMID- 7554449 TI - Elevated serum level and altered glycosylation of alpha 1-acid glycoprotein in hyperimmunoglobulinemia D and periodic fever syndrome: evidence for persistent inflammation. AB - Crossed affinoimmunoelectrophoresis using concanavalin A and Aleuria aurantia lectin as diantennary glycan- and fucose-specific affinocomponents, respectively, was applied to study changes in the concentration and glycosylation of the acute phase protein alpha 1-acid glycoprotein (AGP) in sera obtained from patients with hyperimmunoglobulinemia D and periodic fever syndrome. Increases in concentration of AGP compared to control values were found not only during attacks, but also during remissions. Compared to healthy controls, the presence of diantennary glycan-containing glycoforms of AGP also increased during febrile attacks, while no changes were found during remissions. A continuous high degree of alpha 1-->3 fucosylation was accompanied by a continuous high expression of sialyl Lewisx on AGP. Despite the clinical picture of recurrent febrile attacks with asymptomatic intervals, these studies indicate that hyperimmunoglobulinemia D should be considered a condition of persistent inflammation. PMID- 7554451 TI - Effects of calorie restriction on transforming growth factor beta 1 and proinflammatory cytokines in murine Sjogren's syndrome. AB - The present study was carried out to determine whether restricting dietary calories prevents salivary gland abnormalities and modulates expression of transforming growth factor beta and proinflammatory cytokines, IL-6, and TNF alpha in major salivary glands (SG) of autoimmune lupus-prone (NZB x NZW)F1 (B/W) female mice. These mice develop focal lymphocytic interstitial and periductal round cell infiltrates in salivary glands similar to those of humans with Sjogren's syndrome. Weanling B/W mice were fed a nutritionally adequate semipurified diet either ad libitum (AL) or a calorie-restricted (CR; 40% less calories than AL) diet. The mice were sacrificed at 3.5 months (young) and 8.5 months (old) of age. Histopathologic and histomorphometric analyses as well as growth factor and cytokine protein and mRNA expression were carried out in the SG. Histomorphometric analysis of SG from young mice showed no differences between AL and CR mice, but old AL (vs old CR) had a 7.3-fold higher focus score and a 34-fold increase in percentage area inflammation. mRNA analysis revealed significantly higher levels of TGF beta 1 in SG of old CR (6.8-fold) mice. In contrast, CR reduced mRNA expression of proinflammatory cytokines (IL-6, 2.9-fold for young and 4.8-fold for old; TNF alpha, old 3.9-fold). By immunoblotting, significantly higher levels of TGF beta 1 protein was detected in old CR mice (vs old AL; 13.2-fold). IL-6 and TNF alpha proteins were undetectable in both young and old CR groups, whereas an increase in IL-6 (4.7-fold) and TNF alpha (9.3 fold) was observed in old AL mice. These results indicate that amelioration of the histological severity of disease in SG of B/W mice is paralleled and possibly mediated by increased expression of immunosuppressive TGF beta 1 and decreased expression of proinflammatory cytokines. PMID- 7554453 TI - A novel methodology for quantitating the enhancement of cutaneous delayed-type hypersensitivity by IMREG-1: a measure of the immunopotentiation of cell-mediated immunity. AB - IMREG-1, a low-molecular-weight immunomodulator derived from normal human leukocyte dialysates, has been shown to enhance cutaneous delayed-type hypersensitivity (DTH) responses to recall antigens. Both IMREG-1 and the biologically active peptides (Tyr-Gly[YG] and Tyr-Gly-Gly[YGG]) identified therein are able to accelerate and enhance DTH in a concentration-dependent manner. In this study, we describe a novel methodology for analyzing and quantitating this response and demonstrate its use with data comparing drug to placebo. Subjects demonstrating prior sensitivity to a recall antigen (tetanus toxoid) received intradermal injections of tetanus toxoid alone (control) and either dilutions of IMREG-1 plus antigen, or placebo plus antigen, on the volar surface of the forearm. The response, as measured by area of erythema, was calculated and plotted as a function of time. The area under the resulting curve (AUC) was then determined by use of the trapezoidal rule, whereby the area of a trapezoid formed between each sequential pair of time points was calculated. The AUC computed for each site receiving a dilution of IMREG-1 or placebo (test) was compared with the AUC computed at the site that received antigen alone (control) by means of a test to control (T/C) ratio. The respective T/C ratios for designated dilutions of IMREG-1 or placebo provided a basis of comparison between responses to IMREG-1 and to placebo, while also controlling for individual sensitivity in response to antigen. We demonstrate in this study that the enhanced response to IMREG-1 plus antigen is statistically different from that seen with placebo plus antigen. This response, as a function to time, predominantly appears in the 12- to 24-hr period after injection, illustrating the ability of the immunomodulator to accelerate, enhance, and sustain a DTH response. We further conclude that the effect of IMREG-1 in this context is one of immunopotentiation of cell-mediated immunity. PMID- 7554452 TI - Thymic epithelial cell abnormalities in (NZB x H-2u)F1 mice. AB - Thymic maturation processes including MHC restriction and self-recognition require intimate association of thymocytes and stromal cells. Compared to the thymic architecture of various "normal" control strains of mice, defects in the thymic microenvironment have been demonstrated in New Zealand black (NZB) mice. Moreover, it is well known that NZB(H-dd) mice, when crossed with NZW(H-2u) mice, (NZB x NZW)F1, display a unique spectrum of autoimmune disease manifestations, including murine SLE. Using an extensive panel of monoclonal antibodies that define the thymic microenvironment, we examined two additional strains of (NZB x H-2u)F1 mice: (NZB x C57BL/10.PL)F1 and (NZB x PL/J)F1 mice to investigate the contributions of the H-2 and non-H-2 loci to the thymic abnormalities previously documented to occur in murine lupus. NZB, NZW, and (NZB x NZW)F1 mice were studied concurrently as were two additional control strains C57BL/6 and C57BL/10Sn. NZW mice had a normal thymic architecture as did the other H-2u mice and the control strains. In contrast, (NZB x NZW)F1 mice had a significantly altered thymic microenvironment; mild thymic abnormalities were also found in (NZB x PL/J)F1 but not in (NZB x C57BL/10.PL)F1. As expected, (NZB x NZW)F1 mice developed elevated titers of autoantibodies to DNA, proteinuria, and decreased life span. Interestingly, only (NZB x PL/J)F1 mice had increased levels of IgM antibodies to dsDNA, but did not manifest IgG anti-DNA or reduced survival. Defects in thymic stromal cells are associated directly to autoimmunity and their origin appears to be determined by non-H-2 loci. PMID- 7554454 TI - Complement activation during painful crisis in sickle cell anemia. AB - Previous studies documented complement activation in sickle cell disease patients and suggested that this contributes to increased risk of infection. We have demonstrated alternative pathway activation initiated by membrane phospholipid changes which occur in sickled erythrocytes. The present studies compared complement activation products in serial samples from sickle cell anemia patients at baseline and during hospitalization for painful crisis to examine the correlation between complement activation and disease activity. Plasma concentrations of Bb, C4d, and C3a were measured as well as C3 bound to erythrocytes. Patients were subdivided into those with continuous pain and those with intermittent painful episodes. In patients with intermittent pain, there was little evidence of complement activation at baseline and increased plasma concentrations of Bb and C3a during painful crisis. Elevated C3a and C4d levels were observed in patients with continuous pain regardless of hospitalization status, suggesting a continuous underlying inflammatory process in these patients. PMID- 7554456 TI - Proceedings of the Jeffrey Modell Immunodeficiency Symposium. Advances in Primary Immunodeficiency Disease. Paris, France, October 10-11, 1994. PMID- 7554455 TI - Pretreatment of donors and grafts with rapamycin: the effect on graft survival and the tissue drug retention. PMID- 7554457 TI - T cell differentiation: control by the pre-TCR and alpha beta TCR. PMID- 7554458 TI - Nonreceptor protein tyrosine kinase involvement in signal transduction and immunodeficiency disease. AB - The nonreceptor protein tyrosine kinases (PTKs) have been grouped into 10 different enzyme families based on predicted amino acid sequences. As the number of enzymes belonging to the nonreceptor class of PTK is increasing, one challenge is to determine how these various classes of PTKs interact within the cell to promote signal transduction. Herein, the activation of four classes of nonreceptor PTKs is discussed in relation to their interactions with each other as well as with other signaling molecules during the process of lymphocyte surface antigen receptor-mediated activation. Recent findings of nonreceptor PTK loss-of-function mutations in different immunodeficiency diseases has revealed the important contribution of this group of enzymes to lymphocyte development. PMID- 7554459 TI - Molecular and genetic insights into the role of protein tyrosine kinases in T cell receptor signaling. PMID- 7554460 TI - T cell activation deficiencies. PMID- 7554461 TI - Genetic studies in severe combined immunodeficiency. PMID- 7554462 TI - Interaction of antigenic peptides with MHC and TCR molecules. AB - Recent studies have demonstrated that antigen analogs can act as powerful and specific inhibitors of T cell activation. Analysis of various experimental systems suggests that this effect might be related to engagement of the T cell receptor (TCR) by low-affinity ligands and that the observed inhibition may be mediated by interference with membrane events crucial for signal transduction, such as proper TCR oligomerization. In addition, experimental data obtained by our laboratory and others suggest that TCR antagonism may be of significant value as a therapeutic approach and as a tool to dissect the process of thymic education. PMID- 7554463 TI - Immunodeficiency in IL-2-knockout mice. AB - Interleukin-2-deficient mice (IL-2-/-) generated by targeted mutagenesis proved to be an excellent system to study the function of this lymphokine in the context of whole immune system. In addition, they provide an animal model of a primary immunodeficiency caused by a single-gene defect. Our results indicate that the essential degect in IL-2-/- mice is the lack of a negative regulatory function, which leads to breakdown of self tolerance. The development of autoimmune diseases in IL-2-deficient mice stresses the link between autoimmunity and primary immunodeficiencies. PMID- 7554464 TI - Interleukin-10 gene knock-out mice: a model of chronic inflammation. PMID- 7554465 TI - Analysis of lambda repertoire in kappa-deficient mice. PMID- 7554466 TI - B cell differentiation in bone marrow. PMID- 7554467 TI - The spectrum of mutations in Btk that cause X-linked agammaglobulinemia. PMID- 7554468 TI - Structural basis for X-linked agammaglobulinemia (XLA): mutations at interacting Btk residues R562, W563, and A582. AB - It has been suggested that tryptophan 563 is sandwiched between residues R562 and A582 in Bruton's agammaglobulinemia tyrosine kinase (Btk). Mutations of the surrounding residues have been shown to cause X-linked agammaglobulinemia. Substitutions R562P and A582V were noticed to have impaired kinase activity. However, based on Western blot analysis, the mutant proteins were expressed at normal levels. Molecular modeling of the kinase domain has previously indicated that these residues presumably govern the position of the W563 side chain, which is thought to interact with the catalytic loop. W563 is inside the molecule and too far away from the catalytic center to interact directly with the substrate or cofactors. To prove these model-based conclusions, a conservative substitution with phenylalanine for W563 was made, and the resultant mutant lacked kinase activity. These results confirm our previous assumption that the side chain of W563, invariant in protein tyrosine kinases, is crucial for Btk kinase activity. Mutations in the surrounding residues seem to inactivate Btk by affecting the location of W563. PMID- 7554469 TI - The role of gp39 (CD40L) in immunity. AB - The function of CD40 and its ligand (CD40L;gp39) has provided a molecular understanding for cognate interactions and the basis for hyper IgM syndrome. Studies using an antibody that neutralizes gp39 function in vivo have demonstrated that interactions between CD40 and gp39 are essential for primary and secondary thymus-dependent humoral immune responses, as well as for the generation of memory B cells. In addition, gp39 is critical for B cells to upregulate important costimulatory molecules and acquire professional antigen presenting cell status. If B cells do not receive the gp39 signal, they are rendered tolerogenic for T cells. Finally, gp39-CD40 interactions are important in thymic education since blockade of this interaction interrupts negative selection. PMID- 7554470 TI - CD40-CD40 ligand (CD40L) interactions and X-linked hyperIgM syndrome (HIGMX-1). AB - Interactions between the B cell surface antigen CD40 and its ligand (CD40L) expressed on activated T cells play a critical role in isotype switching. This is illustrated by failure of isotype switching in patients with X-linked hyperIgM syndrome in whom the CD40L gene is mutated and by failure of isotype switching of CD40-deficient mice in response to T-cell-dependent antigens. We review these findings and discuss the signaling mechanisms of CD40 and the developmental control and transcriptional regulation of CD40L expression. PMID- 7554471 TI - The influence of gene deletions and duplications within the IGHC locus on serum immunoglobulin subclass levels. AB - To study the influence of heterozygous gene deletions and duplications in the human IGHC region on serum concentrations of antibodies, we have analyzed the IgG subclass levels in a large number of individuals from three different populations. Our results show that heterozygous gene deletions of the C gamma 1, C gamma 2, or C gamma 4 genes are all associated with a lower mean serum level of the affected subclass whereas duplication of the C gamma 2 or C gamma 4 genes result in either no change (IgG2) or a slightly elevated (IgG4) level of serum immunoglobulins. PMID- 7554472 TI - Adenosine deaminase deficiency: molecular basis and recent developments. PMID- 7554473 TI - PEG-ADA replacement therapy for adenosine deaminase deficiency: an update after 8.5 years. AB - Polyethylene glycol-modified adenosine deaminase (PEG-ADA) has now been used for 8.5 years as enzyme replacement therapy for immunodeficiency due to ADA deficiency. PEG-ADA restores a metabolic environment necessary for recovery of immune function. In most cases, the level of function achieved has been sufficient to protect against opportunistic and life-threatening infections. To date, mortality and morbidity with PEG-ADA have been less than for haploidentical bone marrow transplantation. As a true "orphan drug" used to treat a very small patient population, the cost per patient of PEG-ADA is very high, but it has been well tolerated, free of adverse reactions, and effective as an alternative for patients who lack an HLA-identical marrow donor, but are considered too ill to undergo haploidentical marrow transplantation. Concomitant treatment with PEG-ADA has also permitted investigation of gene therapy to be carried out safely. PMID- 7554474 TI - Interleukin-6 activity in dogs with juvenile polyarteritis syndrome: effect of corticosteroids. AB - Juvenile polyarteritis syndrome (JPS) is an idiopathic febrile disease in dogs. Elevated serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) have been reported in human patients with vasculitis. We investigated whether these cytokines are also elevated in serum of dogs with JPS using sensitive bioassays. Increased levels of IL-6 activity were detected in the serum of 12 acutely ill dogs, whereas the IL-6 activity decreased to low or undetectable levels during convalescence. Treatment of 5 acute JPS dogs with prednisone resulted in a rapid clinical improvement accompanied by a decrease of IL-6 activity. Withdrawal of prednisone treatment caused reappearance of clinical symptoms and high serum IL-6 activity within a few days. TNF activity could not be detected in the samples of normal dogs, convalescent JPS, or acute JPS dogs. These studies support a role for IL-6 in the pathogenesis of JPS. PMID- 7554476 TI - Role of the immune response in interstitial cystitis. PMID- 7554475 TI - Interleukin 6 during active visceral leishmaniasis and after treatment. AB - To determine the utility of the serum concentrations of interleukin 6 (IL-6) as a marker of disease activity and therapeutic efficacy in visceral leishmaniasis (VL), IL-6 levels were measured in 19 patients with active VL from Sudan before and after treatment. IL-6 levels were 30 +/- 6 pg/ml during the active phase of the disease, decreased to levels around the detection limit of the assay (2 pg/ml) directly after successful antimony therapy, and remained low or undetectable for up to 6 months in persistently cured patients (P < 0.005 versus baseline). In 2 patients who had a relapse of VL, IL-6 was elevated at the time the relapse was diagnosed. Two patients with post-kala-azar dermal leishmaniasis did not have detectable IL-6 in their circulation. Sequential measurements of serum IL-6 levels may be useful for monitoring therapeutic efficacy in patients with VL. PMID- 7554478 TI - Immunosuppression in experimental cryptococcosis: variation of splenic and thymic populations and expression of class II major histocompatibility complex gene products. AB - Previous studies from our laboratory have shown that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a nonrelated antigen, the human serum albumin (HSA). In the present study, we determined the variation of thymus and spleen cell populations in rats infected with C. neoformans and immunized with HSA-CFA at the time when suppressor activity was demonstrated. At 21 days postinfection, the number and the percentage of CD4+CD8+ cells were significantly increased in the thymus together with a minor imbalance in other thymocytes subsets. The study of two class II molecules encoded within the major histocompatibility complex, IA and IE, showed that the total number of class II IA-positive cells was increased in the glands of animals infected when compared to the glands of animals only immunized, while the corresponding percentages were lower than those in control rats. On the contrary a significant increase in both the number and the percentage of IE phenotype was observed in the thymus of infected rats, compared to the animals that were only immunized and used as a control. The IE/IA phenotype ratio within each group was increased in rats injected with the fungus. The study of spleen populations revealed an increase in CD4+ and CD8+ cells and a decrease in the B cells. The IE antigen was increased in the spleen of infected animals. The IA molecule expression showed no difference between the infected animals and the control groups. The IE/IA phenotypes ratio was mildly increased in the spleen of infected rats. These findings reveal that the cryptococcal infection can render an important imbalance in the thymus and spleen T-cell compartment together with a significant increase in the expression of the IE molecule at the time when the suppressor activity was demonstrated in this model. PMID- 7554477 TI - Virological markers in cerebrospinal fluid are predictive of ovine lentivirus associated subclinical encephalomyelitis. AB - Encephalomyelitis is a sequela to ovine lentivirus (OvLV) and human immunodeficiency virus infections. Examination of autopsy tissue from 38 naturally infected asymptomatic sheep showed that 7 (18%) had subclinical neurological lesions characterized by perivascular and periventricular infiltrates of lymphocytes and histiocytes in the leptomeninges, cerebral white matter, choroid plexus, and/or cervical spinal cord. Intralesional histiocytes were shown to contain lentiviral capsid proteins or RNA. Infectious virus (2/7), viral proteins (4/7), and antiviral antibody (5/7) were only detected in cerebrospinal fluid (CSF) from animals with central nervous system (CNS) lesions associated with OvLV infection, suggesting that such virologic markers in CSF, when used concurrently, are predictive of pathologic changes specific to the CNS. PMID- 7554479 TI - A possible role of TGF-beta in the formation of malignant effusions. AB - The detailed mechanisms underlying the formation of malignant effusions are incompletely defined. In order to determine whether transforming growth factor beta (TGF-beta) would contribute to the formation of malignant effusions, we investigated the effect of TGF-beta on the morphology, growth, and permeability of human mesothelial cells, which are thought to serve as a permeability barrier in the pleuroperitoneal cavities. Treatment of the mesothelial cells with a TGF beta dose ranging from 0.1 to 10 ng/ml for 96 hr induced distinct morphologic changes in the cells. Each cell increased in size as did the volume of the intercellular spaces. TGF-beta also significantly inhibited the growth of mesothelial cells at a concentration ranging from 0.1 to 10 ng/ml. This growth inhibition was blocked completely by the addition of anti-TGF-beta antibody. Treatment of the mesothelial cells with 2.0 ng/ml TGF-beta significantly increased the permeability of a mesothelial cell monolayer as assessed by a FITC albumin permeability assay. In our clinical analysis using 10 effusion samples obtained from patients with various types of carcinoma cells, considerable level of TGF-beta could be detected by ELISA, ranged from 0.90 to 8.75 ng/ml. Our data suggest that TGF-beta plays an important role in the formation of malignant effusions through structural and functional damage to the mesothelial cells. Malignant effusions may accumulate in the pleuroperitoneal cavity as a result of the mesothelial cell damage caused by this cytokine which is released from disseminated cancer cells. PMID- 7554480 TI - Animal models of multiple sclerosis. PMID- 7554481 TI - During HIV infection, CD4+ CD38+ T-cells are the predominant circulating CD4+ subset whose HLA-DR positivity increases with disease progression and whose V beta repertoire is similar to that of CD4+ CD38- T-cells. AB - Three-color automated flow cytometry was carried out on peripheral blood CD4+ and CD8+ T-lymphocytes of 42 HIV-positive patients using tri-color anti-CD4 or anti CD8, phycoerythrin-anti-CD38, and fluorescein-anti-HLA-DR, mAbs to elucidate further the T-cell activation hypothesis recently proposed to explain CD4+ T-cell abnormalities observed during HIV infection. CD4+ CD38+ T-cells constituted the major part of circulating CD4+ T-cells in HIV-infected patients and their HLA-DR molecule positivity increased as their disease progressed. The level of CD38 and HLA-DR expression on CD4+ T-cells was positively correlated to that of CD8+ T cells and to the level of beta 2-microglobulin. Next, to determine whether CD38 expression was associated with a selective expansion or deletion of V beta gene defined subsets, we compared the V beta gene frequencies between CD38+ and CD38- T-cells from HIV-infected CDC stage II patients using 13 mAbs specific to V beta families. While selective expansion of certain V beta families was observed in CD4+ and CD8+ T-cells the T-cell receptor V beta subset distribution was similar among CD38+ and CD38-, CD4+ and CD8+ T-cells, suggesting that CD38+ expression was either independent of an HIV-encoded antigen-driven process or rather indicative of T-cell immaturity. It is proposed that the phenotype of circulating CD4+ and CD8+ T-cells of HIV-infected patients is a feature of two different mechanisms: (i) an in vitro activation state responsible for increased DR expression and selective expansion of V beta gene-defined subsets, and (ii) T cell immaturity due to an increased turnover of these cells and accounting for increased CD38 expression. PMID- 7554482 TI - Experimental autoimmune encephalomyelitis in rodents as a model for human demyelinating disease. AB - The cause of multiple sclerosis (MS) is unknown, but the pathology is consistent with an immunological etiology. Studies conducted with the animal model of MS, experimental autoimmune encephalomyelitis (EAE), have provided insight into how the immune system can provoke an immunopathological response characteristic of that seen in MS. The use of inbred rats and mice for studies of EAE has been especially rewarding with respect to the identification of the epitopes of encephalitogenic antigens responsible for the induction of this autoimmune disease and in elucidating the effector mechanism underlying EAE. Moreover, it has also been possible to ascertain how EAE can be regulated, leading to therapeutic modalities which have been applied in clinical studies of MS patients. This review briefly summarizes studies of EAE in rodents, drawing comparisons with immunological findings reported in patients with MS. It is clear that important lessons can be learned from the detailed investigation of animal models that may be applicable to human immunological disorders. PMID- 7554483 TI - CD40 expression by human fibroblasts. AB - The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand. PMID- 7554484 TI - Rabbit anti-chromatin antibodies recognize similar epitopes on a histone H1 molecule as lupus autoantibodies. AB - Autoantibodies in systemic lupus erythematosus (SLE) are believed to be produced through an antigen-driven mechanism, i.e., autosensitization with autoantigens. If this is the case, it is conceivable that antigenic determinants reactive with SLE autoantibodies and induced antibodies raised by immunization are quite similar. To examine this issue, rabbits were immunized with purified histone H1 or chromatin. Although immunization with purified histone H1 did not produce detectable amounts of anti-histone H1 antibodies, immunization with chromatin mounted relatively good antibody responses against not only all histone subunits but also ssDNA. Epitopes on a histone H1 molecule reactive with SLE autoantibodies and rabbit antisera were determined using deletion mutants of a histone H1 molecule. At least two epitopes reactive with induced antibodies were found on a histone H1 and these were closely related to or the same as major autoepitopes in SLE. These data might indicate that B cells producing anti histone H1 autoantibodies are quite similar to those induced by immunization and provide additional evidence that autosensitization with an autoantigen might be operative and a particulate antigen chromatin could be one of the immunogens for the production of anti-nuclear antibodies of various kinds in SLE. PMID- 7554485 TI - Effects of an aminosteroid inhibitor of phospholipase C-dependent processes on the TCR-mediated signal transduction pathway in human T cells. AB - Phospholipase C (PLC) is a key enzyme in the T cell antigen receptor (TCR) mediated signal transduction pathway in human T cells. Agonist-induced PLC activation leads to a cascade of intracellular events that ultimately regulate gene transcription and T cell activation. We studied the effects of U-73122, a putative inhibitor of PLC-dependent events, on TCR/CD3 complex-mediated early and late events in human T cells. Both anti-CD3 monoclonal antibody-induced 1,4,5 inositol trisphosphate (IP3) and free intracytoplasmic calcium [Ca2+]i increases were inhibited by U-73122 (0.05-0.1 microM), but not by the related inactive analog, U-73343. U-73122 did not affect thapsigargin-evoked [Ca2+]i increase in T cells, indicating a specific mode of inhibition of CD3 signaling. Late events in T cell activation like CD3-mediated T cell proliferation and mitogen-induced interleukin 2 receptor (IL2-R) expression were also inhibited by this agent. T cell proliferation induced by a combination of a phorbol ester and ionomycin was not affected by U-73122. Although an agonist effect on basal IP3 and [Ca2+]i levels was observed with high concentrations of U-73122, the inhibitor alone did not induce any proliferative effect or IL2-R expression in T cells. Our results demonstrate for the first time that U-73122 is a specific inhibitor of PLC dependent processes in human T cells and could serve as a valuable tool for studying T cell signal transduction pathways. PMID- 7554486 TI - Anti-cardiolipin antibodies are associated with anti-endothelial cell antibodies but not with anti-beta 2 glycoprotein I antibodies in HIV infection. AB - HIV infection is associated with polyclonal increase in serum immunoglobulins and with elevated titers of serum antibodies to a variety of self antigens, including anti-phospholipid antibodies. In the present study, we found a high prevalence of 46.8% of serum IgG anticardiolipin antibodies (ACA) in a group of 111 unselected HIV-seropositive individuals. The presence of ACA was correlated with that of IgG antibodies to endothelial cells (AECA) but not with that of anti-beta 2 glycoprotein I antibodies, that were only found in 7.4% of the patients. The presence of IgG ACA was not associated with detectable lupus anticoagulant activity, nor with a history of thrombosis. Serum titers of ACA were not correlated with absolute numbers of circulating CD4+ cells. We found no relationship between the presence and titers of ACA, hypergammaglobulinemia, and serum titers of natural IgG autoantibodies to a panel of self antigens. Our results suggest that increased titers of ACA in HIV infection result from a biased expansion of B cell clones producing natural autoantibodies. PMID- 7554487 TI - Natural killer (NK) and lymphokine-activated killer (LAK) activities in a patient who recovered from cancer, and the characteristics of LAK cells generated from CD4-CD8- and CD8+ peripheral blood lymphocytes. AB - The natural killer (NK) and lymphokine-activated killer (LAK) activities of a patient who recovered from bladder transitional cell carcinoma were determined. The patient was diagnosed in 1980 and repeated several therapies until 1986. Since 1987, he has been in a tumor-free state. Recently, very high NK and LAK activities were found. The NK activity was recognized not only against the NK sensitive cell line K562, but also against NK-unsensitive Raji and Daudi cell lines and against autotumor cell line H41. The percentage of CD16+ in peripheral blood lymphocytes (PBL) was 52.9%, the CD8+CD16+ was 26.3%, and the CD4-CD8-CD16+ was 26.6% (17.5, 5.4 and 12.1% in healthy blood donor (control), respectively). Because the patient had large numbers of CD4-CD8- cells in the PBL, CD4-CD8- LAK were generated from them. The CD4-CD8- LAK cells exhibited extremely high killer activity, whereas the CD8+ LAK cells generated from the CD8+ cells showed a low level of killer activity. These results suggest that the tumor-free condition of this patient may be related to these high killer activities. In this patient, several cytokines were also determined, although the results were not completely interpreted. PMID- 7554489 TI - Cutaneous delayed-type hypersensitivity responsiveness in patients during and after Plasmodium falciparum and Plasmodium vivax infections. AB - To assess cellular immune function in malaria, 61 patients admitted to the Bangkok Hospital for Tropical Diseases with Plasmodium falciparum (PF) or Plasmodium vivax malaria were examined with the MULTITEST CMI system (Merieux Institute, Florida) to evaluate delayed-type hypersensitivity (DTH) during and after acute disease over 4 weeks. All patients demonstrated significantly decreased responsiveness to seven commonly encountered recall antigens. This deficit was most severe immediately upon admission (prior to therapy). Uncomplicated Pf cases demonstrated significant hyporesponsiveness only during Week 1. Responses in moderate/severe falciparum and all vivax patients gradually increased in Weeks 2 and 3 but remained significantly below control values. This study confirms functional cell-mediated immune deficits in falciparum malaria and, for the first time, shows hyporesponsiveness in vivax malaria. We conclude that malaria causes a pronounced CMI deficit that is still detectable in some individuals for 3-4 weeks after treatment of acute infection. These changes in DTH should be a consideration in future vaccine development and in evaluation of immune status in endemic areas. PMID- 7554488 TI - CD4+ T cells mediate preexposure-induced increases in murine intraabdominal abscess formation. AB - We have previously shown that an increased number of Escherichia coli/Bacteroides fragilis intraabdominal abscesses are produced in mice after preexposure to small numbers of live E. coli or B. fragilis. Splenic lymphocyte subset changes and the importance of different elements of the immune response in this system were studied. Preexposure to bacteria induced a significant increase in the percentage of splenic T cells without altering the CD4/CD8 ratio. The passive transfer of either 10(7) mixed splenic lymphocytes, 5 x 10(6) mixed T cells, or 2.5 x 10(6) CD4+ T cells from preexposed animals to naive siblings 24 hr prior to abscess induction resulted in increased abscess formation. Transfer of serum, B cells, < 10(7) lymphocytes, CD8+ T cells, or any cell type from naive animals did not change abscess number. The bacterial composition of abscesses changed only in animals receiving either serum or B cells from donors preexposed to B. fragilis, where an increased number of B. fragilis per abscess was found. The CD4+ T cell response can be altered by transient infections and is critical to subsequent abscess formation, and a concurrent humoral response may play a role in determining an abscess' ultimate bacterial composition. PMID- 7554490 TI - Crohn's disease is accompanied by changes in the CD4+, but not CD8+, T cell receptor BV repertoire of lamina propria lymphocytes. AB - To identify disease-specific T cell changes that occur in Crohn's disease (CD), the T cell receptor (TCR) BV repertoires of lamina propria lymphocytes (LPL) isolated from the diseased colon of seven CD patients and eight controls were determined by semiquantitative polymerase chain reaction (qPCR). As an internal control for the effects of HLA and other genes on the TCR repertoire, the BV repertoires of peripheral blood lymphocytes (PBL) from the same individuals were similarly determined and used for comparison. It was observed that the BV repertoires of LPL and PBL within the same individual were very different in both the CD and control groups. However, the CD4+, but not CD8+, repertoires of LPL and PBL differed to a much greater extent in the CD group than in the control group. Furthermore, in each CD patient there was a unique pattern of BV segments which were increased in the CD4+ LPL repertoire relative to that in PBL. These observations suggest that the inflammatory process in CD involves responses by specific CD4+ T cells to specific antigens. The isolation of such inflammation specific CD4+ T cells may make it possible to identify the antigens which are responsible for the inflammatory process in CD and provide a better understanding of its pathogenesis. PMID- 7554492 TI - Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. AB - The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. PMID- 7554491 TI - Vertebrate gastrulation and axial patterning: editorial overview, Part 1. PMID- 7554493 TI - Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector. AB - The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. PMID- 7554494 TI - Mesodermal patterning during avian gastrulation and neurulation: experimental induction of notochord from non-notochordal precursor cells. AB - The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate. PMID- 7554495 TI - Translational control of activin in Xenopus laevis embryos. AB - Activin is a potent mesoderm inducing factor present in embryos of Xenopus laevis. Recent evidence has implicated activin in the inhibition of neural development in addition to the well-established induction of mesoderm in ectodermal explants. These diverse effects are critically dependent on the concentration of activin yet little is known about the mechanisms regulating the level of activin in the embryo. We report that the 3' untranslated region (3' UTR) of activin beta B mRNA inhibits the translation of activin in embryos. Micro injection of activin mRNA from which the 3' UTR has been deleted is 8-10-fold more potent in inducing mesoderm than mRNA containing the 3' UTR. Truncation of the 3' UTR also leads to a marked enhancement of activin protein levels in embryos but has no effect when the truncated mRNA is translated in vitro. The 3' UTR also confers translational inhibition on a heterologous mRNA. These data show that a maternal factor(s) present in X. laevis regulates the translation of injected activin beta B mRNA. This factor(s) could be responsible for regulating the levels of endogenous activin beta B protein during mesoderm induction and the specification of ectodermal derivatives such as neural and epidermal tissues. PMID- 7554496 TI - Cell mixing during early epiboly in the zebrafish embryo. AB - Descendants of early blastomeres in the zebrafish come to populate distinctive regions of the fate map. We present a model suggesting that the distribution of cells in the early gastrula (the fate map stage) results from the passive response of cells to reproducible forces that change the overall shape of the blastoderm just prior to gastrulation. We suggest that one of the morphogenetic changes that accompanies epiboly, the upward doming of the yolk cell into the overlying blastoderm, could be responsible for cell mixing. In support of the model, we show that the timing, extent, and directions of cell mixing in the embryo accurately reflect the expectations of the model. Finally, we show that one portion of the gastrula, a marginal region that later gives rise to many of the mesendodermal derivatives, experiences little cell mixing during the doming process. As a result, this region in the gastrula is populated by the descendants of the subset of the early blastomeres that were originally at the margin. The finding that cytoplasm initially at the edge of the 1-celled blastodisc is transmitted specifically to mesendodermal precursors at the fate map stage raises the possibility that maternal determinants may contribute to initiation of embryonic patterning in the zebrafish embryo. PMID- 7554497 TI - Cloning, sequencing, and expressional analysis of the chick homologue of follistatin. AB - Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin beta A and activin beta B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hindbrain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin beta A during early embryogenesis, whereas activin beta B was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins. PMID- 7554498 TI - Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4. AB - We provide a comparative analysis of the expression patterns and ventral mesoderm inducing properties of Xenopus BMP-2 and BMP-4. Transcripts for BMP-2 and BMP-4 are maternally stored in eggs, and zygotic expression of these genes is uniform in the ectoderm and mesoderm in late blastulae. During gastrulation, BMP-2 is expressed at a low level throughout the ectoderm and marginal zone, but at early neurula stages a patch of dorso-anterior cells displays enhanced expression. In contrast, BMP-4 transcripts are restricted to the ventrolateral marginal zone during gastrulation, and in late gastrula and early neurula BMP-4 is expressed in the epidermis but not the neural plate. At post-neurula stages, BMP-2 and BMP-4 transcripts are associated with a variety of mesodermal structures, including the pharyngeal pouches, heart, blood island, and blastopore. At tailbud stages, BMP-2 and BMP-4 are expressed in neural tissues including the neural tube and brain. In mesoderm induction assays, BMP-2 and BMP-4 induce Xhox3, an early ventral posterior mesoderm marker, and larval alpha Tl globin, a marker for red blood cells. Induction of red blood cells in response to BMP-4 was demonstrated by staining with a hemoglobin-specific reagent. Little is known about factors that induce hematopoietic lineages in vertebrates, and these results provide evidence linking BMP activity and blood differentiation. Globin induction by BMP-2 and BMP 4 is blocked by co-expression of a dominant-negative activin receptor, suggesting that either endogenous activin signals are required for BMP-mediated induction, or that the truncated activin receptor interferes with signaling by BMP receptors. In assays on marginal zone explants, we demonstrate that BMP-4 respecifies dorsal mesoderm to form ventral mesoderm, consistent with its ability to induce blood and to ventralize embryos. BMP-2, however, does not display such activity. The findings extend and support evidence that BMP-2 and BMP-4 function in ventral mesoderm induction and patterning in Xenopus. Our data furthermore highlight the multiple functions these factors fulfill during early vertebrate embryogenesis. PMID- 7554500 TI - Epidemiology of the leishmaniases. AB - The leishmaniases are a group of zoonotic infections caused by protozoan parasites of the genus Leishmania. These infections produce a variety of different clinical diseases depending on the virulence or tropism of the parasite and differential host immune responses. Newly recognized clinical presentations, such as viscerotropic leishmaniasis in American military veterans of Operation Desert Storm, continue to challenge clinicians. Epidemics of classic visceral leishmaniasis leading to thousands of deaths are ongoing in Brazil, India, and the Sudan. Epidemics of localized cutaneous leishmaniasis are ongoing in many areas of South America, North Africa, and Central Asia. A marked increase in cases is often associated with an influx of nonimmune populations into newly cleared agricultural populations into newly cleared agricultural areas or population expansion into previously unsettled areas surrounding cities. The emergence of leishmaniasis as an important opportunistic infection in AIDS patients portends an ominous future as the HIV pandemic sweeps into the hyperendemic areas of South America, Africa, and the Indian subcontinent. Parenteral transmission via needle sharing in HIV coinfected individuals in Spain is an epidemiologically significant new mode of transmission. Finally, recent work has elucidated an enzootic transmission cycle involving L. mexicana in Texas. PMID- 7554501 TI - Leprosy. The beginning of an end to a public health problem? AB - Incidence rates of leprosy seem to be falling in most countries around the world, despite the HIV epidemic. Among the reasons for the declining rates are changing socioeconomic conditions and high BCG vaccination coverage. The numbers of people disabled by leprosy, however, are more important than incidence rates of leprosy per se. The issue of the disabled will remain a public health problem for many decades to come. PMID- 7554499 TI - Expression of HGF/SF, HGF1/MSP, and c-met suggests new functions during early chick development. AB - We report the cloning of fulllength cDNAs for a plasminogen-related growth factor, hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor, c-met, and a close member of the same family, hepatocyte growth factor like/macrophage stimulating protein (HGF1/MSP), from the chick. We have used these cDNAs to provide the first report of the expression of this family of growth factors and the c-met receptor at early stages of vertebrate development. RNAase protection and wholemount in situ hybridization were used on chick embryos between formation of the primitive streak and early organogenesis. We find patterns of expression for HGF/SF and its receptor c-met consistent with their known roles in epithelial-mesenchymal transformation and angiogenesis. In addition, these genes and HGF1/MSP are expressed in discrete locations within developing somites, suggesting a role in paraxial mesodermal development. Very strong and early expression of HGF/SF in the elevating limb buds suggests its involvement in limb outgrowth. HGF1/MSP is expressed in the notochord and then in the prospective floor plate region and could play a role in development of the neural tube. Interestingly, c-met is often more closely associated with HGF1/MSP than with its known ligand, HGF/SF, raising the possibility that c-met expression may be induced by HGF1/MSP. PMID- 7554502 TI - Lyme disease. AB - A clear understanding of the epidemiology of Lyme disease is needed to bring about its effective treatment, prevention, and control. Strategies for effective intervention must be based on quantitative descriptions of the complex interactions of B. burgdorferi with its human and other vertebrate hosts, tick vectors, and the environment. Sound decision making on diagnosis and patient management requires an understanding of the prior probabilities of infection and disease, the performance characteristics of laboratory tests, and the cost benefits of various treatments. Lyme disease is still a relatively new disease for which there are many unanswered questions; it is an exciting and challenging model of an emerging vector-borne zoonotic disease. PMID- 7554504 TI - Epidemiology of human papillomaviruses. AB - The inherent difficulties in the study of HPV infection have made the epidemiology of this common infection complex. The early natural history of HPV infection is similar for all genital and nongenital tissues. Genital HPV infection, however, especially cervical infection, is strongly linked to carcinoma. With the advent of molecular diagnosis of infection, and possible future serologic assays, the natural history of HPV-induced disease and its relationship to the development of invasive carcinoma can lead to more appropriate surveillance and treatment of patients at risk for progression. PMID- 7554503 TI - Epidemiology and prevention of measles. AB - Measles is a highly contagious viral illness which once affected more than 95% of persons in the United States. Since licensure of a safe and highly effective vaccine, measles incidence has fallen to less than 1% of prevaccination levels. A relative resurgence of measles during 1989-1991, however, focused attention on the need to maintain high immunization levels, particularly among preschool-aged children in urban areas. Improved vaccine coverage levels and a second-dose strategy to eliminate susceptibilities resulting from vaccine failure can help achieve the goal of elimination of indigenous measles from the United States by 1996. PMID- 7554505 TI - Kaposi's sarcoma. How can epidemiology help find the cause? AB - Kaposi's sarcoma (KS) remains the most commonly diagnosed cancer in AIDS patients. Neither the cause nor a cure for AIDS-related KS is known. KS serves as a striking example of how epidemiologists seek the cause of any disease. Epidemiologic analysis of reported KS cases is revealing but not definitive. The leading hypotheses for the cause of AIDS-related KS are an as-yet-unidentified sexually transmitted infectious agent and exposure to inhalant alkyl nitrites, often called poppers. Epidemiology suggests that persons can reduce their risk of KS by avoiding nitrite inhalants and changing behavior to reduce the risk of sexually transmitted infections. PMID- 7554506 TI - Skin cancer. AB - It is estimated that 92,000 new cases of melanoma and 2,750,000 cases of nonmelanocytic skin cancer occur worldwide each year. Incidence of these cancers varies more than 100-fold from low rates in Asian populations to very high rates in the white population of Australia. Incidence of melanoma has been increasing in white populations by some 3% to 7% per year over the past 30 years; recent very sharp increases in some populations are probably due to early and increasing detection of cancers that were already there. Incidence of nonmelanocytic skin cancers probably is also increasing. Sun exposure is the main cause of skin cancer, accounting for at least 65% of melanomas worldwide and a much higher proportion in white populations. Pattern as well as amount of sun exposure is important in determining the risk of melanoma and probably also of basal cell carcinoma, with an intermittent pattern being associated with the greatest risk. There is increasing evidence that nonsolar sources of ultraviolet radiation, in particular sunlamps and sunbeds, increase the risk of melanoma, and PUVA therapy and exposure to ionizing radiation are established causes of nonmelanocytic skin cancer. PMID- 7554507 TI - The epidemiology of acquired melanocytic nevi. A brief review. AB - Presence of large numbers of acquired melanocytic nevi is a strong risk factor for malignant melanoma in adults. A series of studies conducted over the past 10 to 15 years have shown that the lighter skin color, propensity to burn rather than to tan in the sun, and frequent episodes of childhood sunburn increase nevus prevalence in childhood and adolescence. Solar ultraviolet radiation exposure itself has been implicated in both the genesis of new nevi in children and the disappearance of nevi in older adult life. PMID- 7554508 TI - Genetic epidemiology of familial melanoma. AB - Genetic epidemiology focuses on the role of genetic factors and their interaction with environmental factors in the occurrence of disease. Approximately 8% to 12% of malignant melanoma cases occur in individuals with a familial predisposition. To date, loci on chromosomes 1p and 9p have been implicated in the development of familial melanoma. Genetic epidemiologic techniques provide tools to study the heterogeneity of familial melanoma and to examine the interaction between genetic and nongenetic components of melanoma. Management of high-risk individuals from melanoma-prone families should aim to prevent disease by reducing exposure to known and suspected risk factors and to detect early melanoma by recognizing and performing biopsies on suspicious-looking nevi. PMID- 7554509 TI - Preventing skin cancer through behavior change. Implications for interventions. AB - Sun exposure is the only major causative factor for skin cancer for which prevention is feasible. Both individual and community-based interventions have been effective in changing sun exposure knowledge and attitudes but generally have not been effective in changing behaviors. An integrative model of behavior change is described that has been successful in changing behavior across a wide range of health conditions. This model holds promise for developing a rational public health approach to skin cancer prevention based on sound behavioral science. PMID- 7554510 TI - The current status of melanoma early detection and screening. AB - Multiple strategies are required to combat the increasing incidence and mortality of cutaneous melanoma. Major questions about melanoma and skin cancer are reviewed, with particular attention to education and early detection. PMID- 7554511 TI - Psoriasis. AB - Epidemiology, by imposing methodologic control and a numerate approach, can provide major contributions to"capture" and understand psoriasis. Certainly, more efficient research strategies in many areas are needed, and consensus should be reached on critical issues such as case classification and onset definition. Multidisciplinary research work and large study networks are requested to progress in a systematic way. One of the ultimate goals of epidemiologic investigations is to identify causes, with a view to developing preventive strategies. It is plausible that the possibility of modulating clinical expressiveness of the disease instead of treating active lesions may become available to clinicians (disability prevention) based on future etiologic research. PMID- 7554512 TI - On the definition and epidemiology of atopic dermatitis. AB - Hospital-derived criteria for atopic dermatitis may need to be refined before being applied to epidemiologic studies. Atopic dermatitis is a common and miserable condition, and environmental factors are probably at least as important as genetic factors in determining disease expression. These findings put us one step nearer to our dream of disease prevention. PMID- 7554514 TI - The epidemiology of occupational contact dermatitis. AB - The dermatologist who is aware of the epidemiology of occupational contact dermatitis (OCD) can find this information helpful in making a diagnosis, determining etiology, and recommending preventive efforts. This article reviews some of the available epidemiologic data sources and their limitations. These data sources provide important information on the prevalence and incidence, the public health importance, the risk factors, the common etiologic agents, the prognosis, and the preventive measures for OCD. PMID- 7554513 TI - Epidermolysis bullosa. Application of epidemiologic principles to the study of a group of rare diseases via a disease registry. AB - The study of rare diseases may provide unique insights into new mechanisms of disease. Such research is difficult to perform, however, owing to a variety of problems associated with patient recruitment and classification, and the biases that are frequently associated with the study of rare diseases. Despite its many limitations, establishment of disease registries may facilitate this research, as evidenced by the depth and breadth of findings recently obtained in inherited epidermolysis bullosa via such a mechanism. PMID- 7554515 TI - Epidemiology of adverse cutaneous reactions to drugs. AB - Adverse cutaneous reactions to drugs are among the most frequent adverse reactions to many systemically administered therapeutic agents. In addition to these reactions resulting in discontinuation of drugs important to a patient's treatment, some adverse cutaneous reactions to drugs may result in substantial morbidity or even death. The early recognition of these clinically important reactions is made on clinical grounds. Epidemiologic data do suggest, however, that a relatively small number of drugs are responsible most often for these most serious reactions. PMID- 7554516 TI - Dermatologic health services research. AB - Health services research is a field that uses techniques from many other disciplines to study health care, especially its quality and costs. This article presents examples that illustrate how health services research addresses the delivery of dermatologic care--for example, the development and use of sound quantitative measures of the outcomes of our care, the implementation of strategies to improve these outcomes, the assessment of patients' access to care and their satisfaction with care, and determinations of the cost-effectiveness of our care. Following these examples is a brief review of principles of experimental inquiry and common methodologic pitfalls in health services research, as a guide for readers in the interpretation of published studies. PMID- 7554517 TI - Dermatologic uses of interferons. AB - Interferons are members of the cytokine family, with diverse antiviral, antiproliferative, antifibrotic, and immunomodulatory activities. Over the past 10 years, interferon use in clinical dermatology has extended beyond its historical antiviral efficacy. In this article, FDA-approved indications and non FDA-approved dermatologic uses of interferons are reviewed. PMID- 7554518 TI - Compassionate treatment of Wegener's granulomatosis with rabbit anti-thymocyte globulin. AB - The purpose of this study was to evaluate the effect of treatment with anti thymocyte globulin in patients with Wegener's granulomatosis, untreatable with cyclophosphamide and steroids. MATERIALS AND METHODS: Five patients with active Wegener's granulomatosis, either not responsive to standard therapy, or who could not tolerate alkylating agents received a single course of rabbit anti-thymocyte globulin (ATG). RESULTS: Four patients showed a favourable response to treatment with partial or complete remission of disease activity, during a follow-up period of five to twelve months. One patient had progressive retroorbital granuloma, which resulted in enucleation of the eye. Side effects of the ATG treatment were mild with chills and fever during the first infusion, and development of serum sickness in two patients. Two other patients had labial herpes simplex shortly after the start of treatment, no other infectious complications were seen. CONCLUSIONS: We conclude that ATG treatment seems to be an effective treatment for patients with severe Wegener's granulomatosis. PMID- 7554519 TI - An ultrastructural study of glomerular basement membrane in rheumatoid arthritis patients with urinary abnormalities. AB - We measured the thickness of the glomerular basement membrane (GBM) in 48 rheumatoid arthritis (RA) patients with proteinuria and/or hematuria and studied its relationship to clinical features of RA. Ten cases with minor glomerular abnormalities, renal cancer, donor of renal transplantation, were studied as controls. Secondary glomerular diseases and hereditary thin basement membrane disease (TBMD) were excluded. Mean GBM thickness was 289 +/- 74 nm (mean +/- SD) in RA patients, which was significantly thinner than that of control group (342 +/- 38 nm) (p < 0.01). Mean GBM thickness were 276 +/- 72 nm and 336 +/- 68 nm in RA patients with and without gold sodium thiomalate (GST) treatment, respectively (p < 0.05). Mean GBM thickness of RA patients without GST and controls were not different statistically, but RA patients with GST had significantly thinner GBM, compared with controls (p < 0.01). The mean thickness of GBM were also 274 +/- 69 nm and 344 +/- 72 nm in RA patients with and without hematuria, respectively (p < 0.01). According to these results, we suspect that the thinning of GBM in RA patients may be related to GST treatment. PMID- 7554520 TI - Insulin-like growth factor-1 stimulates production of mesangial cell matrix components. AB - Diabetic nephropathy is characterized by mesangial cell proliferation and expansion of the mesangial matrix. Insulin-like growth factor-1 (IGF-1) increases in the kidney early in experimental diabetes. The effect of IGF-1 on mesangial cell proliferation and synthesis of extracellular matrix (ECM) proteins was examined to test the hypothesis that IGF-1 stimulates mesangial cells to synthesize ECM proteins. ECM proteins were measured by immunoprecipitation after metabolic labeling of rat mesangial cells in culture. IGF-1 caused a 2.4-fold increase in mesangial cell proliferation as measured by 3H-thymidine incorporation. IGF-1 caused an increase in cellular laminin, fibronectin and type IV collagen, 46.8 +/- 15.4%, 31.3 +/- 11.4%, and 27.7 +/- 12.6% increase respectively compared to control cells. IGF-1 did not effect cellular type 1 collagen, decrease of 8.2 +/- 8.7%. There was a trend toward increased total protein synthesis by IGF-1, 36.5 +/- 2.5%. In summary, IGF-1 stimulates ECM component production by mesangial cells. Thus, IGF-1 has the capacity to mediate the histologic changes characteristic of diabetic nephropathy. PMID- 7554521 TI - The efficacy of cyclosporine A in adult nephrotic syndrome with minimal change disease and focal-segmental glomerulosclerosis: a multicenter study in Korea. AB - A multicenter prospective study conducted in four university hospitals in Korea investigated the efficacy and tolerance of cyclosporine A (CyA, Sandimmun) in 30 patients with adult nephrotic syndrome [25 patients with minimal change disease (MCD) and 5 with focal-segmental glomerulosclerosis (FSGS)]. After a 6-week washout period, CyA 5 mg/kg/day and prednisolone 10 mg/day were administered for up to 8 months, depending on responses to CyA. The rate of relapse after withdrawal of CyA was assessed up to 10 months. Of the 30 patients enrolled, 3 withdrew prematurely due to adverse events. Of the 27 patients who completed the study, 22 had MCD and 5 had FSGS. The prior steroid responses of these patients were 19 steroid-dependent (SD), 4 frequent-relapser (FR) and 4 steroid-resistant (SR) type. High rates of complete remission were obtained after CyA treatment in both MCD and FSGS patients, 86.4% (19/22) and 80% (4/5) respectively. Previous steroid responses did not affect the response to CyA; complete remission was obtained in 84.2% (16/19) of SD patients and in 75% (3/4) of SR patients. The mean (+/- SEM) duration of CyA treatment to attain complete remission in SD and SR patients was 3.8 (+/- 0.6) weeks and 10.7 (+/- 2.7) weeks, respectively (not significantly different). Tapering or withdrawal of CyA was followed by release of nephrotic syndrome in many patients, and the cumulative relapse rates at month 10 were 68.4% (13/19) in MCD patients and 50% (2/4) in FSGS patients. When considered according to prior steroid responses, the relapse rate was 73.3% (11/15) in SD patients and 50% (2/4) in SR patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554522 TI - "Non-dipper" hypertensive patients and progressive renal insufficiency: a 3-year longitudinal study. AB - Patients with a blunted or absent nocturnal blood pressure (BP) drop may be subject to increased risk for target organ damage. In this 3-year longitudinal case-control study we tested the hypothesis that an association exists between a reduced or absent night-time fall in BP and a future decline of kidney function in renal hypertensive patients. The case subjects were 48 hypertensives with renal insufficiency, divided into two groups according to the presence (dippers: n 20) or absence (non-dippers: n 28) of a nocturnal diastolic BP decline greater than 10% of daytime values, detected by ambulatory BP monitoring. At the baseline evaluation the two groups did not differ with respect to age, sex, body weight, office systolic and diastolic BP, mean daytime ambulatory BP, creatinine clearance, 24 h proteinuria. In the ambulatory BP profiles over a 3-year follow up the nocturnal reductions of systolic and diastolic BP in the dippers were 14% and 15%, respectively, vs 7% and 5% in the non-dippers (p = 0.002/0.003). The non dippers had a faster rate of creatinine clearance decline than the dippers (0.37 +/- 0.26 vs 0.27 +/- 0.09 ml/min/month; p = 0.002). Urinary protein excretion increase was higher in the non-dipper group than in the dipper group (993 +/- 438 vs 691 +/- 222 mg/24 h; p = 0.009). This longitudinal study suggests that the non dipping pattern of ambulatory BP can be associated with a faster progression of renal insufficiency in renal hypertensives and that a proper nocturnal BP control is an additional aim of antihypertensive therapy. PMID- 7554523 TI - A multicenter, randomized, double-blind, placebo-controlled, two-year trial to study the effect of nitrendipine on chronic renal transplant function. AB - The ongoing multicenter, randomized, double-blind, placebo-controlled trial investigates the effect of nitrendipine on kidney function after renal transplantation. Renal transplant recipients (6th-12th postoperative week, serum creatinine < 3 mg/dl) were divided into a normotensive (diastolic blood pressure < 90 mmHg) and a hypertensive group (diastolic blood pressure > or = 90, < 115 mmHg). Normotensive patients are randomly treated for 104 weeks with nitrendipine 2 x 5 mg daily or placebo, hypertensive patients with 2 x 10 mg - 2 x 20 mg nitrendipine daily or placebo and in case of inefficacy with additional antihypertensive drugs. Primary end point of the study is the renal transplant function. The trial was started in June 1990. One hundred and eight patients were included into the normotensive and 138 patients into the hypertensive group. Renal allograft function, cyclosporine trough levels and the donor characteristics were not different between the normotensive and hypertensive groups at entry into the study. After 12 months there was no significant change of renal transplant function in both groups. Cyclosporine trough levels were also similar in the normotensive and hypertensive group after 12 months. As expected, blood pressure decreased significantly after 12 months from 150 +/- 17/95 +/- 11 mmHg to 141 +/- 16/90 +/- 9 mmHg in the hypertensive group (p < 0.01). In contrast, in the normotensive group blood pressure increased significantly from 128 +/- 12/80 +/- 6 mmHg to 135 +/- 15/86 +/- 8 mmHg (p < 0.001). No normotensive but 4 hypertensive patients developed graft failure during the first 12 months of the study. PMID- 7554524 TI - Cholesterol precursor concentration in plasma from patients with chronic renal failure or kidney grafts. AB - Plasma lathosterol concentration which is an index of cholesterol synthesis rate was measured in 36 patients with chronic renal failure (CRF) including 13 who were dialysis independent, 12 receiving hemodialysis and 11 on continuous ambulatory peritoneal dialysis (CAPD), 17 renal transplant recipients and 27 healthy control subjects. Concentrations of plasma lathosterol were significantly (ANOVA p = 0.03) lower in all categories of renal patients excepting those treated by CAPD, compared with control values. In normolipidemic subjects (cholesterol < or = 6.50 mmol/l and triglycerides < or = 2.0 mmol/l) plasma lathosterol levels in hemodialysis patients and renal transplant recipients were still significantly (ANOVA p = 0.02) lower than control values. In transplant recipients, the plasma lathosterol/cholesterol ratio was significantly (p = 0.02) lower than those treated with azathioprine and prednisone compared with those treated with these drugs and cyclosporin. Significant (p < 0.001) positive correlations were recorded between plasma lathosterol concentration and plasma levels of apolipoprotein B (apoB) and very low density lipoprotein (VLDL) lipids in the renal patients. These data suggest that the rate of cholesterol synthesis may be low in undialysed and hemodialysed patients with CRF and in renal transplant recipients, particularly those treated with double therapy, and may be linked to the metabolism of apoB and VLDL. PMID- 7554525 TI - Low molecular weight heparin does not necessarily reduce lipids and lipoproteins in hemodialysis patients. AB - Recent studies have indicated a beneficial effect of one particular low molecular weight heparin preparation (Fragmin) on lipid metabolism in patients on chronic hemodialysis as compared to unfractionated heparin. We conducted a prospective crossover study with paired comparison of two different anticoagulant agents to examine the effects of a recently released new low molecular weight heparin (Sandoparin) on lipid and lipoprotein parameters in 24 patients starting hemodialysis. During the first six months of observation patients received Sandoparin. Then patients were switched to unfractionated heparin and observed for further six months. After switching from Sandoparin to unfractionated heparin we observed significant decreases in total cholesterol (from 168.6 +/- 42.2 to 154.4 +/- 41.9 mg/dl, p < 0.02), LDL cholesterol (from 106.4 +/- 35.2 to 89.9 +/- 32.3 mg/dl, p < 0.005), triglycerides (from 148.7 +/- 85.0 to 121.4 +/- 88.8 mg/dl, p < 0.05) and apolipoprotein B (from 100.0 +/- 35.3 to 89.9 +/- 30.4 mg/dl, p < 0.05) and a significant increase in HDL cholesterol (from 32.8 +/- 12.5 to 37.7 +/- 17.5 mg/dl, p < 0.02). This is in contrast to earlier results and can possibly be explained by a higher percentage of fractions with high M(r) in the investigated Sandoparin, which results in a more pronounced depletion of lipoprotein lipase. Together with the enhanced hepatic clearance of lipoprotein lipase induced by low molecular weight heparins, this may decrease lipoprotein lipase activity with a subsequent increase in plasma triglycerides, total and LDL cholesterol. We conclude from our data that a general recommendation for clinical use of low molecular weight heparin in hemodialysis patients cannot be given. PMID- 7554526 TI - The pathogenesis of decreased aspartate aminotransferase and alanine aminotransferase activity in the plasma of hemodialysis patients: the role of vitamin B6 deficiency. AB - The mechanisms responsible for the low plasma (p) AST and ALT activity in HD patients remain elusive. This prospective study was undertaken to clarify the relationship of AST, ALT and pyridoxal-5'-phosphate (PLP) levels (active form of vitamin B6) in these patients. A group of 52 HD patients were given oral pyridoxine HCl (30 mg daily), for 5 weeks. Prior to supplementation (Day 0), 17 (33%) patients had deficient pPLP levels (Group 1), the remainder (35 patients) had normal pPLP values (Group 2). A positive correlation was noted between pPLP and pAST (r = 0.57, p < 0.01) or pALT (r = 0.68, p < 0.01). The mean pAST (9.2 +/ 0.3 vs 13.4 +/- 0.7 U/l) and pALT (8.6 +/- 0.6 vs 11.4 +/- 0.9 U/l) were markedly lower in Group 1 than those in Group 2, respectively. These low AST and ALT levels increased to those seen in Group 2 where no change from basal values was seen despite vitamin B6 supplementation (Day 35). The administration of vitamin B6 resulted in a significant increase in pPLP in both groups (Day 35) but pPLP dropped to subnormal levels in 5 patients in Group 1 and 7 in Group 2 three months after supplementation was stopped (Day 125). The mean AST and ALT of these 12 pPLP deficient patients became lower than those of the remaining 40 patients with normal pPLP values (p < 0.05). In conclusion, low pAST and pALT levels in HD patients are in part due to a deficiency of pPLP which serves as a coenzyme for these transaminases. PMID- 7554528 TI - A morphometric study of preeclamptic nephropathy with focal segmental glomerulosclerosis. AB - Focal segmental glomerulosclerosis (FSGS) is observed in severe or atypical preeclamptic patients biopsied postpartum. To further define the structural characteristics that might be related to atypical clinical manifestations in this disorder, we analyzed the postpartum renal biopsies of eight preeclamptic patients with FSGS by morphometry and studied the structural-functional relationships. For comparison, three postpartum biopsies with primary FSGS and eight nonpregnant subjects with primary FSGS were also studied. Mesangial volume fraction was increased in the preeclamptic FSGS group being 2 times that of the pregnant or nonpregnant primary FSGS group. Mean surface density of peripheral glomerular basement membrane (PGBM) per glomerulus was markedly decreased in the preeclampsia group being 1/3 that for the pregnant or nonpregnant primary FSGS group. Volume density of cortical interstitium in the preeclamptic FSGS was smaller than that of the primary FSGS group. In preeclamptic FSGS, mesangial volume fraction correlated inversely with PGBM surface area (r = -0.84; p < 0.01) and with creatinine clearance (Ccr) (r = -0.63; p < 0.05). PGBM surface area per glomerulus in preeclamptic FSGS correlated with Ccr (r = +0.63; p < 0.05). Mean volume density of cortical interstitium was not related to Ccr nor with the percentage of glomeruli with FSGS in preeclamptic FSGS. These results suggest that decreased glomerular filtration function in atypical preeclampsia is linked to decreased PGBM surface area, that results from mesangial expansion and particularly from mesangial interposition. PMID- 7554527 TI - Glomerular filtration rate and kidney size in type 2 (non-insulin-dependent) diabetes mellitus. AB - The objective of the present study was to estimate glomerular filtration rate and kidney size in recently diagnosed and long-term type 2 (non-insulin-dependent) diabetic subjects. The study design comprised of a population-based controlled cross-sectional survey of middle-aged type 2 diabetic subjects in the City of Tampere, Southwest Finland. One hundred and fifty consecutive recently diagnosed and 146 long-term middle-aged type 2 diabetic subjects with a disease duration of at least five years and one hundred and fifty age- and sex-matched (to recent diabetic subjects) non-diabetic control subjects were recruited. The glomerular filtration rate by single-shot 51Cr-EDTA clearance and kidney size by native X ray tomography were measured. The glomerular filtration rate (ml/min/1.73 m2) was increased in both recently diagnosed (males 121 [27] and females 112 [27]) and long-term (males 123 [24] and females 102 [36]) diabetic subjects (corrected for age) compared to control subjects (males 111 [26] and females 93 [17]). The kidney areas (cm2) were greater in both recent diabetic (males 116.6 [15.4] and females 99.1 [15.3] and long-term diabetic (males 118.3 [15.8] and females 100.4 [15.2]) subjects than in the control group (males 104.3 [12.0] and females 88.6 [12.0]). All differences between diabetic subjects and non-diabetic subjects were statistically significant (p < 0.05), except that between long-term diabetic and non-diabetic females for glomerular filtration rate (p = 0.07). Analyzed by linear regression glomerular filtration rate was related to kidney area in all study groups and to hemoglobin A1c in long-term diabetic males.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554530 TI - A mathematical equation to differentiate overload proteinuria from tubulo interstitial involvement in glomerular diseases. AB - Determination of marker proteins like albumin and alpha 1-microglobulin allows to differentiate various types of proteinuria in kidney diseases. In the present communication we calculate the degree of tubulointerstitial involvement by quantitation of the tubular marker alpha 1-microglobulin in urine in relation to albuminuria. A mathematical relation between minimal tubular proteinuria with the degree of albumin excretion was observed. Cases forming this line did not exhibit interstitial fibrosis when analyzed histologically. In contrast most cases exhibiting higher excretion rates of the tubular marker showed various degrees of tubulointerstitial involvement. In order to differentiate interstitial contribution from overload tubular proteinuria in patients with an albumin excretion rate above 3000 mg/g creatinine alpha 1-microglobulin (measured) is suggested to be corrected by the "glomerular" component of alpha 1-microglobulin using the following equation: "tubulo-interstitial alpha 1-microglobulin" = alpha 1-microglobulin (measured) -4.7 exp (2.2 x 10(-4)) [albumin]. Alternatively the correction can be performed graphically. This procedure may be of considerable help in preventing misinterpretations of urinary protein patterns in patients with nephrotic proteinuria. PMID- 7554529 TI - Urinary alpha 1- and beta 2-microglobulin in light chain proteinuria. AB - Thirteen patients with light chain (LC) proteinuria (11 with multiple myeloma and two with monoclonal gammopathy of undetermined significance) were followed up for one to 3.5 (median 2.5) years and studied for urinary excretion of LCs, alpha 1 microglobulin (alpha 1 M), beta 2-microglobulin (beta 2M), albumin, and serum creatinine concentration at intervals of 6 +/- 2 months. At the beginning of the follow-up, urinary alpha 1M excretion correlated with that of beta 2M (r = 0.81, p = 0.0007) and LCs (0.69, p = 0.0084), and with the serum creatinine level (r = 0.56, p = 0.047). During the follow-up period, renal function remained stable in eight patients despite high or fluctuating LC excretion. In seven of them, urinary alpha 1M was repeatedly below 150 mg/24 h and in one it transiently exceeded that level. The remaining five patients had an unstable renal function (deterioration in four, improvement in one) although their urinary LC and albumin excretion during the study period was comparable to those in patients with stable renal function. In the five patients with unstable renal function, high (> 150 mg/24 h) urinary alpha 1M excretion was associated with a rise in the serum creatinine level. alpha 1M excretion was thus found to be a useful indicator of renal damage caused by LCs. PMID- 7554531 TI - Ultrapure polymerized bovine hemoglobin (UPPBHb) improves integrity of the isolated perfused rat kidney (IPRK): effects on function and structure. AB - To test the oxyphoretic properties and potential nephrotoxic side-effects of polymerized hemoglobin solutions, isolated rat kidneys were perfused in a recirculating system for 180 min. Group I was perfused with a substrate enriched Ringer solution containing hydroxyethylstarch (HES) to produce isoncotic conditions. In group II HES was substituted in part by UPPBHb (34 g/l) with a high portion of low molecular weight molecules (= UPPBHb1). In group III 34 g/l of UPPBHb containing an increased fraction of high molecular weight polymers (= UPPBHb2) was used. Only UPPBHb2-perfused kidneys showed a reduced renal perfusate flow (RPF, 13.3 +/- 1.1 ml/min g kw), when compared to HES-perfused controls (15.5 +/- 0.8) and UPPBHb1 (15.1 +/- 1.2). Glomerular filtration rate (GFR) was significantly higher in UPPBHb1-perfused kidneys (902 +/- 107 vs 633 +/- 55 microliters/min g kw for HES). This difference became even more pronounced in the third hour of perfusion (474 +/- 125 vs. 103 +/- 33). In contrast, UPPBHb2 produced low initial GFR levels of 385 +/- 25, which had only a minor tendency to decline with time. Parallel to GFR, absolute reabsorption of sodium (TNa) andoxygen consumption (QO2) showed values of 110 +/- 16 and 5.46 +/- 0.33 mumol/min g kw in UPPBHb1-kidneys vs 83 +/- 6 and 5.09 +/- 0.27 in controls and vs 53 +/- 4 and 3.66 +/- 0.12 in UPPBHb2-kidneys. Fractional excretion of sodium (FENa), of potassium (FEK), and of water (FEH2O) in UPPBHb1 and UPPBHb2-perfused kidneys were not significantly different from HES-perfused controls at any time of perfusion. Urinary flow rate (UFR) was similar in UPPBHb1- and HES-kidneys. Nevertheless, control kidneys tended to render oliguric during the third hour of perfusion (UFR 19.9 +/- 4.1 microliters/min g kw), whereas UPPBHb1 preserved urinary flow in a better way (83.7 +/- 32.4). UFR of UPPBHb2-kidneys was significantly reduced initially (30.2 +/- 5.1 vs. 105 +/- 33 for HES), but increased steadily up to 67 +/- 23. In the UPPBHb1 and HES group, all functional parameters determined declined dramatically within the third hour of perfusion, whereas UPPBHb2 produced functional stability. The in vivo reaction pattern of renal autoregulation was better preserved in UPPBHb-perfused kidneys than in HES perfused controls: 74 +/- 6 vs. 59 +/- 5 vs. 42 +/- 4% (of full autoregulatory response) for UPPBHb1, UPPBHb2, and HES kidneys, respectively. Light- and electron microscopic analysis revealed major alterations only for the outer medulla of HES-kidneys.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554532 TI - Erythrocyte glutathione peroxidase and superoxide dismutase activities in different stages of chronic renal failure. AB - Antioxidant enzyme activities, glutathione peroxidase (GSH-Px) and Cu, Zn superoxide dismutase (SOD-1), were investigated in erythrocytes of non-dialyzed patients with varying degrees of chronic renal failure (CRF), and of patients on regular hemodialysis treatment. The results obtained have shown that GSH-Px and SOD-1 activities were significantly higher in erythrocytes from non-dialyzed CRF patients than in corresponding age-matched healthy controls. Antioxidant enzyme activities increase with the progression of chronic renal insufficiency, and the more pronounced augmentation in GSH-Px and SOD-1 activities were found in erythrocytes from the end-stage renal patients (with Ccr less than 20 ml/min). The increase of GSH-Px and SOD-1 activities seen in non-dialyzed CRF patients were abolished in patients undergoing regular hemodialysis treatment. We propose that the increased GSH-Px and SOD-1 activities could be a protective mechanism for the cells due to the hyperproduction of free radicals in chronic renal failure. The lowering of red blood cell antioxidant activity in uremic patients on chronic dialysis may contribute to the increased oxidative damage in uremia and in the development of uremic complications. PMID- 7554533 TI - Virological characteristics of hepatitis C virus infection in chronic hemodialysis patients: a cross-sectional study. AB - Detection of hepatitis C virus viremia (HCV RNA) in serum of hemodialysis (HD) patients is crucial for documenting ongoing infection because the clinical and epidemiological importance of anti-HCV positivity is not clear. HCV viremia was studied in 104 HD patients by reverse transcription polymerase chain reaction (RT PCR) using primers localized in the 5' non-coding region of the viral genome. We used two different methods to detect HCV RNA: a direct PCR amplification of HCV RNA from human serum, and a standard RT PCR procedure (requiring the RNA extraction step). There were 50 (48%) anti-HCV positive patients in this population. Twenty-two (21.1%) out of 104 patients showed HCV RNA in serum by standard RT PCR technique: they belonged to the anti-HCV positive patient group, whereas all anti-HCV negative patients were HCV RNA negative. Prevalence of HCV RNA was more than doubled when standard RT PCR was used compared to direct RT PCR protocol. There was a good association between serum HCV RNA and circulating anti HCV antibodies, tested by second-generation ELISA and RIBA assays. HCV viremia was not associated with either the presence or the absence of a particular RIBA antibody specificity. AST and ALT levels had no predictive value for HCV viremia, because they were repeatedly normal in the majority of viremic patients (16/22: 73%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554534 TI - Peritoneal clearance of inorganic sulfate. AB - The peritoneal clearance of sulfate was studied in a group of 12 stable CAPD patients and compared to the clearance of other well-studied solutes such as urea, phosphate, and creatinine. For this purpose we obtained 24 h peritoneal dialysate collections and performed a modified 6-hour peritoneal equilibration test. The results showed that the peritoneal clearance of sulfate is very similar to those of creatinine and phosphate but significantly smaller than the clearance of urea. Decreased daily excretion of sulfate in relation to protein catabolic rate was noted and requires further studies to define its mechanisms. PMID- 7554535 TI - Fluconazole treatment of candida peritonitis with delayed removal of the peritoneal dialysis catheter. AB - Candida peritonitis was treated with fluconazole in ten continuous ambulatory peritoneal dialysis (CAPD) patients without immediate removal of the peritoneal catheter. Shortly prior to diagnosis, six patients (60%) had received broad spectrum antibiotics. Gram stain of peritoneal fluid detected yeast in 70% of cases. In eight patients the peritoneal dialysis catheter was removed within one week of diagnosis because of clinical deterioration. In the majority of cases (90%), candida peritonitis resolved only after catheter removal in spite of ongoing fluconazole therapy. Fluconazole was well tolerated by all patients. PMID- 7554536 TI - IgA nephropathy associated with hyper IgAnemia, psoriasis or pustulosis and ossification. AB - This is a report on two cases of IgA nephropathy associated with psoriasis vulgaris, having hyper IgAnemia (above 500 mg/dl) and ossification. Case 1 is a 47-year-old woman with a 7-year history of psoriasis vulgaris, and case 2 is a 57 year-old man with a 17-year history of this disease. IgA was 526 and 1,356 mg/dl, respectively. HLA analysis showed A2, A26 (10), Bw62 (15), Bw46, Cw3, DRw12 (5), and DRw8 in the former, and A2, A11, B13, Bw46, Cw11, DR4, and DRw8 in the latter. Renal biopsy specimens disclosed mild mesangial proliferative glomerulonephritis and moderate mesangial proliferative glomerulonephritis with predominant IgA deposition in mesangial area, respectively. A bone-scintigraphy revealed a high uptake of radioisotopes in the left shoulder, the vertebra, the sacroiliac joint, both sides of the knees and ankles, and the sterno-cost clavicular area. An X-ray study showed ossification of the posterior longitudinal ligament (OPLL) in the former, and ankylosing spinal hyperostosis (ASH) in the latter. A review of the literature discloses three other case reports of hyper IgAnemia, IgA nephropathy, psoriasis or pustulosis, and ossification. The alertness of dermatologists, orthopedic surgeons, rheumatologists, and general practitioners will be required to attain to a more frequent diagnosis of the above combination. PMID- 7554537 TI - The effect of anti-HCV seropositivity on the TNF-alpha level in hemodialysis patients. PMID- 7554539 TI - Viable myocardium, infarct artery patency, and collaterals to the infarct zone. PMID- 7554538 TI - Streptokinase gene variable region classification in streptococci: lack of correlation with post-streptococcal glomerulonephritis. AB - To investigate a possible causal role of streptokinase (SKase) in acute post streptococcal glomerulonephritis (APSGN), the major variable region of SKase genes of Streptococcus pyogenes strains isolated from patients with and without APSGN were analyzed using the polymerase chain reaction, restriction enzyme analysis and the direct sequencing of SKase genes. In the APSGN-associated strains, six of nine revealed mutant classes corresponding to the nephritogenic classes I and II proposed by Johnston et al. [1992], the remaining three belonged to non-nephritogenic classes. In twenty strains not associated with APSGN, seventeen belonged to classes I and II, while three were from other classes. The major variable region of the SKase gene shows no apparent relation with induction of APSGN in humans, suggesting that unique classes of streptococcal SKase do not play a role in the pathogenesis of APSGN. PMID- 7554540 TI - The history of cardiology--is it important today? PMID- 7554541 TI - Electrolyte disturbances in chronic heart failure: metabolic and clinical aspects. AB - The pathophysiology of congestive heart failure (CHF) includes conditions (e.g., activation of the renin-angiotensin-aldosterone system) which, when combined with CHF therapies, make patients afflicted with this syndrome quite susceptible to electrolyte disturbances. The most commonly encountered are hyponatremia, hypokalemia, and hypomagnesemia. These derangements are of vast clinical importance; their development not only represents an immediate threat to the CHF patient (e.g., dysrhythmias secondary to hypokalemia), but are also indicative of underlying pathophysiologic events, an unfavorable clinical course, and occasionally an adverse therapeutic response. The optimal care of CHF patient includes the recognition and management of these electrolyte disturbances. PMID- 7554543 TI - In consecutive patients hospitalized with acute myocardial infarction, infarct location according to routine electrocardiogram is of minor importance for the outcome. AB - Most studies have suggested that patients with anterior myocardial infarction have an adverse prognosis compared with patients with inferior infarction. The objective of this study was to compare the mortality and morbidity in anterior versus inferior acute myocardial infarction (AMI) during 1 year in a consecutive series of patients hospitalized with AMI. All patients fulfilling the criteria for AMI who were admitted to a single hospital during 21 months (n = 921) participated in the study. Patients with anterior infarction (n = 312) had a 1 year mortality rate of 26% versus a rate of 24% for patients with inferior infarction (n = 269) (p > 0.2). The corresponding figures for patients with no previous infarction who developed Q waves were 27 and 21%, respectively (p > 0.2). Reinfarction, thromboembolic events, and other aspects of morbidity during long-term follow-up appeared with similar frequency in the two groups. Thus, in a nonselected group of patients admitted to a single hospital because of AMI, the prognosis was found to be similar among patients with inferior and those with anterior infarction. In the subset of patients with a first myocardial infarction who developed Q waves, there was a trend indicating higher mortality in anterior infarction. PMID- 7554542 TI - Ventricular tachyarrhythmias, myocardial ischemia, and sudden cardiac death in patients with hypertensive heart disease. AB - Hypertensive heart disease is increasingly considered to be a strong and independent risk factor for sudden cardiac death. Ventricular tachyarrhythmias in these patients are common and mainly the result of electrophysiologic abnormalities and increased electrical vulnerability of the hypertrophic myocardium. However, proarrhythmia in the hypertrophic heart often is facilitated and aggravated by electrolyte disturbances, the sympathoadrenergic tone, transient blood pressure crisis, and especially by the occurrence of myocardial ischemia. Myocardial ischemia in the setting of hypertensive heart disease may result from stenotic lesions in large and/or small coronary artery vessels and, in the absence of both, will result from the altered cellular oxygen supply and consumption in the hypertrophic myocardium. Recent studies have shown that acute and transient myocardial ischemia are common in many hypertensives, often fail to be symptomatic, and that the dynamic interaction of left ventricular hypertrophy, transient myocardial ischemia, and ventricular tachyarrhythmias may provide a crucial link for the high incidence of sudden cardiac death in patients with hypertensive heart disease. PMID- 7554544 TI - ST-segment depression on ambulatory electrocardiography in the early in-hospital period after acute myocardial infarction predicts early and late mortality: a short-term and a 3-year follow-up study. AB - A surveillance study was conducted to determine the in-hospital and long-term prognostic value of ST-segment depression assessed by ambulatory electrocardiographic monitoring (AEM) during the early in-hospital period after acute myocardial infarction (AMI). ST-segment depression (STD) was determined by computer analysis of 24-h ECG tapes as a horizontal or downsloping change in ST level by > 0.1 mV from the reference base line. The ST level was measured 80 ms after the J point of all normally conducted complexes for > or = 1 min. All computer-detected ST events were verified by one trained reader. Tapes corresponding to 74 patients were analyzed. In addition, 23 tapes corresponding to age- and gender-matched controls were also analyzed. Patients were divided into two groups: 22 patients (30%) showed STD (Group A), and 52 patients (70%) had no episode of STD (Group B). Among controls, 1 person (4%) showed STD. During the early follow-up period (14 +/- 11 days after hospital admission), cardiac events occurred in 11 patients [7 (32%) in Group A and 4(8%) in Group B, p < 0.01], including 6 cardiac death [5 (23%) in Group A and 1 (2%) in Group B, p < 0.01], 3 acute coronary artery bypass surgeries [2 (9%) in Group A and 1 (2%) in Group B, p = NS], and 2 nonfatal myocardial infractions (both in Group A, p = NS). During a mean follow-up period of 3 years (36 +/- 15 months), 18 patients died [10 (45%) in Group A and 8 (15%) in Group B, p = 0.01]. Eleven deaths were sudden [7 (32%) in Group A and 4 (8%) in Group B, p < 0.01]. Eighteen AMI occurred [11 (50%) in Group A and 7 (13%) in Group B, p < 0.005]. Twenty patients underwent revascularization procedures [7 (32%) in Group A and 13 (25%) in Group B, p = NS].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554545 TI - Evaluation of antihypertensive effects of once-a-day isradipine and fosinopril: a double-blind crossover study by means of ambulatory blood pressure monitoring. AB - We compared the efficacy and tolerability of isradipine (ISR) and fosinopril (FOS) once-a-day administration in 17 outpatients, 9 men and 8 women, aged 35-65 years (mean +/- SD = 58 +/- 10 years), affected by mild to moderate primary systemic hypertension. The patients were given single-blind placebo for 2 weeks and thereafter, in double-blind, randomized, crossover sequence, ISR (5 mg) and FOS (20 mg), both for 4 weeks. At the end of each period, patients underwent 24-h noninvasive blood pressure (BP) monitoring by means of an A&D TM 2420 Monitor Model 7, with readings taken very 10 min during the day (from 7 A.M. to 11 P.M.), and every 20 min during the night (from 11 P.M. to 7 A.M.) Similarly, BP load (BPL) as percentage of systolic and diastolic BP reading > 140 and > 90 mmHg was investigated. Both ISR and FOS induced a highly significant (p < 0.0001) decrease in BP from 158/96 +/- 7/6 mmHg to 133/86 +/- 6/6 and to 132/83 +/- 10/7 mmHg, respectively. Mean BP decreased from 117 +/- 6 mmHg to 102 +/- 6 mmHg (ISR) (p < 0.0001) and to 99 +/- 8 mmHg (FOS) (p < 0.0001). Both ISR and FOS significantly (p < 0.0001) reduced systolic BPL from 78 +/- 16% to 44 +/- 13% and 28 +/- 12%, respectively, and diastolic BPL from 70 +/- 15% to 40 +/- 13% (p < 0.0001) and 35 +/- 13% (p < 0.0001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554546 TI - Long-term denopamine therapy for hemodialysis patients with chronic heart failure. AB - Denopamine was orally administered for more than 12 months to patients with chronic heart failure on maintenance hemodialysis. The plasma level in subjects treated with denopamine at 30 mg/day tended to be higher than that in subjects on 15 mg/day. There was no gradual increase in plasma level as the duration of therapy prolonged. Left ventricular end-diastolic and end-systolic diameters as well as ejection fraction on echocardiography showed a tendency to be improved by denopamine. Similarly, the cardiothoracic ratio was improved temporarily. No adverse effects were detected by electrocardiography and laboratory tests. These observations suggest that denopamine is safe and effective for hemodialysis patients with chronic heart failure. PMID- 7554547 TI - Progression and regression of coronary artery disease--linkage of clinical, pathologic, and angiographic findings. AB - Progression and regression of coronary artery disease are analyzed with respect to clinical, pathologic, and angiographic findings. Clinically, progression of coronary artery disease is highly unpredictable process. The pattern of progression is not linear in time but sometimes is rapid and other times is slow. The atherosclerotic lesion is not pathologically homogeneous. Regression therapies, therefore, might be effective depending on the composition of the lesion and the phase of atherosclerotic evolution. True clinical regression should be concordant with the clinical improvement of symptoms as well as prognosis. Further studies are required to establish the effectiveness of therapy for the prevention of progressive coronary artery disease. PMID- 7554548 TI - Electroconvulsive therapy-induced transient T-wave inversions on ECG. AB - Electroconvulsive therapy (ECT), regarded as safe, well tolerated, and one of the most effective treatments for depression, is used frequently in patients with underlying coronary artery disease. ECT has been associated with ST depression and arrhythmias probably due to increased myocardial demand in patients with coronary artery disease. This report describes a case of transient new T-wave inversions in precordial leads V2 and V3 and flattening of T waves in leads III, V3, V4, V5, and a VF after two courses of ECT in a patient with minimal cardiac risk factors, normal echocardiogram, and normal pre-ECT electrocardiogram (ECG). These T-wave changes may represent increased sympathetic activity induced by ECT. Previous prospective studies of 26 and 21 patients undergoing serial ECT found only one new T-wave inversion and no pathologic Q waves on serial ECGs, suggesting that this is a rare finding. PMID- 7554549 TI - Acute reversal of pheochromocytoma-induced catecholamine cardiomyopathy. AB - Pheochromocytoma is a rare neuroendocrine tumor. We report a case of pheochromocytoma-induced cardiomyopathy in an 18-year old white female. A review of the literature indicated that this is the most dramatic reversal of pheochromocytoma cardiomyopathy reported. With aggressive medical management, there was complete recovery of the hemodynamic, echocardiographic, and electrocardiographic abnormalities within 14 days. This case emphasizes the importance of aggressive and appropriate medical therapy in pheochromocytoma heart disease. PMID- 7554550 TI - Mitral valve prolapse causing severe mitral regurgitation in a patient with absent right pulmonary artery. AB - A 33-year-old man was investigated for dyspnea on exertion and the presence of a pansystolic murmur. Physical examination revealed dextrocardia confirmed by chest radiograph, which also showed oligemic right lung field. Subsequent cardiac catheterization revealed secundum atrial septal defect, persistent left sided superior vena cava, and severe mitral valve prolapse causing severe mitral regurgitation with pulmonary hypertension. The right pulmonary artery was absent. It is the first report of the association between severe mitral valve prolapse and absent right pulmonary artery. PMID- 7554551 TI - William Murrell. PMID- 7554552 TI - Methotrexate: the agent of choice for chronic inflammatory disorders. A perspective ten years later. PMID- 7554553 TI - The role of interleukin-8 and other cytokines in the pathogenesis of Felty's syndrome. AB - OBJECTIVE: Felty's syndrome (FS) is defined as rheumatoid arthritis (RA) with neutropenia and, in some cases, splenomegaly; the outcome is primarily determined by the risk of infection, which is related to the degree of neutropenia. We analysed whether the clinical manifestations of FS, especially neutropenia, could be explained by abnormalities in cytokine production. METHODS: We examined the production in FS of five cytokines involved in the maturation and activation of polymorphonuclear cells (PMNs): IL-1 beta, TNF alpha, IL-8, G-CSF and GM-CSF. Because of the role of systemic IL-8 in neutrophil migration, serum IL-8 levels were also evaluated. RESULTS: Spontaneous and anti-CD16 stimulated cytokine production was similar in FS, RA and healthy controls (NC). However, anti-CD3 stimulated IL-8 production was significantly increased compared to NC in both RA and FS. FS patients who spontaneously produced G-CSF in culture were protected from bacterial infections. Serum IL-8 levels were elevated in FS and RA compared to NC (p < 0.001 for both groups). In FS, serum IL-8 was higher in patients with a history of bacterial infections compared to those without (p < 0.01) and there was a weak inverse correlation between neutropenia and serum IL-8 levels (Kendal's tau B = -0.31, p = 0.05). CONCLUSION: The neutropenia of FS cannot be explained by changes in peripheral blood cytokine production, although changes in the bone marrow microenvironment cannot be excluded. Our data do suggest a possible role for G-CSF and IL-8 in the development of certain FS complications. PMID- 7554554 TI - Vertebral fractures in patients with rheumatoid arthritis treated with corticosteroids. AB - OBJECTIVES: To examine the relationship between roentgenological deformities of the vertebral column and clinical manifestations of vertebral fractures in patients with RA, treated with glucocorticosteroids (Cs). METHODS: In all outpatients of Utrecht University Hospital with RA, who were currently using Cs (n = 52), roentgenograms of the thoracic and lumbar vertebral column were taken. Roentgenological deformities were scored a vue using the Kleerekoper score, and quantitatively using the Heidelberg and Utrecht scoring methods. Clinical manifestation of a vertebral fracture was defined as a vertebral deformity leading to prescription of therapy: bedrest and/or analgesics. Clinical manifestations of fractures were also recorded in a control group of 55 RA patients not on Cs, matched for age and gender. RESULTS: The prevalence of vertebral deformities in the Cs-treated RA patients, assessed with the Kleerekoper score, was 58% (30 of 52 patients). In 7 of the Cs-treated patients clinical manifestations of a vertebral fracture were present, versus 1 in the control group (p = 0.03). No significant correlation between the 3 deformity scores and the cumulative dose of Cs was found. No (inverse) correlation between serum osteocalcin and the daily dosage of prednisone was found. CONCLUSION: Corticosteroids in active RA are associated with fractures, even when low doses are used: the prevalence of vertebral deformities (58%) in the Cs-treated RA patients was much higher than the frequency in postmenopausal women of the same age reported in the literature, and the reported incidence of clinically manifestations of vertebral fractures was higher in the Cs-treated RA patients than in the control group of RA-patients not treated with Cs. PMID- 7554555 TI - Peptide autoantigenicity of the small nuclear ribonucleoprotein C. AB - OBJECTIVE: This study determines the antigenic regions of the nRNP C lupus autoantigen. METHODS: Twenty-one anti-nRNP C sera, six patients with other autoimmune serology, and five normal control sera were assessed for binding with the overlapping octapeptides of the nRNP C protein. Column absorptions were conducted to assess the percentage of the anti-nRNP response which was directed against one major epitode of nRNP C: RESULTS: Eleven regions of nRNP C are bound in different combinations by anti-nRNP C patient sera. Neither normal controls nor patients without anti-nRNP C antibodies significantly bind any regions of nRNP C: The antigenic region spanning amino acids 117-126, PAPGMRPP, is recognized by all twelve Sm, nRNP precipitin positive patients tested. Indeed, from 14 to 32% of the anti-nRNP reactivity in Sm, nRNP precipitin positive patient sera is directed against PAPGMRPP: All of these patients also bind a very similar sequence which is repeated three times in the Sm B/B' protein. Patients sera with only anti-nRNP antibodies, however, do not recognize this proline rich sequence as antigenic. CONCLUSION: This elucidation of the specific areas of nRNP C which are important in the autoantigenicity of nRNP C will hopefully lead to a better understanding of the involvement of these autoantibodies in the etiology and pathogenesis of SLE. PMID- 7554556 TI - Abnormal expression of apoptosis-related antigens, Fas and bcl-2, on circulating T-lymphocyte subsets in primary Sjogren's syndrome. AB - OBJECTIVE: To clarify the pathological role of the apoptosis-related molecules expressed on peripheral blood (PB) lymphocyte subsets in primary Sjogren's syndrome (SS). METHODS: The levels of apoptosis-regulating proteins, Fas and bcl 2, were determined in the PB lymphocyte subsets from 21 patients with SS and 14 healthy controls by 2-color flow cytometry. RESULTS: In the PB from SS patients, lymphocytopenia, especially CD4+ cell-lymphocytopenia, was prominent. As observed in previous studies, the percentages of CD4+ CD45RA+ cells were lower in the SS patients than in the controls, while activated (DR+) cells were increased in CD4+ cells from the patients. Fas+ cells were also increased in the patients' CD4+ cells and CD8+ cells, but not in their B cells or natural killer cells. Furthermore, we observed several positive correlations among the percentages of activated cells (DR+ cells or CD45RA-cells) and Fas+ cells recognized in the CD4+ and/or CD8+ cells from the patients. On the other hand, intra-cellular bcl-2 proteins measured as mean fluorescence intensity were significantly diminished in the CD4+ cells, CD8+ cells, CD19+ cells, CD45RO+ cells and Fas+ cells from 14 SS patients compared with 12 healthy controls. In addition, the numbers and/or percentages of CD4+ cells and Fas+ cells positively correlated with their expression of bcl-2 in SS patients. CONCLUSION: The abnormal balance between Fas and bcl-2 expression detected in the PB lymphocyte subsets from SS patients relates, at least partially, to the lymphocytopenia observed in the patients. PMID- 7554557 TI - Impaired binding capacity of asialyl and agalactosyl IgG to Fc gamma receptors. AB - OBJECTIVE: Autoimmunity in rheumatoid arthritis has been associated with deficient glycosylation of serum and synovial IgG which could potentially be mediated through the binding of the Fc portion of the molecule to its cognate receptor. METHODS: Normal IgG1 and IgG3 were affinity-purified, and sialic acid and galactose were clipped off using neuraminidase and beta-galactosidase, respectively. The binding of these sugar-depleted IgG to the Fc gamma receptors (Fc gamma R)IIIb on polymorphonuclear leukocytes (PMN) was assessed by flow cytometry. RESULTS: The binding of both asialyl and agactosyl IgG1 and IgG3 to PMN was significantly lower than that of the native IgG1 and IgG3. CONCLUSION: These data indicate that agalactosyl and, to a lesser degree, asialyl IgG, do not bind as efficiently as native IgG to Fc gamma R. Such a reduction in the perspective of the heterogeneity of the PMN Fc gamma RIIIb. PMID- 7554558 TI - Yersinia-associated arthritis in rats: effect of 65 kDa heat shock protein, bovine serum albumin and incomplete Freund's adjuvant. AB - OBJECTIVE: We have previously shown that the microbial load of rats has a significant effect on their susceptibility to Yersinia-associated arthritis. In this study our aim was to see whether mycobacterial 65 kDa heat shock protein (hsp) could induce the same suppressive effect in experimental Yersinia associated arthritis as has been reported for arthritides induced by adjuvant, pristane, or streptococcal cell walls (SCW). METHODS: Arthritis was induced by the intravenous injection of Yersinia enterocolitica 0:8 into Lewis rats. Hsp, bovine serum albumin (BSA) or NaCl, administered in incomplete Freund's adjuvant (IFA), was given subcutaneously on day -5 or +5 with regard to the bacterial inoculation. RESULTS: Mycobacterial hsp given in IFA on day -5 significantly suppressed the development of arthritis. However, a similar suppression was observed with BSA or NaCl given in IFA. CONCLUSION: These results, together with those known from the effect of microbial load, suggest that susceptibility to Yersinia-associated arthritis is easily affected by external factors. PMID- 7554559 TI - Prevalence and clinical significance of subungual splinter haemorrhages in patients with the antiphospholipid syndrome. AB - OBJECTIVE: To assess the prevalence and clinical significance of subungual splinter haemorrhages in patients with the antiphospholipid syndrome. METHODS: A group of 147 patients with the antiphospholipid syndrome (124 female, 23 male) were studied. All patient histories were carefully reviewed and each patient was interviewed at least once. Particular attention was paid to clinical manifestations of the antiphospholipid syndrome at the time of subungual splinter haemorrhages. Patients who had evidence of infective endocarditis confirmed by echocardiogram and a positive blood culture were excluded from the study. RESULTS: Six out of 147 patients developed subungual splinter haemorrhages. Amongst 80 patients (54%) with a history of venous thrombosis, only one developed subungual splinter haemorrhages during a lupus flare in the post-partum period. This sign was found in 4 out of 67 patients with arterial occlusions; in 2 of these the occlusion manifested as a cerebrovascular accident. Another patient with a history of lupus and recurrent miscarriages developed subungual splinter haemorrhages during a lupus flare. CONCLUSION: Subungual splinter haemorrhages appeared to be relatively uncommon in our group of patients with the antiphospholipid syndrome. The differences in clinical setting and response to different treatment regimes in these cases suggest varied aetiopathologies and may reflect a number of different types of vascular insult. PMID- 7554560 TI - Ten-year follow up study of patients from a Yersinia pseudotuberculosis III outbreak. AB - A Yersinia pseudotuberculosis serotype III outbreak in 1982 was characterized by a high frequency of post-infectious complications. Ten years later 16 out of the 19 patients originally included in the outbreak were reached for a follow up evaluation. Altogether nine patients suffered from chronic joint symptoms. Four of them were HLA B27-positive. Two of these had ankylosing spondylitis; one with severe erosive polyarthritis and secondary amyloidosis which led to uremia requiring haemodialysis and eventually to death, the other with ankylosis of the lumbar spine and sacroiliitis. None of the patients any longer had detectable anti-Yersinia antibodies. The long-term prognosis of Yersinia-triggered reactive arthritis is discussed. PMID- 7554561 TI - Circulating levels of soluble CD30, a marker of cells producing Th2-type cytokines, are increased in patients with systemic lupus erythematosus and correlate with disease activity. AB - OBJECTIVE: To measure the levels of serum soluble CD30 (sCD30), a marker of cells producing T helper 2(Th2)-type cytokines, in systemic lupus erythematosus (SLE) and undifferentiated connective tissue disease (UCTD), and to determine its value in assessing disease activity. METHODS: Serum levels of sCD30 were measured by ELISA in 21 patients with SLE, in 17 patients with UCTD and in 40 normal donors. Disease activity was evaluated according to the ECLAM scoring system. RESULTS: sCD30 values were 53.84 +/- 58.24 U/mL in SLE, 22.65 +/- 9.82 U/mL in UCTD and 5.3 +/- 5.7 in normal controls (p < 0.0005 SLE vs controls; p < 0.05 SLE vs UCTD). sCD30 levels were directly related to the disease activity (p < 0.002). CONCLUSION: These data support a relationship between the Th2-type immune response and the pathogenesis of SLE, and suggest that sCD30 can be used as a simple marker for the evaluation of disease activity. PMID- 7554562 TI - Blood dehydroepiandrosterone sulphate (DHEAS) levels in pemphigoid/pemphigus and psoriasis. AB - Serum dehydroepiandrosterone sulphate (DHEAS) levels and diagnostic autoantibody titers were measured in patients with pemphigoid/pemphigus [n = 46/4; 21 men and 29 women, 42 to 93 years of age (mean 79)]. Twenty-four patients were either on peroral Prednisolone (n = 11), or topical treatment with betamethasone (n = 13), and the other 26 were either receiving non-steroidal drugs or were untreated. Their DHEAS levels were compared to those of 20 patients with psoriasis, and to 23 patients with secondary osteoarthritis (OA). Assessing the patients by group, the mean DHEAS level was markedly lower in the pemphigoid/pemphigus than in the psoriasis and OA patients (geometric mean 600 vs. 2130 and 2100 nmol/l, respectively; p < 0.001). This difference was independent of steroid treatment. No correlation was found between the DHEAS levels and antibody titers. The low levels found in pemphigoid/pemphigus are concordant with those reported for systemic lupus erythematosus, rheumatoid arthritis and polymyalgia rheumatica/giant cell arteritis. DHEAS deficiency is a permanent feature in these autoimmune diseases, and may contribute to their etiology and/or pathophysiology. PMID- 7554563 TI - Construct validation of the ACR 1991 revised criteria for global functional status in rheumatoid arthritis. AB - In order to assess the construct and discriminatory validity of the 1991 ACR functional status criteria for rheumatoid arthritis (RA), a cross-sectional analysis of 62 consecutive patients with RA (according to the American Rheumatism Association 1987 revised criteria) who were attending the outpatient clinic of rheumatology, University Hospital Zurich, was carried out. A moderate-to-strong association of the ACR criteria with pain (r = 0.54), the tender (r = 0.54) and swollen joint count (r = 0.31), grip strength (r = -0.49), C-reactive protein (r = 0.35), the HAQ disability index (r = 0.76), self-perceived global health (r = 0.58), and the Larsen radiological score (r = 0.32) was found. The mean scores of most disease parameters and all 8 domains of the health assessment questionnaire were significantly different between, and increased regularly across, the 4 classes. We conclude that the ACR 1991 functional status criteria for RA are a valid measure of the consequences of impairment and discriminate well the physical functional status. PMID- 7554564 TI - Serum neopterin concentrations in Wegener's granulomatosis correlate with vasculitis activity. AB - Raised serum neopterin concentrations were found in 13 of 27 patients (47%) with Wegener's granulomatosis. The neopterin concentrations significantly correlated with the Birmingham Vasculitis Activity Score as well as with the erythrocyte sedimentation rate and C-reactive protein. High serum neopterin levels were also associated with infectious complications. The finding of increased neopterin concentrations in patients with Wegener's granulomatosis supports the role of cell-mediated immunity and particularly of activated macrophages in the pathogenesis of the disease. PMID- 7554565 TI - Posture in systemic lupus erythematosus. AB - Systemic lupus erythematosus (SLE) is a connective autoimmune disease that may involve many organ systems and neural function impairment. Postural apparatus dysfunction in SLE may develop in the case of central and peripheral neural involvement. We studied the presence of postural abnormalities in SLE patients with different degrees of disease activity. Twenty-eight subjects (18 SLE patients and 10 normal controls) underwent postural evaluation by means of Static Computerised Posturography. Disease severity or specific clinical problems did not form selection criteria. Lupus activity was assessed using the Systemic Lupus Activity Measure (SLAM). A statistically significant (p < 0.05) increase in the parameters of trace length, trace surface, trace velocity, standard deviation in velocity, and Fast Fourier trX and Y (FET), were found in both the closed and opened eyes tests, when lupus patients were compared to controls subjects. No significant pattern variation in posture was observed between more active and less active SLE patients. Postural control alterations in SLE may be considered a complication of the chronic autoimmune inflammatory process, independent of disease activity as evaluated by the SLAM index. The site of the neuropathy remains uncertain, although we believe it to be peripheral rather than central in origin due to the absence of symptoms or clinical signs of CNS involvement or vascular degeneration. PMID- 7554566 TI - Algodystrophy in conjunction with van der Hoeve's syndrome. AB - Van der Hoeve's syndrome is the all-round variant of Lobstein's disease (late osteogenesis imperfecta) and is characterized by the concurrent presence of osteoporosis, blue sclerae, and deafness. The literature has reported sporadic cases of a combination of algodystrophic syndrome and Lobstein's disease. Our case represents a classic example: a 50-year-old woman with blue sclerae since birth, bilateral deafness, major dental alterations, vitreous osteoporosis and a history of pathological fractures who, two years from the onset of menopause, complained of pain involving the right ankle. After admission to our Department, a diagnosis of algodystrophy combined with van der Hoeve's syndrome was made on the basis of her clinical history, objective signs and x-ray results. Treatment consisted of i.v. clodronate disodium at a dose of 300 mg daily in 250 ml saline solution for 7 consecutive days. At the end of the treatment cycle the patient reported an improvement in her symptoms with nearly total regression of right ankle pain, while swelling disappeared within a few weeks. PMID- 7554567 TI - Orbital pseudotumor as a presenting sign of temporal arteritis. AB - Both unilateral proptosis (2 cases) and radiological evidence of orbital inflammatory pseudotumor in the absence of exophthalmos (1 case) have been separately described as presenting signs of temporal arteritis. We report a patient presenting with bilateral exophthalmos associated with CT and MRI signs of orbital inflammation, who had biopsy-proven temporal arteritis. Our case is unique in view of the association of the above clinical and radiological features, and the bilateral involvement of orbital tissues. PMID- 7554568 TI - Buerger's disease and protein S deficiency: successful treatment with prostacyclin. AB - In this report we describe a female patient who presented with peripheral circulatory impairment. She had previously been thought to have a systemic vasculitis, but had failed to respond to corticosteroid and immunosuppressive therapy. Serologic investigations were negative. The patient was a very heavy smoker, and it became clear that her condition had many of the clinical features of Buerger's disease (thrombangiitis obliterans). The diagnosis was confirmed by angiography, and a coagulation screen showed her to have associated protein S deficiency. Her symptoms responded rapidly to cessation of smoking, warfarin therapy, and a series of intravenous prostacyclin infusions. We discuss in detail the possible pathologic mechanisms which might explain the association of protein S deficiency and thrombangiitis obliterans. PMID- 7554569 TI - Polymyositis and chronic graft-versus-host disease: efficacy of intravenous gammaglobulin and methotrexate. AB - Polymyositis developed in a patient who had received a bone marrow transplant for the treatment of acute lymphoblastic leukemia 20 months earlier. It was a manifestation of chronic graft-versus-host disease associated with sclerodermatous changes of the skin and sicca syndrome. Polymyositis was treated successfully by polyvalent human intravenous immunoglobulin and methotrexate. PMID- 7554570 TI - Treatment of early rheumatoid arthritis: a review of current and future concepts and therapy. AB - This review contrasts the difficulty of differentiating early rheumatoid arthritis (RA) from the many other causes of inflammatory polyarthritis and emphasizes the need for early treatment of RA with drugs that are present considered inappropriate for a mild, self-limiting disease. Pointers to help establish a definitive diagnosis of RA include the use of the American College of Rheumatology (ACR; formerly the American Rheumatism Association) diagnostic criteria, the presence of rheumatoid factor (RF), a raised erythrocyte sedimentation rate (ESR), raised serum levels of C-reactive protein (CRP) and low concentrations of serum sulphydryl (SH) groups. More sophisticated tests that are helpful and indicative of erosive-type disease are genetic markers such as the human leukocyte antigen HLA DR4 (especially the third allelic variable of DR beta 1) as well as an impaired sulphoxidation status. Similarly, the presence of the shared epitope alleles 0401 and 0404 quantitatively increases disease susceptibility and severity as their penetration increases. Experience with treatment indicates that control of the inflammatory process of RA reduces the progression of radiological damage. It is concluded that antimalarials are effective in mild disease, as is auranofin, whilst sulphasalazine is more effective than hydroxychloroquine in patients with disease diagnosed as definitive or classical RA. Intramuscular gold also lessens the development of erosions. The role of corticosteroids has not been defined. A number of combinations of drugs have been used, although they are probably not indicated as the first choice treatment for very early disease. Attitudes towards early treatment are changing; these are briefly reviewed and the authors' opinions on the management of early disease are outlined. Finally some new ideas, both theoretical and practical, on future development are summarized. PMID- 7554571 TI - Relapsing focal myositis: a case report. AB - We present a 9 year old boy with a focal myositis presenting as a pseudotumor on the right calf. Other smaller masses were present in the thigh and pretibial area on the same leg. The principal lesion recurred after two surgical excisions. Corticosteroid treatment was unsuccessful. However, methotrexate therapy resulted in a progressive regression of the lesions. PMID- 7554572 TI - Acute lethal necrotising pancreatitis in childhood systemic lupus erythematosus- possible toxicity of immunosuppressive therapy. AB - We report on a 16 year old girl with a three-year history of systemic lupus erythematosus who developed a case of acute lethal haemorrhagic pancreatitis. She presented with high grade fever, skin rash, malaise, and arthralgias. Laboratory lupus activity parameters were markedly elevated. In the absence of renal, pulmonary, cardiac or cerebral involvement, our patient developed pancreatitis leading to pancreatogenic shock. Until 14 days before the onset of pancreatitis, the patient's medications included prednisolone, azathioprine and methotrexate. At autopsy, no autoimmune vasculitis was found in the affected pancreatic tissue. Therefore, an etiologic role of combination therapy had to be considered. Whereas methotrexate has never been reported to be linked to pancreatitis, a few publications describing prednisolone and azathioprine in connection with pancreatitis do exist. Thus, if pancreatitis is not just termed idiopathic, it must be attributed to a combination regimen of drugs including methotrexate. A review of the literature shows that pancreatitis in SLE is rare and has never been associated with methotrexate therapy before. PMID- 7554573 TI - Coexistence of diffuse idiopathic skeletal hyperostosis and ankylosing spondylitis in two patients. PMID- 7554575 TI - Evaluation of two enzymatic immunoassays for anticardiolipin antibody measurements. PMID- 7554574 TI - Lipid profile and anticardiolipin antibodies in rheumatoid arthritis. PMID- 7554576 TI - Early treatment of systemic onset juvenile chronic arthritis with low-dose cyclosporin A. PMID- 7554577 TI - T cell lymphoma masquerading as adult onset Still's disease. PMID- 7554579 TI - Takayasu's arteritis and Wegener's granulomatosis. A coincidental case. PMID- 7554578 TI - A case of multicentric reticulohistiocytosis. PMID- 7554580 TI - A case of Tbc oligoarthritis with negative cultures: early diagnosis using CT scan and in situ hybridization. PMID- 7554581 TI - Detection of anti-neutrophil cytoplasmic antibodies by immunoprecipitation. AB - Immunoprecipitation (IP) of radiolabeled PMN extracts was used as the gold standard for anti-proteinase 3 (PR-3) autoantibody detection to validate immunofluorescence (IF) and alpha granule (alpha) ELISA. A myeloperoxidase (MPO) ELISA was also used in parallel. We studied 48 patients with strictly defined vasculitic syndromes in the initial active phase of their disease. The 3 methods confirmed the high (> 90%) sensitivity and specificity of anti-PR-3 for patients with Wegener's granulomatosis (WG). Similarly, a high (86%) sensitivity of MPO ELISA was found in microscopic polyarteritis as defined. Using alpha-ELISA, we could not improve the detection rate of anti-PR-3 obtained by IF-cANCA-pattern reading. Moreover, a small proportion (< 15%) of biopsy-proven WG patients had anti-MPO antibodies detected by IF, usually as pANCA but also, even if rarely, as bona fide cANCA (< 5%). Thus, IF would seem to be the most reliable screening method and alpha-ELISA should be used for confirmation. On the other hand, because MPO-ELISA detected twice as many anti-MPO positive sera as did pANCA pattern reading by IF, we suggest that in the clinical context of a vasculitis, MPO-ELISA should also be used as a screening test. Although IP is not designed for routine clinical use, it should be required when reporting the presence of anti-PR-3 in vasculitis-like diseases that are fertile grounds for false positive reactions. PMID- 7554582 TI - Validity of newborn oscillometric blood pressure. AB - The objective of this study was to determine the validity of oscillometric blood pressure in relation to arterial blood pressure. Thirty-one newborns were studied. Clinical characteristics, complications, and treatment interventions were documented. Arterial pressure (every 2 sec) and oscillometric pressure (every 3 min) were concurrently recorded for 1-2 h. Serial observations of oscillometric pressure followed the trend of arterial pressure in the individual newborn. However, the study averaged oscillometric pressures were lower than the arterial pressures: systolic, by 1 mmHg; mean, by 5.3 mmHg (p < 0.0001); and diastolic, by 4.6 mmHg (p < 0.0001). The variance of individual, 15-min averaged, and 1-h averaged observations of oscillometric pressure in relation to arterial pressure was examined. Variance for individual observations may be large. The least variance of oscillometric pressure was in the 1-h averaged mean pressure, in which the difference was +/- 2 mmHg in 77% and +/- 4 mmHg in 95% of observations. Clinical characteristics, with the exception of birth weight and treatment interventions, did not affect the variance of oscillometric pressure. This study implies that the offset in relation to arterial pressure should be established for each oscillometric pressure monitoring system. Hourly averaged mean oscillometric pressure is satisfactory for many newborn assessments and management circumstances. However, arterial pressure may be necessary to accurately document transient hypertension or hypotension or an unstable blood pressure. PMID- 7554583 TI - Microbial surveillance of human islet isolation, in vitro culture, and cryopreservation. AB - The aim of these studies was to investigate microbiological contamination during the isolation and preservation of islets of Langerhans in a low-temperature tissue bank. Islets were isolated from the pancreases of 117 organ donors, then cryopreserved. In the initial 47, microbial culture was completed only after thawing: Enterobacter cloacae (n = 4) and gram-negative bacilli (n = 9) were isolated for a positive culture rate of 27.6%. It was not possible to ascertain the source of the contaminants, since cultures were taken only at the conclusion. A total of 70 consecutive pancreases were then subjected to islet isolation and cryopreservation while prospectively culturing at these steps: step 1, from the pancreas transport media; step 2, after intraductal perfusion of collagenase; step 3, after dissociation; step 4, after purification; step 5, following a 6-48 h in vitro culture; step 6, post-thaw; step 7, after dilution of the cryoprotective agent; and step 8, after final 48-h in vitro culture. The transport fluid was infected with aerobic gram-negative (n = 6), gram-positive (n = 5), and yeast microorganisms (n = 2) for an overall step 1 contamination rate of 19%. These contaminants were found in 9% of our local program vs. 26% from distantly procured pancreases. Contaminants at subsequent steps were 10% (step 2), 9% (step 3), so that only 3% were infected at the conclusion of isolation (step 4), and 0% after initial culture (step 5). Amongst pancreases that were initially sterile, new contaminants could be identified in 12% at step 2 and 8% at step 3; however, these also became undetectable.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554584 TI - Determination of the test performance of less costly methods of Helicobacter pylori detection. AB - Since Helicobacter pylori (Hp) has become so important in the management of peptic ulcers, it is imperative that noninvasive and inexpensive methods of diagnosis be available. The objective of this prospective, double-blind study was to determine the performance of the noninvasive 14C-urea breath test and the less expensive hematoxylin-phloxin-saffron (HPS) and Giemsa stains in the detection of this infection. As well, we set out to determine the minimal number of biopsies required to make an accurate diagnosis. Fifty consenting adults referred for gastroscopy underwent antral biopsies for HPS, Giemsa and Steiner silver staining and a 14C-urea breath test. The ability of HPS, Giemsa, and the breath test to differentiate between the presence or absence of Hp infection as defined by the silver stain was assessed using contingency table analysis. Hp was detected in 24 patients with the silver stain. Half of these patients had evidence of ulcer disease, compared to 3 of 26 patients who were negative for Hp on silver stain (p < 0.01). The Hp positive group were more frequently male (62% vs. 31%, p < 0.05) and more often had chronic active gastritis (96% vs. 7.7%, p < 0.001). The sensitivity of the breath test, HPS, and Giemsa stains were 95.8% (95% confidence interval: 79-100%), 75% (53-90%), and 95.8% (79-100%), respectively. Specificity was 100% (87-100%) for all 3 methods of detection. The breath test was also able to discriminate between heavily infected and moderately infected patients as defined by silver stain (p < 0.05), and could be shortened from 30 to 15 min without any loss of accuracy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554585 TI - Increased membrane-bound protein kinase C in porcine thyroid cells following TSH exposure. AB - Activation of protein kinase C (PKC) follows its translocation from the cytosol to an active membrane-bound form. Previously, we demonstrated that only high concentrations of partially-purified bovine thyroid-stimulating hormone (TSH) could translocate PKC in vitro when measured by histone phosphorylation. Measuring PKC by Western blotting, we assessed whether physiological concentrations of human TSH could translocate PKC in porcine thyroid cells. The known PKC stimulator, phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), caused maximal translocation of PKC at 100 nM with a 84.5% decrease in cytosolic PKC and a corresponding 252.1% increase in membrane-bound PKC. Although TSH did not affect cytosolic PKC, human TSH at 1, 10 and 100 microU/ml produced increases compared to control values in membrane-bound PKC of 80.3 +/- 15.8, 66.8 +/- 9.7 and 74.0 +/- 19.8%, respectively (mean +/- SE, p < 0.01 at all TSH concentrations used). These studies provide evidence that physiological concentrations of TSH increase membrane-bound PKC in vitro, a process linked to its activation. In addition, we observed qualitative differences in phorbol ester and TSH-mediated PKC activation. PMID- 7554586 TI - Medical aspects of ketone body metabolism. AB - Ketone bodies are produced in the liver, mainly from the oxidation of fatty acids, and are exported to peripheral tissues for use as an energy source. They are particularly important for the brain, which has no other substantial non glucose-derived energy source. The 2 main ketone bodies are 3-hydroxybutyrate (3HB) and acetoacetate (AcAc). Biochemically, abnormalities of ketone body metabolism can present in 3 fashions: ketosis, hypoketotic hypoglycemia, and abnormalities of the 3HB/AcAc ratio. Normally, the presence of ketosis implies 2 things: that lipid energy metabolism has been activated and that the entire pathway of lipid degradation is intact. In rare patients, ketosis reflects an inability to utilize ketone bodies. Ketosis is normal during fasting, after prolonged exercise, and when a high-fat diet is consumed. During the neonatal period, infancy and pregnancy, times at which lipid energy metabolism is particularly active, ketosis develops readily. Pathologic causes of ketosis include diabetes, ketotic hypoglycemia of childhood, corticosteroid or growth hormone deficiency, intoxication with alcohol or salicylates, and several inborn errors of metabolism. The absence of ketosis in a patient with hypoglycemia is abnormal and suggests the diagnosis of either hyperinsulinism or an inborn error of fat energy metabolism. An abnormal elevation of the 3HB/AcAc ratio usually implies a non-oxidized state of the hepatocyte mitochondrial matrix resulting from hypoxia-ischemia or other causes. We summarize the differential diagnosis of abnormalities of ketone body metabolism, as well as pertinent recent advances in research. PMID- 7554587 TI - Influence of undergraduate and postgraduate education on recruitment and retention of physicians in rural Alberta. AB - The composition of practising physicians in Alberta, with respect to medical school of graduation, changed between 1986 and 1991. The percentage of graduates of the 2 Alberta medical schools increased, and the percentage of graduates of foreign medical schools decreased. Graduates of the University of Calgary increased their percentage in family practice in urban and rural communities except in Edmonton, while graduates of the University of Alberta increased their percentage almost everywhere except in communities with populations of < 4,000. Although graduates of Alberta medical schools are locating their practices in rural regions, the smaller communities continue to depend on foreign medical graduates. Retention is a problem in communities of < 4,000, with greatest mobility demonstrated by non-Albertan Canadian graduates and foreign medical graduates while Alberta graduates demonstrate less mobility. Overall, 85% of new Alberta physicians have had undergraduate or postgraduate experience in Alberta or Canada. Those with no medical educational experiences in Canada are more likely to locate in small communities. Those with postgraduate training in Alberta or Canada are more likely to locate in urban centres. When both undergraduate and postgraduate influences are considered, Alberta graduates appear to locate in non-urban regions to a greater extent than other Canadian or foreign graduates. For family physicians and specialists, the city where postgraduate training was obtained has a profound influence on the choice of urban practice locations. PMID- 7554588 TI - Canadian Society for Clinical Investigation and The Royal College of Physicians and Surgeons of Canada annual meeting. Montreal, Quebec, September 13-17, 1995. Abstracts. PMID- 7554589 TI - Cervical ripening and labor induction: progress and challenges. PMID- 7554590 TI - Cervical assessment. AB - The relationship between the clinical state of the cervix and spontaneous onset of labor has long been known. The cervix can be characterized by evaluating five parameters: effacement, dilatation, firmness, position, and level of the presenting part. The relative associations between successful labor induction and these factors appears to vary, with the strongest correlation observed with cervical dilation, whereas the association with cervical station and position is less robust. By its nature, cervical examination is somewhat subjective, but the use of objective criteria and numerical scoring of this information has substantially reduced this source of error. The Bishop score and it variations have been used widely and accepted in clinical practice. Its advantage lies in its simplicity, reproducibility, and predictability in successful inductions. Although criticized for the equal weight given to each of the five elements, none of the modifications to the original score has been shown to improve predictability. Although the Bishop score was initially evaluated only in multiparous women, it has been extended to nulliparous women with an equal degree of predictive value. To date, the Bishop score remains as the best and simplest method available to determine the duration and safety of induced labor. PMID- 7554592 TI - Amniotomy to induce labor. AB - Amniotomy to induce labor is used frequently. The potential risks compared with potential benefits of artificial rupture of membranes have caused the popularity of amniotomy to vary in the last two centuries. Although there are little data available from prospective randomized studies regarding the effectiveness of amniotomy alone to induce labor, several series have showed success in its use. In addition, no well-accepted, prospectively randomized study is available comparing the effectiveness of amniotomy to oxytocin for induction of labor. The most effective combination of amniotomy with uterotonic agents to induce labor is still a fertile area for investigation. PMID- 7554591 TI - Oxytocin to induce labor. AB - Oxytocin is a highly successful and safe agent for inducing labor and has a fairly large therapeutic index. It has, however, minimal, but not trivial, antidiuretic and vascular activity when used in large doses. Therefore, to induce labor, low-dose oxytocin dosing regimens that produce efficacious uterine activity and avoid side effects are recommended. Oxytocin should be used in the lowest possible doses necessary to effect a clinical response. Diligence must be exercised when monitoring labor and fetal well-being. Hofbauer, the first to use oxytocin to induce labor, said in 1927 that oxytocin, "with its power of producing regular, rhythmical and forcible uterine contractions, should be regarded as a most beneficent and valuable agent, which, however, should always be employed with care and a realisation of its limitations and dangers." His words remain valid today. PMID- 7554593 TI - Labor induced using methods that do not involve oxytocin. PMID- 7554594 TI - Anatomy and physiology of cervical ripening. AB - The uterine cervix is a unique organ composed predominately of the extracellular matrix proteins, collagen, elastin, and glycosaminoglycans. During pregnancy and labor, this organ is metabolically active, which is rare in adult tissue. The metabolism is under reproductive hormonal control and is more complex than previously appreciated. Smooth muscle cells, which comprise 10-15% of cervical tissue, undergo programmed cell death and play a role in cervical softening. Apoptosis is a genetically timed event and could explain the species-specific length of gestation. Further research in the next several years will reveal more completely the exciting process of cervical ripening. Only when this phenomenon is understood will rational therapy for preterm labor and post-term pregnancy with an unripe cervix be available. Specific defects in cervical ripening will then be diagnosed and treated. For example, if apoptosis is shown to play an important role in the process of cervical ripening, it could be inhibited. Conversely, it could be induced in the unripe cervix. If we would look for it, we would find that it is probably occurring today in the clinical use of cervical ripening agents. The most important contributor to cervical softening, however, is a rearrangement and realignment of the collagen, elastin, and smooth muscle cells, which occurs due to mechanical forces and to a rearrangement of the collagen that occurs as the content of glycosaminoglycans varies in the cervix with time. One form of dermatan sulfate, decorin, may help to separate the collagen fibrils and then open them up. This rearrangement also involves fiber shortening below the critical length for tensile strength, allowing for extensibility of the cervix. Because of its orientation in the cervix, elastin contributes to the ratchet-like mechanism of dilatation. Finally, the cervix undergoes change in two phases--softening, which involves collagen realignment, and dilatation. The proteolytic enzymes in the cervix degrade cross-linked, newly synthesized collagen, and they help activate other enzymes in a cascade. However, the predominate anatomic and physiologic change in ripening is the rearrangement of collagen. PMID- 7554595 TI - Mechanical methods of cervical ripening. PMID- 7554596 TI - Cervical ripening and labor induction after previous cesarean delivery. AB - The preponderance of published data derived from TOL/VBAC patients indicate that (1) if there is no contraindication to spontaneous cervical ripening, there is no contraindication to use of prostaglandin gel or tents to ripen the cervix; and (2) if there is no contraindication to the spontaneous onset of labor, there is no contraindication to use of oxytocin or amniotomy to induce labor. Both conclusions are based on the assumption that treatment and care is provided in a safe, modern obstetric service with staff and resources compatible with national standards. PMID- 7554597 TI - Relaxin used to produce the cervical ripening of labor. AB - Important progress has been made in understanding the physiology of relaxin in human pregnancy. The structure of relaxin and its genetics have been elucidated. Progress has been made into understanding the control of relaxin secretion. Many actions of relaxin have been defined. Some of the effects of too much or too little relaxin have been described. However, the cellular mechanisms of relaxin action must still be determined, and the relationship of relaxin to diseases of pregnancy must be clarified. Although relaxin affects the human cervix, its physiologic role is not clear. Pregnancies can proceed uneventfully in the absence of circulating relaxin. Thus circulating relaxin is not critical for maintaining pregnancy or for cervical ripening. The mechanisms by which pregnancy is maintained and the mechanism of the timely onset of labor at full term are not well understood. There is a plethora of data suggesting many parallel overlapping control mechanisms for uterine control. Most of these systems are not critical because their actions can be compensated for by other systems. Understanding the physiology of human pregnancy must account for these multiple systems. Within this context, the role of relaxin in the physiology of labor must be understood. PMID- 7554598 TI - Outpatient cervical ripening. AB - A limited number of studies have provided preliminary data on the efficacy and safety of PGE2 used as an outpatient cervical ripening agent. Multiple inpatient studies have confirmed the effect of PGE2 in favorably changing the cervical score, reducing the incidence of failed inductions and instrumental deliveries, shortening the induction-to-delivery interval, and reducing oxytocin use with few maternal side effects and no known adverse effects on neonatal outcome. These studies, however, are difficult to compare because of differences in study design, PGE2 formulation, patient selection (including differences in cervical scoring criteria), indications for labor induction, placement of the PGE2 agent, and outcome variables, and they certainly cannot be extrapolated to outpatient use. Intracervically applied Prepidil is the only form of PGE2 approved by the Food and Drug Administration available for cervical ripening. The commercial production of an intravaginal form of PGE2, which would require less effort and expertise in administration and greater patient comfort, may warrant exploration. In an environment of increasing pressures for cost containment, the convenience and cost-effectiveness of outpatient cervical ripening provides a stimulus for further controlled studies in this area to delineate an effective and safe dose and vehicle for widespread application. PMID- 7554599 TI - Training, certification, and credentialing in gynecologic operative endoscopy. PMID- 7554600 TI - What is the role of hysteroscopy in the management of abnormal uterine bleeding? AB - Many otherwise healthy women will experience a significant disruption in lifestyle from abnormal uterine bleeding. Most of those seeking medical attention will not be at risk for developing anemia. In each case, a thorough search for underlying systemic, hormonal, and organic causes should be instituted. The use of blind endometrial sampling to evaluate the uterine cavity, by itself, is an inaccurate technique for diagnosing pathologic conditions commonly associated with menorrhagia, such as endometrial polyps, submucous myomata, and focal endometrial abnormalities including adenocarcinoma and its precursors. The supplementary application of diagnostic hysteroscopy with directed biopsy will ensure the recognition of these intracavitary lesions. The majority of women found to have endometrial polyps and submucous myomata can gain a successful reduction in their menstrual flow without hysterectomy by undergoing hysteroscopic removal of these lesions. Those without other uterine or pelvic pathology and who are closer to perimenopause are more likely to sustain long lasting relief from these procedures. Medical therapy should be the first line of treatment for premenopausal women who are found to have no obvious cause for their abnormal uterine bleeding. Many of those who do not respond to, are unable to tolerate, or are unwilling to attempt this approach will undergo hysterectomy as the final answer. The absence of uterine pathology in most of these cases places an absolute demand on our specialty to innovate, and, whenever suited, to use more conservative surgical solutions. Our efforts to alter this behavior will undoubtedly be closely monitored by agents of managed care aiming to reward measures that reduce cost and improve the quality of care. The use of hysteroscopic ablation and resection to treat women suffering from intractable menorrhagia can safely and effectively reduce menstrual blood flow and should significantly curtail the performance of unnecessary hysterectomy. The comparative benefits and long-term advantages of these techniques beyond hysterectomy await the results of further studies. Furthermore, the risks of these hysteroscopic procedures to produce iatrogenic adenomyosis or to conceal or delay the usual signs of adenocarcinoma have yet to be ascertained. Vigilance for endometrial disease must not dwindle in the face of amenorrhea, as evidenced by a recent case report describing the development of endometrial carcinoma after 5 years of amenorrhea following endometrial electrocoagulation. Future methods of endometrial destruction for the control of abnormal uterine bleeding may include the nonhysteroscopic use of radio frequency, thermal transfer, hyperthermia, and photodynamic therapy.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554601 TI - Medical management should be routinely used as primary therapy for ectopic pregnancy. AB - It does not appear that methotrexate can or should be used to totally replace laparoscopic salpingostomy. However, given its success rate in selected patients, its cost-effectiveness, and its low incidence of side effects, methotrexate therapy certainly can be used as an alternative therapy. The next step in developing this treatment option will be to conduct a randomized clinical trial comparing laparoscopic salpingostomy with intramuscular methotrexate. This type of study will answer questions regarding the patient's health related quality of life and economic impact of the two treatment modalities. For now, it seems prudent to offer methotrexate to those patients with an unruptured ectopic gestational mass 3.5 cm or less in greatest dimension. PMID- 7554602 TI - For tubal pregnancy, surgical treatment is usually best. AB - Laparoscopic treatment of tubal pregnancy is safe and effective. It permits performance of both radical and conservative procedures. Irrespective of the mode of access, conservative procedures carry the risk of retention of trophoblastic tissue. Systemic methotrexate administration is effective in the treatment of retained trophoblastic tissue, especially if the diagnosis is made early, preferably within the first postoperative week. The subsequent reproductive outcome appears to be the same, irrespective of the mode of access (laparotomy or laparoscopy) by which the procedure is done. PMID- 7554603 TI - The case for laparoscopic management of the adnexal mass. AB - Careful patient selection criteria can be used to identify patients who are appropriate for management of an adnexal masses via operative laparoscopy. Proper intraoperative assessment and liberal use of rapid frozen section are also important for optimal clinical outcome. Reported studies show that laparoscopic management of adnexal masses can be safely done. The short hospital stay and rapid return to normal activity combine to potentially reduce the overall cost of patient care. When cancer is unexpectedly found at the time of laparoscopic surgery for an adnexal mass, the surgeon should be prepared to proceed with staging laparotomy for appropriate treatment. PMID- 7554605 TI - Minimally invasive treatment of urinary stress incontinence and laparoscopically directed repair of pelvic floor defects. PMID- 7554604 TI - Laparoscopic removal of the adnexal mass: the case for caution. PMID- 7554606 TI - Myomectomy: laparoscopy or laparotomy? PMID- 7554607 TI - Laparoscopic hysterectomy: does it work?: a bicontinental review of the literature and clinical commentary. PMID- 7554608 TI - Laparoscopic surgery: does it have a role in the management of gynecologic malignancies? PMID- 7554609 TI - Endometrial ablation. PMID- 7554610 TI - Safety and training. AB - Provided progress is made systematically, the surgeon never attempts to perform operations that are too difficult, and all the safety protocols are adhered to, hysteroscopic surgery should be within the capabilities of most gynaecologists. Failure to appreciate the risks and failure to undergo continuous training in the learning period will inevitably lead to complications which should otherwise be avoidable. Hysteroscopic surgery offers many advantages over conventional surgery and, like many other types of minimal access surgery, appears to be the surgery of the future. Endoscopic surgery will not supplant open surgery but will become another essential part of our practice which the patient will expect and, indeed, demand. There will be problems. Training programmes must be established to try to prevent complications which will be encountered if inadequately trained surgeons perform these new forms of surgery. There must be continuous education in existing methods and research into value and results of new techniques and instruments both for laser and electrosurgery. It is possible that in the future new biophysical or pharmacological approaches may render laser and electrosurgery obsolete. Safer forms of physical energy, photosynthesis and new forms of hormone therapy are all undergoing trials with preliminary results which are encouraging. Whichever techniques eventually prove to be the most effective and safe, we must learn to use them because we owe to our patients the opportunity to benefit from them. PMID- 7554611 TI - Endometrial ablation: indications and preliminary diagnostic hysteroscopy. AB - Endometrial ablation is indicated when a woman with excessive uterine bleeding has, completed childbearing, been shown to suffer from dysfunctional bleeding, has exhausted all reasonable nonsurgical alternatives and would previously have been offered a hysterectomy. Diagnostic hysteroscopy is an integral part of the investigation of such a patient. Its primary function is to identify intrauterine lesions which might, if treated alleviate the need for ablation, or if untreatable, constitute contraindications to ablation. This chapter describes in detail the indications for and contraindications to, ablation. A short description of the medical management of dysfunctional bleeding is given. The instruments, technique and hysteroscopic findings which may be encountered are discussed. PMID- 7554612 TI - The tissue and thermal effects of electrosurgery in the uterine cavity. AB - A brief outline of the physics associated with the production of electrosurgical wave-forms has been described. The conversion of electrical to thermal energy in electrosurgical techniques has demonstrable tissue and thermal actions and these were discussed in greater detail. In vitro electroresection was associated with a narrow ZTN underlying the electrode that did not vary with power output, but was related to duration of exposure. For blend 2 cutting output the mean ZTN ranged from 0.97 to 1.40 mm for a single sweep of the electrode. With longer time increments this ZTN increased to 2.20 mm. Coagulation by desiccation, in vitro, resulted in a depth of destruction of 3.24-3.49 mm that did not vary with power output or duration of exposure. Fulguration only achieved a depth of destruction of up to 1.78 mm that was related to time. This confined-ZTN in the fulguration mode was felt to be too narrow to destroy the deeply seated myometrial gland elements. Thermal transmission through blocks of human uterine tissue, in vitro, has been demonstrated. Resection generated a maximum rise of 5.8 degrees C at 12 mm from the electrode, with desiccation, a maximum of 4.0 degrees C. It was also demonstrated that resection and desiccation have differing thermal profiles, the latter being more confined in nature. Both power output and duration of exposure in resection are important factors, in vitro, with regard to heat generation. Duration of exposure during desiccation is important in terms of heat generation, but not power output. Repetitive electrosurgical insults have a cumulative effect on the thermal profile in vitro. The underlying damage to uterine tissue during electrosurgery in vivo has been established. In electroresection the zone of thermal necrosis was related to duration of exposure, whereas in electrodesiccation the damage appeared confined, self-limiting, and not time related. The lack of clinically significant demonstrable thermal transmission through the uterus was important. However, excessively prolonged application of electrosurgical energy (greater than 5 seconds) in the narrow regions of the uterus, especially with electroresection, could still be potentially dangerous. The use of the desiccation mode in the cornual regions is recommended for three reasons; the loop electrode has greater potential for perforation in this thin region (because of its diameter); the desiccating mode appeared to have a clearly defined, consistent ZTN that did not alter appreciably; and the thermal imaging of the ball electrode, in vitro, demonstrated the more confined nature of its thermal profile compared to the loop electrode.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554613 TI - Endometrial resection. AB - The development of hysteroscopy and endometrial resection is reviewed and instrumentation is described. Suitable fluids for uterine distension and methods of delivery are discussed and safe limits to fluid absorption suggested. Selection of patients, preoperative investigation and preparation are described. Anaesthesia and operative technique are discussed with particular reference to the methods used by the authors and some common operative problems and their solutions are described. The results of treatment are discussed with reference to the published literature and two additional large unpublished series; one of 500 resections carried out in Oxford by 19 surgeons, of widely differing experience, with a follow-up period from 1 to 5 years; the other a personal series of 585 cases done by one of the authors (E.M.H.) with a follow-up period of 1 to 4 years. Factors affecting the likely success of the procedure, operative and post operative complications, the difficulty and results of repeated resections and the indications for subsequent hysterectomy are discussed. Finally, the economic aspects of the technique and the author's conclusions as to the place of endometrial resection in the treatment of menorrhagia are given. PMID- 7554614 TI - Rollerball endometrial ablation. AB - Attempts at endometrial destruction to treat abnormal uterine bleeding unresponsive to medical therapy in women at risk to hysterectomy are not new. However, a practical and effective method was not introduced until the early 1980s, when the Nd:YAG laser was used through the hysteroscope for endometrial photocoagulation. Soon after, electrosurgery proved to be a good alternative for endometrial ablation, either by resection, coagulation, or both. Because of its relative simplicity, effectiveness, and low complication rate, the rollerball endometrial ablation has been attractive to physicians and patients alike as a good alternative to endometrial laser ablation. The overall results, particularly in properly selected patients who have been hormonally suppressed to thin the endometrium and permit good tissue penetration of the electrosurgical energy, have been most satisfactory. PMID- 7554615 TI - Endometrial laser ablation. AB - With good techniques endometrial laser ablation can be a safe and effective treatment for dysfunctional uterine bleeding. For effective intrauterine surgery it is important to control the intrauterine environment during the procedure to ensure safe operating conditions. This can best be achieved by using a continuous flow hysteroscope and pressure-controlled fluid infusion pump. The laser energy should be applied with a direct dragging technique to produce a series of parallel furrows. Safe ablation will be achieved if the three golden rules are followed and the laser is activated only when the tip of the fibre is visible, only when it is being drawn towards the operator and only when the fibre is being moved across the endometrial surface. With a high power Nd: YAG laser at 80 W power, a treatment time of around 20 min should be regularly achieved. This approach can control menstrual flow in 90% of cases and result in an overall satisfactory outcome in around 85% of cases. Improved patient selection may further improve these results. These satisfactory results can be achieved with minimum morbidity but the potential for serious complications still exists so these techniques should not be undertaken without adequate training and good equipment. PMID- 7554616 TI - Treatment of dysfunctional bleeding and fibroids by advanced endoscopic techniques with the Nd:YAG laser: from the present to the future. AB - Both the electrical current of the resectoscope and the energy of the Nd:YAG laser have been effective tools in the destruction of endometrial tissue to a sufficient depth to avoid regeneration. GnRH-agonist therapy effects a decrease in the total uterine cavity area which facilitates surgical treatment and reduces the risk of fluid overload syndrome. The recurrence rate of meno/metrorrhagia is higher when the uterine cavity is more than 10 cm2. The use of GnRH-agonists represents an adjunct for preoperative reduction of submucosal myomas so that subsequent hysteroscopic myomectomy is possible. A two-step hysteroscopic therapy combined with GnRH-agonist therapy is performed when the largest portion of the submucosal myoma is located in the uterine wall. In cases of numerous submucosal and intramural myomas, a laparoscopic supracervical hysterectomy is performed because of the high risk of recurrence after the hysteroscopic procedure. PMID- 7554617 TI - Radiofrequency induced endometrial ablation. AB - Radiofrequency endometrial ablation is an experimental technique which requires a minimum of training and appears to be safe so long as a strict protocol is maintained. The success rate in over 1000 patients in multiple centes gives an incidence of amenorrhoea or oligomenorrhoea in excess of 80%. Therefore, as long as patients are strictly selected more than eight out of 10 hysterectomies for dysfunctional uterine bleeding can be avoided. The technique is particularly suitable for women with severe cardiopulmonary disease because there is no risk of pulmonary or cerebral oedema due to fluid intravasation. PMID- 7554618 TI - Audit of currently available endometrial ablative techniques. AB - Techniques for the treatment of menorrhagia have changed rapidly over the last few years. The enthusiastic introduction by a small number of protagonists of a variety of ablation/resection techniques has been followed by an exponential uptake in their use. At the same time there has been a period of critical appraisal by the Royal College of Obstetricians and Gynaecologists and the British Society for Gynaecological Endoscopy. The MISTLETOE Survey was the culmination of this association and has provided useful large data sets of the various techniques under evaluation. It is expected that recommendations concerning; the training of potential operators; various practical aspects of the surgical, medical and anaesthetic techniques required to undertake the procedures safely; types and frequencies of complications of the procedures, and success rates following the widespread use of these procedures will be made available following analysis of the data sets. PMID- 7554619 TI - Alternative treatment. AB - Alternatives to endometrial ablation include re-education of the 20% or more women complaining of menorrhagia whose blood loss is normal, less than 35 ml, and the use of an increasing variety of drugs to find an effective regime with minimal side effects. Endometrial resection is an effective treatment, particularly in the short term. Laparoscopic and vaginal hysterectomy have advantages over endometrial resection in the long term, avoiding failure and retreatment for menorrhagia. Hysteroscopic, laparoscopic and open myomectomy are more suitable in treating fibroids of significant size, although these techniques may be used with endometrial resection to avoid hysterectomy. Myoma reduction may become a less morbid alternative to myomectomy. PMID- 7554620 TI - Aseptic loosening in uncemented total hip arthroplasty in a canine model. AB - An in vivo canine model was developed to investigate the histologic and biochemical parameters associated with aseptic loosening. Thirty-eight canines had cementless total hip arthroplasty. Experimental groups were designed specifically to investigate the relative contributions of implant motion and particulate debris (cobalt chrome alloy, titanium aluminum vanadium, and polyethylene) on the resultant periprosthetic tissues. Tissues from a stable, well-ingrown prosthesis provided a control. Importantly, the histologic and biochemical characteristics of the experimentally induced membranes consistently correlated with previous in vitro reports of tissues retrieved at revision surgery for aseptic loosening. Implant motion and all 3 particulate debris groups resulted in increased numbers of macrophages in the periprosthetic membranes. The histologic findings paralleled the increase in levels of biochemical mediators of bone resorption as measured by collagenase, gelatinase, prostaglandin E2, and interleukin-1 activity. The most striking results were seen in the histology and biochemistry of the particle groups with highly cellular membranes showing increased biochemical activity when compared with controls. The clinical relevance of this work lies in the description of an in vivo model of aseptic loosening that can be used to investigate the effects of numerous variables implicated in aseptic loosening. Ultimately, the model may serve as a basis for developing therapeutic interventions. PMID- 7554621 TI - Biology of grit-blasted titanium alloy implants. AB - This study describes the biologic integration of grit-blasted titanium alloy (Ti 6A1-4V) implants that were press fit into the distal femoral canal of young adult rabbits and evaluated by histologic, histomorphometric, and biomechanical methods. Polished and aluminum oxide grit-blasted (4.2 +/- 0.7 microns surface roughness) solid implants were compared with titanium fibermetal implants. Nondecalcified cross sections were studied by histology, histomorphometry, and electron microscopy in the backscatter mode at 3, 6, and 12 weeks after implantation. Pullout strength was measured at 12 weeks. Data were analyzed by analysis of variance and post-hoc Student-Newman-Keuls and Scheffe's tests. The blasted implants had significantly more bone intimately in contact with the implant surface (31%) than the fibermetal (17%), or solid polished implants (15%). By 3 weeks, woven bone had formed directly on the surface of the blasted implants, whereas there was a discrete space between woven bone and the other implants. Active remodeling of bone was shown by fluorochrome uptake at the surface of the blasted implants at 12 weeks after implantation. The strength of fixation of blasted and fiber-metal implants was significantly greater than polished implants at 12 weeks after implantation. Direct attachment of newly formed bone onto the blasted implant surface was confirmed by backscatter electron microscopy. The results of this study indicate that grit-blasted titanium surfaces provide an excellent surface for bone implant integration. PMID- 7554622 TI - Revision total hip replacement using the S-ROM femoral component. AB - The proximally porous-coated, modular S-ROM femoral component was used in 52 complex total hip revisions done in 48 patients. These patients had severe bone loss, leg length inequality, and instability. Twenty-two patients required structural femoral allografts; 8 had previous resection arthroplasties for sepsis. The mean number of previous hip operations was 3. The stem was press fit, and the metaphyseal sleeve was selectively cemented to the allograft. The preoperative Harris rating was 44 points; at a mean of 3 years, followup was 82 points. Eighty-four percent of the patients were satisfied with their outcomes. No radiographic or histologic evidence of fretting at the modular sleeve-stem junction or along the stem was seen. Significant thigh pain persisted in 2 patients and was directly related to stem diameters > 17 mm. Complications in these complex cases were not infrequent, reflecting the need for allograft augmentation, and included greater trochanter bursitis and nonunion in 20 hips, minor nonpropagating fracture in 13 hips, and 12 dislocations. Mechanical loosening occurred in 5 hips. There were no complications attributable to the S ROM modular femoral component, and the prosthesis has proven to be versatile and did well in these very difficult cases. PMID- 7554623 TI - Femoral revision hip arthroplasty with uncemented, porous-coated stems. AB - One hundred seventy-five cementless femoral hip revision surgeries with extensively porous-coated stems and 161 concomitant acetabular revisions done from 1984 to 1991 were retrospectively reviewed. Average age at surgery was 62.4 years. Followup ranged from 2 to 10 years (average, 5 years). One hundred sixty eight femoral components (96%) remain in place. Two unstable components, 1 stable fibrous component, and 1 bone ingrowth component were painful and required rerevision. Two femoral components were removed as part of resection arthroplasties for repetitive acetabular failure caused by pelvic dissociation. One component was removed for late hematogenous infection. Using the Engh radiographic criteria, femoral stems were judged to have achieved bony ingrowth in 82.8% (n = 174) of the cases. Bony ingrowth occurred more frequently when the canal was filled with the prosthesis and with lesser degrees of bone stock deficiency. Severe stress shielding occurred in 11 (7.6%; n = 144) of the bone in grown cases and correlated with preoperative osteoporosis and larger diameter prosthesis. Significant thigh pain was less common in the bone ingrown (4.2%; n = 144) than in the stable fibrous group (18.5%; n = 27). Significant thigh pain in bone ingrown stems was more likely to occur with osteoporotic and bone stock deficient femurs. To date, significant wear and osteolysis have not been observed. PMID- 7554624 TI - Proximal femoral allografts for reconstruction of bone stock in revision arthroplasty of the hip. AB - Proximal femoral allografts have been used to restore uncontained circumferential defects of the multiply revised total hip arthroplasty. These grafts are used with long stem components that are cemented to the graft but not the host. The junction of host and graft is stabilized by the stem and a step cut with cerclage wires. Autograft bone is placed at the junctions to induce union. Full weightbearing is delayed until union occurs between the graft and the host femur, usually by 3 months. One hundred sixty-eight structural femoral allografts were done; average followup was 4.8 years as of January 1, 1995. Success was defined as an increase in the clinical score of at least 20 points, a stable implant, and no need for further surgery related to the allograft. The success rate in 130 patients with at least 2 years followup is 85%. There have been 17 revisions in 16 patients: 3 revisions for infection, 8 for dislocation, 5 for nonunion, and 1 for pain. The revision rate is 10.1%. Radiographic analysis showed 7 nonunions, minor resorption in 6 patients, and significant resorption in 1 patient. All implants are stable with no lucent lines. The results support using this technique for full circumferential segmental proximal femoral defects in revision hip arthroplasty. PMID- 7554625 TI - Preliminary report of impaction grafting for exchange femoral arthroplasty. AB - Gie and Ling have described a method for femoral component revision using compressed morselized cancellous allograft and a cemented collarless polished taper stem. The authors report their early experience with this technique. Of the first 67 patients who had femoral exchange by impaction grafting, 60 were alive 2 to 5 years after hip revision; 2 hips failed because of late sepsis, and 5 patients were decreased. In 56 individuals available for review, the Harris Hip Score average was 90 points, with > 80% reporting no pain. On radiograph, 48% of the stems showed an average of 2.8 mm of subsidence in the polymethylmethacrylate mantle, but only 7% of the cement graft composites had subsided in the cortical tube. Lucent lines were rare, and in 93% of revised femurs the radiographs showed evidence of graft incorporation and bone remodeling. There were 6 reoperations in the group: 3 for late fracture of the femoral shaft and 3 for cup exchange (2 chronically dislocating, 1 loose). No evidence for femoral component loosening was found in this group. Further study is necessary, but these preliminary findings give rise to cautious optimism that this is a reliable method for femoral revision, reconstruction, and reconstitution. PMID- 7554626 TI - The John Charnley Award. Thrombogenesis during total hip arthroplasty. AB - The activation of the clotting cascade leading to deep venous thrombosis begins during total hip arthroplasty, but few studies have assessed changes in coagulation during surgery. A better understanding of thrombogenesis during total hip arthroplasty may provide a more rational basis for treatment. In 3 separate studies, the following observations were made. Circulating indices of thrombosis and fibrinolysis: prothrombin F1.2, thrombin-antithrombin complexes, fibrinopeptide A, and D-dimer, did not increase during osteotomy of the neck of the femur or during insertion of the acetabular component, but rose significantly during insertion of the femoral component. Thrombin-antithrombin complexes, fibrinopeptide A, and D-dimer were higher after insertion of a cemented component than insertion of a noncemented femoral component. A significant decline in central venous oxygen tension was observed after relocation of the hip joint and after insertions of cemented and noncemented femoral components, providing evidence of femoral venous occlusion during insertion of the femoral component. In patients receiving a cemented femoral component, mean pulmonary artery pressure increased after relocation of the hip joint, indicating intraoperative pulmonary embolism. No changes in mean pulmonary artery pressure were noted with noncemented total hip arthroplasty. Administration of 1000 units of unfractionated heparin before insertion of a cemented femoral component blunted the rise of fibrinopeptide A. The results of these studies suggest that (1) the greatest risk of activation of the clotting cascade during total hip arthroplasty occurs during insertion of the femoral component; (2) femoral venous occlusion and use of cemented components are factors in thrombogenesis during total hip arthroplasty; and (3) measures to prevent deep venous thrombosis during total hip arthroplasty (such as intraoperative anticoagulation) should begin during surgery rather than during the postoperative period and be applied during insertion of the femoral component. PMID- 7554627 TI - Survivorship of uncemented proximally porous-coated femoral components. AB - Three hundred seventy-five consecutive total hip arthroplasty revisions done using proximally porous-coated femoral components of 6 designs were reviewed a mean of 4.7 years after surgery. Fifty-nine hips have been rerevised for aseptic femoral loosening, and 4 for osteolysis. Moderate or severe pain was present in 23% of surviving hips, and radiographic evidence of femoral loosening was present in 38% of surviving hips at the most recent followup. At 8 years, survivorship free of revision for aseptic femoral failure (for loosening or osteolysis) was 58% (95% confidence intervals, 44.3%, 69.6%); survivorship free of aseptic femoral loosening (revision for aseptic loosening or radiographic loosening) was 20% (95% confidence intervals, 12%, 27%); and survivorship free of symptomatic femoral loosening (revision for aseptic loosening or radiographic femoral loosening with moderate or severe pain) was 45% (95% confidence intervals, 32.3%, 56%). More severe preoperative bone loss correlated with poorer survivorship free of aseptic loosening and subsidence of > or = 5 mm. Differences among the prosthetic-type groups with respect to patient's age, gender, and bone loss severity precluded direct comparison of performance for each prosthetic type; however, all the prostheses had a significant rate of rerevision and aseptic loosening. Stable long-term fixation with the proximally porous-coated femoral components used in this series was not achieved on a predictable and reproducible basis. The damaged, weakened bone often present in the proximal femur during revision probably does not provide an optimal environment for sturdy initial or long-term biologic fixation of these devices that rely on the proximal femoral bone for fixation. PMID- 7554628 TI - Results of cemented femoral revision total hip arthroplasty using improved cementing techniques. AB - Seventy-nine consecutive femoral component revision total hip arthroplasties done in 73 patients from 1977 to 1983 using a distal intramedullary cement plug and a cement gun delivery system were evaluated to determine if improved results were obtained with newer cementing techniques. At a minimum 10-year followup interval, only 1 patient was not available for followup and 47 hips had minimum 10-year followup radiographs. The incidence of femoral rerevision for aseptic loosening was 9.5% for those hips with minimum 10-year followup and 5.4% for the entire group. The incidence of radiographic femoral failure (defined as a revision or definite or probable loosening) was 26.1% for those hips followed for a minimum of 10 years and 16.3% for the entire group. Compared with historical controls, these results represent an improvement over those reported with the use of earlier cementing techniques. PMID- 7554629 TI - Morselized allograft in acetabular reconstruction. A postmortem retrieval analysis. AB - Contained acetabular defects often are repaired at revision surgery using morselized bone allograft. The extent to which this graft becomes incorporated is unknown, and the value of radiographs for predicting allograft incorporation is unclear. To better understand the effectiveness of morselized allograft in revision hip arthroplasty, the authors studied postmortem specimens from 3 patients treated with this type of graft. Histologic analysis of acetabular components in situ for 18 months showed allograft fragments invested with loose myxofibrous tissue. Vascularized tissue had penetrated the allograft fragments to a depth of 4 mm. In peripheral areas, the vascularized ingrowth was accompanied by partial osteoclastic resorption of graft trabeculae and application of living bone to allograft fragments. After 53 months in situ, graft fragments had remodeled and showed progressive vascular ingrowth. By 83 months, the graft almost completely had incorporated. Morselized allograft bone is useful for restoring bone deficiency within contained acetabular defects. The graft does incorporate, but the extent of graft incorporation cannot be predicted accurately by postoperative radiographs. PMID- 7554630 TI - Ten years of experience with porous acetabular components for revision surgery. AB - A prospective study was completed for 139 porous-coated revision acetabular components that were implanted with rim fit fixation and had a mean of 4.3 years' followup (range, 2-9.8 years). The Anatomic Porous Replacement component was studied. Harris Hip Scores were used for clinical evaluation, and the pain and limp components of the Harris Hip Score were assessed. Radiographic measurements were recorded as radiolucent lines by Delee and Charnley zones. Clinical results were of lesser value in this study because of the emphasis on the acetabular component. Of these components, 4.3% had revision; only 2 (1.4%) were revised for loosening. Migration occurred in 2.1% of these acetabular components. In the first 2 years postoperatively, 17.4% of components had increased radiolucent lines and 9.4% had fewer radiolucent lines. After 2 years, fixation was stable: 4% had progressively more radiolucent lines and 2% fewer radiolucent lines. The authors could not show any statistical difference in radiographic fixation of these components when adjunctive screw fixation was used or not used. Fixation of the acetabular components was better when slurry bone graft was not used to layer the acetabular surface. Fixation was similar whether the rim fit was obtained with or without protrusion of the medial dome of the cup into the pelvis. The authors did not observe osteolysis in the acetabular bone surrounding these components. PMID- 7554631 TI - Revision total hip arthroplasty using a cementless acetabular component. Technique and results. AB - Are the midterm results (range, 7-11 years) for revision of the acetabulum with a cementless hemispherical component comparable with other published revision techniques at similar followup? One hundred thirty-eight acetabular revisions for aseptic loosening were done in 132 patients using a cementless hemispherical component coated with titanium mesh and inserted with supplemental screw fixation. Twelve patients died, 9 were lost to followup, and 6 could not return for followup, leaving 111 patients (115 hips) with a mean of 100 months of followup. Thirteen hips required revision (11%): 4 were done for recurrent dislocations, and 6 for sepsis; 3 stable cups were revised (at the time of stem revision). No cup was revised for aseptic loosening. Radiographic review was available for 105 patients (109 hips) at mean 98-month followup (78-135 months). A complete radiolucency was seen in 4% of the cups, a partial progressive radiolucency in 3%, and a partial nonprogressive radiolucency in 54%; no radiolucency was present in 39%. A screw radiolucency was seen in 2%, and osteolysis at the cup margin in 4%. Revision of the acetabulum with a cementless porous-coated hemispherical fiber-metal component is superior to the results reported for acetabular revisions with cement at similar followup. PMID- 7554632 TI - Economics of revision total hip arthroplasty. AB - A study was undertaken to determine the relative work input and risk involved in doing primary total hip arthroplasty compared with revision total hip arthroplasty. These data were compared with the reimbursement to the hospital and to the surgeon. Parameters examined included operative time, length of hospital stay, blood loss, use of bone graft, and incidence of complications requiring further treatment. Revision total hip arthroplasty required significantly more work and risk on the part of the surgeon and significantly more hospital resources. The estimated hospital cost was more than the diagnosis-related group reimbursement for primary cases and dramatically more for revision cases. Actual surgeon reimbursement was less than prosthetic cost and was not significantly higher for revision than primary cases. PMID- 7554633 TI - Efficacy and safety of enoxaparin to prevent deep vein thrombosis after hip arthroplasty. AB - Four clinical studies were conducted in the United States and Canada to compare the efficacy and safety of Enoxaparin, a low molecular weight heparin, with low dose unfractionated heparin and placebo for the prevention of deep venous thrombosis after hip arthroplasty. In each study, patients were randomized consecutively into treatment groups of placebo, unfractionated heparin (5000 IU 3 times daily or 7500 IU twice daily), or Enoxaparin (30 mg twice daily, 40 mg once daily, or 10 mg once daily), with treatment started postoperatively. All patients had noninvasive studies and bilateral lower extremity radiocontrast venography at the end of study treatment or on discharge from the hospital (not applicable to the first 24 patients enrolled in the Canada-1 study). One thousand nine hundred forty patients were treated, and 1937 patients were included in efficacy analysis. The incidence of total deep venous thrombosis was as follows: placebo group, 46% (22 of 50 patients); heparin group, 16% (87 of 539 patients); Enoxaparin group, 30 mg twice daily 12% (93 of 785 patients); Enoxaparin 40 mg daily group, 14% (14 of 402 patients); and Enoxaparin 10 mg daily group, 25% (40 of 161 patients). Incidence of proximal deep venous thrombosis was 22%, 5%, 4%, 4%, and 11%, respectively. Major bleeding events were reported in 4% of the placebo group, 2% to 4% in the Enoxaparin group, and 6% in the unfractionated heparin group. In these clinical studies, Enoxaparin, 30 mg twice daily, was shown to be as effective and safe as low dose unfractionated heparin to prevent deep venous thrombosis after hip arthroplasty. PMID- 7554634 TI - Magnetic resonance imaging of the pelvis. New orthopaedic applications. AB - A preliminary study of using magnetic resonance angiography to detect occult proximal thrombi in patients who had hip arthroplasty was done. Despite the presence of susceptibility artifact caused by metallic components, diagnostic visualization of thigh vessels was made in a preliminary series of 15 patients. Confirmation of all previously documented (by contrast venogram via dorsal foot vein cannulation or Doppler study) proximal thrombi was made in all 15 patients. One patient had a thrombus in the contralateral extremity that had been undetected by Doppler study; 4 additional pelvic thrombi occurred in 3 patients, which had been undocumented previously. Because magnetic resonance angiography is noninvasive, requiring no contrast agent, it has advantages over conventional venography to detect occult proximal thrombi. New fast spin echo sequences are discussed that enhance visualization of regional anatomic structures adjacent to metallic prosthetic components. Emphasis was placed on assessing the posterior soft tissue envelope in patients having recurrent dislocations after total hip arthroplasty, despite acceptable component alignment. Preliminary results show a consistent absence of a posterior pseudocapsule in patients having dislocations, as compared with control patients having no dislocations. PMID- 7554635 TI - Recementing a femoral component into a stable cement mantle using ultrasonic tools. AB - Recementing a femoral prosthesis into a retained cement mantle was done during revision total hip arthroplasty in 15 patients using ultrasonic tools to reshape the existing cement mantle. Early complications such as bone perforation or fracture were avoided. The 2-year followup in 4 patients revealed no adverse effect on the bone-cement interface as judged radiographically. One patient had rerevision for instability at 1 year after operation. Indications for this technique include isolated failure of the cement-prosthesis interface, femoral component fracture, or isolated acetabular revision in which exposure needs, instability, leg length inequality, or femoral-head size dictated change of the femoral component. PMID- 7554636 TI - The resistant frozen shoulder. Manipulation versus arthroscopic release. AB - Frozen shoulder is often a self-limited disease, but approximately 10% of patients have long-term problems. Arthroscopy was done in 40 patients with persistent pain, stiffness, and functional loss for at least 1 year without improvement despite conventional treatment. In the first 20 patients, manipulation was done with an arthroscopy before and afterward; in the second 20 patients, the contracted structures were divided through arthroscopy. This was a prospective cohort study; 2 patients were not available for followup. The arthroscopic division procedure was done in 4 sequential steps: (1) resection of the inflammatory synovium in the interval area between the subscapularis and supraspinatus; (2) progressive division of the anterior superior glenohumeral ligament and anterior capsule; (3) division of the subscapularis tendon but not muscle; and (4) division of the inferior capsule. The results were assessed independently on the basis of pain, stiffness, and function. The followup varied from 2 to 5 years after intervention. Patients treated with arthroscopy and manipulation did as well as the patients treated with arthroscopic division for restoration of range of movement. However, the patients in the arthroscopic division group had significantly better pain relief and restoration of function. Fifteen of 20 patients treated with arthroscopic division had an excellent result compared with 7 of 18 patients treated with arthroscopy and manipulation. Patients with diabetes did worse initially, but the outcome was similar to patients without diabetes. Patients with diabetes in particular may benefit from early intervention. PMID- 7554637 TI - Changes on magnetic resonance images after traumatic hip dislocation. AB - Fourteen patients with traumatic hip dislocation had serial magnetic resonance imaging and routine radiographic studies from the time of injury through 24 months after injury. One experienced radiologist interpreted all images prospectively for abnormalities suggesting osteonecrosis of the femoral head and posttraumatic arthritis. Eight hips demonstrated abnormal marrow signals on T1 and T2 weighted images within 6 weeks of injury. These changes progressed in 3 hips, and osteonecrosis was confirmed subsequently by plain radiography. The abnormal marrow signals in the remaining 5 hips proved to be transient, resolving on magnetic resonance images within 3 months in 4 of the 5 patients. Magnetic resonance imaging can be used with confidence for the early detection of osteonecrosis of the femoral head after traumatic hip dislocation or fracture dislocation. The presence of acetabular or femoral shaft hardware did not preclude magnetic resonance imaging assessment of these patients when coronal, sagittal, and axial images were obtained. Magnetic resonance imaging was not reliable for assessing marrow changes within the first week after injury, nor was it helpful in predicting which patients were at risk for posttraumatic arthritis to develop. An algorithm is proposed for using magnetic resonance imaging in the early diagnosis of osteonecrosis of the femoral head after traumatic hip dislocation. PMID- 7554638 TI - Determinants of reduced survival following hip fractures in men. AB - To assess determinants of poor survival after hip fractures in men, a population based cohort study was conducted among 131 men in Rochester, MN, who had their first hip fracture during the period from 1978 to 1989, and an equal number of age-matched control men from the community. One hundred nine patients with fractures died during 373 person-years of followup, but only 75 control men died during 742 person-years of observation. The risk of dying increased with the level of comorbidity among hip fracture cases (hazard ratio 3.2; 95% confidence interval, 1.2-8.2), as well as with age (hazard ratio, 1.4 per 10-year increase; 95% confidence ratio, 1.1-1.8) and mental confusion during hospitalization (hazard ratio, 4.2; 95% confidence interval, 2.5-6.9). Discharge to a nursing home and low activity status also were predictors of death in the univariate analysis. Excess mortality among men with hip fractures can be explained best by interaction of the fracture with serious underlying medical conditions. PMID- 7554639 TI - Rates of tibial osteotomies in Canada and the United States. AB - This study determined the temporal trends and factors associated with the rates of performance of tibial osteotomies from 1985 to 1990 in Ontario, Canada and the United States. The Health Care Financing Administration, Ontario Health Insurance Plan, and National Hospital Discharge Survey databases were used to determine the number of osteotomies from 1985 to 1990. Osteotomy rates decreased in both countries approximately by 11% to 14% per year in patients 65 years and older and by 3% to 4% per year in patients younger than 65 years. Men received twice as many osteotomies as women in both countries. In the United States, the average rate of tibial osteotomies was 2 to 3 times lower than in Ontario. PMID- 7554640 TI - Changes in hip muscles after above-knee amputation. AB - To learn about the changes appearing in hip muscles after an above-knee amputation, 3-dimensional reconstructions of the hip and thigh region of 12 patients with above-knee amputations were made based on transverse magnetic resonance images. In all patients, the amputations were done at least 2 years before the study and were necessitated by trauma or osteosarcoma. The results show that, at higher amputation levels, the geometry of the once-biarticular muscles was changed. The cleaved muscles (40%-60%) and the intact muscles (0-30%) at the amputated side were atrophied. The amount of atrophy of the intact muscles at the amputated side was related to stump length. To avoid an abduction contracture in 8 patients with amputations, the iliotibial tract was not fixed. In 4 of these 8 patients, a flexion contracture was visible. If the tract was not fixed, the hip extension torque of the gluteus maximus, which inserts into the tract, decreased. As a result, the risk of appearance of a flexion contracture increased because the strongest hip flexor (iliopsoas muscle) was not involved in the amputation. Abduction contracture could be avoided only if the hip adductors were fixed accurately, especially at higher amputation levels. PMID- 7554641 TI - The Otto Aufranc Award. Impact of gamma sterilization on clinical performance of polyethylene in the hip. AB - Despite studies to determine their causes, significant variations in polyethylene acetabular component wear rates, radial cracking of component rims, and occasional delamination cannot be explained. A subsurface white band frequently occurs in such damaged components. These damaged components often are gamma sterilized. To date, the origin of the band and its effect on polyethylene chemical and mechanical properties, and hence, clinical performance, have not been confirmed, and correlations between radiation sterilization and clinical wear have not been made. By developing techniques for polyethylene retrieval testing and rating, chemical analysis, and mechanical analysis, this research has determined that gamma sterilization in air alters the chemical and mechanical properties of polyethylene over time, resulting in high subsurface oxidation, reduced ductility, and reduced strength. Gamma sterilization-induced oxidation is found to be most severe in the subsurface region of components, and coincides with zones of significantly reduced strength and ductility. This chemical and mechanical property degradation is time dependent and is not typically visible until after 3 years' postirradiation. The presence of the subsurface white band significantly correlates with clinical cracking and delamination observed in retrieved components. Wear of the retrieved components often is observed to have progressed into this heavily oxidized, weakened, and embrittled zone. A method for accelerated aging shows that irradiating in air causes oxidation damage in polyethylene components that is not seen with other sterilization methods. Modifications of gamma sterilization techniques to minimize this damage are discussed. PMID- 7554642 TI - Long-term outcome of 42 knees with chronic infection after total knee arthroplasty. AB - The outcome of treatment in 40 patients (42 knees) with chronic infections after total knee arthroplasty was reviewed. Eighteen knees were treated with a 2-stage reimplantation. Sixteen of these 18 knees were treated with antibiotic-containing beads between debridement and reimplantation, and 7 of these were also treated with antibiotics in the cement at reimplantation. Infection did not recur in any of these 18 knees. Clinically, the 2-stage reimplantation group averaged a score of 90 points on the Knee Society Clinical Rating System. Average function score was 86.5 points, with average range of motion from 2 degrees to 109 degrees. Sixteen knees were treated with an arthrodesis: 9 with a 1-stage technique with a uniplanar external fixator and 7 with a 2-stage technique with intramedullary nail internal fixation. Infection did not recur in 6 of 9 knees treated with the 1-stage technique, but only 2 had a solid arthrodesis. All 7 treated with the 2 stage intramedullary nail technique had no recurrence of infection and achieved a solid fusion. Reimplantation or arthrodesis was not attempted in 8 other knees because of recalcitrant infection, vascular complications, or medical infirmity. Of the 42 knees, 11 (26%) had a severely morbid outcome. The infection could not be eradicated in 7 knees: 6 required amputation and 1 had a solid fusion but chronic drainage. In 3 knees, the infection was cured but resection arthroplasties were required, and in 1 patient an amputation was needed as a result of an intraoperative vascular complication. PMID- 7554643 TI - Biomechanical study on tibialis posterior tendon transfers. AB - Two methods are used to route the tibialis posterior tendon anteriorly to achieve dorsiflexion: (1) around the medial side of the tibia, or the subcutaneous route; and (2) through the interosseous membrane, or the interosseous route. This study determined the effect of site of tendon insertion on ankle and foot motions and compared the efficacy of both routes. Eleven fresh normal cadaveric legs were used. The detached tibialis posterior tendon was transferred anteriorly through the interosseous membrane and anchored to the first cuneiform along the first metatarsal axis by a barbed staple. The specimen was mounted on a mechanical testing machine. Tension was applied to the tendon and ankle and foot motions were measured. The experimental procedure was repeated with tendon insertion along the second metatarsal axis and serially through to the fifth metatarsal axis. The entire experiment was repeated using the subcutaneous route. The interosseous route was more effective in achieving maximum dorsiflexion with minimal pronation. Shifting the insertion medially caused supination, whereas a more lateral insertion caused pronation. PMID- 7554644 TI - Measurement of polyethylene wear in metal-backed acetabular cups. I. Three dimensional technique. AB - A new technique, the 3-dimensional technique, has been developed. Using a digitzer, points taken from anteroposterior and lateral radiographs are input into a personal computer. A 3-dimensional solid model of the prosthesis is created using custom software. Tilt and anteversion of the acetabular cup are measured, and the model is rotated to a standard frontal view, allowing measurement of femoral head displacement from the center of the acetabular cup. Comparing serial radiographs of the same patient gives the direction and distance of femoral head displacement over time, and this value is used to calculate the minimum volume of polyethylene debris generated. The 3-dimensional technique has been validated by construction of a precision acrylic phantom, and milling of a metal-backed acetabular cup to simulate polyethylene wear. Three-dimensional measurement of femoral head displacement using this technique has an accuracy of +/- 0.15 mm, and volume calculations are within 8% of the true amount of polyethylene removed from the cup. The 3-dimensional technique gives previously unavailable information, and can be used for clinical evaluation of polyethylene wear and evaluation of new prosthetic designs and bearing surfaces. PMID- 7554645 TI - Measurement of polyethylene wear in metal-backed acetabular cups. II. Clinical application. AB - The new 3-dimensional technique was applied to the radiographs of 141 patients who had received Porous Coated Anatomic total hip prostheses. Values were obtained for the position of the acetabular cup, the 3-dimensional distance and direction of femoral head displacement, and the minimum volume of polyethylene debris produced. Mean age of the patients at the time of replacement was 61 years old; mean followup was 5.6 years (range, 4-7.2 years). Change in the position of the femoral head between initial and long-term followup films was assumed to represent polyethylene wear, and formulas were used to calculate the minimal volume of polyethylene debris produced. The overall rate of 3-dimensional femoral head displacement was 0.264 mm per year, almost twice that usually quoted in the current literature for 2-dimensional linear wear. A significant contribution to this value was made by anterior and posterior displacement. Two-dimensional femoral head displacement (measured in the plane of anteroposterior radiographs) on the same patients was 0.149 mm per year. The mean minimum volume of polyethylene debris produced after 5.6 years was 0.448 cm3 (range, 0.00-2.83 cm3), giving a mean rate of 0.079 cm3 polyethylene debris produced each year. Thirteen patients in this series had radiologic osteolysis and a significantly greater femoral head displacement and polyethylene volumetric wear than those with no osteolysis. Patients with a 32-mm femoral head diameter and a polyethylene linear < 1 cm thick had a significantly greater amount of polyethylene wear. This series gives previously unavailable data on 3-dimensional femoral head displacement and is the first report that correlates the minimum volume of polyethylene wear produced with radiologic osteolysis. PMID- 7554646 TI - Hip pain in a 45-year-old woman. PMID- 7554647 TI - Spur formation and heel pain. PMID- 7554648 TI - The long-term results of low-friction arthroplasty of the hip performed as a primary intervention. 1972. PMID- 7554649 TI - The Frank Stinchfield Award. Pulmonary fat embolism in revision hip arthroplasty. AB - Unilateral cemented hip hemiarthroplasty was done on 16 dogs who subsequently had revision arthroplasty and who were divided into 1 control and 3 experimental groups: The first group had cement extraction using osteotomes; the second, using a high speed burr; the third, an ultrasonic tool. Hemodynamic and transesophageal echocardiographic monitoring were done. Postmortem pulmonary specimens were examined for differences in the quantity of fat emboli. There was a significant increase in emboli with the ultrasonic tool as compared with osteotomes and high speed burr. There was no significant difference in emboli between the osteotomes and high speed burr. Fat emboli syndrome is related to mechanical compression of the femoral canal. The ultrasonic instrument was unique in its tendency to cause large embolic showers, especially during extraction of the distal cement plug. In these young dogs, minimal hemodynamic changes and no cardiac dysrhythmias occurred, which in part may be attributed to their good health. These changes may remain subclinical for patients with good cardiorespiratory reserve, or may become life threatening for those with poor reserve. By outlining the mechanisms of fat embolism in revision total hip arthroplasty, it may be possible to decrease future morbidity, especially in patients who frequently have cardiopulmonary disease. PMID- 7554650 TI - Nonconsolidated polyethylene particles and oxidation in Charnley acetabular cups. AB - Nonconsolidated particles of ultra high molecular weight polyethylene are believed to be defects that adversely can affect the wear performance of total joint prostheses. The present study was done to determine the number, size, and distribution of these particles and to determine if their presence correlated with wear performance, as well as with other clinical and implant parameters. Forty retrieved and 7 new, never-implanted acetabular components were examined using light microscopy on thin cross sections. Particles were found in 92% of retrieved components and in all the new components. Particles in the retrieved components were either randomly distributed (32 components) or banded (with particles localized in regions approximately 1 mm below the outer surface of the component). No correlations were found between the number or area of particles and the wear performance or any of the clinical or implant variables. The presence of particles in the new implants was found to correlate with the length of time since the components had been radiation sterilized. For retrieved components, the density (and, therefore, the level of oxidative degradation) was high in the areas of banded particles. For new components, the density was higher the longer the time since sterilization. Nonconsolidated polyethylene particles are prevalent in total replacements but their source and cause are unknown. The results of this study show that they do not appear to affect or correlate with the length of implantation of acetabular cups. However, they still may be expected to adversely affect performance in cases where large numbers of particles are banded together near articulating surfaces of high stress environments such as found in the knee. PMID- 7554651 TI - Preclinical testing of total hip stems. The effects of coating placement. AB - The long-term fixation endurance of noncemented hip stems in total hip arthroplasty is subject to incompatible design goals. To reduce stress shielding and periprosthetic bone loss, proximal fixation and load transfer are indicated. However, to prevent interface motion and promote interface-bonding security, fixation preferably should be maximized over the entire stem surface. In this study, the authors questioned whether hydroxyapatite coatings could be applied in patterns that reduce bone resorption, while maintaining safe interface stress levels. For that purpose, strain-adaptive bone-remodeling theory was applied in 3 dimensional finite element models, to simulate the long-term postoperative bone resorption process. During the process, the adaptation of interface stresses was monitored, and its effects on interface failure probability evaluated. This analysis was done for a fully coated stem, a 1/3 proximally coated stem, a smooth uncoated, press-fitted stem, and a stem with 5 proximal patches of circumferential stripes. The uncoated stem reduced bone loss dramatically, but promoted interface motions and distal pedestal formation. In all cases, the gradual bone-remodeling process increased the interface security of the coated stems. Bone loss and interface failure probability were not very different for the fully and 1/3-coated stems. Stripe coating reduced bone resorption considerably, while increasing long-term interface failure probability only slightly. The investigators concluded that the initial stability and the ingrowth potential of such a stem design are likely to be inadequate. PMID- 7554652 TI - The impact of modularity in total hip arthroplasty. AB - Use of modularity in total hip arthroplasty has been of benefit in terms of allowing inventory reduction while providing surgeon versatility and thus optimal joint reconstruction. As with any advancement, the improvements gained must be weighed against their cost. The authors sought to examine the effects that modularity may have on the results of total hip arthroplasty in terms of bone loss, durability of fixation, time to failure, and incidence of revision. Using data from their total joint registry, the authors examined their experiences with total hip arthroplasties done during the past 25 years. They sought to compare the results of total hip arthroplasties in patients who differed chiefly by the number of modular junctions present in their hip prostheses. With each incremental increase in modularity, an earlier appearance of radiolucencies, an earlier occurrence of aseptic loosening, and an increased incidence of osteolysis in the absence of loosening was found. The authors conclude that suboptimal design rather than the concept of modularity best explains these results. The presence of suboptimal design in a total hip arthroplasty may cause polyethylene to be stressed beyond its performance limits. PMID- 7554653 TI - Complications of femoral and acetabular modularity. AB - The versatility of modular total hip arthroplasties have rapidly extended their applications. However, these new interfaces can lead to complications that were not observed with monolithic components. These problems have been noted with modular femoral and acetabular components and have been associated with the generation of particulate debris. This article reviews the authors' clinical observations and histologic, biomechanic, and spectophotometric evaluations of modular total hip arthroplasties. New data comparing both synovial fluid metal levels in well-fixed and loose monolithic and modular prosthetic hip implants are presented. In modular total hip components, synovial fluid cobalt levels correlated positively with patient weight and length of implantation. The generation of particulate debris in modular total hip components may induce periprosthetic osteolysis. Taper locks for femoral components and locking mechanisms for the polyethylene liner and metallic cup must be designed to avoid the production of particulate debris. PMID- 7554655 TI - False-negative morphine-augmented hepatobiliary scintigraphy with a rim sign. AB - A patient with cholelithiasis and clinically suspected of acute cholecystitis underwent hepatobiliary scintigraphy that demonstrated initial nonvisualization of the gallbladder in association with a subtle rim sign, but subsequent gallbladder visualization after administration of intravenous morphine. After an 8-day period of medical management, an elective cholecystectomy was performed, which revealed a gangrenous gallbladder with a gallstone in the neck and full thickness necrosis. These results suggest that gallbladder visualization on morphine-augmented hepatobiliary scintigraphy should be interpreted cautiously in the presence of a rim sign because acute cholecystitis remains a possibility. PMID- 7554654 TI - Local and distant products from modularity. AB - In this study, the local and distant distribution of solid and soluble products of corrosion from the head and neck junction of modular femoral total hip prosthetic components were characterized. Particulate corrosion products from retrieved implants and surrounding tissues were analyzed. Serum transport and urinary excretion of metal was measured in correlation with the degree of corrosion at the head and neck junction. Particles of metal oxides, metal chlorides, and chromium phosphate corrosion products were identified on implants of 10 designs from 6 manufacturers. The most abundant solid corrosion product on the implant and within the periprosthetic tissues (size range, < 1-200 micrometers) was an amorphous chromium orthophosphate hydrate-rich material. Serum cobalt and urine chromium concentrations were elevated significantly in patients with implants that had moderate to severe corrosion in comparison with those with no to mild corrosion. Solid corrosion products from modular femoral stems may accelerate articular wear via a 3-body mechanism. Phagocytosable particles of these corrosion products may stimulate macrophage-mediated periprosthetic bone loss. Systemic dissemination of metallic corrosion products raises the issue of systemic toxicity; however, no overt evidence of metal toxicity was observed in this study. PMID- 7554656 TI - Investigation of abnormal appearing thyroid glands on Tc-99m sestamibi breast scintimammography. AB - Of 507 breast scintimammograms performed using Tc-99m sestamibi, 34 patients were incidentally found to have abnormal appearing thyroid glands. The authors sought to investigate the clinical significance and/or ascertain an extraneous causative factor for these findings. One cold thyroid nodule was detected and was proven benign through biopsy. Two cases of subacute and chronic thyroiditis were proven, and it is possible that additional patients had this disease process. Focal areas of increased thyroid Tc-99m sestamibi uptake were observed in eight patients and probably represent parathyroid adenomas, nonautonomous hyperfunctioning thyroid nodules, or nontoxic multinodular goiter. The results did not yield an extraneous causative factor, underlying pathology, or clinically significant disease in all of the patients investigated, but the findings suggest a need for careful evaluation of any unusual uptake in the thyroid gland and the rest of the image. The authors do not recommend investigation of all abnormal appearing thyroid glands on Tc-99m sestamibi scintimammography. However, clinical correlation should be recommended. PMID- 7554657 TI - Three-phase bone imaging in bone marrow edema of the knee. AB - Transient bone marrow edema has been thought to be related to other similar entities including transient regional osteoporosis and spontaneous osteonecrosis. Although the findings of three-phase bone imaging have been described in osteonecrosis, scant attention has been paid in the nuclear medicine literature to patients with transient bone marrow edema. The authors report the findings of three-phase bone imaging in two patients with knee pain and a transient bone marrow edema pattern as documented by MRI. PMID- 7554658 TI - Subcutaneous splenosis. AB - Splenosis is the ectopic implantation of splenic tissue, usually as the result of trauma. Intraperitoneal, intrathoracic, and retroperitoneal sites of implantation have been reported. The authors report a case of subcutaneous splenosis involving the abdominal wall that was imaged with CT and scintigraphy. PMID- 7554659 TI - MAI osteomyelitis. 18-year scintigraphic follow-up. AB - Mycobacterium avium-intracellulare osteomyelitis is an infrequently reported entity, especially in immunocompetent patients. When multifocal, the imaging findings can be confusing, simulating metastatic disease or other bone lesions. An immunocompetent patient with multifocal Mycobacterium avium-intracellulare osteomyelitis is presented who has been followed for 18 years and experienced episodes of exacerbations and further dissemination of her disease, followed by periods of partial remission and clinical quiescence. Bone scintigraphy was very useful in evaluating the extent of involvement and monitoring response to treatment. PMID- 7554660 TI - Diffusely discordant In-111 WBC/Tc-99m SC bone marrow uptake. A possible chemotherapeutic effect. AB - In-111 WBC scintigraphy in a woman with relapsed acute lymphoid leukemia demonstrated normal uptake of white blood cells by the liver and spleen, but virtually absent bone marrow activity. Tc-99m SC imaging confirmed normal marrow function and distribution. A bone marrow biopsy revealed mildly hypocellular, regenerating marrow without leukemic infiltration. The effects of systemic cytotoxic chemotherapy on marrow reticuloendothelial function may have been responsible for this discordant uptake. PMID- 7554661 TI - Detection of a 2.1 cm hepatocellular carcinoma in an 11-year-old boy by Tl-201 chloride. Case presentation and correlative imaging findings. AB - Abnormal uptake of Tl-201 chloride in the liver along with correlative radiologic imaging is presented in an unusual case of an 11-year-old boy with a small 2.1 cm hepatocellular carcinoma. Although planar Tl-201 images were normal, triple headed SPECT Tl-201 images showed a focal area of increased liver activity at the site of the tumor which corresponded to a photopenic area on Tc-99m SC SPECT images. The patient successfully underwent partial liver resection of a pathologically proven hepatocellular carcinoma. PMID- 7554662 TI - Evaluation of embolization effect of hepatocellular carcinoma by hepatic arterial flow study with Tc-99m MAA SPECT. AB - The degree of hepatocellular carcinoma vascularity in eight patients was assessed with Tc-99m MAA radionuclide angiography before and after treatment. For chemoembolization therapy, lipiodol and doxorubicin hydrochloride were administered intraarterially along with Gelfoam particles. The ratio of radioactivity in the hepatocellular carcinoma compared to that in the surrounding uninvolved tissue before therapy was 6.5-19.0 with a mean of 12.0. After therapy, the ratio dropped to 0.5-1.3. Because survival improved significantly after the therapeutic regimen used in these patients, radionuclide Tc-99m MAA angiography appears to be an accurate method for demonstrating successful embolization of the vascular supply in hepatocellular carcinoma. PMID- 7554663 TI - In-111 DTPA Ommayagrams in leptomeningeal carcinomatosis. AB - Radionuclide Ommayagrams in 25 patients with leptomeningeal carcinomatosis were performed after injection of 0.5 mCl of In-111 DTPA into Ommaya shunt reservoirs to evaluate cerebrospinal fluid-shunt communication, detect the blockage of the cerebrospinal fluid pathway, evaluate the cerebrospinal fluid flow pattern, and predict the distribution of the chemotherapeutics. All results were correlated with MRI evaluations of the brain and spine, as well as clinical findings. Radionuclide Ommayagrams were found to be 73% sensitive and 100% specific in the evaluation of cerebrospinal fluid space disease. MRI of the spine and head was found to be 100% sensitive, but 86% specific for obstructive cerebrospinal fluid space disease. PMID- 7554664 TI - Scintigraphic assessment of pericardio-peritoneal window patency. Relevance to peritoneal dialysis. AB - To alleviate recurrent pericardial effusion secondary to systemic lupus erythematosus, pericardio-peritoneal window was performed. Subsequently, end stage renal disease developed and the patient required peritoneal dialysis. Patency of the pericardio-peritoneal window was demonstrated by intraperitoneal injection of Tc-99m SC through a Tenckhoff catheter, which prompted special counsel to the patient in order to prevent infectious pericarditis potentially complicating peritoneal dialysis induced-peritonitis. PMID- 7554665 TI - Detection of increased blood flow to the affected arm in repetitive strain injury with radionuclide and Doppler ultrasound studies. A case report. AB - A case of clinically diagnosed repetitive strain injury was referred for investigation to rule out the possibility of an occult bone disease. The patient was a female keyboard operator who had pain and tenderness over the flexor muscles of the right hand and arm. The pain was severe and almost constant. The authors observed an increase in Tc-99m MDP delivery to the affected forearm during the dynamic sequence of a three-phase bone scintigram, indicating increased blood flow compared to the contralateral side. The same result was achieved using quantitative blood flow measurements with the Doppler technique and Tc-99m HMPAO perfusion imaging. These findings agree with recent studies that suggest increased total arm blood flow in repetitive strain injury and may provide an easy screening method. PMID- 7554666 TI - Fe-52 imaging of intrathoracic extramedullary hematopoiesis in a patient with beta-thalassemia. AB - Fe-52 scintigraphy was used to confirm extramedullary hematopoiesis in a patient with beta-thalassemia and intrathoracic masses. Imaging was performed on a standard gamma camera with a high-energy collimator. Tc-99m labeled tin colloid and In-111 chloride scintigraphy failed to reveal uptake by the masses. The exclusion of malignancy obviated the need for invasive diagnostic measures. PMID- 7554667 TI - I-131 MIBG and bone scintigraphy in a patient with MEN. AB - The case of a patient with MEN is described. Emphasis is placed on the role of I 131 MIBG and bone scintigraphy as a part of the diagnostic and follow-up procedures. PMID- 7554668 TI - The change in the distribution of Tc-99m human serum albumin radioaerosols in asthma after a 1-week course of corticosteroid inhalation treatment. AB - This study evaluated the effects of corticosteroid inhalation on the deposition pattern of Tc-99m HSA radioaerosols in 24 patients with asthma. The homogeneous degree of depositing radioaerosol was quantitatively evaluated using a modified standard score system over both lungs. The baseline scores were calculated before inhalation therapy of 0.05 mg beclomethasone dipropionate four times daily for 1 week. The studies were then repeated after treatment to evaluate the effects of inhalation therapy. After treatment, the scores decreased in 16 of 24 cases (67%), which means that the degree of the bronchial obstruction decreased. The statistical results revealed significant differences in the total homogeneity score (P < 0.001) before and after therapy. Thus, a 1-week course of beclomethasone dipropionate inhalation therapy improves the bronchial obstruction in asthma patients as shown by the quantitative homogeneity score system of the Tc-99m HSA radioaerosol inhalation lung scintigraphy. PMID- 7554669 TI - Bilateral subclavian vein thromboses presenting as a superior vena cava syndrome demonstrated by radionuclide blood flow and In-111 WBC imaging. AB - A superior vena cava syndrome was caused by venous thrombosis of both proximal subclavian veins. Collateral pathways were clearly demonstrated by radioisotope venography. Indium-111 labeled platelet thrombography was useful in localizing thrombotic lesions. PMID- 7554670 TI - Supine lung clearance of Tc-99m DTPA and HMPAO aerosols. AB - The speed of Tc-99m DTPA/HMPAO radioaerosol clearance from the lungs that is represented as a slope from lungs to blood was measured in 23 male normal controls using commercial lung radioaerosol delivery units in the supine position in order to avoid the influences of gravity. The right lung was selected and three regions of interest were created for equal subdivisions of the upper, middle, and lower third. The results show that the clearance of Tc-99m DTPA/HMPAO aerosols in the upper lung is slowest. The differences between upper and lower lungs for Tc-99m DTPA/HMPAO aerosol clearances are significant. The clearance of Tc-99m DTPA aerosols was significantly faster than those of Tc-99m HMPAO in any region. The authors conclude that, although the effect of gravity disappears in the supine position in our study, the differences of aerosol clearance in different regions are still significant. Lipophilic Tc-99m HMPAO aerosols were slower than those of hydrophilic Tc-99m DTPA, which suggests there are at least two different mechanisms. PMID- 7554671 TI - Tc-99m DTPA-HSA SPECT in a case of hemangioma of the spleen. PMID- 7554672 TI - Intense muscle uptake of Ga-67 citrate in a patient with hypercoagulable state after exercise. PMID- 7554673 TI - Incidental detection of papillary carcinoma of the thyroid by Tc-99m (V) DMSA tumor scintigraphy. PMID- 7554674 TI - Follow-up of treatment of a cerebral arteriovenous malformation with acetazolamide and positron emission tomography. PMID- 7554675 TI - Visualization of a postoperative bile leak during cholescintigraphy. Morphine augmentation prevents a false-negative study. PMID- 7554676 TI - Accumulation of I-123 IMP in hepatic cell adenoma. PMID- 7554677 TI - The detection of occult phlebitis with Tc-99m stannous fluoride colloid labeled white blood cell scintigraphy. PMID- 7554678 TI - Lung perfusion scintigram in a patient with tetralogy of Fallot and a unidirectional Glenn shunt. A case report. PMID- 7554679 TI - Splenic-perisplenic infected hematoma detected on radiogallium-radiocolloid subtraction study. PMID- 7554681 TI - SPECT in adult mosaic Down's syndrome with early dementia. PMID- 7554682 TI - Gambling among adolescents. AB - Gambling onset during adolescence has been the subject of recent articles in the lay press. This study reports on the incidence of gambling in adolescents. There were 97 males (48.7%) and 102 females (51.3%) between the ages of 12 and 18 years. The mean age of the gamblers was 15.12 +/- 1.83 (SEM), and of the nongamblers 14.36 +/- 1.78. Eighty-three percent of the males and 61% of the females reported gambling. Forty-four percent of those who gambled, but only 26% of the nongamblers, reported having at least one parent who gambled. Our study confirms the high incidence of gambling by teenagers. Of concern is the potential link between gambling and other risk-taking or addictive behaviors. Perhaps we should include gambling as part of our anticipatory guidance for adolescents. PMID- 7554680 TI - Intestinal Tl-201 activity in the thorax mimicking lung metastasis of malignant melanoma. PMID- 7554683 TI - Connatal Leigh disease. AB - Two children are described with pathology-proven Leigh disease. Rather than the typical degenerative course with loss of acquired development, they presented with a static encephalopathy manifested by seizures from birth and failure to acquire any milestones. A similar connatal presentation has been reported in other degenerative disorders, such as Pelizaeus-Merzbacher disease. Heredodegenerative disorders should be considered when no cause is discovered for a severe, congenital, static encephalopathy. PMID- 7554684 TI - Evaluation of vision screening practices of Illinois pediatricians. AB - A survey of vision screening practices of American Academy of Pediatrics physicians in Illinois is described. The response rate was 42%. Sixty percent of physicians tested visual acuity of children 5 years and older, and half of this group tested children 2 to 4 years old. The most common reasons for not testing visual acuity were inadequate time (42%), children too young (18%), or screening done at school (18%). The majority (88%) refer to an ophthalmologist after a single vision screening failure, while about half perform the cover-uncover test on infants and children. The results suggest many Illinois pediatricians do not perform vision screening of preschool children, though screening does occur at other sites. PMID- 7554685 TI - Amphotericin-induced heart-rate decrease in children. AB - We describe six children with acute decreases in heart rate temporally related to amphotericin B administration. All patients had achieved their maximal dose within 3 to 4 days. Heart-rate drops occurred as early as day 3 but could be delayed up to day 7 after start of therapy. The mean heart rate dropped from 104 +/- 8/min (range 96 to 114) to 62 +/- 8/min (range 48 to 72) (P = 0.0001). A slower heart rate than baseline was noted during the entire duration of drug administration, from 60 minutes of starting the infusion to 220 minutes (mean 120 +/- 40) after discontinuation of the infusion. This reaction was noted in six of 90 (6.7%) patients who had amphotericin. These six children were compared with six age-matched children who received the drug but in whom such changes in heart rate did not develop. The method of administration of amphotericin B was similar in both patients and controls, starting with 0.25 mg/kg/day and increasing by 0.25 mg/kg/day up to 1 mg/kg/day. Children with heart-rate drop received amphotericin for 4.6 +/- 1.8 days, significantly shorter than their controls (12.6 +/- 6.9 days) (P = 0.02), suggesting that this adverse effect has led to early discontinuation of amphotericin therapy. Physicians and nurses caring for children receiving amphotericin B should be aware of this potential adverse effect, which can be serious in a patient with an underlying heart condition or in a patient who is already on heart-rate-lowering drugs. PMID- 7554686 TI - Diagnosing Gaucher disease. Early recognition, implications for treatment, and genetic counseling. AB - The diagnosis of Gaucher disease, the inherited deficiency of glucocerebrosidase and the most common inherited disorder of Ashkenazi Jews, can often be missed by clinicians. Medical records from patients with Gaucher disease were reviewed, revealing a wide range of initial misdiagnoses and the frequent use of unnecessary invasive diagnostic procedures. The diagnosis of Gaucher disease is readily established by enzymatic or DNA analyses in conjunction with a thorough history and physical examination. Consequently, greater awareness of the symptoms encountered in these patients could alleviate unnecessary anxiety, testing, and confusion. A definitive diagnosis of Gaucher disease has important implications for genetic counseling and treatment. PMID- 7554687 TI - Therapeutic applications of alpha interferon for chronic viral hepatitis in pediatric patients. PMID- 7554688 TI - Abdominal pain as a presenting symptom of supraventricular tachycardia. PMID- 7554689 TI - Urinary retention related to phenytoin therapy. PMID- 7554690 TI - Pediatricians' preferences for anticipatory guidance topics compared with parental anxieties. PMID- 7554691 TI - Potentially fatal hypersensitivity pneumonitis in a child. PMID- 7554693 TI - A perspective on controversies about neonatal circumcision. PMID- 7554692 TI - More on Kienbock's disease and avascular necrosis. PMID- 7554694 TI - A perspective on controversies about neonatal circumcision. PMID- 7554695 TI - A perspective on controversies about neonatal circumcision. PMID- 7554696 TI - A basis for overheat production in SIDS. PMID- 7554697 TI - Availability of anticancer drugs in the United States, Europe, and Japan from 1960 through 1991. PMID- 7554698 TI - Comparison of chloroguanide and mephenytoin for the in vivo assessment of genetically determined CYP2C19 activity in humans. AB - OBJECTIVES: The main objective of this study was to examine the relations between chloroguanide (proguanil) and mephenytoin metabolic ratios to determine whether or not chloroguanide could replace mephenytoin as a probe for the indirect in vivo measurement of CYP2C19 activity. An additional objective was to examine the interactions between chloroguanide, omeprazole, and mephenytoin, which are three substrates of CYP2C19. METHODS: Twenty healthy volunteers received 200 mg chloroguanide orally on three separate occasions in an open, randomized-sequence crossover design: once alone, once 2 hours before the oral administration of 100 mg mephenytoin, and once after oral administration for 7 days of 40 mg/day omeprazole. During one additional period, 100 mg mephenytoin was administered orally. The chloroguanide to cycloguanil ratio was determined in plasma 4 hours after drug administration; it was determined in urine collected over 4, 8, and 24 hours. The mephenytoin hydroxylation index was also measured in urine. RESULTS: All subjects were extensive metabolizers of chloroguanide and mephenytoin. We found no correlation between the mephenytoin hydroxylation index and the chloroguanide to cycloguanil ratio in any of the urine samples collected or in plasma. In the presence of chloroguanide, mephenytoin hydroxylation index increased from a baseline value of 1.2 +/- 0.2 to 1.7 +/- 1.0 (p < 0.05). In the presence of omeprazole, the chloroguanide to cycloguanil metabolic ratio in 24 hour urine increased from 2.2 +/- 1.0 to 5.6 +/- 3.2 (p < 0.001). CONCLUSION: Chloroguanide inhibits the CYP2C19-dependent 4'-hydroxylation of mephenytoin. The bioactivation of chloroguanide to cycloguanil is inhibited by the CYP2C19 substrate omeprazole. However, the chloroguanide to cycloguanil metabolic ratio does not reflect the same array of S-mephenytoin hydroxylase activities found in extensive metabolizers as that show by the mephenytoin hydroxylation index. PMID- 7554699 TI - Propranolol disposition in Chinese subjects of different CYP2D6 genotypes. AB - Propranolol pharmacokinetics among different genotypes of CYP2D6 was compared in this study. The Chinese (Han) population consisted of 44 healthy unrelated individuals living in southern Taiwan. Endonuclease tests based on polymerase chain reaction were used to determine C/T188 genotypes of CYP2D6 in leukocyte deoxyribonucleic acid. Based on codon 188 genotypes, subjects were categorized into three groups: homozygous C/C188 (n = 13), heterozygous C/T188 (n = 14); and homozygous T/T188 (n = 17). Each subject was given a 40 mg propranolol tablet. Blood samples were drawn before and 12 hours after propranolol administration to measure propranolol and 4-hydroxypropranolol. Three genotypes showed distinct time profiles of plasma propranolol and 4-hydroxypropranolol. The area under plasma concentration curve values (mean +/- SEM), were 322.0 +/- 40.8, 481.6 +/- 77.5, and 766.1 +/- 92.8 nmol.hr/L, respectively, for C/C188, C/T188, and T/T188 subjects (p < 0.05). The 48-hour excreted amount of 4-hydroxy-S-propranolol-O glucuronide, but not 4-hydroxy-R-propranolol-O-glucuronide, was significantly higher for C/C188 than for T/T188 subjects (p < 0.05). This study shows a different propranolol disposition in Chinese subjects of different CYP2D6 genotypes. PMID- 7554701 TI - Effects of felbamate on the pharmacokinetics of phenobarbital. AB - The effects of felbamate on the pharmacokinetics of phenobarbital and one of its main metabolites, parahydroxyphenobarbital, were assessed in a parallel-group, placebo-controlled, double-blind study, in 24 healthy volunteers. Pharmacokinetic parameters of phenobarbital and parahydroxyphenobarbital were determined from plasma and urine samples obtained after 28 days of daily administration of 100 mg phenobarbital and after a further 9 days of phenobarbital plus 2400 mg/day felbamate or placebo. Felbamate increased phenobarbital values for area under the plasma concentration-time curve from 0 to 24 hours and maximum concentration by 22% and 24%, respectively, whereas placebo had no effect. This increase was caused by a reduction in parahydroxylation of phenobarbital and possibly through effects on other metabolic pathways. Because felbamate inhibits the S-mephenytoin hydroxylase (CYP2C19) isozyme in vitro, it appears that phenobarbital hydroxylation is mediated in part by this isozyme. PMID- 7554700 TI - Loratadine administered concomitantly with erythromycin: pharmacokinetic and electrocardiographic evaluations. AB - OBJECTIVE: To evaluate the effects of coadministration of loratadine and erythromycin on the pharmacokinetics and electrocardiographic repolarization (QTc) pharmacodynamics of loratadine and its metabolite descarboethoxyloratadine in healthy volunteers. METHODS: Twenty-four healthy volunteers were studied in a prospective, double-blind crossover design while confined in a Clinical Research Center. The primary pharmacodynamic end point of the study was the difference between baseline and day 10 mean QTc intervals obtained from surface electrocardiograms. Plasma concentrations of loratadine, descarboethoxyloratadine, and erythromycin were measured on treatment day 10 for pharmacokinetic analysis. Subjects received in random sequence the following three treatments for 10 consecutive days during three separate study periods: 10 mg loratadine every morning plus 500 mg erythromycin stearate every 8 hours, or 10 mg loratadine every morning plus placebo every 8 hours, or placebo every morning plus 500 mg erythromycin stearate. RESULTS: Concomitant administration of loratadine and erythromycin was associated with increased plasma concentrations of loratadine (40% increase in area under the plasma concentration-time curve [AUC]) and descarboethoxyloratadine (46% increase in AUC) compared with loratadine alone. Analysis of variance showed no difference between the treatment groups in effect on QTc intervals compared with baseline, and no significant change from baseline was observed. No clinically relevant changes in the safety profile of loratadine were observed, and there were no reports of sedation nor syncope. CONCLUSION: Although concomitant administration of loratadine and erythromycin was associated with increased plasma concentrations of loratadine and descarboethoxyloratadine, no clinically relevant changes in the safety profile of loratadine were observed. In this study, 10 mg loratadine administered orally for 10 consecutive days was well tolerated when coadministered with therapeutic doses of erythromycin stearate. PMID- 7554702 TI - Studies of the oral bioavailability of alendronate. AB - Clinical studies were performed to examine the oral bioavailability of alendronate (4-amino-1-hydroxy-butylidene-1,1-bisphosphonate monosodium). All studies, with the exception of one performed in men, involved postmenopausal women. Short-term (24 to 36 hours) urinary recovery of alendronate after an intravenous dose of 125 to 250 micrograms averaged about 40% in both men and women. In women, oral bioavailability of alendronate was independent of dose (5 to 80 mg) and averaged (90% confidence interval) 0.76% (0.58, 0.98) when taken with water in the fasting state, followed by a meal 2 hours later. Bioavailability was similar in men [0.59%, (0.43, 0.81)]. Taking alendronate either 60 or 30 minutes before a standardized breakfast reduced bioavailability by 40% relative to the 2-hour wait. Taking alendronate either concurrently with or 2 hours after breakfast drastically (> 85%) impaired availability. Black coffee or orange juice alone, when taken with the drug, also reduced bioavailability (approximately 60%). Increasing gastric pH, by infusion of ranitidine, was associated with a doubling of alendronate bioavailability. A practical dosing recommendation, derived from these findings and reflective of the long-term nature of therapy for a disease such as osteoporosis, is that patients take the drug with water after an overnight fast and at least 30 minutes before any other food or beverage. PMID- 7554703 TI - Clinical pharmacokinetics of physostigmine in patients with Alzheimer's disease. AB - OBJECTIVE: To study the pharmacokinetic and pharmacodynamic properties of physostigmine in subjects with Alzheimer's disease. METHODS: Plasma physostigmine concentration and butyrylcholinesterase inhibition were measured in blood samples collected during and after a single high-dose (1 to 1.5 mg for 45 to 60 minutes) and a sustained low-dose steady-state intravenous infusion in nine subjects with Alzheimer's disease. Escalating doses (0.5 to 25 mg/day) were administered during a 2-week period. A dose (2 to 12 mg/day) that optimized cognition in each subject was identified and then administered in a randomized, double-blind, placebo controlled crossover design for 1 week. RESULTS: The elimination half-life of physostigmine was 16.4 +/- 3.2 (SE) minutes. Clearance and volume of distribution were 7.7 +/- 0.9 (SE) L/min and 2.4 +/- 0.6 (SE) L/kg, respectively. Butyrylcholinesterase inhibition half-life was 83.7 +/- 5.2 (SE) minutes. During sustained steady-state infusion, plasma physostigmine concentration (r = 0.95) and butyrylcholinesterase inhibition (r = 0.99) were linearly correlated with the dose. In five cognitive responders, the memory enhancement was significantly correlated (r = 0.86; p < 0.05) with butyrylcholinesterase inhibition. CONCLUSIONS: These results showed that, in cognitive responders, memory enhancement by physostigmine in Alzheimer's disease is correlated directly to the magnitude of plasma cholinesterase inhibition. Furthermore, during single-dose conditions, the dynamic half-life is five-fold longer than the kinetic half-life. PMID- 7554704 TI - Azithromycin and terfenadine: lack of drug interaction. AB - A double-blind placebo-controlled study was conducted in healthy men to determine the effect of coadministration of azithromycin on the pharmacodynamics and pharmacokinetics of terfenadine. Administration of 500 mg azithromycin for 1 day and 250 mg on 4 subsequent days did not affect the pharmacokinetics of the pharmacologically active terfenadine carboxylate metabolite when 60 mg terfenadine was given twice daily for 12 days, starting 7 days before azithromycin administration. Terfenadine alone resulted in a 0.010 msec increase in the rate-corrected QT interval (QTc), but the incremental effects of azithromycin and placebo on QTc in volunteers receiving terfenadine were not statistically different. It is concluded that the potentially life-threatening disorders that have been attributed to a pharmacokinetic interaction between macrolide antibiotics and terfenadine are unlikely to take place in patients treated simultaneously with azithromycin and terfenadine. PMID- 7554705 TI - Pharmacokinetics of intravenous ondansetron in healthy children undergoing ear, nose, and throat surgery. AB - BACKGROUND: To determine the pharmacokinetics of the 5-HT3 antagonist ondansetron in children, informed written consent was obtained from the parents of 21 healthy children aged from 3 to 12 years scheduled for ear, nose, and throat surgery. METHODS: The children were stratified according to age: 3 to 7 years and 7.1 to 12 years, and a single intravenous infusion of 2 or 4 mg ondansetron, respectively, was administered over 5 minutes before induction of anesthesia. After completion of the infusion, anesthesia was induced intravenously and maintained with inhalational anesthesia. Whole blood (3 ml) was obtained before administration of ondansetron, at completion of the infusion, at the beginning and end of surgery, and at 3, 4, 6, 8, 10, and 12 hours after start of the infusion. Pharmacokinetic variables were determined with use of standard noncompartmental techniques. RESULTS: Mean plasma clearance was 0.50 L.hr-1.kg-1 and 0.39 L.hr-1.kg-1, the mean volume of distribution at steady-state was 1.70 L.kg-1 and 1.61 L.kg-1, and the mean plasma terminal half-life was 2.6 hours and 3.1 hours for the 2 mg and 4 mg groups, respectively. On a body surface area basis, mean plasma clearance was 14.0 and 13.7 L.hr-1.m-2 and mean volume of distribution was 47.7 and 55.9 L.m-2 for the 2 and 4 mg groups, respectively. There were no serious adverse events attributable to ondansetron. CONCLUSIONS: These data indicate that the pharmacokinetics of ondansetron in children from 3 to 12 years old are predictable and similar to those in adults. The elimination half-life of ondansetron increases in parallel with age. However, clearance is constant when normalized to body surface area, but the volume of distribution increases over the age range studied. PMID- 7554706 TI - Norfloxacin interferes with cyclosporine disposition in pediatric patients undergoing renal transplantation. AB - Prophylactic treatment with norfloxacin was initiated in a group of pediatric patients undergoing renal transplantation who were receiving cyclosporine and were susceptible to recurrent urinary tract infections. At discharge from the hospital, the mean daily dose of cyclosporine needed to maintain trough cyclosporine blood levels of 150 to 400 ng/ml was 4.5 mg/kg/day for the patients who received norfloxacin compared with 7.4 mg/kg/day for patients who did not receive the antibiotic. This observation suggested that the clearance of cyclosporine was decreased by the concomitant use of norfloxacin. The effect of norfloxacin on specific drug-metabolizing cytochrome P450 isozymes in vitro was examined to determine if the metabolism and subsequent clearance of cyclosporine and possibly other drugs are altered through a metabolic interaction with norfloxacin. In human liver microsomes, the activity of cytochrome P4503A4, the isozyme that metabolizes cyclosporine in humans, was inhibited by norfloxacin. In rat liver microsomes, norfloxacin inhibited the activity of cytochrome P4503A2, the isozyme responsible for cyclosporine metabolism in this species, but did not alter the activity of the rat cytochrome P450 isozymes 1A, 2E1, and 4A1. The in vitro studies suggest that the lower cyclosporine dose required by pediatric patients who were given norfloxacin was caused by inhibition of the metabolism of cyclosporine. PMID- 7554707 TI - Local angiotensin-converting enzyme inhibition blunts endothelin-1-induced increase in forearm vascular resistance. AB - OBJECTIVE: The physiologic role of endothelin-1 is not well established; however, it may have a role in modulation of peripheral vascular tone complimentary to angiotensin II. In vitro and animal studies suggested an interrelationship between angiotensin II and endothelin-1 vasoconstriction. We hypothesized that local vascular or systemic renin-angiotensin II systems must be intact for endothelin-1-mediated vasoconstriction in humans. METHODS: To test this hypothesis, responses to brachial artery infusion of endothelin-1 alone and endothelin-1 plus local low-dose infusion of enaliprilat were studied in seven healthy male and seven healthy female volunteers. RESULTS: In these subjects, baseline forearm vascular resistance (mean +/- SEM; 24 +/- 3.5 mm Hg.ml/dl forearm vol/min) increased with a 38.2 ng/min endothelin-1 infusion (61.8 +/- 6.8 mm Hg.ml/dl forearm vol/min; p < 0.01). Forearm vascular resistance decreased when 38.2 ng/min endothelin-1 was infused concomitantly with a local 5 micrograms/min infusion of enaliprilat (45.5 +/- 5.9 mm Hg.ml/dl forearm vol/min; p < 0.01 compared with endothelin-1 alone). CONCLUSIONS: These data indicate that an endothelin-1-induced increase in forearm vascular resistance is inhibited by local forearm angiotensin-converting enzyme inhibition. PMID- 7554709 TI - Pharmacokinetics and pharmacodynamics of the monoamine oxidase B inhibitor mofegiline assessed during a phase I dose tolerance trial. AB - The safety, pharmacokinetics, and pharmacodynamics of single oral doses of up to 48 mg and daily (for 28 days) doses of up 24 mg mofegiline were investigated in healthy male volunteers. Plasma pharmacokinetics indicated rapid absorption and elimination: time to reach maximum concentration occurred at about 1 hour; half life ranged from 1 to 3 hours. Maximal plasma concentration and area under the plasma concentration-time curve increased and oral clearance decreased disproportionately with dose. Mofegiline rapidly and markedly inhibited platelet monoamine oxidase B (MAOB) activity, which returned to baseline within 14 days. Urinary excretion of phenylethylamine increased proportionately with doses up to 24 mg. No changes in urinary elimination of catecholamines, blood pressure, heart rate, or ECG were observed. A classic maximum tolerated dose was not achieved in these studies. However, the 48 mg single dose and the 24 mg multiple daily dose far exceeded the dose (1 mg) that was associated with > 90% platelet MAOB inhibition. PMID- 7554708 TI - Effects of nabumetone on prostanoid biosynthesis in humans. AB - BACKGROUND: The active metabolite of the anti-inflammatory drug nabumetone has been characterized as a selective inhibitor of the inducible prostaglandin H synthase (PGHS). The aim of this study was to investigate the rate of eicosanoid biosynthesis after oral dosing with nabumetone in nine healthy subjects. METHODS: We measured the urinary excretion of products of platelet (11-dehydro-thromboxane B2 [TXB2]) and renal (prostaglandin IF2 alpha [PGF2 alpha]) arachidonate metabolism as in vivo indexes of the constitutive PGHS-1 pathway. Moreover, the production of TXB2 during whole blood clotting was assessed as an index of the cyclooxygenase activity of platelet PGHS-1 ex vivo. RESULTS: At steady state, nabumetone (500 and 1000 mg daily for 7 days) was associated with statistically significant dose-dependent reduction in the urinary excretion of 11-dehydro-TXB2 and serum TXB2 levels by approximately 50% to 70%. However, the drug did not significantly affect the urinary excretion of PGF2 alpha. After discontinuation of nabumetone, urinary 11-dehydro-TXB2 excretion and whole blood TXB2 production returned to predrug levels with a similar timecourse that was consistent with the elimination half-life of its active metabolite. The daily administration of low dose aspirin (40 mg), a selective inhibitor of platelet PGHS-1, caused a cumulative inhibition of urinary 11-dehydro-TXB2 and whole blood TXB2 production that recovered with a timecourse consistent with platelet turnover. CONCLUSIONS: Nabumetone does dose-dependently inhibit the cyclooxygenase activity of platelet PGHS-1 of healthy subjects both in vivo and ex vivo. Thus it is unlikely that its safety profile in patients may be related to selective inhibition of the inducible PGHS-2. PMID- 7554710 TI - Restoration of normal sperm characteristics in hypoprolactinemic infertile men treated with metoclopramide and exogenous human prolactin. AB - We investigated the effects of induced increase in prolactin levels on spermatogenesis in 20 infertile men with hypoprolactinemia using exogenous human prolactin (hPRL) and metoclopramide. The subjects were selected from a population of 175 infertile men in whom the prevalence of hypoprolactinemia was 33.14%. Mean basal plasma prolactin was 2.79 +/- 0.62 ng.ml-1 in the infertile men and 9.57 +/ 2.14 ng.ml-1 in the normal control subjects. At the sixteenth week, mean plasma prolactin was 9.41 +/- 1.3 ng.ml-1 in subjects treated with exogenous hPRL and 5.2 +/- 0.7 ng.ml-1 in subjects treated with metoclopramide. Mean basal sperm concentration was approximately 8.8 million per milliliter in the infertile men and 41.5 million per milliliter in the normal control subjects. Mean sperm concentration was approximately 37 million per milliliter in subjects treated with exogenous hPRL, whereas the peak mean value was 23 million per milliliter in subjects treated with metoclopramide for 16 weeks. At basal conditions, the mean percentages of abnormal sperm were 66.75% +/- 14.93% and 21.36% +/- 4.78% in infertile and normal subjects, respectively. In subjects treated with exogenous hPRL and metoclopramide, the mean percentage of abnormal sperm were 24.7% and 31%, respectively, at week 16. Mean plasma prolactin, mean sperm concentration and the mean percentage of abnormal sperm were 3.3 +/- 1.4 ng.ml-1, 7 million per milliliter, and 60.5, respectively, in the infertile subjects after drug withdrawal at week 14. In normal control subjects, there was no significant difference (p = 0.01) in the plecebo effect. We therefore conclude that the low prolactin levels in this group of infertile men may be one of the primary causes of their infertility. PMID- 7554712 TI - Embryology and anatomy of the cranial base. AB - This article provides an overview of the complex embryologic development and anatomic relationships of the cranial base. The processes of neural cell migration, and chondrocranial base formation and ossification are discussed. In addition, the relative sectional anatomy is addressed with the goal of providing a functional review for clinicians dealing with cranial base disorders. The complex influences of the surrounding matrix and diffusible factors involved in skull base development are sources of continuing investigation. Through elucidation of the mechanisms of basal cranial development, the understanding of congenital and pathologic disorders of this area as well as cranial base influence on facial growth will be enhanced greatly. PMID- 7554713 TI - Diagnostic radiology of the cranial base. AB - The skull base is a complex interface for bone, nerves, vessels, and musculature. With knowledge of the underlying anatomy, with an understanding of the strengths of CT and MR imaging, and with a familiarity of the commonest lesions, a rational approach to diagnostic imaging can be made, narrowing the clinical differential diagnosis and simplifying presurgical planning. PMID- 7554714 TI - Pathology of tumors of the cranial base. AB - Tumors of the skull base are varied and can be categorized into benign and malignant neoplasms. This article outlines the types of tumors found in this surgically challenging anatomic location. PMID- 7554711 TI - USAN Council. List No. 375. New Names. Dacliximab. PMID- 7554715 TI - Endovascular therapy for surgical diseases of the cranial base. AB - Endovascular therapy for surgical diseases of the cranial base is in many ways a survey of the field of interventional neuroradiology (or endovascular neurosurgery). As the multidisciplinary focus to the treatment of various cranial base diseases has become increasingly complex, the role of endovascular therapy as both a primary and secondary treatment modality has expanded. A comprehensive review of this role is presented. PMID- 7554716 TI - Neurosurgical considerations of cranial base surgery. AB - Several craniotomies have been described that allow extensive resection of skull base and low-lying cranial tumors that involve little disfigurement to the patient. These techniques should be of interest to plastic surgeons as they may be called to aid their neurosurgical colleagues in exposing the anterior skull base or may be involved in combined procedures to resect tumors that involve the face, sinuses, orbit, and cranial vault. PMID- 7554717 TI - Transorbital approaches to the cranial base. AB - Orbital neoplasms and their treatment may have significant negative impact on patient's survival, function, and appearance. It is essential to establish early and accurate diagnosis. The anatomic location of the orbit allows neoplastic processes to threaten structures in the intracranial cavity relatively easily. This adds significantly to the complexity of managing orbital tumors. Improved surgical approaches allow better visualization of the neoplasm and greater protection of essential anatomic structures. This also permits immediate functional and aesthetic reconstruction. A multidisciplinary approach involving the head and neck surgeon, neurosurgeon, reconstructive surgeon, ophthalmologist, radiologist, radiation oncologist, and medical oncologist enhances the quality of management of orbital lesions. Further development of new techniques as well as improved application of adjuvant modalities will, it is hoped, continue to improve the prognosis of patients with orbital neoplasms. PMID- 7554718 TI - Transfacial approaches to the cranial base. AB - This article classifies and describes the anterior transfacial approaches to the cranial base as determined by the anatomic location of the lesions. With the use of craniofacial techniques, many of these lesions now may be resected without any significant functional or anesthetic morbidity. PMID- 7554719 TI - Posterior skull base transfacial approaches. AB - Transfacial approaches to the midline skull base provide direct access for resection of complex lesions. The transfrontal nasal-orbital and transmaxillary (Levels III and V) approaches have become the "work horses" of our skull base team, in that either alone or in combination, they provide access to most parts of the anterior skull base and clivus. The flexibility offered by the ability to select a Level I or Level II approach in place of a Level III approach, or a Level VI approach in place of a Level V approach, however, greatly enhances the individualization of treatment strategy. The overlapping exposure shared by these techniques provides flexibility in the choice of surgical exposure and allows multiple factors to be considered, including tumor location and size. PMID- 7554720 TI - Neurofibromatosis of the skull base. AB - Neurofibromatosis of the skull base is a syndrome that may occur in an isolated fashion or as a component of Von Recklinghausen disease. This article discusses the clinical features of neurofibromatosis, diagnostic methods, and surgical treatment. PMID- 7554721 TI - Vascular lesions of the cranial base. AB - Vascular lesions can be classified broadly into two groups on the basis of cellular proliferation characteristics: hemangiomas and vascular malformations. Additional lesions include a heterogeneous group of vascular tumors such as those represented by hemangiopericytoma and those with a frankly malignant course. Those lesions located around the cranial base remain challenging and difficult clinical problems. The care of patients with these problems is complex; it frequently will need a full spectrum of individuals from many disciplines involved in many diverse aspects of management: medical, radiologic, anesthetic, and surgical. Fortunately, advances in science and technology have markedly expanded our capability in treating these patients and have allowed us increasing safety and ease in dealing with previously untreatable and unresectable lesions. PMID- 7554722 TI - Soft tissue reconstructive choices for craniofacial reconstruction. AB - The extensive blood supply to the scalp enables the use of local flaps based mainly on the superficial temporal artery: these include skin, temporoparietal fascia, subgaleal fascia, and temporal fascia. Also, the temporalis muscle provides flaps based upon the temporal branches of the internal maxillary artery. On top of the skull, scalp flaps, galeal flaps, and pericranial flaps can be used as random flaps or as axial flaps based on the problems or anatomic reasons that would preclude microvascular reconstruction. The pectoralis, latissimus, splenius capitis, and trapezius muscle flaps can dependably reach the base of the skull. The increasing reliability and versatility of microvascular reconstruction render their use standard for craniofacial reconstruction. PMID- 7554723 TI - Reconstruction of the cranial base. AB - The cranial base is a complex boundary between intracranial and extracranial anatomic structures. Adequate reconstruction of the basicranium depends on the location, size, and nature of the defect. A reconstructive ladder can be built on a few basic principles: coverage of vital structures, separation of the intra- and extradural spaces, procedural simplicity, and aesthetics. Individual consideration should be given to reconstruction of bone, soft tissue, dura, and cerebrospinal fluid spaces. PMID- 7554725 TI - Complications of skull base surgery. AB - The increasing number of patients undergoing resection of complex skull base lesions with good outcome is indicative of the interest and expertise in treating tumors of the skull base. Advances in surgical techniques and improved knowledge of the microanatomy have contributed to the ability to manage these tumors aggressively. These procedures are not without morbidity, such as neurologic deficits, infection, and hemorrhage. These complications are expected and, with appropriate and timely management, can be minimized. PMID- 7554724 TI - Microsurgery for cranial base tumors. AB - Local or regional flaps usually are sufficient for reconstruction of defects of the cranial base. In some cases, however, free-tissue transfer may be the best or the only choice. Reasons for choosing free-tissue transfer over other methods of reconstruction include previous radiation, previous surgery, limited arc of rotation of pedicle flaps, lower complication and failure rates of free flaps, and specific tissue requirements that only can be met with a free flap. The anatomy, available flap choices, indications, and technical considerations applicable to cranial base reconstruction with microvascular surgery are discussed. PMID- 7554726 TI - Dietary omega-3 fatty acids and cholesterol modify desaturase activities and fatty acyl constituents of rat intestinal brush border and microsomal membranes of diabetic rats. AB - The diabetes-associated changes in intestinal uptake of nutrients are modified by isocaloric alterations in the type of dietary lipids, and is associated with alterations in the phospholipid and fatty acyl content of the intestinal brush border membrane. We wished to test the hypothesis that diet- and diabetes associated changes in brush border membrane phospholipid fatty acids are due to alterations in the activity of enterocyte microsomal delta-5, delta-6 and delta-9 desaturases. Adult female Wistar rats were divided into two groups. In half of the animals, diabetes was produced with the injection of streptozotocin, and the other half of the animals served as nondiabetic controls. Both groups were raised on chow for two weeks and were then randomized to one of four semisynthetic diets for two weeks: beef tallow low in cholesterol (BT), beef tallow high in cholesterol (BTC), fish oil low in cholesterol (FO), or fish oil high in cholesterol (FOC). Feeding a high cholesterol diet increased the activity of jejunal enterocyte microsomal membrane activity of delta-5 and delta-9 desaturases when fed with FO in non-diabetic control rats, increased delta-5 desaturase in diabetic rats fed FO, and increased the ileal activity of delta-5 and delta-6-desaturases in control and diabetic animals fed FO. Dietary fatty acids, cholesterol and diabetes did not produce the changes in the amount of fatty acids in BBM phosphatidylcholine or phosphatidylethanolamine expected from the measured alterations in delta-5 (20:4 omega 6 and 20:5 omega 3), in delta-6 (18:3 omega 6 and 18:4 omega 3), or in delta-9 desaturase (18:1 omega 9 and 16:1 omega 7). In summary, 1) the activities of enterocyte microsomal membrane delta 5, delta-6 and delta-9-desaturases are independently influenced by dietary fatty acids or cholesterol, or by diabetes; 2) changes in dietary fatty acids, cholesterol and diabetes are associated with alterations in the fatty acyl constituents of brush border membrane phosphatidylcholine and phosphatidylethanolamine, but these fatty acyl changes are not explained on the basis of variations in the activities of the microsomal desaturases. Thus, the intestinal brush border membrane and the enterocyte microsomal desaturases are capable of adapting in response to changes in dietary lipids or diabetes, but the two alterations are not necessarily causally interrelated. PMID- 7554727 TI - Occurrence of islet cell reactive autoantibodies in diabetes-prone BB/OK rats is not associated with the onset of diabetes: a cross-sectional study of BB rats and their diabetes-resistant congenic strains. AB - Diabetes-prone BB rats are one of the extensively studied animal models of human type 1 (insulin-dependent) diabetes. We have detected islet cell reactive autoantibodies (ICRA) in sera of age-matched (120 days of age) diabetic and non diabetic diabetes-prone BB/OK rats compared to sex- and age-matched diabetes resistant LEW.1A control rats by cellular enzyme-linked immunosorbent assay (CELISA) using either desiccated or viable rat insulinoma (RIN) cells as target. The antibody levels measured using desiccated RIN cells (mean O.D. +/- SD; prevalence) of the diabetic group (1.41 +/- 0.59; 94.1%) and the non-diabetic group (1.23 +/- 0.48; 100%) were significantly (p < 0.01) increased compared to those of the diabetes-resistant LEW.1A control rats (0.24 +/- 0.1; 3.2%). Additionally, in a 209-day-old group of non-diabetic diabetes-prone BB/OK rats the autoantibody levels were further significantly (p < 0.01) increased (2.65 +/- 0.48; 100%). Comparing both congenic rat strains BB.1A/OK and LEW.1BB/OK, only the BB.1A/OK rats which bears the genetic background of diabetes-prone BB/OK rats were found to be antibody positive (0.68 +/- 0.43; 63.1%). The results were confirmed by the CELISA using viable RIN cells. We conclude that the prevalence of islet cell autoantibodies in diabetes-prone BB/OK rats and congenic BB.1A/OK rats is closely associated with the genetic susceptibility to diabetes and may be independent of Beta-cell destruction, but they are not a predictive marker for the onset of the disease in this animal model. PMID- 7554728 TI - Anti platelet property of anti-single-stranded DNA antibodies in type 1 (insulin dependent) diabetes mellitus. AB - Nineteen of 50 sera from children with type 1 (insulin-dependent) diabetes mellitus showed anti-platelet reactivity. This reactivity was significantly associated with the presence of proteoglycan cross-reactive anti-ssDNA antibodies. As well as DNA-anti-DNA interaction, increasing salt concentration of the dilution buffer caused a decrease in the binding of positive sera to platelets. Purified ssDNA inhibited also the anti-platelet reactivity of anti ssDNA positive sera. The addition of purified IgG from anti-ssDNA positive but not from anti-ssDNA negative sera to washed platelets caused an increased collagen-induced platelet aggregation similar to that obtained with the addition of polycationic agents. It may be suggested that ionic interaction between anti negative charged molecule antibodies (such as anti-ssDNA antibodies) and platelet surface negative charges may be a pathophysiological mechanism contributing to the altered platelet function observed in diabetes. PMID- 7554729 TI - Bilateral ptosis with ophthalmoplegia in a 72-year-old woman with diabetes. AB - Ophthalmoplegia is common cranial neuropathy of Diabetes. In case of 3rd nerve involvement, usually unilateral extra ocular muscles are affected. However, ptosis is very rare in patients with diabetic neuropathy. In this report, we describe bilateral ptosis with ophthalmoplegia in diabetes. In ophthalmoplegia associated with diabetes, ischemic nerve infarction was reported. We treated this patient with Lipo prostaglandin (PG) E1. Since then, increased platelet aggregation activity was found in this patient. After two months, the symptoms of this patient were improved. PMID- 7554730 TI - Ultrasound training for non-radiologists. PMID- 7554731 TI - Who's for tips? PMID- 7554732 TI - Optimization of prostate carcinoma staging: comparison of imaging and clinical methods. AB - PURPOSE: The diagnostic value of endorectal coil MRI, body coil MRI, transrectal ultrasound, digital rectal examination and PSA levels were prospectively analysed in order to define the most accurate preoperative staging method. METHODS: 33 patients with prostate carcinoma, who underwent subsequent prostatectomy, were enrolled in the study and examined on a 1.5T system using the body coil as well as the endorectal surface coil before and after the administration of contrast material. The results were compared to digital rectal examination, prostate specific antigen levels and endorectal ultrasound. RESULTS: Staging accuracy of endorectal coil MRI was 87.9% with a sensitivity of 88.9% and specificity of 86.7%. For body coil MRI, the staging accuracy was 75.7%, the sensitivity 66.7% and the specificity 87.9%, for transrectal ultrasound 69.6%, 41.7% and 100% and for the digital rectal examination 56.6%, 33.3% and 100%, respectively. Prediction was improved by combining results of endorectal coil MRI with PSA values. CONCLUSION: Endorectal ultrasound and digital rectal examination both had a tendency to underestimate the extent of the lesion. Endorectal coil MRI proved to be the best preoperative staging method. In combination with PSA values, diagnostic accuracy could be further improved. Therefore, local staging of prostate cancer could be based on these two parameters alone. PMID- 7554733 TI - The role of intravenous contrast enhancement in magnetic resonance imaging of prostatic carcinoma. AB - Although magnetic resonance imaging (MRI) has been shown to be a promising technique for staging prostatic carcinoma, its accuracy in detection of extraprostatic spread and seminal vesical invasion is subject to considerable error. The role of dynamic MRI, following bolus intravenous contrast medium, in depicting carcinoma of the prostate was evaluated and the information provided by this technique compared with unenhanced images. Spin-echo T1-weighted, T2 weighted, bolus contrast enhanced and delayed post-contrast enhanced T1-weighted images were obtained in 20 patients with histologically proven adenocarcinoma of the prostate. Tumour was identified in all of the patients studied. When compared with unenhanced and delayed post-contrast enhanced images, dynamic bolus contrast enhanced images enabled the best definition of tumour within the gland in 50% of patients and demonstrated extracapsular spread more clearly in 80% of patients. It is concluded that dynamic bolus contrast enhancement may be useful in selected patients with prostatic carcinoma by enabling tumour margins to be depicted more clearly. PMID- 7554734 TI - Radiographic manifestations of AIDS related lymphoma in the thorax. AB - Intrathoracic disease in AIDS-related lymphoma (ARL) produces a range of radiographic appearances. This study aimed to determine the frequency, distribution and characteristics of these appearances. The case notes and findings on imaging of 116 consecutive cases of ARL were reviewed retrospectively. In 52 cases there were abnormalities present on chest imaging (chest radiography or chest CT scanning). In 20 patients abnormalities were judged to be due to lymphomatous involvement and in 15 cases the thorax was the major site of disease. Correlation with biopsy results from thoracic disease (9), other disease (5), or evidence of response to treatment (6) was made. The most frequent imaging findings were pleural or intrapulmonary masses (7), frequently peripheral and sometimes with cavitation. In one case this simulated mycetoma formation. Pleural effusions and mediastinal lymph nodes were also frequently present, either alone or in combination. AIDS-related lymphoma uncommonly involves the chest as a major site of disease. A pleural or intrapulmonary mass with cavitation may represent the only site of disease and in these cases CT guided biopsy is likely to provide the histological diagnosis. PMID- 7554735 TI - Complications of the ileal pouch: is the pouchogram a useful predictor? AB - A series of ileal pouchograms from 25 consecutive patients has been analysed retrospectively. Ileal pouchography may demonstrate abnormalities which delay closure of the covering ileostomy. The aim was to determine whether disruption of the ileoanal anastomosis and/or leak at pouchography correlated with pelvic sepsis after ileostomy closure. Disruption of the stapled ileoanal anastomosis is a sensitive (88%) but not specific predictor (57%) for subsequent pelvic sepsis. The predictive value of a negative test is high (89%). Leak of contrast from the anastomosis is specific (81%) but not sensitive (56%) for pelvic sepsis. No significant relationship was demonstrated between width of the presacral space and the presence of pelvic sepsis. No significant relationship was demonstrated between diameter of the ileoanal anastomosis and symptoms of stricture. The presence of anastomotic disruption or leak at pouchography prior to ileostomy closure are useful predictors of potential pelvic sepsis. PMID- 7554736 TI - Colour Doppler ultrasound in renal transplant artery stenosis: which Doppler index? AB - A prospective study comparing colour Doppler ultrasound (US) with the 'gold standard' of intra-arterial digital subtraction angiography in the evaluation of renal transplant artery stenosis was performed. Both the intrarenal vessels and the transplant renal artery were examined by Doppler US. Diagnostic arteriography was performed only if, on Doppler, the peak systolic velocity in the transplant renal artery exceeded 1.5 ms-1. Of 109 patients, the transplant artery could not be visualized using colour Doppler US in three, and these were omitted from statistical analysis. Of the remaining 106 patients, 31 had a peak systolic velocity greater than 1.5 ms-1 in the transplant renal artery and were referred for DSA. Of the multiple renal Doppler indices recorded, the peak systolic velocity in the transplant artery was the best discriminating measurement for the detection of renal artery stenosis. A peak systolic velocity of > or = 2.5 ms-1 in the transplant renal artery had a sensitivity of 100% and a specificity of 95% for the detection of renal artery stenosis ( > 50% diameter reduction). Although a significant difference in Pulsatility Index, Resistive Index, Acceleration Index and Acceleration Time was recorded from the intrarenal vessels in the angiographically normal and stenosed groups with Doppler, these measurements were less useful as discriminating diagnostic tests. In conclusion, the peak systolic velocity in the transplant renal artery is the most sensitive Doppler criterion for renal artery stenosis and is sensitive and specific enough to be used as a screening test. The intrarenal acceleration time and index should not be used in isolation. PMID- 7554737 TI - Ultrasound in children with osteomyelitis. AB - Ultrasound (US) is a proven method of identifying subperiosteal abscess formation in osteomyelitis, but its precise role in the management of children with the condition is contentious. We reviewed nine children in whom US was used in the management of long bone osteomyelitis, including ribs. US was helpful in confirming the presence of a subperiosteal abscess in those children with an appropriate history and localizing signs. In these circumstances a bone scan is unnecessary. Problems were encountered when US failed to identify a subperiosteal abscess, because of the early stage of the disease process, and in multifocal abnormalities. In these circumstances a bone scan should still be performed. PMID- 7554738 TI - Reversible superior mesenteric vein thrombosis in acute pancreatitis. The CT appearances. AB - We describe the computed tomography (CT) appearances of four patients with acute or acute on chronic (case 3) pancreatitis which demonstrated isolated superior mesenteric vein (SMV) thrombosis. In three of the four cases follow-up CT scans showed the SMV thrombosis to have resolved with resolution of the underlying pancreatitis without anticoagulation or surgical intervention. PMID- 7554739 TI - Imaging of small cell carcinoma of the oesophagus. AB - Four cases of oesophageal small cell carcinoma are described. Radiological features at presentation included oesophageal strictures in association with bulky intrathoracic and intraabdominal lymphadenopathy. The subject is reviewed and the radiologically relevant features are emphasized. PMID- 7554740 TI - Pre-operative imaging of esthesioneuroblastoma. AB - The pre-operative images of three patients with biopsy proven esthesioneuroblastoma are presented. The role of computed tomography (CT) and magnetic resonance imaging (MRI) in the pre-operative determination of tumour extent is discussed. PMID- 7554741 TI - Intestinal stricturing as a late presentation of appendiceal abscess. AB - We report two patients in whom the primary presentation of an appendix abscess was atypical, and who later developed obstructive symptoms due to extrinsic intestinal stricturing from chronic inflammation. In one case a short ileal stricture was revealed on enteroclysis, in the other double contrast barium enema showed sigmoid stricturing. PMID- 7554742 TI - Wessex regional radiology audit: barium enema in colo-rectal carcinoma. AB - In the first Wessex regional radiology audit, a retrospective comparison of pathology and radiology reports was made to determine rates of demonstration and reporting of colo-rectal carcinoma on barium enema. Barium enema films were analysed by peer review in cases of discrepancy. The standard set was 100% demonstration and 100% diagnosis of colo-rectal carcinoma on a barium enema undertaken in the 12 months preceding histological diagnosis. The carcinoma demonstration rate was 97% and carcinoma reporting rate 85%. Of 1351 patients with carcinoma, 557 had a barium enema in the preceding 12 months. Four hundred and seventy-one enema reports satisfactorily recorded the malignant lesion (85%). The reasons for non-reporting of a carcinoma were failure to demonstrate the lesion (18 cases, 3%), failure to perceive a demonstrated lesion (23 cases, 4%) and failure to analyse a perceived lesion (45 cases, 8%). Any improvement in demonstration cannot be estimated, but 94% of carcinomas should be reported when improved reporting techniques are instituted and the audit loop is closed. PMID- 7554744 TI - Case report: duodenal obstruction and obstructive jaundice due to a large submucosal gallstone--endoscopic and CT appearances. PMID- 7554743 TI - Case report: high flow priapism diagnosed by colour Doppler flow mapping. PMID- 7554745 TI - Case report: critical lower limb ischaemia due to aortic dissection relieved by percutaneous transfemoral fenestration. PMID- 7554746 TI - Case report: giant synovial osteochondromatosis in association with acromegaly. PMID- 7554747 TI - Case report: renal adenocarcinoma with ultrasonographic appearances suggestive of angiomyolipoma. PMID- 7554748 TI - A Mars bar is not an adequate fatty meal--a comparison with Calogen. PMID- 7554749 TI - Implementation of guidelines for radiographic projections. PMID- 7554750 TI - Insulin resistance, hypertension and the insulin-responsive glucose transporter, GLUT4. PMID- 7554751 TI - Luminal epidermal growth factor is trophic to the small intestine of parenterally fed rats. AB - 1. Intestinal atrophy contributes to the clinical difficulties of patients on parenteral nutrition. Systemic administration of epidermal growth factor reverses this effect, but there is concern over the clinical safety of intravenous administration of growth factors. We therefore investigated whether administration of luminal epidermal growth factor could reverse the atrophy induced in a rat model of parenteral nutrition when epidermal growth factor was given alone or in combination with soya bean trypsin inhibitor to reduce proteolytic digestion of the epidermal growth factor. 2. Infusion of soya bean trypsin inhibitor alone decreased intraluminal tryptic activity by about 90% but did not result in increased proliferation. Intragastric infusion of epidermal growth factor (72 micrograms/day per rat) caused a 26% increase in proliferation (determined by 2-h metaphase arrest) in the duodenum (P < 0.01) when compared with animals receiving 'control' intragastric infusion. However, intragastric epidermal growth factor had no effect on more distal regions of the bowel, probably reflecting rapid proteolysis of the epidermal growth factor by luminal proteases. In contrast, a trophic effect of luminal epidermal growth factor was seen in the duodenum (28% increase, P < 0.01) and jejunum (24% increase, P < 0.05) of animals which had received epidermal growth factor with soya bean trypsin inhibitor. This was probably due to the soya bean trypsin inhibitor decreasing the rate of degradation of epidermal growth factor by intestinal proteases, allowing biologically active epidermal growth factor to reach more distal portions of the bowel.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554752 TI - Biliary excretion of chylomicron remnant cholesterol in the rat: responses to the expansion of their plasma pool and promoting role of stimulated bile-acid synthesis. AB - 1. Chylomicron remnants, the intermediate intestinal lipoproteins carrying the bulk of dietary cholesterol, are actively taken up and degraded in the hepatocytes, releasing cholesterol which can be excreted in bile. To study this pathway, a mass of remnants, leading to a consistent rise in hepatic cholesterol, was administered as an intravenous bolus in rats with chronic bile fistula equilibrated by water, electrolyte and taurocholate infusions, and changes in biliary lipids and bile acids were evaluated for up to 24 h in comparison with baseline. 2. A mean 16% increase in the net output of bile acids was observed at each time interval after lipoprotein injection, accounting for a 24h cumulative excretion of approximately one-third of the administered cholesterol mass. These changes did not reach statistical significance however. The cholesterol output and concentrations of all biliary lipids did not vary either. Without taurocholate replacement, remnants injection was followed by a 15-20% decrease in bile acid and bile lipid secretion, presumably due to an insufficient hepatic bile-acid flux. 3. When [3H]cholesterol-labelled remnants were administered at the same mass in the chronic equilibrated bile fistula model, 21% of injected radioactivity was excreted in 24h, distributing mostly in acidic rather than neutral sterols (20.02 +/- 1.85 compared with 1.07 +/- 0.04), with an acidic to neutral sterol mean ratio of 16. 4. To exclude interfering effects from the administered cholesterol mass and chronic bile fistula, 3H-labelled remnants were also studied as a cholesterol trace injected in rats with acute bile fistula.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554753 TI - Compartmental analysis of colonic transit reveals abnormalities in constipated patients with normal transit. AB - 1. The aims of this study were to investigate if compartmental analysis can be used to analyse colonic transit measurements and to search for a relationship between transit time and the parameters deduced from this analysis. In addition, an attempt was made to determine if such analysis could reveal a functional abnormality in patients who complain of constipation but have normal colonic transit. 2. The subjects included 11 healthy controls, 10 patients with chronic diarrhoea and 55 constipated patients. Segmental and total colonic transit time were measured using a previously described method. Compartmental analysis was based on a three-compartment system, with k1, k2 and k3 being the coefficients of diffusion out of the right colon, the left colon and the rectosigmoid area respectively. 3. Patients complaining of constipation who had delayed transit time were the only subjects to have lower values of coefficient k1 than controls. k2 was lower than normal in all patients complaining of constipation, but this decrease was more marked in subjects with delayed colonic transit time than in subjects with normal colonic transit time. All patients complaining of constipation had lower values of k3 than control subjects. 4. This study shows that analysis of colonic transit time is feasible using a simple diffusion law, and that the results are correlated to clinical data. Moreover, this analysis permits detection of abnormalities in two groups of subjects: those who complain of constipation but are labelled as having normal colonic transit time and those who have chronic diarrhoea. PMID- 7554754 TI - Human gastrin and vasoactive intestinal polypeptide responses to endurance running in relation to training status and fluid ingested. AB - 1. Plasma concentrations of gastrin, glucose, vasoactive intestinal polypeptide and non-esterified fatty acid were analysed in six male endurance runners and six male hockey players before, during and 15 min after 90 min treadmill running at 65% maximum oxygen uptake under two conditions: no fluid ingestion (trial A) and ingestion of 8% maltodextrin solution (trial B). 2. Exercise resulted in significantly elevated plasma gastrin concentrations compared with resting values in both groups after trial A (endurance runners, 69.4 ng/l; hockey players 71.4 ng/l) and trial B (endurance runners, 105.5 ng/l; hockey players, 83.2 ng/l). The gastrin response was not significantly different between the trials. 3. Plasma vasoactive intestinal polypeptide levels increased significantly beyond resting levels for both groups during trial A (endurance runners, 76.1 +/- 53.7 ng/l; hockey players 155.6 +/- 41.9 ng/l) and trial B (endurance runners 47.5 +/- 21.3 ng/l; hockey players 132.9 +/- 43.9 ng/l). The vasoactive intestinal polypeptide response to trial B was significantly attenuated compared with trial A. 4. There were no significant differences between endurance runners and hockey players for plasma gastrin, although hockey players produced significantly elevated concentrations of plasma vasoactive intestinal polypeptide after both trials compared with endurance runners. 5. Plasma glucose levels throughout trial B were significantly greater than after trial A irrespective of the group. Plasma glucose concentrations were not significantly different between endurance runners and hockey players. 6. Plasma non-esterified fatty acid concentrations rose significantly for both groups throughout exercise. Trial B resulted in an attenuated non-esterified fatty acid response compared with trial A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554755 TI - Regional sympathetic nervous activation after a large meal in humans. AB - 1. To investigate the link between post-prandial thermogenesis and sympathetic nervous activation we have studied the effects of a single large meal on regional sympathetic nervous activity in healthy, lean subjects. 2. In nine male subjects, noradrenaline spillover was measured from the heart, kidney and liver using isotope dilution, both while fasting and after consumption of a high-energy liquid meal of composition 53% carbohydrate, 32% fat and 15% protein (energy value 2.64-3.51 MJ). Regional oxygen consumption, whole-body oxygen consumption and, in a subset of subjects, muscle sympathetic nerve firing (microneurography) were also measured. 3. Both whole-body oxygen consumption (P < 0.03) and total body spillover of noradrenaline (P < 0.01) rose after the meal, with peak increases of 24% and 56% respectively. Spillover of noradrenaline from the heart was unchanged, that from the hepatosplanchnic circulation increased marginally (0.377 nmol/min to 0.480 nmol/min, P = 0.09), while renal noradrenaline spillover more than doubled (0.440 nmol/min to 0.937 nmol/min, P < 0.05). Skeletal muscle sympathetic nerve activity (peroneal nerve) increased from 7.7 bursts/min at rest to peak at 17.9 bursts/min 60 min after the meal in the three subjects in whom stable recordings were obtained. 4. The meal increased oxygen consumption in the kidneys and liver significantly, from 11.5 +/- 1.6 ml/min to 14.5 +/- 1.1 ml/min and from 46 +/- 7 ml/min to 57 +/- 6 ml/min respectively (P < 0.05), but not in the heart. 5. Consumption of a large meal produces a substantial and relatively selective increase in sympathetic outflow to the kidneys and skeletal muscle. While resting regional oxygen consumptions and noradrenaline spillovers were related, the changes that occurred in each were unrelated, so that no direct relationship could be demonstrated between postprandial thermogenesis and sympathetic activity. PMID- 7554757 TI - Adverse effects of hypoxaemia on diastolic filling in humans. AB - 1. Abnormalities of myocardial relaxation may occur as a consequence of myocyte hypoxia. We have therefore examined the effects of hypoxaemia on right and left ventricular diastolic function in 10 healthy male subjects. 2. After resting to reach baseline haemodynamics, subjects were rendered hypoxaemic by breathing a variable nitrogen/oxygen mixture. Oxygen saturation (SaO2) was maintained at 85 90% for 20 min and then at 75-80% for a further 20 min. Haemodynamic and diastolic filling parameters were measured noninvasively at baseline and at the end of each period of hypoxaemia. 3. Diastolic filling of both ventricles was significantly impaired by hypoxaemia. In comparison with baseline, left ventricular isovolumic relaxation time and transmitral E-wave deceleration time corrected for heart rate were significantly prolonged at SaO2 75-80%: mean difference in corrected relaxation time, 9.8 ms (95% confidence interval 1-19); mean difference in corrected deceleration time, 34 ms (95% confidence interval 11 56). Similarly, right ventricular isovolumic relaxation time and transtricuspid E wave deceleration time were significantly prolonged at SaO2 values of 75-80% compared with baseline: mean difference in relaxation time, 20.3 ms (95% confidence interval 3-38); mean difference in deceleration time, 33 ms (95% confidence interval 11-55). 4. During hypoxaemia there were dose-related increases in heart rate, cardiac output and mean pulmonary artery pressure, but no effects on mean arterial pressure. 5. Hypoxaemia significantly impairs relaxation of left and right ventricles in normal humans. These changes may reflect impairment of intracellular calcium transport secondary to the effects of myocyte hypoxia. PMID- 7554756 TI - Sympathetic stimulations by exercise-stress testing and by dobutamine infusion induce similar changes in heart rate variability in patients with chronic heart failure. AB - 1. Heart rate variability can be used to evaluate autonomic balance, but it is unclear how inotropic therapy may affect the findings. The aim of the study was to assess whether heart rate variability can differentiate between sympathetic stimulation induced by inotrope infusion or by physical exercise. 2. Ten patients with chronic heart failure (64.3 +/- 5.4 years of age) underwent four dobutamine infusions (8-min steps of 5 micrograms min-1 kg-1) and four supine bicycle exercise tests (5-min steps of 25 W). Plasma noradrenaline was evaluated, as well as the SD of R-R intervals, together with low-frequency (0.03-0.14 Hz) and high frequency (0.15-0.4 Hz) components of heart rate variability using autoregressive spectral analysis. 3. Exercise and inotrope infusion produced similar changes in heart rate variability. An exercise load of 50 W and a dobutamine infusion of 15 micrograms min-1 kg-1 gave the following results respectively: heart rate, 120.3 +/- 3.0 beats/min versus 110.2 +/- 3.0 beats/min; SD, 16.0 +/- 1.1 ms versus 16.3 +/- 2.5 ms; low-frequency component, 4.3 +/- 0.3 ln-ms2 versus 4.4 +/- 0.3 ln-ms2 and high-frequency component, 2.6 +/- 0.3 ln-ms2 versus 2.2 +/- 0.3 ln-ms2. All comparisons were nonsignificant. The variables of heart rate variability showed high reproducibility in the same subject during different conditions. Noradrenaline was elevated by exercise from 326.0 +/- 35.2 pg/ml to 860.1 +/- 180.4 pg/ml (P < 0.05), but was unchanged by dobutamine infusion. 4. Heart rate variability changes cannot differentiate between dobutamine infusions and physical exercise, indicating that we should be cautious in evaluating patients undergoing inotropic therapy. The degree of receptor stimulations, rather than the level of sympathetic drive, would appear to determine the changes in heart rate variability. PMID- 7554758 TI - Plasma-mediated neutrophil activation during acute myocardial infarction: role of platelet-activating factor. AB - 1. Polymorphonuclear neutrophils are involved in the development of myocardial injury during ischaemia through the release of free oxygen radicals and by adhesion of activated polymorphonuclear neutrophils to endothelium, resulting in plugging of coronary capillaries. Polymorphonuclear neutrophil activation may be a result of contact with ligands expressed by endothelial cells and/or a response to soluble stimuli released from ischaemic tissue to the plasma. 2. To investigate this we studied plasma-mediated polymorphonuclear neutrophil activation in vitro using plasma samples collected from 14 patients with acute myocardial infarction at time of admission and 6 h and 1, 2, 5 and 7 days later. Plasma samples were incubated with washed polymorphonuclear neutrophils isolated from healthy donors. Expression of adhesion molecules CD18/CD11b integrin and L selectin (Leu-8) were measured by flow cytometry and superoxide anion production in polymorphonuclear neutrophils was measured by chemiluminescence. 3. Plasma samples obtained 6 h and 1 day after admission were capable of inducing CD18/CD11b antigen expression, superoxide anion production and L-selectin shedding in the washed polymorphonuclear neutrophils, and this effect was significant when compared with plasma taken at 5 and 7 days after admission. 4. The plasma-mediated polymorphonuclear neutrophil stimulation was prevented when the PMN were pretreated with platelet-activating factor receptor antagonists BN52021 or BN50739. The platelet-activating factor concentrations detected in the plasma samples were not higher than those detected in plasma from healthy subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554761 TI - Non-invasive measurement of microvascular permeability to a small solute in man: validation of the technique. AB - 1. The purpose of the study was to evaluate a non-invasive technique for measurement of microvascular permeability to a small hydrophilic solute. 2. The technique measures the clearance of 99mTc-labelled diethylenetriaminepenta-acetic acid (99mTc-DTPA) from plasma into interstitial fluid in a limb after intravenous injection and uses a scintillation probe and a technique of graphical analysis called the Patlak plot, the uptake constant of which reflects 99mTc-DTPA transfer from plasma to interstitial fluid. Using deconvolution analysis, the retention function in the limb of intravenous 99mTc-DTPA was also measured. 3. The clearance values given by these two analytical techniques were compared with clearance from the same vascular bed after bolus femoral intra-arterial injection of 99mTc-DTPA. 4. Sixteen patients undergoing routine diagnostic arteriography were studied: six received sequential femoral intra-arterial injections of 99mTc labelled human serum albumin (HSA) and 99mTc-DTPA, two received sequential intra arterial and intravenous injections of 99mTc-HSA and eight received sequential intra-arterial and intravenous injections of 99mTc-DTPA. Tissue uptake and clearance were recorded from the limb with a scintillation probe and plasma clearance by arterial blood sampling. Tracer recirculation was addressed using a second scintillation probe over the contralateral limb. 5. After intra-arterial injection, 99mTc-HSA clearance was monoexponential, reflecting intravascular transit, and was completed by 2-5 min in seven subjects and in about 10 min in one. The corresponding 99mTc-DTPA clearance curves in the six subjects who also received intra-arterial DTPA were biexponential, analysis of which yielded a 99mTc-DTPA extraction fraction of about 0.6. By comparison with 99mTc-HSA clearance, the first exponential clearly corresponded to intravascular transit of unextracted 99mTc-DTPA. 6. In the eight patients given sequential intra-arterial and intravenous injections of 99mTc-DTPA, the second exponential recorded after intra-arterial injection, representing 99mTc-DTPA clearance from the interstitial fluid, agreed well with (a) the Patlak uptake constant recorded over the limb after intravenous injection, representing clearance from plasma into the interstitial fluid and (b) the retention function of 99mTc-DTPA in a limb calculated by deconvolution analysis. The mean clearance following intra-arterial injection (expressed in relation to extracellular fluid volume) was 9.6 (SD 2.4) ml min-1 100 ml-1, while the corresponding mean clearance after intravenous injection was 8.8 (2.1) ml min-1 100 ml-1 calculated by Patlak analysis and 10.5 (2.7) ml min-1 100 ml-1 by deconvolution analysis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7554760 TI - Vascular and hormonal responses to arginine: provision of substrate for nitric oxide or non-specific effect? AB - 1. The vascular and hormonal effects of L- and D-arginine were compared in healthy subjects and in patients with insulin-dependent diabetes mellitus or untreated essential hypertension. 2. Infusion of L- or D-arginine (40 mumol/l) in the forearm vascular bed, sufficient to increase the local concentration approximately 20-fold, had no effect on blood flow or the vasodilator response to acetylcholine (30 and 100 nmol/min) in patients with insulin-dependent diabetes (n = 7) or essential hypertension (n = 7), or in age- and sex-matched control subjects (n = 7 in both groups). 3. Systemic infusion of 10 g of L-arginine (n = 5) or D-arginine (n = 3) increased plasma concentration of arginine approximately 20-fold without altering supine or erect haemodynamics. Increases in plasma insulin, prolactin and glucagon were seen with both enantiomers. The stereopurity of arginine was confirmed in a cell-culture assay system. 4. We conclude that, in healthy subjects and patients with essential hypertension or insulin-dependent diabetes, synthesis of nitric oxide within the vasculature is not limited by substrate availability. At high concentrations of arginine, non-stereospecific effects, including alterations in hormone concentration, occur. It remains to be determined whether these non-stereospecific hormonal changes might contribute to certain haemodynamic effects of arginine. PMID- 7554759 TI - Contribution of vascular nitric oxide to basal blood pressure in conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats. AB - 1. The aim of this study was to clarify the extent to which vascular nitric oxide contributes to basal blood pressure in conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats. 2. The contribution of vascular nitric oxide to maintenance of blood pressure was estimated by measuring the pressor response to an intravenous injection of nitric oxide synthase inhibitor, N omega-L arginine methyl ester, given after serial injections of captopril, vasopressin V1 receptor antagonist (V1-antagonist) and ganglion blocker (pentolinium) in conscious spontaneously hypertensive and Wistar Kyoto rats aged 20-28 weeks. To estimate the 'amplifier property' of hypertrophied vasculature in spontaneously hypertensive rats, which is known to modulate pressor responses, the lower blood pressure plateau after serial injections of captopril, V1-antagonist and pentolinium and the maximum blood pressure elicited by subsequent injection of increasing doses of phenylephrine were also measured. 3. The serial injections of captopril, V1-antagonist and pentolinium decreased mean arterial pressure from 164 +/- 9 mmHg to 67 +/- 2 mmHg and from 117 +/- 2 mmHg to 49 +/- 1 mmHg in spontaneously hypertensive and Wistar Kyoto rats respectively. The subsequent injection of N omega-L-arginine methyl ester restored mean arterial pressure almost to its control levels in both spontaneously hypertensive and Wistar Kyoto rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554762 TI - Intracranial pressure at high altitude and acute mountain sickness. AB - 1. Raised intracranial pressure has been noted in severe forms of acute mountain sickness and high-altitude cerebral oedema, but the role of intracranial pressure in the pathogenesis of mild to moderate acute mountain sickness is unknown. 2. Serial measurements of intracranial pressure were made indirectly by assessing changes in tympanic membrane displacement in 24 healthy subjects on rapid ascent to 5200 m. 3. Acute hypoxia at 3440 m was associated with a rise in intracranial pressure, but no difference was found in pressure changes at 4120 or 5200 m in subjects with or without symptoms of acute mountain sickness. 4. Raised intracranial pressure, though temporarily associated with acute hypoxia, is not a feature of acute mountain sickness with mild or moderate symptoms. PMID- 7554763 TI - Degenerative joint disease. PMID- 7554764 TI - Basal and exercise-induced changes in plasma catecholamines: relationship to age, smoking habits and sampling times. AB - Contrary to earlier investigations, we have previously shown that the initial increase in venous plasma noradrenaline (NA) during exercise did not differ in young and elderly non-smoking subjects exercising at the same relative workload (75%), and that the greatest increase at maximal workload was observed in the young subjects. In order to determine whether the observed discrepancy could be explained by long-term smoking, plasma NA and adrenaline (A) were evaluated in eight elderly male smokers (mean age 67 years). The results were compared with data reported previously [Jensen et al. (1994) Exercise-induced changes in plasma catecholames and neuropeptide Y: relation to age and sampling times. J Appl Physiol, 76, 1269-1273] in seven young (mean age 27 years) and seven elderly (mean age 61 years) non-smoking male subjects. As shown earlier, basal plasma NA was significantly increased in elderly smokers compared to young and elderly non smokers. During exercise at 75% of maximal work capacity, no difference was observed between elderly smokers and non-smokers. No difference in plasma A was found between elderly smokers and non-smokers either at rest or during exercise. These findings indicate that the exercise-induced increase in plasma NA did not differ in elderly smokers and non-smokers despite increased resting levels in the former group, and that the increase in the elderly subjects was not augmented compared to that in young subjects. PMID- 7554765 TI - Comparison of some criteria for diagnosing a myocardial infarction after aorto coronary bypass grafting (CABG). AB - The incidence of myocardial infarction during a 1-year follow-up period after coronary bypass surgery (CABG), i.e. a recent myocardial infarction (RMI), was studied in 86 patients. Different criteria for the diagnosis of a RMI were compared. Clinical observation, including ECG and serum enzyme analysis, diagnosed RMI in 8% of patients. Specific ECG changes indicating RMI (ECGsp) occurred in 12% of cases, and if less specific ECG changes were also taken into account (ECGsp+nonsp) RMI was found in 30% of cases. Asynergy, detected by two plane ventriculography, indicated RMI in 24% of the patients, and was probably the most valid of the criteria examined. The differences in diagnostic accuracy of the various criteria highlight the importance of defining diagnostic criteria. PMID- 7554767 TI - The response of skin microcirculation to hyperthermic stress in tumour patients. AB - Hyperthermia and its effect on tissues are topics of great interest to scientists working in the area of radiation biology and medicine. It has been shown by many workers, that blood flow in malignant tissue displays a different response to heating than that in normal tissue. Initially, the blood flow in tumour tissue is greater than that in normal tissue, and when heat is applied there is an increase in blood flow. The extent of the increase in flow with increasing temperature is greater in normal tissue than in tumour tissue. In our laboratory we studied the effect of temperature on skin blood flow. The skin overlying tumour tissue was compared with skin with no underlying abnormality in cancer patients, and with the skin in healthy control subjects. The instrument used was a Laser Doppler Perfusion Monitor, Pf3 (Perimed, Stockholm, Sweden). We found that the skin overlying tumour tissue showed higher basal perfusion than the skin at the contralateral site with no underlying abnormality. The skin above tumour tissue showed a reduced perfusion response to an increase in temperature (vascular sluggishness) compared to skin at the contralateral site and skin in healthy controls. The reduction in thermal response depends on the size of the tumour. PMID- 7554766 TI - Fat metabolism and its response to infusion of insulin and glucose in patients with advanced chronic obstructive pulmonary disease. AB - In order to investigate fat metabolism and the regulation of lipolysis and blood fuel metabolites by insulin, nine patients with chronic obstructive pulmonary disease (COPD) with chronic hypoxaemia and seven healthy control subjects of similar age were investigated by determination of the turnover rate of free fatty acids (TOR), using 1-14C-oleic acid as a tracer, and arterial concentrations of FFA, glycerol and 3-hydroxybutyrate. The measurements were performed in the basal state and during insulin and glucose infusion, aiming at euglycaemia at insulin levels of 50 and 100 mU l-1. The subjects' ages were 64 +/- 2.7 and 66 +/- 1.1 (mean +/- SEM) years in the COPD and control groups, respectively. TOR was 0.73 +/- 0.06 and 0.52 +/- 0.02 mmol min-1 (P < 0.05) in the basal state, 0.33 +/- 0.04 and 0.30 +/- 0.02 at an insulin level of 50 mU l-1 and 0.32 +/- 0.08 and 0.24 +/- 0.02 at an insulin level of 100 mU l-1, in the COPD and control groups, respectively. Arterial FFA concentration was 0.98 +/- 0.08 and 0.75 +/- 0.06 mmol l-1 (P < 0.05) in the basal state in the COPD and control groups, respectively. During the clamp, the decrease in FFA mirrored that in TOR. The results show that the state of lipolysis is increased in severe COPD patients with chronic hypoxaemia. Furthermore, the results suggest a reduced effect of insulin in lipolysis. PMID- 7554768 TI - Autonomic responses to the Valsalva manoeuvre in healthy subjects. AB - To assess normal autonomic haemodynamic responses to the Valsalva manoeuvre, 158 healthy unmedicated subjects, aged 25-60 years, were examined. For measurement of beat-to-beat blood pressure on a finger, the Finapres instrument was used. Phase to-phase changes in instantaneous blood pressure and heart rate and the latency response between the end of a Valsalva manoeuvre and points on the resultant blood pressure and heart rate were calculated, and the reference limits for various indices were determined. Sex had no or only marginal effect on blood pressure or heart rate responses or latencies. Ageing was accompanied by a smaller decrease and smaller partial recovery of blood pressure during the strain, with attenuation of reflectory bradycardia, and lengthening of the latencies. It is concluded that age-related reference values should be applied in the interpretation of the Valsalva responses. The following responses should be analysed: mean blood pressure decrease and partial recovery during the strain (adrenergic vasoconstrictor function), reflectory bradycardia after the strain (parasympathetic function), and the latencies (sympathetic and parasympathetic function). PMID- 7554769 TI - Cardiovascular autonomic control is disturbed in nocturnal teethgrinders. AB - To examine the hypothesis of disturbed autonomic function, non-invasive cardiovascular reflex tests were performed on 11 sleep bruxists in the waking state. The tests included the Valsalva manoeuvre, a deep-breathing test, and an orthostatic test (standing up). The R-R intervals were monitored continuously, and blood pressure was measured non-invasively and continuously using the Finapres method. In total, 64% of bruxists showed abnormalities in at least two variables reflecting the cardiovascular autonomic function. Abnormalities were found in blood pressure regulation during the Valsalva strain, and in the immediate biphasic heart rate response during standing up, but not in the vagally mediated deep-breathing difference. These findings suggest that bruxism is accompanied by abnormalities in autonomic function, particularly in sympathetic vasoconstrictor function. PMID- 7554770 TI - Spirometry and ventilation-perfusion inequality in patients with mild allergic asthma before and during the pollen season. AB - Several studies on asthma have shown a low correlation between gas exchange and spirometry, especially after treatment with bronchodilators. The aim of the present study was therefore to examine both spirometry results and gas exchange during a pollen-free period and at the end of the pollen season in patients with mild and well-controlled allergic asthma. Pulmonary gas exchange was studied using a modified form of the multiple inert gas elimination technique. Lung volumes and forced expiratory flows were measured by common spirometry. During the non-pollen season, spirometry and forced expiratory flows were within the reference values in all but one patient, who had decreased indices for airway flow. Three other patients showed signs of minor gas exchange impairment. During the pollen season, FRC was slightly increased (P < 0.05) and MEF50 was slightly decreased (P < 0.05) for the group. Two patients had an increased index for gas exchange impairment (log SDQ was 0.64 and 0.59) and four patients had borderline log SDQ (0.50 to 0.56). However, the mean log SDQ was not increased in the pollen season. The results show that, both in the pollen season and in the pollen-free season, low degrees of gas exchange impairment could be present in pollen allergic asthmatic patients despite normal spirometry. The low degree of gas exchange impairment in some patients indicates the presence of airway inflammation with oedema and/or secretion. However, high degrees of ventilation perfusion inequality were not observed in these patients where air flow rates were mainly normal. PMID- 7554771 TI - An increase in micropulmonary red cell mass in hyperlipidaemic patients. AB - It is known that pulmonary microcirculation rheology is partly affected by plasma levels of lipoproteins, but only a few data are available for humans. Therefore, in a sample of 30 normal volunteers and 90 patients with various types of primary hyperlipoproteinaemia, the plasma levels of total cholesterol (Chol), low density cholesterol (LDL), the high density cholesterol (HDL), triglyceride (Tg) and fibrinogen (Fib) were measured in conjunction with determinations of plasma viscosity (PV) and the pulmonary capillary red cell volume (RCVc). RCVc was estimated from measurements of the vascular component of the single-breath diffusing lung capacity for carbon monoxide, using our own modification of the Roughton-Forster's method. By stepwise regression analysis, the variation in RCVc was almost completely accounted for (r2 = 0.87) by variations in PV, Chol, Tg and the anthropometric confounding factors. The proposed explanations for increased pulmonary capillary red cell mass (up to 151% of the predicted value) in hyperlipidaemic patients included the hypothesis of increased pulmonary microhaematocrit, which agrees with the observed in-vitro lipoprotein-dependent increase in erythrocyte aggregability. PMID- 7554772 TI - Metabolic, thermal and circulatory effects of intravenous infusion of amino acids in tetraplegic patients. AB - Metabolic, circulatory and thermal effects of intravenously (i.v.) administered amino acids were studied in eight patients with complete cervical spinal cord injuries, and compared with the effects in eight healthy subjects. Using indirect calorimetry and catheter techniques, whole-body and splanchnic oxygen consumption, blood flow and blood temperatures were measured before and at timed intervals during 2.5 h of i.v. infusion of 600 kJ of a mixture of 19 amino acids. Pulmonary oxygen uptake increased from 209 +/- 11 to 267 +/- 13 ml min-1 in the patients and from 268 +/- 5 to 320 +/- 8 ml min-1 in the controls. The thermic effect of amino acids was 21 +/- 3% and 16 +/- 2% in patients and controls, respectively. In both groups the splanchnic tissues accounted for approximately half of the rise in whole-body oxygen consumption. Cardiac output rose by, on average, 0.5 +/- 0.1 and 0.8 +/- 0.2 1 min-1 in patients and controls, respectively, while the hepatic blood flow remained unchanged in both groups. Pulmonary arterial blood temperature increased by 0.647 +/- 0.100 degrees C in the patients and by 0.244 +/- 0.174 degrees C in the controls (P < 0.05). The whole-body specific heat was low in the patients, its calculated maximum value being approximately 20% below the normal level. During the amino acid infusion the arterial blood concentration of amino acids rose by approximately 170% and 112% of its basal levels in patients and controls, respectively, indicating a significantly reduced capacity for cellular uptake of amino acids in tetraplegic patients. It is concluded that, in tetraplegic patients, i.v. infused amino acids induce prompt thermogenesis of normal magnitude accompanied by supranormal temperatures and amino acid concentrations in the blood, and that low whole-body specific heat contributes to the well-known thermoregulatory instability in tetraplegia. PMID- 7554773 TI - M-mode magnetic resonance imaging: a new modality for assessing cardiac function. AB - Magnetic resonance imaging (MRI) studies of the heart have been used for some years, but there are few tools available to quantify cardiac motion. A method has been developed that creates an M-mode MRI image, analogous to the one used in echocardiography, to display motion along a line as a function of time. The M mode image is created from MRI images acquired with an ordinary gradient echo cine sequence. In a cinematographic display of the images, a cursor line can be positioned in order to determine the orientation of the measurement. A resampling algorithm then calculates the appearance of the M-mode image along the cursor line. The MRI method has been compared to echocardiographic M-mode in a phantom study and by measuring mitral and tricuspid annulus motion in 20 normal subjects. The phantom study showed no significant differences between MRI and echocardiographic M-mode measurements (difference < 1 mm). The annulus motion exhibits a similar pattern using both methods and the measured amplitudes are in close agreement. M-mode MRI provides similar information to echocardiography, but the cursor line can be placed arbitrarily within the image plane and the method is thus not limited to certain acoustic windows. This makes M-mode MRI a promising technique for assessing cardiac motion. PMID- 7554774 TI - Pilocarpine iontophoresis test: an index of physiological sweat secretion? AB - The pilocarpine iontophoresis test (P-test) is used as a predictor of the capacity to produce sweat. Therefore, we studied the reproducibility of this test in 12 normal subjects on 10 consecutive days. Furthermore, we determined whether the P-test reflects whole-body and regional sweat secretion during exercise in the heat. Finally, we determined whether the P-test stimulates the eccrine sweat glands to maximal sweat secretion. Six growth hormone-deficient (GHD) patients who are known to have decreased sweating, and 11 healthy control subjects were studied. To induce maximal sweat secretion, the patients exercised on a bicycle ergometer at a workload corresponding to 40% of their maximal aerobic power (VO2max). The 11 healthy subjects exercised at a workload of 150 W. All subjects exercised for 60 min in ambient air at 35 degrees C, with 50% relative humidity. The P-test showed a mean day-to-day variation of 20.8% between individual subjects. There was a significant positive correlation between the P-test and regional sweat secretion (r2 = 0.74). The correlation coefficient (r2) was 0.50 for the correlation between the P-test and whole-body sweat secretion, and 0.52 for the correlation between regional sweat secretion and whole-body sweat secretion. We conclude that the pilocarpine iontophoresis test reflects heat- and exercise-induced sweating capacity. However, this test does not induce maximal sweating, and it cannot be used as a single reliable predictor of whole-body sweating, due to considerable day-to-day variation. PMID- 7554775 TI - Prevention of macrovascular disease: absolute proof or absolute risk? PMID- 7554776 TI - Is there a seasonal variation in the incidence of gestational diabetes? AB - It has been suggested that the incidence of gestational diabetes (GDM) may vary with different seasons of the year. The fasting and 2 h glucose levels after a 75 g glucose tolerance test were therefore examined in 2749 women being tested for GDM and were correlated with the mean temperature at 0900 h by month and by season. The mean monthly temperature ranged from 22.3 degrees C in January to 13.6 degrees C in July. The monthly incidence of GDM ranged from 3.5% (95% CI, 0.7-6.3) in September to 8.7% (4.7-12.7) in November. The seasonal incidence of GDM ranged from 7.4% (5.6-9.2) in autumn to 5.5% (3.5-7.5) in winter. Chi-squared analysis did not suggest a significant association between the proportion of women diagnosed with GDM and either the month (p = 0.47) or the season (p = 0.39). Multiple regression analysis, after correction for maternal age, parity, BMI, and the week of testing revealed a non-significant association between fasting glucose and monthly temperature (p = 0.36) and an increase in the 2 h glucose of 0.026 mmol l-1 for each increase of 1 degree C in temperature (p = 0.011). In a temperate coastal area of Australia these differences do not appear clinically significant and have not caused a variation in the incidence of GDM with different seasons. PMID- 7554777 TI - The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. AB - A combination of metabolic and vascular defects have been implicated in the pathogenesis of diabetic neuropathy. Animal studies have demonstrated that a reduction in nerve blood flow may be an important early defect and that vasodilators can prevent or ameliorate nerve dysfunction. The potential factors contributing to nerve ischaemia include structural defects in the endoneurial microvasculature together with rheological abnormalities, abnormalities in vasoactive agents which regulate nerve blood flow including nitric oxide and the eicosanoids, and alterations in tone of the autonomic innervation of the nerve vasculature. The principle metabolic defects which have been implicated include disruption of the polyol pathway, altered lipid metabolism, advanced glycosylated end-product formation, increased oxidative stress, and diabetes-induced defects in growth factors. The demonstration that activation of the polyol pathway in experimental diabetes may affect nerve blood flow, and conversely that vasoactive agents appear to be important in regulating some aspects of nerve metabolism, has highlighted the interdependence of the metabolic and vascular defects in the pathogenesis of this condition. Thus, selective intervention aimed at a key defect early in this cascade may subsequently correct a number of later abnormalities offering therapeutic hope in this chronic debilitating complication. PMID- 7554778 TI - The roots of Ariel: Sylvia Plath and her father's foot. PMID- 7554779 TI - Differences in joint mobility and foot pressures between black and white diabetic patients. AB - Limited joint mobility is common in diabetes and is related to high foot pressures and foot ulceration. We have examined the differences in joint mobility and foot pressures in four groups matched for age, sex, and duration of diabetes: 31 white diabetic, 33 white non-diabetic, 24 black diabetic, and 22 non-diabetic black subjects. Joint mobility was assessed using a goniometer at the fifth metacarpal, first metatarsal, and subtalar joints. In-shoe and without shoes foot pressures were measured using an F-Scan system. Neuropathy was evaluated using clinical symptoms (Neuropathy Symptom Score), signs (Neuropathy Disability Score), and Vibration Perception Threshold. There was no difference between white and black diabetic patients in Neuropathy Symptom Score, Neuropathy Disability Score, and Vibration Perception Threshold. Subtalar joint mobility was significantly reduced in white diabetic patients (22 +/- 7 degrees) compared to white controls (26 +/- 4 degrees, black diabetic patients (25 +/- 5 degrees), and black controls (29 +/- 7 degrees), and increased in black controls compared to white controls and black diabetic patients (level of statistical significance p < 0.05). Without shoes foot pressures were higher in white diabetic patients (8.31 +/- 400 kg cm-2) compared to white controls (6.81 +/- 2.31 kg cma2), black diabetic patients (6.2 +/- 2.53 kg cm-2) and black controls (5.00 +/- 1.24 kg cm 2) and lower in black controls compared to white and black diabetic patients (p < 0.05 in all cases). We conclude that racial differences exist in joint mobility and foot pressures between black and white subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554780 TI - The association of fibrinolysis and hyperlipidaemia with quantitative sensory tests in an epidemiological study of Swedish type 1 diabetic patients. AB - Serum levels of cholesterol, HDL-cholesterol, triglycerides, lipoprotein Lp(a), and the fibrinolysis factors tPA (tissue plasminogen activator) and PAI-1 activity (plasminogen activator inhibitor) were compared with sensory thresholds for vibration, electrical current perception, and pain in a population-based study comprising 239 patients with diabetes mellitus Type 1, aged 15-50 years. Univariate regression analyses (n = 180) showed significant correlations between elevated sensory thresholds and age, duration of diabetes, serum cholesterol and triglycerides, and HbA1c. In multivariate regression analysis, age, duration of diabetes, height, and serum triglycerides showed significant independent associations with five or six of the six measured sensory threshold variables. In addition there was a significant association between increased thresholds for vibration and Lp(a) levels. Thus, increased sensory thresholds for vibration, current perception, and pain in patients with Type 1 diabetes are associated with increased serum triglyceride levels, and Lp(a) levels are associated with increased threshold for vibration. Fibrinolytic activity is unrelated to these measures of nerve function in Type 1 diabetic patients. PMID- 7554781 TI - Heat shock protein studies in type 1 and type 2 diabetes and human islet cell culture. AB - Heat shock proteins (HSP) play an important role in auto-immunity and infection. Glutamic acid decarboxylase (GAD) the prime antigen in Type 1 diabetes has similar amino acid sequences to HSP65. An ELISA was developed using a plant derived HSP65 antibody. HSP65 antibody was present in the serum of all normal subjects (median 1.64 AU, IQ range 1.49-1.74). Lower levels were found in established Type 1 diabetes (1.41 AU, 1.32-1.61, p < 0.001) and Type 2 diabetes (1.45 AU, 1.35-1.59, p < 0.006). In Type 1 HSP antibody levels fell with age (p = 0.007) and with duration (p = 0.008) and women with Type 1 had lower levels than men (p = 0.009). Human islet cell culture subjected to heat shock revealed an approximate four fold increase in heat shock protein antigen in the surrounding medium. The release of HSP antigen from stressed islet cells together with the finding of HSP antibody in the serum of all subjects suggest that HSP65 should not be completely discarded as having a possible role in the development of Type 1 diabetes. Low levels of HSP antibody in patients with established diabetes is probably a manifestation of impaired immunity induced by the diabetic state. PMID- 7554783 TI - Short-term mortality in childhood onset insulin-dependent diabetes mellitus: a high frequency of unexpected deaths in bed. AB - Mortality and the causes of death have been studied in a population-based cohort of 4919 childhood onset IDDM cases. Enrolment began in 1977 and at the time of study there had been a maximum duration of disease of 13.5 years, with a total of 33,721 person years at risk. Survival status was ascertained by linkage to the Swedish Cause-of-Death register. Death certificates, autopsy protocols, and hospital records were scrutinized for classification of causes of deaths. Twenty males and 13 females with IDDM died before the age of 28.5 years. This corresponds to a Standardized Mortality Rate for age of 262% (95% confidence limits, 172-400) for the boys and 384% (95% confidence limits, 232-635) for girls. Seven patients died of ketoacidosis, four at onset of diabetes. Nine cases were found 'dead in bed', having been seen apparently healthy 1-2 days before death. One of these cases had signs of cerebral haemorrhages at autopsy and another one had signs of bite marks in the mouth, but otherwise all autopsies were normal and no evidence of alcohol or other intoxication was found. In a well educated population with good access to inexpensive diabetes care, there is still a two- to threefold excess mortality among young onset insulin-dependent diabetic individuals including a high frequency of unexplained deaths in bed. PMID- 7554782 TI - Comparison of the effects on the diurnal blood pressure, glucose, and lipid levels of a diet rich in monounsaturated fatty acids with a diet rich in polyunsaturated fatty acids in type 2 diabetic subjects. AB - The influence on blood pressure, glucose metabolism, and lipid levels of an exchange of polyunsaturated fatty acids with monounsaturated fatty acids in the diet was compared in 16 Type 2 diabetic outpatients. In a cross-over study design the patients were randomly assigned to 3 weeks treatments with a diet containing 30% energy as monounsaturated fatty acids, or an isoenergetic diet with 30% energy as polyunsaturated fatty acids. The contents of total fat, saturated fatty acids, cholesterol, and fibre were similar in the two diets. On the last days of the two diet periods, 24-h ambulatory blood pressure was measured and the responses of glucose, hormones, and lipids to a carbohydrate rich test meal were measured. As compared with the polyunsaturated fatty acids diet, the monounsaturated fatty acids diet reduced 24-h systolic (129 +/- 11 vs 124 +/- 8 mmHg (mean +/- SD), p = 0.02) and diastolic blood pressure (76 +/- 11 vs 73 +/- 8 mmHg, p = 0.02). The two diets had similar, beneficial effects on glycaemic control, and cholesterol and lipoprotein concentrations. A diet rich in monounsaturated fatty acids has beneficial effects on the blood pressure, while similar effects on glucose and lipid levels are observed in normotensive Type 2 diabetic subjects. PMID- 7554785 TI - Diabetes care in South Asian and white European patients with type 2 diabetes. AB - Aspects of diabetes care in South Asian and white European patients with Type 2 diabetes attending a hospital review clinic were explored. Among the clinic population of 1710 patients, 258 (15%) were of South Asian origin. A significantly greater proportion (95% CI for difference in proportions 8-22%) of these patients was treated with oral hypoglycaemic drugs than in white Europeans, in whom there was a correspondingly greater proportion receiving insulin treatment. In a case-control study, where 154 patients in each racial group were stratified according to treatment regimen, significantly more South Asian patients (13/30 vs 6/30, p < 0.05) on insulin were treated with a once-daily regimen. Despite these observed differences in treatment of diabetes, glycaemic control was no worse in South Asian patients when compared to their white European counterparts. South Asian diabetic patients attending hospital diabetes clinics in the UK can experience similar levels of glycaemic control to white Europeans. PMID- 7554784 TI - Psychosocial predictors of acute complications of diabetes in youth. AB - In this paper we determine whether individual and family psychosocial functioning predicts the risk for recurrent acute diabetic complications. An onset-cohort of 61 children and adolescents with Type 1 diabetes received conventional diabetes care. Episodes of ketoacidosis and of severe hypoglycemia were recorded for 8 years, and glycaemic control was measured by glycohaemoglobin. Measures of psychosocial functioning of the patient and parents were obtained during the first year. Over 8 years, 28% of subjects had at least one episode of ketoacidosis, and 21% had at least one episode of hypoglycaemia. The odds of observing recurrent hypoglycaemia versus recurrent ketoacidosis was 14 times greater in boys than in girls (Fisher's exact test p < 0.05). Girls with recurrent ketoacidosis had more behaviour problems and lower social competence, they reported higher levels of family conflict, and their parents reported lower levels of family cohesion, expressiveness and organization in year one. These relationships were independent of any association with poor glycaemic control. Recurrent hypoglycaemia in boys was generally unrelated to individual and family functioning or glycohaemoglobin. Despite our small sample size, our findings are suggestive of relationships that may lead to early identification of patients who are prone to recurrent ketoacidosis, and to the development of early intervention strategies. PMID- 7554786 TI - Frequency of thyroid dysfunction in diabetic patients: value of annual screening. AB - A randomly selected group of 1310 adult diabetic patients attending a diabetic outpatient clinic received annual screening for thyroid disease, by estimating serum free thyroxine and TSH concentrations. The overall prevalence of thyroid disease was found to be 13.4%, and was highest (31.4%) in Type 1 diabetic females, and lowest in Type 2 diabetic males (6.9%). As a direct result of screening, new thyroid disease was diagnosed in 6.8% (89 patients) of the population screened; the commonest diagnosis was subclinical hypothyroidism (4.8%), followed by hypothyroidism (0.9%), hyperthyroidism 0.5%), and subclinical hyperthyroidism (0.5%). Female patients with Type 1 diabetes had the highest annual risk of developing thyroid disease (12.3%), but all patient groups had a higher incidence of thyroid dysfunction, compared to that reported in the general population. This study suggests that thyroid function should be screened annually in diabetic patients to detect asymptomatic thyroid dysfunction which is increased in frequency in a diabetic population. PMID- 7554787 TI - Glycated haemoglobin levels in patients with diabetes in one general practice over a 10-year period. AB - The aim of the study was to evaluate the level of control, as reflected by HbA1c, in patients with diabetes attending one general practice over a 10-year period. The study was based in one general practice in South Tyneside, UK and consisted of an analysis of HbA1c values of all patients with diabetes attending the practice between 1983 and 1992. HbA1c levels were analysed and are presented as multiples of the standard deviation above the mean. In the practice 256 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 76 with insulin dependent diabetes mellitus (IDDM), attended for a total of 1596 doctor/patient contacts in the diabetic clinic over 10 years. The prevalence of diabetes was 1.9%. Over the course of the clinic, in any one year, 25% of patients with NIDDM and 55% with IDDM had levels of HbA1c above those thought to be associated with increased risk of microvascular complications. Significant reduction in glycated haemoglobin (HbA1c) occurred in the first year after diagnosis (p < 0.01) and after changing treatment from diet alone to diet and oral hypoglycaemic agents (p < 0.001). We conclude that a large proportion of patients within this population had levels of glycaemic control that put them 'at increased risk'. PMID- 7554788 TI - Reduction of gangrene and amputations in diabetic renal transplant patients: the role of a special foot clinic. AB - This 4-year prospective study investigated the reasons for high levels of gangrene and major amputation in diabetic renal transplant patients and whether regular multidisciplinary foot care could reduce morbidity. All foot lesions were documented and investigated in 50 diabetic patients, mean age 49.2 +/- 11.0 (SD) years, duration of diabetes 25.3 +/- 9.0 years, time since renal transplantation 60.2 +/- 35.1 months, who attended a special foot clinic monthly for education, vascular and neurological assessment, podiatry and footwear. Foot lesions included: neuropathic ulcers, ischaemic ulcers, traumatic lesions, Charcot's arthropathy, pathological fracture. Treatment included antibiotics, podiatry, footwear, and angioplasty or distal bypass where appropriate. Only 13 patients were deemed ischaemic but peripheral neuropathy was a very common finding (mean VPT 24.8 +/- 12.9 V). Gangrene and major amputations showed a decrease on previous years and healing times for lesions were similar to those previously reported in diabetic patients without renal transplants. The majority of foot lesions, both in soft tissue and bone, were related to neuropathy and trauma and responded well to optimal foot care within the renal unit. Gangrene and major amputations were usually preventable. PMID- 7554789 TI - The Acropolis Affirmation Diabetes Care--St Vincent in progress. Statement from St Vincent Declaration meeting, Athens, Greece, March 1995. PMID- 7554790 TI - Random blood glucose estimation in type 2 diabetes: does it reflect overall glycaemic control? PMID- 7554791 TI - Hypoglycaemic threshold after pancreatic transplantation. PMID- 7554793 TI - Taking the intentional stance at 12 months of age. AB - This paper reports a habituation study indicating that 12-month-old infants can take the "intentional stance" in interpreting the goal-directed spatial behavior of a rational agent. First, we examine previous empirical claims suggesting that the ability to attribute intentions to others emerges during the second half of the first year. It is argued that neither the perceptual evidence (concerning the early ability to discriminate agents), nor the behavioral data (indicating the use of communicative gestures for instrumental purposes) are sufficient to support such claims about the early appearance of a theory of mind, as there are alternative explanations for these phenomena in terms of simpler psychological processes. It is then suggested that to show that an infant indeed attributes an intention to interpret the goal-directed behavior of a rational agent, one needs to demonstrate that the baby can generate an expectation about the most rational future means action that the agent will perform in a new situation to achieve its goal. We then describe a visual habituation study that meets this requirement. The results demonstrate that based on the equifinal structure of an agent's spatial behavior, 12-month-old infants can identify the agent's goal and interpret its actions causally in relation to it. Furthermore, our study indicates that infants of this age are able to evaluate the rationality of the agent's goal-directed actions, which is a necessary requirement for applying the intentional stance. In closing, we discuss some of the theoretical and methodological implications of our study. PMID- 7554792 TI - An abstract to concrete shift in the development of biological thought: the insides story. AB - For more than a century, theorists of cognitive development have embraced some form of the thesis that cognitive development proceeds from concrete to abstract knowledge. In contrast to this view, we suggest an abstract to concrete shift in the development of biological thought. In five studies we examine children's expectations for what could be inside animals and machines and we find that children of all ages respond systematically, revealing abstract expectations for how the insides of animals and machines should differ. By 8 years, children seem to have more concrete expectations for the nature of insides, and are substantially more accurate than preschoolers. More broadly, we suspect that an abstract to concrete progression may capture important features of how knowledge develops in the realm of biological thought and in many other areas of understanding as well. PMID- 7554794 TI - How heritability misleads about race. AB - The Bell Curve revives and elaborates an argument given by Jensen to the effect that facts about heritability of IQ in whites dictate that blacks are genetically inferior in IQ. But clarification of the concept of heritability shows that this reasoning is fallacious. Heritability is an uninteresting measure that only misleads us about race. PMID- 7554795 TI - The resilience of combinatorial structure at the word level: morphology in self styled gesture systems. AB - Combinatorial structure at both word and sentence levels is widely recognized as an important feature of language--one that sets it apart from other forms of communication. The purpose of these studies is to determine whether deaf children who were not exposed to an accessible model of a conventional language would nevertheless incorporate word-level combinatorial structure into their self styled communication systems. In previous work, we demonstrated that, despite their lack of conventional linguistic input, deaf children in these circumstances developed spontaneous gesture systems that were structured at the level of the sentence, with regularities identifiable across gestures in a sentence, akin to syntactic structure. The present study was undertaken to determine whether these gesture systems were structured at a second level, the level of the word or gesture--that is, were there regularities within a gesture, akin to morphological structure? Further, if intra-gesture regularities were found, how wide was the range of variability in their expression? Finally, from where did these intra gesture regularities come? Specifically, were they derived from the gestures the hearing mothers produced in their attempt to interact with their deaf children? We found that all of the deaf children produced gestures that could be characterized by paradigms of handshape and motion combinations that formed a comprehensive matrix for virtually all of the spontaneous gestures for each child. Moreover, the morphological systems that the children developed, although similar in many respects, were sufficiently different to suggest that the children had introduced relatively arbitrary distinctions into their systems. These differences could not be traced to the spontaneous gestures their hearing mothers produced, but seemed to be shaped by the early gestures that the children themselves created. These findings suggest that combinatorial structure at more than one level is so fundamental to human language that it can be reinvented by children who do not have access to a culturally shared linguistic system. Apparently, combinatorial structure of this sort is not maintained as a universal property of language solely by historical tradition, but also by its centrality to the structure and function of language. PMID- 7554796 TI - Through a narrow window: working memory capacity and the detection of covariation. AB - The samples of data that people use in their attempts to detect relationships in the environment are limited in size by working memory capacity. The implications of that constraint are explored by analyzing the sampling distribution of the most common measure of relationship--the product moment correlation (Pearson's rxy). This distribution is skewed when the population correlation differs from zero (i.e., when a correlation exists), and the more so, the smaller the sample. As both the median and the mode of the sampling distribution are more extreme than the population value, it follows that samples likely to be encountered indicate a correlation stronger than that in the population. Thus, the limited capacity of working memory may serve as an amplifier that helps people to avoid missing strong relationships. As the distribution is more skewed the smaller the sample size, the effect suggests an explanation for the fact that young children detect meaningful covariation fairly rapidly. PMID- 7554797 TI - The acquisition of the English past tense in children and multilayered connectionist networks. AB - The apparent very close similarity between the learning of the past tense by Adam and the Plunkett and Marchman model is exaggerated by several misleading comparisons--including arbitrary, unexplained changes in how graphs were plotted. The model's development differs from Adam's in three important ways: Children show a U-shaped sequence of development which does not depend on abrupt changes in input; U-shaped development in the simulation occurs only after an abrupt change in training regimen. Children overregularize vowel-change verbs more than no-change verbs; the simulation overregularizes vowel-change verbs less often than no-change verbs. Children, including Adam, overregularize more than they irregularize; the simulation overregularized less than it irregularized. Interestingly, the RM model--widely criticized as being inadequate--does somewhat better, correctly overregularizing vowel-change verbs more often than no-change verbs, and overregularizing more often than it irregularizes. Although Plunkett and Marchman's (1993) state of the art model incorporated hidden layers and back propagation, used a more realistic phonological coding scheme, and explored a broader range of parameters than Rumelhart and McClelland's model, their results are farther from psychological reality. It is unknown whether any connectionist model can mimic a child's performance without resorting to unrealistic exogenous changes in the training or input, but it is clear that adding a hidden-layer and back-propagation does not ensure a solution. PMID- 7554798 TI - Automation of polymerase chain reaction tests. Reduction of human errors leading to contamination. AB - We compared the performance over 21 months of manually performed polymerase chain reaction (PCR)-based DNA analysis experiments with 25 months of automated PCR performed by a Zymark robotic system. Automation of the PCR technique resulted in a sixfold reduction in the number of experiments reporting carryover contamination and decreased the overall rate of contamination among total reactions 68-fold. Whereas contamination occurrences among manual experiments were evenly dispersed over the study interval and correlated with the lack of experience of laboratory personnel, the contamination that occurred with the robotic system was confined to the first 10 months of operation. In manual experiments, many of the 81 no-target false positives were sufficiently strong to result in the invalidation of 151 samples and positive controls. The seven no target control false positives in the automated system were weak bands that were easily subtracted as background. Because none of the negative samples had DNA bands, no sample on the automated system has ever been invalidated as a result of contamination. Automation of PCR tests appears to offer great promise in reducing contamination to acceptable levels (e.g., < or = 0.1%). PMID- 7554799 TI - Minimum bactericidal concentrations of Propionibacterium acnes isolates from cases of chronic endophthalmitis. AB - Six isolates of Propionibacterium acnes recovered from cases of chronic infectious endophthalmitis following extracapsular cataract extraction were tested for antibiotic susceptibility. All isolates were susceptible to penicillin, cefazolin, and vancomycin with a macrobroth dilution method. Minimum bactericidal concentrations testing at 72 h revealed that six of six isolates were killed by < or = 1.0 micrograms of vancomycin/ml, one of six isolates by < or = 1.0 micrograms of penicillin/ml, and zero of six isolates by < or = 1.0 micrograms cefazolin/ml. PMID- 7554800 TI - Evidence of nosocomial spread of Candida albicans causing bloodstream infection in a neonatal intensive care unit. AB - Candida albicans is an increasingly important bloodstream pathogen. We investigated a cluster of bloodstream infections in the neonatal intensive care unit (NICU) to determine whether nosocomial transmission occurred. Subjects included any patient in the NICU who developed clinically significant bloodstream infection with C. albicans from January 1984 to December 1987 (N = 7). Isolates were typed by restriction fragment length polymorphism analysis using a C. albicans-specific DNA probe (27A). Four of the neonates were infected from June to August 1984 (1.4 infections per 100 admissions) (the epidemic period) versus none in the period from January to May 1984, and three in the period from September 1984 to December 1987 (0.12 infections per 100 admissions) (P = .002). Three of the four patients in the epidemic period were infected with identical strains, readily distinguished from epidemiologically unrelated strains from the NICU. We conclude that nosocomial transmission of C. albicans occurred and that neonates in intensive care units may represent one group at increased risk. PMID- 7554801 TI - Laboratory and clinical evaluation of Mycobacterium xenopi isolates. AB - Mycobacterium xenopi and Mycobacterium avium complex (MAC) are biochemically similar. To define the laboratory characteristics of M. xenopi that distinguish it from MAC, 53 M. xenopi isolates from different areas in the United States and 47 isolates recovered at one hospital were evaluated by 13 biochemical tests, AccuProbe MAC (Gen-Probe, Inc., San Diego, CA, USA), colony morphology, formation of X-colonies, pigmentation in response to light, growth on MacConkey agar without crystal violet, and relative growth rates at 25 degrees C, 36 degrees C, and 45 degrees C on solid media. Relative growth rates of 10 M. xenopi and 11 MAC isolates were measured at 25 degrees C, 36 degrees C, and 42 degrees C in Middlebrook broth processed using the BACTEC TB System. Ten M. xenopi were tested for p-nitro-alpha-acetylamino-beta-hydroxypropiophenone inhibition at 36 degrees C and 42 degrees C. Reevaluation of 81 isolates previously identified as MAC by biochemical tests alone revealed that two were M. xenopi. The most reliable characteristics distinguishing M. xenopi from MAC were the presence of X-colonies (M. xenopi 97% vs MAC 1%), positive 3-day arylsulfatase (M. xenopi 88% vs MAC 1%), growth at 25 degrees C (M. xenopi 0% vs MAC 100%), and AccuProbe MAC test results (M. xenopi 0% hybridized). Retrospective chart review of 37 patients using American Thoracic Society criteria revealed that six (16%) patients had clinically important isolates. At one of our hospitals M. xenopi was the second most common mycobacterial species isolated for 1990-1992, accounting for 27% of all isolates, whereas at our other hospital it accounted for 1% of isolates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554802 TI - Specimen type as a source of variability in the reproducibility and timing of cytomegalovirus identification by culture. AB - Cytomegalovirus (CMV) is a significant contributor to morbidity and mortality among immunocompromised individuals; therefore, rapid and accurate diagnosis is essential. We compared positive cultures (n = 147) from different specimen types as to (a) the incubation time to a positive result and (b) the reproducibility of positive findings in replicate cultures. Five replicate shell vials were inoculated from each specimen: Two vials were stained at 24 h, two at 48 h, and one held and observed for a maximum of 30 days. Positive cultures from tissue biopsy specimens required the shortest incubation (mean = 1.9 days) and urine specimens the longest (mean = 3.9 days) (P < .005). Tissue biopsy specimens were the most reproducible (48.4% of specimens were positive in five of five replicates) and urine specimens the least (no specimens were positive in five of five replicate vials) (P < = .0002). The observed interspecimen variability is important because failure to understand and adjust for these differences could negatively influence the ability to identify CMV in culture. PMID- 7554803 TI - Modifications for disk diffusion susceptibility testing criteria of clinafloxacin (CI-960, AM-1091, PD127371) and fleroxacin (Ro 23-6240, AM-833) following studies with a challenge panel of ciprofloxacin-resistant clinical isolates. AB - A collection of ciprofloxacin-resistant organisms (200 strains) was developed among 300 total test strains to "challenge" the initial interpretive criteria developed for clinafloxacin (formerly CI-960), fleroxacin (formerly Ro 23-6240, AM-833), and ciprofloxacin. Generated results indicate the necessity for modified criteria for disk diffusion tests (5-micrograms disks) as follows: for clinafloxacin, susceptible at > or = 21 mm ( < or = 1 microgram/ml) and resistant at < or = 17 mm ( > or = 4 micrograms/ml), or susceptible at > or = 19 mm ( < or = 2 micrograms/liter) and resistant at < or = 15 mm ( > or = 8 micrograms/ml); for fleroxacin, susceptible at > or = 16 mm ( < or = 2 micrograms/ml) and resistant at < or = 11 mm ( > or = 8 micrograms/ml); and for ciprofloxacin, resistant at < or = 17 mm > or = 4 micrograms/ml). These modifications maximize total absolute interpretive accuracy between the standardized test methods, especially minimizing the potential for false susceptibility (very major errors) when testing truly resistant isolates. The clinafloxacin spectrum was widest against these resistant isolates, and the other two tested fluoroquinolones (ciprofloxacin, fleroxacin) possessed comparable overall spectrums of activity, although ciprofloxacin was generally more active on a weight basis. We urge the rapid acceptance of these criteria to improve the accuracy of the widely used National Committee for Clinical Laboratory Standards method for disk diffusion tests. PMID- 7554804 TI - Moraxella septicemia in children with diarrheal disease. AB - We report 10 cases of Moraxella septicemia associated with diarrheal disease. Their clinical presentations and outcomes are discussed. Recognition of the pathogenicity of these microorganisms in appropriate clinical setting should result in prompt and specific therapy. PMID- 7554805 TI - Fatal endocarditis due to Aerococcus urinae. AB - Aerococcus species are Gram-positive cocci having Gram-stain characteristics of staphylococci (i.e., form clusters), but biochemical and growth characteristics resembling streptococci and enterococci (i.e., catalase-negative, alpha-hemolytic growth on blood agar). A case of fatal endocarditis due to Aerococcus urinae is presented. Endocarditis should be suspected and, if confirmed, treated aggressively when A. urinae is recovered from blood cultures. PMID- 7554806 TI - A survey of beta-lactamase-producing Haemophilus influenzae. An evaluation of 5750 isolates. AB - Previous studies have reported that 15%-34% of Haemophilus influenzae produce beta-lactamase. A surveillance program was developed by SmithKline Beecham Clinical Laboratories to determine the current percentage of beta-lactamase producing H. influenzae from five selected geographic locations in the United States. In 1993, results of 5750 isolates from specimens submitted to five reference clinical laboratories were evaluated. Data were collected from 29 states and the District of Columbia. The percentages of beta-lactamase-producing H. influenzae was 33% and ranged from 22%-40% for the individual states. PMID- 7554807 TI - Reevaluations of disk diffusion susceptibility testing interpretive criteria for lomefloxacin and norfloxacin using fluoroquinolone-resistant isolates. AB - The current National Committee for Clinical Laboratory Standards interpretive criteria for disk diffusion susceptibility testing of lomefloxacin and norfloxacin were reevaluated using a test panel of 298 bacteria (200 with ciprofloxacin minimum inhibitory concentrations (MICs) at > or = 4 micrograms/ml). MICs were correlated with the diameter of zones of inhibition by regression statistics and error-rate bounding methods. Modifications of the interpretive criteria for lomefloxacin disk susceptibility testing are proposed as follows: susceptible at > or = 20 mm and resistant at < or = 16 mm, a 2-mm decrease of existing break-point zones. These criteria result in an absolute interpretive agreement of 95.3% with a very major (false susceptible) error rate of only 0.7%. The currently used criteria for norfloxacin (susceptible at > or = 16 mm and resistant at < or = 13 mm) were validated, and these break-points had an absolute interpretive correlation between methods of 91.9%. The change proposed for lomefloxacin disk interpretations would minimize minor and major errors most often reported for Staphylococcus saprophyticus isolates. PMID- 7554808 TI - Management of congestive heart failure in the elderly patient. AB - Coronary artery disease, hypertension, valvular heart disease, and cardiomyopathies are the commonest causes of CHF in elderly patients. Almost half of elderly patients with CHF have normal LV ejection fraction. LV ejection fraction must be measured before knowing how to treat CHF. Underlying causes of CHF should be treated when possible. Precipitating causes of CHF should be treated. Diuretics are the first-line drug in the treatment of CHF. ACE inhibitors reduce mortality in patients with CHF and should be administered with diuretics to patients with CHF and abnormal or normal LV ejection fraction. Oral isosorbide dinitrate plus hydralazine improves survival in patients with CHF, and should be administered to patients with CHF and abnormal or normal LV ejection fraction who cannot tolerate ACE inhibitor therapy or in whom CHF persists despite therapy with diuretics plus ACE inhibitors. Digoxin should be administered to patients with a rapid ventricular rate associated with supraventricular tachyarrhythmias. Digoxin should be administered to patients with CHF in sinus rhythm and abnormal LV ejection fraction that does not respond to diuretics plus ACE inhibitors or in patients unable to tolerate ACE inhibitors or other vasodilator therapy. Digoxin should not be given to patients with CHF in sinus rhythm with normal LV ejection fraction. Calcium channel blockers are contraindicated in patients with CHF and abnormal LV ejection fraction but may be used to treat CHF with normal LV ejection fraction. The use of beta blockers in the treatment of CHF with abnormal LV ejection fraction is experimental. However, beta blockers might improve clinical symptoms in patients with CHF and normal LV ejection fraction by slowing heart rate, thereby increasing LV diastolic filling time and increasing LV end-diastolic volume. Maintenance of sinus rhythm will also increase LV diastolic filling time in patients with CHF and normal LV ejection fraction. PMID- 7554809 TI - Abdominal aortic aneurysm. PMID- 7554810 TI - Antithrombotic therapy. AB - Antithrombotic therapy has evolved from initially limited available agents into numerous treatment options. Thrombin inhibitors have major therapeutic uses in a myriad of thromboembolic disorders. Standard indirect inhibitors, such as heparin and warfarin, have been troublesome clinically owing principally to nonhemostatic side effects and to relative nonselectivity. These drug options may soon be supplemented and possibly supplanted by more selective, direct inhibitors with a finely honed, exclusive effect on thrombin. This approach may, in turn, provide superior, safer therapies for prevention and propagation of thrombus in many cardiovascular diseases. PMID- 7554811 TI - Long term, implantable ventricular assist devices: what are they and who needs them? PMID- 7554812 TI - Management of transient ischemic attacks. PMID- 7554813 TI - Recognition of mechanical complications of acute myocardial infarction. PMID- 7554814 TI - Inotropic therapy for systolic heart failure. PMID- 7554815 TI - Pericardial effusion and tamponade: evaluation, imaging modalities, and management. AB - Pericardial effusions may be present in a variety of clinical situations, often presenting challenging clinical diagnostic and therapeutic problems. Although several imaging modalities are available, ECHO has become the diagnostic method of choice due to its portability and wide availability. CT and MRI may also be employed and may be more accurate. A pericardial effusion under pressure may result in hemodynamic compromise and tamponade. Although there are several echocardiographic clues to tamponade (including diastolic chamber collapse, Doppler flow velocity paradoxus, and inferior vena cava phlethora), the diagnosis remains a clinical and hemodynamic one. The clinical signs include elevated jugular venous pressure, hypotension, tachycardia, and pulsus paradoxus. Hemodynamic measurements include equalization of diastolic pressures and decreased cardiac output Treatment of tamponade involves drainage of the effusion and prevention of reaccumulation. Needle pericardiocentesis via the subxiphoid approach is a reasonable initial treatment. However, this may need to be accompanied by catheter drainage or surgical pericardial window. A new catheter based technique--percutaneous balloon pericardiotomy-- appears useful in select patients with malignancy in order to avoid more invasive surgical procedures. Occasionally, in patients with recurrent effusions, instillation of sclerosing agents into the pericardial space or even total pericardiectomy may be necessary. PMID- 7554816 TI - Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4. AB - A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol. 66, 1889-1900 (1985); Sabara et al., J. Virol. 53, 58-66 (1985); Sabara et al., J. Gen. Virol. 67, 201-212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed. PMID- 7554817 TI - Novel glycolipids of Mycobacterium avium and related M. paratuberculosis strains of relevance to AIDS and Crohn's disease. AB - The polar glycolipid fractions of several mycobacterial strains of the closely related species M. avium and M. paratuberculosis have been analysed by thin layer chromatography (TLC), high pH anion exchange chromatography (HPAEC), gas-liquid chromatography (GC) and nuclear magnetic resonance (NMR) spectroscopy. The upper phase of a Folch partitioning (rather than the lower phase analysed by others) was subjected to TLC in solvent system chloroform-methanol-water 50:40:10 v/v/v. A major band was purified from each mycobacterial strain. Monosaccharide analysis of that from M. avium A14 (from an AIDS patient) contained Glc, Ara, Man, Gal in ratios 7:4:3:2. whereas one strain of M. paratuberculosis (316F) had low levels of Ara, Gal and Man with major monosaccharides being Glc and two unidentified monosaccharides. A second M. paratuberculosis strain (J10) had a single TLC band containing only Glc. These known strains were compared to two slow growing mycobacterial isolates, one from a Crohn's patient and one isolated from armadillo. These were similar to J10 in only having Glc present: the former also had a similar NMR spectrum to J10, whereas the latter had a different NMR spectrum from any of the other strains analysed. The results therefore indicate that M. paratuberculosis strain 316F is more closely related to M. avium (from an AIDS patient) than it is to the classical M. paratuberculosis strain J10 and a Crohn's isolate. PMID- 7554818 TI - Suppression of transplant immunity in experimental trichinellosis. AB - Skin allograft rejection in Balb/c and C57BL/6J mice following experimental infection with 300 larvae of Trichinella spiralis or Trichinella pseudospiralis was studied. Skin grafts from normal C57BL/6J mice were transplanted to infected Balb/c mice and vice versa at days 3, 10, 20 and 30 post-infection. The clinical criteria for graft rejection, scarring and graft falling, were followed. The results indicated that T. spiralis and T. pseudospiralis infections induced a significant delay in graft rejection when compared to the control groups. A maximum rejection time of 24 days was observed in T. spiralis infected C57BL/6J mice which received skin grafts from Balb/c mice on day 3 post-infection. The rejection in the uninfected control group was on day 7 post transplant. The mean rejection times for transplants on various days post-infection, with both species were very similar. Also, the rejection profiles in Balb/c mice were comparable to that observed in C57BL/6J mice, with a maximum delay of 26 days to rejection again obtained in mice transplanted on day 3 post-infection, for both species. When the skin grafts were performed 5 or 10 days prior to infection, the rejection occurred on day 7, as in the control group. The effect of T. spiralis and T. pseudospiralis soluble larval extracts (TSE or TPE) on graft rejection was also examined. Four intraperitoneal injections of 50 micrograms each of TSE or TPE every 48 h for 7 days did not induce any significant delay in graft rejection. In contrast, secretory antigens prepared from cultured larvae in vitro induced significant delays in graft rejection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554820 TI - Antigenic heterogeneity of avian paramyxoviruses of serotype 2 ("Yucaipa-like") isolated from domestic and wild birds in Israel. AB - Thirty-three viruses of PMV-2 serotype isolated in Israel from domestic and wild birds during epizooty of a respiratory disease in 1979-1981 were studied comprehensively in comparison with a set of reference PMV-2 viruses using cross reaction hemagglutination inhibition tests, elution-hemagglutination pattern and pattern of migration of viral proteins in polyacrylamide gel electrophoresis. The results demonstrated considerable heterogeneity amongst the local isolates by the above three criteria which were not correlated with each other. There was no significant correlation between the differences found and chronology of isolation of the viruses. The results indicated that there may have been simultaneous co circulation of different PMV-2 strains in the local avian reservoir at the beginning of the epizootic rather than temporal accumulation of variants as a result of antigenic drift of an "ancestor" virus which caused the initial infection. PMID- 7554821 TI - Influence of the temperature upon the proliferative response of lymphocytes of tench (Tinca tinca) during winter and summer. AB - The influence of incubation temperature upon proliferation of Tinca tinca lymphocytes induced by phytohemagglutinin (PHA) and E. coli lipopolisaccharide (LPS) mitogens was studied during the summer and the winter. The cultures were performed in vitro at 22 degrees C in both summer and winter, and at 12 and 30 degrees C in winter and summer respectively. The proliferative response at 22 degrees C was higher than that at 12 degrees C during the winter, and a small increase was observed at 30 degrees C respective to 22 degrees C in summer. These results indicate that in vitro lymphocyte proliferation requires temperatures higher than those in the fish environment. PMID- 7554819 TI - Virulence factors associated with strains of Escherichia coli from cases of sudden infant death syndrome (SIDS). AB - Strains of Escherichia coli isolated from cases of Sudden Infant Death Syndrome, healthy infants and infants that died of other causes were subjected to a series of tests with particular reference to serotyping, toxigenicity and adherence factors. E. coli from SIDS infants tended to have a low hydrophobicity and high toxigenicity, compared to those from healthy infants, while no notable differences in haemagglutination patterns were observed between these two groups of strains. PMID- 7554822 TI - Evaluation of different serological tests for detection of antibodies against Serpulina hyodysenteriae in pig sera. AB - Swine dysentery is a mucohemorrhagic diarrheal disease caused by S. hyodysenteriae. The detection of asymptomatic carriers in herds is possible by serological tests. However, cross-reactions between S. hyodysenteriae and S. innocens pose a major problem in serological diagnosis. Several serological tests were evaluated for detection of antibodies to S. hyodysenteriae such as: indirect hemagglutination, passive hemolysis, conglutination and microagglutination tests. Among the tests used, only the microagglutination test was able to detect antibodies to S. hyodysenteriae. 70 to 95% of the pigs were invariably seropositive in a single dilution of 1:10 in actively infected herds whereas the number of seropositives did not exceed 10% in presumably non-infected herds. The test was found to be simple, and reliable to be used with confidence for detection of herd infection using boiled cell suspension as an antigen. PMID- 7554823 TI - Convergence of untutored song in group-reared zebra finches (Taeniopygia guttata). AB - Zebra finches develop abnormal song if they cannot interact with adult song tutors during the 1st few months after hatching. The authors investigated whether untutored juveniles would learn song from each other. The birds were isolated from adult males at 10 days of age and their songs recorded after Day 80. By the authors' measures of syllable sharing and the judgments of human listeners, the songs of untutored brothers were as similar to each other as those of birds reared together with a tutor. The songs of group untutored birds were, however, more variable, and they contained abnormal elements, as did the songs of birds reared apart from all other males. The fact that untutored brothers copied song from each other raises the possibility that juveniles might influence each other's song development, even when adult tutors are present. If this were the case, it would increase the range of social interactions that determine which songs juveniles learn. PMID- 7554824 TI - Orientation invariant pattern recognition by pigeons (Columba livia) and humans (Homo sapiens). AB - The orientation invariance of visual pattern recognition in pigeons and humans was studied using a conditioned matching-to-sample procedure. A rotation effect, a lengthening of choice latencies with increasing angular disparities between sample and comparison stimuli, was replicated with humans. The choice speed and accuracy of pigeons was not affected by orientation disparities. Novel mirror image stimuli, rotation of sample shapes, a delayed display of comparison shapes, and a mixed use of original and reflected sample shapes did not lead to a rotation effect in pigeons. With arbitrarily different odd comparison shapes, neither humans nor pigeons showed a rotation effect. Final experiments supported the possibility that the complete absence of a rotation effect in pigeons is because they are relatively better than humans at discriminating mirror-image shapes compared with arbitrary shapes. PMID- 7554825 TI - Hand preferences for a coordinated bimanual task in 110 chimpanzees (Pan troglodytes): cross-sectional analysis. AB - Hand preference for a coordinated bimanual task was assessed in a sample of 110 chimpanzees (Pan troglodytes). Subjects were presented with opaque tubes, the inside of which was coated with peanut butter. The hand and finger used to extract the peanut butter was recorded in 2 test sessions. A population right hand bias was found. Juvenile and adolescent subjects were less lateralized than adults. All subjects primarily used their index finger to extract peanut butter. Use of the index finger was higher in adults and adolescents compared with juveniles. The results suggest that coordinated bimanual tasks (a) elicit strong hand preferences at the individual level and (b) elicit population level right handedness. PMID- 7554826 TI - Manual laterality in chimpanzees (Pan troglodytes) in complex tasks. AB - To determine the manual laterality of a sample of 24 chimpanzees, 4 problem apparatuses were used, the solution for which (obtaining a food item) required the use of 1 or both hands in sequential, simultaneous, or both sequential and simultaneous actions. The majority of the subjects showed significant and consistent hand preferences, especially in the actions that required a precision grip. The results obtained suggest the existence of factors linked to the specific characteristics of the task to be performed and to the ontogenetic maturation of the subject, which would influence the directionality and degree of the hand preference displayed. PMID- 7554827 TI - Use of social information in the problem solving of orangutans (Pongo pygmaeus) and human children (Homo sapiens). AB - Fourteen juvenile and adult orangutans and 24 3- and 4-year-old children participated in 4 studies on imitative learning in a problem-solving situation. In all studies a simple to operate apparatus was used, but its internal mechanism was hidden from subjects to prevent individual learning. In the 1st study, orangutans observed a human demonstrator perform 1 of 4 actions on the apparatus and obtain a reward; they subsequently showed no signs of imitative learning. Similar results were obtained in a 2nd study in which orangutan demonstrators were used. Similar results were also obtained in a 3rd study in which a human encouraged imitation from an orangutan that had previously been taught to mimic arbitrary human actions. In a 4th study, human 3- and 4-year-old children learned the task by means of imitation. PMID- 7554829 TI - Ethical issues in the application of virtual reality to medicine. AB - Virtual reality (VR) will be increasingly used as a research tool in medicine. Examples include the computer modelling of complex pathological regulatory systems in disease; the integration of massive data bases derived from structural imaging of diseased organs and simultaneous functional mapping of the same organ; the development of neural prosthetic devices in sensory and/or mental impairment and the assessment or modification of major behavioural abnormalities in selected individuals. In time, advances in VR will lead to increasing pressure to introduce patients to a virtual reality environment and it is here that ethical problems will most frequently be encountered. Early examples would include the monitoring of anaesthesia and intensive care procedures but may extend to the support and enrichment of life of those patients who suffer chronic disabilities. In the setting of virtual reality, they may be able to experience much improved educational, recreational or pleasurable pursuits. Whilst the ultimate goal of improvement of the human condition will be cited to justify any human costs encountered in the development, testing and introduction of VR machines to patient care, pertinent ethical problems must simultaneously be addressed. These include the possibility that some researchers place their own advancement above the interests of a particular patient. Some other researchers may be unduly paternalistic especially when making decisions about patients who because of mental impairment or illness are unable to give their informed consent. Potentially, VR machines may be prone to errors such that they introduce into the mental life of susceptible individuals specific distortions that serve to exacerbate the symptoms of mental illness or induce such symptoms when none were previously present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554828 TI - Prenatal auditory experience directs species-typical perceptual responsiveness in bobwhite quail chicks (Colinus virginianus). AB - The present study examined the role of the relationship between pre- and postnatal stimulus cues in directing perceptual preferences of bobwhite quail chicks. Bobwhite quail were exposed to either prenatal auditory or prenatal auditory-visual stimulation. Both prenatal auditory and auditory-visual stimulation resulted in accelerated responsiveness to combined postnatal auditory visual cues. However, whether or not subjects displayed accelerated perceptual responsiveness depended on the relationship between the nature of the pre- and postnatal auditory stimuli used. These results demonstrate the salience of auditory cues in directing early postnatal perceptual behavior in precocial birds and help to explicate why prior research in this area has found that augmented prenatal sensory stimulation can result in either accelerated or decelerated patterns of perceptual functioning. PMID- 7554830 TI - An inventory of computer resources for the medical application of virtual reality. AB - Within the framework of the scientific community, we could define virtual reality (VR) as an effective simulation of complex environmental aspects related to both interaction-dependent and high-risk operations, where mistakes will lead to unacceptable consequences. Surgeons performing open surgery, endoscopists working on the intestine or neurologists working on the brain-all are impressive examples where the VR approach looks potentially quite interesting. In fact the risk of unsatisfactory implementations, too poor with respect to the complexity of the real world, coming from the unsatisfactory performance of the present-day technology, is quite high. Typically, the loss of the real time requirement for the operator interactions can remove much of the effectiveness of what has been built. Some questions about the real need for high performance computational resources and high level graphic resolution need to be answered. This paper presents and discusses an inventory of computational resources used in real applications. PMID- 7554831 TI - Virtual reality in surgical education. AB - Virtual reality (VR) is an emerging technology that can teach surgeons new procedures and can determine their level of competence before they operate on patients. Also VR allows the trainee to return to the same procedure or task several times later as a refresher course. Laparoscopic surgery is a new operative technique which requires the surgeon to observe the operation on a video-monitor and requires the acquisition of new skills. VR simulation could duplicate the operative field and thereby enhance training and reduce the need for expensive animal training models. Our preliminary experience has shown that we have the technology to model tissues and laparoscopic instruments and to develop in real time a VR learning environment for surgeons. Another basic need is to measure competence. Surgical training is an apprenticeship requiring close supervision and 5-7 years of training. Technical competence is judged by the mentor and has always been subjective. If VR surgical simulators are to play an important role in the future, quantitative measurement of competence would have to be part of the system. Because surgical competence is "vague" and is characterized by such terms as "too long, too short" or "too close, too far," it is possible that the principles of fuzzy logic could be used to measure competence in a VR surgical simulator. Because a surgical procedure consists of a series of tasks and each task is a series of steps, we will plan to create two important tasks in a VR simulator and validate their use. These tasks consist of laparoscopic knot tying and laparoscopic suturing. Our hypothesis is that VR in combination with fuzzy logic can educate surgeons and determine when they are competent to perform these procedures on patients. PMID- 7554832 TI - Endoscopic surgery simulation in a virtual environment. AB - The minimally invasive nature of endoscopic surgery allows operations to be performed on patients through small incisions, often under local anaesthesia. Patient recovery times and cosmetic detriment are thus greatly reduced, while overall quality of care is improved. Presently, surgeons are trained to perform endosurgical procedures in a number of ways: practising with surgical training devices, using animal models and assisting experienced surgeons. In this paper, the focus is on answering the key question: "Can virtual environment technology assist surgeons in training and maintaining endoscopic surgery skills?" Initial developments towards surgical simulators have clearly demonstrated the great potential of virtual environment technology for surgical training purposes. Breakthroughs towards surgery are expected within the next 5 to 10 years. PMID- 7554833 TI - Building a hybrid patient's model for augmented reality in surgery: a registration problem. AB - In the field of Augmented Reality in Surgery, building a hybrid patient's model, i.e. merging all the data and systems available for a given application, is a difficult but crucial technical problem. The purpose is to merge all the data that constitute the patient model with the reality of the surgery, i.e. the surgical tools and feedback devices. In this paper, we first develop this concept, we show that this construction comes to a problem of registration between various sensor data, and we detail a general framework of registration. The state of the art in this domain is presented. Finally, we show results that we have obtained using a method which is based on the use of anatomical reference surfaces. We show that in many clinical cases, registration is only possible through the use of internal patient structures. PMID- 7554834 TI - Computer generated visual assistance during retroperitoneoscopy. AB - In this paper we present a system of computer assisted surgery, the function of which is to enhance the real time feedback between the patient's body, the surgical tools and the surgeon. We show using such a system during some surgical operations--in our case, retroperitoneoscopy. After giving the requirements for such a system, we describe the components currently used in our experiments. We detail the different problems and our current solutions; photographs of an experiment carried out on a cadaver and of classical operations are shown in this paper. PMID- 7554835 TI - Ophthalmic microsurgical robot and associated virtual environment. AB - An ophthalmic virtual environment has been developed as part of a teleoperated microsurgical robot built to perform surgery on the eye. The virtual environment is unique in that it incorporates a detailed continuum model of the anatomical structures of the eye, its mechanics and optical properties, together with a less detailed geometric-mechanical model of the face. In addition to providing a realistic visual display of the eye being operated on, the virtual environment simulates tissue properties during manipulation and cutting and the forces involved are determined by solving a mechanical finite element model of the tissue. These forces are then fed back to the operator via a force reflecting master and so the surgeon can experience both the visual and mechanical sensations associated with performing surgery. The virtual environment can be used to enhance the images produced by the camera on the microsurgical slave robot during surgery and as a surgical simulator in which it replaces these images with computer graphics generated from the eye model. PMID- 7554836 TI - Biomechanics for preoperative planning and surgical simulations in orthopaedics. AB - Surgical simulations are particularly appropriate for the large volume and expense of joint replacement procedures in orthopaedics. A first generation surgical simulator has been developed to model the implantation procedure for cementless acetabular and femoral components in total hip replacement surgery. The simulator is based upon finite element analysis and predicts the early postoperative mechanical environment that results from a proposed surgery. Since the short- and long-term clinical success of cementless hip replacement components is very dependent upon the initial mechanics of the bone-implant system, such simulations can help orthopaedic surgeons to develop better preoperative plans. PMID- 7554837 TI - Virtual reality arthroscopy training simulator. AB - This paper describes the result of the interdisciplinary cooperation of traumatologists of the Berufsgenossenschaftliche Unfallklinik (BGU) in Frankfurt am Main and a team of computer graphics scientists of the Franunhofer Institute for Computer Graphics in Darmstadt. We have developed a highly interactive training simulator system by means of computer graphics and virtual reality techniques. PMID- 7554838 TI - Virtual reality for physically disabled people. AB - This paper demonstrates how physically disabled people can benefit from the innovative virtual reality techniques. Several specific examples show the applicability of virtual reality to the therapy and rehabilitation of people with various disabilities. In addition, the paper describes how physicians can use virtual reality as an advanced visualization tool for the diagnosis of physical disabilities. Finally, possible display techniques and input devices for diagnosis and rehabilitation purposes are discussed briefly. PMID- 7554839 TI - Evaluation and retraining of adults' cognitive impairment: which role for virtual reality technology? AB - Immersive virtual reality (IVR) is a technology already developed to assist cognitive psychologists and therapists in their clinical work with brain-damaged patients. The rationale, the software and the hardware of the first application (ARCANA 1) based on affordable technology are discussed here, in order to provide a concrete example of what the authors think may be the role of IVR as a clinical tool. Although prospects are exciting, extensive research is needed to validate this new approach and reveal its limitations and advantages. PMID- 7554841 TI - Three-dimensional video imaging for endoscopic surgery. AB - Recently a number of manufacturers have developed three-dimensional (3-D) video systems which significantly improve visualization and enhance the ability of the surgeon to perform delicate endoscopic dissection and suturing. These 3-D video systems may also improve the education of surgeons-in-training as they would have a better understanding of three-dimensional anatomy during laparoscopic surgery. Most of the 3-dimensional stereoendoscopic video systems currently available have four basic principles of stereoendoscopic image processing in common: image capture, conversion of 60-120 Hz images; presentation of left and right images on a single monitor; and separation of the left and right eye images. Skill tests performed assessing laparoscopic suturing and knot tying have demonstrated a 25% increase in speed and accuracy of these laparoscopic tasks when utilizing a three dimensional video imaging greatly facilitates the efficiency of endoscopic reconstructive procedures. While three-dimensional video imaging systems are more costly than conventional two-dimensional video equipment, the enhanced depth perception produced by 3-D endoscopes has been demonstrated to improve the performance of minimally invasive surgical procedures. PMID- 7554840 TI - Virtual reality and telepresence for military medicine. AB - The profound changes brought about by technology in the past few decades are leading to a total revolution in medicine. The advanced technologies of telepresence and virtual reality are but two of the manifestations emerging from our new information age; now all of medicine can be empowered because of this digital technology. The leading edge is on the digital battlefield, where an entire new concept in military medicine is evolving. Using remote sensors, intelligent systems, telepresence surgery and virtual reality surgical simulations, combat casualty care is prepared for the 21st century. PMID- 7554842 TI - Vision-based object registration for real-time image overlay. AB - This paper presents a computer vision-based technique for object registration, real-time tracking, and image overlay. The capability can be used to superimpose registered medical images such as those from CT or MRI on to a video image of a patient's body. Real-time object registration enables an image to be overlaid consistently on to objects even while the objects and cameras viewing it are moving. Object registration is composed of feature tracking, feature correspondence, and pose calculation of objects. This technique is based on geometric models of objects, but it can be extended so that some image overlay is possible without a prior model of the object. PMID- 7554843 TI - Ray casting and the enclosing-net algorithm for extracting shapes from volume data. AB - One of the most useful applications of virtual reality is to let doctors view the inside of the human body non-invasively and in real time. In this paper, we first survey the area of virtual reality and volume-visualization techniques. We discuss our ray casting implementation for viewing the medical data which is available as a set of slices from NMR and CT scans. Next we present a new method, the enclosing-net algorithm, for extracting iso-surfaces from volume data. A simple implementation of our technique is described. Since the topology of the extracted surface is well defined and non-ambiguous, the enclosing-net algorithm eliminates a major problem of surface extraction techniques. In addition, the number of polygons representing the surface can be controlled to obtain a finer shape. Therefore real-time interaction is feasible on both low-end and high-end graphics machines, making the enclosing-net algorithm suitable for virtual reality experiments. PMID- 7554844 TI - 3D-high resolution imaging of tumors and aneurysms at the cranial base-comparison of CT and MR. PMID- 7554845 TI - Real-time interactions in MRI. AB - With the advent of magnetic resonance fluoroscopic imaging, it is possible to obtain images at a rate which can be considered effectively real-time. Image refresh rates can be as low as 0.5 s. Such rates are fast enough to allow MR to be used for guidance of surgical instruments during micro-surgical procedures. Examples include the localization of biopsy needles within a tumor, localization of surgical tools for application of therapeutic drugs or the proper positioning of optical fibers for application of laser heat. It may also be used for long duration monitoring of a patient in an attempt to diagnose rare and short duration clinical events. PMID- 7554846 TI - The responsive workbench: a virtual working environment for physicians. AB - The paper describes the concept of the Responsive Workbench (RW). This virtual environment was designed to support end users working on desks, workbenches, and tables as physicians, architects and scientists with an adequate human-machine interface. We attempt to construct a task-driven interface for this class of users by working in an interdisciplinary team from the beginning. The system is explained and evaluated along three medical applications: medical education, a cardiological tutorial with a simulation system for ultrasonographic examinations of the heart and surgery planning. Virtual objects are located on a real "workbench". The objects, displayed as computer generated stereoscopic images are projected onto the surface of a table. The participants operate within a non immersive virtual environment. A "guide" uses the virtual environment while several observers can also watch events by using shutter glasses. Depending on the application, various input and output modules can be integrated, such as motion, gesture and speech recognition systems which characterize the general trend away from the classical multimedia desktop interface. The RW is compared with other common virtual reality systems as head mounted displays. First experiences of the collaborators are drawn and future enhancements are proposed. PMID- 7554847 TI - The rural and global medical informatics consortium and network for radiology services. AB - Telemedicine systems that provide health delivery services to rural clinics and hospitals are being demonstrated in clinical settings. This paper summarizes the research and consortia projects at the University of Arizona Medical Center. The paper describes the Rural and Global Picture Archiving and Communications (PACS) environment developed under a National Science Foundation grant. The Rural and Global PACS environment includes many workstations, database, and networking components and is treated as a large distributed system. These components are described in this paper. The multimedia services for radiology provided by the Rural and Global PACS are described and their performance is measured. Finally, the current research work using the Open Software Foundation's distributed computing environment (OSF DCE) services is described. An OSF DCE testbed for the Rural and Global PACS is described and the rationale of using OSF DCE in the project is presented. PMID- 7554848 TI - An approach to validation of a leg simulation by the comparison of two dynamic models. AB - The dynamics of a jointed leg were simulated using two different models, one based on a recursive Newton-Euler method and one on a closed-form Lagrange method. To validate the models, the simulations were run in parallel and the intermediate steps and output of the two methods were compared to one another to reveal the presence and locations of errors. Sources of error and the use of this method for the detection of errors are discussed. Some errors could not have been detected using only a single simulation. PMID- 7554849 TI - Interactive modelling and simulation of biochemical networks. AB - The analysis of biochemical processes can be supported using methods of modelling and simulation. New methods of computer science are discussed in this field of research. This paper presents a new method which allows the modelling and analysis of complex metabolic networks. Moreover, our simulation shell is based on this formalization and represents the first tool for the interactive simulation of metabolic processes. PMID- 7554850 TI - Three-dimensional uniform grid modeling of electrical defibrillation on a data parallel computer. AB - Finite element modeling has played an increasingly important role in the study of defibrillation. In order to model well the complex anatomical details, a large number of elements are required in the finite element grid, leading to a large set of equations that often cannot be solved effectively with the computational power of conventional computers. In this paper, we describe the use of a data parallel computer to provide the memory and reduction in solution time for solving these large finite element problems. Using a uniform grid and a nodal assembly technique, the discretized problem domain can be mapped efficiently to the parallel computer, allowing the solution of problems with over two million unknowns. The finite element algorithm for a three-dimensional inhomogeneous anisotropic body is described together with its parallel implementation. Test results for a canine torso model constructed from CT images are also presented. PMID- 7554851 TI - A spreadsheet program for simulation of bioequivalence and bioavailability studies. AB - This paper presents a spreadsheet for Excel for Windows, which simulates bioequivalence (BE) trials. The program incorporates intersubject and intrasubject variability in drug absorption and disposition as well as assay precision and the uniformity of the administered dose. The output provides confidence intervals and a pass/fail code for each study. This program is useful for simulating BE trials using widely available and simple-to-use spreadsheet programs. An example of the application of the program in assessing the influence of intrasubject variability on the outcome of BE testing of two identical formulations is also presented. PMID- 7554852 TI - Computers and networks in medical and healthcare systems. AB - Information technology has an important and expanding role in the delivery of high quality healthcare services. Until recently health informatics systems have generally been developed as independent centralized databases. With computing communications technologies now being introduced into major hospitals, many new information services can now be provided to enhance the patient-care provider interaction. This paper firstly reviews (existing and currently evolving) information technologies related to text and image-based patient related data. A case study of the introduction of network services at the Austin Hospital, Melbourne, then describes the advantages of computing communication technologies in the healthcare environment, and outlines the strategy adopted to implement these services in a clinically advantageous cost-effective manner. PMID- 7554853 TI - Logistic equations in the analysis of S-shaped curves. AB - A version of the logistic equation that provides useful descriptive parameters for the asymptote, slope and inflection point of the curve is presented and is suggested for use in empirically fitting S-shaped curves to data. A short SAS program that easily calculates the parameter values is shown. PMID- 7554854 TI - Simulation of the uptake of a reactive gas in a rat respiratory tract model with an asymmetric tracheobronchial region patterned on complete conducting airway cast data. AB - Generally, the uptake of reactive gases by the respiratory tract is simulated assuming that all paths from the trachea to the most distal airspaces are equivalent. As this is not the case, especially for nonhumans, the adequacy of this approach to predict doses that can be useful in the fields of toxicology and risk assessment is subject to question. To explore this issue, a dosimetry model is developed which combines the use of one-dimensional convection-dispersion equations in conjunction with multiple path anatomic models so that the dosimetry model simultaneously simulates transport and uptake in all the airways and airspaces of the anatomic model. For this work, the anatomic model of the tracheobronchial (TB) region is patterned on cast data which describe the dimensions and branching network of the 4807 airways of the TB region of a rat. Distal to each of the 2404 terminal bronchioles of the anatomical model, the air space is modeled as a single path. The results presented are preliminary; they focus on the predictions themselves to obtain an understanding of what the model has to say about uptake in a complex set of branching airways. Results include the following predictions: (1) Regardless of path there is a similarity along different paths in the shape of concentration profiles as well as a similarity in the shape of dose profiles. (2) Along a path in the TB or pulmonary region, dose decreases distally. (3) Generally, proximal alveolar region (PAR, a region of major morphological damage due to O3 and NO2) dose decreases the more distal the PAR. (4) There is considerable variation in the doses of the different airways or alveolar surfaces in the same generation. (5) The maximum and minimum PAR doses do not correspond to paths with, respectively, the smallest and largest number of generations from the trachea to the PAR. (6) The ratio of the maximum to minimum PAR dose is very sensitive to tidal volume. These results give a more realistic understanding of respiratory tract gas transport and uptake. The model also predicts aspects that equivalent path models cannot, such as the dose distribution of different but morphologically equivalent sites. PMID- 7554855 TI - Extracting knowledge from large medical databases: an automated approach. AB - Tools which can uncover patterns in patients' records and then make predictions based on that knowledge are and will continue to be high priority in many medical informatics groups. These tools are impacting the performance of outcome studies by discovering patterns which can then be verified with standard statistical tools. This paper demonstrates INC2.5, a general classification system, as a tool for assisting physicians in the decision making process. INC2.5 gathers information from patient records and builds a decision tree which is used to assist physicians in predicting the outcome of new patients. The decision tree will also reveal any patterns which the system found in the data. Successful results of such a system can be used to enhance outcome studies as well as to spread clinical information to areas with fewer resources. PMID- 7554856 TI - Semiautomated quantitation of urinary protein and creatinine. AB - The application of personal computers to routine tasks such as the recording and manipulation of data can greatly increase the speed and accuracy of the analyses. This report describes the use of a Macintosh computer and a BioTek microplate reader for the analysis of urinary protein and creatinine. Previously published assay methods for protein and creatinine were adapted for analysis using 96-well microplates. The adapted assays had excellent linearity (r2 = 0.99), with reduced variability compared to the previous methods. This resulted in increased sensitivity as demonstrated by 95% confidence limits. In addition, reductions of both assay materials and sample sizes were realized. The direct connection of the computer and microplate reader reduced the time needed to record absorbance measurements and perform analyses when compared to the more common protocols and methods of analysis. The assay protocols, equipment and interface, data file management, and methods of analysis are described. PMID- 7554857 TI - Computer-assisted mapping of infarcted and viable regions of gross cardiac sections following experimental myocardial infarction. AB - We have developed a computer-assisted method that creates digital maps of the viable and infarcted regions of tetrazolium-stained gross cardiac sections. Here we describe and test the method, using a canine occlusion-reperfusion infarction model. Quantitative image analysis showed that the method accurately recorded differences between infarcted and viable regions at a spatial resolution of 5-20 pixels per mm2. Microscopic analysis of tissue samples taken from the sections showed that the maps were accurate. In 15 of 15 cases the histology of the samples matched that predicted by the maps. A comparison of infarct area measurements derived from the maps showed that the method was reproducible. The average intraoperator standard deviation was +/- 8% of a slice's infarcted area and the average interoperator standard deviation was +/- 20%. We conclude that the method creates accurate, detailed, and reproducible maps of the infarcted and viable regions of tetrazolium-stained cardiac sections. PMID- 7554858 TI - Automatic analysis of hand radiographs for the assessment of skeletal age: a subsymbolic approach. AB - The assessment of skeletal maturity is crucial for the analysis of growth disorders and plays an important role in paediatrics. For this reason, several methods have been developed for estimating skeletal maturity. Among them, the Tanner and Whitehouse method (TW2), which is based on the analysis of hand radiographs, is usually considered the most accurate and reliable. Nevertheless, TW2 is applied only in a small fraction of cases, due to its complexity and long examination times. Thus, the development of automated systems which reliably implement this method is highly desirable. However, major difficulties have been found in the development of computer-based systems for the assessment of skeletal maturity. In particular the extraction of the bones of interest has proved to be extremely challenging. In this paper, we propose a system architecture for the implementation of the TW2 method, which is based on artificial neural networks. For each bone considered, the maturation stage is determined by means of a two step process which first locates the position of the bone in the radiograph and then analyzes the bone shape. Experimental results obtained with our implementation of the carpal version of TW2 are in good agreement with those provided by trained observers. PMID- 7554859 TI - Lesion size quantification in SPECT using an artificial neural network classification approach. AB - An artificial neural network (ANN) has been developed to determine the size of lesions detected in single photon emission computed tomographic images. The network is the Learning Vector Quantizer and is trained to perform size quantification based on image neighborhoods extracted around the lesions. The ANN is compared to the optimal, Bayesian algorithm developed to perform the same task using the unreconstructed, projection data. The performance of the neural network is evaluated at two different noise levels. The Bayesian algorithm provides the upper bound for size quantification performance against which the ANN is compared. In the ideal case where the Bayesian algorithm has explicit knowledge of the underlying distributions, its performance is superior to that of the neural network. However, in the more realistic case where the distributions need to be estimated from the same learning sample the ANN was trained on, the two algorithms have comparable performances. PMID- 7554860 TI - A model of fluid resuscitation following burn injury: formulation and parameter estimation. AB - A dynamic compartmental model is developed to describe the redistribution of fluid and albumin between the circulation and the intact and injured interstitia following burn injury in humans. Transcapillary fluid and albumin exchange is described by a coupled Starling mechanism, while the effect of the burn is represented by time-dependent perturbations to all three compartments. The unknown model parameters are determined for two groups of patients, having less than and greater than 25% total body surface area burns, by statistical fitting of model predictions to patient data from two sources. The parameters include the perturbations to the fluid filtration coefficients in uninjured and injured tissue, GkF,Tl and GkF,BT, respectively, the relaxation coefficient, r, which describes the exponential decay of the perturbations, and the exudation factor, EXFAC, which relates the protein concentration in the exudate to that in the injured tissue. Perturbations to other parameters, including the membrane permeability-surface area product and the albumin reflection coefficient in the injured and uninjured tissues, are determined based on interrelationships with GkF,Tl and GkF,BT. The values of GkF,BT, when corrected for tissue destruction and decreased post-injury perfusion, are in reasonable agreement with the limited experimental data available from the literature. The model and its parameters are further validated by comparing the simulated patient responses to the clinical data used in the parameter estimation as well as to data available from two additional sources. PMID- 7554861 TI - Three-dimensional reconstruction of biliary tract from biplane projections. AB - Over the past few years, very little has been done in the area of 3-D reconstruction of the bile duct. Since the system in use for 3-D visualization of the biliary tree is built by surgical or autopsy materials, it generally cannot be applied to clinical diagnosis. In this paper, an algorithm for three dimensional reconstruction of the biliary tree from two mutually orthogonal is presented to provide accurate and reproducible 3-D information of the biliary tree structure. It has been proven to be useful in diagnosis prior to operation or non-surgical treatment, particularly, obstructive stones can be visualized by using a transparency technique. As experiments demonstrated, the proposed method can be used as a useful tool for the visualization of 3-D structure of biliary tree in clinical applications. PMID- 7554862 TI - DATAPLOT: a graphical display package for bacterial morphometry and fluorimetry data. AB - A microbiological image processing system has been developed, which is used to determine both morphometry and fluorimetry data on (mainly faecal) bacteria. A special program for display of these data has been developed for this system. Besides probability density estimates of any one or two parameters and scatter plots of any two or three parameters in the distribution, it can generate scatter plots using the actual bacterial shapes, rather than arbitrary symbols. A particularly powerful feature of the program is the possibility to overlay a contour plot of the probability density distribution of the bacteria in parameter space, with a scatter plot of the actual bacterial shapes involved. In this paper, the program's organization, options, and the algorithms used are discussed. PMID- 7554863 TI - CBA--an atlas-based software tool used to facilitate the interpretation of neuroimaging data. AB - CBA, a software tool used to improve quantification and evaluation of neuroimaging data has been developed. It uses a detailed 3-dimensional brain atlas that can be adapted to fit the brain of an individual patient represented by a series of displayed images. Anatomical information from the atlas can then be introduced into the images. If the patient has been imaged in different modalities, adaptation of the atlas to the different images will provide the transformation that brings the images into registration. CBA can thus be used as a tool for fusing multimodality information from the same patient. Furthermore, by applying the inverse atlas transformation, images from a patient can be transformed to conform to the anatomy of the atlas brain. This anatomical standardization, where the atlas brain itself serves as the anatomy standard, brings data from different individuals into a compatible form providing possibilities to perform individual-group and group-by-group comparisons between patients and normal controls. PMID- 7554864 TI - Artificial intelligence programming with LabVIEW: genetic algorithms for instrumentation control and optimization. AB - A genetic algorithm for instrumentation control and optimization was developed using the LabVIEW graphical programming environment. The usefulness of this methodology for the optimization of a closed loop control instrument is demonstrated with minimal complexity and the programming is presented in detail to facilitate its adaptation to other LabVIEW applications. Closed loop control instruments have variety of applications in the biomedical sciences including the regulation of physiological processes such as blood pressure. The program presented here should provide a useful starting point for those wishing to incorporate genetic algorithm approaches to LabVIEW mediated optimization of closed loop control instruments. PMID- 7554865 TI - Development, implementation and a first evaluation of a protocol processing system (ProtoVIEW). AB - In this contribution the protocol information system ProtoVIEW is presented. The system provides the necessary information to physicians about diagnostic procedures and therapies. It is implemented as a stand alone system. The design criteria are discussed and the results of a first evaluation presented. It appears that interns can easily find the required information with the help of the system. The time that they need for accessing the relevant information is relatively short (about 1 min). The users expressed the opinion that the system is easy to use and does support them in the management of their patients. On the basis of this evaluation and evaluations reported elsewhere it is concluded (a) that stand-alone protocol systems can support daily patient management in a positive way and (b) that the design criteria for a protocol information system as presented in this paper are useful for prospective protocol information system developers. PMID- 7554866 TI - Comment on: Updates of bioequivalence programs (including statistical power approximated by Student's t) PMID- 7554867 TI - Prodromal symptoms of schizophrenia in first-episode psychosis: prevalence and specificity. AB - Three hundred thirteen patients with first-episode psychosis were assessed using the Royal Park Multidiagnostic Instrument for Psychosis (RPMIP) to determine differences among seven DSM-III-R diagnoses in the comparative frequencies and diagnostic efficiencies of DSM-III-R schizophrenia prodromal symptoms. Patients with a diagnosis of schizophrenia and schizophreniform disorder were significantly more likely to evince prodromal symptoms. A multinomial logit model suggested that individual prodromal symptoms were relatively poor at distinguishing between diagnoses. This was confirmed when sensitivity, specificity, and positive (PPP) and negative (NPP) predictive power of individual prodromal symptoms were examined. Although DSM-III-R schizophrenia prodromal symptoms do occur more commonly in schizophrenia, they are by no means pathognomonic of that disorder. PMID- 7554868 TI - The predelusional state: a conceptual history. AB - The predelusional state (PDS) is defined as the set of psychopathologic events preceding the crystallization of delusions, and includes strange cognitions, moods, conations, and motor acts that may be fleeting and defy description. This review exclusively deals with the historic aspects of PDS. It is noted that during PDS the patient is expected to report experiences for which, on account of their novelty, he may not even have a name. Thus, it is quite likely that according to culture and personal codes and to the conceptual brief of the interviewer, similar experiences might be reported as depersonalization, bodily sensations, dysphoria, changes in perception of reality or time, dissolution of "ego boundaries," etc. It is therefore not surprising that since the 19th century, PDS has been considered a disorder of cognition, emotions, volition, and consciousness. PMID- 7554869 TI - Clinical profile of comorbid major depression and alcohol use disorders in an initial psychiatric evaluation. AB - This study evaluated the clinical profile of patients who presented with both DSM III major depression (MD) and alcohol use disorder (AUD) as compared with that of patients with either diagnosis alone. Comparison of these three groups (MD without AUD, AUD without MD, and comorbid AUD-MD) selected from a large sample (8,139) of general adult psychiatric patients showed that the historical, cross sectional, and dispositional profile of AUD-MD patients occupied an intermediate position between that of the other two groups. The symptomatology of AUD-MD patients was somewhat closer to that of MD patients, whereas the premorbid personal and social history and dispositional status were more similar to those of AUD patients. AUD-MD patients had a strikingly higher rate of suicidal indicators as compared with AUD or MD patients. PMID- 7554870 TI - Is there a relationship between clozapine and obsessive-compulsive disorder?: a retrospective chart review. AB - The emergence of obsessive-compulsive symptoms during clozapine treatment has been reported in recent case studies, yet the incidence and significance of this finding is still unclear pending reliable data from a larger sample of patients. Hospital records of 142 randomly selected inpatients started on clozapine treatment at McLean Hospital before July 1, 1992, were reviewed retrospectively. Based on a limited retrospective chart review, there were no definitive cases of patients who developed obsessive-compulsive disorder (OCD) or whose OCD worsened as a result of clozapine treatment. Although some fluctuation of OCD symptoms may have occurred in two cases, it is unclear whether those symptoms were related to treatment with clozapine (or other psychotropic drugs) or to undulations in the natural history of OCD. No definitive relationship between OCD symptoms and clozapine treatment could be established in this limited study. Further clarification of this matter awaits outcome research using prospective methodologies. PMID- 7554871 TI - 5-year prospective, naturalistic follow-up study of panic disorder. AB - Ninety-nine patients with panic disorder (PD) not comorbid with other psychiatric disorders were evaluated for 5 years using a naturalistic prospective design. The probability of achieving full remission, albeit transitory, was 37.5%, whereas 72.8% of cases showed a consistent amelioration. However, among patients with an initial positive outcome, the probability of remaining well was 41.4% after 5 years. When the general course of the disorder during the follow-up period was considered, only 12.12% of the subjects had a complete and stable remission of symptoms, whereas 47.47% had a generally positive but not fully satisfactory amelioration either due to infrequent recurrences of the illness or to chronic continuation of symptoms at a mild level. On the other hand, 40.40% of the subjects reported an overall poor outcome because of the presence of a recurrent pattern of illness (11.11%) or because the periods of well-being did not represent more than 40% of the time being considered (29.29%). Among the predictors taken into consideration, only duration of illness before intake showed a strict relationship with long-term outcome, with patients having a lesser duration of illness at the moment of the index episode showing a significantly better outcome. PMID- 7554873 TI - Routine assessment of patient progress: a multiformat, change-sensitive nurses' instrument for assessing psychotic inpatients. AB - A new instrument, the Routine Assessment of Patient Progress (RAPP), is a standardized scale with a unique format that allows trained nurses to incorporate both interview and observational data into a comprehensive assessment of psychiatric inpatients. The scale was developed to accurately document treatment effects, assist in treatment planning, and provide ancillary judgments of psychopathology that may affect diagnosis. The RAPP demonstrates satisfactory interrater reliability and internal consistency. The RAPP total score and its two subscales correlated highly, where expected, with an observation-based nursing scale, global measures, and an interview-based psychopathology scale. RAPP scores demonstrated sensitivity to change over time in patient functioning. It reliably discriminated among patients discharged to settings with varying degrees of independent living. The use of the RAPP over several years on a psychiatric inpatient unit has shown it to be a practical clinical tool that eases nurses' charting demands, helps create care plans, and facilitates interdisciplinary communication. PMID- 7554872 TI - Validity of psychiatric diagnoses in patients with substance use disorders: is the interview more important than the interviewer? AB - Although structured diagnostic interviews are increasingly being used in substance abuse treatment settings, there has been limited systematic evaluation of their ability to enhance reliability and validity of psychiatric diagnoses. The present report provides data on the concurrent, discriminant, and predictive validity of current substance use disorders and common comorbid diagnoses in a sample of 100 substance abuse patients. Diagnoses formulated primarily by master's-level clinicians in the usual course of their duties were compared with diagnoses formulated by research technicians using a semistructured interview. Results indicated that the validity of clinician diagnoses was good for substance use disorders, moderate for personality disorders, and poor for anxiety disorders and major depression. Greater validity was observed for substance abuse diagnoses formulated by research technicians using the semistructured interview. Based on these findings, we conclude that psychiatric diagnosis in substance abuse patients may be improved by adding elements of structured interviews to the clinician's usual assessment. PMID- 7554874 TI - Better than expected: improvements in borderline personality disorder in a 3-year prospective outcome study. AB - We report on the symptomatic outcome, predictors of outcome, and different courses of change in a 3-year prospective, naturalistic study of 37 female patients with borderline personality disorder (BPD). Results show significant improvements in several areas and no significant deterioration. Most patients showed a course of erratic improvement, and only a few showed decline over time. The main baseline predictors of good 3-year outcome were low obsessiveness and phobia symptoms. Results from the Global Assessment Scale (GAS) in this study, like five prior studies, demonstrate that by 3 years BPD patients can be expected to move from a poor to a fair level of functioning. It is suggested that over a 3 year period, improvements may be expected from BPD patients. Prior reports of poor short-term outcome may have been the result of different diagnostic criteria and outcome measurement. Limitations of this study and the need for tentative interpretation of its results are also discussed. PMID- 7554875 TI - Comorbidity of anxiety disorders in a multicenter anxiety study. AB - From 11 sites in New England and Missouri, 711 patients with > or = one of five index anxiety disorders were recruited onto a longitudinal study in which they were interviewed every 6 months regarding symptoms, course, and treatments received. Of the five disorders studied, panic disorder without agoraphobia was the disorder most often found as a sole diagnosis and generalized anxiety disorder (GAD) was least often found alone, both as lifetime diagnoses or when restricted to cases active at intake. Panic disorder with agoraphobia and agoraphobia without history of panic disorder (AWOPD) had three specific diagnoses with which they were frequently comorbid: social phobia, simple phobia, and GAD. AWOPD, social phobia, and GAD were frequently found in the presence of each other. It is possible that the experience of anxiety due to any syndromal cause may decrease the threshold for an individual to experience other anxiety symptoms or disorders. Clinicians should be aware of these patterns of comorbidity in order to formulate accurate differential diagnoses and prescribe treatments in a rational manner. PMID- 7554876 TI - Eating disorders with and without substance use disorders: a comparative study of inpatients. AB - We assessed the co-occurrence of DSM-III-R axis I and II disorders and self reported psychologic distress in inpatients with eating disorders with and without substance use disorders (ED-SUD and ED groups, respectively) and in a matched comparison sample with substance use disorders but no eating disorder (SUD group). The three groups showed similar distributions of axis I disorders but differed in the distribution of axis II disorders. Cluster B personality disorders were diagnosed more frequently in SUD and ED-SUD groups than in the ED group. In contrast, cluster C personality disorders were diagnosed more frequently in the ED group than in SUD and ED-SUD groups. The SUD group reported greater psychologic distress than ED and ED-SUD groups. Possible implications of the observed group differences for psychologic models of why these disorders may be associated are considered. PMID- 7554877 TI - Textile dermatitis: an update. (I). Resins, additives and fibers. AB - Cases of textile-related dermatitis reported in the medical literature after the mid-1980s are reviewed. Part I focuses on cases in which textile resins, fiber additives, or fibers were the causal agent. Studies which provide insight into understanding fabric-induced prickle and itch are included. PMID- 7554878 TI - An outbreak of occupational dermatosis in an electronics store. AB - An outbreak of skin disorders was noted by the primary care physician responsible for the health care of 52 employees in an electronics store. The cases occurred after termite treatment of the store, and the breakdown of the air-conditioning system in several areas in the store. Investigations were initiated to allay worker concern that the chemicals used for termite treatment (in particular, arsenic trioxide) were responsible for the outbreak. 9 of the 52 workers were found to have heat rash (miliaria rubra). 21% (7) of workers who worked in non air-conditioned areas had heat rashes, as compared to 11% (2) of workers who worked in air-conditioned work zones. 2 workers had contact dermatitis due to exposure to greaseless lubricant, and 8 other workers had a non-work-related skin disorder. Indoor wet bulb globe temperature index measurements in the non-air conditioned areas were within threshold limit values, but ventilation in these areas was very poor. 2 environmental samples had non-detectable limits of arsenic. PMID- 7554879 TI - A study on expert reading of patch test reactions: inter-individual accordance. AB - To diagnose allergic or irritant contact dermatitis, a clinically relevant contact allergy has to be demonstrated or ruled out, respectively. Although patch testing has been used for 100 years, it remains the method of choice for diagnosing contact allergy. A disadvantage of patch testing is that reading is subjective, based on inspection and palpation of the test area, implying that the assessment is subject to the reader's knowledge and experience. This study was carried out to investigate the accordance in reading patch test reactions between 5 dermatologists. 4 groups, each with 10 patients, participated. Within each group, all 10 were allergic to one and the same sensitizer; nickel, epoxy resin, ethylenediamine, or Kathon CG. The sensitizers were tested in serial dilutions and applied randomly to the back. The tests were read independently by the dermatologists in a blinded fashion. A protocol was used where the dermatologists had to note the presence of the morphological features erythema, infiltration, papules, vesicles, and bullae. In this way, it was possible to allocate the various readings into 4 classification systems, 3 European and one American, although the definition of the various classifications might differ slightly. Based on the readings and classifications, it was possible to calculate the degree of accordance within the 4 systems used. It was also possible to analyze the degree of accordance for the various morphological features. Total accordance for the 5 reading dermatologists for positive and negative test reactions was noted in 36% and 46%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554880 TI - Effect of sodium lauryl sulfate (SLS) on in vitro percutaneous penetration of water, hydrocortisone and nickel. AB - The dose- and time-related effect of sodium lauryl sulfate (SLS) on in vitro percutaneous penetration was studied using 3 radiolabeled tracer compounds with different physicochemical properties: tritiated water, hydrocortisone and nickel. Human cadaver abdominal skin from caucasian women was used as membrane in static in vitro penetration cells. Simultaneous application of SLS together with 1 of the tracer compounds showed, after 48 h, a significant dose-effect relationship between SLS concentration (0.25%, 2% and 10%) and penetration of tritiated water or nickel (p < 0.001, Spearman), whereas SLS had no significant effect on penetration of hydrocortisone. When 4% SLS was applied as pretreatment, a significant time-effect relationship, after 48 h, was found between pretreatment time (0.5, 2 and 8 h) and penetration of tritiated water. A similar relationship was not found for penetration of nickel or hydrocortisone. Pretreatment of the skin with SLS for 2 h using 3 concentrations (0.25%, 4% and 10%) showed, after 48 h, a significant dose-effect relationship between SLS treatment and penetration of tritiated water or nickel (p < 0.001, Spearman). Pretreatment had no effect on penetration of hydrocortisone. Pretreatment simulates a cleaning-washing situation. The present in vitro skin penetration model, using human cadaver skin, described the dose-effect and time-effect relationships for SLS on the penetration profiles of 3 different compounds. The model may be extended to other compounds with suspected irritant/damaging effect on the skin barrier. It should be kept in mind that the model uses a dead skin membrane without the barrier repair mechanisms of live skin. PMID- 7554882 TI - Exposure characteristics and cutaneous problems in operating room staff. AB - Health care personnel have exposure to a variety of cutaneous irritants and allergens and a high prevalence of cutaneous problems. The objectives of this exploratory study were to characterize the exposures and determine the prevalence of cutaneous symptoms and findings in operating room personnel, and to examine relationships between exposure characteristics and cutaneous outcomes. A questionnaire and standardized hand examination were used to assess the exposures and cutaneous status of operating room personnel. 184 operating room staff (90% of the eligible population) were assessed. Current skin problems were reported by 26%. Hand examination revealed that 9% had findings consistent with eczema and 10% changes of moderate dryness. A variety of preventive practices were being used by those with symptoms or findings. Cutaneous problems are common in operating room personnel. The use of preventive strategies (changing the type of gloves worn or the type of soap used for scrubbing, the use of glove liners and emollients) varied between different groups. These workers might benefit from more education regarding cutaneous hazards, preventive stategies and the importance of appropriate investigation of these problems. PMID- 7554881 TI - Analysis of patients with allergic patch test reactions to a plastics and glues series. AB - 839 patients were patch tested with a series of 31 plastics and glues allergens at a dermatologic clinic over a period of 7 years. 52 (6%) had a positive patch test reaction to 1 or more such allergens. Clinic charts of 47 patients (out of 52) were available for the study. All but 3 patients had dermatitis on their hands: 17 had only hand dermatitis. 25 (53%) patients' test results were of present or past relevance to their skin diseases. 11 patients (68%) with occupational allergic contact dermatitis (ACD) and 14 (45%) with non-occupational dermatitis had relevant reactions. p-tertiary-butylphenol-formaldehyde resin was the most common allergen (9 cases). 7 patients reacted to diaminodiphenylmethane. 5 patients, who had colophony allergy, reacted to abietic acid and 4 to abitol. There was no reaction to 14 test substances. Special series, such as this plastics and glues series, reveal the cause of ACD less frequently than standard series. However, there is no other way to confirm allergy to these usually industrial allergens, which can also sensitize through non-occupational exposure during hobby working or through unpolymerized monomer left in the finished plastic product. PMID- 7554883 TI - Lipstick allergic contact dermatitis from gallates. PMID- 7554884 TI - Contact urticaria syndrome from sorbitan sesquioleate in a corticosteroid ointment. PMID- 7554885 TI - 2 cases of dermatitis from rare sources of sensitization to frequent contactants. PMID- 7554886 TI - Contact urticaria from abietic acid. PMID- 7554887 TI - Contact dermatitis from acrylic nails. PMID- 7554888 TI - Occupational sensitization to diphencyprone in a chemist. PMID- 7554890 TI - Severe contact dermatitis due to natural latex. PMID- 7554889 TI - Allergy to fibrin tissue in dental medicine. PMID- 7554891 TI - A pilot study of pentoxifylline for the prevention of poison ivy/oak reactions. PMID- 7554892 TI - Thimerosal is a frequent sensitizer but is not in the standard series. PMID- 7554893 TI - Contact allergy to dicyclohexyl carbodiimide and diisopropyl carbodiimide. PMID- 7554894 TI - Allergic contact dermatitis from naftifine. PMID- 7554895 TI - Allergic contact dermatitis from sertaconazole with cross-sensitivity to miconazole and econazole. PMID- 7554896 TI - Occupational contact dermatitis from colophony and formaldehyde in banknote paper. PMID- 7554897 TI - Occupational exposure to isothiazolinones. PMID- 7554898 TI - A mammogram coupon program: a collaborative effort to provide mammograms to uninsured women in Connecticut. AB - A collaborative effort to provide free screening mammograms to uninsured women throughout Connecticut using monies raised by the Connecticut RACE FOR THE CURE was launched during National Breast Cancer Awareness Month, October 1994. The program involved 55 American College of Radiology (ACR) accredited mammography facilities and 48 social service agencies. Eligibility criteria stipulated that participants be age 40 or older, be asymptomatic, have no health insurance, and have not had a mammogram within the last year. A total of 531 women received screening mammograms with four breast cancers diagnosed. The typical woman who was screened was white, between age 40 and 59, had a low income, a high school education or less, and no health insurance. Most had had a mammogram at some point in her life, but had not had one in the last two years. It was found that recruiting eligible women was more difficult than initially estimated. Many of the participating social service agencies served clients already on assistance, and, therefore, had mammogram coverage. Mass media, in particular television, proved to be a powerful recruiting tool which reached primarily "working poor" women. Future efforts should allow for a lengthier planning time to recruit hard to-reach women and anticipate the need for case management to ensure follow-up of abnormal findings. PMID- 7554899 TI - Inaccurate medical student introductions: frequency and motivation. PMID- 7554900 TI - Continuous infusion of beta-lactam antibiotics: a rational dosing approach. PMID- 7554901 TI - The trouble with managed care. PMID- 7554902 TI - Credentialing and being fair with MDs. PMID- 7554903 TI - Proceedings of the 5th International Conference on Tooth Morphogenesis and Differentiation. Kerkrade, The Netherlands, May 4-8, 1994. PMID- 7554904 TI - Tooth crown morphogenesis and cytodifferentiations: candid questions and critical comments. AB - Teeth are probably meristic units and crown morphogenesis leads to tooth specific distribution of functional cells. Since heterodonty is derived from homodonty, one way to understand tooth morphogenesis would be to unravel the involved phenomena in homodont species and then to characterize the "put up job" of evolution leading to species specific dentitions with particular functional abilities. Interaction of paleontologists and developmental biologists should be initiated. My naive "developmental" point of view will illustrate only one facet of mouse tooth morphogenesis and cytodifferentiation. The main concern will be to try to discriminate between known facts and speculations, between hypotheses and anticipated or deduced certitudes, to call attention to conflicting data, to suggest some further investigations and to advocate the point of view that molecular interpretations should be founded on indisputable morphological data. PMID- 7554905 TI - Analysis of amelogenin proteins using monospecific antibodies to defined sequences. AB - Amelogenins are the predominant proteins found in the developing enamel matrix and are believed to play a crucial role in normal mineralization. Although the amelogenin gene is found as a single copy in all species in which it has been examined, multiple amelogenin polypeptides ranging in size from 5 to 25 kDa are obtained upon extraction of developing enamel matrix, making identification and characterization of individual components difficult. This heterogeneity may be ascribed to transcription of divergent genes located on the X and Y chromosomes, alternative splicing of the primary transcripts, physiologic degradative processing, and artefactual degradation. In order to characterize individual components, antibodies were produced to the following peptides: (1) QPLQPMQPMQPLQPLQPL (corresponding to the repeat sequence encoded only in the bovine X chromosome gene), (2) IRHPPLPP (corresponding to a unique sequence generated by alternative splicing found in leucine-rich amelogenin peptide (LRAP), (3) LPDLPLEAWPATDKTKREEVD corresponding to the amelogenin carboxy terminus. Amelogenin proteins obtained from fetal bovine molars were subjected to SDS PAGE and Western electrotransfer, and immuno-ultrastructural analysis. These analyses demonstrated that: (1) the distribution of amelogenin polypeptides isolated from male fetuses differed appreciably from that of females, (2) the LRAP junctional peptide sequence can be specifically identified, and (3) the LRAP peptide can be immunolocalized in the enamel matrix of both males and females. PMID- 7554906 TI - Analysis of the regulatory region of the bovine X-chromosomal amelogenin gene. AB - The amelogenin proteins, which are crucial for normal enamel mineral formation, are secreted by ameloblasts during development of tooth enamel. In order to better understand the mechanisms involved in regulation of expression of the amelogenin genes, the bovine X-chromosomal amelogenin gene was cloned and a 3.5 KB fragment upstream of exon 1 was inserted into a beta galactosidase (beta gal) expression vector for production of transgenic mice. When tissues from these mice were treated with Xgal, a substrate for beta gal, only ameloblasts and some of the adjacent stratum intermedium cells contained blue stain. To obtain further information concerning regulation of expression, the 3.5 KB amelogenin gene fragment was evaluated in transfection experiments. Nonoverlapping 1.9 and 1.5 KB fragments of the upstream region were subcloned separately into a vector that contains the SV40 promoter and the CAT reporter gene. Each amelogenin gene fragment was able to suppress CAT activity driven by the heterologous SV40 promoter in transfected HeLa cells. We theorize that each of these gene fragments contains regulatory elements important for the tissue-specific and developmentally-regulated pattern of expression of the X-chromosomal amelogenin gene. PMID- 7554907 TI - Recent advances in amelogenin biochemistry. AB - This paper reviews advances in amelogenin biochemistry in three areas; (i) amelogenin expression; (ii) amelogenin post-translational and post-secretory processing, and (iii) amelogenin structure and function. Recent studies of amelogenin expression have demonstrated that alternative-splicing of mouse amelogenin RNA generates seven distinct mRNAs, coding for amelogenin proteins from 194 to 44 amino acid residues in length. A polyclonal antibody to a sequence of the 194-residue murine amelogenin identified this protein in vivo. While several studies have reported that amelogenins are post-translationally phosphorylated, it has proved difficult to confirm this view. Mass spectrometry studies of bovine and porcine TRAP and LRAP amelogenins have established a phosphoserine residue at position-16 as originally reported by Takagi et al. for a 180-residue bovine amelogenin. Also, we discovered that the detailed mechanism(s) of carboxy-terminal amelogenin proteolytic processing appear different than previously reported. In terms of amelogenin structure, it is well known that amelogenins form aggregated structures. Studies employing a recombinant amelogenin and dynamic light-scattering instrumentation demonstrated aggregate structures of 15-20 nm in radius, corresponding to a mass of 2-3 million daltons. Imaging these aggregates by transmission electron and atomic force microscopy suggested that these structures are equivalent to the "stippled" or "granular" material seen in electron photomicrographs of developing enamel. Collectively, these advances in amelogenin biochemistry lead to a new view of amelogenin structure, processing and functions in enamel biomineralization. PMID- 7554908 TI - A review of the aggregation properties of a recombinant amelogenin. AB - The present paper reviews some recent data on the aggregation properties of a recombinant amelogenin using dynamic light scattering, transmission electron microscopy, atomic force microscopy, and size exclusion chromatography. It was found that the recombinant amelogenin M179 molecules in solution form spherical monodisperse aggregates (15-20 nm) which are predominantly stabilized by intermolecular hydrophobic interactions while their surfaces are charged, carrying the hydrophilic carboxy-terminal sequence. We concluded that the spherical aggregates represent the "stippled materials" secreted by the ameloblasts at the mineralization front. We further speculate that the self assembly process in the formation of amelogenin aggregates may play a primary role in the structural organization of mineralizing enamel. PMID- 7554910 TI - In situ hybridization of calbindin-D 28 k transcripts in undecalcified sections of the rat continuously erupting incisor. AB - Calbindin-D9k and calbindin-D-28k genes are useful systems to investigate the tissue- and stage-specificity as well as the hormonal control of gene expression. Since they regulate cellular calcium mobilization, their study may be of interest in mineralized tissues. However, thus far, immunocytochemical labelling has been mainly realized in these systems. In order to set up methods for mRNA investigation, in situ hybridization of calbindin-D28k mRNAs was performed in the continuously erupting incisor of Sprague-Dawley rats (15-, 30-, and 56-day-old). 35S UTP labelled antisense and sense riboprobes specific for brain calbindin-D 28k were used for in situ hybridization. Specific and non-specific signals could not be discerned when studying decalcified samples. In contrast, on sections not pretreated with EDTA, calbindin-D 28k transcripts (in tooth and kidney) appeared strongly labelled with antisense probes, while sense probes provided a negligible background. In ameloblasts, the signal (i.e., calbindin-D 28k mRNA levels) increased during the presecretory stage. Different mRNA gradients and subcellular distribution patterns characterized the secretory and maturation stages. A nuclear labelling was observed, associated with the highest levels of transcripts. These data suggest a developmental control of calbindin-D28k mRNA transcription. Calbindin-D28k gene expression appears to be up-regulated during the initiation of both secretory and maturation stages of enamel mineralization. PMID- 7554909 TI - Alternative splicing of amelogenins. AB - Amelogenins comprise as much as 90% of the protein in the developing enamel matrix. Separating amelogenins by gel electrophoresis reveals a complex of polypeptides with apparent mobilities ranging from low molecular weight species on up to 28,000 Daltons. A major objective of our research is determine the extent to which alternative RNA splicing contributes to this heterogeneity. We have cloned seven alternatively spliced mouse amelogenin mRNAs. The predicted translation products of these messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. The 194 residue amelogenin is the only mouse amelogenin to include a polypeptide segment encoded by exon 4, which has a deduced amino acid sequence of KSHSQAINTDRTAL. Antibodies were raised against synthetic exon 4 encoded polypeptides and used to immunostain histologic tooth sections. These data indicate that alternatively spliced amelogenin mRNAs are translated into protein and secreted into the enamel matrix. PMID- 7554911 TI - The basic and applied biology of tooth eruption. AB - The dentition and the alveolar process of each jaw develop simultaneously so that, by the time the crown is completed and eruption begins, the crown is enclosed in a crypt within alveolar bone. Thus, the eruption of a tooth to its functional position involves discretely localized, bilaterally symmetrical bone resorption to produce an eruption pathway and bone formation to fill in the space previously occupied by the crown and growing roots. Studies of crypt surfaces during eruption confirm this polarization of alveolar bone metabolism around a tooth with respect to both bone cells and mineralized surface topography. Experimental studies of tooth eruption have shown that the dental follicle, the dense connective tissue investment of the tooth, is necessary for eruption and that neither bone resorption nor bone formation occur without the adjacent part of the dental follicle. Early in eruption the coronal part of the follicle accumulates mononuclear cells which have cytochemical and ultrastructural features of osteoclasts and the apical part of the follicle, a site of intense cell proliferation, binds epidermal growth factor (EGF). The dental follicle contains a variety of proteins and the concentration of several change during eruption. Prominent among them are a reduction in matrix metalloproteinases and an increase in protoglycans as eruption proceeds. The contribution of these changes to those in cell proliferation, migration and differentiation during tooth eruption present experimental opportunities for developmental biologists. The rate-limiting factor of the earliest (intraosseous) stage of tooth eruption is bone resorption and eruption can be accelerated or retarded by the local delivery of factors which increase or decrease the activity of osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554913 TI - The contrasting effects of colony-stimulating factor-1 and epidermal growth factor on tooth eruption in the rat. AB - Both epidermal growth factor (EGF) and colony-stimulating factor-1 (CSF-1) have been shown to accelerate eruption of teeth in rodents. We compared the effects of neonatal injections of EGF (1 micrograms/g body weight) and CSF-1 (10(6) units) alone or together on the eruption of incisors and first molars. EGF accelerated the eruption of incisors with no significant effect on first molars. CSF-1, in contrast, accelerated molar eruption more than incisor eruption. CSF-1, but not EGF, increased the numbers of mononuclear cells in the dental follicle and osteoclasts on adjacent alveolar bone surfaces around the first molar and produced enhanced resorption of crypt surfaces as revealed by scanning electron microscopy. These data suggest that during eruption rodent incisors and molars may preferentially respond to different molecular regulators. PMID- 7554912 TI - Altering tooth eruption by blocking bone resorption--the local delivery of bafilomycin A1. AB - Tooth eruption is a complicated process requiring a coordination of bone resorption and bone formation by a variety of factors in and around the dental follicle proper and bone resorption is the rate-limiting step early in the process. We have recently described a method to deliver to the crypt of erupting dog premolars a reversible blocker of bone resorption, bafilomycin A1, and shown that its delivery for two week blocks bone resorption and eruption during this period without effect on adjacent teeth or on bone formation. In this study we show that delivery of 10(-6) M bafilomycin A1 via a cannulated osmotic minipump for two weeks early in the eruption of premolars delayed the eruption of these teeth for eight weeks. Similar delivery of the vehicle to the contralateral premolar had no effect on eruption. These data are the first clinical application of this potent drug and show that a short term local delivery is reversible and that blocking resorption for two weeks causes a fourfold delay in tooth eruption. Modifications of this approach may have clinical applications in dentistry. PMID- 7554914 TI - Homeobox genes and orofacial development. PMID- 7554915 TI - Microradiography and fluorescence microscopy of bone remodeling on the basal crypt of permanent mandibular premolars in dogs during eruption. AB - Alveolar bone of erupting teeth was studied in order to define the types of calcified tissues deposited as well as the rate of tooth growth. The third (P3) and fourth (P4) mandibular premolars of 30 dogs aged 12-24 weeks were analyzed by microradiography and microscopy in fluorescent and ordinary light. The bone plate separating P3 and P4 from the mandibular canal presented a complex arrangement of lamellar and woven bone, and even of chondroid tissue. During the pre-eruptive phase, this plate shifted towards the base of the mandible by means of selective resorption and apposition activities. As soon as the furcation was formed, bone apposition appeared on the alveolar side and became the main activity under P3 at the outset of eruption. Under the roots of P4 it occurred 4 weeks later. Dynamic morphometry in fluorescence microscopy showed that eruption progressed faster than the radicular growth. The formation of interradicular bone underwent the same acceleration as the eruption. However, though the tissues were formed at a high rate, it cannot be inferred therefrom that they are responsible for tooth shifting. They might just fill the space left by the erupting tooth. PMID- 7554916 TI - Accelerated eruption of rat lower incisor. Relationship between impeded and unimpeded eruption rates, rate of attrition, tooth length, and production of dentin and enamel. AB - The present investigation studies the effects of persistently cutting one lower rat incisor out of occlusion. Within four days, the rate of eruption of the cut (unimpeded) incisor increased to 216% and that of the uncut (impeded) contralateral to 136% of the baseline rate. While the former remained high, the latter decreased gradually to about 90% within three weeks. The rate of attrition of the impeded incisor increased to 233% of the baseline rate within two days, then fell abruptly, and remained at a slightly lower level than the rate of eruption. Accordingly, the length of the erupted part of the impeded incisor decreased initially, but increased gradually after about four days. Measurements made on SEM micrographs of the series of transverse tooth segments obtained when cutting the incisor out of occlusion, showed that growth-related increase in mesiodistal tooth width was arrested from the 10th segment, dentin thickness decreased gradually to about 50% in the 12th segment, and enamel thickness, after an initial increase, decreased to about 80% in the 11th segment. The present study provides experimental evidence that accelerated eruption affects morphogenesis and histogenesis of the rat lower incisor. An impeded incisor, especially the contralateral, may not serve as an ideal control. PMID- 7554917 TI - The distribution of cellular retinoic acid-binding protein I (CRABPI) and cellular retinol-binding protein I (CRBPI) during molar tooth development and eruption in the rat. AB - The distribution of cellular retinoic acid-binding protein (CRABPI) and cellular retinol binding protein (CRBPI) was studied in a series of prenatal and early postnatal rats, covering the main stages of development and eruption of the molar teeth. CRABPI positive cells were found in the mesenchymal cells of the dental follicle from the cap stage and in the dental papilla from the early bell stage. In the dental papilla, CRABPI positive cells were situated adjacent to the enamel organ in the cervical loop region and in the subodontoblastic region. Newly formed odontoblasts were CRABPI positive for a short period of time. The enamel organ was CRBPI and CRABPI negative, except for the presence of CRABPI positive cells in the internal enamel epithelium over the tip of cusps and in parts of the stratum intermedium. During root formation, CRABPI positive cells were found in the developing periodontal ligament, in the dental papilla adjacent to the epithelial root sheath and in the subodontoblastic zone. During crown formation, CRBPI positive cells were mainly localized to the mesenchymal cells of the dental papilla during the cap stage of crown development. The periosteum of the developing mandible contained CRABPI positive cells while some osteoclasts appeared to show a weak but positive reaction to CRBPI. The findings were considered in terms of the possible significance of retinoid-binding proteins during tooth and bone development. PMID- 7554918 TI - C-CAM expression in odontogenesis and tooth eruption. AB - The distribution of the cell adhesion molecule C-CAM was analyzed during tooth development, eruption and formation of the junctional epithelium in rat molars by immunohistochemistry and in situ hybridization. C-CAM could not be detected during odontogenesis until the late bell stage; then only the mRNA was demonstrated in the odontoblasts and ameloblasts. Prior to eruption, a local increase in C-CAM (mRNA and protein) was observed in the reduced enamel epithelium. During eruption, high C-CAM levels were seen in the fusion zone between the oral epithelium and the reduced enamel epithelium. In the adult rat, C-CAM remained strongly expressed in the junctional epithelium. Our study indicates that C-CAM may play a role in odontogenesis and during formation of the epithelial structures involved in tooth eruption and formation of the junctional epithelium. PMID- 7554919 TI - Regulation of bone sialoprotein gene transcription by steroid hormones. AB - During the initial formation of bone, dentine and cementum in tooth morphogenesis, fully differentiated osteoblasts, odontoblasts and cementoblasts express bone sialoprotein (BSP), a mineralized tissue-specific acidic glycoprotein that has been implicated in the nucleation of hydroxyapatite crystal growth. The expression of BSP is regulated by steroid hormones that modulate mineralized tissue formation. Thus, the transcription of the BSP gene is induced by glucocorticoids in association with osteoblast differentiation and glucocorticoids also stimulate the expression of BSP in differentiated osteoblasts. In contrast, however, vitamin D3 suppresses bone formation and abrogates the expression of BSP. Our studies, using the osteoblastic cell lines ROS 17/2.8 and UMR 106-06, have revealed that the glucocorticoid (10(-8) M dexamethasone; dex) effect on BSP mRNA involves both direct and indirect pathways. To determine the molecular basis of the direct pathway on transcriptional regulation of the BSP we have isolated and characterized the promoter regions of both the human and rat BSP genes. The promoters are characterized by a highly conserved region (BSP box) encompassing the immediate promoter region, which includes a unique inverted TATA box overlapped by a putative (DR3) vitamin D3 response element (VDRE). Possible glucocorticoid response elements are present approximately 1 kb and approximately 1.4 kb further upstream. Transient transfection analysis of chimeric constructs linked to a luciferase reporter gene have shown Dex-stimulated expression in constructs that include one or both GREs, whereas vit D3 suppresses expression in a short construct that includes the VDRE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554920 TI - Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell differentiation. AB - A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers. PMID- 7554921 TI - Molecular biology experimental strategies for craniofacial-oral-dental dysmorphology. AB - This paper is a minireview of molecular biology experimental strategies for problems within craniofacial-oral-dental biology. Many of these strategies have already made remarkable contributions towards understanding the complex developmental processes associated with craniofacial biology. For example, the utilization of these strategies has resulted in the successful mapping of approximately 70 genes related to craniofacial anomalies (e.g., Pax, retinoic acid receptors, cadhedrins, aggrecan, cell adhesion molecules, substrate adhesion molecules, etc.), 30 genes related to dental tissue disorders (e.g., BMPs, bone morphogenetic proteins; dentin phosphoproteins, dentin sialoglycoproteins, enamelins, amelogenins), 20 genes related to facial clefting defects (e.g., Hox genes, transforming growth factor alpha), and 3 genes related to craniosynostosis (e.g., Msx-2). This minireview highlights selected examples of scientific progress derived from the following experimental strategies: (i) molecular approaches to the organization of the mouse and human chromosomes, with the mapping of specific gene sequences linked to human diseases (e.g., amelogenesis imperfecta, Boston type craniosynostosis, Rieger's syndrome, Treacher Collins syndrome); (ii) reverse genetic approaches for studies of gene function; (iii) homologous recombinations and the advances from "knock-out" transgenic mouse models for human craniofacial-oral-dental diseases; (iv) mutational analyses of congenital craniofacial-oral-dental dysmorphogenesis; (v) structural biology studies using computer-assisted molecular modeling for protein-protein, protein nucleic acid and protein-inorganic interactions; (vi) computer modeling of genetic paradigms; and (vii) a cluster of newer methodologies including computer assisted morphometry, new microinjection techniques, new cell membrane and intracellular dyes, and a number of new RNA and DNA viral constructs for the delivery of genes to enhance the resolution of cell fate maps, cell lineage studies and gene therapy approaches to human diseases. PMID- 7554922 TI - Understanding enamel formation from mutations causing X-linked amelogenesis imperfecta. PMID- 7554923 TI - The mineral and protein content of enamel in amelogenesis imperfecta. AB - The purpose of this investigation was to characterize the enamel composition of teeth affected with the hereditary enamel disorders known as amelogenesis imperfecta. Teeth from 10 individuals representing all 3 major AI types (hypocalcified, n = 3; hypomaturation, n = 3; hypoplastic, n = 4) and 10 normal teeth were studied. Half of each tooth was used for histological and biochemical studies. The enamel protein content was estimated by amino acid analysis. The enamel mineral content (volume %) was determined from the calcium and/or phosphorus content. Calcium was measured using atomic absorption and phosphorus was determined colorimetrically. The mean enamel mineral content was reduced for all hypomaturation and hypocalcified AI teeth while hypoplastic AI enamel varied from normal to reduced compared with normal enamel. The enamel protein content was increased in all but one AI case (7 cases were examined for protein) compared with the normal enamel. The mineral and protein content in AI enamel showed a significant inverse relationship (R = -0.939, P = 0.001). This study shows that all three of the major AI groups can have subtypes associated with substantial decreases in the enamel mineral content, although hypomineralization appears most severe in the hypomaturation and hypocalcified AI types. The decreased mineral content was associated with an increased protein content in AI enamel. These findings provide further evidence that altered enamel mineralization in AI teeth likely involves abnormal post-secretory processing of the enamel proteins. PMID- 7554924 TI - Ultrastructural study of tooth enamel with amelogenesis imperfecta in AI nephrocalcinosis syndrome. AB - This paper describes the ultrastructure of the affected enamel and the clinical features in two siblings with the syndrome of nephrocalcinosis and amelogenesis imperfecta. Nephrocalcinosis was diagnosed by intravenous pyelography, and confirmed by ultrasonography and CT scan. Amelogenesis imperfecta AI was diagnosed clinically and histologically. Light microscopy showed that the affected enamel surfaces were rough and the enamel was hypoplastic and mainly positively birefringent. Scanning electron microscopy revealed a rough and extensively cracked enamel surface covered with oval shaped blister-like protrusions. TEM showed porous enamel consisting of loosely packed and randomly oriented thin ribbon-like crystals with little or no prismatic structure. Observations showed that hypoplasia together with hypocalcification and/or hypomaturation defects were present in the same tooth, indicating the possibility of an abnormality in interstitial matrix, leading to dystrophic calcification in the kidney and abnormal tooth enamel formation, or alternatively an involvement of two separate but closely linked genes. PMID- 7554925 TI - Effects of thyro-parathyroidectomy and parathyroidectomy upon dentinogenesis: Part I: Light microscopy. AB - In order to determine the differential effects of the thyroid hormones and the parathyroid hormone upon dentinogenesis in the rat incisor one control group (C) and four groups of surgically treated rats were studied: parathyroid autotransplanted (PTT), thyroidectomized (TX), parathyroidectomized (PTX), and thyro-parathyroidectomized group. One month after surgery the incisors were dissected and the tissues were prepared for light microscopy and morphometric measurements. This study revealed modifications in the TPTX rats as well as in the PTX rats: an enlargement of the predentin, alterations in the predentin appearance and the presence of mineralization defects. These results confirm that the effects observed are probably due to a PTH deficiency and/or hypocalcemia and suggest that their occurrence is associated with a determined stage of dentinogenesis in the rat. PMID- 7554926 TI - Effects of thyro-parathyroidectomy and parathyroidectomy upon dentinogenesis: Part II: Electron microscopy. AB - An ultrastructural study was carried out in order to better characterize the findings observed in the first part of our study. The materials and methods are the same as those used in the preceding paper. This study reveals the occurrence of structures which display a symmetrical cross-banded pattern within the predentin and dentin of thyro-parathyroidectomized (TPTX) and parathyroidectomized (PTX) rats. A difference in the distribution of the symmetrical banded structures as dentinogenesis advances, as well as differences in the amount of the symmetrical banded structures between TPTX and PTX rats were observed. The symmetrical banded structures correspond with the so-called symmetrical SLS previously described in the incisor of normal and pathologic rats. The occurrence of these structures at a given stage of the incisor development suggests that the odontoblast is sensitive to the parathyroid hormone deficiency and/or hypocalcemia in a precise stage of its maturation. PMID- 7554927 TI - The spatial localization of Dlx-2 during tooth development. AB - The spatial distribution of Dlx-2 protein during murine tooth development has been investigated using immunohistochemistry with Dlx-2 antibodies. In common with several other homeobox genes expressed in toothgerms, Dlx-2 shows a multiphasic distribution in both epithelially and mesenchymally derived structures. This localization shows a number of similarities with the expression of Msx-2 and suggests a role for Dlx-2 in tooth initiation and tissue patterning. PMID- 7554928 TI - Coronal dentinal nodules induced by single or multiple injections of HEBP in young rats. AB - In the developing tooth, the bisphosphonate HEBP causes hypoplasias and hypomineralization of the enamel and dentine as well as inhibition of acellular cementum formation. Here, we describe a novel effect, associated with dentine mineralization. HEBP was administrated to young rats, and the maxillary molars were analyzed histologically. Localized dentinal nodules, protruding towards the pulp, were found in the developing crown of the molars. They occurred in regions, where the mantle dentine was about to mineralize at the time of the injection, and were more frequent at the mesial cusp side. The nodules accumulated mineral, as evidenced by the fluorescence after calcein and tetracyclin labelling. Histologically, the nodules were separated from the enamel by a layer of mantle dentine and were progressively surrounded by predentine and dentine. The nodules were interpreted to contain transport or metabolism intermediates, which were locally accumulated due to the interruption of the mineralization process by HEBP. PMID- 7554929 TI - Effects of vincristine on the developing hamster tooth germ in vitro. AB - Vincristine is one of the cytostatic drugs present in cocktails commonly used for the treatment of cancer in children. The aim of this study was to evaluate biochemically and histologically the toxic effects of this drug on the developing tooth in vitro using the organ culture model in order to be able to predict what damage the drug can induce in the developing teeth from children undergoing anti neoplastic chemotherapy. The most profound effect of the drug (10(-8)M-10(-4)M vincristine) on the developing tooth germ was the induction of mitotic arrests at the cervical loop and in the inter-cuspal regions. The 10(-4)M-10(-6)M vincristine doses were cytotoxic to most cells in the developing tooth germ. The 10(-7)M vincristine dose apart from induction of mitotic arrests, did not appear to be cytotoxic to the mature differentiated secretory cells. However, this dose induced incomplete nuclear polarization of the differentiating ameloblasts and odontoblasts. At 10(-8)M vincristine, the only effect observed were mitotic arrests; the secretory cells did not appear to have been affected at all. On the other hand, mineralization (TCA-soluble 45Ca and 32P uptake) was dose-dependently decreased from 10(-7)M vincristine upwards. 10(-9)M vincristine, the lowest dose tested, did not induce any changes in the developing tooth germ. The organ culture data indicate that 10(-9)M vincristine is the highest (safe) dose which does not induce any toxic effects in the developing hamster tooth germ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554930 TI - Influence of substrate nature and immobilization of implanted dentin matrix components during induction of reparative dentinogenesis. AB - The biological effects of isolated soluble dentin extracellular matrix components on the induction of reparative dentinogenesis in exposed cavities in ferret canine teeth have been shown to be blocked by immobilizing the extracellular matrix components on nitrocellulose or Millipore membranes during implantation. This contrasts with the picture of induction of odontoblast-like cell differentiation and reparative dentin deposition on existing insoluble dentin matrix of the exposure walls when the extracellular matrix components are implanted in lyophilized form. These data indicate the importance of an existing insoluble dentin matrix in providing a substrate to potentiate the growth factor like activity of soluble isolated dentin extracellular matrix components in the induction of reparative dentinogenesis. PMID- 7554931 TI - Induction of reparative dentinogenesis in vivo: a synthesis of experimental observations. AB - EDTA--and/or guanidine HCl--insoluble dentinal matrix, or demineralized dentin which had been treated with plasma fibronectin, or pieces of Millipore filters coated with a recombinant fibronectin-like engineered polymer, incorporating many RGD sequences, were implanted into central parenchymal sites of young dog molars, via mechanical pulp exposures. Furthermore demineralized dentin and Millipore filters coated with plasma fibronectin were placed into the central pulp of old animals. Histological analysis of buffered formalin-fixed tissues showed that: 1. The dentinogenic activity was retained in the EDTA--and/or guanidine-insoluble dentin matrix. 2. Implantation of Millipore filters supplemented with the recombinant polymer did not induce any odontoblast-like cell differentiation, indicating that the interactions of pulp cells with the exogenous fibronectin are not RGD-dependent. 3. Acid-insoluble dentin matrix or plasma fibronectin (both separately inducing dentinogenesis in dental pulp of young animals) did not show any dentinogenic activity when exposed in pulp sites of old animals. Acid insoluble dentin matrix and plasma fibronectin also failed to induce dentinogenic activity in the young pulpal tissues, when both factors were combined before to their implantation. Synthesizing the present data with previous relevant information it could be suggested that in the mechanism initiating reparative dentinogenesis, growth factors (endogenous or artificially implanted) and fibronectin are involved and this mechanism seems to be more complex than the simple immobilization of pulp cells onto an adhesion substratum. PMID- 7554932 TI - Responses of immunocompetent cells to cavity preparation in rat molars: an immunohistochemical study using OX6-monoclonal antibody. AB - Responses of immunocompetent cells, especially class II major histocompatibility complex (MHC) antigen-expressing cells, were investigated after cavity preparation in the erupted upper first molar teeth of rats, by immunohistochemistry using OX6-monoclonal antibody. In control teeth, OX6 immunopositive cells were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused an acute edematous reaction between the injured odontoblasts and predentin, and most of OX6-immunopositive cells in the affected site shifted away from the pulp-dentin border. After 12-24 hours, many OX6-immunopositive cells accumulated along the pulp-dentin border and extended their cytoplasmic processes into the exposed dentinal tubules. After 72 hours, newly differentiated odontoblasts replaced the degenerated odontoblasts, and few OX6-immunopositive cells remained along the pulp-dentin border. Our data suggest that some of the class II MHC antigen-expressing cells in the dental pulp participate in the initial defense reaction and presumably serve as a biological sensor for the external stimuli arriving through the exposed dentinal tubules. PMID- 7554934 TI - Analysis of tooth development in mice bearing a TGF-beta 1 null mutation. AB - While transforming growth factor-beta 1 (TGF-beta 1) and its related mammalian isoforms TGF-beta 2 and TGF-beta 3 are coexpressed in developing tooth organs, the specific biological role of each isoform is unknown. To delineate the role of TGF-beta 1 in odontogenesis, we have studied tissues from mice that lack a functional TGF-beta 1 gene. Histologic analyses revealed that in TGF-beta 1 (-/-) mice, tooth morphogenesis, cytodifferentiation and histogenesis were unaffected. Using in situ hybridization we studied the patterns of distribution of TGFs-beta 1, beta 2 and beta 3 in the TGF-beta 1 (+/+, +/- and -/-) genotypes. Our results indicate no detectable TGF-beta 1 mRNA in null tissues while TGFs-beta 2 and beta 3 showed normal temporal-spatial patterns of distribution. Using antibodies against TGF-beta 1, we observed immunoreactive TGF-beta 1 in tissues from null mice suggesting that maternally-derived TGF-beta 1 may be involved in the rescue of several developmental events in TGF-beta 1 knockout mice. PMID- 7554933 TI - Dlx and other homeobox genes in the morphological development of the dentition. AB - The dentition is a segmental system whose evolution and morphology bears analogy to the evolution of segmentation in the vertebral column and limb. Combinatorial expression of members of the large "Hox" class of homeobox regulatory genes has been shown to play an important role in positional specification in these skeletal systems. This raises the possibility that homeobox genes are also used for positional specification in the dentition, and several homeobox genes are known to be expressed in developing teeth. To identify additional dentally expressed homeobox genes, cDNA from from murine tooth germs at 9.5, 14.5, and 17.5 days gestational age was amplified by PCR using sets of degenerate primers to the homeodomains of 18 different classes of homeobox genes. Amplification products were cloned and sequenced and compared to known gene sequences. To date this approach has confirmed the presence of Msx1, Msx2, Dlx1, and Dlx2, and identified several other homeobox genes not previously known to be expressed in teeth: Dbx, MHox, and Mox2A, plus an a additional Dlx gene, Dlx7. The Msx and Dlx genes are the best current candidates for a combinatorial mechanism that controls the differentiation of structures within and between teeth, and perhaps also the evolution of those structures. PMID- 7554935 TI - EGF receptor expression in mineralized tissues: an in situ hybridization and immunocytochemical investigation in rat and human mandibles. AB - There is extensive evidence that growth factors play a central part in the autocrine/paracrine regulation of cell growth and differentiation in mineralized tissues. In order to investigate involvement of the EGFr receptor (EGFr) in forming mineralized tissues, its expression was studied by in situ hybridization and immunocytochemistry in mandibles of growing rats, as well as in human embryos. In Hertwig's epithelial root sheath of rat molar, EGFr mRNAs appeared strongly expressed, while dental pulp and dental follicle showed weak labeling. The lingual epithelium of rat incisor showed strong labeling, which decreased after epithelial dislocation. Cells of the adjoining lingual dental pulp and dental follicle, as compared to epithelium, contained a low level of EGFr mRNAs. In contrast, a significant signal with antisense RNA probe was observed in bone. Sense RNA probes provided a regular background or no labeling. Undifferentiated cells located in the periosteum and endosteal spaces were labeled. EGFr mRNAs were also present in osteoblasts and in lesser amounts in some osteocytes. In rat and in human bone, both osteoblasts and osteocytes were positive on immunostaining. Similarly in the Hertwig's root sheath, EGFr immunostaining and in situ hybridization labeling were closely related. These data show that different patterns of EGFr expression in forming mineralized tissues are tissue- and stage-specific. However, in all these cells, the present in situ investigation supports the assumption that EGFr is involved in the early stages of cellular proliferation and differentiation. This report also suggests that EGFr may play a role in differentiated and mature cells of mineralized tissues. PMID- 7554936 TI - Expression of basement membrane components in the dental papilla mesenchyme of monkey tooth germs--an immunohistochemical study. AB - The present work, which employs indirect immunoperoxidase methods, demonstrates electron microscopic localization of three major basement membrane (BM) components--type IV collagen, laminin, and heparan sulfate proteoglycan--at the early stages of odontogenesis in tooth germs of the Japanese macaque (Macaca fuscata). Intense immunostaining for each examined component occurred at the interface between the inner enamel epithelium and the dental papilla mesenchyme. At higher magnification, immunoreaction products were observed both in the lamina densa and lamina fibroreticularis. Fuzzy substances occurring very close to the lamina fibroreticularis manifested moderate immunoreactivity. In addition, immunostaining took place in the dental papilla mesenchyme. The dental papilla cells located close to the BM demonstrated immunoreactive material mainly on plasma membranes facing the BM. Reaction products were also observed in large concavities formed in some areas of the cell surfaces; and small, immunopositive vesicles occurred close to the plasma membrane. Immunoreaction products could be found in the cisternae of the rough endoplasmic reticulum of some mesenchymal cells. These findings suggest that dental papilla mesenchymal cells may produce the three major BM components and those of the components that are incorporated into the dental BM--particularly into the lamina fibroreticularis--during tooth development. PMID- 7554937 TI - Detection of bromo-deoxyuridine- and proliferating cell nuclear antigen immunoreactivities in tooth germ. AB - The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU- and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ. PMID- 7554938 TI - Cytoskeleton of the mesenchymal cells of the rat dental papilla and dental pulp. AB - This study describes the immunolocalization of actin, cytokeratins and vimentin during differentiation of the dental papilla in the rat. Incisors and first molars were sectioned from mandibles of Wistar rats from embryonic day (E)-14 to (E)-21 and weeks 1, 2, 3, 12 and 104 after birth, fixed in 90% alcohol, decalcified in EDTA, infiltrated with 5% sucrose, frozen in dry ice, and cryosectioned at 10 microns. The sections were immunolabelled using indirect immunofluorescence with a panel of monoclonal antibodies and FITC-phalloidin for F-actin localization. F-actin was present in follicular mesenchyme and odontoblast processes. Vimentin labelled dental papilla fibroblasts, differentiating, functional (secretory) and aged odontoblasts. Vimentin was uniformly localized in the cytoplasm of pre-odontoblasts but was redistributed to the apical pole of these cells during polarization. Of the cytokeratins, only cytokeratin 19 was found in differentiating odontoblasts. It was not present in dental papilla fibroblasts, functional or aged odontoblasts. These results suggest that actin and the redistribution of vimentin may be involved in odontoblast differentiation and odontoblast process formation/support and that these events may be preceded by the expression of cytokeratin 19. PMID- 7554939 TI - Epithelial-mesenchymal signaling during tooth development. AB - Classic studies on experimental embryology have shown that organ development in an embryo is largely regulated by so called inductive tissue interactions which mostly take place between epithelial and mesenchymal tissues. Also in the developing tooth, both morphogenesis and cell differentiation are governed by such interactions. Characteristic features of epithelial-mesenchymal interactions are that they are sequential and reciprocal, i.e. "induction" appears to consist of a chain of signaling events between the tissues. During the last decade, the expression patterns of numerous molecules have been studied in developing organs by in situ hybridization and immunohistology. Many of them have been associated with epithelial-mesenchymal interactions, and it is apparent that same molecules participate in regulation of morphogenesis in a number of different organs. Transcription factors such as Msx-1, Msx-2 and Egr-1, growth factors, including TGF beta's, BMPs, and FGFs, and structural proteins such as syndecan and tenascin are expressed in transient, time and space-specific patterns in many organ rudiments, including the tooth. We have shown by tissue recombination studies that the expression of certain molecules is indeed regulated by epithelial mesenchymal interactions in the early tooth germ. In particular, during the early stages of morphogenesis, when the dental epithelium induces the condensation of mesenchymal cells around the epithelial bud, the expression of many genes is upregulated in the condensed mesenchyme. Previous experimental tissue recombination studies have indicated that at the same time the capacity to instruct tooth morphogenesis shifts from the dental epithelium to the dental mesenchyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554940 TI - Enamel matrix proteins and ameloblast biology. AB - The paper reviews the changes in ameloblast ultrastructure, concomitant with the changes in its functions across the major stages of amelogenesis. It describes the mechanisms associated with the major events in biosynthesis and degradation of the major enamel proteins (amelogenins and tuftelin/enamelins) and with the presecretory and postsecretory mechanisms leading to the heterogeneity of these extracellular matrix proteins. The gene structure, chromosomal localization, protein, primary structure and possible function, and the involvement of the different proteins in X-linked (amelogenin) and possibly in autosomally linked (tuftelin) amelogenesis imperfecta, the most common hereditary disease of enamel, are also discussed. PMID- 7554941 TI - Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis. AB - Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis. PMID- 7554942 TI - Lipids in predentine and dentine. AB - Using two histochemical methods, malachite green-aldehyde and iodoplatinate, phospholipids were visualized in the predentine of rat incisors in the spaces located between collagen fibers and in dentine as needle-like structures located along individual or groups of mineralizing collagen fibers. The same staining pattern was seen with phospholipase A2-gold. Autoradiographic investigation using 3H choline as labelled precursor, visualized the incorporation of membrane associated and extracellular choline-containing phosphatidyl choline and sphingomyelin. The cell and membrane-associated labelling decreased gradually between 24 and 4 days, whereas incorporation of the labelled precursor as stable extracellular matrix component was seen in dentine. In addition to these investigations, pharmacologically induced (suramine) and genetically (Krabbe's disease) lysosomal storage pathology was investigated. Defects due to lipid metabolism alterations were seen in predentine and/or in dentine. The major differences visualized here between the non-mineralized and mineralized compartments and interactions between phospholipids and proteoglycans, support the view that phospholipids as matrix components play an important role in the mechanisms of dentine formation and mineralization. PMID- 7554944 TI - Enamel mineralization and the role of ameloblasts in calcium transport. AB - Amelogenesis is a dynamic and unique process of cell-matrix interactions in that matrix synthesis, degradation and resorption all proceed simultaneously, coupled with mineral depositions in a compartment between ameloblasts and dentin or dental papilla. Accumulation of data suggest the role of ameloblasts in tooth morphogenesis and matrix formation, but no fully acceptable explanation has been given concerning the role of ameloblasts in calcium transport. In this article, old and new points of issue raised regarding the role of ameloblasts in calcium acquisition are reviewed and possible mechanisms whereby the ameloblasts prevent the rise of cytosolic calcium while actively or less actively transporting calcium are elaborated upon based on recent findings. PMID- 7554943 TI - Collagen fibrils in the odontoblast layer in the teeth of the rat and the house shrew, Suncus murinus, by scanning electron microscopy using a maceration method. AB - It is not well known whether there are gaps in the tight junctions between odontoblasts and whether the fluid flows from the pulp to the predentin through these gaps. The collagen fibrils in the odontoblast layer were investigated using a maceration method in order to show the existence of the gaps between tight junctions of the odontoblasts. The mandibles containing teeth of the rat and the house shrew were digested by NaOH maceration and revealed the architecture of the collagen fibrils under scanning electron microscopy. The collagen fibrils went from the pulp, through the odontoblast layer, and were woven into the collagen network of the predentin in all teeth used in this study. Thick bundles of collagen were seen in the odontoblast layer at the pulp horn of the rat molars. Because there are many collagen fibrils in the odontoblast layer, it is considered that the tight junction of the odontoblast is of the discontinuous type. PMID- 7554945 TI - Localization of plasma membrane Ca2+ pump mRNA and protein in human ameloblasts by in situ hybridization and immunohistochemistry. AB - The distribution of the plasma membrane Ca(2+)-pump (PMCA) proteins in human ameloblasts was examined immunohistochemically using monoclonal antibodies JA8 and 5F10. Further, the distribution of mRNA transcripts derived from two PMCA genes, PMCA-1 and PMCA-4 was examined using in situ hybridization. In rats, the PMCA-1 gene is purported to code for PMCA proteins with a role in maintaining the intracellular Ca2+ levels in nonepithelial cells. Other genes including the PMCA 4 gene may code for PMCA proteins characteristic of Ca2+ transporting epithelia. The present results show immunohistochemical staining in the Tomes processes and plasma membranes of human ameloblasts. Our studies also demonstrate a gradation of expression of the PMCA-1 and PMCA-4 mRNA transcripts which parallels the onset and progression of enamel mineralization. These studies suggest that PMCA proteins in human ameloblasts may function both in intracellular Ca2+ homeostasis and in regulating the vectorial Ca2+ influx into mineralizing enamel. PMID- 7554946 TI - Mechanistic understanding of enamel mineralization under fluoride regime. AB - In order to learn more about how the microenvironment for enamel mineralization is modified by fluoride at low concentrations (0 through 1 ppm) and how excess fluoride retards the degradation and removal of amelogenins, we studied precipitation reactions in an in vitro model utilizing a dialysis chamber. The results showed that, with the limited supply of Ca ions through the ultrafiltration membrane, the solution composition surrounding the seed crystals showed a proximity to the steady-state condition after 12-24 h equilibration. Major findings were that (a) fluoride overcame partially the inhibition of precipitation and growth reactions by enamel proteins and (b), with this accelerating effect of fluoride, the steady-state Ca concentrations in the media surrounding the seed crystals decreased substantially as a function of fluoride concentration. The overall results support the concept that the presence of fluoride in the mineralizing milieu can modify markedly the steady-state concentrations of mineral lattice ions, particularly decreasing free Ca2+ concentrations, which in turn may modulate protease activities in situ. PMID- 7554947 TI - The control of ingress of albumin into developing enamel from adjacent dentine of the rat incisor. AB - The present study used an immunohistochemical approach to map the distribution of albumin within the dentine adjacent to the developing enamel of both impeded and unimpeded rat incisors to determine if the dentine could be a possible route of entry for this protein into the developing enamel matrix. In dentine adjacent to the secretory and transition stages of the developing enamel, the dentinal tubules were labelled only over approximately the pulpal quarter of their length. The bulk of the dentine showed no labelling. However, labelling within the dentine appeared at the ADJ at a position approximately 2.5 mm occlusal to the distal root of the first molar, adjacent to enamel with no visible residual matrix. The results of this study suggest that adventitious ingress of albumin into enamel from the dentine is restricted during enamel secretion and can only potentially occur once enamel maturation has been initiated. PMID- 7554948 TI - Ultrastructural organic-inorganic relationships in calcified tissues: cartilage and bone vs. enamel. AB - Close organic-inorganic relationships exist in all calcified tissues, the inorganic substance being linked to crystal ghosts (CGs). These are organic, crystal-like structures present in areas of initial calcification. In cartilage and bone, they form aggregates with the same morphology and distribution as the calcification nodules; in enamel, they consist of long filament- and ribbon-like structures, having the same arrangement as untreated crystals. CGs of cartilage and bone are acidic structures with histochemical properties of proteoglycans; CGs of enamel probably correspond to enamelins. The close morphologic similarity between CGs and crystals suggests that the former have a role in the formation of the latter. PMID- 7554950 TI - Structural relationship between the primary crystal formations and the matrix macromolecules in different hard tissues. Discussion of a general principle. AB - For many years we have investigated the earliest crystal formations of different developing hard tissues (matrix vesicle, bone, dentine, enamel, etc.) by different electron microscopic measurements. It was observed that primarily Ca phosphate (apatite) "chains," composed of nanometer sized particles (dots, islands), exist, which coalesce rapidly to needles. For the mineralization of collagen (e.g., bone, dentine) the center to center distances between the dots in the mineral chains represent the distances between nucleating sites, so-called "active sites" of collagen which bind primarily Ca for a subsequent nucleation. For the mineralization of noncollagen macromolecules (e.g., enamel) the same principle of mineral nucleation at such "active sites" exists being represented indirectly by corresponding center to center distances between the dots in the mineral chains. PMID- 7554949 TI - Dentin mineralization and the role of odontoblasts in calcium transport. AB - Dentin is formed by two simultaneous processes, in which the odontoblasts are instrumental--the formation of the collagenous matrix, and mineral crystal formation in this matrix. This pattern of formation is similar to that of bone, another mineralized connective tissue. Dentin and bone also have chemical compositions which are similar but with distinct differences. It is of fundamental importance to understand how the ions constituting the inorganic phase are transported from the circulation to the site of mineral formation and how this transport is regulated. For dentinogenesis, calcium is essentially the only ion for which data are available. Recent evidence suggests that a major portion of the Ca2+ ions are transported by a transcellular route, thus being under cellular control. The cells maintain a delicate Ca2+ ion balance by the concerted action of transmembraneous transport mechanisms, including Ca-ATPase, Na+/Ca2+ exchangers and calcium channels, and of intracellular Ca(2+)-binding proteins. The net effect of this is a maintenance of a submicromolar intracellular Ca2+ activity, and an extracellular accumulation of Ca2+ ions in predentin, at the mineralization front. Predentin can be regarded as a zone of formation and maturation of the scaffolding collagen web of the dentin organic matrix. In addition to collagen, it contains little but proteoglycan. Simultaneous with mineral formation, additional non-collagenous macromolecules are added to the extracellular matrix of dentin, these presumably being transported within the odontoblast process. Among these are highly phosphorylated dentin phosphoprotein (phosphophoryn) and another pool of proteoglycan.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554951 TI - Elemental distributions in predentine associated with dentine mineralization in rat incisor. AB - Electron probe microanalysis was applied to study quantitatively and semi quantitatively the elemental concentrations and distributions that occur in predentine during the dentine mineralization of rat incisor. Apex regions of the continuously growing incisors were rapidly dissected and cryofixed in liquid nitrogen-cooled propane. Ultrathin cryosections were prepared from the dentine tissue. On the average in the extracellular predentine element concentrations of calcium and phosphorus were about 0.5% (w/w) and 0.5-1% (w/w), respectively; so the calcium content in the extracellular predentine is higher while the phosphorus content is much lower than in the odontoblast area. Due to the high content of glycosaminoglycans in the extracellular matrix the concentration of sulfur in the predentine was more than 1% (w/w); the potassium content was found in the range of 0.6-0.8% (w/w) which is quite high for an extracellular area and the concentrations of sodium and chlorine were higher than 2% (w/w). Elemental mapping analysis was carried out to demonstrate the distribution of some important elements at the predentine/dentine border during mineralization. PMID- 7554952 TI - A new method for in vitro calcification using acrylamide gel and bovine serum. AB - To investigate the mechanism of biological calcification in vitro, a model system consisting of an acrylamide gel block (1 x 3 x 3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 microliters of fetal bovine serum at 37 degrees C for 7 days in a CO2 incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO2 and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10-60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO2 the Ca content in the gel block increased, reached the highest level at about 3.5% CO2 and then began to decrease. In 40% gel and at 5% CO2, the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO2 greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro. PMID- 7554953 TI - Cementum specific components which influence periodontal connective tissue cells. AB - We have isolated two polypeptides from cementum one of which promotes the growth and the other the attachment of periodontal cells. One polypeptide, the cementum derived growth factor (CGF), was extracted from healthy human and bovine teeth by 1 M CH3COOH and purified by heparin-affinity chromatography and HPLC. The CGF is a 23 kDa polypeptide which is mitogenic to fibroblasts and smooth muscle cells. It is active alone, but its activity is highly potentiated by plasma-derived serum or EGF. It induces classical mitogenic signaling events, which include Ca++ mobilization, inositol phosphate hydrolysis, activation of phosphokinase C (PKC) and transcription of cellular protooncogenes c-fos and jun-B. The magnitude and pattern of activation of signaling events and their susceptibility to PKC inhibitors and pertussis toxin indicated that the CGF may be a distinct molecular species. The CAP is a 55 kDa polypeptide which promotes the attachment and spreading of fibroblasts, smooth muscle cells, bone cells and endothelial cells, but not epithelial cells. Antibodies to CAP immunostain cementum, but not other tissues. Root surfaces bind CAP. The CGF and CAP do not appear to be present in adjacent periodontal structures. Our data show that the CAP and CGF selectively interact with periodontal cell populations and affect their biological activities, and thus may influence the formation and regeneration of periodontal connective tissues. PMID- 7554954 TI - Thermal stability of mineralized and demineralized dentin: a differential scanning calorimetric study. AB - The purpose of this study was to determine the difference, if any, in the thermal stability of collagen in mineralized and demineralized dentine compared to that in unmineralized tissues, using differential scanning calorimetry, DSC. Human tooth dentin blocks, about 1 x 1 x 2 mm in size, were used in this study. Some dentin blocks were demineralized using a Plank Rychlo solution; others, using EDTA solution. The mineralized dentin showed an exothermic peak at about 310 degrees C and the combustion of organic materials was completed at about 450 degrees C. For the demineralized dentin, the combustion was completed at higher temperature range and showed a strong exothermic peak at about 470 degrees C. An exotherm at the temperature between 450 degrees C and 470 degrees C was also observed in DSC pattern of native type I collagen from calf skin and rat tail tendon. DSC pattern of rat tail collagen showed a close similarity to that of the demineralized dentin. Statistically, the same heat flow value was obtained both from the mineralized dentin and the demineralized dentin and from the native type I collagen. These findings indicated that the thermal stability of collagen in dentin is lower than collagen in uncalcified connective tissue. It is suggested that in calcified collagen, the apatite crystallites may have intruded into spaces of the crosslinks of intra- and inter-fibrils, and in so doing, destroyed the crosslinks. PMID- 7554955 TI - Oriented growth of octacalcium phosphate on and inside the collagenous matrix in vitro. AB - To investigate the factors which regulate the growth of apatite in a collagenous matrix, a calcification experiment was carried out in a model system, where Ca2+ and PO43- ions diffused into a slice of Achilles tendon from mutually opposite grew on the Ca-side of the collagenous matrix, while small plate-like OCP crystals (< 1 micron) grew inside the matrix. The major part of crystals grew with the c-axis parallel to the collagen fibers. Others grew with the c-axis parallel to the collagen bands or perpendicular to the fibrils. Crystals grew with orientation on collagen fibrils from the beginning. It was concluded that collagen fibrils and property of the collagenous matrix played a regulatory role in the deposition and growth of OCP in the collagenous matrix. PMID- 7554957 TI - Ganoine formation in the scales of primitive actinopterygian fishes, lepisosteids and polypterids. AB - The scales of primitive living actinopterygian fishes, lepisosteids and polypterids, have retained ganoine, a hypermineralized layer which covered the scales of the osteichthyan ancestors. To know finally its tissue origin in the actinopterygian lineage, ganoine formation was described in Lepisosteus oculatus, with scales devoid of dentin, and was compared to ganoine formation in two polypterids, Calamoichthys calabaricus and Polypterus senegalus, with scales possessing a dentin layer. The events taking place before, during and after ganoine deposition were studied in experimentally regenerated scales using light and transmission electron microscopy. In spite of differences in tissue composition and in organization of the epidermal cells on the scale surface, ganoine formation is similar in both types of scales. Preganoine is deposited by epidermal cells and constitutes a thick layer which mineralizes progressively to become ganoine, a true enamel. The cellular processes involved in ganoine formation were compared to those described for enamel in mammalian teeth. PMID- 7554956 TI - Synergistic effects of magnesium and carbonate on properties of biological and synthetic apatites. AB - Magnesium (Mg) and carbonate (CO3) are minor elements associated with enamel, dentin and bone apatite. The purpose of this study was to determine the effect of Mg and CO3 on some properties of synthetic apatites to gain insights on their effects on biological apatites. Biological apatites from human enamel and dentin and from bovine bone and synthetic apatites with/without Mg or CO3 were characterized using x-ray diffraction, infrared absorption, thermogravimetry and chemical analyses. Dissolution in acidic buffer was also determined. Results from this study demonstrated: (1) the synergistic effects of Mg and CO3 on reducing the crystallinity and increasing the extent of dissolution of synthetic apatites; (2) dentin and bone, compared to enamel apatite contained higher levels of Mg and CO3; had lower crystallinity and higher extent of dissolution. The lower crystallinity and higher extent of dissolution of dentin and bone compared to enamel apatite may be partly attributed to their higher Mg and CO3 concentrations. PMID- 7554958 TI - Evidence of two types of odontoblasts during dentinogenesis in elasmobranchs. AB - The fine structure of the odontoblasts in the sting rays, Dasyatis akajei, Dasyatidae, and Urolophus aurantiacus, Urolophidae, was examined using light and transmission electron microscopy. In the dentinogenesis stage, the odontoblasts have been classified into two types, that is, dark cells and light cells, based on differences in their fine structure. Many dark odontoblasts found along the predentine displayed well-developed organelles with secretory activity around the nuclei. They contained large amounts of expanded rER, widely distributed Golgi apparatus and secretory granules. In contrast, light odontoblasts showed a relatively clear cytoplasm and extended long processes which passed through the predentine and penetrated into the dentine. They contained large numbers of microtubules in the processes and many mitochondria around the nuclei. It is suggested that the light odontoblasts play an important part in material transport to the dentine and/or act as a sensory organ of the tooth. The dark odontoblasts seem to produce the organic matrix of the dentine and to prepare for mineralization in the dentine. PMID- 7554959 TI - The effect of glycosylaminoglycans on the mineralization of sheep periodontal ligament in vitro. AB - The effect of removal of glycosylaminoglycans on the mineralization of sheep periodontal ligament was determined using enzyme digests followed by incubation in solutions supersaturated with respect to hydroxyapatite at pH 7.4. TEM revealed that control periodontal ligament remained unmineralized. However, tissue from which glycosylaminoglycans had been removed contained plate-like crystals arranged parallel to and within the collagen fibrils. Electron probe and electron diffraction studies suggested that the crystals were apatitic with a similar order of crystallinity to dentine, and a Ca:P ratio of 1.61. In addition, the glycosylaminoglycan content of periodontal ligament, cementum and alveolar bone was compared using cellulose acetate electrophoresis. Periodontal ligament contained predominantly dermatan sulfate while cementum and alveolar bone contained mostly chondroitin sulfate. A role for glycosylaminoglycans in maintaining the unmineralized state of the periodontal ligament is suggested. Control of expression of specific proteoglycan species on a spatially restricted basis is presumably central to this role. PMID- 7554960 TI - The effects of aging upon the connective tissues of the periodontal ligament. AB - The ultrastructural features of the cells and of the oxytalan fibers within the periodontal ligaments of aged rats (2 year old) were quantified and compared with data for tissues obtained from younger animals (8 week old). Sections of the mid root regions of the mandibular first molars were prepared for examination by TEM. The fibroblasts of the aged rats were found to differ in 3 respects: the areas occupied by endoplasmic reticulum were significantly less, the areas occupied by intracellular collagen profiles were also less, and both the numbers and sizes of intercellular contacts were significantly different (p < 0.05). For the oxytalan fibers, no differences were observed between the periodontal ligaments of the aged and control animals both in terms of numbers of fibers per 50 microns and in terms of area of tissue occupied. Thus, in contrast to the apparent lack of age changes so far determined for the extracellular matrix of the periodontal ligament (collagen fibrils and oxytalan), the periodontal fibroblasts exhibit some age changes as perceived at the ultrastructural level. PMID- 7554961 TI - Ultrastructural characterization of mesenchymal and epithelial cells co-cultured from human dental root apical explants. AB - Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium beta glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of beta glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation. PMID- 7554962 TI - Immunohistochemical characterization and localization of MHC class II antigen presenting cells in the periodontal ligament of rat incisors. AB - Dendritic cells have been identified both in lymphoid and nonlymphoid tissues as non-phagocytic antigen-presenting cells, equipped with extensive flamelike cytoplasmic projections. Our immunohistochemical study revealed presence of a large population of dendritic cells and other immunocompetent cells, showing a region-specific distribution, in the lingual periodontal ligament of continuously erupting rat incisors. This study aims to reveal the kinetics and cytological characterization of immunocompetent cells in the periodontal ligament of rat incisors with special reference to their differentiation pathway in the unique local environment. PMID- 7554963 TI - Dentin matrix proteins and dentinogenesis. AB - The precise mechanisms involved in dentinogenesis are not understood; however, the information to date suggests that a number of highly controlled extracellular events are involved. Mature odontoblasts secrete collagen at the cell border into predentin. They synthesize and secrete other non-collagenous proteins (NCPs) at the mineralization front, possibly through odontoblastic processes. A collagen NCP complex is formed at the predentin-dentin border and apatite crystal initiation and growth takes place. One of the research needs is to uncover the nature of this dentin collagen-NCP complex and to understand how it controls mineralization. At least three dentin specific NCPs are known: phosphophoryn(s), dentin sialoprotein (DSP) and AG1 (Dmp1). Other macromolecules are commonly made by osteoblasts and odontoblasts and participate in bone and dentin formation. Some progress in understanding dentin mineralization has been gained by focusing upon the role of phosphophoryns. These highly phosphorylated proteins are secreted at the mineralization front, where a small portion binds in the gap region of type I collagen fibrils. This portion of phosphoproteins probably initiates formation of plate-like apatite crystals. Additional phosphoryns in higher concentrations bind to the growing apatite crystals and slow their growth, possibly influencing their size and shape. Other areas which need careful investigations are those involving the mechanisms involved in odontoblast differentiation, how the synthesis of the dentin specific NCPs is controlled and the precise roles of these macromolecules in dentinogenesis. Future experimentation will focus on the gene structures for these NCPs and the mechanisms of tissue specific gene regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554964 TI - In situ hybridization shows Dmp1 (AG1) to be a developmentally regulated dentin specific protein produced by mature odontoblasts. AB - Acidic phosphorylated proteins are prominent constituents of the extracellular matrix of bone and dentin. It has been postulated that they may have important structural and regulatory roles in the process of tissue mineralization. Studies of a cDNA library, prepared from cells of the rat incisor odontoblast-pulp complex of 3 week old Sprague-Dawley rats, led to the identification of a serine rich acidic protein, designated AG1, which appeared to be a dentin matrix component. In order to determine which cells of the odontoblast-pulp complex were responsible for the making of AG1, in situ hybridization was carried out using digoxigenin-labeled probes. The full length AG1 cDNA was subcloned into the pBluescript vector, which contains two strong promoters, T3 and T7. The sense and antisense complementary RNA (cRNA) hybridization probes were prepared by in vitro transcription using T3 and T7 polymerases in the presence of 11-dUTP. Incisor sections were obtained from rat embryos at days 16, and 20, and newborns at days 2 and 5. No AG1 mRNA was detected in the embryonic sections, but digoxigenin labeling was evident in odontoblasts secreting mineralizing dentin at postnatal days 2 and 5. Sense probes showed no hybridization. Pulp cells, Meckel's cartilage, and alveolar bone were free of hybridization with the antisense probe. Unexpectedly, a low level of digoxigenin staining was seen in the cytoplasm of secretory ameloblasts, but not in the preameloblasts, stratum intermedium or stellate reticulum of the enamel organ. These data show that AG1 expression is regulated developmentally and is restricted to secretory stage mature odontoblasts. PMID- 7554965 TI - Molecular analysis of rat dentin sialoprotein. AB - Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome. PMID- 7554966 TI - Identification of regulatory elements necessary for the expression of the COL1A1 promoter in murine odontoblasts. AB - Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes. PMID- 7554967 TI - Characterization of protein kinases involved in dentinogenesis. AB - Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP. PMID- 7554968 TI - Models for the study of cementogenesis. AB - Cementum is a mineralized tissue that acts to connect the periodontal ligament to the tooth root surface. Its composition is very much like bone, being comprised mainly of type I collagen, inorganic mineral and noncollagenous proteins, however the origin of the cells and factors necessary for cementum formation have yet to be elucidated. Our laboratory has focused on the role that adhesion molecules, and their cell surface receptors, play in the formation of cementum and tooth root. In order to study this, we used a mouse molar as a model system. This system enabled us to study the formation of four distinct mineralized tissues; bone, cementum, dentin and enamel at various stages of their development. For these studies, we initiated experiments to examine potential cementoblast progenitor cells, in vitro. As a first step, we show that dental papilla and dental follicle cells, n vitro, obtained from molar tissues at day 21 of development, induce mineralized nodules, in vitro. In addition, we obtained tissues from mice where defects in root development may exist and determined bone sialoprotein (BSP) protein expression, a mineralized tissue specific adhesion molecule, in such tissues. As discussed here, we found that osteopetrotic (op/op) mice have delayed and/or defective root development and BSP does not localize in the dental tissues, at day 33 of development. In addition, dentin formation was defective and odontoblasts appeared immature, based on morphological examination. In contrast, the day 33 control molars demonstrated positive staining for BSP localized to root cementum, with normal formation of dentin. PMID- 7554970 TI - Combined oral contraceptives: do we know all of their effects? AB - Combined oral contraceptives are undoubtedly popular. By the end of the 1980's, an estimated 63 million married women around the world were using this method of contraception. In Britain, perhaps 95% of all sexually active women have used the pill at some time before their 30th birthday. Commensurate with such widespread usage, huge amounts of money have been spent during the past 35 years investigating the health effects of this method of contraception. Since it appears that all of the potential risks and benefits have now been identified, can we divert resources from pill-related research into new areas of activity. While this proposition may be attractive to funding bodies, and other researchers competing for increasingly scarce resources, it ignores the fact that several major uncertainties remain concerning the safety of combined oral contraceptives. PMID- 7554969 TI - Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line. AB - During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7554971 TI - Ovarian function during low-dose oral contraceptive use. AB - Lowering the total steroid dose in modern oral contraceptives (OCs) has been connected with a higher incidence of ovarian follicle and cyst formation. To investigate the presence of ovarian follicles and cysts by means of vaginal ultrasonography and serum hormone determinations during use of two low-dose OCs, 65 volunteers were randomized to receive either 20 micrograms ethinylestradiol (EE) + 150 micrograms desogestrel (group A) or 35 micrograms EE + 250 micrograms norgestimate (group B) for a 2-month study period. At baseline, 39% of women in group A and 31% in group B exhibited at least one follicle < 35 mm in diameter. By the end of the second treatment cycle, the frequency of these follicles had decreased to 14% in each group. Only one subject in the higher estrogen group developed an ovarian cyst > 35 mm. One subject in each group demonstrated hormone levels characteristic of ovulation; no pregnancy occurred in either group. The 20 micrograms EE preparation was not found to lead more often to ovarian follicles or cysts when compared with a 35 micrograms EE preparation, possibly because of the type and dose of the progestogen used. PMID- 7554972 TI - The effect on blood pressure of a monophasic oral contraceptive containing ethinylestradiol and gestodene. AB - To obtain an overview of the effect of monophasic gestodene on blood pressure and to determine the frequency of "OC elevated BP/hypertension," the results of blood pressure monitoring from four clinical studies of contraceptive efficacy and safety have been retrospectively analyzed. A total of 1930 women took part in the studies, which recorded BP for up to 24 cycles. Analysis of results revealed that 97 women (5.0%) showed an increase in blood pressure from previously normal to elevated values while taking monophasic gestodene, with only 26 (1.35%) fulfilling the criteria of "OC elevated BP/hypertension." Only four women dropped out of the trials due to hypertensive blood pressure values, while 67 women (3.5%) experienced a normalization of previously elevated blood pressure measurements. In conclusion, this analysis has confirmed that gestodene has a negligible effect on blood pressure, with increased BP a relatively rare event. PMID- 7554973 TI - Effect of two oral contraceptives containing ethinylestradiol and gestodene or norgestimate upon androgen parameters and serum binding proteins. AB - The effect of a triphasic oral contraceptive containing ethinylestradiol and gestodene (EE/GSD) on various serum hormonal parameters was compared with that of a monophasic formulation containing 35 micrograms ethinylestradiol and 250 micrograms norgestimate (EE/NGM). Blood samples were collected from 46 women on days 2, 11, and 21 of the preceding control cycle and of the third, sixth and twelfth treatment cycle. There was no significant difference in the influence on any hormonal parameter between both formulations. Both EE/GSD and EE/NGM caused a time-dependent suppression of serum dehydroepiandrosterone sulphate (DHEA-S) by 20-30% (p < 0.01) and a reduction of 5 alpha-androstane-3 alpha, 17 beta-diol glucuronide by 50-60% (p < 0.01) during each treatment cycle, while androstenedione levels were reduced by 25% (p < 0.01). There was also a significant decrease in the levels of total testosterone by 30-35% (p < 0.01) and free testosterone by 60% (p < 0.01), while sex hormone-binding globulin (SHBG) was increased by 200-240% on days 11 and 21 (p < 0.01). During the pill-free interval the SHBG levels were reduced to a certain degree but remained elevated by 100% as compared to the pretreatment values. The serum levels of corticosteroid-binding globulin (CBG) which is known to be influenced only by the estrogenic component of combination pills, increased significantly by 170% (p < 0.01) during each treatment cycle. During the pill-free interval of 7 days, the CBG levels decreased but were still elevated by 90-100% as compared to the control cycle. Similarly, the serum levels of cortisol were significantly elevated by 110-140% (p < 0.01) during treatment with both preparations. The results demonstrate a profound suppression of androgen levels and peripheral androgen metabolism. PMID- 7554975 TI - Depression in users of depo-medroxyprogesterone acetate. AB - Prevalence of depression is high among poor, young, Hispanic inner city women. Depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in this group. DMPA labelling suggests that depression may worsen with use. In order to identify any association of DMPA use with worsening depression, we surveyed an English-speaking subset of DMPA users in a Title-X funded family planning clinic. Eighty women completed the CES-D scale on two occasions: once about four weeks after a DMPA injection when the subject would have been exposed to the highest blood levels, and once immediately prior to an injection when recent blood levels of the drug would be somewhat lower (or absent preceding the first injection). The median CES-D score was 14. The scores were not related to timing of the test (pre- or post-injection). The depression scores were somewhat higher among those women receiving their first DMPA injection during the study period (i.e., unexposed women) and among those women who had received four or more injections. Scores were unrelated to age or parity, but were somewhat higher in women who reported fewer years of education or a recent adverse pregnancy outcome. These data provide little evidence of increasing depression with long-term use of DMPA and no evidence of a short-term effect of dose (within the contraceptive range) on mood. Women at risk of depression should not be denied DMPA as a contraceptive choice. PMID- 7554974 TI - Oral misoprostol versus vaginal gemeprost for cervical dilatation prior to vacuum aspiration in women in the sixth to twelfth week of gestation. AB - The effectiveness of oral misoprostol versus vaginal gemeprost for cervical dilatation prior to vacuum aspiration was compared in women in the 6th to 12th week of pregnancy. Sixty-four nulliparous women requesting termination of pregnancy between 6th to 12th weeks of gestation were randomized to receive either 400 micrograms misoprostol orally or 1 mg vaginal gemeprost at 12 hr or 3 hr prior to vacuum aspiration, respectively. The cervical dilatation at vacuum aspiration, the ease of the subsequent surgical procedure, and the incidence of complications and side effects were compared between these two methods of cervical priming. The median cervical dilatation at vacuum aspiration in the misoprostol group was significantly greater than that in the gemeprost group (8.0 mm versus 7.0 mm, p < 0.02). Preoperative side effects were significantly less frequent in the misoprostol group (p < 0.01). The ease of dilatation assessed subjectively by the operating surgeons was also improved significantly in the misoprostol group (p < 0.01). However, the duration of operation and blood loss were similar in both groups. Since misoprostol is also much cheaper and more convenient to use, we conclude that oral misoprostol is better than vaginal gemeprost for cervical dilatation prior to vacuum aspiration in first trimester pregnancy. PMID- 7554976 TI - Comparative study on intermittent versus continuous use of a contraceptive pill administered by vaginal route. AB - A multicenter, international, randomized, comparative trial was conducted to assess the acceptability, efficacy and safety of two different schedules of a contraceptive pill, containing 250 micrograms levonorgestrel and 50 micrograms ethinyl estradiol, administered by the vaginal route. One schedule of daily administration for 21 days with a seven-day interruption to allow withdrawal bleeding was compared to daily administration without interruption for bleeding. A total of 900 women were recruited in three countries, Brazil, Egypt and China; 7,090 women-months of vaginal pill use were recorded (3,364 using the pills intermittently and 3,726 continuously). Four undesired pregnancies occurred, one in Egypt and three in China, all four in women using the pills intermittently. There was a statistically significant difference (p = 0.486) in pregnancy rate between the two groups. There were no other significant differences in discontinuation rates despite marked differences in bleeding patterns, amenorrhea predominating in the continuous use group. Hemoglobin levels increased significantly in the two groups but hematocrit was significantly higher in the continuous use group. PMID- 7554979 TI - Glycation, oxidation, and glycoxidation reactions in the development of diabetic complications. PMID- 7554977 TI - Effect of levonorgestrel-releasing intrauterine device on hormonal profile and menstrual pattern after long-term use. AB - In the present study 14 women after 6 years' use of levonorgestrel-releasing IUD were investigated for the changes of LH, progesterone (P), estradiol (E2), prolactin (PRL) and serum binding globulin (SHBG) in relation to the levonorgestrel levels throughout a segment of 26-40 days with the aim of comparing the hormonal profiles with those during the first year of use of Lng IUD. Ultrasound scanning was used to follow the development of follicles along with the RIA measurement of hormones. The results of serum LH, P and E2 showed ovulation in 11 cases with either normal menstrual cycles (5 cases), prolonged or irregular cycles (4 cases) or with amenorrhea for 2-3 years (2 cases). One case showed insufficiency of luteal function and 2 cases showed anovulation but with follicular hyperactivity. Higher percentages of ovulatory cycles (78.5%) were found after 6 years of use. No case of complete suppression of ovulation was found. Anovulatory cycles only constituted 14.3%. Clinically, the development of follicles followed by ultrasound scanning further confirmed the hormonal findings. The persistent enlargement of follicles coincided with high levels of E2. After 6 years of use, the serum levels of levonorgestrel were still maintained at mean levels of 314.26 pmol/L and 470.63 pmol/L in the ovulatory and anovulatory groups, respectively. It is concluded that over two-thirds of the cases have ovulatory cycles after long-term use of Lng-IUD; the contraceptive effect is mainly due to its local action on the endometrium, with much less effect on the ovarian function. PMID- 7554978 TI - Time-related effects of a triphenylethylene antiestrogen on estrogen-induced changes in uterine weight, estrogen receptors, and endometrial sensitivity in rats. AB - Time-related estrogen antagonistic action of a single oral contraceptive (1.25 mg/kg) dose of the triphenylethylene antiestrogen centchroman was determined in ovariectomized immature rats. Tamoxifen and nafoxidine were used for comparison. A single oral administration of centchroman followed by three doses of estradiol 17 beta (1 microgram/d, s.c.) caused significant dose-dependent inhibition in estradiol-17 beta-induced increase in uterine weight and nuclear and cytosolic estrogen receptors. But the inhibition at antiimplantation dose was evident only if estradiol-17 beta treatment was initiated not later than 48 h post antiestrogen. Alternatively, when antiestrogen treatment was followed by a single dose of estradiol-17 beta between days 2-7, a synergistic action, typical of antiestrogens possessing weak estrogen agonistic activity, was observed. In immature rats in which a condition mimicking preimplantation was produced by estradiol-17 beta (0.5 microgram/d, s.c.) priming on days -2 and -1, followed by progesterone (1 mg/d, s.c.) and an endometrial sensitizing dose (0.5 microgram/d, s.c.) of estradiol-17 beta at 1600 h on day 4, anti-implantation dose of centchroman administered on day 1, too, failed to inhibit uterine weight gain induced by sensitizing dose of estradiol-17 beta, but caused marked inhibition in endometrial sensitivity to a deciduogenic stimulus and decidualization and weight gain of traumatized uterine horn 96 h post-traumatization over non-traumatized horn was only about 150% (725% in controls). Inhibition in endometrial sensitivity and decidualization was evident when the interval between antiestrogen treatment and sensitizing estradiol was < 126 h. Pinopods were present on endometrial surface on day 5 whether or not priming and/or sensitizing doses of estradiol were administered, but decidual response was mild if either of these doses of estradiol-17 beta was deferred. Findings suggest that: (a) duration of antiestrogenic action of single anti-implantation dose of centchroman in rat was about 126 h, which in ovariectomized immature rats was evident only when a condition mimicking preimplantation was produced and the antiestrogenic response was based on inhibition in estradiol-induced endometrial sensitivity and not uterine weight gain; (b) priming as well as sensitizing estrogen were essential to get optimal decidual responses; (c) appearance of pinopods on endometrial surface may not be related to endometrial sensitivity; and (d) tamoxifen and nafoxidine appear slightly longer acting with duration of antiestrogenic action of approximately 150 h. PMID- 7554981 TI - Oxidative damage of glycated protein. PMID- 7554980 TI - Biochemical and histopathological study of beta 2-microglobulin amyloidosis. PMID- 7554983 TI - Dialysis-related amyloidosis: from raw clinical facts to unresolved questions. PMID- 7554982 TI - Effectiveness of new push/pull hemodiafiltration for arthralgia in long-term hemodialysis patients. PMID- 7554984 TI - Influence of dialysis membranes on the development of dialysis-related amyloidosis. PMID- 7554985 TI - Prevention of dialysis-related amyloidosis using the polyacrylonitrile AN69 membrane. PMID- 7554986 TI - Development of a PMMA membrane which can remove beta 2-microglobulin and its clinical significance. PMID- 7554987 TI - Beta-2-microglobulin removal by different hemodialysis membranes. PMID- 7554988 TI - Effects of a new adsorbent column for removing beta-2-microglobulin from circulating blood of dialysis patients. PMID- 7554990 TI - Formation of methylglyoxal-modified proteins in vitro and in vivo and their involvement in AGE-related processes. PMID- 7554989 TI - Glycation of metal-containing proteins such as Cu,Zn-superoxide dismutase, ceruloplasmin, and ferritin: possible implication for DNA damage in vivo. PMID- 7554991 TI - Structures of advanced glycation end products and their role in pathophysiological states. PMID- 7554992 TI - Crosslines, fluorophores in the AGE-related cross-linked proteins. AB - We can summarize our results as follows: (1) A pair of fluorescent crosslines were isolated from the Maillard reaction mixture; (2) AGE-proteins contained crossline-like structures, and (3) crossline-like immunoreactivities were accumulated in renal tissues of diabetic rats. From these results we concluded that fluorophores in AGE proteins have crossline-like structures and we had the first indication that XLs could be markers for renal disorders. PMID- 7554994 TI - Size heterogeneity of amyloid fibril proteins of beta 2-microglobulin origin in hemodialysis and disease-specific antibodies. PMID- 7554993 TI - Beta 2-microglobulin modified with the AGE products of the Maillard reaction in dialysis-related amyloidosis. PMID- 7554995 TI - Urinary and serum beta 2-microglobulin in living related kidney donors and in renal failure. PMID- 7554996 TI - Glycosaminoglycans in dialysis-related amyloidosis. PMID- 7554997 TI - Clinical aspects of dialysis-related amyloidosis. PMID- 7554998 TI - Role of macrophages in beta 2-microglobulin-related dialysis amyloidosis. PMID- 7554999 TI - Histamine, histamine H2-receptor antagonists, gastric acid secretion and ulcers: an overview. AB - Histamine, a biogenic amine, is involved in allergic reactions and asthma. The involvement of histamine in peptide ulcers is reviewed here. The discovery, distribution, synthesis, catabolism, and pharmacological effects of histamine are briefly described. Histamine actions are mediated by more than one type of receptor. The discovery, development and mode of action of H2-antagonists is discussed. A brief comparison of the clinical profiles (dosage regimen, metabolism and drug interactions) of the four currently used H2-antagonists (cimetidine, ranitidine, nizatidine and famotidine) is given. Furthermore, due to their ability to bind to cytochrome P-450, these compounds have the potential to interfere with the hepatic clearance of other drugs which are also metabolised by the mixed-function oxidase system in man. Therefore, a brief discussion of their adverse effects and drug interactions is included. Modulation of gastric acid secretion, in particular the role of cAMP and the proton pump, is described. Peptic ulcer is a major disease in the Western world and the aetiology and treatment of peptic ulcer are summarised. PMID- 7555000 TI - Inhibitory effects of corticoids on reductive halothane dehalogenation. AB - The effects of corticoids, hydrocortisone, methylprednisolone, betamethasone and dexamethasone, on the reductive metabolism of halothane to produce chloro difluoroethylene (CDE) and chlorotrifluoroethane (CTE) in the liver microsomes of guinea-pig were examined. The substrate differential spectrum for methylprednisolone showed a peak at 412 nm and trough at 395 nm, typical modified type II. The other corticoids showed a similar spectral change. The corticoids had no effect on NADPH-cytochrome P450 reductase. All of these corticoids inhibited the reductive dehalogenation of halothane. The concentrations of hydrocortisone, methylprednisolone, betamethasone and dexamethasone causing 50% inhibition of CDE formation from halothane were 5.1 +/- 0.7 mg/ml, 3.1 +/- 1.2 mg/ml, 2.3 +/- 0.4 mg/ml and 2.4 +/- 0.6 mg/ml, respectively, with no significant differences except for hydrocortisone. The concentrations of hydrocortisone, methylprednisolone, betamethasone and dexamethasone causing 50% inhibition of CTE formation from halothane were 4.9 +/- 0.5 mg/ml, 3.1 +/- 1.0 mg/ml, 2.4 +/- 0.3 mg/ml and 28.0 +/- 9.1 mg/ml, respectively, with no significant differences except for dexamethasone. Our results showed that corticoids inhibit halothane metabolism. PMID- 7555001 TI - Absence of a pharmacokinetic interaction between quinidine and diazepam. AB - Influence of diazepam on the pharmacokinetics of quinidine was studied in eight healthy human volunteers by administering 250 mg of quinidine sulphate either alone or in combination with 10 mg diazepam in a cross-over study. Quinidine in the biological samples was estimated fluorimetrically. Comparison of various pharmacokinetic parameters of quinidine (Cmax 2.165 +/- 0.457 vs 2.239 +/- 0.338 micrograms/ml, Tmax 2 +/- 0.71 vs 2.13 +/- 0.58 h, T1/2 7.004 +/- 1.302 vs 7.82 +/- 1.63 h, Cls/f 153.63 +/- 45.65 vs 134.87 +/- 31 ml/h/kg, AUC0-alpha 22.131 +/ 6.118 vs 24.321 +/- 4.924 micrograms.h/ml) by means of a two tailed t-test revealed no significant (p > 0.05) effect of diazepam on quinidine disposition kinetics. PMID- 7555002 TI - In vitro protein binding and tissue distribution of D(+) usnic acid. AB - In vitro protein binding of D(+) usnic acid in rabbit plasma and purified bovine serum albumin was investigated by equilibrium dialysis. The drug was highly protein bound, approximately 99.2%, and the extent of protein binding remained constant at usnic acid concentrations in the range of 1-5 micrograms/ml. The extent of binding, however, tended to decrease at low albumin concentrations and higher drug concentrations; Scatchard plot analysis indicated the existence of two classes of binding sites with association constants of 34.3 x 10(-6) and 1.43 x 10(-6) M respectively. Tissue distribution studies of usnic acid were undertaken in rats following i.p. administration. Usnic acid was well distributed into well perfused organs. The tissue/plasma ratio in lungs was high, which could be advantageous in a therapeutic agent for pulmonary tuberculosis. PMID- 7555003 TI - Distribution of dipropyl [35S]-sulphoxide in the rat. AB - Dipropyl [35S]-sulphoxide was administered by gavage (4.24 mmol/kg body weight) to adult male Wistar rats and the placement of radioactivity about the animal examined at 4, 8 and 12 hours post-dosing. Widespread and diffuse distribution throughout soft tissues was observed with the largest amounts of radioactivity being found within the liver (3.2% dose at 4 h) and kidney (1.3% dose at 4 h). Activity levels declined over the 12 hour experimental period. This distribution pattern is discussed and compared with results previously reported for dimethyl sulphoxide. PMID- 7555004 TI - Phonological errors in aphasic naming: comprehension, monitoring and lexicality. AB - This paper investigates the production of phonological errors in aphasic naming, examining the relationship between these errors and deficits in comprehension. The predictions of Dell and O'Seaghda's (1991) computational model of speech production were tested by lesioning. The set of lesioned models demonstrated a strong correlation between proportion of phonologically related errors in naming and comprehension accuracy. In contrast, no correlation between proportion of phonological errors in naming and comprehension accuracy was found for a group of fifteen aphasics. This paper also examines monitoring behaviours, such as the presence of self-corrections and interrupted responses, again finding no relationship with auditory comprehension. There was also no evidence for a lexical bias in the phonological errors for these aphasic subjects. Phonologically related errors were argued to be words only by chance; this was supported by the fact that they occurred more frequently on short words than long words, and that the proportion of real word errors was not significantly different to that observed in a pseudocorpus of errors. We conclude that a production-based monitor would be compatible with these results but that any comprehension-based monitor is not reliably employed by some or all of the aphasic subjects examined here. Additionally, these data are incompatible with models of language processing where speech input and output share the same processing components. PMID- 7555005 TI - Word specific impairments in naming and spelling but not reading. AB - A patient, who had suffered a left-hemisphere CVA, is described. He had a major aphasic syndrome which encompassed both his propositional speech and literacy skills. However, his comprehension of both spoken and written language was intact. His superior ability to retrieve nouns as opposed to other word classes was systematically investigated. This superiority of noun processing was restricted to concrete nouns. It was found that he had a significant deficit in the naming of action pictures as compared with object pictures, and a significant deficit in the written spelling of verbs as compared with nouns when matched for concreteness. His single-word reading, for all word classes, was virtually intact. The theoretical significance of these findings is discussed. It is concluded that current information processing models, that postulate a unitary phonological output store, will require elaboration in order to account for these data. We tentatively suggest that there may be exclusive output stores for naming, reading and spelling. PMID- 7555006 TI - Right-left confusion in Gerstmann's syndrome: a model of body centered spatial orientation. AB - Gerstmann's syndrome encompasses the tetrad of finger agnosia, agraphia, acalculia and right-left confusion and is associated with lesions of the dominant angular gyrus. The localizing value of this syndrome has been questioned because multiple mechanisms can account for each of the components of the syndrome. We present the case of a man who developed Gerstmann's syndrome following a focal infarct of the left angular gyrus. The patient's right-left confusion could not be accounted for by either an aphasia or a degraded body schema. A series of experiments that investigated the patient's spatial mapping system by progressively restricting the degrees of freedom for spatial rotation revealed an isolated defect in deriving the relative position of an object along the horizontal axis. Defective horizontal mapping can account for the other components of Gerstmann's syndrome because they all share a common dependency on relative horizontal positioning. PMID- 7555007 TI - Forgetting from long-term memory in dementia and pure amnesia: role of task, delay of assessment and aetiology of cerebral damage. AB - This study investigated the hypothesis that memory disordered patients suffer of an exalted forgetting rate from long-term memory within the first few minutes following acquisition. For this purpose, we administered to groups of Alzheimer's (AD), Multi-infarct demented (MID), pure amnesic and age-matched normal controls a test involving immediate and 15-min delayed word-list recall and a modified version of the Huppert and Piercy's procedure (1978) assessing yes/no Recognition for pictures at 90 sec, 10 min, 1 hr and 24 hr delay intervals. Results showed an abnormal immediate/delayed performance decline for the word-list recall in AD and pure amnesic patients. In the same period, however, their Recognition score in the Huppert and Piercy's procedure decayed at normal rate. In the following intervals (1 hr and 24 hr), both groups disclosed abnormal forgetting slopes. Vascular demented patients, by contrast, disclosed fully normal forgetting rates both on the word-list recall and on the Huppert and Piercy's procedure. These findings are discussed in the light of cognitive mechanisms and neuroanatomical structures presumably underlying memory consolidation. PMID- 7555008 TI - Selective impairments in managerial knowledge following pre-frontal cortex damage. AB - Script generation was investigated in patients with lesions in the prefrontal (n = 9) and posterior (n = 8) cortical regions and in normal subjects (n = 16). Three different activities ranging in degree of familiarity were studied. Frontal patients did not differ from patients with posterior lesion and Normal subjects in the number of actions evoked, mean evocation time, or centrality. Impairments in script information processing were observed only in patients with prefrontal lesions, and for the three types of scripts. Specifically these patients made errors in ordering actions in the correct temporal sequence, failed to close scripts and to remain within the stated boundaries, and made deviant estimates of action importance. The results suggest that pre-frontal cortical lesions provoke a selective impairment in managerial knowledge (Grafman, 1989) that may contribute to difficulties in the formulation and execution of plans. PMID- 7555009 TI - Prosopagnosia: a clinical and anatomical study of four patients. AB - We tested in great detail face perception and face memory in four cases of prosopagnosia. Three of them showed deficits of face perception and/or of familiar faces memory. There was one prosopagnosic patient, however, who showed no abnormality in face perception and discrimination and could recall familiar faces. This case suggests that a form of prosopagnosia may exist, which is due to the disconnection between face perception and face memory. X-ray CT, MRI and PET studies revealed that two patients did not have damage of the left hemisphere, supporting the view that prosopagnosia can arise from unilateral right-sided lesion. PMID- 7555010 TI - Unilateral neglect can be affected by stimuli in the neglected field. AB - Neglect patients' attention is usually pathologically captured by stimuli present on the side ipsilateral to the brain damage. In previous research it was shown that not only the mere presence but also the number of "relevant" stimuli on the right side influenced patient's performance. However, the influence of stimuli whose presence was completely irrelevant for the execution of the task was not previously studied. We asked neglect patients to bisect lines of 28 cm of length, which could be presented alone or with line-drawings of objects placed either only on the left, only on the right or bilaterally on the display. We found that the presence of irrelevant left-side stimuli improved the bisection performance in 3 out of 5 patients with respect to the baseline condition, in which, no stimuli were present on either side of the display. Right-side stimuli did not affect the performance, whereas bilateral stimuli tended to worsen the performance for those patients who presented the beneficial effect of left-side stimuli. These findings were discussed with reference to the hypotheses advanced to explain neglect. PMID- 7555011 TI - Relations between verbal and nonverbal memory performance: evidence of confounding effects particularly in patients with right temporal lobe epilepsy. AB - Confounding left hemisphere verbalization strategies can be suggested as being a major problem in the evaluation of the assumed involvement of right temporo limbic structures in "nonverbal" visual/figural memory processing. We addressed this issue by evaluating the easily-verbalized Benton-visual-retention-test in 60 patients with either left (LTLE) or right temporal lobe epilepsy (RTLE) and 30 healthy controls. We formally estimated the informational (verbal) content of each item which hypothetically would be needed to solely retain the item from verbal memory. The results indicated confounding of verbal learning and figural memory only in the presence of right temporal lobe dysfunctions. Selective visual/figural learning deficits in RTLE patients became obvious when the verbal load of the figural material exceeded their verbal learning capacity. Instead of excluding verbalization by the use of abstract figural items, its inclusion provides a possibility to control compensatory strategies which overshadow the presence of visual/figural memory deficits. PMID- 7555012 TI - Developmental dyscalculia and brain laterality. AB - The correlation between arithmetic dysfunction and brain laterality was studied in 25 children with developmental dyscalculia (DD). The children were tested on a standardized arithmetic battery and underwent a neurological and neuro psychological evaluation. A diagnosis of left hemisphere dysfunction (n = 13) was based on right side soft neurological signs, performance IQ (PIQ) > verbal IQ (VIQ), dyslexia and intact visuo-spatial functions. The criteria for right hemisphere dysfunction (n = 12) were left body signs, VIQ > PIQ, impaired visuo spatial functions and normal language skills. The groups were similar for age, gender, and socio-economic status. Our results showed that both groups scored more than 2 SD below the mean adjusted score on the arithmetic battery, but the left group was significantly worse in 3 areas: mastery of addition/subtraction, complex multiplication and division and visuo-spatial errors (p < 0.05). The data indicate that dysfunction of either hemisphere hampers arithmetic acquisition, but arithmetic impairment is more profound with left hemisphere dysfunction. PMID- 7555013 TI - Within- and between-task dissociations in visuo-spatial neglect: a case study. AB - We describe a patient, J.B., who suffered an infarction of the right internal capsule and thalamus. There was a pronounced left hemiplegia but no visual field deficit. Investigated at four months and four years post-stroke, there were no signs of left visuo-spatial neglect other than a severe impairment of line bisection. The results on a test of perceptual judgement of the left and right extents of pretransected lines showed mild neglect of the left. The magnitude of this deficit did not predict quantitatively the severity of the patient's rightward deviation on a manual bisection task. Additional data are also reported on perceptual judgements and (manual) bisections of radially-oriented lines. PMID- 7555014 TI - Words interact with colors in a globally aphasic patient: evidence from a Stroop like task. AB - Stroop-like manual reaction time tasks were administered to 16 controls and one globally aphasic patient who was unable to match written color words with the corresponding color. Although the patient could not explicitly access the meaning of written color words, he showed a similar pattern of performance in the reaction time tasks as the controls: written words were processed automatically and they interacted with the processing of the color of the letters. Thus, the results of this study suggested that it is possible to process unrecognized words implicitly to a cross-domain semantic level where the processing of words interacts with the processing of colors. The results from this and other studies on implicit language processing indicate that linguistic stimuli can be analysed to relatively high levels without any awareness. PMID- 7555015 TI - Object recognition without knowledge of object orientation. AB - Several theories have been proposed to explain our ability to recognise objects from a number of viewpoints. Orientation-dependent accounts emphasize the position of the object relative to the viewer, while orientation-independent accounts (e.g. Marr) rely on descriptions of an object's component parts relative to its principal axis of elongation. An opportunity to compare the merit of these theories has arisen in a patient (L.G.) who had a rare neuropsychological sign in which knowledge of the canonical upright of object drawings was profoundly disrupted. Such orientation errors were evident in her drawings from memory and to copy, and in an orientation-matching task. In a critical experiment she showed a deficit in providing the canonical upright of individual object drawings that was independent of any difficulty in object recognition. The implications of these data for theories of object recognition are discussed. PMID- 7555016 TI - Two types of phonological alexia. AB - It is hypothesized, on the basis of a lexical model of reading, that there are two different underlying causes of phonological alexia. It is predicted that these two types of phonological alexia will be accompanied by different sets of symptoms. Published cases of phonological alexia are examined for evidence in support of these predictions. Two distinct groups of phonological alexic patients are observed. These results support the notion of two types of phonological alexia. The failure to find any phonological alexic patients who do not fall into one of these two categories provides evidence against non-lexical reading models. PMID- 7555017 TI - The role of the right temporal lobe in card sorting: a case study. AB - It has been demonstrated previously that performance on the modified Wisconsin Card Sorting Task (Nelson, 1976) is compromised by hippocampal sclerosis, particularly of the right hemisphere (Corcoran and Upton, 1993). Further evidence in support of this claim is presented here in the form of a case study. The findings from this case demonstrated that performance on the same task was not impaired following a right sided frontal lobectomy but was found to be deficient consequent to seizure recurrence in the right temporal lobe and following right temporal lobe removal. The clinical and theoretical significance of this case is discussed. PMID- 7555018 TI - The multiple functions of hemoglobin. AB - The aim of this review is to focus and discuss several parallel biological functions of hemoglobin besides its basic function of oxygen transport. In light of the information present in the literature the following possible physiological roles of hemoglobin are discussed: (1) hemoglobin as molecular heat transducer through its oxygenation-deoxygenation cycle, (2) hemoglobin as modulator of erythrocyte metabolism, (3) hemoglobin oxidation as an onset of erythrocyte senescence, (4) hemoglobin and its implication in genetic resistance to malaria, (5) enzymatic activities of hemoglobin and interactions with drugs, and (6) hemoglobin as source of physiological active catabolites. PMID- 7555020 TI - Helpless in the ICU. PMID- 7555019 TI - Intestinal brush border glycohydrolases: structure, function, and development. AB - The hydrolytic enzymes of the intestinal brush border membrane are essential for the degradation of nutrients to absorbable units. Particularly, the brush border glycohydrolases are responsible for the degradation of di- and oligosaccharides into monosaccharides, and are thus crucial for the energy-intake of humans and other mammals. This review will critically discuss all that is known in the literature about intestinal brush border glycohydrolases. First, we will assess the importance of these enzymes in degradation of dietary carbohydrates. Then, we will closely examine the relevant features of the intestinal epithelium which harbors these glycohydrolases. Each of the glycohydrolytic brush border enzymes will be reviewed with respect to structure, biosynthesis, substrate specificity, hydrolytic mechanism, gene regulation and developmental expression. Finally, intestinal disorders will be discussed that affect the expression of the brush border glycohydrolases. The clinical consequences of these enzyme deficiency disorders will be discussed. Concomitantly, these disorders may provide us with important details regarding the functions and gene expression of these enzymes under specific (pathogenic) circumstances. PMID- 7555021 TI - The metamorphosis of critical care: how are you thriving and surviving? PMID- 7555022 TI - Policy on use of neuromuscular blockers discussed. PMID- 7555023 TI - Protocol for prevention of complications of endotracheal intubation. AB - The care of patients with ETs is important, because pulmonary perfusion and oxygen ventilation play vital roles during the recovery process. Although several research studies have been conducted on various aspects of ET care, no standard of care on intubated patients has been developed based on results of these research studies. The value of the research findings should be recognized to ensure effective nursing interventions, minimize complications, and provide optimal nursing care. PMID- 7555024 TI - The effect of patient position on arterial oxygen saturation. PMID- 7555025 TI - Bedside percutaneous tracheostomy: implications for critical care nurses. PMID- 7555026 TI - Challenging diagnosis: adult respiratory distress syndrome. AB - ARDS patients require nursing care on all levels. Basic care such as turning and prevention of fatigue, as well as nursing care necessitated by mechanical ventilation and hemodynamic monitoring, is needed. Because nurses are the only professionals at the bedside around the clock, we are in the unique position to make a difference for ARDS patients. There is no cure yet for ARDS; therefore, appropriate supportive nursing care is of paramount importance. PMID- 7555027 TI - Understanding the oxyhemoglobin dissociation curve. AB - The oxyhemoglobin dissociation curve helps critical care nurses to better understand how various factors affect the oxygenation status of patients. Disease processes or treatment modalities that may cause shifts in the curve should be identified and the effects of the increased or decreased affinity assessed. Knowledge of conditions that affect hemoglobin-oxygen affinity, results of careful patient assessment, and oxygenation monitor readings allow critical care nurses to intervene and attempt to correct tissue hypoxia of critically ill patients. PMID- 7555028 TI - Enhancing bereavement care in the pediatric ICU. PMID- 7555029 TI - Caring for the pneumonectomy patient: challenges and changes. AB - Caring for pneumonectomy patients presents the nurse with challenges such as early detection of potentially fatal complications, promoting postoperative recovery, and preparing the patient for a life with one lung. Changes in healthcare practice and treatment modalities contribute to the challenge of providing quality nursing care for all patients. PMID- 7555030 TI - Computer-based instruction for critical care nurse educators. PMID- 7555031 TI - The patient-driven system. PMID- 7555032 TI - Peter DeBlieux, MD: violence living with the growing shadow. Interview by by Michael Villaire. PMID- 7555033 TI - A cross-over study comparing the efficacy of a combination of atenolol and nifedipine at different doses in angina pectoris. AB - Although the combined administration of atenolol and nifedipine has been shown to be effective in the treatment of angina pectoris the optimum dosage level of the combination has not yet been established. A double-blind, randomly assigned three period cross-over study was carried out therefore to investigate the effects of different daily doses of a fixed combination of 50 mg atenolol and 20 mg sustained-release nifedipine per capsule. Twenty-one patients with stable angina pectoris were randomized, after a 2-week run-in period, to receive treatment for 4 weeks with either 1 capsule twice daily (A), 2 capsules in the morning and 1 in the evening (B), or 2 capsules twice daily (C). A treadmill exercise tolerance test was performed together with a clinical evaluation after the run-in and each of the three treatment periods. Analysis of the results from the 19 patients who completed the study revealed there were no significant differences between the three treatment periods with respect to the general exercise test parameters, number of anginal attacks per week or nitrate consumption. Few side-effects were reported and were only mild in nature. On these findings, therefore, there would appear to be little justification for increasing the dose of the combination above the equivalent of treatment A. PMID- 7555034 TI - Potent activation of nitric oxide synthase by garlic: a basis for its therapeutic applications. AB - Garlic (Allium sativum L.) is thought to have a variety of therapeutic applications including inhibition of platelet aggregation. Many of the therapeutic actions of garlic parallel the physiological effects of nitric oxide and may be explained by its ability to increase nitric oxide synthase activity intracellularly. Our studies showed that both water and alcoholic extracts of garlic are very potent inhibitors of platelet aggregation induced by epinephrine and ADP. Similar dilutions of garlic extract also activated nitric oxide synthase activity in isolated platelets in vitro. The same extract was also very effective in activating nitric oxide synthase activity in placental villous tissue. The addition of garlic extracts increased nitric oxide synthase activity in a dose dependent manner. Nitrite levels in the supernatants of incubated placental villous tissue were similarly increased. Activation of calcium-dependent nitric oxide synthase and the subsequent production of nitric oxide is probably the most novel mechanism yet claimed by which garlic can exert its therapeutic properties. PMID- 7555035 TI - Pharmacokinetics, metabolism and biliary and urinary excretion of oral ramipril in man. AB - In order to evaluate the pharmacokinetics and excretion of ramipril in man, 8 cholecystectomy patients aged between 53 and 68 years received 5 mg ramipril orally as a single dose. All patients had a T-drain inserted to permit bile collection; all gave their informed consent to participate in the trial. Serum samples were collected half-hourly until 2 hours, then hourly until 6 hours, then at 8, 10, 24 and 25 hours after intake. Urine was collected in 2-hour fractions until 8 hours, followed by a 4- and a 12-hour fraction. Bile was collected hourly until 6 hours, followed by a 6- and a 12-hour collecting fraction. Concentrations of ramipril and ramiprilat in serum, and determinations in urine and bile of ramipril, ramiprilat, ramipril glucuronide, ramiprilat glucuronide, diketopiperazine and diketopiperazine acid were made; total amounts excreted were calculated. Peak concentrations of ramiprilat in plasma (8.7 +/- 1.6 ng/ml) were reached after about 8 hours. AUC0-8 and AUC0-24-values were 36.5 and 111.9 ng.h/ml, respectively. Ramiprilat Cmax-concentrations were about 300-fold higher in bile than in plasma, the corresponding difference for ramipril between bile and plasma was about 4-fold. The main fractions excreted in the urine were diketopiperazine acid and ramiprilat amounting to 13.2 +/- 5.6 and 4.4 +/- 2.4%, respectively, of the dose administered. Only a very small fraction of the dose was excreted with urine as unchanged ramipril, on average 0.9 +/- 1.0%. The main fractions excreted in the bile were diketopiperazine acid, ramiprilat glucuronide and diketopiperazine, 9.0 +/- 5.3, 3.4 +/- 4.2 and 2.0 +/- 1.2% in 24 hours, respectively, of the dose administered. Only a negligible fraction of the dose (average 0.1 +/- 0.1%) was excreted with bile as unchanged ramipril. In conclusion, there is strong evidence that circulating ramipril and ramiprilat are eliminated by both the liver and the kidneys. For the patients studied it can be estimated from late collection periods that some 2/3 of circulating ramipril and ramiprilat are eliminated by the kidneys and 1/3 eliminated by the liver. PMID- 7555036 TI - Rifaximin in the treatment of chronic hepatic encephalopathy. AB - A study was performed to assess the efficacy and tolerability of rifaximin in the treatment of encephalopathy during cirrhosis of the liver. Fifty-five patients suffering from grade 1, 2 and 3 portosystemic encephalopathy, with a mean age of 58.9 years (range 30 to 86 years) were evaluated. The patients were treated for 15 consecutive days with rifaximin, an antibiotic which is not absorbed by the intestinal wall, at a dosage of 1200 mg/day in association with sufficient lactulose to induce 2 or 3 evacuations per day. Combined use of the 2 drugs proved an efficient means of controlling the majority of signs and symptoms. After just a few days, an improvement in the signs of encephalopathy was noted in all patients. The treatment was well tolerated and the patients completed the trial without any drug-related side-effects. The results of our trial, although in the context of an open assessment, confirm the clinical efficacy of rifaximin in association with a non-absorbable disaccharide such as lactulose. The 2 compounds have a synergetic effect in reducing ammonia-producing flora. Its efficacy and good tolerability make rifaximin a valid alternative to the use of aminoglycoside antibiotics associated with disaccharides in the treatment of patients with liver disease, particularly in the case of prolonged therapy. PMID- 7555037 TI - Routine intraoperative application of high-dose aprotinin in open heart surgery in adults: antibody formation after first exposure. AB - High-dose aprotinin is now routinely used in cardiac surgery to reduce postoperative blood loss and transfusion requirements, although several cases of anaphylactic reactions to the proteinase inhibitor have been reported. As part of a multi-centre study to evaluate the immunological response to aprotinin after first exposure 61 cardiac surgical patients were treated with the Hammersmith regimen. Patients with previous aprotinin exposure were excluded from the study. To determine specific IgG and IgE antibodies blood samples were taken pre operatively, within 3 to 4 weeks and 6 to 7 months after operation. Determinations were made by using Western Blot and ELISA methods. Fifty-six patients were followed up for a 6-month period, 26 (46.4%) of them developed IgG antibodies to aprotinin determined by Western Blot, whereas only 14 (26.8%) patients with IgG antibodies were found by the ELISA. IgE antibodies were not found in any of the patients. On hospital admission and 6 months post-operatively additional intradermal prick tests were performed. No clear-cut positive reaction to the skin test was found in any patient. PMID- 7555038 TI - The effect of age and liver disease on the pharmacokinetics of the calcium antagonist, nisoldipine. AB - Two studies were performed-one in elderly, hypertensive patients and one in patients with chronic liver disease-to investigate the effect of age and liver disease upon the pharmacokinetics of nisoldipine, a dihydropyridine-type calcium antagonist. The effect of acute and chronic administration of nisoldipine (once and twice daily) was investigated in 17 elderly hypertensive patients. Compared with previously published data from young healthy volunteers, the values for Cmax and AUC appear to be higher in elderly hypertensive patients while Tmax and half life were unchanged. Nisoldipine significantly reduced both systolic and diastolic blood pressure when given acutely to elderly hypertensive patients. Major alterations in the pharmacokinetics of nisoldipine were found in 7 patients with chronic liver disease when compared with the elderly hypertensives and healthy volunteers. The values for AUC, Cmax, half-life and volume of distribution were all higher than expected from the volunteer data while clearance was lower. One patient receiving primidone had very low nisoldipine levels, suggesting that the concomitant administration of agents that may induce the metabolism of nisoldipine should be discouraged. Nevertheless, comparisons of nisoldipine plasma levels after acute and chronic administration showed no evidence of any accumulation in either patient population in the doses used. The drug was generally well tolerated although 1 patient with chronic liver disease was withdrawn due to fluid retention. PMID- 7555039 TI - Cardiac auscultation: a cost-effective diagnostic skill. PMID- 7555040 TI - Strategies for preventing children from becoming victims of violence. PMID- 7555041 TI - Caring for the short, endocrinologically normal child. PMID- 7555042 TI - The role of total parenteral nutrition in the patient with cancer. PMID- 7555043 TI - Sperm--egg recognition mechanisms in mammals. PMID- 7555044 TI - Regulation of oocyte growth and maturation in fish. AB - This chapter has briefly reviewed the current status of investigations on the hormonal regulation of oocyte growth and maturation in fish (see Figs. 4 and 9). Pituitary gonadotropins are of primary importance in triggering these processes in fish oocytes. In both cases, however, the actions of gonadotropins are not direct, but are mediated by the follicular production of steroidal mediators, estradiol-17 beta (oocyte growth) and 17 alpha,20 beta-DP or 20 beta-S (oocyte maturation). Investigators have established that both estradiol-17 beta and 17 alpha,20 beta-DP are biosynthesized by salmonid ovarian follicles via an interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). The granulosa cell layers are the site of production of these two steroidal mediators, but their production depends on the provision of precursor steroids by the thecal cell layers. A distinct steroidogenic shift from estradiol 17 beta to 17 alpha,20 beta-DP, occurring in salmonid ovarian follicles immediately prior to oocyte maturation, is a prerequisite for the growing oocytes to enter the maturation stage, and requires a complex and integrated network of gene regulation involving cell specificity, hormonal regulation, and developmental patterning. The cDNAs for most of the steroidogenic enzymes responsible for estradiol-17 beta and 17 alpha,20 beta-DP biosynthesis have been cloned from rainbow trout ovaries. Our next task is to determine how gonadotropin and other factors act on ovarian follicle cells to turn the expression of these specific genes on and off at specific times during oocyte growth and maturation. Increasing evidence now suggests that a variety of neuromodulatory, autocrine, and paracrine factors may also be involved in the regulation of steroidogenesis in fish ovarian follicles. Molecular biological technologies should be applied to identify these substances. Of considerable interest is the finding that MIH, unlike most steroid hormones, acts on its receptors at the surface of oocytes. Further studies of the association of the MIH-MIH receptor complex with a Gi protein, probably resulting in the inactivation of adenylate cyclase, should lead to a discovery of a new mechanism of steroid hormone action. The early steps following MIH action involve the formation of the major cytoplasmic mediator of MIH, MPF. Fish MPF, like that of Xenopus and starfish, consists of two components: cdc2 kinase and cyclin B. Nevertheless, the mechanism of MIH-induced MPF activation in fish oocytes differs from that in Xenopus and starfish because the appearance of cyclin B protein is a crucial step for 17 alpha,20 beta-DP induced oocyte maturation in fish.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7555045 TI - Nuclear transplantation in mammalian eggs and embryos. PMID- 7555046 TI - Transgenic fish in aquaculture and developmental biology. PMID- 7555047 TI - Molecular basis of mammalian egg activation. PMID- 7555048 TI - Axis formation during amphibian oogenesis: reevaluating the role of the cytoskeleton. PMID- 7555049 TI - Specifying the dorsoanterior axis in frogs: 70 years since Spemann and Mangold. PMID- 7555050 TI - Mechanisms of calcium regulation in sea urchin eggs and their activities during fertilization. PMID- 7555051 TI - Adenovirus--an eternal archetype. PMID- 7555052 TI - Abortive adenovirus infection and host range determinants. PMID- 7555053 TI - Functional similarity between adenovirus E1B 19-kDa protein and proteins encoded by Bcl-2 proto-oncogene and Epstein-Barr virus BHRF1 gene. PMID- 7555054 TI - Adenovirus fiber. PMID- 7555055 TI - Transcription of adenovirus RNA polymerase III genes. PMID- 7555056 TI - Adenovirus endopeptidase and its role in virus infection. PMID- 7555057 TI - E3 transcription unit of adenovirus. PMID- 7555058 TI - Adenovirus structure by X-ray crystallography and electron microscopy. PMID- 7555059 TI - Structure of the receptor binding domain of adenovirus type 5 fiber protein. PMID- 7555060 TI - Virus assembly. PMID- 7555061 TI - Selective encapsidation of adenovirus DNA. PMID- 7555062 TI - Role of adenovirus structural components in the regulation of adenovirus infection. PMID- 7555063 TI - Nuclear organization of replication and gene expression in adenovirus-infected cells. PMID- 7555064 TI - Adenovirus DNA replication. PMID- 7555065 TI - The mechanism of adenovirus DNA integration: studies in a cell-free system. PMID- 7555066 TI - Post-transcriptional control of adenovirus gene expression. PMID- 7555067 TI - Structure, function, and evolution of adenovirus virus-associated RNAs. PMID- 7555068 TI - The complete nucleotide sequence of the DNA of human adenovirus type 12. PMID- 7555069 TI - Molecular interactions during adenovirus DNA replication. PMID- 7555070 TI - Expression, nuclear transport, and phosphorylation of adenovirus DNA replication proteins. PMID- 7555071 TI - Homologous recombination in the replicative cycle of adenovirus and its relationship to DNA replication. PMID- 7555072 TI - Modulation of gene expression by adenovirus transformation. PMID- 7555073 TI - Transcriptional regulation of the adenovirus E1A gene. PMID- 7555074 TI - Functional domains of adenovirus E1A oncogenes which control interactions with effectors of cellular immunity. PMID- 7555075 TI - Assessing the role of E1A in the differential oncogenicity of group A and group C human adenoviruses. PMID- 7555076 TI - Regulation of adenovirus E2 transcription unit. PMID- 7555077 TI - Model systems for studying the effects of adenovirus E3 genes on virulence in vivo. PMID- 7555078 TI - Molecular determinants of adenovirus tropism. PMID- 7555079 TI - Enteric adenoviruses. PMID- 7555080 TI - Adenovirus E1A: transcription regulation and alteration of cell growth control. PMID- 7555081 TI - Adenovirus-augmented, receptor-mediated gene delivery and some solutions to the common toxicity problems. PMID- 7555082 TI - Gene therapy using adenovirus vectors. PMID- 7555083 TI - Regulation of p53-dependent apoptosis by E1A and E1B. PMID- 7555084 TI - Transcriptional modulation by the adenovirus E1A gene. PMID- 7555085 TI - Regulation of viral and cellular gene expression by E1A proteins encoded by the oncogenic adenovirus type 12. PMID- 7555086 TI - Cap-dependent and cap-independent translation: operational distinctions and mechanistic interpretations. PMID- 7555088 TI - Cap-independent translation of heat shock messenger RNAs. PMID- 7555087 TI - Cap-independent translation in adenovirus infected cells. PMID- 7555089 TI - Cap-independent translation and internal initiation of translation in eukaryotic cellular mRNA molecules. PMID- 7555090 TI - Translation of encephalomyocarditis virus RNA by internal ribosomal entry. AB - Picornavirus 5' NCRs contain IRES elements that have been divided into two groups, exemplified by PV (type 1) and EMCV (type 2). These elements are functionally related and have an intriguing level of structural and sequence similarity. Some conserved RNA sequences and/or structures may correspond to cis acting elements involved in IRES function, so that there may also be similarities in the mechanism by which the two types or IRES promote initiation. The function of both types of IRES element appears to depend on a cellular 57 kDa polypeptide, which has been identified as the predominantly nuclear hnRNP protein PTB. However, a specific function for p57/PTB in translation has not yet been established. These two groups can be differentiated on the basis of their requirements for trans-acting factors. The EMCV IRES functions efficiently in a broader range of eukaryotic cell types than type 1 IRES elements, probably because the latter require additional factor(s). A second distinction between these IRES element is that initiation occurs directly at the 3' border of type 2 IRES elements, whereas a nonessential spacer of between 30 nt and 154 nt separates type 1 IRES elements from the downstream initiation codon. PMID- 7555091 TI - Anatomy of the poliovirus internal ribosome entry site. PMID- 7555092 TI - The role of the La autoantigen in internal initiation. PMID- 7555093 TI - Structure and function of the hepatitis C virus internal ribosome entry site. PMID- 7555094 TI - Dermatologic insights. PMID- 7555095 TI - Paint dermatitis: the role of "routine" patch test series in detecting contact allergens in paint. PMID- 7555096 TI - A dermatologic diary. Portrait of a practice. PMID- 7555097 TI - Brucellosis. PMID- 7555098 TI - Impetigo. PMID- 7555099 TI - Coccidioidomycosis: the other great imitator. AB - A "great imitator," like the perhaps more familiar syphilis and tuberculosis, coccidioidomycosis has a wide variety of clinical presentations. A recent dramatic surge in the incidence of this saprobe increases the likelihood that clinicians will encounter an infected patient. Although rare, the disseminated form of the disease is potentially fatal. Vigilance and familiarity with the many faces of this pathogen will permit early detection and treatment. We present two cases of disseminated coccidioidomycosis and briefly review this increasingly common fungal infection. PMID- 7555100 TI - Green hair. AB - Green hair is an unusual dermatologic condition usually due to the deposition of copper from exogenous sources. We report the cases of two patients who presented to our clinic with green discoloration of their hair. This pigmentation of hair has generally been reported in patients with blond hair as a consequence of increased concentrations of copper in domestic or swimming pool water. Although an increased copper content of the affected hair seems to be a prerequisite, other predisposing factors have to be present for this situation to occur. These include previous hair damage (mechanical, sun exposure, bleaching, dyeing, waving), frequent contact with chlorinated water, or use of alkaline shampoos. Several options for treatment have been described for this problem, including application of hot vegetable oil, hydrogen peroxide, edetic acid- or D penicillamine-containing shampoos, or hydroxyethyl diphosphonic acid. PMID- 7555101 TI - Subcutaneous pheomycotic cyst of the finger caused by Exophiala jeanselmei: association with a wooden splinter. AB - A 58-year-old man with mild glucose intolerance noted a subcutaneous cystic nodule on the ventral aspect of his ring finger. He had experienced trauma to the finger while gardening five weeks before the lesion occurred. Exophiala jeanselmei was isolated and identified on culturing of aspirate from the cystic nodule. The nodule was then removed surgically, and a wooden splinter was found close to the subcutaneous fibrous cyst, suggesting that the causal organism had entered the subcutaneous tissue with the wooden splinter. PMID- 7555102 TI - Common and less common gingival overgrowth conditions. AB - Gingival enlargement is caused by some local and systemic pathologic conditions. Clinical manifestations vary and depend on the underlying defects. Diagnosis of these abnormalities and their relationship to underlying systemic conditions is essential prior to establishment of any treatment. Several gingival overgrowth conditions resulting from various causes are included in this review. Some conditions are rare and appear with clinical features interesting to both dentists and dermatologists. A brief review of causes, pathogenesis, and differential diagnosis is presented. PMID- 7555103 TI - Reactivation of oral-lingual herpes by chlorinated swimming pool water: a case report. AB - The authors describe the case of a highly stressed 36-year-old man who experienced ten or more painful episodes per year of recurrent oral-lingual herpes simplex virus 1, which were only partially responsive to acyclovir therapy for three years. A three-year diary of activities, personal stresses, concurrent infections, local trauma, and other possible psychogenic, somatogenic, and environmental events was used systematically to attempt to pair the stresses with the recurrent herpes episodes. Chlorinated swimming pool water seems to have been the triggering agent of the recurrent herpes simplex virus 1 episodes due to its temporal correlation and the greater than twenty-four-month asymptomatic period after the patient discontinued swimming in chlorinated water, but continued to swim in fresh and salt water, along with his normal pursuit of all other activities and habits. PMID- 7555104 TI - Chronic idiopathic urticaria associated with panic disorder: a syndrome responsive to selective serotonin reuptake inhibitor antidepressants? AB - We present two patients with a history of chronic idiopathic urticaria occurring in conjunction with a panic disorder (DSM-IIIR), in whom both the urticaria and panic disorder responded favorably to a course of the selective serotonin reuptake inhibitor antidepressants fluoxetine and sertraline, respectively. Both patients had previously required systemic corticosteroids to manage their urticaria. Panic disorder is an anxiety disorder characterized by the presence of recurrent unexpected panic attacks. Serotoninergic mechanisms play an important role in panic disorder. In contrast to antidepressants such as doxepin, which have previously been found to be effective in the treatment of chronic urticaria, the newer selective serotonin reuptake inhibitor antidepressants are only weakly antihistaminic and anticholinergic. The response of both the urticaria and panic disorder to selective serotonin reuptake inhibitor antidepressants may suggest a common pathogenic factor involving serotoninergic mechanisms. PMID- 7555105 TI - Norwegian scabies in a patient with AIDS: report of a case. AB - A severe form of crusted scabies is described in a patient with acquired immunodeficiency syndrome. He was treated with 1 percent lindane lotion and 5 percent sulfur ointment with 2 percent salicylic acid, which resulted in the clearing of the skin lesions. PMID- 7555106 TI - Silicone gel sheeting in scar therapy. AB - The purpose of this study was to analyze the efficacy of silicone gel sheeting in the treatment of fresh and long-standing hypertrophic and keloid scars. All subjects applied the gel sheeting in the same fashion and wore it for twelve to twenty-four hours per day for at least two months. After at least six months' follow-up, twenty of thirty-six (56 percent) chronic scars were improved. Eleven of fourteen fresh hypertrophic scars (79 percent) did not recur after surgery during a similar follow-up period. Side effects were minimal. Silicone gel sheeting is safe and effective treatment for hypertrophic and keloid scars. The mechanism of action is not completely understood. PMID- 7555107 TI - Morphogenetic effects induced by spermine and ruthenium red in yeasts. AB - Spermine (Sp) produces growth inhibition and wall malformation in Saccharomyces cerevisiae in response to oversynthesis of beta-glucans and chitin. The effect is related to the polycation nature of the molecule. In the present work, to verify this hypothesis, the yeast was treated with the abiogenic polycation ruthenium red (RR). The strict analogy observed between the RR- and Sp-induced alterations reinforced our earlier assumption that Sp interacted with the anionic sites of the plasmalemma determining a spurious activation of the two inserted enzymes beta-glucan and chitin synthases. This view was further confirmed by the aberrant accumulation of beta-glucans in Schizosaccharomyces pombe and of chitin in Rhodotorula glutinis treated with Sp and RR. In these micro-organisms spermidine, which bears three amino groups instead of the four encountered in Sp, was ineffective. It is inferred that at least four cation sites must be present in a compound in order to affect wall morphogenesis in yeasts. PMID- 7555108 TI - Localization of landmarks and bands in the karyotype of Felis catus. AB - Using 5-bromodeoxyuridine and Giemsa solution, representative high resolution RBG banded haploid karyotypes of Felis catus and corresponding idiograms at the 525 band stage were established and the results are discussed. PMID- 7555109 TI - Membrane-associated interleukin-1 on macrophages stimulated with Actinobacillus actinomycetemcomitans lipopolysaccharide induces osteoclastic bone resorption in vivo. AB - The effect of paraformaldehyde-fixed murine macrophage P388D1 cells stimulated with Actinobacillus actinomycetemcomitans Y4 lipopolysaccharide (membrane associated interleukin-1) on osteoclastic bone resorption was investigated. Both the number of osteoclasts and the bone resorption surfaces increased considerably along the inside of the marrow space of mouse calvaria when membrane-associated interleukin-1 mediated by A. actinomycetemcomitans Y4 lipopolysaccharide were injected into the subcutaneous tissues overlying the central calvaria of 4-week old C3H/HeJ mice. Membrane-associated interleukin-1 caused hypercalcaemia when injected into C3H/HeJ mice twice a day for 2 days. The calvaria of C3H-HeJ mice injected with membrane-associated interleukin-1 contained less mineral than those of the control mice. These results suggest that membrane-associated interleukin-1 on macrophages stimulated with A. actinomycetemcomitans Y4 lipopolysaccharide plays an important role in inflammatory osteoclastic bone resorption in periodontal diseases. PMID- 7555110 TI - Measurement of respiratory acoustic signals. Effect of microphone air cavity width, shape, and venting. AB - STUDY OBJECTIVE: We have previously investigated the effects of microphone type and coupler air chamber depth on lung sound characteristics. We now report the results of experiments exploring the effects of air chamber width, shape, and venting on lung sounds. DESIGN: We used a single electret microphone with a variety of plastic couplers. The couplers were identical except for the diameter and shape of the air chamber. We used cylindrical chambers of 5, 10, and 15 mm in diameter at the skin and conical chambers of 8, 10, and 15 mm in diameter. We compared the inspiratory lung sound spectra obtained using each of the couplers. We also examined the tendency of various needle vents to transmit ambient noise into the microphone chamber. SETTING: Anechoic chamber. MEASUREMENTS AND RESULTS: The shape and diameter had little important effect on the lung sound spectrum below 500 Hz. From approximately 500 to 1,500 Hz, the 5-mm diameter couplers showed slightly less sensitivity than the 10- and 15-mm diameter couplers. All conical couplers provided approximately 5 to 10 decibel more sensitivity than the cylindrical couplers. All vents allowed some ambient noise to enter the chamber but the amount was trivial using the narrowest, longest vent. CONCLUSIONS: These data suggest that the optimal electret microphone coupler chamber for lung sound acquisition should be conical in shape, between 10 and 15 mm in diameter at the skin, and either not vented or vented with a tube no wider than 23-g or shorter than 20 mm. PMID- 7555111 TI - Predicting death after CPR. Experience at a nonteaching community hospital with a full-time critical care staff. AB - OBJECTIVE: To identify a series of variables which predict death after in hospital cardiopulmonary resuscitation (CPR). DESIGN: Retrospective observational study. SETTING: A nonteaching community hospital with 24-hr on-site critical care specialists. PATIENTS: Consecutive adults undergoing CPR between August 1989 and July 1991. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Two hundred forty two patients suffered a total of 289 cardiopulmonary arrests. Forty patients (16.5%) survived to discharge. Thirty-nine (16%) patients had more than one cardiopulmonary arrest. Survival of second CPR was 18%. Acute physiology and chronic health evaluation (APACHE) II scores within 24 h of admission and CPR (APACHE[a] and APACHE[b]) were measured. APACHE(a) and (b) scores more than 20 had a 96% predictive value positive and were associated with a five-fold decrease in survival. Besides APACHE, cardiopulmonary arrests on medical floors and after day 4 of hospitalization, duration of CPR more than 15 min, and asystole assumed significance at multivariate levels for predicting death. Ventilatory assistance and Glasgow coma score of less than 9 at 24 h after CPR predicted death for initial survivors at multivariate levels. Survival on telemetry units were similar to the ICU (17 vs 21%) but twice that of the medical floors. CONCLUSIONS: The CPR outcome can be predicted early during hospital course, which may assist physicians to formulate a do-not-resuscitate order. Patients surviving a CPR should be considered candidates for another resuscitation if clinically warranted. Low-risk patients can safely be admitted to telemetry units instead of to more costly ICUs. PMID- 7555112 TI - Work of breathing as a weaning parameter in mechanically ventilated patients. AB - STUDY OBJECTIVE: Predicting patient responses to changes in ventilatory support or weaning is primarily based on bedside measure of respiratory mechanics and clinical judgement. Work of breathing (WOB) has been considered to be the best predictive factor in weaning. This study evaluated patient WOB as a predictive measure of weaning and extubation. DESIGN: Blinded, descriptive evaluation of the accuracy of preextubation values of patient WOB in predicting successful extubation. SETTING: Critical care units of 500-bed major teaching hospital of the University of Hawaii. PATIENTS: Twenty-four patients from the medical and surgical ICU were enrolled in the study. Patient diagnoses included COPD, ARDS, pneumonia, and renal failure. MEASUREMENTS AND MAIN RESULTS: In all patients, weaning from mechanical ventilation was accomplished by the clinical ICU teams who were blinded to the patient WOB. After extubation, patient WOB was compared with extubation attempts. In 14 patients, WOB was elevated above normal levels prior to successful extubation, while only 1 patient whose WOB was abnormal prior to extubation required reintubation within 24 h. WOB and clinical parameters were normal in 10 successfully extubated patients. CONCLUSIONS: In this group of mechanically ventilated patients, measurement of patient WOB was less accurate than conventional weaning parameters and clinical judgement for predicting successful extubation. This clinical evaluation study suggests that WOB alone is inadequate as a weaning parameter. PMID- 7555113 TI - Elevated imposed work of breathing masquerading as ventilator weaning intolerance. AB - OBJECTIVE: To test the hypothesis that, if apparent ventilatory insufficiency observed during a weaning or preextubation trial is due to a significant contribution of imposed work of the endotracheal tube and breathing apparatus (WOBImp), and the patient's actual physiologic work of breathing (WOBPhys) is not excessive, it should be possible to extubate these patients safely. DESIGN: Prospective descriptive study. SETTING: University hospital trauma intensive care unit. PATIENTS: A total of 28 (17% of all ventilated patients) adults intubated for 48 h or longer, who developed tachypnea (40 +/- 9 breaths/min) but whose blood gas exchange met predefined extubation criteria, were evaluated over a 3 month period. INTERVENTIONS: Using a microprocessor-based monitor (Bicore Monitoring Systems Inc, Irvine, Calif) total patient work of breathing (WOBTOT) was determined by integrating the change in intraesophageal pressure with tidal volume measured with a miniature pneumotachograph positioned at the airway opening. If the patient's WOBTOT was equal to or greater than 0.8 J/L, WOBImp was determined by integrating the changes in carinal pressures with tidal volume. If neither the patient's WOBTOT or WOBPhys was excessively greater than that of spontaneous breathing at rest (ie, < 0.8 J/L: normal range, 0.5 to 0.6 J/L), the patient was extubated. MEASUREMENTS AND RESULTS: Breathing frequency, peak inspiratory flow rate (PIFR), auto-Peep (PEEPa), dynamic compliance (CDXN) WOBTOT, WOBImp, resistance to expiratory airway flow (RAWE) were measured, and WOBPhys calculated (WOBTOT) minus WOBImp). The means and SDs were calculated, and data were analyzed by unpaired t test and linear regression. Six patients (5%) were found to have WOBTOT of < 0.8 J/L and were successfully extubated without determination of WOBImp. Twenty-one patients were found to have an elevated WOBTOT (1.6 +/- 0.83 J/L), and had WOBImp measured. In these 21 patients, WOBImp (1.1 +/- 0.64 J/L) was twice WOBPhys (0.5 +/- 0.26 J/L). Extubation was successful in 20 of 21 patients in which WOBPhys was determined not to be excessive (ie, < 0.8 J/L). The last patient had an elevated WOBPhys (1.4 J/L) and was not extubated until his disease improved later. Overall, reintubation rate was 4%. CONCLUSIONS: Increased WOBTOT may be misinterpreted as a patient failure (ie, tachypnea) and weaning halted or extubation not done, prolonging intubation. The ability to measure the contribution of WOBImp to WOBTOT can identify those patients who may be safely extubated when WOBphys (WOBTOT minus WOBImp) is acceptable and the apparent ventilatory insuffiency is related to significant WOBImp. PMID- 7555114 TI - Noise measurements during high-frequency oscillatory and conventional mechanical ventilation. AB - OBJECTIVE: To evaluate the noise levels with high-frequency oscillatory ventilation and conventional mechanical ventilation. DESIGN: An observational, prospective study. SETTING: Pediatric intensive care unit. SUBJECTS: The caretakers and environment of the pediatric intensive care unit. INTERVENTIONS: High-frequency oscillatory and conventional mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Caretakers evaluated noise using a visual analog scale. Noise was measured with a decibel meter and an octave band frequency filter. There was twice as much noise perceived by the caretakers and as measured on the decibel A scale. All measures showed significantly greater noise, especially at low frequencies, with high-frequency oscillatory ventilation. CONCLUSIONS: High frequency oscillatory ventilation exposes the patient to twice as much noise as does the use of conventional mechanical ventilation. PMID- 7555115 TI - Right ventricular function and plasma atrial natriuretic peptide levels during fiberbronchoscopic alveolar lavage in critically ill, mechanically ventilated patients. AB - STUDY OBJECTIVE: To assess the influence of fiberbronchoscopic alveolar lavage on hemodynamics, right ventricular function, and plasma atrial natriuretic peptide (ANP) concentrations in critically ill, mechanically ventilated patients. DESIGN: Prospective investigation. SETTING: Eight-bed ICU of a university hospital. PATIENTS: Fourteen patients with cardiovascular instability due to a systemic inflammatory response syndrome who were mechanically ventilated. INTERVENTIONS: Fiberbronchoscopic alveolar lavage after fluid replacement, deep sedation, and paralyzation. Intervention time: 10 min. After inspection of the endobronchial system, one lavage of 40 mL sterile saline solution was instilled in each lung and recovered. MEASUREMENTS AND RESULTS: The fiberbronchoscopic procedure induced a prompt increase in mean pulmonary arterial pressure after 3 min (median[range]: 25 [13 to 39] to 30 [19 to 45] mm Hg, p < 0.05), which increased further after 6 min (34 [17 to 46] mm Hg, p < 0.01). Cardiac index increased simultaneously (4.25 [3.1 to 5.7] to 4.85 [4.3 to 6.9] L/min.m2 after 6 min, p < 0.01), whereas mean arterial pressure and heart rate remained unchanged. Central venous pressure rose from 12 (3 to 18) mm Hg before procedure to 14 (4 to 20) mm Hg after 6 min (p < 0.01). The right ventricular function was measured using a "fast response" ejection fraction thermodilution catheter: end-diastolic volume increased (238 [137 to 358] to 280 [150 to 4ll] mL after 9 min, p < 0.05), as well as stroke volume (88 [54 to 113] to 103 [67 to 153] mL after 9 min, p < 0.01). Right ventricular ejection fraction (37 [25 to 50] %) did not change significantly during the procedure, but the stroke work index was reinforced (8.2 [4.7 to 15.7] to 13.3 [2.4 to 41.3] gm.M/M2 after 6 min, p < 0.01). Plasma c-ANP concentration rose from 135 (24 to 350) to 196.5 (44 to 830 pg/ml after 20 min (p < 0.05). Systemic vascular resistance decreased from 533 (390 to 1,042) to 429 (281 to 684) dynes.s/cm5 after removal of the bronchoscope (p < 0.01). CONCLUSIONS: Although acute pulmonary hypertension was observed during the fiberbronchoscopic procedure, the right ventricular performance did not deteriorate in hemodynamically unstable patients. To maintain a "hyperdynamic cardiovascular state," the right ventricular stroke work was reinforced, presumably by the "Frank-Starling mechanism." We assume that the acute distention of the right side of the heart resulted in elevated ANP concentrations. The marked decrease in systemic vascular resistance might be due to high ANP levels. PMID- 7555116 TI - Effect of previous antimicrobial therapy on the accuracy of the main procedures used to diagnose nosocomial pneumonia in patients who are using ventilation. AB - We evaluated the effect of antibiotic treatment received before the suspicion of pneumonia on the diagnostic yield of protected specimen brush (PSB), direct examination (BAL D) and culture (BAL C) of lavage fluid on consecutive mechanically ventilated patients with suspected nosocomial pneumonia. Bronchoscopy was always performed before any treatment for suspected pneumonia. One hundred and sixty-one patients with suspected pneumonia underwent PSB and BAL before any institution or change in antibiotic therapy (AB). Sixty-five patients received AB for an earlier septic episode (ON AB group) and 96 patients did not (OFF AB group). All but two strains recovered were highly resistant to previous AB. Sensitivity and specificity of each test were not different between the ON AB and OFF AB groups as well as the percentage of complete agreement between the 3 procedures, 74 and 67% respectively. We conclude that previous AB received to treat an earlier septic episode unrelated to suspected pneumonia do not affect the diagnostic yield of PSB and BAL. PMID- 7555117 TI - Cardiopulmonary effects of positive pressure ventilation during acute lung injury. AB - STUDY OBJECTIVES: To assess the gas exchange and hemodynamic effects of pressure limited ventilation (PLV) strategies in acute lung injury (ALI). We hypothesized that in ALI, the reduction of plateau airway pressure (Paw) would be associated with less alveolar overdistention and thus have better hemodynamic and gas exchange characteristics than larger tidal volume (Vr) ventilation. SETTING: Laboratory. DESIGN: Prospective time-controlled sequential animal study. MEASUREMENTS: Right atrial, pulmonary artery, left atrial, arterial, lateral pleural (Ppl), and pericardial (Ppc) pressures, Paw, ventricular stroke volume, mean expired CO2, and arterial and mixed venous oxygen contents. Airway resistance and static lung compliance were also measured. INTERVENTIONS: Intermittent positive pressure ventilation (IPPV) given before (control) and after induction of ALI by oleic acid infusion (0.1 mL/kg). IPPV at FIO2 of 1, VT of 12 mL/kg, and frequency adjusted to maintain normocarbia. ALI PLV was given during ALI and defined as that VT which gave a similar plateau Paw to that of control IPPV. High-frequency jet ventilation (HFJV) and ALI HFJV were also given and defined as frequency within 10% of heart rate and mean Paw similar to that during control IPPV. RESULTS: After ALI, static lung compliance, PaO2, and pH decreased, whereas airway resistance and PaCO2 increased. For a constant lung volume, Ppl and Ppc were not different between control and ALI. Both absolute dead space (VD) and intrapulmonary shunt fraction increased after ALI, but absolute VD was lower with ALI PLV and ALI HFJV when compared with ALI IPPV. Ventilation did not alter hemodynamics during ALI. CONCLUSIONS: Changes in lung volume determine Ppc and Ppl. PLV strategies do not alter hemodynamics but result in less of an increase in VD/VT than would be predicted from the obligatory decrease in VT. PMID- 7555118 TI - Structural features of tracheal tube biofilm formed during prolonged mechanical ventilation. AB - The dissemination of tracheal tube biofilm into the mechanically ventilated lung has been proposed as a contributory factor in the pathogenesis of ventilator associated pneumonia. In the present study, conventional light microscopy, confocal laser scanning microscopy, and scanning electron microscopy were used to examine luminal tracheal tube biofilm in tubes from ten consecutive medical intensive care patients. Biofilms also were cultured. No tube contained a predominantly microbial aggregate. Microorganisms were either dispersed throughout the biofilm or restricted to the most superficial layer. Neutrophil polymorphonuclear cells were present in all biofilms in a pattern suggesting that a layering or stratification had taken place. The distribution of neutrophils and microorganisms was consistent with a progressive accretion of respiratory secretions, rather than formation of a predominantly microbial biofilm. PMID- 7555119 TI - Recurrent pneumothoraces in ventilated patients despite ipsilateral chest tubes. AB - The incidence of recurrent pneumothoraces was analyzed in mechanically ventilated patients with the adult respiratory distress syndrome (ARDS) or non-ARDS causes of respiratory failure who had ipsilateral chest tubes in place. The radiographs of 39 consecutive patients with 47 initial pneumothoraces were evaluated for pneumothorax recurrence and chest tube positioning, which was prospectively defined as having a "vertical" or "horizontal" orientation. "Horizontal" positioning indicated that the chest tube may have been placed into a major fissure or the posterior hemithorax. Sixteen of the 47 pneumothoraces in all study patients, 14 of the 21 pneumothoraces in patients with ARDS, and 2 of the 26 pneumothoraces in patients without ARDS recurred (p < 0.0001) despite an ipsilateral chest tube; 9 of the 14 ARDS pneumothorax recurrences were tension types. "Horizontal" chest tube positioning in patients with ARDS had a positive predictive value of 86% and 64% for recurrences of pneumothoraces and tension pneumothoraces, respectively. Recurrent pneumothoraces occur commonly in mechanically ventilated patients with ARDS despite ipsilateral chest tubes. Because pneumothorax recurrences appear to be related to horizontal chest tube placement, imaging studies should verify that chest tubes are placed in optimally in the anterior hemithorax away from interlobar fissures in this patient population. PMID- 7555120 TI - Management of pediatric acute hypoxemic respiratory insufficiency with bilevel positive pressure (BiPAP) nasal mask ventilation. AB - OBJECTIVES: To evaluate the efficacy and complications of noninvasive nasal mask bilevel continuous positive airway pressure ventilation in pediatric patients with hypoxemic respiratory insufficiency. DESIGN: Retrospective chart review. SETTING: Intensive care unit, university affiliated tertiary care children's hospital. PATIENTS AND METHODS: The study reviewed all patients admitted to the pediatric ICU with acute hypoxemic respiratory insufficiency who received bilevel noninvasive continuous nasal mask positive airway pressure delivered by a bilevel positive airway pressure system (BiPAP; Respironics Inc; Murrysville, Pa). RESULTS: Bilevel nasal mask positive pressure ventilation was utilized in 28 patients. Median patient age was 8 years (range, 4 to 204 months). The most common primary diagnosis was pneumonia. Nine patients demonstrated severe underlying neurologic disease or immunocompromise. Median duration of nasal mask ventilation was 72 h (range, 20 to 840 h). Clinical and laboratory variables immediately prior to bilevel nasal mask positive airway pressure and approximately 1 h after institution were evaluated. Respiratory rate decreased significantly with nasal mask ventilation (45 +/- 18 breaths per minute to 33 +/- 11, mean +/- SD, p < 0.001). Arterial blood gas PaO2 (71 +/- 13 mm Hg to 115 +/- 55), PaCO2, pulse oximetry saturation, and pH all improved significantly (p < 0.01). Using standard estimates for inspired oxygen, calculated alveolar-arterial gradients (271 +/- 157 to 117 +/- 65, p = 0.001), and PaO2/FIo2 ratios (141 +/- 54 to 280 +/- 146, p < 0.001), both improved significantly with nasal mask ventilation. Only 3 of 28 patients required intubation or reintubation. CONCLUSIONS: We conclude that noninvasive nasal positive pressure mask ventilation can be safely and effectively used in pediatric patients to improve oxygenation in mild to moderate hypoxemic respiratory insufficiency. It may be particularly useful in patients whose underlying condition warrants avoidance of intubation. PMID- 7555121 TI - Nitric oxide administration using constant-flow ventilation. AB - Nitric oxide (NO) gas is known as both a vasodilator and a toxin. It can react with oxygen to form compounds more toxic than itself, such as nitrogen dioxide (NO2). The reactions are time dependent; thus, infusing NO into breathing circuits as close to ventilated subjects as possible may help minimize toxic byproduct exposure. Unfortunately, flow rates commonly used with mechanical ventilation favor laminar gas flow (streaming) within the breathing circuits. Streaming could delay mixing of NO with other inhaled gases. This mixing delay may interfere with accurate monitoring and/or delivery of NO. We tested the hypothesis that streaming of NO infused by constant flow into the inspiratory limb of a constant-flow mechanical ventilation system can lead to NO concentration delivery estimate errors. We then compared the NO2 concentrations at the ventilator Y-piece with three different NO mixing methods: blending the gases before they reach the breathing circuit inspiratory limb, infusing NO directly into the breathing circuit inspiratory limb far enough from the Y-piece to ensure thorough mixing, and infusing NO directly into the breathing circuit inspiratory limb immediately before the gases reach an in-line mixing device placed close to the Y-piece. Our results indicate that streaming can lead to NO concentration delivery estimate errors and that these errors can be characterized by measuring NO concentration variations across the inspiratory tubing's luminal diameter. NO2 concentration measured at the ventilator Y-piece were dependent on NO concentrations (p < 0.0001), NO delivery methods (p < 0.0001), and interactions between NO concentrations and NO delivery methods (p < 0.0001). We conclude that gas streaming and toxic byproduct exposure should be considered together when choosing an NO delivery method. PMID- 7555122 TI - The effects of applied vs auto-PEEP on local lung unit pressure and volume in a four-unit lung model. AB - BACKGROUND: The application of positive end-expiratory pressure (PEEP) and maintenance of increased mean airway pressure (MAP) has been associated with improved oxygenation in adult respiratory distress syndrome. Recently, attention has been directed toward elevating MAP by establishing auto-PEEP when ventilating with an inverse inspiratory to expiratory ratio in opposition to applied PEEP. We theorized that FRC distribution and local lung unit end-expiratory pressure (EEP) would be different when equal levels of PEEP were established by applying PEEP or by producing auto-PEEP. METHODS: Using a four-chamber lung model with each chamber having a different time constant (TC), we applied equal levels of applied PEEP (I:E ratio 1:3) and auto-PEEP (I:E ratio 3:1) and evaluated local lung unit EEP and end expiratory lung volume (EELV). RESULTS: During all trials with applied PEEP, local lung unit EEP was equal to applied PEEP, whereas during auto PEEP local EEP differed (p < 0.01). At a tracheal auto-PEEP level of 12.7 cm H2O, the lung unit with the longest TC (slow lung unit) had an EEP of 15.8 cm H2O, while the shortest TC unit (fast lung unit) had an EEP of 10.1 cm H2O (p < 0.01). Similarly, local EELVs were more maldistributed with auto-PEEP than with applied PEEP. At a tracheal PEEP level of 12.7 cm H2O, the EELV increase in the slow lung unit with auto-PEEP was 1,054 mL vs 918 with applied PEEP (p < 0.01), whereas the fast lung unit's EELV increase with auto-PEEP was 142 mL compared with 212 mL with applied PEEP (p < 0.01). CONCLUSION: Comparing equal levels of the auto-PEEP with applied PEEP, a greater maldistribution of local lung unit EEP and EELV was established with the auto-PEEP. During auto-PEEP, the greatest EEP and EELV occurred in the slow lung unit, and the lowest EEP and EELV developed in the fast lung unit. PMID- 7555123 TI - Comparison of silver nitrate and tetracycline as pleural sclerosing agents in rabbits. AB - The ideal agent to produce pleurodesis has not been identified. Tetracycline, the drug used most commonly in the 1980s, is no longer available. Talc either aerosolized or in a slurry is the agent used just most commonly at the present time, but there are concerns about its safety. Another possibility is silver nitrate, which was widely used in the past, but was abandoned on account of side effects. We hypothesized that lower concentrations of silver nitrate than had been used in the past would be effective in creating a pleurodesis in rabbits. The following medications in a total volume of 2 mL were instilled intrapleurally in three groups of ten anesthetized rabbits: 0.25% or 0.50% silver nitrate and 35 mg/kg tetracycline. Twenty-eight days after the injection, the animals were sacrificed and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The intrapleural injection of 0.50% silver nitrate produced an effective pleurodesis. The mean degree of gross pleurodesis in the rabbits that received 0.50% silver nitrate (3.4 +/- 1.2) did not differ significantly from that of the rabbits that received tetracycline (3.5 +/- 0.7) (scale 0 to 4). The mean degree of microscopic pleural fibrosis in the rabbits that received 0.50% silver nitrate (3.4 +/- 0.7) did not differ significantly from that of the rabbits that received tetracycline (3.9 +/- 0.3). However, 0.25% silver nitrate was ineffective in creating pleural fibrosis, either grossly or microscopically. No rabbits died after the intrapleural injection of the drugs. There were no observed side effects after the injection of silver nitrate. The present study demonstrates that 0.50% silver nitrate instilled into the pleural space is an effective agent for producing pleurodesis in the rabbit; its effect is comparable to tetracycline 35 mg/kg. This agent should be compared with tetracycline derivatives and talc in studies in humans. PMID- 7555124 TI - Assessment of asthma in the workplace. ACCP consensus statement. American College of Chest Physicians. PMID- 7555125 TI - Smoking and health: physician responsibility. A statement of the Joint Committee on Smoking and Health. American College of Chest Physicians. American Thoracic Society. Asia Pacific Society of Respirology. Canadian Thoracic Society. European Respiratory Society, and International Union Against Tuberculosis and Lung Disease. PMID- 7555126 TI - A proposed new international TNM staging system for malignant pleural mesothelioma. From the International Mesothelioma Interest Group. AB - STUDY OBJECTIVE: Investigation of the behavior and treatment of the diffuse malignant pleural mesothelioma (MPM) is hindered by the lack of an accurate universally accepted staging system. To address this problem, the International Mesothelioma Interest Group (IMIG) has developed a new TNM-based staging system. METHODS: The staging system was developed at a consensus meeting of IMIG members involved in clinical research in MPM, including the originators of previously proposed staging systems. The new staging system is based on the analysis of emerging information about the impact of T and N status on survival. RESULTS: In contrast to five previous staging systems, the T descriptors designated as T1, T2, T3, and T4, provide precise anatomic definitions of the local extent of the primary tumor. The N descriptors, designated as N0, N1, N2, and N3, are virtually identical to those used in the International Lung Cancer Staging System. The stage groupings recognize new data about the better prognosis of T1 and N0 tumors and classify those tumors into stages I and II. The adverse impact of nodal metastases on survival noted in some recent surgical series warrants placing node positive tumors in stage III. Locally advanced unresectable (T4) tumors and extrathoracic disease (N3 or M1) are classified as stage IV. CONCLUSION: This proposed staging system reconciles and updates several earlier systems, and can provide the framework for analyzing the results of prospective clinical trials aimed at improving the currently dismal prognosis of MPM. PMID- 7555127 TI - Alice in intensiveland. Being an essay on nonsense and common sense in the ICU, after the manner of Lewis Carroll. PMID- 7555128 TI - Stimulant-induced pulmonary toxicity. PMID- 7555129 TI - Conduit options in coronary artery bypass surgery. AB - The choice of graft conduit is crucial to the success of coronary artery bypass grafting (CABG) because the patency of a coronary conduit is closely associated with an uneventful postoperative course and a better long-term patient survival. The standard conduits used for CABG are the greater saphenous vein (GSV) and the internal thoracic artery (ITA). An excellent substitute conduit for coronary bypass operations that can be taken "off the shelf" is certainly the dream of every practicing cardiac surgeon. However, virtually every synthetic and biologic alternative to arterial conduits or autologous fresh saphenous vein has proved disappointing. Fortunately, patients with absolutely no autologous conduit alternatives are uncommon. Circumstances exist, however, that often necessitate the use of alternative conduits such as young hyperlipemic patients, absent or unsuitable autologous ITAs and GSV as a result of previous myocardial revascularization, peripheral arterial reconstruction, and varicose vein ligation procedures. This review provides an update on the clinical work done with all coronary conduits available for myocardial surgical revascularization. PMID- 7555130 TI - Lingular and middle lobe infiltrates in an elderly woman. PMID- 7555131 TI - Dyspnea in a 30-year-old Hispanic man with albinism. PMID- 7555132 TI - A case of pancreatic carcinoma causing massive bronchial fluid production and electrolyte abnormalities. AB - A 39-year-old man developed massive bronchorrhea (2 to 3.5 L/d) with electrolyte and volume depletion about 2 years after undergoing a Whipple's procedure for pancreatic carcinoma. An open lung wedge biopsy specimen was consistent with metastatic adenocarcinoma with extensive growth along preexisting pulmonary architecture. Chemical analysis of the bronchial fluid revealed markedly elevated levels of amylase confirming the pancreatic origin of the tumor. The mechanism of massive bronchorrhea is not known. Chemical analysis of bronchial fluid in comparison to serum and the temporary response to chemotherapy are most consistent with secretory and transudative mechanisms. PMID- 7555133 TI - Surgical treatment of complications 45 years after extraperiosteal pneumonolysis and plombage using acrylic resin balls for cavitary pulmonary tuberculosis. AB - An infected axillary sinus tract discharged balls made of an acrylic resin consisting essentially of polymerized methyl methacrylate (Lucite) 45 years following performance of an extraperiosteal pneumonolysis and Lucite ball plombage for collapse therapy of right upper lobe cavitary tuberculosis. Surgical extraction of the balls was performed, followed by a partial decortication of the lung and intrathoracic transposition of a pectoralis major muscle flap to fill the residual pleural space. Primary healing was attained, and the patient is well 1 1/2 years after surgery. PMID- 7555134 TI - Pulmonary artery aneurysm presenting as a lung mass. AB - We describe a case of pulmonary artery aneurysm in which clinical clues and conventional imaging suggested a lung tumor, and the actual nature of the lesion was discovered at the time of thoracotomy. This case shows the importance of an awareness of this condition in the formulation of a differential diagnosis for a lung mass. PMID- 7555135 TI - Nephrobronchial fistula and lung abscess resulting from nephrolithiasis and pyelonephritis. AB - There are multiple etiologies reported as causes of lung abscess; however, this differential rarely includes intra-abdominal abnormalities other than extension of a hepatic process. We describe a patient who was found to have a lung abscess and empyema resulting from the development of a nephrobronchial fistula secondary to nephrolithiasis and pyelonephritis. The patient had no urinary symptoms or known abdominopelvic infection and the etiology of lung abscess was only incidentally discovered after chest CT revealed extension of pleural fluid below the diaphragm. PMID- 7555136 TI - Painless ischemia provoked by mental stress in the coronary care unit. AB - A 30-year-old man suffered a cardiac arrest after setting fire to his home. Continuous ST segment electrocardiographic monitoring in the Coronary Care Unit detected painless ST segment depression during an interview with a police officer. At angiography, the patient proved to have severe three-vessel coronary artery disease. This case demonstrates the clinical value of continuous ST segment monitoring in the ICU. In addition, the case illustrates that mental stress is a potential trigger for acute myocardial ischemia in the ICU. PMID- 7555137 TI - Acute respiratory failure due to extramedullary hematopoiesis. AB - Extramedullary hematopoiesis (EMH) associated with myelofibrosis uncommonly occurs within the thorax. We describe the first reported case of acute and rapidly fatal respiratory failure due to pulmonary interstitial EMH associated with myelofibrosis. Interstitial EMH should be considered in the differential diagnosis of patients with interstitial pulmonary infiltrates and respiratory failure accompanying a disease process known to predispose to the development of EMH. PMID- 7555138 TI - Spontaneous pneumothorax in children with AIDS. AB - The incidence of pneumothorax in HIV-infected children has not been reported. In adults with AIDS, pneumothorax has been described exclusively in association with Pneumocystis carinii pneumonia (PCP). We report the cases of three children with AIDS, one with lymphoid interstitial pneumonitis (LIP) without evidence of PCP and two with PCP, all of whom developed spontaneous pneumothorax (SP). On presentation, none of the children had any risk factors for the development of pneumothorax, but all had radiographic evidence of subpleural cystic lesions and bilateral pleural adhesions. None of the patients responded to conservative medical management, which included chest tube thoracostomy and chemical pleurodesis. Two patients underwent pleurectomy that resulted in resolution of the pneumothorax. Both patients with PCP who developed pneumothorax died, but the patient with LIP and SP has had no recurrences of any serious respiratory problems 3 years after pleurectomy and excision of the intrathoracic cysts. PMID- 7555139 TI - Use of cardiopulmonary bypass during bronchoscopy following sand aspiration. A case report. AB - A 6-year-old boy with massive sand aspiration was effectively treated with femoral vein to femoral artery cardiopulmonary bypass (CPB), saline bronchial lavage, and exogenous surfactant. The patient was discharged the 9th hospital day without apparent sequelae. CPB should be considered for cases of sand or gravel aspiration when gas exchange is compromised. PMID- 7555140 TI - The "continuing" education of a physician. PMID- 7555141 TI - Talc pleurodesis for treating malignant pleural effusions. PMID- 7555142 TI - Is flexible fiberoptic pleuroscopy more cost-effective compared with VATS? PMID- 7555143 TI - Application of thoracoscopy for lung metastasis. PMID- 7555144 TI - What is the true impact of crack on the lung? PMID- 7555145 TI - Adverse pulmonary effects of mesalamine. PMID- 7555146 TI - Effects of ipratropium bromide on pulmonary hemodynamics in COPD. PMID- 7555147 TI - Collapsible airways contributing to airflow limitation. PMID- 7555148 TI - Consider other extubation strategies to maintain difficult airways. PMID- 7555149 TI - Effect of moderate altitude on the effect of mouth-to-mouth ventilation on blood gas analysis. PMID- 7555150 TI - "Steering clear" of automobile accidents in patients with sleep disorders. PMID- 7555151 TI - The heart in oxygen debt. PMID- 7555152 TI - Justifying the use of blood cultures when diagnosing community-acquired pneumonia. PMID- 7555153 TI - Predictors of cardiopulmonary resuscitation outcome in a community hospital. PMID- 7555154 TI - Work of breathing measurements. Can they help identify patients who can be successfully extubated? PMID- 7555155 TI - Management of pediatric acute hypoxemic respiratory insufficiency with bilevel positive pressure nasal mask ventilation. PMID- 7555156 TI - Malignant pleural mesothelioma. A proposed new staging system. PMID- 7555157 TI - Age and reversible lung disease. Let's grow old, but not be forgotten or ignored! PMID- 7555158 TI - Left ventricular systolic performance during acute hypoxemia. AB - STUDY OBJECTIVE: Although some of the cardiovascular responses to hypoxemia are well described, effects on myocardial contractility have not been defined. Such effects are readily assessed by noninvasive techniques and we have therefore evaluated Doppler-phonocardiographic parameters of systolic left ventricular contractility in normal humans rendered hypoxemic. DESIGN: Eight healthy male volunteers were studied. Parameters were measured after resting to achieve baseline haemodynamics, after 20 min moderate hypoxemia (SaO2 85 to 90%), and after a further 20 min of severe hypoxemia (SaO2 75 to 80%). Hypoxemia was induced by breathing a variable N2/O2 mixture. MEASUREMENTS: Pulsed-wave Doppler analysis of ascending aortic blood flow was combined with phonocardiography to measure indices of systolic left ventricular function at baseline and at the end of each period of hypoxemia. RESULTS: There was a significant, dose-related increase in cardiac output in response to hypoxemia, from 5.5 +/- 0.26 L/min at baseline to 6.1 +/- 0.08 L/min during moderate hypoxemia and to 7.0 +/- 0.23 L/min during severe hypoxemia. Likewise, heart rate increased significantly in dose-related fashion although stroke volume was not affected by either level of hypoxemia. Hypoxemia had no significant effects on systolic or diastolic blood pressures, but caused a significant reduction in systemic vascular resistance. Aortic peak and mean acceleration and acceleration time were not affected by moderate or severe hypoxemia. Although the systolic time intervals measured shortened significantly during severe hypoxemia, these were no longer significant when appropriate corrections were made for heart rate. CONCLUSIONS: Although cardiac output increases during hypoxemia, this is due to increases in heart rate but not to any effect on stroke volume. Parameters of left ventricular systolic function and myocardial inotropic state were also not affected by severe hypoxemia. Systolic left ventricular function and myocardial contractility are thus well preserved in normal humans during hypoxemia. PMID- 7555159 TI - Changes in wall motion in patients treated for unstable angina. A suggestion of the stunned and hibernating myocardium in humans. UNASEM Collaborative Study Group. Unstable Angina Study Using Eminase. AB - BACKGROUND: A double-blind, placebo-controlled study using anistreplase was performed in 159 patients with unstable angina. All patients had a history of unstable angina combined with typical ECG changes and without evidence of a previous, recent, or ongoing myocardial infarction. The purpose of the present study was to analyze the relationship between the patency of the culprit artery and the behavior of the ischemia-related regional left ventricular (LV) wall motion. METHODS AND RESULTS: On entry to the study, all patients received conventional drug therapy: i.v. nitroglycerin therapy, an oral beta-blocking agent, and a calcium antagonist. Baseline angiography was carried out within 3 h after randomization, a mean of 4.2 +/- 3.0 h (range, 1 to 17 h) after the last attack of chest pain. Treatment with trial medication was withheld in 33 cases. Sixty-five patients with coronary artery disease received anistreplase (30 U/5 min)/heparin and 61 patients heparin-only therapy. Angiography was repeated 20.6 +/- 4.6 h (mean +/- SD; range, 12 to 39 h) after the baseline angiographic study. To assess changes in regional myocardial wall motion, the LV wall was divided into seven segments. The ischemia-related coronary artery stenosis was calculated quantitatively and related to the quantitatively assessed mean regional left ventricular ejection fraction (RLVEF) of the ischemia-related segments. In 118 of 126 patients who received trial medication, we found that anistreplase/heparin therapy leads to a significantly (p < 0.01) greater reduction in coronary artery diameter stenosis than heparin-only therapy (n = 63, mean +/- SD, 11 +/- 22, vs n = 55, mean +/- SD, 3 +/- 11%). Anistreplase/heparin therapy was related to a larger significant improvement of the ischemia-related RLVEF than heparin-only therapy, although the latter association was not statistically significant (n = 63, mean +/- SD, 7 +/- 15, vs n = 55, mean +/- SD, 5 +/- 14%). The effects of change of coronary artery stenosis on regional LV wall motion were also determined. A paradoxical finding was that a persistently occluded vessel or a vessel showing an increase in coronary artery stenosis was associated with a greater improvement of the ischemia-related RLVEF than a reopened vessel or a vessel with a reduction in coronary artery stenosis (n = 15, mean +/- SD, 7 +/- 11, vs n = 41, mean +/- SD, 8 +/- 13, vs n = 15, mean +/- SD, 1 +/- 12, vs n = 47, mean +/- SD, 5 +/- 16%, NS). One day after the last attack of chest pain, the regional LV wall motion was still abnormal in about 20% of patients. CONCLUSION: In these patients with unstable angina, the LV wall motion improved both in the treated and the control group at follow-up angiography 1 day later. Improved coronary arterial anatomy was associated with a lesser improvement of the LV contractile function than when worsening of the coronary angiographic appearance occurred. There is no rational explanation of these results. This is a beginning of an effort to elucidate the clinical significance of the stunned and hibernating myocardium in humans. PMID- 7555160 TI - Color Doppler ultrasonography for the assessment of renal blood flow in heart failure. AB - To validate color Doppler ultrasonography of renal arteries for the assessment of renal blood flow (RBF), we compared left and right RBF estimates and their sum (total RBF) by echo-Doppler with data obtained by iodine 123-123I-p-aminohippuric acid (PAH) scintigraphy in 19 patients with heart failure and 7 normal control subjects. Single-side and total RBF estimates by echo-Doppler ranged, respectively, between 179 and 428 mL/min/m2 and from 378 to 835 mL/min/m2 in patients with heart failure and between 265 and 601 mL/min/m2 and from 564 to 1,182 mL/min/m2 in normal control subjects. Single-side and total RBF estimates by echo-Doppler correlated well with measurements obtained by scintigraphy (r = 0.74 and 0.76, respectively, in patients with heart failure; both: p < 0.001). At Bland and Altman's analysis of correspondence between the 2 techniques, there were 17 disagreements (33.7%) for single-side RBF and 8 disagreements (31.8%) for total RBF. However, the two techniques disagreed markedly in only two single-side and one total RBF estimates. Thus, in patients with heart failure, RBF assessed noninvasively by color Doppler ultrasonography has a good correlation with 123I PAH renal scintigraphy data over a wide range of blood flow. PMID- 7555161 TI - Quality of life in octogenarians after open heart surgery. AB - OBJECTIVES: To determine the quality of life in octogenarians after open heart surgery. BACKGROUND: Despite an increasing number of cardiac operations on octogenarians, the outcome as measured by functional status, independence of living, and psychological parameters of quality of life remain unproved. METHODS: Two groups of octogenarians (group 1, undergoing operation in 1986; group 2 in 1991) were reviewed retrospectively to determine operative mortality and functional results. RESULTS: Group 1 (n = 15, mean age of 83.2 years) and group 2 (n = 53, mean age 83.0 years) were studied. Operations included isolated coronary artery bypass grafting (group 1, 10; group 2, 29) and valve replacements +/- coronary artery bypass grafting +/- other procedures (group 1: 5; group 2, 24). Group 1 had 9% hospital mortality and 53% actuarial survival after a mean follow up of 6.3 years. Group 2 had 17% hospital mortality and 72% actuarial survival after a mean follow-up of 1.5 years. At follow-up, significant improvements were observed in New York Heart Association (NYHA) angina class, congestive cardiac failure class, number of cardiovascular symptoms, and indices for satisfaction with overall life and general affect in both groups. Further, group 2 also showed significant improvements in independence of living, ease of life, and Karnofsky dependency category, but these improvements were less evident in group 1 after a longer period of follow-up. At follow-up, 75% of group 1 and 84% of group 2 octogenarians would definitely have made the same decision to undergo open heart surgery in retrospect. CONCLUSION: This study demonstrates improved quality of life after open heart surgery in octogenarians. PMID- 7555162 TI - Efficacy of combined coronary revascularization and valve procedures in octogenarians. AB - From January 1982 to October 1991, 42 consecutive patients 80 years of age and older underwent a combined cardiac procedure with coronary revascularization and valve repair or replacement. There were 20 women and 22 men. Mean age at operation was 82.8 years (range, 80 to 89.7 years). Twenty-seven patients (64%) were in New York Heart Association (NYHA) functional class III or IV preoperatively. Six patients (14.3%) had undergone previous cardiac procedures. There were six hospital deaths (14.3%). The only significant preoperative risk factor identified for the event hospital death was aortic insufficiency (p = 0.005). The 36 hospital survivors were followed up at a mean of 21.1 months after hospital discharge. There were nine (21%) late deaths occurring at a mean of 21.3 months postoperatively: two from acute myocardial infarctions and seven from chronic heart failure. Survival analysis indicated that higher preoperative NYHA class (p = 0.0003), hypertension (p = 0.015), hypercholesterolemia (p = 0.03), and elevated left atrial/left ventricular gradient (p = 0.04) were incremental risk factors for overall mortality. The actuarial survival at 40 months was 51.9%, with no significant difference as compared with an age-, sex-, and race matched population. Of the 27 late survivors, 26 were in NYHA class I or II. We conclude that octogenarians may undergo complex cardiac surgical procedures with an expectation of an acceptable mortality rate and significant improvement in their functional status. These results must be taken into consideration in light of reported strategies to ameliorate health-care costs by limiting availability of complex medical care to the elderly. PMID- 7555163 TI - Clinical utility of blood cultures in adult patients with community-acquired pneumonia without defined underlying risks. AB - STUDY OBJECTIVE: We retrospectively examined the clinical utility of obtaining routine blood cultures before the administration of antibiotics in certain nonimmunosuppressed patients with community-acquired pneumonia (CAP) admitted to the hospital during 1991. DESIGN: Retrospective review. SETTING: Grady Memorial Hospital (a county hospital primarily serving inner-city Atlanta). PATIENTS OR PARTICIPANTS: Hospital discharge diagnosis listings identified 1,250 adults ( > or = 18 years old) with pneumonia. From this group of patients, we selected patients admitted to the hospital with (1) respiratory symptoms and a lobar infiltrate on chest radiograph that were present at the time of hospital admission, (2) two or more sets of blood cultures obtained within 48 h of hospital admission, and (3) absence of defined risk factors: HIV-related illness, malignancy, recent chemotherapy, steroid therapy, sickle cell disease, nursing home residence, or hospital stays within the past 14 days. MEASUREMENTS AND RESULTS: Five hundred seventeen patients (mean age, 52 years;: age range, 18 to 103 years) qualified. Of these 517 patients, 25 patients (4.8%) had growth in blood cultures considered contaminants while 34 (6.6%) had blood cultures positive for the following pathogens: 29 Streptococcus pneumoniae, 3 Haemophilus influenzae, and 1 Streptococcus pyogenes, 1 Escherichia coli. Antibiotic therapy was changed for 7 of the 34 patients with positive blood cultures (1.4% of study patients). Antibiotic regimens were altered in 48 additional patients based on sputum culture, poor clinical response, and allergic reactions. CONCLUSIONS: Few blood cultures were positive for likely infecting organisms in adult patients with CAP without defined underlying risk factors. Furthermore, a total of $34,122 was spent on blood cultures at $66 per patient. In this carefully defined group of patients, blood cultures may have limited clinical utility and questionable cost-effectiveness. PMID- 7555164 TI - The etiology and antimicrobial susceptibility patterns of microorganisms in acute community-acquired lung abscess. AB - OBJECTIVE: To determine the spectrum and antibiotic susceptibility patterns of microorganisms causing acute community-acquired lung abscess. DESIGN: A prospective survey. SETTING: Medical emergency department and wards of a tertiary teaching hospital. PATIENTS: Thirty-four adult patients with both clinical and radiologic features compatible with a diagnosis of acute community-acquired lung abscess who had received less than 48 h of antibiotic therapy. INTERVENTIONS: Microbiologic specimens obtained by percutaneous lung aspiration and with a protected specimen brush via fiberoptic bronchoscopy were submitted for aerobic and anaerobic culture. MAIN OUTCOME MEASURES: Identification of all microorganisms, including anaerobes, and determination of antibiotic susceptibility. RESULTS: A mean of 2.3 bacterial species per patient was isolated, anaerobes alone being isolated in 44% of cases, aerobes alone in 19%, and mixed aerobic and anaerobic isolates in 22%. Aerobic Gram-negative pathogens were uncommon. In seven patients, Mycobacterium tuberculosis was identified; in two it was associated with other bacteria. In four patients, no organisms were isolated. All the nonmycobacterial isolates were susceptible to amoxicillin clavulanate and in addition the anaerobes were all susceptible to chloramphenicol and almost all to a combination of penicillin and metronidazole. Among the anaerobes, the level of resistance to penicillin, metronidazole, and clindamycin individually was 21%, 12%, and 5%, respectively. CONCLUSIONS: Community-acquired acute lung abscess is usually caused by multiple anaerobic and less frequently aerobic Gram-positive microorganisms, which should respond to empirical therapy with amoxicillin-clavulanate, chloramphenicol, or a combination of penicillin and metronidazole. Tuberculosis, which may be indistinguishable from an acute lung abscess, occurred in 21% of patients in our study. Most bacterial pathogens are sensitive to conventional antimicrobial therapy and further investigation with percutaneous lung aspiration or bronchoscopy is indicated only when there is lack of early response to therapy or there is the presence of atypical clinical features. PMID- 7555165 TI - Interleukin-1 beta in pleural fluids of different etiologies. Its role as inflammatory mediator in empyema. AB - STUDY OBJECTIVES: To measure interleukin-1 beta (IL-1 beta) levels in pleural effusions of different etiologies and their relationship with several pleural inflammatory parameters, and to verify whether IL-1 beta can be used as diagnostic marker in the differential diagnosis of pleural diseases. MATERIAL AND METHOD: One hundred two pleural effusions were analyzed using a monoclonal antibody enzyme-linked immunosorbent assay. Pleural fluids were classified as follows: transudates (n = 28), empyema (n = 14), parapneumonic (n = 13), tuberculous (n = 19), neoplastic (n = 17), and miscellaneous effusions (n = 11). RESULTS: IL-1 beta was above 200 pg/mL in all the patients with empyema but only in three patients with other etiologies. Two of those three had parapneumonic effusions and the remaining one had a tuberculous pleurisy with a previous bacterial empyema. No significant relationships were found between pleural effusion IL-1 beta levels and the different inflammatory parameters analyzed. As a diagnostic criterion for empyema, pleural IL-1 beta concentration greater than 200 pg/mL had a sensitivity of 100%, a specificity of 96%, and a positive and negative predictive value of 0.82 and 1, respectively. CONCLUSIONS: Our data suggest that IL-1 beta has a significant role in pyogenic infections of the pleural space but not in effusions of other etiologies. It could be used as a diagnostic marker of empyema. PMID- 7555166 TI - AIDS-related spontaneous pneumothorax. Risk factors and treatment. AB - OBJECTIVE: To define risk factors and optimum therapy for AIDS-related spontaneous pneumothorax (PTX). DESIGN: Case-control study. SETTING: Tertiary care center. PATIENTS: Thirty-five patients with AIDS who developed spontaneous PTX between January 1, 1988 and December 31, 1991, of whom 27 (77.1%) did so in the setting of Pneumocystis carinii pneumonia (PCP). Forty-one patients who were diagnosed as having PCP and did not develop PTX served as the control group. RESULTS: Using logistic regression, a history of cigarette smoking, aerosolized pentamidine treatment, and the observation of pneumatoceles by chest radiography were associated with an increased risk of PTX. Although not associated with an increased risk of occurrence of PTX, the use of systemic corticosteroids for PCP treatment was associated with a longer requirement for chest tube drainage. Although chest tube drainage alone was often successful, chemical and surgical pleurodesis was often effective in treating prolonged air leaks and was associated with a lower incidence of recurrent PTX, although this difference did not achieve statistical significance (p = 0.07). CONCLUSIONS: Patients at high risk of developing AIDS-related spontaneous PTX can be identified. Systemic corticosteroids may increase the risk of morbidity from AIDS-related PTX. Chemical and surgical pleurodesis may be of value in short-term treatment and in reducing the risk of recurrence. PMID- 7555167 TI - Short-term effects of postural drainage with clapping vs autogenic drainage on oxygen saturation and sputum recovery in patients with cystic fibrosis. AB - To compare the short-term effects of postural drainage with clapping (PD) and autogenic drainage (AD) on oxygen saturation, pulmonary function, and sputum recovery, we studied ten patients with cystic fibrosis (CF) randomly treated with PD or AD on separate days. Pulse oximetry was monitored and sputum was collected during and for 1 h following each treatment. Pulmonary function was measured before and then 1, 15, and 60 min after each treatment. There was no significant difference in the amount of sputum recovered with AD (14.0 +/- 3.5 g) vs PD (10.4 +/- 3.0 g) and no significant differences in pulmonary function occurred. Oxygen saturation during PD fell from 93.3 +/- 0.7% to 91.2 +/- 0.8% (p < 0.01) and required 15 min following treatment to return to baseline. Oxygen saturation did not fall during AD and increased to 94.5 +/- 0.7% by 1 h following treatment (baseline, 93.3 +/- 0.8%; p < 0.01). We conclude that AD is less likely to produce oxygen desaturation and may be better tolerated by patients with CF, while producing similar benefits in sputum clearance. PMID- 7555168 TI - Clinical, pathophysiologic, and microbiologic characterization of bronchiectasis in an aging cohort. AB - STUDY OBJECTIVE: Awareness of bronchiectasis on the part of clinicians has been low in recent years, although it was previously well recognized. We believe that bronchiectasis is underdiagnosed, and that current literature is skewed toward the esoteric etiologies of bronchiectasis. DESIGN: We reviewed the clinical, radiologic, microbiologic, and physiologic findings in 123 well-studied patients with proved bronchiectasis. SETTING: The University of Texas Health Center at Tyler Hospital and Clinics. MEASUREMENTS AND RESULTS: There were 38 men and 85 women with a mean (+/- SD) age of 57.2 +/- 16.7 years; 55% were lifetime nonsmokers. Diagnosis was confirmed with CTs of the chest in 56%, by bronchogram in 28%, and surgery with the remainder. Seventy percent of patients gave a history of an antecedent potentially causative event for the bronchiectasis, usually pneumonia. Symptoms of bronchiectasis included chronic cough with the production of purulent sputum, hemoptysis, recurrent fever, and pleurisy. The finding of crackles on chest examination was the rule (70%) with wheezing present in 34% of the group. Pulmonary function studies documented airway obstruction to be present in 54% of the lifetime nonsmokers. The chest radiographs were abnormal in 91.3%, showing fibrotic stranding and infiltrates. A variety of pathologic microbial flora, particularly Pseudomonas aeruginosa and other opportunistic organisms, were isolated from the sputum. Patients who had smoked had much the same picture as nonsmokers, although they had a greater degree of airway obstruction. CONCLUSIONS: A characteristic clinical picture of bronchiectasis emerges after review and evaluation of these data. Knowledge of this picture should allow ready recognition of the disease. PMID- 7555169 TI - The effect of fiberoptic bronchoscopy on cerebral hemodynamics in patients with severe head injury. AB - STUDY OBJECTIVE: Concerns exist about the effect of flexible fiberoptic bronchoscopy (FFB) on intracranial pressure (ICP). We studied the effect of FFB on cerebral hemodynamics in patients with severe head injury. DESIGN: Prior to FFB, patients were anesthetized and muscle relaxants were given as necessary to eliminate coughing. Comparisons were made of mean arterial pressure (MAP), ICP, and cerebral perfusion pressure (CPP) prior to, during, and after FFB, as well as comparisons of mean cerebral hemodynamic values in an 8-hour period before and after FFB. Observations were made of changes in neurologic status post-FFB. SETTING: Surgical intensive care unit of Level 1 Trauma Center. PATIENT POPULATION: Fifteen patients with severe head injury in whom ICP was monitored and who required FFB for diagnosis of nosocomial pneumonia or treatment of lobar collapse. RESULTS: Pre-FFB ICP averaged 14.3 mm Hg (range, 6 to 26 mm Hg). During FFB, patients experienced a mean increase in ICP of 13.5 mm Hg above basal values (p = 0.0001). At peak ICP, MAP increased from a baseline of 92.3 mm Hg (SD +/- 16.1) to 111.5 mm Hg (+/- 13.9). Mean CPP was 83.7 mm Hg at peak ICP (range, 52 to 121 mm Hg), a 14.0% increase over baseline. The ICP and MAP returned to basal levels following bronchoscopy. No patient had a clinically significant increase in ICP or demonstrated any deterioration in Glasgow Coma Scale score or neurologic examination findings post-FFB. CONCLUSIONS: Although FFB causes an increase in ICP in patients with severe head injury, MAP also rises, and an adequate CPP is maintained. The ICP returns to basal levels after the procedure. When properly performed, FFB does not adversely affect neurologic status in patients with severe head injury. PMID- 7555170 TI - The relationship of sleepiness and blood pressure to respiratory variables in obstructive sleep apnea. AB - As a follow-up to a previous assessment of complications of sleep-disturbed breathing in 265 patients, we have reevaluated measures of sleepiness and hypertension in patients with obstructive sleep apnea (OSA) (n = 518), central sleep apnea (n = 50), and subclinical sleep-disordered breathing (SDB) (n = 107). Both subjective and objective (multiple sleep latency test [MSLT]) measures indicated that OSA patients were sleepier than those with subclinical SDB. The OSA patients weighed significantly more than the patients with central sleep apnea or subclinical SDB. They had a higher proportion of men, described more habitual sleepiness, and had a higher likelihood of feeling unrefreshed in the morning compared with the group with subclinical SDB. Among the OSA patients, there was a significant correlation between subjective and objective assessment of sleepiness, but this relationship was quantitatively very small. A forward stepwise regression analysis revealed that weight, and to a lesser degree waking time after sleep onset, could account for 65.5% of the variance in subjective sleepiness. Seventy-five percent of the variance of the mean sleep latency in the MSLT could be accounted for by the mean minimum arterial oxygen saturation in non REM sleep and the nocturnal sleep latency. Diastolic BP was significantly higher in OSA patients compared with the patients with central sleep apnea and subclinical SDB. When covarying for weight, age, and gender, this effect lost significance. Among OSA patients taken by themselves, 98.3% of the variance in diastolic blood pressure could be accounted for by the mean minimum arterial oxygen saturation in non-REM sleep, with very small additional contributions of apnea/hypopnea index, weight, and age. In summary, among patients across a spectrum of SDB, differences in diastolic BP were primarily associated with weight, age, and gender. Among OSA patients, perhaps because of a more limited variance in weight, diastolic BP was associated with measures of SDB. PMID- 7555171 TI - Coancestry in apparently sporadic primary pulmonary hypertension. AB - OBJECTIVE: To examine sporadic cases of primary pulmonary hypertension of coancestry. DESIGN: An epidemiologic study of families of patients with primary pulmonary hypertension. SETTING: A university-affiliated referral population. PARTICIPANTS: Family members of 13 patients with primary pulmonary hypertension. MEASUREMENTS: Family pedigrees involving grandparents, parents, siblings, and children were supplemented by genealogic records. Coefficients of kinship (Ck) were calculated for the patients with primary pulmonary hypertension who demonstrated coancestry and compared with 500 sets of controls drawn at random from genealogic records. RESULTS: Two patients with sporadic primary pulmonary hypertension demonstrated coancestry. The great-great grandfather and great-great grandmother of one patient were the great-grandfather and great-grandmother of the other patient. No other cases of primary pulmonary hypertension were identified in these two families. The CK of the affected individuals (CK = 10.02 x 10(-5)) suggests strongly that the observed relationship did not occur by chance alone. Among 500 random sets of matched controls, only two sets yielded CK of 10.02 x 10(-5) or greater (p = 0.004). Coancestry could not be identified for the other five families of patients with sporadic primary pulmonary hypertension for whom genealogic records were available. CONCLUSIONS: The finding of coancestry in patients with sporadic primary pulmonary hypertension suggests that a genetic basis exists for some patients with apparently sporadic primary pulmonary hypertension. Familial primary pulmonary hypertension may be more common than previously recognized. PMID- 7555172 TI - Prevalence of acute pulmonary embolism among patients in a general hospital and at autopsy. AB - PURPOSE: The purpose of this investigation is to estimate the prevalence of acute pulmonary embolism (PE) in a general hospital, its frequency among patients who died, and the ability of physicians to diagnose PE antemortem. METHODS: The prevalence of acute PE among 51,645 patients hospitalized over a 21-month period was assessed in 1 of the 6 clinical centers (Henry Ford Hospital) that participated in the collaborative study, prospective investigation of pulmonary embolism diagnosis (PIOPED). The diagnosis of PE was made by pulmonary angiography, or in those who did not undergo pulmonary angiography because they declined or were ineligible for randomization to angiography in PIOPED, the diagnosis was based on the ventilation/perfusion (V/Q) lung scan. Based on data in PIOPED, PE was considered to be present in 87% of patients with high probability V/Q scam interpretations, 30% with intermediate probability interpretations, 14% with low probability interpretations, and 4% with nearly normal V/Q scans. RESULTS: The estimated prevalence of acute PE in hospitalized patients was 526 of 51,645 (1.0%; 95% confidence interval [CI], 0.9 to 1.1%). Based on extrapolated data from autopsy, PE was estimated to have caused or contributed to death in 122 of 51,645 (0.2%; 95% CI, 0.19 to 0.29%). Pulmonary embolism was observed at autopsy in 59 of 404 (14.6%; 95% CI, 11.3 to 18.4%). Among patients with PE at autopsy, the PE caused or contributed to death in 22 of 59 (37.3%; 95% CI, 25.0 to 50.9%) and PE was incidental in 37 of 59 (62.7%; 95% CI, 49.1 to 75.0%). Among patients at autopsy who died from PE, the diagnosis was unsuspected in 14 of 20 (70.0%; 95% CI, 45.7 to 88.1%). Most of these patients had advanced associated disease. In these patients, death from PE occurred within 2.5 h in 13 of 14 (92.9%; 95% CI, 66.1 to 99.8%). CONCLUSION: Pulmonary embolism is common in a general hospital. The prevalence of PE at autopsy has not changed over 3 decades. The frequency of unsuspected PE in patients at autopsy has not diminished. Even among patients who die from PE, the PE is usually unsuspected. Such patients, however, typically have advanced disease. Among moribund patients, incidental PE is rarely diagnosed. Patients who suffer sudden unexplained catastrophic events in the hospital are a group in whom the diagnosis might be suspected more frequently if physicians maintain a high index of suspicion. PMID- 7555173 TI - A modified Abrams needle biopsy technique. AB - STUDY OBJECTIVE: To compare the diagnostic sensitivity of a modified Abrams needle pleural biopsy technique (A1) with the standard Abrams (A2) and Cope needle biopsy methods. The modified Abrams pleural biopsy technique consisted of suctioning each tissue sample into a syringe without removing the needle completely from the chest until the completion of the entire procedure. Both the standard Abrams and Cope needle techniques required needle removal from the chest after each pleural biopsy. DESIGN: Retrospective chart analysis. SETTING: Community teaching hospital affiliated with Stanford University. PATIENTS: Forty seven patients (30 men and 17 women) with a mean age 44.5 years (range, 19 to 81 years) who were referred to a pulmonary consultation service for pleural biopsy. INTERVENTIONS: Two of us (C. M. K. and F. T. K.) used the modified Abrams technique and two of us (W. A. J. and A. C. C.) used the standard Abrams technique. The Cope needle was used as originally described. MEASUREMENTS: We recorded the type of pleural biopsy needle and technique used in each patient. Biopsy specimen diameter and number of tissue samples obtained, final diagnoses, and complications were recorded. RESULTS: The diagnostic sensitivity for tuberculous pleurisy was 82% for the modified Abrams method, 71% for the standard Abrams method, and 88% for the standard Cope technique (p > or = 0.3). There was no difference in size of tissue sample obtained (A1 vs A2), number of biopsies, or complications among the three methods of pleural biopsy. CONCLUSIONS: The modified method of Abrams needle biopsy demonstrates a diagnostic sensitivity for pleural tuberculosis (82%) that is equivalent to that for the standard Abrams or Cope methods. PMID- 7555174 TI - Conservative treatment of overanticoagulated patients. AB - STUDY OBJECTIVE: To determine the outcome of over-anticoagulated patients who were treated with vitamin K and those who were treated conservatively by holding doses and increasing monitoring frequency. A secondary objective was to compare conservative management with American College of Chest Physicians (ACCP) treatment guidelines when followed. DESIGN: Retrospective chart review of all patients with international normalized ratios (INRs) of 6 or greater and concurrently receiving warfarin between November 1993 and February 1994. SETTING: A Veterans Affairs Medical Center providing inpatient and outpatient care. Patients receiving warfarin are managed by an established anticoagulation clinic. PATIENTS: Fifty-one consecutive patients receiving warfarin who had an INR of 6 or greater were reviewed. INTERVENTIONS: Data collection included INR, risks for bleeding, indication for anticoagulation, interventions, and patient outcomes. RESULTS: INRs ranged from 6.1 to 81.8. Forty-eight patients (94%) did not receive vitamin K; they were treated by withholding doses and increasing monitoring frequency. One developed minor bleeding. Three patients (6%) received vitamin K. Two of these patients died of unrelated problems. The third patient required 47 days of heparin therapy prior to achieving therapeutic oral anticoagulation. CONCLUSION: This trial showed that conservative treatment of nonbleeding overanticoagulated patients is safe. A prospective trial comparing the ACCP guidelines with a conservative approach is needed. PMID- 7555175 TI - Chronic cough with a history of excessive sputum production. The spectrum and frequency of causes, key components of the diagnostic evaluation, and outcome of specific therapy. AB - STUDY OBJECTIVE: To determine (1) the spectrum and frequency of causes of chronic cough with a history of excessive sputum production (CCS) and (2) the response of these causes to specific therapy. STUDY DESIGN: Prospective study utilizing the anatomic diagnostic protocol originally developed to diagnose chronic cough. PATIENTS: Seventy-one immunocompetent adults who complained of expectoration of greater than 30 mL of sputum per day. LOCATION: University hospital pulmonary outpatient clinic. RESULTS: Patients were seen an average of 4.2 times over 4.6 months before a specific diagnosis was made. The cause of CCS was determined in 97%. It was due to one cause in 38%, 2 in 36%, and three in 26%. Postnasal drip syndrome (PNDS) was a cause 40% of the time, asthma 24%, gastroesophageal reflux disease (GERD) 15%, bronchitis 11%, bronchiectasis 4%, left ventricular failure 3%, and miscellaneous causes 3%. Among patients with a normal chest radiograph who were nonsmokers and not taking an angiotensin converting enzyme inhibitor; CCS was due to PNDS, or asthma, or GERD, or all three in 100% of cases. Chest radiograph, methacholine inhalation challenge, 24-h esophageal pH monitoring, bronchoscopy, and spirometry with bronchodilator each had a sensitivity and negative predictive value of 100%. Chest radiograph and barium swallow had positive predictive values of only 38% and 30%, respectively. CONCLUSIONS: (1) The anatomic diagnostic protocol for cough is also valid for CCS; (2) the major causes of chronic excessive sputum production and chronic cough are so similar that CCS should be considered a form of chronic cough; (3) the evaluation of CCS is more complicated and takes longer than the evaluation of chronic cough; (4) the major strength of the laboratory diagnostic protocol is that it reliably rules out conditions; (5) the outcome of specific therapy is almost always successful; and (6) the term "bronchorrhea" can be misleading if it is applied to excessive sputum production before a specific diagnosis of its source is made since the most common cause of excessive sputum that is expectorated (PNDS) is a disorder of the upper respiratory tract. Therefore, nonspecific therapies theoretically aimed at reducing mucus production in the lower respiratory tract are not likely to be helpful. PMID- 7555176 TI - Peripheral airspace dimensions in patients with COPD. AB - Monodisperse aerosol particles can be used to assess noninvasively intrapulmonary airspace dimensions. Since emphysematic changes in the peripheral lung are difficult to detect with most of the common lung function tests, aerosol-derived airway morphometry was used to assess the peripheral airspace dimensions (EAD800) in 25 patients with COPD and in 36 healthy volunteers. Spirometric and body plethysmographic measurements were performed in all patients. In ten patients, high-resolution CT-derived mean lung density (MLD) was additionally assessed. In healthy subjects, EAD800 was 0.39 +/- 0.05 mm. In patients, EAD800 was significantly increased (0.82 +/- 0.33 mm). In a subset of nine patients with severe alpha 1-antitrypsin deficiency and clinically severe emphysema, EAD800 was even larger (1.14 +/- 0.32 mm). In patients, EAD800 correlated with MLD (r = 0.82), diffusion capacity (DCO) (r = 0.78), and FEV1 (r = -0.75). Since MLD is considered a valid indicator for lung emphysema, the close correlation between EAD800 and MLD suggests that EAD800 reflects enlarged peripheral airspace dimensions in patients with emphysema. PMID- 7555177 TI - 4th American College of Chest Physicians Consensus Conference on Antithrombotic Therapy. Tucson, Arizona, April 1995. Proceedings. PMID- 7555178 TI - Clinical recommendations using levels of evidence for antithrombotic agents. PMID- 7555179 TI - Oral anticoagulants. Mechanism of action, clinical effectiveness, and optimal therapeutic range. PMID- 7555180 TI - Aspirin and other platelet-active drugs. The relationship among dose, effectiveness, and side effects. PMID- 7555181 TI - Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. PMID- 7555182 TI - Hemorrhagic complications of anticoagulant treatment. PMID- 7555183 TI - Hemorrhagic complications of thrombolytic therapy in the treatment of myocardial infarction and venous thromboembolism. PMID- 7555184 TI - Therapeutic use of n-3 fatty acids for vascular disease and thrombosis. PMID- 7555185 TI - Use of antithrombotic agents during pregnancy. PMID- 7555186 TI - Prevention of venous thromboembolism. PMID- 7555187 TI - Antithrombotic therapy for venous thromboembolic disease. PMID- 7555188 TI - Antithrombotic therapy in atrial fibrillation. PMID- 7555189 TI - Antithrombotic therapy in valvular heart disease. PMID- 7555190 TI - Antithrombotic therapy in patients with mechanical and biological prosthetic heart valves. PMID- 7555191 TI - Antithrombotic agents in coronary artery disease. PMID- 7555192 TI - Coronary thrombolysis. PMID- 7555193 TI - Antithrombotic therapy in patients with saphenous vein and internal mammary artery bypass grafts. AB - In most studies, aspirin has been shown to be effective for a period of 1 year in reducing the frequency of saphenous vein bypass graft occlusion when begun 1 day before surgery, on the day of surgery, or the day after surgery. Effective doses of aspirin range from 100 to 975 mg/d. Aspirin in combination with dipyridamole is effective in the prevention of saphenous vein bypass graft occlusion if aspirin and dipyridamole therapy is started 1 or 2 days before surgery or aspirin therapy is started on the day of surgery but dipyridamole therapy is started before surgery or if treatment with both aspirin and dipyridamole is started 1 day after surgery. Aspirin in combination with dipyridamole is not more effective than aspirin alone in the prevention of saphenous vein graft occlusion. Bleeding is higher among patients treated with aspirin alone than among controls if aspirin therapy is started 1 day before surgery. Bleeding is not greater in comparison to controls if aspirin therapy is started the day of surgery or 1 day after surgery. When aspirin and dipyridamole are used in combination, bleeding is higher than in controls, and bleeding is higher than with aspirin alone. The continued use of aspirin for 2 additional years after an initial year of aspirin therapy for the prevention of saphenous vein bypass graft occlusion showed no additional long-term benefit on graft patency at the end of the third year. PMID- 7555194 TI - Antithrombotic therapy in peripheral arterial occlusive disease. PMID- 7555195 TI - Antithrombotic therapy for cerebrovascular disorders. An update. PMID- 7555196 TI - Making decisions about antithrombotic therapy in heart disease. Decision analytic and cost-effectiveness issues. PMID- 7555198 TI - Antithrombotic therapy in patients undergoing coronary angioplasty. PMID- 7555197 TI - New antithrombotics. PMID- 7555199 TI - Medicolegal implications of consensus statements. PMID- 7555201 TI - Concentrations of ceftibuten in bronchial secretions. AB - Ceftibuten is a broad-spectrum oral cephalosporin exhibiting antimicrobial activity against a wide range of gram-negative and some gram-positive pathogens. Pharmacokinetic studies have shown that the molecule has an oral bioavailability higher than 90% of the administered dose (reaching peak serum concentrations of 5 19 mg/l after a single dose of 200 and 400 mg). Moreover, ceftibuten has been shown to be useful in the treatment of acute lower respiratory tract infections. This study was performed to determine the distribution of ceftibuten in bronchial secretions from patients affected by the exacerbation of chronic bronchitis. Patients were treated with a single 400-mg oral dose of ceftibuten. Blood and bronchial-secretion samples were obtained just before, and at 0.5, 1, 2, 4, 8, 12, 16 and 24 h after dosing. Cells were separated from bronchial secretions by centrifugation. Ceftibuten in duplicate samples of both serum and bronchial secretion was quantified by HPLC. Ceftibuten reached peak levels 2 and 4 h after oral administration in serum and in bronchial secretions, respectively (18.12 +/- 2.13 and 9.19 +/- 3.1 mg/l, respectively). Falling curves after the peaks showed a monoexponential decay. The absorption was very rapid both in serum and bronchial secretions, but elimination was slower in bronchial secretions than in serum. PMID- 7555200 TI - Antithrombotic therapy in children. PMID- 7555202 TI - An in vitro investigation of the bioactivities of ciprofloxacin and the new fluoroquinolone agents clinafloxacin (CI-960) and PD 131628 against Mycobacterium tuberculosis in human macrophages. AB - In this study the intracellular bioactivity of ciprofloxacin and the new fluoroquinolone agents clinafloxacin (CI-960) and PD 131628 against Mycobacterium tuberculosis (H37Rv) was compared with rifampicin using human macrophages. Monocyte-derived macrophages were infected with M. tuberculosis in the presence of 10% autologous serum and treated with the antibiotics for 2 days, either immediately after infection or 3 days post-infection. The survival of the intracellular microorganisms was determined using the BACTEC tuberculosis system. Clinafloxacin, although not as active, compared favourably with rifampicin at concentrations ranging from 0.1 to 5 micrograms/ml in both systems, whereas PD 131628 performed reasonably well only when added directly after infection. However, ciprofloxacin was relatively unimpressive with intracellular bioactivity detected only with the highest concentration used (5 micrograms/ml). The ability of clinafloxacin, but not PD 131628, to inhibit mycobacteria after most of the organisms have escaped from the fused phagosomes emphasizes the importance of using a prolonged incubation time after infection when screening new antituberculosis drugs for intracellular bioactivity. PMID- 7555203 TI - Bactericidal kinetics of an in vitro infection model of once-daily ceftriaxone plus amikacin against gram-positive and gram-negative bacteria. AB - The in vitro efficacy of ceftriaxone plus amikacin combination against gram positive and gram-negative bacteria, clinically isolated from patients affected by pneumonia in intensive care units, was compared to that of the 2 drugs used alone. The study was performed using a dynamic model in which the human kinetics of the drugs after intramuscular administration was simulated. The antibacterial activity was tested by determining the bacterial cell count (CFU/ml). Killing curves came out from plotting the log CFU/ml versus time. In the same way, ceftriaxone and amikacin concentrations were assayed by HPLC and fluorescence polarization immunoassay, respectively. The results show that ceftriaxone plus amikacin combination exert a high killing activity against all tested strains. The two antibiotics alone initially have a good killing activity but this is followed by bacterial regrowth for all tested isolates. This data supports the results of several clinical studies which have shown a good therapeutic efficacy of ceftriaxone plus amikacin combination in the treatment of severe infections caused by organisms intermediately sensitive to these drugs. PMID- 7555204 TI - Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium kansasii. AB - The newly developed E test was compared with a conventional proportion dilution method for determining the sensitivity of Mycobacterium kansasii to amikacin, streptomycin, fusidic acid, rifampicin, clarithromycin, ciprofloxacin, ofloxacin, and fleroxacin. There was no more than one strain with different rating, except for ciprofloxacin. In this case, the breakpoint concentration had an unfavourable position at the top of the strip, and susceptible isolates in the dilution test were defined resistant in the E test. It is concluded that the E test is suitable for testing slowly growing mycobacteria other than tubercle bacilli, and may replace the more laborious dilution methods, particularly for testing M. kansasii. PMID- 7555205 TI - Antibacterial activity of azithromycin against Brucella melitensis. AB - In vitro antibacterial activity of the new macrolide azithromycin was tested against 116 strains of Brucella melitensis, isolated from 115 patients in a major tertiary care referral center. Eighty-seven percent of the strains were inhibited by 1.0 mg/l and all the 116 strains by 2.0 mg/l of azithromycin. Comparison was made with tetracycline, gentamicin, trimethoprim-sulfamethoxazole, rifampicin and ciprofloxacin. All the isolates were susceptible to gentamicin, tetracycline, trimethoprim-sulfamethoxazole, rifampicin. One hundred and fifteen of the 115 strains were also susceptible to ciprofloxacin. PMID- 7555206 TI - In vitro antibacterial activities of FK037: a new parenteral cephalosporin. AB - The antibacterial and bactericidal activities and the stability of FK037 to beta lactamases were investigated and compared with those of cefpirome, flomoxef, ceftazidime, ceftizoxime, and vancomycin. Against clinical isolates of methicillin-sensitive and methicillin-resistant Staphylococcus aureus, FK037 was less active than vancomycin but more potent than the other drugs tested. Among highly methicillin-resistant staphylococci (MIC for methicillin: 200 mg/l or higher), none of the strains were highly resistant to FK037 (MIC > or = 100 mg/l) unlike the other cephalosporins. With the exception of Enterococcus faecalis, FK037 had an activity equipotent to that of cefpirome and far superior to those of flomoxef, ceftazidime and ceftizoxime against other gram-positive clinical isolates. The activity of FK037 against Pseudomonas aeruginosa and Pseudomonas cepacia was equipotent to that of cefpirome but 2- to 4-fold less active than that of ceftazidime. FK037 inhibited 90% of Enterobacter cloacae and Citrobacter freundii growth at a concentration of 6.25 mg/l; it was as active as cefpirome and much more active than ceftazidime. Against the other gram-negative bacteria tested, FK037 was equipotent to cefpirome and ceftazidime and was more effective than flomoxef. FK037, like cefpirome, portrayed a high stability to various beta lactamases except type II penicillinase and oxyimino-cephalosporinase (CXase). FK037 scored an MIC range of 0.013-6.25 mg/l against numerous beta-lactamase producing bacterial strains with the exception of some CXase-producing strains and a cephalosporinase-producing C. freundii; and against strains other than P. aeruginosa FK037 was as active as cefpirome and 2- to 32-fold more active than ceftazidime. FK037 displayed highly potent activities against cephalosporinase producing E. cloacae, C. freundii and Serratia marcescens which were resistant to flomoxef and ceftizoxime. FK037 was highly bactericidal against S. aureus and Escherichia coli at its MIC or higher. PMID- 7555207 TI - Mechanism of enhancement of bactericidal activity of phagocytes against Klebsiella pneumoniae treated with subminimal inhibitory concentrations of cefodizime. AB - The effects of a sub-MIC of cefodizime on the morphology of the capsular structures and on the surface physicochemical properties, such as hydrophobicity and charge, of encapsulated Klebsiella pneumoniae were studied. The enhancement of bactericidal activity of macrophages against bacteria treated with sub-MICs of antibiotics was evaluated as the killing index. Cefodizime treatment gave the highest value of 32. Electron microscope observations revealed that the capsular material layer of cefodizime-treated K. pneumoniae was markedly thinner (32 nm) than that of untreated bacteria (160 nm) or bacteria treated with other antibiotics (75-90 nm). Contact angle measurement revealed that the surface of cefodizime-treated K. pneumoniae was more hydrophobic than that of untreated bacteria or bacteria treated with other antibiotics. Furthermore, the negative charge of the surface of K. pneumoniae decreased significantly with cefodizime treatment compared with the surface of untreated bacteria. These findings suggest that the treatment of K. pneumoniae with a sub-MIC of cefodizime reduced the thickness of the capsular material layer and that these changes increased the surface hydrophobicity of the bacteria and decreased the negative charge of the bacterial surface to render K. pneumoniae more susceptible to phagocytic activity by reducing the physical repulsion between the bacteria and phagocytes. PMID- 7555208 TI - Antifungal activity of LY295337 in vitro against clinical isolates of Candida spp. AB - LY295337 is a new antifungal agent derived from the echinocandins; it shows in vitro and in vivo activity against a great variety of pathogenic fungi. In this study, we evaluated the in vitro activity of LY295337 against 201 strains of Candida spp. using two culture media: Sabouraud dextrose broth (SDB) and RPMI 1640; fluconazole was used as reference antifungal agent. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method for LY295337 in SDB and RPMI 1640, and for fluconazole by an agar dilution method. The fungicidal activity of LY295337 was also determined. The MIC-50 as well as MIC-90 values obtained for LY295337 in SDB (range 0.025-0.6 mg/l) were lower than those obtained for RPMI 1640 (0.035-1.25 mg/l). All the strains resistant to fluconazole were sensitive to LY295337 (MIC < 0.6 mg/l). These results show that LY295337 is an excellent antifungal agent with a fungicidal activity in vitro against various species of Candida. PMID- 7555209 TI - Impact of rufloxacin and ciprofloxacin on the intestinal microflora in a germ free mice model. AB - The impact of rufloxacin and ciprofloxacin on faecal microflora was investigated in an experimental germ-free mice model into which human gastrointestinal flora was introduced and stabilized. Animals received oral doses of 10, 40 or 80 mg rufloxacin/kg, 30 or 60 mg ciprofloxacin/kg once daily for 10 days, or no treatment. Microflora was studied before, during and 1 week after treatment. No significant effect ( < 2 log reduction, p > 0.05) on aerobic microflora was observed after rufloxacin as compared to controls, except for a marked decrease in enterobacteria and a slight decrease in enterococci, staphylococci and anaerobes at the highest dose. Both doses of ciprofloxacin significantly reduced enterobacteria and clostridia, while the lower dose slightly reduced peptococci, bifidobacteria and eubacteria. The depression of the intestinal ecosystem was reversible, except for enterobacteria at the higher ciprofloxacin dose. No bacterial resistance was detected after either treatment. PMID- 7555210 TI - Cefodizime modulates in vitro tumor necrosis factor-alpha, interleukin-6 and interleukin-8 release from human peripheral monocytes. AB - Among third-generation cephalosporins, cefodizime (CFDZ) has shown to modulate many functions of the host defense system against infections. The aim of the present study was to assess the in vitro CFDZ-dependent modulation of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and IL-8 release from lipopolysaccharide (LPS)-stimulated human peripheral mononuclear cells (MNCs). Two other third-generation cephalosporins: ceftriaxone (CFX) and ceftazidime (CFT), were also tested under the same experimental conditions. At concentrations ranging from 200 to 50 micrograms/ml, CFDZ significantly decreased TNF-alpha and IL-6 release from maximally (LPS 1 microgram/ml) stimulated MNCs (42% inhibition of TNF-alpha release with 100 micrograms/ml of CFDZ). On the other hand, CFDZ revealed a marked stimulatory effect on IL-8 release (200 micrograms/ml of CFDZ induced 51.5% enhancement of IL-8 release). On the contrary, both CFX and CFT failed to exert any significant effect on TNF-alpha, IL-6 or IL-8 release. PMID- 7555211 TI - Does P-glycoprotein play a pivotal role in the drug resistance of an MDR variant, K562/Dox? AB - A multidrug-resistant (MDR) variant, K562/Dox, was selected from repeated exposure of human erythroleukemia cell line K562 to doxorubicin (Dox). K562/Dox displayed typical MDR features with respect to its cross-resistance to a variety of functionally and structurally unrelated compounds: vincristine (Vin), Dox, mitomycin C, reduced steady-state intracellular anthracycline accumulation, and elevated P-glycoprotein expression/mdr1 mRNA transcription/mdr1 gene amplification. Nevertheless, by incubation of cells with Dox/epirubicin (Epi)/daunorubicin (Dau) (5-80 micrograms/ml), the initial drug uptake was similar (p > 0.05) in K562/Dox and K562 cells, suggesting P-glycoprotein-mediated drug efflux would not occur unless a relatively high cellular drug concentration was reached. After 8 h incubation of cells with 50 ng/ml Dox (5 times higher than its IC50 to K562 cells), there were only slight differences (p > 0.05) in intracellular drug levels between K562/Dox and K562 cells, clearly indicating that K562/Dox, circumventing drug toxicity in this case, was irrelevant to reduced drug accumulation caused by P-glycoprotein. Similar results were obtained when Epi or Dau was applied. Despite complete restoration of anthracycline accumulation in K562/Dox cells in the presence of 6 mumol/l verapamil, the reversal of their drug resistance was incomplete. These results suggest that P glycoprotein-mediated drug efflux possibly did not play a primary role in the drug resistance of K562/Dox cells. PMID- 7555212 TI - Initial treatment of sepsis in non-neutropenic patients: ceftazidime alone versus 'best guess' combined antibiotic therapy. AB - We conducted a prospective, randomized, multicentric study in community hospitals. Patients with clinical sepsis, rectal temperature > or = 38 degrees C and pulse rate > or = 100 bpm were randomized to receive ceftazidime (group CAZ) or a combination of antibiotics freely chosen by the clinician following his 'best guess' (group COMB). On specified grounds, the clinician could also treat patients in an open group with a free combination of antibiotics (group OPEN). The severity of disease at study admission was assessed by a clinical estimation and an Apache II score. There were 128 patients included: 56 randomized in group CAZ, 50 in group COMB, and 22 in the OPEN group. Ninety-one patients were evaluable: 41 in group CAZ, 30 in group COMB, 20 in OPEN group. At the end of the period of empirical treatment (48-72 h), the clinical success rates (improvement of status) were 93, 93 and 75% (p for group OPEN vs. groups CAZ or COMB: 0.10). The bacteriological success rates (sterile blood cultures) were 91, 88 and 80% (p not significant). mean Apache II score was 16.7 and the score correlated significantly with outcome, as did clinical evaluation. In conclusion, ceftazidime alone was a safe antibiotic therapy in this study and we could not demonstrate a superiority of a combined antibiotic therapy chosen by the clinician following his 'best guess' over ceftazidime. PMID- 7555213 TI - Clinical experience with ceftriaxone treatment in the neonate. AB - The safety of ceftriaxone has been evaluated in 80 neonates who were treated empirically for suspected infection with either ceftriaxone and ampicillin (group A, age 0-72 h) or ceftriaxone and vancomycin (group B, age greater than 72 h). Within 48 h after birth 2 group A patients died from sepsis (Haemophilus influenzae, Streptococcus pneumoniae, 1 case each); 1 group B patient died from sepsis (Pseudomonas aeruginosa). All bacterial isolates from group A patients were susceptible to ceftriaxone, but in 4 of the 8 group B patients with positive cultures a change in antibiotic therapy was required. Eosinophilia, thrombocytosis and an increase in serum alkaline phosphatases were observed in a limited number of patients during and after discontinuation of treatment. Direct hyperbilirubinemia ( > 2 mg/dl) occurred in 2 cases during treatment. Gallbladder sludge was sonographically diagnosed in 6 patients, but disappeared within 2 weeks after detection. One neonate had exanthema. Nurses rated ease of administration as very good. Ceftriaxone appears to be an interesting alternative in the empiric antibiotic treatment in the early neonatal period. PMID- 7555214 TI - The effects of systemic family violence on children's mental health. AB - This study examines the link between different forms of family aggression and children's symptoms of psychopathology. The goal of the study was to understand what forms children's problems might take in violent homes and whether close ties within the family (to the mother or a sibling) buffered children. Interviews with 365 mothers and 1 of their children between the ages of 6 and 12 about abuse in the home, support and closeness within the nuclear family, and mother's and children's mental health formed the basis of this study. Families were recruited from battered women's shelters and the community. We found that different forms of abuse in the home were highly interrelated and that children of battered women were at risk for child abuse. Domestic violence predicted children's general psychopathology, but we uncovered little evidence for the presence of specific sorts of disorders as a result of family dysfunction. Although mothers experiencing conjugal violence were more likely to have mental health problems, their mental health did not mediate the children's response to family conflict. Finally, there was less sibling and parental warmth in families marked by aggression, although when it was present, family social support failed to buffer children. Although the general pattern of results was consistent across respondents (mother and child), there was low agreement on symptoms of child psychopathology. PMID- 7555215 TI - Community level factors and child maltreatment rates. AB - Using census and administrative agency data for 177 urban census tracts, variation in rates of officially reported child maltreatment is found to be related to structural determinants of community social organization: economic and family resources, residential instability, household and age structure, and geographic proximity of neighborhoods to concentrated poverty. Furthermore, child maltreatment rates are found to be intercorrelated with other indicators of the breakdown of community social control and organization. These other indicators are similarly affected by the structural dimensions of neighborhood context. Children who live in neighborhoods that are characterized by poverty, excessive numbers of children per adult resident, populations turnover, and the concentration of female-headed families are at highest risk of maltreatment. This analysis suggests that child maltreatment is but one manifestation of community social organization and that its occurrence is related to some of the same underlying macro-social conditions that foster other urban problems. PMID- 7555216 TI - Alternative pathways to chronic depressive symptoms in young adults: gender differences in developmental trajectories. AB - Personality and intelligence associated with depressive symptoms in 23-year-olds were evaluated using prospective data from preschool through adolescence. Gender moderated the prospective relations between personality/intelligence and age-23 depressive symptoms. Young men with elevated age-23 General Behavior Inventory (GBI) scores manifested as early as in preschool allocentric behaviors: undersocialization and interpersonal antagonism. This pattern remained highly stable over the following 15 years. Prospective correlates of depressive symptoms in young women--not reliably identified until adolescence--were more likely than in young men to express autocentric concerns: oversocialization and introspective concern with self. These gender differences continued to persist but decreased in strength after puberty. Intellectual competence displayed significantly stronger negative correlations with age-23 GBI scores in males than in females. Findings were discussed in terms of gender differences in the development of chronic depressive symptoms, with special emphasis on equifinality in developmental pathways. PMID- 7555217 TI - Only children and children with siblings in the People's Republic of China: levels of fear, anxiety, and depression. AB - In 1979, China implemented its one-child-per-family policy in order to control its burgeoning population. Subsequently, concern has been raised about the policy and its potentially negative effects on children. In the present study, we examined these presumed effects on 202 adolescents born before the policy went into effect, 290 preadolescents born during the period in which the policy was being implemented, and 239 children who were born after the policy went into effect. Measures of fear, anxiety, and depression were obtained. Contrary to our hypotheses, based on concerns raised by the one-child policy, children with siblings reported significantly higher levels of fear, anxiety, and depression than only children, regardless of when they were born. For depression, this effect was qualified by a sibling status x age interaction. Children with siblings born after the policy went into effect, or during its implementation, reported higher levels of depression than did only children; however, only children and children with siblings born before the policy went into effect did not differ significantly from one another. Sociocultural factors associated with these findings are explored. PMID- 7555218 TI - Friends' influence of adolescents' adjustment to school. AB - Adolescents may be influenced both by their friends' behaviors and by the features of their friendships. To examine both types of influence, seventh and eighth graders (N = 297) were asked in the fall of a school year to report their involvement and disruption at school. The students also described the positive and negative features of their best friendships. Teachers reported on the students' involvement, disruption, and grades. These assessments were repeated in the following spring. Students whose friends in the fall described themselves as more disruptive increased in self-reported disruption during the year. Girls' self-reported disruption was more influenced by that of their very best friend than was boys'. Students whose very best friendships had more positive features increased in their self-reported involvement during the year. Students whose friendships had more negative features increased in their self-reported disruption, but only if their friendships also had many positive features. The theoretical and practical implications of these findings, and the adequacy of different methods for estimating friends' influence, were discussed. PMID- 7555219 TI - Friendships and social networks in childhood and adolescence: fluidity, reliability, and interrelations. AB - Social networks and friendships were tracked over a 3-week period for 132 students enrolled in fourth- and seventh-grade classrooms. Individual interviews were employed to collect data on friendships. Social group membership was determined by the composite social-cognitive map (SCM) procedure and by self reports. Considerable overlap was found among the methods for establishing relational patterns (i.e., friendships, self-reported groups, composite social cognitive maps). When loose criteria for stability were employed, high stability was found in friendships (p = .56-.75) and social group membership (p = .90) over a 3-week period. But when stringent criteria were employed, only modest social relationship stability was observed in both methods, suggesting that there is much greater fluidity in peer bonds than has been generally recognized. The relationship between friendship and social network measures, the distinctive information yielded by social networks, and gender and age differences in group structure, fluidity, and friendships are discussed. PMID- 7555220 TI - Prosocial behavior and caring in adolescence: relations to self-understanding and social judgment. AB - The relation of self-understanding and moral judgment to dedicated prosocial behavior is investigated. Participants were African-American and Latin-American adolescents who had been nominated by community leaders for having demonstrated unusual commitments to care for others or the community (care exemplars). The care exemplars, and matched comparison adolescents, were extensively interviewed over the course of 4-6 sessions in order to elicit self-understanding, moral judgment, and implicit personality theories. The care exemplars were more likely than the comparison adolescents to: (1) describe themselves in terms of moral personality traits and goals, (2) view themselves as having closer continuity to their pasts and futures, (3) think of themselves as incorporating their ideals and parental images, and (4) articulate theories of self in which personal beliefs and philosophies are important. There were no differences between the care exemplars and the comparison adolescents in developmental stages of moral judgment nor in the abstractness of their implicit personality theories. PMID- 7555222 TI - Cognitive representations of self, family, and peers in school-age children: links with social competence and sociometric status. AB - Attachment and social-cognitive theories of interpersonal relations have underscored the integral role that internalized cognitive representations may play as mediators of the link between family and peer relationships. 3 predictions consistent with this conceptualization received support in the present study of 161 7-12-year-old school children. In Part 1 of the study, significant connections were found among different components of cognitive representations, including social perceptions, interpersonal expectancies, and schematic organization and processing of social information. Moreover, generalization was found among children's representations across 3 interpersonal domains--that is, family, peer, and self. In Part 2, negative representations of self and others were found to be associated with increased social impairment, including dysfunctional social behavior and less positive status in the peer group. Implications of the findings for theories of interpersonal competence and interventions with socially impaired children are discussed. PMID- 7555221 TI - The role of emotionality and regulation in children's social functioning: a longitudinal study. AB - Multiple measures of children's emotionality (emotional intensity and negative affectivity), regulation (including attentional and behavioral regulation and coping), and social functioning (teachers' reports of nonaggressive/socially appropriate behavior and prosocial/socially competent behavior; and parents' reports of problem behavior) were obtained for 6-8-year-olds. In addition, emotionality, attentional regulation, and coping were assessed 2 years previously. Social functioning was expected to be predicted by low negative emotionality and high levels of regulation. In general, the data supported the predictions, although the findings for parent reports of problem behavior were primarily for boys. Prediction of social functioning from measures of regulation and emotionality occurred primarily within a given context (school vs. home) rather than across contexts, even though there were relations across reporters within the school or home context. In addition, vagal tone, a marker of physiological regulation, was positively related to competent social functioning and emotionality/regulation for boys, but inversely related for girls. PMID- 7555223 TI - Social interactions in the home and the development of young children's conceptions of the personal. AB - Observations and interviews of 20 middle-class 3- and 4-year-olds and their mothers were conducted to examine the emergence of the personal domain. Interviews with children showed that 3- and 4-year-olds make a conceptual distinction between personal, and moral or conventional issues. Interviews with mothers indicated that they viewed it as important for young children to have freedom of choice over personal issues to develop a sense of autonomy and individuality. Observations in the home revealed that mothers tended to give direct social messages to children about moral, conventional, and prudential events, and were more likely to give indirect social messages in the form of offered choices to children in response to personal issues. Mothers were more likely to negotiate with children over personal than other social events. These data revealed a pattern of social interactions concordant with event domain, which included a reciprocal system along the border between the personal and the conventional. PMID- 7555224 TI - Children's gender-based reasoning about toys. AB - The goal of these studies was to investigate how preschool children use gender based reasoning in making judgments about toy preferences for themselves and for others. In Studies 1 and 2, children (n = 22, n = 71) were shown unfamiliar, non sex-typed toys and asked to rate how much they, other girls, and other boys would like each toy. As expected, children made gender-based inferences: "What I like, children of my sex will also like, and children of the other sex will not like." Study 3 was designed to assess how children use gender-based reasoning to make decisions about attractive and unattractive toys when they are given gender labels. Children (n = 91) were shown unfamiliar toys varying in attractiveness that were given explicit gender labels (e.g., "this is a toy girls really like") or no label. With a different experimenter (to avoid demand characteristics), children rated their own and others' liking of the toys. Children used gender labels to guide their own preferences and their expectations for others. Even with very attractive toys, children liked toys less if they were labeled as being for the other sex, and expected other girls and boys to do the same. The role of gender-based reasoning in cognitive theories of gender and on children's play preferences is discussed. PMID- 7555225 TI - Individual differences in young children's pretend play with mother and sibling: links to relationships and understanding of other people's feelings and beliefs. AB - 50 33-month-old children were observed at home with their siblings and mothers. Observational measures of pretend play, observer ratings of the child's, mother's, and sibling's behavior, and measures of family discourse about feelings were collected. At 40 months each child was assessed on Bartsch and Wellman's false beliefs task and Denham's affective perspective taking task. Results revealed individual differences in the amount and sophistication of young children's social pretend play and suggested that these individual differences are related to experiences in the relationships that young children have with their mothers and siblings. Results also indicated that early social pretend play was significantly related to the child's developing understanding of other people's feelings and beliefs. The data are interpreted as providing support for the notion that early experience in social pretense is associated with children's mastery of the relation between mental life and real life. The importance of considering the relationship context of social pretense is also discussed. PMID- 7555226 TI - Maternal working models of attachment, marital adjustment, and the parent-child relationship. AB - This study examined the connection between maternal working models, marital adjustment, and the parent-child relationship. Subjects were 45 mothers who were observed in problem-solving interactions with their 16-62-month-old children (M = 33 months). Mothers also completed the Attachment Q-set, the Adult Attachment Interview, and a marital adjustment scale. As predicted, maternal working models were related to the quality of mother-child interactions and child security, and there was a significant relation between marital adjustment and child security. Maternal working models and marital adjustment were also associated interactively with child behavior and child security. Among children of insecure mothers, child security scores were higher when mothers reported high (vs. low) marital adjustment. No relation between child security scores and mothers' marital adjustment was found among children of secure mothers. These results suggest that maternal working models influence parenting and child adjustment well beyond infancy, to which period the few existing studies of adult attachment have been restricted. The results also suggest that interactions between maternal working models and the marital adjustment on child behavior and attachment security need to be more closely examined. PMID- 7555227 TI - Effects of mothers' simulated withdrawal and depressed affect on mother-toddler interactions. AB - Effects of enacted maternal withdrawal and depression were observed in a sample of 18-36-month-old children during counterbalanced brief (10-min) episodes of simulated depression and normal affect. As predicted, when mothers enacted withdrawal and depressed affect by becoming less positive, expressive, involved, talkative, and responsive, toddlers physically withdrew from them, made more negative physical bids for attention, and became more unfocused and generally negative. Children accepted their unresponsive mothers' infrequent interaction bids, but did not attempt to comfort them when they appeared depressed. In mothers' normal mood episodes, children were more positive, were never unfocused, and played closer to their mothers. As expected, toddlers reacted negatively to transitory maternal withdrawal and depressed affect, displaying their distress in a developmentally appropriate manner. PMID- 7555228 TI - Temperament and craniofacial variation in the first two years. AB - High levels of circulating glucocorticoids in fetal animals are associated with inhibited development of the lateral processes of the maxilla, or upper jaw, as these bones grow toward midline union early in gestation. This inhibition can prevent fusion and lead to the formation of cleft palate. Mouse strains are differentially susceptible to this effect. These strains also differ in behavioral and physiological characteristics that are analogous to those we have observed in 2 temperamental groups of children, analogies that prompted the current study. Infant temperament was assessed using behavioral observation at 4 months, and the ratio of upper facial width (bizygomatic breadth across the cheekbones) to height of the head and face was measured at 14 and 21 months. High reactive 4-month-old infants, who are predisposed to becoming timid, inhibited toddlers, had smaller bizygomatic ratios (narrower faces) at both 14 and 21 months compared to their low reactive peers, who are predisposed to becoming outgoing, uninhibited children. Differences in facial width were modest though significant and not attributable to overall stature. PMID- 7555229 TI - From walking to running: applying a dynamical systems approach to the development of locomotor skills. AB - Developmental transitions of complex systems may be studied by selecting (collective) variables that constrain the degrees of freedom for each developmental state. In a dynamical systems approach, the transitions from state to state are engendered through the scaling of contributing subsystems (control parameters). In this study, the locomotor skills of walking and running were compared in newly running infants by observing several likely collective variables including relative stance, estimated pathway of center of mass, and segmental/joint action. 4 children were filmed longitudinally at 5.5, 7.5, and 9.5 months of independent walking and then at 3 years of age. 3 trials per gait were selected for single stride analysis and compared with data from 4 adults. In general, the proposed collective variables showed transitional forms over the first few months of running, indicating a relatively continuous change between the 2 gait forms. Coordination of the knee joint was very similar between gaits and across age, but the ankle joint was less consistent for both gaits in the infants. Relative stance and stride length data indicated that the children could not generate vertical and horizontal displacement. These findings echo those found in newly walking infants and suggest that similar rate-limiting parameters are present for both gaits. PMID- 7555231 TI - Vasoactive intestinal peptide receptors in brains of hepatic encephalopathy rats. An experimental study. AB - We evaluated the effect of vasoactive intestinal peptide (VIP) receptor, a brain gut peptide receptor which is capable of exciting central neurons, on the pathogenesis of hepatic encephalopathy (HE). By means of radio-ligand binding assay, VIP receptors in the crude synaptosomal membranes of rat brains were investigated in rat models with HE induced by partial hepatectomy following carbon tetrachloride intoxication and in controls. The binding to the VIP receptor was studied, with 125I-VIP as a radioligand. Scatchard analysis of the binding data was linear, yielding a dissociation constant (Kd) of 0.28 +/- 0.01 nM and a maximal binding capacity (Bmax) of 9.56 +/- 0.29 fmol/mg of protein in HE rats. Only decreased Bmax values were observed (P < 0.002), while the Kd values were statistically unchanged (P > 0.20) in HE rats as compared with the controls. The results suggest that the changes of VIP receptors in the brains of HE rats play a significant role in the pathogenesis of HE. The mechanism of HE induced by the alterations of VIP receptors in the brains was discussed. PMID- 7555230 TI - Applicability of prostaglandin E1 indirect portal vein angiography in patients with portal hypertension. AB - The imaging quality of the portal vein was obviously improved with prostaglandin E1 (PGE1) indirect portal vein digital subtraction angiography (DSA) in 23 cases. The time-density curve showed that the occurrence rate of opposite hepatic blood flow of splenic vein (SV) was the highest (17.4%). The total visualization rate of the left gastric vein (LGV) was 78.3%, and the visualization rate of the short gastric vein (SGV) was 36.4%. 38.9% of the LGV and all the SGV were visualized with indirect portal vein DSA through SA. Indirect portal vein angiography through superior mesenteric artery and that through splenic artery were of equal importance. In portal hypertension patients with hemorrhage of the digestive tract, when LGV and SGV could not be visualized in PGE1 indirect portal vein DSA, the possibility of non-varices vein bleeding should be considered. When opposite hepatic blood flow with obvious dilation appeared in LGV and SGV, devascularization of the pericardial blood vessels would be justifiable. PMID- 7555233 TI - Radiotherapy of ectopic ACTH syndrome due to thoracic carcinoids. AB - From 1984 to 1990, six patients with histologically and endocrinologically proven ectopic ACTH syndrome were treated in our department. The lesion in five patients was clinically evident, but in the other was occult. All lesions were located within the chest. Four were thymic carcinoid and the other two were bronchial carcinoids. Most of the patients had typical clinical manifestations of Cushing's syndrome. Laboratory tests including histopathological examination confirmed the diagnosis of ectopic ACTH syndrome. The treatment consisted of surgical removal of the mass supplemented by radiotherapy. The results showed that after treatment all patients had satisfactory remission. Two bronchial carcinoid patients have been living for 40 and 88 months respectively without recurrence. Among 4 thymic carcinoid patients, only one has been living with no tumor for 34 months. One patient had recurrence and the other 2 died 32 and 50 months after the treatment respectively. However, the symptoms of two thymic carcinoid patients had not been relieved at all until radiotherapy was given. This paper discusses the method, dose and indications of radiotherapy. It should be pointed out that the prognosis of bronchial carcinoids is good while that of thymic carcinoids is relatively poor. PMID- 7555232 TI - Enhanced adeno-associated virus vector expression by adenovirus protein-cationic liposome complex. A novel and high efficient way to introduce foreign DNA into endothelial cells. AB - Adeno-associated virus vector (AAV), a non-pathogenic integrating vector, can integrate on chromosome 19 in humans. But its gene transfer efficiency is quite low. In this study we combined adenovirus (Adv-5) capsid protein or the Fiber protein of Adv with liposome, termed adenosome (adenovirus protein-cationic liposome complex). This complex can bring AAV/CMV-LacZ to the endothelial cells, and improve the efficiency of gene transfer. It may be a novel, specific, stable and safe gene delivery system and will be widely used in human gene therapy. PMID- 7555234 TI - Direct preparation and meiotic analysis of human semen. AB - A meiotic study of human semen specimens was carried out in 60 cases. Among them, 20 were normal controls, 20 suffered from infertility, and 20 had a history of recurrent spontaneous abortions. Of the cases of infertility and abortion, 50% were found to have numerical and/or structural chromosomal aberrations in spite of the normal blood karyotype. It is suggested that chromosomal aberrations may be the cause of sterility and abortion. The success rate of semen chromosome G banding in the infertility, abortion and control groups was 70%, 55% and 25% respectively. The semen samples of three cases of heterozygous carriers with reciprocal chromosomal translocation were also studied. The ring quadrivalents were identified in all cases. The results may explain the recurrent abortions in these patients. PMID- 7555235 TI - A case-control study of the etiology of laryngeal cancer in Liaoning Province. AB - A case-control study of laryngeal cancer was conducted in Liaoning Province between January 1991 and April 1992 among 100 subjects with newly diagnosed cancer and 100 controls. The results of one factor analysis, odd ratio (OR), and conditional logistic regression indicated that smoking was the first significant risk factor for laryngeal cancer with an OR value of 16.8. Tobacco smoking ranked the first with an OR value of 30.4. Other factors such as alcoholic indulgence, insufficient intake of vegetable and fruits in winter and spring, air pollution in room and working environment, and fatigue of vocal cords were closely associated with the occurrence of laryngeal cancer. PMID- 7555237 TI - A new method for early diagnosis of carcinoma in bile duct-sonography of common bile duct before and after injection of ceosunin. AB - 42 cases of dilatation (7-15 mm) of extrahepatic bile duct after ceosunin injection (cholagogic method) were observed ultrasonographically. Among the 18 cases of testified obstructive lesions of the bile duct, 13 (72%) showed widening of the bile duct with an increase of calibre from 9.8 mm to 11.7 mm, 3 (16%) had no obvious change, and 2 (12%) shrank. Among the 24 cases of non-obstruction 22 (92%) shrank with a decrease calibre from 9.6 mm to 6.3 mm, and 2 (8%) showed no obvious change. After receiving cholagogue, 9 cases of periampullary carcinoma in the obstructive group were confronted with widening of the bile duct calibre. After longitudinal and transverse rotation scanning method was used, 8 of the 9 cases had their tumor mass visualized. Combined cholagogic and rotation scanning methods raised the lesion manifestation rate of lower bile duct from 41% to 88%. A dilated bile duct unable to show shrinkage of calibre after cholagogue injection is believed to be a criterion for judging the presence of a suspicious case of bile duct obstruction. This criterion has a sensitivity of 89%, specificity of 92%, and accuracy of 90%. PMID- 7555236 TI - Morphometric analysis of spinous cell in oral submucous fibrosis. Comparison with normal mucosa, leukoplakia and squamous cell carcinoma. AB - The morphometric analysis of the spinous cell in 16 specimens of oral submucous fibrosis (OSF) was made by using interactive image analysis system (IBAS-II). 19 parameters of the size and shape were chosen, and compared with normal mucosa, leukoplakia, dysplasia and carcinoma. The results indicated that the cell dimensions (area, perimeter, all kinds of diameter) and nuclear cytoplasmic ratio in OSF were between normal mucosa and dysplasia as well as carcinoma. The former showed a progressive decrease (P < 0.01), and the latter showed a progressive increase (P < 0.01). The dimensions of the nuclei did not show considerable differences among the groups (P < 0.05). A series of discriminant functions had been developed with stepwise discriminant analysis, the agreement ratio for OSF was 93.75%. The decrease of cell area and the increase of nuclear cytoplasmic ratio could reflect a malignant progress. The cell morphometric model could discriminate OSF well from other groups, suggesting that the change of the epithelium in OSF appearing in the spinous cell is specific itself. PMID- 7555239 TI - Treatment of intracranial germinoma. AB - In 31 patients with intracranial germinoma who received radiotherapy, 15 were operated on, and 13 were treated with CSF shunt (7) and external ventricular drainage (6). After radiotherapy, clinical symptoms were improved in 93.5% of the patients. Follow-up of 16 patients for 2 to 14 years showed better results. We consider that initial treatment with radiotherapy may be appropriate for germinomas because of its high radiosensitivity. If hydrocephalus developed, external ventricular drainage combined with radiotherapy was performed, but shunting operation might be avoided because of potential peritoneal seedings of germinomas. In addition to chemotherapy, re-irradiation is an effective method for recurrent germinoma. PMID- 7555238 TI - Biochemical and biophysical characteristics of mitochondria in the hypertrophic hearts from hypertensive rats. AB - A comparative study of the biochemical and biophysical characteristics of myocardial mitochondria was conducted in 16-weeks-old male stroke-prone spontaneously hypertensive rats (SHRsp) and normotensive Wistar-Kyoto control rats (WKY) (n = 6-12). Cardiac and ventricular weights of SHRsp were significantly higher than those of the WKY group. The average amounts of mitochondria (Mit) and sarcoplasmic reticulum (SR) proteins isolated from the ventricle of SHRsp were 37% and 25% greater, respectively, when compared with those of WKY. Mit cytochrome c oxidase activities (mumol/min/mg) were 4.63 +/- 0.71 for SHRsp and 5.55 +/- 0.50 for WKY myocardium (mean +/- s), so that the activity of the former was significantly reduced by 17% (P < 0.01). Mit Ca2+, Mg(2+)-ATPase activity (mumol/min/mg) of SHRsp was 24% lower than that of WKY myocardium (P < 0.01). The fluorescence polarization of myocardial Mit membrane labelled with DPH (1, 6-diphenyl-1, 3, 5-hexatriene) in hypertensive rats was not different from that in control rats. The spectra of intrinsic protein fluorescence in 300-380 nm at an excitation wavelength of 290 nm were also identical in the Mit or SR between SHRsp and WKY myocardium. An ultrastructural analysis showed that there were more new-born Mit with smaller volumes and an increased number of isolated Mit in vitro from SHRsp myocardium, while there were larger Mit volumes isolated from WKY myocardium. These results demonstrated the disorder of mitochondrial energy metabolism in hypertrophied myocardium from SHRsp, which may be involved in the abnormality of myocardial calcium metabolism. PMID- 7555240 TI - A new animal model of coronary thrombosis and effects of antithrombotic agents. AB - The model was established in male rabbits with experimental atherosclerosis, which was induced by diet cholesterol (0.5 g per day for 8 weeks), by means of intravenous injection of one unit of pituitrin (P.P.). To evaluate the effects of aspirin, heparin and viper venom (VV) on this model, 26 male rabbits were divided randomly into four groups: group A (GA) as control, group B (GB) treated with heparin (10mg, i.v.), group C (GC) with VV (0.08 arginine esterase activity units), group D (GD) with both heparin and VV. Aspirin (30 mg) was given orally before experiment. The results showed that the rate of coronary thrombosis was 11.26% in GA, 8.10% in GB, 9.17% in GC, and 7.56% in GD respectively. The difference between each of three treated groups and the control one was significant (P < 0.005, 0.05, 0.001, respectively). Such a difference can also be found between GA and that without oral aspirin (11.26% vs 16.39%, P < 0.001). The beneficial effects of heparin and VV may be due to their inhibitory effects on different steps of thrombosis, i.e., heparin can prolong the coagulation time, and VV can inhibit platelet aggregation and decrease the concentration of plasma fibrinogen. It is concluded that heparin, viper venom, and especially their combination would be useful in the treatment of human acute coronary syndromes. PMID- 7555241 TI - Intravesical instillation of interleukin-2. An effective method for preventing the recurrence of bladder cancer. AB - The result of intravesical instillation with interleukin-2 (IL-2) for preventing the recurrence of bladder cancer and discussion of its mechanism was reported. 20 patients with histologically confirmed bladder transitional cell carcinoma were investigated. 2,000 u IL-2 was intravesically instillated every day for six days after resection of the neoplasm. The serum level of tumor necrosis factor (TNF) was significantly increased in 17 patients after IL-2 therapy. Recurrence occurred in 4 patients during the follow-up period of 10-18 months. The basic level of serum TNF was low and was not increased or even decreased after IL-2 in these patients. On the contrary, recurrence did not occur in another 5 patients with low basic level of TNF, but it was significantly increased after treatment of IL-2. TNF played a key role in preventing the recurrence of bladder cancer. The patients who had low basic level of TNF, which did not markedly increase or even decrease after IL-2 therapy were at high risk of recurrence. PMID- 7555243 TI - Anomalous origin of left coronary artery from pulmonary artery. Successful correction for a 7-year-old patient. AB - A 7-year-old girl with an anomalous origin of the left coronary artery from the pulmonary artery restored connection of the left coronary artery to the ascending aorta by aortocoronary bypass graft. Aortocoronary bypass grafting was performed with a vascular prosthesis. Postoperative angiography showed a patent left coronary artery without narrowing or kinking. Clinical improvement was rapid and the left ventricular function recovered completely. PMID- 7555242 TI - Changes of cellular morphology and nuclear DNA content in different nasopharyngeal epithelium from different patients. AB - Cellular morphology and nuclear DNA content were measured with point-counting technique and flow cytometry (FCM) respectively in normal nasopharyngeal epithelium (NE), tissue adjacent to carcinoma (ATC) and nasopharyngeal carcinoma (NPC). The results showed that all 20 patients with NE showed diploid, whereas 38 (38%) of 101 patients with NPC, 7 (58%) of 12 patients with recurrent NPC, 6 (30%) of 20 patients with ATC and 2 (33%) of 6 patients with NE positive to EBVCA IgA test showed nondiploid. A total of 19 morphometric parameters of the cell were measured and calculated. The results showed that with malignant transformation of NE, most of the cellular morphometric parameters gradually increased in numerical values while no marked difference was shown between normal NE and simple hyperplasia or metaplasia in terms of cytomorphometric characteristics. Most of the parameters in dysplasia changed significantly as compared with those normal NE, simple hyperplasia or metaplasia and NPC groups. Cytomorphometric analysis seems to give strong support to the concept that dysplasia is among the transitional stages of disease process between normal NE and NPC. Our results show that morphometric analysis and FCM may offer valuable and objective criteria in the early diagnosis of NPC and premalignant disease. PMID- 7555244 TI - Recent advances of perinatal medicine in China. PMID- 7555246 TI - A survey of extracorporal shock wave lithotripsy in China. PMID- 7555245 TI - Current status of medical imageology. In memory of the 100th anniversary of the discovery of X-rays by the great physicist Wilhelm Rontgen. PMID- 7555247 TI - Progress in diagnostic and therapeutic techniques of cardiovascular diseases in China 1994. PMID- 7555248 TI - An extensive subfrontal approach to the lesions involving the skull base. AB - A modification of the transbasal approach of Dorome called extensive subfrontal approach and the surgical results with this approach in 22 cases are presented. Bilateral frontal craniotomies incorporated with the removal of orbital ridges and part of the orbital roofs were fashioned en bloc. It may give rise to good exposure of the midline lesions of the anterior, middle and posterior skull base, minimizing the need for the retraction of frontal lobes. There was no surgical mortality in this series of cases. Of the 20 cases with tumors, total resections were achieved in 11 cases, subtotal or large resections in 4 cases and partial resection in one case. Two patients with spontaneous rhinorrhea were successively treated surgically. 21 patients had a follow-up with a time ranging from 1-11 years (a mean of 3 years). 15 patients resumed their jobs with no evidence of recurrence of the original disease, and 5 patients able to live self-care. One patient with an olfactory neuroblastoma died 3 years after the operation owing to relapse of the tumor. PMID- 7555250 TI - Preliminary study on controlled hypotension induced by nimodipine in craniocerebral surgery. AB - Nimodipine, produced in China, was applied to lower the blood pressure (BP) in 20 cases in craniocerebral operation. 0.02% solution of Nimodipine was dripped intravenously (by 0.02% of density), at a preliminary speed of 600-800 micrograms/min, and 4-12 mg of total dose. Mean arterial pressure (MAP) decreased and maintained at 7.3-8.0 kPa, and lasted 30-60 minutes. The scopes of decreasing pressure were 33.0%-37.7% of MAP, 31.6%-35.6% of systolic pressure (SP), and 36.1%-41.9% of diastolic pressure (DP). The results of lowering pressure which reduced the intraoperative bleeding were significant. After withdrawal of drug supply for 15-30 minutes, the BP raised up to the normal level. No "rebounding BP rise" occurred. PMID- 7555249 TI - Percutaneous endovascular embolization of intracerebral arteriovenous malformations. Experience in 72 cases. AB - Endovascular therapeutic embolization of arteriovenous malformations (AVMs) of the brain was performed in 72 patients between October 1986 and March 1993. From October 1986 to May 1991, 38 patients in this series were treated with isobuty1-2 cyanoacrylate (IBCA) glue. After June 1991, in the remaining 34 patients the embolic materials used included surgical silk (5-0), polyvinyl alcohol (PVA), ethanol, and estrogen. After treatment, 29 patients (40.3%) had complete angiographic obliteration of AVM, 15 (21%) transient neurologic postembolization deficit, and 3 (4%) permanent deficit without death. Embolic agents and procedures are discussed as to their curative effects, safety, normal perfusion pressure breakthrough (NPPB) problem, with silk mixture fluid being considered preferable. PMID- 7555251 TI - Laryngeal reconstruction and survival after function-sparing laryngectomy for selected T3 and T4 lesions. AB - Most patients lost their vocal ability after ablative surgery for the laryngeal cancer. Total laryngectomy has a history of over 100 years, hence many surgeons are accustomed to the en bloc organ resection and the loss of ability for communication after surgery. The recent advance of surgery introduces the concept and technique of radical cure and functional preservation of the cancer-afflicted organ at the same setting. For lesions of late T categories (T3, T4), the maintenance of normal function after wide resection to ensure radicality demands a special technique to repair the crippled larynx. For this purpose a local osteomuscular flap was designed to reconstruct a functioning larynx. The surgical manoeuvre was described and the result proved to be successful. The outcome of 138 patients with T3 and T4 laryngeal carcinomas following conservation laryngectomy was analyzed. The five-year survival rate was 69% for supraglottic type and 75% for glottic type. The quality of life was much improved. PMID- 7555252 TI - Changes in the endocochlear potential and cochlear blood flow induced by ATP infusion and arterial occlusion. AB - To assess the relationship between cochlear blood flow (CBF) and auditory function, a procedure of intravital microscopy for observations of the lateral wall vessels of the cochlea coupled with the simultaneous measurement of the endocochlear potential (EP) was established in guinea pigs with gradual ischemia of the cochlea. It was found that occlusions of both common carotid arteries and one of the vertebral arteries produced a minor reduction in CBF with no significant alteration in the EP. When intravenous infusion of ATP induced sharp and severe decreases in CBF, the EP varied only slightly from the baseline in some animals while there were no alteration in others. Furthermore, ATP infusions combined with arterial occlusions caused even more severe declines in CBF and a moderate decrease in the EP. The results indicate that not only does the CBF satisfy the basic needs of the processes of cochlear function, but also has a regulatory mechanism to ensure the normal function of the cochlea in the ischemia condition. It was also found that the changes in the stria vascularis vessels induced by decreases in blood pressure (BP) and heart rates were more severe than those of the spiral ligament vessels. This phenomenon indicated that the stria vascularis vessels were more sensitive to decreases of BP and heart rates. PMID- 7555253 TI - Children's development effecting blood oxygen desaturation following apnea. AB - One hundred and fifty-six children aged from 4 months to 12 years undergoing elective plastic surgery were included in this study. All children were ASA physical status I, under normal development, ranging from 64 to 140 cm in body height and from 6.0 to 41.0 kg in body weight. Following 2-minute preoxygenation, the time when SpO2 dropped to 95% and 90% in apneic period had a close correlation with age, weight and height of children by means of linear and non linear regression analysis. The smaller the children's weight, the higher the incidence of severe arterial desaturation after reinstitution of manual ventilation with 100% oxygen at SpO2 of 90%. It is suggested that younger children are more susceptible to hypoxemia than older ones during apnea and provided no problem existing other than oxygen delivery, an SpO2 of 95% might be the safe limitation of apnea in pediatric anesthesia induction. PMID- 7555254 TI - Reexpression of nerve growth factor receptor in human traumatic injured spinal cord. AB - In this study, the reexpression of nerve growth factor receptor (NGFR) on paraffin sections of the human spinal cord was examined with immunohistochemical method in 18 cases with survival periods of 2 hours to 28 months after trauma. The results were as follow: the reexpression of NGFR in motoneurons of the ventral horn began on the fourth day after trauma and decreased within 30 days after trauma. However, it could still be observed in patients who survived up to 28 months. The axons in funiculus dorsalis reexpressed NGFR 7 hours to 9 weeks after injury, which may be interpreted as axoplasmic transport effect of NGFR in the spinal ganglion cells. NGFR labelled intraspinal microvessels were present in the injured spinal cord. Reexpression of NGFR in motoneurons after injury reflects an increased demand of neurotrophic factors, and an increased access exerting the physiological effects of trophic factors mediated by NGFR. PMID- 7555256 TI - Factors influencing impedance during radiofrequency ablation in humans. AB - Impedance during radiofrequency (RF) catheter ablation procedures is dependent on a variety of parameters related to the catheter, cabling, reference patch, body size, and temperature. To examine the influence of body size, impedance was measured during clinical ablation procedures in 93 patients (Group I) with a wide range of body sizes. In 14 other patients (Group II), impedance was measured during variations in catheter tip size (5, 6 and 7 Fr), reference patch size (120 and 60 cm2), patch location (chest vs. thigh), and catheter tip tissue contact. The average impedance was also compared to average tip temperature in Group II patients. Impedance decreased with increasing catheter tip size, reference patch size and proximity of the patch to the heart. However, the effects of body geometry were complex. For example, using a chest patch, impedance increased with body surface area, but using a thigh patch it decreased, suggesting that lung volume may increase impedance, but body width may actually decrease it. An increase in tip tissue contact, relative to blood contact, increased the impedance, suggesting that impedance may be a useful measure of tip tissue contact. Finally impedance decreased with increasing tip temperature, suggesting that impedance may be useful as a real time measure of tissue and blood heating. The results are interpreted in terms of an electrical analog which suggests further that despite the lower total power when the same voltage is applied to a higher impedance, less voltage should be applied to achieve the same tissue effect when the measured impedance is higher. PMID- 7555257 TI - Experimental study on the model of coronary artery thrombosis in dog. AB - We have developed a model of coronary artery thrombosis in dog by using the method stimulating coronary adventitia with direct current. The results have shown that the thrombi are similar to human artery thrombi in composition and the model of coronary artery thrombosis is satisfactory and the time of forming thrombosis is shorter than that by other methods. PMID- 7555255 TI - Structural and ultrastructural studies of the synovial membrane in temporomandibular joint disturbance syndrome. AB - To gain further knowledge on temporomandibular joint disturbance syndrome (TMJDS), the synovial membranes of 7 patients and 3 cadavers were examined with light microscope and transmission electron microscope. The results showed that the synoviocytes degenerated or disappeared in TMJDS. The cytoplasm contained numerous fibrils, while the organelles diminished in number. The surface of the synovial membrane was covered with a fibrinoid material. The surface of the synovial membrane was covered with a fibrinoid material. The authors consider that these pathological changes reduce or eliminate the normal functions of the synovial membrane, which may lead to damage to the articular cartilage. The articular adhesion found sometimes in TMJDS may be related to the changes in the synovial membrane. PMID- 7555259 TI - Thrombolytic therapy for acute myocardial infarction: where are we now? Where should we go? PMID- 7555258 TI - Randomized case-controlled trial of oral low-dose acyclovir for prevention of virus infections in recipients of renal allografts. AB - A randomized case-controlled trial of oral low-dose acyclovir (600-800 mg per day) has been conducted for the prevention of virus infections in 66 recipients of renal allografts since 1990. In comparison with the untreated controls, acyclovir could prevent herpes virus simplex (HSV), reduce morbidity of pneumonia from 10 cases (30%) to 3 cases (9%) (P < 0.05) and lower CMV-IgM positive rate from 30% to 12%. Serum Cr and BUN in acyclovir group were lower than those in control group. These results strongly suggested that oral administration low-dose acyclovir could prevent virus infections after renal transplantation. PMID- 7555260 TI - Molecular cloning of a cDNA encoding filarial common antigen. PMID- 7555262 TI - Osteomyelitis of sacral spine caused by aspergillus versicolor with neurologic deficits. PMID- 7555263 TI - Hypertension prevalence and status of awareness, treatment and control in China. AB - A nation-wide survey of blood pressure (BP) and high BP was made in China in 1991, using standardized methods and covering 950,356 men and women aged 15 years and above, from 30 provinces, autonomous regions, and municipalities. The purpose of the study was to estimate the prevalence and the status of awareness, treatment, and control of hypertension. The overall prevalence rate of hypertension defined as systolic BP > or = 140 or diastolic BP > or = 90 mmHg or currently on antihypertensive medication was 13.6%, and was higher in urban (16.3%) than in rural (11.1%) setting. The rate was 6.6% if a BP cut-point of 160/95 mmHg was used. Rates were low in younger age-groups but rose sharply after age 45. The total number of hypertensives in 1990 was estimated to be close to 90 millions. Of all hypertensives, about 1/4 were aware of their high BP, half of these were on medication, and only 3% had their BP controlled. All the rates were higher if the BP cut-point was 160/95 mmHg. Higher rates were found in urban than rural setting and, to a lesser degree, for women than men. In conclusion, hypertension was found to be common in China, and the rates of awareness, drug treatment, and controlled BP were low, indicating an urgent need for developing a national program of treatment and prevention of high BP. PMID- 7555264 TI - Research and development of biological products in China. PMID- 7555261 TI - Simultaneous resection of a stage-III primary squamous carcinoma of lung and a carcinoid of cardia. A case report. PMID- 7555266 TI - An experimental study of intravascular stent in abdominal aorta of atherosclerotic miniature pigs. AB - To study the effects of nitinol alloy stent in the treatment of atherosclerotic arterial stenosis, eight miniature pigs were subject to abdominal aortic balloon denudation and high-fatty food. Then 8 nitinol alloy stents were implanted into each abdominal aortas of pigs. The pigs were equally divided into two groups. One group was given captopril (3 mg/kg/d). All animals were sacrified for pathological examination 6 to 10 months after stent implantation. The degree of arterial intima proliferation in the areas of stent implantation not significantly different from that of areas of balloon denudation alone; the atherosclerotic lesions were found at the arterial surface of stent implantation sites. The intima layer was rich in smooth muscle cells, with atherosclerotic plaque formed around the stent wire. On the other hand, significant decrease in arterial intima proliferation was found in group II with no atherosclerotic plaque. The arterial stenosis resulting from atherosclerotic lesion could not be prevented by implantation of intravascular stent, and on the contrary, the mechanical stress of stent wire might worsen the atherosclerosis. Captopril might impede the development of atherosclerotic stenosis after stent implantation. PMID- 7555265 TI - Adenovirus mediated gene transfer of vascular smooth muscle cells and endothelial cells in vitro. AB - Introducing foreign gene(s) into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is the pre-requirement of gene therapy for cardiovascular diseases. We have explored the use of adenoviral vectors (Adv-CMV/LacZ) to transfer LacZ gene into cultured VSMCs and ECs. Our results demonstrated that adenoviral vectors transferred foreign gene into VSMCs and ECs high-efficiently with dose-dependent response pattern. The frequencies of transfection reached 100% at the viral titer of 10(9) pfu/ml. Comparing the sensitivities of VSMCs and ECs to adenoviral vectors, we found that ECs were more sensitive than VSMCs, of which the frequencies of transfection in ECs reached 80% while in VSMCs only 40% for 8 hrs after transfection. In addition, the transfection of ECs and VSMCs with adenoviral vectors was partly blocked by monoclonal antibodies to Fiber and Core protein of the adenoviral capsid, but not by monoclonal antibody to Hexon protein. It is suggested transfection of ECs and VSMCs with adenovirus vectors is mediated by Fiber or Core protein of adenoviral capsid proteins. PMID- 7555267 TI - Evaluation of serum troponin T measurement in acute myocardial infarction. AB - The release kinetics of the cardiac specific troponin T (cTnT), measured by an immunosorbent assay, in 32 patients with acute myocardial infarction (AMI) was evaluated. In 8 AMI patients (25%), the release kinetics of serum cTnT showed biphasic serum concentration curves with a large peak at 10 to 18 hours after onset and the serum cTnT concentrations were up to 30 to 120 times of the normal upper limit-detected (0.2 microgram/ml), and another samll cTnT peak at 70 to 100 hours after onset of pain. The serum cTnT measurements in the remaining 24 AMI patients (75%) gave only one large peak which was around 32 to 108 hours after onset of pain. cTnT appeared in serum as early as 3 to 4 hours and remained elevated until 240 hours after the infarctions. The results indicated that (1) cTnT was a sensitive and specific marker of AMI; (2) the biphasic curve of cTnT releae in general, the ratio of cTnT at around 14 to 32 (14/32) > 1 in particular, may be a useful marker suggesting early reperfusion; (3) the continuous elevation of serum cTnT in AMI course might be a prognostic indicator for unfavorable outcomes. PMID- 7555268 TI - Modification of IFSSH system for evaluation of impairment of hand function. AB - We modified the examination and evaluation standard of impairment of hand function proposed by the International Federation of Societies for Surgery of the Hand (IFSSH). 1) The thumb adduction and opposition examination is changed from measuring distance alone to calculation of the ratio of thumb length to the distance of movement. 2) Sensation grading and two-point discrimination test are included in the sensory function evaluation. 3) In the estimation of anatomic loss of the hand, instead of measuring the length of the loss, calculation of the area of loss is proposed. 4) In the evaluation of joint movement, besides the angle of movement and position of the joint itself, influence of the defect distal to the joint thereon is also considered. 5) In the original evaluation standard, sensory disturb ances of the palmar and dorsal aspects of the hand as well as the anatomic loss of tissues through the palm are not taken into account. The present work pays special attention to this point. The software of the above mentioned work has been developed, approved by experts and adopted nation wide in China. The data of hand function examinations can be fed into the computer and the function loss in percentage can be displayed, or printed out. This software, using a graphic display, can also take care of the filling and retrieving of case history. At present the menu and instructions are in Chinese, which can be changed to suit other language.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555269 TI - Modified distal osteotomy of shortening the first metatarsal and for treatment of hallux valgus. AB - We evaluated the results of distal osteotomy of shortening the first metatarsal head with internal fixation of shape memory metal compression staple for hallux valgus in 26 feet of 17 women with a mean age of 41.0 years (30-65 years). The time for return to light work averaged 18.2 days, and to full work and normal walking 40.3 days after the operation. 16 patients (25 feet) had complete pain relief. Only in one foot, the pain was transferred under the 2nd metatarsal head. The appearance of the feet was markedly improved and the width of the forefeet was decreased by a mean of 0.92 cm after the operation. All of the patients were satisfied with the appearance of their feet. Radiographic analysis of feet showed that all osteotomies united, and the average angle of hallux valgus was improved from 30.1 degrees to 16.0 degrees, with the mean intermetatarsal angle of 11.2 degrees-6.5 degrees. No external fixation of plaster splintage was used and the distal fragment during healing of the osteotomy was stable. The time of bone healing was shortened and patients were allowed to bear weight at early stage. PMID- 7555270 TI - Immunohistochemical study of HNK-1 (Leu-7) antigen in prostate cancer and its clinical significance. AB - Anti-HNK-1 monoclonal antibody (MAb) was reactive with non-cancerous and cancerous prostatic epithelia, as well as natural killer cells, myelinated nerves and cells from APUD systems. The expression of HNK-1 antigen on prostate cancer was investigated immunohistochemically by avidinbiotin-peroxidase complex (ABC) method with anti-HNK-1 MAb to clarify the relationship between anti-HNK-1 immunostaining of the cancerous tissue and the tumor differentiation, and that between the former and the survival rate of patients. Of 52 patients with prostate cancer, 49 were reactive with anti-HNK-1 MAb, the positive rate being 94%. The well differentiated cancer showed the highest percentage of positive cancer cells and the strongest staining, while the poorly differentiated cancer had the lowest percentage of positive cancer cells and the weakest staining. HNK 1 antigen was highly expressed on prostate cancer, and the better differentiated the cancer, the more HNK-1 antigen expressed. We also analyzed the total 5-year survival rate of the 52 patients, and the average 5-year survival rate and non progression rate of 32 patients in stage D2 who had received only endocrinotherapy. Significantly higher survival rate and non-progression rate were observed in the group with more than two-thirds positive cancer cells as compared with the group with less than two-thirds positive cancer cells. The results suggest that the expression of HNK-1 antigen on prostate cancer may be a useful prognostic factor for patients with prostate cancer. PMID- 7555271 TI - Establishment and characterization of two cell lines derived from human transitional cell carcinoma. AB - Two continuous cell lines derived from the neoplastic urothelium had been maintained in culture for more than two years. The first cell line derived from the urothelium of a fusion papillocarcinoma on the left lateral wall of the bladder was designated as TBC-1 and grown in vitro for more than 150 generations. The second cell line derived from the urothelium of a papillocarcinoma in the left renal pelvis was designated as TPC-1 and grown in vitro for more than 100 generations. Characterization studies made on both cell lines showed that the cells had a rapid doubling time, exhibited multilayering and produced tumors in sc of BALB/c. Tumor nodules that produced sc of BALB/c kept similar cellular and pathological features to those of the primary biopsy specimens under light and electron microscopes. TPC-1 cell line exhibited a three-dimensional structure of transitional epithelium on the nylon-mesh disk which was coated with a layer of rat tail collagen. Both TBC-1 and TPC-1 cell lines formed colonies in soft agar. Their forming rates were 35% and 28%, respectively. The chromosome number of TBC 1 cells ranged from 17 to 84, with a modal number of 54; whereas that of TPC-1 cells ranged from 28 to 139, with a modal number of 49. The TBC-1 cells showed mutant p53 and ras p21 protein expression and expressed weakly ABH blood group isoantigens. Analysis of lactic dehydrogenase (LDH) isozymes showed the highest levels of LDH isozyme 4 sonicated cell lysates of TBC-1 and TPC-1 respectively. PMID- 7555272 TI - Sialographic follow-up study of patients with Sjogren's syndrome. AB - Sialographic follow-up studies of 36 cases of Sjogren's syndrome (SS) were performed with the duration of 5-72 months (mean 25 months). Three groups were divided according to the international criteria: secondary group, 14 cases (xerophthalmia, or/and xerostomia, and connective tissue diseases), primary group, 14 cases (xerostomia and xerophthalmia), and possible group, 8 cases (xerophthalmia or xerosptomia only). Sialographic findings at follow-up examination included (1) delayed evacuation, which may occur as early as 5 months later; (2) increased punctate sialectasis; (3) decreased punctate sialectasis with globular increase; (4) punctate, globular, and cavity sialectasis with radiolucent sialolith; and (5) centripetal atrophy. The cases with secondary and possible SS without xerostomia demonstrated little involvement of the parotid gland, without sialectasis. But sialodochitis was shown when radiolucent sialolith formed. The stimulated whole salivary flow rate (SWSFR) was closely related to sialographic appearance, i.e., the cases with lower value of SWSFR showed more siatectasis sialographically, but with higher SWSFR value, and less sialectasis. The relationship between the SS and pyogenic or candidal infection was discussed. PMID- 7555273 TI - An experimental study of spinal cord evoked potential. AB - The characteristics of spinal cord-evoked potentials were investigated by using an in vitro spinal cord preparation. The spinal cord isolated from adult rats was immersed in a bathing chamber filled with oxygenated artificial cerebrospinal fluid (aCSF). The spinal cord-evoked potentials elicited by the stimulation of the spinal cord were recorded by using bipolar platinum electrodes. The potentials recorded consisted of early and late negative components (N1, N2). The inhibitory effect of lowered temperature on the N1 potential was clearly demonstrated. The oxygen deprivation of aCSF showed the higher sensitivity of N2 potential to hypoxia than that of N1. It was also possible to analyze the effects of potassium and magnesium ion concentration and pH on the evoked potentials. These results showed that the pure effects of various physiological and chemical factors on the spinal cord-evoked potentials can be analyzed by this experimental model. PMID- 7555275 TI - Radiotherapy of midline malignant reticulosis. AB - The results of radiotherapy made on 23 patients with midline malignant reticulosis showed that the 1-, 3-, 5- and 8-year survival rates were 91.3%, 63.2%, 50.0% and 40.0%, respectively. The optimum radiation dose was 50-60 Gy / 5 6 weeks. Chemotherapy with CCNU following the radiotherapy facilitated the healing of lesion, reduced local recurrence and distant metastasis, improved the therapeutic effect and raised the survival rate. PMID- 7555274 TI - Multiple subpial transection for treatment of intractable epilepsy. AB - On the basis of experimental study, we applied multiple subpial transection (MST) to treat 50 patients with intractable epilepsy in which epileptigenic lesion involved functional areas such as pericentral gyrus, postcentral gyrus, Broca's area, Wernicke's area, visual cortex, etc. They were followed up for 6 to 40 months. Complete control of seizures was obtained in 32 patients, significant reduction of seizure (more than 50%) in 13, reduction (less than 50%) in 3, and no effect in 2. The total effective rate was 96%. No functional defect was found in all patients. The mechanism of the disease and surgical technique were discussed in detail. We consider that MST could replace some standard excisional therapy for local epilepsy. PMID- 7555276 TI - Effects of ginsenoside Rb1 and Rg1 on synaptosomal free calcium level, ATPase and calmodulin in rat hippocampus. AB - Calcium homeostasis in synaptosomes is altered during aging. The intrasynaptomal free calcium concentration ([Ca2+]i) was determined in 3- and 24-month-old rats treated with or without Rb1 and Rg1. As expected, the [Ca2+]i level increased with age. Treatment with Rbl and Rgl elicited an obvious decrease of [Ca2+]i content, especially in aged rates. In addition, Rb1 and Rgl significantly stimulated the activity of Na+, K+-ATPase while Rbl inhibited the activity of Ca2+, Mg2+-ATPase and calmodulin. In view of the close relationship of [Ca2+]i level with aging, the changes of [Ca2+]i induced by Rbl and Rgl, as shown by our results, might provide an explanation of the mechanisms of their antiaging function. PMID- 7555277 TI - Endotoxin-induced liver injury and changes in the levels of plasma tumor necrosis factor-alpha and interleukin-6 in rabbits. AB - ELISA was employed to detect changes in plasma TNF-alpha and IL-6 level in rabbits having endotoxin-induced generalized Shwartzman reaction. Liver injury was assessed by the increase of serum ALT and hepatic histopathologic changes. The results showed that plasma TNF-alpha and IL-6 levels increased remarkably 12h after intravenous injection of endotoxin twice at a 24h interval, and these changes corresponded with the degree of liver injury. Coinjection of dexamethasone and endotoxin partly prevented the elevation of TNF-alpha and IL-6 levels and reduced liver injury. These results indicated that TNF-alpha and IL-6 were involved in endotoxin-induced liver injury. PMID- 7555279 TI - A case of blindness caused by acute iodine poisoning. PMID- 7555278 TI - A further study on liver alveolar echinococcosis. Report of 70 cases. PMID- 7555282 TI - [Congenital hereditary anhidrotic ectodermal osteo-chondrodystrophy]. AB - Two boys aged 5 and 9 treated because of congenital, hereditary anhidrotic ectodermal osteo-chondrodystrophy have been presented. Slipped epiphyses were found among other coexisting articular disturbances. PMID- 7555283 TI - [The influence of microwave monochromatic radiation on bone fracture union in rabbits]. AB - The influence of 53.57 GHz microwave radiation of 10mW/cm2 power density on fresh radial bone fracture in 16 rabbits has been investigated. After osteotomy, the occipital area was irradiated one hour daily during two weeks. The development of bony union in experimental and control group (12 rabbits) were compared on the basis of radiographic and physical assessments on the 7-th, 14-th, 21-st and 28 th day after osteotomy. Favourable effect of microwave radiation on bone union was found, especially in the first 14 days after osteotomy. An intense periosteal reaction accompanied the healing process in experimental animals. Biochemical and morphological blood tests run simultaneously were standard. PMID- 7555280 TI - Rhinomanometric study in China. PMID- 7555284 TI - [Burst fractures of the cervical vertebrae]. AB - A series of 237 patients with burst fracture of the cervical spine was treated between 1965 and 1991. These fractures usually result from head-on dive or fall from the height leading to quadriplegia. An early surgical decompression of the spinal cord and fusion of the spine at the site of injury from the anterior approach is the treatment of choice in author's opinion. It improves neurological and functional results of treatment, shortens hospitalisation time. PMID- 7555281 TI - [Surgical treatment of familial hypophosphatemic rickets]. AB - In 20 children with tibial and femoral bones deformities due to hypophosphatemic rickets a corrective osteotomy/osteoclasis at 2 or 4 sites was performed. Both preoperative and postoperative pharmacological treatment was introduced. Good results were achieved in 13 cases, satisfactory an 4 and bad in 3 cases. PMID- 7555287 TI - [Antigravitational spondylometer--a device for the measurement of lumbo-sacral spinal instability]. PMID- 7555286 TI - [Ultrasonography--selective use for diagnosis in spine pathology]. AB - A series of 5 patients diagnosed with a meaningful influence of ultrasound has been presented. In 2 cases the changes within iliopsoas muscle were found, sacral anterior meningocele, neoplastic infiltration of the great retro-perineal vessels from the fourth lumbar vertebral body metastasis and neurofibromatosis of cervical spinal canal were diagnosed in the remaining cases. PMID- 7555285 TI - [Conservative operative procedure in surgery of the spine]. AB - An operative treatment within the spine due to the traumatic, neoplastic, vascular or other indications frequently result in complications as disturbance of the shape, flexibility, strength, mobility of the spine and injury of neural structures of a various degree. These complications may be prevented by minimizing surgical injury to the bone, ligaments, nerves and vessels and by adequate intra- and postoperative stabilization of the spine. The management will be effective only if the level, range and type of injury as well as spine tolerance against mechanical loads is diagnosed accurately. The authors included case reports. PMID- 7555288 TI - [Surgical treatment of funnel chest]. AB - Fourty one patients (33 males and 8 females) with pectus excavatum were operated by Ravitch method modified by Politowski or Drews (36 cases and 5, respectively) from 1980 to 1989. The age of patients ranged from 5 to 25 years (mean 11 years), they were operated because of cosmetic reasons exclusively. Twenty nine of them reappeared to be examined after follow up of 15 months to 10 years (mean 3.5 years). Subjective opinion of the patient, clinical examination and radiological appearance were taken into consideration. There were 72.5% of excellent and good results. No recurrence of the deformity was observed, the most suitable age for the surgery is from 8 to 10 years. PMID- 7555289 TI - [Treatment of funnel chest by a modified Brown method]. AB - A series of 19 patients operated on because of pectus excavatum has been analyzed to assess results of Zuk modification of the Brown method. Vital lung capacity, funnel volume, physical fitness, and cosmetic result have been assessed. An asymmetry and developmental disturbances within insertions of pectoralis major, rectus and oblique abdominal muscles were found as well as developmental disturbances of costal cartilage. In 95% of cases a positive patient opinion along with satisfactory functional and cosmetic result have been achieved. PMID- 7555290 TI - [What determines treatment results of the Frejka pillow treatment for congenital hip dislocation?]. AB - The results of treatment of 1008 hips in 779 children were analyzed and their dependence on objective factors established. The major influence is attributed to the initial displacement of the femoral head; the greater it is the worse is the result. The age at the onset of treatment influences results only indirectly with displacement increasing in time. No other factor as sex, side involved (left, right or both), or family history seems to play a role. PMID- 7555291 TI - [Functional treatment of central hip dislocation--personal modifications]. AB - A slow, lasting for several hours method for reduction of central dislocation of the hip with the use of bidirectional traction, with Rush pin introduced in the trochanter major and Steinmann pin in supracondylar area, with 18 degrees of the lateral tilt of the bed, with standard upper frame of Gruca bed mounted laterally and great traction force against the hip is presented. The universal Gruca splint was employed in the after-treatment. In 12 patients with grade II and III central dislocation of the hip excellent early anatomical and functional results were achieved. PMID- 7555294 TI - [Van Neck disease or a variation of ischio-pubic synchondrosis development?]. AB - Diagnostic problems in the case of 9 years old boy with flared ischio-pubic synchondrosis are presented. The condition is differentiated between Van Neck disease and variation od ischio-pubic synchondrosis development. PMID- 7555293 TI - [Personal experience in treatment of trochanteric fractures of the hip by the Ender method]. AB - The authors have presented results of treatment of 150 patients with trochanteric fractures of the hip operated by the Ender method. Hundred twenty of them were capable of weight bearing at the discharge from the hospital. Fifty patients showed up for the final examination, 48 walking unaided or with a walking stick. Follow up ranged from 0.5 to 5 years. The fracture was united in all cases. PMID- 7555292 TI - [Bioelectric activity of the gluteus medius muscle after total McKee-Farrar hip replacement]. AB - Electromyography of the gluteus medius muscle in arthritic patients after total McKee-Farrar hip replacement has been presented. The control group consisted of nonoperated arthritic hips and normal hips. It was found that arthritis of the hip or total hip replacement do not significantly influence the average score of the integrated gluteus medius electomyogram. The gluteus medius strength on Trendelenburg sign testing is 1.5-2 times greater than on acting against maximal resistance with patient in supine position. PMID- 7555295 TI - [Long-term results of knee joint rehabilitation after fascia interposition]. AB - The method, schedule and results of rehabilitation in patients with the chondral defects in knee joints treated by partial or total fascial grafting were presented. In the series of 114 patients followed up from 1 to 19 years after operation there were 56.1% good and fair results in the group with partial grafting and 26.6% in the group with total fascial grafting. An aggravation of rheumatoid process, coincidence of deformities in adjacent joints, weakening of muscles and ligaments in the knee and Hoffa pad grafting during the development of the method yielded 17.7% bad or unsatisfactory results. PMID- 7555297 TI - [Use of supra-malleolar traction in treatment of closed fractures of the crural shafts]. AB - The results of treatment of 36 closed fractures of the crural shafts reduced with supra-malleolar traction have been presented and evaluated with own scale. The best results were achieved with traction maintained for 5-6 weeks. Most of the failures were due to the disturbed union and restricted range of movement in the ankle. The authors conclude, that traction prevents secondary displacement and should be recommended for treatment of unstable fractures of the crural shafts. PMID- 7555296 TI - [Genu recurvatum--etiopathogenesis and treatment]. AB - A case of 12 years old girl operated on because of genu recurvatum and paretic clubfoot after buttock and thigh phlegmon in infancy has been presented. There were 2 stages. In the first one the correction of clubfoot was performed, m.quadriceps was elongated in the second one. Good cosmetic and functional result has been achieved. PMID- 7555298 TI - [Clinical evaluation of surgical treatment of congenital clubfoot by peri-talar release]. AB - Clinical assessment of the results of surgical treatment of 105 congenital clubfeet by peri-talar release has been carried out. There were 58% of soft and 42% of stiff clubfeet in this group. The average patient age at the operation was 17.8 months. The Wynne-Davies point scale modified by Marciniak was used to assess the results. Forty nine feet (46.4%) were rated good, 28 (26.7%) satisfactory and 28 (26.7%) poor. Fifty percent of stiff feet were rated poor and 68.9% of soft ones were rated good. Plantocontourography revealed marked decrease of internal rotation in the operated feet. The authors conclude peri-talar release is an effective method of treatment of soft congenital clubfoot. PMID- 7555300 TI - [Transoral approach to the anterior aspect of C1-C3 cervical vertebrae]. AB - Transoral approach to C1-C3 vertebrae has been presented. The approach has been used in 2 patients with tumor of the upper cervical spine and in 4 patients with pseudoarthrosis of the dens. Surgical technique and 2 typical cases have been reported. PMID- 7555299 TI - [Open total dislocation of the talus]. AB - A case of 35 years old patient with open, total, fixed dislocation of the talus reduced with heel traction is presented. The Sudeck syndrome subsided after one year. Weight bearing was not allowed for 7 months. An examination after 2.5 years revealed minor osteoarthritis, full range of movement in the foot but limited walking capability. PMID- 7555301 TI - [Treatment of idiopathic scoliosis with Texas Scottish Rite Hospital instrumentation (TSRH)--preliminary report]. AB - General principles of construction of TSRH instrumentation and its use for treatment of idiopathic King type III scoliosis have been presented. Surgical technique has been described on the basis of procedure performed in the case of 13 years old girl with primary right sided thoracic adolescent scoliosis. PMID- 7555302 TI - [Vascularization of the lumbar spine in the human fetus]. AB - The vascularization of the lumbar spine has been studied in 19 human fetuses of both sexes after filling of the arterial system with Micropaque. Structural radiographs were taken and lumbar arteries with their branches were marked in every segment examined. The frequency of branches incidence was described. The differences in vascularization between left and right side of the lumbar spine within medullar arteries and articular processes arteries amounted to 20 and 5 per cent respectively. PMID- 7555305 TI - [A comparative analysis of tension properties of the coracoclavicular ligament and a carbon fiber implant (preliminary investigation)]. AB - The results of comparative analysis of tension properties between 10 preparations of coracoclavicular ligament harvested from the cadavers and 3 samples of carbon fiber prosthesis have been presented. INSTRON 1195 apparatus has been employed. The results obtained indicate superiority of carbon fiber ligamentous prosthesis over natural ligament in respect to the elastic properties. PMID- 7555304 TI - [A case of epidural lumbar spine abscess]. PMID- 7555303 TI - [Two cases of congenital defects within the transverse processes of the lumbar spine]. AB - Two cases of congenital defects of transverse processes of lumbar spine causing lumbalgia are described. The first one was produced by direct contact of the elongated processes with subsequent pseudoarthrosis. Hypertrophied processes caused stenosis around intervertebral foramen and irritated posterior neural branch in the second case. Surgical resection of the hypertrophied transverse processes has brought the cure. PMID- 7555306 TI - [A case history regarding a 14-year old girl with acromio-clavicular dislocation and tear of the coracoclavicular ligament]. AB - A rare injury in children and its surgical treatment is presented. The coraco acromial ligament was used to restore acromio-clavicular junction. Its acromial end has been transferred to the coracoid tuberosity of the clavicle and placed subperiosteal with 2-point suture fixation. An excellent clinical and radiological result has been achieved. PMID- 7555307 TI - [Experimental femoral neck fractures in rats after prolonged steroid therapy]. AB - The method of the experiment has been presented. It was found, that after 8 weeks of hydrocortisonum hemisuccinatum administration, 20 mg per day in two doses the braking force decreased by 15 per cent. The calculation of the measurements at 150 g of animal body weight proved to be useful. PMID- 7555308 TI - [A case of stress fracture of the femoral neck]. AB - A case of 28 years old male with stress fracture of the femoral neck is presented. Heterogeneous clinical picture is blamed for diagnostic problems in the initial stage of the disease. Bone scintigraphy aids in the early diagnosis. The use of low frequency electromagnetic field has had unquestionable effect on shortening of the treatment time in the case reported. PMID- 7555309 TI - Use of Ilizarov distraction modified by Cattaneo and Catagni for femoral lengthening. AB - Ilizarov distraction device with proximal fixation by Cattaneo and Catagni has been used for femoral lengthening in 13 patients. Planned femoral elongation, fast remodeling of the newly formed bone and intended correction of the anatomical and mechanical axis of the limb have been achieved in all cases independently of the level of corticotomy. Full weight bearing was maintained during entire period of treatment. PMID- 7555311 TI - [Percutaneous method for achilles tendon lengthening in treatment of spastic equinus deformity with progressive muscular dystrophy]. AB - Surgical technique of percutaneous lengthening of Achilles tendon has been presented. Thirty-seven feet in 26 children have been operated on. There was no intraoperative inadvertent tenotomy or excessive lengthening, no complications occurred in the postoperative course. An average follow-up was of 18 months. The procedure resulted in 7 degrees of dorsal flexion on an average. The authors recommend this method in the treatment of the spastic pes equinus. PMID- 7555310 TI - [Results of treating comminuted fractures of the distal end of the tibia]. AB - Fifty-seven cases of comminuted fractures of the distal end of the tibia, inclusive of 9 cases of open fractures have been treated between 1987 and 1992. The fractures were rated according to the Ruedi-Allgower classification. Modalities of treatment used by the authors have been discussed. Long-term results have been assessed in 40 cases. Twenty of them were rated good, 13 fair and 7 bad. In 20 cases osteoarthritis of the talocrural joint has been found. Conservative treatment or minimal surgery (fixation of the fibula) rendered good and fair results in grade I and II fractures. Majority of bad results was found in grade III fractures due to the severe destruction of the articular surfaces of the talocrural joint. PMID- 7555312 TI - [Dynamic fracture stabilization with the DYBASTAB--with the help of dynamic compensating stabilizers--DYNASTAB D-K--new outlook on treating fractures]. AB - Dynamic stabilizer DYNASTAB D-K for functional, ambulatory treatment of fractures is presented. The possibility to include an active hinge enabling physiological movement of the injured or remaining in the injured zone joint allows for an early mobilization of the limb concerned. Solid, spatial screws anchoring enables early axial dynamization within the union zone. Plastic cubes reduce the weight and prevent disturbance of electrophysiology of the fracture healing. The hydraulic chamber pressure measurement taken at the load will permit the assessment of bony union consolidation. PMID- 7555313 TI - [Orthopedic terminology of the knee--general terminology]. PMID- 7555314 TI - [Orthopedic terminology of the knee--terminology for instability]. PMID- 7555315 TI - [Moore hemiarthroplasty of the hip for treatment of femoral neck fracture]. AB - A series of 158 hemiarthroplasties of the hip with the use of Moore prosthesis has been performed in patients with recent femoral neck fractures or its complication (nonunion, necrosis) between 1980 and 1992. From 73 patients operated between 1986 and 1990 32 showed up for clinical assessment, 11 responded in writing to the questionnaire. In 26 cases the result was rated good, in 9 fair and 8 results were poor. Twenty-five patients died. The authors explain small number of patients who reported themselves for evaluation by high mortality rate due to the old age and general medical problems. Authors' findings supported by the literature indicate, that hemiarthroplasty of the hip remains as a useful mode of treatment for femoral neck fracture or its complications. PMID- 7555316 TI - [Results of treating fractures of the femur and tibia with closed intramedullary fixation]. AB - The paper presents the technique and results of treatment of 450 fractures of the femur or tibia. Closed intramedullar fixation has been employed. There was no surgical approach to the fracture site. The intramedullar nail was inserted through small incision remote from the fracture. Flexible reamers were used to prepare the medullar cavity. X-ray intensifier was used to monitor the procedure. If the fracture was located outside the middle third the nail was interlocked. Fixation achieved was stable, no additional immobilization was needed. Mobilization was started at the first post-operative day. No wound infection or osteomyelitis occurred. Weight bearing was allowed after 2 months for 80 per cent of the patients. In all cases the union was achieved before the end of the third month. PMID- 7555317 TI - [Polfix fixator in treating fractures and disorders of long bone healing- introductory report]. AB - Polfix, a new method for stable osteosynthesis is presented. This is an evolution of Zespol method used for last 10 years but is mechanically stronger with better elastic properties. Six operated cases allow for initial conclusions only. PMID- 7555318 TI - [Partial release of the ilio-tibial band as a treatment for snapping hip]. AB - Partial release of the iliotibial band has been performed in 3 patients with bilateral snapping hip. The technique of surgical procedure has been described. In all cases very good both functional and cosmetic results have been achieved. PMID- 7555319 TI - [Outcome of treating osteoarthritic degenerative changes in the knee with high tibial osteotomy]. AB - We reviewed a series of 47 patients-43 female and 4 male-of whom 41 were available for follow-up, the average age of patients was 61 years (range, 50-72 years)-with osteoarthritic knee that were treated by high tibial osteotomy and external fixation. Patients complained of knee varus deformity-10/20 degree, pain, effusion, ligaments laxity and flexion contracture about 10-15 degrees. At a mean follow up 3 years (range, 1-5 years) the results in 15 were rated very good, the results in 17 as good, results in seven as fair and in two as poor. Complications included 5 cases of transient peroneal nerve neuropraxia and superficial infection in two patients. High tibial osteotomy is most effective in the treatment of osteoarthritis of the knee with varus deformity, when correction is made to 10 degrees valgus position. The stable external fixation enables immediate mobilization, dispenses with the use of the cast and have not required second operation for removing fixation frame. PMID- 7555320 TI - [Magnetic resonance imaging and arthroscopy in meniscal and ligamentous injuries of the knee]. AB - A series of 37 patients after knee injury underwent magnetic resonance imaging, in 21 cases subsequent arthroscopy has been performed. All patients had previous clinical diagnosis of meniscal and/or anterior cruciate injury. The changes within the menisci and anterior cruciate ligament have been confirmed by MRI in 76 per cent and 81 per cent respectively. In conclusion magnetic resonance imaging and arthroscopy are complementary methods in the knee joint diagnostics. PMID- 7555321 TI - [Treatment of traumatic spondylolisthesis of the axis]. AB - Classification of hangman's fracture according to Levine has been presented. The classification enables individualization of the management depending on the mechanism of the injury. A series of 37 patients with traumatic spondylolisthesis of the axis has been analyzed. Skull traction in flexion injury cases led to the increased dislocation. PMID- 7555322 TI - [Evaluation of surgical treatment for congenital over-riding fifth toe]. AB - Results of treatment for congenital varus fifth toe in 18 patients (20 feet) are discussed. The age at operation ranged from 2 to 28 years, different procedures were performed. The patients were followed-up for 1-8 years. Long extensor of the fifth toe transfer (Assman and Lapidus procedures) rendered better both cosmetic and functional results then corrective subcapital osteotomy of the fifth metatarsal. PMID- 7555323 TI - [Diagnostic problems in a case of benign chondroblastoma]. AB - A case of benign chondroblastoma is described. Atypical radiographic picture with broad zone of sclerotic bone around the tumor has created diagnostic problem. PMID- 7555324 TI - [Results of treating giant cell tumor with emphasis on local recurrence]. AB - The paper presents results of treatment of 16 patients operated on because of giant cell tumor. Frequent local recurrence after curettage makes the authors to suggest more common use of the thermal or chemical destruction of the neoplastic cells to achieve oncologic sterility. PMID- 7555325 TI - [Evaluation of prognostic factors in malignant synovioma]. AB - Prognostic factors in 78 patients (36 males and 42 females) aged 9 to 68 treated for malignant synovioma in Warsaw Institute of Oncology between 1950 and 1988 have been assessed. Case history ranged from 1 month to 18 years. The neoplasm was found primarily within lower limb- 45 cases (58%), upper limb- 23 (29%), head, neck and trunk were the sites in 10 patients (13%). In 37 cases tumor diameter was less then 5 cm (T1) and in 37 more (T2 and T3). The diameter remained unknown in 4 patients. Monophase structure was found in 50 cases, biphase structure in 28. Five years survival constituted 48%, 10 years- 28%. The best prognostic value had size of the tumor and the duration of the symptoms. The size below 5 cm gave 58% years survival, above 5 cm only 34%. The history longer then 12 months offered 70% of 5 years survival and shorter one only 27%. Patients with longer history and smaller size of the tumor had significantly better prognosis then those with short history and tumor exceeding 5 cm in size. Females with tumor larger than 5 cm had much better changes then males. Remote metastases worsened the prognosis markedly. The age, localization and histological type of the neoplasm had no prognostic value. PMID- 7555326 TI - [The value of computed tomography in evaluation of newly formed bone remodeling after lengthening]. AB - The paper presents evaluation of the newly formed bone remodeling and measurement of its density with the use of computed tomography in 7 children who underwent bone lengthening. The author concludes that CT is a precise tool in determination of cortical bone quantity, generation of medullar cavity and degree of regenerate mineralization. PMID- 7555327 TI - [Computerization of the orthopedic-traumatology department]. AB - The authors the scheme of the computerization of their orthopedic-traumatologic department. An everyday work has been markedly advanced. PMID- 7555328 TI - [Ultrasonographic diagnostics and the organization of treating congenital dysplasia of the hip in Torun]. AB - A series of 9348 children (18696 hips) has been screened with ultrasound between 1990 and 1992; congenital dysplasia of various degree was found in 459 hips. All children were treated nonoperatively. The early results of conservative treatment are encouraging. Since the introduction of the new prophylaxis-treatment system only 4 children with persistent dislocation required surgery. PMID- 7555330 TI - [Pedicular-process and laminar-process pseudoarthroses in computed tomography and their clinical significance]. AB - CT has been performed in 7 patients with process-pedicular and process-laminar pseudoarthroses present on the radiographs. CT analysis showed pseudo-acetabular area within the lamina protruding into the spinal canal thus producing stenosis. Pseudo-acetabulum within the pedicle decreased the volume of the lateral recess. Both phenomena gain clinical significance in case of primarily narrow spinal canal or combined with secondary stenosis. They were found in 5 patients; in 2 cases the changes were unilateral due to the unequal length of articular processes. PMID- 7555331 TI - [Fracture of the dens--conservative or surgical treatment?]. AB - An analysis of 28 cases of dens fracture has shown that conservative treatment is generally sufficient in Anderson type I and type III fractures. In type II fractures surgical treatment is indicated, especially if the fracture is dislocated, complicated by neurological impairment or has occurred in the elderly. PMID- 7555329 TI - [30 years of operation at the Cathedral and Orthopedic Clinic of the Medical Academy in Lodz]. AB - University Orthopedic Department in Lodz was founded on January 1, 1964. The first head of the Department was prof. E. Bartkowiak M.D., Ph.D. and prof. H. Zwierzchowski M.D., Ph.D. took over in 1979. During past years out of 38 surgeons employed 8 became head of other orthopedic-traumatologic departments. An orthopedic training completed 22 graduates, Ph.D. was received by 20. The Department took part in pre- and post-graduate teaching. 14,570 operations have been performed on 31,091 admitted patients (12,969 children, 18,122 adults). Out Patient Department helped 139,493 persons. The employees published 232 papers, 35 of them abroad. They participated in 21 congresses, meetings and symposia abroad as well as in 15 domestic PTO I TR congresses with 72 presentations and organized two of them in 1966 and 1984. They were also included in General Board of PTO I TR as well as its Lodz branch. PMID- 7555332 TI - [A case of giant lipoma in the lumbosacral area]. PMID- 7555334 TI - [Use of the Ilizarov method in treatment of forearm deformity due to multiple exostoses]. AB - The use of Ilizarov distraction device in complete treatment of the forearm deformity due to multiple exostoses is presented. The method allowed for one stage correction of all components of the deformity. PMID- 7555335 TI - [Is the giant cell tumor in the distal part of the radius a tumor with a high potential malignancy?]. AB - The effects of the cure of four patients with the giant-cell tumor in the distal part of the radius have been resumed by the authors. In their opinion positive effects of the cure depend on suitable radical of surgery but not on primary location of the tumor. PMID- 7555333 TI - [Errors and complications in treatment of lateral humeral condyle fractures in children]. AB - The long-term results of the lateral humeral condyle fracture treatment in 35 children are presented. The mean follow-up was 8.2 years. There were 31 good results and 4 poor. The most common errors and complication are discussed. PMID- 7555336 TI - [Early results of surgical treatment for congenital hip dislocation in children using Degi's method in children under 18 months of age]. AB - Early results of surgical treatment of congential hip dislocation by the Dega method in 18 children (20 hips) aged from 6 to 17 months are presented. Following the excision of the soft tissue obstructions and Dega transiliac osteotomy stable reduction of the hip has been achieved in all cases. Proximal femoral osteotomy has been performed in 6 cases only. Clinical results were markedly superior over radiological ones. Fair radiological outcomes resulted from surgical errors or avascular necrosis of the femoral head due to the previous conservative treatment. Avascular necrosis after operative treatment has not been noted. PMID- 7555338 TI - [Elasticity modulus of lyophilized cortical bone in the human femur]. AB - The visco-elastic properties of the lyophilized cortical bone of the human femur has been studied by means of the balance rheometer. The samples of bone were harvested from healthy individuals who suddenly died or committed suicide. The donor age ranged from 35 to 45 years. The measurements were taken with the use of the balance rheometer at 20 degrees Celsius and sample deformation frequency of 12.5 Hz. It was found, that elasticity modulus of the lyophilized human cortical bone is about 30 per cent higher than the modulus of fresh cortical bone. In the elastic range the modulus depended on an amplitude of deformation. The compact lyophilized bone turned out to possess to a small degree also viscous properties; thus it should be considered as an elastic-viscous material with considerable superiority of elastic properties. PMID- 7555339 TI - [Treatment of lateral knee instability during lengthening of the leg by the Ilizarov method]. AB - The leg lengthening by the Ilizarov method has been performed concurrently with treatment for lateral instability of the knee in three children. The treatment included lowering of the fibular head along with distal fibular collateral ligament attachment. PMID- 7555337 TI - [Distant migration of a broken Nystrom-Szulc nail used for hip arthrodesis]. AB - A series of 14 arthritic patients who underwent Dickson hip arthrodesis with the use of Nystrom-Szulc nail has been reviewed. In three patients these solid nails were broken and the fragments have migrated distantly. These cases are reported. PMID- 7555340 TI - [Avulsion fracture of the tibial tuberosity]. PMID- 7555341 TI - [37-year survival of a patient with osteosarcoma of the proximal tibia after biological resection according to Grucy]. AB - A case of patient with osteosarcoma of the tibia resected by the Gruca method who survived 37 years is presented. In spite of the neoplastic recurrence (resected anatomically), long-lasting osteomyelitis, pathological fracture and increasing stiffness of the knee joint the patient has managed to carry out his life plans. This proves the Gruca method to be valuable although being presently replaced by polyvalent chemotherapy. PMID- 7555342 TI - [Ultrasonographic evaluation of the hip joint in newborns and infants using Graf's method]. PMID- 7555343 TI - [The author's principles for treating small angle scoliosis in view of current concepts about scoliosis etiology and pathogenesis]. AB - Hypothetical assumption, based on phylogenetic, anatomical and epidemiological studies as well as on clinical findings led the author to the conclusion, that regardless of an etiologic factor and the duration of its action the chief determinant for preserving the curve is the contracture of the short muscles and ligaments of the spine backed by scoliosis action of the long spinal muscles. Taking this into consideration the author has developed a method for small angle scolioses treatment by means of gravitational counteraction and removal of mainly ligamentous contracture of the spine. That can be accomplished by flexion rotation exercises supplemented by "segmental gymnastics". The principles are validated by 30 years of clinical experience and advantageous results. PMID- 7555344 TI - [The value of radiculography in light of intraoperative evaluation]. AB - Radiculographic assessment and intraoperative findings in patients with single level lumbar disc nucleus pulposus herniation have been compared with each another. Radiculography has been confirmed in 81 per cent of cases. PMID- 7555345 TI - [Personal experience in the use of closed reduction and transcutaneous K-wire fixation for displaced supracondylar fractures of the humerus in children]. AB - The method of closed reduction and percutaneous K-wire fixation has been used for treatment of 20 children with displaced supracondylar fracture of the humeral bone. No infection, vascular or neural complication has been observed. In all cases good clinical and radiological results have been achieved. The authors conclude the method presented above should be used as a treatment of choice for displaced supracondylar fracture of the humerus in children. PMID- 7555346 TI - [Treatment outcome for fracture of the radial head]. AB - Clinical, radiological and dynamometric assessment served to evaluate results of treatment for the radial head fracture in 51 patients out of 89 managed between 1983 and 1993. Comminuted fractures, especially if complicated by ligament capsular injury of the elbow have bad prognosis. Results of treatment are poor in this type of injury regardless the mode of management. PMID- 7555347 TI - [Remodeling of the hip joint with avascular necrosis during nonoperative treatment of congenital dysplasia, subluxation or dislocation]. AB - Radiological analysis of 61 hips in 32 children served to present natural history of the nonoperatively managed dysplastic hip affected with avascular necrosis of the proximal femur that appeared during treatment. An attempt to establish relationship between the degree of avascular necrosis and radiological state of the hip after growth termination has been made. Ossification disturbances within the femoral neck of some hips and its prognostic value for proximal femur deformity have been assessed. The final radiological assessment revealed 18 per cent excellent results, 34.4 per cent good, 31.2 per cent fair and 16.4 per cent poor results. In hips with changes limited to the femoral head fair and poor results combined constituted less then 25 per cent. If the femoral neck was also involved the percentage amounted to 71. The results accomplished allowed for creating an own prognostic classification of avascular necrosis of the hip. PMID- 7555348 TI - [Treatment for congenital dislocation of the hip at the orthopedic department of the Institute of Orthopedics and Rehabilitation in Poznan from 1946 to 1991]. AB - During forty-five years (1946-1991) over 6000 children were treated conservatively and over 6000 surgical procedures for congenital dislocation of the hip were carried out at Orthopedic Department of Institute of Orthopedics and Rehabilitation in Poznan. Modalities of treatment, types and numbers of operations are presented. PMID- 7555349 TI - [Unstable pelvic fracture with concomitant displacement of the uterus--case report]. AB - The paper deals with the displacement of the uterus observed during management of Malgaigne type pelvic fracture in a 33 years old female. An intrauterine device seen on the radiographs revealed displacement of the uterus towards contralateral side of the pelvis. The reduction of the fracture stabilized with Molski metal clamp reconstituted normal anatomy. PMID- 7555350 TI - [Arthroscopic diagnostics and treatment for cysts of the anterior cruciate ligament]. AB - From among over 500 knees examined arthroscopically in 5 cases operated on because of painful limitation of the range of motion besides an inveterate ACL injury a cyst located around injured fibers was found. Arthroscopic removal of the cyst rendered permanent improvement in all cases. No recurrence has occurred. PMID- 7555351 TI - [Lateral release of the patella. I--Diagnostics, indications]. AB - Basic principles of diagnostics of the patello-femoral joint are presented. Indications for arthroscopic subcutaneous patellar lateral release are discussed. PMID- 7555352 TI - [Lateral release of the patella. II. Arthroscopic technique of subcutaneous lateral release, complications]. AB - Principles of operative technique of arthroscopic patellar lateral release are presented. The most common complications and measures to avoid them are discussed. PMID- 7555353 TI - [Combined treatment of crural shortening and calcaneus-malleolar conflict using the Ilizarov distractor]. AB - The complex mode of treatment for crural shortening and calcaneus-malleolar conflict with Ilizarov distractor in 3 children is presented. The management resulted in fast ossification of the distal tibial epiphysis, adequate position of displaced lateral malleolus and correction of the tarsal varus as well as angular deformity of the tibia. The importance of timing for this kind of treatment is emphasized by the author. PMID- 7555354 TI - [Abdominal abscess. Apropos of 530 cases]. AB - A pathophysiological study of intra-abdominal abscess and their systemic effects were reported. Different methods of diagnosis were examined together with their respective value. Possible operative procedures were presented and analyzed. A certain number of practical points designed to avoid postoperative intraabdominal abscess and to reduce their gravity and systemic effects were developed from a series of 530 observations. PMID- 7555355 TI - [Treatment of spastic equinovarus deformity. Apropos of 60 operations]. AB - The authors evaluate the efficiency of the surgical technique of split anterior tibialis transfer, describe the long term functional results of the operation, and discuss the indications of this method of treatment. The results were very satisfactory: after surgery, 46 of 53 patients had normal shoes, 33 patients had improvement of their level of autonomy. The morbidity was very low, no patient had aggravation of the neurologic disease or general complications. The authors conclude that this operation should be employed more frequently in these disabled patients who can find definite improvement. PMID- 7555356 TI - [Treatment of rhizomelic arthrosis of the thumb by partial resection of the trapezium and rib cartilage autograft. Preliminary results]. AB - The authors have studied the results of a personal technique concerning the treatment of trapezo-metacarpal arthritis: a partial trapezectomy is associated with a antogenous rib cartilage graft. 13 cases were treated using the procedure. 9 patients are reviewed with a follow up mean one year. 4 patients are free of pain, 5 are improved. Strength is improve in 4 cases (pinch), similar or inferior to the pre-operative status in 5 cases. Increase of motion concern Kapandji test and first web space. Follow up is insufficient to allow a conclusion about the long term behaviour of cartilage graft. PMID- 7555357 TI - [Lateral lumbar disk herniation]. AB - A retrospective study of 41 lateral discal hernias observed between 1984 and 1991 were studied among the 1080 discal hernias treated during this period. CT scan, performed in all cases, distinguished several different types of hernia: foramen hernias (26), extraforamen hernias (12), mixed forms (5) associated with canal component (11). Thirteen disco scans were required. Nucleolysis was performed in 24 patients (58%) and surgical treatment was the first intention choice in 17 (41%). Outcome, evaluated with a function score developed in the unit were good in the 17 surgery cases (100%). In the nucleolysis patients results were good or excellent in 13, average in 4, and poor in 7. Five of the nucleolysis failures were later operated leading to good results in 3, average in 1 and no change in 1. Indications for surgery are more frequent in this type of discal hernia and results in our surgical series were better than those for chemonucleolysis. PMID- 7555358 TI - [Current status of carotid surgery]. AB - Introduced in the fifties, reconstructive surgery of the carotid arteries has undergone an extensive development in Europe and the United States. Prospective randomized multicentric clinical trials have clearly demonstrated its effectiveness in stroke prevention for both symptomatic and asymptomatic patients with tight carotid stenosis. The diminution of relative risk secondary to surgery is at least 40%; it is 80% for major ipsilateral stroke. There is no benefit if the vessel narrowing is less than 30%. For the 30 to 70% stenosis a benefit has not yet been clearly established. The prophylactic effect of carotid surgery is highly dependent of a low postoperative morbidity. The 30 day stroke plus death rate should not exceed 3% for asymptomatic patients and 5% for symptomatic patients. Postoperative morbidity-mortality has greatly improved over the last twenty years. Several factors are involved; the main improvement may be related to better surgical technique assessed by intra-operative angiographic control rather than the many methods of monitoring or cerebral protection. Ten years patency of arterial reconstruction is greater than 90%. Stroke protection is effective since the annual rate of cerebral events in an operated population is about 2% while it is about 10% in a control population. Inversely, late mortality is still high, primarily due to cardiac disease. PMID- 7555359 TI - [Current status of reconstructive surgery of the sub-inguinal artery]. AB - Saphenous vein bypass is the optimal operation for arterial reconstruction in the subinguinal region. There has been no demonstrated advantage to using a devalvulated saphenous vein in situ rather than an inverted dissected vein. Prosthetic bypass may be preferred over saphenous bypass with a vein < or = 4 mm if the lower anastomosis is situated on the upper popliteal artery, if the life expectancy is short or if on the contrary the downstream bed is normal. Permeability after percutaneous transluminal angioplasty does not last as long as after bypass surgery for obliterations, but the operation is less aggressive and does not compromise a subsequent procedure. The best indications are short stenoses in patients with a long walking distance in whom surgery would be excessive. Angiography or ultrasound angioscopy after a bypass procedure or endoluminal dilatation can check the technical quality of the operation and help improve permeability duration. Adjuvant treatment such as a venous patch on a prosthetic anastomosis or various drugs are highly important in improving the duration of permeability. A prospectively planned follow-up should be an integral part of arterial reconstruction surgery in order to identify subsequent stenoses and programme their treatment before occlusion occurs. PMID- 7555360 TI - [Current role of surgery in chronic venous diseases]. AB - Non invasive explorations of chronic venous disease, particularly echo-doppler techniques, have greatly improved our understanding of the underlying pathophysiological mechanisms. The findings of the physical examination are however still the basis of all indications for surgery. The most frequently performed procedures involve treatment of superficial venous insufficiency. Classical stripping of the large superficial veins with crossectomy has largely been replaced by more individualized procedures. The long-term effectiveness of certain revolutionary methods such as the Chiva procedure have not yet been proven. Indications for surgery of the perforating veins is a matter of debate but could benefit from the development of less traumatic endoscopic techniques. Surgery of deep vein insufficiency is another field of development. The follow-up in recently reported series is now long enough for an assessment of the therapeutic value of these procedures. Deep vein surgery has given a variable degree of success for post thrombotic syndrome but its effectiveness is proven in Primary deep vein insufficiency. PMID- 7555361 TI - [Present status in the study of placenta]. PMID- 7555362 TI - [Stereoptical study of the microvilli in intrauterine growth retardation placenta with different ponderal indexes]. AB - Two groups of 6 intrauterine growth retardation (IUGR) placentae each from normal ponderal index (NPI) or low ponderal index (LPI) and 10 placentae from normal pregnancy as control were quantitatively analyzed with light and electron microscopic morphometry. The results showed that the placental weight, volume, surface area of the villi and villous fetal capillary in IUGR were significantly reduced compared with those in the control group. Not only were the placental weight, volume, surface area of villi, villous capillary, intermicrovillous surface density and absolute values surface area of the microvilli in NPI group significantly lower than that in the control group, but also lower than in LPI group. This suggests that there was a functional deficiency of placenta in IUGR placentae, nonetheless, the changes of functional structures in IUGR placentae with different ponderal indexes were not the same. The stereoptical study with light microscopy and with the electron microscopic level in confirming that NPI group has a more severe deficiency in placental function than in the LPI. PMID- 7555363 TI - [Placental tissue histomorphometry in hydrops fetalis]. AB - Placentae from 10 hydrops fetalis with hemoglobin Bart's disease were studied by tissue volumetry on the relative percentage of placental proper to its components, the surface area of villi and villi capillaries, and by comparing with 6 placentae from premature deliveries of 30-36 weeks. The placental weight, volume, percentage of placental proper and the villi volume, as well as the surface area of villi were significantly increased as compared with the controls. The relatively decreased volume percentage of intervillous space volume and surface area of villi capillaries resulted in a reduction of maternal-fetal exchanging surface area. We suggest careful prenatal scanning for Thalassemia and avoiding marriage between local peoples to reduce the incidence of hemoglobin Bart's disease. PMID- 7555364 TI - [Correlation of fetal umbilical artery velocity waveforms with placental pathology and biochemistry]. AB - The ratio of peak systolic to least diastolic flow velocity (S/D) of the umbilical artery was measured in 119 pregnant women and its relation with placental pathology were studied. Results obtained indicated that S/D ratio decreased gradually with advanced gestational age and showed a negative correlation (r = -0.972 8, P < 0.01). A ratio of < 3 was defined as normal value. Patients were divided into 2 groups according to the S/D ratio and their placental histological findings were compared. The number of small arteries in the tertiary villi stem and syncytial vascular membranes were significantly less in group 2 (S/D > or = 3) than that in group 1 (S/D < 3, P < 0.01). The counts of villous syncytial knots and stroma fibrosis of villi were more significant in the placenta of group 2 as well. The mean value of molondialdehyde in placenta of group 2 was significantly higher (P < 0.01) than that of group 1, while superoxide dismutase value is to the contrary (P < 0.01). These indicated that there was a reduced circulation resistance in placenta during normal pregnancy and remained stable till term. When S/D > or = 3, it indicated that physiological and biochemical changes occurred in placenta and the outcome of pregnancy might be affected. PMID- 7555365 TI - [Uteroplacental blood flow monitoring by color Doppler flow imaging in pregnancy induced hypertension]. AB - The uteroplacental blood flow was monitored using color Doppler flow imaging (CDFI) in 31 cases of pregnancy induced hypertension (PIH) and 71 cases of normal pregnancy. At the same time the values of serum estriol (E3), human placental lactogen (hPL), thromboxane (TXB2), 6-KP and TXB2/6-KP ratio were measured. The results showed that time average velocity (TAV) and volume of blood flow (Q) of uterine and umbilical arteries in PIH cases were much lower than those in normal pregnant women and fetuses (P < 0.05). After treatment with magnesium sulfate and qingxintong there was a marked improvement in placental blood flow. TAV and Q of uterine and umbilical arteries was also increased (P < 0.05). In PIH cases the E3 and hPL levels were much lower, while the ratio of TXB2/6-KP was significantly higher than that in normal pregnancy. Using CDFI to observe the velocity and volume of uteroplacental blood flow can monitor the blood supply of uterus and placenta. It is a valuable predictive index of fetal and placental function. PMID- 7555366 TI - [Placental ureaplasma urealyticum infection and low birth weight]. AB - Ureaplasma urealyticum (UU) infection during pregnancy may result in poor outcome. Placentae from 116 normal healthy pregnancies were cultured for UU with a recovery rate of 28.45%. 94 cases ended by normal vaginal delivery had a UU positive rate of 29.79%. The UU positive rate of 22 placenta from cases by cesarean section was 22.73%. The 2 positive rates showed no difference of significance (P > 0.05). This result indicated that UU infection of the placenta was present before passing through the birth canal and not due to contaminant during delivery. The birth weight of the neonates with positive UU infection was on the average 2,919g, while that in the negative UU cases was 3,490g, being 571g higher than in the positive group (P < 0.01). This study showed that placental infection with UU is associated with a low birth weight. PMID- 7555367 TI - [Impact of placental hormone withdrawal on postpartum depression]. AB - Prenatal and postpartum measurements of serum beta-hCG, estradiol (E2) and progesterone (P) concentration were done by radioimmunoassay in 20 cases of postpartum depression (PD) and 20 cases without postpartum depression (NPD). The results showed significantly higher serum levels of P and beta-hCG (at 36-37 gestational week) in PD, but no difference in the serum P and beta-hCG concentration between the PD and NPD after childbirth. The falling of the two hormones was faster in PD than in NPD. In neither prenatal nor postpartum test was any difference seen in serum E2 concentration between PD and NPD. These results suggest that supposely, the endogenous risk factor, the rapid withdrawal of the placental hormones (P and beta-hCG) after delivery, leads to PD. PMID- 7555368 TI - [Influence of dihydroetorphine hydrochloride and tramadol on labor pain and umbilical blood gas]. AB - Ninety primigravide were randomly allocated into three groups at the beginning of active phase of labor. Dihydroetorphine hydrochloride (DHE) was administered to group A (n = 30), tramadol to group B (n = 30), and group C (n = 30) was a blank control. Various parameters about analgesia effects, progress of labour and fetal and neonatal well-being were investigated and also umbilical artery and vein blood gases analyzing were carried out in all cases. As a result, the effective rate of pain relief in group A was 67% and in group B 63% (P > 0.05). The average time of onset of action in DHE group was 16.5 +/- 2.9 minutes which was significantly shorter than 26.1 +/- 5.4 minutes in tramadol group (P = 0.0001). There were higher rate (36.7%) of operative intervention (forceps and vaginally or cesarean section) and a higher average amount of postpartum hemorrhage in group A, as compared with the control group, but no significant difference was shown between group B and group C. No difference was found in other parameters (among the three groups duration of labour, Apgar scores of neonates, cord blood gases, etc.). The conclusion is that, both DHE and tramadol may have a good effect of pain relief in labour. The time of onset of action in DHE group is significantly shorter than that in tramadol group. Neither analgesics cause circulation and respiratory depression in the mother and neonates, but DHE may have influence on uterine contraction. To achieve an excellent pain relief by higher dosage of DHE and tramadol need further randomized investigation. PMID- 7555369 TI - [Analysis of 29 maternal deaths caused by amniotic fluid embolism]. AB - Twenty nine maternal deaths caused by amniotic fluid embolism (AFE) during 1989 1993 in Beijing area were reviewed. The results show that the average maternal mortality ratio due to AFE was 5.9/100,000 live births responsible for 15.5% of total maternal deaths. In 13 of the 29 patients presented the clinical signs were of postpartum hemorrhage and coagulation disorders. Oxytocics had been used in 12 of the 29 cases during labor. In more than half of the patients there was delay in diagnosis. This was that the percentage of AFE which may present with the signs of coagulation disorder was higher than previously thought, and unreasonable use of oxytocics during labor may be associated with AFE. This kind of AFE is easy to misdiagnose. PMID- 7555370 TI - [Pathological findings of proximal tubal occlusive infertility]. AB - Exicised tubal segments (including interstial and isthmic portions) from 33 women with proximal tubal occlusive infertility were examined pathologically. We found that pathologic entities included chronic tubal inflammation (70.59%), tubal endometriosis (9.80%), obliterative fibrosis (7.84%), salpingitis isthmica nodosa (5.88%), tubal tuberculosis (3.92%). Only one of the tubes (1.96%) showed no obvious pathological change. These observations on proximal tubal occlusion in combination with clinical features and pelvic changes indicate that: (1) These are multiple distinct pathologic patterns, and pelvic findings do not predict the pathologic changes of the tubes. (2) Induced abortion and uterine curetage is an important factor for the proximal tubal occlusive infertility. Pelvic inflammatory disease and intrauterine device are other factors. PMID- 7555371 TI - [Epidemiological study on risk factors of pelvic endometriosis]. AB - In order to study the risk factors associated with pelvic endometriosis, a case control study was carried out from Mar. 1991 to Feb. 1993 in our gynecological ward. A total of 190 cases with endometriosis and 192 patients with non endometriosis diseases (control group) were interviewed. Single factor analysis indicated that risk factors related to endometriosis were menorrhagia (OR = 3.09), short menstrual cycle (< 27 days) (OR = 1.89), complicated with uterine leiomyoma and (or) adenomyosis (OR = 1.84), occupation as cader (OR = 1.71), all with statistical significance (P < 0.05). The artificial abortion rate of married patients was similar between the two groups, but the abortion frequency was fewer in endometriosis patients (P < 0.05). Multifactor analysis confirmed that the risk factors associated with endometriosis were menorrhagia and occupation as cadre only. PMID- 7555372 TI - [Androgen receptors and effects of sex hormones on growth of a human ovarian carcinoma cell line]. AB - Previous study had showed that ovarian carcinoma cell line (HO-8910 cells) contained estrogen receptors (ER) and progesterone receptors (PR). In this experiment, androgen receptors (AR) in these cells were determined by a whole cell binding assay with a synthetic nonmetabolizable androgen-methyltrienolone (3H-R1881). The results showed that there existed high affinity and low capacity, specific binding sites for 3H-R1881 in these cells, the apparent maximum binding capacity and dissociation constant of these binding sites revealed by scathchard analysis being 6.52 fmol/10(6) cells and 2.96 nmol/L respectively, indicating the presence of AR in addition to ER and PR in HO-8910 cells. Furthermore, the effects of sex hormones, i.e., estrodiol, progesterone and testosterone on the growth of HO-8910 cells were also investigated. The results showed that testosterone at 10 nmol/L slightly increased the number of HO-8910 cells, but the increase was not dose-dependent and not statistically significant. Estrodiol and progesterone had no growth-stimulatary or inhibitary effects on these cells. PMID- 7555374 TI - [Clinical analysis of 6 cases of gestational trophoblastic neoplasms complicated by cardiotoxicity after chemotherapies]. AB - Gestational trophoblastic neoplasm treated by large dosage of 5-fluorouracil and actinomycin could cause cardiotoxicity. In 6 cases of gestational trophoblastic neoplasms admitted to our hospital from March to December 1993, cardiotoxicity was found to have occurred after chemotherapies in 10/21 courses. There were 2 cases with heart failures, 4 cases with palpitation and 4 cases with feelings of oppression and (or) chest pains. In 7 cases the electrocardiogram (EKG) findings were abnormal (70.0%) and in 5 cases there were increased sera glutamic oxalacetic transaminase enzyme (AST). 90.0% of these complications were brought under control after decreasing the dosages and changing the methods of administration. If the proper managements were not given on time, patient may even expire. So attention should be paid to this complication. It is suggested that attention should be paid to patients' subjective symptoms, and proper surveillance by EKG and serum AST determinations should be conducted to minimize deaths due to chemotherapeutic complications. PMID- 7555373 TI - [Hypomagnesemia in patients of gynecologic neoplasms following chemotherapy with cisplatin]. AB - Serum magnesium in 79 patients of gynecologic neoplasms treated with cisplatin and their controls was measured. The results showed: (1) the average value of serum magnesium in patients following chemotherapy with cisplatin was significantly lower than in the controls; (2) the incidence of hypomagnesemia was positively correlated with the number of the courses and the total dosages of chemotherapy, being 52.9% after one to three courses of treatment and 92.0% after more than six courses of treatment; (3) the incidence of hypomagnesemia was directly related with the severity of gastrointestinal disorders; (4) serum magnesium following cisplatin chemotherapy was not correlated with serum blood urea nitrogen (BUN). Hypomagnesemia is clinically characterized by symptoms of the nervous system which are found in 14.0% of hypomagnesemic patients. PMID- 7555376 TI - [Current status and prospect of spine surgery in China]. PMID- 7555375 TI - [Prevention and treatment of toxic and adverse effects of cisplatin]. PMID- 7555377 TI - [Lumbar spinal stenosis syndrome: classification, pathology and operative consideration]. AB - A total of 168 cases of lumbar spinal stenosis syndrome were operated on in recent 6 years. They included 13 cases of developmental spinal stenosis and 155 cases of acquired spinal stenosis. All patients were followed up for an average of 2 years and 7 months, ranging from 3 months to 5 years and 10 months. The results of treatment were not satisfactory in developmental spinal stenosis (excellent 46.2%, good 30.7%, fair 15.4%, poor 7.7%), but good in acquired spinal stenosis (excellent 72.2%, good 22.6%, fair 5.2%, poor 0). Moreover, with different classification and pathology, acquired spinal stenosis had different results. The authors stressed that it is very important to consider the methods and the range of operation according to the pathological characteristics of myelography and CTM of lumbar spinal stenosis. PMID- 7555378 TI - [Operative treatment for cervical vertigo caused by foramen arcuale]. AB - The foramen arcuale of vertebral artery of altas is one of the causes of vertigo. Leven cases of the cervical vertigo caused by foramen arcuale were operated on. The patients were followed up for 3-6 years. The results were satisfactory. The mechanism of inducing vertigo by foramen arcuale was discussed. We demonstrated that excision of the foramen arcuale is available for cervical vertigo. PMID- 7555379 TI - [The pathogenetic factors for neurogenic intermittent claudication caused by degenerative lumbar spinal stenosis]. AB - The pathogenetic factors in 89 patients with typical neurogenic intermittent claudication caused by lumbar spinal stenosis were studied retrospectively. 75 patients had degenerative lumbar spinal stenosis, and 67 had unilateral nerve root canal stenosis. In most patients, the stenotic location was found at the entrance zone of nerve root canal (75/89), involving only a single level (60/89) mostly at L4.5. The pathogenetic factors for degenerative lumbar spinal stenosis were degenerative changes of lumbar disc only (55/89), and those for hypertrophy of facet joints (22/89), thickened ligamentum flavum (11/89), and spondylolis thesis (10/89). All patients were operated on: 57 of them had less invasive procedures. The excellent and good early follow-up results were noted in 73% of the patients (65/89). The authors emphasized that the surgical treatment of lumbar spinal stenosis should be as less invasive as possible rather than a tranditional extensive lumbar laminectomy. PMID- 7555380 TI - [The results of operative treatment of diastematomyelia]. AB - From May 1978 to July 1993, 31 cases of diastematomyelia were treated surgically. There were 7 males and 24 females, 2 to 34 years old. The follow-up period varied from 2 month to 14.4 years. All patients were couplicated by congenital spinal deformity. Three of them had two septums. The results of operative treatment of the 31 cases were: recovery in 10 cases, remarkable effectiveness in 5, effectiveness in 11, noneffectiveness in 5. The effective rate was 84%. The operative indications and key points were also discussed. PMID- 7555381 TI - [An experimental study on the relationship between disc nutrition and disc degeneration]. AB - Micro-angiography, scanning electron microscopy and hydrogen washout technique were used to investigate the disc nutrition condition and the nutritional pathway of endplate in bipedal rats with agting. Compared with the controls, the nutrition condition of bipedal rats underwent deterioration and the number of vascular distribution under the endplate decreased significantly, but no increase in thickness of endplate caused by microfractures of subchondral bone and callus formed during the healing process was observed. The abnormal stress may bring about vertebral body intra-osseous circulation disorder and then consequent decrease of vascular channels which related to the disturbance of disc nutrition. This strongly suggested that nutrition conditions of nucleus pulposus were not deteriorated by impaired diffusion capacity, but by decrease of vasculature that contacts with endplate. PMID- 7555382 TI - [Dynamec changls in dimension of cross-sectional areas of lumbar canal and dural sac: an experimental study]. AB - The variation in the cross-sectional areas (CSA) of lumbar canal and dural sac under extension and flexion was studied by using CT scans in ten human cadaver lumbar spine specimens. CSA at different levels or same level in varying positions were distinctive. CSA in flexion position was increased, while in extension position reduced. Dynamic changes of CSA of canal (CSAC) at L4,5,L5-S1 were very significant (P < 0.01), and those of CSA of dural a (CSADS) at all levels but L5-S1 were significant (P < 0.05). It was considered that dynamic changes of CSAC at intervertebral levels were determined by the range of lumbar motions, but those of CSADS were influenced by changes of canal and biomechanical factors of intracanal soft-tissue and changes of dural sac length. Measurement of CSADS was more significant than that of CSAC. PMID- 7555383 TI - [Biomechanical evaluation of cervical stability with Halifax clamp after bilateral facetectomy]. AB - With nine fresh adult cervical spine specimens (C3-T1), the three-dimensional motions of C4-5 segment were analysed to evaluate the immediate stability following bilateral facetectomy, Halifax clamp and wire fixation. The results showed that the stability with Halifax clamp was greater than that of the intact state in flexion, extension and lateral bending, but the same as in axial rotation. The stability with wire fixation was greater than that in flexion and equal to that in extension, lateral bending and axial rotation. The ROMs with Halifax Clamp were less than those with wire fixation in flexion, extension and lateral bending, but close to in axial rotation. PMID- 7555384 TI - [Progress in the study on occipitocervical biomechanics]. PMID- 7555385 TI - [Surgical treatment of colonic and rectal cancer with hepatic metastasis]. AB - In 568 patients with colonic and rectal cancer treated in our hospital from January 1970 to January 1991, 266 had colonic cancer and 302 rectal cancer. 469 patients (263 rectal and 233 colonic) were operated on. A total of 382 patients (186 and 196 colonic) were resectable. The average resection rate was 76.6%. 68 patients (30 rectal and 38 colonic) had hepatic metastasis with a rate of 11.97%. 13 patients (19.1%) had hepatic metastasis focus resected. PMID- 7555386 TI - [Surgical treatment of carcinoma of low rectum]. AB - 1230 cases of low rectal cancer were treated surgically from 1954 through 1990 with a resectability rate of 79.92% and a curative resectability rate of 63.98%. Among curative resections, abdomino-perineal excision accounted for 71.16% and sphincter-saving resection for 28.84%. Before 1980, 19.19% cases were treated with SSR and since 1980 it was raisen to 41.04%. In comparison of the results of group before 1980 with those since 1980, the mortality rate of curative resection was 1.56% and 0.99% respectively. The 5-year survival rate of curative resection (life table method) was 58.96% +/- 2.87% in APR and 80.39% +/- 5.06% in SSR before 1980, and 71.65% +/- 3.28% in APR and 83.82% +/- 3.46% since 1980. The local recurrence after curative resection was 13.57% in APR and 12.33% in SSR. The data showed that in properly selected cases, the outcome was better in SSR than in APR either in survival or in quality of life. The feasibility and reasonality of the SSR and the rules for selecting the operative procedures in low rectal cancer were discussed in detail. PMID- 7555388 TI - [The transphincteric approach excision of rectal villous adenomas]. AB - Twenty-four patients with rectal villous adenomas were operated on which posterior transphincteric approach. They had benign villous adenona in (13 patients), villous adenomas showing atypia (2), and villous adenomas developed malignancy (9). All the patients gained excellent results, except one with wounded infection after operation. No patient died at operation. No patient developed rectal fistula and incontinence of feces. The different methods of operation with excised villous adenoma of the rectum were discussed and compared. We conclude that the posterior transsphincteric approach is better than others. PMID- 7555387 TI - [Interference of selenium germanium and calcium in carcinogenesis of colon cancer]. AB - We studied DMH induced colon cancer in 120 wistar rats, which were divided into 8 groups based on different diets. They were killed and autopsied on 4 weeks after the last injection of DMH. The tumors in various organs including its characteristics, number, site, histological types and ultrastructural changes were observed. The results showed that high fat diet has a significant effect on DMH induced colon cancer. Selenium and calcium can inhibit the effect of DMH and decrease the incidence of colon cancer. Selenium can also interfere the effect of high fat diet but germanium has no effect on colon carcinogenesis. PMID- 7555390 TI - [A preliminary study of surgical therapy and combined therapy for cervical esophageal carcinoma]. AB - 52 patients with carcinoma of cervical esophagus were treated surgically. Their 3 and 5-year survival rate of was 38.5% and 35.1% respectively. The 3- and 5-year survival rate for surgery alone was 15.4% (2/13) and 9.1% (1/11). The 3- and 5 year survival rate for combined therapy (pre- or postoperative radiation and surgery) was 48.1% (13/27) and 52.9% (9/17). The 3- and 5-year survival rate for surgical salvage after failure of radical radiotherapy was 41.7% (5/12) and 33.3% (3/9). Our data suggest that the result of combined therapy is much better than the result of surgery or radiotherapy. The invation of recurrent nerve, hypopharynx, trachea, larynx and thyroid gland by tumor is contracindicated to surgery. PMID- 7555389 TI - [Manometric study of the esophagus and intrathoracic stomach after partial esophagectomy for carcinoma]. AB - The pressure in the residual esophagus and in the thoracic stomach was measured by using SG-II computer manometer in 25 patients who underwent resection of esophageal cancer. Thirty normal mean were chosed as controls. The results indicated that a high pressure zone was present above the esophago-gastric anastomosis. It may be helpful in preventing and reducing reflux esophagitis. We stress that the prevention of gastroesophageal reflux after the operation is related to the operative method and the topography of the stomach. PMID- 7555391 TI - [Tumor recurrence prevention using photodynamic therapy after treatment of bladder cancer]. AB - Photodynamic therapy with hematoporphyrin derivative was given to 21 patients with recurrent transitional cell carcinoma of the bladder. Each patient received red light (630nm) derived from a gold-vapor laser machine into the bladder. The light dose was given to the whole bladder by using the bulb diffuser with power density ranging from 36 to 72J/cm2. All patients had a complete response at 6 months. Three of the 21 patients had tumor recurrence at 12 months. This new light delivery system appears to be effective in the prevention of bladder cancer. Photochemotherapy is as useful as bladder chemotherapy. Its preventive effect will be elevated if it is repeated 6-12 months after first treatment. PMID- 7555392 TI - [Surgical management of severe hemorrhage and retroperitoneal perforation after endoscopic sphincterotomy]. AB - From 1990 to 1993, 5 patients with severe hemorrhage and retroperitoneal perforation following endoscopic sphincterotomy (EST) were surgically treated. Two patients with hemorrhage and one with perforation survived. The remaining two died of hemorrhage plus perforation. Early diagnosis and surgical intervention was very important to lower the mortality, especially in those with common bile duct stones retention, acute pancreatitis, or cholangitis. For patients with late stage perforation or recurrent hemorrhage, palliative procedure aimed at isolating the EST incision from the digestion of bile and pancreatic juice should be performed. PMID- 7555393 TI - [Storage temperature and skin xenograft survival]. AB - Pieces of guinea pig skin, 0.3-0.4mm in thickness, 1.5cm in diameter, were stored at different temperatures. The skin fragments were divided into five groups: (1) fresh skin; (2) skin stored at 4 degrees C for 48hr; (3) skin stored at -20 degrees C for 48hr; (4) skin stored at -80 degrees C for 48hr; (5) skin stored at -196 degrees C for 48hr. The cryoprotective agent was the same in all groups except group 2. The experimental skin was grafted on both sides of the back of anesthetized mice, two pieces of skin of the same group for one mouse. The grafted skin survival was determined by daily observation. The skin was considered as rejected if necrosis took place in 80% of the grafted skin. Grafted skin biopsy was performed for pathological examination. The results showed that the survival days of stored skin xenografts were prolonged as the stored temperature lowered. The pathological changes (neutrophil infiltration, thrombosis of small vessels, necrosis of epidermal and dermal cells) were mild and delayed as the stored temperature lowered. So low temperature may decrease the antigenicity of the stored skin. PMID- 7555394 TI - [Gallbladder contraction and microscopi observation of the gallbladder wall in gallstone patients]. AB - On the basis of oral cholecystography (OCG), we selected gallstone patients for nontoperative intervention. Their gallbladder function was considered as normal when gallbladder contracted over 50%-75% after a fat meal at 1 hour. We collected 99 gallstone patients and 19 normal controls; gallbladder function was normal in 77 patients and poor in 22. After a fat meal gallbladder volumes were calculated by B ultrasonography. The results showed that both fasting and residual volume in patients with so-called normal contraction were significantly greater than that in the controls (P < 0.001), though the contraction rate was not different. The combination of gallbladder contraction rate measurement and residual volume calculation by ultrasonography was superior over traditional OCG. PMID- 7555395 TI - [Precautions in conducting refractive corneal surgery]. PMID- 7555396 TI - [Strict control of refractive corneal surgery]. PMID- 7555397 TI - [Taking precautions to the complications of radical keratotomy]. PMID- 7555398 TI - An analysis on follow-up results of photoreactive keratectomy for treatment of myopia. AB - A prospective study of excimer laser photorefractive keratectomy (PRK) was performed with the aim of correcting a range of myopic errors between -1.00 and 10.00 diopters on 422 myopic eyes that were followed for more than one year. At postoperative one year, 91.4% of the eyes had uncorrected visual acuity of 20/20 or better, and 97.4% were within +/- 1.00 diopter of the desired emmetropia, and the corneal haze disappeared in most of the eyes. Elevated intraocular pressures were observed in 20.4% of the eyes. The decrease of one line of the best corrected visual acuity occurred in 7.6% and of two lines in 0.5% of the eyes. We think that the excimer laser PRK is a very predictable, safe, stable, and effective method to correct myopia up to -10.00 D, and longer follow-up will be necessary. PMID- 7555399 TI - Corneal topography analysis before and after radial keratotomy. AB - Corneal topographic analyses were conducted on 58 eyes of 42 cases with mild or moderate myopia by computer-assisted photokeratography before and after radial keratotomy (RK). The results indicate that the corneal surfaces of most examined eyes (76%) are positive aspheric shape before RK, while after RK they are changed to negative aspheric shape. Before RK, the corneal topography of most examined eyes (67%) is symmetric bow tie pattern or asymmetric bow tie pattern; after RK, the forms of central flattened regions are mainly round or approximately round, dumbbell and belt-shaped. Before and after RK there is no significant change in surface regularity index (SRI), but surface asymmetry index (SAI) is changed significantly. RK makes the central and central peripheral parts of the cornea flattened. The most significant RK effect is found at the region 1.140 +/- 0.090 mm away from the corneal optical center. Examination and analysis on the corneal topography before and after RK not only provide an accurate and objective basis for RK operational plan, but also make it possible to objectively and quantitatively evaluate the RK effect, to accurately predict the RK clinical effects, and to effectively improve the RK clinical quality. PMID- 7555400 TI - [A study on five-year postoperative results of radial keratotomy]. AB - Radial keratotomy was performed on 285 myopic eyes (155 cases) with a mean of 5.48D. 1-2 pairs of flag-shaped transverse incisions were also made to correct corneal astigmatism over 1.00D. After a postoperative follow-up of 4-5 years, the mean keratometric corneal refractive power, corneal astigmatism and spherical equivalent refraction of the eyes were reduced by 3.98D, 0.36D and -4.74D, respectively. The rates of emmetropia, overcorrection and undercorrection were 70.7%, 15.5% and 13.8%, respectively. The decrease of the best corrected visual acuity by over two lines occurred in 3.4% of the eyes. The percentage of eyes with postoperative uncorrected visual acuity > or = 1.0 was 63.8% and that > or = 0.6, 89.7%. The complications included corneal microperforation in 4.6%, visual fluctuation in 34.1% and glare in 8.8% which decreased to 1.8% after postoperative 6 months. The mean rate of endothelial cell loss at the central optical zone was 6.2% in the postoperative 1-2 years. PMID- 7555401 TI - Repeated radial keratotomy. AB - 38 cases 59 eyes received repeated radial keratotomy in which the secondary incisions were performed along the scars of old incisions. The average follow-up after the reoperation was 13 months. Before reoperation, the mean spherical equivalent of the refraction was -3.78D and the mean keratometry was 40.15D. 13 months after the reoperation, the mean spherical equivalent of the refraction decreased by 2.30D, and the mean keratometry decreased by 2.04D. After the reoperation, the mean uncorrected visual acuity increased by 5 lines. There was no relationship between the therapeutic effect of the reoperation and the degree of initial myopia or pre-reoperative keratometry. The repeated radial keratotomy can decrease the degree of astigmatism and its complications are few. PMID- 7555402 TI - Astigmatism analysis after radial keratotomy. AB - Radial keratotomy without T-cutting was performed on 245 eyes of 124 patients with 6-8 months of post-operative follow-up. The results showed that post operatively astigmatism was increased in 74 eyes (30.2%) with the increased range from +0.50 to +3.50DC, decreased in 27 eyes (11.0%) and not changed in 144 eyes (58.8%). The main causes of astigmatic change included microperforations, irregular incisions, incisions across the central optical zone and various depths of incisions, etc. PMID- 7555403 TI - [An analysis of severe complications of radial keratotomy]. AB - Seven cases with severe complications threatening vision, including intraocular infection, corneal leucoma, secondary glaucoma, cataract and delayed rupture of corneal incisions following radial keratotomy (RK) were presented. The causes of the complications are related to corneal perforation and decreased tensile strength of the eye. Correct selection of indications, perfection of technique and instruments, avoidance of corneal perforation and prevention of infection are the measures to prevent severe complications following RK. PMID- 7555404 TI - [Clinical observation of posterior chamber lens implantation in children]. AB - Extracapsular cataract extraction with posterior chamber intraocular lens (IOL) implantation was performed on 22 eyes of 16 patients with congenital or traumatic cataract and with ages ranging from 4 to 7 years old. The periods of follow-up were 1-2 years with a mean, 18 months. The postoperative reaction was mild, no untoward complications occurred in the remote follow-up and the postoperative visual acuities recovered well. The youngest age of IOL implantation, the choice of diopter of refraction and the management of intra- and post-operative complications were discussed. PMID- 7555405 TI - [A comparison analysis on methods of astigmatism examinations for children]. AB - 70 children 140 eyes with astigmatic refractive error were reported. The methods of examination included retinoscopy, autorefraction and keratometry. The data of eye astigmatism and keratic astigmatism measured by the methods were analyzed. It revealed that there existed a good identity among the three methods, and the relationship between eye astigmatism (EAs) and keratic astigmatism (KAs) shown by the formula EAs = 0.94 KAs - 0.13 was similar to that of previous reports. It is suggested that the keratometry be necessary as an assistant method of refraction to assure to get a reliable astigmatism result for children. PMID- 7555406 TI - [A study on expression of epidermal growth factor receptor in pleomorphic adenoma of lacrimal gland]. AB - Expression of epidermal growth factor (EGF) receptor was examined in 32 cases of pleomorphic adenoma of lacrimal gland by means of an immunohistochemical method. While normal lacrimal glands were all negative for the antigen, EGF receptor was positive in 10 pleomorphic adenomas and the positive staining was mainly limited in the tumor cells in trabecular or duct-like arrangement or squamous metaplastic epithelium. These data suggest that the expression of EGF receptor be significantly higher in pleomorphic adenoma than in normal lacrimal gland, and also suggest that EGF receptor be expressed in the neoplastic cells which are considered to be of duct origin. PMID- 7555407 TI - [The clinical study of local treatment of ocular malignant tumors with IL-2 LAK cells]. AB - The authors reported the method of treatment of ocular malignant tumors at early and middle stages with local injections of self lymphokine-activated killing (LAK) cells which were induced by gamma IL-2 in vitro. Satisfactory therapeutic results were obtained. Among the 25 cases of ocular malignant tumors, 10 cases were treated only with IL-2 LAK cells, nine of them got complete regression (CR) and one got partial regression (PR); the other 15 cases were treated with operation and IL-2 LAK cells and they all obtained complete remission. In the follow-up, except the contact of 1 case with PR was lost, all the other 24 cases did not relapse after 1-5 years of observation. The above results show that the treatment of malignant tumors with local injection of self IL-2 LAK cells is a very effective immunotherapy. PMID- 7555409 TI - [Clinical progress in the therapy of myopia with excimer laser keratotomy]. PMID- 7555408 TI - [Effect of interferon on filtering blebs after trabeculectomy in rabbit eyes]. AB - A study on the suppressive effect of interferon on the formation of filtering surgical scar was carried out. Trabeculectomy was performed on 6 rabbit eyes. 100,000 Units of interferon were subconjunctivally injected at the filtering bleb during the operation and on the 3rd and 7th post-operative day. The filtering blebs, filtering function and the scanning electron microscopic examinations of the surgical areas of the walls of the globes were observed. The results show that interferon can reduce the scar, improve filtering function and can be tried clinically. PMID- 7555410 TI - Sites of attachment and density assessment of female Ixodes ricinus (acari: ixodidae) on dairy cows. AB - A sample of 110 lactating dairy cows was examined to describe the attachment sites of female Ixodes ricinus ticks and to assess whether counts on one or two body areas can predict infestation and its level. Seventy per cent of the cows were infested by I. ricinus and the average tick burden on the infested cows was 15, ranging from 1 to 136. Preferred attachment sites were the axilla, udder/groin, neck, dewlap and flank. Udder/groin and axilla carried respectively 35.3% and 44.1% of the total tick burden. To predict infestation (yes/no), sensitivity of counts on axilla, on udder/groin and on both areas was respectively, 76.6%, 80.6% and 94.8%. Total burden prediction equations based on count on axilla and udder/groin are presented. PMID- 7555411 TI - Susceptibility of immature Ixodes scapularis (Acari: Ixodidae) to desiccants and an insecticidal soap. AB - Efficacy of commercially available formulations of desiccants (Drione, Dri-die, diatomaceous earth) and insecticidal soap (Safer's) comprised of active ingredients were evaluated against Ixodes scapularis Say immatures in petri dish and grass turf bioassays. In petri dish bioassays at label rate, all products, except diatomaceous earth, provided a high degree of control by 24 and 48 h (71 100% mortality). Mortality of larvae exceeded 94% by 4 h after treatment with Drione (10 x label rate held at 85% RH), Dri-die (label rate held at both 85% and 98% RH) and Safer's insecticidal soap (10 x label rate held at 98% RH). Nymphal mortality was highest by 4 h after treatment with Safer's insecticidal soap (up to 96%). Larvae were more susceptible than nymphs to desiccants, however, nymphs were more susceptible than larvae to Safer's insecticidal soap. Immature ticks treated with Safer's insecticidal soap exhibited sublethal effects with significantly decreased attachment to hosts and no engorgement. In grass turf bioassays, mortality of nymphs was equivalent (85-95%) between treatments of chlorpyrifos (0.6 kg [AI]/ha, Safer's insecticidal soap, and Drione. In the same bioassays, nymphal mortality was moderate after treatment with Dri-die (23-29%) and low following application with diatomaceous earth (16%) and for untreated controls (6%). PMID- 7555412 TI - The effects of tick numbers and intervals between infestations on the resistance acquired by guinea-pigs to adults of Rhipicephalus evertsi evertsi (Acari: Ixodidae). AB - Guinea-pigs were infested three times at short (1 week) intervals or long (10 week) intervals between successive infestations with low (1), medium (8) and high (15) numbers of adult Rhipicephalus evertsi evertsi. To compare the resistance developed by the hosts at short and long intervals between infestations, a challenge (fourth) infestation was performed with the high number of ticks. Resistance was assessed by comparing the mean weight of engorged female ticks that dropped from the hosts. Concentrations of beta globulins in the host sera were also monitored. Different tick loads (low, medium and high) and infestation intervals had a similar influence on the reduction in weight of the females which was between 60-70% after the final challenge. However, the concentration of beta globulins, was highest in the guinea-pigs exposed to medium and high numbers of ticks with long infestation intervals, probably because of an increase in the production of protective antibodies in response to more intensive antigenic challenge. The relationship between engorgement weight and beta globulin levels in host sera is discussed. PMID- 7555413 TI - Can manometric parameters predict response to biofeedback therapy in fecal incontinence? AB - PURPOSE: Biofeedback therapy may improve fecal control in up to 50 percent to 92 percent of patients with fecal incontinence. Identification of favorable manometric parameters before biofeedback therapy may help in selection of patients suitable for such therapy. METHODS: Twenty-eight patients with fecal incontinence (idiopathic, 11; iatrogenic trauma, 8; obstetric trauma, 9) who underwent biofeedback therapy were studied to determine whether manometric parameters could predict the result of therapy. Biofeedback was given using a computer software program designed to strengthen the external anal sphincter with auditory and visual feedback. RESULTS: Thirteen patients (46.4 percent) achieved excellent results; eight patients (28.6 percent) had good results, but seven patients (24.5 percent) failed to improve after biofeedback therapy. Resting or squeeze anal canal pressure, pressure volume, sphincter length, sphincter fatigue rate, and cross-sectional asymmetry of the entire sphincter before biofeedback failed to reveal any statistically significant differences between responders and nonresponders. However, the cross-sectional asymmetry of the high-pressure zone within the sphincter at rest was greater in nonresponders than in responders (not improved, 25.8 percent; good result, 20.2 percent; excellent result, 15.4 percent; P < 0.07). This difference was even greater on squeeze (not improved, 21 percent; good result, 17.6 percent; excellent result, 13.2 percent; P < 0.04). The number of biofeedback sessions, response on bearing down, and quality of rectoanal excitatory reflex were not reliable indicators of outcome. No statistical difference was found in mean resting and squeeze pressures after biofeedback between responders and non-responders. CONCLUSIONS: Except for increased cross-sectional asymmetry in the high-pressure zone, which may be a forerunner of adverse outcome, manometric parameters before biofeedback do not predict response to biofeedback therapy. Improvement in continence may be independent of resting and squeeze pressures achieved after biofeedback therapy. PMID- 7555414 TI - Proliferative activity at rectal anastomoses performed with various suture materials. An experimental study. AB - BACKGROUND: Suture line recurrence continues to be an important cause of failure following curative resection for colorectal carcinoma. Our aim was to determine whether the nature of the suture material used affected the proliferative activity of colonic crypt cells at the anastomosis. METHODS AND MATERIALS: Sprague-Dawley rats were randomized into one control and three experimental groups. In each experimental group the proximal 2 cm of rectum were resected and anastomoses constructed with titanium clips, interrupted 6-0 Vicryl (Ethicon, Inc., Somerville, NJ), and interrupted 6-0 silk. Control animals had a sham operation. One-quarter of each group of rats were killed at 14, 30, 60, and 90 days. Each animal received intraperitoneal tritiated thymidine 30 minutes before death. Each anastomosis was harvested, and longitudinally oriented crypts were analyzed for the total number and position of labeled cells at five equal distances from the anastomosis. Random crypts were studied in the control group. RESULTS: Labeling indices were increased in almost all experimental groups at days 14, 30, 60, and 90. There were no persistent, statistically significant differences in labeling indices among the various suture materials. CONCLUSION: The type of suture material used did not significantly affect the proliferative rate in this animal model. PMID- 7555415 TI - Toxic megacolon complicating pseudomembranous enterocolitis. AB - PURPOSE: Toxic megacolon is a rare complication of pseudomembranous enterocolitis. We reviewed our recent experience with this complication. METHODS: The first five patients of the series were studied retrospectively, and six others were followed prospectively. RESULTS: Between June 1992 and May 1994, 11 patients (8 male, 3 female) developed toxic megacolon secondary to pseudomembranous enterocolitis. Mean age was 60.7 +/- 11.8 (range, 40-79) years. Presenting symptoms and signs included diarrhea, 100 percent; malaise, 91 percent; abdominal pain, 82 percent; abdominal distention, 82 percent; abdominal tenderness, 72 percent; anemia less than 12 gm, 72 percent; albumin less than 3 gm, 64 percent; tachycardia greater than 100, 55 percent; fever greater than 38.5 degrees celsius, 45 percent; shock or hypotension, 45 percent. Predisposing factors included antibiotics, 64 percent; immunosuppressants or chemotherapy, 36 percent; antidiarrheals, 27 percent; and barium enema in one patient. Five patients (45 percent) had more than one predisposing factor. X-rays showed transverse colon dilation and loss of haustrations in eight patients (72 percent), with a mean diameter of 9.9 +/- 3.4 cm. Flexible proctosigmoidoscopy showed pseudomembranes in all scoped patients, and toxin assay for Clostridium difficile was positive in all patients. One patient had emergency surgery. Ten patients were initially treated medically with nasogastric suction and intravenous resuscitation (90 percent) and antibiotics (100 percent), usually in the intensive care unit (80 percent). Four patients did not respond and underwent surgery; two others improved, then deteriorated, and also underwent surgery. Altogether, 7 of 11 patients (64 percent) underwent surgery. Three patients (27 percent) responded well to medical treatment. One patient was deemed too ill to undergo surgery and died. Mean delay to surgery was 3.0 +/- 1.3 days. No sealed or overt perforation was found at laparotomy. All patients who underwent surgery had a subtotal colectomy, with either a Hartmann's stump (71 percent) or a mucous fistula (29 percent). Eventually, five of seven patients who were operated on and two of four medically treated patients died (overall mortality, 64 percent). Only one patient underwent closure of ileostomy and anastomosis. CONCLUSION: Toxic megacolon complicating pseudomembranous enterocolitis is a serious problem that carries a high morbidity and mortality rate, regardless of treatment. PMID- 7555416 TI - Effect of rectosigmoid stump length on restorative proctocolectomy after subtotal colectomy. AB - BACKGROUND: The length of the rectosigmoid stump left after subtotal colectomy and ileostomy is believed to affect postoperative complications, including sepsis, success of future restorative proctocolectomy, and long-term functional outcome. METHODS: We reviewed the charts of 60 patients with toxic ulcerative colitis who were treated with subtotal colectomy leaving either a short (25) or long (35) rectosigmoid stump and who eventually underwent restorative proctocolectomy between 1983 and 1992 at a large tertiary care center. Data were collected on preoperative disease duration and steroid use, operative time, blood loss, transfusion requirements, length of stay, stool frequency, fecal incontinence, and sexual dysfunction. RESULTS: There were no statistically or clinically significant differences between groups. CONCLUSIONS: Rectosigmoid stump length does not appear to affect complications or long-term outcome in patients with toxic ulcerative colitis treated with subtotal colectomy and restorative proctocolectomy. PMID- 7555417 TI - Gradient of pressure and time between proximal anal canal and high-pressure zone during internal anal sphincter relaxation. Its role in the fecal continence mechanism. AB - PURPOSE: The normal response to rectal distention is a relaxation of the proximal anal canal (PAC). We hypothesized that this mechanism would require a gradient of pressure and time to preserve continence. METHODS: Sixteen volunteers (10 male), mean age, 41.5 (range, 24-60) years, were studied using an eight port axial catheter with a compliant balloon at its tip. Relaxation was induced by a small volume of rectal distention (15-30 ml of air) and was recorded until recovery of resting anal pressure (RAP). Duration of relaxation was measured until recovery of RAP. Amplitude of relaxation was determined between RAP before rectal distention (RAP-BR) and pressure at the point of maximum relaxation (RAP-PMR). Gradient of pressure was determined by comparing RAP-PMR in the high-pressure zone (HPZ) and PAC. Contraction in the distal anal canal was interpreted as external anal sphincter contraction (EASC) and was compared with RAP-PMR in the HPZ. RESULTS: Relaxation was significantly greater in PAC than in HPZ (50 vs. 36 percent; P = 0.001). RAP-PMR was significantly higher in HPZ than in PAC (30.7 vs. 12.6 mmHg; P = 0.001). EASC was observed in six patients and did not show significant difference with RAP-PMR in HPZ (39.7 vs. 36.3 mmHg; not significant). Relaxation began at the same time in all levels but lasted significantly longer in PAC compared with HPZ (13.5 vs. 9.4 sec; P = 0.003). CONCLUSION: Anal relaxation induced by small volume rectal distention involves a gradient in the pressure and time of relaxation between PAC and the HPZ. PMID- 7555418 TI - Ultrasonographic examination for lateral lymphatic spread and local recurrence of rectal cancer. Preoperative detection and evaluation. AB - PURPOSE: A preoperative evaluation of the lateral lymphatic spread is essential to decide the indication of autonomic nerve preserving operation for rectal cancer. For this evaluation, we used common ultrasonographic examination of the lower abdomen. We also performed this examination on postoperative patients in whom local recurrence had been suspected. Results and effectiveness of examination were evaluated. METHOD: First, we identified arteries in the pelvic cavity (common, external, and internal iliac arteries), and then we sought lymph nodes in the spaces between these arteries, urinary bladder, and rectum. RESULTS: We performed this examination on 40 preoperative patients with rectal cancer and could detect lymph nodes in 12 patients. By the size and features of these lymph nodes, five patients were considered lateral lymphatic spread-positive. After the operation, resected specimens revealed three patients were pathologically positive, and the two others were false-positive. Only one patient was false negative. Total accuracy of the examination was 92.5 percent; however, the positive lymph node around middle rectal artery and obturator artery had been overlooked. We also performed this examination on postoperative patients in whom local recurrence had been suspected. In one postoperative patient, a recurrent lesion of 3.0 x 3.5 cm in size was detected along the right external iliac artery and could be resected by the retroperitoneal surgical approach. CONCLUSION: We consider this examination very effective for detecting lymph node metastasis preoperatively and recurrent lesions in the pelvic cavity. PMID- 7555419 TI - Impact of family history of colon cancer on development of multiple primaries after diagnosis of colon cancer. AB - PURPOSE: The purpose of this study was to assess risk of developing multiple primaries after a diagnosis of colon cancer and to determine the impact that having a family history of cancer has on cancer risk. METHODS: Data from the Utah Cancer Registry and the Utah Population Database were used. A cohort of 2,236 first primary colon cancers were observed for the subsequent development of additional primary cancers. RESULTS: We observed a greater than expected incidence of colon, rectal, and pancreatic cancers among the cohort. The standardized incidence ratios were 2.77 (95 percent confidence interval (CI), 2.07-3.70), 2.26 (95 percent CI, 1.34-3.81), and 2.38 (95 percent CI, 1.32-4.30), respectively. Having a family history of colon or rectal cancer did not greatly influence risk of having a multiple primary. However, there was a trend toward increased risk of pancreatic cancer (hazard rate ratios, 1.99; 95 percent CI, 0.67-5.90) and bladder cancer (hazard rate ratios, 2.35; 95 percent CI, 0.77 7.18) among patients with a family history of rectal cancer. We also observed that risk of uterine cancer in the cohort was positively associated with family history of uterine cancer, risk of breast cancer was positively associated with family history of breast cancer, and risk of prostate cancer was positively associated with family history of prostate cancer. CONCLUSIONS: People with colon cancer are at a greater risk of developing colon, rectal, and possibly pancreatic cancer. Although a family history of colon or rectal cancer did not have a large impact on developing other cancers, a family history of other primary cancers did influence risk of other cancers. PMID- 7555420 TI - Inducibility of endogenous tumor necrosis factor by tumor cells from colorectal tumor patients at Dukes stage C as a novel prognostic factor following curative operation. AB - PURPOSE: It is well known that tumor cells secrete endogenous tumor necrosis factor (en-TNF) as a cytokine when stimulated with lipopolysaccharide (LPS). We, therefore, analyzed the biologic role of en-TNF secreted by tumor cells themselves to learn the prognosis of patients and the susceptibility of tumors to biologic response modifiers in particular. METHODS: Patients with Dukes C colorectal tumors were studied after curative operation to determine the inducibility of en-TNF by their primary cultured cells in response to LPS. RESULTS: Irrespective of the known clinicopathologic factors of the tumors, en TNF was produced in 21 of a total of 44 patients. The group of patients with clear en-TNF production showed a significantly lower incidence of recurrence and/or metastasis and a higher survival rate than the group without en-TNF production, suggesting a strong correlation between patient prognosis and inducibility of en-TNF. CONCLUSION: Inducibility of en-TNF by tumor cells themselves can be a novel prognostic factor for patients with colorectal tumor, especially of Dukes Stage C. PMID- 7555421 TI - Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate. AB - PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked to N-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing the in vitro and in vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. The in vitro toxicity was highly specific because the conjugate (MAAC) inhibited de novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 microgram/ml and 0.06 microgram/ml, respectively. Colon survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis. PMID- 7555423 TI - Internal rectal intussusception--fact or fancy? AB - PURPOSE: There is still considerable debate whether internal intussusception represents a functional disorder. We have reviewed our results in an effort to define its symptomatology and to assess defecography. METHODS: Rectopexy has been performed for internal intussusception in 37 patients. Eighteen had solitary rectal ulcer syndrome (SRUS), and 31 had anterior rectal wall prolapse. Defecography demonstrated anterior wall prolapse in 13, circular prolapse in 21, and no disorders in 3 patients. Pelvic floor function was normal. Follow-up varied from one to nine years. RESULTS: Twenty-six patients became asymptomatic. Anterior wall prolapses could not be palpated anymore. All SRUS lesions healed. Patients with SRUS (P < 0.001) or circular prolapse (P < 0.001) became significantly more asymptomatic. Results in patients with anterior rectal wall prolapse were significantly worse (P < 0.001). CONCLUSIONS: Internal intussusception is a distinct functional rectal disorder. Its symptomatology and findings during physical examination are aspecific. Characteristic defecographic features and presence of SRUS are indications for surgery, provided pelvic floor function during straining is normal. PMID- 7555422 TI - Flat adenomas and flat adenocarcinomas of the colorectal mucosa in Japanese and Swedish patients. Comparative histologic study. AB - PURPOSE: In recent years, flat adenomas of the colorectal mucosa have been intensively investigated by Japanese pathologists. Results of that work indicate that flat adenomas may antedate the development of colorectal carcinomas. Because of differences in the histologic definition of flat adenomas with severe dysplasia and with intramucosal carcinoma within the group, one single observer having both Western and Asian training in pathology reviewed the material. METHODS: A total of 287 flat colorectal lesions were reviewed: 109 from the Karolinska Hospital, Stockholm, 137 from the Tokyo Medical and Dental University (TMDU) (which included 5 cases from the Nagoya City University), and 41 from the Cancer Institute (CI), Tokyo. Lesions were histologically classified following strict histologic criteria. Thus, flat adenomas were divided into those having low-grade dysplasia (LGD; having dysplastic cells in the deeper half of the epithelium), high-grade dysplasia (HGD; dysplastic cells were found even in the superficial half of the epithelium), intramucosal carcinoma (dysplastic glands displayed molding with buddings and often a cribriform pattern), and adenocarcinoma (braking through the muscularis mucosa, with neoplastic cells in the submucosal layer or deeper). RESULTS: Whereas in Stockholm only 14.7 percent of lesions had HGD, as much as 56.9 percent and 56.1 percent, respectively, had HGD at the two Tokyo Hospitals. Intramucosal carcinomas were not found in the Stockholm material but occurred in 2.2 percent of lesions seen at TMDU and in 4.9 percent of those seen at the CI. Notably, only 2.7 percent of the specimens at Karolinska Hospital had invasive adenocarcinoma, but it was seen in as many as 4.4 percent at TMDU and 21.9 percent at the CI. CONCLUSIONS: This study indicates that there were histologic differences between flat neoplasias of the colorectal mucosa harvested in Stockholm and Tokyo. In Japan lesions were obviously more advanced (in terms of HGD) and more aggressive (in terms of intramucosal and submucosal invasion). The cause for the differences found in those two disparate geographic regions remains poorly understood. The results, however, may help us understand some of the unclear points and discussions appearing in the literature on this subject. PMID- 7555424 TI - Surgical management of ileosigmoid fistulas in Crohn's disease: role of preoperative colonoscopy. AB - PURPOSE: Surgical treatment of ileosigmoid fistulas in Crohn's disease remains controversial and can be radical (resection of both segments) or conservative (ileal resection with suture or wedge resection of the sigmoid). At our institution, the sigmoid defect is sutured if the sigmoid is not affected by primary Crohn's disease or by important stricture; otherwise, the sigmoid is resected. We reviewed our experience to evaluate our results with this procedure. METHODS: Thirty patients with ileosigmoid fistulas underwent operation. Among them, 15 had a preoperative colonoscopy, whereas others had no endoscopic work up. In nine patients, the sigmoid was thought to be affected by Crohn's disease (n = 7) or stricture (n = 2) and was resected. In 21 patients, the sigmoid was thought to be affected by proximity, and a simple suture (n = 15) or wedge resection (n = 6) was performed. Eleven patients had a temporary stoma (37 percent). One had coloprotectomy. RESULTS: One patient died postoperatively. One patient had postoperative sigmoidocutaneous fistula after conservative treatment. Histology of the sigmoid specimen showed Crohn's disease in 8 patients (27 percent), including 5 of 9 resected specimens, and 3 of 21 conservative procedures. All patients with Crohn's misdiagnosis did not have preoperative colonoscopy. Nine of 11 stomas were closed in a median delay of four months. With a median delay of nine years, four patients have again undergone surgery for recurrent colonic Crohn's disease, all of whom underwent surgery initially without preoperative colonoscopy. CONCLUSION: Preoperative endoscopic assessment of the colon is a reliable guide to use when choosing between sigmoid resection or a conservative approach and can result in reduced morbidity and improved long term results. PMID- 7555425 TI - Whole abdominal radiotherapy and concomitant 5-fluorouracil as adjuvant therapy in advanced colon cancer. AB - PURPOSE: This analysis was undertaken to assess whole abdomen radiation therapy and concurrent 5-fluorouracil for toxicity and patterns of failure in high-risk colon cancer patients after curative surgical resection. METHODS: Eighteen patients were treated adjuvantly after curative resection. Four patients (22 percent) had Stage B and 14 (78 percent) had Stage C disease. Histology was poorly differentiated in 4 (22 percent) and moderately differentiated in 14 (78 percent) patients. Four patients received whole abdominal radiation only, 30 Gy at 1 Gy/day. Fourteen patients had an additional locoregional boost of 9.6 to 16 Gy at 1.6 Gy/day. The liver received 19.8 Gy at 0.67 Gy/day. 5-Fluorouracil was given as a continuous infusion during therapy. RESULTS: With a median follow-up of three years, 6 of 18 (33 percent) patients have relapsed. Failure occurred locally in 3 of 18 (17 percent) and distantly in 4 of 18 patients (22 percent). Four of six (67 percent) failures occurred in the liver. The five-year actuarial survival and disease-free survival were 78 percent and 66 percent, respectively. Median elapsed time on radiotherapy was 73 days, with 5 of 18 patients (28 percent) requiring two or more weeks of unplanned treatment breaks. Acute Grade 3 to 4 toxicity (diarrhea, leukopenia) occurred in 3 of 18 patients (17 percent), with late complications (bowel obstruction) occurring in 2 of 18 patients (11 percent). CONCLUSIONS: Whole abdominal radiotherapy with concomitant 5 fluorouracil appears to improve local control but not to prevent liver metastases. Significant toxicity resulted in frequent interruption of therapy and protracted its course. Whether this adjuvant regimen impacts on survival or offers an advantage over locoregional irradiation remains to be studied. PMID- 7555426 TI - Endorectal ultrasonic detection of malignancy within rectal villous lesions. AB - PURPOSE: The ability of endorectal ultrasonography (EU) to detect the presence of a malignant focus within rectal villous adenomas was studied. METHODS: Clinical charts were reviewed of 62 consecutive patients undergoing EU of rectal villous adenomas, in whom histologic confirmation was available. RESULTS: Twelve lesions were found to contain cancer, of which only two demonstrated clinical signs of induration. Positive predictive value of EU for detecting a malignant focus was 66.7 percent, negative predictive value was 88.7 percent, sensitivity was 50 percent, and specificity was 94 percent. There was moderate overall agreement between pathologic and ultrasound staging (kappa statistic, 0.48). When an optimal image was obtained, all cancers that penetrated the submucosa were detected. Sensitivity of the technique was compromised in some large exophytic lesions and those at the level of the anal sphincter because of artefacts produced in the ultrasonographic image. CONCLUSION: A clear EU image can detect a malignant focus within a villous adenoma and direct the surgeon to the appropriate plane of surgical resection. In lesions with an ambiguous image, a malignancy cannot be excluded. PMID- 7555427 TI - Colonic subepithelial collagenous thickening in diabetic patients. AB - PURPOSE: This study was designed to investigate the effect of intestinal subepithelial collagenous thickening on diabetic diarrhea because one of the seven patients diagnosed with collagenous colitis was diabetic. METHODS: Rectosigmoidoscopic rectal biopsies were taken from 50 diabetic patients (8 with and 42 without diarrhea), 20 nondiabetic patients with diarrhea, and 10 healthy patients. Histopathologic examinations and measurements of subepithelial collagen layers were performed on these biopsies. RESULTS: In diabetic patients who had diarrhea, the subepithelial collagen layer (SCL) was thicker than it was in diabetics without diarrhea (P < 0.05). In diabetic groups, the SCL was thicker than it was in both nondiabetics with diarrhea and those without diarrhea (P < 0.05). There was no statistical difference between nondiabetics with diarrhea and those without (P > 0.05). There was no correlation between collagen thickness, age, and diabetes duration (P > 0.05). CONCLUSIONS: It was concluded that there was a thickening of the colonic SCL in diabetic patients. PMID- 7555428 TI - Current therapy for recurrent and extensive anal warts. AB - PURPOSE: This study was undertaken to review the literature regarding the current therapy for recurrent and extensive anal warts. METHODS: The available treatments for condyloma acuminatum are reviewed with particular regard to their efficacy for recurrent or extensive anal lesions. Topical agents, surgical methods, and the use of interferon are discussed. Treatment of anal warts in the immunocompromised patient is also addressed. CONCLUSIONS: Although small lesions may be responsive to repeated applications of topical agents, more extensive lesions require surgical or combination treatment. Intralesional interferon may be a useful adjunct to surgical methods to decrease recurrence. PMID- 7555430 TI - Cancer recurrence following laparoscopic colectomy. Report of two patients treated with heated intraperitoneal chemotherapy. AB - PURPOSE: Use of laparoscopic techniques for resection of colon and rectal cancer has raised considerable controversy. There is increasing concern that wound recurrence and peritoneal dissemination may represent a potentially fatal complication of this technique. METHODS: The surgical literature was reviewed, and clinical course of two patients is presented. RESULTS: Our two patients had tumor recurrence in the laparoscopy port sites within one year after laparoscopic assisted colectomy for Dukes B adenocarcinoma of the colon. At laparotomy, diffuse peritoneal carcinomatosis without lymph node or liver metastases were found in both patients. They were treated by surgical resection of recurrent disease combined with heated intraoperative intraperitoneal mitomycin C chemotherapy and five days of early postoperative intraperitoneal 5-fluorouracil. These patients are clinically free of disease at 1.5 years after treatment of peritoneal implants. CONCLUSIONS: Cancer recurrence in abdominal wall incisions after laparoscopic colectomy has been reported in an increasing number of patients. It is possible that this technique should be abandoned. Cytoreductive surgery combined with intraperitoneal chemotherapy may represent the most adequate treatment of recurrent cancer that occurs following laparoscopic colectomy. PMID- 7555429 TI - New intraluminal bypass tube for management of acutely obstructed left colon. AB - PURPOSE: A surgical technique is described for a new intracolonic bypass procedure in patients who underwent colonic surgery with an unprepared colon. METHODS: Resection and primary anastomosis was performed. The intraluminal bypass tube used was a latex condom. RESULTS: No clinical anastomotic leakage was noted. CONCLUSIONS: This is a safe, low cost, and uncomplicated procedure that decreases the risk of dehiscence and permits the performance of a high number of primary anastomosis. PMID- 7555432 TI - Laparoscopic colorectal surgery. PMID- 7555431 TI - Femoral neuropathy secondary to the use of a self-retaining retractor. Report of three cases and review of the literature. AB - PURPOSE: Three recent cases of femoral neuropathy at our institution following colorectal surgery have been ascribed to the use of the self-retaining Bookwalter retractor. The pathophysiology of neural injury includes compression, stretch, transection, ligation, iliopsoas hematoma, ischemia, and cement encapsulation. The aim of this study is to provide a comprehensive review of femoral nerve anatomy and mechanism of retractor injury. METHODS: The relationship of the femoral nerve to the lateral blade of the Bookwalter retractor was evaluated during colorectal surgery and in cadaveric dissections. RESULTS: The lateral blade of the self-retaining retractor was observed to either compress or impinge the intrapelvic portion of the femoral nerve. CONCLUSION: The incidence of postoperative femoral neuropathy is likely underestimated because a majority of cases are self-limited. This debilitating iatrogenic injury can be prevented with a thorough understanding of femoral nerve anatomy and careful placement of self retaining retractor blades. PMID- 7555434 TI - NO chi generation by cultured small intestinal epithelial cells. AB - The effect of cytokines, growth factors, mitogens, and bacterial products on nitric oxide (NO) generation by monolayers of small intestinal epithelial cells-6 (IEC-6) cells was evaluated. Subconfluent IEC-6 cells were maintained in DMEM containing 5% fetal calf serum and after 16-24 hr of incubation, the medium was replaced with fresh medium in the presence or absence of calcium ionophore (CaI), L-NAME, L-NNA, individual growth factors, cytokines, or mitogens. After 72 hr of culture, the media supernatant was collected and NO chi generation was determined. NO synthase activity was determined in sonicated supernatants of IEC 6 cells by [14C] arginine conversion to citrulline. NO chi generation in subconfluent cultures was greater than in fully confluent cultures, suggesting contact inhibition. NO chi generation by IEC-6 cells was significantly increased by CaI and inhibited by L-NAME and L-NNA. LPS, IL-1 beta, IL-2, IL-8, IFN-8, TFN alpha, EGF, TGF-alpha, bFGF, and PHA significantly increased NO chi generation. NO synthase activity in IEC-6 cells (4.2 +/- 1.7 pmol/min/10(6) cells) was NADPH dependent. These results suggest that stimulation of NO chi generation by intestinal epithelial cells through cytokine bacterial products and mitogens may be one of the mechanisms responsible for their effects in the intestinal tract. PMID- 7555436 TI - Validation of a new measure of diarrhea. AB - Adequate measures of diarrheal disease are important to assess severity for clinical use and outcomes research. We developed a questionnaire to assess diarrhea severity and complications, and administered it to 205 HIV positive patients with diarrhea, fever, or weight loss. Noteworthy variations in stool form were reported by individuals and across subjects. Self-reported diarrhea correlated with the occurrence of any stool pictured without form. However, verbal descriptors "loose" and "semiformed" had little value in assessment of diarrheal disease. Both verbal and pictorial stool descriptors correlated well with diarrhea complications (pain, urgency, tenesmus, incontinence, and nocturnal diarrhea). By factor analysis, discomfort and nondiscomfort diarrhea complications loaded on different factors, consistent with clinical experience that discomfort is a distinct problem in diarrheal disease. In summary we have developed an instrument to precisely characterize diarrhea severity that correlates well with clinically important events such as incontinence and abdominal pain. PMID- 7555433 TI - The scientific growth of gastroenterology during the 20th century. The 1994 G. Brohee Lecture. AB - Energized by the growth of the basic sciences during the latter half of the 20th century, gastroenterology advanced from a modest clinical activity to an increasingly scientific discipline. The decisive change followed World War II, when the Office of Scientific Research and Development transferred university and industry wartime research contracts to the National Institutes of Health (NIH), followed by establishment of the National Science Foundation and the General Medicine Study Section (NIH). Other factors contributing to the progress of gastroenterology included: (1) the increasing body of scientific knowledge; (2) innovative technological advances; (3) philanthropic, pharmaceutical, and governmental (NIH) support of research; (4) emphasis on controlled clinical and laboratory studies; and (5) the enlarging global scientific communication network. Selected highlights on the evolving knowledge of gastrointestinal hormones and the discovery of cholecystography illustrate some of the pathways of gastroenterology's 20th century advance. PMID- 7555435 TI - Perinuclear anti-neutrophil cytoplasmic antibody and refractory pouchitis. A case control study. AB - Refractory pouchitis (RP) is a debilitating complication of ileal pouch reservoirs that affects approximately 2.5% of patients. Although the cause of RP is unknown, it is frequently hypothesized that it reflects underlying Crohn's disease. Since perinuclear anti-neutrophil cytoplasmic antibody (pANCA) is found in approximately 70% of ulcerative colitis patients but only rarely in Crohn's disease patients, it may help distinguish Crohn's disease from ulcerative colitis. Therefore, to test whether RP reflects "missed" Crohn's disease, we determined the ANCA status of 26 patients with RP. The pANCA was positive in 42% of cases [50% of Kock pouch cases and 33% of ileoanal pull-through (IAPT) cases] and 57% of matched control subjects without pouchitis (N = 42, P = NS). Moreover, 3/6 (50%) of IAPT RP subjects whose signs and symptoms most suggested Crohn's disease tested positive for pANCA. When compared to controls, IAPT cases exhibited significantly more preoperative extraintestinal manifestations (EIMs) of inflammatory bowel disease (P < 0.05). The presence of preoperative EIMs was 100% predictive of postoperative EIMs (P < 0.05). Review of pouch biopsies from cases of RP revealed no pathognomonic histologic features of Crohn's disease. These data confirm our previous suggestion that RP does not reflect underlying Crohn's disease but may be associated with the EIMs of inflammatory bowel disease. PMID- 7555438 TI - Management of Syphacia muris infestation in rat colonies. PMID- 7555437 TI - Prothrombotic state and signs of endothelial lesion in plasma of patients with inflammatory bowel disease. AB - Recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in inflammatory bowel disease (IBD) pathogenesis. To evaluate the implication of the hemostatic system during these thrombotic events, we analyzed plasmatic values of prothrombotic state markers, physiologic inhibitors of coagulation, and endothelial lesion markers in 112 IBD patients. We found an increase in thrombin-antithrombin complexes and a decrease in antithrombin III, probably due to consumption, demonstrating an increase in thrombin generation. High levels of D-dimer reflect increased fibrin formation, but there is no correlation between thrombin generation markers and D-dimer, possibly suggesting the presence of inadequate fibrinolysis. Levels of tissue factor pathway inhibitor were higher in patients than in controls. Nine patients with Crohn's disease (35% of our sample) had levels of this marker under 70% (range 37-69%). Von Willebrand factor values were increased and those of thrombomodulin only in active patients. Most of the changes were detected in patients with inflammatory activity, and there were no differences between ulcerative colitis and Crohn's disease. In conclusion, these results support the hypothesis that there is an endothelial lesion with sustained coagulation activation in IBD patients. PMID- 7555439 TI - Effect of octreotide and erythromycin on idiopathic and scleroderma-associated intestinal pseudoobstruction. AB - Treatment of chronic intestinal pseudoobstruction with prokinetic agents has been disappointing. Our study was designed to determine if octreotide and erythromycin would provide sustained relief from nausea, abdominal pain, and bloating in pseudoobstruction. Using gastrointestinal manometry, quantitative parameters of the activity front of the migrating motor complex at baseline and after prokinetic therapy with erythromycin and octreotide were determined in 14 patients with intestinal pseudoobstruction who had nausea, abdominal pain, and bloating. Patients were treated with erythromycin and octreotide for 20-33 weeks. Octreotide increased the frequency, duration, and motility index of activity fronts (AFs) from 1.2 +/- 0.3 AFs/4 hr, 2.7 +/- 0.7 min, and 85 +/- 23 min mm Hg to 4.1 +/- 0.8 AFs/4 hr, 5.5 +/- 0.7 min, and 152 +/- 24 min mm Hg, respectively (P < 0.05). Antral activity was decreased from 63 +/- 14 to 23 +/- 8% by octreotide (P < 0.05). Erythromycin induced antral activity; however, small intestinal motor activity was suppressed. While on erythromycin and octreotide, five patients had long-term improvement of nausea and abdominal pain. All responders had at least 5 AFs/4 hr induced by octreotide. We conclude that octreotide and erythromycin relieve abdominal pain and nausea in pseudoobstruction. Patients who have at least 5 AFs/4 hr after octreotide administration are most likely to clinically respond. PMID- 7555440 TI - IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys. AB - Since celiac disease screening by traditional IgA anti-endomysial antibody test is limited by high costs of monkey esophagus commercial kits as well as by rising ethical problems related to the endangered species, the identification of an inexpensive and commonly available substrate for this antibody determination is urgently required. To achieve this goal, we compared the prevalence of IgA anti endomysial antibodies detected on monkey esophagus with that on human umbilical cord. Fifty-seven (95%) of 60 untreated adult celiacs were positive for these antibodies on monkey esophagus as well as on human umbilical cord. IgA anti endomysial antibodies, detected on both tissues, were negative in all 200 disease and healthy controls tested, displaying a 100% specificity for gluten-sensitive enteropathy. These data suggest that human umbilical cord can replace monkey esophagus for IgA anti-endomysial antibodies test. Human umbilical cord allows unlimited testing for celiac disease screening on wide series of high-risk subjects, permitting identification of greater numbers of asymptomatic celiac patients with a remarkable saving of money and bypassing the ethical problems related to killing monkeys. PMID- 7555441 TI - Adult celiac disease is frequently associated with sacroiliitis. AB - No data are available on the presence and frequency of peripheral or central joint disease, routinely determined by bone scintigraphy with 740 MBq of [99mTc]MDP, in adult celiac disease. Bone scintigraphy was carried out to detect early acute inflammatory lesions in 22 adult celiac patients (15 females and seven males; mean age 36.72 years, range 17-63). Bone scintigraphy was positive for sacroiliitis in 14 cases (63.6%). Except in the case of one patient suffering from rheumatoid arthritis, laboratory data were normal. Our data suggest that as in other chronic intestinal diseases, celiac disease in adults, is frequently associated with central joint disease. This high incidence of sacroiliitis, the joint disease most frequently found in our patients, has not been previously reported in other series. We believe, therefore, this difference could be explained by the different methodology used for the screening of joint disease. PMID- 7555442 TI - Tuberculous mesenteric lymphadenitis presenting as pyloric stenosis. AB - A 17-year-old Filipina with a three-year history of intermittent, projectile vomiting and weight loss was admitted. A diagnosis of peptic ulcer disease was made, but she was unresponsive to antiulcer therapy. Fever, anorexia, cough, and exposure to tuberculosis were denied. Chest x-ray was normal. On barium swallow, the stomach and duodenal bulb were dilated. Endoscopic antral biopsy showed chronic inflammation. Computed tomography revealed enlarged periportal and peripancreatic lymph nodes and an intrahepatic mass. Liver biopsy failed to show any acid-fast bacilli. On laparotomy, the pyloroduodenal area was extrinsically compressed by surrounding lymph nodes, which, on biopsy, contained granulomatous inflammation with caseation necrosis and Langhan's giant cells. Gastrojejunostomy was done and antituberculous drugs were given. Pyloric stenosis due to tuberculosis is rare, but it should be considered in patients who come from areas where the disease is endemic. Medical management for such cases may suffice. PMID- 7555443 TI - Influence of bradykinin in gastrointestinal disorders and visceral pain induced by acute or chronic inflammation in rats. AB - This work investigated the role of bradykinin in viscerosensitivity before and during inflammation in two models of visceral pain induced by rectal distension (RD) or "abdominal distension" (AD) in rats. RD induced both inhibition of colonic motility and an increase of abdominal spike bursts. Bradykinin receptor antagonist, Hoe 140 did not affect any of the RD-induced responses. After TNB induced rectal inflammation, colonic inhibition and the number of abdominal contractions were enhanced. Hoe 140 selectively reduced the abdominal response to the highest distension volume, without affecting the colonic response. In AD group, acetic acid inhibited gastric emptying and increased the number of abdominal contractions, whereas the same volume of saline did not affect any of the responses. Before inflammation, Hoe 140 (1-5 mg/kg, intraperitoneally) did not affect per se abdominal and gastric emptying responses; in contrast, at 5 mg/kg, intraperitoneally, it reduced significantly (P < 0.05) both acetic acid induced responses. We conclude that bradykinin is involved in viscerosensitivity changes related to abdominal and rectal distension in inflammatory conditions. PMID- 7555444 TI - Recurrent pseudo-Crohn's disease. PMID- 7555445 TI - Graft-versus-host disease after small bowel transplantation is associated with host colonic injury. AB - The present studies were undertaken to evaluate the histologic effects of graft versus-host disease on the host colon after small bowel transplantation. Graft versus-host disease was produced in six Lewis x Brown Norway F1 rats by performing vascularized, out-of-continuity small bowel transplants from parental Lewis donors. Host proximal and distal colon were sampled 14 days after operation when signs of graft-versus-host disease, including weight loss and splenomegaly, were present. Tissue was assessed histologically by blinded observer and compared to eight sham-operated controls. Three histologic features were noted to be statistically increased in diseased animals: (1) mucin loss; (2) crypt abscesses; and (3) large lymphoid aggregates in the mucosa and submucosa. These features were more commonly noted in the distal rather than the proximal colon. Another group of five grafted animals treated with cyclosporine A (10 mg/kg/day intramuscularly) still lost weight but did not display overt signs of graft versus-host disease and had normal-sized spleens. There was normal mucin content and no evidence of crypt abscesses in these treated animals, although large lymphoid aggregates were present. It is concluded that mucin loss, crypt abscesses, and large lymphoid aggregates are characteristics of graft-versus-host disease-induced colonic injury in this model and that these changes are most evident in the distal colon. Cyclosporine A therapy does not completely reverse the histological changes of colonic graft-versus-host disease. This model may be useful in studying the mechanisms by which immune mediated colitides preferentially affect the distal colon. PMID- 7555446 TI - FK-506 and cyclosporine A (CsA) immunomodulation of the human gut mucosal immune system. AB - FK-506 and cyclosporine A (CsA) are two immunosuppressive drugs used in the treatment of patients after liver and small intestine transplantation. A clinical advantage of FK-506 over CsA has been observed in these patients. Although the immunomodulation of both drugs has been well documented in the circulatory immune system, their effect on the mucosal immune system is not well established. In this study, the effect of FK-506 on the human gut mucosal immune system was compared to CsA. Proliferation of human colonic lamina propria lymphocytes (LPL) was measured by DNA synthesis and ornithine decarboxylase (ODC) activity. Results show that FK-506 and CsA suppress LPL DNA proliferation in a dose-dependent manner. FK-506 had a stronger antiproliferative effect compared to CsA. Moreover, the antiproliferative effect of both drugs was not dependent on monocytes or monocyte-associated factors (IL-1 beta, IL-6). In addition, exogenous addition of IL-2 did not restore the suppressive effect of either drug on LPL DNA synthesis. We conclude that: (1) both drugs have an antiproliferative effect on the human mucosal immune system; and (2) the stronger effect of FK-506 on human LPL compared to CsA may explain its superior clinical response observed in patients after liver/small intestine transplantation. PMID- 7555447 TI - Hemodynamic effects of hypothyroidism induced by methimazole in normal and portal hypertensive rats. AB - Portal hypertension is accompanied by a hyperdynamic circulatory state that shares some similarities with thyrotoxicosis. This study was conducted in order to investigate the hemodynamic effects of hypothyroidism in a rat model with portal hypertension induced by partial portal vein ligation (PVL). Four groups of 10 rats each were studied: normal control and hypothyroid rats, and PVL control and hypothyroid rats. Hypothyroidism was induced by methimazole 0.04% in drinking water. Hemodynamic measurements were performed using the radioactive microsphere technique. Induction of hypothyroidism was confirmed by elevated TSH levels. In the PVL groups, hypothyroidism ameliorated the hyperdynamic circulation. Portal venous inflow and portal pressure dropped significantly: 7.1 +/- 0.2 vs 4.8 +/- 0.3 ml/min/100 g body wt (P < 0.01) and 13.4 +/- 0.9 vs 10.9 +/- 0.8 mm Hg; (P < 0.01), respectively. In normal rats, hypothyroidism was manifested by a hypodynamic circulatory state. These results demonstrate that hypothyroidism induced by methimazole is followed by amelioration of the hyperdynamic circulation, normalization of portal venous inflow, and reduction of portal pressure. PMID- 7555448 TI - Temporary use of peritoneovenous shunting for treatment of tense ascites following a Whipple procedure. AB - Tense, nonchylous ascites following a Whipple procedure has not been reported to date. We describe the course of such a patient successfully treated by a peritoneovenous shunt. A 49-year-old male developed tense ascites following pancreaticoduodenectomy. Despite conservative measures, abdominal distension developed to the point of dyspnea and orthopnea for over a six-month period. Because the physicochemical characteristic of the ascitic fluid was consistent with that of hepatic lymph, ascites was considered due to injury to the lymphatics around the porta hepatis. A peritoneovenous shunt was established for the treatment of ascites and was removed when the ascites had resolved at nine months after shunting. Our experience suggests that, in case conservative measures fail in the control of ascites, either direct repair of the lymphatic leak by laparotomy or the temporary use of peritoneovenous shunting may be considered. PMID- 7555449 TI - Abnormal serum transaminases following therapeutic doses of acetaminophen in the absence of known risk factors. AB - J.M., a healthy, 25-year-old male, volunteered for a study involving warfarin and acetaminophen. Acetaminophen 1 g four times a day was started for 21 days. Liver function tests taken at regular intervals for the first 12 days were unremarkable. On day 18, however, aspartate aminotransferase (AST) was 527 IU/liter and alanine aminotransferase (ALT) was 166 IU/liter. Acetaminophen was discontinued and serum transaminase levels returned to baseline levels two weeks later (AST = 26, ALT = 20). Analysis of J.M.'s urine samples over the first 18 days showed excretion patterns of glucuronide, sulfate, and glutathione derived cysteine and mercapturic acid conjugates were similar to the other subjects in the study. Acetaminophen causes hepatotoxicity in overdose or malnourished or alcoholic patients, none of which applied to our subject. Differences in metabolic activation and capacity for glutathione synthesis can predispose individuals given therapeutic doses of acetaminophen to adverse effects. Failure to detoxify a highly reactive metabolite, formed by P-450 metabolism, via glutathione conjugation is responsible for the development of acute hepatic necrosis. Accumulation of the toxic metabolite due to depleted glutathione stores may have occurred with prolonged high dosing in our subject and been responsible for his abnormal rise in liver enzymes. PMID- 7555450 TI - Prospective, multicenter study on value of computerized tomography (CT) in gallstone disease in predicting response to bile acid therapy. AB - The aim of the study was to assess the value of quantitative attenuation values (Hounsfield units) and of gallstone pattern by computerized tomography in predicting response to bile acid therapy. We carried out a prospective study in a multicenter setting on 90 consecutive outpatients with radiolucent gallstones. All received bile acid therapy (UDCA 10 mg/kg/day or UDCA + CDCA 5 mg/kg/day of each) up to two years. Hounsfield units for gallstones were recorded using standardized criteria and six categories of patterns were defined: hypodense, isodense, homogenously dense, laminated, rimmed and speckled. We assessed gallstone dissolution rate (percent reduction in volume), response to therapy (> 25% reduction in volume), and final outcome of therapy. Eighty-one percent of patients with hypodense/isodense and all four patients with speckled stone pattern responded to therapy, whereas none of the 10 patients with laminated/rimmed and only 45% of patients with homogenously dense stone pattern did. Complete dissolution was achieved by 68%, 50%, 35%, 0% of the hypodense/isodense, speckled, homogenously dense, rimmed/laminated gallstones, respectively. The use of Hounsfield units did not show an advantage over gallstone pattern for predicting either response or final outcome to bile acid therapy. We conclude that computerized tomography analysis of gallstones is of value in predicting response to bile acid therapy and that gallstone pattern alone predicts response in most cases without the need for quantitative assessment. PMID- 7555452 TI - Endoscopic approach to pancreas divisum. AB - Pancreas divisum has been claimed to be a harmless congenital variant or to occasionally cause acute relapsing pancreatitis (ARP), chronic pancreatitis (CP), or a chronic abdominal pain (CAP) syndrome. Both surgical and endoscopic approaches to accessory papilla decompression have been promulgated and widely disparate results reported in the literature. We retrospectively reviewed a five year experience with dorsal pancreatic duct decompression at our institution utilizing a variety of endotherapeutic techniques. Data collected included procedural complications; patient interpretation of pre- and posttherapy pain, frequency, and intensity graded on an analog pain scale; frequency of hospitalization; and patient perception of "global" improvement to endotherapy. At a mean follow-up of 20 months, there was a statistically significant decrease in pancreatitis incidence in 15 patients with ARP (P = 0.016) and 19 patients with CP (P = 0.025). The frequency and intensity of chronic pain was also significantly improved (P < 0.001) in the latter group. In contrast, only one of five patients with CAP and normal dorsal pancreatography and secretin tests experienced global improvement, and there was no improvement utilizing an analog pain scale (P = 0.262) in the group as a whole. There was a 20% incidence of mild procedure or subsequent stent-related pancreatitis and an 11.5% accessory papilla restenosis rate. It is concluded that a subset of carefully selected patients with pancreas divisum may respond to endotherapy but that long-term follow-up will be required to define its ultimate place in the management of symptomatic patients with this anomaly. PMID- 7555453 TI - Radioactive choline metabolism in guinea pig gallbladder. Is there measurable acetylcholine release? AB - Acetylcholine may be released from gallbladder intrinsic nerves in response to cholecystokinin stimulation. This study characterized metabolites of [14C]choline produced in the gallbladder and released during incubation, with or without cholecystokinin-octapeptide. Radiolabeled [14C]choline was applied to the mucosal or muscle surface of intact guinea pig gallbladders in an organ bath. After radiolabeling, gallbladders were incubated with or without the contractile agonist cholecystokinin-octapeptide. Metabolites of [14C]choline were identified in gallbladder tissue and incubation buffers using HPLC and thin-layer chromatography. The major metabolites of [14C]choline were betaine and phosphocholine. [14C]Phosphocholine was incorporated slowly into [14C]phosphatidylcholine. [14C]Choline was released into buffers during incubation. [14C]Acetylcholine constituted less than 1% of radiolabel in the gallbladder. There was no identifiable [14C]acetylcholine released in buffers. Cholecystokinin-octapeptide did not affect choline metabolism. These studies showed that choline in the gallbladder is metabolized along pathways similar to those in the liver. Gallbladders released mostly choline, rather than acetylcholine, even during hormonally induced contraction. PMID- 7555451 TI - Cholelithiasis in Taiwan. Gallstone characteristics, surgical incidence, bile lipid composition, and role of beta-glucuronidase. AB - The nature and occurrence of gallstones in Taiwan and their etiologic factors might not be the same as in Western countries and warranted a systematic investigation. Gallbladder biles and gallstones were obtained at surgery from 100 and 74 patients, respectively. Common duct bile and stones were either drained through an indwelling common duct T-tube or aspirated through a nasobiliary catheter in 108 patients. Gallstones were analyzed for bilirubin, cholesterol, bile acid, calcium, and residue, and biles for bile acid, cholesterol, phospholipid, bilirubin, and beta-glucuronidase. There were four major kinds of gallstones in Taiwan: cholesterol/mixed stones, high-residue black formed pigment stones, low-residue brown formed pigment stones, and muddy pigment stones. The surgical incidence of all types of stones increased steadily during the past four decades. During the past 15 years the relative frequencies for mixed, formed pigment, and muddy pigment stones had been roughly 40, 40, and 20%, respectively, with a further increase in the mixed stones and a decrease in the muddy pigment stones in recent years. Improvement of nutritional status and living standards might contribute to such changes. Cholesterol content in the common duct and gallbladder biles was higher in the mixed stone group than in other groups. Bacterial beta-glucuronidase activity was detected in 53% of patients with muddy pigment stones. Endogenous beta-glucuronidase activity and concentration were also highest in this group, intermediate in the formed pigment and mixed stone group, and lowest in the control. We concluded that hypercholesterobilia was responsible for increasing incidence of mixed stones during the past two decades, while both bacterial and human beta-glucuronidase might contribute to pigment cholelithiasis. PMID- 7555454 TI - Lysophosphatidylcholine-stimulated protein and glycoprotein production by human gallbladder mucosal cells. AB - It has been demonstrated in experimental cholecystitis in cats produced by lysophosphatidylcholine that the development of inflammation is associated with the exsorption of a large amount of protein into the gallbladder lumen. It was subsequently demonstrated that in feline experimental cholecystitis the protein produced was albumin and that its production was decreased by vesicular transport inhibitors, suggesting an active secretory process. In the present study, the effect of lysophosphatidylcholine on protein production by fresh, isolated human gallbladder mucosal cells was evaluated. Isolated gallbladder mucosal cells were incubated with [14C]leucine for 24 hr in tissue culture medium. The cells readily incorporated the radioactive label into cellular protein, a process inhibited by cycloheximide. Exposure of the cells to lysophosphatidylcholine for 1 hr in buffer solution resulted in loss of intracellular protein into the buffer solution. Exposure of the cells for 1 hr prior to lysophosphatidylcholine administration to vesicular transport inhibitors, colchicine, and cytochalasin B and to 4 degrees C culture conditions failed to alter the lysophosphatidylcholine produced passage of the 14C label extracellularly. SDS-PAGE evaluation of the protein produced demonstrated that human gallbladder mucosal cells continuously produced a 66-kDa protein that was not increased by increasing concentration of lysophosphatidylcholine and a 14-kDa protein that increased with increasing concentrations of lysophosphatidylcholine. Employing Western blotting with specific antibodies, the 66-kDa protein was demonstrated to not be albumin but a 66-kDa glycoprotein, and the 14-kDa protein was demonstrated to contain phospholipase A2. Human gallbladder mucosal cells produced a protein and glycoprotein in response to lysophosphatidylcholine by a mechanism not related to vesicular transport. PMID- 7555455 TI - Alpha 1-antitrypsin deficiency and chronic pancreatitis. AB - Alpha 1-antitrypsin deficiency is a genetic disorder commonly associated with pulmonary and hepatic injury. Low serum levels of this glycoprotein result in an imbalance between circulating protease and protease inhibitors, which is thought to play a role in the development of emphysema. In recent studies, a protease-to protease inhibitor imbalance in patients with alpha 1-antitrypsin deficiency was thought to be a mechanism contributing to the development of chronic pancreatitis. The heterozygous phenotype and low levels of this glycoprotein have been reported to occur more frequently in patients with chronic pancreatitis than in healthy controls. We report a patient with Pi-SS phenotype alpha 1-antitrypsin deficiency and chronic pancreatitis complicated by recurrent pancreatic pseudocysts and chronic abdominal pain. Our case supports the association between chronic pancreatitis and alpha 1-antitrypsin deficiency. Furthermore, this case provides support for the use of pancreatic stent drainage in the management of intractable abdominal pain in patients with chronic pancreatitis and a dominant stricture. PMID- 7555456 TI - Prevalence of fatty liver in Japanese children and relationship to obesity. An epidemiological ultrasonographic survey. AB - The prevalence of fatty liver in children is unknown and its relationship to obesity is poorly defined. The present study of 810 northern Japanese children (4 12 years old) determined the prevalence of fatty liver in the pediatric population and its relationship to obesity. Diagnosis of fatty liver was based on established real-time ultrasonographic criteria. The overall prevalence of fatty liver was 2.6% and was higher for boys (3.4%) than for girls (1.8%), although not statistically significant (P = 0.15). Fatty liver was found in children as young as 6 years of age. There was no significant association between the prevalence of fatty liver and height (physical growth). There was a strong positive correlation between fatty liver prevalence and established obesity indices: Rohrer's Index- chi 2 linear trend = 59.2, P < 0.0001; body mass index--chi 2 linear trend = 91.6, P < 0.0001; and age-gender-adjusted Japanese standard index of weight for height--chi 2 linear trend = 93.2, P < 0.0001. However, direct measurement of abdominal subcutaneous fat thickness by ultrasonography was the best predictor of fatty liver: chi 2 linear trend = 159, P < 0.0001. These results indicate that fatty liver may develop very early in life, and there is a direct relationship between degree of obesity and fatty liver in children. PMID- 7555457 TI - Plasma and tumor prolactin in colorectal cancer patients. AB - Fifteen consecutive patients with recently diagnosed colorectal cancer were studied for plasma and tumor tissue prolactin content. In eight patients (four men and four postmenopausal females), preoperative high plasmatic prolactin was found (mean 1553 nmol; range 516-3677 nmol). In three of them, prolactin was also present in the tumor cells. All plasma prolactin levels returned to normal after tumor resection and remained so during a three-month follow-up. The tumor stage by Duke distribution was similar for both high and normal plasmatic prolactin patients. The role of prolactin in the pathogenesis of colorectal cancer, and as a marker of the tumor, remains to be established. This is the first time that prolactin has been detected in human colon cancer. PMID- 7555458 TI - Healing of gastric body ulcer with gastroprotective versus antisecretory treatment. AB - The aim of this study was to compare the healing effect of a gastroprotective agent and antisecretory drugs in gastric body ulcer where failure of the mucosal defense might be an important factor. Eighty-five patients with benign gastric ulcer were divided into four groups: treated with antacids (I), cimetidine (II), ranitidine (III), and colloidal bismuth subcitrate (De-Nol) (IV). Endoscopically confirmed complete healing was achieved in 57, 61, and 63% in groups I, II, and III, respectively, and in 88% in group IV (P < 0.05). Gastric secretion did not change significantly. Relapses during the next three years occurred several times more frequently in groups I, II, and III than in group IV. Helicobacter pylori was positive in about half the relapsing patients in groups I, II, and III but negative in those of group IV. It is concluded that De-Nol treatment of gastric body ulcer was more efficient than antisecretory drugs both initially and in reducing relapses. PMID- 7555459 TI - Effects of free radical scavengers on indomethacin-induced aggravation of gastric ulcer in rats. AB - Effects of treatment with free radical scavengers in the healing process of acetic acid-induced gastric ulcer on the ulcer aggravation induced by indomethacin were investigated. Gastric ulcers were produced on the anterior wall of the stomach of male Sprague-Dawley rats by submucosal injection of 20% acetic acid. To investigate the role of oxygen radicals, rats with gastric ulcer were treated with scavengers for six weeks and then treated with indomethacin (1 mg/kg/day). While superoxide dismutase (10,000 units/kg/day) did not affect the ulcer area after indomethacin treatment, allopurinol (50 mg/kg/day) slightly inhibited the increase in ulcer area. Dimethyl sulfoxide (1% solution, ad libitum) produced a significant decrease in size of the ulcer after indomethacin treatment. Increased lipid peroxides in the gastric mucosa after indomethacin treatment decreased significantly in the rats of the dimethyl sulfoxide and allopurinol groups. These results indicate that lipid peroxidation mediated by oxygen radicals plays an important role in the mechanism of ulcer aggravation induced by indomethacin. PMID- 7555460 TI - Role of calcium in thromboxane B2-mediated injury to rabbit gastric mucosal cells. AB - A sustained increase in cytosolic Ca2+ can damage gastric mucosal cells. The present study has examined the role of Ca2+ in thromboxane B2 (TXB2)-mediated damage of rabbit isolated gastric mucosal cells. Cells were isolated from rabbit oxyntic mucosa by collagenase-EDTA digestion. Cell metabolic activity and cell damage were estimated by alamar blue dye absorbance and trypan blue uptake, respectively. Cellular Ca2+ was monitored by indo-1 dye fluorescence. Addition of TXB2 (10(-6) and 10(-8) M) to the cell suspension resulted in a decrease in metabolic activity, and this effect was reduced when Ca2+ was removed from the incubation medium. TXB2 addition to the incubation medium resulted in an increase in cytosolic Ca2+ and incubation of cells with the intracellular Ca2+ chelator, BAPTA-AM (20 microM), reduced cell injury in response to TXB2. Incubation of cells with the Ca2+ ionophore A23187 (1-25 microM) resulted in a dose-dependent increase in trypan blue uptake and a reduction in cell metabolism. Cell injury in response to A23187 were exacerbated by addition of TXB2 (10(-8) M) to the cell suspension. TXB2 treatment reduced cellular content of reduced glutathione (GSH), while exogenous GSH addition (10 mM) reduced TXB2-mediated cell injury. These data demonstrate that TXB2 can directly injure gastric mucosal cells. Gastric mucosal cellular damage in response to TXB2 is mediated in part by a disruption of Ca2+ homeostasis as well as a reduction in cellular GSH content. PMID- 7555461 TI - Intragastric polyethylene glycol-400 protects against ethanol-induced gastric mucosal lesions despite pretreatment with indomethacin or iodoacetamide. AB - It has been shown that intragastric administration of polyethylene glycol-400 (PEG-400) by gavage needle protected the rat gastric mucosa from 96% ethanol induced lesions in a dose-dependent fashion. The inhibitions of the lesions were 10.5, 53.5, 94.6, and 99.2% at doses of 275, 1375, 2750, and 5500 mg/kg, respectively. The duration of the protective effect was approximately 12 hr. The gastroprotection offered by PEG-400 was not modified by pretreatment with either subcutaneous indomethacin (25 mg/kg) or iodoacetamide (100 mg/kg). Gastric motility, measured by a balloon method, was dose-dependently inhibited by intragastric administration of PEG-400. The inhibited gastric motility (amplitude gastric contraction) induced by PEG-400 was not modified by pretreatment with either indomethacin or iodoacetamide. The gastric emptying rate, investigated by measuring the disappearance of intragastrically administered [99mTc]DTPA from the stomach of rats treated with PEG-400 (5500 mg/kg) was markedly retarded. There was an increase in both the fluid volume and the mucus volume retained in the gastric lumen only for PEG-400 (5500 mg/kg) at 1, 2, and 4 hr after administration. The rats treated with 0.7 ml of vehicle plus 96% ethanol had significantly less damage than those treated with 0.2 ml of vehicle plus 96% ethanol. These results indicate that intragastric PEG-400-protective effect was not mediated by endogenous prostaglandins, sulfhydryl compounds of the gastric mucosa, or changes in gastric contractile patterns. We conclude that the protective effect of intragastric PEG-400 may be the result of retarded gastric emptying together with an osmotic pull of fluid into the stomach and the increase in gastric mucus volume.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555462 TI - Compliance of the proximal stomach and dyspeptic symptoms in patients with type I diabetes mellitus. AB - In the present study the function of the proximal stomach and its role in eliciting dyspeptic symptoms were evaluated in patients with diabetes mellitus. Eight type I diabetics with cardiovascular autonomic neuropathy and dyspeptic symptoms, and 10 healthy volunteers were studied using an electronic barostat device connected to a intragastric bag. The intragastric bag was inflated and deflated by stepwise pressure increments, creating pressure-volume curves. During the experiment the blood glucose concentrations were maintained within the euglycemic range in the diabetics. The volume-pressure curves showed a larger volume during the pressure increase in the diabetics than in the controls (P < 0.01). This resulted in a significant difference in compliance (dV/dP), 57.2 +/- 4.2 ml/mm Hg in diabetics and 43.7 +/- 3.5 ml/MM Hg in controls (P < 0.014). The volume-pressure curves during deflation of the intragastric balloon were different from the curves during inflation, creating a hysteresis loop. The area between the inflation and deflation curves was 827 ml/mm Hg in diabetics and 627 ml/mm Hg in the controls (P = 0.21). Gastric distension induced more upper gastrointestinal sensations in the patients than in the volunteers: nausea (P < 0.002), bloating (P < 0.003), upper abdominal pain (P < 0.001). IN CONCLUSION: this study showed that the compliance of the proximal stomach is increased in diabetic patients with autonomic neuropathy and gastrointestinal symptoms. This abnormality, probably due to autonomic neuropathy, is associated with increased symptom generation during gastric distension. PMID- 7555463 TI - Nizatidine accelerates gastric emptying of a solid meal in rats. AB - Nizatidine, a new histamine-2-receptor antagonist, stimulates gastrointestinal motility in dogs and gastric emptying of liquids in rats. Effect of nizatidine on gastric emptying of a solid meal was investigated using a novel gastric emptying model in rats. Male Wistar rats (weighing 200-300 g) were supplied with powdered food containing 30 w/w% barium 14 hr before the beginning of the experiment and x ray photography of rat stomach was taken under light ether anesthesia. Gastric emptying was assessed by percentage of a decrease in area 30 min after drug was injected intraperitoneally. There was a positive correlation between the area of the gastric outline and the weight of the gastric contents (r = 0.94, P < 0.01). Ether anesthesia itself did not affect gastric emptying. Nizatidine increased gastric emptying dose-dependently (emptied percentage; vehicle: 4.9 +/- 1.5%, 1 mg/kg: 7.2 +/- 0.4%, 3 mg/kg: 10.4 +/- 2.0%, 10 mg/kg: 16.7 +/- 4.9%, 30 mg/kg: 25.7 +/- 7.4%). N-Desmethyl nizatidine (NDM) also stimulated gastric emptying, but nizatidine S-oxide, cimetidine, an famotidine had no significant effects on gastric emptying. Nizatidine and neostigmine, but not NDM, at a subthreshold dose accelerated gastric emptying treated with a low dose of acetylcholine (0.1 mg/kg). Atropine (2 mg/kg, -30 min) did not modulate the gastroprokinetic action of nizatidine, but blocked that of NDM. These findings suggest that this noninvasive method may allow measurement of gastric emptying of solids accurately and that nizatidine and NDM facilitate gastric emptying probably mediated by a direct and/or an indirect (acetylcholinesterase inhibition) cholinergic mechanism. PMID- 7555465 TI - Famotidine, a histamine-2-receptor antagonist, inhibits the increase in rat gastric H+/K(+)-ATPase mRNA induced by intravenous infusion of gastrin 17 and histamine. AB - We examined the effects of gastrin and histamine on rat gastric H+/K(+)-ATPase, the enzyme responsible for H+ secretion, gene expression in vivo. Gastrin 17 (G 17) or histamine dihydrochloride (histamine) was continuously infused through the femoral vein of anesthetized rats. Gastric H+/K(+)-ATPase mRNA levels were measured using northern blot analysis. Infusion of G 17 and histamine increased the H+/K(+)-ATPase mRNA level significantly compared with basal control level or vehicle control level (P < 0.01). However, pretreatment with famotidine, a potent histamine-2 (H2)-receptor antagonist, inhibited the increase of rat gastric H+/K(+)-ATPase mRNA following G 17 and histamine infusion. These findings indicate that both histamine and G 17 increase expression of H+/K(+)-ATPase mRNA by activating H2 receptor on the parietal cell. PMID- 7555464 TI - An update on histamine H3 receptors and gastrointestinal functions. AB - The distribution and functions of histamine H3 receptors in the gastrointestinal tract is reviewed with particular reference to the effects on gastric acid secretion, mucosal protection, and intestinal motility. Histamine H3 receptor activation has negative effects on acid secretion induced by indirect secretagogues in cats, dogs, and rabbits; less clear effects were found in rats. An inhibitory effect on histamine release induced by different stimuli was observed in rats, rabbits, and dogs after H3 receptor agonists, thus supporting the idea that H3 receptors occur in ECL cells. (R)-alpha-methylhistamine has a marked protective effect against gastric lesions induced by ethanol in rats, being slightly less effective against aspirin and stress. H3 receptor activation decreases the intestinal motility induced by electrical stimulation in a variety of gut preparations, reducing both cholinergic and NANC neurotransmitter release. In this tissue the inhibitory effects mediated by histamine H3 receptors seem to be coupled, via a G protein, to a restriction of Ca2+ access into the nerve terminal; other mechanisms, however, have been suggested in the gastric mucosa. Histamine H3 receptors have already been subdivided into two receptor subtypes, H3A and H3B, the former being the subtype predominant in the gastrointestinal tissue. The increasing availability of selective agonists and antagonists of H3 receptors will unravel possible novel actions and physiological roles of histamine. PMID- 7555466 TI - Conventional dose of omeprazole alters gastric flora. AB - Quantitative cultures were carried out on samples from gastric juice obtained from 12 ambulatory patients with esophagitis before and one month after omeprazole therapy. An increase in the number of patients in whom gastric juice was culture-positive, as well as an increment in the bacterial counts were noted. The spectrum of microorganisms isolated from gastric juice was identical to the normal flora of the oral cavity, mainly alpha-hemolytic streptococci, corynebacteria, and Candida species. Thus, the counts of organisms within gastric contents are simply a reflection of swallowed oral microflora that were able to survive due to the less acidic environment. PMID- 7555468 TI - Achalasia-like syndrome presenting after highly selective vagotomy. AB - Vagotomy is reported as a secondary cause of achalasia. Highly selective vagotomy, however, has rarely been reported to cause an achalasia-like syndrome. We suspect that periesophageal trauma accounted for the LES abnormalities seen at manometry in our patient but cannot explain the aperistalsis of the body of the esophagus. Pneumatic dilation improved his dysphagia only slightly but allowed him to maintain his nutrition with oral liquid enteral supplements. We recommend barium swallow, endoscopy, sounding the esophagus with a 50- to 60-French dilator, and manometry in evaluating patients with dysphagia after highly selective vagotomy. If an achalasia-like syndrome is demonstrated, then conservative management with observation and liquid nutritional supplements for four to eight weeks is appropriate. If this fails, pneumatic balloon dilation may be considered. Clearly, a preoperative history of dysphagia should prompt evaluation before highly selective vagotomy. This case represents a transient achalasia-like syndrome after highly selective vagotomy and signifies the importance of conservative management. PMID- 7555467 TI - Severe gastric mucosal damage induced by NSAIDs in healthy subjects is associated with Helicobacter pylori infection and high levels of serum pepsinogens. AB - Helicobacter pylori infection and NSAIDs are considered the two most important exogenous factors in ulcer disease. The interrelation between the two factors is not, however, clear. Moreover, serum pepsinogen has been suggested as a risk marker for the development of NSAID-induced gastrointestinal lesions. Fifty-one healthy volunteers, enrolled in a prospective, double-blind study carried out to evaluate gastrointestinal side effects of meloxicam and piroxicam, were analyzed to determine whether: (1) the prevalence of H. pylori correlates with the occurrence and severity of NSAID-induced gastrointestinal lesions, and (2) serum pepsinogen A and C levels could be used as markers of NSAID-induced mucosal damage. Upper endoscopy was performed by the same investigator before and after 28 days of treatment with placebo, meloxicam (7.5 mg/day and 15 mg/day), or piroxicam (20 mg/day). NSAID-induced damage was graded separately for hemorrhages and erosion ulcers according to Lanza's scale. There were no statistically significant differences in the prevalence of H. pylori in subjects with and without NSAID-induced mucosal lesions. However, there was a positive association between H. pylori infection and the severity of mucosal damage: total mean endoscopic score was 2.9 +/- 0.3 in H. pylori-positive subjects versus 1.6 +/- 0.5 in H. pylori-negative subjects (P < 0.05). Pepsinogen A and C levels increased from 55.3 +/- 3 to 149.4 +/- 15 and from 6.3 +/- 0.5 to 11.5 +/- 2.2, respectively (P < 0.05) in subjects who developed severe endoscopic injury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555469 TI - Muscle sympathetic nerve activity is higher in intensively versus conventionally treated IDDM subjects. AB - OBJECTIVE: To determine whether poor long-term glycemic control may play a role in the lower muscle sympathetic nerve activity (MSNA) levels in insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: Intraneural electrodes were used to record MSNA from the peroneal nerve at baseline and during euglycemic insulin infusion (120 mU.m-2.min-1) in 16 IDDM subjects enrolled in the Diabetes Control and Complications Trial (DCCT), 8 intensively treated (HbA1c 7.1 +/- 1.2%) and 8 conventionally treated (HbA1c 9.0 +/- 1.5%; P < 0.05). RESULTS: Fasting plasma glucose levels tended to be higher at baseline in the conventionally treated group (11.3 +/- 1.7 mmol/l) than in the intensively treated group (7.4 +/- 1.1 mmol/l, P < 0.1), but did not differ during insulin infusion (conventional, 5.0 +/- 0.3 mmol/l; intensive, 5.1 +/- 0.4 mmol/l). Plasma free insulin levels did not differ between groups either before or during insulin infusion. The intensively treated group had significantly high MSNA levels than the conventionally treated group both in the fasting state (16.2 +/ 2.7 vs 10.5 +/- 4.4 bursts/min, P < 0.05 and during insulin infusion with euglycemia (27.8 +/- 2.1 vs 17.5 +/- 5.2 bursts/min. CONCLUSIONS: MSNA levels in intensively treated IDDM subject are higher than in conventionally treated subjects. These results suggest that improved long-term glycemic control is associated with increased sympathetic neural outflow to muscle. The mechanism for this effect remains unclear. PMID- 7555470 TI - Relation of steroid hormones to glucose tolerance and plasma insulin levels in men. Importance of visceral adipose tissue. AB - OBJECTIVE: Low plasma testosterone levels are associated with hyperinsulinemia and glucose intolerance in men. However, it is unclear whether these abnormalities are related to the concomitant alteration in regional adipose tissue (AT) accumulation associated with reduced androgen levels. RESEARCH DESIGN AND METHODS: We measured plasma steroid levels in a sample of 79 men, ranging from lean to obese (aged 29-42 years), for whom an oral glucose tolerance test (OGTT), anthropometric and computed tomography (CT) measurements of body fatness, and AT distribution were performed. Sex hormone binding globulin (SHBG) and the following steroids were measured after extraction from plasma and chromatography: dehydroepiandrosterone, androstenedione, androst-5-ene-3 beta,17 beta-diol, testosterone, estrone, and estradiol (E2). RESULTS: Several significant negative correlations were found between adrenal C19 steroid precursors, testosterone, SHBG, and fasting insulin levels, as well as between plasma glucose and insulin concentrations measured during the OGTT (-0.25 < or = r < or = -0.35, 0.05 > or = P > or = 0.001). The best steroid correlate of plasma glucose and insulin homeostasis indexes was the E2: testosterone ratio (0.34 < or = r < or = 0.42, 0.005 > or = P > or = 0.001). However, after correction of steroid levels for either fat mass, body mass index (BMI), or visceral AT area, as measured by CT, no significant residual associations were noted between testosterone, adrenal C19 steroid, SHBG, and estrogen levels and indexes of plasma glucose-insulin homeostasis, although the positive association between the E2: testosterone ratio and glucose area remained significant after adjustment for total body fat mass and BMI. Furthermore, 15 pairs of obese subjects, matched for visceral AT area, showing either low or high levels of the steroids studied, did not differ in fasting insulin and postglucose plasma insulin levels or in glucose tolerance. CONCLUSIONS: These results suggest that the previously reported relationships between androgen levels and indexes of plasma glucose-insulin homeostasis are mediated, to a large extent, by concomitant alterations in levels of total body fat and visceral AT in men. PMID- 7555471 TI - Catheter complications associated with implantable systems for peritoneal insulin delivery. An analysis of frequency, predisposing factors, and obstructing materials. AB - OBJECTIVE: To evaluate catheter survival and identify mechanisms involved in catheter obstructions during a 109.8 patient-year experience with implanted pumps for peritoneal insulin delivery. RESEARCH DESIGN AND METHODS: Fifty-one type I diabetic patients were recruited in feasibility studies of two models of implanted systems for peritoneal insulin delivery. Both systems had a silicone coated polyethylene catheter and infused Hoechst 21 PH neutral insulin (U400 or U100). Catheter obstruction was suspected each time the increase of insulin flow rate over 50% of usual need was insufficient to correct an impairment of glycemic control in the absence of of intercurrent factors. A laparoscopic examination was then systematically performed under general anesthesia. The disclosed material occluding the catheter was submitted to a pathological analysis. By actuarial analysis, we examined the estimated effects of the potential determinants of the catheters' duration of proper operation on catheter survival. RESULTS: Over an implantation duration of 25.8 +/- 14.0 months (mean +/- SD), 34 catheter obstructions were diagnosed in 24 patients, resulting in an incidence of 31 events per 100 patient-years. Fifty percent survival rate of the first implanted catheter was 27 months (95% confidence interval [CI]: 19-32) on actuarial analysis. Six catheters were cleared under laparoscopy and 24 were replaced, while 2 systems were definitively explanted and 2 combined replacements of pump and catheter were performed because of an associated pump slowdown. In five cases, an alkaline rinse procedure of the pump was necessary after catheter replacement to restore usual insulin needs, suggesting an associated insulin aggregation in the pump. Twenty obstructions were due to a fibrin clot at the catheter tip, and 14 obstructions were created by a tissue encapsulation around the catheter. A previous experience of peritoneal insulin infusion from portable pumps or a longer duration of diabetes ( > 21 years) both appeared as conditions significantly reducing the time of a catheter's proper operation (P < 0.01 and P < 0.05, respectively) either by tip obstructions or by encapsulations. Pathological analysis of catheter encapsulations showed a collagen fibrosis in all studied patients (n = 11), which was associated with a lymphocytic infiltrate in five patients and also with anti-insulin immunoreactive amyloid deposits in four patients. Catheter tip clots were composed of fibrinlike material, nonreactive to anti-insulin antibodies. CONCLUSIONS: Catheter obstruction is a frequent adverse technical event occurring with implanted insulin pumps. Progress is expected in the biocompatibility of catheter material and more specifically in the stability of insulin preparations to prevent immuno-inflammatory reactions and insulin amyloid deposits that appear to be involved in catheter failures. PMID- 7555473 TI - Incidence and prevalence of childhood diabetes in Slovakia (1985-1992). Slovak Childhood Diabetes Epidemiology Study Group. AB - OBJECTIVE: To provide reliable data on the incidence and prevalence of insulin dependent diabetes mellitus (IDDM) in children in Slovakia. RESEARCH DESIGN AND METHODS: A total of 754 Slovak children with IDDM, aged 0-14 years, were identified during an 8-year study period. There were 369 boys and 385 girls from an average population of 1,363,499 children. All patients were prospectively recorded. The primary ascertainment was based on notifications by physicians from outpatient departments. The second independent source of information was from all hospitals where the diabetic patients were hospitalized. The completeness of ascertainment was based on the capture-recapture procedure. RESULTS: The completeness of the ascertainment was 95%. In 1992, the annual incidence rate of IDDM (per 100,000) in the 0- to 14 year-old age-group was 8.92 for both sexes (7.95 for boys and 9.93 for girls). In the youngest age-group, an increase in the incidence rates from 2.89 in 1985 to 6.54 in 1992 was observed. Seasonality, with a higher incidence in autumn and winter, was observed in children older than 4 years. The prevalence rates of IDDM (per 1,000) were 0.28-0.50% in the 8-year period. CONCLUSIONS: The incidence of IDDM in Slovak children (0-14 years) is similar to what has been found in other Central European countries. In children younger than 4 years of age, in the last 2 years (1991 and 1992) more patients with diabetes were found than in any period during the preceding 6 years. The recent conspicuous increase in the incidence of IDDM among the younger children in Slovakia urges us to study its mechanisms, so far unknown, and to search for possible means of prevention. PMID- 7555472 TI - Comparison of insulin with or without continuation of oral hypoglycemic agents in the treatment of secondary failure in NIDDM patients. AB - OBJECTIVES: Optimal insulin regimens for non-insulin-dependent diabetes mellitus (NIDDM) patients with secondary failure are controversial. We evaluated the efficacy, side effects, and quality of life of patients receiving insulin either alone or in combination with their previous oral hypoglycemic agents (OHAs). RESEARCH DESIGN AND METHODS: Fifty-three Chinese patients with NIDDM (mean age 53.9 +/- 12.6 years, duration of diabetes 9.0 +/- 4.9 years, body wt 60.4 +/- 13.3 kg with corresponding body mass index 24.2 +/- 4.3 kg/m2, receiving the maximum dose of sulfonylurea and/or metformin) were confirmed to have OHA failure. Twenty-seven patients were randomized to continue OHAs and were given additional bedtime insulin (combination group); 26 patients were randomized to insulin therapy alone with twice-daily insulin (insulin group). Insulin doses were increased incrementally, aiming at fasting plasma glucose (FPG) < 7.8 mmol/l during a stabilization period of up to 8 weeks. Insulin dosage, body weight, glycemic control, and quality of life were assessed before and at 3 and 6 months after stabilization. RESULTS: Both groups showed similar improvement of glycemic control. For the combination group, FPG decreased from 13.5 +/- 2.7 to 8.9 +/- 3.0 mmol/l at 3 months (P < 0.0001) and to 8.6 +/- 2.5 mmol/l at 6 months (P < 0.0001). For the insulin group, FPG decreased from 13.5 +/- 3.6 to 7.5 +/-3.0 mmol/l at 3 months (P < 0.0001) and to 9.8 +/- 3.5 mmol/l at 6 months (P < 0.0001). No significant differences were observed between the groups. Similarly, both groups had significant improvement of fructosamine and glycosylated hemoglobin (HbA1c). Fructosamine fell from a mean of 458 to 365 mumol/l at 3 months (P < 0.0001) and to 371 mumol/l at 6 months (P < 0.0001) and from 484 to 325 mumol/l at 3 months (P < 0.0001) and to 350 mumol/l at 6 months (P < 0.0001) for the combination and insulin groups, respectively. HbA1c decreased from 10.2 to 8.4% at 3 months (P < 0.0001) and to 8.7% at 6 months (P < 0.0001) in the combination group and from 10.7 to 7.8% at 3 months (P < 0.0001) and to 8.4% at 6 months (P < 0.0001) in the insulin group. Despite similar improvement of glycemia, insulin requirements were very different. At 3 months, the combination group was receiving a mean of 14.4 U/day compared with 57.5 U/day in the insulin group (P < 0.0001). Similar findings were observed at 6 months (15.0 vs 57.2 U/day, P < 0>0001). Both groups gained weight. However, for the combination group, weight gain was 1.6 +/- 1.8 kg at 3 months and 2.1 +/- 2.5% kg at 6 months (both P < 0.0001 vs baseline), whereas for the insulin group, weight gain was 3.5 +/- 4.3 and 5.2 +/- 4.1 kg, respectively (both P < 0.0001 vs baseline). Weight gain was significantly greater in the insulin group (P < 0.05 at 3 months, and P < 0.005 at 6 months). Fasting plasma triglyceride decreased in the insulin group (1.8 +/- 1.0 to 1.4 +/- 0.8 mmol/l at 3 months [P < 0.005] and to 1.4 +/ 0.7 mmol/l at 6 months [P < 0.02] but not in the combination group. No changes were observed in total and high-density lipoprotein cholesterol. No severe hypoglycemic reactions were recorded in either group. Mild reactions occurred with similar frequency in both groups. Well-being and quality of life improved significantly in both groups. The majority of patients (82.7%) wanted to continue insulin beyond 6 months, irrespective of the treatment group. CONCLUSIONS: In NIDDM patients with secondary OHA failure, therapy with a combination of OHAs and insulin and with insulin alone was equally effective and well tolerated. However, combination therapy was associated with a lower insulin dose and less weight gain. Combination treatment may be considered when OHA failure occurs as a potential intermediate stage before full insulin replacement. PMID- 7555474 TI - Improvement of impaired counterregulatory hormone response and symptom perception by short-term avoidance of hypoglycemia in IDDM. AB - OBJECTIVE: To test the hypothesis that impaired counterregulatory hormone response and symptom perception, induced by recurrent hypoglycemic episodes over 2 days, may be improved by short-term (2-day) avoidance of hypoglycemia. RESEARCH AND DESIGN: We examined two groups of insulin-dependent diabetes mellitus (IDDM) patients (n = 16), none of whom exhibited signs of peripheral or autonomic neuropathy. Two sequential euglycemic-hypoglycemic clamp studies were performed applying stable glycemic plateaus of 5.6, 3.3, 2.2, and 1.7 mmol/l, at which the patients' awareness of and responses to hypoglycemia were evaluated. In the intervention group (n = 11), three short-term hypoglycemic ( < 2.2 mmol/l) episodes (days 1-3) preceded the first clamp study (day 4), whereas the second clamp study (day 6) followed a 2-day interval of strict avoidance of hypoglycemia. A control group (n = 5) was introduced to detect adaptation effects caused by the study procedure per se. RESULTS: This short-term avoidance of hypoglycemia caused improvement of the impaired counterregulatory hormone response during insulin-induced hypoglycemia involving adrenaline (P < 0.05), adrenocorticotrophic hormone (P < 0.03), and cortisol (P < 0.05). Improvement of hypoglycemia symptom awareness encompassed overall symptom perception (multiple analysis of variance, P < 0.04) and the automatic symptoms of heart pounding (P < 0.05) and sweating (P < 0.05). CONCLUSIONS: The previously reported compromised neuroendocrine counterregulation and symptom awareness, occurring as a consequence of repetitive hypoglycemic episodes over 2 days, may be improved by a single 2-day interval of strict avoidance of hypoglycemia PMID- 7555475 TI - The reliability and repeatability of the blood glucose response to prolonged exercise in adolescent boys with IDDM. AB - OBJECTIVE: To determine the intrasubject reliability and repeatability of the blood glucose response to prolonged exercise in adolescents with insulin dependent diabetes mellitus (IDDM) when pre-exercise meal, exercise, and insulin regimens are kept constant. RESEARCH DESIGN AND METHODS: Nine IDDM adolescent boys with diabetes duration of 9.7 +/- 4.8 years participated in two testing sessions 5-17 days apart. Carbohydrate intake, subcutaneous insulin injections, exercise bouts, and their timing were identical in both sessions. Exercise started 1 h after breakfast and consisted of six 10-min cycling bouts at moderate intensity (heart rate 145-150 beats/min), separated by 5-min rest periods. During rest periods, blood samples for glucose were taken and a supplemental carbohydrate beverage was consumed. Subjects were asked periodically to guess their own blood glucose levels. RESULTS: Intersession plasma glucose levels for each time period from the start of exercise to the end of the final recovery period were unchanged between sessions (P > 0.05) and highly correlated (r = 0.88 to r = 0.96, P < 0.01). The intraclass correlation for plasma glucose was 0.95. Decreases in plasma glucose from the start of exercise to the end of exercise were, at most 5 mmol/l, and intersession decreases did not differ significantly (P > 0.05). One subject experienced hypoglycemia. The decrease in plasma glucose did not differ significantly between fair and poor glycemic control (glycosylated hemoglobin < or = 10% and > 10%, respectively). Subjects markedly underestimated their blood glucose levels when they were high, but their guesses were closer to the measured values at lower levels. CONCLUSIONS: --Intersession observations demonstrated that the intrasubject blood glucose responses to prolonged moderate intensity exercise were reliable and repeatable when pre-exercise meal, exercise, and insulin, regimens were kept constant. This is an important finding for implementing and evaluating educational strategies for improving the metabolic control associated with prolonged exercise, as well as for future research on means for preventing exercise-induced hypoglycemia. PMID- 7555476 TI - Relationship of low-density lipoprotein particle size to plasma lipoproteins, obesity, and insulin resistance in Japanese men. AB - OBJECTIVE: To determine the clinical characteristics of patients with small low density lipoprotein (LDL) particles among Japanese men with mild glucose intolerance and to investigate the relationship of LDL particle size to the levels of other plasma lipoproteins, obesity, insulin resistance, and blood pressure (BP). RESEARCH DESIGN AND METHODS: The subjects were 40 men with impaired glucose tolerance or diabetes treated by diet alone, and 40 healthy men matched for age and body mass index (BMI) were used as control subjects. LDL particle size was measured using gradient gel electrophoresis. RESULTS: Of the 40 patients with glucose intolerance, 19 had small LDL (particle size < 25.5 nm) compared with only 4 of the 40 control subjects. In the patients with small LDL, the plasma levels of cholesterol, triglycerides, and apolipoprotein B, the fasting serum immunoreactive insulin, and the waist-to-hip ratio were all higher than in the patients with normal LDL (particle size > or = 25.5 nm), while the high-density lipoprotein cholesterol level was lower. However, there were no significant differences in BMI, BP, or insulin sensitivity in a euglycemic clamp study between the small-LDL and normal-LDL subgroups. CONCLUSIONS: Japanese men with glucose intolerance frequently have small LDL, and this abnormality is associated with other dyslipoproteinemias and increased waist-to-hip ratio. PMID- 7555477 TI - Body sway in diabetic neuropathy. AB - OBJECTIVE: To evaluate the influence of peripheral neuropathy on body sway assessed by posturography. RESEARCH DESIGN AND METHODS: The age-matched study subjects included 10 insulin-dependent diabetes mellitus (IDDM) patients with peripheral neuropathy (DN), 23 IDDM patients without peripheral neuropathy (D) according to the San Antonio Consensus Conference guidelines, and 21 control subjects (C). All subjects with symptoms and/or clinical signs of postural instability were excluded from the study. RESULTS: The trace surface was significantly larger in the DN than in the C and D groups (P < 0.05), and the trace length was longer in the DN than in the C and D groups (P < 0.01). Mean velocity was faster in the DN than in the other two groups (P < 0.001). A direct relationship was found between the parameters of posturography and some parameters of the nerve conduction velocity. CONCLUSIONS: Diabetic patients with peripheral neuropathy demonstrate a relative deficit in their ability to maintain posture. Posturography allows an early disclosure of the failure of postural control. PMID- 7555479 TI - Impaired left ventricular diastolic function and vascular postischemic vasodilation associated with microalbuminuria in IDDM patients. AB - OBJECTIVE: Microalbuminuria is considered an important predictor of cardiovascular events in diabetic patients. In this study, a possible association of microalbuminuria with significant changes in left ventricular (LV) morphology and function and generalized vascular dysfunction was analyzed in insulin dependent diabetes mellitus (IDDM) patients without hypertension, coronary artery disease, or autonomic dysfunction. RESEARCH DESIGN AND METHODS: Thirty-four young long-term IDDM patients, 16 with and 18 without microalbuminuria, and 20 control subjects were studied. LV systolic function and wall thickness were evaluated by M-mode echocardiography. LV diastolic function was studied using a combined echo Doppler and phonocardiographic technique. The hyperemic response to forearm ischemia was measured by strain-gauge plethysmography. All patients underwent 24 h ambulatory blood pressure monitoring. RESULTS: LV mass index and wall thickness:radius ratio were significantly higher in microalbuminuric patients. LV relaxation was significantly impaired in both diabetic groups compared with control subjects; moreover, this impairment was significantly greater in microalbuminuric than in normoalbuminuric patients. In microalbuminuric patients, forearm postischemic vasodilation was also significantly lower and mean awake diastolic blood pressure (dBP) was significantly higher than in the other two groups. CONCLUSIONS: Our data suggest that microalbuminuria is associated with significant changes in LV morphology, a more severe impairment of cardiac diastolic function, altered vascular dilatory capacity, and higher daytime dBP. Therefore, microalbuminuric patients should be considered to have a higher risk of cardiovascular complications and be kept under closer surveillance. PMID- 7555478 TI - Trial of pentoxifylline for diabetic impotence. AB - OBJECTIVE: To test the effectiveness of pentoxifylline as a therapy for diabetic impotence. RESEARCH DESIGN AND METHODS: A 3-month placebo-controlled double-blind treatment study was conducted at a single center. Therapeutic response was assessed subjectively by serial self-appraisals of erectile function and objectively by nocturnal penile tumescence (NPT) monitoring. RESULTS: This cohort of impotent diabetic men displayed substantial neurological and penile vascular dysfunction. Age, body mass index, duration of diabetes, duration of impotence, glycemic control, mode of therapy for non-insulin-dependent diabetes, mean testosterone, mean prolactin, and penile brachial indexes were similar in the treatment (n = 34) and placebo groups (n = 26). NPT monitoring revealed infrequent erectile events, diminished average rigidity, and decreased tumescence. Pentoxifylline did not alter overall glycemic control and did not improve neurological, vascular, or erectile function. In fact, a slight decrease in average percent rigidity from 22 +/- 3 to 17 +/- 2% (mean +/- SE, P < 0.05) was observed in the pentoxifylline-treated group. Although eight (24%) pentoxifylline-treated and five (19%) placebo-treated men reported benefit, no objective improvement was observed in nocturnal tumescence. CONCLUSIONS: Pentoxifylline is not an effective treatment for diabetic erectile dysfunction. PMID- 7555480 TI - Implementation of treatment protocols in the Diabetes Control and Complications Trial. AB - OBJECTIVE: To describe the methods used to implement intensive and conventional therapies in the Diabetes Control and Complications Trial (DCCT) and the metabolic results that occurred with the different treatment regimens. RESEARCH DESIGN AND METHODS: The DCCT was a controlled clinical trial that demonstrated the beneficial effect of intensive therapy on the long-term complications of insulin-dependent diabetes mellitus (IDDM). A total of 1,441 volunteers with IDDM, aged 13-39, from 29 centers in the U.S. and Canada, were randomly assigned to conventional or intensive diabetes therapy. Intensive therapy, which used multiple daily injections (MDI) of insulin ( > or = 3 injections/day) or continuous subcutaneous insulin infusion (CSII), was implemented by a team that included diabetes nurses, dietitians, behavioral experts, and diabetologists. Volunteers in the intensive treatment group could use MDI or CSII, based on patient and clinic preference, and could switch between therapies over the course of the study. The volunteers were followed for a mean of 6.5 years (range 3-9 years). RESULTS: A detailed analysis of implementation of the two treatments indicates that intensive and conventional treatment subjects adhered to their respective insulin injection regimens > 97% of the time. Adherence to other elements of intensive treatment was similarly high and resulted in median HbA1c values between 6.7 and 7.2, compared with 8.7-9.2 with conventional therapy, over the course of the study. Severe hypoglycemia occurred three times more often in intensively treated subjects. Although subjects on intensive treatment were not randomly assigned to MDI or CSII, we compared those subjects who used either of these methods for > or = 90% of the study time. CSII-treated patients maintained a mean HbA1c of 6.8 vs. 7.0 in MDI-treated subjects during the trial (P < 0.05). The frequency of hypoglycemia with coma and seizure and diabetic ketoacidosis was modestly higher with CSII than with MDI. CONCLUSIONS: Intensive therapy was implemented successfully in the DCCT. The detailed description herein will serve to facilitate translation of the DCCT results to the clinical setting. PMID- 7555481 TI - Lower limb venous pressure in diabetic neuropathy. AB - OBJECTIVE: Distended turgid veins have been noted in the feet of patients with diabetic neuropathy. This study assessed the supine venous pressure and its correlation with diabetic neuropathy and arteriovenous (AV) shunting in the feet of type I and type II diabetic subjects. RESEARCH DESIGN AND METHODS: Seventeen patients with chronic Charcot joints, 11 patients with a history of neuropathic foot ulceration, 14 diabetic control subjects, and 11 normal control subjects were studied. Neuropathy was assessed by vibration and thermal thresholds and standard cardiovascular autonomic neuropathy tests. Supine venous pressure was measured by insertion of a 21-gauge needle connected to a strain gauge manometer into the veins on the dorsum of the foot. Venous PO2 was used as a measure of AV shunting. RESULTS: Venous pressure was raised in the group with chronic Charcot joints (18.9 +/- 4.2 mmHg, P < 0.001 vs. the diabetic control group [11.3 +/- 2.6 mmHg]), the normal control group (12.3 +/- 2.3 mmHg, mean +/- SD), and the diabetic group with a history of neuropathic foot ulceration (16.0 +/- 3.7 mmHg, P = 0.008 vs. diabetic control subjects and P = 0.04 vs. normal control subjects), with a maximum of 28 mmHg. Venous pressure was correlated with heart rate variation to deep breathing, r = -0.61 (P = 0.001), vibration threshold, r = 0.56 (P = 0.001), Valsalva ratio, r = -0.64 (P -= 0.001), warm threshold, r = 0.69 (P = 0.001), and venous PO2, r = 0.43 (P = 0.02). There was no correlation with skin temperature, duration of diabetes, age, HbA1c, random blood glucose, ankle/brachial Doppler index, height, or serum creatinine. CONCLUSIONS: Venous pressure is much higher in the neuropathic diabetic limb and is correlated with the severity of neuropathy and with AV shunting. PMID- 7555482 TI - A new and simple questionnaire to identify people at increased risk for undiagnosed diabetes. AB - OBJECTIVE: To develop a simple questionnaire to prospectively identify individuals at increased risk for undiagnosed diabetes. RESEARCH DESIGN AND METHODS: People with newly diagnosed diabetes (n = 164) identified in the Second National Health and Nutrition Examination Survey and those with neither newly diagnosed diabetes nor a history of physician-diagnosed diabetes (n = 3,220) were studied. Major historical risk factors for undiagnosed non-insulin-dependent diabetes were defined, and classification trees were developed to identify people at higher risk for previously undiagnosed diabetes. The sensitivity, specificity, and predictive value of the classification trees were described and compared with those of an existing questionnaire. RESULTS: The selected classification tree incorporated age, sex, history of delivery of a macrosomic infant, obesity, sedentary lifestyle, and family history of diabetes. In a representative sample of the U.S. population, the sensitivity of the tree was 79%, the specificity was 65%, and the predictive value positive was 10%. CONCLUSIONS: This classification tree performed significantly better than an existing questionnaire and should serve as a simple, noninvasive, and potentially cost-effective tool for diagnosing diabetes in the U.S. PMID- 7555483 TI - Feasibility of intraperitoneal insulin therapy with programmable implantable pumps in IDDM. A multicenter study. The EVADIAC Study Group. Evaluation dans le Diabete du Traitement par Implants Actifs. AB - OBJECTIVE: To report the overall French experience, obtained through the collaboration of seven centers (EVADIAC [Evaluation dans le Diabete du Traitement par Implants Actifs] register), on the safety, feasibility, and efficacy of intraperitoneal insulin therapy by programmable implantable pumps, using three different devices. RESEARCH DESIGN AND METHODS: This is a multicenter prospective study involving 224 type I diabetic patients implanted with a programmable implantable pump (cumulative follow-up: 353 patient-years; mean duration: 1.5 +/- 0.9 years [mean +/- SD]. The Infusaid and the Promedos devices are equipped with a side port and refilled with U100 insulin (Hoechst 21 PH); the Minimed pump is not equipped with a side port and is refilled with U400 insulin (Hoechst 21 PH). Metabolic data and adverse events were recorded in a central register run by EVADIAC. RESULTS: A total of 29 local pump-pocket events (8/100 patient-years) and 9 pump failures (2.5/100 patient-years) occurred. The major technical problems were 1) pump flow rate reduction related to insulin aggregates, reversible after alkaline rinsing of the pump, and 2) 47 catheter obstructions requiring laparoscopic or conventional surgery. Pump therapy was abandoned in only 11 patients. HbA1c (7.4 +/- 1.8 vs. 6.8 +/- 1.0%, P < 0.001), mean glycemia (8.7 +/- 1.5 vs. 7.8 +/- 1.0 mmol/l, P < 0.001), and blood glucose SDs (3.8 +/- 0.8 vs. 3.3 +/- 0.8 mol/l, P < 0.001) decreased significantly after 6 months and remained lower than baseline thereafter. CONCLUSIONS: Intraperitoneal insulin infusion using an implantable programmable pump is a feasible and relatively safe technique that may improve metabolic control and glycemic stability. Long-term studies, however, are needed to demonstrate whether or not the improvement in glycemic control could be sustained for several years. PMID- 7555484 TI - Treatment of hypercholesterolemia in NIDDM with policosanol. AB - OBJECTIVE: To determine whether elevated levels of cholesterol and low-density lipoprotein (LDL) cholesterol in non-insulin-dependent diabetes mellitus (NIDDM) patients could be decreased by policosanol, a new cholesterol-lowering drug. NIDDM predisposes patients to coronary artery disease (CAD) through the direct action of hyperglycemia on the arteries as well as the dyslipidemia induced by NIDDM. RESEARCH DESIGN AND METHODS: This double-blind placebo-controlled trial was performed in 29 patients with NIDDM and hypercholesterolemia. After stable glycemic control was achieved by diet and/or oral hypoglycemic drugs, patients were instructed to follow a cholesterol-lowering diet for 6 weeks. Patients who met entry criteria received, under double-blind conditions, policosanol (5 mg) or placebo tablets twice a day for 12 weeks. RESULTS: Policosanol (10 mg/day) significantly reduced total cholesterol by 17.5% and LDL cholesterol by 21.8% compared with baseline and placebo. Furthermore, high-density lipoprotein (HDL) cholesterol was raised by 11.3% (not significant), and triglycerides showed a statistically nonsignificant decrease of 6.6%. These changes in lipid profile were similar to those induced by policosanol in nondiabetic patients with type II hyperlipoproteinemia. CONCLUSIONS: Glycemic control was unaffected by treatment. No clinically or biochemically adverse effects attributable to treatment were observed. Only one patient (placebo) withdrew from the trial because of an adverse experience (erythema). We concluded that policosanol is effective and safe in patients with NIDDM and hypercholesterolemia. PMID- 7555486 TI - Dietary composition, body weight, and NIDDM. Comparison of high-fiber, high carbohydrate, and modified-fat diets. AB - OBJECTIVE: To examine the effects of a high-carbohydrate low-fat (HCLF) and a modified-fat (MF) diet on body weight and metabolic control in subjects with non insulin-dependent diabetes mellitus (NIDDM) living at home. RESEARCH DESIGN AND METHODS: Twenty-four NIDDM patients followed HCLF and MF diets alternately and in random order for a 3-month period while at home, with a 1-month baseline and washout between diets. Before and after each diet, fasting glucose and lipids, HbA1c, blood pressure, and body weight were measured. Dietary preferences were assessed by questionnaire. RESULTS: Subjects consumed 50% of energy as carbohydrate and 23% as fat on the HCLF diet and 40% of energy as carbohydrate and 36% as fat (over half of which was monounsaturated fat) on the MF diet. Subjects lost weight on both HCLF and MF diets (mean loss 0.7 and 1.3 kg, respectively). Although the MF diet resulted in a small decrease in fasting glucose levels, there was no significant change in HbA1c. Similarly, there was no significant difference between the diets in changes in blood pressure or fasting blood lipids. Most subjects (65%) preferred the MF diet. CONCLUSIONS: Although the MF diet is not a low-fat diet, it did not appear to facilitate weight gain in subjects with NIDDM living at home. The MF diet provides an alternative for individuals unable to comply with HCLF diets. PMID- 7555485 TI - Glucokinase gene variants in subjects with late-onset NIDDM and impaired glucose tolerance. AB - OBJECTIVE: To investigate the frequency of variants of the glucokinase (GCK) gene in subjects with late-onset non-insulin-dependent diabetes mellitus (NIDDM) and in subjects with late-onset impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: The study population included 36 Finnish patients with late-onset NIDDM who were treated with diet for > 8 years or who were newly diagnosed and 40 subjects with late-onset IGT who had low or normal insulin levels when tested by an oral glucose tolerance test. All exons, exon-intron junctions, and islet and liver promotor regions of the GCK gene were amplified with the polymerase chain reaction and screened for mutations using single-strand conformation polymorphism analysis. RESULTS: A silent third-base substitution (TAC: >TAT) in codon 215 of exon 6 was found in 2.8% of NIDDM patients and in 5.0% of IGT subjects. Polymorphisms were found in islet exon 1 at nucleotide 403 (C-->G) in 16.7% of NIDDM patients and in 17.5% of IGT subjects and in the noncoding region of the islet promotor at nucleotide -30 (G-->A) in 13.9% of NIDDM patients and in 25.0% of IGT subjects. Furthermore, in liver intron 1 a variant (C-->T), 12 base pairs upstream from the splice acceptor site, was found in 5.6% of NIDDM patients and in 7.5% of IGT subjects. CONCLUSIONS: These results indicate that the mutations in the coding region of the GCK gene are not likely to play a major role the pathogenesis of late-onset NIDDM or IGT in the Finnish population. PMID- 7555487 TI - Muscarinic cholinergic antagonism does not enhance recovery from hypoglycemia in IDDM. AB - OBJECTIVE: Because muscarinic cholinergic agonism in the absence of an increase in glucagon secretion inhibits hepatic glucose production, we tested the hypothesis that muscarinic cholinergic antagonism enhances glucose recovery from hypoglycemia in insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: Eight (initially euglycemic) patients with IDDM received overnight infusions of insulin and were studied on three occasions in random order. Hypoglycemia was induced by low-dose insulin infusion (4.0 pmol.kg-1.min-1) from 0 through 80 min; observations were continued through 240 min. At 0 and 80 min, intravenous injections of atropine only (1.0 mg), placebo and then atropine, respectively, or placebo only were administered. RESULTS: Increments in heart rate (P < 0.001) and prevention of the pancreatic polypeptide response to hypoglycemia (P = 0.042) after atropine administration documented muscarinic cholinergic antagonism. The absent glucagon response to hypoglycemia was unaltered, but the epinephrine response was increased (P = 0.010). Nonetheless, rates of glucose production and utilization and plasma glucose concentrations were unaltered. CONCLUSIONS: We conclude that muscarinic cholinergic antagonism does not enhance glucose recovery from hypoglycemia in patients with IDDM. PMID- 7555488 TI - Cecum erosion: unusual but serious complication of an implanted catheter for peritoneal insulin delivery. PMID- 7555489 TI - Captopril-induced acute pancreatitis. AB - OBJECTIVE: To increase awareness concerning abdominal pain associated with the use of captopril, which is a molecule frequently used in diabetic patients. CASE: One case of acute pancreatitis associated with captopril, confirmed by a reintroduction, is described in a type II diabetes patient. CONCLUSIONS: Captopril and other angiotensin-converting enzyme inhibitors are very frequently prescribed in diabetic patients. Therefore, it is important to be aware of the possibility of pancreatitis linked to these molecules and to withdraw the treatment in case of severe unexplained abdominal pain. PMID- 7555490 TI - Patient empowerment and the traditional medical model. A case of irreconcilable differences? PMID- 7555491 TI - DCCT results and diabetes care in developing countries. PMID- 7555492 TI - A call for specific codes for diabetes foot and eye care. AB - In this report, we propose new International Classification of Diseases (ICD) codes that could be incorporated into computer-based patient records or administrative data to monitor and improve diabetes care. Neither the ICD, 9th Revision, nor its imminent replacement, the ICD, 10th Revision, has specific codes for foot examinations and funduscopic examinations in the asymptomatic person, high-risk diabetic foot status, or clinically significant macular edema. Adoption of official codes for these procedures and conditions implemented in conjunction with computerized databases could be used for surveillance, program planning, and quality of care assessment. Computerized medical records could use the codes to monitor care and issue reminders to patients and providers. Payors could offer reimbursement incentives to encourage compliance with standard recommendations. These codes for care procedures could be linked to outcomes, such as amputations and blindness, to improve our understanding of the etiology of blindness and the relationship between process and outcome. The uniform adoption of these codes would facilitate comparison between health care systems, geographic regions, and nations. The diabetes community should encourage the National Center for Health Statistics to adopt new codes that could be used to monitor diabetes preventive care practices. PMID- 7555493 TI - An increase in newly onset IDDM admissions following the Los Angeles earthquake. PMID- 7555494 TI - Regarding the role of growth hormone in the development of diabetic retinopathy. PMID- 7555495 TI - Atmospheric pressure effects on glucose monitoring devices. PMID- 7555497 TI - Obesity, diabetes, and insulin resistance: implications from molecular biology, epidemiology, and experimental studies in humans and animals. Synopsis of the American Diabetes Association's 29th Research Symposium and Satellite Conference of the 7th International Congress on Obesity, Boston, Massachusetts. AB - The number of genetic features potentially contributing to the development of obesity and of NIDDM continues to expand, even while evidence from epidemiological studies and prospective longitudinal evaluations of risk factors continue to argue strongly for nongenetic factors in the expression of these disorders. Even as the intensive search goes on for the diabesity gene(s), it is imminently clear that treatment initiatives can and must be oriented to reducing the well-established risk factors, most likely through multiple simultaneous interventions and alterations. Clearly restriction of calories is the most immediately effective of the various tools currently available. The recent results of long-term minimal restriction suggest extraordinary benefits to health can result for those predisposed to the diabesity or insulin resistance syndrome. Changes in diet composition may facilitate maintenance of a lower calorie intake, but in themselves they are not likely to significantly alter the risks of the syndrome, with the exception of lower cholesterol diets for reducing elevated cholesterol levels. Exercise and increased general physical activity must be seen as potentially beneficial; however, because of the magnitude of the physical training activities likely to be effective in lowering insulin resistance on a chronic basis, the expected outcomes from the usual types of relatively limited exercise regimen are less likely to have powerful preventive effects. Nevertheless, the combination of exercise and moderate alterations in caloric intake is likely to have a synergistic beneficial effect. Pharmacological interventions in the insulin resistance syndrome are in their early developmental phases, and we look forward to new developments in this area. Effective anti obesity agents are greatly needed, and new understanding of the excess risk carried by obesity for many features of the diabesity or insulin resistance syndrome suggest the urgency of greater investment in research toward new modes of therapy. We look forward with great anticipation to the next Obesity, Diabetes, and Insulin Resistance Conference, where new progress in prediction, new understanding of pathogenesis, new genetic markers and modifiers, and new treatment candidates are sure to be forthcoming. PMID- 7555496 TI - NIDDM treatment. PMID- 7555498 TI - Earlier appearance of impaired insulin secretion than of visceral adiposity in the pathogenesis of NIDDM. 5-Year follow-up of initially nondiabetic Japanese American men. AB - OBJECTIVE--To identify risk factors for development of non-insulin-dependent diabetes mellitus (NIDDM) during a 5-year longitudinal follow-up of second generation Japanese-American (Nisei) men. RESEARCH DESIGN AND METHODS--For 5 years, 137 initially nondiabetic Nisei men were followed with 75-g oral glucose tolerance tests at the initial visit and at 2.5- and 5-year follow-up visits. Body fat distribution was assessed by computed tomography (CT) and body mass index (BMI) calculated at each visit. Fasting insulin and C-peptide, the increment of insulin and C-peptide at 30 min after the oral glucose load, intra abdominal and total subcutaneous fat by CT, and BMI were compared between those who remained nondiabetic (non-DM) and those who had developed NIDDM at 2.5 years (DM-A) and 5 years (DM-B). RESULTS--At baseline, the DM-A group had significantly increased intra-abdominal fat, elevated fasting plasma C-peptide, and lower C peptide response at 30 min after oral glucose. At the 2.5-year follow-up, this group had markedly increased fasting plasma insulin and decreased 30-min insulin and C-peptide response to oral glucose. The DM-B group also had significantly lower insulin response at 30 min after oral glucose at baseline but no significant difference in intra-abdominal fat or fasting plasma insulin and C peptide levels. When this group developed NIDDM by 5-year follow-up, however, an increase of intra-abdominal fat was found superimposed on the pre-existing lower insulin response. Fasting plasma insulin and C-peptide remained low. CONCLUSION- In DM-A, lower 30-min insulin response to oral glucose (an indicator of beta-cell lesion) and increased intra-abdominal fat and fasting C-peptide (indicators of insulin resistance) were the risk factors related to the development of NIDDM. DM B subjects had a lower 30-min insulin response to oral glucose at baseline and increased intra-abdominal fat at 5-years, when they were found to have NIDDM. Thus, both insulin resistance and impaired beta-cell function contribute to the development of NIDDM in Japanese-Americans, and impaired beta-cell function may be present earlier than visceral adiposity in some who subsequently develop NIDDM. PMID- 7555500 TI - Mortality and morbidity associated with body weight in people with IDDM. The WHO Multinational Study of Vascular Disease in Diabetes. AB - OBJECTIVE: Strict glycemic control in people with insulin-dependent diabetes mellitus (IDDM) reduces the risk of microvascular complications, but improvements in control are also associated with weight gain. Fears about the mortality risks of obesity may limit the acceptability of tight control. Therefore, we examined morbidity and mortality risks associated with body weight in people with IDDM. RESEARCH DESIGN AND METHODS: This was a cohort study of 644 men and 576 women with IDDM from nine centers worldwide. Baseline examinations were performed in 1975-1977, and mortality follow-up continued until 1988. RESULTS: Body weight was positively associated with blood pressure and, in men, with cholesterol. Fasting blood glucose was higher in the most obese groups in women only. There were 204 deaths among the men and 148 among the women. There was a reverse J-shaped relationship between body weight and all-cause mortality, with the highest mortality rates occurring in the leanest body mass index (BMI) category. The age , duration-, and center-adjusted mortality rate ratio (95% confidence interval) comparing BMI category < 20 kg/m2 with BMI category > or = 22 and < 24 kg/m2 was 2.64 (1.59-4.38) in men and 1.54 (0.77-3.06) in women. Additional adjustment for smoking, blood pressure, glucose, cholesterol, and proteinuria did not qualitatively alter these findings. CONCLUSIONS: We conclude that except in very lean people with IDDM, body weight is not significantly associated with mortality. Thus, efforts to improve glycemic control should not be restricted by concerns about the effects of weight gain on mortality. PMID- 7555499 TI - Assessment of diabetes-related distress. AB - OBJECTIVE: To describe a new measure of psychosocial adjustment specific to diabetes, the Problem Areas in Diabetes Survey (PAID), and to present initial information on its reliability and validity. RESEARCH DESIGN AND METHODS: Before their routine clinic appointments, 451 female patients with type I and type II diabetes, all of whom required insulin, completed a self-report survey. Included in the survey was the PAID, a 20-item questionnaire in which each item represents a unique area of diabetes-related psychosocial distress. Each item is rated on a six-point Likert scale, reflecting the degree to which the item is perceived as currently problematic. A total scale score, hypothesized to reflect the overall level of diabetes-related emotional distress, is computed by summing the total item responses. To examine the concurrent validity of the PAID, the survey also included a series of standardized questionnaires assessing psychosocial functioning (general emotional distress, fear of hypoglycemia, and disordered eating), attitudes toward diabetes, and self-care behaviors. All subjects were assessed for HbA1, within 30 days of survey completion and again approximately 1 2 years later. Finally, long-term diabetic complications were determined through chart review. RESULTS: Internal reliability of the PAID was high, with good item to-total correlations. Approximately 60% of the subject sample reported at least one serious diabetes-related concern. As expected, the PAID was positively associated with relevant psychosocial measures of distress, including general emotional distress, disordered eating, and fear of hypoglycemia, short- and long term diabetic complications, and HbA1, and negatively associated with reported self-care behaviors. The PAID accounted for approximately 9% of the variance in HbA1. Diabetes-related emotional distress, as measured by the PAID, was found to be a unique contributor to adherence to self-care behaviors after adjustment for age, diabetes duration, and general emotional distress. In addition, the PAID was associated with HbA1 even after adjustment for age, diabetes duration, general emotional distress, and adherence to self-care behaviors. CONCLUSIONS: These findings suggest that the PAID, a brief, easy-to-administer instrument, may be valuable in assessing psychosocial adjustment to diabetes. In addition to high internal reliability, the consistent pattern of correlational findings indicates that the PAID is tapping into relevant aspects of emotional distress and that its particular feature, the measurement of diabetes-related emotional distress, is uniquely associated with diabetes-relevant outcomes. These data are also consistent with the hypothesis that diabetes-related emotional distress, separate from general emotional distress, is an independent and major contributor to poor adherence. Given that the study was limited to female patients using insulin, further examination of the clinical usefulness of the PAID will need to focus on more heterogeneous samples. PMID- 7555501 TI - Mortality risk by body weight and weight change in people with NIDDM. The WHO Multinational Study of Vascular Disease in Diabetes. AB - OBJECTIVE: Care guidelines for people with non-insulin-dependent diabetes mellitus (NIDDM) emphasize the importance of weight loss in reducing mortality risk. However, existing evidence regarding the relationship between weight and mortality and the effects of weight change is conflicting. We examined these relationships in the World Health Organization Multinational Study of Vascular Disease in Diabetes. RESEARCH DESIGN AND METHODS: This was a cohort study of 1,416 men and 1,544 women. Baseline examinations were performed in 1975 through 1977, a morbidity follow-up was performed in 1983, and a mortality follow-up continued until 1988. Data were analyzed according to geographical groups: Europeans, East Asians, and Native Americans. The relationship between weight change and mortality was analyzed for Europeans only. RESULTS: Generally, body mass index (BMI) was positively associated with age, blood pressure, and cholesterol but was negatively associated with duration of diabetes, prevalence of retinopathy, and use of insulin. There was no clear relationship between BMI and mortality across the geographical groups. In Europeans, weight loss in the leanest subjects at baseline (BMI < 26 kg/m2) was associated with a threefold increase in mortality risk compared with those who had maintained a steady weight (relative risk [RR] 3.05, 95% confidence interval [CI] 1.26-7.36). Only in the most obese group was weight loss associated with a reduction in mortality risk (BMI > 29 kg/m2, RR 0.84, 95% CI 0.40-1.74). CONCLUSIONS: The positive association of BMI with age, blood pressure, and cholesterol and the negative association with duration of diabetes, retinopathy, and use of insulin may explain why there is no strong relationship between BMI and mortality in NIDDM. Weight loss, particularly in the relatively lean diabetic person, may be associated with an increased mortality risk. PMID- 7555502 TI - Daily walking combined with diet therapy is a useful means for obese NIDDM patients not only to reduce body weight but also to improve insulin sensitivity. AB - OBJECTIVE: To evaluate the effects of walking combined with diet therapy (1,000 1,600 kcal/day) on insulin sensitivity in obese non-insulin-dependent diabetes mellitus (NIDDM) patients. RESEARCH DESIGN AND METHODS: Subjects were divided into two groups: 10 patients were managed by diet alone (group D), and 14 patients were placed in the diet and exercise group (group DE). Group DE was instructed to walk at least 10,000 steps/day on a flat field as monitored by pedometer (19,200 +/- 2,100 steps/day), and group D was told to maintain a normal daily routine (4,500 +/- 290 steps/day). A glucose clamp procedure at an insulin infusion rate of 40 microU.min-2.min-1 was performed before and after the 6- to 8 week training program. Mean serum insulin concentrations ranged from 720 to 790 pmol/l. RESULTS: While body weight (BW) in groups D and DE decreased significantly (P < 0.01) during the study, the amount of BW reduction in group DE was greater than that in group D (7.8 +/- 0.8 vs. 4.2 +/- 0.5 kg, P < 0.01). After training, glucose infusion rate (GIR) and metabolic clearance rate (MCR) in group D did not significantly increase; however, GIR and MCR increased significantly in group DE, from 17.21 +/- 1.11 to 26.09 +/- 1.11 mumol.kg-1.min-1 (P < 0.001) and from 3.0 +/- 0.3 to 5.3 +/- 0.4 ml.kg-1.min-1 (P < 0.001), respectively. The analysis of variance showed significant effects of exercise (time x exercise, P = 0.0005) for the improvement of MCR. Significant correlations were also observed between delta MCR and average steps per day (r = 0.7257, P < 0.005) in group DE. CONCLUSIONS: Walking, which can be safely performed and easily incorporated into daily life, can be recommended as an adjunct therapy to diet treatment in obese NIDDM patients, not only for BW reduction, but also for improvement of insulin sensitivity. PMID- 7555503 TI - Role of metformin accumulation in metformin-associated lactic acidosis. AB - OBJECTIVE: To investigate the role of metformin accumulation in the pathophysiology of metformin-associated lactic acidosis. RESEARCH DESIGN AND METHODS: We used high-performance liquid chromatography to measure plasma metformin concentrations in 14 patients who experienced lactic acidosis (pH < 7.35 and lactate concentration 5 > mmol/l) while receiving chronic metformin treatment. Their treatment was generally based on alkalinization and dialysis therapy. RESULTS: Clinical shock and/or evidence of tissue hypoxia was found in all patients with the exception of one who had a nonsteroidal anti-inflammatory drug-induced anuria. Ten patients had significant metformin accumulation (plasma metformin concentrations 4.1-84.9 mg/l, normal value 0.6 +/- 0.5 mg/l before drug intake), generally because of failure to withdraw metformin despite intercurrent pathological conditions affecting its renal elimination (serum creatinine concentrations ranging from 269 to 1,091 mumol/l). There was no metformin accumulation (plasma metformin 0.03-0.7 mg/l) in the four other patients, who had less severe renal failure (serum creatinine 140-349 mumol/l). The severity of the patient's general condition did not predict early hospital mortality (death before discharge from the intensive care unit) even in patients in shock. Whereas it was high in those without metformin accumulation (only 1 of 4 patients recovered), early hospital mortality was low in the 10 patients with metformin accumulation and was not related to its extent (3 patients died with end-stage hepatic failure or cardiac failure). Correlation studies showed a positive correlation between serum creatinine and plasma metformin and between plasma metformin and arterial lactate but, for the latter correlation, only in patients with metformin accumulation. CONCLUSION: Metformin-associated lactic acidosis is not necessarily due to metformin accumulation; true type B (aerobic) lactic acidosis, i.e., without an apparent associated hypoxic factor, seems exceptional. Neither the severity of the clinical picture nor the degree of metformin accumulation predicted survival; rather, the prognosis was dependent upon the severity of the associated pathological conditions. PMID- 7555505 TI - Prevalence and associated features of albuminuria in Koreans with NIDDM. AB - OBJECTIVE: To determine the prevalence and the associated features of microalbuminuria and overt proteinuria in Korean subjects with non-insulin dependent diabetes mellitus (NIDDM) attending a hospital clinic. RESEARCH DESIGN AND METHODS: A total of 631 Korean outpatients with NIDDM were studied cross sectionally for the presence of albuminuria and other micro- and macrovascular complications. Urinary albumin excretion rate (AER) was determined in timed overnight urine samples. Subjects were divided into three groups: no nephropathy (AER < 20 micrograms/min), microalbuminuria (AER 20-200 micrograms/min), and overt proteinuria (AER > 200 micrograms/min). RESULTS: Increased AER was present in 34% of our patients: 20% had microalbuminuria and 14% had overt proteinuria. Of patients with diabetes duration > or = 15 years, 35% had overt proteinuria. Most (82%) patients with overt proteinuria had retinopathy. Although the prevalence of microalbuminuria as a whole did not differ according to diabetes duration, the prevalence of microalbuminuria in the patients with retinopathy increased with diabetes duration. The microalbuminuric patients without retinopathy (diabetes duration < 5 years) were characterized by higher prevalence of hypertension and previous obesity, higher plasma triglyceride level, and lower plasma high-density lipoprotein cholesterol level. CONCLUSIONS: The prevalence of overt proteinuria in Korean NIDDM patients with a long diabetes duration was higher than that reported in Caucasians. Our data also suggest that the clinical meaning of microalbuminuria may be different based on the presence or the absence of retinopathy. Microalbuminuria in patients with retinopathy most probably would reflect diabetic nephropathy. In contrast, some recent-onset NIDDM patients with microalbuminuria in the absence of retinopathy had features of syndrome X. PMID- 7555504 TI - The relationship between smoking and microvascular complications in the EURODIAB IDDM Complications Study. AB - OBJECTIVE: To examine the relationship between smoking and both glycemic control and microvascular complications in patients with insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: This was a prevalence survey of 3,250 men and women aged 15-60 years with IDDM from 31 diabetes centers in 16 European countries. Participants completed a questionnaire, had retinal photographs taken, and performed a 24-h urine collection. HbA1c, frequency of hypoglycemic and ketoacidotic episodes, urinary albumin excretion rates, and retinopathy were compared by smoking category. RESULTS: The prevalence of smoking was 35% in men and 29% in women. Current smokers had poorer glycemic control and, among men, were more likely to have had a ketoacidotic episode than were those who never smoked. Ex-smokers had equivalent glycemic control and marginally more hypoglycemic episodes did than those who never smoked. Current smokers had a higher prevalence of microalbuminuria and total retinopathy than did those who never smoked. Ex-smokers had a higher prevalence of macroalbuminuria and proliferative retinopathy than did those who never smoked, but both had a similar prevalence of microalbuminuria. Adjustment for either current or long-term glycemic control could not fully account for these differences. CONCLUSIONS: Smoking is associated with poorer glycemic control and an increased prevalence of microvascular complications compared with not smoking. Ex-smokers can achieve glycemic control equivalent to and have a prevalence of early complications similar to that of those who never smoked. We suggest that poorer glycemic control can account for some of the increased risk of complications in smokers, and that quitting smoking would be effective in reducing the incidence of complications. Urgent action is required to reduce the high smoking rates in people with IDDM. PMID- 7555506 TI - Hormonal contraception in women with IDDM. Influence on glycometabolic control and lipoprotein metabolism. AB - OBJECTIVE: Safe and effective contraceptive methods are essential for women with insulin-dependent diabetes mellitus (IDDM), but opinions on the use of hormonal oral contraceptives by these women are conflicting. We evaluated the effects on glycometabolic control and lipoprotein metabolism in women with IDDM treated with an oral contraceptive not previously studied in a diabetic population. RESEARCH DESIGN AND METHODS: A total of 22 women with IDDM received a monophasic combination of ethinyl estradiol and gestodene for 1 year; 20 women of comparable diabetic status using nonhormonal contraception were selected as control subjects. Evaluation was performed before and after 1, 3, 6, and 12 months of hormonal intake using nonparametric statistical methods. RESULTS: Except for a higher median age of the control group, the baseline values for all clinical and metabolic variables were similar in the two groups, and in neither of the groups were changes in blood pressure, body mass index, or glycemic control observed. In the oral contraceptive group, decreased serum levels of low-density lipoprotein (LDL) cholesterol and increased levels of triglycerides and lipoprotein A were noted, whereas total cholesterol and high-density lipoprotein cholesterol levels were unchanged. In the control group, a decrease of LDL cholesterol was observed. No effect of tobacco smoking on glycometabolic control or lipoprotein metabolism could be demonstrated during hormonal intake. CONCLUSIONS: No evidence of impaired glycometabolic control or adverse changes in serum levels of lipoproteins known to be associated with atherosclerosis was observed in women with well-controlled IDDM during 1 year of oral contraception with ethinyl estradiol and gestodene. PMID- 7555507 TI - Effects of Vitamin E on susceptibility of low-density lipoprotein and low-density lipoprotein subfractions to oxidation and on protein glycation in NIDDM. AB - OBJECTIVE: To evaluate the effect of vitamin E supplementation on the susceptibility of low-density lipoprotein (LDL) and LDL subfractions to oxidation and on protein glycation in non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Twenty-one men with NIDDM (HbA1c = 6-10%), ages 50 70, were randomly assigned to either 1,600 IU/day of vitamin E or placebo for 10 weeks after a 4-week placebo period. LDL and LDL subfractions were isolated after 4 weeks of placebo and after 6 and 10 weeks of therapy. Susceptibility of LDL to copper-mediated oxidation was measured by conjugated diene formation (lag time) and formation of thiobarbituric acid-reactive substances (TBARS). Fasting serum glucose, mean weekly blood glucose, HbA1c, and glycated plasma protein concentrations were also determined at these time points. RESULTS: Vitamin E content in plasma and LDL increased 4.0- and 3.7-fold, respectively, in the vitamin E-treated group. Vitamin E decreased the susceptibility of LDL to oxidation in comparison with placebo (lag time, 243 +/- 46 vs. 151 +/- 22 min, P < 0.01; 3 h TBARS, 24 +/- 12 vs. 66 +/- 18 nmol malondialdehyde/mg LDL, P < 0.05). Vitamin E content also increased significantly in both buoyant and dense LDL subfractions, and their oxidation was dramatically reduced. The lag time of LDL oxidation correlated well with the content of vitamin E in both LDL and its subfractions (r = 0.69-0.92). Glycemic indexes did not change significantly in either group during the study. Protein glycation, including glycated hemoglobin, glycated albumin, glycated total plasma proteins, and glycated LDL were unchanged in the vitamin E group. CONCLUSIONS: Supplementation of vitamin E in NIDDM leads to enrichment of LDL and LDL subfractions and reduced susceptibility to oxidation. Despite a greater percentage increase in vitamin E content in small dense LDL, it remained substantially more susceptible to oxidation than was buoyant LDL. This suggests that dense, LDL may gain less protection against oxidation from antioxidant supplementation than does larger, more buoyant LDL. In contrast to previous reports, vitamin E supplementation did not reduce glycation of intracellular or plasma proteins. PMID- 7555509 TI - Insulin sensitivity is not impaired in Mexican-American women without a family history of diabetes. AB - OBJECTIVE: The purpose of this research was to compare insulin sensitivity in Mexican-Americans and non-Hispanic whites without a family history of diabetes to establish whether insulin resistance is a defect intrinsically related to subjects of Mexican origin. RESEARCH DESIGN AND METHODS: In study A, we compared insulin sensitivity in 12 Mexican-American and 12 non-Hispanic white women with normal glucose tolerance and no family history of diabetes. In study B, we compared insulin sensitivity in two groups of normal glucose-tolerant Mexican Americans, nine with a positive (FHD+) and nine with a negative (FHD-) family history of diabetes. In both studies, the groups were closely matched for age, total body fat content, and fat topography. Insulin sensitivity was assessed with the euglycemic insulin clamp (20 microU.min-1.m2 surface area) which was performed in combination with tritiated glucose infusion and indirect calorimetry. Total fat mass and fat-free mass (FFM) were assessed by a tritiated water dilution technique, and regional fat distribution was evaluated by anthropometry and magnetic resonance imaging. RESULTS: During a 4-h euglycemic insulin clamp (study A), rates (mg.min-1.kg FFM-1) of total (6.32 +/- 0.64 vs. 6.62 +/- 0.81), oxidative (3.54 +/- 0.24 vs. 3.51 +/- 0.19), and nonoxidative (2.78 +/- 0.48 vs. 3.11 +/- 0.75) glucose utilization were similar in Mexican Americans and non-Hispanic whites; hepatic glucose production (0.33 +/- 0.13 vs. 0.35 +/- 0.13) was suppressed similarly in both groups. During a 2-h euglycemic insulin clamp (study B), Mexican-Americans with FHD+ had lower rates of insulin mediated total (3.55 +/- 0.39 vs. 5.93 +/- 0.59, P < 0.001), oxidative (3.31 +/- 0.25 vs. 4.32 +/- 0.17, P < 0.01), and nonoxidative (0.24 +/- 0.28 vs. 1.61 +/- 0.49, P < 0.01) glucose disposal than subjects with FHD-; suppression of hepatic glucose production (0.24 +/- 0.14 vs. 0.18 +/- 0.12) was similar in both groups. CONCLUSIONS: These results indicate that in the absence of a family history of non-insulin-dependent diabetes mellitus, Mexican-American women are not less sensitive to insulin than non-Hispanic white women. PMID- 7555508 TI - Reduction of glycosylated hemoglobin and postprandial hyperglycemia by acarbose in patients with NIDDM. A placebo-controlled dose-comparison study. AB - OBJECTIVE: To compare the safety and efficacy of three doses of acarbose (100, 200, and 300 mg three times daily) with placebo for the treatment of non-insulin dependent diabetes mellitus (NIDDM) in patients maintained on dietary therapy alone. RESEARCH DESIGN AND METHODS: This multicenter double-blind placebo controlled trial was 22 weeks in duration. The trial consisted of a 2-week screening period, a 4-week placebo run-in period, and a 16-week double-blind treatment period. The primary measure of drug efficacy was the mean change from baseline in HbA1c levels. Additional efficacy variables included the mean change from baseline in fasting and postprandial plasma glucose and serum insulin levels. RESULTS: After 16 weeks of treatment, acarbose-treated patients had statistically significant reductions in mean HbA1c levels of 0.78, 0.73, and 1.10% (relative to placebo) in the 100-, 200-, and 300-mg t.i.d. groups, respectively. Significant reductions in fasting and postprandial plasma glucose levels, glucose area under the time-concentration curve, and maximum glucose concentration were also observed in acarbose-treated patients. Although there were no statistically significant differences among the 100-, 200-, and 300-mg treatment groups, there was a trend toward a dose-response relationship for most plasma glucose variables that were measured. Gastrointestinal side effects (e.g., abdominal pain, flatulence, and diarrhea) and serum transaminase elevations (e.g., aspartate aminotransferase [AST] and alanine aminotransferase [ALT] were more frequently reported in the acarbose-treated patients than in the placebo treated control patients. Transaminase elevations occurred only at the 200-, and 300-mg dosages and were readily reversible on discontinuation of treatment. CONCLUSIONS: Acarbose at doses of 100, 200, and 300 mg administered three times daily for 16 weeks significantly reduced HbA1c levels and postprandial hyperglycemia. Treatment with acarbose is a safe and effective adjunct to dietary therapy for the treatment of NIDDM. PMID- 7555510 TI - Similar insulin sensitivity in NIDDM patients with normo- and microalbuminuria. AB - OBJECTIVE: To investigate whether insulin resistance and microalbuminuria are associated in non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Insulin sensitivity was assessed using a hyperinsulinemic euglycemic clamp in 11 normoalbuminuric and 9 microalbuminuric NIDDM patients matched for sex, age, body composition, glycemic control, diabetes duration, and therapy. RESULTS: Isotopically determined glucose disposal was similar in normo- and microalbuminuric patients in the basal state (mean +/- SD; 3.30 +/- 1.01 vs. 3.46 +/- 0.82 mg.kg lean body mass [LBM]-1.min-1; NS) and during hyperinsulinemia (7.16 +/- 2.65 vs. 6.63 +/- 2.88 mg.kg LBM-1.min-1;NS). No difference was observed in nonoxidative glucose disposal or lipid oxidation. Endogenous glucose production was equally suppressed by insulin (-0.08 +/- 0.99 vs. 0.30 +/- 1.12 mg.kg-1 LBM.min-1; NS). Glucose oxidation tended to be lower in the normoalbuminuric patients in the basal state (1.16 +/- 0.37 vs. 1.41 +/- 0.36 mg.kg LBM-1.min-1) and during hyperinsulinemia (2.35 +/- 0.72 vs. 2.90 +/- 0.77 mg.kg LBM-1.min-1; both P < 0.15). Urinary albumin excretion rate correlated with the insulin-stimulated glucose oxidation rate (r = 0.59, P = 0.0064), and a similar trend was seen in the basal state (r = 0.42, P = 0.063). Protein oxidation was higher in normoalbuminuric patients (1.6 +/- 0.5 vs. 1.0 +/- 0.4 mg.kg LBM-1.min-1; P = 0.017) and correlated inversely with albuminuria (r = 0.70, P = 0.0007). Serum growth hormone increased during insulin infusion; however, the increase was significantly greater in microalbuminuric patients. Plasma lipoproteins, maximal aerobic capacity, and 24-h ambulatory blood pressure were similar in the two groups. CONCLUSIONS: Basal and insulin-stimulated glucose uptakes are comparable in carefully matched normo- and microalbuminuric NIDDM patients, and glucose oxidation may be positively related to albuminuria. The inverse relation between protein oxidation and albuminuria may be due to higher growth hormone levels during daily life perturbations in glucose in microalbuminuric patients. PMID- 7555512 TI - Clinical onset characteristics of familial versus nonfamilial cases in a large population-based cohort of childhood-onset diabetes patients. AB - OBJECTIVE: To compare characteristics at clinical onset of childhood-onset diabetes patients with and without a first-degree relative with childhood-onset insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: In a nationwide continuous incident diabetes register covering patients from 0 to 14 years of age with a high level of ascertainment, we compared 687 patients who at onset had at least one first-degree relative with insulin-treated diabetes with 5,137 patients without such relatives. RESULTS: The pattern of change over the 15 year period was similar among familial- and sporadic-case patients. The seasonal pattern, with a lower incidence during the warmer period of the year, was similar in both groups. Age at clinical onset was also similar in both groups in either sex. When the proband had a sibling who already had the disease, the mean age at onset was significantly higher when compared with sporadic-case or other familial case patients. CONCLUSIONS: This analysis of a very large set of population-based cases of childhood diabetes showed that patients who had one first-degree relative with insulin-treated diabetes at onset shared the onset characteristics of those without such family members, including age at onset, sex ratio, seasonality, and secular trend. The findings may indicate that the complex interactions between genetic and nongenetic risk factors subsequently leading to IDDM are mainly shared by familial- and sporadic-case patients. PMID- 7555511 TI - Safety and efficacy of normalizing fasting glucose with bedtime NPH insulin alone in NIDDM. AB - OBJECTIVE: To examine the safety and overall clinical effects of normalizing the fasting plasma glucose (FPG) level with bedtime NPH insulin alone in patients with non-insulin-dependent diabetes mellitus (NIDDM) that is poorly controlled with maximal doses of sulfonylureas. RESEARCH DESIGN AND METHODS: Twelve obese male NIDDM subjects were treated for 16 weeks with bedtime insulin after a 4-week sulfonylurea washout. The insulin dosage was increased until the FPG level was normalized. The 24-h plasma glucose profiles and lipid and HbA1c levels were measured at the beginning and end of the study, and the incidence and severity of hypoglycemic episodes were closely monitored. In addition, hyperglycemic clamp studies were performed to assess insulin secretion and provide an indirect measurement of insulin sensitivity. RESULTS: FPG (14.6 +/- 0.9 mmol/l at week 0) was normalized ( < 6.4 mmol/l) within 6 weeks (5.9 +/- 0.6 mmol/l) and remained at target levels until the end of the study (4.0 +/- 0.03 mmol/l at week 16, P < 0.001). The insulin dose was 80 +/- 9 U/day (0.86 +/- 0.10 U/kg). Improved glycemic control was confirmed by a reduction in HbA1c (10.9 +/- 0.05 vs. 7.2 +/- 0.2%, P < 0.001) and mean 24-h glucose (17.2 +/- 0.2 vs. 7.4 +/- 0.2 mmol/l, P < 0.001). The incidence of mild or moderate hypoglycemic episodes was 3.4 +/- 1/patient for the entire 16-week study, and no patient experienced severe hypoglycemia. Bedtime insulin significantly improved total cholesterol, low density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and triglyceride levels (P < 0.01). Weight gain was 2.4 +/- 0.7 kg, and blood pressure was unchanged. During the hyperglycemic clamp, there was an improvement in the first phase (P < 0.001) and in the second phase (P < 0.01) of insulin secretion. There also was an increase in the rate of exogenous glucose infused (M) (P < 0.01) and in the M/C-peptide ratio (P < 0.02), suggesting enhanced insulin sensitivity. CONCLUSIONS: NPH insulin given at bedtime in amounts sufficient to achieve a normal FPG level does not cause excessive or severe hypoglycemia and does lead to good glycemic and lipid control in NIDDM. Bedtime insulin therapy also is accompanied by improved insulin secretion and insulin sensitivity. We conclude that a single dose of insulin alone at bedtime merits consideration as a therapeutic strategy in patients with poorly controlled NIDDM. PMID- 7555513 TI - A comparison of premixed insulin preparations in elderly patients. Efficacy of 70/30 and 50/50 human insulin mixtures. AB - OBJECTIVE: To compare the postprandial hyperglycemic response to a standard breakfast of two premixed humulin insulin mixtures, 50/50 (50% NPH human insulin and 50% regular human insulin) and 70/30 (70% NPH human insulin and 30% regular human insulin) in elderly non-insulin-dependent diabetes mellitus (NIDDM) patients. RESEARCH DESIGN AND METHODS: On two mornings, each patient (n = 20) consumed a standard breakfast after a single dose of 50/50 or 70/30 insulin (0.3 U/kg) was administered in a randomized crossover fashion. Plasma glucose and serum free insulin concentrations were measured before and for 4 h after insulin administration. RESULTS: Plasma glucose reached a peak at 60 min and a nadir at 240 min for both types of insulin. No differences in maximum and minimum glucose concentrations, time to maximum and minimum glucose concentrations, or areas under the curve were noted. Free insulin levels did not differ significantly. CONCLUSIONS: These results suggest that small changes in the composition of premixed insulin mixtures in NIDDM patients may not result in improved postprandial glycemic control. PMID- 7555514 TI - Symptoms of hypoglycemia in children with IDDM. AB - OBJECTIVE: To examine the symptoms of hypoglycemia in children with insulin dependent diabetes, from the perspective both of the child and of the child's parents, and to compare the symptom reporting of the diabetic children with that of adult diabetic patients. RESEARCH DESIGN AND METHODS: Interviews were conducted with 100 parents and 43 of their children. The frequency and intensity of symptoms of hypoglycemia were documented using a structured interview and classified into groups using Principal Components Analysis (PCA). RESULTS: Diabetic children and their parents showed close agreement concerning the relative frequency and the intensity of symptoms reported. PCA of the symptom reports showed that diabetic children and their parents identified the same distinct subgroups of hypoglycemia-related symptoms: behavioral disturbance and autonomic-neuroglycopenic subgroups. CONCLUSIONS: Hypoglycemic symptoms in children with diabetes clearly differ from those experienced by insulin-treated adults and, in particular, include behavioral changes as primary features of a low blood glucose. These observations have important implications for parental education on hypoglycemia. PMID- 7555516 TI - Neuropathic arthropathy of the spine in diabetes. PMID- 7555515 TI - Diabetes care policies and practices in Michigan nursing homes, 1991. AB - OBJECTIVE: To describe local standards of care for nursing home patients with diabetes, to characterize the care that nursing home patients with diabetes receive in Michigan, and to determine if the care provided meets local and national standards. RESEARCH DESIGN AND METHODS: In March 1991, a questionnaire was administered and chart reviews were conducted as part of the Medical Review and Nursing Evaluation conducted by the Michigan Department of Public Health. The questionnaire was completed by the head nurses at 17 skilled nursing homes to learn about local institutional standards of care. Chart reviews were conducted on a sample of five patients with diabetes from each nursing home to describe the care provided and to compare it with local and national standards. RESULTS: Almost all nursing homes had some diabetes care orders or protocols. Standing orders were most often present to guide nutritional and nursing care (e.g., diet, blood glucose monitoring, foot care). Standing orders were less often present to guide medical care (e.g., blood glucose parameters to contact physician) and surveillance of complications (e.g., eye exams). In general, the care provided did not meet local or national standards for diabetes care. Care practices were closer to national standards when registered dietitians (RDs) participated in meal planning and written institutional policies existed. CONCLUSIONS: In this sample of Michigan nursing homes, those with RDs and standing orders provided care more in keeping with guidelines. There is room for improvement in diabetes care practices in nursing homes. It may be time for diabetes-related organizations to re-examine standards for diabetes care in nursing homes. PMID- 7555517 TI - Inpatient management of adults with diabetes. PMID- 7555518 TI - Serum lipoprotein(a) is increased in hypertensive NIDDM patients. PMID- 7555519 TI - Unpleasant alcohol effect in diabetes associated with 3243 bp mitochondrial tRNALeu(UUR) mutation. PMID- 7555520 TI - Insulin pump treatment for recurrent ketoacidosis in adolescence. PMID- 7555521 TI - The clinical relevance of fetal hemoglobin in IDDM and NIDDM patients. PMID- 7555522 TI - Hyperglycemia and cardiovascular risk in NIDDM. PMID- 7555523 TI - Prevalence of IDDM in adults in the community. PMID- 7555524 TI - Is it time to modify the glucose tolerance test for the diagnosis of gestational diabetes. PMID- 7555525 TI - Insulin sensitivity in patients with NIDDM and the A-to-G mutation at nucleotide 3,243 of the mitochondrial tRNALeu(UUR) gene. PMID- 7555527 TI - Diabetes and the Internet. PMID- 7555526 TI - Five cases of hyperthyroidism in type I diabetic patients treated with intraperitoneal insulin infusion. PMID- 7555528 TI - Tests of glycemia in diabetes. PMID- 7555529 TI - A diabetes potpourri, Part 2. PMID- 7555530 TI - Factors determining the blood pressure response to enalapril and nifedipine in hypertension associated with NIDDM. AB - OBJECTIVE: To examine the factors that determine the blood pressure response to enalapril and nifedipine monotherapy in the treatment of hypertension associated with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: After a 6-week placebo baseline period, 102 hypertensive NIDDM patients were randomly assigned, double-blindly, to treatment with nifedipine retard (slow release) (n = 52) or enalapril (n = 50). The daily dosage of enalapril was increased, if required, from 10 to 20 to 40 mg and that of nifedipine from 40 to 60 to 80 mg at 4-week intervals during the 12-week titration period. Blood pressure, 24-h urinary albumin excretion (UAE), biochemical data, and serum angiotensin-converting enzyme (ACE) activity were measured at weeks -6, -4, 0, 4, 8, and 12. At week 0, venous blood was also sampled for baseline plasma atrial natriuretic peptide, renin, aldosterone, and serum insulin concentrations. RESULTS: At week 12, the mean daily dose of enalapril was 35 +/- 11.4 mg, and 27 (57%) patients were receiving the maximum daily dose of 40 mg. In the nifedipine group, the mean daily drug dose was 50 +/- 12.9 mg, and 4 (8%) were receiving the maximum daily dose of 80 mg. Despite a dose-dependent fall in the serum ACE activity in the enalapril group, the mean arterial pressure (MAP) was reduced by only 8 mmHg throughout the 12-week titration period compared to a decline of 15, 18, and 19 mmHg at weeks 0, 4, and 12, respectively, in the nifedipine group (P = 0.01 between groups). In the enalapril group, changes in MAP between weeks 0 and 12 correlated significantly with baseline plasma glucose (r = 0.45, P = 0.001) and aldosterone concentrations (r = -0.32, P = 0.02) and UAE (r = 0.3, P = 0.04). There was no statistically significant correlation between the changes in MAP and baseline plasma renin concentration. On multivariate analysis, the baseline renal function, glycemic control, and plasma aldosterone and serum insulin concentrations were all independently related to the changes in blood pressure in the enalapril-treated patients. No such statistical associations were observed in the nifedipine group. CONCLUSIONS: In hypertensive NIDDM patients, the activity of the renin-angiotensin-aldosterone system, the level of serum insulin, glycemic control, renal function, and proteinuria may be important determinants of the blood pressure response to ACE inhibition. Good glycemic control may optimize the antihypertensive efficacy of concomitant ACE inhibitor therapy. PMID- 7555531 TI - An assessment of the association between functional edentulism, obesity, and NIDDM. AB - OBJECTIVE: To assess the association between a measure of extensive tooth loss (functional edentulism) and obesity with non-insulin-dependent diabetes mellitus (NIDDM) in a racially heterogeneous sample. RESEARCH DESIGN AND METHODS: A cross sectional survey (370 subjects) was performed by reviewing the medical and dental records of dependently or independently living individuals who were treated as inpatients and/or outpatients at a Department of Veterans Affairs facility. Frequencies and descriptive measures were derived; univariate and multiple logistic regression analyses were conducted to test for associations, confounding, effect modification, and interaction using functional edentulism and obesity as the independent variables and NIDDM as the dependent measure. RESULTS: Functionally edentulous individuals were at significantly greater risk for NIDDM (estimated odds ratio [OR] = 4.06), than the obese (OR = 3.29). These relationships were not confounded by age or race in this sample. Obesity did confound functional edentulism in the multivariable model, suggesting that they act independently on the outcome variable (NIDDM). CONCLUSIONS: Dentist-assessed functional edentulism and physician-diagnosed obesity were significantly associated with NIDDM in this sample of predominantly older men. This finding ought to be considered by primary care providers in formulating dietary strategies in order to facilitate the realization of their therapeutic goals. PMID- 7555532 TI - Positive methane-producing status associated with increased serum cholesterol in subjects with impaired glucose tolerance. AB - OBJECTIVE: To determine if those who produce methane (i.e., have presence of methane in breath) have higher serum cholesterol than those who do not produce methane in subjects with impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: We measured breath gases and fasting serum total and high-density lipoprotein (HDL) cholesterol and triglyceride (TG) levels in 21 subjects with IGT. RESULTS: The 11 methane-producers were well matched to the 10 non-methane producers for age, sex, and body mass index. Methane-producers had higher fasting serum total (6.5 +/- 0.3 vs. 5.5 +/- 0.2 mmol/l; P < 0.02) and low-density lipoprotein (4.3 +/- 0.3 vs. 3.4 +/- 0.2 mmol/l; P < 0.05) cholesterol concentrations with no difference in TG or HDL levels. CONCLUSIONS: The results suggest that in subjects with IGT, positive methane-producing status may be associated with increased serum cholesterol levels. PMID- 7555534 TI - IDDM in the province of Pavia, Italy, from a population-based registry.A descriptive study. AB - OBJECTIVE: To report the incidence of insulin-dependent diabetes mellitus (IDDM) in the Province of Pavia, Italy, in the 0- to 29-year-old age-group between 1988 and 1992. Urban versus rural residence, socioeconomic level, and family size of IDDM cases were also investigated. RESEARCH DESIGN AND METHODS: A prospective register was established in 1988 to collect all newly diagnosed IDDM patients with onset before 30 years of age. The primary data source was based on notification of new cases by hospitals, out-patient clinics, family doctors, and pediatricians. The secondary and independent data source consisted of the registries of prescriptions for insulin syringes in the health districts of the province. RESULTS: In 5 years (1988-1992), 66 cases of IDDM in the 0- to 29-year old age-group were identified. The completeness of ascertainment was 100% for the combined sources. Age-adjusted (world-standardized) incidence rates per 100,000 (95% confidence interval) were 9.52 (6.42-13.61), 6.72 (4.68-9.34), and 8.27 (6.42-10.58), respectively, for the age-groups 0-14, 15-29, and 0-29. The rates were higher for residents in urban areas. The number of children in the families of IDDM patients was significantly higher than in the reference population. CONCLUSIONS: Our data indicate the concordance of IDDM incidence rates with the North-Italian rates and a possible association of the disease with environmental factors. These factors might enhance the susceptibility to IDDM in genetically predisposed individuals. PMID- 7555533 TI - Abnormal albuminuria as a predictor of mortality and renal impairment in Chinese patients with NIDDM. AB - OBJECTIVE: Microalbuminuria predicts mortality in non-insulin-dependent diabetes mellitus (NIDDM), but its association with deterioration of renal function remains more controversial than in insulin-dependent diabetes mellitus (IDDM). Using albumin-to-creatinine ratios (ACRs) in random spot urine samples is a convenient method for evaluating albuminuria. We studied prospectively the predictive values of albuminuria in NIDDM when assessed by this urine measurement. RESEARCH DESIGN AND METHODS: Between 1991 and 1992, we restudied the clinical and biochemical status of 403 Chinese NIDDM patients recruited in 1989 after a follow-up period of 26.6 +/- 3.2 months (mean +/- SD). Spot urine ACR was measured on two occasions and microalbuminuria was defined as a mean ACR between 5.6 and 38 mg/mmol. RESULTS: From the original cohort, 29 patients had died mostly because of cardiovascular events with or without renal failure. The overall relative risk of death in patients with abnormal albuminuria was 7.1 (P < 0.001) (microalbuminuria: 3.7, P = 0.04; macroalbuminuria: 11, P < 0.001). On multivariate analysis, the independent predictive factors for mortality were plasma creatinine (wald = 12.1, P < 0.001) and glucose concentrations (wald = 10.4, P < 0.001) in the normo- and microalbuminuric patients (n = 11) and age (wald = 4.4, P = 0.03) and plasma creatinine (wald = 8.2, P < 0.01) in the macroalbuminuric group (n = 18). In the survivors (n = 374), baseline spot urine ACR was independently associated with 2-year spot urine ACR in the normo- (P < 0.001), micro- (P < 0.01), and macroalbuminuric groups (P = 0.01). In addition, baseline spot urine ACR was independently related to 2-year plasma creatinine (P = 0.01) in the macroalbuminuric group. The rates of change of the reciprocal of plasma creatinine ( delta [Cr]-1) were -27.3 +/- 62.5, -43.4 +/- 68.6, and -108.8 +/- 98.81.mumol01.month-1 in the normo-, micro-, and macroalbuminuric groups, respectively (P < 0.001). The delta [Cr]-1 was independently and inversely related to the baseline spot urine ACR (P < 0.001) and 2-year systolic blood pressure (P < 0.001). CONCLUSIONS: Abnormal albuminuria as indicated by a random spot urine ACR > 5.6 mg/mmol predicts increased mortality and is associated with the progression of albuminuria and deterioration of renal function in Chinese NIDDM patients. PMID- 7555535 TI - Features of syndrome X in first-degree relatives of NIDDM patients. AB - OBJECTIVE: To determine whether the features of syndrome X are more common in first-degree relatives of non-insulin-dependent diabetes mellitus (NIDDM) patients than in control subjects with no family history of diabetes. RESEARCH DESIGN AND METHODS: A total of 154 first-degree relatives from 60 families with two or more NIDDM patients and 154 age- and sex-matched control subjects were studied. All subjects underwent a 75-g oral glucose tolerance test and baseline lipid blood and anthropometric measures. The features of syndrome X that were studied were obesity, hypertension, dyslipidemia (high triglyceride levels and low high-density lipoprotein [HDL] cholesterol concentrations), impaired glucose tolerance (World Health Organization criteria), and insulin resistance (as assessed by the homeostasis model assessment). RESULTS: Relatives were heavier than control subjects (body mass index 27.5 +/- 5.2 vs. 25.2 +/- 4.6 kg/m2, respectively [mean +/- SD], P < 0.0002), had lower HDL cholesterol concentrations (1.2 +/- 0.3 vs. 1.4 +/- 0.4 mmol/l, P < 0.001), were more insulin-resistant (2.3 [0.7-7.6] vs. 1.6 [0.5-5.1], geometric mean [95% confidence intervals], P < 0.0001), and had more individuals classified as having impaired glucose tolerance (28 of 154 [18%] vs. 7 of 154 [7%], chi 2, P < 0.001). The differences in insulin resistance and HDL cholesterol concentrations between the groups were independent of obesity. CONCLUSIONS: Features of syndrome X occur more frequently in relatives of NIDDM patients than in control subjects with no family history of diabetes. PMID- 7555536 TI - Extra-pancreatic manifestations in diabetes secondary to mitochondrial DNA point mutation within the tRNALeu(UUR) gene. AB - OBJECTIVE: A point mutation in the mitochondrial genome has been identified as a cause of diabetes and deafness. We report two pedigrees with and A-to-G transition at nucleotide 3243 of mtDNA within the tRNALeu(UUR) gene and focus our investigations on other localizations of the anomaly, particularly muscle and retina. RESEARCH DESIGN AND METHODS: Muscular localization has been studied in probands by invasive and noninvasive methods, including muscle biopsy (evaluation of the proportion of mutated mtDNA in comparison to blood cells, measurement of respiratory chain complex activities and histological and histochemical aspects) and 31P-nuclear magnetic resonance (NMR) spectroscopy. Ophthalmic and angiographic examination of retina, electroretinography, and visual evoked potentials were performed in five subjects. RESULTS: This mutation, previously described in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), was expressed more abundantly in muscle than in nucleated blood cells. This low expression in blood cells could hamper the diagnosis for some patients. In addition, despite poor clinical expression, muscle was found to be highly affected. Ragged red fibers and dystrophic mitochondria were observed in muscle biopsy. Histochemical assays showed decreased activity of respiratory chain complexes, and 31P-NMR in vivo data further confirmed the defect of muscle oxidative processes. Exercise-induced lactate production was increased. Finally, in both families, an atypical "salt and pepper" pigmentary retinopathy was observed without consequences on visual acuity. CONCLUSIONS: In diabetes secondary to 3243 mtDNA mutation, infraclinical muscular and ocular lesions are frequent. These two locations of the disease, which are easily investigated by simple methods, can help in the diagnosis of this new type of diabetes. PMID- 7555537 TI - The epidemiology of diabetes and pregnancy in the U.S., 1988. AB - OBJECTIVE: To determine the prevalence of pregnancy complicated by diabetes in a representative sample of the U.S. population. RESEARCH DESIGN AND METHODS: We analyzed data from a multistaged cross-sectional probability sample of live births recorded in the U.S. in 1988 for women 15-49 years of age. The main outcome measure was pregnancy complicated by diabetes. RESULTS: Diabetes was present in congruent to 154,000 (4%) of all pregnancies in the U.S. Gestational diabetes mellitus (GDM) accounted for 135,000 of such pregnancies (88%), non insulin-dependent diabetes mellitus (NIDDM) for 12,000 (8%), and insulin dependent diabetes mellitus for 7,000 (4%). On average, the mothers with NIDDM (29.6 years) and GDM (29.3 years) were older than mothers whose pregnancies were not complicated by diabetes (26.2 years; P < 0.05). In multivariate analyses, the odds of having a pregnancy complicated by GDM increased significantly with maternal age and body mass index. CONCLUSIONS: Pregnancy is complicated by diabetes more often than was previously believed. More frequent testing may further increase the apparent prevalence of GDM. PMID- 7555539 TI - Beyond glycemia. PMID- 7555538 TI - Diabetes complications: the renal-retinal link. An epidemiological perspective. PMID- 7555543 TI - An alternative approach to the diagnosis of diabetes with a review of the literature. PMID- 7555541 TI - Why use the oral glucose tolerance test? PMID- 7555540 TI - Which test for diagnosing diabetes? PMID- 7555542 TI - The pathogenesis and prevention of diabetes in adults. Genes, autoimmunity, and demography. PMID- 7555545 TI - Scuba diving and diabetes. Practical guidelines. PMID- 7555544 TI - Is the oral glucose tolerance test obsolete? PMID- 7555546 TI - Measurement of D-chiro-inositol in clinical studies. PMID- 7555547 TI - The effect of hyperglycemia on glipizide absorption in NIDDM patients. PMID- 7555549 TI - Metformin. PMID- 7555550 TI - High incidence of childhood-onset IDDM in Kuwait. Kuwait Study Group of Diabetes in Childhood. AB - OBJECTIVE: To determine the incidence of insulin-dependent diabetes mellitus (IDDM) in children aged 0-14 years in Kuwait, as part of the World Health Organization Multinational Collaborative Study (DIAMOND), and to determine if the incidence rates have increased. RESEARCH DESIGN AND METHODS: All cases of IDDM diagnosed before the child's 15th birthday between 1 January 1992 and 31 December 1993 were recorded. Prospective notification of all children with newly diagnosed diabetes who were admitted to hospitals and periodic review of hospital medical records provided the primary source; notification by physicians working in diabetic clinics, in which registry of all new cases is mandatory, provided the secondary source of ascertainment. RESULTS: The degree of ascertainment was 92.2%. The annual incidence of IDDM for children aged 0-14 years over the 2-year period was 15.4/100,000 (95% confidence interval, 12.4-19), with a male:female ratio of 1.2:1. The age-specific annual incidence rates for the age-groups 0-4, 5 9, and 10-14 years were 12.8, 15.1, and 18.3/100,000, respectively, with a male:female ratio of 1.45:1 in the 0- to 4-year-old age-group and an equal sex ratio in the 5- to 9- and 10- to 14-year-old age-groups. No significant difference was detected between incidence rates of IDDM in boys and girls in the three age-groups. There was no significant linear trend toward an increase in IDDM incidence rates as age advanced. Compared with a previous study by Taha et al. (Taha T, Moussa M, Rashed A, Fenech F: Diabetes mellitus in Kuwait: incidence in the first 29 years of life. Diabetologia 25:306-308, 1983), there was a nearly fourfold increase of IDDM in the age-group 0-14 years, mainly in those children < 5 years old, suggesting a rapid increase in a short period of time. CONCLUSIONS: Kuwait has the highest incidence of IDDM in children in the region, and an apparently increasing incidence has been demonstrated over the last decade. PMID- 7555548 TI - Impaired glucose tolerance of pregnancy by WHO and NDDG criteria National Diabetes Data Group. PMID- 7555553 TI - Feasibility and acceptability of a proposed infant feeding intervention trial for the prevention of type I diabetes. AB - OBJECTIVE: To determine the feasibility of a randomized double-blind controlled trial of an infant formula without intact cow's-milk protein for preventing type I diabetes in high-risk children. RESEARCH DESIGN AND METHODS: We surveyed 83 people who either were parents of a child with type I diabetes or were pregnant women with type I diabetes in the ambulatory diabetes and obstetrics clinics in a university hospital. After a written and verbal description of the cow's milk diabetes hypothesis, participants were asked to sign a sham consent form. A questionnaire designed to explore factors affecting their decision to either sign or not sign the consent form, as well as infant-feeding patterns, was subsequently administered. RESULTS: Overall, 69.9% (95% confidence interval, 60.0 79.8%) consented to participation in the proposed randomized trial. The decision to consent was not affected by the degree of belief in the cow's milk-diabetes hypothesis, the child's risk of diabetes, the respondent's demographic data, or infant feeding habits. CONCLUSIONS: A randomized feeding intervention study is an acceptable and feasible way to determine whether avoidance of cow's-milk protein during the first 6 months of life prevents type I diabetes in North American children. PMID- 7555554 TI - Patient empowerment. Results of a randomized controlled trial. AB - OBJECTIVE: The purpose of this study was to determine if participation in a patient empowerment program would result in improved psychosocial self-efficacy and attitudes toward diabetes, as well as a reduction in blood glucose levels. RESEARCH DESIGN AND METHODS: This study was conducted as a randomized, wait listed control group trial. The intervention group received a six-session (one session per week) patient empowerment education program; the control group was assigned to a wait-list. At the end of 6 weeks, the control group completed the six-session empowerment program. Six weeks after the program, both groups provided follow-up data. RESULTS: The intervention group showed gains over the control group on four of the eight self-efficacy subscales and two of the five diabetes attitude subscales. Also, the intervention group showed a significant reduction in glycated hemoglobin levels. Within groups, analysis of data from all program participants showed sustained improvements in all of the self-efficacy areas and two of the five diabetes attitude subscales and a modest improvement in blood glucose levels. CONCLUSIONS: This study indicated that patient empowerment is an effective approach to developing educational interventions for addressing the psychosocial aspects of living with diabetes. Furthermore, patient empowerment is conducive to improving blood glucose control. In an ideal setting, patient education would address equally blood glucose management and the psychosocial challenges of living with diabetes. PMID- 7555552 TI - Association between lipoprotein(a) and insulin-like growth factor I during puberty and the relationship to microalbuminuria in children and adolescents with IDDM. AB - OBJECTIVE: To study pubertal changes in serum lipoprotein(a) [Lp(a)] and insulin like growth factor I (IGF-I) in insulin-dependent diabetes mellitus (IDDM) and the relationship to microalbuminuria. RESEARCH DESIGN AND METHODS: Seventy-nine children and adolescents (59 with normoalbuminuria, 20 with microalbuminuria) with > or = 5 years of IDDM were investigated together with 54 healthy control subjects in a cross-sectional study. Fasting serum Lp(a); apolipoprotein (apo) A 1 and B; total, low-density lipoprotein (LDL), and high-density lipoprotein cholesterol; triglycerides; and IGF-I were analyzed as were HbA1c and overnight albumin excretion rate (AER). Pubertal development was assessed by Tanner staging. RESULTS: Lp(a), apoB, triglycerides, and total and LDL cholesterol were higher (P < 0.001) and apoA-1 was lower (P = 0.03) in normoalbuminuric IDDM patients than in healthy control subjects. Lp(a) was increased during puberty (stages 2-4) in IDDM patients but not in healthy subjects, whereas IGF-I was significantly increased during puberty in healthy control subjects only. In IDDM patients Lp(a) correlated to insulin dose, total cholesterol, and LDL cholesterol, but not to IGF-I, HbA1c, systolic and diastolic blood pressure, diabetes duration, age, or sex. In multiple regression analysis with Lp(a) as the dependent variable, puberty was the only significant contributor to the regression (r2 = 0.33, P = 0.008). Microalbuminuria was seen only in the pubertal stage 4-5. Lp(a) tended to be higher (P = 0.06) as did apoB, whereas IGF-I was lower (P < 0.001) in this group than in normoalbuminuric patients of the same pubertal stages. In multivariate analysis, with log AER as the dependent variable, apoB/apoA-1, systolic blood pressure, age, and IGF-I but not Lp(a) added to the regression (r2 = 0.47, P < 0.0001). CONCLUSIONS: Lp(a) is elevated during puberty in normoalbuminuric subjects with IDDM, independent of metabolic control and IGF-I. Lp(a) tends to be further increased in microalbuminuria but does not seem to be a contributing determinant of log AER whereas low IGF-I does. Prospective studies are required to establish the temporal relationship between increased Lp(a) and microalbuminuria in children and adolescents with IDDM. PMID- 7555551 TI - A double-blind placebo-controlled trial evaluating the safety and efficacy of acarbose for the treatment of patients with insulin-requiring type II diabetes. AB - OBJECTIVE: To determine whether a forced titration of acarbose (from 50 to 300 mg three times daily) administered over a 24-week period, in conjunction with diet and insulin therapy, improves glycemic control and reduces daily insulin requirements in insulin-requiring type II diabetes. RESEARCH DESIGN AND METHODS: This multicenter, randomized, double-blind, placebo-controlled trial was 36 weeks in duration. The trial consisted of a 6-week pretreatment period, a 24-week double-blind treatment period, and a 6-week post-treatment follow-up period. The primary efficacy variables were the mean change from baseline in HbA1c levels and the mean percentage change from baseline in total daily insulin dose. RESULTS: Treatment with acarbose was associated with significant reductions in HbA1c levels of 0.40% (P = 0.0001) and in total daily insulin dose of 8.3% (P = 0.0015). There were also significant reductions in all plasma glucose variables measured, including a 0.9 mmol/l reduction in fasting glucose (P = 0.0440), a 2.6 mmol/l reduction in glucose Cmax (P = 0.0001) and a 270 mmol.min-1.l-1 reduction in glucose area under the curve (P = 0.0002). Although acarbose treatment was associated with a greater incidence of adverse events than was placebo treatment, primarily flatulence and diarrhea, these events did not generally prevent patients from completing the study. CONCLUSIONS: The results of this study suggest that acarbose is a safe and effective adjunct to diet and insulin therapy for the management of insulin-requiring type II diabetes. PMID- 7555555 TI - The influence of pregnancy on IDDM complications. AB - OBJECTIVE: Although pregnancy has been associated with an increased progression of certain insulin-dependent diabetes mellitus (IDDM) complications, particularly retinopathy, both the short- and long-term relationships between pregnancy and both neuropathy and macrovascular disease are poorly documented. This study was conducted to comprehensively examine the influence of pregnancy on the development and progression of IDDM complications. RESEARCH DESIGN AND METHODS: Using the Pittsburgh Epidemiology of Diabetes Complications Study population (childhood-onset IDDM), two nested, pair-matched case-control studies were conducted. Women who had completed at least one successful pregnancy (n = 80) were matched to women with no history of pregnancy by age, duration of IDDM, race, and marital history. The first nested study (study 1) compared the prevalences of five IDDM complications between case and control groups. The second nested study (study 2) compared the incidences of the same five complications over an approximate 2-year interval during which the case subjects (n = 30) completed a successful pregnancy. RESULTS: There were no significant differences in the prevalence rates of coronary heart disease, neuropathy, proliferative retinopathy, lower extremity arterial disease, and overt nephropathy by case-control status, while parity did not predict any complication in multiple logistic analysis (study 1). In study 2, there were small but nonsignificant differences in incidence rates of overt nephropathy and lower extremity arterial disease between the groups, whereas case subjects had almost 3 times the incidence rate of proliferative retinopathy (P = 0.58) and 10 times the incidence rate of neuropathy (P < 0.001) as did other matched control subjects. In multivariate analysis, parity predicted neuropathy incidence but did not predict the incidence of any other complication, including proliferative retinopathy. CONCLUSIONS: Women with IDDM who experience a pregnancy may not be at an increased risk of diabetes complications later in life. However, in the short term, pregnancy may accelerate the development of some complications, such as neuropathy. PMID- 7555557 TI - Physiological modulation of plasma free fatty acid concentrations by diet. Metabolic implications in nondiabetic subjects. AB - OBJECTIVE: To determine the effect of varying the amount of carbohydrate and glycemic index (GI) of breakfast test meals on plasma free fatty acid (FFA) responses of nondiabetic subjects and to see whether the glycemic response at lunch was related to the plasma FFA response to breakfast. RESEARCH DESIGN AND METHODS: We studied eight subjects over a 6-h period on four separate occasions using a randomized Latin-square design. They received isocaloric breakfast test meals that were either high (84 g) or low (41 g) in carbohydrate and had either a high (approximately 100) or a low (approximately 70) GI, followed by a standard lunch 4 h later. RESULTS: The initial fall in plasma FFAs after breakfast was similar for all four test meals, but the extent of rebound differed significantly. The mean plasma FFA concentration just before the start of lunch (4 h) was highest after the low-GI, low-carbohydrate breakfast (418 +/- 42 mumol/l), followed by high-GI, low-carbohydrate (277 +/- 48 mumol/l), high-GI, high carbohydrate (227 +/- 32 mumol/l), and low-GI, high-carbohydrate (149 +/- 23 mumol/l) (P < 0.01). The concentration of plasma FFAs at 4 h was directly related to the total area under the glycemic response curve to lunch (r = 0.691, n = 32, P < 0.0001). CONCLUSIONS: In nondiabetic subjects, the type and amount of carbohydrate eaten at breakfast influences the plasma glucose, insulin, and FFA responses to breakfast and also affects the glucose, insulin, and FFA responses to a subsequent standard lunch. The glycemic responses after lunch were closely related to the plasma FFA concentration 4 h after breakfast, which we speculate is due to the inhibitory effect of FFAs on insulin action. PMID- 7555556 TI - Diabetes in urban African-Americans. II. High prevalence of microalbuminuria and nephropathy in African-Americans with diabetes. AB - OBJECTIVE: African-Americans with diabetes have an increased risk of endstage renal disease, but underlying mechanisms are poorly understood. We undertook this study to evaluate prevalence and risk factors for renal disease in an African American population with diabetes. RESEARCH DESIGN AND METHODS: We measured urine albumin excretion in 578 consecutive patients presenting for the first time to the Grady Memorial Hospital Diabetes Unit in Atlanta, GA. The unit serves an urban population that is predominantly African-American; 85% of patients have non insulin-dependent diabetes mellitus (NIDDM). Subjects provided 24-h and/or approximately 3-h urine collections for measurement of albumin and creatinine. RESULTS: Correlation of the albumin/creatinine ratio (micrograms/mg) with the 24 h albumin excretion rate was 0.89 (P < 0.001, n = 123). Although the median duration of diabetes was only 1 year, among all subjects, the estimated prevalence of microalbuminuria (30-300 mg albumin/24 h) was 25% and that of nephropathy ( > 300 mg albumin/24 h) was 11%. Among African-Americans with NIDDM (n = 466), the estimated prevalence of microalbuminuria was 24% and that of nephropathy was 12%; prevalence remained high (25 and 5%, respectively) among 219 patients with < 1 year known duration of diabetes. Metabolic control was not associated with disease. However, among all subjects with NIDDM, the odds ratio for nephropathy among subjects with disease duration > 5 years compared with those with disease duration < 1 year was 4.65 (95% confidence interval [CI] 2.24 9.79), and the odds ratio for nephropathy among subjects with hypertension compared with those without hypertension was 2.64 (CI 1.42-4.93). Odds ratios were comparable among African-Americans with NIDDM. Trends were similar but less significant for subjects with microalbuminuria. CONCLUSIONS: Albuminuria can be identified reliably and conveniently by the albumin/creatinine ratio in brief urine collections. In our patients, clinically significant albuminuria occurred in 36% of persons at first presentation. Since increased risk was associated with hypertension and control of hypertension can slow progression of renal disease, screening for albuminuria and treatment of hypertension should be aggressive in urban populations of African-Americans with diabetes. PMID- 7555558 TI - The prevalence and severity of microvascular complications in pancreatic diabetes and IDDM. AB - OBJECTIVE: To investigate and compare the prevalence, associations, and severity of retinopathy and nephropathy in patients with pancreatic diabetes (PD) and insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: Thirty patients with PD due to alcohol-induced chronic pancreatitis were matched for age, sex, and duration of diabetes with 30 patients with IDDM. Retinopathy was assessed by fluorescein angiography using the Wisconsin classification. Renal function was assessed by albumin excretion rates (AERs) in at least two timed overnight urine collections and glomerular filtration rates (GFRs) by single injection of 51Cr-EDTA. Microalbuminuria was defined as AER 20-200 micrograms/min and nephropathy as AER > 200 micrograms/min. RESULTS: Retinopathy was found in 33% of patients with PD and in 40% with IDDM. The spectrum of disease was similar in the two groups. The geometric mean of AER was 15 micrograms/min (range 1 1,541) in the PD group and 24 micrograms/min (2-2,288) in the IDDM group. Nephropathy was found in 7 PD and in 5 IDDM patients, and a reduced GFR was present in 8 (26%) and 4 (13%) of the two groups, respectively. Microalbuminuria occurred in 9 (33%) and hyperfiltration in 3 (10%) in each group. These differences were insignificant. Retinopathy correlated with AER in both groups. Retinopathy and AER correlated with duration of diabetes in the IDDM but not in the PD group. CONCLUSIONS: Microvascular complications are equally common and severe in PD and IDDM, and improved glycemic control should be the goal in both. PMID- 7555559 TI - Noninvasive detection of functional alterations of the arterial wall in IDDM patients with and without microalbuminuria. AB - OBJECTIVE: To test endothelial function in a group of 10 normoalbuminuric and eight microalbuminuric insulin-dependent diabetes mellitus patients (ages 28 +/- 3 [mean +/- SE] and 28 +/- 1 years, respectively), in comparison with 16 control subjects (age 35 +/- 2 years, normal subjects vs. diabetic subjects P = NS), to identify prestructural abnormalities of the arterial wall. An early stage of vascular involvement seems in fact to be characterized by functional alterations of endothelial control on vascular tone and wall interaction with circulating cells. Furthermore, many recent studies suggest the importance of microalbuminuria as an early marker not only of nephropathy but also of retinopathy and macroangiopathy. RESEARCH DESIGN AND METHODS: Endothelium mediated flow-dependent vasodilation and endothelium-independent vasodilation (induced by glyceryl trinitrate administration) were evaluated in the right common femoral artery by echo-Doppler ultrasound. Arterial wall distensibility was evaluated at the common femoral artery by an echo-tracking system. RESULTS: In spite of a comparable increase in flow velocity, endothelium-mediated vasodilation was significantly reduced in diabetic subjects, particularly in microalbuminuric patients. Endothelium-independent vasodilation was also significantly impaired in diabetic subjects, particularly in microalbuminuric subjects; whereas arterial wall distensibility, an index of the viscoelastic properties of the wall, was similar in the three groups. CONCLUSIONS: These results confirm a reduced vasodilatory capacity in diabetes mellitus, with a more marked alteration in microalbuminuric diabetic subjects. This reliable, noninvasive evaluation of arterial function is particularly useful for early diagnosis of vascular involvement. PMID- 7555561 TI - Absorption of rapid-acting insulin in obese and nonobese NIDDM patients. AB - OBJECTIVE: To study the absorption rate of rapid-acting insulin from subcutaneous injection sites in nonobese and obese non-insulin-dependent diabetes mellitus (NIDDM) patients. RESEARCH DESIGN AND METHODS: Ten nonobese and 10 obese NIDDM patients (body mass indexes 24.1 +/- 0.4 and 31.4 +/- 0.8 kg/m2, respectively) received four subcutaneous injections of 125I-labeled rapid-acting insulin (Actrapid Human, 5 U): three in the abdominal wall above, lateral to, and below the umbilicus; and one in the thigh. The depth of the subcutaneous fat layer was measured using ultrasound techniques. The residual radioactivity was monitored externally for 270 min. RESULTS: The disappearance half-life of 125I-insulin was between 4 and 6 h from all injection sites, with the exception of the upper abdominal area in the nonobese subjects, where it measured approximately 3 h. The residual radioactivity did not differ between nonobese and obese patients measured from any of the sites. In the nonobese group, the most rapid absorption of 125I-insulin was found from the upper abdominal area and the slowest from the thigh. In the obese group, the absorption rates did not differ between sites. No correlation was found between the depth of the fat layer and the residual radioactivity when measured at any site. CONCLUSIONS: Our results indicate that the absorption of rapid-acting insulin is markedly slow in both obese and nonobese NIDDM patients compared with IDDM patients and healthy subjects studied previously. In the nonobese group, the most rapid absorption of 125I-insulin is obtained after injection into the upper abdominal area. Inter- and intraregional differences are small in the obese patients. Consequently the choice of injection site is of little importance in this group. PMID- 7555560 TI - Angiotensin I-converting enzyme gene polymorphism is associated with myocardial infarction, but not with retinopathy or nephropathy, in NIDDM. AB - OBJECTIVE: To clarify the relationship between the angiotensin I-converting enzyme (ACE) gene polymorphism and diabetic micro- and macroangiopathy in patients with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: We examined 267 NIDDM patients with various stages of diabetic retinopathy, 61 patients with myocardial infarction (MI), and 136 patients without MI. An insertion/deletion polymorphism of the ACE gene was typed by polymerase chain reaction. RESULTS: Although no association was found between ACE gene polymorphism and diabetic retinopathy or nephropathy, this polymorphism was associated with MI in the patients with NIDDM. Homozygotes for the deletion polymorphism (DD genotype) were found more frequently in diabetic patients with MI (31.1%) than in diabetic patients without ischemic heart disease (16.9%), with a relative risk of 2.22 (95% confidence interval 1.11-4.46, P = 0.024). CONCLUSION: These data indicate that ACE gene polymorphism is associated with MI, but not with retinopathy or nephropathy, in patients with NIDDM and suggest that the ACE gene confers susceptibility to diabetic macroangiopathy but not to microangiopathy. PMID- 7555562 TI - Apolipoprotein E polymorphism and insulin levels in a biethnic population. AB - OBJECTIVE: To study the association of apolipoprotein E (apoE) polymorphism with important cardiovascular risk factors other than cholesterol levels, such as insulinemia and insulin resistance. RESEARCH DESIGN AND METHODS: In this report, we study the association of three major apoE phenotypes (apoE 3/2, apoE 3/3, and apoE 4/3) with indicators of insulin resistance such as fasting insulin, glucose, and lipid levels in 320 nondiabetic Mexican-Americans and non-Hispanic whites from San Antonio, TX. RESULTS: The two ethnic groups differed in the frequencies of the three major apoE phenotypes. However, the associations of these phenotypes with lipid and insulin levels were similar in both ethnic groups and in both sexes. Compared with the other two major apoE phenotypes, the apoE 3/2 phenotype was associated with lower levels of total and low-density lipoprotein cholesterol and lower levels of fasting and 2-h postload insulin. CONCLUSIONS: In addition to the association with cholesterol levels, the variability at the apoE locus may be associated with a much broader set of metabolic factors that relate to insulin resistance. PMID- 7555563 TI - Retinopathy in animal models of diabetes. PMID- 7555564 TI - Twin studies in insulin dependent diabetes and other autoimmune diseases. PMID- 7555566 TI - Treatment with insulin infusion pumps and ketoacidotic episodes: from physiology to troubleshooting. PMID- 7555565 TI - Heparan sulphate in the pathogenesis of diabetic nephropathy. PMID- 7555567 TI - A new look at viruses in type 1 diabetes. PMID- 7555568 TI - Percussion and physical diagnosis: separating myth from science. AB - There are three percussion sounds, which are easily distinguishable by objective measures: tympany (heard with percussion over the intestines), resonance (heard over the normal lung), and dullness (heard over the liver or thigh). The percussion sound that is produced reflects the ease with which the body wall vibrates, which in turn is influenced by many variables, including the strength of the stroke, the condition and state of the body wall, and the underlying organs. Underlying organs or disease may cause dullness to occur at distant sites. There is good interobserver agreement among clinicians with regard to calling a particular percussion sound dull, resonant, or hyperresonant. In contrast, there is very poor interobserver agreement among clinicians using percussion to measure the span of a particular organ. The use of comparative percussion can detect most large pleural effusions, but this method is able to detect only a few pneumonias. Shifting dullness is a reliable and fairly accurate sign for the detection of ascites. Both of these techniques can still be recommended after a review of the literature. Topographic percussion (e.g., using percussion to locate the heart, liver, and spleen borders or dimensions) has poor reproducibility, is significantly inaccurate in many patients, and has little clinical utility; it should be abandoned. Its fundamental principle--that sound waves penetrate only several centimeters of tissue, resulting in a note reflecting abnormalities only in this layer of tissue--is incorrect. Auscultatory percussion offers no advantage over conventional percussion, with the possible exception of auscultatory percussion of the shoulder. Auscultatory percussion should be abandoned as a bedside diagnostic technique. PMID- 7555569 TI - Cogan and Verhoeff: a friendship of genius. PMID- 7555571 TI - The origins of the National Eye Institute 1933-1968. The Fifth Charles B. Snyder Lecture. AB - Many organizations and individuals prompted the authorization of the National Eye Institute by the United States Congress. The United States Marine Hospital and Public Health Service established a Laboratory of Hygiene in 1887, which became the research center of the National Institute of Health in 1930. The Albert D. and Mary Lasker Medical Foundation was mainly responsible for the 1945 conversion of the American Society for the Control of Cancer to the American Cancer Society, dedicated to medical research. Mildred Weisenfeld, a patient with retinitis pigmentosa, founded The Fight for Sight! in 1946 to provide funds for eye research. The Laskers invited Miss Weisenfeld to testify in support of a National Institute of Neurology, and her appeal was so persuasive that it emerged as the National Institute of Neurological Diseases and Blindness. Congressman John Fogarty and Senator Lister Hill directed the attention of the Congress and the public to the need for the Federal support of medical research. In 1960 Jules Stein, the legendary founder of the Music Corporation of America (MCA), established a new philanthropy, Research to Prevent Blindness, which provided skilled leadership in detailing the need for research in blinding disease and obtaining Congressional and Presidential approval of a new institute. The Committee for Research in Ophthalmology and Blindness was instrumental in bringing the groups interested in the welfare of the blind into harmony with groups concerned with medical research in blinding disease. The Association of University Professors of Ophthalmology was the first medical society to support the need for a National Eye Institute. The National Eye Institute was formally approved 8 August 1968 and celebrated its 25th anniversary in 1993. PMID- 7555570 TI - Toichiro Kuwabara on Dave Cogan. An interview. Interview by Sally Smith Hughes. AB - In this interview, Dr. Kuwabara recounts his first meeting with Dr. Cogan and describes his work at the Howe Laboratory, their pioneering research on diabetic retinopathy, histochemical and electronmicroscopical studies. This warm recollection exemplifies the mutual respect and deep friendship between Drs. Kuwabara and Cogan. PMID- 7555572 TI - A history of low vision and blind rehabilitation in the United States. AB - The author presents a history of services for the blind and those with low vision in the United States focusing on the areas of orientation and mobility, and rehabilitation teaching. The controversies involving the development of these specialties are discussed. Emphasis is placed on the role of Father Thomas Carroll in the establishment of low vision and blind services for the military and veterans administrations. Published data regarding the past role of ophthalmologists in obtaining low vision services for patients is reviewed. The opportunity for a more active role for ophthalmologic involvement in the rehabilitation of the low vision and blind patient is discussed. PMID- 7555573 TI - The blindness of John Milton. AB - John Milton (1608-1674) often has been regarded as the greatest poet of his time, yet he did not compose his most famous work, Paradise Lost, until after he had become blind in both eyes. On the basis of clues in Milton's writings, several possible diagnoses have been advanced to explain his loss of vision. The evidence for and against each theory is presented. PMID- 7555575 TI - Pioneers in the history of orbital decompression for Graves' ophthalmopathy. R.U. Kroenlein (1847-1910), O. Hirsch (1877-1965) and H.C. Naffziger (1884-1961). AB - Orbital decompression for Graves' ophthalmopathy began in 1890 when Dollinger adapted Kroenlein's technique for removal of an orbital dermoid cyst to decompress into the subtemporal fossa. Modifications occurred over the years to decompress the orbit into the paranasal sinuses and/or the anterior cranial fossa. The lives of three surgeons who initially performed these operative procedures are reported. PMID- 7555574 TI - The tarsectomy operation of A.P.L. Gillet de Grandmont (1837-1894) and its periodic rediscovery. AB - During the past two centuries, several methods of ptosis repair have been described and refined, but each method has typically been subject to cycles of popularity, neglect and reincarnation. Repair of ptosis through partial resection of the tarsus is one such example. Although Sir William Bowman included tarsectomy with his historic levator resections, the French ophthalmologist, A.P.L. Gillet de Grandmont was the first to specifically emphasize resection of tarsus for the correction of ptosis. Yet contemporary advocates of tarsectomy for ptosis repair have generally failed to recognize Gillet de Grandmont's contribution and his role in the history of ptosis surgery. For this reason, a review of this subject is presented. PMID- 7555576 TI - Brain wars: passion and conflict in the localization of vision in the brain. AB - David Ferrier and Hermann Munk, both pioneers in early brain research, engaged in a running controversy regarding the primary vision center in the brain. Both misinterpreted their experimental observations, but in different ways. Ferrier placed vision in the parietal lobe, Munk in the occipital lobe, However, Munk also felt he had observed a 'psychic blindness' in his animals, which was in reality only a central scotoma or other primary vision defect, and not the impairment of higher visual capacities that he proposed. Munk's concept has been repeatedly cited as the experimental correlate of visual agnosia, but is inherently fallacious. PMID- 7555577 TI - Jaeger, about glaucoma. AB - E. Jaeger published the first illustration of the glaucomatous cup. Throughout his clinical career he published multiple observations about findings in glaucoma. In his book Ueber Glaucoma he praised and advocated A. von Graefe's iridectomy but contradicted Graefe's concept of the etiology being a localized choroiditis in favor of systemic gout. PMID- 7555578 TI - Optical uses of fans. PMID- 7555579 TI - Neurology of the ocular muscles; emergence of a neuro-ophthalmologist. A personal history and tribute to David Cogan. PMID- 7555581 TI - William Fisher Norris: a Philadelphia ophthalmologist. AB - In 1870, William Fisher Norris returned from studies abroad to find himself at the forefront of new developments in American ophthalmology. Recognition of the specialty was just beginning and Norris was named Professor of Ophthalmology in the department just established at the University of Pennsylvania. Norris was well-qualified for the job. As a member of a prominent Philadelphia family he had the right connections. He was dedicated to the science of ophthalmology and to medical education. He was a born leader, bending his efforts toward the development of a new department, and recruiting a faculty all dedicated to the creation of a new and better specialty. PMID- 7555582 TI - E.J. Curran, M.D., D. Ophth. An historical vignette. AB - An historical vignette recounting the life of E.J. Curran, M.D., from his beginnings in Australia to his medical education at some of the most prestigious institutions of that era. His significant contributions as chairman of the University of Kansas Department of Ophthalmology are discussed. He became a national figure in ophthalmology when he elucidated the pathophysiology behind pupillary block glaucoma and the reason peripheral iridotomy would permanently relieve this condition. PMID- 7555580 TI - William Thomson and Joseph Janvier Woodward. AB - William Thomson and Joseph Janvier Woodward were two of several exceptionally versatile and highly productive young physicians who volunteered for service with the Union Army at the outbreak of the Civil War, and then were subsequently assigned to the Washington Area where they played significant roles and made major contributions towards the development of the Army Medical Museum. Both pioneered in photomicrography. While Thomson deserves priority, Woodward was the more prolific contributor whose work and publications helped draw attention to the Army Medical Museum as a center for excellence in pathology. After the War Thomson returned to Philadelphia where his interests in photography stimulated his pursuit of optics and eventually his becoming one of the first American physicians to specialize in ophthalmology. He became Professor of Ophthalmology at Jefferson Medical College. PMID- 7555583 TI - Pope John XXI, ophthalmologist. AB - Pope John XXI was an ophthalmologist before becoming Pope. He was born in Lisbon and trained at University of Paris and was part of the early medical school faculty in Siena. He became physician for Pope Gregory X and shifted to church duties. He rose quickly in the ranks of the Catholic church and was elected Pope in 1276 AD. His term was short. He died when construction in the Papal Palace at Viterbo, collapsed on him. His text book is in two parts with introductions to the eye followed by descriptions of diseases with their mostly medical treatments. He concludes with a section about his own wonderful waters. The work survived and a copy was even found amongst Michelangelo's papers. Unfortunately the book turns out to be a plagiarism of two earlier text books. His most notable achievement supposedly was discovering that glaucoma was a disease with a hard eye. It turns out that this section is referring only to a suppurative external disease with indurated lids called sclerophthalmia. PMID- 7555584 TI - Did George McClellan commit malpractice? AB - In several sources, it is stated that Dr. George McClellan was one of the first surgeons to lose an ophthalmic malpractice case in the United States. Examining the original sources, one can conclude that he actually won a libel suit. The trial testimony gives a useful insight into cataract surgery in the 1820s as well as the medical climate of that period. This trial revealed to the public the existence of the secret medical society, Kappa Lambda. PMID- 7555587 TI - Standard for clinical electroretinography (1994 update). PMID- 7555586 TI - Alex E. Krill: a brief biography of his life and final days. AB - At the time of his death in 1972 at 44 years, Alex E. Krill had an established reputation as an authority on the topic of inherited retinal and choroidal dystrophies. He lived to see the publication of the first volume of his two volume textbook on Hereditary Retinal and Choroidal Diseases but perished in an airline crash before the completion of the second volume of this text. Although Dr. Krill had completed a substantial portion of the material for this second volume, the publication of a second volume would unlikely have resulted without major additional efforts from two of his colleagues Drs. Desmond Archer, Belfast, Northern Ireland, and August Deutman, Nijmegen, the Netherlands. This work is a testimonial to the scope of Alex Krill's knowledge of various retinal disorders and their proper investigation and classification by electrophysiologic and psychophysical procedures. It is also a lasting tribute to his memory by two generous and devoted colleagues. PMID- 7555585 TI - Life events and visual symptoms of Adolf Hitler (with personal anecdotes). PMID- 7555588 TI - Visual dysfunction in patients with mitochondrial myopathies. I. Electrophysiologic impairments. AB - Seventeen patients with biopsy-confirmed mitochondrial progressive external ophthalmoplegia underwent electroretinography and visual evoked potential testing to checkerboard-reversal stimuli to investigate subclinical visual dysfunction. Seven patients (41%) had impaired Snellen visual acuity that was never less than 0.6. Thirteen patients (76%) showed electroretinographic and/or visual evoked potential alterations, whereas six (35%) showed impairment on both tests. Two patients showed delayed VEP P100 latency without fundus, electroretinographic or visual acuity anomalies. Visual dysfunctions were not related to age at onset and course of the disease. PMID- 7555590 TI - Does corneal grafting alter the electroretinogram? AB - Ten patients who required corneal graft operations and who had normal retinal function were examined by standardized electroretinography preoperatively and on two occasions postoperatively. No significant change was observed when the various preoperative and postoperative responses were compared. Thus, corneal grafting per se does not affect the electroretinogram. Electrophysiologic tests have been used to assess retinal function in the presence of opaque media. An important consideration in any such measurements is the effect of the opaque media themselves on the response. PMID- 7555589 TI - Visual dysfunction in patients with mitochondrial myopathies. II. Contrast sensitivity function. AB - Contrast sensitivity function with stationary and temporally modulated (8 Hz) black and white vertical sinusoidal gratings was investigated in 11 patients with biopsy-confirmed mitochondrial progressive external ophthalmoplegia. To evaluate contrast sensitivity function results, the contrast sensitivity versus spatial frequency experimental data for each subject were fitted with a second-order polynomial, and the coefficients of the best-fitting function from normal and patient groups were compared. Patients with progressive external ophthalmoplegia, as a group, showed a decreased sensitivity at the intermediate spatial frequencies (i.e., around 1.4 c/deg) with stationary gratings and through a wider range of spatial frequencies with temporally modulated gratings. These findings were confirmed by analyzing contrast sensitivity functions from each individual. The study showed that contrast sensitivity function detects visual function abnormalities noninvasively in a number of patients with mitochondrial progressive external ophthalmoplegia with unaffected Snellen visual acuity. PMID- 7555592 TI - An improved holder for the DTL fiber electrode in electroretinography. PMID- 7555593 TI - Adrenergic effects on the corneal and intraretinal direct-current electroretinogram and on the standing potential of albino rabbit eyes. AB - This study was undertaken to investigate further the responsiveness of the albino rabbit retinal pigment epithelium and the inner retina to adrenergic agents as reflected in changes of the direct-current electroretinogram and of the standing potential of the eye. After unilateral vitrectomy on albino rabbits, a continuous intraocular perfusion with a reference solution was established. The reference solution was then alternated with the test solution. The direct-current electroretinogram and the standing potential were recorded from both eyes with a scleral contact lens and a reference electrode connected to matched calomel half cells. An in vivo experimental technique that allows intraocular perfusion of a test substance and simultaneous intraretinal microelectrode measurements was also used. The alpha-adrenergic agonist phenylephrine (0.04 microM, n = 8) produced a reversible increase in c-wave amplitude (48%, p < 0.001) and also a small increase in b-wave amplitude (12%, p < 0.002). There was no significant influence on the a-wave amplitude. The standing potential was elevated at 1694 +/- 362 microV (mean +/- SEM) (p < 0.002). The alpha 2-adrenergic agonist clonidine caused similar effects on the electroretinogram, although at a higher concentration (40 microM, n = 5), with an elevation of the c-wave (25%, p < 0.001) and a small b-wave increase (12% p < 0.002). No significant influence on the a-wave or on the standing potential was found. Intraretinal direct-current electroretinogram-recordings during intraocular perfusion with phenylephrine showed an increase in transepithelial potential (p < 0.004; n = 6), accompanied by a reduction of the slow PIII (p < 0.0035; n = 6). The c-wave increase resulting from alpha-adrenergic stimulation seems to be generated partly across the retinal pigment epithelium, with an increase in transepithelial potential, combined with a reduction of the slow PIII. The elevation of the b-wave amplitude, together with the influence on the slow PIII, suggests alpha adrenergic effects also on the inner retina. The experimental technique used in this study with intraocular perfusion after vitrectomy and simultaneous intraretinal direct-current recordings seems to be a practicable method for studies of the influence of pharmacologic agents on the retina and the retinal pigment epithelium. PMID- 7555591 TI - Amplitude scaling relationships of Burian-Allen, gold foil and Dawson, Trick and Litzkow electrodes. AB - The bipolar Burian-Allen electrode represents the International Society for Clinical Electrophysiology of Vision standard for recording the electroretinogram. With prolonged recording there is a high risk of corneal abrasion from the electrode, while alternatives such as gold foil electrodes or fibers represent less risk. The standards require that alternative electrodes be demonstrated to give equivalent waveform and amplitudes. Electroretinograms were recorded with the bipolar Burian-Allen electrode and four alternative electrode configurations: a unipolar Burian-Allen electrode, a bipolar and monopolar gold foil electrode and a Dawson, Trick and Litzkow fiber electrode with all other recording conditions identical. The results represent a guide for comparisons of electroretinograms between studies using these electrodes. Recordings were made from two subjects for all five electrode configurations and six additional subjects with unipolar gold foil and bipolar gold foil electrodes alone. Flash stimuli over a range of intensities from full intensity to -1.5 log units were used. Recordings were repeated in the one session and on a subsequent session to provide test-retest reliabilities. Significant (p < 0.0001) differences in b-wave amplitude resulting from electrode type and intensity were demonstrated. The unipolar Burian-Allen and unipolar gold foil electrodes produced the greatest amplitude responses. The alternatives to the bipolar Burian-Allen electrode were equally or more reliable. The Dawson, Trick and Litzkow electrode produced lower amplitude response than the bipolar Burian-Allen electrode but was the only one with significantly greater between-session reliability. PMID- 7555595 TI - Flash visual evoked response binocular summation in normal subjects and in patients with early-onset esotropia before and after surgery. AB - Flash visual evoked responses were recorded and visual evoked response binocular summation was assessed in normal children between the ages of 1 and 58 months, in normal adults and in children with early-onset esotropia before and longitudinally for 1 year after surgical binocular alignment. Normal flash visual evoked response binocular summation started in the range of facilitation (> 2.0) at 1 month of age and decreased to adult levels by 3.7 months of age. The shape of the flash visual evoked response binocular summation function obtained from the patients with early-onset esotropia, appeared similar to that of normal subjects; however, the rapid decrease in flash visual evoked response binocular summation from facilitation to normal adult levels occurred after surgical binocular alignment. In normal adults, flash visual evoked response binocular summation was significantly reduced by a 40-diopter base-in prism, suggesting that binocular misalignment was not the reason for the facilitation in flash visual evoked response binocular summation in either childhood population. It is proposed that this facilitation may reflect a process that leads to binocularity and that develops rapidly with binocular alignment. PMID- 7555596 TI - Simultaneously recorded retinal and cerebral potentials to windmill stimulation. AB - Visual evoked retinal and cerebral potentials were recorded to onset rotation of an isoluminant sectored disc. While the retinal potentials recorded to onset rotation closely resembled the electroretinogram to a checkerboard or stripe pattern of fixed element size, the visual evoked potential changed interindividually and intraindividually from a fast positive wave at high contrasts, velocities and number of windmill segments to a later negative component at low contrasts, velocities and windmill segments. With change in luminance, contrast, speed and extent of rotation field size and number of disc segments, the visual evoked potential was generally less affected than the electroretinogram. PMID- 7555594 TI - Light adaptation of the human photopic oscillatory potentials: influence of the length of the dark adaptation period. AB - Photopic electroretinograms recorded immediately after a period of dark adaptation show a regular increase in amplitude with time spent in light. The retinal mechanisms at the origin of this light adaptation effect remain obscure. The purpose of this study was to investigate the duration of the dark adaptation period needed to produce an optimal light adaptation effect as demonstrated by photopic oscillatory potential recordings. Our results indicate that the light adaptation effect can be separated into two distinct processes. The first one, activated early in the dark adaptation process, reduces the amplitude of the fourth oscillatory potential to 32% of control after less than 5 min of dark adaptation, while the second process, activated after more than 10 min of dark adaptation, appears to impact solely the amplitude of the earlier oscillatory potentials 2 and 3. Our results suggest that the light adaptation effect is mediated by two distinct retinal pathways or mechanisms. PMID- 7555597 TI - The effect of non-enzymatic glycation on recombinant human aldose reductase. AB - It has been demonstrated that activation of aldose reductase (AR; EC 1.1.1.21) in diabetic tissues plays an important role in the pathogenesis of diabetic complications. In the present study, the effects of non-enzymatic glycation of recombinant human AR (rhAR) on enzyme activity and affinity for its substrate (glyceraldehyde), co-factor (NADPH) and inhibitors (ARI; Sorbinil, Tolrestat, AL 1576 and Statil) were examined. Although rhAR was successfully non-enzymatically glycated with HPLC-purified [3H]D-glucose, the Michaelis constant (Km) and catalytic efficiency (Kcat/Km) for glyceraldehyde, the Km for NADPH and the inhibitor constant (Ki) for ARI did not change. These results suggest that the mechanism of AR activation and its insensitivity to inhibition observed in diabetic tissues cannot be attributed to its non-enzymatic glycation. PMID- 7555598 TI - Glucose tolerance in rural diabetic Thais, first-degree relatives and non diabetic controls. AB - To determine whether non-insulin-dependent diabetes mellitus (NIDDM) in a rural Thai population is characterised by insulin resistance and hyperinsulinaemia, 17 unselected diabetic outpatients from a regional hospital, five first-degree relatives and 10 healthy controls were studied. Subjects in these groups were matched as closely as possible for age and sex, and mean body mass indices were similar (mean +/- S.D.; 21.8 +/- 5.5, 20.6 +/- 1.4 and 21.8 +/- 2.3 kg/m2, respectively, P > 0.5). Beta-cell function (%B) and insulin sensitivity (%S), expressed relative to values for non-diabetic Caucasians, were assessed mathematically using the 'CIGMA' model and plasma glucose and insulin achieved after a standard 1-h glucose infusion. The diabetic patients had higher fasting plasma glucose concentrations than the controls (8.6 +/- 4.0 vs. 4.6 +/- 0.4 mmol/l, P < 0.01) but plasma insulin levels were comparable (geometric mean [ S.D.-+S.D.]; 4.0 [1.7-9.4] vs. 4.0 [1.7-9.2] mU/l, P > 0.1). %B in the diabetic group (21% [10-41]) was lower than in the controls (128% [88-187], P < 0.001) while %S tended to be higher (185% [86-400] vs. 111% [49-251], 0.1 > P > 0.05). Relatives had intermediate values of both variables. %S and %B correlated poorly in the diabetic group (P > 0.1) but together accounted for 90% of the variation in basal plasma glucose (multiple r = 0.95, n = 17, P < 0.0001). Beta-cell dysfunction appears the primary defect in diabetic patients from a Thai subsistence farming population. Insulin resistance may not always characterise NIDDM in geographical areas where a 'thrifty genotype' would be expected; other factors associated with diabetes in developing countries (such increased susceptibility to serious infections) may also influence diabetes prevalence. PMID- 7555600 TI - Effects of an individual intensive educational control program for insulin dependent diabetic subjects with poor metabolic control. AB - The aim of our study was to evaluate the efficiency of an individual intensive educational control program on improving the metabolic control of insulin dependent diabetic patients at short- and long-term follow-up. Fifteen insulin dependent diabetic subjects with poor metabolic control (hemoglobin A1c > 9%) were included. At entry, their knowledge of diabetes (DKQ2 test), total energy intake and its distribution, insulin schedule, technical skill for insulin administration and self monitoring of blood glucose were evaluated. According to the initial evaluation, individual goals were stipulated and monitored in weekly visits. Individual life-style was particularly kept in mind. Thereafter, patients were switched to our ambulatory clinic for outpatients. At 1, 6, 12 and 24 months of follow-up, the items analyzed at the beginning were reevaluated. After 1 month, the program produced a significant decrease in hemoglobin A1c and an increase in knowledge of diabetes. The same beneficial effects were present at 6, 12 and 24 months evaluation compared to those values recorded at entry. There were neither major changes in dietary intake nor insulin schedule nor any increase in the frequency of hypoglycemic episodes. In conclusion, our program (5.2 +/- 0.8 weekly visits) significantly reduced and sustained hemoglobin A1c values close to those levels recommended by multicenter controlled trials. We consider that our program produced two major changes: a long-lasting improvement in knowledge of diabetes and an increase in self-monitoring blood glucose which provided the key for optimal self-regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555599 TI - Serum 25-hydroxyvitamin D3 levels decreased in impaired glucose tolerance and diabetes mellitus. AB - A cross-sectional survey was carried out in a New Zealand Polynesian and Caucasian workforce of 5677 staff aged 40-64 years to determine whether serum concentrations of 25-hydroxyvitamin D3 are altered in people with newly diagnosed diabetes mellitus and impaired glucose tolerance (IGT). Serum 25-hydroxyvitamin D3 concentration was significantly lower in newly detected cases with diabetes and IGT (n = 238) compared with controls individually matched by sex, age (+/- 2 years), ethnicity, and date of interview (mean (S.D.): 69 (31) vs. 76 (34) nmol/l; P = 0.0016). Among controls, serum concentrations were significantly lower in Maori (mean (S.E.) = 65 (5) nmol/l; P = 0.0013) and Pacific Islanders (59 (4) nmol/l; P = 0.0001) compared with Europeans (82 (3) nmol/l), after adjusting for age, sex, and time of year. We conclude that diabetes and IGT are associated with low serum concentrations of 25-hydroxyvitamin D3 and that low concentrations of this hormone in New Zealand Polynesians may partly explain their increased prevalence of diabetes/IGT compared with Europeans. PMID- 7555601 TI - Metabolic control quality and free radical activity in diabetic patients. AB - The role of oxidative/reductive balance derangement in the pathogenesis of diabetic microangiopathy has often been discussed in the last few years. Therefore, we decided to evaluate the influence of intensive insulin therapy on selected indicators of free radical production. The levels of plasma hydrogen peroxide (H2O2) and serum malonyldialdehyde (MDA) were estimated in 15 patients with Type 1 and 15 with Type 2 diabetes before and after 2 weeks of intensive treatment. The initial H2O2 and MDA levels in all cases were significantly higher than in controls. After 2 weeks of treatment, the values for both estimated parameters were significantly lower; however, they were still higher than in the control group. Our results seem to confirm the previous suggestions concerning the relation between metabolic disturbances and oxidative stress in diabetic patients. PMID- 7555602 TI - Continuous subcutaneous insulin infusion (CSII) in children and adolescents with chronic poorly controlled type 1 diabetes mellitus. AB - This study was undertaken to determine if continuous subcutaneous insulin infusion (CSII) could improve control, diminish episodes of diabetic ketoacidosis (DKA), decrease number of hospitalizations and save health care expenditure in children and adolescents with long-standing poorly controlled diabetes mellitus. A retrospective analysis was done of six patients with type 1 diabetes for 1-8 years, of whom 4 were non-adherent to the diabetic regimen (ages 12-16.5 years) and 2 of whom had brittle diabetes (ages 8.5 and 10 years). These patients were non-randomly placed on the MiniMed (Sylmar, CA) CSII system. The year prior to CSII was compared with the year during pump use. Glycoslyated hemoglobin (HbA1c), spot urinary microalbumin, total cholesterol, insulin dose, growth velocity, number of convulsions and hypoglycemic events, number of episodes of DKA, number of hospitalizations and total inpatient costs were compared for the 2 years. The year prior to CSII, mean HbA1c was 9.02% (S.D. = 0.86%), mean number of hospitalizations was 5.2/patient (S.D. = 4.6), mean number of hospital days was 20.8/patient (S.D. = 14.7) and mean cost was $29330/patient (S.D. = $22804). During 1 year of CSII, mean number of hospital days decreased to 5 days/patient (S.D. = 0.8, P = 0.016), mean number of hospitalizations (including DKA and pump initiation) decreased to 1.7/patient (S.D. = 0.7, P = 0.31), mean inpatient costs decreased to $12762/patient (S.D. = $5.950, P = 0.047). HbA1c, urinary microalbumin, cholesterol, insulin dose and growth velocity did not change in a statistically significant manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555603 TI - Renin-aldosterone axis in normoalbuminuric insulin-dependent diabetes mellitus patients with glomerular hyperfiltration. AB - The renin-aldosterone axis was evaluated by captopril test in 22 normotensive normoalbuminuric insulin-dependent diabetes mellitus (IDDM) patients with and without glomerular hyperfiltration. Patients were divided into those with glomerular hyperfiltration (Hf-IDDM) and with normal glomerular filtration rate (GFR; Nf-IDDM) according to the upper limit of GFR (134.7 ml/min per 1.73 m2). Sixteen normal individuals were also studied. GFR was measured by the 51Cr-EDTA single injection method, extracellular fluid volume as the distribution volume of 51Cr-EDTA, and blood volume using 51Cr-sodium chromate-labelled red blood cells. Twenty-five mg of captopril were administered per os and plasma renin activity (PRA; RIA), plasma aldosterone (RIA) and blood pressure were measured at 0 and 120 min post-captopril. PRA at time zero (Hf-IDDM = 2.4 +/- 1.7; Nf-IDDM = 2.5 +/ 1.9; controls = 1.0 +/- 0.6 ng/ml/h) and at 120 min (Hf-IDDM = 9.9 +/- 8.3; Nf IDDM = 11.2 +/- 8.9; controls = 5.4 +/- 5.7 ng/ml/h) was higher in IDDM patients than in controls (P = 0.01). The increase of PRA was similar in patients (Hf-IDDM = 7.5 +/- 7.3, and Nf-IDDM = 8.7 +/- 7.2 ng/ml/h) and controls (4.4 +/- 5.3 ng/ml/h). There was no difference in PRA levels between Hf-IDDM and Nf-IDDM patients. PRA did not correlate with GFR, aldosterone, blood pressure, blood volume, duration of diabetes, 24-h urinary sodium and metabolic control indexes. Plasma aldosterone and the magnitude of its decrease after captopril was similar among patients and controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555604 TI - Determinants of vibration perception thresholds in IDDM and NIDDM patients. AB - The relative influence of diabetes mellitus-related and physiological factors on vibration perception thresholds was assessed in 353 patients in a hospital-based setting (173 insulin-dependent and 180 non-insulin dependent patients, aged 51.1 +/- 15.9 years) and 80 healthy controls (aged 43.3 +/- 15.2 years) employing a Biothesiometer. Vibration perception thresholds were bilaterally measured at the thumbs, medial malleoli and halluces. Sixty (17.0%) older patients had off-scale thresholds (> 50 V). As no systematic side differences were found, values of contralateral sites were averaged. Considering the effects of age, height, gender and skin temperature in controls, age accounted for 46.7 and 52.2% threshold variance at the ankles and halluces, respectively, while height explained 5.1 and 5.1%, respectively. At the thumbs, only age was of relevance. Age relationships with vibration thresholds in health did not differ from published reports at any site. In the patient group, influences of age, height, gender, skin temperature, years from diagnosis, HbAlc, serum creatinine, drop in systolic blood pressure on standing, and ankle/arm blood pressure indices were assessed for each type of diabetes. For both types, age and height again had relevant effects at the lower extremities as did age and gender at the thumbs. Skin temperature was only marginally significant at the halluces of NIDDM patients. Of the disease-related factors, HbAlc had the strongest effect: for both IDDM and NIDDM higher levels were associated with lower vibration sensitivity. Increasing disease duration led to significantly higher thresholds in IDDM patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555605 TI - The cerebral vascular response to a rapid decrease in blood glucose to values above normal in poorly controlled type 1 (insulin-dependent) diabetes mellitus. AB - The effect of rapid lowering of blood glucose on cerebral blood flow (CBF) was studied in 10 Type 1 (insulin-dependent) diabetic patients (age 23.5 +/- 3.8 years; mean +/- S.D.) with longstanding, poor metabolic control (HbAlc 11.2 +/- 1.0%; normal value 4.0-5.3%) using an intravenous xenon 133 single photon emission computed tomography technique. After a fall in blood glucose, during 81 +/- 11 min (mean +/- S.E.M.), from 18.2 +/- 1.4 mmol/l to 9.2 +/- 0.9 mmol/l CBF was unchanged, but increased from its initial value of 48.8 +/- 2.9 ml/100 g per min to 57.1 +/- 2.4 ml/100 g per min (P < 0.001) when the blood glucose level was restored. The CBF was higher in the right compared to the left hemisphere at all measurements (1.8 +/- 0.5 ml/100 g per min, P < 0.01; 1.9 +/- 0.5 ml/100 g per min, P < 0.05; 2.1 +/- 0.7 ml/100 g per min, P < 0.05, respectively). The change in CBF was inversely correlated with time for fall of blood glucose, but there was no correlation with absolute levels of blood glucose. The respiratory end tidal PCO2 decreased during the low blood glucose level, but there was no correlation between the PCO2 and CBF. The cerebral volume was unchanged during the study. The results indicate that in patients with chronic hyperglycemia a rapid fall in blood glucose may cause a rise in CBF of the same magnitude as previously shown during absolute hypoglycemia in patients with well controlled diabetes mellitus and in normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555606 TI - Anemia due to reduced serum erythropoietin concentration in non-uremic diabetic patients. AB - We encountered two patients with non-insulin-dependent diabetes mellitus (DM) who developed normocytic normochromic anemia. Routine hematological examinations revealed no specific causes except for the reduced serum levels of erythropoietin. Since their renal functions were preserved, the anemias may not have been due to chronic renal failure. Treatment with human recombinant erythropoietin (rHuEPO) improved anemia, ascribing the cause of anemia to low levels of erythropoietin in these patients. Underlying common clinical features of the two patients were longstanding poorly controlled diabetes mellitus accompanied with advanced neuropathy. Since erythropoietin production is regulated in part by autonomic nervous system, the results suggest that erythropoietin production could be prematurely impaired in patients with severe diabetic autonomic neuropathy. PMID- 7555607 TI - Peripheral vascular disease in non-insulin-dependent diabetes mellitus in south India. AB - The prevalence of peripheral vascular disease (PVD) was assessed in terms of ankle/brachial index by doppler studies in a large cohort of non-insulin dependent diabetes mellitus (NIDDM) patients in South India. One hundred and ninety-two out of 4941 patients (3.9%) had evidence of PVD. There was a slight female excess in PVD patients. There was a linear increase in prevalence of PVD with increasing duration of diabetes. Multiple logistic regression analyses showed that serum cholesterol, serum creatinine, systolic BP, duration of diabetes and ishaemic heart disease are strong predictive factors for PVD. The prevalence of PVD in South Indians is lower than that reported in European populations. PMID- 7555609 TI - Neltenexine: morphological investigation of protection against elastase-induced emphysema in rats. AB - The instillation of elastase into airways is a widely adopted experimental method to quickly produce emphysematous lesions that mimic human disease anatomically and physiologically. Experiments were undertaken to determine whether of not neltenexine, a new drug active on surfactant production, would diminish the severity of this disease. Anaesthetized rats were instilled tracheally with porcine pancreatic elastase (46 U/mg) dissolved in saline, in a single instillation of 0.33 mg/100 microliters. Neltenexine was administered in one experiment at the dose of 25 mg/kg i.p. daily for 30 days. In a second test, neltenexine was given at the same dose six days before the elastase instillation and then by the same schedule as in the first experiment; this was done in search of a possible preventive action. At the end of the treatment, lungs were removed and fixed, and slices were dehydrated, critically point dried, coated with gold and observed by scanning electron microscopy (SEM). Rats that were both pretreated and treated with neltenexine showed a significant reduction in the alveolar deformation induced by elastase. There were no differences between pretreated and treated animals. These experimental findings suggest that neltenexine might prove to be useful for preventing pulmonary emphysema. Biochemical studies in man are needed to confirm the clinical application of neltenexine. PMID- 7555608 TI - Effects of delapril on stroke, kidney dysfunction and cardiac hypertrophy in stroke-prone spontaneously hypertensive rats. AB - This study was performed to investigate the beneficial effects of prolonged treatment with an angiotensin converting enzyme (ACE) inhibitor, delapril, on the appearance of symptoms of hypertensive cardiovascular disease in stroke-prone spontaneously hypertensive rats (SHRSP). Cardiovascular disease symptoms: stroke, kidney dysfunction and cardiac hypertrophy, were evaluated by monitoring the incidence of stroke signs, urinary excretion of protein and the heart weight, respectively. The SHRSP that were kept under salt-loaded conditions (1% NaCl drinking solution) from six weeks of age developed severe hypertension, showed an increased incidence of stroke signs and increased urinary excretion of protein. Long-term treatment with delapril (10mg/kg/day, p.o. for four weeks) decreased the blood pressure and completely inhibited the incidence of stroke signs and the increase in urinary excretion of protein. In SHRSP that were kept under normal conditions (without 1% NaCl drinking solution), long term treatment with delapril at the same dose decreased the heart weight and, after five weeks of treatment, left ventricular weight was decreased significantly and the wall/lumen ratio of small coronary arterioles and the thickness of the left ventricular wall were decreased slightly. These results indicate that delapril can prevent the development of symptoms of hypertensive cardiovascular diseases: stroke, kidney dysfunction and cardiac hypertrophy, with antihypertensive activity in SHRSP. PMID- 7555610 TI - Prophylactic effect of ornidazole on experimental tetanus in mice. AB - Tetanus is still responsible for many deaths, especially in the developing parts of the world. In this study, the prophylactic effect of ornidazole on experimental tetanus in mice was investigated. The initial minimum lethal dose (MLD) of Clostridium tetani spores was determined in mice and 100 MLD was applied to mice in experiments. Ornidazole was then administered at the dose of 20 mg, 40 mg and 80 mg/kg. In addition, penicillin and horse tetanus serum were also administered to other groups. According to the present results, ornidazole decreased the number of deaths in mice significantly in a dose and drug administration time dependent manner. These results suggest that, together with essential wound care and active immunization, ornidazole (or another nitroimidazole) may be a useful and supportive therapeutic agent in tetanus prophylaxis. PMID- 7555611 TI - Evaluation of in vitro and in vivo antimicrobial activity of a new topical antiinfective agent. AB - ST 1103 (Undecyl [4-N,N,N-trimethylammonium-(R)-3- isovaleroyloxy]-butanoate methanesulfonate) is a novel compound endowed with a broad antimicrobial spectrum. ST 1103 is able to inhibit the in vitro growth of Gram-positive bacteria (mean MIC value of 2.60 micrograms/ml), Gram-negative bacteria (mean MIC value of 27.00 micrograms/ml), yeasts and yeast-like fungi (mean MIC value of 3.76 micrograms/ml), filamentous and dermatophytic fungi (mean MIC value of 18.33 micrograms/ml). Since indirect evidence indicates a poor oral absorbtion, ST 1103 was topically administered to mice with skin infections caused by mixed inocula. In these conditions, ST 1103 was able to cure mice infected with T. quinckeanum, S. aureus as well as immunodepressed mice infected with T. quinckeanum, S. aureus and C. albicans. Conversely, miconazole (reference compound) appeared inadequate, in our experimental conditions, for a definitive therapy of the skin mycosis superinfected by staphylococcus. By using an in vitro 3D-human skin model, ST 1103 was fairly well tolerated in terms of both cell viability and release of inflammatory mediators. In a dermal tolerance study in mice, ST 1103 at a concentration of 1% did not show any sign of local irritation on both intact and abraded skin after an 8-day topical treatment. In conclusion, ST 1103 appears to be a promising candidate for treatment of cutaneous infections caused by mixed microbial pathogens. PMID- 7555612 TI - Effects of lysine-arginine association on immune functions in patients with recurrent infections. AB - Combined L-lysine-L-arginine therapy is capable of inducting recovery in age related decline of thymic activity in mice and in elderly humans. The clinical usefulness of the association has also been shown in children with recurrent respiratory infections, while an increase in the number of CD3+ lymphocytes has been shown in patients with chronic lymphatic leukaemia. Recently, in vitro effects of the association on neutrophil function have been reported. In particular, the association was able to increase random migration, chemotaxis, phagocytosis-associated- and f-MLP-induced chemiluminescence. In this paper the authors evaluate the effects of L-lysine-L-arginine combination (lisargin) on several humoral and cell-mediated immunologic parameters in patients with recurrent infection. An increase of neutrophil random migration and chemotaxis (evaluated by a new technique, based on a computer assisted image processing system) was found. Furthermore an increase in the absolute number of lymphocytes involved in cytotoxic activity and IgG levels was observed. PMID- 7555613 TI - Ciprofloxacin in the management of pulmonary tuberculosis in the face of hepatic dysfunction. AB - Twenty-five patients who had extensive pulmonary tuberculosis and hepatitis induced by antituberculosis drugs were treated with ciprofloxacin together with other relatively non-hepatotoxic drugs, either during the interim phase awaiting recovery of liver function in some, or as definitive therapy as required by the compromised hepatic status of others. Only 22 patients were assessable. All tolerated ciprofloxacin well during the phase of hepatic dysfunction. All patients improved with these ciprofloxacin-containing regimens, but the optimal dosage, specific efficacy and long-term safety of the drug in such cases require further evaluation. PMID- 7555615 TI - RP 59500 postantibiotic effect defined by bacterial ultrastructure. AB - Staphylococcus aureus was exposed to 0.3 microgram/ml RP 59500, an injectable streptogramin, 0.75 minimum inhibitory concentration (MIC) for six hours, washed, resuspended in fresh medium and incubated for ten hours. The postantibiotic effect (PAE) was determined by growth kinetics and by bacterial ultrastructure. The PAE was eight hours when determined by changes in the cell diameters and three hours when determined by the count of colony forming units (CFU). PMID- 7555614 TI - Design features of a five-year Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT). AB - Young survivors of myocardial infarction represent a poignant challenge to clinical research on atherogenic mechanisms and factors predisposing to and precipitating coronary thrombosis. Young male postinfarction patients are characterized by heavy smoking, dyslipoproteinaemias involving very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), a family history of premature coronary artery disease, hyperinsulinaemic responses to oral and intravenous glucose challenges, an elevated plasma fibrinogen concentration and defective fibrinolytic function. Based on the multiplicity of metabolic and haemostatic disturbances present in these patients, a double-blind, randomized, placebo-controlled angiographic trial was initiated to determine whether bezafibrate, a clofibrate analogue, retards the progression or facilitates regression of premature coronary atherosclerosis. Men under the age of 45 years who survived a first myocardial infarction were screened for participation in the study. A fasting serum cholesterol value > or = 5.2 mmol/l and/or serum triglycerides > or = 1.6 mmol/l after three months of dietary treatment and angiographically demonstrable lesions in at least one coronary segment were required for inclusion. Treatment with diet and bezafibrate (200 mg t.i.d.) or matching placebo is continued for five years during which time re angiography is performed after two years and at the end of the study. The primary aim of the trial is a comparison between the bezafibrate and placebo groups for change in mean minimum luminal diameter between the baseline and five-year coronary angiograms. This report presents the design features of the Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT) and a review of current knowledge of mechanisms underlying premature coronary atherosclerosis and myocardial infarction at a young age. PMID- 7555616 TI - Ulcerogenicity and effect on inhibition of prostaglandin generation of indometacin farnesil, a prodrug of indomethacin, in rat gastric mucosa: comparison with indomethacin or loxoprofen. AB - Indometacin farnesil was compared with indomethacin and loxoprofen in rats to ascertain whether it caused less gastric mucosal damage than the two older drugs. Damage was evaluated in terms of the size of ulcers that formed after oral administration and the changes in concentrations of prostaglandins E2 and I2 in the mucosa. Indometacin farnesil caused less damage than indomethacin and tended to cause less damage than loxoprofen. Indometacin farnesil was less potent than indomethacin in inhibiting prostaglandin generation by gastric mucosa. This property of indometacin farnesil may contribute to the low ulcerogenicity of this compound. PMID- 7555617 TI - Comparison ketoprofen, ibuprofen and naproxen sodium in the treatment of tension type headache. AB - The safety and efficacy of treatment of tension-type headache with either ketoprofen or ibuprofen and naproxen sodium were evaluated in a prospective, randomized, double-blind parallel-group-study in 345 subjects. All patients were valid for evaluation of efficacy and safety. Headache pain intensity and pain relief were measured on categorical verbal scales 30, 45, 60, 120, 180 and 240 min after ingestion of a single dose of 12.5 mg or 25 mg ketoprofen, 200 mg ibuprofen and 275 mg naproxen sodium. At no time in four hours observation the efficacy of the four treatments differed, neither in pain intensity difference nor in the pain relief scale. A statistical comparison test was performed only once analysing the primary efficacy variable, the sum of pain intensity differences. There was no statistically significant difference among all treatments in this respect. The results of this clearly indicate that ketoprofen in a dosage of 12.5 or 25 mg, compared to 200 mg ibuprofen and 275 mg naproxen sodium, is an effective and safe treatment in tension-type headache. PMID- 7555618 TI - Somatostatin 14 and joint inflammation: evidence for intraarticular efficacy of prolonged administration in rheumatoid arthritis. AB - Previous studies with intraarticular administration of somatostatin (SST14) in rheumatoid arthritis showed an antiinflammatory and analgesic effect. The aim of the present study was to demonstrate the efficacy and tolerability of SST14 in rheumatoid arthritis (RA) patients for a longer period of treatment than previously scheduled. Forty-one patients with RA of the knee were treated with a cycle of intraarticular injection of 750 micrograms of SST14, every 15 days. The efficacy of SST14 was evaluated by determining acute phase parameters (erythrocyte sedimentation rate, C-reactive protein [CRP]) and by clinical assessment (pain at rest and on movement, joint tenderness, morning stiffness, spontaneous pain). Additionally, telethermography was performed to evaluate the intensity of the joint inflammation. The tolerability of the treatment was also assessed both by patients and physicians. SST14 produced a reduction in all parameters; this was already statistically significant after the second injection in terms of pain at rest and on movement, and after the third injection for all other symptoms. The treatment showed an excellent tolerability, both local and systemic. Our results indicate the analgesic property of SST14 and demonstrate its capacity to reduce progressively joint inflammation confirmed by thermography and by reduction of pain, after a month of therapy. PMID- 7555619 TI - Why publish three negative articles on carbamazepine as a medication for the treatment of cocaine dependence? PMID- 7555620 TI - Carbamazepine treatment of cocaine dependence: a placebo-controlled trial. AB - Cocaine-induced kindling, which has been hypothesized to underlie cocaine-induced craving, is reversed by carbamazepine treatment. Though preliminary studies showed carbamazepine to be useful for relapse prevention in cocaine-dependent subjects, more recent studies have failed to replicate those findings. We conducted a randomized, double-blind, placebo-controlled trial of carbamazepine 600 mg/day in 40 cocaine-dependent males. During active treatment there were no significant effects of carbamazepine on cocaine use, alcohol consumption, anxiety or depressive symptoms. At three months post-treatment carbamazepine-treated subjects reported fewer drinking days. We conclude that carbamazepine at this dose level is probably not efficacious for treatment of cocaine dependence. PMID- 7555621 TI - Double-blind comparison of carbamazepine and placebo for treatment of cocaine dependence. AB - This study was conducted to determine the effectiveness of carbamazepine (CBZ) for treatment of cocaine dependence. Sixty-two (CBZ = 28, placebo = 34) cocaine dependent (DSM-III-R criteria) volunteers consented to be treated for eight weeks with standardized outpatient individual counseling twice a week plus double-blind CBZ or inactive placebo. During the 8-week trial, both groups showed increased number of urine samples negative for cocaine, significantly (P < 0.01) decreased self-reported cocaine use (money spent and grams used), and decreased Beck Depression Inventory and Symptom Check List-90-Revised (SCL-90-R) total scores. However, there were no significant differences between CBZ and placebo. This study does not support the effectiveness of CBZ for outpatient treatment of cocaine dependence. PMID- 7555622 TI - Carbamazepine treatment for cocaine dependence. AB - We report on a double-blind, placebo-controlled study of carbamazepine (CBZ) treatment for cocaine dependence. A previously reported uncontrolled study found CBZ to be a beneficial pharmacotherapy for cocaine dependence. Statistical analyses were performed on data from 82 subjects who were randomized to 10 weeks' treatment with either CBZ, titrated to 4-12 micrograms/ml, (n = 37) or placebo (n = 45). The two treatment groups did not differ for primary outcome measures of retention time in treatment, urine samples positive for cocaine metabolite, subject reported desire for cocaine or for subject reported side-effects. CBZ was not an effective treatment in this study. PMID- 7555624 TI - Measures of cocaine-seeking behavior using a multiple schedule of food and drug self-administration in rats. AB - Animal models of human drug abuse measuring (i) cocaine-seeking behavior maintained by primary or secondary reinforcers, (ii) the time to extinction of cocaine-seeking behavior, and (iii) following extinction, reinstatement of operant responding resulting in presentation of a cocaine-associated stimulus were developed in rats. Animals were trained to respond on a multiple schedule of food and intravenous cocaine reinforcement during which either food or cocaine paired with auditory or visual stimuli were available during four alternating 30 min schedule components. At the beginning of each experimental session, a 10-min component (stimulus component) was introduced during which the stimuli associated with the primary reinforcers were made available response-contingently. Subsequent non-contingent presentation of food or cocaine at the beginning of the stimulus component produced a significant increase in lever-pressing resulting in presentation of the respective reinforcer-associated stimulus. Removal of cocaine and the associated stimulus during all schedule components led to extinction of drug-seeking behavior within six days. Lever-pressing resulting in presentation of the drug-associated stimulus was subsequently by non-contingent delivery of cocaine, but not by non-contingent presentation of the stimulus alone. These results suggest that different aspects of cocaine-seeking behavior, such as operant responding resulting in presentation of a cocaine-associated external stimulus, time to extinction in the absence of primary reinforcement, and reinstatement of responding following extinction can be measured in the rat. These tests may provide useful tools for the assessment of potential treatment drugs for human cocaine abuse. PMID- 7555623 TI - Factors associated with coping in persons undergoing alcohol and drug detoxification. AB - In order to identify factors associated with the coping styles of detoxifying alcohol and drug patients, the coping styles of 137 alcohol and drug patients drawn from three city, suburban, and rural rehabilitation programs were examined in relation to various indices, including depression and anxiety. The results showed that depression was a moderately useful predictor of wishful thinking--the highest coping style utilized by patients. The relatively infrequent use of problem-focused coping across a range of stressful situations suggests that alcohol and drug patients may benefit from therapeutic programs designed to train them in the use of problem-focused coping strategies. PMID- 7555625 TI - Acute tolerance to the cardiovascular effects of volatilized cocaine free base in rats. AB - The cardiovascular effects elicited by volatilized cocaine were compared to those produced by i.v. drug administration. All of the subjects exposed to volatilized cocaine exhibited an initial increase in mean arterial pressure (MAP), profound bradycardia, and heart blocks. Intravenous administration of cocaine (1.5 mg/kg i.v.), a dose equivalent to the amount of drug subjects received from inhalation exposure, led to a transient increase in MAP but failed to produce any obvious ECG abnormalities or bradycardia; however, increasing the dose to 4.5 mg/kg resulted in considerable toxicity. Finally, the hemodynamic effects elicited by inhalation cocaine were found to undergo rapid tolerance during the first 5-min exposure period and the three subsequent drug exposures. PMID- 7555626 TI - Socio-demographic correlates and pattern of drug abuse in eastern Saudi Arabia. AB - This is a preliminary study of the socio-demographic characteristics and pattern of substance abuse in the eastern region of Saudi Arabia. Clinico-statistical analysis of 116 patients was based on the Diagnostic and Statistical Manual for Mental Disorder, Third Edition, Revised (DSM-III-R) definitions of drug abuse, a semi-structured psychiatric interview and examination, and laboratory investigations. Eighty-three percent of the patients were below the age of 32 years, 52.6% were unemployed and the majority were of intermediate education. Eighty-four percent of the patients abused heroin either alone or in combination with other drugs. Of the patients 31% used alcohol, 26% used cannabis, and 10% used stimulants. The use of other drugs was rare. Heroin abusers were younger with short duration and less polydrug abuse than alcoholics. In comparison with heroin abusers studied abroad, more of our patients were employed and drug use was more chronic, i.e., the mean duration of drug abuse was much longer. PMID- 7555627 TI - Analysis of the antinociceptive actions of the kappa-opioid agonist enadoline (CI 977) in neonatal and adult rats: comparison to kappa-opioid receptor mRNA ontogeny. AB - Earlier reports indicate that kappa-opioid agonists may be especially potent in the formalin test of tonic nociception, and that neonatal rat pups are more sensitive to mu-agonists, when compared to adults. We have assessed the potency of enadoline (CI-977), a novel and selective kappa-opioid agonist, in the formalin and tail-flick nociceptive tests in 3-day-old rat pups and compared their responses to adults in the same tests. In addition, the recent cloning of the kappa-opioid receptor has allowed us to make the first evaluation of the ontogeny of kappa-opioid receptor mRNA in an effort to elucidate a possible mechanism for differences in sensitivity to kappa-opioid agonists. Enadoline was found to be a potent antinociceptive agent in the formalin test; the neonates were eight times more sensitive than the adults. kappa-Opioid receptor mRNA, however, is present in whole brain at adult levels as early as postnatal day 3. Previous studies have shown kappa-opioid receptor levels, as measured by radioligand binding and receptor autoradiography, to be present at postnatal day 3 as well. Consequently, it is unlikely that gross differences in receptor number subserved the modest increase in sensitivity to enadoline observed in the neonates in the formalin test. Enadoline was less potent and less effective in the tail-flick test in the neonates. The adults were similarly insensitive to the antinociceptive effects of enadoline in the tail-flick test. Additional studies indicated that enadoline significantly increased locomotor activity, as assessed by the open-field test, in neonates, while decreasing activity in the adults.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555628 TI - [Preclinical resuscitation--which factors influence the long-term course? A 5 year follow-up]. AB - OBJECTIVE: The factors that influence long-term survival after out-of-hospital resuscitations were investigated. PATIENTS AND METHODS: Between 1985 and 1989, out of a total of 8403 responded emergency calls, 505 resuscitations were undertaken out of hospital by the emergency medical service in Goppingen. All emergency calls were recorded uniformly. Of the 505 resuscitations, 154 were primarily successful (30.5%), and 58 were secondarily successful, i.e. the patients were ultimately discharged from hospital. In 56 of them the further course could be followed at least 5 years after the resuscitation (45 males, 11 females; mean age 57 [10-83] years). The patients' charts were analysed; in 51 cases data could be obtained from the family doctor, from ambulant care or from home visits. RESULTS: 34 patients (60.4%) were still alive 5 years after the resuscitation. The highest death rate (16%) was in the first post-resuscitation year. Prognostically unfavourable factors were: advanced age (P < 0.01), underlying cardiac disease (n = 49; P < 0.025), especially coronary heart disease (n = 34; P < 0.01). Patients with primary ventricular fibrillation and previous myocardial infarction (n = 10) had a poorer prognosis than those with acute infarction (n = 24; P < 0.05). Reduced survival chances occurred in those with impaired left ventricular function, as measured echocardiographically (P < 0.05), or with cardiac arrhythmias, Lown classes III and IV (P < 0.05), as well as in those with severe neurological sequelae (n = 8; P < 0.08). CONCLUSION: Long-term prognosis depends, in the first instance, on the resuscitated patient's basic condition and not so much on the circumstances of the resuscitation. PMID- 7555629 TI - [ACE inhibitor-associated interstitial lung infiltrates]. AB - HISTORY AND CLINICAL FINDINGS: A 60-year-old woman was prescribed Captopril, 37.5 mg daily, for severe left-ventricular dysfunction with associated mitral regurgitation. Left ventricular function improved markedly in the subsequent 6 months and the mean pulmonary capillary wedge pressure became normal. However, exertional dyspnoea unexpectedly did not improve but rather increased and she also developed severe dry cough. TESTS: The chest radiogram and computed tomography revealed multiple small interstitial infiltrates in both lungs without signs of congestion. Body plethysmography did not demonstrate any obstructive or restrictive ventilatory abnormalities. TREATMENT AND COURSE: When captopril was discontinued, the cough improved and the exertional dyspnoea quickly disappeared without any further treatment. All immunological tests, including those for antibodies against bird antigens and for antinuclear antibodies, were negative. The interstitial infiltrates had already markedly regressed 9 days after captopril had been discontinued and they had disappeared after 2 months. CONCLUSION: This case indicates that angiotensin-converting enzyme inhibitors should be considered as a cause of interstitial pulmonary infiltrates associated with dyspnoea. PMID- 7555631 TI - [The folic acid requirement and metabolism]. PMID- 7555632 TI - [Metastatic heart tumors]. PMID- 7555633 TI - [Regulations for clinical trials of drugs and medical products--a stroke of genius of the legislator]. PMID- 7555630 TI - [Necrotizing herpes simplex encephalitis as the cause of a progressive dementia syndrome]. AB - HISTORY AND FINDINGS: A 65-year-old woman was twice hospitalized because of disorientation and insomnia with depression. In the course of antidepressive treatment the symptoms regressed each time so that the diagnosis was made of pseudo-dementia during depression. About a year after the first admission the symptoms recurred, despite continued antidepressive treatment. Clinical criteria now suggested Alzheimer-type dementia as the diagnosis. COURSE AND TREATMENT: The symptoms of dementia increased markedly and generalized seizures requiring anticonvulsive drugs occurred after 4 months. Although IgG antibodies against Herpes simplex virus (HSV) were demonstrated in cerebrospinal fluid (CSF), no antiviral treatment was instituted because HSV encephalitis appeared unlikely at this stage. But the patient's mental and physical state further deteriorated in subsequent months. During an attack of pneumonia the patients became somnolent and went into status epilepticus. Repeat virological examination of CSF now revealed IgG and IgM antibodies against HSV, confirming encephalitis. Her condition markedly improved over 12 days on aciclovir, 750 mg 3 times daily intravenously. 6 weeks later the pneumonia recurred, as did the encephalitis, with apnoeic phases and massive left heart failure. The patient died a few days later. Immunohistochemical tests of brain tissue were positive for HSV type II. CONCLUSIONS: This case report demonstrates that an investigation on dementia must include a complete liquor analysis. If HSV encephalitis is suspected, an antiviral therapy must be administered immediately. Even in adults who seem to have normal immunity HSV type II can cause HSV encephalitis. PMID- 7555634 TI - [Retinal arterial occlusions and vasculitis]. PMID- 7555635 TI - [A new multiligature applicator for the endoscopic therapy of esophageal varices]. PMID- 7555636 TI - [The procedure for elimination in potentially lethal thallium poisoning]. PMID- 7555637 TI - [Is travel prophylaxis worthwhile? An assessment of the economy of preventive measures against malaria, hepatitis and typhus in travelers]. PMID- 7555638 TI - [The recanalization of the chronically occluded infarct vessel in single-vessel coronary disease. The reduction of cardiac events in long-term clinical follow up]. AB - OBJECTIVE: The prognostic significance of recanalisation of a chronically occluded infarct vessel in single-vessel coronary disease remains controversial, in contrast to early re-opening of the infarct vessel in the acute state of infarction. It was the purpose of this prospective study to discover whether successful recanalisation in the former influences the incidence of cardiac events (death, infarction, by-pass operation) and clinical symptoms in the long term. PATIENTS AND METHOD: Recanalisation procedures were successful in 58, unsuccessful in 41 of 99 patients (81 men, 18 women; mean age 55 [28-79] years) with anterior wall (n = 53) or posterior wall (n = 46) myocardial infarction (AMI and PMI, respectively). The two groups were similar with respect to age, sex, left-ventricular function, indication, exercise capacity and premedication. But the interval between infarction and recanalisation was shorter in the patients who had successful recanalisation (5.1 +/- 5.3 vs 7.8 +/- 7.6 months; P < 0.05). Mean follow-up period for all patients was 55.8 +/- 8.9 months after the recanalisation procedure. RESULTS: There were significantly fewer cardiac events after successful than failed recanalisation, both for the total group of patients (5% vs 23%; P < 0.01) and those with AMI (9 vs 36%; P = 0.012). In the patients with PMI there was only a trend in favour of those with successful recanalisation (0% vs 14%; P = 0.058). Symptomatic improvement was reported by 73% of patients after successful but only 40% after failed recanalisation (P < 0.01). CONCLUSION: The results provide pointers towards prognostic indications of recanalisation even after chronic occlusion of the infarct vessel. The procedure should therefore be attempted if the occlusion is morphologically suitable. PMID- 7555639 TI - [Pseudomesotheliomatous carcinoma in HIV infection]. AB - HISTORY AND FINDINGS: A 32-year-old man, known to have been HIV-positive for 6 years, was admitted to hospital because of dyspnoea and dry cough. For 8 months he had an HIV illness in stage C3 (Center for Disease Control and Prevention, Atlanta) with fever, weight loss and obviously reduced general and nutritional state. There was almost total auscultatory dullness over the right hemithorax and the chest radiogram showed massive right pleural effusion, as well as broad pleural deposits which were suspicious of tumours. The first pleural aspiration recovered 1000 ml exudate. An infectious cause could not be identified. As the effusion was refractory to treatment (301 of effusion were drained over several days), thoracoscopy was performed despite the patient's poor general condition. It revealed extensive tumour masses over the right pleura. COURSE: After the endoscopy the patient's condition deteriorated rapidly and he died 3 weeks after admission, only 1 1/2 months after onset of the first symptoms. Histological examination of the endoscopically obtained biopsy revealed large-cell carcinoma. The form of growth indicated that it was a pseudomesotheliomatous carcinoma. CONCLUSION: No previous case of this type of tumour in an HIV-positive patient has been reported. This patient's course was less favourable than is expected in those without HIV infection. PMID- 7555640 TI - [The torsion of an accessory liver lobe as the rare cause of acute abdomen]. AB - HISTORY AND CLINICAL FINDINGS: A 35-year-old woman was admitted because of cramp like abdominal pain with nausea and vomiting since the previous evening. There was no history of previous abdominal symptoms of jaundice. Physical examination suggested atypical appendicitis with pain on palpation of the right mid-abdomen, localized paraumbilical guarding tension and diffuse pressure pain, more on the right, on rectal examination. Body temperature was 37.8 degrees C. EXAMINATIONS: There were leucocytosis (12000/microliters) and abnormally high liver function tests (GOT 95 U/l, GPT 110 U/l, LDH 368 U/l, alkaline phosphatase 335 U/l). Ultrasonography of the abdomen demonstrated free fluid in the Douglas pouch. TREATMENT AND COURSE: Laparotomy, performed because atypical appendicitis was suspected, revealed a torsioned pedicle of a severely bleeding accessory liver lobe positioned near the gall bladder bed. The accessory lobe was removed and the patient became symptom-free. This is the 15th reported case of clinical manifestations of an accessory liver lobe. PMID- 7555641 TI - [Hyperthyroxinemia in nonsuppressed TSH. Its differential diagnosis and therapy]. PMID- 7555642 TI - [Transplant vasculopathy following heart transplantation]. PMID- 7555644 TI - [The radiation risk for the personnel in the heart catheterization laboratory]. PMID- 7555643 TI - [Friedrich Theodor Frerichs in Berlin]. PMID- 7555645 TI - [Combined therapy with azithromycin and omeprazole for the eradication of Helicobacter pylori]. PMID- 7555647 TI - [Laparoscopic surgery versus laparotomy in extrauterine pregnancy: an analysis of the cost-benefit relationship]. PMID- 7555648 TI - [Ambulatory-access echocardiography--"service" for practitioners. Its importance for the diagnosis and therapy planning of heart failure]. PMID- 7555646 TI - [Liver transplantation in familial amyloidosis]. PMID- 7555649 TI - [Adenocarcinoma of the cervix and the use of oral contraceptives]. PMID- 7555654 TI - [Bones and bone diseases. Part V: Pathogenesis of type-II osteoporosis]. PMID- 7555650 TI - [Self-expanding metal endoprosthesis in palliation of stenosing tumors of the upper gastrointestinal tract. Comparison of experience with three stent types in 82 implantations]. AB - INTRODUCTION AND AIM OF STUDY: As there are no reports of studies comparing different self-expanding metal stent types the results obtained with three stent types in 73 patients (58 men, 15 women; mean age 65 [43-90] years who had a total of 82 implantation were analysed. PATIENTS AND METHODS: 31 Wallstents were implanted in 23 patients, 31 Ultraflex stents (all without covering) in 30 patients, and 20 Gianturco-Z stents (with covering) in 20 patients. The three groups of patients did not differ significantly with respect to degree of dysphagia, frequency of previous treatment, length of stenosis by tumour, tumour localization and histological tumour type. Six patients, treated with the Gianturco-Z stent, had an tracheo-oesophageal fistula. RESULTS: All 82 implantations were technically successful. Complete occlusion was achieved in all patients with fistulas, as proven radiologically. Serious early complications, namely stent migration, occurred in two patients (2.4%). The degree of dysphagia improved equally in all three groups (Wallstent, from 2.0 to 0.7; Ultraflex from 2.2 to 0.9, Gianturco-Z from 2.1 to 0.5 (not significant). The median follow-up time differed: 89 days for the Wallstent group, 129 for the Ultraflex group and 77 days for the Gianturco-Z group. Tumour ingrowth was the main problem with the non-covered stents. Accordingly, there were 43% reinterventions with the Wallstent, 37% with the Ultraflex and 20% with the Gianturco-Z stents. CONCLUSIONS: Self-expanding metal stents improve palliative treatment of stenosing tumours of the oesophagus and cardia, particularly in view of the low complication rate (< 3%) in the early postimplantation phase. But technical improvements are needed to reduce the number of reinterventions for restenosis in the follow-up period. PMID- 7555652 TI - [Cholestatic jaundice after ingestion of amoxicillin and clavulanic acid]. AB - HISTORY AND FINDINGS: A 61-year-old woman fell ill with recurrent nausea, loss of appetite and tiredness. Five days later she noted increasing jaundice of skin and sclerae, pale stools and dark urine, and she developed itching over the whole body. Among biochemical tests alkaline phosphatase (537 U/l) and bilirubin (32.0 mg/dl) were markedly increased, while both GOT (102 U/l) and GPT (39 U/l) were only slightly elevated. Ultrasonography was normal and extrahepatic cholestasis appeared unlikely on endoscopic retrograde cholangiopancreatography. Cholestasis due to virus hepatitis was also excluded. It was only on repeated and direct questioning that the patient reported having taken three tablets daily of Augmentin (amoxicillin and clavulanic acid) for 12 days 5 weeks before the onset of symptoms. TREATMENT AND COURSE: After symptomatic treatment with Colestyramine (4 g three times daily), ursodeoxycholic acid (250 mg twice daily) and the antihistaminic Clemastine (1 g two times daily) the severe pruritus decreased quickly and alkaline phosphatase concentration became normal. At follow-up examination 4 and 8 weeks later the laboratory tests were normal. CONCLUSIONS: This report of Augmentin-induced cholestasis is not unique. It shows that previous drug intake must be carefully investigated in any case of cholestasis of unknown cause. PMID- 7555653 TI - [Pain management in cancer patients]. PMID- 7555651 TI - [Cyclosporin in severe steroid-requiring bronchial asthma]. AB - BASIC PROBLEM: Treatment of chronic severe bronchial asthma with corticosteroids is inadequate in a minority of patients and is often accompanied by considerable side effects. Additional specific immunosuppression appears to be therapeutically promising. PATIENTS AND TREATMENT: Three patients (2 women, aged 44 and 29, a man aged 57 years), all with chronic severe asthma requiring corticosteroids, were given cyclosporin (mean dose 1.8 mg/kg; serum level 72 +/- 35 ng/ml) additional to conventional bronchospasmolytic drugs for 9 to 20 months. COURSE: The frequency and intensity of asthmatic attacks markedly decreased in all three patients. The mean peak-flow measurements in the mornings before broncholysis had increased by 23% over the precyclosporin level of the calculated normal value. Peak flow variability improved by 13%. The mean one-second forced expiratory volume (FEV1) rose from 37 to 66% of the normal value (P < or = 0.05) and correlated with the serum cyclosporin level (correlation coefficient 0.58-0.97). The frequency of acute severe asthmatic attacks (FEV1 < or = 40%) requiring additional hospitalization with intravenous administration of glucocorticoids fell by 30%. The systemic corticosteroid maintenance dosage could be significantly reduced or the drug discontinued in two patients. CONCLUSION: These observations indicate that cyclosporin can be useful in the treatment of selected cases of chronic severe steroid-refractory asthma. Prospective studies are needed to judge its long-term efficacy. PMID- 7555656 TI - [No additional 10% tax for chief physicians with old contracts based on the Health Care Law. Ruling of the Baden-Wurttemberg District Labor Court of 12 April 1995]. PMID- 7555657 TI - [Vibration artefacts in the electrocardiogramm]. PMID- 7555655 TI - [Bradycardia in sleep-related respiratory disorders. Clinical relevance, pathogenesis and therapy]. PMID- 7555659 TI - [Value of the serum lipase content in diagnosing pancreatitis]. PMID- 7555660 TI - [Exercise-induced urticaria and anaphylaxis]. AB - AIM: The study was designed to characterise more exactly the complex syndrome of exercise-induced urticaria and anaphylaxis in order to obtain guidelines for their management. METHODS: 30 patients (18 women and 12 men with physical exercise-induced urticaria and anaphylaxis were investigated by questionnaire. The following items were of particular interest: age and sex; age at first manifestation; type, duration and intensity of the precipitating activity; type, duration and sequence of symptoms; prophylactic or therapeutic measures as cofactors. RESULTS: Initial symptoms occurred at an average age of 22 (7-50) years. Atopy was present in 70%. Jogging (60%), ball games (40%) and walking (27%) were the most frequent precipitating activities. On each occasion symptoms began a few minutes to hours after the start of the exercise. During a typical episode an average of eight symptoms were observed, most frequently affecting the skin (pruritus, angiooedema, erythema and urticaria), dyspnoea and gastrointestinal manifestations. Syncope occurred in nine patients: before they lost consciousness they noted at least two prodromal symptoms. The most common co factors were humid-warm weather, severe sweating and eating certain foods shortly before the exercise. Prophylactic measures were quite different between individuals. CONCLUSION: Providing detailed information on how to avoid possible cofactors and manage prodromal symptoms should be at the forefront of looking after such patients, most of whom lead a rather active life. PMID- 7555661 TI - [Ectopic thyroid tissue in the heart--two case reports]. AB - A 61-year-old previously healthy woman had been suffering from a dry cough for several weeks. On auscultation she had a 3/6 holosystolic murmur. The electrocardiogram (ECG) showed incomplete right bundle branch block (IRBBB) and abnormal repolarisation. Conventional and oesophageal echocardiography revealed a right ventricular tumour and significant coronary heart disease. At operation a tumour, arising from the interventricular septum and histologically consisting of thyroid tissue, was resected. In addition, the left mammary artery was anastomosed to the anterior interventricular branch of the left coronary artery. A 34-year-old woman developed increasing exertion-independent dyspnoea. For years she had had occasional attacks of palpitations and restlessness, once even a syncope. The ECG showed IRBBB, echocardiography demonstrated a tumour attached to the right side of the interventricular septum. The tumour was removed and found histologically to consist of colloid-containing thyroid follicles. Both tumours were benign. 45 and 36 months, respectively, later both patients are free of symptoms. PMID- 7555658 TI - [Hyperventilation syndrome]. PMID- 7555662 TI - [Significance of serum troponin T in the differential diagnosis of myocardial infarct vs rhabdomyolysis vs renal insufficiency]. AB - HISTORY AND FINDINGS: A 30-year-old man in renal failure due to focal sclerosing glomerulonephritis was, after a seizure with subsequent rhabdomyolysis, found to have an increase not only of creatinekinase (CK) activity (> 100,000 U/l) but also of cardiac troponin T (cTnT) concentration in serum (13 micrograms/l; normal: < 0.1 microgram/l), in the absence of any cardiac symptoms. COURSE: Raised concentration of cTnT is known to occur in patients with renal failure but no cardiac symptoms. In our patient, despite increased creatinine levels, serum cTnT gradually fell to normal within 3 weeks, i.e. the raised creatinine played no part in the elevation of cTnT, which in this patient was probably due to crossreaction between cardiac and skeletal TnT, or transient fresh synthesis of cTnT in regenerating skeletal musculature. CONCLUSIONS: In patients with extensive rhabdomyolysis and renal failure the assessment of the clinical status and the ECG, to exclude myocardial infarction, are of crucial importance. The cTnT level by itself is of only limited value in such cases. PMID- 7555664 TI - [Effectiveness and acceptance of powder inhalants]. PMID- 7555663 TI - [Herpetic eczema in pregnancy]. AB - HISTORY AND FINDINGS: In a 32-year-old 26-week pregnant woman with atopic eczema densely packed blisters, ca. 1 mm in diameter, developed within the eczema areas, typical of herpetic eczema. This diagnosis was confirmed by identifying the virus under the electronmicroscope and demonstrating herpes simplex virus DNA in the polymerase chain reaction. TREATMENT AND COURSE: During intravenous aciclovir administration (5 mg/kg three times daily for 9 days) the blisters quickly healed. As a bacterial superinfection was suspected (later confirmed by culturing Staphylococcus aureus from a skin swab) the patient was also given erythromycin (500 mg four times daily by mouth for 7 days), as well as fusidic acid cream locally. She delivered a healthy child in the 37th week of pregnancy. CONCLUSION: Herpetic eczema is a serious complication of chronic eczema. If it occurs during pregnancy, the risk or fetal damage by aciclovir must be balanced against the risk of intrauterine herpes simplex infection. PMID- 7555665 TI - [Porphyria cutanea tarda (chronic hepatic porphyria)]. PMID- 7555667 TI - [Remission of diarrhea of unknown origin in omeprazol therapy]. PMID- 7555666 TI - [Scope and timing of risk disclosure for diagnostic interventions. Ruling of the Federal Court of Justice, 4 April 1995]. PMID- 7555669 TI - [Molecular diagnosis of the Hippel-Lindau syndrome]. PMID- 7555668 TI - [Differential diagnosis of post-infection arthralgias]. PMID- 7555670 TI - [Stent implantation for the palliative therapy of upper inflow obstruction in bronchial carcinoma]. AB - AIM OF STUDY: To analyse results obtained in the palliative treatment of obstruction of the upper inflow veins by stent implantation in patients with bronchial carcinoma. PATIENTS AND METHODS: Stents were implanted in 14 patients with upper venous obstruction due to bronchial carcinoma (three women, eleven men: mean age 64 [48-74] years), 4 of the superior vena cava (SVC), 6 of the SVC and brachiocephalic vein, 4 of the SVC and subclavian vein. To prevent thromboembolic phenomena all patients received heparin in therapeutic doses for 1 3 days from the time of stents implantation. RESULTS: Treatment was successful (regression of the clinical signs) in 12 of the 14 patients. Six patients died, without recurrence of the clinical signs, an average of 141 (10-420) days after the procedure. Mean survival time of the six patients who remained without symptoms was 3 months (longest interval 9 months). In one patient thrombotic occlusion of the SVC occurred 6 days after stent implantation. However, local thrombolysis successfully re-opened the vessel. Renewed upper vein obstruction occurred in two patients during follow-up. None of the stent filaments fractured and there was no stent dislocation. CONCLUSION: With a success rate of 86% stent implantation proved to be a sparing procedure in the palliative treatment of upper inflow vein obstruction, especially in patients with extensive malignant disease. PMID- 7555671 TI - [Immunogenicity and tolerance of a combined hepatitis A/B vaccine. Preliminary results with a candidate vaccine]. AB - AIM OF STUDY: Active immunization against hepatitis A having been undertaken in Germany since January 1993, a multicentre study was conducted to test, for the first time, immunogenicity of and tolerance to a candidate vaccine against hepatitis A and B. SUBJECTS AND METHODS: 50 healthy volunteers aged 18-40 years, negative for antibodies against hepatitis A (HAV) and B (HBs), received three intramuscular injections of the candidate vaccine (720 EIU of strain HM 175 and 20 micrograms recombinant HBsAg) in a total volume of 1 ml, on day 1 and then, one month and 6 months later. RESULTS: Four weeks after the first injection the seroprotection rate (percentage of subjects with protective antibody titres) was 90% for HAV and 28% for HBs. The second injection produced seroconversion rates of 98% and 50%, respectively, and after the third one of 100% and 98%. All reported side effects were minor, of short duration and decreased after each injection. After the first injection, effects at the site of injection occurred in 50% of subjects, decreasing to 6% after the third one. The only systemic side effect, headache, occurred in only 4% of subjects and only after the first injection. CONCLUSION: The test vaccine against hepatitis A and B proved to be highly immunogenic, safe and well tolerated. PMID- 7555672 TI - [Collagenous sprue]. AB - HISTORY AND FINDINGS: A 45-year-old man with type I diabetes mellitus was admitted to hospital because of colicky abdominal pain and 5-6 watery stools daily. Upper gastrointestinal endoscopy showed nearly total atrophy of the villi in the duodenum and jejunum suggesting coeliac disease. However, gluten-free diet for 2 weeks brought no improvement. Another examination of the biopsy 6 weeks after the first endoscopy revealed extensive collagen deposition in the lamina propria of the small intestine, giving the diagnosis of collagenous sprue. TREATMENT AND COURSE: Parenteral nutrition, lactulose, cisapride, cholestyramine, doxycycline, paromomycin, vancomycin and octreotide failed to affect the loss of fluid from the gut which 12 weeks after admission had increased to 221 daily. However, it was stopped after prednisolone was administered (100mg daily). 7 months after starting the steroid treatment the collagen layer had disappeared and the villous atrophy had partially regressed. Over the next 6 months the prednisolone dosage was decreased to 10 mg daily. Shortly thereafter a perimembranous glomerulonephritis occurred, with proteinuria (up to 60 g/d) and oedema. It regressed to 6 g/d when the steroid dose was increased and cyclosporin, 0.5 g/d, had been added. On maintenance dosage of cyclosporin the histological and clinical remission of the collagenous sprue has now lasted for over 2 years. CONCLUSIONS: This case suggests that steroid administration is an effective treatment of collagenous sprue. The presence of diabetes and other immune-related diseases in this case also suggests that an immunological mechanism may play a causative role in collagenous sprue. PMID- 7555674 TI - [Analysis of the individual quality of an oral anticoagulant using the ratio F1+2/INR]. PMID- 7555675 TI - [The bones and their diseases. VI. The origin of "secondary" osteoporosis]. PMID- 7555673 TI - [MALT lymphoma of the ileum as the cause of an enterocolic invagination]. AB - HISTORY AND FINDINGS: A 44-year-old woman who had moved to Germany from Kazakhstan 2 years previously developed right upper abdominal pain. A cylindrical mass was palpable in the area. There were strong intestinal sounds. INVESTIGATIONS: There was an iron-deficiency anaemia (haemoglobin 6.3 g/dl, serum iron 4 micrograms/dl) and occult blood was found in the stool. Coloscopy revealed a lumpy mass external to the transverse colon but fixed to it, partially obstructing the lumen. However, on contrast infusion the mass could be displaced in to the caecum, suggesting enterocolic invagination. TREATMENT AND COURSE: At operation the cause of the investigation was found to be a large jagged tumour of the terminal ileum. A right hemicolectomy and excision of the terminal ileum were performed. Histologically the tumour was a centroblastic non-Hodgkin lymphoma of the MALT type (mucosa-associated lymphatic tissue). PMID- 7555676 TI - [Intestinal non-Hodgkin's lymphoma]. PMID- 7555677 TI - [Malignant melanoma in adolescents]. PMID- 7555678 TI - [Vascular endothelial growth factor in the aqueous humor in diabetic retinopathy and other diseases of the retina]. PMID- 7555679 TI - [Patient-controlled analgesia with pethidine in acute intermittent porphyria]. PMID- 7555680 TI - [Requirements of raising game pheasants (Phasianus colchicus sp.) in respect to animal welfare laws]. AB - The present study works for a list of requirements of animal protection on the commercial rearing of common pheasant. Therefore universally valid tags and requirements are evolved for feeding, illumination regiment in the rearing compartments and housing densities and for pheasants freedom from injury. The duty of the veterinary authorities for powerful checkups is pointed out. PMID- 7555681 TI - [Comparative studies of the release and ability to survive of game pheasants (Phasianus colchicus sp.) from intensive and extensive husbandry]. AB - Artificially reared pheasants had been released at an age of 10 weeks on two stands in a suitable habitat with a wild pheasant population in Lower Saxony, Germany. Two groups (200 pheasants reared in large fly-aviaries, 200 pheasants reared in dark stables) were bought from commercial breeders who offer pheasants for restocking wild populations. Some of the released pheasants were marked with radio-transmitters. There was no significant difference in survival rate between the two rearing groups. Moreover a significant difference in survival rate between the two releasing points could be find out. Within 32 days 114 (30%) of the 380 released pheasants died mainly by predators e.g. fox/martens (44%) and goshawk (12%). 59% of these refound dead pheasants died already within the first week. 20 pheasants died in the releasing-aviary. Most of the released pheasants alive were re-observed within a diameter of 1000 metres from the releasing points. Only some pheasants dispersed more than 2000 metres. No pheasant released could be observed longer than 5 weeks. We suppose that most of pheasants released died within short time. PMID- 7555683 TI - [Feasibility for the influence of official veterinarians on the improvement of animal husbandry]. AB - The present article reports on the possibilities to improve keeping conditions of laboratory animals. Such improvement are urgent because international laws provide only a very poor minimal living standard and prove to be incapable of insuring those living conditions that are considered to be acceptable for laboratory animals. PMID- 7555682 TI - ["Breeding for distress" in fowl]. AB - During the last hundred years the fancying and breeding of exhibition poultry became very popular as free time activity. Certain breeding standards were set, and the aim of actual breeding endeavours is to meet these standards through selective breeding. Not only were serious defects established as standard qualities (e.g. domestic ducks with feather crests, rumplessness, and ear-tufts in the domestic fowl), but certain distinctive marks were changed into excessive formations (e.g. pigeon breeds with large, cauliflower-like wattles, or enlarged crops and severe behaviour changes). Accurate analysis of such breeding efforts shows that these breeds are to be called defective and abnormal in the sense of section 11b of the German protection of animals legislation. Whether an increased embryonic mortality, especially during the hatching period, constitutes a condemnable state of cruelty against is recently being discussed. Criticised are also standard features which handicap, in varying degrees, certain functional systems of the natural behaviour patterns. Section 11b of the German protection of animal legislation in its present wording is definitely no sufficient mean to control the contemporary excess in breeding of domestic animals. PMID- 7555684 TI - [Feasibility for the influence of the animal welfare commissioner and cooperation with the responsible authorities]. AB - The possibilities of the Animal Protection Commissioner in assuming influencing control in the planning and performance of animal experiments will be discussed by giving consideration to the LAW on Animal Protection, and its paragraph 9 in particular. At the same time, attention will be drawn to the significance of the position and authority that the Animal Protection Commissioner holds in assuming his tasks, whereby the cooperation with responsible authorities appointed in accordance with municipal law will be another point of discussion. PMID- 7555685 TI - [Electrofishing from animal welfare aspects]. AB - Through improper application of electrofishing techniques fish may be harmed severely. The injuries can range from inner bleeding to broken vertebrae. Due to these possible injuries each electrofishing procedure requires approval of the fisheries authorities. Only persons with a special license are allowed to fish with electricity. The fisheries authorities only approve electrofishing for reasons stated in the fisheries laws. If an application to fish with electricity is approved, the permission is restricted to certain persons, certain equipment units, certain stretches of water and clearly outlined goals. Damages to fish can be reduced to a minimum by the strict regulations of the fisheries authorities and by the training of the fishermen. PMID- 7555686 TI - [Preventative tail amputation in fattening bulls]. AB - In 5 cattle fattening farms with 900 to 3000 bulls tail docking was practised prophylactically in consequence of losses due to tail necrosis. Following detailed consultations in the farms temporary exceptional permission of the ban of amputation according to article 6 of the German Animal Welfare Law was given to 4 of the 5 farms, associated with real instructions to improve the housing conditions. By that means communication between farmer and veterinary authority was intensified and the contrariety between legislation and practice was pointed out clearly. The farmers were concerned with consequences of the ban of tail docking intensively. A process of understanding began, resulting in renunciation of prophylactic tail docking in 4 of the 5 farms. PMID- 7555687 TI - [Aspects of animal welfare relevant to poultry exhibitions]. AB - After a summary of the organized poultry racebreeding and the ways of exhibitions in the FRG, we point out the most frequent collisions with the law for animal protection nowadays: The forbidden clipping of parts about the head at certain game fowl e.g. Modern and Old English Game and the bantams of these races, dealings to avoid flying and the acute strain through transports. Because of his competence the civil servant vet is asked for to stop grievances primarily by advice. PMID- 7555688 TI - [Supervision of animal welfare regulations in animal markets--an official veterinary stepchild?]. AB - The authors report about problems in animal welfare during commercial animal exhibitions. A list of claims in accordance to animal welfare for these forms of animal presentation is given. Remaining problems are described. PMID- 7555689 TI - [The total efficiency of stunning traps for the capture of stone martens and red foxes in hunting situations]. AB - The actual paper represents the pathomorphologic changes on martens and foxes hunted in instant killing traps under huntsmanlike conditions. The results are critically discussed concerning the manner of death, the moment of death of the killed animals regarding the relevant legislation. PMID- 7555690 TI - Structural responses of Japanese quail intestine to different diets. AB - Japanese quail were fed five diets of different nutrient composition and the structural responses of the intestinal tract were measured. Six morphometric variables were recorded. Depending on the diet, we found drastic changes in those factors affecting surface dimensions of the mucosa epithelium and the thickness of the muscle layers of the intestinal wall. We performed a principal component analysis to describe the interrelationship among the different variables and the response of the intestine to different diets. Two principal components were found accounting for surface and muscle layers respectively. Cytokinetic experiments (TdR/BrdU double labelling of S-phase nuclei) were performed to estimate the epithelial turnover time. We found a labelling index between 1.5% and 3% depending on the segment of the intestine. The length of the S-phase was found to be constant along the gut at 6.5 (+/- 2.5) hours. The epithelial turnover time, necessary to rebuild and adjust the intestine, ranged between 10 and 17 days. We discuss evolutionary ecology perspectives as well as applied poultry aspects of the data. PMID- 7555691 TI - Selective retention of fluid digesta in the hindgut of bandicoots and other marsupial caecum fermenters. AB - Many small hindgut fermenters have a mechanism in the proximal colon that separates fluid and/or bacteria from large particles, and excretes the large particles relatively rapidly. The fluid and/or bacteria are retained in the caecum, which concentrates digestive effort in that region of the hindgut and improves overall digestive efficiency. Previously observed among marsupials only in arboreal marsupials, selective retention of fluid digesta has recently been reported also in bandicoots, small terrestrial omnivores. The separation mechanism operated independently of the nature of the diet, indicating that it is probably an important factor in the ability of bandicoots to switch between insect and plant foods, and thus to exploit nutritionally unpredictable environments. Results are discussed in relation to possible locations in the marsupial hindgut of the pacemaker that in eutherians has been shown to initiate retrograde movement of fluid and small particles in the proximal colon towards the caecum. PMID- 7555692 TI - Intestinal methanogenesis in primates--a genetic and evolutionary approach. AB - The presence of significant numbers of intestinal methanogens among vertebrates does neither depend on elaborated morphological structures nor on predominantly plant-based diets. Phylogenetic rather than dietary restrictions limit the occurrence of methanogenic bacteria also in the hindguts of primates. The Old World monkeys are methanogenic--with the only exceptions of Cheirogaleid lemurs and bush-babies. In contrast, among New World monkeys the lack of intestinal methanogens is observed frequently in capuchins and marmosets. Since the absence of methanogens does not parallel distinct morphological, physiological, or ethological characteristics, it is likely that methanogenesis depends on a hereditary predisposition. In humans, methane-producers account for approximately 50% of the European populations. In this study, 56 individuals belonging to 5 families were studied for the occurrence of methane in the breath. The results of this screen are compatible with the assumption that the trait "methane emission" segregates as an autosomal, dominant character. Our findings suggest a high specificity of the symbiosis between primates and methanogenic bacteria. Therefore, the persistence of significant numbers of methanogens in the hindgut might be facilitated by a specific receptor for methanogenic bacteria. PMID- 7555693 TI - Sulphate reduction and methanogenesis in the ovine rumen and porcine caecum: a comparison of two microbial ecosystems. AB - A series of experiments was conducted to study the relationship between methanogenesis and sulphate reduction in ovine rumen and porcine caecum. Effect of 2-bromoethane sulphonate on hydrogen production by digesta suggested that the most important H2-disposal system in the rumen is methanogenesis and that methanogenesis is not predominant H2-disposal system in the porcine caecum. This inference was supported by the difference in predominant H2-utilizers in these two microbial ecosystems; Methanogenic bacteria (MB) were predominant in the rumen and sulphate reducing bacteria (SRB) were predominant in porcine caecum. Free sulphate levels in digesta appear to affect the relationship between MB and SRB. Sulphate levels in the rumen were likely to be insufficient for SRB to outcompete MB. PMID- 7555694 TI - Comparative views of electrophysiological parameters of large intestinal segments in pig, sheep, pony, guinea pig and rat. AB - Short circuit current (ISC) and transepithelial conductance (gt) across sheets of epithelia were measured in the caecum, the proximal and the distal colon of pig, sheep, pony, rat and guinea pig. The electrical parameters underline the basic segmental and species differences. The diversity of ISC demonstrates the different nature of electrogenic transport mechanisms, and data clearly show the heterogeneity with respect to transport mechanisms along the large intestine in the various species. The great differences in amiloride sensitive ISC indicate the variabilities in the electrogenic Na transport. Whereas in the pig, sheep and pony caecum, in the guinea pig proximal colon and in all segments of the rat hindgut no indications for a major electrogenic Na transport was seen, in all other segments amiloride caused a marked decrease in ISC. Electrogenic Na transport seems to be highest in sheep distal colon and in pig proximal and distal colon, somewhat less in guinea pig and in pony distal colon. The epithelium with the lowest Powest transepithelial conductance clearly is that of the pony caecum. Except in sheep, gt-values are not much different from those in pony and also pig and guinea pig. By far the epithelium with the highest conductance is that of the rat proximal colon. gt was similar in the proximal and the distal colon of pig, sheep and pony; in guinea pig values were slightly, in rat much lower. PMID- 7555696 TI - Regulation of intracellular pH in the colonic epithelial cell line HT29 clone 19A. AB - Intracellular pH (pHi) of the colonic tumor cell line HT29 cl. 19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer grown in a polarized manner on permeable supports were examined. An amiloride-sensitive Na(+)-H+ exchange and a stilbene-insensitive Cl( )-HCO3- exchange mechanism have been identified in the basolateral membrane. This Na(+)-H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the Cl(-)-HCO3- exchanger is an acid loader. Both of these in the basolateral membrane located mechanisms are important for the maintenance of intracellular pH in HT29 cl. 19A cells. PMID- 7555695 TI - Transepithelial transport of short chain fatty acids and their metabolism in pig hindgut. AB - In vitro experiments in Ussing chambers were performed in order to study the transport as well as the intraepithelial metabolism of short chain fatty acids in the caecum, proximal and distal colon. Stripped epithelial tissues were incubated in isotonic buffer solutions. SCFA were only present in the mucosal solutions at a concentration of 60 mmol/l consisting of 60% acetate, 25% propionate and 15% butyrate at the beginning of each flux rate measurement. For osmotic reasons SCFA were completely replaced by gluconate in serosal buffer solutions. The tissues were incubated under short-circuit current conditions for 1 h. In addition, with epithelia from the proximal colon flux rate measurements were also performed after setting the transepithelial potential difference (PD) to 25 mV with the serosa being positive. At the end of each 1h flux period samples were taken from the mucosal and serosal solutions for HPLC analysis of SCFA concentrations. SCFA transport and rate of intraepithelial metabolism were calculated from mucosal uptake and serosal release during the experimental periods. In the presence of 2 cm2 serosal area of the tissues, mucosal SCFA uptake ranged between 53 and 75 mumol with no change of molar SCFA proportions irrespective of hindgut segment. Voltage clamp condition did not influence mucosal SCFA uptake indicating the presence of electroneutral transport mechanisms. Serosal SCFA release ranged between 105 and 126 mumol thus exceeding mucosal loss. In serosal solutions molar butyrate proportions were significantly lower in comparison with mucosal solutions. This was accompanied by slight increases of molar acetate proportions indicating intraepithelial butyrate cleavage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555698 TI - Expression of estrogen and androgen receptor in the bovine gastrointestinal tract. AB - Reproductive and maturational nutritive needs are examples for situations in which alterations in circulating concentrations of sex steroids are associated with changes in gastrointestinal function. In order to investigate whether there is a causal relationship between sex steroids and gastrointestinal function, we aimed to investigate the responsiveness for androgens and for estrogens of the bovine gastrointestinal tract. Using Northern blot analysis, estrogen receptor (ER) mRNA was detected in rumen tissue. Comparing the ER expression in rumen from females of different reproductive stages, we found that no differences related to cycle stage, pregnancy or parturition could be detected. In contrast, the ER expression rates in the uterus of the respective animals showed the same dependency of reproductive stage as demonstrated earlier for the ER protein, indicating that there might be a tissue specific regulation of ER. By in-situ hybridization of rumen tissue sections the expression of ER was localized in the epithelium of the papillae. In the muscular layer no positive signals for ER mRNA were observed. Above rumen, the presence of ER and androgen receptor (AR) mRNA was determined in various intestinal tissues using reverse transcription (RT) and polymerase chain reaction (PCR). Primers were selected from the bovine androgen and estrogen receptor sequence to amplify parts of the sequence coding for the hormone binding part of the respective receptor. The PCR amplifies were subsequently electrophoresed on 1% agarose gels and visualized by ethidium bromide staining. ER mRNA expression was demonstrated in reticulum, omasum, abomasum, duodenum, jejunum, ileum, caecum and colon. AR mRNA expression was not determined in the forestomaches, but was present in all intestinal segments investigated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555697 TI - Effect of n-butyric acid on epithelial cell proliferation of pig colonic mucosa in short-term culture. AB - Short-chain fatty acids (SCFA) such as acetic, propionic and n-butyric acids produced by hindgut bacteria stimulate gut epithelial cell proliferation through afferent neural and efferent non-neural systemic transmissions beside a probable local mechanism. In the present study, we developed an experimental system using pig hindgut mucosa in short term culture to clarify the mechanism of the local trophic effect of SCFA. Pig mucosal tissue pieces of the distal colon were cultured in RPMI 1640 medium containing glutamine, 20% (v/v) newborn calf serum and n-butyric acid (0, 0.5, 1.0 or 5.0 mmol/L). Crypt cell production rate from 0.5 to 3.5 and from 21 to 24 hours of culture was measured. Butyric acid increased crypt cell production rate of pit distal colon only at 5 mmol/L. The effect of butyric acid did not differ between samples of different length of n butyric acid exposure. The effect of n-butyric acid in this study resembled to that found in human biopsied specimens of the colon. The present results also indicated that epithelial cells of pig colonic mucosa in short-term culture presented here retained the proliferative activity and the responsiveness to n butyric acid. PMID- 7555699 TI - Retrograde flow of urine from cloaca to caeca in laying hens in relation to different levels of nitrogen intake. AB - Retrograde flow of urine into the caeca provides a more efficient recovery of nutrients and supplies a nitrogen source for the caecal microbes. 30 laying hens of Leghorn type were used in the present experiments. Hens were randomly divided into three treatment groups and fed a high nitrogen (19.8% crude protein in dry matter), medium (15.0% crude protein in dry matter), or low (10.6% crude protein in dry matter) isocaloric diet for fourteen days prior to and during the experimental period (8 h). Results from the study show that the retrograde flow of urine from cloaca to caeca is significantly higher in birds given a low protein diet compared to medium or high levels. It also shows that the produced amount of urine is significantly higher in hens given a low protein diet compared to the medium level diet. PMID- 7555700 TI - Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications. AB - In this study we have analysed the expression of Hoxb-4, Hoxb-1, Hoxa-3, Hoxb-3, Hoxa-4 and Hoxd-4 in the neural tube of chick and quail embryos after rhombomere (r) heterotopic transplantations within the rhombencephalic area. Grafting experiments were carried out at the 5-somite stage, i.e. before rhombomere boundaries are visible. They were preceeded by the establishment of the precise fate map of the rhombencephalon in order to determine the presumptive territory corresponding to each rhombomere. When a rhombomere is transplanted from a caudal to a more rostral position it expresses the same set of Hox genes as in situ. By contrast in many cases, if rhombomeres are transplanted from rostral to caudal their Hox gene expression pattern is modified. They express genes normally activated at the new location of the explant, as evidenced by unilateral grafting. This induction occurs whether transplantation is carried out before or after rhombomere boundary formation. Moreover, the fate of the cells of caudally transplanted rhombomeres is modified: the rhombencephalic nuclei in the graft develop according to the new location as shown for an r5/6 to r8 transplantation. Transplantation of 5 consecutive rhombomeres (i.e. r2 to r6), to the r8 level leads to the induction of Hoxb-4 in the two posteriormost rhombomeres but not in r2,3,4. Transplantations to more caudal regions (posterior to somite 3) result in some cases in the induction of Hoxb-4 in the whole transplant. Neither the mesoderm lateral to the graft nor the notochord is responsible for the induction. Thus, the inductive signal emanates from the neural tube itself, suggesting that planar signalling and predominance of posterior properties are involved in the patterning of the neural primordium. PMID- 7555701 TI - The REVOLUTA gene is necessary for apical meristem development and for limiting cell divisions in the leaves and stems of Arabidopsis thaliana. AB - The form of seed plants is determined by the growth of a number of meristems including apical meristems, leaf meristems and cambium layers. We investigated five recessive mutant alleles of a gene REVOLUTA that is required to promote the growth of apical meristems and to limit cell division in leaves and stems of Arabidopsis thaliana. REVOLUTA maps to the bottom of the fifth chromosome. Apical meristems of both paraclades (axillary shoots) and flowers of revoluta mutants frequently fail to complete normal development and form incomplete or abortive structures. The primary shoot apical meristem sometimes also arrests development early. Leaves, stems and floral organs, in contrast, grow abnormally large. We show that in the leaf epidermis this extra growth is due to extra cell divisions in the leaf basal meristem. The extent of leaf growth is negatively correlated with the development of a paraclade in the leaf axil. The thickened stems contain extra cell layers, arranged in rings, indicating that they may result from a cambium-like meristem. These results suggest that the REVOLUTA gene has a role in regulating the relative growth of apical and non-apical meristems in Arabidopsis. PMID- 7555702 TI - Localization of oskar RNA regulates oskar translation and requires Oskar protein. AB - The site of oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for oskar RNA localization and translational repression map to the oskar 3'UTR, while sequences involved in the correct temporal activation of translation reside outside the oskar 3'UTR. Upon localization of oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. We propose that initially localization of oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism. PMID- 7555703 TI - A sea urchin homologue of the chordate Brachyury (T) gene is expressed in the secondary mesenchyme founder cells. AB - Chordates are thought to have emerged from some common ancestor of deuterostomes by organizing shared anatomical and embryological features including a notochord, a dorsal nerve cord and pharyngeal gill slits. Because the notochord is the most prominent feature of chordates and because the Brachyury (T) gene is essential for notochord formation, the T gene is a key molecular probe with which to explore the origin and evolution of chordates. We investigated whether the sea urchin (echinoderm) conserves the T gene and, if so, where the sea urchin T gene is expressed. A cDNA clone for the sea urchin T (HpTa) gene contained a long open reading frame that encodes a polypeptide of 434 amino acids. Although the overall degree of amino acid identity was not very high (52%, sea urchin/mouse), in the T domain of the N terminus the amino acid identity was 73% (sea urchin/mouse). The HpTa gene is present as a single copy per haploid genome. As with the chordate T gene, the expression of HpTa is transient, being first detected in the swimming blastula, maximally transcribed in the gastrula, decreasing at the prism larval stage and barely detectable at the pluteus larval stage. HpTa transcripts were found in the secondary mesenchyme founder cells, vegetal plate of the mesenchyme blastula, extending tip of the invaginating archenteron and, finally, the secondary mesenchyme cells at the late-gastrula stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555704 TI - Distribution of tissue progenitors within the shield region of the zebrafish gastrula. AB - The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish. PMID- 7555705 TI - four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila. AB - Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs. PMID- 7555706 TI - Cloning and characterization of a second AP-2 transcription factor: AP-2 beta. AB - AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues. PMID- 7555707 TI - Properties of the dorsal activity found in the vegetal cortical cytoplasm of Xenopus eggs. AB - The Xenopus egg contains a maternal dorsal determinant that is specifically localized to the vegetal cortex. We have previously shown that vegetal cortical cytoplasm can generate a full dorsal axis when it is injected into ventral vegetal blastomeres of a cleavage-stage embryo. In this study, we have defined further the properties of the dorsal activity. The cortical dorsal activity arises during oocyte maturation after germinal vesicle breakdown. When injected into the four extreme animal pole blastomeres of ultraviolet-ventralized 32-cell embryos, vegetal cortical cytoplasm partially rescued dorsal axial structures. As revealed by lineage tracing, these axial structures formed ectopically from the progeny of the cells that were injected. Injection of animal cortical cytoplasm had no effect. When mid-blastula (stage 8) animal caps from these injected embryos were isolated and cultured, both vegetal cortex-enriched and animal cortex-enriched animal caps produced only epidermis. Therefore vegetal cortex, on its own, is not a mesoderm inducer. Between stage 8 (blastula) and stage 10 (gastrula), a ventral mesoderm-inducing signal spreads from vegetal cells towards the animal pole. We tested whether this natural mesoderm-inducing factor interacts with the activity found in the vegetal cortex. Injection of vegetal cortex enhanced the formation of neural tissue and cement gland when animal caps were isolated at stage 10. When cultured from stage 8 in the presence of the ventral mesoderm-inducing fibroblast growth factor, animal caps enriched in vegetal cortex developed significantly more neural tissue and cement gland than ones enriched in animal cortex. These results indicate that the dorsal activity localized to the egg vegetal cortex alters the response of cells to mesoderm inducers. PMID- 7555708 TI - Homeotic response elements are tightly linked to tissue-specific elements in a transcriptional enhancer of the teashirt gene. AB - Along the anterior-posterior axis of animal embryos, the choice of cell fates, and the organization of morphogenesis, is regulated by transcription factors encoded by clustered homeotic or 'Hox' genes. Hox genes function in both epidermis and internal tissues by regulating the transcription of target genes in a position- and tissue-specific manner. Hox proteins can have distinct targets in different tissues; the mechanisms underlying tissue and homeotic protein specificity are unknown. Light may be shed by studying the organization of target gene enhancers. In flies, one of the target genes is teashirt (tsh), which encodes a zinc finger protein. tsh itself is a homeotic gene that controls trunk versus head development. We identified a tsh gene enhancer that is differentially activated by Hox proteins in epidermis and mesoderm. Sites where Antennapedia (Antp) and Ultrabithorax (Ubx) proteins bind in vitro were mapped within evolutionarily conserved sequences. Although Antp and Ubx bind to identical sites in vitro, Antp activates the tsh enhancer only in epidermis while Ubx activates the tsh enhancer in both epidermis and in somatic mesoderm. We show that the DNA elements driving tissue-specific transcriptional activation by Antp and Ubx are separable. Next to the homeotic protein-binding sites are extensive conserved sequences likely to control tissue activation by different homeodomain proteins. We propose that local interactions between homeotic proteins and other factors effect activation of targets in proper cell types. PMID- 7555710 TI - Shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-beta-responsive gene. AB - Differentiation in the Drosophila eye imaginal disc traverses the disc as a wave moving from posterior to anterior. The propagation of this wave is driven by hedgehog protein secreted by the differentiated cells in the posterior region of the disc. Hedgehog induces decapentaplegic expression at the front of differentiation, in the morphogenetic furrow. We have identified a gene, shortsighted, which is expressed in a hedgehog-dependent stripe in the undifferentiated cells just anterior to the furrow and which appears to be involved in the transmission of the differentiation-inducing signal; a reduction in shortsighted function leads to a delay in differentiation and to a loss of photoreceptors in the adult. shortsighted is also required for a morphogenetic movement in the brain that reorients the second optic lobe relative to the first. shortsighted encodes a cytoplasmic leucine zipper protein with homology to a mouse gene, TSC-22, which is transcriptionally induced in response to TGF-beta. PMID- 7555711 TI - Function of the Polycomb protein is conserved in mice and flies. AB - A key aspect of determination--the acquisition and propagation of cell fates--is the initiation of patterns of selector gene expression and their maintenance in groups of cells as they divide and develop. In Drosophila, in those groups of cells where particular selector genes must remain inactive, it is the Polycomb Group of genes that keep them silenced. Here we show that M33, a mouse homologue of the Drosophila Polycomb protein, can substitute for Polycomb in transgenic flies. Polycomb protein is thought to join with other Polycomb-Group proteins to build a complex that silences selector genes. Since members of this group of proteins have their homologues in mice, our results suggest that the molecular mechanism of cell determination is widely conserved. PMID- 7555709 TI - Notch regulates wingless expression and is not required for reception of the paracrine wingless signal during wing margin neurogenesis in Drosophila. AB - In the developing wing margin of Drosophila, wingless is normally expressed in a narrow stripe of cells adjacent to the proneural cells that form the sensory bristles of the margin. Previous work has shown that this wingless is required for the expression of the proneural achaete-scute complex genes and the subsequent formation of the sensory bristles along the margin; recently, it has been proposed that the proneural cells require the Notch protein to properly receive the wingless signal. We have used clonal analysis of a null allele of Notch to test this idea directly. We found that Notch was not required by prospective proneural margin cells for the expression of scute or the formation of sensory precursors, indicating Notch is not required for the reception of wingless signal. Loss of Notch from proneural cells produced cell-autonomous neurogenic phenotypes and precocious differentiation of sensory cells, as would be expected if Notch had a role in lateral inhibition within the proneural regions. However, loss of scute expression and of sensory precursors was observed if clones substantially included the normal region of wingless expression. These 'anti-proneural' phenotypes were associated with the loss of wingless expression; this loss may be partially or wholly responsible for the anti-proneural phenotype. Curiously, Notch- clones limited to the dorsal or ventral compartments could disrupt wingless expression and proneural development in the adjacent compartment. Analysis using the temperature-sensitive Notch allele indicated that the role of Notch in the regulation of wingless expression precedes the requirement for lateral inhibition in proneural cells. Furthermore, overexpression of wingless with a heat shock-wingless construct rescued the loss of sensory precursors associated with the early loss of Notch. PMID- 7555712 TI - Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells. AB - A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation. PMID- 7555713 TI - Functional analysis of the Antirrhinum floral homeotic DEFICIENS gene in vivo and in vitro by using a temperature-sensitive mutant. AB - Flowers of the temperature-sensitive DEFICIENS (DEF) mutant, def-101, display sepaloid petals and carpelloid stamens when grown at 26 degrees C, the non permissive temperature. In contrast, when cultivated under permissive conditions at 15 degrees C, the morphology of def-101 flowers resembles that of the wild type. Temperature shift experiments during early and late phases of flower development revealed that second and third whorl organ development is differentially sensitive to changes in DEF expression. In addition, early DEF expression seems to control the spatially correct initiation of fourth whorl organ development. Reduction of the def-101 gene dosage differentially affects organogenesis in adjacent whorls: at the lower temperature development of petals in the second whorl and initiation of carpels in the centre of the flower is not affected while third whorl organogenesis follows the mutant (carpelloid) pattern. The possible contribution of accessory factors to organ-specific DEF functions is discussed. In situ analyses of mRNA and protein expression patterns during def 101 flower development at 15 degrees C and at 26 degrees C support previously proposed combinatorial regulatory interactions between the MADS-box proteins DEF and GLOBOSA (GLO), and provide evidence that the autoregulatory control of DEF and GLO expression by the DEF/GLO heterodimer starts after initiation of all organ primordia. Immunolocalisation revealed that both proteins are located in the nucleus. Interestingly, higher growth temperature affects the stability of both the DEF-101 and GLO proteins in vivo. In vitro DNA binding studies suggest that the temperature sensitivity of the def-101 mutant is due to an altered heterodimerisation/DNA-binding capability of the DEF-101 protein, conditioned by the deletion of one amino acid within the K-box, a protein region thought to be involved in protein-protein interaction. In addition, we introduce a mutant allele of GLO, glo-confusa, where insertion of one amino acid impairs the hydrophobic carboxy-terminal region of the MADS-box, but which confers no strong phenotypic changes to the flower. The strong mutant phenotype of flowers of def 101/glo-conf double mutants when grown in the cold represents genetic evidence for heterodimerisation between DEF and GLO in vivo. The potential to dissect structural and functional domains of MADS-box transcription factors is discussed. PMID- 7555714 TI - Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. AB - In the nematode, Caenorhabditis elegans, VA and VB motor neurons arise from a common precursor cell but adopt different morphologies and synapse with separate sets of interneurons in the ventral nerve cord. A mutation that inactivates the unc-4 homeodomain gene causes VA motor neurons to assume the VB pattern of synaptic input while retaining normal axonal polarity and output; the disconnection of VA motor neurons from their usual presynaptic partners blocks backward locomotion. We show that expression of a functional unc-4-beta galactosidase chimeric protein in VA motor neurons restores wild-type movement to an unc-4 mutant. We propose that unc-4 controls a differentiated characteristic of the VA motor neurons that distinguishes them from their VB sisters, thus dictating recognition by the appropriate interneurons. Our results show that synaptic choice can be controlled at the level of transcription in the post synaptic neuron and identify a homeoprotein that defines a subset of cell specific traits required for this choice. PMID- 7555715 TI - Genetic ablation of petal and stamen primordia to elucidate cell interactions during floral development. AB - Two models have been proposed to explain the coordinated development of the four whorls of floral organs. The spatial model predicts that positional information defines the four whorls simultaneously, and that individual organs develop independently of surrounding tissues. The sequential model suggests that inductive events between the outer and inner whorl primordia are required for appropriate organogenesis. To test these models we have genetically ablated second and third whorl floral organ primordia to determine if organ identity, number or position are perturbed in the first or fourth whorls. We used diphtheria toxin to specifically ablate floral cells early in development in Nicotiana tabacum and in Arabidopsis thaliana. Second and third whorl expression of the diphtheria toxin A chain coding sequence (DTA) was conferred by the Arabidopsis APETALA3 (AP3) promoter. Both Nicotiana and Arabidopsis flowers that express the AP3-DTA construct lack petals and stamens; it appears that the second and third whorl cells expressing this construct arrest early in floral development. These results show that first and fourth whorl development is normal and can proceed without information from adjacent second and third whorl primordia. We propose that positional information specifies the establishment of all four whorls of organs prior to the expression of AP3 in the floral meristem. PMID- 7555716 TI - HGF/SF: a potent cytokine for mammary growth, morphogenesis and development. AB - The mammary gland is a renewing tissue in which morphogenetic processes and differentiation occur cyclically during the menstrual cycle, pregnancy and lactation. These events have been shown to be dependent upon epithelial mesenchymal interactions. Studies of the effects of individual factors, their cellular source and their target cell populations in the different developmental stages of the mammary gland are greatly facilitated by the accessibility of this organ and the application of new techniques that allow purification of the major epithelial and stromal components of this tissue. Here we demonstrate that HGF/SF and its cellular receptor, c-met, are expressed and regulated temporally during mouse mammary development and differentiation. We show that human and mouse mammary fibroblasts produce HGF/SF and that HGF/SF is not only mitogenic but morphogenic and motogenic for both human and mouse mammary epithelial cells. We have found that human luminal and myoepithelial cells express c-met differentially and that HGF/SF has different effects on these two mammary epithelial cell populations. HGF/SF is mitogenic for luminal cells but not myoepithelial cells, and morphogenic to myoepithelial cells but not luminal cells. This is discussed in the context of the proliferative compartments in the normal mammary gland and the potential role of the myoepithelial cells to act as the skeleton for ductal development. PMID- 7555717 TI - The Hox11 gene is essential for cell survival during spleen development. AB - The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of beta-galactosidase from the recombinant hox11 allele in +/- mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11 -/- mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11 -/- mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis. PMID- 7555718 TI - Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death. AB - The goosecoid gene encodes a homeodomain-containing protein that has been identified in a number of species and has been implicated in a variety of key developmental processes. Initially suggested to be involved in organizing the embryo during early development, goosecoid has since been demonstrated to be expressed during organogenesis-most notably in the head, the limbs and the ventrolateral body wall. To investigate the role of goosecoid in embryonic development, we have inactivated the gene by gene targeting to generate mice mutant for the goosecoid gene. Mice that are homozygous for the goosecoid mutation do not display a gastrulation phenotype and are born; however, they do not survive more than 24 hours. Analysis of the homozygotes revealed numerous developmental defects affecting those structures in which goosecoid is expressed during its second (late) phase of embryonic expression. Predominantly, these defects involve the lower mandible and its associated musculature including the tongue, the nasal cavity and the nasal pits, as well as the components of the inner ear (malleus, tympanic ring) and the external auditory meatus. Although the observed phenotype is in accordance with the late expression domains of goosecoid in wild-type embryos, we suggest that the lack of an earlier phenotype is the result of functional compensation by other genes. PMID- 7555720 TI - A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. AB - Cell differentiation commonly dictates a change in the cell cycle of mitotic daughters. Previous investigations have suggested that the Drosophila bag of marbles (bam) gene is required for the differentiation of germline stem cell daughters (cystoblasts) from the mother stem cells, perhaps by altering the cell cycle. In this paper, we report the preparation of antibodies to the Bam protein and the use of those reagents to investigate how Bam is required for germ cell development. We find that Bam exists as both a fusome component and as cytoplasmic protein and that cytoplasmic and fusome Bam might have separable activities. We also show that bam mutant germ cells are blocked in differentiation and are trapped as mitotically active cells like stem cells. A model for how Bam might regulate cystocyte differentiation is presented. PMID- 7555719 TI - Origin and specification of type II sensory neurons in Drosophila. AB - The peripheral nervous system (PNS) of Drosophila is a preferred model for studying the genetic basis of neurogenesis because its simple and stereotyped pattern makes it ideal for mutant analysis. Type I sensory organs, the external (bristle-type) sensory organs (es) and the internal (stretch-receptive) chordotonal organs (ch), have been postulated to derive from individual ectodermal precursor cells that undergo a stereotyped pattern of cell division. Little is known about the origin and specification of type II sensory neurons, the multiple dendritic (md) neurons. Using the flp/FRT recombinase system from yeast, we have determined that a subset of md neurons derives from es organ lineages, another subset derives from ch organ lineages and a third subset is unrelated to sensory organs. We also provide evidence that the genes, numb and cut, are both required for the proper differentiation of md neurons. PMID- 7555721 TI - Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo. AB - Using immunofluorescent labeling and laser-scanning confocal microscopy, we show that isoforms of histone H4 acetylated on lysine 5, 8 and/or 12 (H4.Ac5-12), as well as RNA polymerase II, become enriched at the nuclear periphery around the time of zygotic gene activation, i.e., the 2-cell stage, in the preimplantation mouse embryo. In contrast, DNA and H4 acetylated on lysine 16 are uniformly distributed throughout the cytoplasm. Culture of embryos with inhibitors of histone deacetylase trichostatin A and trapoxin results in an increase in the (1) amount of acetylated histone H4 detected by immunoblotting, (2) intensity and sharpness of the peripheral staining for H4.Ac5-12, and (3) relative rate of synthesis of proteins that are markers for zygotic gene activation. The enhanced staining for H4.Ac5-12 at the nuclear periphery seems to require DNA replication, but appears independent of cytokinesis or transcription, since its development is inhibited by aphidicolin but not by either cytochalasin D or alpha-amanitin. Lastly, the restricted localization of H4.Ac 5-12 is not observed in the 4-cell embryo or at later stages of preimplantation development. These results suggest that changes in chromatin structure underlie, at least in part, zygotic gene activation in the mouse. PMID- 7555722 TI - Transformation of the germ line into muscle in mes-1 mutant embryos of C. elegans. AB - Mutations in the maternal-effect sterile gene mes-1 cause the offspring of homozygous mutant mothers to develop into sterile adults. Lineage analysis revealed that mutant offspring are sterile because they fail to form primordial germ cells during embryogenesis. In wild-type embryos, the primordial germ cell P4 is generated via a series of four unequal stem-cell divisions of the zygote. mes-1 embryos display a premature and progressive loss of polarity in these divisions: P0 and P1 undergo apparently normal unequal divisions and cytoplasmic partitioning, but P2 (in some embryos) and P3 (in most embryos) display defects in cleavage asymmetry and fail to partition lineage-specific components to only one daughter cell. As an apparent consequence of these defects, P4 is transformed into a muscle precursor, like its somatic sister cell D, and generates up to 20 body muscle cells instead of germ cells. Our results show that the wild-type mes 1 gene participates in promoting unequal germ-line divisions and asymmetric partitioning events and thus the determination of cell fate in early C. elegans embryos. PMID- 7555723 TI - How the Hox gene Ultrabithorax specifies two different segments: the significance of spatial and temporal regulation within metameres. AB - In Drosophila, the Hox gene Ultrabithorax (Ubx) specifies the development of two different metameres--parasegment 5, which is entirely thoracic, and parasegment 6, which includes most of the first abdominal segment. Here we investigate how a single Hox gene can specify two such different morphologies. We show that, in the early embryo, cells respond similarly to UBX protein in both parasegments. The differences between parasegments 5 and 6 can be explained by the different spatial and temporal pattern of UBX protein expression in these two metameres. We find no evidence for multiple threshold responses to different levels of UBX protein. We examine in particular the role of Ubx in limb development. We show that UBX protein will repress limb primordia before 7 hours, when Ubx is expressed in the abdomen, but not later, when UBX is first expressed in the T3 limb primordium. The regulation of one downstream target of UBX, the Distalless gene, provides a model for this transition at the molecular level. PMID- 7555724 TI - Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail. AB - To analyse the roles of FGF activity and brachyury during gastrulation we have directly compared the consequences of inhibition of FGF-receptor signalling with the phenotype of the zebrafish brachyury mutant, no tail (ntl). We show that expression of ntl is regulated by FGF and that inhibition of FGF receptor signalling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. We propose a model to explain the FGF-dependent regulation of ntl and ntk that accounts for the above phenotypes. Consistent with this model, over-expression of eFGF led to suppression of anterior fates and development of trunk and tail derivatives only. In addition, widespread activation of convergence and extension movements resulted in the formation of multiple axis-like structures. Expression of eve1 and cad1 was also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorising centre during AP patterning, mediated by FGF activity in this tissue. PMID- 7555725 TI - A predicted membrane protein, TRA-2A, directs hermaphrodite development in Caenorhabditis elegans. AB - The nematode C. elegans naturally develops as either an XO male or XX hermaphrodite. The sex-determining gene, tra-2, promotes hermaphrodite development in XX animals. This gene encodes a predicted membrane protein, named TRA-2A, which has been proposed to provide the primary feminising activity of the tra-2 locus. Here, we show that transgenic TRA-2A driven from a heat shock promoter can fully feminise the somatic tissues of XX tra-2 loss-of-function mutants, which would otherwise develop as male. TRA-2A is thus likely to provide a component of the tra-2 locus that is both necessary and sufficient to promote female somatic development. Transgenic TRA-2A driven by the heat shock promoter can also transform XO animals from male to self-fertile hermaphrodite. This result establishes the role of tra-2 as a developmental switch that controls somatic sexual cell fate. We show that a carboxy-terminal region of TRA-2A, predicted to be intra-cellular, can partially feminise XX tra-2 loss-of-function mutants and XO tra-2(+) males. We suggest that this intra-cellular domain of TRA 2A promotes hermaphrodite development by negatively regulating the FEM proteins. PMID- 7555726 TI - Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. AB - Goosecoid (gsc) is an evolutionarily conserved homeobox gene expressed in the gastrula organizer region of a variety of vertebrate embryos, including zebrafish, Xenopus, chicken and mouse. To understand the role of gsc during mouse embryogenesis, we generated gsc-null mice by gene targeting in embryonic stem cells. Surprisingly, gsc-null embryos gastrulated and formed the primary body axes; gsc-null mice were born alive but died soon after birth with numerous craniofacial defects. In addition, rib fusions and sternum abnormalities were detected that varied depending upon the genetic background. Transplantation experiments suggest that the ovary does not provide gsc function to rescue gastrulation defects. These results demonstrate that gsc is not essential for organizer activity in the mouse but is required later during embryogenesis for craniofacial and rib cage development. PMID- 7555727 TI - Gratuitous mRNA localization in the Drosophila oocyte. AB - Many of the genes that control pattern formation in Drosophila encode mRNAs that are localized to discrete regions of the oocyte during oogenesis. While such localization is generally assumed to be important for the pattern-forming activities of these genes, this has been rigorously demonstrated in only a few cases. Here we address the role of mRNA localization for the dorsoventral patterning gene K10. K10 mRNA is localized to the oocyte's anterior cortex following its transport into the cell during early stages of oogenesis. We show that mutations in cappuccino and spire, which permit K10 mRNA transport, but prevent subsequent anterior localization, do not disrupt the synthesis or localization of K10 protein. We also show that modified K10 transgenes that produce transcripts which are uniformly distributed throughout the oocyte, or which are mislocalized to the oocyte's posterior pole, produce localized and functional K10 protein. We conclude that the anterior localization of K10 mRNA is not important for K10 protein targeting or gene function. We propose that the anterior localization of K10, and probably other mRNAs, is a by-product of mRNA transport and does not necessarily reflect a requirement for localization per se. PMID- 7555729 TI - Expression of the cell cycle control gene, cdc25, is constitutive in the segmental founder cells but is cell-cycle-regulated in the micromeres of leech embryos. AB - The identifiable cells of leech embryos exhibit characteristic differences in the timing of cell division. To elucidate the mechanisms underlying these cell specific differences in cell cycle timing, the leech cdc25 gene was isolated because Cdc25 phosphatase regulates the asynchronous cell divisions of postblastoderm Drosophila embryos. Examination of the distribution of cdc25 RNA and the zygotic expression of cdc25 in identified cells of leech embryos revealed lineage-dependent mechanisms of regulation. The early blastomeres, macromeres and teloblasts have steady levels of maternal cdc25 RNA throughout their cell cycles. The levels of cdc25 RNA remain constant throughout the cell cycles of the segmental founder cells, but the majority of these transcripts are zygotically produced. Cdc25 RNA levels fluctuate during the cell cycles of the micromeres. The levels peak during early G2, due to a burst of zygotic transcription, and then decline as the cell cycles progress. These data suggest that cells of different lineages employ different strategies of cell cycle control. PMID- 7555728 TI - Mutations in the Drosophila gene bullwinkle cause the formation of abnormal eggshell structures and bicaudal embryos. AB - Subcellular localization of gene products and cell migration are both critical for pattern formation during development. The bullwinkle gene is required in Drosophila for disparate aspects of these processes. In females mutant at the bullwinkle locus, the follicle cells that synthesize the dorsal eggshell filaments do not migrate properly, creating short, broad structures. Mosaic analyses demonstrate that wild-type BULLWINKLE function is required in the germ line for these migrations. Since the mRNA for gurken, the putative ligand that signals dorsal follicle cell fate, is correctly localized in bullwinkle mutants, we conclude that our bullwinkle alleles do not affect the dorsoventral polarity of the oocyte and thus must be affecting the follicle cell migrations in some other way. In addition, the embryos that develop from bullwinkle mothers are bicaudal. A KINESIN:beta-GALACTOSIDASE fusion protein is correctly localized to the posterior pole of bullwinkle oocytes during stage 9. Thus, the microtubule structure of the oocyte and general transport along it do not appear to be disrupted prior to cytoplasmic streaming. Unlike other bicaudal mutants, oskar mRNA is localized correctly to the posterior pole of the oocyte at stage 10. By early embryogenesis, however, some oskar mRNA is mislocalized to the anterior pole. Consistent with the mislocalization of oskar mRNA, a fraction of the VASA protein and nanos mRNA are also mislocalized to the anterior pole of bullwinkle embryos. Mislocalization of nanos mRNA to the anterior is dependent on functional VASA protein. Although the mirror-image segmentation defects appear to result from the action of the posterior group genes, germ cells are not formed at the anterior pole. The bicaudal phenotype is also germ-line dependent for bullwinkle. We suspect that BULLWINKLE interacts with the cytoskeleton and extracellular matrix and is necessary for gene product localization and cell migration during oogenesis after stage 10a. PMID- 7555730 TI - frizzled regulates mirror-symmetric pattern formation in the Drosophila eye. AB - Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. We have investigated the mechanisms by which this pattern formation occurs and our results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. We propose that frizzled mediates a bidirectional signal emanating from the equator. PMID- 7555731 TI - pRb is necessary for inhibition of N-myc expression by TGF-beta 1 in embryonic lung organ cultures. AB - The beta type transforming growth factors (TGF-beta) are potent inhibitors of epithelial cell proliferation, and data suggest that growth inhibition by TGF beta 1 is mediated through suppression of Myc family genes in certain cell types. Indirect evidence has indicated that the product of the retinoblastoma gene (pRb) may also be involved in this pathway. Previously, we have shown that TGF-beta 1 inhibits branching morphogenesis and N-myc expression in mouse embryonic lung cultures. The purpose of this study was to determine the role of pRb in the inhibition of branching morphogenesis and N-myc expression by TGF-beta 1. Treatment with TGF-beta 1 was shown to inhibit development of lungs from homozygous Rb null (Rb-/-) and heterozygous null (Rb+/-) mouse embryos to the same extent as lungs from wild-type (Rb+/+) embryos. However, TGF-beta 1 treatment did not suppress N-myc expression in Rb-/- as it did in Rb+/+ embryonic lung explants as determined by in situ hybridization and quantitative RT-PCR. The effect of TGF-beta 1 treatment on N-myc expression in lungs from Rb+/- embryos was intermediate between that seen in Rb+/+ and Rb-/- embryos. Embryonic lungs derived from transgenic mice expressing the SV40 large T-antigen in lung epithelium under the control of the surfactant protein C promoter also showed inhibition of development in response to TGF-beta 1 treatment. The data demonstrate that pRb is necessary for TGF-beta 1 suppression of N-myc expression but not for TGF-beta 1 inhibition of branching morphogenesis; therefore, suppression of N-myc is not necessary for inhibition of branching morphogenesis by TGF-beta 1. PMID- 7555732 TI - Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element. AB - The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid. PMID- 7555733 TI - Sex-specific differentiation of a male-specific abdominal muscle, the Muscle of Lawrence, is abnormal in hydroxyurea-treated and in fruitless male flies. AB - A prominent sex-specific abdominal muscle in male Drosophila is the Muscle of Lawrence (MOL), which is induced by male-specific innervation. We have examined MOL development in wild-type males, in males fed hydroxyurea to ablate the muscle precursors and in fruitless mutants, in which the MOL muscle develops aberrantly. One striking feature of MOLs in wild-type males was the presence of additional muscle nuclei compared with neighboring muscles or MOL-homologues in females. We tested whether muscle length and the sex-specific expression of a reporter gene depended critically on the number of nuclei present within a MOL fiber. MOL fibers developing from a reduced myoblast pool in hydroxyurea-affected hemisegments were recognizable by their attachment points and still contained more nuclei than did neighboring medial fibers, suggesting that these MOL fibers were able to actively recruit myoblasts nearly as well as wild-type MOLs. However, many of the hydroxyurea-affected MOL fibers were incapable of the normal male-specific expression of a muscle-specific reporter gene. We suggest that early events in MOL development, such as finding the correct muscle attachment points, are relatively insensitive to the number of MOL nuclei compared with later events, such as the sex-specific expression of a reporter gene. In fruitless mutant males, MOL-position fibers are smaller and had substantially fewer nuclei compared to wild-type MOLs. Since the number and distribution of muscle precursors was the same in fruitless mutant and wild-type animals, we propose that one fru+ function is to direct the male-specific recruitment of myoblasts into MOL-myotubes. However, fruitless+ must have more than one role in MOL fiber development, since simple reduction in the number of muscle nuclei, as demonstrated by the hydroxyurea ablations, is insufficient to account for all of the MOL muscle phenotypes in fruitless mutant males. PMID- 7555734 TI - PDGF signalling is required for gastrulation of Xenopus laevis. AB - During Xenopus gastrulation, platelet-derived growth factor (PDGF) receptor-alpha is expressed in involuting marginal zone cells which migrate over ectodermal cells expressing PDGF-A. To investigate the role of PDGF signalling during this process, we have generated a novel point mutant of PDGF receptor-alpha analogous to the W37 mutation of c-kit. This molecule is a specific, potent, dominant inhibitor of PDGF signalling in vivo. Injection of RNA encoding this protein into Xenopus embryos prevents closure of the blastopore, leads to abnormal gastrulation and a loss of anterior structures. Convergent extension is not inhibited in these embryos, but rather, involuting mesodermal cells fail to adhere to the overlying ectoderm. PDGF may therefore be required for mesodermal cell-substratum interaction. PMID- 7555735 TI - Genetic determinants of sense organ identity in Drosophila: regulatory interactions between cut and poxn. AB - Two genes involved in defining the type of sense organ have been identified in Drosophila. The gene cut differentiates the external sense organs (where it is expressed) from the chordotonal organs (where it is not); among the external sense organs poxn differentiates the poly-innervated organs (where it is expressed) from the mono-innervated organs (where it is not). Here we show that the expression of poxn in normal embryos does not depend on cut, and that poxn is capable of inducing the expression of cut. We have identified a small domain of the very large cut regulatory region as a likely target for activation by poxn. PMID- 7555736 TI - bFGF as a possible morphogen for the anteroposterior axis of the central nervous system in Xenopus. AB - Vertebrate neural development is initiated during gastrulation by the inductive action of the dorsal mesoderm (Spemann's organizer in amphibians) on neighbouring ectoderm, which eventually gives rise to the central nervous system from forebrain to spinal cord. Here we present evidence that bFGF can mimic the organizer action by inducing Xenopus ectoderm cells in culture to express four position-specific neural markers (XeNK-2, En-2, XIHbox1 and XIHbox6) along the anteroposterior axis. bFGF also induced the expression of a general neural marker NCAM but not the expression of immediate-early mesoderm markers (goosecoid, noggin, Xbra and Xwnt-8), suggesting that bFGF directly neuralized ectoderm cells without forming mesodermal cells. The bFGF dose required to induce the position specific markers was correlated with the anteroposterior location of their expression in vivo, with lower doses eliciting more anterior markers and higher doses more posterior markers. These data indicate that bFGF or its homologue is a promising candidate for a neural morphogen for anteroposterior patterning in Xenopus. Further, we showed that the ability of ectoderm cells to express the anterior markers in response to bFGF was lost by mid-gastrula, before the organizer mesoderm completely underlies the anterior dorsal ectoderm. Thus, an endogenous FGF-like molecule released from the involuting organizer may initiate the formation of the anteroposterior axis of the central nervous system during the early stages of gastrulation by forming a concentration gradient within the plane of dorsal ectoderm. PMID- 7555737 TI - Evidence for target regulation of the development of androgen sensitivity in rat spinal motoneurons. AB - Specific neuronal circuits within the vertebrate nervous system express high levels of steroid receptors and are sensitive to the effects of steroid hormones. The mechanisms by which these neuronal circuits develop their unique steroid sensitivity are unknown. One intriguing hypothesis is that retrograde influences during early postnatal life play a role in determining which central nervous system (CNS) neurons become sensitive to steroids. We now present evidence that during a critical period in early postnatal development, axonal injury disrupts the normal development of steroid sensitivity. The spinal nucleus of the bulbocavernosus (SNB) is a neuromuscular system that is highly androgen-sensitive at the level of both the motoneurons and their target muscles. Testosterone levels regulate the size of SNB motoneurons and their muscles in adult rats. Cutting the axons of SNB motoneurons on postnatal day 14 (P14) caused permanent decreases in SNB motoneuronal soma size, as well as in SNB target muscle weight. Interestingly, SNB motoneurons that survived axotomy on P14 failed to develop their normal ability to respond to testosterone in adulthood. That is, they did not respond to changes in testosterone levels with changes in soma size. The same effect was not seen after axotomy 1 week later in development, suggesting a critical period for this effect. Thus, separation from the target muscles during an early critical period in development blocked the differentiation of androgen sensitivity by SNB motoneurons, consistent with a role for the target in the normal development of steroid sensitivity by CNS neurons. PMID- 7555738 TI - Intradermal implants of histotypic adrenal gland rotary cultures. AB - The implantation of histotypic adrenal gland cultures is described in the present study. The cultures were prepared from mice at birth and were rotary incubated for 10 days. When the cells reached phenotypic maturation, they were implanted in the dermal bed of the auricular pavilion of young adult mice. This location was ideal for implantation because transillumination enabled daily inspection of the progress of implant survival. Twenty days after implantation, the implants and associated tissues were removed to study the degree of maturation and the viability of the implants. Some cultures showed a moderate percentage of steroidal cells, while others demonstrated a high predominance of the chromaffin cells. All the implanted cultures successfully survived a postoperative period of 20 days. Both adrenergic and noradrenergic cells reached their maximum grade of cytological differentiation and neither inflammatory reactions nor adverse immunological effects were observed. On the contrary, the implants were profusely vascularized by the host tissue, particularly in those implants with a higher content of chromaffin cells. PMID- 7555739 TI - Lactate transport in cultured glial cells. AB - Uptake of L-lactate was investigated with a radioactive tracer method in cultured rat glioma cells and in astroglia-rich primary cultures derived from rat brain. In the glioma cells, a saturable component of uptake was identified with half maximal uptake occurring at 1.0 +/- 0.4 mM lactate. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. In astroglia rich primary cultures, no saturable component of uptake could be detected in the concentration range studied (0.1-15 mM). In conclusion, lactate uptake at physiological concentrations is predominantly mediated in the glioma cells by a carrier-dependent process, whereas in astroglial cells, simple diffusion appears to be the dominant way of lactate transport. PMID- 7555740 TI - Expression of cell surface markers and myelin proteins in cultured oligodendrocytes from neonatal brain of rat and mouse: a comparative study. AB - Dissociated brain cell cultures are a useful model for investigating development and differentiation of oligodendrocytes in vitro. The current studies compare the developmental patterns of expression for oligodendrocyte lineage/myelin markers in both primary and secondary oligodendrocyte cultures derived from mouse and rat neonates. The rat and mouse dissociated brain cell cultures express the same myelin-specific antigens, but mouse oligodendrocytes produce a larger and more elaborate sheet-like membrane than rat oligodendrocytes, and some of the myelin markers (O4, GC, and MBP) show more intense membrane staining in mouse cultures. GD3 appears to be a good oligodendrocyte marker for rat cells, but it is nonspecific in mouse cells. There are fewer oligodendrocytes in mouse cultures, and they appear to require a longer differentiation time than rat oligodendrocytes. These same results are also observed in secondary oligodendrocyte cultures, although in general late myelin markers such as MBP and PLP are expressed at a much lower level in mouse cells than rat cells. PMID- 7555741 TI - Cellular localization of gap junction mRNAs in developing rat brain. AB - We investigated the developmental expression and cellular resolution of connexin32 and 43 mRNA in the rat brain using in situ hybridization. Utilizing 35S-labelled probes, in situ hybridization was performed on sections of embryonic day 20 and postnatal days 3, 10, 15, 30 and adult brain. Connexin32 mRNA was first detected in brainstem nuclei at postnatal day 3 and in the midbrain at postnatal day 15. The level of this message continued to increase to postnatal day 30 where the level of message reached a plateau or slightly decreased by adulthood. The distribution of signal included the medial vestibular nucleus, dorsal paragigantocellular nucleus and fibre tracts of the midbrain and brainstem such as the cerebellar white matter, spinal trigeminal tract, and decussation of the superior cerebellar peduncle, areas containing predominantly neurons or oligodendrocytes. Connexin43 mRNA was first detected much earlier than connexin32 and was found in the leptomeniges of the E20 brain. It was found at all ages examined and distributed homogenously in the regions of the brain examined, suggesting its presence in astrocytes. The connexin43 riboprobe also hybridized strongly to the ependymal cells of the fourth ventricle and cerebral aqueduct. These results describe the cellular resolution of connexin mRNAs during development and that a differential pattern of expression exists in the various cell populations in the central nervous system. PMID- 7555742 TI - Tumor necrosis factor-alpha production by human fetal microglial cells: regulation by other cytokines. AB - Tumor necrosis factor (TNF)-alpha has been postulated to play an important physiologic as well as pathologic role within the developing brain. In the present study, we found that human fetal microglial cells released abundant amounts of TNF-alpha upon stimulation with lipopolysaccharide (LPS). Treatment of microglial cell cultures with antibodies specific to interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-beta augmented LPS-stimulated release of TNF alpha. Each of these cytokines dose-dependently suppressed TNF-alpha release. Also, TNF-alpha mRNA expression was inhibited by each of these cytokines. By way of contrast, treatment of microglial cell cultures with IL-alpha or IL-1 beta alone or in the presence of LPS, resulted in increased release of TNF-alpha, and IL-1 stimulated the expression of TNF-alpha mRNA. These findings suggest that these cytokines are likely to modify the beneficial and harmful effects of TNF alpha within the developing brain. PMID- 7555743 TI - [Is extensive diphtheria vaccination necessary?]. PMID- 7555744 TI - [Glucokinase: the first hit in the hunt for a gene in late-onset diabetes]. PMID- 7555745 TI - [Glucagon peptide--new treatment for late-onset diabetes?]. PMID- 7555746 TI - [Relationship between intestinal inflammation and arthritis]. PMID- 7555747 TI - [Prevention of fractures of the proximal end of the femur]. PMID- 7555748 TI - [Is the mind located in the brain?]. PMID- 7555750 TI - [Low back pain in adolescents]. PMID- 7555752 TI - [Sarcoidosis-associated kidney changes]. PMID- 7555749 TI - [Severe epilepsy in children less than 2 years old: does surgery help?]. PMID- 7555751 TI - [Citalopram in the treatment of panic disorders and school phobia in children and adolescents]. PMID- 7555753 TI - [Can "psychopathy" be cured?]. PMID- 7555754 TI - [Penicillin-resistant pneumococci are coming--are you prepared?]. PMID- 7555755 TI - [Troponin--current possibility for the diagnosis of myocardial infarction]. PMID- 7555756 TI - [Is now the time to evaluate the function of child health clinics?]. PMID- 7555758 TI - [Development of electrical function of the brain in light of quantitative EEG]. PMID- 7555757 TI - [Does coitus have an effect on the course of pregnancy?]. PMID- 7555760 TI - [Loss of teeth in a 1-year-old child]. PMID- 7555759 TI - [Cardiac pacemaker infections in Helsinki University Central Hospital 1989-93]. PMID- 7555761 TI - [Anomaly of the first brachial cleft]. PMID- 7555762 TI - [Asbestos-associated cancer as an occupational disease]. PMID- 7555763 TI - [The diagnosis and treatment of hypercholesterolemia and other hyperlipidemias in children--recommendations of the Finnish Pediatricians' Association]. PMID- 7555764 TI - [Discontinuation of epilepsy drug therapy]. PMID- 7555766 TI - [Unknown patient]. PMID- 7555765 TI - [Intracranial hypertension cannot be excluded with computerized tomography]. PMID- 7555767 TI - [Ski racing and asthma]. PMID- 7555768 TI - [Ozone depletion--does the incidence of skin cancer increase?]. PMID- 7555769 TI - [Top groups in science in Finland]. PMID- 7555770 TI - [HIV and family planning]. PMID- 7555773 TI - [Realization of immunization in child health centers]. PMID- 7555772 TI - [3-dimensional imaging--new computerized tomography technique]. PMID- 7555771 TI - [Psychological factors associated with organ transplantation]. PMID- 7555776 TI - [Persistent ear infection, impaired hearing and fever]. PMID- 7555774 TI - [Vitamin B 12 in zoster neuralgia]. PMID- 7555775 TI - [An unusual cause for prolonged cough and exercise-induced shortness of breath]. PMID- 7555777 TI - [Slipped capital femoral epiphyses]. PMID- 7555780 TI - [Does vaccination prevent Rotavirus gastroenteritis?]. PMID- 7555778 TI - [Mothers and iron]. PMID- 7555781 TI - [Is legislation needed in the treatment of infertility?]. PMID- 7555779 TI - [Are there grounds for iron supplementation?]. PMID- 7555782 TI - [Nocturnal paroxysmal dystonia--epilepsy or sleep disorder?]. PMID- 7555783 TI - [Assessment of the adequacy of anesthesia]. PMID- 7555784 TI - [Mental well-being of postpartum women]. PMID- 7555785 TI - [Hallucination as a conversion symptom in mental disorders]. PMID- 7555786 TI - [Disulfiram therapy]. PMID- 7555787 TI - [Acute heart failure in a young man (clin conference)]. PMID- 7555788 TI - [Thromboplastin time, INR reporting and recommendations for anticoagulant therapy]. PMID- 7555789 TI - [Immunological novelties for clinicians]. PMID- 7555792 TI - [Cytokine measurements from patient specimens]. PMID- 7555791 TI - [Complement deficiencies]. PMID- 7555790 TI - [Antibody avidity in immune response and in the diagnosis of infectious diseases]. PMID- 7555793 TI - [Superantigens]. PMID- 7555794 TI - [Autoimmune diseases and autoantigens]. PMID- 7555795 TI - [Endoscopy today]. PMID- 7555796 TI - [An expanding spectrum of hepatitis viruses]. PMID- 7555797 TI - [Esophageal reflux disease]. PMID- 7555799 TI - [Endoscopic ultrasonography--a new tool for gastroenterology]. PMID- 7555798 TI - [Cause-targeted treatment of ulcer disease]. PMID- 7555801 TI - [Laparoscopy in gastroenterology]. PMID- 7555800 TI - [When should gallstones be treated?]. PMID- 7555803 TI - [Who has alcoholic liver disease?]. PMID- 7555804 TI - [Spare parts for the abdominal region]. PMID- 7555802 TI - [Treatment and prevention of chronic viral hepatitis]. PMID- 7555806 TI - [Screening for colon cancer]. PMID- 7555805 TI - [Fulminant pancreatitis]. PMID- 7555807 TI - [Irritable bowel syndrome]. PMID- 7555808 TI - [Enteral vs parenteral nutrition?]. PMID- 7555809 TI - [Changing treatment of inflammatory bowel diseases]. PMID- 7555810 TI - [Depression]. PMID- 7555811 TI - [How many of us are depressed?]. PMID- 7555813 TI - [Central concepts and problems in the diagnosis of depression]. PMID- 7555812 TI - [Depression and creativity]. PMID- 7555814 TI - [Depression amd depressive feelings in childhood and adolescence--problems in diagnosis and assessment]. PMID- 7555815 TI - [Severe mood disorders and heredity]. PMID- 7555818 TI - [Shame as a cause of psychological depression]. PMID- 7555817 TI - [The development of susceptibility to depression and its psychodynamics]. PMID- 7555816 TI - [Biological factors in severe depression]. PMID- 7555819 TI - [Course and prognosis of severe depression]. PMID- 7555820 TI - [Drug therapy and other biological therapies in mood disorders]. PMID- 7555821 TI - [Psychotherapy in depressive conditions]. PMID- 7555822 TI - [Problems in psychotherapy in a severely depressed patient in light of a case report]. PMID- 7555823 TI - [Depressed patients and their family]. PMID- 7555825 TI - [Are current views of handling and classification of psychiatric problems discouraging?]. PMID- 7555824 TI - [Depression and suicide]. PMID- 7555826 TI - [Is ubiquinone worth noticing?]. PMID- 7555827 TI - [New pulmonary symptoms by killer Hantavirus]. PMID- 7555828 TI - [Nuclear hormone receptors]. PMID- 7555829 TI - [Beta blockers, calcium channel blockers and psyche]. PMID- 7555830 TI - [Women and capital offense]. PMID- 7555831 TI - [Can risk factors for pressure sores be decreased with a special mattress?]. PMID- 7555832 TI - [Prevention of pulmonary embolism using inferior vena cava filters]. PMID- 7555834 TI - [Xanthogranulomatous cholecystitis--an unusual cause of gallbladder rupture]. PMID- 7555833 TI - [Significance of the helmet respirator in the treatment of farmer's occupational asthma]. PMID- 7555835 TI - [Stress fractures]. PMID- 7555836 TI - [Reasons for diphtheria vaccination campaign]. PMID- 7555838 TI - [Euthanasia--contempt for life or respect for life?]. PMID- 7555837 TI - [McArdle disease and specialization]. PMID- 7555839 TI - [Old and new red blood cell indices]. PMID- 7555840 TI - [Adenosine in the treatment of supraventricular arrhythmias]. PMID- 7555841 TI - [Is the risk of leukemia increased for children near nuclear installations?]. PMID- 7555842 TI - [Exposure to heavy meals in Lapland]. PMID- 7555843 TI - [Multi-injection treatment of diabetes--does the balance of treatment improve?]. PMID- 7555844 TI - [Surgical treatment of unilateral vocal cord paralysis]. PMID- 7555845 TI - [Small bowel endoscopy during surgery in the treatment and diagnosis of angiodysplasias]. PMID- 7555846 TI - [Treatment of subarachnoid hemorrhage]. PMID- 7555848 TI - [Transesophageal echocardiography]. PMID- 7555847 TI - [Specificity of magnetic resonance spectroscopy]. PMID- 7555849 TI - [Referee system in scientific journals]. PMID- 7555850 TI - [DNA analysis and personal insurance]. PMID- 7555851 TI - [New tools in coronary artery angioplasty]. PMID- 7555853 TI - [Domestic animals and pets as sources of infection]. PMID- 7555852 TI - [From nociception to pain: to feel or not to feel]. PMID- 7555854 TI - [Results of endoscopic treatment of tubal pregnancy]. PMID- 7555856 TI - [Who needs an adder bite treatment kit in winter time?]. PMID- 7555855 TI - [Clinical significance of fetal cardiac arrhythmias]. PMID- 7555857 TI - [Development regression in a child caused by vitamin B12 deficiency]. PMID- 7555858 TI - [Unknown patient--psychogenic amnesia treated with hypnosis]. PMID- 7555859 TI - [Medical staff within the social network of the psychiatric patient]. PMID- 7555860 TI - [Hodgkin disease and amyloidosis]. PMID- 7555861 TI - [Use of anti-inflammatory drugs in children]. PMID- 7555862 TI - [Is polymerase chain reaction practical in the diagnosis of microbes?]. PMID- 7555864 TI - The discovery of the pathogenesis of Meniere's disease. PMID- 7555863 TI - [Lower limb pain and leaping progressive paraparesis]. PMID- 7555865 TI - Glomus tumor of the left ear. PMID- 7555866 TI - Recurrent laryngeal granulomas. PMID- 7555867 TI - Preoperative evaluation. PMID- 7555868 TI - Endoscopic excision of an antral lesion via middle meatal antrostomy. PMID- 7555870 TI - Audiological profiles and Meniere's disease. AB - Pure-tone audiograms of 501 patients with preoperative Meniere's disease were analyzed. Age of onset and incidence of bilaterality were studied. The most common audiogram was the peak-type (50.26%), next the falling-type (26.26%), and then the dip-type (9.24%). Although few papers mention this peak audiogram, we suggest it is a diagnostic feature of Meniere's disease. Average age of onset was 42.59 years; this did not differ from males to females. Prevalence in females exceeded that in males by about 4:3. The disease was bilateral in 31.13%. Generally, those with better hearing thresholds at the first examination had less tendency to develop bilaterality. Age of onset was not significantly correlated with bilaterality. PMID- 7555869 TI - Environmental controls: dust mite--Part I. PMID- 7555871 TI - Meniere's syndrome: an approach to therapy. AB - There are patients with symptoms similar to those of Meniere's disease who do not have Meniere's disease and therefore do not respond to conventional medical or conservative surgical management. Some have subtle disorders of carbohydrate and lipid metabolism and inner ear otosclerosis, which are responsible for the production of their symptoms. The response to dietary therapy confirms that glucose tolerance levels which are not diabetic nor hypoglycemic may be sufficiently significant in relation to inner ear function to produce the symptoms like those of Meniere's Disease. Management of inner ear otosclerosis with supplemental sodium fluoride, calcium carbonate, and Vitamin D was also effective in controlling the symptoms in some patients. PMID- 7555872 TI - Permanent restoration of hearing and vestibular function by the endolymphatic subarachnoid shunt operation. AB - In cases of surgically treatable Meniere's disease, the endolymphatic subarachnoid shunt operation, when successful, can eliminate endolymphatic hydrops and restore function which has not been permanently damaged. This paper will demonstrate the restoration to normal of cochlear and vestibular function following successful endolymphatic subarachnoid shunt procedures. A series of 454 Endolymphatic Subarachnoid Shunt Operations performed on patients with Meniere's disease is reported. Twenty-six patients had surgery on their only hearing ear. Overall, the Endolymphatic Subarachnoid Shunt Operation offered success in sixty five percent of operations performed. Cases of failure can often receive significant benefit from progressively more destructive procedures. The use of the Endolymphatic Subarachnoid Shunt procedure performed early in the course of Meniere's disease can significantly reduce the risk of permanent hearing loss and disability. PMID- 7555874 TI - Invasive fungal sinusitis in the immunocompromised pediatric patient. AB - Invasive fungal sinusitis in the pediatric population appears to be a relatively rare entity. An increasing incidence has been noted in accordance with the widespread use of antibiotics, steroids, antineoplastic drugs and radiation therapy. Three illustrative cases are described which outline the initial presenting symptoms, findings on physical examination, computed tomography (CT), intraoperative findings, and histopathologic findings. A review of the literature is presented and a treatment protocol is proposed once a diagnosis is made. Early suspicion and diagnosis are the most important prerequisites for successful treatment which consists of control of the underlying disease, surgical debridement, and systemic antifungal therapy. Cooperation between the otolaryngologist, pediatric specialists, and pathologists are key to the survival of children with invasive fungal sinusitis. Survival in this series was dependent upon return of an immune competent state. PMID- 7555873 TI - Historical development of surgery for pediatric laryngeal stenosis. PMID- 7555875 TI - Spindle cell carcinoma of the pyriform sinus: a case report. PMID- 7555876 TI - Osteoradionecrosis of the hyoid induced by combined modality therapy for laryngeal carcinoma. AB - Contemporary management of laryngeal carcinoma often incorporates multiple modalities of therapy. We report a case of osteoradionecrosis of the hyoid bone in a patient treated with surgery, chemotherapy, and radiation therapy for a supraglottic squamous cell carcinoma. A discussion regarding pathophysiology, radiation dosimetry and treatment options is also presented. PMID- 7555877 TI - A new kind of rays. PMID- 7555878 TI - Lung cancer in tropical Africa. PMID- 7555879 TI - Lung cancer at the University College Hospital, Ibadan, Nigeria. AB - A 30-year retrospective review of cases of lung cancer from the Cancer Registry of the University College Hospital (UCH), Ibadan, Nigeria was carried out. A total of 142 cases were analysed with a male:female ratio of 1.7:1. Peak age incidence in females was 2 decades (4th) earlier than for males. Squamous cell carcinoma was found most commonly but adenocarcinoma predominated in females. Mucoepidermoid and adenoidcystic carcinomas were absent. About 27% of patients were under 40 years old and the majority of these (82%) showed histological variants which are associated with the bad prognosis (i.e. anaplastic carcinoma, adenocarcinoma, small cell tumours and sarcomas. At autopsy regional lymph nodes were found to be most commonly involved by secondary metastases while the spinal cord was least involved. The epidemiology of malignant lung tumours in Ibadan probably differs from that in the more industrialized countries and this suggests a different view of risk factors for this environment. Additionally there is an urgent need for developing methods for earlier diagnosis if the mortality associated with this disease is to be reduced especially as it occurs predominantly in young people. PMID- 7555880 TI - Primary pulmonary carcinoma in Lagos, Nigeria: a clinicopathological study. AB - A retrospective clinicopathological study of bronchogenic carcinoma in patients admitted to Lagos University Teaching Hospital (LUTH) during a 17-year period (1975-1991) is presented. Out of the 148 cases, 88 were confirmed histopathologically and in the remaining 66 cases, diagnosis was based on a combination of clinical and radiological features. Epidermoid carcinoma was the commonest histopathological variant and the male/female ratio was 2:1. Sixty five percent of the patients admitted to being heavy smokers. The majority of patients presented with weight loss, chronic cough with chest pain and haemoptysis in that order. With an average of nine cases yearly, this study shows that this disease is still uncommon in Nigerians. PMID- 7555881 TI - Breast cancer during pregnancy and lactation in Zaria, Nigeria. AB - As most African women with breast cancer are premenopausal, the probability of pregnancy and lactation co-existing with breast cancer is higher than among the Caucasian patients who are usually post-menopausal. However, because of the relative rarity of breast cancer in black women compared with the Caucasians, experience with gestational breast cancer in African women is very limited. Among 95 women younger than 50 years of age who had the diagnosis of breast cancer over a 12 1/2 year period in Zaria, 25 (26.3%) were either pregnant (n = 14) or lactating (n = 11) at the time of presentation. Comparison of the clinical feature of these 25 with those of the remaining 70 who were neither pregnant nor lactating showed no difference in the clinical presentation, histological tumour type, and the advanced stages of the breast cancer at presentation between the two groups. PMID- 7555882 TI - Epidemiological and clinical aspects of incidental carcinoma of the prostate in Africans: experience at the Lagos University Teaching Hospital, Lagos and the Kenyatta National Hospital, Nairobi. AB - Twenty patients in Lagos and 24 patients in Nairobi with incidental carcinoma of the prostate in resected glands for benign hyperplasia were studied at the Lagos University Teaching Hospital (1978-1982) and at Kenyatta National Hospital (1988 1992), respectively. The age range for the Lagos group was 45-78 years with a mean age of 61 years and a peak incidence in the seventh decade (60-69 year age group) compared to the Nairobi group with age range of 50-89 years with a mean age of 66 years and a peak incidence in the eighth decade (70-79 year age group). Both groups had predominantly well differentiated low grade stage TIA (AI) incidental carcinoma of the prostate, being 80% in the Lagos group and 79.2% in the Nairobi group. High grade undifferentiated stage TIB (A2) incidental carcinoma of the prostate was present in 20% of the Lagos group and 20.8% of the Nairobi group. In both groups, the majority of patients (80%) in the Lagos group and (79.2%) in the Nairobi group presented late with similar symptoms of prostatic obstruction. PMID- 7555883 TI - Enteric pathogens in malnourished children with diarrhoea. AB - Enteric pathogens were determined from stools of 273 children aged less than 5 years at Kenyatta National Hospital (KNH), 43.6% (119/273) of whom were malnourished according to the Wellcome criteria. Rotavirus was detected by ELISA test, Salmonella, Shigella and E. coli by culture on MacConkey and Salmonella Shigella agar at 37 degrees C overnight and Campylobacter on Skirrow's selective media at 42 degrees C for 48 hrs. These were identified by biochemical tests and serotyping using specific antisera. Whereas isolation rate for Campylobacter (0.0% vs 5.0%, p = 0.006), well malnourished ETEC-LT (0.6% vs 5.0%, p = 0.003) and T. hominis (0.0% vs 3.4%, p = 0.03) was higher in the malnourished children, EPEC (30.5% vs 10.1%, p < 0.001) and Salmonella+ETEC-LT (7.8% vs 1.7%, p = 0.02) was higher in children. The other enteric pathogens were equally isolated from normal and malnourished children. A larger proportion of malnourished children had diarrhoea of unknown aetiology compared to the well nourished (26.6% vs 50.4%, p < 0.001). Campylobacter and T. hominis may be opportunistic infections due to immuno-suppression in malnutrition. Diarrhoea of unknown aetiology may be due to aetiological agents that were not determined in this study. PMID- 7555884 TI - Environmental risk factors for acute respiratory infections among children of military personnel in Uganda. AB - A community-based, cross-sectional survey was done in five Army camps in the central area of Uganda to explore relationships between environmental household conditions and the presence or absence of acute respiratory infections. A total sample of 122 homes with 152 children of both sexes were studied. Each child was medically examined and diagnosis recorded. A check-list was filled out for each household that had at least one child aged 5 years or less; the list recorded conditions of crowding, roofing materials, house ventilation, cooking fuels and cooking place, family income and immunization status. Cross-tabulation tables were prepared and Chi-square values calculated for the various forms of ARI and environmental characteristics. Significance level was put at 5% (p = < 0.05). Using standard tables, significant associations were found between ARI diagnosed at the examination of a child and the following: number of persons per house (p = 0.01); bed sharing (p = 0.027); house ventilation (p = > 0.01) and; presence or absence of a smoke vent in a house (p = 0.002). Crowding and increased indoor air pollution were rife in Ugandan Army camps and seem to have contributed to the local incidence of ARI in children. It is recommended that health education activities be started to promote improved environmental sanitation and to reduce crowding. Simple smoke vents should be installed over fireplaces to reduce indoor air pollution. The use of gas or electricity as cleaner cooking fuels and making better constructed houses are preferable strategies but both are not likely to be achieved at the moment. PMID- 7555885 TI - Dermatological problems of onchocerciasis in Nebbi District, Uganda. AB - A cross-sectional survey of 770 subjects in onchocerciasis hyperendemic villages and 223 subjects from a control community in Nebbi District in the West Nile region of Uganda revealed a high prevalence of onchocercal skin disease of 48% in endemic villages. The most common skin problem was troublesome itching (40%); and the prevalent skin lesions were chronic papular onchodermatitis (16%), depigmentation (4%), lichenified onchodermatitis (2%) and acute papular onchodermatitis (1%). Other typical varieties of onchocercal skin diseases such as, hanging groin, lymphoedema and marked lymphadenopathy were infrequent, and considered rare. Pityriasis vergicolor was the most common non-onchocercal skin lesion in both control and endemic communities, accounting for 37% of all non onchocercal skin lesions. These skin lesions were associated with a variety of psycho-social and economic impact; and there was a positive correlation between the prevalence of troublesome itching and the prevalence of modules (correlation coefficient r = 0.62, p = 0.00). Given the prospects of onchocerciasis control based on mass ivermectin distribution in communities where blindness is common, we recommend that treatment be extended to communities where blindness is less common, but skin disease known to be predominant. PMID- 7555886 TI - Road traffic accidents in Kenya: an epidemiological appraisal. AB - A descriptive analysis of road traffic accident (RTA) and injury data in Kenya was done using routine accident reports, official statistical abstracts, published and unpublished surveys. The characteristics of injury-producing accidents examined included trends, distribution patterns, risk factors, types of vehicles involved, and road-users injured or killed. The numbers killed increased by 578%, while non-fatal casualties rose by 506% between 1962 and 1992. Fatality rate per 10,000 vehicles increased from 50.7 to 64.2, while fatality per 100,000 population ranged between 7.3 and 8.6. 66% of the accidents occurred during daytime. 60% of the reported RTAs occurred on rural roads and had a higher case fatality rate (CFR) of 16% compared to those occurring in urban areas (11%). Human factors were responsible for 85% of all causes. Vehicle-pedestrian collisions were most severe and had the highest CFR of 24%, while only 12% of injuries resulting from vehicle-vehicle accidents were fatal. Utility vehicles, 'matatus' and buses were involved in 62% of the injury producing accidents. Of all traffic fatalities reported, pedestrians comprised 42%, passengers 38%, drivers 12%, and cyclists 8%. The high pedestrian and passenger deaths imply the need to investigate the underlying risk factors, operational and policy issues involved in the transport system, and to develop and implement appropriate responsive road safety interventions. PMID- 7555887 TI - Clinical features of Black African neonates with Down's syndrome. AB - Early reports indicated a low prevalence of Down's syndrome (DS) in black African children. More recent research demonstrates an incidence similar to, or higher than that reported to occur in First World populations. One of the possible reasons for underreporting of DS in Africa, appears to be the lack of recognition of the problem at birth. In this study, the musculoskeletal, central nervous system and craniofacial features are documented in 40 black DS neonates and 50 black control neonates without DS, and the findings are compared with those from a reported series of 37 caucasian DS and 40 healthy newborns. Musculoskeletal and central nervous system features were markedly similar in black and caucasian infants. However, the craniofacial features of the African DS newborns approximated more closely those of the normal African neonates, than was the case in the caucasian DS and normal neonates. This finding may partially explain the underreporting of DS in this population, and it emphasizes the need for a clinical awareness of DS and for complete clinical examination to identify affected infants. PMID- 7555888 TI - Lagos "area boys and girls" in rehabilitation: their substance use and psychosocial profiles. AB - We report on the psychosocial and substance use profiles of the 55 "Area Boys and Girls" (ABG) admitted to a Rehabilitation Camp in Lagos between March and May 1993 under the main auspices of the People's Bank of Nigeria. The majority were males (73%); young adults (mean age/SD: 30.6/6.3); unemployed (73%); not married (80%); of low educational standing (87%) and from low socio-economic background (over 70%). Almost all were current users of heroin (89%) and cocaine (87%), procured with a substantial amount of money and administered mainly through smoking and "chasing the dragon". None of the clients reported injecting the drug. These findings have highlighted the intricate nature of the problems of ABG and the need for a comprehensive rehabilitation package encompassing general medical, psychological and psychiatric care. Such programmes, ideally free, should be a joint venture between the government, international agencies, non governmental organizations and the community as a whole. The key emphasis should be on equipping the clients with occupational skills for life-long economic sustenance. To curtail the growing numbers of displaced young people in the country, the government should give due attention to the root causes of the multiple social ills in the society. PMID- 7555890 TI - Experience with ventriculo peritoneal shunts at the University of Nigeria Teaching Hospital, Enugu. AB - In a study of 212 children at the University of Nigeria Teaching Hospital, Enugu who received ventriculo peritoneal shunt for hydrocephalus over a 13-year period (1977-1989, 14 had infected shunts and one developed shunt nephritis. Staphylococcus aureus was responsible for 36.4% of the positive cultures and was responsible for the only case of shunt nephritis. This is about the fourth case of shunt nephritis associated with Staphylococcus aureus shunt infection, in the literature. The pertinent features of this case were intraventricular haemorrhage as the cause of the hydrocephalus, frequent revision of shunts before the onset of nephritis and full recovery following intravenous antibiotic therapy and reimplantation of a new shunt. PMID- 7555889 TI - Monitoring progress towards health for all by the year 2000: indicators from Iganga District, Uganda. AB - A sample of 5018 inhabitants in three counties of Iganga district, Uganda was selected by means of a multi-stage cluster sampling procedure and interviewed in 1984. A sub sample was re-interviewed, the following year to study population dynamics. 50.2% of the population were under 15 years of age and 4.2% were aged one year or less. About 80% of both sexes aged 6-15 years were in school or had primary education. Male adult literacy rate was 62% and female adult literacy rate was 38%. The average number of people per sleeping room was 2.4. 82.9% of households used well or unprotected spring as main source of water and 30% of households did not have pit latrine. Infant mortality rate was estimated to be 126 per 1000 livebirths and measles was the major cause of mortality (38%) in the under fives. The crude birth rate estimate was 51 per 1000. The estimate of lameness due to polio was 6.6 per 1000 children aged 15 years or below. The mean weight and the mean height of the children were both below 50th percentile of the NCHS standards. In the re-survey, the crude birth rate and infant mortality rate estimates corresponded well to the initial values for the total sample obtained in the previous survey. 5% of the population had moved out of the village and 1.8% had moved to settle in the village. The growth rate in both weight and height of the under five children was satisfactory compared to the rate of the standard. From the result of the study, only six years after adoption of PHC in Uganda, the relatively high educational level of the younger population should be taken as a positive indicator of better health indices in the future. PMID- 7555891 TI - Socio-economic client characteristics and consequences of abortion in Nairobi. AB - In Nairobi, 281 women presenting with alleged miscarriage were subdivided in the following subgroups, 91 spontaneous abortions (group 1), 152 suspected induced abortions (group 2) and 38 admittedly induced abortions (group 3). In the statistical analysis women in group 1 were compared with women from groups 2 and 3 combined (here referred to as group 4). Group 4 differed significantly from group 1 in the following respects: they were younger (p = 0.03) although they had previously born more babies (p = 0.008), lived more often together with their parents (p = 0.024), were less often married (p = 0.000012), worked more often as housewives (p = 0.00079), and lived in bigger households (p = 0.000015). No significant differences were encountered regarding religious beliefs. Group 4 women were more informed about contraceptives than group 1 (p = 0.04), particularly regarding injectables (p = 0.000096) and oral contraceptives (p = 0.0013) but also intra uterine devices (p = 0.024). Almost 90% of group 4 women indicated post abortion contraceptive interest, while only 1/3 of group 1 women did so. Group 3 women needed extensive parenteral treatment with a total hospital treatment cost amounting to 300 times that of group 1. The total cost of intravenous infusions was almost 10 times as high in group 3 as in group 1. The total number of days in hospital was significantly higher in group 4 than in group 1 (p = 0.0098).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555893 TI - Peer review and refereeing in science. PMID- 7555892 TI - Goldenhar syndrome (oculo-auriculo-vertebral dysplasia): report of two cases. AB - A report of two cases of oculo-auriculo-vertebral dysplasia (Goldenhar syndrome) from Benin City, Nigeria is presented. Both cases were seen in males with normal anthropometric parameters of head circumference, length and birth weight. The diagnosis was made in the neonatal period. Both babies had the consistent features of epibulbar dermiods, pre-auricular tags, and vertebral anomalies, though the lesions were seen on the left side of the body as against the expected right. Suddenly, within the first five weeks of life, both babies died of unknown cause. PMID- 7555894 TI - Is prophylactic popliteal node dissection necessary in malignant melanoma of the foot? PMID- 7555895 TI - A new, simple method for the rapid quantification of malarial parasitaemia in stained thick blood films. PMID- 7555897 TI - [Can we cure the state institutions?]. PMID- 7555898 TI - [The disjunctive in human resource training in dentistry in Mexico]. PMID- 7555896 TI - [Nursing research]. PMID- 7555899 TI - [Information system on human resources as strategic device to achieve efficiency and democratization in local health management]. PMID- 7555900 TI - [Factors associated with medical productivity]. PMID- 7555901 TI - [Action learning. Is this the way to train health managers in developing countries?]. PMID- 7555902 TI - [Development of an observed and evaluation model of health personnel supply in the municipality of Buenos Aires]. PMID- 7555903 TI - [Successful experience with the vector control courses in the provinces of Cuba]. PMID- 7555904 TI - [Extracurricular programs at dental education institutions in Latin America++ and the United States of America. Contribution to the study]. PMID- 7555905 TI - Correlates of sleep latency on the multiple sleep latency test in a clinical population. AB - The multiple sleep latency test (MSLT) is commonly used as an objective measure of sleepiness. We retrospectively correlated MSLT scores from 147 patients with other information relating to sleepiness, namely demographic information, data from nocturnal polysomnograms (PSGs), and subjective assessments. The only variable that showed a valid and statistically significant correlation with the MSLT score was sleep latency on the PSG. The results were largely similar within subgroups focusing on (1) individuals with the ability to fall asleep on every nap, (2) subjects with abnormally short MSLT scores, (3) nap attempts that were successful, and (4) patients with particular diagnoses. We conclude that the mean sleep latency on the MSLT, in a clinical population, does not correlate well with a number of variables expected to influence sleepiness. While the validated utility of the MSLT in separating patients from normals or in identifying narcolepsy is not disputed, the accuracy of the MSLT as a measure of sleepiness appears to be limited. PMID- 7555906 TI - Role of the hypothalamic hamartoma in the genesis of gelastic fits (a video stereo-EEG study). AB - Patients having a hypothalamic hamartoma frequently present epileptic attacks of laughter, and they later experience multiple additional seizure types, which invariably lead to a severe drug-resistant epilepsy. If this association is now well-known, relationships between the hypothalamic mass and the different types of seizures remain still mysterious. We report the case of a 16-year-old girl suffering from this peculiar epileptic picture, in whom a stereo-EEG study was performed, allowing us to record both the hamartoma, the neighboring hypothalamic structures, and other bilateral cortical areas. It showed that gelastic fits were strictly linked to ictal discharges which began and remained well localized in the hamartoma. Conversely, atonic seizures, which might result from a secondary epileptogenesis, admitted a widely extended bilateral frontal cortical origin, sparing the lesion, and slightly involving the posterior hypothalamus. Stereotactic radiosurgery of the hamartoma proved to be ineffective on both types of seizures, probably because of the too low dose of X-rays delivered (18 grays), as suggested by the absence of hypothalamic mass changes on MRI. Such data, never reported to our knowledge, seem able to contribute to a better understanding of this very peculiar epileptic syndrome, and perhaps to a better adapted therapeutic management. PMID- 7555908 TI - Interelectrode coherences from nearest-neighbor and spherical harmonic expansion computation of laplacian of scalp potential. AB - Interchannel coherence is a measure of spatial extent of and timing relationships among cerebral electroencephalogram (EEG) generators. Interchannel coherence of referentially recorded potentials includes components due to volume conduction and reference site activity. The laplacian of the potential is reference independent and decreases the contribution of volume conduction. Interchannel coherences of the laplacian should, therefore, be less than those of referentially recorded potentials. However, methods used to compute the laplacian involve forming linear combinations of multiple recorded potentials, which may inflate interchannel coherences. WE compared 3 methods of computing the laplacian: (1) modified Hjorth (4 equidistant neighbors to each electrode), (2) Taylor's series (4 nonequidistant neighbors), and (3) spherical harmonic expansion (SHE). Average interchannel coherence introduced by computing the laplacian was less for nearest-neighbor methods (0.0207 +/- 0.0766) but still acceptable for the SHE method (0.0337 +/- 0.0865). Average interchannel coherence for simulated EEG (random data plus a common 10 Hz signal) was less for laplacian than for referential data because of removal of the common referential signal. Interchannel coherences of background EEG and partial seizure activity were less with the laplacian (any method) than with referential recordings. Laplacians calculated from the SHE do not demonstrate excessively large interchannel coherences, as have been reported for laplacians from spherical splines. PMID- 7555909 TI - Signal-space projections of MEG data characterize both distributed and well localized neuronal sources. AB - We describe the use of signal-space projection (SSP) for the detection and characterization of simultaneous and/or sequential activation of neuronal source distributions. In this analysis, a common signal space is used to represent both the signals measured by an array of detectors and the underlying brain sources. This presents distinct advantages for the analysis of EEG and MEG data. Both highly localized and distributed sources are characterized by the components of the field patterns which are measured by the detectors. As a result, a unified description of arbitrary source configurations is obtained which permits the consistent implementation of a variety of analysis techniques. The method is illustrated by the application of SSP to auditory, visual and somatosensory evoked-response MEG data. Single-trace evoked responses obtained by SSP of spontaneous activity demonstrate that a considerable discrimination against both system noise and uncorrelated brain activity may be achieved. Application of signal-space projections determined in the frequency domain to spontaneous activity illustrates the possibility of including temporal relationships into the analysis. Finally, we demonstrate that SSP is particularly useful for the description of multiple sources of distributed activity and for the comparison of the strengths of specific neuronal sources under a variety of different paradigms or subject conditions. PMID- 7555907 TI - EEG coherence has structure in the millimeter domain: subdural and hippocampal recordings from epileptic patients. AB - Subdural recordings from 8 patients and depth recordings from 3 patients via rows of electrodes with 5-10 mm spacing were searched for signs of significant local differentiation of coherence calculated between all possible pairs of loci. EEG samples of 2-4 min were taken during 4 states: alertness, stage 2-3 sleep, light surgical anesthesia permitting the patient to respond to questions and electrical seizures. Coherence was computed for all frequencies from 1 to 50 Hz or 0.3-100 Hz; for comparisons the mean coherence over each of 6 or 7 narrower bands between 2 and 70 Hz was used. Whereas the literature supports the view that EEG coherence is usually substantial over many centimeters, the hypothesis here tested--and found to be well above stochastic expectations--is that significant structure occurs in the millimeter domain for EEG recorded subdurally or within the brain. In both the subdural surface samples and those from temporal lobe depth electrode arrays coherence declines with distance between electrodes of the pair, on the average quite severely in millimeters. This is nearly the same for all frequency bands. For middle bands like 8-13 and 13-20 Hz, mean coherence typically declines most steeply in the first 10 mm, from values indistinguishable from 1.0 at < 0.5 mm distance to 0.5 at 5-10 mm and to 0.25 in another 10-20 mm in the subdural surface data. Temporal lobe depth estimates decline about half as fast; coherence > or = 0.5 extends for 9-20 mm and > or = 0.25 for another 20-35mm. Low frequency bands (1-5, 5-8 Hz) usually fall slightly more slowly than high frequency bands (20-35, 35-50 Hz but the difference is small and variance large. The steepness of decline with distance in humans is significantly but only slightly smaller than that we reported earlier for the rabbit and rat, averaging less than one half. Local coherence, for individual pairs of loci, shows differentiation in the millimeter range, i.e., nearest neighbor pairs may be locally well above or below average and this is sustained over minutes. Local highs and lows tend to be similar for widely different frequency bands. Coherence varies quite independently of power, although they are sometimes correlated. Regional differentiation is statistically significant in average coherence among pairs of loci on temporal vs frontal cortex or lateral frontal vs. subfrontal strips in the same patient, but such differences are usually small.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7555910 TI - Frontal inhibitory spike component associated with epileptic negative myoclonus. AB - The aim of this study was to characterize paroxysmal EEG activities associated with epileptic negative myoclonus (ENM) in an epileptic patient presenting with ENM. ENM was predominant in the right upper limb and was correlated to a spike in the left central region. Spikes associated with ENM (SaENM) and spikes unrelated to ENM (SuENM) were identified by the temporal relation between the left central spike and the EMG silent period in the right wrist extensor. SaENM showed a significantly longer duration than SuENM (128 +/- 27 msec versus 92 +/- 21 msec, respectively; P < 0.01). SaENM and SuENM were submitted to spike averaging and topographic mapping. Spike averaging was performed averaging the EEG 640 msec before and after the peak of the spike. Both averaged SaENM and SuENM consisted of a negative spike with highest amplitude at C3 and similar topographic characteristics. The discriminant feature between the two types of spikes was the presence, in averaged SaENM, of a second smaller negative spike, 40 msec after the peak of the spike at C3, whose maxima were distributed over the left frontal region. We labeled this second spike as ENM-related component. We conclude that, in our patient, ENM was associated with a frontal cortical potential suggesting the involvement of frontal areas in the generation of negative myoclonus. PMID- 7555911 TI - Origin and distribution of P13 and P14 far-field potentials after median nerve stimulation. Scalp, nasopharyngeal and neck recording in healthy subjects and in patients with cervical and cervico-medullary lesions. AB - We studied median nerve SEPs in 10 healthy subjects, by means of simultaneous recording over the scalp, around the neck and near the ventral surface of the medulla using a nasopharyngeal (NP) electrode. This recording technique enabled us to clearly differentiate P13 and P14 potentials. The former was always found in NP records, while the latter was more evident in scalp traces. The same technique was used to study 9 patients with various lesions of the cervical cord or cervico-medullary junction. Patients with high cervical lesions demonstrated abnormalities of both P13 and P14 potentials, while patients with lesions of the cervico-medullary junction demonstrated a clear dissociation between normal P13 in scalp and NP traces, and abnormal scalp P14. Patients with lower cervical lesions, selectively involving the central grey matter, showed normal P13 and P14 potentials, in spite of abnormal N13 cervical responses. Our findings strongly suggest that both scalp and NP P13 have the same generators in higher segments of the cervical cord, and that NP more than scalp records are effective in analyzing the P13 response. We suggest that the selective recording of the P13 potential could be useful in the assessment of focal lesions of the higher cervical cord or of the cervico-medullary junction. PMID- 7555912 TI - Effect of sleep stage on somatosensory evoked potentials by median nerve stimulation. AB - The effects of sleep stage on early cortical somatosensory evoked potentials (SEPs) and short-latency components elicited by median nerve stimulation were studied in 12 normal volunteers. The latency of P13 in the awake stage was not significantly different from that in any sleep stage. The latencies of N16, N20 and P20 were significantly prolonged while the amplitude of N20 was decreased during the non-rapid eye movement (NREM) sleep stage. P22, P23 and N24 components showed double peaks (P23a, P23b, N24a, N24b) during the NREM sleep stage in 6 subjects, while N24 showed a single peak and only P22 and P23 showed double peaks in 5 other subjects. The latencies and morphologies of SEPs during rapid eye movement sleep stage were almost the same as those during the awake stage. These findings suggest that NREM sleep affects the latency, amplitude and morphology of N16 and early cortical components. PMID- 7555913 TI - Sequential analysis of the brain's transfer properties during consecutive REM episodes. AB - Classical analysis of the spontaneous sleep EEG has revealed alterations of REM sleep in psychiatric diseases and under the influence of drugs. In order to elucidate possible functional differences between different REM episodes even in healthy subjects we investigated in 10 volunteers the transfer properties of the brain by measuring auditory (AEP) and visual evoked potentials (VEP) from scalp positions Fz, Cz and Pz during the night. According to linear system theory we computed the so-called amplitude-frequency characteristics (AFC) from averaged AEPs and VEPs during the first and each of the following 3 REM episodes. These functions describe the relationship between the input and output of the investigated system. A 3-factorial analysis of variances with the independent factors frequency band, REM episode and electrode position revealed a statistically significant main effect for the factor REM episode under auditory stimulation (P = 0.05), whereas no significant main effect for REM episode was found under visual stimulation (P = 0.88). Applying a 2-factorial analysis of variance with the independent factors REM episode and electrode position in the case of auditory stimulation we could demonstrate a statistically significant main effect (P = 0.029) for the factor REM episode in the beta range (12.5-20 Hz). A subsequent analysis of contrasts revealed that the first REM episodes could be differentiated from each other. For auditory stimulation the beta resonance during the first REM episode appears enhanced compared to each of the later REM episodes. These findings point to a functional difference of the brain's transfer functions between the first and the 3 following REM episodes, indicating different information processing during consecutive paradoxical sleep. PMID- 7555914 TI - Auditory event-related potentials to deviant stimuli during drowsiness and stage 2 sleep. AB - Twelve subjects were tested using a 3-tone auditory oddball paradigm consisting of a standard 1000 Hz tone (P = 80%) and two deviants, namely, a 1200 Hz tone and a 2000 Hz tone (both P = 10%). Testing took place in 3 conditions: (1) attend, in which the subject had to count one of the deviant tones; (2) ignore, in which the subject read a book; and (3) sleep, in which the subject was encouraged to go to sleep during presentation of the tones. In the awake conditions stimulus deviance elicited mismatch negativity (MMN) and P3. During drowsiness, no separate mismatch negativity (MMN) could be detected, but the 2000 Hz tone evoked a broad fronto-central early negative deflection, suggesting an overlap of N1 and MMN. In the same condition, P210, N330 and P430 appeared, all being sensitive to magnitude of deviance. During stage 2, the P210, N330 and P430 amplitudes increased, most notably to the large deviant. These data indicate that differential processing of auditory inputs is maintained during drowsiness and stage 2 sleep, but do not support the notion that MMN or P3 activity comparable to the waking state occurs to oddball stimuli during this stage. It is hypothesised that during light sleep, scanning of the environment is performed by a different system than in the awake state and that during drowsiness a gradual switch between these two systems takes place. PMID- 7555915 TI - Brain-stem auditory evoked potential monitoring. Variations of stimulus artifact in brain death. AB - Brain-stem auditory evoked potentials (BAEPs) were recorded in 20 subjects with brain death (mean age, 33.2 +/- 15.1 years) and 20 healthy volunteers (mean age, 29.8 +/- 6.8 years). Brain death was due to head injury (n = 14), encephalitis (n = 3), brain-stem hemorrhage (n = 1), cerebellar hemorrhage (n = 1) or cerebral infarction (n = 1). The presence, latency and amplitude of the individual BAEP components and variations of the stimulus artifact were evaluated. The mean (+/- S.D.) amplitude of the stimulus artifact was 0.26 +/- 0.12 microV in the brain dead subjects and 0.09 +/- 0.05 microV in the control group (P < 0.001, t test). The causes of the phenomenon of increasing stimulus artifacts in the evolution of brain death remain unclear. PMID- 7555916 TI - Auditory steady-state responses to multiple simultaneous stimuli. AB - Steady-state responses can follow multiple simultaneous auditory stimuli. If the stimuli are modulated at different rates, responses specific to each stimulus can be assessed by measuring in the frequency domain response the spectral component corresponding to the rate of modulation. When each stimulus has a different carrier frequency or different ear of presentation, the responses when 8 stimuli are presented simultaneously are not significantly different than when each stimulus is presented alone. Since significant responses can be recognized down to intensities that average 14 dB above behavioral threshold, this technique may be useful in objective audiometry. It is also possible to record steady-state responses to multiple modulations of the same carrier frequency. In this case, the amplitude of the responses when the stimuli are combined is smaller than when the stimuli are presented alone. The decrease in amplitude depends upon the number of concomitant stimuli and their relative intensities. These effects are probably due to the compressive rectification occurring during cochlear transduction, and the data may be used to model cochlear processing of auditory stimuli. PMID- 7555917 TI - Information processing during cardiac surgery: an event related potential study. AB - The aim of this study was to investigate whether information processing persists during general anesthesia, and if so, to determine the relationship between the degree of cognitive processing measured during anesthesia and the presence or absence of intraoperative memories measured after anesthesia. Subjects were 12 patients, undergoing cardiac surgery with propofol/alfentanil anesthesia. During several periods of the operation, event related potentials (ERPs) to frequent and infrequent tones of different pitch were analyzed. After the operation, a word recognition task with ERP recording was administered to determine whether intraoperatively presented words would elicit a (covert) recognition reaction in the brain. ERP wave forms could be obtained during the intraoperative recording periods but differed substantially from those in the awake state. The presence of ERP components up to 500 msec after stimulus presentation suggests that auditory information processing continued during anesthesia up to a certain level of cognition. Intraoperative ERPs to frequent and infrequent tones were not different from each other implying that differences in pitch could not be detected. The postoperative results demonstrated evidence for intraoperative memories in 3 patients. For 2 of these 3 patients, low propofol levels as well as reliable ERPs with large amplitudes were found close to the moment of information presentation. The results emphasize the importance of combining intra- and postoperative measurements and suggest that late ERP components might be used as indicators of an increased risk of auditory perception. PMID- 7555918 TI - Aging, caffeine, and information processing: an event-related potential analysis. AB - Structural and energetic processes in information processing were studied in young and elderly subjects. A visually focussed selective search task was used, in which subjects had to select relevant information, followed by controlled memory search processes to locate a target item. Caffeine was used to manipulate the energetic state of the subjects. During task performance event-related potentials (ERPs) and reaction time (RT) were recorded. Subjects were 15 young and 15 elderly healthy, non-smoking, moderate caffeine consumers (250-600 mg/day). Before the experimental sessions they abstained from caffeine for > or = 12 h. In the experiment subjects received 250 mg caffeine or placebo dissolved in decaffeinated coffee. RT data seem to indicate that aging effects are at least partly due to a shift in the speed-accuracy trade-off. ERP results provide evidence for decreased levels of energy resources in the elderly. The identification of relevant information and stimulus evaluation processes were delayed in the elderly, suggesting an additional effect of aging on structural processes. Caffeine improved performance and increased the amplitude of the N1, N2b, and P3b, in both young and old subjects. These results suggest that caffeine makes more energy resources available for task performance. The effects of aging on P3b latency were counteracted by caffeine. Other caffeine effects did not differ significantly between young and elderly subjects. PMID- 7555919 TI - Data dependent weighted averages for recording of evoked potential signals. AB - Two new filters are proposed to replace conventional on-line artifact rejection routines. They are based on an algorithm that computes a weight for each trial according to its similarity to the rest. The robustness of the two filters and their capacity to reduce recording time significantly were verified experimentally. PMID- 7555920 TI - P300 in response to the subject's own name. AB - The P300 component is influenced by task relevance which is typically achieved by employing an active task involving the evoking stimulus. This study explored the possibility of making use of the subject's name, which is innately relevant to the subject, to achieve stimulus relevance. Dependence of P300 amplitude on auditory presentation of the subject's name was assessed in two experiments in which no response was required: (1) a passive 2-word "oddball" paradigm where the low probability word was the subject's first name; (2) a passive 3-word "oddball" paradigm consisting of two low probability words, one of which was the subject's name, and a third, high probability word. In the first experiment, a typical active 2-word "oddball" paradigm, which did not include the subject's name, was compared with the passive paradigm. P300 amplitude was larger in response to the subject's name compared to the other word in the two-word paradigm, indicating that the name can be used to evoke P300 in a passive paradigm. It was also larger than either of the other two words in the 3-word paradigm, suggesting that stimulus relevance has an additional effect on P300 amplitude beyond rarity. PMID- 7555921 TI - Recovery functions of early cortical median nerve SSEP components: normative data. AB - The recovery functions of parietal P14-N20, N20-P27 and frontal P22-N30 amplitudes were assessed in 17 healthy controls aged 20-50 years by means of the paired stimulus technique. One unpaired and 4 paired stimuli with interstimulus intervals (ISIs) of 25, 50, 75 and 100 msec were cyclically presented in a single run. Responses to the unpaired stimulus were subtracted off-line from paired stimulus responses. The highest suppression was reached at shorter ISIs for components with shorter latencies. The mean suppression of P22-N30 was influenced by the subject's age, being greater in younger subjects. Normative data are reported. PMID- 7555922 TI - Recovery after surgery of the spinal N24 SEP in dural arteriovenous malformation of the dorsal cord. AB - We studied somatosensory evoked potentials (SEPs) to tibial nerve stimulation in two patients suffering from dorsal dural arteriovenous malformation (AVM). We found in both patients abnormalities in the lumbar N24 potential and in the cortical P40 response. After surgical removal of the AVM, the N24 recovered in both patients. Cord lesions probably occur in patients with dural AVM because of a theft of blood through the fistula; N24 recovery may therefore be associated with a restoration of blood supply after surgery. The N24 recovery in our patients with dural AVM suggests that the abnormality of this potential does not necessarily reflect irreversible damage to the lumbo-sacral cord and that the N24 recording can be useful in post-surgical monitoring. PMID- 7555923 TI - Early detection of neurological involvement in systemic lupus erythematosus patients. AB - In lupus the neurological abnormalities usually mimic many primary neurological diseases and often represent the only manifestation of SLE for several years before the onset of other features of the disease. The aim of this study was to investigate lupus patients to obtain electrophysiological data of possible neurological abnormalities even in the absence of neuropathy symptoms. Thirty eight SLE patients, twenty healthy volunteers and 10 steroid-using control subjects were included. Eighteen were asymptomatic but 20 of them had neuropathy symptoms. Median, ulnar and peroneal nerve motor, median, ulnar and sural nerve sensory conduction velocities, median and tibial somatosensory evoked potentials (SEP) were recorded. 23.6% of all lupus patients had peripheral nerve conduction slowing and 39.5% had SEP abnormalities; almost half of them being asymptomatic. 44.7% of the total had carpal tunnel syndrome; again almost half of them (47%) were asymptomatic. There was no significant correlation between the electrophysiologic parameters and the clinical and laboratory parameters of the disease. In lupus patients, SEP abnormalities were more pronounced than peripheral findings, reflecting the CNS involvement more common than the peripheral neuropathy. Therefore, we suggest performing the electrophysiological studies in lupus patients, even in the asymptomatic ones, early in the course in order to detect the nervous system involvement. PMID- 7555926 TI - Effects of stimulus intensity on frontal, central and parietal somatosensory evoked potentials after median nerve stimulation. AB - Effects of the intensity of electrical median nerve stimulation were previously reported for the subcortical and first cortical somatosensory evoked potentials (SEPs) but not for later cortical waves whose applications in neurology have gained growing interest in recent years. This paper therefore describes the stimulus intensity effects on frontal, central and parietal SEP waveforms up to 90 msec after stimulus. The intensities were 1.5 and 2 times sensory threshold (ST), motor threshold (MT), and 1.5 and 2 times MT. Between 1.5 x ST and MT all SEP components grew in amplitude, except N60 which was essentially saturated already at 1.5 x ST. The growth was most marked for P14 and N20 whereas later potentials changed less, i.e. the slopes of the intensity-amplitude curves progressively flattened with increasing latency of SEP component. Between MT and 2 x MT no significant further alterations occurred in the early cortical potentials up to 30 msec. However, subtle changes occurred in the P40-N60 waveforms and subtraction of responses revealed a small centroparietal P35-N45 difference wave elicited only by high-intensity (2 x MT) stimulation. It is concluded that for practical purposes stimulation slightly above MT yields maximum cortical SEPs. The results are not generally compatible with the proposition that P40 and N60 are conveyed by higher threshold, small-diameter afferent fibers compared with N20. However, the P35-N45 difference wave at 2 x MT indicates that small-diameter afferent components may be embedded in the waveforms obtained at high intensity. PMID- 7555924 TI - Radial sensory nerve entrapment in carpal tunnel syndrome. AB - In 100 subjects with Carpal Tunnel Syndrome (CTS) and any other illness, the conduction velocity of the radial sensory nerve (RSN) was studied on the hypothesis that, because of the relation with the tendon of the brachioradialis and extensor carpi radialis longus muscles, microtrauma can injure the RSN. In 13 patients, for a total of 15 limbs involved, the SCV is reduced less than 2.5 SD, in 5 the distal latency is prolonged and in 1 there is both a slowing of SCV and prolonged distal latency. The involvement of RSN is more frequent in the dominant hand. There is no relation between the severity of CTS and damage of RSN or between clinical and electrophysiological data. It therefore seems that the entrapment of RSN is quite frequent and can explain some symptoms and modify the therapy. PMID- 7555925 TI - Hemispheric asymmetries of early cortical somatosensory evoked potentials revealed by topographic analysis. AB - Scalp topography of the early cortical SEPs was analysed in 50 normal right handed subjects. The following hemispheric asymmetries of the potentials were found: 1) The maximal amplitude of N20 was located predominantly at the P electrode after right sided stimulation, but varied between the CP, P and O electrode after left sided stimulation. 2) The N20 amplitudes were higher after right sided stimulation in approximately 70% of the subjects and the P25 and N30 amplitudes were higher after left sided stimulation in again approximately 70% of the subjects. No side difference of amplitudes was found for the subcortical N14. The N20 amplitude and location asymmetries were attributed to an anatomical asymmetry of the somatosensory cortex. PMID- 7555927 TI - Stimulus induced repetitive muscle potentials in the gracile axonal dystrophy (GAD) mouse. AB - Gracile axonal dystrophy (GAD) is an inherited neurodegenerative disease in the mouse with progressive sensory ataxia and motor paresis. Electromyographic examination was conducted in 25 unanaesthetized GAD mice and 24 controls of the same strain during 6, 9 and 12 wk of postnatal life. Among 9 and 12 wk old mice, about 75% showed resting spontaneous activities--either fibrillation or fasciculation or both. On stimulation of the tibial nerve at ankle, with single pulse, the EMG showed repetitive muscle potentials of large amplitude, following "M" response. The frequency and duration of the train of these stimulus--induced repetitive muscle potentials (SIRMP) were almost constant in a given animal. The SIRMP failed to reappear in response to the second stimulus within 5 s when twin pulses were applied or within 5 min when tetanic stimuli were applied, indicating their fatigability. It is concluded that the SIRMP originate from immature, supplementary motor endplates that develop at ultraterminal nerve sproutings induced by denervation and reinnervation and possibly are due to hyperexcitable trigger points in the peripheral nerve endings. The EMG abnormalities in the GAD mouse are very much similar to those observed in human syndromes with hyperexcitable peripheral nerves that result in sustained muscle activity like neurotonia and hence the GAD mouse is a good model of such motor abnormalities in man. PMID- 7555928 TI - The electrophysiological study of differential diagnosis between amyotrophic lateral sclerosis and cervical spondylotic myelopathy. AB - Sternocleidomastoid muscle EMG (SCM-EMG) and dermatomal somatosensory evoked potential (DSSEP) were evaluated as diagnostic aids in differentiation between amyotrophic lateral sclerosis (ALS) and cervical spondylotic myelopathy (CSM) in this study. 104 inpatients were divided into three groups: those diagnosed clinically as either (A) ALS (36 cases), or (B) CSM (32 cases) and (C) those needed further investigation (36 cases). EMG of limb muscles and sternocleidomastoid of both sides in all, and of tongue muscles in 58/104 of three groups; DSSEP in 56/104 of all three groups; MRI/CTM in 12/36 of group A and all patients of group B and C. SCM-EMG abnormal in 34/36 of group A but normal in all 32 of group B. SCM-EMG also abnormal in 32/36 of group C and 8 of them showed postoperative aggravation. DSSEP normal in 12/12 of group A while abnormal in 19/20 of group B. DSSEP abnormal only in 6/24 of group C. MRI/CTM showed spinal compression in all cases presenting abnormal DSSEP and close correspondence between upper margins of pathological involvement as revealed using these two methods. Both SCM-EMG and DSSEP have proved to be valuable differential aids between ALS and CSM. PMID- 7555929 TI - Electromyographic validation of basic exercises for physical conditioning programmes. I. Analysis of the deltoid muscle (anterior portion) and pectoralis major muscle (clavicular portion) in rowing exercises with middle grip. AB - The electromyographic activity of the shoulder muscles Deltoid--anterior portion (DA) and Pectoralis major--clavicular portion (PMC) was tested on 24 male volunteers using a 2 channel TECA TE4 electromyograph and Hewlett Packard surface electrodes during the execution of four different modalities of rowing exercises with middle grip. The results showed that rowing exercises are not appropriate for physical fitness programmes for PMC but some of them are significantly useful for DA. The authors present some suggestions to the use of the tested exercises. PMID- 7555930 TI - Neurophysiological examination of end-plate function in patients with lung cancer. AB - Twenty-eight patients affected by lung cancer were studied with neurophysiological and clinical examinations to show the prevalence of alteration of neuromuscular transmission. During repetitive stimulation at 20 Hz we found in 6 cases (22%) an increment (> 20%) and in 4 cases a decrement ( > 10%) of the size of muscle action potentials. In 15 patients (57%) we found low sensory action potential amplitude (< 11 uv). In 3 (11%) of the 6 cases with incremental response the overall examination suggested a disorder of neuromuscular junction such as Eaton-Lambert syndrome. Our results suggest that the peripheral nervous system involvement in patients affected by lung cancer is higher than usually estimated. PMID- 7555931 TI - Effects of visual and auditory stimuli on median nerve somatosensory evoked potentials in man. AB - To demonstrate electrophysiological evidence of neural connections of somatosensory with visual and auditory pathways, interactions of median nerve somatosensory evoked potentials (SEPs) with visual evoked potentials (VEPs) and auditory evoked potentials (AEPs) were analyzed in 12 healthy subjects. In Experiment I (SEP vs. VEP), three patterns of stimulation, namely, electric stimulation of left median nerve, binocular LED flash, and simultaneous electric and flash stimulation, were applied with random interstimulus intervals of 2-4 sec until more than 100 responses had been obtained and averaged for each pattern of stimulation. Simultaneous electric and visual responses were subtracted from arithmetical sums of SEPs and VEPs to show interactions between somatosensory and visual responses. In Experiment II (SEP vs. AEP), binaural short-duration tone bursis instead of LED flashes were used in the same manner as in Experiment I, and interactions between somatosensory and auditory responses were analyzed. Results indicated that the effects of both visual and auditory interaction on SEPs were significant around 120-130 msec of latency. In other words, cross-modal sensory interaction occurred in the late phase of sensory processing. PMID- 7555932 TI - Colorectal mini-de novo carcinoma: a reality in Germany too. AB - BACKGROUND AND STUDY AIMS: Based on Japanese case studies, we examined whether colorectal mini-de novo carcinoma also occurs outside Japan. We defined mini-de novo carcinomas as carcinomas infiltrating the submucosa, with a maximum diameter of 10 mm, and with no evidence of precursive adenomatous tissue. PATIENTS AND METHODS: Between 1988 and 1994, we diagnosed carcinomas of this type in polypectomy and surgical resection specimens from 155 patients. These mini-de novo carcinomas did not differ from carcinomas arising from adenomas in terms of patient age (median 67.1 years), sex distribution (men: women 0.96:1), or location--they occurred primarily in the sigmoid (53%) and rectum (27.3%). RESULTS: Most of the mini-de novo carcinomas were macroscopically of the polypoid type (59.4%); flat, elevated carcinomas were also relatively frequent, including those with a central concave depression (21.9%) and those without a depression (12.3%). Histologically, all of the lesions without exception were adenocarcinomas (grade 1: 28.4%, grade 2: 65.8%, grade 3: 5.8%). There was carcinomatous invasion of submucosal lymphatic or blood vessels in 20%. CONCLUSIONS: Our analysis shows that colorectal mini-de novo carcinoma is not a purely Japanese phenomenon, and that these carcinomas are being diagnosed with increasing frequency as the awareness of their existence and macroscopic growth characteristics increases. PMID- 7555933 TI - Comparison of injection sclerotherapy and laser photocoagulation for bleeding peptic ulcers. AB - BACKGROUND AND STUDY AIMS: The most widely used endoscopic procedures in the management of patients with bleeding peptic ulcer are at present sclerotherapy and thermal methods. In an attempt to assess the most effective method of achieving hemostasis, we compared injection sclerotherapy and laser photocoagulation in terms of the efficacy of initial hemostasis, rebleeding, need for surgery, mortality, and the appearance of the ulcer after the hemostatic procedure. PATIENTS AND METHODS: In this prospective, randomized trial, 160 patients were treated with injection sclerotherapy (1% polidocanol), and 155 patients with laser photocoagulation (Nd:YAG laser) in cases of Forrest I, Forrest IIa, and Forrest IIb hemorrhage. The bleeding activity was classified according to the modified Forrest criteria. Polidocanol injection and Nd:YAG laser photocoagulation were not preceded by epinephrine administration. RESULTS: There were no significant overall differences between the groups in the outcome in terms of definitive hemostasis, rebleeding, urgent surgery, and death (p = 0.487). In the case of the subgroup with Forrest I lesions, laser photocoagulation was more efficacious than sclerotherapy (p = 0.0078). In the Forrest IIa and Forrest IIb subgroups, the two methods were equally effective (p = 0.202 and 0.513 respectively). In the sclerotherapy patients, definitive initial hemostasis in Forrest IIa was achieved in 100%, whereas in the laser group this rate was 92%, with 28% of patients initially developing hemorrhage after one or two laser pulses. Ulcer healing was slower following sclerotherapy than after photocoagulation. CONCLUSION: Injection sclerotherapy and laser photocoagulation are equally effective in achieving definitive hemostasis in bleeding peptic ulcers. Laser photocoagulation is more efficacious in patients with active bleeding, whereas injection sclerotherapy is more effective in patients with a nonbleeding visible vessel. PMID- 7555934 TI - Success of endoscopic injection therapy in correlation with maximal one-day transfusion requirement. AB - BACKGROUND AND STUDY AIMS: The rate of blood transfusion is related to blood flow and the diameter of the bleeding vessel. Therapeutic endoscopy is less effective in larger vessels. To determine the effect of therapeutic endoscopy with pure ethanol injection in massive peptic ulcer bleeding, we conducted a retrospective study using the maximal one-day blood requirement as an indicator of the required blood transfusion. PATIENTS AND METHODS: The maximal one-day blood requirement was defined as the total amount of blood transfusion needed within a day to keep hemodynamics stable and hemoglobin above 8.0 g% before therapeutic endoscopy. From January 1986 to May 1993, 283 patients with high-risk signs of the stigmata of hemorrhage on endoscopy, who received pure ethanol injection therapy, were included in this study. There were 214 men and 69 women with a mean age of 58.4 years (ranging from 16 to 93 years). One hundred forty-three had gastric ulcers; 125 had duodenal ulcers; and 15 had stomal ulcers. Patients whose maximal one-day blood requirement was less than 1000 ml were assigned to Group I. Patients without, and patients with, major organ diseases whose maximal one-day blood requirement was more than 1000 ml were assigned to Group IIa and Group IIb, respectively. RESULTS: In Group I, 87.1% attained permanent hemostasis; 51.3% in Group IIa; and 49.4% in Group IIb. Temporary hemostasis and failure rates were 8.9% and 4.8% in Group I; 14.5% and 33.8% in Group IIa; and 21.2% and 29.4% in Group IIb. The rate of permanent hemostasis was significantly lower in patients with massive bleeding (p < 0.001) but did not differ between patients with and without major organ diseases (p > 0.05). CONCLUSION: The success rate for pure ethanol injection therapy was lower in patients with a large maximal one-day blood transfusion requirement. PMID- 7555935 TI - Transcatheter arterial embolization for massive bleeding from duodenal ulcers not controlled by endoscopic hemostasis. AB - BACKGROUND AND STUDY AIMS: We evaluated the efficacy of transcatheter arterial embolization (TAE) in patients in whom endoscopic hemostasis of a massively bleeding duodenal ulcer failed. PATIENTS AND METHODS: TAE was performed in 11 patients with endoscopically uncontrollable massively bleeding duodenal ulcers, and the results and long-time outcome were studied. Two additional cases of failed endoscopic hemostasis were treated surgically without TAE. The entire group of 13 patients represented 5% of endoscopically treated duodenal ulcers and 0.6% of all cases with upper gastrointestinal bleeding who underwent emergency endoscopy (n = 2073). All but one of these 13 patients had concomitant disease. RESULTS: Arteriograms performed before TAE revealed extravasation of contrast material around the gastroduodenal artery (GDA), the anterior superior pancreaticoduodenal artery (ASPD), or the posterior superior pancreaticoduodenal artery (PSPD) in six of 11 cases. We failed to stop the bleeding in one patient, in whom only the common hepatic artery side of the GDA bleeding site was embolized; this patient died. TAE was successful in the other ten patients, in whom the long stretch of the GDA, including the ASPD and PSPD, was embolized around the bleeding site. Two surgically treated patients died within a week. CONCLUSIONS: Our findings indicate that TAE may induce hemostasis in 90% of patients with serious concomitant diseases who have endoscopically uncontrollable massive bleeding from duodenal ulcers. PMID- 7555936 TI - Omeprazole versus ranitidine as adjunct therapy to endoscopic injection in actively bleeding ulcers: a prospective and randomized study. AB - BACKGROUND AND STUDY AIMS: Although high rates of initial hemostasis can be achieved with endoscopic injection therapy in actively bleeding ulcers, the incidence of rebleeding is not negligible. Optimal conditions for clotting may require achieving deep and sustained acid inhibition to avoid the deleterious effect of acid and pepsin secretions on the hemostatic process. The aim of this study was to assess whether omeprazole could improve the efficacy of ranitidine as an adjunct treatment in endoscopic injection therapy to avoid rebleeding. PATIENTS AND METHODS: Eighty-six patients with active arterial bleeding from a peptic ulcer disclosed at emergency endoscopy were included in this prospective trial. All patients received injections of 1:10,000 adrenaline. Subsequently, they were randomized to receive either intravenous omeprazole (n = 45), with an initial dose of 80 mg followed by 40 mg every eight hours for four days and thereafter with oral administration; or ranitidine (n = 41), 50 mg every six hours for 12 to 24 hours and thereafter with oral administration. RESULTS: The two groups were well matched in terms of clinical and endoscopic data. There were no statistically significant differences between the groups with regard to: further bleeding (29% in both groups), need for emergency surgery (20% in the omeprazole group vs. 22% in the ranitidine group), transfusion requirements (2.4 +/- 2.2 vs. 2.2 +/- 2.1 units), length of hospital stay (14.1 +/- 13.9 vs. 15.3 +/- 15.4 days), or mortality (7% vs. 2%). CONCLUSIONS: Our results suggest that omeprazole does not improve the efficacy of ranitidine after endoscopic injection therapy in patients with an active arterial bleeding ulcer. PMID- 7555937 TI - Percutaneous endoscopic gastrostomy (PEG): comparison of push and pull methods and evaluation of antibiotic prophylaxis. AB - BACKGROUND AND STUDY AIMS: Infection of the gastrostomy opening after placement of a percutaneous endoscopic gastrostomy (PEG) catheter has been reported to occur quite often, especially when the pull method is used. We therefore compared complications occurring with the pull and push methods, and evaluated the role of antibiotic prophylaxis. PATIENTS AND METHODS: In a prospective study, 100 consecutive patients were randomly assigned to group A (pull plus antibiotic prophylaxis: amoxycillin-clavulanic acid 3 x 1.2 g i.v. over 24 hours; 37 patients), group B (pull without antibiotic prophylaxis; 34 patients) and group C (push without antibiotic prophylaxis; 29 patients). The indications for PEG placement were dysphagia due to oropharyngeal tumors (56%), neurological disease (32%), or other (12%). Patients were evaluated twice weekly for one month after the PEG placement. RESULTS: PEG catheters were successfully placed in 96% of the patients. The total procedure-related complication rate was significantly lower in group A than in groups B and C (28%, 58%, and 70%, respectively; p < 0.01). Major complications occurred in one patient in group A (seeding metastasis of a hypopharyngeal carcinoma in the gastrostomy tract), and in four patients in group B (three cases of peritonitis and one aspiration, resulting in two deaths), but in none of the group C patients. Group A patients experienced fewer peristomal infections than the other two groups (14%, 30%, and 41%, respectively: p = 0.05). The risk of peristomal pain was similar (11%, 15%, and 11%, respectively; p = n.s.). In three patients in group C, the PEG catheter had to be replaced by the pull method, due to repeated dislocation of the balloon catheter. CONCLUSIONS: The complication rate with PEG placement is high with both the push and pull methods. The complication rate with the pull method is significantly reduced when antibiotic prophylaxis is used. PMID- 7555938 TI - Percutaneous endoscopic gastrostomy as a decompressive technique in bowel obstruction due to abdominal carcinomatosis. AB - BACKGROUND AND STUDY AIMS: Percutaneous endoscopic gastrostomy (PEG) is a simple method of achieving nonsurgical gastric decompression in patients suffering from metastatic abdominal tumors and upper gastrointestinal tract obstruction. The aim of this prospective study was both to evaluate the efficacy of PEG for intestinal decompression in patients with disseminated abdominal cancer and to compare two catheters with different diameters. PATIENTS AND METHODS: Over a one-year period, 22 consecutive female patients (mean age 53.7, range 29-73) were referred to us and a PEG was successfully placed in 21. In four patients with unsatisfactory endoscopic trans-illumination of the anterior abdominal wall, an ultrasound unit was used to identify an adequate site for PEG placement. RESULTS: All patients experienced substantial symptomatic relief after a few days: vomiting and nausea completely resolved, and abdominal pain persisted in one patient only. No gastrostomy-related additional morbidity was noticed. We randomly inserted a 15 French or a 20-French tube: no statistically significant difference was noticed between the two in the symptomatic relief provided. CONCLUSIONS: Our data support the hypothesis that PEG is an effective, safe, and well-tolerated method of achieving gastric decompression in cancer patients; ultrasound guidance was an interesting option in positioning a tube in difficult situations; a standard nutritional tube, namely 15 or 20 French in diameter, may be large enough to obtain excellent clinical results. PMID- 7555939 TI - What should be done when initial endoscopic therapy for bleeding peptic ulcer fails? PMID- 7555940 TI - Endoscopic drainage of pancreatic pseudocysts: patient selection and evaluation of the outcome by endoscopic ultrasonography. PMID- 7555941 TI - Persistence of the gallbladder following laparoscopic cholecystectomy: a case report. AB - Laparoscopic cholecystectomy is the technique of choice for cholecystectomy. No experience with congenital abnormalities of the gallbladder has been reported. We report here the case of a 42-year-old woman who developed recurrent symptoms seven months after laparoscopic cholecystectomy and was found to have the majority of her gallbladder still intact. We speculate that the patient had a bilobate gallbladder as the explanation for the mishap. She subsequently underwent open repeat surgery. PMID- 7555942 TI - Laparoscopic diagnosis of peritoneal serous papillary carcinoma. AB - Peritoneal serous papillary carcinoma is a rare primary tumor of the peritoneum. We report here a case diagnosed by laparoscopy, and summarize the clinicopathological features previously reported in patients with this tumor. Laparoscopy was performed in a 66-year-old woman with high-protein ascites and a three-month history of lower abdominal pain. Macroscopically, the parietal and visceral peritoneum was studded with prominent white nodules up to 5 mm in diameter. Multiple biopsies revealed a tubulopapillary serous adenocarcinoma. After exclusion of metastatic peritoneal carcinomatosis (especially ovarian cancer) and malignant mesothelioma, the diagnosis of peritoneal serous papillary carcinoma was established. The patient was placed on chemotherapy (first-line: 5 fluorouracil, adriamycin, and mitomycin C; second-line: paclitaxel). She died 22 months after diagnosis. The present case is also remarkable for the coexistence of granulomatous peritonitis overshadowing the malignant nature of the process. This case report emphasizes the importance of laparoscopy with multiple biopsies in the workup of undetermined exudative ascites. PMID- 7555943 TI - Esophageal bezoar: report of five more cases. PMID- 7555945 TI - Combined hydrostatic and pneumatic balloon dilatation of the post-gastrectomy "hourglass" stomach. PMID- 7555944 TI - Severe arterial bleeding after percutaneous transhepatic cholangiographic drainage. PMID- 7555946 TI - Severe complications arising from oesophageal varix ligation with the Stiegmann Goff set. PMID- 7555947 TI - An unusual solution in a case of dislodgement of a cuffed esophageal prosthesis. PMID- 7555948 TI - Endosonographic features of a calcified mucinous gastric carcinoma. PMID- 7555949 TI - Endoscopic clipping: a helpful tool for positioning self-expanding esophageal stents. PMID- 7555950 TI - Kentuckians' attitudes toward children with epilepsy. AB - We explored Kentuckians' attitudes toward children with epilepsy. Questions compared respondents' attitudes about children with epilepsy, asthma, hyperactivity, and AIDS. Random digit dialing led to 617 completed interviews. The key questions asked concerned (a) how a pupil with each illness would alter the classroom environment, and (b) how the condition would affect the child's quality of life (QOL) at age 21 years. Respondents used a 0-10 rating scale (0 = worst, 5 = normal, 10 = best). A dichotomous variable divided respondents into those who rated below and those who rated at or above the norm: 24% predicted a deterioration of the classroom environment with the addition of a pupil with epilepsy (similar to AIDS at 26%); 41% predicted a lessened QOL at age 21 years (a worse rating than either asthma or hyperactivity). We created summary indexes, using difference scores between epilepsy ratings and ratings for the other conditions: Relative Educational Distance (RED) and Relative Quality of Life Distance (RQLD) measures. On the RED index, rural and Appalachian respondents showed the greatest prejudice toward pupils with epilepsy. Conversely, we noted the greatest prejudice in RQLD among urban residents (the best educated group). Our data do not support contentions that prejudices against persons with epilepsy are disappearing. PMID- 7555951 TI - Spatial and temporal characteristics of neonatal seizures. AB - Thirty-two neonates (26 term and 6 premature) having seizures were prospectively recruited and studied. Using prolonged video/EEG monitoring, we quantified seizure variables (electrographic and clinical seizure durations, interictal periods and electrographic seizure spread) for all 1,420 seizures recorded. The effects of time and antiepileptic drug (AED) therapy were analyzed statistically. Seizures were generally frequent, with limited electrographic spread. However, some neonates had consistently longer interictal periods and 13% had mean interictal periods > 60 min. Seizure variables were relatively stable over time, but they changed with AED therapy. There was a trend to decreased seizure duration, increased length of interictal periods, and decreased electrographic spread. Furthermore, there was evidence of reduced clinical features after sequential AED infusions. Seizures ceased during the monitoring period in 22 neonates. Eighty-five percent of all seizures had no clinical manifestations. Among neonates with clear clinical correlates, clinical observations underestimated electrographic seizures in individual neonates by a mean of 54% (range 0-95%). Seizures generally had limited electrographic spread. Use of only four recording electrodes, characteristic of some portable EEG systems, underestimated seizures in 19 neonates, and missed all seizures in 2. PMID- 7555952 TI - Migrating partial seizures in infancy: a malignant disorder with developmental arrest. AB - Fourteen infants of both sexes had a previously unreported epileptic condition characterized by nearly continuous multifocal seizures. The first seizures occurred at a mean age of 3 months, without antecedent risk factors. At 1 to 10 months, the seizures became very frequent. They were partial with variable clinical expression, and the EEG showed that the discharges randomly involved multiple independent sites, moving from one cortical area to another in consecutive seizures. Although their topography varied, the EEG ictal pattern of each seizure was very similar. It consisted of rhythmic alpha or theta activity which spread to involve an increasing area of the cortical surface. Patients regressed developmentally and became quadriplegic with severe axial hypotonia. Three patients died at age 7 months and at age 7 and 8 years, respectively. Seizures were controlled in only 2 patients, and only 3 children resumed psychomotor development. Extensive investigation failed to determine an etiology, and there was no familial recurrence. Neuropathological examination of the brain in two cases showed only severe hippocampal neuronal loss and accompanying gliosis. PMID- 7555954 TI - Predictive value of the first ictal recording in determining localization of the epileptogenic region by scalp/sphenoidal EEG. AB - The number of seizures recorded during a noninvasive evaluation for epilepsy surgery varies across centers. We retrospectively studied the accuracy of the first recorded seizure in predicting the final localization of the epileptogenic region. Sixty-six consecutive patients undergoing continuous EEG monitoring with scalp and sphenoidal electrodes were studied. The first recorded seizure was determined to be either well localized or nonlocalized and was compared with the ultimate localization after multiple seizures were recorded. The first seizure was well localized in 28 and nonlocalized in 38 patients. In the localized group, the first seizure correctly predicted the final localization of the EEG monitoring study in 26 patients, whereas 2 patients had bilateral independent temporal seizures. In the nonlocalized group, ictal onset remained nonlocalized in 34 patients despite recording of multiple (median of five) seizures. This gave a sensitivity of 87% and specificity of 94% for the first recorded seizure to predict the final results of noninvasive EEG monitoring. We conclude that the first recorded seizure is highly predictive of the final results of prolonged noninvasive ictal EEG recordings but it cannot exclude the possibility of multiple epileptogenic foci. PMID- 7555953 TI - Seizures involving the supplementary sensorimotor area in children: a video-EEG analysis. AB - Most reports of supplementary sensorimotor seizures have been of adults with medically refractory epilepsy. Typically, supplementary sensorimotor seizures have onset in childhood. We describe the electroclinical features in 12 children. Cases were selected from an EEG laboratory population in whom video-EEG was performed routinely on all children. Supplementary sensorimotor seizures were diagnosed when there was bilateral tonic posturing of the upper or lower extremities, preserved consciousness, and lack of postictal confusion. Sensory auras were reported by 8 of 9 children aged > 3 years who had daytime seizures. Speech arrest occurred in all patients in whom it could be assessed, and abnormal vocalization was observed in 7 children. Interictal EEGs were often normal (49% of recordings), and ictal EEG changes could be subtle. An etiology was demonstrated in only 2 children, and brain imaging studies were normal in the other 10 patients. Seizures were controlled with antiepileptic drugs (AEDs) in 6 of the 12 children. The clinical manifestations of supplementary sensorimotor seizures in children are similar to those reported in adults; misdiagnosis is common at all ages. PMID- 7555955 TI - Monitoring sodium methohexital distribution with [99mTc]HMPAO with single photon emission computed tomography during Wada test. AB - Twenty-five consecutive patients being considered for surgery for intractable epilepsy had intracarotid sodium methohexital procedures (ISM) as part of their evaluation. The lipophilic brain SPECT agent, [99mTc]hexamethylpropylene aminoxime (HMPAO), was administered intravenously during the procedure to determine the intracerebral distribution of methohexital. The validity of the ISM depends on consistent delivery of the anesthetizing agent to one hemisphere, including the mesial structures of the temporal lobe. To prevent postoperative language and memory deficits, correct interpretation of the test results supposes a reliable knowledge of which regions of the brain have been anesthetized. Currently, no absolute criteria allow determination of the level and topographical extent of the anesthesia. We compared results of HMPAO-SPECT with clinical and EEG video-monitoring data and with results of digital subtraction angiography (DSA) performed during the test. In all patients, the effect of SM was ipsilateral cerebral hypoperfusion on SPECT and crossed cerebellar diaschisis. The distribution of HMPAO varied from patient to patient. The delivery of SM to mesial temporal lobe structures was not constant. Using nonparametric tests, we demonstrated a statistically significant relationship between hypoperfusion on SPECT and duration of hemiplegia but not with the duration of aphasia. Hypoperfusion on SPECT was also related to the onset and duration of drug-induced delta activity on EEG. SPECT showed a statistically different distribution of SM in the brain from that predicted with DSA. We present our experience with HMPAO-SPECT use for mapping the distribution of methohexital's effects during the ISM (Wada test). We confirm the results of previous studies that SPECT assessment may be an excellent way of determining the distribution of barbiturate during the examination. It increases confidence in interpreting results of speech and memory testing by detecting either contralateral diffusion of the drug due to crossflow between hemispheres or insufficient quantitative delivery to the homolateral hemisphere. PMID- 7555957 TI - Epilepsy in twentieth century literature. AB - Literature can be viewed as a mirror that reflects society's attitude toward epilepsy. This article examines how epilepsy is portrayed in selected twentieth century literary works. Although these works were written by intellectuals, they still offer an image of epilepsy as a phenomenon characterized by supernatural features. PMID- 7555956 TI - Acquired epileptiform opercular syndrome: a second case report, review of the literature, and comparison to the Landau-Kleffner syndrome. AB - A 5-year-old girl developed recurrent prolonged episodes of severe oral apraxia, dysarthria, and drooling, similar to the opercular syndrome in children. Each episode lasted several weeks to > 6 months and was associated with exacerbation of epileptiform activity in her EEG. Electrographic status epilepticus during slow wave sleep (ESES) was recorded during three of the exacerbations. The EEG improved markedly when clinical symptoms subsided. Antiepileptic drugs (AEDs) were not effective, although there was some improvement when they were combined with a ketogenic diet. A similar case was described by Roulet et al. We believe that this is a distinct epileptic syndrome, equivalent to the Landau-Kleffner syndrome (LKS). PMID- 7555958 TI - Public perceptions about epilepsy. PMID- 7555960 TI - NBQX blocks acute and late epileptogenic effects of perinatal hypoxia. AB - Clinically, and in experimental models, perinatal hypoxic encephalopathy is commonly associated with seizures. We previously described a rat model in which hypoxia induces seizures and permanently increases in seizure susceptibility in immature rats [postnatal day (P) 10-12] but not in older rats. In the present study, we compared the effect of pretreatment with the excitatory amino acid antagonists MK-801 and NBQX versus lorazepam in our rat model of perinatal hypoxia. Animals exposed to hypoxia at P10 without treatment have frequent seizures during hypoxia and subsequently exhibit increased seizure susceptibility to flurothyl. Treatment with 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX 20 mg/kg) effectively suppressed hypoxia-induced seizures in immature rats and also protected against permanent changes in flurothyl threshold in adulthood, whereas treatment with MK-801 (1 mg/kg) or lorazepam (LZP 1 mg/kg) did not prevent these hypoxia-related epileptogenic effects. These results suggest that activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) receptors may partly mediate the age-dependent epileptogenic effect of hypoxia in the perinatal period. PMID- 7555959 TI - Age-related differences in the effects of GABAA agonists microinjected into rat substantia nigra: pro- and anticonvulsant actions. AB - GABAergic transmission in the substantia nigra pars reticulata (SNR) has an important role in the control of experimental seizures. In the flurothyl seizure model, SNR microinjection of the selective GABAA receptor agonist muscimol results in a biphasic dose-response curve in adults: Intermediate doses are anticonvulsant, but high doses have proconvulsant effects. Another GABAA agonist, THIP (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol), also produces anticonvulsant effects at lower doses, whereas higher doses tend to produce a proconvulsant effect. In 16-day-old rat pups, no anticonvulsant but only proconvulsant effects of muscimol occur, and at lower doses than in adults. These data suggest that the immature SNR is significantly more sensitive to the proconvulsant effects of GABAA receptor agonists than is the SNR of adults. We hypothesize that the age-related differences in nigral GABAergic response may be due to ontogenic changes in GABAA-sensitive neuronal circuits in the SNR. PMID- 7555961 TI - Differential impairments of spatial memory and social behavior in two models of limbic epilepsy. AB - To explore memory impairments in temporal lobe epilepsy, we used two experimental models in the rats: (a) kainate-induced status epilepticus (SE) resulting in excitotoxic damage and in later spontaneous seizures; and (b) amygdala kindling, known to induce no lesions (or only minor) and neuronal reorganization. Long-term effects of these models on memory were investigated with a spatial learning task in a radial-arm maze, and a social interaction test that implies degree of short term memory. An histological analysis was made to determine neuronal damage or loss caused by epileptic activity in brain regions that could be related to memory functions. Kainate-induced epilepsy produced large memory deficits in animals tested 5 months after the injection. The rats showed severe lesions in amygdala and hippocampus and piriform and entorhinal cortex. Spatial memory was strongly diminished. The social memory test was severely impaired, probably due to the extent of amygdala injury, which is known to disturb social behavior. On the contrary, kindled rats showed no evident lesion in any brain region and displayed performances as good as those of controls in both tests. These experiments demonstrated that memory deficits appear to be related to the severity of neuronal damage in limbic areas, and the ability to develop seizures (permanence) is not solely responsible for these memory disturbances. PMID- 7555962 TI - Effects of the anticonvulsant losigamone and its isomers on the GABAA receptor system. AB - We conducted in vitro studies to clarify the possible involvement of GABAA receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+ losigamone) and AO-294 (- losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, < or = 100 microM losigamone did not affect the specific binding of [3H]GABA, [3H]flunitrazepam, or [35S]t-butyl-bicyclophosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10(-8)-10(-5) M, stimulated 36Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10(-5) M potentiated the effect of a suboptimal concentration of exogenous GABA 10(-5) M on 36 Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS-induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating 36Cl influx in the spinal cord neurons by a GABA sensitive mechanism and at a side distant from the GABA channel. PMID- 7555963 TI - Action of GP 47779, the active metabolite of oxcarbazepine, on the corticostriatal system. I. Modulation of corticostriatal synaptic transmission. AB - Oxcarbazepine (OCBZ) is the keto-analogue of carbamazepine (CBZ). In humans, OCBZ is rapidly and almost completely metabolized to 10, 11-dihydro-10-hydroxy-CBZ (GP 47779), the main metabolite responsible for the drug's antiepileptic activity. The corticostriatal pathway is involved in the propagation of epileptic discharges. We characterized the electrophysiological effects of GP 47779 on striatal neurons by making intracellular recordings from corticostriatal slices. GP 47779 (3-100 microM) produced a dose-dependent inhibition of glutamatergic excitatory postsynaptic potentials (EPSPs). This effect was not coupled either with changes of the membrane potential of these cells or with alterations of their postsynaptic sensitivity to excitatory amino acids (EAA) suggesting a presynaptic site of action. GP 47779 reduced the current-evoked firing discharge only at concentrations > 100 microM. GP 47779 did not affect the presynaptic inhibitory action of adenosine, showing that presynaptic adenosine receptors were not implicated in the GP 47779-mediated reduction of corticostriatal EPSPs. Our data indicate that GP 47779 apparently acts directly on corticostriatal terminals to reduce the release of EAA, probably by inhibiting high-voltage-activated (HVA) calcium (Ca2+) currents (described in the accompanying article). The inhibitory action of GP 47779 on corticostriatal transmission may contribute to the antiepileptic effects of this drug. PMID- 7555964 TI - Action of GP 47779, the active metabolite of oxcarbazepine, on the corticostriatal system. II. Modulation of high-voltage-activated calcium currents. AB - GP 47779, the active metabolite of oxcarbazepine (OCBZ) inhibits glutamatergic excitatory postsynaptic potentials (EPSPs) in rat striatum (described in the accompanying article). This effect was presumed to involve the modulation of the calcium (Ca2+) signals at either pre- or postsynaptic level. Therefore, we directly tested whether GP 47779 could modulate Ca2+ conductances in cortical as well as in striatal neurons. GP 47779 produced a reversible dose-dependent decrease in high-voltage-activated (HVA) Ca2+ currents evoked by membrane depolarization in isolated cortical pyramidal cells. GP 47779-mediated reduction in HVA Ca2+ currents, if occurring also at corticostriatal axon terminals, might explain the reduction of glutamate release in the striatum. An inhibitory action of GP 47779 on HVA Ca2+ currents was also observed in isolated striatal neurons. The effect of HVA Ca2+ currents in cortical and striatal neurons persisted in the presence of nifedipine, suggesting that dihydropyridine-sensitive channels were not involved in the GP 47779-mediated responses. We propose that the modulation of HVA Ca2+ channels by this carbamazepine (CBZ) analogue may account for its inhibitory action on transmitter release. PMID- 7555965 TI - Influence of epilepsy and temporal lobe resection on olfactory function. AB - Olfactory auras accompany some cases of epilepsy. Several aspects of olfactory function, including sensitivity, also may be altered. We reviewed the literature on these topics, as well as studies evaluating the influences of temporal lobe resection and other seizure management procedures on olfactory function. We concluded that: (a) despite several studies, the prevalence of olfactory auras in epilepsy is unknown, with estimates ranging from < 1% to > 30%; (b) epilepsy appears to cause a generalized decrease in olfactory functioning, although increased sensitivity may occur in some epileptic patients at some time in the preictal period; (c) other sensory modalities are also affected by the epileptic process which, in some cases, involve limbic-related temporal lobe structures; (d) many of the olfactory deficits previously attributed to temporal lobe resection actually exist preoperatively; (e) a taste/flavor confusion exists in the reporting of taste auras; (f) unpleasant auras are associated with hyperresponsiveness of neurons, which may explain why most epilepsy-related olfactory auras are described as "bad"; and (g) interesting parallels exist between the effects of the neuroendocrine system on seizure activity and olfactory function. PMID- 7555966 TI - Physiologic and morphologic characteristics of granule cell circuitry in human epileptic hippocampus. AB - Morphological and electrophysiological techniques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 microM) of bicuculline; bursts were sensitive to the N-methyl-D-aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced hyperexcitability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of gamma-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus. PMID- 7555967 TI - Comparative anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6 dimethylphenyl)phthalimide and prototype antiepileptic drugs in mice and rats. AB - We compared the anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6 dimethylphenyl)phthalimide (ADD 213063) with those of phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), ethosuximide (ESM), valproate (VPA), and felbamate (FBM). Evaluation of anticonvulsant properties performed according to well-established procedures in rats and mice showed that ADD 213063 is most effective in protecting animals against maximal electroshock seizures (MES). This anti-MES activity is achieved with nontoxic doses, with the optimal effect recorded in rats dosed orally with anti-MES ED50 and protective index (PI) values of 25.2 mumol/kg and > 75, respectively. ADD 213063 protects to a lesser extent against seizures induced by subcutaneous (s.c.) picrotoxin and subcutaneous pentylenetetrazol (PTZ) in mice dosed intraperitoneally and orally, respectively. The profile of anticonvulsant action of ADD 213063 closely parallels that of CBZ. PMID- 7555968 TI - Anticonvulsant efficacy of ADCI (5-aminocarbonyl-10,11-dihydro-5H dibenzo[a,d]cyclohepten-5,10-imine) after acute and chronic dosing in mice. AB - ADCI (5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine), a low-affinity uncompetitive N-methyl-D-aspartate (NMDA) antagonist, is a broad spectrum anticonvulsant with a favorable side-effect profile. In the present study, we sought to determine if tolerance develops to the anticonvulsant activity of ADCI, using the maximal electroshock (MES) test to assess seizure protection. Mice were treated with three daily injections of a 2 x ED50 dose for MES protection (18 mg/kg, intraperitoneally, i.p.) or vehicle for 7 or 14 days. On the day after the chronic treatment protocol, all animals received a challenge dose of ADCI (18 mg/kg) and 15 min later were evaluated in the MES test. In control animals, 83-94% of animals were protected and the ADCI plasma levels immediately after the MES test were 5.5-9.7 micrograms/ml. In treated animals, 29 and 0% of animals were protected at 7 and 14 days, respectively, and the ADCI plasma levels were 77 and 52% of the control values. [3H]Dizocilpine binding to brain NMDA receptors was unaltered by the chronic drug treatment. In subsequent experiments, we determined that 14-day chronically treated animals could be completely protected by increased doses of ADCI (ED50 28.9 mg/kg). In both naive and chronically treated animals receiving a challenge dose of ADCI, plasma drug levels decreased in two phases, the first with a time constant of approximately 55 min and the second with a much slower rate. The estimated plasma concentrations of ADCI reflecting threshold (3-5 micrograms/ml) and 50% protection (5-7.5 micrograms/mg) were similar in naive and chronic animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555969 TI - Epidemiology of infantile spasms in Sweden. AB - We performed a retrospective study of infantile spasms (IS) in 57 children (19 girls and 38 boys) from the east-central region of Sweden for the period 1987 1991. The incidence was 0.45/1,000 newborns a year, with a spread of 0.17-0.76 between the seven counties included. Forty-three cases (75%) were symptomatic and 14 (25%) were idiopathic. Cerebral malformations were diagnosed in 8 children, and chromosomal aberrations were diagnosed in 5. In addition, 2 children each were diagnosed as having tuberous sclerosis and neurofibromatosis. At follow-up, 57% in the idiopathic group had normal development, whereas 91% in the symptomatic group had died or developed a multihandicap. The value of modern neuroradiological techniques for etiological diagnosis is stressed. PMID- 7555970 TI - Secular trends and birth cohort effects in unprovoked seizures: Rochester, Minnesota 1935-1984. AB - The incidence of idiopathic/cryptogenic epilepsy and isolated unprovoked seizures has been relatively stable in the population of Rochester, Minnesota, for the 50 year period 1935 through 1984. In each decade, the age-specific rates exhibited a consistent U-shaped pattern of decreasing rates from infancy to age 40-49 and a progressive increase thereafter to a second peak at age 70 years. Males had a 15% higher incidence of cryptogenic unprovoked seizures than females. The most pronounced secular trend was a decrease in the incidence in children aged < 10 years for the first 4 decades of the study; however, this trend was interrupted by a slight rebound in the decade 1975-1984. There has been a progressive decrease in the incidence of cryptogenic unprovoked seizures in individuals aged > or = 50 from 1965 through 1984. This decrease paralleled the decrease in cerebrovascular disease in the community. The decrease in idiopathic unprovoked seizures may be related to a concurrent trend in "silent stroke." Plots and Poisson regression analysis did not show patterns in the incidence of idiopathic unprovoked seizures related to successive birth cohorts. However, significantly lower incidence rates were observed for the 1930-1934 birth cohort, about half that of all others, between the ages of 5 and 54 years. PMID- 7555971 TI - Induction of pseudoseizures with intravenous saline placebo. AB - For a 2-year period, all patients admitted to the inpatient adult EEG videotelemetry unit of the University of Miami School of Medicine underwent attempted event induction with intravenous normal saline placebo. Of 175 patients monitored during that period, 101 underwent attempted placebo saline induction, whereas 58 patients were either in the pediatric age group, were undergoing a repeat hospitalization (i.e., depth electrode monitoring), or refused induction. The final diagnosis in each patient was established after review of the history; physical, interictal, and ictal EEG findings; brain imaging studies; interictal and postictal brain single photon emission computed tomography (SPECT) and serum prolactin levels; psychiatric and psychological evaluations; and detailed neuropsychological testing. Final diagnoses were separated into epilepsy alone, pseudoseizures, epilepsy and pseudoseizures, and other (neither epilepsy nor pseudoseizures). No patient with an eventual diagnosis of epilepsy alone was inducible. Forty-one patients with a diagnosis of epilepsy were not inducible. Of 32 patients with an eventual diagnosis of pseudoseizures, 29 were inducible. One of these 29 was also diagnosed with epilepsy. Three patients with an eventual diagnosis of pseudoseizures were not inducible; 90.6% of patients with an eventual diagnosis of pseudoseizures were inducible, i.e., had events identical to those reported by history, after injection of saline placebo. Placebo saline injection is a safe and effective means of distinguishing epilepsy from pseudoseizures. PMID- 7555972 TI - Self-injury and incontinence in psychogenic seizures. AB - Two patients who incurred significant injuries during psychogenic seizures prompted us to do a telephone survey of self-injury and incontinence in 102 consecutive patients diagnosed with psychogenic seizures by EEG-closed-circuit TV (EEG-CCTV) monitoring. Seventy-three patients (or a close family member or friend) were reached by telephone and responded to our survey. During typical attacks of psychogenic seizures, 40% reported injuries, 44% reporting tongue biting, and 44% reported urinary incontinence. Suicide attempts were reported by 32% and were more common in those with self-injury and urinary incontinence. We compared the results of patients with psychogenic seizures with those of 30 patients with refractory epilepsy documented by ictal recordings, using a similar telephone survey. Injuries of all types were more commonly reported by epilepsy patients. Burn injuries were reported only by patients with epilepsy. Suicide attempts were more commonly reported by the psychogenic seizure group. Self injury and incontinence are commonly reported by psychogenic seizure patients. In view of their significant association with suicide attempts, they may indicate an underlying depression. PMID- 7555973 TI - Benzodiazepine-GABAA receptor binding during absence seizures. AB - The role of benzodiazepine (BZD)-gamma-aminobutyric acidA (GABAA) receptors in the pathogenesis of absence seizures is uncertain. In this study, we examined the effect of absence seizures on the binding of flumazenil to the BZD binding site of the GABAA receptor. Five patients with idiopathic generalized epilepsy (IGE) were studied at rest and during absence seizures with [11C]flumazenil and positron emission tomography (PET). Normalized regional cerebral time-activity curves from the resting and ictal scans were compared with each other and with computed simulations showing the effects of changes in cerebral blood flow (CBF) and [11C]flumazenil binding. No evidence was found for a change in [11C]flumazenil binding with absence seizures. This result, together with those of a recent study showing no abnormality of [11C]flumazenil binding interictally in patients with childhood and juvenile absence epilepsy (JAE) does not support a primary role for the BZD binding site of the GABAA receptor in the pathogenesis of absence seizures. PMID- 7555974 TI - Plasma trace element, plasma glutathione peroxidase, and superoxide dismutase levels in epileptic children receiving antiepileptic drug therapy. AB - Some antiepileptic drugs (AEDs) may alter trace element metabolism and free radical scavenging enzyme activities in humans and experimental animals. We investigated the effect of long-term AED therapy on copper (Cu), zinc (Zn), manganese (Mn), selenium (Se), magnesium (Mg), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) in the plasma in children with epilepsy. During treatment with valproate (VPA) or carbamazepine (CBZ) monotherapy plasma Cu, Zn, Mn, Se, and Mg concentrations of patients were not statistically different from those of control subjects. The level of serum VPA weakly correlated with the increase in plasma Zn level. Recent studies suggest that membrane lipid peroxidation may be causally involved in some forms of epilepsies, and the decreased free radical scavenging enzyme activity is believed to cause the increased risk of an idiosyncratic drug reaction encountered in the management of epilepsy. Because GSH-PX and SOD are the most important members of antioxidant defense mechanisms, we quantitated the activities of these enzymes in plasma of children with epilepsy receiving VPA or CBZ. Only plasma GSH-PX activities in VPA group were higher than those of the control group, and the difference was statistically significant. PMID- 7555975 TI - Pharmacokinetics of tiagabine, a gamma-aminobutyric acid-uptake inhibitor, in healthy subjects after single and multiple doses. AB - Tiagabine (TGB) HCl, a new antiepileptic compound, is a potent and specific inhibitor of gamma-aminobutyric acid (GABA) uptake. In conjunction with three phase I studies, the pharmacokinetics of TGB were examined in 58 healthy male volunteers. Study I involved single increasing doses (2-24 mg TGB HCl); study II involved doses of 2-10 mg given once daily for 5 days; study III explored one dose daily (6 or 12 mg) for 14 consecutive days. Plasma TGB concentrations were measured by high-performance liquid chromatography (HPLC). Pharmacokinetic parameters were calculated by standard noncompartmental methods. Pharmacokinetic profiles were similar in all three studies and indicated that the processes of absorption and elimination of TGB were linear. The drug was rapidly absorbed, and half-life (t1/2) averaged 5-8 h. The accumulation ratio was fairly low: < 1.4 in most subjects. Secondary peaks in plasma concentration-time profiles suggested enterohepatic recycling. Lack of significant effects on antipyrine clearance indicated that TGB does not induce or inhibit hepatic microsomal enzyme systems. PMID- 7555976 TI - A review of the preclinical pharmacology of tiagabine: a potent and selective anticonvulsant GABA uptake inhibitor. AB - We review the neurochemical and behavioral profile of the selective gamma aminobutyric acid (GABA) uptake inhibitor, (R)-N-(4,4-di-(3-methylthien-2-yl)but 3-enyl) nipecotic acid hydrochloride [tiagabine (TGB), previously termed NNC 05 0328, NO 05-0328, and NO-328], which is currently in phase III clinical trials for epilepsy. TGB is a potent, and specific GABA uptake inhibitor. TGB lacks significant affinity for other neurotransmitter receptor binding sites and/or uptake sites. In electrophysiological experiments in hippocampal slices in culture, TGB prolonged the inhibitory postsynaptic potentials (IPSP) and inhibitory postsynaptic currents (IPSC) in the CA1 and CA3 produced by the addition of exogenous GABA. In vivo microdialysis shows that TGB also increases extracellular GABA overflow in a dose-dependent manner. Together these biochemical data suggest that the in vitro and in vivo mechanism of action of TGB is to inhibit GABA uptake specifically, resulting in an increase in GABAergic mediated inhibition in the brain. TGB is a potent anticonvulsant agent against methyl-6,7-dimethyoxy-4-ethyl-B-carboline-3-carboxylate (DMCM)-induced clonic convulsions (mice), subcutaneous pentylenetetrazol (PTZ)-induced tonic convulsions (mice and rats), sound-induced convulsions in DBA/2 mice and genetically epilepsy-prone rats (GEPR), and electrically induced convulsions in kindled rats. TGB is partially efficacious, against subcutaneous PTZ-induced clonic convulsions, and photically induced myoclonus in Papio papio. TGB is weakly efficacious in the intravenous PTZ seizure threshold test and the maximal electroshock seizure (MES) test and produces only partial protection against bicuculline (BIC)-induced convulsions in rats. The overall biochemical and anticonvulsant profile of TGB suggests potential utility in the treatment of chronic seizure disorders such as generalized clonic-tonic epilepsy (GTCS), photomyoclonic seizures, myoclonic petit mal epilepsy, and complex partial epilepsy. PMID- 7555977 TI - Neocortical temporal lobe epilepsy: characterizing the syndrome. PMID- 7555979 TI - Zonisamide: electrophysiological and metabolic changes in kainic acid-induced limbic seizures in rats. AB - We studied the pharmacological mechanism of zonisamide (ZNS) using an electrophysiological and autoradiographical method in a limbic seizure model in rats. Limbic seizure status epilepticus was induced by a unilateral microinjection of kainic acid (KA) into the amygdala. Initially, observed seizures were limited to the side of the injected amygdala and then propagated to bilateral sensorimotor cortex. Eighty minutes after injection, secondarily generalized seizure status epilepticus was induced, with each seizure lasting approximately 30 s and recurring every 5 min. ZNS 100 mg/kg was administered intravenously (i.v.) during the generalized seizure. Forty minutes after ZNS administration, epileptic activity was observed only at the KA-injected amygdalar site and spikes were not observed in the bilateral sensorimotor cortex. We studied local cerebral glucose utilization (LCGU) after ZNS or saline administration using an autoradiographical method in the same limbic seizure preparation. In the ZNS group, LCGU decreased in the ipsilateral sensorimotor cortex and hippocampus, whereas in the controls LCGU increased in these structures. On the other hand, ZNS did not suppress the epileptic activity of the primary focus and no decrease in LCGU was observed in the KA-injected amygdala. ZNS inhibited seizure propagation from the epileptogenic focus but did not suppress the epileptic activity of the focus. Our results suggest that ZNS is effective for the treatment of secondarily generalized seizure. PMID- 7555978 TI - Age-dependent effects of gamma-aminobutyric acid agents on flurothyl seizures. AB - Behavioral characteristics of seizures have age-dependent features, which suggests that effective treatment of seizures may be age-specific as well. In experiments that used the flurothyl seizure model, we examined the effects of several drugs that affect GABAergic neurotransmission in rats of various ages. Systemic administration of phenobarbital (PB, a drug that enhances GABAA receptor mediated inhibition) was anticonvulsant in most age groups. In contrast, gamma vinyl GABA (VGB, a drug that increases endogenous GABA levels and enhances both GABAA and GABAB receptor transmission) did not have anticonvulsant effects. Baclofen (a GABAB receptor agonist) was proconvulsant in 9-day-old rat pups, and anticonvulsant in 15-30-day-old rats and lost its anticonvulsant activity in 60 day-old rats. CGP 35348 (a GABAB receptor antagonist) was proconvulsant in developing rats but not in 60-day-old rats. A novel GABAB receptor antagonist, CGP 36742, was proconvulsant in 9- and 15-day-old rats but had no effects in 30- and 60-day-old rats. These results indicate that the effects of presumed GABAergic agents are not uniform across the age span. The differences may reflect age-dependent maturational changes of GABA receptor subtypes, differential action of the drugs on pre- and postsynaptic sites and possible non-GABAergic effects. PMID- 7555980 TI - Recurrent seizures alter renal function and plasma atrial natriuretic peptide levels in rats. AB - Status epilepticus can lead to impaired renal function, which has been attributed to complications of myoglobinuria. We confirmed changes in renal function in the absence of myoglobinuria by measuring renal hemodynamics, fluid and electrolyte excretions, and plasma levels of renin and atrial natriuretic peptide (ANP) before and after a 30-min period of recurrent generalized seizures in anesthetized, paralyzed rats. Renal plasma flow (RPF), renal blood flow (RBF) and glomerular filtration rate (GFR) decreased by approximately 60% after seizures. In contrast, urinary sodium excretion, urine flow, and plasma ANP levels increased approximately threefold. Urinary potassium excretion and plasma renin levels were unchanged. Renal function is profoundly altered after 30 min of seizures, primarily due to intense renal vasoconstriction precipitating a dramatic reduction in GFR. The concomitant increases in sodium and urine excretion may be mediated by the marked increase in plasma ANP levels. The decreases in GFR and RBF might contribute to the renal failure observed in some patients after status epilepticus. PMID- 7555981 TI - Neuroepidemiological study of childhood epilepsy by application of international classification of epilepsies and epileptic syndromes (ILAE, 1989). AB - A population-based survey of childhood epilepsy was made in 1975 on the total population of children aged < 10 years living in Okayama Prefecture (n = 2,378 patients). Using the data obtained, we attempted to reclassify the various types of epilepsy according to the international classification (ILAE, 1989). Reclassification was possible in 1,872 (78.7%) of the 2,378 cases. The 1,872 cases consisted of 1,045 (55.8%) with localization-related epilepsies, 824 (44.0%) with generalized epilepsies, and 3 (0.2%) with epilepsies undetermined whether focal or generalized. Classification of the epilepsies in a population based survey using the international classification involves difficulties, because both clinical and EEG findings are essential. However, if an appropriate area is selected, classification of epilepsies and epileptic syndromes in a population-based survey is possible by referring to all medical records stored at every hospital and practitioner's clinic that administers treatment to patients with epilepsy in the area. PMID- 7555982 TI - Comparison of mesial versus neocortical onset temporal lobe seizures: neurodiagnostic findings and surgical outcome. AB - We compared historical features, surface EEG findings, results of intracarotid sodium amobarbital memory testing (IAT), and outcome after anterotemporal lobectomy (ATL) in patients with mesiotemporal lobe seizure onset with those with more diffuse temporal lobe seizure onset (intracranial EEG). Forty-eight patients evaluated consecutively between July 1985 and October 1991 with both scalp/sphenoidal and intracranial EEG were shown to have seizures originating in one temporal lobe. No patients had temporal lobe tumor or vascular malformation. Thirty-seven of the 48 patients had seizure onset in the amygdala/hippocampus (amyg/hipp). Eleven of the 48 had either temporal neocortical onset or simultaneous amyg/hipp and neocortical onset. Patients with mesial onset seizures were more likely to have lateralized memory impairment on IAT (p = 0.05). We noted a trend toward a difference in age of first risk for epilepsy between the two groups (p = 0.09) but not for a difference in any specific risk factor. There were no significant differences in surface EEG interictal findings. Unlike in previous studies, comparison of outcome between the two groups showed no difference in seizure-free outcome. Sudden unexpected death (SUD) was more frequent in neocortical seizure patients who were not seizure-free (p < 0.05). PMID- 7555983 TI - Ictal contralateral paresis in complex partial seizures. AB - Certain behaviors that occur during a complex partial seizure (CPS) are useful in lateralizing the side of seizure onset. In 5 (5.3%) of 94 consecutive patients with partial epilepsy, we observed ictal unilateral arm and hand paresis during 27 of 34 CPS. In all these seizures, this behavior occurred contralateral to an epileptogenic temporal lobe, as determined by video-EEG monitoring and surgical outcome. In 5 of the 27 seizures, an observer demonstrated that the paretic arm and hand were flaccid. None of these patients had postictal (Todd's) paralysis. In most of the seizures, the arm ipsilateral to seizure onset had simultaneous purposeful movements or automatisms, sometimes with awkward posturing. Ictal unilateral paresis is distinctly different from ictal dystonia or postictal paralysis and consistently lateralizes seizure onset to the contralateral temporal lobe. Recognition of this particular ictal behavior and comparison to other simultaneous behaviors can aid in the lateralization and possibly localization of the epileptogenic zone. PMID- 7555984 TI - Menstrual disorders in women with epilepsy receiving carbamazepine. AB - We measured concentrations of serum sex hormones and sex hormone binding globulin (SHBG) in relation to regularity of the menstrual cycles in 8 women before carbamazepine (CBZ) treatment was initiated and after 1 and 5 years of CBZ therapy. In addition, we evaluated menstrual cycle regularity and related endocrine changes in 56 women receiving CBZ treatment for > 5 years. Serum SHBG levels increased, and serum concentrations of 17 beta-estradiol (estradiol) and estradiol/SHBG ratio decreased during CBZ treatment. Two of the 8 patients (25%) in the prospective study group developed menstrual irregularities during the first 5 years of therapy. In the cross-sectional study group of patients treated with CBZ for > 5 years, the frequency of menstrual disturbances was also 25.0% (14 of 56 patients). Concentrations of serum sex hormones and SHBG were measured in 13 women with menstrual disorders and in 11 randomly selected women with regular cycles. In most cases, menstrual disorders were associated with increased serum SHBG and decreased serum estradiol levels and low estradiol/SHBG ratio. Long-term CBZ treatment results in increased serum SHBG levels and decreased estradiol effect, which correlate with the frequency of menstrual disorders in CBZ-treated women with epilepsy. PMID- 7555985 TI - Serum prolactin in neonates with seizures. AB - We studied serum prolactin (PRL) in 28 newborn infants with acute encephalopathy. Six patients had electrographically confirmed seizures. Twenty-two patients comprised the nonictal group. In the seizure group, PRL was determined at the first onset of the seizure (baseline) and at 15 and 30 min postictal. In the nonseizure group, PRL was determined at the end of the EEG and 15 min later. EEGs were visually analyzed for the presence of seizures and background abnormality (normal or mildly, moderately, or markedly abnormal). Etiologic diagnoses included congenital heart disease (12), hypoxic-ischemic encephalopathy (4), sepsis (4), respiratory distress syndrome (5) meconium aspiration (1), and metabolic disease (2). Serum PRL was significantly higher (p < 0.05) at baseline and 15 min postictally in the patients with seizures than in the nonictal group. However, PRL levels 15 and 30 min postictally were not statistically different from baseline values. Baseline PRL correlated significantly (p < 0.001) with EEG background abnormality in both groups; therefore, patients with the most abnormal EEG backgrounds had higher levels of PRL than those with a relatively normal EEG background. We conclude that newborns with EEG-confirmed seizures, particularly if seizures are not associated with clinical signs, have high baseline serum PRL levels that do not increase significantly in the immediate postictal period. Serum PRL levels correlate with the severity of the brain insult as evaluated by EEG background. Further studies are needed to enhance our understanding of the dynamics of PRL secretion in newborns with seizures and acute encephalopathy. PMID- 7555987 TI - Electrocorticography and temporal lobe epilepsy: relationship to quantitative MRI and operative outcome. AB - We investigated the relationship between electrocorticography (ECoG), quantitative magnetic resonance imaging (MRI), and surgical outcome in 165 patients with intractable nonlesional temporal lobe epilepsy (NLTLE). A standard mesial temporal resection was performed in all patients. Patients with an operative follow-up < 1 year were excluded from the study. The extent of the lateral temporal neocortex resection (LCR) was guided by ECoG and the side of surgery. The extent of the LCR was not predictive of seizure outcome in patients with or without hippocampal formation atrophy (p > 0.5). Patients undergoing a right anterior temporal lobectomy had a larger LCR (p < 0.0001), but the side of surgery was not of predictive value in determining seizure outcome (p > 0.1). The topography of the acute intracranial spikes did not correlate with operative outcome (p > 0.5) and was independent of hippocampal volumetric studies (p > 0.5). The postexcision ECoG was also shown not to be of prognostic importance (p > 0.5). Our results indicates that the extent of the lateral temporal cortical resection and the ECoG findings are not important determinants of surgical outcome in patients with NLTLE. PMID- 7555986 TI - Efficacy and tolerability of vigabatrin in children with refractory partial seizures: a single-blind dose-increasing study. AB - The efficacy and tolerability of vigabatrin (VGB) in children with refractory partial epilepsy were assessed in a single-blind, add-on, fixed-sequence, placebo controlled trial. After 1-month observation, the patients entered a 7-month treatment period that involved administration of placebo for 1 month followed by VGB at the initial dosage of 40 mg/kg/day, to be increased to 60 and 80 mg/kg/day at 2-month intervals if seizures persisted. Of the 46 children enrolled in the study, 7 dropped out prematurely due to lack of efficacy of the drug (n = 6) or increased seizure frequency (n = 1). In 11 patients who either became seizure free (n = 3) or improved markedly (n = 8), treatment was completed at a dose < 80 mg/kg/day. The average number of seizures per month in the 39 patients who completed the study decreased from 97 during placebo to 21, 12, and 9 after 2, 4, and 6 months of VGB treatments respectively (p < 0.0001 at each time). Response to VGB remained statistically significant when dropouts were included in the evaluation. The number of patients who had > 50% reduction in seizure frequency after 2, 4, and 6 months was 28, 33, and 35, respectively. Eight patients became seizure-free during the last 2 months of VGB treatment (3 at 40, 3 at 60, and 2 at 80 mg/kg/day, as compared with none during placebo treatment). Serum levels of associated antiepileptic drugs (AEDs) showed no significant changes, except for serum phenytoin (PHT) concentration, which significantly (p < 0.01) decreased after VGB treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7555988 TI - Frequency of the electroencephalographic discharge in seizures of focal and widespread onset in intracerebral recordings. AB - The information provided by the EEG during an epileptic seizure is of critical importance in the localization of an epileptic focus. Localizing information is usually provided by the region of onset, by the predominance of the discharge during the seizure, and by postictal slow waves. In patients with intracerebral electrodes, we investigated another aspect of the discharge: its highest frequency. We divided seizures into those with a focal onset and those with a regional onset. Results indicated clearly that fast frequencies (15-30 Hz) were much more frequent during seizures of focal onset than during seizures of widespread onset. Highest frequencies were not necessarily observed at the onset of focal seizures but could be observed at any time during a seizure of focal onset. High frequencies appear to characterize small epileptogenic zones (EZ). Our results may be helpful in the interpretation of intracerebral EEGs when large brain areas remain unexplored and one is often uncertain of the extent of an EZ. PMID- 7555989 TI - Visual learning on a selective reminding procedure and delayed recall in patients with temporal lobe epilepsy. AB - We administered a visual selective reminding procedure for the learning of designs to healthy controls and to left or right temporal lobe epilepsy (TLE) patients without demonstrable brain lesions. In comparison with the other groups, right TLE patients showed a deficit of learning characterized by an impairment in storing visual material and consistently retrieving it from long-term memory. Patients with left TLE performed similarly to controls. These data are consistent with the hypothesis of right hippocampal involvement in the learning of nonverbal material. Delayed retrieval of learned material was similar in all the subject groups tested at long time intervals, suggesting the impairment of learning in patients with right TLE does not necessarily imply a subsequent deficit in the retrieval of stored information. This finding may agree with the opinion that distinct functional systems subserve learning and memory. PMID- 7555990 TI - Positron emission tomography with [11C]deuterium-deprenyl in temporal lobe epilepsy. AB - We performed positron emission tomography (PET) with [11C]deuterium-deprenyl in 9 patients with temporal lobe epilepsy (TLE) undergoing evaluation for possible epilepsy surgery. Seven patients had unilateral and 2 had bilateral mesiotemporal epileptic foci based on the preoperative investigation including ictal EEG discharges and PET with 2-[18F]fluoro-2-deoxyglucose (FDG). Deprenyl is an irreversible inhibitor of monoamine oxidase type B (MAO-B) with a very high affinity for the enzyme. In the brain, MAO-B is preferentially located in astrocytes, and a previous in vitro study showed increased binding of the ligand in sclerotic hippocampi. Dynamically acquired N-[methyl-11C]-a,a-di-deutero-L deprenyl distribution in PET images were analyzed graphically, and the focus regions were assessed visually on the PET images. In addition, the accumulation rate and distribution volume of the tracer relative to the cerebellar cortex were measured in standardized homologous temporal regions by semiquantitative methods. Uptake of [11C]deuterium-deprenyl was significantly increased in the epileptogenic temporal lobes, both apparently and semiquantitatively. By calculating mean interlobar ratios, we identified the temporal lobe containing the epileptic focus in six unilateral cases. One case was ambiguous but was not falsely localized. The two bilateral cases were correctly identified as such. Our results suggest that PET with [11C]deuterium-deprenyl might be a useful method for identification of epileptogenic temporal lobes. PMID- 7555992 TI - Self-induced photogenic seizures in a child with severe myoclonic epilepsy in infancy: optical investigations and treatments. AB - In a 2-year-old patient with severe myoclonic epilepsy in infancy, we studied self-induced photogenic seizures using optical filters and blue-tinted contact lenses. The patient induced absences and/or myoclonic jerks by two kinds of behaviors: flickering hand movement (FHM) and forced eye closure (FEC). The placebo inhibitory effect on FHM by a blank goggle frame indicated that acquisition of learning affected the frequency of FHMs per se. Optical studies suggested that the degree of absorption from approximately 600-700 nm might determine the inhibitory effect of filters on FHM. Investigations using contact lenses showed that blue-tinted lenses gradually reduced photosensitivity and inhibited FHMs. PMID- 7555991 TI - False lateralization of temporal lobe epilepsy with FDG positron emission tomography. AB - We report 2 patients in whom visual interpretation of interictal positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) suggested false lateralization of an epileptic focus. PET scans were interpreted as showing diffuse left temporal lobe hypometabolism in 1 patient and lateral temporal hypometabolism in the other. However, seizures began in the right mesial temporal lobe in both patients, and both responded favorably to right temporal lobectomy. In 1 patient, the intracranial EEG showed continuous asymptomatic subclinical seizure activity emanating from the right amygdala. These limbic discharges probably caused unrecognized right temporal lobe hypermetabolism. In the other case, quantitative analysis of metabolic rates showed conflicting mesial and lateral metabolic indexes. Frequent mesial interictal discharges might have increased lateral temporal metabolism. We conclude that asymptomatic epileptiform activity may alter temporal lobe metabolism and that quantitative PET analysis helps clarify contradictory visual PET interpretations. PMID- 7555993 TI - Hematoma-related seizures detected during subdural electrode monitoring. AB - A 28-year-old woman with epilepsy developed the new onset of paroxysmal tongue tingling during subdural electrode monitoring. Her symptoms coincided with electrographic seizure activity arising from an area of the perisylvian region that had not previously been involved in her habitual seizures. At electrode removal, a focal 2 x 2 cm hematoma was detected and evacuated from beneath these electrode contacts. Unexpected episodic events may represent nonhabitual seizure activity related to the surgical procedure. PMID- 7555995 TI - Annual meeting of the American Epilepsy Society. Baltimore, Maryland, December 1 6, 1995. Abstracts. PMID- 7555994 TI - The medical-moral account on epilepsy by Pedro de Horta: a historical review. AB - We bring to reader's attention the book Medical-Moral Account of the Most Troublesome and Rigorous Illness of Epilepsy, written in 1754 by the Mexican physician Pedro de Horta. This book is the earliest text on epilepsy written in the New World. We examine Pedro de Horta's medical and religious concepts of epilepsy and analyze concepts of epilepsy in Hispanic communities during the eighteenth century. PMID- 7555996 TI - Ecosystem services: an essential component of sustainable use. PMID- 7555997 TI - COMPACT predictions: is there a catch? PMID- 7555999 TI - Burning border health issues. PMID- 7555998 TI - Drinking water and leukemia. PMID- 7556000 TI - Drink and diet. PMID- 7556001 TI - Nature's medicine cabinet. PMID- 7556002 TI - Russian rivers of radiation. PMID- 7556003 TI - As the cell cycles. PMID- 7556004 TI - Molecular pathology: unlocking the cell's secrets. PMID- 7556005 TI - What's hiding under the sink: dangers of household pesticides. PMID- 7556007 TI - Making headlines in the lab. PMID- 7556006 TI - What's eating us about what we're eating. PMID- 7556008 TI - Like sugar for poison: glucose as a substitute for benzene. PMID- 7556009 TI - Ethylenebisdithiocarbamates and ethylenethiourea: possible human health hazards. AB - Humans are exposed to ethylenebisdithiocarbamates (EBDCs) from environmental sources. Exposure to EBDCs is chronic for workers in a variety of industries, where EBDCs are used for their properties as slimicides, vulcanization accelerators, antioxidants, and scavengers in waste-water treatment. EBDCs, and particularly the EBDC metabolite ethylenethiourea, have clearly defined, important toxic effects in various animal species, and there is reason to suspect they are carcinogenic in humans. In the absence of definitive information regarding human risk, further studies need to be done. In the interim, regular surveillance of workers with high levels of exposure to EBDCs, with specific attention to markers of thyroid and hepatic pathology, should be considered. PMID- 7556010 TI - Effects of coumestrol on estrogen receptor function and uterine growth in ovariectomized rats. AB - Isoflavonoids and related compounds such as coumestrol have classically been categorized as phytoestrogens because these environmentally derived substances bind to the estrogen receptor (ER) and increase uterine wet weight in immature rats and mice. Assessment of the binding affinities of isoflavonoids for ER and subsequent effects on uterine growth suggest these compounds are less active estrogens than estradiol and therefore may reduce the risk of developing breast or prostate cancer in humans by preventing estradiol binding to ER. With the renewed interest in the relationships between environmental estrogens and cancer cause and prevention, we assessed the effects of the phytoestrogen coumestrol on uterotropic response in the immature, ovariectomized rat. Our studies demonstrated that in this animal model, coumestrol is an atypical estrogen that does not stimulate uterine cellular hyperplasia. Although acute (subcutaneous injection) or chronic (multiple injection or orally via drinking water) administration of coumestrol significantly increased uterine wet and dry weights, the phytoestrogen failed to increase uterine DNA content. The lack of true estrogenic activity was characterized by the inability of this phytoestrogen to cause cytosolic ER depletion, nuclear ER accumulation, or the stimulation of nuclear type II sites which characteristically precede estrogenic stimulation of cellular DNA synthesis and proliferation. In fact, subcutaneous or oral coumestrol treatment caused an atypical threefold induction of cytosolic ER without corresponding cytosolic depletion and nuclear accumulation of this receptor, and this increased the sensitivity of the uterus to subsequent stimulation by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556011 TI - A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. AB - Sewage, a complex mixture of organic and inorganic chemicals, is considered to be a major source of environmental pollution. A random screen of 20 organic man-made chemicals present in liquid effluents revealed that half appeared able to interact with the estradiol receptor. This was demonstrated by their ability to inhibit binding of 17 beta-estradiol to the fish estrogen receptor. Further studies, using mammalian estrogen screens in vitro, revealed that the two phthalate esters butylbenzyl phthalate (BBP) and di-n-butylphthalate (DBP) and a food antioxidant, butylated hydroxyanisole (BHA) were estrogenic; however, they were all less estrogenic than the environmental estrogen octylphenol. Phthalate esters, used in the production of various plastics (including PVC), are among the most common industrial chemicals. Their ubiquity in the environment and tendency to bioconcentrate in animal fat are well known. Neither BBP nor DBP were able to act as antagonists, indicating that, in the presence of endogenous estrogens, their overall effect would be cumulative. Recently, it has been suggested that environmental estrogens may be etiological agents in several human diseases, including disorders of the male reproductive tract and breast and testicular cancers. The current finding that some phthalate compounds and some food additives are weakly estrogenic in vitro, needs to be supported by further studies on their effects in vivo before any conclusions can be made regarding their possible role in the development of these conditions. PMID- 7556012 TI - Fertility in mice after prenatal exposure to benzo[a]pyrene and inorganic lead. AB - Experimental evidence suggests that inorganic lead and benzo[a]pyrene (BaP) suppress the development of primordial oocytes during fetal life. We examined the single and combined effects of prenatal exposure to BaP and moderate doses of lead. The fertility and ovarian morphology of F1 female NMRI mice in four treatment groups (nine mice per group) were investigated: control; lead (F0 given 1 g PbCl2/L in drinking water until mating); BaP (10 mg/kg body weight daily by oral intubation on days 7-16 of F0 pregnancy); and combined lead and BaP. F1 groups exposed prenatally to BaP either alone or in combination with inorganic lead showed markedly reduced fertility with few ovarian follicles compared to controls, whereas the group exposed to lead only had measures comparable to the controls. Mice exposed to both lead and BaP had a significantly longer gestation period (days to litter) compared to mice exposed only to BaP, lead, or controls. There is a nonsignificant indication that the compounds together further reduce number of offspring, number of litters, and litter size. These results suggest that lead and BaP have synergistic effects on impairment of fertility. The possibility of synergism may be of human relevance as inorganic lead and BaP are ubiquitous environmental pollutants. PMID- 7556013 TI - Drinking water and pregnancy outcome in central North Carolina: source, amount, and trihalomethane levels. AB - In spite of the recognition of potentially toxic chemicals in chlorinated drinking water, few studies have evaluated reproductive health consequences of such exposure. Using data from a case-control study of miscarriage, preterm delivery, and low birth weight in central North Carolina, we evaluated risk associated with water source, amount, and trihalomethane (THM) concentration. Water source was not related to any of those pregnancy outcomes, but an increasing amount of ingested water was associated with decreased risks of all three outcomes (odds ratios around 1.5 for 0 glasses per day relative to 1-3 glasses per day, falling to 0.8 for 4+ glasses per day). THM concentration and dose (concentration x amount) were not related to pregnancy outcome, with the possible exception of an increased risk of miscarriage in the highest sextile of THM concentration (adjusted odds ratio = 2.8, 95% confidence interval = 1.1-2.7), which was not part of an overall dose-response gradient. These data do not indicate a strong association between chlorination by-products and adverse pregnancy outcome, but given the limited quality of our exposure assessment and the increased miscarriage risk in the highest exposure group, more refined evaluation is warranted. PMID- 7556015 TI - Imported seabass as a source of mercury exposure: a Wisconsin case study. AB - The Wisconsin Division of Health investigated mercury exposure in a 40-year-old man, his 42-year-old wife, and their 2.5-year-old son. At the time of our investigation, these individuals had blood mercury levels ranging from 37 to 58 micrograms/L (normal < 5 micrograms/L) and hair samples from the adults contained 10-12 micrograms mercury/g dry weight. A personal interview and home inspection failed to identify any occupational or household sources of mercury exposure. The family's diet included three to four fish meals per week. The fish was purchased from a local market and included Lake Superior whitefish, Lake Superior trout, farm-raised trout and salmon, and imported seabass. Analysis of these fish found that only one species, the imported seabass, contained significant mercury levels. Two samples of the seabass obtained from the vendor on different days contained mercury concentrations of 0.5 and 0.7 mg/kg. Based on consumption estimates, the average daily mercury intakes for these individuals ranged from 0.5 to 0.8 micrograms/kg body weight. Six months after the family stopped consuming the seabass, blood mercury levels in this man and woman were 5 and 3 micrograms/L, respectively. Analysis of sequential blood samples confirmed that mercury elimination followed first-order kinetics with a half-life of approximately 60 days. PMID- 7556017 TI - Environmentally Induced Alterations in Development: a Focus on Wildlife. Proceedings of a meeting. Racine, Wisconsin, December 10-12, 1993. PMID- 7556014 TI - Mortality study of workers in 1,3-butadiene production units identified from a chemical workers cohort. AB - The International Agency for Research on Cancer has given the designations of "sufficient evidence" of carcinogenicity of 1,3-butadiene in experimental animals and "limited evidence" of carcinogenicity in humans. To investigate the carcinogenic effect in humans, we conducted a cohort mortality study among 364 men who were assigned to any of three 1,3-butadiene production units located within several chemical plants in the Kanawha Valley of West Virginia, including 277 men employed in a U.S. Rubber Reserve Plant which operated during World War II. The butadiene production units included in this study were selected from an index developed by the Union Carbide Corporation, which listed for each chemical production unit within their South Charleston, West Virginia and Institute, West Virginia, plants all products, by-products, and reactants. Departments included in the study were those where butadiene was a primary product and neither benzene nor ethylene oxide was present. A total of 185 deaths were observed; the standardized mortality ratio (SMR) for all causes of death was 91, reflecting lower mortality among the study population than the U.S. population. The study found a significantly elevated standardized mortality ratio (SMR) for lymphosarcoma and reticulosarcoma based on four observed cases (SMR = 577; 95% CI = 157-1480), which persisted in an analysis using county referent rates. An excess of lymphosarcoma and reticulosarcoma among all workers and among workers with routine exposure to 1,3-butadiene was also observed in the only other cohort of 1,3-butadiene production workers previously studied.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556016 TI - Xenoestrogens released from lacquer coatings in food cans. AB - We present data showing that some foods preserved in lacquer-coated cans and the liquid in them may acquire estrogenic activity. Hormonal activity was measured using the E-screen bioassay. The biological activity of vegetables packed in cans was a result of plastic monomers used in manufacturing the containers. The plastic monomer bisphenol-A, identified by mass spectrometry, was found as a contaminant not only in the liquid of the preserved vegetables but also in water autoclaved in the cans. The amount of bisphenol-A in the extracts accounted for all the hormonal activity measured. Although the presence of other xenoestrogens cannot be ruled out, it is apparent that all estrogenic activity in these cans was due to bisphenol-A leached from the lacquer coating. The use of plastic in food-packaging materials may require closer scrutiny to determine whether epoxy resins and polycarbonates contribute to human exposure to xenoestrogens. PMID- 7556018 TI - Possible interrelations among environmental toxicants, amphibian development, and decline of amphibian populations. AB - Many amphibian populations are declining in a number of geographical locations throughout the world. In most cases, the cause or causes are unknown, but are assumed to result from man-made alterations in the environment. We review existing evidence concerning how environmental xenobiotics could contribute to declines of amphibian populations by impacting growth and development of the young. This paper examines the potential roles of toxicants in: a) affecting the susceptibility of young to disease; b) retarding growth and development of amphibian young; c) affecting the ability of larvae to avoid predation; d) affecting the development of physiological, morphological, or behavioral processes in a manner that subsequently impairs the ability of the young for future reproduction; and e) directly causing mortality of young. These issues are not well studied, and more studies are needed before the roles of environmental xenobiotics in amphibian declines are fully understood. PMID- 7556019 TI - Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed frog. AB - Many environmental pollutants have estrogenic activity in animals. Xenobiotic estrogens include many pesticides and industrial chemicals that biocumulate. The impact of these common pollutants on the reproductive success of wildlife may be considerable, particularly in threatened or endangered species. This research examined the use of plasma vitellogenin in males as a biomarker for estrogenic xenobiotics in reptiles and amphibians. Adult male turtles (Trachemys scripta) and frogs (Xenopus laevis) were given ip injections of estradiol-17 beta (E2), diethylstilbestrol (DES), or o,p'-DDT (1-chloro-2-[2,2,2-trichloro-1-(4 chlorophenyl)ethyl)benzene) daily for 7 days, and plasma was collected on day 14. The estrogenic activity of each compound was determined by measuring the induction of plasma vitellogenin. Vitellogenin was identified by precipitation, electrophoresis, Western blot, and enzyme-linked immunosorbant assay (ELISA). In both species, estradiol and DES treatments induced the most vitellogenin, whereas DDT treatments induced smaller amounts of vitellogenin in a dose-dependent fashion. These data indicate that induction of plasma vitellogenin in males may be a useful biomarker of xenobiotic estrogen activity in wild populations of reptiles and amphibians. PMID- 7556020 TI - Green turtle fibropapillomatosis: challenges to assessing the role of environmental cofactors. AB - Green turtle fibropapillomatosis (GTFP) is a growing threat to the survival of green turtle (Chelonia mydas) populations worldwide. Recent transmission studies point to an infectious etiology. Several field studies suggest that high GTFP prevalence is associated with marine habitats that have been impacted by agricultural, industrial, or urban development. Environmental contaminants could be involved in GTFP through several plausible mechanisms including cocarcinogenesis and contaminant-induced immune suppression. However, an association of contaminants with GTFP has not been established. A broader perspective is needed when studying infectious diseases such as GTFP in complex ecosystems. Alternative explanations for high GTFP prevalence in some near-shore habitats include the following: a) these habitats provide an optimum physical environment for survival and transmission of the infectious agent; b) these habitats attract a high density of susceptible turtles or harbor a higher density of potential vectors, facilitating transmission of the pathogen in a density dependent fashion; and c) these habitats may contain other stressors that render turtles more susceptible to GTFP. Application of scientifically rigorous criteria in the epizootiology of GTFP in free-ranging populations remains a formidable challenge. PMID- 7556021 TI - Gonadal steroidogenesis in vitro from juvenile alligators obtained from contaminated or control lakes. AB - The ubiquitous distribution of many contaminants and the nonlethal, multigenerational effects of such contaminants on reproductive, endocrine, and immune systems have led to concerns that wildlife worldwide are affected. Although the causal agents and effects are known for some species, the underlying physiological mechanisms associated with contaminant-induced reproductive modifications are still poorly understood and require extensive research. We describe a study examining the steroidogenic activity of gonads removed from juvenile alligators (Alligator mississippiensis) obtained from contaminated or control lakes in central Florida. Synthesis of estradiol-17 beta (E2) was significantly different when ovaries from the contaminated and control lakes were compared in vitro. Additionally, testes from males obtained from the contaminated lake. Lake Apopka, synthesized significantly higher concentrations of E2 when compared to testes obtained from control males. In contrast, testosterone (T) synthesis from all testes examined in this study displayed a normal pattern and produced concentrations greater than that observed from ovaries obtained from either lake. Interestingly, the pattern of gonadal steroidogenesis differs from previously reported plasma concentrations of these hormones obtained from the same individuals. We suggest that the differences between the in vivo and in vitro patterns are due to modifications in the hepatic degradation of plasma sex steroid hormones. PMID- 7556022 TI - Effects of polychlorinated dibenzo-p-dioxins and dibenzofurans on nesting wood ducks (Aix sponsa) at Bayou Meto, Arkansas. AB - Wood ducks (Aix sponsa) nesting along Bayou Meto downstream from a hazardous waste site in central Arkansas were contaminated with polychlorinated dibenzo-p dioxins (PCDDs) and dibenzofurans (PCDFs). Residues in eggs, based on 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ), ranged up to 611 parts per trillion (ppt), and egg arithmetic means were 90-fold higher at the site nearest the point source compared with a reference site. We monitored productivity of wood ducks in artificial nest boxes at three sites on the bayou and at a reference site on a separate drainage during 1988-1990. Productivity was suppressed (p < 0.05) at the bayou sites compared with the reference site, and egg TCDD-EQs were inversely correlated (p < 0.001) with productivity in corresponding nests. The threshold range of toxicity, where reduced productivity was evident in wood ducks (based on TCDD-EQs), was > 20 to 50 ppt. Oxidative stress and teratogenic effects occurred in ducklings at the more contaminated nesting sites nearest the point source. These findings suggest that wood ducks may be more sensitive to PCDD and PCDF contamination than some other aquatic birds and could serve as an indicator species for monitoring biological impacts from these contaminants. PMID- 7556023 TI - Contaminants and sea ducks in Alaska and the circumpolar region. AB - We review nesting sea duck population declines in Alaska during recent decades and explore the possibility that contaminants may be implicated. Aerial surveys of the surf scoter (Melanitta perspicillata), white-winged scoter (M. fusca), black scoter (M. nigra), oldsqaw (Clangula hyemalis), spectacled eider (Somateria fischeri), and Steller's eider (Polysticta stellei) show long-term breeding population declines, especially the latter three species. The spectacled eider was recently classified threatened under the Endangered Species Act. In addition, three other diving ducks, which commonly winter in coastal areas, have declined from unknown causes. Large die-offs of all three species of scoters during molt, a period of high energy demand, were documented in August 1990, 1991, and 1992 at coastal reefs in southeastern Alaska. There was no evidence of infectious diseases in those scoters. The die-offs may or may not be associated with the long-term declines. Many scoters had elevated renal concentrations of cadmium (high of 375 micrograms/g dry weight [dw]). Effects of cadmium in sea ducks are not well understood. Selenium concentrations in livers of nesting white-winged scoters were high; however, the eggs they laid contained less selenium than expected based on relationships for freshwater bird species. Histological evaluation found a high prevalence of hepatocellular vacuolation (49%), a degenerative change frequently associated with sublethal toxic insult. Cadmium and selenium mean liver concentrations were generally higher in those birds with more severe vacuolation; however, relationships were not statistically significant. We do not know if sea duck population declines are related to metals or other contaminants. PMID- 7556024 TI - A review of factors affecting productivity of bald eagles in the Great Lakes region: implications for recovery. AB - The bald eagle (Haliaeetus leucocephalus) population in North America declined greatly after World War II due primarily to the eggshell thinning effects of p,p' DDE, a biodegradation product of DDT. After the banning of DDT in the United States and Canada during the early 1970s, the bald eagle population started to increase. However, this population recovery has not been uniform. Eagles nesting along the shorelines of the North American Great Lakes and rivers open to spawning runs of anadromous fishes from the Great Lakes still exhibit impaired reproduction. We have explored both ecological and toxicological factors that would limit reproduction of bald eagles in the Great Lakes region. Based on our studies, the most critical factors influencing eagle populations are concentrations of environmental toxicants. While there might be some continuing effects of DDE, total PCBs and most importantly 2,3,7,8-tetrachlordibenzo-p dioxin equivalents (TCDD-EQ) in fishes from the Great Lakes and rivers open to spawning runs of anadromous fishes from the Great Lakes currently represent a significant hazard to bald eagles living along these shorelines or near these rivers and are most likely related to the impaired reproduction in bald eagles living there. PMID- 7556025 TI - Morphometric abnormalities in brains of great blue heron hatchlings exposed in the wild to PCDDs. AB - Great blue heron hatchlings from colonies in the Strait of Georgia, British Columbia, Canada are being monitored for environmental contaminant exposure and effects by the Canadian Wildlife Service. The contaminants of concern are polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), primarily derived from kraft pulp mill effluent. The levels of PCDDs and PCDFs in eggs from the most contaminated colonies peaked in 1988 and 1989 and dropped dramatically through 1990 to 1992. Brains of heron hatchlings (taken as eggs from the wild and hatched in the laboratory) were analyzed for gross morphological abnormalities. Brains from highly contaminated colonies (Crofton, British Columbia and University of British Columbia Endowment Lands) in 1988 exhibited a high frequency of intercerebral asymmetry. The frequency of this abnormality decreased in subsequent years as the levels of TCDD and TCDD-TEQs (toxic equivalence factors) decreased. The asymmetry was significantly correlated with the level of TCDD and TCDD-TEQs in eggs taken from the same nest. Yolk-free body weight negatively correlated and the brain somatic index positively correlated with the TCDD level in such pair-matched eggs. These results indicate that gross brain morphology, and specifically intercerebral asymmetry, may be useful as a biomarker for the developmental neurotoxic effects of PCDDs and related chemicals. PMID- 7556026 TI - Decreased lymphocyte responses in free-ranging bottlenose dolphins (Tursiops truncatus) are associated with increased concentrations of PCBs and DDT in peripheral blood. AB - Since 1987, large-scale mortalities of dolphins have been reported along the Atlantic coast of North America, in the Gulf of Mexico, and in the Mediterranean Sea. Autopsied bottlenose dolphins, Tursiops truncatus, which were collected from the large-scale mortality along the Atlantic coast in 1987 to 1988, exhibited opportunistic infections indicative of immune dysfunction. Further, these animals had high levels of chlorinated hydrocarbons, such as PCBs and DDT, that can suppress immune functions. The purpose of this study was to determine whether there is a relationship between chemical contaminant exposure and immune response in free-ranging dolphins. In June of 1991, peripheral blood was obtained from members of a bottlenose dolphin population that resides along the west coast of Florida. Peripheral blood lymphocyte responses to Concanavalin A (Con A) and phytohemagglutinin (PHA) were determined in vitro and compared by regression analysis with contaminant concentrations in whole blood from a small subset of these animals (n = 5). These data indicate that a reduced immune response in these bottlenose dolphins was correlated with increasing whole blood concentrations of several contaminants. Specifically, inverse correlations were found between Con A-induced lymphocyte proliferation and tetrachlorinated to octachlorinated biphenyls (r2 values ranged from 0.70 to 0.87). Con A-induced lymphocyte responses also correlated inversely with p,p'DDT (r2 values of 0.73 and 0.79); o.p'-DDE (r2 values of 0.93 and 0.96); and p,p'-DDE (r2 values of 0.73 and 0.81). PMID- 7556027 TI - Commentary on effects of anthropogenic and natural organic chemicals on development, swimming behavior, and reproduction of Daphnia, a key member of aquatic ecosystems. AB - Because of their trophodynamic role, small invertebrates are often critical components of ecosystems. An especially important group of freshwater invertebrates is the water fleas of the genus Daphnia. These animals are often the dominant herbivores in lakes and ponds. They play a key role in determining water clarity (by grazing on algae) and they are an important part of the diet of fish. Natural chemical signals (kairomones) produced by predators affect the development, life history strategy, and behavior of zooplankton. Laboratory studies of anthropogenic chemicals that have biological activity (xenobiotics), such as the insecticide carbaryl, have demonstrated effects of concentrations in the 1 to 5 ppb range on Daphnia development, growth rate, and swimming behavior in our laboratory experiments. Low concentrations of carbaryl inhibit growth and reproduction and delay maturation, whereas survivorship was not effected. These sublethal exposures to carbaryl reduced Daphnia population growth rate (productivity) by about 15% (at 5 ppb), enough to have significant ecological effects on the rest of the lake community. The insecticide carbaryl showed synergistic interactions with natural chemicals associated with predators (kairomones) that modify Daphnia development and life history characteristic. In addition, there were complex synergisms between carbaryl, the predator odors, and oxygen concentration (low oxygen concentration can be either a natural environmental stress or an anthropogenic stress). Daphnia produce males facultatively, usually in late fall; at other times, reproduction is asexual.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556030 TI - Defining the role of pollutants in the disruption of reproduction in wildlife. AB - Although chemical exposure has been associated with reduced reproduction in certain North American fish, reptiles, and mammals, definitive cause-and-effect data are lacking in many instances. Because the increasing use and global transport of industrial chemicals pose significant risk to successful reproduction, methods should be developed that can define the geographic extent and magnitude of injury and risk to wildlife. Because industrial chemicals are articles of commerce, information about injury to wildlife has been contentious and too often ineffective in changing societal behavior. The following strategies are advocated for inferring causal relationships. First, a balanced and comprehensive assessment of the data is necessary to determine the geographic extent of exposure and reproductive effects associated with environmental pollution. Initial efforts to document reproductive injury should focus on specific ecosystems in which detrimental effects have been observed, but lack sufficient causal data. Model systems (including experimental mesocosms or field ecosystems) should be identified or designed that can adequately test multigenerational reproductive effects. Mechanistic data from supportive laboratory studies on reproductive toxicity, quantitative structure-activity relationships, and bioaccumulation can be used to predict effects of related pollutants and to determine risk. Such information is essential to prevent future injury to wildlife and to prioritize the numerous remediation decisions facing our society. PMID- 7556028 TI - Possible mechanisms of action of environmental contaminants on St. Lawrence beluga whales (Delphinapterus leucas). AB - A small isolated population of beluga whales (Delphinapterus leucas) that are highly contaminated by pollutants, mostly of industrial origin, resides in the St. Lawrence estuary, Quebec, Canada. Overhunting in the first half of the century was the probable cause for this population to dwindle from several thousand animals to the current estimate of 500. The failure of the population to recover might be due to contamination by organochlorine compounds, which are known to lead to reproductive failure and immunosuppression in domestic and laboratory animals and seals. Functional and morphological changes have been demonstrated in thyroid gland and adrenal cortex in many species exposed to organochlorinated compounds, including seals. Morphological lesions, although different, were also found in belugas. Functional evaluation of thyroid and adrenal glands of contaminated (St. Lawrence) versus much less contaminated (Arctic) belugas is currently under way. Necropsy of St. Lawrence belugas showed numerous severe and disseminated infections with rather mildly pathogenic bacteria, which suggests immunosuppression. Organochlorine compounds and other contaminants found in beluga whales cause immunosuppression in a variety of animal species including seals. Thirty-seven percent of all the tumors reported in cetaceans were observed in St. Lawrence beluga whales. This could be explained by two different mechanisms: high exposure to environmental carcinogens and suppression of immunosurveillance against tumors. Overall, St. Lawrence belugas might well represent the risk associated with long-term exposure to pollutants present in their environment and might be a good model to predict health problems that could emerge in highly exposed human populations over time. PMID- 7556029 TI - Reproductive impairment in the Florida panther: nature or nurture? AB - Many of the remaining members of the endangered Florida panther (Felis concolor coryi) population suffer from one or more of a variety of physiological, reproductive, endocrine, and immune system defects including congenital heart defects, abnormal sperm, low sperm density, cryptorchidism, thyroid dysfunction, and possible immunosuppression. Mercury contamination, determined to be the cause of death of a female panther in 1989, was presented as the likely cause of thyroid dysfunction. As genetic diversity in the species was less than expected, all of the other abnormalities have been attributed to inbreeding. However, exposure to a variety of chemical compounds, especially those that have been identified as environmental endocrine disrupters (including mercury, p,p'-DDE, and polychlorinated biphenyls), has elicited all of the listed abnormalities in other species. A number of these contaminants are present in South Florida. An exposure pathway has been identified, and evidence presented in this paper, including the fact that there appears to be no significant difference between serum estradiol levels in males and females, suggests that many male panthers may have been demasculinized and feminized as a result of either prenatal or postnatal exposure. Thus, regardless of the effects of inbreeding, current evidence seems to indicate that environmental contaminants may be a major factor contributing to reproductive impairment in the Florida panther population. PMID- 7556031 TI - Tinkering with the tinkerer: pollution versus evolution. AB - Pollutants can act as powerful selective forces by altering genetic variability, its intergenerational transfer, and the size, functional viability, adaptability, and survival of future generations. It is at the level of the cell and the individual that meiosis occurs, that genetic diversity is maintained, and behavior, reproduction, growth, and survival occur and are regulated. It is at this level that evolutionary processes occur and most pollutants exert their toxic effects. Chronic exposure to chemicals contributes to the cumulative stress on individuals and disrupts physiological processes and chemically mediated communication thereby threatening the diversity and long-term survival of sexually reproducing biota. Regional or global effects of pollution on the atmosphere, hydrosphere, and lithosphere have indirectly altered Earth's life support systems, thereby modifying trace metal balance, reproduction, and incidence of UV-B-induced DNA damage in biota. By altering the competitive ability and survival of species, chemical pollutants potentially threaten evolutionary processes and the biodiversity and function of intercepting ecosystems. PMID- 7556032 TI - Resistance-skills training and onset of alcohol use: evidence for beneficial and potentially harmful effects in public schools and in private Catholic schools. AB - Recent research suggests that the success of social influence prevention programs is due to enhancing an adolescent's ability to resist passive social pressure (e.g., social modeling and overestimation of peer use), and is not due to teaching refusal skills for combating active social pressure (i.e., alcohol and drug offers). Using 4 waves of longitudinal data (collected in the 5th, 6th, 7th, and 8th grades) from 11,995 students participating in the Adolescent Alcohol Prevention Trial, resistance-skills training was found to be an effective strategy for preventing the onset of alcohol use when program assumptions were met. However, a counterproductive effect was found for adolescents attending public school who received a resistance training only condition. PMID- 7556033 TI - Nicotine withdrawal in chippers and regular smokers: subjective and cognitive effects. AB - From previous studies, chippers (very light, long-time cigarette smokers) seem not to be nicotine dependent, despite decades of smoking. The effect of tobacco deprivation on chippers' withdrawal reactions was examined. Matched groups of 26 chippers and 25 regular smokers were studied while abstaining or smoking for 2 day blocks, with assessments administered 5 times daily by palm-top computers. As hypothesized, chippers showed no changes as a result of nicotine deprivation. In contrast, regular smokers demonstrated distinct changes in craving, mood, arousal, and sleep disturbance. The computers also tested participants' cognitive performance. Unlike chippers, regular smokers' performance on complex tasks was slower under deprivation; the effect could not be explained by changes in motor performance or simple reaction time. Results suggest performance may have been improved by nicotine rather than by worsened by withdrawal. PMID- 7556034 TI - Are job characteristics related to fibrinogen levels in middle-aged women? AB - This study examined whether employment status or job characteristics thought to be stressful were related to fibrinogen level in a sample of 161 healthy middle aged women. Employed women had higher fibrinogen levels than did nonemployed women. Moreover, among employed women, those who perceived high levels of job stress or low support from their bosses had elevated fibrinogen, independent of menopausal status. Perception of low support from one's boss was related to higher fibrinogen levels only among premenopausal women or postmenopausal women who were not using hormone replacement therapy. These results are consistent with the notion that stress associated with some job characteristics influences levels of fibrinogen in women. PMID- 7556035 TI - Stress-induced hemoconcentration of blood cells and lipids in healthy women during acute psychological stress. AB - This study examined the effects of psychological stress on hemoconcentration in women. Hematologic and hemodynamic variables were assessed in 17 women before and after a 3-min speech task. Significant changes in hematocrit, hemoglobin levels, red and white blood cell (WBC) count, and calculated plasma volume occurred during psychological stress (all ps < .05). Significant increases were also observed for total cholesterol, triglycerides, high density lipoprotein cholesterol, low density lipoprotein cholesterol, and free fatty acid (FFA; all ps < .05) during stress. After statistically correcting for the hemoconcentration effects of decreased plasma volume during stress, only WBC count and FFA concentration remained significantly elevated during the stress task (p < .006 and p < .05, respectively). In sum, acute stress alters hemoconcentration in women, which in turn can account for most stress-induced changes in lipids. PMID- 7556036 TI - Injury risk factors in children with attention deficit hyperactivity disorder. AB - Processes related to injury in children with Attention Deficit Hyperactivity Disorder (ADHD) were examined. Two groups of 7-11-year-old boys (14 ADHD and 16 controls) watched a videotape simulating play activities in order for them to identify risky behaviors and then answered questions about risky scenes. Groups did not differ in ability to identify hazards, but children with ADHD anticipated less severe consequences following risky behavior and reported fewer active methods of preventing injury than did controls. Cognitive factors, including lower expectations of personal risk in hazardous situations and less ability to generate prevention strategies and safety rules, may contribute to increased injury liability in boys with ADHD. PMID- 7556037 TI - Relationships of self-esteem and efficacy to psychological distress in mothers of children with chronic physical illnesses. AB - This study examined relationships of children's illness-related functional limitations and 2 maternal psychological resources, self-esteem and efficacy, to symptoms of psychological distress in 365 urban mothers of 5- to 9-year-old children with diverse chronic illnesses. Multiple regression controlling for sociodemographic variables indicated that presence of functional limitations in the child and lower resources each were associated with higher maternal scores on a psychological symptom scale. Self-esteem had a main effect on maternal distress; however, a significant Efficacy x Functional Status interaction term suggested that mothers experienced greater distress when their children had illness-related functional limitations and maternal efficacy was low. Interventions aimed at enhancing maternal psychological resources may reduce the likelihood of distress in mothers of children with chronic illness. PMID- 7556038 TI - Association between daily coping and end-of-day mood. AB - Research indicates that self-reports of coping with stressful occurrences are associated with psychological and health outcomes. However, measures of coping may be biased by retrospective distortion as they assess coping over relatively long periods. In this study, a sample of 79 men completed a coping assessment daily for several weeks about the day's most "bothersome" problem. Repeated daily measurement of coping allowed analysis of within-subject effects of coping efforts. Same-day mood reported by the men (targets) and reports of the men's mood by their spouses (observers) were outcome variables. Within-subject analyses indicated that catharsis and social supports were associated with increased negative affect, whereas use of acceptance was associated with less negative affect. Use of distraction, acceptance, and relaxation were associated with increased positive affect. These findings held for target- and observer-reported mood. PMID- 7556039 TI - Effects of baseline responses, in-task feelings, and duration of activity on exercise-induced feeling states in women. AB - This study investigated the hypothesis that the effects of acute aerobic exercise on feeling states may be influenced by the objective dose of activity, subjective responses during exercise, and preexisting levels of feeling states. College-age women (N = 80) completed baseline measures and were then randomly assigned to 1 of 4 conditions: attention control for 10 min, or exercise for 10 min, 25 min, or 40 min. Levels of exertion and affect were assessed during exercise, and posttesting occurred 20 min following activity. Exercise enhanced revitalization in comparison with the control condition; however, this effect occurred only for participants scoring low to moderate on the pretest. In addition, in-task feeling states predicted postexercise revitalization even after we controlled for the treatment, the pretest, and the Pretest x Treatment interaction. PMID- 7556040 TI - Tumour necrosis factor, equine arthritis and clinical research. PMID- 7556041 TI - Twenty years of equine scintigraphy--a coming of age? PMID- 7556042 TI - 'No hoof no horse?'. PMID- 7556043 TI - Effects of prerace exercise, frusemide, sex and ambient temperature on blood sodium, bicarbonate and pH values in standardbred horses. AB - Analysis of data collected at racetracks showed that temperature, the diuretic drug, frusemide, exercise, temperature/exercise interaction and sex/age had significant (P < 0.05) effects on pH and bicarbonate ion concentration (P < 0.01). Sodium concentrations were significantly (P < 0.001) affected by temperature and frusemide. We suggest that the normal range limits for blood sodium, bicarbonate and pH used in prerace testing procedures should be adjusted for ambient temperature and for horses given frusemide and/or prerace exercise. These adjustments should improve the precision of detecting illegal alkalogenic agents and incidence of false positive and false negative test results. PMID- 7556044 TI - Hoof horn abnormalities in Lipizzaner horses and the effect of dietary biotin on macroscopic aspects of hoof horn quality. AB - This study involved a macroscopic evaluation of hoof quality in 152 Lipizzaner horses (130 from Austria and 22 from other countries) and a controlled double blind trial of the effects of biotin on hoof horn growth and quality over 19 months in 42 stallions from the Spanish Riding School (SRS) in Vienna. Using a grading system that incorporated evaluation of horn wall, white line, sole and frog, the macroscopic study revealed the following: 90% of the Austrian Lipizzaners had soft white lines and crumbling, fissured horn at the bearing border of the walls; 39% of the stallions of the SRS, > 4-years-old, had medium to severe hoof horn changes. Daily administration of 20 mg biotin to a test group of horses (n = 26) and a placebo to a control group (n = 16) showed that after 9 months the test group had significantly improved compared to the beginning of the trial and the placebo group (P < 0.01). In the test group, further improvement was observed during the following 5 months and, subsequently, the same good level of hoof condition was maintained over 3 further years of observation. Growth rate of the horn wall was equal in the biotin and placebo group, being 7 mm/28 days, giving a wall renewal period of 11 months. Mean plasma biotin level of untreated horses was 350 ng/l; plasma levels of biotin supplemented horses were > 1000 ng/l. It was concluded that continuous dietary supplementation with biotin at a daily dose of 20 mg is indicated to improve and maintain hoof horn quality in horses with less than optimum quality hoof. PMID- 7556045 TI - Histological and physical assessment of poor hoof horn quality in Lipizzaner horses and a therapeutic trial with biotin and a placebo. AB - This paper represents the second part of a study searching for factors which could be responsible for an inferior hoof horn quality of the Lipizzaner horses of the Viennese Spanish Riding School (SRS) noticed in the late 1980s. It includes an evaluation of a treatment with biotin and 42 Lipizzaner horses were tested in a double blind study. The following parameters were evaluated: 1) the histology of the hoof horn in samples from the bearing border 2) the tensile strength of the coronary horn in bearing border samples and 3) the influence of biotin upon the histological changes and the tensile strength after application of 20 mg/day of biotin during 38 months. The histological alterations of bearing border specimens were assessed by use of a grading system (Grade 0 = unchanged; Grade 1 = slight changes; Grade 2 = moderate changes; Grade 3 = severe changes). Initially more than two thirds of the horses showed moderate to severe changes: microcracks visible in the transition from the middle to the inner zone of the coronary horn; separation of the sole from the coronary horn in the region within the white zone. The tensile strength of the coronary horn, with a mean of 39 N/mm2, was 13 N/mm2 lower compared with the unchanged hooves of 10 Warmblood horses, which were investigated by Kung (1991). After 19 months of biotin treatment, the horn quality showed a small but significant improvement. This could be shown by comparing the histological tissue structure before and after treatment, as well as treated animals vs. a placebo group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556046 TI - Confidential enquiry of perioperative equine fatalities (CEPEF-1): preliminary results. AB - The Confidential Enquiry into Perioperative Equine Fatalities (CEPEF-1) is an observational multi-institutional prospective study of recovery outcome at 7 days post operatively, as called for by Steffey (1991). Data from 6,255 general anaesthetics (February 91-March 93) were submitted confidentially by 62 clinics. The outcomes of 333 cases which were subjected to euthanasia and which were not classified 'alive' or 'died' at 7 days, were excluded from the analysis. The remaining 5922 cases were analysed to identify risk ratios (RR) between survivors and nonsurvivors for a variety of factors. These preliminary results indicate an overall death rate, for equine patients dying or being subjected to euthanasia within 7 days of a general anaesthetic because of perioperative complications, of 102/6255 (1.6%). This mortality rate decreased to 46/5220 (0.9%) when all colic surgery and delivery of foals under general anaesthesia were excluded. There was an increased risk for mares in the last trimester of pregnancy (RR = 6.4). Patients undergoing emergency abdominal procedures (colic patients and pregnant mares undergoing controlled or caesarean section delivery of foals) under general anaesthesia were at increased risk (RR = 12.9) compared to ear, nose and throat surgery. Within orthopaedic surgery, patients requiring internal fixation were at increased risk (RR = 3.2) compared to those undergoing miscellaneous orthopaedic surgery. There was no difference in risk between breeds. Patients not placed in dorsal recumbency were at reduced risk (RR = 0.3-0.5) compared to those which were in dorsal recumbency.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556047 TI - Age related changes in biochemical markers of bone metabolism in horses. AB - Biochemical markers of bone metabolism were analysed in serum samples obtained from 60 horses with no history of orthopaedic disease (age 3 months-20 years). Serum levels of the carboxyterminal propeptide of type I procollagen (PICP), a marker of bone formation and the pyridinoline cross linked telopeptide domain of type I collagen (ICTP), a putative marker of bone resorption, were measured by radioimmunoassay (RIA). Serum levels of the bone specific isoenzyme of alkaline phosphatase (BALP), another marker of bone formation, were measured by a wheatgerm agglutinin affinity (WGA) method. Total alkaline phosphatase levels were also determined. Serum levels of PICP were significantly correlated with bone ALP (r = 0.78, P < 0.0001) and ICTP (r = 0.87, P < 0.0001). ICTP levels also correlated significantly with bone ALP (r = 0.81, P < 0.0001). However, total alkaline phosphatase did not correlate significantly with PICP, ICTP and BALP in horses over 1 year of age. There was an inverse correlation between serum levels of all biochemical markers and age of animals, with the most significant changes seen over the first 2 years. In animals less than 1 year of age, the reference ranges (mean +/- s.d. 1.96) were as follows: PICP 1216-2666 micrograms/l, ICTP 13.8-26.7 micrograms/l, bone ALP 134-288 u/l and total ALP 223-498 u/l. In 2-year olds, the equivalent reference ranges were: PICP 550-1472 micrograms/l, ICTP 7.96 22.8 micrograms/l, bone ALP 32.7-125 u/l and total ALP 134-238 u/l.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556049 TI - Variations in urinary gamma glutamyl transferase/urinary creatinine ratio in horses with or without pleuropneumonia treated with gentamicin. AB - The urinary GGT/urinary creatinine (uGGT/uCR) ratio was measured on Days 1, 3 and 10 in 4 adult, healthy horses; in 6 adult, healthy horses treated with gentamicin at recommended dosages and 9 adult horses treated for pleuropneumonia with gentamicin at recommended dosages. Plasma creatinine and gentamicin trough concentrations were measured on the same days. The uGGT/uCr ratio was higher in the normal horses (mean +/- s.d. 22.85 +/- 13.69) than previously reported normal values (10.5 +/- 6.8) (Adams and McClure 1985). Analysis of variance for repeated measures was used to compare the ratio in the 3 groups while controlling for the effect of time. Sick horses had a significantly higher uGGT/uCr ratio than either of the 2 groups of normal horses. Both groups of horses that were treated with gentamicin had similar percentage increases in uGGT/uCr ratio over the treatment period with the most marked increases found between treatment Days 1 and 3. The increase in uGGT/Cr ratio was predominantly a result of an increase in uGGT activity rather than a decrease in uCr concentration. The increase in uGGT activity and uGGT/uCr ratio occurred without abnormalities in serum creatinine or gentamicin trough concentrations. These findings demonstrate that urine GGT activity and uGGT/uCr ratio should be expected to increase in response to gentamicin therapy at recommended dosages without measurable changes in serum creatinine. This suggests that an elevation of the uGGT/uCr ratio in horses being treated with gentamicin would not necessarily require changes in, or withdrawal of, the gentamicin treatment as long as increases in the plasma creatinine do not exceed 0.3 g/l and gentamicin trough concentrations are < 2 micrograms/l. PMID- 7556048 TI - Induction of intra-articular tumour necrosis factor during acute inflammatory responses in equine arthritis. AB - Synovial fluid (SF) was collected at 2, 12 and 26 h post racing from 5 Thoroughbred horses (6 joints) with degenerative joint disease. The effects of serial arthrocentesis on SF TNF alpha levels were controlled for by testing, in parallel, site- and time-matched samples from clinically normal horses (i.e. without arthritis). A significant induction in TNF alpha bioactivity was detected in SF from arthritic joints (compared to the control joints) over the 26 h following racing. After subtraction of values for the arthrocentesis control SF, TNF alpha and protein levels and WBC and mononuclear cell numbers each peaked at 12 h in the SF from the degenerative joints, although there were no statistically significant correlations between any of these parameters. The presence in the SF of TNF alpha, as well as immunoreactive IL-1 beta and IL-6, was confirmed through use of specific anti-human cytokine IgG antibodies in neutralisation and slot blot radioimmunoassays. TNF beta was not detected in the SF by slot-blot radioimmunoassay. These results suggest that a significant increase in intra articular TNF alpha occurs during acute inflammatory arthritis in horses. The lack of correlation between infiltrating inflammatory cells and SF TNF alpha levels further suggests that the source of TNF alpha may be resident cells of the joint, as opposed to infiltrating cells found within the joint fluids. SF from clinically normal and arthritic joints of racing and hospitalised horses were also screened for bioactive TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556050 TI - Transection of the accessory ligament of the superficial digital flexor tendon for treatment of tendinitis: long term results in 61 standardbred racehorses (1985-1992). AB - Medical records and lifetime race results were obtained for 61 Standardbred racehorses treated consecutively from 1985 to 1992 with transection of the accessory ligament of the superficial digital flexor tendon (AL-SDF) for tendinitis of one or both forelimbs. A clinical diagnosis of tendinitis was confirmed in all cases by ultrasonographic examination. After surgery, 50 (82%) horses raced and 42 (69%) completed 5 or more starts. Eight horses (13%) failed to start and/or complete 5 or more races due to a recurrence of tendinitis. Six (10%) horses did not start and/or complete 5 or more starts for reasons unrelated to tendinitis and 5 (8%) horses were lost to follow-up. The median time between surgery and the first post operative start was 39 weeks. Fifty-seven per cent of those horses to start after surgery completed 20 or more starts and 40% raced on 40 or more occasions. Of the 42 horses to complete 5 or more races, the median number of post operative starts was 37 at time of follow-up. Thirty-two of 50 (64%) horses to start after surgery established or lowered lifetime racemarks. Thirty-six horses had raced prior to surgery. Thirty-one of 36 (86%) horses returned to racing after treatment of SDF tendinitis with accessory desmotomy (AD) and 27 horses (75%) completed 5 or more starts. Median earnings per start decreased 25% after surgery. Twenty-five horses had not raced prior to surgery. Nineteen (76%) horses in this group were age 2 years at time of injury.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556051 TI - Vascular phase scintigraphic evaluation of equine distal limb perfusion following trauma: 3 cases. PMID- 7556052 TI - Bilateral synovial chondromatosis of the metatarsophalangeal joints in a pony. PMID- 7556053 TI - Solution structure of porcine pancreatic phospholipase A2. AB - The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme. PMID- 7556054 TI - Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids. AB - The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins. PMID- 7556056 TI - Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus. AB - The sequence and crystal structure at 2.75 A resolution of the homodimeric glycyl tRNA synthetase from Thermus thermophilus, the first representative of the last unknown class II synthetase subgroup, have been determined. The three class II synthetase sequence motifs are present but the structure was essential for identification of motif 1, which does not possess the proline previously believed to be an essential class II invariant. Nevertheless, crucial contacts with the active site of the other monomer involving motif 1 are conserved and a more comprehensive description of class II now becomes possible. Each monomer consists of an active site strongly resembling that of the aspartyl and seryl enzymes, a C terminal anticodon recognition domain of 100 residues and a third domain unusually inserted between motifs 1 and 2 almost certainly interacting with the acceptor arm of tRNA(Gly). The C-terminal domain has a novel five-stranded parallel-antiparallel beta-sheet structure with three surrounding helices. The active site residues most probably responsible for substrate recognition, in particular in the Gly binding pocket, can be identified by inference from aspartyl-tRNA synthetase due to the conserved nature of the class II active site. PMID- 7556057 TI - The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype. AB - NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product. PMID- 7556055 TI - Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate. AB - The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. PMID- 7556060 TI - The molecular basis for the recognition of misfolded glycoproteins by the UDP Glc:glycoprotein glucosyltransferase. AB - The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N-acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N-acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase. PMID- 7556059 TI - Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae. AB - Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE-containing S. typhimurium expressed a YopE mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia. PMID- 7556058 TI - A novel iron uptake mechanism mediated by GPI-anchored human p97. AB - The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI) linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals. PMID- 7556061 TI - MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19. AB - Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface in a reversible fashion. Interaction specifically and directly occurs with the N-terminal presequence and is abolished after inactivation of either MOM22 or MOM19. Binding is salt sensitive, suggesting that recognition involves electrostatic forces between the positive charges of the presequence and the acidic cytosolic domain of MOM22. MOM19 and MOM22 can be cross-linked with high efficiency. We propose that the two proteins form a complex which functions as the presequence receptor at the mitochondrial surface and facilitates the movement of preproteins into the translocation pore. PMID- 7556063 TI - The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization. AB - Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions. PMID- 7556062 TI - tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin. AB - The mitochondrial genome of Trypanosoma brucei does not encode any identifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. In order to analyse the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episomally and their import into mitochondria assessed by purification and nuclease treatment of the mitochondrial fraction. Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA(Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosolic tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5'-flanking sequence of the trypanosomal tRNA(Tyr) gene. In all cases efficient import of each of the tRNAs was observed. We independently confirmed the mitochondrial import of the yeast tRNA(His), since in organello [alpha-32P]ATP-labelling of the 3'-end of the tRNA was inhibited by carboxyatractyloside, a highly specific inhibitor of the mitochondrial adenine nucleotide translocator. Import of heterologous tRNAs in their own genomic contexts supports the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T. brucei, but rather that the tRNA structure itself is sufficient to specify import. PMID- 7556065 TI - Telomere elongation in immortal human cells without detectable telomerase activity. AB - Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism. PMID- 7556064 TI - Granzyme A-deficient mice retain potent cell-mediated cytotoxicity. AB - Granzyme A, a granule-associated serine proteinase of activated cytotoxic T cells and natural killer cells, has been reported to play a critical role in DNA fragmentation of target cells. To address the question of the biological role of granzyme A, we have now generated a granzyme A-deficient mouse mutant by homologous recombination. Western blot analysis, enzyme assays and reverse transcription-PCR confirmed the absence of granzyme A in activated T cells. In addition, deletion of granzyme A does not alter the expression patterns of other granule components, such as granzymes B-G and perforin. Granzyme A-deficient mice are healthy and show normal hematopoietic development. Most notably, their in vitro- and ex vivo-derived cytotoxic T cells and natural killer cells are indistinguishable from those of normal mice in causing membrane disruption, apoptosis and DNA fragmentation in target cells. Furthermore, granzyme A deficient mice readily recover from both lymphocytic choriomeningitis virus and Listeria monocytogenes infections and eradicate syngeneic tumors with kinetics similar to the wild-type strain. These results demonstrate that granzyme A does not play a primary role in cell-mediated cytotoxicity, as has been assumed previously. PMID- 7556066 TI - Phosphorylation in halobacterial signal transduction. AB - Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. PMID- 7556068 TI - Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3. AB - Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies. PMID- 7556067 TI - ErbB3 and ErbB2/neu mediate the effect of heregulin on acetylcholine receptor gene expression in muscle: differential expression at the endplate. AB - Motor neurons modulate acetylcholine receptor (AChR) gene expression in skeletal muscle by two signalling pathways: the transmitter-evoked depolarization of muscle membrane inhibits AChR gene transcription throughout the myofibre presumably via activation of a serine/threonine kinase, while the transcription rates of AChR genes in the synaptic region are increased by nerve-derived trophic factors including AChR-inducing activity (ARIA). To gain further insight into these interactions we characterized the receptor for heregulin (HRG)/ARIA in muscle. We showed that HRG increases AChR alpha-subunit mRNA levels via tyrosine phosphorylation of ErbB3 and ErbB2/neu in myotubes. The protein tyrosine phosphatase inhibitor, pervanadate, potentiated the responses to HRG that were in turn blocked by the tyrosine kinase inhibitor erstatin, indicating the relevance of tyrosine phosphorylation to these events. The effects of HRG were inhibited by enhanced cellular serine/threonine phosphorylation which has been implicated in the repression of AChR genes by electrical activity. Immunocytochemical analysis of adult rat muscle revealed that while ErbB2/neu is present throughout the entire surface of the myofibre membrane, ErbB3 expression is exclusively restricted to the endplate suggesting its involvement in synapse-specific transcription of AChR genes by HRG/ARIA. PMID- 7556072 TI - The rat hepatic leukemia factor (HLF) gene encodes two transcriptional activators with distinct circadian rhythms, tissue distributions and target preferences. AB - Hepatic leukemia factor (HLF) is a member of the PAR family of transcription regulatory proteins. We have characterized the rat HLF gene and studied its expression and activity. The rat HLF gene is transcribed from two alternative promoters, alpha and beta, with different circadian amplitudes and tissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liver and kidney, like the previously described human HLF RNA. The beta RNA HLF isoforms use a CUG codon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminus relative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundant HLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudes and have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43 stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently than from the albumin promoter, while the converse is true for HLF36. PMID- 7556070 TI - Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. AB - In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases. PMID- 7556074 TI - RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes. AB - The ada and aidB genes are part of the adaptive response to DNA methylation damage in Escherichia coli. Transcription of the ada and the aidB genes is triggered by binding of the methylated Ada protein (meAda) to a specific sequence located 40-60 base pairs upstream of the transcriptional start, which is internal to an A/T-rich region. In this report we demonstrate that the Ada binding site is also a binding site for RNA polymerase. RNA polymerase is able to bind the -40 to -60 region of the ada and the aidB promoters in the absence of meAda, and its binding is mediated by the alpha subunit. This region resembles the UP element of the rrnB P1 promoter in location, sequence and mechanism of interaction with RNA polymerase. We discuss the function of UP-like elements in positively controlled promoters and provide evidence that Ada does not act by enhancing RNA polymerase binding affinity to the promoter region. Instead, Ada stimulates transcription by modifying the nature of the RNA polymerase-promoter interaction, allowing RNA polymerase to recognize the core promoter -35 and -10 elements in addition to the UP-like element. PMID- 7556069 TI - Activating point mutations in the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of beta subunit dimerization and cell type-specific molecules in signalling. AB - We have combined retroviral expression cloning with random mutagenesis to identify two activating point mutations in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 by virtue of their ability to confer factor independence on the haemopoietic cell line, FDC-P1. One mutation (V449E) is located within the transmembrane domain and, by analogy with a similar mutation in the neu oncogene, may act by inducing dimerization of h beta c. The other mutation (I374N) lies in the extracellular, membrane-proximal portion of h beta c. Neither of these mutants, nor a previously described mutant of h beta c (FI delta, which has a small duplication in the extracellular region), was capable of inducing factor independence in CTLL-2 cells, while only V449E could induce factor independence in BAF-B03 cells. These results imply that the extracellular and transmembrane mutations act by different mechanisms. Furthermore, they imply that the mutants, and hence also wild-type h beta c, interact with cell type-specific signalling molecules. Models are presented which illustrate how these mutations may act and predict some of the characteristics of the putative receptor-associated signalling molecules. PMID- 7556073 TI - CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells. AB - We have analysed the molecular basis for the function of the C/EBP alpha transactivation domain. We have previously found that the three C/EBP alpha transactivation elements (TEs) synergistically activate transcription in mammalian cells. We now report that two of these elements, TE-I and -II, co operatively mediate in vitro binding of C/EBP alpha to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP alpha coincide, and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and II in both yeast and mammalian cells. Our data demonstrate a biochemical basis for the modular buildup of transactivation domains, and indicate that this modularity is conserved in eukaryote evolution. We also show that the same amino acid motifs in a cellular activator can co-operate to mediate contacts between the activator and two distinct basal transcription factors. These results suggest that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related, and support the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process. PMID- 7556071 TI - The Polycomb-group gene, extra sex combs, encodes a nuclear member of the WD-40 repeat family. AB - We have delimited the extra sex combs (esc) gene to < 4 kb that include a single transcript and are able to rescue both the maternal and zygotic esc phenotypes. Several mutations have been identified within the esc transcript. In agreement with earlier genetic studies, esc is expressed maternally and its product is most abundant during the early embryonic stages. It encodes a protein of the WD-40 repeat family, which localizes predominantly to the nucleus. During germ band extension, it is expressed in a stereotypic pattern of neuroblasts. We propose a model in which Esc is recruited by gap proteins both to act as a corepressor that competes with the TAFII80 coactivator to block transcription and also to mediate the transition to permanent repression by Polycomb-group proteins. PMID- 7556076 TI - Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. AB - The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation. PMID- 7556075 TI - Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. AB - SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45. PMID- 7556078 TI - The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. AB - The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction. PMID- 7556077 TI - Identification of two KH domain proteins in the alpha-globin mRNP stability complex. AB - Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family. PMID- 7556079 TI - Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition. AB - The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double strand cleavage. PMID- 7556080 TI - A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20. PMID- 7556081 TI - The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C. AB - The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far. PMID- 7556082 TI - (Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima. AB - To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase. PMID- 7556083 TI - A mouse model for the cystic fibrosis delta F508 mutation. AB - Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. PMID- 7556085 TI - Glycosylinositol-phosphoceramide in the free-living protozoan Paramecium primaurelia: modification of core glycans by mannosyl phosphate. AB - Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1 6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide. PMID- 7556084 TI - A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery. AB - The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mutant, which has a Mal-phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG. PMID- 7556086 TI - dad-1, an endogenous programmed cell death suppressor in Caenorhabditis elegans and vertebrates. AB - Programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary or potentially harmful cells in multicellular organisms. Recent studies demonstrate that the molecular control of this process is conserved phylogenetically in animals. The dad-1 gene, which encodes a novel 113 amino acid protein, was originally identified in a mutant hamster cell line (tsBN7) that undergoes apoptosis at restrictive temperature. We have identified a dad-1 homologue in Caenorhabditis elegans (Ce-dad-1) whose predicted product is > 60% identical to vertebrate DAD-1. A search of the sequence databases indicated that DAD-1-like proteins are also expressed in two plant species. Expression of either human dad-1 or Ce-dad-1 under control of a C.elegans heat-shock-inducible promoter resulted in a reduction in the number of programmed cell death corpses visible in C.elegans embryos. Extra surviving cells were present in these animals, indicating that both the human and C.elegans dad-1 genes can suppress developmentally programmed cell death. Ce-dad-1 was found to rescue mutant tsBN7 hamster cells from apoptotic death as efficiently as the vertebrate genes. These results suggest that dad-1, which is necessary for cell survival in a mammalian cell line, is sufficient to suppress some programmed cell death in C.elegans. PMID- 7556087 TI - Negative feedback regulation of wild-type p53 biosynthesis. AB - When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage. PMID- 7556088 TI - Calmodulin binding to Drosophila NinaC required for termination of phototransduction. AB - The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin-binding proteins in the retina and the NinaC-calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo-transduction. In the current report, we present evidence for two calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, we generated transgenic flies expressing ninaC genes deleted for either C1 or C2. We found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction. PMID- 7556089 TI - Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells. AB - We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin. PMID- 7556092 TI - Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase. AB - Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5 phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell. PMID- 7556091 TI - Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons. AB - Neurotrophins promote neuronal survival by signalling through Trk receptor tyrosine kinases: nerve growth factor signals through TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)4 through TrkB and NT3 through TrkC. Although studies in some, but not all, cell lines indicate that NT3 can also signal through TrkA and TrkB, it is not known if such signalling can occur in neurons. We show that NT3 can promote the in vitro survival of sensory and sympathetic neurons isolated from embryos that are homozygous for a null mutation in the trkC gene. During the mid-embryonic period, NT3 promoted the survival of as many trigeminal and nodose neurons as the preferred neurotrophins, NGF and BDNF. However, later in development, these neurons lost their ability to respond to NT3. NT3 also promoted the survival of almost all sympathetic neurons, but no decrease in effectiveness was observed during development. Trigeminal neurons from trkC-/- trkA-/- embryos did not respond to NT3 and nodose neurons from trkB /- embryos likewise failed to respond to NT3. These results show that NT3 can signal through TrkA and TrkB in neurons at certain stages of development and may explain why the phenotype of NT3-/- mice is more severe than that of trkC-/- mice. PMID- 7556090 TI - Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. AB - We have examined the mechanism of signal transduction by the hemidesmosomal integrin alpha 6 beta 4, a laminin receptor involved in morphogenesis and tumor progression. Immunoprecipitation and immune complex kinase assays indicated that antibody- or laminin-induced ligation of alpha 6 beta 4 causes tyrosine phosphorylation of the beta 4 subunit in intact cells and that this event is mediated by a protein kinase(s) physically associated with the integrin. Co immunoprecipitation and GST fusion protein binding experiments showed that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit was found to be phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupted association of alpha 6 beta 4 with hemidesmosomes, but did not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. These results indicate that signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and that phosphorylation of distinct sites in the beta 4 tail mediates assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2. PMID- 7556093 TI - IgM receptor-mediated transactivation of the IgH 3' enhancer couples a novel Elf 1-AP-1 protein complex to the developmental control of enhancer function. AB - The function of the temporally regulated B lymphocyte-specific immunoglobulin heavy chain (IgH) 3' enhancer has been linked to the IgH class switch machinery, but the physiological mechanism(s) of activation has not been discerned. Following crosslinking of the IgM receptor, we demonstrate that the enhancer is transactivated in the B lymphoma cell line BAL-17. In both induced primary B lymphocytes and BAL-17 cells, the enhancer activation is concomitant with the recruitment of a novel DNA binding complex, nuclear factor of activated B cells (NFAB). NFAB contains the tissue-restricted Ets protein Elf-1 and the AP-1 factors Jun-B and c-Fos, which bind to a novel 3' enhancer ETS-AP-1 motif. IgM receptor-mediated activation or stimulation by phorbol-ester in BAL-17 cells demonstrates that the ETS-AP-1 motif, when linked to a heterologous gene, can confer a ligand/receptor-dependent response. In NIH 3T3 cells, Elf-1 expression is required for efficient ETS-AP-1 promoter activity in response to stimulation by 12-O-tetradecanylphorbol 13-acetate. Our results suggest a biological role for Elf-1 in the regulation of IgH gene expression, attribute a functional role for receptor-induced AP-1 proteins in B lymphocytes and provide evidence for a direct link between IgM receptor-mediated signalling and 3' enhancer activation. PMID- 7556094 TI - Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression. AB - The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element'; CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. We now show that CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation of all three genes. In addition, the cdc25C, cyclin A and cdc2 genes share an identical 5 bp region ('cell cycle genes homology region'; CHR) starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting showed the periodic occupation of the cyclin A CDE in the major groove, and of the CHR in the minor groove. Finally, all three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556095 TI - Repression and activation of transcription by Gal and Lac repressors: involvement of alpha subunit of RNA polymerase. AB - Gal or Lac repressor binding to an upstream DNA segment, in the absence of DNA looping, represses the P1 promoter located on the same face and activates the P2 promoter situated on the opposite face of the DNA helix in the gal operon. Both inhibition and stimulation of transcription requires the physical presence of the C-terminal domain of the alpha subunit of RNA polymerase although the latter is not required for transcription itself. We propose that Gal and Lac repressors inhibit or stimulate transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the C-terminal alpha domain to an unfavorable state at P1 or a more favorable state at P2, respectively. PMID- 7556097 TI - Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants. AB - In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation. PMID- 7556096 TI - Interaction of the sex-lethal RNA binding domains with RNA. AB - Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo. PMID- 7556098 TI - Selection of novel Mg(2+)-dependent self-cleaving ribozymes. AB - Four RNA motifs are known that catalyse site-specific cleavage in the presence of Mg2+ ions, all discovered in natural RNAs. In a single in vitro selection experiment we have isolated representatives of five novel classes of Mg(2+) dependent ribozymes. Small versions of three of these showed that a very simple internal loop type of secondary structure is responsible for the activity. One of these was synthesized in a bimolecular form, and compared directly with the hammerhead ribozyme; for the new ribozyme, the cleavage step of the reaction is much faster than the spontaneous rate of phosphodiester bond cleavage, yet substantially slower than that for the hammerhead. The results suggest that many more Mg(2+)-dependent self-cleaving RNA sequences can be found. PMID- 7556099 TI - Two group I ribozymes with different functions in a nuclear rDNA intron. AB - DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI. PMID- 7556100 TI - Activation of alpha-toxin translation in Staphylococcus aureus by the trans encoded antisense RNA, RNAIII. AB - The synthesis of virulence factors in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, encoded by the agr locus. Transcription of genes coding for secreted toxins and enzymes is stimulated, while transcription of cell surface protein genes is repressed by RNAIII. In the case of staphylococcal alpha toxin, RNAIII also seems to stimulate translation by an independent mechanism. In this report we show that in a mutant lacking RNAIII the rate of alpha-toxin (hla) production relative to the cellular concentration of hla mRNA was reduced 10-fold as compared with the wild-type strain. A 75% complementarity between the 5' end of RNAIII and the 5' untranslated region of the hla transcript suggests a direct interaction between the RNAs. A complex of RNAIII and hla mRNA was demonstrated in extracts of total RNA from the wild-type strain, and also with in vitro synthesized RNAs. Ribonuclease T1 digestion experiments revealed that the ribosome binding site of the hla transcript is blocked by intramolecular base pairing. Hybridization with RNAIII prevents this intramolecular base-pairing and makes the hla mRNA accessible for translation initiation. This is, to our knowledge, the first example of an 'antisense RNA' that stimulates translation of the target mRNA. PMID- 7556102 TI - The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae. AB - Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results. PMID- 7556101 TI - Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies. AB - We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif- directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA. PMID- 7556103 TI - The location and structure of double-strand DNA breaks induced during yeast meiosis: evidence for a covalently linked DNA-protein intermediate. AB - We have determined the precise location and structure of the double-strand DNA breaks (DSBs) formed during Saccharomyces cerevisiae meiosis. Breaks were examined at two recombination hot spots in both wild-type and rad50S mutant cells. At both loci, breaks occurred at multiple, irregularly spaced sites in a approximately 150 nucleotide interval contained within an area of nuclease hypersensitive chromatin. No consensus sequence could be discerned at or around break sites. Patterns of cleavage observed on individual strands indicated that breaks initially form with a two nucleotide 5' overhang. Broken strands from rad50S mutant cells contained tightly bound protein at their 5' ends. We suggest that, in S.cerevisiae, meiotic recombination is initiated by a DSB-forming activity that creates a covalently linked protein-DNA intermediate. PMID- 7556104 TI - A mutant T7 RNA polymerase as a DNA polymerase. AB - We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction. PMID- 7556105 TI - Human immunodeficiency virus type 1 reverse transcriptase tG:T mispair formation on RNA and DNA templates with mismatched primers: a kinetic and thermodynamic study. AB - The relationship between human immunodeficiency virus (HIV) type 1 reverse transcriptase tG:T mispair formation and base pair stability was investigated using DNA and RNA templates with 15 bp matched or mismatched DNA primers. tG:T mispair formation during primer elongation was undetectable on tDNA-DNA duplexes but occurred with a frequency of 10(-4) on matched tRNA-DNA duplexes. The frequency increased to 7.0 x 10(-4) and 1.3 x 10(-3) on tRNA-DNA duplexes with tG:T mismatches located 6 and 9 bp beyond the polymerization site. From Km values at 37 degrees C, the free energy change upon dissociation (delta G degrees 37) of the tG:T mispair increased from matched to mismatched tRNA-DNA duplexes by 0.36 1.21 kcal/mol. delta G degrees 37 for a correct tG:C pair decreased by 0.06-1.00 kcal/mol. In comparison with DNA-DNA duplexes, thermal melting measurements on RNA-DNA duplexes demonstrated smaller enthalpy (delta delta H degrees = -17.7 to 28.1 kcal/mol) and entropy (delta delta S degrees = -59.3 to -83.4 cal/mol/K) components. A strong entropy-enthalpy compensation resulted in small free energy differences (delta delta G degrees 37 = 0.8 to -2.2 kcal/mol). Thus, although DNA DNA and RNA-DNA duplexes are of comparable stability in solution, the RNA-DNA duplex presents more facile base pair opening and higher conformational flexibility. The release of helical strain at constant helix stability in RNA-DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G-->A hypermutation. PMID- 7556106 TI - The structural repertoire of the human V kappa domain. AB - In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa-J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed. PMID- 7556108 TI - Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation. AB - Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed. PMID- 7556107 TI - Induction of micronuclei and sister chromatid exchanges by polycyclic and N heterocyclic aromatic hydrocarbons in cultured human lymphocytes. AB - Many natural environments are contaminated with carcinogenic polycyclic aromatic hydrocarbons (PAHs) and N-heterocyclic aromatic hydrocarbons (NHAs) as complex mixtures of coal tar, petroleum, and shale oil. These potentially hazardous substances are prevalent at many former tar production and coal gasification sites. Three polycyclic [benzo(a)pyrene (BaP), benz(a)anthracene (BAA), and 7,12 dimethylbenz(a)anthracene (DMBA)] and two N-heterocyclic [7H dibenzo(c,g)carbazole (DBC), and dibenz(a,j)acridine (DBA)] aromatic hydrocarbons were analyzed for cytotoxic and genotoxic effects on human lymphocytes. All of these polyaromatic compounds are normally present in the environment, except for DMBA. Lymphocytes from healthy donors were isolated from whole blood. The 5-ring polycyclic aromatic BaP consistently induced micronuclei in a linear dose dependent manner with doses from 0.1-10.0 micrograms/ml, whereas the 4-ring compounds (BAA and DMBA) had no effect on the induction of micronuclei above controls except at 5 and 10 micrograms/ml. Of the two N-heterocyclic compounds, DBC produced a significant increase in micronuclei in lymphocytes, but the dose response tended to plateau above 0.1 microgram/ml. DBA showed an effect on the frequency of micronuclei above controls only at high doses of 5 and 10 micrograms/ml. The average background frequency of micronuclei for 7 lymphocyte donors averaged 3.1 per 1,000 stimulated cells, whereas the average frequency of micronuclei at 10 micrograms/ml BaP was 36.8 per 1,000 stimulated cells. The lowest effective dose in 2 donors for BaP occurred at 0.1 microgram/ml. At a challenge dose of 1 microgram/ml (4 microM) of BaP, considerable variation in micronuclei induction between 7 individuals was observed, ranging from 2-6-fold increases above spontaneous frequency. Over a dose range of 1-10.0 micrograms/ml (4-40 microM), BaP also induced sister chromatid exchanges (SCEs) in lymphocytes, whereas BAA had no effect above controls. Parallel studies of both cytogenetic endpoints showed that the micronucleus assay is a more sensitive indicator of BaP exposure at equivalent doses. Mitotic and replication indices of BaP-exposed lymphocytes showed that cell proliferation is only moderately inhibited even at the highest dose; this shows that bulky DNA-adducts are generally compatible with cell survival. The cytogenetic data are consistent, first-off, with reports that individuals in the population vary widely with respect to the inducibility of the CYP1A1 gene, which is known to be involved in polycyclic aromatic hydrocarbon metabolism, in particular, in BaP. Secondly, the data support the fact that polyaromatic compounds differ with regard to micronucleus induction within the same sample(s) of human lymphocytes, indicating selective metabolism of polyaromatic compounds that may reflect carcinogen sensitivity of the individual.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556110 TI - Analysis of mutations induced by replication of UV-damaged plasmid DNA in HeLa cell extracts. AB - We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C-->A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell. PMID- 7556111 TI - Improved sensitivity of the unscheduled DNA synthesis assay in primary rat hepatocytes following culture in serum-free defined media. AB - The unscheduled DNA synthesis (UDS) assay has been used extensively for the in vitro detection of DNA damage caused by compound exposure. However, the in vitro UDS assay has been insensitive for the detection of certain chemicals, particularly nitroaromatic compounds, that are positive in bacterial mutation assays. Recently, studies have been reported which describe alterations in the hepatocyte membrane following collagenase perfusion. Independently, a method for serum-free tissue culture has been developed which results in the up-regulation of cell surface receptors and which may restore membrane functions. Fourteen compounds, including seven nitroaromatics, were evaluated in the in vitro UDS assay employing a serum-free procedure. Five compounds that were previously reported positive in the standard in vitro UDS assay were also found positive using the serum-free method. In addition, five of the nitroaromatic compounds produced positive results with the serum-free method. 1-Methyl-3-nitro-1 nitrosoguanidine and 2-acetylaminofluorene, routinely used as positive controls in the UDS assay, showed greater activity in the serum-free assay. These results suggest that the use of serum-free media improves the sensitivity of the in vitro UDS assay and that the serum-free procedure potentially offers an effective alternative to the more labor intensive and more costly in vivo UDS assay. PMID- 7556109 TI - Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells. AB - The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. PMID- 7556113 TI - Genotoxic effects of p-aminophenol in Chinese hamster ovary and mouse lymphoma cells: results of a multiple endpoint test. AB - p-Aminophenol (PAP), a metabolite of aniline and acetaminophen, has been reported to be mutagenic in the L5178Y mouse lymphoma assay, but not in the CHO/HGPRT assay. In the present study, the effects of PAP in these two cells lines were examined to determine if the difference in activity is related to an intrinsic difference in the cell lines. CHO and L5178Y tk +/- mouse lymphoma cells were treated with PAP for 4 hr and assayed for 3 genetic endpoints: gene mutation at the HGPRT or TK locus, respectively; chromosomal aberrations at approximately 20 hr after initiation of treatment; and single-strand DNA breaks as detected by the single cell electrophoresis assay immediately after treatment. All treatments were conducted in the absence of S9. There was a dose-related, significant increase in TFT-resistant mouse lymphoma cells at dose levels that reduced survival to < or = 50% of concurrent controls. In CHO cells, however, there was no increase in thioguanine-resistant cells at dose levels that reduced cell survival to < 20%. These results are consistent with published reports on PAP. While the CHO cells were slightly more resistant to the toxic effects of PAP, the dose levels used in the two cell lines did not differ by more than 2-fold. At equivalent survival levels, PAP induced a significant (up to 20% aberrant cells) number of aberrations, primarily complex rearrangements, in both cell lines. In the single cell electrophoresis assay, there was a reproducible dose-related increase in cells with single-strand DNA breaks with both the L5178Y cells and the CHO cells. The induction of single-strand breaks and chromosome aberrations by PAP suggests that, mechanistically, PAP produces similar genetic damage in both CHO and L5178Y cell lines, but intrinsic differences between assay systems are responsible for the divergent gene mutation results. PMID- 7556114 TI - Genotoxicity of m-phenylenediamine and 2-aminofluorene in Salmonella typhimurium and human lymphocytes with and without plant activation. AB - The promutagenic arylamines, m-phenylenediamine (mPDA) and 2-aminofluorene (2 AF), were evaluated for their genotoxicity in Salmonella typhimurium strain YG1024 and in human lymphocytes. These agents were assayed with and without TX1MX plant activation mix. Both arylamines without activation were refractory in S. typhimurium, demonstrating that plant activation was required for the generation of their ultimate mutagenic metabolites. However, using the alkaline single-cell gel/Comet assay, both mPDA and 2-AF directly induced DNA damage in human lymphocytes. This effect was reduced when the human cells were treated with the arylamine plus TX1MX. mPDA with or without plant activation was not toxic to the exposed cells. However, at concentrations over 80 microM, 2-AF was toxic to lymphocytes. This toxic response was eliminated by incubation with TX1MX. mPDA and 2-AF were plant-activated into mutagens for S. typhimurium. However, these plant-activated products had a reduced genotoxic potency in human lymphocytes. PMID- 7556115 TI - Atrazine treatment potentiates excretion of mutagenic urine in 2,6-dinitrotoluene treated Fischer 344 rats. AB - Atrazine (ATZ), an s-triazine herbicide, is a widespread environmental contaminant. The hepatocarcinogenic component of technical grade dinitrotoluene, 2,6-dinitrotoluene (2,6-DNT, 19.5%), is a byproduct of trinitrotoluene synthesis and is found at production sites. This study explores the effect of ATZ treatment on the bioactivation of the promutagen, 2,6-DNT. Male Fischer 344 rats (5 weeks old) were administered 50 mg/kg of ATZ by gavage for 5 weeks. At 1, 3, and 5 weeks, both DMSO-control and ATZ-pretreated rats were treated p.o. with 75 mg/kg of 2,6-DNT and were housed in metabolism cages for urine collection. Sulfatase- and beta-glucuronidase-treated, concentrated urine was bioassayed for urinary mutagens in a microsuspension modification of the Salmonella assay with and without metabolic activation. No significant change in mutagen excretion was observed in ATZ-treated rats; however, an elevation in direct-acting urine mutagens from rats receiving ATZ and 2,6-DNT at weeks 1 (359 +/- 68 vs. 621 +/- 96 revertants/ml) and 5 (278 +/- 46 vs. 667 +/- 109 revertants/ml) of treatment was observed. The increase in production of urinary mutagens was accompanied by an elevation in small intestinal nitroreductase activity. Increases in large intestinal nitroreductase and beta-glucuronidase were observed after 5 weeks. There was no apparent effect of ATZ following 5 weeks of treatment on the production of 2,6-DNT-derived hepatic DNA adducts. ATZ treatment modifies intestinal enzymes responsible for promutagen bioactivation, and potentiates the excretion of mutagenic urine in 2,6-DNT-treated animals. PMID- 7556112 TI - Relationships among in vitro mutagenicity assays: quantitative vs. qualitative test results. AB - In previous investigations, we studied the relationships between the profiles of the qualitative responses of in vitro short-term tests (mutation in Salmonella typhimurium, chromosomal aberrations in CHO cells, sister chromatid exchanges in CHO cells, and mutation in mouse lymphoma cells) and common sets of chemicals. In this paper, we address the study of the quantitative responses (potency). We show that two analyses point to similar patterns of relationships: the mutation in mouse lymphoma cells assay is most similar to the CHO sister chromatid exchange assay, and the Salmonella assay is most similar to the CHO chromosomal aberrations assay. PMID- 7556116 TI - Clastogenic effects of 1,3-butadiene and its metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane in splenocytes and germ cells of rats and mice in vivo. AB - Clastogenicity of 1,3-butadiene (BD), 1,2-epoxybutene (EB), and 1,2,3,4 diepoxybutane (DEB) was studied in splenocytes and germ cells of rats and mice by means of micronucleus assays (cytokinesis-block method for splenocytes, suspension method for germ cells). Inhalation exposure of mice to 200, 500, or 1,300 ppm BD (6 h/d; 5 days) induced significant chromosome damage in spermatocytes at the preleptotene stage. EB and DEB induced significant amounts of clastogenic damage in splenocytes and spermatocytes of rats and mice. The lowest tested effective doses for mice and rats were, respectively, 40 and 80 mg/kg for EB, and 15 and 30 mg/kg for DEB. In splenocytes, 80 mg EB/kg induced 3.6 times more MN in mice than in rats, whereas 30 mg DEB/kg induced the same amount of damage in both species. Damage in germ cells of mice was induced in early spermatocytes treated with 40 and 80 mg EB/kg, and in late spermatocytes exposed to 30 mg DEB/kg. In rats, 40 mg EB/kg induced damage in early spermatocytes, whereas 80 mg EB/kg induced chromosomal damage in early and late spermatocytes. In rats treated with DEB, clastogenic damage was induced in spermatocytes at preleptotene, zygotene, diplotene, and diakinesis stages. When the clastogenic potential of EB and DEB in splenocytes and germ cells of mice and rats was compared, DEB always showed a stronger effect than EB. Body weight, testis weight, ratio of testis weight to body weight, and ratio of Golgi to Golgi + cap phase spermatids were used as parameters for toxicity. Exposures to 500 and 1,300 ppm BD were somewhat toxic to mice. Doses of 80 mg EB/kg and 30 mg DEB/kg exhibited toxic effects in mice and rats. PMID- 7556117 TI - Acute mountain sickness relates to sea-level partial pressure of oxygen. AB - The aim of this study was to clarify the relationships between acute mountain sickness (AMS), studied during an expedition in the Andes, and some physiological parameters determined before the expedition, i.e. biometrical characteristics of the subjects [maximal oxygen consumption (VO2max), body fat content, body mass index], functional pulmonary tests (forced vital capacity, forced expiratory volume at the first second), ventilatory or cardiac responses measured at 4,500 m [hypoxic ventilatory responses (HVR) 4,500 and hypoxic cardiac responses (HCR) 4,500, respectively), cold pressor responses. To achieve this objective, 11 subjects were firstly submitted to a hypobaric poikilocapnic hypoxic test (589 hPa, 4,500 m) at rest and during exercise to study minute volume, respiratory frequency, end tidal partial pressure of O2 (PETO2) and CO2, HVR 4,500, HCR 4,500 and to a cold pressor test of the hand (5 min in 5 degrees C cold water) to study heart rate, blood pressure and skin temperature changes. The AMS was assessed daily by questionnaire during a 12-day expedition in the Andes following both Hackett's method and Environmental Symptoms Questionnaire (modified ESQ II). Maximal AMS-Hackett score, maximal AMS-ESQ score and mean AMS-ESQ score were defined. The quantifications of AMS following the two methods were correlated. No significant relationships were observed between mean AMS-ESQ score and the biometrical characteristics of the subjects, the functional pulmonary tests, HVR 4,500, HCR 4,500 or the cold pressor responses. However, it appeared that the mean AMS-ESQ score was correlated with PETO2 measured at rest and during exercise (50% VO2max) both in hypoxia and normoxia. A closer linear relationship was observed during the exercise in normoxia (r = -0.92, P < 0.0001). These results could suggest that AMS was related to a relative alveolar hypoventilation more in relation to breathing pattern than HVR. PMID- 7556118 TI - New evidence from magnetic resonance imaging of brain changes after climbs at extreme altitude. AB - The aim of the present study was to look for anatomical changes in climbers' brains, using magnetic resonance imaging (MRI), after extremely high-altitude climbs and to relate them to possible associated risk factors. Clinical history, neurological examinations and MRI were carried out on a group of nine climbers before and after climbing to over 7500 m without the use of supplementary oxygen. None of the subjects showed any neurological dysfunctions. In five climbers MRI abnormalities (high signal areas, cortical atrophy) were observed before the expedition. After the descent, two of them showed new high intensity signal areas recorded by MRI. Both subjects suffered severe neurological symptoms during the climb. The present study suggested that the brain changes observed by MRI could be related to the severity of clinical events at high altitude. However, we do not know the exact meaning of such MRI findings or the reason for their location, predominantly in posterior regions of the brain. The new evidence that a high percentage of climbers show MRI brain abnormalities, and especially the appearance of changes after the ascent, reinforces the possibility of a potential neurological risk in high-altitude climbing. PMID- 7556120 TI - Changes in skeletal muscle oxygenation during incremental exercise measured with near infrared spectroscopy. AB - To determine the change in muscle oxygenation in response to progressively increasing work rate exercise, muscle oxyhemoglobin + oxymyoglobin saturation was measured transcutaneously with near infrared spectroscopy in the vastus lateralis muscle during cycle ergometry. Studies were done in 11 subjects while gas exchange was measured breath-by-breath. As work rate was increased, tissue oxygenation initially either remained constant near resting levels or, more usually, decreased. Near the work rate and metabolic rate where significant lactic acidosis was detected by excess CO2 production (lactic acidosis threshold, LAT), muscle oxygenation decreased more steeply. As maximum oxygen uptake (VO2max) was approached, the rate of desaturation slowed. In 8 of the 11 subjects, tissue O2 saturation reached a minimum which was sustained for 1-3 min before VO2max was reached. The LAT correlated with both the VO2 (r = 0.95, P < 0.0001) and the work rate (r = 0.94, P < 0.0001) at which the rate of tissue O2 desaturation accelerated. These results describe a consistent pattern in the rate of decrease in muscle oxygenation, slowly decreasing over the lower work rate range, decreasing more rapidly in the work rate range of the LAT and then slowing at about 80% of VO2max, approaching or reaching a minimum saturation at VO2max. PMID- 7556119 TI - Are oxygen uptake kinetics at the onset of exercise speeded up by local metabolic status in active muscles? AB - To assess the rate-limiting factor of oxygen uptake (VO2) kinetics at the onset of exercise, six healthy male sedentary subjects performed repeated one-legged constant-load cycle exercise. The one-legged constant-load exercise test consisted of two 5-min periods of pedalling at an exercise intensity of 50 W, with a 5-min rest between periods (these exercise periods, i.e. first and second exercises, were performed by the same leg). The exercise was then repeated using the other leg. In addition, two-legged incremental exercise was investigated to establish whether VO2 kinetics were affected by slower heart rate kinetics. The incremental exercise test consisted of two-legged pedalling first with the cycle unloaded as a warm-up for 5 min followed by 50-W exercise for 5 min. The exercise intensity was then increased to 100 W for 5 min. During exercise, gas exchange parameters were determined by the breath-by-breath method and cardiac output (Qc) was determined continuously by an impedance technique with a computer-based automated system. To determine the kinetics of heart rate (HR), Qc, and VO2, a best fit procedure was employed using least-squares criteria with a time delay, except during the initial increase. During the one-legged constant-load exercise test, VO2 kinetics were significantly accelerated by repeated exercise using the same leg. On the other hand, when the exercise was changed to the other leg, VO2 kinetics were significantly slower, although Qc kinetics continued to be faster. During the incremental exercise test, although the HR response was slower at the transition from 50-W to 100-W exercise than at the transition from warm-up to 50 W exercise, there were no significant changes in VO2 kinetics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556123 TI - Mechanical step variability during treadmill running. AB - The present study was designed to study intra-individual step variability measured both on vertical displacement of the body (delta Z) and on step time (delta t) parameters by means of a kinematic arm and during treadmill running. A group of 17 subjects ran successively at 60%, 80%, 100% and 140% of their maximal aerobic velocity (Vamax). The total number of steps analysed was 6116. The absolute delta Z step variability (sigma delta Z) ranged between 5 mm and 21 mm while the absolute delta t variability (sigma delta t) ranged between 6 ms and 40 ms. Step variabilities were due to step asymmetry (from 38.5% to 48.5% of the step variability) and to stride variability. For submaximal velocities (60%, 80%, and 100% Vamax) both sigma delta t and sigma delta Z were independent of velocity or body dimensions whereas differences between subjects were significant (P < 0.01) for sigma delta Z. On the other hand, variabilities were significantly increased when velocity was changed from submaximal to the 140% Vamax level. Furthermore, at submaximal levels sigma delta Z was linked to the subject's energy cost of running (P < 0.05). Therefore, the intra-individual step variability should not be neglected in future studies on mechanical efficiency of running and it is suggested that, to obtain a good accuracy (better than 1%, P < 0.05) on mean value and variability of the mechanical parameters, measurements should be performed on at least 32-64 consecutive steps, which corresponds to about 15 to 20s of running. PMID- 7556121 TI - Motor unit recruitment strategy of knee antagonist muscles in a step-wise, increasing isometric contraction. AB - The purpose of this study was to determine if differences exist between the control strategies of two antagonist thigh muscles during knee flexion and extension muscular coactivation. Surface myoelectric signal (MES) of the quadriceps (rectus femoris) and the hamstrings (semitendinosus) were obtained from both muscles while performing step-wise increasing contractions during flexion and extension with the knee at 1.57 rad of flexion (90 degrees). The median frequency of the power density spectrum, which is related to the average muscle fiber action potential conduction velocity and therefore to motor unit recruitment, was calculated from each MES. The results suggest that, in all the subjects tested, when the muscle acts as antagonist most motor units are recruited up to 50% of the maximal voluntary force, whereas when the muscle acts as antagonist motor units are recruited up to 40% of the maximal voluntary force. The force range past 40-50% of the maximal force is also characterized by differences between the agonist/antagonist. PMID- 7556122 TI - The haematological, biochemical and immunological profile of athletes suffering from the overtraining syndrome. AB - To help clarify the overtraining syndrome (OTS), a combination of parameters were measured in ten athletes who were suffering from OTS. Blood samples were obtained at rest and a range of haematological, biochemical and immunological tests were carried out on the samples. For each parameter, the mean value for the group was compared to an established normal range amongst age-matched controls. The subjects were also asked to complete a questionnaire to establish the severity of their condition. The data indicated that the debilitating fatigue experienced by the OTS sufferers was not related to any of the blood parameters traditionally associated with chronic exercise stress, since levels were normal in OTS. The only parameter measured which deviated significantly from the normal range for both the sedentary controls and the athletes was the plasma concentration of glutamine. Although the data in this study would suggest that plasma glutamine concentrations represented an objective, measurable difference between OTS subjects and normal controls, it remains to be shown that there is any correlation between glutamine concentrations and other clinical symptoms of OTS such as physical capability. PMID- 7556125 TI - Effects of chronic low frequency stimulation on structural and metabolic properties of hindlimb suspended rat soleus muscle. AB - The use of chronic low frequency stimulations (CLFS, 10 Hz bipolar current 8 h.day-1) as a countermeasure against unweighting-induced muscle alterations was investigated in rat soleus muscle during 21 days of hindlimb suspension (HS). It was shown that CLFS was able to minimize the soleus muscle atrophy induced by suspension (-29% in stimulated muscles compared to -56% in the non-stimulated soleus muscle). In parallel, CLFS partly prevented the HS-induced decreases in the cross-sectional area of type I fibres and in the total and myofibril protein contents. Stimulation at low frequency reduced the increase in the fast-myosin expression recorded with unweighting. Moreover, the HS-induced increase in glycolytic capacity was counteracted to a considerable extent by CLFS. In conclusion, the results of this study showed that CLFS can only partly prevent the HS-induced modifications in the soleus muscle. However, the limited effectiveness of CLFS to prevent muscle atrophy emphasized the critical role of reduced load bearing in the induction of soleus muscle atrophy. PMID- 7556126 TI - Effect of exercise and adrenal insufficiency on urea production in rats. AB - Experiments on Wistar rats were designed to study the effect of exercise on urea production in the liver of intact and adrenalectomized rats. The urea production rate was assessed by the 14C-urea content in liver tissue after administration of NaH14CO3. In intact rats swimming caused increases in 14C-urea content in the liver compared to the resting concentrations in intact control rats: by 45% after 30 min of swimming carrying an additional load of 10% body mass by, 35% after 3 h of swimming without an additional load and by 103% after 10 h of swimming. Concentrations of urea in liver and blood were elevated simultaneously. The specific activity of 14C-urea did not change significantly as a result of the exercise performed. In adrenalectomized rats the basal rate of urea production was reduced by an insignificant amount, but swimming for 3 h resulted in a decrease in liver 14C-urea (by 24%). The results confirmed the exercise-induced increase in urea production and indicated as essential role for adrenal hormones in this response. PMID- 7556124 TI - Neuromuscular performance in voluntary bilateral and unilateral contraction and during electrical stimulation in men at different ages. AB - A group of 33 men divided into three different age groups, M30 years (n = 11), M50 years (n = 12) and M70 years (n = 10) volunteered as subjects for examination of their maximal voluntary isometric bilateral and unilateral forces and force time curves of the knee extensor muscle group as well as electromyogram activity of the vastus lateralis, vastus medialis and rectus femoris muscles of the right and/or left leg contractions. Electrical stimulation (ES) of 50 Hz was also given by two surface tin electrodes for each subject and each leg separately with the maximal tolerable intensity for recording the isometric force evoked. The maximal force produced by the voluntary isometric unilateral knee extension combined with ES was also measured. Maximal voluntary bilateral force of 1142 (SD 82) N in M30 was greater (P < 0.001) and the force of 1094 (SD 228) N in M50 was also greater (P < 0.05) than that of 962 (SD 70) N recorded for M70. The shapes of the isometric force-time curves, especially in absolute values, differed also among the groups so that the force produced during the early positions of the curve were in M30 greater (P < 0.05-0.001) than the force produced M50 and in M70. Neither the maximal voluntary bilateral force per the summed unilateral force nor the average integrated EMG between the bilateral and unilateral conditions differed significantly from each other either in M30, M50 or in M70. The force produced by pure ES was significantly greater in M30 (P < 0.05) than in M50 and M70, but the latter two groups were not significantly different. When ES was combined with the voluntary contractions, the absolute force values declined (P < 0.05-0.001) with increasing age similarly to those forces produced by the voluntary contractions alone. The present results suggest that increasing age results in great decreases both in the maximal voluntary strength and explosive force characteristics of the neuromuscular system but no bilateral deficit may necessarily be observed either in neural activation or in force production in a simple single joint isometric force production task of the knee extensors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556129 TI - Effect of arm cranking exercise on skin blood flow of lower limb in people with injuries to the spinal cord. AB - The purpose of this study was to examine whether arm cranking exercise induces changes in skin blood flow in the paralyzed lower limbs of people with injuries to the spinal cord (PISC). Ten PISC with lesions located between Th5 and L5 and six control subjects performed arm cranking exercise for 6 min at three intensities, 10, 30 and 50 W, at a room temperature of 25 degrees C. Oxygen uptake (Vo2) and heart rate (HR) were measured for the last 2 min of each exercise period. The skin blood flow at the anterior thigh (BFsk,t) was continuously monitored using laser Doppler flowmetry for the whole 6-min period and for the first 10 min of recovery following exercise. During exercise, the PISC showed lower Vo2 and greater HR than the control subjects. No increase in BFsk,t was found in six of the PISC with lesions at or above Th12, irrespective of the exercise intensity. On the other hand, in PISC with lesions at L1 or below, BFsk,t increased significantly (P < 0.05) with an increase in Vo2 and HR, although the BFsk,t at a given Vo2 and HR was lower than that in the control subjects. These results would suggest that arm exercise can promote the blood circulation in the skin of the lower limbs if the injury level is below L1. PMID- 7556127 TI - Anaerobic threshold in children: determination from saliva analysis in field tests. AB - The purpose of this study was to determine the anaerobic threshold of children by the analysis of saliva collected during field tests. A group of 25 children (mean age, 10.5 years) performed an incremental exercise test on a track, consisting of 4-min stages at increasing running velocities. Before each test (at rest) and at the end of each stage, both blood (via finger pricks) and saliva samples (for measurement of salivary concentrations of Na+ and Cl-) were collected to determine lactate threshold (Thla-) and saliva threshold (Thsa), respectively. There were no significant differences between values of Thla- and Thsa when expressed either as running velocity [mean Thla-, 10.73 (SD 1.96) km.h-1; mean Thsa, 10.89 (SD 1.69) km.h-1)] or heart rate [Thla-, 182(SD 14) beats. min-1 Thsa 183 (SD 11) beats.min-1]. In addition, correlations between Thsa and Thla were high, when both values were expressed as running velocity in kilometres per hour (r = 0.89; P < 0.001), or heart rate in beats per minute (r = 0.90; p < 0.001). In conclusion, these findings suggested that saliva analysis would be a valid method for anaerobic threshold determination in field tests. PMID- 7556131 TI - Physical conditioning in rats influences the central and peripheral catecholamine responses to sustained exercise. AB - We have investigated the effect of treadmill running in rats (25m.min-1 using a 3% gradient; for 1 h or 2 h) on the cortical extracellular concentrations of noradrenaline (NA) and its main metabolites-3,4-dihydroxyphenylglycol and 3 methoxy-4-hydroxyphenylglycol- and the plasma adrenaline (A) and NA concentrations in relation to prior physical conditioning (1 or 2-h running.day-1 for 12 days). Cortical microdialysates and peripheral blood were collected during 1-h resting, 1-h or 2-h running and for 1 h after exercise. Catecholamines and their metabolites were quantitated using high performance liquid chromatography with electrochemical detection. Treadmill running stimulated concomitantly peripheral catecholamine secretion and central noradrenergic activity, i.e. NA turnover and release. The effect extended into the recovery period even more as the duration of the run increased. Prior physical conditioning greatly influenced the central and peripheral catecholamine responses: the 1-h trained rats experienced the 2-h run as a stressful new event eliciting great long-lasting catecholamine responses, whereas the 2-h trained rats exhibited a progressive sustained catecholamine increase with an earlier onset of the central NA release. The data are discussed in relation to the psychological and intellectual effects of exercise and physical fitness in humans. In addition, the positive correlation found between the central noradrenergic activation and peripheral A secretion confirmed and extended our previous observations in exercising men and gave support to the hypothesis that the elevation of circulating A can be a relevant factor mediating--directly or indirectly--the exercise-induced central effects. PMID- 7556132 TI - Effect of short-term strenuous exercise on erythrocyte 2,3-diphosphoglycerate in untrained men: a time-course study. AB - The literature on the response of erythrocyte 2,3-diphosphoglycerate (2,3-DPG) following exercise is replete with inconsistencies, and recent studies have shown that the time of blood sampling during and following exercise, as well as the duration of exercise, are important in evaluating the response of 2,3-DPG. Experiments were designed to measure the response of 2,3-DPG following short-term strenuous exercise in two groups of untrained men. Twelve men, 19-22 years old (study 1), exercised on a bicycle ergometer at 122.5 W for 10 min and red blood cell (RBC) 2,3-DPG was measured at 0 and 50 min following exercise. The level of 2,3-DPG (mumol.ml-1 RBC) increased after exercise (P < 0.05), but this increase was not significant when 2,3-DPG was expressed as mol.mol-1 hemoglobin (Hb). However, following 50 min of rest, 2,3-DPG (mol.mol-1 Hb) decreased significantly. In a second group (study 2), nine other men, aged 18-19 years, exercised at the same workload for 15 min and 2,3-DPG was measured at 0, 30, 60, 180, and 330 min respectively after exercise, and no significant mean changes in the level of the phosphate were observed. Findings from these studies suggest that 2,3-DPG does not provide a compensatory adjustment to facilitate oxygen delivery in the hypoxia of short-term strenuous exercise in untrained males immediately following exercise and when recovery intervals of up to 330 min are also examined. It is suggested that 2,3-DPG be reported as mol.mol-1 Hb, since the phosphate exists on Hb in an equimolar ratio in normal physiological states. PMID- 7556130 TI - Cutaneous vasodilatation responses synchronize with sweat expulsions. AB - To examine whether cutaneous active vasodilatation is mediated by sudomotor nerve fibres we recorded cutaneous blood flow and sweat rates continuously with laser Doppler flowmetry and capacitance hygrometry, respectively, from the dorsal and plantar aspects of the foot in 11 male subjects at varying ambient temperatures (Ta) between 22 and 40 degrees C (relative humidity 40%). In a warmer environment (Ta 29-40 degrees C), predominant responses of the blood flow curve from the sole of the foot were transient depressions (negative blood flow responses, NBR), whereas those from the dorsal foot were transient increases (positive blood flow responses, PBR). The PBR on the dorsal foot occurred spontaneously or in response to mental or sensory stimuli, and when PBR did not fuse with each other the rate of PBR was linearly related to tympanic temperature. When dorsal foot sweating was continuous, PBR on the dorsal foot almost entirely synchronized with sweat expulsion. When dorsal foot sweating was intermittent PBR sometimes occurred on the dorsal foot without corresponding sweat expulsions, but these PBR showed a complete correspondence with subthreshold sweat expulsion seen on a methacholine treated area. The amplitude and the duration of PBR showed a significant linear relationship with the amplitude and the duration of the corresponding sweat expulsion. In a thermoneutral or cooler environment (Ta 22-29 degrees C), PBR occurred on the sole of the foot when mental or sensory stimuli elicited sweating in that area. Thus, PBR occurred when and where sweating appeared. Atropine failed to abolish PBR on the dorsal foot. Blockade of the peroneal nerve eliminated both PBR and NBR on the dorsal foot.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556133 TI - A simplified procedure of direct calorimetry for bedside monitoring of the resting metabolic rate. AB - A simplified procedure of direct calorimetry (SPDC) for determination of resting metabolic rate of respiratory uncompromised subjects in a supine position is presented. This procedure was based on computer-assisted measurements of heat losses due to evaporation, radiation, conduction, and convection. The subject's total loss of mass was recorded hydraulically with a beam scale and afterwards transformed into a digital electric signal. Differences between dry bulb temperature and mean skin temperature were measured by semiconductor thermistors and also transformed into digital signals. With special software an interfaced personal computer assisted in performing SPDC and in calculating heat losses due to evaporation, radiation, and conduction. In a thermoneutral environment, six healthy volunteers were investigated to determine the mean convective heat transfer coefficient (hc) from the difference in an individual between the metabolic energy transformation (M) measured by indirect calorimetry (IC) and the sum of heat losses by radiation, conduction, and evaporation. The room-specific value of hc of 2.12 (SD 0.22) W.m-2.degrees C-1 was in good agreement with data in the literature. Compared to the results of M from a second series of IC, the total heat loss (THL) measured by SPDC in a thermoneutral environment was calculated as 100.5 (SD 6.0)%. The THL by SPDC performed three times at 3-h intervals on ten other volunteers revealed a mean difference of 0.22 (SD 1.74) W.m-2. Thus, SPDC would seem to be a valid and reproducible method under conditions of thermal neutrality. PMID- 7556134 TI - An aid to the determination of the ventilatory threshold. AB - Detection of the ventilatory threshold during an incremental load exercise test by eye can be difficult. Although various alternative methods employing information other than the ventilation can be used to assist in determining the ventilatory threshold, they rely on underlying assumptions about the physiological basis for the ventilatory threshold. The method presented here (CUSUM) uses only the ventilation data, and therefore avoids such assumptions. Twelve subjects performed a total of 47 incremental exercise tests to exhaustion. Determinations of the ventilatory thresholds made by eye from the ventilation data (mean of three independent observers) were used as a standard for comparison with determinations using the modified V-slope method and the CUSUM method. A mean (SD) difference of 0.6 (2.84) ml.min-1.kg-1 was found between the standard ventilatory thresholds and those determined using the modified V-slope method. A similar comparison between the standard ventilatory thresholds and those determined using the CUSUM method yielded a difference of -0.11 (2.35) ml.min 1.kg-1. It was concluded that the CUSUM method was a useful aid for the detection of the ventilatory threshold using the ventilation data alone. PMID- 7556135 TI - Core temperature thresholds for hyperpnea during passive hyperthermia in humans. AB - Humans have higher ventilation when they are hyperthermic but it is not known whether core temperature thresholds for ventilation exist, nor has a physiological rationale been presented for this response. To examine this question, ventilation was studied in relation to core temperatures in humans rendered hyperthermic in a warm bath. Seven subjects [mean (SE), 23.3 (1.4) years] wearing only shorts and a thick felt hat with ear flaps were immersed to the neck in a bath at 41 (0.5) degrees C for 25 min. Tympanic (Tty), esophageal (Tes), thigh skin and forehead skin temperatures, heart rate, inspired minute ventilation (V1 at body temperature and pressure, saturated), ventilation frequency and oxygen consumption (VO2 at standard temperature and pressure, dry) were recorded at 30-s intervals. At immersion V1 briefly increased to 18.6 (3.0) l.min-1, returned to about the pre-immersion value, and significantly increased to 19.3 (3.0) l.min-1 by the end of immersion. VO2 increased significantly from the pre-immersion value of 0.27 l.min-1 to 0.67 l.min-1 by the first 0.5 min of immersion, but then returned to its pre-immersion value. Tty increased to 38.7 (0.2) degrees C and Tes increased to 39.0 (0.2) degrees C by the end of immersion. Core temperature thresholds for increases in V1 were evident at 38.1 degrees C when expressed against Tty and at 38.5 degrees C when expressed against Tes. The results indicated that during body warming core temperature thresholds for V1 are reached and subsequently a hyperpnea was evident, despite VO2 remaining at a resting value.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556128 TI - A review of the control of breathing during exercise. AB - During the past 100 years many experimental investigations have been carried out in an attempt to determine the control mechanisms responsible for generating the respiratory responses observed during incremental and constant-load exercise tests. As a result of these investigations a number of different and contradictory control mechanisms have been proposed to be the sole mediators of exercise hyperpnea. However, it is now becoming evident that none of the proposed mechanisms are solely responsible for eliciting the exercise respiratory response. The present-day challenge appears to be one of synthesizing the proposed mechanisms, in order to determine the role that each mechanism has in controlling ventilation during exercise. This review, which has been divided into three primary sections, has been designed to meet this challenge. The aim of the first section is to describe the changes in respiration that occur during constant-load and incremental exercise. The second section briefly introduces the reader to traditional and contemporary control mechanisms that might be responsible for eliciting at least a portion of the exercise ventilatory response during these types of exercise. The third section describes how the traditional and contemporary control mechanisms may interact in a complex fashion to produce the changes in breathing associated with constant-load exercise, and incorporates recent experimental evidence from our laboratory. PMID- 7556138 TI - Identification of the electrophilic substrate-binding site of glutathione S transferase P by photoaffinity labeling. AB - We determined the electrophilic substrate-binding site of rat glutathione S transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G site) and the electrophilic substrate-binding (H-site) regions. The procedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose. The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P. PMID- 7556136 TI - M-wave modulation at relative levels of maximal voluntary contraction. AB - Frequency (mean and median power frequency, f and fm) and amplitude (average rectified and root mean square values, ARV and rms), parameters of the M-wave, and the dorsiflexor force parameters of the anterior tibial muscles were measured in seven healthy human subjects. Intermittent, voluntary contractions at relative intensities (40%, 60%, and 80%) of maximal voluntary contraction (MVC) were performed in conjunction with electrical stimulation. The M-wave parameter changes were measured over the course of the isometric contractions. At higher force levels, M-wave potentiation was observed as increases in both ARV and rms. The ARV augmentation attained levels as high as 206.1 (SD 7.4)% of resting values after both initial and final contractions of 80% MVC, reaching statistical significance (P < 0.01). The f and fm failed to show a significant difference at any level of contraction. It was surmised that potentiation of the M-wave was the result of an increased contribution of muscle fibre type IIb recruited during higher contraction levels, reflecting the change to larger, deeper innervating motoneurons as the intensity of contraction, as a percentage of MVC, rose. Recruitment of type IIb fibres, which have been reported to have a higher energy potential and frequency content, were thought to reflect changes in the local excitability threshold of some motor units as the force intensity increased during the intermittent voluntary contractions. It is suggested that the M-wave elicited after contractions has the potential to reflect, to some extent, motor unit recruitment changes resulting from the preceding contractions, and that through comparisons of M-wave amplitude parameters contributions of varying fibre types over the course of a contraction may be indicated. PMID- 7556137 TI - Effects of intracerebroventricular administration of aminophylline on hyperbaric induced increase in carotid blood flow. AB - Carotid blood flow was measured in rats by implanted transit-time ultrasonic flowprobes during hyperbaric experiments at up to 70 bar (7 MPa) using an helium oxygen hyperoxic (partial pressure of O2 = 400 mbar) mixture. Before the hyperbaric experiment, an intracerebroventricular injection of phosphate saline buffered solution (PBS) or aminophylline, an adenosine receptor blocker, in PBS was given. Throughout the hyperbaric experiment carotid blood flow increased with ambient pressure in both PBS, i.e. control, and aminophylline treated rats. The increase in carotid blood flow was significantly attenuated in aminophylline treated rats. Additional experiments showed that the increased carotid blood flow was independent of hyperoxia as well as of temperature. The hypothesis that the hyperbaric dependent increase in carotid blood flow was mediated by brain adenosine receptors and its implication regarding a cerebral vasodilatation are discussed. PMID- 7556140 TI - Thermal sensitivity in NIH 3T3 fibroblasts transformed by the v-fos oncogene. Correlation with reduced accumulation of 68-kDa and 25-kDa stress proteins after heat shock. AB - The effect of v-fos transformation on the cellular response to heat shock has been investigated. NIH 3T3 fibroblasts were transfected with the FBR p75gag-fos gene fusion under the control of the long terminal repeat (LTR) promoter of Finkel-Biskin-Reilly (FBR) murine sarcoma virus and with the gene encoding hygromycin resistance. Several hygromycin-resistant clone isolates, that expressed various levels of p75gag-fos oncoprotein, were analyzed as they displayed properties of transformed cells, such as altered morphology, shorter doubling time, serum-independent growth and foci formation in soft agar. The thermal response of these clones was compared to that of the control cells expressing the hygromycin-resistance gene only. Here, we report that the v-fos transformed clones displayed an enhanced thermosensitivity which resulted in a reduced tolerance to thermal stress. Heat-treated v-fos-transformed cells displayed a decreased expression and accumulation of the major stress proteins Hsp68 (68-kDa heat-shock protein) and Hsp25 which probably resulted of a reduced accumulation of the corresponding mRNAs. This effect was particularly intense at the level of Hsp25. These alterations in cell survival and stress-protein expression appeared correlated to the level of p75gag-fos. At least for Hsp68, the transcription of this gene was not found altered by v-fos expression suggesting that this oncogene increases the turn-over of Hsp68 mRNA. After the heat-shock treatment, v-fos transformation also reduced the time period during which the constitutively expressed stress protein Hsc70 redistributes inside the nucleus. Since Hsp68 and Hsp25 are molecular chaperones that in vivo protect cells against the deleterious effects of heat shock, it is conceivable that their reduced accumulation and altered cellular distribution following heat shock may contribute, at least in part, to the thermosensitivity of v-fos-transformed NIH 3T3 fibroblasts. PMID- 7556139 TI - The catalytic subunit of cAMP-dependent protein kinase from Ascaris suum. The cloning and structure of a novel subtype of protein kinase A. AB - A complete cDNA clone encoding the catalytic subunit of cAMP-dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 amino acids is 48% identical with the sequence of mouse C alpha subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N-terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N-terminal region and an acidic amino acid near the C terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N terminal and C-terminal parts of the protein. There is strong evidence that a single gene encodes cAMP-dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X-ray structure of the mammalian enzyme suggest a high degree of conservation in the three-dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952-957] and the mammalian enzyme. PMID- 7556143 TI - Conserved alternative splicing in the 5'-untranslated region of the muscle specific enolase gene. Primary structure of mRNAs, expression and influence of secondary structure on the translation efficiency. AB - We report here the isolation and characterization of cDNAs covering the 5'-end region of mouse and rat mRNAs that encode the beta or muscle-specific isoform of the glycolytic enzyme enolase. As previously determined for humans, two classes of beta-enolase transcripts with distinct sequences in their 5'-untranslated regions are present in both mouse and rat muscles. A mechanism of alternative splicing, conserved from mouse to man, generates the two forms of mRNA. Secondary structure predictions indicated that, in all cases, a more stable secondary structure could exist in the 5' end of the message with the longer leader. In vitro transcripts containing defined human or mouse 5'-untranslated sequences were obtained by fusion of the different cDNA clones and tested for their relative translational efficiencies in rabbit reticulocyte lysates. Transcripts containing the human long and short leader sequences showed differences in the translational rate, suggesting a role for the 5'-untranslated region in the regulation of translation. No detectable difference was found between transcripts with the two distinct mouse leader sequences. In addition, both transcripts are bound to polysomes and are equally distributed along differently sized polysomes in C2C12 myogenic cells. The relative expression of the two spliced forms in developing and adult muscle tissues by means of reverse transcription and polymerase chain reaction did not show a stage-specific or a tissue-type-specific pattern. A putative functional role for the 5'-untranslated sequences of beta enolase transcripts is discussed. PMID- 7556141 TI - Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake. AB - The aim of the present study was to determine if human N-terminal half transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor independent mechanism for accumulation of iron from transferrin. PMID- 7556142 TI - Enhanced phosphorylation of nuclear 21-kDa and 34-kDa proteins in hepatoma cell death induced by tumor-necrosis factor-alpha. AB - The role of nuclear protein phosphorylation in intracellular signal transduction of tumor-necrosis factor-alpha (TNF-alpha) in the human hepatoma cell line PLC(PRF/5) was investigated. TNF-alpha, which displays cytolytic activity against PLC hepatoma cells, elevated the in vitro phosphorylation of two nuclear proteins (21 kDa and 34 kDa) 16 h after treatment. The cytotoxicity and enhanced nuclear protein phosphorylation by TNF-alpha treatment decreased in the presence of dexamethasone. Both the 21-kDa and 34-kDa proteins were extracted with 2.2 M NaCl from nuclear pellets and phosphorylated in kinase reaction mixtures containing a high concentration of salt. By phosphoamino acid analysis, the specificity of the nuclear kinase was found to be directed toward serine residues. The protein kinase inhibitors H7, staurosporine and herbimycin A, inhibited the phosphorylation of the 21-kDa and 34-kDa proteins in vitro, but calphostin C and heparin did not. The treatment of cells with 4 beta-phorbol 12-myristate 13 acetate or okadaic acid did not affect the in vitro phosphorylation of the two nuclear proteins. An anti-Fas antibody increased the phosphorylation of the 21 kDa and 34-kDa proteins in PLC cells. DNA fragmentation was observed in PLC cells treated with TNF-alpha and anti-Fas antibody after 24 h treatment. These data suggest an involvement of nuclear protein kinase in signal-transduction pathways of apoptotic cell damage triggered by TNF-alpha in PLC hepatoma cells. PMID- 7556145 TI - Interaction of melanosomal proteins with melanin. AB - Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink-eyed-dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell-free melanosomal melanization assay. Upon incubation of a melanosome-rich fraction with the melanin precursor L-3,4-dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinase-related family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane-bound p locus protein may also interact closely with melanin. PMID- 7556144 TI - Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein. AB - The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved. PMID- 7556146 TI - Alpha 2-macroglobulin receptor mediates binding and cytotoxicity of plant ribosome-inactivating proteins. AB - It has been proposed that unconjugated type I ribosome-inactivating proteins (RIP) enter cells through passive mechanisms such as fluid-phase pinocytosis. However, some observations, such as the difference in sensitivity to type I RIP among different cell types, and the organ-specific toxicity of type I RIP, indicate a specific mechanism for the entry of these proteins into target cells. The alpha 2-macroglobulin receptor (alpha 2MR) is responsible for the binding and endocytosis of several ligands, including alpha 2-macroglobulin/proteinase complexes, plasminogen-activator-inhibitor complexes, apoE-enriched beta-very low density lipoproteins, and lipoprotein lipase. Here we demonstrate that saporin, a potent type I RIP, binds specifically to purified alpha 2MR and the binding is prevented by some alpha 2MR ligands. Moreover, the occupancy of specific ligand binding sites on cell surface alpha 2MR decreases the cytotoxicity of saporin. The A chain of ricin, a type II RIP, also interacts with alpha 2MR. This, and the fact that saporin and ricin A chain both interact also with alpha 2 macroglobulin, indicates a general mechanism of complex interactions between RIP and cellular membranes that is mediated by alpha 2-macroglobulin and the alpha 2MR system. PMID- 7556147 TI - Does the overexpression of pro-insulin-like growth factor-II in transfected human embryonic kidney fibroblasts increase the secretion of lysosomal enzymes? AB - Insulin-like growth factor-II (IGF-II) and lysosomal enzymes bearing the mannose 6-phosphate (Man6P) recognition marker, bind to two distinct binding sites of the IGF-II/M6P receptor. The two classes of ligands reciprocally modulate the binding of the other class of ligand to the receptor [Kiess, W., Thomas, C. L., Greenstein, L., Lee, L., Sklar, M. M., Rechler, M. M., Sahagian, G. G. & Nissley, S. P. (1989) J. Biol. Chem. 264, 4710-4714]. We asked whether or not overexpression of pro-IGF-II by cells in culture leads to missorting of lysosomal enzymes. Human embryonal kidney fibroblasts were transfected with the full-length human IGF-II cDNA or a control cDNA. Solution hybridization/RNase protection experiments using a human IGF-II riboprobe showed that two transfectants expressed large quantities of IGF-II mRNA, whereas the non-transfected cells did not. The analysis of conditioned media revealed that these cells secrete approximately 0.15 micrograms and 1.0 micrograms immunoreactive IGF-II/ml and 22 x 10(6) cells and 24 x 10(6) cells within 24 hours. Immunoreactive IGF-II was shown by Western blotting to represent 17-kDa pro-IGF-II. The amount of the lysosomal enzyme, beta-hexosaminidase, was approximately twofold increased in the conditioned media from pro-IGF-II overexpressing cells compared with control media, as shown by Western-blot analysis and immunoprecipitation of media extracts of metabolically labeled cells. The synthesis rate of beta hexosaminidase was not affected by pro-IGF-II overexpression. In addition, the basal amount of another newly synthesized lysosomal enzyme, the cathepsin D precursor, was also twofold higher in pro-IGF-II overexpressing cells than in control cells. In contrast, the surface binding and cellular uptake rate of a Man6P-containing neoglycoprotein did not differ between the cell lines. The results indicate that the overexpression of pro-IGF-II doubles the secretion and/or reduces the re-uptake of beta-hexosaminidase and cathepsin D to approximately 20% of the total synthesized enzymes in human embryonal kidney fibroblasts compared to control cells. We hypothesize that, in cells synthesizing high amounts of pro-IGF-II, the growth factor may modulate the targeting of a portion of lysosomal enzymes, mainly by partially enhancing the secretion of newly synthesized enzymes and, in addition, possibly by affecting the re-uptake mechanism. PMID- 7556148 TI - Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II and acyl-coenzyme A oxidase gene expression by fibrates and dietary fatty acids. AB - The regulation by fibrates and dietary fatty acids of the hepatic gene expression of apolipoproteins (apo) A-I and A-II, the major protein constituents of high density lipoproteins, as well as of acyl-CoA oxidase, the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, was studied in vivo in the rat and in vitro in primary cultures of rat hepatocytes. In primary hepatocytes, different fibrates decreased apo A-I and increased acyl-CoA oxidase mRNA levels, whereas apo A-II mRNA only decreased in level after treatment with fenofibric acid, but not after bezafibrate, gemfibrozil or Wy-14643 treatment. Treatment with fenofibric acid counteracted the increase in apo A-I mRNA levels observed after dexamethasone or all-trans retinoic acid treatment, whereas simultaneous addition of fenofibric acid together with all-trans retinoic acid or dexamethasone resulted in a superinduction of acyl-CoA oxidase mRNA. Addition of the n-3 polyunsaturated fatty acids (PUFAs), docosanohexaenoic acid and eicosanopentaenoic acid, or the fatty acid derivative alpha-bromopalmitate, decreased apo A-I and increased acyl-CoA oxidase mRNA in a dose-dependent and time-dependent manner, whereas apo A-II mRNA did not change significantly. Nuclear run-on experiments demonstrated that fenofibric acid and alpha bromopalmitate decreased apo A-I and increased acyl-CoA oxidase gene expression at the transcriptional level. When rats were fed isocaloric diets enriched in saturated fat (hydrogenated coconut oil), n-6 PUFAs (safflower oil) or n-3 PUFAs (fish oil), a significant decrease in liver apo A-I and apo A-II mRNA levels was only observed after fish oil feeding. Compared to feeding low fat, liver acyl-CoA oxidase mRNA increased after fat feeding, but this effect was most pronounced (twofold) in rats fed fish oil. Results from these studies indicate that fish oil feeding reduces rat liver apo A-I and apo A-II gene expression, similar to results obtained after feeding fenofibrate. Fibrates and n-3 fatty acids (and the fatty acid derivative, alpha-bromopalmitate) down-regulate apo A-I and induce acyl-CoA oxidase gene expression through a direct transcriptional action on the hepatocyte. In contrast, only fenofibric acid, but not the other fibrates or fatty acids tested, decrease apo A-II gene expression in vitro. PMID- 7556149 TI - Reversible binding and inhibition of catalase by nitric oxide. AB - The interactions between nitric oxide (NO), H2O2, and catalase were investigated. H2O2 did not cause detectable breakdown of NO in the absence of catalase, but did cause NO breakdown in the presence of catalase. Catalase bound NO, and NO rapidly and reversibly inhibited catalase with a Ki of 0.18 microM. The significance of these results for NO cytotoxicity is discussed. PMID- 7556150 TI - Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization. AB - Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80 + 90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII. In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues. After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80 + 90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N- and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII. PMID- 7556153 TI - In vitro folding of Escherichia coli outer-membrane phospholipase A. AB - Recombinant outer-membrane phospholipase A (OMPLA) of Escherichia coli was expressed without its signal sequence from the T7 phi 10 promoter. As a result of the cloning strategy the protein had an N-terminal extension of six amino acid residues. The protein accumulated in the cytosol in inclusion bodies. Conditions were established for the efficient folding of OMPLA in vitro in the presence of Triton X-100. After in vitro folding, the protein was present as a mixture of folded and unfolded forms. Ion-exchange chromatography was used for the purification of OMPLA and the separation of correctly folded, enzymically active enzyme from unfolded inactive protein. The final protein preparation was pure and fully heat-modifiable based on SDS/PAGE. The recombinant enzyme had a specific activity of 71 U/mg, which is similar to the value of the wild-type enzyme, purified from the membrane. The final yield of active enzyme was 35 mg protein/l culture of an A600 of 6. Circular dichroism spectroscopy revealed a high content of beta strand, in good agreement with a predicted beta-barrel structure of this outer-membrane protein. PMID- 7556152 TI - Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2 pH dependent conformational states. AB - The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5 11, the E degree values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 1H-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pKa most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E degree values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s-1 at pH 9.2. PMID- 7556151 TI - Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum. AB - Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duee, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region. PMID- 7556154 TI - Mutation of the metal-bridging proton-donor His63 residue in human Cu, Zn superoxide dismutase. Biochemical and biophysical analysis of the His63-->Cys mutant. AB - The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co-ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O2/O2- and O2-/H2O2 pairs so that copper can both oxidize and reduce the O2- substrate, a requirement for dismutase activity. The enzyme binds the substrate analogue azide (N3-), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O2- substrate is reduced to less than 1% of wild-type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide-copper electron transfer process and to the catalytic mechanism. PMID- 7556155 TI - Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish. AB - Tropomyosin (TM) has been isolated from the cardiac muscle, and fast and slow trunk (myotomal) muscles of the mature salmonid fish Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri). When examined electrophoretically, isoforms of TM were detected which were specific, and exclusive, to each type of muscle. Cardiac and fast muscles contained single and distinct isoforms, while slow muscle contained two distinct isoforms, closely related in terms of apparent M(r), and pI. There was no detectable difference between the same TM type from either salmon or trout. On a variety of gel systems, the cardiac and slow isoforms migrated in close proximity to each other and to rabbit alpha-TM. The fast isoform comigrated with rabbit beta-TM. In developing salmon fry, a more acidic (unphosphorylated) variant of TM was present in addition to, and of similar M(r) to, the fast adult isoform. This TM declined in steady-state level during maturation and was virtually undetected in adult muscle. All of the isolated TMs contained little or no covalently bound phosphate and were blocked at the N-terminus. The amino acids released by carboxypeptidase A, when ordered to give maximal similarity to other muscle TMs, were consistent with the following sequences: fast (LDNALNDMTSI) and cardiac (LDHALNDMTSL). The C-terminal region of the slow TM contained His but was heterogeneous. In viscosity measurements, performed as a function of increasing protein concentration, at low ionic strength (t = 5 degrees C, pH 7.00), fast TM exhibited the highest relative viscosity values. Lower and equivalent levels of polymerisation occurred with the cardiac and slow TMs. Polymerisation of all three isoforms was temperature dependent, with cardiac TM being least sensitive and fast TM being most sensitive. Determination of the complete coding sequence of adult fast TM confirmed the findings of the carboxypeptidase analysis, but the remainder of the sequence more closely resembled alpha-type TMs than beta-type TMs. Overall, salmon fast TM contains 20 (mostly conservative) substitutions compared to rabbit striated muscle alpha-TM and 40 (mostly conservative) substitutions compared to rabbit striated muscle beta-TM. This demonstrates that electrophoretic mobility is not, in all instances, a suitable method to assess the isomorphic nature of striated muscle TMs. PMID- 7556156 TI - Immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain. AB - Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin. PMID- 7556157 TI - Fasciola hepatica cathepsin L proteinase cleaves fibrinogen and produces a novel type of fibrin clot. AB - A cathepsin L proteinase secreted by the parasitic helminth Fasciola hepatica can cleave fibrinogen and produce a fibrin clot with a specific activity of 4.7 National Institutes of Health thrombin-equivalent U/mg. This is the first report of a fibrinogen-clotting activity aside that of thrombin and the snake venom proteinases, which are all serine proteinases. Clot formation by cathepsin L is not inhibited by the thrombin inhibitor hirudin or by the anti-polymerant H-Gly Pro-Arg-Pro-OH. The enzyme exerts its activity on fibrinogen in a unique manner. Although the cleavage of fibrinogen may involve the initial removal of fibrinopeptides, additional proteolysis of the alpha, beta and gamma fibrinogen polypeptides takes place. SDS/PAGE analysis of the cathepsin-L-produced clots revealed that cleavage of the alpha polypeptide (66 kDa) precedes that of the beta (52 kDa) and gamma (46.5 kDa) polypeptides. Concurrent with the cleavage of these polypeptides is the appearance of components of 120, 100 and 25 kDa. The appearance of higher molecular-sized components in the cathepsin L clots suggests that polymerisation involves the formation of molecular interactions that are resistant to boiling in mercaptoethanol and SDS. PMID- 7556159 TI - Melatonin antagonizes alpha-melanocyte-stimulating hormone enhancement of melanogenesis in mouse melanoma cells by blocking the hormone-induced accumulation of the c locus tyrosinase. AB - Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24-48 h treatment and at concentrations of melatonin higher than the physiologic levels of the circulating hormone. Although these effects on the melanogenic activities were modest, incubation of melanocytes with melatonin prior to the addition of the melanotropin mediated a dramatic inhibition of alpha-melanocyte-stimulating-hormone-(alpha-MSH)-induced melanogenesis. This inhibitory effect was evident at melatonin concentrations as low as 10 nM. Inhibition was nearly total at 0.1 mM melatonin, even at high concentrations of alpha-MSH (1 microM). The inhibitory effect of melatonin on alpha-MSH stimulation of melanogenesis was investigated. Melatonin appeared to act at least at two stages. Pharmacological concentrations of melatonin diminished the number of alpha-MSH receptors to about 75% of the control values without an apparent effect on receptor affinity, as determined by receptor binding studies using 125I-[N-Leu4-D-Phe7]alpha-MSH as a probe. Physiological concentrations of melatonin also appeared to interfere with the intracellular events coupling increased cAMP levels and induction of the c locus tyrosinase, since it strongly inhibited the theophylline-mediated stimulation of melanogenesis. The inhibition of tyrosinase stimulation was higher in the microsomal than in the melanosomal fractions of cells which were treated with melatonin, then exposed to either alpha-MSH (1 microM) or theophylline (1 mM), suggesting that one of the main effects of melatonin might be inhibition of the induction of tyrosinase de novo synthesis. PMID- 7556158 TI - Inhibition of bovine cytochrome P-450(11 beta) by 18-unsaturated progesterone derivatives. AB - The last step of aldosterone biosynthesis, an 11 beta-hydroxylation followed by two 18-hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-450(11 beta) (deoxycorticosterone (DOC)-->corticosterone-->18 hydroxycorticosterone-->aldosterone). The 11 beta- and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 11 deoxy[14C]corticosterone and [3H]corticosterone, respectively, as substrates. The inhibition of 11 beta-hydroxylase activity by corticosterone was competitive (Ki = 60 microM) showing that transformation of both substrates occurs at the same site. Double-label/double-substrate experiments, using an equimolar mixture of 11 deoxy[14C]corticosterone and [3H]corticosterone, suggested that 18 hydroxycorticosterone is directly formed from 11-deoxycorticosterone without the intermediate corticosterone leaving the enzyme. Inhibitions by 18 vinylprogesterone and 18-ethynylprogesterone, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Perard, S., Piffeteau, A. & Marquet, A. (1989) J. Steroid Biochem. 33, 119-124], were characterized for both activities (11 beta- and 18-hydroxylase). The value of reversible Ki for the 18 hydroxylation (Ki = 5 microM for 18-vinylprogesterone and 30 microM for 18 ethynylprogesterone) is lower than that for the 11 beta-hydroxylation (30 microM and 100-150 microM, respectively); the former inhibitor is stronger than the latter for both steps. The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18 Vinylprogesterone gave rise to a type I spectrum with a Ks value of 35 microM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher Ks value (140 microM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed. PMID- 7556161 TI - In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by beta 2GlcNAc-transferase I is an essential prerequisite for the action of beta 2GlcNAc-transferase II and beta 2Xyl transferase. AB - Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (beta 2Xyl-T), beta 1 2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N acetylglucosaminyltransferase II (beta 2GlcNAc-T II) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = 4GlcNAc beta 1-4GlcNAc or O-(CH2)7CH3] with UDP-Xyl and connective tissue microsomes have been purified and characterized by 1H-NMR spectroscopy in conjunction with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specificity studies focused on connective tissue beta 2Xyl-T show that the minimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)7CH3. The enzyme activity can therefore be characterized as UDP-Xyl:Glc-NAc beta 1-2Man alpha 1-3Man beta-R (Xyl to Man beta) beta 1-2 xylosyltransferase. In substrate specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xyl residue at C2 of beta Man totally abolishes the enzyme activity. Xylose-containing oligosaccharides are not acceptors for beta 2GlcNAc-T I. In combination with the substrate specificity of beta Xyl-T, this shows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective tissue is proposed. PMID- 7556160 TI - The primary structure of Hyphomicrobium X dimethylamine dehydrogenase. Relationship to trimethylamine dehydrogenase and implications for substrate recognition. AB - The gene encoding dimethylamine dehydrogenase from Hyphomicrobium X has been cloned and over-expressed in Escherichia coli. Using the chemically determined protein sequence, primers were designed to amplify DNA fragments encoding the proximal and distal parts of the gene. These fragments were used to synthesise two probes and the dmd gene was cloned as part of two BamHI fragments isolated from digested genomic DNA. The sequence of the complete open reading frame was determined on both strands and contained 2211 bp coding for a protein of 736 amino acids, including the N-terminal methionine residue that is removed when expressed in the native host. The molecular mass of the processed apoprotein predicted from the DNA sequence is 82,523 Da. Dimethylamine dehydrogenase is closely related to the trimethylamine dehydrogenase of Methylophilus methylotrophus W3A1 (63.5% identical) and other class I FMN-binding beta 8 alpha 8 barrel flavoproteins. Residues in the active site of trimethylamine dehydrogenase that are known, or implicated, to be important in catalysis are conserved in dimethylamine dehydrogenase. Sequence alignment of dimethylamine and trimethylamine dehydrogenases suggests that the specificity for secondary and tertiary amines resides in a single amino acid substitution in a substrate binding aromatic bowl located in the active site of the enzymes. PMID- 7556163 TI - Molecular modelling of the interaction between the catalytic site of pig pancreatic alpha-amylase and amylose fragments. AB - A stereo chemical refinement of the crystalline complex between porcine pancreatic alpha-amylase and a pseudopentasaccharide from the amylostatin family has been performed through molecular mechanics calculations, using a set of parameters appropriate for protein and protein-carbohydrate interactions. The refinement provided a starting point for docking a maltopentaose moiety within the catalytic site, in the absence of water. A thorough exploration of the different orientations and conformations of maltopentaose established the sense of binding of the amylosic substrate in the amylase cleft. After optimising the geometry of the binding site, the conformations adopted by the four contiguous linkages could be rationalised by considering the environment, either hydrophobic or hydrophilic, of the different glucose moieties. Seemingly, details of the non bonded interactions (hydrogen bonds, van der Waals and stacking interactions) that underlie this molecular recognition have been established. In particular, it was confirmed that the three acidic amino acids of the catalytic site (Asp197, Asp300 and Glu233) are close to their glucosidic target, and that there is no steric reason to propose an alteration of the 4C1 conformation of the glucose residue prior to hydrolysis. However, in the absence of water molecules, it is difficult to elucidate the details of the catalysis. Additional macroscopic information has been gained, such as the impossibility to fit a double-helical arrangement of amylose chains in the amylasic cleft. This explains why some native starches containing such motifs resist amylolytic enzymes. Tentative models involving longer amylosic chains have been elaborated, which extend our knowledge of the interaction and orientation of starch fragments in the vicinity of the hydrolytic sites. PMID- 7556162 TI - Activation of the double-stranded-RNA-activated protein kinase and induction of vascular cell adhesion molecule-1 by poly (I).poly (C) in endothelial cells. AB - Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1 beta (IL-1 beta), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1 beta. Incubation of HUVE cells with the synthetic dsRNA, poly (I).poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2 alpha, and increased activation of the transcription factor NF-kappa B. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I).poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-kappa B sites, and deletion of the NF-kappa B sites eliminates basal and poly (I).poly (C)-induced CAT activity, supporting the importance of NF-kappa B in the poly (I).poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating I kappa B alpha (the inhibitory subunit of NF-kappa B) in a dsRNA dependent manner. This suggests that phosphorylation of I kappa B alpha by PKR could be an initial step in the activation of NF-kappa B by dsRNA. NF-kappa B is also activated by IL-1 beta in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2 alpha phosphorylation. Poly (I).poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1 beta. Although phosphorylation of eIF2 alpha interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I).poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1 beta. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I).poly (C) compensate for the partial translational block resulting from increased eIF2 alpha phosphorylation. These studies indicate that transcriptional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells. PMID- 7556164 TI - Spectroscopic and mutagenesis studies on the CuA centre from the cytochrome-c oxidase complex of Paracoccus denitrificans. AB - Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: gparallel = 2.18; gperpendicular = 2.03. gparallel exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556166 TI - Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM). AB - The essential mitochondrial inner-membrane protein, Mim44, is involved in the translocation of preproteins across the mitochondrial inner membrane. Two other putative components of this protein-translocation system are the integral inner membrane proteins, Mim23 and Mim17. Here, we present genetic evidence for functional co-operation of all three proteins. Furthermore, we show that Mim23 and Mim17 are associated in a protein complex that also contains two proteins of 55 kDa and 20 kDa. We speculate that this subcomplex forms the proteinaceous import channel of the inner-membrane which transiently interacts with a less abundant peripheral complex of Mim44 and mitochondrial heat-shock protein Hsp70. PMID- 7556165 TI - Induction of the Escherichia coli cytochrome d by low delta mu H+ and by sodium ions. AB - Regulation of synthesis of cytochrome d in Escherichia coli has been studied using mutants with cytochrome-d--beta-galactosidase gene fusions. It was shown that various protonophorous uncouplers, when added to the growth medium, cause induction of the cytochrome d synthesis. The cytochrome-d-inducing activity of uncouplers correlates with their ability to inhibit such a delta mu (H+)-driven function as motility of the E. coli cells. An increase in the Na+ concentration in the growth medium from 1.5 mM to 25 mM results in induction of the cytochrome d synthesis. The cytochrome-d-inducing effect of uncouplers is much more pronounced when the Na+ concentration is high than when it is low. These data are in agreement with the assumption that cytochrome d is involved in the Na+ energetics substituting for the H+ energetics when the latter appears to be inefficient. Mutations in arcA or arcB genes (but not in fnr gene) completely prevent the increase in the cytochrome d level induced by uncouplers but are without effect on that induced by Na+. It is assumed that in the control of the cytochrome d synthesis, the Arc system is involved in the delta mu H+ sensing whereas sensing of delta mu Na+ (or of the Na+ concentration) is mediated by some other receptor system. PMID- 7556167 TI - Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli. AB - Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227 Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum. PMID- 7556168 TI - Studies on the regulation and localization of 5-lipoxygenase in human B lymphocytes. AB - Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bis[dimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5 lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocytochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes. PMID- 7556169 TI - Intracellular levels and secretion of insulin-like-growth-factor-binding proteins in MCF-7/6, MCF-7/AZ and MDA-MB-231 breast cancer cells. Differential modulation by estrogens in serum-free medium. AB - The synthesis and secretion of insulin-like growth factor binding proteins (IGFBPs) were studied in MDA-MB-231 (estrogen-receptor-negative), MCF-7/6 (estrogen-receptor-positive, invasive) and in MCF-7/AZ (estrogen-receptor positive, non-invasive) human breast carcinoma cell lines. Cells were grown or maintained in a chemically defined medium. Under these conditions, we found different patterns of IGFBPs in the three cell types. MDA-MB-231 cells secrete most of the IGFBPs they produce whereas MCF-7/6 and MCF-7/AZ cells maintain a high intracellular level. In MDA-MB-231 cells, the major IGFBP is IGFBP-4 which is the minor form in MCF-7/6 and MCF-7/AZ cells. IGFBP-2 and IGFBP-5 are predominant in MCF-7/6 cells while MCF-7/AZ cells produce far less IGFBPs and do not contain detectable amounts of 29-32-kDa forms (IGFBP-5). In MCF-7/6 cells, estradiol-17 beta specifically decreases both the intracellular content and secretion of IGFBP-2 and IGFBP-5. Estrogen regulation of IGFBPs cell content and secretion was found to be tamoxifen-resistant, and only slightly antagonized by ICI 182,780, a pure antiestrogen. The function of these regulations relative to the invasive phenotype and proliferation has now to be determined. PMID- 7556170 TI - Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. AB - Two proteins with seven transmembrane-spanning domains typical of guanosine nucleotide-binding-protein-coupled receptors have been identified as cannabinoid receptors; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR-based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10-100-fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B-cells > natural killer cells >> monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern-blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti-(human CB2) IgG; this experiment showed that CB2 expression was restricted to B-lymphocyte-enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor-mediated actions on the immune system through the CB2 receptor. PMID- 7556171 TI - Enhanced cAMP accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain. AB - Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by 125I-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease. PMID- 7556172 TI - Localized chemical reactivity in double-stranded DNA associated with the intercalative binding of benzo[e]pyridoindole and benzo[g]pyridoindole triple helix-stabilizing ligands. AB - Footprinting with methidiumpropyl-EDTA.FeII has been used to map the binding sites on duplex DNA of two closely related benzopyridoindole derivatives which selectively stabilize triple-helical DNA-oligonucleotide complexes. Both ligands bind to many sites, including certain oligopurine.oligopyrimidine tracts, with a weak preference for some (but not all) sequences rich in A.T base pairs. This indifference to primary sequence, with evidence of binding to the commonly disfavoured (A)n.(T)ntracts, may at least partially explain why the ligands stabilize triplex structures composed of T.A.T pairings. Neither 3-methoxy-7H-8 methyl-11- [(3'amino)propylamino]benzo[e]pyrido[4, 3-b]indole (BePI) nor 3- methoxy-7-[3'-diethylamino)propylamino]-10-methyl-11H- benzo[g]pyrido[4,3 b]indole (BgPI) affect the reaction of dimethyl sulphate or potassium tetrachloropalladinate with the N7 of purines in the major groove, but both enhance the reactivity of purines (mostly adenine residues) towards diethylpyrocarbonate, both proximal and distal to their identified binding sites. With potassium permanganate and osmium tetroxide/pyridine, probes for the accessibility of the 5,6 double bond of pyrimidine residues, BgPI has a more potent effect than BePI and, generally, the reaction with KMnO4 is more pronounced than that with OsO4. BgPI conspicuously potentiates the oxidation of pyrimidines in the triplet sequences 3'-ATA, 3'-GTA and 3'-GCA, whereas BePI enhances the reactivity of OsO4 towards thymine in sequences 3'-ATYR, with no effect on cytosine residues. Thus, despite their structural homology and common lack of specific sequence preferences, the two benzopyridoindole derivatives induce distinct conformational changes in duplex DNA, not just within the sites where footprints can be detected. PMID- 7556173 TI - The molecular biology of multidomain proteins. Selected examples. AB - The aim of this review is to give an overview of the contribution molecular biology can make to an understanding of the functions and interactions within multidomain proteins. The contemporary advantages ascribed to multidomain proteins include (a) the potential for metabolite channelling and the protection of unstable intermediates; (b) the potential for interactions between domains catalysing sequential steps in a metabolic pathway, thereby giving the potential for allosteric interactions; and (c) the facility to produce enzymic activities in a fixed stoichiometric ratio. The alleged advantages in (a) and (b) however apply equally well to multi-enzyme complexes; therefore, specific examples of these phenomena are examined in multidomain proteins to determine whether the proposed advantages are apparent. Some transcription-regulating proteins active in the control of metabolic pathways are composed of multiple domains and their control is exerted and modulated at the molecular level by protein-DNA, protein protein and protein-metabolite interactions. These complex recognition events place strong constraints upon the proteins involved, requiring the recognition of and interaction with different classes of cellular metabolites and macromolecules. Specific examples of transcription-regulating proteins are examined to probe how their multidomain nature facilitates a general solution to the problem of multiple recognition events. A general unifying theme that emerges from these case studies is that a basic unitary design of modules provided by enzymes is exploited to produce multidomain proteins by a complex series of gene duplication and fusion events. Successful modules provided by enzymes are co opted to new function by selection apparently acting upon duplicated copies of the genes encoding the enzymes. In multidomain transcription-regulating proteins, former enzyme modules can be recruited as molecular sensors that facilitate presumed allosteric interactions necessary for the molecular control of transcription. PMID- 7556176 TI - Characterization of the binding of factor Xa to fibrinogen/fibrin derivatives and localization of the factor Xa binding site on fibrinogen. AB - The binding of human factor Xa to fibrinogen and its derivatives was characterized. Factor Xa bound to immobilized fibrin with a concentration at half maximal binding (C50) of 100 nM. The 4-carboxyglutamic acid (Gla) domain of factor Xa is important in factor Xa binding to fibrin monomer, based on the following observations; the binding requires Ca2+; Gla-domain-lacking factor Xa could not bind to fibrin; factor Xa binding was significantly reduced by prior treatment of factor Xa with factor IX/factor-X-binding protein from the venom of Trimeresurus flavoviridis which specifically binds to the Gla domain of human factors IX and X. Factor Xa also bound to fibrinogen, fibrinogen degradation products (FDP)-D and FDP-E, with a similar affinity (C50 = 75-131 nM). In a solution-phase equilibrated binding assay, approximately 0.76 mol factor Xa bound to 1 mol fibrinogen with a dissociation constant of 180 nM. The binding of 125I labeled factor Xa to the fibrin monomer was inhibited markedly by unlabeled factor Xa, but only slightly by thrombin, suggesting that the binding site of factor Xa on fibrin monomer differs from that of thrombin. We localized the binding site of factor Xa on fibrinogen: factor Xa bound strongly to the A alpha chain, but weakly to the B beta and gamma chains of fibrinogen. The A alpha chain was then digested with lysyl endopeptidase and separated by reverse-phase HPLC. Among resulting peptides, factor Xa bound specifically to a peptide corresponding to residues Asp82-Lys123 of the A alpha chain. This factor-Xa-binding site is located in the boundary between the central E domain and the terminal D domain of fibrinogen and is apparently distinct from the reported thrombin-binding site. PMID- 7556175 TI - Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. AB - We previously reported the molecular cloning of a mouse guanosine-nucleotide binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located. PMID- 7556177 TI - The oxidation of hemocyanin. Kinetics, reaction mechanism and characterization of met-hemocyanin product. AB - The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050-1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43-108, J. Wiley, New York]. Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and preceds the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M-1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M-1), where a second azide ligand also binds in the terminal mode (K = 20 M-1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes. PMID- 7556178 TI - Structure of rat annexin V gene and molecular diversity of its transcripts. AB - We report here the isolation and characterization of the entire rat annexin V gene, which is shown to span about 40 kbp, and to consist of 14 exons, 13 introns and two promoters. Previous studies found that both human and chicken annexin V genes bear 13 exons and one promoter, which possess no TATA sequence or CCAAT box, and have a high GC content. In the present study, we found that one of the promoters for rat annexin V shares these features, which are typical among promoters for housekeeping genes, and that this promoter, as expected, exerted its transcription in all of tissues we examined. In contrast, another promoter was found to possess not only a TATA box but also a glucocorticoid-responsive element, and was shown to express messages in a relatively limited number of tissues, including brain. We also demonstrated the presence of at least four species of rat annexin V transcripts that were generated by distinct transcription initiation and alternative splicing. PMID- 7556174 TI - Rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases. AB - The gene organization and nucleotide sequence of an aspartic proteinase (AP) of plant origin were first disclosed by cDNA and genomic DNA cloning of a rice AP (oryzasin). The deduced amino acid sequence of oryzasin 1 was significantly similar to those of other APs (34-85%), with highest similarity (85%) to barley AP (HvAP). Oryzasin 1, as well as HvAP, is distinct from animal and microbial APs in that the plant APs contain a unique 104-amino-acid insertion in the C-terminal region. The oryzasin 1 gene spans approximately 6.6 kbp and is composed of 14 exons and 13 introns. The exon-intron organization of the oryzasin 1 gene is totally different from those of genes for animal and microbial APs such as human cathepsin D, rat renin, bovine chymosin, aspergillopepsin A of Aspergillus awamori, proteinase A of Saccharomyces cerevisiae and rhizopuspepsin of Rhizopus niveus, despite the fact that oryzasin 1 shows overall sequence similarity to these APs. PMID- 7556179 TI - Primary structure, sequence-specific 1H-NMR assignments and secondary structure in solution of bromelain inhibitor VI from pineapple stem. AB - One of the bromelain inhibitors, isoinhibitor VI (BI-VI), was purified from pineapple stem powder and its complete amino acid sequence was determined by conventional protein sequencing. These results revealed that the protein consists of an 11-residue light chain and a 41-residue heavy chain, cross-linked to each other by disulfide bonds to form the native inhibitor of 52 residues (M(r) = 5888). The secondary structure of BI-VI was analyzed based on the sequence specific 1H resonance assignment of its two-dimensional NMR spectra. BI-VI was shown to be composed of two domains (A and B) which are formed by antiparallel beta-sheets, but has no alpha-helix. These results were consistent with the CD spectra of BI-VI. Residues Lys27-Ile29 (heavy chain) form a triple-stranded antiparallel beta-sheet with residues Asp9-Tyr11 and Lys22-Glu24 (heavy chain) in the A domain and residues Cys5-Cys7 (heavy chain) form another triple-stranded beta-sheet with residues Cys6-Cys8 (light chain) and Asp32-Ile34 (heavy chain) in the B domain. The secondary structure as well as the primary structure of BI-VI was distinctly different from that of the other cysteine protease inhibitor, cystatin, and from that of basic pancreatic trypsin inhibitor. PMID- 7556180 TI - Hypothyroidism affects the expression of the beta-F1-ATPase gene and limits mitochondrial proliferation in rat liver at all stages of development. AB - In order to analyze the role of thyroid hormones in mitochondrial biogenesis, we have studied the expression pattern of the beta subunit of the mitochondrial ATP synthase complex in liver and in isolated mitochondria during postnatal development of hypothyroid rats. Chemically induced hypothyroidism promoted a significant reduction in body and liver masses at all stages of development. Furthermore, plasma 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L thyronine (T4) concentrations were significantly reduced in hypothyroid animals when compared to euthyroid animals. Remarkably, steady-state beta-F1-ATPase mRNA levels in livers of hypothyroid animals showed an approximately 50% reduction when compared to age-matched euthyroid rats at all stages of development. The relative amounts of beta-F1-ATPase protein determined in isolated mitochondria of 1-day-old and adult hypothyroid animals were similar to those determined in mitochondria of age-matched euthyroids, indicating that hypothyroidism does not affect organelle differentiation in the liver of suckling and adult rats. In contrast, the relative amount of beta-F1-ATPase protein in liver homogenates varied (0-30% reduction) due to the hypothyroid condition during development. These findings suggest the existence of compensatory mechanisms operating at the translational and/or post-translational levels which promote proliferation of mitochondria in the hypothyroid liver. However, when the liver mass was considered, hypothyroidism significantly reduced overall mitochondrial proliferation in rat liver. Interestingly, the effects of thyroid hormones on the biogenesis of the ATP synthase complex at latter stages of development provide an example in which the hypothyroid condition limits the expression of the nuclear encoded gene with no apparent effect on the expression of the mitochondrial encoded genes (ATP synthase subunits 6-8). PMID- 7556181 TI - The (2'-5')oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges. Demonstration of the existence and identification of its reaction products. AB - We have proved the presence of (2'-5')oligoadenylates [(2'-5')An] and oligoadenylate synthetase [(2'-5')An synthetase] in the marine sponge Geodia cydonium. (2'-5')An isolated from sponge crude extract competed with authentic (2'-5')An for binding to polyclonal antiserum against (2'-5')An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2'-5')A oligomers. The biological activity of sponge (2'-5')An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2'-5')An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2'-5')An synthetase from sponge extract binds to poly(I).poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43-kDa (2'-5')An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2'-5')An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control. PMID- 7556184 TI - Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus. AB - Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid. PMID- 7556183 TI - The macromolecular structure of type-VI collagen. Formation and stability of filaments. AB - Type-VI collagen microfilaments were directly isolated from human amnion without using strong denaturing reagents. The microfilaments were characterized by electron microscopy, SDS/PAGE and immunoprecipitation. There was no evidence of other components bound to the isolated filaments and no covalent bonds between adjacent tetramers. The association between tetramers was further analyzed by studying the affinity between globular domains and the helix of type-VI collagen. Solid-phase-binding assays and conventional column chromatography showed that the globular domains have a high affinity for each other and for the helices of type VI collagen, indicating that filaments may be assembled and stabilized in the absence of additional components. Hyaluronan did not stabilize the filaments nor facilitate the assembly of tetramers into filaments. The interaction between domains was also studied after modifying the sugar moieties of type-VI collagen globular domains and monomeric triple-helical domains. The oligosaccharides are involved in helix-helix interactions but not in interactions of the globular domains with each other or with the triple helix. PMID- 7556182 TI - Nitric-oxide-mediated activation of iron-regulatory protein controls hepatic iron metabolism during acute inflammation. AB - The molecular regulation of intracellular iron metabolism has been studied in the livers of rats undergoing an acute inflammatory reaction following turpentine injection. Treatment induced an increase in the steady-state level of the transferrin receptor (TfR) mRNA, peaking 18 h after treatment and returning to control levels 24 h after treatment, with no change in TfR gene transcription. RNA band-shift assays documented an activation of the cytoplasmic RNA-binding protein called the iron-regulatory protein (IRP), in parallel with a rise in the amount of TfR transcripts. A 2-3-fold increase in the amount of H and L ferritin subunit mRNAs was found 12-18 h after turpentine treatment. Surprisingly, higher accumulation of ferritin mRNAs did not result in appreciable differences in the liver ferritin content. This might be due to the concomitant rise of IRP activity, which is known to prevent ferritin mRNA translation. The absence of significant changes in the total iron and ferritin contents prompted us to investigate the role of nitric oxide (NO), an inflammatory mediator which is also known to modulate the activity of IRP. Northern-blot analysis showed a marked enhancement in the expression of the inducible form of nitric oxide synthase mRNA in turpentine-treated rats. Furthermore, the activation of IRP and the increase of the TfR mRNA content that occur in turpentine-treated rats were abolished by treatment with N5-nitro-L-arginine, a specific nitric oxide synthase inhibitor. The present data suggest that NO-mediated activation of IRP regulates alterations of hepatic iron homeostasis that occur in acute inflammation. PMID- 7556186 TI - Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose 4,6-cyclophosphate. AB - The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6 dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6 cyclophosphate and having the following structure [structure: see text] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate. PMID- 7556187 TI - Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region. AB - The product of the c-myc proto-oncogene, Myc, has been implicated in the transcriptional regulation of several genes, acting either as an activator or repressor of gene expression. To determine whether Myc is involved in the modulation of the expression of the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene, we used both stable cell lines overexpressing Myc and transient co transfection assays. We show that the endogenous C/EBP alpha protein level is repressed in stable cell lines overexpressing Myc. We also show that enforced expression of Myc in mouse hibernoma HIB-1B cells dramatically repressed the expression of C/EBP alpha--promoter-reporter fusion genes. This effect of Myc was mediated through the core promoter region. Mutation of the initiator site could not abolish this affect, indicating that Myc may interact with some component(s) of the basal transcription machinery. PMID- 7556188 TI - Antisense effects of cholesterol-oligodeoxynucleotide conjugates associated with poly(alkylcyanoacrylate) nanoparticles. AB - Oligonucleotides covalently attached to a cholesteryl moiety are more stable in biological media and better taken up by eukaryotic cells. However, their anchoring in hydrophobic cellular membranes and endosomes after endocytosis restricts their access to cellular nucleic acids. New methods of cellular delivery and the biological activity of the conjugates were studied. The cholesteryl residue was conjugated via disulfide bond to the 5' or 3' terminal phosphate group of two oligodeoxyribonucleotide dodecamers complementary to the mutated region of Ha-ras oncogene mRNA. The conjugates were able to form complementary duplexes with the mutated 27-b target fragment of mRNA but not with the wild-type sequence. Efficient sequence-specific RNase H cleavage of complementary mRNA was induced with low (< or = 500 nM) concentrations of the conjugates. At higher concentrations, this cleavage was progressively inhibited, probably due to an interaction between RNase H and the cholesterol residue. The hydrophobic conjugates could be adsorbed onto poly(isohexylcyanoacrylate) nanoparticles via their cholesteryl moieties and delivered to eukaryotic cells. Cholesterol-conjugated oligonucleotides were able to sequence-specifically inhibit the proliferation of T24 human bladder carcinoma cells in culture. PMID- 7556185 TI - The pH dependence and modification by diethyl pyrocarbonate of isocitrate lyase from Phycomyces blakesleeanus. AB - We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaxpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units. Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second-order rate constant of 18.58 M-1 s-1 (at pH 6.0 and 20 degrees C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N-carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O-carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N-carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data protection analyses indicate that the reactive histidine residues are within the active site of the enzyme. PMID- 7556190 TI - Na+/K(+)-ATPase with a blocked E1ATP site still allows backdoor phosphorylation of the E2ATP site. AB - The role of simultaneously existing ATP-binding sites in the catalytic process of Na+/K(+)-ATPase is unclear. In order to learn whether blocking the E1ATP site affects the properties of the E2ATP site, the E1ATP site was inactivated by either fluorescein 5'-isothiocyanate, the non-phosphorylating Cr(H2O)4AdoPP[CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring 'backdoor phosphorylation' in the presence of ouabain, or K(+)-activated hydrolysis of p-nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5' isothiocyanate or the non-phosphorylating Cr(H2O)4AdoPP[CH2]P, backdoor phosphorylation and the activity of K(+)-activated p-nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5'-isothiocyanate or Cr(H2O)4AdoPP[CH2]P decreased the affinity of the p-nitrophenylphosphatase activity of the E2ATP site for the substrate p nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5'-isothiocyanate-inactivated Na+/K(+)-ATPase has a lowered sensitivity for ATP [Scheiner-Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592-9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K(+)-dependent p-nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K(+)-ATPase, and binding of ATP to one site affects the property of the other site [Scheiner-Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173-178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site. PMID- 7556189 TI - Tissue-type transglutaminase from red sea bream (Pagrus major). Sequence analysis of the cDNA and functional expression in Escherichia coli. AB - A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43% identity to the sequence of guinea pig liver TGase, revealing a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28 degrees C), E. coli cells transformed with pTTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of epsilon-(gamma-glutamyl)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials. PMID- 7556193 TI - Elicitors and suppressors of hydroxyproline-rich glycoprotein accumulation are solubilized from plant cell walls by endopolygalacturonase. AB - Treatment of bean cell walls with a pure endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum race beta released oligogalacturonides and pectic fragments which were separated according to their charge and size. Among galacturonic-acid-containing components, elicitors and suppressors of the plant cell wall hydroxyproline-rich glycoprotein (HRGP) were recovered. Two active small oligogalacturonides with degrees of polymerization of 2 and 3 were characterized by high-performance anion-exchange-chromatography pulsed amperometric detection and fast-atom-bombardment mass spectrometry; they elicited 40-70% hydroxyproline increase within 48 hours at 450 nmol/bean cutting. In contrast, pectic fragments of higher molecular mass, predominantly composed of galacturonic acid and containing sugars typical of the rhamnogalacturonan II domain of pectic polysaccharides, had the ability to substantially suppress hydroxyproline deposition. Maximum suppressor activity, 30-40% below the activity of the control, occurred in 48 hours. In view of the low one-cycle turnover of these proteins in the cell wall and of their structural role, these changes might significantly affect cell wall properties. Elicitation and/or suppression of hydroxyproline were correlated to modifications of HRGP-extensin gene expression. Northern-blot analysis of RNA showed that changes in the transcript intensity became clearly visible within the first 12 hours after the start of either treatment. The results show that pectic components of the plant extracellular matrix have the potential to regulate wall matrix biogenesis. Implications of this finding in plant defense and development are discussed. PMID- 7556191 TI - Involvement of retinoic acid receptor alpha in the stimulation of tissue-type plasminogen-activator gene expression in human endothelial cells. AB - Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids. PMID- 7556192 TI - Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. AB - Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. In conjunction with the relatively low costs for isotopes, manpower and NMR instrument time, this makes biosynthetic fractional 13C labeling of proteinogenic amino acids particularly attractive to support process design and metabolic engineering in biotechnology, since screening procedures become feasible which enable a systematic characterization of the cell's metabolic state as a function of parameters that are involved in the optimization of biotechnological processes. PMID- 7556194 TI - Point mutations in the hydrophobic C-terminal region of ricin A chain indicate that Pro250 plays a key role in membrane translocation. AB - A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps. PMID- 7556195 TI - Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding protein-coupled receptors. AB - beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide binding proteins (G-protein) subsequent to receptor phosphorylation by the beta adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors. PMID- 7556196 TI - A nuclear protein, synthesized in growth-arrested human hepatoblastoma cells, is a novel member of the short-chain alcohol dehydrogenase family. AB - We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed. PMID- 7556197 TI - Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation. AB - The biosynthesis of Pep5, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Pep5 synthesis in the homologous host Staphylococcus epidermidis 5 which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pepI [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H.-G. (1994) Appl. Env. Microbiol. 60, 2876-2883], a gene pepT coding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Pep5 biosynthesis. Deletion of PepT reduced Pep5 production to about 10%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly processed Pep5 fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Pep5 but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine. PMID- 7556198 TI - NMR studies of the major coat protein of bacteriophage M13. Structural information of gVIIIp in dodecylphosphocholine micelles. AB - The membrane-bound form of the major coat protein (gVIIIp) of bacteriophage M13 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J-coupling constants. The protein consists of two alpha-helices which are connected by a hinge region around residue 21. From spin-label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. Comparison of the data of gVIIIp in DodPCho micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski, M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322-8328; Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M. (1994) Biochemistry 33, 12,990-12,997] reveals that the structures of the protein in the two detergent micelles are very similar. They differ only in the arrangement of the detergent molecules around the protein. For gVIIIp in SDS micelles, we found a micellar structure which is distorted near the C-terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules. PMID- 7556199 TI - Characterization of the prismane protein from Desulfovibrio vulgaris (Hildenborough) by low-temperature magnetic circular dichroic spectroscopy. AB - The prismane protein of Desulfovibrio vulgaris (Hildenborough) contains a putative [6Fe-6S] cluster. This novel iron-sulfur cluster has been characterized here by magnetic circular dichroism (MCD) spectroscopy. Three paramagnetic redox states of the cluster, [6Fe-6S]5+, [6Fe-6S]4+ and [6Fe-6S]3+, each show a distinctive low-temperature MCD spectrum which is unlike that observed for any other iron-sulfur clusters. Magnetization data for the prismane protein in these three redox states indicate ground state spins that are in accordance with previous EPR assignments. For the protein as isolated, with the [6Fe-6S]5+ form of the cluster, magnetizations show an exceptionally steep initial slope that can be fit to a ground state of spin S = 9/2. For the semi-reduced protein, the cluster in the [6Fe-6S]4+ form, magnetizations show an initial slope characteristic for a ground state of spin S = 4. For the dithionite-reduced protein, with the [6Fe-6S]3+ form of the cluster, magnetizations are typical for a ground state of spin S = 1/2. PMID- 7556200 TI - Refined solution structure of the Tyr41-->His mutant of the M13 gene V protein. A comparison with the crystal structure. AB - The three-dimensional solution structure of mutant Tyr41-->His of the single stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been refined in two stages. The first stage involved the collection of additional NOE-based distance constraints, which were then used in eight cycles of back-calculations and structure calculations. The structures of the gene V protein dimers were calculated using simulated annealing, employing restrained molecular dynamics with a geometric force field. In the second stage of the refinement procedure, solvent was explicitly included during the dynamic calculations. A total of 30 structures was calculated for the protein, representing its solution structure in water. The first calculation step significantly improved the convergence of the structures, whereas the subsequent simulations in water made the structures physically more realistic. This is, for instance, illustrated by the number of hydrogen bonds formed in the molecule, which increased considerably upon going to aqueous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wild-type molecule, except for the DNA-binding loop (residues 16-28). This antiparallel beta-hairpin is twisted and partially folded back towards the core of the protein in the NMR structure, whereas it is more extended and points away from the rest of the molecule in the X-ray structure. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution. PMID- 7556202 TI - Determination of haem electronic structure in cytochrome b5 and metcyanomyoglobin. AB - The paramagnetic shifts of 13C nuclei positioned alpha to the haems in the A and B forms of rat cytochrome b5 and in metcyanomyoglobin have been analysed in terms of molecular orbitals based on D4h symmetry with a rhombic perturbation. The contribution to the 13C shifts from pseudocontact interactions is calculated from parameters obtained for a metal-centred dipolar shift tensor by fitting 1H shifts. The effect of electron delocalisation onto the vinyl groups of these haems b is separated with reference to the shifts of the vinyl beta carbons. In each case, it was found that the orientation of the magnetic axes in the plane of the haem is rotated away from the iron-nitrogen vectors in the opposite sense to the rotation of the rhombic perturbation and the molecular orbitals. The orientation of the orbitals is closely aligned with the normal to the single His ligand in metcyanomyoglobin, and with the average of the two normals in the bis His cytochrome b5. It is concluded that the in-plane anisotropy of haems b is dominated by the orientation of the axial ligands in a similar manner to that in haems c and that the approximations used are weakened, but not invalidated, by the presence of partially conjugated vinyl groups. PMID- 7556201 TI - Conformational studies of osteocalcin in solution. AB - 1H-NMR and circular dichroism studies have been carried out on osteocalcin, a 49 residue, calcium-binding protein, the sequence of which contains a disulphide bridge, a proline-rich segment and three gamma-carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an alpha helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the alpha helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca(2+) free protein. 1H-NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca(2+)-bound form) and have the gamma-carboxyglutamic acid side chains exposed on one face of the molecule. PMID- 7556203 TI - Template-directed protein folding into a metastable state of increased activity. AB - The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P'1Ser-->Pro variant in the trypsin-reactive subdomain differ before and after refolding on trypsin-Sepharose. A heterogenous and thermodynamically dominant population of conformers was attained in solution. However, the enzyme inhibitory activity of the variant was dramatically increased in the presence of trypsin-Sepharose and a stoichiometric ratio of the two subdomains was obtained as expected for a single conformation. The subsequent return for the initial mixture of conformers in solution reveals a high kinetic barrier late in the folding process. The template facilitates folding kinetically, as shown by a rate acceleration of more than four orders of magnitude. The final state was also the thermodynamically favoured one on the template, due to its increased affinity for the enzyme. The long-range effects on folding kinetics and the partial activity, and the absence of free sulfhydryl groups after refolding in solution indicate rearrangements between closely related conformers late in folding. The importance of minor structural distortions in immobilized trypsin suggests a close structural analogy between the final and the transition state of protein folding. PMID- 7556204 TI - Quinoline 2-oxidoreductase and 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase from Comamonas testosteroni 63. The first two enzymes in quinoline and 3 methylquinoline degradation. AB - The enzymes catalysing the first two steps of quinoline and 3-methylquinoline degradation by Comamonas testosteroni 63 were investigated. Quinoline 2 oxidoreductase, which catalyses the hydroxylation of (3-methyl-)quinoline to (3 methyl-)2-oxo-1,2-dihydroquinoline, was purified to apparent homogeneity. The native enzyme, with a molecular mass of 360 kDa, is composed of three non identical subunits (87, 32, and 22 kDa), occurring in a ratio of 1.16:1:0.83. Containing FAD, molybdenum, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins. Molybdopterin cytosine dinucleotide is the organic part of the pterin molybdenum cofactor. Comparison of N-terminal amino acid sequences revealed similarities to a number of procaryotic molybdenum-containing hydroxylases. Especially the N termini of the beta-subunits of the quinoline 2-oxidoreductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec. B1, and of quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec. 1B showed striking similarities. Further degradation of (3-methyl-)2-oxo-1,2 dihydroquinoline proceeds via dioxygenation at the benzene ring, i.e. at 5,6 position [Schach, S., Schwarz, G., Fetzner, S. & Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 175-181]. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially purified; NADH and oxygen are required for the reaction, and the enzymic activity is enhanced 1.5-fold by addition of Fe2+ ions. Unexpectedly, this aromatic ring dioxygenase did not separate into distinct protein components, but is apparently a single-component enzyme. The molecular mass was estimated to be about 260 kDa. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is very thermolabile. However, dithioerythritol and low concentrations of substrate had a moderately stabilizing effect. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-blocking agents, by metal-chelating agents, and by the flavin analogues quinacrine and acriflavin. PMID- 7556205 TI - Phytanic acid alpha-oxidation in rat liver peroxisomes. Production of alpha hydroxyphytanoyl-CoA and formate is enhanced by dioxygenase cofactors. AB - Patients with generalized peroxisomal disorders, rhizomelic chondrodysplasia punctata, and Refsum disease are all unable to alpha-oxidize 3,7,11,15 tetramethylhexadecanoic (phytanic) acid. The exact cause of the oxidation defect in these patients is not well characterized, in part because there is only limited knowledge of the biochemical pathway. In 1969, the alpha-oxidation of phytanic acid was reported [Tsai, S.-C., Avigan, J. & Steinberg, D. (1969) Studies on the alpha-oxidation of phytanic acid by rat liver mitochondria, J. Biol. Chem. 244, 2682-2692] to involve the formation of an alpha-hydroxyphytanic acid intermediate prior to removal of the alpha carbon. Subsequently, most researchers have had difficulty detecting this intermediate. In the present study, cofactors known to form hydroxy intermediates by both monooxygenase and dioxygenase reaction mechanisms were incubated with purified rat liver peroxisomes and either [2,3-3H]phytanic acid or [1-14C]phytanic acid. Reaction products were separated by reverse-phase HPLC. A single reaction product, identified as alpha-hydroxyphytanoyl-CoA rather than the free fatty acid, was detected when 2-oxoglutarate/Fe+2/ascorbate, cofactors associated with a dioxygenase reaction mechanism, were present. Concomitant with alpha hydroxyphytanoyl-CoA production, there was an increased accumulation of formate and CO2. This increase in alpha-oxidation products is evidence that alpha hydroxyphytanoyl-CoA is a true pathway intermediate and that the entire pathway functions in peroxisomes. In contrast, alpha-hydroxyphytanoyl-CoA was not formed in any quantity in mitochondria. These studies suggest that the alpha hydroxylation step of phytanic acid oxidation, which has been shown to be defective in Refsum disease, is located in peroxisomes. PMID- 7556206 TI - Structures of the O-specific polysaccharide chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis lipopolysaccharides. AB - Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6-deoxy-L-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 1H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O-deacylated polysaccharide, which was studied by methylation analysis, 1H-NMR and 13C-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear 13C, 1H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the O-specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6-deoxy-L-altrofuranose built up of tetrasaccharide-repeating units having the following structure: [sequence: see text] Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glucose and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy showed that the O-specific polysaccharide of P. cerevisiiphilus has the following structure: -->2)-beta-D-Fucf-(1-->2)-alpha-D-Glcp-(1-->. PMID- 7556207 TI - The structure and serological specificity of Proteus mirabilis O43 O antigen. AB - On the basis of sugar analysis and NMR spectroscopy, including selective spin decoupling, one-dimensional NOE, two-dimensional homonuclear and 13C,1H heteronuclear correlation spectroscopy, the following structure of the acidic O specific polysaccharide of Proteus mirabilis O43 was established: -->4)-alpha-D GalpA-(1-->3)-alpha-D-GalpA-(1-->3)-alpha-D-Gl cpNAc-(1-->4)-alpha - D-Glcp-(1- >, where GalA is galacturonic acid and Galp is galactopyranose. No serological cross-reactivity was observed between lipopolysaccharides of P. mirabilis O43 and other studied Proteus strains, except for P. mirabilis O10. The O-specific polysaccharide of P. mirabilis O43 was serologically active in precipitation and inhibition tests but the activity was lost after periodate oxidation. These data suggest that the O43 specificity is determined by a wide epitope with the immunodominant role of 4-substituted D-Glc or/and D-GalA, which are destroyed by periodate oxidation. PMID- 7556208 TI - EPR characterization of an archaeal succinate dehydrogenase in the membrane-bound state. AB - The membrane-bound succinate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum was characterized by EPR spectroscopy and its functional properties were determined. The highest turnover values of succinate dehydrogenase activity were observed at pH 7.4, which is somewhat above the internal pH value of T. acidophilum. The temperature optimum of the reaction was determined as 78 degrees C and the Km value for succinate using phenazine methosulfate as the electron acceptor at 53 degrees C was 0.32 mM. The membrane bound enzyme was able to reduce the artificial electron acceptors phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,6 dichloroindophenol. Succinate oxidation was coupled to oxygen consumption in a completely 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive manner. In the oxidized state, T. acidophilum membranes exhibited an almost isotropic EPR spectrum with g-values at gz = 2.017, gy = 2.000, and gx = 1.968 that were assigned to a [3Fe-4S]1+ cluster (S3). Upon reduction with succinate, the membranes displayed a spectrum characteristic of 2Fe-2S clusters (S1), with g values at gz = 2.029, gy = 1.935, and gx = 1.915. In the dithionite-reduced state, additional resonances can be observed. An axial component, with g-values at gz = 2.057, gy = 1.917, and gx = 1.917 was assigned to a [4Fe-4S]1+ cluster. The saturation behaviour of the S1 cluster was strongly altered in the dithionite reduced form, thus indicating spin-spin interaction between the S1 center and another paramagnetic center, possibly cluster S2. In both the succinate and the dithionite-reduced membranes, parallel-mode EPR spectra displayed a resonance at g = 14, which may be due to a transition of the S = 2 multiplet of the reduced 3Fe-4S cluster. Spin quantitation yielded a relative stoichiometry of cluster S1 to cluster S3 of 1:1. The results obtained by EPR spectroscopy indicated that the characteristic iron-sulfur cluster S1 [2Fe-2S], S2 [4Fe-4S], and S3 [3Fe-4S], were also present in this archaeal succinate dehydrogenase. EPR redox titrations of T. acidophilum membranes at pH 5.5 yielded a reduction potential of +60 +/- 20 mV for cluster S3 and of +68 +/- 20 mV for cluster S1. The axial [4Fe-4S]2+/1+ center had a reduction potential of -210 +/- 20 mV. PMID- 7556209 TI - Macromolecules increase the channeling of ADP from externally associated hexokinase to the matrix of mitochondria. AB - Macromolecules can restore the morphological changes in the outer mitochondrial compartment that occur upon isolation of the organelle. They decrease the volume of the intermembrane space and increase the number of intermembrane contact sites. In this study, we investigated the effects of macromolecules on one of the processes occurring in the mitochondrial outer compartment and for which the native structure might be important, i.e. the ADP supply from outer-membrane bound hexokinase-I to oxidative phosphorylation. With the use of a reconstituted system in which rat liver mitochondria and extramitochondrial pyruvate kinase compete for ADP generated by hexokinase, it was shown that (a) part of the ADP generated by mitochondrially associated hexokinase is not accessible to pyruvate kinase and is channeled into the mitochondrion, (b) in the presence of 10% (mass/vol.) macromolecules (i.e. dextran M20 or BSA) the pyruvate kinase inaccessible fraction increases from 19% to 31% of the ADP produced by hexokinase, (c) the ADP channeling is a characteristic property of bound hexokinase, and (d) the increased channeling induced by macromolecules can neither be explained by direct effects of these macromolecules on the basic respiratory properties of rat liver mitochondria, nor by direct effects of the kinetic properties of hexokinase-I. ATP and ADP determinations were performed in hexokinase/mitochondria incubation mixtures in the presence of macromolecules. These determinations showed that an important consequence of the channeling capacity of bound hexokinase is that lower extramitochondrial ADP levels and consequently higher extramitochondrial ATP/ADP ratios are maintained than when hexokinase is not bound. The experimental data demonstrate that the ADP channeling activity associated with bound hexokinase leads to the formation of two ADP concentration gradients, one across the outer membrane and one between bound hexokinase and the bulk phase. PMID- 7556211 TI - Allosteric regulation of proton translocation by a vacuolar adenosinetriphosphatase. AB - The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosine-triphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reconstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6-chloro-2-methoxyacridine; fluorescence data were collected and stored in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence of initial rates of proton translocation on substrate concentration were fitted well by the Michaelis-Menten equation, as were the kinetics of ATP hydrolysis. ADP and other nucleoside diphosphates were potent inhibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves which could be fitted by the empirical Hill equation, the Hill coefficient approaching 2 at high inhibitor concentrations. Data sets containing initial-rate estimates were collected over a wide range of independently varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on the concerted-transition model of allosteric inhibition proposed by Monod, Wyman and Changeux. These equations were fitted to the data by weighted non-linear regression, using an iterative computer program to obtain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions: that the ATPase exists in two conformational states, R and T; that only the R state is catalytically active; that each state contains three kinetically equivalent catalytic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind both to the catalytic sites and to the regulatory site. The optimized values of the kinetic parameters indicate that in the absence of nucleoside diphosphate, the enzyme is almost completely in the R state; that nucleoside triphosphates bind more tightly to the R than to the T state; that binding of nucleoside diphosphates to the regulatory site is very tight, but occurs only in the T state; and that competitive binding of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nucleoside diphosphates are much stronger inhibitors than the free nucleotides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the catalytic sites.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556210 TI - Interaction of mitochondrial F1-ATPase with trinitrophenyl derivatives of ATP. Photoaffinity labeling of binding sites with 2-azido-2',3'-O-(4,6 trinitrophenyl)adenosine 5'-triphosphate. AB - It was shown recently that ATP present at near saturating concentrations did not prevent binding and hydrolysis of submicromolar concentration of trinitrophenyl adenosine triphosphate (Tnp-ATP) by F1-ATPase [Murataliev, M. B. & Boyer, P. O. (1994) J. Biol. Chem. 269, 15431-15439]. To explore F1-ATPase binding sites that bind Tnp-ATP a new photoreactive analog of ATP, 2-azido-trinitrophenyl adenosine triphosphate (2-N3-Tnp-ATP) has been synthesized and used for photoaffinity labeling of mitochondrial F1-ATPase. The analog shares many properties of the parent non-azido Tnp-ATP as shown from spectral characteristics, binding with F1 ATPase, and kinetic and inhibition studies. 500 microM ATP does not prevent binding and hydrolysis of low concentrations of 2-N3-Tnp-ATP by F1-ATPase. Photoirradiation of the enzyme-analog complex formed under such conditions results in the labeling of the catalytic-site peptide. This shows that in the presence of near saturating ATP, Tnp-ATP can enter the catalytic cycle and inhibit ATP hydrolysis by initial binding at a third catalytic site. The results give strong evidence that only two catalytic sites need to have bound substrate for near maximal turnover rate, and that three catalytic sites of F1-ATPase participate equally in catalysis. When F1-ATPase binds substoichiometric 2-N3-Tnp ATP in the presence of Mg2+, illumination of the inactive complex formed results in the covalent labeling of a catalytic site. This shows that F1-ATPase forms similar inactive complexes when ADP or Tnp-ADP is bound at a catalytic site in the presence of Mg2+. Exposure of the nucleotide-depleted F1-ATPase to 20 microM 2-N3-Tnp-ATP followed by a short incubation with excess of Tnp-ATP results in binding, and, upon illumination, in a covalent labeling of a non-catalytic-site peptide. PMID- 7556212 TI - In vivo synthesis of ATPase complexes of Propionigenium modestum and Escherichia coli and analysis of their function. AB - Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole unc operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F1 part was not functional. Transformants expressing an E. coli F1-P. modestum F0 hybrid exhibited considerable ATPase activities. Binding of the F1 part to the membrane was very weak, however, and the coupling between ATP hydrolysis and Na+ transport was impaired. After combining the genes for E. coli ATPase subunits alpha, beta, gamma, delta and epsilon and the hydrophilic part of subunit b with P. modestum ATPase subunits a and c and the hydrophobic part of subunit b on a plasmid, a non functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was solubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F0 subunits detectable in the purified enzyme. A stable F0 complex is thus not formed with the hybrid b subunit. The absence of a functional F0 complex was in accord with proton-conduction measurements with bacterial vesicles. The only functional Na(+)-translocating ATPase expressed in E. coli thus far consists of E. coli subunits alpha, beta, gamma and epsilon, and P. modestum subunits delta, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem. 218, 937-944]. During the cloning conducted in our present study, errors in the sequence entry into the EMBL data bank (accession no. X58461) for the P. modestum ATPase alpha and beta subunits became evident, which are corrected in this paper. PMID- 7556213 TI - Annexin 1 is overexpressed and specifically secreted during experimentally induced colitis in rats. AB - Annexin 1 is a protein induced by glucocorticoids endowed with extracellular anti inflammatory properties. In this study, the local expression and secretion of annexins 1-6, in rat proximal colon, were studied at different times after intracolonic administration of 30 mg trinitrobenzenesulfonic acid in 50% ethanol. Secretion was identified by incubating colonic tissues in a culture medium. The expression of annexins was detected by immunoblotting in tissue homogenates and incubation media. Inflammatory stages were evaluated by measuring myeloperoxidase activity. Annexin 1 expression in colons increased after trinitrobenzenesulfonic acid treatment and was maximal between days 1 to 9, during the cellular stage of the inflammation that corresponded to maximal myeloperoxidase activity. From 12 h to 9 days after trinitrobenzenesulfonic acid/ethanol treatment, annexin 1 was specifically secreted. Annexin 3 was also overexpressed during the cellular stage, but the expression of annexins 2, 4, 5, and 6 was unchanged; none of these annexins were secreted. Annexin 1 was shown to be physiologically secreted because its release was specific, abundant, and not correlated with cellular lysis. Annexin 1 may be considered as a putative candidate in the control of the gut inflammatory processes. PMID- 7556214 TI - Anti-La monoclonal antibodies recognizing epitopes within the RNA-binding domain of the La protein show differential capacities to immunoprecipitate RNA associated La protein. AB - The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti-La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre-tRNA, pre-5S rRNA, hY RNAs, pre-U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7-2 RNA. Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross-competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3. Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA-binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts. PMID- 7556215 TI - A new approach to isolate genomic control regions. Application to the GATA transcription factor family. AB - We have designed a new strategy to isolate unknown DNA regions interacting with one or several related regulatory proteins. It involves trapping such DNAs by their cognate binding proteins followed by PCR amplification, as described previously [Kinzler, K. & Vogelstein, B. (1989) Nucleic Acids Res. 17, 3645 3653]. To overcome the inability of such a procedure to discriminate between functional and non-functional binding sites as well as to specifically trap short DNA motifs from the whole higher eukaryotic genome, we have used as starting material DNA isolated from transcriptionally competent chromatin fractions, instead of total genomic DNA. To test our strategy, we selected human DNA sequences that bind members of the GATA family, known to recognize similar WGATAR motifs. These proteins are expressed in different cell types in which they regulate the transcription of different sets of genes; thus, transcriptionally active chromatin containing GATA motifs should differ according to the cell type. We have trapped and analyzed DNA fragments isolated from an active chromatin fraction, from erythroid cells and lymphoid cells, using GATA-1 and GATA-3 proteins, respectively. We show that regulatory GATA sequences known to be in open chromatin in erythroid cells (typified by the HSIII fragment of the beta globin locus control region) or in lymphoid cells (typified by a fragment of the CD2 locus control region) are dramatically enriched in a cell-specific manner, demonstrating the potency of the method. The sequences of the erythroid or lymphoid DNA fragments isolated through the procedure described here were determined and display subset-site preference for GATA-1 and GATA-3. PMID- 7556216 TI - A silencer and an adjacent positive element interact to modulate the activity of the human insulin promoter. AB - A negative regulatory element (NRE) is located between positions -279 and -261 relative to the transcription start site of the human insulin gene. The NRE contains at least three distinct overlapping binding sites for several nuclear proteins. These proteins may be distinguished by their interaction with mutant variants of the NRE. Mutagenesis of two of these protein-binding sites within an insulin gene fragment containing sequences from position -361 to position +112 attenuated the negative activity of the NRE, confirming the importance of these sites in the function of the NRE. When placed in isolation upstream of the herpes simplex-virus thymidine-kinase promoter, the NRE exhibited stimulatory activity in non-beta (BHK) and beta (HIT) cells. The positive activity within the NRE was mapped to a sequence that resembled the binding site for the ubiquitous factor Oct-1. These results indicated that the negative activity of the NRE was dependent on its interaction with other regulatory sites within the insulin gene. Thus, the NRE exhibited negative activity in transfected HIT cells when placed upstream of an insulin gene fragment (positions -261 to +112). However, its activity was modulated in a positive manner by inclusion of additional sequences from position -279 to -341. This region contains the CT3 box that binds the homeodomain protein IUF1 (insulin upstream factor 1). The NRE resembled a silencer, being at least partly independent of precise location and orientation, and being able to operate upon a variety of promoters. The role of the NRE is unclear, although it may be involved in restricting expression of the insulin gene to cells of the islets of Langerhans. PMID- 7556217 TI - The sulfakinins of the blowfly Calliphora vomitoria. Peptide isolation, gene cloning and expression studies. AB - The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide. Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His- Met-Arg Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)- Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide. His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurons of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous. PMID- 7556218 TI - Thermodynamic analysis of the binding of 5-fluoro-2'-deoxyuridine 5' monophosphate to thymidylate synthase over a range of temperatures. AB - The binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16-35 degrees C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases. A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16-35 degrees C. The enthalpy changes become more negative as the temperature increases, with delta Cp = -170 +/- 20 J.K-1.(mol FdUMP bound)-1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer. PMID- 7556219 TI - Properties of rabbit liver aldehyde oxidase and the relationship of the enzyme to xanthine oxidase and dehydrogenase. AB - The properties of the molybdenum iron-sulfur flavoprotein, aldehyde oxidase from rabbit livers, have been further investigated in comparison with bovine milk xanthine oxidase. In agreement with earlier work, the ultraviolet/visible spectra indicate that the flavin and iron-sulfur centres of the enzymes are quite similar to one another. The molybdenum centres have been compared by EPR spectroscopy of molybdenum(V) and regarding re-insertion of the sulfido ligand of molybdenum into the desulfo enzyme forms. The pH optimum for sulfide insertion is approximately 2 lower for aldehyde oxidase than for xanthine oxidase. A detailed comparison of molybdenum(V) EPR signals has been made for the signals known as Arsenite, Slow and Rapid. Computer simulation of spectra in 1H2O and 2H2O, at 9 and 35 GHz was used. Slow signals from the two enzymes are scarcely distinguishable from one another. Under the conditions used, aldehyde oxidase yielded only the Rapid type 2 signal, whereas xanthine oxidase gives both the Rapid type 1 and 2 signals. The nature of the structural difference between the Rapid type 1 and type 2 signal giving species is discussed. It is concluded that the molybdenum centres of xanthine oxidase and aldehyde oxidase are indeed similar to one another and that such differences as exist between their molybdenum(V) EPR signals and re sulfuration properties are related to differences only in the substrate-binding sites. N-terminal amino acid analyses have been performed on peptides obtained by trypsin cleavage of aldehyde oxidase. Comparison with a sequence previously deduced [Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S. & Repine, J. E. (1993) Proc. Natl Acad. Sci. USA 90, 10690-10694] makes it clear that the latter is not, as was assumed, that of a xanthine dehydrogenase but of an aldehyde oxidase. In contrast to the situation with xanthine oxidase, attempts to convert non-proteolysed aldehyde oxidase to a dehydrogenase form by treatment with dithiothreitol were unsuccessful. The reason for this is considered in the light of sequence data in the literature. The location of the NAD(+)-binding site is discussed, and the sequence data are also discussed in relation to the molybdenum, iron-sulfur and substrate-binding sites. PMID- 7556220 TI - Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling with synthetic inhibitors. AB - Kinetic parameters for the inactivation of the 6-phospho-beta-galactosidase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of 2,4-dinitrophenyl-2 deoxy-2-halogeno- galactoside-6-phosphates have been determined. These inhibitors function by the formation of a stabilised glycosyl-enzyme intermediate. Inactivation and reactivation studies indicate that the fluoro derivative is formed most rapidly, but is also hydrolysed fastest. The chloro derivative forms the most stable covalent intermediate. HPLC profiles of V8-protease digestion of native and inhibited protein show significant differences, whereas the inhibited 6-phospho-beta-galactosidase and a point mutant of 6-phospho-beta- galactosidase (E375Q) yield the same proteolytic fragments. The suggestion that E375 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two peptides, residues 336-375 and 376-383, are not produced, due to the absence of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of the respective V8 cleavage site due to the chemical reaction of the inhibitor at position 375. Specific modification of the glycosyl bond between the inhibitor and E375 by aminolysis with benzylamine generated a glutamatic-acid-5-benzylamide complex at that position in the peptide. The Edman derivative of the modified E375 appears to be stable and was isolated by Edman degradation of trypsin-digested V8-peptide. It was shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-galactosidase, consistent with previous findings for enzymes in this family. PMID- 7556221 TI - Characterization of a cAMP-binding protein from the bivalve mollusc Mytilus galloprovincialis. AB - Three cAMP-binding proteins have been identified by photoaffinity labeling with 8 azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two cAMP-binding sites/monomer, was judged to be a regulatory (R) subunit of cAMP-dependent protein kinase since it re-associated with and inhibited purified catalytic (C) subunit of this enzyme from mussel, in the absence but not in the presence of cAMP. The molecular mass of the complex between Mytilus cAMP-binding protein and C subunit, estimated by analytical gel-filtration, was 220 kDa, a value which agrees with a R2C2 stoichiometry for the mussel cAMP-dependent protein kinase holoenzyme. On the basis of the elution pattern from DEAE-cellulose chromatography and its ability to be phosphorylated by purified C subunit of cAMP dependent protein kinase, the 54-kDa protein could be classified as a type II regulatory subunit. Furthermore, no mobility shift on SDS/PAGE upon phosphorylation/dephosphorylation of Mytilus protein was observed, a similar behaviour to that shown by the mammalian RII beta isoform. The 42-kDa and 37-kDa proteins, which were recognized by a specific antiserum against the 54-kDa protein and fail to be phosphorylated by Mytilus C subunit, are probably products generated by proteolysis of the 54-kDa protein, although they were shown even when inhibitors of the major types of proteases were used. PMID- 7556222 TI - Expression of the subunits of protein kinase CK2 during oogenesis in Xenopus laevis. AB - Northern-blot analysis of RNAs from different tissues demonstrated that the mRNA for the protein kinase CK2 alpha subunit is very abundant in the ovary of Xenopus laevis. The competitive reverse-PCR technique has been used to quantitate the mRNA for both CK2 alpha and CK2 beta subunits during oogenesis. The results obtained using eight different animals consistently show an increment of 2-3-fold in the mRNA for both subunits in vitellogenic oocytes (stages II-VI). Each stage VI oocyte contains approximately 5 x 10(-7) molecules CK2 alpha mRNA and 1 x 10( 7) molecules CK2 beta mRNA. These amounts are considerably higher than many other mRNAs analyzed in these cells. Activity measurements of CK2 using casein or a specific model peptide revealed increments of about 10-12-fold during oogenesis, and also indicated that the amount of enzyme in the nucleus accounted for 15-30% of the total enzyme in the oocyte at all stages. Western-blot analysis of CK2 alpha indicated that the amount of this protein subunit also increased during oogenesis in a parallel fashion with the increment of enzymic activity. PMID- 7556223 TI - Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon. AB - Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins. PMID- 7556224 TI - Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme. AB - The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000 fold increase in the ratio of beta-decarboxylase/transaminase activity. PMID- 7556225 TI - Risk factors for systemic fungal infections in liver transplant recipients. AB - The risk factors for systemic fungal infections were analysed retrospectively in 186 orthotopic liver transplant procedures performed in 152 patients between June 1985 and January 1993. The total incidence of systemic fungal infections was 16.5% (25/152). The incidence of disseminated candidiasis, aspergillosis, and combined candidiasis and aspergillosis was 6.5% (n = 10), 7.2% (n = 11) and 2.6% (n = 4), respectively. Mortality associated with systemic fungal infections was 80% (20 of 25 patients). There were ten cases of disseminated candidiasis, with 4 patients surviving, and 11 cases of invasive aspergillosis, with 1 patient surviving. All patients with combined systemic fungal infection died. To identify perioperative risk factors, 39 variables were used to compare patients with systemic fungal infections versus those without fungal infections. Fourteen variables were significantly associated with systemic fungal infections by univariate analysis. A consecutive logistic regression analysis revealed that the amount of fresh frozen plasma transfused due to poor initial function of the allograft and acute renal failure requiring hemofiltration were independently significant risk factors for systemic fungal infections. There was no statistical correlation between systemic fungal infections and the underlying liver disease, previous long-term corticosteroids and the postoperative immunosuppressive therapy. Risk factors identified in this study should be considered in the postoperative care of the individual liver transplant recipient. In our study a poor initial function of the hepatic allograft substantially increased the risk of systemic fungal infection. PMID- 7556227 TI - Antibacterial properties of lansoprazole alone and in combination with antimicrobial agents against Helicobacter pylori. AB - The activities of various types of antiulcer agents against Helicobacter pylori strains were determined by an agar dilution method. Among the compounds tested, benzimidazole proton pump inhibitors were found to have significant activity against this organism. The activity of lansoprazole was fourfold more potent than that of omeprazole and bismuth subsalicylate, with MICs ranging from 1.56 to 25 micrograms/ml. Exposure of Helicobacter pylori to lansoprazole led to an extensive loss of viability as well as suppression of virulence factors such as motility, adhesiveness to epithelial cells and urease activity. The combination of lansoprazole with antimicrobial agents such as penicillins, cephalosporins, macrolides, tetracyclines, aminoglycosides, quinolones, metronidazole and bismuth subsalicylate generally had an additive effect on inhibition of Helicobacter pylori growth. PMID- 7556229 TI - Differential and enrichment media for selective culture and recognition of yeast species from clinical material. AB - An enrichment medium was developed and evaluated for isolation of fluconazole resistant minority yeast species in a hospital setting. The enrichment medium was made by adding fluconazole (10 micrograms/ml) to yeast nitrogen base/glucose broth. Under laboratory conditions the broth permitted detection of 20 of 20 Candida krusei isolates and 20 of 20 Candida glabrata isolates in mixtures with Candida albicans even when the Candida albicans cells outnumbered those of the other species by 1000:1. Results of culture on the enrichment medium were compared with those obtained on routine agar media and on a yeast differential agar which facilitates detection of mixed yeast species by their colony colours. Only one Candida glabrata isolate was detected on the enrichment broth but not found on routine culture of 68 yeast-positive clinical specimens. However, bacterial over-growth in some broths may have retarded the appearance of other yeast isolates. On the yeast differential agar, 20 clinical specimens were found to contain mixtures of yeast species compared with only 2 on routine culture. PMID- 7556228 TI - Prospective study of antigenemia, plasma viremia and lymphocytic viremia in HIV infected hemophiliacs. AB - A total of 186 blood samples from 24 HIV-1 seropositive hemophiliac patients, monitored every four months for 29 months, were investigated for the presence of viral antigen in plasma. In addition, peripheral blood mononuclear cells (PBMC) were cultured for HIV-1, using normal PBMC as a target for replication. Antigenemia was detected in 51% of the patients and from PBMC in 87.5% of the patients. The incidence of HIV isolation in asymptomatic patients (42.8%) was similar to that found in symptomatic patients (51.4%). Patients with opportunistic infections had a higher incidence of lymphocytic viremia (p < 0.05). Plasma viremia was closely associated (p < 0.05) with low CD4+ counts and infection progression. The persistence of antigenemia was also a marker of a poor clinical course. In treated patients, plasma viremia was the marker that better correlated with the clinical course, and it did not appear during the first nine months of therapy. Zidovudine doses of > 500 mg/day significantly lowered the appearance of antigenemia and lymphocytic viremia (p < 0.05). PMID- 7556226 TI - Pharmacodynamic effects of meropenem on gram-negative bacteria. AB - The in vitro initial killing and post-antibiotic effect (PAE) of meropenem on five gram-negative reference strains were evaluated by bioluminescence assay of bacterial adenosine triphosphate (ATP) and viable count. Morphology studies were performed in parallel. Meropenem showed concentration-dependent long (2-5 h) PAEs on Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens when assayed by bioluminescence and induced spheroplasts at almost all concentrations. The bioluminescence PAEs reached a maximum response at 4 x MIC. These PAEs of meropenem on Escherichia coli, Klebsiella pneumoniae and Serratia marcescens were longer than corresponding PAEs of imipenem shown in previous studies. The higher affinity of meropenem than imipenem for PBP 3 might explain the longer PAEs obtained with meropenem. However, there was only a very short PAE, no PAE or even a negative PAE when viable count was used as the initial value for the PAE calculation. A strong initial decrease in viability but a less pronounced change in intracellular ATP was registered. Since this initial change in cell numbers is the initial value for the PAE calculation, the length of PAE was highly method dependent. In summary, a strong initial killing and no PAE were shown using viable count as the initial value for the PAE calculation, but a weak initial killing and long PAEs were shown when bioluminescence was used throughout the experiments. PMID- 7556231 TI - Validation of an enzyme immunoassay for serodiagnosis of acute Q fever. AB - An enzyme immunoassay was validated for the serodiagnosis of acute Q fever. Minimum positive tests were determined for both serial dilutions and a single dilution of patient sera. To establish the specificity of the test, 152 serum samples were tested from individuals with no evidence of past Coxiella burnetii infection. Diagnostic titers were set at > or = 128 for the IgM and IgG responses to phase I, at > or = 512 for the IgM response to phase II and at > or = 1,024 for the IgG response to phase II Coxiella burnetii. These titers gave a false positive rate of < or = 1%. Alternatively, testing a single dilution of sera (1:128) gave specificities ranging from 97.3 to 98.7%. Tests with the greatest sensitivities, using serially diluted early convalescent-phase sera, were the IgM (84%) and IgG (80%) responses to phase II Coxiella burnetii. At a single serum dilution, 92% of early convalescent sera had a positive IgG response to phase II Coxiella burnetii. With a high specificity and good sensitivity, the EIA can be used to diagnose acute Q fever with a single convalescent serum specimen. The duration of a positive response was greater than five years. PMID- 7556234 TI - Use of norfloxacin for prevention of symptomatic urinary tract infection in chronically catheterized patients. AB - Thirty-four elderly inpatients with long-term urethral catheters were randomly assigned to receive either norfloxacin (200 mg/day) or placebo in a double-blind study with cross-over after three months. Twenty-three patients completed the entire study. Norfloxacin treatment was associated with a persistent decrease in gram-negative isolates (p < 0.005) and the acquisition of gram-positive norfloxacin-resistant flora. It also resulted in a highly statistically significant reduction of symptomatic urinary tract infections (1 vs. 12, p < 0.02), a decrease in catheter-associated local complications, obstructions and leakage (p < 0.05) and an improvement in the patients' general condition (p < 0.001). In conclusion, within the conditions of the present study, long-term suppression of gram-negative bacteriuria by norfloxacin reduced the incidence of catheter-related urinary tract infection and associated morbidity. PMID- 7556230 TI - Use of gas-liquid chromatography for subgrouping coagulase-negative staphylococci during a nosocomial sepsis outbreak. AB - Gas-liquid chromatographic (GLC) fatty acid profile correlation analysis was applied for the subgrouping of 169 coagulase-negative staphylococci collected during an outbreak of nosocomial sepsis in a hematologic unit. The fatty acid profile similarity index between six ciprofloxacin-resistant Staphylococcus epidermidis septicemia strains was as high as 98.39 +/- 0.68, indicating a high degree of resemblance. This finding corroborated the finding by conventional typing methods that the isolates shared the same strain characteristics and, therefore, could be derived from the same epidemiological origin. Further, the GLC fatty acid profiles were analyzed for coagulase-negative staphylococcal cutaneous isolates recovered from colonization cultures of the patients and personnel in that same unit. The similarity index between 88 ciprofloxacin resistant Staphylococcus epidermidis skin isolates with similar plasmid profiles was as high as 95.47 +/- 3.78, whereas the correlation coefficient between 45 ciprofloxacin-susceptible Staphylococcus epidermidis skin isolates with different plasmid profiles was only 85.23 +/- 10.82. Cluster analysis grouped the ciprofloxacin-resistant Staphylococcus epidermidis isolates into one distinct cluster, while most of the ciprofloxacin-susceptible Staphylococcus epidermidis isolates were grouped into two separate clusters. When compared with the plasmid profiling, the GLC method congruously grouped 127 (87%) of the 146 Staphylococcus epidermidis isolates, thereby suggesting its potential value in subgrouping coagulase-negative staphylococci during nosocomial outbreaks. PMID- 7556232 TI - Multi-laboratory comparison of eight commercially available Helicobacter pylori serology kits. Helicobacter pylori Serology Study Group. AB - The performance of eight commercially available EIA kits in detecting antibody to Helicobacter pylori was evaluated by a panel of 17 laboratories using serum from 59 patients selected from endoscopy clinics in Belgium, Ireland, Italy, the Netherlands and Switzerland. Each laboratory received a randomly numbered set of sera and was ignorant of the culture results of the patients. The performance of the kits was assessed in terms of diagnostic accuracy compared to culture (measured by sensitivity and specificity), the inter-laboratory variability in diagnostic accuracy and the number of laboratories that experienced problems in using the kits. Grey zone results, which are routinely used to highlight the uncertain interpretation of results that lie near the cut-off point between positive and negative diagnoses, were accounted for in the analysis. Laboratories experienced practical problems in using some kits, whilst other kits were found to have high inter-laboratory variation or low diagnostic accuracy. There was no single kit that performed better on every criterion than the others. The Orion kit was a good all-round performer, whilst the Roche kit was excellent at detecting positive results, although it had a slightly raised false-positive rate. PMID- 7556233 TI - Diagnosis of Toxoplasma gondii infection in AIDS patients by a tissue-culture technique. AB - Toxoplasma gondii infection was investigated in 14 AIDS patients with neurological involvement who showed clinical and computer tomographic scan signs suggestive of toxoplasmic encephalitis or extraneuronal localization suggestive of toxoplasmic infection. Blood, lung secretion (bronchoalveolar lavage and/or induced sputum sample) and cerebral spinal fluid (CSF) samples were cultured, and the results compared with the results of direct examination by Giemsa and by immunofluorescence of lung secretions and CSF. Toxoplasma gondii were observed directly in only four patients by immunofluorescence in bronchoalveolar lavage, induced sputum and CSF samples, and in three of these patients by Giemsa staining of bronchoalveolar lavage and CSF smears. In contrast, parasites were detected after 48 h in blood cultures from 11 of the 14 AIDS patients, in CSF cultures from eight of them and also in cultures of bronchoalveolar lavage and induced sputum from the six patients with respiratory and radiological features indicative of lung tissue damage. The findings indicate the value of tissue culture for diagnosis of toxoplasmosis as well as for monitoring the effects of treatment and also indicate that culture of induced sputum could be a powerful tool in establishing the incidence of pulmonary toxoplasmosis in AIDS patients. PMID- 7556235 TI - Successful treatment of a brain abscess due to Trichoderma longibrachiatum after surgical resection. AB - A case of brain abscess due to Trichoderma longibrachiatum in a leukemic patient with prolonged neutropenia is reported. Definitive cure was achieved after neurosurgical resection of the abscess and prolonged antifungal therapy. Trichoderma is a filamentous fungus species, which is only exceptionally pathogenic in humans. This genus and particularly the species Trichoderma longibrachiatum should be added to the growing list of fungi causing infection in immunocompromised patients. PMID- 7556237 TI - Isolation of a Bordetella avium-like organism from a human specimen. AB - The isolation of a strain of Bordetella for which the species could not be determined but which most closely resembled Bordetella avium is reported. The strain was isolated in mixed culture from an ear swab of a patient suffering from chronic otitis media. The bacterium showed the typical biochemical reactions of Bordetella avium but differed in antimicrobial resistance pattern, protein and fatty acid composition, and DNA-DNA and DNA-rRNA hybridization. Further studies will clarify the taxonomic status of this strain within the Bordetella Alcaligenes ribosomal RNA cluster. PMID- 7556238 TI - Systemic infections in three infants due to a lactose-fermenting strain of Salmonella virchow. AB - Three previously healthy children developed gastroenteritis which led within a few days to systemic infections, two cases of bacteremia and one of meningitis. A lactose-fermenting Salmonella virchow strain was isolated from cerebrospinal fluid and blood cultures. In one case, this strain was also isolated from stool cultures. All the children had been fed the same milk formula. There was no other relationship between them. The batch of dried-milk formula was confirmed as the source of the infection by isolation of an identical lactose-fermenting Salmonella virchow strain by the Centro Nacional de Alimentacion. PMID- 7556236 TI - Extra-intestinal infections with multiply drug-resistant Salmonella typhimurium in hospitalized patients in Jordan. AB - During the 12-year period from 1978 to 1989, Salmonella typhimurium was the most frequently isolated serotype (592/1,500; 39.5%) among all clinical Salmonella isolates at Jordan University Hospital. Extra-intestinal infections due to Salmonella typhimurium accounted for 68 (11.5%) isolates. A high percentage of Salmonella typhimurium strains (52-90%) were resistant to commonly used drugs in Jordan. Most of the antibiotic-resistant strains of Salmonella typhimurium (10/12) examined which were from extra-intestinal sources contained a large plasmid (55 MDa) in addition to two to four small plasmids. These strains were also able to transfer most or part of their drug resistance in vitro. It is concluded that the invasive potential of Salmonella typhimurium isolates is probably associated with the presence of a large virulence plasmid and multiple antibiotic resistance. PMID- 7556239 TI - Multicenter Spanish study of ciprofloxacin susceptibility in gram-negative bacteria. The Spanish Study Group on Quinolone Resistance. AB - The susceptibility of 2,426 gram-negative bacteria obtained from 18 Spanish hospitals to ciprofloxacin was evaluated. Among different medical centers, susceptibility to ciprofloxacin ranged from 83 to 100% for Enterobacteriaceae, from 35 to 100% for Pseudomonas aeruginosa, from 0 to 100% for Xanthomonas maltophilia, Acinetobacter spp. and other gram-negative non-fermenting bacilli, and from 33 to 100% for Campylobacter spp. All clinical isolates of Haemophilus influenzae, Moraxella catarrhalis and Neisseria gonorrhoeae were susceptible to ciprofloxacin. PMID- 7556242 TI - Development of resistance to imipenem among nosocomial isolates of Pseudomonas aeruginosa. PMID- 7556241 TI - Detection of hepatitis B virus DNA in chronic carriers of hepatitis B surface antigen in southwestern Greece. AB - The presence of hepatitis B virus (HBV) DNA in sera of 56 chronic carriers of hepatitis B surface antigen (HBsAg) was determined by three methods: the Abbott hybridization assay, the polymerase chain reaction (PCR) followed by gel electrophoresis and UV visualization (PCR-GE), and PCR followed by DNA enzyme immunoassay (PCR-DEIA). HBV DNA was detected in four samples positive for hepatitis Be antigen (HBeAg) by all methods used. Both PCR-GE and PCR-DEIA detected viraemia in two anti-HBe, anti-HBc IgM positive samples. In the group of 50 anti-HBe positive samples the sensitivity of the three methods was 10%, 24% and 32%, respectively. PCR-GE and PCR-DEIA results correlated well with the patients' clinical status; of 20 patients with elevated ALT levels, 12 (60%) were found to be positive in the PCR-GE and another 2 were found to be positive in the PCR-DEIA (70%). These data indicate that PCR-DEIA is the most sensitive method for detection of HBV DNA. This method can be relatively easily applied in the clinical laboratory for monitoring the progression of disease and/or interferon therapy in patients with chronic hepatitis B. PMID- 7556240 TI - Association between hepatitis C virus seroreactivity and HIV infection in non intravenous drug abusing prostitutes. AB - Two hundred and ninety-four non-transfused prostitutes from Spain, who denied intravenous drug abuse, were tested for hepatitis C virus (HCV) antibodies. Seventeen (5.78%) of them were seropositive. Both in univariate and correspondence analyses, serological results for hepatitis C were associated with the HIV-1 serostatus (p < 0.001), number of sex partners (p < 0.05) and a history of genital ulcers (p < 0.05). In logistic regression analysis, hepatitis C seropositivity was associated only with HIV-1 infection (adjusted odds ratio = 13.6; 95% confidence interval = 3.3-55.2). These results show that hepatitis C seropositivity is associated with HIV-1 infection in female non-intravenous drug abusing prostitutes. These findings are also consistent with the hypothesis that HCV can be sexually transmitted with low efficiency. PMID- 7556244 TI - Detection of Chlamydia trachomatis in a low prevalence population. PMID- 7556243 TI - No evidence of Chlamydia trachomatis infection in children with wheeze. PMID- 7556245 TI - Abdominal aortic aneurysms: assessment with gadolinium-enhanced time-of-flight coronal MR angiography (MRA). AB - A prospective study was performed to evaluate the efficacy of a gadolinium chelate-enhanced MR angiography (MRA) using a coronal acquisition in the preoperative assessment of aneurysms of the abdominal aorta (AAA). Twenty patients with AAA were explored with MR using a two-dimensional (2D) time-of flight MRA technique with a coronal acquisition, before and after intravenous administration of 0.1 mmol/kg of a gadolinium-chelate. Gadolinium-chelate enhanced MRA with a coronal acquisition decreased deleterious saturation effects within aorta and iliac arteries in 16 of 20 patients (80%) and improved the overall quality of the images of the upper and lower parts of the AAA (i.e. extent of the aneurysm). Furthermore, gadolinium-chelate-enhanced MRA with a coronal acquisition allowed a better differentiation between slow flow and mural thrombus. Gadolinium-chelate-enhanced MRA had a sensitivity of 100% and a specificity of 96% for evaluation of renal artery involvement, and a sensitivity of 93% and a specificity of 100% for iliac artery involvement. The results of this study show that gadolinium-chelate-enhanced MRA obtained with a coronal acquisition is efficacious for the preoperative assessment of AAA. PMID- 7556250 TI - Transrectal and transvaginal ultrasonography in the preoperative staging of rectal carcinoma. AB - To evaluate the diagnostic ability of transrectal and transvaginal ultrasonography (TRUS and TVUS), 132 patients with rectal carcinoma were examined. Analysis of the data obtained has shown that the high quality of endosonographic imaging allows the performance of detailed staging of rectal carcinoma. In the great majority of patients (91%) the staging was carried out correctly. Neoplastic invasion was overstaged in only five cases and understaged in another five cases. Altered pararectal lymph nodes could be visualized by endosonographic examination in 54.5% of patients. PMID- 7556246 TI - Magnetic resonance imaging of lung signal intensity and dimensions in patients with advanced lung disease before and after single lung transplantation. AB - A retrospective analysis of lung signal intensity normalized for fat and lung cross-sectional area normalized for body surface area was obtained from cardiac gated spin-echo (echo time 40 ms) images before and after single-lung transplantation in 12 patients with pulmonary airway disease (seven emphysema, two bronchiectasis and three obliterative bronchiolitis) and 14 with interstitial lung disease (three pulmonary fibrosis, four fibrosing alveolitis and seven lymphangioleiomyomatosis). Nine healthy volunteers were studied for comparison. The native lung in pulmonary airway disease without inflammatory processes has normalised signal intensity (79 +/- 12%) similar to that of the control (100 +/- 6%) while interstitial lung disease had a higher normalised signal intensity (172 +/- 33%; P = < 0.05). The transplanted lung had a similar normalised signal intensity (103 +/- 12%) to that of the control except when there was a rejection reaction (one patient) or infection (two patients). Compared with the control, the native lung was smaller in pulmonary fibrosis (50 +/- 8%) and fibrosing alveolitis (78 +/- 8%), while larger in lymphangioleiomyomatosis (136 +/- 6%) and pulmonary airway disease (165 +/- 8%). The cross-sectional area of the transplanted lung was comparable to that of the control (100 +/- 13%). Distinctive features of lung normalised signal intensity and cross-sectional area were demonstrated in patients with pulmonary airway disease and interstitial lung disease before lung transplantation. These measurements could be useful noninvasive indices for assessment of the transplanted lung and for frequent follow-up of patients without exposure to ionizing radiation. Future developments are required to enhance lung signal sufficiently to detect less severe diseases. PMID- 7556247 TI - Preoperative CT and MR imaging of inferior vena cava leiomyosarcoma. AB - We investigated the radiological findings of five patients with primary leiomyosarcoma of the inferior vena cava (IVC) comparing cavography, computed tomography (CT), and magnetic resonance imaging (MRI). The radiographic presentation ranged from an intraluminal lesion with obstruction of the IVC to a tumor mass extending from the media musculature into the surrounding perivascular tissue with only slight protrusion into the lumen. The lesions were associated with late or few symptoms. Pathognomonic radiological findings could not be established. In cases with an extraluminal growth a lobulated, well-defined, encapsulated tumor with an inhomogeneous contrast uptake is characteristic in CT studies. Additional information was provided by MRI. Leiomyosarcomas with extraluminal involvement showed homogeneous intermediate signal intensity (SI) on T1-, and mixed intermediate/high SI on T2-weighted images. In cases with predominantly intraluminal growth, both CT and MRI demonstrated the IVC to be dilatated and allowed to differentiate between tumor extent and obliterating thrombosis. Modern imaging modalities allow an early and accurate pre-operative diagnosis resulting in a higher rate of surgical resection and improvement of survival. PMID- 7556249 TI - Sonographic demonstration of intestinal involvement in Henoch-Schonlein syndrome. AB - We evaluated the efficacy of intestinal sonography in the diagnosis of gastrointestinal involvement of Henoch-Schonlein syndrome (HSS). Intestinal sonography was performed in 20 children who were clinically diagnosed as HSS and sonographic findings of the intestinal system were reviewed. Out of 20 patients, 10 who suffered from abdominal pain demonstrated sonographic findings consistent with small intestinal involvement (dilatation of intestinal segments, hypomotility, and eccentric thickening of the intestinal wall). Our results reveal that sonography of the intestine may be useful in the evaluation of the involvement of HSS. PMID- 7556251 TI - Computed tomography in the evaluation of cutaneous T-cell lymphoma. AB - The computed tomography (CT) scans performed in 28 patients with cutaneous T-cell lymphoma (CTCL) were reviewed. Fifteen patients had clinically advanced cutaneous mycosis fungoides, six patients Sezary syndrome and seven variant CTCL. Of the 40 scans available 12 were normal, 15 indeterminate and 13 abnormal. Indeterminate and abnormal nodes showed a predilection for inguinal and axillary sites with a relative sparing of deep nodal regions. Visceral involvement was infrequent. In six patients CT detected abnormalities not obvious clinically and upstaged the disease. CT should be performed as part of the initial staging and as a baseline for follow-up in patients with advanced mycosis fungoides, Sezary syndrome and variant CTCL. PMID- 7556253 TI - An unusual CT and MR appearance of a posterior fossa dermoid. PMID- 7556248 TI - Paratubal cystic leiomyoma: radiologic and pathologic analyses. PMID- 7556252 TI - Multifocal synchronous osteosarcoma. PMID- 7556255 TI - Computed tomographic evaluation of surgically significant vascular variations related with the temporal bone. AB - Variations of the vascular structures related with the temporal bone may cause important problems in diagnosis, treatment planning and surgery. High resolution computed tomography (CT) scans of 700 temporal bones of 350 patients were retrospectively examined for the incidence of dehiscent jugular bulb, high jugular bulb, diverticulum of jugular bulb, anteriorly located sigmoid sinus and dehiscent internal carotid artery. Dehiscent jugular bulb was seen in 27 (3.9%), high jugular bulb was seen in 142 (20.3%), jugular bulb diverticulum was seen in 55 cases (7.9%). The average distance between external acoustic canal and sigmoid sinus was found to be 13.3 mm and in 12.4% of the cases this distance was < 10 mm. Of 700 temporal bones, 10 (1.4%) showed dehiscent carotid canal. To aid diagnosis, treatment planning and surgery, CT scanning is currently a very reliable tool in determining these conditions. Special attention should be paid to the position of the vascular structures in the preoperative temporal bone CT scans. PMID- 7556254 TI - Basal ganglia infarcts with calcifications in children. PMID- 7556256 TI - Assessment of the safety and efficacy of iobitridol, an iodinated contrast medium (30% iodine), in cranial CT. AB - In a single-centre comparative randomised double-blind trial, 40 patients undergoing head CT were examined using iobitridol and compared with 40 examined using iohexol. The contrast media were assessed for clinical safety and diagnostic efficacy. No significant difference was observed between the two groups and there were no significant reactions. It is concluded that iobitridol is a safe and effective contrast medium. PMID- 7556257 TI - Percutaneous catheter drainage of amebic liver abscesses with and without intrahepatic biliary communication: a comparative study. AB - Influence of communication with the intrahepatic biliary system on the clinical picture of amebic liver abscesses in 33 consecutive patients resistant to medical therapy, and their response to percutaneous catheter drainage was evaluated. Abscess-biliary communication was found in 27% of the sample. Patients with abscesses communicating with the biliary tree presented more frequently with jaundice (67% vs. 0%, P < 0.005), with a longer duration of illness (median 20 vs. 12 days, P < 0.001), had larger lesions (median 600 vs. 320 ml, P < 0.001) and required catheter drainage for longer periods (median 17 vs. 6.5 days, P < 0.000001). However the presence of a biliary communication did not materially affect the cure rate with catheter drainage (89% vs 100%, P > or = 0.05). In conclusion, an abscess-biliary communication is not uncommon in refractory amebic liver abscesses, and can be clinically detected by the presence of jaundice. Though a prolonged period of drainage may be necessary in the presence of this complication, catheter drainage can be expected to result in cure. PMID- 7556258 TI - Complex ruptured amebic liver abscesses: the role of percutaneous catheter drainage. AB - The failure of medical therapy for amebic liver abscess may be followed by its perforation, a complication associated with high mortality. We assessed the role of percutaneous catheter drainage in management of the sequelae of ruptured amebic abscesses in 13 critically ill patients; 22 intrahepatic lesions, three of which were multiloculated, were drained. Catheters were also placed in 17 extrahepatic collections: pleural space (n = 5), subphrenic (n = 7), perihepatic/subhepatic (n = 3), greater sac of peritoneum (n = 2). No attempt at percutaneous drainage failed. Prompt resolution of clinical features following drainage was a uniform feature. Successful resolution of the abscesses occurred within 20 days in 11 patients. In the remaining two, catheters needed to be retained in situ for 35 and 50 days. The mean hospital stay was 15 days (range 10 20 days). 100% patient survival was achieved, without a single morbid episode. Our results suggest that patients with ruptured amebic abscesses can be effectively and safely managed by percutaneous catheter drainage irrespective of the extent of extrahepatic contamination. PMID- 7556259 TI - CT of minor intubation trauma with clinical correlations. AB - Even when performed by an experienced physician, endotracheal intubation is more traumatic than previously supposed. Following emergency intubation, patients have little probability of having a normal larynx. One-hundred patients underwent CT scan of the larynx 6 months or more following endotracheal intubation of short duration (up to 8 h). Ten patients (Group 1) with respiratory arrest underwent emergency intubation; 90 surgical patients (Group 2) underwent anesthesia with endotracheal intubation. Indirect laryngoscopy was performed in 59 symptomatic patients. Abnormal CT findings were present in 86 out of 100 patients. CT irregularities, which included tears, scars and small laryngoceles, were noted on indirect laryngoscopy in 59 symptomatic patients. The laryngeal damage following endotracheal intubation is surprisingly high. PMID- 7556261 TI - A cost saving technique for the introduction of biliary stents. PMID- 7556262 TI - Ectopic blind ureter with ipsilateral renal agenesis: a case report. PMID- 7556260 TI - CT-guided aspiration in potentially infected total hip replacements complicated by heterotopic bone. AB - Aspiration of the hip joint in the setting of a potentially infected total hip replacement is occasionally unsuccessful under fluoroscopic guidance because of periarticular heterotopic bone. Of 136 consecutive patients with total hip replacements who were referred to the radiology department for hip aspiration because of suspected infection, successful intraarticular needle placement was achieved in 132 (97%) under fluoroscopic guidance. In four (3%) cases, the attempted aspiration was unsuccessful because heterotopic bone prevented the needle tip from reaching the joint; a second attempt was performed successfully under CT guidance. CT confirmed that the anterior approach was completely blocked by heterotopic bone and that there was a gap in this bone elsewhere through which a needle could be passed into the joint. The increased cost of CT was modest because it was necessary in only a small percentage of patients. PMID- 7556263 TI - Pulmonary MR angiography at 1.0 T: early results with k-space segmented and post contrast TurboFLASH two-dimensional time-of-flight sequences. AB - PURPOSE: To evaluate the combined performance of two time-of-flight methods in imaging the pulmonary arteries. MATERIALS AND METHODS: This study was prospectively conducted in 28 patients suspected for pulmonary embolism (PE). Sixteen patients were free of pulmonary vascular disease, and 12 had pulmonary vascular disease as demonstrated by pulmonary angiography. To reduce artifacts caused by cardiac and respiratory motion, MR images were acquired in all subjects using bi-dimensional (2D), gradient-recalled echo (GRE), breath-hold techniques. Sagittal thin (6-mm) sections obtained with ECG gating, k-space segmentation and incremented flip-angles (TONE), and coronal thick (15-mm) sections obtained after a unique injection of Gadolinium chelate were used. RESULTS: High quality images were obtained in all 16 (100%) subjects free of pulmonary disease with both techniques, and in 10 and 12 (87% and 100%) patients suspected for pulmonary artery disease with sagittal and coronal Gd-enhanced MRA, respectively. In patients free of pulmonary disease, TONE images exhibited distal pulmonary arteries with 2.1 subsegmental divisions on average, whereas Gd-enhanced TurboFLASH images were the most accurate to identify proximal pulmonary arteries within the mediastinum, even if only 0.8 subsegmental divisions were seen on average. A correct diagnosis of pulmonary embolism was obtained in all cases but one, with use of both MRA techniques, with an overall accuracy of 86%. CONCLUSION: The association of segmented sagittal GRE images and coronal first pass Gd-enhanced GRE images can provide information upon normal and diseased pulmonary arteries within the mediastinum until subsegmental pulmonary branches, even in patients with short-breathing. Further studies of patients with various pulmonary artery diseases will confirm whether this technique makes pulmonary MRA feasible in clinical routine situations. PMID- 7556264 TI - Hormonal induction of an immediate-early gene response in myogenic cell lines--a paradigm for heart growth. AB - Cardiac hypertrophy is characterized by growth of myocardial cells without proliferation. Many endo- paracrine stimuli such as angiotensin II, endothelin, alpha 1-adrenergic agonists, and insulin have been shown to be able to induce cardiac hypertrophy either in vivo or in vitro. We have used the myoblast model of differentiation and proliferation to determine nuclear signal transduction mechanisms in muscle and (by analogy) cardiac growth. The first nuclear event known to occur when a growth stimulus acts upon a cell is induction of a family of immediate-early genes. Our group focused on the role of one of these genes, the early growth response gene-1 (Egr-1). We have shown that this gene is induced in isolated adult cardiac myocytes in the presence of endothelin. An anti-sense oligonucleotide complementary to the first six codons of the Egr-1 mRNA abolishes the stimulation of protein synthesis induced by endothelin. In the present study we further characterized paracrine growth stimuli in the myogenic cell line Sol8, which was used as a paradigm to further investigate mechanisms of paracrine growth induction. We demonstrated that a variety of candidate endo- paracrine stimuli for the induction of cardiac hypertrophy induced the Egr-1 messenger RNA in the myogenic cell line Sol8. Among these are endothelin, insulin, basic fibroblast growth factor, and platelet-derived growth factor BB (PDGF BB). We conclude: (1) In analogy to the myocardium, these growth factors act upon myoblasts. (2) This line appears to be a suitable model for the molecular characterization of Egr-1 target genes. PMID- 7556265 TI - Differential influences of carnitine palmitoyltransferase-1 inhibition and hyperthyroidism on cardiac growth and sarcoplasmic reticulum phosphorylation. AB - To characterize interventions resulting in 'physiological' growth of the heart, Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) had hyperthyroidism induced (0.05 mg.kg-1.day-1 triiodothyronine for 6 days) or were treated with a high dose of the carnitine palmitoyltransferase-1 inhibitor, etomoxir (15 mg.kg-1.day-1 for 5 weeks). Etomoxir increased cardiac growth evenly, but hyperthyroidism resulted in an over-proportional higher right ventricular weight. Both interventions increased the proportion of the myosin isozyme V1. The rate of sarcoplasmic reticulum (SR) Ca2+ uptake was increased to a greater extent in hyperthyroid rats than in etomoxir-treated rats (P < 0.05). Left ventricular levels of immunoreactive phospholamban (semiquantitative ELISA) were moderately decreased (P < 0.05) in hyperthyroid rats but not in etomoxir treated rats. The protein kinase A-catalyzed in vitro 32P-incorporation into the SR Ca2+ pump modulator phospholamban was greatly reduced (P < 0.05) in hyperthyroid rats, indicating an increased in vivo phosphorylation. Etomoxir did not affect phospholamban phosphorylation in WKY rats. Thus, both a higher in vivo phospholamban phosphorylation state and a greater number of active Ca2+ pumps contributed to an increased rate of SR Ca2+ uptake in hyperthyroidism. The etomoxir treatment primarily increased the number of active Ca2+ pumps. A scheme is proposed focusing on long-term vs short-term regulation of the SR Ca2+ pump/phospholamban system in diseased states. PMID- 7556266 TI - Myocyte reorganization in hypertrophied and failing hearts. AB - In hypertrophied and failing hearts there are major changes in the overall contractile performance. We present a review of our previous work relating the alterations in myocardial force, work, power and relaxation, that lead to changes in overall ventricular performance, to changes in the actin-myosin cross-bridge cycle characteristics along with the degree of activation and inactivation (calcium cycling). Tissues from hypertrophied rabbit and failing human (volume overload, dilated cardiomyopathy) heart were used in these studies. Myocardial peak twitch tension (mN.mm-2) was reduced in dilated cardiomyopathy (human) (25.9 +/- 3.9 vs 13.9 +/- 2.0, 37 degrees C), volume overload (human) (44.0 +/- 11.7 vs 19.9 +/- 3.7, 21 degrees C) and pressure overload (rabbit) (46.1 +/- 2.6 vs 41.7 +/- 5.0, 21 degrees C). We used myothermal and mechanical data to analyse the average cross-bridge force time integral and the amount of calcium cycled per gram per beat. Tension-dependent Heat (mJ.g-1) (TDH) (cross-bridge cycling) and tension-independent heat (mJ.g-1) (TIH) were reduced in all of the experimental preparations (dilated cardiomyopathy, human, 37 degrees C: TDH, 3.39 +/- 0.59 vs 1.34 +/- 0.22; TIH 1.51 +/- 0.02 vs 0.16 +/- 0.03) (volume overload, human 21 degrees C: TDH, 7.23 +/- 2.22 vs 1.92 +/- 0.25; TIH, 0.75 +/- 0.19 vs 0.39 +/- 0.04) (pressure overload, rabbit, 21 degrees C: TDH, 6.60 +/- 0.75 vs 3.05 +/- 0.46; TIH, 1.00 +/- 0.17 vs 0.41 +/- 0.08).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556267 TI - Myocardial phenotypic changes in Na+, K+ ATPase in left ventricular hypertrophy: pharmacological consequences. AB - Cardiac adaptation to permanent overload induces several phenotypic changes which finally result in a system which works more economically, together with a slower Vmax. The molecular target of digitalis is the NA+, K+ ATPase, which is a polymorphic molecule. We have recently demonstrated that during cardiac hypertrophy this target is modified and that a shift occurs in the alpha 1 subunit, from the normally present alpha 2 isosubunit to alpha 3, which is a fetal isoform with a lower affinity for sodium and a higher affinity for ouabain. Such a shift explains why, in rat cardiac hypertrophy ouabain is less toxic than normal and is released from its target more slowly. It may also explain at least in part the discrepancies observed in clinical trials on the efficacy of digitalis. PMID- 7556268 TI - Myocardial fibrosis in hypertensive heart disease: an overview of potential regulatory mechanisms. AB - Myocardial fibrosis in hypertensive heart disease (HHD) can present as a reactive process, involving intramyocardial coronary arteries and arterioles with extensions of collagen into the neighbouring interstitial space, and as a replacement for necrotic cardiac myocytes. Fibrosis adversely affects myocardial stiffness and therefore regulatory mechanisms are of considerable interest. Mechanisms responsible for scarring (reparative fibrosis) are based on factors that adversely influence myocyte survival. This topic is not covered in this brief review. Mechanisms responsible for the perivascular/interstitial fibrosis that appear in both the normotensive, non-hypertrophied right and the pressure overloaded, hypertrophied left ventricule in HHD are addressed herein. They include: (a) angiotensin II (Ang II)-mediated coronary vascular hyperpermeability with subsequent fibrosis; (b) direct hormonal regulation of fibroblast collagen turnover, whereby Ang II, aldosterone and/or endothelins may be involved; (c) autocrine and paracrine signalling between fibroblasts and/or endothelial cells that alters collagen synthesis and degradation and which includes an angiotensin converting enzyme found in fibrous tissue. Collagen turnover in the myocardium is a dynamic process and fibrous tissue is anything but inert. PMID- 7556269 TI - Endothelium-modulated proliferation of medial smooth muscle cells: influence of angiotensin II and converting enzyme inhibition. AB - This study investigated the role of the endothelium and angiotensin II (Ang II) in regulating medial smooth muscle cell (SMC) proliferation. [3H]-thymidine incorporation into medial SMC of rat arteries was examined in vivo, using ballooned rat carotid arteries, as well as in vitro, using cultures of aortic tissue rings (organoids). In vivo, maximal medial [3H]-thymidine incorporation occurred within 3 days post-ballooning. In endothelium-denuded organoids, maximum medial DNA synthesis was achieved after 7 days of culture. [3H]-thymidine labelling of SMC in intact organoids (with endothelium) increased minimally during culture, indicating that the endothelium provided protection with respect to medial proliferation under basal conditions (culture in the presence of 1% plasma-derived serum). Inclusion of 10(-7) M Ang II significantly elevated medial [3H]-thymidine incorporation above that in control cultures. The stimulatory effect of Ang II was much more pronounced in intact organoids that in endothelium denuded organoids, indicating synergistic growth regulation by Ang II and endothelium-derived factors. When organoids were cultured in the combined presence of Ang II and the ACE inhibitor cilazaprilat, labelling indices of intact organoids were also significantly increased above control, but to a lower level than those obtained in the presence of Ang II alone. However, for endothelium-denuded organoids, medial [3H]-thymidine incorporation in the combined presence of Ang II and cilazaprilat was not significantly different from that in untreated controls. Thus, cilazaprilat exerts both endothelium-dependent and endothelium-independent negative regulatory effects on medial SMC proliferation. PMID- 7556271 TI - Collagen turnover and its regulation in the normal and hypertrophying heart. AB - Eighteen different collagen types have now been identified, at least five of which are found in the extracellular matrix of the heart. The interstitial collagens, types I and III are the most abundant components. Due to their tensile strength and arrangement in a network, surrounding and inter-connecting myocytes and capillaries, these collagens transmit forces throughout the myocardium, maintain cardiac structure during the cardiac cycle and contribute to the visco elastic properties of the myocardium. Early application of isotopic techniques to assess turnover suggested that heart collagen, in contrast to cytoplasmic proteins, was virtually inert. The later development of more accurate methods for measuring its synthesis and degradation rates revealed that collagen turnover was quite rapid and that heart cells synthesize and degrade collagens throughout life. Thus, changes in the balance between synthesis and degradation may lead to changes in the composition of the collagen network, which may have marked effects on compliance, resulting in changes in heart function. Current research is focused on the role of mechanical forces and growth factors in the regulation of collagen metabolism. An understanding of the regulatory mechanisms involved in controlling collagen metabolism is fundamental to the development of agents that may reverse or prevent excess collagen deposition in cardiovascular diseases. PMID- 7556270 TI - Extracellular matrix deposition in hypertensive hearts antifibrotic effects of ramipril. AB - Hypertension induced in rats by suprarenal banding has a blood pressure elevating effect that is accompanied by the occurrence of cardiac hypertrophy and fibrosis. This phenomenon is already present at 2 weeks after banding and persists up to 1.5 years. The increase in cardiac weight is mostly due to the development of fibrosis, since myocytes are only slightly increased in size. The fibrotic tissue consists mainly of fibronectin and collagen and contains numerous cellular elements. The occurrence of fibrosis can be completely inhibited by the administration of the specific ACE inhibiting drug, ramipril, which indicated that angiotensin II may directly stimulate fibroblasts to produce fibronectin and collagen. The antifibrotic effect of ramipril was also present in a low dosage that did not lower blood pressure, confirming the hypothesis that angiotensin II has a direct effect on connective tissue cells and their ability to produce extracellular matrix proteins. The direct effect of the renin-angiotensin system on the activity of interstitial cells was further proven by molecular biology techniques showing an upregulation of transcription for collagen I and III which is prevented by ACE inhibition. PMID- 7556272 TI - Hormonal regulation of cardiac fibroblast function. AB - In arterial hypertension or congestive heart failure, myocardial fibrosis is associated with an activated renin-angiotensin-aldosterone system (RAAS). This reactive fibrosis presents as an excessive accumulation of fibrillar collagen within the normal connective tissue structures of the myocardium in either ventricle, irrespective of its haemodynamic load. It therefore would appear that circulating (hormonal) and not haemodynamic factors are responsible for this adverse fibrous tissue response. The cardiac fibroblast expresses mRNA for types I and III collagens, the major fibrillar collagens in the heart, and for collagenase or matrix metalloproteinase 1 (MMP 1), the key enzyme for interstitial collagen degradation. Therefore, adult rat cardiac fibroblasts were cultured to ascertain whether the RAAS effector hormones angiotensin II (Ang II) or aldosterone (Aldo) directly stimulate collagen synthesis or inhibit MMP 1 production. Collagen synthesis, determined by 3H-proline incorporation and MMP 1 activity determined by degradation of 14C-collagen, were measured under serum free conditions in confluent, quiescent fibroblasts after 24 h incubation with Ang II or Aldo over a wide range of concentrations (10(-11) -10(-6) M). In addition, collagen synthesis was measured after incubation with the mineralocorticoid, dexoycorticosterone (DOC), or the prostaglandin, PGE2. Collagen synthesis, normalized per total protein synthesis, increased significantly in a dose-dependent manner after incubation with either mineralocorticoid hormone, Aldo or DOC, or after incubation with Ang II compared with untreated control cells. In contrast, collagen synthesis was significantly decreased with PGE2 treatment. This increase in collagen synthesis in Ang II or mineralocorticoid-stimulated fibroblasts could be completely abolished by Ang II type 1 or mineralocorticoid receptor antagonists, respectively. (ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556273 TI - Local regulation of the coronary circulation in health and disease: role of nitric oxide and endothelin. AB - Coronary artery disease is the leading cause of morbidity and mortality in western countries. Its pathogenesis is unknown, but involves enhanced vasoconstriction, increased interaction of platelets and monocytes with the vessel wall, as well as proliferation, migration and extracellular matrix formation of vascular smooth muscle. The endothelium lies in a strategic anatomical position between circulating blood and vascular smooth muscle cells. This supports the concept that dysfunction of these cells significantly contributes to coronary artery disease. Besides other mediators, endothelial cells are a source of nitric oxide and endothelin. Nitric oxide is a vasodilator, an inhibitor of both platelet function and proliferation and migration of vascular smooth muscle. Endothelin is a potent vasoconstrictor that facilitates proliferation. Under pathological conditions, in particular the presence of cardiovascular risk factors, endothelial dysfunction occurs and is a major contributor to the increase in platelet vessel wall interaction, vasoconstriction and proliferation in the coronary system. Endothelium-dependent vasodilation is usually reduced and endothelium-dependent constrictor responses, as well as endothelin production, are augmented. Hence, endothelial cells are important targets and mediators of coronary artery disease. PMID- 7556274 TI - Endothelium-mediated vasodilation during ACE inhibition. AB - ACE inhibitors are superior to other vasodilators in the treatment of congestive heart failure and may be advantageous in patients with myocardial infarction and hypertension. The mechanisms mediating these beneficial effects are not clear. The present article discusses the mechanisms leading to augmented release of endothelium-derived nitric oxide during ACE inhibition. Acute potentiation of bradykinin (Bk)-induced vasodilation was studied in rings of bovine and human coronary arteries mounted in organ chambers for recording of isometric force. The ACE inhibitors captopril, enalaprilat, fosinoprilat, lisinopril, or ramiprilat alone did not affect vascular tone in isolated coronary tone in isolated coronary artery preparations with intact endothelium. However, in the presence of exogenous Bk, kallidin, or one of the slowly degradable Bk2-receptor agonists D Arg(Hyp3)-Bk or [Hyp3-Tyr(Me)8]-Bk they elicited potent concentration-dependent relaxations. Relaxations in response to lisinopril were not observed in the presence of other vasodilators. They were prevented by mechanical removal of the endothelium, inhibition of nitric oxide synthase or Bk2-receptor blockade. The data indicate that ACE inhibitors potentiate the effects of Bk on endothelial cells by a local mechanism, probably independent of the degradation of bradykinin. The chronic effects of ACE inhibitors on endothelial function were compared with those of selective angiotensin(AT)1-receptor blockade in cyclosporin A (CsA) treated rats. Chronic AT blockade alone does not affect endothelium-dependent relaxation and increases contractions to ATII in the rot aorta. Combination of CsA with either an ACE-Inhibitor or an AT2 receptor antagonist prevented the endothelial dysfunction in the rat arta observed after CsA alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556275 TI - Control of coronary angiogenesis. AB - We investigated the development of coronary collaterals in canine and porcine models using either progressive experimental coronary occlusion or microembolism of small vessels. Muscular arteries developed on the epicardial surface of canine -but not of porcine hearts, where large, thin-walled intra-myocardial capillaries developed irrespective of the type of vascular occlusion. The common motif for both types of vascular adaptation is the part played by inflammation, i.e. the involvement of monocytes/macrophages that are probably the producers of growth factors. Emphasis is placed on the importance of remodelling by controlled proteolysis to create the space for the new vascular cells. PMID- 7556276 TI - Hypertensive heart disease: cardioreparation by reversal of interstitial collagen in patients. AB - Impairment of coronary flow reserve in arterial hypertension and hypertrophy is caused by multiple mechanisms. Medial wall thickening of the small intramural arteries and rarification of capillarization are the major structural alterations in hypertensive hypertrophy. Since the small intramural resistance arteries contribute more to coronary resistance than capillaries, medial wall thickening of the intramyocardial arterioles has to be considered the most relevant mechanism for causing impaired coronary flow reserve in arterial hypertension. Additionally, qualitative changes in myocardial tissue composition and structure, such as reactive interstitial fibrosis, rather than the mere augmentation of the myocardial muscle mass, impair flow reserve extravascularly. Except for the structural vascular and myocardial changes, in some hypertensive patients an impaired endothelium-mediated vasodilation of the microvessels occurs. Preliminary clinical data indicate that myocardial interstitial collagen content can be regressed in some hypertensive patients by clinical antihypertensive therapy over a period of 9-12 months. PMID- 7556283 TI - Nodal involvement in cancer of the uterine cervix: value of lymphography and MRI. AB - In this study the preoperative staging of lymph node involvement in cervical cancer was analyzed retrospectively in 155 cases. All results were correlated with lymph node histology. One hundred and three patients underwent preoperative lymphography. Twelve of them were investigated by lymphography and MRI (magnetic resonance imaging). One patient was staged Ia, 10 patients were staged Ib and one patient was staged IIb. All patients underwent surgery; 18.5 lymph nodes were removed on average. Histologically lymph node involvement was found in 3 cases. Lymphography was able to detect lymph node involvement correctly in one case (33% sensitivity). We got two false positive results by lymphography (67% specificity). By MRI all cases were classified as nodal negative. MRI did not have advantage over bipedal lymphography in detecting lymph node involvement in our study, even though the results of lymphography were also poor. PMID- 7556277 TI - Directions in antihypertensive treatment--our future from the past. AB - Future directions in antihypertensive treatment will rely on our present experience with antihypertensive drugs, on new concepts of cardiovascular regulation and on novel antihypertensive agents. At present, we seek early detection of hypertension; treatment should focus on normalization of blood pressure, the reversal or prevention of left ventricular hypertrophy, associated coronary artery disease and on the prevention of, or reparation of, myocardial fibrosis and microangiopathy. Therefore, combination therapy is advisable in severe cases, and any monotherapy should focus on the pharmacological principles compatible with these goals. ACE inhibitors and calcium antagonists appear to meet these requirements. There are, in addition, novel drugs i.e. angiotensin II receptor antagonists and renin inhibitors, as well as therapeutic stimulation of endothelial nitric oxide by L-arginine, the inhibition of endothelin-1 mediated vasoconstriction, and potassium-channel openers. All are examined in this contribution to delineate the perspectives in antihypertensive therapy. PMID- 7556281 TI - Extra-pelvic metastasis of endometrioid carcinoma; resistance to chemo-radiation therapy--response to paclitaxel treatment. Case report. AB - We present a case report of a 56 year old female suffering from recurrence of simultaneous endometrial and ovarian carcinomas, confined to distant neck and axillary lymph nodes. Resistance of the tumor to conventional chemotherapy and radiotherapy was followed by experimental Paclitaxel treatment which led to complete regression of all metastatic foci. PMID- 7556282 TI - Cell dynamics of undifferentiated carcinomas of the ovary. Immunohistochemical estimation of their growth fraction and apoptotic status. AB - The proliferative and apoptotic status of undifferentiated carcinomas of the ovary was estimated immunohistochemically by antibody for Ki-67 proliferative antigen and Le(Y) apoptotic antigen. Well-differentiated types of ovarian carcinoma were examined for comparison. In 8 cases of undifferentiated carcinomas, the proliferative activity in terms of the growth fraction expressed as percentage of positive nuclei for Ki-67 antigen ranged from 28.2 to 81.2% (mean 60%). In well-differentiated carcinomas, which included 8 serous, 8 mucinous, 4 endometrioid and 4 clear cell adenocarcinomas, the growth fraction ranged from 8.2 to 33.4% (mean 20%). Thus, statistically significant differences were found in the proliferative activity between undifferentiated and well differentiated ovarian carcinomas. On the other hand, apoptotic status as estimated by Le(Y) staining varied considerably among the cases in both undifferentiated and differentiated carcinoma groups. Nevertheless, in general, undifferentiated carcinomas seemed to be more prone to apoptosis than well differentiated carcinomas. It is concluded that in ovarian undifferentiated carcinomas, more active cell proliferation takes place with a concomitant cell loss compared to their differentiated counterparts. PMID- 7556280 TI - Cyclophosphamide, methotrexate, 5-fluorouracil, and folinic acid (CMFF) as first line chemotherapy for treatment of advanced breast cancer. A pilot study. AB - In order to evaluate the maximum tolerable dose of a combination chemotherapy consisting of cyclophosphamide, methotrexate, 5-fluorouracil, and folinate (CMFF), 30 female patients with histopathologically confirmed, previously untreated advanced breast cancer were entered into this pilot study. Chemotherapy consisted of fixed doses for methotrexate (40 mg/m2 i.v. on day 1), 5 fluorouracil (500 mg/m2 i.v. on day 2 to 4) and folinic acid (2 x 200 mg/m2 i.v., 0 + 2 h on day 2 to 4). The dose of cyclophosphamide was escalated stepwise, starting with 200 mg/m2 i.v. on day 2 to 4, to 240 mg/m2, 290 mg/m2, 360 mg/m2 and 400 mg/m2, respectively, for each subsequent five patients. Treatment was repeated every four weeks. A total of 92 treatment cycles was given. Myelosuppression was the dose-limiting toxicity: leukopenia WHO grade III or IV was observed after a total of 28 cycles and anemia of equal intensity after 1 cycle. No thrombocytopenia WHO grade III or IV was recorded. Myelotoxicity increased with higher doses of cyclophosphamide. Among non-hematologic toxicities, alopecia was reported in two-thirds of the patients. Nausea and vomiting was noted in 25% of treatment cycles, but in one cycle only WHO grade III was recorded. No other toxicities exceeding WHO grade II occurred.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556278 TI - Protein kinase cascade activated by mechanical stress in cardiocytes: possible involvement of angiotensin II. AB - Mechanical stress induces cardiac hypertrophy and expression of specific genes in the cardiac myocytes. External stimuli are generally transduced into the nucleus through the activation of a protein kinase cascade. We have previously shown that stretching cardiomyocytes stimulates the activity of protein kinase C (PKC), mitogen-activated protein (MAP) kinase and S6 protein kinase. In the present study, we examined two other kinases, Raf-1 kinase and MAP kinase kinase, which are supposed to lie between PKC and MAP kinase in the protein kinase cascade. Stretching cardiocytes by using the in vitro system induced hyperphosphorylation of Raf-1 kinase and activation of MAP kinase kinase. The protein kinases activated by mechanical stress are similar to those activated by growth factors. We examined the possible involvement of angiotensin II (Ang II) in the protein synthesis and gene expression induced by mechanical stress. CV11974, an Ang II receptor antagonist, partially suppressed the increases in amino acid incorporation, c-fos gene expression and MAP kinase activity induced by stretching. These results suggest that a variety of protein kinases are activated by mechanical stress and that locally produced Ang II may in part play important roles in converting mechanical stimuli into biochemical signals. PMID- 7556279 TI - Absence of an association between G-CSF support and clinical response in heavily pre-treated ovarian cancer patients receiving salvage chemotherapy with paclitaxel (Taxol). AB - This study was undertaken to determine if there was an association between the use of granulocyte colony-stimulating factor (G-CSF) and the clinical response to paclitaxel in patients with advanced ovarian cancer. Seventy-seven heavily pre treated patients were given 135 mg/m2 paclitaxel every 21 days, with G-CSF support as necessary according to standard criteria. Patients were stratified into two subgroups according to the presence or absence of G-CSF use. The clinical response, progression-free interval (PFI) and survival were determined for each patient and analyzed relative to G-CSF use. Thirty-three patients received G-CSF support. The overall response rate was 32% (one complete and 24 partial) with similar patient numbers from each subgroup. The mean PFI and survival were not significantly different between the subgroups; 170.9 +/- 21.2 and 401 +/- 45.5 days (G-CSF) and 182.4 +/- 30.2 and 367 +/- 36 days (no G-CSF), respectively. We conclude that G-CSF use is not associated with response to paclitaxel in heavily pre-treated ovarian cancer patients. PMID- 7556284 TI - Copper, zinc, and magnesium tissue and serum levels in patients with cervical carcinoma. AB - Serum and cervical tissue copper (Cu), zinc (Zn) and magnesium (Mg) levels were determined by atomic absorption spectrophotometry in 65 women with cervical carcinoma and compared with levels in 30 healthy women. The patients mean serum Cu level (184.8 +/- 12.3 mugr/dl) was significantly higher than the control group (p < 0.05). However, in relation to the control group, the differences in Mg, Zn and Cu levels in cancerous tissues of patients with cervical carcinoma were not statistically significant (p > 0.05). There was also no significant difference between FIGO Stage I and IIA patients according to their serum and tissue concentrations of these trace elements. We concluded that serum and tissue copper, zinc and magnesium determinations have no use in cervical carcinoma management. PMID- 7556285 TI - Cervical intraepithelial neoplasia: carbon dioxide laser vaporization and conization. Our experience. AB - Between 1984 and 1992, the CO2 laser was used to treat 341 patients affected by CIN of various degrees. On the basis of specific indications these patients underwent vaporization or conization. Two hundred and twenty eight patients underwent vaporization of intraepithelial lesions, 221 (96.92%) of whom were free of disease after one laser treatment, as observed during the subsequent years of follow-up. In 105 out of the 113 patients treated with conization the cone margin was free of disease. One hundred and one (96.19%) of the these 105 patients had no evidence of disease after one year of follow-up. In the remaining 8 (7.07%) cases examination of the cone revealed the presence of invasive carcinoma. The major complication was vaginal discharge observed in 115 (33.72%) patients. The CO2 laser is a very effective therapeutic procedure for the treatment of intraepithelial lesions of the cervix and permits to preserve reproductive function and anatomic integrity. PMID- 7556286 TI - Placental-site trophoblastic tumor. Principles of diagnosis, clinical behaviour and treatment. AB - Placental site trophoblastic tumor (PSTT) is a rare form of the gestational trophoblastic disease (GTD). Seventy four cases have been reported in the English literature up to now. Based on all this data, the death rate is 20% in PSTT. There are many unknown aspect of PSTT on the principles of diagnosis and on the management as well. In this paper, all of the literature related to PSTT has been reviewed and according to these data, diagnosis, clinical behavior and therapy of PSTT have been discussed. PMID- 7556287 TI - A colposcopic scoring system for grading cervical lesions. AB - Colposcopic scoring systems have been used for distinguishing low-risk from high risk cervical lesions. We studied 159 HPV DNA positive patients (7 with HPV 6/11, 51 with HPV 16/18, 52 with HPV31/33/35, 25 with mixed HPV types, and 24 with untypable HPV) (mean age 25.7 years, SD 7.4), and 69 age matched HPV-DNA negative control, patients, drawn from the same clinical setting. A dot-blot hybridization technique (ViraPap and VyraType, Digene Diagnostics, MD, USA) was used for HPV DNA hybridizations types 6/11, 16/18, and 31/33/35. Each patient underwent cytologic and colposcopic evaluation at four-month intervals. A colposcopic score for grading cervical lesions was used. It included features of lesion borders, tone of acetowhitening, and vascular atypia. Each feature was graded using three categories, so that an increasing score would be associated with gradually more severe lesions. The mean follow-up time was 12.2 months (SD 8.7) for the cases, and 12.8 months (SD 6.9) for the controls. The study end point was defined as the presence of cytologic changes consistent with CIN, in which case colposcopically directed biopsy and endocervical curettage were performed. The mean colposcopic score derived from the features of ATZ was 4.9 in the HPV DNA positive patients and 4.3 in the HPV DNA negative patients. The mean colposcopic score was 3.3 for those with HPV 6/11, 4.5 for HPV 16/18, 5.3 for HPV 31/33/35, 5.0 for mixed HPV types, and 4.0 for untypable HPV.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556288 TI - Ascitic fluid-associated lymphocytes in patients with metastatic ovarian cancer. Analysis of lymphocyte subsets and of phenotypical changes induced by "in vitro". AB - Ascitic fluid and peripheral blood samples, from seven patients affected by peritoneal carcinomatosis secondary to ovarian tumors, were analyzed to examine the phenotypic properties of ascitic fluid-associated lymphocytes (AFAL) and then asses T-cell activities in the presence of different interleukins. The AFALs showed a relative enrichment of CD2+, CD3+, CD5+, CD4+ T cells compared to the peripheral blood samples, while the contrary was true for CD56+ and CD57+ cells. The same phenomenon was observed in both CD3+, CD56+ and CD2+, CD56+ cell populations. Fewer CD71+ and CD25+ cells were found in the ascitic fluid suspensions whilst the opposite was true for HLA-DR+ lymphocytes. The data obtained from the in vitro incubation of AFALs in the presence of IL-1 (or), IL 2, IL-4, IL-6 or IFN showed that IL-1, IL-2 and IFN- can induce a two-fold increase in the percentages of HLA-DR+ T lymphocytes. IL-6, IL-2 and IL-1 seemed to have a fairly modest effect on CD25. A better survival of CD56+ cells was found only in the presence of IL-4 and IL-6. Even though preliminary and based on a limited number of patients, our results would seem to invite caution in instituting intraperitoneal administration of one or more cytokines for the treatment of such lesions in humans. PMID- 7556289 TI - From hyperplasia to frank breast neoplasia. Carcinogenesis. Immunoprevention. AB - There is strong evidence that in advanced cases of breast fibrocystic disease, the risk of cancer is elevated. Cyclic breast glandular hyperplasia is commonly associated with mastodynia and/or breast fibrocystic disease. The administration of progestins, antiestrogens and/or local progesteron, results in some cases in a desensibilisation, accompanied by loss in responsiveness to hormonal therapy. Out of 167 patients (pts) suffering from mastodynia and/or breast fibrocystic disease with positive delayed-type hypersensitivity (DTHS) reactions to a pharmaceutical Placenta Suspension (PS), when injected intradermally, in 87 pts. who failed to respond to hormonal therapy, a vaccine preparade from PS admixed with an adjuvant (BCG), was administrated in one intradermal injection (0.1). In all the pts. recruited into the study, a complete remission of the symptoms occurred and in the majority of cases lasted throughout the 12 month follow-up period. The essential factor of relative hyper-estrinism, initiates breast epithelial hyperplasia and also increases stromal ground substance, which has the propensity to fibrous reorganisation. A true or relative hypoxia results, as a consequence of connective tissue sclerosis and epithelial thickness, constituting a supplementary factor for further epithelial proliferation. The risk of gene faults is greater when hypoxia operates at cell viability level and for long enough duration. Within the frame of persistent multicellular proliferative potential, a basic shift in energy metabolism is accompanied by appearance of fetal isoenzymes and of membrane glycoproteins, that induces a host immunological reaction (emphasised by PS).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556290 TI - Epidemiology and survival of endometrial cancer. Valuation of data of National Cancer Institute in Naples 1987-1992. AB - Cancer of the endometrium is a neoplasm which essentially strikes postmenopausal women and in which the predominant risk factors are recognized as an excessive imbalance of estrogen and obesity. The survival rate is good in the first stage and is acceptable in the second, while in the third and especially the fourth stage is unfavorable. To verify and evaluate such facts we have carried out a study of survival rates on patients admitted in our Institute surgery division from 1987 to 1992. The results confirm the importance of the stage and histologic grading of the determining variables, while there appears to be more controversy regarding the role of myometrial invasion. In conclusion, our data show that intervention by pelvic-aortic lymphadenectomy contributes to an improvement in survival rates independently of stage. PMID- 7556291 TI - New staging investigations for lung cancer: what will they have to offer to be clinically useful? PMID- 7556292 TI - Scintigraphic imaging of oncogenes with antisense probes: does it make sense? AB - Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity. PMID- 7556294 TI - Imaging metastatic testicular germ cell tumours with 18FDG positron emission tomography: prospects for detection and management. AB - The aim of this study was to investigate the role of positron emission tomography (PET) with [18F]fluoro-2-deoxyglucose (18FDG) in metastatic testicular germ cell tumours. Twenty-one patients with stage II-IV testicular germ cell tumours were imaged by PET with a multiwire proportional chamber PET system and 18FDG. Avid 18FDG uptake was seen in metastatic disease from primary seminoma and malignant teratoma. Normal tissue uptake was seen in differentiated teratoma or necrotic, fibrotic tissue. 18FDG standard uptake values and tumour to normal tissue ratios were 6.0 +/- 1.4 and 1.7 +/- 0.4 (mean +/- 1SD), respectively, for malignant tissue. Reduction of 18FDG tumour to normal tissue ratios from pre-treatment to on-treatment scans was predictive of response (n = 3). No significant reduction in 18FDG uptake was seen in patients not responding to therapy (n = 2). These results suggest a role for 18FDG PET in the detection and management of metastatic testicular germ cell tumours. PMID- 7556293 TI - Rhenium-188 sulphur colloid as a radiation synovectomy agent. AB - Radiation synovectomy has been shown to be an effective treatment for the rheumatoid arthritic knee. In this study, we evaluated the suitability of rhenium 188 as a radiation synovectomy agent. In addition, we were successful in labelling sulphur colloid with 188Re. In vitro stability tests revealed that more than 95% of the 188Re remained in colloid form over a 3-day period. Intra articular injection of 188Re sulphur colloid into arthritic rabbit joints was followed by gamma camera imaging to quantify the leakage. The mean retention percentages of 188Re colloid in arthritic knees were 93.7% (+/- 1.4%), 90.8% (+/- 1.7%) and 87.2% (+/- 0.6%) at 1 h, 1 day and 2 days, respectively. A biodistribution study of the arthritic rabbits revealed that the highest activity outside the knees was in the liver and the kidneys. Our preliminary results indicate that 188Re sulphur colloid may be an effective radiopharmaceutical for radiation synovectomy. PMID- 7556296 TI - Feasibility of first-pass radionuclide angiocardiography with a 10-mCi technetium bolus using a single-crystal digital gamma camera: implications for technetium sestamibi single-day protocols. AB - To test the feasibility of rest first-pass radionuclide angiocardiography (FPRNA) using a 370-MBq (10-mCi) bolus and a single-crystal gamma camera, 40 patients underwent both FPRNA and equilibrium radionuclide angiocardiography (ERNA). Ejection fraction (EF) and regional wall motion (RWM) were assessed by three observers. The interobserver reproducibility was good: the intraclass correlation coefficient was 0.97 for both techniques. The correlation coefficient between FPRNA and ERNA EFs ranged between 0.90 and 0.92. FPRN EFs were significantly higher (P < 0.003) by less than 5 percentage points, this difference having no clinical implications for patient classification. The study provides arguments as to why this difference may be explained self-attenuation rather than counts statistics problems. Both techniques were concordant for RWM analysis. We conclude that FPRNA with 10 mCi is a reliable tool to assess rest left ventricular function, which makes it possible to perform simultaneously myocardial perfusion and function assessment in a single-day protocol using a single-crystal gamma camera. PMID- 7556295 TI - Assessment of skull base involvement in nasopharyngeal carcinoma: comparisons of single-photon emission tomography with planar bone scintigraphy and X-ray computed tomography. AB - The diagnostic contribution of single-photon emission tomography (SPET) to the detection of bone lesions of the skull base was explored in 200 patients with nasopharyngeal carcinoma (NPC). Comparison of SPET with planar bone scintigraphy showed that SPET improved the contrast and better defined the lesions in 107 out of the 200 patients. Comparison of SPET with X-ray computed tomography (CT) showed that SPET did not miss the lesions detected by CT while CT missed 49% of the lesions detected by SPET. The only false-positive lesion with SPET was detected in the mastoid bone. SPET detected skull base lesions in all of the 35 patients with cranial nerve involvement, while CT missed eight and planar bone scintigraphy missed four. The findings suggest that SPET should be included in the routine check-up examinations of patients with NPC. PMID- 7556301 TI - Cardiac single-photon emission tomography with a 90 degrees dual-head system. AB - Dual-head single-photon emission tomography (SPET) systems which permit a 90 degrees orientation of the heads provide a twofold increase in sensitivity for 180 degrees SPET compared with single-head systems. Since such systems have additional mechanical and electrical alignment and stability requirements, clinical equivalency for myocardial perfusion SPET must be demonstrated. Accordingly, we studied 26 subjects who underwent exercise thallium scintigraphy consecutively with both single-head and dual-head systems. Image acquisition was similar, except that the dual-head study took one-half the time. Image reconstruction was identical. Images were interpreted in a blinded fashion, and rated for technical quality. All 26 studies were equivalently interpreted for the presence or absence of perfusion defects. Fourteen of the studies were judged to be technically equivalent; seven were judged to be slightly better with the single-head system; three were judged to be slightly better with the dual-head system; and two were judged to be definitely better with the dual-head system. We conclude that dual-head 90 degrees systems provide equivalent clinical performance in half the acquisition time. PMID- 7556298 TI - Estimation of glomerular filtration rate using chromium-51 ethylene diamine tetra acetic acid and technetium-99m diethylene triamine penta-acetic acid. AB - Simultaneous measurements of the clearance rate of chromium-51 ethylene diamine tetra-acetic acid (51Cr-EDTA) and technetium-99m diethylene triamine penta-acetic acid (99mTc-DTPA) were performed in 54 patients with a range of function between 9 and 176 ml/min. Using multiple blood samples the two clearance values correlated well (r = 0.97, SEE 8.6 ml/min) and DTPA clearance was higher by 2.9%. For each radiopharmaceutical the plasma clearance rates obtained using multiple blood samples were compared with those obtained with simplified methods, i.e., the 60-180 min two-sample method of Russell and the mono-exponential method with the Brochner-Mortensen correction. For both radiopharmaceuticals the clearance values correlated well with the Russell method (r = 0.99, SEE = 4.1 ml/min for EDTA; r = 0.99, SEE 4.9 ml/min for DTPA) and the mono-exponential method (r = 0.99, SEE 3.6 ml/min for EDTA; r = 0.99, SEE 3.9 ml/min for DTPA). The mean plasma clearance obtained using multiple blood samples did not differ significantly from that obtained with the Russell method, either in patients with a glomerular filtration rate (GFR) < 30 ml/min or in patients with GFR > or = 30 ml/min. The mean plasma clearance obtained using multiple blood samples differed significantly from that obtained with the mono-exponential method because of the great difference observed in patients with GFR > or = 30 ml/min. It is concluded that the Russell two-sample method after injection of 99mTc-DTPA is accurate enough for routine clinical use. PMID- 7556300 TI - Increased sensitivity through use of overlapping 180 degrees orbits in clinical myocardial perfusion imaging. AB - Clinical cardiac imaging is hindered by noise due to limited activity and imaging time. Use of 90 degrees dual-detector systems with 90 degrees gantry rotation may provide the best sensitivity/resolution for cardiac imaging, but this option is not readily available to those using a triple-detector system with detectors at 120 degrees intervals. This study utilizes a cardiac/chest phantom to compare several triple-detector orbits, with assessment of sensitivity and resolution. A 180 degrees rotation with reconstruction of two of the three heads was evaluated, resulting in overlapping 180 degrees orbits; use of a starting angle of 165 degrees for the first head placed the overlapping portion of the orbits over the LAO myocardial region, where camera-cardiac distance is most favorable. Use of this overlapping orbit yielded resolution equivalent to a conventional (single head) 180 degrees rotation. Sensitivity was 87%-90% of that of a 90 degrees dual detector system, and 16%-20% better than the common practice of using a 120 degrees orbit with reconstruction of 1 1/2 heads to achieve 180 degrees of data. Use of 360 degrees acquisition with reconstruction of all three heads provided the greatest sensitivity, though at the expense of some loss in image quality. Thus, for those centers performing cardiac imaging using a triple-detector system, use of overlapping 180 degrees orbits is the preferred acquisition choice. PMID- 7556299 TI - Validation of automated brain contour determination in normal and abnormal cerebral single-photon emission tomography. AB - A contour detection algorithm for cerebral studies, using the method of Tomitani, has been implemented on a single-photon emission tomographic (SPET) system. It is based on the detetion by threshold of the brain edge in the sinogram and does not depend on the reconstruction algorithm. Thirteen normal subjects underwent an examination on both computed tomography (CT) and SPET using a head holder to ensure the reproducibility of the positioning. The CT scan contour of the brain was drawn manually according to the brain parenchyma limits. The SPET brain contour was obtained by use of the Tomitani algorithm after the threshold had been determined on an active cylindrical phantom. Using a threshold of 37% of the maximum uptake, the length of the contour as well as the area obtained with SPET and CT were not found to be statistically different. The method of Tomitani, which is simpler and faster then previous methods, provides contours which superimpose very well with CT scan images. Application to patients with unilateral pathological defects is possible by requiring that the contour is symmetrical. PMID- 7556297 TI - Measurement of left ventricular ejection fraction from gated technetium-99m sestamibi myocardial images. AB - Left ventricular ejection fraction (LVEF) and single-photon emission tomographic (SPET) imaging of the myocardium can be performed after a single technetium-99m sestamibi (MIBI) injection. Sixty patients underwent SPET imaging with MIBI. Immediately after SPET acquisition ECG-gated 99mTc-MIBI perfusion images were acquired using 24 planar images per R-R interval. A new method for measurement of LVEF from the ECG-gated 99mTc-MIBI perfusion images was developed. To validate the method, LVEF derived from MIBI perfusion images was compared with that from conventional radionuclide ventriculography in all 60 patients. Forty patients had evidence of myocardial infarction and 20 had normal perfusion on MIBI imaging. There was no statistically significant difference between LVEF computed from 99mTc-MIBI perfusion images and that from radionuclide ventriculography (r = 0.7062, P < 0.001). There was little difference associated with the technique (intraobserver variability r = 0.9772, P < 0.001). Interobserver variability was also good (r = 0.8233, P < 0.001). LVEF from 99mTc-MIBI perfusion images can be obtained at the same time as assessment of myocardial perfusion and in the same orientation and metabolism of the myocardium, thereby permitting more accurate and realistic prognosis and diagnosis in patients with coronary artery disease. PMID- 7556302 TI - Quantitative evaluation of thallium-201 uptake in predicting chemotherapeutic response of osteosarcoma. AB - Thallium-201 has been shown to be useful in predicting tumour viability in patients undergoing neoadjuvant chemotherapy for osteogenic sarcoma. Early studies relied upon qualitative assessment of analog images to obtain predictive results. Recently, the lesion to normal tissue uptake ratio of 201Tl has been used in evaluating bone and soft tissue sarcomas. This study attempts to quantitate changes in tumour to normal tissue ratio following chemotherapy. Eight consecutive patients with classical osteosarcoma received standard preoperative chemotherapy with a combination of cisplatin, adriamycin and high-dose methotrexate. 201Tl gamma scintigraphic images were obtained both before and after chemotherapy. The average counts taken over the tumour divided by that from the contralateral normal tissue area yielded a tumour-to-normal tissue (T/N) ratio. The percentage change in the T/N ratio before and after preoperative chemotherapy was correlated with the percentage of tumour necrosis from pathological section. The median post-chemotherapy T/N ratio was 1.85 (range 0.5 7.7). The median percentage change in T/N ratio after chemotherapy was -58% (range +26% to -83%). The median percentage of necrosis from pathological section was 80% (range 0%-95%). There was a good correlation between the percentage of tumour necrosis and the percentage change in T/N ratio (rank correlation coefficient r = 0.84, P = 0.0085). Quantitative assessment of changes in 201Tl uptake by osteosarcoma correlates well with tumour necrosis after preoperative chemotherapy. This method may be used to predict response to chemotherapy at an earlier stage, enabling the clinician to consider alternative chemotherapeutic regimens or salvage surgery. PMID- 7556303 TI - Fluorine-18-fluorodeoxyglucose positron emission tomography for preoperative parathyroid imaging in primary hyperparathyroidism. AB - Fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging was performed in seven consecutive patients with primary hyperparathyroidism to preoperatively locate parathyroid adenomas. Foci of FDG accumulation corresponding to abnormal parathyroid tissue were observed in two out of nine surgically excised parathyroid adenomas. It was concluded that FDG PET imaging demonstrated a too low sensitivity for systematic preoperative detection and localization of parathyroid glands causing primary hyperparathyroidism. PMID- 7556304 TI - Oxygen bubbling can improve the labelling of pentavalent technetium-99m dimercaptosuccinic acid. AB - It has previously been reported that almost all of the trivalent technetium-99m dimercaptosuccinic acid (99mTc (III) DMSA) present in the labelling product of pentavalent technetium-99m DMSA (99mTc (V) DMSA) can be changed into 99mTc (V) DMSA by bubbling with pure oxygen. We therefore performed studies in animals (mice) and humans to investigate the effect of such oxygen bubbling on the labelling efficiency of and on the renal uptake of 99mTc. The method of labelling of 99mTc (V) DMSA was that of Hirano. It was found that oxygen bubbling oxidized the contaminated 99mTc (III) DMSA into 99mTc (V) DMSA in vitro and decreased the uptake of radioactivity in the kidney in both animals and humans. PMID- 7556305 TI - Radiopharmaceuticals 1994. Nil desperandum. European Association of Nuclear Medicine Committees on Radiopharmaceuticals and Positron Emission Tomography. AB - On the basis of the discussions at a symposium held in Dusseldorf and attended by representatives of various interested bodies, European legislation as it affects radiopharmaceuticals is reviewed. Due consideration is given to the new, centralised and decentralised, registration procedures, effective since 1 January 1995. The dossier required to support an application for marketing authorisation is discussed, separate consideration being given to single-photon emitters, therapeutic radio-nuclides and positron-emitting radiopharmaceuticals. The role of the European Pharmacopoiea is also considered. It is concluded that the new, modified procedures for the registration of medicinal products in the European Union may actually inhibit free availability of radio-pharmaceuticals within the Community, and that there is a strong case for modification of the European Directives so that radiopharmaceuticals are placed in a separate category to therapeutic drugs, with less stringent registration requirements. PMID- 7556307 TI - The use of technetium-99m sestamibi to indicate breast cancer invasiveness. PMID- 7556308 TI - Estimation of brain 5HT2 receptor binding potential with a single-dose PET paradigm. PMID- 7556309 TI - Failure to label red blood cells adequately in daily practice using an in vivo method: methodological and clinical considerations. PMID- 7556306 TI - Radiolabelled monoclonal antibodies in tumour imaging and therapy: out of fashion? AB - The initial enthusiasm for the development of diagnostic and therapeutic studies involving the use of monoclonal antibodies was replaced by scepticism as hopes remained unfulfilled. Against this background one needs to ask whether immunoscintigraphy (IS) serves clinical needs effectively and whether radioimmunotherapy (RIT) has a future. The current review considers these questions by reference to relevant studies. Taking colorectal cancer as an example, an appraisal is offered of the ability of IS to detect disease at an early stage and thereby to reduce mortality, and of the influence of the results of IS on patient management. It is concluded that in a limited number of cases of colorectal cancer and other solid tumors, IS will allow surgery to be performed at a stage where cure is still possible because of its ability to detect early recurrence. Turning to RIT, the results of studies in respect of various tumour types are reviewed, with due attention to reported toxicity. As regards colorectal cancer, no consistent therapeutic effects have been achieved, and myelotoxicity is typically the dose-limiting factor. Thus many questions remain to be answered, regarding antigens to be targeted, fractionation schedule, the use of "humanised" antibodies, choice of radionuclide and the use of intact immunoglobulins or fragments. These questions are considered. Overall it is concluded that the most promising application of RIT is as adjuvant therapy in patients with minimal residual disease, and a controlled multicentre trial is recommended. The development of more potent radio-immunoconjugates for therapeutic and ultimately diagnostic purposes will contribute to the improvement and development of IS by increasing its potential to influence prognosis. PMID- 7556310 TI - Simple purification and quality control of radio-iodinated Hippuran for the estimation of effective renal plasma flow. PMID- 7556311 TI - The U.S. Advisory Committee on Human Radiation Experiments. PMID- 7556313 TI - Recurrent ventricular tachycardia in asymptomatic young children with an apparently normal heart. AB - Ventricular tachycardia without underlying heart disease is rare in infancy and childhood. Four young children (median age 8 months at initial presentation) with frequently recurrent episodes of asymptomatic and self-limiting ventricular tachycardia are reported. By noninvasive investigation no apparent heart disease has been found in all patients. Initially three of the four children had been treated with an anti-arrhythmic drug. Treatment was soon stopped in two patients for lack of symptoms and for lack of efficacy of therapy; one patient remained on beta-blocker therapy. One child did not receive anti-arrhythmic therapy. After a mean follow up of 32 months all patients continued to be asymptomatic despite frequently recurrent episodes of self-limiting ventricular tachycardia. CONCLUSION: Ventricular tachycardia in asymptomatic children with an otherwise normal heart carries a good prognosis. Invasive investigation (cardiac catheterization with electrophysiological study and right ventricular biopsy) can be withhold, as long as there are no symptoms. For lack of efficiency of antiarrhythmic drugs in suppressing ventricular tachycardia in asymptomatic children with apparently normal hearts, these patients may be left without therapy but have to be followed closely. PMID- 7556312 TI - Craniovertebral junction anomalies in inherited disorders: part of the syndrome or caused by the disorder? AB - Patterns of skeletal abnormality at the craniovertebral junction in the normal population and in syndromes such as Down, Morquio etc, are compared and the recent embryological data and comparative anatomy reviewed. The authors' view based on their own clinical and radiological experience is that the os odontoideum is the product of excessive movement at the time of ossification of the cartilaginous dens and is exactly analogous to the unfused Type II odontoid fracture. True hypoplasia of the odontoid peg is part of a wider segmentation defect associated with Klippel Feil, occipitalised atlas and/or basilar invagination; it is hardly ever associated with instability. PMID- 7556315 TI - Intractable diarrhoea in infancy in the 1990s: a survey in Italy. AB - A "quick" prevalence study of intractable diarrhoea (defined as diarrhoea lasting more than 3 weeks and dependent on parenteral nutrition [PN] for more than 50% of daily caloric intake) was conducted by FAX. All 35 paediatric gastroenterology services which had been contacted answered questionnaire sent by FAX. 20 cases of intractable diarrhoea were identified in 9 centres. In 12 cases PN was administered at home, the other 8 cases being treated as inpatients for an average duration of 9.5 months. A diagnosis had been established in 11 out of 20 cases. Auto-immune enteropathy was the most frequent diagnosis (5 cases); congenital microvillous atrophy (3 cases); chronic pseudo-obstruction (2 cases) and multiple food intolerance (1 case). Undefined 9/20 cases presented atrophy of intestinal mucosa. The age of the beginning of diarrhoea varied from 2 days to 12 years, but was more than 16 months only in some cases with auto-immune enteropathy. CONCLUSION: Intractable diarrhoea has a low prevalence in Italy and remains a rare but very intricating problem. Long-term PN is recommended in most cases: autoimmune enteropathy is the most frequent cause but in about half of the cases the aetiopathogenetic diagnosis is still not defined. PMID- 7556314 TI - Re-occlusion of residual leaks after transcatheter occlusion of patent ductus arteriosus. AB - Between March 1990 and November 1993 175 patients underwent successful closure of patent ductus arteriosus using the Rashkind double umbrella device. Of those patients seen on follow up, 13 (9 female, 4 male) had a residual leak and were admitted for implantation of a second device alongside the first device. The interval between the procedures ranged from 6 to 22 months. The mean age at the first procedure was 73.9 months (range 24-204 months) and the mean weight was 18.9kg (range 8.4-64). The mean age at the second procedure was 86.9 months (30 213) and the mean weight was 21.4 kg (8.6-64). The first device was 17 mm in 11 patients and 12 mm in two. The second device was 17 mm in four patients and 12 mm in nine. Four patients required two 17 mm devices in total. There was no difference in the two procedures regarding the fluoroscopy time, procedure time, complications and length of hospital stay. The second procedure was uneventful; however, in one patient the residual ductus had to be dilated before successfully deploying a 12 mm device. Ten patients had immediate total occlusion and three had a trivial leak on echocardiography 24 h later. One of the three patients was found to have total occlusion on Doppler echocardiography 1 year later. The other patients are yet to be seen for follow-up. Hence a total occlusion rate was in 11/13 patients (85%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556316 TI - Patent ductus venosus associated with a hyperintense globus pallidum on T1 weighted magnetic resonance imaging and pulmonary hypertension. AB - We report the case of a 13-year-old Japanese boy with a patent ductus venosus. He experienced mild disorientation and hallucination at age 8 years. Hyperammonaemia was discovered at age 12 years. Brain MRI demonstrated multiple intracranial hyperintense lesions, mainly in the globus pallidum, which suggested portosystemic encephalopathy. Patent ductus venosus was demonstrated by abdominal ultrasonography and angiography. Cardiopulmonary investigation revealed pulmonary hypertension. An intracranial hyperintense lesion observed on T1-weighted MRI may be an initial clue for discovering a patent ductus venosus in asymptomatic patients. CONCLUSION: When patent ductus venosus is disclosed, pulmonary hypertension should be sought, as in cases with other portosystemic shunts. PMID- 7556317 TI - Fulminant hepatic failure in a child as a potential adverse effect of trimethoprim-sulphamethoxazole. AB - Trimethoprim-sulphamethoxazole (TMP-SMZ) is considered a safe drug for treatment of infectious bacterial diseases in children. Side-effects are rare and generally take the form of a hypersensitivity reaction to the sulphamethoxazole component of the drug. Hepatic injury usually presents as a transient elevation of liver enzymes, which is of little clinical relevance. Fulminant liver failure due to TMP-SMZ has been reported in only six adults and never in children. We here report a 5-year-old girl who developed fulminant liver failure 3 weeks after her third exposure to TMP-SMZ. After a biphasic clinical course she underwent successful liver transplantation. CONCLUSION: Trimethoprim-Sulphamethoxazole may cause fulminant liver failure in children. The disease can run a biphasic clinical course and liver transplantation must be considered as the therapeutic option for these patients. PMID- 7556318 TI - Homozygous protein C deficiency--management with protein C concentrate. AB - Two unrelated female infants with homozygous protein C (Pr C) deficiency are reported. Both are of U.K. Pakistani origin and in each case the parents are consanguinous. A previous sibling had died in each family. Both sets of parents were shown to be carriers. The concentration of Pr C in both infants was low at birth. Both developed necrotic skin lesions (purpura fulminans) and responded well to Pr C concentrate. Both are developing normally although one has visual impairment due to retinal artery thrombosis which occurred before treatment was commenced. Both infants are treated with intravenous Pr C concentrate administered daily by the parents at home. Studies of the half-life of exogenous Pr C in one of the patients has shown an increase from 2.7 to 10.8 h during the course of treatment thus enabling it to be administered once daily while still maintaining effective plasma concentrations. In the other patient half-life has fluctuated but Pr C is also given once daily. This is the first report of this condition being treated in this way in the United Kingdom. CONCLUSION: Infusion of Pr C is a safe and efficient way of treating infants with homozygous Pr C deficiency in the medium term. PMID- 7556320 TI - Mother-to-child transmission of human immunodeficiency virus type 1: influence of parity and mode of delivery. Paediatric AIDS Group of Switzerland. AB - In a national prospective study of risk factors for mother-to-child transmission of human immunodeficiency virus (HIV), 316 children of HIV-positive mothers were followed up for at least 6 months. Infection status was determined in 254 of them and 46 were found to be infected giving a transmission rate of 18.1%. Univariate analysis of potential risk factors for mother-to-child transmission showed an association between primiparity and increased transmission rate: odds ratio 2.2, 95% confidence interval (CI) 1.1-4.6, P < 0.05. Analysis by logistic regression confirmed this association (adjusted odds ratio 2.4) and showed, in addition, a negative association between transmission rate and elective Caesarean section (adjusted odds ratio 0.36, 95% CI 0.13-0.97, P < 0.05). The effect of primiparity was less pronounced in combination with elective Caesarean section (odds ratio 1.7) than with other delivery modes (odds ratio 2.5, difference not significant). HIV-infected children were less likely to experience the birth of a younger sibling during the observation period than their uninfected counterparts (2 of 46 vs 27 of 208, P < 0.05 by logrank test). CONCLUSION: Primiparous women appear to transmit HIV to their children at a higher rate. This could be explained by increased intrapartum transmission because of longer and more complicated labour in primiparas and/or by a self-selection of women with lower risk of transmission among those deciding to have additional children. PMID- 7556319 TI - Histiocytic haemophagocytosis in a patient with Kawasaki disease: changes in the hypercytokinaemic state. AB - A 32-month-old Japanese boy exhibited haemophagocytic syndrome (HPS) during the recurrent course of Kawasaki disease. Despite repeated gamma-globulin therapy, he developed cytopenia with marked hepatomegaly and evidence of histiocytic haemophagocytosis in the bone marrow. Serum levels of interferon-gamma and tumour necrosis factor, but not of interleukin-1 beta, increased in parallel with his symptoms. No confirmation was obtained of the association of toxic reactions to the used drugs. No coronary lesions remained as sequelae. CONCLUSION: Cytopenia in Kawasaki disease could herald HPS, and the hypercytokinaemia involved in the two febrile syndromes might be of distinct nature. PMID- 7556321 TI - Intracranial tuberculosis in The Netherlands: four paediatric cases. AB - Since 1987 a resurgence of tuberculosis is occurring in the Netherlands in analogy to the situation in other industralised countries. So far this has not been associated with an increased incidence of tuberculous meningitis, which is still a rare complication of the disease. Four cases of intracranial tuberculosis were recently diagnosed in our hospital. All children were from middle-class Dutch families with no identified risk for tuberculosis. An extensive contact identification search could identify a contact in two patients. Two children were in an advanced stage of disease before diagnosis. Skin tests were repeatedly negative in all cases which delayed treatment in two cases for almost 2 weeks. One patient developing a cerebral tuberculous focus during therapy responded well to a second course of corticosteroids. Three children developed severe neurological sequelae. CONCLUSION: This report illustrates that suspicion of intracranial tuberculosis is warranted in the case of an ill-defined inflammatory neurological syndrome. PMID- 7556322 TI - In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: characterization of seven common mutations. AB - Mutations in the gene encoding phenylalanine hydroxylase (PAH) cause persistent hyperphenylalaninaemia. To date, more than 200 point mutations and microdeletions have been characterized. Each mutation has a particular quantitative effect on enzyme activity and recessive expression of different mutant alleles results in a marked interindividual heterogeneity of metabolic and clinical phenotypes. In this paper we demonstrate how a simple clinical test can be used to evaluate the correlation between mutation genotype and phenylalanine metabolism. In hyperphenylalaninaemic patients with known PAH mutation genotype, we have investigated phenylalanine turnover in vivo by measuring the ability to eliminate a test dose of L-phenylalanine. All patients could be considered functionally hemizygous for one of their mutant alleles by carrying on the other allele a mutation that is known to completely abolish PAH activity and encode a peptide with no immunoreactivity. Seven mutations (R408W, IVS-12nt1, R261Q, G46S, Y414C, A104D, and D415N) were characterized by oral phenylalanine loading, each mutation being represented by at least three patients. The elimination profile determined for a 3-day period provides a measure to compare residual activity of the mutant proteins and to assign each mutation to a particular metabolic phenotype. The established relation between genotype and phenotype may enable prediction of the severity of the disease by genotype determination in the newborn period. This will aid in the management of hyperphenylalaninaemia and may improve prognosis. CONCLUSION: The possibility of predicting the residual enzyme activity by DNA analysis performed already in the newborn period allows the prompt implementation of a diet that is adjusted to the degree of PAH deficiency. This may improve management and prognosis of hyperphenylalaninaemia. PMID- 7556324 TI - Streptococcus pneumoniae invasive disease in the neonatal period: an increasing problem? AB - A series of 11 cases of invasive infection with Streptococcus pneumoniae, occurring over an 11-year period, is reported. Eight of the 11 cases occurred during the final 2 years of the study suggesting that the incidence of infection may be increasing. Infection carries a high mortality (3/11). Morbidity includes meningitis, convulsions and respiratory failure. In one case S. pneumoniae meningitis occurred in both mother and newborn. Most mothers who carried the organism were asymptomatic at the time of delivery. CONCLUSION: S. pneumoniae should be specifically sought in swabs taken from the pregnant mother and newborn and if isolated, even in the absence of symptoms, antibiotic therapy against the organism should be strongly considered. PMID- 7556325 TI - The efficacy of heparin in maintaining peripheral infusions in neonates. AB - The study set out to determine the survival times of peripheral total parenteral nutrition (TPN) infusion sites in neonates using a prospective, single blind, randomised trial design. The effects of various concentrations of co-administered heparin was measured using survival analysis, and of other continuous variables using multivariate analysis, against a non-heparinized control group. The study was conducted in special care baby unit located within a specialist maternity hospital in London, United Kingdom. Heparin at 0.1, 0.25, 0.5 and 1 IU/ml was added to TPN infusions delivered through peripheral veins and the survival times of the infusions determined. For infusion sites receiving heparinized fluids, the relative risk of failure decreased and the median survival time increased as the heparin concentration increased, with a maximal effect at a heparin concentration of 0.5 IU/ml (P < 0.001). Multivariate analysis using the Cox proportional hazard model confirmed the efficacy of heparin and highlighted a history of infusion therapy and the co-administration of gentamicin (from a range of drugs analysed) as being risk factors associated with infusion site failure. CONCLUSION: Intravenous infusion survival time can be prolonged using heparin additive at an optimal concentration of 0.5 IU/ml. This should also be of additional interest to paediatricians as heparin is an ubiquitous drug on neonatal units and its clinical use needs to be rationalised. PMID- 7556326 TI - Erythrocyte sodium-potassium transport in hyperkalaemic and normokalaemic infants. AB - One of the causes of early onset hyperkalaemia in very low birth weight infants is presumed to be the dysfunction of K+ transport across the cell membrane. Sodium-potassium adenosine triphosphatase(Na(+)-K+ ATPase) is known to play a major role in K+ transport. We compared the concentrations of erythrocyte Na(+) K+ ATPase (Vmax levels) for hyperkalaemic and normokalaemic infants of matched gestational age. In hyperkalaemic infants, the highest levels of Vmax were reached at 24-48 h after birth, but in normokalaemic infants, there were no significant changes in Vmax levels during the 1st week after birth. At 12-72 h after birth, erythrocyte K+ concentrations for hyperkalaemic infants were higher than those of normokalaemic infants. For both groups of infants, the highest levels of plasma K+ during the 1st week after birth showed a positive correlation with those of Vmax. CONCLUSION: Na(+)-K+ ATPase on the cell membrane is activated to compensate for hyperkalaemia; however, when this compensation is incomplete, hyperkalaemia occurs. PMID- 7556328 TI - Hyponatraemia and hyperkalaemia in acute pyelonephritis without urinary tract anomalies. AB - Three children with severe hyponatraemia and hyperkalaemia associated with acute pyelonephritis are reported. All were very young male infants in a poor general condition and seriously dehydrated. Diagnostic procedures did not detect obstructive uropathy or vesico-ureteric reflux. CONCLUSION: Hyponatraemia and hyperkalaemia occurs in young infants with severe acute pyelonephritis in the absence of obstructive uropathy or vesico-ureteric reflux. The severe inflammation of the kidney itself may explain the electrolyte disturbance by a transient resistance of the distal tubule to aldosterone. PMID- 7556327 TI - Nephropathy with Wilms tumour or gonadal dysgenesis: incomplete Denys-Drash syndrome or separate diseases? AB - We report three children, one presenting with nephropathy, bilateral Wilms tumour (WT) and cryptorchism, one with combined nephropathy and gonadal dysgenesis and one with nephropathy which developed 13 years after a WT. The first case was recognized as typical Denys-Drash syndrome (DDS) which is characterized by the combination of nephropathy, intersex disorders and WT. The two other patients, who did not express the full spectrum of the syndrome, were older than 10 years, when they reached and stage renal failure. The fact that nephropathy in childhood is combined with such rare diseases like gonadal dysgenesis and/or WT, supports the concept of a common aetiology with DDS. Therefore, the patients were analysed for possible Wilms tumour suppressor gene (WT1) mutations. In all three individuals mutations in the heterozygous configuration could be demonstrated. CONCLUSION: These results provide evidence that incomplete and complete DDS are diseases of the same spectrum. WT1 analysis of more children with two symptoms of the triad of DDS should be helpful in establishing genotype-phenotype correlations and in understanding differences in the clinical picture of DDS. PMID- 7556323 TI - Early-onset fatal encephalomyopathy associated with severe mtDNA depletion. AB - We studied a 3-month-old girl who was admitted to hospital because of respiratory distress. The clinical course was characterized by a rapidly progressive generalized hypotonia with lactic acidosis and she died at 4 months of age. A muscle biopsy showed few ragged-red fibres and lack of histochemical cytochrome c oxidase reaction in all fibres. Enzyme activities of the respiratory chain complexes containing subunits encoded by the mitochondrial DNA (mtDNA) were markedly decreased. A quantitative Southern blot analysis revealed 99% depletion of mtDNA in muscle and normal amounts in blood. There was no family history and the dizygotic twin sister of the patient was no symptomatic. CONCLUSION: This new case confirms the rapidly fatal evolution associated with severe depletion of muscle mtDNA. PMID- 7556330 TI - Are patients with homocystinuria being missed? PMID- 7556329 TI - Dental amalgam and Feer disease. PMID- 7556331 TI - Cardiac tumours in tuberous sclerosis. PMID- 7556333 TI - Congenital pneumococcal sepsis in one of a set of premature twins. PMID- 7556332 TI - Ceftriaxone-associated gallbladder lithiasis in children. PMID- 7556334 TI - Growth retardation as a long-term side-effect of alpha-interferon therapy. PMID- 7556335 TI - Effects of indomethacin on jejunal mucosal blood flow in the infant rat. PMID- 7556336 TI - Rats, I've been bitten! PMID- 7556337 TI - Neurological deterioration in patients with urea cycle disorders under valproate therapy--a cause for concern. PMID- 7556339 TI - Arthrotec in general practice. Proceedings from the clinical investigator meeting held May 1994, Leeds, UK. PMID- 7556338 TI - Combination therapy with tacrolimus and betamethasone for a patient with X-linked auto-immune enteropathy. PMID- 7556340 TI - Emergency hospital admissions. PMID- 7556342 TI - Review of Arthrotec clinical data. PMID- 7556341 TI - NSAIDs and the gut--where are we now? PMID- 7556343 TI - Rheumatological audit--a general practice perspective. PMID- 7556344 TI - Arthrotec, diclofenac and ibuprofen in general practice. PMID- 7556345 TI - Rheumatological audit--a hospital perspective--the acute hot joint. PMID- 7556347 TI - Exercise rehabilitation for patients with peripheral arterial disease. AB - Intermittent claudication, resulting from PAD, impairs functional status. Reducing the disability result from the disease is therefore an important goal of treatment. To evaluate the efficacy of an intervention designed to improve functional status requires that appropriate outcome measures be developed to assess all treatments. Such outcome measures include graded treadmill testing and questionnaire assessment. These methodologies are important because they have a high degree of precision and accuracy and are practical and reproducible. Thus, functional status changes resulting from any intervention can be evaluated, and interventions can be compared with one another using the same methodologies. Currently, interventional therapies are often used to treat a portion of patients with claudication [49]. Such therapies restore blood flow and improve functional status, but with a high associated cost: morbidity and mortality. Pharmacological therapies currently are being developed, but the role of drugs in the overall management of claudication needs further study. Importantly, exercise therapy has been shown in numerous studies to be efficacious and very well tolerated by patients. Patients improve both their walking ability in the laboratory and their community-based functional status. Because of the efficacy of this treatment, in addition to the low associated morbidity, exercise therapy is recommended as a major treatment option for persons with intermittent claudication due to PAD. PMID- 7556348 TI - Influence of exercise on human sleep. AB - Several research paradigms have been used to examine the influence of exercise on sleep. Epidemiological studies show that exercise is perceived as helpful in promoting sleep and suggest that regular physical activity may be useful in improving sleep quality and reducing daytime sleepiness. Additional epidemiological inquiry is clearly warranted based on the available evidence. Acute exercise experiments that have measured sleep physiology directly from subjects who either performed, or refrained from, daytime exercise indicate that exercise is associated with a small, but reliable increase in Stage 2 and slow wave sleep. The mechanism(s) that underlie exercise-associated increases in SWS is unknown. However, there is evidence that links elevations in daytime core body temperature to increases in SWS. Acute exercise experiments were found to be associated with a reduction in REM sleep and a delay in REM onset latency that were larger in magnitude than the effects observed for Stage 2 and SWS. These REM sleep observations highlight the need for continued study of the consequences of exercise on both circadian and homeostatic aspects of sleep. The delay in REM onset latency observed in the naturalistic acute exercise studies was consistent with the results of experiments in which environmental factors were more rigorously controlled and showed that physical exercise, or a concomitant, can induce a phase delay in markers of the human circadian pacemaker. It is worth pointing out that the most sophisticated and rigorous experiments from a standpoint of understanding sleep, such as those involving constant routines, bed rest, and temporal isolation, have for the most part used exercise in a crude manner. Because exercise is a stressor with diverse psychophysiological consequences that depends in part upon the interaction of multiple factors (e.g., the setting; degree of environmental heat stress; the activity history and fitness of the subject; the duration, intensity, and timing of the exercise bout; body position, etc.), understanding the influence of exercise on sleep will be stymied until carefully designed sleep studies also use exercise in an equally sophisticated and systematic way. Exercise is widely believed to have large effects on sleep. However, the scientific evidence does not strongly support this common belief. This incongruity may well be explained in part by considering the paradigms that have been used to study exercise and sleep.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556346 TI - The Tayside record linkage system. PMID- 7556350 TI - Functional morphology and motor control of series-fibered muscles. PMID- 7556352 TI - The evolution of research on motor development: new approaches bringing new insights. PMID- 7556351 TI - The impact of exercise on the immune system: NK cells, interleukins 1 and 2, and related responses. PMID- 7556349 TI - Exercise and oxidative stress: role of the cellular antioxidant systems. PMID- 7556353 TI - Integration of the physiological factors determining endurance performance ability. AB - This model is used to understand the interrelationships of the physiological factors determining endurance performance ability during prolonged exercise. Early studies found that marathon runners maintain a velocity in competition that corresponds to the intensity at which lactate begins to accumulate in blood and muscle [7, 8, 19]. From this observation, the concept developed that this blood lactate threshold (LT Vo2) reflects the degree of muscular stress, glycogenolysis and fatigue. However, it was not clear whether the lactate accumulation was a result of cardiovascular limitations linked to oxygen delivery, as reflected by Vo2max [54], as opposed to metabolic factors in the exercising muscle related to the extent to which mitochondrial respiration is disturbed to maintain a given rate of O2 consumption [29, 30]. Two studies were performed to determine whether LT Vo2 was tightly coupled to Vo2max. In one study, endurance-trained ischemic heart disease patients were observed to possess a Vo2max that was 18% below that of normal master athletes who followed the patient's training program and who displayed the same performance ability as the patients. Both the patients and the normal men displayed an identical LT Vo2 (i.e., 37 ml/kg/min) (Fig. 2.5). Therefore, performance was determined primarily by LT Vo2 instead of Vo2max in this situation, albeit with abnormal subjects. In a second study we assembled two groups of competitive cyclists who were identical in Vo2max but differed by having a high or low LT Vo2 (82% vs. 66% Vo2max) [13]. When cycling at 80-88% Vo2max, the low LT group displayed more than a 2-fold higher rate of muscle glycogen use and blood lactate concentration, and as a result were able to exercise only one-half as long as the high LT group. Performance time for a given Vo2 was clearly related to LT Vo2 instead of Vo2max (Fig. 2.6). This is not to say that Vo2max plays no role in determining LT Vo2, because as in heart disease patients, it clearly sets the upper limit. Indeed, we have seen that much of the variance (i.e., 31-72%) in LT Vo2 is related to Vo2max. (Fig. 2.11.) However, improvements in performance after the first 2-3 yr of intense training are associated with improvements in LT Vo2, whereas Vo2max generally increases very little thereafter (Table 2.3). The next question concerns the factors responsible for further increases in LT Vo2 and Performance. Another major factor determining LT Vo2 is the muscle's Aerobic Enzyme Activity or mitochondrial respiratory capacity, as discussed in previous reviews [29, 30].(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556354 TI - Physical activity, body weight, and adiposity: an epidemiologic perspective. AB - Overweight is a common problem in the United States, especially among minority women and persons of lower socioeconomic status and lower educational attainment. Moreover, the prevalence of reported inactivity may be highest in these same population subgroups. Both overweight and sedentary behavior are important risk factors for chronic disease morbidity and mortality; however, there is encouraging evidence that moderate to higher levels of physical activity may provide protection from certain chronic diseases, even among persons with established risk factors. Several methodological issues preclude the ability to determine accurately the impact of physical activity on body weight and adiposity. These issues include (a) a low prevalence of higher-intensity physical activity in the general population, (b) measurement error with regard to self reported activity, especially that of lower-intensity, (c) inappropriate time frame of the physical activity assessment, (d) effect modification by age and gender, and (e) failure to adjust for important statistical confounders. Despite these methodological issues, the inverse association between physical activity and weight has been reported in several cross-sectional epidemiologic studies, which consistently report lower body weight, or more favorable distribution of body fat, with higher categorical levels of self-reported physical activity. Directionality of the physical activity and weight relation cannot be determined from these studies, however, and the few longitudinal epidemiologic studies that have assessed the influence of physical activity on the risk of weight gain report inconclusive results. This is possibly because a one-time assessment of physical activity if not adequate in describing the contribution of habitual physical activity on long-term weight maintenance. Therefore, longitudinal population-based studies with multiple assessments of physical activity over long follow-up periods are necessary to determine this relationship. In any case, the evidence suggests that persons concerned with overweight, or especially the prevention of overweight and obesity, should increase their physical activity. Sociodemographic characteristics such as age, gender, educational level, and weight are associated with physical activity patterns and choices. Therefore, these characteristics should be considered by professionals when implementing physical activity interventions for weight control. Walking is accessible to all segments of the U.S. population. Because walking is convenient, low cost, and safe, and can result in weight loss if done regularly for durations of at least 20-30 min, its relative merits should be stressed in weight reduction and maintenance programs. Furthermore, to reduce the morbidity and mortality associated with overweight, obesity, and sedentary behavior, priority for intervention programs should be directed at persons in the most vulnerable sectors of the population. PMID- 7556356 TI - Role of exercise in the cause and prevention of cardiac dysfunction. PMID- 7556357 TI - Free radicals and exercise: effects of nutritional antioxidant supplementation. PMID- 7556355 TI - Human skeletal muscle metabolism in health and disease: utility of magnetic resonance spectroscopy. PMID- 7556358 TI - Sports medicine aspects of cervical spinal stenosis. PMID- 7556359 TI - Aging and body composition: biological changes and methodological issues. AB - There is no doubt that body composition changes with aging. Some general trends have been described, including an increase in body weight and fat mass in middle age followed by a decrease in stature, weight, FFM, and body cell mass at older ages. Losses in muscle, protein, and bone mineral contribute to the decline in FFM; however, the onset and rates of decline remain controversial. Most data are available for men and women < 80 yr and we know relatively little about the normal status and the changes that occur in body composition in elderly men and women. This situation has developed in part because the changes that occur in various body constituents with aging confound the estimation of body composition by traditional techniques. Hence, there is a need for longitudinal reference data in persons 80 yr of age, both to describe the normal status and to develop valid prediction equations for estimating body composition in older men and women in settings outside the laboratory. This should be possible using new technologies and approaches based on multiple component models of body composition. An understanding of the normal changes in body composition with increasing age, the normal variation in these changes, and their health implications is important for the health, nutritional support, and pharmacologic treatment of elderly men and women in the United States. The information is especially important because elderly men and women, in terms of both numbers and health care dollars, represent the most rapidly expanding segment of the U.S. population. PMID- 7556360 TI - Effects of diving and hyperbaria on responses to exercise. PMID- 7556362 TI - The fetal and infant origins of disease. PMID- 7556363 TI - Aminophylline: could it act as an antioxidant in vivo? AB - Potential antioxidant properties of aminophylline and theophylline were investigated. We have found that these drugs, though ineffective against superoxide anion and hydrogen peroxide, are scavengers of hydroxyl radical (OH.). Indeed, second-order rate constants (k) of aminophylline and theophylline with OH. are about 1.9 x 10(10) mol-1 s-1 and 4.5 x 10(9) mol-1 s-1, respectively. Ethylenediamine, which is present in the aminophylline molecule, significantly contributes to this marked OH. scavenging activity, since it is characterized by a high k value, i.e. about 8 x 10(9) mol-1 s-1. However, when using therapeutically relevant concentrations of the methylxanthines (9 and 13 micrograms mL-1), significant antioxidant effects against OH.-induced oxidant injury are evident only with aminophylline. Although all three substances can apparently bind and inactivate iron, only aminophylline is effective at 9 and 13 micrograms mL-1; also this action is favoured by ethylenediamine. Moreover, therapeutic concentrations of aminophylline, but not of theophylline, are capable of antagonizing hypochlorous acid (HOCl); this effect is entirely due to the presence of ethylenediamine. Oxidant species, such as OH. and HOCl, have been implicated in the pathophysiology of asthma; it could be hypothesized, therefore, that some therapeutic effects of aminophylline may be related to its antioxidant properties, which are partly or totally attributable to ethylenediamine, depending on the chemical identity of the prooxidant antagonized (e.g. iron/OH. or HOCl). Aminophylline antioxidant capacity should be taken into account when investigating the lung epithelial lining fluid antioxidant capacity and oxidative stress indices in humans. PMID- 7556364 TI - Alzheimer amyloid beta-peptides exhibit ionophore-like properties in human erythrocytes. AB - There is growing evidence that the amyloid beta-peptide (beta 1-40) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta 1-40 has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether beta 1-40 (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2 h and 4 h at 37 degrees C together with 6 mumol L-1 of beta 1-40 or of fragments beta 1-28 and beta 25-35, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05 mumol L-1). Moreover, beta 1-40 and its two fragments induced a significant alteration of 45Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of beta 1-40 and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca(2+)-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease. PMID- 7556361 TI - Changes in movement capabilities with aging. PMID- 7556365 TI - Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear and polymorphonuclear phagocytes. AB - Isolated human mononuclear and polymorphonuclear phagocytes were stimulated in the presence and absence of serotonin (5-HT), the major secretory product of activated platelets, and the release of reactive oxygen metabolites during the respiratory burst was assessed by luminol-enhanced chemiluminescence. In the presence of 5-HT, a dose-dependent suppression of the chemiluminescence signal occurred, irrespective of the stimulus used to elicit the respiratory burst. A similar suppression of luminol-enhanced chemiluminescence was also seen in a radical generating cell free system. 5-HT was found to be oxidized by the reactive oxygen species released by stimulated phagocytes. This oxidation is prevented in the presence of other antioxidants. The major 5-HT oxidation product was isolated by gel chromatography and identified by mass-spectrometry as a 5-HT dimer, probably 5,5'-dihydroxy-4,4'-bitryptamine. It is concluded that the 5-HT released from activated thrombocytes at sites of inflammation and endothelial cell damage acts as a true scavenger of reactive oxygen species generated during the respiratory burst of stimulated phagocytes and may thus modulate various aspects of cell-mediated defence reactions. PMID- 7556366 TI - Relationship of apolipoproteins AI, B and lipoprotein Lp(a) to hepatic function of liver recipients during the early post-transplant period. AB - To evaluate serum apo AI, apo B, Lp(a) and the ratio of unesterified cholesterol to total cholesterol as markers of hepatic synthetic capacity after orthotopic liver transplantation, serial measurements of these variables were performed on post-transplant days 1, 3, 5, 7, 10 and 14 in 70 patients. Liver function was assessed by a quantitative dynamic test based on the hepatic conversion of lidocaine to monoethylglycinexylidide (MEGX). Patients were divided into two groups on the basis of clinical and laboratory findings, those with evidence (n = 46) and those without evidence (n = 24) of hepatic dysfunction. Apo AI levels fell in both groups to day 5, but then began to increase in the group with good hepatic function, a highly significant (P < 0.001) positive correlation being found with the results of the MEGX test on post-transplant days 7, 10 and 14. The ratio of unesterified cholesterol to total cholesterol rose in both groups from days 1 to 7 and then began to fall in the group without hepatic dysfunction; a highly significant (P < 0.001) negative correlation was observed with the results of the MEGX test on days 10 and 14, Apo B levels rose in both groups from days 1 to 10, with no significant differences between the two groups; no correlation was observed with the results of the MEGX test on any study day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556367 TI - Improved metabolic control in patients with non-insulin-dependent diabetes mellitus is associated with a slower accumulation of glycation products in collagen. AB - Twenty-one patients with non-insulin-dependent diabetes in poor metabolic control were subjected to intensified therapy, in most cases with insulin, to investigate whether it is possible to slow down the accumulation of advanced glycosylation end products of collagen by improving glycaemic control. Fasting and mean daily blood glucose, serum fructosamine and glycohaemoglobin levels, as well as glycation of collagen were measured before and after 1.5 years of intensified therapy. All these parameters except for fructosamine correlated significantly with fasting blood glucose and glycohaemoglobin when measured before the insulin therapy was started, when the patients had had poor but stable metabolic control for a long period of time. After 1.5 years of intensified therapy the level of glycation of collagen did not significantly correlate with the fasting blood glucose or glycohaemoglobin levels, suggesting that the non-enzymatic glycosylation of collagen reflects a longer period of metabolic control of diabetes than the glycohaemoglobin level. Intensified treatment improved previously poor metabolic control in patients with non-insulin-dependent diabetes, and this improvement was reflected in a decrease in fasting and mean daily blood glucose levels, serum fructosamine and glycohaemoglobin concentrations, and in the level of early products of glycation of collagen. The average content of advanced glycosylation end products of collagen, assayed in terms of collagen-linked fluorescence did not decrease. However, they accumulated more slowly in the patient tercile with the greatest decrease in the level of fasting blood glucose than in the tercile with the smallest decrease, and even a decrease in fluorescence was observed in the patients with the greatest improvement in the metabolic control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556370 TI - The influence of ulnar nerve blockade on skin microvascular blood flow. AB - Microvascular research is seriously hampered by the great temporal and spatial variability of the measured skin blood flow and variation in sympathetic vasomotor reflexes within and between persons. Therefore skin vasomotor reflexes were studied before and after ulnar nerve blockade within the same person, resulting in a temporal complete denervation of the fifth finger and partial denervation of the fourth finger. Skin temperature and laser Doppler flux (LDF) were registrated to measure predominantly arteriovenous shuntflow. Measurements were performed on the palmar tip of the second and fifth finger in nine healthy volunteers, at baseline, and during a sympathetic reflex test (i.e. inspiratory gasp) and postural response test. Beat-to-beat digital blood pressure was recorded from the third and fourth finger by a Finapres device. Baseline capillary blood cell velocity (CBV) was measured at the nailfold of the second and the fifth finger. After ulnar blockade baseline skin temperature, LDF and CBV increased significantly, with respectively (mean +/- SE) 3.2 +/- 0.9 degrees C, 20.9 +/- 5.9 relative perfusion units and 0.79 +/- 0.40 mm-1 s. The percentage LDF decrease of the fifth finger during inspiratory gasp was 48.2 +/- 5.3% before and 3.1 +/- 0.9% after blockade. The postural response test showed a decrease in LDF of the fifth finger with no significant difference before and after blockade, respectively 12.3 +/- 14.7% and 8.0 +/- 2.7%, while no difference was found in the increase in digital blood pressure in the denervated fourth finger compared to both the same finger before blockade and to the third non-blocked finger.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556371 TI - Albumin affects erythrocyte aggregation and sedimentation. AB - Erythrocyte aggregation and sedimentation is determined by the concentration of high molecular plasma proteins such as fibrinogen and immunoglobulins. The role of albumin, the most abundant plasma protein, is controversial. We analysed the influence of human albumin (0-80 g L-1) on sedimentation behaviour of erythrocytes suspended (haematocrit 35%) in fibrinogen-enriched normal plasma, plasma from patients with hypoalbuminaemia, solutions of dextran 70 in buffer, and mixtures of fibrinogen, immunoglobulins and albumin in buffer. Sedimentation was measured by the Westergren method, viscometry was performed at low and high shear rate. The addition of albumin to normal plasma, hypoalbuminaemic plasma, or dextran solutions in buffer decreased the erythrocyte sedimentation rate. In contrast, in a mixture of fibrinogen and immunoglobulins in buffer, albumin increased erythrocyte sedimentation. When either fibrinogen or immunoglobulins were omitted, or fibrinogen was replaced by dextran, albumin did not increase sedimentation, which indicates that increased sedimentation was due to an interaction of all three compounds. The viscosity of erythrocyte suspensions was increased by albumin. It is concluded that the influence of albumin on erythrocyte aggregation is complex and depends on the technique used; low shear viscosity is increased, but erythrocyte sedimentation, i.e. under no flow conditions, is decreased. The inhibitory action of albumin on the erythrocyte sedimentation rate has clinical implications and must be known by physicians. It may help to interpret sedimentation rates more correctly and prevent misinterpreting presumed therapeutic successes or failures. PMID- 7556368 TI - Intracoronary magnesium is not protective against acute reperfusion injury in the regional ischaemic-reperfused dog heart. AB - Intravenous magnesium lowers mortality in patients with suspected myocardial infarction. We tested the hypothesis that the protective effect may be due to a direct, local influence of magnesium on myocardial reperfusion injury in a dog model of ischaemia/reperfusion. Ten anaesthetized open chest dogs underwent 1 h of left anterior descending artery (LAD) occlusion and 6 h of reperfusion. The animals received intracoronary (i.c.) magnesium aspartate (Mg, n = 5) or vehicle infusion (n = 5) for the first hour of reperfusion. Mg infusion was adapted to actual LAD flow (ultrasonic flow probe) to increase regional plasma concentration by 4 mmol L-1. Regional myocardial function was measured as percent systolic wall thickening (sWTh, sonomicrometry). Intracoronary Mg increased LAD flow during application (at 15 min reperfusion; Mg, 194 +/- 44 (mean +/- SD); control, 116 +/ 41 mL min-1 100 g-1, P < 0.01). sWTh decreased during coronary occlusion from 14.3 +/- 7.1% to -4.7 +/- 2.7% in the control group and from 14.8 +/- 2.5% to 4.1 +/- 3.1% in the Mg group. Throughout the reperfusion period wall function remained depressed in both groups to a similar degree (control, -3.5 +/- 1.8%; Mg, -3.0 +/- 1.9% at 6 h reperfusion). Global haemodynamics were not different. Infarct size after 6 h reperfusion (TTC staining) was similar in both groups (Mg, 20.6 +/- 5.0; control, 24.4 +/- 8.7% of area at risk). Regional magnesium application (i.c.) to post-ischaemic reperfused myocardium had no influence on infarct size or post-ischaemic regional wall function in this model.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556372 TI - Left ventricular hypertrophy is associated with a stronger impairment of non oxidative glucose metabolism in hypertensive patients. AB - Hypertensive patients with left ventricular hypertrophy (LVH) have a higher degree of hyperinsulinaemia than hypertensive patients without LVH. Obese patients with LVH have also been demonstrated to have a very low glucose disappearance rate after an intravenous glucose bolus. No studies have investigated the difference in insulin action and substrate oxidation in hypertensive patients with and without LVH. For this reason 36 subjects were enrolled for our study: (1) healthy control subjects (n = 10); (2) hypertensive patients without LVH (n = 12); and (3) hypertensive patients with LVH (n = 14). All subjects underwent an oral glucose tolerance test (OGTT, 75 g of glucose) and a euglycaemic hyperinsulinaemic glucose clamp (insulin infusion rate, 7.1 pmol (kg min)-1 for 120 min). In this latter test indirect calorimetry allowed substrate oxidation determination. Echocardiographic methods allowed LVH assessment. Hypertensive patients with LVH had the lowest insulin-mediated nonoxidative glucose metabolism compared to hypertensive patients without LVH (P < 0.01) and to healthy subjects (P < 0.001). In the whole group of hypertensive patients (n = 26), partial correlations showed left ventricular mass index (LVMI) associated with fasting plasma insulin levels (r = 0.44 P < 0.005), insulin mediated whole body glucose disposal (r = -0.41 P < 0.01) and nonoxidative glucose metabolism (r = -0.33 P < 0.04) independently of age, body weight, systolic blood pressure and plasma catecholamines levels. In conclusion, our data provide evidence that LVH in hypertensive patients is associated with a worsening in nonoxidative glucose metabolism. PMID- 7556369 TI - Liposoluble vitamins and naturally occurring carotenoids in porphyria cutanea tarda. AB - The authors consider two groups of patients with overt sporadic porphyria cutanea tarda (PCT) from different continents, with the aim of evaluating the possible impairment of the liposoluble antioxidative system, given the possible synergic effect of porphyrins and iron in promoting oxidative cellular damage. Twenty three Italian outpatients with overt sporadic PCT and 11 outpatients with PCT from Buenos Aires (Argentina) were matched with 60 patients with liver cirrhosis and 52 healthy Italian controls. Serum levels of alpha- and beta-carotene, cryptoxanthin, zeaxanthin, lutein, lycopene, retinol and alpha-tocopherol were detected by a high-performance liquid chromatographic technique devised in our laboratory, which afforded an accurate and simultaneous resolution of all these compounds. The results point to a significant reduction in plasma levels of alpha and beta-carotene in both the PCT populations with respect not only to controls, but also to the cirrhotic population, which had more severe liver damage. Moreover, other carotenoids with proven antioxidative properties, like cryptoxanthin and lycopene, are greatly reduced in our PCT populations. This confirms the suggested synergic effect of iron and porphyrins in the oxidative intracellular damage with consequent depletion of antioxidative liposoluble molecules. PMID- 7556375 TI - Ibogaine and its congeners are sigma 2 receptor-selective ligands with moderate affinity. AB - Ibogaine (12-methoxyibogamine) exhibited moderate affinity for sigma 2 sites (Ki = 201 nM) and low affinity for sigma 1 sites (Ki = 8554 nM), thus showing 43-fold selectivity for sigma 2 receptors. Tabernanthine (13-methoxyibogamine) and (+/-) ibogamine had sigma 2 Ki = 194 nM and 137 nM, respectively. However, they showed 3- to 5-fold higher sigma 1 affinity compared to ibogaine, resulting in about 14 fold selectivity for sigma 2 sites over sigma 1. A potential ibogaine metabolite, O-des-methyl-ibogaine, had markedly reduced sigma 2 affinity relative to ibogaine (Ki = 5,226 nM) and also lacked significant affinity for sigma 1 sites. (+/-) Coronaridine ((+/-)-18-carbomethoxyibogamine) and harmaline (1-methyl-7-methoxy 3,4-dihydro-beta-carboline) lacked significant affinity for either sigma subtype. Thus, sigma 2 receptors could play a role in the actions of ibogaine. PMID- 7556374 TI - Reverse transcription-polymerase chain reaction detection of collagen transcripts in healing human wounds. AB - The aim of this study was to analyse the expression of COl1A1, COl1A2 and COl3A1 in 6 mm diameter punch biopsies obtained from human wounds. Total RNA was isolated from biopsies taken from Sacrococcygeal pilonidal sinus excision cavities at weekly intervals between surgery and clinical closure. cDNAs were generated from the RNA using reverse transcriptase and polymerase chain reaction (PCR) amplifications performed with oligonucleotide primer pairs specific for regions of the COl1A1, COl1A2 and COl3A1 genes. The expression of these three genes was demonstrated throughout the course of healing on 36 biopsies taken from nine patients between surgery and clinical closure. Amplification bands demonstrated on cDNAs generated from 6 mm diameter biopsies were comparable in intensity and specificity with those generated from 50 mg excised scar tissue and cultured fibroblasts. The RT-PCR technique described here allows the rapid 'routine' detection of specific gene expression in 6 mm biopsies obtained from healing wounds. PMID- 7556373 TI - Effect of colectomy with ileo-anal anastomosis on the biliary lipids. AB - Total colectomy with ileo-anal anastomosis is an effective treatment for ulcerative colitis and familial adenomatous polyposis. The absence of the colon and the coexistence of bile acid malabsorption may increase bile lithogenicity, but data on biliary lipid composition in patients with this operation is lacking. Our aim was to assess bile lithogenicity, bile composition and mass of biliary lipids within the gallbladder. We studied 11 patients with total colectomy and ileo-anal anastomosis and 16 healthy controls. We measured the percentage composition of conjugated bile acids and the masses within the gallbladder of the three main biliary lipids. This method, in contrast with measurement of cholesterol saturation index, can determine the cause of bile lithogenicity in terms of absolute modifications of the biliary lipids. There was no difference in the cholesterol saturation index between patients and controls. Colectomy patients had reduced masses of all three biliary lipids (medians and ranges, mmol): cholesterol 0.11 (0.03-0.24) vs. 0.36 (0.02-0.96), P < 0.02; bile acid 1.62 (0.75-5.21) vs. 3.95 (1.27-8.70), P < 0.01; phospholipids 0.35 (0.07-0.69) vs. 1.14 (0.14-3.00), P < 0.002. They also had reduced per cent deoxycholic acid: 3.8 (0.0-27.6) vs. 17.4 (6.4-44.7), P < 0.005, and increased percent cholic acid: 44.9 (23.3-71.4) vs. 34.3 (19.2-57.9), P < 0.05. We conclude that, despite having bile acid malabsorption, patients with colectomy and ileo-anal anastomosis have a normal cholesterol saturation index, caused by a concomitant reduction in the masses of all three biliary lipids. The reduced per cent biliary deoxycholic acid may help explain the reduced cholesterol and phospholipid masses in these patients. Total colectomy with ileo-anal anastomosis does not seem to predispose to the formation of cholesterol gallstones. PMID- 7556376 TI - Possible involvement of nitric oxide in red nucleus stimulation-induced analgesia in the rat. AB - There is considerable evidence that nitric oxide (NO) plays a role in synaptic transmission in both central and peripheral nervous systems. Recent studies have suggested the involvement of the L-arginine-NO pathway in nociceptive transmission/modulation. Electrical stimulation of the red nucleus in the rat evokes potent analgesia. Microinjection of different concentrations of L-arginine (1 nmol-1 mumol), but not of D-arginine, produced quick and long-lasting analgesia. Pretreatment with N-nitro-L-arginine methyl ester (1 mumol), a nitric oxide synthase inhibitor, significantly prevented L-arginine-induced analgesia. Further, pretreatment of animals with methylene blue, a known guanylate cyclase inhibitor, also attenuated the development of analgesia. Our results suggest that L-arginine caused production of NO, which in turn activated the red nucleus analgesic system. PMID- 7556377 TI - Is endothelin-1 release at reperfusion of the ischaemic human heart due to cold induced displacement of endothelin from binding sites? AB - Following reperfusion of ischaemic human hearts subjected to cold (4 degrees C) cardioplegia during coronary bypass surgery, there was an increase in cardiac outflow of endothelin-1 but not the pro-peptide big endothelin-1. Furthermore, specific endothelin-1 binding in human lung membrane preparations was displaced by incubation in buffer medium at 4 degrees C. The present results thus indicate that cold-induced displacement of endothelin-1 binding, rather than increased synthesis, may explain the cardiac release of endothelium-1 following ischaemia during heart surgery in which cold cardioplegia has been used. PMID- 7556378 TI - Angiotensin II receptor subtypes and contractile responses in portal vein smooth muscle. AB - The selective biphenylimidazole and tetrahydroimidazopyridine antagonists exemplified by losartan (DuP 753) and PD 123319 have been shown to bind selectively to angiotensin AT1 and AT2 receptor subtypes, respectively. To characterize which subtypes of angiotensin II receptors are expressed in mammalian portal vein smooth muscle, we performed, using both membrane and strip preparations, [3H]angiotensin II binding experiments and then contraction experiments to investigate the functional relevance of these binding sites. Specific binding of [3H]angiotensin II was of high affinity, saturable and reversible. Specific binding of [3H]angiotensin II was completely displaced by angiotensin II and the peptide antagonist [Sar1,Ile8]angiotensin II. The inhibition of [3H]angiotensin II binding by losartan (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)- methyl]imidazole, potassium salt) and DuP 532 (2-n-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5 yl)biph enyl-4-yl)- methyl]imidazole-5-carboxylic acid) was biphasic and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation represented 75% of the total binding and showed high affinity for angiotensin II, losartan and DuP 532, but low affinity for the peptide angiotensin AT2 receptor antagonist CGP 42112A (N-alpha-nicotinoyl-Tyr-Lys-[N alpha-CBZ-Arg]-His-Pro-Ile-OH) and thus appeared identical to the cloned angiotensin AT1 receptor subtype. The remaining 25% of the sites showed nearly 1000-fold lower affinity for losartan, 6500-fold lower affinity for DuP 532 and high affinity for PD 123319 (S-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5 diphenylacetyl- 4,5,6,7-tetrahydro-1H-imidazo-[4,5-c] pyridine-6-carboxylic acid, difluoroacetate monohydrate) and CGP 42112A, with values of Ki in the same range (nM) as those found for losartan and DuP 532 at angiotensin AT1 binding sites. These sites appear to be angiotensin AT2 receptors. Only the angiotensin AT1 receptor subtype interacted with G-proteins, as indicated by the 80% inhibition of [3H]angiotensin II binding in the presence of guanosine 5'-O-(3-thiophosphate) or fluoroaluminates. Although the angiotensin II-induced contraction was completely inhibited by losartan with a pA2 value of 8.8, PD 123319 reduced the angiotensin II-induced contraction by 20-25%, indicating that both angiotensin AT1 and AT2 receptor subtypes are functional in portal vein smooth muscle. PMID- 7556379 TI - SR 33589, a new amiodarone-like antiarrhythmic agent: anti-adrenoceptor activity in anaesthetized and conscious dogs. AB - We have assessed the ability of amiodarone and the new amiodarone-like antiarrhythmic agent, SR 33589 (N,N-dibutyl-3-[4-((2-butyl-5 methylsulphonamido)benzofuran-3-yl-c arbonyl) phenoxy]propylamine), to inhibit the effects of adrenoceptor stimulation in anaesthetized and conscious dogs. In anaesthetized, atropinized dogs, adrenoceptor stimulation was achieved (i) by i.v. administration of adrenaline and measurement of increased blood pressure (ii) by i.v. administration of isoprenaline and measurement of increased heart rate and decreased blood pressure. In conscious dogs, adrenoceptor stimulation was achieved by i.v. administration of isoprenaline and measurement of increased heart rate. In anaesthetized, atropinized dogs, both amiodarone and SR 33589 inhibited to similar extents, alpha-adrenoceptor stimulation (as indicated by attenuation of adrenaline-induced increases in blood pressure). The beta 1 adrenoceptor inhibitory activity of SR 33589 (as demonstrated by blockade of isoprenaline-induced increases in heart rate) was significant, but less marked than amiodarone (heart rate elevation reduced by 39%, P < 0.001 with 10 mg/kg SR 33589 and by 52%, P < 0.01 with 10 mg/kg amiodarone). In contrast, its beta 2 adrenoceptor antagonistic activity (as demonstrated by blockade of isoprenaline induced reduction in blood pressure) was more marked (mean blood pressure decrease reduced by 69%, P < 0.01 with 10 mg/kg SR 33589 and by 31%, P < 0.05 with 10 mg/kg amiodarone). In conscious dogs, both SR 33589 and amiodarone (12.5, 25 and 50 mg/kg p.o.) inhibited isoprenaline-induced increases in heart rate by approximately the same amount for varying durations depending on the dose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556380 TI - Pharmacological evaluation of N-methyl-actinodaphnine, a new vascular alpha adrenoceptor antagonist, isolated from Illigera luzonensis. AB - The pharmacological activity of N-methyl-actinodaphnine, isolated from Illigera luzonensis, was determined by functional and binding experiments with peripheral tissues. In a functional study, N-methyl-actinodaphnine was a simple competitive antagonist of contractions elicited by phenylephrine (pA2 = 7.11) in rat thoracic aorta; it also competitively antagonised the clonidine-induced inhibition of the twitch response of rat vas deferens (pA2 = 5.01). In addition, [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat isolated thoracic aorta was concentration dependently inhibited by N-methyl-actinodaphnine (1 and 10 microM); however, it had no effect on cyclic AMP and cyclic GMP contents. Additionally, N-methyl-actinodaphnine had extremely low affinity for thromboxane receptors, prostaglandin receptors, Ca2+ channels, muscarinic receptors, histamine receptors, beta-adrenoceptors, neurokinin and leukotriene receptors in vitro. However, N-methyl-actinodaphnine also possessed 5 hydroxytryptamine (5-HT) receptor blocking activity. Its potency for blocking 5 HT receptors was about 14 times less than that for blocking alpha 1 adrenoceptors. In binding experiments, N-methyl-actinodaphnine displaced biphasically the binding of 0.2 nM [3H]prazosin to cultured A10 cells. The selectivity for alpha 1-adrenoceptor subtypes was also investigated in rat vas deferens and spleens. The contractile response in rat vas deferens to noradrenaline was competitively inhibited by N-methyl-actinodaphnine with a pA2 value of 6.58; N-methyl-actinodaphnine also competitively antagonized the phenylephrine-induced contraction in rat spleen with a pA2 value of 7.38. These results indicate that N-methyl-actinodaphnine is a selective alpha 1-adrenoceptor antagonist. Furthermore, it is more selective for the alpha 1B- than for the alpha 1A-adrenoceptor subtype. PMID- 7556381 TI - Stimulatory effect of muscimol on gastric acid secretion stimulated by secretagogues in vagotomized rats under anesthesia. AB - The effect of intravenous administration of muscimol, a GABAA receptor agonist, on gastric acid secretion from perfused stomach was studied in vagotomized rats anesthetized with urethane. Muscimol did not stimulate acid secretion by itself. In contrast, muscimol dose dependently potentiated acid secretion induced by pentagastrin, bethanechol and direct vagal stimulation, but not histamine. Muscimol-potentiated acid secretion induced by pentagastrin and bethanechol was not influenced by pretreatment with atropine or cimetidine, respectively. Muscimol-potentiated acid secretion evoked by direct vagal stimulation was prevented by pretreatment with proglumide, a gastrin receptor antagonist. Muscimol-potentiated acid secretion evoked by bethanechol was dose dependently prevented by bicuculline methiodide, suggesting an involvement of peripheral GABAA receptors. These results suggest that muscimol stimulates acid secretion under certain conditions, and that two mechanisms are involved in this effect. The effects of muscimol on acid secretion may be mediated by increasing the release of histamine by pentagastrin, bethanechol and direct vagal stimulation. In addition, muscimol would also be effective if muscarinic agents were already occupying muscarinic acetylcholine receptors on parietal cells. PMID- 7556382 TI - Phorbol esters elicit Ca(2+)-dependent delayed contractions in diabetic rat aorta. AB - To determine whether diabetes alters vascular effects mediated by activation of protein kinase C, the contractions induced by phorbol esters were examined in aortic rings from rats with 8- to 12-weeks streptozotocin-induced diabetes and compared with those from age-matched control rats. In diabetic rat aorta, phorbol 12,13-dibutyrate (PDB) (> or = 30 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (300 nM) elicited a delayed, sharply developing rise in tension following an initial gradually developing contraction. In control rat aorta, these agents produced only an initial slowly developing contraction. Both the initial and the delayed contractile responses observed in diabetic aorta were completely abolished by pretreatment with 20 nM staurosporine, and the delayed phase of contraction was not seen in Ca(2+)-free medium or in the presence of 1 microM nifedipine. The concentration-response curves for the contractions induced by PDB revealed that PDB at concentrations > or = 30 nM produced significantly greater responses in diabetic aorta than in control aorta. In control aorta, exposure to Ca(2+)-free medium and pretreatment with 1 microM nifedipine shifted the concentration-response curves for PDB to the right without changing the maximal response. Under these conditions, there were no differences in the curves for PDB in control and diabetic aortas. These results suggest that the appearance of the delayed phase of contraction, possibly due to a delayed opening of Ca2+ channels, during activation of protein kinase C may be responsible for the enhanced contractile responses to phorbol esters in diabetic rat aorta. PMID- 7556383 TI - Genetic selection for nicotine activity in mice correlates with conditioned place preference. AB - Genetically heterogenous stock (HS) mice are being used to develop lines which have differential locomotor response to subcutaneously administered (0.75 mg/kg) nicotine. These groups of nicotine-depressed, nicotine-activated or randomly bred control mice were tested as to conditioned place preference using the same dose of nicotine employed to determine their locomotor performance in activity tests. Results indicate that the nicotine-activated mice showed a significantly greater preference to nicotine when compared to the nicotine-depressed mice; this effect was seen in the first generation and continued in the more recently tested third generation. Evidence is offered to support the hypothesis that it is the stimulatory effects of drugs (of abuse) that can be directly correlatable with the strength of their reinforcing effect upon behavior. PMID- 7556384 TI - Effects of tetrahydroaminoacridine and nicotine in nucleus basalis and serotonin lesioned rats. AB - The present study was designed to investigate the hypothesis that concurrent degeneration of serotonin and acetylcholine cells may decrease the therapeutic effects of cholinergic drugs on cognitive functioning in Alzheimer dementia. Therefore, we compared the effects of pretraining injections of a cholinesterase inhibitor, tetrahydroaminoacridine (1, 3 and 5 mg/kg i.p.), and nicotine (0.03, 0.1 and 0.3 mg/kg i.p.) on spatial navigation (water maze) and passive avoidance in nucleus basalis- and nucleus basalis+p-chlorophenylalanine-lesioned rats. Nicotine (0.1 and 0.3 mg/kg) promoted passive avoidance performance of nucleus basalis-lesioned rats, but nicotine did not improve performance of combined lesioned rats. Tetrahydroaminoacridine (3 mg/kg) facilitated passive avoidance performance of nucleus basalis- and combined-lesioned rats. However, tetrahydroaminoacridine-treated nucleus basalis+p-chlorophenylalanine-lesioned rats were not performing better than vehicle-treated nucleus basalis-lesioned rats. Spatial navigation of nucleus basalis and nucleus basalis+p chlorophenylalanine-lesioned rats was slightly impaired during the first training day and tetrahydroaminoacridine 3 mg/kg restored the performance of combined lesioned rats. Combined-lesioned rats performed as well as the controls during the other training days. The present results suggest that, in Alzheimer's disease, combined degeneration of nucleus basalis cholinergic and brainstem serotonergic cells decreases the therapeutic effect of nicotine, but not that of tetrahydroaminoacridine. PMID- 7556386 TI - Dual inhibitory action of enadoline (CI977) on release of amino acids in the rat hippocampus. AB - The effect of the kappa-opioid receptor agonist enadoline (CI977, (5R)-(5 alpha,7 alpha,8 beta)-N-methyl-N-[7-(1-pyrrilidinyl)-1-oxaspiro [4,5]dec-8-yl-4 benzofuranacetamide monohydrochloride), on the release of amino acids was studied in the hippocampus of freely moving rats. K+, 100 mM, or veratrine, 100 microM, were applied for 10 min via the dialysis probe, either alone (control groups) or together with CI977 (after a 10 min pretreatment with CI977 in the perfusion medium). To test the specificity of the response to CI977, nor-binaltorphimine, a selective kappa-opioid receptor antagonist, was delivered together with CI977 in two groups of animals. To test the effect of systemic injection, CI977 was given subcutaneously 30 min prior to either stimulus. K(+)-induced release of glutamate and aspartate was significantly reduced by CI977, 2.5 mM; release of gamma aminobutyric acid (GABA) was reduced by 250 microM CI977 in the probe. The effect of CI977 on release of glutamate and aspartate, but not of GABA, was reversed by nor-binaltorphimine (45 microM). Systemic treatment with CI977, 1 or 10 mg/kg, did not reduce K(+)-induced release of glutamate. Veratrine-induced release of aspartate and glutamate was significantly inhibited by 25 microM and release of GABA by 250 microM CI977 in the probe, and this effect was not modified by nor binaltorphimine (58 microM). Systemic injection of CI977 1 mg/kg significantly reduced veratrine-induced release of glutamate. These results indicate that CI977 regulates release of amino acids by two independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556385 TI - Salicylaldoxime blocks K+ and Ca2+ currents in rat cardiac myocytes. AB - The effects of salicylaldoxime, 2-(OH)C6H4CH = NOH, on the action potential duration, transient outward K+ current and slow inward Ca2+ current were studied in isolated rat ventricular myocytes. The application of salicylaldoxime (0.1-2.0 mM) reversibly increased the action potential duration and reduced in a dose dependent manner both the transient outward K+ and the slow inward Ca2+ currents. The effect of salicylaldoxime on these two ionic currents was similar to that of 2,3-butanedione monoxime, but was about ten times more potent. Compounds which block both K+ and Ca2+ currents may represent a new type of Class III antiarrhythmic agent which counteracts arrhythmias initiated by re-entry with reduced proarrhythmic risk via triggered activity. PMID- 7556387 TI - Role of nitric oxide and neuropeptides in neurogenic vasodilatation of the guinea pig mesenteric artery. AB - Although dense networks of adrenergic nerves are present and noradrenaline causes vasoconstriction, electrical field stimulation failed to elicit any constriction of the isolated ring preparation of the guinea pig mesenteric artery. In the presence of an active tone, a vasodilator response was elicited by electrical field stimulation in endothelium-removed tissues. Nonadrenergic, noncholinergic nerves mediate the electrical field stimulation-induced vasodilator response, since guanethidine and atropine did not affect while tetrodotoxin abolished it. Multiple mediators seem to participate in this vasodilatation. NADPH-diaphorase positive nerves, calcitonin gene-related peptide (CGRP)- and vasoactive intestinal peptide (VIP)-immunoreactive nerves were present in the mesenteric artery. Nitro-L-arginine but not nitro-D-arginine suppressed the electrical field stimulation-induced vasodilator response with rapid onset and L-arginine restored it. VIP and CGRP relaxed the tissue in a dose-dependent manner. Pretreatment of the animals with capsaicin partly reduced the electrical field stimulation induced vasodilator response. CGRP-(8-37), a CGRP antagonist, slightly attenuated the vasodilator response induced by both electrical field stimulation and CGRP. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, decreased the nitro-L arginine- and capsaicin-insensitive component of the electrical field stimulation induced vasodilator response. Zinc protoporphyrin IX, an inhibitor of CO formation, did not affect the electrical field stimulation-induced response. In the presence of nitro-L-arginine without an active tone, electrical field stimulation induced a vasoconstrictor response that was sensitive to bunazosin and guanethidine. The results show that the electrical field stimulation-induced vasodilator response of the mesenteric artery of guinea pigs is mediated by nitric oxide (NO), CGRP and some yet unidentified substance(s). Elimination of the vasodilator response unmasked the adrenergic vasoconstrictor response to electrical field stimulation. PMID- 7556388 TI - Ca2+ channel blocker, diltiazem, prevents physical dependence and the enhancement of protein kinase C activity by opioid infusion in rats. AB - The influence of an L-type Ca2+ channel blocker, diltiazem [(2S-cis)-3 (acetyloxy)-5-[2-(dimethylamino)-ethyl]-2,3-dihydro-2- (4- methoxyphenyl)-1,5 benzothiazepin-4(5H)-one], on the behavioral signs of naloxone (opioid receptor antagonist)-precipitated withdrawal syndrome and the enhancement of protein kinase C activity in the pons/medulla regions of rats rendered dependent on morphine (mu-opioid receptor agonist) or butorphanol (mu/delta/kappa mixed opioid receptor agonist) was investigated. The expression of physical dependence produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microliters per h) or butorphanol (26 nmol/microliters per h) for 3 days, as evaluated by naloxone (5 mg/kg, i.p.)-precipitated withdrawal signs, was dose dependently attenuated by concomitant infusion of diltiazem (10 and 100 nmol/microliters per h). Furthermore, diltiazem (100 nmol/microliters per h) completely inhibited the enhancement of cytosolic protein kinase C activity in the pons/medulla regions in rats rendered dependent by continuous infusion with morphine or butorphanol. These results suggest that the augmentation of intracellular Ca2+ concentration mediated through L-type Ca2+ channels during continuous opioid infusion leads to the enhancement of cytosolic protein kinase C activity in the pons/medulla region which is intimately involved in the development and/or expression of physical dependence on opioids. PMID- 7556389 TI - Isolation rearing prevents the reinforcing properties of amphetamine in a conditioned place preference paradigm. AB - Social isolation has been demonstrated to alter individual reactivity to addictive drugs. The present experiments compared the effects of isolation rearing and socially rearing rats on the reinforcing properties of amphetamine. Male Lister hooded rats were housed from 21 days (weaning) either alone (isolation reared rats) or in groups of four rats per cage (socially reared rats). Six weeks later, the rats were tested for their sensitivity to d amphetamine (1.5 and 5 mg/kg) using a conditioned place preference paradigm. The treatment quadrant was selected as that in which the rat spent least time during a preconditioning trial. After saline conditioning, the socially reared rats showed a significant (P < 0.05) aversion for the treatment quadrant relative to the opposite quadrant. Following amphetamine (1.5; 5 mg/kg) conditioning, socially reared rats spent significantly more time (P < 0.05) in the treatment quadrant relative to the opposite quadrant while isolation reared rats failed to display either the aversion effect of saline conditioning or amphetamine induced place preference. These results demonstrate isolation rearing prevents the reinforcing properties of amphetamine. PMID- 7556390 TI - Effects of a histamine H2 receptor agonist and antagonist on restraint-induced antinociception in female mice. AB - Restraint for 1 h induced significant antinociceptive activity, as assessed by the hot plate test, in female mice. The antinociceptive activity was significant throughout the 1 h period of observation starting immediately after restraint. Prior administration of the histamine H2 receptor agonist dimaprit (1.5-6.0 mg/kg s.c.) 15 min before restraint further enhanced the restraint-induced antinociceptive activity. Furthermore, the induction of antinociceptive activity by restraint was antagonised by prior administration of histamine H2 receptor antagonists, cimetidine (2.5-10.0 mg/kg s.c.) or zolantidine (2.5-10.0 mg/kg s.c.). However, when these drugs were administered immediately after restraint for 1 h, the antinociceptive activity observed was similar to those restrained animals receiving saline injection. The histamine receptor agonist and antagonists, at the doses used in the present study, did not affect the response of unrestrained animals to the hot plate test. These results demonstrate that the effect of a histamine H2 receptor agonist and antagonists on restraint-induced antinociception is dependent upon their time of administration and may act by altering the intensity of stress, thus affecting the antinociceptive activity induced. PMID- 7556392 TI - Novel 5-HT2-like receptor mediates neurogenic relaxation of the guinea-pig proximal colon. AB - The aim of the current investigation was to characterize the 5-HT receptors that mediate neurogenic relaxation of the guinea-pig proximal colon. After blockade of 5-HT2A, 5-HT3 and 5-HT4 receptor-mediated contractions, 5-hydroxytryptamine (5 HT) induced relaxations yielding a biphasic concentration-response curve. Other tryptamines were also agonists with the following rank order of potency: 5-HT > 5 carboxamidotryptamine = 5-methoxytryptamine > or = alpha-methyl-5-HT (partial agonist) > tryptamine (partial agonist). 5-Hydroxytryptophan, 2-methyl-5-HT and N methyltryptamine were virtually inactive as agonists. The curve to 5-HT was not affected by pargyline, citalopram, phentolamine, or by the 5-HT4 receptor antagonists 2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino)ethyl ester (SDZ 205-557) and (1-butyl-4-piperidinylmethyl)-8-amino-7-chloro-1,4-benzodioxan+ ++-5-carboxylate (SB 204070). 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH DPAT), 5-methoxy-3[1,2,3,6-tetrahydroxypyridin-4-yl]-1H-indole (RU 24969), 2-(2,6 dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 1-(3 chlorophenyl)piperazine (mCPP), 1-(m-trifluoromethylphenyl)-piperazine (TFMPP), flesinoxan, sumatriptan and 6-chloro-2-(piperazinyl)-pyrazine (MK212) were inactive as 5-HT receptor agonists. The first phase of the curve to 5-HT was inhibited by: metergoline (pA2 = 8.8 +/- 0.3, against 5-methoxytryptamine 9.3 +/- 0.3), methysergide (non-surmountable), methiothepin (non-surmountable), spiroxatrine (non-surmountable), MK212 (non-surmountable), mesulergine (7.8 +/- 0.3), mCPP (7.1 +/- 0.1), mianserin (7.0 +/- 0.4), ritanserin (8.9 +/- 0.2), rauwolscine (7.0 +/- 0.2), yohimbine (6.2 +/- 0.2), 1-(1-naphthyl)-piperazine (7.7 +/- 0.2) and RU 24969 (6.4 +/- 0.1), but not by 1-(2-methoxyphenyl)4-[4-(2 phthalimidobtyl]-piperazine (NAN-190), spiperone, sumatriptan, 8-OH-DPAT and flesinoxan. It is suggested that the 5-HT receptor under study could be considered an unknown 5-HT2-like receptor. PMID- 7556391 TI - Antithrombotic effect of a humanized anti-GPIIb/IIIa monoclonal antibody, YM207, in a photochemically induced thrombosis model in monkeys. AB - We investigated the antithrombotic effects of a F(ab')2 fragment of a humanized anti-platelet glycoprotein (GP) IIb/IIIa monoclonal antibody, YM207, in a platelet-rich thrombosis model. Thrombus was induced in a mesenteric venule of squirrel monkeys by irradiation with filtered light in combination with i.v. administration of a fluorescent dye (sodium fluorescein). Time to occlusive thrombus formation was significantly prolonged by i.v. bolus injection of 0.3 mg/kg of YM207. At doses of 1 mg/kg and 3 mg/kg, complete occlusion did not occur during 60 min of the observation. Platelet aggregation in platelet-rich plasma was inhibited by YM207 at doses 0.3 mg/kg or more. A good correlation was obtained between inhibition of platelet aggregation and the percentage of GPIIb/IIIa receptors blocked by YM207. Bleeding time was significantly prolonged at a dose of 3 mg/kg. Platelet counts showed no significant change. These results suggest that YM207 is effective in preventing platelet-rich thrombus formation without inducing the prolongation of bleeding time or a significant decrease in platelet count. YM207 may be a useful therapeutic agent for the treatment of thrombotic disorders. PMID- 7556393 TI - Methamphetamine plus scopolamine potentiates behavioral sensitization and conditioning. AB - The effects of repeated methamphetamine (4.0 mg/kg) plus scopolamine (0.5 mg/kg) treatment on behavioral sensitization and drug conditioning in rats were compared with the effects of repeated methamphetamine treatment. Behavioral sensitization induced by repeated methamphetamine plus scopolamine treatment was more vigorous than that induced by repeated methamphetamine treatment. Repeated methamphetamine plus scopolamine treatment produced sensitized responses, not only to methamphetamine plus scopolamine and methamphetamine but also, to a lesser extent, to scopolamine. Methamphetamine plus scopolamine-sensitized rats but not methamphetamine-sensitized rats exhibited conditioned responses to a low frequency tone (300 Hz, 100 dB) associated with the drug state, suggesting that robust methamphetamine plus scopolamine-induced behavioral sensitization may lead to enhanced conditioning. It is plausible that robust behavioral sensitization might operate via a reciprocal balance between the dopaminergic and cholinergic systems in favor of dopaminergic dominance. Conditioning to the drug-associated tone may be mediated via a reciprocal balance between the two transmitter systems. PMID- 7556394 TI - Effect of thielocin A1 beta on bee venom phospholipase A2-induced edema in mouse paw. AB - Several investigators have reported that inactivation of secretory phospholipase A2 purified from bee venom with p-bromophenacyl bromide, an irreversible inhibitor, before injection resulted in attenuation of the subsequent inflammatory reaction in the mouse paw edema model. Recently, thielocin A1 beta, a novel secretory phospholipase A2 inhibitor from fungi, was found to suppress histamine release from mast cells stimulated with secretory phospholipase A2. These observations led us to examine the effect of thielocin A1 beta against secretory phospholipase A2-induced paw edema. Thielocin A1 beta inhibited bee venom phospholipase A2 in a dose-dependent manner (IC50 = 1.4 microM). In addition, the inhibition of bee venom phospholipase A2 was noncompetitive (Ki = 0.57 microM) and reversible. Subplantar injection of bee venom phospholipase A2 produced a rapid but transient edematous response. Coinjection of thielocin A1 beta (1 microgram/paw) with bee venom phospholipase A2 resulted in a 44.7 +/- 4.6% reduction of edema formation. This anti-edema action was not enhanced by cyproheptadine (antihistamine/antiserotonin). These results suggest that thielocin A1 beta shows edema-reducing activity via inhibition of the phospholipase A2 activity which participates in histamine release by mast cells. PMID- 7556396 TI - Possible mechanisms of beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum. AB - The mechanisms of the beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum were investigated. Noradrenaline caused graded relaxation of this preparation. However, the concentration-response curves for noradrenaline were unaffected by propranolol (approximately 10(-5) M) or phentolamine (approximately 10(-5) M). The responses to noradrenaline were antagonized in a concentration-dependent manner by bupranolol, and Schild plots of the data revealed a pA2 value of 5.53. Also, bupranolol antagonized responses to isoprenaline, and Schild plots of the data revealed the pA2 value to be 8.53. Noradrenaline significantly increased the cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP response elicited by noradrenaline, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to noradrenaline in guinea pig taenia caecum is mainly mediated by beta 3-adrenoceptors (or atypical beta-adrenoceptors) and that in guinea pig taenia caecum noradrenaline behaves as a beta 3-selective adrenoceptor agonist. PMID- 7556395 TI - Alteration of neutrophil trafficking by a lipocortin 1 N-terminus peptide. AB - Sialidase, fucoidin and a peptide corresponding to most of lipocortin 1 N terminus, termed LC1-(Ac2-26)-peptide, induced an intense 2 h neutrophilia whereas a monoclonal antibody to murine CD11b induced an effect by 1 h. The neutropenic response stimulated by platelet-activating factor (PAF) was significantly reduced in the presence of sialidase, fucoidin, LC1-(Ac2-26) peptide and monoclonal antibody anti-CD11b. Neutrophil migration into a 6-day-old mouse air-pouch induced by interleukin-1 was inhibited by all the pharmacological agents. In vitro, PAF up-regulated CD11b expression on the neutrophil surface but neither human or mouse LC1-(Ac2-26)-peptide inhibited this response. CD11b up regulation on neutrophils occurred after PAF administration in vivo and was maximal at 2 min. LC1-(Ac2-26)-peptide mimics the action of agents interfering with leucocyte rolling and adhesion in vivo, however, does not inhibit CD11b up regulation in vitro suggesting other phenomena are important in the activity of this peptide. PMID- 7556398 TI - Evidence for an anxiogenic action of AMPA receptor antagonists in the plus-maze test. AB - The effects of the non-NMDA receptor antagonists, the new alpha-amino-3-hydroxy-5 methyl-4-isoxazole-propionic acid (AMPA)-selective receptor antagonist, LY326325, and the AMPA/kainate-selective receptor antagonist, NBQX (6-nitro-7 sulfamoylbenzo(f)quinoxaline-2,3-(1H,4H)dione), on plus-maze behavior and locomotor activity were examined. LY326325 induced a dose-dependent decrease in the per cent time spent in open arms as well as in the per cent entries into the open arms. NBQX caused a dose-dependent reduction in the per cent time spent in open arms but had no effect on the per cent entries into the open arms. The behavioral actions of the AMPA receptor antagonists were observed at doses which had no influence on the locomotor activity of the animals. Based upon the current findings it is suggested that AMPA receptor antagonists produce a dose-dependent increase of anxiogenic behavior in the plus-maze test situation. PMID- 7556397 TI - Inflammation enhances peripheral mu-opioid receptor-mediated analgesia, but not mu-opioid receptor transcription in dorsal root ganglia. AB - mu-Opioid receptor agonist [D-Ala2,NMe-Phe4,Gly5-ol]enkephalin (DAMGO)-induced peripheral analgesic effects occur early in hindpaws inoculated with Freund's complete adjuvant and increase in parallel to the development of inflammatory signs. Antagonism of these effects by beta-funaltrexamine, an irreversible mu opioid receptor antagonist, suggests that the effective number of peripheral opioid receptors does not increase during early stages, but does so at later stages of the inflammation. As determined by a ribonuclease protection assay, mu opioid receptor mRNA in dorsal root ganglia is abundant in untreated animals, but does not significantly increase following inflammation. Thus, peripheral analgesic efficacy of DAMGO is not correlated with transcription or number of mu opioid receptors at early inflammatory stages. At later stages, however, the number of peripheral mu-opioid receptors appears to increase and may enhance opioid efficacy. PMID- 7556399 TI - Effects of a cannabinoid on spontaneous and evoked neuronal activity in the substantia nigra pars reticulata. AB - Single unit electrophysiology was used to explore the role of cannabinoid receptors in the substantia nigra pars reticulata. Intravenous and intraperitoneal injections of the potent and selective synthetic cannabinoid (R) (+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3- de]-1,4 benzoxazin-6-yl](1-naphthalenyl) methanone (WIN 55,212-2) produced modest but significant increases in the spontaneous firing rate of neurons in the substantia nigra pars reticulata. In a second set of experiments, WIN 55,212-2 (up to 1.0 mg/kg i.v.) antagonized the inhibition of firing produced in the substantia nigra pars reticulata by electrical stimulation of the striatum. The pharmacological specificity of this effect was demonstrated using the inactive enantiomer WIN 55,212-3. The possibility that WIN 55,212-2 exerts its effects by regulating gamma-aminobutyric acid (GABA) release from striatonigral fibers was suggested by the observation that bicuculline (up to 0.5 mg/kg i.v.) reversed the effect of striatal stimulation. It thus appears that cannabinoid receptors on striatonigral neuron terminals may regulate movement by disinhibiting the activity of substantia nigra pars reticulata neurons, perhaps by inhibiting the release of GABA into the substantia nigra pars reticulata. PMID- 7556400 TI - Behavioral studies on the putative gamma-type endorphin receptor using different antibodies. AB - To investigate the significance of endogenous, neuroleptic-like gamma-type endorphins and their putative receptors, polyclonal and monoclonal antibodies against gamma-type endorphins, which may bio-inactivate the ligands for the receptors, and monoclonal anti-idiotype antibodies, which presumably bind to the receptors, were injected into the nucleus accumbens of the rat brain. The desenkephalin-gamma-endorphin-induced antagonism of the hypomotility response elicited by challenge with apomorphine injected into the nucleus accumbens was used as test system. Both the anti-desenkephalin-gamma-endorphin antibodies and anti-idiotype antibodies blocked the action of exogenous desenkephalin-gamma endorphin. Thus, the anti-idiotype antibodies may serve as receptor antagonists. Chronic treatment (injection into the nucleus accumbens) with the anti-idiotype antibodies induced sustained hypermotility, decreased habituation and impaired passive avoidance behavior. In such treated animals local treatment with apomorphine did not elicit hypomotility. It is suggested that gamma-type endorphins influence the setpoint for feedback regulation in dopaminergic neurons equipped with gamma-type endorphin receptor systems. PMID- 7556401 TI - kappa-Opioid receptor agonist protects against ischemic reduction of 2 deoxyglucose uptake in morphine-tolerant rats. AB - We examined the effects of mu-opioid receptor agonist and antagonists, and kappa opioid receptor agonist on the hypoxia/hypoglycemia-induced reduction in 2 deoxyglucose uptake of rat hippocampal slices. Naloxone, a mu-opioid receptor antagonist and (5,7,8)-(+)-3,4-dichloro-N-methyl-N-(7,8,1-pyrrolidinyl)-1 oxaspirol+ ++ (4,5)dec-8-yl)-benzeneacetamide methanesulfonate, U-62,066E, a kappa-opioid receptor receptor agonist, showed neuroprotective actions against the hypoxia/hypoglycemia-induced deficit in glucose uptake. In contrast, morphine exhibited an exacerbating action. These results suggest that blockade of mu opioid receptor- and stimulation of kappa-opioid receptor-mediated functions has a protective role against the hypoxia/hypoglycemia-induced decreases in glucose metabolism in hippocampal slices. Chronic administration of morphine (10 mg/kg) for 9 days affected neither the basal nor the hypoxia/hypoglycemia-induced reduction in 2-deoxyglucose uptake. Rats treated with morphine chronically exhibited not only tolerance to the analgesic effect but also tolerance to the exacerbating action. However, chronic morphine did not modify U-62,066E-induced neuroprotection. These findings indicate that the receptor mechanisms of neuroprotection produced by the activation of kappa-opioid receptors may not be involved in mu-opioid receptor function. PMID- 7556402 TI - Regulation of ion transport by histamine in human colon. AB - Histamine, added to the basolateral side of voltage clamped human colon in vitro, induced a rapid onset, transient inward short circuit current which was concentration dependent over the range 0.01-3 mM. This response was largely due to electrogenic chloride section since it was virtually abolished by bumetanide or by chloride replacement in the bathing solutions. Responses were unaffected by amiloride or acetazolamide. Neither the histamine H2 receptor agonist dimaprit (1 mM) nor the histamine H3 receptor agonist S-(+)-alpha-methyl histamine (1 mM) altered short circuit current. Responses to histamine were significantly reduced by the histamine H1 receptor antagonist mepyramine (1-10 microM) but not altered by the histamine H2 receptor antagonist cimetidine (100 microM) or by the histamine H3 receptor antagonist thioperamide (1 microM). Short circuit current responses to histamine were not altered by tetrodotoxin (1 microM). Piroxicam (10 microM) and nordihydroguaiaretic acid (100 microM) were without effect when used individually but significantly reduced responses to histamine when used simultaneously. These results indicate that histamine stimulates chloride secretion across human colonic epithelium by a mechanism which is mediated exclusively via histamine H1 receptors. This action does not involve intrinsic nerves but appears to be dependent upon eicosanoid synthesis. PMID- 7556403 TI - Effects of bradykinin and bradykinin analogues on spleen cells of mice. AB - The present study was undertaken to examine the effects of bradykinin and selected bradykinin analogues on mononuclear cells derived from mouse spleen. Bradykinin as well as des-Arg9-bradykinin, a bradykinin B1 receptor agonist, were able to induce the release of so-called charge-changing lymphokines, which could be identified as interleukin-1, interleukin-6, interleukin-2 and as interleukin-2 receptor. The cytokine release evoked by bradykinin and all analogues showed a bell-shaped dose dependence in a range of 10(-8) M to 10(-6) M and could be inhibited by the specific bradykinin receptor antagonist, D-Arg0[Hyp3,Thi5,D Tic7,Oic8]bradykinin (HOE140), and by bradykinin analogues with N-methyl phenylalanine at position 2 in concentrations as low as 10(-12) M and 10(-13) M, respectively. Obviously the N-terminus of bradykinin seems to be responsible for the interaction with the mononuclear cells concerning all peptides investigated. PMID- 7556404 TI - A novel non-xanthine adenosine A1 receptor antagonist. AB - FK453, (+)-(R)-[(E)-3-(2-phenylpyrazolo[1,5-alpha]pyridin-3-yl) acryloyl]-2 piperidine ethanol, was examined for adenosine receptor antagonistic activity using isolated guinea-pig atria and aorta and for affinity for adenosine receptors in the rat cerebral cortex and striatum in comparison with FR113452 (S enantiomer of FK453), PD116948 (1,3-dipropyl-8-cyclopentylxanthine), theophylline (1,3-dimethylxanthine) and CGS15943 ([1,2,4]triazolo[1,5-c]quinazolone). FK453 showed potent inhibition of the negative inotropic activity elicited by 10 microM adenosine with an IC50 of 560 pM in guinea-pig atria. However, FK453 was less potent in inhibiting the relaxation induced by 3.2 microM adenosine and had an IC50 of 1.18 microM in guinea-pig aorta. The IC50 values for FR113452, PD116948, theophylline and CGS15943 were 1.18 microM, 1.31 nM, 20.2 microM and 74.2 nM in atria and > 100 microM, 656 nM, 239 microM, 127 nM in aorta respectively. In the binding study, FK453 antagonized [3H]N6-cyclohexyladenosine binding to the rat cortical adenosine A1 receptor with an IC50 of 17.2 nM. The IC50 values for FR113452, PD116948, theophylline and CGS15943 were 10.1 microM, 4.7 nM, 67.7 microM and 241 nM respectively. FK453 inhibited [3H]5'-N ethylcarboxamideadenosine binding to rat striatum adenosine A2 receptor with an IC50 of 11.3 microM. FK453 had no adenosine A1 receptor agonistic activity, since it had no negative inotropic activity up to 100 microM in isolated guinea-pig atria. These results demonstrate that FK453 is a novel non-xanthine adenosine receptor antagonist and is potent and selective for the adenosine A1 receptor subtype. PMID- 7556405 TI - Histamine-induced edema in the rat paw--effect of capsaicin denervation and a CGRP receptor antagonist. AB - Histamine is known to cause edema and excitation of small-diameter primary afferent neurons. In the present study we wanted to investigate to which extent afferent neurons participate in histamine-induced edema and, subsequently, determine possible inhibitory effects of a tachykinin NK1 receptor and CGRP receptor antagonist on the histamine response. Intraplantar injection of histamine (0.5 mumol) into the rat hind paw caused a 34% increase of paw volume. In capsaicin-denervated rats, this effect of histamine was nearly abolished. The calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37), but not the tachykinin NK1 receptor antagonist SR140333, caused significant inhibition of the edema response. Further indication that CGRP can promote the histamine action was obtained in capsaicin-denervated rats, where co-injection of CGRP (0.3 pmol) increased the edema response to intraplantar histamine. In additional experiments, plasma protein extravasation in the paw skin was evaluated after close arterial infusion of histamine. Also in these experiments CGRP-(8-37), but not SR140333, significantly reduced the histamine effect. The observation that in the rat hind paw a CGRP receptor antagonist, but not a tachykinin NK1 receptor antagonist, attenuates histamine-induced vascular leakage raises the possibility that in some tissues CGRP receptor antagonists may be superior to tachykinin NK1 receptor antagonists in reducing histamine-induced neurogenic inflammatory responses. PMID- 7556408 TI - BN-063, a newly synthesized adenosine A1 receptor agonist, attenuates myocardial reperfusion injury in rats. AB - To assess the efficacy of the newly synthesized selective adenosine A1 receptor agonist, BN-063 (1-cyclopropylisoguanosine), against myocardial reperfusion injury, 31 rats underwent 45 min of left coronary artery occlusion and 1 h of reperfusion. Animals were randomly assigned to four groups: control, I0.5-R0.5, in which BN-063 (0.5 mg/kg i.v. bolus) was administered during both ischemia and reperfusion, R-0.5 and R-1.0, in which BN-063 was administered only during reperfusion at 0.5 and 1.0 mg/kg, respectively. The area at risk was determined by intravascular injection of blue dye during coronary artery occlusion, which was performed by retightening the ligature at the end of reperfusion, and infarct size was determined by incubation of heart slices in nitro blue tetrazolium chloride. A significant reduction in infarct size, as a percentage of the area at risk, was noted with all three BN-063 treatment groups (control: 63.5 +/- 4.0%, I0.5-R0.5: 39.6 +/- 3.7%, R-0.5: 37.5 +/- 3.5%, R-1.0: 38.1 +/- 5.2%). However, the I0.5-R0.5 group did not shown a more beneficial effect than the other two BN 063-treated groups. In addition, BN-063 exerted a protective effect on the number of ventricular premature contractions associated with reperfusion (control: 906 +/- 52, I0.5-R0.5: 325 +/- 61, R-0.5: 321 +/- 95, R-1.0: 340 +/- 46). The results of this study demonstrate that BN-063, through activation of adenosine A1 receptors, exerts antiarrhythmic and anti-infarct effects during myocardial ischemia-reperfusion. Therefore, BN-063 would be useful clinically in the treatment and prevention of acute myocardial infarction. PMID- 7556407 TI - In vitro and in vivo evidence for a tachykinin NK1 receptor antagonist effect of vapreotide, an analgesic cyclic analog of somatostatin. AB - Vapreotide, a long-acting somatostatin analog, possesses an analgesic effect. The purpose of this work was to determine a tachykinergic involvement. Vapreotide reduced substance P-induced biting and scratching in mice. This inhibitory effect of substance P action was confirmed by experiments performed on the bronchial apparatus of guinea-pigs known to possess tachykinin NK1 and NK2 receptors. (i) Vapreotide reduced the substance P-induced plasmatic exudation. (ii) It inhibited selectively the tachykinin-dependent second contractile phase induced by electrical field stimulation of isolated bronchi. (iii) It shifted to the right the concentration-effect curve of substance P-induced contraction of isolated main bronchi. The peptide displaced [3H]substance P (IC50 = 3.3 +/- 1.8 x 10(-7) M) from guinea-pig bronchial tachykinin NK1 sites. The displacement of [125I]neurokinin A, a specific tachykinin NK2 receptor ligand, needed higher concentrations (IC50 = 4.5 +/- 0.6 x 10(-6) M). It is concluded that vapreotide possesses an antagonist activity on guinea-pig tachykinin NK1 receptors; the involvement in its analgesic action is discussed. PMID- 7556410 TI - Effects of ageing and adrenergic stimulation on alpha 1- and beta-adrenoceptors and phospholipid fatty acids in rat heart. AB - The purpose of this study was to examine the influence of ageing on the alterations in binding characteristics of adrenoceptors and membrane phospholipid fatty acids in rat heart following repeated administration of epinephrine. The maximal number of binding sites (Bmax) and dissociation constant (Kd) of [3H]prazosin and [3H]dihydroalprenolol binding to alpha 1- and beta adrenoceptors, respectively, changed significantly during ageing. The downregulation of alpha 1- and beta-adrenoceptors after repeated epinephrine administration for one week, did not differ with age, but the response of the affinity (1/Kd) of both alpha 1- and beta-adrenoceptors to epinephrine treatment was age dependent. In 3-month-old rats the affinity of alpha 1-adrenoceptors was decreased after epinephrine treatment but the affinity of beta-adrenoceptors was unchanged. In 10- and 23-month-old rats the affinity of beta-adrenoceptors decreased after epinephrine treatment but the affinity of alpha 1-adrenoceptors did not change. During ageing the linoleic acid (18:2(n-6)) level decreased in phosphatidylcholine and the arachidonic acid (20:4(n-6)) level increased in phosphatidylcholine and phosphatidylethanolamine. After epinephrine administration the 18:2(n-6) level decreased and the docosahexaenoic acid (22:6(n 3)) level increased in phosphatidylcholine and phosphatidylethanolamine and those changes were not age dependent. The 20:4(n-6) level increased in phosphatidylcholine after epinephrine administration, but that increase was smaller in old than in young rats. The results show that both ageing and epinephrine administration simultaneously modify the fatty acid composition of membrane phospholipids and the binding properties of alpha 1- and beta adrenoceptors in rat heart. PMID- 7556409 TI - Effects of kappa-opioid receptor agonists on stimulated phosphoinositide hydrolysis in rat kidney. AB - To determine the effects of kappa-opioid receptor agonists on phosphoinositide metabolism in rat renal cortex, tissue slices labelled with [3H]inositol were stimulated with norepinephrine or carbachol alone or in combination with the kappa-opioid receptor agonists, ethylketocyclazocine, trans-3,4-dichloro-N-methyl N-[2-(pyrrolindinyl)-cyclohexyl)- benzeneacetamide (U50,488) and nalorphine. Both norepinephrine and carbachol stimulated phosphoinositide hydrolysis (measured in a LiCl buffer) concentration- and time-dependently. The EC50 and maximal stimulation of phosphoinositide hydrolysis for norepinephrine and carbachol were approximately 3 microM and 0.15 dpm released/dpm incorporated, respectively. Concentrations up to 1 mM of ethylketocyclazocine, U50,488 or nalorphine alone did not affect phosphoinositide hydrolysis. However, ethylketocyclazocine and U50,488 decreased 10 microM norepinephrine-stimulated phosphonositide hydrolysis concentration-dependently, each with an approximate IC50 of 30 microM. In contrast, nalorphine had no effect on norepinephrine-stimulated phosphoinositide hydrolysis. In addition, concentrations of up to 1 mM ethylketocyclazocine or U50,488 did not alter carbachol-stimulated phosphoinositide hydrolysis. The inhibitory effect of U50,488 and ethylketocyclazocine on norepinephrine stimulated phosphoinositide hydrolysis was blocked by the selective kappa-opioid receptor antagonist, nor-binaltorphimine. These results indicate that kappa 1 opioid receptor stimulation may affect phosphoinositide metabolism in rat renal cortex by modulating the subcellular effects of renal alpha 1-adrenoceptor activation. PMID- 7556406 TI - Recombinant interleukin-1 beta inhibits gastric acid secretion by activation of central sympatho-adrenomedullary outflow in rats. AB - The inhibitory mechanism of gastric acid secretion induced by human recombinant interleukin-1 beta was investigated in bilaterally vagotomized, urethane anesthetized rats. Intracerebroventricular administration of interleukin-1 beta (10, 50 and 100 ng/animal) dose dependently inhibited the gastric acid secretion induced by electrical stimulation of the vagus nerve at 3 Hz. Inhibition of gastric acid secretion induced by interleukin-1 beta (50 ng/animal) was abolished both by splanchnectomy and by phentolamine (5 mg/kg i.m.). Greater splanchnic nerves ramify into the adrenal branch and gastric sympathetic preganglionic branch. The interleukin-1 beta (50 ng/animal)-induced inhibition was also abolished by intracerebroventricular pretreatment with indomethacin (500 micrograms/animal), while pretreatment with the same dose of this reagent by the intraperitoneal route was without effect. These results suggest that centrally administered interleukin-1 beta induces a prostaglandin-mediated central excitation of the sympatho-adrenomedullary system, and the resultant activation of gastric alpha-adrenoceptors inhibits the vagally stimulated gastric acid secretion in rats. PMID- 7556412 TI - Cyclosporin A is a substance P (tachykinin NK1) receptor antagonist. AB - The immunosuppressive cyclic undecapeptide, cyclosporin A, inhibited the binding of [125I]substance P to tachykinin NK1 receptors expressed by human IM-9 lymphoblastoid cells, U-373 MG human astrocytoma cells and guinea pig lung parenchyma with IC50 values of 425 +/- 58, 783 +/- 180, and 784 +/- 163 nM respectively. The dihydro derivative of cyclosporin A (dihydro-cyclosporin A) was an equally effective inhibitor, but the O-acetylated derivative (cyclosporin A OAc) was 3-4 fold less potent. The cyclosporin compounds also inhibited [125I]neurokinin A binding to human NK2 receptors with potencies slightly less than at NK1 sites. In contrast, they were 8-20-fold less effective inhibitors of [125I]MePhe7-neurokinin B binding to guinea pig NK3 receptors (p < 0.001). Thus, the cyclosporin A compounds showed selectivity for NK1 and NK2 receptors. The structure-activity pattern for the effects of cyclosporin A compounds at tachykinin receptors differs from the pattern previously described for their immunosuppressive activity. All three compounds inhibited substance P induced interleukin-6 (IL-6) secretion from U-373 MG astrocytoma cells with potencies similar to their NK1 receptor binding affinities. In addition, cyclosporin A blocked substance P induced phosphatidylinositol (PI) turnover in U-373 MG cells without blocking the corresponding response to histamine. This novel pharmacological profile of the cyclosporin A compounds as NK1 receptor antagonists does not appear to correlate with other known in vitro cyclosporin A functions. PMID- 7556413 TI - Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells. AB - (+)-Anatoxin-a is a neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. In this study synthetic (+/-)-anatoxin-a was tested on isolated bovine adrenal chromaffin cells to determine its ability to evoke secretion of endogenous catecholamines through neuronal-type nicotinic receptor activation. Anatoxin-a was found to act as a potent agonist of the secretory response of chromaffin cells with an EC50 of 1-2 microM, compared with an EC50 of 4-5 microM for nicotine. The cells responded to anatoxin-a and nicotine with bell-shaped concentration-response curves consistent with desensitisation at concentrations of anatoxin-a greater than 5 microM and of nicotine greater than 20 microM. The secretion of catecholamines stimulated by anatoxin-a was completely inhibited in a non-competitive manner by the nicotinic antagonist mecamylamine with an IC50 of 0.4-0.5 microM. In the presence of depolarising concentrations of K+ (15 or 50 mM), anatoxin-a increased the secretion of catecholamines in a concentration dependent manner up to the same maximum as that achieved by anatoxin-a alone. It is concluded that anatoxin-a acts as a potent and selective nicotinic agonist, capable of evoking secretion of endogenous catecholamines from chromaffin cells via their neuronal-type nicotinic receptor. PMID- 7556411 TI - The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis. AB - Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands. PMID- 7556414 TI - Synergistic interaction of adenylate cyclase activators and nitric oxide donor SIN-1 on platelet cyclic AMP. AB - The molecular mechanism of the synergistic platelet inhibition by activators of adenylate cyclase and guanylate cyclase in human platelets was investigated. The adenylate cyclase activators iloprost and prostaglandin E1 and the guanylate cyclase activator 3-morpholino-syndnonimine (SIN-1) dose-dependently inhibited thrombin-induced aggregation of washed human platelets. Furthermore, SIN-1 at a concentration inhibiting platelet aggregation by only 10% shifted the IC50 values of iloprost and prostaglandin E1 by one order of magnitude to the left, indicating a synergistic action of adenylate cyclase and guanylate cyclase activators. Iloprost and prostaglandin E1 dose-dependently elevated platelet cAMP without a significant influence on cGMP. In contrast, the platelet cGMP level was dose-dependently elevated by SIN-1. In addiiton, SIN-1 markedly increased cAMP level induced by low concentrations of adenylate cyclase activators (0.1-0.3 nM iloprost or 10-150 nM prostaglandin E1). In contrast, the rise in cAMP induced by higher adenylate cyclase activator concentrations (3 nM iloprost or 30 microM prostaglandin E1) was significantly reduced in the presence of SIN-1. The same biphasic mode of action of SIN-1 was observed with forskolin, an adenylate cyclase stimulator acting receptor independently, indicating a prostacyclin receptor independent mechanism. The cAMP elevating effect of SIN-1 in the presence of low prostanoid concentrations was completely abolished by piroximone, a selective inhibitor of phosphodiesterase type III. Therefore, the inhibition of phosphodiesterase III by cGMP seems to be the mechanism for the elevation of cAMP levels by SIN-1 in the presence of low concentration of adenylate cyclase activators in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556415 TI - The role of inositol 1,3,4,5-tetrakisphosphate in internal Ca2+ mobilization following histamine H1 receptor stimulation in DDT1 MF-2 cells. AB - Receptor-activated formation of inositol phosphates results in mobilization of intracellular stored Ca2+ in a variety of cells, including vas deferens derived DDT1 MF-2 cells. Stimulation of the histamine H1 receptor on these cells caused a pronounced formation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) with respect to that of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). In this study, the role of inositol phosphates, in particular Ins(1,3,4,5)P4 on the internal Ca(2+)-releasing process was investigated in permeabilized and histamine stimulated intact DDT1 MF-2 cells. In permeabilized cells. Ins(1,4,5)P3 induced a concentration-dependent release of intracellular stored Ca2+. Addition of Ins(1,3,4,5)P4 did not cause Ca2+ mobilization, but its presence enhanced the amount of Ca2+ released by Ins(1,4,5)P3, thereby increasing the total Ca(2+) releasing capacity. The effect of both inositol phosphates was inhibited by heparin, known to block Ins(1,4,5)P3-sensitive receptors. Thus, the additional amount of Ca2+ released by Ins(1,3,4,5)P4 is mediated, either via Ins(1,4,5)P3 sensitive Ca2+ channels, or via different heparin-sensitive Ca2+ channels activated by both Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Histamine H1 receptor stimulation in intact cells induced a Ca(2+)-dependent K+ current, representing Ca2+ release from internal stores if receptor-activated Ca2+ entry from the extracellular space was prevented under Ca(2+)-free conditions or in the presence of La3+. This transmembrane current was abolished in the presence of intracellularly applied heparin. Depletion of Ins(1,4,5)P3-sensitive Ca2+ stores by internal application of Ins(1,4,5)P3 reduced the histamine evoked K+ current to some extent if the contribution of external Ca2+ was excluded.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556417 TI - Spectral analysis of body weight, food and water consumption and spontaneous motor activity in male Sprague-Dawley rats. AB - Spectral analyses by maximum entropy method were performed on the time-series data of body weight, food and water consumption and spontaneous motor activity in Sprague-Dawley rats. These parameters were recorded by a monitoring system collected every 30 min for 10 days. All the parameters had circadian (24 +/- 4 hr) and ultradian (less than 20 hr) rhythms. From the logarithmic transformation of spectral frequencies and their power spectral densities, the characteristics of the spectrum of body weight showed 1/f fluctuation and those of food and water consumption were white noises. The spectrum of the spontaneous motor activity showed complex characters. The range of frequencies lower than 0.1 Hz showed white noise while the range of those higher than 0.1 Hz were 1/f or 1/f2 fluctuations, especially in the range between 0.5 and 1.0 Hz (1-2 hours cycle). The auto-correlation coefficients of body weight did not declined suggesting that 24 hours cycle is only periodic phenomenon in body weight changes. Though the auto-correlation coefficients of food and water consumption also did not declined, they had not enough auto-correlations. This was consistent with the appearance of white noise in spectral analyses. The auto-correlation coefficients of spontaneous motor activity declined remarkably, and this suggests that there are various periodic changes in spontaneous motor activity including one to two hours cycles. These results suggest that the possibility of further extension of research is expected by using rhythmicity. It is also indicated that the time of measurement of body weight must be taken into consideration in experiments because of the presence of the circadian rhythmic changes. PMID- 7556419 TI - Analysis of sexual behavior in rams (Ovis aries). AB - In this study, a matured ram was paired with an estrus ewe and sexual behavior was investigated. All the behavior was recorded by a time-lapse video tape recorder from the start of pairing. The series of unit movements comprising the sexual behavior, the mounting series (MS), was extracted from the record and analyzed quantitatively (n = 774). The MS starts from ram's approaching or following a ewe and ends by ram's mounting on a ewe. The sexual behavior was consisted of the following eight unit movements, following or approaching (F/A), chin resting (CR), flehmen (Fl), mounting (Mo), nosing (No), nudging (Nu), pushing (Pu) and twisting (Tw). Analysis of a frequency of any combinations of two unit movements consisted of preceding and succeeding ones, observed in the MS was conducted by cell-by-cell test using the transitional matrix to make a flow diagram of the ram's sexual behavior. As a result, the pattern of unit movements, F/A-Tw-Nu-CR-Mo, formed a main route of the MS, whereas the repertoire of a sequential pattern increased by diverging. Though Fl and No didn't lead to mounting directly, these movements associated with olfactory perception also belonged to sexual behavior and may have independent function from other six unit movements that constitute the MS. PMID- 7556418 TI - Tissue distribution of deoxyribonuclease I (DNase I) activity level in mice and its sexual dimorphism. AB - The distribution of deoxyribonuclease I (DNase I) in mouse tissue and body fluids, the effects of sex on the enzyme activity, and the postnatal developmental regulation of the enzyme were examined in C3H/He mice. DNase I activity was high in the salivary gland and urine; and moderate in the liver, kidney, spleen, preputial gland and seminal vesicle. Weak activity was detected in the pancreas, heart, lung, brain, small intestine, thymus, coagulating gland and serum. Sexual dimorphism, of which males had significantly higher activity than females, was observed in the kidney, urine and liver. This sexual dimorphism in renal DNase I activity was eliminated by gonadectomy to males and testosterone treatment to females. These results reveal sex hormonal control of DNase I production in the kidney. DNase I activity in the liver of infant mice was almost equivalent to that of adult mice while the enzyme activity in the salivary gland, kidney and spleen remained at a low level; less than 5% at one week old and less than 20% at four weeks old. These results suggest tissue specific control of DNase I activity levels during postnatal development. PMID- 7556421 TI - Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos. AB - Mice entirely derived from ES cells were obtained from aggregates of TT2 ES cells and cytochalasin B induced tetraploid embryos. Tetraploid embryos were cocultured with ES cells in a well on the Multiplate-Terasaki. After embryo transfer of the aggregates, the male newborns were recovered normally after Cesarean section and reached adulthood. The male mice exhibited complete pigmentation of the eye and coat, suggesting ES cell contributions alone. Alkaline phosphatase-1 analysis yielded no evidence of tetraploid cells in the kidney or liver. The TT2-derived males were fertile, produced normal offspring, and exclusively transmitted the TT2 genotype to their progeny. This result clearly shows that ES cells are able to support complete fetal development. PMID- 7556420 TI - Retinal projections to the subcortical nuclei in the Japanese field vole (Microtus montebelli). AB - Retinal projections in the Japanese field vole (Microtus montebelli) were determined by anterograde transport of horseradish peroxidase (HRP). Injection of HRP into the unilateral vitreous body demonstrated that the terminal labeling of the optic projections was seen bilaterally in the suprachiasmatic nucleus (SCH), the ventral (GLv) and dorsal (GLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the medial pretectal nucleus (NTOM) of the pretectum (PT) and the superficial layer of the superior colliculus (CS), with contralateral predominance, and only contralaterally labeled terminals were found in the lateroposterior thalamic nucleus (LP), the lateral pretectal nucleus (NTOL) of the PT, the dorsal (DTN) and medial (MTN) terminal nuclei of the accessory optic system (AOS). The distribution area of the retinofugal terminals was divided into a three laminar arrangement in the GLd, i.e., layers 1 and 3 and layer 2, received the retinal input from contralateral and ipsilateral eye, respectively, as in arboreal squirrels. The contralateral CS received retinal fibers in the superficial layer, while ipsilateral optic fibers projected sparsely to the stratum opticum of the colliculi. Retinal connections to the DTN and MTN of the AOS were clearly discerned but no lateral terminal nucleus with retinal afferents was found. In addition, the AOS had no inferior fasciculus. These findings indicate that the vole has a contradictory features of a well- and a less developed sense of vision. Namely, the image forming visual system such as the retino-GLd was as well-developed as in a squirrel, on the other hand, the non image forming visual system such as the retino-AOS was less-developed as in an insectivore's brain. PMID- 7556416 TI - A photoaffinity probe for the polyamine site regulating the NMDA receptor. AB - Polyamines produce on the NMDA-receptor channel activity a regulatory effect subsequent to their binding to specific sites on the receptor-complex. The photoactivatable polyamine derivative L-azidophenylspermine shows properties which suggest its potential as a photoaffinity probe to investigate the nature and topology of these sites. In the dark, its effect on the binding of tritiated N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) to synaptosomal plasma membranes is similar to that of diaminodecane. Arcaine antagonizes both the effects of L azidophenylspermine and diaminodecane on [3H]TCP binding. L-Azidophenylspermine competes in post-synaptic densities with tritiated spermidine for a unique binding site with an EC50 similar to that of spermine. Upon irradiation, L azidophenyl-spermine incorporates into this material with a high efficiency to a level consistent with both the Bmax for tritiated spermidine and the estimated density of NMDA receptors in this fraction. PMID- 7556423 TI - Development of intestinal flora of human-flora-associated (HFA) mice in the intestine of their offspring. AB - Development of intestinal flora in newborn human-flora-associated (HFA) mice was compared with that in newborn conventional (CV) mice. Facultative anaerobes were detected from the first day after birth in both CV and HFA mice but anaerobes were not detected in the first week. Anaerobes rapidly increased from the 2nd week after birth and became predominant in newborn intestine. Most of the intestinal bacteria in adult CV and HFA mice were colonized in the intestine of CV and HFA mice, respectively, within 3 weeks after birth. The human intestinal flora established in the intestine of HFA mice finally reproduced without any remarkable change in composition in the intestine of newborn HFA mice. The development of intestinal flora in HFA mice was similar to that in CV mice but not that in human infants. These results indicated that human flora associated in HFA mice could be transferred from mothers to their offspring although HFA mice could not simulate the development of intestinal flora of the human infant. PMID- 7556422 TI - Characterization of insulin-dependent diabetes mellitus induced by a new variant (DK-27) of encephalomyocarditis virus in DBA/2 mice. AB - A murine diabetes mellitus induced with a new diabetogenic variant (DK-27) which we isolated from the M variant of the encephalomyocarditis (EMC) virus was characterized. Male DBA/2 mice (9.5 weeks old) were infected with various infectious doses of DK-27 intraperitoneally. Blood glucose and insulin levels were examined in association with the viral replication. Pancreatic pathology and hormone contents and stable hemoglobin A1c (St-A1c) levels were also examined on the final day of observation (35 days of post-infection). In infected mice, blood glucose levels rapidly elevated at 72 hr, slightly decreased between 7 and 10 days and finally became sustained hyperglycemia. On the other hand, blood insulin levels elevated at 48 hr, promptly decreased, and subsequently became sustained hypoinsulinemia. Viral replication in pancreases reached the highest titers at 48 hr and rapidly disappeared with all infectious doses used. Pancreatic insulin contents in infected mice were not detectable, and glucagon contents were not affected. In pathological examination, atrophy of islets and marked diminution of B-cells were observed, and A-cells occupied the major part of an infected islet. St-A1c levels reflected lasting hyperglycemia. These findings show that DK-27 causes insulin-dependent diabetes mellitus by the specific and direct destruction of pancreatic B-cells in susceptible mice. Such a diabetic model mouse will be useful for therapeutic studies. PMID- 7556424 TI - Diurnal variations in blood chemical items in Sprague-Dawley rats. AB - Diurnal variations in 23 blood chemical items were investigated in Sprague-Dawley rats of both sexes at 6 weeks of age. Blood samples were obtained every two hours. One to three periodic components detected by the maximum entropy method from the observed time-series and the optimum fitting parameters were calculated by the least squares method. The fitting curves were compared with the observed data for the evaluation of systematic and fluctuating parts of the time domain as circadian variations. The periodic components were detected from all the blood chemical items examined. This suggests that all items have systematic parts as basic rhythmic changes. The optimum fitting curves corresponded to the observed time-series in the following items: glucose, total protein, A/G ratio, alkaline phosphatase, Na, K, Cl and BUN of both sexes and total cholesterol, triglyceride, cholinesterase, CPK, inorganic phosphorus, direct and total bilirubin, creatinine and uric acid in males. Others showed insufficient correspondence to many fluctuating parts. This randomness was more frequently seen in females than in males. PMID- 7556425 TI - Survey of Pseudomonas aeruginosa contamination in human beings and laboratory animals. AB - Several serotypes of Pseudomonas (P.) aeruginosa were isolated from the oral cavities of researchers, but no positive cases were found among the animals they had contacted or in the environment. These results indicated that researchers are not a source of P. aeruginosa infection for animals. However, P. aeruginosa was detected on the hands of researchers and animal caretakers after they finished their work. The same serotype of P. aeruginosa was found in the animals and the environment. These findings demonstrated that the researchers and the animal caretakers were contaminated with P. aeruginosa by the animals, and then became infective vehicles. PMID- 7556426 TI - Effects of gonadotropin administration on follicular growth and in vitro fertilization in female hypogonadal mice. AB - Folliculogenesis in female hypogonadal (hpg) mice was examined after treatment with exogenous gonadotropins. The female mutant mice were characterized by a deficiency of hypothalamic gonadotropin-releasing hormone (GnRH), leading to the absence of estrus and ovulation. Gonadotropin administration induced resumption of gonadal development and vaginal opening. The follicles that developed with gonadotropin treatment were very similar to those in normal littermates. The oocytes from hpg mice showed the capacity for fertilization and development to produce viable young after in vitro fertilization and embryo transplantation. Thus, the combination of the exogenous gonadotropin administration, in vitro fertilization and embryo transfer method appear to be helpful to breed mutant hpg mice efficiently. PMID- 7556427 TI - Comparative histomorphology between isoproterenol-induced early lesions and postmortem artifacts in the rat heart. AB - Isoproterenol-induced early myocardial lesions were compared histomorphologically to commonly observed postmortem artifacts in the rat hearts which were collected up with 40 min after death. Isoproterenol-induced early lesions were characterized by multifocal myocardial eosinophilia restricted to the inner one third of the left ventricle and septum. These very early lesions were similar to the artifacts which were also characterized by myocardial eosinophilia and increased with time after death. The dilation of intercellular spaces which was also an artifact occurred at 40 min. Contraction bands, fragmentation of fibers and inflammatory cellular infiltration noted only in the isoproterenol-induced lesions were the differentiating criteria from artifacts. The postmortem artifacts were not observed in the hearts fixed immediately after death. PMID- 7556428 TI - Maternal placenta-like endometrial hyperplasia in a beagle dog (Canine deciduoma). AB - Three thumb-sized segmental swellings were found in both uterine horns of a 15 month-old, non-pregnant and non-pseudopregnant female beagle dog. Histopathological examination of the uterus revealed a marked proliferation of the endometrium that was characterized by two distinct parts, an inner tightly folded banded layer and an outer dilated spongy layer, quite similar to the maternal placenta except for the fetus and chorion in the lumen. Because the plasma level of progesterone was unusually high at autopsy, this hormonal disorder might be related to the pathogenesis of the endometrial hyperplasia in the present case. PMID- 7556430 TI - Detection of metrial gland cells in rat cervix after delivery by smear method. AB - It has been reported that the metrial glands, which form yellowish-white masses on the mesometrial triangle after pregnancy, coincide with the number of implants in the rat. In this study, the migration of the metrial gland cells (MGC) from the metrial glands to the endometrium after placental desquamation was investigated by the smear method. In smeared specimens, MGC appeared as large round cells with an unstained nucleus (a large binucleate cell was frequently seen), surrounded by a pinkish rim of cytoplasm, with PAS-staining; or appeared as a large reddish violet nucleus surrounded by dark violet cytoplasm with Giemsa staining. Numerous MGC were observed on the endometrium on the mesometrial side after artificial desquamation of the placenta on day 20 of pregnancy and day 0 of delivery. Further, MGC were observed on the endometrium on the antimesometrial side or in the cervix (including the internal and external orifice of the uterus) on day 0 of delivery, but no MGC could be detected in the cervix or on the antimesometrial side either during the period of placental signs (days 11-13) or on day 20 of pregnancy. Although MGC disappeared rapidly after delivery, slight traces of MGC remained on the mesometrial side until day 3 after delivery. In the case of abortion, MGC were observed in the cervix on the day of vaginal bleeding. In view of these results, it is considered that confirmation of MGC on the endometrium or the cervix provides evidence of abortion or delivery. PMID- 7556429 TI - Deodorization of laboratory animal facilities by ozone. AB - Deodorizing effect of ozone was investigated comparing two types of compact ozonizing apparatus made on an experimental basis. The concentrations of ammonia and trimethylamine were examined as an indicator for deodorizing effect of ozone in animal rooms of rats and guinea pigs at laboratory animal facilities of three different universities. Both of the ozonizing apparatus were able to remove ammonia and trimethylamine in animal rooms, with no significant difference in the performance of the two apparatus. PMID- 7556431 TI - Cadherin expression in the developing vertebrate CNS: from neuromeres to brain nuclei and neural circuits. AB - Cadherins are a family of cell surface glycoproteins which mediate cell-cell adhesion by a Ca(2+)-dependent mechanism. Results from in vitro studies with cadherin-transfected cell lines show that cadherins preferentially bind to each other in a homophilic fashion. In the developing vertebrate brain, at least 10 cadherins are found. Some of these cadherins are expressed in a restricted fashion in particular developing brain nuclei and neural circuits. Based on these results, specific morphogenetic roles for cadherins during CNS development have been proposed. This review focuses on the possible role of cadherin-mediated sorting and aggregation of early neurons and neurites in the formation of brain nuclei, fiber tracts, and neural circuits. Moreover, at least 1 cadherin is also expressed in a segmental ("neuromeric") fashion in the early chicken forebrain, suggesting that this cadherin regulates developmental processes involved in the transformation from the neuromeric organization of the early neuroepithelium to the functional organization of the mature brain. PMID- 7556432 TI - Transforming growth factor-beta promotes epidermal growth factor-induced thyroid cell migration and follicle neoformation in collagen gel separable from cell proliferation. AB - The regulation of growth and migration of thyroid follicular cells by epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta 1) were studied in primary cultures of porcine thyroid follicles embedded in collagen gel. Cultures were exposed to growth factors and [3H]thymidine (1 microCi/ml) for 3 days and then examined by light microscopic autoradiography. EGF at 1 ng/ml increased the [3H]thymidine labeling index from approximately 1% (control value) to 60% and stimulated fivefold the number of cells invading the collagen matrix. Intermediate responses were seen after treatment with 0.1 ng/ml of EGF. EGF stimulated [3H]thymidine incorporation was reduced by TGF-beta 1 at concentrations above 0.1 ng/ml. In contrast, TGF-beta 1, which alone only had a minor stimulatory effect on cell motility, markedly promoted the migratory response to EGF (1 ng/ml). Thus, 0.1 ng/ml TGF-beta 1 doubled the fraction of migrating cells without changing the level of [3H]thymidine incorporation. Moreover, cell migration was still fourfold over control values in cultures exposed to 1.0 ng/ml EGF and 1.0 ng/ml TGF-beta 1, despite a strongly inhibited [3H]thymidine labeling. The number of microfollicles located peripherally to the mother follicles was increased synergistically by EGF and TGF-beta 1. The epithelium of mother follicles became grossly discontinuous in regions of intense cell migration induced by EGF and TGF-beta 1 in combination. In conclusion, TGF beta 1 modulates the response of porcine thyrocytes to EGF in collagen gel cultures by promoting cell migration along with inhibition of [3H]thymidine incorporation. This suggests that EGF stimulates cell motility independent of the mitogenic signal. The persistent loss of epithelial integrity during enhanced cell migration indicates that mechanisms of intercellular adhesiveness are down regulated by TGF-beta 1 and EGF in cooperation. PMID- 7556433 TI - Tyrosine kinase inhibitors inhibit multiple steps of the cell cycle of vascular smooth muscle cells. AB - Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the mode of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxycinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [3H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF induced DNA synthesis in a dose-dependent fashion, with IC50 values of 4.7 +/- 1.4 microM for methyl 2,5-dihydroxycinnamate, 6.7 +/- 2.5 microM for genistein, and 0.17 +/- 0.07 microM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. the lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5 dihydroxycinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle. PMID- 7556434 TI - Plasmin abrogates alpha v beta 5-mediated adhesion of a human keratinocyte cell line (HaCaT) to vitronectin. AB - At cellular surfaces, urokinase-type plasminogen activator (uPA) is bound to a specific receptor (uPA-R). When bound to this receptor, uPA activates plasminogen, which is derived from plasma or the interstitial fluids. Thus, plasmin is provided for proteolysis of pericellular proteinaceous substrates. Here we demonstrate by immunocytology and laser scan microscopy that in the human keratinocyte cell line HaCaT uPA-R and uPA are localized together with the integrin alpha v beta 5 in focal contacts. Via the integrin alpha v beta 5, HaCaT cells adhere to vitronectin in a RGD-dependent manner. Plasmin interfered with the alpha v beta 5-mediated keratinocyte adhesion to vitronectin, most likely via cleavage of vitronectin and destruction of its cell binding function. Our findings demonstrate that plasmin, when generated by the uPA-dependent cell surface-associated pathway of plasminogen activation, can abrogate the cell binding function of vitronectin and can thus disturb the adhesive interaction with this matrix molecule. In focal contacts molecules are assembled that are crucial for adhesion to vitronectin (i.e., the integrin alpha v beta 5), as well as for the generation of plasmin (i.e., uPA-R and uPA), which can negatively influence the binding interaction. We suggest that the plasmin-mediated abrogation of the interaction between the integrin alpha v beta 5 and vitronectin is a pathway of negative regulation; the codistribution of uPA-R/uPA and alpha v beta 5 in focal contacts may restrict this process to areas of cell/matrix contact. PMID- 7556435 TI - Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells. AB - The treatment of NIH3T3 cells with L-asparaginase causes a complete and reversible growth arrest with a decrease of cell number in the first 2 days. The enzyme induces impressive morphological changes that have been studied exploiting eosin in fixed cells and calcein in intact cells as sources of fluorescence for confocal microscopy. The first changes are observed after 12 h of treatment and the process is complete after 48 h. Both nucleus and cytoplasm shrink, while cells round and lose processes. Eventually most cells break; several debris include strongly hematoxylinic bodies negative for eosin fluorescence. Some cells neither round nor break in fragments. Throughout the process cells and fragments retain calcein fluorescence, thus indicating the integrity of the cell membrane. A rapid depletion of the intracellular pools of both glutamine and glutamate occurs in treated cells, followed by a decrease in DNA and protein syntheses, while the cell content of ATP, the transmembrane gradient of sodium, and the active transport of amino acids are scarcely affected. It is concluded that (i) L asparaginase induces an apoptotic process in NIH3T3 cells that is forerun by a marked intracellular depletion of glutamate and glutamine; and (ii) although the enzyme completely suppresses cell proliferation, only a subset of cells undergoes apoptosis upon treatment. These findings provide a model for the characterization of factors that determine cell sensitivity to the effects of L-asparaginase. PMID- 7556436 TI - Cell cycle regulation and subcellular localization of the major human uracil-DNA glycosylase. AB - The subcellular localization of the human DNA-repair enzyme uracil-DNA glycosylase from the UNG gene has been studied using flow cytometry and laser scanning confocal microscopy of freely cycling HeLa S3 cells. A two-parameter flow cytometric analysis using propidium iodide and UNG-specific antibodies demonstrated that total cellular UNG increased during the G1-phase and was approximately doubled in early S-phase compared to early G1. The UNG level was stable during the S-phase and increased further during G2, reaching a 2.8-fold level compared to early G1. This factor included differences in cell size and staining variabilities. These findings were confirmed using two-parameter confocal analysis of UNG/DNA and UNG/mitochondria at different stages of the cell cycle. Although the major fraction of UNG was associated with nuclei, we also observed distinctive staining associated with mitochondria and a more diffuse staining probably reflecting UNG in the cytosol. Furthermore, very little UNG staining was observed in nucleoli. The UNG level in different cell compartments varied at different stages of the cell cycle, and this variation was most pronounced in the nuclei. These results demonstrate that the gene product from the UNG gene is located within three subcellular compartments and that the distribution between these compartments varies during the cell cycle. PMID- 7556437 TI - IL-1 alpha is constitutively expressed in the rat intestinal epithelial cell line IEC-6. AB - The rat small intestinal epithelial cell line, IEC-6, was examined for the presence of IL-1 mRNAs using the reverse transcription/polymerase chain reaction method. IL-1 alpha but not IL-1 beta transcripts were detected in plastic adherent cells. The levels of IL-1 alpha transcripts were similar in cells cultured at different densities. IL-1 activity was not detected, by bioassay, in the culture supernatant of the cells, nor was it membrane associated. IL-1 activity was detected in cell lysates, although its measurement was made difficult by the presence of an inhibitor of the bioassay. Intracellular IL-1 alpha was detectable using Western blots. Interleukin-1 receptor antagonist mRNA was also detectable in plastic adherent cells. Treatment with TGF-beta 1, IL-1 beta, IL-6, TNF-alpha, or combinations of any two of these cytokines failed to induce the secretion of the IL-1 alpha or to significantly change the levels of mRNA. The IEC-6 cell line is similar to other epithelial cells in having intracellular pools of IL-1 alpha. PMID- 7556438 TI - Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. AB - In the S-phase of the eukaryotic cell cycle, newly replicated DNA is assembled into chromatin. I used indirect immunofluorescence microscopy to localize the sites of chromatin assembly in respect to DNA replication. Replication foci in the nuclei of permeabilized HeLa cells were labeled by incorporation of biotin-16 dUTP and detected by fluorescent streptavidin. Prelabeling of replication foci in vivo with bromodeoxyuridine showed that replication in permeabilized cells proceeds at preexisting replication forks. The localization of chromatin assembly factor 1 (CAF-1) was determined with subunit-specific monoclonal antibodies. CAF 1 is not detectable in mitotic cells and is detectable only at background levels in about 60% of all interphase nuclei. The other interphase nuclei show an intense punctate immunostaining of CAF-1. These sites of CAF-1 colocalize with replication foci during all stages of the S-phase. No other discrete sites of CAF 1 are observed. Human replication protein A (RPA) colocalizes with these replication/chromatin assembly sites. In addition, extra nuclear sites of RPA are observed that probably represent prereplication foci, poised for initiation of DNA replication. PMID- 7556439 TI - Recycling of the 67-kDa elastin binding protein in arterial myocytes is imperative for secretion of tropoelastin. AB - We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab')2 fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A1, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A1 inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin. PMID- 7556441 TI - Localization and in situ phosphorylation state of nuclear tau. AB - The localization and phosphorylation state of tau in LA-N-5 neuroblastoma cells was examined. Our results demonstrate that there are two populations of tau in LA N-5 cells: cytosolic tau and nuclear tau. Indirect immunofluorescent microscopy revealed that nuclear tau is specifically localized to the nucleolus while cytosolic tau is diffusely distributed. To localize and quantitate tau in LA-N-5 cells by subcellular fractionation, a method was developed to extract tau from the nucleus while preserving the endogenous state of the protein. These studies revealed that 16% of the total tau protein in LA-N-5 cells is located in the nucleus and more specifically was found predominantly in the chromatin fraction containing DNA, chromatin, and associated proteins. The phosphorylation state of nuclear and cytosolic tau was examined by labeling LA-N-5 cells with 32Pi and immunoprecipitating tau from the different fractions. These data demonstrated that nuclear tau and cytosolic tau are phosphorylated approximately to the same extent. To determine if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-5 nuclei were isolated, incubated with [gamma-32P]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were 32P-labeled, tau was not phosphorylated. These results suggest that nuclear tau is not phosphorylated in the nucleus but rather in the cytosol prior to transport into the nucleus. The specific localization of nuclear tau strongly suggests that it has a functional role in the nucleus. However, further studies are necessary to determine the function of nuclear tau and how it may be regulated by phosphorylation. PMID- 7556442 TI - SARs, ARSs, and A,T-rich regions evidenced by restriction mapping on an 835-kb DNA fragment from Drosophila. AB - In Drosophila, sequences anchoring the DNA molecule to the scaffold (SARs) and sequences able to replicate autonomously (ARSs) had been shown to comap on an 835 kb DNA fragment (Brun et al. (1990) Mol. Cell. Biol. 10, 5455-5463). To investigate the question of whether this comapping results from the coincidental recruitment of SARs and ARSs in A,T-rich regions, A,T-rich regions of the 835-kb DNA fragment have been identified by restriction analysis with enzymes recognizing motifs made exclusively of A and T. Within the limits of sensitivity of this approach, the obtained data favor the idea of a noncoincidental recruitment: obviously a SAR and an ARS subpopulation are preferentially localized in the A,T-rich regions, but not every A,T-rich region displays a SAR activity, or an ARS activity, or both, nor are all SARs or ARSs localized in the A,T-rich regions. In addition, the data support the idea that a statistical assessment of base composition using restriction analysis might be developed into a general useful approach to genome organization. PMID- 7556440 TI - Fertilization in Dictyostelium: pharmacological analyses and the presence of a substrate protein suggest protein kinase C is essential for gamete fusion. AB - The role of protein kinase C (PKC) during fertilization in the model eukaryote Dictyostelium discoideum was studied. Inhibition of PKC activity using staurosporine, chelerythrine, and bisindoylmaleimide resulted in a dose-dependent decrease in gamete fusion without any detectable effect on cell morphology or growth. At 1.0 microM, staurosporine led to a greater than 90% inhibition of gamete fusion. In support of this, chelerythrine and bisindoylmaleimide at 10 microM inhibited gamete cell fusion by 98 and 99%, respectively. In all cases, subsequent removal of the inhibitor allowed for the completion of sexual development in a manner indistinguishable from untreated, control cultures. In contrast, the stimulation of PKC by the addition of the phorbol ester 12-O tetradecanoylphorbol-13-acetate at 5 nM resulted in a 56% enhancement of cell fusion. In order to identify PKC substrates that may regulate fertilization in D. discoideum, in vitro phosphorylation was carried out followed by SDS-PAGE. A number of proteins were phosphorylated, only one of which, a protein of about 50,000 M(r), appears to be a PKC substrate. In total, these results coupled with earlier work suggest that PKC functions as part of a calcium-mediated signaling pathway that regulates fertilization in D. discoideum, suggesting that the dual signaling pathway that regulates fertilization in higher eukaryotes may have evolved very early. PMID- 7556444 TI - Sequence data and chromosomal localization of human type I and type II hair keratin genes. AB - A cDNA library constructed with poly(A)+ RNA from human scalp was screened with selected fragments of both murine type I and type II hair keratin cDNAs. Two keratin clones, one type I, phKI-2, and one type II, phKII-1, were isolated and sequenced. In Northern blots, cDNA probes containing the 3'-noncoding sequences of the clones specifically hybridized to scalp mRNA species. Based on sequence homology comparisons with the four known murine type I hair keratins mHa1-4, the phKI-2 encoded keratin could be identified as human hair keratin hHa2. Similarly, sequence comparison with the four type II sheep wool keratins K2.9-12 revealed an orthologous relationship between the largest member of the type II wool keratin subfamily, K2.9 (i.e., sHb1) and the phKII-1 encoded human hair keratin (hHb1). The specific 3'-noncoding sequences of hHa2 and hHb1 were also used to isolate genomic fragments for both keratins from human genomic libraries which were than used for fluorescence in situ hybridization to human metaphase chromosomes. The hHa2 gene could be mapped to the long arm of chromosome 17, whereas the hHb1 gene was found on the long arm of chromosome 12. DAPI banding of the chromosomes allowed sublocalization of the hHa2 gene to 17q12-q21 and the hHb1 gene to 12q13, i.e., gene loci that have also been previously determined for human type I and type II epithelial keratins. PMID- 7556445 TI - Biochemical analysis of murine Wnt proteins reveals both shared and distinct properties. AB - The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b. PMID- 7556443 TI - Reversible inhibition of gap junctional intercellular communication, synchronous contraction, and synchronism of intracellular Ca2+ fluctuation in cultured neonatal rat cardiac myocytes by heptanol. AB - We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca2+ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca2+ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca2+ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca2+ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca2+ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca2+ fluctuations, which facilitate synchronized contraction of cardiac myocytes. PMID- 7556447 TI - The fate of myoblasts following transplantation into mature muscle. AB - Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme beta galactosidase (beta-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (beta-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and beta-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest beta-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell transplantation and for the application of such studies to cell therapy and cell mediated gene therapy. PMID- 7556446 TI - Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis. AB - Changes in chromatin supraorganization defined in terms of patterns of chromatin texture were studied by video image analysis in Feulgen-stained revertants of LTR ras-transformed NIH 3T3 cells and in cell lines obtained by transfection of these revertants with sense and antisense constructs of the lysyl oxidase gene (also named Lox or "ras recision gene"). The objective was to determine whether changes in expression of the Lox gene, which have been assumed to modulate cell transformation by ras, could also affect the chromatin supraorganization changes known to be elicited in NIH 3T3 cells by ras transformation. The image analysis results revealed that, although a nuclear phenotype visually similar to the most frequent one (III) in ras-transformed NIH 3T3 cells also appeared in the revertant, it contained a remarkably less tight chromatin packing state. This situation was also found in the revertant transfected with the sense construct of the Lox gene, but in the revertant transfected with the Lox antisense constructs the chromatin texture of the III phenotype was equal to or close to that of the ras-transformed cells. With regard to the nuclear phenotype characterized by abundant loosely packed chromatin and less represented in the transformed cell lines (I'), changes in the various cell lines, although detectable, were not as drastic as those reported for the III phenotype. The enhancement in chromatin condensation of the type III nuclei, which affects euchromatin, is probably associated with a limited transcription of the genome. Although the image analysis results are mostly in agreement with previously published data on the molecular biology and tumorigenicity of the same cell lines, it appears that the phenomenon of chromatin condensation once established in NIH 3T3 cells by LTR-ras transformation could not be totally reverted by simply affecting Lox expression. PMID- 7556448 TI - Transforming growth factor beta regulation of epidermal growth factor receptor in ectocervical epithelial cells. AB - Transforming growth factor beta (TGF beta) is a pluripotent modulator of cell function and an important suppressor of cervical epithelial cell proliferation. In the present study, we examine the effects of TGF beta 1 on the level and activity of the epidermal growth factor receptor (EGFR) in HPV-16 immortalized cervical epithelial cells. In ECE16-1 cells, increased EGFR levels are observed within 24 h after initiation of TGF beta 1 treatment and levels continue to increase with time. This increase is correlated with a TGF beta 1-dependent decrease in proliferation rate. Scatchard analysis indicates that the population of EGFR sites induced by TGF beta 1 have a low affinity for EGF (Kd = 4.08 nM) compared to the receptors present prior to TGF beta 1 treatment (Kd = 0.3 and 1.6 nM). TGF beta 1 treatment also reduces EGFR kinase autophosphorylation activity. Cell cycle studies indicate that TGF beta 1-treated cells arrest in the G1 phase of the cell cycle and that regulation of EGFR level was independent of cell cycle stage in both TGF beta 1-treated and untreated cells. However, EGFR level was related to the G1 phase time. Parallel studies indicate that a TGF beta 1 dependent increase in p53 level is also correlated with increased time spent in G1. These results suggest that TGF beta 1 inhibition of ECE16-1 cell proliferation may act both by the replacement of high affinity/high kinase activity EGFR sites with low affinity/low kinase activity EGFR sites and a p53 mediated cell cycle arrest. PMID- 7556449 TI - Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative analyses by immunofluorescence and immunoelectron microscopy. AB - The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli. PMID- 7556450 TI - Scleroderma-derived human fibroblasts retain abnormal phenotypic and functional characteristics following retroviral transduction with the SV40 tsT antigen. AB - In this study an amphotropic retrovirus has been used to efficiently transduce normal human (NF) and scleroderma (systemic sclerosis; SSc) dermal fibroblasts (SScF) with a sequence encoding a temperature-sensitive mutant of the SV40 large T antigen (tsA58-U19). From the primary outgrowths of skin explants, cultures were generated whose growth was stringently temperature-dependent. When grown at a low, permissive temperature (35 degrees C), both normal and SSc-transduced cells continuously divided with similar doubling times, whereas at a high, nonpermissive temperature (39.5 degrees C), division of both the NF and SScF cells was rapidly arrested. These cells have been passaged more than 50 times, have the typical morphological appearance of fibroblasts, and have retained an anchorage-dependent phenotype. The transduced normal cells (tsT-NF) synthesized the matrix molecules collagen and fibronectin and expressed phenotypic antigens characteristic of their nontransduced counterparts, including MHC Class I, VLA beta 1 (CD29), Hermes 1 (CD44), VLA-4 alpha (CD49d), ICAM-1 (CD54) and LFA-3 (CD58) and the cell surface ectoenzymes neutral endopeptidase (CD10), aminopeptidase N (CD13), and dipeptidyl peptidase IV (CD26). Analysis of the transduced SSc fibroblasts (tsT-SScF) showed that these cells exhibited certain major features of the SSc pathology, notably the abnormally high synthesis of type I collagen, increased expression of ICAM-1, and depressed levels of CD26. Moreover, these phenotypic characteristics were retained even after prolonged culture in vitro. The tsT-SScF cells also retained their responsiveness to cytokines, since interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) both produced a marked increase in ICAM-1 expression. Our findings show that infection of SScF with the SV40 tsT antigen extends the life span of these cells and does not ablate their abnormal phenotypic and functional characteristics. PMID- 7556451 TI - Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system. AB - Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system. PMID- 7556453 TI - Molecular cloning of a murine cDNA encoding a novel protein, p38-2G4, which varies with the cell cycle. AB - Proliferating cells express genes active in cell cycle control. The modulation of control genes and factors are required to maintain critical cell cycle activities. We used a set of monoclonal antibodies prepared against DNA-binding proteins from Ehrlich ascites tumor cells in immunofluorescent microscopy to screen for proteins showing cell cycle-specific staining patterns. Here, we report cloning and characterizing of a novel mitogen-inducible gene from murine macrophages that predicts a cell cycle-specifically modulated nuclear protein of 38 kDa, designated p38-2G4. p38-2G4 displayed a speckled pattern of varying fluorescence intensity confined to the nucleus, but sparing the nucleoli. Strongly stained granules were observed between G1 and mid S phase, followed by a less abundant punctated arrangement toward the end of S phase, and negative fluorescence at the S/G2 transition. Thereafter, the nuclear staining reappeared. Additionally, p38-2G4 expression vanished in G0-arrested cells and was restored after release from growth arrest. p38-2G4 conserved in vertebrates by means of immunofluorescence data contains a number of putative phosphorylation sites, a cryptic nuclear localization signal, and an amphipathic helical domain. Our cDNA and its deduced amino acid sequence is related to a Schizosaccharomyces pombe gene encoding a 42-kDa protein that associates with curved DNA, suggesting that we have cloned the murine homologue of the S. pombe gene which defines a novel cell cycle-specifically modified and proliferation-associated nuclear protein in mammals. PMID- 7556452 TI - Inhibition of HGF/SF-induced membrane ruffling and cell motility by transient elevation of cytosolic free Ca2+. AB - HGF/SF (0.5-100 ng/ml) induced rapid membrane ruffling, formation of microspikes, and increased motility of HT115 cells within 5 min of addition. These effects were not accompanied by any change in cytosolic free Ca2+ concentration. However, ATP (0.5-10 mM) induced a transient rise in cytosolic free Ca2+ concentration in HT115 cells from a resting concentration of 100 nM to a peak of 400 nM before returning to baseline within 3 min. The addition of ATP to cells treated with HGF/SF inhibited both membrane ruffling and cell movement. The effect of ATP was attributed to the transient rise in cytosolic free Ca2+ concentration, because cytosolic BAPTA, which prevented the rise in cytosolic free Ca2+ concentration, also abolished the inhibitory effect of ATP. Raising cytosolic free Ca2+ concentration with ionomycin and ADP also inhibited membrane ruffling. It was thus concluded that transiently raised cytosolic free Ca2+ concentration inhibited HGF/SF-induced membrane ruffling of HT115 cells. PMID- 7556454 TI - Cell division does not increase transepithelial permeability of LLC-PK1 cell sheets. AB - The transepithelial electrical resistance (TER) across LLC-PK1 cell sheets is unstable for several days postseeding, even when the cells are trypsinized from a previously confluent culture and replated at confluent density. We therefore followed the TER of LLC-PK1 cells plated at confluent density to elucidate characteristics of the TER fluctuations after plating of the cells. Control cultures reached a maximum TER of 1800 omega.cm2 approximately 24 h after plating. TER then declined sharply, reaching generally stable values (approximately 175 omega.cm2) only after 4 days. Cell cycle activity ([3H] thymidine incorporation) peaked approximately 22 h after plating, prior to the peak in TER values, and then followed a decline similar to that of the TER. Treatment of cells with EGF at 24 h after plating caused the TER values reached at 3-4 days postseeding to exceed time matched controls by approximately 100%. This EGF-treated group showed a concomitant increase in [3H]thymidine incorporation and cell density compared to control. Transepithelial fluxes of [14C]D-mannitol across control vs EGF-treated cell sheets were not, however, significantly different at the 4-day time point, indicating that a change in tight junction sieving on the basis of size had not occurred. Diffusion and bi ionic potential studies indicated that the change in TER in EGF-treated cells was instead due to altered charge selectivity at the tight junction and/or intercellular space. We conclude: (1) TER across LLC-PK1 cell sheets does not stabilize until 4 days after seeding at confluent density and (2) cell division and resultant increased cell density in LLC-PK1 cell sheets can correlate with elevated TER values, due to altered ion permeability of the paracellular pathway. Permeability to both Na+ and Cl- decreased as a result of EGF treatment but the decline in chloride permeability was significantly greater. Not only was there a decrease in the permeabilities of all halide anions after exposure of cell sheets to EGF, but the permeability sequence changed after EGF exposure. PMID- 7556455 TI - TPA increases conductance but decreases permeability in neonatal rat cardiomyocyte gap junction channels. AB - Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (gj) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltage clamp, gj increased by 15.5 +/- 7.2% (mean +/- SD, n = 9) and by 46.3 +/- 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca2+ concentration in the electrode solution did not change the TPA-induced increase in gj observed under WC conditions. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (alpha PDD), did not affect gj. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas alpha PDD did not change this distribution, TPA shifted the gamma j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6 CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 +/- 7.2 cells (mean +/- SD, n = 18) and 6-CF labeled 8.4 +/- 2.2 cells (n = 20). Whereas alpha PDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 +/- 1.3 cells (n = 11) and of 6-CF to 2.4 +/- 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA. PMID- 7556457 TI - Expression of the amyloid precursor protein of Alzheimer's disease on the surface of transfected HeLa cells. AB - The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa beta A4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP695 isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4 degrees C followed by incubation at 37 degrees C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membrane-matrix stabilization. PMID- 7556458 TI - The propagation of a zone of activation along groups of flagellar doublet microtubules. AB - The complexity of the 9 + 2 flagellar axoneme has made it difficult to discover the mechanism of bend propagation. We have studied a simplified preparation of mechanically "opened-out" groups of doublet microtubules that adopt a helical, ribbon-like form. From the very long sperm of the quail, ribbons of doublets up to 130 microns long have been obtained. We reactivated them photolytically by releasing ATP from caged ATP, thus observing the reactivation from the beginning. The response to ATP was a reduction in the pitch and diameter of the helix, in what we refer to as an "active zone" (AZ). Ahead of the AZ was a short region of increase in helical pitch and diameter, the pre-AZ. We ascribe these two altered geometries to the development of tension between the doublets, actively (in the AZ) and passively (in the pre-AZ). The AZ/pre-AZ complex established itself at one end of a helix--almost certainly the proximal end--then it propagated toward the other end of the helix at a mean velocity of 15 microns s-1 (using 1 mM caged ATP), maintaining or increasing its length as it traveled. This is the same velocity as that for bend propagation on cylindrical axonemes detached from their basal structures. Successive propagations on the same helix were seen. Thus, active and inactive segments of the same doublet assembly can coexist, even though all parts are exposed to ATP. The motor response is seen to be a localized event that is transmitted metachronally. The propagation of activation is an intrinsic property of structures in the interdoublet gap and does not require constituents of the cytosol other than ATP and Mg2+. Since it occurs in helical ribbons (3 + 0, 4 + 0, etc.), the propagation of activity must be independent of central axonemal structures; furthermore, it cannot be dependent on the integrity of the 9 + 2 cylinder nor on any feedback from large-scale features of the waveform. PMID- 7556456 TI - Biosynthetic transport of a major lysosomal membrane glycoprotein, lamp-1: convergence of biosynthetic and endocytic pathways occurs at three distinctive points. AB - We studied the kinetics of the biosynthetic transport of lysosome-associated membrane glycoprotein-1 (lamp-1) to the endocytic compartments in cultured rat hepatocytes. Newly synthesized lamp-1 (NS-lamp-1) was transported to the trans Golgi from rough endoplasmic reticulum with a half time (t1/2) of 13 min. From the trans-Golgi, at least 25% of NS-lamp-1 was delivered to the cell periphery: to the cell surface and early endosomes with t1/2 s of 32 and 33 min, respectively. A comparison of the kinetics of the biosynthetic transport of lamp 1 to both compartments demonstrated that NS-lamp-1 takes two peripheral routes from the Golgi apparatus; it is delivered to early endosomes directly and after reaching the cell surface. A major portion of NS-lamp-1 follows a direct intracellular pathway to late endosomes (t1/2 = 45 min) and subsequently to lysosomes (t1/2 = 85 min). The kinetic data of the biosynthetic transport to these endocytic vacuoles suggested that a significant fraction of NS-lamp-1 returns to the late endosomes immediately after its arrival at lysosomes and that there is a unique retrograde delivery of NS-lamp-1 from late to early endosomes prior to its transport to lysosomes. Thus, in cultured rat hepatocytes, the lamp 1 biosynthetic and the endocytic pathways converge at the three distinctive points. Late endosomes are centrally situated in the complex biosynthetic route of lamp-1. PMID- 7556459 TI - TGF-beta modulates the expression of retinoic acid-induced RAR-beta in primary cultures of embryonic palate cells. AB - We have previously shown that both transforming growth factor-beta (TGF-beta) and retinoic acid (RA) regulate the expression of cellular retinoic acid binding proteins (CRABP) I and II and TGF-beta 3 mRNAs in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. We now describe additional cross-talk between the RA and TGF-beta signal transduction pathways--the ability of TGF beta, including the endogenous form(s), to modulate the expression of the nuclear retinoic acid receptor-beta (RAR-beta). Northern blot hybridization revealed that RA induced the expression of RAR-beta mRNA, there being little or no detectable expression in untreated MEPM cells. Induction by 3.3 microM RA was abrogated by simultaneous treatment with TGF-beta 1 (5 ng/ml). TGF-beta 1 alone had no effect on RAR-beta mRNA expression. Determination of RAR-beta mRNA half-life by treatment with actinomycin D indicated that TGF-beta 1 did not alter the stability of RAR-beta mRNA. Conditioned medium (CM) from MEPM cells contained little active TGF-beta protein; heat treatment of the CM dramatically increased the amount of active TGF-beta as assessed by the mink lung epithelial cell bioassay. Furthermore, heat- or acid-activated CM also inhibited CRABP-I and RA induced RAR-beta expression. The effect of heat-activated conditioned medium could be abrogated with panspecific neutralizing antibodies to TGF-beta, confirming that endogenous TGF-beta is the biologically active factor in heat activated CM. These results provide evidence for complex interactions between TGF beta and RA in the regulation of gene expression in embryonic palatal cells and suggest a role for endogenous TGF-beta in the regulation of expression of genes encoding elements of the RA signal transduction pathway. PMID- 7556461 TI - Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells. AB - Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells. PMID- 7556460 TI - Fetal-adult phenotype transition, in terms of the serum dependency and growth factor requirements, of human skin fibroblast migration. AB - Since the migration of human fetal skin fibroblasts into a denuded area of a cell monolayer has been demonstrated to be serum-independent (fetal phenotype), whereas that of adult-donor skin fibroblasts is serum-dependent (adult phenotype), a study was carried out to determine at which life stage the fetal adult phenotype transition, in terms of serum dependency of human skin fibroblast migration, occurs. The results demonstrated that this phenotype transition occurred before birth, and, therefore, the fetal skin fibroblast phenotype differed from those of infant-, child-, adult-, and elderly-donor skin fibroblasts. But, the fetal-, child-, and adult-donor skin fibroblast phenotypes did not change during in vitro aging. On the other hand, the growth factor requirement for human skin fibroblast migration changed similarly to the serum dependency for fibroblast migration: the signal for fibroblast migration changed from basic fibroblast growth factor for the fetal phenotype to platelet-derived growth factor for the adult phenotype. PMID- 7556462 TI - Comparison of fluorescein angiography with microvascular anatomy of macaque retinas. AB - Recent anatomic work has shown that the capillary network of the fovea is multilaminar. We have identified the elements of this network that are visualized by fluorescein angiography and those that are missed. Fluorescein angiograms of monkey retinas (Macaca fascicularis) with good visualization of individual capillaries were obtained by standard clinical techniques. Retinal whole mounts were prepared from the same animals. Anatomic drawings made from the whole mounts were used to identify which parts of the capillary network were visualized angiographically. Angiographic estimates of dimensions of the foveal avascular zone corresponded closely to the anatomy. Capillary visibility declined rapidly from near perfect visualization at the edge of the foveal avascular zone to less than 40% by 900 microns eccentricity. While all the widest capillary segments (diameter 6.1-7.0 microns) were visualized, only 43% of the modal group of capillary segments (diameter 4.1-4.5 microns) were detected. When a relatively homogeneous population of capillaries was analyzed (diameters limited to the narrow range of 4.0-5.0 microns), visualization declined monotonically with depth in the retina. Capillary segments in the nerve fiber plane were visualized more than four times as effectively as segments of comparable diameter in the deepest vascular plane. High quality angiograms accurately delineate the foveal avascular zone, but they visualize only a fraction of the adjacent multilaminar network. Therefore, current techniques may not detect the earliest nonperfusion of capillaries in vaso-occlusive diseases. Capillary visibility is a joint function of diameter and of retinal depth. The decline in visualization with retinal depth implies that light scattering in the retina degrades the angiographic image. PMID- 7556463 TI - Chloride binding in the stroma of cultured human corneas. AB - In the ox cornea, more than half of the non-diffusible, matrix negative charge is derived from the binding of free chloride ions. Because the magnitude of the net matrix charge is the dominant factor which determines the degree of stromal swelling, we investigated whether this phenomenon, stromal chloride binding, also occurs in human corneal stroma. Intrastromal ion concentrations were measured with radio-isotopes when human (outdated Eye Bank) corneas or (fresh) bovine corneas, physically clamped to maintain a constant hydration, were incubated in buffered 154 mM NaCl. The intrastromal chloride ion concentration was compared to the normalized concentrations of trace quantities of radio-labelled acetate and lactate ions. For human corneas, the intrastromal chloride ion concentration was found to be significantly higher (P < 0.001, t-test) than the normalized concentrations of both acetate and lactate ([Cl]i = 142.5 +/- 0.9 mM, (n = 9); [acetate]i = 131.2 +/- 1.2 mM, (n = 8); [lactate]i = 131.9 +/- 1.5 mM, (n = 5); all values are mean +/- S.E.M.). The sodium ion concentration was elevated ([Na]i = 176.0 +/- 1.8 mM, (n = 9)). These results demonstrate that chloride binding occurs to a significant extent in cultured human corneal stroma and suggest that chloride binding may be evident in the native human cornea. PMID- 7556464 TI - Phosphorylation of alpha-crystallin in rat lenses is stimulated by H2O2 but phosphorylation has no effect on chaperone activity. AB - Alpha crystallin (alpha), a phosphorylated structural protein of the lens, has been shown to be a chaperone preventing other lens proteins from aggregating. It is now demonstrated that with oxidative stress imposed on cultured rat lenses, the incorporation of labeled phosphate into the alpha polypeptide chains increased by two to four times over a 90-min period in comparison to control experiments. The phosphorylation rate of the B chain, alpha B, was twice that of the A chain, alpha A. However, phosphorylation of the alpha chains has an insignificant effect on the chaperone activity of alpha or the individual alpha A and alpha B chains as measured by suppressing the thermally induced aggregation of beta low or gamma crystallins. It was also found that the alpha A aggregates are more effective chaperones than the alpha B aggregates. The size of the macromolecules resulting from reaggregation of the isolated non-phosphorylated or phosphorylated alpha B chains are not markedly effected by phosphorylation. However, phosphorylation of the alpha A chain leads to a heterogeneous population with two major species, one similar in size to alpha A and another approximately twice as large. It is concluded that the phosphorylation of alpha is associated with some other function of the protein than that of chaperone activity and that this function may be linked to a protective response to oxidative stress. PMID- 7556465 TI - Analysis of alpha-crystallin expression in cultured mouse and rabbit lens cells. PMID- 7556466 TI - Rod outer segment (ROS) renewal as a mechanism for adaptation to a new intensity environment. I. Rhodopsin levels and ROS length. AB - This study outlines the time course of cellular changes which occur within the renewal process of the rod cell during a switch to a new cyclic intensity environment. In the following experiments we demonstrate that by using the renewal process. Sprague-Dawley rats switched to a new cyclic intensity can adjust both the length of the outer segment and the amount of rhodopsin per retina. Previously it was established that the rod outer segment (ROS) length is not constant when animals are exposed to an intensity different from their normal environment. In the present study, we investigated how changes in ROS length were achieved by the rod cell. We noted that soon after a change in intensity, the ROSs were always shortened. This occurred when rats were switched either to a higher or lower light intensity than their rearing level. A final ROS length was achieved within 21 days (approximately two turnover periods). This length change required decreased disk removal in animals switched to a low light to achieve a lengthened ROS. In animals moved to a higher light intensity, however, disk removal rate did not change but ROS length did shorten, suggesting a change in disk addition. It is known that rhodopsin levels are up- and down-regulated with changes in environmental lighting. In this study, rhodopsin levels of animals switched from a low, cyclic intensity of 3 lx into a more intense cyclic light, 200 lx, dropped dramatically within 7 days to the rhodopsin value of an animal reared in the higher intensity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556467 TI - Rod outer segment (ROS) renewal as a mechanism for adaptation to a new intensity environment. II. Rhodopsin synthesis and packing density. AB - Using intraocular injection of a 3H-phe and 3H-leu mixture, we found that the net synthesis of rhodopsin is light-intensity-dependent and can adjust when an animal encounters a new lighting environment. Rhodopsin net synthesis dropped dramatically in day 1, 200-lx immigrants; however, preliminary studies show that the opsin mRNA levels in these animal were the same as that found in the 200-lx native group. This suggests that the changes in the net synthesis of rhodopsin found when an animal is moved to a higher intensity light may be controlled at some point post-transcriptionally. Microspectrophotometric measurements on single rod cells revealed that the differences in whole-eye rhodopsin levels between the two cyclic intensity groups, 3 lx and 200 lx, were also present at the single cell level. This supports previous studies suggesting that the density of rhodopsin per disc varies according to the intensity of light to which the animal is exposed. Individual rods also had differences in packing density of rhodopsin at the base compared to the tip of the same outer segment when the animal had been switched to a new intensity for one half of a turnover period (5 days). This indicates the amount of rhodopsin per disc is regulated and suggests that renewal is the process responsible for creating a modified outer segment. Animals, when switched to a new intensity, can adjust the synthesis of rhodopsin and the number of molecules added per disk to ensure the appropriate packing density of rhodopsin in the rod outer segment (ROS) disk membranes for that level of light intensity. PMID- 7556468 TI - Programmed cell death of retinal ganglion cells during experimental glaucoma. AB - The death of retinal ganglion cells during glaucoma is thought to result from damage to their axons as they exit the eye through the lamina cribrosa. In this study, intraocular pressure in the rat was increased to twice the normal average by cauterizing two limbal-derived veins. To investigate whether retinal ganglion cells in the glaucomatous eye follow an apoptotic type of death, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase to the 3'-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in numbers of biotin-labeled nuclei after administration of the protein synthesis inhibitor, cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when the intraocular pressure is markedly increased. This raises new possibilities in the treatment of glaucomatous damage to the retina, by the potential interruptibility of a program for neuronal death. PMID- 7556469 TI - Degradation of differentially oxidized alpha-crystallins in bovine lens epithelial cells. AB - There is a growing consensus that altered proteins are more susceptible to degradation than native proteins. The enhancement of degradation of damaged proteins may be of significance since it prevents the accumulation of damaged proteins in cells. Several proteolytic pathways have been discovered in the lens. These include ATP-independent, ATP-dependent and ATP/ubiquitin-dependent proteolytic pathways. However, the extent of involvement of these proteolytic pathways in degradation of damaged proteins is not well described. alpha Crystallin was oxidized by exposure to 0.03-3.2 mol.OH (mol protein)-1. Modifications to the oxidized alpha-crystallin and proteolytic susceptibility of the oxidized alpha-crystallin were studied. Exposure to > 0.32 mol.OH per mole of subunit produced aggregates and fragments of alpha-crystallin. Changes in isoelectric points of the proteins were observed after exposure to 0.64 mol.OH (mol protein)-1. The extent of loss of tryptophan and sulfhydryl groups was related to the level of .OH-exposure. Carbonyl content increased progressively with increasing oxidation. When incubated with a supernatant of bovine lens epithelial cells, the .OH-modified proteins were proteolytically degraded up to three times faster than untreated alpha-crystallin. ATP stimulated the degradation of native alpha-crystallin and alpha-crystallin which was exposed to 1.6 mol.OH (mol subunit protein)-1 (alpha 1.6). Sixty-seven per cent and 100% of the ATP-dependent degradation of native alpha-crystallin and alpha 1.6 was ubiquitin-dependent, respectively. The data indicate that alpha-crystallins oxidized by .OH are recognized and degraded rapidly by cytoplasmic proteolytic systems in bovine lens epithelial cells. Both ATP-independent and ATP/ubiquitin dependent proteolytic pathways are involved in the degradation of native and oxidized alpha-crystallin. PMID- 7556470 TI - Dietary restriction delays cataract and reduces ascorbate levels in Emory mice. AB - Dietary restriction can effectively extend lifespan and retard many age-related debilities. One hypothesis to explain the beneficial effects of dietary restriction is that it prolongs maintenance of cellular homeostasis by limiting endogenous oxidative stress and preserves oxidative defense mechanisms during aging. Ascorbate, a primary antioxidant, may play a major role in preventing oxidative damage. Ascorbate levels were determined in dietary restricted (R) and control (C) Emory mice, a strain which develops age-related cataract due in part to oxidative damage to lens proteins. Mice which consumed a diet restricted by 40% in calories had lower ascorbate concentrations in plasma, liver and kidney. Nevertheless, R animals showed significantly delayed progression of cataract which extended over the entire second half of life. The R diet did not result in different ascorbate levels in this lens. Aging was associated with a decrease in ascorbate in all the examined tissues except lens of both the R and C groups. It is not clear from these data that ascorbate is a prominent factor in the delay of cataract formation or other debilities in R Emory mice. However, it also appears unlikely that lens ascorbate is cataractogenic. PMID- 7556471 TI - Oxygen consumption in the isolated toad retina. AB - Retinal O2 utilization was studied to identify the O2 consuming processes in the retina and the spatial distribution of those processes. Neural retina, retinal pigment epithelium and choroid were dissected from the toad eye and superfused with an oxygenated Ringer's solution. Double-barreled microelectrodes were used to measure O2 and local voltage simultaneously within the retina in both light and dark adaptation. The profile of PO2 was measured during a withdrawal of the electrode tip across the retinal pigment epithelium and through the neural retina. The PO2 decreased through the distal retina, reaching a minimum in the inner segment or outer nuclear layer, and then increased steadily through the proximal retina. From fitting PO2 profiles measured in the dark-adapted retina to a three-layer diffusion model, O2 consumption was found to be 1.0 +/- 0.4 and 0.4 +/- 0.3 ml O2 (100 g min)-1 in the outer and inner halves of the retina, respectively. Light decreased consumption in both halves of the retina. In steady illumination (500 nm) that saturated the ERG b- and c-waves, O2 utilization decreased significantly to 48% and 68% of the dark values in the outer and inner retina, respectively. When Na+ was removed from the superfusate to inhibit the photoreceptor Na+/K+ pump, O2 consumption in the outer retina decreased by about the same amount as in light, but O2 consumption in the inner retina was not significantly affected. PMID- 7556472 TI - Light-evoked oxygen responses in the isolated toad retina. AB - Transient changes in retinal oxygen in response to light stimuli were studied to further understand the light-evoked change in oxygen consumption. Double-barreled microelectrodes, which measured oxygen and local voltage simultaneously, were positioned near the photoreceptor inner segments of the toad neural retina retinal pigment epithelium-choroid preparation. Light-evoked oxygen responses were measured in a normal [Na+] solution, and in a test solution with lowered extracellular [Na+] to inhibit Na+/K+ pumping. Under the normal [Na+] condition, retinal oxygen tension increased in response to light indicating that oxygen utilization had decreased. When the Na+ concentration was lowered in the retina, the oxygen tension decreased in response to light, indicating an increase in oxygen utilization which was smaller than the Na+/K+ pump effect and therefore masked under normal conditions. The increase in oxygen utilization in lowered [Na+] was suppressed by adding 0.7 mM 3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor, suggesting that the response was largely due to hydrolysis and subsequent resynthesis of cyclic GMP. Results of fitting the light evoked responses to exponential functions suggested that the decrease in oxygen consumption caused by slowing of the photoreceptor Na+/K+ ATPase had a time constant between 130 and 180 sec and that the increase in oxygen utilization from increased cyclic GMP synthesis was faster. PMID- 7556473 TI - Prolonged bilateral carotid artery occlusion induces electrophysiological and immunohistochemical changes to the rat retina without causing histological damage. AB - Reduction of the retinal blood flow by occlusion of both common carotid arteries suppressed the b-wave of the rat's electroretinogram. Transient occlusion of the carotids for 45 min reduced the b-wave by 46% without affecting the amplitude of the a-wave. The normal ERG activity returned 30 min after restoration of blood flow. Prolonged carotid occlusion for 7 days totally abolished the b-wave but enhanced the a-wave amplitude. Although b-wave amplitude suppression has been considered as an indicator of retinal ischaemia, no histological changes were seen in retinas of rats subjected to 45 min or 7 days of two-vessel occlusion, when observed by light microscopy. Moreover, GABA, GABAA receptor, calretinin and PKC-alpha immunoreactivities were unaltered. Carotid artery occlusion did, however, induce the expression of the cytoskeletal protein, glial fibrillary acidic protein (GFAP), in retinal Muller cells. The increase in the Muller cell GFAP immunoreactivity was related to how long the carotids were occluded as well as the reperfusion time. Prolonged occlusion for 7 days resulted in a 356% increase in retinal GFAP. These findings show that a reduction of retinal blood flow by occlusion of the carotids causes a metabolic stress to the retina and elicits events associated with gliosis without resulting in 'ischaemic-like' morphological changes. PMID- 7556474 TI - Calcimycin-induced lens epithelial cell apoptosis contributes to cataract formation. AB - Previous studies have shown that calcimycin induces cataract in organ culture. To investigate the mechanism of this induction, the viability of lens epithelial cells in calcimycin (calcium ionophore, A23187)-treated rat lenses were examined. During incubation of lenses with 5 microM calcimycin, apoptotic epithelial cells were found after a 2-hr treatment as determined by terminal deoxynucleotidyl transferase (TdT) labeling. The percentage of apoptotic cells quickly rose as the incubation time increased. After a 12-hr incubation, more than 60% of the lens epithelial cells underwent apoptosis. Prolonged c-fos expression, previously shown to be an indicator of programmed cell death, was also observed during this treatment. DNA fragmentation assays further confirmed that the TdT labeled cells were indeed apoptotic. Under the same incubation conditions, the cultured lenses gradually lost transparency and became completely opaque in about 30 hr. Since the vertebrate lens contains only a single layer of epithelial cells, apoptotic death of these cells activated by calcimycin quickly destroys the lens epithelium, impairs homeostasis of the underlying fiber cells and initiates development of lens opacification. PMID- 7556475 TI - Development of the blood-retinal barrier in vitro: formation of tight junctions as revealed by occludin and ZO-1 correlates with the barrier function of chick retinal pigment epithelial cells. AB - To elucidate the molecular mechanisms of the blood-retinal barrier (BRB), we examined chick retinal tissues histochemically using antibodies against tight junction proteins such as ZO-1, 7H6 antigen, and occludin. Retinal pigment epithelial (RPE) cells in situ in chickens and late chick embryos expressed all of the tight junctional proteins examined, showing that tight junctions seal the cell borders of chick RPE cells in vivo. On the other hand, RPE cells isolated from late chick embryos and transferred in vitro did not express occludin, ZO-1 and 7H6 antigen. The effects of differentiation-inducing agents, such as retinoic acid, dexamethasone and dimethyl sulfoxide (DMSO) were tested. Only DMSO induced an increase in transepithelial electrical resistance (TER) in a time-dependent manner. Under supplementation with DMSO, immunofluorescently demonstrable occludin and ZO-1 were induced progressively at cell borders in parallel with the increase in TER that occurred with decreases in inulin and dextran permeability. Electron microscopically tight junction-like junctional apparatus were induced in RPE cells. These results indicated that tight junctions of RPE cells play an important role in the formation of the BRB. PMID- 7556477 TI - Precipitation of crystallins from young rat lens by endogenous calpain. AB - The purpose of these experiments was to study the mechanism for precipitation of lens crystallins in cataract. An in vitro model was developed to activate the endogenous protease calpain II in the soluble proteins from young rat lens by addition of calcium in the presence of 120 mM KCl. Light-scattering, insoluble proteins were produced approximately 4-6 days after calpain II activation. Results showed that proteolysis was caused by activation of lens calpain II, proteolysis preceded precipitation by several days, and alpha-crystallin acted as a molecular chaperone against precipitation of crystallins caused by proteolysis. These data supported our hypothesis that calpain-induced proteolysis of the N terminal arms of beta-crystallin polypeptides leads to a loss of normal oligomerization of beta-crystallin polypeptides and formation of abnormal insoluble aggregates, possibly stabilized by hydrophobic interactions. PMID- 7556476 TI - Acetylcholine modulation of the short-circuit current across the rabbit lens. AB - Rabbit lenses were bathed within a bicameral Ussing-type chamber under short circuit conditions. In this situation the short-circuit current (Isc) reflects, across the anterior aspect, the presence of anteriorly facing K+ conductance(s) plus the Na(+)-K+ pump current. Across the posterior surface the Isc is primarily carried by the movement of Na+ from the posterior bathing solution to the lens. Addition of acetylcholine (ACh) to the posterior hemichamber did not affect the translens electrical parameters; but, its introduction to the anterior bath at 1 microM immediately reduced the Isc from 8.91 +/- 1.47 to 5.84 +/- 1.28 microA cm 2 and increased the translens resistance from 1.50 +/- 0.08 to 1.59 +/- 0.09 K omega cm2 (+/- S.E.S; P < 0.05 as paired values, n = 25 lenses). The suppressed Isc gradually recovered and reached 75% of the control value 5 min after the introduction of the neurotransmitter. In six cases the recovery was nearly complete (> or = 95% of control) within this time. The preaddition of 0.1 microM atropine prevented an effect by 1 microM ACh. When atropine was added within 1 min of ACh, the suppressed Isc immediately recovered. The ACh-elicited Isc suppression was averted in lenses pre-exposed to either K+ channel blockers (quinidine or barium) or to the endoplasmic reticular Ca(2+)-ATPase inhibitor thapsigargin (Tg: 0.1 microM), which in itself produced Isc inhibitions similar to those seen with ACh under control conditions. Similarly comparable were the ACh-evoked Isc inhibitions garnered upon introduction of the agonist to lenses bathed in the absence of extracellular Ca2+. In these cases, however, the Isc recovered fully within 2-3 min. This condition also revealed that the anterior removal of medium Ca2+ increased the Isc by about 50%, a completely reversible phenomenon; Ca2+ restoration in the presence of the Ca2+ channel blocker, nifedipine (10 microM), blunted markedly the reversal to the control Isc. Overall, these results suggest that ACh receptor activation induces the release of intracellularly stored Ca2+, which in turn leads to the temporary deactivation of a K+ conductance(s); in addition, secondary Ca2+ inflow may further extend the observed inhibition. During this study, the Isc of about 30% of the lenses used spontaneously oscillated (common duration of 30 min, with a mean peak frequency of 0.76 +/- 0.32 cycle min-1 and mean amplitude of 4.07 +/- 2.65 microA cm-2; +/- S.D.S, n = 24). Experiments attempted to determine the sensitivity of the oscillatory activity to ACh. Tg, nifedipine, and the phorbol ester PMA.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556478 TI - Exploring the role of histidines in the catalytic activity of duck delta crystallins using site-directed mutagenesis. AB - The duck delta 2-crystallin gene encodes an enzymatically-active argininosuccinate lyase while the delta 1-crystallin gene product, although 94% identical, is enzymatically inactive. Four histidine residues in the duck delta 2 crystallin. His91, His110, His162 and His178, were converted to asparagine residues in an effort to define the role of histidines in the catalytic process of this enzyme-crystallin and to explain the lack of enzyme activity in the delta 1-crystallin protein. The recombinant mutant proteins were expressed in E. coli and purified to homogeneity for analysis. These four residues were chosen because they fall within highly conserved regions of argininosuccinate lyases from several species. This analysis revealed that change of His91 or His162 for asparagine resulted in complete loss of activity. The His110 enzyme had a reduced Vmax and the His178 enzyme was near normal in its kinetic properties. These data confirm the roles of histidine in the catalytic process of this enzyme-crystallin and suggest that the change of His91 to Gln91 observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins. PMID- 7556479 TI - Effect of germanium-132 on galactose cataracts and glycation in rats. AB - Germanium compounds have been shown to be effective in preventing the formation of advanced glycation end-products and for reversible solubilization of glycated proteins. As protein glycation has been proposed to play a role in lens opacification, we initiated studies to evaluate the effects of 2-carboxyethyl germanium sesquioxide (germanium compound 132 or Ge-132) on galactose-induced cataractogenesis. For this study young Sprague-Dawley rats were fed a 50% galactose diet. One group of rats received topical saline and another group was administered Ge-132 in saline four times a day. The lenses were periodically examined with an ophthalmoscope and at desired intervals processed for light and scanning electron microscopy. Our observations, beginning at 3 days and continuing to 21 days of galactose feeding, exhibited the characteristic galactose-induced morphological alterations, which include the formation of vacuoles, cysts, membrane disruption and swelling of fibers and epithelial cells as well as disorganization of the bow in lenses of rats in both groups. However, in the majority of rats administered Ge-132 these alterations were delayed as compared to the lenses of rats administered saline. Our findings show that, although the initiation, progression and pattern of lens opacification in rats receiving saline and Ge-132 were similar, in the majority of lenses the progression and establishment of mature cataracts in the Ge-132 group of rats were delayed. Analysis of the water-soluble and water-insoluble lens-protein fractions for glycated proteins showed increased levels of the Amadori products and advanced glycation related fluorescent products in galactosemic rats treated with saline eye drops. In rats receiving the topical Ge-132 treatment the levels of these glycation products were substantially reduced to levels lower than control values. Prevention of glycation seems to be a mechanism by which cataract progression is delayed. PMID- 7556480 TI - Thermal control of rod outer segment length and shedding in a fish, Fundulus zebrinus. AB - The effects of temperature on rod outer segment (ROS) length and membrane shedding were studied in a cyprinodont fish, Fundulus zebrinus. After 30 days in 14L/10D cyclic light and 17 degrees C, ROS length averaged 41.2 microns. Fish were then exposed to 7, 17 or 27 degrees C for 10 and 25 days before being sampled 5 hr before and 1-4 hr after light onset. In 7 degrees C ROS shortened to 83.5% of initial controls within 10 days, then only 4.1% further, to 79.4% by day 25 (34.4, 32.7 microns). ROS length did not change significantly in fish remaining at 17 degrees C (39.7 and 40.7 microns at day 10 and 25) or in fish moved to 27 degrees C (41.7 and 41.6 microns). Phagosomes were most numerous in 7 degrees C and least numerous in 17 degrees C, but varied in overall size among the largest phagosomes being more common after light onset. After light onset at day 25, the estimated volume per phagosome was 1.14, 4.73 and 5.75 microns 3 in 7, 17 and 27 degrees C. Total phagosome volume per 100 microns RPE at 27 degrees C was generally double that at 17 degrees C. Apparently, in F. zebrinus, the number of disks shed from ROS is adjusted during thermal acclimation to stabilize ROS length. PMID- 7556481 TI - Sex differences in chick eye growth and experimental myopia. AB - Stimulated by reported sex differences in eye size and refraction in humans, we compared refractions and ocular size measurements of male and female chicks. Chicks were reared under a 12-hr light/dark cycle with one eye open and the other occluded by a goggle or lid suture, each well-established means of inducing ipsilateral myopia. While the open eyes of the two sexes emmetropized equally well, both ultrasound and caliper measurements demonstrated that on average males have larger eyes. Although statistically significant for defined populations, the sex differences in open eyes were to small to be diagnostic for individual birds. The visually-deprived eyes of male chicks were more reactive than those of females, especially following deprivation by a goggle. Consistently, the anterior chamber of visually-deprived eyes of males was deeper than those of the females regardless of the means of impairing vision. With a goggle, males also developed more myopia and a deeper vitreous chamber than females similarly occluded. When goggles were removed, rapid recovery occurred in 2 weeks irrespectively of sex. PMID- 7556482 TI - alpha-Crystallin polypeptides in developing chicken lens cells. AB - We provide evidence that the different cells that form the chicken lens have isoelectric variants of alpha-crystallins at early and late developmental stages. We separated the alpha A and alpha B-crystallin subclasses by sodium dodecylsulphate polyacrylamide gel electrophoresis and then further resolved each by isoelectric focusing and assays with specific anti alpha-crystallin antibodies. We found that the annular pad, cortical and nuclear fibers, as well as the epithelial cells, contain alpha A and alpha B native chains and their respective isoelectric variants. These results on adult and embryonic lenses obtained a short time after the onset of alpha-crystallin expression suggest that lens cells, having different phenotypes, are able to produce post-translational modifications of the alpha A and alpha B chains as a part of their developmental program. PMID- 7556483 TI - Reorganization of actin microfilaments and microtubules in regenerating retinal pigment epithelium. AB - Retinal pigment epithelium (RPE) regenerating after experimental damage in rabbits exhibits major changes in cell shape, polarity and junctions--features that depend on the cytoskeleton. This report correlates these changes with the redistribution of actin microfilaments and microtubules, using electron microscopy and confocal laser scanning microscopy. We compare immature cells with the more mature cells that form the new epithelial monolayer. Two populations of immature RPE cells are interspersed at the edge of the regenerating RPE sheet. One population of immature cells makes few junctions with their neighbors or the basement membrane. They form pseudopodia and exhibit a prominent network of actin microfilaments beneath the plasma membrane. These cells are probably motile and advance the epithelial sheet. Another population of immature cells contains numerous stress fibers that insert into large basement membrane attachments. The cells make focal adhesions with their neighbors, rather than the junctional complexes characteristic of mature RPE cells. These cells are probably not motile and mature into the cells forming the new monolayer--cuboidal cells with numerous basal folds and apical villi and a complete belt of intercellular junctions. Stress fibers are lost as the circumferential bundle associated with the zonula adherens re-forms. Microtubules, which form prominent longitudinal bundles running through the processes of immature cells, take on the meshwork organization characteristic of mature RPE as the immature cells differentiate. PMID- 7556484 TI - Influence of diltiazem on the ocular irritative response to nitrogen mustard. AB - Application of nitrogen mustard to the eye of rabbits causes an anterior segment irritation and a biphasic elevation of intraocular pressure. This intraocular pressure response is composed of an initial peak, produced by neuropeptides released by excited sensory nerves, and a second, slower rise due to prostaglandins. We studied the effect of diltiazem, a calcium antagonist that selectively blocks chemical excitation of sensory nerves, on the inflammatory response to nitrogen mustard. In adult rabbits, intraocular pressure was determined by pneumatonometry; pupil diameter and palpebral opening were measured with a ruler while conjunctival vasodilation, edema and secretion were scored in subjective units (0-8). Aqueous humor protein content was analysed at the end of the experiment. Bilateral application of 1% nitrogen mustard evoked within the first 6 hr an intraocular pressure elevation followed by ocular hypotony, miosis, palpebral closure, conjunctival vasodilation, edema and an elevation of aqueous proteins. Topical application of 10 mM diltiazem, prior to administration of nitrogen mustard elicited by itself a transient, small intraocular pressure increase and reduced significantly the acute intraocular pressure elevation and conjunctival vasodilation evoked by the irritant; delayed conjunctival edema and palpebral closure were also attenuated by 10 mM diltiazem. The decrease of miotic response and of aqueous humor protein content was not significant. Diltiazem at 2.8 mM was effective only in reducing significantly conjunctival edema and vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556485 TI - Effect of pH on the ocular distribution of a topical carbonic anhydrase inhibitor. AB - Ampholyte carbonic anhydrase inhibitors (including MK-927) have previously been shown to elicit greater intraocular pressure reduction when applied in ionized form at moderately acidic or alkaline pH compared to application of un-ionized drug at neutral pH. Since ionized solutions should be far more membrane impermeant than un-ionized solutions, we attempted to solve this apparent paradox. We studied the distribution at 0.5-18 hr of MK-927 in eye tissues and fluid after topical instillation of one drop of a 0.5% solution at pH 4.9, 7.0 and 9.1. Measured drug concentrations at 30 min in corneal epithelium and stroma are approximately 4.5-fold greater after acidic or alkaline application than after neutral application, with the highest levels being found in corneal epithelium. The same pattern is seen in ciliary process, the site of aqueous humor production. Here drug concentration is approximately 60% of that in cornea at 30 min. Free drug concentrations in ciliary process were used to compute the time course of maximal carbonic anhydrase inhibition for the three modes of application. Drug concentration in sclera, uvea and aqueous humor at all times are all low by comparison, suggesting drug movement is from cornea to ciliary process via the corneo-scleral junction. The k(in) for proton movement across the corneal epithelium was measured (k(in) = 12.4 hr-1) from which a permeability coefficient (P = 2.7 x 10(-2) cm sec-1) was computed. Separate analysis was made of the pH status of the cornea 30 min and 1 hr following instillation of 1 drop 2% acidic (pH 4.9) and alkaline (pH 9.1) MK-927, with sodium sulfadiazine and pilocarpine.HCl as pH controls. Stromal bicarbonate at 30 min was approximately halved after acidic drops and doubled after alkaline drops consistent with pH decrease or increase of nearly 0.3 U. The results are in accord with classical schemes for amine permeation of the cornea at acidic pH when consideration is given to movement of acid equivalents and corneal pH. Thus drug inside the eye is in relatively (> 95%) lipophilic form except that trapped in the cornea shortly after acidic or alkaline applications. PMID- 7556486 TI - Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye. AB - Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6 8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow. PMID- 7556488 TI - Computer-generated model of the quaternary structure of alpha crystallin in the lens. PMID- 7556487 TI - Effects of Muller cell disruption on mouse photoreceptor cell development. AB - Muller cells have been proposed to play an important role in photoreceptor cell development during the final stages of retinal maturation. The effect of disrupting Muller cells during mouse retinal development was investigated using the specific glial cell toxin, DL-alpha-aminoadipic acid (AAA). By giving multiple systemic injections over several days, impairment of Muller cell function was maintained during the period of photoreceptor migration and differentiation. Following three consecutive days of AAA treatment [commencing on post-natal (P) day 3, 5, 7 or 9, and examined at P8-P14], clumps of photoreceptor nuclei were displaced through the inner segments, lying immediately beneath the retinal pigment epithelium (RPE). Apart from the scalloped appearance of the outer retina, the overall lamination pattern of the retina was relatively well preserved. Even when AAA treatment commenced as early as P3, several days prior to the formation of the outer nuclear layer, the majority of photoreceptors migrated to their correct position and formed inner and outer segments. Therefore, the signals for photoreceptor migration are either provided by the Muller cells prior to P3, or, alternatively, are derived from different intrinsic or extrinsic cues. Disruption of Muller cell function was evidenced by decreased glutamine synthetase activity as well as by increased glial fibrillary acidic protein (GFAP) and decreased cellular retinaldehyde-binding protein (CRALBP) immunoreactivity. Immunocytochemistry with an antibody to CD44, which labels the microvilli of Muller cells at the outer limiting membrane, coupled with electron microscopic analysis, demonstrated that the zonulae adherentes between Muller cells and photoreceptors were either irregular or absent in areas adjacent to displaced clumps of photoreceptors. Thus AAA treatment of early post-natal mice results in localized disruption of the contacts between Muller cells and photoreceptors. These pathologic changes persist into adulthood since at P28, while short stretches of photoreceptors appeared relatively normal with fully developed outer segments, periodic clumps of displaced photoreceptor nuclei were still present adjacent to the RPE. In conclusion, Muller cell processes at the outer limiting membrane appear to play a critical role in providing a barrier to aberrant photoreceptor migration into the subretinal space. PMID- 7556489 TI - Laudatio to Sohan Singh Hayreh recipient of the Von Sallmann Prize. New Delhi, India, November 1994. PMID- 7556490 TI - The 1994 Von Sallman Lecture. The optic nerve head circulation in health and disease. AB - This is a brief overview of multifaceted anatomical, experimental and clinical studies conducted by the author since 1955 on the optic nerve head circulation in health and disease. Conclusions, based on the accumulated information provided by these studies, are summarized. The studies on the pattern of blood supply of the optic nerve head have shown that: (a) its main source of blood supply is the posterior ciliary artery circulation, with retinal circulation supplying only the surface nerve fiber layer, (b) there is marked interindividual variation in the blood supply pattern, and (c) the blood supply in the optic nerve head has a sectorial distribution. The various factors which produce interindividual variation in the blood supply of the optic nerve head are discussed, particularly those in the posterior ciliary artery circulation; this is because all available evidence indicates that it is derangement in the posterior ciliary circulation in the optic nerve head that is primarily responsible for the common ischemic disorders of the optic nerve head, e.g. anterior ischemic optic neuropathy and glaucomatous optic neuropathy. Factors that may derange the blood flow in the optic nerve head include defective autoregulation of blood flow in it, vascular changes in its feeding arteries, hematologic abnormalities, systemic arterial hypertension and hypotension, and intraocular pressure; their roles are discussed. For better understanding and management of optic nerve head ischemic disorders, there is an urgent need for an accurate clinical method of assessment of blood flow in the posterior ciliary circulation in optic nerve head, since no satisfactory method is currently available. Redness or pallor of the optic disk on ophthalmoscopy is not a true guide to the optic nerve head vascularity as it gives no information about the state of the posterior ciliary circulation. Fluorescein fundus angiography, though far superior to the optic disk color for evaluation of optic nerve head vascularity, has a number of limitations. All these topics and various controversies about them are discussed briefly. PMID- 7556491 TI - Oxygen consumption in the rat outer and inner retina: light- and pharmacologically-induced inhibition. AB - Biochemical, physiological and histological data have established that 55-65% of retinal mitochondria are located in the photoreceptor inner segments and suggested that photoreceptors have at least a two-fold greater oxygen consumption (QO2) than the remaining inner retina. QO2 in isolated whole rat retina (QWR), outer retina (QOR) and inner retina (QIR) was measured during dark and rod saturating light adaptation. The effects of function-specific chemical agents on QWR, QOR and QIR during dark and light adaptation were determined. In addition, the oxidation-reduction (redox) potential of cytochrome a3 of whole, outer and inner retina was measured during dark and light adaptation. During dark adaptation, the mean QWR was 1.62 mumol O2 (mg dry wt)-1 hr-1 and whole retinal level of reduced cytochrome a3 was 19%. They decreased by 24% and 37% during light adaptation, respectively. To determine QOR and QIR during dark and light adaptation, the outer retina was pharmacologically-isolated from inner retina using L-2-amino-4-phosphonobutyric acid plus kynurenic acid (APB/Kyn). Experiments in the presence or absence of APB/Kyn revealed that: (i) QOR, but not QIR, of the dark-adapted retina was decreased 37% during light adaptation, (ii) the outer and inner retina consumed 65% and 35% of the QWR during dark adaptation, respectively, and 54% and 46% of the QWR during light adaptation, respectively, (iii) the level of reduced retinal cytochrome a3 in the outer, but not inner, retina was decreased 34% during light adaptation, (iv) during light adaptation, the rate of QO2 was equal in the outer and inner retina, and (v) the effects of APB/Kyn were reversible. These results establish that the mean rate of QIR and retinal cytochrome a3 are unchanged during dark or light adaptation. In addition, they suggest that QOR:QIR in the rat may be modeled using a 65%:35% model during DA and a 55%:45% model during LA. All the function-specific agents- IBMX, lead, diltiazem, ouabain, CO2+ plus Mg2+ and verapamil--significantly decreased QWR during dark and light adaptation. A more detailed analysis revealed that IBMX and lead each selectively reduced (> or = 90%) QOR during dark adaptation whereas CO2+ plus Mg2+ and verapamil each selectively reduced (> or = 93%) QIR during dark and light adaptation. These results are consistent with the known pharmacological sites and mechanisms of these agents. Additional experiments determined that the IBMX- and lead-induced inhibition of QOR during dark adaptation resulted, either wholly or partially, from the influx of extracellular Ca2+. During dark adaptation in Ca(2+)-free medium: (i) QWR and QOR increased while QIR was unchanged, (ii) QOR was not decreased in the presence of IBMX and (iii) QOR was only partially decreased in the presence of lead.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556492 TI - Mechanisms of the biphasic effects of peroxides on the retinal vasculature of newborn and adult pigs. AB - We tested whether the ontogenic differences in the constrictor effects of peroxides on the retinal vasculature were modulated by dilator cyclo-oxygenase products. Retinal arteriole (100-200 microns) vasomotor response to H2O2, t-butyl hydroperoxide, and cumene hydroperoxide were studied in isolated eyecup preparations using video camera monitoring of vessel diameter. A time- and dose dependent biphasic retinal vasomotor response to all peroxides was observed on tissues of newborn and adult pigs. A rapid vasoconstriction (first 2 min) was followed by a relaxation which was greater in the adult than in the newborn tissues. The constrictor as well as the dilator response to peroxides and the observed increase in prostanoids were blocked by the cyclo-oxygenase inhibitor indomethacin. The peroxide-induced relaxation was inhibited or markedly attenuated by the prostaglandin I2 synthase blockers, trans-2-phenyl cyclopropylamine and minoxidil on tissues of newborn and adult animals. These agents also prevented the increase of the prostaglandin I2 receptor-coupled second messenger, cyclic 3',5'-adenosine monophosphate. Our data indicate that prostaglandin I2 plays a major role in counteracting the initial constrictor effects of peroxides in the retinal vasculature, and that the reversal of this constriction is greater in the adult than the newborn. These findings suggest that reduced reversal of vasoconstriction by the dilator prostaglandin I2 during an oxidative stress in the newborn may facilitate vasoconstriction by the dilator prostaglandin I2 during an oxidative stress in the newborn may facilitate neovascularization in retinopathy of prematurity. PMID- 7556495 TI - The repair response following Nd:YAG laser sclerostomy ab interno in rabbits. AB - This study was undertaken to examine ultrastructurally the course of tissue regeneration after thermally-induced laser sclerostomy and compare it with the post-operative clinical events in a rabbit model. Applying a continuous wave neodymium:YAG (Nd:YAG) laser, two sclerostomies were created ab interno on one eye in each of ten pigmented rabbits using a 200-microns-diameter quartz optical fiber; the unoperated fellow-eyes served as controls. Intraocular pressure (IOP) was measured daily before and after surgery. Animals were observed post operatively for up to 16 days. Well-defined filtering blebs and a low complication rate demonstrated the success of the procedure. IOP was significantly lowered during the entire course of the observation period but after the fifth day, the conjunctival blebs had disappeared. The morphology of the changes in the sclerostomy fistulas were analysed at 2-day intervals by light and electron microscopy. Immediately after surgery, the canal was ensheathed by an approximately 100-microns-thick layer of coagulated collagenous tissue in which two zones could be distinguished according to the intensity of damage. Within 5 days, the inner and outer canal openings were invaded by macrophages and fibroblasts originating from the iris root and episclera, respectively. The former cells were engaged in the phagocytosis of disintegrated collagen adjacent to the lumen. More distally located fibrils which had incurred less severe damage, had retained their fibrillar structure but had lost banding periodicity. After thermal stress had faded, they appeared to undergo a process of repolymerisation. By day 10, the lumen had become occluded by a loose meshwork of phagocytes, fibroblasts and proliferating capillaries. These new vessels and the loose nature of the canal-occluding framework and of the surrounding regenerating collagenous tissue could have further permitted percolation and transport of aqueous humor, since IOP remained low, despite the disappearance of filtering blebs. Although the time course of repair is more rapid in rabbits than in humans, the data gleaned nonetheless yield valid information respecting the sequence of events following thermally-induced scleral fistula. PMID- 7556493 TI - Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses. AB - In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22,000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide. PMID- 7556494 TI - Calcium-mediated disintegrative globulization of isolated ocular lens fibers mimics cataractogenesis. AB - Single viable fiber cells have been isolated from the cortex of rat ocular lens by proteolytic digestion of the intact lens in calcium-free media. In isomolar sucrose, the isolated cells maintain their fiber-shaped morphology and exclude trypan blue. The surface morphology of the isolated fiber cells appears to be largely unaffected by the isolation procedure. The concentrations of adenine nucleotides, GSH, GSSG and the rate of glycolysis in the isolated fiber cells were comparable to those in the cortex. Upon perfusion of the tissue chamber with Ringer's solution, the fiber cells undergo a series of transformations, beginning with cell swelling, periodic blebbing along the longitudinal cellular axis, and eventual disintegration of the fiber into a number of resealed globules or round cells which resemble light-scattering areas in human cortical and supranuclear cataract. This disintegrative globulization of the fiber cells appears to be mediated by calcium influx, as it was prevented or delayed by a reduction in extracellular calcium concentration, verapamil or lanthanum. Since disturbances in calcium homeostasis are associated with various forms of cataract, such Ca(2+) mediated disintegrative globulization of the fiber cells may be responsible for the formation of light scattering centers during cataractogenesis. PMID- 7556496 TI - Descemet's membrane in the iridocorneal-endothelial syndrome: morphology and composition. AB - The iridocorneal-endothelial syndrome is a disease of the ocular anterior segment characterized by corneal failure, glaucoma and iris destruction. Specular photomicroscopical and histological studies suggest the disorder is caused by a population of abnormal corneal endothelial cells. In other corneal endotheliopathies Descemet's membrane, the basement membrane underlying the endothelial cells, is disfigured by the presence of an abnormal region of extracellular matrix termed a posterior collagenous layer, which is laid down by the diseased endothelial cells. In this study we sought to establish the typical morphology and composition of Descemet's membrane in the iridocorneal-endothelial syndrome. Ultrastructural examination of Descemet's membrane in 27 keratoplasty specimens identified three morphologic patterns. In the majority there was a posterior collagenous layer which in all cases consisted of an anterior layer of wide-spaced collagen and a posterior layer of microfibrils embedded in an amorphous matrix. In four specimens which did not possess a posterior collagenous layer the anterior banded zone of Descemet's membrane was absent. In five corneas Descemet's membrane was normal. The composition of the posterior collagenous layer was examined by immunoelectron microscopy (five corneas) and histochemistry (six corneas). Collagen Types I, III, V, VI and VIII, fibronectin, tenascin and oxytalan were microfibrillar components, collagen Type VIII formed wide-spaced collagen whilst laminin was present in the amorphous matrix. The stereotyped derangements of structure and composition identified in the endothelial basement membrane may significantly influence the pathobiology of this disorder. PMID- 7556497 TI - Three-dimensional membrane crystals in amphibian cone outer segments: 2. Crystal type associated with the saddle point regions of cone disks. AB - In light-adapted, perfusion fixed retinas of the Congo eel salamander, Amphiuma, we have observed distinctive 3-D crystalline domains within the axial array of cone outer segment disks. These crystalline domains, each involving 2-12 disks, have been observed in the distal half of cone outer segment, and are associated with saddle point regions and immediately adjacent segments of the cone disk perimeter. In longitudinal sections, the crystals typically display an axially oriented array of cytoplasmic filaments with lateral spacings in the range of 12 13 nm. The width of the intradiskal compartment is expanded to 8-10 nm within the crystal, and approximates the width of the cytoplasmic compartment. In some sections, the cytoplasmic filaments are axially aligned with intradiskal filaments of similar length and width. In transverse sections, the projected lattice appears to be approximately rectangular, with unit cell dimensions of approximately 12 nm x 12.5 nm. In shape, orientation, dimension, location, projection symmetry and associated membrane spacing relationships, the cone outer segment crystal filaments share a strong resemblance with lattice filaments located along the perimeters and preincisures of rod disks. These similarities suggest that the cone crystal filaments may be related to the rim protein of rod disks. Lastly, the preferential association of these crystals with saddle point regions indirectly supports the hypothesis that reductions in COS disk area with apical displacement are accomplished by resorption of disk membrane components through the saddle points. PMID- 7556498 TI - Ion, water and neutral solute transport in Xenopus oocytes expressing frog lens MIP. AB - We have expressed frog (Rana pipiens) lens major intrinsic protein (MIP) in Xenopus oocytes and observed its effect on ion conductance, water permeability and neutral solute transport. SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels. Immunolocalization indicated the expressed MIP migrated to the plasma membrane. MIP had no effect on the slope of oocyte I-V relations in the range -50 to +10 mV, although the averaged I-V curve was shifted 10 mV positive to control. MIP increased oocyte water permeability by a factor of 1.9 +/- 0.2, whereas the permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione or urea was unchanged. Glycerol permeability was enhanced in oocytes expressing MIP. In contrast to control oocytes, 3H-glycerol radioactivity accumulation did not follow first order kinetics. Radioactivity continued to accumulate even after 19 h of uptake and went beyond equilibrium with the bath. The time course of MIP mediated glycerol uptake was modeled assuming metabolic trapping with good results. Based on this model, MIP increased oocyte glycerol permeability by a factor of 2.7. PMID- 7556500 TI - Chronic ocular hypertension following episcleral venous occlusion in rats. PMID- 7556501 TI - Proceedings of the 2nd International Symposium on the Neurobiology and Neuroendocrinology of Aging. Bregenz, Austria, July 24-28, 1994. PMID- 7556499 TI - Ultrastructural features of retinal dystrophy in mutant vitiligo mice. AB - Ultrastructural features of the retinal pigment epithelium and photoreceptor cells were studied in vitiligo (C57BL/6 mivit/mivit) mice. Eyes from 12-day- to 56-week-old animals were analysed. Abnormal photoreceptors were seen in 12-day old mice. By 3 weeks malformed outer segments were evident in the posterior and equatorial retina, but normal photoreceptors were present in the periphery. By 28 weeks, a marked gradient in cell loss was evident, with a progressive increase in cell viability along the posterior-peripheral axis. Viable intact photoreceptors were still present in the peripheral retina of 56-week-old mice. Melanosome content varied between adjacent pigment epithelium cells in both the posterior and peripheral retina. In the choroid, however, a steep posterior-peripheral gradient in melanosome content was evident with highest pigmentation in the periphery. In the optic nerve head region abnormal development of photoreceptors was correlated with proliferation of abnormal pigment epithelium cells. Accumulation of rod outer segment debris in the posterior and peripheral subretinal space preceded photoreceptor cell death. Short pigment epithelial microvilli without proper attachment to photoreceptors are suggestive of alterations in pigment-epithelium-photoreceptor interaction, which might affect photoreceptor differentiation and phagocytosis of rod outer segments by pigment epithelium cells. PMID- 7556503 TI - The pineal gland and melatonin in relation to aging: a summary of the theories and of the data. AB - Within recent years, many investigators have implicated the pineal gland and melatonin in the processes of both aging and age-related diseases. These theories stem from the importance of melatonin in a number of biological functions and the fact that melatonin production in the organism is gradually lost throughout life, such that in very old individuals of any species the circadian melatonin rhythm is bearly discernible. In most species, from algae to humans, where it has been investigated, melatonin has been shown to exhibit a strong circadian rhythm in production and secretion, with high levels of the indole always being associated with the dark period of the light:dark cycle. One theory states that when the melatonin rhythm deteriorates during aging, other circadian rhythms are likewise weakened and rhythms become dysynchronized. This dysynchronization is believed to contribute significantly to aging and to render animals more susceptible to age related diseases. Another theory assumes that the waning melatonin cycle provides an important switch for genetically programmed aging at the cellular level; furthermore, because all cells in the organism are exposed to the same gradually dampening melatonin signal throughout life, all cells age more or less at the same rate. In this theory, it is presumed to be the duration of the nocturnally elevated melatonin (which, like the amplitude, is reduced during aging), which, when coupled to a time-gating signal, is consequential in determining the rate of aging. Another compelling argument that the reduction in melatonin with age may be contributory to aging and the onset of age-related diseases is based on the recent observation that melatonin is the most potent hydroxyl radical scavenger thus far discovered. A prominent theory of aging attributes the rate of aging to accumulated free radical damage. Inasmuch as melatonin can markedly protect macromolecules, especially DNA, against free radical attack, it could, indeed, be a major factor in determining the rate at which organisms age. Besides its ability to directly scavenge the highly toxic hydroxyl radical, melatonin also promotes the activity of the antioxidative enzyme glutathione peroxidase, thereby further reducing oxidative damage. These actions may be manifested more obviously in the central nervous system, which is highly susceptible to damage by oxygen based radicals and, because of its inability to regenerate and its high vulnerability to oxidative attack, its deterioration may be especially important in aging.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556502 TI - Education, homeostasis, and longevity. AB - The effects of education in lengthening life expectancy and reducing disability and disease in old age are examined for neural and hormonal correlates. Hormones and local factors influencing growth, in regulating neural cell death, proliferation, and differentiation, support the view of a "brain reserve capacity" built up during early learning years and capable of affording a protective action (increased threshold to damage) against the losses of normal and abnormal aging. Observation from studies on the effects of estrogen, dexamethasone, and a number of local factors on growth and differentiation of human neuroblastoma cells and of glial cells are supportive of a role for these factors in maintenance of homeostasis at an advanced age. Continuing education and learning is proposed as an effective means to promote longevity and improve life in old age. PMID- 7556504 TI - Oxygen free radicals, melatonin, and aging. AB - The recognized peripheral and central biochemical, neuroendocrinological, behavioral, pharmacological, and clinical actions of melatonin are involved at different levels of an overall concept of the pathophysiology of aging. This conceptual approach combines the biochemistry of oxygen free radicals, the social behavior of the individual and the resulting cellular alterations. This has allowed us to propose antioxidant properties of melatonin that we have experimentally demonstrated. PMID- 7556506 TI - Estrogen-induced hypothalamic beta-endorphin neuron loss: a possible model of hypothalamic aging. AB - Over the course of normal aging, all female mammals with regular cycles display an irreversible arrest of cyclicity at mid-life. Males, in contrast, exhibit gametogenesis until death. Although it is widely accepted that exposure to estradiol throughout life contributes to reproductive aging, a unified hypothesis of the role of estradiol in reproductive senescence has yet to emerge. Recent evidence derived from a rodent model of chronic estradiol-mediated accelerated reproductive senescence now suggests such a hypothesis. It has been shown that chronic estradiol exposure results in the destruction of greater than 60% of all beta-endorphin neurons in the arcuate nucleus while leaving other neuronal populations spared. This loss of opioid neurons is prevented by treatment with antioxidants indicating that it results from estradiol-induced formation of free radicals. Furthermore, we have shown that this beta-endorphin cell loss is followed by a compensatory upregulation of mu opioid receptors in the vicinity of LHRH cell bodies. The increment in mu opioid receptors presumably renders the opioid target cells supersensitive to either residual beta-endorphin or other endogenous mu ligands, such as met-enkephalin, thus resulting in chronic opioid suppression of the pattern of LHRH release, and subsequently that of LH. Indeed, prevention of the neuroendocrine effects of estradiol by antioxidant treatment also prevents the cascade of neuroendocrine aberrations resulting in anovulatory acyclicity. The loss of beta-endorphin neurons along with the paradoxical opioid supersensitivity which ensues, provides a unifying framework in which to interpret the diverse features that characterize the reproductively senescent female. PMID- 7556507 TI - Mitochondrial DNA mutations associated with aging and degenerative diseases. AB - Accumulating evidence has emphasized the role of genetic factors in the development of aging and degenerative diseases. Mitochondrial DNA (mtDNA), that codes for protein subunits essential for the maintenance of mitochondrial ATP synthesis, acquires mutations at a much higher rate than that of nuclear DNA. Recent studies have shown that somatically acquired mutations such as deletions in mtDNA are caused by oxygen damage during the life of an individual. Accumulation of these somatic mutations in postmitotic neuromuscular cells causes bioenergetic deficiency leading to age-associated dysfunction of cells and organs. The base sequencing of the entire mtDNA from individuals revealed that inherited germ-line point mutations accelerate the somatic oxygen damage, and the fragmentation in mtDNA leads to phenotypic expression such as premature aging and degenerative diseases. This article reviews the concept, molecular genetics, pathology, clinical symptoms, diagnosis, and therapy of mitochondrial aging and related diseases. PMID- 7556508 TI - Dietary restriction. AB - Dietary restriction (DR) slows the rate of actuarial aging of rats and mice and in addition retards and/or delays many phenotypic characteristics of aging such as the age-associated deterioration of physiological systems and the occurrence and progression of age-associated disease. These antiaging actions result from a reduction of energy intake by the animal but are not due to a decrease in metabolic rate per unit of lean body mass. However, there is evidence that altered characteristics of fuel use (but not the intensity of fuel use) may underlie the antiaging action of DR. There is also evidence that DR has a general protective action in regard to damage caused by acute stressors; possibly a similar protective action in regard to damage caused by prolonged, low intensity aging processes plays a major role in the antiaging action of DR. PMID- 7556505 TI - Individual differences in hypothalamic-pituitary-adrenal activity in later life and hippocampal aging. AB - Variation in magnitude of cognitive decline in later life is a central feature of human aging. The more severe forms of dementias, such as Alzheimer's disease, clearly define one end of the spectrum. However, among those showing no obvious signs of clinical dementia there are considerable individual differences. Thus, although evidence for learning, memory, and language loss appears in some individuals as early as 50-55 years of age, many people continue to function alertly well into their 90s. These individuals exemplify what Rowe and Kahn (1987) have termed "successful" aging. The wide variability in CNS aging, often a nuisance factor in studies, are becoming a major focus for brain aging research (e.g., Gage et al., 1984;Gallager and Pelleymounter, 1988; Aitken and Meaney, 1990; Issa et al., 1990). Our studies over the past few years have added support to the idea that individual differences in hypothalamic-pituitary-adrenal (HPA) activity can account for part of the variation seen in neurological function among the elderly. In this article we discuss the evidence for the idea that adrenal glucocorticoids can compromise hippocampal function and, thus, produce cognitive impairments, as well as the potential mechanisms for these effects. PMID- 7556510 TI - Sex-specific life table aging rates in large medfly cohorts. AB - Life table aging rate (LAR) is defined as the age-specific rate of mortality change with age and corresponds to the first derivative or slope of the mortality schedule at each age. We computed the sex-specific LARs for 167 Medfly cohorts, containing a total of approximately 600,000 individuals of each sex. We found that: (a) the LAR is not constant for either sex; at the youngest ages, mortality changes by approximately 1.4-fold daily, whereas at the older ages (>30 days), mortality levels off and decreases; (b) female LAR is higher than male LAR from 0 to 12 days, at which time the rates cross over and male LAR is higher than female LAR; however, at about 30 days, both male and female LARs are less than or equal to unity; (c) mortality in each of the 167 cohorts leveled off for both sexes. The average age for this leveling off was around 23 days for females and 31 days for males; and (d) the mortality rate at the age of leveling off was approximately 1.4-fold higher in males than in females--0.13/day for females and 0.19/day for males. Implications of these findings are briefly discusses, including the use of summary measures of senescence, the arbitrariness of selecting the age of senescence onset, and test of hypotheses concerned with explanations of mortality patterns such as leveling off at older ages. PMID- 7556509 TI - Nutrition and its relationship to aging. AB - The aging process alone has no significant adverse consequences for the caloric intake and the nutritional status of healthy elderly individuals. Epidemiological data suggest that in humans, in contrast to rodents, undernutrition reduces the life span. In the Western World, malnutrition in old age has become uncommon and is, for the most part, the result of physical illness and/or of psychological and socio-economic factors, such as depressive disorders, social isolation, smoking, alcohol abuse, and poverty. Body weight shows a U- or J-shaped relationship to mortality risk with the highest survival rates found at normal to moderate overweight. However, studies that have controlled for disease already present, smoking status, serum cholesterol level, or hypertension, suggest an increased mortality risk for lower and upper extremes of body weight, only. Populations with healthy lifestyles have significantly greater life expectancy that the average normal population. Even in the very old, exercise has been shown to improve muscle strength and function. The studies suggest that nutritional intake and nutritional status in old age is multifactorial and dependent not only on appetite and availability of diverse food, but also on physical activity, body mass, education, and an involved social lifestyle. PMID- 7556511 TI - Semiconduction of proteins as an attribute of the living state: the ideas of Albert Szent-Gyorgyi revisited in light of the recent knowledge regarding oxygen free radicals. AB - Oxyradicals have been considered as harmful byproducts causing molecular damage during aging. However, evidence is accumulating to show that the actual situation is more complex: the living state implicitly involves the production of oxyradicals. (1) Blast type cells produce much less oxyradicals than the differentiated ones, and an increased production of OH radicals induce differentiation of various lines of leukemia cells; meanwhile, their superoxide dismutase expression increases to a very high extent. (2) The supramolecular organization of the cells is developed by means of "useful" crosslinking effects OH radicals. (3) Repiratory inhibition of oxyradical production (KCN intoxication, suffocation, etc.) would kill living organisms prior to the exhaustion of energy reserves. It is assumed that the continuous flux of OH radicals is a prerequisite for a electron delocalization on the proteins, which is a semiconduction of p-type, proposes already in 1941 by Albert Szent-Gyorgyi, and refuted on a "theoretical" basis. It has become clear by now that the carbon based semiconduction is possible because diamond transistors are known to exist. The recently developed atomic force microscopy offers some real possibilities for experimental testing of this assumption. This concept may lead us to new horizons in interpretation of living functions, such as the basic memory mechanisms in brain cells and their impairment during aging. PMID- 7556512 TI - The role of aging in the clinical expression of essential tremor. AB - Essential tremor is the most common form of abnormal tremor. It is a monosymptomatic disorder characterized by action tremor but no other sign of motor dysfunction. More than half of all cases are inherited through a Mendelian dominant gene. Mild essential tremor is probably the cause of tremulousness that is frequently attributed to aging. The prevalence of essential tremor increases with age, and aging appears to have an independent effect on the clinical characteristics of essential tremor. Autopsies have revealed no discernible pathology. Essential tremor probably emerges from subtle pathologic transformation of a physiologic neuronal oscillator, possibly the inferior olive. A more complete characterization of the effects of aging on motor pathways may be necessary to fully understand the natural history and pathogenesis of this disorder. PMID- 7556515 TI - Gonadotropin secretion during aging in women: review article. AB - Biological aging during the postmenopause markedly affects the neuroendocrine control of gonadotropin release. The determination of the age-related dynamics on gonadotropin secretion in postmenopausal women have proven to be a valid approach for delineating changes as a function of progressive age. As a result, major functional derangements, primarily at a hypothalamic rather than a pituitary site, have been determined as concomitants of aging in women. Furthermore, aging may impair the negative feedback sensitivity to ovarian sex steroids, and interfere with the central neurotransmitter activity governing gonadotropin secretion. The data reviewed on gonadotropin secretion in postmenopausal women support the view that the age-related processes are related to a hypothalamic rather than to a pituitary hypofunction. PMID- 7556514 TI - Changes in tissue responsiveness to hormones and neurotransmitters during aging. AB - Responsiveness to many hormones and neurotransmitters by target tissues is altered during aging. Three model systems that are representative of these changes are reviewed. These are (1) striatal dopaminergic regulation of motor function, (2) IP3 and calcium-mediated electrolyte and neurosecretion by the alpha1-adrenergic and muscarinic cholinergic systems in parotid and corpus striatum, and (3) beta adrenergic and epidermal growth factor (EGF) stimulated DNA synthesis in hepatocytes. PMID- 7556516 TI - Increased glial fibrillary acidic protein (GFAP) levels in the brains of transgenic mice expressing the bovine growth hormone (bGH) gene. AB - Transgenic mice, expressing the gene for bovine growth hormone (bGH), exhibit increased body size, reduced reproductive capacity, and high basal levels of several hormones including corticosterone. Their shortened life span may be indicative of accelerated aging. As prominent astrogliosis of the CNS accompanies aging in rodents, bGH transgenic mice were examined for astrogliosis, as quantified by an ELISA for the astrocyte-localized protein, glial fibrillary acidic protein (GFAP). Transgenic mice were produced by mating C57BL/6 x C3H F1 hybrid females with male descendants of animals produced by microinjection of fertilized eggs with phosphoenolpyruvate carboxykinase (PEPCK)/bGH-hybrid gene. Transgenic mice (approximately 3.5 and approximately 12 months of age) weighed significantly more than same age or older (approximately 20 month) controls. Most of their internal organs, including the heart, kidneys, adrenals, liver, and spleen, were also heavier. In contrast, the thymus was heavier only in the younger transgenic mice. Serum corticosterone was highest in the older transgenic mice. A small but significant increase in whole brain, cortex, and cerebellar weight, relative to controls and the older transgenic mice, was found in the younger transgenic mice. Control mice exhibited large, significant age-related increases in GFAP. Increases of 35, 70, 68, 89, 79, and 95% for cortex, cerebellum, striatum, hippocampus, midbrain, and brain stem, respectively, were found when comparing the oldest (approximately 20 months) control mice to the youngest (approximately 3.5 months). In contrast, in the olfactory bulbs and the hypothalamus there were no age-related changes in the levels of GFAP in control mice. Transgenic mice (approximately 3.5 months) had significantly elevated GFAP levels relative to the same-age controls in all brain areas examined. In some brain areas, the GFAP levels found in the younger transgenic mice were equivalent to those found in the oldest controls. No differences between controls and transgenics were found in tyrosine hydroxylase protein levels of striatum or hypothalamus. The elevated GFAP levels of transgenic mice may reflect increased neural damage due to accelerated aging processes or damage associated with high circulating levels of bGH or corticosterone. Alternatively, the increased expression of GFAP in the transgenic mice may reflect altered regulation of GFAP rather than an increase signaled by neural damage. PMID- 7556513 TI - Central auditory aging: GABA changes in the inferior colliculus. AB - Age-related hearing loss (presbycusis) is a complex state that reflects pathologic changes along the entire auditory neuraxis. Loss of speech understanding, decreased ability to localize sounds, and a decreased ability to detect and extract signals in noise are characteristic problems encountered by the elderly. Central (neural) presbycusis frequently results in a dramatic loss in speech understanding without a parallel change in pure-tone thresholds. In spite of evidence that suggests these deficits cannot be fully explained by peripheral changes alone, few studies have examined the neurochemical basis of central auditory dysfunction in aging. Age-related alterations in neural circuits involved in the processing of acoustic information could reflect changes in the synthesis, degradation, uptake, release, and receptor sensitivity of neurotransmitters, perhaps secondary to cell loss and/or progressive deafferentation. A series of studies designed to test this hypothesis has examined aging in the central auditory system of the F344 rate. Age-related changes associated with GABA neurotransmitter function in an important auditory midbrain structure, the inferior colliculus, have been investigated. These studies found: (1) decreased numbers of GABA immunoreactive neurons; (2) decreased basal levels (concentrations) of GABA; (3) decreased GABA release; (4) decreased glutamic acid decarboxylase activity; (5) decreased GABAB receptor binding; (6) decreased numbers of presynaptic terminals; and (7) subtle GABAA receptor binding changes. Collectively, these age-related changes suggest altered GABA neurotransmitter function in the IC. Identification of specific neurotransmitter changes in structures important in speech processing could eventually lead to the development of pharmacotherapy for selective types of age related hearing loss. PMID- 7556518 TI - Limbic control of endocrine glands in aged rats. AB - Two weeks after bilateral lesions in the dorsal and ventral hippocampus, septum, and basolateral amygdala, plasma concentrations of ACTH, TSH, corticosterone, aldosterone, and thyroxine were determined in 5-6 month-old (adult) and 24-26 month-old (aged) male Wistar rats. The results indicate that with age, limbic system control over the hypothalamic-pituitary- adrenal/thyroid axes changes, although these changes are not uniform. Thus, the inhibitory influences of the dorsal/ventral hippocampus and septum on ACTH release tend to diminish as age increases. However, the inhibitory influence of the amygdala on ACTH appears to become enhanced during the aging process. The data also support an enhanced sensitivity of the adrenal cortex to ACTH during aging. In adult animals, lesions of the dorsal/ventral hippocampus and amygdala result in significantly decreased plasma concentrations of TSH consistent with a stimulatory influence of these regions on TSH release. Lesions in the septum, however, lead to significantly increased TSH secretion in these animals. To these finding, lesions of the dorsal/ventral hippocampus and amygdala in aged animals resulted in significantly increased TSH secretion, while results of septal lesions were not significant. PMID- 7556517 TI - The role of "invertors" (intracellular activators) in age-related changes in cell response to hormones. AB - It has been shown that a number of hormones (insulin, testosterone, thyroxine, and adrenaline) can stimulate synthesis of plasma membrane regulators-- "invertors" in a cell. The synthesis of "invertors," which is under genomic control, decreases with age. As a result, insulin, testosterone, and thyroxine do not provoke Na,K-ATPase activation and plasma membrane hyperpolarization of hepatocytes and myocardiocytes in old rates. Also, due to desensitization of the heart to adrenaline, a smaller decrease occurs in adenylate cyclase activity in old animals. The Na,K-ATPase of plasma membranes of old animals maintain their ability to respond to the "invertors." The decrease in the synthesis of "invertors" represent an important mechanism of change in the cell's reaction to hormones. PMID- 7556521 TI - Hematopoiesis occurs in rhythms. PMID- 7556522 TI - Separation of graft-vs.-host disease and graft-vs.-leukemia effect against chronic myelogenous leukemia. PMID- 7556520 TI - The neuropsychology of aging. AB - There are three general categories of causes of the cognitive decline associated with aging: disuse, disease, and aging per se. People tend to use certain skills or abilities less with age and, thus, those skills decline due to the disuse. Physical illnesses tend to increase with age, which will tend to compromise cognitive functioning. Further, there are actual neurobiological changes with age that will contribute to deterioration of cognitive abilities. Variability of performance between different individuals within an age group increases with age due to each of these three major contributing factors to age decline. The best defense against age-related cognitive deterioration is practice. Practice tends to mitigate the effects of aging by not allowing disuse to occur. In addition, practice can overcompensate for age effects by building a larger reserve capacity to offset any real neurobiological effects of age. Practice can also lead to compensatory strategies in which alternative way of maintaining performance levels are found. PMID- 7556519 TI - Psychorehabilitation aspects in older age groups. AB - Throughout medical history, suggestion is the oldest and most common method of relieving human distress and treating physical disease. One of the oldest Egyptian documents, the Ebers Papyrus (1552 BC) states, "Lay your hands upon him to quiet the pain in the arms and say that the pain will disappear." The psychogenic and therapeutic potency of Native American trance dances are also well documented. In a North American tribe practicing ancient traditions, such dances were found to produce an altered feeling for time, loss of conscious control, drastic emotional outbreaks, illusions, hypersuggestibility, and a deep feeling of restored youthfulness (Jilek, 1982). These and other historical documents show the deep historical connection between somatic and mental processes. From the viewpoint of a neurologist or a psychiatrist, a combination of somatic illness, depressive syndromes, organic psychosyndromes, and multimorbidity is frequent in elderly patients (Kortus, 1992). Thus, integrated psychotherapy in geriatric rehabilitation is necessary and useful. Practical psychotherapeutic methods are discussed in this manuscript. PMID- 7556523 TI - The Belgrade laboratory (b/b) rat and the role of hypoxia in the maintenance of hematopoietic stem cells. PMID- 7556525 TI - Stage- and lineage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells. AB - There is growing evidence that the HOX homeobox-containing transcription factors are differentially expressed during hematopoiesis. We have previously demonstrated that the HOXA10 gene is expressed in unfractionated normal marrow and in immortalized leukemic cell lines with myelomonocytic features, but not in cell lines with lymphoid or erythroid features. To gain insights into the patterns of activation of this gene during hematopoietic differentiation, we have examined HOXA10 expression in CD34+ and CD34- subfractions of normal marrow and normal peripheral blood, as well as samples from patients with a variety of acute and chronic leukemias. HOXA10 is strongly expressed in CD34+ normal marrow cells, markedly downregulated in CD34- marrow cells, and inactive in mature neutrophils, monocytes, and lymphocytes. HOXA10 is expressed in all types of acute myelogenous leukemia (AML) with the notable exception of acute promyelocytic leukemia (AML M3). HOXA10 message is observed in chronic myelogenous leukemia (CML) but appears to be reduced in accelerated phase and blast crisis, particularly lymphoid blast crisis. With rare exception, HOXA10 expression is not observed in samples of acute or chronic lymphoid leukemias. Normal marrow and patient samples appear to contain a single transcript which encodes a full-length homeobox-containing protein, while immortalized cell lines contain an additional alternatively spliced transcript. These studies indicate that HOXA10 expression is restricted to early stages of myeloid differentiation. PMID- 7556524 TI - Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR ABL oncoprotein. AB - Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines. PMID- 7556526 TI - Cellular and cytokine-mediated effects of CD4-positive lymphocyte lines generated in vitro against chronic myelogenous leukemia. AB - Leukemia cells from a patient with chronic myelogenous leukemia (CML) in accelerated phase were used to generate CD4+, CD8- T lymphocyte lines from an unrelated normal subject sharing HLA-A2 and DR4 with the patient. In chromium release cytotoxicity assays, lines showed specificity for patient cells and were unreactive against third-party CML and K562 cells. Cytotoxicity was blocked by anti HLA-DR on target cells. Some lines showed preferential cytotoxicity to PHA induced lymphoblasts and some to CML cells. There was a broad correlation between cytotoxicity to CML cells by 51Cr release and CFU-CM inhibition. However, even weakly cytotoxic lines were inhibitory to CML CFU-GM. This effect was partly mediated by the T cell line supernatant: four of five supernatants tested inhibited the growth of CFU-GM. Antibody neutralization studies demonstrated the presence of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF alpha) in these supernatants. There was a greater suppression of CML CFU-GM when compared with CFU-GM from normal individuals. One supernatant from a noncytotoxic T cell line stimulated CFU-GM and was demonstrated by antibody neutralization studies to contain interleukin-3 (IL-3) and GM-CSF. These data indicate that alloreacting CD4 cells exert both cytotoxic and cytokine-mediated antileukemia effects which may relate to the graft-vs.-leukemia (GVL) effect in CML following bone marrow transplantation. PMID- 7556527 TI - Functional and phenotypic upregulation of CD13/aminopeptidase-N on precursor-B acute lymphoblastic leukemia after in vitro stimulation. AB - Acute lymphoblastic leukemia (ALL) cells of precursor-B type were assessed for expression of the cell surface peptidase CD13 (aminopeptidase-N) after 72 hours' culture in 10% B cell growth factor (BCGF), TPA, or medium alone. CD13 was analyzed phenotypically using a specific monoclonal antibody (mAb) by flow cytometry, and also with a spectrophotometric enzyme assay to measure the cleavage of specific peptide substrates. CD13 antigen was induced in all 10 cases of precursor-B ALL after culture with BCGF, with weaker expression seen in cells incubated with TPA or in medium alone. Aminopeptidase-N-like enzymatic activity was also demonstrated in cultured cells, particularly after BCGF exposure. Using the mAb WM-15, which specifically inhibits aminopeptidase-N function, we demonstrated that induction of true aminopeptidase-N activity was largely restricted to BCGF-treated cells, in which approximately 20% of total aminopeptidase activity was due to aminopeptidase-N. Phenotypic expression of the peptidase CD10 (neutral endopeptidase) was not altered on cultured cells. These findings indicate that CD13 expression can be selectively upregulated on ALL cells in response to proliferative stimuli. This peptidase, in cooperation with CD10 and perhaps other surface enzymes, may act to regulate the concentration of molecules at the cell surface which influence the growth of precursor-B ALL cells. PMID- 7556528 TI - Hematopoietic progenitor cell mobilization into the peripheral blood of mice using a combination of recombinant rat stem cell factor (rrSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF). AB - A significant increase in the hematopoietic stem cell (HSC) concentration has been observed in the peripheral blood and spleen of mice treated with rhG-CSF alone or with a combination of rhG-CSF plus rrSCF. The longer the duration of cytokine treatment, the higher the stimulatory effect on stem cell mobilization. In addition, cytokine administration led to a reduced stem cell concentration in the bone marrow. The total amount of HSCs in the body did not change following cytokine administration. These data support the theory that stem cells are mobilized from the bone marrow into circulation, as opposed to expanding in blood and spleen. In sharp contrast to rhG-CSF alone, a combination of rhG-CSF plus rrSCF produced a strong increase in the self-maintenance ability of peripheral blood day 11 colony-forming units-spleen (CFU-S-11). After 10 days of cytokine treatment, long-term culture initiating cells (LTC-IC) were seen in the peripheral blood; in normal mice, the content of LTC-IC in the blood was below the detection level. The activation of stromal progenitors (cells capable of transferring the hematopoietic microenvironment) by cytokine treatment was observed here for the first time. The results suggest that a combination of rhG CSF plus rrSCF is more effective than rhG-CSF alone in obtaining a large amount of transplantable HSCs. PMID- 7556529 TI - Natural killer (NK) cells inhibit human umbilical cord blood erythropoiesis. AB - To examine the role of NK cells on in vitro human umbilical cord (HUC) blood erythropoietic progenitor growth, 25 normal HUC blood samples were depleted of CD56+ cells by using immunomagnetic beads coated with CD56 monoclonal antibodies (mAb). When stimulated by erythropoietin (Epo) to form colonies in plasma clot medium, the CD(56+)-depleted preparations demonstrated a two-fold increase in the number of early erythropoietic progenitors (BFU-E) over nondepleted preparations. This stimulatory effect of CD56+ depletion on BFU-E growth was not due to artifactual stimulations of other accessory cells by the mAb or the dynabeads used in the depletion procedure, since separate addition of these materials to culture did not exert any stimulatory effect on BFU-E growth. Direct co-culture of purified NK and autologous cord blood mononuclear cells (MNC) in plasma clot medium resulted in a dose-dependent decrease in BFU-E population. In addition, when NK and MNC were cocultured separately in double-layer cultures, the expansion of BFU-E was significantly decreased. Because direct cell-to-cell contact is prohibited in double-layer cultures, the observed inhibition of BFU-E proliferation could be mediated at least in part through soluble factors. To test this hypothesis, NK cell supernatant fluid obtained 24 hours after NK cell incubation was added to plasma clot culture medium. A significant decrease in BFU E number was again observed. In conclusion, our results indicate that HUC blood BFU-E proliferation is inhibited by NK cells, and that the mechanism of this inhibition is mediated, at least in part, by one or more humoral factors. PMID- 7556530 TI - Fully and long-term xenogeneic hematopoietic chimeras created in poorly concordant rat-mouse combinations: expression of hereditary donor characteristics. AB - We created fully and stable xenogeneic hematopoietic chimeras in "poorly concordant" rat-mouse strain combinations defined by their high histocompatibility-antigen disparity and by the high titer of mouse-serum natural cytotoxic antibodies (NcAb) to rat donor bone marrow cells (BMC). Recipients were adult male (C57BL/6 x DBA/2)F1 (BDF1) mice, and donors of untreated BMC were adult male WAG strain rats. We tried several approaches to improve the quality and the stability of the rat-cell engraftment and to avoid the risk of graft-vs. host reaction (GVHR). Best results were obtained when: 1) BDF1 recipients were previously thymectomized and then heavily irradiated to lower their immunocompetence; 2) irradiated recipients were implanted with a newborn BDF1 thymus, which allows maturing rat T lymphoid cells to be made tolerant to mouse antigens in vivo, which lowered the risk of GVHR; and 3) recipients were given a high number of untreated rat BMC (4-5 injections of 1.6 x 10(7) cells) to reduce the risk of rejection of rat BMC by mouse NcAb. We found that rat BMC engraftment was highly effective (75 to 100% rat hemoglobin and 100% rat IgG) and long lasting (more than 10 months). The grafted rat cells were very tolerant toward host histocompatibility antigens but maintained all their immunological potentialities. Moreover, using the "poorly concordant" Wistar Furth (WF)-BDF1 combination, we showed that a genetically controlled characteristic of the hematopoietic system of the WF rat donor was maintained and functionally expressed in the xenogeneic environment of BDF1 mice. PMID- 7556531 TI - Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities. AB - In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P) GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities. PMID- 7556532 TI - Marrow-derived heparan sulfate proteoglycan mediates the adhesion of hematopoietic progenitor cells to cytokines. AB - Heparan sulfate proteoglycan (HS-PG), an important component of the human bone marrow extracellular matrix (ECM), is believed to influence hematopoietic progenitor cell development by binding and localizing growth factors to specific niches within the hematopoietic microenvironment. We utilized a model ECM system, which uses immobilized ECM proteins and/or cytokines and bone marrow populations enriched for human hematopoietic stem cell (HSC), to assess the effects of HS-PG on the development of primitive hematopoietic progenitor cells. HS-PG alone failed to bind hematopoietic progenitor cells cloned from bone marrow CD34+CD15 HLA-DR- cells, which are enriched for HSC. HS-PG alone failed to function as a mitogen. In sharp contrast, the interaction of HS-PG with either growth factors (interleukin-3 [IL-3] or stem cell factor/Kit ligand [KL] or an ECM protein (thrombospondin [TSP]) markedly influenced progenitor cell adherence. The binding of either IL-3 or KL to HS-PG resulted in a two-fold increase in attachment of the colony-forming unit-granulocyte/macrophage (CFU-GM), a 1.5-fold increase in attachment of the burst-forming unit-erythroid (BFU-E) and the high-proliferative potential colony-forming cell (HPP-CFC), and a two- to three-fold increase in attachment of the colony-forming unit granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) compared to localized growth factor alone. Attachment of the BFU-megakaryocyte (BFU-MK), however, was slightly reduced by the interaction of either IL-3 or KL with HS-PG. The interaction of HS-PG with TSP resulted in a two-fold increase in CFU-GM and CFU GEMM attachment, while the attachment of BFU-E, HPP-CFC, and BFU-MK was unaltered. We conclude that HS-PG cooperatively interacts with both growth factors and ECM proteins to augment progenitor cell localization within the hematopoietic microenvironment. PMID- 7556533 TI - Hematopoietic stem cells in the hereditarily anemic Belgrade laboratory (b/b) rat. AB - The Belgrade (b/b) rat has hereditary hypochromic microcytic anemia as the consequence of intracellular iron deficiency. Studies in the b/b rat have also demonstrated alteration in hematopoiesis at the progenitor cell level. In the present study, investigations were extended to the bone marrow hematopoietic stem cells as determined by measurements of marrow repopulating ability (MRA) and day 8 spleen colony-forming units (CFU-S-8). A reduced number of CFU-S-8 per femur was found, together with a low incidence per 10(5) bone marrow cells and a nondetectable proliferation rate. The proliferation rate did not increase after treating b/b rat bone marrow cells in vitro with a stimulator for CFU-S proliferation, indicating a proliferative block. The treatment of b/b rats with iron enhanced the proliferation rate and partially increased the number of CFU-S 8 in bone marrow. Chronic transfusion of b/b rats with washed RBC from non-anemic animals restored both the number and the proliferative response of bone marrow CFU-S-8. The MRA of b/b rats was reduced, but in proportion to the decrease in the bone marrow cellularity of these animals. MRA (pre-CFU-S) of b/b rats recovered completely after both iron treatment and chronic transfusions, suggesting that changes in the pre-CFU-S pool are secondary rather than directly induced by the genetic defect. These results indicate the importance for stem cell proliferation of normal oxygenation--the arterial oxygen content in b/b rats is six times lower than in (+/+, b/+) rats--which recovered after the iron treatment and was completely restored after chronic transfusions. High-dose iron therapy abrogated the proliferative block of CFU-S-8, but number of CFU-S-8 was not completely recovered in spite of normalized oxygenation, indicating a possible suppressive effect of iron overload on the marrow microenvironment. PMID- 7556534 TI - A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CFU-MK. AB - A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5 x 10(4) cells/mL (10(4) cells/well) with test samples in 96-well tissue culture plates for 4 days at 37 degrees C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples--including a wide variety of cytokines and unknown growth factors, alone or in combinations--and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway. PMID- 7556535 TI - A marrow harvest procedure under local anesthesia. AB - This report details a bone marrow harvest procedure performed outside the hospital setting under local anesthesia, thereby avoiding many of the risks associated with the traditional surgical procedure. In approximately 30 minutes, 450 milliliters of marrow can be collected from eight bone punctures, containing a median of 4.18 x 10(9) cells and 33 x 10(6) progenitor cells as defined by CD34 expression. Reinfusion of a median 1.2 x 10(6) CD34+ cells/kg in 10 breast cancer and lung cancer patients after dose-intensive chemotherapy resulted in the recovery of granulocytes > 100/mm3 by day 14 and platelets > 20,000 by day 21. Without progenitor cell support, such recoveries could take 30 and 40 days, respectively. Collection of marrow using this protocol does not compromise the engraftment capability of the progenitor cells, seldom necessitates blood product support, is safer for the patient, and reduces the cost of harvesting by 75% compared to inpatient or day surgery procedures. PMID- 7556536 TI - Postnatal development of cortical acetylcholinesterase-rich neurons in the rat brain: permanent and transient patterns. AB - The development of acetylcholinesterase (AChE) activity within cortical neurons of the rat brain was investigated using a histochemical method. The fate of these neurons in later stages of development was studied in animals in which AChE within cortical axons (mostly cholinergic) had been depleted by lesions of the cholinergic neurons of the basal forebrain or by injections of diisopropyl fluorophosphate. We designated neurons with medium to high intensity of reaction product as AChEH and neurons with a low intensity of reaction product as AChEL. Four groups of AChEH cortical neurons were detected: (1) AChEH Cajal-Retzius cells were present in layer I at birth (P0) and decreased steadily in number until none could be detected at P17 or thereafter. (2) AChEH neurons within layer VI and underlying white matter were present at P0, peaked in number and staining intensity at P8-P9, showed a moderate decrease in number at P11-P13 and a further decrease into adulthood. (3) AChEH polymorphic intracortical neurons appeared at P3-P4 in deep cortical layers and by P9 were present in layers II-VI. They continued to increase in number through P11-P14 at which time they displayed the adult pattern and were found in all cortical areas. (4) A large population of AChEH pyramidal neurons appeared at P1-P4, peaked at P8-P10 and was no longer visible at P21. In the adult cerebral cortex, few pyramidal neurons displayed AChE activity and these were almost always of the AChEL type. These results indicate that the AChE within cortical neurons is developmentally regulated and that the content of this enzyme helps to differentiate cortical neurons into distinct populations. The transient expression of AChE activity within cortical neurons suggests a role for this enzyme in the development of the cerebral cortex. PMID- 7556537 TI - Schwann cells and fetal tectal tissue cografted to the midbrain of newborn rats: fate of Schwann cells and their influence on host retinal innervation of grafts. AB - Schwann cells transplanted into the adult central nervous system (CNS) can exert powerful growth-promoting effects on damaged axons. An important issue is whether central axons induced to regrow by Schwann cells retain the capacity to recognize and selectively innervate their appropriate target cells. To examine how Schwann cells may influence the specificity of neuron-neuron interactions in CNS neuropil, we cultured neonatal rat Schwann cells and mixed them with dissociated fetal tectal cells. In some instances, Schwann cells were prelabeled with Hoechst dye 33342. Schwann cells comprised between 2.5 and 15% of the combined cell population. After reaggregation, cografts were injected onto the midbrain of newborn rats. One to 6 months later, grafts were examined for the presence of Schwann cells and the pattern and density of host retinal innervation of the cografts was assessed. Immunohistochemical studies showed that areas of the transplants containing large numbers of surviving Hoechst-labeled Schwann cells were strongly immunoreactive for the low-affinity nerve growth factor receptor (p75), S-100, GFAP, and laminin. Very little peripheral (Po positive) myelin was seen. As in pure fetal tectal grafts, host retinal axons were sometimes observed to innervate superficial, localized areas in the cografts known to be homologous to the retinorecipient layers of the superior colliculus. Unlike pure tectal grafts, however, optic axons were not confined to these regions and fibers were often dispersed within the cograft neuropil. Dense growth was seen in association with Hoechst-labeled Schwann cells and, in some cases, optic axons were observed to grow toward Schwann cells and away from nearby target areas. These observations suggest that, under certain circumstances, Schwann cells can stimulate retinal axons to grow into inappropriate (nontarget) regions in the CNS, presumably by producing growth promoting factors which mask or compete with signals released from the target neurons themselves. PMID- 7556538 TI - Differences in electrophysiological properties of angiotensinergic pathways from the subfornical organ to the median preoptic nucleus between normotensive Wistar Kyoto and spontaneously hypertensive rats. AB - Electrophysiological properties of angiotensinergic pathways from the subfornical organ (SFO) to the median preoptic nucleus (MnPO) were investigated in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) under urethane anesthesia. The activity of SFO neurons that were antidromically activated by electrical stimulation of the MnPO was compared between WKY (n = 28) and SHR (n = 27). No significant differences were observed between WKY and SHR in the latency, conduction velocity, or threshold of antidromic activation. The firing rate was significantly shorter in SHR. The activity of MnPO neurons was tested for a response to microiontophoretic application of angiotensin II (ANG II) and electrical stimulation of the SFO. Sixteen of 46 MnPO neurons tested in WKY and 15 of 47 MnPO neurons tested in SHR were excited by both ANG II applied iontophoretically and SFO stimulation, and the excitatory responses were prevented by iontophoretically applied saralasin, a specific ANG II antagonist. In these MnPO neurons that demonstrated the excitation to both SFO stimulation and ANG II, the firing rate was significantly higher and the threshold current required to evoke the SFO stimulus-induced excitation was significantly lower in SHR. The sensitivity to SFO stimulation was much greater in SHR than in WKY. These results provide evidence that there are marked alterations in the physiological properties of the angiotensinergic circuit from the SFO to the MnPO between WKY and SHR and imply that a disorder in the neural circuit may contribute, in part, to hypertension. PMID- 7556539 TI - YM90K, an AMPA antagonist, has no neurotoxic effects on cerebrocortical neurons in rats. AB - The neuroprotective properties of glutamate receptor antagonists arise from their ability to antagonize the excitotoxic actions of endogenous excitatory amino acids. However, J. W. Olney et al. (1989, Science 224: 1360-1362) have reported that MK-801, an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, induced morphological damage in neurons in the cerebral cortex of rats. YM90K is a potent alpha-amino-3-hydroxy-5-methylisoxazole propionic acid receptor antagonist which has high neuroprotective efficacy against delayed neuronal injury. The purpose of this study was to investigate whether YM90K induces a vacuolar reaction in the cytoplasm of neurons similar to that seen after the administration of MK-801. All experiments were performed on female F344 rats. YM90K was administered by iv infusion for 3 h at the dose of 40 mg/kg/h. MK-801 was given by single sc injection at the dose of 1 mg/kg. All rats receiving MK 801 showed neuronal vacuolation. The affected neurons were recognized as medium sized pyramidal-shaped neurons which were distributed between layers II and IV in the posterior cingulate and retrosplenial neocortices. Most of these vacuoles contained multiple small and round structures that appeared to be remnants of mitochondria. Other vacuoles were recognized as enlarged sER or those present within the bilaminar nuclear membrane. MK-801 also induced heat shock protein immunoreactivity in the same neurons. In contrast, no such pathomorphological changes could be detected in the YM90K-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556540 TI - The "campus syndrome" in pigs: neurological, neurophysiological, and neuropharmacological characterization of a new genetic animal model of high frequency tremor. AB - Inherited neurological diseases in animals are of interest to a wide range of scientific disciplines, particularly because such animals may be suited as genetic animal models for respective human disorders. Because the pig has a number of anatomic and physiologic features similar to those of human beings, this species is becoming increasingly popular in biomedical research. The usefulness of pigs as genetic models of neurological diseases is illustrated by the porcine model of malignant hyperthermia (MH), i.e., a frequently fatal myopathic disease in both pigs and humans. In the present study, we describe a new hereditary movement disorder in Pietrain pigs, which may represent a useful genetic animal model of high-frequency tremor. Because the disorder was first detected in the offspring of a boar named "Campus," we use the term "Campus syndrome" in this respect. Segregation analysis of breeding studies indicates that the syndrome is inherited as a monogenic dominant trait. DNA-based testing of the mutation involved in MH myopathy showed that expression of the Campus syndrome in pigs is not dependent on homozygosity for the MH mutation. In affected pigs, the Campus syndrome develops at an average age of 27 days. The syndrome is characterized by muscular weakness and a very intense tremor of the legs when standing and walking but not when at rest in a lying position. The intensity of tremor and muscular weakness progressively increases with age, resulting in pronounced postural instability. Despite these neurological abnormalities, body weight gain in affected pigs does not differ from that in unaffected siblings.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556542 TI - Neurotoxicity of dopamine and protective effects of the NMDA receptor antagonist AP-5 differ between male and female dopaminergic neurons. AB - Neurodevelopmental and neurodegenerative disorders related to dopaminergic transmission typically exhibit a sex-specific prevalence. In order to investigate the underlying cellular mechanisms, primary cultures of dissociated embryonic rat midbrain were subject to a 24 h treatment with dopamine in concentrations between 1 and 1000 microM. Dopamine caused a dose-dependent loss of neurons and reduction of neurites immunoreactive to tyrosine hydroxylase with a LD50 of about 100 microM. Application of D1-like or D2-like receptor agonists instead of dopamine did not induce cell loss. Neither D1-like or D2-like receptor antagonists nor the nitric oxide synthase inhibitor N-nitro-L-arginine were capable of blocking dopamine-induced cell death. When tissue from male and female donors was cultured separately, a twofold sex difference was consistently present: (1) Survival rates of female dopaminergic neurons in the presence of LD50 concentrations of dopamine were about twice those of male neurons. (2) The N-methyl-D-aspartate-receptor antagonist AP-5 was capable of rescuing female but not male dopaminergic neurons from dopamine-induced cell death. It is concluded that dopamine neurotoxicity is not mediated by dopamine receptors and is aggravated by glutamate excitotoxicity but not by nitric oxide. The male-specific vulnerability is the first direct evidence that the prevalence of certain neurodevelopmental or neurodegenerative disorders may have a basis in the biology of the single dopaminergic neuron. PMID- 7556541 TI - Cultured rat microglia express C1q and receptor for C1q: implications for amyloid effects on microglia. AB - Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds beta 1-42, the major constituent of senile plaques, and since beta 1-42 is toxic to microglia in vitro, we asked if preincubation of beta 1-42 with C1q alters either metabolic indices of amyloid induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that pre-incubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to beta 1-42 on beta 1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-gamma. While these data exclude a role for the C1q receptor in beta 1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-gamma would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques. PMID- 7556543 TI - Conditions that may determine blood vessel phenotype in tissues grafted to brain. AB - The hypothesis, that a blood vessel's phenotype is determined by the tissue it vascularizes and not by the vessel's source, does not hold for tissue beyond a certain period of development. In mature skeletal muscle grafted to choroid plexus of adult and 2-week-old rats, some vessels were choroidal or fenestrated (FV) rather than, according to the hypothesis, continuous (CV), like those of muscle. In E14 fetal muscle placed on the choroid plexus, approximately 80% of the grafts' capillaries were CV, like those of muscle. Most choroidal FV that entered the grafts were apparently changed to CV. By E16, about 70% of the graft vessels remained as FV rather than being converted. Thus, FV were changed to CV by a hypothetical conversion factor made, apparently, by E14 grafts but not by E16 grafts. In grafts from [3H]thymidine-labeled donors, an appreciable number of CV in E14 grafts were identified as intrinsic to the muscle. When hosts were labeled to verify the origin of FV in their nonlabeled donor grafts, only a few FV, to date, were tagged. The FV must have come from host choroid plexus, the only available source of graft FV. Vascular endothelial growth factor (VEGF) might convert CV into FV, yet VEGF and the mRNA for its receptor were present in choroid plexus but not in the grafts. Therefore, VEGF is not a conversion factor. The purported factor that changes FV to CV may be expressed in E14 muscle grafts but diminishes by fetal age E16 and beyond. PMID- 7556544 TI - Chronic administration of malonic acid produces selective neural degeneration and transient changes in calbindin immunoreactivity in rat striatum. AB - Adult rats received chronic dialytic delivery devices that exposed the striatum to a 100 mM, 400 mM, or 4 M solution of the reversible succinate dehydrogenase inhibitor malonic acid (MA). Three weeks of exposure to 100 or 400 mM MA produced no significant reduction in striatal cytochrome oxidase staining, whereas striata chronically exposed to 1 and 4 M MA showed a significant and dose-related reduction in cytochrome oxidase staining. In striata exposed to 1 M MA, analysis of regions radial to the necrotic core revealed significant reduction of nissl cell staining with relative sparing of NADPH-diaphorase-containing neurons. Although 100 and 400 mM MA failed to produce lesions, both of these concentrations significantly decreased the number of striatal calbindin (CALB) immunoreactive perikarya. The reduction in CALB immunoreactivity was partly reversed in animals allowed to survive 4 weeks after cessation of exposure to 400 mM MA. These results indicate that, like striatal lesions produced by quinolinic acid, lesions produced by chronic exposure to MA possess a Huntington's disease like pattern of selective neurodegeneration. In addition, exposure to subthreshold MA concentrations (100 and 400 mM) produce widespread transient changes in striatal CALB that may be associated with a premorbid state of neuronal dysfunction. PMID- 7556545 TI - Evidence for the involvement of histamine in the antidystonic effects of diphenhydramine. AB - Although diphenhydramine hydrochloride is known to eliminate or reduce the symptoms of dystonia in human patients with acute dystonic reactions and idiopathic torsion dystonia, its mechanism of action is still unclear. In the present study, we show that the antihistamine properties of diphenhydramine may contribute to its beneficial effects. Acute dystonic reactions were produced in rats with unilateral microinjection of haloperidol into the red nucleus as previously described. Similar to the pattern in humans, this effect could be attenuated by coadministration of diphenhydramine. Unilateral microinjection of histamine itself into the rat red nucleus produced dystonic postures (torticollis) in a dose-dependent manner, demonstrating that a histamine dysfunction could contribute to the pathophysiology of dystonia. The torticollis produced by histamine could be significantly attenuated with coadministration of the H1 antagonists diphenhydramine or pyrilamine or the H2 antagonist cimetidine. These effects are thought to be mediated through the red nucleus because significantly more torticollis was observed when histamine was injected into the red nucleus rather than surrounding mid-brain areas, the substantia nigra, or the lateral ventricle. The present data, taken together with studies in humans, suggest the involvement of histamine in some types of dystonia. Furthermore, the red nucleus and related motor pathways may have a more important role in dystonia than previously thought. PMID- 7556546 TI - A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord. AB - We previously demonstrated that Schwann cells (SCs) in semipermeable guidance channels promote axonal regeneration in adult rat spinal cord transected at the mid-thoracic level. Propriospinal but not supraspinal axons grew into these channels. Here, we tested the ability of exogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to promote axonal regeneration in this novel model. The two neurotrophins were delivered simultaneously into the channel by an Alzet minipump at a rate of 12 micrograms/day for each neurotrophin for 14 of 30 days tested; phosphate-buffered saline, the vehicle solution, was used as a control. Significantly more myelinated nerve fibers were present in SC/neurotrophin grafts than in SC/vehicle grafts (1523 +/- 292 vs 882 +/- 287). In the graft, at least 5 mm from the rostral cord-graft interface, some nerve fibers were immunoreactive for serotonin, a neurotransmitter specific to raphe derived axons in rat spinal cord. Fast blue retrograde tracing from SC/neurotrophin grafts revealed labeled neurons in 10 nuclei of the brain stem, 67% of these being in the lateral and spinal vestibular nuclei. The mean number of labeled brain stem neurons in the SC/neurotrophin group (92; n = 3) contrasted with the mean in the SC/vehicle group (6; n = 4). Our results clearly demonstrate that BDNF and NT-3 infusion enhanced propriospinal axonal regeneration and, more significantly, promoted axonal regeneration of specific distant populations of brain stem neurons into grafts at the mid-thoracic level in adult rat spinal cord. PMID- 7556549 TI - Target influences on the morphology of trigeminal axons. AB - Axons grow in two stages: First, they exhibit rapid, target-directed extension; then they begin to collateralize and elaborate terminal arbors in their targets. To investigate possible regulatory influences on these phases of axon growth, we have used an in vitro paradigm in which we cocultured embryonic or postnatal rat trigeminal ganglion explants with isochronic, heterochronic, and/or heterotypic targets. Cultures were fixed after 5 days and ganglion cell processes were labeled with DiI. Trigeminal processes were able to regenerate into several peripheral targets as well as into CNS explants from trigeminal or nontrigeminal regions of the brain. In peripheral tissues, the processes showed target-specific growth patterns. In CNS tissue, the type of growth (unbranched extension versus collateralization/arbor formation) varied markedly with the explant age: If trigeminal ganglia were harvested at a time (E15) when their axons would be elongating in the embryo and cocultured with isochronic tissues, their processes had a simple morphology, were loosely bundled, and reconstituted a distinct fiber tract, mimicking their in vivo growth pattern. If, challenged by more mature tissue, axons of E15 ganglion cells formed discrete arbors. Finally, if trigeminal ganglia were harvested at an age (E20, PND 5) when their axons had already formed arbors in the brain and induced to innervate younger (E15) targets, their axons reverted back to the elongation stage. These results demonstrate that the target environment sets specific, developmentally regulated constraints on the patterns of growth manifested by primary sensory axons. PMID- 7556550 TI - Increased amyloid protein precursor and apolipoprotein E immunoreactivity in the selectively vulnerable hippocampus following transient forebrain ischemia in gerbils. AB - The postischemic time course of amyloid protein precursor (APP), beta-amyloid protein (beta-AP), and apolipoprotein E (APO-E) immunoreactivity were examined in comparison to neuronal necrosis in the selectively vulnerable hippocampal CA1 region of gerbils subjected to 10 min of bilateral carotid occlusion-induced forebrain ischemia. Loss of 90% of the CA neurons occurred between 24 and 72 h after ischemia, after which no further neuronal necrosis was observed. At 24 h postischemia, there was a decrease in APP and beta-AP immunostaining in the CA1 region. However, beginning at 2 days, there was a dramatic increase in the staining for both proteins. This coincided with a progressive increase in the expression of APO-E and glial fibrillary acidic protein (GFAP) staining between Days 2 and 6, indicative of an activation of astrocytic protein synthesis. Each of the immunocytochemical markers also increased in the less vulnerable CA3 region. However, the peak increase in that region was much less than that in CA1 and, by 7 days, only the GFAP staining remained significantly above the sham level. It has been shown that the E4 isoform of APO-E, when oxidized, avidly binds to beta-AP and thus increases the likelihood of co-beta-AP/APO-E deposition. Therefore, it is postulated that the increased levels of amyloid proteins coincident with an increased production of APO-E in response to ischemic neuronal necrosis may provide conditions that are favorable for the postischemic formation of amyloid deposits. PMID- 7556547 TI - Neocortical grafts placed in the infarcted brain of adult rats: few or no efferent fibers grow from transplant to host. AB - The present study examines the capacity of fetal neocortical grafts placed in a brain infarct to exchange axonal projections with the host brain. Five to 7 days after a middle cerebral artery occlusion in adult spontaneously hypertensive rats, dissociated neocortical primordium from fetuses of gestational age 15-16 days was implanted into the infarcted area. Four to 11 months later, the neural tracers Phaseolus vulgaris-leucoagglutinin and Fluoro-Gold were injected in the grafts and host neocortex. An extensive axonal network was present in the transplants but only one of eight rats with appropriate placed injections displayed efferent connections from transplant to host. The sparse axonal outgrowth indicates major limitations for fetal rat cortical grafts to form connections with host neural circuitries after an ischemic insult. PMID- 7556551 TI - Magnitude of GAP-43 induction following peripheral axotomy of adult rat dorsal root ganglion neurons is independent of lesion distance. AB - Regenerative axon growth in peripheral neurons is accompanied by increased expression of the growth-associated protein GAP-43. We examined the increase of GAP-43 immunoreactivity in DRG neurons following lesions at different distances along the sciatic nerve, using immunocytochemistry. To control for the variable involvement of DRG axons following injury at different sites, injured neurons were identified by retrograde labeling with Fluoro-Gold. GAP-43 labeling was similar for proximal, distal, and far-distal injuries when only injured neurons are considered. Our results stand in contrast to studies which show that GAP-43 upregulation in neurons of the central nervous system occurs only when lesions are made close to the cell body. This suggests that the mechanisms which control GAP-43 expression following injury differ between central and peripheral neurons. PMID- 7556548 TI - Analgesia induced by morphine microinjection into the lateral hypothalamus of the rat. AB - Earlier studies have demonstrated that electrical stimulation of the lateral hypothalamus (LH) produces analgesia in tests of brief, phasic pain and more prolonged tonic pain. The present experiment examined the effect of morphine microinjection into the LH on formalin-induced pain. The results show that a 10 micrograms morphine microinjection significantly decreases pain responses. Moreover, the decrease persists for the duration of the 25-min test period, which began 25 min after the formalin injection. These findings suggest that electrical stimulation of the LH engages an endogenous opiate system which is capable of producing prolonged analgesia. PMID- 7556553 TI - The effect of thalamic lesions on primate taste preference. AB - Taste preferences and aversions were examined before and after the placement of bilateral, electrophysiologically guided electrolytic lesions of the thalamic taste area in five Old World monkeys. The only consistent changes shown by all lesioned monkeys on the 30-min, single-bottle intake test were a significantly attenuated consumption of 0.003 M quinine hydrochloride and a significantly elevated intake of 0.1 M sodium chloride. These specific taste deficits are contrary to the global hypogeusia predicted on the basis of previous research. A revised definition of the boundaries of the gustatory thalamus is suggested and the merits of the thalamic monkey preparation are compared with those of the decerebrate rat model. PMID- 7556552 TI - Regional differences in responsiveness of adult CNS axons to grafts of cells expressing human neurotrophin 3. AB - Neurotrophin 3 (NT3) belongs to the neurotrophin family, which also includes nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 4/5. NT3 mRNA is widely expressed in the rodent nervous system, but the physiological function of the native protein is still unclear. Genetically modified cell lines that produce physiological amounts of NT3 can provide a useful tool in the elucidation of the NT3 effects in the adult central nervous system (CNS). Genetically modified rat primary skin fibroblasts expressing and secreting human NT3 (hNT3) were prepared and characterized. In vitro, cell lines derived from different retroviral constructs expressed hNT3 mRNA, as determined by PCR and RNA blot analysis. Secretion of biologically active hNT3 was confirmed by specific elicitation of neurite outgrowth from cultured chick primary sympathetic and sensory neurons and from rat fetal locus coeruleus neurons in the presence of hNT3-producing cell conditioned media. In vivo, implanted fibroblasts survived well up to the maximal experimental time points of 6 weeks (brain) and 4 weeks (spinal cord) and continued to express hNT3 mRNA in vivo. As early as 2 weeks postgrafting, specific sprouting of host sensory neurites in response to hNT3 producing grafts was observed in the spinal cord. In contrast, hNT3-producing cerebral grafts did not induce a sprouting response different from that observed with control grafts. These findings establish the existence of a regionally different responsiveness of the CNS axons to local hNT3 overexpression. PMID- 7556554 TI - Corticospinal motor neurons in the adult rat: degeneration after intracortical axotomy and protection by ciliary neurotrophic factor (CNTF). AB - Little is known about the neurotrophic factors that may regulate maintenance, growth, and/or repair of corticospinal motor neurons (CSMN) in the developing or the adult mammal. We have developed an adult rat in vivo model of CSMN injury involving (i) bilateral prelabeling of CSMN with a cervical spinal cord injection of cholera toxin B subunit (CTB), (ii) unilateral axotomy close to the cell bodies by an intracortical lesion between cell layer V and the corpus callosum, and (iii) implantation of a continuous infusion device in the cortical parenchyma near the lesion. Two weeks later, coronal sections of the cortex are immunostained for CTB, and CTB-stained neurons are counted over defined section areas and compared to those on the contralateral (nonlesioned, noninfused) side. CTB-stainable neurons were 30-40% of the control side when the lesion was about 200 microns from the deeper face of the cell layer, and survival increased with increasing lesion depths. The model can be used to assess protective effects of potential CSMN trophic factors. The low survival achieved with the more superficial lesions (200 and 300 microns) was markedly improved by continuous infusion of ciliary neurotrophic factor at 0.1 to 1.0 microgram/day. PMID- 7556555 TI - Hypophysectomy produces analgesia and paraventricular lesions have no effect on formalin-induced pain. AB - Chemical destruction of the pituitary gland has been shown to alleviate severe cancer pain in a substantial proportion of patients. The underlying mechanisms, however, remain controversial. The present study investigated the effects of hypophysectomy in the formalin test, which provides an animal model of tonic pain, and attempted to determine a possible neural mechanism to explain the effectiveness of the procedure. Hypophysectomized rats displayed significantly less pain behavior in the formalin test than control rats that underwent a sham hypophysectomy, implicating the pituitary gland in the modulation of tonic pain. Since the paraventricular nucleus of the hypothalamus (PVN) provides a major source of input to the pituitary gland, the effects of electrolytic lesions of the PVN on tonic pain were examined. The results failed to show a significant effect of PVN lesions on pain responses in the formalin test. The results suggest that the pituitary gland modulates tonic pain in the formalin test and that the test provides an animal model to study possible mechanisms which underlie the relief of severe cancer pain by hypophysectomy. However, since PVN lesions did not affect the response of rats in the formalin test, it is possible that the PVN is only one of multiple interacting neural and endocrine structures that influence the functions of the pituitary. PMID- 7556556 TI - Loa loa: structural diversity of a 15-kDa repetitive antigen. AB - A ladder antigen of Loa loa was identified on Western blots of all life cycle stages probed with loaisis sera. The smallest subunit has a relative M(r) of about 15 kDa and larger subunits represent size increments of 15.0 kDa. An 1800 bp genomic clone encoding this antigen was characterized further by restriction mapping. Southern blot analysis, and nucleotide sequencing. The antigen is encoded by multiple copies of a gene, linked in tandem repeats of 396 bp, each of which encodes 132 amino acids. These repeats have diverged sufficiently to produce distinct restriction enzyme sites and Southern blot hybridization patterns. The 1764-bp insert contains no introns and encodes 588 amino acids, representing one incomplete and four complete repeats. At the 3' end of three repeats, there are consensus proteolytic cleavage sites; one repeat has no cleavage site. Two perfect repeats show a 93.9% amino acid identity with one another; the rest of the repeats, despite being adjacent to one another, show only 31-42% identical amino acids. Putative asparagine N-linked glycosylation sites are expressed by only two of the repeats. Despite this structural diversity, each L. loa repeat showed homology to Ascaris suum allergen and the homologue protein described in Dirofilaria immitis and Brugia pahangi. PMID- 7556558 TI - Plasmodium falciparum: characterization of a 0.7-kbp, moderately repetitive sequence. AB - A 692-bp-long repetitive sequence of Plasmodium falciparum was cloned. Two contiguous repeats were sequenced. The homology between them was 85%, the differences being due only to base replacements. The sequence was found 60 times repeated in the genome and was detected in 11 of 14 chromosomes. When used as a probe, the cloned fragment detected the parasite with the same sensitivity the 21 bp repeat (pRepHind) did. PCR amplification detected 0.02 pg of DNA, equivalent to a single parasite, in strains from America, Asia, and Africa. The restriction patterns were polymorphic and different among the strains. Analysis of lambda EMBL clones that contain the repetitive sequence confirms that it is present in various genomic contexts and is located subtelomerically. PMID- 7556557 TI - Trypanosoma congolense: proliferative responses and interleukin production in lymph node cells of infected cattle. AB - T-cell-mediated immune responses to defined antigens of Trypanosoma congolense were measured in cattle undergoing primary infection. The antigens used were the variable surface glycoprotein and two invariant antigens, a 33-kDa cysteine protease (congopain) and a recombinant form of a 69-kDa heat-shock protein. Proliferative responses were highest during the second week postinfection and were detected in cells obtained from the lymph node draining the site of infection but not in peripheral blood mononuclear cells. Production of IL-2 and IFN-gamma was measured in supernatants from antigen-stimulated lymph node cell cultures. Expression of IL-2, IL-4, and IFN-gamma mRNA was detected in antigen stimulated lymph node cells by reverse transcription-polymerase chain amplification. PMID- 7556559 TI - Toxoplasma gondii: effect of sublethal irradiation on host resistance in mice. AB - The effect of sublethal whole-body irradiation (500 rad) on the resistance of C57BL/6 mice to infection with Toxoplasma gondii was studied. Mice irradiated 1 day before or 4 days after infection via the intraperitoneal or peroral route with cysts of the mildly virulent ME49 strain of T. gondii died sooner than did unirradiated controls. The time to death of irradiated mice was suggestive of impaired acquired immunity. Irradiated mice infected intraperitoneally with T. gondii cysts exhibited reduced levels of Thy-1+CD4-CD8- cells, less natural killer cell activity against YAC-1 targets, and lower levels of IFN-gamma than controls. Numbers of CD4+ and CD8+ cells were also lower in infected, irradiated mice. Irradiated mice immunized with a vaccine strain of T. gondii, and later challenged with a highly virulent strain, were less well protected than unirradiated controls. Irradiation appears to impair early, CD4(+)- and CD(8+) independent resistance to T. gondii, as well as acquisition of immunity. PMID- 7556562 TI - Tritrichomonas foetus: comparison of isolate virulence in an estrogenized mouse model. AB - Previous studies indicated that Tritrichomonas foetus isolate ATCC 30003 was capable of maintaining only short-term genital infection in estrogenized BALB/c mice. In the present study, the ability of eight T. foetus isolates to establish and maintain infections in intravaginally inoculated estrogenized BALB/c mice was examined. All isolates were found to be equally capable of establishing genital infections but varied greatly in ability to maintain infections. One isolate maintained infection for less than 7 weeks, four isolates maintained intermediate infections lasting less than 13 weeks, and three isolates maintained chronic infections of greater than 26 weeks. Varying the number of trophozoites inoculated intravaginally decreased the ability of isolates to establish infection but did not affect maintenance of infection. Prolonged passage of T. foetus isolates either in vivo in an estrogenized nu/nu BALB/c mouse or by in vitro culture failed to affect their ability to maintain infection, suggesting that virulence was parasite-dependent and not related to environment-induced changes. Co-infection of estrogenized mice with isolates ATCC 30003 and MU Y32 failed to increase the length of ATCC 30003 infections or decrease the length of isolate MU Y32 infections. Taken together these results indicate that T. foetus isolates vary greatly in virulence in estrogenized BALB/c mice and provide evidence suggesting that maintenance of infection is a parasite-controlled factor. PMID- 7556561 TI - Plasmodium falciparum: effects of membrane modulating agents on direct binding of rhoptry proteins to human erythrocytes. AB - We studied the effects of membrane modulation on the interaction of Plasmodium falciparum rhoptry proteins of 140/130/110 kDa (Rhop-H) with human and mouse erythrocytes. Cells treated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n octylcyclopropyl)octanoate, myristoleyl alcohol, and proteins extracted with sublytic concentrations of membrane solubilizing detergents were used in erythrocyte binding assays. Protein binding was evaluated by immunoblotting using Rhop-H- and SERA-specific antisera, 1B9, K15, and 5E3, respectively. Protein binding to liposomes prepared with dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) or dilauroyl-L-alpha-phosphatidylcholine (DLPC) was also examined. Our results show that erythrocyte membrane modulation markedly enhanced direct Rhop-H binding to intact human erythrocytes. Binding of SERA to intact human erythrocytes appeared unaffected. Both DPPC and DLPC liposomes had similar Rhop-H and SERA protein binding activities. However, binding to DLPC liposomes was reduced. Rhop H and SERA extracted with the detergents octanoyl-N-methylglucamide, decanoyl-N methylglucamide, sodium deoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio] 1-propane sulfonate bound directly to intact human erythrocytes, probably by partitioning hydrophobically into the membranes. Sodium carbonate treatment demonstrated a nonintegral association of Rhop-H with the erythrocyte membrane during invasion. Membrane modulation may expose cryptic phospholipid binding sites in the bilayer. PMID- 7556563 TI - Schistosoma mansoni: the presence and potential use of opiate-like substances. AB - The present study demonstrates that morphine- and codeine-like molecules are present in Schistosoma mansoni following HPLC separation and identification with an appropriate commercially available antibody. Furthermore, the endogenous material, corresponding to morphine, mimics authentic morphine in its ability to induce immunocyte rounding and immobility, an action that is naloxone sensitive. The codeine-like material is not found at high concentrations compared to the morphine-like material, indicating, as in mammals and Mytilus edulis, the potential rapid conversion of codeine to morphine. Coincubation with human leukocytes increases the endogenous level of this material in adult worms, indicating the presence of a positive feedback loop. Last, EDTA, a chelator of divalent cations, has a strong stimulating effect in the synthesis of morphine like material by the worm as noted by higher levels of this material in its presence. Taken together, the results suggest that this parasite may utilize this immune downregulating molecule in its effort to escape host immunosurveillance as well as in inhibiting an immune response directed against itself. PMID- 7556564 TI - Developmentally regulated sensitivity of Trypanosoma brucei brucei to the cytotoxic effects of human high-density lipoprotein. AB - The bloodstream developmental stages of the protozoan parasite Trypanosoma brucei brucei are lysed by normal human serum. The cytotoxic factor is a minor sub-class of human high-density lipoprotein termed trypanosome lytic factor (TLF). T.b. brucei rapidly develops resistance to TLF when incubated at 26 degrees C under conditions which allow differentiation to the procyclic, insect midgut developmental stage. This in vitro differentiation system allowed us to correlate loss of TLF sensitivity with other parameters of differentiation to the procyclic form. The onset of resistance to TLF occurs within 2 hr after shifting bloodstream forms to differentiation conditions. TLF resistance is correlated with a rapid but transient decrease in protein synthesis by the parasite, is acquired prior to cell division at 26 degrees C, and precedes the loss of variant surface glycoprotein. In addition, we found binding and uptake of TLF by established procyclic trypanosomes was reduced approximately fivefold relative to bloodstream trypanosomes and the TLF binding observed in procyclics was nonspecific. No TLF was bound to the procyclic flagellar pocket membrane and the procyclics failed to endocytose any of the surface-bound TLF. In contrast, bloodstream forms bind TLF via a flagellar pocket-localized protein and bound TLF is taken up by endocytosis. These findings suggest that resistance of procyclic T. b. brucei to TLF-mediated lysis is due to a reduction in the endocytosis of TLF by this developmental stage of the parasite. PMID- 7556565 TI - Plasmodium falciparum: parasites defective in early stages of gametocytogenesis. AB - Some molecular characteristics of Plasmodium falciparum lines which do not produce gametocytes are described. Parasites carrying a subtelomerically deleted chromosome 9 cannot form even the earliest forms of gametocytes, detectable with antibodies against the gametocyte-specific antigen Pfg27. In a parasite culture of clone HB3, in which both intact and deleted forms of chromosome 9 are present, full-length chromosome 9 molecules are retained mainly in gametocytes. These data suggest that the subtelomeric portion of chromosome 9 is required at an early stage of gametocytogenesis. Parasite subclones derived from gametocyte producing clone 3D7, which completely lost ability to produce gametocytes, are also described. Unlike the previous gametocyteless lines, these parasites stably maintain a full-length chromosome 9 and the ability to cytoadhere to C32 melanoma cells after prolonged asexual propagation. Their defect in sexual development is therefore genetically and functionally distinct from that of parasites carrying a deleted chromosome 9. Gametocyteless subclones derived from 3D7 do not produce any Pfg27 mRNA, while this gene is anomalously expressed in asexual stage parasites of two lines of a different genetic background, 1776sel8 and C10, one able and the other unable to produce gametocytes. PMID- 7556560 TI - Plasmodium: genus-conserved primers for species identification and quantitation. AB - Stable RNAs have regions of primary sequence that are nearly identical in every member of the Plasmodium genus and not found in the host or in other common pathogens. Several "genus-conserved" sequences, which flank hypervariable regions, were identified within the small subunit ribosomal RNA of Plasmodium species. Primers based on these conserved sequences permit amplification of species- or possibly even strain-specific sequences from samples of unknown composition. As an example of this approach, sequences from the four human malaria species were successfully recovered from Giemsa-stained blood smears, including two different sequences for Plasmodium ovale (of 91.5% similarity). This type of information is useful for epidemiological and phylogenetic analysis of any malaria species. We show that amplification of rRNA-derived sequences behaves in a competitive fashion during the cycles of polymerase amplification and therefore target sequences from Plasmodium species are amplified in proportion to their abundance in the sample. There are several implications of this finding. (1) The proportion of different products resulting from amplification from samples with mixed infections is closely related to the proportion of infecting species. (2) Direct quantitation of parasite nucleic acids within a sample can be derived when known amounts of competitor RNA are added to the RT/PCR reaction. (3) Amplification of rRNA sequences, using genus specific primers, allows one to monitor the development of the parasite in the mosquito. PMID- 7556566 TI - Extracellular matrix in animal development. Role of extracellular matrix in animal development--an introduction. PMID- 7556567 TI - Tenascins, a growing family of extracellular matrix proteins. AB - The tenascins are a family of large multimeric extracellular matrix proteins consisting of repeated structural modules including heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III repeats, and a globular domain shared with the fibrinogens. The tenascins are believed to be involved in the morphogenesis of many organs and tissues. To date three members of the tenascin family have been described, tenascin-C, tenascin-R, and tenascin-X. Tenascin-R seems to be specific for the central and peripheral nervous system, tenascin-X is most prominent in skeletal and heart muscle, while tenascin-C is present in a large number of developing tissues including the nervous system, but is absent in skeletal and heart muscles. Tenascin-C was the original tenascin discovered, partly because of its overexpression in tumors. Inferring from cell biological studies, it has been proposed that tenascin-C is an adhesion modulating protein. PMID- 7556568 TI - Syndecan family of cell surface proteoglycans: developmentally regulated receptors for extracellular effector molecules. AB - Syndecans are a family of integral membrane proteoglycans with conserved membrane spanning and intracellular domains but with structurally distinct extracellular domains (ectodomains). They are known to function as heparan sulphate co receptors in fibroblast growth factor signalling as well as to link cells directly to the extracellular matrix. These and other biological activities of syndecans involve specific interactions of the heparan sulphate side chains of syndecans with cytokines and extracellular matrix proteins. Four different vertebrate syndecans, designated as syndecans 1-4 (or syndecan, fibroglycan, N syndecan and amphiglycan, respectively), are known. During embryonic development, syndecans have specific and highly regulated expression patterns that are distinct from the expression in adult tissue, suggesting an active role in morphogenetic processes. The developmental expression of syndecans is particularly intense in mesenchymal condensates and at epithelium mesenchyme interfaces, where a number of heparan sulphate-binding cytokines and matrix components are also expressed in a regulated manner, often spatially and temporally co-ordinated with the syndecan expression. Recent evidence indicates that the regulation of heparan sulphate fine structure (mainly the number and arrangement of sulphate groups along the polymer) provides a mechanism for the cellular control of syndecan-protein interactions. Furthermore, morphogenetically active cytokines such as fibroblast growth factor-2 and transforming growth factor-beta participate in the regulation of syndecan expression and glycosaminoglycan structure. This review discusses the developmental expression and binding functions of syndecans as well as the molecular regulation of specific heparan sulphate-protein interactions. PMID- 7556574 TI - Length-tension dependence in vascular smooth muscle: possible participation of endothelin. AB - The results of experiments with isolated strips of rat portal vein, and of 'sandwich' experiments involving strips of the portal vein or aorta, revealed the involvement of endothelin in the development of myogenic reactions of the vascular smooth muscle, observed on distension of the vascular wall. Released by the endothelial cells, endothelin is capable of stimulating the contraction of these muscles. PMID- 7556573 TI - Effect of royal jelly on serum lipids in experimental animals and humans with atherosclerosis. AB - The primary objective of this review was to assess the size and consistency of Royal Jelly (RJ) effect on serum lipids in experimental animals and humans. The data from animal studies were pooled, where possible, and statistically evaluated by Student's t-test. Meta-analysis was used for the evaluation of human trials. It was found that RJ significantly decreased serum and liver total lipids and cholesterol levels in rats and rabbits and also retarded the formation of atheromas in the aorta of rabbits fed a hyperlipemic diet. Meta-analysis of the controlled human trials of RJ to reduce hyperlipidemia showed a significant reduction in total serum lipids and cholesterol levels and normalization of HDL and LDL as determined from decrease in beta/alpha lipoproteins. The best available evidence suggests that RJ at approximately 50 to 100 mg per day, decreased total serum cholesterol levels by about 14%, and total serum lipids by about 10% in the group of patients studied. PMID- 7556569 TI - The extracellular matrix during heart development. AB - The embryonic extracellular matrix, which is comprised of glycosaminoglycans, glycoproteins, collagens, and proteoglycans, is believed to play multiple roles during heart morphogenesis. Some of these ECM components appear throughout development, however, certain molecules exhibit an interesting transient spatial and temporal distribution. Due to significant new data that have been gathered predominantly in the past 10 years, a comprehensive review of the literature is needed. The intent of this review is to highlight work that addresses mechanisms by which extracellular matrix influences vertebrate heart development. PMID- 7556575 TI - The influence of prostacyclin (PGI2) on contractile properties of isolated right ventricle of rat heart. AB - The mechanism of the positive inotropic effect of prostacyclin (PGI2) (2.6 x 10( 6) mol/l) on the isolated right ventricle of rat heart was studied. Our results show that the positive inotropic effect of prostacyclin is produced indirectly through beta adrenoceptors and slow Ca2+ channels, because blockade of slow Ca2+ channels with verapamil (10(-6) mol/l) and beta adrenoceptors with propranolol (10(-6) mol/l) abolishes this effect. Alpha adrenoceptors do not mediate the action of PGI2. PMID- 7556576 TI - The distribution of TM-316-associated surface antigen on polymorphonuclear leucocytes: an immunoelectron microscopic study. AB - It has been established that MoAb TM-316 recognizes an epitope on leucocytes and specifically inhibits the chemotactic behavior of leucocytes. In the present paper, the distribution of this epitope on the cell surface and in intracellular organelles was studied by immunoelectron microscopy. Leucocytes separated from the blood of healthy men and from synovial fluid from patients suffering from rheumatoid arthritis were used. They were fixed with a mixture containing paraformaldehyde, glutaraldehyde and picric acid. As the second antibody, goat anti-mouse IgM conjugated to 10 nm gold colloids was employed. In normal specimens, the epitope was found to some extent on the cytoplasmic membrane of neutrophilic leucocytes, but it was only sparsely distributed on eosinophilic and basophilic leucocytes. On activated neutrophilic leucocytes, obtained from the synovial fluid of rheumatoid arthritis patients, the immunolabeling was markedly increased. The number of sites where the epitope occurs on the surface of leucocytes is thus associated with the cell type, and also with the level of activation of the leucocytes. In order to investigate the processing of the antigen, the intracellular localization of the epitope in the neutrophilic leucocytes was also studied. The epitope recognized by TM-316 was also detected in/on the characteristic granules and Golgi stacks. PMID- 7556577 TI - IgE-binding trypsin inhibitors in plant pollen extracts. AB - In an attempt to access the possible role of protease-antiprotease mechanisms of non-immune defence in pollinosis, only low levels of trypsin-, kallikrein- or plasmin-like proteinases could be detected in aqueous pollen extracts. In contrast, several pollen species displayed appreciable trypsin inhibitory activity, e.g. Parietaria, Olea, Ambrosia, Rumex, Chenopodium, Holcus and Poa spp. These proteins of the serpin family of anti-proteinases were found to bind specific IgE-antibodies from the serum of hay fever patients. As examples, the IgE-binding trypsin inhibitors from the pollen of Parietaria judaica and Ambrosia elatior were purified and characterized as acidic proteins with pI 4.2 and a molecular weight of 20-24 kDa. PMID- 7556578 TI - Pentoxifylline attenuates acute lung injury induced by microemboli. AB - Pentoxifylline (PTX), a methylxanthine derivative, effectively prevents acute lung injury in different animal models. To investigate whether PTX would attenuate acute lung injury induced by microemboli resulting from treatment with calcium chloride (CaCl2) suspension, an isolated blood-perfused rat lung model was used. Pretreatment with PTX prevented the increase in pulmonary arterial pressure (PAP), lung weight gain and protein concentration in the lavage fluid after CaCl2 treatment. PMID- 7556570 TI - Extracellular matrix components in intestinal development. AB - Intestinal morphogenesis and differentiation are dependent on heterotypic cell interactions between embryonic epithelial cells (endoderm) and stromal cells (mesenchyme). Extracellular matrix molecules represent attractive candidates for regulators of these interactions. The structural and functional diversity of the extracellular matrix as intestinal development proceeds is demonstrated by 1) spatio-temporal specific expression of the classically described constituents, 2) the finding of laminin and collagen IV variants, 3) changes in the ratio of individual constituent chains, and 4) a stage-specific regulation of basement membrane molecule production, in particular by glucocorticoids. The orientation/assembly of these extracellular matrix molecules could direct precise cellular functions through interactions via integrin molecules. The involvement of extracellular matrix, and in particular basement membrane molecules in heterotypic cell interactions leading to epithelial cell differentiation, has been highlighted by the use of experimental models such as cocultures, hybrid intestines and antisense approaches. These models allowed us to conclude that a correct elaboration and assembly of the basement membrane, following close contacts between epithelial and fibroblastic cells, is necessary for the expression of differentiation markers such as digestive enzymes. PMID- 7556571 TI - Role of laminin-nidogen complexes in basement membrane formation during embryonic development. AB - Laminin and nidogen (entactin) are major glycoprotein components of basement membranes. At least seven different isoforms of laminin have been identified. Laminin and nidogen form high affinity complexes in basement membranes by specific binding between the laminin gamma 1 chain and the G3 globule of nidogen. Additional interactions between nidogen and collagen IV, perlecan and other basement membrane components result in the formation of ternary complexes between these matrix components. Nidogen is highly susceptible to proteolytic cleavage, and binding to laminin protects nidogen from degradation. Nidogen is considered to have a crucial role as a link protein in the assembly of basement membranes. Basement membrane components are synthesized at high levels during tissue growth and development, and sites of morphogenesis correlate with localized remodelling of basement membranes. The formation of distinct basement membrane matrices in the developing embryo is influenced by the laminin isoforms produced and by whether laminin and nidogen are co-expressed and secreted as a complex or are produced by cooperation between two cell layers. The potential roles of laminin nidogen complexes, cell-matrix interactions, and other intermolecular interactions within the matrix in basement membrane assembly and stability are discussed in this review. PMID- 7556572 TI - The extracellular matrix of the hematopoietic microenvironment. AB - The bone marrow microenvironment plays an important role in promoting hematopoietic progenitor cell proliferation and differentiation and the controlled egress of these developing hematopoietic cells. The establishment of long-term bone marrow cultures, which are thought to mimic hematopoiesis in vitro, and various stromal cell lines has greatly facilitated the analysis of the functions of this microenvironment. Extracellular matrix (ECM) molecules of all three categories (collagens, proteoglycans and glycoproteins) have been identified as part of this microenvironment and have been shown to be involved in different biological functions such as cell adhesion and anti-adhesion, binding and presentation of various cytokines and regulation of cell growth. It is suggested that these matrix molecules in combination with cytokines are crucial for compartmentalization of the bone marrow. Although many cell adhesion molecules have been characterized on the surface of hematopoietic progenitor cells, the nature of cellular receptors for the ECM components is less well defined. During leukemia, many immature blood cells are released from bone marrow, but it is not yet known whether these abnormalities in hematopoiesis are also caused by an altered microenvironment or altered composition of its extracellular matrix. The elucidation of the involvement of specific ECM-isoforms and as yet not characterized ECM components and their receptors in the bone marrow will certainly help towards a better understanding of these phenomena. PMID- 7556579 TI - Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea. AB - The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10 phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN. PMID- 7556581 TI - Chronic overcrowding decreases cytoplasmic free calcium levels in T lymphocytes of aged CBA/CA mice. AB - Intracellular calcium concentration is a sensitive marker of the homeostasis of living cells, and its increase is an essential step of T lymphocyte activation. Changes in the environment provoke an adaptive stress-response of the organism. In our present work we have investigated the effect of chronic overcrowding on resting and lectin-stimulated cytoplasmic free calcium concentration of splenic T lymphocytes from young and aged CBA/CA mice (50 animals total). The animals were kept under 'normal' (68 cm2/animal) or 'overcrowded' (22 cm2/animal) conditions for 3 months. Young animals showed no change in resting and stimulated calcium after overcrowding. T cells from aged mice, however, displayed significantly smaller levels of both resting and lectin-stimulated intracellular calcium concentration (p < 0.01 each), as compared to those of the non-stressed, aged animals. This inadequate adaptation in the calcium metabolism of T lymphocytes may significantly contribute to the diminished immune response of the aged in stress. PMID- 7556580 TI - Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo. AB - The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching. PMID- 7556582 TI - Nitroglycerin inhibits the phosphorylation of intermediate filament proteins rather than myosin light chain on porcine coronary artery sustained contraction. AB - The smooth muscle relaxation induced by nitroglycerin is hypothesized to be mediated by an increase in the cytoplasmic concentration of guanosine 3',5' monophosphate (cGMP) and subsequent dephosphorylation of the 20-kilodalton myosin light chain (MLC). We investigated this hypothesis in procine coronary arterial smooth muscle stimulated with histamine (3 microM) or K+ (30 mM). Stimulation of [32P]Pi-labeled muscle with histamine or K+ for 2 min resulted in a four- or 6.2 fold increase, respectively, in the incorporation of 32P into MLC. After 48 min of exposure to histamine, MLC phosphorylation decreased to the basal level and the phosphorylation of desmin, synemin, and of three unidentified cytosolic proteins was increased. K+ stimulation resulted in a sustained increase of MLC phosphorylation but had no effect on the phosphorylation of desmin, synemin, or the three unidentified cytosolic proteins. Application of nitroglycerin (1 microM) 48 min after histamine stimulation inhibited the phosphorylation of desmin, synemin, and the three cytosolic proteins. The sustained phase of histamine-induced contraction was also inhibited to a greater extent then the acute phase of histamine-induced contraction and both the acute and sustained phases of K(+)-induced contraction. These results suggest that MLC phosphorylation is required for both phases of K(+)-induced contraction, whereas phosphorylation of intermediate filament proteins is required for the sustained phase of histamine-induced contraction. Intermediate filament proteins, rather than MLC, may also be the target for the relaxant action of nitroglycerin during histamine-induced sustained contraction. PMID- 7556583 TI - The sterilization of honey with cobalt 60 gamma radiation: a study of honey spiked with spores of Clostridium botulinum and Bacillus subtilis. AB - Unprocessed honey is a recognized wound-healing remedy. However, to make clinical use of honey acceptable, it should be sterile. To find the lowest dose of irradiation needed for sterilization, six batches of honey (a-f) were gamma irradiated with 6, 12, 18, 22 and 25 kGy Cobalt-60. After a dose of 25 kGy the antibacterial activity was not altered. Presumably glucose oxidase (EC 1.1.3.4), which produces hydrogen peroxide, is not easily damaged by irradiation. Amylase activity on the other hand was significantly reduced to 19%, 19%, 21%, 22%, 43% in batches a), b), c), d) and f) respectively, whereas no decrease was observed in batch e). All batches spiked with approximately 10(6) spores from Cl. botulinum or B. subtilis per 50 g honey proved to be sterile after irradiation with a dose of 25 kGy. Honey was also spiked with Cl. botulinum at up to 5000 spores per 50 g honey, which is the upper limit of natural contamination. The sterilizing dose in this case was 18 kGy. PMID- 7556585 TI - Hepatic hydroxylation of melatonin in the rat is induced by phenobarbital and 7,12-dimethylbenz[a]anthracene--implications for cancer etiology. PMID- 7556584 TI - Hormonal control of sexual receptivity in cockroaches. AB - Many animals exhibit specific behaviors associated with sexual receptivity only when they are reproductively competent. In insects with gonadal maturation cycles, these behaviors usually coincide with ovarian maturation. In the cockroach Blattella germanica, juvenile hormone (JH), produced by the corpora allata (CA), regulates female reproductive physiology. Various experimental manipulations, including ablation of the CA, therapy with JH analogs, CA denervation, ovariectomy, and changing nutrient quality, coupled with time-lapse video recording, support the hypothesis that JH also controls female sexual receptivity. A re-examination of the role of the CA in the maturation of male sexual readiness shows that, while sexual behavior develops in the absence of JH in both B. germanica and Supella longipalpa, JH accelerates the expression of sexual readiness. PMID- 7556587 TI - In vivo and in vitro reversibility of chlorphentermine-induced phospholipidosis in rat alveolar macrophages. AB - Chlorphentermine (CP) is a cationic, amphiphilic drug (CAD) that has been studied widely for its ability to induce phospholipidosis, a disorder characterized by excessive accumulation of cellular phospholipid and ultrastructural development of lysosomal lamellar bodies (LLBs) in the cell. The accumulation of inducing drug correlates with increasing phospholipids. In the present study, we examined the reversibility of this disorder in rat alveolar macrophages (AMs) following a 7-day treatment (30 mg/kg/day, ip). The reversibility of phospholipidosis was examined under in vivo conditions and under in vitro conditions in cell cultures for a period of up to 12 days. There was a marked reduction in cellular CP levels and phospholipid content after 4 days of recovery, both in vivo and in vitro; however, there was no indication of significant loss of LLBs. Beyond this time point, ultrastructural recovery from phospholipidosis lagged behind the biochemical recovery temporally and was somewhat less rapid in vitro than in vivo. By 12 days of recovery, AMs from both groups had recovered biochemically, but a moderate level of LLBs was still present in some AMs in the in vitro recovery group. The results of this study indicate that there are more similarities than differences when comparing the recovery of phospholipidotic cells in vitro to that occurring in vivo. We conclude that the use of cell cultures may prove valuable in studying the reversibility of CAD-induced phospholipidosis. PMID- 7556586 TI - Effect of glucose on the respiratory burst of circulating neutrophils from asthmatics. AB - The respiratory burst is dependent on a source of glucose. We wished to investigate the effect of glucose on the superoxide production of circulating neutrophils. Superoxide production of neutrophils was significantly enhanced by glucose concentration of from 1 to 50 mmole/liter in the medium. The neutrophils from asthmatics in both the acute and remission phases showed greater production of superoxide than those of controls. When the neutrophils were made to undergo the respiratory burst, initially in the absence of glucose, and thereafter in the presence of 5 and 20 mmole/liter glucose, the rate of superoxide formation with higher glucose medium was decreased in the control cells but significantly increased in the cells of the acute asthmatics in remission. It is concluded that glucose as an energy source is potentially critical in determining the rate of the respiratory burst and that the neutrophils from asthmatic subjects in some way have an enhanced uptake or metabolism of this substrate. Glycemic status may then have some role in determining the amount of superoxide production, and therefore airway inflammation, in asthma. PMID- 7556588 TI - Role of ischemia in rats with spinal cord injury induced by decompression sickness. AB - The microsphere technique was used to determine whether blood flow to the central nervous system and various organs is impaired in rats with spinal cord injury induced by decompression sickness. For this purpose cannulas were placed in the left ventricle of the rats for the injection of microspheres and in the tail artery as the reference site for withdrawal of blood for the calculation of cardiac output (CO) and blood flow (BF) and for measurement of blood pressure (BP) and heart rate (HR). The rats were then subjected to a simulated dive that by electrophysiologic criteria rapidly (within 60 min after diving) induces severe neurologic deficits in the cord. Microspheres were used to determine CO and BF before and at 10, 60, and 180 min after diving. CO, BP, and HR were not affected by diving. BF to various regions of the brain, heart, bone, and fat was also not affected by diving. BF decreased in the lung (40%) and skeletal muscle (50%) and increased in spinal cord (20%) at 10 min after diving. At 60 and 180 min after diving the only alterations seen were increases in hepatic arterial and portal BF. Analysis of the distribution of cardiac output showed that diving induced changes that essentially paralleled the BF changes described above. We conclude that perfusion in the central nervous system is maintained in rats with spinal cord injury induced by decompression sickness. These results indicate that ischemia does not play a role in the pathophysiology of neuronal injury in this model of decompression sickness. PMID- 7556589 TI - Skeletal muscle necrosis and regeneration after injection of BaH1, a hemorrhagic metalloproteinase isolated from the venom of the snake Bothrops asper (Terciopelo). AB - The effects of BaH1, a hemorrhagic metalloproteinase isolated from Bothrops asper venom, on mouse gastrocnemius muscle was investigated. The toxin induced severe hemorrhage within minutes after injection. Groups of necrotic muscle fibers were observed after the sixth hour, with evident disorganization of myofibrillar material. At the ultrastructural level myofibrils in these cells lost their regular arrangement, Z lines were absent, and sarcomeres were disoriented, although there was no evidence of myofilament clumping or hypercontraction. Plasma membrane was interrupted in many portions. Mitochondrial alterations included swelling and the formation of flocculent densities and dense intracristal plates. At 12, 24, and 48 hr necrotic cells had amorphous masses of myofilaments and abundant phagocytic cells were observed both within necrotic fibers and in the interstitial space. Fraction D-1, which contains the three hemorrhagic metalloproteinases BaH1, BH2, and BH3, did not cause direct muscle damage when incubated with gastrocnemius muscle in vitro. Upon intramuscular injection in mice this fraction induced a small but significant increment in muscle lactic acid levels. Observations carried out 7 and 14 days after BaH1 injection revealed some regenerating muscle fibers with centrally located nuclei. However, other areas had few regenerating fibers of reduced diameter, surrounded by abundant fibroblasts, fibrosis, calcification, and remnants of necrotic muscle cells. When BaH1 was injected together with B. asper myotoxin III, a myotoxic phospholipase A2 that induces myonecrosis but does not affect blood vessels, a poor muscle regeneration was observed. In contrast, if B. asper myotoxin III was injected alone, regeneration proceeded normally and successfully. PMID- 7556590 TI - Intercellular adhesion molecule-1 expression in experimental alcoholic liver disease: relationship to endotoxemia and TNF alpha messenger RNA. AB - We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship among intercellular adhesion molecule-1 (ICAM-1) expression, tumor necrosis factor-alpha (TNF-alpha), plasma endotoxin, and inflammatory changes in the liver. Rats were fed different dietary fats (saturated fat, corn oil, and fish oil) with ethanol; control rats were fed isocaloric amounts of dextrose instead of ethanol. At sacrifice the following were evaluated: liver pathologic changes, TNF-alpha mRNA by reverse transcription PCR, plasma endotoxin, and ICAM-1 by immunohistochemistry and immunoblot analysis. Upregulation of ICAM-1 in endothelial lining cells in central and portal veins was observed in rats showing evidence of pathologic changes. Rats fed fish oil and ethanol, which exhibited the most severe inflammation, also showed hepatocyte ICAM-1 staining. The presence of ICAM-1 staining, in general, correlated with the level of TNF-alpha mRNA expression and plasma endotoxin levels. Upregulation of ICAM-1 in rats fed ethanol may contribute to the inflammatory changes seen in this model. The association between ICAM-1 upregulation and endotoxin and TNF-alpha mRNA suggests a role for these mediators in the inflammatory process in alcoholic liver injury. PMID- 7556592 TI - Effects of thrombin and the thrombin receptor activating peptide, SFLLRN, on redistribution of platelet alpha-granule contents are similar and independent of the extent of thromboxane formation. AB - Immunocytochemistry with gold-labeled antibodies was used to compare the effects of stimulation of human platelets with thrombin (1 U/ml) and the thrombin receptor activating peptide, SFLLRN (20 microM). After 3 min, redistribution of fibrinogen, von Willebrand factor, and P-selectin (GMP-140, CD62) was examined, the percentages of [14C]serotonin and beta-thromboglobulin released from pre labeled platelets were measured, and the amount of thromboxane B2 formed was assayed. Upon stimulation with either thrombin or SFLLRN, the platelets had changed from their normal disc shape to spheroidal forms with short pseudopodia. Few alpha-granules remained, the open canalicular system was expanded (more so with SFLLRN) and contained most of the fibrinogen and von Willebrand factor, although small amounts were evident on the platelet surface. Most of the P selectin was on the surface. Both thrombin and SFLLRN caused complete release of beta-thromboglobulin and 88.3 and 77.5% release of [14C]serotonin, respectively. However, formation of TXB2 caused by thrombin was 10 times greater than that caused by SFLLRN (969 +/- 173 vs 76 +/- 22 ng/10(9) platelets). Thus, the redistribution of platelet alpha-granule contents is similar with thrombin or SFLLRN stimulation and is unaffected by the extent of thromboxane formation. PMID- 7556591 TI - Nuclear p53 immunoreactivity in papillary thyroid cancers is associated with two established indicators of poor prognosis. AB - The tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic alterations. Cells harboring mutant or inactivated wild type p53 protein are at risk for the development of genomic instability. Nuclear accumulation of p53 protein is associated with the stepwise dedifferentiation of papillary carcinoma. We asked whether nuclear p53 accumulation is associated with two known indicators of poor prognosis in papillary carcinoma. We studied 55 consecutive papillary cancers (28 from Russia, and 27 from upstate New York). Nuclear p53 immunoreactivity was assessed using a monoclonal antibody, DO-1, on Formalin-fixed paraffin-embedded specimens. The DNA index was determined by computerized image analysis of Feulgen-stained sections. Nearly all cases were well differentiated and none were associated with distant metastases or extrathyroidal invasion. All primary lesions were less than 4 cm in diameter, and almost all patients were female. Nuclear p53 immunoreactivity was associated with a high-risk group characterized by two known indicators of poor prognosis: age > 50, aneuploid DNA content, or both. In the high-risk group (N = 24) 33% of cases displayed nuclear p53 positivity, compared with only 6% in a low-risk group (N = 31) which lacked both features (P = 0.015, two-tailed Fisher exact test). Nuclear accumulation of immunoreactive p53 protein is associated with two established indicators of poor prognosis in papillary carcinoma of the thyroid. This result is consistent with the idea that aberrations in p53 function are associated with the stepwise loss of differentiation in this cancer. PMID- 7556594 TI - HPV16 E7 oncoprotein induces expression of a 110 kDa heat shock protein. AB - Heat shock protein genes are induced by various kinds of stress. Besides stress, the heat shock family gene hsp70 has been shown to be induced by growth stimulating agents such as the DNA virus oncoproteins and serum. Here, we report cloning of a novel cDNA that encodes a 100 kDa heat shock protein-related polypeptide as a human papillomavirus oncoprotein E7-inducible gene. E7 induces expression of this heat shock protein at the level of RNA synthesis. Moreover, the induction of this heat shock protein-mRNA was dependent on the conserved region 2 of the E7 protein, which is essential for binding to the proteins of the retinoblastoma family. PMID- 7556593 TI - Differentially expressed mRNAs as a consequence of oxidative stress in intact cells. AB - Intracellular redox conditions influence the activity of several transcription factors leading to a modulation of the expression of the genes controlled by these factors. We examined the changes in cell transcription patterns after oxidative stress induced by diethylmaleate (DEM). Using the differential display technique we identified several differentially expressed sequence tags, four of which are identical or highly homologous to sequences contained in the human cDNAs encoding vimentin, c-fos, cytochrome oxidase IV and ribosomal protein L4; another one corresponds to a transcript of the mitochondrial genome of unknown function. The remaining five cDNAs are not recorded in any sequence data bank. One of these, named Rox3, lights up two mRNA species of approximately 3400 and 3600 bp, significantly increased after treatment with DEM or with other oxidizing agents. This increase appears precociously after exposure to DEM and it is completely prevented by pretreatment with N-acetylcysteine. The Rox3 fragment was used to screen a cDNA library; one fully sequenced clone showed 100% homology with the putative human guanine nucleotide regulatory protein nep1. PMID- 7556595 TI - RNA duplex unwinding activity of alfalfa mosaic virus RNA-dependent RNA polymerase. AB - An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic virus infected tobacco is capable of synthesizing in vitro full-size RNAs of minus and plus polarities. However, the enzyme is not able to perform a complete replication cycle in vitro. The products were found to be completely base-paired to their templates. The enzyme was able to use double-stranded RNA as a template for RNA synthesis if it could initiate from a single-stranded promoter. The inability (of most) of our enzyme preparations to create a single-stranded initiation site could explain why they could not perform a complete replication cycle in vitro. This is the first report on duplex RNA unwinding activities by a plant viral RdRp. PMID- 7556600 TI - Acylation of adenylyl cyclase catalyst is important for enzymic activity. AB - Incubation of human thrombocytes in the presence of [3H]palmitic acid leads to incorporation of this fatty acid into the alpha subunit of Gs as described [Linder et al., Proc. Natl. Acad. Sci. USA 90 (1993) 3675-3679; Degtyarev et al., Biochemistry 32 (1993) 8057-8061] but also into the catalyst of adenylyl cyclase which has not been recognized before. Treatment of labeled membranes with hydroxylamine released the label from both components. Label incorporated into the catalyst could be identified as [3H]palmitate. At the same time chemical deacylation caused partial loss of adenylyl cyclase activity. PMID- 7556599 TI - The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1. AB - The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or Leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to protein serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning. PMID- 7556598 TI - Detection of alpha/beta-hydrolase fold in the cell surface esterases of Acinetobacter species using an analysis of 3D profiles. AB - The primary sequence of esterases from Acinetobacter lwoffii RAG-1 and A. calcoaceticus BD413 were compared with linearized structural sequences of two hundred proteins selected from Brookhaven Protein DataBank using a modified version of the Bowie et al. algorithm [3]. Significant structural homology was found to alpha/beta proteins and specifically to those with the alpha/beta hydrolase fold for which the crystal structure was reported. No such homology was detected using common primary sequence alignment programs such as FASTA or BLAST. PMID- 7556597 TI - The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120. AB - A major acute phase protein (pig-MAP) has been isolated from the sera of pigs after turpentine injection. The protein is the pig counterpart of a recently cloned human serum protein denominated PK-120, which is a putative substrate for kallikrein [Nishimura et al., 1995 FEBS Lett. 357, 207-211]. The protein exists in other mammalian species and it is also an acute phase protein, at least in the rat. Pig-MAP shows homology, as PK-120, with the heavy chain 2 (HC-2) of the inter-alpha-trypsin inhibitor superfamily but does not possess trypsin inhibitory activity. PMID- 7556601 TI - Tumorigenicity of EBNA2-transfected cells. AB - The Epstein-Barr virus nuclear antigen 2 (EBNA2) gene is thought to be important for transformation by Epstein-Barr virus (EBV), but the mechanism of this transformation is little understood. Here, to examine the transforming ability of EBNA2, we transfected a rat fibroblast cell line F2408 with a recombinant EBNA2 expression plasmid and examined cell morphology, colony formation in soft agar, and tumorigenicity in nude mice. The morphology of transfected clones was similar to those of untransfected cells, but two of seven clones grew in soft agar, and four clones of seven clones reproducibly formed tumors in nude mice. These four clones showed EBNA2 expression, but non-tumorigenic clones did not. These results indicate that the expression of EBNA2 is correlated with tumorigenicity. PMID- 7556596 TI - Modulation of soybean lipoxygenase expression and membrane oxidation by water deficit. AB - The modulation of the activity and expression of soybean lipoxygenases 1 (LOX-1) [Vliegenthart, J.F.G. and Veldink, G.A. (1982) in: Free Radicals in Biology (Pryor, W.A., Ed.) pp. 29-64, Academic Press, New York] and 2 (LOX-2) by water deficit (osmotic stress) has been investigated, by following gene expression at the transcriptional and translational levels. Osmotic stress enhanced the transcription of the genes of both isoenzymes, leading to increased levels of the corresponding mRNAs, protein contents and specific activities. Abscisic acid (ABA) did not mediate enhancement of LOX expression, but caused a decrease of LOX 2 activity and was ineffective on LOX-1. Water deficit also caused oxidative modification of soybean membrane pool lipids [Schmidt, W.E. and Ebel, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4117-4121], attributable to the increase of conjugated hydroperoxides in the esterified fatty acids of the lipid bilayer. PMID- 7556602 TI - Dramatic effects of external alkalinity on neuronal calcium recovery following a short-duration glutamate challenge: the role of the plasma membrane Ca2+/H+ pump. AB - Alkalinization of the external medium has been shown to suppress Ca2+ extrusion from neurons due to inhibition of the plasmalemmal Ca2+/H+ pump. In our experiments on fura-2-loaded rat cerebellar granule cells and mouse hippocampal neurons, an increase in pHo from 7.4 to 8.5 following a 1-min glutamate or NMDA challenge caused a dramatic delay in [Ca2+]i recovery which in some cases was accompanied by an additional increase in [Ca2+]i. Normalization of pHo, or removal of Ca2+ from the alkaline solution allowed [Ca2+]i to decrease rapidly again. External alkalinity did not affect the initial rapid decline in [Ca2+]i following a 25 mMK+ pulse. In cerebellar granule cells, the alkaline pHo considerably increased the 45Ca2+ uptake both at rest and following a 2-min GLU pulse. A comparison of these effects of alkaline pHo with those produced by removal of the external Na+ led us to conclude that the Ca2+/H+ pump plays a dominant role in the mechanism of the fast Ca2+ extrusion from glutamate- or NMDA treated neurons. PMID- 7556603 TI - Identification of functional ionotropic glutamate receptor proteins in pancreatic beta-cells and in islets of Langerhans. AB - The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of beta-cell membranes was performed with subunit specific antibodies. We identified NMDAR1 subunits of the NMDA and KA-2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. beta-cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic beta-cells. PMID- 7556604 TI - Effect of cyclosporin A on energy coupling in pea stem mitochondria. AB - Effect of cyclosporin A on energy coupling in pea stem mitochondria is studied. It is found that incubation of mitochondria with 100 nM FCCP and/or CAtr, oligomycin, CaCl2, palmitate and ADP results, after some lag phase, in a collapse of delta psi generated by succinate oxidation in the presence of rotenone. Cyclosporin A (0.2-0.8 nmol/mg mitochondrial protein) markedly increases the lag phase. The cyclosporin A effect requires dithioerythritol to be added to the isolated medium. Metabisulphite fails to substitute for dithioerythritol. The relationships between these effects and cyclosporin A-sensitive mitochondrial permeability transition in animal mitochondria are discussed. PMID- 7556605 TI - The difference between ADP-beryllium fluoride and ADP-aluminium fluoride complexes of the spin-labeled myosin subfragment 1. AB - Electron paramagnetic resonance (EPR) spectroscopy was used for investigation of the structure of spin-labeled myosin subfragment 1 (S1) containing ADP and phosphate analogues, such as orthovanadate, aluminium fluoride (AlF4), and beryllium fluoride (BeFx). It has been shown that the local conformational changes in the region of Cys-707, induced by formation of the S1-ADP-BeFx complex, differ from those of S1 containing ADP-AlF4 or other phosphate analogues but are similar to the changes which occur in the presence of ADP or ATP gamma S. It is suggested that S1-ADP-AlF4 and S1-ADP-BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**-ADP-P(i) and S1*-ATP, respectively. PMID- 7556606 TI - Complete amino acid sequence of the protease inhibitor from buckwheat seeds. AB - The complete amino acid sequence of protease inhibitor BWI-1 from buckwheat (Fagopyrum esculentum Moench) seeds has been established by automatic Edman degradation and mass spectrometry. The molecule of the inhibitor consists of 69 amino acid residues, with a molecular mass calculated as 7743.8 Da. The active site of the inhibitor contains an Arg45-Asp46 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the proteinase inhibitor I family. PMID- 7556607 TI - Reversed electron transfer through the bc1 complex enables a cytochrome c oxidase mutant (delta aa3/cbb3) of Paracoccus denitrificans to grow on methylamine. AB - In Paracoccus denitrificans four classes of redox proteins are involved in the electron transfer from methylamine to oxygen:methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome c oxidase. MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. At least three different cytochromes c and two types of cytochrome c oxidase, cytochromes aa3 and cbb3, have previously been proposed to participate in the electron transfer pathways from methylamine to oxygen. In this study, participation of both cytochrome c oxidases and of the quinol oxidase (cytochrome bb3) has indeed been confirmed by analysis of a series of oxidase mutants. Interestingly, a P. denitrificans cytochrome c oxidase mutant (delta aa3/cbb3) retains the capacity to oxidise methylamine. It is demonstrated that the oxidation of the cytochrome c pool in this mutant does not proceed via an alternative cytochrome c oxidase, but rather via an 'uphill' electron transfer through the bc1 complex to ubiquinone, driven by the membrane potential. The subsequent oxidation of ubiquinol proceeds via the only remaining terminal oxidase, the bb3-type quinol oxidase. PMID- 7556609 TI - Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C terminal fragment containing both a copper and quino-cofactor. AB - Limited proteolysis of recombinant Hansenula polymorpha yeast amino oxidase produces a 48 kDa fragment which corresponds to the C-terminal two-thirds of the protein. The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase. PMID- 7556608 TI - The S100 family protein MRP-14 (S100A9) has homology with the contact domain of high molecular weight kininogen. AB - The heterodimeric molecule MRP-8/MRP-14 (S100A8/S100A9) is abundantly expressed in circulating monocytes and neutrophils. We report here an homology between the C-terminal 'tail' region of MRP-14 (S100A9) and sequences within the plasma protein, high molecular weight kininogen (HMWK) which are involved in binding to negatively charged surfaces such as kaolin. MRP-14 also binds to kaolin and is competitively inhibited by HMWK and by peptides corresponding to MRP-14 tail and the HMWK 'contact' regions. Furthermore both MRP-14 and the tail peptide inhibit the coagulation cascade in vitro giving functional relevance to the homology between MRP-14 and HMWK. At inflammatory sites, MRP-8/14 is localised to areas of close contact between myeloid cells and endothelium. The results of this study identify a potential binding region in MRP-14 and suggest that it could function by interfering with fibrin formation at sites of leukocyte transendothelial migration. PMID- 7556610 TI - High-resolution atomic-force microscopy of DNA: the pitch of the double helix. AB - Using a cationic lipid bilayer, we show that DNA can be reliably adsorbed to the bilayer surface for atomic force microscopy (AFM) in aqueous buffers at high resolution. The measured width of the dsDNA is close to 2 nm, and a periodic modulation on dsDNA is reproducibly detected by the AFM. The measured period is 3.4 +/- 0.4 nm, in excellent agreement with the known pitch of the double helix. The right-handedness of the double helix is directly discernible in high resolution AFM images. Thus, this approach can be readily applied to the study of DNA-protein interactions, as well as sequence mapping at high resolution. PMID- 7556611 TI - Water-soluble fatty acid derivatives as acylating agents for reversible lipidization of polypeptides. AB - A novel method allowing the conjugation of a fatty acid to a peptide or protein in aqueous buffer is described in this paper. L-Cysteinyl 2-pyridyl disulfide (CPD) (III), which was obtained by reacting L-cysteine (I) with 2,2 dithiopyridine (II), was reacted with the N-hydroxysuccinimide ester of palmitic acid (IV) to yield a water-soluble derivative of palmitic acid, termed Pal-CPD (V). Pal-CPD (V) could be reacted with a sulfhydryl-containing peptide or protein in aqueous buffer to yield the palmitic acid-derivatized conjugate (VI). The palmitic acid-derivatized Bowman-Birk protease inhibitor (BBI), synthesized using this conjugation method, was demonstrated to have 140-fold higher uptake into Caco-2 cell monolayers compared to native-BBI. The biological activity of the conjugate, as assessed using an in vitro transformation assay, was retained. PMID- 7556614 TI - Impact of erythrocytes on mouse embryonal development in vitro. AB - To elucidate the role of reactive oxygen species (ROS) in the development of mouse embryo, effects of superoxide dismutase (SOD), catalase and erythrocytes were studied in vitro. Oocytes were fertilized and 2-cell-cleaved embryos were cultured in the presence or absence of either erythrocytes, SOD or catalase. Under standard culture conditions, the fertilization and cleavage rates were 77.4 and 12.5%, respectively. In the presence of xanthine and xanthine oxidase, those rates decreased to 28.2 and 4.5%, respectively. The hazardous effect of ROS was completely inhibited by erythrocytes. These results suggested that small amounts of erythrocytes might effectively degrade ROS during the development of cultured embryos. PMID- 7556615 TI - Inhibition of degranulation of human polymorphonuclear leukocytes by complement factor D. AB - A degranulation inhibiting protein could be isolated from human plasma ultrafiltrate by a three-step purification method including ion-exchange chromatography, gelfiltration and affinity-chromatography. The protein was identified as complement factor D by means of sequence analysis. Its degranulation inhibiting activity was determined with regard to its effect on the FNLPNTL-induced lactoferrin secretion of human polymorphonuclear leukocytes. Complement factor D caused a dose-dependent decrease of the FNLPNTL-stimulated lactoferrin degranulation down to 34% of stimulated controls. PMID- 7556612 TI - Identification of a Xenopus glutamine synthetase gene abundantly expressed in the embryonic nervous system but not in adult brain. AB - We used a PCR-based subtraction cloning procedure with concanavalin A-treated and -untreated animal caps from stage 9 Xenopus embryos to search for genes up regulated during early neural development. One such gene was found to encode a protein homologous to several known glutamine synthetases, and we named it xGS. Molecular hybridization studies revealed that xGS mRNA is maternally transmitted and abundantly expressed in neuroectoderm-derived tissues during the gastrula and neurula stages. The expression of xGS mRNA in the nervous system continues until the larval stages, but declines thereafter and becomes undetectable in adult brain. Considering its metabolic activity and potential neuroprotective effect against the neurotoxic substances such as glutamate and ammonia, the glutamine synthetase may play an important role in the early stages of vertebrate neural development. PMID- 7556613 TI - Stable expression of maize auxin-binding protein in insect cell lines. AB - To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and carboxy-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells. PMID- 7556616 TI - Interaction of the bacterial protein toxin alpha-haemolysin with model membranes: protein binding does not always lead to lytic activity. AB - alpha-Haemolysin interaction with model membranes has been investigated by a 2 fold procedure. First, protein binding has been measured, by a direct method as well as through changes in the intrinsic fluorescence of the protein when incubated with liposomes and divalent cations. Then, the above results have been correlated with the protein lytic activity. The extent of protein binding is not significantly modified by the presence or absence of Ca2+, or by changes in lipid composition, although these factors influence greatly the membrane lytic activity of the protein. Moreover, Ca2+ binding to the toxin must occur prior to protein binding to the bilayer, for a lytic effect to take place. PMID- 7556617 TI - Slow inactivation conserved in heteromultimeric voltage-dependent K+ channels between Shaker (Kv1) and Shaw (Kv3) subfamilies. AB - Single K+ channels were recorded under the cell-attached mode in Xenopus oocytes injected with an equal amount of mRNAs coding for NGK1 (Kv1.2) and NGK2 (Kv3.1a) voltage-dependent K+ channels. A new class of channels of 20 pS in conductance with three degrees of inactivation was observed. The results suggest that voltage dependent NGK1 Shaker and NGK2 Shaw K+ channels, from different subfamilies, assemble to form heteromultimeric K+ channels in Xenopus oocytes and show characteristics inherited from two parental channels. PMID- 7556619 TI - Comparison of recombinant cyclooxygenase-2 to native isoforms: aspirin labeling of the active site. AB - The search for isoform-specific enzyme inhibitors has been the focus of much recent research effort. Towards this goal, human recombinant cyclooxygenase-2 (EC 1.14.99.1, prostaglandin H synthase) was expressed in insect cells and purified to > 98% purity. Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX-2 and COX-1. After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX-1 and corresponded to the enzyme dimer as measured with size-exclusion chromatography. Peptide mapping via Lys-C protease identified a site of N-linked glycosylation and the aspirin covalent modification site. In the presence of heme, aspirin specifically acetylated Ser-516. The enzyme will be suitable for biophysical studies and may lead to isoform-specific enzyme inhibitors. PMID- 7556618 TI - Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta. AB - The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu-Leu-Tyr-Ala-Phe Pro-Arg-Val-amide, has a molecular weight of 974.6 and is blocked at both the amino and carboxyl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila. PMID- 7556621 TI - Diet- and diabetes-induced changes of ob gene expression in rat adipose tissue. AB - ob gene regulation is as yet unknown. We first examined whether the ob gene is under physiological control by the nutritional state. Fasting produced a sharp (95%) decrease of ob mRNA in epididymal and inguinal fat pads from 24 h onward. Refeeding rapidly (3-6 h) re-induced ob gene expression and corrected it within 24 h. Similar changes in fatty acid synthase (FAS) and GLUT4 mRNAs were observed, whereas phosphoenolpyruvate carboxykinase (PEPCK) mRNA showed an opposite evolution. We next examined the potential role of insulin. In adipose tissue of streptozotocin-diabetic rats, ob mRNA levels were decreased by 80%. Insulin treatment (4 days) only marginally increased ob mRNA, but restored euglycemia and overcorrected FAS, GLUT4 and PEPCK expression. In conclusion, we provide evidence for a physiological regulation of ob gene by variations in the nutritional state. We also show that ob expression is impaired in streptozotocin-diabetic rats and only slightly restored by insulin treatment, which suggests that ob gene is not or only minimally regulated by the hormone. PMID- 7556620 TI - The death domain motif found in Fas (Apo-1) and TNF receptor is present in proteins involved in apoptosis and axonal guidance. AB - The interaction of Fas (Apo-1) and TNF receptor-1 with their respective ligands can lead to cell death. The so-called death domain, a sequence motif present in the cytoplasmic portion of the two receptors, has been identified as a critical structural element involved in signal transduction that leads to apoptosis. Here we describe several additional proteins which contain a death domain. Novel members of this family include proteins known to be implicated not only in apoptosis but also in neuron guidance. PMID- 7556623 TI - Herbimycin A suppresses NF-kappa B activation and tyrosine phosphorylation of JAK2 and the subsequent induction of nitric oxide synthase in C6 glioma cells. AB - Herbimycin A, a potent tyrosine kinase inhibitor, suppressed nitric oxide synthase (NOS) induced by lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) in C6 glial cells. LPS activated NF-kappa B, and this effect was inhibited by pretreatment with herbimycin A. In addition, IFN-gamma activated the tyrosine protein kinase, JAK2, and tyrosine-phosphorylation by itself was also inhibited by herbimycin A. These results suggest that herbimycin A suppresses iNOS induction by inhibition of both NF-kappa B activation caused by LPS, and tyrosine phosphorylation of JAK2 caused by IFN-gamma in C6 glioma cells. PMID- 7556624 TI - Prothymosin alpha mRNA levels vary with c-myc expression during tissue proliferation, viral infection and heat shock. AB - Expression of prothymosin alpha, an acidic nuclear protein implicated in cellular proliferation, has been reported to be regulated by c-myc in vitro. We have studied the correlation of expression levels between prothymosin alpha and c-myc, using three different in vivo systems, viz. normal ontogenic process of placental development, lytic viral infection and heat shock treatment. The two genes have been found to share a similar expression pattern, although prothymosin alpha mRNA remains always detectable, indicating the existence of yet another mechanism, in addition to c-myc, which regulates its expression in vivo. PMID- 7556622 TI - Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance. AB - We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice. PMID- 7556625 TI - Expression and disulfide-bond connectivity of the second ligand-binding repeat of the human LDL receptor. AB - The human LDL receptor (LDLR) has a binding domain which consists of seven contiguous ligand-binding (LB) repeats, each approximately 40 amino acids long with three disulfide bonds. The second LB repeat, which is required for full binding of LDL, has been expressed, purified and folded to yield a single, fully oxidized isomer. By selective reduction and alkylation, we have shown that the cysteine residues have a I-III, II-V, IV-VI connectivity, matching that recently determined for the amino-terminal repeat. We suggest that the first two LB repeats of the LDLR, with their unique disulfide-bonding pattern, serve as a structural paradigm for other LB repeats. PMID- 7556626 TI - Characterization of RNA with unusual electrophoretic mobility from tissues of patients with Crohn's disease. AB - RNAs from tissues of patients with Crohn's disease that migrate off the diagonal in a two-dimensional gel electrophoresis system were partially characterized. One of the RNA species was a discrete cleavage product of region V2-9 of 28S rRNA; another is a conformer or variant of 5.8S rRNA; and a third is a mixture of unidentified fragments with mobility similar to that of 7S RNA. The yield of these species from resected tissue and their visualization by silver staining was very sensitive to the details of the preparative procedure. No evidence of viroid like RNA was found within the range of molecular sizes (< 7S) that we examined. PMID- 7556628 TI - Molecular flexibility in wheat gluten proteins submitted to heating. AB - Prolamin proteins are responsible for the network that gives wheat dough its viscoelastic properties. Non-prolamin depleted gluten was prepared under conditions that preserve its functionality. Electron Spin Resonance (ESR) was used to provide information about the dynamics of the protein at temperatures between 5 and 90 degrees C by specific spin labelling of its cysteine residues. The spectra were of a composite type, resulting from at least two populations of spin labels largely differing in molecular mobility. The correlation time of the less mobile nitroxide radicals was determined by saturation transfer ESR. Upon heating there was a transfer from the slow to the fast moving population of radicals, and an increase of mobility of this last catagory that followed the Arrhenius law. The effect of temperature on molecular flexibility was reversible. This was not the case for purified, polymerised glutenin subunits extracted from gluten. Urea created similar modifications on gluten as heat. PMID- 7556627 TI - Oxidized LDL induce hsp70 expression in human smooth muscle cells. AB - Heat shock protein 70 (hsp70) has been detected in atherosclerotic lesions, in which endothelial cells and smooth muscle cells are involved. In a previous report we showed that Ox-LDL, a causal factor in atherosclerosis, could induce hsp70 expression in cultured human endothelial cells [Zhu et al. B.B.R.C. 1994, 200:389]. Here, with immunofluorescence and immunoblotting techniques, we show that Ox-LDL are capable of inducing hsp70 expression also in human smooth muscle cells, and that this induction is dependent on cell density and on the concentration of Ox-LDL. The induced expression of hsp70 was higher in human umbilical vein smooth muscle cells than in a human smooth muscle cell line. Conversely, Ox-LDL was cytotoxic to both types of cells, more so to the human smooth muscle cell line. These observations indicate that Ox-LDL may be a stress responsible for hsp70 expression in atherosclerotic plaques and the presence of hsp70 in plaques may be a useful marker for continuous oxidative damage in the arterial wall. PMID- 7556629 TI - Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures. AB - Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 micrograms/ml increased the formation of thromboxane A2, prostaglandin D2, E2 and F2 alpha 6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 microM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes. PMID- 7556631 TI - Mouse sperm membrane potential: changes induced by Ca2+. AB - Mouse sperm resting membrane potential (Er) (-42 +/- 8.8 mV), determined with a potential sensitive dye, depended on extracellular K+ and, in the absence of extracellular Ca2+ ([Ca2+]e), on external Na+ ([Na+]e). Ca2+ addition (> 5 microM) to sperm in Ca-free media induced a transient hyperpolarization (Ca-ith) which strongly depended on [Na+]e and less on external Cl- ([Cl-]e). Cd2+ and Mn2+ (microM) mimicked the Ca2+ effect, but not Ba2+. The Ca-ith was partially inhibited by ouabain (74%, IC50 = 5.8 microM) and niflumic acid (38%, IC50 = 240 microM), indicating the participation of the Na-K ATPase and Cl- channels. In Ca free low-Na+ media, Ca2+ addition caused a depolarization sensitive to: nimodipine (25 microM), trifluoperazine (12.5 microM) and Mg2+ (1.2 mM), suggesting the participation of Ca2+ channels. Since some inhibitors of the sperm Ca-ith block the acrosome reaction (AR), both processes may share transport systems. PMID- 7556630 TI - IL-2-induced gene expression of protein-tyrosine phosphatase LC-PTP requires acidic and serine-rich regions within IL-2 receptor beta chain. AB - A protein-tyrosine phosphatase LC-PTP is preferentially expressed in hematopoietic cells and is an early response gene in lymphokine stimulated cells. Here, we found the LC-PTP mRNA induction by IL-2 was markedly inhibited by several tyrosine kinase inhibitors. The induction required both the acidic and serine-rich regions of the IL-2 receptor beta chain (IL-2R beta) in mouse IL-3 dependent pro-B BAF-B03 transfectants. This is strikingly different from the induction of c-myc gene expression, which requires the serine-rich region alone. In addition, overexpression of activated-Lck or -Raf kinases resulted in augmented LC-PTP mRNA expression in myeloid cell line 32D transfectants. Considering the previous findings that the acidic region of the IL-2R beta is responsible for association with Lck and activation of Raf kinase, IL-2-induced expression of LC-PTP mRNA may be primarily transduced through a Lck-Raf mediated signaling pathway. PMID- 7556632 TI - Oxygen equilibrium and electron paramagnetic resonance studies on copper(II) iron(II) hybrid hemoglobins at room temperature. AB - Copper(II)-iron(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunits are substituted with copper(II) protoporphyrin IX, have been prepared. The affinities of the ferrous-subunits in both hybrids for the first binding oxygen are as low as the affinity of deoxyhemoglobin under various solution conditions, indicating the equality of behavior in copper(II) protoporphyrin IX and deoxyheme. Electron paramagnetic resonance (EPR) examinations on these hybrids at room temperature show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the alpha subunits within a low affinity conformation of hemoglobin. These results suggest that copper(II) protoporphyrin IX is a useful EPR probe at room temperature for investigating the deoxyheme environment in hemoglobin. PMID- 7556633 TI - Isolation, characterization, and chromosomal location of a gene encoding the delta 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana. AB - A full-length cDNA and the corresponding At-P5S gene encoding the first enzyme of the proline biosynthetic pathway, the delta 1-pyrroline-5-carboxylate (P5C) synthetase, were isolated in Arabidopsis thaliana. The At-P5S cDNA encodes a protein of 717 amino acids showing high identity with the P5C synthetase of Vigna aconitifolia. Strong homology is also found at the N-terminus to bacterial and yeast gamma-glutamyl kinase and at the C-terminus to bacterial gamma-glutamyl phosphate reductase. Putative ATP- and NAD(P)H-binding sites are suggested in the At-P5S protein. The transcribed region of the At-P5S gene is 4.8 kb long and contains 20 exons. Southern analysis suggests the presence of only one At-P5S gene in the A. thaliana genome mapped at the bottom of the chromosome two. Expression analysis of At-P5S in different organs reveals abundant At-P5S transcripts in mature flowering plant. Rapid induction of the At-P5S gene followed by accumulation of proline was observed in NaCl-treated seedlings suggesting that At-P5S is osmoregulated. PMID- 7556635 TI - The voltage-dependent K+ channel (Kv1.5) cloned from rabbit heart and facilitation of inactivation of the delayed rectifier current by the rat beta subunit. AB - We have isolated a cDNA coding for a delayed rectifier K+ channel (RBKV1.5) from rabbit heart. The amino acid sequence of RBKV1.5 displays a homology to that of other K+ channels of Kv1.5 class. Overall amino acid identity between RBKV1.5 channel and Kv1.5 channel of other species is about 85%. RNA blot analysis revealed the expression of the primary transcript in various rabbit tissues, at the highest level in both the atrium and ventricle. When expressed in Xenopus oocytes, RBKV1.5 current showed a delayed rectifier type characteristics, which was converted to rapidly inactivating currents upon coexpression with a beta subunit. PMID- 7556634 TI - Activation of bee venom phospholipase A2 through a peptide-enzyme complex. AB - Phospholipase A2 activation by membrane-bound peptides was investigated in order to understand the role of the membrane-induced conformation on activation, and to examine the occurrence of a peptide-enzyme complex at the lipid/water interface. For the peptides studies, bee venom phospholipase A2 was stimulated regardless of the membrane-bound conformation (alpha-helix, beta-sheet or random coil). Using antisera raised against melittin, we were able to demonstrate the occurrence of a calcium-dependent complex involving the enzyme, phospholipid substrate, and peptide. PMID- 7556636 TI - Participation of rhop21 in serum-dependent invasion by rat ascites hepatoma cells. AB - Rat ascites hepatoma cells (MM1 cells) penetrate through a cultured mesothelial cell monolayer (MCL) in the presence of fetal calf serum (FCS), but scarcely do so in its absence. Inactivation of rhop21 of MM1 cells by ADP-ribosyltransferase C3 resulted in the suppression of this serum effect on the penetration, suggesting that the serum effect was mediated by rhop21. To ascertain this assumption MM1 cells were transfected with an activated (Val14) human rhoA cDNA (Neo/RhoA 1-7). The transfectants penetrated MCL extensively even in the absence of FCS and became largely independent of serum for the penetration. These results suggest that serum-induced invasion by MM1 cells is mainly mediated by rhop21. PMID- 7556637 TI - Import of sterols into mitochondria of the yeast Saccharomyces cerevisiae. AB - An in vitro assay was designed to study the import of radiolabeled ergosterol and cholesterol from unilamellar vesicles into isolated mitochondria of the yeast Saccharomyces cerevisiae. Supply of ergosterol to the mitochondrial surface was enhanced by a cytosolic fraction containing a lipid transfer protein, whereas no such additive to the assay was required for cholesterol transport. Both sterols reached the inner mitochondrial membrane. During import, they were detected in contact sites between the outer and the inner mitochondrial membrane supporting the idea, that these zones are sites of intramitochondrial lipid translocation. Transport of ergosterol between the outer and the inner mitochondrial membrane was not affected by addition of ATP, depletion of ATP caused by treatment of mitochondria with apyrase and oligomycin, and incubation with the uncoupler CCCP, indicating that this process is energy-independent. PMID- 7556638 TI - Protein expression of the alpha, gamma, delta and epsilon subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum. AB - Total protein kinase C (PKC) activity and protein expression of the alpha and delta subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contact-inhibited quiescent state (also known as G(o)) in the presence of serum. At the same time protein expression of PKC subspecies gamma and epsilon decreases while the beta I, beta II, iota and zeta subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the G(o) states brought about by contact inhibition or serum deprivation. PMID- 7556639 TI - A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+) dependent exocytosis in chromaffin cells. AB - SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca(2+)-evoked catecholamine release from digitonin permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca(2+)-dependent catecholamine release with an IC50 = 20 microM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca(2+)-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex. PMID- 7556640 TI - Transfection of cells with basic fibroblast growth factor and Kaposi fibroblast growth factor genes induce resistance to and receptor modulation of tumor necrosis factor. AB - Tumor necrosis factor (TNF) has been shown to inhibit the growth of some cell types and stimulate the proliferation of others by a mechanism that is not understood. In the present study, we investigated the effect of transfection of NIH-3T3 cells with either the basic fibroblast growth factor gene (bFGF) or the kaposi FGF gene (K-fgf) on the growth-modulatory effects of TNF. Our results show that transformation of cells with either gene leads to resistance to the growth inhibitory effects of TNF. The K-fgf gene was found to be a more potent inducer of cellular resistance than the bFGF gene. The cellular resistance correlated with the inhibition of TNF-induced activation of phospholipase A2 and down modulation of TNF receptors. Overall, our results indicate that both K-fgf and bFGF play an important role in suppression of antiproliferative effects of TNF. PMID- 7556641 TI - PDGF receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase. AB - Low M(r) phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW-PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW-PTP substrate/s we have expressed in NIH-3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW-PTP expressing cells has enabled us to demonstrate an association between LMW-PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW-PTP on the phosphorylated platelet derived growth factor receptor. PMID- 7556642 TI - RK-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G1phase. AB - A specific inhibitor of protein tyrosine phosphatase (PTPase), RK-682 (3 hexadecanoyl-5-hydroxymethyl-tetronic acid) was isolated from microbial metabolites. In vitro, RK-682 inhibited dephosphorylation activity of CD45 and VHR with IC50 54 and 2.0 microM, respectively. In situ, sodium orthovanadate and RK-682 enhanced the phosphotyrosine level of Ball-1 cells, a human B cell leukemia, but not the phosphoserine/threonine level. The PTPase inhibitors, however, had the different arrest point on the cell cycle progression. Sodium orthovanadate inhibited the cell cycle progression at G2/M boundary phase, on the other hand, RK-682 inhibited the G1/S transition. PMID- 7556644 TI - Isolation and partial cloning of ryanodine-sensitive Ca2+ release channel protein isoforms from human myometrial smooth muscle. AB - Partial cDNAs of the ryanodine receptor were cloned using PCR analysis from reverse transcribed total and mRNA, extracted from freshly isolated pregnant, non pregnant, and cultured human myometrial smooth muscle. The identity of these clones was confirmed by nucleotide sequencing of the fragments and indicate the expression of both the skeletal and brain ryanodine receptor isoforms in these preparations. In freshly isolated non-pregnant myometrial tissue, membrane fractions displaying specific [3H]ryanodine binding activities were isolated using density gradient centrifugation. SDS-PAGE of the sucrose gradient fractions indicated the specific comigration of a polypeptide with a molecular mass of approximately 544 kDa with the ryanodine binding activity. PMID- 7556645 TI - Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine 262 in the presence of heparin (or tubulin). AB - Tau protein, the major component of the aberrant structures termed paired helical filaments (PHFs) present in the brain of Alzheimer's disease patients, is pathologically phosphorylated in sites in and around the tubulin-binding sites. A single protein kinase, glycogen synthase kinase 3 (GSK 3), is able to phosphorylate tau at the flanking regions and, additionally, at the tubulin binding motifs if heparin or tubulin is present. Serines-262 and -324 have been found to be modified at the tubulin-binding region of tau protein by GSK 3 in the presence of heparin or tubulin. PMID- 7556646 TI - Pancreatic secretory trypsin inhibitor gene is highly expressed in the liver of adult-onset type II citrullinemia. AB - Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS is deficient specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there are no apparent abnormalities in the amount, translational activity, and nucleotide sequence of hepatic ASS mRNA. Recent results of homozygosity testing indicate that the primary defect of type II citrullinemia is not within the ASS gene locus. In this present work, to understand the pathogenesis and pathophysiology of type II citrullinemia, we have characterized the alterations of gene expression in the liver of type II patients using the recently developed mRNA differential display method. Some cDNA bands expressed differently in type II citrullinemia patients and control were selected, cloned, and sequenced. Nucleotide sequence analysis and homology searching revealed an interesting clone which has 99% homology with the human pancreatic secretory trypsin inhibitor (hPSTI). Northern blot and RT-PCR analyses showed that the expression of hPSTI mRNA increased significantly in the liver of all type II patients tested. Furthermore, the concentration of hPSTI protein was found to be higher in the liver of type II citrullinemia than in control. These results suggest that hPSTI may be related to the primary defect of type II citrullinemia and may be useful as a diagnostic marker, although the detailed mechanism of the high expression of hPSTI mRNA in type II liver is not yet known. PMID- 7556643 TI - A novel tau-tubulin kinase from bovine brain. AB - During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II. PMID- 7556647 TI - Isolation of a novel ras-recision gene that is induced by hydrogen peroxide from a mouse osteoblastic cell line, MC3T3-E1. AB - Hydrogen peroxide appears to mediate growth factor actions, and it inhibits DNA synthesis in normal mouse osteoblastic cells (MC3T3-E1) at non-toxic doses. However the sensitivity of cells to H2O2 is greatly decreased in their ras transformants. To understand the molecular basis of this sensitivity to H2O2, we attempted to identify H2O2-inducible cDNA clones from MC3T3 cells by differential screening of cDNA libraries, and one of such genes, named HIC-53, was isolated. The level of HIC-53 mRNA was moderately increased by H2O2 as well as by calcium ionophore or dexamethasone, but was not increased by the addition of serum, tumor promoting phorbol ester, or epidermal growth factor. Among mouse organs, HIC-53 mRNA levels were higher in the kidney and lung, but were almost undetectable in the brain, heart, bone, muscle or spleen. In MC3T3 cells transformed with v-Ki ras, the HIC-53 mRNA level was markedly decreased, and effect of H2O2 was abolished. Although the biological function of HIC-53 is unknown at present, the predicted amino acid sequence exhibited some similarity with bovine cardiac Na+/Ca+ exchanger. The nucleotide sequence of HIC-53 cDNA showed no significant similarity with other known gene sequences. PMID- 7556648 TI - Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro. AB - Three new cell lines of human glioblastoma have been established. These cells co expressed hepatocyte growth factor (HGF) and its receptor, c-Met, genes in vitro. Reverse-transcriptase/polymerase-chain reaction study revealed that the cells also expressed gene for HGF activator, a recently cloned serine proteinase, suggesting that HGF might have a role in glioma cells in vitro as an autocrine factor. The activator mRNA was also detected in other well-established glioma cell lines, glioma tissues and normal brain. The concomitant expression of HGF, HGF activator and c-met was also detected in one glioblastoma case in vivo out of five tested. PMID- 7556652 TI - The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution. AB - Cellulose-binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose-binding affinities of the CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reesei. The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose binding face. PMID- 7556650 TI - Somatostatinergic phenotype markers in the human neuroblastoma cell-line LA-N-2. AB - We have characterized somatostatinergic phenotype markers of the human neuroblastoma, LA-N-2. A single mRNA-transcript (approximately 850bp) and two cellular somatostatin immunoreactivity forms, a high molecular weight form (M(r) 15,000) and a fragment corresponding to somatostatin-28 was found, while the somatostatin-14 peptide was absent. Saturation binding experiments demonstrated a single class of high-affinity somatostatin receptors with Kd and Bmax of 0.27 +/- 0.03 nM and 45 +/- 1 fmol/mg protein. Partial G-protein uncoupling (30%) was demonstrated, using GTP gamma S, with an affinity of 9.7 nM. The LA-N-2 cell line, previously shown to be cholinergic, may serve as a simplified system to elucidate heterologous neurotransmittor interactions. Such studies are of interest since dysfunctions of the cholinergic basal forebrain neurons and somatostatin immunoreactive interneurons have been consistently observed in Alzheimer's disease. PMID- 7556651 TI - Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine alpha 2-macroglobulin. AB - The receptor-binding domains (RBDs) of human and bovine alpha 2-macroglobulin (alpha 2M) have been isolated after limited proteolysis of methylamine-treated alpha 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine alpha 2M give diffraction patterns suitable for X-ray analysis, and a complete dataset with a maximum resolution of 2.3 A has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3(1)21 or P3(2)21 with cell parameters a = b = 106.8 A, c = 72.2 A. PMID- 7556653 TI - Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins. AB - The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose-dependently inhibited the endogenous adenylyl cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi-like proteins as they were blocked by pertussis toxin. Upon co-transfection with the alpha subunit of Gz, the ability of melatonin to regulate both type II and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin-insensitive G proteins such as Gq, G12 and G13 were unable to interact with the melatonin receptor. PMID- 7556649 TI - Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. AB - Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin-1 (IL-1) induce angiogenesis by yet undefined mechanisms. We demonstrate that PGE2 and IL 1 induces the expression of vascular endothelial growth factor (VEGF), a selective angiogenic factor by rheumatoid synovial fibroblast cells. Transcripts for the EP1 and EP2 subtypes of PGE receptors are expressed in synovial fibroblasts. Activators of protein kinase A pathway stimulated the expression of VEGF whereas down-regulation of protein kinase C did not influence the PGE effect, suggesting that signalling from the EP2 receptor via the protein kinase A pathway is important. The induction of VEGF expression by PGE2 and interleukin-1 alpha may be an important mechanism in inflammatory angiogenesis. PMID- 7556654 TI - 1H-NMR and photo-CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus. AB - Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli [Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P. and Vanoni, M. (1995) Gene 154, 99-103]. NMR and photo-CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster consisting of Phe5, Tyr7, Phe31 and Tyr33 while Trp23 is fully exposed to solvent. Phe31, Tyr33 and Trp23 are located within a triple stranded antiparallel beta-sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding. Phe31 and Trp23 are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA-binding proteins. PMID- 7556655 TI - The first observation of O2- generation at real time in vivo from non-Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication. AB - Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2- which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2- in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol-induced O2- generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2- generation was completely abolished by GdCl3. Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non-Kupffer cell population, probably liver sinusoid endothelial cell to produce O2-. PMID- 7556656 TI - Analogue versus digital recognition of DNA by bleomycin: an effect of the carbohydrate moiety. AB - We have sought to determine the influence of the carbohydrate moiety of the antitumour antibiotic bleomycin on the sequence-specific cleavage of DNA. Both bleomycin A2 and deglycobleomycin A2 produce different cleavage patterns with DNA in which the 2-amino group has been removed from guanine, added to adenine, or both, as well as on a designed DNA fragment containing a few defined cleavage sites. Although each drug cleaves DNA primarily at GpT and GpC sites, the cleavage at these sites is frequently found to be stronger with deglycobleomycin compared with bleomycin A2. Conversely, in most cases the cleavage at secondary sites, in particular at ApT steps, is significantly reduced or even abolished with deglycobleomycin. The results indicate that the gulose-mannose moiety of bleomycin A2 plays a significant role in the recognition of preferred nucleotide sequences and confirm the view that both secondary structure and interaction with guanine are involved in determining sequence-specific cleavage of DNA by bleomycin. PMID- 7556657 TI - Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus. AB - The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates 4 methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2MU) and p nitrophenyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2PNP). The kinetic isotope effect on Vmax (beta DV), at pH 6.0, as revealed by direct comparison of rates obtained with Neu5Ac alpha 2MU and the [3,3-2H]-substituted substrate analogue, was shown to be inverse. This indicates that sialidase-catalysed hydrolysis of Neu5Ac alpha 2MU proceeds with substantial positive charge development at the reaction centre in the transition state for the formation of the glycosyl cation-enzyme intermediate. However, no such inverse effect on Vmax at pH 6.0 was observed when using Neu5Ac alpha 2PNP and the [3,3-2H]-substituted substrate. A mechanism by which hydrolysis proceeds through an alpha-lactone intermediate has been proposed by Guo et al. [8]. We propose that the differences in beta DV for the substrates investigated are due primarily to the differing properties of the aglycon leaving groups, which may result in influenza virus sialidase catalysing substrate hydrolysis by a similar mechanism with alternative stabilisation of transition state. PMID- 7556659 TI - Rat renin gene transcription is initiated at a single start site. AB - The promoter region of the renin gene in man, mouse and rat contains several putative transcription start sites, which in mouse have been shown to be tissue specific and differently regulated. To investigate which of these start sites are used during stimulation of renin gene transcription by the major physiological control factors, we determined the transcription start sites of rat renin in the kidney and adrenal glands by RNase protection using a cRNA probe spanning 387 bases upstream and 121 bases downstream from the canonical transcription initiation site. To stimulate renin gene expression, we used renal artery stenosis, angiotensin II antagonists, furosemide and isoprenaline infusions and low sodium diet. Our results suggest that only one TATA box is functional in rat kidney and adrenal. PMID- 7556658 TI - The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis. AB - A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 >> iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC 19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach. PMID- 7556660 TI - ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils. AB - Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility. PMID- 7556661 TI - Resistance against multiple plant viruses in plants mediated by a double stranded RNA specific ribonuclease. AB - Many plant viruses have single-stranded RNAs as their genomes. During the course of replication, genomic RNAs and their complementary template RNAs are likely to form double-stranded (ds) RNA at least transiently. To attack replication intermediates of many plant RNA viruses specifically, we introduced a yeast derived ds-RNA specific RNase gene called pac 1 into plants. The transformed plants showed a decrease in lesion numbers when they were challenged with tomato mosaic virus, and a delay in the appearance of symptoms when inoculated with cucumber mosaic virus or potato virus Y. PMID- 7556662 TI - Small-angle X-ray solution scattering study on the dimerization of the FKBP25mem from Legionella pneumophila. AB - The dimerization of the FK506-binding peptidyl-prolyl cis/trans-isomerase (PPIase) FKBP25mem (Mip (macrophage infectivity potentiator) protein) from Legionella pneumophila was studied by small-angle X-ray solution scattering. A value of 44 kDa, independent on the protein concentration between 2 and 13 mg/ml, confirming that FKBP25mem is a dimer was found for the molecular mass of the protein. The radius of gyration of the protein is 3.3 nm and the Porod volume 87 nm3. A model of the shape of FKBP25mem was evaluated from the scattering curve. Each monomer consists of a proximal and a peripheral domain, which are perpendicular to each other. The envelope of the crystallographic model of human FKBP12 fits well into the peripheral domain. The contact regions between the two monomers in the dimeric protein are probably located between the N-terminal parts of the monomers. PMID- 7556664 TI - A new hepatocyte stimulating factor: cardiotrophin-1 (CT-1). AB - Recently, a novel cytokine, cardiotrophin-1 (CT-1), was cloned and found to induce cardiac myocyte hypertrophy in vitro. Amino acid sequence similarity showed CT-1 to be a member of the IL-6/LIF/CNTF/OSM/IL-11 cytokine family. Since all known members of the IL-6 cytokine family induce an hepatic acute phase protein (APP) gene expression, we investigated the ability of CT-1 to induce a liver acute phase response. Upon stimulation of rat hepatoma cells, CT-1 and LIF induced the strongest rat fibrinogen mRNA expression, OSM and IL-6 induced a less pronounced response. When human hepatoma cells and primary rat hepatocytes were stimulated with CT-1, the expression of human haptoglobin and rat alpha 2 macroglobulin mRNA was induced. The induction of the acute phase response was dose- and time-dependent. In this study we demonstrate that CT-1, a novel cytokine belonging to the IL-6 cytokine family, is a hepatocyte stimulating factor. PMID- 7556663 TI - Silencing of the epidermal growth factor receptor in the absence of the ligand requires phospholipase C activity. AB - The possible involvement of phospholipase C beta (PLC beta) in a crosstalk mechanism between G-protein coupled receptors and receptor tyrosine kinases was investigated in HeLa-S3 and A-431 cells. A basic activity of the receptor for epidermal growth factor (EGF) in the absence of its ligand was found only in A 431 cells overexpressing this receptor. Inhibition of PLC drastically increased EGF receptor activity in both cell lines, suggesting that PLC activity is necessary for the silencing of the EGF receptor in the absence of its ligand. Activation of PLC beta and protein kinase C (PKC) via G-protein-linked ATP receptors greatly diminished the basic EGF receptor activity in A-431 cells. This negative regulation was prevented by the protein tyrosine phosphatase inhibitor, vanadate. The results suggest a crosstalk between a G-protein-linked receptor and a receptor tyrosine kinase, involving signalling via PLC beta and PKC to a downstream protein tyrosine phosphatase functioning in the control of EGF receptor activity. PMID- 7556667 TI - Identification of the primary growth response gene, ST2/T1, as a gene whose expression is differentially regulated by different protein kinase C isozymes. AB - Individual protein kinase C isozymes have been shown to play different roles in mediating proliferation, differentiation and transformation, but it is not known to what extent these effects involve induction of expression of particular genes. To explore the differential gene expression that might be induced by activation of different PKC isozymes, we stably transfected NIH 3T3 cells with expression vectors that encode the isozymes PKC-alpha, -beta II, -gamma, -delta, -epsilon, sigma and -eta. Using differential display-reverse transcription-polymerase chain reaction we isolated a small cDNA that encodes a portion of the primary response gene, ST2 (also referred to as T1 or DER4), and we confirmed by RNA blot studies that ST2/T1 expression is differentially regulated by PKC isozymes. ST2/T1 mRNA is undetectable in the unstimulated parental NIH 3T3 cells that express only the alpha isozyme of PKC, but it can be induced by phorbol ester treatment. Clones that overexpress PKC-alpha, -delta or -epsilon similarly do not express ST2/T1 until they are stimulated with phorbol esters, which induces expression of ST2/T1 with kinetics similar to wild-type NIH 3T3 but to different extents. In contrast, ST2/T1 mRNA is already present in unstimulated cells that overexpress PKC-beta II, -gamma, -sigma and -eta, but phorbol ester greatly enhances ST2/T1 expression in these cells. These results suggest a differential role for PKC isozymes in mediating the ST2/T1 expression that is induced by growth stimuli. PMID- 7556666 TI - Casocidin-I: a casein-alpha s2 derived peptide exhibits antibacterial activity. AB - Here we report the isolation and characterization of an antibacterial peptide from bovine milk inhibiting the growth of Escherichia coli, and Staphylococcus carnosus. The primary structure of the peptide was revealed as a 39-amino-acid containing fragment of bovine alpha s2-casein (position 165-203) by means of Edman amino acid sequencing and mass spectrometry. Since human milk does not contain any casein-alpha s2, these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling. PMID- 7556665 TI - Heat shock activation of NFkB in rat liver is mediated by interleukin-1. AB - Exposure to high temperature (heat shock) activates the transcription factor NFkB in the liver of the living rat, but is not effective in hepatoblastoma cells in culture: on the contrary, activation of the heat shock transcription factor (HSF) occurs under both conditions. Pre-treatment of the rat with IL-1 receptor antagonist suppresses the activation of NFkB, which seems to be mediated by the release of this cytokine, but does not hamper the activation of HSF and the concurrent induction of hsp 70 mRNA. IL-1 activity actually shows a strong, albeit transient, increase in the blood of heat shocked rats. PMID- 7556669 TI - Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating zeta-carotene desaturation. AB - We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of zeta-carotene and neurosporene to lycopene when expressed in E. coli cells accumulating zeta-carotene or neurosporene. This enzyme is unable to dehydrogenate either phytoene or lycopene. The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca. 59 kDa and which is synthesized as a precursor with a NH2-terminal extension resembling transit peptides for plastid targeting. Sequence comparison reveals 33 35% similarity with previously cloned plant or cyanobacterial phytoene desaturases. In contrast, only limited sequence similarity is found with a zeta carotene desaturase from the cyanobacterium Anabaena. PMID- 7556671 TI - Molecular cloning of the cDNA encoding a novel protein disulfide isomerase related protein (PDIR). AB - We isolated the cDNA of a novel protein disulfide isomerase (PDI)-related protein, designated PDIR, from a human placental cDNA library. Deduced from its nucleotide sequence, PDIR has the three CXXC-like motifs (Cys-Ser-Met-Cys, Cys Gly-His-Cys and Cys-Pro-His-Cys), which are found in proteins within the PDI superfamily and are responsible for oxidoreductase activity. PDIR has a hydrophobic stretch at its amino terminus, which may serve as a signal sequence, and the putative endoplasmic reticulum (ER) retention signal 'Lys-Glu-Glu-Leu' at its carboxy terminus, indicating that PDIR is an ER resident protein. Northern blots showed that PDIR is preferentially expressed in cells actively secreting proteins and that the expression of PDIR is stress-inducible. These results suggested that PDIR has oxidoreductase activity of disulfide bonds against polypeptides and that it acts as a catalyst of protein folding in the lumen of the ER. PMID- 7556670 TI - Novel disulfide-constrained pentapeptides as models for beta-VIa turns in proteins. AB - The conformational behavior of cyclic peptides of the amino acid sequence Cys Phe/Ala-Pro-Ala-Cys has been investigated through the combined use of molecular simulation methods and NMR experiments to find models for beta-VIa turns of proteins. Both oxidized (cyclic) peptides and reduced (linear) forms were investigated. At least 95% of the cyclic peptides show a cis conformation of the Xaa-Pro bond in solution in DMSO or water, whereas all other peptide bonds are trans. Furthermore, we observed a hydrogen bond between the NH group of residue Ala4 and the C = O group of residue Cys1. Both properties are indicative of beta VIa turns. After reduction of the disulfide bridge, the all-trans form of the peptide bonds predominates. PMID- 7556672 TI - Classification of multi-helical DNA-binding domains and application to predict the DBD structures of sigma factor, LysR, OmpR/PhoB, CENP-B, Rapl, and Xy1S/Ada/AraC. AB - We have systematically compared structures of multi-helical DNA-binding domains (DBDs) which have been determined by crystallography or NMR spectroscopy. All the known multi-helical DBDs are very similar. The core of these structures consists of two alpha-helices in the helix-turn-helix combination, associated with one or two other helices. The structures can be classified according to either additional structural compositions or the configuration of the helices. Many DBDs, whose structures are currently unknown, have sequences which resemble those of known structures, permitting outlines of the new structures to be predicted. PMID- 7556668 TI - Interleukin-4 induces endothelial vascular cell adhesion molecule-1 (VCAM-1) by an NF-kappa b-independent mechanism. AB - While all features of the inflammatory response induced by IL-1 are not observed following IL-4 stimulation, suboptimal concentrations both cytokines result in synergistic VCAM-1 expression in HUVEC. We have shown that, while IL-1 stimulated HUVEC express GM-CSF, tissue factor and VCAM-1, only VCAM-1 is detectable after exposure to IL-4. While kB was found essential for both basal and IL-1-mediated activity of VCAM-1, IL-4 induction was kB-independent. Inducible kB-binding proteins were identified in IL-1-, but not IL-4-stimulated nuclear extracts. Our results indicate that IL-4 exerts its transcriptional effects on the VCAM-1 gene through element(s) which do not require kB. PMID- 7556673 TI - Inhibition of aromatase expression by a psoralen-linked triplex-forming oligonucleotide targeted to a coding sequence. AB - The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20-base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence-specific triple helix (triplex) with a purine-rich tract in the P450arom coding sequence. The psoralen-linked ODN (Pso20T) formed photo-induced cross-linked products with target double-stranded DNA. Cross-linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF-7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T-treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex-mediated interruption of transcription within intact cells. PMID- 7556674 TI - Peroxynitrite-mediated oxidation of dihydrorhodamine 123 occurs in early stages of endotoxic and hemorrhagic shock and ischemia-reperfusion injury. AB - To quantify peroxynitrite production during shock, we measured oxidation of dihydrorhodamine 123 in rats. In endotoxic and hemorrhagic shock and splanchic ischemia-reperfusion, dihydrorhodamine oxidation rapidly increased, which was prevented by inhibition of endothelial nitric oxide (.NO) synthase (ecNOS). Thus, peroxynitrite is already formed at early stages of shock from ecNOS-derived .NO. Overproduction of .NO by the inducible NOS at late shock was not associated with additional increases in dihydrorhodamine oxidation. ecNOS inhibition enhanced dihydrorhodamine oxidation in control rats. These latter findings may be explained by .NO-mediated inhibition of peroxynitrite-induced dihydrorhodamine oxidation, a phenomenon also observed in vitro. PMID- 7556675 TI - Hypoxia prolongs neutrophil survival in vitro. AB - Neutrophil apoptosis represents a major mechanism involved in the resolution of inflammation. Since hypoxia induces apoptosis in several cell lines and is of particular relevance in many disease states, we studied the effect of oxygen concentration on neutrophil survival in vitro. Hypoxia caused a dramatic decrease in neutrophil apoptosis (% apoptosis 20 h: 78.7 +/- 2.2% in 21% O2, 61.4 +/- 6.5% in 2.5% O2, 23.1 +/- 3.2% in 0% O2, n = 5). This was additive to the effect of GM CSF (50 U/ml), not associated with induction of bcl-2 expression, and was not mimicked by methionine (5 mM), superoxide dismutase (200 micrograms/ml) or Trolox (10 mM) but was mimicked by catalase (250 micrograms/ml). Hence, hypoxia has a bcl-2-independent effect on neutrophil apoptosis that may adversely affect the clearance of these cells from an inflammatory focus. PMID- 7556677 TI - A beta-subclass phosphatidylinositol-specific phospholipase C from squid (Loligo forbesi) photoreceptors exhibiting a truncated C-terminus. AB - A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid (Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M(r) 98,181, and has marked similarity with PLC beta isoforms, including conservation of the 'X' and 'Y' regions. It is unique in having a major C-terminal truncation. A major protein of apparent M(r) 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision. PMID- 7556676 TI - Histidine and tyrosine phosphorylation in pea mitochondria: evidence for protein phosphorylation in respiratory redox signalling. AB - A 37 kDa protein in pea mitochondria was found to contain phosphorylated residues. Phosphorylation was acid-labile but stable in alkali solution, a unique property of phosphorylation on histidine, indicating that a signal transduction pathway with homology to bacterial two-component systems might exist in plant mitochondria. We also describe the first example of tyrosine phosphorylation in plant organelles and the first indication of protein phosphorylation as part of a redox signalling mechanism in mitochondria. Labelling of three proteins (28, 27 and 12 kDa) was found to be dependent on the redox state of the reaction medium. Their phospho-groups were resistant to alkali as well as acid treatment and labelling was inhibited by the tyrosine kinase inhibitor genistein. PMID- 7556678 TI - Mutations in the NKXD consensus element indicate that GTP binds to the gamma subunit of translation initiation factor eIF2. AB - Initiation factor eIF2 binds GTP and promotes the binding of methionyl-tRNA to ribosomes. Biochemical and sequence evidence suggests that the GTP might bind to either the beta- or gamma-subunit of eIF2. Mutations were made in the NKXD consensus elements found in both subunits and individual mutant forms were overexpressed in transiently transfected COS-1 cells. The effect on the translational efficiency of a reporter mRNA for dihydrofolate reductase was monitored. Mutations in the gamma-subunit cause severe repression of protein synthesis, whereas those in the beta-subunit are only mildly inhibitory. The results support the view that GTP binds exclusively to the gamma-subunit. PMID- 7556679 TI - The functioning of the SRP receptor FtsY in protein-targeting in E. coli is correlated with its ability to bind and hydrolyse GTP. AB - In this study, we have established that FtsY, the E. coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY. These observations suggest that in E. coli GTP is important for proper functioning of FtsY in protein-targeting. PMID- 7556680 TI - Synthesis and biological evaluation of substituted phosphate triester alkyl lyso phospholipids (ALPs) as novel potential anti-neoplastic agents. AB - Phosphate triester derivatives of the anti-neoplastic alkyl lyso phospholipid (ALP) have been prepared as novel potential therapeutic agents. In particular, symmetrical phosphate triesters have been prepared, using phosphorochloridate chemistry. The compounds have been fully characterised by a range of techniques, and assayed for their inhibition of DNA synthesis by mammalian cells in culture. The compounds are generally inhibitory towards DNA synthesis in the microM range. However, the magnitude of the effect varies greatly with the phosphate structure; alkynyl and glycol substituted phosphates being especially potent. PMID- 7556681 TI - Effect of the PufQ protein on early steps in the pathway of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. AB - The addition in trans of the pufQ gene to a strain of Rhodobacter capsulatus from which the entire puf operon had been deleted, increased its ability to synthesize coproporphyrinogen from both delta-aminolevulinic acid and porphobilinogen. Studies at the enzyme level indicated that the conversion of porphobilinogen to uroporphyrinogen III had about a 2-fold higher level of activity in the anaerobically-grown pufQ-containing strain. This increase in activity over the puf-deletion strain appeared to occur during transitions from aerobic to semiaerobic growth conditions. These results indicated that the PufQ protein may exert a stimulatory effect quite early in the pathway of bacteriochlorophyll biosynthesis. PMID- 7556682 TI - Human colon produces fully processed glucagon-like peptide-1 (7-36) amide. AB - The human colon contains many open-type endocrine cells which express the preproglucagon gene and possess glucagon-like peptide-1 (GLP-1) immunoreactivity, but the molecular form of the peptide is unknown. Acid ethanol extracts of human colon (n = 4) were subjected to gel filtration and successive purification by high-pressure liquid chromatography, monitored by specific RIAs. A single GLP-1 immunoreactive peak was isolated and identified as GLP-1 (7-36)amide by amino acid sequence analysis and mass spectrometry. We conclude that proglucagon is processed in the large intestine in the same manner as in the small intestine, and results in the formation of fully processed biologically active GLP-1. PMID- 7556683 TI - NOR-2 (neuron-derived orphan receptor), a brain zinc finger protein, is highly induced during liver regeneration. AB - Zinc-finger proteins are involved in several cellular processes. Some of these proteins are implicated in the primary cellular response in regenerating liver and mitogen-stimulated cells. Using a rat cDNA brain library, we have isolated a clone designated NOR-2, encoding a protein containing two zinc-finger motifs and whose expression is highly induced during G0/G1 transition. We analysed the expression of NOR-2 mRNAs during early growth in regenerating liver and in both insulin-stimulated H4-II cells and pheochromocytoma-derived cell line PC12 treated by NGF. In these systems, there is an early, rapid and transient accumulation of NOR-2 mRNAs. The induction of NOR-2 mRNAs does not require de novo protein synthesis, since it is not prevented by cycloheximide treatment. Mobility shift assays show that NOR-2 protein binds to NBRE, a target sequence for r-NGFI-B family. Structurally, NOR-2 is closely related to the recently identified NOR-1 factor. Therefore, like NOR-1, NOR-2 belongs to the r-NGFI-B sub family of nuclear receptors superfamily. PMID- 7556684 TI - Involvement of protein kinase in delta 12-prostaglandin J2-induced expression of rat heme oxygenase-1 gene. AB - We recently identified the cis-regulatory element and its specific nuclear binding factors for delta 12-prostaglandin (PG) J2-induced expression of the rat heme oxygenase, HO-1 [Koizumi, T., Odani, N., Okuyama, T., Ichikawa, A. and Negishi, M. (1995) J. Biol. Chem. 270, in press]. Here we further examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 gene expression. Protein kinase inhibitors, 2-aminopurine and staurosporine, suppressed the delta 12-PGJ2-induced HO-1 mRNA and the nuclear protein binding to the delta 12-PGJ2 responsive cis-regulatory element in rat basophilic leukemia cells. Furthermore, the nuclear protein binding to the element was suppressed by in vitro phosphatase treatment of the nuclear proteins from delta 12-PGJ2-treated cells. These findings suggest that delta 12-PGJ2 induces the expression of the HO-1 gene through phosphorylation of the nuclear proteins which bind to the delta 12-PGJ2 responsive element. PMID- 7556685 TI - alpha-Crystallin quaternary structure: molecular basis for its chaperone activity. AB - alpha-Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an 'open' micellar structure composed of alpha A subunits is used to simulate chaperoning of partially heat denatured soluble gamma-crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 alpha/gamma molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601-13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311 21]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509-16]; i.e. the bound gamma-crystallin monomers are not in a central cavity, but are separated by alpha A subunits. PMID- 7556686 TI - Salt-stabilized globular protein structure in 7 M aqueous urea solution. AB - A 7 M aqueous urea solution of the 63-residue N-terminal domain of the 434 repressor at pH 7.5 and 18 degrees C contains a mixture of about 10% native, folded protein and 90% unfolded protein. Interconversion between the two conformations is slow on the NMR chemical shift time scale, so that observation of separate resonances can be used to monitor the equilibrium between folded and unfolded protein when changing the solution conditions. In this paper we describe the influence of various salts or non-ionic compounds on this conformational equilibrium. Solution conditions are described which contain a homogenous preparation of the folded protein in the presence of 6 to 7 M urea, providing a basis for an NMR structure determination in concentrated urea and for studies of the solvation of the folded protein in mixed water/urea/salt environments. PMID- 7556688 TI - The practice environment in the next century. PMID- 7556689 TI - Changes in nursing through the years: thank God for disposables! PMID- 7556687 TI - Cross-linking of at least three binding sites mediates signal transduction in a CR2-positive Burkitt lymphoma derived cell line (Raji). AB - In this study we demonstrate that Raji cells, a CR2-positive Burkitt lymphoma derived cell line, during cell growth, need the cross-linking of multiple OKB7 binding sites or C3d determinants to mediate signal transduction. The loss of one of these affects the cellular response. Moreover, OKB7, the anti-CR2 MoAb, recognizes C3d determinants on the cell surface and inhibits signal transduction induced by anti-C3d polyclonal antibody. Since Raji cells are always CR2 positive during cell growth, we suppose that at least another protein, along with CR2, may be involved in setting up a cell surface complex able to receive and transduce the signal triggered by OKB7. In our experimental system the protein that offers a third binding site to OKB7, may be represented by a 33 kDa protein bearing C3d determinants. PMID- 7556690 TI - Computer applications: technology supporting today's staff nurse. PMID- 7556691 TI - Contemporary nursing education: why and hows. PMID- 7556693 TI - The effect of individualized prescriptions for nursing on stress of cardiovascular surgery patients. PMID- 7556695 TI - Caring for our colleagues can make a difference. PMID- 7556692 TI - Utilization and satisfaction with health care services for chronically ill elderly at home. PMID- 7556696 TI - Ethics rounds. PMID- 7556697 TI - Like a bridge over troubled waters. PMID- 7556694 TI - Career advancement trends of African-Americans in nursing administration. PMID- 7556698 TI - Sally Hutchinson, RN, PhD, FAAN: an exemplar of a nurse researcher. Interview by Pamela S Chally and Funmi Borisade. PMID- 7556699 TI - A bridge to growing. PMID- 7556700 TI - Smile and accept the challenge--restructuring is here to stay. PMID- 7556702 TI - Council on cultural diversity: performing a culturally competent child health assessment. PMID- 7556701 TI - Florida stumbles on block grant ... patient lives and nurses jobs take the fall. PMID- 7556703 TI - I want to go home. PMID- 7556705 TI - The wisdom of one, the wisdom of many, the value of one, the value of many. The responsibility of one, the responsibility of many. PMID- 7556704 TI - Users of AHCPR-supported guidelines report improvements in quality of care. PMID- 7556706 TI - Action not just talk. PMID- 7556707 TI - Transplantation and the eye. PMID- 7556708 TI - From the forehead of Zeus: the ontogeny of the immune response. AB - Many of the most important developments that result in a fully functioning vertebrate immune system take place in the developing fetus. From a variety of gene segments there is assembled in B cells a congeries of antibody combining sites, one to a cell, which form the greater part of the large repertoire of immunological specificities that characterise the system. This capability is further expanded later by somatic mutations. Just as immunoglobulin isotypes are produced sequentially (IgM, IgD, IgG, IgE, IgA) as they are read along the chromosome, so does the fetus and neonate manifest immunological competence sequentially to different antigens by employing variable region germline genes as they appear along the chromosome. The generation of T cell receptor diversity is accomplished by a similar mechanism of gene segment translocations. Each stage in the lineage of T and B cells is associated with the appearance of unique combinations of surface molecular markers, which in T cells characterise also the specialised functions of different subsets. If the immune system does not spring forth quite fully formed from the evolved vertebrate genome, as Athena did from the forehead of Zeus, ontogenetic mechanisms have made it very nearly complete. PMID- 7556709 TI - The laws of transplantation: a modern perspective. PMID- 7556710 TI - The humoral response to an allograft. AB - Antibodies are known to damage grafted tissues by a variety of means, so it is important to know how the humoral response is initiated. In this paper we summarise the cellular events in B cell activation, the mechanisms of antibody mediated rejection and the evidence that antibody apparently does not contribute to early corneal graft rejection. The role of antibodies in chronic graft loss is discussed. PMID- 7556711 TI - Pathways of dendritic cell differentiation and development. PMID- 7556712 TI - Role of vascular endothelial cells in the allograft response. AB - Following transplantation endothelial cells lining an allograft come into contact with immune cells of the recipient. Activation of an immune response, by graft endothelial or other cells, will lead to local increases in cytokine production and cell-mediated lysis. Inflammatory cytokines have been shown, mainly in vitro, to have marked effects on endothelial function and act to produce a pro thrombotic, pro-adhesive and promitogenic phenotype. These data are reviewed and ways in which these changes could lead to rejection due to graft lysis or vascular occlusion are discussed. PMID- 7556713 TI - Histocompatibility antigens. PMID- 7556714 TI - The role of human minor histocompatibility antigens in graft failure: a mini review. PMID- 7556716 TI - Rejection reactions to different organ transplants. PMID- 7556717 TI - Immunomodulation for transplantation tolerance. PMID- 7556719 TI - Non-MHC antigens and their relative resistance to immunosuppression after corneal transplantation. AB - We have used a high responder rat model to examine the role that non-MHC antigens play in corneal graft rejection. Recipients were backcross animals derived from a cross between two inbred strains, which mimicked the human outbred population in that donor and recipient could be matched or mismatched for MHC antigens, while non-MHC mismatches were variable and unknown. All mismatched grafts and 87% of matched grafts were rejected (median survival 11 and 17 days respectively). The high incidence of rejection of matched grafts indicates that several independently segregating non-MHC genes play a role in rejection. Moreover, the immune response to matched grafts appeared resistant to immunosuppression, suggesting that matching does not permit reduced dosage of immunosuppressants. A mechanism is discussed whereby matching at the class II locus may enhance presentation of mismatched histocompatibility antigens or viral peptides derived from infected graft cells, thereby prejudicing graft survival. PMID- 7556715 TI - The control of hyperacute rejection by genetic engineering of the donor species. AB - Activation of endogenous complement is inhibited both in the soluble phase and at the membrane surface by a group of structurally similar proteins. A possible solution to hyperacute rejection is to produce donor animals transgenic for human complement regulators. Mouse cells expressing the human complement regulatory proteins decay accelerating factor (DAF) or membrane cofactor protein (MCP) were produced both by hybridoma technology and by transfection with the appropriate cDNAs. The expression of either or both of these products protected the mouse cell from lysis by human (though not rabbit) complement in the presence of naturally occurring human anti-mouse antibody. This effect could be abrogated by the addition of monoclonal antibody against DAF or MCP. Hyperacute rejection of discordant organ xenografts is mediated by human complement. A 6.5 kilobase minigene for DAF has been microinjected into porcine fertilised ova. Forty-five pigs transgenic for human DAF have been produced. Of these, 65% transcribe message. The amount of message produced varied substantially from animal to animal and was independent of copy number integrated. Expression of human DAF on the porcine lymphocyte surface could be detected and this was able to downregulate human complement activation. Amounts of protein expressed on different tissues varied both from pig to pig and within animals from tissue to tissue. The pigs grow and develop normally with no evidence of ill effects due to possession of the transgene. PMID- 7556718 TI - The mechanisms of corneal graft failure in the rat. AB - The success rate of corneal transplantation is very similar to that of other organ transplantations because corneal transplants can induce an allograft rejection similar to other organ transplants. Only the centre of the cornea can be considered an immunologically privileged site. The reasons for this are considered in this review of experimental corneal grafting which has led to an understanding the immunological mechanisms behind corneal graft rejection. The topics discussed include the role of antigen presenting cells (APCs) in experimental corneal graft rejection and their distribution in the cornea. PMID- 7556722 TI - The effects of storage of corneal tissue on Langerhans cells. PMID- 7556726 TI - Immunosuppression in corneal transplantation. AB - This paper reviews the clinical post-operative management of keratoplasty and the management of corneal graft rejection. In both instances corticosteroids remain the mainstay of treatment; however, the literature shows a wide range for both route and frequency of administration. Grafts at 'high risk' require more immunosuppressive therapy, but no universally accepted definition of high risk exists and consequently different treatment regimens are difficult to compare and evaluate. Studies using univariate and multivariate survival analysis suggest that recipient corneas can be divided into low, medium and high risk depending on the number of quadrants of vascularisation (avascular, 1-2 quadrants and 3+ quadrants respectively). This wider classification would make the devising and comparing of treatment regimens more consistent. In high-risk cases, corticosteroids alone provide insufficient immunosuppression and systemic cyclosporine is needed in exceptional cases. When managing rejection episodes, a severe reaction involving the endothelium often does not respond to topical steroids alone, and systemic corticosteroids are required. Instead of oral steroids, we now prefer to use an intravenous 'pulse' of 500 mg methylprednisolone: this is at least as effective, avoids prolonged medication, and may confer some long-term benefit. PMID- 7556721 TI - How successful is corneal transplantation? A report from the Australian Corneal Graft Register. AB - Corneal graft outcome was assessed within a large, prospectively collected database of 4499 records. Penetrating corneal graft survival was 91% at 1 year, 72% at 5 years and 69% at 7 years. The three most common indications for graft were keratoconus (30%), bullous keratopathy (25%) and failed previous graft (18%); the three most common causes of graft failure were rejection (34%), infection (18%) and glaucoma (9%). The vast majority of grafts were performed for improved visual acuity. About four-fifths of recipients achieved at least one line of better acuity on the Snellen chart post-operatively; of the remainder with unchanged or worse acuity, only 21% had failed grafts. Overall, 43% of recipients achieved a best corrected Snellen acuity of 6/12 or better, 52% achieved 6/18 or better, and 20% had acuitities of less than 6/60. Reasons for poor post-operative acuity (recorded as less than 6/60) included graft failure (41%) and comorbidities in the grafted eye (43%). A number of risk factors for graft failure were examined: in most instances, there was little room for decision-making or expert intervention. PMID- 7556723 TI - When can neural tissue be transplanted? PMID- 7556720 TI - Effect of cytokine-induced migration of Langerhans cells on corneal allograft survival. AB - The unique paucity of Ia+ Langerhans cells (LCs) in the central cornea contributes to the immunological privilege of corneal allografts. A variety of stimuli can induce the centripetal migration of peripheral LCs. At least one of these stimuli (i.e. latex bead instillation) induces interleukin-1 (IL-1) secretion by corneal cells which acts as a potent chemoattractant for LCs. Within 30 minutes of intracorneal injection of IL-1, centripetal migration of LCs can be detected. The presence of donor-derived LCs in corneal allografts doubles the incidence of rejection of fully allogeneic corneal allografts as well as MHC matched, multiple minor H mismatched corneal allografts. Although the presence of donor-specific LCs greatly jeopardises corneal allograft survival, migration of host-derived LCs into corneal allografts does not appear to increase the risk of rejection. PMID- 7556724 TI - Immunological non-responsiveness and acquisition of tolerance in relation to immune privilege in the eye. AB - Immune privilege is a dynamic, physiological process that enables the eye to accept foreign tissue grafts in an unprecedented fashion. Privilege is actively acquired and maintained by immune regulatory forces that represent an important form of antigen-specific immunological tolerance. Privilege in the eye results from eye-dependent modifications in the induction (afferent limb) and expression (efferent limb) of immunity to intraocular antigens. The eye-dependent features that are important in privilege include integrity of the blood-ocular barrier, the virtual absence of lymphatics, an afferent drainage pathway that is almost exclusively via the blood vasculature, and an immunosuppressive intraocular microenvironment. This microenvironment is comprised of a variety of cytokines and neuropeptides that (1) impair antigen-driven activation of primed and alloreactive T cells, (2) suppress effector functions of activated macrophages, and (3) modify the antigen processing and presenting properties of indigenous, bone-marrow-derived professional antigen presenting cells. Eye-derived antigenic signals, which escape when local antigen presenting cells migrate via the blood to the spleen, selectively activate regulatory T cells that impair the development of antigen-specific delayed hypersensitivity and complement fixing antibodies, a phenomenon termed anterior chamber associated immune deviation (ACAID). ACAID has been implicated in the extraordinary success of orthotopic corneal allografts, as well as the prolonged intraocular survival enjoyed by transplants of retinal tissues. The active features of immune privilege can be exploited to secure successful corneal and retinal transplantation. PMID- 7556725 TI - Regulators of immunological responses in the cornea and the anterior chamber of the eye. AB - Although the eye is considered to be immunologically privileged, this privilege is not absolute. This is well demonstrated by, for example, the fate of corneal transplantations. Clinical studies in man and experimental studies in animals have shown that survival of a corneal transplant depends on the local condition of the cornea and the anterior chamber of the eye. The presence of neovascularisation or Langerhans cells in the recipient cornea endangers the graft, while the intracorneal production of immunosuppressive factors may inhibit the development of rejection. The balance between suppressive and stimulatory factors determines whether a local immune response will develop. PMID- 7556728 TI - Perfluorocarbon heavy liquids. PMID- 7556730 TI - Retinal detachment and giant retinal tears in aniridia. AB - Clinical findings in aniridia may include corneal epitheliopathy, glaucoma, lens subluxation and cataract, and hypoplasia of the fovea and optic nerve. We report the occurrence of retinal detachment due to giant tears in four eyes of three children with aniridia. All eyes were buphthalmic and none had undergone lens or posterior segment surgery. All operated eyes underwent vitreolensectomy and silicone oil injection; useful vision was restored in two eyes. The pathogenesis and management of this previously unreported complication are discussed. PMID- 7556727 TI - The host response in experimental corneal xenotransplantation. AB - In addressing the worldwide shortage of human donor cornea for transplantation, animal cornea may be a substitute if mechanisms of xenogeneic (cross-species) rejection can be identified and controlled. Xenotransplantation of solid organs is followed by hyperacute rejection with minutes due to humoral graft rejection. In an experimental model corneal xenografts in rats survived for 2-3 days, depending on the phylogenetic disparity of the donor animal. Endothelial injury was the specific cause of graft failure, probably mediated by humoral rejection mechanisms. A later cell-mediated rejection response was seen. The potent humoral response is the most important feature differentiating xenograft from allograft rejection. PMID- 7556732 TI - Retinal detachment in AIDS-related cytomegalovirus retinitis. AB - Patients with acquired immune deficiency syndrome (AIDS) and cytomegalovirus retinitis (CMVR) are surviving longer due to the use of virostatic medicines and improved treatment of opportunistic infections. As a result, retinal detachment is likely to become an increasingly common cause of visual morbidity in these patients. The incidence and outcome of retinal detachment complicating CMVR was studied at two London AIDS centres. Patients with CMVR were identified prospectively and underwent standard treatment. Retinal detachments were diagnosed during regular follow-up. If retinal reattachment surgery was performed, a standard procedure of vitrectomy and silicone oil internal tamponade was employed. Of 147 patients with CMVR, 41 (28%) developed retinal detachments (47 eyes). Forty-three detachments were rhegmatogenous and 4 were exudative. Fifteen eyes of 9 patients with rhegmatogenous detachments underwent retinal reattachment surgery. Of these, visual acuity remained stable or improved in 12 eyes (80%) in the immediate post-operative period. At the last clinic visit, 8 eyes (53%) maintained a visual acuity of 6/60 or better. The visual results of surgery are good in selected patients, bearing in mind the progressive nature of the underlying disease and poor life expectancy. PMID- 7556733 TI - A novel conjunctival incision for horizontal strabismus surgery. AB - We present a randomised, controlled study of a new approach to conjunctival incision in horizontal strabismus surgery. A peritomy is performed from 2 to 10 o'clock inferiorly allowing free access to the horizontal recti from below. No limbal stay sutures are required and the conjunctiva is left unsutured on completion of surgery. Results of 13 patients are presented. Each patient is paired with a control on whom conventional surgery was performed. The pairs were matched for age and surgeon. Results show a decrease in the time taken for the procedure and decreased discomfort in the post-operative period. The external appearance of the operated eye and the incidence of complications over a 6 week period were the same for both techniques. PMID- 7556731 TI - Progressive outer retinal necrosis (PORN) in AIDS patients: a different appearance of varicella-zoster retinitis. AB - Retinal infections caused by the varicella-zoster virus (VZV) have been reported in immunocompetent and immunocompromised individuals. Two cases of a VZV-related retinitis are described with the characteristic features of the recently described progressive outer retinal necrosis (PORN) syndrome. Both patients suffered from the acquired immunodeficiency syndrome (AIDS) with greatly reduced peripheral blood CD4+ T lymphocyte counts, and presented with macular retinitis without vitritis. The disease was bilateral in one case and unilateral in the other. The clinical course was rapidly progressive with widespread retinal involvement and the development of rhegmatogenous retinal detachment with complete loss of vision in the affected eyes despite intensive intravenous antiviral therapy. VZV DNA was identified in vitreous biopsies, by molecular techniques based on the polymerase chain reaction (PCR), in both patients. At present, the use of very high-dose intravenous acyclovir may be the best therapeutic option in these patients for whom the visual prognosis is poor. Intravitreal antiviral drugs could also contribute to the management of these cases. PMID- 7556734 TI - Factors affecting visual outcome in children following uniocular traumatic cataract. AB - The correction of paediatric traumatic aphakia remains a controversial topic. This study examines retrospectively the visual outcome in 32 children with uniocular traumatic cataracts. Fifteen received intraocular lens implants following lensectomy, and 17 received aphakic contact lenses. Age range was 2-14 1/2 years at the time of injury. The maximum follow-up time was 13 years. Twenty four children obtained a good visual result (6/5 to 6/18). These were equally divided between those receiving intraocular lens implants and those with contact lenses. Factors adversely affecting visual outcome are discussed. These include complex trauma, delay in referral for lensectomy, inadequate postoperative correction of aphakia, contact lens difficulties and problems with occlusion therapy. Although aphakic correction with intraocular lens implants may require several subsequent surgical procedures such as capsulotomy, we advise early lensectomy and intraocular lens implantation where possible, particularly in young children with traumatic cataracts. This eliminates contact-lens-associated problems and maximises the chance of good visual outcome and retention of stereoscopic vision. PMID- 7556729 TI - The ocular surface as part of the mucosal immune system: conjunctival mucosa specific lymphocytes in ocular surface pathology. AB - The mucosal immune system includes mucus membranes of the gut, respiratory and urogenital tracts. Mucosa-specific, intraepithelial lymphocytes (IELs), that correspond to the suppressor/cytotoxic subset and also express the human mucosal lymphocyte antigen (HML-1), are a unique component of this system. We have recently demonstrated these cells in the human conjunctiva, establishing the ocular surface as an integral part of the mucosal immune system. In this study we examined the distribution of lymphocyte subsets, with particular attention to mucosa-specific lymphocytes, in two ocular surface disorders, namely conjunctival intraepithelial neoplasia (CIN) and ocular cicatricial pemphigoid (OCP). Cryosections of biopsy specimen were immunostained using a panel of monoclonal antibodies against different lymphocyte subsets. In CIN, the CD8/HML ratio was decreased (1 +/- 0) and CD8/CD4 ratio was reversed (0.54 +/- 0.21). HML-1+ cells were distributed throughout the epithelial layers of dysplastic tissue. Biopsy specimens of OCP showed normal ratios of CD8/HML (1.4 +/- 0.16) but the CD8/CD4 was low (1.29 +/- 0.88). Association of HML-1+ cells with the basal layer of normal epithelium and with all layers of dysplastic epithelium suggests that expression of HML-1 antigen may be induced by actively dividing cells. HML-1+ cells may have a role in immune mechanisms associated with ocular surface disorders. PMID- 7556735 TI - The effect of trabeculectomy on refraction, keratometry and corneal topography. AB - After successful trabeculectomy patients often complain of reduction in vision even after several months. Amongst other factors, corneal astigmatism appears to be altered. A pilot study measuring, pre- and post-operative corneal topography indicated three types of astigmatic change: some patients develop a relative superior corneal steepening, others a superior flattening and yet others complex regional changes that do not conform to either of these patterns. The present study was designed to evaluate further these patterns of variation in corneal curvature and to look for corresponding refractive and keratometry changes. Twenty-nine patients admitted for trabeculectomy had pre-operative assessment of subjective and automated refraction, manual keratometry and corneal topography from which simulated keratometry values were calculated. A standard trabeculectomy procedure was performed and post-operative measurements of the same parameters were taken at 1 and 3 months after surgery. Similar patterns of corneal topographic change to those found in our pilot study were noted. In both the superior steepening and superior flattening groups there was an increase in vertical keratometry and a shift towards 'with-the-rule' astigmatism. Furthermore a 1 year analysis of 13 patients from our pilot study indicated that the topographic changes lasted for at least 12 months after surgery. We conclude that computer-assisted corneal topography reveals complex regional changes in corneal curvature that are not readily detected from alterations in refraction or keratometry. These changes are sufficiently great to have a significant effect on visual function in some patients. PMID- 7556736 TI - Intraocular pressure control following microtrabeculectomy. AB - The use of the Kelly Descemet's membrane punch enables the glaucoma surgeon to perform smaller trabeculectomies. The outcome of 'microtrabeculectomy' employing a 2 x 2 mm superficial scleral flap and a 0.75 mm internal osteum was evaluated on 65 eyes of 50 patients (mean age 70 years). The operation site was nasal in left eyes and temporal in right eyes. In the 56 eyes where 5-fluorouracil was not used, the mean intraocular pressure (IOP) on diagnosis was 33.4 mmHg, the mean pre-operative IOP being 25.1 mmHg. After a mean follow-up of 13.4 months (minimum 3 months) following surgery, the mean IOP was 13.4 mmHg with 88% of eyes controlled at < 21 mmHg on no medications. Nasally sited microtrabeculectomies resulted in lower IOPs than temporally sited procedures (11.8 vs 14.9 mmHg, p = 0.003) at last follow-up visit. PMID- 7556738 TI - Cataract extraction following vitrectomy and silicone oil tamponade. AB - Surgery for cataract resulting from pars plana vitrectomy combined with fluid/silicone oil exchange is often rewarding in terms of visual acuity improvement. Careful choice of surgical method and suitable modifications of standard technique reduce the incidence of serious complications. Per-operative posterior capsulotomy is a safe method of maintaining a clear visual axis after ECCE. PMID- 7556739 TI - Visual acuity following extracapsular cataract extraction in diabetes: a meta analysis. AB - Although pre-operative retinopathy severity appears to be a major factor in determining the visual outcome of diabetic extracapsular cataract extraction, its precise relationship to post-operative visual acuity is ill defined. A meta analysis was therefore carried out, and studies were included if pre-operative maculopathy and retinopathy status was sufficiently defined to permit discrimination of visual outcome between subgroups. Weighted mean proportions of eyes achieving a post-operative visual acuity > or = 6/12 were as follows: no retinopathy, 87%; non-proliferative retinopathy with no maculopathy, 80%; quiescent proliferative retinopathy with no maculopathy, 57%; non-proliferative retinopathy with maculopathy, 41%; quiescent proliferative retinopathy with maculopathy, 11%; active proliferative retinopathy, 0. Differences in visual outcome between groups were significant (chi 2 = 119.9, p < 0.0005), attributable mostly to the trend across groups (chi 2 for trend = 115.4, p < 0.0005). Logistic regression indicated that maculopathy was a more potent predictor of post operative visual acuity < or = 6/12 (odds ratio 6.4, 95% CI 4.13-9.94, p < 0.0005) than quiescent proliferative retinopathy (odds ratio 3.33, 95% CI 2.04 5.42, p < 0.0005). The severity of retinopathy and maculopathy prior to cataract surgery in diabetics are the major determinants of post-operative visual acuity. Further study of the relationship between pre-operative retinopathy severity and the incidence of post-operative complications, progression of retinopathy and maculopathy is required to optimise the management of cataract in diabetes. PMID- 7556737 TI - Polarising light biomicroscopy and the relation between visual acuity and cataract. AB - The use of polarised light in biomicroscopy allows discrimination of lenticular features which are not discernible using standard biomicroscopic techniques. However, just as is true of most brightness-dependent photographic methods testing the effect of filters, past uses of polarised light did not involve any control of illumination. The present study involved the use of polarised light in the photo-biomicroscopy of the anterior segment in cataract patients, the consequential loss in illumination being mimicked with different neutral density filters. This served to distinguish between polarisation and attentuation. The patients were refracted and their corrected visual acuity was recorded. A qualitative scale was drawn up ranking weights of lenticular opacities. Spearman's rank correlation was applied to the photographs obtained with conventional and polarised-light biomicroscopy in turn. Brightness control served to confirm that biomicroscopy with polarised light offers some advantages over conventional methods, and correlates with visual acuity better than is true of conventional biomicroscopy. The Spearman ranks derived from the results obtained with polarised light correlate significantly with the patient's visual acuity. It is concluded that polarised light biomicroscopy, in conjunction with the standard examination, can improve the diagnosis of cataract and assist in relating it to a patient's visual acuity. PMID- 7556740 TI - On the pathology of the iridocorneal-endothelial syndrome: the ultrastructural appearances of 'subtotal-ice'. AB - The iridocorneal-endothelial syndrome (ICE syndrome) is characterised by corneal failure, glaucoma and iris destruction. Specular photomicroscopical and histological studies of the corneal endothelium in this disease show a population of abnormal cells named 'ICE-cells'. In many patients some areas of the endothelium are occupied by ICE-cells and others by normal cells, an appearance described as 'subtotal-ICE'. Specular photomicroscopical observations suggest that ICE-cells and normal endothelial cells may actively interact at the boundary zone where they meet. The purpose of this study was to examine the ultrastructural appearances of the boundary zone to gain insight into the cellular pathology of this region. Thirty-five corneas taken from patients with the ICE syndrome were examined by light, transmission and scanning electron microscopy. The subtotal-ICE appearance was demonstrated in four specimens. The morphology of ICE-cells at the boundary zone suggests that they are non-motile but also implies a general state of high metabolic activity. Many of the normal endothelial cells in this region are damaged, an appearance which may result from a toxic effect from the nearby ICE-cells. PMID- 7556742 TI - The development of adaptive head movements following enucleation. AB - Recent work in our laboratory has revealed that enucleated patients produce large lateral and vertical head movements during visually guided grasping. These movements may allow them to maximise the use of retinal motion cues in planning and controlling their grasp. The aim of the present study was to determine whether the tendency to produce these adaptive head movements increases as a function of time since enucleation. We tested a group of 12 enucleated patients in whom the time between surgery and testing varied from 2 weeks to 35 years (mean = 11.2 years). These patients were required to reach out and grasp oblong blocks of different sizes at different distances. Correlational tests revealed an increase in the proportion of self-generated lateral and vertical head movements versus forward head movements as a function of post-enucleation time (r(s)(12) = 0.68, p < 0.025 and r(s)(12) = 0.65, p < 0.025, respectively). This suggests that enucleated patients may be adapting to living with one eye by learning to increase the proportion of their lateral and vertical head movements during the performance of skilled motor acts. PMID- 7556743 TI - A pre-operative mydriatic regime using a single application of tropicamide Novel Ophthalmic Delivery System and guttae phenylephrine 10%. AB - Tropicamide Novel Ophthalmic Delivery System (NODS) and guttae (g.) phenylephrine 10% was applied once, 2 hours before cataract surgery, as an alternative to our standard pre-operative regime of g. cyclopentolate 1% and g. phenylephrine 10% used four times before surgery. The two treatment groups were compared using a randomised, prospective, observer-masked study design. The horizontal pupil diameter was significantly smaller using the new regime but the per-operative decrease in diameter was not significantly different, suggesting that surgical miosis was not influenced by reducing the number of mydriatic applications. There was no difference in the subjective grading of the pupil, suggesting that adequate mydriasis suitable for cataract surgery was obtained using the alternative regime. No major complications were documented in relation to the use of NODS. We discuss why less intensive mydriasis is likely to be as efficacious and describe its potential advantages. PMID- 7556741 TI - Indocyanine green angiographic findings in central serous chorioretinopathy. AB - The purpose of this study is to better characterise, on the basis of a large number of cases and follow-up evaluations, choroidal abnormalities recently observed with indocyanine green (ICG) angiography in central serous chorioretinopathy (CSC). Digital ICG videoangiography was performed in 145 patients with active or inactive, acute or chronic CSC. Forty-eight patients were re-examined in a follow-up period of 6-22 months (mean 10 months). Areas of choroidal leakage attributable to hyperpermeability of the choriocapillaris were found in 98.6% of patients in association with active or resolved pigment epithelial leaks and pigment epithelial detachments. Diffusion of ICG into the choroid was characterised by rapid centrifugal spreading of the dye with a wash out pattern which was particularly evident in areas corresponding to pigment epithelial detachments. In patients with a long-standing disease, when choroidal hyperfluorescence faded, hypofluorescent spots became increasingly evident revealing pigment epithelial alterations not shown by fluorescein angiography. Areas of choroidal leakage remained unchanged in each patient during the follow up period, even when subretinal exudation resolved either spontaneously or after photocoagulation. In 5 eyes we observed the appearance of leakage points on pre existing areas of choroidal leakage. Zonal hyperpermeability of the choriocapillaris characterises all the evolutional stages of CSC and seems to be the primary alteration of this disease. When it corresponds to pigment epithelial detachments choriocapillaris hypermeability is probably associated with local hyperperfusion. PMID- 7556744 TI - Pupil dilatation in the pseudoexfoliation syndrome. PMID- 7556745 TI - The effect of topical beta-adrenoceptor blocking agents on pulsatile ocular blood flow. AB - Thirty-three ocular hypertensive patients (21 with primary open angle glaucoma and 12 glaucoma suspects) were randomly assigned to receive either timolol, levobunolol or betaxolol in one eye. Pulsatile ocular blood flow (POBF) was measured before treatment (baseline) and 2 hours after drop administration. After 1 week of regular twice-daily dosage, POBF was measured again both immediately before and 2 hours after drop instillation. All measurements were made by an investigator masked to treatment. POBF increased by 11% (p = 0.09) at week 0 after levobunolol administration, and by 22% (p = 0.20) at week 1 before drop administration compared with baseline. It dropped by 23% and 25% (p = 0.04 and 0.06, respectively) before and after betaxolol administration at week 1. Although POBF was reduced in the timolol group, this change was not significant. These results can not be explained uniformly by changes in intraocular pressure or blood pressure. The relevance of these measurements to visual function in glaucoma is not known. PMID- 7556747 TI - Automated achromatic contrast and chromatic discrimination sensitivity testing in dysthyroid optic neuropathy. AB - Our experience of patients with dysthyroid eye disease shows that normal chromatic discrimination sensitivity precludes the diagnosis of optic nerve compression (31 patients), and that clinically confirmed optic nerve compression is invariably associated with decreased chromatic discrimination sensitivity thresholds (8 patients). Dysthyroid patients enrolled in this study underwent automated achromatic contrast and chromatic discrimination sensitivity testing on presentation, with repeat assessment of those patients suspected of developing optic nerve compression. If chromatic discrimination sensitivity was decreased, patients were followed up more frequently. If abnormal chromatic discrimination sensitivity was accompanied by a relative afferent pupillary defect (RAPD) or decreased Snellen visual activity (VA), then optic nerve decompression was performed. The automated chromatic discrimination sensitivity test described represents a quick, reproducible and cheap clinical test which we feel is of value in assessing patients with dysthyroid eye disease. We suggest that sequential chromatic discrimination sensitivity assessment is a sensitive and effective way of monitoring patients at risk of dysthyroid optic neuropathy. PMID- 7556748 TI - The geographical epidemiology of ocular diseases: some principles and methods. AB - With the increasing availability of geographically referenced data in health research the time is ripe to review the use of particular geographical and spatial analysis techniques in ophthalmic research. Analysis of the geographical distribution of ocular diseases, particularly in Britain, has not had a high profile, but there are certain diseases, such as congenital eye malformations in children, where such analysis methods are particularly appropriate. We review the data requirements and then a variety of analytical techniques, some of which partition geographical space into areal units (such as counties or electoral wards), others of which treat space as continuous. We conclude with some comments on software that is available for such analyses. PMID- 7556746 TI - Uniocular fields of fixation in thyroid eye disease. AB - Immunosuppressive therapy is well established in the treatment of thyroid eye disease (TED). The best response has been observed in those with active (wet phase) disease of short duration. A prospective study was designed to observe the effects of orbital radiotherapy and oral immunosuppression on patients with TED, and to assess whether any pre-treatment parameters were predictive of the outcome. Significant improvements in uniocular fields of fixation (UFOF) and in the Mourits' disease activity scale were seen after treatment. The degree of improvement in UFOF was positively correlated with the level of initial disease activity. The use and technique of UFOF in assessing disease phase and activity are discussed. PMID- 7556749 TI - Surgical repair of medial ectropion. PMID- 7556751 TI - Metastatic colonic adenocarcinoma of the orbit with intraneural extension from the brow to the brainstem. PMID- 7556750 TI - Microwave ovens may cause serious ocular injury. PMID- 7556752 TI - Bilateral panuveitis in a patient with colonic adenocarcinoma and Streptococcus milleri liver abscesses. PMID- 7556753 TI - Corneal keloid: aetiology and management in Lowe's syndrome. PMID- 7556754 TI - Localised giant papillary conjunctivitis secondary to a dermolipoma. PMID- 7556755 TI - Ectopic retinal tacks. PMID- 7556756 TI - Encysted Tenon's bleb over superiorly placed single-plate Molteno implant causing proptosis and strabismus. PMID- 7556758 TI - Superior oblique myokymia masquerading as an inferior rectus palsy. PMID- 7556757 TI - Prevalence of chronic hypokalaemia amongst elderly patients using acetozolamide and diuretics. PMID- 7556760 TI - Patent foramen ovale in occipital cerebrovascular accident. PMID- 7556759 TI - Intralenticular haemorrhage complicating trabeculectomy. PMID- 7556762 TI - Dacryocystitis. PMID- 7556761 TI - Penetrating ocular fish-hook injury. PMID- 7556763 TI - Myasthenia gravis with pupillary involvement. PMID- 7556764 TI - Extraocular compression prior to cataract surgery. PMID- 7556765 TI - Oral acyclovir in herpes zoster ophthalmicus. PMID- 7556766 TI - Optic nerve lesions in raised intraocular pressure. PMID- 7556767 TI - A modified method allows for correlation between NADPH-diaphorase histochemistry and immunohistochemistry for the demonstration of neuronal nitric oxide synthase (nNOS). AB - Results obtained with the conventional nitro blue tetrazolium salt method for the visualization of the NADPH-diaphorase (NADPH-d) activity of nitric oxide synthase (NOS) are not specific for this particular enzyme, since this activity represents only a fraction of the total cellular NADPH-d pool. Therefore, the standard NADPH d procedure was modified by performing the incubation in the presence of formaldehyde. Parallel application of the modified NADPH-d staining technique and the indirect immunofluorescence using an antibody against the neuronal isoenzyme (nNOS) on rat, mouse and guinea-pig tissues showed a correlation between histochemical and immunocytochemical staining. It can thus be concluded that the modified NADPH-d procedure allows for a more specific detection of the histochemical nNOS activity than the conventional method. PMID- 7556768 TI - Cytochemical staining of vacuolar compartments in the developing small intestine of mice. AB - Osmium impregnation, nicotinamide adenine dinucleotide phosphatase (NADPase) and thiamine pyrophosphatase (TPPase) localisation have been applied as a cytochemical tool for the labelling of the endomembranes in the small intestinal cells of mice during embryonic development. Only cisternae of the endoplasmic reticulum were stained by prolonged osmification in the undifferentiated cells. At later developmental stage, Os-black was found in cis Golgi cisternae, small vesicles and also in the endoplasmic reticulum of some epithelial cells. At the early embryonic stage the product of NADPase reaction was visible in medial saccules of the Golgi complex and in lysosomes, while at the later stages positive staining appeared also on the apical cell surface and within the structures probably belonging to endosomal compartment. TPPase activity was constantly present in trans Golgi cisternae of the cells during the studied period of fetal life. These results indicate that the cytochemical markers for Golgi staining can also be found in several other endomembrane systems. They give the insight into various connections between the particular intracellular compartments occurring at different developmental stages. PMID- 7556769 TI - The entrance of surface active material to pulmonary capillaries in the lungs of newborn rats. AB - In newborn rats we investigated the entrance of surfactant to the pulmonary capillaries a few minutes after the birth. Time is crucial for the process which can be called "drying" of the lung, when a large amount of intraalveolar fluid enters the pulmonary circulation. Ultrastructural studies have revealed bulk transport and endocytosis of surfactant via alveolar septa to the blood vessels, but it is still questionable whether the entrance of surfactant to pulmonary capillaries is related to its clearance or to "drying" of lung's alveoli. PMID- 7556772 TI - Biotinylated L-selectin ligand analogs as cytochemical probes in cytospin preparations. AB - Amino-derivatives of L-selectin ligand analogs: phosphonoester core polysaccharide (PPME) and fucoidin were biotinylated with the use of biotinyl-N succinimide ester, and these biotinylated analogs b-PPME and b-fucoidin were demonstrated as useful tools to investigate the functional activity of L selectins in cytospin preparations obtained from healthy human donors and from patients with chronic lymphocytic leukemia (CLL). The avidin/biotin system adds a new alternative to the application of the L-selectin ligand analogs (PPME, fucoidin) which have been formerly used as fluoresceinated, solid phase or immobilized probes. PMID- 7556771 TI - Peripheral blood leukocytes and lymphocyte subpopulations as determined by flow cytometric measurements in healthy children. AB - Peripheral venous blood leukocyte subpopulations were determined in 69 healthy children and compared in two age groups: 8 to 12 years and 13 to 17 years, the period of pubescence and intensive growth of the organism. As compared to work of other authors, more leukocyte subpopulations were recognized in the examined large group of children. Whole blood monocyte and total lymphocyte absolute counts decreased with age as well as the absolute counts of T lymphocytes and CD8+ T lymphocytes. Percentage of CD4+ cells and CD4/CD8 ratio were higher in the older age group. Percentage as well as absolute counts of CD19+ CD5+ cells and CD19+ CD10+ cells are given. PMID- 7556773 TI - Calcium in pollen-pistil interaction in Petunia hybrida Hort. II. Localization of Ca2+ ions and Ca(2+)-ATPase in unpollinated pistil. AB - The localization of Ca2+ and Ca(2+)-ATPase activity in the unpollinated pistil of Petunia hybrida has been studied. Free or loosely sequestered Ca2+ ions have been found: (1) in the stigma exudate, (2) in the pistil transmitting tract, (3) on the surface of ovule, (4) in the cell wall of the embryo sac. The sites of Ca2+ sequestration in the transmitting cells were the plastids and vacuoles. In some cells, the Ca2+ ions were associated with the tonoplast and plasmalemma underlying the transverse walls which connect the transmitting cells. The sites of particular accumulation of Ca2+ were the surfaces of the placenta and of the ovule. In the ovule, numerous precipitates occurred in the extended walls connecting the embryo sac with the cells of the nucellus. Ca(2+)-ATPase was commonly localized on the plasmalemma (1) of the cells of the transmitting tract of the stigma and style, (2) of the placenta and (3) of the somatic cells of the ovule. The plasmalemma of the embryo sac cells was nearly completely devoid of the reaction product. PMID- 7556770 TI - Nonspecific alkaline phosphatase activity can be responsible for staining of NADPH-diaphorase activity in certain non-neural cells. AB - The NADPH-diaphorase (NADPH-d) reaction is frequently used to visualize the diaphorase activity of nitric oxide synthase (NOS). However, this tetrazolium salt procedure can be of limited specificity at sites where non-specific alkaline phosphatase (alP) and NADHd activity co-exist. This is shown in the present paper using methods of catalytic histochemistry for these three enzymes and levamisole as alP inhibitor for certain mouse tissues. In the urothelium, portio, vaginal and endometrial epithelium as well as in some smooth muscle cells alP hydrolyzes NADPH to NADH which in turn serves as substrate for NADHd leading to false positive formazan production. To exclude this possibility, it is recommended always to include levamisole in the incubation medium if the NADPHd activity of NOS has to be investigated. PMID- 7556774 TI - The influence of the disappearance of plasmodesmal connections between antheridia and thallus on 3H-GA3 transport. AB - Autoradiograms of antheridial filaments labelled after tritiated gibberellic acid (3H-GA3) administration at various developmental stages of Chara contraria (in control and after plasmolysis) show that intensity of gibberellin binding depends mainly on symplasmic connection of antheridia with thallus. Young antheridia before spontaneous symplasmic isolation which precedes spermiogenesis, contain antheridial filaments labelled more intensively than the older ones. This difference disappears if antheridia were plasmolyzed and symplasmic isolation was induced before incubation. These findings are in agreement with the previous results obtained in Chara vulgaris after 14C-GA3 treatment. PMID- 7556775 TI - [Progress in pineal research]. PMID- 7556776 TI - [Effect of estradiol on 5 alpha-reductase activity in osteoblast-like cell (UMR106-01)]. AB - Bone is known to be a target organ of not only estrogens, but also androgens. The mechanism by which these steroids exert their action on bone cells is still poorly understood. In the present study, the effect of 17 beta-estradiol (E2) on 5 alpha-reductase activity, converting testosterone (T) to a more potent biological androgen, dihydrotestosterone (DHT), was assessed in an osteoblast like cell line of rat origin (UMR106-01). Cells were incubated under standardized conditions with varying concentrations of E2 (10(-12)-10(-6) M) for 48 hours. Incubation medium was replaced when the cells were preconfluent and thereafter at 24 hour intervals. Then the cells were harvested. Each cell homogenate was incubated with [4-14C]-T. DHT was detected as a single metabolite on silicagel thin layer chromatography. 5 alpha-reductase activity was determined by measuring the amount of labeled DHT from T. The radiochemical purity of DHT recovered after incubation was confirmed by recrystallization to constant specific activity. Under the conditions used, no estrogen was detected. Production of insulin-like growth factor-I and alkaline phosphatase in UMR106-01 was increased when E2 was added into the culture medium, however, 5 alpha-reductase activity was significantly decreased by the addition of 10(-12) M to 10(-6) M of E2. Maximum inhibition was noticed at 10(-10) M. Our results demonstrate that UMR106-01 cells have a capacity to transform T into the biologically more potent androgen, DHT. The result, that the enzyme activity was influenced by E2, suggests the regulatory mechanism of both sex steroids on the steroid metabolism in osteoblasts. PMID- 7556778 TI - [Changes of specific and non-specific immunological functions before and after transsphenoidal tumor excision--a case of Cushing disease]. AB - The interrelationship between the hypothalamic-pituitary-adrenal axis and the immune system has been becoming clear. However, most research about this interrelationship has been performed by in vitro experiments and by using animal models. To know the effect of hypercortisolism on human immune systems in vivo, we report at 32-year-old man with typical Cushing disease whose specific and non specific immunological functions were estimated before and after successful transsphenoidal surgery. We made a diagnosis of Cushing disease with dexamethasone suppression test, CRF stimulation test, venous sampling, and MRI scan. Before transsphenoidal surgery, both plasma ACTH (100pg/ml) and urinary free cortisol (567 micrograms/day) were higher than the normal range, and the parameters of specific (CD4/CD8 ratio, serum Ig A, PHA/Con-A induced T cell blast formation, and NK cell activity) and non-specific (neutrophil phagocytosis and bactericidal function) immunological functions were clearly impaired. However, at 6 weeks and 6 months after the tumor excision when hormonal abnormalities were changed to normal, every impaired immunological function was improved to the normal range. These data suggest that impaired specific and non-specific immunological functions were induced by hyercortisolism not only in vitro but also in vivo (a state of Cushing disease). PMID- 7556781 TI - Radiograph of the month. Pulmonary cryptococcosis. PMID- 7556777 TI - [Peripheral plasma corticotropin-releasing hormone (CRH) in an aged patient with fasting hypoglycemia associated with an insufficient secretion of insulin: an implication of plasma CRH in glucose metabolism]. AB - We present a 66-year-old man with morning fasting hypoglycemia from an unknown cause associated with markedly suppressed levels of insulin. In this patient we examined the diurnal changes of plasma corticotropin-releasing hormone (CRH). ACTH, cortisol, glucose, insulin and body temperature, and the correlations among them. We also discussed an implication of plasma CRH in glucose metabolism by taking these findings together with results from previous studies on plasma CRH in diabetic or hypoglycemic animals and human beings. In this case, the stress induced by severe spontaneous hypoglycemia in the morning fasting state increased CRH in plasma compared to the euglycemia state and simultaneously activated the hypothalamic-pituitary-adrenal system as well as the sympathetic nervous system remarkably. The daily intravenous infusion of glucose brought the fasting hypoglycemia to normal and hypothermia to normothermia in the morning, and improved no or blunt responsiveness of insulin to glucose. On the 50th day of therapy, the i.v. infusion of glucose quickly produced moderate hyperglycemia and an increase in plasma insulin, and inhibited secretions of CRH, ACTH and cortisol. The source of plasma CRH remains obscure. However, the positive correlations of plasma CRH with both plasma ACTH and cortisol and several lines of evidence indicate that CRH in peripheral plasma is derived from both the hypothalamus and extrahypothalamic peripheral tissue and that during stressed conditions, in particular, the CRH increase in plasma is derived mainly from the paraventricular nucleus of the hypothalamus. The role of CRH not only in the systemic circulation but also in the endocrine pancreases for glucose metabolism remains to be clarified. PMID- 7556779 TI - [The effects of antiandrogen TZP-4238 on plasma testosterone and LH and steroidogenesis in rat and canine testis]. AB - TZP-4238 suppresses plasma testosterone in humans, but its action on the androgen biosynthesis pathway has not been established. Therefore, we researched the testicular testosterone level and the testosterone biosynthesis pathway in vitro in rats before and after receiving a single or continuous oral dose of TZP-4238. The total testosterone fell to 60% of the basal level within 3-8 hr (p < 0.05) and then returned to the control concentration by 24 hr after a single administration of 32 mg/kg. The alteration of the plasma testosterone level correlated well with that of the intratesticular level, which was decreased to 50% at 3-8 hr and recovered to the control level by 24 hr. However, the decrement of the plasma LH level at 3-8 hr after a single oral administration was slight and it then returned to the original level at 12 hr. During the 8 weeks of daily administration of 0.5 mg/kg of TZP-4238 or chlormadinone acetate to dogs, the plasma testosterone levels were slightly lower than the basal extent. In vitro experiments were conducted on the rat testis using the exogenous precursor steroids 20 alpha-hydroxycholesterol, pregnenolone and progesterone, in various steps leading to the biosynthesis of testosterone. Trilostane acted at 3 beta hydroxysteroid dehydrogenase (50% inhibition concentration, IC50 was 1 microM), ketoconazole inhibited the 17 alpha-hydroxylase, and C20, 22- and C17, 20-lyase activities, with an IC50 of 1-50 microM. Cyproterone acetate inhibited both the 3 beta-hydroxysteroid dehydrogenase (IC50;50 microM) and C17, 20-lyase. On the other hand, TZP-4238 exhibited a weaker inhibition of 3 beta-hydroxysteroid dehydrogenase (IC50; 100 microM) than cyproterone acetate, but not of hydroxylase and lyase. Though TZP-4238 did not inhibit the increased testosterone level induced by hCG, trilostane markedly inhibited the effect induced by hCG.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556783 TI - National Practitioner Data Bank. PMID- 7556784 TI - Malaria paradigms in India and control strategies. AB - The paper gives a brief history of malaria control in India through the National Malaria Control Programme (NMCP), National Malaria Eradication Programme (NMEP), implementation of the Modified Plan of Operation (MPO), strengthening of malaria control by launching P. falciparum Containment Programme (PfCP) and the Urban Malaria Scheme (UMS). Making reference to various evaluations of the NMEP, the paper analyses the present malaria situation and brings out reasons demanding change in the strategy of malaria control in consonance with the global malaria control strategy of the World Health Organization (WHO). The epidemiological analysis has revealed that the present adverse malaria situation concentrates mostly under the following five epidemiological paradigms viz. (i) tribal malaria, (ii) rural malaria, (iii) urban malaria, (iv) industrial malaria, and (v) border malaria. Malaria control requires specific approaches and control strategies for each paradigm. We have suggested changes/augmentation in the organizational set-up beginning from NMEP Directorate to the most peripheral health units. The primary responsibility of malaria prevention and control including cost in developmental projects should be shared by the corporate sectors through intersectoral coordination. Residual problems during maintenance phase of the project would come under the general health services. International and bilateral cooperation increases resources availability. The available tools and their rational use for malaria control in different epidemiological paradigms has been discussed with emphasis on integrated control, selective use of chemical insecticides and adoption of cost-effective and sustainable malaria control methods. In this context, intersectoral collaboration, community participation, training, operational research and health education have been discussed as the vital components for effective malaria control. PMID- 7556785 TI - Assessment of the medical emergency. AB - The role of the dentist in an office emergency is reviewed in this article. Emergency decision making, emergency preparedness, and emergency recognition are discussed. Clinical findings that allow one to gauge the level of illness are listed and described. A standard resuscitation scheme, providing a framework for an effective emergency response, is examined in detail. Determination of the need to activate the emergency medical services system and transfer of the patient to a hospital emergency department is discussed. A checklist for presenting the patient's case to other health care providers and documenting the event is provided also. PMID- 7556782 TI - A practical office flow sheet for cancer screening in adults. PMID- 7556786 TI - Emergency drug therapy. Drugs and routes of administration. AB - Management of medical emergencies requires a thorough knowledge of specific medications and their routes of administration. This article briefly discusses the medications that should be included in a basic emergency cart and routes in which to administer them. PMID- 7556787 TI - Anxiety. Its manifestation and role in the dental patient. AB - Emergencies that arise out of psychophysiologic responses are rarely life threatening and can be managed readily by the alert dental office team. It is imperative, however, to use all available information during the emergency assessment. Inaccurate diagnosis and treatment as a result of confusion between presyncope and hyperventilation may result in an accelerated onset of the emergency episode. Rapid recognition of an impending alteration of consciousness should minimize the progression toward or reduce any period of unconsciousness. If unconsciousness occurs, basic life support and supine positioning with supplemental oxygen should be provided immediately. Finally, the dental team should be prepared to treat other life-threatening emergencies that might initially present as syncope, such as cardiac arrest, stroke, or anaphylactic reactions. PMID- 7556780 TI - [Three thyroid patients showing fluctuation of thyroid hormone autoantibody titers during long-term treatment]. AB - Development and fluctuation of thyroid hormone autoantibody (THAA) titers were observed during long-term treatment of thyroid diseases in three patients. The presence of THAA was noticed by spuriously high serum free thyroid hormone levels measured with an analog tracer RIA (Amerlex-M FT3, FT4) in all three patients. Amerlex-M FT3 or FT4 levels gradually decreased to appropriate values for the clinical status according to the decreasing titers of THAA. Free thyroid hormone levels with radiolabeled antibody radioassay (Amerlex-MAB FT3, FT4) were not affected by the THAA and always reflected actual thyroid function. Case 1 was a 46-year-old man with untreated primary hypothyroidism. Auti-T4 autoantibody was detected in his serum. The 125I-T4 analog binding to the autoantibody (125I-T4 analog binding ratio) gradually declined after L-T4 therapy and finally almost disappeared two years and four months later. Amerlex-MAB FT4 level rose to the normal range two months after T4 therapy, but TSH level remained slightly elevated (5.4-13 microU/ml) for five months during T4 therapy. The 125I-T4 analog binding ratio and anti-Tg autoantibody (TgAb) titer were inversely correlated. Case 2 was a 72-year-old woman had received desiccated thyroid for a long time. Sequential changes of 125I-T4 analog binding ratio were very similar to those of TgAb titer. Case 3 was a 74-year-old woman with Graves' disease. She had been treated with methimazole (MMI) and desiccated thyroid for three years and five months. Ten months after stopping both drugs, anti-T3 autoantibody was detected. The 125I-T3 analog binding ratio was transiently elevated and gradually declined to reference range for four years during L-T4 therapy. 125I-T3 analog binding ratio and TgAb titer changed in a similar way. These results suggest that desiccated thyroid hormone therapy and TgAb formation are related to the development of THAA and that L-T4 therapy reduces the THAA titer. PMID- 7556788 TI - Respiratory emergencies in the dental office. AB - Respiratory emergencies are among the most common problems encountered in dental practice and are potentially among the most devastating. Therefore, they must be recognized rapidly and treated promptly. This article focuses on the clinically significant pathophysiology of respiratory emergencies, such that the practitioner can effectively identify the patients with a risk of developing a respiratory crisis and use this information to help provide rapid, effective therapy. Simple protocols are presented for the treatment of the most common respiratory emergencies. PMID- 7556789 TI - Acute cardiac care in dental practice. AB - Acute cardiac care in the dental office is always based on a limited patient data base and limited ability to intervene. The dentist must be aware that the parameters of these conditions are always the same. The dentist must recognize a problem in cardiac function, ensure tissue perfusion and oxygenation, restore cardiac function, influence the underlying cause, recognize promptly when additional acute medical intervention is necessary, and be able to activate the emergency medical system. PMID- 7556790 TI - Neurologic emergencies. AB - Neurologic emergencies are rare, and they usually occur in easily identifiable patients, provided that a thorough medical history has been previously obtained. Rare as these may be, however, they occur without warning and are potentially life threatening. Consequently, the dentist should be prepared by virtue of knowledge of the pathophysiology and therapy and by formal training and certification in basic life support. PMID- 7556791 TI - Management of immediate allergic reactions. AB - Allergic and pseudoallergic reactions can be associated with all drug classes used in dental practice. A thorough medical history is essential to avoid challenging a patient with an agent for which they have proven intolerance. Despite this precaution, the dentist must be prepared to manage an immediate reaction, should it occur. In all cases, management should begin with standard ABC assessment and oxygen supplementation. The administration of either diphenhydramine or epinephrine is predicated on the severity of the reaction. Suggestions regarding dosages and routes of administration are summarized in Table 4. The duration of action for epinephrine is relatively brief (10 to 30 minutes), and dosages may need to be repeated if symptoms recur. Following stabilization, patients who have experienced anaphylactoid reactions should be transported by EMS to the closest emergency room for definitive management. A treatment algorithm summarizing management of allergic reactions is presented in Figure 1. PMID- 7556792 TI - Emergency management of hypoglycemia and hyperglycemia. AB - The majority of diabetic medical emergencies can be prevented by proper preoperative instructions. New or emergency patients unable to receive such instructions are at higher risk, occasionally necessitating the prudent use of various agents to treat hypoglycemia. The signs and symptoms of hypoglycemia and hyperglycemia are reviewed, allowing one to make an educated differential diagnosis of the altered physiological state. Finally, some conditions are not preventable or treatable in the dental office, with notification of the emergency medical system being the indicated treatment choice. PMID- 7556793 TI - The poisoned patient. Toxicologic emergencies. AB - Local anesthetics with and without vasoconstrictors, analgesics, antibiotics, and antianxiety medications are used routinely in dentistry. Most agents used in the dental office have a safe therapeutic dose range between effective and toxic so that the potential for adverse reactions is easily ignored. This article discusses toxicity, allergy, idiosyncrasy, drug interactions, local anesthesia, and the use of recreational drugs and alcohol. PMID- 7556794 TI - The abused patient. AB - Domestic violence is an ever-increasing problem in modern day society and includes physical abuse directed against children, spouses, and the elderly. Dentists are frequently the first health professionals to render treatment to an abused patient and must consequently be knowledgeable in recognizing the tell tale signs of abuse and be aware of their reporting obligations and requirements. Early recognition with timely referrals to appropriate agencies can possibly help prevent more significant injuries and even death from occurring in abused patients. PMID- 7556795 TI - Occupational health and safety emergencies. AB - When considering potentials emergencies in the dental office, one usually first thinks about potential drug reactions or adverse response to underlying systemic diseases. The rare, but potential, emergencies arising from the office environment itself also exist. Toxic reactions to various chemicals found in the office must be considered also. Adequate ventilation helps prevent the long-term consequences of breathing nitrous oxide and chemclave exhaust. Care must be taken in obtaining complete medical histories. Identify patients with communicable diseases such as TB. The potential for transmission of these infections to office staff and other patients exists. Health histories must include the ability to identify patients with latex sensitivity. The increased use of latex products among health care workers has resulted in a higher incidence of latex sensitivity. The office staff must be prepared to recognize and quickly treat anaphylactic reactions. A latex-free environment must be provided for high-risk patients. Even with the use of universal precautions, blood contamination exposures and needle sticks will still occur. Protect office staff against hepatitis through the administration of a hepatitis prevention vaccine. Establish a protocol in advance for handling blood exposure incidents. Update the patient history to determine potential risk. The exposed individual must receive counseling as to the potential risk of HIV infection. If there is a potential risk of HIV contamination, the exposed individual must be offered the opportunity to initiate prophylactic chemotherapy within 1 hour of exposure. Even though occupational health and safety emergencies are rare, they must be considered and planned for. Contingency plans, such as providing a latex-free environment, must be available for preventing emergencies. The office staff must be prepared to treat immediate emergencies such as anaphylaxis and caustic material spills. Arrangements must be available to quickly handle exposure to communicable infections. PMID- 7556796 TI - Hemostasis for dental surgery. AB - Uncontrolled hemorrhage following dental surgery is one of the most serious emergency situations dentists may encounter. Uncontrolled hemorrhage following a surgical procedure requires prompt and appropriate action, or significant morbidity or mortality can result. Through a thorough understanding of hemostasis, the causes of uncontrolled bleeding and the means to control hemorrhage, risks can be minimized and morbidity averted. All dentists performing dental surgery should be well versed in the management of bleeding emergencies. PMID- 7556797 TI - Resuscitation of the pediatric patient. AB - Fortunately, the incidence of pediatric cardiopulmonary arrest is extremely low in the outpatient dental office setting. Because most cardiopulmonary arrests in children result from a progressive deterioration in respiratory function, outcome critically depends on rapid diagnosis and evaluation of the adequacy of ventilation and the pediatric airway. This holds true for any pediatric medical emergency. Our goal must be instituting simple resuscitative measures before full cardiopulmonary arrest develops. Whatever the nature of the medical emergency, caring for a child under these circumstances is challenging. Pediatric Advanced Life Support (PALS) and continual review of the American Heart Association guidelines should be considered by those specializing in the treatment of infants and children. This training will not only bolster practitioner confidence, but enable prompt, effective response for any pediatric medical emergency. PMID- 7556798 TI - Emergency cardiac care. Moral, legal, and ethical considerations. AB - According to the American Heart Association, "Successful completion of an ACLS course means in accordance with the cognitive and performance standards of the American Heart Association. It does not warrant performance, nor does it, per se, qualify or authorize a person to perform any procedure. It in no way related to licensure, which is a function of the appropriate legislative, health or educational authority." The same can be said for BLS; however, with the recent revisions in ECC programs, even this disclaimer has been eliminated from the most recent textbooks in BLS and ACLS. This is in keeping with "...the American Heart Association's reaffirmation of its role as an educational resource rather than as a certifying agency." Lay public course participants in BLS (which technically includes dentists) will now receive course participation cards. Health care provider course participants will continue to receive "course completion cards" if all criteria have been satisfied by the student. The trend is to categorize what was previously termed testing as evaluation. Certification has become a thing of the past. It is unclear at this time what the impact this policy change will have on agencies who rely upon documentation from the American Heart Association to satisfy requirements that have been imposed upon dentists and other health professionals for initial licensure and relicensure. Semantics aside, one thing that remains clear is the expectation of the dentist with regards to emergency management. Expertise (for lack of a better term) in specific aspects of ECC remains a standard. For all dentists, expertise in BLS is that standard. For dentists administering deep sedation and general anesthesia, expertise in ACLS is the community standard. Although there is some ambiguity for those dentists administering conscious sedation, at the very least, they should have expertise in BLS. In addition, they are strongly encouraged to have expertise in ACLS, particularly because the limited hours of training in conscious sedation provide less medical background than is acquired during training in deep sedation and general anesthesia. In addition, the dentist is ultimately responsible for the demeanor of his or her office and staff. In the prehospital dental office setting, the matter of converting a dental office team geared to efficient delivery of dental procedures, into a team primed to perform emergency cardiac care seems daunting. This is especially so if the dentist has little undergraduate or clinical preparation for managing life-threatening emergencies. Therefore, an emergency management plan (with oversight for its implementation by the dentist) is of paramount importance. PMID- 7556801 TI - Regeneration of the tunic cuticle in the compound ascidian, Botrylloides simodensis. AB - The tunic cuticle of Botrylloides simodensis, a colonial ascidian, is an electrondense sheet overlaying the tunic in which zooids are embedded. When the margins of colonies were cut off and the colonies were incubated in running seawater, the tunic cuticle regenerated at the cut surface of the tunic matrix by the following process: 1) Electron-dense fibers appeared at the cut surface within a few minutes. 2) Fiber formation proceeded during the next several hours, and the number of fibers greatly increased. 3) In 12 to 24 hours, the fibers aggregated and formed a continuous layer, which was the newly regenerated cuticle. 4) Minute protrusions appeared in the new cuticle after 3 to 5 days. Fiber formation occurred even in fragments of colonial margins that were incubated in a bottle of seawater. It was inhibited under some experimental conditions including low pH and the presence of some proteases or protease inhibitors. PMID- 7556799 TI - Fish major histocompatibility complex genes: an expansion. AB - The advent of polymerase chain reaction technology has provoked a large amount of progress in the field of fish major histocompatibility complex (MHC) research. Many new teleost sequences have been reported in the last four years, including representatives of all classes of MHC genes. While the intron-exon structure of teleost MHC genes is now becoming clear, the organisation of the genes within the teleost MHC is still unclear. The sequences reported to date have been used for phylogenetic analysis and, due to their evolutionary position, are discussed in relation to hypotheses regarding the origin of the MHC. Teleost MHC gene sequences are also examined to see if conserved features of the both the nucleotide and amino acid sequences of higher vertebrate MHC genes are present. Differences in these features will reflect functional differences between teleost and mammalian MHC genes and may also have evolutionary implications. PMID- 7556800 TI - Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus. AB - Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the host's defense. PMID- 7556802 TI - cDNA sequences and organization of IgM heavy chain genes in two holostean fish. AB - Immunoglobulin M heavy chain (mu) sequences of two holostean fish, the bowfin, Amia calva, and the longnose gar, Lepisosteus osseus, were amplified from spleen mRNA by RACE-PCR, cloned, and sequenced. Each mu chain showed the conserved four constant domain structure typical of a secreted mu chain. Southern blot analyses with specific heavy chain variable (VH) and constant (CH) region probes suggest that both fish possess an IgH locus that resembles that of the teleosts, amphibians, and mammals in its organization. The overall sequence similarity of gar and bowfin mu chains was 60% and 48% at the nucleotide and amino acid levels, respectively, while similarity to the mu chains of teleosts and elasmobranchs was lower. The bowfin mu chain possesses a distinctive proline-rich sequence at the C mu 1/C mu 2 boundary; a shorter proline-rich sequence is present at this position in the gar mu chain. Both gar and bowfin show, in their C mu 4 sequences, motifs that could serve as cryptic splice donor sites for the production of mRNA encoding the membrane-bound form of the mu chains, and the bowfin also shows a potential cryptic splice donor site in the C mu 3 exon. PMID- 7556803 TI - Alternate pre-mRNA processing pathways in the production of membrane IgM heavy chains in holostean fish. AB - A single gene encodes two forms of the IgM heavy chain (mu) in vertebrates: one (microseconds) present in serum as secreted IgM and the other (microns) as the antigen receptor form of IgM present on the B-lymphocyte membrane. The mRNAs encoding microseconds and microns are derived from a single primary transcript by alternate pathways of RNA processing. In all vertebrates so far examined, with the exception of teleosts, microns mRNA is produced by splicing the transmembrane (TM) encoding exon 1 into a cryptic donor site near the 3' end of the C mu 4 exon. In contrast, teleost species splice the TM exon 1 into the regular splice donor site at the 3' boundary of the C mu 3 exon. We have examined micron mRNAs in two species of primitive bony fish, the holostean bowfin and the longnose gar. These fish utilize both the C mu 3 to TM1 (teleost) pathway and the typical cryptic C mu 4 to TM1 pathway. In addition the bowfin possesses a cryptic splice donor site near the middle of C mu 3. This is used in the production of a third species of microns-encoding mRNA, but does not participate in the production of an alternate form of the microseconds mRNA. The structure and patterns of expression of their mu genes suggest that the gar and bowfin may be more closely related than implied by the current view of fish evolution. PMID- 7556804 TI - Retroviruses and diabetes in animal models: hypotheses for the induction of the disease. AB - This review examines the association between retroviruses and diabetes in the mouse model, the role of retroviruses in the pathogenesis of Type 1 diabetes and the mechanisms by which retroviruses can induce an autoimmune reaction. Three putative mechanisms are considered: the expression of retroviral protein(s) on the beta-cell surface as the first step in immune response against beta cells; the homology of a retroviral product with a self antigen inducing a cross reacting autoimmune response (molecular mimicry); and a retroviral product showing homology with interleukin-2 and inducing T-cell activation against beta cell antigens and loss of tolerance. These findings are discussed for their possible implications in the pathogenesis of human Type 1 diabetes. PMID- 7556805 TI - Testosterone and risk factors for cardiovascular disease in men. AB - It has been assumed for years that male testosterone levels play a central role in worsening lipoprotein patterns and causing greater susceptibility to ischemic heart disease. Yet most clinical trials of quasi-physiologic doses of intramuscular testosterone in older men show no effect on high-density lipoprotein (HDL)-cholesterol, while cross-sectional epidemiologic studies almost uniformly find that endogenous testosterone levels are positively associated with HDL-cholesterol levels. Further work is required to determine whether and why physiologic testosterone levels in the high normal range appear to be conducive to optimal cardiovascular health for adult men. PMID- 7556806 TI - Comparison of miglitol and glibenclamide in diet-treated type 2 diabetic patients. AB - The efficacy of the new intestinal alpha-glucosidase inhibitor, miglitol, and glibenclamide were compared in a 6-month double-blind controlled protocol involving 100 non-insulin dependent diabetic patients under diet alone. HbA1c levels (initially between 7 and 11%) were reduced (p < 0.05): -0.78 +/- 0.21% after miglitol and -1.18 +/- 0.20% after glibenclamide. The difference between the two treatments was not significant, although glibenclamide appeared to be more active than miglitol at 8 (p = 0.002) and 16 weeks (p = 0.01) but not at 24 weeks. Fasting glycaemia decreased after miglitol (8.7 +/- 0.3 vs 9.6 +/- 0.3 mmol/l, p = 0.005) and after glibenclamide (8.0 +/- 0.3 vs 9.1 +/- 0.3, p = 0.007). After miglitol, a decrease was noted after breakfast (p < 0.001) and lunch (p < 0.001). The same was true for glibenclamide (p = 0.004 and p < 0.001 respectively). A significant reduction in glucose incremental area during a standard meal test was noted at the end of miglitol (p = 0.008) or glibenclamide treatment (p = 0.04). Subgroups of nonresponders to both treatments were identified (10/49 with miglitol, 9/47 with glibenclamide). Side effects were recorded in 10 patients treated with miglitol (flatulence and meteorism, diarrhoea, 1 discontinued therapy) and in 10 treated with glibenclamide (asthenia, sensation of hunger). This study indicates that miglitol is suitable for initial application in diet-resistant Type 2 diabetic patients, providing, a persistent effect and acceptable side effects. PMID- 7556809 TI - Locus of control and metabolic control. AB - Previous studies designed to establish in diabetic patients the relationship between metabolic control and locus of control are controversial. The aim of the present study was to find answers to the following questions: is there a link between an internal locus of control and improved metabolic control? Is this true for type I and type II diabetic subjects? Is this improved metabolic control linked directly, or even indirectly, with the locus of control by types of behaviour, such as for example a greater desire for information concerning the disease (knowledge) and closer adherence to doctors' recommendations (compliance)? Sixty-one patients (36 type I and 25 type II) on insulin therapy were compared according to the type of their locus of control using two different questionnaires (Rotter and Wallston). The extent of their knowledge about diabetes was also assessed; self-monitoring of blood glucose (SMBG) was considered to be a measure of compliance, while the HbA1 level was considered to be an indicator of metabolic control. The study compared the influence of the type of locus of control on the various parameters. The results indicate that, irrespective of the questionnaire, type I "internals" exhibited better metabolic control (p < 0.05) than type I "externals", even with a lower level of knowledge of diabetes (p < 0.01) and less frequent SMBG (p < 0.05). However, the benefits of internality as regards metabolic control were not as great when this internality was extreme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556807 TI - Relationship between fat intake and glomerular filtration rate in normotensive insulin-dependent diabetic patients. AB - Glomerular hyperfiltration is a candidate marker for diabetic nephropathy in insulin-dependent diabetic patients since it can reflect elevated glomerular capillary pressure, a cause of glomerulosclerosis. We studied the potential contribution of several dietary components to glomerular hyperfiltration during a cross-sectional study of 110 consecutive normotensive, non-proteinuric insulin dependent patients with respect to glomerular filtration rate (GFR) and food intake. GFR was measured using the 51Cr-EDTA plasma disappearance technique. Glomerular hyperfiltration was defined as GFR > 137 ml.min-1 1.73 m-2 (mean +2 SD of age-matched healthy controls). Food intake was recorded with a computer assisted programme. Thirteen patients displaying glomerular hyperfiltration ingested more protein (1.60 +/- 37 vs 1.38 +/- 0.34 g.kg-1 body weight.day-1; p = 0.032) and more fat (1.70 +/- 0.54 vs 1.39 +/- 0.44 g.kg-1 body weight.day-1; p = 0.022) than other subjects, although their total energy intakes were similar. Univariate regression analysis showed that GFR was positively related to both protein (r = 0.28; p = 0.003) and fat (r = 0.25; p = 0.007) intakes and negatively related to age (r = -0.29; p = 0.002). Stepwise multivariate regression analysis indicated 2 independent determinants for GFR: age (F = 15.26) and fat intake (F = 13.15). Excess fat intake may contribute to glomerular hyperfiltration in insulin-dependent diabetes. PMID- 7556808 TI - Human diabetic cataract: role of lipid peroxidation. AB - To test whether impaired glutathione redox status may be related to lens oxidative damage in humans, we measured glutathione (total and oxidised forms) and malondialdehyde, a lipid peroxidation product, in clear lenses and diabetic and non-diabetic cataracts. Diabetic cataracts were divided into 2 subgroups with either intact or abnormal haemo-ocular barrier as evaluated by preoperative iridography. Decreased total glutathione values were observed in cataractous (diabetic and non-diabetic) as compared to clear lenses (p < 0.001), whereas enhanced oxidised glutathione levels were found in diabetic caracts as compared to non-diabetic ones and clear lenses (p < 0.001). Malondialdehyde concentrations were significantly higher in all types of cataracts, especially myopic and diabetic ones, than in clear lenses (p < 0.001). Moreover, malondialdehyde levels in diabetic lenses were inversely correlated with total glutathione (r = 0.80; p < 0.001) and linearly correlated with oxidised glutathione values (r = 0.76; p < 0.001). Finally, glutathione redox status was found to be more seriously impaired in lenses from diabetic patients with abnormal than intact haemo-ocular barrier. These data suggest a contributory role of lipid peroxidation and glutathione oxidation and consumption in the pathogenesis of cataract, especially in diabetic lenses with haemo-ocular barrier abnormality. PMID- 7556811 TI - Lack of reproducibility of low-dose dopamine-induced microalbuminuria in type 1 diabetic patients. PMID- 7556810 TI - [Modification of hemorheological parameters in microvascular complications of diabetes]. AB - The aim of the present study was to assess the possible modifications in the parameters of red cell aggregation and blood and plasma viscosity in 92 diabetic patients compared to 82 non diabetic control subjects. Based on the presence of microalbuminuria (> 30 mg/24 h) and/or retinopathy each group of diabetic patients was divided into two subgroups. This study shows increased red cell aggregation and blood viscosity among diabetic patients with microangiopathy. There was a very good correlation between fibrinogen level and the different rheological measurements. The results of this study confirm the importance of the blood rheology abnormalities observable in diabetes. These disorders increase peripheral vascular resistances and ischemia and therefore worsen diabetic nephropathy and retinopathy. PMID- 7556812 TI - [Diabetes and anesthesia: care of diabetics during the intraoperative period. Recommendations of ALFEDIAM (French Language Association for the Study of Diabetes and Metabolic Diseases)]. PMID- 7556814 TI - [Obesity, The ob gene product and control of adipose mass]. PMID- 7556813 TI - [Nutrition and diabetes. Recommendations of ALFEDIAM (French Language Association for the Study of Diabetes and Metabolic Diseases)]. PMID- 7556815 TI - [Is insulin therapy sufficiently used in type II diabetes? Minimal insulin therapy, or intensified insulin therapy?]. PMID- 7556816 TI - Risk factors and their identification second part: study designs for identification of risk factors. AB - This is the second a series of three articles which reviews the identification of risk factors of a disease, here: diabetes or complications of diabetes. In the first of the series [1], we gave the definition of a risk factor, along with measures of its force-relative risk and odds ratio, followed by the epidemiological definitions of the diseases: diabetes, coronary heart disease and hypertension. Risk factors were further discussed and we completed the discussion by some observations on the bias which can arise from a study or from its analysis, which can lead the researcher to the wrong conclusion. In this second article we define the three types of epidemiological studies which are used to determine whether factors are associated with a disease: observational or cross sectional studies, cohort studies and casecohort studies. Examples are provided of each of these study types; their advantages and disadvantages are discussed. The final paper will provide some examples of the identification of risk factors from the literature. The first example involves diabetes and pancreatic cancer, the second birth weight and non-insulin dependent diabetes. Having found an association between a risk factor and diabetes, we will discuss whether it can be considered to be a risk factor, and if so whether it is likely to be a cause of the disease. PMID- 7556817 TI - Pain during childbirth. PMID- 7556819 TI - Some aspects on the mechanism of human labor and delivery. PMID- 7556818 TI - Influence of epidural analgesia on fetal and neonatal well-being. AB - Epidural analgesia is a frequently used method to reduce the pain of child bearing. Concerns regarding the safety and potential hazards still persist in the medical community. This review intends to examine how epidural analgesia determines the various factors of fetal and neonatal well-being. Placental drug transfer of opiates like morphine, pethidine and fentanyl is rapid and can lead to neonatal depression. Sufentanil seems to be the safest opiate to administer epidurally. Local anaesthetics are transferred to the fetus in substantial amounts, but the reported effects are subtle and are probably inconsequential. Utero- and fetoplacental blood flow seems to be improved by epidural analgesia with local anaesthetics. Even when using stronger solutions for more extensive blockade in patients for caesarean section, no adverse effects could be demonstrated using pulsed Doppler technique as long as prolonged hypotension (> 2 min) is avoided. Hypotension is best prevented with 20-25 ml/kg crystalloid preload and prompt treatment with ephedrine or etilephrine. Addition of adrenaline to local anaesthetics is considered to be safe for the healthy mother and fetus but it should best be avoided in mothers with pregnancy induced hypertension. Fetal and neonatal acid-base balance and gas-exchange are not adversely affected by epidural analgesia. Many studies show that epidural analgesia can indeed protect the fetus if hypotension is prevented. Neonatal well being evaluated by APGAR, BRAZELTON, SCANLON and NACS scores is not significantly influenced by local anaesthetics. Neonatal depression can occur however with epidural use of morphine, fentanyl and alfentanil. Sufentanil, again in doses up to 30 micrograms in association with bupivacaine seems to be devoid of depressive effects on the neonate. In summary, the anaesthetist has good arguments to reassure his obstetrical colleagues that providing epidural analgesia for pregnant women in labour is a justifiable intervention to support the natural process of child-bearing. PMID- 7556820 TI - Effects of epidural analgesia on the progress of labor and the mode of delivery. PMID- 7556821 TI - Opioids in combination with local anesthetics for epidural analgesia during labor. PMID- 7556822 TI - Role of patient-controlled epidural analgesia in obstetrics. PMID- 7556823 TI - Labor pain relief using bupivacaine and sufentanil: patient controlled epidural analgesia versus intermittent injections. AB - OBJECTIVES: To determine whether the use of patient-controlled epidural analgesia (PCEA) versus intermittent injections (CIT) resulted in local anesthetic dose reduction. STUDY DESIGN: PCEA and CIT using a mixture of 0.125% bupivacaine with sufentanil 1 or 0.75 microgram/ml were compared in 60 and 195 parturients, respectively. Assessments included pain scores, local anesthetic consumption, degree of motor blockade, type of delivery and neonatal outcome. Statistical analysis was done using Student's t test and Chi-squares. RESULTS: PCEA and CIT provided effective analgesia during labor and delivery. A higher dose of opioid significantly reduced the use of local anesthetic solution in PCEA-patients. There was no difference in motor blockade, type of delivery and neonatal outcome. CONCLUSION: Patient-controlled epidural analgesia is an effective, safe and acceptable alternative to conventional intermittent epidural injections for pain relief during labor and delivery. PMID- 7556825 TI - Is an epidural test dose mandatory? PMID- 7556824 TI - Epidural analgesia during labor: continuous infusion or patient-controlled administration? AB - Patient-controlled epidural analgesia (PCEA) has several advantages over continuous epidural infusion of bupivacaine during labor: it produces a good analgesia with a limited sensory spread; generally, less bupivacaine is administered and maternal satisfaction with pain control is increased. However, the quality of analgesia is similar to that obtained with other forms of epidural administration. Moreover, PCEA is only a particular form of epidural and, as such, has the same safety requirements. PCEA does not appear to reduce the workload of the anesthetic team. The cost of the PCA pump will need to be included in future evaluation of the cost/benefit ratio. PMID- 7556826 TI - Combined spinal and epidural blockade for analgesia in labour. PMID- 7556829 TI - The grand multipara. AB - Grandmultiparity (GMP) has long been considered an obstetric complication for both mother and fetus, although recent studies indicate that, with proper perinatal care, women with high-parity rates are no longer at high risk. The current study examines the outcome of delivery in 1700 women in their fifth or more delivery, as compared with two control groups: 622 primiparas and 735 multiparas (two to three previous deliveries). Excellent prenatal care was available free of charge to all parturients. Our objectives were to evaluate the management of GMP in contemporary obstetrics and to assess whether grand multiparas are still high-risk patients. The age of the grandmultiparas was significantly higher compared with with the control groups, which may explain the higher incidence among them of antenatal medical disorders, such as diabetes mellitus and hypertensive disease. No significant differences were found among the three groups for preterm or post-term births, small-for-gestational-age infants, polyhydramnios, oligohydramnios, perinatal death, fetal distress, multiple births, placenta previa, abruptio placentae or cord prolapse. Macrosomia was markedly higher in the grandmultiparas and multiparas than in nulliparas. Thus, our results indicate that good perinatal care can ensure better results in grandmultiparas, and that grandmultiparity no longer needs to be considered a high-risk obstetric category in our population. PMID- 7556828 TI - The nature and consequences of childbirth pain. AB - For most women, childbirth is associated with very severe pain often exceeding all expectations. Some childbirth education groups and popular texts on the subject, however, seem disposed to encourage unrealistic expectations: claiming that labour is other than painful and that pharmacological analgesia is both unnecessary and harmful. All too often, those who promote such views witness women in labour only occasionally and are rarely responsible for patient care. Pain associated with uterine contractions should be distinguished from that associated with delivery: for there are important differences in the clinical characteristics, neural pathways and physiological responses. In the first stage of labour pain is largely visceral in origin, whereas during the transitional and second stages somatic pain becomes more pronounced. As described in this review, it is now well established that uterine contraction pain evokes a generalised neuroendocrinal stress response producing widespread physiological effects during the first stage of labour. They include increased oxygen consumption, hyperventilation and respiratory alkalosis; increased cardiac output, systemic peripheral resistance and blood pressure; delayed gastric emptying; impaired uterine contractility and diminished uterine perfusion; and metabolic acidaemia. While other factors (such as anxiety, starvation and physical exertion) are also partly responsible for inducing some of these effects, pain appears to be the most potent source because they are all obtunded by effective epidural analgesia. PMID- 7556830 TI - Breech presentation and cesarean section in term nulliparous women. AB - OBJECTIVE: To examine pregnancy outcome in nulliparous women with single term breech presentation. METHODS: Two departments of Obstetrics and Gynecology at the same hospital used different approaches to deliver nulliparous women with singleton breech presentation at term. One department (A) delivered by trial of labor and the other (B) delivered by elective cesarean section. Prospectively and blinded to obstetric condition, parturients were assigned to either department in a systematic alternate fashion. The study period covered 8 years (1985-1992). The pregnancy outcome parameters examined were: Apgar score, intra- and post-partum death and maternal and neonatal morbidity. Neonatal morbidity was classified in three major categories: non-neurological trauma, neurological signs and respiratory problems. RESULTS: The study included 264 women of whom 135 delivered in department A and 129 in department B. Department A had 35 vaginal and 100 cesarean births and department B 10 vaginal and 119 cesarean births. There was no intra-partum death and the only post-partum death occurred among vaginal deliveries. The Apgar score was significantly worse at 1 and 5 min in vaginally delivered babies of department B. Neonatal morbidity was significantly more frequent after vaginal births (P < 0.01). Maternal morbidity was significantly higher following cesarean sections (P < 0.01). Babies of vaginal deliveries had significantly higher non-neurological trauma (P < 0.01) and pathological neurological signs (P < 0.01) than those delivered by the abdominal route. CONCLUSION: The level of risk for mother and child in the nulliparous with term singleton breech, suggests cesarean section as the preferred route of delivery. PMID- 7556827 TI - Anaesthesia for caesarean section (epidural and general): effects on the neonate. AB - Anaesthesia may impair the condition of the neonate either directly (largely mechanically in utero or due to the influence of drugs) or indirectly via alteration of placental perfusion. These influences may further differ during normal caesarean section from those under complicated conditions. The criteria with which neonatal conditions are estimated need to be carefully distinguished with respect to results obtained. Neurobehavioural test results may give different information to the observer. A variety of drugs, not simply one, is used during general anaesthesia (GA). Even the consequences of maternal stress fetal and neonatal levels of catecholamines or endogenous peptides may play a role. Local anaesthetics are known to cross the placenta as general anaesthetics do; in most cases, their effects are clinically irrelevant. Fetal and neonatal deliterious effects of regional anaesthesia (RA) are mainly related to maternal hypotension and the administration of large doses of local anaesthetics. If adequate doses of local anaesthetics and/or opioids are used, alterations in neurobehavioural scores are subtle and transient. Under normal maternal and fetal conditions, GA and RA are almost identically useful with respect to neonatal well being after caesarean section; subtle and inconsistent neurobehavioral residua may be present for a short period of time following GA. Under conditions of a compromised fetus, the neonate may however benefit from epidural anaesthesia more than from GA. PMID- 7556831 TI - Reflectance pulse oximetry in neonates. AB - OBJECTIVE: To test the feasibility and reliability in neonates of an in-house reflectance pulse oximetry (RPOX) system as an alternative method of non-invasive oxygen saturation monitoring when transmission pulse oximetry (TPOX) cannot be used, as in fetal scalp oxygen saturation monitoring during delivery. STUDY DESIGN: The study population consisted of 31 intensive care neonates. The RPOX sensor was attached by suction to the forehead, cheek, occiput and back; recordings were under simultaneous TPOX control. RESULTS: There was close agreement between RPOX and TPOX oxygen saturation and heart rate values, even in periodic breathing. RPOX signals from the back were unreliable because of breathing artifacts. Differences in mean absolute RPOX and TPOX oxygen saturation values were due to different calibrations. Both systems were equally sensitive to motion artifacts. CONCLUSION: As a feasible and reliable method of non-invasive oxygen saturation monitoring in neonates, RPOX has potential applications in fetal scalp monitoring. PMID- 7556832 TI - Neuronal death after perinatal asphyxia. AB - During perinatal asphyxia several mechanisms aim to limit cerebral damage. However, when the degree of asphyxia passes beyond a certain threshold, brain damage is inevitable. This review focuses on the various factors determining the final cerebral outcome. Metabolic and biochemical events, such as the intracellular level of calcium, the formation of oxygen derived free radicals, the release of excitotoxic neurotransmitters and the interrelationship of these parameters are discussed. Furthermore, steps possibly useful to pharmacologic intervention aiming to reduce cerebral damage are presented. PMID- 7556833 TI - Provider-associated factors in obstetric interventions. AB - OBJECTIVE: To assess which factors influence provider-associated differences in obstetric interventions. STUDY DESIGN: A survey of obstetricians and co-workers in a sample consisting of 38 Dutch hospitals was taken, using a questionnaire that contained questions about personal and hospital-policy data, and 19 clinical problems with a choice between intervention and non-intervention. From the clinical problems an Intervention Score was assembled. The influence of the personal and hospital-policy items on this Intervention Score was studied by analysis of variance. RESULTS: Overall the Intervention Score was low, with considerable interindividual variation. Four personal/hospital items influenced the Intervention Score: the teacher could affect the score in either direction; the increasing age of the obstetrician and routine electronic fetal monitoring had an increasing effect; employment of midwives had a decreasing effect. Other factors, including litigation, had no effect. CONCLUSION: Supplier-induced differences do exist in obstetric interventions and are influenced by personal and hospital-policy factors. PMID- 7556835 TI - Single dose ceftriaxone versus single dose cefuroxime plus metronidazole for preventing febrile morbidity and urinary tract infection in vaginal hysterectomy. AB - OBJECTIVE: To compare the prophylactic efficacy of 1 g ceftriaxone with 1.5 g cefuroxime + 0.5 g metronidazole for febrile morbidity and urinary tract infection in patients undergoing vaginal hysterectomy. STUDY DESIGN: A prospective, randomized, comparative, non-blinded study with at least 100 patients on each side. The two-sample t-test was used for testing equality of two means and the chi 2 test was used for pair differences. RESULTS: Single dose cefuroxime + metronidazole and ceftriaxone were equally effective in preventing febrile morbidity postoperatively. Additionally ceftriaxone was more effective in sterilizing pre-operatively existing bacteriuria. PMID- 7556834 TI - Randomized study of vaginal chlorhexidine disinfection during labor to prevent vertical transmission of group B streptococci. AB - OBJECTIVE: To evaluate the effect of vaginal disinfection with chlorhexidine gel during labor on vertical transmission of group B streptococcus, as a method to prevent vertical transmission and subsequent neonatal early onset group B streptococcal disease. STUDY DESIGN: A prospective study with randomization of 1020 parturients to one of three groups as soon as labor started. In all parturients, anus, introitus and cervix were cultured semiquantitatively. Two groups were treated double-blindly with 10 ml of either a 0.3% chlorhexidine gel or a placebo gel, applicated around the portio and into the fornices. If labor still continued, a second application was given after 10 h. The third group received no treatment. Ear, pharynx and umbilicus of all newborns were also cultured semiquantitatively. RESULTS: Nine hundred and eighty one women were evaluated. The overall incidence of group B streptococcal carriership was 19.4%. Vertical transmission was 52.4% in the chlorhexidine group, 71.4% in the placebo group and 66.7% in the control group (P = 0.069). When testing the transmission rates for the chlorhexidine versus the combined placebo plus control group (69.3%), the difference was 16.9% (P = 0.026). CONCLUSION: Vaginal disinfection with a chlorhexidine gel during labor modestly reduces group B streptococcal vertical transmission. Because the method is cheap, simple and safe, it should be considered for routine use. Our results indicate that it may reduce the incidence of early onset group B streptococcal sepsis by 2-32%. PMID- 7556836 TI - Laparoscopic management of malignant ovarian cysts: a 78-case national survey. Part 2: Follow-up and final treatment. AB - This paper reports a retrospective multi-institutional French survey carried out in 1992 to determine the incidence of laparoscopic management of malignant ovarian cysts. Of 5307 ovarian lesions treated endoscopically, 78 were malignant (1.47%) including 60 borderline tumours (77%) and 18 ovarian cancers (23%). Laparoscopic treatment was puncture in 23% of cases, partial exeresis in 51% and total removal in 26%. Laparotomy was immediately performed in 25% of the cases and as a second stage procedure in 58% (mean delay: 78 days). Laparotomy was not performed in 16% of the cases. Our findings suggest that laparoscopic management of ovarian lesions that subsequently prove to be malignant is not uncommon. To prevent the risk of metastasis, thorough pre-operative and per-operative evaluation is mandatory. In 22.4% of the patients presenting lesions in this study, laparoscopic tampering resulted in an upgrading of FIGO stage. PMID- 7556837 TI - Preserved pituitary response under ovarian stimulation with HMG and GnRH antagonists (Cetrorelix) in women with tubal infertility. AB - OBJECTIVE: To examine the pituitary response in patients undergoing short-term application of the GnRH antagonist Cetrorelix in the mid-cycle phase for hypophysial suppression of premature LH surges within an IVF-program. DESIGN: Twenty patients suffering from primary or secondary tubal infertility were stimulated with hMG from cycle day 2. From day 7 till ovulation induction Cetrorelix was administered in two different dose regimens (15 patients 3 mg s.c. daily; 5 patients 1 mg s.c. daily). Three hours before ovulation induction a GnRH test was performed using 25 micrograms of native GnRH and the pituitary response examined by measurement of the serum LH concentration after 30 min. RESULTS: Premature LH surges could be avoided in the 3-mg group and in the 1-mg group, respectively. Due to this, none of the cycles had to be cancelled. Oestradiol profiles and ultrasound demonstrated a satisfactory follicular maturation. All patients showed pronounced suppression of the serum LH levels before ovulation induction. The mean increase of serum LH due to the performed GnRH test was 10 mIU/ml for the 3-mg group, while the average maximum in the 1-mg group was about 32.5 mIU/ml. CONCLUSIONS: The pituitary response is preserved by the treatment with the GnRH antagonist Cetrorelix. The extent of suppression of the adenohypophysis, as expressed by the different reactions on GnRH test, can be modulated by the dosage administered. This should allow ovulation induction by GnRH or one of its agonists instead of hCG, which could be beneficial in patients at high risk of Ovarian Hyperstimulation Syndrome (OHSS) and those suffering from Polycystic Ovary Disease (PCOD). PMID- 7556839 TI - Lack of positive correlation between placental blood flow and mean arterial pressure in the non-anaesthetised guinea pig near term; an indication for autoregulation? AB - The objective of the present study was to evaluate whether placental blood flow (PBF) decreases or increases, in response to a fall or rise in mean arterial pressure (MAP), respectively, in an unanaesthetised animal model with a haemochorial placenta. Such a response would support the absence of autoregulation of placental blood flow for changes in mean arterial pressure. To this end, 31 late-pregnant guinea pigs were subjected to isovolemic haemodilution, a sham procedure or isovolemic haemoconcentration, after adequate recovery from instrumentation. The percentage change from baseline in PBF (delta PBF, %), was compared between animals with a fall in MAP, in response to haemodilution or the sham procedure (MAP-fall, n = 8, mean delta MAP -11 +/- 7%) and those with a rise in MAP, following haemoconcentration (MAP-rise, n = 10, mean delta MAP +15 +/- 8%). In addition, the percentage change in the blood flow to the myocardium, gastrointestinal tract, adrenals, kidneys, brain, skin, carcass and myometrium was compared between the MAP-fall and the MAP-rise group. The direction of the change in PBF did not differ between the MAP-fall and the MAP-rise group. In this respect, the distribution of delta PBF over the two groups was comparable with that observed for the myocardium, gastrointestinal tract, adrenals, skin, carcass and myometrium. The blood flow to the kidneys and the brain varied in response to the change in haematocrit. The results of this study show that PBF varies independently from MAP. This observation supports the idea that the blood flow to the labyrinthine placenta of the awake and healthy guinea pig near term may be autoregulated for hydrostatic inflow pressure, at least over the spectrum of arterial pressures observed in the present study. PMID- 7556838 TI - Serum levels of insulin-like growth factor-binding protein-3 in normal pregnancy. AB - The aim of this study was to investigate circulating levels of insulin-like growth factor-binding protein-3 (IGFBP-3) in women with normal pregnancy. A total of 468 pregnant women at various gestational ages and 30 non-pregnant females were recruited in this study. Serum concentration of IGFBP-3 was determined by radioimmunoassay. Maternal serum levels of IGFBP-3 increased as pregnancy progressed. The concentration of IGFBP-3 in pregnancy serum was positively associated with the gestational age. The mean values of circulating IGFBP-3 in the second and third trimesters of pregnancy were higher than those in the first trimester of pregnancy and in non-pregnant females. In addition, there was a highly significant correlation between IGFBP-3 and IGF-I in pregnancy serum. We conclude that, during pregnancy, IGFBP-3 appears to be the principal binding protein for circulating IGF-I and may be involved in the regulation of fetal growth. PMID- 7556840 TI - Vaginal foreign bodies in women in postmenopause and in senium. AB - We report on three women, 81, 73 and 69 years of age, in whom foreign bodies had been introduced into the vagina either by the patients themselves or by somebody else. In all cases, medical help was needed to remove the object. In one patient the foreign body had remained within the vagina for 7 years. Only two of the three women admitted that the object had been introduced for sexual stimulation. Some papers are quoted that report on similar events, however, in younger patients. Our observations demonstrate the often problematical sexual situation of many old people. PMID- 7556842 TI - Early prenatal diagnosis of holoprosencephaly: the value of transvaginal ultrasonography. AB - Holoprosencephaly is a rare condition characterized by different degrees of fused ventricles resulting from the failure of the prosencephalon to cleave during embryonic life. In this report we present a case of alobar holoprosencephaly diagnosed prenatally at 15 weeks' gestation in a woman with a family history of open neural tube defect. The diagnosis was initially missed because only suboptimal views of the fetal brain were obtained with transabdominal ultrasonography due to fetal position; however, a better depiction of brain anatomy was obtained using the transvaginal approach, allowing the detection of a single ventricle and fused thalami. We conclude that transvaginal ultrasonography is not only important for the early prenatal diagnosis of central nervous system abnormalities but also for enhancing brain visualization when transabdominal examination of the fetal head is limited due to maternal and fetal factors. PMID- 7556841 TI - Critical limb ischemia after accidental subcutaneous infusion of sulprostone. AB - A 34-year-old patient was treated with constant intravenous infusion of sulprostone because of postpartum hemorrhage from a hypotonic uterus. The arm in which sulprostone had been infused was painful 23 h after infusion. A day later, the arm was found to be blueish, edematous and extremely painful as a result of arterial spasm. The vasospasm was probably caused by accidental subcutaneous infusion of sulprostone as a result of a displaced intravenous catheter. A diagnosis of critical limb ischemia was made. Treatment with the prostacyclin analogue iloprost resulted in full recovery. Critical limb ischemia as a serious complication of sulprostone has not been previously reported. PMID- 7556845 TI - Benzodiazepine use during pregnancy: a prospective study. PMID- 7556847 TI - How to handle breech presentation and delivery? PMID- 7556844 TI - Comment: physiology of cervical change over the course of labor. PMID- 7556846 TI - Hook effect in immunoradiometric assay for human chorionic gonadotropine as a marker for trophoblastic disease. PMID- 7556849 TI - The regionalization of perinatal care in France--description of a missing policy. AB - This paper reviews published data on the organization and evaluation of regionalized perinatal care programs in countries where they exist and describes those elements presently lacking in France. The advantage of maternal compared to neonatal transport is described vis-85-vis its effect on perinatal deaths as well as neonatal and developmental handicaps. Most studies describe a major advantage of maternal transfers, with less perinatal deaths (including stillbirths and neonatal deaths) and lower rates of severe developmental handicaps. In contrast, French publications show that widespread regionalization of perinatal care does not exist in spite of proposals advocating this concept made more than 20 years ago. Thus, many deliveries of less than 33 weeks gestation length or/and less than 1500 g birth weight take place in maternity sites without adequate neonatal care, despite ample evidence that such infants are at much greater risk of perinatal death or severe handicaps at 2 years of age after transfer than if they had been born in a perinatal center. For these reasons, professional organizations in France should choose to implement a policy of maternal transfers as a major effort in the near future. PMID- 7556848 TI - Pre-eclampsia: physiology and immunological aspects. AB - Pre-eclampsia is a frequent, unpredictable syndrome which is dangerous for both mother and foetus. The concept of placental ischemia has gained wide acceptance among the numerous theories put forward to explain the illness. The setting up of preeclampsia seems to be scheduled in two steps: (1) an absolute or relative placental ischemia due to vascular diseases or hypertrophic placenta, or most often secondary to implantation defect, particularly anomaly with the invasive trophoblast; (2) a diffuse endothelial disease. The connection between these two steps is incompletely disclosed. The authors demonstrate that the maternal immune system which is strongly stressed during all the stages of normal gestation is implicated in pre-eclampsia. Its role is probably not univocal. Foeto trophoblastic antigens could be poorly recognised. This defect of recognition could lead to the abnormalities of trophoblastic invasion observed in pre eclampsia. Pre-eclampsia does not seem to be accompanied by an immunological rejection of the foetus. Some genetically predisposed patients do not have a sufficiently competent immune system to neutralise one or more of the toxic products released by the ischemic placenta. Certain types of pre-eclampsia could be auto-immune, with the auto-antibodies directed against certain types of phospholipids or trophoblastic constituents. A disequilibrium between oxidation and anti-oxidation mechanisms involving neutrophils could lead to aggression of the endothelium which is observed in pre-eclampsia. Pre-eclampsia could represent a form of immuno-dystrophy, with the excessive production of adverse cytokines locally, directed against the trophoblast. Without directly implicating the immune system as the trigger of pre-eclampsia, it seems that its role is unclear. In some cases it develops protective mechanisms which, when overwhelmed or inadequate, allows pre-eclampsia to occur. In other cases it can form part of the cascade of aggressions leading to the abnormalities encountered. The integration of these abnormalities in the pathophysiological models, could help improve the classification of pre-eclampsia. This attempt will lead to a more adapted preventive and therapeutic management of pre-eclampsia. PMID- 7556850 TI - Pharmacological and genetic approaches for the study of circadian rhythms in mammals. AB - Two different approaches have been utilized to study the controlling mechanisms that underlie the generation and entrainment of circadian rhythms in mammals. The use of specific drugs to alter the period and/or the phase of circadian rhythms has provided new insights into both the pathways by which environmental information reaches the mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) and the cellular and neurochemical events within the SCN itself which are involved in circadian rhythmicity. A second approach, which seeks to exploit genetic differences in the properties of the circadian system, holds the promise of eventually defining the cellular and molecular events that are part of the clock itself, the events that underlie the entrainment of the circadian clock by environmental factors, and the expression of overt rhythms driven by the clock. It is anticipated that the pharmacological and genetic approaches to the study of circadian rhythms will complement each other as the underlying physiological mechanisms of the circadian clock system become defined. PMID- 7556851 TI - Cytochrome P450 in the brain: neuroendocrine functions. AB - The effectiveness of steroid hormone metabolites as sedatives and anesthetics has been known for many years. More recently, their interaction with neurotransmitter receptors has helped to elucidate their mechanism of action, but their physiological functions and their role in disturbances of behavior, anxiety, and sleep/wakefulness have yet to be elucidated. Until 1981 it was assumed that metabolites of steroid hormones arose from the adrenals and gonads and that their action on neurotransmitter receptors was a mechanism of communication between the brain and the periphery. The evidence that the brain could accumulate steroids independently of the adrenals and gonads in 1981 and later the evidence for the presence of the cholesterol side chain cleavage enzyme (P450scc) in the brain have challenged this concept and stimulated a great deal of interest in the possibility that the brain could be making its own steroids from cholesterol for some as yet undefined purpose. In this review we examine the data pertaining to the role of brain P450 in the synthesis and degradation of neurosteroids. We summarize the data on the presence of P450scc in the brain and try to answer the following questions: (1) Does P450scc in the brain contribute significantly to the synthesis of GABAA receptor active steroids? (2) Can the P450scc in the brain account for the accumulation of pregnenolone in the brain? (3) Is there evidence for special functions of the pregnenolone synthesized in the brain? (4) Is there a role for other forms of brain P450 in neurosteroid action? PMID- 7556852 TI - The neurohypophysial endocrine regulatory cascade: precursors, mediators, receptors, and effectors. AB - The neurohypophysial endocrine regulatory cascade has been described as a molecular model of neuroendocrine control of organismal functions. Any physiological function can be analyzed in molecular terms as a succession of interactions occurring either in a solution or in a membrane system. The key mechanism in the ordering of the cascade is the conformational recognition of the two partners at each step. Each interaction results in a change of conformation of a recognized protein that in turn becomes a recognizer for the following molecule. The cascade starts within the secretory cell by the processing of the expressed precursor along the secretory pathway until the storage of the mature mediator in vesicles and its subsequent exocytic secretion in blood. The circulating mediator recognizes the target cell through specific membrane receptors that transduce the message within this target cell. A second intracellular cascade leads to activation of the effector, the protein fulfilling the physiological function. The complexity of the messages is, in part, due to the duplication propensity of the genomic DNA, the frequent occurrence of multiple copies for precursors, mediators, receptors, and effectors, and therefore, a combinatorial diversity that increases during the course of evolution. Vertebrate neurohypophysial hormones can be ordered in two main evolutionary lineages, culminating in oxytocin and vasopressin in placental mammals. In this field, diversification of the messages was made by differential processing of the precursors, secondary gene duplications, the emergence of several types of receptors for each hormone, and a variety of effectors triggered by the second messengers within differentiated target cells. This review is an attempt to integrate neurohypophysial functions at the molecular, cellular, and organismal levels. PMID- 7556843 TI - Colchicine is excreted at high concentrations in human breast milk. PMID- 7556853 TI - Fetal biophysical profile and vibratory acoustic stimulation in high-risk pregnancies. AB - OBJECTIVES: To determine the influence of the non-stress test (NST) on the efficiency of the fetal biophysical profile (FBP) and to test the clinical usefulness of the FBP and its combination with vibratory acoustic stimulation (VAS) in managing high-risk pregnancies. METHODS: One hundred twenty fetuses of preeclamptic patients were included in a prospective study. Five standard variables of the FBP were observed ultrasonically following NST. In cases of non reactive NST, external VAS was applied and the FBP score calculated and compared with the FBP score before VAS. RESULTS: Of 120 calculated FBPs, 102 (85%) had normal profile scores before VAS and 104 (86.7%) after VAS. No statistically significant difference was found. The sensitivity, specificity, and positive and negative predictive values of the FBP score in predicting poor perinatal outcome were 94.7%, 94.4%, 75% and 99%, respectively. VAS produced a high conversion (58.8%) of non-reactive NST to reactive fetal heart rate pattern. The false negative rate of the FBP score was 9.8 per 1000, which did not increase after VAS. CONCLUSIONS: The efficiency of the FBP score was not significantly improved by VAS, although a high conversion of non-reactive to reactive NST was produced. The FBP with its three 'acute biophysical variables' was found to be an accurate method of antepartum assessment even without an NST. The low incidence of perinatal complications among patients with normal FBP scores, permits the conservative management of preterm high-risk pregnancies. PMID- 7556854 TI - Great grand multiparity--beyond the 10th delivery. AB - OBJECTIVE: To investigate the perinatal outcome and obstetric complications of women delivering for the 10th time or more. METHODS: Four hundred twenty women of great grand multiparity were analyzed in a modern health care setting and compared with our general population of obstetric patients, with regard to past history, maternal age, gestational age, mode of delivery, fetal outcome and intercurrent medical/obstetric problems. RESULTS: The study group showed significantly lower rates of low birth weight infants and instrumental delivery. No significant difference was seen in the incidence of cesarean section, pathologic fetal presentation, maternal hypertension, gestational diabetes, hemorrhage, or perinatal morbidity or mortality. There was a slightly higher incidence of twin births compared with the general population. CONCLUSION: It is probable that women capable of reaching their 10th delivery are basically healthy. If offered adequate perinatal care, they are not a high-risk group during subsequent deliveries. PMID- 7556855 TI - Cost-benefit analysis of laparoscopic adnexectomy. AB - OBJECTIVE: To determine the economic feasibility (from the patient's viewpoint) of laparoscopic adnexectomy (LA) in a developing country. METHOD: Forty-four LA cases of benign ovarian cysts or tubal pregnancy were compared with 44 conventional open adnexectomy (OA) cases treated during the same period and matched by diagnosis, procedure and degree of difficulty. RESULT: The postoperative hospital stay was 1.3 +/- 0.6 days (mean +/- S.D.) and 5.3 +/- 1.1 days (P < 0.001) for the LA and OA groups, respectively. LA cases had a shorter recovery period (5.8 +/- 2.2 days vs. 27.2 +/- 6.6 days, P < 0.001), but higher hospital charges (US$463.3 +/- 84.9 vs. US$229.8 +/- 92.2, P < 0.001), caused mainly (89.2%) by disposable laparoscopic instruments. Incremental cost-benefit analysis revealed a possible saving with LA for higher income patients (US$9.2 per day). CONCLUSIONS: LA in Thailand may have economic benefit only to the higher income patients. Benefits to other patients in developing countries will depend on future cost reductions of disposable instruments and/or more effective use of reusable instruments. PMID- 7556856 TI - Transvaginal trucut biopsy in patients with abdominopelvic mass. AB - OBJECTIVE: To perform an abdominopelvic or pelvic mass biopsy as an office procedure, as an alternative to laparoscopic biopsy. METHODS: A transvaginal trucut biopsy using a disposable biopsy needle was carried out in 5 cases. RESULTS: The biopsy gave a clear diagnosis of ovarian cancer in 2 cases, non tubercular inflammation in 2 cases and tuberculosis in 1 case. CONCLUSIONS: In the developing world, transvaginal trucut biopsy is an important method for diagnosis. PMID- 7556857 TI - Postmenopausal bone loss in Japanese women. AB - OBJECTIVES: Our purpose was to detect anatomic variations in postmenopausal bone loss and to determine the risk factors most affecting bone mass in Japanese women. METHODS: One hundred sixty-six Japanese women (54 premenopausal and 112 postmenopausal), aged 40-68 years, were enrolled in the study. Bone mineral densities (BMD) of the lumbar spine (L2-4) and total body were measured by dual energy X-ray absorptiometry and the results were subjected to statistical analysis. RESULTS: Among the various sites studied, the lumbar spine (L2-4) showed the fastest bone loss after menopause. Multiple regression analysis indicated that two factors, number of years since the menopause and body mass index (BMI), affected lumbar spine (L2-4) BMD. CONCLUSION: Measurement of lumbar spine (L2-4) BMD is suitable for evaluating postmenopausal osteoporosis. The risk factors for postmenopausal bone loss were a long period after menopause and a low BMI. PMID- 7556858 TI - Pain control in abortion. AB - OBJECTIVE: To reduce the pain experienced during abortion. METHODS: A four-phase study was conducted at a free-standing abortion clinic. Phase 1: a double-blind, randomized, placebo-controlled trial of 600 mg ibuprofen given preoperatively was carried out in 193 women. Pain during the procedure and 30 min postoperatively was compared. Phase 2: a double-blind, randomized trial compared 1% lidocaine, buffered 1% lidocaine and 0.25% bupivacaine in terms of the pain of the injection and the pain of the procedure in 200 women. Phase 3: waiting times of 0, 3 and 10 20 min between the end of the injection and the beginning of the procedure were compared in terms of pain during the procedure in 139 women. Phase 4: local anesthetic was injected into one side of the cervix slowly and one side quickly and the pain of the injections was compared in 87 women. RESULTS: Phase 1: ibuprofen reduced pain scores more than placebo with the improvement being greatest for the postoperative period. Phase 2: buffered lidocaine was significantly less painful to inject than plain lidocaine or bupivacaine. Phase 3: waiting between the injection and the procedure did not improve pain control. Phase 4: slower injections were found to be less painful than fast injections. CONCLUSIONS: Several methods were found to improve pain control during abortions. PMID- 7556860 TI - Diabetes in pregnancy. AB - OBJECTIVE: To determine whether there is an association between the level of glycemic control and perinatal complications in pregnant diabetic patients. METHODS: Two hundred sixty confirmed cases of pre-existing diabetes, gestational diabetes and impaired glucose tolerance were analyzed to assess risk factors, modality of treatment, level of blood sugar control and effect on perinatal morbidity and mortality. RESULTS: Risk factors for the development of diabetes included age ( > 25 years), East Indian ethnic origin, glycosuria, and a history of diabetes in a first-degree relative. Treatment consisted of diet alone or in combination with soluble insulin. There is a marked increase in perinatal mortality and maternal morbidity if normoglycemia is not maintained. CONCLUSIONS: A greater emphasis must be placed on the detection of diabetes in pregnancy and effective treatment should be instituted as early as possible to achieve normoglycemia if associated complications are to be reduced. PMID- 7556861 TI - Dysgerminoma of the ovary in a patient with triple-X syndrome. AB - A 24-year-old patient with dysgerminoma of the right ovary and hypoplastic left ovary was screened for chromosomal aberration. Triple-X syndrome was found in the oral epithelium and lymphocyte culture, while chromosomal in situ hybridization of the tumor itself showed a normal XX karyotype. Although trisomy X and dysgerminoma are coincidental findings in our case report, women with malignant germ cell tumors and abnormal genitalia should be checked for chromosomal aberrations to exclude anomalies carrying a Y chromosome. PMID- 7556862 TI - Primary squamous cell carcinoma of the endometrium. AB - All reported cases of primary squamous cell carcinoma of the endometrium and the effectiveness of the different treatment modalities in the world literature are reviewed. We present the first reported case of primary squamous cell carcinoma of the endometrium treated by radical hysterectomy, pelvic and para-aortic lymphadenectomy. The patient is without signs of disease 4 years after surgery. This is the longest follow-up of all reported cases. There are very few cases from which is to conclusively predict the best mode of therapy. The current standard of treatment is total abdominal hysterectomy and bilateral salpingo oophorectomy with or without pelvic and para-aortic lymph node sampling, depending on the histology and depth of the myometrial invasion. Adjuvant therapy includes radiation therapy according to the extent of disease. PMID- 7556859 TI - Massive fetomaternal hemorrhage. AB - We evaluated the potential prognostic factors for fetomaternal hemorrhage (FMH). Sinusoidal heart rate pattern and decrease in fetal movement are considered important signs of FMH. However these findings were detected in only two of six cases of FMH and were thought to be late signs of FMH. Fetal hemoglobin levels in the maternal circulation for infants born alive following FMH were less than 5%. Our findings suggest that this amount of blood loss could cause fetal death. PMID- 7556865 TI - Monoclonal antibody-defined lymphocyte subsets in cervical carcinoma. PMID- 7556864 TI - Prolonged retention of fetal bones with osseous metaplasia of the endometrium. PMID- 7556866 TI - Misdiagnosis of tuberculosis in semen by the IS6110 PCR assay. PMID- 7556863 TI - Genital burns and vaginal delivery. AB - Obstetric complications may result from burn scarring in the genital area. Women in developing countries typically squat around cooking fires, and burns are common. This recent case in Nepal describes obstructed labor in a young woman whose genital area had extensive scarring from a cooking fire injury. Proper antenatal assessment by health care providers can reduce the risk to mothers and infants of the consequences of a birth canal damaged or obstructed by burn scarring. PMID- 7556867 TI - ACOG technical bulletin. Septic shock. Number 204--April 1995 (replaces no. 75, March 1984). Committee on Technical Bulletins of the American College of Gynecologists. AB - Septic shock remains an important cause of death and permanent disability in obstetric and gynecologic patients. Prompt recognition and diagnosis are critical to optimizing outcome. A thorough understanding of the pathophysiologic mechanisms operative in septic shock is important in treating these critically ill women. Immediate attention to oxygenation and hemodynamic stability is of paramount importance in effecting the best possible outcome. The use of sophisticated technology, including pulmonary artery monitoring and mechanical ventilation is frequently necessary, requiring intensive monitoring. Consultation with practitioners experienced in the management of septic shock is often helpful. PMID- 7556868 TI - ACOG committee opinion. Premenstrual syndrome. Number 155--April 1995 (replaces no. 66, January 1989) Committee on Gynecologic Practice. American College of Obstetricians and Gynecologists. PMID- 7556869 TI - Standards of education in obstetrics and gynecology. Report of the Committee on Education in Obstetrics and Gynecology and Human Reproduction. PMID- 7556870 TI - Anterior pituitary cells: getting to know their neighbors. AB - We review in this article recent findings on the effects of cell proximity (nearness to each other) on anterior pituitary cell function in vitro. Important cellular processes, including protein synthesis, basal hormone release, response to hypothalamic regulatory factors and signal transduction can change as a function of the distance between adjacent cells. These findings, together with a growing body of evidence on intrapituitary intercellular interactions, form a basis for proposing a model of anterior pituitary cell regulation. In this scheme extrapituitary, intracellular and intercellular signaling pathways all contribute and interact to control secretory processes in cells. PMID- 7556871 TI - Cellular localization of 11 beta-hydroxysteroid dehydrogenase 2 gene expression in the ovine adrenal gland. AB - The cellular localization of 11 beta-hydroxysteroid dehydrogenase 2 (11 beta HSD2) gene expression in the ovine adrenal gland was determined by in situ hybridization histochemistry. 11 beta-HSD2 mRNA was localized exclusively to the adrenal cortex of the adult sheep, and within the cortex the mRNA was highly expressed in the zona fasciculata and zona reticularis with relatively low expression in the zona glomerulosa. Radiometric conversion assay using adrenal cortical tissues revealed extremely high levels of 11 beta-HSD activity which was characteristic of 11 beta-HSD2 in that it was NAD-dependent and displayed a Km for cortisol of 41 +/- 4 nM. This indicates that 11 beta-HSD2 mRNA within the ovine adrenal gland is translated and functional with respect to enzymatic activity. In marked contrast, 11 beta-HSD1 mRNA was undetectable in either the cortex or medulla of adult sheep adrenal glands. In conclusion, we have demonstrated, for the first time, the zonal localization of 11 beta-HSD2 mRNA and the presence of 11 beta-HSD2 activity in the adult sheep adrenal cortex. The adrenal 11 beta-HSD2 may function to (1) regulate the rate of cortisol secretion by adrenocortical cells; (2) protect these cells from high levels of locally produced glucocorticoids; and/or (3) provide an important source of circulating cortisone, which can be activated by the action of 11 beta-HSD1 reductase in organs such as the liver. PMID- 7556872 TI - Structure of the human luteinizing hormone-choriogonadotropin receptor gene: unusual promoter and 5' non-coding regions. AB - The complete organization of the human luteinizing hormone-choriogonadotropin (LH/CG) receptor (LH/CGR) gene and the structure of 1591 bp of its 5' flanking region have been determined. This gene spans over 70 kbp and contains 11 exons. The first ten exons and part of the last exon encode the extracellular domain of the receptor while the transmembrane and intracellular domains are encoded by the remaining part of the last exon. The gene encodes a 701 amino acids long preprotein, contrary to a previous report of 699 amino acids. Primer extension experiments and polymerase chain reaction (PCR) mapping allowed definition of the transcription initiation site, which is located 1085 bp upstream from the initiation codon. The 5' non-coding region is thus unusually long. The promoter region which is different from the murine LH/CG receptor promoter, contains two putative TATA boxes at positions -34 and -47 and a CAAT box consensus sequence at position -89. A consensus sequence corresponding to a cAMP responsive element is found at position -697. Seven API consensus sequences are also found in the 5' flanking region of the gene. Southern blot experiments demonstrated an informative biallelic polymorphism within the human LH/CG receptor gene locus using BglII endonuclease. The cloning of the human LH/CGR gene and the determination of the organization and structure of its 5' flanking region allow the study of its hormonal, developmental and tissue-specific regulation. Primers and PCR conditions are described for the direct genomic sequencing of all the exons of the gene. This information should facilitate the study of pathological mutations of the receptor. PMID- 7556873 TI - Insulin secretion from islets of GK rats is not impaired after energy generating steps. AB - Insulin secretory responses of intact and electrically permeabilised islets of Goto-Kakizaki (GK) rats, a novel model of non-insulin dependent diabetes mellitus, and Wistar (control) rats were compared to investigate the mechanism of the impairment of insulin secretion from pancreatic islets of GK rats. Insulin secretion from intact islets in response to glucose, glyceraldehyde, succinate monomethylester and tetramethyl p-phenylenediamine, which reduces cytochrome c directly, was significantly impaired in GK rats compared to control rats (P < 0.05, P < 0.01, P < 0.05 and P < 0.05, respectively). However, Ca(2+)-induced insulin release from electrically permeabilised islets of GK rats was higher than that of control rats. Moreover, insulin secretion from intact islets in response to 50 mM KCl, which depolarises islet cells, was not impaired in GK rats. These results indicate that insulin secretion from islets of GK rats is not impaired after energy generating steps of metabolism. PMID- 7556875 TI - Quantitative analysis of messenger ribonucleic acid encoding natriuretic peptide receptors in aldosterone-producing adenoma. AB - There exist conflicting data regarding the inhibitory effect of atrial natriuretic peptide on aldosterone production from aldosterone-producing adenoma (APA). Natriuretic peptides mediate their actions through natriuretic peptide receptors (NPRs). Whether or not NPRs are present in the tumors remains controversial. To elucidate this paradox, gene expression of NPRs was examined by Northern blot analysis and competitive polymerase chain reaction in tumorous and non-tumorous portions of APA, and in normal adrenal gland from patients with renal cell carcinoma. The results of Northern blot analysis showed the presence of messenger ribonucleic acid (mRNA) of three NPRs in all adrenal tissues, including APA. The proportional expression of NPR gene transcripts in APA was type A (0.6%), type B (18.7%), and type C (80.7%). The levels, but not the proportions, of type C and possibly type B NPR mRNAs were lower in tumorous and non-tumorous portions of APA compared to those in normal adrenal gland (type C 190.2 +/- 24.5 [means +/- SEM, normal adrenal gland] > 168.1 +/- 20.8 [non tumorous portion] > 112.2 +/- 15.5 [tumorous portion] pg/10 micrograms total RNA, F = 3.82, P < 0.05; type B 45.2 +/- 8.5 [normal adrenal gland] > 30.0 +/- 5.2 [non-tumorous portion] > 25.1 +/- 4.1 [tumorous portion] pg/10 micrograms total RNA, F = 3.03, P = 0.065). The mRNA levels of type C, rather than type A or type B, NPR were correlated with the percentage of zona fasciculata-like cells in APA (r = 0.90, P < 0.05). In conclusion we have demonstrated the presence of mRNA encoding the three NPRs in APA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556876 TI - Molecular and biochemical characterization of the mouse brain corticotropin releasing hormone-binding protein. AB - A 37 kDa corticotropin-releasing hormone-binding protein (CRH-BP), distinct from the CRH receptor, is expressed in rat anterior pituitary corticotrophs and many regions of the brain, suggesting that CRH-BP may modulate the biological activity of CRH. In these studies a mouse brain CRH-BP (mCRH-BP) cDNA has been isolated and characterized. The 1666 nucleotide mCRH-BP cDNA is expressed in brain and pituitary and encodes a 322 amino acid protein that is highly homologous to human and rat CRH-BPs. Recombinant mCRH-BP, expressed in cultured mammalian cells, binds human CRH (Kd(app) = 0.56 nM and Ki(app) = 0.37 nM) and the alpha-helical (9-41) CRH antagonist (Ki(app) = 0.28 nM) with high affinity, but exhibits much weaker affinity for ovine CRH (Ki(app) = 206 nM). Recombinant mCRH-BP also blocks CRH-induced adrenocorticotropin release from AtT-20 cells. Additional biochemical characterization of the binding activity of mCRH-BP indicates that CRH-BP and CRH receptor utilize different molecular interactions to bind CRH. PMID- 7556874 TI - The D2 receptor: blocked transcription in GH3 cells and cellular pathways employed by D2A to regulate prolactin promoter activity. AB - Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556878 TI - Functional analysis of a variant of the thyrotropin receptor gene in a family with Graves' disease. AB - Nucleotide sequence analysis of PCR fragments corresponding to the TSH-receptor (TSHR) amplified from genomic DNA collected from the four members of a family, two of which had Graves' thyrotoxicosis, revealed a nucleotide substitution in the first position of codon 36 of the TSH-receptor gene in the two patients. The nucleotide substitution was from G to C, leading to a 36D-->36H change (D36H) in the predicted amino acid sequence of the receptor. The altered sequence was also found in DNA obtained from their mother, but not in DNA from their father. We stably expressed the two receptor variants in NIH 3T3 cells, by transfection of cDNA encoding the wildtype (WT) and D36H variants of the TSHR. Neither the binding of 125I-TSH nor the responsiveness to TSH measured as cAMP formation, appeared to be different in the TSHR-D36H compared to the TSHR-WT. Furthermore, the D36H-receptor also became desensitized when exposed to TSH as did the WT receptor. PMID- 7556877 TI - Interleukin-1 beta-induced stimulation of insulin release in mouse pancreatic islets is related to diacylglycerol production and protein kinase C activation. AB - The aim of the present study was to investigate the mechanisms responsible for the acute, stimulatory effects of interleukin-1 beta (rIL-1 beta; 1 ng/ml) on insulin release from mouse pancreatic islets. For this purpose, mouse islets were exposed for 60-120 min to rIL-1 beta and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 mM glucose, but both in the presence of 5.6 or 16.7 mM glucose, or 10 mM leucine + 2 mM glutamine, it induced a 60-100% increase in insulin release. Moreover, rIL-1 beta also enhanced the effects of 1 mu/ml glipizide on insulin release, but failed to increase insulin release induced by 30 mM KCl or by glucose plus phorbol ester (TPA; 100 nM). These early stimulatory effects of rIL-1 beta on insulin release were neither accompanied by major increases in glucose or amino acid metabolism, nor by modifications in islet cAMP content, and they were prevented by mannoheptulose, diazoxide or verapamil. rIL-1 beta potentiation of glucose-induced insulin release was not accompanied by modifications in [Ca2+]i, but the cytokine increased diacylglycerol production and induced protein kinase C (PKC) activation. Down-regulation of PKC completely prevented the stimulatory effects of rIL-1 beta on glucose-induced insulin release. In conclusion, rIL-1 beta induces an early stimulation of insulin release in mouse beta-cells by a mechanism independent of glucose metabolism, cAMP generation or modifications in [Ca2+]i. This effect is probably related to diacylglycerol formation and stimulation of PKC. PMID- 7556880 TI - Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line. AB - The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/intracellular domain of the human GH receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution. PMID- 7556881 TI - Changes in 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein-, fura-2 and autofluorescence in intact rat pancreatic islets in response to nutrients and non nutrients. AB - Intracellular pH (pHi) was measured in intact rat islets loaded with the dye 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein. Raising the concentration of glucose from 3 to 13 mM caused a modest, gradual increase in pHi (500:450 fluorescence ratio). The addition of 20 mM lactate caused a gradual decline in pHi which reversed upon withdrawal of lactate. In contrast, the weak acids propionate and acetate (20 mM) induced a rapid, pronounced fall in pHi followed by a gradual recovery. Upon removal of the weak acid, a marked, reversible alkalinization occurred. The addition of 20 mM NH4Cl caused a pronounced intracellular alkalinization, followed by recovery. The subsequent removal of NH4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pHi. Epifluorescence at 350 and 380 nm excitation, and the 350:380 fluorescence ratio, an index of cytosolic [Ca2+] ([Ca2+]i), were measured in islets loaded with the calcium indicator fura-2. Approximately 30% of the total fluorescence was estimated to be derived from NAD(P)H autofluorescence. Addition of KCl or acetylcholine to fura-2 loaded islets raised and lowered, respectively, the 350 and 380 signals, thereby causing marked increases in the 350:380 ratio. Neither KCl nor acetylcholine affected cellular NAD(P)H autofluorescence in non loaded islets. An increase in glucose concentration caused an increase in both the 350 and 380 fluorescence signals and also in the 350:380 ratio. Qualitatively similar, although smaller changes were observed when Ca2+ was omitted from the medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556879 TI - Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers. AB - Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo. PMID- 7556882 TI - Growth hormone amplifies insulin-like growth factor I induced progesterone accumulation and P450scc mRNA expression. AB - The interaction of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the acquisition of progesterone biosynthetic capacity were examined in cultured porcine granulosa cells. Basal progesterone production was not affected (P > 0.05) by GH treatment. However, concurrent treatment with GH produced a 4.1 fold increase (539 versus 2214 ng/culture) in the IGF-I-stimulated accumulation of progesterone. GH potentiated IGF-I induced progesterone production in a dose and time dependent manner, with a time requirement of 48 h or less. The amplified effect of GH was not attributable to changes in cellular protein, DNA content, cell number, plating efficiency or cell viability. Moreover, Northern blot analyses revealed that GH enhanced IGF-I induced expression of the gene encoding cytochrome P450 side chain cleavage. These observations provide further evidence to support the role of GH in the regulation of ovarian steroidogenesis. PMID- 7556886 TI - Parathyroid hormone-related peptide and 8701-BC breast cancer cell growth and invasion in vitro: evidence for growth-inhibiting and invasion-promoting effects. AB - It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone-related peptide (PTHrP) gene and that N-terminal PTHrP immunoreactivity can be found in cell medium. Here we have firstly measured immunoreactive PTHrP in 8701-BC cell medium using antibodies raised against midregion and C-terminal fragments, and also demonstrated the expression of PTH/PTHrP receptor by 8701-BC cells. Secondly, we have examined the role, if any, elicited by diverse PTHrP domains on 8701-BC cell proliferation, and invasive behaviour in vitro related to production of extracellular proteolytic enzymes. Our data show that PTHrP [1-34], and, to a minor extent, [67-86] and [107-139], are anti-mitogenic but 'invadogenic' for 8701-BC cells, and suggest that diverse enzymatic activities may contribute to cell invasion in response to different PTHrP fragments. In light of the present data on a chemoattractive role for PTHrP in vitro, we hypothesize that this protein might intervene in local control of the invasive process in breast carcinoma. PMID- 7556884 TI - Hydrogen peroxide suppresses low-density lipoprotein (LDL) uptake and LDL supported steroidogenesis by porcine luteal cells. AB - The present study tested the hypothesis that hydrogen peroxide (H2O2) inhibits low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by luteal cells. LDL uptake: dispersed porcine luteal cells from mid-cycle (days 6-11, estrus = day 0) were incubated for 0-120 min at 37 degrees C in F-10 medium + 0.1% BSA containing various concentrations of H2O2 (0-1000 microM). Cells were washed with catalase (2800 U/ml), and then with fresh medium. Cell viability based on trypan blue exclusion was unaltered by H2O2 exposure through 60 min. H2O2-exposed cells were incubated with fluorescent-tagged-LDL (Dil-LDL; 1 microgram/ml) for 10 min at 37 degrees C. Fluorescence of small (SLC) and large (LLC) luteal cells was analyzed by flow cytometry (n = 6 experiments). H2O2 (> or = 10 microM) caused a progressive reduction (P < 0.01) in mean fluorescence intensity (MFI) of SLC and LLC indicative of up to a 30-35% decline in LDL uptake. Progesterone (P) production: dispersed luteal cells (4 x 10(4)/0.2 ml) were pre-cultured in DMEM/F-12 medium overnight (approximately 18 h) in 96-well culture plates. Wells were rinsed and fresh media (0.2 ml) containing H2O2 (0-500 microM) was added. After 30 min, the following treatments were added: human(h)LDL (0 or 50 micrograms/ml), human chorionic gonadotropin (hCG; 0 or 100 ng/ml), hCG + LDL, or 22R-hydroxycholesterol (22[OH]-C; 0 or 25 micrograms/ml). Cells were incubated for an additional 4 h, and P concentrations in final media samples were measured by RIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556885 TI - Alteration of prostaglandin E receptors in advanced breast tumour cell lines. AB - We studied the direct effects of PGE2, often produced at high levels by mammary tumours, on three human breast cancer cell lines diversely advanced in malignancy regarding differentiation and tumorigenicity in nude mice. We evaluated PGE2 effect on cell growth, PGE2 receptor level and functionality. Our results show that PGE2 induces cAMP accumulation and inhibits the growth of the most differentiated breast cancer cells. We also demonstrate that loss and probably dysfunction of PGE2 receptors is related to an advanced tumorigenic phenotype of the cells. Thus, it seems that during progression of breast cancer, the cell growth escapes from control by PGE2. Nevertheless, it is possible to control the growth of advanced breast cancer cells in vitro by direct induction of intracellular cAMP accumulation. PMID- 7556887 TI - Migration of oligodendrocyte precursors on astrocytes and meningeal cells. AB - Oligodendrocytes populate developing white matter and repopulate demyelinated regions of the CNS by migration. Although little is known about their migratory routes, the environment through which these cells migrate, whether during development, disease, or injury, is packed with astrocytes infiltrated with or bounded by meningeal cells. In the present study, the migration of oligodendrocyte precursors from primary cultures and of the precursor cell lines (CG4 and Oli-neu) on astrocytes and meningeal cells was investigated using tissue culture migration assays and time lapse video microscopy. Oligodendrocyte precursors and the cell lines were found to migrate poorly on astrocytes and meningeal cells compared to migration on laminin even though both astrocytes and meningeal cells express cell surface laminin. The migration-inhibitory activity was not detected in conditioned media derived from either astrocytes or meningeal cells, nor was it detected from matrix deposited by these cells. Analyses of the events immediately following cell-cell contacts revealed that oligodendrocyte precursor-astrocyte contacts were typically long-lasting and appeared to be adhesive, whereas precursor-meningeal cell contacts usually resulted in rapid withdrawal of the precursor cell process. No correlation was found, however, between general adhesiveness and the rate of migration. Our results suggest that both astrocytes and meningeal cells retard migration of oligodendrocyte precursors, consistent with the view that they may impede the movement of oligodendrocyte precursors into CNS lesion sites. PMID- 7556888 TI - Calcium-independent, meiotic spindle-dependent metaphase-to-interphase transition in phorbol ester-treated mouse eggs. AB - Mouse eggs, arrested at metaphase II of meiosis, form pronuclei as a result of fertilization or exposure to parthenogenetic agents, such as the phorbol ester phorbol myristate acetate (PMA). Exposure of eggs to the microtubule inhibitor colcemid caused the disappearance of the meiotic spindle and prevented the PMA induced release from metaphase. However, colcemid- and PMA-treated eggs which lacked spindles formed nuclei when treated in addition with the protein synthesis inhibitor cycloheximide or the protein kinase inhibitor 6-dimethlyaminopurine. To prevent an increase in intracellular calcium concentration ([Ca2+]i), eggs were exposed to the cell permeant acetoxymethyl ester (AM) form of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA). Nuclei formed in 45% of eggs treated with BAPTA AM and PMA and in 90% of eggs treated with BAPTA AM, PMA, and cycloheximide, suggesting that an increase in [Ca2+]i is not necessary for the PMA-induced release from metaphase. The [Ca2+]i did not change in eggs which formed nuclei in response to PMA, providing additional evidence that PMA activates eggs without elevating the [Ca2+]i. PMID- 7556883 TI - Retinoic acid induction of mouse cellular retinoic acid-binding protein-I gene expression is enhanced by sphinganine. AB - Cellular retinoic acid-binding protein-I (CRABP-I) gene expression is induced in mouse embryonal carcinoma P19 cells specifically by retinoic acid (RA) and the induction is enhanced by sphinganine. The effects of retinoic acid and sphinganine on CRABP-I gene expression can be accounted for by a stimulation of its transcription rate. Using a lacZ reporter system, it was determined that a DNA fragment containing a putative AP-1 binding site in the promoter region of CRABP-I gene is required for the up-regulation of CRABP-I gene transcription. PMID- 7556889 TI - The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. AB - The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GFP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition. Fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. Fluorescence is most visible between 24 36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos. PMID- 7556892 TI - Follicle cell regulation of amino acid uptake and polyphosphoinositide metabolism in oocytes of the limpet Patella vulgata. AB - We investigated the role of follicle cells during meiosis reinitiation in the oocytes of Patella vulgata. Germinal vesicle breakdown, chromosome condensation, and metaphase-1 spindle formation were artificially induced by treating oocytes with 10 mM NH4Cl. By following the accumulation of 32PO4 and [3H]inositol in polyphosphoinositides (PPI) and by measuring chemical amounts of these lipids, we found that the presence of follicle cells caused PPI turnover to remain at a low level in the immature oocyte. On the other hand, the presence of these cells was necessary for maximal activation of the metabolism of these lipids during maturation. We also examined the nutritive role of follicle cells by studying the transport of leucine and found that the presence of these cells was necessary for optimal transport of this amino acid. We conclude that follicle cells can exert either a stimulatory or an inhibitory effect on the oocyte metabolism according to its state of maturity. PMID- 7556890 TI - Neurogenic cells inhibit the differentiation of cardiogenic cells. AB - In this study, we present evidence that neurogenic cells inhibit the differentiation of cardiogenic cells. When cells of the entire area pellucida at stage 5 were dissociated and reaggregated, the aggregates differentiated into neural tissues and other structures of any germ layer origin, except for heart tissues, despite the fact that the cardiogenic cells are already committed to differentiate. The phenomenon also occurs from stages 6 to 8, during which cells are in a higher state of commitment. Using combinations of different regions of the area pellucida, it was confirmed that neurogenic cells are responsible for the inhibition of cardiogenic cell differentiation. The inhibition is not species specific because quail and chick neurogenic cells inhibit each other's cardiogenic cell differentiation. Direct contact between cardiogenic and neurogenic cells seems to be necessary for inhibition because cardiogenic cell differentiation takes place if cardiogenic and neurogenic cells are separated by a porous membrane. Dissociated neural tubes from early stages, but not dissociated telencephalon from 2-day-old chicks, also inhibit cardiogenic cell differentiation. These results suggest that neurogenic cells may sequester molecules necessary for the differentiation of cardiogenic cells rather than produce inhibitory molecules. PMID- 7556891 TI - Regulation of Dictyostelium early development genes in signal transduction mutants. AB - The secreted cyclic nucleotide phosphodiesterase (PDE) and its glycoprotein inhibitor (PDI) are among the first genes expressed when Dictyostelium amoebae begin their development. We used a series of mutants with defects in signal transduction to ask whether cAMP receptors 1 and 3, G alpha2, G beta, adenylyl cyclase (ACA), or the protein kinase A catalytic subunit (PKAcat) are required for the initial appearance or later regulation of the PDE and the PDI transcripts. The PDE gene produces a 1.9-kb transcript during vegetative growth and then a 2.4-kb transcript during the early hours of development. Regulation of the 2.4-kb transcript in CAR1, G alpha2, G beta, and ACA mutants is similar to that of isogenic parental strains, although its level is reduced in strains that carry the CAR1 mutation. CAR1/CAR3 double mutants also produce less PDE transcript, but the PDE gene remains inducible by cAMP. The PKAcat mutant produces the 2.4-kb PDE transcript, but in this mutant the vegetative transcript is retained in development. CAR1 and CAR3 are not required for transcription of the PDI gene, but deleting CAR1 leads to overproduction of the PDI transcript. Induction or repression of the PDI gene does not require G alpha2, G beta, or ACA. PKAcat is required for synthesis of the PDI transcript. The results suggest a two-stage regulation of these early genes through a G alpha2/G beta-independent mechanism and an absolute dependence of PDI on the PKAcat. PMID- 7556893 TI - Maturation of fast and slow motor units during synapse elimination in the rabbit soleus muscle. AB - In adult mammalian skeletal muscles, fast and slow muscle fibers are selectively innervated by single inputs from corresponding motor neuron types, giving rise to fast and slow motor units. At birth, however, muscle fibers are polyinnervated, and connections between motor neurons and muscle fibers are not as specific as those found in adults. Excess synapses are removed during the first few postnatal weeks. In addition to changes in the degree of polyinnervation, motor unit types undergo maturation in their contractile properties. In this study, we have investigated the maturation of motor unit types during postnatal synapse elimination in the rabbit soleus muscle. The ratio of twitch tension to tetanic tension in a motor unit is an indication of its contractile type. Our results indicate that during synapse elimination, the twitch/tetanus ratios for fast motor units increase while those for slow motor units decrease. The ratio of motor unit tension at polyinnervated ages to that at singly innervated ages has previously been used to estimate the degree of polyinnervation for fast versus slow muscle fibers. We found that twitch and tetanic tension yield conflicting estimates of polyinnervation. This discrepancy was resolved on the basis of intracellular recordings of endplate potentials. Using latencies to endplate potentials as an indicator of muscle fiber type, we found that fast and slow muscle fibers are polyinnervated to a similar extent during both early and intermediate stages of synapse elimination, suggesting that specific tension, and not polyinnervation, changes differently in fast versus slow muscle fibers. These changes are consistent with those we found in twitch/tetanus ratios. Furthermore, these intracellular recordings suggest a high degree of specificity at birth, which is further refined during synapse elimination. PMID- 7556896 TI - Characterization of the SpHE promoter that is spatially regulated along the animal-vegetal axis of the sea urchin embryo. AB - To understand how the maternally determined animal-vegetal polarity of the sea urchin embryo is established, we have begun to examine the regulatory apparatus of the gene encoding the Strongylocentrotus purpuratus hatching enzyme (SpHE). Previous studies have shown that the pattern of SpHE mRNA accumulation reflects the animal-vegetal developmental axis in that transcription is strongly upregulated during early cleavage in more animal blastomeres, but not in those around the maternally specified vegetal pole of the 16-cell embryo [Reynolds et al., Development 114, 769-786 (1992)]. Tests of SpHE promoter function in vivo using chloramphenicol acetyltransferase and beta-galactosidase enzymatic reporters define a regulatory region within several hundred nucleotides of the transcription initiation site. This region is sufficient to mediate both strong expression in the early blastula and spatially correct transcription. However, neither this region nor longer upstream sequences are sufficient to reproduce the transcriptional downregulation after very early blastula stage that is observed for endogenous genes. Biochemical assays of protein-DNA interactions within the regulatory region identify at least nine sites binding at least six different factors. These cis elements include Otx (an orthodenticle homologue), CCAAT, ets related, and three unidentified motifs. Deletions and/or replacements of these cis-elements, alone and in combination, indicate that no single factor is essential for SpHE promoter activity, but instead that various combinations of subsets of these elements are capable of eliciting levels of transcription similar to those of the unaltered regulatory region. This density of regulatory elements is consistent with the intense transcription of endogenous SpHE genes during cleavage. PMID- 7556895 TI - Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos. AB - We have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division. PMID- 7556897 TI - Developmental changes in the prothoracicotropic hormone content of the Bombyx mori brain-retrocerebral complex and hemolymph: analysis by immunogold electron microscopy, quantitative image analysis, and time-resolved fluoroimmunoassay. AB - The insect brain neuropeptide, prothoracicotropic hormone (PTTH), elicits the molting process by stimulating ecdysteroidogenesis in the prothoracic glands. Changes in the subcellular distribution of PTTH during the fifth larval instar and larval-pupal metamorphosis of Bombyx were investigated using immunogold electron microscopy and computerized image analysis. A time-resolved fluoroimmunoassay was employed to quantify the hemolymph PTTH titer and the hemolymph ecdysteroid titer was determined by radioimmunoassay. Immunoreactivity was apparent only in the cytoplasm of two pairs of dorsal-lateral neurosecretory cells (La) in the brain, the prothoracicotropes, and their axons that traverse the peripheral area of the brain neuropil and then enter the neurohemal lobes of the corpora cardiaca and end in the corpora allata. Immunospecificity was evident as noted by the restriction of the 5-nm gold particles to the neurosecretory granules. Synaptic contacts were noted frequently between the dendritic collaterals of the prothoracicotropes and neighboring collaterals from other neurons. The morphological data suggest that PTTH is synthesized and released throughout the fifth larval instar. The concentration of gold particles in the neurosecretory granules and the number of neurosecretory granules increased five times during the fifth instar, and these increases were correlated with surges in the hemolymph PTTH and ecdysteroid titers. The data suggest that PTTH biosynthesis, secretory granule formation, and secretion are three continuous and almost synchronized events. PMID- 7556894 TI - A series of mutations in the D-MEF2 transcription factor reveal multiple functions in larval and adult myogenesis in Drosophila. AB - The D-mef2 gene encodes a MADS domain transcription factor expressed in differentiated muscles and their precursors in the Drosophila embryo. Embryos deficient for D-MEF2 protein due to a deletion of upstream transcriptional control sequences fail to form muscle, suggesting that the gene is required for muscle cell differentiation. To directly demonstrate a role for D-mef2 in embryonic myogenesis, we isolated gene mutants containing EMS-induced point mutations, characterized the effects of these mutations on D-MEF2 protein stability and nuclear localization, and analyzed the resulting muscle phenotypes. Our results show that in the somatic muscle lineage, D-mef2 is required for both the formation and patterning of body wall muscle. In the absence of somatic myogenesis, there is extensive apoptosis among the myoblast cell population. In contrast, in the cardiac muscle lineage, morphogenesis of the dorsal vessel occurs normally but the three myosin subunit genes are not expressed. Mutant embryos also exhibit an abnormal midgut morphology, which correlates with the absence of alpha PS2 integrin gene expression and muscle-specific enhancer function, suggesting that D-mef2 regulates the inflated locus which encodes this integrin subunit. D-MEF2 is also expressed in adepithelial cells and rare D-mef2 transheterozygous mutant adults fail to fly, consistent with defects observed in the indirect flight muscles. These results demonstrate that the D-mef2 gene has multiple functions in myogenesis and tissue morphogenesis during Drosophila development. PMID- 7556900 TI - Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8. AB - In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF. PMID- 7556899 TI - Chicken perinatal troponin Ts are generated by a combination of novel and phylogenetically conserved alternative splicing pathways. AB - During perinatal development, avian and mammalian skeletal muscles express a novel set of troponin T (TnT) isoforms with higher M(r)s and more acidic pIs than their adult counterparts. In mammals, these TnTs result from the incorporation of a developmentally regulated 5'-fetal exon into fast TnT mRNAs. To determine whether chicken perinatal TnTs are generated by a similar mechanism, we sequenced 40 chicken TnT 5'-cDNAs from perinatal and adult pectoralis. Evidence for five 5' exons not present in the mammalian gene was found, including one, y, whose splicing is developmentally regulated. Although none of these new exons are homologous with the mammalian fetal exon, their alternative splicing, superimposed on three conserved splicing patterns of the phylogenetically shared 5'-exons, generates the chicken perinatal TnT isoforms. These observations indicate that chicken, and most likely other avian species, evolved an independent 5'-alternative splicing mechanism to generate perinatal TnTs that have the same biophysical, and presumably functional, properties as their mammalian homologs. PMID- 7556898 TI - The gene encoding murine alpha 1,3-galactosyltransferase is expressed in female germ cells but not in male germ cells. AB - An essential step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked glycans on zona pellucida glycoprotein 3 (ZP3). While there is agreement on the primary role of O-linked glycans in sperm-ZP3 binding, there is a striking lack of consensus on both the terminal monosaccharide(s) required for a functional binding site and the cognate protein on the sperm cell surface that recognizes this glycan. Much current debate centers on the essential role of nonreducing terminal N-acetyl-glucosaminyl or alternatively, alpha-galactosyl residues, to form a functional sperm binding ligand. Relevant to this debate, we demonstrated that alpha 1,3-galactosyltransferase (alpha 3-GT), which adds nonreducing terminal alpha-galactosyl residues to glycans, is not expressed in murine spermatocytes or spermatids. The objectives of this study were to determine whether alpha 3-GT is expressed in female germ cells and to compare the pattern of expression of two other terminal glycosyltransferases, beta 1,4 galactosyltransferase (beta 4-GT) and alpha 2,6-sialyltransferase (alpha 6-ST), between male and female germ cells. Total RNA was isolated from growing oocytes obtained from 15-day-old animals, fully grown oocytes, and eggs as well as spermatogonia, spermatocytes, and spermatids. The presence of alpha 3-GT, beta 4 GT, and alpha 6-ST mRNAs was analyzed by an RT-PCR-based assay. Our data demonstrate that the alpha 3-GT gene is expressed in female germ cells, but not in male germ cells. In contrast, both beta 4-GT and alpha 6-ST are expressed during oogenesis and spermatogenesis. This differential expression of alpha 3-GT in female germ cells is consistent with the model of sperm-egg binding in which a nonreducing terminal alpha-galactosyl residue is required for a functional determinant on ZP3 and with our hypothesis that the biological significance for the suppression of alpha 3-GT expression in male germ cells is to prevent sperm sperm aggregation. PMID- 7556904 TI - Efficient hormone-inducible protein function in Xenopus laevis. AB - A major problem in analyzing gene function during Xenopus development has been the inability to induce gene expression in a temporally controlled manner. We have attempted to solve this problem with a system of hormone-activated protein function, using the myogenic gene MyoD as a paradigm. We show that microinjection of RNA for MyoD fused to the ligand-binding domain of either the estrogen or glucocorticoid receptor results in hormone-dependent activation of MyoD function, as assayed by ectopic induction of muscle-specific actin mRNA. Induction is tightly regulated in both isolated animal caps and intact embryos, with ectopic muscle-specific actin expression inducible after 2 hr of hormone treatment. Higher levels of MyoD-receptor fusion proteins that native MyoD protein are present in embryos, apparently a result of increased fusion protein stability. This is the first demonstration that hormone-inducible fusion proteins can work effectively in a complex embryo. PMID- 7556902 TI - Spatial redistribution of poly(A)+ RNA during polarization of the Fucus zygote is dependent upon microfilaments. AB - Asymmetrical distribution of mRNA has been associated with polarization and cell fate determination during early development of animal embryos. In this report we determine the distribution pattern of poly(A)+ RNA during early embryogenesis of the brown alga Fucus. Poly(A)+ RNA is symmetrically distributed in the egg and early zygote. Shortly after the polar axis is established, poly(A)+ RNA becomes segregated to the thallus pole of the zygote. Following cytokinesis, most of poly(A)+ RNA is partitioned into the thallus cell. We show that the spatial redistribution of poly(A)+ RNA requires intact microfilaments and the fixation of the polar axis, but is not dependent upon polarized growth of the rhizoid, intact microtubules, or orientation of the division plane. PMID- 7556905 TI - The distal human myoD enhancer sequences direct unique muscle-specific patterns of lacZ expression during mouse development. AB - Transgenic mice carrying the bacterial lacZ reporter gene under the control of the regulatory elements of the human myoD gene have been produced. The developmental expression of the myoD reporter transgene in somites, limb buds, visceral arches, and cephalocervical regions was studied in transgenic embryos by beta-gal staining. In somites, the spatiotemporal pattern of transgene expression was different from other muscle-specific regulatory and structural genes and revealed that myoD-expressing cells arise in distinct patterns in somites that are dependent on position along the anterior-posterior (AP) body axis (occipital and cervical vs thoracic and more posterior myotomes). Transgene expression did not follow a strict anterior to posterior sequence of activation and therefore was not strictly correlated with somite developmental age. Moreover, the pattern of transgene expression along the dorsal-ventral myotomal axis was dependent on somite position along the anterior-posterior axis. While myoD expression is first detected after the myotome is well-formed, transgene expression in the dorsal and ventral medial lips of the dermatome suggests a function for myoD in the expansion of the myotome. Whole-mount in situ hybridization confirmed that these unique patterns of transgene expression in somites, as well as expression in limb buds, visceral arches, and other myogenic centers, are concordant with the distribution of endogenous myoD transcripts. These results shed new light on the developmental differences between myotomes at different positions along the AP and DV axis and demonstrate a unique axial pattern of somitic myoD expression, suggesting a specific role of myoD in myotome lineage determination and differentiation. PMID- 7556901 TI - Green fluorescent fusion proteins: powerful tools for monitoring protein expression in live zebrafish embryos. AB - The recent development of transgenic technology in zebrafish has opened an exciting new avenue in which to explore vertebrate development. However, as in other species, the inability to easily identify live transgenic fish severely limits the potential of this promising technology. To determine whether the recently described green fluorescent protein (GFP) might provide a convenient live staining method in zebrafish, we constructed a glutathione S-transferase/GFP fusion protein (GST-GFP). GST-GFP cRNA, when injected into individual blastomeres of early zebrafish embryos, resulted in the rapid development (3 hr) of easily detectable green fluorescence which persisted for up to 4 days. GFP fluorescence was restricted to progeny of the injected cell and appeared to have no adverse effects on embryonic development despite widespread expression. Our findings demonstrate that GFP fusion proteins will provide a simple yet powerful means of monitoring production of heterologous proteins in live zebrafish. PMID- 7556903 TI - Whole-mount in situ hybridization of cell-type-specific mRNAs in Dictyostelium. AB - We have been able to hybridize nonradioactive probes from cell-type-specific genes to fixed whole-mounts prepared at the mound, slug, and culminant stages of Dictyostelium development. The cellular patterns of labeling with probes from the prespore gene, cotB, and the prestalk genes, ecmA and ecmB, confirmed the patterns seen in strains carrying reporter constructs in which the regulatory regions of these genes drive beta-galactosidase. This technique permits the direct observation of protein synthetic capacity from characterized genes without the need of generating transformed lines carrying specific reporter constructs. Moreover, the pattern is not complicated by a previous developmental history of gene expression. PMID- 7556906 TI - The matured eye of Xenopus laevis tadpoles produces factors that elicit a lens forming response in embryonic ectoderm. AB - Previous studies have indicated that the outer cornea can undergo transdifferentiation to form a lens in the tadpole larva of Xenopus laevis following removal of the original lens. This transformation appears to require an interaction with the neural retina. In the present study, we carried out a series of experiments to determine if the matured tadpole eye can also elicit lens formation in embryonic ectoderm. Labeled embryonic ectoderm was removed from the presumptive lens-forming region, or from the belly region (ventral ectoderm), at various stages of development (stages 11-19, gastrula to neural tube stages) and implanted into the eye cavity (posterior chamber) of advanced stage 52-55 tadpoles. After 3 days, we examined the tadpoles and their implanted tissues for lens cell formation using lens-specific antibodies. Implanted presumptive lens ectoderm differentiated lens cells in a large number of cases. The percentage of cases forming lens cells and the extent of morphological differentiation increased with increasing age of the implanted tissue. Implanted ventral ectoderm also formed lens cells, although at a reduced frequency and with limited morphological differentiation. These results indicate that the environment of the matured tadpole eye cavity stimulates lens cell formation in both presumptive lens and nonlens ectoderm. The development of the implanted tissues was compared to that found in previous studies where these tissues were cultured as explants or transplanted to lens-forming regions during early development and subjected to various periods of embryonic lens induction. Together, these findings suggest that the process of embryonic lens formation is related to that involved in transdifferentiation of the tadpole cornea during "lens regeneration." However, the inductive effect of the matured tadpole eye is qualitatively different from that of the early period of embryonic lens induction and, while more intense, may be more closely related to that which takes place via the optic vesicle during the later phase of embryonic lens induction. PMID- 7556907 TI - Identification of a complex between centrin and heat shock proteins in CSF arrested Xenopus oocytes and dissociation of the complex following oocyte activation. AB - Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90 kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested. PMID- 7556908 TI - A portable regulatory element directs specific expression of the Caenorhabditis elegans ubiquitin gene ubq-2 in the somatic gonad. AB - The Caenorhabditis elegans ubq-2 gene encodes a fusion of ubiquitin and a 52 amino-acid ribosomal protein. This single copy gene is both cis- and trans spliced. It is expressed in all life stages of the worm and its transcript abundance is unaffected by heat stress. Transgenic analysis shows that expression of ubq-2 is regulated by an upstream promoter and a downstream element. The downstream element is required for ubq-2 promoter activity in embryos and in cells of the somatic gonad, including the distal tip cells, sheath cells, spermathecal cells, and cells of the uterus. The gonad-specific activity of the downstream regulator is transferable to a stress gene promoter such that heat inducible expression of the transgene occurs in the somatic gonad. Stress inducible beta-galactosidase expression in the gonad does not occur in all life stages, but is initiated late in the second or early in the third larval stage, when differentiation of gonadal tissues begins. Expression of a beta galactosidase fusion protein from constructs containing the downstream ubq-2 regulator causes abnormalities of the gonad and embryonic lethality. Gonad abnormalities include arrested development and general disorganization. These abnormalities may be related to the overexpression of ubiquitin in the gonad. PMID- 7556911 TI - Tissue boundaries and cell behavior during neurulation. AB - We have analyzed the dynamics of the boundaries between the neural plate and the epidermis and between the neural plate and the notoplate. Our experiments confirm that these two boundaries have important roles in neurulation. Measurements of the lengths of neural fold (the boundary between epidermis and neural plate) in embryos of axolotls and newts reveal that neural folds abutting the prospective brain decrease in length while neural folds abutting the prospective spinal cord increase in length during neurulation. We tested the proposition that boundaries of the neural plate with epidermis and with notoplate are essential for proper neurulation. Cuts made along the boundaries with epidermis or with notoplate stop, or greatly diminish, neural plate elongation and tube formation. Explanting the axolotl neural plate without any bordering epidermis stops plate elongation and prevents neural tube closure, but neural plates explanted with a rim of epidermis elongate and close into tubes. Cutting the notoplate boundary stops midline elongation in the newt embryo or diminishes it in the axolotl embryo. We conclude that the notoplate boundary and part of the boundary of the epidermis that abuts the prospective spinal cord organize cell behavior to elongate the neural plate and help close the neural tube. The boundary of the neural plate with the epidermis is essential for tube closure both because it organizes plate elongation in the spinal cord region and because cell behavior becomes organized at the boundary such that neural folds are raised and a rolling moment is produced that helps form the neural tube. PMID- 7556909 TI - Segment-specific expression of the neuronatin gene during early hindbrain development. AB - The developing hindbrain is segmented in a series of repetitive bulges called neuromeres or rhombomeres. In the mouse, first molecular evidence for segmentation of the hindbrain came from rhombomeres 3- and 5-specific expression of the Krox-20 gene. The hindbrain segments are linked with the expression of different Hox genes which have a role in patterning the hindbrain and branchial region of the vertebrate head. Here we identified by subtractive hybridization a gene, mouse neuronatin, that is downregulated in P19 embryo carcinoma cells that have undergone a partial differentiation process. Neuronatin encodes putative transmembrane proteins of 54, 55, and 81 amino acids that might serve as protein ligands, cofactors, or small cell adhesion molecules. The neuronatin gene is transiently expressed in rhombomeres 3 and 5 during early hindbrain development and in the floor of the foregut pocket. In addition, expression is observed in the early Rathke's pouch, in the derived adenohypophysis, and in the developing inner ear. During later embryogenesis the neuronatin gene is strongly expressed in the major part of the central and peripheral nervous system. These results suggest that neuronatin participates in the maintenance of segment identity in the hindbrain and pituitary development and maturation or maintenance of the overall structure of the nervous system. PMID- 7556914 TI - Regulation of the Hoxb-8 gene: synergism between multimerized cis-acting elements increases responsiveness to positional information. AB - Hox genes play a key role in the specification of regional development in the vertebrate embryo. They are expressed in regionally restricted domains along the anterior-posterior axis, generally extending from a sharp rostral boundary toward the posterior end. We have studied the regulation of the murine Hoxb-8 gene in vivo using reporter constructs and found that 11 kb of genomic sequences upstream of Hoxb-8 confer a Hox-like pattern of expression on a lacZ reporter gene fused in-frame to the first exon of Hoxb-8. Reporter gene expression was detectable from early stages onwards, but reached rostral expression boundaries in mesoderm and neurectoderm that were more posterior than those of the endogenous gene. Within the upstream region, we have identified several cis-acting elements which are individually capable of driving regionally restricted expression in combination with the Hoxb-8 promoter, and we have investigated their relative contributions to the expression pattern. The results suggest that, in this experimental context, these upstream elements, as well as previously identified elements located within the Hoxb-8 gene, cooperate in setting the rostral expression boundaries. Furthermore, we show that multiple identical copies of cis acting elements can cooperate, causing a pronounced anterior shift in the expression boundaries. This indicates that the rostral extent of expression is limited by the activity of a transcription factor binding to these elements and that this activity was, at some point in development, higher in precursors of more posterior structures than in precursors of anterior structures. This factor is thus very likely to be involved in the transduction of positional signals. PMID- 7556913 TI - Migrating interstitial cells differentiate into neurons in hydra. AB - Interstitial cell migration has been observed numerous times in grafted hydra, but the extent to which graft injuries might stimulate the migration of otherwise nonmotile cells was unknown. The present study describes the migration and differentiation of vital dye-labeled interstitial cells in intact, normal hydra. Interstitial cells, stained with a fluorescent vital dye, migrated away from a labeled patch of ectodermal cells and subsequently were found throughout the body column. Shortly thereafter, labeled neurons began to appear among the migrating cells. The number of migrating interstitial cells remained constant over 5 days, whereas the number of labeled neurons increased. Labeled interstitial cells and neurons accumulated primarily in the head and peduncle, as observed in previous studies of short-term migration patterns in grafted hydra. The present study shows that migration of interstitial cells occurs in normal nongrafted hydra and that the accumulation patterns parallel results from graft experiments. The population of migrating cells appears to be limited to neuron precursors. PMID- 7556912 TI - Regulation of the appearance of division asynchrony and microtubule-dependent chromosome cycles in Xenopus laevis embryos. AB - Divisions of animal-cap blastomeres dissociated from Xenopus laevis embryos are synchronous mostly up to 12th cleavage or the 13th cell cycle, but become asynchronous afterward, during the midblastula transition (MBT), and at the same time, chromosome cycles become microtubule-dependent and are arrested in mitosis if treated with nocodazole. To investigate causes for these changes in cell-cycle control, we observed division synchrony in animal-cap blastomeres dissociated from embryos whose nucleocytoplasmic ratio (N/C) had been altered by constriction of zygotes or by delaying nucleation into zygote halves and compared their mitotic indices in the presence and absence of nocodazole. Thus, we found that asynchronous divisions always commenced when N/C reached the value of 128 to 256 times that of an animal blastomere of the 32-cell embryo, corresponding to the 12th and 13th cycles of a normal embryo, while the number of synchronous cycles became variable, ranging from 9 to 14, depending on the initial N/C. Treatment with alpha-amanitin or cycloheximide did not alter the number of synchronous cycles. However, the time at which the mitotic index of nocodazole-treated blastomeres first exceeded that of control remained constant, at 3 to 5 hr after 5th cleavage, regardless of the initial N/C. Thus, chromosome cycles of blastomeres first became sensitive to nocodazole at a variable N/C, ranging from 8 to 1024 times that of an animal blastomere of the 32-cell embryo. The timing of the appearance of nocodazole sensitivity was unaffected by alpha-amanitin treatment, whereas it was markedly delayed following cycloheximide treatment. These results suggest that the commencement of division asynchrony is N/C dependent, whereas the development of microtubule-dependent cell cycles is age dependent, most likely being programmed by the translation of stored mRNAs. PMID- 7556915 TI - Dorsal-ventral patterning genes restrict NK-2 homeobox gene expression to the ventral half of the central nervous system of Drosophila embryos. AB - Genes that affect the expression of the NK-2 homeobox gene were identified by comparing the patterns of NK-2 mRNA in wild-type Drosophila embryos with patterns in mutant embryos that have defects in genes that are required to establish the ventral-dorsal pattern of primordia in the early embryo. The NK-2 gene was shown to be activated by dorsal in the ventral half of the embryo during the syncytial blastoderm stage of development. However, expression of the NK-2 gene is restricted to the ventral half of the ventrolateral neurogenic anlagen and part of the procephalic region. The NK-2 gene is not expressed in the mesodermal anlage due to repression by snail, in mesectodermal cells due to repression by single-minded, or in the lateral neuroectodermal and/or dorsal epidermal anlagen due to repression mediated indirectly by decapentaplegic. Twist activates the NK 2 gene in the posterior portion of the embryo or is a coactivator with dorsal. The stripes of medial neuroectodermal cells that synthesize NK-2 mRNA are converted into clusters of neuroectodermal cells that contain NK-2 mRNA by segmentally repeated decreases in NK-2 mRNA. Medial neuroblasts, neuroblasts in the posterior portion of segments, and some ganglion mother cells and neurons express the NK-2 gene. These results suggest that the NK-2 gene receives and integrates information from ventral-dorsal and anterior-posterior gradients of gene regulators and that ventral, dorsal, anterior, and posterior boundaries of each cluster of neuroectodermal cells that express NK-2 are determined independently. PMID- 7556916 TI - A transgenic neuroanatomical marker identifies cranial neural crest deficiencies associated with the Pax3 mutant Splotch. AB - The murine Pax3 gene encodes a transcription factor containing a paired domain as well as a paired-type homeodomain. Its expression during embryonic development is temporally and spatially restricted, including mainly the dorsal part of the neural tube, the mesencephalon, the neural crest derivatives, and the dermomyotome. Development in the absence of Pax3 can be studied in Splotch mutant mice, which bear mutations within the Pax3 gene. Various alleles have been phenotypically and molecularly characterized. Abnormalities have been observed in the brain, the neural tube, the trunk neural crest derivatives and in muscles of these mutants. The importance of PAX3 during human embryonal development is readily seen in Waardenburg patients, who present a dominant inherited syndrome consisting mainly of craniofacial abnormalities, pigmentation deficiencies, and deafness, consecutive to PAX3 mutations. In order to analyze the nervous system of Splotch embryos in more detail, we employed the transgenic mouse line L17. These transgenic mice harbor a beta-galactosidase marker gene under the control of Hoxa-7 promoter elements. Probably in combination with cis-elements adjacent to the integration site of the L17 transgene, the Hoxa-7 elements drive the expression of the marker gene in major parts of the peripheral nervous system, as well as in more restricted parts of the central nervous system. These structures can be visualized during embryonic development, allowing detailed neuroanatomical studies in midgestation embryos. We describe the beta-galactosidase expression in wild-type L17 mice and demonstrate the applicability of L17 mice to the study of the nervous system. We then apply this experimental system to the analysis of Splotch embryos. Our findings underline the importance of Pax3 in the development of neural crest-derived structures, especially of cranial ganglia and nerves. We suggest the use of L17 mice as a valuable tool to perform similar analysis for other embryonal mutant phenotypes. PMID- 7556917 TI - Growth cone neurotransmitter receptor activation modulates electric field-guided nerve growth. AB - We have studied the interactions between two nerve guidance cues, which alone induce substantial growth cone turning: endogenous neurotransmitters and small dc electric fields. d-tubocurarine, a nicotinic AChR (acetylcholine receptor) antagonist, inhibited field-induced cathodal orientation of cultured neurites, whereas atropine, a muscarinic AChR blocker, and suramin, a P2-purinoceptor antagonist, markedly enhanced the guidance properties of the applied field. These experiments implicate the activation of growth cone nicotinic AChRs by self released acetylcholine in the mechanism underpinning electric field-induced neurite orientation and raise the possibility that growth cones release neurotransmitter prior to target interaction in order to assist their own pathfinding. Additionally, they provide the first evidence that coactivation of several neurotransmitter receptors may interact to regulate directed nerve growth. Such interaction in vivo, where guidance signals coexist, would add further levels of control to neurite guidance. PMID- 7556919 TI - The MLC1f/3f gene is an early marker of somitic muscle differentiation in Xenopus laevis embryo. AB - cDNAs clones encoding the MLC1f and MLC3f proteins of Xenopus laevis have been isolated from a stage 42 cDNA library. Sequence analysis reveals that the amphibian MLC1f and MLC3f isoforms are similar to the mammalian and avian cognates. The two isoforms share a common 141-amino-acid carboxy-terminal regions. These are 49 and 9 residues long for the MLC1f and MLC3f isoforms, respectively. This suggests a genomic organization similar to the mammalian and avian genes, with two promoters and alternative splicing. The developmental expression of the MLC1f/3f mRNAs was studied by Northern blot and RNase protection and their spatial expression analyzed by in situ hybridization. Both the MLC1f and MLC3f mRNAs can be detected in the developing embryo from the end of gastrulation and accumulate rapidly in the somitic mesoderm. Expression of the MLC1f/3f gene can also be detected in animal cap explants which have been induced to form mesodermal derivatives by exposure to activin A or bFGF. However, unlike other muscle-specific markers, neither transcript from the MLC1f/3f gene can be detected in embryonic or adult cardiac muscle, their expression being restricted to somitic muscle. Together, these data demonstrate that expression of the MLC1f/3f gene provides a sensitive and specific marker for skeletal muscle differentiation. Ectopic expression of myogenic factors in animal caps induces the expression of the MLC1f/3f gene, suggesting that the amphibian gene, like its mammalian and avian counterparts, is a regulatory target for members of the MyoD family of transcription factors. PMID- 7556918 TI - Calcium channel subtypes and intracellular calcium stores modulate electric field stimulated and -oriented nerve growth. AB - In culture, embryonic spinal neurites from Xenopus laevis show striking growth responses to steady dc electric fields, at a time when endogenous electric fields of similar size impinge on the developing nervous system. A high proportion of cultured neurites reorient, with both turning and branching directed cathodally. Neurite growth rates are increased and growth is differential (faster cathodally than anodally). Voltage-dependent calcium channels and calcium release from intracellular stores are shown to control these events. However, the pharmacological sensitivities of these phenomena differ, indicating different control mechanisms. PMID- 7556910 TI - The Drosophila E93 gene from the 93F early puff displays stage- and tissue specific regulation by 20-hydroxyecdysone. AB - Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors. We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse. PMID- 7556921 TI - Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes. AB - The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration. PMID- 7556920 TI - Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin. AB - Gastrulation in Xenopus laevis transforms a ball of undifferentiated cells into a well-organized elongated embryo with a distinct body axis, as a result of extensive cell rearrangements and movements. Since cell adhesion is thought to play an important role during tissue morphogenesis, we investigated the potential role for C-cadherin, a primary cell-cell adhesion molecule in Xenopus, in gastrulation movements. A deletion mutant consisting of the extracellular and transmembrane domains but lacking the cytoplasmic tail (Ctrunc) was constructed to act as a dominant-negative inhibitor of the endogenous protein. Injection of in vitro transcribed Ctrunc mRNA into the prospective dorsal involuting marginal zone, the region that undergoes the most extensive movements, caused a reproducible defect in gastrulation. The defect, an inability to complete involution and close the blastopore, was cadherin-specific, because it could be rescued by expressing full length C-cadherin. Ctrune also seems to inhibit morphogenetic movements of animal cap explants isolated from injected embryos. Expression of Ctrunc did not appear to interfere with inductive processes in the embryos that developed gastrulation defects, because they formed complete axial structures, including a complete head and notochord. These results demonstrate that C-cadherin plays a critical role in Xenopus laevis gastrulation movements, and they support the hypothesis that cadherins can mediate cellular rearrangements during tissue morphogenesis. PMID- 7556922 TI - The regulation of MyoD gene expression: conserved elements mediate expression in embryonic axial muscle. AB - To analyze the transcriptional regulatory mechanisms of the myoD gene, we generated transgenic mice bearing a lacZ gene driven by a 6-kb 5'-flanking sequence of the mouse myoD gene including a proximal regulatory region (PRR) and a distal regulatory region (DRR), which are sufficient for activation of muscle specific transcription in vitro. The expression of the reporter lacZ gene was detected from 10.5 days post coitum in the myotomes, consistent with endogenous myoD mRNA. However, in limb buds and branchial arches, the appearance of the lacZ positive cells was delayed for one day compared with the endogenous myoD mRNA, suggesting the existence of a different control mechanism among muscle cell lineages. Further, a subset of cells in the central nervous system (CNS), where endogenous myoD mRNA was not detected, expressed the transgene transiently. The same 6-kb MyoD-lacZ gene injected into Xenopus embryos was expressed in the myotomes and in CNS cells at the tailbud stage. Deletion analyses in both transgenic mice and Xenopus embryos indicated that the DRR and PRR were together sufficient for the expression pattern in skeletal muscle. In addition, analysis in Xenopus indicated that a third enhancer region between -1.3 kb and -275 bp could substitute for the DRR. The functional conservation of the regulatory region of the mouse myoD gene in Xenopus embryos suggests that the regulatory pathway that activates myoD gene transcription in axial muscles is conserved among vertebrates. PMID- 7556924 TI - Integrins and the development of three-dimensional structure in the Drosophila compound eye. AB - A stereotyped, three-dimensional network of cell-cell contacts mediated by adherens junctions and cell-extracellular matrix contacts mediated by focal adhesions defines the architecture of the Drosophila ommatidium. Developmental reconstruction shows that this network is built in an incremental and generally conservative sequence; contacts established early in eye development typically persist into adulthood. Reconstructions show that photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabdomeres. Rhabdomeres become aligned to the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell-cell and cell-extracellular matrix contacts. Parallel reconstructions of retinal development in integrin mutants show that several eye phenotypes trace their origin to the structural failure of the cone cell plate. PMID- 7556923 TI - Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation. AB - Vascular endothelial growth factor (VEGF)--also known as vascular permeability factor--has been implicated in the regulation of blood vessel formation, i.e., vasculogenesis and angiogenesis. High amounts of VEGF mRNA and protein have been detected during embryonic and tumor angiogenesis, but it remained unclear whether the level of VEGF correlated with the extent of vascularization in a given organ or tissue. We examined the role of VEGF and the high affinity, signal-transducing VEGF receptor-2 (flk-1) in the avian embryo. In a gain of function transgene-like approach the retroviral expression vector RCAS was used to increase the level of quail VEGF during critical periods of avian limb bud growth and morphogenesis. In contrast to basic fibroblast growth factor, which recently was demonstrated to induce morphogenetic alterations when overexpressed in this system, overexpression of VEGF in the limb bud exclusively resulted in hypervascularization as reflected by an increase in vascular density. However, cartilage expressing the construct was not vascularized prematurely. Thus hypervascularization was probably due to the augmentation of the VEGF signaling mechanism in a permissive environment. In addition to hypervascularization, vascular permeability was dramatically increased, leading to local and in some cases to general edema. This is the first indication of a link between the functions of VEGF as a vascular growth factor and as a permeability factor. VEGF receptor-2 (flk-1) was found to be upregulated only in those areas where VEGF was overexpressed. This implies a positive feedback system of the VEGF receptor on its own synthesis and would provide a basis for a paracrine system in which ligand concentration is critical for the extent of tissue vascularization. Our results show that the VEGF/VEGF-receptor system is specific and sufficient for the formation of new blood vessels. They also have implications for somatic gene therapy of diseases which are characterized by a lack of blood vessels such as chronic ischemic diseases of heart and brain. PMID- 7556925 TI - NGF receptor expression in sensory neurons develops normally in embryos lacking NGF. AB - Numerous in vivo and in vitro studies have shown that NGF increases the expression of its receptors, p75 and TrkA, in NGF-responsive cell lines and in NGF-responsive neurons of the developing and mature nervous system. To determine if endogenous NGF is required for the normal developmental increase in p75 and TrkA expression that occurs in sensory neurons shortly after they innervate their targets, we used quantitative RT/PCR to compare the levels of p75 and trkA mRNAs in the trigeminal ganglia of normal mouse embryos and embryos that are homozygous for a null mutation in the NGF gene. We show that the marked increase in p75 and trkA mRNA expression that occurs between E11 and E13 in normal embryos takes place on time and to the same extent in NGF-/- embryos. We also show that trigeminal neurons from E13 NGF+/+ and NGF-/- embryos have very similar dose responses for survival induced by NGF. These findings clearly show that the expression of both p75 and TrkA and the sensitivity of developing sensory neurons to NGF do not require and are not modulated by target-derived NGF during the early stages of target field innervation. PMID- 7556926 TI - A novel homeobox cluster expressed in repeated structures of the midgut. AB - A gene cluster (LOX3-C) containing three duplicated homeobox sequences (Lox3A, Lox3B, and Lox3C) was characterized in the leech Hirudo medicinalis. The leech homeoboxes have a limited homology to those of Antennapedia-class genes, but do not have homologs among currently characterized insect genes. The Lox3 genes belong to a new family, named Xlox, that also includes genes from mouse, rat, frog, and a distantly related leech, Helobdella triserialis. All members of the Xlox family are expressed in specific regions of the embryonic gut, where they seem to affect morphogenesis and cell differentiation. The three homeoboxes of LOX3-C described here are contained within nearly identical direct tandem repeats. The LOX3-C region produces at least two transcripts, one present in both embryos and adults and the other only in early embryos. Early Lox3 expression is restricted to specific regions of the midgut primordium, in 12 segmentally repeated, transverse stripes of fusiform cells found at the positions where the midgut will constrict to form the 11 diverticula of the crop. A role in the development of segmentally iterated structures in an initially homogeneous midgut is proposed for LOX3-C. PMID- 7556928 TI - The organizer-associated chick homeobox gene, Gnot1, is expressed before gastrulation and regulated synergistically by activin and retinoic acid. AB - Gnot1, a Not (for notochord) family homeobox gene, is expressed in the chick pregastrulation blastoderm. Gnot1 expression in the epiblast is upregulated as the posteriorly derived hypoblast moves forward anteriorly to form a layer beneath it, which is of particular interest considering the known inductive role of the hypoblast in axis formation in the chick. Both activin and retinoic acid are able to activate Gnot1 expression in cultured blastodermal cells and show a strong synergistic effect when applied in combination. Strong superinduction of Gnot1 transcripts in the presence of cycloheximide also indicates the presence of a potent and labile intracellular inhibitor capable of modulating Gnot1 expression. During gastrulation, Gnot1 transcripts become localized specifically to tissues associated with "organizer" function (Hensen's node, head process, notochord). The expression data and the response to mesoderm inducing factors and axial "caudalizing" signals suggest that Gnot1 may be involved in specification of the embryonic body axis and could play a part in regulating features of the trunk/tail organizer in the chick embryo. PMID- 7556929 TI - Peripheral neurons depend on CNS-derived guidance cues for proper navigation during leech development. AB - In leech, major nerve pathways are pioneered by CNS neurons and evidence from dye injection and antibody experiments suggest that they serve as guides for later differentiating neurons. In this study we have directly tested this hypothesis by examining the consequences of CNS ablation on the navigation in the periphery of a well-defined population of afferent sensory neurons. We show that in the absence of CNS-derived axons the axonal growth cones of this population of peripheral neurons extend with little directionality and instead of forming orderly projections, default into forming circular fasciculated pathways with each other. This suggests that CNS-derived guidance cues are absolutely required for the correct navigation of these peripheral sensory neurons. PMID- 7556930 TI - Serotonin-induced meiosis reinitiation from the first prophase and from the first metaphase in oocytes of the marine bivalve Hiatella flaccida: respective changes in intracellular Ca2+ and pH. AB - In the marine bivalve Hiatella flaccida, full-grown oocytes in ovaries are arrested at the first prophase (prophase-I) of meiosis, whereas spawned oocytes have reinitiated meiosis from prophase-I and are again arrested at the first metaphase (metaphase-I). The neurohormone serotonin (5-hydroxytryptamine, 5-HT) was able to trigger meiosis reinitiation both from prophase-I and from metaphase I. Exposure of prophase-I oocytes to 5-HT caused an increase in intracellular Ca2+ ([Ca2+]i) composed of an initial towering transient and a following lower but sustained elevation. 5-HT-stimulated prophase-I oocytes also showed a gradual rise in intracellular pH (pHi), reaching a plateau level. None of these 5-HT induced responses was affected by the complete absence of external Ca2+. On the other hand, these responses were suppressed by preinjection of heparin, an antagonist of inositol 1,4,5-trisphosphate-sensitive receptors. Metaphase-I oocytes also exhibited a [Ca2+]i increase in response to 5-HT; the initial [Ca2+]i transient was larger than that in prophase-I oocytes when stimulated with the same 5-HT concentration. Furthermore, after the initial transient, the elevated [Ca2+]i was not sustained but sometimes returned to the prestimulus level and then increased again. Metaphase-I oocytes had higher resting pHi levels than prophase-I oocytes and showed no significant pHi changes after addition of 5 HT. These results suggest that both a [Ca2+]i increase and a pHi rise are responsible for the release from prophase-I arrest, while a [Ca2+]i increase alone is concerned with the release from metaphase-I arrest. PMID- 7556927 TI - Androgen regulation of a laryngeal-specific myosin heavy chain mRNA isoform whose expression is sexually differentiated. AB - The larynx, the vocal organ of Xenopus laevis, is sexually differentiated; male laryngeal muscle fibers are entirely fast twitch while female fibers are predominantly slow twitch. In adults, all male laryngeal muscle fibers express the mRNA for a laryngeal-specific myosin heavy chain (MHC), LM; female laryngeal muscle expresses LM in a subset of fast-twitch fibers. In juvenile females, LM expression is increased by exposure to exogenous androgen, suggesting that sexually differentiated expression of this laryngeal-specific MHC is regulated by exposure to male sex hormones. Here we examine hormonal regulation of LM expression in juvenile male and female frogs. Exposure to exogenous androgen increases LM expression in both sexes. In situ hybridization analyses of larynges reveal upregulation of LM expression within 8 hr of androgen treatment in males; upregulation is not evident until after 48 hr in females. The upregulation in juvenile females includes both an increase in the number of muscle fibers expressing LM and an increase in expression in fibers already expressing LM. In juvenile males, all fibers express LM from the end of metamorphosis on; levels of LM expression are increased in all fibers by androgen treatment. Androgen-induced increases in LM expression are prevented by treatment with cycloheximide and are thus dependent upon protein synthesis. Castration of juvenile males results in diminished LM expression relative to intact animals. We conclude that expression of LM is regulated by exposure to androgen and that this regulation accounts for the sexually differentiated phenotype. PMID- 7556931 TI - The effects of lithium chloride on pattern formation in Tetrahymena thermophila. AB - Lithium ions have long been known to exert dramatic effects on the specification of cell fates in multicellular systems. We have analyzed the effects of Li+ on intracellular patterning in a complex unicellular organism, the ciliate Tetrahymena thermophila. LiCl does not affect the locations of major structural landmarks in the cortical region of wild-type cells and does not modify the phenotype of pattern-mutant cells. However, in all strains studied LiCl differentially affects early stages of oral development. It initially triggers a slow regression of oral primordia, which is followed by an excessive proliferation of basal bodies that leads to a hypertrophy of the ciliature of the cell's feeding organelle. This hypertrophy mimics the effects of the membranellar pattern-D mutation, the phenotype of which is enhanced in the presence of LiCl. These effects were partially reversed by myo-inositol; however, neomycin failed to mimic the effects of LiCl. Thus, although lithium ions have major cellular effects on Tetrahymena, they do not influence the specification of the body plan in a manner analogous to that observed in multicellular organisms and may work in part through mechanisms other than the now-classical inositol-phosphate cycle. PMID- 7556932 TI - Effect of FGF on gene expression in chick limb bud cells in vivo and in vitro. AB - Fibroblast growth factors (FGFs) are central to signaling in the developing limb. FGF-2 and FGF-4 can substitute for the apical ectodermal ridge to maintain both limb bud outgrowth and polarizing region signaling. Here, we have repeated and extended previous studies and investigated local effects of the apical ectodermal ridge on gene expression of Msx-1, 5' members of the HoxD complex, and Bmp-2 in the limb bud mesenchyme and tested whether members of the FGF family can substitute for the ridge to maintain their expression patterns. We found that expression of Msx-1, Hoxd-13, and Bmp-2 in posterior limb bud mesenchyme is dependent on a local signal from the apical ectodermal ridge. When the apical ectodermal ridge of young chick wing buds is removed, or when posterior cells are taken from the bud and placed in culture, expression of Msx-1, Hoxd-13, and Bmp-2 is not detectable in posterior mesenchymal cells. Local application of FGF-soaked beads to posterior limb mesenchyme following ridge removal or addition of FGF to cultured cells maintains expression of Msx-1, Hoxd-13, and Bmp-2. In contrast, expression of Hoxd-11 in posterior mesenchyme appears to be stable in the absence of either the apical ectodermal ridge or FGF. Expression of Msx-1 in anterior and apical cells is also locally maintained by the apical ectodermal ridge and this effect can be reproduced by local application of FGF. Furthermore, the addition of FGF to cultured anterior limb bud cells maintains their ability to respond to positional cues when grafted back into limb buds. PMID- 7556934 TI - Development of the Xenopus pronephric system. AB - The pronephros serves as the embryonic kidney of the lower vertebrates. In this report we describe the development of the pronephric system of Xenopus laevis utilizing scanning electron microscopy and novel monoclonal antibodies that specifically recognize different parts of the pronephros. Antibody 3G8 recognizes the tubules and nephrostomes of the pronephroi only and does not react with the duct. Antibody 4A6 stains only the duct and the nephrostomes. These antibodies thus allow the positive identification of these two intermediate mesoderm derivatives. Both reagents detect antigens expressed some time after the pronephric structures first form and probably represent markers of terminal differentiation. When the tubules and duct first form they are separate structures that can easily be distinguished; the connective tubules have a distinctive organization, the collecting (or common) tubule is broader than other tubules, and the narrow pronephric duct has a specific shape and position. In later stages the collecting tubule and the rostral portion of the duct undergo a considerable amount of convolution, and both contribute to the final coiled tubular body of the pronephros. The ability of 3G8 and 4A6 to distinguish these two elements of the nephric system was used to reexplore classical experiments on the interaction between these two structures during development of the pronephric system. The use of whole-mount analysis has allowed us to examine large numbers of embryos from different stages and dissected in a variety of planes. These experiments demonstrate the dynamic nature of the intermediate mesoderm and indicate that although the pronephros may be specified by mid-neurula stages, patterning is not complete until tailbud stages. PMID- 7556935 TI - Formation of the female pronucleus and reorganization and disassembly of the first interphase cytoskeleton in the egg of the glossiphoniid leech Theromyzon rude. AB - Eggs of the leech Theromyzon rude were studied under dissecting, fluorescence, light, and electron microscopes. The sperm-derived centrosome divides and separates at early first interphase. The female pronucleus forms beside the male pronucleus at the centrally located perinuclear plasm domain, by fusion and remodelling of karyomeres that have descended from the animal pole at the tip of an ooplasmic process (centripetal ooplasmic flow). Retraction of this process (centrifugal ooplasmic flow) appears to return ooplasm to the animal pole. The reversible flow of ooplasm occurs along a subset of monaster microtubules that exhibit delayed depolymerization, and organelles thus move in both directions. Depolymerization of the first interphase monaster fibers seems to occur as a regular wave, moving from the internally located perinuclear plasm to the peripherally situated egg poles. However, disassembly of the ectoplasmic monaster fibers is preceded by furrowing and reorganization of both microtubules and microfilaments along polar rings and meridional bands. Bipolar contraction of microfilaments and shortening of microtubules lead to organelle concentration at the egg poles. PMID- 7556936 TI - Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. AB - Membrane potential (VM) was investigated in mouse and bovine sperm populations. VM was determined from the fluorescence emission of the lipophilic anion, bis(1,3 diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), and from the lipophilic cation, 3,3'-dipropylthiodicarbocyanine iodide (DiSC3(5)). Fluorescent signals were corrected for contributions of mitochondrial potentials and apparent VM values were obtained by calibrations in sperm selectively permeabilized with valinomycin or with gramicidin D. The calculated VM values of uncapacitated mouse and bovine sperm were approximately -35 and -30 mV, respectively. In contrast, capacitated populations of mouse and bovine sperm have VM values of -50 to -60 mV. Membrane hyperpolarization is due in part to an enhanced K+ permeability. The development of zona pellucida-activated signal transducing mechanisms during capacitation is dependent upon hyperpolarization. It is suggested that VM alterations regulate the activation state of sperm, thereby suppressing premature acrosome reactions in uncapacitated sperm and permitting capacitated sperm to respond to zona pellucida stimuli. PMID- 7556933 TI - Upregulation of the 72-kDa type IV collagenase in epithelial and stromal cells during rat tracheal gland morphogenesis. AB - Submucosal glands secrete most of the mucus that lubricates the tracheal surface and protects it from irritants and infection. These glands develop postnatally in the rat, permitting convenient study of the mechanisms controlling this process. One such mechanism involves degradation of the supportive connective tissue matrix at the front of the growing glands. We recently showed that tracheal gland cell invasion of collagen gels in vitro is dependent on secretion of a 72-kDa type IV collagenase. In the present study, we show that the activity of this enzyme (also referred to as matrix metalloproteinase-2 or gelatinase A) is elevated at the time of gland development in vivo. That this increase is at least partly mediated at the level of steady-state mRNA was indicated by semiquantitative PCR analysis of gland-enriched, microdissected tissue samples. Immunohistochemistry revealed that the enzyme was present at the interface between the glands and extracellular matrix. In situ hybridization revealed that the cognate mRNA was present in epithelial cells of glands undergoing morphogenesis (particularly Postnatal Day 7) but not in those of adult glands or the surface epithelium. At all ages, stromal cells below the surface epithelium were labeled; labeling intensity was highest at the time and location of gland morphogenesis. These findings suggest that the 72-kDa type IV collagenase is developmentally regulated in gland and stromal cells at the level of steady-state mRNA and plays a role in the degradation of extracellular matrix during tracheobronchial gland morphogenesis. PMID- 7556937 TI - The inositol 1,4,5-triphosphate receptor expression in Drosophila suggests a role for IP3 signalling in muscle development and adult chemosensory functions. AB - Inositol 1,4,5-triphosphate (IP3) is generated as a second messenger in many diverse cellular signalling pathways. In general these signalling pathways activate a membrane-bound phospholipase C, which cleaves the phospholipid phosphatidylinositol bisphosphate to generate IP3 and diacylglycerol. IP3 binds to a specific intracellular receptor, which is a membrane protein and a ligand gated Ca2+ channel, that causes Ca2+ release from intracellular stores. The inositol 1,4,5-triphosphate receptor (IP3R) is thus an integral part of the IP3 signalling pathway and can be used as a marker to identify biological processes that use IP3 as a second messenger. We have used an affinity-purified antibody, directed against a bacterial fusion protein and containing 339 amino acids of the Drosophila IP3R, to detect this protein in adult heads and during embryonic and pupal development. Our results suggest that in Drosophila the IP3 signalling pathway is used during muscle development, primarily when myoblasts undergo rapid multiplication, in both embryos and pupae. In adults, IP3 is probably a second messenger in more than one sensory transduction pathway, as well as in other as yet undefined brain and muscle functions. PMID- 7556938 TI - Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish. AB - Zebrafish embryos have three or four identifiable primary motoneurons per hemisegment. We previously reported that, while several ventral cells initially express the zebrafish Islet-1 (Isl-1) gene, a member of the LIM/homeobox gene family, the expression of this gene becomes restricted to a single or a pair of cells slightly anterior to each segment border by 16 hr after fertilization. Double staining by in situ hybridization and immunohistochemistry strongly suggested that these cells were mainly rostral primary motoneurons. Here, we have isolated two novel zebrafish cDNA clones for more Isl-1 family genes, termed zfIsl-2 and zfIsl-3. zfIsl-2 mRNA starts to be expressed in the ventral midsegmental cells per hemisegment around 15 hr. Double labeling experiments have shown that these midsegmental cells are the caudal primary motoneuron (CaP) and its variant equivalence pair. Our results revealed the heterogeneity in the expressed genes among primary motoneurons before the fates of the primary motoneurons are irreversibly determined, and further suggest the involvement of the Isl-1 and zfIsl-2 genes in the determination of cellular identities by primary motoneurons in embryonic zebrafish. zfIsl-3 mRNA is not expressed in motoneurons but is expressed at 17 hr, mainly in the ventral myotomes. This suggests that zfIsl-3 may be involved in the regional specification of the myotome and also in target recognition by CaP. zfIsl-2 is also expressed throughout the developing eye and tectal region of the midbrain, the target for the retinal axons. In the ventral spinal cord of the spadetail mutant embryo, which has defects in the somites, the cells expressing zfIsl-2 mRNA significantly decreased in number in contrast to the increase in cells expressing Isl-1 mRNA, suggesting the influence of the somites on the expression of both genes. PMID- 7556939 TI - Trigeminal sensory neurons require extrinsic signals to switch neurotrophin dependence during the early stages of target field innervation. AB - Sensory neurons of the embryonic mouse trigeminal ganglion are transiently supported by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 during the earliest stages of target field innervation before they become dependent on nerve growth factor (NGF) for survival. To determine whether the switch from BDNF/NT3/NT4/5 dependence to NGF dependence occurs autonomously in these neurons or whether extrinsic signals are required, we studied the survival of trigeminal neurons in vitro before, during, and after the switchover period. Trigeminal neurons cultured before they show any response to NGF survived with BDNF, NT3, or NT4/5 well beyond the switchover period. When these early neurons were switched from BDNF, NT3, or NT4/5 to NGF after various times in culture they died as rapidly as neuro-trophin-deprived neurons. Neurons that were switched from BDNF, NT3, or NT4/5 to NGF in cultures set up at stages throughout the switchover period exhibited an NGF survival response that improved with age. Moreover, the ability of NGF to promote long-term survival also increased with embryonic age. These results show that, unlike the onset of BDNF dependence, which is controlled by an intrinsic timing mechanism in early sensory neurons, the switch to NGF dependence relies on extrinsic signals acting on the neurons during the switchover period and that in vivo signals are also required for the maturation of the NGF survival response from a transient to a long-term response. Retinoic acid, which induces NGF dependence in early sympathetic neurons, was ineffective in promoting NGF dependence in early sensory neurons. PMID- 7556940 TI - The 220-kDa vitelline coat glycoprotein mediates sperm binding in the polarized egg of Unio elongatulus through O-linked oligosaccharides. AB - In previous studies we found that two glycoproteins of 220 and 180 kDa account for 80-90% of the material dissolved from the vitelline coat (VC) of Unio elongatulus egg (Focarelli and Rosati, 1993). The two glycoproteins were purified by electroelution and used to raise the corresponding polyclonal antibodies. Immunofluorescence experiments showed that the 180-kDa protein species is ubiquitous in the VC, whereas the 220-kDa protein is concentrated in a restricted region of the vegetal pole, the crater region, where the sperm-egg interaction occurs. Binding assays indicated that only the 220-kDa protein interacted with the sperm and that the protein bound in the apical region and triggered acrosomal changes in sperm. Competition binding assays showed that O- and not N-linked oligosaccharides derived from the 220-kDa protein competed with the binding of the protein to sperm and that fucose is involved in the ligand role of the 220 kDa protein. PMID- 7556941 TI - A novel regulatory region is required for trophoblast-specific transcription in transgenic mice. AB - The trophectoderm epithelium of the blastocyst is the first tissue to differentiate in the mammalian embryo and gives rise to all cells of the trophoblast lineage. These cells form the majority of the fetal component of the placenta, which is critical for intrauterine development. Our interest in the molecular determinants of trophoblast lineage development has led us to undertake an analysis of the regulatory region of a trophoblast-specific gene termed 4311. The transcription start site was mapped by both primer extension and RNase protection analyses. We show that cloned 4311 upstream sequences are able to confer the correct temporal and spatial expression pattern of 4311 on a reporter gene in transgenic mice. In addition, we have characterized a 340-bp region, 3.7 kb upstream of the transcription start, that is sufficient for the specific expression of lacZ in cells of the developing spongiotrophoblast in transgenic mice. Delineation of a diploid trophoblast-specific regulatory region will facilitate the identification of factors that may regulate the development of this lineage. PMID- 7556942 TI - Normal expression and the effects of ectopic expression of the Drosophila muscle segment homeobox (msh) gene suggest a role in differentiation and patterning of embryonic muscles. AB - Myogenesis is a several step process that requires genes involved in specifying mesoderm lineage and genes involved in determining muscle identity, differentiation, and patterning. We report here on the isolation, characterization, and expression pattern of a cDNA clone encoded by the previously uncharacterized Drosophila muscle segment homeobox (msh) gene and its possible role in myogenesis. The amino acid sequence of the msh homeobox domain is highly homologous to the homeodomains of the Drosophila S59 and empty spiracles genes and the Hox 7 and Hox 8 family of vertebrate homeobox genes. In addition, the 5' end of msh has 52% sequence identity to the 5' end of the empty spiracles gene and encodes several stretches of amino acids rich in serine, alanine, proline, glutamine, and acidic amino acids, indicating potential domains of regulatory activity. The expression of msh is initially detected at about stage 6 in the dorsal lateral ectoderm of the embryo and later in the developing central (CNS) and peripheral nervous systems. During germ band retraction (stage 12), msh continues to be expressed in cells of the nervous system as well as cells of the somatic mesoderm corresponding mostly to the developing dorsal and lateral somatic body wall muscles. These mesodermal cells, which continue to express msh in daughterless mutant embryos, undergo an increase in cell number in neurogenic mutants. By late stage 14 of embryonic development, msh expression is greatly reduced or absent in most or all mesoderm and muscle but continues in CNS until hatching. Ectopic expression of msh in the mesoderm results in altered expression of the S59 and nau/Dmyd genes leading to a loss of some muscles and defects in the patterning of others, suggesting that the muscle defects are at the level of recruitment and/or patterning of muscle precursor cells. Thus the similarity of Drosophila msh expression to that of the homologous vertebrate Hox 7 and Hox 8 genes together with the effects of ectopic expression of msh in the mesoderm suggest a role for the msh-like family of genes in mesodermal and muscle differentiation and patterning. PMID- 7556943 TI - Initiation of anterior head-specific gene expression in uncommitted ectoderm of Xenopus laevis by ammonium chloride. AB - The role of homeobox-containing genes in the regional specification of the vertebrate embryo has been an area of intense research over the last decade. Whereas it appears that the homeobox genes of the Hox gene family play an important role in the specification of the trunk, the genes and processes involved in the specification of the head are less well understood. We have isolated a new head-specific homeobox gene, XANF-2, that appears to be involved in the regional specification of the anterior head of Xenopus embryos. This gene is initially expressed in the anterior dorsal region of early embryos and later exclusively in the primordium of the anterior pituitary gland. XANF-2 represents the earliest marker for the anterior pituitary lineage. Ammonium chloride is able to induce the expression of XANF-2 in uncommitted ectoderm. These and other data indicate that ammonium chloride is capable of inducing a large portion of the anterior dorsal region of the embryo which includes, but is not limited to, the anterior pituitary gland and cement gland anlagen. This implies that changes in intracellular ionic conditions play an important role in the formation of the anterior head region. In addition to NH4Cl, injection of follistatin RNA can induce transcription of XANF-2, suggesting that these two unrelated compounds can activate a chain of events leading to the formation of the amphibian head. Furthermore, we demonstrate that planar induction in Keller sandwiches can induce XANF-2 expression as well as the expression of the cement gland-specific gene, XCG 13, indicating that planar signaling can account for induction of even the most anterior regions of the embryo. PMID- 7556944 TI - The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene. AB - Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices. PMID- 7556947 TI - Male and female cooperate in the prohormone-like processing of a Drosophila melanogaster seminal fluid protein. AB - Acp26Aa is a Drosophila seminal fluid protein that plays a role in the elevation of egg-laying by the mated female and has structural features of a prohormone. The protein, which has a region of sequence similarity to the egg-laying hormone of Aplysia, is transferred to the Drosophila female during mating. Acp26Aa is processed in the mated female's genital tract. We show here that the processing involves sequential proteolytic cleavages, and we map the position of these cleavages. Although Acp26Aa is not cleaved in the male, its processing in the mated female requires activities donated by the male. Acp26Aa ectopically expressed in unmated females is not processed. Processing of Acp26Aa in wild-type females mated to males with altered seminal fluid is dependent on the presence and amount of male accessory gland secretions. The need for molecular cooperation between the sexes for processing of Acp26Aa could restrict its activity to the mated female. PMID- 7556945 TI - Sequence and expression of amphioxus alkali myosin light chain (AmphiMLC-alk) throughout development: implications for vertebrate myogenesis. AB - The lower chordate amphioxus, widely considered the closest living invertebrate relative of the vertebrates, is a key organism for understanding the relationship between gene duplications and evolution of the complex vertebrate body plan. In tetrapod vertebrates, the alkali myosin light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large family with stage-, tissue-, and fiber-type-specific expression of different isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and nematodes, only one MLC-alk gene has been found, but the number of such genes in deuterostome invertebrates and lower vertebrates is unknown. The present report, describing the sequence and expression throughout development of the amphioxus gene for alkali myosin light chain (AmphiMLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A full-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphioxus cDNA library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot analysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated that this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed expression initially in the myotomes of somites 2-5 of neurula embryos and soon thereafter in the myotomes of somite 1 and of newly forming somites progressively added posteriorly. Myotomal expression continues throughout larval development and into the adult stage as the myotomal cells differentiate into striated, mononucleate muscle cells--unlike vertebrate striated muscle cells, those of amphioxus never become multinucleate. In late larvae and adults myotomal expression of AmphiMLC alk is localized along the medial edge of the myotome and at the ends of the cells. This is the first demonstration of intracellular localization of MLC transcripts in muscle cells of any animal. Expression of AmphiMLC-alk was also detected in smooth muscles as well as in striated muscles not derived from the myotome. These expression data are consistent with the Southern blot analysis in suggesting that there is only one MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebrate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7556946 TI - Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis. AB - The molecular signaling of secondary induction is a fundamental process in organogenesis during embryonic development. To study the signal transduction pathways involved, we used developing chicken skin as a model and focused on the roles of intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of forming dermis around H and H stage 30. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. The presence and activity of PKC, PKA, and P-CREB in developing chicken skin are further characterized by immunoblot, kinase activity, and gel shift assays. To explore their physiological significance, embryonic chicken dorsal skin explants were treated with different modulators in medium or in beads for localized effects. The results showed that PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. We observed that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented. PMID- 7556949 TI - Skeletal muscle mitogen-activated protein kinases and ribosomal S6 kinases. Suppression in chronic diabetic rats and reversal by vanadium. AB - The mitogen-activated protein (MAP) kinases and ribosomal S6 protein kinases in the skeletal muscle of insulin-resistant long-term (2 and 6 months' duration) diabetic rats were investigated to understand further the changes in insulin intracellular signaling pathways that accompany diabetes. The effects of insulin mimetic vanadium compounds on the activity of these kinases were also examined. In the insulin-resistant 2-month diabetic rats, the basal activities of MAP kinases were relatively unchanged, while the basal activities of S6 kinases were significantly increased. Intravenous injection of insulin moderately activated both the 42-kDa MAP kinase (p42mapk) and a 44-kDa MAP kinase (p44erk1) in the 2 month control rats but not in the 2-month diabetic rats. Insulin treatment markedly stimulated the activity of a novel 31-kDa S6 kinase and the previously described 90-kDa ribosomal S6 kinase encoded by one of the rsk genes (p90rsk) in the 2-month control rats, while the effect was substantially reduced in the diabetic rats. In the 6-month diabetic rats, the basal phosphotransferase activities of both MAP kinases were depressed threefold or greater. This correlated with reductions in the amount of immunoreactive p42mapk and p44erk1 proteins in extracts from the diabetic rats. The basal activity of the 31-kDa S6 kinase activity was also reduced fourfold in the 6-month diabetic rats. Treatment of the 2-month diabetic rats with vanadyl sulfate resulted in euglycemia, prevented the increase in the basal activity of S6 kinase, and improved the activation of S6 kinase by insulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556948 TI - Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-beta as a key mediator. AB - Renal cells are a rich source of transforming growth factor (TGF)-beta, and they serve as targets for its actions. Our hypothesis that activation of the TGF-beta system in the kidney is implicated in the development of diabetic renal disease stems from the close similarity of actions of TGF-beta and high ambient glucose on renal cell growth and extracellular matrix metabolism. Proximal tubule cells and glomerular mesangial cells cultured in high glucose concentration express increased TGF-beta 1 mRNA and protein levels, and treatment with anti-TGF-beta antibodies results in prevention of the effects of high glucose to induce cellular hypertrophy and stimulate collagen biosynthesis. Several in vivo studies by different groups of investigators have reported overexpression of TGF-beta in the glomeruli in human and experimental diabetes. We have also observed that the development of renal hypertrophy in the insulin-dependent diabetic BB rat and NOD mouse is associated with increased expression of TGF-beta 1 in the kidney and that short-term administration of antibodies capable of neutralizing the activity of TGF-beta in the streptozotocin mouse model of diabetes results in attenuation of whole kidney and glomerular hypertrophy and overexpression of mRNAs encoding matrix components. Together, these findings are consistent with the hypothesis that the diabetic state stimulates TGF-beta expression in the kidney and that in turn this growth factor may mediate, in an autocrine/paracrine manner, some of the principal early manifestations of diabetic renal disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556950 TI - Higher proinsulin and specific insulin are both associated with a parental history of diabetes in nondiabetic Mexican-American subjects. AB - Both insulin resistance and decreased insulin secretion have been hypothesized to be precursors of non-insulin-dependent diabetes. An elevated proinsulin concentration reflects abnormal proinsulin processing and could indicate abnormal insulin secretion. We examined fasting insulin (measured by a radioimmunoassay that does not cross-react with proinsulin), as a marker of insulin resistance, and proinsulin and the fasting proinsulin-to-insulin ratio, as markers of impaired proinsulin processing, in 597 nondiabetic Mexican-Americans from the San Antonio Heart Study. Fasting insulin, proinsulin, and the fasting proinsulin-to insulin ratio were higher in subjects with a parental history of diabetes than in subjects without such a history. These differences remained statistically significant after adjustment for obesity, body fat distribution, and glucose tolerance. A parental history of diabetes in nondiabetic Mexican-Americans is associated with an increase in fasting specific insulin and a disproportionate increase in proinsulin relative to insulin. These data suggest that both increased insulin resistance and abnormal processing of proinsulin are present in offspring of parents with diabetes. PMID- 7556952 TI - Contribution of blood flow to leg glucose uptake during a mixed meal. AB - Insulin has important effects to increase skeletal muscle (leg) blood flow under euglycemic hyperinsulinemic clamp conditions and after oral glucose tolerance testing. The present studies examined the effects of mixed meal consumption on the components of leg glucose uptake (LGU) in lean, healthy adults. Seventeen men and women underwent measures of leg plasma flow and arteriovenous (AV) glucose difference before and for 6 h after a mixed meal providing one-third of daily energy expenditure. Another eight men and women underwent the same studies before and during the consumption of the same-size meal administered in small frequent feedings over 6 h. After the bolus meal, peak leg AV glucose gradient increased approximately fivefold (P < 0.001), whereas the peak increase in leg plasma flow was 20% (NS). No significant contribution of increased leg blood flow to the increase in postprandial LGU was apparent. Over the last 100 min of the frequent feedings meal, the leg AV difference increased approximately fourfold (P < 0.001 vs. basal), whereas leg blood flow increased only by 16% (NS vs. basal). We conclude that after a mixed meal, leg (primarily skeletal muscle) blood flow does not increase enough for blood flow to be a major contributor to glucose uptake. These findings raise questions regarding the relative importance of insulin's hemodynamic effects in modulating glucose tolerance under more usual conditions. PMID- 7556951 TI - Treatment of IFN-gamma transgenic mice with anti-IFN-gamma reveals the remodeling capacity of the adult pancreas. AB - Pancreatic expression of gamma-interferon (IFN-gamma) initiates a cascade of pathogenic changes that include pancreatic inflammation, islet cell destruction, hyperglycemia, and islet regeneration. In this study, we explore the developmental plasticity of the adult pancreas and particularly its ability to return to normoglycemia and to remodel itself from an advanced pathogenic state. This was approached by treating adult transgenic mice with a pulse of anti-IFN gamma antibody and determining the functional and morphological status of the pancreas. We demonstrated that anti-IFN-gamma antibody administration led to the reduction of hyperglycemic blood glucose levels in transgenic mice. We also observed that the pancreas returned from a profoundly perturbed state toward normality. Analysis of the mitotic index indicated that cell proliferation previously associated with islet cell regeneration was greatly reduced after anti IFN-gamma administration. Our results highlight the ability of the adult pancreas to remodel itself and return from a complex pathological state to normalcy once the trophic signal inducing this pathology is removed. These data also suggest that anti-IFN-gamma administration may have important clinical implications for treatment of chronic pancreatitis in humans. PMID- 7556953 TI - Role of phosphodiesterase III in the antilipolytic effect of insulin in vivo. AB - The effect of three types of phosphodiesterase (PDE) inhibitors on in vivo antilipolysis was investigated in healthy subjects using a 2-h euglycemic, hyperinsulinemic (40 mU.m-2.min) clamp together with microdialysis of abdominal subcutaneous adipose tissue. During hyperinsulinemia (approximately 330 pmol/l), the circulating glycerol concentration was reduced to approximately 50% of the basal level of 53.2 +/- 3.6 mumol/l, indicating an antilipolytic effect. The decrease in adipose tissue dialysate glycerol, which mirrors the change in interstitial glycerol concentration, was about 40% during hyperinsulinemia when Ringer's solution alone was perfused. Local perfusion with a selective PDE IV inhibitor, rolipram (10(-4) mol/l), did not influence the insulin-induced decrease in dialysate glycerol (F = 0.8 vs. perfusion with Ringer's solution by two-factor analysis of variance [ANOVA]), although rolipram increased the dialysate glycerol level by 144 +/- 7% of the baseline value. However, local perfusion with a selective PDE III inhibitor, amrinone (10(-3) mol/l), or a nonselective PDE inhibitor, theophylline (10(-2) mol/l), abolished the ability of insulin to lower dialysate glycerol (F = 16.5, P < 0.01 and F = 8.5, P < 0.01, respectively, as compared with perfusion with Ringer's solution). The findings could not be explained by changes in the local blood flow (as measured by a microdialysis--ethanol escape technique), which was not affected by hyperinsulinemia in the presence or the absence of PDE inhibitors in the dialysis solvent. We conclude that PDEs play an important role in mediating the antilipolytic effect of insulin in vivo and that PDE III is the dominant isoenzyme modulating this effect. PMID- 7556954 TI - Late progression to diabetes and evidence for chronic beta-cell autoimmunity in identical twins of patients with type I diabetes. AB - Previous studies suggest that after 6 years of discordance, identical twin pairs rarely become concordant for type I diabetes. With up to 39 years of follow-up from the onset of diabetes in the index twin, we determined how many discordant twins have evidence of beta-cell autoimmunity and how many develop overt diabetes. We longitudinally followed 23 pairs of identical twins (or triplets) that were selected from a total group of 30 pairs because they were discordant for type I diabetes when first ascertained. Seven developed diabetes after 3, 3, 7, 8, 9, 31 and 36 years of discordance. By survival analysis, the concordance after 10 years from the onset of diabetes in the index twin was estimated as 23% (95% confidence interval, 5-40%), increasing to 38% (95% confidence interval, 8 69%) after 31 years. Among 16 twins remaining nondiabetic at last follow-up (8-39 years of discordance), 12 were assessed with serial intravenous glucose tolerance tests and a total of 407 measurements by radioassay of antibodies against three defined autoantigens (glutamic acid decarboxylase, insulin, and the recently cloned molecule ICA512). Two-thirds (8 of 12) had evidence of beta-cell autoimmunity (persistently positive autoantibody levels) and/or first-phase insulin release less than the 1st percentile of control subjects. In summary, identical twins may develop diabetes after a prolonged period of discordance and approximately two-thirds of long-term discordant twins have evidence of persistent beta-cell autoimmunity and/or beta-cell damage. The concordance for beta-cell autoimmunity, therefore, is much higher than for overt diabetes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556955 TI - Phosphorylation and activity of Na+/H+ exchanger isoform 1 of immortalized lymphoblasts in diabetic nephropathy. AB - In both essential hypertension and diabetic nephropathy (DN), the ubiquitous cellular Na+/H+ exchanger (NHE) exhibits altered kinetics with increased transport activity. The mechanism for this phenotype and its dependence on the presence of serum are unknown, but increased lymphoblast NHE activity in DN has been attributed to a defect in post-translational processing of NHE-1 rather than an increased cellular exchanger number. Phosphorylation of NHE-1 has been proposed to play a role in its activation in a variety of cell models. We have examined, therefore, the role of NHE-1 phosphorylation and the effect of serum in determining the increased NHE-1 activity in lymphoblasts from patients with DN. Cells from these patients exhibited increased NHE activity in the presence and absence of fetal calf serum (range 42-59%, P < 0.005, analysis of variance) and an increased proliferation rate (P < 0.01) when compared with cells from both normoalbuminuric diabetic patients and non-diabetic control subjects. However, NHE-1 abundance was very similar among all groups in the presence and absence of serum, indicating that increased NHE activity in cells of nephropathy patients was due to an increased turnover number. This nephropathy phenotype was not accompanied by an increased net phosphorylation of NHE-1 in the presence or absence of serum. Our findings suggest that increased NHE-1 activity in cells of DN patients is independent of the presence of serum and is not attributable to altered NHE-1 phosphorylation. Additional post-translational mechanisms for activation of NHE-1, therefore, may be involved. PMID- 7556957 TI - Regulation of insulin receptor mRNA splicing in rat tissues. Effect of fasting, aging, and diabetes. AB - Recent findings suggested that alterations in insulin receptor isoform expression might be involved in the molecular mechanism of insulin resistance. Using reverse transcription reaction followed by competitive polymerase chain reaction, we measured the level of the receptor mRNA variants in rat insulin-sensitive tissues, under conditions of decreased insulin effectiveness (fasting, aging, and diabetes). The liver expressed the mRNA variant with exon 11 predominantly, and the hind limb skeletal muscles expressed the mRNA without exon 11. The heart and epididymal adipose tissue expressed both variants. Fasting and streptozocin induced diabetes increased the level of receptor mRNAs in the liver but did not modify the repartition between the two variants. The modification of the expression ratio, in favor of the form with exon 11, found by some authors in the skeletal muscle of insulin-resistant patients was not observed in rat muscles that expressed > 99% of the form without exon 11 under all the conditions tested. In adipose tissue, the proportion of both mRNA variants was never altered (45% of exon 11-positive [Ex11+]), while the total receptor mRNA concentration changed markedly during fasting or aging. The only modification observed in the isoform distribution was a significant decrease in Ex11+ mRNA concentration in the liver, muscle, and heart of old rats. We conclude that alternative splicing of insulin receptor mRNA is not involved in the impairment of insulin action during fasting or diabetes. Its potential role in the insulin resistance of old animals remains to be defined. PMID- 7556956 TI - Crosses of NOD mice with the related NON strain. A polygenic model for IDDM. AB - Chromosome locations of non-major histocompatibility complex (MHC) genes contributing to insulin-dependent diabetes mellitus (IDDM) in mice have been determined by outcrossing NOD mice to other inbred strains congenic for the NOD MHC haplotype (H2g7). At least nine non-MHC IDDM susceptibility genes (Idd) were previously identified at first backcross (BC1) after outcross of NOD to C57BL/10.H2g7 congenic mice (B10.H2g7). We investigated whether the same set of Idd loci segregated with IDDM susceptibility after outcross of NOD to NON.H2g7 congenic mice. Since the outcrosses to NON.H2g7 and B10.H2g7 were performed in the same vivarium, direct comparisons were made of the chromosomal locations and relative strengths of Idd alleles in diabetic progeny from the two different outcrosses. In comparison with the NOD x B10.H2g7 outcross, the NOD x NON.H2g7 outcross produced significantly higher IDDM frequencies in F1, F2, and BC1 generations. The high F2 diabetes frequency allowed evaluation of the effects of homozygous expression of both the susceptibility and the resistance allele at Idd loci. This analysis demonstrated that no single non-MHC Idd locus was essential for the onset of diabetes in this cross. After outcross to NON.H2g7, Idd4 (chromosome [Chr] 11), Idd5 (Chr 1), and Idd8 (Chr 14) did not segregate with IDDM in either the BC1 or the F2 generation. Diabetogenic NOD-derived alleles at Idd2 (Chr 9), Idd3 (Chr 3), and Idd10 (Chr 3) were segregating in the BC1. An NON derived allele contributing to susceptibility on Chr 7 (Idd7) was also detected. Dominant traits, detectable only in the F2 cross, were encoded by Chr 4 (Idd9) and two newly mapped loci on Chr 13 (Idd14) and 5 (Idd15). A third dominant trait was encoded by Chr 6 (possibly Idd6), but here, in contrast to Idd9, Idd14, and Idd15, the NON allele was diabetogenic. Stepwise logistic regression analysis of the BC1 and F2 data confirmed that the ability to identify certainty of the non MHC Idd loci was contingent on the extent of homozygosity for NOD background genes. This study shows that the diabetogenic phenotype can be achieved through the actions of variable combinations of MHC-unlinked genes and a diabetogenic MHC haplotype. PMID- 7556959 TI - Treatment with insulin-like growth factor I alters capillary permeability in skin and retina. AB - Treatment with insulin-like growth factor I (IGF-I) is accompanied by mild generalized and reversible edema. These changes may be due to increased capillary permeability. Therefore, we studied the effects of subcutaneous IGF-I treatment in healthy subjects on capillary permeability of the skin and the retina. Eight healthy subjects were treated with saline or recombinant human IGF-I (rhIGF-I) (10 micrograms.kg-1.h-1 s.c.) in a randomized crossover fashion. Transcapillary diffusion of sodium fluorescein (NaF) was quantitated by video densitometry in the skin in all subjects on the 4th treatment day. In seven subjects, plasma penetration of NaF at the blood-retinal barrier was investigated using vitreous fluorometry (VF) on days 3 and 5. Fluorescent light intensities of the skin and plasma penetration determined by VF were significantly higher during the IGF-I treatment as compared with those during the control situation. In conclusion, IGF I treatment at the above dose is accompanied by increased transcapillary diffusion of NaF in skin and in retinal vessels. PMID- 7556960 TI - A monitor of secretion from single pancreatic beta-cells. AB - ATP is known to be coreleased with insulin from pancreatic beta-cells. To monitor insulin secretion from single beta-cells, a single beta-cell was surrounded in culture by Fura 2-loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP. CPAE cells did not respond with an elevation in cytoplasmic calcium concentration ([Ca2+]i) to either tolbutamide (100 mumol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+]i to ATP (0.1-10 mumol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 mumol/l) increased [Ca2+]i in both the beta-cell and the adjacent CPAE cells in co-culture. Suramin (100 mumol/l), an ATP-receptor blocker, inhibited the tolbutamide-induced elevation in [Ca2+]i in the CPAE cells but did not inhibit the elevation in [Ca2+]i in the beta-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the beta-cell. When co-culture of the beta-cell and CPAE cells was stimulated by kainate (1 mmol/l) and then tolbutamide (10 mumol/l), the CPAE cells showed elevations in [Ca2+]i in response to kainate and tolbutamide during elevation in [Ca2+]i in the beta-cell. This strongly suggests that insulin secretion as well as an increase in [Ca2+]i in response to different agents, i.e., kainate and tolbutamide, occurs in a single beta-cell. A long exposure of tolbutamide (100 mumol/l, 4 min) resulted in a long-lasting elevation in [Ca2+]i in the beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556958 TI - Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor. AB - Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by < 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor. PMID- 7556961 TI - Levels of lipoprotein(a), apolipoprotein B, and lipoprotein cholesterol distribution in IDDM. Results from follow-up in the Diabetes Control and Complications Trial. AB - Levels of lipoprotein(a) [Lp(a)], apolipoprotein (apo) B, and lipoprotein cholesterol distribution using density-gradient ultracentrifugation were measured as part of a cross-sectional study at the final follow-up examination (mean 6.2 years) in the Diabetes Control and Complications Trial. Compared with the subjects in the conventionally treated group (n = 680), those subjects receiving intensive diabetes therapy (n = 667) had a lower level of Lp(a) (Caucasian subjects only, median 10.7 vs 12.5 mg/dl, respectively; P = 0.03), lower apo B (mean 83 vs. 86 mg/dl, respectively; P = 0.01), and a more favorable distribution of cholesterol in the lipoprotein fractions as measured by density-gradient ultracentrifugation with less cholesterol in the very-low-density lipoprotein and the dense low-density lipoprotein fractions and greater cholesterol content of the more buoyant low-density lipoprotein. Compared with a nondiabetic Caucasian control group (n = 2,158), Lp(a) levels were not different in the intensive treatment group (median 9.6 vs. 10.7 mg/dl, respectively; NS) and higher in the conventional treatment group (9.6 vs. 12.5 mg/dl, respectively; P < 0.01). No effect of renal dysfunction as measured by increasing albuminuria or reduced creatinine clearance on Lp(a) levels could be demonstrated in the diabetic subjects. Prospective follow-up of these subjects will determine whether these favorable lipoprotein differences in the intensive treatment group persist and whether they influence the onset of atherosclerosis in insulin-dependent diabetes. PMID- 7556962 TI - Differential immunogenetic determinants of polyclonal insulin autoimmune syndrome (Hirata's disease) and monoclonal insulin autoimmune syndrome. AB - The insulin autoimmune syndrome (IAS), or Hirata's disease, is characterized by the combination of fasting hypoglycemia, high concentration of total serum immunoreactive insulin, and presence of autoantibodies to native human insulin in serum. Autoantibody production is classified as monoclonal or polyclonal, with the majority of IAS cases classified as polyclonal. Previously, we observed a striking association between the human leukocyte antigen (HLA) class II alleles DRB1*0406/DQA1* 0301/DQB1*0302 and Japanese IAS patients with polyclonal insulin autoantibodies (IAAs) and T-cell recognition of human insulin in the context of DRB1*0406 molecules. Because of such a strong HLA association in IAS, we performed intra- and interethnic studies on IAS-associated DRB1 alleles and searched for the critical amino acid residue(s) for IAS pathogenesis. Glutamate at position 74 in the HLA-DR4 beta 1-chain was presumed to be essential to the production of polyclonal IAA in IAS, whereas alanine at the same position of the HLA-DR beta 1-chain might be important in the production of monoclonal IAA. PMID- 7556964 TI - Effects of a 48-h fat infusion on insulin secretion and glucose utilization. AB - To determine the effects of prolonged elevation of plasma free fatty acids (FFAs) on insulin secretion, we infused Liposyn II (4.3 mumol.kg-1.min-1) plus heparin (0.4 U.kg-1.min-1) intravenously into six healthy volunteers for 48 h. Another six volunteers received saline infusions and served as control subjects. In all 12 subjects (11 men and 1 woman), plasma glucose was clamped at approximately 8.6 mmol/l. Liposyn/heparin infusion resulted in a 9.4-fold increase in plasma FFA concentration (from 132 to 1,237 mumol/l), a 46% increase in insulin secretion rates (from 241 to 352 pmol/min, P < 0.05) (determined by deconvolution of plasma C-peptide concentration), and a 30% decrease, during the initial 24 h, in the rate of glucose infusion needed to maintain hyperglycemia (from 55.5 to 39.1 mumol.kg-1.min-1, P < 0.02). This decrease disappeared during the second 24 h. In summary, we found that physiologically elevated plasma FFAs 1) potentiated glucose-stimulated insulin secretion for 48 h and 2) initially caused peripheral insulin resistance that disappeared during the 2nd day, probably as a result of elevated circulating insulin levels. We conclude that in healthy volunteers under hyperglycemic conditions, fat infusion produced insulin resistance that was compensated for after approximately 24 h by persistent hypersecretion of insulin. PMID- 7556965 TI - Linkage analyses of the MODY3 locus on chromosome 12q with late-onset NIDDM. AB - Non-insulin-dependent diabetes mellitus (NIDDM) is a clinically and genetically heterogeneous disorder. Maturity-onset diabetes of the young (MODY), an autosomal dominant form of NIDDM, has been used as a model for genetic studies of NIDDM. We recently reported linkage between markers on chromosome 12q and diabetes in 25% of our French MODY families. To evaluate if this gene is also implicated in late onset NIDDM, we performed linkage studies between two markers of the MODY region and diabetes in 172 families with late-onset NIDDM. Both parametric and nonparametric methods were used in a total of 600 affected sib-pairs. Linkage was rejected in this population by all methods, implying that the MODY gene on chromosome 12q is not a major gene for late-onset NIDDM in this population. However, we cannot exclude a modifying role in a polygenic disorder or an important role in some families. PMID- 7556966 TI - What is the secretory potential of submucosal mucous glands within the human gullet in health and disease? AB - Histology of the human esophageal mucosa reveals numerous submucosal mucous glands, scattered along the esophagus and especially accumulated below the upper and above the lower esophageal sphincters. Mucin remains a major organic component of human esophageal secretion, collected using our newly developed esophageal perfusion catheter. Esophageal mucin is accompanied by nonmucin proteins, epidermal growth factor (EGF) and prostaglandin E2 (PGE2). Bicarbonate is the major inorganic component of esophageal secretion in humans. Mucosal exposure to an HC1/pepsin solution, mimicking the natural gastroesophageal scenario, significantly changed the secretory profile of all esophageal secretion components. The rate of secretion of esophageal mucin, EGF and PGE2 under the impact of HC1/pepsin in patients with reflux esophagitis appeared to be significantly impaired, although the basal rate of esophageal PGE2 output remained higher than in controls. These data indicate that a significant impairment in esophageal components of the preepithelial mucosal barrier, paralleling the severity of mucosal inflammation, may have a detrimental impact on the protective potential of the esophageal mucosal barrier, facilitating the development of reflux esophagitis. PMID- 7556967 TI - What role do salivary inorganic components play in health and disease of the esophageal mucosa? AB - The integrity of the esophageal mucosa depends upon an equilibrium between aggressive factors and protective mechanisms. Esophageal mucosal protective mechanisms operate at three overlapping levels: (1) preepithelial, (2) epithelial and (3) postepithelial. Since aggressive factors always operate on the luminal side of the esophagus, preepithelial defense remains as the first line in mucosal barrier protection. Salivary secretion quantitatively and qualitatively contributes to the protective potential of the preepithelial barrier. Salivary volume and its buffering capacity are elaborated by lowering intraluminal pH within the esophagus, and are key factors in restoration of physiologic pH within the esophagus. Salivary secretory response to esophageal mechanical (bolus) and chemical (intraluminal pH) stimuli, mediated by the esophagosalivary reflex pathway, is impaired in patients with reflux esophagitis. PMID- 7556963 TI - In situ hybridization of interleukin 6 in diabetic nephropathy. AB - Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowman's capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN. PMID- 7556971 TI - Oesophageal defence mechanisms. AB - Various defence mechanisms are found in the oesophagus which can be elicited by reflux damage. Premucosal defence includes bicarbonate ions and epidermal growth factor (EGF) secreted by salivary and oesophageal glands. The mucosa can respond by increasing epithelial cell turnover and upregulating EGF receptor and endocytosis. The intercellular barrier can be increased by the contents of membrane-coating granules. Local pH can be regulated by carbonic anhydrase. The whole viscus can exhibit peristalsis to effect a mechanical clearance of the refluxed gastric and duodenal material. PMID- 7556969 TI - Systemic factors in esophageal mucosal protection. AB - Blood bicarbonate is an important systemic factor in esophageal protection due to its capacity for buffering H+ either in the lumen, at the epithelial surface, within the intercellular space, or within the cell. The ability to buffer within the lumen is in part dependent on submucosal gland secretion, while buffering in the intercellular space depends on bicarbonate diffusion from blood, and intracellular protection depends on the presence of basolateral membrane bicarbonate transport via a mechanism such as the Na(+)-dependent Cl-/HCO3- exchanger. PMID- 7556968 TI - Do salivary organic components play a protective role in health and disease of the esophageal mucosa? AB - Aggressive factors operating within the esophageal lumen during gastroesophageal reflux are balanced by adequately mobilized protective mechanisms. Esophageal mucosal protection operates at three different although complementary dimensions: (1) preepithelial, (2) epithelial and (3) postepithelial. Since aggressive factors predominantly operate within the esophageal lumen, preepithelial defense is pivotal in mucosal protection. The preepithelial barrier is significantly enhanced by the quantity and the quality of salivary organic components such as salivary mucin, nonmucin protein, salivary epidermal growth factor (EGF) and salivary prostaglandin E2. The rate of secretion of salivary mucin, nonmucin protein and EGF under the impact of intraesophageal mechanical (bolus) and chemical (HCl/pepsin) stimulation, mimicking the natural gastroesophageal reflux scenario, is significantly impaired in patients with RE, whereas the rate of salivary PGE2 output remains essentially unchanged. Salivary secretory response to esophageal mechanical and chemical stimuli in terms of organic components, mediated by the esophagosalivary reflex pathway, exhibits a significant impairment in patients with reflux esophagitis. PMID- 7556972 TI - Transmucosal potential difference as an index of esophageal mucosal integrity. AB - All the epithelia lining the gastrointestinal (GI) tract, including that of the esophagus, exhibit a transmucosal electrical potential difference (PD). The luminal surface of the GI mucosa is indeed electrically negative when compared with the serosal one. Although it was initially felt that the body of the esophagus exhibits a PD near 0 or slightly positive, recent studies, using parenteral reference electrodes, have shown a negative PD of around -15 mV. Measurement of esophageal PD has been mainly used to locate both the lower and the upper esophageal sphincters but very rarely to evaluate esophageal mucosal integrity in clinical settings, most probably due to the difficulties encountered during measurement of mucosal PD. Reliable techniques to measure esophageal PD simultaneously with esophageal pressure or mucosal pH are now available. Application of these recently developed methodologies showed that measurement of esophageal PD during either manometry or endoscopy provides meaningful information about mucosal integrity. Indeed, tissue injury, either neoplastic of inflammatory, usually results in a less negative PD. In contrast, an abnormally high negative PD is very often observed in patients with columnar-lined lower esophagus. In patients with microscopic reflux esophagitis, PD exhibits less negative values which are significantly correlated with the degree of the mucosal damage. Normalization of the altered PD after either medical or surgical treatment makes it an additional parameter to evaluate the effect of a given therapy. PMID- 7556973 TI - Structure and role of Langerhans' cells in the human oesophageal epithelium. AB - Oesophageal Langerhans' cells (LC) are bone marrow-derived dendritic cells situated suprabasally in most stratified squamous epithelia, such as the epidermis and the epithelium of oesophageal mucosa. LC function as antigen presenting cells. Associated with relatively numerous intra-epithelial lymphocytes present in the normal state in oesophageal mucosa, they play a major role in the immunologic defense system of the oesophagus. Structure and role of LC are summarized in this review. Moreover, the implications of LC in different pathologic oesophageal reactions are discussed. PMID- 7556970 TI - How does the esophageal epithelium maintain its integrity? AB - The esophageal epithelium maintains its integrity by both structural and functional epithelial defenses. Structural components are cell membranes and intercellular junctional complexes. Functional components are cellular defenses against acidification consisting of intracellular buffering by basic proteins and bicarbonate as well as epithelial transport (Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange). Epithelial repair is also a functional component and can be carried out by cell replication and possibly by epithelial restitution. PMID- 7556974 TI - Glyburide crosses the placenta in vivo in pregnant rats. AB - Non-insulin-dependent diabetes mellitus (NIDDM) is normally treated by oral hypoglycaemic agents, but their use is excluded during pregnancy because of their potential teratogenic and hypoglycaemic effects on the fetus. This caveat was recently questioned as glyburide was shown to cross an isolated cotyledon in vitro in insignificant amounts. In the present study, placental transport of glyburide in vivo was examined as an indispensable step towards clinical trials. Tritiated glyburide, C14 albumin or C14-labelled diazepam were injected into 13, 9 and 11 pregnant rats, respectively and the radioactivity was measured thereafter in maternal blood and in whole fetal extracts. The ratios between radioactivity in fetal tissue to that in maternal blood for glyburide (0.535 +/- 0.068) were similar to those of diazepam (0.641 +/- 0.057) which readily crosses the placenta. However, they differed significantly from those for albumin (0.048 +/- 0.0004) which does not cross. Moreover, glyburide in fetal tissue consistently reflected its concentration in maternal blood when measured at consecutive intervals after intravenous injection in the mother. In contrast, albumin in fetal tissue was low at all time points regardless of its levels in maternal blood when measured at different times after injection. These data suggest that glyburide crosses the placenta of pregnant rats and should therefore be considered with caution as a hypoglycaemic agent in the treatment of gestational diabetes. PMID- 7556975 TI - Binding of human, porcine and bovine insulin to insulin receptors from human brain, muscle and adipocytes and to expressed recombinant alternatively spliced insulin receptor isoforms. AB - Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain. PMID- 7556976 TI - Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture. AB - Tumour necrosis factor alpha (TNF alpha) has been found to cause a delipidation of fat cells and a decrease of the adipose tissue mass. In the present study, we tried to elucidate some of the mechanisms responsible for this phenomenon by investigating the action of TNF alpha on specific pathways which are involved in lipid storage. Cultured stromal cells from human adipose tissue were induced to differentiate into adipose cells by exposure to adipogenic factors and subsequently used for studying the effects of TNF alpha on fat cell metabolism. Presence of 5 nmol/l TNF alpha for 24 h resulted in a complete loss of the stimulatory effect of insulin on 2-deoxy-glucose transport. This inhibitory action was paralleled by a decrease of GLUT4 protein and mRNA levels. The amount of cellular GLUT4 protein was reduced by 49 +/- 3% after a 24-h exposure and by 82 +/- 18% after a 72-h exposure to 5 nmol/l TNF alpha. GLUT4 mRNA was almost undetectable after a 24-h incubation with 5 nmol/l TNF alpha. In a similar time dependent manner, TNF alpha dramatically reduced the lipoprotein lipase mRNA content of the cells. Furthermore, incubation of cultured human fat cells with TNF alpha resulted in a marked dose-dependent stimulation of lipolysis, assessed by glycerol release, by up to 400% above controls, which became apparent after a 6-h exposure at the earliest.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556977 TI - Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetics GK rats and its defective augmentation by glucose. AB - In spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of KATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca2+]i in the diabetic condition. At 30 nmol/l Ca2+ (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 mumol/l Ca2+ (maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 mumol/l Ca2+ from permeabilized control islets (p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4 dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca2+ in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca2+]i rise and energy state in response to glucose in diabetic beta cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus. PMID- 7556978 TI - Interleukin-1 beta inhibition of insulin release in rat pancreatic islets: possible involvement of G-proteins in the signal transduction pathway. AB - In vitro exposure of rat pancreatic beta cells to interleukin-1 beta (IL-1 beta) inhibits glucose-stimulated insulin release (2140 +/- 239 and 323 +/- 80 pg.islet 1.h-1 at glucose levels of 16.7 mmol/l in control and IL-1 beta-exposed islets, respectively, n = 7, p < 0.001). Cholera toxin (2 micrograms/ml) or pertussis toxin (0.5 microgram/ml) potentiated, as expected, glucose-induced insulin release in control islets, but, in addition, when added together with IL-1 beta, were able to prevent the IL-1 beta mediated inhibition of glucose-stimulated insulin secretion (2087 +/- 301 and 1662 +/- 173 pg.islet-1.h-1, respectively, p < 0.05 vs islets exposed to IL-1 beta alone). To investigate the mechanism by which the toxins prevent the IL-1 beta effect, we then measured nitrite levels, glucose oxidation and Ca2+ uptake. Nitrite levels in the culture medium were 4.2 +/- 1.4 and 24.0 +/- 5 pmol.islet-1.24 h-1 in control islets and in IL-1 beta exposed islets, respectively (n = 6, p = 0.05). In islets exposed to IL-1 beta and cholera or pertussis toxins, nitrite levels were 9.1 +/- 3 and 12.4 +/- 6 pmol.islet-1.24 h-1, respectively (n = 6, NS vs control islets). Glucose oxidation at 16.7 mmol/l glucose was 31.1 +/- 2.9 pmol.islet-1.120 min-1 in control islets and 16.8 +/- 2.7 pmol.islet-1.120 min-1 in IL-1 beta-treated islets (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556982 TI - Electrophysiological assessment of visual function in newly-diagnosed IDDM patients. AB - Electrophysiological tests (electroretinogram, oscillatory potentials, visual evoked potentials, in the basal condition and after photostress) reveal an abnormal function of the visual system in insulin-dependent diabetic (IDDM) patients. The aim of our work was to assess whether electrophysiological abnormalities in visual function exist in newly-diagnosed diabetic patients free of any fluorangiographic signs of retinopathy. Ten control subjects (age 28.7 +/- 2.44 years) and then IDDM patients (age 25.2 +/- 6.78 years; disease duration 5.3 +/- 3.5 months) in stable metabolic control (HbA1C 7.5 +/- 1.1%) were evaluated. Flash-electroretinograms and oscillatory potentials were similar in both groups. Visual evoked potentials (VEP) recorded under basal conditions showed that P100 latency was significantly increased in the diabetic patients compared to control subjects (p < 0.01), while N75-P100 amplitude was similar in both groups. The recovery time of VEP after photostress was equivalent in diabetic patients and control subjects. The impaired basal VEPs suggest an early involvement of the nervous conduction in the optic nerve. However, the preserved flash electroretinogram and the normal recovery time after photostress indicate that a short disease duration does not induce physiopathological changes in the outer retinal layers or in the macular function. PMID- 7556981 TI - Insertion/deletion polymorphism in the angiotensin-I-converting enzyme gene is associated with coronary heart disease in IDDM patients with diabetic nephropathy. AB - Insulin-dependent diabetic (IDDM) patients with diabetic nephropathy have a highly increased morbidity and mortality from coronary heart disease. An insertion (I) /deletion (D) polymorphism in the angiotensin-I-converting enzyme (ACE) gene has been shown to be associated with coronary heart disease. Therefore, we have investigated the role of this ACE/ID polymorphism in 198 IDDM patients with diabetic nephropathy and 190 normoalbuminuric IDDM patients. The prevalence of myocardial infarction and other coronary heart disease was significantly elevated in patients with nephropathy, 19% (38/198) vs 8% (15/190), p < 0.001. In the nephropathic group 12 of 63 (19%), 23 of 95 (24%), and 3 of 40 (7.5%) patients with the DD, ID and II genotypes, respectively had a history of coronary heart disease, II vs DD and ID, p < 0.05 when compared to nephropathic patients without coronary heart disease. Multiple logistic regression analysis of the risk factors associated with coronary heart disease in univariate analysis revealed that the II genotype acts as an independent protective factor against coronary heart disease, odds ratio II/DD + ID 0.27 (95% confidence interval 0.07 0.97, p < 0.05). There was no difference in genotype or allele frequency (D/I) between patients with and without nephropathy, 0.56/0.44 in both groups, but plasma ACE concentration was elevated in patients with nephropathy 609 (151-1504) ng/ml as compared to patients with normoalbuminuria, 428 (55-1630) ng/ml, p < 0.001. We suggest that ACE/ID polymorphism may influence the frequency of life threatening cardiac complications in IDDM patients suffering from diabetic nephropathy, a condition characterized by increased plasma ACE concentration. PMID- 7556979 TI - Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose. AB - Establishing whether high ambient glucose affects the plasma membrane Na+/H+ exchanger is relevant to understanding the adverse effects of high glucose on cell replication and the mechanisms of the increased exchanger activity encountered in diabetic patients with nephropathy. In 8 primary and 15 first passage isolates of human endothelial cells cultured in 30 mmol/l glucose for 8.7 +/- 2.3 and 15.8 +/- 2.3 days, respectively, we determined Na+/H+ exchanger activity and mRNA levels. Activity was determined by measuring 22Na+ influx in the presence or absence of dimethylamiloride (DMA) after intracellular acidification. We also measured fibronectin mRNA because fibronectin provides signals for cell replication through the Na+/H+ antiporter. Control cells grown in 5 mmol/l glucose showed at morphologic confluency a total Na+ influx (in nmol.mg protein-1.min-1) of 10.1 +/- 3.2 in primary and 11.7 +/- 2.2 in first subculture, which was reduced to 5.3 +/- 0.3 in the presence of DMA. Paired cultures exposed to 30 mmol/l glucose and exhibiting pHi and cell densities identical to controls showed in both primary and first subculture a reduction in total Na+ influx (delta = -0.98 +/- 0.93 nmol.mg protein-1.min-1 p < 0.005) whereas DMA-resistant Na+ influx was identical to that of control. Neither chronic hypertonicity nor acute exposure to high glucose mimicked the effects of chronic high glucose. The level of the Na+/H+ exchanger isoform 1 (NHE-1) mRNA was unchanged by high glucose whereas fibronectin mRNA levels were increased 1.5 fold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556980 TI - Insulin resistance is associated with high plasma ouabain-like immunoreactivity concentration in NIDDM. AB - The aim of the present study was to elucidate the pathophysiologic significance of circulating ouabain as a link between insulin resistance (IR) and hypertension (HT) in NIDDM. Euglycaemic (4.5 mmol/l) hyperinsulinaemic (360-580 pmol/l) clamping was performed using an artificial endocrine pancreas. Plasma ouabain like immunoreactivity (OLI) was determined by radioimmunoassay using a highly specific antibody to ouabain. HT was defined as systolic blood pressure > 140 mm Hg and/or diastolic > 90 mm Hg or being treated with antihypertensive agents. The values (mean +/- SEM) of glucose infusion rate (GIR) and plasma OLI were compared among the four groups classified using IR and HT as factors. Group I (IR-/HT-, n = 15): GIR 7.20 +/- 0.36 mg.kg-1.min-1, OLI 130.8 +/- 20.9 pmol/l, which was not different from that in eight normal control subjects (7.69 +/- 0.40 mg.kg-1.min-1 and 142.6 +/- 32.3 pmol/l, respectively); Group II (IR-/HT+, n = 13): 5.89 +/- 0.36 mg.kg-1.min-1, 172.5 +/- 35.0 pmol/l; Group III (IR+/HT-, n = 14) 1.91 +/- 0.28 mg.kg-1.min-1, 576.6 +/- 161.5 pmol/l (p < 0.01 vs Group I and II); Group IV (IR+/HT+, n = 15) 1.79 +/- 0.22 mg.kg-1.min-1, 703.1 +/- 170.1 pmol/l (p < 0.01 vs Group I and II), respectively. Six of 57 NIDDM patients studied exhibited very high (> 1500 pmol/l) plasma OLI concentrations, showed marked insulin resistance and were all hypertensive. When analysed as a whole, plasma OLI was negatively correlated with GIR (p < 0.001), but was not correlated with arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556983 TI - Mutation in the mitochondrial tRNA(leu) at position 3243 and spontaneous abortions in Japanese women attending a clinic for diabetic pregnancies. AB - Mitochondrial DNA is exclusively maternally inherited. We recently found the prevalence of diabetic patients with an A to G transition at position 3243 of leucine tRNA (3243 base pair (bp) mutation) to be nearly 1% in randomly selected Japanese subjects. Here, we report the higher prevalence of diabetic patients with the 3243 bp mutation in a specific Japanese population of women attending a diabetic pregnancy clinic. Of 102 patients with non-insulin-dependent diabetes mellitus 6 (5.9%) were positive for the mutation, 1 (8.3%) of 12 patients with gestational diabetes and 2 (5.9%) out of 34 borderline diabetic patients. In contrast, none of 64 patients (0%) with insulin-dependent diabetes mellitus had the 3243 bp mutation. Moreover, there was a difference in the prevalence of spontaneous abortions between patients with and without this mutation (27.3 vs 12.4%). Among nine probands with the mutation, four had a history of one spontaneous abortion (p = 0.0518) and two had a history of two abortions (p = 0.0479). Two probands had a spontaneous abortion even while under strict diabetic metabolic control. The 3243 bp mutation thus may cause spontaneous abortion during pregnancy. PMID- 7556984 TI - Islet autoantibody markers in IDDM: risk assessment strategies yielding high sensitivity. AB - Identification of islet autoantigens offers the possibility that antibody tests other than islet cell antibodies may be used for assessing risk of insulin dependent diabetes mellitus (IDDM). The aim of this study was to determine the combination of islet autoantibody markers that could identify most future cases of IDDM. Islet cell antibodies, antibodies to glutamic acid decarboxylase (GAD)65, 37,000/40,000 M(r) islet tryptic fragments, carboxypeptidase-H, and islet cell autoantigen (ICA)69 were measured in sera from 100 newly-diagnosed IDDM patients, 27 individuals prior to onset of IDDM, and 83 control subjects. Islet cell antibodies were detected in 88% of IDDM patients and 81% with pre IDDM, GAD65 antibodies in 70% of IDDM patients and 89% with pre-IDDM, and antibodies to 37,000/40,000 M(r) islet tryptic fragments in 54% of IDDM patients and in 48% with pre-IDDM. The latter were found only in conjunction with islet cell antibodies and were more frequent in young onset cases. All 20 IDDM patients and the 3 pre-IDDM subjects who had islet cell antibodies without GAD65 antibodies had antibodies to 37,000/40,000 M(r) islet tryptic fragments, and all but one had disease onset before age 15 years. No sera strongly immunoprecipitated in vitro translated ICA69 or carboxypeptidase-H; 4% of patients had anti-ICA69 and 11% anti-carboxypeptidase-H levels above those of the control subjects. The findings suggest that none of the single antibody specificities are as sensitive as islet cell antibodies, but that a combination of GAD65 antibodies and antibodies to 37,000/40,000 M(r) islet tryptic fragments has the potential to identify more than 90% of future cases of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556986 TI - Estimates of Krebs cycle activity and contributions of gluconeogenesis to hepatic glucose production in fasting healthy subjects and IDDM patients. AB - Normal subjects, fasted 60 h, and patients with insulin-dependent diabetes mellitus (IDDM), withdrawn from insulin and fasted overnight, were given phenylacetate orally and intravenously infused with [3-14C]lactate and 13C bicarbonate. Rates of hepatic gluconeogenesis relative to Krebs cycle rates were estimated from the 14C distribution in glutamate from urinary phenylacetylglutamine. Assuming the 13C enrichment of breath CO2 was that of the CO2 fixed by pyruvate, the enrichment to be expected in blood glucose, if all hepatic glucose production had been by gluconeogenesis, was then estimated. That estimate was compared with the actual enrichment in blood glucose, yielding the fraction of glucose production due to gluconeogenesis. Relative rates were similar in the 60-h fasted healthy subjects and the diabetic patients. Conversion of oxaloacetate to phosphoenolpyruvate was two to eight times Krebs cycle flux and decarboxylation of pyruvate to acetyl-CoA, oxidized in the cycle, was less than one-30th the fixation by pyruvate of CO2. Thus, in estimating the contribution of a gluconeogenic substrate to glucose production by measuring the incorporation of label from the labelled substrate into glucose, dilution of label at the level of oxaloacetate is relatively small. Pyruvate cycling was as much as one-half the rate of conversion of pyruvate to oxaloacetate. Glucose and glutamate carbons were derived from oxaloacetate formed by similar pathways if not from a common pool. In the 60-h fasted subjects, over 80% of glucose production was via gluconeogenesis. In the diabetic subjects the percentages averaged about 45%. PMID- 7556985 TI - Variation by age group and seasonality at diagnosis of childhood IDDM in Europe. The EURODIAB ACE Study Group. AB - Recent data provided by the EURODIAB ACE study group have confirmed wide variation in the incidence of insulin-dependent diabetes mellitus (IDDM) across Europe. The aim of this report is to compare age-specific incidence and seasonality at clinical onset of IDDM between study regions. Using a uniform methodology, the EURODIAB ACE framework ascertained 3,168 newly-diagnosed cases of IDDM in children under the age of 15 years during 1989-1990. Eighteen percent of the cases were age 0-4 years at diagnosis, 34% were age 5-9 years and 48% were age 10-14 years. Poisson regression analysis suggested that there were highly significant statistical differences in incidence between the three age groups and between the 24 regions. Although incidence rates in the 0-4 year and 5-9 year age groups varied from region to region in a similar fashion, the pattern of variation in the older age group was different. Seasonality of diagnosis conformed to a sinusoidal model with a peak occurring in winter, a feature which was consistently observed in both sexes and in all age groups. However, a statistically significant heterogeneity in the seasonal distribution was present among regions, those in Scandinavia showing the smallest relative amplitude. The first insulin injection was given the same day or the day after diagnosis in 93% of the cases for whom data were available. PMID- 7556987 TI - Hypertension and overweight associated with hyperinsulinaemia and glucose tolerance: a longitudinal study of the Finnish and Dutch cohorts of the Seven Countries Study. AB - To elucidate the role of hypertension as part of the insulin resistance syndrome, the longitudinal relationships of hypertension and overweight with hyperinsulinaemia and glucose tolerance were examined in the Dutch and Finnish cohorts of the Seven Countries Study (Zutphen, and east and west Finland). Three cohorts of men, born between 1900 and 1919, were first examined in 1959/1960. At the 30-year follow-up survey a 2-h glucose tolerance test was carried out on 619 of the surviving men, and fasting insulin was also measured. Blood pressure and body mass index (BMI) were measured several times during the entire 30-year follow-up period. In cross-sectional analyses, men with diabetes and impaired glucose tolerance at the 30-year follow-up examination had a significantly higher systolic blood pressure and a higher prevalence of hypertension than men with normal glucose tolerance, independent of age, cohort and BMI (p < 0.01). These differences had already been seen 5, 20 and 30 years earlier. Subjects with hyperinsulinaemia (fasting insulin > or = 9.2 mU/l) had a higher BMI and a higher prevalence of hypertension. This cross-sectional association with hypertension was independent of age, cohort and BMI. BMI levels of men with hyperinsulinaemia had been shown to be higher 5, 20 and 30 years earlier, but blood pressure levels had not. These results indicate that hypertension is independently associated with glucose tolerance and insulin resistance in three Caucasian cohorts. Changes in blood pressure precede abnormal glucose tolerance but not hyperinsulinaemia; therefore, glucose tolerance appears to be a stronger correlate of hypertension than hyperinsulinaemia. PMID- 7556988 TI - Double blind trial of nicotinamide in recent-onset IDDM (the IMDIAB III study). AB - Nicotinamide has been recently introduced, in addition to intensive insulin therapy for patients with recent-onset insulin-dependent diabetes mellitus (IDDM) to protect beta cells from end-stage destruction. However, available data are conflicting. A double blind trial in 56 newly-diagnosed IDDM patients receiving nicotinamide for 12 months at a dose of 25 mg/kg body weight or placebo was designed in order to determine whether this treatment could improve the integrated parameters of metabolic control (insulin dose, glycated haemoglobin and C-peptide secretion) in the year after diagnosis. In addition to nicotinamide or placebo, patients received three to four insulin injections daily to optimize blood glucose levels. Patients treated with nicotinamide or placebo received similar doses of insulin during follow-up and 1 year after diagnosis with comparable glycated haemoglobin levels 6.7 +/- 1.8% nicotinamide vs 7.1 +/- 0.6% placebo). Basal and glucagon stimulated C-peptide secretion detectable at diagnosis were similarly preserved in the course of 12 months follow-up both in nicotinamide and placebo treated patients. No adverse effects were observed in patients receiving nicotinamide. When age at diagnosis was taken into account, nicotinamide treated older patients ( > 15 years of age) showed significantly higher stimulated C-peptide secretion than placebo treated patients (p < 0.02). These results suggest that nicotinamide can preserve and improve stimulated beta cell function only in patients diagnosed after puberty.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556991 TI - Failure of GLP-1(7-36)amide to affect glycogenesis in rat skeletal muscle. AB - Glucagon-like peptide-1(7-36)amide has been described as exerting potent glycogenic action and as stimulating glycolysis in skeletal muscle. We exposed isolated rat soleus muscle strips to various concentrations of glucagon-like peptide-1(7-36) amide (10(-11) - 10(-6) mol/l) or insulin (10(-10) - 10(-7) mol/l) and determined the respective effects on glucose metabolism. Insulin markedly increased the rate of glucose incorporation into glycogen with a maximal effect at 10(-8) mol/l insulin (348 +/- 46% of intraindividual control experiment, p < 0.005), while glucagon-like peptide-1(7-36)amide was without an effect (e.g. 10(-11) mol/l, 96 +/- 10%; 10(-9) mol/l, 104 +/- 9%; 10(-7) mol/l, 121 +/- 13%; not significant). Likewise, glucagon-like peptide-1(7-36)amide did not affect the rate of 3H-2-deoxy-glucose transport or glycogen content of soleus muscle strips. The rates of aerobic or anaerobic glycolysis were also not increased. The findings were independent of peptide source and of employed muscle size. Our results do not suggest any effect of glucagon-like peptide-1(7-36)amide on skeletal muscle glucose metabolism and, hence, are in contrast to data derived from similar experiments by others. PMID- 7556989 TI - A comparison of the effects of low- and conventional-dose thiazide diuretic on insulin action in hypertensive patients with NIDDM. AB - In conventional doses, thiazide diuretics impair glucose tolerance and decrease insulin sensitivity, making them an unpopular choice for treating diabetic patients with hypertension. However, use of low-dose thiazide diuretics may avoid the adverse metabolic effects seen with conventional doses. In a double-blind, randomised crossover study we assessed peripheral and hepatic insulin action in 13 hypertensive non-insulin-dependent diabetic patients after a 6-week placebo run-in and following two 12-week treatment periods with either low (1.25 mg) or conventional (5.0mg) dose bendrofluazide. There were no differences between doses in their effects on systolic and diastolic blood pressure. Bendrofluazide 1.25 mg had significantly less effect on serum potassium, uric acid, fasting glucose and HbA1C concentrations than the 5.00 mg dose. Exogenous glucose infusion rates required to maintain euglycaemia were significantly different between doses (p < 0.05) with conventional-dose bendrofluazide worsening peripheral insulin resistance compared to baseline (23.8 +/- 2.9 vs 27.3 +/- 3.5 mumol.kg-1.min-1, p < 0.05) and low-dose bendrofluazide producing no change compared to baseline (26.8 +/- 3.6 vs 27.3 +/- 3.5 mumol.kg-1.min-1, p = NS). Postabsorptive endogenous glucose production was higher on treatment with bendrofluazide 5.0 mg compared to 1.25 mg (11.7 +/- 0.5 vs 10.2 +/- 0.3 mumol.kg-1.min-1, p < 0.05) and suppressed to a lesser extent following insulin (4.0 +/- 0.7 vs 2.0 +/- 0.4 mumol.kg-1.min-1, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7556992 TI - Mitochondrial DNA, diabetes and pancreatic pathology in Kearns-Sayre syndrome. AB - Mitochondrial DNA (mtDNA) mutations are associated with diabetes mellitus but their role in the onset of hyperglycaemia is unclear. A patient presented with diabetes requiring insulin therapy at the age of 7 years, followed by diagnosis of Kearns-Sayre syndrome (KSS). Beta-cell function was absent at age 19 years as shown by lack of glucose-stimulated C-peptide secretion. Following development of a cardiac conduction defect the patient died aged 21 years. Analysis of mtDNA in blood and several tissues revealed related re-arranged deletions, duplications and deletion dimers in addition to normal mtDNA with the highest levels of duplications in kidney and blood. Pancreatic tissue from the KSS patient was compared with tissue from an insulin-dependent diabetic patient with a similar clinical history of diabetes. Islets in KSS were small, regular in shape and contained predominantly glucagon-containing cells with no evidence of beta cells. In comparison, a small number of beta cells were present in some of the larger more irregularly-shaped islets from the insulin-dependent diabetic patient. These data together suggest that in KSS the loss of beta cells at the onset of diabetes is less disruptive to islet architecture: a small proportion of beta cells or their gradual destruction over a long period would allow retention of islet shape. Abnormal function of the re-arranged mtDNA could affect both development and function of pancreatic islet cells since glucose-stimulated insulin secretion is energy dependent. PMID- 7556990 TI - Role of syntaxin in mouse pancreatic beta cells. AB - The role of syntaxin 1, a protein involved in the docking of synaptic vesicles at presynaptic active zones, has been investigated in pancreatic islet cells. Using two different monoclonal antibodies we have shown that syntaxin 1 is present in the pancreatic islet cell microsomal fraction. Furthermore, functional experiments demonstrate that anti-syntaxin antibodies inhibit CA(2+)-dependent insulin secretion in permeabilized islet cells. These data indicate that syntaxin 1 is present in the pancreatic beta cell and it is likely to play a functional role in the exocytosis of secretory granules. PMID- 7556993 TI - Vascular factors in diabetic neuropathy: comment. PMID- 7556997 TI - 31st Annual meeting of the European Association for the Study of Diabetes. Stockholm, Sweden, 12-16 September 1995. Abstracts. PMID- 7556995 TI - Shared amino acid sequences between glutamic acid decarboxylase 65 and 67 and alpha-2-macroglobulin. A focus for cross-reactive autoantibodies? PMID- 7556994 TI - The cumulative incidence of childhood diabetes mellitus in Sweden unaffected by BCG-vaccination. PMID- 7556996 TI - Absence of effect of culture duration on glucose-activated alterations in intracellular calcium concentration in mouse pancreatic islets. PMID- 7556998 TI - Family-centered birthing. PMID- 7556999 TI - More precise definitions needed? PMID- 7557000 TI - Specialists teaching generalists. PMID- 7557001 TI - The need to teach chaos theory. PMID- 7557002 TI - Mexican family physicians. PMID- 7557003 TI - Treating an HIV-positive male patient. PMID- 7557005 TI - Enhancing community support for family practice residencies. AB - BACKGROUND: The self-interest of employers, insurers, and the public in maintaining a healthy workforce and containing health care costs can be tied directly to primary care. Therefore, primary care training programs have a sound basis on which to solicit financial, political, and moral support from employers, insurers, and the public. METHODS: Our residency program director invited community and business leaders interested in the supply of primary care physicians to join a newly formed advisory board. The work of the advisory board has focused on four areas: public relations, political influence, recruiting, and fund-raising and has developed creative ways to generate political and financial support for the residency. RESULTS: During the board's 3 1/2 year life span, it has influenced political decisions affecting the residency and generated more than $100,000 in support for the program. CONCLUSIONS: Soliciting local nonmedical leaders to participate on an advisory board could bring important benefits to any residency. Those community leaders who directly or indirectly benefit from a residency program should be recruited into a formal support network. PMID- 7557006 TI - The value of a residency. PMID- 7557004 TI - Economic analysis of family practice residency programs: a report from the Northeastern Ohio Network. AB - BACKGROUND: The financial impact of a family practice residency program on a sponsoring institution is poorly understood. This study intended to describe as fully as possible all the expenses and revenues from five community-based family practice residency programs in northeastern Ohio. METHODS: Direct and indirect expenses, revenues, and demographics were evaluated for 1992. Similarities and differences among the participating programs were examined. RESULTS: Overall expenses per resident were similar in all five programs, with a range from $162,000 to $203,000. Revenues reflected the number of residents in the program, although collection ratios varied. Inpatient collections ranged from 53%-76% and outpatient collections ranged from 60%-76%. An average of 30% of graduates from the past 10 years were on the active medical staff of their sponsoring institution, with a range of 21%-36%. CONCLUSION: Based on the expenses, revenues collected, and reasonable assumptions made about cost of care in the hospital setting, the family practice residencies are probably a break-even operation, excluding the benefit of providing primary care physicians to the community. PMID- 7557007 TI - Family medicine interest groups at US medical schools. AB - BACKGROUND AND OBJECTIVES: Family medicine interest groups (FMIGs) are student organizations that help students learn about family practice and foster interest in the specialty. To date, their activity has not been studied. The goal of this study was to characterize FMIG activity in US medical schools and FMIG relationship to specialty selection. METHODS: A questionnaire was mailed to all US medical school family medicine predoctoral directors. Results were correlated with specialty selection data. RESULTS: Of the 99 campuses that responded, 88 had FMIGs. Participation by students in FMIGs has increased from 1,944 in 1987 to 6,248 in 1992 (P < .001). Also, participation in FMIGs at medical schools is positively correlated with selection of family practice by graduating seniors (r = .33). DISCUSSION: FMIGs are increasingly popular and may lead to increased student interest in family practice. FMIGs are weakly correlated with increased selection of family practice by graduating seniors in a simple bivariate analysis, but this association does not necessarily imply a causal relationship. Also, this study is limited by its basis on a retrospective survey. PMID- 7557010 TI - Using ICPC in a computer-based primary care information system. AB - INTRODUCTION: Although the framework of the episode of care offers much promise in examining the process of primary health care, the development of episode oriented, automated data sources has proceeded slowly. Experience with the International Classification of Primary Care (ICPC) in the European Community has confirmed its usefulness in creating and analyzing episodes of care, but it has seen little use in the United States. We describe the development of a Primary Care Information System (PCIS), which employs ICPC to create episodes of care from routinely collected clinical data. METHODS: The PCIS is a partially computerized medical information system running on a standard Macintosh microcomputer. The PCIS integrates ICPC, ICD-9-CM, and CPT-4 coding structures to provide episode-oriented data for clinical, administrative, research, and reimbursement needs. The performance of the PCIS was assessed based on five major issues: clinician cooperation, data-entry accuracy, validity of episode data, cost, and perceived value to users. RESULTS: The data collection and entry process required minimal additional effort from clinicians and data-entry personnel, and data-entry accuracy exceeded published estimates for other primary care data sources. Data management costs of about $1 per encounter compare favorably to published estimates for other office-based clinical information systems. The major problem seen during pilot testing was inaccurate tracking of episode boundaries through changes in providers and consequent changes in labels for problems and diagnoses. This problem has been addressed in development of the second-generation PCIS. CONCLUSION: The combination of an episode-oriented framework such as ICPC and a flexible medical information system provides a promising platform for the study of the content and process of primary health care. PMID- 7557008 TI - Gender concordance between family practice residents and diagnoses in an ambulatory setting. AB - BACKGROUND: Female physicians are more likely than male physicians to treat female patients. This study extends the scope of previous studies by scrutinizing gender concordance with specific diagnoses. METHODS: A total of 90,407 physician patient encounters handled by 90 family practice residents from 1984-1993 in an ambulatory care setting were analyzed. In addition to genitourinary problems, the analysis included the 20 most frequently recorded diagnoses. RESULTS: Patients' encounters with residents differed between male and female residents. Female residents handled more encounters (P < .001) for vaginitis, prenatal care, menstrual disorders, menopausal symptoms, cervicitis, abnormal Pap smear, breast disease, breast lump, and general medical exam (female patient). Female residents handled a greater percentage (P < .001) of female patients within the 20 most frequent diagnoses. There was little difference with male patients. CONCLUSIONS: Gender concordance is suggested between male and female patients with specific diagnoses and their physicians. Educators must balance patient preferences and satisfaction with adequate exposure to the gamut of medical problems. PMID- 7557009 TI - The doctor, the patient: a first-year course for personal and professional growth. AB - BACKGROUND AND OBJECTIVES: Medical education has tended to focus primarily on the biomedical aspects of care while neglecting the human side of patients and students. To help students become aware of student, patient, and family issues that may impact the doctor-patient relationship, a first-year course was developed. METHODS: This course consists of 12 didactic sessions and 10 small group meetings led by family medicine faculty. Topics include self-assessments, analysis of videotaped doctor-patient interactions, genogram development, and family systems. Students have evaluated the course for content, strengths, weaknesses, and impact. RESULTS: This course was highly rated by the students. The students reported gaining insight into personal issues that might impact their interaction with patients. Students also became aware of the importance of family issues to their patients' health and well-being. CONCLUSIONS: Through courses like this, students may gain deeper insight into issues that impact them, their patients, and the doctor-patient relationship. PMID- 7557011 TI - Accuracy of on-site data entry in a rural primary care research network. AB - OBJECTIVE: This study examined how accurately research data can be entered into a database by rural research network members with little computer training and no data-entry experience. METHODS: A rural primary care research network in the Upper Peninsula of Michigan collected data from a study, and each practice entered its data on a standard database. The data were then sent electronically to the main research center where they were downloaded and analyzed. Accuracy of data entry was evaluated by comparing data received at the center with data on the original questionnaire. RESULTS: Accuracy of the data, entered by practice coordinators (nurses) with little or no computer experience, was completed with an error rate of one error per 220 keystrokes or .22 errors per 1,000 keystrokes. CONCLUSIONS: Results indicate that remote data entry in research networks can be accomplished accurately and reliably with little training. PMID- 7557013 TI - Breaking through. PMID- 7557012 TI - Managing patients. PMID- 7557017 TI - The biochemistry of programmed cell death. AB - Programmed cell death (PCD) is involved in the removal of superfluous and damaged cells in most organ systems. The induction phase of PCD or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD inducing stimuli converge into a few stereotypical pathways and cells pass a point of no return, thus becoming irreversibly committed to death. It is only during the successive degradation phase that vital structures and functions are destroyed, giving rise to the full-blown phenotype of PCD. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations commonly considered to define PCD (apoptotic morphology of the nucleus and regular, oligonucleosomal chromatin fragmentation) have to be ascribed to the late degradation phase. The decision as to whether a cell will undergo PCD or not may be expected to be regulated by "switches" that, once activated, trigger self-amplificatory metabolic pathways. One of these switches may reside in a perturbation of mitochondrial function. Thus, a decrease in mitochondrial transmembrane potential, followed by mitochondrial uncoupling and generation of reactive oxygen species, precedes nuclear alterations. It appears that molecules that participate in apoptotic decision-making also exert functions that are vital for normal cell proliferation and intermediate metabolism. PMID- 7557015 TI - How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase. AB - The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved. PMID- 7557016 TI - Mechanism and structure of thioredoxin reductase from Escherichia coli. AB - The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH. Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase. The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data. In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both. The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear. To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain. The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer. Reverse rotation allows reduction of the redox active disulfide by the reduced flavin. This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed. PMID- 7557018 TI - Oligonucleotide-mediated modulation of mammalian gene expression. AB - The notion that oligonucleotides can modulate gene-specific expression was established more than a decade ago. Recent advances in molecular genetics have broadened the armamentarium used to manipulate gene expression in biological systems including triplex DNA, antisense RNA/DNA, and ribozymes (catalytic RNA). These oligonucleotides demonstrated important early application to the elucidation of cellular signaling pathways. More recently, studies with these agents have probed their utility as potential therapeutic agents, especially in the realm of cancer. With the implementation of gene therapy in early clinical trials, oligonucleotide-mediated suppression of gene expression has emerged as an important strategy for gene therapy. This review will discuss the current knowledge in this field, focusing on the biology of triplex DNA, antisense oligonucleotides, and ribozymes. PMID- 7557014 TI - Serendipity, science, and a new hantavirus. AB - This essay follows a team of scientific investigators step by intriguing step as it pursues the cause of the mysterious 1993 deaths of healthy young adults in the southwestern United States. Using the science of the day, the team unravels the elusive origin of a potentially widespread killer--tracking a new hantavirus to its home, tracing its lineage, and differentiating its DNA from the large hantavirus family. This is the first in the "Breakthroughs" series. PMID- 7557020 TI - The purification of membranes by affinity partitioning. AB - We describe affinity partitioning as a preparative method for membranes and membraneous structures such as organelles, cells, and viruses. Biospecific affinity partitioning is carried out in aqueous polymer two-phase systems, commonly with polyethylene glycol and dextran as phase polymers, in an environment compatible with membrane structures. Ideally, two-phase conditions are chosen to partition the bulk of membrane material into one phase, while the affinity ligand, conjugated to the second phase polymer, will selectively pull the membranes to be isolated into this phase. Suitable ligands include lectins, antibodies, and receptor-specific agents. Because the method has so far been used successfully in rather few instances, all using high ligand receptor densities in target membranes, the discussion focuses on factors to be considered when developing affinity partitioning conditions using new ligands. PMID- 7557019 TI - Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers. AB - Various external stimuli regulate cellular Ca2+ fluxes. Information from these stimuli is transmitted to the cell interior by signal transducing systems that involve interactions between receptors, G-proteins, and enzymes that generate second messengers. The possible mechanisms by which "cross-talk" within and between these second messenger-generating systems provides an intricate molecular avenue for fine modulation of intracellular Ca2+ are reviewed. PMID- 7557021 TI - Ras target proteins in eukaryotic cells. AB - The 21 kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. Less well understood are the downstream signal transduction cascades initiated by Ras in response to external stimuli. Only recently have many diverse studies in lower eukaryotes and vertebrates converged to demonstrate that Ras directly regulates multiple signaling pathways. In most eukaryotes, Ras functions as a positive regulator of an ERK/MAPK signal transduction cascade through the activation of a MEKK. In mammalian cells the primary Ras-responsive MEKK is the protein kinase Raf. Although Raf remains the most significant mediator of Ras signaling in most model systems, it does not explain all the biochemical responses observed in cells with activated Ras proteins. Yeast two hybrid and GST-fusion protein binding studies have identified new proteins distinct from Raf that could interact with Ras in other signaling pathways. In addition to Raf, other potential Ras target proteins include MEKK1, PI(3)K, p120GAP, ralGDS, and PKC zeta. This review will attempt to summarize the current literature of accepted and potential Ras-dependent signaling proteins in both lower eukaryotes and vertebrates. PMID- 7557022 TI - Molecular mechanisms and therapeutic strategies related to nitric oxide. AB - The formation of nitric oxide (NO) from L-arginine is now recognized as a ubiquitous biochemical pathway involved in the regulation of the cardiovascular, central, and peripheral nervous systems, as well as in other homeostatic mechanisms. The L-arginine:NO pathway comprises a substrate, L-arginine, a family of enzymes, the NO synthases, and at least one physiological effector system, the soluble guanylate cyclase. NO also inhibits enzymes in target cells and can interact with oxygen-derived radicals to produce other toxic substances. Thus, NO also plays a role in immunological host defense and in the pathophysiology of certain clinical conditions. Several steps in the L-arginine:NO pathway are amenable to manipulation. Some substances will change the concentration and/or actions of NO with consequences that, in certain cases, may be therapeutic. In addition, other agents themselves generate NO and thus mimic the actions of the endogenous mediator. This brief overview will discuss some possible interventions in the pathway and the potential benefits as well as undesirable side effects that might arise from them. PMID- 7557023 TI - High-affinity binding of melatonin by human circulating T lymphocytes (CD4+). AB - This paper shows the presence of high-affinity binding sites for melatonin in human circulating T lymphocytes, but not in B lymphocytes. The binding of melatonin to T cells was dependent on time, stable, reversible, saturable, specific, and inversely correlated to the production of melatonin, expressed as the nocturnal 12 h production of its urinary metabolite 6-sulfatoxymelatonin. The affinity of these binding sites (Kd = 0.27 nM) suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, among the lymphocyte subpopulations studied, binding of melatonin was mostly found in CD4+ cells rather than in CD8+ cells. Results suggest that CD4+ cells may be the target of melatonin among the human circulating lymphocytes. PMID- 7557025 TI - Trolox inhibits apoptosis in irradiated MOLT-4 lymphocytes. AB - MOLT-4 cells, a human lymphocytic leukemia line, undergo apoptosis in response to a variety of stimuli, including exposure to ionizing radiation. Very little is known of the molecular mechanisms by which radiation induces apoptosis. Morphology changes and chromatin cleavage at internucleosomal sites accompany apoptosis in these cells. We found that trolox, a water-soluble derivative of vitamin E that penetrates biomembranes and protects mammalian cells from oxidative damage, blocks DNA fragmentation in irradiated MOLT-4 cells. Levels of DNA fragmentation in cells not treated with trolox were directly related to both radiation dose and time postirradiation. Preincubation of cells with trolox or incubation with trolox only during irradiation did not protect cells. A 4 h postirradiation incubation with trolox was sufficient to completely block fragmentation measured at 24 h, indicating the processes triggered by radiation to induce DNA fragmentation occur early after irradiation. Removal of cells from trolox earlier than 4 h resulted in progressively less inhibition. Trolox preserves the integrity of irradiated cells as judged by increased viability and thymidine incorporation. Radiation induces an uptake of extracellular Ca2+ into MOLT-4 cells that was blocked by a postirradiation incubation with trolox. These results suggest that membrane-associated oxidations triggered by radiation are responsible for radiation-induced apoptosis in MOLT-4 cells. PMID- 7557024 TI - Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat. AB - Restenosis after angioplasty is believed to result from stimulation of smooth muscle cells (SMC) by various growth-promoting factors as a consequence of endothelial injury. In this study we have tested the hypothesis that insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) interaction is a rate-limiting step for SMC replication by blocking this interaction with a synthetic D-amino acid peptide structurally resembling the D-domain of IGF-1. After rat carotid artery denudation, semiquantitative PCR analysis demonstrated a significant elevation of IGF-1, platelet-derived growth factor B, transforming growth factor beta 1, and epidermal growth factor mRNAs 10 days after endothelial injury, concomitantly with the induction of intimal SMC proliferation and intimal thickening. Administration of 10-30 micrograms.kg-1.day-1 of D-analog of IGF-1, devoid of proteolytic degradation in body fluids, reduced intimal SMC replication by 60-70%. The peptide also inhibited [3H]TdR incorporation and [3H]glycine incorporation in cultured SMCs by 60-80%, whereas a "scrambled" control peptide consisting of the same amino acids had no effect. The results suggest that IGF 1/IGF-1R interaction is a rate-limiting step for SMC replication. Blocking of this interaction with stabile D-peptide analog of IGF-1 at the level of IGF-1R may offer an entirely new approach for the prophylaxis and treatment of restenosis after cardiac revascularization procedures. PMID- 7557026 TI - The gerontogenes age-1 and daf-2 determine metabolic rate potential in aging Caenorhabditis elegans. AB - Mutations in the genes age-1 and daf-2 extend life span of Caenorhabditis elegans by 100 and 200%, respectively, in axenic culture. Adult worms that are mutant in either of these genes have higher metabolic capacities, called metabolic rate potentials, at all ages and the extension of their life expectancies are positively correlated with the increases of metabolic rate potential. The activities of catalase, superoxide dismutase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase are all higher relative to those in worms that are wild type for these genes, but acid phosphatase is down-regulated and alkaline phosphatase activity is lowered to 10% of the activity measured in age 1(+) and daf-2(+) worms. These results suggest that genes that regulate metabolic activity may play central roles in longevity and senescence. PMID- 7557027 TI - Deleted in oral cancer-1 (doc-1), a novel oral tumor suppressor gene. AB - We have identified, isolated, and partially characterized doc-1, a novel cDNA sequence whose activity is consistent with a suppressor of hamster oral carcinogenesis. Doc-1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in malignant hamster oral keratinocytes. The full-length doc-1 cDNA encodes an 87 amino acid product that shows a significant homology to one of the seven novel genes induced in mouse fibroblasts by TNF-alpha. Transfection of the full-length doc-1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage-independent growth, suggesting reversion of transformation phenotypes. We propose that doc-1 is a novel tumor suppressor gene in oral cancer development. PMID- 7557029 TI - Cholesterol homeostasis: role of the LDL receptor. PMID- 7557031 TI - [Clinical usefulness of transesophageal echocardiography in heart surgery complications]. AB - The role of monoplane transesophageal echocardiography (TEE) in rapid decision making process was investigated in 115 critically ill patients (pts) with early postoperative complications after cardio-thoracic surgery (hypotension, central venous pressure and/or wedge pressure elevation, electrocardiographic S-T segment elevation). Systolic and diastolic function of left ventricle, left ventricular wall motion abnormalities, right ventricular function, valves or prosthetic valves function, left ventricular outflow tract and morphologic changes were evaluated. Echocardiographic diagnoses were classified as: useful, incomplete, not diagnostic, misleading, unexpected. Echocardiographic diagnoses were confirmed by surgical or pathologic findings in all patients operated or dead. All but one patients, who needed surgical therapy, were operated on the basis of echo-diagnosis alone. Therapeutic changes induced by echo-diagnosis were evaluated and classified as major and minor. Diagnosis was fast (7 +/- 2 m) and sure (no complication). TEE was useful in 91% of cases (105/115 pts), incomplete in 2.3% (3/115 pts), not diagnostic in 2.3% (3/115 pts) and misleading in 3.4% of cases (4/115 pts). TEE findings made major therapeutic changes necessary in 66.9% (77/115 pts); there was a shift from medical to surgical therapy in 28% (41/115 pts); in 14.7% (17/115 pts) minor changes in drug therapy were made. TEE was also useful in quick and safe placement of devices (Swan-Ganz catheter, intra aortic balloon pump, endocardial pace maker, ventricular assist device) and in guiding urgent pericardiocentesis. The effects of medical therapy and evolution of ventricular dysfunction were well monitored by TEE. In our experience TEE was a very useful tool for management of early complications after cardio-thoracic surgery. PMID- 7557030 TI - [Coronary angioplasty in chronic total occlusion: angiography results, complications, and predictive factors]. AB - The results of coronary angioplasty of chronic total occlusions are still controversial. We examined baseline clinical and angiographic parameters and the success and complication rates of 142 consecutive procedures of coronary angioplasty of chronic occlusions selected among 1084 total procedures performed between January 1989 and May 1993. Procedural success was obtained in 109 procedures (77%) with 4 major cardiac complications (2.8%) consisting in one cardiac death caused by left main artery occlusion following an attempt of dilatation of an occluded left anterior descending artery, two non Q wave myocardial infarctions caused by distal embolization and one cardiac tamponade following artery perforation, successfully treated by pericardiocentesis. Extensive coronary dissection occurred in 24 procedures (17%): 5 caused vessel reocclusion and 19 were successfully treated by prolonged balloon inflation and/or use of long balloons or stent implantation. Among 15 baseline clinical and angiographic variables, none was predictive of cardiac complications or coronary artery dissection. In contrast, procedural success was significantly related with absence of bridging collaterals, duration of occlusion < 3 months and a tapered or thrombotic morphology of occlusion. Presence or absence of collaterals was the most significant variable selected by multivariate discriminant analysis. When patients with bridging collaterals were excluded from the analysis, occlusion morphology was the most important determinant. Success rates were 95% and 82% in patients with a tapered morphology lesion with < 3 months and > 3 months duration, respectively (ns); in contrast, success rates were 79% and 36% in patients with abrupt occlusion morphology of < and > 3 months duration (p = 0.001). PMID- 7557033 TI - [Estimate of lung capillary pressure and identification of the "pseudonormal" transmitral flow pattern in patients with severe systolic dysfunction of the left ventricle]. AB - OBJECTIVES: Aim of this study was to investigate whether Doppler-derived transmitral flow velocity variables would allow a reliable estimate of pulmonary capillary wedge pressure in patients with left ventricular systolic dysfunction and normal mitral flow velocity pattern, and would then predict the presence of a "pseudonormal" transmitral flow velocity profile. BACKGROUND: Abnormal relaxation and increased chamber stiffness have opposing effects on the left ventricular filling pattern. When both abnormalities are present at the same time, as often occurs in patients with systolic dysfunction, the mitral pattern may appear to be normal: in this case the ability of Doppler recording to assess diastolic function and predict left ventricular filling pressure may be significantly compromised. METHOD: Pulmonary capillary wedge pressure (PWP) and Doppler transmitral flow velocity profile were simultaneously recorded in 70 postinfarction patients with ejection fraction < or = 35% and mitral flow velocity pattern apparently normal (E/A ratio between 1 and 2). Doppler traces were analyzed using a microcomputer-based digitizing system, and the following variables were measured: peak flow velocity in early diastole (E) and during atrial contraction (A), peak E/A wave velocity ratio and the deceleration time (Dec T) of early diastolic flow. RESULTS: Mean E/A ratio was 1.3 +/- 0.2 and mean PWP was 17 +/- 7 mm Hg. Forty-two patients had a PWP < 20 mm Hg and the remaining 28 patients (40%) had PWP > or = 20 mm Hg. Peak E, peak A and E/A ratio were similar in the two groups, whereas Dec T was significantly (p < 0.001) lower in patients with > or = 20 mm Hg PWP (93 +/- 15 vs 146 +/- 19 msec). A very weak correlation was found between PWP and both E and A velocity (r = 0.14 and r = 0.09, respectively). Furthermore, no correlation was found between E/A ratio and PWP (r = 0.08). Conversely, a very close negative correlation was observed between Dec T of early filling and PWP (r = -0.96). Sensitivity and specificity of < or = 120 ms in Dec T in predicting > or = 20 mm Hg in PWP were 100% and 99%, respectively. CONCLUSIONS: These data point out the relevant role of Doppler derived deceleration time of early diastole in predicting left ventricular filling pressure. In patients with left ventricular systolic dysfunction an apparently normal diastolic filling pattern is often associated with elevated PWP. Through the estimate of increased PWP, early deceleration time seems to provide an important means of differentiating a real normal from a pseudonormal or masked abnormal filling pattern caused by increased left ventricular filling pressure. PMID- 7557032 TI - [Inverse relationship between fetal growth and arterial pressure in children and adults]. AB - BACKGROUND: Increased prevalence of hypertension, ischaemic heart disease and stroke has been reported in subjects with impaired growth during fetal life and infancy. Blood pressure could mediate this relation. Indeed, reduced growth in fetal life and infancy has been associated with a raised blood pressure in children and adults. However, there is controversy about the relative importance of intrauterine environment and extrauterine adverse environment which can act throughout the life course. We therefore studied the relation between birth weight, which is known to be an indicator of fetal growth, and blood pressure in children and their parents. This association could thus be assessed in childhood before the external environmental influences became important, and in adulthood. METHODS: Seven hundred and fifteen healthy schoolchildren (379 boys) aged 3-12 years from primary schools, and 448 parents (252 women) aged 20-44 years, born at term, without hypertension or diabetes, were studied. Blood pressure and birth weight were measured. Birth weight was taken from the hospital records. Data were analysed by tabulation of means and linear regression and correlation techniques. Mean systolic and diastolic blood pressure were calculated according to birth weight and current weight as fourths of their distributions. RESULTS: There was a significant inverse relation between birth weight and systolic blood pressure both in children and adults. Current weight standardised regression coefficient showed a change of -2.68 mm Hg (95% Cl - 2.0 to 3.26, p = 0.027) for each Kg increase in birth weight in children, and -3.82 mmHg (95% Cl -3.21 to -4.39, p = 0.011) in adults. Within each current body weight group the reduction in mean systolic blood pressure from the lowest to the highest birth weight group was larger in adults (10.4 mmHg) than in children (4.1 mmHg). Adults but not children showed also an inverse relation between birth weight and diastolic blood pressure. Weight standardised regression coefficient was -3.0 mm Hg (95% Cl -2.45 to -3.62, p = 0.036). CONCLUSIONS: Blood pressure in inversely related to birth weight in childhood. This relation becomes stronger in adulthood. Therefore, reduced growth during fetal life may be linked with an increased risk of developing hypertension and cardiovascular disease. PMID- 7557035 TI - [Revision and optimization of processes: a fundamental timing for adequate use of the resources and technological innovation. An example of intervention in the cardiology field and considerations on "total quality" in medicine]. AB - The Cardiology Unit of the Este General Hospital began its activity in 1988. We soon identified a mismatch between a good, up-to-date diagnostic instrumentation with growing customers' demand and an inadequate utilization of the instruments. Waiting lists were getting longer, customers were not satisfied, no-shows at the appointments increased and we had a progressive loss in image. We therefore decided to intervene on our processes, starting the project "TOTAL QUALITY IN CARDIOLOGY". We focused our attention on two main fields, namely (1) electrocardiography, (2) other diagnostic techniques, separately analyzed because of important differences. Point (1) is basically worked out by paramedical personnel, in high numbers and with stable demand, while point (2) is determinantly linked to medical activity, although with concomitant need for paramedical support. The figures are lower for point (2) but are steadily growing. In the two operating fields we further identified two separate adverse effects: 1). ELECTROCARDIOGRAPHIC EXAMINATIONS (ECG) ARE TOO TIME CONSUMING, 2) THE NUMBER OF DIAGNOSTIC PROCEDURES IS TOO LOW FOR THE INSTRUMENTS AVAILABLE. We used preliminary analysis with process flow diagrams and our interventional methods were policy deployment and daily routine work. ELECTROCARDIOGRAPHIC EXAMINATIONS ARE TOO TIME CONSUMING. From cause-effect diagram for cause classification and subsequent Pareto analysis we identified two groups of main causes: 1. the paramedical-patient team is not able to optimise usage of the instrument; in particular, total time for undressing of the patient, lead attachment, dressing of the patient, change in ECG conductivity cream and, if necessary, repetition of ECG for mistakes in procedure is much longer than operative time of the instrument; 2. the necessary copy of the ECG done by the instrument was too time consuming (2') as compared to total procedure time. Implementation plans have been as follows: activity was concentrated in one single room at constant temperature (20 degrees); we augmented the number of dressing rooms and nurses (from 1 to 2 unit); we substituted the ECG conductivity cream with a water-alcoholic solution and the copy with a photocopy. RESULTS: decrease in mean time for ECG from 6'52" to 3'25" (for example: total ECGs 1992: 14,827, total spared time: 852 working hours); reductions in dead times; capability to cope rapidly with high demand; consequent possibility to utilise paramedical personnel for other activities; reduction in copy costs from 156 to 50 Lit each (total reduction 1,571,662 Lit). EXPECTATIONS. further revision of the procedures to keep pace with new electrocardiographic instruments and to achieve shorter operative times. PMID- 7557034 TI - [Planar determination of the aortic valve area with transesophageal echocardiography with multiplanar probe in patients with aortic stenosis and insufficiency. Comparison with transthoracic Doppler echocardiography]. AB - BACKGROUND: The simple determination of transaortic pressure gradient does not accurately assess the severity of an aortic valve stenosis. Thus, estimating the aortic valve area (AVA) is vital for clinical decision-making. Cardiac catheterization has been considered the "gold-standard" for the quantification of the stenotic valve area, but this technique may underestimate the actual valve area when aortic regurgitation is associated. Doppler transthoracic echocardiography (TTE) with the continuity equation method is usually employed for AVA estimation. Recently, in pure aortic stenosis, transesophageal echocardiography (TEE) has provided AVA values well-correlated to hemodynamic invasive results. METHODS: In this study, we correlated AVA values by TTE and multiplane TEE in 18 patients with combined aortic valve stenosis and regurgitation. RESULTS: The mean values of AVA by TEE and TTE were 0.74 +/- 0.12 and 0.68 +/- 0.55 cm2, respectively (p = NS). TEE-derived AVA correlated well to TTE-derived AVA (r = 0.816; p < 0.0001). Critical aortic stenosis was predicted by TEE with 100% sensitivity and specificity. Total time of examination was significantly longer for TTE (p < 0.00001). CONCLUSIONS: In conclusion, direct planimetry by multiplane TEE is a reliable method for AVA determination in aortic stenoinsufficiency. For this purpose, when the technical quality of TTE study is poor or when the patient is critically ill and does not tolerate a longer lasting TTE, multiplane TEE should be considered. PMID- 7557028 TI - Inhibition of collagenase type I expression by psoralen antisense oligonucleotides in dermal fibroblasts. AB - Type I collagenase plays an important role in both tumor metastasis and the remodeling of connective tissue in normal human skin, during wound healing, for example, and may participate in the pathophysiology of some dermatological diseases such as skin cancer and a chronic blistering disease, recessive dystrophic epidermolysis bullosa. In an effort specifically to inhibit collagenase expression, we have designed phosphorothioate antisense oligonucleotides, linked at the 5' ends with photoreactive 4' (hydroxyethoxymethyl)-4,5',8-trimethyl-psoralen (HMT), and directed them against the 5' end of the collagenase mRNA. Two antisense-HMT molecules targeting a region overlapping the initiation codon were compared. Only one contained the HMT moiety targeting a 5'TpA on its complementary sense strand, and we observed greater than 50-fold improvement on the cross-linking of this antisense oligonucleotide to its target sequence after ultraviolet A (UVA) irradiation. Likewise, sequence complementary to the 5'TpA target was also required to demonstrate specific inhibition of in vitro translation of collagenase mRNA. Tissue culture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in monolayer or in 3-dimensional dermal equivalents, showed lowered collagenase levels 24 h after UVA irradiation as compared to controls. Initial screening of antisense oligomers for specific hybridization and photo-cross-linking is a useful step in the design of antisense oligonucleotides, and allowed us to design an HMT-linked antisense phosphorothioate oligonucleotide that specifically inhibits the expression of fibroblastic collagenase. PMID- 7557037 TI - [Congenital arterio-venous malformations: an unusual cause of heart failure at birth and during the first months of life]. AB - BACKGROUND: Congenital arteriovenous malformations represent a rare cause of heart failure in neonates. MATERIALS: Four neonates with arteriovenous malformations (cerebral in one, hepatic in two and pulmonary in one) are reported. RESULTS: In the first three cases Color Doppler echography was able to image the arteriovenous malformations, to identify the afferent and efferent vessels and to quantify the arteriovenous shunt. In the neonate with pulmonary malformation, a cardiac cause of heart failure was excluded by echocardiography, and the diagnosis was stated by selective angiography. The neonate with cerebral great arteriovenous malformation died at 4th day of life. The two neonates with hepatic malformations improved both with medical therapy; in the last neonate, with multiple arteriovenous pulmonary fistulae, selective embolization was performed, but the child died a few months later. CONCLUSION: Congenital arteriovenous malformations are rare, and an early diagnosis is the base for a correct therapy; Color Doppler echography represents the gold standard for cerebral and hepatic malformations, while selective angiography should be reserved to patients who might profit of embolization or surgical resection. PMID- 7557039 TI - [White-coat hypertension]. AB - White-coat hypertension may be broadly defined by the coexistence of normal ambulatory, or self-measured, blood pressure (BP) with persistently increased clinic BP. White-coat hypertension should not be considered as a separate clinical entity, but, rather, as a low-risk stratum of essential hypertension which may be identified using ambulatory BP monitoring or self-measured BP. One possible cause of white-coat hypertension may be a conditioned response to the clinical visit, with consequent alerting reaction and rise in BP. Specifically, white-coat hypertension may be defined by an average daytime ambulatory BP ideally less than 130 mm Hg systolic and 80 mm Hg diastolic (or, at least, 134 mm Hg systolic and 90 mm Hg diastolic) in untreated subjects with essential hypertension and clinic BP above 140 mm Hg systolic or 90 mm Hg diastolic for at least three visits. Several cross sectional studies have shown that hypertensive target organ damage (TOD) is not dissimilar between clinically normotensive subjects and subjects with white-coat hypertension but this finding is still controversial. Moreover, nearly all studies have shown that TOD is lesser in subjects with white-coat hypertension than in those with higher levels of ambulatory BP. Evidence is accumulating that white-coat hypertension is a condition of low cardiovascular risk. In this setting, a prospective study from our center and preliminary data from another center suggest that the incidence of serious cardiovascular complications of hypertension (myocardial infarction, sudden death, stroke, etc) is lower in subjects with white-coat hypertension than in those with ambulatory hypertension, and not dissimilar between subjects with white-coat hypertension and clinically normotensive subjects. Further prospective studies are needed to clarify the very-long term history of white-coat hypertension and to test the hypothesis that antihypertensive drug treatment may not be necessary, as unable to improve an already good prognosis, in uncomplicated subjects with white-coat hypertension and absence of concomitant risk factors. PMID- 7557036 TI - [A case of radiation-induced coronary occlusion treated with elective and emergency PTCA]. AB - We describe the case of a patient (pt) treated with radiotherapy for Hodgkin's lymphoma at the age of 17. Two years later he presented an apical AMI and underwent coronary angiography (CA) for postinfarction angina. A 40% stenosis of the left anterior descending (LAD) was found in the proximal portion and the vessel was occluded at the middle level. Septal and diagonal branches supplied collaterals to the distal LAD and left ventricular function was only mildly reduced (EF angio-ventriculographic = 52%). We successfully performed a first PTCA, but the pt was re-admitted to our hospital few days later for a new large anterior myocardial infarction with refractory hypotension and low output condition. An intraaortic balloon catheter was inserted and CA demonstrated proximal LAD occlusion; a new PTCA was then performed and the opening of the vessel was obtained after 90' from symptoms' onset. The subsequent course was uneventful and the pt was discharged after 20 days. The ejection fraction was 39%. Thirty days after, a third PTCA with Palmaz-Schatz stent implantation was necessary for unstable angina due to a restenosis of the proximal LAD. After ten months follow-up the pt is asymptomatic with negative exercise test and an angioscintigraphic EF = 47%. CA and intravascular ultrasound demonstrated nor restenosis or progression of the disease, with a good minimal luminal diameter (MLD). A review of the literature on this topic is presented. Moreover we discuss the mechanism of coronary stenosis and occlusion and the reasons for choosing PTCA in the various settings. PMID- 7557038 TI - [Acute myocardial ischemia: ECG or echocardiography? Considerations on a clinical case]. AB - We present a clinical case and a review of the literature on the usefulness of two-dimensional echocardiography for the diagnosis of acute myocardial ischemia in patients presenting with chest pain but without diagnostic electrocardiographic changes. The case refers to a patient admitted a few hours after an episode of prolonged chest pain without diagnostic electrocardiographic changes, but with a severe wall motion abnormality assessed by two-dimensional echocardiogram. The electrocardiogram obtained 24 hours after admission showed giant negative T waves in the anterior chest leads when the patient was asymptomatic and the echocardiogram showed disappearance of wall motion abnormalities. Coronary angiography demonstrated a thrombotic occlusion of the left anterior descending coronary artery, that was treated by transluminal angioplasty. The considerations refer to the following points: 1) a substantial portion of patients with chest pain examined in the emergency room has initially a normal or non-diagnostic electrocardiogram; 2) in these patients two dimensional echocardiography provides a highly sensitive and specific noninvasive tool for the establishment of the correct diagnosis of acute myocardial ischemia when a severe wall motion abnormality is shown; 3) if successive amelioration of regional myocardial dysfunction is assessed, this was probably due to stunned myocardium; 4) it is probably not correct to consider T wave inversion as the electrocardiographic equivalent of myocardial stunning, as the two phenomena are not chronologically correlated. PMID- 7557040 TI - [Physiopathology of vasovagal syncope: review of the most endorsed theories and recent findings]. AB - Vasovagal syncope is associated with an abnormal reflex and the physiopathological mechanisms of the phenomenon overall are only partially known. Experimental and clinical studies suggest that the main factor which triggers the syncope is the brusque interruption of the alpha-adrenergic tone with marked, sudden peripheral vasodilation. Although documented, vagal hypertony, with consequent bradycardia and asystolia, is only occasional and is almost always a secondary phenomenon. The most commonly suggested cause of vasovagal syncope is a Bezold-Jarish reflex starting from the cardiac receptors in the walls of the ventricle, mediated by the paradoxical activation of afferent vagal fibres. However, recent studies are suggesting that there may be other pathogenetic mechanisms such as the paradoxical activation of the venous-atrial baroceptors and other "extracardiac" vascular receptors. The neuro-endocrine aspect of the vasovagal reaction is very complex and in spite of the many studies carried out on the catecholamine, renal-angiotensive system, arginine-vasopressin, and b endorphine trends, there are still many points awaiting clarification. The response of the autonomous nervous system linked to age also require further research. PMID- 7557041 TI - [Knowing how to read a scientific article. 2. Knowing how to single out a risk factor]. PMID- 7557042 TI - [Stratification of risk at discharge after acute myocardial infarction: prognostic significance of advanced age]. PMID- 7557043 TI - [Guidelines on the diagnostic assessment of patients with syncope]. PMID- 7557044 TI - [Cytogenetic findings in chronic myelocytic leukemia and their prognostic correlation in patients of the National Institute of Cancer of Mexico]. AB - This study describes the cytogenetic alterations in patients with chronic myelogenous leukemia (CML) seen at the Instituto Nacional de Cancerologia (INCAN), Mexico City, whether they have been treated previously or not. It correlates these findings with the prognosis. We studied 31 patients seen during June 1987 and June 1990. Philadelphia (Ph+) chromosome was present in 61% of all specimens. In 10 cases it was the only anomaly, resulting in a survival greater than 40 months. Aneuploidies were seen in 50% of patients with previous treatment and in 31.5% of those without treatment. Patients with numerous abnormalities and double Ph+ (with or without Ph+ chromosome) had a mean survival of 32 months. The worst prognosis was seen in the a cases with no growth, with a mean survival of 19 months. This study suggests that the prognosis of patients with CML correlates with the cytogenetic anomalies whether patients have been treated previously or not. It also seems that the group of patients whose cytogenetic study does not grow or cannot be evaluated upon direct exam have a worse prognosis which may be secondary to intrinsic defects of the neoplastic cells that do not grow in vitro, resulting in a more aggressive disease in vivo. PMID- 7557046 TI - [Diabetic neuropathy. Current concepts on etiopathogenesis, diagnosis, and treatment]. AB - Diabetic neuropathy (DN) is chronic complication which occurs in 50% of long standing diabetes mellitus. DN is a consequence of hyperglycemia probably through the following mechanisms: a) activation of aldose-reductase, intracellular sorbitol accumulation and myoinositol depletion, reduced activity of Na+/K+ATPase, loss of Na+ channels and demyelination; b) proteins glycation; c) microangiopathy; the first mechanism being the best known and the most reliable. DN may be subclinical or clinical. The main clinical picture is a peripheral, bilateral, symmetric polyneuropathy with a "socks and gloves" sensory impairment, muscular weakness, hyporeflexia, plantar ulcers and arthropathy. Less frequent syndromes are proximal motor neuropathy and mononeuropathy of cranial nerves or thoraco-abdominal roots. Diagnosis is based on clinical data, and may be sustained on impaired nerve conduction velocity, abnormal evoked somatosensory potentials, or sural nerve biopsy. These methods are highly sensitive but unspecific. Etiopathogenic treatment is based on glycemic control and aldose reductase inhibitors. Improvement in clinical, electrophysiologic and histopathologic data have been obtained with the latter. Symptomatic treatment includes carbamazepin, phenytoin, tricyclic antidepressives and a phenotiazin. Mononeuropathies tend to complete recovery in less than 6 months. Polyneuropathy is thought to be irreversible and progressive; however, with excellent glycemic control or with aldose reductase inhibitors nerve damage may be stabilized or even reversed. PMID- 7557045 TI - [Vestibular and oculomotor disorders in patients with congenital strabismus]. AB - A group of patients with congenital endotropia or exotropia was investigated in their capacity to perform reflex eye movements. Some patients had a successful surgical correction of their eye defect. The functions measured were the vestibulo-oculomotor reflex, the feedback control loop for searching and following eye movements and the optomotor reflex. A loss in the functional capacity was displayed in all the tests done. The average value of the losses found for the whole group, according to the test used, were from 8 to 27% for the vestibulo-oculomotor reflex; 22 to 42% for the eye tracking feedback loop, and 48% for the optomotor reflex. The deficit was not due to the incorrect eye position, because the successfully operated subjects had the same magnitude of the motor defect than the non-operated patients. PMID- 7557047 TI - [The clinical laboratory in the diagnosis, classification, and follow-up of patients with HIV/AIDS infection]. PMID- 7557048 TI - [Indications and risks of the ovarian hormonal therapy with replacement purposes]. PMID- 7557049 TI - [Medications and breastfeeding]. PMID- 7557050 TI - [Advances in the treatment of acute myocardial infarction]. PMID- 7557051 TI - [A passion added to others: doctor Juan Somolinos Palencia's passion for the National Academy of Medicine]. PMID- 7557052 TI - [Epilepsy from a metaphysical perspective: an interpretation of the biblical story of the epileptic boy and Raphael's Transfiguration]. AB - Raphael's last painting reveals, in the upper half of the picture, Christ's transfiguration on Mount Tabor and, in the lower half, the young boy's epileptic seizure at the foot of the mountain in the presence of the other disciples. Raphael depicts both events, which are told in succession in the Gospels, as if they took place at the same time. By synchronizing both scenes, Raphael demonstrated a significant correspondence between Christ and the epileptic boy which reveals the epileptic seizure as a symbolic representation of a transcendental event. This metaphysical aspect of epilepsy depicted by Raphael can also be found in the corresponding biblical passages. In the Gospels, the metamorphosis caused by the epileptic seizure is used as a simile for Christ's transfiguration through suffering, death and resurrection. PMID- 7557053 TI - [Diagnosis and classification in psychiatric disorders]. AB - This paper provides a general overview of the evolution of the diagnostic concepts in Psychiatry, of the research methods for future improvement, of the goals reached and of the deterrants that have to be surpassed. Classification, standardization and systematization of mental disorders has not been an easy task. For this reason, during a long period of time a wide diversity in the classification of the mental illness that ran along with the thinking trend of each and everyone of the different authors has been observed. This resulted in a division of the specialty and in the contamination of its ideologies and also, it has damaged the reputation of the diagnosis. The development of psychopharmacology and of the epidemiological systems made necessary the implementation of a strict diagnostic system that could make possible the planning of treatments, the establishment of prognosis and the design of preventive measurements. England was the cradle of this change due to the fact that the situation of the specialty availed itself of the influence of the prestigious and noteworthy German professors exiled in this country. Since 1972, some of the material that has been published illustrates the new tendency of nosology. Since then, there has been a great proliferation of works in this field. The culminating point of these investigations is represented by the Diagnosis and Statistical Manual of the American Psychiatric Association and by the International Classification of Illnesses (ICD-10). From this point of view, diagnosis is just a provisional hypothesis of a work that can be identified because of its pathological expressions and which is also of help in the communication between specialists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557054 TI - [The role of the Ministry of Health in the prevention of diseases caused by blood transfusion]. PMID- 7557055 TI - [Mortality and possible lost years of life due to tobacco consumption in Mexico]. AB - An analysis was performed to estimate mortality and YPLL attributable to 20 smoking related diseases, based on the prevalence rates of current and former smokers obtained from a national survey; mortality data from vital statistics, and their relative risks and attributable mortality fractions due to tobacco consumption of the 20 diagnoses set for 1986. Numerical estimates of national mortality and YPLL attributable to cigarette consumption for the diagnosis set were made. Among the population of 20 years of age and older on 1986, a total of 17,405 deaths where attributable to smoking cases (47.0/100,000 individuals of 20 to 74 years of age), and takes the 9th place of grouped general mortality. Cardiovascular disease rated first place with 33 per cent of total cases; respiratory disease came second with 31.6 per cent. All 20 diagnoses someans 146.299 YPLL between 20 to 65 years (1.4 per cent from total ypll 0-70 years). PMID- 7557057 TI - [Modulation of the cell membrane function by cholesterol and its relation to disease]. PMID- 7557059 TI - [In vivo experimental models of amebiasis]. AB - In vivo experimental models for amebiasis have provided important information about the mechanisms of host-parasite interaction which determine the production of disease. In the laboratory, several species of rodents have been used to study the intestinal and hepatic amebiasis. For the former, the model of "washed-closed cecal loop" in guinea-pig and hamster has been useful to study the early invasive events. In gerbils we also produced early intestinal lesions by intracecal inoculation with monoxenically cultured amebas. Hamsters and gerbils have been used as susceptible animals for hepatic amebiasis, and rats and guinea-pigs as resistant animals. Morphological analysis of hepatic lesions of susceptible animals showed the role of host inflammatory cells in the process of liver damage. The resistance in the production of liver abscess in rats and guinea-pigs is due in part to the action of polymorphonuclear leukocytes in the rat and the macrophages in the guinea pigs. Complete characterization and standardization of the various models of amebiasis in rodents constitute the bases for other important biomedical studies aimed to the disease control. PMID- 7557056 TI - [Mitotic index and kinetics of lymphocyte proliferation in biologic monitoring]. AB - Xenobiotics exposure generates concern, mostly because the effects on DNA and cancer generation are not well known. Identification of dangerous agents are difficult due to the large latency period between exposure and clinic manifestations (from years to several generations). Biomarkers are proposed for human biomonitoring of individuals that due to natural, occupational, medical or accidental exposure are considered of "high risk". The present work evaluates the use of biomarkers of cell proliferation in lymphocyte cultures of individuals exposed to hydro-arsenicism, dangerous industrial products (DIP), neuro cysticercosis patients under treatment with praziquantel and individuals treated with metronidazole. The slowing of cell proliferation in arsenic exposed individuals and in neuro-cysticercosis patients, in which higher cancer frequencies han been reported, evidence the usefulness of these parameters. We did not find lymphocyte proliferation changes due to DIP exposure although we did find an increased proliferation due to metronidazole treatment. The biological meaning of these data opens new research interests. PMID- 7557058 TI - [Demonstration of clinical bioequivalency of teldane and feliberal using a bioassay with intradermal histamine challenge in healthy volunteers]. AB - To demonstrate the bioequivalence of two terfenadine formulations, a clinical equivalence trial was performed. The antihistaminic's ability to prevent the response to intradermal histaminic challenge was herein assessed. Twelve healthy volunteers underwent a randomized, double blind, cross over trial with seven-day treatments. Dermal response to histamine concentrations of 0, 1, 2, 5 and 10 mcg was determined by measuring the wheal produced on the deltoid area. Measurements were made 72 hours prior to terfenadine administration, 1 hour after the first dose and 11 hours after second and last doses of each treatment. Both formulations showed the same latency, extent and duration in protective effect against histaminic challenge. This confirms the clinical equivalence of the two formulations and suggests that they have a similar bioavailability. PMID- 7557063 TI - [Observer variation in diagnostic procedures related to cardiothoracic surgery]. AB - The results reliability is a predominant condition in the validity of clinical research. The observer agreement determination is a useful procedure in the confirmation of the obtained results in clinical research. The present work is a presentation of the basis about observer agreement and a review of national publications in cardiology, pneumology and cardiothoracic surgery between january 1980 and july 1993, where we found just two publications with the application of observer agreement determination in echocardiography, electrocardiography, microscopy and hemodynamics. It was concluded that is necessary the increase in determination of observer agreement and inform this results in our publications, excluding the variability attributed to patients, diagnostics equipment or the circumstances. PMID- 7557061 TI - [Relations between delayed diagnosis and forms of onset in Duchenne muscular dystrophy]. AB - Duchenne Muscular Dystrophy (DMD) is usually diagnosed several years after the onset of symptoms. The relatives of the patients with DMD frequently consult family physicians when they notice the first symptom. The purpose of this study was to determine the cause that influence the delayed diagnosis of DMD. Twenty two patients with confirmed diagnosis of DMD were interviewed at two Neurology Centers (Mexican Social Security Institute) in Monterrey, Nuevo Leon, Mexico. Two forms of onset of DMD: Retarded development and locomotion problems were found, confirming other studies. The mean age of onset of symptoms for 22 patients was 2.4 years. The mean age for DMD diagnosis was 4.9 years. Retarded development occurred in 12 (54.4%) of all cases and the age of diagnosis was between 1 and 6 years of age. In 10 cases of the group with locomotion problems (45.4%) the diagnosis was made between 3 and 11 years of age. The serum Creatine Kinase was increased in all patients and in the early stages these levels were much higher than late stages. Family physicians have opportunities to make early diagnosis of DMD if they are aware of the two forms of onset of the disease: Retarded development and locomotion problems and of the changes in serum CK levels. The findings of this study confirm the importance of family physicians in that respect and also in making recommendations for routine determination of serum Creatine Kinase (CK) as early as possible in a child with symptoms suggestive of DMD. PMID- 7557062 TI - [Primary or of unknown etiology pulmonary hypertension: survival and its determinants]. AB - The main objective of the present study is to characterize mortality in PPH and the factors associated with survival. Our series comprises 61 patients diagnosed of PPH and prospectively followed at the Cardiopulmonary Department between the years of 1977 and 1992. Univariate and multivariate analysis were used to examine relations between survival and selected demographic, medical-history, pulmonary function, laboratory, and hemodynamic variables. We used the date of initial diagnostic catheterization as an index for determining survival. The method of Kaplan-Meir was used to estimate overall survival distribution. RESULTS: the mean age of the group was 22.6 +/- 11 years with a female to male ratio of 3.06:1. Pulmonary hemodynamics at entry were: mean pulmonary artery pressure (PAP) = 65.5 +/- 17.5 mmHg; right atrial pressure (RAP) = 7.5 +/- 5 mmHg; cardiac index (CI) = 3.15 +/- 1.31.mi.m2; pulmonary vascular resistance (RP) = 24 +/- 11 U. The pulmonary to systemic resistance ratio (Rp/Rs) was 0.8 +/- 0.2. The median survival (MS) of the whole group 4.04 years. There was a significant difference in MS between the groups with and without vasodilator treatment (5.04 and 2.12 years respectively), however, the hemodynamic profile at entry was also different, with higher RAP, PAP, PVR, and higher Rp/Rs in the group without treatment (p < 0.05). Factors associated with poor survival (univariate) were: increased RAP, RP, and Rp/Rs and decreased forced vital capacity (FVC), decreased CI, and stroke volume index and decreased mixed venous PO2. The absence of vasodilator treatment was also associated with an increased risk of death. On the multivariate analysis, survival was associated mainly to FVC and to pulmonary hemodynamic variables. We conclude that survival in PPH is related to pulmonary hemodynamic factors at diagnosis. PMID- 7557060 TI - [Differential diagnosis of Becker and Duchenne muscular dystrophy]. AB - The Becker's Muscular Dystrophy (BMD) is a disease with similar aspect and distribution to the Duchenne Muscular Dystrophy (DMD), although it is usually less severe. The main purpose of this investigation was to outline the most important clinical characteristics that can help in the differential diagnosis between these two diseases. Thirty eight patients were studied; 16 with BMD and 22 with DMD. Clinically both are very similar, and the best criteria for the differentiation of this two diseases is the inability to walk. The age of symptomatology onset was 10.5 +/- 7.2 years in BMD and 2.3 +/- 13 years in DMD showing an overlapping of 18.42% of DMD and DMB at the age of 4, this overlapping difficult the precise diagnosis between both diseases. The creatine kinase (CK) study was not relevant. PMID- 7557067 TI - Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease. AB - BACKGROUND & AIMS: Transmural perivascular mononuclear cell infiltrates are a feature of Crohn's disease. The aim of this study was a molecular characterization of the mechanisms leading to the formation of these infiltrates. METHODS: Endothelial cell and leukocyte expression of the adhesion molecules directing leukocyte transendothelial migration were studied in situ by immunohistochemical analysis of 10 samples from patients with Crohn's disease and 10 samples from normal controls. Double-staining methods were used to characterize the cells forming the infiltrates. RESULTS: CD11a+ and L-selectin positive mononuclear cells seemed to be the major component of perivascular infiltrates. The vast majority of these cells were CD68+, CD31+ monocytes/macrophages surrounded by CD3+, L-selectin-positive, CD31+, CD45RA+, and/or CD45RO+ T lymphocytes. T lymphocytes within the vessels expressed both CD45RA and CD45RO markers. Endothelial cells were intercellular adhesion molecule 1 positive and mostly CD34+. Strong adhesion between L-selectin-positive and CD11a+ intravascular mononuclear cells and CD34+ and intercellular adhesion molecule 1-positive endothelial cells were observed. CONCLUSIONS: Data indicate that peripheral mononuclear cells are actively recruited in the submucosa of Crohn's disease tissue; endothelial cells express adhesion molecules highly permissive for transendothelial migration of monocytes and both naive and memory T cells contributing to infiltrates generation; and close membrane contact between migrated macrophages and naive T cells leads to the T-cell transition from naive to memory phenotype within Crohn's disease. PMID- 7557065 TI - [Historical course of surgical techniques in pancreas transplantation]. AB - Since the original description of the pancreas transplant technique in animals, initially as a pedicle transplant in 1892, free graft in 1913, and then as a vascularized organ in 1927, there have been several changes in the handling of the exocrine secretions, as well as the vascular anastomoses, and the posttransplant function in relation to the immunosuppressive therapy. The surgical technical changes have included the ligature of the duct, the free drainage to the abdominal cavity, the pancreas transplant with a duodenal segment, the chemical occlusion of the duct, and its drainage to the intestine or to the bladder with a duodenal segment. This last technique remains as the standard technique currently used for pancreas transplantation, along with the combined transplant of the kidney. This work reviews the historical aspects, the evolution of the experimental and clinical surgical techniques, and the current results associated with the surgical management of pancreas transplantation. PMID- 7557064 TI - [Effects levocabastine ophthalmic solution (H1 receptor antagonist) on symptoms of vernal conjunctivitis. A prospective, randomized double-blind study]. AB - A double blind, prospective and randomized study was carried out in forty patients with clinical diagnosis of vernal conjunctivitis and eosinophilia in conjunctival scrapings. They were divided in two randomized groups of 20 patients each. One of the groups received levocabastine ophthalmic solution and the other saline solution drops. The mean age of the study group was 9.1 years old with a vernal conjunctivitis history of 44 months. The control group mean age was 10.1 years old and a history of vernal conjunctivitis of 48 months prior the enter of the study. One group received levocabastine 0.5 mgrs/ml while the other balanced saline solution, one drop every 12 hours per seven days. The patients evaluated their symptoms through a visual analogic scale (every day). Data collected was analyzed, for the first, third and seventh day, through U-Mann Whitney statistical analysis. Epiphora and photophobia showed a significant improvement. Itching, edema and foreign body sensation showed improvement in the levocabastine group but without statistical significance. Hyperthermia remained the same in both groups studied. PMID- 7557066 TI - [Origins of medical specialties in Mexico]. PMID- 7557068 TI - Localization and biosynthesis of aminopeptidase N in pig fetal small intestine. AB - BACKGROUND & AIMS: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS: This study was performed using histochemistry and immunoelectron microscopy and [35S]methionine labeling of cultured mucosal explants. RESULTS: Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg(2+)-precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing in the apical endocytic structures. CONCLUSIONS: In comparison with the adult enzyme, fetal aminopeptidase N has a more widespread subcellular distribution with substantial amounts present in apical endocytic compartments characteristic of the fetal enterocyte. PMID- 7557069 TI - Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa. AB - BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB EGF) has been recently identified as a member of the EGF family. EGF receptors to which HB-EGF can bind have been detected in some types of gastric epithelial cells. The aim of this study was to investigate whether HB-EGF is produced in gastric epithelial cells to maintain normal gastric mucosa. METHODS: Gene expression and production of HB-EGF protein were investigated using Northern hybridization and immunohistochemistry, and the types of cells producing this protein were determined in human gastric mucosa. RESULTS: HB-EGF messenger RNA was detected in the body and antrum. Immunohistochemical staining showed that HB EGF was localized mainly in parietal cells of fundic glands and in gastrin cells of pyloric glands. Also, the immunoreactivity of EGF receptors was observed in parietal cells and gastrin cells and faintly in surface epithelial cells and mucous neck cells of the proliferative zone. CONCLUSIONS: The results suggest that HB-EGF is synthesized mainly in parietal cells and gastrin cells and may act in an autocrine and/or paracrine manner in the regulation of proliferation and differentiation of the gastric mucosal cells through their surface EGF receptors. PMID- 7557070 TI - Gastrin-stimulated changes in Ca2+ concentration in parietal cells depends on adenosine 3',5'-cyclic monophosphate levels. AB - BACKGROUND & AIMS: The parietal cell has secretory receptors for histamine and acetylcholine, whereas the functional nature of the gastrin/cholecystokinin B receptor is controversial. This study in isolated gastric glands investigates the cholecystokinin B receptor-induced intracellular calcium concentration ([Ca]i) response in enterochromaffin-like (ECL) and parietal cells as a function of adenosine 3',5'-cyclic monophosphate pathways. METHODS: The responses of [Ca]i in ECL and parietal cells of perfused rabbit or rat calcium orange-loaded gastric glands were determined using confocal microscopy. ECL cells were identified by position, size, and autofluorescence and parietal cells by position and size. RESULTS: Gastrin (1 mumol/L) produced an elevation of [Ca]i levels in both ECL and parietal cells. In the presence of 100 mumol/L cimetidine, the ECL cell response to gastrin was not affected but the [Ca]i response of the parietal cell was abolished. With dibutyryl adenosine 3',5' phosphate in addition to cimetidine, the response of the parietal cell [Ca]i to gastrin was restored in both the rat and rabbit. CONCLUSIONS: The [Ca]i response of the parietal but not the ECL cell to the addition of gastrin seems to depend on the presence of normal or elevated intracellular adenosine 3',5'-cyclic monophosphate levels. Therefore, H2 receptor activity may be permissive for the effect of gastrin on parietal cell function. PMID- 7557074 TI - Flow cytometric analysis of the DNA content in colorectal adenomas with focal cancers. AB - BACKGROUND & AIMS: Variable results have been reported on the nuclear DNA content of colorectal polyps. The significance of DNA aneuploidy in the malignant transformation of colorectal polyps was evaluated. METHODS: We analyzed by flow cytometry the nuclear DNA content of freshly frozen samples of 50 colorectal adenomas with or without focal cancers, analyzing separately the adenomatous and cancerous regions of the polyps. RESULTS: In the adenomatous regions of the 50 polyps, the DNA was diploid in 43 and aneuploid in 7; the adenomas with DNA aneuploidy in the adenomatous regions were more frequently accompanied by focal cancers than were the DNA-diploid adenomas (P < 0.01). In 60% of the polyps with DNA aneuploidy in the cancerous regions, the DNA was also aneuploid in the adenomatous region and had similar DNA indices; this result suggests that the DNA aneuploidy had already occurred during the adenomatous stage, which lends support to the concept of the adenoma-carcinoma sequence. DNA aneuploidy in the adenomatous region was significantly correlated with the size of colorectal polyps (P < 0.05). CONCLUSIONS: DNA aneuploidy may be an important indicator for the early diagnosis of malignant transformation of colorectal polyps. PMID- 7557073 TI - Long-term histomorphological surveillance of the pelvic ileal pouch: dysplasia develops in a subgroup of patients. AB - BACKGROUND & AIMS: Little is known about the long-term morphology of the pelvic ileal pouch after restorative proctocolectomy in patients with ulcerative colitis. This study analyzed the mucosal adaptation in the pouch during a long term follow-up. METHODS: Mucosal biopsy specimens were obtained from 87 patients during a follow-up of 6.3 years (SD, 2.7; range, 3-14 years). The villous surface density, degree of inflammation, and type of mucin were determined from glycolmethacrylate-embedded sections. RESULTS: Three basic patterns of mucosal adaptation were observed: regular response with normal mucosa or mild villous atrophy and no or mild inflammation (type A, 51%), transient atrophy response with temporary moderate or severe villous atrophy followed by normalization of architecture (type B, 40%), and constant atrophy response with permanent subtotal or total villous atrophy developing from the early functioning period accompanied by severe pouchitis (type C, 9%). Low-grade dysplasia occurred in 3 patients with type C response. CONCLUSIONS: In a small group of patients with ulcerative colitis, the mucosa of the pelvic pouch adapts with constant severe villous atrophy accompanied by long-standing pouchitis. This group of patients should be identified and undergo regular endoscopic and histomorphological surveillance because of risk of developing neoplasia in the pouch mucosa. PMID- 7557072 TI - Abnormal intestinal motor patterns explain enteric colonization with gram negative bacilli in late radiation enteropathy. AB - BACKGROUND & AIMS: Bacterial overgrowth and intestinal pseudo-obstruction may succeed abdominal radiotherapy, and absence of intestinal migrating motor complex (MMC) has been reported in bacterial overgrowth. The aims of this study were to address the relationship between intestinal patterns of motility and gastrointestinal microflora and to elucidate the pathogenesis of late radiation enteropathy. METHODS: Forty-one consecutive female patients with symptoms of late radiation enteropathy were examined by prolonged ambulatory manometry, culture of gastric and duodenal samples with quantification of gram-negative bacilli (GNB) by the glucose gas test, the [14C]D-xylose breath test, and determination of pH and short-chain fatty acids in gastric juice. RESULTS: The intensity of MMC explained 61% (P < 0.001) and 71% (P < 0.001) of the variability of GNB in the stomach and duodenum, respectively, corresponding to the severity of disease. Abnormal MMC index and presence of irregular bursts were the best predictors of GNB (86%; P < 0.001, multiple regression). Fasting gastric pH explained gastric bacterial counts (63%; P < 0.001) but did not predict GNB. CONCLUSIONS: Impaired motility emerges as a causal factor for gastrointestinal colonization with GNB, whereas hypochlorhydria facilitates unspecific gastric colonization. Abnormal motility and GNB in the proximal small intestine are essential factors in the pathogenesis of severe late radiation enteropathy. PMID- 7557071 TI - Gene expression of keratinocyte and hepatocyte growth factors during the healing of rat gastric mucosal lesions. AB - BACKGROUND & AIMS: The factors that stimulate the growth of gastric mucosal cells during gastric mucosal healing are not completely understood. This study was designed to investigate the gene expression of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) in the healing of gastric mucosal lesions. METHODS: Northern blot analysis and reverse-transcription polymerase chain reaction were used to show HGF and KGF messenger RNA. RESULTS: Transcripts of HGF and KGF were not shown in rat gastric mucosal epithelium but were found in submucosal and muscular layers under normal conditions. When acute gastric mucosal lesions were induced by indomethacin treatment, expression of HGF messenger RNA was augmented in submucosal, muscular, or serosal layers, whereas the transcript of KGF was not influenced. When rat gastric mucosal epithelial cell line RGM1 and rat gastric mucosal primary culture cells were incubated with HGF or KGF, their proliferation was enhanced. CONCLUSIONS: The results showed increased gene expression of HGF together with constant production of KGF during gastric mucosal healing. PMID- 7557075 TI - Interaction between atrial natriuretic peptide and vasoactive intestinal peptide in guinea pig cecal smooth muscle. AB - BACKGROUNDS & AIMS: The role of atrial natriuretic peptide (ANP) in gastrointestinal motility is still unclear. The aim of this study was to investigate the relationship between ANP and vasoactive intestinal peptide (VIP) in guinea pig cecal circular smooth muscle cells. METHODS: The inhibition of 125I ANP binding or 125I-VIP binding to cecal smooth muscle cells was assessed using unlabeled peptides (i.e., ANP, ANP fragments, VIP, secretin, and peptide histidine isoleucine); the effect of ANP, ANP fragments, and VIP on muscle contraction stimulated by 1 mumol/L carbachol was assessed; and the inhibitory effects of ANP 1-11 on VIP-induced relaxation, ANP 1-11 and VIP 10-28 (a VIP antagonist) on ANP-induced relaxation, and nitric oxide production inhibitors on ANP-induced relaxation were assessed. RESULTS: The specific binding of 125I-ANP was inhibited completely by unlabeled ANP and VIP in a dose-dependent manner but only slightly inhibited by secretin and peptide histidine isoleucine. ANP 1-11 and C-atrial natriuretic factor inhibited the binding of 125I-ANP with a lower affinity than ANP. ANP only partly inhibited 125I-VIP binding. ANP and VIP inhibited 1 mumol/L carbachol-induced contraction in a dose-dependent manner. ANP 1-11 significantly inhibited VIP-induced relaxation. ANP 1-11, VIP 10-28, and NO production inhibitors completely inhibited ANP-induced relaxation. CONCLUSIONS: The results of the study showed that ANP 1-11 antagonized ANP-induced relaxation and that ANP stimulated NO production and subsequently induced relaxation via a receptor to which VIP binds. PMID- 7557076 TI - Intravital demonstration of sequential migration process of lymphocyte subpopulations in rat Peyer's patches. AB - BACKGROUND & AIMS: Although recirculation of lymphocytes through Peyer's patches is important for specific immune defense, the intraorgan migration of lymphocyte subpopulations has not been clearly understood. The aim of this study was to compare the spatial distributions of labeled lymphocytes among various subpopulations in rat Peyer's patches. METHODS: Lymphocytes collected from intestinal lymph were separated into CD4+, CD8+, and T and B cells, labeled with a fluorochrome carboxyfluorescein diacetate succinimidyl ester, and injected into the jugular vein. Peyer's patches of recipient rats were observed by intravital fluorescence microscopy. RESULTS: No significant difference was found in the percentage of lymphocytes in transit or in the rolling velocity among different subpopulations. Lymphocytes sticking to the venules increased in number at 10-20 minutes, with preferential adherence of CD4+ cells to venules of 25-50 microns and preferential adherence of B cells to the venules of a wider size range. After 30 minutes, extravasated lymphocytes moved into the interstitium. B cells migrated from venules more quickly than CD4+ cells. CD8+ cells showed an intermediate pattern between CD4+ and B cells in sticking and migratory behaviors. Subsequently, CD4+ and CD8 cells preferentially appeared in parafollicular microlymphatics. CONCLUSIONS: Significant differences were observed among lymphocyte subpopulations in terms of spatial distribution of lymphocytes sticking to venules, migration into the interstitium, and their lymphatic transport. PMID- 7557079 TI - Expression, processing, and secretion of gastrin in patients with colorectal carcinoma. AB - BACKGROUND & AIMS: The relationship between gastrin and the development of colorectal carcinoma (CRC) remains controversial. Problems with previous studies include failure to measure all forms of gastrin, lack of comparison between stored and secreted gastrin, and not controlling for Helicobacter pylori infection (a known cause of hypergastrinemia). The aim of this study was to quantify progastrin and progastrin-derived peptides in the resected tumor and plasma of patients with CRC and in the antrum and plasma of normal subjects. METHODS: Four region-specific gastrin antisera were used to measure progastrin, glycine-extended gastrin, amidated gastrin, and total gastrin. RESULTS: Progastrin, amidated gastrin, total gastrin, and glycine-extended gastrin were detected in 100%, 69%, 56%, and 44% of tumors, respectively (n = 32). When allowing for H. pylori infection, circulating amidated gastrin levels were not significantly elevated in patients with CRC. However, compared with control H. pylori-positive and H. pylori-negative subjects, fasting plasma total gastrin levels were increased in H. pylori-positive (5.2-fold) and H. pylori-negative (2.3-fold) patients with CRC. CONCLUSIONS: Gastrin or its processing intermediates are present in a high proportion of CRCs. Nonamidated gastrin levels are elevated in the circulation of patients with CRC regardless of H. pylori status. We conclude that gastrin should continue to be assessed as a circulating or autocrine growth factor in the development of CRC. PMID- 7557077 TI - Risk factors associated with refractory peptic ulcers. AB - BACKGROUND & AIMS: The risk factors associated with refractory peptic ulcers are still undefined. The purpose of this study was to identify these factors in a multivariate context. METHODS: Clinical and endoscopic findings as well as Helicobacter pylori status, gastric secretion analysis, serum gastrin levels, nonsteroidal anti-inflammatory drug (NSAID) use, and objective testing of aspirin use by platelet cyclooxygenase activity were studied in 60 consecutive refractory patients with peptic ulcer and 54 matched nonrefractory controls. RESULTS: Refractory patients had a longer history of symptomatic ulcer, had an earlier onset, had more frequent relapses, and smoked more during the episode of refractoriness. H. pylori status was similar in both groups, but H. pylori eradication in a subset of refractory patients (23 of 26) was highly effective in healing these ulcers (14 of 23). Globally, NSAID-analgesic abuse (including > 1500 mg/day paracetamol) was present in 40% of refractory patients (P < 0.006). Objective testing showed that 43.7% of NSAID use was surreptitious. Multivariate logistic regression analysis identified only NSAID and analgesic abuse and the number of relapses as individually affecting refractoriness. CONCLUSIONS: NSAID and analgesic abuse is the single most important exogenous factor associated with refractoriness. H. pylori infection emerges as an important intrinsic factor, but almost a quarter of refractory patients cannot be linked to either NSAID use or H. pylori infection. PMID- 7557078 TI - Turnover of the gastric H+,K(+)-adenosine triphosphatase alpha subunit and its effect on inhibition of rat gastric acid secretion. AB - BACKGROUND & AIMS: The rate of turnover and the effect of inhibition of acid secretion on the turnover of gastric H+,K(+)-adenosine triphosphatase (ATPase) is unknown. The aim of this study was to determine the turnover of the alpha subunit of gastric H+,K(+)-ATPase in rats under control conditions and during inhibition of acid secretion by ranitidine or omeprazole. METHODS: The turnover of the alpha subunit of the ATPase was determined by measuring the loss of incorporated 35S methionine. This was compared with the rate of recovery of K(+)-stimulated ATPase activity in the omeprazole-treated animals. RESULTS: The half-life of the alpha subunit was 54 hours. A 1-week treatment with omeprazole had no significant effect, but the half-life increased to 125 hours (P < 0.01) after continuous ranitidine infusion. After omeprazole treatment, K(+)-stimulated ATPase activity recovered with a half-time of 15 hours. CONCLUSIONS: The turnover of the gastric ATPase subunit was independent of omeprazole inhibition but was prolonged by ranitidine. The effect of ranitidine suggests that the resting pump in tubulovesicles may turn over more slowly than the stimulated pump in the secretory canaliculus. The rapid recovery of ATPase activity compared with turnover after omeprazole is caused by both H+,K(+)-ATPase synthesis and loss of covalently bound drug. PMID- 7557080 TI - Human intestinal intraepithelial lymphocytes have potent chemotactic activity. AB - BACKGROUND & AIMS: Inflammation is created by the movement of leukocytes toward soluble attractants (chemokines). The aim of this study was to identify the chemokines that attract intestinal lymphocytes. METHODS: Intraepithelial and lamina propria lymphocytes were isolated from human jejunal mucosa of healthy individuals and cultured in interleukin (IL) 2 for 3 days. Migration was assessed using the Boyden transwell assay. Increases in cytoplasmic calcium ion concentration ([Ca2+]i) were measured from changes in fluorescence in Fura-2 loaded lymphocytes on exposure to the cytokines. RESULTS: A large number of intraepithelial lymphocytes migrated toward IL-8 and RANTES (regulated on activation, normal T cell expressed and secreted), and a small number migrated toward IL-10. In contrast, only a few lamina propria lymphocytes migrated toward IL-8, and none responded to the other chemokines tested. The chemokines IL-8 and IL-10, but not RANTES, increased [Ca2+]i in intraepithelial lymphocytes; this calcium mobilization was not affected by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicating that calcium is released from intracellular sources. None of the factors caused calcium mobilization by lamina propria lymphocytes. CONCLUSIONS: This study shows that intraepithelial lymphocytes have greater migratory and calcium-mobilizing activity than lamina propria lymphocytes and that these two activities do not correlate with each other. PMID- 7557081 TI - Higher proximal duodenal mucosal bicarbonate secretion is independent of Brunner's glands in rats and rabbits. AB - BACKGROUND & AIMS: Duodenal bicarbonate secretion is impaired in patients with duodenal ulcer. Before characterization of any cellular transport defect is possible, the origin of duodenal bicarbonate (epithelial cells and/or Brunner's glands) must be determined. The aim of this study was to determine the role of Brunner's glands in duodenal bicarbonate secretion. METHODS: Rats, which have Brunner's glands only in the proximal duodenum, and rabbits, which have Brunner's glands throughout the duodenum, were anesthetized. Basal and stimulated (with HCl, prostaglandin E2, and vasoactive intestinal polypeptide [VIP]) bicarbonate secretion was measured in three isolated intestinal segments: proximal duodenum, distal duodenum, and proximal jejunum. Mucosal surface area and Brunner's gland thickness was quantitated in each segment. RESULTS: Secretion rates in proximal and distal duodenum and proximal jejunum were significantly different. Normalized proximal-to-distal duodenal gradients in bicarbonate secretion were similar in the two species despite significantly different gradients of Brunner's gland thickness. In rabbits, gradients of bicarbonate secretion and Brunner's gland thickness were not correlated. In both species, HCl, prostaglandin E2, and VIP stimulated secretion in all three segments. If the agonists specifically stimulated Brunner's gland bicarbonate secretion, relationships between gradients of bicarbonate secretion and Brunner's gland thickness would have been anticipated. This was not observed. CONCLUSIONS: The higher rates of bicarbonate secretion in the proximal duodenum than in the distal duodenum and proximal jejunum are independent of Brunner's glands. PMID- 7557083 TI - Indomethacin-induced gastric injury and leukocyte adherence in arthritic versus healthy rats. AB - BACKGROUND & AIMS: Arthritic patients are at greater risk of developing nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers than other NSAID users. Leukocyte adherence to the vascular endothelium has been suggested to play an important role in the pathogenesis of experimental NSAID-associated gastropathy. The aim of this study was to examine the role of leukocyte adherence in NSAID-induced gastric injury in healthy and adjuvant-induced arthritic rats. METHODS: Leukocyte adherence was examined using intravital microscopy before and after indomethacin administration. The role of CD18 and intercellular adhesion molecule 1 (ICAM-1) in indomethacin-induced gastric injury and leukocyte adherence was examined using specific monoclonal antibodies against these molecules. RESULTS: Indomethacin (5-10 mg/kg) caused significantly more gastric damage in arthritic than normal rats, and the former group had significantly greater levels of leukocyte adherence to mesenteric postcapillary venules under basal conditions. Dexamethasone markedly reduced basal and indomethacin-induced leukocyte adherence and the extent of gastric damage. In arthritic rats, pretreatment with a monoclonal antibody directed against ICAM-1 significantly reduced gastric damage and leukocyte adherence to the levels observed in healthy rats, whereas an antibody directed against CD18 had no effect. CONCLUSIONS: Indomethacin-induced damage in healthy and arthritic rats is largely dependent on ICAM-1 expression. The increased susceptibility of arthritic rats to indomethacin induced gastric injury may be partly related to elevated levels of ICAM-1 dependent leukocyte adherence. PMID- 7557084 TI - Cholecystokinin-B receptor ligands of the dipeptoid series act as agonists on rat stomach histidine decarboxylase. AB - BACKGROUND & AIMS: The effect of gastrin on the enterochromaffin-like cells in the rat stomach is mediated by cholecystokinin (CCK)-B receptors and manifested as activation of histidine decarboxylase (HDC). The dipeptoids PD 136450, PD 135158, and PD 134308 are thought to be selective CCK-B receptor antagonists. The effects of the dipeptoids and of gastrin-17 and sulfated CCK-8 on rat stomach HDC activity were examined. METHODS: Drugs were infused intravenously or subcutaneously to fasted rats, and the HDC activity was determined. RESULTS: The dipeptoids activated HDC with maximal responses (50%-60% of the maximal response to gastrin) at 1 mumol.kg-1.h-1. Rat gastrin-17 activated HDC maximally at 3 nmol.kg-1.h-1, and sulfated CCK-8 produced maximal response at 20 nmol.kg-1.h-1. The CCK-B receptor antagonist L-365,260 inhibited the HDC activation induced by gastrin, sulfated CCK-8, or the dipeptoids. The dipeptoids did not inhibit the gastrin-induced HDC activation. CONCLUSIONS: Gastrin, sulfated CCK-8, and the dipeptoids activated rat stomach HDC. L-365,260 but not devazepide inhibited the HDC activation. Thus, the dipeptoids, which failed to inhibit the gastrin-induced HDC activation, act as CCK-B receptor agonists and not as antagonists. It is important to recognize this to ensure appropriate interpretation of data obtained with these drugs. PMID- 7557082 TI - Regional chemoprevention of carcinogen-induced tumors in rat colon. AB - BACKGROUND & AIMS: Rat colon neoplasms are distributed 60% in the distal colon (DC) and 40% in the proximal colon (PC), similar to distribution of colon cancers in the industrialized world. The effects of chemopreventive agents that affect colon tumor incidence on the distribution of colon tumors were studied. METHODS: Colon tumor distribution, numbers, and volumes were measured in the DC and PC of rats administered azoxymethane (15 mg/kg subcutaneously 2x) as an initiating agent and fed diets containing various chemopreventive agents. RESULTS: In control rats, azoxymethane-induced tumor incidence in the DC exceeded that in the PC, but tumor volume was greater in the PC than the DC. Ellagic acid showed no chemopreventive effect and maintained the PC-DC colon tumor gradient. Oltipraz, a modestly effective chemopreventive agent, principally reduced the incidence of DC tumors. DL-d-difluoromethylornithine also greatly altered tumor number in the DC compared with the PC. In contrast, piroxicam (400 ppm) reduced PC tumors by 82% but DC tumors only by 57%. With all regimens, tumor volume remained greater in the PC than the DC. CONCLUSIONS: Chemopreventive agents have a selective regional effect on colon tumorigenesis in the rat. Elucidation of the mechanism for these differences may help clarify the modes of action of chemopreventive agents in colon cancer. PMID- 7557086 TI - Intragastric capsaicin stimulates colonic motility via a neural reflex in conscious dogs. AB - BACKGROUND & AIMS: Capsaicin is known to contract smooth muscles and to have an excitatory action on thin, primary afferent neurons. The aim of this study was to test the hypothesis that intragastric capsaicin may have an excitatory effect on colonic motility. METHODS: Mongrel dogs equipped with five strain-gauge force transducers on the colon (C1-C5) were used. In 5 dogs, the effect of intragastric capsaicin (0.05, 0.1, 0.3, and 0.5 mg/kg) on colonic motility in the presence or absence of the cholinergic blockers atropine and hexamethonium was studied. In 4 dogs, the effect of capsaicin (0.5 mg/kg) administration into a vagally innervated or denervated gastric pouch on colonic motility was investigated. RESULTS: Motility at C2-C3, C1-C3, and C1-C5 was significantly increased by 0.1, 0.3, and 0.5 mg/kg of capsaicin, respectively. The excitatory effect of capsaicin on colonic motility was inhibited by atropine and hexamethonium. Colonic motility at C1-C4 was significantly increased by capsaicin administration into a vagally innervated pouch but was not affected by capsaicin administration into a vagally denervated pouch. CONCLUSIONS: These results indicate that intragastric capsaicin predominantly stimulates motility in the proximal half of the colon through neural pathways, probably through a vagovagal reflex, but sympathetic involvement cannot be excluded. PMID- 7557085 TI - Prophylactic colectomy or surveillance for chronic ulcerative colitis? A decision analysis. AB - BACKGROUND & AIMS: The treatment of patients with long-standing ulcerative colitis involving the entire colon is controversial. The aim of this study was to examine the effectiveness of surveillance colonoscopy or prophylactic colectomy on colon cancer mortality in patients with chronic ulcerative colitis. METHODS: Using decision analysis, computer cohort simulation of patients with ulcerative colitis was performed to evaluate 17 strategies including no colonoscopic surveillance, surveillance at varying intervals, and prophylactic proctocolectomy with ileal pouch-anal anastomosis. The model examined which biopsy results (low grade dysplasia, high-grade dysplasia, or cancer) should lead to proctocolectomy and ileal pouch-anal anastomosis. Published data on the incidence of cancer with ulcerative colitis, the sensitivity and specificity of colonoscopy with biopsy, the risks of colonoscopy and surgery, and the prognosis with colon cancer were used. RESULTS: For a 30-year-old patient with pancolitis for 10 years, the model suggests that prophylactic colectomy would increase life expectancy by 2-10 months compared with surveillance and by 1.1-1.4 years compared with no surveillance. Surveillance would improve life expectancy by 7 months to 1.2 years compared with no surveillance. In sensitivity analysis, results were most affected by the cumulative incidence of cancer in patients with chronic ulcerative colitis. CONCLUSIONS: Either surveillance or prophylactic colectomy should increase life expectancy in patients with ulcerative colitis. PMID- 7557087 TI - Paracellular intestinal transport of six-carbon sugars is negligible in the rat. AB - BACKGROUND & AIMS: Active D-glucose absorption has been theorized to increase convective flow and enhance tight junction permeability such that paracellular transport becomes the major mechanism of D-glucose absorption. This concept was tested in rats by measuring the absorption of four gavaged, nonmetabolizable six carbon sugars (L-glucose, L-galactose, L-mannose, and D-mannitol) thought to be absorbed solely by the paracellular route. METHODS: Uptake of gavaged probes was measured by recovery in 24-hour urine specimen collections. RESULTS: L-glucose (71.2% +/- 2.4%) absorption exceeded that of the other probes (1.4%-9%). Coadministration of 3.0 mol/L D-glucose, 0.22 mol/L D-glucose, or chow significantly reduced the absorption of L-glucose to 38.1% +/- 7.2%, 61% +/- 3.3%, and 53.6% +/- 3.5%, respectively, but did not influence the absorption of the other six-carbon probes. CONCLUSIONS: (1) L-glucose seems to have a weak affinity for a D-glucose carrier and is not a marker of paracellular transport, and (2) paracellular transport accounts for a minimal fraction of D-glucose uptake; this fraction is not enhanced by ingestion of D-glucose or chow. PMID- 7557091 TI - The effects of recombinant human hemoglobin on esophageal motor functions in humans. AB - BACKGROUND & AIMS: Nitric oxide controls lower esophageal sphincter (LES) relaxation and esophageal peristalsis in opossums, but its role in the control of esophageal motility in humans is not defined. Hemoglobin inactivates NO by binding it. Recombinant human hemoglobin (rHb1.1) was used to test the hypothesis that NO mediates esophageal motor functions in humans. METHODS: rHb1.1 or human serum albumin was administered intravenously to fasting male volunteers. Esophageal manometric studies were performed before, during, and up to 6 hours after the infusion. RESULTS: rHb1.1 increased the velocities of peristaltic contractions to produce simultaneous contractions in 6 of 9 subjects. It increased the amplitude and duration of contractile waves in the esophagus. There was no consistent effect on the resting tone of the LES, but LES relaxation was inhibited. Spontaneous, simultaneous high-pressure contractions occurred in 8 of 9 subjects. Lower retrosternal chest pain during swallowing was observed in 4 subjects. CONCLUSIONS: rHb1.1 interfered with esophageal peristalsis and LES relaxation. It precipitated esophageal spasm in some subjects. These data support the hypothesis that the timing of smooth muscle esophageal peristalsis and LES relaxation are mediated by NO. They suggest that some disorders of esophageal motor function may result from defects in NO neuromuscular communication. PMID- 7557090 TI - Verification of the lactase site of rat lactase-phlorizin hydrolase by site directed mutagenesis. AB - BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity. PMID- 7557088 TI - Nitric oxide modulates pepsinogen secretion induced by calcium-mediated agonist in guinea pig gastric chief cells. AB - BACKGROUND & AIMS: Nitric oxide, a putative cellular messenger synthesized from L arginine, is a powerful modulator of gastric motility and secretions. The aim of this study was to investigate whether (1) guinea pig gastric chief cells express NO synthase, (2) NO modulates the pepsinogen secretion and guanosine 3',5'-cyclic monophosphate (cGMP) generation induced by calcium (Ca2+)-mediated agents, and (3) NO donors and cGMP analogues stimulate pepsinogen release. METHODS: Chief cells were prepared by sequential digestion with collagenase and Ca2+ chelation. NO generation was measured by determining the NO coproduct citrulline. RESULTS: NO synthase immunoreactivities were constitutively expressed in approximately 70% chief cells. Carbachol (10 mumol/L) caused a 4- 6-fold increase in pepsinogen release, citrulline generation, intracellular Ca2+ concentration ([Ca2+]i) and cGMP concentration. These effects were concentration dependently inhibited by NG monomethyl-L-arginine (L-NMMA). As gastrin, cholecystokinin, thapsigargin, and Ca2+ ionophore increased NO generation, [Ca2+]i seemed to regulate NO synthase activity. [Ca2+]i chelator and calmodulin antagonist inhibited the carbachol induced pepsinogen secretion and NO generation. Preincubating the cells with L NMMA had no effect on carbachol-stimulated inositol triphosphate generation or [Ca2+]i or Ca(2+)-dependent adenosine triphosphatase levels. Nitrovasodilator agents and 8-bromo-cGMP stimulated pepsinogen release. CONCLUSIONS: Gastric chief cells express a Ca2+/calmodulin-dependent NO synthase. NO modulates the stimulatory effect of Ca(2+)-mediated agonists on pepsinogen release. PMID- 7557089 TI - Cytoprotective effects of CI-959 in the rat gastric mucosa: modulation of leukocyte adhesion. AB - BACKGROUND & AIMS: CI-959 is an anti-inflammatory agent that inhibits neutrophil adhesion, respiratory burst, and mast cell histamine release in vitro. In view of the emerging role of neutrophils in gastric erosive damage, the goals of this study were to assess the gastric cytoprotective effects of CI-959 and identify the mechanism responsible for this action. METHODS: Cytoprotective effects in the rat nonsteroidal anti-inflammatory drug and ethanol erosion models were assessed using image analysis. The in vivo effects of CI-959 on gastric acid secretion, arachidonic acid metabolism, and intracellular sulfhydryl and leukocyte adhesion were also examined. RESULTS: CI-959 protected prophylactically against the erosive damage induced by aspirin, indomethacin, and ethanol with 50% effective doses (ED50s) of 0.05, 1.0, and 0.07 mg/kg administered orally, respectively. When administered after indomethacin or ethanol, CI-959 had no effect on the healing of erosive damage. CI-959 did not alter gastric acid secretion, arachidonic acid metabolism, or intracellular sulfhydryl levels. In vivo, CI-959 blocked leukocyte adhesion in intravital microscopy studies using indomethacin (ED50, < 5 mg/kg orally) or platelet-activating factor (50% inhibiting concentration, approximately 10 mumol/L) as the adhesion stimulus. CONCLUSIONS: The most likely mechanism responsible for the cytoprotective effects of CI-595 is its inhibitory effects on leukocyte trafficking and/or adhesion. PMID- 7557093 TI - Continuous prazosin administration in cirrhotic patients: effects on portal hemodynamics and on liver and renal function. AB - BACKGROUND & AIMS: Hepatic vascular resistance is influenced by alpha-adrenergic tone. The aim of this study was to investigate the effects of continuous blockade of alpha-adrenoceptors with prazosin on hemodynamics, liver function, and renal function and whether the association of propranolol or furosemide enhances the portal pressure lowering effect of prazosin. METHODS: Cirrhotic patients with portal hypertension were studied at baseline and after a 3-month course of prazosin (n = 18) or placebo (n = 10). RESULTS: No changes were observed in the placebo group. Prazosin decreased the hepatic venous pressure gradient (HVPG) while increasing hepatic blood flow. Liver function improved as shown by an increase in hepatic and intrinsic hepatic clearances of indocyanine green and galactose elimination capacity. A significant reduction in mean arterial pressure and systemic vascular resistance was associated with increases in plasma renin activity and aldosterone concentration and a decrease in glomerular filtration rate. The plasma volume increased significantly, and 6 patients developed edema. The association of propranolol (n = 8) but not furosemide (n = 7) to prazosin increased the reduction in HVPG and attenuated the increase in plasma renin activity. CONCLUSIONS: In cirrhotic patients, continuous prazosin administration reduces portal pressure and improves liver perfusion and function but favors sodium and water retention. The association of propranolol enhances the decrease in portal pressure, suggesting a potential benefit from this combined therapy. PMID- 7557092 TI - Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. AB - BACKGROUND & AIMS: The mechanisms causing liver injury in cholestatic diseases are unclear. The hypothesis that accumulation of hydrophobic bile acids in hepatocytes during cholestasis leads to generation of oxygen free radicals and oxidative injury was tested. The aim of this study was to determine if hydrophobic bile acid toxicity is associated with increased hydroperoxide generation in isolated rat hepatocytes and mitochondria. METHODS: Hepatocytes were exposed to taurochenodeoxycholic acid (TCDC; 0-2000 mumol/L) or taurocholic acid (TC; 1000 mumol/L), and cellular injury, intracellular hydroperoxide generation, and thiobarbituric acid-reacting substances (TBARS) were measured. Isolated mitochondria were incubated with 400 mumol/L chenodeoxycholic acid or 400 mumol/L cholic acid, and hydroperoxide generation was measured fluorometrically. RESULTS: Hepatocyte injury, hydroperoxide generation, and TBARS increased over 4 hours on exposure to TCDC but not TC. Hydroperoxide generation preceded hepatocyte injury and accumulation of TBARS. Preincubation of hepatocytes with the antioxidant, d-alpha-tocopheryl succinate, completely abrogated cellular injury, hydroperoxide, and TBARS generation. Hydroperoxide generation was increased in mitochondria exposed to chenodeoxycholic acid. CONCLUSIONS: Intracellular generation of hydroperoxides by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity. Antioxidants may be of benefit in cholestasis. PMID- 7557094 TI - Cytochrome P450 2E1 and glutathione S-transferase M1 polymorphisms and susceptibility to hepatocellular carcinoma. AB - BACKGROUND & AIMS: Genetic polymorphisms in enzymes involved in carcinogen metabolism have been found to influence susceptibility to cancer. The aim of this study was to examine whether cytochrome P450 2E1 (CYP2E1) and/or glutathione S transferase M1 (GSTM1) genetic polymorphisms were related to susceptibility to hepatocellular carcinoma (HCC). METHODS: Genotyping of CYP2E1 and GSTM1 was performed using the polymerase chain reaction on peripheral white blood cell DNA from 30 patients with HCC and 150 controls nested in a cohort study. RESULTS: The c1/c1 genotype of CYP2E1, detected by PstI or RsaI digestion, was found in 83.3% of patients with HCC and in 63.3% of controls (P = 0.034). Homozygosity for the c1/c1 genotype significantly increased the risk of developing HCC in cigarette smokers (P = 0.001) but posed no increased risk in those who never smoked. The HCC risk associated with cumulative exposure to cigarette smoke was also more striking in individuals who carried the c1/c1 genotype. Habitual alcohol drinking modified the HCC risk of cigarette smoking among those with the c1/c1 genotype. No association with the risk of HCC was observed for the DraI polymorphism of CYP2E1 or for the GSTM1-null genotype. CONCLUSIONS: Polymorphisms of CYP2E1 may play an important role in cigarette smoking-related hepatocarcinogenesis. PMID- 7557097 TI - A meta-analysis of somatostatin versus vasopressin in the management of acute esophageal variceal hemorrhage. AB - BACKGROUND & AIMS: Although sclerotherapy is the current standard therapy for bleeding esophageal varices, the best method for initial control is unclear. The aim of this meta-analysis was to compare the efficacy and toxicity of somatostatin and vasopressin in short-term treatment of hemorrhage from esophageal varices. METHODS: Using MEDLINE, all randomized trials comparing somatostatin with vasopressin in subjects with endoscopically documented acute esophageal variceal bleeding were identified. The quality of each study was critically and independently evaluated, and quantitative data for initial cessation of bleeding, sustained control of bleeding, and major adverse effects were abstracted. The relative risk (RR) and number needed to be treated were calculated. RESULTS: The RR or likelihood of achieving initial control of bleeding with somatostatin vs. vasopressin was 1.62 (95% confidence interval [CI], 1.37-1.93), and the number needed to be treated was 3.7, i.e., between 3 and 4 patients would have to be treated with somatostatin for 1 patient to derive additional benefit over vasopressin. For trials that measured sustained control of bleeding, somatostatin was superior to vasopressin (RR, 1.28 [95% CI, 1.00 1.65]; number needed to be treated, 8.8). The risk of adverse effects was greater for subjects given vasopressin (10% vs. 0%; P = 0.00007). CONCLUSIONS: This meta analysis suggests that somatostatin is more efficacious in controlling acute hemorrhage from esophageal varices and has a lower risk of adverse effects than vasopressin. PMID- 7557095 TI - Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. AB - BACKGROUND & AIMS: Based on a recently cloned rat liver organic anion transporter, we attempted to clone the corresponding human liver organic anion transporting polypeptide. METHODS: A human liver complementary DNA library was screened with a specific rat liver complementary DNA probe. The human liver transporter was cloned by homology with the rat protein and functionally characterized in Xenopus laevis oocytes. RESULTS: The cloned human liver organic anion transporting polypeptide consists of 670 amino acids and shows a 67% amino acid identity with the corresponding rat liver protein. Injection of in vitro transcribed complementary RNA into frog oocytes resulted in the expression of sodium-independent uptake of [35S]bromosulfophthalein (Michaelis constant [Km], approximately 20 mumol/L), [3H]cholate (Km, approximately 93 mumol/L), [3H]taurocholate (Km, approximately 60 mumol/L), [14C]glycocholate, [3H]taurochenodeoxycholate, and [3H]tauroursodeoxycholate (Km, approximately 19 mumol/L). Northern blot analysis showed cross-reactivity with messenger RNA species from human liver, brain, lung, kidney, and testes. Polymerase chain reaction analysis of genomic DNA from a panel of human-rodent somatic cell hybrids mapped the cloned human organic anion transporter to chromosome 12. CONCLUSIONS: These studies show that the cloned human liver organic anion transporter is closely related to, but probably not identical to, the previously cloned rat liver transporter. Furthermore, its additional localization in a variety of extrahepatic tissues suggests that it plays a fundamental role in overall transepithelial organic anion transport of the human body. PMID- 7557096 TI - Diagnostic utility of contrast echocardiography and lung perfusion scan in patients with hepatopulmonary syndrome. AB - BACKGROUND & AIMS: Two modalities, contrast echocardiography and lung perfusion scan, are used to identify intrapulmonary vascular dilatation and diagnose hepatopulmonary syndrome (HPS), but a comparison of these two procedures has not been performed. The aim of this study was to compare the use of these diagnostic modalities in detecting intrapulmonary vascular dilatation and diagnosing HPS. METHODS: Forty consecutive outpatients with biopsy-proven cirrhosis had contrast echocardiography, a lung perfusion scan, and arterial blood gases analyzed. RESULTS: Fifteen of 40 cirrhotics (38%) had positive contrast echocardiogram results. Seven patients with positive echocardiogram results had gas exchange abnormalities and could be considered to have HPS (7 of 40 [17.5%]). Three of these patients were hypoxemic and had no concurrent cardiopulmonary disease, and each had positive contrast echocardiogram and lung perfusion scan results and were readily diagnosed as having HPS. The other 4 patients (3 hypoxemic and 1 normoxemic with an elevated alveolar-arterial gradient) had coexisting intrinsic lung disease and/or chest radiograph abnormalities complicating the diagnosis of HPS, and each had positive echocardiogram and negative lung scan results. The remaining 8 patients with positive echocardiogram results had normal lung scan and normal gas exchange results. No patient had positive lung scan and negative contrast echocardiogram results. CONCLUSIONS: Contrast echocardiography is the most useful screening test for intrapulmonary vasodilatation and may be positive more frequently than lung perfusion scans in patients with HPS. PMID- 7557100 TI - Characterization and response to interleukin 1 and tumor necrosis factor of immortalized murine biliary epithelial cells. AB - BACKGROUND & AIMS: Biliary epithelial cells are the target of numerous immune mediated liver diseases, yet their role in pathogenesis remains unclear because of difficulties in obtaining pure preparations. The aim of this study was to establish pure clones of immortalized murine intrahepatic biliary epithelial cells. METHODS: The transgenic mouse harboring the SV40 thermosensitive immortalizing mutant gene TsA58 under the control of the major histocompatibility complex class I promoter was used to establish conditionally immortalized intrahepatic bile duct cells by countercurrent centrifugal elutriation and clonal dilution. RESULTS: Immortalized clones of cells expressing cytokeratin 19, which organized themselves into ductlike structures, were obtained. On electron microscopic sections, cells were well differentiated and polarized. Cells proliferate in response to epidermal growth factor, interleukin 1 alpha, and tumor necrosis factor alpha. Using the reverse-transcriptase polymerase chain reaction technique, these cells were found to contain messenger RNA, which encodes for the interleukin 1 and tumor necrosis factor receptors. CONCLUSIONS: The availability of unlimited numbers of pure bile duct cells that behave in an identical fashion to biliary epithelial cells from "normal" mice will allow for more rigorous studies of the behavior and function of this epithelium. PMID- 7557098 TI - Hypoaminotransferasemia in patients undergoing long-term hemodialysis: clinical and biochemical appraisal. AB - BACKGROUND & AIMS: It has been reported that patients with chronic renal failure have low serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels as a result of vitamin B6 deficiency. Chronic hepatitis C is common among patients undergoing hemodialysis, and low aminotransferase levels cause diagnostic problems. The aim of this study was to determine the cause of hypoaminotransferasemia. METHODS: Serum levels of vitamin B6 and its coenzyme were reassessed in relation to AST and ALT levels in patients undergoing long term hemodialysis using high-performance liquid chromatography. RESULTS: The mean (+/- SD) serum AST and ALT levels in 304 patients negative for hepatitis B surface antigen and anti-hepatitis C virus were 9.2 +/- 2.4 and 7.4 +/- 1.7 IU/L, respectively. In 556 normal adults, they were 22.7 +/- 5.4 and 18.0 +/- 4.0 IU/L, respectively (P < 0.001). Mean serum vitamin B6 and pyridoxal-5'-phosphate levels in patients undergoing dialysis were not reduced compared with the control, although occasionally patients had low levels. The AST and ALT assay reagents in Japan do not contain added pyridoxal-5'-phosphate; addition of pyridoxal-5' phosphate (0.1 mmol/L) to the reagent significantly increased measurements to similar extents in both groups. CONCLUSIONS: Serum AST and ALT levels in patients undergoing dialysis are very low, but they are not a result of vitamin B6 deficiency. The upper normal limits of AST and ALT levels in patients undergoing dialysis should be reduced considerably, and these levels should be interpreted with caution in the diagnosis of liver disease. PMID- 7557101 TI - Human biliary epithelial cell line Mz-ChA-1 expresses new isoforms of calmodulin dependent protein kinase II. AB - BACKGROUND & AIMS: Calmodulin-dependent protein kinase II is a family of closely related multimeric enzymes that regulate a wide variety of cellular processes. In biliary epithelial cells, this kinase seems to regulate Ca(2+)-dependent CI- currents. The aim of this study was to identify isoforms of this kinase expressed in biliary cells. METHODS: Sequencing of reverse-transcription polymerase chain reaction products identified multiple isoforms in Mz-ChA-1 cells. RESULTS: Two previously identified isoforms (gamma B and gamma C) and three new isoforms (gamma D, gamma E, and gamma F) of calmodulin-dependent protein kinase II were identified. Each of the novel isoforms contains a unique insert of 114 base pairs in the association region. This insert lies outside the previously identified variable region. In addition, gamma D and gamma F contained other deletions (42 and 69 base pairs, respectively) in the variable region. These isoforms are expressed in a variety of tissues, including biliary epithelial and gallbladder cells, but only gamma C is expressed in rat hepatocytes. CONCLUSIONS: Identification of these biliary kinase isoforms paves the way for future studies that will elucidate the role of individual isozymes in agonist-stimulated biliary Cl- and fluid secretion. PMID- 7557099 TI - Treatment of the cholesterol biosynthetic defect in Smith-Lemli-Opitz syndrome reproduced in rats by BM 15.766. AB - BACKGROUND & AIMS: The Smith-Lemli-Opitz syndrome is a recessive inherited disorder characterized by neurological developmental defects and dysmorphic features with a defect in cholesterol synthesis at the conversion of 7 dehydrocholesterol to cholesterol. BM 15.766 inhibits 7-dehydrocholesterol-delta 7-reductase and reproduces the biochemical defect. The aim of this study was to investigate the effects of cholesterol, cholic acid, and lovastatin feeding on rats fed BM 15.766. METHODS: Plasma cholesterol and 7-dehydrocholesterol concentrations were related to the hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. RESULTS: With the inhibitor treatment, plasma cholesterol concentrations decreased 67%; 7-dehydrocholesterol concentrations increased from trace to 17 mg/dL; and hepatic HMG-CoA reductase activity and messenger RNA levels were stimulated 74% and two times, respectively. In inhibitor-treated rats, feeding cholesterol increased plasma cholesterol concentrations 3.7 times, decreased 7-dehydrocholesterol concentrations 88%, and reduced elevated HMG-CoA reductase activity and messenger RNA levels 74% and 49%. Feeding cholic acid increased plasma cholesterol without reducing 7-dehydrocholesterol concentrations. The combination of cholic acid and cholesterol enhanced plasma cholesterol 9.5 times without decreasing 7-dehydrocholesterol levels. Feeding lovastatin depressed plasma cholesterol further without reducing 7 dehydrocholesterol levels. CONCLUSIONS: Cholesterol is essential to correct abnormal cholesterol synthesis induced by BM 15.766 in rats by expanding the pool and inhibiting HMG-CoA reductase. Neither cholic acid nor lovastatin are effective separately, but cholic acid plus cholesterol may offer some additional benefit. PMID- 7557102 TI - Subclinical Addison's disease: a cause of persistent abnormalities in transaminase values. AB - A common reason for referring patients to hepatologists is persistently abnormal serum transaminase levels with vague constitutional symptoms. In the United Kingdom, these abnormalities are most often caused by a fatty liver either related to obesity or alcohol abuse; they are less commonly caused by chronic liver disease, particularly chronic viral hepatitis, autoimmune hepatitis, or chronic biliary disease. Endocrine disease is rarely a cause of these abnormalities, although hypothyroidism and hyperthyroidism are well-recognized causes. Addison's disease has been only reported once in the literature by R. G. Olsson as a cause of increased transaminase levels associated with constitutional symptoms; it is not mentioned in textbooks on hepatology. Three patients with Addison's disease are reported here, all of whom had increased serum transaminase levels for more than 6 months before the recognition of the hypoadrenalism with resolution to normal after steroid replacement. Hepatologists should consider subclinical Addison's disease as a cause of persistently increased transaminase levels with constitutional symptoms in the absence of evidence for fatty liver as well as viral and autoimmune markers. PMID- 7557103 TI - Esophageal achalasia and coexistent upper esophageal sphincter relaxation disorder presenting with airway obstruction. AB - Acute airway obstruction associated with esophageal achalasia is an uncommon but life-threatening complication. The pathophysiology of this phenomenon has not been fully defined. A fully documented case of coexistent esophageal achalasia and upper esophageal sphincter relaxation abnormality presenting with airway obstruction is reported. The patient was initially treated with Heller's myotomy but had a recurrence of respiratory distress. She was successfully treated by cricopharyngeal myotomy. The causes of gas entrapment and respiratory distress are likely to be due to failure of both swallow- and distention-induced upper esophageal sphincter relaxation. Cricopharyngeal myotomy is an effective treatment for this complication, probably by facilitating esophagopharyngeal gas venting. PMID- 7557104 TI - Celiac disease and alopecia areata: report of a new association. AB - Celiac disease is frequently associated with other autoimmune disorders but has never been reported in association with alopecia areata. In a routine clinical practice, 3 patients with such an association were observed. In one of the patients, celiac disease was diagnosed after the occurrence of malabsorption symptoms. In the youngest patient, a 14-year-old boy, gluten-free diet resulted in complete regrowth of scalp and body hair. A prospective screening program for celiac disease using antigliadin and antiendomysial antibodies was therefore set up in 256 consecutive outpatients with alopecia areata. Three patients, all completely asymptomatic for intestinal diseases, were found to be positive and underwent biopsy. Histological analysis showed a flat intestinal mucosa consistent with the diagnosis of celiac disease. The results show that alopecia areata may constitute the only clinical manifestation of celiac disease and that the association between these two conditions is a real one because the observed frequency of association is much greater than can be expected by chance. It is suggested that antigliadin and antiendomysial antibodies should be included in the work-up of patients with alopecia areata. PMID- 7557105 TI - Cholangitis associated with paroxysmal nocturnal hemoglobinuria: another instance of ischemic cholangiopathy? AB - Ischemia is increasingly recognized as a cause of cholangiopathy. The aim of this study was to report a case of association of paroxysmal nocturnal hemoglobinuria with abrupt-onset cholangiopathy. Anomalies resembling sclerosing cholangitis were documented in a patient suffering from recurrent biliary pain. None of the conditions that have been associated with primary sclerosing cholangitis or other forms of cholangiopathy was present, but shortly thereafter, paroxysmal nocturnal hemoglobinuria occurred. Hepatic vein thrombosis later complicated the course of the disease. Because the fortuitous coincidence of these uncommon conditions is unlikely, this case indicates that paroxysmal nocturnal hemoglobinuria is a cause of ischemic cholangiopathy. Other thrombogenic conditions may also be implicated in some instances of apparently idiopathic cholangiopathy. PMID- 7557106 TI - Experimental models of inflammatory bowel disease. PMID- 7557109 TI - Pouchitis and neoplasia in the pelvic ileal reservoir. PMID- 7557108 TI - Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity. AB - BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients. PMID- 7557107 TI - In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer. AB - BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer (HNPCC) has been linked recently to a defect in repairing mismatched nucleotides in DNA. The aim of this study was to screen for germline mutations that result in prematurely truncated proteins in two of the mismatch repair genes identified at this time, hMLH1 and hMSH2, in a consecutive series of patients belonging to familial aggregations of colorectal cancer. METHODS: Nineteen individuals with colorectal cancer from 19 families were consecutively referred because of a strong positive family history of colorectal cancer. Premature truncation mutations in hMLH1 and hMSH2 were sought from lymphocyte RNA by using an in vitro transcription/translation (IVTT) assay. RESULTS: Protein truncating mutations in the hMLH1 or hMSH2 genes were found in 50% of families with HNPCC (6 of 12) but were not observed in any of the remaining familial aggregations that did not fulfill the standard criteria for HNPCC. In some of the IVTT-positive samples, the mutations were characterized by genomic sequencing. CONCLUSIONS: IVTT may be a practical method to accomplish primary screening of germline mutations in DNA mismatch pair genes in HNPCC; however, a broader approach is necessary to obtain a more complete picture of the mutational spectrum in HNPCC and other familial aggregations of colorectal cancer. PMID- 7557110 TI - Relationship of gastrin processing to colon cancer. PMID- 7557113 TI - Stem cell factor and intestinal lymphoepithelial communication. PMID- 7557112 TI - Physiological regulation of intestinal epithelial tight junctions as a consequence of Na(+)-coupled nutrient transport. PMID- 7557111 TI - Colitic cancer: supervision, surveillance, or surgery? PMID- 7557115 TI - Hepatocellular carcinoma and hepatitis B. PMID- 7557114 TI - Stent or surgery for the palliation of malignant biliary obstruction: is the choice clear now? PMID- 7557116 TI - Conscious sedation for gastroscopy. PMID- 7557118 TI - Remodeling the hypergastrinemia model. PMID- 7557117 TI - Photodynamic therapy for early esophageal cancer. PMID- 7557119 TI - Complications of parenteral nutrition. PMID- 7557121 TI - NHE-1 isoform of the Na+/H+ antiport is expressed in the rat and rabbit esophagus. AB - BACKGROUND & AIMS: Rabbit esophageal cells show an amiloride-sensitive Na+/H+ antiporter activity. Several distinct molecular isoforms of the Na+/H+ exchanger family (NHE) are reported to be present in the gastrointestinal tract of rats and rabbits. The aim of this study was to examine which isoforms are present in rabbit and rat esophageal cells and whether this isoform could be up-regulated by serum factors. METHODS: Specific primers designed from the rat NHE-1-4 and the rabbit NHE-1-3 isoform sequences were used for reverse-transcription polymerase chain reaction analysis with RNA from rabbit and rat esophageal cells. Ribonuclease protection assay was used to determine the serum-induced up regulation of NHE-1. Antibodies raised against the NHE-1 C-terminal fragment were used for Western blotting with rabbit esophageal membranes. RESULTS: In both the rat and rabbit esophagus, only the NHE-1 isoform messenger RNA could be detected. The NHE-1 messenger RNA, detected in rabbit esophageal cells grown from tissue explants, was up-regulated by serum factors. The antibody detected a 95 kilodalton protein in esophageal cell membranes. CONCLUSIONS: The rabbit and rat esophagus exclusively express the NHE-1 isoform, hypothesized to be involved in cytoplasmic pH regulation. Therefore, the results of this study suggest a role for NHE-1 in protecting cells against gastric acid that is refluxed into the esophagus. PMID- 7557120 TI - Effect of endogenous hypergastrinemia on gastrin receptor expressing human colon carcinoma transplanted to athymic rats. AB - BACKGROUND & AIMS: The effect of endogenous hypergastrinemia on growth of human colon carcinoma is not known. Our aim was to study the growth of human colon carcinoma in an animal model with endogenous hypergastrinemia. METHODS: Human colon carcinoma was transplanted to the colon of 40 athymic rats. Of these, 25 underwent gastric fundectomy to accomplish endogenous hypergastrinemia, and 15 were sham operated to serve as controls. The duration of the study was 8 weeks. During the last week, 12 fundectomized animals received a gastrin (cholecystokinin B) receptor antagonist. Metaphase arrest index, local invasion, and distant spread of the tumor were investigated. Expression of gastrin and cholecystokinin B receptor messenger RNA was examined by reverse-transcription polymerase chain reaction. RESULTS: Tumor spread by direct extension outside the colon was observed in all animals, and liver metastases were observed in 10 of the 25 fundectomized animals. Sham-operated animals showed none of these features. The metaphase arrest index of the tumor did not differ between fundectomized animals given the cholecystokinin B receptor antagonist and sham operated animals, whereas it was significantly increased in fundectomized animals not given the antagonist. The tumor expressed both gastrin and cholecystokinin B receptor messenger RNA. CONCLUSIONS: The results indicate that endogenous hypergastrinemia may promote proliferation and spread of human colon carcinoma expressing cholecystokinin B receptor. PMID- 7557123 TI - Modulation of esophageal responses to magnetic stimulation of the human brain by swallowing and by vagal stimulation. AB - BACKGROUND & AIMS: Although magnetic stimulation of the human cerebral cortex activates corticoesophageal pathways, it is not known whether these pathways are mediated via brain stem neurons activated during swallowing and by vagal afferents. The aim of this study was to determine the effect of swallowing and vagal stimulation on esophageal electromyographic responses evoked by magnetic stimulation of the human cortex. METHODS: Magnetic stimulation of the cortex was performed in 7 subjects at variable intervals after swallows and at intervals ranging from 10 to 200 milliseconds after vagal stimulation in 5 subjects. RESULTS: Between swallows, cortical stimulation induced both early (10.1 +/- 0.5 milliseconds) and late (52.3 +/- 2.3 milliseconds) esophageal electromyographic responses with amplitudes of 66.9 +/- 23.4 microV and 32.9 +/- 7.4 microV, respectively. During swallowing, both responses were facilitated because their latencies decreased (8.9 +/- 0.5 milliseconds and 47.3 +/- 4.8 milliseconds, respectively), whereas their amplitudes increased (203.1 +/- 54 microV and 79.8 +/- 22.5 microV, respectively). Vagal stimulation facilitated the early cortically evoked response. Its latency decreased from 10.2 +/- 0.2 milliseconds to 8 +/- 0.2 milliseconds, whereas its amplitude increased from 145.1 +/- 44 microV to 230.5 +/- 41.6 microV. CONCLUSIONS: Corticoesophageal pathways activated by magnetic stimulation of the human cortex share the same population of brain stem motor neurons to the upper esophagus that are activated during swallowing and vagal stimulation. PMID- 7557125 TI - Corticosteroids increase procollagen gene expression, synthesis, and secretion by human intestinal smooth muscle cells. AB - BACKGROUND & AIMS: Collagen synthesis by smooth muscle cells plays an important role in intestinal fibrosis. Corticosteroids inhibit collagen synthesis in fibroblasts. The aim of this study was to examine the effect of corticosteroids on the expression of collagen by human intestinal smooth muscle (HISM) cells in vitro. METHODS: Collagen synthesis was determined by the sensitivity of radiolabeled protein to collagenase. Secretion was determined by polyacrylamide gel electrophoresis of radiolabeled procollagen in the medium. Procollagen messenger RNA was determined by Northern blot analysis. RESULTS: Collagen synthesis by confluent HISM cells was not affected by corticosteroids at 10(-10) to 10(-5) mol/L but, in subconfluent cultures, was nonspecifically increased 50% at 10(-5) mol/L. Procollagen secretion was nonspecifically increased 60% at 10( 6) mol/L dexamethasone without any effect on the type III/I ratio. Procollagen I and III messenger RNA levels responded in a biphasic manner: a 45%-65% increase at 10(-10) mol/L and a 15% and 30% decrease at 10(-8) and 10(-6) mol/L. In fibroblasts, collagen synthesis was inhibited 85% by dexamethasone, procollagen secretion was decreased 70%, the type III/I ratio decreased from 70:1 to 18:1, and procollagen messenger RNA was inhibited 25% and 60% (types I and III). CONCLUSIONS: Collagen expression by HISM cells is refractory to corticosteroids and, at certain concentrations, is augmented. PMID- 7557124 TI - Bacterial adaptation in patients with short bowel and colon in continuity. AB - BACKGROUND & AIMS: Long-term carbohydrate malabsorption in patients with short bowel and colon in continuity (SBC) could result in a more efficient fermentation. The bacterial fermentation capacity in patients with SBC was assessed. METHODS: Eleven fasting patients with SBC ingested 60 g lactulose with 10 g polyethylene glycol. Stool specimens were analyzed. Patients were compared with 8 normal subjects who ingested 60 g lactulose on two occasions, separated by 8 days during which 20 g lactulose was taken twice daily. Moreover, the daily amount of bacteria excreted in stools was measured in 6 patients with SBC and 6 normal subjects. RESULTS: Despite fast transit time, patients fermented more lactulose and hexoses and had a higher activity of beta-galactosidase in stools than nonadapted normal subjects (P < 0.01); these parameters were roughly similar in patients and adapted normal subjects. The fecal output of short-chain fatty acids was significantly lower in patients than in nonadapted normal subjects (P < 0.03). Patients excreted a significantly greater amount of bacteria in stools than normal subjects (P < 0.05). CONCLUSIONS: In patients with SBC, the capacity of bacterial flora to ferment lactulose and fecal bacterial mass is spontaneously increased, suggesting that hyperfermentation may affect other carbohydrates. Moreover, hyperfermentation is associated with efficient removal of extra short chain fatty acids from fecal water. PMID- 7557122 TI - Manganese superoxide dismutase and reduced nicotinamide adenine dinucleotide diaphorase colocalize in the rat gut. AB - BACKGROUND & AIMS: Superoxide and other free radicals participate in inflammatory bowel disease and ischemia-reperfusion injury. Manganese superoxide dismutase (SOD) scavenges superoxide. Mn SOD is colocalized with reduced nicotinamide adenine dinucleotide (NADH) diaphorase in some tissues. NADH diaphorase histochemistry selectively stains enteric nerves. The aim of this study was to seek colocalization of Mn SOD with NADH diaphorase in the gut, especially in enteric nerves. METHODS: Indirect immunofluorescent staining and histochemistry were used to localize Mn SOD and NADH diaphorase in rat gut. RESULTS: Strong Mn SOD immunoreactivity was found in parietal cells, most intramural nerve cell bodies, the colonic interstitial cells of Cajal (at the submucosa-circular muscle layer interface), and intestinal epithelium cells. Weak to moderate Mn SOD immunoreactivity characterized smooth muscle cells, small submucosal arteries, esophageal striated muscle, esophageal epithelium, gastric epithelium, and intestinal glands. NADH diaphorase histochemistry (with Triton X-100) resulted in identical staining. CONCLUSIONS: Mn SOD and NADH diaphorase are colocalized throughout rat gut with strong activity in enteric nerves and colonic interstitial cells of Cajal. PMID- 7557126 TI - HLA-linked susceptibility and resistance genes in Crohn's disease. AB - BACKGROUND & AIMS: Previous studies have shown a positive association of HLA-DR4 DQ4 with Crohn's disease in the Japanese population, but the association between Crohn's disease and HLA genes has yet to be fully elucidated. The aim of this study was to determine the Crohn's disease/HLA association using a DNA typing method. METHODS: A total of 90 unrelated patients with Crohn's disease and 336 healthy controls were typed for HLA class II genes including DP using DNA typing with the polymerase chain reaction-sequence-specific oligonucleotide probes method. RESULTS: Allelic analysis showed that DRB1*0405, DRB1*0410, DQA1*03, DQB1*0401, and DQB1*0402 are positively associated and DRB1*1501, DRB1*1302, and DQB1*0602 negatively associated with Crohn's disease. DP genes showed no significant association with Crohn's disease. Haplotype analysis showed positive associations with DRB1*0405-DQA1*03-DQB1*0401, DRB1*0410-DQA1*03-DQB1* 0402, and DRB1*0802-DQA1*03-BQB1-0402 haplotypes and negative associations with DRB1*1501 DQA1*0102-DQB1*0602 and DRB1*1302-DQA1*0102-DQB1*0604 haplotypes. CONCLUSIONS: In Crohn's disease in the Japanese population, the HLA-linked disease susceptibility gene is primarily associated with DQB1*04, in which leucine at the 56th position is a unique amino acid, and the disease resistance allele is suggested to be DQA1*0102. PMID- 7557127 TI - A morphological study on the histogenesis of human colorectal hyperplastic polyps. AB - BACKGROUND & AIMS: Little is known about the histogenesis of human colorectal hyperplastic polyp, although this polyp is clinically very common. Therefore, the structural features of the polyp and their implications regarding histogenesis were studied. METHODS: A total of 261 foci were examined using scanning electronmicroscopic observation of the isolated crypt and surface structure, NaOH cell maceration and scanning electron microscopy, dissecting microscopy, and standard histological analysis. RESULTS: In surface view, each polyp crypt was discretely demarcated as in the normal crypt, suggesting that the crypt epithelium had not replaced the adjoining crypt. Notches at the base and various stages of branching, observed in 21.8% of the isolated crypts, were considered to reflect crypt fission. Several polyps with a single crypt mouth consisting of fissioned multiple crypts suggested polyp origin from a single crypt and growth by fission. Juxtaposition of small polyps and their fusion suggested polycentric origin. Almost all polyps showed increased stromal inflammatory cell infiltration and/or a lymphoid follicle at the base. CONCLUSIONS: Hyperplastic polyps originate by the apparent fusion of single abnormal crypts within a small region of mucosa. The polyps grow by fission of the crypt and fusion of the polycentrically originated polyps. Chronic inflammation has some relation to this process. PMID- 7557129 TI - Functional evidence for the presence of nitric oxide synthase in the dorsal motor nucleus of the vagus. AB - BACKGROUND & AIMS: Histochemical studies indicate that reduced nicotinamide adenine dinucleotide phosphate diaphorase, the nitric oxide synthase-related enzyme, is present in the dorsal motor nucleus of the vagus of the cat. We have previously shown in vitro that NO synthase is present in this nucleus in the rat and that the excitatory effect of N-methyl-D-aspartate on these neurons is in part caused by NO formation. The aim of this study was to obtain functional evidence for the presence of NO synthase in the cat dorsal motor nucleus of the vagus. METHODS: L-Glutamate, L-arginine, D-arginine, the NO donor S-nitroso-N acetyl-penicillamine, and the NO synthase inhibitor NG-nitro-L-arginine-methyl ester were unilaterally microinjected into the rostral dorsal motor nucleus of anesthetized cats, and antral and pyloric motility were monitored using extraluminal force transducers. RESULTS: Microinjection of L-arginine increased gastric motility, whereas D-arginine had no effect. Vagotomy eliminated the L arginine-induced increases. Microinjection of S-nitroso-N-acetyl-penicillamine increased antral motility. NG-Nitro-L-arginine-methyl ester prevented L-arginine from exerting an effect on gastric motility. CONCLUSIONS: Motility increases obtained after microinjection of L-arginine into the dorsal motor nucleus and prevention of these motility increases with microinjection of a NO synthase inhibitor provide functional evidence for the presence of NO synthase in the dorsal motor nucleus of the vagus in the cat. PMID- 7557130 TI - Immunohistochemistry of gap junctions in normal and diseased gastric mucosa of humans. AB - BACKGROUND & AIMS: Intercellular communication through gap junctions has been proposed to be an important mechanism for the maintenance of tissue homeostasis. However, few studies have considered the role of gap junctions in gastric mucosa. The purpose of this study was to evaluate the distribution of gap junction specific proteins in normal and diseased gastric mucosa of humans. METHODS: Biopsy specimens were obtained endoscopically. Immunohistochemical staining was performed by standard immunoperoxidase techniques using an anti-connexin 32 monoclonal antibody. RESULTS: In normal gastric mucosa, connexin 32 was present chiefly in the foveolar cells and in a decreasing staining gradient extending down to the necks of the glands. Connexin 32 also was observed in the epithelial cells of atrophic mucosa in a pattern similar to that observed in normal controls. Conversely, the majority of the foveolar cells adjacent to erosions had reduced or absent staining. Connexin 32 also was reduced significantly or absent from metaplastic epithelial cells. No malignant cells from patients with carcinoma contained detectable connexin 32. CONCLUSIONS: Intercellular communication likely is impaired in precancerous or paracancerous lesions of the stomach. Abnormal intercellular communication thus may play an important role in the progression from mucosal injury to intestinal metaplasia and/or gastric carcinoma. PMID- 7557128 TI - Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis. AB - BACKGROUND & AIMS: Inflammatory bowel disease is characterized by increased synthesis of nitric oxide. The aim of this study was to determine if inducible NO synthase (iNOS) was responsible for tissue injury, potentially via peroxynitrite formation, in the guinea pig model of gut inflammation. METHODS: Inflammation was induced in guinea pig ileum by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. iNOS gene expression was assessed by reverse-transcriptase polymerase chain reaction and Western blotting, immunohistochemistry was determined by its localization, and activity was inhibited with the specific inhibitor aminoguanidine administered via the drinking water for 7 days. Nitration of tyrosines was assessed by immunohistochemistry. RESULTS: In control animals, iNOS gene expression was minimal to absent, whereas, in hapten, inflammation-marked iNOS gene expression was evident from day 1 to 7. Nitrotyrosine and iNOS immunohistochemistry were colocalized, and positive staining was most intense in epithelia and neurons. Inhibition of NO formation prevented nitrotyrosine formation. Aminoguanidine inhibited the inflammatory response and restored morphology. CONCLUSIONS: The colocalization of tyrosine nitration with iNOS immunoreactivity suggests that iNOS may be responsible for tissue injury and the formation of NO-dependent nitrating species, potentially peroxynitrite. Inhibition of iNOS may afford a new therapeutic approach to the treatment of inflammatory bowel disease. PMID- 7557131 TI - Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon. AB - BACKGROUND & AIMS: Colonic inflammation may lead to motility disturbances, including severe atony. Nitric oxide is released by inflamed tissue and induces smooth muscle relaxation. The aim of this study was to analyze NO generation pathways in colonic tissue from patients who had ulcerative colitis with or without toxic megacolon and in tumor-free samples from patients with colonic neoplasm. METHODS: Enzymatic activity was determined by transformation of [14C]arginine to [14C]citrulline in mucosa and muscular layer samples. Immunostaining of tissue sections with antibody against inducible NO synthase was investigated. The effects of endotoxin on NO synthase activity was tested in muscle strips from human colon. RESULTS: Ca(2+)-independent NO synthase was undetectable or very low in muscularis propria from tumor and colitis controls. In contrast, specimens from patients with toxic megacolon had high activity (P < 0.05). Positive immunostaining for inducible NO synthase was found in muscular layers from patients with megacolon but not in tumor and colitis controls. Finally, endotoxin induced Ca(2+)-independent NO synthase activity in colonic muscle. CONCLUSIONS: Toxic megacolon is associated with the appearance of inducible NO synthase in the colonic muscularis propria. Local generation of excessive amounts of NO may be responsible for the colonic dilatation that is the hallmark of this syndrome. PMID- 7557132 TI - Eosinophilic esophagitis attributed to gastroesophageal reflux: improvement with an amino acid-based formula. AB - BACKGROUND & AIMS: Treatment for gastroesophageal reflux may be ineffective in patients with an eosinophilic infiltration of the esophagus. The aim of this study was to investigate whether unremitting symptoms of gastroesophageal reflux and biopsy abnormalities of the esophagus may be associated with the ingestion of certain foods. METHODS: Ten children previously diagnosed with gastroesophageal reflux by standard testing with long-standing symptoms (median, 34.3 months; range, 6-78 months) despite standard antireflux therapies, including Nissen fundoplication in 6 patients, were fed the elemental formulas Neocate or Neocate 1-Plus (Scientific Hospital Supplies Inc., Gaithersburg, MD) for a minimum of 6 weeks. Each child had repeat endoscopy followed by open food challenges. RESULTS: While receiving the formulas, patients had either resolution (n = 8) or improvement (n = 2) of symptoms. On follow-up esophageal biopsy, the maximal intraepithelial eosinophil counts decreased significantly before (median, 41; range, 15-100) to after (median, 0.5; range, 0-22) the formula trial (P = 0.005). Other reactive epithelial changes of the esophageal mucosa also improved significantly. All patients redeveloped their previous symptoms on open food challenges. CONCLUSIONS: Chronic gastrointestinal symptoms and histological changes of the esophagus unresponsive to standard treatments for gastroesophageal reflux were improved by the use of elemental formulas. Symptoms recurred when specific dietary proteins were reintroduced during open food challenges. The mechanism of these observations is unknown. PMID- 7557134 TI - The expression of tumor-rejection antigen "MAGE" genes in human gastric carcinoma. AB - BACKGROUND & AIMS: The genes MAGE-1 and MAGE-3 both encode melanoma peptide antigens recognized by major histocompatibility complex-restricted cytotoxic T lymphocytes. The antigens may be a target for immunotherapy. There is, however, little information on the expression of these genes in gastric carcinomas. Therefore, the expression of MAGE genes in gastric carcinomas was evaluated. METHODS: The expression of MAGE-1, MAGE-2, and MAGE-3 genes in tumors and corresponding normal tissue specimens was studied using a reverse-transcription polymerase chain reaction. The results were analyzed according to clinicopathologic factors of the tumor. RESULTS: In the 68 gastric carcinomas studied, MAGE-1, MAGE-2, and MAGE-3 messenger RNA were detected in 41%, 31%, and 38%, respectively. Fifty percent of the gastric carcinomas expressed at least one of the MAGE genes. Messenger RNA for the three MAGE proteins was not detected in normal gastric tissue. MAGE gene expression in gastric carcinomas was not associated with a significant clincopathology of the tumor. However, gene expression was lower in mucinous carcinomas (3 of 10). CONCLUSIONS: MAGE-1, MAGE 2, and MAGE-3 are expressed in a high percentage of gastric carcinomas. These tumor rejection antigens may provide tumor-specific targets for immunotherapy. PMID- 7557133 TI - Stimulatory mechanism of EM523-induced contractions in postprandial stomach of conscious dogs. AB - BACKGROUND & AIMS: EM523, a motilin agonist, is intended to be used as a gastroprokinetic during the postprandial period, but the mechanism(s) by which EM523 stimulates postprandial contractions in the stomach has not been studied before. The aim of this study was to examine the mechanism of contraction stimulating activity by EM523 in fed dogs. METHODS: Contractile activity in the gastric antrum of 5 dogs was monitored using a long-term implanted force transducer and measured by integrating the area under the curve. Test materials were continuously infused or injected intravenously. RESULTS: EM523 (1-30 micrograms/kg) induced a dose-dependent increase in fed-type contractions. EM523 induced contractile activity was partially inhibited by atropine, hexamethonium, dopamine, 5-hydroxytryptamine 3 (5-HT3) receptor antagonist, and substance P antagonist. Atropine-resistant and EM523-induced contractions were further inhibited by 5-HT3 receptor antagonist and substance P antagonist, and the combined use of the two antagonists completely eliminated the atropine-resistant and EM523-induced contractions. CONCLUSIONS: EM523-induced contractions in the fed stomach are quite different from phase III contractions in the fasted state and are mediated partially through the cholinergic pathway. The noncholinergic pathway involves 5-HT3 and neurokinin 1 receptors. PMID- 7557135 TI - Mucus and alkali secretion in the rat duodenum: effects of indomethacin, N omega nitro-L-arginine, and luminal acid. AB - BACKGROUND & AIMS: Duodenal mucus and bicarbonate secretion appear to play an essential role in the protection of the duodenum. The aim of this study was to examine duodenal bicarbonate and mucus secretion and the effects of cyclooxygenase inhibition, nitric oxide synthase inhibition, and luminal acid. METHODS: Duodenal mucus gel thickness was measured using microelectrodes during intravital microscopy in anesthetized rats. Bicarbonate secretion was measured using back-titration. RESULTS: A continuous layer of mucus with a mean thickness of 284 +/- 11 microns (n = 35) and a mean alkaline secretion of 0.18 +/- 0.01 mumol.cm-2.min-1 were found in untreated animals. Indomethacin decreased both mucus and bicarbonate secretion by about 35%. NO synthase inhibition with N omega nitro-L-arginine reduced mucus secretion by about 21% but increased bicarbonate secretion by 39%. Exposure of the mucosal surface to 10 mmol/L HCI increased mucus secretion by 44% and bicarbonate secretion by 22%. CONCLUSIONS: The duodenal mucus layer is continuous. It can be easily removed, and new secretion can be followed. Duodenal mucus secretion is strongly stimulated by luminal acid and endogenous prostanoids and less markedly by NO, whereas bicarbonate secretion is stimulated by acid and endogenous prostanoids and inhibited by endogenous NO. PMID- 7557136 TI - Micrometastases in bone marrow of patients undergoing "curative" surgery for gastrointestinal cancer. AB - BACKGROUND & AIMS: Immunohistochemical detection of bone marrow micrometastases has been reported as a prognostic marker in colorectal cancer. The aims of this study were to evaluate the potential advantage of flow cytometry as an objective method of identifying and quantifying micrometastatic deposits within bone marrow and to determine the prevalence and quantity of micrometastases in patients undergoing surgery for gastrointestinal cancers. METHODS: Flow cytometry was first validated by a controlled "spike" experiment in which varying numbers of neoplastic epithelial cells were added to bone marrow, and cytometry was performed in a blinded fashion. Three neoplastic cell lines (colonic and esophageal) with varying degrees of expression of cytokeratin-18 were used. Epithelial cells were detected by dual staining with fluorescence-labeled, monoclonal anti-cytokeratin, and propidium iodide. RESULTS: Cytometry reproducibly detected the presence of > or = 10 neoplastic cells per 10(5) marrow cells. Micrometastases were found in 20%-30% of patients undergoing potentially curative resection of colorectal and gastroesophageal adenocarcinomas. There was a trend toward increasing positivity for marrow deposits with advanced Dukes' staging of colorectal cancer. CONCLUSIONS: Flow cytometric assessment of bone marrow is a reliable, objective, and quantitative method of detecting micrometastatic deposits found in a substantial subset of patients undergoing surgery for gastrointestinal adenocarcinomas. PMID- 7557137 TI - Adenocarcinoma of the esophagogastric junction and Barrett's esophagus. AB - BACKGROUND & AIMS: Barrett's esophagus is associated with adenocarcinoma of the esophagus. The aim of this study was to find the prevalence of Barrett's esophagus in patients with adenocarcinoma of the esophagogastric junction. METHODS: Consecutive, freshly resected surgical esophagogastrectomy specimens were examined, and multiple histological sections were made around the tumor periphery. Barrett's esophagus was defined as specialized columnar epithelium above the esophagogastric junction. Tumors centered < or = 2 cm from the junction were defined as junction cancers. RESULTS: Barrett's esophagus was found in 9 of 9 (100%) esophageal adenocarcinomas compared with 0 of 8 (0%) squamous carcinoma controls (P < 0.001). Ten of 24 (42%) junction adenocarcinomas had a Barrett's esophagus. A Barrett's esophagus was found in 8 of 12 (67%) junction cancers < or = 6 cm in length but only 2 of 12 (17%) larger tumors (P < 0.05). Barrett's esophagus was significantly associated with junction tumors < 6 cm compared with squamous carcinoma controls (P < 0.02). In 5 specimens with junction cancer, the length of Barrett's esophagus was < 3 cm, and in 5 specimens it was > or = 3 cm. Specialized epithelium was often found below the esophagogastric junction in controls. CONCLUSIONS: Adenocarcinomas of the esophagogastric junction are associated with short and long segments of Barrett's esophagus. Larger cancers probably overgrow and conceal the underlying specialized columnar epithelium from which they arise. PMID- 7557139 TI - Immunization against a rat colon carcinoma by sodium butyrate-treated cells but not by interleukin 2-secreting cells. AB - BACKGROUND & AIMS: Vaccination of patients with colon cancer with irradiated autologous tumor cells and bacille Calmette-Guerin (BCG) was reported to augment mean survival. It was recently observed that a local treatment combining recombinant interleukin 2 and the differentiation agent sodium butyrate cured rats with colon cancer peritoneal carcinomatosis. To optimize vaccination protocols, the comparison of the efficacy of irradiated tumor cells mixed with BCG with that of interleukin 2-gene-transfected cells and of tumor cells pretreated with sodium butyrate was performed. METHODS: The poorly immunogenic rat colon carcinoma cells PROb were used in a vaccination assay. Interleukin 2 transfected PROb cells, either proliferating or irradiated, were used. The efficiency of irradiated PROb cells mixed with BCG, of interleukin 2-transfected cells, or of cells pretreated with sodium butyrate was tested. RESULTS: Vaccination with irradiated parental cells and BCG did not provide protection. Irradiated interleukin 2-transfected cells were poorly efficient in the vaccination assay. Conversely, vaccination with irradiated parental cells pretreated with sodium butyrate before injection provided good protection. CONCLUSIONS: Interleukin 2-secreting cells efficiently vaccinated animals when injected while replicating but not after irradiation. Conversely, sodium butyrate pretreatment provided a simple and efficient vaccination scheme that generated a long-term immune memory and allowed the use of irradiated cells. PMID- 7557138 TI - Effect of atropine on the frequency of reflux and transient lower esophageal sphincter relaxation in normal subjects. AB - BACKGROUND & AIMS: Low basal lower esophageal sphincter (LES) pressure is believed to be an important mechanism of reflux. The effects of atropine on the frequency and mechanisms of gastroesophageal reflux under the experimental conditions of a low basal LES pressure in 13 normal subjects were studied. METHODS: LES pressure, esophageal pressures, esophageal pH, and crural diaphragm electromyogram were recorded simultaneously in the postprandial period for 30 minutes before and two 30-minute periods after the injection of atropine. RESULTS: Atropine reduced the basal LES pressure from 16.4 +/- 3 to 8.7 +/- 2 mm Hg. The frequencies of reflux in the control and two postatropine periods were 3.5 +/- 0.5, 0.4 +/- 0.2, and 0.8 +/- 0.3, respectively (P < 0.05). The frequencies of transient LES relaxations decreased from 3.5 +/- 0.5 in the control to 0.4 +/- 0.2 and 1.5 +/- 0.4 in the two postatropine periods (P < 0.05). Transient LES relaxation and associated inhibition of the crural diaphragm was the major mechanism of reflux under conditions of low LES pressure induced by atropine. CONCLUSIONS: Atropine-induced low LES pressure does not predispose to reflux in normal healthy subjects. Atropine reduces the frequency of reflux by its inhibitory effect on the frequency of transient LES relaxation. PMID- 7557143 TI - The interleukin-2 receptor down-regulates the expression of cytochrome P450 in cultured rat hepatocytes. AB - BACKGROUND & AIMS: Interleukin (IL) 2 is used in advanced cancers, but its effects on cytochrome P450 remain unknown. Other cytokines down-regulate hepatic cytochrome P450, but it is not known whether this involves cytokine receptors. The aim of this study was to determine whether the IL-2 receptor is expressed on hepatocytes and whether its activation by IL-2 depresses cytochrome P450 in cultured rat hepatocytes. METHODS: A monoclonal antibody specific for the rat IL 2 receptor alpha chain was used to label the receptor, whereas effects on cytochrome P450 were determined after 24 hours of culture with human recombinant IL-2 (5000 U/mL). RESULTS: The presence of the IL-2 receptor in hepatocytes was shown by immunoblots, flow cytometry, and scanning confocal microscopy. IL-2 caused a 46% decrease in total cytochrome P450; a 35%, 35%, 36%, 26%, and 56% decrease in immunoreactive cytochrome P4501A1, 2B, 2C11, 2D1, and 3A, respectively; and a marked decrease in cytochrome P4503A2 and 2C11 messenger RNAs. Addition to the culture medium of the anti-receptor antibody or the tyrosine kinase inhibitor genistein prevented the IL-2-mediated decrease in cytochrome P450. CONCLUSIONS: IL-2 down-regulates the expression of cytochrome P450 genes in cultured rat hepatocytes by interacting with its receptor expressed on hepatocytes. PMID- 7557141 TI - Esophagopharyngeal distribution of refluxed gastric acid in patients with reflux laryngitis. AB - BACKGROUND & AIMS: A variety of otolaryngological abnormalities have been attributed to the contact of gastroesophageal refluxate with respective structures of the aerodigestive tract. The aim of this study was to determine and compare the pharyngoesophageal distribution of gastric acid refluxate between patients with proven laryngitis attributed clinically to gastroesophageal reflux and three control groups. METHODS: An ambulatory 24-hour simultaneous three-site pharyngoesophageal pH monitoring technique was used to measure reflux parameters in the pharynx, proximal esophagus, and distal esophagus. RESULTS: Between-group comparison showed no significant difference in the reflux parameters in the distal esophagus between the studied groups. A significantly higher percentage of distal reflux episodes reached the proximal esophagus in the laryngitis group than in the control groups (P < 0.01), and the number of pharyngeal reflux episodes and time of acid exposure were significantly higher in the laryngitis group than in the control groups (P < 0.001). CONCLUSIONS: Compared with normal controls and patients with gastroesophageal reflux disease, pharyngeal reflux of gastric acid is significantly more prevalent and the ratio of proximal to distal esophageal acid reflux episodes is significantly increased in patients with posterior laryngitis. Simultaneous three-site ambulatory pharyngoesophageal pH monitoring may provide supporting evidence when the diagnosis of reflux-induced aerodigestive tract lesions is considered. PMID- 7557140 TI - Dual inhibitory pathways link antral somatostatin and histamine secretion in human, dog, and rat stomach. AB - BACKGROUND & AIMS: The secretion and function of antral histamine are not known. The aims of this study were to characterize the mechanisms of histamine release from the gastric antrum of humans, dogs, and rats and to determine whether histamine can influence the secretion of somatostatin and gastrin. METHODS: Somatostatin, gastrin, and histamine secretion from superfused antral segments was measured using radioimmunoassay. RESULTS: Superfusion with thioperamide (H3 antagonist) increased somatostatin and decreased gastrin and histamine secretion in all three species; superfusion with (r)-alpha-methylhistamine (H3 agonist) had the opposite effect. The pattern implied that endogenous histamine, acting via H3 receptors, exerts an inhibitory paracrine influence on somatostatin secretion, which in turn regulates gastrin secretion. Superfusion with somatostatin antibody increased histamine secretion; the increase was not affected by the gastrin antagonist L-365,260, implying that it was not mediated by the concurrent increase in gastrin but by suppression of an inhibitory pathway linking somatostatin and histamine. Superfusion with methacholine alone and in the presence of either the H3 agonist or antagonist confirmed the existence of reciprocal inhibitory pathways linking somatostatin and histamine. CONCLUSIONS: Antral histamine in humans, dogs, and rats is linked to antral somatostatin via reciprocal inhibitory paracrine pathways that serve to amplify the regulatory influence of somatostatin. PMID- 7557144 TI - Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets. AB - BACKGROUND & AIMS: Plasma membrane solute transport may be regulated in many epithelial cells by vesicle traffic to and from the site of residence of the transporter. The aim of this study was to determine if this phenomenon may also play a role in the regulation of canalicular transport of bile acids. METHODS: Confocal microscopy and image analysis were performed to quantitatively assess changes in secretory capacity and vesicle targeting in isolated rat hepatocyte couplets that had been exposed to fluorescent bile acid after pretreatment with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and/or nocodazole. RESULTS: DBcAMP stimulated bile acid secretion by 240% while significantly increasing canalicular circumference. Nocodazole decreased secretion by 410% and significantly decreased canalicular circumference. When DBcAMP was added to nocodazole-treated couplets, a slight but significant increase was found in both fluorescent bile acid secretion and canalicular circumference as compared with nocodazole alone. Finally, DBcAMP stimulated translocation of vesicles to the canalicular membrane as determined by immunocytochemical localization of a putative bile acid transporter, Ca2+, Mg2+-ecto-adenosine triphosphatase. CONCLUSIONS: The findings support the view that apical membrane transport activity in the rat hepatocyte is highly regulated by the insertion of vesicles into this domain and that this process involves both microtubule-dependent and independent mechanisms. PMID- 7557142 TI - Mechanism of action of intracisternal apolipoprotein A-IV in inhibiting gastric acid secretion in rats. AB - BACKGROUND & AIMS: We recently showed that intracisternal injection of apolipoprotein A-IV (apo A-IV), a protein produced by the small intestine in response to fat, inhibits gastric acid secretion. The aim of this study was to investigate the mechanism of acid inhibition by central apo A-IV. METHODS: Gastric acid secretion was determined in pylorus-ligated conscious rats. The effect of intracisternal injection of apo A-IV on gastric acid secretion stimulated by pentagastrin, bethanechol, or intracisternal thyrotropin-releasing hormone (central vagal stimulant) was examined. The effects of vagotomy, indomethacin, and adrenergic blockers on the acid inhibition of apo A-IV were examined to investigate the role of the vagal system, prostaglandin pathways, and adrenergic system. RESULTS: Intracisternal apo A-IV significantly inhibited pentagastrin-, bethanechol-, and thyrotropin-releasing hormone-stimulated gastric acid secretion in a similar fashion. Inhibition of pentagastrin-stimulated acid secretion by apo A-IV still occurred even in vagotomized rats. Yohimbine but not indomethacin or propranolol eliminated apo A-IV--induced inhibition of acid. CONCLUSIONS: Intracisternal apo A-IV inhibits gastric acid secretion through alpha 2-adrenergic receptors. The vagal pathway and the prostaglandin system are not involved in apo A-IV--induced acid inhibition. PMID- 7557146 TI - Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. AB - BACKGROUND & AIMS: It was recently reported that two point mutations within the hepatitis B virus (HBV) core promoter region (A to T at position 1762 and G to A at position 1764) are associated with fulminant hepatitis and lead to hepatitis B e antigen (HBeAg)-negative phenotype. The aim of this study was to correlate core promoter sequence variations with HBeAg status and clinical outcome in various forms of HBV infection. METHODS: Core promoter region of HBV was amplified by polymerase chain reaction and directly sequenced in 94 patients: 37 patients with fulminant hepatitis, 20 with acute self-limited hepatitis, 30 with chronic hepatitis, and 7 patients with end-stage cirrhosis. RESULTS: Core promoter region was found to be heterogenous and no specific changes correlated with HBeAg/anti HBeAg status or survival in patients with fulminant hepatitis. Substitutions at positions 1762 and 1764 were found in HBV strains from 4 patients (10%) with fulminant hepatitis, 2 patients (10%) with self-limited hepatitis, 8 patients (27%) with chronic hepatitis, and in 5 of 7 patients with end-stage cirrhosis. The majority of these patients were HBeAg positive. CONCLUSIONS: Mutations at positions 1762 and 1764 are rarely observed in HBV strains from patients with fulminant hepatitis B in the United States but are common in patients with chronic hepatitis. Even when present, they seem to be insufficient to lead to the HBeAg-negative phenotype. PMID- 7557145 TI - Ki-ras mutations and p53 protein expressions in intrahepatic cholangiocarcinomas: relation to gross tumor morphology. AB - BACKGROUND & AIMS: We previously reported that intrahepatic cholangiocarcinomas (ICCs) can be divided into three categories according to their gross appearance with possible links to biological behavior. Ki-ras and p53 gene alterations are thought to be involved in early and late phases of carcinogenesis, respectively. This study was performed to investigate the relationship between the gross appearance and genetic alterations of ICC. METHODS: We examined 21 patients with ICC. Ki-ras point mutations were assessed by polymerase chain reaction/single strand conformation polymorphism methods followed by direct DNA sequencing. Expressions of p53 protein were immunohistochemically assessed. RESULTS: Ki-ras point mutations were found in 10 patients (48%), and expressions of p53 protein were detected in 4 (19%). Applying the gross classification that we previously proposed, Ki-ras mutations were prominent in the periductal extension type (4 of 6; 67%) and the spicula-forming type (6 of 10; 60%). On the other hand, none of the five mass-forming-type tumors harbored Ki-ras mutations. Expressions of p53 protein did not show any clear association with gross appearance. CONCLUSIONS: Ki ras gene alterations may be involved in the cholangiocarcinogenesis of periductal extension and spicula-forming but not mass-forming types, suggesting that the underlying processes of development are different. PMID- 7557148 TI - Massive ascitic fluid loss and coagulation disturbances after liver transplantation. AB - BACKGROUND & AIMS: A unique syndrome in which massive ascitic fluid loss developing 1-4 weeks after orthotopic liver transplantation (OLT) was associated with a hypercoagulable state and thrombotic complications is described. After OLT, severe coagulation abnormalities (international normalized ratio, 4.6) developed in a patient with ascitic losses of up to 12 L/day. The patient developed hypovolemia and severe systemic antithrombin III deficiency and venoocclusive disease in the graft. The aim of this study was to determine the prevalence of this syndrome after OLT. METHODS: Coagulation studies were performed in 25 additional patients with large ascitic fluid losses after OLT and in 7 cirrhotic patients not undergoing transplantation. RESULTS: All transplant recipients developed systemic deficiencies of multiple coagulation factors including antithrombin III. Markers of prothrombin activation were significantly elevated in both ascites and serum in all patients, and thrombotic complications subsequently developed in 5 patients. In the 7 cirrhotic patients, markers of prothrombin activation remained normal or minimally elevated and thrombotic complications were not found. CONCLUSIONS: Massive ascitic losses after OLT may lead to a hypercoagulable state from unreplaced losses of plasma coagulation factors into ascites and accumulation of thrombin in the systemic circulation. Hypovolemia and major coagulation abnormalities should be corrected with fresh frozen plasma, which may prevent the development of thrombotic complications. PMID- 7557149 TI - Pancreatic interstitial pH in human and feline chronic pancreatitis. AB - BACKGROUND & AIMS: Advanced chronic pancreatitis is associated with a reduction in pancreatic blood flow. To determine the physiological significance of this decrease, pancreatic interstitial pH was measured in a model of obstructive chronic pancreatitis in cats and in patients with chronic pancreatitis. METHODS: In cats, pancreatic interstitial pH and blood flow were measured serially under basal conditions and after secretory stimulation as chronic pancreatitis evolved. Basal pancreatic interstitial pH was also measured in patients undergoing an operation for chronic pancreatitis or periampullary cancer (controls). RESULTS: In normal cats, pancreatic interstitial pH was 7.41 +/- 0.01 and blood flow was 124 mL.min-1.(100 g pancreas-1). With the evolution of chronic pancreatitis, interstitial pH and blood flow progressively decreased to 7.21 +/- 0.04 (P < 0.007) and 75 +/- 11 (P < 0.007), respectively. From 1 to 2 weeks, secretory stimulation reduced pancreatic interstitial pH and blood flow further, but as secretory function was lost, this effect disappeared. In patients with chronic pancreatitis, the interstitial pH was lower (7.02 +/- 0.06) than in controls (7.25 +/- 0.04; P < 0.05). CONCLUSIONS: These observations are consistent with the hypothesis that, in chronic pancreatitis, acidic metabolites associated with pancreatic secretion accumulate within the pancreas, probably because of impaired blood flow. PMID- 7557150 TI - Retinoids: effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines. AB - BACKGROUND & AIMS: Advanced pancreatic carcinoma has a dismal prognosis despite extensive chemotherapeutic trials. The aim of this study was to evaluate the role of retinoids as an experimental therapeutic approach for pancreatic cancer. METHODS: Four ductal and one acinar pancreatic tumor cell lines were investigated. Growth was determined by cell number and a human tumor clonogenic assay. In vivo growth was assessed by xenografts transplanted into nude mice. Differentiation was characterized by immunofluorescence microscopy and carbonic anhydrase II gene expression. Retinoid receptors were characterized by Northern blotting and reverse-transcriptase polymerase chain reaction. RESULTS: Retinoid treatment results in a time- and dose-dependent growth inhibition in vitro and in vivo of ductal but not acinar pancreatic tumor cells. Retinoid treatment induces a more differentiated phenotype in ductal tumor cells as shown by morphological criteria and increased expression of carbonic anhydrase II. All pancreatic tumor cell lines expressed a broad panel of cellular retinoid binding proteins and nuclear retinoid receptors. Retinoic acid receptor gamma and cellular retinoic acid binding protein II were found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines. CONCLUSIONS: Detailed knowledge of nuclear retinoid receptor expression may provide rational strategies for retinoid treatment of pancreatic cancer. PMID- 7557147 TI - Normalization of nitric oxide production corrects arterial vasodilation and hyperdynamic circulation in cirrhotic rats. AB - BACKGROUND & AIMS: Recent studies suggest that production of nitric oxide is increased in cirrhosis. This study determines to what extent this increased production contributes to arterial vasodilation and hyperdynamic circulation in cirrhosis. METHODS: Mean arterial pressure (MAP), cardiac index, and systemic vascular resistance (SVR) were determined in cirrhotic rats with ascites undergoing long-term treatment with different doses of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (3 mg or 0.5 mg.kg-1.day-1). Untreated cirrhotic rats with ascites and controls were also studied. The vascular production of NO was estimated by the aortic concentration of guanosine 3',5' cyclic monophosphate (cGMP). RESULTS: Untreated cirrhotic rats had significantly lower MAP and SVR and higher cardiac index and aortic cGMP concentration than controls. When administrated to cirrhotic rats, an L-NAME dose of 3 mg.kg-1.day-1 induced a reduction of cGMP concentration less than normal levels. In these rats, MAP and SVR increased to greater than and cardiac index decreased to less than values in controls. By contrast, cirrhotic rats treated with 0.5 mg.kg-1.day-1 L NAME had similar aortic cGMP concentrations as controls, suggesting a normalization of NO production. This was associated with a normalization of MAP, cardiac index, and SVR and a reduction in the elevated plasma renin activity and vasopressin concentration. CONCLUSIONS: Normalization of vascular NO production corrects systemic hemodynamic abnormalities in cirrhotic rats with ascites. PMID- 7557151 TI - Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation. AB - BACKGROUND & AIMS: Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. METHODS: Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8 anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. RESULTS: CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. CONCLUSIONS: These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP. PMID- 7557152 TI - Insulin autoimmune syndrome as a cause of spontaneous hypoglycemia in alcoholic cirrhosis. AB - Hypoglycemia in fulminant hepatic failure and hyperinsulinemia in cirrhosis are well-described phenomena. A patient with alcoholic cirrhosis who developed fasting hypoglycemia with an extremely high immunoreactive insulin level and a mildly elevated C-peptide level is reported. An insulinoma was excluded by detailed radiological imaging of the pancreas and by endoscopic ultrasonography. Detection of very high levels of insulin autoantibodies with no prior exposure to exogenous insulin confirmed the diagnosis of insulin autoimmune syndrome. During his hospital course, the patient developed another rare syndrome, acquired inhibitors to factor V, which led to the fatal coagulopathy that resulted in his death. Insulin autoimmune syndrome is the third leading cause of spontaneous hypoglycemia in Japan, where it has been associated with a variety of diseases and drugs. Outside of Japan, only approximately 20 cases have been reported and usually have been found in the context of an underlying autoimmune disorder or prior exposure to sulfhydryl drugs. It is believed that this is the first case reported outside Japan occurring in association with alcoholic liver disease, and the first in the world with coexisting acquired inhibitors to factor V. PMID- 7557154 TI - Ischemic hepatitis due to obstructive sleep apnea. AB - The case of an obese patient who developed massive centrilobular liver cell necrosis, severe coagulopathy, acute renal failure, and encephalopathy is presented. Hypovolemia and heart failure were absent, but the acute liver disease was associated with severe arterial hypoxemia due to obstructive sleep apnea that was shown by the nocturnal blood oxygen desaturation, the results of the polysomnographic study, and normal baseline pulmonary function tests. In this obese patient, liver cell necrosis was caused by severe liver cell hypoxia secondary to severe arterial hypoxemia as a consequence of obstructive sleep apnea associated with a Pickwickian syndrome. This observation is consistent with the hypothesis that liver ischemia was directly related to severe arterial hypoxemia. PMID- 7557153 TI - Mast cell cholangiopathy: another cause of sclerosing cholangitis. AB - A 75-year-old woman with known systemic mastocytosis presented with abdominal pain, ascites, and bile duct thickening on computed tomography and ultrasonography. A liver biopsy specimen showed infiltration with mast cells. Endoscopic retrograde cholangiography showed ductal changes compatible with those found in primary sclerosing cholangitis. Brush cytology of the intrahepatic bile ducts confirmed mast cell infiltration. Systemic mastocytosis can infiltrate the biliary system, producing a cholangiopathy radiographically similar to primary sclerosing cholangitis. PMID- 7557155 TI - DNA mismatch repair and cancer. AB - The genetic basis of cancer involves certain classes of genes, particularly oncogenes, tumor-suppressor genes, and DNA mismatch repair genes. Originally identified in bacteria and yeast, the human homologues of DNA mismatch repair genes have been implicated in the pathogenesis of the hereditary nonpolyposis colorectal cancer syndromes, as well as a variety of different sporadic cancers. An appreciation of their role in cancer is predicated on an understanding of their function in the processes of DNA repair. This article reviews the recent developments and advances in the biology of the human DNA mismatch repair genes and their involvement in the pathogenesis of cancer. PMID- 7557156 TI - A glimpse at the central mechanism for swallowing? PMID- 7557157 TI - Spontaneous transient lower esophageal sphincter relaxations: a target for treatment of gastroesophageal reflux disease. PMID- 7557158 TI - Regulation of secretion and absorption by recruitment and recycling of primary transport proteins. PMID- 7557159 TI - Irritable bowel: it helps to have a friendly physician--or so it would seem. PMID- 7557160 TI - The impact of immunonutrition. PMID- 7557161 TI - Infection outcome in hemodialysis patients and kidney recipients. PMID- 7557162 TI - Endoscopic surveillance in ulcerative colitis still does not work. PMID- 7557163 TI - Hypercalcemia, calcium in bile, and calcium in gallstones. PMID- 7557165 TI - PEG in a patient with ascites and varices. PMID- 7557164 TI - Percutaneous trans-hepatic cholangioscopic lithotomy for hepatolithiasis: long term results. AB - A prospective study was undertaken to evaluate the long-term results of percutaneous trans-hepatic cholangioscopic lithotomy in 48 patients with hepatolithiasis during a 4- to 10-year follow-up period. Complete clearance of the intrahepatic stones was achieved in 40 patients (83.3%). In these 40 patients, long-term results during the follow-up period were as follows: free of symptoms and without evidence of recurrent stones, 22 patients (55%); free of symptoms with recurrent stones, 2 patients; recurrent stones with cholangitis, 14 patients (35%); symptoms of cholangitis without evidence of recurrent stones, 2 patients. Of those patients with recurrent stones, secondary biliary cirrhosis developed in 1, and in 9 of the patients in whom secondary cholangitis developed, subsequent laparotomy or percutaneous drainage was required. Three of the 40 patients (7.5%) died within the 4- to 10-year follow-up period. The procedural failure rate in terms of stone retention was 20.5% for patients with bile duct strictures. No stones were retained in patients without strictures. The postprocedural rate of stone recurrence for patients with bile duct strictures was 51.6%; no recurrence was seen in patients without strictures. PMID- 7557167 TI - The migrating gastrostomy tube. PMID- 7557168 TI - Randomized trial of prevention of biliary stent occlusion by ursodeoxycholic acid plus norfloxacin. PMID- 7557166 TI - Colorectal ulcers associated with sodium phosphate catharsis. PMID- 7557170 TI - Percutaneous trans-hepatic cholangioscopy and lithotripsy in the treatment of intrahepatic stones: a study with 5 year follow-up. AB - BACKGROUND: Intrahepatic stones are characterized by high treatment failure and recurrence rates. In the past, surgery played a major role in the management of this disease, but surgical intervention may not be feasible in previously operated patients or those classified as poor surgical risks. The development of percutaneous trans-hepatic cholangioscopy and lithotripsy has played an important role in the treatment of these patients. METHODS: We reviewed our results in 165 patients with intrahepatic stones treated by percutaneous trans-hepatic cholangioscopy and lithotripsy in the past 12 years. RESULTS: The rate of complete stone removal was 80% (132 cases) and the rate of recurrent biliary stones after a mean 58 month follow-up interval was 32.6% (43 of 132 cases). There were 2 deaths during the treatment period and 10 deaths during follow-up. CONCLUSIONS: Percutaneous trans-hepatic cholangioscopy and lithotripsy are good alternatives in treating primary intrahepatic stones, especially for those patients unsuitable for surgery. PMID- 7557169 TI - Self-expanding metal stents for palliative treatment of malignant biliary and duodenal stenoses. PMID- 7557171 TI - Duplex Doppler endosonography in the diagnosis of splenic vein, portal vein, and portosystemic shunt thrombosis. AB - BACKGROUND/AIMS: Endoscopic ultrasonography is a promising procedure for imaging mesenteric vascular structures. METHODS: Duplex and color Doppler endosonography were used to prospectively evaluate 20 asymptomatic paid volunteers. Subsequently, 11 patients with nondiagnostic transabdominal ultrasound and suspected thrombosis of the splenic and/or portal veins or a portosystemic shunt were evaluated with duplex endosonography. The final diagnosis was based on CT, angiography, and/or surgery or autopsy findings in 9 of 11 patients. RESULTS: In normal volunteers, mesenteric vessel flow velocities and diameters were similar to previously described values. In 10 of the 11 patients with failed transabdominal ultrasound, duplex endosonography was able to provide the correct diagnosis (accuracy of ultrasound 0% versus EUS 91%, p < .001). Mean portal vein diameter was greater in the patient group than in the normal volunteers (18.5 mm versus 10.7 mm, p < .001) and all of the normal volunteers had a portal vein diameter less than 13 mm. No complications were experienced. CONCLUSION: Duplex endosonography allows visualization of the intra-abdominal vasculature and can be considered when transabdominal ultrasound is nondiagnostic in patients with suspected thrombosis of their splenic vein, portal vein, or portosystemic shunt. EUS is able to identify indirect findings of portal hypertension including portal vein enlargement and venous collaterals. PMID- 7557173 TI - Pneumatic dilation in achalasia with a low-compliance balloon: results of a 5 year prospective evaluation. AB - To evaluate long-term efficacy and side effects after pneumatic dilation with low compliance balloons, 40 patients with achalasia were prospectively studied during a 5-year period. All patients were investigated before, 4-6 weeks after, and at 28 +/- 15 months (final visit) after pneumatic dilation. Additionally, 12 patients underwent 24-hour esophageal pH-metry before and 26 +/- 14 months after dilation. Initial symptomatic success was obtained in 35 of 40 patients (87.5%). The remaining five and another seven patients with recurrent dysphagia underwent a second dilation, and two of these patients finally underwent esophagomyotomy. Effective dilation was reflected by a significant decrease of the symptom scores (p < 0.01) and an increase of the gastric cardia diameter both at 4-6 weeks after dilation and at the final visit (p < 0.01). Dilation reduced the lower esophageal sphincter pressure from 28.3 +/- 0.8 mmHg to 16.4 +/- 6.4 mmHg at the 4-6 weeks exam and to 14.7 +/- 5.5 mmHg at the final visit, respectively (p < 0.01). Neither the diameter of the esophageal body nor the motility of the tubular esophagus was affected by pneumatic. Esophageal pH-metry showed an increase of the number and duration of reflux episodes (pH < 4) after dilation (p < 0.05), whereas only one patient reported heartburn and another asymptomatic patient revealed esophagitis I0 at endoscopy. Therefore, pneumatic dilation with low compliance balloons proved to be safe and long lasting treatment of achalasia. Although prolonged esophageal acid exposure was measurable after dilation, clinically relevant gastroesophageal reflux occurred in only 5% (n = 2). PMID- 7557172 TI - An experimental study of optimal parameters for bipolar electrocoagulation. AB - The role of artery diameter, probe force application (94, 250 or 491 g), coagulation time (2, 10, or 20 secs) and BICAP generator output (5, 10) were studied in rabbit arteries of 3 to 5 mm hemicircumference. Arterial welds were then tested to destruction by increasing intraluminal pressure. The best arterial welds were achieved by the highest force and coagulation time; high generator output did not improve weld strength. These in vitro experiments suggest that when treating a bleeding peptic ulcer, BICAP therapy should be done with a low generator output of 5 or less, for at least 20 seconds per site, and with as great a compression force as possible. PMID- 7557175 TI - Morbidity in neurologically impaired children after percutaneous endoscopic versus Stamm gastrostomy. AB - Neurologically impaired children frequently require a feeding gastrostomy. Few reports are available comparing the incidence of postoperative complications and symptomatic gastroesophageal reflux after endoscopic versus operative Stamm gastrostomy in this group of children. We undertook a retrospective study of 63 consecutive neurologically impaired children requiring a feeding gastrostomy, with an average of 23 months of follow-up. No child had symptomatic gastroesophageal reflux. Thirty children had a percutaneous endoscopic gastrostomy and 33 had a Stamm gastrostomy, depending on the preference of the surgeon. The two groups were comparable in age range, cause of neurologic impairment, and indication for gastrostomy. Minor complications occurred in 30%. All three major complications occurred after Stamm gastrostomy, including two postoperative deaths. Symptomatic gastroesophageal reflux developed in 60%. The incidence of fundoplication after gastrostomy was 10% in the percutaneous endoscopic gastrostomy group and 39% after Stamm gastrostomy (p < .025). Morbidity was lower after percutaneous endoscopic gastrostomy than after Stamm gastrostomy in this group of neurologically impaired children. Fundoplication for symptomatic gastroesophageal reflux was infrequent after percutaneous endoscopic gastrostomy and significantly more common after Stamm gastrostomy. Percutaneous endoscopic gastrostomy is recommended as the initial procedure in neurologically impaired children without symptomatic gastroesophageal reflux who require a feeding gastrostomy. PMID- 7557174 TI - Felinization of the esophagus. AB - Transient transverse folds of the esophageal mucosa as seen by barium esophagography and on endoscopic examination have been termed "felinization." This finding has been considered a normal variant or has been associated with reflux esophagitis. No symptoms have been associated with its presence. In 280 unselected patients undergoing upper gastrointestinal endoscopy in our clinic, felinization was identified in 24% by prospective visual examination. It was not correlated with sex, age, symptom complex, or common coexistent endoscopic findings. These transverse esophageal folds, although visually interesting, appear to have no clinical significance. The endoscopist should recognize and differentiate them from fixed or persistent folds that can be associated with mucosal disease. PMID- 7557176 TI - The effect of droperidol on objective markers of patient cooperation and vital signs during esophagogastroduodenoscopy: a randomized, double-blind, placebo controlled, prospective investigation. AB - We investigated the effect of droperidol on objective markers of cooperation and vital signs in 140 patients undergoing elective diagnostic esophagogastroduodenoscopy. Procedure duration and the total doses of midazolam and meperidine required during the procedure were evaluated as objective markers of patient cooperation. The droperidol group comprised 66 patients and the placebo group 74 patients. Patient and procedure characteristics were similar for both groups. Droperidol produced a 10% reduction in procedure duration. Linear multiple regression modeling revealed droperidol to be a significant predictor of procedure duration (p = .036). Droperidol significantly reduced midazolam and meperidine requirements (p < .01). Nonetheless, four patients in the droperidol group received naloxone to reverse prolonged, excessive drowsiness. Droperidol produced a significant reduction in procedure-associated increase in pulse rate but did not exacerbate procedure-associated reduction in mean arterial pressure. Droperidol favorably influences markers of patient cooperation during elective, diagnostic esophagogastroduodenoscopy. However, the clinical significance of these changes is unclear. PMID- 7557179 TI - Initial results using low-dose photodynamic therapy in the treatment of Barrett's esophagus. PMID- 7557177 TI - Prolonged evaluation of epinephrine and normal saline solution injections in an acute ulcer model with a single bleeding artery. AB - BACKGROUND: Animal studies of epinephrine or normal saline solution injection for bleeding ulcers do not consistently demonstrate local tamponade effect. METHODS: We studied the change of bleeding rates of 28 acute gastric ulcers with a single bleeding artery in 10 dogs. Four injections of 1 mL epinephrine 1:10000 at 1 mm from the spurting artery (n = 7) were compared to four injections of normal saline solution 1 to 5 mL (n = 12) and to four dry needle sticks (n = 9). Bleeding rates were measured at initial arterial incision and at minutes 1, 5, 10, 15, 20, 25, and 30 after treatment. RESULT: Reductions in early blood loss to 24.3% +/- .05 of baseline occurred with saline solution, to 17.7% +/- .03 with epinephrine, and to 66.0% +/- 1.8 in controls (p < .05 for epinephrine and saline solution vs control). A tendency for saline solution injected ulcers to resume bleeding was identified, with late blood loss increasing to 26.9% +/- .05 of baseline, (saline solution vs control) compared to 7.7% +/- .02 in epinephrine injected ulcers (p < .05 vs control). CONCLUSIONS: The early acute hemostatic effect of injection therapy depends on local tamponade. The prolonged hemostatic effect is a combination of tamponade and vasoconstriction, with advantage of epinephrine over saline solution. PMID- 7557178 TI - A prospective study of fungal infection of gastric ulcers: clinical significance and correlation with medical treatment. AB - A prospective study was performed to evaluate the prevalence of fungal infection in gastric ulceration and its effect on ulcer healing in 178 benign and 97 malignant gastric ulcers. Fungal infection was defined as presence of fungal hyphae or spores in the biopsy forceps specimen. For patients with benign gastric ulcers, sucralfate 1 g q.i.d. was prescribed and a second panendoscopy examination was carried out after 6 weeks to evaluate ulcer healing. Fungal colonization was found in 36 (20.2%) patients with benign gastric ulcers and 26 (26.8%) patients with gastric cancers (p > 0.2). The mean age of patients with benign gastric ulcer with fungal infection (group I) was 64.2 +/- 11.4 years, whereas the mean age of those without fungal infection (group II) was 56.2 +/- 13.1 years (p < 0.01). Follow-up panendoscopy after 6 weeks of sucralfate therapy revealed 6 of 24 patients (25%) in group I and 19 of 81 patients (23%) in group II with unsatisfactory healing (difference not significant). Comparison of confounding factors such as smoking, daily tea or coffee intake, underlying disease, ulcer location, and endoscopic appearance between these two groups revealed no significant differences. In conclusion, the presence of fungus in gastric ulcers is a secondary phenomenon and it does not affect ulcer healing. PMID- 7557181 TI - Barrett's esophagus: photodynamic therapy for ablation of dysplasia, reduction of specialized mucosa, and treatment of superficial esophageal cancer. PMID- 7557180 TI - Yield of prospective, noninvasive evaluation of the common bile duct combined with selective ERCP/sphincterotomy in 1390 consecutive laparoscopic cholecystectomy patients. AB - The role of noninvasive evaluation of the common bile duct combined with selective preoperative endoscopic retrograde cholangiography and sphincterotomy was prospectively evaluated in 1390 consecutive patients subjected to laparoscopic cholecystectomy. Preoperative common bile duct testing included liver chemistries, transcutaneous ultrasonography, and intravenous cholangiography. When indicated by various sets of abnormal studies, prelaparoscopic endoscopic retrograde cholangiography was attempted in 129 patients (9.3%) and successfully accomplished in 122 (94.6%). Seventy-six patients (62.3%) had duct stones or a papillary stenosis, and 73 of them (96%) were treated successfully by endoscopic duct clearance and subsequent laparoscopic cholecystectomy. No deaths occurred, and the morbidity rate was 6.2% (8/129), including 2 cases of pancreatitis and 1 case of a retained duct stone after sphincterotomy. Predicting the presence of common duct pathology was 60% accurate when based on abnormal laboratory test results alone, 69% when based on abnormal laboratory test results and concomitant radiologic abnormalities, and 42% when based on radiologic criteria alone (p < .05). We conclude that patients with altered serum liver chemistries with or without concomitant positive radiologic criteria should undergo endoscopic cholangiography before laparoscopic cholecystectomy. However, in patients with radiologic duct dilatation as the sole indicator for duct stones, the frequency of normal findings in endoscopic examinations is high. This latter group is probably better managed with intraoperative cholangiography and postlaparoscopic sphincterotomy if needed. PMID- 7557185 TI - Vascular-enteric fistula: diagnosis by colonoscopy. PMID- 7557182 TI - Release of entrapped delivery devices from wire mesh stents. PMID- 7557183 TI - Marking clips for the accurate positioning of self-expandable esophageal stents. PMID- 7557186 TI - Expandable metal coil stent for treatment of broncho-esophageal fistula. PMID- 7557187 TI - Recurrent esophagorespiratory fistula in a patient with metastatic breast cancer: long-term palliation with endoprostheses and hormonal therapy. PMID- 7557184 TI - Localization of neuroendocrine tumors utilizing linear-array endoscopic ultrasonography. PMID- 7557188 TI - Endoscopic ultrasound in the placement of a percutaneous endoscopic gastrostomy tube in the non-transilluminated abdominal wall. PMID- 7557189 TI - Diagnosis of colonic pneumatosis cystoides intestinalis by endosonography. PMID- 7557190 TI - Management of intrahepatic stones. PMID- 7557191 TI - Photodynamic therapy: a new light on Barrett's esophagus. PMID- 7557193 TI - Combined endoscopic-percutaneous--assisted sphincterotomy in the treatment of choledochocele. PMID- 7557192 TI - Esophageal intubation with a duodenoscope. PMID- 7557195 TI - [Psychological status of tumor patients. Observations and recommendations from the viewpoint of inpatient cancer after-care and oncologic rehabilitation]. PMID- 7557194 TI - [Genetic predisposition for breast carcinoma: overview of molecular genetic principles from long familial epidemiologic observation]. PMID- 7557196 TI - [Legal questions on sterilization of mature mentally handicapped patients]. PMID- 7557197 TI - ["Inhuman practices in gynecology in national socialization and its victims"]. PMID- 7557198 TI - [Improved control of HIV transmission from mother to child]. PMID- 7557200 TI - [The clinical picture of endoscopically treated extrauterine pregnancy. Postoperative follow-up of 677 endoscopically operated extrauterine pregnancies 1983-1993]. AB - 677 ectopic pregnancies were treated by laparoscopy from 1983-1993. Complications in 26 cases led to a second laparoscopic intervention; these complications are analysed to access the advantages and risks of this new procedure. The postoperative HCG blood levels were observed and salpingectomy vs. organ preserving techniques compared. 30 cases of postoperative HCG-increases and 19 cases with operative treatment of residual trophoblast are also analysed. Endoscopic salpingectomy of tubal pregnancies is a safe procedure with a very low complication rate. Organ-preserving treatment of tubal pregnancies via laparoscopy has a critical complication range of 7-9%. This should be considered for the choice of the operating procedure and the patient should be informed of the risks and their frequency. PMID- 7557199 TI - [Introduction of laparoscopic surgery in extrauterine pregnancy]. AB - Top evaluate laparoscopic surgery in extrauterine pregnancy, the period from 1990 1993 was analysed, and the first 44 operations including only one laparoscopy (2%), were compared with the following 70 operations with 60 laparoscopic procedures (86%). Except for a younger mean gestational age in the second interval, both groups showed no differences in the history and preoperative findings. During the second period, however, the intraoperative blood loss was smaller and the rate of conservative procedures was higher. Operation times and complication rates were not different. But in laparoscopy, duration of the procedure significantly decreased with advancing experience. Postoperative hospital stay was half as long in the second group. Subsequent procedures were necessary in 3% of 61 laparoscopies due to adhesion ileus and persistent trophoblast, respectively. Persistent pregnancy was seen in 2% of 42 conservative laparoscopies. Laparotomy due to failed laparoscopy was done in 2 of 63 patients (3%) with bleeding complications. Laparoscopy in the hand of the experienced surgeon is a suitable method to treat ectopic pregnancy and allows also organ preservation. PMID- 7557202 TI - [Pelviscopic myoma enucleation: technique, limits, complications]. AB - Between 1992 and 1993 surgery conserving the organ was undertaken in 215 patients with uterine myomas. Only myomas of more than 2 cm in diameter were included. It was possible to conserve the organ in 207 cases (90%). Myomectomy by pelviscopy was performed in 131 cases. The procedure was successful in 117 cases (89%), secondary laparotomy had to be done in 14 of these patients. On average the myomas removed by pelviscopy measured 5.2 cm in diameter. The S.E.M.M. (Serrated Edged Macro Morcellator) was used in the procedure. It did not take long to morecellate even larger myomas (the largest one removed by pelviscopy weighed 418 g) and to remove them by means of a 15 mm-trocar. An average of 2 myomas were removed per patient (r: 1-5). The mean Hb drop amounted to 1.5 g%. Repeat pelviscopy had to be done in one patient because of a secondary haemorrhage, a laparotomy and hysterectomy for subilius had to be performed in one case on the 3rd postoperative day. An intestinal loop has adhered to the uterine wound dehiscence. No other complications were observed after pelviscopic myomectomy. A 41-year-old patient wanting children suffered a late complication, namely a ruptured uterus in the 28 w of pregnancy. It is therefore imperative to inform patients who are still in the reproductive phase about the possibility of an uterus rupture after pelviscopic myomectomy. PMID- 7557201 TI - [Histology and cytology of laparoscopically operated "simple ovarian cysts"]. AB - In a retrospective study the histopathological findings of 127 laparoscopically operated unilocular anechoic smooth-walled ovarian cysts have been correlated with clinical characteristics (age, duration of observation, complaints, hormonal treatment), size by ultrasound, kind and colour of cysts content as well as cytological findings. The age of patients differed from 16-61 years (mean +/- s: 36 +/- 16). The histopathologic findings yielded 15 (11.8%) functional cysts, 30 (23.6%) persistent corpus luteum cysts, 9 (7.1%) endometriomas, 7 (5.5%) cystic teratomas, 9 (7.1%) undifferenciated cysts and 57 (44.9%) cystadenomas. There were no differences between histopathologic diagnosis groups according to age and cysts size by ultrasound. Functional cysts with complaints (n = 6) may explain that the observation time in 60% of all functional cysts was smaller than 6 weeks, whereas persistent corpus luteum cysts, endometriomas, cystic teratoma and cystadenomas had been observed for longer than 6 weeks in more than two thirds. Intraoperative evaluation of cysts content as "chocolate"-like was suspicious of endometriomas, but was also present in cysts of other histopathological findings. By means of cytology, endometrioma (siderophages) was suspected in 44.4% and a cystadenoma in 42.1% of all histopathologically verified cases. In all, the cytologic findings were useful for correct histopathological diagnosis in only 33.9% of all 127 cases. It is concluded that differential diagnosis of simple ovarian cysts is not possible by clinical characteristics, neither by ultrasound nor by cytological evaluation. Ovarian cysts should be observed for at least two hormonal cycles. A hormonal treatment by combination preparations containing high doses of oestrogen is also recommended. In cases of persisting ovarian cysts laparoscopic removal is necessary. PMID- 7557203 TI - [Laparoscopic ante-fixation of the uterus--modification of the Menge operation]. AB - Surgical antefixation of the retroflected uterus is today for several reasons a rare event. We report on a forty-year old patient with dyspareunia, dysmenorrhoea and lumbalgia. Elevation of the uterus by means of a Hodge pessary relieved her symptoms. This prompted laparoscopical suspension of the diagnosed retroflexio uteri mobilis. We used a modification of Menge's technique stitching the rotund ligament to the anterior surface of the uterus. This modified technique is especially useful because it can be performed easily by laparoscopy. In addition shortening of the round ligaments achieves a physiological position of the uterus in the pelvis without major changes in the pelvic anatomy. PMID- 7557204 TI - [Total pelviscopic removal of ovarian tumors in a bag bag posterior colpotomy]. AB - Treatment of suspicious ovarian masses requires the oophorectomy in toto without opening the tumor or cyst wall. We describe a laparoscopic technique for the in toto-removal of clinically suspicious ovarian tumours without puncture or morcellation of the tumour before it is entirely brought outside of the abdominal wall and without performing any abdominal incision longer than 25 mm: Oophorectomy is performed by means of bipolar coagulation and the CO2-laser. A nylon bag (Lapsac, Cook Inc.) is inserted into the abdomen and the ovary is enclosed in this bag by pulling the drawstring. The drawstring is held with a needleholder while the posterior vaginal fornix is opened. The needleholder drives the drawstring out of the pelvis through the vagina; this manoeuvre only takes a short amount of time, therefore preventing loss of CO2-gas and of visibility. By pulling the drawstring from outside the vagina the bag can be easily removed with its unmorcellated content. There is no danger of the intestines being damaged by a grasping forceps. Such a problem could occur in the event of extraction through a posterior culdotomy under impaired visibility due to loss of CO2-gas. Because the bag itself serves as a closing valve of the vaginal opening, there is a good visibility all the time. PMID- 7557205 TI - [Regression of functional cysts: high dosage ovulation inhibitor and gestagen therapy has no added effect]. AB - To investigate the need for hormonal treatment in patients with functional ovarian cysts (FOC), the efficacy of this treatment was evaluated in a retrospective and also in a randomised prospective study. By retrospective analyses the resolution of FOC with a mean diameter larger than 2.0 cm at the beginning of a cycle was determined in 113 patients (31.6 +/- 4.6 years). Fifty seven women received an oral contraceptive (ethinylestradiol 50 micrograms/d for 7 days, ethinylestradiol 50 micrograms and desogestrel 125 micrograms/d for 15 days), the others had no therapy. In a second study 59 patients (32.3 +/- 4.6 years) were randomised to receive a combination of ethinylestradiol 50 micrograms and levonorgestrel 250 micrograms/d for 21 days (Group 1, n = 24), or lynestrenol 10 mg/d continuously (Group 2, n = 14) or a third group (Group 3, n = 21) without treatment. In both studies no differences were found between those patients who had hormonal treatment and those who had not. The prospective study revealed that spontaneously appearing FOC and FOC evolving after ovulation induction during the cycle prior to study enrolment, resolved equally well within 12 weeks independent of contraceptive or gestagen treatment. FOC persisted in only one woman (group 2) who had a surgically proven endometrioma. In conclusion, hormonal treatment does not produce regression of FOC in women of reproductive age. PMID- 7557206 TI - [Serum lipids in treatment with transdermal estradiol and oral norethisterone acetate]. AB - With transdermal estradiol substitution the so called "primary liver passage" is avoided. Taking into account also the low dose of estradiol the risk of hepatic side effects can be reduced. On the other hand, it was assumed that for the same reason desirable lipid effects regarding cardiovascular protection may also not be possible, in contrast to oral estrogen treatment. Treating 26 postmenopausal women with the estradiol patch releasing 0.05 mg daily and with 1 mg oral norethisterone acetate, added at least during 10 days in each cycle, a significant reduction was observed in total cholesterol as well as in LDL- and VLDL-cholesterol of about 15-20%. HDL-cholesterol first showed a decrease and thereafter it increased again to basic level. It is supposed that the reason for this may be different effects on subfractions of HDL-cholesterol. The triglycerides were lowered to about 20%. This result is thought to be important because oral estrogens have been associated with increases in triglycerides. By lowering LDL-cholesterol as well as triglycerides, both serum lipids, most important with respect to cardiovascular protection, are shown to be influenced positively. PMID- 7557207 TI - [Successful outcome of "intracytoplasmic sperm injection" (ICSI) within the scope of in vitro fertilization]. AB - Poor function of spermatozoa accounts for half of all human infertility. "Intracytoplasmic sperm injection" (ICSI) is a new micromanipulatory method for the treatment of male infertility. "ICSI" was performed in cases of oligoasthenozoospermia in 12 IVF cycles, obtaining a fertilisation rate of 71.2% of all micromanipulated oocytes. So far four ongoing pregnancies occurred. Thus "Intracytoplasmic sperm Injection" represents a new encouraging technique to conceive, even in cases of severe male infertility. PMID- 7557209 TI - [Doppler ultrasound of the middle cerebral artery: pre-final normalization of cerebrovascular circulation?]. AB - We report on the circulatory changes of a severely growth-retarded fetus at 26 weeks of gestation with intrauterine death after 6 days of observation and a birth weight of 480 g. On admission the fetus already showed signs of circulatory centralisation; furthermore, we found pulsation in the umbilical vein and diastolic reverse flow in the umbilical artery. During the days of observation this reverse flow increased and the compensatory phase of fetal circulatory centralisation was followed on the last day by a phase of circulatory decompensation with an apparently normal middle cerebral artery waveform, mimicking preterminal normalisation of fetal cerebral blood flow. PMID- 7557210 TI - [Hemorrhagic shock caused by intraperitoneal hemorrhage after amniocentesis]. AB - This case report its on a haemorrhagic shock developing after amniocentesis during early pregnancy. Initially caused by a lesion of a small subserous artery in the upper uterine fundal wall, the patient consequently developed a protracted haemorrhagic shock that required emergency treatment and surgery. Thirteen days later fetal death was diagnosed by ultrasonographic examination, apparently caused by an abnormality of the umbilical cord without any connection with the previous complication. PMID- 7557211 TI - [Writing and reading scientific texts]. PMID- 7557208 TI - [Torsion of the pregnant uterus]. AB - Pathological version of the pregnant uterus is a rare complication. A case of 180 degrees sinistrotorsion of the uterus in the 39th gestational week is reported. The case was asymptomatic with the exception of preterm labor in the 30th week of pregnancy and was discovered during a cesarean section carried out because of myoma obstructing the birth canal. Mother and child were discharged in perfect health. PMID- 7557214 TI - [Limits of assistant liability in delivery by an assisting physician in residency]. PMID- 7557213 TI - [31 years experiences with triplets. 13 years self-help for parents with the ABC Club e.V., International Triplet and Multiple Birth Initiative]. PMID- 7557212 TI - [Pregnancy, labor and partnership in a family with an IVF child]. PMID- 7557215 TI - [Deficits in gynecologic education in patient examination for suspected sex offenses]. PMID- 7557216 TI - [Histopathologic correlates of false-positive cytologic findings in the uterine cervix]. AB - Between 1984 and 1992, we reinvestigated 170 cases of cervical smears obtained in conization or hysterectomy which had produced false positive cytologic findings. We looked for morphological changes that could explain the cytologic findings. Such changes were found in half of the cases. These cases included 54 (63.6%) specimens with marked cellular polymorphism as a result of inflammation, tissue regeneration, or atrophy. In 51.9% of the cases showed histologic signs of possible HPV infection. The initial findings required reclassification in 12.9% of the cases, and in fifteen cases (17.6%) the biopsy matched the cytologic findings, without being validated by the final findings. We observed histologic phenomena corresponding to the cytologic findings in 30.8% of the cases in group IIID (n = 26) using Papanicolaou's classification system. In groups IV a (n = 72), IV b (n = 44), and V (n = 28), we found a histologic explanation in 54.2%, 47.7%, and 60.7% of the cases, respectively. In most cases we observed polymorphic cellular changes primarily associated with HPV. PMID- 7557217 TI - [Occurrence of suspicious changes in cervix cytology in women after liver transplantation]. AB - After transplantation the necessary immunosuppressive therapy predisposes to the development of de novo cancers. From January 1989 to April 1994 we collected PAR smears of 98 women before and repeatedly after liver transplantation. After surgery all women received as immunosuppressive agents either Cyclosporin A or FK 506 as long-term medication. In seven patients (8.5%) who had a normal cervical cytology before transplantation a suspicious PAP smear was found on average 11 months after transplantation. In five cases a histological verification (exploratory excision, coning of the cervix) was made. In two cases we found a CIN III. Possible causes for the higher incidence of dysplasia of the cervix observed in the transplant patients are discussed. Similar to kidney transplant recipients female liver transplant recipients are at higher risk of developing cervical dysplasias and neoplasias. Accelerated development of malignancy seems likely. The influence on the cervical cells of both immunosuppressive drugs Cyclosporin A and FK 506 seems to be of a similar nature. Recommendations for the gynaecological care of female liver transplant recipients treated with immunosuppressive therapy are given. PMID- 7557218 TI - [The Bethesda system--an improvement in classification of cervix cytology?]. AB - Cervical cytology screening programmes have significantly reduced cervical cancer mortality. In 1988 the National Cancer Institute developed a new classification for reporting cervical cytology--"The Bethesda System". The aim of our study was to compare the clinical usefulness of this new classification with the classification used in German-speaking countries (which discriminates cytologically between moderate and severe dysplasia) including a verbal classification predicting the histological grade of cervical intraepithelial neoplasia (CIN I, II, III). 671 patients with abnormal cervical cytology results and subsequent conisation or hysterectomy were included. We correlated cytological and histological diagnosis (gold standard). In cases of cytologically suggested moderate dysplasia we found histologically CIN I, CIN II and CIN III in 11%, 41% and 42%, respectively. Our results show that discrimination of CIN II and CIN III by means of cytology is limited. Using our therapy schedules we found that adequate therapy is possible by each of the cytological classification systems. The cervical smear is used as a screening test to determine which women require further examination using colposcopy. Under these circumstances an adequate therapy is possible with both classifications. The results underline the importance of colposcopy in the management of cervical dysplasia. As long as conisation is commonly used in German-speaking countries as the only diagnostic tool after an abnormal cervical smear the Bethesda System increases the risk of overtreatment. PMID- 7557219 TI - [The prognostic effect of scintigraphy-guided lymphadenectomy in therapy of stage Ib cervix carcinoma]. AB - In a retrospective study the survival rates of 161 patients with stage Ib cervical cancer after radical operation (Latzko, Wertheim-Meigs) including complete or incomplete pelvine lymphadenectomy were compared. To increase radicality of lymphadenectomy, preoperative targeting of pelvic lymph nodes was done in all the patients using 99mTc-Sb2S3 radiocolloid. Intraoperatively, a gamma-camera being integral part of an operating table allowed delineation and scintigraphy-guided resection of pelvic lymph nodes. Dependent on the evidence of remaining pelvine foci of radioactivity at the end of the operation, lymphadenectomy was assessed as complete or incomplete. Mean observation time of completely lymphadenectomised patients (n = 117,72.67%) were 80 months (5-169 months) and 42 months (1-149 months) of the incompletely lymphadenectomised patients (n = 44,27.33%). In 28 (23.93%) completely lymphadenectomised patients against only 5 (11.36%) patients with incomplete lymphadenectomy, lymph node metastases were proved histologically. Five year-survival rate of completely lymphadenectomised patients was 85.47% and 88.64% of incompletely lymphadenectomised patients (not significant, Mantel-Test). Also, selective comparison of lymph node-positive patients did not suggest a divergent trend indicated by 13 (46.43%) deaths of completely and 3 (60.0%) deaths of the incompletely lymphadenectomised patients after an observation of five years. The technique of scintigraphy-guided pelvic lymphadenectomy using 99mTc-Sb2S3 radiocolloid cannot be expected to improve prognosis of patients with cervical cancer stage Ib. PMID- 7557220 TI - [Pregnancies in HIV infected women in Switzerland]. AB - In a national multicentre study, 229 pregnancies in 219 HIV-positive women were prospectively followed up between January 1, 1990, and October 30, 1993. 69.8% were infected by intravenous drug abuse and 91.5% were asymptomatic (CDC classes II and III) in early pregnancy. 48 (21.0%) were first discovered to be HIV infected during the index pregnancy: 46 of these had risk factors. The present epidemiologic development does not seem to warrant a general HIV-screening in pregnancy at this time. 71 pregnancies (31%) were terminated; 158 children were born, 17 (23.3%) of the 73 definitely classified are HIV-infected. An asymptomatic HIV infection with a sufficiently high (> 200/microliters) CD4 cell count has no proven influence on the pregnancy. Otherwise, however, maternal infectious diseases can lead to prematurity. For mothers with i.v. drug abuse, there is a significantly higher incidence of prematurity and fetal growth retardation. The maternal HIV infection can be transmitted to the child either during pregnancy or at delivery. The incidence of vertical transmission in our study was 23.3%; the most predictive parameter for a prenatal HIV transmission is a low anti-p24 antibody titre. The risk of intrapartum transmission seems to be somewhat, but not significantly, reduced for primary Caesarean sections. Recently, prophylaxis with Zidovudin during pregnancy, beginning after the 14th GW, was found to reduce vertical HIV-transmission by 66%. Since the virus can also be transmitted through mothers' milk, HIV-positive mothers should not nurse their babies. Maternal infections are significantly more frequent in HIV-positive women, and are a risk factor for prematurity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557221 TI - [Immune status of pregnant patients especially in possible premature labor]. AB - In this study it could be shown that pregnant women having a normal course of pregnancy had significantly higher levels of T-lymphocytes and T-helper cells compared with non-pregnant women. Also other parameters belonging to cellular immune defence showed a similar--in the sense of an improved immune function- trend (even though this was not statistically significant). In pregnant women with symptoms of threatened prematurity significantly lower levels for lymphocytes. T-lymphocytes and T-helper cells were measured than in women who had a normal course of pregnancy. The ratio was also clearly reduced--in those cases where later preterm labour did actually occur it was particularly low at 1.1. In the group of pregnant women with premature labor the number of those who found the situation "very stressful" amounted to 65% and in the group whose course of pregnancy was normal, the percentage was 26%. The results of this study point to the fact that in pregnant women with premature labor the immune function is often impaired and it can be assumed that this provides favourable conditions for ascending infections which then cause a higher risk of prematurity. Further studies should be made concerning the causal connections which we have postulated between physical and psychological overstrains and impairment of the immune function. PMID- 7557222 TI - [Initial experiences with ambulatory irrigation hysteroscopy]. AB - During one year, 120 patients were referred for outpatient diagnostic hysteroscopy. The most common indications were abnormal uterine bleeding (67%), followed by sterility and infertility, respectively (18%). The uterine cavity was visualised in 103 patients, and about half of the patients (50.5%) had demonstrable uterine pathology. 75 patients (72.8%) did not experience any pain during or after the examination and 28 women (27.2%) reported varying levels of pain. In 17 cases (14%) passage through the internal cervical os was either not possible for anatomical reasons or was not tolerated by the patients. However, diagnostic hysteroscopy, combined with directed biopsy where appropriate, is now considered the method of choice for identifying intrauterine pathology. Performed in an outpatient setting, diagnostic hysteroscopy using a fluid distention medium is largely pain-free and hardly accompanied by side effects, provided patients are thoroughly counseled beforehand. Essential preliminaries for a successful examination are careful patient selection, a clear clinical indication and, above all, the skilled coordination between eye and hand. More widespread use of diagnostic hysteroscopy may spare numerous women the stress of curettage while at the same time facilitating optimal selection of those cases really requiring further medical examination or therapy. PMID- 7557223 TI - [Initial experiences with laparoscopic intraoperative ultrasound]. AB - Preoperative ultrasound as well as intraoperative laparoscopic diagnosis have both their limits, a fact that might be significant for laparoscopic surgical management. In particular, since in this surgical domain, where almost everything has become technically feasible, the operator must decide what is to the benefit of the patient. A possible solution could be laparoscopic ultrasound, i.e. ultrasound diagnosis per laparoscopy at the very site of the lesion. The advantages are evident. Due to the closeness to the organ to be examined, the frequency of the scan head can be extremely high, resulting in better resolution. Also, structures could be visualized by ultrasound which e.g., due to adhesions cannot be seen with the laparoscope. Moreover, ultrasound offers a view into the structures which can be seen only from the exterior with the laparoscope. To perform these examinations we used a specially designed scanhead (Toshiba): Instead of the optical system a crystal array was inserted into a conventional gastroscope. The result was a high resolution 7.5 MHz linear array at the distal end of a freely movable gastroscope. Colour doppler sonography is feasible with this scanhead. We examined 19 patients, 16 of them with ovarian tumours, and attempted visualization of the uterine myometrium/endometrium as well as of the liver. In six cases of ovarian tumours in which only cystic structures were found by preoperative transvaginal sonography, laparoscopic ultrasound additionally revealed solid inner structures. In 5 cases direct laparoscopic view of the ovarian lesions was impossible due to severe adhesions. They were, however, easily detected by intraoperative ultrasound.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557224 TI - [Diagnostic microlaparoscopies with 2 mm optics]. AB - Within a period of 5 months a serious of 42 small diameter laparoscopies (2 mm) (SDL) were performed with a modified technical approach. 28 (SDL). Procedures were done under general anaesthesia and the findings were controlled by concurrent conventional laparoscopy. In 26 out of 28 patients we could confirm the SDL diagnosis. 14 patients had SDL in analgosedation. Optical information gained by SDL was sufficient and, as experience in handling the system increased, also competitive. The procedures performed in analgosedation were tolerated and highly appreciated (reduced incisional pains, reduced complaints due to pneumoperitoneum). SDL procedures seem to have possible indications in gynaecology. PMID- 7557225 TI - [Cranial sinus thrombosis in puerperium]. AB - Sinus venous thrombosis is a rare and severe disease, clinically characterised by headache, nausea, vomiting, focal deficits and epileptical seizures. We describe a case of sinus venous thrombosis 7 days post partum in a 21-year old woman. The pathogenesis symptoms, diagnosis and therapy are discussed. Advice for obstetric and gynaecological treatment after sinus venous thrombosis is given. PMID- 7557226 TI - [Special suction tube holder for a laser handpiece in effective smoke suction evacuation. On the problem of smoke suction in external CO2 laser therapy]. AB - Treatment with a CO2 laser causes high smoke production, which leads to reduced view to the surgical field, to bad smell and has potential health hazards. Hitherto the problem of the external smoke evacuation had not been solved sufficiently. Therefore a special sucking-tube holder for a laser handpiece is presented. The end of the sucking tube exactly follows all movements of the handpiece and can be positioned close to the source of smoke. Through a defined angle the smoke can be removed quickly out of the way of the laser beam. According to the practical experiences so far, this special equipment on a laser handpiece guarantees an effective evacuation of smoke, allows the reduction of assisting staff, reduces irritation through bad smell and additionally minimizes the potential risks of smoke caused by an external CO2 laser application. PMID- 7557227 TI - [Immunophenotypic heterogeneity of CD7+CD4-CD8--acute leukemia]. AB - Fourteen cases of lymphoid and myeloid acute leukemia (AL) were studied for expression on blast cells of CD7 antigen, a cell surface marker found early during T lineage differentiation. This heterogenic group of CD7+ CD4-CD8- AL includes distinct cytological subvariants with: myeloid (AML MO, M1, M4, M5) and lymphoid (pre-T-cell) commitment, biphenotypic or mixed lineage AL and AL with minimal signs of blast cell differentiation, which appear not to follow lineage restriction. The latter subset of AL may represent the transformed counterpart of an early stem cell prior to lineage commitment. PMID- 7557228 TI - [Use of a new antimacrophage monoclonal antibody D11 in diagnosis of hemoblastosis]. AB - Expression of new Mab D11 on blood and bone marrow cells was investigated in 85 hemoblastosis patients. In normals, antigen D11 is expressed on some monocytes and all tissue macrophages. D11 was noted on lymphoblasts of 5 out of 10 cases with B-cell ALL and of 1 case in B-lymphoid blast crisis of chronic myeloid leukemia. In T-ALL, ANLL, non-Hodgkin's lymphomas in leukemization stage, hairy cell leukemia and chronic lymphoid leukemia the cells were nonresponsive to Mab D11. Unlike D11 which have round nuclei, lymphoblasts D11+ have folded nuclei and more pronounced cytoplasmic basophilia. There were both B and myeloid antigens on D11+ blasts. ALL D11+ patients had extramedullary foci, more suppressed granulocytic and thrombocytic components of hemopoiesis, shorter remissions than those with ALL D11-. PMID- 7557229 TI - [Blood lymphocyte ultrastructure in chronic lymphoproliferative diseases]. AB - The authors have found that in chronic B-cell lymphoid leukemia and splenic lymphocytoma the proportion of irregular nuclei diminishes, in some patients split nuclei emerged. The proportion of irregular nuclei in lymphocytoma is significantly higher than that in chronic leukemia. The above diseases are characterized by disordered position of mitochondria in relation to cell center and of this center against nuclear invaginations. Microvilli on the cell surface become less numerous. It is suggested that in chronic lymphoproliferative diseases cytoskeleton in tumor cells may undergo structural and functional alterations. PMID- 7557230 TI - [Experience in using plasmapheresis in comprehensive therapy of myelotoxic agranulocytosis in cancer patients]. AB - Plasmapheresis was applied in 10 patients given chemotherapy and radiation treatment for myelotoxic agranulocytosis. After 1-3 sessions the patients improved, recovered hemopoiesis and normal protein metabolism. Of 10 patients 9 survived. PMID- 7557231 TI - [Feasibility of correction of blood aggregation properties in the course of extracorporeal hemosorption in atherosclerosis patients]. AB - Aggregation and rheological properties of blood were evaluated in 20 atherosclerotic patients after cryoplasmosorption. The change of the patients' plasma for extracorporeally modified autoplasma reduced coagulation potential, enhanced fibrinolysis and suppressed anticoagulation systems. Clinical efficacy manifested in decreased frequency and duration of anginal episodes, intensity of intermittent claudication. Biochemically, cholesterol levels and atherogenic index lowering was evident. PMID- 7557233 TI - [Induction of human platelet aggregation by nerve tissue antigens and immune complexes]. AB - It is shown that antigens of the peripheral nerve and immune complexes (IC) are able to induce platelet aggregation. The highest IC aggregation activity was seen in the excess of the antigens. Specific antiserum had no aggregation capacity, but IC produced in its participation displayed higher aggregation activity than free antigens. The involvement of antigens and IC of the peripheral nerve in impairment of platelet function is suggested. PMID- 7557234 TI - [Restoration of hematopoiesis following allo- and automyelotransplantation in patients with hemoblastosis]. AB - The early stages of hemopoiesis repair as indicated by histologic examinations of trephine biopsy specimens (visual assessment and morphometry) are characterized in 39 patients with hemoblastoses, 33 of these with acute leukemia, 4 with chronic myeloleukemia, and 2 with lymphoblastic lymphosarcoma, following transplantation of allogeneic (n = 19), syngeneic (n = 5), and autologous (n = 15) bone marrow after conditioning with cyclophosphamide plus total radiation exposure in 27 patients and myelosan with cyclophosphamide in 12 ones. Hemopoiesis repair was found to be slower after autologous transplantation of bone marrow than after allogeneic transplantation and in patients following conditioning with cyclophosphamide and total irradiation in comparison with those pretreated with myelosan and cyclophosphamide. Patients' age over 25, diagnosis of the disease more than 12 months before transplantation, and variant of leukemia (nonlymphoblastic) were the factors exerting negative influence on the rate of hemopoiesis recovery. PMID- 7557232 TI - [Effect of phorbol ester on the proliferation and differentiation of blast cells from leukemia patients and blast secretion of biologically active products]. AB - The influence of tetra phorbol diesther (TPA) on primary blast cells of patients with acute leukemia and blastic crises of chronic myelogenous leukemia and the influence of the condition medium (CM) of the primary and TPA-treated blast cells on the proliferation of HL-60 cell line has been studied. The level of interferon alpha (IFN-alpha) in CM was tested. TPA inhibited proliferation and induced macrophage-like differentiation of primary AML blast cells and these changes were accompanied by modulation of IFN-alpha expression in CM. The effect of blast CM on proliferation of HL-60 was both inhibitory and stimulating and depended on the time of treatment and individual characteristics of patients. It has been shown that the level of IFN-alpha in CM was not correlated with antiproliferative effect of CM. The role of individual differences in capability of primary blast cells to be induced by differentiated agents and the nature of these differences are discussed. PMID- 7557235 TI - [H2O2 generation and human neutrophil aggregation as affected by lectins]. AB - The effects of eight plant lectins on human neutrophils aggregation and H2O2 release were studied. Both processes were stimulated by lectins from Viscum album, Triticum vulgaris, Phaseolus vulgaris and Canavalia ensiformis while lectins from Solanum tuberosum, Sambucus nigra and Glycine mas displayed only aggregating activity and Arachis hypogaea lectin was not effective. Heating (46 degrees C) and UV-radiation during 4-5 min were found to suppress completely H2O2 release from neutrophils keeping the capacity of cells to be aggregated. The findings indicate that lectin-induced human neutrophils aggregation is necessary but not sufficient condition of respiratory burst of cells. The comparison of the aggregating activity of lectins with their ability to induce the H2O2 release is supposed to be the basis of screening of lectins with antitumor and immunomodulatory activity. PMID- 7557236 TI - [Use of fractionated soy phospholipids in a test of activated partial thromboplastin time]. AB - Fractionated phospholipids (PL) from soya beans were tried as partial thromboplastin (PT) in the activated partial thromboplastin time test. PL fractions were investigated for content of individual phospholipid classes, acidity and procoagulant activity as were PT from human brain and chromatographically pure PL. Procoagulant activity of phospholipid mixtures is higher than that of chromatographically pure PL and is unrelated to PL variety. Soya PL mixtures with human brain PT demonstrating high procoagulant activity are characterized by 0.3-0.8 ratio of total acid to total neutral PL and acid number 40-60 mg KOH. Isolated from soya PL procoagulants accelerate clotting of plasma from healthy subjects 2.2-fold being just a little less active than brain PL. This makes the above procoagulants usable as test-reagent for evaluation of hemostasis. PMID- 7557238 TI - [Comparative analysis of causes of error in determining blood groups of the ABO system with isohemagglutinating sera and monoclonal antibodies]. PMID- 7557237 TI - [Quantitative and cytologic changes in various types of blood leukocytes in liquidators 5-6 years after the accident at the Chernobyl power plant]. AB - Radiation-induced effects were studied in leukocytes from 300 males 5-6 years after the Chernobyl accident. Radiation dose received ranged from 5 to 100 sGy. Leukocyte composition of blood was determined in blood smears stained according to Pappenheim and at flow cytometry on Facs tar laser cell sorter. Compared to controls, the majority of the patients had moderate relative and absolute lymphocytosis, eosinophilia and neutrophilopenia. Nuclear structure was changed more frequently in lymphocytes and neutrophils from the exposed subjects, especially in those who had received large dose. The findings evidence that 5-6 years after radiation accident the exposed subjects develop quantitative and qualitative shifts in leukocytes suggesting probable radiation effects on genetic system of the baseline hemopoietic cells. PMID- 7557240 TI - [Evaluation of the effectiveness of immunotherapy and prediction of pregnancy outcomes on the basis of parameters of cellular immunity in women with habitual abortion]. PMID- 7557239 TI - [Creation and evaluation of the effectiveness of a computer database based on results of screening of donors for markers of viral hepatitis B and C]. PMID- 7557241 TI - The effect of lithium on endothelial-dependent relaxation in rat isolated aorta. AB - 1. In endothelium-containing rings of rat aorta, precontracted by phenylephrine, addition of acetylcholine (Ach), resulted in a concentration-dependent relaxation through the release of endothelial dependent relaxing factors, including nitric oxide (IC50 = 8.41 microM). 2. Pretreatment of the tissues with 20 microM indomethacin, significantly decreased the relaxation. 3. Preincubation of the preparations in medium solution in which sodium has been partially replaced by 0.5 mM lithium, significantly reduced Ach-induced endothelial dependent relaxation (EDR). 4. Lithium (2 mM) in medium, significantly increased Ach induced relaxation. 5. As is shown in this study, lithium has two opposite actions of EDR, with the dose of 0.5 mM inhibiting, while the dose of 2 mM potentiates EDR. Thus it seems that the action of lithium on EDR is mediated through two separate mechanisms. PMID- 7557242 TI - Alteration by ouabain of rat submandibular glands function. AB - 1. Effects of various doses of intraperitoneal ouabain (1,2 and 5 mg/kg) on rat submandibular saliva were investigated in this study. 2. Potassium and calcium and their product (K+ x Ca2+) were found to be elevated in all groups. 3. Changes in salivary flow were not the major cause of the alterations in electrolytes. 4. Protein concentrations were elevated in the doses of 1 and 2 mg/kg of the drug and somewhat reduced in the dose of 5 mg/kg of ouabain but still above the base line. 5. The results show that there is an ouabain-induced close parallelism between magnesium and total protein secretion from rat submandibular glands. PMID- 7557243 TI - Effects of chronic lithium pretreatment on apomorphine-induced penile erection. AB - 1. The effects of chronic lithium pretreatment (600 mg/l in drinking rats, 30 days) on penile erection (PE) induced by apomorphine were investigated in rats. This treatment resulted in a serum Li concentration after 30 days of 0.31 +/- 0.01 mmol/l. 2. Subcutaneous (s.c.) administration of mixed D1/D2 dopamine receptor agonist apomorphine (0.05-0.5 mg/kg) induced PE in a biphasic manner. The maximum effect was obtained with 0.1 mg/kg of the drug while the response decreased with increasing doses of apomorphine from 0.1 to 0.5 mg/kg. 3. Pretreatment of animals with 0.0125-0.1 mg/kg of D1 dopamine receptor antagonist SCH 23390 or D2 dopamine receptor antagonist sulpiride (12.5-100 mg/kg) decreased apomorphine-induced PE. Combination of SCH 23390 (0.025 mg/ kg) with sulpiride (12.5 mg/kg) caused a stronger inhibitory effect on apomorphine response. This indicates that both D1 and D2 dopamine receptors may be involved in PE induced by apomorphine. 4. The response induced by apomorphine (0.05-0.05 mg/kg) was decreased in animals pretreated with chronic lithium. The inhibitory effect of sulpiride on apomorphine response, increased in animals pretreated with lithium, in contrast the inhibitory effect of SCH 23390 did not change in this condition. However, a combination of SCH 23390 with sulpiride increased inhibitory effect on apomorphine response in lithium pretreated rats. 5. It is concluded that chronic lithium inhibits PE induced by dopaminergic mechanism(s). PMID- 7557244 TI - Gastroprotective activity of the novel proton pump inhibitor lansoprazole in the rat. AB - 1. The protective activity of lansoprazole was evaluated on gastric mucosal lesions induced by intragastric 25% NaCl (1 ml/rat for 1 hr) and by indomethacin (20 mg/kg for 6 hr) in the rat and compared with that of omeprazole. 2. Lansoprazole (3, 10 and 10 mumol/kg i.g.) dose-dependently prevented the formation of indomethacin-induced lesions, the inhibition being 99% at the highest dose. Omeprazole, 10 mumol/kg i.g., enhanced the damage by indomethacin while higher doses caused a reduction, lesion index being reduced by 98% at 100 mumol/kg. 3. Histologically in lansoprazole- as well as in omeprazole-pretreated rats, indomethacin-induced necrosis of the mucosa was absent, luminal epithelium being intact. 4. Lansoprazole (30, 100 and 300 mumol/kg) and omeprazole (30, 100 and 300 mumol/kg) dose-dependently reduced the formation of lesions by hypertonic saline. 5. Present results indicated that lansoprazole and omeprazole protect the gastric mucosa in different experimental models of gastric ulceration. PMID- 7557246 TI - Effect of bunazosin, alpha 1-adrenoceptor blocker, on multidirectional contractile response and localization of bunazosin binding sites in human hypertrophied prostate. AB - 1. Effects of bunazosin, an alpha 1-adrenoceptor blocker, on the contraction induced by norepinephrine in human hypertrophied prostate were examined in vitro. 2. Prostatic specimens showed maximum contraction at 10(-4) M norepinephrine in longitudinal and circumferential directions to the urethra. 3. Bunazosin (10(-7) M) blocked norepinephrine-induced contraction with a parallel shift of the dose response curve in both directions (pA2: 8.76 +/- 0.15; pA2: 8.90 +/- 0.08, respectively). 4. Serial sections of prostates were also evaluated by autoradiography. The binding sites were diffusely distributed in the interstitium. 5. We concluded that bunazosin affects multidirectional contraction in prostates. PMID- 7557247 TI - Enhancement in the protective qualities of gastric mucus by ebrotidine during duodenal ulcer healing. AB - 1. Gastric mucus from duodenal ulcer patients before and following therapy with a new antiulcer agent, ebrotidine, at 400, 600 and 800 mg dose was examined for changes in the physicochemical qualities and anti-H. pylori activity. 2. The results of physical measurements revealed that successful therapy with ebrotidine was accompanied by a 25% increase in gastric mucus viscosity, and a 20% increase in H+ retardation capacity, while its hydrophobicity increased by 11%. 3. The enhancement in the physical properties of mucus with ebrotidine therapy were also reflected in a marked (2.6-2.9-fold) increase in the proportion of the high molecular weight form of mucin. Furthermore, following therapy with ebrotidine, the gastric mucins showed a 36% higher content of sulfate as compared to that before the therapy. 4. Assays on the H. pylori aggregating titer of gastric mucin revealed that ebrotidine therapy at all three doses evoked a 4-fold increase in mucin anti-H. pylori activity. 5. The data demonstrate that duodenal ulcer therapy with ebrotidine leads to a marked improvement in the protective qualities of gastric mucus essential for the maintenance of mucosal integrity and enhances the inherent mucosal defense against H. pylori infection. PMID- 7557245 TI - Gastric antisecretory activity of lansoprazole in different experimental models: comparison with omeprazole. AB - 1. The activity of the novel proton pump with inhibitor lansoprazole was examined in different gastric secretion models in vitro and in vivo, in comparison with omeprazole. 2. In the conscious cat with gastric fistula lansoprazole (0.25-2 mumol/kg i.v.) caused a dose-dependent reduction of the acid secretion induced by dimaprit, pentagastrin, 2-deoxy-D-glucose and bombesin, being approximately as potent as omeprazole (0.25-1.5 mumol/kg i.v.). Similar to omeprazole, lansoprazole was also more effective when administered in hyperacidic states. 3. In the anaesthetized rat with lumen perfused stomach lansoprazole (0.03-1 mumol/kg i.v.) was approximately 3 times more potent than omeprazole (0.1-3 mumol/kg i.v.) in inhibiting the acid secretion induced by histamine, 2-deoxy-D glucose and forskolin. 4. In the isolated gastric fundus from the immature rat lansoprazole (1-30 microM) reduced basal acid secretion and the acid response to histamine and forskolin, with a potency not significantly different from that of omeprazole. 5. No significant differences were found in the different species between lansoprazole and omeprazole as for the duration of action. 6. In conclusion, lansoprazole exerts a marked antisecretory effect in a variety of gastric secretion models from different species. However, it did not significantly differ from omeprazole when considering either the potency or the duration of action. PMID- 7557248 TI - D-lysergic acid reduces microtubule-associated tau protein in SH-SY5Y human neuroblastoma cells. AB - 1. SH-SY5Y, an adrenergic human neuroblastoma cell line, was used to examine the hypothesis that D-lysergic acid (LSD) affects the metabolism of microtubule associated tau protein, thus affecting microtubule assembly and the transport of neurotransmitters. 2. After 48 hr treatment LSD (10(-5) and 10(-7) M) decreased 50 kDa tau protein in the membrane (pellet) fraction. The drug (10(-5) M) also decreased in the cytoplasmic (supernatant) fraction. 3. This reduction in tau protein was accompanied by a 65% increase (P < 0.05) in total protein after LSD (10(-7) M) in the cytoplasmic fraction. PMID- 7557250 TI - Non-competitive antagonism of N-methyl-D-aspartate receptor inhibits tolerance to the analgesic action of U-50,488H, a kappa-opiate receptor agonist in the rat. AB - 1. The effect of dizocilpine (MK-801), a non-competitive inhibitor of N-methyl-D aspartate (NMDA) receptor on the development of tolerance to the analgesic and hypothermic actions of U-50,488H, a highly selective kappa-opiate receptor agonist, was determined in the rat. 2. Male Sprague-Dawley rats were made tolerant to the pharmacological actions of U-50,488H by twice daily intraperitoneal (i.p.) injections of the drug (25 mg/kg) for 4 days. 3. Multiple injections of U-50,488H resulted in the development of tolerance to its analgesic and hypothermic actions in the rat. MK-801 injected 10 min before each injection of U-50,488H, dose-dependently inhibited the development of tolerance to the analgesic action but the tolerance to the hypothermic action of U-50,488H was unaffected. 4. Multiple injections of U-50,488H decreased the body weight gain. MK-801 dose-dependently decreased the gain in body weight further. 5. The results indicate that non-competitive antagonism of the NMDA receptor by MK-801 can selectively inhibit the tolerance to the analgesic action of U-50,488H in the rat, however, such an effect was associated with significant decreases in normal gain in body weight. PMID- 7557249 TI - Attenuation of tolerance to, and physical dependence on, morphine in the rat by inhibition of nitric oxide synthase. AB - 1. The effect of NG-monomethyl-L-arginine (NMMA), an inhibitor of nitric oxide synthase (NOS), on the development of tolerance to and physical dependence on morphine was determined in the rat. 2. Male Sprague-Dawley rats were rendered tolerant to and dependent on morphine by the subcutaneous implantation of four morphine pellets (each containing 75 mg morphine base) during a 3 day period. Placebo pellet implanted rats served as controls. 3. Chronic administration of morphine resulted in the development of tolerance to the analgesic action of morphine. Twice daily injections of NMMA (4 or 8 mg/kg) attenuated the tolerance to morphine as evidenced by higher analgesic response in NMMA treated than in vehicle treated morphine tolerant rats. 4. Chronic administration of morphine also resulted in the development of physical dependence as evidenced by the appearance of a variety of symptoms including stereotyped jumping response following naltrexone injection. Concurrent treatment with NMMA inhibited naltrexone-induced jumping response but other responses like fecal boli formation, wet dog shakes, teeth chattering, rearing and ejaculations were not modified. 5. It is concluded that inhibition of NOS can attenuate the development of tolerance to, and physical dependence on, morphine in the rat. However, it appears that higher doses of NOS inhibitors are required in the rat than in the mouse for blockade of both tolerance and physical dependence processes. PMID- 7557252 TI - Effects of NMDA receptor modulation on hippocampal type 2 theta activity in rats. AB - 1. The present study was designed to investigate whether pharmacological modulation of N-methyl-D-aspartate (NMDA) receptor function could modify hippocampal type 2 theta activity in the dentate gyrus of rats. 2. The effects of pre-recording administration of d-cycloserine (DCS: 1.0, 3.0 and 9.0 mg/kg, i.p.), a partial agonist at the NMDA receptor associated glycine site, and MK-801 (0.1 mg/kg, i.p.), a noncompetitive NMDA receptor antagonist, were examined in freely moving rats. 3. Using adult Wistar rats, which had recording electrodes implanted unilaterally into the hilus of dentate gyrus, we recorded five 4 sec epochs of awake-immobility-related hippocampal EEG activity bands (1-20 Hz) 40 min after d-cycloserine and 2 hr after administration of MK-801. 4. In the off line analysis, the spectral power and the frequency at the maximal theta power were calculated. 5. D-cycloserine (1.0-9.0 mg/kg) did not affect the frequency at the maximal theta power. However, the dose of 3.0 mg/kg, though not the 1.0 or 9.0 mg/kg doses, significantly increased the spectral power of the hippocampal immobility-related EEG activity. 6. In line with the previous findings, 0.1 mg/kg MK-801 decreased both the frequency at the maximal theta power as well as the spectral power of hippocampal type 2 EEG activity. 7. The present data show a clear relationship between NMDA receptors and hippocampal type 2 theta activity and suggest that the pharmacological modulation of the receptor function, using appropriate doses of glycine binding site agonist, d-cycloserine, may be a possible means to positively modulate the immobility-related hippocampal EEG activity. PMID- 7557253 TI - Inhibition by ginseng total saponin of the development of morphine reverse tolerance and dopamine receptor supersensitivity in mice. AB - 1. Ginseng total saponin (GTS), 200 mg/kg i.p. 3 hr prior to morphine, inhibited the development of reverse tolerance to the ambulatory-accelerating effect of morphine. 2. GTS, 200 mg/kg, also prevented the development of dopamine receptor supersensitivity induced by the chronic administration of morphine, 10 mg/kg a day for 7 days. 3. These results suggest that GTS may be useful for the prevention and therapy of the adverse action of morphine. PMID- 7557251 TI - CI-966, a GABA uptake inhibitor, antagonizes ischemia-induced neuronal degeneration in the gerbil. AB - 1. Cerebral ischemia of 5 min duration was induced in unanesthetized gerbils by bilateral occlusion of the carotid arteries. 2. The extent of cerebral damage was assessed by the elevation of motor activity in comparison with pre-ischemic levels and by a histological assessment of the extent of neuronal degeneration of the CA1 area of the hippocampus. 3. The GABA transport inhibitor CI-966 (10 mg/kg i.p.) was tested for cerebroprotective activity in a gerbil stroke model. CI-966 reduced the extent of stroke injury as assessed by locomotor activity and measurement of hippocampal CA1 pyramidal cell injury. 4. It is proposed that enhancement of extracellular GABA levels during ischemia accounts for the cerebroprotective actions of CI-966. PMID- 7557255 TI - Antinociceptive effects of clebopride in the mouse. AB - 1. The effects of the substituted benzamide clebopride, an orthopramide, on nociception of chemical and thermal stimuli were investigated. 2. Clebopride (0.5, 1.0 and 2.0 mg/kg) promoted significant analgesia in the tail-flick and hot plate tests and against abdominal constrictions produced by acetic acid or acetylcholine. 3. The analgesic effects of clebopride were not influenced by pretreatment with naltrexone (1-3 mg/kg). 4. The results suggest that clebopride induces analgesia against both thermal and chemical nociceptive stimuli, which is not mediated via opioid mechanisms. PMID- 7557257 TI - The disposition of new arabinosylcytosine derivative-- 5'-chloro-5'-deoxy arabinosylcytosine--in rats. AB - 1. Pharmacokinetic properties of a new derivative of the widely used and very potent antileukemic agent arabinosylcytosine (araC)--5'chloro-5'-deoxy arabinosylcytosine (5'-Cl-araC)--were investigated after intraperitoneal (i.p.) and oral routes of administration in rats and compared with the equimolar dose of araC administered orally. 2. It was found that substitution of the hydroxyl group at position 5' resulted in a change of pharmacokinetic parameters. 3. There is a large difference in average serum concentrations of 5'-Cl-araC administered by the i.p. and oral routes; the average serum concentration obtained after i.p. injection being several times higher in comparison to those after oral administration. 4. However, the latter are, at the same time, lower than the average serum concentrations of araC administered by the same route in an equimolar dose. 5. On the other hand, the apparent volume of distribution is much larger, and the area under the curve of serum concentration of 5'-Cl-araC is smaller, after oral as compared to the i.p. route of administration indicating more extensive tissue distribution together with higher tissue binding of 5'-Cl araC when compared to the parental drug araC. PMID- 7557254 TI - Effect of omega-conotoxin GVIA on tetrodotoxin-insensitive acetylcholine release by nicotine in guinea-pig bladder. AB - 1. Contractile responses and acetylcholine release evoked by nicotine and electrical field stimulation (EFS) were determined by isotonic transducer and radioimmunoassay, respectively. 2. Nicotine-induced contraction was reduced to 30% by nicotinic receptor antagonist, hexamethonium but was insensitive to tetrodotoxin. EFS-induced contraction was abolished by tetrodotoxin but was insensitive to hexamethonium. Replacement of external Na by choline completely abolished the contractile responses evoked by nicotine and EFS. 3. Both contractions evoked by nicotine and EFS were inhibited by omega-conotoxin GVIA, and inhibitory effects of the toxin were greater in low Ca concentrations. 4. In the condition that external Na or Ca is omitted from physiological solution, acetylcholine release evoked by nicotine was not observed. Nicotine-induced acetylcholine release was partially inhibited by omega-conotoxin but was insensitive to tetrodotoxin. 5. In conclusion, nicotine interacts with nicotinic receptors located on nerve terminals and produces transmitter release which depends on external Na through tetrodotoxin-insensitive mechanisms. It is suggested that voltage-dependent omega-conotoxin sensitive Ca channels are partially involved in the nicotine-induced transmitter release. PMID- 7557256 TI - The effect of nickel chloride on the isolated hemidiaphragm of the rat. AB - 1. NiCl2 (cumulative concentrations of 0.56-1.91 mmol 1(-1)) produced concentration-dependent depression of tension developed (Td) and the maximum rate of rise of tension (dT/dt max) of isometric contraction of the isolated rat hemidiaphragm, during direct subtetanic (DST) electrical stimulation, only. EC50 values for NiCl2-induced depression of Td and Dt/dt max were 0.88 +/- 0.06 and 0.83 +/- 0.13 mmol 1(-1), respectively. NiCl2 did not significantly change either parameter of the isometric contraction during direct single-pulse (DSP) electrical stimulation. 2. Maximal depression of Td and dT/dt max, produced by a single concentration of NiCl2 (1 mmol 1(-1)) during DST electrical stimulation was obtained 20 min after addition of the drug in the bathing medium. 3. In the normal Tyrode solution, addition of CaCl2 (final concentration of 5.86 mmol 1( 1)) almost completely antagonized the depressant effect of NiCl2 (1 mmol 1(-1)) on Td and dT/dt max during DST electrical stimulation. In the calcium-free solution, the depression both of Td and dT/dt max produced by NiCl2 (1 mmol 1( 1)) was significantly more pronounced in comparison with the effect of NiCl2 in the normal Tyrode solution. 4. L-calcium channel activator, Bay K 8644 (25 mumol 1(-1)), significantly potentiated both Td and dT/dt max during DST electrical stimulation, but NiCl2 (1 mmol 1(-1)) decreased both parameters of the isometric contraction even in the presence of this concentration of Bay K 8644. On the other hand Bay K 8644 (25 mumol 1(-1)) did not antagonize NiCl2-induced depression of Td and dT/dt max. 5. Verapamil (2.5 mumol 1(-1); 45 min of incubation) and lidocaine (0.10 mmol 1(-1); 30 min of incubation) significantly potentiated the depression of Td and dT/dt max, produced by NiCl2 (1 mmol 1(-1), during DST electrical stimulation. The addition of CaCl2 (final concentration of 7.20 mmol 1(-1)) in the bathing medium only partially antagonized the depressant synergistic action of both verapamil or lidocaine and NiCl2 on Td and dT/dt max. 6. Forskolin (cumulative concentrations of 2.60-44.20 mumol 1(-1)) fully antagonized NiCl2-induced depression of both Td and dT/dt max; propranolol (1 mumol 1(-1)) did not abolish this antagonizing action of forskolin. Also, NiCl2 (cumulative concentrations of 0.56 -1.54 mmol 1(-1)) did not change potentiating effect of forskolin (23.4 mumol 1(-1)).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7557258 TI - Comparison of the pharmacological response of human corpus cavernosal tissue with the response of rabbit cavernosal tissue. AB - 1. This study directly compares the response of cavernosal tissue obtained from sexually mature rabbits with the response of human corpus cavernosal tissue obtained during implant surgery for psychogenic impotence (five individual samples) to field stimulation and specific autonomic agonists. 2. At 2 g basal tension, field stimulation of the rabbit corpus cavernosal tissue produced a frequency dependent biphasic response consisting of an initial relaxation followed by contraction. Low frequency stimulation induced primarily relaxations whereas high frequency stimulation induced primarily contractions. FS of human corpus cavernosal tissue induced a frequency dependent contraction. 3. In general, the rabbit corpus cavernosal strips showed a significantly greater degree of spontaneous activity than the strips of human cavernosal tissue. 4. Phenylephrine stimulated a rapid and sustained increase in basal tension in both tissues. Although the isolated strips weighed the same, the magnitude of the response of the rabbit tissue was significantly greater than the response of the human tissue. 5. For both tissues, FS relaxations were completely inhibited by L NAME showing that the relaxations were mediated by nitric oxide. Similarly, for both tissues, nitroprusside, ATP, and bethanechol induced similar dose-response relaxations of pre-stimulated tissue. 6. In conclusion, the major difference between the response of human and rabbit tissue to various forms of stimulation was that isolated strips of human corporal tissue responded to FS with contractions at all frequencies whereas the rabbit tissue responded to the relaxations at low frequencies of stimulation; a clear bi-phasic response at intermediate frequencies; and contraction at high frequencies. PMID- 7557259 TI - Changes induced by pineal indoles in post-implantation mouse embryos. AB - 1. A subcutaneous injection of hydroxyindoleacetic acid (HIAA), melatonin (MEL), methoxytryptophol (MTP) or methoxytryptamine (MTA) (1 mg/25 g body wt/injection) at 8.5 days post-coitum (p.c.) did not produce any effect on the development of embryos in utero at 10.5 days p.c. 2. Two subcutaneous injections (3 mg/25 g body wt/injection) of MTP, but not HIAA, MEL nor MTP, at 7.5 and 8.5 days p.c. brought about a decrease in gravid uterine weight and number of live fetuses and an increased incidence of early resorptions at 17.5 days p.c. 3. MEL at doses 100 and 200 micrograms/ml, MTP at doses 50 and 100 micrograms/ml and MTA at 25, 50 and 100 micrograms/ ml brought about an increase in the number of abnormal embryos cultured in vitro and an increased incidence of abnormal yolk sac circulation, body axis, optic and otic placodes, branchial apparatus, forelimb buds and cranial neural tube. 4. The findings show than melatonin, methoxytryptamine and methoxytryptophol produced an embryotoxic effect on embryos at the early somite stage in vitro while only methoxytryptamine had an adverse effect on embryonic development in vivo. PMID- 7557260 TI - Effects of adenosine drugs on apomorphine-induced licking in rats. AB - 1. In the present work, the effect of adenosine agonists and antagonists on apomorphine-induced licking has been studied. 2. Subcutaneous (s.c.) injection of apomorphine (0.125, 0.25 and 0.5 mg/kg) produced dose-dependent licking in rats. 3. Adenosine agonists 5'-N-ethylcarboxamide-adenosine (NECA) and N6 cyclohexyladenosine (CHA) decreased or increased the apomorphine response respectively. 4. Adenosine antagonists theophylline and 8-phenyltheophylline (8 PT) decreased the response induced by apomorphine. Potentiation of licking induced by CHA was decreased by 8-PT pretreatment. 5. It is concluded that adenosine receptors may be involved in the licking behaviour. PMID- 7557261 TI - Carbachol-induced desensitization of rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors. AB - 1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3). 2. Exposure of RBL-m3 cells to 100 microM carbachol for 30 min in Ca2+-free medium inhibited the secretion induced by the subsequent addition of 10 microM carbachol plus Ca2+. 3. Desensitized cells bound [3H]quinuclidinyl benzilate with a similar Bmax and Kd to those of control cells. 4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization. 5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal alpha-toxin. PMID- 7557262 TI - Protein kinase C involvement in apoptosis. AB - 1. Conflicting observations on the involvement of PKC in apoptosis point to a great variability depending on cell type, agent or condition causing apoptosis, phase of cell cycle and intracellular signaling pathway. 2. Inhibition by PKC of store-operated calcium entry mechanisms, which are sensitive to the oncoprotein bcl-2, should block the activation of calcium-dependent enzymes triggering the apoptotic cell death. 3. Activation of phosphatases by ceramide and inhibition of PKC by sphingosine seem to mediate the sphingomyelin pathway to apoptosis. 4. A putative target protein appears to be p34cdc2 which is regulated by a network of kinases and phosphatases. The uncoupling of timing for p34cdc2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that shares some features with apoptosis. PMID- 7557263 TI - Effects of aluminum on neuronal signal transduction: mechanisms underlying disruption of phosphoinositide hydrolysis. AB - 1. Aluminum is neurotoxic in humans and animals and alters formation of inositol phosphate (IP) second messengers following in vivo or in vitro exposure. 2. Several components of the IP signalling system including G-proteins, phosphatidylinositol-specific phospholipase C (PI-PLC), protein kinase C (PKC) and Ca2+ homeostasis are susceptible to inhibition/disruption by aluminum compounds. 3. Recent evidence suggests that, despite its effects on other components, competitive inhibition by aluminum of phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis by PI-PLC underlies its effects on agonist stimulated IP generation. PMID- 7557264 TI - Influence of etomoxir on the expression of several genes in liver, testis and heart. AB - 1. The effect of ethyl-2-[6-(4-chlorophenoxy)hexyl) oxirane-2-carboxylate (etomoxir) and its oxirane analogues on the expression of several genes from liver and testis as well as the beneficial effect of etomoxir on heart performance and myosin isozyme expression is reviewed. 2. In liver, the effect of etomoxir, alone or in combination with fat or di-(2-ethylhexyl)phthalate (DEHP) on the expression of several genes related to lipid metabolism has been studied. The simultaneous addition of etomoxir and a fat diet produces an increase in the expression of carnitine palmitoyl transferase (CPT) I, cytochrome P-450 4A1 omega hydroxylase and fatty acid binding protein (L-FABP). The mRNA levels of other genes such as CPT II, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and fatty acid synthase (FAS) are increased by etomoxir alone. Neither cytosolic nor mitochondrial HMG-CoA synthase have any significant effect on the mRNA levels induced by etomoxir. A probably frequent mechanism for the action of etomoxir may involve the overload of non-metabolized fatty acids produced after the inhibition of CPT I by the oxirane compounds. There is some speculation as to whether the peroxisome proliferator activated receptor (PPAR) increases its participation in the expression, under the action of etomoxir. 3. In testis, the changes in several genes related to cholesterogenesis, ketogenesis, fatty acid synthesis and transport of fatty acids into mitochondria have also been reviewed. Etomoxir in testis does not appear to produce any effect either alone or in combination with DEHP or a fat diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557265 TI - Neutrophil-mediated platelet activation: a key role for serine proteinases. AB - 1. Neutrophils and platelets interact in vitro through multiple biochemical pathways in both directions, resulting in an inhibition or a potentiation of their reactivity, depending on the experimental conditions. 2. Under some conditions, a full stimulation of platelets (aggregation and degranulation) can be induced by neutrophils. The present review is focused on this aspect for which serine proteinases released from the azurophilic granules of neutrophils activate surrounding platelets. 3. The different facets of this process at the cellular and molecular levels, are presently depicted and their relevance to the in vivo situation suggested. PMID- 7557266 TI - The mammalian tachykinin receptors. AB - The tachykinins (TKs) are a family of small peptides which share the common C terminal sequence Phe-X-Gly-Leu-MetNH2. Three peptides of this family, substance P, neurokinin A and neurokinin B, have an established role as neurotransmitters in mammals. 2. Three receptors for TKs have been cloned: they are G-protein coupled receptors with seven putative transmembrane spanning segments and have been termed NK1 (substance P-preferring), NK2 (neurokinin A-preferring) and NK3 (neurokinin B-preferring). 3. Synthetic agonists are available to selectively stimulate only one receptor, while natural TKs can act as full agonist at each one of the three receptors, albeit at different concentrations. 4. A number of potent and selective antagonists, both peptide and nonpeptide in nature, have recently been developed. 5. The introduction of these ligands has revealed an unforeseen pharmacological heterogeneity of NK1, NK2 and NK3 receptors which appears largely, if not exclusively, linked to the existence of species homologues of the three receptors. PMID- 7557267 TI - Actions of manganese ions in contraction of smooth muscle. AB - 1. Manganese ions (Mn2+) have been used in the last few decades as one of a number of inorganic Ca2+ channel blockers to investigate Ca2+ channels in excitable smooth muscle cells. 2. It has been recently reported that in addition to its inhibitory effects on the Ca2+ channels, Mn2+ in millimolar concentrations also produces contraction in the state of cell membrane depolarization. 3. Mn2+ has been shown to be able to permeate the cells via voltage-operated L-type Ca2+ channels in the membranes and directly activates contractile proteins in smooth muscles. 4. Intracellular sites of action have been proposed for Mn2+ in smooth muscles. PMID- 7557268 TI - Adrenocorticotropin activates glycerol-3-phosphate acyltransferase in bovine adrenal glomerulosa cells. AB - 1. The mechanism whereby ACTH activates the synthesis of diacylglycerol (DAG) in isolated adrenal glomerulosa cells was investigated. 2. ACTH activates glycerol-3 phosphate acyltransferase (G3PAT) in intact and cell-free preparations of adrenal glomerulosa cells. Whereas activation of G3PAT by ACTH was observed in homogenates and membrane fractions, no activation was observed by angiotensin II (AII) at the same concentration. 3. ACTH effects were mimicked by nonspecific phospholipase C (PLC). 4. Our preliminary results suggest that ACTH activation of G3PAT may account for ACTH-induced increases in DAG via de novo synthesis of phosphatidic acid (PA). PMID- 7557269 TI - Effects of vasoactive intestinal peptide and its homologues on the acetylcholine mediated secretion of fluid and protein from the rat submandibular gland. AB - 1. Acetylcholine-mediated (ACh-mediated) secretion of fluid and protein from rat submandibular glands was enhanced by intravenous injection of vasoactive intestinal peptide (VIP) and of secretin but not of peptide histidine isoleucine (PHI) or gastric inhibitory peptide (GIP). 2. When VIP and ACh were administered together, the enhancement of fluid secretion was inhibited by pretreatment with atropine or 4-DAMP and the enhancement of protein secretion was inhibited by pretreatment with atropine or phentolamine. 3. The enhancement of the ACh-induced secretion of fluid by secretin was strongly inhibited by pretreatment with atropine, and it was weakly inhibited by pretreatment with phentolamine or haloperidol. 4. These results suggest that the synergistic effects of VIP, PHI, secretin and GIP on the ACh-mediated secretion of fluid and protein from the rat submandibular gland do not reflect the extent of the structural homology of each peptide to VIP. PMID- 7557270 TI - Quinidine enhances intracellular Ca2+ accumulation during rapid stimulation. AB - 1. The quinidine-induced modification of intracellular Ca2+ concentration ([Ca2+]i) was studied in guinea-pig myocardium using fura-2. Quinidine reduced the systolic fluorescence signal level for [Ca2+]i and enhanced the end-diastolic signal level during a stimulation train. 2. The diastolic decay of [Ca2+]i fitted 2 exponential curves. Quinidine distorted the stimulation frequency-dependent acceleration of rapid [Ca2+]i decay, and prolonged the mean time constant of rapid decay after 2 Hz stimulation, from 154.4 to 205.3 msec (20 microM), and to 259.7 msec (60 microM quinidine). The time constant of slow recovery from [Ca2+]i accumulation after the stimulation train was not affected by stimulation frequency, or by quinidine, or caffeine. 3. These results suggest that quinidine modulates [Ca2+]i via a balance between the slowing of rapid [Ca2+]i decay and the reduction of the systolic [Ca2+]i. This effect may contribute to the anti arrhythmic and pro-arrhythmic effects exerted by quinidine in some conditions. PMID- 7557271 TI - Inhibitory effects of d-nicotine on the responses evoked by 1-isomer in trachea and bronchus isolated from guinea-pig and rabbit. AB - 1. The effects of d-nicotine on the responses induced by 1-isomer were studied in tracheae and bronchi isolated from guinea-pigs and rabbits. In guinea-pigs trachea 1-nicotine produced a biphasic response consisting of initial contraction and following relaxation. In other airway preparations 1-nicotine produced only contraction. 2. d-Nicotine did not produce any responses except for the case of guinea-pig trachea. d-isomer produced only relaxation and relative potency was approximately 0.44 in guinea-pig trachea. 3. Pretreatment with d-nicotine (30-300 microM) reduced concentration response curves for 1-isomer in a non-competitive manner in all preparations used in this study. 4. 1-nicotine at the concentration of 3 microns, which did not produce any response itself, reduced the concentration response curve of 1-nicotine in guinea-pig trachea. 5. Inhibition by d-nicotine or 1-nicotine (3 microM) of the concentration-response curve of 1 nicotine may be due to desensitization of nicotine receptors. PMID- 7557272 TI - Protection by selective amino acid solutions against doxorubicin induced growth inhibition of Escherichia coli. AB - 1. Incubation of Escherichia coli with 0.7 mM doxorubicin in MBS-glucose medium resulted in complete growth inhibition, an inhibition that was blocked by placing specific amino acids (AA) in the medium. 2. The mechanism of protection by AA was similar to that reported previously for cells poisoned by hyperoxia and by paraquat, e.g. of 20 common AA, ten percent, ten do not and the branched-chair AA are among those required for inhibition. 3. Unlike hyperoxia and paraquot stringency which caused elevation of intracellular concentrations of guanosine tetraphosphate (ppGpp), doxorubicin inhibition did not elevate ppGpp. 4. Concentrations of ppGpp were increased by isoleucine starvation as expected, and the subsequent addition of doxorubicin did not abolish that increase; however, pretreatment with doxorubicin prevented the induction of stringency by isoleucine starvation. 5. This suggests that doxorubicin directly inhibits ppGpp synthesis or protein biosynthesis to leave tRNA loaded as is the case with chloramphenicol. PMID- 7557273 TI - Effect of Ca2+ agonist Bay K 8644 in human placental arteries. AB - 1. Bay K 8644 (0.1 microM induced weak contractions in human placental artery segments that were increased in the presence of 7.5 mM K+. K+ and serotonin (5 HT) induced contractions that were enhanced by preincubation of segments with Bay K 8644. These enhancements were reduced by nifedipine (0.1 microM) and diltiazem (1 microM). 2. Bay K 8644 induced a 45Ca2+ uptake increase which was potentiated by depolarization with K+ (less than 30 mM) and antagonized by nifedipine. K+ (15 and 30 mM) and 5-HT (1 microM) induced 45Ca2+ uptake that was enhanced by Bay K 8644. 3. These results suggest that Bay K 8644: (1) is unable to activate the quiescent potential-operated Ca2+ channels (POCs) of these arteries, and (2) activates receptor (5-HT)-operated Ca2+ channels or facilitates Ca2+ influx through POCs activated by 5-HT. PMID- 7557274 TI - Neuronal regulation of ornithine decarboxylase induced by androgens in the mouse kidney. AB - 1. Neuronal, but not circulating catecholamines, regulate the induction of ornithine decarboxylase (ODC) by testosterone in the mouse kidney. 2. Central and peripheral catecholamine-depleting agents, such as reserpine or alpha-methyl-p tyrosine, exerted a more pronounced effect on renal ODC than the selective agents tetrabenazine or guanethidine. 3. Benserazide and haloperidol decreased the induction of renal ODC produced by testosterone in female mice. 4. Renal denervation produced a partial inhibition of renal ODC in male mice and decreased the induction of ODC elicited by testosterone in female mice. 5. These results suggest that both peripheral sympathetic neurons as well as central related factors can modulate the effect of androgens on renal ODC activity. PMID- 7557276 TI - [Long term study of the treatment with recombinant alfa 2b interferon in chronic active hepatitis due to B virus in children and adolescents]. AB - To assess the efficacy of recombinant alfa 2b-interferon treatment "Heberon alfa R" in children with chronic active hepatitis (CAH) B virus, we conducted a long term study (three years) in 22 children infected with hepatitis B virus (17 males and 5 females), age range 3 to 15 years. Diagnostic criteria included the clinical picture, laboratory tests, virus markers (HBeAg, HBsAg), laparoscopy and liver biopsy. Children under 12 years received 3 million IU of interferon per day whereas those older than 12 years received 6 million IU of interferon per day by intramuscular injection, three times per week for four months. Alanine aminotransferase (ALT) levels had been elevated for six months in all patients and hepatitis B viral infection was replicative. A variance analysis was made to evaluate ALT response to interferon administration and the Mc Nemar test was used to analyze HBeAg/anti-HBe behavior. Seventeen (77%) out of 22 patients responded to treatment (clearance of HBeAg and ALT levels returned to normal. HBeAg seroconversion (anti-HBe) occurred in 36% of patients during the first year (p < 0.01) and it increased to 50% by the third year follow-up. ALAT levels also decreased and the difference was statistically significant (p < 0.01). This occurred during and after treatment with a steady and increasing tendency to return to normal levels within the first and third year. Side effects were scarce, transient and tolerable and they only appeared during the initial phase of treatment; symptoms were mainly influenza-like and they disappeared very soon. There were no late side effects such as medullar depression, renal toxicity and glycemia alterations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557275 TI - [Gastrointestinal fistulas. Treatment with a somatostatin analogue (SMS 201 995)]. AB - Post surgical gastrointestinal fistulas are a frequent problem around the world. We present our experience with a synthetic analog of somatostatin (SMS 201-955) used in 19 patients with gastrointestinal fistulas between January 1992 [corrected] and November 1993. We compare them with 50 patients with gastrointestinal fistulas randomized selected between 1980. The fistulas were classified according to their topographic location in five groups and to their flow in two groups: high and low fistulas. All patients were treated with nutritional support, antibiotic and octreotide (SMS 201-995). In the first 24 hours the fistulas flow where reduced in 51.63% and 72 hours later the reduction was of 70.62%. In 13 patients the fistulas where closed between 36.7 + 0 - 13.94 days (p < 0.01). This difference represent a cost reduction of 1,220,673.96 Bs. for each patient or 45,581.55 Bs. for each day. Two patients died in the study (10.52%) and two patients stop the drug because of adverse reaction. The octreotide reduced the fistulas flow and reduced the time needed to close. PMID- 7557277 TI - [Morphology of Blastocystis hominis in feces and evaluation of parasitological methods]. AB - A study of the morphology of Blastocystis hominis in stool in ninety-four cases humans is described as central body, ameba and granular form were found as previously described. In addition a "Globulose" form as a variation of granular form is first described. The central body form (96.8%) was the most abundant form. Three parasitological methods as direct microscopical examination sample with saline solution 0.85%, lugol, Sudan III, stained with Quensel, Iron hematoxylin and culture are evaluated to detection the Blastocystis hominis forms. Our results show that the direct microscopical examination (saline solution 0.85%, lugol, Sudan III, stained with Quensel) is the most sensitive and specific method than culture. The identification of Blastocystis hominis in stool difficult due to the diversity of shapes and size, which generate confusion with other intestinal protozoa and host cells. PMID- 7557278 TI - [Macroscopic and histopathological aspects of atypical gastric epithelium]. AB - Atypical Epithelium is a localized lesion, which has histological and macroscopic findings similar to gastric cancer. In this paper we evaluated 44 cases of atypical epithelium studied and treated in our institute. We found 3 cases of atypical epithelium associated with early gastric cancer. That is 2.66% of the total of all cases. We established the relationship of these lesions and following variables: age, size, localization, macroscopic shape and intestinal metaplasia. We found atypical epithelium in patients older than 50 years old, most of then smaller than 2 cm., localized in antrum and body gastric, and macroscopically were slight elevated lesions. Histologically all of them had intestinal metaplasia. PMID- 7557280 TI - [Complications of laparoscopic cholecystectomy]. AB - The laparoscopy cholecistectomy is a surgical procedure described in 1987, and it has had an important apogee and it's had substitute to open procedure. Between its advantages is notable, the postoperatory evolution without pain, paralytic ileus and the short time of hospitalization besides the patient can go away the same day in some cases. In Venezuela, the procedure has been received with enthusiasm by the surgeons with prefer this procedure however, there are complications derived in part for the lack of experience in the first cases, but occur non related of the experience. The quantization of the complications is not simple to realize for different reasons. We present twelve complications, two belong to patients intervened in the Hospital Universitario de Caracas and ten belong to patient transferred from other center. The complications were: pseudoaneurism of hepatic artery, hematoma of the wall, cutting of common bile duct, section the right bile duct, abscess of vesicular bed in two cases, retropneumoperitoneum, ascites by biliary fistula and four biloma. The treatment varied in every case and the utilization of radiologic technique with percutaneous drainage were useful in five cases. We conclude: first, this technique is not free of complications. Second, the percutaneous drainage (abscess, biloma), endoscopic procedure (biliary prosthesis) in fistulaes and arteriographics (arterial embolization), are useful in some cases, and could prevent the surgical reintervention. PMID- 7557279 TI - [Intestinal microflora in children with acute and persistent diarrhea]. AB - The etiology and pathogenesis of persistent diarrhoea is usually multifactorial and sometimes can not be identified. It is necessary to define if an alteration of the enteric microflora is a risk factor that influence the duration of the diarrhoea. 30 infants with acute diarrhoea and 30 with persistent diarrhoea were studied. A sample of duodenal content was taken by a doble-lumen tube and processed microbiologically in search for enteric microorganisms, anaerobic and Candida. These result were correlated with nutritional status, the previous use of Metroonidazol and the results of the stool culture. The presence of bacterial overgrowth and the identification of the duodenal microflora is an important factor for the persistence of the diarrhoea. There was a quantitative and qualitative alteration of the duodenal flora in both groups of patients. PMID- 7557281 TI - [Hepatic tolerance of aceclofenac]. AB - NSAID's are largely used drugs. Among the reported side effects of this type of drugs is liver damage. 73 patients suffering from diverse rheumatological pathologies (arthrosis 46.6%, rotators 9.68%, rheumatoid arthritis 21.92%, lumbalgia 5.48%, other diagnosis 6.85%) were evaluated. Aceclofenac (AC) 100 mg. dose every 12 hours was administered. A Transaminase value determination was taken both at the beginning and end of medication for each patient. Average age of patients was 52.71 years, 78% being female. Treatment was administered as follows, 42.47% of the patients received a 60 days treatment, 28.8% a 45 days treatment, 23.3% a 30 days treatment and 5.47% received a 15 days treatment. Reported mean GOT value at the beginning was 16.6 U, at the end 17.01 U. GPT initial value was 15.84 U and final 16.53 U. A GOT increase, 20 U to 50 was observed in a single patient. GPT was also increased from 32 U to 47 U in one case. Results led us to a no liver damage report of the NSAID's AC in these group of patients. PMID- 7557282 TI - [Autoimmune diseases of the liver. Autoimmune hepatitis]. PMID- 7557284 TI - [Chagasic esophageal disease]. PMID- 7557283 TI - [Etiology of colorectal cancer]. AB - Colorectal cancer affect the 15% of general population in developed countries. Cancer is a multistep process in which multiple genetic alterations must usually occur in several years. The premalignant step consists of one or multiple aberrant crypts due to hyperproliferation of cells and its shift from the deep third of the crypt to its surface. It has been suggested that abnormality in the APC gene is responsible for this. Furthermore, there exists DNA hypometilation, activation of the gene K-ras and ornithine decarboxylase activity. There is also a loss of MCC gene, that seems to interact with the APC gene. Entire alterations described make possible the Class I adenoma formation. This adenoma, needs the loss of the DCC gene (late stage in the carcinogenesis process), to become a Class II adenoma. The following alteration is deleted and mutation of the p53 gene. There is also an activation of the c-myc oncogene. These two genes are important mechanisms for the conversion of a benign adenoma to a malignant one, adenoma with in situ carcinoma or Class III adenoma. This type of adenoma becomes carcinoma and metastatic stage, throughout inactivation of several tumor suppressor genes. Besides the hereditary APC alteration and other acquired genetic changes as described above there are other associated genetics, antigenics, and enzymes that have an important role in the adenoma-carcinoma sequence. Several carcinogenic factors have been described which also contribute in the adenoma and carcinoma formation: ulcerative colitis, acromegaly, familial history of colonic neoplasia, certain professions, smoking and drinking, consumption of red or processed meat, etc.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557285 TI - [Endoscopic retrograde cholangiopancreatography in hepatic fascioliasis]. AB - We present a 60 years old female with a history of upper right quadrant pain, fever and eosinophily. We made the diagnosis of fasciolasis on clinical, parasitology studies and radiological findings. In the endoscopic retrograde cholangiopancreatography (ERCP) we found the bile duct of normal caliber, but with many filling defects. Fasciola eggs were found in the bile. This is the first case with endoscopic retrograde cholanglopancreatography and the fourth case of fasciolasis in Venezuela. PMID- 7557286 TI - [Adenosquamous carcinoma of the colon]. PMID- 7557287 TI - [Early gastric cancer staging by radiology]. PMID- 7557289 TI - Buffering capacity and H+ membrane conductance of gram-negative bacteria. AB - Buffering capacity and membrane H+ conductance were examined in seven Gram negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceae studied here showed a decrease in both parameters as the external pH increased, over the pH range studied. The other four species presented an increase in buffering capacity and membrane conductance to protons as the external pH increased from 5.5 to 7.0. PMID- 7557288 TI - Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis. AB - The molecular effect of lipopolysaccharides (LPS) from porphyromonas gingivalis as well as Escherichia coli on the tyrosine protein phosphorylation in the splenic B lymphocytes from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ mice was examined. P. gingivalis LPS induced tyrosine phosphorylation of selected membrane proteins that included the phosphoproteins with apparent molecular masses of 24.8 kDa and 26.0 kDa (p24.8 and p26.) in the B lymphocytes from both strains of mice, while E. coli LPS induced p24.8 and p26.0 in C3H/HeN B Lymphocytes only. These findings suggest that through the same tyrosine phosphorylation pathway as observed in C3H/HeN B lymphocytes, P. gingivalis LPS induced the activation of C3H/HeJ B lymphocytes in which a trigger signal by E. coli LPS could not be transduced to initiate tyrosine protein phosphorylation. PMID- 7557290 TI - Dr fimbriae coding region associated hemolytic activity of Escherichia coli. AB - We investigated the hemolytic activity of Escherichia coli strain EC901 carrying plasmid pBJN406 containing genes draA-E involved in expression of the mannose resistant Dr hemagglutinin, and in its isogenic insertion mutants devised with Tn5, Tn3, and TnphoA. While E. coli BN406 displayed rapid hemolytic activity against equine erythrocytes, insertion mutations in draD and draE, but not in draA, draB, and draC, abolished all hemolytic activity. These data suggest a role for draD and draE in the expression of hemolysis. PMID- 7557291 TI - Cloning and sequence analysis of the aminopeptidase My gene from Mycoplasma salivarium. AB - The complete nucleotide sequence of a major component of aminopeptidase My purified from Mycoplasma salivarium was determined. The protein gene encoded a protein consisting of 520 amino acids with a molecular mass of 58079 Da. The protein contained two tryptophan residues, one of which was encoded by UGA. A computer-aided homology search suggested that aminopeptidase My had properties similar to those of leucine aminopeptidase (EC 3.4.11.1). PMID- 7557292 TI - Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals. AB - Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins. The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains. Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants. This communication reports that the 159-261 amino acids of external flagellum (FliCi-1), 285-331 amino acids of hook protein (FlgE-2), and 309-391 amino acids and 440-537 amino acids of hook associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study. PMID- 7557294 TI - Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis. AB - The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive. PMID- 7557293 TI - Drastic changes in the peptidoglycan composition of penicillin resistant laboratory mutants of Streptococcus pneumoniae. AB - The penicillin MIC of 2 Streptococcus pneumoniae clinical isolates was increased 100-fold (from 0.02 to 2.0 micromilligrams) and 20-fold (from 0.5 to 10.0 micromilligrams) through gradual exposure of the bacteria to increasing concentrations of penicillin in the laboratory. In both mutants the affinity of all four high molecular mass penicillin binding proteins (PBPs) for penicillin was drastically reduced accompanied by major changes in the composition of peptidoglycan as resolved by HPLC. The ratio of crosslinked to monomeric peptides became virtually inverted in the resistant cell walls with monomers representing two-thirds of the muropeptide species. The proportion of the crosslinked tri tetra dimer, a major component of the cell wall of the original isolates, decreased to one-third or one-sixth of its normal representation, while the amounts of tripeptide monomers with an alanyl-serine substitution on the lysine epsilon amino group increased by close to a factor of two. The growth rates of both mutants decreased by a factor of approximately two, as compared to the original bacteria. PMID- 7557295 TI - An unstable small-colony variant of a noninvasive mutant of Salmonella typhimurium is highly invasive for MDCK cells. PMID- 7557296 TI - Virulence factors (aerobactin and mucoid phenotype) in Klebsiella pneumoniae and Escherichia coli blood culture isolates. AB - We examined the presence of two virulence factors in 241 blood isolates of Klebsiella pneumoniae from patients hospitalized during 1989 and 1990 in 7 French hospitals, and 125 blood isolates of Escherichia coli from one hospital. Aerobactin was scored phenotypically and genotypically with an intragenic DNA probe of 2 kb. The mucoid phenotype was assessed by culture on trypticase soy agar and by genotypic analysis (intragenic DNA probe of 235 bp). Only 6% K. pneumoniae isolates were aerobactin-positive with no significant variation according to geographical location while 20% of K. pneumoniae isolates displayed the mucoid phenotype, with a significant variation according to hospital. Aerobactin was always associated with the mucoid phenotype. The frequency of aerobactin production but not mucoid phenotype (14%) was higher among E. coli isolates (48%). They harbored two types of large plasmids. Intraperitoneal injection into mice of 10(3) cfu of K. pneumoniae producing both virulence factors demonstrated that capsular serotype K2 was the more virulent K23 and K28. PMID- 7557297 TI - Effects of mutations and deletions on expression of the Enterobacter cloacae ompX gene. AB - The Enterobacter cloacae outer membrane protein OmpX is involved in resistance to beta-lactams, and possesses virulence characteristics. To gain more insight into the genetic elements that are important for OmpX expression, several mutations were introduced at, and immediately upstream of, the N-terminus of the OmpX coding sequence. These mutations enabled us to delete the 5' untranslated region and the signal peptide coding sequence. The former led to decreased ompX expression, indicating an unexpected and hitherto unexplained role for this region. Deletion of the signal peptide coding sequence blocked transport across the cytoplasmic membrane, indicating that translocation of OmpX across the cytoplasmic membrane is mediated by the general secretory pathway. PMID- 7557299 TI - Most probable number enumeration of H2-utilizing acetogenic bacteria from the digestive tract of animals and man. AB - A method is proposed that allows the enrichment and most probable number estimation of H2/CO2(-)utilizing acetogenic bacteria. It is based on the difference in acetate production for serial dilutions incubated under either a test H2/CO2 (4:1), or a control N2/CO2 (4:1) headspace atmosphere. A nutritionally non-selective medium was used, containing bromoethane-sulfonic acid as inhibitor of methanogenic archaea and 10% pre-incubated clarified rumen fluid. Acetogenic bacteria were enumerated in rumen and hindgut contents of animals and in human feces. They ranged from below 10(2) to above 10(8) per gram wet weight gut content and their population levels were the highest in the absence of methanogenesis. The method described therein should prove useful to better understand the diversity and ecological importance of dominant gut acetogens. PMID- 7557298 TI - Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus. AB - Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene. PMID- 7557302 TI - Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus. AB - To study the correlation between emetic toxin and HEp-2 vacuole activity produced by Bacillus cereus isolated from an outbreak of vomiting-type food poisoning, some properties and emetic activities of both purified HEp-2 factor (cereulide) and partially purified factor to rhesus monkeys were determined. The results indicate that both cereulide and partially purified factor were very stable to digestion with proteolytic enzymes, different pH, and heating. Vomiting was induced in the rhesus monkeys orally administered with both substances. From these findings, cereulide (or HEp-2 vacuole factor) is strongly suggested to be an emetic toxin itself. PMID- 7557301 TI - Cloning, nucleotide sequence determination and expression of the Staphylococcus aureus hyaluronate lyase gene. AB - The Staphylococcus aureus 8325-4 hyaluronate lyase gene (hysA) was identified after detecting hyaluronate lyase activity expressed by phages from a genomic library. The hysA open reading frame, capable of encoding a protein of 91 980 Da, was identified by Tn5 mutagenesis and nucleotide sequencing. HysA shares 35 and 36% amino acid sequence identity with group B streptococcal hyaluronate lyase and pneumococcal hyaluronidase, respectively. A 94-kDa protein was expressed in Escherichia coli minicells, a result consistent with the coding capacity of hysA. Identification of the S. aureus 8325-4 hyaluronate lyase gene will allow the regulation of this putative virulence determinant to be studied. PMID- 7557303 TI - Linear DNA plasmids of yeasts. AB - Linear DNA plasmids are common among filamentous fungi and plants in which they are generally associated with mitochondria. In contrast, the linear DNA plasmids of yeast appear to be cytoplasmic and to have a specific genetic organization. In these respects the yeast linear plasmids can be regarded as a separate group of plasmids. A survey of plasmids among about 1800 strains covering practically all the currently accepted species of yeast (about 600) has provided a preliminary, overall view of the nature and frequency of yeast plasmids. Linear DNA plasmids were found at a frequency of 1-2%, with more than 20 cases of new linear plasmids discovered in a large variety of species. The same survey showed that circular DNA plasmids are characteristically confined to a few genera and species. The linear DNAs have many features in common with the linear killer DNAs of Kluyveromyces lactis and form a large family of multicopy, small DNAs disseminated among yeast species. PMID- 7557300 TI - Phylogenetic analysis of polycyclic aromatic hydrocarbon degrading mycobacteria by 16S rRNA sequencing. AB - Mycobacterium sp. PYR-1 was previously isolated in our laboratory and was shown to be able to mineralize high molecular mass polycyclic aromatic hydrocarbons (PAHs) [Heitkamp and Cerniglia, (1988) Appl. Environ. Microbiol. 54, 1612-1614]. In this research, the 16S rRNA gene (rDNA) of this strain was amplified by polymerase chain reaction (PCR) and directly sequenced by cycle sequencing method. We compared this sequence with all known mycobacterial 16S rDNA sequences available from GenBank and found that Mycobacterium sp. PYR-1 16S rDNA differs from the other mycobacteria, especially in the region of nucleotides 168-200 (in the Escherichia coli numbering system). Using the 16S rDNA sequences of the mycobacteria, a phylogenetic tree was constructed. The data from the phylogenetic tree and similarity values suggest that Mycobacterium sp. PYR-1 is closer to M. aurum and M. vaccae. Using the same approach, we also determined the 16S rDNA from an another PAH-degrading Mycobacterium sp. PAH135, isolated by Grosser and colleagues (1991) (Appl. Environ. Microbiol. 57, 3462-3469). Mycobacterium sp. PAH135 was found to be closer to M. aichiense, and different from our Mycobacterium sp. PYR-1. PMID- 7557304 TI - A membrane-like structure envelopes substrate mycelium during colony development in Streptomyces. AB - Colonies of Streptomyces antibioticus were studied by transmission and scanning electron microscopy. The micrographs show that substrate mycelium growth takes place among an intercellular material and this mycelium is covered by a surface film. This structure could be a boundary between the aerial mycelium and the substrate mycelium. PMID- 7557305 TI - Strain typing of Lentinula edodes by random amplified polymorphic DNA assay. AB - Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shittake mushroom, Lentinula edodes. Seven primers produced polymorphisms in all 15 strains tested, producing 12-19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applicatiosn in mushroom breeding and strain improvement programs. PMID- 7557306 TI - Non-physiological expression of UhpT does not lead to uncontrolled leakage of sugar phosphates out of Escherichia coli cells. AB - De-regulated expression of uhpT under control of the tac promoter, by increasing concentrations of isopropyl-thio-beta-D-galactoside, progressively inhibited the growth rate of Escherichia coli cells, to such an extent that growth was fully inhibited at 1 mM of the inducer. Significantly, additio of glucose 6-phosphate to the growth medium of the cells did not protect against this inhibition. Furthermore, efflux of sugar phosphates from the cells did not take place under these conditions, unless protonophoric uncouplers were added. We therefore conclude that the regulation of uhpT expression, i.e. via extracellular induction through a two-component system, did not evolve in order to prevent loss of essential metabolites from the cytoplasm under conditions when extracellular sugar phosphates are not available. PMID- 7557307 TI - Giant linear plasmids of beta-lactam antibiotic producing Streptomyces. AB - A survey of the total cellular DNA from five beta-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and beta-lactam antibiotic synthesis gene clusters were located on the chromosome. PMID- 7557308 TI - Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers. AB - An internal fragment representing approximately 85% of sod genes from seven Gram positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens. PMID- 7557309 TI - Production of trehalose phosphorylase by Catellatospora ferruginea. AB - A range of microorganisms was screened for new and high producer strains of trehalose phosphorylase (EC 2.4.1.64). Trehalose phosphorylase activity was found in cells of actinomyctes of the genera Actinomadura, Amycolata, Catellatospora, Kineosporia, and Nocardia. Among them, Catellatospora ferruginea showed the highest enzyme activity. Trehalose phosphorylase from C. ferruginea was able to catalyse both the phosphorolysis of trehalose into beta-glucose 1-phosphate and D glucose and the synthesis of trehalose from beta-glucose 1-phosphate and D glucose. PMID- 7557310 TI - Expression of DnaK and GroEL homologs in Leuconostoc esenteroides in response to heat shock, cold shock or chemical stress. AB - The mechanism of adaptation of bacteria to survive at elevated temperature in the human host and the expression of heat-shock proteins in response to stress was examined by labelling with [35S]methionine. An increase in culture temperature from 26 degrees C to 37 degrees C induced expression of certain bacterial proteins (70 and 60 kDa). Heat shock at 40 degrees C, cold shock (10 degrees C), ethanol treatment or arsenite treatment also led to an increased expression of heat shock proteins of 70 and 60 kDa. Actinomycin D completely blocked the induction, indicating that transcription is required for the overexpression of stress proteins in Leuconostoc mesenteroides. N-terminal sequence analysis showed that these proteins were homologous to the highly conserved chaperone proteins DnaK and GroEL of Escherichia coli, respectively. PMID- 7557311 TI - Saprophytic Pseudomonas syringae strain M1 of wheat produces cyclic lipodepsipeptides. AB - A saprophytic fluorescent bacterium (strain M1) isolated from wheat was identified as Pseudomonas syringae and shown to produce the cyclic lipodepsipeptides, syringomycin E and syringopeptin SP25A. M1 grew in planta but did not affect germination or cause disease symptoms in wheat. The findings show that the production of these metabolites, generally regarded as plant virulence factors, does not correlate with plant pathogenicity. PMID- 7557312 TI - Characterization of phage phi O139, a Vibrio cholerae O139 temperate bacteriophage with cohesive DNA termini. AB - A temperate bacteriophage isolated from Vibrio cholerae O139, the new epidemic strain of cholera, was found to have a polyhedral head 65 nm in diameter and a rigid contractile tail 120 nm in length. The phage chromosome was a double stranded DNA of 35 kb, with unique cohesive ends and had a G + C content of 58.8%. A restriction map of the phage DNA was constructed using the restriction endonucleases AvaI and BstEII. The phage, whose presence could be detected in nine out of 13 V. cholerae O139 isolates tested, was found to have identical chromosomal integration sites in all the strains. The phage attachment site (attP) was found to be located very close to one end of the genome. PMID- 7557314 TI - Use of an enhanced Escherichia coli opal suppressor strain to screen a Mycoplasma hyopneumoniae library. AB - Many mycoplasma genes contain internal UGA (opal) codons because of their use as tryptophan coding codons. This results in a lack of expression of many cloned mycoplasma antigenic epitopes in Escherichia coli. It has been shown that opal suppressors can be used to enhance expression of defined mycoplasma gene sequences, but no studies have been published using E. coli suppressor strains to screen mycoplasma gene libraries for immunoreactive epitopes. The E. coli suppressor strain ISM612 was used to screen a Mycoplasma hyopneumoniae Lambda gene library. This strain contained an inducible opal suppressor, trpT, as well as the release factor 2 mutation prfB3. Strain ISM612 was shown to enhance antibody recognition of cloned mycoplasmal gene sequences. PMID- 7557313 TI - The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis. AB - Lactococcus lactis cells secreting the lantibiotic nisin, commercially used for food preservation, must protect their cell membrane against the pore-forming activity of extracellular nisin. The nisI gene product has been suggested to be a lipoprotein, which due to the location on the extracellular surface would be an ideal candidate for an immunity protein. In vivo labelling of NisI from L. lactis N8 expressed in Escherichia coli proved that NisI is a lipoprotein. Expression of nisI in the nisin-sensitive L. lactis MG1614 strain resulted in immunologically active protein on the cytoplasmic membrane in comparable amounts to the immune strain L. lactis N8, but only to slightly increased nisin immunity, suggesting that additional proteins are needed for full immunity. PMID- 7557315 TI - Utilization of the leucocin A export system in Leuconostoc gelidum for production of a Lactobacillus bacteriocin. AB - The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 (ATCC 11506) and is active against various lactobacilli and Enterococcus faecalis. The genetic determinants encoding the lactacin F peptides, LafA and LafX, are organized in a chromosomal operon comprised of genes lafA, lafX, and ORFZ. The lactacin F operon was introduced into Leuconostoc (Lc.) gelidum UAL187-22 which produces leucocin A. Leucocin A, a plasmid-encoded bacteriocin, inhibits E. faecalis, Listeria monocytogenes, and other lactic acid bacteria. The culture supernatant of the Leuconostoc transformant containing the lactacin F operon inhibited both lactacin F-and leucocin A-sensitive indicators. Concurrent expression of both bacteriocins did not alter the production of native leucocin A. Additive inhibitory effects due to the presence of both bacteriocins were not observed. An isogenic derivative of UAL187-22, which has lost the leucocin-encoding plasmid, was unable to produce active lactacin F when transformed with the appropriate recombinant plasmid. The ability of Lc. gelidum UAL187-22 to produce lactacin F demonstrates that the export system for leucocin A is capable of producing both bacteriocins simultaneously. PMID- 7557316 TI - Control of ilvIH transcription during amino acid downshift in stringent and relaxed strains of Escherichia coli. AB - Transcription of the ilvIH operon was reduced during amino acid starvation of wild-type Escherichia coli. The effect was abolished by a relA mutation and was enhanced by a spoT mutation, thus suggesting a possible negative role of ppGpp accumulation on ilvIH transcription. No effect of amino acid downshift was observed on the synthesis of lrp mRNA, encoding the positive regulator (Lrp) of ilvIH transcription. PMID- 7557317 TI - The construction of a reporter system and use for the investigation of Clostridium perfringens gene expression. AB - A reporter system was constructed to enable the study of gene expression in Clostridium perfingens. The system was based on plasmid shuttle vector pJIR410, which contained the C. perfringens erythromycin resistance gene. The vector was modified by the introduction of a DNA fragment comprising the open reading frame of the C. perfringens chloramphenicol acetyltransferase gene and flanking transcriptional terminators. The presence of a unique restriction site, engineered into the extreme 5' end of the open reading frame enabled a promoter region to be inserted to form an in-fram transcriptional fusion with catP. The system was tested by inserting the promoter region of the alpha-toxin gene of C. perfringens. The production of chloramphenicol acetyltransferase in C. perfringens was monitored during growth and the pattern of expression was shown to reflect levels of plc mRNA and alpha-toxin in the parent strain. PMID- 7557318 TI - Regulation of hydroperoxidase (catalase) expression in Escherichia coli. AB - As part of its adaptive response to oxidative stress, Escherichia coli produces two inducible hydroperoxidases called HPI and HPII. Upon exposure to sublethal levels of hydrogen peroxide, HPI expression is induced at the transcriptional level by OxyR, a member of the LysR family of autoregulators. OxyR, functioning as both a sensor and transducer, contains a critical redox-sensitive Cys residue that is oxidized by hydrogen peroxide. This is thought to induce a conformational change in the tertiary structure of the OxyR tetramer altering its DNA-binding specificity and resulting in an increase in the transcription of katG and several other OxyR-dependent genes. In contrast, synthesis of the HPII enzyme is not induced by hydrogen peroxide. Expression of both HPI and HPII is growth phase dependent levels of HPI and HPII are 10-fold higher in stationary phase than exponential phase cultures. These growth phase-dependent increases are largely dependent on RpoS, a stationary phase specific sigma factor that is itself subject to complex transcriptional and post-transcriptional controls. Several metabolic signals have been proposed to activate the RpoS regulon including hyperosmolarity, weak acids, homoserine lactone and UDP-glucose. Since both HPI and HPII are members of the RpoS regulon, elucidation of the mechanism of regulation of RpoS should contribute to our general understanding of hydroperoxidase regulation. PMID- 7557320 TI - The ligase chain reaction distinguishes hepatitis B virus S-gene variants. AB - A novel method for the identification of hepatitis B virus (HBV) variants was developed. The ligase chain reaction (LCR) distinguished the sequences of two isolates from the Gambia showing a change at nucleotide 421 within the region coding for the surface protein a antigenic determinant. One sequence was derived from a child previously immunized with HBV vaccine, while the other, reported here for the first time, was from a chronic carrier. Nucleotide variations within the target sequence at, or up to 3 bases from the point of ligation inhibited the reaction. The LCR recognised variations in as little as 0.9 fmol DNA and will permit the rapid detection of HBV variants in molecular epidemiological studies. PMID- 7557321 TI - In vitro colonization ability of human colon mucosa by exogenous Lactobacillus strains. AB - We have tested five Lactobacillus strains for their in vitro colonization ability in a human colon epithelium culture model. Positive colonization occurred in different degrees in the presence of four Lactobacillus strains. We have found that L. casei GG and L. acidophilus NCFB 1748 induce an intermediate colonization. These two strains, largely investigated by others, have been reported to be beneficial to humans. The use of human intestinal tissue in our study partially reconstitutes the complex architectural specificity of human epithelium with the mucus layer and more closely simulates the in vivo situation. PMID- 7557322 TI - Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia. AB - A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92-94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface exposed epitopes on the C-terminus. PMID- 7557319 TI - Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli. AB - A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength. PMID- 7557325 TI - Interaction of Porphyromonas gingivalis with transferrin. AB - In this study, we characterized the binding of transferrin to Porphyromonas gingivalis using a classical receptor-binding assay, and examined the relationship between the binding and availability of transferrin for the growth of P. gingivalis. The binding of 125I-labeled human transferrin to P. gingivalis occurred rapidly, reversibly and specifically. Scatchard analysis yielded a Kd of 1.37 +/- 0.16 microM and an apparent number of 1.13 +/- 0.26 x 10(5) receptors per cell. The binding of transferrin was much increased when organisms were grown in iron-limited conditions. Among the species of black-pigmented anaerobic.rods, those strains of P. gingivalis which had high transferrin-binding activity exhibited unrestricted growth following the addition of transferrin to the hemin free culture medium. On the other hand, the presence of transferrin in the culture medium did not support unrestricted growth of organisms that had low transferrin-binding activity. These results suggest that the binding of transferrin to P. gingivalis cells may be a preliminary step in iron acquisition, which allows them to survive in the healthy periodontal environment. PMID- 7557323 TI - Molecular analysis of OmpR binding sequences involved in the regulation of ompF in Escherichia coli. AB - OmpR, the transcriptional regulatory protein of ompF, had not been previously shown to specifically bind to the -70 to -60-bp region of ompF. We show that the 102 to -76-bp sequence of ompF has a high affinity binding site for OmpR and produced a single OmpR/ompF complex (complex b). Extension of this DNA fragment to include an inverted repeat sequence located between the -71 and -64-bp region resulted in the formation of a second, slower migrating complex (complex a). A 102 to -58-bp fragment containing a substitution of the -70 CG bp was able to form complex b, but not complex a. A mutant OmpR protein derived from a strain that can not repress ompF was unable to form complex a, while complex b was formed normally. Deletion of the -70 CG bp resulted in incomplete repression of OmpF. These results suggest that OmpR binds to the -71 to -64-bp region and that this sequence plays a role in the regulation of ompF in Escherichia coli. PMID- 7557324 TI - Interference between Salmonella serotypes in intestinal colonisation of chickens: correlation with in vitro behaviour. AB - The effect of colonisation of the alimentary tract of newly hatched chicks by different Salmonella serotypes on the establishment in the gut by other Salmonella strains inoculated afterwards was assessed. Although profound inhibition of colonisation had been found previously to be genus-specific, considerable variation was found within the Salmonella genus. Some strains were found to be much more inhibitory than others and some were more easily inhibited than were others. There was not an absolute relationship between inhibitory activity and colonisation ability. No relationship was seen between inhibition and serotype or phage types within serotypes. There was no correlation between in vivo inhibition and the extent of inhibition that occurred in early stationary phase cultures in rich, undefined broth cultures. PMID- 7557326 TI - Silent genes in bacteria: the previously designated 'cryptic' ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates. AB - Gene ilvG in Escherichia coli K-12 and ilvI in 'Salmonella typhimurium LT2' (S. enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively. These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance. We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain 'S. typhimurium LT2'. All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvI sequences. Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme. Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates. We suggest that the mutations leading to inactivation of both ilvI in 'S. typhimurium LT2' and ilvG in E. coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation. PMID- 7557327 TI - The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13. AB - The pCloDF13 encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murecin lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF 13. PMID- 7557328 TI - A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans. AB - A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis. PMID- 7557329 TI - The use of PCR to isolate a putative ABC transporter from Saccharopolyspora erythraea. AB - A gene (ertX) encoding a putative ABC transporter was cloned from the erythromycin producer Saccharopolyspora erythraea, using PCR. The primers were based on regions of homology from ABC transporters which confer resistance to macrolide antibiotics. While ertX encodes a protein with a strong degree of similarity to other macrolide ABC transporters from streptomycetes and staphylococci, it did not confer resistance to erythromycin, tylosin, spiramycin, oleandomycin, josamcin, chalcomycin or midecamycin when subcloned into sensitive streptomycete hosts. Southern blot analysis suggested that ertX did not constitute part of the erythromycin gene cluster as identified to date. PMID- 7557330 TI - Variability of Leishmania (Leishmania) infantum among stocks from immunocompromised, immunocompetent patients and dogs in Spain. AB - Leishmania (Leishmania) infantum is the causative agent of both the cutaneous and visceral forms of leishmaniasis in southwest Europe; the dog is the main reservoir. In order to identify the L. (L.) infantum zymodemes present in Spain, a total number of 85 Leishmania stocks isolated from dogs (31), HIV-positive patients (46) with visceral or cutaneous leishmaniasis, a patient with visceral leishmaniasis complicating renal transplantation (1) and immunocompetent patients (7) with visceral or cutaneous leishmaniasis, have been characterized by isoenzyme typing. All canine stocks were MON-1, which is the most widespread zymodeme in the Mediterranean area. In immunocompetent patients three zymodemes were found: MON-1 (2), MON-24 (2) and MON-34 (3). Nine different zymodemes were obtained in stocks from HIV co-infected patients, indicating a higher variability of L. (L.) infantum amongst them: MON-1 (in 21 stocks), MON-24 (7), MON-28 (1), MON-29 (3), MON-33 (7), MON-34 (1) and MON-183 (4). Two new zymodemes, MON-198 (1) and MON-199 (1), were described among HIV patients from Spain. The stock from the renal transplanted patient was MON-1. The exclusive presence of certain zymodemes in immunocompromised patients and their absence in typical cases of cutaneous and visceral leishmaniasis and in infected dogs suggests two possibilities: (i) an anthroponotic pattern of leishmaniasis where intravenous drug user-infected patients act as potential reservoir for these new zymodemes. In the latter, syringes could act as the vehicles for infected monocytes; (ii) the cellular immune system could select virulent from non-virulent zymodemes in immunocompetent visceral leishmaniasis patients. PMID- 7557331 TI - Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations. AB - Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo'-deficient mutants of Escherichia coli K-12 with the following order of potency: Zn(II) > Cd(II) >> Co(II). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities. PMID- 7557332 TI - Immunological characterization and localization of a Porphyromonas gingivalis BApNA-hydrolyzing protease possessing hemagglutinating activity. AB - A monoclonal antibody (mAb-PC) was produced against a BA pNA-hydrolyzing protease possessing hemagglutinating activity (Pase-C) from Porphyromonas gingivalis. Other P. gingivalis BA pNA-hydrolyzing enzymes (Pase-B and Pase-S) did not react with this antibody. By ELISA or SDS-PAGE and Western immunoblotting analysis, mAb PC recognized all P. gingivalis and P. endodontalis strains tested but did not recognize other members of the Porphyromonas genus nor other putative periodontopathogenic organisms. Pase-C, extracellular vesicles (ECV) and human strains of P. gingivalis showed two major immunoreactive bands (44 kDa and 40 kDa), whereas a different pattern was obtained with animal strains of P. gingivalis. Biotinylarginyl chloromethane, an irreversible inhibitor of trypsin like proteases, did not affect the reactivity of Pase-C with mAb-PC on immunoblot. By reversed-phase electronmicroscopy following immunogold labeling, the antibody was shown to bind to the cell surface of P. gingivalis. mAb-PC inhibited the hemagglutinating activity of both P. gingivalis cells and ECV whereas a monoclonal antibody against LPS of P. gingivalis did not. These results suggest that Pase-C is located on the cell surface of P. gingivalis and may participate in erythrocyte binding. PMID- 7557333 TI - The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. AB - The Gram-positive soil bacterium Bacillus subtilis, generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe. Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth. Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium. The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions. Surprisingly, anaerobically grown B. subtilis lost the capacity for sporulation. An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B. subtilis. Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes. Four open reading frames with significant homology to the E. coli respiratory nitrate reductase operons (narGHIJ, narZYWV) were isolated and termed narGHJI. A chromosomal knock-out mutation of the B. subtilis nar operon totally abolished nitrate respiration. PMID- 7557334 TI - In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes. AB - The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyrl-CoA and isobutyrl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyrl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA. PMID- 7557336 TI - Purification and characterization of 3-isopropylmalate dehydrogenase from a thermoacidophilic archaebacterium Sulfolobus sp. strain 7. AB - 3-Isopropylmalate dehydrogenase was purified (about 2000-fold) to homogeneity for the first time from an archaebacterium, Sulfolobus sp. strain 7. The enzyme showed an apparent molecular mass of about 110 kDa by gel filtration and a single 36-kDa polypeptide band on SDS-PAGE, suggesting tri- or tetrameric structure. The pI value was 6.9. The N-terminal amino acid sequence was similar to enzymes from other sources. The enzyme activity was greatly stimulated by the presence of Mn2+, Cd2+, Mg2+, or Co2+. In contrast to 3-isopropylmalate dehydrogenase from other sources, monovalent cations such as K+ and Na+ were neither essential for activity nor stability of the protein. The enzyme was extraordinarily thermostable. PMID- 7557335 TI - Structure and partitioning of bacterial DNA: determined by a balance of compaction and expansion forces? AB - The mechanisms that determine chromosome structure and chromosome partitioning in bacteria are largely unknown. Here we discuss two hypotheses: (i) the structure of the Escherichia coli nucleoid is determined by DNA binding proteins and DNA supercoiling, representing a compaction force on the one hand, and by the coupled transcription/translation/translocation of plasma membrane and cell wall proteins, representing an expansion force on the other hand; (ii) the two forces are important for the partitioning process of chromosomes. PMID- 7557337 TI - DnaK expression in response to heat shock of Streptococcus mutans. AB - The oral pathogen, Streptococcus mutans, persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans, pulse-chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans. PMID- 7557338 TI - Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains. AB - The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe. The S. carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature sensitive B. subtilis secA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism. PMID- 7557339 TI - Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum. AB - Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-beta-D N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69,716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKT10) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56. PMID- 7557340 TI - High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae. AB - Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species. PMID- 7557341 TI - Identification of a chromosomally encoded kanamycin acetylase in Porphyromonas gingivalis. AB - Sonic extracts from the test strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens were tested for the ability to inactivate kanamycin sulfate in the presence of ATP or acetyl coenzyme A (acetyl-CoA). Residual kanamycin activity was determined by a bio-assay using Escherichia coli JM109 as the assay organism. Sonic extracts of all test strains of P. gingivalis inactivated kanamycin. All strains tested in this study required the presence of acetyl-CoA for inactivation, indicating that inactivation was by acetylation. The gene of a kanamycin-inactivating protein from P. gingivalis 16-1 was cloned into E. coli utilizing the plasmid vector PTZ18R. The resultant kanamycin-resistant clone, harboring plasmid pPG16 with a P. gingivalis insert, expressed a kanamycin inactivating activity, which was enhanced by the addition of acetyl-CoA, confirming that the kanamycin was inactivated by acetylation. Southern blot analysis indicates that the gene was conserved among all P. gingivalis strains tested. PMID- 7557343 TI - Effect of concentration of compounds containing iron on the growth of Porphyromonas gingivalis. AB - We examined the effect of the concentration of various types of iron molecules on the regulation of growth of Porphyromonas gingivalis. Bacterial growth was monitored spectrophotometrically. The hemin-depleted cells of P. gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron compounds. The relationship between the specific growth rate of organisms and the concentration of iron-containing compounds was determined. The value of Ks, a parameter analogous to the Michaelis Menten constant, was estimated. P. gingivalis 381 showed a Ks value of 3.85, 4.91 and 0.0017 microM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support growth of P. gingivalis. These findings suggest that P. gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron-limited environment. PMID- 7557342 TI - The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein. AB - The phosphoglycerate kinase (pgk), triosephosphate isomerase (tpi), and enolase (eno) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli. In high copy number, the pgk gene complemented an E. coli pgk- strain. In T. neapolitana, the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima, T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima, which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes. PMID- 7557345 TI - Resistance to fluconazole in Candida albicans from AIDS patients correlated with reduced intracellular accumulation of drug. AB - Mucosal candidosis is an almost inevitable consequence of AIDS. Resistance to fluconazole therapy associated with enhanced tolerance, detectable in microbiological estimation of sensitivity, occurs in up to 10% of cases with late stage AIDS. We report here our biochemical analysis of the basis of resistance in a study of two susceptible and two resistant isolates. Resistance was not associated with a change in the target enzyme sterol 14 alpha-demethylase, as indicated by equivalent levels of fluconazole inhibition of activity in extracts from all four isolates, or by mutations in sterol delta desaturase as previously observed in Saccharomyces cerevisiae and Ustilago maydis. Reduced cellular content of fluconazole in the resistant isolates of between six to ten-fold was observed which could account for their resistant phenotype. PMID- 7557346 TI - Complex segregation analysis of familial diseases with variable age of onset: comparison of different methods by a simulation study. AB - Standard methods of segregation analysis, such as originally developed in the unified model (UM) or the regressive logistic model (RLM) do not account for age of onset, and use instead age at examination. To take into account age of onset, models should be formulated using survival analysis concepts, as it was recently proposed with a model based on a logistic hazard function (LHM). A simulation study was conducted to compare the performances of the three methods (UM, RLM, and LHM) in analyzing generated familial data with variable age of onset. When the data were simulated under a polygenic hypothesis, all analysis models were robust with respect to the false conclusion of a major gene, if the tests of transmission probabilities were performed properly. When the data were generated under a major gene hypothesis, two main results were observed: 1) the use of the LHM markedly increased the power to detect a major gene, in particular when a genotype by age interaction was introduced in the model; and 2) in the situation of disease-specific mortality, the use of either UM (whether specific mortality was accounted for or not) or RLM led to both spurious conclusions and bias in parameter estimates. These latter results obtained with the UM and the RLM can be explained by the violation of one constraint of both models observed in a situation of disease-specific mortality, i.e., given all covariates, the probability of being affected and that of not being affected should sum to 1. The use of methods based on survival analysis concepts is recommended in the study of familial diseases with variable age of onset, especially in the case of a correlation between age of onset and age at examination which is induced by disease-specific mortality. PMID- 7557347 TI - Sex-specific effects for body mass index in the new Norwegian twin panel. AB - Sex-specific effects for body mass index (BMI) were explored in a newly established, population-based Norwegian twin panel. The sample includes 5,864 individuals, aged 18-25 years, who responded to a questionnaire containing items for zygosity classification, height, weight, health, health-related behaviors, well-being, and demographic information. Among the 2,570 intact pairs who returned the questionnaire there were 416 identical (MZ) male pairs, 387 fraternal (DZ) male pairs, 528 MZ female pairs, 443 DZ female pairs, and 796 unlike-sexed pairs. Alternate sets of models testing for either sex-specific genetic or environmental parameters were evaluated using structural equation analysis. Results from the most parsimonious model indicated that the genes contributing to variation in BMI are not identical for men and women; rather, some genetic effects were shared by the sexes and some were unique to each sex. Total variation in BMI could be explained by sex-specific additive genetic effects, as well as genetic and non-shared environmental effects common to men and women. Estimates of heritability were .708 for men and .789 for women, and the male-female genetic correlation was 0.622. The series of models specifying sex-specific shared environment also fit the data and suggests that shared environmental factors may be important for males but not for females. The findings raise questions concerning the relationship between sex-specific effects for BMI and sex differences in health outcomes. PMID- 7557348 TI - Statistical methodology for estimating twin similarity with respect to a dichotomous trait. AB - Statistical methodology is presented for the estimation of twin similarity with respect to a dichotomous trait. The methodology focuses on the intraclass correlation as the parameter of interest and is analogous to methodology commonly applied to continuous outcome data. For inference problems involving a single sample, confidence interval construction and significance testing are discussed. For two sample problems, test procedures are provided that are an alternative to an approach recently presented by Ramakrishnan et al. [(1992) Genet Epidemiol 9:273-287 (1)]. Two examples based on published data sets are given to illustrate the proposed techniques. PMID- 7557344 TI - Evaluation of pig lungs following an experimental challenge with Actinobacillus pleuropneumoniae serotype 1 and 5 in pigs inoculated with either hemolysin protein and/or outer membrane proteins. AB - Histopathological changes were compared in pigs challenged with Actinobacillus pleuropneumoniae serotype 1 and serotype 5 after inoculation with subunit vaccines. The vaccines consisted of outer membrane protein and/or hemolysin protein isolated from Actinobacillus pleuropneumoniae serotype 1 or both subunits combined. Twenty-seven cross-bred pigs were separated into six groups: Groups I and IV were vaccinated and boostered with 1500 micrograms outer membrane protein; Groups II and V were vaccinated and boostered with 250 micrograms hemolysin protein; Groups III and VI were vaccinated and boostered with a combination of 1500 micrograms outer membrane protein and 250 micrograms hemolysin protein. Groups I, II and III were challenged with A. pleuropneumoniae serotype 1; and Groups IV, V and VI were challenged with A. pleuropneumoniae serotype 5. Groups III and VI demonstrated the least severe lung tissue damage, with significantly lower (P < 0.05) lung involvement as compared to the other groups. Lesions were noted in all six groups. These results showed that complete protection against A. pleuropneumoniae infection was not feasible using a subunit vaccine consisting of just outer membrane protein and hemolysin protein, and that some cross-protection did occur. PMID- 7557349 TI - Use of robust statistical methods to determine the effect of fragile X on means and variance components of a quantitative trait. AB - Owing to the presence of outliers, an estimated 3.5% in the ridge breadth data and 1.7% in the height data, the effect of fragile X on height and ridge breadth was examined using robust statistical techniques for data collected from 54 families afflicted with this disorder. It is shown that fragile X affects ridge breadth and height in a different manner. Fragile X women had a greater mean ridge breadth than normal women, whereas there was a similar trend, but no significant difference, between normal and fragile X men. Fragile X men were shorter than normal men, but no significant difference between the mean height of normal and fragile X women was observed. However, fragile X girls were shown to grow more quickly and to stop growing earlier than normal girls. An examination of the covariance between relatives classified according to fragile X status showed that for both traits the effect of fragile X was to reduce the covariance between parents and offspring, which produced the effect of departure from an additive polygenic model of inheritance. PMID- 7557350 TI - Method for calculating risk associated with family history of a disease. AB - A method is described for estimating excess relative risks of a disease from familial factors. Beginning with population-based series of cases and controls, a cohort of each subject's relatives is formed and checked for disease against a population based registry. The disease experience of the cohort formed from each subject's relatives is summarized as a kinship-weighted familial standardized incidence ratio (FSIR). The FSIR's are used as exposure estimates in conditional linear excess relative risk models, which may be used not only to screen for significant familial disease aggregations, but also to estimate relative risks, population attributable risks, and gene-environment interactions. The method is demonstrated on 4083 breast cancer cases from Utah and a set of matched controls. PMID- 7557352 TI - Dementia among elderly apolipoprotein E type 4/4 homozygotes: a prospective study. AB - The E*4 allele of the apolipoprotein E (APOE) gene on chromosome 19 has been shown to be an age- and dose-related risk factor for Alzheimer's disease. Of 870 elderly women participating in an osteoporosis study, 13 were previously found to be homozygous for the APOE*4 allele; 1 was deceased and the rest were assessed for dementia in a "piggyback" study of dementia. One had moderate [clinical dementia rating (CDR = 2], 2 had mild dementia (CDR = 1), and 2 had possible dementia (CDR = 0.5). All 3 women over 80 years were definitely demented (CDR > or = 1). Typically, genetic studies of Alzheimer's and other dementias require the identification and diagnosis of large numbers of demented subjects, at considerable expense, followed by genotyping or phenotyping with a relatively low yield of individuals with rare alleles. We demonstrate a more cost-effective approach in which the population is first phenotyped and then stratified by phenotype, so that diagnostic evaluation can be restricted to individuals with the phenotype of interest. PMID- 7557351 TI - Population genetic study of the human dopamine transporter gene (DAT1). AB - The human dopamine transporter gene, DAT1, acts to transport released dopamine into presynaptic terminals of the brain. The possibility that the DAT1 gene plays a role in genetic diseases of the brain has led to studies of DAT1 in several psychiatric and neurological disorders. Previous sequence analysis of DAT1 revealed a 40-bp repeat in the 3' end of the gene. In order to identify all potential alleles for this VNTR marker a population database was established. One thousand seventy-four unrelated individuals were screened by PCR for the region containing the 40 bp repeat. Allele frequency differences were found between black Americans and Caucasians or Hispanics but no differences were observed between Caucasians and Hispanics. A previously unreported allele was detected in all three populations. Thus, we have shown that screening a large population identifies new alleles and generates more accurate allele frequencies. PMID- 7557354 TI - Mother-child dialogue about emotions and preschoolers' emotional competence. AB - Conversations about emotions between preschoolers and their mothers constitute an important form of shared meaning. As groundwork for a child's developing theory of mind, such conversations are expected to predict aspects of children's emotional competence. Forty-seven preschoolers and their mothers looked at a picturebook in which characters displayed emotions on almost every page; although the emotions were central to the plot, it was up to each mother and child (dyad) whether to discuss them or not. Conversations were transcribed and coded for frequency and function of emotional language as well as for specific emotions to which both mother and child referred. In general, mothers talked more than their children did about emotions, yet the frequency of emotional utterances that served as simple comments or to guide the other's behavior did not differ for mothers and children. As expected, patterns of emotional language of mothers and children were related to indices of positive social-emotional development. PMID- 7557353 TI - Taiwanese love styles and their association with self-esteem and relationship quality. AB - This study is an examination of how Lee's (1973, 1977, 1988) love styles are related to Taiwanese attitudes toward romantic relationships. Traditional literature was explored to ascertain Chinese beliefs about love and customs surrounding courtships and marriage. Taiwanese students at the University of Texas completed the Love Attitudes Scale (C. Hendrick & S. Hendrick, 1990), a measure of Lee's love styles. Six factors were extracted, using a principle components analysis, that were similar in many ways to the love styles derived from American samples, but that reflected Chinese beliefs and attitudes. The relationships of Taiwanese love styles to self-esteem and relationship quality (i.e., satisfaction, the probability of breaking up, and the number of conflicts experienced in a month) suggested the significance of Chinese traditions as well as current social changes in Taiwan. PMID- 7557355 TI - Framework for a comprehensive description and measurement of emotional states. AB - The rationale for a general three-dimensional approach to measuring emotions (the Pleasure-Arousal-Dominance, PAD, Emotional-State Model) is described. Evidence bearing on two early versions of the PAD scales is reviewed, and three studies report additional development and refinement of a final set of PAD scales. Factor analysis of 58 PAD items in Study 1, 41 PAD items in Study 2, and 34 PAD items in Study 3 consistently yielded three nearly orthogonal Pleasure, Arousal, and Dominance factors. The final 16-item State Pleasure, 9-item State Arousal, and 9 item State Dominance scales had alpha reliability coefficients of .97, .89, and .80, respectively. Across Studies 2 and 3, intercorrelations among the latter three scales did not exceed .09 in absolute value, showing that the scales provided a parsimonious base for the general assessment of emotional states. Discriminant and construct validity evidence for the scales were reviewed. Potential applications of the scales were noted. PMID- 7557356 TI - Restriction fragment length polymorphisms in polymerase chain reaction amplified ribosomal DNAs of three Trichogramma (Hymenoptera: Trichogrammatidae) species. AB - Restriction fragment length polymorphisms from PCR amplified ribosomal DNAs of three Trichogramma species, T. minutum, T. brassicae, and T. near sibiricum, were studied. Length variation in the internal transcribed spacer (ITS) region was observed. The ITS region of T. brassicae is about 1350 base pairs (bp) in length and those of T. minutum and T. near sibiricum are 1300 bp. These three species also differ in the size of their ITS1 and ITS2 regions. Restriction enzyme digestions of these regions showed unique banding patterns for each species. The amplified 18S region of ribosomal DNA is about 1800 bp in length and showed no length variation between the three species of Trichogramma. Restriction enzyme digestion of this region by BamHI differentiated T. brassicae from the other two species. Restriction site maps of the ITS and 18S regions were constructed for each species. The amplified 28S region is about 1700 bp for these three species. Restriction of this region by RsaI and SacII differentiates these three species. The reported results indicate that these species of Trichogramma can be clearly differentiated from one another by nuclear ribosomal DNA markers. PMID- 7557359 TI - The role of B chromosomes in the population dynamics of yellow-necked wood mice Apodemus flavicollis (Rodentia, Mammalia). AB - Seasonal changes in the frequency of animals with B chromosomes were studied during 1989 in the population of yellow-necked wood mice, Apodemus flavicollis, on the mountain Jastrebac in Yugoslavia. During the study the estimated density of animals reached a very large value (378.8 individuals/ha for the April sample), which produced stress conditions leading to population decline. Six samples with a total of 247 specimens were karyotypically analyzed. Animals with Bs were present in all samples in frequencies that differed significantly at the 5% level. This difference was produced by the reduction in frequencies of specimens with Bs between the April and May samples (0.53 versus 0.29; chi 2(1) = 9.44; p < 0.002) and appeared mostly as a result of increased elimination of B carrying animals from the prereproductive group. Young animals with Bs were inferior in stress conditions produced by overabundance of animals. From the first to the last sample, the frequency of animals with one B among B-containing animals constantly decreased, while the frequency of animals with more than one B increased. The possible role of Bs in regulating population dynamics is discussed. PMID- 7557360 TI - Molecular characterization of a tandem repeat, Afa family, and its distribution among Triticeae. AB - We have characterized a so-called D genome specific repetitive DNA sequence (pAs1) of Aegilops squarrosa L. (2n = 14, genome DD) with respect to its DNA sequence and its distribution among Triticeae species. The clone consisted of three units of a repetitive DNA sequence of 336 or 337 base pairs, and was AT rich (65.2%). DNA analyses revealed the presence of the pAs1-like sequences in other genomes of Triticeae species, although the repetition was greatly (as much as 100-fold) variable among the genomes. The repetitive sequences from 10 diploid species were amplified using PCR with specific primers, and the sequential variability was analyzed by the digestion pattern obtained with five restriction enzymes. Since the AfaI site was the most conservatively present in the unit of the repetitive sequences, we named them "Afa family." The analysis clearly displayed the variation of the repetitive sequences regardless of the uniformity of the size of the amplified product. These results indicated that plural amplification events of these repetitive sequences happened independently in the genome evolution of Triticeae. PMID- 7557357 TI - On the mechanism of homologous synapsis in lycosid spiders. AB - Lycosid spiders have 13 pairs of telocentric chromosomes and 2 nonhomologous sex chromosomes in males. At leptotene, the kinetochore ends are attached to the nuclear envelope via thickened attachment plaques. Homologous synapsis begins at the attachment plaques and proceeds zipper-like through the length of the synaptonemal complex. We have tested whether or not this simple form of homologous synapsis is obligatory by inducing reciprocal translocations. Since we find many irradiated cells with quadrivalents at diakinesis-prometaphase and metaphase I, clearly a backup system exists that can bring together homologous segments disparate from each other in the nucleus. This mechanism apparently does not depend on end-initiated synapsis. Furthermore, we have found in previous studies that wolf spider bivalents are always unichiasmate, with either proximally or distally placed chiasmata. Since many chain quadrivalents, but no ring quadrivalents, were seen in this study, crossing over and chiasma placement obey a different set of rules when homologous segments are switched between chromosomes. PMID- 7557358 TI - Localization of a new ferritin heavy chain sequence present in human brain mRNA to chromosome 11. AB - Two types of ferritin heavy (H) chain clones have been isolated from cDNA libraries of human fetal and adult brain: one corresponds to the ferritin H chain mRNA that is abundant in liver and is called "liver-like" brain cDNA; the other contains an additional 279 nucleotide (nt) sequence in the 3' untranslated region and is called brain ferritin H chain cDNA. To map the 279-nt sequence, polymerase chain reaction (PCR) amplification was carried out using DNA from rodent x human hybrid cell lines containing single human chromosomes as templates, and oligomeric primers homologous to the 3' end of the 279-nt sequence (primer A) and to a coding sequence just 5' to the 279-nt sequence. Significant PCR product of the size expected from analysis of the brain ferritin H chain cDNA clones and a genomic ferritin H chain clone (487 bp) was generated only from hybrid-cell DNA containing human chromosome 11. This PCR product and the "liver-like" brain cDNA (lacking the 279-nt sequence) both hybridized to chromosome 11 fragments that are known to define the well-characterized functional liver ferritin H chain gene and a putative pseudogene. Preliminary data indicate that primer A (and thus the 279 nt sequence) maps to the functional ferritin H chain gene fragments, but binding to the pseudogene has not been ruled out. PMID- 7557361 TI - Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush). AB - Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes. PMID- 7557362 TI - Identification of fat-cell enhancer activity in Drosophila melanogaster using P element enhancer traps. AB - To identify genes important in fat-cell metabolism and development, we have screened Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements. We have identified those "enhancer-trap lines" that contain transposable P elements integrated near fat cell specific enhancer elements. We anticipate that the genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. Furthermore, the identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors svp, a steroid-hormone receptor, and Kr, a zinc-finger protein, are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. And finally, in a single line transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development. PMID- 7557363 TI - Chromosomal distribution of a repeated DNA sequence from C-genome heterochromatin and the identification of a new ribosomal DNA locus in the Avena genus. AB - Satellite DNA specific to the oat C genome was sequenced and located on chromosomes of diploid, tetraploid, and hexaploid Avena ssp. using in situ hybridization. The sequence was present on all seven C genome chromosome pairs and hybridized to the entire length of each chromosome, with the exception of the terminal segments of some chromosome pairs. Three chromosome pairs belonging to the A genome showed hybridization signals near the telomeres of their long arms. The existence of intergenomic chromosome rearrangements and the deletions of the repeated units are deduced from these observations. The number of rDNA loci (18S 5.8S-26S rDNA) was determined for the tetraploid and hexaploid oat species. Simultaneous in situ hybridization with the satellite and rDNA probes was used to assign the SAT chromosomes of these species to their correct genomes. PMID- 7557364 TI - Structure and expression of histone H3.3 genes in Drosophila melanogaster and Drosophila hydei. AB - We demonstrate that in Drosophila melanogaster the histone H3.3 replacement variant is encoded by two genes, H3.3A and H3.3B. We have isolated cDNA clones for H3.3A and cDNA and genomic clones for H3.3B. The genes encode exactly the same protein but are widely divergent in their untranslated regions (UTR). Both genes are expressed in embryos and adults; they are expressed in the gonads as well as in somatic tissues of the flies. However, only one of them, H3.3A, shows strong testes expression. The 3' UTR of the H3.3A gene is relatively short (approximately 250 nucleotides (nt)). H3.3B transcripts can be processed at several polyadenylation sites, the longest with a 3' UTR of more than 1500 nt. The 3' processing sites, preferentially used in the gonads and somatic tissues, are different. We have also isolated the Drosophila hydei homologues of the two H3.3 genes. They are quite similar to the D. melanogaster genes in their expression patterns. However, in contrast to their vertebrate counterparts, which are highly conserved in their noncoding regions, the Drosophila genes display only limited sequence similarity in these regions. PMID- 7557366 TI - Preventing fatalities from anaphylaxis: an allergist-immunologist's perspective. AB - A patient with asthma related to his work in a greenhouse with Lathyrus odoratus was studied. Specific IgE against pollen of this cultivar of the Leguminosae family could be demonstrated, indicating a type I allergic mechanism. Monitoring of serial PEFR measurements and nonallergic bronchial responsiveness warrant diagnosis of occupational asthma. PMID- 7557367 TI - Preventing fatalities from anaphylaxis: an emergency medicine physician's perspective. PMID- 7557365 TI - Fatal and near fatal idiopathic anaphylaxis. AB - OBJECTIVE: To document that idiopathic anaphylaxis may have a fatal or near fatal outcome. DESIGN: Review of selected cases seen personally by authors during the past 16 years. SETTING: University faculty practice and private practices. PATIENTS: All cases of idiopathic anaphylaxis seen by the authors are not presented, but 10 cases were selected to demonstrate two fatalities and eight cases of near fatalities. RESULTS: Two fatal cases had expired before emergency service therapy. Eight near fatal cases responded to acute therapy and subsequently were controlled. Remission of idiopathic anaphylaxis was then induced. Idiopathic anaphylaxis may be fatal or potentially fatal and must be treated to prevent a fatal outcome. CONCLUSIONS: The documentation of fatalities and near fatalities should help patients and their physicians accept intense management of idiopathic anaphylaxis that will result in control and induction of a remission in idiopathic anaphylaxis. PMID- 7557368 TI - Recurrent dermopathy after remission of Stevens-Johnson syndrome secondary to mild dermal trauma. AB - We report on three patients who had an apparent recurrence of the dermatitis of Stevens-Johnson Syndrome (SJS) after remission had been induced with corticosteroids. The recurrences were related to mild trauma to the skin, including the Red Man Syndrome, after vancomycin in two patients. Both responded to corticosteroids, and vancomycin could be continued with modification in the rate of infusion. The third patient had dry, pruritic skin and the exacerbation of SJS appeared related to the trauma associated with intense scratching. A post SJS inflammatory dermatitis may occur after remission of SJS secondary to cutaneous trauma. This recurrent SJS dermopathy is rapidly responsive to moderate dose corticosteroid therapy. PMID- 7557370 TI - A comparison of imaging techniques in patients with chronic sinusitis (X-ray, MRI, A-mode ultrasound). AB - Thirty-five patients age 9 to 67 were evaluated for chronic sinusitis by history, physical and laboratory examination, and imaging techniques (X-ray, magnetic resonance imaging (MRI) and flexible rhinoscopy). MRI was the most predictive. To establish the diagnosis of sinusitis, it was more sensitive than plain X-ray for intrasinus disease. Findings of edema, erythema, and drainage on flexible rhinoscopy were consistent with chronic sinusitis and were confirmed by MRI and sinus X-rays in 41% of the cases. Nasal smears for polymorphonuclear cells and eosinophils were suggestive of a diagnosis of chronic sinusitis, but other laboratory tests (CBC, sedimentation rate, quantitative immunoglobulins, total IgEs) were of very limited value in the diagnosis of chronic sinusitis. PMID- 7557369 TI - Determination of inflammatory markers in allergic reactions to drugs. AB - Serum tryptase (Tryp) and eosinophil cationic protein (ECP) and urine N methylhistamine (N-MH) were quantitated in a group of 13 subjects who had experienced immediate allergic reactions to different drugs. Results indicated that both Tryp and N-MH were involved and the levels were related to the severity of the reaction. Results of serum ECP levels failed to provide relevant information concerning the participation of eosinophils in immediate reactions to drugs. PMID- 7557373 TI - Commentary on the article "Management of the adult asthma patient" (Jan.-Feb., 1995) PMID- 7557372 TI - Occupational asthma in horticulture caused by Lathyrus odoratus. PMID- 7557371 TI - The usefulness of questionnaire-derived information to predict the degree of nonspecific bronchial hyperresponsiveness. AB - Nonspecific bronchial hyperresponsiveness (BHR) is a hallmark of clinical asthma, but can be present in nonasthmatics as well. The diagnosis of asthma is based on clinical grounds, and no laboratory procedure can definitely establish its presence. This poses a problem in studies of asthma. If epidemiological studies are to provide valid information, the tools used must have a relative degree of predictive or diagnostic ability. This report determined whether the American Thoracic Society-Division of Lung Disease (ATS-DLD) respiratory questionnaire has the ability to predict different degrees of non-specific BHR. In the years 1983 1990, when the ATS-DLD questionnaire was used in our Natural History of Asthma study, 192 subjects completed the ATS-DLD questionnaire and underwent a standardized methacholine challenge. A recursive partitioning analysis of the ATS DLD questionnaire was able to predict which questions would likely be answered if the subject had nonspecific bronchial reactivity to inhaled methacholine of 100 and 200 breath units. Positive responses for questions concerning treatment for asthma, wheezing, or shortness of breath, and emergency treatment for asthma predicted the presence of increased bronchial reactivity. PMID- 7557374 TI - Allergists as directors of medical departments: a demonstration of leadership. PMID- 7557376 TI - Altering specific telomerase RNA template residues affects active site function. AB - The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying a template sequence in the telomerase RNA. We studied the functional roles of specific residues in the Tetrahymena telomerase RNA template region. Unexpectedly, mutation of certain templating residues caused dramatic effects on specific aspects of the enzyme reaction, including loss of enzymatic fidelity and premature product dissociation. None of these fundamental changes in enzymatic action are explainable by altered base-pairing between the telomerase RNA and DNA substrate. These influences of specific template bases of the telomerase RNA on enzymatic properties of telomerase provide evidence for critical roles of these RNA residues in two active site functions--fidelity and DNA substrate/enzyme interaction. PMID- 7557375 TI - Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. AB - The beta-globin locus control region (LCR) is a complex regulatory element that is essential for the appropriate red cell-specific expression of all cis-linked beta-globin genes. Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice. Thus, 5'HS2 has been assumed to be an important element for the function of the LCR in vivo. We have utilized homologous recombination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chromosomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (delta HS2 + neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12 fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expression of the globin genes. When homozygous, the deletion/replacement mutation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenotypic effects resulting from the deletion of 5'HS2 from those attributable to insertion of the selectable marker, the selectable marker was removed by expressing the FLP site-specific recombinase in ES cells harboring the homologous recombination event. Mice derived from these ES cells (delta HS2 delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results indicate that although 5'HS2 demonstrates significant regulatory activities in a variety of assays, deletion of this element from the endogenous beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elements in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation. PMID- 7557377 TI - Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. AB - Telomerase is a DNA polymerase fundamental to the replication and maintenance of telomere sequences at chromosome ends. The RNA component of telomerase is essential for the synthesis of telomere repeats. In vitro, the template domain (5'-CAACCCCAA-3') of the Tetrahymena telomerase RNA dictates the addition of Tetrahymena-specific telomere repeats d(TTGGGG)n, onto the 3' end of G-rich or telomeric substrates that are base-paired with the template and alignment regions of the RNA. Using a reconstituted in vitro system, we determined that altering the sequence of the alignment and template domains affects processivity of telomerase without abolishing telomerase activity. These results suggest that alternative template/alignment regions may be functional. In the ciliate telomerase RNAs, there is a conserved sequence 5'-(CU)GUCA-3', located two residues upstream of the template domain. The location and sequence of this conserved domain defined the 5' boundary of the template region. These data provide insights into the regulation of telomere synthesis by telomerase. PMID- 7557378 TI - The basis for a heat-induced developmental defect: defining crucial lesions. AB - Because lethal heat shocks perturb a multitude of cellular processes, the primary lesions responsible for death from heat stress remain to be defined. In Drosophila, sublethal heat treatments produce developmental anomalies that frequently mimic the effects of known mutations and are hence referred to as phenocopies. Mutations subject to phenocopy mimicry provide signposts to those biological processes most sensitive to heat and most important for the function and survival of the organism as a whole. We have analyzed a particular developmental defect inducible in early embryos of Drosophila melanogaster. By molecular, phenotypic, and genetic criteria, we have found extensive parallels between this phenocopy and certain dominant mutations in the segmentation gene fushi tarazu (ftz). Our analysis of this phenocopy indicates that the crucial lesion is interference with proper turnover of ftz protein, resulting in ftz overexpression. Our results provide a novel explanation for a heat-induced developmental defect. Perturbations in relative amounts of important regulatory proteins may be a common mechanism by which heat-shock phenocopies arise. PMID- 7557379 TI - sur-2, a novel gene, functions late in the let-60 ras-mediated signaling pathway during Caenorhabditis elegans vulval induction. AB - We describe here a new gene acting downstream of let-60 ras in the vulval signaling pathway of Caenorhabditis elegans. The sur-2 (suppressor of ras) gene is defined by eight mutations identified in a genetic screen for suppressors of the Multivulva phenotype of let-60(n1046), an activated let-60 ras mutation. sur 2 mutations result in pleiotropic, incompletely penetrant phenotypes that include a Vulvaless phenotype in hermaphrodites, defects in development of the male tail, gonadal abnormalities, and larval lethality, indicating a role for the sur-2 gene product in multiple developmental events. Genetic epistasis analyses suggest that sur-2 is required late in the vulval signaling pathway, downstream of let-60 Ras, and is likely to act downstream of the Raf/MAP Kinase cascade. We cloned the sur 2 gene by DNA-mediated transformation and have shown that it encodes a novel protein. We also show that a sur-2::lacZ transgene is expressed in the vulval precursor cells at the time of vulval determination. PMID- 7557380 TI - Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. AB - Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival. PMID- 7557381 TI - fyn tyrosine kinase is involved in keratinocyte differentiation control. AB - Induction of tyrosine phosphorylation is an early and specific event which is required for mouse keratinocyte differentiation to occur, in response to both calcium and TPA (12-0-tetradecanoylphorbol-13-acetate). We report here that there is an increase of tyrosine kinase activity immunoprecipitable with anti phosphotyrosine antibodies specifically in response to calcium--and a number of other divalent cations--within 2 min of exposure. Such an activity does not correspond to any of the known tyrosine kinases that were tested. A second tyrosine kinase activity is induced in response to both calcium and TPA, and has been identified as fyn, a nonreceptor tyrosine kinase of the src family. fyn activation is induced in keratinocytes within 6 hr of calcium exposure, but already within 2 min of TPA treatment. Cortactin, a p80-85 substrate of src- and fyn-related kinases that localizes with actin at cell adhesion sites, is increasingly tyrosine phosphorylated in calcium- and TPA-induced differentiation, with a time course which parallels that of fyn activation. Keratinocytes with a specific disruption of the fyn, but not yes kinase gene show no induction of phosphorylation of p80-85 proteins, and are significantly altered in their differentiation response both in vitro and in vivo. Thus, at least two tyrosine kinase activities are induced in keratinocyte differentiation, one of which has been identified as fyn and shown to be specifically involved in this process. PMID- 7557383 TI - A novel bacterial transcription cycle involving sigma 54. AB - sigma 54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that sigma 54 is attached selectively to the template strand, which must be single-stranded for transcription initiation. sigma 54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established sigma 70-holoenzyme transcription cycle. Permanganate footprinting experiments show that the bound sigma 54 and the elongating core RNA polymerase downstream of it are each associated with a single strand DNA region. Template commitment assays show that the promoter-bound sigma 54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation. PMID- 7557382 TI - Ectopic expression of the knox homeo box gene rough sheath1 alters cell fate in the maize leaf. AB - Rough sheath1 (Rs1) is a dominant mutation that alters cell fate and causes unregulated cell division and expansion in the maize leaf. A knox (Kn1 like-homeo box gene) sequence closely linked to the Rs1-O mutation was cloned and shown by transposon mutagenesis to encode the rs1 gene. The deduced amino acid sequence of the RS1 protein is highly similar to KN1 in the homeo domain but contains a unique amino-terminal region. rs1 is expressed in the shoot apical meristem in a circular pattern preceding leaf initiation, but is not detectable in leaf primordia or mature leaves in normal plants. Rings of rs1 expression subtend leaf insertion sites in the shoot, and lateral organ primordia in inflorescence and floral meristems. The timing and position of rs1 expression in meristems suggests a possible role for rs1 in patterning the placement of lateral organs along the axis of the shoot. In contrast to wild type, rs1 is expressed in early leaf primordia of Rs1 mutant plants, suggesting that ectopic expression causes the mutant phenotype. Ectopic expression in Rs1-O plants suggests the ligular [corrected] region is more competent to respond to rs1 than blade or sheath tissues. PMID- 7557385 TI - Loss of methylation activates Xist in somatic but not in embryonic cells. AB - The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation. PMID- 7557386 TI - Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. AB - mRNA transcription, DNA amplification, and progeny production of human papillomaviruses (HPVs) are closely linked to squamous epithelial differentiation in patient papillomas. Because suprabasal, differentiated keratinocytes have exited the cell cycle for days or weeks and because viral DNA synthesis requires the host DNA replication machinery, HPVs must have a mechanism to reactivate the essential host genes. In this study, we show via acute recombinant retrovirus infection that an intact E7 gene of either high-risk or of low-risk HPV genotypes, under the control of its respective native enhancer-promoter, induced proliferating cell nuclear antigen (PCNAs) expression in the suprabasal cells of epithelial raft cultures of primary human foreskin keratinocytes (PHK). The cellular differentiation program was unaltered by the viral oncoprotein; it was essential for high HPV promoter activity. Furthermore, extensive host chromosomal DNA replication took place in differentiated cells of HPV-18 E7-expressing raft cultures and of patient laryngeal papillomas caused by HPV-6. These results indicate that the main function of the E7 protein is to reactivate host DNA replication machinery to support viral replication in differentiated, noncycling cells. PMID- 7557384 TI - A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. AB - The full set of consensus sequences at the 5' splice site is recognized during splicing of pre-mRNA in extracts depleted of U1 snRNP. High concentrations of HeLa SR proteins or purified SC35 alone promote the splicing of specific RNA substrates, bypassing the requirement for U1 snRNP in formation of the U2 snRNP pre-mRNA complex. Under these conditions, mutations in the substrate that increase the sequence complementarity between U6 snRNA and the 5' splice site region can facilitate splicing. This provides additional strong evidence that U1 snRNP is not essential for splicing. Thus, the consensus sequence at the 5' splice site is probably recognized twice during splicing of most introns; however, some pre-mRNAs could potentially be processed in the absence of interactions with U1 snRNP in regions of the nucleus containing high concentrations of SR protein. PMID- 7557388 TI - Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. AB - Using homologous recombination, mice lacking cyclin D1 were generated by replacing most of the first exon of the Cyl-1 gene with sequences encoding neomycin resistance. Cyl-1(-1-) mice were viable and fertile but consistently smaller than their heterozygous or wild-type littermates. The nullizygous animals also showed two distinctive abnormalities: a severe retinopathy caused by impaired development of all layers of the retina and, in the mammary gland during pregnancy, a marked reduction in acinar development accompanied by a failure to lactate. Approximately 50% of animals also had a malformation of the jaw that manifested itself as a misalignment of the incisor teeth. Mouse embryo fibroblasts isolated from 14 day nullizygous, heterozygous, or wild-type embryos and grown under standard conditions showed similar cell-cycle and growth characteristics. Thus although cyclin D1 kinase activity may facilitate G1 progression, it is not essential for the development of most tissues and organs, and only a few specialized cell lineages are demonstrably sensitive to its absence. PMID- 7557387 TI - Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis. AB - Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes. PMID- 7557389 TI - Lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis. AB - The mechanisms that underlie the segregation of muscle founder cells in the Drosophila embryo are undefined. We show that the proneural gene lethal of scute (l'sc) is expressed in clusters of cells in the somatic mesoderm, from which individual muscle progenitors are singled out by progressive restriction of l'sc expression. Coexpression of l'sc and S59 (a putative muscle identity gene) in a subset of muscle progenitors shows that muscle founders are produced by division of muscle progenitors. In neurogenic mutant embryos the restriction of l'sc expression fails and all cells in a cluster coexpress l'sc and S59. Loss-of function and overexpression phenotypes indicate a role for l'sc in the segregation of muscle progenitors and the formation of the muscle pattern. PMID- 7557390 TI - scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development. AB - The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices. PMID- 7557391 TI - Disassembly of the Mu transposase tetramer by the ClpX chaperone. AB - Mu transposition is promoted by an extremely stable complex containing a tetramer of the transposase (MuA) bound to the recombining DNA. Here we purify the Escherichia coli ClpX protein, a member of a family of multimeric ATPases present in prokaryotes and eukaryotes (the Clp family), on the basis of its ability to remove the transposase from the DNA after recombination. Previously, ClpX has been shown to function with the ClpP peptidase in protein turnover. However, neither ClpP nor any other protease is required for disassembly of the transposase. The released MuA is not modified extensively, degraded, or irreversibly denatured, and is able to perform another round of recombination in vitro. We conclude that ClpX catalyzes the ATP-dependent release of MuA by promoting a transient conformational change in the protein and, therefore, can be considered a molecular chaperone. ClpX is important at the transition between the recombination and DNA replication steps of transposition in vitro; this function probably corresponds to the essential contribution of ClpX for Mu growth. Deletion analysis reveals that the sequence at the carboxyl terminus of MuA is important for disassembly by ClpX and can target MuA for degradation by ClpXP in vitro. These data contribute to the emerging picture that members of the Clp family are chaperones specifically suited for disaggregating proteins and are able to function with or without a collaborating protease. PMID- 7557392 TI - Formation and resolution of double-strand break intermediates in V(D)J rearrangement. AB - A recently described pre-B cell line can be induced at high temperature to actively rearrange its immunoglobulin light-chain loci. We used this cell line to determine the fate of double-strand breaks generated by V(D)J rearrangement. After induction, 30%-40% of K loci had broken JK1 signal ends. JK1-coding ends were detectable, but 10- to 100-fold less frequent. Both covalently closed (hairpin) and open, blunt, processed coding ends were observed. Coding junctions involving JK1 accumulated with similar kinetics as JK1 signal ends, arguing that coding ends can be resolved quickly and efficiently to coding junctions, whereas signal ends remain mostly unjoined. Signal ends are then joined rapidly when cells are returned to the low temperature. These results support the model that broken signal ends and hairpin coding ends are authentic intermediates in V(D)J recombination. It appears that hairpin coding ends are rapidly opened, processed, and resolved to coding junctions, whereas joining of signal ends is clearly uncoupled from the joining of coding ends and can be much slower. Efficient formation of signal junctions may require cell cycle progression, or down regulation of the recombination machinery. PMID- 7557393 TI - Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast. AB - The first step in the decay of many eukaryotic mRNAs is shortening of the poly(A) tail. In yeast, deadenylation leads to mRNA decapping and subsequent 5' --> 3' exonucleolytic degradation of the transcript body. We have determined that the major poly(A)-binding protein Pab1p plays at least two critical roles in this pathway. First, mRNAs in pab1 delta strains were decapped prior to deadenylation. This observation defines a new function for Pab1p as an inhibitor of mRNA decapping. Moreover, mutations that inhibit mRNA turnover suppress the inviability of a pab1 delta mutation, suggesting that premature mRNA decapping in pab1 delta strains contributes to cell death. Second, we find that Pab1p is not required for deadenylation, although in its absence poly(A) tail shortening rates are significantly reduced. In addition, in the absence of Pab1p, newly synthesized mRNAs had poly(A) tails longer than those in wild-type strains and showed an unexpected temporal delay prior to the initiation of deadenylation and degradation. These results define new and critical functions for Pab1p in the regulation of mRNA decapping and deadenylation, two important control points in the specification of mRNA half-lives. Moreover, these results suggest that Pab1p functions in additional phases of mRNA metabolism such as mRNP maturation. PMID- 7557394 TI - U14 base-pairs with 18S rRNA: a novel snoRNA interaction required for rRNA processing. AB - U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA. PMID- 7557396 TI - Cloning and characterization of the gene encoding translation elongation factor 1 alpha from Aureobasidium pullulans. AB - The gene (TEF1) encoding translation elongation factor 1 alpha from the dimorphic fungus Aureobasidium pullulans (Ap) strain R106 has been cloned using Candida albicans TEF1 as a heterologous hybridization probe. Electrophoretic molecular karyotype/hybridization analysis of Ap revealed eight chromosomal bands and suggested that TEF1 resides on chromosome VI. Comparison of the genomic DNA sequence with the cDNA sequence of TEF1 verified the presence of three introns, the first one occurring five nucleotides from the start of translation. The deduced amino acid (aa) sequence encoded a protein consisting of 459 aa (49,663 Da) possessing high identity to a variety of TEF1-encoded proteins. A strong codon bias, similar to that observed in highly expressed yeast genes, was evident in A. pullulans TEF1. The ApTEF1 promoter region showed some sequence similarity to the well-studied TEF1 promoter from Saccharomyces cerevisiae, including a region from -23 to -11. This region exhibited high homology to a promoter upstream activating sequence (UAS) in S. cerevisiae. Such sequences have been shown to be essential promoter elements in genes coding for the highly expressed components of the yeast translation apparatus. PMID- 7557397 TI - Sequence of the met-10+ locus of Neurospora crassa: homology to a sequence of unknown function in Saccharomyces cerevisiae chromosome 8. AB - We have determined the sequence of the Neurospora crassa met-10+ gene and its flanking regions, and have isolated and analyzed cDNA clones for this region. We have identified two closely linked genes transcribed in the same orientation. The met-10+ gene is the downstream gene; an open reading frame (ORF) derived from five exons encodes a 475-amino-acid protein. The deduced protein lacks similarity to other characterized proteins. However, it exhibits a strong similarity to the product of an ORF of unknown function on Saccharomyces cerevisiae chromosome 8. This sequence similarity suggests functional equivalence and should facilitate identification of the function of met-10+ using gene disruptions in S. cerevisiae. PMID- 7557395 TI - In memoriam Alexander A. Bayev, 1904-1994. PMID- 7557398 TI - The Aspergillus niger (ficuum) aphA gene encodes a pH 6.0-optimum acid phosphatase. AB - We have used the Aspergillus niger (An) aphA gene as a probe and cloned the A. ficuum (Af) SRRC 265 gene encoding an extracellular pH 6.0-optimum acid phosphatase (APase6) from a genomic library. The identity of the Af aphA gene was confirmed and its nucleotide (nt) sequence verified by comparing its deduced amino acid (aa) sequence to that of purified Af APase6. A comparison of the nt sequences of the An and Af genes suggested that errors were made in the previously reported An aphA sequence. Several regions of the An aphA were resequenced and the mistakes corrected. With its nt sequence corrected, the An aphA is nearly identical to the cloned Af gene encoding APase6, and in 90.4% agreement in the coding regions. Both genes have three conserved introns and when translated, both nt sequences code for a polypeptide of 614 aa. There is now evidence that the two cloned genes are homologous and code for acid phosphatases that are 96% identical. PMID- 7557399 TI - Sequence and developmental regulation of the gene that encodes the Dictyostelium discoideum L3 ribosomal protein. AB - We have isolated and characterized genomic and cDNA recombinant plasmids that encode the Dictyostelium discoideum (Dd) ribosomal protein L3 (rpL3). Genomic plasmids were identified using a probe derived from the Saccharomyces cerevisiae TCM1 gene, that encodes the yeast rpL3. The DdL3 gene contains two introns and encodes a protein 398 amino acids in length that shows a high degree of homology to the conserved rpL3 protein of both lower and higher eukaryotes. During development, both the pattern of accumulation of DdL3 mRNA and changes in its translational activity are identical to those observed for other r-protein mRNAs. PMID- 7557400 TI - Cloning vectors for the production of proteins in Dictyostelium discoideum. AB - We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products. PMID- 7557402 TI - A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain. AB - The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles. PMID- 7557401 TI - Upstream promoter elements are sufficient for selenocysteine tRNA[Ser]Sec gene transcription and to determine the transcription start point. AB - The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA[Ser]Sec gene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3'-trailer sequence. Replacement of the tRNA[Ser]Sec coding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA[Ser]Sec promoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA[Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point (tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp. PMID- 7557405 TI - The 3'-untranslated regions from the Trypanosoma brucei phosphoglycerate kinase encoding genes mediate developmental regulation. AB - The phosphoglycerate kinase (PGK)-encoding genes of Trypanosoma brucei are transcribed in a polycistronic fashion, but the mRNAs encoding the three PGK isozymes show differing developmental regulation. We demonstrate here that the 3' untranslated regions of the major cytoplasmic and glycosomal PGK isozymes are capable of conferring the anticipated types of regulation on a transfected reporter gene. PMID- 7557403 TI - A gene encoding gamma-adaptin is required for apical extension growth in Ustilago maydis. AB - The nucleotide (nt) sequence of an Ustilago maydis (Um) gene that complemented a partially osmotic-remedial temperature-sensitive (or-ts) mutant defective in apical extension growth has been determined. It contained a continuous open reading frame (OFR) predicted to encode a protein of 853 amino acids (aa). The deduced aa sequence was homologous to gamma-adaptin, a component of clathrin coated vesicles derived from the Golgi. PMID- 7557404 TI - A family of trypanosome cdc2-related protein kinases. AB - The cyclin-dependent kinases, most notable of which is cdc2, are key regulators of the cell cycle, and are highly conserved in evolution. We have cloned and analysed three cdc2-related kinase-encoding genes (tbcrk1-3) from the protozoan parasite Trypanosoma brucei. tbcrk1 encodes a 34-kDa protein with 54% amino acid (aa) identity to the human cdc2, tbcrk2 a 39-kDa protein with 49% identity and tbcrk3 a 35-kDa protein with 54% identity. tbcrk1-3 have substitutions in the 16 aa sequence, the 'PSTAIRE' domain, that characterises the cdc2-related kinase family, to give PCTAIRE, PSTAVRE and PQTALRE motifs, respectively. The three kinases have conserved Tyr and Thr residues that are sites of phosphorylation in cdc2 and are important for regulating kinase activity. Southern blot analysis revealed that each tbcrk is a single copy gene. Pulse-field electrophoresis located the tbcrk genes to some of the largest of the trypanosome chromosomes at greater than 3 Mb. Western blots with anti-PSTAIRE polyclonal antibody detected proteins of 32, 43 and 65 kDa in all life-cycle stages and a 90-kDa protein in bloodstreams forms, implying the presence of a family of cdc2-related kinases. Trypanosomes have a remarkably large gene family of cdc2-related kinases for such a primitive organism. The crk genes may be involved in controlling aspects of the cell cycle which are linked to the differentiation of the parasite during its complex life cycle. PMID- 7557406 TI - Construction of a series of pACYC-derived plasmid vectors. AB - A series of cloning vectors has been constructed from pACYC177 and pACYC184. These vectors contain a MCS, the lacZ alpha reporter gene, and P15A ori. Plasmids pMBL18, pMBL19 and pMBLcos express the bla gene encoding ampicillin resistance, while pMCL200 and pMCL210 contain the cat gene encoding chloramphenicol resistance. In addition, the cosmid pMBLcos was constructed with the P15A ori. These vectors are useful for cloning or subcloning genes which are unstable and/or toxic in high-copy-number vectors. PMID- 7557407 TI - New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers. AB - Recently, we described pKIL18/19 positive-selection cloning vectors which rely on the inactivation of the cytotoxic ccdB gene [Bernard et al., Gene 148 (1994) 71 74]. They are new tools for simplifying gene cloning/sequencing procedures, as only recombinant DNA allow the formation of viable colonies. To enhance positive selection capabilities, putative translational reinitiation codons located within the pKIL18/19 ccdB were modified by site-directed mutagenesis. New pKIL-derived vectors with kanamycin (KmR) or chloramphenicol (CmR) resistance-encoding genes were also constructed. In addition, a new host derived from the DH2 strain was developed. PMID- 7557408 TI - The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12. AB - The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (sigma 70) subunit of RNA polymerase) from Escherichia coli C was determined. This gene differs from that of E. coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a sigma 70 subunit with three amino acid (aa) changes and a deletion of ten acidic aa. PMID- 7557409 TI - A cassette vector for protein engineering the lantibiotic nisin. AB - Expression vectors are described that make use of a plasmid-encoded nisA cassette encoding the peptide component of nisin. Specific mutations can readily be incorporated throughout the coding region of nisA. Using this vector in a nisA mutant host, three variant nisins, with dehydroalanine (Dha) residues changed to Ala residues, were generated. PMID- 7557410 TI - Cloning and sequence of the Bordetella bronchiseptica adenylate cyclase-hemolysin encoding gene: comparison with the Bordetella pertussis gene. AB - The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule. PMID- 7557411 TI - Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis. AB - Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC. PMID- 7557412 TI - Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity. AB - Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102- >Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage. PMID- 7557413 TI - New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. AB - A set of antibiotic-resistance and promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions was constructed. The cassettes contain the aacC1 gene of transposon Tn1696 conferring resistance to gentamicin in a large variety of Gram- and Gram+ bacteria. In addition to the antibiotic-resistance gene, a promoterless Escherichia coli lacZ gene was included in the cassettes, allowing the determination of the transcriptional activity at the insertion site. The cassettes can be excised from a plasmid mediating ampicillin resistance by many commonly used restriction enzymes. The new constructs have been successfully used for mutagenesis and studies of gene transcription in Rhizobium meliloti. PMID- 7557414 TI - Genetic analysis of the terminal 8-bp inverted repeats of transposon Tn7. AB - Mutations in the terminal 8-bp (5'-T1G2T3G4G5G6C7G8-3') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C2 at both ends of Tn7, no transposition is detected. C6 mutations have no effect on transposition frequency. Replacement with 5'-A3C4G5C6G7C8-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-end mutants, C2 and C6, sometimes yielded a 6-bp target duplication. This observation implies that cleavage of the target site might also involve interaction with the donor ends which, when mutant, relax the specificity for target-site cleavage. PMID- 7557415 TI - Molecular characterization of the Azotobacter vinelandii recF gene. AB - The recF gene from Azotobacter vinelandii (Av) has been cloned by complementation in an Escherichia coli (Ec) recF mutant. The sequence of 1568 bp has been determined and analyzed. It showed an open reading frame of 1092 nt coding for a 364-amino-acid (aa) polypeptide. The comparison of the deduced aa sequence of the recF of Av with those of other bacteria has elicited the presence of the four conserved domains thought to be essential for RecF function. A transcriptional fusion of a DNA fragment containing the promoter sequence of recF with the lacZ gene of Ec was constructed and 3-4-fold enhancement of promoter activity was observed upon UV induction. PMID- 7557418 TI - Construction of a Streptococcus pyogenes recA mutant via insertional inactivation, and cloning and sequencing of the complete recA gene. AB - To facilitate future genetic studies with Streptococcus pyogenes (Sp), a recA mutant (Rec11) was constructed using a streptococcal integration vector carrying a PCR-derived internal recA fragment. The insertion of the plasmid in the mutant chromosome was identified by Southern hybridization. Resistance to UV and the ability to accept linear DNA transformation by Rec11 were greatly decreased, confirming its RecA phenotype. Using the PCR-derived fragment as a probe, we cloned and sequenced the complete Sp recA gene, which is highly homologous to the recA of S. pneumoniae and Lactococcus lactis. PMID- 7557417 TI - Identification of a gene encoding a bacteriophage-related integrase in a vap region of the Dichelobacter nodosus genome. AB - Dichelobacter nodosus is the principal causative agent of ovine footrot. Nucleotide (nt) sequences from the D. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [Katz et al., J. Bacteriol. 176 (1994) 2663 2669]. In the present study, the limits of the virulence-associated (va) DNA around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to probe genomic DNA from the D. nodosus virulent strain A198 and the benign strain C305. This va region was found to be approx. 11.9 kb in length, and to be interrupted by a short DNA segment which is also found in the benign D. nodosus strain. Sequence analysis of the entire region revealed an ORF, intA, which is very similar to the integrases of bacteriophages phi R73, P4 and Sf6. Bacteriophages phi R73 and P4 integrate into the 3' ends of tRNA genes, with the integrase genes adjacent to the tRNA genes. A similar arrangement was found in the D. nodosus va region. A 19-bp nt sequence was found to be repeated at the ends of the va region, and may represent the bacteriphage attachment site. These findings suggest that D. nodosus may have acquired these DNA sequences by the integration of a bacteriophage, or an integrative plasmid that contains a bacteriophage-related integrase gene. The high similarity of the D. nodosus integrase to integrases from coliphages suggests that these va sequences may be transferred between distantly related bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557416 TI - Protection from proteolysis using a T4::T7-RNAP phage expression-packaging processing system. AB - DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta-galactosidase) triple fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558, an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20 kDa protein is produced intact.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557421 TI - Nucleotide sequence analysis of the 23S ribosomal RNA-encoding gene of Bartonella bacilliformis. AB - We report the cloning and characterization of the 23S ribosomal RNA (rRNA) encoding gene (rDNA) of Bartonella bacilliformis (Bb). The 2821-bp gene is preceded by an 11-nucleotide (nt) inverted repeat (IR) located 81 nt upstream in the tRNA(Ala)-23S rDNA intergenic spacer. The gene is followed by an 8-nt IR, five nt downstream in the 23S-5S rDNA intergenic spacer. The nt sequence of the Bb 23S rDNA is most similar to the 23S rDNA of Rhodopseudomonas palustris (Rp), with 85.4% sequence identity. The Bb 23S rDNA has 77.8% identity to the same gene from Escherichia coli (Ec). Secondary structure predictions indicate that the large subunit (LSU) Bb rRNA contains two smaller stem-loops at nt 1459-1544 and 1658-1685, as compared to the corresponding loops from Ec (nt 1405-1597 and 1707 1751, respectively). In addition, the Bb 23S rRNA has a large 72-nt stem-loop at nt 130-201, as compared to the Ec 18-nt stem-loop (nt 131-148). There is no Bb homologue of the 15-nt stem-loop at the 3' end of the Ec molecule (nt 2791-2805). The Bb 23S rRNA lacks the large stem-loop present in the LSU rRNA of closely related Rhodobacter sphaeroides (Rs) (nt position 1225-1331) which is thought to be involved in cleavage of 23S RNA precursor molecules into 16S and 14S rRNA species. This is the first LSU rDNA nt sequence for any member of the Bartonellaceae family. PMID- 7557419 TI - Sites for co-integration of large staphylococcal plasmids. AB - Site-specific recombination is thought to play an important role in the evolution of multi-resistant plasmids in bacteria, including the human pathogen Staphylococcus aureus (Sa). A mechanism for site- and orientation-specific recombination between large Sa plasmids was identified in Sa strain 1054. A replication-thermosensitive derivative of plasmid pI9789::Tn552 (called pS1) was found to form stable co-integrates with the large plasmid pOX1054 in the Sa strain 1054. Two closely related recombination sites were identified on these plasmids at which recombination occurred to form co-integrates. The sites (rs9789 from plasmid pI9789::Tn552 and rs1054 from pOX1054) were cloned and studies with them showed that the recombination at these sites occurs by a new method. The site rs1054 (27 bp) is deleted and rs9789 (26 bp) is duplicated during recombination. The data show that plasmid pS1 contributes the site for recombination and that the gene(s) encoding the protein(s) involved in recombination are encoded on either pOX1054 or the 1054 chromosome. PMID- 7557422 TI - The NAM9-1 suppressor mutation in a nuclear gene encoding ribosomal mitochondrial protein of Saccharomyces cerevisiae. AB - The nuclear gene NAM9 from Saccharomyces cerevisiae (Sc) codes for a protein which, on the basis of sequence homology, was previously postulated to be a mitochondrial (mt) equivalent of the Escherichia coli (Ec) S4 ribosomal protein (r-protein) [Boguta et al., Mol. Cell. Biol. 12 (1992) 402-412]. The mt-r character of the NAM9 product is now confirmed by cross-reaction with the antisera for the Sc mt r-proteins. The NAM9-1 mutation, characterized previously as the nuclear suppressor of some ochre mt mit- mutants, is found to be a single nucleotide substitution changing Ser82 to Leu within the part of NAM9 corresponding to the S4 region involved in interaction with the 16S rRNA. This indicates that the mechanism of NAM9-1 suppression could be analogous to the suppression due to ram (ribosomal ambiguity) mutations in the Ec structural gene encoding r-protein S4. The NAM9-1 mutation leads also to defect in respiratory growth in the background of the wild-type mit+ genome. PMID- 7557424 TI - Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis. AB - The identification of a xylose reductase (XR)-encoding gene (XYL1) from the xylose-assimilating yeast Kluyveromyces lactis (Kl) is described. XYL1 was isolated as a highly expressed fusion clone from a 'lacZ translational fusion library. DNA sequence analysis revealed an open reading frame (ORF) of 987 bp capable of encoding a polypeptide of 329 amino acids (aa). The deduced aa sequence displays a 62% overall identity to that of XR from Pichia stipitis. Gene disruption studies indicate that XYL1 exists as a single copy in the yeast genome and is essential for growth on xylose. Northern blot analysis of the XYL1 transcript and measurement of the XR enzymatic activities show, in contrast to other known XR-encoding genes, a constitutive expression of Kl XYL1. PMID- 7557423 TI - Cloning and characterization of the Saccharomyces cerevisiae LYS7 gene: evidence for function outside of lysine biosynthesis. AB - The Saccharomyces cerevisiae LYS7 gene has been cloned based on its genetic map position and complementation of a lys7 mutant. The 1453-bp sequence contains an open reading frame (ORF) that predicts a unique 249 amino acid (aa) protein. A Northern blot experiment demonstrated that LYS7 transcription was not regulated by lysine-specific or general aa control mechanisms. To investigate the effects of total loss of LYS7 function, we created a complete deletion of the gene and introduced this allele into wild-type yeast. The lys7 delta mutant requires lysine and simultaneously displays an array of phenotypes that include pH and temperature sensitivity. The pleiotropic phenotypes of the lys7 delta mutant and the constitutive transcription pattern are at odds with the hypothesis that Lys7p functions solely in the lysine biosynthesis pathway. PMID- 7557425 TI - Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica. AB - NAD-dependent formate dehydrogenase (FDH) was isolated from Candida methylica (Cm) grown on 0.5% methanol. Its N-terminal amino acid (aa) sequence was determined, as was that of a commercial FDH from Candida boidinii. Degenerate oligodeoxyribonucleotides were made to the 5' region of the fdh gene from Cm using this information and to the 3' region using C-terminal aa sequence data from the methylotropic yeast, Hansenula polymorpha. An almost complete 1.1-kb fragment was amplified from Cm genomic DNA via the polymerase chain reaction (PCR). This fragment was cloned, sequenced and used to probe a Southern blot, from which a 3.4-kb EcoRI fragment containing the fdh open reading frame (ORF) was isolated. The complete nucleotide sequence of this fdh ORF was determined and corresponds to a protein of 364 aa (40,343 Da). The ORF of fdh was cloned into pKK223-3 using PCR and transformed into Escherichia coli. Enzymatically active FDH was produced to 15% of soluble E. coli protein. The deduced aa sequence of this FDH is compared to the aa sequences of four known FDH, from bacteria, yeast, fungi and plant mitochondria. PMID- 7557426 TI - Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain). AB - Dengue virus type 2 (DEN-2), a member of the Flaviviridae family, has a positive strand RNA genome, 10,723 nucleotides (nt) in length and encoding a single polyprotein precursor consisting of 3391 amino acids (aa). In order to construct a full-length cDNA clone, the viral genome was cloned into 5' (nt 1-2203 under the control of the T7 promoter (pT7)) and 3' (nt 2203-10,723) constructs. A full length DEN-2 cDNA under pT7 control was assembled in vitro after excising the two cDNA inserts from the 5' and 3' constructs, and joining them with T4 DNA ligase. The RNA produced by in vitro transcription of the cDNA using T7 RNA polymerase was infectious, as shown by transfection of permissive BHK-21 and Vero cells, and propagation of the virus particles released into the culture media. The virus particles stably maintained the conservative mutation introduced into the 5' construct, and the cells infected with the infectious RNA-derived virus synthesized virus-specific DEN-2 antigens, as shown by immunofluorescence and immunoprecipitations. The full-length infectious clone for DEN-2 should be useful for the study of molecular mechanisms involved in viral RNA replication and virus assembly. PMID- 7557420 TI - Regulation of expression of the valine (branched-chain amino acid) dehydrogenase encoding gene from Streptomyces coelicolor. AB - Expression of the Streptomyces coelicolor (Sc) valine (branched-chain amino acid) dehydrogenase-encoding gene (vdh) is regulated by valine, glucose and NH+4 at the transcriptional level. The results of assays for the level of accumulated vdh mRNA in the Sc J802 strain by primer extension experiments and for the level of catechol dioxygenase (XylE) activity in Sc J802 (vdh::xylE) transformants show that transcription of the vdh gene is induced approx. 2.5-fold by valine, as compared to asparagine as the sole nitrogen source in the presence of glucose as carbon source. Valine induction is repressed by glucose, as compared to glycerol as the carbon source, and by NH+4. Glucose catabolite repression is relieved in Sc M480, a glucose kinase (glkA) deletion mutant. This suggests that glucose repression of vdh and carbohydrate metabolism are due to the same mechanism in Sc, which involves glucose kinase. PMID- 7557427 TI - Characterization of a putative molluscan insulin-related peptide receptor. AB - In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor. PMID- 7557428 TI - The chicken beta A4- and beta B1-crystallin-encoding genes are tightly linked. AB - Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4 crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1 cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1 cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens. PMID- 7557429 TI - AP-4- and AP-5-like proteins from mouse L cells are trans-activators and bind to the GT-II region of SV40 early TRE in a mutually exclusive manner. AB - Plasmids containing the cat reporter gene, transcription of which is directed by deletion mutants of the SV40 early region transcriptional regulatory element (SV40E TRE), were transfected into mouse L cells to determine the DNA motifs of SV40E TRE responsible for maximal gene expression. One deletion mutant, pSVE305, demonstrated a 50% reduction in CAT activity as compared to pSVE338, suggesting the importance of these 33 bp in directing efficient gene expression in mouse L cells. Introduction of triplet point mutations in this region and subsequent transfection studies in mouse L cells revealed three sites which were responsible for the reduction of CAT activity. These three mutations were located in the middle of the binding sites of three trans-activators: AP-3, AP-4 and AP-5. While the levels of CAT activity directed by SV40E TRE deletion mutants were similar in both HeLa and mouse L cells, the profiles of point mutants were different, suggesting that the activating ability of each nuclear factor is different from that of its counterpart in these two cell lines. Electrophoretic mobility shift assays (EMSA) demonstrated that binding of AP-4- and AP-5-like proteins of mouse L and HeLa cells to the GT-II motif occurs in a mutually exclusive manner. Furthermore, we observed a 'reverse competition' binding phenomenon which suggested a unique relationship between AP-4- and AP-5-like proteins of mouse L cells to the GT-II motif. Proteolytic mobility-shift analyses showed that an AP-5 like protein was more resistant to proteolytic digestion than an AP-4-like protein of mouse L cells. PMID- 7557430 TI - cis-regulatory elements within the proximal promoter of the rat gene encoding corticosteroid-binding globulin. AB - Corticosteroid-binding globulin (CBG) transports and modulates the bioavailability of glucocorticoids in blood plasma. It is produced predominantly by the liver, but is also produced in a complex spatial and temporal pattern during development and is regulated hormonally. The rat Cbg promoter (pCbg) has therefore been cloned to allow identification of cis-acting sequence elements that could contribute to its regulation. Five protein-binding sites (P1 to P5) were identified within 236 bp immediately 5' of the transcription start point by DNase I footprinting with rat liver nuclear extracts. These P1-P5 sites are highly conserved in the human pCbg, and resemble recognition sequences for HNF-1, CP-2, DBP, HNF-3 and C/EBP or NF-1L6, respectively. Electrophoretic mobility shift assays indicted that the P1 element most likely binds HNF-1, and transient transfection assays with luciferase reporter plasmids demonstrated that P1-P5 represent a positive component of rat pCbg activity, whereas additional 5' sequences repressed promoter activity 2-4-fold in H4IIEC3 rat hepatoblastoma cells. PMID- 7557431 TI - The chimpanzee alpha-fetoprotein-encoding gene shows structural similarity to that of gorilla but distinct differences from that of human. AB - The chimpanzee (Pan troglodytes) alpha-fetoprotein (AFP)-encoding gene (AFP) spans 18,867 bp from the transcription start point to the polyadenylation site, and the nucleotide (nt) sequence reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of AFP. In addition, we report 3121 bp of 5'-flanking sequence and 4886 bp of 3'-flanking sequence. The entire 26,874 bp of contiguous DNA reported here was determined from three overlapping lambda phage clones. The deduced polypeptide chain is composed of a 19-amino-acid (aa) putative leader peptide, followed by 590 aa of the mature protein. The sequence of chimpanzee AFP was compared with those of the previously published human AFP [Gibbs et al., Biochemistry 26 (1987) 1332-1343] and gorilla AFP [Ryan et al., Genomics 9 (1991) 60-72]. At the aa level, the human AFP differs from the chimpanzee at 6 aa positions and from the gorilla at 4 aa positions; the chimpanzee and gorilla differ at 8 aa positions. There are four types of repetitive sequence elements (X, Alu, Xba and Kpn) in the introns and flanking regions of chimpanzee AFP, and they are located in orthologous positions in the human and gorilla AFP. However, one specific Alu and one Xba repeat in introns 4 and 7, respectively, found in human AFP, are absent from orthologous positions in chimpanzee and gorilla AFP. These two repeats represent human specific novelties that arose from recent DNA transpositions in primate phylogeny. PMID- 7557432 TI - Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli. AB - A papaya cystatin (Cst)-encoding cDNA clone was isolated from a papaya leaf cDNA library and the active protein produced in Escherichia coli. The amino-acid sequence reveals a protein of 11,262 Da with over 40% identity to other published plant Cst. Unique features of the papaya Cst include a single Cys residue, variation in the papain-binding region, and the first reported inhibition of papaya proteinase IV by a Cst. PMID- 7557433 TI - The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins. AB - A novel baculovirus expression vector (pBSV-8His) directs secretion of recombinant proteins into the culture medium of infected insect cells. By providing a vector-encoded signal peptide upstream from a multiple cloning site, the product of the inserted cDNA is directed to the secretory pathway. In addition, a C-terminal His-tag allows convenient purification of the native protein directly from the culture medium in less than 5 h. The His-tag can be cleaved off the purified protein by utilizing an enterokinase cleavage site located directly N-terminal to the His sequence. By insertion of a coding sequence representing the human complement regulatory factor H-like (FHL-1) plasma protein into pBSV-8His, a high level of protein synthesis was demonstrated (9 micrograms/ml). The high level of production and the ease with which native protein can be purified almost to homogeneity, makes pBSV-8His particularly suitable for protein synthesis and purification. The combination of a vector encoded signal peptide and a C-terminal His-tag allows production and direct purification of individual domains of secretory proteins in eukaryotic cells. This feature makes this novel vector extremely useful for structure function analyses of secretory proteins. PMID- 7557435 TI - Tools for the production and purification of full-length, N- or C-terminal 32P labeled protein, applied to HIV-1 Gag and Rev. AB - We have constructed two new vectors for the production of foreign proteins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce protein fused to glutathione S-transferase (GST) at the N- and C-termini, respectively, allowing one-step purification on glutathione-Sepharose. Furthermore, they carry the recognition sequence (RRASV) for the catalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the terminus distal to the GST tag, enabling specific 32P labeling in vitro. By positioning the GST and HMK sequences at opposite ends of the introduced gene, only full-length fusion protein becomes radiolabeled after purification. Avoiding the labeling of shorter fusion protein species, often observed in bacterial expression of foreign genes, is particularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology. PMID- 7557434 TI - A splice donor site mutation results in the insertion of five extra amino acids into P53 from SEWA mouse sarcoma cells. AB - The status of the p53 gene in SEWA-AS12-ADH (S-ADH) cells, a subline of the mouse sarcoma cell line SEWA, was examined. Immunoprecipitation with wild-type (wt) or mutant P53-specific monoclonal antibodies (mAb) showed that both wt and mutant P53 were produced. Sequence analysis of the p53 cDNA and genomic DNA revealed a single nucleotide (nt) substitution at a splice donor site at the beginning of intron 7. As a result of this mutation, an alternative splice site 15 nt further 3' in intron 7 is used. The P53 protein translated from this aberrantly spliced mRNA carries an Arg258-->Ser substitution, followed by an insertion of 5 extra amino acids. This is the first example of a splice-site mutation in the mouse p53 gene. PMID- 7557436 TI - Stress- and sequence-dependent release into the culture medium of HIV-1 Nef produced in Saccharomyces cerevisiae. AB - We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress. By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef. Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures. This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site. Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS. If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers. PMID- 7557437 TI - The Drosophila melanogaster homologue of the human BBC1 gene is highly expressed during embryogenesis. AB - We have previously reported the isolation and preliminary characterisation of a full-length cDNA sequence derived from the human BBC1 gene, a gene which displays differential expression in tumours of the female breast [Adams et al., Hum. Mol. Genet. 1 (1992) 91-96]. Here, we report the isolation and characterisation of the Drosophila melanogaster (Dm) homologue of this human gene. The Dmbbc1 cDNA is 62% identical to the human BBC1 cDNA within a conserved open reading frame and the encoded proteins share 74% sequence similarity. The Dmbbc1 mRNA is expressed at all stages of Dm development, with the highest levels of expression occurring during embryogenesis. In addition, the Dm and human BBC1 proteins share remarkable degrees of identity with the products of recently isolated plant and avian bbc1 cDNAs. The sequences of all the predicted BBC1 proteins are highly similar to that of the rat ribosomal subunit protein L13 [Olvera and Wool, Biochem. Biophys. Res. Commun. 201 (1994) 102-107], strongly indicating that the BBC1 protein is ribosomal protein L13. PMID- 7557438 TI - Identification and expression of a homologue of the murine HoxA5 gene in the Mexican axolotl (Ambystoma mexicanum). AB - An excellent model for studying heart development in vertebrates is the cardiac non-function lethal mutant (gene c) Mexican axolotl, Ambystoma mexicanum. In order to facilitate our analyses of the mutant system, we have undertaken a search for stage-specific molecular markers during embryonic development of the axolotl. We have concentrated on homeobox genes as suitable candidates for monitoring molecular changes during development. A 270-bp probe encoding a portion of the axolotl homeobox gene Ahox-1 was generated by PCR from a stage-18 axolotl embryonic cDNA library. 32P-labelled PCR-amplified Ahox-1 DNA was used as the probe for screening a lambda AM18 cDNA library using moderately stringent conditions. We isolated six clones and determined their partial nucleotide (nt) sequences. One of the clones, which has very high homology to human, mouse and rat Hox A5 (83 and 99% at the nt and amino-acid levels, respectively, in the homeodomain region), was analyzed further. RT-PCR analyses show that the level of expression of HoxA5 is very low at stage 11 of embryonic development (gastrula). The level of expression reaches maximum at stage 25 (tailbud) and then plateaus at stages 30 and 35 (heartbeat onset). Although the expression of Ahox-1 was also found to start at stage 11, it reaches a maximum level at stage 25 and declines at stage 35. We have also studied, using RT-PCR, the tissue-specific expression of HoxA5 and Ahox-1 in juvenile axolotl. PMID- 7557439 TI - Cloning of the barramundi growth hormone-encoding gene: a comparative analysis of higher and lower vertebrate GH genes. AB - In this work the growth hormone-encoding gene (GH) from the fish barramundi (Lates calcarifer) was characterized by nucleotide (nt) sequence analysis and comparative studies on higher and lower vertebrate GH were performed. The barramundi GH contains six exons and five introns. A putative transcription start point 21 nt downstream from a potential TATAAA box was determined, with a modified primary transcript size of 1615 nt predicted. cis-Acting elements potentially important for transcriptional regulation at the basal, hormone responsive and pituitary gland-specific levels were identified. Several microsatellite and minisatellite repetitive sequences were shown to be present within noncoding portions of this gene. Repeat sequences similar to the deca- and undeca-minisatellites of the barramundi GH were observed in the corresponding introns of the tilapia, but not other teleost GH. Comparative studies on the Osteichthyes, Mammalia and Aves vertebrate class GH promoters suggested that the TATAAA box was the only conserved region between these sequences. Conserved sequences, however, were identified within the GH promoters of different species from the Osteichthyes or Mammalia classes. The Osteichthyes A + T-rich sequence (consensus GATRMATYWAAWCA, where R = A or G; M = A or C, Y = C or T and W = A or T) is the only conserved region identified between teleost GH promoters, and is most likely involved in the pituitary gland-specific expression of these genes. PMID- 7557440 TI - The cDNA cloning and transient expression of a chicken gene encoding a 3 beta hydroxysteroid dehydrogenase/delta 5-->4 isomerase unique to major steroidogenic tissues. AB - Two cDNA clones, on containing a 2.7-kb and the other a 1.6-kb insert, were obtained from a chicken adrenal gland cDNA library by screening with a partial chicken cDNA fragment generated by PCR. Primers were used that corresponded to conserved regions among several published cDNA sequences of mammalian 3 beta hydroxysteroid dehydrogenase/delta 5-->4 isomerase (3 beta-HSD). Both clones contained the same open reading frame (ORF) encoding 377 amino acids (aa). The difference in their sizes was due to the use of different polyadenylation sites. The deduced aa sequence showed 54-57% overall identity with those of the human, macaque, bovine, mouse, rat and rainbow trout 3 beta-HSD. A 9-aa stretch (FYYISDDTP) is conserved among the chicken, mammalian and rainbow trout 3 beta HSD, suggesting that it is essential for enzymatic activity. Northern blot hybridization demonstrated the presence of two types of mRNA, corresponding to the above two cDNA classes, in the adrenal gland, ovary and testis. Transient expression of the chicken ORF in COS-7 cells demonstrated coupled dehydrogenase and isomerase activities of 3 beta-HSD on three different substrates; pregnenolone, 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Among the Km values determined for the three substrates, that for dehydroepiandrosterone was the lowest, which was different from the case for human 3 beta-HSD. PMID- 7557441 TI - Identification of DNA recognition sequences for the Pax3 paired domain. AB - The Pax gene family, encoding transcription factors, has been classified into four subfamilies according to their genomic organization, the sequences of the paired domains (PD) and the expression pattern. Pax1 and Pax9 constitute one subfamily, Pax2, Pax5 and Pax8 another, Pax3 and Pax7 another one and Pax4 and Pax6 the fourth subfamily. The PD exhibits DNA-binding activity, and is the most conserved functional motif in all Pax proteins. A high-resolution analysis of a PD structure has been performed [Xu et al., Cell 80 (1995) 639-650] and the DNA binding characteristics of members of two Pax subfamilies (Pax2, Pax5 and Pax6) have been determined. Here, we have utilized a PCR-based selection approach to identify the DNA-binding sequences of the Pax3/PD, a member of a subfamily which has not yet been characterized. Comparison of the Pax3/PD-binding sequences with those of other PD proteins revealed both similarities and differences in the DNA recognition sequence. This suggests that different Pax proteins can regulate the expression of the same target gene, but they can also regulate the expression of completely unrelated genes by binding to their DNA regulatory regions. PMID- 7557442 TI - Molecular characterization of a high-affinity mouse glutamate transporter. AB - The complete coding sequence of a mouse glutamate transporter (mEAAT2) has been cloned by polymerase chain reaction (PCR) from adult whole-brain total RNA. Southern hybridization analysis of PCR products amplified from templates derived from various murine adult tissues demonstrated that the transcript for mEAAT2 was specific to the central nervous system. High-affinity transport of D-aspartate, Km value (17 +/- 5 microM), was determined in a vaccinia/T7 RNA polymerase expression system. The deduced amino-acid sequence of mEAAT2 shares 96 and 93% identity with the rat and human EAAT2 homologues, respectively. PMID- 7557443 TI - Sequence of the promoter region of the mouse gene encoding ornithine aminotransferase. AB - We have isolated and sequenced the promoter region of the mouse gene (mOAT) encoding ornithine aminotransferase. A comparison of these mOAT sequences with previously reported sequences for the rat and human genes encoding OAT, rOAT and hOAT, respectively, revealed a 256-bp region flanking the transcription start point that is highly conserved between the three genes. This region contains sequence motifs resembling binding sites for general transcription factors, as well as other trans-acting regulatory proteins. PMID- 7557444 TI - Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase. AB - A novel cDNA encoding PTP (protein tyrosine phosphatase) was cloned from PC12h cells and designated as PCPTP1 (gene encoding PC12 protein Tyr phosphatase). The longest open reading frame (ORF) of this clone encodes a 656-amino-acid (aa) protein with a single PTP catalytic domain. Western blot analysis using a polyclonal Ab (antibody) raised against the cytoplasmic region of PCPTP1 detected two products, a major 65-kDa and minor 42-kDa protein, designated PCPTP1-MFI and PCPTP1-MVQ, respectively, in PC12h cells. These two proteins correspond to the products translated from the second and fifth methionine of PCPTP1, respectively. The bacterially expressed GST::PCPTP1-MVQ fusion protein had phosphatase activity with pNPP (p-nitrophenyl phosphate) as a substrate. Alignment of the aa sequence of PCPTP1-MVQ with those of other PTP showed the highest similarity to STEP and LC-PTP/HePTP, with 54 and 51% identity, respectively. Northern blot analysis showed only one 3.9-kb transcript in PC12h cells, indicating that PCPTP1 corresponds to this 3.9-kb transcript. The 3.9-kb PCPTP1 mRNA was detected in the brain and adrenal gland, but not in other non-neuronal tissues in adult rats. Two other transcripts of 3.3 and 1.7 kb were also detected in brain. NGF (nerve growth factor) and glucocorticoid are known to bimodally regulate the cell fate decision of sympathoadrenal precursors like PC12 cells, with NGF promoting the neuronal phenotype and glucocorticoid promoting the chromaffin phenotype. Still, both agents decreased the level of PCPTP1 mRNA in PC12h cells. Therefore, it is likely that the decrease in the level of PCPTP1 mRNA might be associated or correlated with cell differentiation in PC12h cells. PMID- 7557446 TI - Various transcripts are generated from the VCSA1 gene by alternative splicing and poly(A) processing in the rat submandibular gland. AB - The members of the VCS (variable coding sequence) multigene family display extensive evolutionary divergence in the protein-coding region. The first described gene (VCSA1) was found to encode a major 0.7-kb mRNA (VCSA1*1T1) coding for a prohormone-like preproprotein, SMR1-VA1, in the submandibular gland (SMG) of Rattus norvegicus. We report here the cloning of four other VCSA1 cDNAs corresponding to mRNAs (VCSA1*1T2 to *1T5) expressed in the SMG. VCSA1*1T1 to *1T4 mRNAs share the three exons previously described and differ in their 3' untranslated regions (UTR). Their differences originate from the alternative utilization of four polyadenylation sites. Comparison of the tissue levels of VCSA1*1T1 and VCSA1*1T4 during post-natal development of the male rat SMG suggests that the poly(A) addition sites are both used at each stage. The fifth RNA transcript (VCSA1*1T5) contains only the first two exons. The nucleotide sequence of the cDNA reveals that VCSA1 has an additional exon (exon 4) which is spliced to exon 2 in VCSA1*1T5. In addition to VCSA1*1T1, at least VCSA1*1T4 and VCSA1*1T5 are actively translated in vivo, as revealed by their association to the polysomal fractions. The protein, P2-VA1, coded by VCSA1*1T5 is 68 amino acids in length and it is likely to be a glycosylated secretory protein. The putative mature P2-VA1 protein completely differs from the SMR1-VA1 pro-protein and very likely has a different function. VCSA1*1T1 is accumulated in the male rat SMG 200-1000-fold more than the other transcripts. Run-on experiments reveal that almost all transcription proceeds several hundred bp downstream from the poly(A) site corresponding to VCSA1*1T1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557447 TI - cDNA cloning and characterization of the transcriptional activities of the hamster peroxisome proliferator-activated receptor haPPAR gamma. AB - We have isolated a cDNA corresponding to the hamster peroxisome proliferator activated receptor haPPAR gamma, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR gamma mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR gamma may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal beta-oxidation. haPPAR gamma binds efficiently in the presence of retinoid X receptor alpha (RXR alpha) to a peroxisome proliferator response element (PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the rate limiting enzyme of peroxisomal beta-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR gamma/RXR alpha on the PPRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR alpha, we show that the transcriptional activities of mPPAR and haPPAR gamma are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR. PMID- 7557449 TI - Cloning of the bovine interleukin-3-encoding cDNA. AB - Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction was used to amplify an IL-3 cDNA from first-strand cDNAs prepared from RNA isolated from 4- and 5-hour concanavalin-A-stimulated peripheral blood lymphocytes (PBL) from N'Dama cattle. An analysis of the cDNA sequence reveals that it contains a 432 nucleotide (nt) open reading frame which codes for 144 amino acids (aa). Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine IL-3 sequence with the sheep, human and mouse IL-3 sequences show that the bovine sequence shares 90.7, 55.8 and 51.9% nt identity, respectively, in the coding region, and 85.4, 35 and 27.7% aa identity, respectively. PMID- 7557450 TI - Isolation and characterization of the functional gene encoding bovine cytochrome c oxidase subunit IV. AB - The structure and expression of the gene (COX4) encoding bovine cytochrome c oxidase subunit IV (COX IV) was studied in order to identify conserved DNA sequence elements involved in the control of mammalian nuclear respiratory genes. The functional bovine COX4 gene consists of five exons and four introns and is similar in organization to rat and mouse COX4. The domain encoded by exon 3 is the most highly conserved among the three species, suggesting it may encode a key functional domain of COX IV. Transcription of bovine COX4 begins at multiple sites, as has been seen previously for rat and mouse COX4 and other TATA-less genes. Comparative analysis of bovine, rat and mouse COX4 promoters identified multiple binding sites for the regulatory proteins Sp1 and GABP (NRF-2). The varied arrangements of multiple Sp1 and GABP sites in mammalian COX4 promoters suggests flexibility in the positioning of regulatory factors in controlling COX4 expression. PMID- 7557451 TI - Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+) dependent 15-hydroxyprostaglandin dehydrogenase. AB - We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15 hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis. PMID- 7557452 TI - Temporal and tissue-specific expression of the MET ORF driven by the complete transcriptional unit of human A1AT gene in transgenic mice. AB - We inserted the sequence coding for the cytoplasmic portion of the human MET receptor into an 18-kb genomic fragment containing the entire human A1AT gene (encoding alpha-1-antitrypsin). Stringent control of gene expression, at the transcriptional, post-transcriptional and translational levels, was ensured by insertion of the MET open reading frame into A1AT, thus maintaining: (i) all the elements that confer tissue-specific transcription initiation, (ii) all the sequences involved in transcript processing and (iii) all the sequences which influence messenger stability and translational efficiency. The expression pattern of this vector in transgenic mice was identical to that of the human A1AT transgene, as well as to that of A1AT in humans with regard to both temporal and tissue-specific regulation. PMID- 7557454 TI - The deduced amino-acid sequence of opsin from rabbit rod photoreceptors. AB - The amino acid (aa) sequence of rabbit opsin from rod photoreceptor cells was determined by direct aa sequencing and conceptual translation from the cDNA. The cDNA (1198 bp) containing the complete coding region encodes a 348-aa opsin protein. Of the 16 rod cell opsins that are known, rabbit opsin is most similar to human opsin (96.3% identity at the aa level). PMID- 7557448 TI - Cloning of the mink plasminogen activator inhibitor type-1 messenger RNA: an mRNA with a short half life. AB - In mink lung CCL64 epithelial cells the rate of synthesis of plasminogen activator inhibitor type I (PAI-1) increases 10-100-fold within 3 h in response to 12-O-tetradecanoyl phorbol-13-acetate (PMA). The PAI-1 gene is regulated transcriptionally. Parallel studies of the time-courses of PAI-1 synthesis and secretion and of mRNA accumulation indicate that the amount of secreted PAI-1 produced by the cells is tightly coupled to the level of its transcript. The half life of the PAI-1 mRNA was found to be 25 min which is much shorter than previously reported for PAI-1 in other cells. Actinomycin D, which is commonly used to determine mRNA half-life, stabilized the PAI-1 mRNA. Cycloheximide also stabilized the mRNA. The short half-life and the superinducibility of PAI mRNA are properties shared with rapidly degraded mRNAs encoding protooncoproteins. A 2.97-kb cDNA clone containing the entire coding sequence of PAI-1 was isolated from a cDNA library made from mink lung CCL64 epithelial cells stimulated with PMA. The PAI-1 cDNA contains a long 3'-untranslated region (UTR) of 1720 bp whose sequence is highly conserved among PAI-1 mRNAs from different species. The PAI-1 mRNA also contains several AUUUA pentamer sequences which are the features of an A+U-rich regulatory element such as is found on the fos protooncogene mRNA. Upstream of one of these AUUUA pentamers are several highly conserved sequences that are also found in the 3' UTR of the fos and integrin receptor alpha-subunit mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557453 TI - Expression of the Bombyx mori beta-tubulin-encoding gene in testis. AB - We have isolated a beta-tubulin-encoding cDNA clone of Bombyx mori from testes and determined the nucleotide sequence. Northern analyses showed that its expression is testis-specific and most active in the pupal stage. PMID- 7557445 TI - Interleukin-6 and ciliary neurotrophic factor trigger janus kinase activation and early gene response in rat hepatocytes. AB - Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) share a signal transducing molecule called gp130. Previous studies showed that CNTF regulates fibrinogen gene expression in rat hepatocytes by competitive binding to the IL-6 receptor. This report explores the post ligand-binding events in the control of fibrinogen and early response gene production stimulated by IL-6 and CNTF in primary rat hepatocytes. Metabolic labeling, using [32P]orthophosphate or anti phosphotyrosine antibody (Ab) blot experiments revealed that both IL-6 and CNTF induced tyrosine phosphorylation of gp130, and the Jak1 and Jak2 kinases in a dose- and time-dependent manner. Additional experiments revealed that only one of the early response genes, junb, but not c-myc or c-fos, was stimulated by the addition of either IL-6 or CNTF. These data suggest that activation of Jak kinases and stimulation of junb reflect a divergence of the IL-6/CNTF signaling pathway and further suggest that junb may participate in cytokine control of acute-phase protein production in the inflammatory response. PMID- 7557455 TI - An unexpected transforming gene in calf-thymus carrier DNA: bovine hst. AB - During a search for transforming genes by transfecting a human cDNA expression library together with calf thymus carrier-DNA into NIH/3T3 cells, we found a focus which was induced by a plasmid containing a sequence highly homologous to human HST (a transforming gene from Human STomach cancer). However, PCR analysis identified the source of this sequence as calf thymus DNA. The deduced amino acid (aa) sequence of bovine HST shows 91 and 81% identity to the human and mouse HST aa sequences, respectively. These data suggest that the hst of calf thymus carrier-DNA could induce transformation of NIH/3T3 cells. PMID- 7557456 TI - Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains. AB - Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work. PMID- 7557457 TI - A novel insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene. AB - A novel insertion sequence (IS)-like element was found in the 5'-upstream region of the alanine carrier protein-encoding gene (acp) in the thermophilic bacterium PS3 chromosomal DNA. The sequence contained an open reading frame (ORF) encoding a polypeptide of 369 amino acids which revealed high similarity with ORFs from IS891 from the cyanobacterium Anabaena and IS1136 from Saccharopolyspora erythraea. The direction of transcription was the same as that of acp, and typical inverted and direct repeats characteristic of IS were found in both the 5' and 3' region of the ORF. Southern hybridization analysis of the chromosomal DNA revealed that multiple copies of the ORF sequence were contained in the PS3 genome. This element might well be a member of a new IS family including IS891 and IS1136, and we have designated this element IS1341. The analysis of acp expression in Escherichia coli cells indicated that IS1341 promotes the expression of acp. PMID- 7557458 TI - Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease. AB - A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism, Thermus filiformis (Tf), using an indicator strain containing a dinD::lacZ fusion. The gene, designated nuc17, has been mapped within a 2300-bp fragment. The 55-kDa Tf nuclease was purified to over 95% homogeneity. Single stranded (ss) DNA is the preferred substrate for the Tf nuclease, although double stranded (ds) DNA can also be digested. Nuclease activity increases with increasing temperature up to 80 degrees C and requires the metal ions Ca++ or Mg++ for catalysis. Tf nuclease is primarily an endonuclease that leaves 5' phosphates in the digested products. The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the Tf nuclease, making it a useful reagent to generate unidirectional deletions. PMID- 7557459 TI - The citrate synthase-encoding gene of Rickettsia prowazekii is controlled by two promoters. AB - The transcripts of the citrate synthase-encoding gene (gltA) in Rickettsia prowazekii (Rp), an obligate intracellular parasitic bacterium, were analyzed by RNase protection (RP), primer extension (PE) and in vitro transcription assays. Analysis of the 5' end of the gltA mRNA by RP and PE assays revealed that there were two gltA mRNAs with the 5' ends located at 16 bp and 307 bp upstream from the gltA coding region. Since these two mRNAs might represent two species of mRNA transcribed from two different promoters or a single transcript that was processed to give two mRNAs, an in vitro transcription analysis with purified Rp RNA polymerase (RNAP) was performed to distinguish these two possibilities. Purified Rp RNAP catalyzed the formation of two transcripts initiated from the same nucleotides indicated by RP and PE. Sequence analysis identified Escherichia coli (Ec) promoter-like sequences immediately upstream from both transcription start points (tsp). The first promoter (promoter P1) had the core sequence TTCTAA N17-TATACT, was 6 bp upstream from the tsp (base A) and was centered at 37 bp upstream from the coding region. The second promoter (promoter P2) had the core sequence ATGAAA-N17-TAAAGT, was 7 bp upstream from the tsp (base T) and was centered at 329 bp upstream from the coding region. This is the first demonstration of multiple promoters in this obligate intracellular parasite which has implications concerning transcriptional regulation. PMID- 7557462 TI - Transcription of the region encoding the ferric dicitrate-transport system in Escherichia coli: similarity between promoters for fecA and for extracytoplasmic function sigma factors. AB - We have analyzed the molecular mechanism of regulation of the ferric dicitrate transport system in Escherichia coli (Ec), by studying the transcription of the regulatory and structural genes under various environmental conditions, and by determining the location of their transcriptional start points and promoter regions. We report here that the main species observed in Northern hybridization analyses were a 2.5-kb mRNA, encoded by the outer membrane protein receptor gene fecA, and a 1.5-kb mRNA encoded by a region including the fecIR genes. The synthesis of the 2.5-kb fecA mRNA is regulated by both citrate and iron. Furthermore, transcription of fecA is dependent on the presence of FecI. The promoter region for the fecA mRNA, a likely site of action for FecI, is not related to the consensus promoter region for sigma 70 RNA polymerase in Ec K-12. However, it shows greatest similarity with promoters of genes regulated by a new sub-family of sigma factors, i.e., the extracytoplasmic function (ECF) sigma factors, which are associated with the expression of genes involved in extracytoplasmic functions, suggesting that FecI may act as a specialized sigma factor. We also show that the fecB,C,D,E transport genes are linked in operon fashion to fecA. Since the levels of the fecB,C,D,E RNAs are extremely low, as compared to the level of fecA mRNA, it is likely that processing from the 3' end must occur and stop near the end of fecA where a hairpin structure is located. PMID- 7557461 TI - Cloning and characterization of three Aspergillus niger promoters. AB - An Aspergillus niger (An) genomic library was constructed using the promoter-trap vector, pLX2A, which contains a hygromycin B (Hy) phosphotransferase-encoding gene (hph) for selection of DNA fragments with promoter activity. This library was transformed in Escherichia coli and 80,000 colonies were obtained, 94% of which contained inserts. Transformations of plasmid DNA from the library into An resulted in 53 Hy-resistant (HyR) colonies. Southern blot analysis of 21 transformants confirmed the integration of hph into the An genome. Using the sib selection procedure, three functional promoters, PX6, PX18 and PX21, were identified from this library. Both DNA strands of all three fragments were sequenced and their sequences showed no significant homology to those in the database. Comparison of the sequences of all known promoters from An suggested that C+T-rich stretches are probably important for promoter structures. The promoter activity was analysed further using beta-galactosidase (beta Gal) as a quantitative marker. The results suggest that while PX21 is a much stronger promoter than the known alpha-amylase promoter of A. oryzae, PX6 promotes only weak expression of beta Gal. PMID- 7557460 TI - Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus. AB - O-methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap). Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Af strain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative S adenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region. PMID- 7557463 TI - Cloning and characterization of a G protein alpha-subunit-encoding gene from the basidiomycete, Coprinus congregatus. AB - Complementary DNA (cDNA) clones encoding two G protein alpha-subunit proteins (CGP alpha 1 and CGP alpha 2) were isolated from a Coprinus congregatus (Cc) hyphal tip cell (HTC) library using PCR-generated biotinylated G protein probes. Sequence analysis of the Cc cgp alpha 1 gene indicates that the gene contains an open reading frame (ORF) that translates into a putative 353-amino-acid (aa) product. The predicted CGP alpha 1 protein exhibits similarity to all known G protein alpha-subunits (it has all of the consensus regions for a GTP-binding protein), especially the mammalian retinal G protein, transducin. The CGP alpha 1 aa sequence is 50% identical overall to the transducin subfamily, cgp alpha 1 shares the same aa size grouping as transducin alpha-subunits and, unlike many other G proteins, both CGP alpha 1 and transducin seem to possess a cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive site. Preliminary reverse transcription PCR (RT-PCR) analysis of cgp alpha 1 and cgp alpha 2 mRNA expression revealed that, unlike cgp alpha 2 which seems to be constitutively expressed, cgp alpha 1 is expressed only in HTC that are competent in responding to light. Thus, the cgp alpha 1 product, CGP alpha 1, is a likely candidate for regulating the blue light-induced signal transduction photomorphogenesis system found in Cc. PMID- 7557464 TI - The fumR gene encoding fumarase in the filamentous fungus Rhizopus oryzae: cloning, structure and expression. AB - The filamentous fungus Rhizopus oryzae (Ro) is known for its ability to overproduce and accumulate high levels of fumaric acid (FA) under stress conditions. In order to study the molecular mechanisms involved in the increased biosynthesis of FA, the gene (designated fumR) encoding Ro fumarase was cloned and analysed for its structure and expression. Nucleotide (nt) sequence and comparison of the fumR product with fumarases from various sources established that fumR contains nine introns and encodes a deduced product of 494 amino acids (aa), related to class-II fumarases. A fumarase protein of 50 kDa was immuno detected in crude Ro extracts. Primer extension experiments mapped the 5' end of the fumR RNA 159 nt upstream from the putative translation start codon. Both primer extension and Northern analysis showed the existence of one transcript of fumR. The level of fumR RNA increased in cells producing FA under stress conditions (high carbon and low nitrogen levels in the medium), suggesting that transcriptional regulation of fumR might be involved in the overproduction and accumulation of FA by Ro cells under stress conditions. The possibility that additional mechanisms are responsible for this phenomenon is discussed. PMID- 7557465 TI - Chromosomal organisation of a repeated gene cluster expressed in mammalian stages of Leishmania. AB - The genomic organisation of a large Leishmania gene cluster, expressed predominantly in intracellular, infective parasite stages, has been determined. Using cosmid cloning, parasite DNA fingerprinting, partial digestion and mapping with 'end-specific' probes, the Lm cDNA2 gene array has been localised to a 55-kb ClaI fragment within the L. major genome. Six tandemly linked gene copies are transcribed to produce an abundant 6-kb transcript; the seventh and last copy of the cluster is truncated at its 3' end. It is likely that these genes encode one or more proteins specific to infective stages of the parasite life cycle. PMID- 7557466 TI - The gene encoding topoisomerase I from the human malaria parasite Plasmodium falciparum. AB - Part of the topoisomerase I (TopoI)-encoding gene from Plasmodium falciparum (Pf) was isolated by PCR from cDNA using oligodeoxyribonucleotides modelled on the highly conserved regions of sequence from other species. The entire TopoI gene was obtained by screening a Pf K1 HindIII-EcoRI genomic library in lambda NM1149 with a random-labeled heterologous probe from the Saccharomyces cerevisiae TopoI gene. DNA sequence analysis revealed an open reading frame of 2520 nt encoding a deduced protein of 839 amino acids (aa) with no detectable introns. The Pf TopoI aa sequence has about 40% identity with most eukaryotic TopoI homologues. The gene is located as a single copy on chromosome 5 and Northern analysis identified a transcript of 3.8 kb. PMID- 7557467 TI - Amino acids of the cholera toxin from Vibrio cholerae O37 strain S7 which differ from those of strain O1. PMID- 7557468 TI - Divergence of a flagellin protein in Serratia marcescens. AB - A gene (hag) encoding the flagellin (Fla) protein was cloned from Serratia marcescens (Sm) 8000, the wild-type strain of Sr41. The hag gene codes for a 348 amino-acid (aa) protein of 36.7 kDa. The predicted aa sequence showed 79% homology compared with the Fla of Sm 274 which has been reported previously [Harshey et al., Gene 79 (1989) 1-8]. Dot-matrix analysis of the Sm 8000 Fla showed that the N- and C-terminal regions of this protein were highly similar to those of other bacterial Fla. However, the aa sequence of the middle portion was quite different from that of a variant strain of the same species, Sm 274. PMID- 7557469 TI - Sequence of an ammonia monooxygenase subunit A-encoding gene from Nitrosospira sp. NpAV. AB - One of three gene copies encoding ammonia monooxygenase subunit A (AmoA) and flanking sequences was isolated from genomic DNA of the chemolithotrophic soil bacterium Nitrosospira sp. NpAV using oligodeoxyribonucleotide primers and the polymerase chain reaction (PCR). The gene (amoA) encodes a 274-amino-acid polypeptide which has similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea. PMID- 7557470 TI - Sequence and transcriptional analysis of the genes encoding the class-II topoisomerase of Mycoplasma gallisepticum. AB - The gyrAB genes encoding the entire B and a portion of the A subunit of DNA gyrase (E.C. 5.99.1.3) from Mycoplasma gallisepticum (Mg), strain S6, were cloned and sequenced. These gyrAB genes are co-transcribed as a single, polycistronic mRNA transcript. The Mg gyrB appears unique among prokaryotic gyrB in its use of GUG as a start codon. PMID- 7557471 TI - Cloning of a cDNA encoding a novel heat-shock protein from Dictyostelium discoideum. AB - We have cloned, from Dictyostelium discoideum, a cDNA encoding a new heat-shock (HS) protein (Hsp) with a predicted molecular mass of 31,447 Da. Outside of its low molecular mass, this Hsp does not share any similarity with the small Hsp currently identified or with alpha-crystallins. Northern blot analysis indicates that this HS-inducible gene is also developmentally regulated

. PMID- 7557472 TI - Recombinant enrichment by exploitation of the wobble bases in the recognition site of long-cutters: design, synthesis and incorporation of zero-background linkers in cloning and expression vectors. PMID- 7557473 TI - An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product. AB - To develop the budding yeast Pichia pastoris (Pp) as a model system for the study of protein secretion, we have characterized a secreted acid phosphatase (Pho1p) from this yeast. Pho1p can be induced 100-fold by starvation for phosphate. The enzyme was purified to homogeneity from a cell-wall extract by DEAE-Sepharose chromatography. We selected mutants that lacked extracellular phosphatase activity and the gene (PHO1) encoding Pho1p was isolated from a recombinant plasmid library of Pp DNA by complementation of the mutant defect. PHO1 encodes a protein of 468 amino acids (aa) with homology to repressible acid phosphatases from other yeast species. The sequence contains a 15-aa N-terminal signal sequence and six potential N-linked glycosylation sites. Antiserum to Pho1p was used to show that Pho1p transits the Pp secretory pathway in less than 5 min. PMID- 7557474 TI - Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity. AB - Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA. PMID- 7557475 TI - Construction of lacIts and lacIqts expression plasmids and evaluation of the thermosensitive lac repressor. AB - To characterize a thermosensitive lacIts mutant isolated by Bukrinsky et al. [Gene 70 (1989) 415-417] and to adapt it as a convenient control element, we have (i) mapped the mutation to the inducer-binding domain of the LacIts repressor, (ii) determined that the mutation resulted in the loss of a HaeIII site in lacIts due to a G-->A transition, leading to a Gly187-->Ser substitution, (iii) removed extraneous lacZop DNA downstream of lacI, and (iv) cloned lacIts (in plasmids based on ori of either ColE1 or P15A) under control of the wild-type or lacIq promoters. The LacIts repressor is insensitive to IPTG. The repression of lacZop by LacIts is very efficient at 30 degrees C and total induction was achieved at 42 degrees C, providing that the LacIts concentrations are not excessive and that repressor-to-operator ratios are not too high. PMID- 7557476 TI - A new set of useful cloning and expression vectors derived from pBlueScript. AB - A new set of cloning vectors derived from pBlueScript (Stratagene, La Jolla, CA, USA) is presented. The ampicillin-resistance-encoding gene (ApR) of pBlueScript has been replaced by genes encoding resistance to either kanamycin (KmR) or tetracycline (TcR). The origin of DNA replication (ori), conferring to pBlueScript a very high-copy-number (500-700 copies/chromosome), has been replaced by the pBR322 ori (15-20 copies/chromosome) or the P15A ori (10-12 copies/chromosome) [Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989]. Therefore, eight new vectors with different drug selection markers and low, medium or high plasmid copy-number were created which are compatible with each other (ColE1 ori and P15A ori) and can be selected to replace one another. These vectors were further modified by the insertion of an expression cassette based on the promoter and AraC repressor/activator of the ara operon, which allows high level expression, extremely tight regulation and very inexpensive induction. High level expression of one or two genes within the same cell is demonstrated. PMID- 7557477 TI - Site-directed mutagenesis of the HtrA (DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures. AB - The HtrA(DegP) 48-kDa serine protease of Escherichia coli is indispensable for bacterial survival at elevated temperatures. It contains the amino-acid sequence Gly208AnsSerGlyGlyAlaLeu, which is similar to the consensus sequence GlyAspSerGlyGlyProLys surrounding the active Ser residue of trypsin-like proteases. Mutational alteration of Ser210 eliminated proteolytic activity of HtrA. An identical effect was observed when His105 was mutated. The mutated HtrA were unable to suppress thermosensitivity of the htrA bacteria. These results suggest that Ser210 and His105 may be important elements of the catalytic domain and indicate that the proteolytic activity of HtrA is essential for the survival of cells at elevated temperatures. PMID- 7557478 TI - Secretion of streptokinase fusion proteins from Escherichia coli cells through the hemolysin transporter. AB - The hemolysin (HlyA) secretion system was used to achieve the sec-independent secretion of streptokinase (Skc) originating from Streptococcus equisimilis into the medium by Escherichia coli cells. The in-frame fusions of the skc gene, either possessing or lacking a region encoding the signal peptide (SP) with the 3'-end of the hlyA gene of various lengths were analysed. All hybrids retained Skc activity. Hybrid proteins devoided of the N-terminal SP, regardless of length of the hlyA secretion signal (62 vs. 194 amino acids), were secreted into the medium by the E. coli HlyA transporter at similar levels. Considerable amounts of hybrid proteins were still, however, associated with E. coli cells, mainly in the degraded form. PMID- 7557480 TI - Cloning and complete sequence of the DNA polymerase-encoding gene (BstpolI) and characterisation of the Klenow-like fragment from Bacillus stearothermophilus. AB - A fragment of the DNA polymerase I-encoding gene (polI) from Bacillus stearothermophilus (Bst) was obtained by PCR. This was used as a probe to obtain a full-length gene from a Bst genomic DNA (gDNA) plasmid library. Comparison of the sequence to B. caldotenax (Bca) showed about 93% homology at the amino acid (aa) level. A Klenow-like (BstpolIk) clone was developed and the recombinant protein displayed DNA polymerase activity similar to the wild-type BstPolI enzyme. PMID- 7557479 TI - The insertion sequence element ISRm2011-2 belongs to the IS630-Tc1 family of transposable elements and is abundant in Rhizobium meliloti. AB - The insertion sequence (IS) element ISRm2011-2 of Rhizobium meliloti (Rm) is characterized by 19-bp imperfect terminal inverted repeats (three mismatches) and a size of 1053 bp. Upon transposition, ISRm2011-2 generates a putative target duplication of 2 bp. ISRm2011-2 carries two major overlapping open reading frames (ORFA and B) with a coding capacity of 135 and 201 amino acids (aa), respectively. A potential translational frameshifting window (5'-AAAAAAAG) is located in the overlapping region of both ORFs. The putative fusion product of both proteins, which probably represents the mature transposase, has a predicted molecular mass of 35.8 kDa and a pI of 10.5. Comparison of the deduced aa sequence of ORFA with database entries revealed homology to putative transposases of some IS elements of the IS3 family, as well as to eukaryotic transcription factors. The protein encoded by ORFB shows homology to transposases (Tps) of the recently proposed IS630-Tc1 family which includes Tps of both prokaryotic and eukaryotic transposable elements. Analyses of the distribution of ISRm2011-2 in natural Rm populations showed that this IS element is abundant in Rm strains. PMID- 7557481 TI - Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation. AB - A series of gusA transcriptional fusion vectors is described for Bacillus subtilis (Bs). The series includes a vector for use with the amyE system of Shimotsu and Henner [Gene 43 (1986) 85-94], an integrative vector and vectors that provide gusA or gusA neo cassettes. The gusA fusions are compatible with lacZ fusion vectors that are widely used with Bs, and gusA and lacZ fusions are expressed at similar levels. beta-Glucuronidase (beta Glu) and beta-galactosidase (beta Gal) do not exhibit any cross-reactivity, there is very little endogenous beta Glu activity in Bs, and there is no indication of mutation to high-level expression. We have use strains containing both gusA and lacZ fusions to compare the times of expression of different genes during sporulation. PMID- 7557482 TI - Amplification and cloning of the Mycobacterium tuberculosis dnaA gene. AB - To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector. PMID- 7557483 TI - Cloning, characterization and expression of the gene encoding cytochrome P-450sca 2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. AB - Pravastatin, a drug for treating hypercholesterolemia, is produced by hydroxylation of ML-236B-Na in Streptomyces carbophilus (Sc) catalyzed by the cytochrome P-450sca (CytP-450sca) monooxygenase system. The gene (cytP-450sca-2) encoding CytP-450sca-2 was cloned from Sc. The gene had an open reading frame of 1233 bp, encoding a 410-amino-acid protein. The partial sequencing of the purified CytP-450sca-2 revealed that the N-terminal Met had been removed. CytP 450sca-2 contained the heme-binding HR2 region characteristic of all CytP-450, as well as the putative oxygen-binding site proposed in CytP-450cam from Pseudomonas putida. ML-236B.Na enhanced transcription of cytP-450sca-2, suggesting that substrate induction in Sc is transcriptionally regulated. S. lividans (Sl) transfected with cytP-450sca-2 converted ML-236B.Na to pravastatin, indicating the cloned gene to be functional in Sl. PMID- 7557484 TI - Cloning and sequencing of a secY homolog from Streptomyces scabies. AB - The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined. The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family. The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family. The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF. Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein. Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence. Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans. The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome. PMID- 7557485 TI - Cloning and sequence of ftsZ and flanking regions from the cyanobacterium Anabaena PCC 7120. AB - Using degenerate oligodeoxyribonucleotide primers based on conserved regions of the cell-division protein FtsZ, a 220-bp fragment of DNA was amplified by the polymerase chain reaction from Anabaena PCC 7120 (Ana). This fragment, which showed significant homology with Escherichia coli ftsZ, was used as a probe to isolate a 15-kb genomic clone containing ftsZ from an Ana DNA library. Sequence analysis revealed an open reading frame (ORF) encoding a protein of 379 amino acids, with 49% identity with E. coli FtsZ. Upstream of Ana ftsZ is a small, unidentified ORF, transcribed in the same direction. An ORF lying downstream of the ftsZ coding region and transcribed in the opposite orientation, shows homology with bacterial glutathione synthetase-encoding genes. Single copies of ftsZ have been identified in Ana and two other cyanobacteria. Multiple transcripts hybridising to ftsZ were detected by Northern hybridisation. PMID- 7557487 TI - The female face of Medicare. Cost-cutting without reform would harm the two thirds of beneficiaries who are female. PMID- 7557486 TI - Genetic evidence for host specificity in the adhesin-encoding genes hxaA of Helicobacter acinonyx, hnaA of H. nemestrinae and hpaA of H. pylori. AB - Gastric and non-gastric species of Helicobacter were examined for the presence of the adhesin-encoding gene, hpaA, from the human-associated gastric Helicobacter H. pylori (Hp), and for adhesin subunit protein HpaA. Amplification of a 375-bp internal DNA fragment of hpaA by PCR demonstrated the presence of the gene in Hp and in two closely related gastric Helicobacters, H. nemestrinae (Hn) and H. acinonyx (Hx), but not in the more distantly related H. felis (Hf) and H. mustelae (Hm). The non-gastric Helicobacters, H. canis (Hc), H. muridarum (Hr), H. fennelliae (He) and H. cinaedi (Hi), were all negative for hpaA. An immunoblot assay of water extracts with adhesin-specific antibody confirmed these results. The deduced amino acid (aa) sequences of Hp HpaA and Hn adhesin A (hereafter termed HnaA) are very similar, having identical receptor-binding motifs (rbm); also, the hemagglutination (HA) properties of Hn and Hp cells were indistinguishable. In contrast, the rbm of Hx adhesin A (hereafter termed HxaA), compared to that of Hp, contained a non-conservative aa substitution (Ile to Thr); also, there was variance in five consecutive aa from 10 to 14 residues upstream from the rbm. We conclude that these aa substitutions in HxaA are probably responsible for the difference in receptor recognition of this adhesin, as evidenced by the resistance of Hx HA to inhibition with N-acetylneuraminyl alpha(2,3)-lactose. These results are consistent with the biological similarity between the natural host(s) of Hp and Hn; i.e., human and non-human primates, and the dissimilarity between these hosts and the feline host, the cheetah. PMID- 7557488 TI - 'Beauty parlor stroke': the injuries continue. PMID- 7557489 TI - MI survivors: using drug therapies to protect the heart. AB - Survivors of myocardial infarction are at increased risk for another MI, congestive heart failure, ventricular arrhythmias, and sudden death. Beta blockers reduce the rate of sudden death, reinfarction, and recurrent ischemia, particularly in elderly patients. Chronic aspirin therapy has shown significant reductions in cardiovascular morbidity and mortality and is recommended for all post-MI patients who can tolerate it. ACE inhibitor therapy has shown benefit in patients with impaired LV function. Identifying post-MI patients at risk for sudden death and preventing fatal arrhythmias has proven difficult. Class I antiarrhythmics should be avoided. Amiodarone and perhaps sotalol appear promising, but large-scale trials are still ongoing. PMID- 7557490 TI - Prescribing for older patients: how to avoid toxic drug reactions. AB - As a group, persons age 65 and older are the largest consumers of pharmaceuticals, accounting for 30% of prescription drugs and 40% of over-the counter medications. Many elderly patients take multiple medications for a variety of concurrent medical conditions. The use of two or more drugs, combined with widely varying degrees of disease-related and physiologic impairment of function, can lead to unintended adverse reactions and even death. The physician can help to minimize adverse drug reactions and improve outcomes by being aware of the principles of clinical pharmacology, the characteristics of specific drugs, and the special physical, psychological, and social needs of older patients. PMID- 7557491 TI - Unexplained syncope: when to suspect pulmonary thromboembolism. PMID- 7557493 TI - Collecting up front from Medicare patients. PMID- 7557492 TI - Hormone replacement therapy for men: has the time come? PMID- 7557494 TI - Effect of maternal diabetes on longevity in offspring of spontaneously hypertensive rats. AB - We studied the effect of maternal diabetes induced by neonatal streptozotocin treatment on the longevity of the male offspring in spontaneously hypertensive rats (SHR). Maternal diabetes significantly decreased the survival in the offspring as compared with the control (p < 0.01). Mean age at death was 14.9 +/- 0.6 months in the offspring from the diabetic dams and 17.9 +/- 1.1 months in that from the control. The life span was significantly correlated with the birth weight (rs = 0.55, p = 0.009). These findings suggest that a diabetic pregnancy may accelerate an age-related degenerative process of the offspring in SHR. PMID- 7557495 TI - Reduced aerobic metabolic efficiency in postischemic myocardium dysfunction in rats: role of aging. AB - It has been suggested that aging may enhance the deleterious effects of myocardial ischemia-reperfusion. This study evaluates the relationship between oxygen consumption and functional impairment during reperfusion following myocardial ischemia in adult and senescent rat hearts. Global ischemia induced a marked impairment of contractile function which was significantly higher in senescent than in adult hearts. During reperfusion postischemic dysfunction was more evident in senescent hearts: at the 10th minute, the developed pressure recovered less (p < 0.05) and end-diastolic pressure increased more (p < 0.05) in senescent than in adult hearts. However, oxygen consumption per unit of work was significantly higher throughout 60 min of reperfusion when compared to controls with no significant difference between adult and senescent hearts. This study demonstrates that following ischemia and reperfusion depression of function and inappropriately high oxygen consumption were observed in both adult and senescent hearts. However, aging was associated with greater contractile impairment, which occurred in the absence of further deterioration of metabolic efficiency of contraction. PMID- 7557496 TI - Effectiveness of immunotherapy in aged leukemic mice. AB - We previously showed that immunotherapy using indomethacin combined with rIL-2 in vivo was very effective in stimulating natural killer (NK) cells and in increasing the life span of young adult mice bearing a tumor of hemopoietic origin. The aim of the present study was to test the efficacy and universality, with respect to age, of this treatment in tumor-bearing mice. DBA/2 mice (10-16 months old) were injected with 5 x 10(6) erythroleukemia cells and remained either: (i) untreated (control); (ii) treated with indomethacin (5 micrograms/ml drinking water) for 9 days from tumor onset; (iii) treated with rIL-2 (24 x 10(3) U/injection) twice a day for the last 4 days of the 9-day tumor-bearing period, or (iv) treated with both indomethacin and rIL-2 concomitantly. Some mice from each group (above) were killed after 9 days of tumor growth, while the others were allowed to survive. Spleen and bone marrow cells were collected from the mice of each group and NK (ASGM-1+) cells were quantitated using an immunoperoxidase technique combined with light microscopy. NK cell-mediated activity was assessed using a standard chromium release assay. The results show that although NK cell numbers increase in the presence of the growing tumor, neither indomethacin alone, rIL-2 alone, nor the combination could further increase the numbers of these cells. Furthermore, indomethacin and/or rIL-2 could not induce NK cell-mediated activity in such mice. Moreover, tumor-bearing aged mice treated as above did not have a significantly longer life span than untreated (control) tumor-bearing mice. The present results indicate an age dependent resistance to a form of immunotherapy already proven very effective in young adult mice. Furthermore, the results of this and our previous studies suggest that immunotherapy, which may be highly effective in one age group, should not be presumed effective throughout life. PMID- 7557497 TI - Diet, not aging, causes skeletal muscle insulin resistance. AB - The purpose of this study was to compare the effects of raising female Fischer rats on a low-fat, high-complex-carbohydrate diet (LFCC) versus a high-fat, sucrose diet (HFS) on serum glucose and insulin as well as skeletal muscle glucose transport. No significant differences were observed between 6- and 24 month-old rats raised on the LFCC diet for serum glucose (3.6 +/- 0.1 vs. 3.7 +/- 0.2 mM) and insulin (88 +/- 6 vs. 98 +/- 10 pM) or for basal (35 +/- 3 vs. 39 +/- 6 pmol/mg protein/15 s) or insulin-stimulated (74.2 +/- 7.6 vs. 69.4 +/- 3.8 pmol/mg protein/15 s) glucose transport. These data indicate that aging per se does not lead to insulin resistance. When the 24-month-old animals raised on the HFS diet were compared with those on the LFCC diet, major differences were observed. Fasting serum insulin was significantly higher in the HFS group (437 +/ 118 vs. 98 +/- 10 pM) and insulin-stimulated glucose transport was significantly reduced (52.5 +/- 3.7 vs. 69.4 +/- 3.8 pmol/mg protein/15 s). Fasting glucose (3.7 +/- 0.2 vs. 3.6 +/- 0.1 mM) and basal glucose transport (38 +/- 6 vs. 39 +/- 6 pmol/mg protein/15 s) were unchanged. These results indicate that diet and not aging per se caused insulin resistance. PMID- 7557498 TI - Aspirin-induced conformational changes in platelet membrane in subjects with stroke. AB - Objectives of this case-control study were (1) to examine membrane conformational changes and dynamics in platelets of patients with stroke prior to and following short-term aspirin administration and (2) to investigate changes in platelet membrane fluidity which may be associated with the ageing process. The study population consisted of 10 patients with ischaemic stroke (age range 61-92, mean 76 years; 6 females and 4 males), 9 age- and sex-matched controls (age range 64 88, mean 73 years; 5 females and 4 males), and 8 healthy young controls (age range 22-39, mean 32 years; 4 females and 4 males). The patients were recruited from the medical and geriatric wards, whereas both young and old controls were recruited from hospital staff and the local community. Aspirin, at a dose of 150 mg/day, was administered to all three groups for 7 days. The order parameter S, which is an index of membrane fluidity and conformation, was measured by the techniques of spin labelling and electron spin resonance spectroscopy on platelets from venous blood samples from patients prior to (within 48 h of admission) and following 7 days of aspirin therapy and from venous blood samples from both young and old controls before and after 7 days of oral aspirin therapy. There was an age-related decrease in platelet membrane fluidity. There was no significant difference in the value of 'S' between patients with ischaemic stroke and old controls. Treatment with aspirin led to a significant decrease in membrane fluidity in both patients and old controls, but had no apparent effect on the platelet membrane fluidity of younger controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557499 TI - Persistent grossly elevated erythrocyte sedimentation rate in elderly people: one year follow-up of morbidity and mortality. AB - The significance of a very elevated erythrocyte sedimentation rate (ESR) in elderly patients is debated. In a retrospective study, we searched the records of a laboratory providing the sole service to a health district for ESR measurement and identified all non-surgical and non-psychiatric patients over the age of 65 who had had an ESR above 50 mm/h. Diagnoses and mortality in a 1-year follow-up were determined from case notes. Four hundred and nine subjects (median age 75; range 65-99) were identified and data on 401 of these (155 male, 246 female; median ESR 80 mm/h, range 50-148) were adequate for 1 year follow-up. Forty-eight percent had a persistently raised ESR (two values > 50 mm/h separated by at least 14 days; group 1); 39% had a single ESR measurement only (group 2), and 13% had a transiently raised ESR (group 3). The commonest diagnosis in group 1 patients was rheumatological disease (51.8%), followed by infection (31.9%) and non haematological malignancy (11%). Infection was the commonest diagnosis in groups 2 (47.4%) and 3 (43.7%), followed by non-haematological malignancy (19.9%) in group 2 and rheumatological disease (20.4%) in group 3. In only 1 in 20 cases was no diagnosis apparent at 1 year. The standardised mortality ratio (SMR) of the combined groups 1 and 2 (482; CI: 421-544) was strikingly raised, and even more so if patients with rheumatoid arthritis were excluded (542; CI 458-625). Where there were sufficient numbers of deaths to make SMR estimations valid, a gradient of mortality against the level of the ESR could be observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557500 TI - Cell adhesion molecules CD11a and CD18 in blood monocytes in old age and the consequences for immunological dysfunction. Preliminary results. AB - Adhesion molecules, such as leukocyte-function-associated antigen (LFA-1 or CD11a/CD18), intercellular adhesion molecule 1 (ICAM-1 or CD54) and Hermes antigen (HCAM or CD44), have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations were present in aged subjects, we studied the expression and density of these molecules on peripheral blood lymphocytes and monocytes from healthy old subjects. A decrease in monocyte subpopulations bearing CD11a/CD18 and an increase in CD11a/CD18 and and CD44 antigen density on lymphocytes and on monocytes, respectively, were observed. These changes might be an event in the mechanism leading to the decreased lymphocyte proliferative response in vitro and to other immunological dysfunctions reported in old subjects. PMID- 7557501 TI - Influence of age and health on immune functions and trace elements. AB - Apparently healthy elderly donors were screened according to a simple protocol that included clinical examination and the determination of hematological and biochemical values. This screening was performed to detect subclinical alterations which might interfere with immune responses and trace element status. The elderly were divided into two groups. The first group consisted of 22 (age 76 +/- 1 years) positively selected elderly (PSE), i.e. healthy subjects with no hematological and laboratory alterations, the second one comprised 13 (age 75 +/- 1 years) negatively selected elderly (NSE). Data were then compared with those obtained from 40 (age 35 +/- 2 years) healthy young controls. In both groups of elderly donors, plasma zinc levels were normal, while plasma copper concentrations were increased. Intracellular values of zinc and copper in mono- and polymorphonuclear cells from both groups of elderly were within reference limits. After in vitro activation, granulocyte chemiluminescence activity was impaired only in NSE. A decrement in the number of circulating CD3 lymphocytes and an increase in CD8d, CD57 cells were found in PSE, while NSE showed an increased number of CD3,DR cells and CD8d, CD57, CD8b,CD57 and CD16,CD56 positive cells. Our results indicate that only plasma copper levels were affected by age, whereas subclinical alterations in hematological or biochemical values appear to impair immune responses in the elderly. PMID- 7557502 TI - AIDS-related optic neuropathy: a histological, virological and ultrastructural study. AB - BACKGROUND: Clinical and histopathological evidence of optic nerve axonal loss has been reported in AIDS patients without retinitis. The study was carried out to investigate the possible involvement of HIV-infected cells in the development of optic nerve degeneration. METHODS: Optic nerves were obtained from eight AIDS patients and four normal controls. These nerves were morphologically and immunohistochemically analyzed. Additionally, using PCR amplification techniques, the retina and optic nerve samples obtained from three HIV-seropositive patients and one control were examined for the presence of HIV and cytomegalovirus antigens. RESULTS: We noted various stages of axonal degeneration in the optic nerves obtained from AIDS patients in whom there was an absence of retinal findings. Characteristic glial changes involving hypertrophic astrocytes, vacuolated oligodendrocytes, and mononuclear phagocyte series cells were also seen in the AIDS optic nerves. HIV DNA was present in at least four of five optic nerves but in only one of five retinas. Control specimens were each negative for all cytomegalovirus and HIV antigens. CONCLUSIONS: Degeneration in the optic nerve may be mediated by HIV-infected macrophages rather than by direct viral infection of neurons. Axonal degeneration due to AIDS at the level of the optic nerve can occur independently of retinal infection. PMID- 7557504 TI - Visual acuities and scotomas after 3 weeks' levodopa administration in adult amblyopia. AB - BACKGROUND: Previous studies have shown that both a single dose of levodopa and a 1-week administration of levodopa improve visual functions in adult amblyopic patients. In the present study, we investigated the effect of increased dosage and duration of levodopa on amblyopes' visual functions. METHODS: Visual acuity and visual fields were examined before and after 3 weeks of daily administration of levodopa/carbidopa as well as 1 month and 2 months after completion of drug therapy in a double masked-design. RESULTS: A significant increase in visual acuities and a decrease in fixation point scotomas were found. Changes were of comparable dimension to those found after 1 week of levodopa administration. Improvement of visual functions persisted 2 months after the levodopa/carbidopa administration was completed. CONCLUSION: The present study confirms previous findings of improvement in visual function in amblyopia after levodopa/carbidopa administration. However, increasing the dosage and the duration of levodopa did not enhance the effect in adults. PMID- 7557503 TI - Echographic pseudoextension of uveal melanomas. AB - BACKGROUND: Standardized echography is routinely utilized to assess uveal melanomas. Echographic pseudoextension is defined as normal structures mimicking intrascleral or extrascleral extension of tumor on echography. METHODS: The records of 151 consecutive uveal melanoma patients evaluated with standardized echography over a 6-year period (1986-1991) were reviewed to identify those in which pseudoextension or true extension was diagnosed. RESULTS: Fourteen (9%) cases of pseudoextension were noted, with causes including juxtapapillary tumor location (seven cases), extraocular muscle insertion (five cases), vortex ampullae (one case), and post-brachytherapy changes (one case). Clinical, echographic, and/or histopathologic follow-up confirmed absence of true extension. Six (4%) cases of true extrascleral extension were identified and confirmed histopathologically. CONCLUSION: Differentiating extraocular tumor extension from pseudoextension is critical, and use of standardized A-scan and contact B-scan echography is integral in this assessment. PMID- 7557505 TI - Immunohistochemical evaluation of the integrity of the blood-aqueous barrier in normal and rubeotic human eyes. AB - BACKGROUND: The integrity of the blood-aqueous barrier (BAB) was analyzed using an immunohistochemical technique for the demonstration of albumin. METHODS: Paraffin sections of 36 normal eyes obtained from eye banks or at autopsy (mean age 62.8 +/- 15.2 years) and 46 eyes with marked iris neovascularization (mean age 54.6 +/- 25.3 years) were formalin-fixed and examined using rabbit anti-human albumin. RESULTS: In normal eyes, albumin staining was found in the iris stroma inside and outside the iris vessels but was not detected across the anterior iris border; albumin was present in the anterior chamber in one eye, but not internal to the nonpigmented ciliary epithelium. In rubeotic eyes, albumin staining extended along the anterior iris in all 46 cases; albumin was demonstrated in the anterior chamber in 31 eyes and along to the nonpigmented ciliary epithelium in 13 eyes. Differences between normal and rubeotic eyes were significant for intensity of albumin staining in the iris stroma and for presence of albumin along the anterior iris, within the anterior chamber, and along the ciliary epithelium (P < 0.001, chi 2 test). CONCLUSION: Our findings indicate that the BAB may be less resistant to leakage in the iris stroma than at the ciliary epithelium. BAB breakdown in rubeotic eyes occurred mainly in the iris; the ciliary epithelium was much less involved. Immunohistochemical staining for albumin appears to be useful for evaluating the integrity of the BAB in human pathologic specimens. PMID- 7557506 TI - Optic nerve fiber count and diameter of the retrobulbar optic nerve in normal and glaucomatous eyes. AB - BACKGROUND: The caliber of the retrobulbar optic nerve and the count of optic nerve fibers vary considerably in normals. The diameter of the retrobulbar optic nerve also decreases with optic nerve atrophy. This study aimed to determine the relationship between the caliber of the optic nerve and the optic nerve fiber count. METHODS: We counted the optic nerve fibers and measured the diameter of histological cross sections of the optic nerve for 56 normal subjects and 23 patients with absolute glaucoma. RESULTS: The optic nerve fiber count increased significantly (P < 0.0001) by 777,000 fibers for every millimeter increase in retrobulbar optic nerve diameter, starting at a baseline diameter of 1.89 mm. CONCLUSION: Using this linear regression equation, the optic nerve fiber count can be estimated in routine histology by measuring the optic nerve diameter. Taking into account a fixation-induced tissue shrinkage, this method may also give some indication of the optic nerve fiber count intravitally, when, for eyes with opaque optic media, the diameter of the retrobulbar optic nerve has been measured by imaging techniques. Greater retrobulbar optic nerve caliber may indicate greater structural reserve capacity. PMID- 7557507 TI - Increase in choroidal blood flow in rabbits with endothelin-1 induced transient complete obstruction of retinal vessels. AB - BACKGROUND: In a previous paper, we reported that retinal blood flow (RBF) ceased immediately after injection of 1 nmol endothelin-1 (ET-1) and no recovery of RBF was detected for at least 50 min. In this study, we confirmed the same duration of RBF cessation and measured choroidal blood flow (CBF) for 180 min. METHODS: We measured CBF in a rabbit model of transient complete obstruction of retinal vessels induced by intravitreal injection of a high dose of ET-1, using the hydrogen clearance method. We also investigated the effects of intravitreal injection of ET-1 on intraocular pressure (IOP), blood pressure, pulse rate and blood gases. RESULTS: CBF was significantly greater in the ET-1-injected eyes than in the control eyes 40-130 min after injection of ET-1 (P < 0.05). The maximal CBF ratio in the ET-1-injected eyes was 128 +/- 7.4% at 40 min. CBF decreased to the pre-injection level at 140 min after the injection of ET-1. There was no significant change in blood pressure, pulse rate and blood gases throughout this experiment, and there was no significant difference in IOP between ET-1-injected eyes and control eyes. CONCLUSION: It seems likely that the increase in CBF resulted from some local mechanisms of control that compensated for the decrease in RBF induced by intravitreal injection of ET-1. This model may be useful for investigation of the regulatory system of intraocular circulation, including endothelin receptors. PMID- 7557509 TI - Corneal neovascularization in rats as a model for photothrombotic therapy using bacteriochlorin a and an argon laser. AB - BACKGROUND: The presence of vessels has a negative influence on corneal transplant survival. Closure of such vessels prior to transplantation may improve the transplant results, and this might be achieved by irradiating the vessels with argon laser light after intravenous administration of a photosensitizer, e.g. bacteriochlorin a (BCA). A suture-induced corneal neovascularization model in rats was set up to test this hypothesis. METHODS: Suture-induced vessels in the cornea of male Wistar rats were irradiated with argon laser light after intravenous administration of BCA. We applied irradiation of varying energy levels and duration and assessed the changes in the vessels by slit-lamp examination, fluorescein angiography and histology. RESULTS: Suture-induced corneal vessels in the rat could be used effectively to study photothrombosis therapy. Intravenous administration of BCA prior to irradiation (lambda = 514.5 nm) of the corneal vessels led to vessel closure at lower energy levels and of longer duration than occurred with laser treatment alone. CONCLUSIONS: Suture induced corneal neovascularization in the rat can be used as a model to study the efficacy of photothrombosis therapy. BCA can be used to enhance the rate and duration of vessel closure. PMID- 7557510 TI - Communication between the subretinal space and the vitreous cavity in the morning glory syndrome. AB - BACKGROUND: The aim was to describe a pathogenic mechanism for a rhegmatogenous retinal detachment in a 69-year-old man with the morning glory syndrome. METHODS: During vitreous surgery for a retinal detachment, a membrane was removed that covered the optic disc anomaly and produced traction on the peripapillary retina. A retinal hole was found in tissue lying within the optic cup, and the hole was sealed using a autologous plasma-thrombin mixture. Silicone oil was used for retinal tamponade. RESULTS: A retinal hole in tissue lying within the optic cup provided a fluid pathway between the vitreous cavity and the subretinal space. Following vitrectomy surgery, bubbles of silicone oil passed through the retinal hole into the subretinal space of the macula. CONCLUSION: This case demonstrates that a retinal hole in tissue lying within the optic disc anomaly of the morning glory syndrome provides a communication for fluid between the subretinal space and the vitreous cavity, resulting in a rhegmatogenous retinal detachment. Vitreous replacement with silicone oil resulted in the migration of silicone bubbles into the subretinal space. PMID- 7557508 TI - Serum-induced collagen gel contraction. AB - PURPOSE: To understand the molecular events underlying disease-related vitreous gel contraction, the effect of serum components on collagen was investigated. METHODS: Bovine vitreous or dermal collagen was incubated with a mixture of transglutaminase (TG; factor XIIIa) and fibronectin (FN), and the biochemical changes of collagen were monitored by gel electrophoresis. In addition, serum induced changes in the volume of the collagen gel were monitored. RESULTS: Gel electrophoresis revealed a new high-molecular-weight band (M(r) 240,000) presumably due to intermolecular cross-links of collagen peptides and FN. The serum components also were shown to cause a significant decrease in the volume of the collagen gel. CONCLUSION. Collagen gel contraction could be attributed to the collagen-FN-collagen cross-links catalyzed by TG. PMID- 7557513 TI - Dental expenditures and insurance coverage among older adults. AB - Using data on 975 elderly persons from the 1990 Health Supplements to the Panel Study of Income Dynamics, we describe the predictors of expenditures for dental services. Forty-four percent of elderly persons reported using some dental services within a year. Thirteen percent had private dental insurance, and 8% had a separate dental policy. The average total expenditure for those who used any dental services was $378, 88% of which was paid out-of-pocket. Persons with a separate dental insurance policy, younger and better educated persons, and those with greater financial resources were more likely to use dental services. PMID- 7557512 TI - The additive miotic effects of dapiprazole and pilocarpine. AB - BACKGROUND: Since dapiprazole on alpha-adrenergic agent, produces miosis by paralyzing the dilator muscle, and pilocarpine, a parasympathetic drug, causes miosis by affecting the sphincter, we speculated that the two drugs might have additive effects. METHODS: The additive miotic actions of pilocarpine 2% and dapiprazole 0.5% were evaluated by comparing the effects of two drugs given together and alone on the reversal of mydriasis induced by tropicamide (0.5%) and phenylephrine (10%) in one eye each of 60 healthy volunteers. RESULTS: Dapiprazole and pilocarpine together induced miosis significantly faster than each drug alone, showing additive effects. CONCLUSION: Co-administration of dapiprazole and pilocarpine at the end of the eye examination will induce fast pupillary constriction, which might be useful in preventing the development of an acute attack of angle-closure glaucoma in patients with anatomically narrow angles. PMID- 7557514 TI - Medical conditions differentially affect the development of IADL disability: implications for medical care and research. AB - Using The Longitudinal Study of Aging, we determined the independent effects of nine self-reported medical conditions on the likelihood of developing specific instrumental activities of daily living (IADLs) disabilities at three points in time. We controlled for demographic factors and self-reported health status. The various medical conditions differentially affect each specific IADL disability, and each IADL disability has its own set of predictors which, in general, do not vary over time. The differential effects of thse predictors need to be taken into consideration by researchers, clinicians, and policymakers when studying disability and when implementing and evaluating programs to reduce disability. PMID- 7557515 TI - Antecedents and consequences of physical activity and exercise among older adults. AB - The antecedents and consequences of four markers of physical activity and exercise are examined for the 6,780 baseline self-respondents to the Longitudinal Study on Aging. These dichotomous markers reflect having a level of physical activity greater than one's peers (45.8%), getting as much exercise as needed (58.9%), having a regular exercise routine (28.4%), and walking a mile or more at least once a week (29.9%). The major factors associated with engaging in these behaviors are having fewer lower body limitations, better perceived health, more non-kin social supports, not worrying about one's health, and having a sense of control over one's health. When added to traditional models predicting subsequent (over the next 6 to 8 years) mortality, nursing home placement, hospital resource consumption, and changes in functional status, the four markers of physical activity and exercise have numerous statistically and substantively significant associations, all of which involve better health outcomes. PMID- 7557511 TI - Recurrent retinal artery obstruction as a presenting symptom of ophthalmic artery aneurysm: a case report. AB - BACKGROUND: Retinal artery obstruction is an ophthalmic emergency requiring immediate treatment. Recurrent episodes are the result of thromboembolic seeding and necessitate diagnostic efforts to find a possible source for this seeding. The most common sources of such seeding are the valves of the heart and the carotid arteries. CASE REPORT: We describe a case of a 25-year-old man who had recurrent episodes of retinal artery obstruction due to embolic spread. DISCUSSION: We suggest the origin of the embolic spread to be a post-traumatic ophthalmic artery aneurysm. The immediate and long-term treatment given to the patient are described, the present case is compared to earlier reports, and the unusual clinical picture and suggested treatment are discussed. PMID- 7557517 TI - Racial and educational differences in physical activity among older adults. AB - This study utilized a nationally representative sample of older adults to investigate the association of race and education with five different measures of self-reported physical activity. Rates of physical activity were low among white Americans and even lower among African Americans. However, a majority of the racial variation in activity was accounted for by racial differences in educational attainment. Those with 8 or fewer years of education were found to be less active than those with 9 or more, despite having controlled for educational differences in income, health compared to one year ago, functional status, body mass, and chronic disease. Discussion focuses on self-efficacy and social and cultural contexts. PMID- 7557516 TI - Activity participation and well-being among older people with arthritis. AB - This study tests the hypotheses that (a) severity of arthritis is inversely associated with frequency of activity participation, and (b) arthritis sufferers who maintain higher levels of participation, particularly in activities which are social in nature, are less likely to experience a decline in well-being. Three activity types are considered: social, physical, and solitary. Results indicate that well-being is influenced by social activity, whereas solitary and physical activity have minimal impact. This suggests that elders with arthritis need not remove themselves from the pursuit of activity and should be encouraged to develop new interests when physical functioning fails. The study also demonstrates the utility of considering activity as multidimensional. PMID- 7557518 TI - Decision making, responsibility, and advocacy in geriatric medicine: physician dilemmas with elderly in the community. AB - This descriptive anthropological study explores the nature of physician decision making in the case of old people who reside in the community. Focusing on physician narratives of actual dilemmas, this article contributes toward a phenomenology of clinical medicine and provides examples of how decisions in geriatrics are influenced by professional values, institutional constraints, and cultural forms. In-depth interviews with 40 physicians revealed dilemmas to have three sources: (1) unresolved questions about how much to intervene in patients' lives to reduce risk; (2) structural, moral, and medical limits to patient advocacy; and (3) the problem of assessing vulnerability and quality of life when pondering placement. PMID- 7557519 TI - Who cares? The size, scope, and composition of the caregiver support system. AB - The informal care networks of 242 impaired older people were examined in terms of the primary caregiver and secondary helpers. Most primary caregivers had at least one secondary helper; 88% identified helpers who provided hands-on help, 19% reported that helpers contributed financially, and 44% had helpers involved in care decisions. The size of the network was larger for direct care compared to financial or decision-making assistance. Almost half of primary caregivers reported that their spouse helped. It was expected that after controlling for characteristics of the primary caregiver and of the older person, that characteristics of the secondary caregiver network would influence the amount and type of primary caregiver assistance and formal care used by the older person. However, both hours and type of care by the primary caregiver were independent of size, scope, and composition of the secondary helper network. This independence suggests that efforts to alter the secondary caregiver network may have limited effects on the primary caregiver. PMID- 7557520 TI - Social class differences in social support among older adults. AB - The purpose of this study is to test for social class differences in social support among older adults. Data on a comprehensive range of social support measures provided by a nationwide sample of elderly people suggests that social class differences emerge when measures of contact with friends, support provided to others, and satisfaction with support are examined. However, significant differences fail to emerge with indicators of contact with family, support received from others, and negative interaction. PMID- 7557521 TI - The relationship between volunteer long-term care ombudsmen and regulatory nursing home actions. AB - This study assesses the relationship between the presence of Oregon volunteer long-term care ombudsmen and externally handled abuse complaints, survey reports, and regulatory sanctions. In 1987, new amendments to the Older Americans Act mandated long-term care ombudsmen access to nursing homes. No studies have systematically examined the relationship between these empowered ombudsmen programs and regulatory abuse investigations, survey findings, or sanction activities. Contrary to pre-1987 studies, this research found that the presence of ombudsmen was related to increased abuse reporting and abuse complaint substantiations, more survey deficiencies, and higher sanction activity. PMID- 7557522 TI - Reminiscence: a continuity theory framework. AB - The goal of this article is to provide theoretical underpinnings for reminiscence by reviewing and evaluating previous research, and integrating it within a theoretical framework. Using a continuity theory perspective (Atchley, 1989), propositions are generated which may facilitate reminiscence research across the life span. Three global functions of reminiscence are derived from continuity theory (private, social, and cognitive functions) and are used as a foundation for the creation of seven propositions. PMID- 7557523 TI - The Alzheimerization of aging. AB - The NIA invests a disproportionately large share of its resources in research on Alzheimer's Disease at the expense of other interests of the broader scientific community in gerontology. Complex social forces that continue to shape this outcome embrace discipline-specific traditions of science advocacy, as well as science-driven intellectual growth. PMID- 7557524 TI - A contextual approach for understanding individual autonomy in managed community long-term care. AB - This article develops a framework for identifying community long-term care program and policy features that support or hinder client autonomy. After introducing the topic, the authors develop a contextual approach for understanding individual autonomy and discuss its relevance to community long term care. They also illustrate how current financing, organization, and delivery of community long-term care provide clients with opportunities for meaningful choice. The authors summarize the implications that a contextual autonomy approach has for both community long-term care policy and program features and make specific policy recommendations. PMID- 7557526 TI - A note on viewing functional change in later life as migration. AB - Empirical research on functional decline in later life is expanding our understanding of this aspect of aging. To increase the value of future research it will be necessary to develop more adequate theories of functional transitions. Toward this goal, a conceptual model that views change as mobility between states is proposed. Exploring the analogy between geographic migration and functional change suggests several migration concepts that might be borrowed by aging researchers. These concepts are useful for organizing existing findings, generating new research questions, and identifying the types of data that need to be collected. PMID- 7557527 TI - Quality of survey informants' reports about death: verification of dates through a record check. AB - To determine whether relatives and others can provide valid information about the dates of survey respondents' deaths, informants' reports were compared with death certificates for 328 deceased respondents from the MESA study of older adults. About two-thirds (64.6%) of the informants accurately reported the complete date of death. A somewhat larger percentage (70.4%) correctly reported the day of death, whereas 86.9% and 89.6% remembered the year and month, respectively. The percentage of correct reports varies by the relationship between informant and respondent. Also, the findings suggest that women are more accurate reporters than are men, and that the percentage of accurate reports is greater for longer term relationships. PMID- 7557525 TI - The effects of poverty environments on elders' subjective well-being: a conceptual model. AB - This article presents a conceptual model, the Urban Ecological Model of Aging, to be used by researchers to assess the impact of living in neighborhoods of concentrated and prolonged poverty on elderly residential satisfaction and subjective well-being. Specifically, the suprapersonal environment characteristics, or aggregated people characteristics, of impoverished urban neighborhoods are explicated and included in a predictive model of community dwelling elders' affective state, i.e., positive and negative affect. Demographic and personal characteristics are also included in the model as predictors. PMID- 7557529 TI - Service directories: reinvigorating a community resource for self-care. AB - This article reviews the development, distribution, and effectiveness of a free, comprehensive mass-mailed community service directory designed for older people. While only half of a pre/post-distribution sample remembered receiving it, recipients of the directory indicate it increased service awareness and prompted various types of use. Alternatives for design and distribution of community directories are discussed. PMID- 7557528 TI - Biobehavioral rehabilitation for older adults with essential tremor. AB - Essential tremor (ET), the most prevalent movement disorder, has a peak prevalence in the sixth decade of life. ET primarily affects the hands and head. Persons with ET are often significantly disabled in communication, work, leisure, domestic activities, and psychosocial adjustment. Medical intervention for ET is often limited in effectiveness. A biobehavioral rehabilitation model for older adults with ET is described. PMID- 7557531 TI - [Cytologic-colposcopic-histopathologic correlations in preinvasive cervical lesions and cervical Human Papillomavirus infections]. AB - Cervico-uterine cancer is the most frequent gynecological neoplasia in Mexico and cervico-vaginal cytology is the most practical and dependable resource in lesions detection. During the last years precursory lesions detection (NIC and HPV infection) has increased. So, every patient presenting with an abnormal cytology should be included in an evaluation program, that includes a colposcopic study with biopsy of suspicious lesions, in order to know cellular abnormality degree, as these studies combination increases diagnosis certainty. Ninety three patients were evaluated by colposcopy, as the Papanicolaou showed abnormality ICN type in any degree or HPV infection data, during the first three years of the Unidad de Colposcopia de la Beneficiencia del Hospital ABC. In 49 patients histopathological study, was done. A correlation of all studies was carried out. There was a correlation cytology-histopathology of 59.18%; and colposcopy histopathology of 89.79%. It was concluded that evaluation by cytology is insufficient to establish a final diagnosis and treatment, and that colposcopic study is fundamental in the evaluation of the patient with abnormal exfoliative cytology. PMID- 7557530 TI - [5th National Congress on Obstetrics and Gynecology. 6-10 November 1994. Abstracts]. PMID- 7557532 TI - [Analysis of the predictive value of prolactin concentration in the umbilical cord as an evaluation parameter for respiratory insufficiency in the newborn]. AB - In fetal pulmonary development several substances and hormones which directly participate, have been involved. One of these is prolactin, which by raising concentrations through a complex mechanism seems to participate in pulmonary maturation. Due to the importance of product's pulmonary maturity producing respiratory failures of the newborn, some progress has been made in opportune diagnosis, and mainly in prophylaxis of this condition. In this study, serum samples were taken from the umbilical chord to know perinatal evolution after analysis of the evolution of each of the 37 cases, the results corresponded to previous results as published in literature for different conditions as toxemia, diabetes mellitus or prematurity, as well as the relationship between hormonal serum concentrations, and gestational age and weight. PMID- 7557533 TI - [Chlamydia trachomatis and cervix dysplasia]. AB - Fifty patients between 18 and 70 years of age from Gynecology and Obstetrics Department, Hospital General "Gonzalo Castaneda" ISSSTE, were studied. Patients were referred for bearing positive cytology with mild, moderate and severe dysplasia; also intentional search for Chlamydia trachomatis was made, both in cytology as well as with the immunofluorescence method, and also directed biopsy. A positive association was found in 10 patients (20%) proving that Chlamydia trachomatis is a promotor and modifier of cervical atypia. PMID- 7557534 TI - [A case of Prune Belly Syndrome. Prenatal diagnosis]. AB - Prune Belly Syndrome is a rare and complicated condition affecting the genitourinary organs and abdominal wall, it was named after the aspect of the abdomen after the bladder has been drained. In its fully developed form presents with the triad: megalocyst, abdominal muscle deficiency and cryptorchidism. We present a case of a patient with 22 weeks of gestation with ultrasonographic diagnosis of a large thoracoabdominal cyst. The delivery was by cesarean operation. We analyzed the literature. PMID- 7557535 TI - [Analysis of the use of the Salinas forceps at the Gynecologic-Obstetric Hospital of Garza Garcia, N.L. (Nuevo Leon)]. AB - Six hundred and thirteen cases of Salinas forceps application at Hospital de Ginecoobstetricia de Garza Garcia, N.L. from November 1992 to April, 1993, were reviewed. The largest patients group, 20 to 29 years of age with 54.5%; primiparae were predominant with 55.9%, the largest amount of applications in term products, 80.8%; elective forceps with 72.5%; low application with 83.0%; medium 2.5%; episiotomy, medium, right lateral in all the cases; epidural block anesthesia in all the patients, and only one complication 0.1%; most frequent position variety OIA with 50%; and the smaller OIP with 2.6%; 96.3% of products weighted more than 2,500 g; and 87.1% with Apgar 8-9 at one minute. Maternal morbidity, 30.1%; fetal morbidity, 6.1%, with one case with facial paralysis (0.1%) by medium forceps. There were no maternal deaths; 3 antepartum fetal deaths; none postpartum. PMID- 7557536 TI - [Hydatidiform mole. Clinical aspects, incidence and risk factors]. AB - From September 1st, 1992, to August 31st, 1994 35 patients with molar pregnancy were studied comparing them with 70 patients with normal pregnancy attended in the Hospital General de Zona 1 IMSS San Luis Potosi with the purpose of establishing the incidence of molar pregnancy to know the risk factors and to study the clinical aspects of it. The found incidence was: 1 molar pregnancy for each 415 deliveries, 22.85% of the mole patients were in a low socioeconomic position against 8.57% of the control group, suggesting that such condition is a risk factor. The average age was 26.28 years old in the mole patients and 26.42 years old in the control group. There were no differences in the number of first pregnancy in both groups: 17.14% against 18.57% in the control group. The predominant symptoms and signs were: bleeding, vesicle expulsion, nausea and vomit. In all of them, a uterine curettage was performed, four needed blood transfusion, one needed subsequent curettage, there were no deaths. It is concluded that the incidence is similar to reports of other national authors, the low socioeconomic level is a factor for the aparition of this disease and it requires a precocious diagnostic to avoid complications. PMID- 7557537 TI - [Spontaneous hepatic rupture during pregnancy. Report of a case. Review of the literature]. AB - The spontaneous hepatic rupture during pregnancy or puerperium has been reported as a rare complication, with an extraordinary severity. Usually it's a pregnancy hypertensive disease complication. Since 1990 it had been reported 120 cases from this pathology in the world literature. We reported a case from a patient who presented spontaneous hepatic rupture during the immediately puerperium and a world literature revision. PMID- 7557538 TI - [Heterologous adenosarcoma of the Mullerian ducts in adolescence. Presentation of clinical case and review of the literature]. AB - This is the case of a 16 years old female with an histopathologic diagnosis of mullerian heterologous adenosarcoma, a non-frequent entity in the adolescence. We carried out a bibliographic review about uterine sarcomas, clinic aspect, classification, epidemiology, prognosis and treatment. PMID- 7557539 TI - [Endometriosis. Analysis of laparoscopic findings at the A.B.C. (American-British Cowdray) Hospital]. AB - Endometriosis approximately affects 10 to 15% of premenopausic women. Diagnosis mainly is made by laparoscopy. We revised all the laparoscopies which were made in labor service at the American British Cowdray Hospital from July 1st 1989 to June 30th 1994 in order to evaluate the incidence of endometriosis in the institution. They were made from 882 laparoscopies done, in 447 cases (50.68%) endometriosis was the main finding, this was similar to the reports published by other authors. The group of age mainly affected was from 20 to 29 years-old. Sterility was found in 53% of these patients, slightly above the results in other series. The 85% of the cases were in hospital for less than 24 hours. Complications were in 7% of them, none required a laparotomy, however. PMID- 7557540 TI - Products of gamma-tocopherol reaction with NO2 and their formation in rat insulinoma (RINm5F) cells. AB - gamma-Tocopherol, commonly found in seed oils, is the major tocopherol in the U.S. diet, is superior to alpha-tocopherol in preventing neoplastic transformation, and demonstrates unique reactivity toward NO2. This article describes the products of reaction between gamma-tocopherol and low concentrations of gaseous nitrogen dioxide (NO2), as well as their endogenous formation in NO-producing RINm5F cells. gamma-Tocopherol in hexane reacts with NO2 to yield two products identified as 2,7,8-trimethyl-2(4,8,12 trimethyltridecyl)-5,6-chromaquinone++ +, "tocored," and 2,7,8 trimethyl-2(4,8,12 trimethyltridecyl) 5-nitro, 6-chromanol, "tocoyellow." Physical data for these two compounds and reaction characteristics are described. The formation of tocored is consistent with a proposed mechanism of gamma-tocopherol-mediated reduction of NO2 to NO involving initial reaction by NO2 at the C-5 position to form an intermediate nitrite ester tocopheryl radical, which then reacts internally to release NO and form 5,6 epoxy gamma-tocopherol. Tautomerization and further oxidation of the latter intermediate by NO2 yields tocored as the main product observed. The reaction of gamma-tocopherol with NO2 to form NO occurs independently of light, whereas alpha-tocopherol requires light to generate NO from NO2. gamma-Tocopherol and aminoguanidine, an NO synthase inhibitor, were superior to alpha-tocopherol in preventing RINm5F cell toxicity induced by Interleukin-1 beta (IL-1 beta). Both tocored and tocoyellow were observed to form in RINm5F cells loaded with gamma-tocopherol and producing NO constitutively, although a consistent increase in these products as a result of induced NO synthesis was not observed. PMID- 7557542 TI - Structural identification of valine hydroperoxides and hydroxides on radical damaged amino acid, peptide, and protein molecules. AB - We have previously demonstrated the formation of two reactive moieties on proteins during free radical attack: hydroperoxides, and 3,4 dihydroxyphenylalanine (DOPA). Here we have undertaken the structural elucidation of the hydroperoxides of valine, the amino acid which is most susceptible to peroxidation. Exposure of L-valine to free radicals generated by radiolysis in an oxygen-saturated system produced three valine hydroperoxides. Upon treatment with sodium borohydride these were reduced to their corresponding hydroxides, which can be separated and purified by high performance liquid chromatography (HPLC). Based on spectroscopic data from high resolution chemical ionization (CI) mass spectrometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton (1H) nuclear magnetic resonance (NMR) and carbon-13 (13C) NMR studies, the three valine hydroxides have been identified as beta-hydroxyvaline [(2S)-2-amino-3 hydroxy-3-methyl-butanoic acid], (2S,3S)-gamma-hydroxyvaline [(2S,3S)-2-amino-3 hydroxymethyl-butanoic acid], and (2S,3R)-gamma-hydroxyvaline [(2S,3R)-2-amino-3 hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyvalines provides a sensitive and accurate method for quantitative measurement. This method enabled hydroxyvalines to be detected in the hydrolysates of a tripeptide (glutamyl-valinyl-phenylalanine) and a protein (bovine serum albumin) that had been gamma-radiolysed and treated with sodium borohydride. Hydroxyvaline may be useful as a marker in studying protein oxidation in some biological systems under oxidative stress. PMID- 7557541 TI - Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation. AB - Commonly used spectrophotometric methods for determining the extent of lipid peroxidation in animal tissue extracts, such as measurements of diene conjugation and thiobarbituric acid reactive substances (TBARS), have been criticized for their lack of specificity. This study shows that lipid hydroperoxides can be effectively quantified in animal tissue extracts using an assay based on the formation of a Fe(III)xylenol orange complex. Addition of H2O2, cumene hydroperoxides, or methanolic tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)xylenol orange complex absorbance peak at 560-580 nm with a corresponding decrease in the xylenol orange peak at 440 nm. Complex formation measured at 580 nm was saturable with both xylenol orange and Fe (II) concentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) caused a saturable reduction of the Fe(III)xylenol orange complex. Formation of the Fe(III)xylenol orange complex was linear with the amount of tissue extract added. A significant correlation (r = 0.88, p < 0.005) existed between the xylenol orange method of estimating lipid peroxidation and the conventional TBARS assay in a series of animal tissues tested. The time course of increase in A580nm in tests using tissue extracts was typical of a free radical reaction; a lag phase was followed by a log phase. No increase in A580nm was observed up to 24 h when highly peroxidizable arachidonic acid was assayed. These results indicate that the formation of the Fe(III)xylenol orange complex reflects a chemical amplification of the original level of lipid hydroperoxides present in tissue extracts and that peroxidizable lipids do not influence the assay. The potential usefulness of the xylenol orange assay for comparative biochemical and toxicological studies of oxidative stress is discussed. PMID- 7557543 TI - DNA methylation by tert-butyl hydroperoxide-iron (II). AB - Reduction of tert-butyl hydroperoxide (TBHP) by iron (II) at pH 4.0 or pH 7.0 in the presence of calf thymus DNA led to generation of high yields of methyl radicals and to DNA methylation. Methyl radicals were identified by spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and alpha-(4-pyridyl-1 oxide)-N-tert-butylnitrone (POBN). The methylated DNA-base adducts were identified in treated DNA hydrolysates by high pressure liquid chromatography (HPLC) and photodiode array UV spectroscopy. In the case of DNA several methylated adducts were identified, namely N7-methylguanine, C8-methylguanine, N3 methyladenine, and O6-methylguanine. By contrast, 2'-deoxyguanosine is alkylated almost exclusively to C8-methyl-2'-deoxyguanosine. These results constitute the first evidence that TBHP is able to alkylate DNA. PMID- 7557544 TI - Oxidative stress by acute acetaminophen administration in mouse liver. AB - Acetaminophen was given to mice at a single dose of 375 mg/kg. In situ liver chemiluminescence, H2O2 steady-state concentration, and the liver concentrations of total and oxidized glutathione were measured 15, 30, and 60 min after acetaminophen administration. Increases of 145% and 72% in spontaneous chemiluminescence and H2O2 concentration were observed 15 min after the injection, respectively. Total glutathione was decreased by acetaminophen administration at all the times studied. The maximal decrease, 83%, was found 60 min postinjection. The ratio GSH/GSSG was found significantly decreased at all the times studied. Microsomal superoxide production was increased by 2.4-fold by addition of acetaminophen. The activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase were determined. Catalase was slightly inhibited (30%) 15 min after acetaminophen administration. No significant changes were found in superoxide dismutase activity. Se and non-Se glutathione peroxidase activities were decreased by 40% and 53% respectively, 15 min after acetaminophen administration. The decrease in catalase and glutathione peroxidase would result in an increased steady state level of H2O2 and hydroperoxides, contributing to cell injury. Damaged hepatocytes were observed, and severe lesions and necrosis appeared 60 min after acetaminophen administration. Our results indicate the occurrence of oxidative stress as a possible mechanism for acetaminophen-induced hepatotoxicity. PMID- 7557545 TI - Oxidative damage in human liver transplantation. AB - The aim of this study was to evaluate oxygen-dependent hepatic reperfusion injury in humans following orthotopic liver transplantation. To this end, a number of blood indices of impaired tissue redox balance were monitored in 19 adult patients for 3 weeks after liver transplantation. Both red cell malonaldehyde and plasma lipid peroxides increased significantly soon after organ reperfusion. This finding was consistently accompanied by decreased plasma vitamin E and red cell total glutathione. A peak of oxidative stress, as measured by the parameters monitored, was evident within 24 h after reperfusion, together with a maximum expression of cytolysis, as measured by plasma alanine aminotransferase. The occurrence of redox imbalance after hepatic reperfusion was shown to be linearly related to irreversible cell damage. As regards the low plasma levels of the two antioxidants after reperfusion, only that of vitamin E appeared statistically related to oxidative stress. With the background of an increasing body of proof, mainly from animal models, the involvement of toxic oxygen metabolites in hepatic cytolysis following orthotopic liver transplantation appears likely. The statistical correlation among the markers of redox imbalance monitored indicates their combined use in further investigation. PMID- 7557546 TI - Plant defense metabolism is increased by the free radical-generating compound AAPH. AB - Effects of the free radical-generating substance 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) on defense systems in plant tissue cultures were investigated. Exposure of Catharanthus roseus, C. tricophyllus, and Pisum sativum cultures to AAPH caused altered levels of reduced and oxidized glutathione. An increased total glutathione content in C. roseus was prevented by the glutathione biosynthesis inhibitor buthionine-sulfoximine. The specific phenylalanine ammonia lyase activity in a C. roseus culture was increased from 4 to 34 mukat(kg protein)-1 by 1 mM AAPH. 5 mM AAPH increased the excretion of phenolic substances into the culture medium of a Pisum sativum culture, from 18 to 67 micrograms ml 1. The level of thiobarbituric acid reactants in a C. tricophyllus culture was increased from 46 to 93 nmol(g fresh weight)-1 by 0.4 mM AAPH. The present results, which constitute the first report on effects of the radical-generator AAPH on plant tissue, were achieved with cultures of various plant species and various types of tissue differentiation and demonstrate that AAPH is a suitable agent for the stimulation of the defensive and secondary metabolism in plant tissue cultures. It is proposed that the effects caused by AAPH are mediated by the generation of free radicals and oxidative stress, and that this agent may be used as a model substance for ozone and UV-B exposure. PMID- 7557547 TI - Reduction of ferrylmyoglobin by dietary phenolic acid derivatives of cinnamic acid. AB - The reaction of dietary phenolic acid derivatives of cinnamic acid with high valence states of horse myoglobin was studied. When metmyoglobin was oxidized by hydrogen peroxide (H2O2) in the presence of the phenolic acids, ferrylmyoglobin was reduced to metmyoglobin. However, addition of the phenolic acids to a ferrylmyoglobin solution resulted in a modified metmyoglobin spectrum characterized mainly by a blue shift of the 631 nm peak and a new isosbestic point at 613 nm suggesting an irreversible modification of the hemeprotein. The efficiency and the kinetic profile of ferrylmyoglobin reduction were dependent on both the concentration and the structure of the phenolic acid. Electron-donating substituent groups in the phenol ring increased the efficiency of ferrylmyoglobin reduction whereas electron-withdrawing groups decreased it. The phenolic acids exhibiting a catechol structure were the most efficient in reducing ferrylmyoglobin. Caffeic and chlorogenic acids react faster than trolox, and caffeic acid faster than ascorbate. During the electron-transfer reactions, the phenolic acids were oxidized to quinoid forms and, in some cases, to polymer products as indicated by comparison of the ultraviolet (UV) spectra obtained in the presence of metmyoglobin/H2O2 with those recorded after oxidation of phenolic acids by horseradish peroxidase/H2O2 and catechol oxidase. The results suggest that dietary phenolic derivatives of cinnamic acid may counteract deleterious oxidations initiated by ferrylmyoglobin. PMID- 7557548 TI - Antioxidant inhibition of thymocyte apoptosis by dihydrolipoic acid. AB - Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis, and that this type of cell death can be inhibited by various thiol containing antioxidants such as N-acetyl cysteine. To study the effects of a physiologically important thiol reductant, rat thymocytes were preincubated with either lipoic acid, dihydrolipoic acid, or lipoamide and then exposed to methylprednisolone or etoposide, two stimuli known to induce apoptosis in these cells. Dihydrolipoic acid and lipoamide both exerted an inhibitory effect on apoptosis induced by the two stimuli, while lipoic acid was inactive. Inhibition of apoptosis was evident as (a) reduced formation of condensed, pyknotic nuclei; (b) a prevention of cell shrinkage; and (c) decreased chromatin degradation. Furthermore, the depletion of reduced glutathione that occurs as thymocytes undergo apoptosis was also prevented in the presence of DHLA. Investigation of the pattern of chromatin fragmentation revealed that DNA in the antioxidant loaded thymocytes remained above 50 kb pairs in size, indicating that inhibition by DHLA was operative at an early step in the apoptotic pathway. These results suggest that intracellular oxidation is an obligate, early component of thymocyte apoptosis. PMID- 7557550 TI - On the mechanism of thyroid hormone-induced respiratory burst activity in rat polymorphonuclear leukocytes. AB - Administration of single doses of 0.1 mg L-triiodothyronine (T3)/kg for 3 consecutive days to fed rats produced a drastic increase in the respiratory burst activity of isolated polymorphonuclear leukocytes (PMN), stimulated with serum opsonized zymosan. This effect was evidenced by the 3.8-fold increment in the integrated chemiluminescence, and seems to be primarily related to the enhanced activity of NADPH oxidase elicited by T3 treatment, with the observed higher myeloperoxidase activity playing a contributory role. In these conditions, hyperthyroidism determines a net enhancement in the oxidant capacity of PMN, as the increased rate of O2.- generation found occurs in the absence of changes in the activity of superoxide dismutase. PMID- 7557551 TI - Action spectrum for UV-induced lipid peroxidation in cultured human skin fibroblasts. AB - Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) into the supernatant of cultured human skin fibroblasts. This process is triggered by ultraviolet A (UVA) and ultraviolet B (UVB) radiations. For UVA irradiances and irradiation times up to 40 W.m-2 and 90 min, respectively, the peroxidation response is linear and obeys the reciprocity law. Corresponding values for UVB are 12 W.m-2 and 30 min, respectively. The action spectrum of the peroxidation process shows a continuously increasing response from about 425 to 275 nm. Whereas the UVB to UVA effectiveness ratio lies in the range of 10(3) to 10(4) for most in vitro or in vivo UV-induced responses, the ratio is only 10 to 100 for the peroxidation process. Given the solar spectral distribution, solar UVA radiation is by far the most effective in triggering the peroxidation response. PMID- 7557549 TI - 21-aminosteroid and 2-(aminomethyl)chromans inhibition of arachidonic acid induced lipid peroxidation and permeability enhancement in bovine brain microvessel endothelial cell monolayers. AB - Selected 21-aminosteroids (U74500A, U74006F, and U74389G) and a 2 (aminomethyl)chromans (U78517F) were tested for their efficacy in preventing arachidonate-induced lipid peroxidation and permeability alterations in brain microvessel endothelial cells (BMECs). The 21-aminosteroids and 2 (aminomethyl)chromans were effective in varying degrees in inhibiting (U74500A = U78517F > U74006F = U74389G) concentration- and time-dependent arachidonate induced thiobarbituric acid reactive substances (TBARS) production by BMECs. Arachidonate produced a corresponding concentration-dependent increase in BMEC monolayer permeability to the membrane impermeant marker, sucrose. Pretreatment of BMEC monolayers with either the 21-aminosteroids or the 2 (aminomethyl)chromans completely blocked the arachidonate-induced increase in permeability to sucrose. Our results demonstrated that these membrane-associating antioxidants were particularly effective in preventing both arachidonic acid induced lipid peroxidation and permeability changes in BMEC monolayers. However, concentrations of some antioxidants that only partially inhibited TBARS production, completely inhibited the arachidonic acid-induced enhancement in BMEC monolayer permeability. Therefore, arachidonic acid-induced effects on BMEC permeability were likely due in part to both lipid peroxidation and direct or indirect effects of the fatty acid on membrane integrity. This study provides further support for the application of primary cultures of BMECs as a useful in vitro system to evaluate mechanisms through which mediators of disease or injury states compromise blood-brain barrier integrity. PMID- 7557552 TI - 7,12-dimethylbenz[a]anthracene induces oxidative DNA modification in vivo. AB - Initiation and promotion are major stages in the multistage carcinogenesis process. Formation of initiating carcinogen-DNA base adducts leads to heritable genetic changes, but the tumor-promoting events induced by complete carcinogens have not, as yet, been elucidated. Oxidant production and oxidative DNA damage induced by phorbol esters (i.e., 12-O-tetradecanoyl-phorbol-13-acetate) are associated with tumor promotion, while antioxidants and inhibitors of oxidative DNA damage suppress promotion and carcinogenesis. Our goal was to establish whether a carcinogen that requires oxidative metabolism for its activity can also induce oxidant production and DNA base oxidation. We found that topical treatment of SENCAR mice with 7,12-dimethylbenz[a]anthracene, which induces tumors in 40 50% of the mice, also causes hydrogen peroxide production and formation of oxidized bases (i.e., 8-hydroxyl-2'-deoxyguanosine and 5-hydroxymethyl-2' deoxyuridine) in epidermal DNA. The levels of oxidized bases were of comparable magnitude to those mediated by the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. The oxidized bases persisted over several weeks in epidermal DNA. These oxidative events appear to be temporally associated with inflammatory responses that include edema and polymorphonuclear leukocyte infiltration, which remained elevated over longer periods of time and at higher levels than those induced by phorbol ester. Because these processes are usually associated with tumor promotion, our results support the conjecture that oxidative events may be involved in what is operationally referred to as the tumor promotion process by 7,12-dimethylbenz[a]anthracene. PMID- 7557554 TI - Molecular basis of neuronal plasticity to gonadal steroids. PMID- 7557553 TI - How do Ca2+ and 5-aminolevulinic acid-derived oxyradicals promote injury to isolated mitochondria? AB - The biosynthetic heme precursor 5-aminolevulinic acid (5-ALA) is a generator of oxygen radicals in vitro and possibly in vivo during pathologic situations of 5 ALA overload, for example, acute intermittent porphyria and saturnism. It has been observed that 5-ALA induces, in isolated rat liver mitochondria, permeabilization of the inner mitochondrial membrane (a phenomenon called permeability transition) as verified by the elimination of the transmembrane electrical potential, Ca2+ release, mitochondrial swelling, and increase in state 4 respiratory rate. The damaging process is primarily attributed to .OH radicals as elucidated by the protection by catalase, superoxide dismutase, and the Fe(II) chelator o-phenanthroline. Ruthenium red, EGTA, and dithiothretol (DTT) have been observed to prevent the action of 5-ALA-generated oxyradicals, suggesting the participation of both Ca2+ and the oxidation of critical thiol membrane proteins in the process of permeability transition. 5-ALA-induced polymerization of thiol membrane proteins has also been demonstrated by SDS-PAGE electrophoresis of the mitochondrial suspensions, a process similar to that observed in mitochondria treated with tert-butyI hydroperoxide. EGTA addition, in contrast with DTT or antioxidants, restores the previously eliminated electrical potential. Furthermore, EGTA prevents the 5-ALA-mediated polimeryzation of thiol proteins. These observations suggest that Ca2+ participates in a later stage of the permeability transition, after the oxidation of the thiol proteins. The effects of 5-ALA-derived oxyradicals in isolated mitochondria could be used as a tool for more general studies of oxidative stress, such as the mitochondrial injury that follows processes of ischemia and reperfusion or xenobiotic poisoning. PMID- 7557555 TI - Neurographic studies in hemiplegic patients. AB - The aim of this investigation was to re-establish the nature of the involvement of the peripheral nervous system after lesions of the central nervous system. An examination of 120 patients with spastic hemiparesis after stroke was performed. The motor conduction velocities, M-response latencies and amplitudes, as well as F-wave latencies were evaluated in median, ulnar, peroneal and tibial nerves. The minimal latency of soleus H-reflex was obtained. Our results revealed normal motor conduction velocities, and distal M-response and H-reflex latencies on the involved side. However the amplitudes of the M-responses were decreased and the latencies of the F-waves prolonged on the spastic side. In conclusion primary axonal degeneration with secondary segmental demyelination, involving predominantly proximal parts of the peripheral nerves, occurs after central nervous system injury. PMID- 7557556 TI - May there exist specific MRI findings predictive of dementia in multiple sclerosis patients? AB - Cognitive deficits are present in a substantial number of Multiple Sclerosis (MS) patients, particularly in those with the chronic-progressive type of the disorder. We assessed cognitive decline and its relationship with T2-weighted images on magnetic resonance imaging (MRI). We submitted a group of 26 patients with progressive MS to both MRI and a battery of neuropsychological tests. Cognitive impairment did not correlate with duration of illness or severity of neurological disability, but rather with the presence of extensive periventricular demyelination on MRI, evaluated as area of confluent lesions. These results suggest that cognitive deficits in MS represent a symptom of disease and not a parallel occurrence. PMID- 7557557 TI - Effect of picotamide and aspirin, combined or alone, on platelet aggregation in patients with cerebral infarction. AB - After 7 and 90 days of treatment, we studied the effect of picotamide, a thromboxane synthase inhibitor (450 and 900 mg/day), aspirin (150 mg/day), and aspirin plus picotamide (150 and 450 mg/day respectively) on platelet aggregation, evaluated in platelet rich plasma of 48 patients affected by ischemic stroke. Platelet aggregation, induced by collagen (1.0 and 2.0 micrograms/ml) and adenosine diphosphate (1.0 and 10 micrograms/L), was significantly increased in patients in comparison with healthy controls. Aspirin (150 mg/day) reduced collagen-induced platelet aggregation (1.0 microgram/ml) after 7 days of treatment. Picotamide (450 mg/day) reduced platelet aggregation induced by both concentrations of collagen, while the higher dose (900 mg/day) had no significant effect. Aspirin plus picotamide reduced the aggregation induced by 1.0 microgram/ml collagen and by 10 mumol/L adenosine diphosphate after 90 days of therapy. This study has shown that patients during the acute phase of stroke are characterized by an increased in vitro platelet aggregation. Aspirin may be beneficial in the acute phase of the cerebral ischemic event. Picotamide and picotamide plus aspirin could be useful for reducing platelet aggregation in long term treatment. PMID- 7557558 TI - Short-latency median nerve somatosensory evoked potentials in three cases of parkinsonism and dopa responsive dystonia. AB - We studied somatosensory evoked potentials after median nerve stimulation in a sporadic case of dopa responsive dystonia and in two brothers with different combinations of dystonia and parkinsonism. The latencies of all potentials were normal. The amplitude of the P20-N30 frontal complex showed a significant reduction in all cases. Our results suggest a common neurophysiopathological pattern underlying these two conditions. PMID- 7557559 TI - Endoscopic intervention in bleeding peptic ulcer. PMID- 7557560 TI - Necrosis and apoptosis in the gastrointestinal tract. PMID- 7557563 TI - Late mortality in elderly patients surviving acute peptic ulcer bleeding. AB - Acute peptic ulcer bleeding is associated with a substantial short term mortality but it is generally assumed that in the modern era of effective medical treatment the longer term prognosis is good. This study evaluated 487 patients aged over 60 years who were discharged from Nottingham University and City Hospitals after admission for acute peptic ulcer bleeding during 1986-91 and 480 age and sex matched community controls. Follow up information was obtained from hospital and general practitioner records and from the National Health Service central register. Mortality was compared with control mortality and with rates expected for England and Wales. During a mean follow up of 34 months 142 (29%) of 487 patients died compared with 58 (12%) of 480 community controls and with 81.5 deaths expected (observed/expected (O/E) = 1.74, 95% confidence limits (CL) 1.5 to 2.1). Six years after admission the actuarial survival estimate was only 50% for ulcer patients compared with 76% for community controls and 69% expected. The increased mortality was similar in men and women and was greatest in the 60-74 year age group. Much of the excess mortality was accounted for by deaths from cancer (O/E 34/19.7 = 1.73; CL 1.2 to 2.4), from respiratory disease (O/E 28/10.9 = 2.57; CL 1.7 to 3.7), and in men from vascular disease (O/E 31/22.4 = 1.38; CL 0.9 to 2.0). Eight deaths resulted from recurrent ulcer complications and four deaths from gastric cancers undetected at the index admission. In conclusion, patients discharged after peptic ulcer bleeding had a substantially reduced life expectancy. The increased mortality was predominantly due to a variety of smoking related diseases rather than recurrent peptic ulcer complications. Deaths from recurrent peptic ulcer complications were infrequent and were less than reported in earlier years possibly reflecting prolonged and widespread used of H2 receptor antagonists. PMID- 7557562 TI - Combined radiochemotherapy for postoperative recurrence of oesophageal cancer. AB - Postoperative recurrences are common after resection for oesophageal cancer. From January 1986 to September 1993 31 patients (30 males, one female, mean (SD) age: 57.5 (8.8) years) were treated for locoregional recurrence (n = 24), metastases (n = 6) or both (n = 1) occurring 15.0 (12.6) months after initial surgery. Radiotherapy and chemotherapy were combined in all cases. Symptomatic improvement was seen in 23 cases (74%) and lasted (excluding treatment period) for 6.3 (4) months. Objective tumoral response was seen in 20 patients (65%) including eight (26%) complete responses. Survival rates were at respectively six months, one, two, and three years: 70.7%, 47.1%, 17.1%, 4.3%. In conclusion, these results show that combined therapy could have a beneficial symptomatic effect and can be associated with prolonged survival in patients with postoperative recurrences of oesophageal cancer. PMID- 7557561 TI - Mucosal reactive oxygen species production in oesophagitis and Barrett's oesophagus. AB - Reactive oxygen species (ROS) produced by inflammatory cells can contribute to tissue destruction. ROS have been implicated in various gastrointestinal abnormalities, including the acid related peptic diseases. Although the development of oesophagitis and Barrett's columnar epithelium is associated with prolonged reflux of gastric acid, the exact mechanism by which tissue damage occurs is not known. To discover if ROS are involved in damage to the oesophageal mucosa, this study measured in vitro the mucosal ROS concentrations of biopsied mucosal samples taken from patients with reflux oesophagitis using luminol enhanced chemiluminescence (LECL). Mucosal biopsy specimens were taken from 83 patients: 19 with normal oesophageal mucosa (group I); 20 with macroscopic oesophagitis (group II); 20 with biopsy confirmed Barrett's epithelium without macroscopic oesophagitis (group III); and 24 with Barrett's epithelium with macroscopic oesophagitis (group IV). The mucosa from patients exhibited significantly higher LECL values than the mucosa from controls. But, there were no significant differences between groups II, III, and IV. Addition of the myeloperoxidase inhibitor, azide, or the hydrogen peroxide scavenger, catalase, to the tissue suspension caused a decrease in LECL values of 32% and 45%, respectively, suggesting that neutrophils--although important--are not the only source of mucosal LECL. These data are consistent with the proposal that ROS play an important part in the tissue injury associated with oesophagitis and Barrett's columnar epithelium. PMID- 7557564 TI - Morbidity and treatment in elderly patients surviving hospital admission with bleeding peptic ulcer. AB - Bleeding peptic ulcer in the elderly is associated with use of non-steroidal, anti-inflammatory drugs (NSAIDs) and aspirin, presenting a therapeutic dilemma for the treatment of survivors. To determine the longterm morbidity of patients surviving bleeding peptic ulcer and their treatment, with particular reference to NSAID use, this study followed up 487 patients aged over 60 years, who survived an episode of bleeding peptic ulcer in Nottingham during 1986 to 1991. Information was gathered by evaluation of general practitioner and hospital records, scrutiny of death certificates, and postal questionnaire to surviving patients. Of 487 patients surviving a bleeding peptic ulcer, 345 were alive at the follow up, a mean 34.2 months after discharge. Of these 201 (58%) had taken NSAIDs before their ulcer bleed (104 non-aspirin NSAIDs, 77 aspirin, 20 both). Compared with non-users, the patients using NSAIDs at the time of presentation were significantly more likely subsequently to consult their general practitioner with locomotor symptoms (56% v 35%, p < 0.001) and receive further NSAIDs (34% v 13%, p < 0.001), but significantly less likely to consult their general practitioner for dyspepsia (31% v 54%, p < 0.001). Antiulcer drug use was widespread in both groups, with an overall point prevalence of 44%. Seventy five patients received NSAIDs during the follow up period (69% with coprescription of antiulcer drugs, usually H2 antagonists). Recurrent peptic ulcer, ulcer complications, and ulcer related deaths were uncommon. Antiulcer drug use was lower in those with ulcer recurrence compared with the point prevalence in those without (17% v 44%, p < 0.05). Patient questionnaire responses confirmed a high level of locomotor symptoms especially in patients previously admitted with NSAID associated ulcer bleeding. In conclusion, in patients surviving peptic ulcer bleeding, recurrent ulcer complications and death seem to be lower than reported by studies that preceded the advent of antiulcer drugs. It is suggested that this low incidence may be a consequence of their use. Granted the low level of recurrent ulceration and the high level of locomotor symptoms in patients studied, current treatment may unduly emphasise the importance of stopping NSAIDs in this group of patients. PMID- 7557565 TI - Value of laparotomy in the diagnosis of obscure gastrointestinal haemorrhage. AB - Over a nine year period a total of 137 patients were investigated for obscure gastrointestinal bleeding on one surgical unit. In 20 patients visceral angiography strongly suggested the presence of caecal or right colonic angiodysplasia. These patients were treated by an appropriate colectomy and they are not considered further in this study. Similarly lesions of the small bowel detected by preoperative investigations are not considered here. Fifty five patients were offered diagnostic laparotomy after the failure of other investigations to establish a diagnosis. Two patients refused. A diagnostic laparotomy was performed in the remaining 53. At operation if no visible lesion was seen an on table enteroscopy was performed using a colonoscope passed per oram and, if necessary, per anum. In nine (17%) patients no cause for bleeding was found. In 18 (34%) patients there was a small bowel vascular anomaly, in 14 (26%) a small bowel tumour, in four (7.5%) a bleeding Meckel's diverticulum, and in eight (15%) other miscellaneous lesions. Laparotomy, with on table enteroscopy where indicated, elucidated the cause of bleeding in 44 patients (83%). It was associated, however, with a postoperative death rate of 7.5% (four patients). After seemingly appropriate surgery, rebleeding occurred in 14 patients (26%). Of 18 patients with small bowel vascular anomalies seven rebled (39%), at an average follow up interval of 32 months. PMID- 7557566 TI - Angiogenesis in gastric ulcers: impaired in patients taking non-steroidal anti inflammatory drugs. AB - Non-steroidal anti-inflammatory drug (NSAID) therapy is associated with delayed gastroduodenal ulcer healing. In rats the degree of angiogenesis (new vessel formation) within the ulcer bed correlates strongly with the extent and speed of ulcer healing and may be inhibited by NSAIDs. This study therefore assessed the vascularity of 38 antral gastric ulcers immunohistochemically, using CD31 a vascular endothelial cell marker, in 17 patients taking NSAIDs and 19 control patients. In the superficial granulation tissue NSAID therapy was associated with a significant reduction in the median number of capillaries (13.5 (IQR: 9.5-18) v 23.5 (14-31) (p < 0.005)), number of vessel buds (6 (4-12.5) v 17 (12-23) (p < 0.05)), and maximum vessel diameter (29 (20.75-30.75) v 33.75 (24-45) (p < 0.05)) when compared with controls. In deep granulation tissue NSAID therapy was similarly associated with a significant reduction in the number of capillaries (9 (6.5-12) v 14 (9-19.25) (p < 0.04)), number of vessel buds (5 (3.5-8.5) v 13 (7 16.5) (p < 0.01)), and maximum vessel diameter (23 (18-20.5) v 33 (21.5-45) (p < 0.02)). There were no differences in vascularity in the adjacent glandular mucosa. Impairment of angiogenesis may be an important mechanism of NSAID related delayed ulcer healing. PMID- 7557567 TI - Effect of longterm misoprostol coadministration with non-steroidal anti inflammatory drugs: a histological study. AB - Prostaglandins are widely used in the prevention and healing of non-steroidal anti-inflammatory drug (NSAID) induced gastric and duodenal ulcers, but their longterm effect on the human gastric mucosa is unknown. This study assessed the effect of coadministration of prostaglandins with NSAIDs on the histology of the gastroduodenal mucosa. Histological appearances (using the Sydney system) of gastric biopsy specimens from 180 patients receiving longterm NSAID treatment of whom 90 had been receiving misoprostol (400-800 micrograms/day) for one to two years were studied. Both groups of patients were comparable with regard to clinical and demographic details. There was no significant difference (p > 0.1) in the prevalence of chronic gastritis (total, corpus or antrum only) between patients receiving (36 of 90 (40%)) or not receiving misoprostol (35 of 90 (39%)). Chronic gastritis was equally associated with the presence of Helicobacter pylori, 86% and 73% (p > 0.1), respectively, in the two groups. Significantly fewer patients receiving misoprostol had reactive gastritis than those receiving only NSAIDs (8 (9%) versus 27 (30%), p < 0.01). Reactive gastritis was not associated with H pylori. Thirty nine (43%) of the misoprostol treated patients had normal histology compared with 16 (18%) receiving only NSAIDs (p < 0.01). These results show two different patterns of gastric damage in patients receiving NSAIDs, namely chronic and reactive gastritis. Misoprostol treatment was associated with a significantly reduced prevalence of reactive gastritis and it is suggested that this, along with its antisecretory action, may explain the reduced prevalence of gastroduodenal lesions when coadministered with NSAIDs. PMID- 7557570 TI - Behaviour of acid secretion, gastrin release, serum pepsinogen I, and gastric emptying of liquids over six months from eradication of helicobacter pylori in duodenal ulcer patients. A controlled study. AB - The behaviour of basal and stimulated acid secretion, gastrin release, serum pepsinogen I, and gastric emptying of liquids was studied in 19 consecutive patients with Helicobacter pylori positive duodenal ulcer, over a follow up period of six months. Eleven patients were studied before and at three and six months after eradication with lansoprazole plus amoxicillin and tinidazole (case group), whereas the remainder, with persistent H pylori infection, were studied before and after three and six months from ulcer healing, thus constituting the control group. In the case group, three months after eradication, fasting serum pepsinogen I fell from (mean (SEM)) 91.9 (6.9) (pretreatment) to 72.2 (5.1) ng/l and the integrated gastrin response to a meal reduced from 11,470 (1174) (pretreatment) to 8130 (608) pg/ml/h (p < 0.05). Fasting serum gastrin concentrations and maximal acid output reduced significantly only six months after eradication. In contrast, no significant change of any of these measurements was seen in the control group either at three or six months from healing compared with the pretreatment values. Gastric emptying of liquids did not change over the entire period of follow up in both study groups. In conclusion, eradication of H pylori in duodenal ulcer patients is accompanied by a rapid fall in serum pepsinogen I and plasma gastrin concentrations, whereas a slight but significant reduction of maximal acid secretion takes place later on. In contrast, gastric emptying of liquids does not seem to be influenced by H pylori status. PMID- 7557569 TI - Expression of trefoil peptides pS2 and human spasmolytic polypeptide in gastric metaplasia at the margin of duodenal ulcers. AB - Duodenal ulcers are associated with gastric metaplasia in the duodenum, both at the ulcer margin and at more distant sites in the duodenal bulb. pS2 and human spasmolytic polypeptide (hSP) are secretory peptides expressed in gastric epithelial cells and in gastric metaplasia. As these peptides may be important in ulcer healing, this study investigated the possibility that the expression of pS2 and hSP is increased in gastric metaplasia at the margin of duodenal ulcers. Duodenal bulb biopsy specimens from 12 duodenal ulcer patients were assessed. Sections were immunostained with monoclonal antibodies for pS2 and hSP. Cytoplasmic stain intensities were measured by an image analysis system and expressed as integrated optical density (IOD) units, In situ hybridisation for pS2 and hSP mRNA was carried out on parallel sections. Duodenal sections were also stained with diatase periodic acid Schiff/alcian blue to localise areas of gastric metaplasia. pS2 antigen staining in the duodenum was restricted to surface epithelial cells, and hSP to acinar and ductular components of Brunner's gland. mRNA localisation corresponded to immunostaining cells. In gastric metaplasia, pS2 expression was greater at the ulcer margin than away from the ulcer, as judged by the intensity of antibody staining (mean IOD units (SEM), 20.6 (3.3) v 9.5 (3.0); p < 0.001). There was a trend towards greater hSP staining at the ulcer margin but this did not achieve statistical significance. These findings support the putative role of pS2 and possible hSP in mucosal healing and providy further evidence for an autocrine 'ulcer-gastric metaplasia repair' loop involving these trefoil peptides. PMID- 7557568 TI - Seroepidemiology of gastritis in Japanese and Dutch working populations: evidence for the development of atrophic gastritis that is not related to Helicobacter pylori. AB - Serological markers of gastritis, like pepsinogen A, pepsinogen C, gastrin, and Helicobacter pylori antibodies, can be used to explore the state of the gastric mucosa in populations with contrasting cancer risks. A decreasing pepsinogen A:C ratio and an increasing serum gastrin are known to reflect an increasing severity of atrophic corpus gastritis, which is a precursor of gastric cancer. In 723 subjects (without gastroduodenal surgery) from Japanese (n = 225) and Dutch (n = 498) working populations, which had a similar composition of age (mean 48 years), sex (male to female ratio 6:1), and type of occupation, fasting serum samples were analysed for IgG antibodies to H pylori, pepsinogen A, pepsinogen C, and gastrin in the same laboratory. H pylori infection was significantly more prevalent in the Japanese than in the Dutch (74.7% and 31.3%); as was a very low pepsinogen A, indicative of severe mucosal atrophy (4.4% and 1.6%). Among subjects with and without severe mucosal atrophy the H pylori seropositivity rate was similar. Between the Japanese and the Dutch there were significant differences in mean gastrin (31.8 and 13.4 pmol/l) and pepsinogen A:C ratio (1.7 and 2.9). These intercountry differences were as great for H pylori negative subjects (gastrin: 23.7 and 10.3 pmol/l, pepsinogen A:C ratio: 2.4 and 3.2) as for H pylori positive subjects (gastrin: 34.6 and 20.1 pmol/l, pepsinogen A:C ratio: 1.5 and 2.5). The intercountry difference in gastrin nearly disappeared after stratification into categories of pepsinogen A:C ratio. In conclusion, the intercountry differences in pepsinogen A:C ratio and gastrin reflect a higher prevalence of mild and severe mucosal atrophy of the corpus in the Japanese than in the Dutch, both among H pylori positive and negative subjects. Thus, these findings suggest that in the Japanese the development of atrophic gastritis is in part unrelated to H pylori. PMID- 7557574 TI - Basal secretion and anaphylactic release of rat mast cell protease-II (RMCP-II) from ex vivo perfused rat jejunum: translocation of RMCP-II into the gut lumen and its relation to mucosal histology. AB - The kinetics of the release of rat mast cell protease-II (RMCP-II) from mucosal mast cells in the jejunum of Nippostrongylus brasiliensis primed (immune) rats was investigated using ex vivo perfusion of a segment of jejunum through the cranial mesenteric artery. The aim of the study was to assess the role of the protease in anaphylaxis and in particular to ascertain whether it is responsible for the histological changes, which include widespread epithelial shedding, seen in the mucosa in in vivo models of anaphylaxis. Perfusion of the jejunal vasculature with a Krebs-Ringer buffer showed that there was basal secretion of RMCP-II by jejunal mast cells in all rats studied. The baseline concentration of RMCP-II was significantly greater (p < 0.05) in immune rats (> 7 ng/ml) previously exposed to nippostrongylus infection than in control, naive animals (< 2 ng/ml). Challenge of immune rats with 100 or 400 worm equivalents of whole worm antigen resulted in an immediate (within 40 seconds) and significant (p < 0.02) increase in the concentration of RMCP-II (to > 3 micrograms/ml) in the vascular perfusate, which was not seen in naive rats or immune rats challenged with an irrelevant antigen. Greater amounts of RMCP-II were also recovered from the jejunal lumen of immune rats compared with naive rats after challenge of both groups with worm antigen. Despite the release of microgram quantities of RMCP-II into the gut lumen and vascular perfusate, however, there were no significant changes seen in the mucosal histology. These results suggest that RMCP-II alone is not responsible fore the loss of gut epithelium seen during anaphylaxis in the rat. PMID- 7557572 TI - Detection of low bone mineral density by dual energy x ray absorptiometry in unsuspected suboptimally treated coeliac disease. AB - Patients with coeliac disease may present with calcium malabsorption but it is unclear whether this results in longterm impairment of bone mineralisation. Dual energy x ray absorptiometry (DXA) was used to study bone mineral density in 34 asymptomatic coeliac disease patients, treated with a gluten free diet for at least two years, and also in 10 newly diagnosed or untreated patients. As expected, untreated patients had low bone mineral density in all regions. In the 29 treated female coeliac disease patients, overall mean values for age adjusted bone mineral density expressed as Z scores were normal although there were many patients with low values, particularly of the lumbar spine and total body. Scores in the postmenopausal patients were significantly worse than in the premenopausal patients and low mean Z scores were found in the five treated male patients. The subjects who had reduced bone mineral density could not be predicted clinically but, despite being asymptomatic, were more likely to have subtotal or partial villous atrophy on small intestinal biopsy (p < 0.0275). In conclusion, although many treated coeliac disease patients have normal bone mineral density, suboptimally treated and newly diagnosed or untreated patients have osteopenia. To reduce the risk of osteoporotic fractures, it is recommended that bone mineral density be measured in all treated coeliac disease patients and those with osteopenia have a repeat intestinal biopsy to assess disease activity. PMID- 7557571 TI - Effect of bran particle size on gastric emptying and small bowel transit in humans: a scintigraphic study. AB - Bran is an effective treatment for constipation but its use is often limited by heartburn and bloating. This study examined the effect of fine and coarse bran (15 g) on the gastric emptying and small bowel transit of a 325 kcal rice test meal. Twelve healthy volunteers underwent a three way cross over study, ingesting the technetium-99m labelled rice meal with or without 15 g of indium-111m labelled fine or coarse bran, in random order. Serial scintigraphic images were obtained to define gastric emptying and colonic arrival of label. Compared with control values (99 (9) minutes) (mean (SEM)), the time to 50% gastric emptying was significantly delayed by coarse but not fine bran, being 121 (6) and 104 (9) minutes respectively, p < 0.05, n = 12. Fundal emptying was unchanged but both brans seemed to increase the proportion of isotope in the antrum at 90 minutes. Small bowel transit was slightly faster with both bran types but in this study the difference was not significant. Both the bran and rice labels moved down the gut without significant separation. Fine bran causes less disturbance of gastric physiology than coarse bran. PMID- 7557573 TI - Isolation and longterm culture of human intestinal microvascular endothelial cells. AB - Microvascular endothelial cells play an important part in inflammation as well as in organ specific leucocyte traffic, and may be functionally different from large vessel endothelium in this respect. This study therefore established a method for isolation and longterm culture of human intestinal microvascular endothelial cells (HIMEC). After dissociation by collagenase/dispase/DNase of mucosal and submucosal tissue obtained from normal adult jejunum, cells were plated and cultured to subconfluence in endothelial serum free medium containing 2.5% fetal calf serum, hydrocortisone, and N6, O2-dibutyryladenosine cyclic monophosphate. Primary cultures were trypsinised and endothelial cells were isolated by paramagnetic beads armed with monoclonal antibody to CD31. Optimal growth conditions for HIMEC cultures were established, allowing up to nine passages (three months in vitro). The cells contained Weibel-Palade bodies, expressed von Willebrand factor, CD31, and VE-cadherin; and bound Ulex Europaeus lectin I. A method to establish longterm cell cultures of HIMEC will facilitate further investigation of the function of intestinal endothelial cells and their participation in physiological and pathological events in the gut. PMID- 7557575 TI - Activated eosinophils and interleukin 5 expression in early recurrence of Crohn's disease. AB - Endoscopic recurrences after radical surgery for Crohn's disease are useful for studying the pathogenesis of initial lesions of Crohn's disease. Factors predisposing to recurrence are poorly understood, but it has been shown that eosinophilic infiltration of the neoileum may occur within a few weeks of resection. The aim of this study was to compare, in nine patients having an ileocolectomy, the infiltration of eosinophils and their activation state in normal and diseased areas of the neoileum, three months after surgery. Tissue eosinophils were studied by histochemical methods and electron microscopy. Mucosal expression of interleukin 5 (IL 5), an important eosinophil activating factor was studied using in situ hybridisation. Sixty per cent of patients had endoscopic recurrence at three months. Eosinophil infiltration was more pronounced in diseased than in endoscopically normal areas and was associated with a high expression of IL 5 mRNA. Ultrastructural analysis showed features of eosinophil activation, but no cytotoxic lesions of surrounding inflammatory or epithelial cells. This study suggests that local synthesis of IL 5 associated with eosinophil activation in the tissues could participate in early mucosal damage in Crohn's disease. PMID- 7557576 TI - Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. AB - Enhanced nitric oxide (NO) generation by stimulated NO synthase (NOS) activity may, through its oxidative metabolism contribute to tissue injury in experimental colitis. In this study the possible amelioration of experimental colitis by NG nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS activity, was evaluated. Colitis was induced in rats by intracolonic administration of 30 mg trinitrobenzene sulphonic acid (TNB) dissolved in 0.25 ml 50% ethanol or by flushing the colon of capsaicin pretreated rats with 2 ml of 5% acetic acid. In several experiments, L-NAME 0.1 mg/ml was added to the drinking water at the time of colitis induction with TNB or seven days before acetic acid treatment. Rats were killed at various time intervals after induction of colitis. A 10 cm distal colonic segment was isolated, weighed, lesion area measured, and explants organ cultured for 24 hours for determination of NO generation by the Greiss reaction. The rest of the mucosa was scraped for determination of myeloperoxidase and NOS activities and leukotriene generation. In TNB treated rats mean arterial pressure was also determined up to 72 hours after damage induction, with or without cotreatment with nitroprusside. L-NAME significantly decreased the extent of tissue injury in TNB treated rats. Seven days after TNB treatment lesion area was reduced by 55%, colonic weight by 37%, and myeloperoxidase and NOS activity by 59% and 42%, respectively. Acetic acid induced colitis in capsaicin pretreated rats was also significantly decreased by L-NAME. Twenty four hours after acetic acid treatment lesion area was reduced by 61%, colonic weight by 21% and NOS activity by 39%. Mean (SEM) arterial blood pressure in TNB+L-NAME treated rats was 37.6 (8.1) mm Hg higher than in TNB treated rats, an effect that was only partially abolished by nitroprusside. These results show that inhibition of NO synthesis by an L-arginine analogue significantly ameliorates the extent of tissue injury in two models of experimental colitis, an effect that is not due only to its vasoconstrictor properties. Modulation of NO generation may be a novel therapeutic approach in inflammatory bowel disease. PMID- 7557577 TI - Breath hydrogen analysis in patients with ileoanal pouch anastomosis. AB - The possible influence on functional outcomes of hydrogen production in the ileoanal pouch after restorative proctocolectomy was investigated by means of lactulose H2 breath tests. Eight of 15 patients had significant increases in breath hydrogen after 10 g lactulose. One patient declined to participate in further investigations, the remaining seven responders had no evidence of small bowel bacterial overgrowth after glucose H2 breath tests. The ability to produce hydrogen by anaerobic fermentation of lactulose in the pouch was unrelated to the age of the patients or of the pouch. Seven of eight responders had successive breath tests after ingestion of lactulose 20 g and wheat starch 100 g. Five of seven had significant increases after lactulose but none after wheat starch. The overall function of the pouch continence, spontaneity of defecation, and 24 hour stool frequency was significantly better in responders than in non-responders. The absence of H2 production of 100 g wheat starch may indicate either increased absorption or defective fermentation. PMID- 7557578 TI - Soluble tumour necrosis factor receptors p55 and p75 in the urine monitor disease activity and the efficacy of treatment of inflammatory bowel disease. AB - The aim of the study was to discover if soluble tumour necrosis factor receptors (sTNF-R p55 and p75) in the urine of patients with inflammatory bowel disease (IBD) could be used to monitor the different stages of the activity of the diseases. Twenty five patients with either Crohn's disease or ulcerative colitis were followed up during a longterm study. The 16 patients who become acutely ill with either Crohn's disease or ulcerative colitis had significantly higher concentrations of sTNF-R p55 and p75 in their urine compared with those who were in remission, or those who were normal controls. There was a significant correlation between increased concentrations (> 20 ng/ml) of both sTNF-R p55 and p75 in the urine and a high Crohn's disease activity index (CDAI) and colitis activity index (CAI). Therefore, determination of sTNF-R is a good non-invasive parameter that can be used to assess the activity of disease and the efficacy of treatment. PMID- 7557579 TI - Differences in colonic tone and phasic response to a meal in the transverse and sigmoid human colon. AB - It is not yet clear whether the regional differences in the physical properties of the colon influence its motor responses. Tonic and phasic colonic motility and compliance of the transverse and sigmoid colon were therefore assessed using a combined barostat-manometry assembly in 22 healthy subjects. Measured colonic compliance was corrected by subtraction of the compliance of the closed barostat system. The mean (SEM) preprandial colonic volumes in the transverse and sigmoid colon were similar (150 (12) and 128 (13) ml, p = NS), corresponding to calculated mean (SEM) colonic diameters of 4.3 cm and 4.0 cm respectively. The mean increase in colonic tone postprandially was significantly greater in the transverse (24.1% (3.5)) than in the sigmoid colon (13.1% (3.0), p < 0.01). The mean increase in phasic contractility was significantly greater, however, in the sigmoid than in the transverse colon (1270 (210) and 425 (60) mm Hg/90 min respectively, p < 0.01). Compliance was greater in the transverse than sigmoid colon (7.6 (0.44) and 4.1 (0.15) ml/mm Hg, p < 0.001). The fasting volume of the colon was significantly correlated with the magnitude of the tonic response to the meal in the transverse and sigmoid colon (p < 0.001 for both). In conclusion, there are quantitatively different but qualitatively similar phasic and tonic responses to the meal in the two colonic regions. Differences in the viscoelastic and luminal dimensions may partly account for these differences in tonic responses. PMID- 7557580 TI - Acute diarrhoea induces rectal sensitivity in women but not men. AB - Some patients with diarrhoea predominant irritable bowel syndrome have increased rectal sensitivity. It is uncertain, however, whether the diarrhoea is a consequence of the rectal sensitivity or if it is sensitising the rectum in some way. The aim of this study was to assess whether inducing diarrhoea in normal healthy volunteers can sensitise the rectum and therefore be a potential or partial cause of the sensitive rectum seen in some patients with diarrhoea predominant irritable bowel syndrome. The anorectal responses to balloon distension were measured in 20 healthy volunteers (aged 20-43 years, 10 female) eight hours after laxative induced diarrhoea or under control conditions. Ingestion of an isoosmotic laxative increased stool output from 1.1 (0.7-2.3) (median (range)) to 8 (5-19) bowel movements per day with no significant differences between men and women. In women rectal sensitivity was significantly increased after diarrhoea compared with control conditions (vol to induce discomfort (ml): 116 (96, 136) v 153 (137, 168), mean (95% CI); p < 0.001). This was associated with a reduction in the volume to induce internal anal sphincter relaxation (16 (12, 20) v 28 (21, 36); p < 0.005), and volume to induce sustained internal anal sphincter relaxation (70 (56, 84) v 90 (67, 113); p < 0.03), but no significant change in rectal compliance (ml/cm H2O at 100 ml) 4.8 (3.5, 6.1) v 4.1 (3.0, 5.1) or distension induced motility (motility index) 994 (341, 1647) v 735 (46, 1424). Conversely, in men diarrhoea had no significant effect on anorectal physiology and their control values were not significantly different from those of the women. In conclusion, the results of this study taken with the finding that irritable bowel syndrome is more common in women, suggests that the male or female sex hormonal environment may be an important factor in allowing the gut to be sensitised to noxious stimuli. PMID- 7557581 TI - Liver histology in hepatitis C infection: a comparison between patients with persistently normal or abnormal transaminases. AB - Forty two cases of confirmed hepatitis C virus (HCV) infection with available liver histology were studied. Most patients, 23 of 42 (55%) had abnormal liver function tests but 19 of 42 (45%) had persistently normal liver transaminases (mean aspartate transaminase (AST) 24.1 IU/l, mean follow up 10 months). Histological examinations in the group with normal AST activities were normal in two of 19 (11%), showed non-specific reactive hepatitis in eight of 19 (42%), chronic persistent hepatitis in six of 19 (31%), and chronic active hepatitis in three of 19 (16%). Twenty three of 42 (55%) had either persistently or temporary raised liver transaminases (mean AST 96.2 IU/l, mean follow up 16 months). Histological examinations in this second group with abnormal liver biochemistry showed reactive hepatitis in five of 23 (22%), chronic persistent hepatitis in six of 23 (26%), chronic active hepatitis in 10 of 23 (43%), and cirrhosis in two (9%). Average alcohol intake was significantly higher in the group within abnormal liver function (17.8 v 6.4 units, p = 0.01). Although serious pathology was more frequent in the abnormal transaminase group, significant liver pathology (chronic persistent hepatitis or chronic active hepatitis) was found in nine of 19 (47%) of cases with repeatedly normal transaminases. Liver biopsy is advised in all cases of chronic hepatitis C infection to accurately assess both the degree of fibrosis and the current activity of the disease. PMID- 7557584 TI - Endoscopy and computed tomography in the diagnosis and follow up of oesophageal leiomyoma. AB - Leiomyoma of the oesophagus, although the commonest benign oesophageal tumour, is still rare compared with malignant tumours of the oesophagus. Leiomyomas of the oesophagus are usually diagnosed on barium swallow or histological examination after section. Five cases of leiomyoma of the oesophagus are reported where diagnosis was made by the combination of barium swallow, upper gastrointestinal endoscopy, and computed tomography and all but one patient were followed up for one to four years. The endoscopic biopsy specimens were non-specific in all five patients but none showed any evidence of malignancy. None of the five patients had a history of dysphagia. This paper describes a conservative approach to medical treatment in asymptomatic oesophageal leiomyoma rather than surgical excision as commonly published. It also emphasises the importance of negative endoscopic pinch biopsy specimens and the role of computed tomography in the diagnosis of oesophageal leiomyoma. PMID- 7557585 TI - Small intestinal stricture complicating superior mesenteric vein thrombosis. A study of three cases. AB - Mesenteric vein thrombosis associated with intestinal stricture, as a consequence of intestinal ischaemia, has only been mentioned twice in published works. The clinical, biological, and morphological aspects as well as the treatment of this morbid association were studied in three patients. In all, a two stage clinical course (initial acute abdominal pain and fever, followed by chronic intestinal obstruction), corresponding to the sequence thrombosis/stricture, was found. x Ray studies showed a regularly contoured intestinal stricture. Surgical resection was required in all three cases for stricture, associated in one case with mesenteric infarction. Anticoagulation treatment was used to preclude recurrence. Increased clinical awareness could lead to the diagnosis of intestinal stricture secondary to mesenteric vein thrombosis more often and at an earlier stage. Treatment consists of evaluation of predisposing features, intestinal resection when necessary, and anticoagulation therapy, as indicated. PMID- 7557586 TI - Effect of Helicobacter pylori on the gastric mucus gel. PMID- 7557587 TI - Audit of percutaneous liver biopsy. PMID- 7557582 TI - Endoscopic biliary stenting in a district general hospital. AB - During a 48 month period to December 1990, 367 patients, median age 75 years, with obstructive jaundice caused by common bile duct stones (201), malignant biliary obstruction (148), and benign biliary strictures (18), underwent therapeutic endoscopic retrograde cholangiopancreatography. Endoscopic biliary stenting and drainage was achieved in 343 of 367 patients attempted (93%), seven patients requiring a combined percutaneous endoscopic approach. Endoscopic stenting failed in 24 patients because of malignant duodenal infiltration (10), Billroth 2 gastrectomy (6), tight and extensive biliary strictures (6), peripapillary diverticulum (1), and technical failure (1). Prolonged follow up was available in 91% (311 of 343). The 30 day mortality was 5% (17 of 343), which included two procedure related deaths (0.6%) from fulminant pancreatitis and major sphincterotomy site bleeding. Early complications occurred in 14% (48 of 343) and late complications occurred in 11.9% (35 of 294) patients, as of the original 343, 17 had died within 30 days and another 32 were lost to follow up. Eighty patients with incomplete bile duct clearance and eight patients with benign biliary strictures had biliary stents inserted for 12-48 months (median 30). Endoscopic biliary stenting services are necessary in a district general hospital with technical success, death and morbidity rates comparable to other studies. PMID- 7557583 TI - High resolution magnetic resonance imaging of the anal sphincter using an internal coil. AB - An internal receiver coil was used to obtain high resolution transverse and oblique coronal magnetic resonance images of the anal sphincter in five normal volunteers and five patients. The internal sphincter had a high signal intensity on T1 weighted, T2 weighted, and STIR sequences whereas the conjoined longitudinal muscle and external sphincter had a low signal intensity. The internal sphincter (but not the external sphincter) showed contrast enhancement after administration of intravenous gadopentetate dimeglumine. The oblique coronal plane was particularly useful for showing the thickness and the relations of the external sphincter. Sphincteric abscesses as well as muscle defects, hypertrophy, and atrophy were clearly shown. The coil was well tolerated by most subjects. It has considerable potential for improving the diagnosis of anorectal disease. PMID- 7557589 TI - Increased CA 125 in tuberculous peritonitis. PMID- 7557588 TI - Audit of percutaneous liver biopsy. PMID- 7557590 TI - Adenomas and a family history of colorectal cancer. PMID- 7557592 TI - Diverticular distraction. PMID- 7557593 TI - Adrenal function following high-dose steroids in ovarian cancer patients. AB - PURPOSE: Steroid doses similar to those used to prevent paclitaxel-associated hypersensitivity reactions and cisplatin-induced nausea have been associated with hypothalamic-pituitary-adrenal (HPA) axis suppression. We assessed HPA function in patients receiving high-dose steroids as part of their chemotherapy regimen for epithelial ovarian cancer. PATIENTS AND METHODS: From January to July 1994, a cross-sectional study of HPA function was performed on patients receiving dexamethasone (DEX) as part of their paclitaxel and cisplatin chemotherapy regimen (n = 9). Patients received 20 mg of DEX orally, 6 and 12 hr prior to paclitaxel (135 mg/m2) and 10-20 mg intravenously before cisplatin (50-100 mg/m2). In addition, patients received approximately 12 mg/day of DEX orally for 4 days after their chemotherapy as an antiemetic. HPA integrity was evaluated by the administration of synthetic adrenocorticotropic hormone (ACTH). The ACTH stimulation test was performed 11-19 days after the completion of the course of DEX. Patients had fasting baseline cortisol levels drawn at approximately 0800 followed by a 25-unit intravenous injection of ACTH. Post-ACTH cortisol levels were repeated at 30 and 60 min. RESULTS: The mean (+/- SEM) fasting baseline level of cortisol was 12.4 +/- 2.3 micrograms/dl (normal, 7-23 micrograms/dl). At 30 min following ACTH administration, the mean cortisol level rose 17.1 micrograms to 29.5 +/- 1.8 micrograms/dl; at 60 min it rose 21.4 micrograms to 33.8 +/- 2.5 micrograms/dl [P < 0.001] (normal increase 9-39 micrograms). All patients demonstrated a sufficient increase in their plasma cortisol after ACTH stimulation, indicating normal HPA function on the days tested. However, there was a significant trend toward lower increases in plasma cortisol at 30 and 60 min as the interval from ACTH stimulation testing to the DEX regimen decreased (r = 0.986; P < 0.0001). The chemotherapy cycle number had no impact on cortisol response in the multivariate analysis. Based on multiple linear regression, HPA function may be suppressed for approximately 8 days, but up to 14 days from the start of this DEX regimen. CONCLUSION: Current steroid regimens prescribed with chemotherapy transiently decrease HPA function, but do not appear to inhibit the HPA axis long term. HPA function may be suppressed for approximately 8 days from the commencement of chemotherapy cycles involving DEX. Patients presenting within the first 8 days of a chemotherapy cycle using steroids with symptoms attributable to HPA suppression may benefit from HPA axis testing. PMID- 7557591 TI - Cholecystokinin and transient lower oesophageal sphincter relaxation. PMID- 7557594 TI - A phase II trial of neoadjuvant chemotherapy prior to radical hysterectomy and/or radiation therapy in the management of advanced carcinoma of the uterine cervix. AB - OBJECTIVE: This prospective trial was designed to access the efficacy and safety of neoadjuvant chemotherapy followed by radical hysterectomy and/or radiation therapy in women with advanced carcinoma of the uterine cervix. METHODS: Thirty women, clinical stages IIb-IVa, were enrolled in this clinical trial. Initial treatment consisted of three cycles of bleomycin, cisplatin, and vincristine administered every 10 days. Depending on the extent of disease after chemotherapy, patients then either underwent radical hysterectomy with bilateral pelvic and periaortic lymphadenectomy or surgical staging. Following review of the surgical findings, tailored radiotherapy was administered. RESULTS: Only 10 women (34%) had tumor regression from neoadjuvant chemotherapy sufficient to allow radical hysterectomy prior to tailored adjuvant radiotherapy; the remainder received primary radiotherapy after surgical staging. Two-year disease-free survival was 68, 43, and 0% for women with clinical stages II, III, and IV, respectively. Four women experienced acute toxicity from chemotherapy requiring medical intervention and eight women suffered chronic toxicities requiring hospitalization and/or surgery. CONCLUSION: The neoadjuvant chemotherapy utilized in this trial was generally ineffective in converting patients from inoperable to operable, had no apparent effect on survival, and was associated with significant toxicity. PMID- 7557595 TI - Combinations of multiple serum markers are superior to individual assays for discriminating malignant from benign pelvic masses. AB - To determine whether measurement of the levels of multiple tumor markers in the preoperative serum of women presenting with a pelvic mass distinguished benign from malignant disease better than the assay of CA 125 alone, sera from 429 patients, 192 of whom had malignant histology, were assayed for 8 different markers: CA 125, macrophage colony-stimulating factor, OVX1, lipid-associated sialic acid (LASA), CA15-3, CA72-4, CA19-9, and CA54/61. The sensitivity and specificity of CA 125 alone (> 35 U/ml) was 78.1 and 76.8%, respectively. A panel consisting of CA 125, OVX1, LASA, CA15-3, and CA72-4 had a sensitivity of 83.3% and specificity of 84.0% when two or more markers were elevated. Using the concentrations of these five markers, logistic regression analysis had a sensitivity of 85.4% and a specificity of 83.1%. Considering the values of markers in different sequences, classification and regression tree analysis substantially improved the sensitivity to 90.6% and the specificity to 93.2%. When applied in clinical practice this approach could improve the management of women presenting with a pelvic mass and may also have application in screening for ovarian cancer. PMID- 7557596 TI - Primary squamous cell carcinoma of the endometrium: a first report of adjuvant chemoradiation. AB - Primary squamous cell carcinoma of the endometrium is very rare, with only 49 cases reported in the literature. In 1928 Fluhmann proposed three criteria to establish the diagnosis: (1) no coexisting endometrial adenocarcinoma, (2) no connection between the endometrial tumor and the squamous epithelium of the cervix, and (3) no squamous cell carcinoma of the cervix present. The median age at presentation is similar to adenocarcinoma of the endometrium. Although most patients have presented with FIGO stage I disease, long-term survival has been dismal despite surgery and radiation therapy. We report a new case treated with postoperative radiation and cisplatin and review the literature. PMID- 7557597 TI - Clinical implications of metastases to the ovary. AB - Predictive factors for ovarian metastases were analyzed in 313 autopsy cases of women dying of malignancy. Genital, hematopoietic, brain tumors, and double primaries were excluded. Ovarian metastases were found in 60 of 313 (19.2%) cases. The primary sites of origin were stomach (18 cases), breast (13), pancreas (7), bile duct (7), large intestine (4), kidney (3), liver (2), lung (1), and other (5). The percentages of ovarian metastasis in each primary site were 50% (3/6) in the kidney, 38.3% (18/47) in the stomach, 30.8% (4/13) in the large intestine, 22.9% (13/57) in the breast, 22.6% (7/31) in the bile duct, 19.4% (7/36) in the pancreas, 5.4% (2/37) in the liver, and 3.4% (1/29) in the lung. Seventy-five percent (75%) of ovaries with metastasis were 5 cm or less in the greatest dimension. Seventy-eight percent (78%) of ovarian metastases were bilateral. Fifty-one percent (51%) of the involved ovaries were cystic. Twenty four percent (24%) of the metastasis were occult in which microscopic disease was present in normal appearing ovaries. The mean age of death in the cases with ovarian metastasis was 53.8 +/- 16.8 years. This was significantly younger than the mean age without ovarian metastasis that mainly was attributable to the younger ages for breast and bile duct cancer. The number of other extraovarian metastatic sites for positive ovarian cases was significantly greater than for the negative ovarian cases (7.6 +/- 2.4 and 5.8 +/- 2.5, respectively). This difference was dominant in gastric and bile duct cancers. The other metastatic sites that accompanied ovarian metastases were the adrenal gland in gastric cancer, the spleen and small intestine in breast cancer, and the adrenal gland and colon in bile duct cancer. The frequency of peritoneal dissemination was essentially the same between cases with positive and negative ovarian metastasis. Scirrhous adenocarcinoma of the stomach more commonly metastasized to the ovary than any other stomach histological type. These findings suggest that although the impact upon outcome of bilateral salpingo-ophorectomy at the initial operation is unknown, the discovery of ovarian metastases in a large proportion of patients may dramatically alter initial management in patients otherwise thought to have only local disease. PMID- 7557598 TI - Three-dimensional ultrasound evaluation of ovarian masses. AB - Seventy-six women with ovarian masses first detected with two-dimensional (2D) ultrasound were then evaluated with three-dimensional (3D) ultrasound. Two dimensional ultrasound detected four of five ovarian malignancies. One additional ovarian carcinoma was diagnosed by 3D scanning. Observation of papillary projections, characteristics of cystic walls, and the extent of capsular infiltration of tumors was superior with 3D ultrasound as was the calculation of ovarian volume. Fifty-seven of the women studied underwent surgery within 7 days of 3D ultrasound evaluation. Histologic examination of surgical specimens confirmed the 3D sonographic diagnoses. There was one false positive and one false negative diagnosis with 2D ultrasound. All cases of ovarian malignancy were diagnosed correctly with 3D ultrasound. PMID- 7557599 TI - Comparative study of the patients with locally advanced stages I and II cervical cancer treated by radical surgery with and without preoperative adjuvant chemotherapy. AB - Ninety-two patients with locally advanced stages IB, IIA, and IIB cervical cancers, who had completed 4 years of follow-up after treatment with preoperative adjuvant chemotherapy prior to radical surgery, were compared with 138 patients in same disease status who were treated with radical surgery without preoperative adjuvant chemotherapy. In the patients treated with preoperative adjuvant chemotherapy (VBP scheme), the chemotherapeutic response was more favorable in squamous cell carcinomas (87%) than that in adenocarcinomas (38%). The detection rate of pelvic lymph node metastasis from the surgical specimens of radical operation was higher in the patients of radical surgery without preoperative chemotherapy (34%; 47/138) than that in the preoperative adjuvant chemotherapeutic patients (17%; 16/92, P = 0.005). Recurrence occurred in 50 of 138 patients (35.5%) who were treated by radical surgery without preoperative chemotherapy and in 17 of 92 patients (18.5%) who were treated with preoperative adjuvant chemotherapy followed by radical surgery. The difference of recurrence rate between two groups was statistically significant (P = 0.004). The tumor-free survival of preoperative adjuvant chemotherapeutic patients was significantly longer than those in the patients without preoperative chemotherapy (P = 0.0067). Tumor response to chemotherapy was a valuable prognostic factor in management of patients and preoperative adjuvant chemotherapy was beneficial in reducing pelvic lymph node metastases, reducing recurrences, and prolonging the survival of the patients with locally advanced cervical cancers, especially in stage IIA. PMID- 7557600 TI - The reliability of preconization diagnostic evaluation in patients with cervical intraepithelial neoplasia and microinvasive carcinoma. AB - The accuracy of preconization cytology and histology was evaluated in 536 patients undergoing combination laser conization. Exact agreement between cytology and cone diagnosis was observed in 41.8% of the patients. The lowest agreement, 13.6% was demonstrated in cytologic cervical intraepithelial neoplasia (CIN) I, the highest in cytologic CIN III, 64.3%. Concerning microinvasive disease, the positive predictive value of cytology was only 27.3%; the negative predictive value 94.6%. When cytology showed CIN II or worse, the cone biopsy showed CIN or invasive disease in 92.8%. Exact agreement between preconization histology and the cone biopsy was found in 59.5%. Highest agreement was observed in CIN III lesions, 67.1%, and the lowest agreement in CIN II lesions, 42.7%. When preconization showed CIN II, a higher grade of lesion was found in the cone biopsy in 29.1%. Cone biopsy revealed invasive disease in 38 cases. In 24 cases, invasive disease was not demonstrated prior to conization, corresponding totally to 4.7% of patients not suspected to have invasive disease prior to conization. Regarding invasive disease, the sensitivity of preconization histology was 36.8%, the positive predictive value 58.3%, and the negative predictive value 95.3%. Kappa statistics demonstrated rather low agreement between cone diagnoses and preconization diagnoses. These results confirm the potential risk of overlooking invasive disease by conventional preconization evaluation and demonstrate the need for excisional methods in the management of cervical intraepithelial neoplasia to provide a sufficient specimen for diagnostic purposes. Combined with the therapeutic results, combination laser conization was a reliable diagnostic and therapeutic method in the management of patients with CIN and microinvasive cancer of the cervix. PMID- 7557601 TI - Recurrent endometrial adenocarcinoma presenting as a solitary humeral metastasis. AB - A 66-year-old woman was admitted because of postmenopausal vaginal bleeding. Diagnostic workup revealed a poorly differentiated endometrial adenocarcinoma. A total abdominal hysterectomy and bilateral salpingo-oophorectomy was carried out (FIGO stage Ia, G3). One and a half years later she developed a solitary humeral metastasis which was treated by local radiotherapy and progesterone acetate. Because osseous metastases in endometrial adenocarcinoma are rare, the literature is reviewed. In analogy to the treatment of pulmonary metastases the option of disarticulation of the patient's arm is discussed. PMID- 7557602 TI - Surgical predictors of para-aortic metastases in early-stage cervical carcinoma. AB - Radical pelvic surgery for cervical carcinoma is contraindicated in the presence of para-aortic node metastases. However, the incidence of para-aortic nodal involvement is very low in early-stage disease. Therefore, it may not be necessary to subject all patients to para-aortic lymphadenectomy prior to radical hysterectomy. Medical records for 408 patients with early-stage cervical carcinoma treated at the Pennsylvania State University-M.S. Hershey Medical Center were reviewed to ascertain if clinical factors can be utilized intraoperatively to accurately predict those patients at minimal risk for para aortic lymph node metastases. The presence of clinically suspicious (abnormally enlarged or firm) pelvic or para-aortic lymph nodes or extracervical spread of tumor at the time of exploration were significant predictors of para-aortic metastases (P < 0.001). The majority of patients (85%) had none of these risk factors, and no patient had para-aortic metastases in the absence of these predictors. Suspicious pelvic or para-aortic lymph nodes were present in the minority of patients (15%) and identified all patients with para-aortic metastases. Therefore, para-aortic lymphadenectomy may be safely omitted at the time of exploration for radical hysterectomy in the absence of enlarged or abnormally firm pelvic or para-aortic lymph nodes. In the presence of either of these factors or extracervical spread of disease a para-aortic lymphadenectomy is necessary to rule out metastases. PMID- 7557603 TI - Adenocarcinoma of the cervix in a renal transplant patient. AB - Treatment of cervical carcinoma in a renal transplant patient with a single pelvic kidney presents several management dilemmas. Standard treatment modalities are complicated by the patient's immunosuppressed state and the location of the transplanted kidney in the standard pelvic field, as well as other surgical risks. No information on the best approach to this clinical situation is available in the English language literature. We present this case to discuss factors considered in selecting a treatment plan and possible reasons for the rapid recurrence and demise of this patient. PMID- 7557604 TI - Myxoid leiomyosarcoma of the uterus with unusual malignant histologic pattern--a case report. AB - A case report of myxoid leiomyosarcoma of the uterus is presented as a rare variant of uterine sarcoma. This case, contrary to most of the 11 cases previously described in the literature, is characterized by highly atypical pleomorphic cells and numerous mitoses. Furthermore, myxoid material is also found in the cytoplasm of pleomorphic tumor cells, a feature, that to our knowledge, has not been previously described. All these tumors, no matter what the microscopic appearance is, exhibit a highly malignant behavior. The best therapy for this tumor is unknown, since there are too few cases from which to draw conclusions. PMID- 7557605 TI - Recurrent squamous cell carcinoma in a rectus abdominis neovagina. AB - The development of a carcinoma in a neovagina is rare. Most neovaginal neoplasias have been carcinomas in situ in the skin graft. To date, there has not been a report of a neovaginal carcinoma in a myocutaneous flap. This report is the first of a squamous cell carcinoma arising in a neovagina constructed from a rectus abdominis myocutaneous flap. PMID- 7557607 TI - Vaginal melanoma. PMID- 7557606 TI - Radical hysterectomy for recurrent carcinoma of the uterine cervix following radiotherapy. PMID- 7557608 TI - Vaginal melanoma. PMID- 7557609 TI - Prospective randomized trial with neoadjuvant chemotherapy in stage IB bulky squamous carcinoma of the uterine cervix. PMID- 7557610 TI - Postsurgical surveillance of patients with FIGO stage I/II endometrial adenocarcinoma. AB - OBJECTIVE: To examine the effect of postsurgical surveillance on survival of patients with FIGO stage I/II endometrial adenocarcinoma. METHODS: We examined the records of 354 patients who underwent primary surgical therapy for FIGO stage I/II endometrial adenocarcinoma. In patients who developed recurrent disease, we determined whether symptoms or signs of disease were present at recurrence and whether there was evidence of disease on Pap smear or chest radiograph. RESULTS: Among the 354 patients in this study, 44 (12%) developed recurrent disease. Sites of recurrence included 12 (27%) isolated vaginal, 12 (27%) pelvic with vagina or abdomen, 4 (10%) isolated lung, 13 (29%) pelvic/abdominal with other distant sites, and 3 (7%) other distant sites. At diagnosis of recurrence 61% of patients had symptoms related to their cancer, 68% had physical exam findings suggestive of recurrence, and 84% had symptoms and/or signs. Findings consistent with recurrent cancer were detected by Pap smear in 25% and on chest radiograph in 20%. Among the 44 patients who developed recurrent disease, 8 (18%) remain alive without evidence of disease, including 6/12 (50%) with isolated vaginal disease and 2/34 (6%) with other patterns of recurrent disease (P = 0.01). Among the 12 patients with isolated vaginal recurrence, 1/3 (33%) in whom recurrent disease was diagnosed by Pap smear alone was salvaged compared to 5/9 (56%) who had symptoms or signs of vaginal recurrence. None of the three patients in whom an abnormal chest radiograph was the only evidence of recurrence survived. CONCLUSIONS: Because of the low recurrence rate of FIGO stage I/II endometrial cancer and the paucity of effective second-line treatment, surveillance Pap smears and chest radiographs appear to have little impact on survival. Although few asymptomatic potentially curable recurrences were detected due to surveillance examinations, the value of psychological reassurance associated with a normal examination is difficult to quantitate. PMID- 7557611 TI - Laparoscopic surgical staging of ovarian cancer. AB - The feasibility of laparoscopic staging in patients with ovarian cancer was undertaken prospectively to determine the ability to adequately evaluate both the intraperitoneal cavity and the retroperitoneal lymph nodes. Two groups of ovarian cancer patients were evaluated: those with optimally debulked advanced disease undergoing second-look procedures and those with presumed stage I disease undergoing surgical staging. Twenty-four of the 44 laparoscopic second-look procedures (56%) were positive for persistent disease. Five of these patients had microscopic disease only, in the omentum, washings, pelvic peritoneum, and in para-aortic lymph nodes (2 patients). In the group of 14 patients undergoing staging for presumed early ovarian carcinoma, metastatic disease was discovered in 8 (57%) patients. Two patients had peritoneal washings positive for adenocarcinoma; 3 had pelvic disease, 1 confined to a fallopian tube and 2 to the pelvic peritoneum; and 3 patients had para-aortic lymph nodes positive for metastatic adenocarcinoma. There were no serious complications in this category. The average hospital stay was 1.6 days. Laparoscopic staging appears to be an accurate staging technique, and further investigation into the validity of this approach is warranted. PMID- 7557612 TI - Staging and recurrence of disease in squamous cell carcinoma of the vulva. AB - In order to determine the prognostic significance of applying the revised FIGO staging system and identify factors contributing to survival after documentation of recurrent disease, a retrospective chart review of our vulvar cancer population was performed. Over a 17-year interval 135 patients were uniformly treated with primary surgical treatment consisting of radical vulvectomy and bilateral groin dissection. Factors contributing to disease-free survival were analyzed using a Cox proportional hazards model. Covariates of survival after recurrence of disease were analyzed using the log-rank method. Neither the clinical assessment of the groin nodes, nor the presence or absence of perineal involvement were related to outcome. Only lesion size and surgical status of the inguinal nodes were significant predictors of disease-free survival (P = 0.02 and P = 0.03, respectively). In addition, there was a statistically significant relationship between the extent of groin involvement (negative, unilateral positive, and bilateral positive nodes) and associated decrement in disease-free survival (P = 0.01). Thirty patients developed recurrence of disease from 2.0 to 47.3 months following surgery. The location of the recurrence, interval from primary therapy to recurrence, and status of the groin nodes at initial surgery were significant prognostic factors in subsequent survival. The revised staging system demonstrated an improvement in patient stratification compared to the criteria of the prior classification. The data are also consistent with the distinction made between Stage III and IV disease in the new classification. The status of the groin nodes at original surgery remained an important prognostic factor even in those patients who later demonstrated recurrence of disease. PMID- 7557613 TI - Adenocarcinoma as an independent risk factor for disease recurrence in patients with stage IB cervical carcinoma. AB - Between 1960 and 1989 1538 patients were treated with initial radiation therapy for FIGO Stage IB squamous cell carcinoma (SCC) of the cervix and 229 patients were treated for Stage IB adenocarcinoma (AC). The overall 5-year survival rates for patients with SCC and AC were 81 and 72%, respectively (P < 0.01). Patients with AC more often had a maximum cervical diameter < 4 cm than those with SCC (53% versus 47%). For 903 patients with tumors > or = 4 cm, 73% of those with SCC survived > or = 5 years compared with only 59% of those with AC (P < 0.01). Although there was no significant difference in the rate of pelvic disease recurrence for patients with AC or SCC tumors > or = 4 cm (17% versus 13%, P = 0.16), the rate of distant metastases was greater for patients with AC (37% vs 21%, P < 0.01). For patients with tumors > or = 4 cm, prognosis was strongly correlated with tumor size (P < 0.01) and lymphangiogram findings (P < 0.01) but not with age (P = 0.58) or tumor morphology (exophytic versus endocervical) (P = 0.33); a trend toward better survival in 165 patients who underwent adjuvant hysterectomy (78% versus 71%) was not significant (P = 0.09). Multivariate analysis confirmed a highly significant independent association between histology and survival; patients with tumors > or = 4 cm in diameter that were AC had an estimated risk of death 1.9 times that of patients with SCC (P < 0.01). These results provide strong evidence that patients with AC of the cervix have a poorer prognosis than those with SCC of similar stage and tumor diameter, reflecting primarily a higher rate of distant metastases in patients with AC. PMID- 7557614 TI - A survey of blood transfusion practices of gynecologic oncologists. AB - OBJECTIVE: To survey the attitudes of gynecologic oncologists about indications for blood transfusion, and compare them to current recommendations about transfusion practice. METHODS: With the permission of the Society of Gynecologic Oncologists (SGO), a study questionnaire was mailed to 548 members and candidates in their directory. The questions asked about transfusion threshold before chemotherapy, following ovarian debulking, and after endometriosis surgery. Indications for transfusion were assessed using a qualitative/quantitative scale. Responses were analyzed with respect to years of SGO membership and practice setting using the chi 2 test and analysis of variance. Results were compared to the 1992 guidelines for transfusion of the American College of Physicians (ACP). RESULTS: Two hundred eighty-five forms were returned (52% of total). Fifty-one percent of respondents were in full-time academic positions; 36% were in private practice. Sixty-one percent were full SGO members, and 35% were candidates. The average prechemotherapy transfusion threshold (TT) was a hemoglobin concentration (hgb) of 7.9 +/- 0.7 g/dl (g). At that level the mean transfusion was 1.9 +/- 0.5 units of packed red blood cells (U). For the ovarian debulking scenario, the mean postoperative TT was 8.1 +/- 0.8 g with a mean transfusion of 1.9 +/- 0.4 U. For the endometriosis case, the mean TT was 7.0 +/- 0.8 g, with a mean transfusion of 1.9 +/- 0.5 U. Analysis showed no difference in practice based on years of SGO membership or practice setting. CONCLUSION: A majority of the gynecologic oncologists surveyed offers transfusion at levels of anemia similar to ACP recommendations, but frequently transfuse more units than recommended. A substantial minority does not follow the guidelines at all. PMID- 7557615 TI - Prolonged continuous infusion cisplatin and 5-fluorouracil with radiation for locally advanced carcinoma of the vulva. AB - Twelve patients ages 37-85 years (mean, 59 years) with locoregionally advanced vulvar carcinoma were treated with a combination of irradiation and chemotherapy using prolonged continuous infusions of 5-fluorouracil (5-FU) and cisplatin. Eleven patients had advanced vulvar disease with tumors 5-18 cm in maximum diameter (mean, 8.7 cm), eight had palpable inguinal nodes with biopsy-proven metastatic carcinoma, and five had fixed nodes. Patients received weekly 96-hr infusions of cisplatin (4 mg/m2/day) and 5-FU (250 mg/m2/day) for a total of 64 mg/m2 of cisplatin and 4 g/m2 of 5-FU in 4 weeks. Concurrent radiation therapy was delivered to the lower pelvis, vulva, and inguinal nodes to a total dose of 40-50 Gy at 2 Gy per fraction in 11 patients. One patient who had been previously treated for cervical cancer received radiation only to the vulva with an appositional electron beam field. Chemoradiation was well tolerated with virtually no hematologic toxicity and no unscheduled breaks in treatment. Eleven of 12 patients had at least a partial clinical response; one patient had a minimal response of unresectable vulvar disease. Of eight patients who underwent vulvar resection 6 weeks after chemoradiation, four had no residual disease in the resected vulvar specimen and remain disease-free 17, 20, 25, and 37 months, respectively, after surgery. Another patient is disease-free 28 months after a complete clinical response without vulvar resection. However, of four patients who had residual disease in the vulvar surgical specimen, disease has recurred within the irradiation field in three. Overall, 6 of 12 patients treated with this chemoradiation regimen remain disease-free 17-30 months after treatment. This is a well-tolerated outpatient regimen that yields a high response rate in patients with massive vulvar carcinomas. PMID- 7557616 TI - Tumor vascularity--a novel prognostic factor in advanced cervical carcinoma. AB - OBJECTIVE: In the search for the optimal treatment of advanced cervical cancer, the identification of valid prognostic factors obtainable without histopathologic investigation of the entire tumor and the locoregional lymph nodes is of paramount interest. Tumor microvessel density has recently been demonstrated to correlate strongly with disease aggressiveness in breast cancer and other malignancies. METHODS: We established a computerized image analysis system to quantify tumor microvascularity by using the closest-individual method, which determines the distribution of distances from random points within the tumor to the closest microvessel (DTCMV). Tumor microvascularity was assessed in paraffin sections of two cylindrical 2 x 20-mm core biopsies obtained transvaginally from the 12 and 6 o'clock positions of each tumor and then immunohistochemically stained for Factor VIII-related antigen. The oncologic relevance of tumor vascularity is studied in an open prospective trial. RESULTS: Tumor vascularity was quantified in 42 patients with cervical cancers > 3 cm in largest diameter, FIGO stages Ib-IVa. This new parameter representing pathophysiological tumor-host interactions was independent of various other patient and tumor characteristics, including age, FIGO stage, tumor size, differentiation, lymph node metastases and lymphatic space involvement. Thirty-nine patients were treated with curative intent either by primary surgery (n = 22) or radiation (n = 17). After a median observation time of 18 months (range 4-41 months), the patients with higher tumor vascularity (mean DTCMV < 83 microns) had significantly shorter disease-free (P = 0.025) and overall (P = 0.032) survival probabilities than patients with lower tumor vascularity (mean DTCMV > or = 83 microns). Cox regression analysis identified tumor vascularity as the strongest independent prognostic factor in this group of patients. CONCLUSIONS: The assessment of tumor microvascularity by computerized image analysis of defined tumor biopsies could become a novel means of predicting tumor aggressiveness in non-early cervical cancer. PMID- 7557617 TI - Intracellular single-chain Fv antibodies--a knockout punch for neoplastic cells? PMID- 7557619 TI - A decision analysis of practice patterns used in evaluating and treating abnormal Pap smears. AB - OBJECTIVE: The purpose of this study was to investigate contemporary methods of evaluating and treating abnormal Pap smears, in terms of their potential for excessive treatment and financial impact on health care delivery systems. METHODS: Clinical algorithms for the evaluation and treatment of abnormal Pap smears were constructed, taking into consideration different philosophies on the need for colposcopic biopsy, the role of cryotherapy, and LLETZ. The algorithms employed (1) colposcopy of all patients with cryotherapy of mild dysplasia and LLETZ of moderate to severe dysplasia; (2) colposcopy with observation of mild dysplasia, treatment of moderate dysplasia by cryotherapy, and severe dysplasia by LLETZ; (3) colposcopy of LGSIL Paps before treatment and immediate LLETZ of HGSIL; and (4) immediate LLETZ of LGSIL and HGSIL Paps. Each algorithm was theoretically applied to a cohort of colposcopy clinic patients based upon referral Pap smear, with excessive treatment and costs calculated. The cohort's repeat Pap smear, colposcopic biopsy, and candidacy for cryotherapy were included in the analysis. The decision to use repeat Pap smear in treatment planning, submit only diagnostic LLETZ pathology, and select immediate LLETZ candidates by HGSIL/severe dysplasia Pap smear was considered. Financial impact was calculated using nationwide fiftieth-percentile reimbursement costs for procedures and related pathology. RESULTS: Colposcopy provided little opportunity for excessive treatment. In contrast, 49.3% of cases subjected to immediate LLETZ would theoretically not have required treatment, if initially evaluated by colposcopy. The use of the subset of HGSIL cases encompassing severe dysplasia only identified patients suitable for immediate LLETZ, with an excessive treatment rate of only 2.8%. Traditional colposcopy (algorithm 2) would have been least expensive at $718 per patient. Algorithms 1 and 3 were intermediate at $785 and $754 per patient, respectively. Immediate LLETZ of all abnormal Paps (algorithm 4) would have been most costly at $838 per patient. CONCLUSIONS: The abandonment of colposcopy and reliance on immediate LLETZ for evaluation and treatment of cervical lesions would have been expensive and had significant potential for excessive treatment. Traditional colposcopic evaluation, coupled with observation of mild dysplasia, appeared to be the most cost-effective means of treating cervical dysplasia and had a low potential for excessive treatment. PMID- 7557620 TI - Targeted eradication of ovarian cancer mediated by intracellular expression of anti-erbB-2 single-chain antibody. AB - OBJECTIVE: Overexpression of the tyrosine kinase receptor erbB-2 is important in the pathogenesis of a variety of neoplasms including ovarian cancer. As a strategy to selectively eradicate erbB-2-overexpressing tumor cells, an anti-erbB 2 single-chain immunoglobin (sFv) gene was constructed to direct expression of intracellular anti-erbB-2 antibody. The purpose of this study is to establish the antitumorigenicity of this strategy in in vitro and in vivo models. METHODS: An anti-erbB-2 sFv construct containing an endoplasmic reticulum (ER)-directed leader sequence was transiently expressed in the human ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine vector. SKOV3 cells transfected with a non-ER form of an anti-erbB-2 sFv construct or an irrelevant plasmid DNA served as controls. Antitumorigenicity, as measured by anchorage-independent growth in soft agar and subcutaneous (sc) tumor formation, was analyzed. The ability to achieve a biological effect with relevant sFv in murine orthotopic xenograft models and in primary human ovarian cancer cells was also evaluated. RESULTS: The ER form of anti-erbB-2 sFv was found to exert a marked antineoplastic effect on the SKOV3 cell line resulting in an arrest of anchorage-independent growth. A significant increase in sc tumor volume was noted in animals challenged with control constructs. In marked contrast, complete tumor eradication was noted at necropsy 80 days after sc transplantation in the group challenged with the ER directed anti-erbB-2 sFv gene. Intraperitoneal treatment of malignant ascites in human tumor xenograft models with the ER form of the anti-erbB-2 sFv gene resulted in profound downregulation of cell surface erbB-2 in retrieved ovarian cancer cells. The ER-directed anti-erbB-2 sFv also elicited a significant cytotoxic effect in transfected primary ovarian cancer cells obtained from a patient with malignant ascites. CONCLUSION: The ability to selectively "knock out" erbB-2 demonstrates that this strategy can induce a significant antineoplastic effect in ovarian cancer cells overexpressing this growth factor receptor. In addition, the ability to accomplish selective abrogation of erbB-2 expression in animal treatment models and to transfect and eradicate primary ovarian cancer cells justifies further investigation of this novel strategy in ovarian cancer patients. PMID- 7557621 TI - Endometrioid adenocarcinoma of the ovary and its relationship to endometriosis. AB - In order to better understand the clinical presentation and biologic behavior of ovarian carcinomas arising in endometriosis, we performed a historical cohort study of all women with endometrioid adenocarcinoma of the ovary (ECO) diagnosed between 1979 and 1991 at our institutions. A review of pathology reports determined the presence or absence of coexisting endometriosis. Cancers adjacent to endometriosis on the same ovary or arising within endometriosis were labeled endometriosis-associated endometrioid adenocarcinoma (EAEA), while all others were considered typical endometrioid adenocarcinoma (TEA). Associations between tumor type and clinicopathologic variables were analyzed by chi 2 and Fisher's exact tests as indicated. Disease-free interval (DFI) and overall survival were estimated using the Kaplan-Meier method. The independent prognostic significance of clinicopathologic variables was determined by multivariate analysis using the Cox proportional hazards regression model. Of 91 ECO patients identified, 63 (69%) had TEA and 28 (31%) had EAEA. Significant differences between TEA and EAEA existed for age at diagnosis (greater than 55 years; 56 vs 32%, P = 0.039), nulliparity (19 vs 46%, P = 0.007), stage (I and II combined; 37 vs 70%, P = 0.004), and disease status at completion of primary surgery (complete tumor resection; 47 vs 70%, P = 0.04). Synchronous atypical endometrial hyperplasia or uterine carcinoma was found in 7/63 (11%) TEA versus 7/28 (25%) EAEA cases (P = 0.054). Estimated 5-year DFI by life table analysis was significantly longer in the EAEA than in the TEA cohorts (57 vs 25%, P = 0.02); however, the 5-year survival difference was not significant (59 vs 45%, P = 0.18). Multivariate analysis identified only stage as an independent prognostic factor in predicting both DFI and survival. In conclusion, women with EAEA are significantly younger, present with earlier stage disease, and have a longer disease-free survival than those with TEA. These factors may reflect a more favorable biologic behavior of ECO when arising in association with endometriosis. PMID- 7557618 TI - Secreted ovarian stromal substance inhibits ovarian epithelial cell proliferation. AB - OBJECTIVE: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. METHODS: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. RESULTS: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in > 10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh Dyer organic extraction placed the activity in the aqueous phase. CONCLUSION: A heat-stable, non-heparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis. PMID- 7557622 TI - Combination anti-gene therapy targeting c-myc and p53 in ovarian cancer cell lines. AB - Gene therapy clinical trials targeting p53 and other genes are underway in nongynecologic cancer systems. To explore the potential for antigene therapy in gynecologic oncology, we examined the in vitro effects of oligonucleotides targeting c-myc and p53 in the ovarian cancer cell lines CAOV-3, SKOV-3, and BG 1. The ATP cell viability assay was used to measure growth effects after 6-day treatments with 27-mer antisense phosphorothioate oligodeoxyribonucleotides (oligos) targeting the Puf/nm23 binding region of c-myc and promoter/ATG region of p53. A random sequence of the p53 27-mer was used as a control, and an untransformed fibroblast cell line was used for comparison. IC50 was defined as the oligo concentration required for 50% growth reduction compared to untreated controls. Synergistic vs antagonistic effects of oligo combinations were quantitated by combination indexes (CI) as calculated from median effect parameters by the methods of Chou and Talalay. Mean +/- SE IC50's of c-myc and p53 antisense oligos in CAOV-3 and SKOV-3 ranged from 1.0 +/- 0.2 to 9.7 +/- 1.3 microM. The IC50's of c-myc oligos were consistently lower than corresponding p53 oligos in all cell lines (P < 0.034, t test). The fibroblast cell line was sensitive to anti-c-myc and combination anti-c-myc/p53 oligos (IC50 = 1.5 +/- 0.6 and 1.4 +/- 0.2 microM, respectively), but not to anti-p53 oligos alone (IC50 > 16 microM). Nonspecific toxicity was observed at concentrations of 16 microM for all cell lines except in BG-1, where maximal growth stimulation occurred at this concentration with anti-p53 oligos. Growth stimulation was also observed in BG-1 with anti-c-myc and anti-c-myc/p53 combinations at intermediate doses, with inhibition at higher doses. While c-myc/p53 combinations in CAOV-3 were synergistic (CI < 0.8), they were antagonistic in SKOV-3 (CI > 3.2). Phosphorothioate oligos directed against c-myc and p53 in different cell lines were shown to have both antiproliferative and stimulatory activity, as single agents and in combination, at concentrations that are achievable in vivo. Because of the complex patterns of effects, further in vitro studies are warranted before considering clinical trials with these agents in gynecologic cancers. PMID- 7557624 TI - [Sleeping Beauty may not sleep anymore. Development of girls between chronology and commerce]. PMID- 7557623 TI - Meta-analysis comparing cisplatin total dose intensity and survival. AB - OBJECTIVE: To determine the relationship between total dose intensity (TDI) of cisplatin/carboplatin, total dose intensity of all chemotherapy drugs (GDI) and median survival (MS) in stage III-IV ovarian cancer patients. METHODS: Over 700 studies from the English literature were reviewed. Prospective clinical trials that had at least one arm treating patients with cisplatin/carboplatin and provided data on both MS and dose and schedule of chemotherapy were included. Dose intensity (DI), TDI, and GDI were calculated for each study arm. To explore possible relationships, several linear regression models were fitted with MS as the dependent variable and DI, TDI, GDI, and other known prognostic factors as the independent variables. RESULTS: Sixty-one study arms were analyzed including 4118 patients. No significant correlation was found between DI and TDI of cisplatin and MS (P = 0.90 and P = 0.11 respectively). Of the 10 variables tested, proportion of patients optimally debulked (%OD), proportion of mucinous tumor, and GDI were found to have a significant correlation to MS. All studies with a GDI less than 20 and %OD < 20 had a MS less than 20 months. On the other hand 77% of studies have a MS greater than 20 months when GDI is > 20 and %OD > 20%. We found single-agent chemotherapy had the worst outcome and there was very little difference between two or more drugs for percentage of studies with > 20 months MS, (56.5% vs 63.3% respectively). CONCLUSIONS: From this meta-analysis we believe that both GDI and %OD are important factors that determine outcome in terms of median survival. PMID- 7557625 TI - [Basic principles of vaginal ultrasound]. PMID- 7557626 TI - [Uterine diagnosis]. PMID- 7557627 TI - [Transvaginal diagnosis of adnexal diseases]. PMID- 7557629 TI - [Vaginal ultrasound diagnosis of tubal diseases]. PMID- 7557630 TI - [Transvaginal Doppler/color Doppler in gynecologic diagnosis]. PMID- 7557628 TI - [Diagnosis of sterility and IVF]. PMID- 7557631 TI - [Vaginal ultrasound of surgical gynecologic complications]. PMID- 7557632 TI - [Transvaginal 3 D ultrasound in gynecology]. PMID- 7557633 TI - [Pre-vesicle ureteral obstruction as a sequela of Bruch colposuspension for urinary stress incontinence and partial prolapse. A case from the expert assessment files]. PMID- 7557634 TI - Inhibitory effect of transferrin on progesterone production in the granulosa cell of humans in vivo and porcine granulosa cell in vitro. AB - We evaluated the effect of transferrin on the regulation of granulosa cell function in humans by evaluating the production of progesterone (P) in the preovulatory phase in vivo, and in cultured porcine granulosa cells in vitro. Twenty-five women treated for in vitro fertilization and embryo transfer had their serum levels of 17 beta-estradiol (E2) and P determined on the day of administration of human chorionic gonadotropin. Transferrin concentrations were also determined in ovarian follicular fluid. In an in vitro study, porcine granulosa cells were cultured in the presence of follicle-stimulating hormone (FSH) and transferrin. Serum levels of P showed a significant negative correlation with those of transferrin (r = -0.53, p < 0.01), whereas serum levels of E2 did not (r = 0.14). When the subjects were divided into two groups by serum P concentration (low P < 1 ng/ml, high P > or = 1 ng/ml) serum concentrations of transferrin were significantly increased in the group with the low versus the high level of p (p < 0.01). Production of P by porcine granulosa cells was suppressed by transferrin in the presence of various concentrations of FSH. Increasing the dose of transferrin significantly suppressed the production of P by those cells in a dose-dependent fashion. The production of P during the preovulatory phase may be suppressed by transferrin in the granulosa cells. PMID- 7557635 TI - Subendometrial myometrial contractions associated with transcervical chorionic villous sampling. AB - Thirty-two consecutive patients, referred for prenatal diagnosis by chorionic villous sampling, were examined by transvaginal ultrasound for the presence of myometrial contractions before and after the procedure. Increased myometrial activity was observed in 78% of patients. The mean frequency increased by 3.5 times and the amplitude increased by 4.2 times compared to the preprocedure condition. Cramping was associated with the increased amplitude and/or frequency of contractions. No significant correlation was found between the direction of propagation of the contraction wave and its frequency or amplitude. The sensitivity of ultrasonographically detected myometrial contractions in predicting cramping is 70%, but only 31% in predicting vaginal bleeding or spotting. Further studies are required to elucidate the prognostic significance of this uterine activity. PMID- 7557636 TI - Endothelin has a role in early pathogenesis of amniotic fluid embolism. AB - The early pathogenesis of amniotic fluid embolism is not completely understood. The entrance of amniotic fluid (AF) into the systemic circulation leads to an initial phase of pulmonary vasospasm, pulmonary hypertension and cor pulmonale. We studied the effect of AF on endothelin (ET) production in vivo and in vitro. Injection of rabbits (pregnant and nonpregnant) with meconium-stained AF, raw AF and supernatant AF led to a significant increase in serum ET. This result was confirmed by using human umbilical vein endothelial cell cultures incubated with the same types of AFs. After infusion of rabbits with AFs, we observed that the lung, heart and kidney were positively stained for ET and von Willebrand factor. AF was found to have an injurious effect on endothelial cells measured by using fura-2. The maximal injurious effect of AFs was observed for the meconium-stained AF. We hypothesized that the early pathological changes in AF embolism may be mediated by ET. PMID- 7557637 TI - The clinical value of umbilical artery Doppler velocimetry in the management of intrauterine growth-retarded fetuses before 32 weeks' gestation. AB - The objective of this study was to investigate the clinical utility of umbilical artery Doppler velocimetry in the management of very premature growth-retarded fetuses. The study comprised 34 fetuses between 26 and 34 weeks' gestation, diagnosed ultrasonically as suffering from intrauterine growth retardation. Based on the umbilical artery Doppler findings, patients were divided into 3 groups: group 1 (10 fetuses) with normal Doppler systolic/diastolic (S/D) ratios (within 2 SD of the mean for gestational age); group 2 (9 fetuses) with significant abnormal umbilical artery S/D ratios (above 2 SD of the mean for gestational age), and group 3 (15 fetuses) with absent or reverse end-diastolic flow. Fetal outcome was assessed in terms of neonatal mortality and morbidity, i.e. low umbilical artery pH (pH < 7.20) and Apgar scores. In group 1, all fetuses had a stable, normal S/D ratio; in group 2, 2 fetuses (22%) showed deterioration in the Doppler findings and were transferred to group 3, while in group 3, 3 of 15 fetuses (20%) showed improvement in the absence of end-diastolic flow and were thus transferred to group 2. Emergency cesarean section due to antenatal deterioration of the biophysical profile occurred in only 1 patient (10%) in group 1, compared to 33 and 87% in groups 2 and 3, respectively (p < 0.05). The other 9 patients in group 1 had normal vaginal deliveries at 36-37 weeks' gestation, with no perinatal complications. The mean expectant interval from admission to delivery in group 3 was 8.2 days, while in groups 1 and 2 it was 23.8 and 22.2, respectively (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557638 TI - Changes in uterine artery Doppler flow velocity waveforms during the third stage of labor. AB - Transabdominal Doppler velocity waveform measurements of the uterine arteries during the third stage of labor were performed upon 25 patients with uncomplicated vaginal deliveries and 5 with prolonged third stage in order to further assess third-stage mechanisms. Based on Doppler flow measurements, three phases of the third stage of labor were observed: (1) a latent phase with a systolic/diastolic ratio (S/D) and a pulsatility index (PI) of 2.14 +/- 0.49 and 0.89 +/- 0.17, respectively; (2) a contraction and detachment phase with an S/D and PI of 2.53 +/- 0.53 and 1.28 +/- 0.37, respectively, and (3) an expulsion phase with an S/D and PI of 2.20 +/- 0.34 and 0.91 +/- 0.20, respectively (p < 0.05). During the third stage of labor, it was shown that uterine contractions squeezed the uterine vessels as they traversed the myometrium, leading to an increase in the extrinsic resistance, which was reflected by high resistance flow. Following placental separation and changes in the placental-site wall, there was slight uterine relaxation resulting in decreased uterine vessel resistance. This observation encourages the authors to continue investigating the contribution of ultrasound and Doppler flow for studies of the postpartum period. PMID- 7557639 TI - Management of amniocentesis in women with oligohydramnios due to membrane rupture: evaluation of a cervical adapter. AB - Thirty-five pregnant women with gestational age 22-37 completed weeks and with prelabour rupture of the membranes in the preterm period (PROM) volunteered to participate in the study. Sixteen women had a sterile cervical adapter, stopping the leakage of amniotic fluid, applied to the ectocervix for 24 h. The remaining 19 women were managed accordingly to prevailing expectant routine without the cervical adapter. All 35 women were assessed initially and after 24 h by ultrasound to obtain amniotic fluid index (AFI) registered. The results show that the average AFI value in the adapter group almost doubled while it changed insignificantly in the group without adapter. The difference in change between the two groups is significant (p < 0.001). There were no infectious complications in the newborn or in the mother. It is concluded that, in case amniocentesis is indicated to enable improved diagnostic accuracy in women with oligohydramnios due to PROM, application of the cervical adapter designed in this study will increase AFI and facilitate a less complicated amniocentesis. PMID- 7557640 TI - Ophthalmic artery velocimetry in preeclampsia. AB - Our objective was to compare ophthalmic artery pulsatility index values from normal pregnant women with those from preeclampsia patients. The ophthalmic artery of 20 normotensive pregnant women, 7 mildly preeclamptic and 2 severely preeclamptic patients was studied once with color Doppler flow imaging and pulsed Doppler ultrasonography after 32 weeks gestation. The peak systolic velocity [49.0 +/- 11.8 (SD) cm/s] in mild preeclampsia was significantly higher (p < 0.0001) than that (32.1 +/- 9.5 cm/s) in normotensive pregnant women, as were the end-diastolic velocity (14.1 +/- 7.7 cm/s vs. 3.7 +/- 1.4 cm/s, p < 0.0001) and time-averaged mean peak velocity (24.4 +/- 10.2 cm/s vs. 10.5 +/- 2.9 cm/s, p < 0.0001). The pulsatility index (1.58 +/- 0.47) in mild preeclampsia was significantly lower (p < 0.0001) than that (2.75 +/- 0.66) in normotensive pregnant women. In the 2 cases of severe preeclampsia, pulsatility index values (case 1: 1.86; case 2: 2.44) in the late stage of the disease process were significantly higher than those (case 1: 1.19; case 2: 1.20) in the early stage. We conclude that mild preeclampsia was associated with a significant decrease in ophthalmic artery vascular resistance, whereas ophthalmic artery vascular resistance in severe preeclampsia increased as the disease process advanced. However, in view of the small number of severe preeclamptic patients, these observations must be considered preliminary. PMID- 7557641 TI - Toxoplasma infection and pathological outcome of pregnancy. AB - To better evaluate the role of infection with Toxoplasma gondii in the outcome of pregnancy, 2,108 women with unsuccessful pregnancies (1,747 with spontaneous abortion, 187 with preterm delivery, 126 who delivered stillborn infants, and 48 whose offspring presented with clinical signs at birth) were tested for toxoplasma IgG and IgM antibodies during late pregnancy and/or up to 6 weeks after its termination. A nonpregnant group consisting of 157 women and a group of 568 women with a normal pregnancy were included as controls. The parameters observed were the prevalence of infection and the rates of acute and chronic infections. A causal relationship was established between acute infection during pregnancy and preterm delivery, stillbirth, and birth of infants with abnormal cranial volume, whereas there was no such association between acute toxoplasmosis and spontaneous abortion, or chronic infection and any of the pathological entities. PMID- 7557643 TI - Laser versus loop electrosurgical excision in vulvar condyloma for eradication of subclinical reservoir demonstrated by assay for 2'5' oligosynthetase human papillomavirus. AB - We studied, in an open clinical trial, 28 women with giant solitary human papillomavirus (HPV) lesions of the vulva that were treated by both laser and the loop electrosurgical excision procedure (LEEP), and analyzed the biopsy specimens of normal skin adjacent to the lesions for papillomavirus sequences by Southern blot hybridization and 2'-5' oligo-A synthetase (interferon) activity. The patients were reexamined after 3 weeks when a second treatment to the still infected areas was reapplied. They were reexamined for recurrence after 3 months: 4 patients harbored HPV-DNA genomes on the laser-treated side, adjacent to the original lesion, compared to 1 patient who developed recurrence on the LEEP side in a more distant, previously untreated, area. The optimal extent of laser or LEEP treatment necessary to reduce recurrence of vulvar condyloma remains to be established. Good in situ hybridization tests combined with the polymerase chain reaction and measurements of oligosynthetase activity to determine the types of cells involved at different sites of infection and to assess harboring of latent HPV genomes in histologically normal tissue are necessary in order to plan proper HPV treatment modality strategies. PMID- 7557642 TI - Effects of a dopamine antagonist (metoclopramide) on the release of beta endorphin, ACTH and cortisol in hyperprolactinemic-amenorrheic women. AB - Responses of LH, TSH, PRL, beta-endorphin-like immunoreactivity, ACTH and cortisol to metoclopramide were evaluated in hyperprolactinemic-amenorrheic women and normal women. Augmented TSH responses and blunted PRL responses to metoclopramide were found in hyperprolactinemic women compared to normal women. A significant increase in serum LH levels was found only in hyperprolactinemic women after metoclopramide. Thus, the hyperprolactinemic women showed hormonal changes compatible with increased central dopaminergic tone. A significant increase in circulating levels of beta-endorphin-like immunoreactivity, ACTH and cortisol was found in hyperprolactinemic women but not in normal women after metoclopramide administration. Thus, beta-endorphin and ACTH secretion appear to be tonically inhibited by increased central dopaminergic tone in hyperprolactinemic-amenorrheic women. PMID- 7557644 TI - Plasma granulocyte colony stimulating factor concentrations in pregnant women. AB - We measured the plasma granulocyte colony stimulating factor (G-CSF) concentrations in pregnant women to evaluate the association between G-CSF and increased neutrophil counts that has been observed during pregnancy. We examined 96 pregnant and 10 nonpregnant women. The G-CSF concentrations were assayed using a sandwich enzyme immunoassay. The G-CSF concentrations in pregnant women were significantly higher at all times than those of nonpregnant women. The G-CSF concentrations were also significantly higher during than before labor. The plasma G-CSF concentrations were positively correlated with the neutrophil counts during the 3rd trimester only. In conclusion, increased G-CSF concentrations may be related to the increases in neutrophil counts in pregnant women, especially during the 3rd trimester. PMID- 7557647 TI - Relationship between abnormally high levels of plasma CA 125 and resolution of acute pelvic pain in two women with endometrioma. AB - Two premenopausal women presented with endometrioma and slightly elevated CA 125 plasma levels (42.5 and 39.0 U/ml). After initial evaluation, both patients had an episode of sudden onset of pelvic pain with rapid resolution after a few hours. During the pain episode, CA 125 plasma levels were similar to the initial evaluation (41.7 and 40.3 U/ml), while 12 h after resolution of the pain, very high levels were measured in both patients (630 and 575 U/ml). The absence of tubal abnormalities and the negative peritoneal fluid cultural evaluation demonstrated by laparoscopy suggested a retrospective clinical diagnosis of partial ovarian torsion with sudden spontaneous untwisting. Therefore, an abnormal elevation of CA 125 plasma levels in patients with benign adnexal masses may be correlated with episodes of partial ovarian torsion with subsequent detorsion. PMID- 7557646 TI - Growth inhibition of uterine cervical cancer cells by a sequential administration of anticancer drugs and irradiation. AB - We investigated the growth-inhibitory effects of anticancer drugs and irradiation in relation to the cell cycle of cervical cancer. Irradiation 0 and 24 h after cisplatin administration significantly decreased cell growth. The number of cells in the G0G1 phase decreased 0-24 h and recovered 48 h after cisplatin or bleomycin administration. The present study suggests that irradiation for cervical cancer is preferable when the cancer cells accumulate during the proliferation phase after administration of cisplatin combined with bleomycin. PMID- 7557645 TI - Cervical dysplasia in HIV-seropositive women: role of human papillomavirus infection and immune status. AB - OBJECTIVE: In the present study we investigated the cytologic and colposcopic characteristics of a cohort of HIV-infected women, with the aim to determine a relationship between immunologic status and frequency and/or severity of cervical abnormalities. MATERIALS AND METHODS: 21 women, who tested positive for the HIV antibody and who were admitted as outpatients because of various gynecologic complications or because of an HIV infection that was under regular clinical surveillance. A pelvic examination was performed and Papanicolaou smears were obtained from endocervix and ectocervix before colposcopic examination. Cytologic samples for human papillomavirus (HPV) detection by polymerase chain reaction were also collected. Results obtained in the group of HIV-infected women were compared with findings in a group of 473 seronegative women recruited consecutively from our outpatient population. Serum samples for T lymphocytes were drawn within 2 weeks of cytologic and colposcopic examination. CD4 and CD8 monoclonal antibodies were purchased from Becton Dickinson (Mountain View, Calif., USA). RESULTS: HIV-infected women had a significantly higher percentage of HPV DNA positivity with respect to the outpatient population (67 vs. 7%, respectively, p < 0.001). Analysis of cytologic specimens revealed 9 women (43%) with cytologic evidence of cervical dysplasia in the HIV-seropositive group vs. 23 (5%) of 473 in the outpatient population (p < 0.001). All the HIV-seropositive women with cervical dysplasia presented an associated HPV DNA positivity; in particular, the percentage of associated HPV DNA type 16 in cervical dysplasia was 78% (7/9 cases). In HIV-infected women, the evaluation of T lymphocyte subset distribution suggested a significant relationship between CD4+ cell decrease and severity of cytologic findings (p = 0.03). DISCUSSION: The HIV-infected women had a tenfold higher prevalence of both HPV infection and cervical dysplasia than the outpatient population; this increased risk seems to be limited mainly to those who also had genital HPV infection. The analysis of immunologic status confirmed previous observations that an impaired immune system results in increased cervical disease. PMID- 7557648 TI - Vaginal scan for identifying endometrial abnormalities: limitations in clinical management. AB - It is generally recognized that an endometrial thickness of 4-8 mm is considered a definite cutoff point for detection of endometrial malignancy by transvaginal sonography. We report on 2 patients with endometrial cancer and on 1 with endometrial hyperplasia, in each of whom endometrial thickness was < 4 mm. PMID- 7557651 TI - Changes in platelet volume and count during pregnancy: a longitudinal study. PMID- 7557649 TI - Ureteral obstruction caused by intrinsic ureteral endometriosis with adenomyosis but without pelvic endometriosis. PMID- 7557650 TI - A case of adenocarcinoma arising within a urethral diverticulum diagnosed only by the surgical specimen. AB - We report an extremely rare case of a 50-year-old woman with an adenocarcinoma arising within a urethral diverticulum. She could not be diagnosed correctly before operation and the microscopic pathological examination of the surgical specimen was the only indication showing the cancer origin. PMID- 7557652 TI - Is removal of the tube mandatory after coagulation in ectopic pregnancies. AB - Laparoscopic treatment of ectopic pregnancy is gaining wide acceptance. We report a new simple laparoscopic technique involving grasping the tube with forceps and coagulating on both sides of the ectopic pregnancy site, leaving the fertilized ovum in situ. This method is suitable for multiparous women and women who request tubal sterilization. Sixteen patients (mean age 32.6 +/- 4.4 years) were treated by using this method. The mean duration of hospitalization was 1.94 +/- 1.12 days. No complications occurred. PMID- 7557653 TI - Modifications of platelet shape change and ATP release during cardiopulmonary bypass. AB - Platelet activation is accompanied by characteristic morphological changes: smooth-disc platelets become more spherical in shape and develop psudopods. The purpose of this study is to investigate whether platelets change after extracorporeal bypass. Twenty-two patients undergoing cardiopulmonary bypass (CPB) were studied prior to anesthesia and immediately after the operation. Platelets activated by different agonists were monitored simultaneously for morphological changes, ATP release and aggregation. While shape change measured before surgery was large, it was significantly reduced after bypass surgery (p < 0.01); morphological changes were quicker postoperatively. Several other parameters also changed: the time lapse between administration of the agonist and the start of ATP secretion decrease significantly (p < 0.01). After activation with high concentrations of ADP, ATP release was significantly increased (p < 0.01). On the other hand, less ATP was released after platelet activation with collagen and arachidonic acid, suggesting a change in platelet adhesion or a downregulation of endoperoxide synthesis. In our study, the importance of preactivation change in shape, estimated quantitatively by percent loss in ability of changing shape, can be compared using various agonists, with proportional defects in release and aggregation. These data provide evidence for different intrinsic levels in platelet defects after CPB. PMID- 7557654 TI - Unstimulated and thrombin-stimulated platelets binding to immobilized fibrinogen and fibrin on polystyrene supports. AB - The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 micrograms/tube, which represents 2.35 micrograms/cm2. Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated 111In-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 +/- 0.3 x 10(6) and 3.1 +/- 0.4 x 10(6) platelets/micrograms fibrin(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 x 10(6) platelets/micrograms fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated 111In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 x 10(6) platelets/micrograms fibrin(ogen)/min, respectively). In saturation conditions, 1 microgram fibrinogen binds 5.7 +/- 0.6 x 10(6) thrombin-stimulated platelets and 1 microgram fibrin binds 4.6 +/- 0.5 x 10(6) thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen. PMID- 7557656 TI - Effects of ionic and nonionic contrast media on clot structure, platelet function and thrombolysis mediated by tissue plasminogen activator in plasma clots. AB - Various radiographic contrast agents have anticoagulant or prothrombotic properties. Ionic agents are reported to have greater antithrombotic potential while nonionic agents are considered more thrombogenic. Some agents after fibrin structure and bind to platelets in purified systems. This study compared the effects of iohexol, a nonionic agent, and iothalamate, an ionic agent, on fibrin assembly, clot structure, platelet function and clot dissolution in plasma. Plasma gels containing increasing concentrations of iothalamate were composed of thinner fibers with decreased fiber mass/length ratios (mu) and reduced gel turbidity. Such clots were more rigid and more resistant to fibrinolysis induced by tissue plasminogen activator (tPA). Gel elastic modula increased from 10,000 to 27,000 dyn/cm2 as iothalamate concentration increased from 0 to 20 mM. 50% lysis time increased from 800 to 1,250 s with the addition of 10 mM iothalamate. At 20 mM, iothalamate had no effect on ADP-induced platelet aggregation but prolonged the lag phase seen with collagen-induced aggregation. Platelet force development increased from 15,300 to 20,400 dyn with 20 mM iothalamate. The effect of iohexol were similar. Gel optical density dropped from 0.50 to 0.32, mu fell from 3.3 to 2.2 x 10(13) D/cm, and elastic modulus rose from 11,000 to 24,000 dyn/cm2 as iohexol concentration was increased from 0 to 20 mM. Clots formed in the presence of 60 mM iohexol and tPA did not dissolve in 72 h while control clot 50% lysis time was 450 s. At concentrations > or = 40 mM, iohexol completely blocked collagen-induced platelet aggregation. Platelet force development increased from 7,660 to 19,600 with 40 mM iohexol. Contrast media possess profound fibrin-altering activities in plasma. Fibrin formed in the presence of some agents may be significantly more resistant to fibrinolysis. PMID- 7557657 TI - Investigation of activated prothrombin complex concentrate as potential hirudin antidote in animal models. AB - The specific thrombin inhibitor r-hirudin (HBW 023) has been demonstrated to be effective in preventing thrombosis in preclinical models. Up to now, no bleeding complications have been observed using therapeutically effective doses in animals studies. However, in case of inadvertent overdosing the occurrence of undesired impairment of coagulation cannot be excluded. As a potential antidote an activated prothrombin complex concentrate (APC) was tested on its ability to normalize blood coagulation. APC given s bolus injections 5 min and 3.0 mg/kg neutralized the r-hirudin-induced prolongation and 3.0 mg/kg neutralized the r hirudin-induced prolongation of whole blood coagulation time in rabbits completely within 5 min without any clot formation in the blood vessels or capillaries of the heart, kidneys, or lungs. Furthermore, bleeding time prolongation induced by bolus application of 3.0 and 30.0 mg/kg r-hirudin was significantly inhibited by APC within 5 min. These results suggest that administration of APC may be an effective way to reverse the effects of r-hirudin in the coagulation system in case of inadvertent overdosing of r-hirudin. PMID- 7557655 TI - Effects of ditazol on the vascular retinal pattern in experimental diabetes in rats. AB - Platelets from diabetic patients are hypersensitive to aggregating agents. An imbalance in thromboxane/prostacyclin synthesis has been postulated as an antecedent to the development of diabetic retinopathy. We studied 3-month streptozotocin-diabetic rats (SDR) treated with 200 mg/kg/day p.o. of ditazol, an antiplatelet drug that inhibits thromoboxane formation. Thromboxane B2 (TxB2) production was higher and 6-keto-PGF1 alpha synthesis lower in SDR than nondiabetic rats (NDR). In treated animals, ditazol inhibited platelet aggregation by 66%, and TxB2 production by 58%, and increased vascular 6-keto-PGF 1 alpha by 45%. Furthermore, 13% of the retinal surface was covered by peroxidase labelled vessels in NDR, 2.1% in nontreated SDR, and 8.9% in SDR treated with ditazol. We postulate that ditazol may prevent or reduce diabetic retinopathy. PMID- 7557658 TI - In vitro characterization of antithrombin III concentrates--a single-blind study. AB - Twenty-three lots of five antithrombin III (AT III) concentrates from four manufacturers were analyzed in a single-blind study. All the preparations had been virus-inactivated by pasteurization, and one concentrate had also been treated with solvent/detergent (S/D). AT III activities were determined using two thrombin-based and one factor Xa-based chromogenic substrate assays. AT III antigen was measured by kinetic nephelometry. All AT III assays were tested against the first international reference preparation coded 72/1. In addition, AT III was characterized by crossed immunoelectrophoresis in the presence of heparin and by gel filtration. The following were quantified: heparin cofactor II activity and antigen content, heparin activity, thrombin-AT III complexes, AT III protease complexes, total protein, albumin, immunoglobulins, glucose and pH. The AT III concentrates differed markedly in terms of their purity and potency. The specific activities of AT III and the ratios of AT III activity to antigen content ranged from 3.4 to 6.9 and from 0.63 to 0.84, respectively. The highest values were found in five lots of the concentrate that had been treated by both pasteurization and S/D. This preparation was the only one that was virtually free of denaturated AT III, as judged by crossed immunoelectrophoresis. Marked batch to-batch variation in AT III potencies was found in two out of the five preparations analyzed. In two out of five lots from one manufacturer, the measured potencies were more than 10% lower than the declared potencies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557659 TI - [Long-term follow-up in polycythemia vera]. AB - 67 patients with polycythemia vera, diagnosed and treated between 1963-1990, were analyzed retrospectively. The mean follow-up was 7.4 +/- 5.1 years and median actuarial overall survival 23.6 years. 50% were treated with a single chemotherapeutic agent, and 46% with combinations of agents. In only 4% was phlebotomy the sole treatment. There were hematological complications in 18%, thromboses in 18%, and bleeding in 21%. PMID- 7557661 TI - [Familial hemolytic uremic syndrome]. AB - Hemolytic uremic syndrome (HUS) consists of an apparently heterogenous group of disorders resulting from a variety of diseases. Outcome and prognosis are largely determined by the basic disease. In the familial type there is frequently progressive deterioration and poor prognosis. The classic epidemic type usually has a better prognosis. The pathophysiology of familial HUS is unknown. Treatment includes transfusion of fresh frozen plasma or plasma exchanges. We report 2 siblings, male and female infants, who developed HUS at ages 7 and 8.5 months, respectively (but they were born 5 years apart). Their courses were characterized by slow onset, gradual deterioration, hypertension and fatal outcome in 1, and end-stage renal failure in the other. PMID- 7557662 TI - [The milk-rejection sign and earlier detection of breast cancer]. AB - 2 cases of infants who refused to nurse from a breast with a cancerous tumor are reported. The first 6 cases of this nature were described 28 years ago. Though this sign could lead to earlier detection of breast cancer in lactating women, it has not been accepted as such by the medical profession. Had the milk-rejection sign been recognized in our cases, breast cancer could have been diagnosed months earlier. We suggest that lactating women should be asked about the nursing habits of their babies and if there is milk-rejection, the breasts should be examined with special care. PMID- 7557660 TI - [Benign meningioma following scalp irradiation in north African immigrants]. AB - Many Jewish immigrants from North Africa in the late 1950s were treated with low dose irradiation of the scalp for tinea capitis. In 2 such patients, a 47-year old man and a 43-year-old woman, both with long histories of headaches and dizziness, meningiomas were found. There is a well-known correlation between low dose irradiation of the scalp and development of meningioma. Therefore, in immigrants from North Africa with prolonged headaches and dizziness, there should be a careful history with regard to irradiation of the scalp and investigation to exclude meningioma. PMID- 7557663 TI - [Retinopathy and serum uric acid in diabetics]. AB - Low serum uric acid (UA) levels have been reported in diabetics. In a group followed for 15 years it was reported that low UA levels preceded the onset of diabetic retinopathy. We studied 95 consecutive diabetic clinic patients between July and September 1992. There was no significant difference in UA levels between those with or without retinopathy: 5.0 +/- 1.2 (SD) mg--vs. 5.3 +/- 1.3, respectively. Multiple regression analysis showed that higher UA levels were related independently to high body mass index (overweight, obesity), and male gender, but not to age, duration of diabetes, glycosylated hemoglobin, diabetic treatment or retinopathy. PMID- 7557664 TI - [Disturbed elderly patients in psychiatric hospitals]. AB - There are few active psychogeriatric units in Israel. We studied several demographic characteristics, source of referral, psychiatric diagnosis, physical characteristics, length of hospital stay, and discharge referral in 214 disturbed patients over the age of 65. They were admitted for psychiatric hospitalization during 1993 and were relatively well physically, their diagnoses were heterogenous, and length of hospital stay was slightly less than the national average. Most were admitted from and discharged back to the community. These data should dissipate prevailing fears and prejudices regarding admission of elderly patients to psychiatric hospitals. More active psychogeriatric units are needed for the specific needs of this population. PMID- 7557665 TI - [Rehospitalization of very-low-birth-weight infants after discharge from neonatal intensive care units]. AB - The higher survival rate of very-low-birth-weight (VLBW) infants is associated with an increasing rehospitalization load, especially during the first year after discharge from neonatal intensive care units. This was evaluated in a sample of 86 VLBW infants born during 1989-1990, compared with 120 normal term newborns delivered during 1990. 34 (39.5%) VLBW infants were rehospitalized, compared with 16 (13.3%) term newborns (2.9:1; p < 0.001). The ratio of hospitalizations between the groups was even higher, 4:1. Of the 49 VLBW infants hospitalized, 25 had respiratory complications (in 15 bronchiolitis), 15 required inguinal herniorrhaphy, 6 had acute gastroenteritis and in 4 there were other causes. Rehospitalized VLBW infants were more frequently of birth weight less than 1000 g and their initial stay in the neonatal intensive care unit tended to be longer. There was no significant correlation between rehospitalization and: gender, gestational age below or above 28 weeks, multiple pregnancy, need for mechanical ventilation, presence of bronchopulmonary dysplasia, or parental socioeconomic status. Hospitals should be adequately prepared to meet the special challenge of frequent rehospitalization of VLBW infants. PMID- 7557667 TI - [Hebrew knowledge of new immigrant physicians]. AB - Since 1989, 10,000 physicians have immigrated to Israel, mostly from the former Soviet Union. Lack of a reasonable command of Hebrew, the language of the Israeli medical system, is likely to adversely affect doctor-patient communication and cause critical errors. We report the development of a structured, written examination to assess knowledge of medical Hebrew. It was administered to 67 Russian immigrant physicians and the results indicated low proficiency in Hebrew in general, and in medical Hebrew in particular. There was a significant correlation with extent of general medical knowledge. PMID- 7557668 TI - [ATLS course in the development of trauma care in Israel]. PMID- 7557666 TI - [Falls in the geriatric department: responsibility of the care-giver and the hospital]. AB - During a 4-year prospective study there were 94 falls in 60 patients hospitalized in our geriatric department, mostly in dependent women. There was a "responsibility group," in which 19 of the falls occurred (20.2%). These included 14 while the patient was being cared for by nurses or relatives, and 5 due to defective fixation of bed side-walls. In the comparison group there were 75 falls while the patient was alone. Risk of falling was increased by cardiovascular conditions, including low blood pressure and arrhythmias, and by dementia, paralysis and low score for activity of daily living (ADL). Impulsiveness of patients was an important cause, making falls unpredictable and difficult to prevent even while under proper care. Diazepam (Assival, Valium) in those with a low ADL (27.5 +/- 16.5) and a relatively high mental test (7 +/- 2.1) seemed dangerous in the responsibility group, possibly due to increased impulsiveness. Nitrazepam (Numbon) appeared to be dangerous in ambulatory patients of the comparison group when both ADL and mental tests were relatively high (53.5 +/- 17.5 and 8.2 +/- 2.3, respectively). Mean ADL (33.7 +/- 20.8) in all patients decreased after falls by -2.8 +/- 9.4 (p < 0.00001). In the responsibility group it decreased by -5 +/- 13.2 (p < 0.16), and after falls during visits of relatives, by -14 +/- 7.1 (p < 0.23). In the comparison group it decreased by 1.9 +/- 7.1 (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557669 TI - [Contribution of molecular genetics to the diagnosis of Charcot-Marie-Tooth disease]. PMID- 7557670 TI - [Endothelin-1 and liver disease]. PMID- 7557671 TI - [Underwater diving during pregnancy]. PMID- 7557673 TI - [Mechanical trauma as a cause of cancer--a continuing dispute]. PMID- 7557672 TI - [In utero vaccination to prevent neonatal infections]. PMID- 7557675 TI - [Guidelines for diagnosis, treatment and follow-up of high blood pressure]. PMID- 7557674 TI - [Iodinated contrast media: adverse reactions, prevention and treatment]. PMID- 7557676 TI - [Zinc deficiency in children]. PMID- 7557677 TI - [How should informed consent for an invasive radiologic procedure be phrased in order that responsibility for contrast agent reactions be blamed on the patient himself]. PMID- 7557678 TI - [Our friend the cough]. PMID- 7557679 TI - [Epidemiology and prognostic factors in cutaneous malignant melanoma]. AB - 207 patients with primary cutaneous malignant melanoma were admitted between August 1, 1988 and July 31, 1992 for local excision and treatment. The female to male ratio was 1.4:1 and the peak age was in the seventies. The most frequent site in males was the back and in females the legs. Superficial spreading melanoma was the most frequent type (40%); there was also a high rate of nodular melanoma (20%), particularly in males. Thin melanomas accounted for most of the cases. On follow-up 27 (13%) developed metastases in transit or in regional lymph nodes and 10 (5%) developed distant metastases; 2 (1%) had locally recurrent melanoma and 2 died of metastatic melanoma. There was a significant positive correlation between Breslow thickness, Clark's level of invasion, histopathological ulceration, nodular and acral lentiginous type of lesion and development of metastases. These data may be valuable for public and professional education and in the prediction of outcome of melanoma. PMID- 7557680 TI - [Multidisciplinary approach to counseling in Huntington's disease]. AB - The recent cloning of the Huntington's disease gene now allows definitive presymptomatic and even prenatal testing. This achievement has posed a considerable challenge for the genetic counselor. We present the multidisciplinary approach implemented in our center, and experience in our first 13 diagnoses. Individuals at risk undergo neuropsychiatric evaluation, genetic counseling, molecular studies, post-counselling follow-up and support according to the protocols suggested by the International Huntington's Disease Association. Molecular studies of cytosine-arginine-guanine repeats showed that 4 individuals and 1 at-risk fetus were unaffected and 6 at-risk individuals, including the mother of the fetus, had inherited the disease mutation. The diagnosis was excluded in 2 individuals clinically suspected of Huntington's disease. All but 1 of those investigated had accepted, and adapted to the new reality. The exception was a carrier who asked to discontinue the contact a week after being informed of the results. PMID- 7557681 TI - [Potential effect of the Russian immigration on cancer incidence in Israel]. AB - Cancer incidence patterns of Jews from the former USSR differ from those among Israeli Jews originating from Europe who arrived with previous waves of immigration. Projected estimates of cancer incidence in Israel following the current Russian immigration indicate that the total cancer rate in the country will grow in proportion to this immigration. In addition, changes are anticipated in the distribution of cancer sites. These include increases in the incidence of esophageal cancer of 50%, stomach and cervical 33%, pulmonary 21%, and laryngeal 18%. In contrast, malignant melanoma, breast and rectal cancer, and leukemia and lymphomas, will decrease slightly. These changes mandate appropriate adjustments with regard to both curative and preventive measures. PMID- 7557682 TI - [Frequency of the Gaucher mutation among recent Russian immigrants]. AB - Gaucher's disease is frequent in the Ashkenazi Jewish population of Israel, with a gene frequency by molecular analysis of approximately 0.032, corresponding to a birth rate of 1:850. The recent immigration from the former Soviet Union brought more than 400,000 Jews, mostly of Ashkenazi descent. However, only a few cases of Gaucher's disease have been diagnosed. Possible explanations are lack of awareness of the signs of Gaucher's disease among Russian health workers and family practitioners in Israel, and a significantly lesser frequency of the Gaucher gene among Russian immigrants than among other Ashkenazi Jews in Israel and the US. We studied the frequency of the 1226G (N370S) mutation in a cohort of 202 recent immigrants from the former Soviet Union. We found 10 carriers (4.95%), indicating a decreased frequency of the Gaucher gene, but by extension, the probability of unidentified cases in need of treatment. Family practitioners should be aware of the possibility that heretofore misdiagnosed symptoms may be those of Gaucher's disease. PMID- 7557683 TI - [Epidemiology of developmental disorders in children in Tel Aviv]. AB - We assessed the prevalence of developmental disorders, the need for intervention, potential for rehabilitation, and also characterized the risk factors, from files of 1,944 children referred during 1981-1990. The study group consisted of 4.3% of the children born in Tel Aviv during this period. Referral was highest between the ages 3-4 years and the causes for referral were language, speech and communication disorders (38%), global developmental delay (20%), motor disorders (16%), and behavioral and emotional problems (15%). Developmental disorders were more prevalent in boys (M/F ratio 1.8:1). 84% had at least 1 risk factor, either pre/perinatal, social or genetic. 66% had a social risk factor requiring involvement of a community social worker, or a parent with a chronic disease. The principal genetic risk factors were a developmental problem in a sibling, and parental consanguinity. The most common perinatal risk factors were birth weight under 2500 grams, hyperbilirubinemia of the newborn and severe asphyxia. Prognosis was good: 83% had normal intelligence and only 10% had severe disability. Upon discharge from the development center at the age of 5 years only 18% required special schooling, but 63% were referred for continuation of rehabilitation services. This study provides multi-disciplinary information that allows planning of requirements for diagnostic, therapeutic and rehabilitation services in the human, logistic and economic fields. PMID- 7557684 TI - [Consequences of misdiagnosis and labeling in psychiatry]. AB - Differences in diagnostic approaches between psychiatry and other medical specialties were examined and problems resulting from misdiagnosis are presented. The labelling and stigma resulting from misdiagnosis have severe implications and there is inherent difficulty in correcting misdiagnoses of major psychiatric disorders. We present a 38-year-old man who underwent numerous psychiatric and psychological examinations in order to change a previous misdiagnosis. The difficulties examiners had in accepting the possibility of misdiagnosis, and its severe consequences, are described. PMID- 7557685 TI - [The stria vascularis of the inner ear and its normal structural variations]. AB - The ultrastructural effects of gentamicin on the stria vascularis of the inner ear of guinea pigs were studied by transmission electron microscopy. In a single specimen of an isolated, inner ear we found an unexpected variation in the structure of the stria vascularis in a normal, pigmented, guinea pig not treated with ototoxic drugs. There were prominent, wide protrusions from the apical surfaces of marginal cells into the endolymphatic space. This finding has not been previously reported and was seen in only 1 of 7 animals studied. It may be considered a normal structural variation, and is not pathological change in the stria vascularis due to ototoxic drugs. PMID- 7557686 TI - [Hazards of infectious diseases in acupuncture]. PMID- 7557687 TI - [Ebola disease]. PMID- 7557688 TI - [Human umbilical cord blood as a source of hematopoietic progenitor cells for transplantations]. PMID- 7557689 TI - [Prenatal alloimmune thrombocytopenia]. PMID- 7557691 TI - [Estrogen-progesterone "add-back" for long-term GNRH analogue therapy]. PMID- 7557692 TI - [The etiology of vulvar carcinoma--still an enigma]. PMID- 7557690 TI - [Andropause: myth or reality?]. PMID- 7557693 TI - [Microalbuminuria in clinical practice]. PMID- 7557694 TI - [Management of N-O neck]. PMID- 7557695 TI - [Acrocyanosis]. PMID- 7557696 TI - [Drug treatment after a first unprovoked epileptic seizure]. PMID- 7557698 TI - [Flumazenil]. PMID- 7557697 TI - [Helicobacter pylori as a cause of benign malignant peptic ulcer]. PMID- 7557699 TI - [Resistance to activated protein C--a novel cause of thrombophilia]. AB - A point mutation in coagulation factor V which causes resistance to cleavage of factor Va by activated protein C (APC), was recently found to underlie thrombotic events. We examined 20 consecutive patients, under the age of 40, who suffered from idiopathic venous or arterial thrombosis. In 8 (40%) there was resistance to APC manifested by absence of the expected prolongation of activated partial thromboplastin time (aPTT). In 3, the addition of normal plasma corrected the anomaly in the patient's plasma, although the addition of factor V- deficient plasma caused no change. In a family of a 17-year-old boy with idiopathic deep venous thrombosis we found a mutation in factor V which was responsible for APC resistance. The patient and 4 family members showed a single G to A transition in position 1691 in their cDNA, resulting in substitution of arginine (506) for glutamine. The mutation in this area, which is the cleavage site for APC, is associated with thrombotic episodes and is frequently observed in patients with familial thrombophilia. PMID- 7557700 TI - [Cystinuria and urolithiasis]. AB - Cystinuria is a hereditary metabolic disorder which causes urinary lithiasis. Patients with cystinuria present at an earlier age (17 years), and with larger stones than patients with other types of urinary stones. As a result of frequent formation of new stones and multiple operations, many patients suffer from obstructive nephropathy and deterioration of kidney function. During the past decade we treated and followed 51 patients with cystine urolithiasis from 39 families. Screening disclosed another family member with cystinuria in 56% of the cases. The diagnosis of cystinuria was only made an average of 2 years after a patient first presented. In the group 12 kidneys had been removed due to various complications and in 7 kidneys there was significant deterioration of function. Each patient underwent a mean of 5.8 surgical interventions, including open operations, extra-corporeal shock wave lithotripsy (ESWL), percutaneous nephrolithotripsy (PCNL) and urethroscopy, but most still had residual stone fragments. To prevent complications from cystine urolithiasis every effort should be made to detect the disease early, including screening of families of patients with cystinuria and close follow-up of all family members. Early detection of stones, before they become large makes noninvasive treatment with ESWL possible, whereas large stones require PCNL. Patients should be urged to maintain high urine outputs and to continue uninterrupted treatment with penicillamine or tiopronin, and urinary alkalinization. PMID- 7557701 TI - [Misdiagnosis and labeling in psychiatry and their consequences: Part II]. AB - The complexity of arriving at a correct psychiatric diagnosis in cases in which physical and mental disorders are interrelated is discussed. A case is presented in which a psychiatric diagnosis had been made and malingering suspected, although the patient actually had a severe neurological disorder. The psychiatric diagnosis had not been changed despite recurrent medical and psychological examinations which clearly indicated a physical disorder. The difficulties that follow misdiagnosing organic disorders as psychiatric disorders are illustrated. Various aspects of the effects of psychiatric misdiagnosis on functional, legal and civil aspects of life are discussed. Emphasis is given to the problems facing those discharged from military service for medical reasons, especially mental disorders. Important measures are currently being applied to overcome some of these problems. It is strongly emphasized that there is need for greater openness and tolerance among psychiatrists when making psychiatric diagnoses. PMID- 7557702 TI - [Improvement of communication between consultants and family practitioners: a controlled trial]. AB - Communication between primary care physician and consultants is essential for continuing patient care. Several studies have noted failures of consultants to provide follow-up information to the referring physician. We studied the referral pattern in a family practice clinic, and tried to increase written communication between consultants and primary care physicians. 306 patients referred to medical consultants during a 6-month period received either a routine referral form with the patient's name, age and reason for consultation, or an experimental referral form with a special area for the consultant to fill out and return to the referring family physician via the patient. The overall referral rate was 6.4%, the most frequently consulted specialists being ophthalmologists (27%), followed by surgeons (26.4%), internists (24.7%), otolaryngologists (10%) and gynecologists (8%). Consultation reports were returned to the referring physician in 55% of the cases. The return rate was significantly higher (62.5%) for the experimental form than the standard form (48%), (p < 0.004, X2 = 15.18). This improvement in response rate was seen for all specialists and for the different types of clinics. The proposed referral form is a simple and inexpensive method of improving communication between family practitioners and specialists, thus ensuring continuity of patient care. PMID- 7557703 TI - [Hereditary hemorrhagic telangiectasia presenting with hematuria and severe anemia]. AB - A patient with hereditary hemorrhagic telangiectasia was admitted with hematuria and severe anemia after mild recurrent episodes of epistaxis. Telangiectasias were found in the skin and buccal and nasal mucosa. No defect in the coagulation mechanism was found; thrombocyte count and function were normal. On cystoscopy, tortuous engorged vessels, some actively bleeding, were seen in the trigonal mucosa. Biopsy showed enlarged vessels in the lamina propria. Electrocoagulation of the bleeding vessels stopped hematuria, but 6 months later it recurred. This time Nd-YAG laser was used to stop the bleeding after electrocoagulation was ineffective. PMID- 7557704 TI - [A multicenter study of lisinopril in the treatment of mild to moderate hypertension]. AB - The efficacy, tolerability and impact on quality of life of the ACE inhibitor lisinopril were evaluated in a 12-week open, multicenter post-marketing surveillance study. 233 patients, 45 years and over with diastolic blood pressure (DBP) 95-105 mm Hg were followed after a washout period of 7 days. 22 withdrew due either to adverse reactions, mainly cough (4.3%) and dizziness (3%), or noncompliance. DBP of 90 mm Hg or less was achieved in 50.7% with once-a-day 10 mg lisinopril, in 26.1% with 20 mg and in 16.1% with 20 mg plus addition of hydrochlorothiazide, 12.5 mg; in only 7.1% was BP not controlled. Adverse reactions other than cough or dizziness were experienced by fewer than 1%. Dartmouth COOP Functional Health Assessment Charts/WONCA were used to evaluate quality of life and were found useful in the study. Compared to baseline assessments, all functional status indicators (physical fitness, feelings, daily activities, social activities, change in health and overall health) scored significantly better in all treated patients after 12 weeks. Thus, in more than 90% of patients lisinopril was well-tolerated, highly effective in lowering blood pressure and possibly (no control group) improved the quality of life of all patients. PMID- 7557705 TI - [Dengue fever]. AB - Dengue fever is a viral disease, transmitted to man via mosquito bites. It is endemic in tropical regions (10 million infected annually) and is characterized by high fever, headache, myalgia, lethargy, vomiting, rash and neutropenia. The upward trend in the number of young Israelis visiting tropical countries increases the number of those potentially exposed to this disease. We present 4 Israelis who returned with dengue fever from Thailand. PMID- 7557706 TI - [Reduction in frequency of pneumonia after stroke, following programmed swallowing evaluation]. AB - Aspiration pneumonia is a frequent complication of cerebrovascular accidents (CVA). It occurs mainly in those with post-CVA swallowing disorders, which can be diagnosed by bedside swallowing evaluation. Evaluation is based on observation of components of the oral and pharyngeal stages of swallowing a drink of 50 ml of clear liquid. Changing the consistency of the diet and the mode of swallowing, following its evaluation, can reduce significantly the frequency of post-CVA aspiration pneumonia. In 180 patients admitted for stroke rehabilitation, aspiration pneumonia occurred in 10% and swallowing disorders were found in 28%. Planned swallowing evaluation followed by the above regimen was associated with a gradual reduction of pneumonia from 16% in the first group of 60 patients to 3% in the second group of 60; and of patients with dysphagia, from 27% in the first group to none in the second. PMID- 7557707 TI - [Missed injuries]. PMID- 7557708 TI - [Screening for prostatic cancer]. PMID- 7557709 TI - [Effect of pregnancy and delivery on genuine stress incontinence]. PMID- 7557710 TI - [Pneumonia in patients on the mechanical respirator]. PMID- 7557711 TI - [Vagus nerve stimulation for partial seizures]. AB - Cerebellar and thalamic stimulation has been known for many years to improve control of epileptic seizures. In the past few years electrical vagus nerve stimulation (VNS) has been introduced and has been effective in controlling seizures in animal models. These encouraging results led to the development of a transcutaneous programmable pulse generator and electrode lead for human use. 2 pilot studies and a multicenter, prospectively-randomized, parallel, double-blind study of patients with refractory partial seizures were performed. In a 3-22 month follow-up, in about 50% of patients seizures were reduced by 30-50%. There were no significant complications of the implant. Side-effects associated with VNS included intermittent hoarseness, coughing and throat pain. Additional controlled clinical trials with many patients and long follow-up are needed. We report 2 patients, the first in Israel, who underwent VNS. PMID- 7557712 TI - [Measles in here to stay]. PMID- 7557713 TI - [Anaphylactoid reactions to radiocontrast media]. PMID- 7557714 TI - [Adverse reactions to amiodarone]. PMID- 7557715 TI - [Electrocution and lightning strikes--pathophysiology and treatment]. PMID- 7557716 TI - [Cavitary lung lesion and chronic renal failure in a 76-year-old man]. PMID- 7557717 TI - [Muscle-sparing thoracotomy: a renewed approach in thoracic surgery]. AB - Lateral thoracotomy has been the traditional approach in general thoracic surgery for several decades, and with anterior and posterior extension, gives excellent exposure for almost all intrathoracic procedures. However, division of the chest wall muscles, usually with diathermy, contributes to prolonged pain and morbidity. A muscle-sparing thoracotomy which preserves chest wall muscles and gives adequate exposure has been used by us for the past 2 years as an alternative to the standard thoracotomy. PMID- 7557719 TI - Shaking the kaleidoscope. PMID- 7557718 TI - [Head injuries from falls in children of 2 ethnic groups]. AB - The highly prevalent problem of falls, the main cause of head injury in children, responds to preventive intervention. To aid in designing fall-prevention programs, we defined the pre- and post-hospitalization characteristics of children of 2 predominant Israeli ethnic groups: Jews (Group A) and non-Jews (Group B). In a prospective study conducted over a 10-month period in our emergency room during the morning shift, data from 274 patients aged 0-14 who presented with head injury due to a fall were collected. In Group B falls and damage were significantly more severe. Based on these differences in injury and in referral patterns between the 2 groups, a high-risk profile for head injury from falls in children was compiled to aid in designing an effective prevention program. PMID- 7557721 TI - Job satisfaction among social work discharge planners. AB - Eighty social workers working with elderly patients in 36 acute care hospitals provided information about their overall job satisfaction as discharge planners and identified sources of satisfaction and dissatisfaction in their jobs. Twenty eight percent were very satisfied and 50 percent somewhat satisfied with their jobs. Sources of satisfaction were the ability to help patients and families, concrete resource provision, job challenge, and autonomy. Dissatisfaction resulted from organizational constraints and lack of support from other health care professionals. Educational and theoretical implications are discussed. PMID- 7557720 TI - Jail recidivism in a forensic case management program. AB - This article explores the factors related to jail recidivism among clients receiving case management services who were seriously mentally ill, homeless, and former inmates in a large urban jail. The authors interviewed six case managers about 51 clients and examined the use of treatment stipulations as a condition of probation, clients' major diagnostic and behavioral problems, and case management strategies. Clients whose case managers actively sought legal stipulations to case management as a condition of probation and parole were more likely to return to jail. Implications for service planning and future research are discussed. PMID- 7557722 TI - Decision-making situations in health care. AB - Social workers in health care settings are constantly required to make clinical decisions about patient care and treatment. Although much attention has been devoted to the normative or ethical aspects of decision making in such settings, little attention has been given to the typical situational aspects of decisions social workers must make in health care. This article discusses four types of clinical decision situations--operational, strategic, authoritative, and crisis- and presents a model to assist in analyzing their components and requirements. Case vignettes drawn from practice experience illustrate each type of decision making situation. The article concludes that knowledge of the situational aspects of practice decision making can be helpful to practitioners by enabling them to sort out courses of action and intervention. PMID- 7557723 TI - Health conditions and perceived quality of life in retirement. AB - This study investigated the effects of specific health conditions on perceived quality of life in retirement as measured by dimensions of retirement satisfaction. Respondents were 252 men and 199 women who participated in the retirement substudy of an ongoing epidemiological investigation of people age 65 and over in two rural Iowa counties. Retirement satisfaction was measured by the Retirement Descriptive Index, which assesses satisfaction with activities and work, finances, health, and people. Pulmonary disease was a predictor of dissatisfaction with health for both sexes. Pulmonary disease and heart attack were the strongest predictors of dissatisfaction with health for men, followed closely by stroke. Arthritis was the strongest predictor of dissatisfaction with health for women. Implications for social work practice are discussed. PMID- 7557724 TI - Cancer pain management and the role of social work: barriers and interventions. AB - Quality of life has become increasingly important in the management of cancer, because patients with the disease are surviving longer. Although effective therapies exist, more than half of all cancer patients suffer unrelieved pain during illness and treatment. Undertreated and unnecessary pain represents a failure of the multidisciplinary team, including the social worker, to respond appropriately to the needs of cancer patients. This article increases social workers' awareness of the prevalence of cancer-related pain, identifies the barriers that undermine effective pain relief, and develops a model for social work intervention. Skills such as communication, assessment, problem solving, and psychological support are explored in detail. PMID- 7557725 TI - Mental health problems and alcohol abuse: co-occurrence and gender differences. AB - A growing body of research has demonstrated that alcohol abuse co-occurs with a broad range of mental health problems. To date, however, there is a scarcity of data concerning the co-occurrence of alcohol abuse and other problems in people who seek mental health outpatient services and, consequently, about gender differences among them. The present study surveyed 376 clients receiving outpatient services at a mental health center. Results demonstrate that women self-reported significantly more psychophysiological distress and family pathology and men reported more community problems and health concerns. Men drank significantly more than women, were more likely to have had a problem with alcohol over the past year, and were more likely to have been treated for substance abuse. For both men and women, the level of alcohol consumption varied directly with the severity of psychophysiological symptoms and community and health problems. PMID- 7557726 TI - Decision-making influence: an empirical study of social workers in nursing homes. AB - Health care research on social workers' organizational influence has focused almost exclusively on the hospital setting. Little is known about social workers' influence in nursing homes, in particular organizational factors that support or constrain their impact on resident care. This article presents findings from a survey of 90 social workers employed in 53 nursing homes who identified their level of influence in five spheres of organizational decision making. With the exception of personnel decisions, there were few differences between the influence levels of the directors of social services and social workers without administrative responsibilities. Number of full-time-equivalent social workers, type of ownership, and length of employment of the director of social services were significantly related to the level of perceived influence held by the director of social services. The implications of these findings for social work practice in nursing homes are discussed. PMID- 7557727 TI - Older people's responses to education about advance directives. AB - As advances in medical technology increase the life span, social workers committed to quality of life for elderly people are faced with practice challenges. Preserving the autonomy and civil rights of elderly people in guiding their own health care even if they are unable to articulate preferences has become an important area of social work intervention. This article evaluates a social work program in a rural New England community designed to promote autonomous decision making by educating elderly people about advance directives. Results revealed a continuum of willingness among elderly people to address future decision making. Interventions to promote autonomy in health care decision making need to address the diversity of elderly people. The primacy of the family emerged throughout the study, suggesting that advance directives may be a family issue rather than solely an individual one. Further needs assessment research is needed to guide program development in this essential area of social work practice. PMID- 7557730 TI - [Ryanodine receptors in the central nervous system]. AB - Ryanodine receptor (RyR) is a calcium release channel protein on the intracellular Ca(2+)-store. While inositol 1,4,5-trisphosphate receptor (IP3R), another intracellular calcium release channel protein, is mainly found in non muscle cells, such as neurons and hepatocytes, and smooth muscles, RyR is the Ca(2+)-release channel protein in skeletal and cardiac muscles. At least three genetically distinct isoforms of RyR are identified: isoform proteins Ryr1, Ryr2, and Ryr3 expressed by ryr1, ryr2 and ryr3, respectively. In the central nervous system where IP3R is much more abundant than RyR, the main isoform of RyR is Ryr2, which is specific to the cardiac ventricular muscle. Recently, ryr3 was detected in specific regions of the brain. In this paper, the heterogeneous distribution and localization of RyR isoforms in the brain are summarized. The discussion extends into their putative functions, especially potential involvement in neuronal plasticity. PMID- 7557728 TI - Denial of death and taxes: America at middle age. PMID- 7557729 TI - Themes and variations among seven comprehensive perinatal drug and alcohol abuse treatment models. PMID- 7557731 TI - [Analysis of GTP-binding protein function with a photoaffinity GTP analog]. AB - The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems using purified components. However, the regulation of receptor-G protein signaling appears to be substantially more complex in the cell and several additional components participate in this event. To study the relationship among G proteins receptors, and effectors in complex systems, such as membranes of permeable cells, it is necessary to employ methods that selectively allow the examination of G protein activation. One such method is photoaffinity labeling using a hydrolysis resistant, photoaffinity GTP analog, P3 (4-azidoanilido)-P1-guanosine 5' triphosphate (AAGTP). Here we describe the synthesis and purification of [32P]AAGTP as well as a procedure suitable studying the G protein function in membrane preparations. Photoaffinity labeling in rat cerebral cortex membranes showed that at least four G proteins (GsH, GsL, Gi, and Go) were labeled by [32P]AAGTP. [32P]AAGTP labeling on Gs and Gi was altered in concert with the activation states of those G proteins. An agonist-specific increase in [32P]AAGTP labeling of the G protein alpha-subunit in a membrane preparation has also been demonstrated. Thus, the photoaffinity labeling method with [32P]AAGTP makes it possible to investigate the behavior of individual G proteins in complex systems such as membrane preparations. PMID- 7557735 TI - [Pharmacological study on the dry distillation tar of delipidated soybean (Glyteer) (5): Antimicrobial activity]. AB - Glyteer (GL) possessed a broad antimicrobial spectra against bacteria and fungi. The antimicrobial activity of GL was bactericidal action, but not bacteriostatic action. GL was more effective against fungi than bacteria. GL ointment also showed antimicrobial activity equal to that of GL. Furthermore, GL had an effect on methicillin-resistant Staphylococcus aureus (MRSA). Resistance to GL was not induced in broth cultures of Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, and Trichophyton mentagrophytes. These results suggest that GL applied externally exerts a potent effect as an anti microbial drug for dermopathy with various microbialpathogens. PMID- 7557734 TI - [Cisapride, a gastroprokinetic agent, binds to 5-HT4 receptors]. AB - The affinity of cisapride for 5-HT4 receptors was investigated in comparison with those of other 5-HT4 receptor agonists and antagonists, such as 5-HT, 5-MeOT, mosapride, zacopride, metoclopramide, BIMU8, GR113808, SDZ 205-557 and ICS 205 930. Cisapride and the other compounds dose-dependently inhibited specific 3H GR113808 binding to 5-HT4 receptors in guinea pig striatal membranes, and complete inhibition of specific 3H-GR113808 binding was achieved at the high concentrations of these compounds. Cisapride was 1.9-, 7.3-, 4.3-, 11- and 26 fold more potent than 5-HT, 5-MeOT, mosapride, zacopride and metoclopramide, respectively, in competing for 5-HT4 receptors. To determine the manner of interaction between cisapride and 5-HT4 receptors, Scatchard analysis of 3H GR113808 specific binding to striatal membranes was performed. Cisapride increased the Kd value of 3H-GR113808 in striatal membranes in a dose-dependent manner without any influence on the binding density (Bmax) of 3H-GR113808. These findings indicate that cisapride binds to 5-HT4 receptors competing with 3H GR113808 in guinea pig striatal membranes. PMID- 7557732 TI - [Effect of efonidipine hydrochloride, a calcium channel blocker, on the experimental cerebral ischemia/anoxia]. AB - The anti-ischemic and anti-anoxic effects of efonidipine, a dihydropyridine calcium antagonist, were studied in several models for cerebral ischemia and anoxia in mice and rats, and the effects were compared with those of nicardipine and flunarizine. Both efonidipine and flunarizine showed protective effects in the models of KCN-induced anoxia and complete ischemia induced by decapitation in mice 6 hr after the treatment, while nicardipine did not show such a long-lasting effect. Efonidipine (1 mg/kg, i.p.), but not nicardipine and flunarizine, prolonged the tolerance times in the asphyxic anoxia model. In mice, efonidipine (4 mg/kg, i.p.) significantly reduced the cumulative mortality rate after bilateral carotid artery ligation. The survival rates at 20 hr after bilateral carotid artery ligation were 33% in the group treated with efonidipine, significantly higher than that in the control group, 0%. On the other hand, the treatment with nicardipine or flunarizine did not increase the rates at 20 hr after the ligation. Moreover, efonidipine attenuated the disturbance of cerebral energy metabolism induced by decapitation in rats. These effects of efonidipine observed in this study were on the whole superior to those of the reference drugs, strongly suggesting the improving effect of efonidipine on cerebral ischemia and anoxia. PMID- 7557733 TI - [Study on the mechanisms of diarrhea induced by a new anticancer camptothecin derivative, irinotecan hydrochloride (CPT-11), in rats]. AB - We investigated the mechanisms of CPT-11-induced diarrhea. 1) CPT-11 (80 mg/kg, i.v.) induced watery diarrhea within 1 hr after dosing in saline-loaded (10 ml/kg, p.o.) rats. This was partially inhibited by subcutaneous injection of atropine (1 mg/kg) or ondansetron (1 mg/kg) and almost completely inhibited by a combination of atropine and ondansetron or by clonidine (0.3 mg/kg) or morphine (10 mg/kg) alone. 2) CPT-11 at the same dose reduced intestinal fluid absorption, which was blocked by the anti-diarrheal agents mentioned above. Intraluminal injection of CPT-11 (20 mg/2 ml) inhibited fluid absorption and induced fluid secretion. 3) CPT-11, 60 mg/kg, by single intravenous injection induced fewer enzymological and histological changes in the small intestine than 5-FU at 270 mg/kg, while 4 consecutive dosings of CPT-11 induced delayed diarrhea (days 5-7) associated with disruption of intestinal integrity. Co-administration with anti diarrheal agents, except for ondansetron, protected against watery diarrhea appearing within 1 hr after CPT-11 on days 3 and 4, but worsened delayed diarrhea. These results suggest that single injection of higher doses of CPT-11 causes watery diarrhea at an acute phase at least partly by reducing fluid absorption or increasing secretion, and that while conventional anti-diarrheal agents protect against watery diarrhea, their co-administration in repeated CPT 11 administration has no ameliorative effect on CPT-11-induced delayed diarrhea. PMID- 7557737 TI - [Pharmacological properties of chitosan-coated dialdehyde cellulose (chitosan DAC), a newly developed oral adsorbent (I). Effect of chitosan DAC in normal rats]. AB - The effects of chitosan-coated dialdehyde cellulose (chitosan DAC), a newly developed oral adsorbent of urea and ammonia, were examined in an in vitro adsorption study and in normal rats. Chitosan DAC showed high adsorption capacity for urea and ammonia in an in vitro study using the diluted supernatant of rat gastrointestinal fluid. In contrast, Kremezin, an oral charcoal adsorbent (AST 120), had little influence on these substances. In normal rats fed diets containing chitosan DAC (1, 2, 3, 4, 5, 7, and 10% content) for three weeks, increases in fecal wet weight, fecal dry weight and fecal water content were observed in a dose-dependent manner. In addition, chitosan DAC feeding increased fecal excretion of nitrogen and electrolytes (sodium, potassium and chloride ions) and decreased the apparent protein ratio in a dose-dependent manner. There were no obvious effects in serum parameters except that increased levels of protein and albumin and decreased levels of blood urea nitrogen, cholesterol and glucose were observed in rats fed a high concentration of chitosan DAC. In conclusion, these findings suggest the possibility that chitosan DAC treatment might be effective for improving chronic renal failure. PMID- 7557736 TI - [Effects of chronic losartan treatment on myocardial, vascular structure and reactivity with aging in spontaneously hypertensive rats (SHR): its effects compared with those of captopril]. AB - To compare the effects of an angiotensin II (Ang II)-receptor antagonist and an angiotensin-converting enzyme (ACE) inhibitor on myocardial, vascular structure and reactivity, SHR (5-week-old) were treated with losartan of captoril for 16 weeks. Losartan (10 mg/kg/day, p.o.) and captopril (30 mg/kg/day, p.o.) significantly prevented the development of hypertension with aging, and the effects by these drugs were almost similar. Losartan and captopril significantly reduced the ventricular weight and the thickening of the coronary artery. Maximal coronary flow (MCF) induced by adenosine in the isolated heart was significantly higher in both the losartan and captopril groups. However, MCF in the captopril group was significantly lower than that in the losartan group. The pressor response to exogenous norepinephrine in the mesenteric arterial bed was significantly lower in the losartan group, whereas that in the captopril group was not. These findings suggest that the local Ang II produced by enzymes other than ACE also may play a role in vascular hypertrophy and hyperresponsiveness in SHR. PMID- 7557739 TI - [Neurobiology of learning and memory and anti-dementia drug]. AB - Discoveries of long-term potentiation and immediate early gene in the central nervous system have enabled new developments in experiments on learning and memory. These experiments are conducted in many kinds of animals with different procedures, physiology, chemistry and pharmacology. However, there is still some confusion when these various procedures are discussed. Memory is defined as information storage of an animal's previous experiences. The memory induces changes in behavioral performance. This means that memory must be observed in whole animals, and one question that can occur is how does long-term potentiation, for example, correlate with memory. Furthermore, memory has been divided into two major classifications, declarative and non-declarative, from the comparison of amnesias observed in humans and animals. The declarative memory can be observed in human subjects, but not in animals. This article presents a neuronal circuit concerning memory formation and some results obtained from benzodiazepines, and it discusses some problems encountered executing when experiments on learning and memory. In addition, the discussion speculates over the possibility for an "anti-dementia drug". PMID- 7557740 TI - [Three simple maze tests employing mice housed in maze apparatuses]. AB - This report deals with three different maze methods using spontaneous learning behavior. Forty-eight mice housed in an apparatus with a multiple maze mastered the maze task on the 5th day after the start of housing. Then they were divided into four groups, and two of the groups were treated with AF64A (3.5 nmol, i.c.v.) or trimethyltin (TMT, 3 mg/kg, p.o.), and the other two groups were treated with the vehicle as the respective control. Seven days after the treatment, their memory retrieval was tested. Subsequently, the same mice were housed in the apparatus with a T-maze. After the finish of the experiment using the T-maze, they were housed in the apparatus with an eight arm radial maze. The control groups mastered the T-maze task with a 3-sec delay on the 4th day after the start of housing and the radial maze task on the 10th day after the start of housing. Both the treatments lowered the performance in all maze tasks. These results show that the mice housed in the apparatus with maze learned to negotiate the maze spontaneously, and the apparatuses are useful for estimating memory in mice with little effort. PMID- 7557738 TI - [Pharmacological properties of chitosan-coated dialdehyde cellulose (chitosan DAC), a newly developed oral adsorbent (II). Effect of chitosan DAC on rats with chronic renal failure induced by adriamycin]. AB - The effects of chitosan-coated dialdehyde cellulose (Chitosan DAC), a newly developed oral adsorbent of urea and ammonia, were examined in rats with progressive chronic renal failure (CRF) induced by adriamycin. CRF rats induced by repeated injections of adriamycin were fed a diet containing chitosan DAC (5% content) or Kremezin (5% content), an oral charcoal adsorbent (AST-120) under strict paired-feeding for four months. CRF rats that received both a normal diet and Kremezin showed progressive azotemia, hyperphosphatemia, hyperlipidemia, proteinuria, and anemia, and began to die from 9 weeks after feeding started. In contrast, chitosan DAC-treatment showed marked prolongation of the survival period and decreases in blood urea nitrogen, serum creatinine, and serum phosphate. In addition, chitosan DAC-treatment ameliorated anemia in CRF rats, although hyperlipidemia and proteinuria were not improved. Furthermore, fecal weight, fecal water content, fecal nitrogen and fecal sodium were markedly increased, and the apparent protein ratio was decreased in CRF rats fed a diet containing chitosan DAC for 9 weeks. In contrast, none of these effects were observed in CRF rats receiving Kremezin. These observations suggest the further possibility of using oral adsorbent therapy for CRF patients. PMID- 7557741 TI - [Experimental studies of physiological and pathological effects induced by systemic hypoxia and the hypoxia-reoxygenation model in rats]. AB - We attempted to make a basic model to investigate a series of factors that induce histological changes in systemic hypoxia-reoxygenation injuries. At first, we set the experimental conditions for hypoxia and the hypoxia-reoxygenation models as follows: respiration volume: 1.5 ml/stroke, respiratory frequency: 80 times/min, oxygen concentration: 14%. Next, Male SPF Wistar rats were anesthetized with pentobarbital sodium. For artificial ventilation, a cannula was inserted in the trachea and connected to the rodent ventilator through two flow meters to allow mixing of 100%N2 and 95%O2-5%CO2 gases at a desired ratio. The influence of hypoxia-reoxygenation was studied and evaluated histologically and biochemically. The rats were placed under the hypoxic condition for either 3 or 6 hr. Then, oxygen partial pressure was restored to 21% followed by reoxygenation for either 3 or 6 hr. Then the rats were sacrificed, and the pituitary, adrenals, heart, stomach and kidneys were removed. The results were as follows: 1) GPT activities were increased by a load of hypoxia, but no influence of reoxygenation was detected. 2) Under the condition of experimental hypoxia, the weights of the pituitary and adrenals increased significantly. 3) The histological findings indicated that 6-hr hypoxia followed by 3-hr reoxygenation induced hypoxia reoxygenation injuries mostly affecting the anterior pituitary and adrenal medulla. PMID- 7557743 TI - Report of the Rodent Bladder Carcinogenesis Working Group. PMID- 7557742 TI - [Analgesic and anti-inflammatory effect of (+/-)-N,N- dimethylcarbamoylmethyl 2 [7-(2-methyl-5H-[1] benzopyrano [2,3-b] pyridyl)] propionate (Y-23023), a new non steroidal anti-inflammatory drug]. AB - The analgesic and anti-inflammatory activities of Y-23023, a new nonsteroidal analgesic and anti-inflammatory compound, were investigated in acute, subacute and chronic pain models in rats. In the carrageenin paw edema test, Y-23023 (0.3 3 mg/kg, p.o.) dose-dependently inhibited hyperalgesia assessed by the Randall Selitto method and paw edema. Y-23023 (1 mg/kg, p.o.), when administered at 2 hr after carrageenin injection, significantly elevated the reduced pain threshold without affecting paw edema. Therefore, the antinociception activity induced by Y 23023 was not the result of its anti-inflammatory activity. In addition, Y-23023 induced antinociception was resistant to post-treatment with naloxone (2 mg/kg, s.c.), but was antagonized by intraplantar injection of prostaglandin E2 (1 microgram/paw). These results suggest that Y-23023 produces a peripheral analgesic effect mediated by inhibition of prostaglandin production. Y-23023 (0.3 10 mg/kg, p.o.) also had a potent inhibitory effect on the silver nitrate-induced arthritic pain. In suppressing acute and subacute pain, Y-23023 was more potent than diclofenac sodium, indomethacin and loxoprofen sodium. The analgesic and anti-inflammatory activities of Y-23023 (0.1-1 mg/kg/day, p.o.) on the adjuvant induced hyperalgesia and the paw swelling were nearly equipotent to diclofenac sodium and indomethacin, but were more potent than loxoprofen sodium. Therefore, Y-23023 would be regarded to show predominantly strong analgesic activity in acute and subacute pain when compared with reference drugs. The above results suggest that Y-23023 is a novel analgesic compound with an anti-inflammatory activity in the clinical field. PMID- 7557744 TI - Urinary bladder carcinogenesis: a working group approach to risk assessment. PMID- 7557745 TI - Anatomical relationships of urinary bladders compared: their potential role in the development of bladder tumours in humans and rats. PMID- 7557746 TI - Role of urinary physiology and chemistry in bladder carcinogenesis. AB - Urine is a complex mixture of numerous substances, only some of which are described above. Literally thousands of substances have been identified in normal urine, including a variety of ions, non-ionic substances and macromolecules. Their presence and concentrations are highly variable, dependent on fluid intake and on nutritional, physiological and biochemical influences. Marked diurnal variations exist. Methodologies involved in the collection and analysis of these components can greatly influence the interpretation of the results. The influence of these various parameters in the urine on bladder carcinogenicity can be either direct or indirect. A major difficulty in studying this aspect of urothelial carcinogenesis is that it is essentially impossible to alter only one variable in the urine at a time. Alteration of any one variable results in physiological alteration of several other of the constituents in the urine. In addition, the processes involved in urothelial carcinogenesis frequently involve a complex interaction of multiple variables, such as volume, osmolality, cationic concentration, anionic concentration, quantitative and qualitative differences in protein, and generation of precipitate, crystals or calculi. Thus, it is likely that the actual mechanisms involved in the carcinogenic process with many of these chemicals, particularly those that are non-genotoxic, will involve a complex interaction of several constituents of the urine. Although this poses a formidable obstacle to our understanding in experimental situations as well as in extrapolating to humans, the role of specific factors appears to be discernible and should offer insight into the risk assessment process (Cohen and Ellwein, 1991 a,b and 1992). PMID- 7557747 TI - Species comparison of the content and composition of urinary proteins. PMID- 7557748 TI - Epithelial tumours of the lower urinary tract in humans and rodents. AB - Bladder neoplasms that are morphologically similar to those occurring in the human bladder can be induced in rats and mice. Thus, these animal models can provide useful information in elucidating the histogenesis of the human bladder cancer. It is unknown whether human and rodent bladder cancers share similar molecular mechanism(s). There are species-specific differences in the type of neoplastic lesions. In rats, irrespective of the carcinogen used, the lesions tend to be polypoid exophytic masses and invasion is a late event, occurring only after a large dose of carcinogen and after an extended period of observation. On the other hand, mice tend to develop nodular invasive carcinomas readily preceded by the development of CIS. In both species, the neoplasms tend to show squamous differentiation, and metastatic spread is rare. Nevertheless, both species provide a useful model for the assessment of human risk of test chemicals because the principal neoplastic lesions that develop in response to test carcinogens are transitional cell neoplasms. Further study of cases of chemically, induced bladder cancer in rodents is essential to determine the applicability of the mode of action to humans. PMID- 7557749 TI - Genotoxicity in the rodent urinary bladder. AB - Elucidation of the mechanisms by which a chemical may induce urinary bladder tumours in rodents can be expected to provide insight into the relative risk from that agent. The methodologies for exploring whether tumour induction may be a response to direct genotoxic effect of the compound have been successfully applied to the bladders of both mice and rats. Thus, with experimental approaches that utilize adduct detection through the use of immunochemical and postlabelling techniques, unscheduled DNA synthesis and mutagenicity as studied with transgenic animals it is possible to obtain fundamental information on the genotoxic potential of carcinogens in the target bladder. Application of these experimental approaches to carcinogens for which the mechanisms of action are not known should permit assessment of the likelihood that genotoxic or non-genotoxic mechanisms are involved in the tumour induction process. Moreover, such studies may provide knowledge of the molecular pathways that are involved in the action of genotoxic agents, thus enabling judgements to be made as to whether humans are subject to tumour induction by the chemical. PMID- 7557750 TI - Effects of stones and other physical factors on the induction of rodent bladder cancer. PMID- 7557751 TI - Human bladder cancer: evidence for a potential irritation-induced mechanism. AB - Bladder cancer is one of the most common human cancers, constituting about 6% and 2% of all cancers among males and females, respectively. Over 90% of all bladder cancers are transitional cell carcinomas, with most of the remainder being squamous cell carcinomas. Smoking and occupational exposure to aromatic amines and other agents are most prominent among the risk factors identified. Inflammation of the bladder, largely by infection but also by stones or a combination of the two, may play some role in human bladder cancer development. The association between inflammation and cancer appears to be stronger for squamous cell than for transitional cell carcinoma. Stones and infection can be important factors in the development of bladder tumours in rodents, but the tumours are predominantly transitional cell rather than squamous cell carcinomas. PMID- 7557752 TI - Urinary bladder carcinogenesis: implications for risk assessment. Rodent Bladder Carcinogenesis Working Group. PMID- 7557753 TI - A simple method for extracting DNA from old skeletal material. AB - Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents. PMID- 7557754 TI - Age estimation of adults from dental radiographs. AB - Previous studies have shown that with advancing age the size of the dental pulp cavity is reduced as a result of secondary dentine deposit, so that measurements of this reduction can be used as an indicator of age. The aim of the present study was to find a method which could be used to estimate the chronological age of an adult from measurements of the size of the pulp on full mouth dental radiographs. The material consisted of periapical radiographs from 100 dental patients who had attended the clinics of the Dental Faculty in Oslo. The radiographs of six types of teeth from each jaw were measured: maxillary central and lateral incisors and second premolars, and mandibular lateral incisors, canines and first premolars. To compensate for differences in magnification and angulation on the radiographs, the following ratios were calculated: pulp/root length, pulp/tooth length, tooth/root length and pulp/root width at three different levels. Statistical analyses showed that Pearson's correlation coefficient between age and the different ratios for each type of tooth was significant, except for the ratio between tooth and root length, which was, therefore, excluded from further analysis. Principal component analyses were performed on all ratios, followed by regression analyses with age as dependent variable and the principal components as independent variables. The principal component analyses showed that only the two first of them had significant influence on age, and a good and easily calculated approximation to the first component was found to be the mean of all the ratios. A good approximation to the second principal component was found to be the difference between the mean of two width ratios and the mean of two length ratios, and these approximations of the first and second principal components were chosen as predictors in regression analyses with age as the dependent variable. The coefficient of determination (r2) for the estimation was strongest when the ratios of the six teeth were included (r2 = 0.76) and weakest when measurements from the mandibular canines alone were included (r2 = 0.56). Measurement on dental radiographs may be a non invasive technique for estimating the age of adults, both living and dead, in forensic work and in archaeological studies, but the method ought to be tested on an independent sample. PMID- 7557756 TI - [44th Congress of the German Association of Oral-Maxillo-Facial Surgery. Proceedings]. PMID- 7557755 TI - Concerning the article by Charles L. Winek, Jr. et al., entitled: 'The role of trauma in postmortem blood alcohol determination' (Forensic Sci. Int., 71 (1995) 1-8) PMID- 7557757 TI - [Bimaxillary osteotomy in osseous malocclusion. Dentoalveolar surgery. Opening address of the congress president]. PMID- 7557758 TI - [Corrective movement of a palatal impacted canine tooth: methodology and outcome of a combined oral surgery/orthodontic procedure]. AB - In 24 patients, 31 palatal impacted maxillary canines were brought in position using the same surgical and orthodontic concept. After temporal surgical exposure a little silver chain was attached at the crown of the canine, the mucoperiostal flap was repositioned, and the canine was moved into its position by innovative orthodontic treatment means. The combined method is described intensively. With respect to the applied technique the results indicate that according to the criteria of periodontal status, vitality, bone support, root resorption and occlusion no differences exist to free erupted canines despite a mean orthodontic correction of 36.7 degrees. PMID- 7557759 TI - [The "incompletely operated" surgical patient as an emergency in the oral surgery clinic]. AB - The evaluation of a large series of cases with uncompleted surgical treatment should lead to recommendations concerning the correct management of these patients. From 1983 to 1992, cases with uncompleted surgical treatment (n = 741) represented 1.6% of all patients treated. 715 cases were related to the removal of teeth: in 431 patients the completion of treatment was sufficient; in 284 cases complications had to be treated. In the majority of cases, documentation and patient management by the primary therapist were incomplete and needed improvement. With respect to this, recommendations are made on the basis of evaluation of the data. PMID- 7557760 TI - [Odontogenic diseases of the maxillary sinuses]. AB - Odontogenic maxillary sinusitis is aetiologically different from rhinogenic maxillary sinusitis. Accordingly, there are distinct and--for the oral maxillofacial surgeon--very specific therapeutic consequences. The general clinical diagnostical principles have scarcely changed in the last years. In diagnostic imaging, computed tomography and magnetic resonance tomography are of great importance, especially concerning pre-operative diagnostics. Endoscopical techniques have been enhanced with the introduction of microcamera-supported endoscopy, a technical improvement. Therapeutical principles are: complete treatment of odontogenic factors in the whole quadrant; conservative and operative therapeutical procedures are to be combined. Other than in the ENT field, where the maxillary sinus is approached by an endo-nasal technique- infundibulotomy-in oral and maxillofacial surgery the transalveolar approach through the fossa canina is used when treating odontogenic maxillary sinusitis. However, cautious methods assuring the preservation of tissue in all respects are treatment of choice. PMID- 7557761 TI - [Orthodontic pre- and postoperative treatment in bimaxillary osteotomy]. PMID- 7557762 TI - [Historical development of surgical wisdom tooth extraction]. AB - Surgical removal of the wisdom teeth is a routine procedure nowadays. Only at the end of the nineteenth century the use of local anesthesia together with the development of radiology led to the establishment of surgical dentistry. Especially the technique of removal of the lower third molar was totally changed and modified many times, depending on the position of the wisdom teeth. First hand instruments were used; later, mechanical devices for bone resection and tooth splitting were employed. Since the 1950s, highly dangerous infections have become rare, thanks to the use of antibiotics. Many publications concerning incision procedure, bone resection and tooth splitting marked the following years. PMID- 7557764 TI - [Lingual osteotomy--a little regarded technique for removal of the displaced mandibular wisdom tooth]. AB - Between 1980 and 1982, 769 wisdom teeth of the lower jaw were removed from 410 patients. The lingual bone splitting technique was used 813 times and vestibular osteotomy 586 times. Statistics concerning intra- and postoperative complications using both methods were gathered and compared. Hypoeasthesia resulting to permanent damage was observed once in the area of the N. lingualis and three times in the area of the N. alveolaris inferior. These results were similar to those obtained by English-speaking authors and indicate the suitability of the lingual bone splitting technique as an alternative to the vestibular approach. The indication for lingual bone splitting osteotomy in our sample was above all the horizontal and vertical impacted wisdom tooth. The decision to use lingual osteotomy should follow radiographic and clinical assessment of the relation between crown and lingual corticalis. This technically and anatomically convincing approach can be unconditionally recommended to the experienced surgeon as a time-saving alternative. PMID- 7557763 TI - [Long-term roentgenologic follow-up of asymptomatic impacted wisdom teeth in former orthodontic patients]. AB - Long-term follow-up of orthopantomographs of 251 adults orthodontically treated former patients showed 113 clinically asymptomatic impacted 3rd molars in 58 patients. Radiographic assessment revealed contact of impacted 3rd molars with the 2nd molars, resorption of upper and lower 2nd molars, reduced bone height on the distal side of the maxillary and mandibular 2nd molars, as well as pathologically widened pericoronal spaces of the upper and lower 3rd molars. The comparison of the sagittal 3rd molar position in 52 impacted teeth revealed a sagittal change in all 3rd molars from post-treatment to follow-up: maxillary and mandibular 3rd molars had rotated to a more upright position and showed an increase in mesio- or distoangulation or a change of axial inclination. The lack of predicting factors such as age, period of impaction, extent of space deficiency, developmental stage, level of eruption, and bone conditions leads the authors to recommend that former orthodontic patients be recalled at regular intervals for assessment of changes in the condition and position of erupting or impacted 3rd molars. PMID- 7557766 TI - [Complications of surgical wisdom tooth removal of the maxilla. A clinical and roentgenologic study of 1,013 patients with statistical evaluation]. AB - In a retrospective study the condition of the alveolar process in the region of the maxillary tuberosity after operative removal of impacted upper third molars was evaluated. Basically the clinical records of 1013 patients were explored. 371 patients were checked clinically, from 355 patients X-ray examinations were taken. There was at least one year between operation and follow-up examination. Pain was the reason for removal of the upper wisdom teeth in 32.9% of the patients. The most common intraoperative complications were oro-antral fistulae (11.3%). Most of the patients 70.9%) showed regular wound healing. Postoperative complications were seen more often and for a longer period in young female patients. In most cases (87.7%) there was a complete bony regeneration of the alveolar process in the area of the maxillary tuberosity. Contrary to our expectations cyst formations were very rare (0.59%). PMID- 7557767 TI - [Results of a comparative prospective randomized study of surgical removal of mandibular wisdom teeth with and without rubber drainage]. AB - This investigation compared the results of 2 types of wound closure - primary closure technique with and without Penrose drains (Naturallatex) - after mandibular third Molar removal. In both the test group (n = 27) and control group (n = 25), the molar were removed using a buccal mucoperiosteal flap. The test group received a drain partially submerged into the socket to secure more drainage of woundsecrete. Examination were performed 1 day, 3 days and 7 days after surgery, and swelling, trismus, pain and analgetic consommation were recorded. Analysis of Variance indicated that there was significant difference between the 2 methods. The drain method appears to minimize postoperative edema, trismus, pain and analgetic consommation, and thus contributes to enhanced patient comfort. PMID- 7557769 TI - [Indications and results of transdental fixation with the Wirz Syntacoben screw in frontal tooth reconstruction]. AB - In a one-armed prospective study including 60 patients (27 male, 33 female; age 17-75 years), 109 teeth were treated by means of a screw system made of Syntacoben and Niob according to Wirz. 103 teeth were transfixed successfully: 48% of them were provided with a partial removable denture; 34% were used as a bridge pier; 11% underwent conservative treatment; in 7% prosthetic treatment is planned. The use of the Wirz-System for primary transfixation combines in an ideal way the advantages of endodontic conservation and the ready-made post with the improved stability of the prosthetic pier. PMID- 7557768 TI - [Wound management after wisdom tooth osteotomy]. AB - Closure and dressing techniques in wound treatment after surgical removal of the mandibular third molars were compared. Large abscesses were found after third molar removal in 8 percent. An extraoral incision was made in 58 percent of these cases. On the other side, in a group of 1189 third molar removals treated by a gauze strip no abscess was observed. In a further investigation the subjective disturbance of patients treated with a dressed gauze were the slightest. Also, the dressing technique in the treatment after third molar removal could decrease the number of postoperative infections. PMID- 7557765 TI - [Severe complications after surgical removal of wisdom teeth]. AB - Between 1988 and 1992, 14,765 impacted third molars were removed in the Department for Maxillofacial Surgery of the University Hospital and Dental Clinic in Zurich. A report on significant complications is given and compared to previous studies. Special attention is given to major complications which needed hospitalisation and/or surgical intervention, like secondary infection of the head and neck region, mediastinal emphysema, mandibular fractures and dislocation of third molars, which could not be removed under local anaesthesia. Mediastinitis, excessive bleeding or permanent damage to cranial nerves were not encountered in these 5 years, but are reviewed. Serious postoperative complications can be life-threatening if not diagnosed correctly and managed accordingly. PMID- 7557770 TI - [Prospective comparative randomized study of tooth apex resection and orthograde root filling with Guttapercha and titanium pins]. AB - The prospective randomised study on the root apex resections with orthograde root filling comparing Guttapercha with Titan pins encompasses 500 patients with 633 operations on all front teeth and premolars. The patients were divided into three preoperative stages so that for this broadly defined diagnoses comparable collectives could be built. After an average postoperative interval of 21 months, 258 patients with 298 resected teeth could be evaluated. From these, 188 root apex resections had been treated with Guttapercha and 110 with Titan pins. 73.5% of the root apex resections were successful, whereby the group treated with Guttapercha was more successful by 7%. The comparatively high failure rate of 26.5% is attributable to the broadly defined diagnosis and the long follow-up period. PMID- 7557773 TI - [Results of follow-up after apicoectomy with 2 different root canal filling materials]. AB - Clinical and radiological findings in two groups of patients in whom apicectomies were done are presented. In one group root canal filling was by means of phosphate cementum; in the other, Diaket and guttapercha points were used. Evaluation was according to the methods applied already by Luders in 1990. Preoperative diagnoses most often had been chronic periapical periodontitis followed by traumatic injuries to frontal teeth and complications encountered during endodontic treatment. In those persons in whom phosphate cementum had been applied, treatment was successful in 210 teeth (77.2%) and a failure was noted in 62 teeth (22.8%). In the Diaket/guttapercha group the success rate was 167 teeth (80.7%), while treatment was without success in 40 teeth (19.3%). PMID- 7557771 TI - [Clinical and roentgenologic outcome evaluation after apicoectomy with zirconium oxide ceramic pins--a prospective study of 112 apicoectomy teeth]. AB - We post-examined 112 apicectomies in 93 patients after an average placement period of 4.8 months (3-15.3 months) both clinically and radiologically in a prospective study to evaluate the security of success using zirconoxide ceramic pins. Clinical functioning was examined by means of the clinical mobility test, the periotest method, the sulcusfluidflowrate (periotrontest), measurements of the depths of parodontal pockets at 6 points and the papillary bleeding index in comparison to the contralateral comparative tooth. To assess the osseous regeneration of the resection defect, we carried out planimetric evaluations of standardized X-ray tooth film exposures during surgery as well as at the post examination. The resected teeth revealed an average loosening of 10.2 in periotest. The sulcusfluidflowrate came to an average of 26.1 (control group: 22.7); the parodontal pockets were an average of 0.2 mm deeper than those of the control teeth. After 4.8 months, 63.54% of the resection defect showed complete osseous regeneration. Considering the clinical and the radiological criteria, the rate of success was 85%. Clinical use of zirconoxide ceramic pins is recommended. The high radiolucency of zirconoxide ceramics permits a safe assessment of the osseous regeneration and therefore a safe assessment of resection success. PMID- 7557774 TI - [Comparative studies with apicoectomy using various surgical techniques and filling materials]. AB - Follow-up examinations were made retrospectively on 799 apicoectomized teeth from a group of patients with a total of 3,524 apicectomies, the mean postoperative interval of observation being 4.2 years. An intraoperatively orthograde root canal filling was performed in 51.3%, an intraoperatively retrograde in 23.7% and a preoperative filling in 25.0% of the patients. Apart from 126 teeth that had already been extracted, a further 160 teeth had been clinically and/or radiologically conspicuous, so that a total of 286 failures was to be recorded. The dentally related success graphs computed according to Kaplan and Meier (1958) showed a significantly worse success probability for the preoperative filling compared to the two other techniques. With regard to the different materials employed in orthograde application, the silver filling had a higher and the guttapercha filling a less favourable success quota compared to a titanium filling. Moreover, in the retrograde method, amalgam resulted in a significantly higher success probability than glass ionomer cement. With an attendant cyst there was a significantly improved success quota over apical parodontitis or a renewed operation. With regard to the localization of apicectomies, maxillary front teeth attained the highest success rate. The present results point unequivocally to an unfavourable outcome after preoperative endodontics and underscore the good prognosis of an intraoperatively orthograde filling. Due to the fact that biocompatibility is in the forefront today, time-tested materials, such as silver and amalgam, are clearly being used less. PMID- 7557772 TI - [Biocem--a new material for retrograde tooth root filling]. AB - This paper describes the application of a new material, Biocem, for retrograde root-canal filling. The series included 222 teeth, including 234 roots treated during the period 1987-1992. This material consists of two components and once mixed, the autopolymerisation results in a three-dimensional structure. The biomechanical, histological and clinical longterm follow-up confirms the optimal qualities of this material, fulfilling the requirements in regard to biocompatibility, watertight sealing and application. The material proved to be superior in comparison to conventional materials and ceramics. The significant reduction of complications and the new apposition, as well as the evaluation of the results are most encouraging. PMID- 7557775 TI - [Advantages of microsurgical apicoectomy and retro-preparation with ultrasound compared with current apicoectomy]. AB - From September 1992 to March 1994 at the Department of Oral Surgery of the Dental School of the University of Vienna a total of 218 microsurgical apicoectomies and ultrasonic retro-preparations were carried out in 142 patients. Among other indications for operation was the failure of previous surgical-endodontic procedures in 62 cases. In 51 teeth (82%) of these patients the cause of failure could be evaluated with the help of the microscope. Twelve teeth (19%) extracted afterwards showed a longitudinal root fracture which had not been diagnosed before. In all other teeth retro-preparation and filling with zincoxide-eugenol cement was possible, even in cases of difficult anatomic approach. Due to the excellent experiences with microsurgical technique in apicoectomies, the use of the microscope in other fields of dental surgery remains only a matter of time. PMID- 7557776 TI - [Dento-alveolar surgery of the irradiated jaw: is infected osteoradionecrosis a preventable complication?]. AB - In spite of preventive procedures before, during and after radiotherapy, profound caries and periodontal breakdown of healthy teeth occur. In the course of dentoalveolar surgery in the region of irradiated bone, the risk of development of an infected osteoradionecrosis can be reduced by prophylactic antibodies, prevention of extensive detaching of the periosteum careful osteotomy or extraction, complete smoothing of sharp alveolar ridges and epiperiosteal plastic coverage. When extensive dentoalveolar surgery is necessary, the application of prophylactic pre- and postoperative hyperbaric oxygen may reduce the rate of complications even more. PMID- 7557777 TI - [The RTOG score (Radiation Therapy Oncology Group) as a guide to treatment of the irradiated field]. AB - The RTOG score, which describes radiation-caused side effects in a reproducible way, was introduced as a guide for the treatment of irradiated patients. By means of a prospective study (27 patients; acute effects) and a retrospective study (81 patients; long-term effects) the score was evaluated. Special importance was placed on the recording of dental and maxillofacial findings as well as on the therapeutic outcome. It was shown that frequency of dental lesions increased with the score of radiation effects while dental treatment decreased inverse to the score of radiation effects. Evaluation of radiation reactions of the lower jaw showed better results concerning preventive treatment and osteoradionecrosis than have been described in the literature. PMID- 7557778 TI - [Surgical tooth reconstruction in conjunction with irradiation of malignant tumors]. AB - An infected osteoradionecrosis (IORN) is one of the most problematic sequelae of radiation in the course of cancer treatment in the head and neck. To prevent this, prophylactic dental extractions have been demanded before radiation, as well as specific measures (epiperiostalplastic cover, perioperative antibiotic treatment) if a tooth has to be extracted after the radiation treatment has been carried out. From 1980 until 1993, 812 Patients who had to be irradiated underwent a prophylactic dental extraction programme. Only in 0.5% of these cases an infected osteoradionecrosis developed later on. 167 patients were treated after the radiation had been applied, using perioperative antibiotic treatment and meticulous soft tissue cover following epiperiostal preparation of the gingiva. In spite of 25% of localised dehiscences no immediate infection of the bone was observed. At the same time 53 infected osteoradionecrosis were treated, 43% of which were of dental origin. Most of these were seen in the lower molar area. The results show the high value of prophylactic dental extractions before radiation as well as the benefit of careful extractions with epiperiostal gingival cover, after the radiation treatment, especially in the area of the posterior lower alveolus. PMID- 7557780 TI - [Opening address by the president on the occasion of the 44th Annual Congress of the German Society of Oromaxillofacial Surgery in Kiel]. PMID- 7557781 TI - [Indications, technique and results of bimaxillary surgery]. PMID- 7557779 TI - [Dental treatment before radiotherapy of carcinoma of the mouth floor]. AB - Two collectives of patients who required radiation therapy were built. In order to determine the adequate occasion and extension of surgical teeth restoration in dependence on radiation therapy, both collectives were compared. The first group implicated 21 manifest ORN. The triggers of these ORN were evaluated. In the second collective the ORN occurrence after 225 generous surgical teeth restorations before radiation therapy were prospectively fixed and analysed. In comparison of both collectives the concept of generous removing of all potential dentogene inflammations before radiation therapy was successful in prevention of the septic ORN. PMID- 7557782 TI - [Bimaxillary segmental osteotomies]. PMID- 7557785 TI - [Predictive soft tissue factors in bimaxillary operations]. PMID- 7557783 TI - [Complications in bimaxillary surgical interventions]. PMID- 7557784 TI - [Anatomic drawings by Leonardo da Vinci]. PMID- 7557786 TI - [Bone stability after bimaxillary surgery]. PMID- 7557788 TI - [Surgery of craniofacial abnormalities]. PMID- 7557787 TI - [Surgical treatment of obstructive sleep apnea by maxillary and mandibular osteotomy]. PMID- 7557789 TI - [Outcome of wisdom tooth germ transplantation]. AB - In 68 patients, 79 tooth germs of the third molar were transplanted. Out of this group, 43 patients with 50 transplantations under-went a follow-up examination. All surgery was performed by one surgeon. Retrospectively, we tried to establish whether the success of postoperative healing depends on the donor and receiver regions. Crossing the jaw border clearly worsens the prognosis for transplantation. Altogether 9 transplantations turned out to be failures, 7 of which had been transplanted from the upper to the lower jaw. The results of this investigation showed incomplete root development in 34%, the necessity of endodontic treatment in 6% and enlarged periodontal pockets in 8%. Ankylosed teeth were found in 10%; we did not see any root resorptions. PMID- 7557791 TI - [Long-term follow-up of tooth transplantation from the functional and periodontal viewpoint]. AB - The outcome of 81 of 99 patients being treated from 1980 to 1993 by transplantation of autologous teeth was investigated. The overall transplant survival rate was 91% after 1 year, 81% after 5 years and 70% after 9 years. The early loss of transplanted teeth was due to healing disorders. The long-term losses were due to caries and replacement resorption. Caries in tooth transplants has to be treated very early, because deep caries or deep drilling leads to an infection of the nutrient canals of the osteodentine. If the pulp chamber of a transplant is once obliterated by osteodentine, endodontic treatment can no longer be performed in many cases. The periodontal condition of the transplants did not differ from that of normal teeth. In 23% there was a decreased tooth mobility, caused by ankylosis of the transplant. 51% of the transplants showed occlusal disturbances but none of the patients had signs of occlusal traumatization of the teeth or of the temporomandibular joint. In comparison to success rates taken from the literature it is pointed out that tooth transplantation has a better long-term prognosis than endodontic treatment. Therefore, in young patients requiring root canal treatment, tooth transplantations should be considered. PMID- 7557790 TI - [Follow-up of tooth vitality with laser Doppler flowmetry in transplanted teeth]. AB - 21 autotransplanted teeth were examined by using laser Doppler flowmetry (LDF) at 1, 2, 3, 6 and 12 postoperative weeks. 20 teeth showed statistically significantly higher perfusion units up to the 6th weeks as compared to the control group. At 12 postoperative weeks, the perfusion values of the autotransplanted teeth were at the same level as those of the control group. It was possible to show a non-revascularisation of one tooth with LDF. Currently, LDF can be considered as the only practicable, non-invasive method to evaluate the revascularization of autotransplanted teeth. PMID- 7557792 TI - [Premolar autotransplantation after front tooth loss in the maxilla]. AB - The general rules for autotransplantation of human premolars in cases of traumatic loss of upper incisors are given. The basic conditions for tooth transplantation concerning the donor side as well as the recipient side are presented. Technical details and the principles of indication are outlined. If a correct technique is combined with a precise indication, the long-term results after tooth transplantation are excellent, with a more than 90% rate of success. Thus the concept of premolar transplantation to the front of the upper jaw in cases of traumatic loss of the upper incisors in children will need more attention in the future. PMID- 7557793 TI - [Desmodont remains after tooth extraction in digital image processing]. AB - Breakage of the dentoalveolar gomphosis during tooth extraction results in varying amounts of desmodont remaining at the surface of the root. By dying the remnants of desmodont with toluidine blue, the surface of the root can be made accessible to computerized optical analysis. After clinically induced extraction of 102 human teeth, and after the loosening of 24 sheep teeth by mechanical vibration, the remaining area of desmodont was compared to that of the entire surface. Except in one case, no parameters were found to influence the amount of desmodont. However, tooth extraction in sheep previously treated with ultrasound or low-frequency vibrations demonstrate more desmodont-free areas. PMID- 7557794 TI - [Effect and sequelae of supernumerary tooth in the premaxilla]. AB - Although uncommon, anterior maxillary supernumerary teeth (mesiodentes) are the most frequent supernumerary teeth. A disturbed or delayed eruption of the adjacent permanent incisors may be due to a mesiodens and should lead to a further radiographic examination. In some cases the exact radiographic location of the mesiodens may prove difficult. The surgical removal of mesiodentes is especially difficult in extreme cranial displacement. The records of 90 patients with 113 mesiodentes are discussed with special reference to their degree of retention, topographical location, width of pericoronal space and surgical removal, and incidence of retention and malposition of the adjacent permanent incisors, respectively. PMID- 7557795 TI - [Clinical and roentgenologic evaluation of the outcome of therapeutic tooth movement for occlusal adjustment]. AB - The indication of therapeutic tooth luxation in partly impacted teeth as the only method of occlusal adjustment has up to the present been rare. It was therefore the aim of this retrospective, long-term study to include clinical and X-ray results following therapeutic molar luxation (OK 6, UK 7). After an average observation period of 5.3 years, all teeth (7) with incomplete root formation at the time of luxation were rated "successful" following molar luxation. On the other hand, only 2 out of 4 teeth were hand, only classified as "successful" following molar luxation with complete root formation. The results have shown that therapeutic tooth luxation for occlusal adjustment of partly retained molars has a high rate of success when the indication is correctly handled. Because the prognosis is determined according to the stage of root formation, the surgical approach of tooth luxation should be used solely in teeth whose root development has the 1/2 to 3/4 of the expected definitive root length and whose Foramen apicale is open at the time of operation. When an atraumatic operation technique is used, further root formation and a maintained sensibility of the pulp can be reckoned with. PMID- 7557796 TI - [The relationships between the EMG excitation pattern of the masseter muscle and the facial skeletal morphology]. AB - The purpose of this study was to investigate the correlations between malocclusions and the electromyographic activity of the orofacial muscles as this activity reveals itself through multi-channel, simultaneous recording of surface potentials from the masseter muscle. Thirty adults with class II or class III malocclusion formed the study's patient base. Surgical correction of the anomaly was planned for all patients. Ten patients served as the control group. In addition to the standard diagnosis, EMG were recorded during various stress situations such as maximal intercuspidations, chewing, swallowing, and mandible protrusion. We obtained a so-called "myomap" by means of the simultaneous multi channel recording of the surface potential of the right masseter. Comparison of the EMG revealed a higher activity from the masticatory muscles in the class II cases than in the class III cases. The analysis of the masseter excitation pattern brought to light interesting differences. A higher activity on the lower margin of mandible in the area of the superficial part of masseter was found in the class II cases, whereas contrary to this an increase in EMG activity was found above the deep part of masseter in class III cases. The differing distribution of excitation of the 2 parts of the muscle in class II and class III malocclusions confirms the correlation between muscle function and morphology. PMID- 7557797 TI - [Muscle relaxation by transcutaneous electric nerve stimulation (TENS) in bruxism. An electromyographic study]. AB - In recent years transcutaneous electrical neuromuscular stimulation (TENS) has become increasingly more common in the treatment of functional diseases of the masticatory muscles and currently the practitioner can choose among a variety of stimulators. In an electromyographic study of 17 adults suffering from nocturnal bruxism and of a control group consisting of 18 adults without any functional disturbances of the masticatory muscles, the effect of this kind of neuromuscular stimulation on the temporal and masseter muscle was examined. The myoelectric signals were registered before and after TENS treatment in 3 different positions of the mandible. Each person was treated 3 times with both the Myo-Monitor (Myo Tronics, Seattle) and the TNS SM2 MF stimulator (schwa-medico, Giessen). The different effects of continuous low frequency and intermittent high frequency muscular stimulation were studied simultaneously. Muscular activity was determined by computer aided integration of the electromyographically measured raw signals. A fourier analysis of the power spectrum yielded information on the frequency behavior of the studied muscles resulting from the TENS treatment. A statistical analysis of the results led to the following significant conclusions: 1. TENS treatment decreased the values of the registered integrated signals on all test persons, however, the treatment increased the median frequency and the mean power frequency (MPF). Since this effect is contrary to muscle fatigue, these electromyographic results can be interpreted as providing objective proof of a relaxation in the treated muscles. 2. The electromyographic changes after TENS treatment were similar when using either the Myo-Monitor or the TNS SM2 MF stimulator. In addition, the 2 different types of stimulation (high or low frequency) showed the same effects. 3. Compared to the persons in the control group, there was no significant increased muscular activity in patients with nocturnal bruxism. Following TENS treatment both groups showed the same alterations in the electromyograms. The results of the study provide further evidence that TENS treatment is an adequate supportive procedure in the treatment of nocturnal bruxism. PMID- 7557798 TI - [The significance of preventive CNS irradiation during antineoplastic therapy in childhood with regard to late odontogenic and mandibulofacial injuries]. AB - The aim of the present study was to determine the adverse effects of antineoplastic chemotherapy and radiotherapy on tooth development, i.e., on dentition in general and on craniofacial growth. The patients studied consisted of 26 children with solid tumors treated with chemotherapy alone and 34 patients with acute lymphoblastic leukemia (ALL) who were treated with intensive and follow-on radiation therapy (18 and 24 Gy) to the neurocranium as a constituent of central nervous system prophylactics in treatment protocols for ALL. The treatment protocol employed in these children is known as the BFM (Berlin, Frankfurt, Munster) protocol. At the time of diagnosis the children ranged in age from 3 to 52 months. All treated patients had microdontia, enamel dysplasia, thinning and tapering of apical roots portions, and marked shortening of the roots. These defects were more pronounced in children treated for ALL, but it was not possible to establish statistically relevant evidence on the influence of adjuvant radiotherapy in this group. This indicates that there is a catch up in craniofacial growth 9 years after therapy and that the most serious long-term adverse effects are on dentition. PMID- 7557799 TI - [The motivation and experiences of patients in connection with orthodontic-oral surgery combined therapy. A patient survey with consideration of the clinical and psychosocial factors]. AB - In a retrospective study 140 patients, 97 females and 43 males who had undergone combined orthodontic surgical therapy between 1984 and 1992, were interviewed using a questionnaire. The "closed form" questions were designed to provide insight into the patients' motivations for seeking treatment and into their perceptions of its course and outcome. Nine working hypotheses taken from the literature served as the study's starting point. The evaluation of the data gathered revealed that, contrary to other studies, the motivation for patients to seek treatment was not directly related to sex, i.e., was not sex specific, although this must be qualified. The unequal distribution of the sex of the patients who had sought treatment, and who were covered in the survey, points to differences between them in their decision making processes. When the results were evaluated, the data showed 92.2% patient satisfaction and even high satisfaction with treatment outcome. 86.4% of the patients stated that they would recommend the operation to others and 82.9% said that they would undergo the operation again. This high level of patient satisfaction was to a great degree directly attributable to, above all, the positive aesthetic result, the quality of the consultation and care, and to the absence of persisting postoperative complications. When the patients were divided into subgroups by means of using single criteria, significant varying degrees of satisfaction could be distinguished among segments of the subgroups. It was not possible, however, through the use of cluster analysis to separate out with precision the small group of patients who were not satisfied with the overall results of their treatment. PMID- 7557802 TI - [Ambulatory surgery in orthopedics--nothing new!]. PMID- 7557800 TI - [A functional orthodontic magnetic appliance (FOMA) after Vardimon. 1. A three dimensional analysis of the force system of the attractive magnets]. AB - The functional magnetic system (FMS) is a removable functional appliance which induces mandibular advance by means of mandibular and maxillary magnets in an attracting configuration. The maxillary and mandibular plates are each equipped with 2 cylindrically shaped cobalt-samarium magnets, 4 mm in diameter and 3 mm in height, which are welded into stainless steel housings. The force system of this magnetic configuration was analyzed using the orthodontic measurement and simulation system (OMSS). OMSS simulated the mandibular jaw movements by separating the installed magnets vertically, corresponding to a mouth opening of X = -10 mm, transversally (right excursion, +/left excursion, -) at Y = +/- 10 mm and sagittally (anterior displacement, +/posterior displacement, -) at Z = +/- 10 mm. The resulting 2D and 3D force/displacement diagrams elucidate the outstanding centripetal-spatial orientation characteristics of the functional magnetic appliance in reference to the full overlap brought about by the attraction of the mandibular magnet by the maxillary magnet. The maximum centripetal forces reached a value of approximately FY, max = 0.65 N for the vertical attracting force at full overlap of the mandibular and maxillary magnets (X = 0.55 mm, Y = Z = 0 mm), a value of FY, max = 0.65 N for the medial shearing force at a partial transversal overlap Z = 0, Y = +/- 2 mm and Y = +/- 6 mm), and for the sagittal shearing force a value of FZ, max = 1.2 N at a partial sagittal overlap of the magnets (Y = 0 mm, Z = +/- 2 mm).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557801 TI - [Ambulatory operations in orthopedics]. AB - Outpatient surgery in orthopedics, which ist becoming increasingly popular and is promoted by present-day health care legislation, is associated with a whole range of ifs and buts. Patient-related factors, including age, readiness and ability to cooperate, and home care, need just as much consideration as physician-related factors (experience of the surgeon, anesthesist und surgical team). Architectural and equipment-related facilities need to be investigated. Not every intervention that is theoretically possible on an outpatient basis can be recommended--for example if intensive aftercare, which is better performed on an inpatient basis, should be necessary. The usual postoperative risks (e.g. thrombosis) must be taken into account, and in such cases, proper care must be guaranteed. PMID- 7557804 TI - [Antineoplastic chemotherapy in metastatic Wilm's tumor]. AB - Wilms tumors may, rarely, also occur in adulthood. A 32-year-old male who, following the removal of a Wilms tumor, developed recurrent disease and metastases to the lungs and liver, was treated with a polychemotherapeutic regimen. Primarily, this was based on the protocol usually applied in children that comprises a combination of actinomycin D, vincristine, and adriamycin. Treatment initially achieved a six-month partial remission. Further treatment became necessary when a recurrence again occurred, and resulted in a "no change" situation that lasted for two months. The subsequent deterioration of the patient prompted monotherapy with vincristine, resulting in a transient clinical improvement. The patient finally died 25 months and two weeks after chemotherapy was first initiated. PMID- 7557805 TI - [Addresses of tumor centers in Germany]. PMID- 7557807 TI - [The value of intrauterine insemination in fertility treatment]. AB - Intrauterine insemination and the use of appropriately processed sperm is an mildly invasive, readily performed means of treating infertility. Particular indications are a poor cervical factor in the female, reduced fertility in the male, and immunological disorders or idiopathic sterility. Under optimal conditions (stimulated menses, case-oriented preparation of sperm) a treatment duration covering 4-5 cycles usually suffices; if success is not achieved within this time, other methods should be tried. PMID- 7557803 TI - [Garlic (Allium sativum)--a potent medicinal plant]. AB - A good deal of evidence suggests beneficial effects of the regular dietary intake of garlic on mild hypertension and hyperlipidemia. Garlic seems to have anti microbial and immunostimulating properties, enhance fibrinolytic activity, and exert favorable effects on platelet aggregation and adhesion. Standardised preparations guarantee exact dosing and minimize the problem of the strong odour of raw garlic. Thus, a traditional folk remedy has established its usefulness for many patients with less severe forms of cardiovascular disease as a medical drug with very few side effects. The available evidence gives rise to the hope that the list of indications may even be considerably extended in the future. PMID- 7557806 TI - [Radiotherapy of degenerative inflammatory joint diseases in the elderly. A worthwhile therapeutic measure]. PMID- 7557808 TI - [Treatment endpoints in reproductive medicine. Presentation from the andrologic viewpoint]. PMID- 7557809 TI - [Dermatomycoses. 5: Mycoses of the scalp]. PMID- 7557810 TI - [Prevention of thrombosis with fraxiparin 0.3 after arthroscopic interventions]. AB - METHODS: During a drug monitoring 101 men and women between 13 and 67 years underwent arthroscopic interventions on the knee on an outpatient basis and received the low-molecular-weight heparin, Fraxiparin 0.3, as prophylaxis of thromboembolism. In general the drug was administered the day before operation, two hours prior to operation and for five to six days postoperatively as a single daily s.c. dose. RESULTS: During the period of treatment, one patient developed a deep venous thrombosis of the leg; on completion of observation time four more cases of venous thrombosis of the leg occurred. In contrast, no pulmonary embolisms were observed. Fraxiparin 0.3 has proved to be very well tolerated, undesired side effects did not occur, and in no case was the preventive treatment interrupted. PMID- 7557811 TI - [Kaposi sarcoma apparently induced by a new oncogenic herpesvirus]. PMID- 7557812 TI - [Pathography and biography--background on the Schreber and Klug cases]. PMID- 7557813 TI - [Differential pharmacologic therapy of generalized anxiety disorders--results of a study with 30 individual case experiments]. AB - In pharmacotherapy of generalized anxiety disorders (GAD) different psychopharmacological agents (neuroleptics, diazepines, antidepressants) proved to be effective. However, there is a lack of predictors of therapeutic response. The present study was designed to address this question in 30 patients with treatment refractory GAD (12 male, 18 female). Amitriptyline 30 mg/d, flupentixol 1.5 mg/d, clotiazepam 15 mg/d, and placebo were administered double-blind and randomly. Each agent was given 4 times for one week. To avoid carry-over effects, all treatment weeks were interrupted by one week's wash-out periods. Thus each single-case experiment comprised 31 weeks. Primary efficacy criterion was Hamilton total score at the end of each treatment week. Statistical analysis (U test) showed that in 19 patients one agent was significantly (p < 0.05) superior to the other substances (clotiazepam n = 11, flupentixol n = 3, amitriptyline n = 5). Placebo was not superior in any of these 30 patients. There was no significant difference between the drugs in 11 patients. However, metaanalysis showed that in chronic GAD, by means of single-case experiments, differences in efficacy between different drugs can be found (p < 0.01). ANOVA showed no drug x time interaction. PMID- 7557816 TI - Pharmacological characteristics and species-related variations of beta 3 adrenergic receptors. AB - Beta-adrenergic receptors (beta-AR) belong to the large multigenic family of receptors coupled to GTP-binding proteins. Three subtypes have been identified: beta 1-, beta 2- and beta 3-AR. Much of the work delineating the precise pharmacological comparison of the three beta-ARs has come from investigations with stably transfected Chinese hamster ovary cells (CHO cells). This review discusses the structure and function of beta 3-AR in various species and presents new findings on a number of beta 3-AR ligands including carazolol, tertatolol and CL 316,243 which were found to be selective and potent beta 3-AR agonists and ZD 2079 and salmeterol which appear to display full but non-subtype selective agonistic activity. Species-related variations of the beta 3-AR pharmacology have been shown for propranolol and bupranolol. With the ongoing characterization of the beta 3-AR at the molecular and cellular level, and with the advent of computer-assisted molecular modelling to aid in the determination of the three dimensional structure of the receptor, it is thought that novel beta 3-AR compounds will become available with improved selectivity and potency. PMID- 7557817 TI - Regulation of 5-HT receptors by corticosteroids: where do we stand? AB - Twenty five years ago, experimental procedures such as adrenalectomy and corticosteroid administration (to intact rats) allowed the recognition of direct and indirect controls of central 5-HT synthesis rate by corticosteroids. These effects indicated that the activity of the hypothalamo-pituitary-adrenal (HPA) axis, whether under basal conditions or during stress, is endowed with a modulatory action upon serotonergic neurons. Nowadays, in situ hybridisation, in vitro autoradiography, and radioligand binding on the one hand, and electrophysiological, behavioural, and neuroendocrinological responses on the other hand, are tools that allow the analysis of direct corticosteroid effects upon 5-HT receptors. Among the dozen of 5-HT receptors identified so far, four receptors (namely the 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2C receptors)--and the 5-HT uptake system--have been the focus of studies aimed at detecting corticosteroid modulatory effects. The results that are reviewed herein indicate that hippocampal 5-HT1A receptors are under the tonic inhibitory control of corticosterone. This control is directly exerted at the level of the 5-HT1A receptor gene, essentially through mineralocorticoid receptors; as well, electrophysiological findings bring support for an additional modulation of hippocampal 5-HT1A receptor-mediated functions by indirect (ie 5-HT1A receptor gene-independent) genomic actions of corticosteroids. In keeping with the respective effects of stressful stimuli and psychotropic drugs upon the HPA axis and central serotonergic systems, it is likely that these corticosteroid-5-HT1A receptor interactions in the hippocampus have consequences in the pathophysiology of mood disorders. However, because the data regarding a corticosteroid control of other 5-HT receptors are either scarce and contradictory (eg 5-HT1B, 5-HT2A, 5 HT2C receptors and 5-HT uptake systems) or lacking, it is at the present time unknown whether corticosteroids exert other effects on 5-HT receptor-mediated functions, including those related to homeostasis. PMID- 7557815 TI - [Value of old and new anticonvulsants in treatment of psychiatric diseases]. AB - Besides the classical application in the treatment of different forms of epilepsies antiepileptic drugs are also used for the therapy of psychic diseases. In the present review the elementary mechanisms of action, pharmacology, indications as well as side effects of antiepileptic drugs are described. Successively, the wide used drugs carbamazepine, valproate, phenytoin and ethosuximide and of new substances which have been launched or will be launched in the near future in Germany such as lamotrigine, vigabatrin, gabapentin and oxcarbazepine, are initially presented with regard to epilepsies, and thereafter in the context of prophylaxis and treatment of psychic diseases. Several uncontrolled and controlled clinical studies indicating the efficacy of carbamazepine and valproate in the treatment of the acute manic syndrome and the prophylaxis of bipolar affective disorders have been presented. An effect on the acute depressive syndrome is, however, uncertain. Because of the strong side effects of lithium and the non-response to lithium in some patients, antiepileptic drugs are an important alternative in treating affective disorders. PMID- 7557814 TI - [Psychoses in multiple sclerosis--a reevaluation]. AB - With the aim of reaching a new classification of psychoses with multiple sclerosis we reviewed the twentieth century literature for observations with regard to the subject, as well as 688 medical records of our patients, looking for the occurrence of paranoid and hallucinatory psychoses in the course of multiple sclerosis. Special attention was paid to the occurrence of cycloid psychoses. With multiple sclerosis, psychoses on the whole--but cycloid psychoses in particular--occur more frequently than in the general population. Women are affected just as frequently as men. Cycloid psychoses occur earlier in the course of the multiple sclerosis than the other psychoses; here, hallucinations occur with a higher frequency. Similar as in the case of HIV-infection, multiple sclerosis can act as a trigger of a cycloid psychosis. The results of our study indicate that men and women experience this disease as similarly threatening. A shortcoming of critical faculties based on the organic disease is an additional factor that favours the outbreak of such a psychosis. PMID- 7557818 TI - Beta 3-adrenoceptors in the cardiovascular system. AB - Behind the classic beta 1 and beta 2-adrenoceptors, recent molecular and pharmacological studies have described a new receptor, called the beta 3 adrenoceptor, in various mammalian tissues (brown and white adipose tissue, digestive smooth muscle). Few authors have investigated the putative existence of the beta 3-adrenoceptor in the cardiovascular system. This paper reviews the available data. In vitro studies show that beta 3-adrenoceptor agonists (BRL 37344, CGP 12177) induce a relaxation of fragments of rat carotid artery which is not antagonized by propranolol. In dogs, these drugs elicit a decrease in blood pressure due to peripheral vasodilation and an increase in heart rate which is of baroreflex origin. The vasodilating effects are mainly observed in cutaneous and adipose tissue vessels and cannot be explained by any known transductional mechanism. Activation of this vascular beta 3-adrenoceptor requires higher doses of catecholamines than for beta 1- or beta 2-adrenoceptors. In humans, the cardiovascular effects of beta 3-adrenoceptor agonists are explained by the activation of beta 1- or beta 2 (and not beta 3-)-adrenoceptors. These studies suggest the presence of vascular (but not cardiac) beta 3-adrenoceptors in dogs. In other species, including man, the presence of such cardiac beta 3 adrenoceptors remains to be resolved. Their physiological relevance remains unknown. PMID- 7557819 TI - Proarrhythmic effects of a quinidine analog in dogs with chronic A-V block. AB - The proarrhythmic effects of 3-hydroxy-hydroquinidine (3-OH-HQ) and quinidine were compared in a canine model of QT-dependent ventricular arrhythmias. Eight hypokalemic ([K+] < or = 3.2 mmol/l) dogs with AV block (around 45 bpm) were given either drug in a randomized order at 2-day intervals. Each drug was given as two 1 hour doses, with a bolus (low dose: 5 mg/kg or high dose: 10 mg/kg) plus infusion (25 or 50 micrograms/kg/min) protocol. Propranolol infusion was combined with a third hour of the high dose infusion. Electrophysiologic measurements were performed at baseline and 30 minutes after the beginning of each dose and propranolol infusion, and proarrhythmic events were recorded 30 minutes before and during the experiment. Neither drugs altered the ventricular cycle length. Quinidine and 3-OH-HQ prolonged the QT interval similarly and significantly when paced at 60 bpm after the low dose (+39 +/- 18 and +28 +/- 22 msec, respectively) and after the high dose (+51 +/- 29 and +50 +/- 22 msec). Quinidine was more arrhythmogenic than 3-OH-HQ: 7/8 dogs (p < or = 0.05) developed ventricular arrhythmias (isolated, repetitive ventricular beats, or polymorphic ventricular tachycardias) during quinidine infusion (low dose: 4 dogs) compared to 3/8 dogs (NS) during 3-OH-HQ infusion (low dose: 1 dog). Addition of propranolol-induced bradycardia (around 30 bpm) caused torsades de pointes (wave burst arrhythmias) or polymorphic ventricular tachycardias after both drugs (in 3 dogs after quinidine and in 2 dogs after 3-OH-HQ). Thus 3-OH-HQ was slightly less arrhythmogenic than quinidine in this model of torsades de pointes, but the addition of an extra favouring factor (bradycardia) reduced that difference. PMID- 7557820 TI - Evidence for activation of both adrenergic and cholinergic nervous pathways by yohimbine, an alpha 2-adrenoceptor antagonist. AB - Adrenoceptors are involved in the control of the activity of the autonomic nervous system and especially the sympathetic nervous system. Activation of alpha 2-adrenoceptors decreases sympathetic tone whereas their blockade has an opposite effect. However, previous investigations have shown that yohimbine (a potent alpha 2-adrenoceptor antagonist) increases salivary secretion through activation of cholinergic pathways. The aim of the present experiment was to investigate the involvement of both the sympathetic and the parasympathetic system in several pharmacological effects of yohimbine. For this purpose, salivary secretion and various endocrino-metabolic parameters (noradrenaline and insulin secretions, lipomobilization) were evaluated in conscious fasting dogs before and after blockade of either the sympathetic (with the beta-adrenoceptor antagonist agent nadolol) or the parasympathetic (with the anticholinergic agent atropine) systems. Yohimbine alone (0.4 mg.kg-1, i.v.) increased within 5-15 minutes, plasma noradrenaline (600%), insulin levels (300%), free-fatty acids (79%) and salivary secretion (143%). Atropine (0.2 mg.kg-1, i.v.) suppressed yohimbine induced salivary secretion (90%) but did not significantly modify the yohimbine induced changes in noradrenaline (312%), insulin (277%) and free-fatty acids (102%) plasma levels. Administration of nadolol (1 mg.kg-1, i.v.) did not change the magnitude of the increase in both noradrenaline plasma levels (550%) and salivary secretion (300%) induced by yohimbine. However, nadolol totally blunted the increase in insulin (15%) and free-fatty acids (4%) plasma levels. These results show that yohimbine-induced increase in salivary secretion is a cholinergic effect whereas the increase in insulin and free fatty acids can be explained by an increase in sympathetic tone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557821 TI - Ontogeny of the cell outward dopamine transporter in canine renal tissues. AB - The present work has determined the activities of aromatic L-amino acid decarboxylase (AAAD) and evaluated the presence of an active transport system for dopamine in renal tissues of developing dogs (newborn puppies less than 24 hours after birth, animals at the age of 10 days and 2 months) and adult animals. AAAD activity (Vmax, in pmol/mg protein/h) as determined in kidney homogenates was found to be in the adult dog kidney (Vmax = 3216 +/- 268) higher (p < 0.05) than that occurring in the three other groups of animals; no significant difference on AAAD activity was observed between the 10 day-old (Vmax = 1139 +/- 185) and the 2 month-old dogs (Vmax = 783 +/- 23). AAAD activity in newborn puppies (Vmax = 259 +/- 40) was markedly lower than in the three other groups. A considerable amount of the total dopamine formed from added L-DOPA in kidney slices, depending on the age, was found to escape into the incubation medium. The application of the Michaelis-Menten equation to the net transport of newly-formed dopamine has allowed the identification of a saturable (carrier-mediated transfer) and a non saturable component (diffusion). The Vmax (nmol/g/15 min), Km (microM) values for the saturable component and diffusion constant (mumol-1) were as follows: adult (Vmax = 112 +/- 16; Km = 319 +/- 35; diffusion constant = 0.0009 +/- 0.0001), 2 month-old (Vmax = 19 +/- 5; Km = 48 +/- 14; diffusion constant = 0.0007 +/- 0.0002), 10 day-old (Vmax = 25 +/- 3; Km = 69 +/- 20; diffusion constant = 0.0033 +/- 0.0007) and newborn (Vmax = 6 +/- 1; Km = 16 +/- 6; diffusion constant = 0.0095 +/- 0.0010). In conclusion, renal AAAD develops with age, though some AAAD activity can already be detected at birth.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557822 TI - Sudden infant death syndrome and diphtheria-tetanus-pertussis-poliomyelitis vaccination status. AB - Because diphtheria, tetanus, pertussis and poliomyelitis vaccine is routinely given during the period of highest incidence of sudden infant death syndrome (SIDS), we carried out a retrospective case-control study to assess whether such vaccination increased the risk of SIDS. The vaccination status of 118 SIDS and 332 control children, matched for sex, date of birth and age of the victims at death, was compared: the victims of SIDS were not significantly more often vaccinated than control children, the odds ratio was estimated at 1.9 with a 95% confidence interval from 0.9 to 3.9. There was a statistical difference between vaccination status of SIDS cases and controls aged less than three months. Nine percent of SIDS cases under 3 months had been vaccinated whereas the matched controls had not. In our study DTCP vaccination was not a risk factor for SIDS; although more of the SIDS infants less than 3 months of age had been vaccinated. This result however, concerns only one subgroup of the population studied and needs to be confirmed with another study of only SIDS infants less than 3 months of age, because DTCP vaccination was not a risk factor for SIDS when considering the total sample of the study. PMID- 7557823 TI - Interest of modafinil, a new psychostimulant, during a sixty-hour sleep deprivation experiment. AB - Modafinil, a new psychostimulant, was evaluated in eight healthy volunteers subjected to 60 hours of sleep deprivation. During continued wakefulness, vigilance was evaluated by self-assessment questionnaires, analogue visual scales, multiple sleep latency tests (MSLT), sleep logs, and continuous ambulatory electroencephalographic recordings (EEG). Modafinil (200 mg) or a placebo was given every 8 hours for three days; the sessions were separated by a 15 day wash out period. Results indicated a satisfactory level of vigilance, both subjective and objective, after the administration of modafinil, characterised by the quasi total absence of microsleep episodes which gradually occurred under placebo conditions. The confirmed wakening potency of modafinil makes this substance suitable for therapeutic use in patients with sleep disorders such as Gelineau's syndrome and hypersomnia. PMID- 7557824 TI - Colloid bismuth versus famotidine in the treatment and prevention of duodenal ulcer relapse: results of a double-blind, double dummy randomized study. AB - Fifty-three consecutive patients with active duodenal ulcer (DU) were randomly included in a double-blind, double-dummy study to test the healing and relapsing rate of two treatment regimens: famotidine 40 mg nocte for 4-8 weeks, followed by 20 mg for 12 months after healing of the ulcer and colloidal bismuth (CBS) (240 mg bid) for 4-8 weeks, followed by placebo maintenance treatment. The results of the short term period confirmed the efficacy of CBS in healing DU (24/25 in CBS group and 19/23 in famotidine group). However, the relapse rate in the CBS treated group was higher (77.8% at 12 months) than in the famotidine group (35.7%) (p = 0.041). Only 7 patients (41.2%) were cleared from Helicobacter pylori (HP) after CBS treatment. In conclusion, the high relapse rate observed in CBS treated patients may be related to the high percentage of patients with HP infection in the tested group and support the hypothesis that lack of efficacy of CBS in preventing DU recurrence is related to its poor eradication of HP. PMID- 7557826 TI - [3D echocardiography. Mathematical principles and technical realization]. AB - The ultimate goal of any imaging technique for the investigation of the anatomy of the beating heart is a 3D-display of the cardiac morphology throughout a complete heart cycle. The reason for this interest is quite clear: 3D-imaging has the potential for a better understanding of the individual morphology under normal and pathological conditions and especially, if complex therapeutic decisions have to been made. In the clinical practice, the echocardiographer attempts to obtain a spatial information by a mental reassembling of the 2D echocardiographic images, that are obtained from different imaging planes. This procedure, however, is very subjective and, thus, highly susceptible for errors. Therefore, the 3D-echocardiography has been developed to replace this mental process by an "objective" and reproducible computerized reconstruction. Prerequisite for such a 3D-surface reconstruction is a cubic, isotropic digital data set with cubic data volumes, so called "Voxels" (Figure 1). The term "isotropic" means, that the resolution is identical in all directions, and that the data density within the cube is homogeneous. Those cubes are the mathematical basis for any 3D-reconstruction. At the first step on the way to 3D-images, the data cubes have to be filled with 2D echo information. So far, three principal modalities of image acquisition are available for the clinical routine: parallel scanning from the esophagus (Figure 2), rotational scanning (transesophageal- Figure 3a--or transthoracic--Figure 3b). In all cases, the imaging planes are incremented by an external stepper motor using a dedicated computer logic for gated image acquisition. At the present time, despite geometrical shortcomings, the TEE omniplane probe with rotational scanning is the most widely used system. It can be applied for standard investigations as well as for "3D"-data acquisition after only minor modifications. The process of 3D-reconstruction is a sequence of repeated steps of image processing. The first step is the elimination of a problem, that is common to all image reconstruction techniques from tomographic information: the imaging planes are recorded at different time points, and mostly under varying conditions. Although several gating techniques are implemented into the image acquisition, some variability is unavoidable, simply because neither the heart nor the surroundings can be frozen during image acquisition.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7557825 TI - New developments in the conduct and management of multi-center trials: an international review of clinical trial units. AB - There is an urgent need for the performance of more, better designed, and better managed randomized clinical trials. After visits to 43 leading organizations and units involved in clinical trials in Europe and North America during 1993, the way of conducting randomized clinical trials was analyzed. By using a structured questionnaire, information on recent improvements in the way randomized clinical trials are performed was identified. These developments encompass human aspects (better information, collaboration, and communication) as well as non-human aspects (press button phone randomization and data management systems). By employing such developments, randomized clinical trials can run much more efficiently. This facilitates faster and better answers to the questions addressed by randomized clinical trials, thereby also making them more ethical. PMID- 7557828 TI - [Trans-thoracic 3D echocardiography reconstruction for left ventricular volume determination]. AB - 2D-echocardiography is a safe and inexpensive method for accurate left ventricular volume quantification. However, accuracy of measurements is limited by the requirement of geometric assumptions and volume computation algorithms. Reproducibility is diminished by erroneous image plane positioning, which is due to the necessity of standardized reference images. The degree of variation can influence clinical decision making. 3D-echocardiography has overcome those problems. Volume quantitation is performed without the need of geometric assumptions, computation algorithms or reference images. The shape of a ventricle is defined by acquired data, not by calculated data. Since the recent development of sophisticated hardware- and software techniques the method can be utilized under clinical circumstances. For data acquisition tomographic planes are acquired either in a random or in a sequential order. For random acquisition the transducer is positioned at various acoustic windows at the thoracic wall and spatial orientation of the corresponding plane is localized by a spark gap device. By this way various, non-parallel cross-sectional images of the left ventricle are acquired. The spatial position of the planes is displayed by lines in a parasternal longitudinal view, each representing a "line of intersection". When tomographic planes are acquired in a sequential manner, mostly rotational scanning is utilized. Here, multiple apical views of the left ventricle are acquired during ECG- and respiration gated rotation of the transducer over a span of 180 degrees.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557829 TI - [Diagnosis of coronary heart disease using echocardiography 3D reconstructions. Diagnosis of global and regional left ventricular function]. AB - The technique of tomographic 3D-echocardiographic reconstructions allows, beside the morphological description of cardiac structures, a left ventricular quantification concerning the diagnosis of the coronary heart disease. The advantage of the tomographic 3D-reconstruction and tomographic quantification is that the left ventricular volume is determined directly by manual planimetry during the whole heart cycle and will not be approximated by geographical algorithms as used for 2D-techniques and alternative 3D-techniques. We worked with the Echoscan reconstruction unit (TomTec) equipped with a pentium-processor and a 64 RAM memory for the acquisition and digitalization of a 3D echocardiographic image data set (Figures 2a and 2b). Automatic image acquisition is performed using a unit existing of a common ultrasound transducer and a special rotational motor device. All the examinations described in this publication are performed by a transthoracic approach. Examining asymmetric model hearts with aneurysm an experimental study sustentiated the high accuracy of determining asymmetric ventricle volumes (mean of difference [3D--true dimensions] = 0.91 ml; SD of differences = +/- 6.17 ml) and aneurysm volume (mean of difference = 0.43 ml; SD of difference = +/- 2.14 ml). Also high reproducibility of 2 repeated 3D-measurements was documented (ventricular volumetry: mean of difference = -0.09 ml; SD of difference = 3.15 ml. Aneurysm volumetry: mean of difference = 0.24 ml; SD of difference = 0.17 ml). The principle of quantification by tomographic planimetry is usable the same way in vivo (Figures 3 and 4).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557827 TI - [Three-dimensional reconstruction of heart valves]. AB - The reconstruction of three-dimensional data sets from two-dimensional echocardiographic images offers several fundamental advantages: 1. more complete data than present in the few standard 2D-view; 2. off-line generation of any desired plane, cut, or perspective after the data set has been acquired; 3. access to quantitative parameters like surface areas (e.g., of valve leaflets or portions of leaflets), volumes, and others, without geometric assumptions. The mitral valve has been the focus of several studies using various techniques of reconstruction of transthoracic or transesophageal images. These studies have shown the mitral annulus to be a non-planar, "saddle-shaped" structure, with an average distance of highest to lowest points of 14 mm in normals. This recognition of mitral annular non-planarity has led to a more stringent echocardiographic definition of mitral valve prolapse. Further studies have shown systolic shrinkage of mitral annular area by about 30% and systolic apico-basal translation of the annulus by approximately 1 cm in normals. In patients with dilated cardiomyopathy, the annulus is flattened, and both cyclic change in annular area and apico-basal translation are significantly reduced. 3D-studies of the left ventricular outflow tract in hypertrophic obstructive cardiomyopathy allow measurement of outflow tract and leaflet surface areas and dynamic spatial visualization of systolic anterior motion of the anterior mitral leaflet. Automated techniques to reconstruct the full grey value data set from a high number of parallel or rotational transesophageal planes allow impressive visualization of normal and diseased mitral and aortic valves or valve prostheses, with special emphasis on generating "surgical" views and perspectives, which cannot be obtained by conventional tomographic imaging.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557830 TI - [Reconstruction methods for 3D imaging and quantification of intracoronary ultrasound cross-sectional images using biplanar fluoroscopy. Initial experiences in vitro and in vivo]. AB - In this investigation, we studied the feasibility of 3D-reconstruction from 2D cross-sectional intracoronary ultrasound images. A computer-aided, automated 3D reconstruction was used to generate cylindrical and sagittal format of vessels in vitro (n = 9) and in vivo (n = 48). Ultrasound 2D-images were acquired with a 20 MHz mechanical intracoronary ultrasound catheter (Boston Scientific). A slow pullback (about 1 mm/s) of the catheter was performed during continuous recording of the ultrasound images. The recorded 2D-images were then fed to an image processing computer. Correction and scaling of the axial vessel dimensions was performed by the assessment of the catheter position in the simultaneously recorded biplane fluoroscopy. Digitized data were then processed to 3D-images with use of voxel space modeling. The 3D-views from any plane can be reconstructed. The in-vitro study demonstrated that the reconstructed images were able to correctly portray the pathological changes of the vessel wall in all specimen as proved by pathologic examination. In the in-vivo study, 3D reconstruction provided not only a spatial visualization of the coronary arterial disorders (such as coronary aneurysm, coronary dissection, spontaneous plaque rupture etc.) but also provides the potential to quantify the mass of lesions. By combining sagittal and cylindrical views, 3D-reconstruction enables longitudinal and orthogonal imaging of the both the vessel lumen and vessel wall, therefore, it has the advantage of cross-sectional viewing as obtained from intracoronary ultrasound and the longitudinal viewing as derived from coronary angiography. These preliminary results of the study indicate that 3D-reconstruction of coronary segments is a promising technique for studying coronary artery disease. Analysis can be based not only on single or multiple cross-sectional images but also on vessel segments facilitating serial studies as for instance studies assessing the progression and regression of atherosclerosis. PMID- 7557831 TI - Three-dimensional reconstruction of coronary arteries with intravascular ultrasound. AB - Three-dimensional (3D-) reconstruction of intravascular ultrasound (IVUS) images is a recently introduced technical method which has rapidly grown in science and clinical practice. In the catheterization laboratory it is particularly important to obtain the 3D-reconstruction on-line, since the dimensions measured and the plaque attributes displayed may guide the therapeutic decision. Off-line reconstruction, however, provides very accurate and reproducible area and volume measurements of lumen and plaque and is thus exceptionally qualified for studying progression/regression of atherosclerosis or restenosis after catheter-based interventions. Complementary 3D-reconstruction methods, revealing specific advantages and limitations, meet the requirements by slightly different technical approaches, but each 3D-reconstruction of two-dimensional IVUS images requires some basic procedural steps. The IVUS images can be acquired during continuous or ECG-gated withdrawals of the IVUS imaging catheter. The latter permits even to visualize the cyclic pulsation of the reconstructed arteries. As an alternative approach a sensing device recognizes the insertion depth of the IVUS catheter and permits reliable measurements even during manual handling of the IVUS catheter. A discrimination between the blood-pool and structures of the vascular wall, performed in the digitized images, can be achieved by the application of different techniques. This processing step which is called segmentation and the image acquisition are particularly crucial with regards to the final quality of the 3D-reconstruction. Currently there are still limitations of 3D-IVUS, but a new approach combining data obtained from 3D-IVUS and biplane angiography offers a promising potential to solve most of the remaining problems in the future. Thus, three-dimensional IVUS offers a great clinical and scientific potential since it provides spatial visualization of vascular pathology, longitudinal and volumetric measurement of luminal and plaque dimensions, and facilitated guidance of catheter-based interventions. Assuming a technical development similar to the progress of the previous years. 3D-IVUS has a realistic chance to gain significant importance and to become a routine technique in the future. PMID- 7557832 TI - [ACE inhibitor after myocardial infarct: are there differences?]. PMID- 7557833 TI - [Therapy of heart failure with calcium channel blockers?]. PMID- 7557834 TI - [Amlodipine in heart failure: initial results of the PRAISE study]. PMID- 7557835 TI - Stimulation of insulin release by non-sulfonylurea hypoglycemic agents: the meglitinide family. AB - Several new non-sulfonylurea hypoglycemic agents such as A-4166, KAD-1229 and repaglinide are structurally related to meglitinide, previously known as the non sulfonylurea moiety of glibenclamide, or its analog S 3075. There is no parallelism between the ionophoretic and insulinotropic efficiency of these compounds. They are all able, however, to decrease K+ conductance in islet cells as documented by a decrease in 86Rb outlfow from prelabelled pancreatic islets. In terms of concentrations of equal insulinotropic capacity, a difference of at least two orders of magnitude is observed between the weakest (meglitinide) and most potent (S 3075) compound. All these agents exert little effect upon insulin release in nutrient-deprived islets, but markedly augment glucose-stimulated insulin release. The hexose can be replaced, however, by a non-glucidic nutrient such as the methyl ester of succinic acid to support the insulinotropic action of the meglitinide analogs. The question is raised as to the potential advantage of these non-sulfonylurea insulinotropic agents over hypoglycemic sulfonylureas. PMID- 7557836 TI - The functional significance of the second extracellular loop of thyrotropin receptor in thyrotropin- and thyroid stimulating antibody-dependent signal transduction. AB - In order to determine the functional significance of the extracellular loop of human thyrotropin receptor (hTSHR), two peptides composed of eight amino acids were inserted into hTSHR by ligating synthetic oligonucleotides into +1811 NCol site of hTSHR cDNA. Mutant hTSHR cDNAs which encode a hydrophobic peptide (ATVLVVPM) and a hydrophilic peptide (GTTRTVAM) between +572 Met and +573 Asp were transfected into Chinese hamster ovary (CHO) cells to develop F-cell lines and R-cell lines, respectively. Of the resulting cloned cell lines, F-29 and R-9 were shown to express mutant hTSHs at the protein level by Western blotting and at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR). We show that neither thyrotropin (TSH) nor IgGs from patients with Graves' disease stimulated cAMP production by F-29 and R-9 cells. 125I-TSH binding study revealed that F-29 and R-9 cells do not bind TSH. Our data demonstrate that the mutations impaired TSH-binding and incapacitated the cells from responding to TSH. The evidence suggests that the second extracellular loop of hTSHR has an important role in TSH and thyroid stimulating antibody (TSAb)-dependent signal transduction. PMID- 7557837 TI - Genistein inhibits Ca2+ influx by extracellular ATP in PC12 pheochromocytoma cells. AB - We previously showed that extracellular ATP-induced Ca2+ influx is inhibited by self-activated protein kinase C in rat PC12 pheochromocytoma cells. In the present study, we examined whether tyrosine kinase is involved in the ATP-induced Ca2+ influx in PC12 cells. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on 45Ca2+ influx, significantly suppressed the ATP-induced 45Ca2+ influx in a dose-dependent manner in the range between 1 and 30 micrograms/ml. Tyrophostin, another inhibitor of tyrosine kinases chemically distinct from genistein, also inhibited the 45Ca2+ influx. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, markedly enhanced the ATP-induced 45Ca2+ influx. These results suggest that tyrosine kinase regulates Ca2+ influx induced by extracellular ATP in PC12 pheochromocytoma cells. PMID- 7557839 TI - Pubertal benzpyrene exposition decreases durably the sexual activity of the adult male and female rats. AB - Single benzpyrene treatment of 5 week old male and female rats significantly decreased their sexual activity at 3 months of age. Among the hormone preparations used nandrolone was comparatively practically ineffective, while estradiol decreased the lordosis quotient of females. In males benzpyrene produced a total failure of ejaculation. These results draw attention to the wide time-scale of receptorine and behavioral effects of the aromatic hydrocarbon, benzpyrene. These effects are detectable following fetal, neonatal or pubertal treatments, especially in females. In males, the negative effect was manifested after neonatal and pubertal treatments only. The experiments suggest that in some cases hormonal imprinting does not develop solely in the perinatal period but it might develop later. PMID- 7557840 TI - The relationships among serum uric acid, plasma insulin, and serum lipoprotein levels in subjects with spinal cord injury. AB - Relationships were investigated among serum uric acid (UA), the insulin response to a standard oral glucose load (75 g), and serum lipoprotein levels in 197 individuals with chronic spinal cord injury (SCI). All subjects had normal liver and renal function. None had a prior history of diabetes mellitus or gout. The mean age of subjects was 50 +/- 1 years, duration of injury (DOI), 18 +/- 1 years, and body mass index (BMI), 25 +/- 0.4 kg/m2. No significant differences were found between those with paraplegia or quadriplegia for any of the parameters measured. The mean serum UA values were not significantly different among the subgroups of subjects with normal glucose tolerance, impaired glucose tolerance, or diabetes mellitus (5.6 +/- 0.2 mg/dl, 5.6 +/- 0.2 and 5.7 +/- 0.3, respectively). Approximately one-half of the subjects had an abnormality in oral glucose tolerance. The levels of serum UA (p < 0.001) and serum triglycerides (TG) (p < 0.01) in the subgroup with hyperinsulinemia were significantly higher than in the subgroup with normal insulin levels. By linear regression analyses, the serum UA concentration was positively correlated with peak plasma insulin level (r = 0.31, p < 0.001), and BMI (r = 0.20, p < 0.01), but not with age, DOI, or peak glucose. The data suggest that in subjects with chronic SCI, as in the healthy able-bodied population, hyperuricemia is associated with hyperinsulinemia, obesity and abnormal lipoprotein metabolism. PMID- 7557838 TI - Effect of testosterone administration, pre- and postnatally, on the immune system of rats. AB - This study was undertaken to investigate and compare in vitro and in vivo immune parameters between female and male rats. We analysed the T-cell proliferative responses to syngeneic and allogeneic cellular antigens (syngeneic and allogeneic mixed lymphocyte reaction), as well as the IgG levels in the sera of our study groups. It has also been studied the influence of gonadectomy and the effect of testosterone administration pre- and postnatally on the above parameters. Our findings showed that hormonal manipulations, can alter the differences observed in immune response between female and male rats. In addition, it is demonstrated that pre- and postnatal sexual steroid manipulations could provoke long lasting immunological effects. PMID- 7557841 TI - Serum testosterone and growth hormone/insulin-like growth factor-I in adults with spinal cord injury. AB - Aging is associated with relative growth hormone and/or testosterone (T) hormone deficiency, and those with SCI may have a premature deficiency of these two hormones. The effects of SCI, duration of injury (DOI), and advancing age with that of human growth hormone (hGH) and insulin-like growth factor I (IGF-I), as well as potential associations between them, were studied. Data were obtained from 20 male subjects with SCI and 16 gender- and age-matched controls. Serum total and free T were lower in subjects with SCI compared with controls (mean +/- SEM, 3.12 +/- 0.29 versus 4.68 +/- 0.28 ng/ml, p < 0.001 and 1.89 +/- 0.18 versus 2.46 +/- 0.22 ng/ml, p < 0.05, respectively). Nine of the 20 subjects with SCI, but none of the controls, had abnormally low serum total T. Arginine-stimulated values for hGH were lower in the group with SCI compared with controls (198 +/- 18 versus 267 +/- 27 ng/ml, p < 0.05). Serum luteinizing hormone and follicular stimulating hormone, as well as body mass index, were not significantly different between the groups. Serum total and free T were correlated with advancing age in controls (r = 0.62, p < 0.01 and r = 0.51, < 0.05, respectively) but not in SCI (r = 0.19, p > 0.43 and r = 0.39, p = 0.09). However, serum total and free T declined with increasing DOI in SCI (r = 0.56, p < 0.01 and r = 0.44, p = 0.05, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557842 TI - A rapid radioimmunoassay for the measurement of antibodies to glutamic acid decarboxylase in human serum. PMID- 7557843 TI - Effects of parathyroid hormone related peptide (1-34) and 1,25-dihydroxyvitamin D on bone alkaline phosphatase activity in fetal rat calvaria cultures. PMID- 7557845 TI - Urinary excretion rate and clearance of IgG4 and alpha 2-macroglobulin in type 2 diabetic patients. PMID- 7557844 TI - Glucose-induced insulin release by pancreatic islets is enhanced in rats with naturally occurring obese non-insulin-dependent diabetes. PMID- 7557846 TI - Expression of transforming growth factor alpha in the liver before and after interferon alfa therapy for chronic hepatitis B. AB - The effect of interferon alfa (IFN-alpha) therapy on the expression of transforming growth factor alpha (TGF-alpha) in the liver during chronic hepatitis B was investigated. Serial liver biopsy specimens were evaluated from 35 patients who had participated in a randomized, controlled trial of recombinant human IFN-alpha for the treatment of chronic hepatitis B. Percutaneous liver biopsy specimens obtained before and 1 year after entry in the trial were sectioned and stained with a monoclonal antibody to TGF-alpha in an avidin-biotin peroxidase-complex system. The expression of TGF-alpha in each section was evaluated blindly (with respect to treatment group and order of biopsies) and was numerically scored. There was no significant difference in TGF-alpha expression before or after therapy between 13 patients receiving daily IFN-alpha, 13 receiving alternate-day IFN-alpha, and 9 receiving no therapy. Sustained clearance of HBV-DNA and DNA polymerase activity occurred in 8 of 26 treated patients ("responders"); the 18 other patients were "nonresponders." Expression of TGF-alpha before IFN-alpha therapy was significantly higher in responders than in nonresponders; after IFN-alpha therapy, TGF-alpha expression decreased significantly among responders compared with nonresponders and untreated controls. Thus, the level of expression of TGF-alpha in the liver, which was correlated with the severity of inflammation in the liver in this study, appeared to be predictive of the response to IFN-alpha therapy in chronic hepatitis B, with a higher level of expression indicating a greater likelihood that the patient would respond. PMID- 7557847 TI - Characteristic difference of hepatocellular carcinoma between hepatitis B- and C- viral infection in Japan. AB - Characteristics of 205 consecutive patients with hepatocellular carcinoma (HCC) admitted during 1990 to 1993 have been analyzed from the standpoint of hepatitis viral infection in Japan. Among 205 HCC patients, 71% of the patients showed positivity for hepatic C virus (HCV) antibody alone, 13% showed positivity both for HCV and HBV (HCV/HBV) antibody, 11% demonstrated HBsAg alone, and negativity of both HCV and HBV antibody in 4% only. Positivity to both HCV antibody and HBsAg was demonstrated in 1% only. Mean detection age of HCVAb-positive HCC as well as both HCV/HBV antibody-positive HCC was 62 +/- 7 years, in contrast to 52 +/- 13 years in HCC with HBsAg (P < .05). Male-to-female ratio among HCVAb positive HCC was 3.3:1, in contrast to 5.5:1 among the HCV/HBVAb-positive HCC and 7:1 among HBsAg-positive HCC, but there was no significant difference in the gender distribution between these groups. More than 60% of HCVAb-positive HCC and HCV/HBVAb-positive HCC were classified into the stage of Child B and C, whereas 65% of HBsAg-positive HCC was at the stage of Child A. The severity of liver disease was confirmed by liver histology, indicating that more than 70% of the HCVAb-positive HCC and the HCV/HBVAb-positive HCC showed cirrhosis, in contrast to 50% among the HBsAg-positive HCC. Three-year survival rate of HCV Ab-positive HCC and HBV/HCVAb-positive HCC was 68% and 56%, respectively, in contrast to 47% in HBsAg-positive HCC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557849 TI - Induction of T-helper cell response to hepatitis B core antigen in chronic hepatitis B: a major factor in activation of the host immune response to the hepatitis B virus. AB - The T helper (Th) cell response to hepatitis B core antigen (HBcAg) was analyzed in 76 chronic hepatitis B virus (HBV) carriers with varying degrees of hepatic inflammation and HBV replication. Fifty-five patients had active viral replication, 28 with minimal histological changes and normal alanine transaminase (ALT) and 27 with active hepatic inflammation and elevated ALT. The remaining 21 chronic hepatitis B surface antigen (HBsAg) carriers had undetectable HBV replication, minimal histological activity, and normal ALT. In addition, 34 chronic HBV carriers were studied prospectively during treatment with alpha interferon. The HBcAg-specific Th cell response was evaluated by a proliferative assay using 3H-thymidine uptake and gamma-interferon production by peripheral blood mononuclear cells. The proliferative response and gamma-interferon production of patients with active hepatic inflammation were significantly higher than in patients with minimal histological changes and in controls. In the longitudinal analysis during alpha-interferon treatment, 22 of 34 patients sustained an ALT flare accompanied by a parallel, significant Th cell response, which preceded or coincided with the ALT flare. The elevation in the Th cell response and the ALT flare were followed by a significant rise in the serum immunoglobulin (Ig) M anti-HBc index. Ten of twenty-two patients with an enhanced Th cell response and an ALT flare seroconverted after alpha-interferon treatment. The Th cell activity in the 10 responders rapidly subsided after hepatitis B e antigen (HBeAg) to anti-HBe seroconversion, whereas in the 12 nonresponders it remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557848 TI - The influence of a maternal chronic hepatitis B virus infection on the repertoire of transcribed T-cell receptor beta chain variable region genes in human cord blood. AB - We used an anchor polymerase chain reaction method to compare the repertoires of transcribed T-cell receptor beta chain variable region (V beta) genes in cord blood T cells from neonates of hepatitis B surface antigen (HBsAg) positive (n = 40) and HBsAg negative (n = 40) women. Fifteen of the HBsAg positive women were hepatitis B e antigen (HBeAg) positive, and 25 were HBeAg negative. The percentage of V beta 7.4 transcripts was lower in cord blood T cells from neonates of HBsAg-positive relative to HBsAg-negative women (9.7% +/- 0.5% vs. 12.7% +/- 0.6%, P = .002). The percent of V beta 5.1 transcripts was higher in cord blood T cells from neonates of HBeAg-positive relative to HBeAg-negative women (9.3% +/- 0.7% vs. 7.0% +/- 0.3%, P < .001). There were no correlations between neonatal maturity at birth and V beta repertoire. In summary, a maternal chronic hepatitis B virus (HBV) infection is associated with changes in the repertoire of transcribed T-cell receptor genes in neonatal cord blood T cells. It is possible that the T-cell response to the HBV is associated with a limited repertoire of V beta genes. The mechanism of vertical chronic HBV infection in human neonates may involve changes in the T-cell response to the virus that are induced in utero. PMID- 7557850 TI - Pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype are the main and independent prognostic factors of sustained response to interferon alfa therapy in chronic hepatitis C. AB - The aim of the study was to determine the respective influence of pretreatment serum hepatitis C virus (HCV) RNA levels and HCV genotype on the response to interferon (IFN) alfa in patients with chronic hepatitis C. We retrospectively studied 141 patients with chronic hepatitis C included in two consecutive controlled trials of IFN alfa. A sustained response was observed in 28, a response followed by relapse in 43, and no response in 70 patients. Pretreatment serum HCV RNA quantitation with the branched DNA (bDNA) assay and HCV genotyping with reverse hybridization assay (LiPA) were performed in all patients. Seventy four percent of the patients had detectable serum HCV RNA (43%, 77% and 84%) in the three groups of patients with sustained response, relapse, and no response, respectively (P = .005). Mean serum HCV RNA level were 1.4 +/- 6 x 10(6), 4.8 +/- 6 x 10(6), and 3.9 +/- 5 x 10(6) genomes/mL in patients with sustained response, response and relapse, and no response, respectively (P < .01). Genotype 1b was found in 7%, 47%, and 46% of the patients in the three response groups, respectively. By univariate analysis, age, source, and duration of HCV infection, serum HCV RNA levels, and HCV genotypes were significantly different in the three response groups. By multivariate analysis, the only independent factors associated with sustained response were low serum HCV RNA levels and HCV genotype other than 1b. Pretreatment serum HCV RNA levels and HCV genotype are the main and independent factors associated with sustained response to IFN therapy. PMID- 7557851 TI - Interferon gamma production by peripheral blood lymphocytes to hepatitis C virus core protein in chronic hepatitis C infection. AB - Evidence suggests that cellular immunity to hepatitis C virus (HCV) core protein may be important in the pathogenesis of viral infection. Therefore, interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) derived from patients with chronic HCV infection (genotype 1b) was examined. The cellular immune response was evaluated with a recombinant HCV core fusion protein derived from a patient with genotype 1b. To identify the immunodominant epitopes, IFN gamma production in responders was also assessed with a panel of nine synthetic peptides that covered the entire core region. It was found that mononuclear cells from 24 (52%) of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate (14%, P < .05). More important, individuals who had received IFN-alpha treatment and went into clinical and virological remission had a higher response rate (75%, P < .05) compared with those with ongoing hepatitis whose treatment failed (31%). Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 141 to 160 was recognized in 9 patients. Interestingly, 7 of 8 patients bearing HLA DR 4 and w53 haplotypes recognized the peptide sequence 141 to 160. Thus, IFN gamma production of the mononuclear cell response appeared to be HLA DR restricted, and the responding cells were identified as CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557852 TI - Antipyrine clearance and response to interferon treatment in patients with chronic active hepatitis C. AB - To determine whether hepatic metabolic function affects the response to interferon treatment, we measured antipyrine clearance (APC) in 85 patients with chronic active hepatitis C and compared the results with treatment outcome. Among 55 patients who responded to interferon by normalization of alanine transaminase (ALT), median APC before treatment was 0.47 (range, 0.12 to 0.98; normal range, 0.34 to 1.02 mL/min/kg body wt), a value that was significantly greater than in 30 nonresponders (0.23; 0.08 to 0.67 mL/min/kg body wt, P < .001). APC was closely associated with response to interferon. The response rate among cases with values > 0.25 mL/min/kg body weight was 79%, the same as in cases without cirrhosis. Cases without cirrhosis and with APC of > 0.25 mL/min/kg body weight had an 85% chance of responding to interferon; this was unlikely a simple reflection of histological activity, because the correlation with Scheuer score was poor in this subgroup (r = -.31, P < .05). A second, independent group of 43 patients was used to test the predictive value of APC (using 0.25 mL/min/kg body wt as a cut-off) for response to interferon treatment. In this group, APC correctly predicted positive response to interferon in 75% of cases. APC was also used to measure the effects of treatment on hepatic metabolic function. Regardless of outcome, there was no change in APC at the end of a 6-month course of interferon treatment. Six months later, however, improvement in APC (14%; P < .05) was evident among responders but not in those who had failed to respond to interferon.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557853 TI - Inhibition of woodchuck hepatitis virus replication by adenine arabinoside monophosphate coupled to lactosaminated poly-L-lysine and administered by intramuscular route. AB - We prepared a hepatotropic conjugate, suitable for intramuscular (IM) injection, of lactosaminated poly-L-lysine with adenine arabinoside monophosphate (ara-AMP), a drug active against hepatitis B virus (HBV). We studied its organ distribution in mice and its antiviral activity in woodchucks that are carriers of woodchuck hepatitis virus (WHV). In mice, after IM administration of a conjugate tritiated in the drug moiety (5.2 micrograms/g equal to 2 micrograms/g of ara-AMP) radioactivity in liver was three times greater than in kidney, spleen, and intestine. On the contrary, after IM injection of unconjugated, tritiated, ara AMP (5 micrograms/g) the amounts of radioactivity in liver, spleen, and kidney were similar. Unconjugated ara-AMP and the conjugate were administered IM to woodchucks for 13 days. Unconjugated ara-AMP decreased viremia at the daily dose of 5 mg/kg but was ineffective at 2.5 mg/kg. The conjugate at the daily doses of 4.2 and 7 mg/kg (equal to 1.5 and 2.5 mg/kg of ara-AMP, respectively) markedly lowered the viremia, which decreased to undetectable levels in the animals treated with the higher dose. Assuming that in HBV-infected patients the same doses will be active, then the amount of conjugate (soluble at 200 mg/mL) required by a 70-kg patient will be contained in a volume of 1.5 to 2.5 mL, compatible with the IM route. Compared with a similar ara-AMP complex with lactosaminated human albumin, currently being studied in clinical trials for the treatment of chronic type B hepatitis, which must be infused intravenously, the present conjugate might provide more patient compliance because of IM administration. PMID- 7557854 TI - Anti-hepatitis B virus activity of a mixture of two monoclonal antibodies in an "inhibition in solution" assay. AB - Two human monoclonal antibodies with anti-hepatitis B activity were investigated separately and as a mixture by means of an "inhibition in solution" assay. With this assay the capacity of anti-HBs antibodies to inhibit the binding of hepatitis B surface antigen (HBsAg) with solid-phase anti-HBs (Ausria II, Abbott Laboratories) was studied. Both HBsAg of different subtypes and purified Dane particles were used. One of the monoclonal antibodies (9H9) was directed against a conformational epitope (anti-"a" like) but induced incomplete inhibition (80% to 90%) of all HBsAg subtypes; the other (4-7B), active against a linear epitope, caused full inhibition of all HBsAg subtypes except one (HBsAg/adw4). In vitro, a mixture of these two monoclonal antibodies was active against HBsAg from liver transplant recipients, including those with suspected variant viruses. PMID- 7557857 TI - Role of hepatitis C virus in dual and triple hepatitis virus infection. AB - Hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) share same transmission routes, thus dual or triple infection may occur and even persist in the same patients. A significant amount of literature has accumulated since the advent of HCV assays. It is pertinent to review and evaluate the clinical and virological significance of HCV in multiple hepatotropic viral infection. The reported series on seroprevalence of HCV indicate that HCV is found in more than 10% of HBV- or HDV-infected patients worldwide. Of the patients with dual or triple infection involving HCV, those having coreplication of viruses tend to have severe and progressive liver disease that is resistant to interferon therapy, in contrast with patients having a single virus infection. Paradoxically, dual or triple hepatitis virus infections are associated with viral interference. In particular, HCV exerts a suppressive effect on HBV and HDV and may enhance seroclearance of HBV antigens or even usurp the role of preexisting virus as the agent for continuing hepatitis. Although HBV and HDV may also suppress HCV, it appears to be less effective. These findings clearly suggest the necessity of monitoring patients with HBV or HDV infections. In view of complex dynamism of viral interaction in multiple hepatotropic virus infection, the importance of HCV assay in the clinical studies can not be overemphasized. The basic mechanisms that regulate the viral interactions, in particular the impact of HCV in dual or triple virus infections, remain to be investigated. PMID- 7557856 TI - Subcellular distribution of large and small hepatitis delta antigen in hepatocytes of hepatitis delta virus superinfected woodchucks. AB - Hepatitis delta virus (HDV) encodes only a single protein, the hepatitis delta antigen (HDAg), which is expressed as two molecular forms (large and small) with different functions in viral replication. Compared with small antigen, large antigen has a 19 residue carboxyl terminal extension. Antibodies that recognize a large antigen-specific epitope within this carboxyl extension, or an epitope shared by both large and small antigens (total antigen), were used in immunohistochemical studies of liver sections from superinfected woodchuck carriers of woodchuck hepatitis virus. There were no differences in the subcellular distributions of large and total antigens, with both generally present only in nuclei of hepatocytes. Rare cells demonstrated cytoplasmic staining. Complete or partial granular nucleoplasmic staining with stained nucleoli was the most common pattern observed. Within 31 days of infection, 0.1% to 19% (mean = 7.4%) of all hepatocytes contained antigen. The proportion of these nuclei containing large antigen ranged from 0 to 100% (mean, 39%), and increased during the first month of infection. The number of antigen-positive nuclei and the proportion staining for large antigen were reduced with progression to chronicity, correlating with reductions in the level of viremia. Thus, the large hepatitis delta antigen shares a common subcellular distribution with small antigen and is found in an increasing proportion of the nuclei of infected cells during the course of acute infection. PMID- 7557855 TI - An open trial of interferon alfa recombinant for hepatitis C after liver transplantation: antiviral effects and risk of rejection. AB - The aim of this open trial was to assess the efficacy and the safety of interferon (IFN) alfa therapy in liver transplant recipients with chronic active hepatitis caused by hepatitis C virus. In July 1991, among 447 liver recipients regularly observed at our institution, 46 had developed HCV-related chronic active hepatitis defined by piece meal necrosis. Fourteen of these 46 patients received IFN alfa 3 mIU three times weekly for a planned duration of 6 months and were compared to the 32 untreated patients. Genotyping and quantification of viremia were performed using type-specific amplification and branched DNA assay. Histological follow-up was available in all patients and routinely before and after IFN therapy. Treated and untreated patients did not differ regarding gender, age, length of follow-up, maximum histological score, and genotypes (41 of 46 were of type 1b). Induction of chronic rejection was observed in 5 of 14 treated patients leading to retransplantation in 3. In contrast, chronic rejection occurred in 1 of 32 untreated patients (P < .005) during the posttransplantation follow-up. Among the 9 treated patients without rejection, a decrease of transaminases or of HCV RNA levels of more than 50% were observed in 8 and 4, respectively; 2 patients had a complete response, and 1 did not relapse after discontinuation of IFN. Histological improvement occurred in 2 of the treated patients and in none of the untreated patients. IFN therapy in liver transplant recipients has poor antiviral effect and can induce chronic rejection. Its use in this setting should be cautious. PMID- 7557859 TI - The liver-spleen scan as a quantitative liver function test: correlation with liver severity at peritoneoscopy. AB - Sulfur colloid distribution on liver-spleen scan is determined by the perfused Kupffer cell mass. The perfused Kupffer cell mass is proportional to the perfused hepatocyte mass, but is less affected by acute changes in hepatocyte function. Thus, sulfur colloid distribution parameters (precisely measured by quantitative liver-spleen scan [QLSS]) may be an excellent test of the perfused hepatic mass. Although no gold standard exists for confirmation, a close correlation should exist between liver disease severity assessed at peritoneoscopy and sulfur colloid distribution. Peritoneoscopy severity (scored as total peritoneoscopy score [PS]; range, 0-5) was assessed in 76 patients who also had QLSS. Multivariate equation were generated to estimate liver disease severity from the QLSS. These were then applied prospectively in 20 consecutive patients to validate these equations. In 76 patients, 62 were evaluated because of chronic liver disease (CLD) and included those with micronodular (20) and macronodular (20) cirrhosis with various degrees of severity (Child's A, 16; B, 29; C, 17). Multivariate analysis yielded a number of combinations of QLSS parameters that correlated with peritoneoscopic severity. These equations were used to estimate liver disease severity. Estimates of liver disease severity (estimated PS [EPS]) correlated well with the PS in these 76 patients (r = .9064; r2 = .8216; P < .0001). Adding histological fibrosis to the QLSS parameters yields an equation for estimating PS that was even more effective (r = .9462; r2 = .8953; P < .001). However, validation of multivariate equations requires confirmation of their value in a second population.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557858 TI - Utility of hepatitis C virus RNA determinations in hepatic tissue as an end point for interferon treatment of chronic hepatitis C. AB - A total of 41 patients with chronic hepatitis C virus (HCV) defined as abnormal liver injury test results for 6 months or more and HCV RNA positivity in plasma were studied to determine if the liver might not be the only focus of HCV infection in individuals treated with interferon alfa (IFN-alpha). All patients were examined for the presence of confounding liver disease and tested negatively for such findings. All tested positively for HCV RNA and had an abnormal hepatic histology. All were treated with IFN for 6 months at a dosage of 5 million units daily. After 6 months of therapy, 29 (71%) had normal alanine transaminase (ALT) values, and 25 (61%) tested negatively for HCV RNA. After 6 months of follow-up, without IFN therapy, 17 (41%) still had normal ALT values, and 16 (39%) still tested negatively for HCV RNA in serum. Patients who continued to test negatively for HCV RNA in serum after 6 months of follow-up also tested negatively for HCV RNA in the liver at the end of IFN therapy. Only 2 subjects who tested negatively for HCV RNA in the liver at the end of treatment relapsed after discontinuing IFN therapy. In contrast, patients who tested positively for HCV RNA in the liver after 6 months of therapy relapsed and tested positively for HCV RNA in serum during the 6 months of follow-up.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557860 TI - Donor selection limits use of living-related liver transplantation. AB - The purpose of this investigation was to assess the applicability of living related liver transplantation in an established regional transplant program by determining the frequency of acceptable living donors from an unselected population of pediatric transplant candidates and identify specific factors limiting application of this technique. During the period May 1992 to May 1994, all children accepted as transplant candidates at the University of California San Francisco were evaluated for potential living-related liver transplantation. Indications for transplantation and patient demographics represented the spectrum anticipated at a regional center. Donor evaluation was performed using a three phase evaluation process we have previously reported. Retrospective analysis identified 75 potential donors for 38 pediatric candidates (age range, 17 days to 14.5 years; mean, 5.1 years). Twenty-three percent of potential donors declined evaluation. Of the 75 potential donors, only 10 (13%) were found to be acceptable for donation. The leading causes for donor declination were significant medical history (23%), ABO blood group incompatibility (23%), and psychosocial history (20%). Of the 38 recipient candidates, 9 (23%) were offered living-donor transplantation. Five patients have received living-donor transplantation, and 4 patients await the procedure when medical indications exist. Seventy-seven percent of recipient candidates received or are awaiting cadaveric transplantation. These results suggest that current donor criteria markedly limit the application of living-related liver transplantation. PMID- 7557862 TI - Reduced serum levels of immunoreactive erythropoietin in patients with cirrhosis and chronic anemia. AB - Chronic anemia is frequently observed in patients affected by cirrhosis. To investigate the possible role of erythropoietin (Epo) in the pathogenesis of anemia in cirrhosis, we measured the immunoreactive Epo levels and the respective hemoglobin (Hb) concentrations in 48 anemic and nonanemic cirrhotic patients and in a control group of healthy subjects and patients with iron-deficiency anemia. Epo concentrations were determined in serum using a sensitive enzyme immunoassay. The regression curve between Epo values and Hb concentrations showed a significant inverse exponential trend both in cirrhotic patients (r = -.55; P < .0001) and controls (r = -.92; P < .0001). In a semilogarithmic plot, the line slope obtained in cirrhotic patients was significantly lower (P < .005) than that of controls, suggesting a blunt Epo response to anemia in cirrhosis. Moreover, covariance analysis showed that the Epo levels for a given degree of anemia were further reduced in the patients with a more severe disease, suggesting a close relation between cirrhosis and the mechanisms involved in the derangement of the Epo feedback system. Finally, the Epo concentrations measured in the cirrhotic patients without anemia did not significantly differ from Epo values obtained in healthy subjects. An impaired Epo response may play a role in maintaining low Hb concentrations in cirrhotic patients with anemia. However, the evidence of a residual Epo response to anemia in cirrhosis and the presence of normal basal Epo levels in nonanemic cirrhotic patients do not support an inadequate Epo secretion as one of the primary causes of anemia in cirrhosis. PMID- 7557861 TI - Portal hypertension and iron depletion in patients with genetic hemochromatosis. AB - Clinically, portal hypertension has been considered to be less common and less severe in patients with cirrhosis resulting from iron overload in homozygotes for genetic hemochromatosis than in patients with cirrhosis of other causes. To characterize the prevalence and progression of portal hypertension in genetic hemochromatosis (GH), 120 cirrhosis and iron-overloaded patients were compared with a control group of 120 patients with postnecrotic cirrhosis (PNC) who were matched for gender, age, Child's class, and alcohol abuse. Gastroesophageal endoscopy and abdominal ultrasonography were performed at diagnosis and repeated every 12 months and every 6 months, respectively, to evaluate the presence and severity of varices, the caliber of the portal vein and its collaterals, and splenic size. At diagnosis a similar frequency of varices was observed in patients with GH (25%) and in PNC (24%), as well as of portal vein abnormalities and spleen enlargement. During the follow-up period, all but two of the patients with GH were treated by phlebotomy and depleted of excess iron. After a mean of 6 +/- 4.3 (SD) years of observations (range, 2 to 10 years), varices were improved or completely reversed in 26% of patients with cirrhosis and GH but in only 5% of those with PNC (P < .01). Bleeding from varices was observed in only one patient with GH but in five patients with PNC. Of 22 patients with GH in whom portal hypertension was unmodified or worsened, 16 had coexistent hepatic viral infection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557863 TI - Polymorphisms in alcohol metabolizing enzyme genes and alcoholic cirrhosis in Japanese patients: a multivariate analysis. AB - Alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and P450IIE1 are the primary enzymes that catalyze the conversion of ethanol to acetaldehyde and then to acetate. Genetic polymorphisms have been reported in ADH2, ADH3, ALDH2, and the 5'-flanking region of P450IIEI. In this study, we used multivariate analysis to determine which genetic polymorphisms in alcohol metabolizing enzymes were independently associated with the development of alcoholic cirrhosis. Thirty-four noncirrhotic alcoholic patients, including 27 with fatty liver and 7 with nonspecific changes, and 46 patients with alcoholic liver cirrhosis were studied. Restriction fragment length polymorphisms (RFLPs) in the ADH2 and P450IIE1 genes were detected by digestion of polymerase chain reaction (PCR)-amplified DNA with MaeIII and RsaI, respectively. In the ALDH2 gene, RFLPs were detected by differences in the MboII site after PCR amplification. By multivariate analysis of four significant factors including total alcohol intake, ADH, ALDH, and P450IIE1 using the multiple logistic regression model, genotype ADH2(2)/ADH2(2) (P = .029) and genotype c1/c1 of P450IIE1 (P = .013) were found to be independently associated with alcoholic cirrhosis. The odds ratios for ADH2(2)/ADH2(2) genotype and the type A genotype of P450IIE1 (c1/c1) were 4.600 and 4.006, respectively. These results suggest that ADH2 and P450IIE1 gene polymorphisms may be independently associated with the development of alcoholic liver cirrhosis in Japan. PMID- 7557865 TI - Plexiform neurofibromatosis of the liver: case report and review of the literature. PMID- 7557866 TI - The combination of ursodeoxycholic acid and methotrexate for patients with primary biliary cirrhosis: the results of a pilot study. AB - Ursodeoxycholic acid (UDCA) and methotrexate (MTX) have both been proposed as treatments for patients with primary biliary cirrhosis (PBC). It has been suggested that a combination of the two drugs may offer advantages over either used separately. In this pilot study, we sought to evaluate the safety and efficacy of this combination for patients with PBC. Thirty-two patients with antimitochondrial antibody positive PBC were prospectively entered into a pilot study and received UDCA, 13 to 15 mg/kg/d, in conjunction with MTX, 0.25 mg/kg/wk, for a period of 2 years. The results of this treatment were compared with those obtained from 180 patients with PBC studied in a placebo-controlled trial of UDCA alone conducted during the same period. Patients in the pilot study and randomized study were comparable with regard to age, gender, and liver biochemistries. The UDCA/MTX-treated patients were of earlier histologic stage and had a lower mean Mayo risk score. During this period, seven patients in the UDCA/MTX group were withdrawn, four for pulmonary toxicity (two who required hospitalization), and one each with mouth ulcer, extreme fatigue, and hair loss. The use of UDCA/MTX was not associated with improvement in symptoms. In the patients receiving UDCA/MTX, biochemical changes were comparable to those of patients receiving UDCA alone but superior to those in the placebo group (P < .05). Histological changes were comparable in all groups at 2 years. Cessation of MTX while UDCA was continued led to no deterioration in liver biochemistries.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557864 TI - Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. AB - We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures. PMID- 7557867 TI - Hepatic involvement in mastocytosis: clinicopathologic correlations in 41 cases. AB - Mastocytosis is a disease of mast cell hyperplasia that may involve several organ systems, including liver. Between 1988 and 1991, we conducted a retrospective prospective study of 41 patients with mastocytosis and found 61% had evidence of liver disease. Hepatomegaly was detected in 24%, splenomegaly in 41%, and elevated serum alkaline phosphatase, serum aminotransaminases, 5'nucleotidase, or gamma-glutamyltranspeptidase (GGTP) in 54% of the patients. Alkaline phosphatase levels directly correlated with GGTP levels, hepatomegaly, splenomegaly, and liver mast cell infiltration and fibrosis. Elevated alkaline phosphatase levels and splenomegaly were observed more frequently in patients with categories II and III mastocytosis. Five patients in combined disease categories II or III developed ascites or portal hypertension and died of complications of mastocytosis; three had hypoprothrombinemia at the time of death. Thirty-five liver biopsy specimens from 25 patients were examined. Mast cell infiltration was commonly observed in the biopsy specimens, more severe in those patients with either category II or III disease, and correlated with hepatomegaly, splenomegaly, alkaline phosphatase levels, and GGTP levels. Mast cells were often only detected by using special stains (toluidine blue and chloracetate esterase). Increased portal fibrosis was seen in 68% of the biopsy specimens and correlated with mast cell infiltration and portal inflammation. Cirrhosis was not observed. Nodular regenerative hyperplasia, portal venopathy, and venoocclusive disease was observed in eight biopsy specimens and may have been the cause of the portal hypertension or ascites in four patients. These findings demonstrate that liver disease with mast cell infiltration is a common finding in patients with mastocytosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557868 TI - Ciprofloxacin and long-term prevention of spontaneous bacterial peritonitis: results of a prospective controlled trial. AB - The aim of this prospective double-blind study was to evaluate the value of long term antibiotic prophylaxis using ciprofloxacin for the prevention of spontaneous bacterial peritonitis (SBP) in 60 cirrhotic patients with low ascitic fluid protein levels (< 15 g/L). The patients were assigned to two groups: group I (n = 28) ciprofloxacin 750 mg per os once a week for 6 months, group II (n = 32) placebo. The two groups were similar for clinical and laboratory characteristics. Twelve patients developed an intercurrent disorder, and 10 patients died during the trial. There were no adverse effects in the treated group. There was a significant decrease in the incidence of SBP (3.6 vs. 22%) (P < .05) and duration of hospitalization (9.3 +/- 4.5 vs. 17.6 +/- 6.2 days) (P < .05) in the treated group as compared with the placebo group. The bacteriological study showed no acquired resistance to ciprofloxacin after 6 months' treatment. These results suggest that long-term preventive antibiotic prophylaxis based on the weekly administration of 750 mg of ciprofloxacin is effective in the prevention of SBP in cirrhotic patients. PMID- 7557870 TI - Serum 7 alpha-hydroxycholesterol as a new parameter of liver function in patients with chronic liver diseases. AB - To examine bile acid synthesis in chronic liver diseases, serum total 7 alpha hydroxycholesterol level was measured by gas-liquid chromatography-mass spectrometry in patients with cirrhosis (n = 23), patients with chronic hepatitis (n = 21), and control subjects (n = 18). The serum 7 alpha-hydroxycholesterol levels were significantly lower in patients with cirrhosis than the controls (78 +/- 59 pmol/mL vs. 237 +/- 97 pmol/mL; mean +/- SD). However, in patients with chronic hepatitis, the level was fully retained (262 +/- 102 pmol/mL). Serum 7 alpha-hydroxycholesterol levels of 17 patients with cirrhosis classified as Child B and C ranged from 33 to 69 pmol/mL, and all were less than the normal range (between 104 and 466 pmol/mL), however, those levels of some patients classified as Child A were within the normal range. Serum 7 alpha-hydroxycholesterol levels significantly correlated with serum albumin, cholinesterase, total bile acid, direct bilirubin, alkaline phosphatase, indocyanine green (ICG) retention rate, hepaplastin test, and lecithin-cholesterol acyltransferase activities. We conclude that bile acid synthesis is well preserved in patients with chronic hepatitis and that it is decreased in most patients with cirrhosis. Serum 7 alpha hydroxycholesterol may be a new parameter of liver function testing to assess hepatic bile acid synthesis in patients with chronic liver diseases. PMID- 7557869 TI - Intrahepatic mast cells in chronic liver diseases. AB - Mast cells are known to be present in human liver but their distribution and density in normal livers and in chronic liver diseases have not previously been examined. In this study, we quantified mast cell numbers and examined their distribution in percutaneous biopsy specimens from normal livers (n = 8) and in two chronic progressive liver diseases: primary biliary cirrhosis (PBC) (n = 40) and alcoholic liver disease (n = 33). We compared differences in mast cell density between these two forms of chronic liver disease because it had been suggested that mast cells may play a role in the development of liver fibrosis, particularly in patients with chronic cholestatic liver disease who frequently have increased plasma histamine levels. Mast cells were identified by immunohistochemistry using a specific monoclonal antibody (AA1) raised against mast cell tryptase after an initial study showed this to be more sensitive for the detection of mast cells than the conventional histochemical stain, toluidine blue. Our results showed that small numbers of mast cells (3.9 +/- 3.3/mm2) are present within the portal tracts and sinusoids of normal livers. In progressive chronic liver disease, increased numbers of mast cells were present, which correlated with the increasing amounts of liver fibrosis present. We found significantly more mast cells in the PBC group compared with the alcoholic group for a given amount of fibrosis. Our findings suggest that mast cells and their mediators may play a role in liver fibrogenesis. PMID- 7557871 TI - Carrier-mediated uptake and excretion of bromosulfophthalein-glutathione in perfused rat liver: a multiple indicator dilution study. AB - The hepatic removal of the glutathione conjugate of bromosulfophthalein (BSPGSH) was studied in the single-pass perfused rat liver with the multiple indicator dilution (MID) technique against various background concentrations of BSPGSH (20 to 214 mumol/L) over which nonlinear binding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing 51Cr labeled red blood cell (a vascular reference), [125I]albumin and [14C]sucrose (large and small molecular weight interstitial references, respectively), D2O (a cellular space reference), and [3H]BSPGSH was injected into the portal vein during steady-state. The eliminated fraction of dose, obtained by subtracting the survival fraction of [3H]BSPGSH in plasma from one, corresponded to the steady state extraction ratio (E) with bulk data, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentration. The major portion of the tracer outflow profile was a throughput component, which is the proportion of tracer that did not enter liver cells during its transit through the liver. The influx, efflux, and sequestration coefficients, evaluated with previously developed barrier-limited models, provided the corresponding influx (k1), efflux (k-1) and excretion (kseq) rate constants. Concentration-dependent influx (Vmax = 83 nmol min-1 g-1 and Km = 3.7 mumol/L), efflux (Vmax = 15 nmol min-1 g-1 and Km = 1.8 mumol/L), and excretion (Vmax = 94 nmol min-1 g-1 and Km = 1.8 mumol/L) were obtained for BSPGSH, when Km values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the Vmax and Km for efflux and excretion because of overestimation, because of the presence of highly concentrated BSPGSH in ductular elements present in liver homogenates; rather, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated entry, the unbound tissue concentration does not equal the unbound plasma concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss of BSPGSH from plasma was regressed against the estimated tissue unbound concentration. This yielded a Vmax of 97 nmol min-1 g-1 and a Km of 3.6 mumol/L, values similar to those obtained from MID.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7557872 TI - Sequential acetaldehyde production, lipid peroxidation, and fibrogenesis in micropig model of alcohol-induced liver disease. AB - The pathogenesis of alcohol-induced liver disease involves the adverse effects of ethanol metabolites and oxidative tissue injury. Previous studies indicated that covalent protein adducts with reactive aldehydes may be formed in alcohol consumers. To study the role of such protein adducts in the development of liver injury, we examined the sequential appearances of adducts of the ethanol metabolite acetaldehyde (AA) and of two products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenol (HNE), in ethanol-fed micropigs. Immunohistochemical stainings using specific antibodies that recognize epitopes of each adduct were performed from liver biopsy specimens obtained at 1, 5, and 12 months from micropigs fed either control diet (n = 5) or ethanol-containing diets (n = 5). After 1 month on the ethanol diet, AA and MDA adducts were observed primarily in the perivenous regions co-localizing with each other and coinciding with increased concentrations of serum aminotransferase markers of liver injury. HNE adducts were usually less intense and more diffuse, and were also seen in some biopsy specimens from control animals. Although the most intense staining reactions at 5 months remained in zone 3, a more widespread distribution was usually seen together with increased evidence of steatonecrosis and focal inflammation. In terminal biopsies at 12 months, perivenous fibrosis was present in three of five biopsy specimens. More extensive pericentral and intralobular fibrosis was noted in one micropig fed ethanol for 21 months. These studies demonstrate that covalent adducts of proteins with reactive aldehydes are formed in early phases of alcohol-induced liver disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557873 TI - Regulation of bile acid synthesis by deoxycholic acid in the rat: different effects on cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase. AB - We examined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combination with ursodeoxycholic acid, on serum and biliary bile acid concentrations, hepatic morphology, and the activities and steady-state messenger RNA (mRNA) levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in the rat. Feeding 1% deoxycholic acid increased serum bile acid concentrations (cholestasis), produced portal triad inflammation, bile duct proliferation, and severe hepatocyte necrosis with nuclear pleomorphism. Hepatic damage was prevented when ursodeoxycholic acid (1%) was combined with the deoxycholic acid (1%), or when deoxycholic acid intake was reduced to 0.4%. HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities were markedly inhibited (-56% and 55%, respectively) with either 1% or 0.4% deoxycholic acid. Ursodeoxycholic acid alone produced an insignificant decline in HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities, and when combined with 1% deoxycholic acid did not lessen the inhibitory effect of the latter. Steady-state mRNA levels increased 20 fold for HMG-CoA reductase and 53-fold for cholesterol 7 alpha-hydroxylase in rats fed 1% deoxycholic acid. In contrast, 0.4% deoxycholic acid decreased HMG CoA reductase mRNA levels 76%, and cholesterol 7 alpha-hydroxylase mRNA levels 82%. Ursodeoxycholic acid alone did not affect HMG-CoA reductase or cholesterol 7 alpha-hydroxylase steady-state mRNA levels. Steady-state mRNA levels and activities of sterol 27-hydroxylase, a key enzyme in the alternative acidic pathway of bile acid synthesis, did not change with either high or low doses of deoxycholic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557874 TI - Elevated mitochondrial gene expression during rat liver regeneration after portal vein ligation. AB - We explored the molecular basis of mitochondrial energy production during rat liver regeneration after portal vein ligation. Ligation of the left branch of the portal vein induces an increase in the weight of the nonligated lobe, counterbalancing the reduced weight of the ligated lobe. Using this model, we investigated changes in mitochondrial DNA-binding proteins, mitochondrial DNA, and mitochondrial messenger RNA (mRNA) in rat hepatocytes of the nonligated lobes. The amount of mitochondrial DNA-binding protein increased maximally (200% to 300% of the preoperative level) at 12 hours after the operation, before an increase (390%) in mitochondrial DNA content at 24 hours, and parallel to an increase (240%) in mitochondrial mRNA levels at 12 hours. These results suggest that the energy supply for liver regeneration is achieved through enhancement of mitochondrial DNA replication as well as transcription, in which the mitochondrial DNA-binding proteins probably play regulatory roles. We also found that in the nonligated lobes, mRNA levels of hepatocyte growth factor increased to a detectable level only 12 hours after the operation. These molecular biochemical data help explain why preoperative portal vein embolization, which is a modification of portal vein branch ligation, is an effective method to prevent posthepatectomy liver failure. PMID- 7557876 TI - Inactivation of Kupffer cells after prolonged donor fasting improves viability of transplanted hepatic allografts. AB - Data from recent studies suggest that donor fasting imparts a beneficial effect on the viability of transplanted hepatic allografts. Because starvation may temporarily inactivate Kupffer cells, and because these cells are the likely mediators of liver injury after prolonged preservation-reperfusion, the purpose of this study is to establish a link between improved organ viability and Kupffer cell inactivation caused by donor allograft fasting. In an in vivo rat liver transplant model, 48 hours of donor fasting (1) improved allograft viability, (2) significantly decreased Kupffer cell phagocytosis, and (3) significantly decreased cytokine (tumor necrosis factor [TNF]) production postrevascularization. These data validate work from previous studies demonstrating that donor fasting improves allograft viability and furthermore support our previous research implicating activation of Kupffer cells as a causative agent of cold ischemia-preservation injury. PMID- 7557877 TI - Oval cell differentiation into hepatocytes in the acetylaminofluorene-treated regenerating rat liver. AB - When hepatocyte regeneration after a two-thirds partial hepatectomy (PH) in rats is blocked by oral gavage of acetylaminofluorene, a proliferation of ductular cells ensues that results in a profusion of neoductules radiating from each portal tract. To examine the possibility that this population of newly emerging cells harbors cells capable of differentiating into hepatocytes, we have looked in these cells for expression of functional markers of hepatocyte commitment at both the RNA and protein levels. Expression of albumin and alpha-fetoprotein (alpha-FP) messenger RNA (mRNA) transcripts were sought in situ using antisense riboprobes, and the expression of a number of cytochrome P450 enzymes was examined immunohistochemically. Before any signs of differentiation the ductular cells strongly expressed cytokeratins 7, 8, 18, and 19 in the same manner as authentic bile ducts, but unlike the latter also expressed vimentin. In situ hybridization studies showed that small bile ducts close to the limiting plate, as well as the newly formed ducts, expressed albumin and alpha-fetoprotein messenger RNAs, and immunocytochemistry showed that the distribution of the respective proteins was similar. Beginning at 1 week after partial hepatectomy, areas of differentiation could be found in the new ducts, with cells resembling either columnar intestinal-type epithelia or hepatocytes. Intestinal-like cells expressed neither albumin, alpha-FP, nor cytochrome P450 enzymes, whereas ductular cells appearing like hepatocytes with the typical membranous distribution of cytokeratin 8 strongly expressed a variety of cytochrome P450 enzymes normally associated with functional hepatocytes. These observations further support the belief that reactive ductules, sprouted from small ducts, can represent an adaptive response of the liver to replenish lost hepatocytes, although some of the newborn cells appear to differentiate along intestinal lines. PMID- 7557875 TI - Nuclear scaffold-associated protease: in situ nuclear localization and effects of a protease inhibitor on growth and morphology of a ras-transformed hepatocyte cell line. AB - We have previously identified a multicatalytic protease (MCP) activity associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts. When we used the chloromethylketone protease inhibitor AAPFcmk, which is targeted to chymotrypsinlike protease activity, we observed a dramatic inhibition of transformation of fibroblasts, with effects that were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPFcmk on Simian Virus 40-immortalized CWSV1 cells compared with a ras transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotinAAPFcmk and fluorescent reagents to demonstrate a nuclear chymotrypsinlike protease activity, which is most prominent at the nuclear envelope. The ras-transformed NR4 cells were highly susceptible to growth inhibition in a dose-dependent manner, showing 85% growth inhibition at 50 mumol/LAAPFcmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 mumol/L) of AAPFcmk tested. In subcellular fractionation studies, the inhibitory effects of AAPFcmk were confined to the NS fraction. The AAPFcmk-induced growth inhibition was accompanied by marked morphological changes in ras-transformed cells, without evidence of overt toxicity. No change in DNA content was observed, but a marked increase in organization of actin cytoskeletal elements was seen. These results suggest that a protease activity associated with the nuclear scaffold has important functions in controlling cytoskeletal filament organization and cell replication. PMID- 7557878 TI - Effects of liver transplantation on bile formation and biliary lipid secretion in the Sprague-Dawley rat. AB - Altered hepatic secretory function after orthotopic liver transplantation constitutes a major perioperative clinical problem. Cholestasis and cholesterol gallstone formation are among the most frequent complications reported. Such changes in the allograft secretory function can be secondary to many factors like graft injury due to preservation and marked rejection, surgical complications, immunosuppressive therapy, and sepsis. The effects of liver transplantation per se on bile formation and biliary lipid secretion are unknown. The rat model of orthotopic liver transplantation was used to characterize better the true effect of transplantation without the influence of these confounding variables. Twenty four-hour bile collections were performed on nine transplanted versus nine liver denervated (sham) rats 4 weeks after surgery, and nine normal Sprague-Dawley rats. The liver allografts showed mild lymphocytic infiltration in portal tracts and the serum alanine transaminase levels were not significantly elevated. Bile flow and the secretion of bile salts and bilirubin under basal conditions were unchanged. Bile salt pool size, synthesis rate, and bile acid composition did not differ among the three groups. However, cholesterol secretion was dramatically reduced (50%) in the transplanted rats and decreased 31% in the liver-denervated rats (P < .001 and .01, respectively), resulting in a more favorable cholesterol saturation index (CSI = 0.29 for transplanted and 0.32 for sham versus 0.45 for normal controls; P < .01). Thus, liver transplantation with its attendant denervation did not impair hepatic secretory function, but rather improved biliary lipid composition despite mild rejection. PMID- 7557879 TI - Differential expression of A and B laminin chains during rat liver regeneration. AB - We have studied the distribution and expression of laminin during rat liver regeneration by immunofluorescence and immunoblotting using affinity-purified laminin antibodies. Laminin was localized on sinusoidal surfaces in normal and regenerating hepatic parenchyma, but enhanced expression was detected during regeneration from 6 hours to 7 days after a partial hepatectomy. A and B laminin chains were specifically detected by Western blotting in highly purified plasma membrane fractions derived from the sinusoidal domain of hepatocytes. Analysis of laminin expression in whole homogenates from hepatectomized rats showed a differential expression of A and B laminin chains during regeneration: The A chain showed a progressive increase, whereas the B chain exhibited an early, prereplicative increase and then gradually declined to control levels at the end of restorative growth. The results suggest differential temporal requirements for A and B laminin chains during hepatic growth and tissue formation. PMID- 7557880 TI - The effect of partial hepatectomy on tumor growth in rats: in vivo and in vitro studies. AB - Residual tumor in the remnant liver after partial hepatectomy (PH) for colorectal liver metastases is a serious clinical problem. This fact is reflected by the high number of recurrences after potentially curative liver resections. Liver regeneration, it appears, might influence the growth of remaining micrometastases in the liver. Using rats, we demonstrated enhancement of growth of a syngeneic colon carcinoma (CC 531) in the remnant liver after 70% PH. Fourteen days after PH, tumor weights in the liver were twice as high as those of sham-operated rats. This difference in tumor weight was not found in extrahepatic tumors. In vitro experiments did not show stimulation of cultured CC 531 cells by portal or systemic serum withdrawn 24 hours or 14 days after hepatectomy as compared with sera obtained after sham operation. Co-cultures of CC 531 cells and hepatocytes (in ratios of 1:10 or 1:1) demonstrated a higher 3H-thymidine incorporation than was the case in separately cultured cells. In co-cultures, bromodeoxyuridine (BrdU) incorporation in DNA was found primarily in CC 531 cells and rarely in hepatocytes. Cell density appeared to be of influence on 3H-thymidine incorporation in co-cultures. Hepatocytes were found to have a stimulating effect on CC 531 cells in low-density cultures, whereas high-density cultures exhibited an inhibiting effect after a culture time of 120 hours. These results show that, depending on cell density in co-cultures, a paracrine stimulating influence of hepatocytes on this type of colon carcinoma cells (CC 531) might be responsible for the increased tumor growth in vivo. PMID- 7557881 TI - Tumor necrosis factor-alpha decreases hepatocyte bile salt uptake and mediates endotoxin-induced cholestasis. AB - Tumor necrosis factor-alpha (TNF alpha), a cytokine that is produced in a variety of inflammatory diseases associated with cholestasis, is believed to be the primary mediator of the systemic effects of endotoxin. Thus, we have investigated the role of TNF alpha in the pathogenesis of endotoxin-induced cholestasis in intact animals, and in the uptake of taurocholate by cultured hepatocytes. Male Sprague-Dawley rats received either intravenous (IV) endotoxin (7.5 mg/kg) or monoclonal anti-TNF alpha antibody followed by endotoxin. Basal bile flow and bile salt excretion were measured for a 2-hour period, after which all animals received an IV bolus of taurocholate (10 mumol/100 g body weight). Endotoxin decreased basal bile flow by 41% and bile salt stimulated bile flow by 38% (n = 12; P < .01). Basal bile salt excretion was decreased 86% after endotoxin administration. Passive immunization with anti-TNF alpha antibody blocked this endotoxin-associated cholestasis. In addition, rat hepatocytes were isolated and cultured in the presence of either endotoxin (10 micrograms/mL) or TNF alpha (100 ng/mL) for 24 hours. These primary hepatocyte cultures exhibited a dose- and time dependent, noncompetitive, inhibition of taurocholate uptake. We postulate that TNF alpha is an important mediator of the cholestasis of sepsis. PMID- 7557883 TI - Parenchymal and nonparenchymal uptake of technetium-99m, indium-111, and iodine 125 low-density lipoprotein in the normal and estradiol-stimulated rat liver: tracer validation for quantitative low-density lipoprotein scintigraphy. AB - This study quantifies the parenchymal and nonparenchymal uptake of technetium-99m (99mTc)- and indium-111 (111In)-low-density lipoprotein (LDL) in different states of hepatic LDL-receptor activity to validate quantitative LDL scintigraphy. Iodine-125 (125I)-LDL was used as reference tracer. Four Sprague-Dawley rats with 17-alpha-ethinyl estradiol (EE)-stimulated LDL-receptor activity and five controls received all three tracers simultaneously 90 minutes before collagenase liver perfusion and metrizamide gradient cell separation. Total liver uptake of 99mTc-, 111In-, and 125I-LDL was 1.8 +/- 1.0, 1.6 +/- 0.8, and 0.2 +/- 0.2% injected dose/g organ weight, respectively. The contribution of nonparenchymal cells to total hepatic tracer uptake was 5.4 +/- 4.7%, 11.6 +/- 10.3%, and 9.6 +/ 7.6% in controls. Estradiol treatment increased total liver uptake to 2.4 +/- 0.5, 2.0 +/- 0.2, and 0.5 +/- 0.3% injected dose/g and reduced nonparenchymal cell contribution to 2.3 +/- 2.6%, 4.2 +/- 4.8%, and 2.6 +/- 2.9%, respectively. Dual-isotope scintigraphy in EE-treated and control rats confirmed these data, with a lower total hepatic uptake of 111In-LDL in comparison with 99mTc-LDL but a comparative degree of increase by EE treatment. Both behave quantitatively comparable as residualizing tracers, yet 99mTc-LDL shows a higher affinity to the LDL receptor pathway of parenchymal cells. However, the nonspecific uptake of both tracers can be neglected for quantitative LDL scintigraphy, and external imaging of hepatic tracer uptake primarily reflects LDL-receptor activity of parenchymal cells. PMID- 7557882 TI - Introduction of a murine p53 mutation corresponding to human codon 249 into a murine hepatocyte cell line results in growth advantage, but not in transformation. AB - The p53 gene is frequently mutated in human tumors; in hepatocellular carcinomas, there is a high frequency of a specific mutation at codon 249 in regions with significant aflatoxin exposure. To assess the role of this p53 mutation in the development of hepatocellular carcinoma, a mutant murine p53 gene, p53ser246, which corresponds to human codon 249, was transfected into a differentiated, nontransformed hepatocyte cell line AML12. Expression of p53ser246 in this line resulted in a growth advantage when compared with either a control vector (which contains a large p53 deletion) or with a different p53 mutant, val135, not found in hepatocellular carcinoma. Overall, there was a threefold increase in colony formation after transfection with p53ser246 as compared with the control or p53val135 vectors, and the p53ser246 plates developed consistently larger colonies. Whereas clones expressing the control or p53val135 constructs showed no significant morphological changes, clones expressing p53ser246 showed increased heterogeneity (large multinucleated cells and areas of small crowded cells) without focus formation. In addition, the ser246 mutation imparted a growth advantage in serum-free media, suggesting less dependence on specific factors present in serum. None of the mutant p53 or control lines were capable of growth in soft agar or tumor formation in nude mice. Thus in this model, in which endogenous wild-type p53 expression is retained, a high level of mutant p53 expression is not sufficient to transform hepatocytes. Our findings indicate that p53ser246 has effects on hepatocytes that may result in a clonal growth advantage and suggest that additional factors are required for the development of hepatocellular carcinoma. PMID- 7557886 TI - Carbon tetrachloride-induced hepatic injury is associated with global DNA hypomethylation and homocysteinemia: effect of S-adenosylmethionine treatment. AB - Carbon tetrachloride (CCl4) administration to rats produces hepatic cirrhosis and supplementation with S-adenosylmethionine (SAM) can partially prevent CCl4 induced liver injury. These effects are thought to be caused by oxidative stress and the subsequent formation of free radicals, but the mechanism whereby this occurs and the accurate nature of the mechanisms by which SAM exerts its protective action are not well understood. The effect of short-term administration of CCl4 on hepatic DNA methylation and on SAM and S adenosylhomocysteine (SAH) were assessed. CCl4 administration to rats for 3 weeks resulted in hypomethylation of liver DNA, determined by comparing the extent to which DNA from livers of control or treated animals could be methylated in vitro using [3H-methyl] SAM as methyl donor. This CCl4 effect on DNA methylation was corrected by the administration of SAM (10 mg/kg/d, intramuscularly), with values of methyl groups incorporation comparable with those observed in the control animals. hepatic SAM was decreased by CCl4 (65.3 +/- 5.27 vs. 102.2 +/- 4.89 nmol/g; P < .05) and SAH was increased (69.5 +/- 14.6 vs. 29.4 +/- 3.83 nmol/g; P < .05). This led to a marked reduction of the SAM/SAH ratio (the methylation ratio) from 3.47 in control rats to 0.94 in CCl4-treated animals (P < .05). SAM treatment partially prevented (P < .05) the reduction of the ratio SAM/SAH induced by CCl4. CCl4 also induced a marked elevation of serum homocysteine levels (more than 20-fold; P < .001), which was partially prevented by SAM administration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557884 TI - Tyrosine kinase growth factor receptors but not seven-membrane-spanning receptors or phorbol esters activate mitogen-activated protein kinase in rat hepatocytes. AB - The response of rat hepatocytes to hormones and growth factors has been extensively studied with respect to phospholipase regulation and calcium mobilization. However, the mitogen-activated protein (MAP) kinase cascade which integrates signals from a wide variety of extracellular stimuli has not been examined in these cells. Thus, in the present study the pathways leading to activation of MAP kinase in primary cultures of adult rat hepatocytes were investigated. Growth factors acting through tyrosine kinase receptors (epidermal growth factor and hepatocyte growth factor) increased Raf and MAP kinase activity through a protein kinase C and calcium-independent pathway. Agonists acting through seven-membrane-spanning receptors (arginine vasopressin and angiotensin II) increased intracellular calcium concentration but did not stimulate Raf or MAP kinase activity. Arginine vasopressin, however, stimulated MAP kinase activity in rat 1a fibroblasts transfected with the hepatic V1a receptor and in rat aortic vascular smooth muscle cells. Phorbol 12-myristate 13-acetate (PMA) was also unable to stimulate Raf and MAP kinase in hepatocytes in spite of a marked activation of protein kinase C. We conclude that only signals arising from tyrosine kinase receptors are able to activate MAP kinase in hepatocytes. Neither agonists acting through seven-membrane-spanning receptors nor phorbol esters stimulate MAP kinase in hepatocytes. The results suggest that specific cellular components that link seven-membrane-spanning receptors with MAP kinase activation in tissues such as vascular smooth muscle are absent in rat hepatocytes. PMID- 7557887 TI - Hepatitis B virus transgenic mice: insights into the virus and the disease. AB - It should be apparent from the foregoing that the transgenic mouse model system has contributed substantially to our understanding of many aspects of HBV biology, immunobiology, and pathogenesis in the past several years. We have learned that HBV can replicate within the mouse hepatocyte, as well as other mouse cell types, suggesting that there are probably no strong tissue or species specific constraints to viral replication once the viral genome enters the cell. However, the failure thus far to detect viral cccDNA in the hepatocyte nucleus in several independently derived transgenic lineages suggests that other, currently undefined, constraints on host range and tissue specificity may also be operative. Thanks to the transgenic mouse model, we now understand the pathophysiological basis for HBsAg filament formation and "ground glass" cell production, and we have learned that at least this viral gene product can be toxic for the hepatocyte, first by compromising its ability to survive the hepatocytopathic effects of lipopolysaccharide and gamma interferon, and eventually by causing it to die in the absence of any obvious exogenous stimulus. In recent studies, it has been shown that preformed nucleocapsid particles do not cross the nuclear membrane, in either direction, at least in the mouse hepatocyte. If this is confirmed, it will have two important implications: first, that nucleocapsid disassembly must occur in the cytoplasm before the nascent viral genome can enter the nucleus; second, that the intranuclear nucleocapsid particles are empty, and therefore serve no currently defined purpose in the viral life cycle. This should stimulate new interest in the analysis of the function of these particles that are a prominent feature of mammalian hepadnavirus infection. The transgenic mouse model has also established definitively that HBV-induced liver disease has an immunologic basis, and that the class I restricted CTL response plays a central role in this process. Additionally, the mouse studies have taught us that when the CTL recognize their target antigen on the hepatocyte they cause them to undergo apoptosis, forming the acidophilic Councilman bodies that are characteristic of viral hepatitis. Further, we have learned that although the CTL initiate the liver disease, they actually contribute more to disease severity indirectly by recruiting antigen nonspecific effector cells into the liver than by directly killing the hepatocytes themselves; and that by releasing gamma interferon when they recognize antigen, the CTL can destroy enough of the liver to cause fulminant hepatitis in mice whose hepatocytes overproduce the large envelope protein and are hypersensitive to the cytopathic effects of this cytokine.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7557888 TI - Predictors of a sustained beneficial response to interferon alfa therapy in chronic hepatitis C. PMID- 7557885 TI - Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. AB - Kupffer cell-derived cytokines are believed to play pivotal paracrine roles in the pathogenesis of alcoholic liver disease (ALD). To evaluate this hypothesis, Kupffer cell gene expression of tumor necrosis factor-alpha (TNF alpha), interleukin (IL)-6, and transforming growth factor-beta 1 (TGF beta 1) were directly examined in the rat model of ALD. Kupffer cells were isolated from the model after 10 and 17 weeks of intragastric ethanol infusion. These two durations resulted in focal hepatocellular injury and liver fibrogenesis, respectively. Oxidative stress as assessed by the hepatic level of thiobarbituric acid reacting substances, was evident at 10 weeks but more pronounced at 17 weeks. The steady state messenger RNA (mRNA) levels of the cytokines were examined by Northern blot analysis using RNA samples from freshly isolated Kupffer cells, and the release of the cytokines was quantitated ex vivo using a 3-day culture. The mRNA levels of TNF alpha and TGF beta 1 were significantly increased by 183% and 204% at 10 weeks and 231% and 295% at 17 weeks in the ethanol-fed rats, respectively. Ex vivo release of TNF activity by control Kupffer cells was undetectable or very low (< 2U/10(5) cells/18 hours) at both time points, but the cells from the ethanol-fed animals secreted appreciably more TNF (27.8 +/- 7.6 U at 10 weeks and 40.4 +/- 10.3 U at 17 weeks). The release of the latent TGF beta 1 protein was also coordinately increased by 143% at 10 weeks and 238% at 17 weeks.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7557889 TI - p53 proteins and aflatoxin b1: the good, the bad, and the ugly. PMID- 7557890 TI - Does interferon precipitate rejection of liver allografts? PMID- 7557892 TI - The physiology of jaundice: molecular and functional characterization of the Crigler-Najjar syndromes. PMID- 7557891 TI - Does interferon therapy for chronic hepatitis B reduce the risks of developing cirrhosis and hepatocellular carcinoma? PMID- 7557893 TI - In vitro modeling of liver membrane copper transport. PMID- 7557894 TI - Identification of chromosomal rearrangements in the human myeloid leukemia cell line GF-D8 by dual-colour fluorescence in situ hybridization. AB - Fluorescence In Situ Hybridization (FISH) studies with chromosome-specific libraries and repetitive probes were performed on the human acute myeloid leukemia cell line GF-D8 in order to define the complex chromosomal rearrangements observed by conventional cytogenetic analysis. Two-colour FISH with whole chromosome painting probes 8 and 11 showed that the add(8) chromosome had an 11-derived region inserted at q24, whereas the add(11) chromosome had an 8 derived region translocated onto q23. It also demonstrated that no normal chromosome 11 is present in GF-D8 cells, since a translocation involving chromosomes 11 and 17q was detected in addition to the add(11). The der(7) chromosome with extra material in its long arm, identified by QFQ and GTG banding, turned out to have a chromosome 15-derived segment translocated to q22. The deletion of 7q was proved to be interstitial, as the 7q-specific telomere as well as a tiny 7-specific band were observed on an unknown chromosome. Fine mapping of the breakpoints involved in the multiple chromosomal rearrangements of the GF-D8 cell line might provide insights into the mechanisms of myeloid leukaemogenesis. PMID- 7557897 TI - Cell kinetic analysis of non-Hodgkin's lymphomas using in vivo iododeoxyuridine incorporation and flow cytometry. AB - The aim of this study was to analyse dynamic cell proliferation parameters in non Hodgkin's lymphomas. Sixty-one patients with newly diagnosed or with recurrent disease were given iododeoxyuridine (IdUrd) intravenously near 4 h prior to tumour biopsy. After staining with an IdUrd reactive antibody and propidium iodide, S-phase fraction (SPF), labelling index (LI), S-phase duration time (Ts) and potential tumour doubling time (Tpot) were determined by flow cytometry. Thirty-eight samples, 15 low grade (LGM) and 23 high grade (HGM) malignant lymphomas, were possible to evaluate. Twenty-three cases were excluded due to aneuploidy, insufficient amount of material or technical problems. Tpot values varied between 0.8-32.9 days (mean 7.0 days). HGM lymphomas had shorter mean Tpot times than LGM lymphomas (4.8 versus 10.4 days, p = 0.05). For Ts the range was 4.2-20.1 h (mean 9.1 h), and a difference between the two histological groups was demonstrated with a longer mean Ts for HGM compared with LGM cases (10.0 versus 7.8 h, p = 0.04). Tpot showed a negative correlation with SPF (P = 0.003), and Ts demonstrated a positive correlation to SPF (p = 0.02). The clinical significance of the dynamic cell proliferation parameters studied remains to be clarified, but the interrelationships between Ts/SPF and Ts/morphologic subtype might be factors of interest for future prognostic studies in malignant lymphomas. PMID- 7557899 TI - Current awareness in hematological oncology. PMID- 7557898 TI - Transplantation of T-lymphocyte depleted marrow with an addback of T cells. AB - Depletion of T-lymphocytes from allogeneic marrow prior to transplantation decreases the risk of serious graft versus host disease (GvHD), but also increases the risk of relapse. We have used a partial T-cell depletion technique, in the hope of controlling GvHD without compromising relapse risk. Allogeneic marrow was fully T-cell depleted ex vivo with Campath 1M, and infused together with an 'addback' of 2 x 10(5) donor peripheral blood T-cells/kg in 16 consecutive patients undergoing BMT from HLA-identical family members. Post transplant immunosuppression was with cyclosporin A in conventional dosage. Eight cases developed grade I or II GvHD, limited to the skin in all cases. Only the two grade II cases required steroid therapy; the remainder were easily controlled with simple emollients. No cases of more advanced GvHD were seen. All patients engrafted promptly. With a median follow-up for the survivors of 976 days, seven cases have relapsed, with an overall projected actuarial disease-free survival of 37.5 per cent at 36 months. The overall outcome (good control of GvHD but high relapse rate) is therefore similar to that expected in patients receiving fully T depleted marrow without post-transplant immunosuppression. PMID- 7557896 TI - Molecular evidence for central nervous system involvement in children with newly diagnosed acute lymphoblastic leukemia. AB - Central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) would have profound implication for the prognosis and accurate stratification of CNS prophylactic therapy. Using PCR technique with specific primers for V, D and J segments of TCRD gene, the pattern of TCRD gene rearrangements in bone marrow lymphoblasts and in cells from cerebrospinal fluid (CSF) have been investigated. The study involved 21 children at the time of diagnosis with B-lineage ALL. In nine of 21 patients incomplete TCRDVD gene rearrangement has been found in CSF cells, which was identical to that observed in bone marrow of the same children. It can be concluded that at least in 43 per cent of all analysed cases, there were signs of CNS involvement in newly diagnosed ALL patients. PMID- 7557895 TI - Comparison of the performance of anti-CD7 and anti-CD38 bispecific antibodies and immunotoxins for the delivery of saporin to a human T-cell acute lymphoblastic leukemia cell line. AB - We have investigated the cytotoxic performance of two different anti-CD7/anti saporin BsAb's (HB2 x DB7-18 and Q1.1), three anti-CD38/anti-saporin BsAb's (OKT10 x RabSap, OKT10 x DB7-18 and Q4.1) and an anti-CD7 (HB2-Sap) and anti-CD38 saporin (OKT10-Sap) immunotoxin for delivering the ribosome inactivating protein (rip) to the human T-cell acute lymphoblastic leukemia cell line HSB-2. In the case of CD7 as target molecule the immunotoxin outperformed both anti-CD7 BsAb's being six times more effective than HB2 x DB7-18 and 98 times more so than Q1.1 at effectively inhibiting protein synthesis in a dose dependent manner. The chemically constructed HB2 x DB7-18 BsAb was more effective at inhibiting protein synthesis and cell growth in target HSB-2 cells in a dose dependent manner than the quadroma produced BsAb Q1.1. Both BsAb demonstrated a prozone effect used at concentrations above 0.1 nM though this was more pronounced for Q1.1 than for HB2 x DB7-18. The prozone effect was partially though not completely reversed by increasing the concentration of saporin in the system. In the case of CD38 as target molecule the anti-CD38 IT OKT10-Sap performed poorly, never actually achieving its IC50. Two BsAb's constructed with monoclonal anti-saporin Fab arms each recognizing a different epitope on the saporin molecule also performed poorly. In contrast the BsAb OKT10 x RabSap constructed with Fab derived from a rabbit polyclonal anti-saporin antiserum performed in a dose dependent manner achieving its IC50 at a concentration of 1.3 nM. This BsAb also exhibited a prozone effect. These results exemplify the importance of cross linking adjacent target molecules on the cell surface in order to achieve effective delivery of saporin to the cell interior. PMID- 7557900 TI - Benign myoepithelioma of the salivary glands: a true entity? AB - Myoepithelioma is a rare neoplasm of the salivary glands which is now recognized as an individual entity in the revised WHO classification. In this study, eleven benign tumours are presented. Most patients gave a history of a slowly enlarging mass, which was cured by surgical excision. However, one case recurred several times over 50 years, and another still has residual tumour and removal is not possible. The histological appearances included solid, myxoid and reticular growth patterns, composed predominantly of spindle shaped or plasmacytoid (hyaline) cells. Many of the tumours also contained occasional small ducts. All 11 tumours were positive for S-100 protein, variable reactions being seen for various other antigens--keratins, human milk fat globulin, carcinoembryonic antigen, alpha smooth muscle actin and vimentin. It is probable that myoepithelioma constitutes one end of a biological spectrum which also includes pleomorphic adenoma and some (non-membranous) basal cell adenomas. In practice, however, we still advocate retention of myoepithelioma as a separate diagnostic category, on the grounds that it has a range of distinctive microscopic appearances and poses its own unique problems in correct identification. PMID- 7557901 TI - Solitary and generalized variants of spindle cell xanthogranuloma (progressive nodular histiocytosis). AB - Twelve cases of solitary spindle cell xanthogranuloma, seven of which had originally been misdiagnosed as dermatofibroma/benign fibrous histiocytoma, were clinicopathologically compared with four cases of progressive nodular histiocytosis, a rare generalized non-X histiocytic disorder. Clinically, a single brown-yellowish papule or nodule is characteristic of solitary spindle cell xanthogranuloma, multiple generalized lesions of progressive nodular histiocytosis. Solitary spindle cell xanthogranuloma occurs with decreasing frequency on the head, neck, upper trunk, or occasionally the extremities of young adults (aged 20-40 years), progressive nodular histiocytosis mostly on the trunk of older patients (aged 40-60 years), both without sex predilection. Histologically, both entities are characterized by predominance (> 90%) of spindle-shaped histiocytes arranged in a storiform pattern. Other mononuclear (vacuolated, xanthomatized, scalloped, oncocytic) and multinucleate (Touton) histiocytes are also regularly seen. Immunohistochemically, both entities exhibit a macrophage/dendritic cell lineage positive for KP1/Ki-M1p (CD68), HAM 56 and factor XIIIa as well as for smooth muscle specific actin and HHF35. Ultrastructurally, dense, regularly laminated, myeloid or pleomorphic cytoplasmic inclusions may be found, but no Birbeck granules are present. This study documents that both solitary spindle cell xanthogranuloma and progressive nodular histiocytosis are distinct entities within the spectrum of a xanthogranulomatous reaction characterized by predominance of spindle-shaped histiocytes. PMID- 7557902 TI - Evaluation of clonal immunoglobulin heavy chain rearrangements in Hodgkin's disease using the polymerase chain reaction (PCR). AB - We have correlated histologic type of Hodgkin's disease, degree of Hodgkin and Reed-Sternberg cell infiltration, percentage of Hodgkin and Reed-Sternberg cell positivity for latent membrane protein, immunophenotype of Hodgkin and Reed Sternberg cells, and immunoglobulin heavy chain (IgH) gene rearrangements detected by polymerase chain reaction (PCR) in 56 unselected Hodgkin's disease cases. Two protocols were used for amplification of IgH gene using Fr2 or Fr3 V region primers, in conjunction with nested primers directed to the JH region. PCR products were run on polyacrylamide gels. Immunohistochemical studies were performed on paraffin sections using monoclonal antibodies for CD20 and latent membrane protein, and polyclonal antibody to CD3. Using both primer combinations we detected a definitive clonal band in 23.2% of the Hodgkin's disease cases. Clonal IgH rearrangements were detected in 23.6% of nodular sclerosis type and in 28.5% of mixed cellularity type. Using a highly sensitive method such as PCR, more than 20% of unselected cases of Hodgkin's disease were found to contain B cell clonal proliferations, but there was no correlation between histological and immunological parameters and molecular analysis results. PMID- 7557903 TI - The prognostic significance of vascular invasion in stage T1 bladder cancer. AB - Transurethral resection specimens from 170 T1 bladder carcinomas were reviewed for the presence of vascular (blood vessel and/or lymphatic) invasion by tumour cells. Such a finding was noted in 17 cases (10%), and occurred most frequently in high grade tumours. Tumour recurrence was documented in 11 of these cases (65%), including seven patients who showed progression to more invasive disease (T2-T4) and six patients (55%) who died of disease. Five-year survival for cases without vascular invasion was 81% versus 44% for those with. This was a statistically significant difference (log-rank, P = 0.004). Neoplasms of high grade (grades 2 to 3), without a papillary configuration, and exceeding 5 cm were associated with vascular invasion (chi-squared; P < 0.001, P = 0.043, and P = 0.061 respectively). In multivariate analysis vascular invasion proved to be an independent prognostic factor (Cox's regression, P = 0.015). We therefore stress the clinical relevance of a thorough evaluation of the state of vascular invasion in stage T1 bladder cancer. PMID- 7557905 TI - Vesical clear cell adenocarcinoma. V. Nephrogenic adenoma: a diagnostic problem. AB - Following the diagnosis of nephrogenic adenoma in a bladder lesion, which was later interpreted as early clear cell adenocarcinoma, the morphological and immunocytochemical features of these two lesions were reviewed to see if differences could be established for future diagnostic application. The architecture, extent, cell type, nuclear pleomorphism, presence of mitotic figures and glycogen content were recorded in 28 nephrogenic adenomas and the clear cell carcinoma. Similarly, the immunoreactivity for CAM 5.2, LP34, EMA and CEA of 10 nephrogenic adenomas and the clear cell carcinoma were compared. Proliferation rate in five nephrogenic adenomas and the carcinoma was assessed by antibody M1B1. Many of the features showed differences in degree or extent (clear cell change, nuclear pleomorphism, CAM 5.2 and CEA positivity). The only features distinct to clear cell carcinoma were the presence of solid islands, mitoses greater than 1/10 HPF (HPF area = 0.4 mm2) and M1B1 counts in excess of 29/200 in clear cell carcinoma (range 30/200-83/200). Only the high M1B1 count was present in the first biopsy of the clear cell carcinoma. PMID- 7557904 TI - Atypical polypoid adenomyomas of the uterus. AB - We performed a clinicopathological immunohistochemical, ultrastructural, and flow cytometric study on six cases of atypical polypoid adenomyoma of the uterus including one with an adenocarcinoma within it. The tumours occurred in nulliparous women aged 22-48 years (average, 33.0 years); three arose in the uterine corpus, and three in the endocervix. Histologically, they were composed of endometrial glands admixed with a stromal component of interlacing bundles of smooth muscle cells. The glands exhibited varying degrees of architectural and cytological atypia. Most of the stromal cells showed strong staining for HHF35, alpha-smooth muscle actin, and vimentin, and some cells contained desmin. Electronmicroscopy, in one case, confirmed the presence of a well-differentiated smooth muscle component. The stromal component may arise as a result of extensive metaplasia of endometrial stromal cells. Uninvolved endometrium showed ciliated cell metaplasia in three patients, and atypical complex hyperplasia in two. One patient had a well-differentiated adenocarcinoma of endometrioid type arising in an endocervical atypical polypoid adenomyoma. All tumours had a diploid DNA content and relatively small S phase fraction (average, 6.23%). The follow-up periods ranged from 4 to 42 months (average, 13.5 months), and all patients were alive and well. Although the histogenesis of atypical polypoid adenomyoma of the uterus remains uncertain, it is suggested that it may arise because of oestrogen related factors. PMID- 7557907 TI - The value of PE-10, a monoclonal antibody against pulmonary surfactant, in distinguishing primary and metastatic lung tumours. AB - A new monoclonal antibody (PE-10) raised against components of pulmonary surfactant has been assessed for its ability to distinguish primary from secondary carcinomas in the lung. We applied this antibody to a series of 107 primary lung carcinomas, 40 adenocarcinomas of other sites, and 26 cases of adenocarcinoma metastatic to lung and pleura. Of the primary lung carcinomas, all the non-mucinous bronchiolo-alveolar carcinomas were positive whereas all the mucinous cases were negative; 60% of other types of adenocarcinoma were positive and 10% of large cell undifferentiated carcinomas, 20% of small cell carcinomas and 40% of atypical carcinoids also showed focal positivity. Squamous cell carcinomas were all negative. Adenocarcinomas of the breast, kidney, large bowel and ovaries were all negative, as were all 26 cases of adenocarcinoma metastatic to the lung and pleura. We conclude that this antibody is highly specific and moderately sensitive for primary tumours of the lung. PMID- 7557906 TI - Giant cell rich parosteal osteosarcoma. AB - A parosteal osteosarcoma of the distal femur with a typical radiographic and macroscopic appearance is reported. On histology the tumour was dominated by large numbers of osteoclast-like giant cells in a fibro-osseous background and islands of immature bone. Most of the neoplastic bone lacked osteoblastic rimming. There was no spatial relationship between the giant cells and areas of haemorrhage. Signs of 'dedifferentiation' were lacking. Cytogenetic a analysis revealed telomeric associations which are frequently found in giant cell tumours of bone. Parosteal osteosarcoma may, on histological appearances, hardly be recognizable as malignant. In addition, unusual changes such as abundant giant cells may be misleading in the absence of clinico-pathological correlation. PMID- 7557908 TI - Keratin immunoreactivity in melanoma of soft parts (clear cell sarcoma). AB - Seven of 24 soft tissue melanomas were shown to express keratins using antibodies CAM 5.2, LP34 and MNF116. No clinical or histological differences were seen in these seven cases when compared with the 17 keratin negative cases. PMID- 7557909 TI - Hepatocellular prolapse of hepatic portal tracts and subendothelial space of central veins in idiopathic portal hypertension. AB - We report a case of idiopathic portal hypertension (IPH) with unusual liver pathology. The liver showed changes similar to these previously reported in IPH and, in addition, we observed the unusual features of prolapse of hepatocytes into portal tracts and also into the subendothelial space of hepatic veins. Hepatocyte prolapse into hepatic veins has previously been reported only in patients with a history of androgenic steroid therapy and immunosuppressive therapy. We speculate that, in our case, prolapse of hepatocytes could be related to the abnormal intrahepatic blood flow or to intrahepatic vasculopathy. PMID- 7557910 TI - Botryomycosis of the liver. AB - A case of visceral botryomycosis of the liver in a 50-year-old man is reported. The clinical diagnosis was hepatocellular carcinoma. Pathological examination of the surgically resected specimen demonstrated microabscesses containing gram- positive microorganisms with surrounding fibrosis replacing liver parenchyma. An immune deficiency state was not demonstrated. Recognition of this condition is important because of its clinical confusion with malignancy and its histological similarity to actinomycosis, nocardia, and eumycotic infections. PMID- 7557912 TI - Sinonasal neuroendocrine carcinoma exhibiting amphicrine differentiation. PMID- 7557911 TI - Plasminogen activator inhibitor-2 expression in inflamed appendix. AB - Plasminogen activator inhibitors are thought to be responsible for the abolition of fibrinolytic activity in inflamed peritoneum. This reduction in the fibrin clearing capacity of the peritoneum promotes the formation of intra-abdominal adhesions. High concentrations of plasminogen activator inhibitor-2 (PAI-2) have been previously found in inflamed peritoneal tissue using immunoassays, but it is undetectable in normal peritoneum. The aim of this study was to localize plasminogen activator inhibitor-2 production in tissue by in situ mRNA hybridisation. Sections of normal and inflamed human appendix were hybridised with a digoxigenin labelled cDNA probe. In normal appendix staining was confined to macrophages in the mucosa. Macrophage staining was also seen in inflamed tissue but with a wider distribution throughout the appendix wall. PAI-2 was also localized to mesothelial cells of inflamed but not normal appendix. Cell identities were confirmed using immunohistochemistry directed against cell specific markers. Staining was absent from control slides incubated with plasmid DNA or PAI-2 probe following ribonuclease digestion. The identification of the cells expressing the PAI-2 gene in peritoneum increases our understanding of the pathophysiological process leading to fibrin deposition within the abdomen during peritonitis. PMID- 7557913 TI - Mucinous eccrine carcinoma of the vulva with neuroendocrine differentiation. PMID- 7557915 TI - Low grade gastric B-cell MALT lymphoma progressing into high grade lymphoma. Clonal identity of the two stages of the tumour, unusual bone involvement and leukemic dissemination. PMID- 7557914 TI - Pigmented eccrine porocarcinoma: a mimic of malignant melanoma. PMID- 7557916 TI - Hodgkin's disease: a monoclonal lymphoproliferative disorder? PMID- 7557917 TI - Frank A. Beach Award. Male-female dominance relations at three ontogenetic stages in the spotted hyena (Crocuta crocuta). PMID- 7557920 TI - Role of the nutritional status of the litter and length and frequency of mother litter contact bouts in prolonging lactational diestrus in rats. AB - Food restricting lactating rat dams over the first 2 weeks of lactation results in a prolongation of the period of lactational diestrus. Such food restriction has not only a direct effect on the dam but also the pups are undernourished, and the pattern of dam-litter contact is also changed. In a series of studies, we investigated the effects of nursing undernourished pups and the change in dam litter interaction on the prolongation of lactational diestrus. While nursing undernourished pups in the last 2 weeks of lactation is sufficient to extend lactational diestrus in ad lib-fed dams nursing well-nourished pups in the last 2 weeks of lactation is not necessary for the prolongation of lactational diestrus seen in food-restricted dams. Further, neither nursing underfed pups nor increased nest time in the first 2 weeks postpartum are necessary factors for the prolongation of lactational diestrus in food-restricted dams. PMID- 7557918 TI - Frank A. Beach Award. Functional significance of steroid modulation of GABAergic neurotransmission: analysis at the behavioral, cellular, and molecular levels. PMID- 7557919 TI - Ontogenetic and social factors affect the endocrinology and timing of reproduction in the female leopard gecko (Eublepharis macularius). PMID- 7557921 TI - Social effects and circadian rhythms in squirrel monkey pituitary-adrenal activity. AB - Simultaneous measures of plasma cortisol and ACTH were collected at the morning peak (AM) and evening nadir (PM) of the circadian rhythm in group-housed and individually housed squirrel monkeys (Saimiri sciureus). Pronounced AM-PM differences in cortisol were evident in both conditions, but morning measures of cortisol in monkeys housed without companions were 32% higher than baseline control values observed when the same monkeys were sampled in groups. Consistent AM-PM differences in cortisol were not associated with consistent AM-PM differences in ACTH, and for monkeys housed without companions, plasma ACTH concentrations were consistently and significantly reduced (23% lower in the morning, 42% lower in the evening). All monkeys were subsequently pretreated overnight when dexamethasone to temporarily suppress the secretion of endogenous ACTH and then challenged the following morning with a bolus injection of synthetic ACTH. Monkeys housed without companions responded to the challenge with greater, more prolonged elevations in cortisol relative to monkeys housed in groups. These observations together suggest that squirrel monkeys housed without companions hypersecrete cortisol at the morning peak of the rhythm because adrenal responsiveness to ACTH is enhanced. Low circulating ACTH levels in turn are maintained by robust glucocorticoid feedback mechanisms that inhibit the synthesis or release of pituitary ACTH. PMID- 7557923 TI - Influence of the opposite sex on photoperiodically induced LH and gonadal cycles in willow tit (Parus montanus). AB - In early January photosensitive willow tits (Parus montanus) were transferred from natural to long days (20L;4D). The study included three groups of birds: one group consisted of pairs (one male and one female kept together in a cage), a second group consisted of males, and a third group included only females. Birds from the separate groups could neither see nor hear each other. Gonadal and LH cycles were followed over a 2-month period. The reproductive systems of male and female willow tits were activated by the long day exposure. This response was, however, affected by the presence/absence of a mate. Ovarian follicles in females caged together with a male grew faster than did follicles in females kept alone in their cages. Ovarian regression started at the same time in both female groups. Contrary to females, gonads in males caged together with females grew slower and also regressed much earlier than did tests in males kept alone. LH cycles differed only slightly between the two groups of females. In both groups maximum LH levels were reached already after 3 days of long day exposure. Maximum LH level was attained earlier in paired males (Day 3) than in single male (Day 7). Males without female company however maintained high plasma levels of LH for a much longer period of time (about 4-5 weeks) than did paired males. The observed differences are discussed in relation to the ecology of the willow tit. PMID- 7557922 TI - Sexual preference and feminine and masculine sexual behavior of male rats prenatally exposed to antiandrogen or antiestrogen. AB - Male rats were prenatally (Day 10-19 of pregnancy) exposed to an antiestrogen, nitromifene citrate (CI628, 1 mg/rat), or an antiandrogen, cyproterone acetate (CA, 10 mg/rat), and in adulthood were examined for their exhibition of male typical and female-typical behavior pattern. Treatment with CI628 abolished the capacity of the adult intact male to ejaculate, enhanced his potential to exhibit feminine sexual behavior, and decreased the intensity of the level of female oriented behavior in a two-choice stimulus situation (estrous female vs active male). The administration of testosterone (T) did not alter these behaviors. Males exposed to CA showed low levels of lordosis behavior and normal levels of female-oriented preference. Further, they showed increased frequency of mounts and decreased number of intromissions, and only a few males ever ejaculated. Macroscopic inspection of the genital organs of the CI628-treated males revealed complete absence of the prostate. The dissections of the CA-treated males revealed a poorly developed penis and a blind-ending vagina. It was concluded that prenatal estrogen (E) is involved (1) in determining the development of mechanisms destined to mediate the display of male-typical behaviors in adulthood, (2) in suppressing the development of mechanisms of female-typical behaviors, and (3) seems to stimulate neural mechanisms influencing sexual preference behavior in the adult. PMID- 7557928 TI - Report from the National Task Force on Access to Hospice Care by Minority Groups. AB - The NHO National Task Force on Access to Hospice Care by Minority Groups, formed in 1987, has undertaken a wide range of activities. In addition to reviewing the research literature, this report summarizes ongoing activities, future goals, and recommended actions for NHO and hospice programs. PMID- 7557925 TI - Behavioral and hormonal changes in female naked mole-rats (Heterocephalus glaber) following removal of the breeding female from a colony. AB - Colonies of naked mole-rats (Heterocephalus glaber) contain a single dominant, breeding female, or "queen", which suppresses reproduction in subordinate females. In this study, the queen and breeding male were removed from a colony at Brookfield Zoo. We examined behavioral and endocrine changes in the remaining colony females. Behavioral observations were conducted weekly and urine samples were collected beginning 3 months prior to the planned removal of animals. Prior to the removal, only the queen displayed the high frequencies of aggressive shoving typical of breeding females. In the first 2 months following removal of the queen, three non-breeding females markedly increased their frequencies of shoving. Urinary progesterone showed that one of three females had probably ovulated both before and after queen removal. Histological examination following fatal agonistic interactions confirmed ovulatory function in two of these three females. Similar behavioral and endocrinological results were obtained for three more females, following the combat deaths of the first three females. These findings suggest that ovarian activation facilitates intrasexual aggression in female naked mole-rats and might contribute to attainment of breeding status. PMID- 7557929 TI - Death and dying from a Native American perspective. PMID- 7557926 TI - Preoptic area lesions disrupt prolactin-induced parental feeding behavior in ring doves. AB - The role of the preoptic area (POA) in mediating PRL-induced parental responsiveness toward young was investigated in nonbreeding-doves with previous breeding experience. Birds received microinjections of the axon-sparing excitotoxin ibotenic acid to destroy neuronal cell bodies in the POA. Sham lesioned controls received POA injections of vehicle. Upon recovery, birds received subcutaneous injections of ovine PRL for 7 days, followed by a 2.5-hr test with a foster squab to assess parental responsiveness. Lesioned doves displayed significantly fewer parental feeding invitations, parental regurgitation feeding bouts, and total regurgitation movements than did sham lesioned animals. As a result, nestlings given to sham-lesioned birds gained significantly more weight than those given to lesioned birds. A significant negative correlation was obtained between the extent of POA damage induced by ibotenic acid infusion and the amount of parental behavior displayed by lesioned animals. These deficits in parental behavior were apparently a selective effect of POA damage because another PRL-mediated behavior, hyperphagia, was unaffected. The results suggest that the POA is an important component of the neural circuitry underlying the expression of PRL-induced parental feeding behavior in non-breeding ring doves. Because elevated PRL levels are temporally associated with parental activity during the breeding cycle, these results also implicate the POA in the spontaneous display of parental feeding activity that occurs naturally at the time of hatching. PMID- 7557927 TI - Prostaglandin E2-9-ketoreductase and prostaglandin F2 alpha activate brain aromatase to induce courtship in the male crested newt, Triturus carnifex. AB - Testosterone, 17 beta-estradiol, prostaglandin E2 (PGE2), and prostaglandin F2 alpha (PGF2alpha) were monitored in plasma and in vitro brain incubations during the main phases of male Triturus carnifex courtship. Plasma and in vitro results were similar: testosterone was highest in inactive males, while 17 beta-estradiol and PGF2 alpha were highest and PGE2 lowest during approach. PGF2 alpha in vitro treatments decreased testosterone and increased 17 beta-estradiol, while 9 ketoreductase inhibitor in vitro treatments increased testosterone and decreased estradiol. In addition, in vitro aromatase and PGE2-9-ketoreductase activities were highest during approach. PGF2 alpha in vitro treatments increased aromatase, while 9-ketoreductase inhibitor decreased aromatase. These results suggest that 9 ketoreductase and PGF2 alpha enhance aromatase activity to trigger male Triturus carnifex courtship. PMID- 7557930 TI - The African American experience: breaking the barriers to hospices. AB - The lack of understanding of the historical perspectives and cultural factors impact all aspects of hospice care for African Americans. This article addresses these areas in a broad context as well as the skills, behavior, and performance required to provide appropriate, competent, and culturally sensitive care to the terminally ill and dying African American and other culturally diverse population groups. PMID- 7557924 TI - Human placental lactogen infusions into the medial preoptic area stimulate maternal behavior in steroid-primed, nulliparous female rats. AB - The effects of central administration of human placental lactogen (hPL) on the onset of maternal behavior were measured in steroid-primed, adult ovariectomized, nulliparous rats. Rats were fitted with bilateral cannulas directed at the medial preoptic area (MPOA) and gonadectomized 1 week before being implanted sc with progesterone (P)-filled Silastic implants (treatment Day 1). On Day 11 P capsules were removed, and each female was implanted sc with a single estradiol (E2) capsule. On Days 11 to 13 animals were infused bilaterally with 40 ng of hPL/infusion or given 0.4 microliter vehicle. Subjects were given hormone or vehicle infusions five times during this period, twice each on Days 11 and 12 (1000 and 1600 hr) and once on Day 13 (1000 hr). Behavioral testing began on day 12 after the 1000 hr infusions and continued daily for 6 days. All females were injected sc twice daily with bromocriptine (2 mg/ke) to suppress endogenous PRL secretion from Day 11 to the completion of testing. The results showed that central infusions of hPL stimulated a fast onset of maternal behavior relative to controls. Latencies to display specific components as well as complete maternal behavior toward foster young were about 1 day in the hPL-treated group and 4 days in vehicle-infused controls. Infusions of hPL into the MPOA between 7.8 and 8.0 AP resulted in the fastest rate of onset of maternal behavior. These findings demonstrate that central infusion of a heterologous placental lactogen, hPL, is capable of stimulating maternal behavior may normally be brought about by exposure to placental lactogens as well as prolactin. PMID- 7557931 TI - Maria: developing a culturally-sensitive treatment plan in pre-hospice south Texas. AB - This is a case study of a young Mexican-American woman who suffered from end stage renal disease and severe depression in Texas, before the availability of hospice care. The patient, while struggling to retain her cultural identity in a renal care unit which stressed efficiency, was labeled noncompliant by the medical team. Through knowledge gained from staff discussions, the medical team was able to integrate cultural sensitivity and hospice principles into the treatment plan. With an increased focus on her psychosocial needs, the patient was able to die with a sense of dignity and cultural integrity. PMID- 7557932 TI - Hispanic American elders: caregiving norms surrounding dying and the use of hospice services. AB - The purpose of this review is to (1) provide an overview of health and demographic characteristics common to the Hispanic elder population, (2) address family caregiving issues surrounding the terminal illness of a loved one, (3) understand resource utilization by Hispanic elderly and their family caregivers, and (4) make recommendations for the provision of information and education about hospice services. Case examples will illustrate patterns and themes unique to Hispanic caregiving. PMID- 7557933 TI - Childhood bereavement among Cambodians: cultural considerations. AB - For many children bereavement services are not available until they develop extreme symptoms of chronic grief, posttraumatic stress disorder or depression, if then. This is especially true in some ethnic/racial communities where language and cultural barriers exist. The author uses a review of the literature and interviews with service providers and religious leaders in the Cambodian community of Long Beach to explore those unique cultural factors that have an impact on healthy childhood grief resolution among Cambodian children. Some suggestions for service provision are offered. PMID- 7557934 TI - Deterrents to access and service for blacks and Hispanics: the Medicare Hospice Benefit, healthcare utilization, and cultural barriers. AB - The Medicare Hospice Benefit may limit access for Blacks and Hispanics because of its requirement of continuity of care, entailing the availability of a primary caregiver. The literature on utilization of healthcare services by Blacks and Hispanics shows these groups were likely to receive too little care, too late. Kalish and Reynolds' (1976) research on attitudes of Blacks, Mexican-Americans, and Whites toward dying shows cultural differences that could affect acceptance of hospice philosophy. In other research reviewed in this paper distrust of White service providers was a significant cultural barrier for Blacks in using health services. Lack of familiarity with the health care system and language barriers were barriers most often for Hispanics. Black caregivers are more likely than Whites to have dying persons living with them, to be extended family members or nonrelated, and to be more limited in their ability to provide caregiving support because of a lack of economic resources. Hispanics appear to have a circumscribed support system, narrowly defined by blood kinship, with females as the expected caregivers. PMID- 7557936 TI - Training in molecular pathology. PMID- 7557935 TI - Identifying and meeting needs of ethnic minority patients. AB - In an effort to meet the unique needs of terminally ill patients and their loved ones within underserved minority populations, hospices are attempting to learn more about the diverse cultures represented in their communities. This paper will discuss one hospice's experience and perceptions of barriers to providing hospice care to individuals of diverse cultural backgrounds and the steps taken to more effectively serve them. PMID- 7557937 TI - Pathogenesis of calciphylaxis: study of three cases with literature review. AB - Calciphylaxis is characterized by ischemic necrosis, primarily of skin. The early phase of the ischemia has not been studied, and the pathogenesis is uncertain. In this study of early calciphylaxis, the vessels responsible for the ischemia seem to be within the material available for microscopic review, and the various stenosing vascular lesions are quantified. A distinctive and previously described small vessel calcification with superimposed endovascular fibrosis is most common, and is much more frequent than two other lesions proposed to cause the ischemia (thrombosis and global calcific obliteration). The calcified stenotic vessels average 100 microns in diameter. Calcification precedes the endovascular fibrosis. Vessels with early endovascular fibroblastic activation are found statistically to be strongly associated with the presence of a giant cell reaction. This endovascular giant cell reaction has not been previously described in calciphylaxis. Two additional cases show similar findings. The histology resembles the reaction to calcium in a variety of other extraosseous calcification syndromes, for example, pseudogout, as if calciphylaxis were an endovascular form of calcium crystal-induced inflammatory disease. The literature is reviewed, and the clinicopathologic, radiographic, and therapeutic implications are discussed. PMID- 7557938 TI - Congenital heart disease and sudden death in the young. AB - Sudden death is a frequent mode of fatal outcome in cardiac disease and does not exclude young people. The aim of this investigation was to establish whether and to what extent sudden death in the young may be ascribable to the substrate of underlying congenital heart disease. Among 182 young people (< or = 35 years) who died of cardiac sudden death and underwent postmortem examination, 58 (32%) had congenital heart disease. Seven showed an intrapericardial rupture of aortic dissection, in the setting of Marfan syndrome in two, isolated bicuspid aortic valve in two, and bicuspid aortic valve and isthmic coarctation in three; all exhibited equally severe degeneration of the aortic wall. Sixteen cases had conduction system anomalies, mostly bypass tracts; 15 coronary artery anomalies (three ostial valve-like stenosis, five origin from the wrong aortic sinus, and seven deep intramyocardial course); 12 hypertrophic cardiomyopathy; five postoperative congenital heart disease including scar following ventriculotomy, conduction system injury, and defects left unrepaired; and three congenital aortic valve stenosis. One third of sudden deaths in the young was ascribable to structural defects present since birth. A large spectrum of congenital heart disease involves the risk of sudden death, but most structural defects are usually not considered to be life threatening. Some of these concealed defects are potentially detectable in life by clinical imaging techniques. PMID- 7557940 TI - Hemosiderin deposition in portal endothelial cells: a novel hepatic hemosiderosis frequent in chronic viral hepatitis B and C. AB - We have recently noted a hitherto undescribed hepatic hemosiderosis confined to endothelial cells of the portal tract in chronic viral hepatitis. In this study, this lesion was surveyed in 156 liver biopsy specimens from patients with chronic hepatitis C and in 21 liver biopsy specimens from patients with chronic hepatitis B. As controls, we examined 110 liver biopsy specimens from patients with primary biliary cirrhosis (PBC), 36 from patients with alcoholic liver injury, nine from patients with autoimmune hepatitis (AIH), and five from patients with primary hemochromatosis. Hemosiderin deposition was found in the endothelial cells of venous vessels in portal tracts regardless of the presence or degree of hemosiderin deposition in hepatic parenchyma. This phenomenon was observed in 65 of 156 cases (42%) of chronic hepatitis C and in eight of 21 (38%) cases of chronic hepatitis B. In controls, this lesion was frequent in AIH (78%), but infrequent in PBC (8.1%) and alcoholic liver injury (11%). The incidence of this lesion showed significant differences between chronic hepatitis C, B, and AIH, and between PBC and alcoholic liver injury. There was a positive correlation between the progression of disease and the incidence of this feature in chronic viral hepatitis; the incidence was 18.3% and 11.1% in milder chronic hepatitis C and B, respectively, and 61.2% and 58.3%, respectively, in more severe cases. However, this correlation was not evident in either PBC or alcoholic liver injury. This hemosiderin deposition was positively correlated with the degree of piecemeal necrosis in chronic hepatitis C, and to a lesser degree, the positive correlation was shown in chronic hepatitis B. These findings suggest that the progression of chronic hepatitis and the piecemeal necrosis in chronic hepatitis C and B, followed by the release of hepatocellular iron to portal and periportal areas, are directly or indirectly responsible for endothelial hemosiderosis. Further studies focusing on this peculiar phenomenon in relation to choice of therapy and evaluation of chronicity of viral hepatitis are encouraged. PMID- 7557939 TI - Adenovirus pneumonia in lung transplant recipients. AB - Although Adenovirus (ADV) pneumonia has been documented in bone marrow, kidney, and liver transplantation recipients, it has only been sporadically reported in lung transplantation recipients. Among our 308 lung transplantation recipients, we identified four who developed ADV pneumonia. Formalin-fixed paraffin-embedded biopsy and autopsy specimens on all cases were studied by routine histology, immunohistochemistry (IHC), and by in situ hybridization (ISH) for evidence of ADV, and the results were correlated with the patients' clinical progression. Three of the four patients were children, and all four had a progressive and rapidly fatal course within 45 days posttransplantation. The lungs showed necrotizing bronchocentric pneumonia with tendency to spread diffusely to produce alveolar damage and organizing pneumonia. The occurrence of this rapidly fatal ADV pneumonia mainly affecting the pediatric population, early in the posttransplantation course, suggests that the infection is primary to the recipient with ADV either originating and reactivating in the donor lung or acquired from the upper respiratory tract of the recipient. The characteristic smudgy intranuclear inclusions of ADV, as well as IHC and ISH positivity, were observed in the lungs of all autopsies. Antemortem biopsy demonstration of ADV by inclusion formation, IHC, and ISH was observed in two patients. In another patient, antemortem ADV was shown only by ISH, and the recognition of inclusions was made difficult by coexistent CMV infection. Although IHC and ISH may have the potential for detecting early infection, recognition of the characteristic clinical setting with necrotizing bronchocentric pneumonia and smudgy intranuclear inclusions should alert one to the diagnosis of ADV pneumonia. PMID- 7557941 TI - Comparison of the prognostic value of four methods to assess mitotic activity in 186 invasive breast cancer patients: classical and random mitotic activity assessments with correction for volume percentage of epithelium. AB - Proliferation markers and especially the Mitotic Activity Index (MAI) are strong and reproducible prognosticators in invasive breast cancer. Traditionally, the MAI has been defined as the total number of mitoses counted in 10 consecutive high-power fields (objective, x40; numeric aperture, .75; field diameter, 450 microns), in the most cellular area at the periphery of the tumor, with the subjectively highest mitotic activity. No correction for epithelial percentage or cellularity was applied. This study investigates whether the prognostic value of mitotic activity could be improved by a random sampling procedure or correction for percentage of epithelium present. For this purpose the prognostic value of four methods used to assess mitotic activity in invasive breast cancer was compared in 4-microns-thick hematoxylin-eosin (H&E)-stained sections of 186 primary invasive breast cancer patients. These were the MAI, the random MAI (rMAI), the Mitosis per Volume (M/V) Index, and the random M/V Index (rM/V Index). The rMAI was defined as the total number of mitotic figures counted in 10 random fields through the whole outlined tumor at x400 magnification. A correction for the volume percentage of epithelium assessed with stereology yielded the M/V Index and the rM/V Index, respectively. The results of all four methods showed moderate to high correlations. Univariate survival analysis (Kaplan-Meier curves; Mantel-Cox test) confirmed that all four methods had a strong prognostic value (P < .001). The MAI, however, produced the best results (Mantel-Cox value, 17.1). Multivariate analysis showed that all four methods had additional prognostic value to tumor size and lymph node status. The M/V Index provided most additional prognostic information, followed by the MAI. Assessment of rMAI took 20 to 30 minutes on average, about two times longer than MAI. The correction for volume percentage of epithelium took about 10 minutes longer for both methods than the uncorrected methods. In conclusion, the rMAI gives an impression of the mitotic activity through the whole tumor, with almost similar prognostic value as the traditional MAI, especially when correcting for percentage of epithelium. Nevertheless, the MAI is still to be preferred, because the assessment is easy to apply and less time consuming. PMID- 7557943 TI - Primary mucoepidermoid carcinoma of the thyroid gland: a report of six cases and a review of the literature of a follicular epithelial-derived tumor. AB - Primary mucoepidermoid carcinomas of the thyroid gland are uncommon tumors of low grade malignant potential. We report six cases of thyroid mucoepidermoid carcinoma, four occurred in women and two in men with an age range of 29 to 57 (median, 46 years). The clinical presentation was that of a painless mass. Radiographic studies showed a single, solid, "cold" nodule in either the right or left lobe, or isolated to the isthmus. There was no history of a mucoepidermoid carcinoma developing in more typical locations (eg, salivary gland) in any of the patients. Histologically, the tumors were characterized by an intimate admixture of squamoid/epidermoid cells and mucocytes. The tumors were delineated but not encapsulated with prominent cyst formation and a variable amount of associated fibrosis. The squamoid component showed horny pearl formation, individual cell keratinization and/or the presence of intercellular bridges. Intracytoplasmic and luminal epithelial mucin was observed in all cases. In three of the cases a prominent eosinophilic cellular infiltrate was intimately identified within the neoplastic proliferation. One other case was noteworthy for the presence of ciliated epithelium. In all of the cases the uninvolved thyroid tissue showed the presence of lymphocytic thyroiditis. Rare foci of squamous metaplasia were observed in two of the cases. A microscopic focus of thyroid papillary carcinoma was observed adjacent to the mucoepidermoid carcinoma in one case. Immunohistochemical evaluation of the mucoepidermoid carcinoma showed the following antigenic profile: cytokeratin (five of five), CAM 5.2 (four of four), thyroglobulin (five of six), calcitonin (none of six), chromogranin (none of six), polyclonal carcinoembryonic antigen (four of four), and monoclonal carcinoembryonic antigen (none of five). Surgical excision was the treatment of choice. All of the patients reported are alive without disease (recurrence or metastasis) over periods ranging from 1 to 15 years. Based on our findings, we believe that these tumors are of low-grade malignant potential and originate from thyroid follicular epithelial cells rather than from solid cell nests of the ultimobranchial body. PMID- 7557944 TI - Comparative morphometric studies of benign and malignant intraductal proliferative lesions of the breast by computerized image analysis. AB - Using computerized image analysis, we newly devised a method for automatic quantitative evaluation of nuclear arrangements in variable proliferative intraductal (cribriform) lesions of the breast, by calculating angles of longest nuclear diameter to a horizon (LNDA). Internuclear correlation in the cribriform lesions was thus examined, comparing 22 benign and 62 malignant cases. In the malignant lesions, nuclear arrangements tended to be multidirectional, probably because of vertical nuclear arrangements toward acinar lumens as reflected by the wide distribution of LNDAs. Conversely, in the benign lesions, groups of nuclei tended to arrange toward one certain direction, forming a complex streaming pattern with the distribution of LNDAs usually producing a distinctive peak. Other conventional nuclear features, such as nuclear area, nuclear perimeter, shortest nuclear diameter (SND), and leading variables were also quantitatively assessed for multivariate analysis. It was noted that nuclear arrangements could represent a favorable discriminator. Linear discriminant function could classify 66 of the total 84 cases (78.6%) as being consistent with the diagnosis of pathologists. We conclude that internuclear arrangement could be a useful discriminating variable in benign and malignant breast lesions for the further development of an automatic analytic system. PMID- 7557946 TI - Nodules of less-differentiated tumor within or adjacent to hepatocellular carcinoma: relative expression of transforming growth factor-alpha and its receptor in the different areas of tumor. AB - Expression of transforming growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR), in hepatocellular carcinomas (HCCs) and adjacent nontumorous livers from 25 Japanese patients were examined using immunoperoxidase staining of paraffin-embedded sections. TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. TGF-alpha and EGFR were not detected by immunohistochemical staining in normal livers. Fifteen of 25 HCCs contained an apparent area of a second tumor (two of the 15 also contained a third tumor) that had a less-differentiated histological grade developing within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis. PMID- 7557945 TI - Focal nodular hyperplasia of the liver: composition of the extracellular matrix and expression of cell-cell and cell-matrix adhesion molecules. AB - We studied by immunohistochemistry 25 cases of focal nodular hyperplasia (FNH) to evaluate the composition of the extracellular matrix and the expression and distribution of endothelial cell-cell adhesion molecules and integrin receptors. The extracellular matrix of FNH retained the overall organization of that of normal liver. The matrix of central scars resembled that of portal tracts. The main difference was the presence of large vitronectin deposits, which might indicate the existence of local hemodynamic disturbances. The matrix lining the sinusoid-like vessels running in the hyperplastic parenchyma retained characteristic features of the normal perisinusoidal matrix, such as the presence of tenascin. In the zone surrounding the central scars, it contained large amounts of laminin, von Willebrand factor, and thrombospondin, suggesting the development of perisinusoidal fibrosis. Laminin deposition was accompanied by the induction of cell-cell adhesion molecules on adjacent endothelial cells and by the up-regulation of specific integrin receptors on both hepatocytes and sinusoidal endothelial cells. In conclusion, our study: (1) reinforces the hypothesis that FNH is merely a hyperplastic response of liver parenchyma to local vascular abnormalities, and (2) shows that the lesions of perisinusoidal fibrosis associated with FNH are accompanied by the induction of integrin receptors on hepatocytes and sinusoidal endothelial cells. PMID- 7557942 TI - Frequent presence of the Epstein-Barr virus in inflammatory pseudotumor. AB - Inflammatory pseudotumor is a presumably nonneoplastic, hematopoietic, and spindled fibrous proliferation that may occur at a variety of anatomic sites. The origin of these proliferations is generally unknown. To evaluate the role of the Epstein-Barr virus (EBV) in inflammatory pseudotumor, 18 specimens from 17 patients were studied by in situ hybridization for EBV ribonucleic acid (RNA), and the morphological and immunologic characteristics of the infected cells were evaluated. These specimens included 10 lymph nodes, six splenic masses, and two hepatic masses. Overall, EBV RNA was detected in 41.2% (seven of 18) of the cases. These included two of 10 (20%) lymph nodes, four of six (66.7%) splenic pseudotumors, and one of two (50%) hepatic lesions. The degree of EBV infection was significantly greater within the tumors in comparison with the surrounding, uninvolved tissue. Two morphologically different EBV-positive cell types, spindled and round cells, were evident, and the infected cell type differed significantly when the nodal and extranodal cases were compared. All of the positive extranodal cases shown, numerous EBV-positive spindled cells, whereas no positive spindle cells (only positive round cells, morphologically consistent with lymphocytes) were noted in the two EBV-positive lymph node pseudotumors. Double-labeling immunohistochemical and in situ hybridization studies in some cases identified rare EBV-positive B cells and rare EBV positive T cells in four and three cases, respectively. Most EBV-positive cells in all cases failed to immunoreact with any B- or T-cell markers. Three of five cases studied, however, did show a subpopulation of smooth muscle actin/EBV-positive spindled cells, five of seven cases showed vimentin/EBV-positive spindled cells, and one of four cases had EBV-positive spindled cells that immunoreacted as follicular dendritic cells. These results suggest that EBV plays a role in a significant number of cases of inflammatory pseudotumor with differences in the incidence of EBV infection and the cell type (spindled vs round cell) infected when extranodal and nodal cases are compared, suggesting a difference in pathogenesis. The cell type infected in extranodal cases seemed to be of mesenchymal origin but could not be clearly defined. PMID- 7557947 TI - Antigen levels of urokinase plasminogen activator and its receptor at the tumor host interface of colorectal adenocarcinomas are related to tumor aggressiveness. AB - The distributions of urokinase and tissue plasminogen activators (uPA, tPA), uPA receptor (uPAR), and plasminogen activator inhibitors (PAI-1, PAI-2) were studied immunohistochemically in two subsets of colorectal adenocarcinomas with low and high aggressiveness, respectively: nine Dukes' stage A tumors with additional other good prognostic markers and 13 Duke's stage C tumors with also other poor prognostic markers (referred to as Dukes' stage A and Dukes' stage C tumors). The results showed that these components of the tissue destructive plasminogen activation system were accumulated at the invading front of the tumors. Both tumor groups showed accumulations of uPA, uPAR, and PAI-1 at the tumor-host interface compared with the location within the tumor epithelium and the adjacent normal mucosa and muscularis propria (all P < .05). However, the uPA level at the tumor-host interface in the Dukes' stage C tumors was twice the level in the Dukes' stage A tumors (P < .05). The uPAR level was also significantly higher in the Dukes' stage C tumors (P < .05), whereas the PAI-1 level was not significantly higher. This may indicate that uPA in more aggressive tumors exceeds the inhibitory capacity represented by PAIs, resulting in enhanced tissue destructive potential that promotes tumor invasion. uPA and uPAR antigen levels and the uPA/PAI-1 ratio at the tumor-host interface appeared to be related to tumor aggressiveness in colorectal cancer. PMID- 7557948 TI - Discordant CD3 expression in lymphomas when studied on frozen and paraffin sections. AB - The results of CD3 staining in the T or putative natural killer (NK) cell lymphomas of nasal and extranasal sites as reported in the literature have been confusing, with some studies reporting a low rate of CD3 positivity and others a high frequency of CD3 staining. The former studies were performed on fresh or frozen tissues, whereas the latter were performed on paraffin sections using a polyclonal antiserum (poly-CD3). Although previous studies have suggested a high concordance rate of CD3 staining between fresh/frozen tissues and paraffin sections, many CD3- cases have not been studied, and the more reliable antigen retrieval techniques have not been applied. In this study, we addressed the question of discordant CD3 expression by comparing the results of CD3 staining in lymphomas as studied on frozen sections and as studied on paraffin sections (with antigen retrieval effected by pressure cooking). This series was biased toward inclusion of a high percentage of cases of putative NK cell lymphomas, which are prevalent among Asians and usually show a CD2+ CD3(Leu4)- CD56+ immunophenotype. Among 35 cases of CD3(Leu4)- T- and T/NK-cell lymphomas, 30 (86%) showed staining with poly-CD3 on paraffin sections. All 15 CD3(Leu4)+ T-cell lymphomas showed positive staining with poly-CD3 on paraffin sections. None of 60 B-cell lymphomas were stained by poly-CD3, confirming no loss of specificity of staining with this antiserum despite use of an effective antigen-retrieval technique. The discordance rate of CD3 staining in T- and T/NK-cell lymphomas in this series was 60%, and this phenomenon was most commonly observed in the CD56+ T/NK-cell lymphomas: CD3(Leu4)- in frozen sections but poly-CD3+ in paraffin sections. Therefore, to avoid confusion, we propose designating the results based on fresh/frozen tissues CD3(f) and those based on poly-CD3 application on paraffin sections CD3(p) in future reporting of CD3 immunophenotype. PMID- 7557949 TI - Role of CD44 in the invasiveness of glioblastoma multiforme and the noninvasiveness of meningioma: an immunohistochemistry study. AB - CD44 is a polymorphic family of cell adhesion molecules that seems to be instrumental in the mechanism of tumor invasion and metastasis. Tumor cell expression of CD44, or lack thereof, may be one of the factors conditioning the highly disparate ability to penetrate the brain extracellular matrix (ECM) exhibited by glioblastoma multiforme (GM) and conventional meningioma. To assess the presence of CD44 in these two tumor types we have immunohistochemically investigated the expression of CD44 standard form (CD44s) and the variant isoforms containing the domain encoded by variant exon 3 (CD44v3) and variant exon 6 (CD44v6) in paraffin-embedded tissue from 10 conventional meningiomas and 10 GMs. A CD44s-/CD44v-phenotype was discerned in the meningioma cases, whereas GMs featured a CD44s+/CD44v- expression profile. Consequently, the growth patterns of meningioma and GM seem to be, at least in part, a reflection of their CD44 expression status. Paucity of CD44 in meningioma cells would render them unable to infiltrate the brain ECM, whereas CD44-rich glioma cells would successfully migrate through it. Conversely, lack of CD44v expression would contribute to explain the lack of metastatic potential characterizing both conventional meningioma and GM. PMID- 7557952 TI - Septic pulmonary thrombosis in streptococcal toxic shock syndrome. AB - A 60-year-old woman who was previously in good health presented with a sore throat, fever, and a flu-like syndrome. Treated initially with acetaminophen and fluids for a presumed viral infection, she had a syncopal episode 4 days later, was admitted to the hospital, and died 3 hours after admission. Laboratory test results suggested sepsis with disseminated intravascular coagulation (DIC), whereas blood cultures grew group A beta-hemolytic streptococci. A postmortem diagnosis of streptococcal toxic shock syndrome was established. It was of particular interest that the pulmonary microcirculation was filled with thrombi that contained numerous gram-positive cocci. Although death from sepsis with DIC is not uncommon, septic pulmonary thrombosis has not been previously described. We speculate that this paradox may reflect unique properties of the virulent strains of Streptococcus pyogenes that are associated with streptococcal toxic shock syndrome. PMID- 7557951 TI - Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy. AB - Adrenal 4 binding protein (Ad4BP) is a transcription factor that regulates the expression of the steroidogenic enzymes and is expressed primarily in steroidogenic cells. We immunolocalized Ad4BP in adrenocortical carcinoma (eight cases) and various malignancies that histologically simulate an adrenocortical carcinoma to evaluate the value of Ad4BP as an immunohistochemical marker of adrenocortical carcinoma. These malignancies examined were renal cell carcinoma (20 cases), hepatocellular carcinoma (10 cases), malignant melanoma (eight cases), ovarian (six cases) and uterine (three cases) clear cell carcinoma, large cell carcinoma of the lung (five cases), and pheochromocytoma (three cases). Nuclear Ad4BP immunoreactivity was observed only in adrenocortical carcinoma cases but not in other tumors examined. Almost all of the adrenocortical carcinoma cells were immunohistochemically positive for Ad4BP including cells associated with bizarre nuclei. These results show that application of Ad4BP immunostain can contribute greatly to the differential diagnosis of adrenocortical carcinoma. PMID- 7557953 TI - Pure red cell aplasia associated with true thymic hyperplasia. AB - Pure red cell aplasia was diagnosed in a 35-year-old otherwise healthy woman. Chest computed tomography (CT)-imaging investigation, detected an upper mediastinal mass corresponding to the thymus gland. A thoracotomy was performed and an enlarged thymus mass was removed, rapidly followed by a full hematologic recovery. Thymic histology confirmed a significant degree of hyperplasia. We conclude that not only thymomas but also other types of thymic pathology may be associated with this type of hematologic dyscrasia. PMID- 7557950 TI - Hepatic amyloidosis in Japan: histological and morphometric analysis based on amyloid proteins. AB - To investigate the relationship between the tissue distribution and the types of amyloid proteins, the detailed histopathologic features of the available liver in 284 cases of amyloidosis were examined. We classified hepatic amyloidosis into three types, namely, the vascular pattern, parenchymal pattern, and stromal pattern according to the topographic distribution pattern of amyloid. Of the 152 amyloid A (AA) cases, all but one exhibited the vascular pattern; the single exception had the parenchymal pattern. Among 117 amyloid light chain (AL) cases, 51.3% exhibited the vascular pattern and 43.6% the parenchymal pattern. The stromal pattern was observed in 5.1% of the cases but was found only in AL amyloidosis. The parenchymal and stromal patterns in the liver seemed to be characteristic morphological distributions of AL amyloidosis. Routine histochemical study is useful to distinguish AL from AA, although some ethnic differences were apparent. Morphometric results showed that the walls of the hepatic arteries with amyloid deposition were significantly thicker than walls in arteries from the control group. The arterial walls in AA amyloidosis, especially, were significantly thicker than walls in AL amyloidosis of any pattern. PMID- 7557954 TI - Autoimmune enteropathy with anti-goblet cell antibodies. AB - A 9-year-old boy with a 5-year history of severe protracted diarrhea requiring home parenteral nutrition and a 1 year history of abnormal liver function tests was admitted for duodenal, rectal, and liver biopsy. Duodenal biopsy results showed mild villus blunting, a mild lymphocytic infiltrate, and absent goblet cells. Paneth cells and endocrine cells could not be identified. Review of several previous biopsies showed an almost total absence of goblet cells by light microscopy. Anti-goblet cell antibodies of the immunoglobulin (Ig)G class were shown by immunofluorescence with a titer of 1:512. Histological examination of rectal mucosa also showed a total lack of goblet cells, orderly surface epithelial cells, and infiltration of the colonic crypts by lymphocytes. Immunoperoxidase staining of rectal mucosa showed increased numbers of lymphocytes with an excess of CD3+, CD45RO+ T cells, and increased numbers of B cells labeling with B1 and L26. Increased numbers of CD25+ (activated) lymphocytes were also observed. HLA/DR expression was striking and observed in both the crypt and surface enterocytes, as well as in the lamina propria. Immunological assessment of the patient showed an inverted CD4/CD8 ratio and IgA/IgG4 deficiency. The liver biopsy and radiological investigation were in keeping with chronic sclerosing cholangitis. Although a slight and transient improvement in histological appearances was observed with prednisolone there was no significant improvement of diarrhea. Trials of azothiaprine and oral cyclosporin did not result in clinical or histological improvement. PMID- 7557955 TI - Autoimmune alterations in choroid plexus basement membrane of some patients with Alzheimer's disease. PMID- 7557956 TI - An update on the diagnostic relevance of CD26 antigen expression in CD30 positive anaplastic large cell lymphomas. PMID- 7557957 TI - Mapping eight new polymorphisms in 11q13 in the vicinity of multiple endocrine neoplasia type 1: identification of a new distal recombinant. AB - Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disorder that predisposes affected individuals to neoplasms of the parathyroid glands, endocrine pancreas, anterior pituitary, and carcinoids. The MEN1 locus has been localized by family studies to 11q13, flanked by markers PGA and D11S97. Eight new polymorphisms located in three separate radiation-reduced somatic cell hybrid segregation groups were developed. The order of the new markers, within the context of previously described loci, was determined by linkage analysis on the Venezuelan reference pedigree. Four independent MEN1 families, consisting of 57 affected individuals, and 70 individuals at-risk for the disease were genotyped. Sixteen people inherited a chromosome that shows recombination between a linked marker and the disease. The nearest proximal and distal markers that show recombination with the disease are D11S822 and GSTP1, respectively, thereby narrowing the candidate region for MEN1 by 50% on the distal side. Using these loci in haplotype analysis, an accurate presymptomatic molecular diagnostic test has been developed. These new markers in 11q13 linked to MEN1 also facilitate the genetic and physical characterization of this very gene-rich region. PMID- 7557959 TI - Linkage of the long QT syndrome to the short arm of chromosome 11: use of five highly polymorphic markers towards more detailed localization of the mutant gene. AB - The long QT syndrome is an autosomally dominantly inherited cardiac disorder characterized by abnormalities of myocardial repolarization, exercise- or stress related syncopal attacks and risk of sudden death due to cardiac arrhythmias. Genetic linkage studies have defined three LQT loci on chromosomes 11p15.5, 3q21 24 and 7p35-36. We performed linkage analyses in three Finnish LQT families using five amplifiable markers assigned to chromosome 11p15. By multipoint linkage analyses we obtained a maximal lod score of 5.503, suggesting that the LQT1 locus maps between D11S922 and D11S1338 on chromosome 11. Our data provide a step towards closer definition of the exact borderlines of the LQT1 locus in chromosome 11 and demonstrate markers with high utility in identification of gene carriers in the affected families. PMID- 7557958 TI - Effects of the mutant von Willebrand factor gene in von Willebrand disease. AB - Von Willebrand disease (vWD) is a common inherited bleeding disorder in humans, and can be divided into a mild (type 1) and severe (type 3) form. Previous linkage studies identified one subject with vWD type 1 who transmitted different alleles of the von Willebrand factor (vWF) gene to his two affected children, one having vWD type 3 and the other having type 1. By screening the promoter and coding sequence (52 exons) of the vWF gene, three missense mutations were detected in this family. The type 1 individual who transmitted different alleles of the gene to his two sick children carries two substitutions, one in exon 5 and the other in exon 18 on the respective alleles. The relationship between the genotype (mutations) and the phenotype in this family is complex. In order further to correlate the relationship in vWD type 1 individuals, fifty-five subjects who carry one null allele of the vWF gene were collected. All these subjects are from vWD type 3 families with known mutations. Biochemical data of these 55 subjects indicate that gene dosage and other factors, such as blood group, age, and environment factors, play a critical role in the development of the phenotype of the disease. PMID- 7557960 TI - Two novel frameshift mutations in the low density lipoprotein receptor gene generated by endogenous sequence-directed mechanisms. AB - DNA samples from 60 unrelated Belgian hypercholesterolemic patients were subjected to heteroduplex analysis of exon 4 of the low density lipoprotein receptor (LDLR) gene. Aberrant mobility bands were detected in 2 patients and the underlying mutations were characterized by DNA sequence analysis. Both mutations, a 19-bp insertion at codon 141 and a 23bp deletion at codon 168, produce premature stop codons in the highly conserved ligand binding domain of the mature LDLR. Sequence data indicated that mispairing between short direct repeats during DNA replication is the most probable mechanism by which these mutations could have arisen. Our observations are consistent with an endogenous sequence-directed mechanism of mutagenesis. PMID- 7557961 TI - Genetic mapping of Xp22.12-p22.31, with a refined localization for spondyloepiphyseal dysplasia (SEDL). AB - Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder characterized in affected males by short stature resulting from a growth defect of the vertebral bodies. We have extended our earlier studies by analyzing 15 families with newly identified microsatellite DNA markers; analysis of recombination events with these markers indicates that the gene responsible for SEDL is located in Xp22 between DXS 16 and DXS 987 on an interval spanning approximately 2 Mb. PMID- 7557962 TI - ICF syndrome (immunodeficiency, centromeric instability and facial anomalies): investigation of heterochromatin abnormalities and review of clinical outcome. AB - A further patient with the ICF syndrome (immunodeficiency, centromeric heterochromatin instability of chromosomes 1, 9 and 16 and facial anomalies) is described. This case is the second to be reported with consanguinity of the parents. This lends support to the theory of autosomal recessive inheritance. The features of the 15 published cases are reviewed. The clinical and cytogenetic characteristics of the syndrome are discussed, and new evidence provided as to the role of centromeres and centric heterochromatin in the production of chromosome aberrations. Correspondence with other authors has made possible a review of the clinical outcome in this condition. PMID- 7557964 TI - Mapping dystrophin gene recombinants in Greek DMD/BMD families: low recombination frequencies in the STR region. AB - A systematic study of 42 Greek DMD/BMD families using 14 polymorphic markers that span the dystrophin gene was performed in order to assess the position and frequency of recombinants in the Greek population and to test whether "hot spots" of recombination and deletions coincide when exclusively studying DMD/BMD families. We report a low percentage of recombination between markers STR44 and STR50; otherwise, the distribution of recombination events in other parts of the gene is largely in agreement with previously published data on Centre d'Etude du Polymorphisme Humaine families. We therefore propose that recombination frequencies and the correlation between recombination and deletion "hot spots" should be evaluated on DMD/BMD families exclusively. PMID- 7557963 TI - A common beta hexosaminidase gene mutation in adult Sandhoff disease patients. AB - beta-Hexosaminidase gene mutations were analyzed in two adult-onset Sandhoff disease Italian patients by PCR analysis of a common known mutation (delta 5') and by heteroduplex analysis of genomic and RT-PCR DNA fragments, covering the whole gene. The patients' genotypes were delta 5'/C1214%, and G890A/C1214T, respectively. As mutation C1214T (Pro405Leu) is also present in the other two late-onset cases so far described, we suggest that C1214T is a common mutation in this type of Sandhoff disease. Mutation G890A (Cys297Tyr) is a novel mutation which presumably causes altered processing of the pro beta chain. PMID- 7557965 TI - Screening for mutations causing X-linked severe combined immunodeficiency in the IL-2R gamma chain gene by single-strand conformation polymorphism analysis. AB - Mutations in the common gamma chain (gamma c or IL2RG) of the interleukin-2, -4, 7, -9 and -15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the gamma c proteins produced as a result of these mutations are discussed. PMID- 7557966 TI - Tumor necrosis factor alpha (TNF-alpha) gene polymorphism in alopecia areata. AB - Tumor necrosis factor alpha (TNF-alpha) phenotypes of two polymorphic systems were determined in 50 patients with alopecia areata, a common inflammatory disease of the skin. The distribution of TNF-alpha T1, T2 phenotypes differed between patients with the patchy form of disease and patients with totalis/universalis disease. There was no significant difference in the distribution of TNF-alpha G,A phenotypes between patient groups. The results of this study provide evidence of genetic heterogeneity between the two forms of alopecia areata, and suggest that the TNF-alpha gene or a closely linked locus within the major histocompatibility complex may play a role in the pathogenesis of the patchy form of disease. PMID- 7557967 TI - Linkage analysis and allelic imbalance in human breast cancer kindreds using microsatellite markers from the short arm of chromosome 3. AB - Eight Icelandic breast cancer kindreds were subjected to linkage analyses with respect to 28 microsatellite loci dispersed along the short arm of chromosome 3. Breast tumors derived from these kindreds were concurrently scored for allelic imbalance with ten of the markers. Linkage to most markers could be excluded on the basis of negative LOD scores and haplotype analyses, although some moderately positive LOD scores resulted. A high frequency of imbalance in the familial tumors was seen with two of the markers in comparison with results obtained from sporadic material. The highest frequency (68%) of imbalance was detected with the marker D3S1217, which is located on 3p14.2-p14.1. Imbalance at the D3S1211 locus, which is more telomeric (3p24.2-p22), was not significantly elevated in the familial tumors. We suggest that the genetic defect responsible for breast cancer susceptibility in these families either promotes instability in the 3p14.2-p14.1 region or enhances the selective advantage of such changes. PMID- 7557968 TI - Analysis of the elastin gene in 60 patients with clinical diagnosis of Williams syndrome. AB - Williams syndrome (WS) is caused by deletion of the elastin (ELN) gene. We have analyzed an intragenic restriction fragment length polymorphism (RFLP) and the gene dosage of ELN using a new probe (FP4) in a series of 60 sporadic patients with a clinical diagnosis of WS. Deletion of the ELN gene was shown in 54 cases, while clinical revaluation of the 6 patients without the deletion did not confirm the diagnosis of WS. These results support the genetic homogeneity of WS, and the high accuracy of ELN molecular analysis, which can be confidenty used for providing genetic counselling to WS families. PMID- 7557969 TI - Improved carrier testing for multiple endocrine neoplasia, type 1, using new microsatellite-type DNA markers. AB - Familial multiple endocrine neoplasia, type 1 (FMEN1), is an autosomal dominant trait generated by hyperfunction of various endocrine glands. The gene for MEN1 has been mapped to chromosome 11q13 by genetic linkage and deletion mapping in tumors. Eight Finnish families, including 46 individuals carrying the risk haplotype, have been typed for four polymorphic microsatellite DNA markers spanning the MEN1 chromosomal region. Three of the loci concerned, D11S913, D11S987, and D11S1337, displayed maximum lod scores (Zmax) 6.70, 9.88, and 2.54, respectively, with no recombinations with the disease gene, whereas a Zmax of 8.43 was obtained for D11S971 at a recombination fraction of 0.03. Our results indicate that the use of this set of markers considerably improves the diagnostic value of genotyping patients at risk of developing MEN1. PMID- 7557970 TI - Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia. AB - Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6(-2) (a transition of A to G at the second nucleotide position within the 3' splice cleavage site of intron 6) and IVS-13(+5) (a transition of G to A at the fifth nucleotide position within the 5' splice cleavage site of intron 13), respectively. The IVS-6(-2) mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6(-2) mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing. PMID- 7557971 TI - Testicular development in an SRY-negative 46,XX individual harboring a distal Xp deletion. AB - A case of a true hermaphrodite presenting with a karyotype of 46,X,del(X)(p21.1- >pter) is described. The testis-determining gene, SRY, was not detected in DNA prepared from either peripheral blood lymphocytes or from a gonad biopsy. The patient also presented with a series of discrete somatic abnormalities, including abnormal skin and retinal pigmentation, and mental retardation. The extent of the Xp deletion was mapped by Southern blotting. X chromosome replication studies of lymphoblast cells prepared from the patient indicated that the deleted X chromosome was inactivated in all cells examined. It is suggested that the phenotype of the patient is caused by the unmasking of a recessive allele(s) on the grossly intact X chromosome. The relationship between the Xp deletion, the intersex phenotype, and the possible role of an Xp locus involved in human sex determination is discussed. PMID- 7557972 TI - A novel coding exon of the human adenomatous polyposis coli gene. AB - We report the genomic cloning and sequence analysis of a novel and (so far) smallest coding exon of the human adenomatous polyposis coli (APC) gene. This additional exon of 54 nucleotides in length, designated APC exon 10A, is located 1.6 kb downstream from exon 10. It resides on a 0.5-kb genomic EcoRI restriction fragment. APC exon 10A is alternatively spliced and inserted in-frame into mature transcripts; it gives an APC protein with an additional 18 amino acids. This highly conserved exon is expressed in a tissue-independent fashion. APC exon 10A flanking sequences are presented so that this novel exon can be included in future mutation screening procedures. PMID- 7557973 TI - Phenylketonuria mutations and their relation to RFLP haplotypes at the PAH locus in Czech PKU families. AB - A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism/variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and 165T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification. PMID- 7557974 TI - Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409. AB - We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hopital Saint-Louis, Paris, France) by the use of the polymerase chain reaction. We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409. The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively. This location was further established by chromosomal in situ hybridization. PMID- 7557975 TI - Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization. AB - The human thyroxine-binding globulin (TBG) gene has been localized to X chromosome (Xq22.2) by in situ hybridization using a biotinylated gDNA probe. This is consistent with previous mapping of the TBG gene to chromosome Xq21-q22. PMID- 7557976 TI - Role of genetic factors in bronchial cancer. Based upon a case of anaplastic lung carcinoma in identical twins. AB - Having observed homozygotic identical twin brothers suffering simultaneously from anaplastic bronchial cancer leading rapidly to death in both cases, the authors assessed the frequency of such cases. The available literature failed to reveal any identical observations, although four cases of twins suffering from bronchial cancer featuring different histologies (three epidermoidal and one bronchiolar alveolar) were noted. Statistics show that, in the area where the observed twins were living, anaplastic cancer occurs each year in 0.39% of 53-year-old men. The case of these twins therefore supports the idea of the role of genetic factors in the determination of bronchial cancer. PMID- 7557979 TI - Detection of a C-insertion polymorphism within the human tumor necrosis factor alpha (TNFA) gene. AB - We have identified a C-insertion polymorphism in the 5'UTR of the first exon of the human tumor necrosis factor alpha (TNFA) gene. TNFA is a cytokine that plays an important role in the inflammatory response. PMID- 7557977 TI - Mosaic loss of 15q11q13 in a patient with hypomelanosis of Ito: is there a role for the P gene? AB - We report a patient with mental retardation, behavioral disturbances, and pigmentary anomalies, consistent with the phenotype of hypomelanosis of Ito (HMI), and in whom cytogenetic analysis revealed mosaicism for an unbalanced translocation. His karyotype is 45, XY,-7,-15,+der(7)(7:15)t(q34:q13)/46,XY. He is therefore monosomic for 7q34 to qter and 15pter to q13 in the cells containing the translocation. The human homolog (P) of the p gene (the product of the mouse pink-eyed dilution locus) maps to 15q11q13. Loss of this locus is believed to be associated with abnormalities of pigmentation, such as the hypopigmentation seen in patients with deletions of 15q11q13, and the Prader-Willi and Angelman syndromes. Mutations within the P gene have also been associated with tyrosinase positive (type II) oculocutaneous albinism. Using fluorescence in situ hybridization, we confirmed that our patient is deleted for one copy of a P gene probe in the cells with the unbalanced translocation, and for loci within the region critical for the Prader-Willi/Angelman syndromes. Although hypomelanosis of Ito is a heterogeneous disorder, we postulate that, in our case and potentially in others, this phenotype may result directly from the loss of specific pigmentation genes. PMID- 7557980 TI - An intragenic TaqI polymorphism in the faciogenital dysplasia (FGD1) locus, the gene responsible for Aarskog syndrome. AB - A Taq1 polymorphism, located in intron 4 of the faciogenital dysplasia (FGD1) gene, the gene responsible for Aarskog syndrome, is described. FGD1 encodes a putative Rho/Rac guanine nucleotide exchange factor involved in mammalian morphogenesis. The identification of an intragenic polymorphism will facilitate the accurate carrier detection of individuals at risk for Aarskog syndrome. PMID- 7557981 TI - Polymorphic (AAT) in trinucleotide repeats derived from a human brain cDNA library. AB - Seven cDNA fragments containing polymorphic (AAT)n trinucleotide repeats were isolated from a human brain cDNA library and mapped by linkage to specific loci. These repeats may serve as gene markers or as candidates for diseases caused by expansion mutation. PMID- 7557978 TI - A region of primer binding variation at the D6S265 locus associated with HLA-A25 and HLA-A26 antigens. AB - D6S265 is a polymorphic dinucleotide repeat, mapped within 70 kb centromeric of HLA-A, on chromosome 6p21.3. While genotyping families for genetic linkage analysis, allele non-amplification resulting in apparent non-Mendelian inheritance was observed at the D6S265 locus in 15 individuals, on chromosomes carrying the HLA-A25 and HLA-A26 antigens. The D6S265 locus was sequenced in a variant individual homozygous for allele non-amplification, and in a non-HLA-A25/ A26 individual, homozygous for D6S265 allele 1. Five base changes were identified in the reverse primer binding region of the variant individual, effectively preventing annealing of the 3' primer to the template. PMID- 7557982 TI - Dinucleotide repeat polymorphism at the D5S99 locus on chromosome 5q33-34. AB - We report a highly polymorphic, sequence-tagged microsatellite site (STMS) at the D5S99 locus that was previously identified by a less informative restriction fragment length polymorphism (RFLP). This marker, which was also localized to the physical map of chromosome 5q by fluorescent in situ hybridization (FISH), should assist in the precision mapping of genes in the area 5q33-34. PMID- 7557984 TI - Autosomal dominant retinitis pigmentosa: a new multi-allelic marker (D3S621) genetically linked to the disease locus (RP4). PMID- 7557983 TI - The importance of clinical documentation in genetic studies of male infertility. PMID- 7557986 TI - Structural organization of the human gene (LMNB1) encoding nuclear lamin B1. AB - We have determined the structural organization of the human gene (LMNB1) that encodes nuclear lamin B1, an intermediate filament protein of the nuclear envelope. The transcription unit spans more than 45 kb and the transcription start site is 348 nucleotides upstream from the translation initiation codon. Lamin B1 is encoded by 11 exons. Exon 1 codes for the amino-terminal head domain and the first portion of the central rod domain, exons 2 through 6 the central rod domain, and exons 7 through 11 the carboxyl-terminal tail domain of this intermediate filament protein. Intron positions are conserved in other lamin genes from frogs, mice, and humans but different in lamin genes from Drosophila melanogaster and Caenorhabditis elegans. In the region encoding the central rod domain, intron positions are also similar to those in the gene for an invertebrate nonneuronal cytoplasmic intermediate filament protein and the genes for most vertebrate cytoplasmic intermediate filament proteins except neurofilaments and nestin. PMID- 7557989 TI - Epidermal surface antigen (MS17S1) is highly conserved between mouse and human. AB - A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, high-lighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5' and 3' untranslated sequences are conserved. Similar RNA folding patterns of the 5' untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. PMID- 7557988 TI - Genomic sequences and structural organization of the human nidogen gene (NID). AB - Nidogen/entactin is a ubiquitous 150-kDa multidomain basement membrane protein. Since in vitro binding studies indicated that nidogen may function as a major mediator in basement membrane organization and assembly, analysis of gene structure and regulation of gene expression will help us to understand many biological processes that involve degradation and reorganization of the basement membrane zone. An approximately 100-kb region of genomic DNA encoding the human nidogen gene (NID) including 5' and 3' flanking sequences has been cloned and characterized by restriction mapping and sequencing. The entire gene is more than 90 kb in length and contains 20 exons. All introns interrupt protein coding sequences. The size of individual introns varies significantly, ranging from 0.6 to 18 kb. Its exon/intron structure revealed that the protein domains of human nidogen are organized in a domain-specific manner with various subdomains being encoded by individual exons, indicating that exon duplication and shuffling have played an important role in determining the present structure of the protein. Comparison of the exon organization with the recently published ascidian nidogen amino acid sequence strongly suggests that vertebrate nidogen might have evolved from a common ancestral precursor resembling ascidian nidogen. PMID- 7557987 TI - Structural organization and mapping of the human TCF11 gene. AB - We recently cloned human cDNA representing several mRNA isoforms of human TCF11, a transcription factor of the basic-region, leucine-zipper (bZIP) family located on chromosome 17q22 as well as a genomic clone of this gene. We have now determined the complete genomic organization of the TCF11 gene, which consists of 9 exons distributed over 15 kb of genomic DNA. Pulsed-field gel electrophoresis was used to construct a physical map around TCF11, to characterize a 450-kb YAC clone that contains the gene, and to link TCF11 physically to BTR, a marker on chromosome 17. PMID- 7557985 TI - Localization of the gene (SYM1) for proximal symphalangism to human chromosome 17q21-q22. AB - Proximal symphalangism, or Cushing symphalangism (MIM 185800), is an autosomal dominant disorder characterized by ankylosis of the proximal interphalangeal joints. Conductive deafness and reduced flexibility of the ankles have also been observed in affected individuals. We have used polymorphic markers throughout the genome to perform genetic linkage analysis in subsequent generations of the family originally described by Harvey Cushing. We have established linkage for this disorder to markers on chromosome 17 (17q21-q22), with Zmax = 6.98 at theta = 0.05 with marker D17S790. PMID- 7557991 TI - Relative efficiencies of chi 2 models of recombination for exclusion mapping and gene ordering. AB - In multilocus linkage analysis, it is common to assume chiasma interference is absent. While this assumption provides mathematical tractability, there is substantial biological evidence contradicting it, particularly when the loci are closely spaced. The chi 2 class of recombination models, recently described by Foss et al. (Genetics 133: 681-691, 1993), has a plausible biological basis and provides a dramatically improved fit over virtually all other models currently in use. Here, a simulation study is performed to assess the relative efficiency of a no interference model analysis to an analysis with a chi 2 model which allows for interference. The results presented show that analysis with the no interference model is inefficient in the presence of interference. PMID- 7557990 TI - Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders. AB - We have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Kruppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Kruppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21 q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes. PMID- 7557993 TI - Human gene for the large subunit of ribonucleotide reductase (RRM1): functional analysis of the promoter. AB - Ribonucleotide reductase comprises two nonidentical protein subunits R1 and R2, both of which are required for enzyme activity and show cell cycle-dependent regulation. The TATA-less promoter of the human RRM1 gene (encodes R1 protein) was examined with reference to regulatory domains upstream of the transcription start site. A region from nt -195 to +3 was found to give maximal expression of a reporter gene when transfected into the human cell line K562. Overall, this 198 bp region shows 58% identity with the equivalent region of the murine promoter; however, it contains two 22-bp domains that were 81 and 91% identical between species. Electrophoretic mobility shift assays were performed using a fragment of the domain closest to the transcription start site. These experiments revealed that several factors were able to bind this region in a sequence-specific manner. One of these factors was shown to be Sp1 by specific competition and supershift using antibody to Sp1. The data presented suggest that Sp1 is involved in the transcription of RRM1. PMID- 7557992 TI - Identification, expression, and pharmacology of a Cys23-Ser23 substitution in the human 5-HT2c receptor gene (HTR2C). AB - The function of brain serotonin-2C (5-HT2C) receptors, including behavioral and neurochemical responses to 5-HT2C agonist challenge, has been suggested to be abnormal in individuals with neuropsychiatric disorders. Thus, it is important to identify polymorphisms and functional variants within this gene. Using SSCP analysis, we identified a Cys23-Ser23 substitution (designated 5-HT2Ccys and 5 HT2Cser) in the first hydrophobic region of the human 5-HT2C receptor. Allele frequencies in unrelated Caucasians were 0.13 and 0.87 for 5-HT2Cser and 5 HT2Ccys, respectively. DNAs from informative CEPH families were typed for this polymorphism and analyzed with respect to 20 linked markers on the X chromosome. Linkage analysis placed the 5-HT2C receptor gene (HTR2C) on Xq24. To evaluate whether this amino acid substitution causes a variant function of this receptor, recombinant human 5-HT2Ccys and 5-HT2Cser receptors were expressed in Xenopus oocytes and tested for responses to 5-HT using electrophysiological techniques. Concentration-response curves for 5-HT were not significantly different in oocytes expressing either form of the receptor, suggesting that the 5-HT2Ccys and 5-HT2Cser receptor proteins may not differ in their responses to serotonin under baseline physiological conditions. PMID- 7557994 TI - Linkage disequilibrium utilized to establish a refined genetic position of the Salla disease locus on 6q14-q15. AB - Salla disease (SD), an inherited free sialic acid storage disorder, is caused by impaired transport of free sialic acid across the lysosomal membrane. Clinical characteristics of the disease include severe psychomotor retardation and some neurological abnormalities. We report here detailed linkage analyses of 50 Finnish SD families that localize the SD disease gene to a refined chromosomal area on 6q14-q15. The highest lod score of 17.30 was obtained with a microsatellite marker of locus D6S280. When linkage disequilibrium was adopted in the linkage analyses, we could further assign the SD locus to the immediate vicinity of marker locus D6S406. Linkage disequilibrium facilitated further restriction of the critical chromosomal region to approximately 80 kb, well within the limits of positional cloning techniques. PMID- 7557995 TI - Human chromosome 17 comparative anchor loci are conserved on bovine chromosome 19. AB - Eight comparative anchor loci on human chromosome 17, TP53, CHRNB1, THRA1, CRYB1, NF1, MPO, MYL4, and P4HB, were mapped to bovine chromosome 19 using bovine x hamster and bovine x mouse hybrid somatic cell lines. This completes the synteny mapping of human chromosome 17 comparative anchor loci in cattle, all of which have been mapped to bovine chromosome 19 and mouse chromosome 11, with the exception of CSH1. It is likely that the suggested homologue of human CSH1, PL1 on cattle chromosome 23, is a not true homologue of the human gene. This study reveals the largest conserved synteny segment among human, cattle, and mouse autosomes described to date. While all of the genes mapped to cattle chromosome 19 are on human chromosome 17, genes on mouse chromosome 11 are distributed on 7 human chromosomes, supporting the hypothesis that there is greater conservation of synteny between human and bovine chromosomes than between human and mouse. PMID- 7557996 TI - Allele-dependent recombination frequency: homology requirement in meiotic recombination at the hot spot in the mouse major histocompatibility complex. AB - Meiotic recombination break joints in the mouse major histocompatibility complex (MHC) are clustered within short segments known as hot spots. We systematically investigated the requirement for sequence homology between two chromosomes for recombination activity at the hot spot next to the Lmp2 gene. The results indicated that a high rate of recombination required a high degree of similarity of overall genome structure at the hot spot. In particular, the same copy number of repetitive sequences within the hot spot was essential for a high frequency of recombination, suggesting that recombination in mouse meiosis is more sensitive to heterozygous deletion or insertion of DNA than to mismatches of single-base substitutions. PMID- 7557997 TI - 41 kilobases of analyzed sequence from the pseudoautosomal and sex-determining regions of the short arm of the human Y chromosome. AB - Determination of 41.2 kb of Y chromosome genomic sequence has been made from a cosmid that spans the Yp pseudoautosomal boundary and includes 18.5 kb of sequence from the patient-defined sex-determining region of the Y chromosome. An AceDB database of the sequence and the analysis data have been produced as a resource for studies of the evolution and population genetics of the Y chromosome. Comparison of the 18.5 kb from the sex determining region to the sex determining region of mouse does not locate any areas of similarity outside SRY/Sry. Indeed, no coding regions other than those previously reported can be detected anywhere in the 41 kb. The Y-specific and pseudoautosomal portions of this sequence have different repeat sequence and GC contents: this may have relevance both to the events defining the pseudoautosomal boundary and to the course of sequence evolution in the absence of recombination. PMID- 7557998 TI - The structures of the human calcium channel alpha 1 subunit (CACNL1A2) and beta subunit (CACNLB3) genes. AB - Calcium influx in pancreatic beta-cells is regulated mainly by L-type voltage dependent calcium channels (VDCCs) and triggers insulin secretion. The alpha 1 subunit (CACN4) and the beta subunit (beta 3) of VDCCs, both of which are expressed in pancreatic islets, are major components for the VDCC activity, and so they may play a critical role in the regulation of insulin secretion. We have determined the structures of the human CACN4 (CACNL1A2) and the human beta 3 (CACNLB3) genes. The CACNL1A2 gene spans more than 155 kb and has 49 exons. Most of the positions interrupted by introns are well conserved between the CACNL1A2 gene and the previously reported L-type VDCC alpha 1 subunit, CACNL1A1, gene. On the other hand, the CACNLB3 gene distributes in approximately 8 kb and comprises 13 exons, most of which are located together within approximately 5 kb. Comparisons of the genomic sequences of CACNL1A2 with the previously reported cDNA sequences indicate that there are a number of polymorphisms in the human CACNL1A2 gene. In addition, the PCR-SSCP procedure of exon 1 of CACNL1A2 revealed a change from 7 to 8 ATG trinucleotide repeats in a patient with non-insulin dependent diabetes mellitus (NIDDM), resulting in an addition of methionine at the amino-terminus of CACN4. The determination of the structures of the human CACNL1A2 and CACNLB3 genes should facilitate study of the role of these genes in the development of NIDDM and also other genetic diseases such as long QT syndrome. PMID- 7558000 TI - Structure and organization of the human thrombospondin 3 gene (THBS3). AB - The promoter/5' flank sequence, cDNA sequence, and exon/intron structures of the human thrombospondin 3 (THBS3) gene have been determined. THBS3 cDNA clones were obtained by PCR amplification of human fetal lung cDNA using THBS3-specific primers. Analysis of cDNA and genomic sequences showed the THBS3 gene to be composed of 23 exons, 1 more than the number of exons in the previously characterized mouse TSP3 gene. The additional exon results from the division of mouse exon F into exons F1 and F2. The cDNA encodes a polypeptide of 956 amino acids that is highly acidic, with a clustering of acidic side chains in the third quarter of the polypeptide. This region corresponds to seven type III (Ca(2+) binding) repeats, a feature shared with other thrombospondins. In addition to these type III repeats, four type II (EGF-like) repeats and NH2- and COOH terminal domains are present in thrombospondin 3. The THBS3 and mouse TSP3 genes differ in intron sizes, but exon sequences and sizes and positions of insertion of introns are conserved to a high degree. The structural organization of the THBS3 gene is of interest because of its close proximity to that of metaxin, with which it shares a common promoter sequence, and to the gene encoding glucocerebrosidase, a deficiency in which causes Gaucher disease. PMID- 7558001 TI - Cloning and mapping of the U2af1-rs2 gene with a high transmission distortion in interspecific backcross progeny. AB - We have cloned and analyzed the mouse U2af1-rs2 gene based on its sequence similarity to the imprinted gene U2af1-rs1 (SP2). Sequence analysis of this U2af1 rs2 cDNA revealed that it contained an open reading frame encoding a protein of 462 amino acid residues. The predicted amino acid sequence showed 72.7 and 35.8% identity to the U2af1-rs1 and U2 small nuclear ribonucleoprotein auxiliary factor, respectively. Interspecific backcross analysis showed this gene to map to the distal region of the X chromosome and also indicated that there was significant distortion of transmission ratio of the U2af1-rs2 allele in the backcrossed progeny from (C57BL/6J x Mus spretus)F1 females mated to Mus spretus males. PMID- 7557999 TI - Complete sequence of the 43-kb human ribosomal DNA repeat: analysis of the intergenic spacer. AB - We have sequenced the remaining 12.4 kb of the 30-kb human ribosomal DNA intergenic spacer (IGS), which allows us to piece together both a complete IGS and a 43-kb rDNA unit. The sequence of the complete IGS reveals a collection of sequence motifs that can be correlated with functions known or expected to reside in the rDNA repeat: modulation of transcription, recombination, initiation of DNA replication, and chromosomal organization. Finally, we find that IGS accumulates variation at a much higher rate than the transcribed regions. This finding leads us to correlate sequence character with types of mutation and sequence context with rate of mutation. PMID- 7558002 TI - Linkage and candidate gene analysis of X-linked familial exudative vitreoretinopathy. AB - Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disorder characterized by avascularity of the peripheral retina, retinal exudates, tractional detachment, and retinal folds. The disorder is most commonly transmitted as an autosomal dominant trait, but X-linked transmission also occurs. To initiate the process of identifying the gene responsible for the X linked disorder, linkage analysis has been performed with three previously unreported three- or four-generation families. Two-point analysis showed linkage to MAOA (Zmax = 2.1, theta max = 0) and DXS228 (Zmax = 0.5, theta max = 0.11), and this was further confirmed by multipoint analysis with these same markers (Zmax = 2.81 at MAOA), which both lie near the gene causing Norrie disease. Molecular genetic analysis further reveals a missense mutation (R121W) in the third exon of the Norrie's disease gene that perfectly cosegregates with the disease through three generations in one family. This mutation was not detected in the unaffected family members and six normal unrelated controls, suggesting that it is likely to be the pathogenic mutation. Additionally, a polymorphic missense mutation (H127R) was detected in a severely affected patient. PMID- 7558003 TI - Physical mapping of 30 CA repeats on human chromosome 22. AB - We report the physical mapping of 30 microsatellite markers specific for chromosome 22 by PCR amplification of DNA from hybrids that divide the long arm into 27 subregions. This work permits further refining of the genetic linkage ordering previously published. PMID- 7558005 TI - Structure and chromosomal localization of a human water channel (AQP3) gene. AB - A cDNA encoding rat AQP3, a water channel and a member of the MIP family, that is expressed predominantly in kidney medulla and colon was cloned recently. To determine the structure, tissue distribution, and chromosomal localization of the human AQP3 gene, we screened a human kidney cDNA library with rat AQP3 probe and isolated a cDNA coding for human AQP3 protein. The deduced amino acid sequence of human AQP3 was 91% identical to rat AQP3. Human AQP3 mRNA was expressed in colon, kidney, liver, pancreas, lung, peripheral leukocytes, spleen, and prostate. The human AQP3 gene was mapped to 7q36.2-q36.3 by chromosome fluorescence in situ hybridization. PMID- 7558004 TI - CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19. AB - Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic alpha-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses. PMID- 7558006 TI - Mapping of the gene for the Mel1a-melatonin receptor to human chromosome 4 (MTNR1A) and mouse chromosome 8 (Mtnr1a). AB - The pineal hormone melatonin elicits potent circadian and reproductive effects in mammals. We report the chromosomal location of the gene for the Mel1a-melatonin receptor that likely mediates these circadian and reproductive actions. PCR analysis of human-rodent somatic cell hybrids showed that the receptor gene (MTNR1A) maps to human chromosome 4q35.1. An interspecific backcross analysis revealed that the mouse gene (Mtnr1a) maps to the proximal portion of chromosome 8. These loci may be involved in genetically based circadian and neuroendocrine disorders. PMID- 7558007 TI - Precise localization of aphidicolin-induced breakpoints on the short arm of human chromosome 3. AB - The common fragile site at 3p14.2 (FRA3B) has been described as the most active fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. We have previously described induction of breakage around FRA3B using aphidicolin in a somatic cell hybrid whose only human component was a single intact chromosome 3. That work led to the isolation of hybrids with breakpoints in the 3p13-p21.1 region with loss of all sequences distal to their respective breakpoints. In this report we describe the further characterization of the breakpoints in many of these cell lines using newly available molecular markers. We also report the identification of YAC clones that span the breakpoints present in many of these hybrids. PMID- 7558008 TI - beta-1,4-N-Acetylgalactosaminyltransferase involved in ganglioside synthesis: cDNA sequence, expression, and chromosome mapping of the mouse gene. AB - beta-1,4-N-Acetylgalactosaminyltransferase (EC 2.4.1.92; GalNAc-T) is a glycosyltransferase involved in the synthesis of gangliosides GM2 and GD2 as well as glycolipid GA2. We have isolated and sequenced the mouse Gal-NAc-T cDNA, studied GalNAc-T mRNA expression in adult tissues and in embryos, and determined the chromosomal location of the GalNAc-T gene, Ggm2. In comparison with the human cDNA, the mouse sequence was 83 and 87% identical at the nucleic acid and amino acid levels, respectively. The GalNAc-T transcript was most abundantly expressed in brain, liver, lung, spleen, and testis among the eight adult tissues examined. Relatively high levels of expression were seen early in mouse development (7-day embryos) compared to later times (11, 15, and 17 days). The Ggm2 gene was mapped to a distal position on mouse chromosome 10 that is homologous to a portion of human chromosome 12. PMID- 7558011 TI - Cloning and chromosomal localization of the human A2b adenosine receptor gene (ADORA2B) and its pseudogene. AB - To determine the chromosomal localization of the human A2b adenosine receptor, the corresponding genomic clone was isolated and used as a probe for fluorescence in situ hybridization to metaphase chromosomes. Partial sequence analysis of the A2b gene (AD-ORA2B) revealed an intron that interrupted the coding region corresponding to the second intracellular loop similar to that reported for A1 and A2a adenosine receptor genes. A pseudogene for the A2b receptor was also identified; it exhibited 79% identity to the A2b adenosine receptor cDNA coding sequence and contained multiple deletions, point mutations, and frame shifts and two in-frame stops. These changes would result in the inability to encode a functional receptor. The genomic clones were utilized to localize the A2b receptor to chromosome 17p12 and the A2b pseudogene to chromosome 1q32. PMID- 7558009 TI - Mapping of retrotransposon sequences in the unstable region surrounding the spinal muscular atrophy locus in 5q13. AB - The mutation that underlies the autosomal recessive disorder spinal muscular atrophy (SMA) is located on chromosome 5q13. Recent studies show that SMA patients frequently have deletions and rearrangements in this region compared to normal controls. During the isolation of candidate cDNAs for the disease, we identified a sequence that shows high homology to the THE-1 retrotransposon gene family. Using YAC fragmentation techniques, we have refined the localization of this sequence to the domain known to show instability in SMA patients. The implication of these results for the mechanism of the mutation in SMA is discussed. PMID- 7558012 TI - The genes for the alpha-type HC3 (PMSA2) and beta-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization. PMID- 7558013 TI - Assignment of human alpha-synuclein (SNCA) and beta-synuclein (SNCB) genes to chromosomes 4q21 and 5q35. PMID- 7558010 TI - A human homologue of the Drosophila polarity gene frizzled has been identified and mapped to 17q21.1. AB - The frizzled (fz) locus in Drosophila is required for the transmission of polarity signals across the plasma membrane in epidermal cells, as well as to their neighboring cells in the developing wing. The identification of a tissue polarity gene from the fz locus in Drosophila melanogaster has been reported. The fz gene encodes a protein (Fz) with seven putative transmembrane domains, which was suggested to function as a G-protein-coupled receptor. Here we report the identification of a human homologue for the fz gene (FZD2). The FZD2 gene was isolated from a human ovarian cDNA library and mapped to 17q21.1 by fluorescent in situ hybridization (FISH) with a corresponding cosmid. The full-length cDNA of human FZD2 encodes a protein (FZD-2) of 565 amino acids that shares 56% sequence identity with Drosophila Fz. The expression of the FZD2 gene seems to be developmentally regulated, with high levels of expression in fetal kidney and lung and in adult colon and ovary. The structure of FZD-2 suggests that it has a role in transmembrane signal transmission, although its precise physiological function and associated pathways are yet to be determined. PMID- 7558015 TI - Human growth hormone-related iatrogenic Creutzfeldt-Jakob disease: search for a genetic susceptibility by analysis of the PRNP coding region. PMID- 7558014 TI - The HIP116 SNF2/SW12-related transcription factor gene (SNF2L3) is located on human chromosome 3q25.1-q26.1. PMID- 7558016 TI - A heterozygous putative null mutation in ROM1 without a mutation in peripherin/RDS in a family with retinitis pigmentosa. PMID- 7558017 TI - Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene. PMID- 7558019 TI - Integration of the physical and genetic linkage map for human chromosome 13. AB - We have used a panel of somatic cell hybrids containing different rearrangements of human chromosome 13 to integrate genetic and physical maps of this chromosome. The positions of 17 translocation/deletion breakpoints on human chromosome 13 have been determined relative to the microsatellite markers on the genetic linkage map compiled by Genethon. Because markers on maps from several other Consortium groups have also been analyzed using many of the same hybrids, it was possible to relate these with the Genethon map. The position of all of the chromosome breakpoints have been placed, wherever possible, between two adjacent markers on the genetic linkage maps using PCR analysis for the presence/absence of the markers in the somatic cell hybrids. The positions of the breakpoints have already been determined cytogenetically, and some of these breakpoints are located at landmark positions on the chromosome. The relative density of markers along the chromosome differs between independently derived maps, and, based on the known locations of certain breakpoints in the physical map, inconsistencies in the genetic maps have been identified. PMID- 7558018 TI - cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs. AB - We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a subunit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. PMID- 7558021 TI - Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes. AB - Macrophage migration inhibitory factor, MIF, is a cytokine released by T lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5' untranslated region of the cDNA, a 3' poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. PMID- 7558020 TI - Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes. AB - The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human genome contains no pseudogenes; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage. PMID- 7558022 TI - The chromosomal location of the mouse mammary tumor gene Int6 and related pseudogenes in the mouse genome. AB - The Int6 gene is a common insertion site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors. We have determined that this gene is located centromeric of the Myc protooncogene on mouse chromosome 15. In the mouse genome there are several other Int6-reactive restriction fragments that are located on mouse chromosomes 6, 11, 14, 17, and 18. Nucleotide sequence analysis of four of six of these additional Int6 fragments showed that they contain processed Int6 pseudogenes. Comparisons between the Int6 genes of the inbred mouse laboratory strains and the wild mouse species Mus spretus and Mus mus musculus indicate that some pseudogenes were present before divergence of these species and others were acquired since their separation. PMID- 7558023 TI - Construction of a panel of transgenic mice containing a contiguous 2-Mb set of YAC/P1 clones from human chromosome 21q22.2. AB - Libraries of the entire human genome, or regions of the genome, have been made in bacteria, yeast, and somatic cells. We have expanded this strategy using overlapping YACs and P1s from human 21q22.2 (the Down syndrome region) to create a panel of transgenic mice containing DNA that encompasses this region of the human genome. Together the members of the in vivo library, each with a unique transgene (four YACs and four P1s), contain approximately 2 Mb of contiguous DNA. The integrity, stable inheritance, and expression of a coding sequence for each member of the YAC panel are demonstrated, and the uses of the panel are described. PMID- 7558024 TI - Genetic mapping of Cmv1 in the region of mouse chromosome 6 encoding the NK gene complex-associated loci Ly49 and musNKR-P1. AB - The Cmv1 resistance gene controls splenic replication of murine cytomegalovirus (MCMV) and confers natural killer (NK) cell-mediated resistance to otherwise lethal infection. The Cmv1 phenotypes of 13 inbred mouse strains have been assessed, and it was found that the Cmv1r resistance phenotype was restricted to the C57BL/6J and Ma/MyJ strains. We have further analyzed the linkage of Cmv1 to the NK gene complex (NKC) mapping to distal mouse chromosome 6 in 99 (BALB/c x C57BL/6J)F1 x BALB/c backcross mice using cloned gene probes and microsatellite markers from this region. No recombinants were observed between Cmv1 and the NKC associated Ly49 and musNKR-P1 multigene families, nor the Kap locus, nor with 7 microsatellite markers, indicating that Cmv1 is closely linked (< 1 cM) to all of these markers. Analysis of the genotype of the MCMV-susceptible BXD8 RI strain around the NKC region revealed that it had C57BL/6J alleles at microsatellite markers immediately proximal and distal to Cmv1. This suggests that the Cmv1s phenotype of this strain is due to a germ-line mutation. Thus, the close linkage of Cmv1 to the Ly49 and musNKR-P1 multigene families suggests that it may represent an NK cell recognition structure encoded in the NKC region. PMID- 7558025 TI - Genomic structure and nucleotide sequence of the p55 gene of the puffer fish Fugu rubripes. AB - The p55 gene, which codes for a 55-kDa erythrocyte membrane protein, has been cloned and sequenced from the genome of the Japanese puffer fish Fugu rubripes (Fugu). This organism has the smallest recorded vertebrate genome and therefore provides an efficient way to sequence genes at the genomic level. The gene encoding p55 covers 5.5 kb from the beginning to the end of the coding sequence, four to six times smaller than the estimated size of the human gene, and is encoded by 12 exons. The structure of this gene has not been previously elucidated, but from this and other data we would predict a similar or identical structure in mammals. The predicted amino acid sequence of this gene in Fugu, coding for a polypeptide of 467 amino acids, is very similar to that of the human gene with the exception of the first two exons, which differ considerably. The predicted Fugu protein has a molecular weight (52.6 kDa compared with 52.3 kDa) and an isoelectric point very similar to those of human p55. In human, the p55 gene lies in the gene-dense Xq28 region, just 30 kb 3' to the Factor VIII gene, and is estimated to cover 20-30 kb. Its 5' end is associated with a CpG island, although there is no evidence that this is the case in Fugu. The small size of genes in Fugu and the high coding homology that they share with their mammalian equivalents, both in structure and sequence, make this compact vertebrate genome an ideal model for genomic studies. PMID- 7558026 TI - Physical localization of eed: a region of mouse chromosome 7 required for gastrulation. AB - In the mouse, the embryonic ectoderm development (eed) region is defined by deletions encompassing the albino (c) locus of chromosome 7. The region is located 1-2 cM distal to the c locus and was of undetermined size. Embryos homozygous for deletions removing eed display defects in axial organization during gastrulation. Two loci, identified by chemical mutagenesis, are known to map within the eed interval. One, l7Rn5, probably represents the gene required for gastrulation. The second, l7Rn6, is required for survival after birth. fit1, a third locus identified by chemical mutagenesis, maps distal to the eed interval and is also required for survival after birth. A 900-kb YAC contig has been constructed, and deletion breakpoints defining the limits of the regions containing these loci have been localized. Their positions place the eed region within a maximum 150-kb interval at the proximal end of the contig, while fit1 maps to a 360-kb interval within the middle of the contig. Several clusters of rare-cutting restriction sites map within these regions and represent potential locations of candidate genes. PMID- 7558027 TI - Isolation and characterization of rat and human cDNAs encoding a novel putative peroxisomal enoyl-CoA hydratase. AB - We have used a PCR-based subtractive hybridization method to identify upregulated cDNAs in the livers of rats treated with a peroxisome proliferator [clofibrate or di(2-ethylhexyl) phthalate]. After four rounds of subtractive hybridization 62 differentially hybridizing clones were partially sequenced and analyzed by sequence homology searching. Of 62, 49 were identical to 14 different upregulated rat sequences in the databank (mostly genes encoding microsomal or peroxisomal enzymes), 4 of 62 were fragments of three previously unknown genes, and 9 of 62 were false positives. Two of the unknown fragments hybridized to a single novel cDNA that was found to be more than 20-fold induced by both peroxisome proliferators. The 36-kDa predicted protein product of this cDNA shows a high degree of sequence homology to enoyl-CoA hydratases of several different species and has a C-terminal peroxisomal targeting sequence. An epitope-tagged protein product of a full-length cDNA was targeted to peroxisomes in a human cell line. We named this gene, which encodes an apparent peroxisomal enoyl-CoA hydratase, ECH1. We have also identified human ECH1 cDNA and mapped its structural gene to 19q13, 3' to the ryanodine receptor, by hybridization to somatic cell hybrid DNA and chromosome 19-specific cosmid arrays. Possible roles for the ECH1 protein product in peroxisomal beta-oxidation are discussed. PMID- 7558028 TI - Construction of a 3.7-Mb physical map within human chromosome 20p12 ordering 18 markers in the Alagille syndrome locus. AB - Alagille syndrome (AGS, MIM 118450) is associated with human chromosome band 20p12. To study this region, we constructed a 3.7-Mb physical map using 36 YACs isolated from the CEPH YAC library with three sequence-tagged sites (STS): D20S503, D20S41, and D20S188. New STSs were obtained from 6 isolated YAC end fragments. Eighteen markers were ordered on the contig as follows:20ptel-D20S177 D20S175-D20S509- D20S5/D20S503-D20S506-D20S162-D20S504- D20S505-D20S507-D20S188 (D20S6-D20S27- D20S189)-D20S186-D20S41-D20S61-D20S492- D20S508-20pcen. A restriction map with the enzymes AscI, MluI, NotI, SacII, and SfiI was generated, revealing seven putative CpG islands. We established a YAC contig that spans the AGS region and thus will be valuable for cloning candidate genes and searching for DNA polymorphisms segregating with this syndrome. PMID- 7558029 TI - Alternative splicing of the tuberous sclerosis 2 (TSC2) gene in human and mouse tissues. AB - The recently isolated gene for tuberous sclerosis 2 (TSC2) encodes a 5.5-kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. We have detected a novel variant of the TSC2 mRNA lacking 129 nucleotides, predicting an in-frame deletion of 43 amino acids spanning codons 946-988 of tuberin. This 129-bp deletion precisely corresponds to exon 25 of the TSC2 gene suggesting that alternative splicing leads to production of two forms of transcripts designated isoforms 1 and 2. Further molecular analysis revealed a third isoform exhibiting a deletion of 44 amino acids spanning codons 946-989 of tuberin. Amino acid 989 is a Ser residue encoded by the first codon of exon 26. The two isoforms also exist in newborn and adult mouse tissues, reinforcing the potential functional importance of these alternatively spliced products. These alternative isoforms should have implications for efforts aimed at identifying mutations in TSC patients. The distinct polypeptides encoded by the TSC2 gene may have different targets as well as functions involved in the regulation of cell growth. PMID- 7558032 TI - Allelotyping in Wilms tumors identifies a putative third tumor suppressor gene on chromosome 11. AB - An analysis of loss of heterozygosity for markers on both the short and the long arm of chromosome 11 was performed in 24 sporadic Wilms tumors. Six cases (25%) showed allelic losses involving the entire chromosome. In one case (4%) the loss was restricted solely to the WT1 gene on band p13. Two cases (8%) displayed allelic losses for WT1 and for markers on band p15.5, where the putative tumor suppressor gene WT2 has been mapped, but retained heterozygosity for markers on the long arm. In three tumors (13%) the loss of heterozygosity involved markers mapped to chromosomal regions p15.5 and q23.3-qter, but did not affect WT1 and markers on q12-q13. Altogether, the proportion of cases showing allelic losses at the distal region of 11q (37%) was comparable to that of cases with LOH affecting the WT1 (37%) or the WT2 (46%) loci, thus suggesting the existence of a third chromosome 11 tumor suppressor gene involved in the pathogenesis of Wilms tumors. PMID- 7558030 TI - Primary structure of human lumican (keratan sulfate proteoglycan) and localization of the gene (LUM) to chromosome 12q21.3-q22. AB - A human corneal fibroblast cDNA library was screened with a bovine lumican cDNA probe to obtain three clones. Sequencing of the longest clone (1.75 kb) yielded an open reading frame of 1014 bp coding for a 338-amino-acid core protein. Amino acid sequencing of a tryptic peptide resulted in a 9-amino-acid match with the derived primary structure, confirming the identity of these clones. Human lumican displays all of the features of small interstitial proteoglycans: N- and C terminal domains with highly conserved cysteines and a central domain containing nine repeats of slight variations of the leucine motif LXXLXLXXNXL. Like bovine lumican, the human core protein contains four possible N-glycosylation sites in the central domains, all or some of which are substituted with keratan sulfate side chains. At the amino acid level, it is 90% identical with bovine and 72% identical with the chicken core protein. The gene (LUM) was localized to human chromosome 12 by hybridizing a cDNA probe to a Southern blot containing a human/hamster monochromosomal mapping panel DNA. Further sublocalization to 12q21.3-q22 was performed by the fluorescence in situ hybridization technique using a lumican P1 genomic clone. By immunohistochemical staining, we show lumican's presence, not only in the corneal stroma as shown previously, but also in the dermal area of the skin, indicating a wider distribution of this proteoglycan. PMID- 7558033 TI - A radiation hybrid map of 95 STSs spanning human chromosome 13q. AB - We have constructed a high-resolution physical map of the long arm of human chromosome 13 using a panel of 94 radiation hybrids. A comprehensive map of 95 chromosome 13-specific sequence tagged sites (STSs) spanning 13q from the presumed centromere at D13Z1 to the known telomere was obtained by multipoint maximum likelihood statistical methods. The 95 markers have an average retention frequency of 10%, with markers closer to the centromere having much greater retention frequencies (22-49%) than distal 13q markers (2-12%). The most likely radiation hybrid map localized the 95 STSs into 54 unique map positions, 34 with odds of 1000:1 or greater; the comprehensive map localized all but 17 STSs with odds exceeding 10:1. The total map length of 13q was 1302 cR9000 (range 6.4-94.4 cR9000) and a physical distance of 98 Mb, so that 1% breakage in the RH panel corresponds to 75 kb. A comparison of the comprehensive RH map to genetic maps of chromosome 13q shows identical locus orders for the common markers, with two exceptions over 1-cM distances. We discuss the possible relationships between the genetic and the radiation hybrid maps. PMID- 7558031 TI - Comparative genome map of human and cattle. AB - Chromosomal homologies between individual human chromosomes and the bovine karyotype have been established by using a new approach termed Zoo-FISH. Labeled DNA libraries from flow-sorted human chromosomes were used as probes for fluorescence in situ hybridization on cattle chromosomes. All human DNA libraries, except the Y chromosome library, hybridized to one or more cattle chromosomes, identifying and delineating 50 segments of homology, most of them corresponding to the regions of homology as identified by the previous mapping of individual conserved loci. However, Zoo-FISH refines the comparative maps constructed by molecular gene mapping of individual loci by providing information on the boundaries of conserved regions in the absence of obvious cytogenetic homologies of human and bovine chromosomes. It allows study of karyotypic evolution and opens new avenues for genomic analysis by facilitating the extrapolation of results from the human genome initiative. PMID- 7558034 TI - Molecular cloning of a human protein that binds to the retinoblastoma protein and chromosomal mapping. AB - We have isolated distinct clones for cellular proteins that bind to the retinoblastoma protein by direct screening of cDNA expression libraries using purified pRB as a probe. The total nucleotide sequence of one of these clones, RBQ-3, was determined and found to encode a protein of 66 kDa localized in the nucleus. The RBQ-3 preferentially binds to underphosphorylated pRB. The region used for binding to this protein was mapped to the E1A-binding pocket B of pRB, which has sequence similarity to the general transcription factor TFIIB. We have mapped the gene to 1q32 using polymerase chain reaction analysis on a human hamster hybrid cell panel and chromosomal fluorescence in situ hybridization. PMID- 7558035 TI - Human fructose-1,6-bisphosphatase gene (FBP1): exon-intron organization, localization to chromosome bands 9q22.2-q22.3, and mutation screening in subjects with fructose-1,6-bisphosphatase deficiency. AB - Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6 bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6 bisphosphatase deficiency. Four nucleotide substitutions were identified, two of which were silent mutations in the codons for Ala-216 (GCT-->GCC) and Gly-319 (GGG-->GGA). The other substitutions were in intron 3, a C-->T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A-->T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity. PMID- 7558036 TI - Three members of the nitric oxide synthase II gene family (NOS2A, NOS2B, and NOS2C) colocalize to human chromosome 17. AB - Nitric oxide synthases (NOSs) are a family of enzymes responsible for the synthesis of nitric oxide from L-arginine and molecular oxygen. Three human NOS enzymes (I, II, and III) with differing cellular distribution and regulatory mechanisms have been identified. To determine whether additional NOSs are encoded in the human genome, a bovine NOS II-related cDNA was used to screen two human genomic libraries. Clones containing three independent genes were isolated. One clone encoded the previously identified NOS II gene (NOS2A). The two other genes specified amino acids homologous, but not identical, to human NOS II (NOS2B and NOS2C). Southern blot hybridization demonstrated that all three genes are present in the human genome. DNA from human-mouse somatic cell hybrids were used to determine the chromosomal location of the NOS II-related genes. All three NOS II related genes colocalized to human chromosome 17 between bands p13.1 and q25. These observations suggest that there is more than one NOS II-related gene in the human genome. This finding may have important implications for the design of NOS isoform-specific inhibitors. PMID- 7558037 TI - A new family of retroviral long terminal repeat elements in the human genome identified by their homologies to an element 5' to the spider monkey haptoglobin gene. AB - A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire GenBank databank and an element, SPHP, located 5' to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence. PMID- 7558038 TI - Sequence and evolution of the blue cone pigment gene in Old and New World primates. AB - The sequences of the blue cone photopigments in the talapoin monkey (Miopithecus talapoin), an Old World primate, and in the marmoset (Callithrix jacchus), a New World monkey, are presented. Both genes are composed of 5 exons separated by 4 introns. In this respect, they are identical to the human blue gene, and intron sizes are also similar. Based on the level of amino acid identity, both monkey pigments are members of the S branch of pigments. Alignment of these sequences with the human gene requires the insertion/deletion of two separate codons in exon 1. The silent site divergence between these primate blue genes indicates a separation of the Old and New World primate lineages around 43 million years ago. PMID- 7558040 TI - The frequency and position of Alu repeats in cDNAs, as determined by database searching. AB - The Alu repeat sequence is estimated to account for 5% of human genomic DNA. The precise relationship of Alu sequences to human fully spliced cDNA has yet to be determined, although many new protocols for cloning cDNAs either depend on the presence of Alus or--more usually--rely on their absence in a population of messages. Previous estimates of the percentage of fully spliced human transcripts that contain Alu repeats have relied on hybridization procedures. Here we have gone directly to the DNA sequence by extracting over 1600 entries from GenBank that are described as human complete cDNAs, and we have assessed the frequency with which the Alu repeat sequence occurs in these sequences. We find that 5% of fully spliced human cDNAs contain Alu sequences. In addition, we have quantified the appearance of Alus in the different cDNA regions, 5' untranslated region (UTR), coding region, and 3' UTR. The vast majority of Alus are found in the 3' UTR, but 14% lie in the 5' UTR, and very rarely an Alu sequence is present within, or partially within, the coding region of the transcript. PMID- 7558039 TI - A high-resolution interval map of the q21 region of the human X chromosome. AB - In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region. PMID- 7558041 TI - Mapping of the gracile axonal dystrophy (gad) gene to a region between D5Mit197 and D5Mit113 on proximal mouse chromosome 5. AB - The gracile axonal dystrophy (gad) mouse, which shows hereditary sensory ataxia and motor paresis, has been morphologically characterized by the dying back type of axonal degeneration in the nerve terminals of dorsal root ganglion cells and motor neurons. In the present study, using an intraspecific backcross between gad and C57BL/6J mice, the gracile axonal dystrophy (gad) gene was mapped to a region between D5Mit197 and D5Mit113. Estimated distances between gad and D5Mit197 and between gad and D5Mit113 are 0.4 +/- 0.3 and 5.0 +/- 1.0 cM, respectively. The gene order was defined: centromere-D5Mit81-D5Mit233-D5Mit184/D5Mit254- D5Mit256 D5Mit197-gad-D5Mit113-D5Mit7. The mouse map location of the gad locus appears to be in a region homologous to human 4p15-p16. Our present data suggest that the nearest flanking marker D5Mit197 provides a useful anchor for the isolation of the gad gene in a yeast artificial chromosome contig. PMID- 7558042 TI - Coexisting amplifications of the chromosome 1p32 genes (PTPRF and MYCL1) encoding protein tyrosine phosphatase LAR and L-myc in a small cell lung cancer line. PMID- 7558043 TI - Assignment of the human gene encoding heterogeneous nuclear RNA ribonucleoprotein I (PTB) to chromosome 14q23-q24.1. PMID- 7558045 TI - A Crouzon syndrome synonymous mutation activates a 5' splice site within the IIIc exon of the FGFR2 gene. PMID- 7558044 TI - The gene (NFE2L1) for human NRF-1, an activator involved in nuclear-mitochondrial interactions, maps to 7q32. PMID- 7558046 TI - Fine mapping of the human bone morphogenetic protein-4 gene (BMP4) to chromosome 14q22-q23 by in situ hybridization. PMID- 7558047 TI - Molecular cloning, cDNA analysis, and localization of a monomer of the N acetylglucosamine-specific receptor of the thyroid, NAGR1, to chromosome 19p13.3 13.2. PMID- 7558049 TI - HLA antigens in five Amerindian groups (Yuko, Bari, Tunebo, Guane and Paez) of Colombia: results of 'Expedicion Humana'. AB - The serological HLA types (A, B, C and D loci) were studied in five different Indian groups in Colombia. The range of polymorphism was not very restricted in these groups, but there was significant genetic heterogeneity among the five populations in all the HLA loci. The gene frequency data, when converted into a kinship matrix and a two-dimensional eigen vector plot, showed a closer affinity between Bari and Yuko Indians, while Guane, Tunebo and Paez Indians were not only genetically different from the former but also well-differentiated from each other. It seems therefore from this study that geographical proximity may play a greater role than linguistic similarities in the genetic affinities of Colombian Amerindians. PMID- 7558048 TI - Orosomucoid phenotyping with monoclonal antibodies:polymorphic occurrence of ORM1*Q0 in aboriginal Taiwanese populations. AB - Three monoclonal antibodies (OR35, OR40 and OR48) against orosomucoid (ORM) were prepared for the phenotyping of the human ORM system. The OR35 and OR48 antibodies recognized ORM1 and ORM2 products, respectively. OR40 reacted strongly to the products of ORM1 but poorly to those of ORM2. With the help of these monoclonal antibodies, ORM phenotyping was performed on 658 individuals from nine subpopulations of aboriginal Taiwanese, with close attention to two individuals with an ORM1 Q0 homozygous phenotype. The ORM1*Q0 allele was found to be at a polymorphic frequency in eight of the nine subpopulations. PMID- 7558050 TI - Linkage disequilibrium between two loci (5' untranslated exon 1 and intron 5 DdeI) of the antithrombin III gene in three ethnic groups in Singapore. AB - The distribution of two common DNA polymorphisms (5' untranslated exon 1 and intron 5-DdeI) of the antithrombin III (ATIII) gene was studied in three ethnic groups in Singapore: 251 Chinese, 221 Dravidian Indians and 102 Malays. The polymorphisms were identified by the polymerase chain reaction and size fractionation in agarose gels. The 5' untranslated to exon 1 polymorphism is a length polymorphism while the intron 5 polymorphism is a restriction site (DdeI) polymorphism. The frequency of the short fragment (S) of the 5' to exon 1 length polymorphism of the ATIII gene was found to be 0.37 in the Chinese, 0.54 in the Malays and 0.65 in the Dravidian Indians. For the Chinese, this was significantly lower compared to the Caucasians and Indians (p < 0.0001) and the Malays (p < 0.01). On the other hand, the frequencies of DdeI+ did not vary significantly among these three populations (p > 0.05). The distribution of different genotypes at these two loci of the ATIII gene was in Hardy-Weinberg equilibrium in all three ethnic groups. A strong linkage disequilibrium between these two polymorphisms was observed in all the ethnic groups and the estimated correlation coefficient (delta) was 0.42 in the Chinese (p < 0.001), 0.61 in the Dravidian Indians (p < 0.001) and 0.43 in the Malays (p < 0.001). The frequencies of haplotype S+, L+ and L- were, respectively, 0.37, 0.40 and 0.23 in the Chinese, 0.65, 0.18 and 0.16 in the Dravidian Indians and 0.54, 0.37 and 0.09 in the Malays.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558052 TI - Refined genetic mapping of juvenile X-linked retinoschisis. AB - Juvenile X-linked retinoschisis (RS) is an eye disease that causes acuity reduction and peripheral visual field loss typically beginning early in life. In further work towards positional cloning of the RS gene, we restudied our previously reported seven large American families and one additional new family, with a total of 63 affected males. RS linkage analysis using microsatellite repeat markers gave the following results: DXS207 (Z = 24.89, theta = 0.01), DXS987 (Z = 24.04, theta 0.01) and DXS999 (Z = 14.70, theta = 0.00). Recombination events in four individuals were studied further with additional markers (AFM291wf5, DXS443, DXS1052, DXS274 and DXS1226), and a flanking interval was obtained (DXS43, DXS207, DXS987)-RS-(AFM291wf5, DXS443). This study moves the RS centromeric boundary to (AFM291wf5, DXS443), about 5.5 cM closer than the previously reported boundary at DXS274 and narrows the RS inclusion interval to about 3.7 cM (using distances from CEPH family data). PMID- 7558051 TI - An evaluation of FASTMAP with emphasis on fine-mapping. AB - We present an evaluation of the FASTMAP method with special reference to the issue of fine-mapping, using both 'real-life' and constructed data, and demonstrate some of the shortcomings of the method. Since the advantages of FASTMAP in terms of savings in computer resources compared to LINK-MAP are enormous, further improvements of the FASTMAP method would be highly desirable. PMID- 7558053 TI - A survey for the rare blood group antigen variants, En(a-), Gerbich negative and Duffy negative on Espiritu Santo, Vanuatu in the South Pacific. AB - The people of Vanuatu exhibit several different genetic red cell polymorphisms. Some of these, such as alpha thalassaemia, are thought to have reached a high frequency as a result of selection pressure by malaria. In this study three rare blood group antigen variants, En(a-), Gerbich negative and Duffy negative, which are thought to confer a protective effect against malaria were sought in a sample of 214 (187 in the case of Duffy) from Espiritu Santo, Vanuatu. No individuals bearing these rare variants were found. The original settlers in Vanuatu are thought to have migrated from Papua New Guinea some 5,000 years ago, so it is of interest to note that no individuals were found to be Gerbich negative despite a high frequency in Melanesians living on some coastal parts of Papua New Guinea. PMID- 7558054 TI - GC and C3 serum groups in ulcerative colitis. AB - The phenotypes and allele frequencies of two serum protein systems (GC and C3) were studied in 91 consecutive patients with ulcerative colitis and compared with healthy controls. No significant differences were found as far as GC was concerned. However, significant differences were observed in C3: the C3*F allele and C3FS phenotype were more frequent in patients than in controls. PMID- 7558058 TI - Possible genetic heterogeneity in the Roberts-SC phocomelia syndrome. PMID- 7558057 TI - A method for rapid detection of arylsulfatase A pseudodeficiency mutations. AB - Pseudodeficiency of arylsulfatase A is a complicating factor in the determination of metachromatic leukodystrophy risk and carrier status. A method using polymerase chain reaction and restriction enzyme digestion to detect the presence of both the mutations that contribute to arylsulfatase A pseudodeficiency is described using DNA from blood or buccal cells. Application of this technique should facilitate determination of metachromatic leukodystrophy status and counseling in families where the pseudodeficiency allele is present. PMID- 7558056 TI - Spectrum of beta-thalassaemia mutations in the population of Saudi Arabia. AB - A study of beta-thalassaemia alleles in 29 beta-thalassaemia homozygotes and HbS/beta-thalassaemia patients from western Saudi Arabia using the allele specific priming technique of the polymerase chain reaction revealed a total of five mutations. The spectrum of mutations found in the western region is significantly different to that previously reported in the eastern region, and brings the total number of mutations observed in Saudi Arabian nationals to ten. A comparison of the types and frequencies of the Saudi Arabian mutations with those found in neighboring Arab countries is made. PMID- 7558059 TI - A real-time electrical impedance tomograph. AB - Electrical properties of tissues in the human body can be imaged using a technology known as Electrical Impedance Tomography. In this modality, sinusoidal electrical currents are applied to the body using electrodes attached to the skin, and voltages that are developed on the electrodes are measured. Using these data, a reconstruction algorithm computes the conductivity and permittivity distributions within the body. This paper describes the reconstruction algorithm, image display algorithm, and hardware of a real-time Electrical Impedance Tomograph known as the Real-Time Imaging System. The reconstruction algorithm, executed by a commercially available coprocessor board that resides in a 386 based personal computer, is a modification of the Newton's One Step Error Reconstructor (NOSER) that minimizes algorithm execution time by precomputing many quantities. The image display algorithm, also executed by the coprocessor board, maps the output of the reconstruction algorithm into an image which is displayed using a video graphics board. The architecture of the system and execution times of algorithms implemented by the system are discussed. Using the continuous data acquisition mode of the Real-Time Imaging System, data from the thorax of a normal human subject were collected. Admittivity changes in the chest, as a result of respiration and the cardiac cycle, are presented. Data that were collected from the leg of a normal subject are shown which demonstrate capabilities of the triggered data acquisition mode of the system, allowing data acquisition synchronization with an electrocardiogram. PMID- 7558060 TI - A computer-controlled research ventilator for small animals: design and evaluation. AB - The understanding of the mechanical properties of the mammalian respiratory system and how they change under the influence of drugs and in disease are frequently pursued in small animals, since they can be easily obtained in large numbers as pure-bred strains. However, conventional experimental set-ups for studying small animals are generally limited in their ability to measure gas flow into the lungs. In this paper, we present a computer-controlled research ventilator for small animals which can provide conventional mechanical ventilation as well as arbitrary flow perturbations with a bandwidth from 0-55 Hz. Respiratory impedance is estimated from the displacement of the piston and the pressure it generates, thereby obviating the need for a direct flow measurement. The performance of the device was tested on mechanical loads whose impedances were calculated theoretically. The measured and predicted loads agreed within less than 5% up to 30 Hz. Furthermore, the measured impedance of two mechanical loads in series precisely matched the sum of their individual impedances. PMID- 7558061 TI - Curvature-based nonfiducial registration for the Frameless Stereotactic Operating Microscope. AB - The Frameless Stereotactic Operating Microscope permits information from CT and MRI scans to be displayed in the operating microscope in the proper scale and perspective without using a mechanical frame in the process, when the microscope is positioned over the surgical field. This registration is currently done using fiducials that must be present when the imaging scans are taken. A new technique, based on the theory of curvatures, has been developed as an alternative to the use of fiducials for the registration of a patient's head position during surgery with diagnostic images from CT or MRI. Surface curvatures are estimated from a trace of points of the surface of the skin, obtained from both the diagnostic images and an intraoperative nonimaging ultrasonic rangefinder. The point traces need not be identical or evenly spaced. Plots of the resulting curvature fields are compared visually and the alignment determined. Phantom testing, using a human skull, has resulted in a median alignment error of 1.95 mm. Testing using a human subject with clinically obtained data has resulted in alignment errors on the order of 7 mm. PMID- 7558062 TI - Three-dimensional finite element analysis of current density and temperature distributions during radio-frequency ablation. AB - This study analyzed the influence of electrode geometry, tissue-electrode angle, and blood flow on current density and temperature distribution, lesion size, and power requirements during radio-frequency ablation. We used validated three dimensional finite element models to perform these analyses. We found that the use of an electrically insulating layer over the junction between electrode and catheter body reduced the chances of charring and coagulation. The use of a thermistor at the tip of the ablation electrodes did not affect the current density decreased more slowly with distance from the electrode surface. We analyzed the effects of three tissue-electrode angles: 0, 45, and 90 degrees. More power was needed to reach a maximal tissue temperature of 95 degrees C after 120 s when the electrode-tissue angle was 45 degrees. Consequently, the lesions were larger and deeper for a tissue-electrode angle of 45 degrees than for 0 and 90 degrees. The lesion depth, volume, and required power increased with blood flow rate regardless of the tissue-electrode angle. The significant changes in power with the tissue-electrode angle suggest that it is safer and more efficient to ablate using temperature-controlled RF generators. The maximal temperature was reached at locations within the tissue, a fraction of a millimeter away from the electrode surface. These locations did not always coincide with the local current density maxima. The locations of these hottest spots and the difference between their temperature and the temperature read by a sensor placed at the electrode tip changed with blood flow rate and tissue-electrode angle. PMID- 7558063 TI - The feasibility of using ultrasound for cardiac ablation. AB - The feasibility of using ultrasound to induce cardiac tissue necrosis for the treatment of arrhythmias was investigated. A theoretical model was used to optimize the operating frequency for necrosis of highly perfused muscle tissue. From these simulations it appeared that frequencies from 10-15 MHz produce the deepest lesions at ultrasound intensities between 15 and 30 W/cm2. Test catheters with a planar ultrasound transducer (diameter 2.3 mm = 7 F) were also constructed and in vitro and in vivo tests with canine heart muscle were performed. Both of these tests showed that the ultrasound catheters could deliver adequate energy to necrose cardiac tissue. The in vivo lesion depths of 5-9 mm indicated that ultrasound has significant potential for cardiac ablation for the treatment of arrhythmias. PMID- 7558055 TI - Scanning chromosome 17 for psoriasis susceptibility: lack of evidence for a distal 17q locus. AB - Evidence for a genetically heterogeneous psoriasis susceptibility locus on distal human chromosome 17q has recently been reported [Science 1994;264:1141]. Making use of an independently ascertained collection of 24 multiplex psoriasis kindreds, we have performed a genotyping scan of chromosome 17 using 12 microsatellite markers and analyzed the data using parametric (lod score) as well as novel nonparametric methods. Pairwise lod scores revealed no evidence for linkage to the previously implicated marker D17S784 under any of eight models varying in mode of inheritance, penetrance, and sporadic cases. Homogeneous linkage to D17S784 could be excluded under all four autosomal dominant models tested (Z < - 5.8 at theta = 0.05), and there was no evidence for genetic heterogeneity. All other chromosome 17 markers tested also failed to detect evidence for linkage in any of the kindreds under either a dominant or a recessive model. Although further analysis using affected sib pair methods provided no statistically significant evidence for linkage to any chromosome 17 marker, a cluster of three distal 17q loci displayed a trend towards greater than expected allele-sharing values (observed/expected = 1.10-1.14). These results do not formally confirm the existence of a psoriasis susceptibility locus on the distal long arm of human chromosome 17, but are suggestive of its possible involvement under a polygenic model, warranting its further investigation in familial psoriasis. PMID- 7558064 TI - Predicting patterns of epicardial potentials during ventricular fibrillation. AB - Ventricular fibrillation (VF) is a fatal cardiac arrhythmia, characterized by uncoordinated propagation of activation wavefronts in the ventricular myocardium. Short-term predictions of epicardial potential fields during VF in pigs were attempted using linear techniques, and prediction accuracy was measured at various stages during sustained episodes. VF was induced in five pigs via premature electrical stimulation. Unipolar electrograms were recorded from an epicardial array of 506 electrodes in a 22 x 23 array with 1-mm spacing. Optimal spatial basis functions (modes) and time-varying weighting coefficients were found using the Karhunen-Loeve decomposition. Linear autoregressive (AR) models incorporating the dynamics of only a few spatial modes led to predicted patterns that were qualitatively similar to observed patterns. Predictions were made 0.256 s into the future, based on 0.768 s of past data, over an area of approximately 5 cm2 on the ventricular epicardium. The mean squared error of predictions varied from as much as 1.23 to as little as 0.14, normalized to the variance of the actual data. Inconsistency in long-term forcasts is partly due to the limitations of linear AR models. Changes in predictability, however, were consistent. Predictability varied inversely with spatial complexity, as measured by the mean squared error of a five-mode approximation. Predictability also increased significantly during the first minute of VF. PMID- 7558065 TI - Calculation of the signal-to-noise ratio for simple surface coils and arrays of coils. AB - This paper describes a new method to calculate the signal-to-noise ratio (SNR) of MR signals obtained from single receiver coils and arrays of receiver coils. The coils are assumed to be place on the surface of a conducting half-space and the SNR is sample-noise dominated. While in conventional methods line integrals over the electric currents in the coils are chosen to calculate the electric and magnetic fields, this new method uses surface integrals over magnetic dipoles covering the area enclosed by the antenna to derive these fields. Using this method, the SNR for simple circular and square coils was analytically calculated. The calculations show that the theoretical difference in SNR between circular and square antennas is very low. Furthermore, based on the new method, a derivation of the ultimate gain in SNR for arrays of surface coils is presented. The SNR of such an array approaches a limit even if the total number of coils is increased to infinity. This ultimate SNR of a coil array is 35.8% above that of a single circular-shaped, size-optimized and linear polarized coil. PMID- 7558066 TI - A novel electric design for electromagnetic stimulation--the Slinky coil. AB - A novel coil design for inductive electromagnetic stimulation of neural cells has been simulated and experimentally tested. This coil improves the focal effect of a magnetic stimulator, and it reduces its inductance, hence the efficiency of the system is improved. The basic structure of the device is derived from the popular "Slinky" toy. The actual device is formed by winding different numbers of loops forming a helical coil on a half torus. The loops are bunched at the axis of the torus. The coil, due to its geometry, generates a unique distribution of eddy currents in nearby tissues which is favorable compared to a solenoid type stimulator. This renders the Slinky coil more selective than conventional coils used for magnetic stimulation. The distribution of eddy currents was analyzed using Matlab, following Faraday's Law of Induction. Improved focality permits the current through the coil to be reduced for the same effect. In addition, the reduced inductance of the Slinky coil decreases the power requirement; thus, the improved efficiency of the system may allow the generation of bursts of pulses, and expand the utilization of the system to possible functional activation of certain neuro-muscular structures when peripheral nerves are stimulated. PMID- 7558067 TI - Analysis of magnetic stimulation of a concentric axon in a nerve bundle. AB - In this paper, we present an analysis of magnetic stimulation of an axon located at the center of a nerve bundle. A three-dimensional axisymmetric volume conductor model is used to determine the transmembrane potential response along an axon due to induced electric fields produced by a toroidal coil. We evaluate four such models of an axon located in: 1) an isotropic nerve bundle with no perineurium, 2) an anisotropic nerve bundle without a perineurium, 3) an isotropic nerve bundle surrounded by a perineurium, and 4) an anisotropic nerve bundle surrounded by a perineurium. The transmembrane polarization computed along an axon for the above four models is compared to that for an axon located in an infinite homogeneous medium. These calculations indicate that a nerve bundle with no sheath has little effect on the transmembrane potential. However, the presence of a perinerium around the nerve bundle and anisotropy in the bundle significantly affects the shape of the transmembrane response. Therefore, during magnetic stimulation, nerve bundle anisotropy and the presence of perineurium must be taken into account for calculation of stimulus intensities for threshold excitation. PMID- 7558068 TI - Cylindrical tissue model for magnetic field stimulation of neurons: effects of coil geometry. AB - An analytical solution for the electric field induced in a homogeneous cylindrical conductor under quasi-static conditions from current in a coil is used to model peripheral nerve stimulation with magnetic fields. A variety of coil geometries is analyzed in terms of the spatial derivative of the induced electric field along a long straight nerve, parallel to the axis, in a human-arm model. The results of these computations are found to be in general agreement with conclusions regarding the optimization of stimulating coils derived from analyses of the semi-infinite tissue model. The differences and their physical basis are pointed out. PMID- 7558069 TI - Hand-held electronic area planimeter. AB - The focus of this paper is to describe the development and fabrication of a portable hand-held electronic area planimeter to measure wheal areas during dermatitis assays. The planimeter straddles over the wheal and the scientist traces the wheal's perimeter using an optical beam splitter/cross-hair and a miniature x-y table. Transducers connected to the x-y table and an electronic circuit performs the area integration and displays the data on a digital panel meter. The apparatus permits noncontact measurement of areas up to 1000 mm2 with a resolution of 1 mm2. PMID- 7558075 TI - The origins of HLA-A,B,C polymorphism. PMID- 7558073 TI - Quantifying the degree of convergence in primate Mhc-DRB genes. PMID- 7558071 TI - Tissue spectroscopy with electrical impedance tomography: computer simulations. AB - A method is proposed by which bioelectrical spectroscopy could be combined with electrical impedance tomography (EIT) to provide noninvasive characterization of tissue. Multifrequency (2-200 kHz) EIT measurements were simulated with a numerical model for a volume of porcine liver immersed in an electrolytic tank. From the reconstructed EIT images the tissue characterization method was then applied enabling a plot of complex resistivity to be drawn for any selected pixel in the image. Simulations were performed for a small volume of degraded tissue embedded in the normal tissue to examine its effect on the derived spectroscopic parameters. The method could have an application in transplant surgery for screening organs for tissue degradation. PMID- 7558074 TI - Evolution of Mhc class II polymorphism: the rise and fall of class II gene function in primates. AB - The substitution rate at the codons implicated at ARS of Mhc class II genes has previously been shown to be heavily biased towards nonsynonymous substitutions, indicative of positive selection for polymorphism. Based on our analysis of the number of synonymous changes at codons outside putative ARS in primates, the average age of the polymorphism at class II loci was found to increase in the following order: DPB1, DRB3, DRB5, DRB1, DRB4, DQB1, DQA1. For DRB loci, nonsynonymous changes were found to exceed synonymous changes at HLA-DRB1, DRB3 and DRB5, while no evidence of deviations from equal rates of synonymous and nonsynonymous substitutions were found for DRB6. The pattern of substitutions at the DRB loci of most Catarrhini species indicates constant positive selection at ARS codons over the evolutionary period examined. An exception to the relatively stable selection pattern between species exhibited by most loci is the appearance of polymorphism under positive selection at DRB4 only in the regular chimpanzee. The ds/dn ratios for DQA1 and DQB1 alleles are lower than for the most polymorphic DRB genes. Since the dn/ds ratio of ARS codons may be positively correlated to the ds for non-ARS codons, at least for DQB1, caution must be exercised in interpreting the low ratio for the DQ genes as an indication of weaker selection. The DQA1 allelic lineages show different dn/ds ratios, consistent with the hypothesis that the lineages are constrained from evolving in relation to the diversity of the interacting DQB1 alleles. In contrast to all other class II loci, DPB1 appears to have been subjected to strong positive selection only in the human lineage, and may represent the most conspicuous example of an Mhc locus acquiring an altered function in antigen presentation. PMID- 7558070 TI - A video-based system for acquiring biomechanical data synchronized with arbitrary events and activities. AB - A video-based data acquisition and interactive multimedia data extraction system are described for measuring and synchronizing large quantities of biomechanical analog data with arbitrary events and activities. Analog signals from up to 32 channels are digitized, frequency-shift key (FSK) coded, and recorded directly onto the audio tracks of a video tape in synchronization with the video information. The data acquisition system includes an A/D converter that digitizes up to 16 multiplexed channels of 8-b data at a fixed sample rate between 60 and 960 Hz, and an FSK modem that transfers the data onto one of two VHS high fidelity (20 Hz-20 kHz bandwidth) audio tracks. Twenty megabytes of digitized data and time codes, along with associated video and normal audio are contained on a conventional 120-min video tape. An analyst interactively reviews the video tape off-line using a computer-controlled VCR and identifies specific events that divide arbitrary activities into time segments. The computer automatically extracts the biomechanical data corresponding to each time segment for further processing or analysis. This system is useful for ergonomics, gait analysis, sports medicine, sleep laboratory, biomechanics, or any application where complex visual events are synchronized with low-frequency analog data. PMID- 7558077 TI - Exonic MHC-DRB polymorphisms and intronic simple repeat sequences: Janus' faces of DNA sequence evolution. AB - The evolution of highly polymorphic gene loci is following routes that cannot be extrapolated from the existing knowledge of single copy genes. In addition, interpreting the evolution of the most polymorphic loci in vertebrates requires a plethora of data from different taxa. We evaluate here the rules for the evolution of Major Histocompatibility Complex (MHC-)DRB genes recently established in humans and other primates on the basis of sequences from several artiodactyl species. MHC genes encode essential molecules for self/altered self/non-self discrimination in the interaction of the organism with its environment. The necessity to effectively present various different antigens to immunocompetent cells causes positive selection pressure on the variability of these genes in the population. Artiodactyls represent the third mammalian order in which this phenomenon was evidence independently. A further incentive to investigate also the surroundings of MHC-DRB loci was the presence of a particular repetitive sequence stretch in the vicinity of the polymorphic exon- in addition to the evolutionarily old alleles, ancient polymorphisms and the mechanisms for their generation and/or maintenance. Besides their utility for indirect gene diagnosis (MHC-DRB typing), the closely linked stretches of simple repetitive DNA in the neighborhood of the highly polymorphic MHC-DRB genes are also interesting remains of the evolutionary history. Evolutionary development is different in genetically inert intronic DNA compared to the exonic counterparts, despite their close vicinity. The persistence of these simple repeats over nearly 100 million years in one location preserving the same basic motif structure is startling. Indirect evidence is weighed that biological meaning should be considered for these elements. The combined analysis of the polymorphic DRB genes and the (highly variable but persistent) simple repeat stretches deepen our understanding of the complexities within a unique genomic compartment encoding essential molecules for self/non-self differentiation in the interaction of the organism with its environment. PMID- 7558078 TI - MHC diversity and selection. PMID- 7558076 TI - Pathogen-based models favoring MHC genetic diversity. AB - We present six models that are currently the most likely ways that pathogens might favor the evolution of MHC genetic diversity. Although each model makes one or more unique predictions, the current lack of crucial data prevents distinguishing the relative importance of each model. However, this first-time organization of these models should contribute to the design of critical experiments. This synthetic review yields at least three essentially new ideas. First, MHC-dependent immune recognition may be sufficiently redundant to render it essentially escape-proof by pathogens. Second, the four models based on pathogen escape do not work (or work weakly) for diversifying class II genes, unless class II-restricted cytotoxic T-cells are important, an idea that is controversial. Third, pathogen-escape events have traditionally been thought to result in only frequency-dependent selection but here we show that heterozygote advantage is an inevitable consequence of such pathogen evasion. Therefore, the controversy over the relative importance of these two forms of balancing selection is largely a false dichotomy. PMID- 7558072 TI - Evolution of MHC class II allelic diversity. PMID- 7558079 TI - Evolution of class I promoter sequences: relationship to function and diversity. PMID- 7558080 TI - A genetic herd-immunity model for the maintenance of MHC polymorphism. PMID- 7558081 TI - Evolution of major histocompatibility complex polymorphisms and T-cell receptor diversity in primates. PMID- 7558082 TI - Generation of MHC class II diversity by intra- and intergenic recombination. AB - The occurrence and significance of recombination events in the generation of MHC class II diversity were discussed. Evidence that intragenic recombination has contributed to the generation of allelic diversity at a DRB locus in the moose was presented. Intergenic recombination (i.e. exchange of sequence information between nonallelic genes) is expected to be rare and only to play a minor role in the generation of class II diversity. Exchange of sequence information between the major isotypic forms of class II genes (DQ, DR and DP) is restricted to a segment encoding a major part of the a-helical region of polymorphic class II beta-chains. In this segment there is no or only weak locus divergence and the frequency of synonymous substitutions between nonallelic genes (DQB vs. DRB) within species is remarkably low, implying that exchange of sequence information has occurred repeatedly during the course of evolution. PMID- 7558083 TI - A "minimal essential Mhc" and an "unrecognized Mhc": two extremes in selection for polymorphism. AB - The high polymorphism of classical Mhc molecules found in mammals is not simply the result of strong selection for pathogen resistance in the recent past, since there are virtually no examples of diseases caused by infectious pathogens for which resistance is determined by particular Mhc haplotypes, and in the best studied case, a particular aspect of malaria in humans, the selection is remarkably weak. We discuss three possibilities to explain high polymorphism in mammals: accumulating, merging and boosting. The mammalian Mhc is complicated and redundant, so that every Mhc haplotype may give some level of resistance due to multiple classical Mhc genes as well as other disease resistance genes; this frustrates the attempts to demonstrate selection for disease resistance. We have looked at two vertebrate groups that may represent two extreme examples of selection for Mhc polymorphism. Birds, like mammals, have highly a polymorphic Mhc that determines strong allograft rejection. However, chickens have a much smaller, compact and simpler Mhc than mammals, as though the Mhc has been stripped down to the essentials during evolution. The selection on a single Mhc gene should be much stronger than on a large multigene family and, in fact, there are a number of viral diseases for which resistance and susceptibility are determined by particular chicken Mhc haplotypes. We have determined the peptide motifs for the chicken class I molecules from a number of haplotypes, which may explain some disease associations quite simply. On the other hand, salamanders have very low Mhc polymorphism and slow allograft rejection. We have isolated axolotl Mhc molecules and shown that they cosegregate with the locus that determines graft rejection in the axolotl, have only a few alleles and only weakly stimulate axolotl T lymphocytes in mixed lymphocyte culture. We believe that salamanders have classical Mhc molecules but most T cells do not recognize them, so that there is no strong selection for polymorphism. PMID- 7558085 TI - Thrombolysis for acute myocardial infarction: how late is too late? PMID- 7558086 TI - Ventricular tachycardia: prevalence, mechanisms and approaches to diagnosis and management. PMID- 7558087 TI - Late rhythm abnormalities after surgical repair of tetralogy of Fallot: noninvasive and invasive evaluation. AB - Twenty five patients aged 6 to 35 years (mean +/- SD: 14.2 +/- 7.1 years), surgically corrected for tetralogy of Fallot (TOF) more than one year ago, were assessed for rhythm abnormalities by resting electrocardiogram (ECG), twenty-four hours ambulatory ECG monitoring, signal averaged ECG and electrophysiological studies. An X-ray chest for assessing heart size, two dimensional and Doppler echocardiography for residual defects and radionuclide angiography for resting left ventricular ejection fraction were also done. Eight out of 25 (32%) patients were found to have significant rhythm abnormalities. Ventricular arrhythmias were detected in four patients. In two, ventricular tachycardia was induced during electrophysiologic studies, being sustained in one. Multiform ventricular ectopics were observed in two other patients. All these patients were operated, after the age of 10 years, and three of them are presently more than 20 years old. Cardiomegaly was present in the chest X-ray in three patients, and significant residual pulmonary stenosis was seen in one patient. Left ventricular ejection fraction was reduced in two, while signal averaged ECG was positive in one of the three cases subjected to this investigation. Supraventricular tachycardia (SVT) was observed in three patients. This was diagnosed by ambulatory ECG monitoring in all but in addition, was also inducible in one patient on electrophysiologic testing. There was no correlation of the occurrence of SVT with age or age at correction for TOF. None of these patients had any residual defect, cardiomegaly or subnormal left ventricular ejection fraction. Transient complete heart block requiring temporary pacing was documented in one patient with a large residual ventricular septal defect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558084 TI - Class II B Mhc motifs in an evolutionary perspective. PMID- 7558088 TI - Clinical, angiographic and histopathological predictors of restenosis after directional coronary atherectomy. AB - Out of 127 directional coronary atherectomy (DCA) procedures done in our laboratory, there were 81 patients who had completed a minimum of six months of follow-up. To study the factors predisposing restenosis after successful DCA, we analysed the clinical and angiographic profile of the patients and the histopathological findings of the excised tissue in 44 patients whose complete follow-up data was available with us. The indication of DCA was an extremely eccentric significant stenosis located in the proximal or midsegment of a large vessel (> or = 3 mm size) in 97 percent of the cases. Angiographic restenosis, defined as more than 50 percent luminal diameter stenosis was absent in 24 (54.5%, Group A) and present in 20 (45.5%, Group B) patients. On univariate analysis, the factors which predisposed to restenosis were: (i) left anterior descending location, (ii) longer lesion length (9.6 +/- 3.1 mm vs 5.2 +/- 1.6, p < 0.01) and (iii) greater post-procedure residual luminal diameter stenosis (13.1 +/- 10.8% vs 4.3 +/- 6%, p < 0.01). No significant difference was found between the two groups for other variables like unstable angina, the location and the morphological characteristics of the lesion and the ratio of the vessel diameter to the size of the Atherocath. Histopathological examination of the retrieved tissue revealed the presence of media with or without external elastic lamina in 8 (33%) patients in Group A--without restenosis compared to only 1 (5%) patients in Group B--with restenosis (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558089 TI - Congenital heart disease among school children in Shimla hills. AB - A stratified random sample of 15,080 school going children out of 40,950 children in the age group of 5 to 16 years were screened for the prevalence of congenital heart disease and rheumatic fever/rheumatic heart disease. Thirty children were found to be suffering from definite congenital heart disease giving a prevalence of 2.25 per thousand. Atrial septal defect was the commonest lesion (38.2%) with a prevalence of 0.87 per thousand followed by ventricular septal defect (32.2%) with a prevalence of 0.73 per thousand. The prevalence of congenital heart disease was found to be significantly more in female (3.3 per thousand) than in male children (1.4 per thousand) (p < 0.05). Three (8.8%) cases had a family history of congenital heart disease. Rheumatic fever/rheumatic heart disease was found to have a significant prevalence among cases with congenital heart disease (8.8%) as compared to children without congenital heart disease (0.3%) (p < 0.001). The findings suggest the need for screening of family members of those suffering from congenital heart disease and that special attention be paid to the occurrence of rheumatic fever/rheumatic heart disease in cases of congenital heart disease. PMID- 7558092 TI - Significance of ST alternans during percutaneous transluminal coronary angioplasty. AB - Repolarisation ST alternans was observed during percutaneous transluminal coronary angioplasty (PTCA) in nine patients. Six had ST alternans on surface electrocardiogram (ECG) while three patients developed ST alternans only on intracoronary ECG recordings. The pattern of occurrence of ST alternans in successive inflations varied. Ventricular arrhythmias occurred in 6 of 28 (21.42%) of inflations associated with ST alternans while in a control group of 50 patients undergoing PTCA who did not have ST alternans, ventricular arrhythmias were noted in only 6 of 342 inflations (1.75%; p < 0.001). It is suggested that ST alternans is a regional ischemic phenomenon. PMID- 7558091 TI - Association of plasma lipoproteins with angiographically defined coronary artery disease. AB - Lipoprotein (a) and other lipoprotein variables have been correlated with angiographic severity of coronary artery disease. However, pattern of lipoprotein (a) distribution in various racial groups has been found to be different. To study this relationship in the Indian patients, plasma levels of lipoprotein (a) and other lipoprotein variables were examined in 144 consecutive patients undergoing coronary angiography. Total cholesterol and cholesterol content of various lipoproteins were assayed by chemical methods, and in 116 patients lipoprotein (a) was detected by gel electrophoresis. Lipoprotein (a) was positive in a total of 78 (67%) patients, and its positivity in the four angiographic groups with normal coronaries, single vessel, double vessel, and triple vessel disease was 69, 78, 61 and 67 percent respectively. Interestingly, lipoprotein (a) greater than 75 percentile tended to be more frequent in patients with increasing severity of coronary artery disease: 15%, 21%, 25% and 28% in the four groups, respectively. However, this was not statistically significant. Plasma levels of total cholesterol, cholesterol content of other lipoproteins and triglycerides were not significantly different in these four groups of patients. This study, therefore, shows that there is no significant correlation of lipoprotein (a) positivity with angiographic severity of coronary artery disease in our group of Indian patients, although higher values are more frequently present in patients with severe disease. PMID- 7558090 TI - Electrophysiologic effects and therapeutic efficacy of intravenous flecainide for termination of paroxysmal supraventricular tachycardia. AB - Flecainide is a class IC antiarrhythmic agent with a controversial role in the treatment of ventricular arrhythmias following myocardial infarction after the publication of the Cardiac Arrhythmia Suppression Trial (CAST). To assess its utility in paroxysmal supraventricular tachycardia (PSVT), we evaluated the electrophysiologic effects and therapeutic efficacy of intravenous flecainide, administered in a dose of 2 mg per kg body weight in 26 patients of PSVT, studied by programmed electrical stimulation. The patients' age ranged from 18-49 years (mean: 27 +/- 8) and none had organic heart disease. The mechanism of PSVT was atrioventricular nodal reentry (AVNRT) with anterograde conduction through slow pathway and retrograde through fast pathway in 14, and atrioventricular reentry (AVRT) utilizing an accessory pathway in 12 patients. Flecainide was successful in terminating the tachycardia in all (100%) patients of AVNRT and 11 (92%) patients with AVRT. In both the types, the tachycardia was terminated by selective block in conduction through the retrograde limb of the reentry circuit. The drug also produced a complete anterograde block with abolition of preexcitation in 6 out of 8 patients with WPW syndrome. After the drug, the tachycardia was reinducible in one patient of AVNRT and 4 with AVRT. The cycle length of inducible tachycardia increased from 295 +/- 25 ms to 389 +/- 24.5 ms after flecainide (p < 0.001). There were no adverse haemodynamic effects of the drug. Our results, thus, showed that intravenous flecainide is a highly effective and safe antiarrhythmic drug for termination of PSVT mediated by atrioventricular nodal and atrioventricular reentry mechanisms without producing any adverse effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558093 TI - Endomyocardial biopsy causing coronary artery-right ventricular fistula after cardiac transplantation. AB - We present 3 cases of coronary artery-right ventricular fistulae diagnosed on yearly check-ups on 160 cardiac transplant patients between September 1982 and January 1995. The fistula was communicating with the ventricular branch of the right coronary artery, the septal branch of the left anterior descending artery and the distal right coronary artery in the three cases being reported. Histological examination of the endomyocardial biopsy specimen showed the presence of small calibre arteries which were not seen in other cases. Although endomyocardial biopsy carries a risk of less than 0.5 percent, the possibility of causing coronary artery-ventricular fistula should be added to the list of the complications of this procedure. The shunt caused by these fistulae is small and has no hemodynamic significance and usually does not warrant any treatment. PMID- 7558094 TI - Aneurysm of the membranous septum with multiple complications. PMID- 7558095 TI - Multiple renal artery stenting with half (disarticulated) Palmaz-Schatz coronary stents and simultaneous coronary angioplasty. PMID- 7558096 TI - Long Q-T interval and Torsade de Pointes in hypothyroidism. PMID- 7558097 TI - Mitral annular abscess in Salmonella endocarditis: resolution with medical therapy. PMID- 7558099 TI - Coronary angioscopy: exploring new frontiers. PMID- 7558098 TI - Aspergillus terreus, an uncommon fungus causing aortic root abscess and pseudoaneurysm. AB - Aspergillus Terreus is a rare fungus which can cause opportunistic infection in humans. A fifteen year old girl had undergone open aortic valvotomy for aortic stenosis and post-stenotic dilatation of the ascending aorta. She was discharged after an uneventful post-operative period. She was reoperated later for para aortic abscess. The pus on culture yielded pure growth of Aspergillus Terreus from multiple media. To our knowledge, this is the first report from India of Aspergillus Terreus causing human infection. Clinical awareness of the rising importance of fungal infections is alerted. PMID- 7558101 TI - Importance of R wave height in right sided chest leads (V1 and V4R) in cases of mitral stenosis. PMID- 7558100 TI - Should angiotensin converting enzyme inhibitors be routinely used in patients with acute myocardial infarction? PMID- 7558102 TI - Pharmacologic treatment for myocardial infarction: role of aspirin. PMID- 7558104 TI - Therapy for opportunistic fungal infections: past, present and future. AB - The field of antifungal chemotherapy is presently rapidly moving. It began in 1903, with the successful use of potassium iodide (KI). Then there was little progress for 50 years, when in 1951, nystatin was introduced, the first useful polyene. Four years later amphotericin B followed, which is still the historical standard against which new systemic antifungals are compared. Except for the development of flucytosine, there was little progress until the early 1970s and the development of the azole drugs. The present era, which is characterized largely by the modifications of azole drugs, began with ketoconazole and brought agents which can be given orally and have increasing potency, decreasing toxicity and a broader spectrum of activity. Recent studies have examined ways to ameliorate the well-known toxicities of amphotericin B. A new approach has been to complex the drug with lipids or entrap it in liposomes. Itraconazole is a broad-spectrum oral triazole whose greatest advantages over the imidazoles are in its activity against aspergillosis and cryptococcosis, though it is also efficacious against the endemic deep mycoses. Fluconazole is a broad-spectrum triazole. It has been shown to be efficacious in various forms of superficial candidosis, including esophageal disease. We have shown in a randomized, double blind, placebo-controlled study that maintenance therapy can completely prevent thrush in AIDS patients with recurrent thrush and possibly prevent all deep and superficial mycoses. Other studies have shown efficacy in cryptococcal meningitis in AIDS comparable to conventional therapy and with far less toxicity, and also in prevention of relapse of cryptococcal disease. Early diagnosis of fungal infections in cancer patients is problematic.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558105 TI - Cytogenetic and viral studies in nasopharyngeal carcinomas in patients of Indian origin. AB - Four cases of undifferentiated nasopharyngeal carcinomas (NPC) (grade III-IV) in patients of Indian origin were investigated for specific chromosome markers and evidence of Epstein-Barr virus (EBV) positivity. Abnormalities involving chromosome #3, like del (3) (p24-pter) and 3q+(q27-qter) were found in these patients, similar to earlier reports in patients of Chinese and Kenyan origin2,4,13 who however were EBV positive, unlike the patients in this study who were EBV negative. Implications of the cytogenetic and serological data in Indian patients with NPC, available for the first time, may throw some light on the etiology of the disease in this ethnic group where nasopharyngeal carcinoma is also endemic. PMID- 7558106 TI - N-acetyltransferase activity--a susceptibility factor in human bladder carcinogenesis. AB - A higher proportion of slow acetylator phenotypes has previously been found among bladder cancer patients. In the present study carried out among 77 male bladder cancer patients and 80 non-cancer controls, 59.74% of the patients and 35% of the controls were slow acetylator phenotypes (p < 0.01). The odds of developing bladder cancer was also observed to be significantly higher among smokers than non-smokers (p < 0.01). The findings suggest that slow N-acetyl phenotype is a susceptibility factor in bladder carcinogenesis. PMID- 7558103 TI - Utility of pericardial contrast echocardiography in haemorrhagic pericardial effusion. PMID- 7558107 TI - Solitary multilocular cyst of kidney (cystic nephroma). PMID- 7558108 TI - Thymolipoma--a rare mediastinal tumour--report of two cases and review of literature. AB - Thymolipoma is a rare mediastinal tumour. We report two cases of thymolipoma. Our first case, a woman of 60 years, presented with an extremely rare association of myasthenia gravis with thymolipoma. Second case presented with dyspnoea productive cough in a male of 62 years. The relevant literature is briefly reviewed. PMID- 7558109 TI - Segmental renal dysplasia--a case report. AB - A case of segmental renal dysplasia presenting with loin pain, a renal mass and hypertension in a young girl of 16 years is being reported. Clinical and histological features of this rare renal anomaly are discussed. PMID- 7558110 TI - Hepatocellular carcinoma metastatic to bones (Case report with review of literature). AB - A seventy year old man presented with bone pains. Investigation revealed a metastatic bone lesion in the humerus. The primary was found in the liver. The patient achieved good palliation with chemotherapy and radiotherapy and survived for one year. PMID- 7558111 TI - Histopathological and histogenetic study of carcinoma stomach in a high risk area. AB - Seventy gastrectomy specimens of carcinoma-stomach received at the Department of Pathology, Medical College, Calicut were prospectively studied. The carcinomas were classified according to Lauren (1965) and adjacent mucosa was studied for evidence of atrophic gastritis and intestinal metaplasia (IM). The scoring system of Lauren was applied in all cases to analyses histogenesis. In cases with histological evidence of intestinal carcinoma, the adjacent mucosa showed IM, suggesting that "intestinal carcinomas" arise from areas of intestinal metaplasia. However, nine of the histollogically "diffuse mucous cell" carcinomas also showed intestinal metaplasia, and four of them were "intestinal" and others "gastric" when Lauren's scoring was applied. In this study type I and type II metaplasia were seen with a total absence of type III colonic metaplasia, raising the question, whether complete small intestinal type metaplasia also has to be viewed as a pre-malignant condition, in high risk areas. PMID- 7558113 TI - [The safety of immunoglobulin preparations]. PMID- 7558112 TI - Laryngeal squamous carcinoma associated with longstanding localised amyloidosis. A case report. AB - An unusual case of squamous carcinoma arising in the background of longstanding localised tracheolaryngeal amyloidosis is reported and the relevant literature is briefly reviewed. PMID- 7558116 TI - [Polio vaccination today: critical remarks]. AB - Vaccination against poliomyelitis remains an absolutely mandatory measure to prevent resurgence of this dreadful viral infection. Today, however, when the chance to get infected is extremely low, one has to reconsider much more the inherent risk of such a living vaccine which is principally able to induce neurologic disease especially in immunocompromised host the number of which is increasing in our population. Since these attenuated vaccine strains multiply largely in the orointestinal tract of a vaccine, those viruses are shed and easily spread into surroundings so that other persons which are not aware of this event are exposed. But also in normal hosts the vaccine strains are able to produce disease because the genetic mutation leading to reduced virulence is not absolutely stable. Back mutations with increased virulence develop during multiplication in the vaccinee and may threaten the vaccinee as well as contact persons. For the sake of security these consequences should be respected much more. Since a dead vaccine of polioviruses is available, one should much more often profit from this choice. PMID- 7558115 TI - [Efficacy of acellular pertussis vaccines]. AB - Acellular vaccines against pertussis could be developed because various virulence factors of B. pertussis have been characterized. Acellular pertussis vaccines should retain the efficacy but have lower side effects, as compared to the conventional whole-cell vaccine. Lacking any correlate of antibacterial resistance, the efficacy of the vaccines had to be tested in large field trials. Such trials have been conducted and are being conducted in various European and in one African country. These trials used different designs, and various different vaccines were tested. All available efficacy data show that acellular pertussis vaccine can effectively protect against typical pertussis. It also seems probable that the efficacy of vaccines, which contain more than two pertussis components may be better than a vaccine containing pertussis toxoid or pertussis toxoid with filamentous hemagglutinin. A three-component acellular pertussis vaccine has been licensed for use in primary vaccination in infants in Germany in early 1995. PMID- 7558114 TI - [Immunization against tuberculosis: new vaccination strategies or is there an alternative to BCG?]. AB - Consistently high tuberculosis rates in many developing nations, the surprising increase in tuberculosis cases in numerous industrialized countries, together with the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis have sharpened public interest in this ancient scourge. Improved tuberculosis control could best be achieved by an efficacious vaccine. The available attenuated vaccine strain, Mycobacterium bovis BCG, has only limited efficiency. This vaccine is capable of protecting against disseminated miliary tuberculosis in the newborn, but it is unable to prevent stable infection and to cause sterile pathogen eradication. Hence, adult tuberculosis, representing the majority of all tuberculosis cases, is not preventable by BCG vaccination. Due to the extraordinarily high rate of asymptomatic M. tuberculosis infection (1/3 of the total world population) any novel vaccination strategy has to fulfil two major tasks: first, prevention of stable infection, second, eradication of already established infection. T lymphocytes represent the major target for any vaccine strategy, because they serve as central mediators of acquired immunity. They segregate into distinct populations, characterized by different activation conditions and biological functions. These T cell populations do not act independently from, but rather interact with, each other mostly through cytokines. Although CD4 T lymphocytes of T helper 1 type are essential for protection, CD8 T cells expressing cytolytic functions are required, in addition. Perhaps other T cell populations, such as gamma/delta T cells and double negative alpha/beta T cells, also participate. An effective vaccine has to stimulate the precise combination of T cells and cytokines required for the different tasks. It remains to be clarified in how far this can be achieved by a single vaccine. PMID- 7558117 TI - [Streptococcus B screening with GBS medium in obstetrics]. AB - Pregnant women and newborn infants were screened for group B streptococcal (GBS) colonization by obtaining paired swabs from the cervix and urethra for the former group and from the ear, nose, umbilical cord, gastric juice and membranes for the latter. One swab was cultured on blood agar; the other was inoculated into serum starch broth (GBS medium), which allows identification of GBS by production of a characteristic orange-colored pigment. From the 2105 paired swabs obtained, a total of 158 were GBS positive by either method; of these, 154 (97.5%) were recovered by the GBS medium and 89 (56.3%) by blood agar plate. No false positive color reactions were observed with GBS medium. 75% of the positive GBS media could be read within 24 h of incubation. The use of GBS medium proved to be an easy and reliable method for screening of maternal and neonatal GBS colonization. PMID- 7558119 TI - [Immunologic therapy for glomerulonephritis with combined immunoadsorption and IVIG therapy]. AB - Immunoadsorption is an improvement of extracorporeal therapy which differs from plasmapheresis. After plasma perfusion on a special adsorption filter the patient's plasma is reinfused. Meanwhile more than 270 immunoadsorptions have been performed. 7 patients with SLE and glomerulonephritis as well as 3 patients suffering from Wegener's vasculitis and glomerulonephritis had been treated with combined immunoadsorption and IVIG therapy. After three sessions of immunoadsorption (2-3 1 plasma filtrate) the patients received 10 g 7S immunoglobulin infusions on three consecutive days. Patients with SLE and end stage kidney involvement (2 patients) did not show any benefit by the above mentioned treatment regimen. Patients with Wegener's vasculitis and glomerulonephritis showed no significant improvement, except in one of three patients who showed a remarkable decrease in proteinuria. On the other hand, patients with SLE and "moderate" kidney involvement (short course of disease, no fixed structural changes) showed improvement on combined immunoadsorption and IVIG therapy. PMID- 7558118 TI - [Genomic DNA fingerprints of Legionella pneumophila serogroup 2 strains as an epidemiologic marker]. AB - Using pulsed-field gel electrophoresis, DNA fingerprints of eleven Legionella pneumophila isolates of serogroup 2 were generated. It was shown that two strains from a patient suffering from pneumonia as well as three environmental strains isolated from the shower in the hotel where the patient stayed 5 days before his illness were identical. Six strains of the same serogroup isolated from other sources were clearly separated. Thus, DNA fingerprints by pulsed-field gel electrophoresis are excellent epidemiological markers for the rarely occurring serogroup 2 of Legionella pneumophila. PMID- 7558120 TI - The induction of respiratory syncytial virus-specific cytotoxic T-cell responses following immunization with a synthetic peptide containing a fusion peptide linked to a cytotoxic T lymphocyte epitope. AB - Previously published work has shown that a cytotoxic T-cell epitope (CTL) representing residues 82-90 of the M2 protein of respiratory syncytial virus (RSV) is the target for a protective response against the virus. In this report, we demonstrate that a synthetic peptide representing residues 81-95, when covalently linked to a fusion peptide derived from the conserved N-terminal 19 residues of the F1 protein of measles virus efficiently primes RSV-specific CTLs in vivo following immunization without adjuvant. PMID- 7558123 TI - Cytokine (IL-8, IL-6, TNF-alpha) and soluble TNF receptor-I release from human peripheral blood mononuclear cells after respiratory syncytial virus infection. AB - During the initial phase of respiratory syncytial virus (RSV) infection, when a low virus-cell ratio is most probable, signs of inflammation are detectable in the infected respiratory tissue. Therefore we analysed the release of the proinflammatory cytokines interleukin-6 (IL-6), IL-8, tumour necrosis factor alpha (TNF-alpha), and the soluble form of the TNF receptor-I (sTNFR-I), from peripheral blood mononuclear cells (PBMC) after exposure to low infectious RSV doses (multiplicity of infection, MOI, 0.001-1) and incubation times of up to 24 hr. The PBMC secreted IL-8 in a time- and virus dose-dependent fashion. As was verified by Northern blot analysis, the increased IL-8 secretion rate was accompanied by an enhanced IL-8 mRNA steady-state level. The infection of the PBMC after 4 hr post-RSV exposure was verified by detection of RSVSH genomic RNA and mRNA after reverse transcription and polymerase chain reaction (PCR) amplification. In addition, after 24 hr post-infection we determined the percentage of infected cells by specific immunofluorescence using monoclonal antibodies directed against the F- and G-proteins. After exposure of PBMC to inactivated RSV, we observed only RSVSH genomic RNA and a reduced IL-8 release. Thus, even the binding and/or phagocytosis of RSV by PBMC induced an IL-8 synthesis to some extent. Following an incubation time of 24 hr, PBMC exposed to small RSV doses synthesized and released high amounts of IL-6 into the cell supernatant. In contrast, only low amounts of TNF-alpha were released from PBMC. In addition to the release of the proinflammatory cytokines, an enhanced level of the sTNFR-I was measured in the cell supernatants at a MOI of 0.1. However, there was no correlation between TNFR-I membrane expression and cell supernatant concentration. Co-culture experiments performed with PBMC and human epithelial cells (A549) revealed that the enhanced IL-8 secretion profile observed in the coculture was partially dependent on the cytokines TNF-alpha, IL-1 beta and TNF beta/lymphotoxin released by the cells themselves. PMID- 7558122 TI - Interleukin-4 enhances interferon-gamma synthesis but inhibits development of interferon-gamma-producing cells. AB - Interleukin-4 (IL-4) is antagonistic for many of the activities of interferon gamma (IFN-gamma) and, as well as suppressing the development of T-helper type-1 (Th1) cells, has been reported to block directly the synthesis of IFN-gamma in human lymphocytes. However, IL-4 transgenic mice produce increased amounts of IFN gamma as well as IL-4. We have compared the ability of rat IL-4 to regulate IFN gamma secretion in short-term cultures of spleen cells with its effect on the differentiation of T lymphocytes into IFN-gamma-producing, or Th1-type, cells. Normal rat spleen cells were stimulated using a variety of mitogens and ovalbumin antigen, with or without IL-4, for 12-24 hr and the levels of IFN-gamma in the supernatants measured by enzyme-linked immunosorbent assay (ELISA). The results show that when normal rat splenocytes were stimulated with phytohaemagglutinin (PHA) or concavalin A (Con A), IL-4 enhanced secretion of IFN-gamma after 12-24 hr. This enhancement was also apparent when splenocytes from animals immunized 10 days previously with alum-precipitated ovalbumin were stimulated with ovalbumin in vitro, and appeared to be mediated primarily via CD+ T cells. In contrast, when spleen cells were maximally stimulated with phorbol myristate acetate (PMA) and ionomycin, addition of IL-4 had no effect on the amount of IFN-gamma secreted. When splenocytes were stimulated with Con A for 4 days in the presence of IL-4, and restimulated with PMA and ionomycin, IFN-gamma secretion was greatly suppressed. Our results indicate that IL-4 exerts differential effects on IFN gamma secretion and on the development of IFN-gamma-producing lymphocytes. PMID- 7558121 TI - IL-10 and IL-3 synergize to cause proliferation of human T cells. AB - Interleukin-3 (IL-3) is a well-documented haemopoietic growth factor, but its effects on T-cell function are less clear. Although this cytokine is a potent growth factor for CD4- CD8- alpha beta T-cell receptor-expressing cells, it has few known effects on single-positive T cells. In this report it is shown that IL 3 synergizes with IL-10 to induced proliferation of T-cell lines and single positive clones. Specificity was verified using blocking anti-IL-10 and anti-IL-3 neutralizing antibodies. This induction of proliferation was dose dependent. Taken together the results imply that IL-3 can act as a growth factor for T-cell lines and single-positive T-helper type-1 (Th1) CD4+ clones in the presence of a cofactor, IL-10, a cytokine that has been documented as being predominantly a T cell inhibitory, and not proinflammatory, cytokine. PMID- 7558124 TI - Allergen-stimulated interleukin-4 and interferon-gamma production in primary culture: responses of subjects with allergic rhinitis and normal controls. AB - The balance of interleukin-4 (IL-4) to interferon-gamma (IFN-gamma) production that is induced following exposure to common environmental antigens is believed to be instrumental in determining whether hypersensitivity or clinical unresponsiveness results to that antigen. To date, evaluation of cytokine (protein) production has been based predominantly on allergen-reactive CD4 T-cell clones or activation of fresh unselected peripheral blood mononuclear cell (PBMC) populations with non-physiological stimuli such as phorbal myristate acetate (PMA) and calcium ionophore, phytohaemagglutinin (PHA), anti-CD3 or anti-CD2/anti CD28 monoclonal antibodies (mAb). Here, ultrasensitive IL-4 and IFN-gamma assays were optimized to allow direct analysis of antigen-stimulated cytokine production by fresh human PBMC. Primary cultures of cells from grass pollen-sensitive allergic rhinitis subjects and non-atopic controls were stimulated using a range of grass pollen allergen concentrations in the absence of exogenous cytokines or polyclonal activators. The majority of subjects (45 of 52) exhibited chloroquine sensitive, CD4-dependent cytokine production in allergen-stimulated, short-term primary culture. Median IL-4 production was substantially greater among atopics (13.0 pg/ml versus < 1 pg/ml, Mann-Whitney U test, P < 0.0000001) and IFN-gamma was lower (P = 0.008), providing direct evidence for an imbalance in both IL-4 and IFN-gamma production among circulating, pollen-reactive cells in individuals with seasonal allergic rhinitis. The distinction in the allergen-driven cytokine responses elicited from normal and atopic donors was underscored by examination of the ratios of IFN-gamma:IL-4 synthesis. Non-atopic individuals exhibited intense IFN-gamma dominance of the T-cell response, in marked contrast to that observed among grass pollen-sensitive individuals (median IFN-gamma:IL-4 ratios of 14.0 versus 0.096, P = 0.000002). The observation that essentially all individuals produced IFN-gamma (+/- IL-4) following antigen stimulation in vitro argues that the most relevant consideration in determining susceptibility to immediate hypersensitivity versus clinical tolerance to environmental allergens is not a genetically defined capacity to recognize the antigen (i.e. if allergen reactive T cells are present in that individual) but the nature of the cytokine response. PMID- 7558125 TI - Effects of in vivo administration of anti-IL-10 monoclonal antibody on the host defence mechanism against murine Salmonella infection. AB - Interleukin-10 (IL-10) is a cytokine that regulates various macrophage functions. To elucidate the involvement of endogenous IL-10 in the early stage of murine salmonellosis, we examined the effect of anti-IL-10 monoclonal antibody (mAb) administration on the host defence mechanism against Salmonella choleraesuis infection. The in vivo administration of anti-IL-10 mAb significantly enhanced host resistance at the early stage of Salmonella infection, as assessed by bacterial growth in the peritoneal cavity and the liver. Enhanced levels of monokine mRNA, including IL-1 alpha, tumour necrosis factor-alpha (TNF-alpha) and IL-12, were observed from day 1 after infection in the peritoneal macrophages in anti-IL-10 mAb-treated mice compared with those in control mAb-treated mice. Mice treated with anti-IL-10 mAb exhibited significantly higher levels of interferon gamma (IFN-gamma) in the peritoneal exudates and major histocompatibility complex (MHC) class II expression on the peritoneal macrophages on days 3 and 5 after infection. Notably, in vivo anti-IL-10 mAb brought about an increment of gamma delta T cells in the peritoneal cavity at the early phase of infection, which was correlated with the expression of endogenous heat-shock protein 60 (HSP60), which is implicated as a potential ligand for gamma delta T cells, in the infected macrophages. Our results suggest that the neutralization of endogenous IL-10 accelerates some macrophage functions and, consequently, the activation of immunocompetent cells, including gamma delta T cells, at the early stage of infection, resulting in an enhanced host defence against Salmonella infection. PMID- 7558126 TI - Immunoregulatory activities of eicosanoids in the rainbow trout (Oncorhynchus mykiss). AB - Eicosanoids such as prostaglandins (PG), leukotrienes (LT) and lipoxins (LX) have been shown to be potent immunoregulatory molecules in mammals. To determine if they have similar roles in 'lower' animals, rainbow trout (Oncorhynchus mykiss) were immunized with either sheep erythrocytes or Aeromonas salmonicida in the presence or absence of the stable analogue of PGE2, 16,16-dimethyl-PGE2, and the number of plaque-forming cells (PFC) or specific antibody levels determined. The higher dose of 16,16-dimethyl-PGE2 (200 micrograms/kg body weight) caused a significant reduction in both PFC number and antibody titre compared with the control. The effect of PGE2, PGE3, 16,16-dimethyl-PGE2, LTB4, LTB5, LXA4, 12-HETE and 12-HEPE on PFC generation following the in vitro challenge of trout splenocytes with sheep erythrocytes was also determined. All of the prostaglandins tested showed a dose-dependent inhibition of PFC after 11 days in culture, while of the remaining eicosanoids only LXA4 had any effect on PFC number, with a dose-dependent stimulatory effect. The cyclo-oxygenase inhibitor, indomethacin, also caused a stimulation in the number of PFC generated, with a maximal effect at c. 25 microM, while the lipooxygenase inhibitors, esculetin and nordihydroguaiaretic acid (5-100 microM), had no significant effect on PFC generation at all concentrations tested. The present results show that, as in mammals, prostaglandins and the cyclo-oxygenase pathway are also important in the regulation of the piscine humoral immune response. Of the lipoxygenase products tested, however, only LXA4 had any significant effect on PFC generation, suggesting that these compounds have only a limited role to play in immune regulation in this organism. Overall this work shows that eicosanoids have a long evolutionary history in immunoregulation, probably dating back at least to the appearance of bony fish some 400 million years ago. PMID- 7558127 TI - Expression of functional insulin-like growth factor-1 receptor on lymphoid cell subsets of rats. AB - It has become evident that insulin-like growth factor-1 (IGF-1) acts as a growth factor for immune cells, yet the precise regulatory role of IGF-1 in the immune system is unknown. The aim of this study was to examine the distribution of IGF-1 receptors on rat lymphoid cells. A flow cytometric method was used, with a biotinylated and functionally active IGF-1 analogue, namely des(1-3)IGF-1, which binds well to IGF-1 receptor but poorly to IGF binding proteins, followed by phycoerythrin-conjugated streptavidin (PE-SA) staining. Our results showed that IGF-1 receptors were readily detectable on a wide variety of the immune cells, including T cells, B cells and monocytes, but the binding capacity for IGF-1 was monocytes > B cells > T cells, as determined by titration experiments. Furthermore, the level of expression on resting CD4+ T lymphocytes was greater than on CD8+ cells, and the concentration of biotin-des(1-3)IGF-1 required to demonstrate the binding to IGF-1 receptor on CD8+ cells (68 nmol/l) was 200-fold higher than for CD4+ cells (0.34 nmol/l), indicating that most of the IGF-1 receptor on CD8+ cells represented lower affinity sites. The level of IGF-1 receptor expression was increased several-fold after concanavalin A stimulation on both CD4+ and CD8+ T-cell subsets. Kinetic analysis of the expression of IGF-1 receptor and its association with interleukin-2 receptors (IL-2R) following activation showed a similar pattern, with no significant differences in the ratio of IGF-1 receptor: IL-2R per cell during the 3 days of cell culture. Our studies suggest that biological activities of IGF-1 include direct stimulation of immune cells, and that expression of IGF-1 receptor may have a role in regulation of T cell function. PMID- 7558128 TI - Transforming growth factor-beta 1 primes macrophages for enhanced expression of the nitric oxide synthase gene for nitric oxide-dependent cytotoxicity against Entamoeba histolytica. AB - Nitric oxide (NO) produced by activated macrophages is the major cytotoxic molecule for in vitro cytotoxicity against Entamoeba histolytica trophozoites. Transforming growth factor-beta 1 (TGF-beta 1) is a potent negative regulator of several macrophage functions, including NO production. In this study, we investigated the effect of TGF-beta 1 on macrophage nitric oxide synthase (mac NOS) mRNA expression and NO production for macrophage cytotoxicity against E. histolytica trophozoites. TGF-beta 1 by itself was incapable of inducing mouse bone marrow-derived macrophage (BMM) amoebicidal activity and NO production (as measured by nitrite). In contrast, TGF-beta 1 pretreatment (4 hr) primed BMM for an enhanced amoebicidal activity of 15% and 23% in response to (interferon-gamma) IFN-gamma+tumour necrosis factor-alpha (TNF-alpha) or IFN gamma+lipopolysaccharide LPS, concomitant with increased NO production of 85% and 27%, respectively. TGF-beta 1 pretreatment increased NO production in response to IFN-gamma+TNF-alpha/LPS stimulation in a time- and dose-dependent manner. By Northern blot analysis, the increased NO production of TGF-beta 1-pretreated BMM was preceded by markedly enhanced expression of mac-NOS mRNA. The priming effect of TGF-beta 1 on NO production was critically dependent on both a TNF-alpha (> or = 100 U) and a LPS (> or = 100 ng) triggering dose in the presence of IFN-gamma. TGF-beta 1 pretreatment enhanced TNF-alpha mRNA expression, but had no effect on TNF-alpha production in culture supernatants after 4 hr of stimulation with IFN gamma+TNF-alpha/LPS; however, at a later time-point (16-48 hr), even though the levels of TNF-alpha mRNA expression were unaffected, TNF-alpha production was reduced. These data demonstrate that TGF-beta 1 priming for increased mac-NOS mRNA expression for NO-dependent cytotoxicity against E. histolytica in response to IFN-gamma+TNF-alpha/LPS stimulation may be involved in the modulation of a TNF alpha triggering signal by TGF-beta 1. PMID- 7558129 TI - Dynamics of the intracerebral and splenic cytokine mRNA production in Toxoplasma gondii-resistant and -susceptible congenic strains of mice. AB - Oral infection with a low-virulence strain of Toxoplasma gondii (Tg) induced a persisting encephalitis in resistant strains of mice. In the present study we have examined transcripts of various cytokines during acute and chronic stages of murine Tg encephalitis. In the brain of infected animals, interferon-gamma (IFN gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL 10 and IL-12 mRNA were induced to a significant extent, but only low levels of IL 4 mRNA were detectable. A similar cytokine profile was observed in the spleen. However, in contrast to the brain, the increase of IL-2 mRNA was particularly pronounced in the spleen, whereas the opposite was found for IFN-gamma and TNF alpha mRNA. Thus, cytokines involved in T-cell proliferation were more prevalent in the spleen, but in the brain, where Tg actively multiplies, the effector molecules IFN-gamma and TNF-alpha were preferentially up-regulated. In addition, a detailed analysis of cytokine mRNA levels in major histocompatibility complex (MHC)-congenic strains of BALB and B10 mice revealed that the genetically regulated susceptibility to Tg was correlated with the amount of intracerebrally produced cytokine mRNA for IFN-gamma, TNF-alpha and IL-6. Mice with a strong increase of these cytokine mRNA were significantly better protected against Tg. This indicates that the outcome of toxoplasmosis may be critically dependent on an adequately regulated intracerebral immune response. PMID- 7558132 TI - CD40-activated human naive surface IgD+ B cells produce IgG2 in response to activated T-cell supernatant. AB - Human IgG2 is an isotype associated with immune responses to carbohydrates. While interleukin-10 (IL-10) induced CD40-activated naive surface (s)IgD+ human B cells to secrete IgG1 and IgG3, none of 20 recombinant cytokines tested alone, or in combination with IL-10, was able to induce these cells to produce IgG2. This was not due to a specific inability of these sIgD+ B cells, as they could be induced to secrete microgram amounts of IgG2, as well as the three other IgG subclasses, when cultured with an anti-CD3-activated CD4+ T-cell clone. The supernatant of this activated CD4+ T-cell clone contained a soluble factor(s) able to induce the secretion of IgG2 by CD40-activated sIgD+ B cells. Following activation, blood T cells also produced a factor(s) inducing CD40-activated naive B cells to secrete IgG2. This CD4+ T-cell clone will thus permit us ultimately to define the presently uncharacterized cytokine(s) inducing naive B cells to secrete IgG2. This will provide a new insight for the study of immunodeficiencies involving a selective defect in IgG2. PMID- 7558133 TI - Increased number of CD4-CD8+ MHC class II-specific T cells in MHC class II deficient mice. AB - Targeted disruption of the A beta-encoding gene of H2b mice abolishes major histocompatibility complex (MHC) class II expression and results in a failure to develop CD4+8- T cells. Besides this major effect, the lack of class II expression affects the level of T-cell receptor (TCR) and CD4 expression on differentiating thymocytes. Moreover, there is no class II-mediated negative selection of thymocytes. All this could result in TCR repertoire changes of the CD4-8+ T-cell subpopulation, which apparently develops normally in these mice. To test this hypothesis, the class II reactivity of CD4-8+ T cells from class II deficient (class II0) mice was analysed. It was found that CD4-8+ T cells from class II0 but not from class II-expressing mice developed a significant level of cytotoxicity against class II-expressing target cells. These results demonstrate an influence of MHC class II molecules on the TCR repertoire of CD4-8+ T cells. PMID- 7558131 TI - Canine IgE monoclonal antibody specific for a filarial antigen: production by a canine x murine heterohybridoma using B cells from a clinically affected lymph node. AB - Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nematodes demonstrated binding of the canine IgE monoclonal antibody to a single 35,000 MW peptide from B. pahangi but not Dirofilaria immitis; immunochemistry using frozen sections of adult worms, microfilariae and fourth stage larvae revealed focal binding of the monoclonal IgE to worm tissue adjacent to dorsal and ventral cords of only Brugia adults. PMID- 7558134 TI - The effects of changes at peptide residues contacting MHC class II T-cell receptor on antigen recognition and human Th0 cell effector function. AB - Cytokines can influence the selection of functional subsets (Th1 or Th2) of CD4+ T cells. However, quantitative changes in affinity of peptide/major histocompatibility complex (MHC) class II/T-cell receptor (TCR) interactions may alter antigen density and modulate T-cell effector function. The possibility exists to use peptide analogues to induce a partial signal to dissociate production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) by T-helper type-0 (Th0) cells and, consequently, to regulate T-cell function. Based on binding assays and resolution of the crystalline structure of an influenza virus haemagglutinin peptide (HA 306-318) bound to the human MHC class II molecule DRB1*0101, we synthesized HA peptide analogues with amino acid substitutions predicted to modify either MHC class II/peptide density or TCR/peptide interactions. When we examined their antigenicity using cloned human Th0 cells, the analogues, in general, elicited a gradation in potency reflected by a reduction in both proliferation and cytokine production (IL-2, IL-4 and IFN gamma). Although the analogue HA-R309 diminished IL-2 production, none of the analogues tested could selectively induce only IL-4 or IFN-gamma. Since, in general, the effector functions of the Th0 cells examined here were resistant to selective manipulation by the peptide analogues, this suggests that for some clones of chronically activated T cells modulation of selected functions may be difficult to achieve. PMID- 7558135 TI - Induction of diabetes with islet-specific T-cell clones is age dependent. AB - To investigate the effects of recipient age on the induction of diabetes by CD4+ islet-specific T-cell clones, we tested four different clones in non-obese diabetic (NOD) mice of different ages. Transfer of each of the T-cell clones resulted in insulitis and full development of diabetes in unirradiated 8-18-day old NOD mice within 2-3 weeks. A small gender bias in disease susceptibility was seen in 8-14-day-old female NOD mice, which showed a twofold increase in disease incidence over both age-matched males and slightly older (15-18-day-old) females. In contrast, both male and female NOD mice, 19 days or older, were highly resistant to T-cell clone-induced insulitis and completely resistant to the development of diabetes. In mice of an intermediate age range (15-18 days old), two of the clones showed a three- to fourfold reduction in transfer of overt diabetes regardless of gender. These results suggest that an important developmental event occurs in both male and female NOD mice between the age of 15 and 19 days, leading to the inhibition of disease induced by T-cell clones. PMID- 7558130 TI - Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis. AB - Control of resistance to cyst burden following per-oral infection with Toxoplasma gondii has been mapped previously to a region of mouse chromosome 17 of approximately 140 kb. This region is contiguous with and contains the class I gene, Ld. Resistance to development of toxoplasmic encephalitis has also been reported to be controlled by genes in this region of H-2. TNF-alpha, D and L genes, as well as unidentified genes, are also in this region. The work described here was performed to identify definitively the gene(s) in this 140 kb region that confers resistance to cysts and encephalitis. The study demonstrates that relative resistance to T. gondii organisms and cyst burden in brain, and toxoplasmic encephalitis, 30 days following per-oral T. gondii infection is correlated absolutely with the presence of the Ld gene in inbred, recombinant, mutant and C3H.Ld transgenic mice. Mice with the Ld gene had lower cyst burdens and less encephalitis than those without the Ld gene. Specifically, 30 days after infection mice with the Ld gene had minimal perivascular inflammation and meningeal inflammation and very few Toxoplasma cysts or organisms in immunoperoxidase-stained preparations of their brains. Mice without the Ld gene had a similar pattern of inflammation, but in addition they had collections of inflammatory cells in the brain parenchyma. Free tachyzoites were found within these foci of inflammation and cysts were present in these areas as well as in contiguous areas without inflammatory cells. There were CD4+ and CD8+ T lymphocytes in the areas of inflammation and throughout the brain parenchyma. Mice that were resistant to cysts and encephalitis had little detectable brain cytokine mRNA expression, while mice that were susceptible had elevated levels of mRNA for a wide range of cytokines, consistent with their greater amounts of inflammation. The present work definitively demonstrates that a Ld-restricted response decreases the number of organisms and cysts within the brain and thereby limits toxoplasmic encephalitis and levels of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL-10, transforming growth factor-beta (TGF-beta), IL-1 alpha, IL1 beta and macrophage inhibiting protein (MIP) mRNA in the brain 30 days after per-oral infection. PMID- 7558136 TI - Synovial fibroblasts as accessory cells for staphylococcal enterotoxin-mediated T cell activation. AB - Rheumatoid arthritis (RA) is thought to be the result of T-cell-mediated autoimmune phenomena. So far, a critical autoantigen has not been identified. Recently, superantigens have been implied in the pathogenesis of RA. In the present study it was tested whether major histocompatibility complex (MHC) class II-positive synovial fibroblast cells (SFC) function as superantigen-presenting cells. SFC were stimulated with interferon-gamma (IFN-gamma) to express class II antigens; then they were cultivated in the presence of T cells with or without staphylococcal enterotoxins (SE). T-cell activation was measured as proliferation and interleukin-2 (IL-2) production. Depending on the dose and type of SE, activation of T-cell clones and also of peripheral T cells was seen. T-cell activation was inhibited by antibodies to MHC class II antigens and also by antibodies to intracellular adhesion molecule type-1 (ICAM-1). The data suggest that class II-positive SFC have the capacity to serve as accessory cells for superantigen-mediated T-cell activation. Thus SFC may participate in the propagation of a T-cell dependent immune response. PMID- 7558139 TI - Polymorphonuclear leucocyte migration through human dermal fibroblast monolayers is dependent on both beta 2-integrin (CD11/CD18) and beta 1-integrin (CD29) mechanisms. AB - Accumulation of leucocytes in inflammation involves their migration through vascular endothelium and then in the connective tissue. We investigated human polymorphonuclear leucocyte (PMNL) migration through a biological barrier of human dermal fibroblasts grown on microporous filters, as a model of PMNL migration in the connective tissue. PMNL did not migrate through a fibroblast monolayer unless a chemotactic factor, e.g. C5a, interleukin-8 (IL-8) or zymosan activated plasma (ZAP; C5adesArg), was added. This migration was partially inhibited (35-70%, depending on the stimulus) by treatment of PMNL with monoclonal antibody (mAb) to CD18 (beta 2-integrins). Most of the CD18 independent migration was inhibited by mAb to beta 1-integrins (CD29). Inhibition by mAb to beta 1 was observed when the PMNL, but not the fibroblasts, were treated with mAb. The role of beta 1-integrins in PMNL transfibroblast migration was detectable only when the function of the CD11-CD18 complex was blocked, because mAb to beta 1-integrin alone had no significant effect on PMNL migration. Migration induced by C5a was more CD18-independent compared to IL-8 or C5adesArg. The CD18-independent migration was also inhibited by mAb to the beta 1-integrin subunits alpha 5 (of very late antigens-5; VLA-5) and alpha 6 (of VLA-6). Treatment of the fibroblasts (4 hr) with tumour necrosis factor-alpha (TNF-alpha) or IL-1 alpha enhanced C5a-induced PMNL transfibroblast migration and increased the proportion of migration utilizing the CD11-CD18 mechanism. However, TNF-alpha treatment had no effect on the degree of beta 1-integrin-dependent migration. These findings suggest that in response to the chemotactic factors C5a, IL-8 and C5adesArg, PMNL migration in the connective tissue is mediated by both CD11-CD18 (beta 2) and beta 1-integrins on the PMNL. The VLA-5 and VLA-6 members of beta 1 integrins are involved in this process. This is in contrast to PMNL migration across endothelium in this system, which is virtually all CD18 dependent with no significant role for beta 1-integrins. PMID- 7558137 TI - Role of B70/B7-2 in CD4+ T-cell immune responses induced by dendritic cells. AB - Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation. PMID- 7558138 TI - Comparative investigation of Langerhans' cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. AB - Human immunodeficiency virus (HIV) is commonly transmitted, during homosexual and heterosexual intercourse, through the rectal and cervicovaginal mucosa, foreskin and urethral epithelia. However, there is uncertainty about HIV transmission through the oral mucosa by oral sex. We have carried out a comparative immunohistological investigation of primate oral, cervicovaginal, foreskin, urethral and rectal epithelia for potential HIV receptors. We investigated epithelial tissues for CD4 glycoprotein, which is the principal receptor for HIV, Fc receptors of IgG for binding HIV-IgG antibody complexes, and HLA class II, which might enable HIV-bound CD4+ cells to gain access to the epithelial cells. CD4 glycoprotein was not found in oral, foreskin, urethral, vaginal or rectal epithelial cells, although CD4+ mononuclear cells were present in the lamina propria of each epithelium. Fc gamma II and Fc gamma III receptors were found in urethral, endocervical and rectal epithelia, and Fc gamma III and Fc gamma I receptors in the foreskin. However, Fc gamma receptors were not found in oral epithelium (buccal, labial, lingual or palatal) and only Fc gamma III receptors were detected in the gingival epithelial cells. HLA class II antigen was also not detected in foreskin, oral or rectal epithelium, but it was expressed by endocervical cells from most human specimens and in male urethral epithelia of non-human male primates. Langerhans' cells were found in all epithelia except those of the urethra and rectum, and they can express CD4 glycoprotein, Fc gamma receptors and HLA class II antigen. The mean number of Langerhans' cells expressing CD4 in the upper third of oral epithelium was significantly lower compared with vaginal epithelium or foreskin. The HIV-binding V1 domain of CD4 was significantly decreased in Langerhans' cells present in oral compared with vaginal epithelium. The results suggest that the foreskin in uncircumcised men and the cervicovaginal epithelium in females might become infected via the CD4+ Langerhans' cells. However, urethral infection might be mediated by HIV-antibody complexes binding to urethral epithelial Fc gamma receptors. The paucity of Langerhans' cells expressing the V1 domain of CD4, the absence of Fc gamma receptors, and a lack of expression of HLA class II antigens in most oral epithelial cells, argue against transmission of HIV through the normal intact oral mucosa. PMID- 7558140 TI - Shedding and enrichment of the glycolipid-anchored complement lysis inhibitor protectin (CD59) into milk fat globules. AB - Protectin (CD59) is a glycolipid-anchored inhibitor of the membrane attack complex (MAC) of human complement (C) that protects blood cells, endothelial cells and various epithelial cells from C-mediated lysis. Because of its activities protectin is a candidate molecule for use in the treatment of paroxysmal nocturnal haemoglobinuria or conditions where MAC causes tissue damage. Soluble, phospholipid-free forms of protectin have been isolated from human urine and produced in recombinant form, but they have only a relatively weak C lysis-inhibiting activity. In the present study we have looked for functionally active protectin in human breast milk. Milk is rich in fat droplets, milk fat globules (MFG), that are enveloped in a plasma membrane derived from secretory cells of the mammary gland. The membranes of MFG contain a variety of glycoproteins expressed by the mammary epithelial cells. Both immunofluorescence and immunoblotting analysis demonstrated that protectin was strongly expressed on human MFG. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) analysis, MFG protectin (CD59M) appeared as distinct bands with apparent molecular weights of 19,000-23,000 MW, similar to protectin extracted from MCF7 breast carcinoma cells. CD59M in breast milk was functionally active and had a glycophospholipid anchor, as judged by its ability to incorporate into guinea-pig erythrocytes and inhibit their lysis by human complement. These results indicate that functionally active protectin becomes enriched in MFG and imply that secretion of glycophospholipid-anchored molecules, e.g. into cow milk and colostrum, could be exploited as a means of producing bioactive molecules that need to be targeted into cell membranes. PMID- 7558142 TI - Attenuated immunogenic parasites are essential in the transfer of immunity to virulent Plasmodium berghei. AB - A less virulent parasite of Plasmodium berghei K173 was isolated that induced immunity against the more virulent parasite. Immunity to this parasite but not to the virulent one, could be transferred by immune spleen cells but not by immune lymph node cells. However, the immune spleen cells did transfer immunity to the virulent strain if accompanied by infection with viable parasites of the less virulent strain, but only if they were allowed to proliferate for a period of 1 week before challenge with the virulent strain. Immune spleen cells could survive two cycles of mouse to mouse transfer. The induction of immunity by transfer of immune spleen cells was associated with the production of anti-parasitic antibody. PMID- 7558143 TI - Differential activation of mouse hepatitis virus-specific CD4+ cytotoxic T cells is defined by peptide length. AB - In this study we have characterized the core epitope recognized by the MHV-A59 specific CD4+ cytotoxic T lymphocyte (CTL) clones HS1 and B6.1, derived from BALB/c and C57/BL6 mice, respectively. These CD4+ clones respond to the promiscuous peptide fragment S-329-343 of the glycoprotein S of MHV-A59. The results indicate that the core peptides of both clones overlap but are not identical. The core region of the HS1 clone is an 8-mer, and comprises the amino acid residues S-332-339, whereas the minimal epitope for clone B6.1 is a 9-mer and comprises the amino acid residues S-334-342. The peptide fragment S-329-343 activates all T-cell effector functions, including proliferation, cytokine secretion and cytolysis. However, in the present study we show that T-cell activation is not an all-or-none phenomenon, in which T-cell stimulation leads to activation of all T-cell effector functions. It appears that changes in the length of a peptide ligand can differentially activate the cytolytic machinery from proliferation and cytokine secretion. Furthermore, the results indicate that, in our case, modulation of the flanking residues of the core epitopes did not convert the cytokine profile of polarized T-helper type-1 (Th1) clones into a Th2-type pattern. PMID- 7558144 TI - Low abundance of naturally processed T-cell determinants following antigen pulsing of antigen-specific human B-cell lines. AB - An attempt was made to recover naturally processed T-cell determinants following antigen pulsing of tetanus toxin-specific human B-cell clones with microgram/ml amounts of antigen. Class II major histocompatibility complex (MHC) molecules were isolated from cells pulsed under optimal conditions and the eluted peptides displayed by reverse-phase high-performance liquid chromatography (HPLC). Antigen pulsed and control-unpulsed cells showed virtually identical optical density (OD)215 profiles, although multiple peptides derived from the input antigen could be identified at the radiochemical level. At least four distinct HPLC fractions contained naturally processed versions of the determinant 830-844, detected using the specific T-cell clone Mix 111. Quantification of the most active fraction indicated that approximately 1 pmole of this determinant was recovered from approximately 5 x 10(9) antigen-pulsed cells. Based on the amount of antigen processed following uptake on membrane immunoglobulin, and quantification of the biologically active material recovered, it was estimated that the efficiency of determinant capture was no greater than 1-2%. Further method development and a considerable increase in the number of cells used (> 10(10)) would appear to be necessary before naturally processed determinants from exogenously pulsed antigens can be reliably and fully characterized. Finally, a theoretical analysis showed that accurate (+/- 0.01%) mass information alone could identify a limited number of candidate peptides from known or putative antigens. PMID- 7558141 TI - Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. AB - In this study highly purified culture filtrate proteins obtained from Mycobacterium tuberculosis strains Erdman and H37Rv were tested for their capacity to stimulate immune T cells in vitro, and to immunize mice in vivo. Analysis of the culture filtrate antigen pool revealed a complex mixture of proteins; after separation of this pool into fractions of defined molecular size using an electrophoretic method, it was found that multiple fractions strongly stimulated interferon-gamma (IFN-gamma) secretion by immune CD4 T cells in vitro. In a further series of experiments mice were given multiple immunizations with the culture filtrate protein pool suspended in emulsions of incomplete Freund's adjuvant. Such mice were as resistant as mice given live bacillus Calmette-Guerin (BCG) vaccine to a low dose aerosol challenge infection with M. tuberculosis, but this resistance waned to low levels by 5 months post-vaccination. Furthermore, experiments using the filtrate antigens to boost or augment immunity induced by the BCG vaccination itself were unsuccessful. These data therefore support the hypothesis that the culture filtrate proteins of M. tuberculosis contain multiple antigens that are strongly recognized by T cells acquired during the initial expression of protective immunity to tuberculosis. Conventional immunization with these purified protein antigens can engender a strong degree of protective immunity, but this immunity is apparently not sustained at the same level as that induced by the live vaccine, perhaps suggesting a lack of suitable stimulation of memory immunity. PMID- 7558145 TI - Immune responsiveness in mutant mice lacking T-cell receptor alpha beta+ cells. AB - Immune responses of mice with T-cell receptor (TCR)gamma delta+ T cells but lacking TCR alpha beta+ cells because of a disruption in the TCR alpha gene, were analysed against alloantigens, soluble protein antigen, killed Mycobacterium tuberculosis and exogenous superantigen. Rejection of skin allografts mismatched for classical major histocompatibility complex (MHC) plus multiple minor H antigens was virtually abrogated but the presence of mismatched Qa-1 non classical MHC antigens on donor tissue resulted in a significant proportion of TCR alpha-/- mice rejecting such grafts. In view of the proposed role for gamma delta T cells in mycobacterial responses, and particularly against self- or mycobacterial heat-shock protein HSP 65, we examined these responses in TCR alpha /- mice. Local responses after immunization were low in lymph nodes and no component of these was directed against mycobacterial HSP 65. However, splenic T cells from mutant mice responded strongly to either purified protein derivative (PPD) or M. tuberculosis. Our findings indicate that TCR alpha-/- mice are selectively compromised: while responses to (undefined) mycobacterial antigens were substantial, responses to some other target antigens such as MHC alloantigens and HSP 65, believed to be preferentially recognized by gamma delta receptors, were poor or absent. However, the fact that the mutant mice more readily rejected allografts that are mismatched for the non-classical MHC antigen Qa-1 in addition to classical MHC and minor-H incompatibility, indicates that in some mice the residual immune response, presumed to be by gamma delta cells, is sufficient to cause skin graft rejection and that recognition of non-classical MHC antigens may play an important part in the response. PMID- 7558146 TI - A novel pathway for Ca2+ signalling in neutrophils by immune complexes. AB - The data presented here demonstrates that immune complexes use novel routes for stimulating a two-phase rise in cytosolic-free Ca2+ concentration. The initial transient Ca2+ rise resulted from release of Ca2+ from intracellular stores, by a route which, unlike f-met-leu-phe, was inhibited by bromophenacyl bromide. The second phase resulted from transmembrane influx and occurred in the absence of store release and by Ca2+ channels that were inhibited by Ni2+ but not SKF 96365 or econazole. Stimulation by immune complexes therefore involves novel routes for both the release of stored Ca2+ and the opening of Ca2+ channels in the plasma membrane. PMID- 7558147 TI - Inhibition of lymphocyte activation by catecholamines: evidence for a non classical mechanism of catecholamine action. AB - The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta adrenergic antagonists propranolol and nadalol did not affect the catecholamine mediated inhibition of lymphocyte activation. Although lymphocytes contain beta adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors. PMID- 7558148 TI - Tyrosine phosphorylation participates in peripheral T-cell activation and programmed cell death in vivo. AB - T-cell antigen receptor (TCR), which is not itself a protein tyrosine kinase (PTK), is thought to be associated with at least two SRC-like PTKs, P59fyn and ZAP-70. Activation of these PTKs is required for T-cell signal transduction. The aim of the present study was to determine the roles of PTKs in peripheral T-cell activation, induced by in vivo bacterial superantigen administration. We demonstrated that in vivo staphylococcal enterotoxin B (SEB) administration induced an enhanced tyrosine phosphorylation in peripheral spleen T cells undergoing a programmed cell death. In vitro immunecomplex kinase assay using antibody against P59fyn showed increased fyn kinase activity in SEB-stimulated spleen T cells. We examined the effect of PTK-specific inhibitors on DNA fragmentation and programmed cell death of V beta 8 positive T cells following in vitro culture of SEB-primed spleen T cells. Our results indicated that pretreatment of SEB-activated T cells with PTK inhibitors reduced DNA fragmentation and programmed cell death of V beta 8 positive T cells. These findings suggest that PTK plays an important role in activation and apoptosis of peripheral T cells induced by in vivo SEB administration. PMID- 7558149 TI - The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice. AB - Resistance to Mycobacterium avium depends on both genetically encoded macrophage functions and acquired T-cell immunity. Cytokines may play a role in either type of resistance. We studied the expression of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) in naturally susceptible BALB/c (Bcgs) and naturally resistant C.D2 (Bcgr) congenic mice infected with two strains of M. avium (one highly virulent and another of low virulence). We observed that cytokine expression patterns correlated better with the virulence of the micro-organism than with the genetic background of the host. The control of the infection by the low virulence strain in either mouse strain was associated with an increased expression of IFN-gamma and IL-2. Only Bcgs mice infected with a virulent strain of M. avium were unable to restrict bacterial growth. An increased expression of IL-4, early during infection, was detected in the course of the latter infection but played no role in determining the susceptibility to infection. Neutralization of IFN-gamma or IL-2 with specific monoclonal antibodies led to an exacerbation of the infection in Bcgr mice by the two strains of M. avium and in Bcgs mice infected with the low virulence strain of M. avium. PMID- 7558150 TI - Interaction of interleukin-6, tumour necrosis factor and interleukin-1 during Listeria infection. AB - Injected recombinant interleukin-6 (IL-6), tumour necrosis factor (TNF) and IL-1 all protect mice against experimental infection with Listeria monocytogenes. We have therefore investigated the interaction of these cytokines during infection. Treatment with recombinant (r)IL-6 enhanced TNF production by spleen cells during the first 2 days of infection. Anti-TNF antibody could totally abolish the protective effect of rIL-6, while the optimal protective function of TNF could not be achieved when IL-6 was neutralized by anti-IL-6 antibody. IL-1 induced a high level of IL-6 in the serum a short time after its administration, and neutralization of IL-6 totally abolished the protective function of rIL-1. The results thus provide further evidence for the complexity of cytokine interaction. PMID- 7558152 TI - Different migration patterns of antigen-presenting cells correlate with Th1/Th2 type responses in mice. AB - Antibodies are produced when antigen-presenting cells (APC) pulsed with an antigen are injected intravenously (i.v.) into BALB/c mice, but subcutaneous (s.c.) injection of such APC causes delayed-type hypersensitivity (DTH). To identify the anatomic sites where T and B cells are activated, we labelled splenic dendritic cells (DC) with a fluorochrome, PKH 26, injected them i.v. or s.c., and used the label to locate them. When the DC were injected i.v., germinal centres in the spleen were hyperplastic on day 1. Most DC moved to T-dependent areas of the white and red pulp on day 1 and remained there at least until day 5, but no DC migrated into the lymph nodes. When the DC were injected s.c., they were in the sinus on day 1 and had entered T-dependent area of draining lymph nodes only by day 3; hyperplasia of germinal centres in the spleen and migration of DC into the spleen were not found. We used the polymerase chain reaction (PCR) to study which mice had spleen cells and lymph node cells that produced the cytokines interleukin (IL)-2, IL-4, IL-10, and interferon-gamma (IFN-gamma). In the sensitization phase, day 1 after DC injection i.v., almost all IL-10 transcript was found in spleen cells, but after DC injection s.c., IL-2 message was most abundant in lymph node cells. The expression of mRNA for IL-4 and IFN gamma in mice that received DC i.v. was not different from that in mice that received DC s.c. in this phase. Immunohistochemical staining showed that cells stained for IL-10 were in the T-dependent area of the spleen from mice that received DC i.v. 1 day after the injection. Three days after the injection of DC i.v., cells stained for IL-10 were in the germinal centres as well. The number of such cells in the spleen of mice that received DC i.v. was significantly more than that in mice that received DC s.c. IL-10 may be important in development of TH2 response. PMID- 7558151 TI - Cytokine production by cats infected with feline immunodeficiency virus: a longitudinal study. AB - The immune responsiveness of cats naturally or experimentally infected with feline immunodeficiency virus (FIV) was studied. Peripheral blood mononuclear cells (PBMC) from naturally infected, symptomatic animals displayed depressed proliferation and interleukin-2 (IL-2) production in response to mitogens, which was accompanied by a significant increase in IL-1, IL-6 and tumour necrosis factor (TNF) production. Longitudinal studies were performed over a period of 4 years in experimentally infected animals. The responses of cells from these cats to concanavalin A (Con A) were consistently less than those from uninfected cats throughout the period but, owing to variation between cats, were significantly lower on only a few occasions. By contrast, the responses of cells to pokeweed mitogen (PWM) were severely affected and declined progressively throughout the 4 year period. In general, responses to Con A but not PWM could be restored by the addition of exogenous IL-2. The decline in immune responsiveness was concurrent with a decline in feline (f)CD4+ cells and an inversion in the CD4:CD8 ratio. Peak production of IL-1, IL-6 and TNF coincided with periods of depressed immune responses. Additionally, immunodeficient responses and elevated levels of proinflammatory cytokines were concurrent with the presence of clinical signs. We conclude that, like human immunodeficiency virus (HIV), FIV infection results in significant perturbation of the immune response. Responses to PWM appear to correlate with disease progression which suggests that the CD3 pathway is affected in the earlier stages of the disease and that additional activation pathways such as CD2 may not be affected until the animal enters the acquired immune deficient syndrome (AIDS) stage of the disease. PMID- 7558154 TI - T-cells subsets and activation in bronchial mucosa of sensitized Brown-Norway rats after single allergen exposure. AB - We have investigated the relationship between changes in T-cell activation in the bronchial mucosa, airway responsiveness and eosinophilic inflammation in sensitized Brown-Norway rats exposed to ovalbumin (OVA). Rats sensitized intraperitoneally with OVA and exposed to OVA aerosol 21 days later showed an enhanced increase in lung resistance (RNL) to acetylcholine (P < 0.05), and a significant increase in the number of eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BAL) (P < 0.05), compared with sensitized but saline-exposed controls. There was a significant increase in cells expressing the T-cell activation marker CD25 (P < 0.05) and the numbers of CD8+ T cells (P < 0.05), but not in the numbers of CD2+ and CD4+ cells. Eosinophil counts in airway submucosal tissue, as assessed by staining with BMK-13; a monoclonal antibody that binds to eosinophil major basic protein (MBP), were increased in rats receiving sensitization and exposure to OVA compared with naive controls (P < 0.002). There were significant positive correlations between the increase in RL to acetylcholine and the numbers of CD25+ (r = 0.92, P < 0.001), CD4+ (r = 0.77, P < 0.05), CD8+ (r = 0.71, P < 0.05) and MBP+ (r = 0.72, P < 0.03) cells in the OVA-sensitized and exposed group, but not in saline-exposed or naive animals. The number of MBP+ cells also correlated with CD25 expression (r = 0.71, P < 0.05). We conclude that airway hyper-responsiveness and inflammatory cell infiltration caused by OVA exposure of sensitized animals is associated with the presence of activated T cells in the airway mucosa. CD8+ T cells may play a role in the regulation of events leading to eosinophil inflammation and airway hyper responsiveness. PMID- 7558153 TI - Induction of T-cell hyporesponsiveness by intrahepatic modulation of donor antigen-presenting cells. AB - In this study, we examined the ability of varying populations of donor cells from B6 mice to induce hyporesponsiveness in T lymphocytes from C3H mice in vitro and in vivo. Small, resting B lymphocytes were inefficient stimulators of T lymphocyte proliferation compared to splenic mononuclear cells (SMNC) and lipopolysaccharide (LPS)-induced B-cell blasts in vitro (P < 0.05). Pretreatment of SMNC with anti-B7-1 or anti-intracellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) similarly resulted in inefficient stimulation of T cell proliferation in vitro (P < 0.05). However, in vivo, only intrahepatic, but not intravenous, injection of donor cells into C3H mice resulted in decreased T lymphocyte proliferation in response to restimulation by alloantigen. This effect was most pronounced following intrahepatic injection of resting B lymphocytes or SMNC pretreated with anti-ICAM-1 mAb compared to uninjected or intravenously injected mice (P < 0.05). The hyporesponsiveness was associated with an increased production of interleukin-4 (IL-4) by the responder T lymphocytes and correlated with enhanced skin allograft survival. These data demonstrate that intrahepatic injection of donor-derived cells induces T-lymphocyte hyporesponsiveness. The mechanism appears to be modulated by an ICAM-1-mediated signal resulting in expansion of an IL-4-producing T-lymphocyte population. PMID- 7558155 TI - Airway hyperresponsiveness, elevation of serum-specific IgE and activation of T cells following allergen exposure in sensitized Brown-Norway rats. AB - T lymphocytes may play a regulatory role in the development of allergic airway hyperresponsiveness (AHR). We have studied the relationship between airway responsiveness and a number of immunological changes in Brown-Norway rats sensitized intraperitoneally and repeatedly exposed to ovalbumin (OVA) aerosol. Acetylcholine provocation concentration (PC)150 (the concentration of acetylcholine causing a 150% increase of base-line lung resistance) was measured and peripheral blood and bronchoalveolar lavage (BAL) cells were collected 18 24hr after the final exposure. Total and OVA-specific IgE in serum was measured by enzyme-linked immunosorbent assay (ELISA). Mononuclear cells were analysed by flow cytometry after labelling with monoclonal antibodies against CD2 (pan T-cell marker), CD4, CD8 (T-cell subsets) or CD25 (interleukin-2 receptor). There were significant differences in PC150 (P < 0.05) and in OVA-specific IgE levels in serum (P < 0.002); CD4+ T cells expressed a significantly increased level of CD25 immunoreactivity in BAL, but not in peripheral blood, of rats sensitized and exposed to OVA, compared with saline-exposed controls (P < 0.02). There was a significant correlation between CD25 expression and BAL eosinophil numbers (r = 0.74, P < 0.001), PC150 (r = 0.63, P < 0.003) and OVA-specific IgE (r = 0.77, P < 0.001). These data suggest that activated T cells may be involved in the regulation of allergen-induced AHR in a relevant animal model of allergic asthma. PMID- 7558157 TI - The phenotype of freshly isolated and cultured human bone marrow allostimulatory cells: possible heterogeneity in bone marrow dendritic cell populations. AB - Putative dendritic cells (DC) and their precursors have been obtained from human bone marrow but their origin and relationship to other myeloid cells remains obscure. A minor bone marrow mononuclear cell (BMMC) population, which contains the most potent allostimulatory cells and lacks mature cell lineage markers (CD3, CD11b, CD14, CD15, CD16, CD19, CD57 and glycophorin A; lineage-negative) was enriched by immunoselection. These preparations, which contain cells with similar characteristics to freshly isolated human blood DC, were further subdivided by serial fluorescent-activated cell sorting (FACS). Potent allostimulatory cells were detected in the CD34, CD33 and CD4 positive and negative subpopulations. Cells with putative DC morphology were present in both the CD33 and CD4 positive and negative fractions. No significant CD13 or Thy-1 staining was seen in the lineage-negative population. In vitro culture of lineage-negative BMMC for 7 days in conditioned medium resulted in a up to fivefold expansion of cells and generated many lineage-positive progeny. This lineage-positive population was as allostimulatory as the negative progeny. Likewise, the CD14-positive and the CD14 negative cell progeny were equally allostimulatory. In contrast, the freshly isolated lineage-positive BMMC (containing CD14-positive monocytes) remained poor stimulators of the mixed lymphocyte reaction (MLR), even after culture in the presence of cytokines. These data suggest that there are at least two phenotypically diverse forms of potent allostimulatory cells in the lineage negative fraction of human BM, at least some of which express the early haemopoietic precursor antigens CD34 or CD33. Some of these precursors generate CD14-positive allostimulatory cells upon in vitro culture, suggesting an intimate link between DC ontogeny and myeloid differentiation. PMID- 7558158 TI - Role of F4/80+ cells during induction of hapten-specific contact hypersensitivity. AB - F4/80, a monoclonal antibody that binds to a surface molecule on mature macrophages and certain dendritic cells, has been used to explore the role of epidermal and dermal cells as antigen-presenting cells (APC) during the induction of contact hypersensitivity (CH) in mice. Systemic administration of the antibody appeared to have little or no physical or functional effect on intraepidermal Langerhans' cells, even though a subpopulation of these cells expressed the F4/80 ligand. None the less, systematically administered F4/80 antibodies were able to impair CH induction when dinitrofluorobenzene (DNFB) was painted on normal body wall skin of BALB/c mice [an ultraviolet B (UVB)-resistant strain]. Interestingly, systemic F4/80 antibodies did not affect CH induction in C57BL/10 mice (a UVB-susceptible strain). When a sensitizing dose of hapten was injected intracutaneously (i.c.) into F4/80-treated BALB/c and C57BL/10 mice, CH induction was impaired in both inbred strains, although the severity of impairment was greater in BALB/c mice. Following UVB radiation of body wall skin, anti-F4/80 treated BALB/c mice displayed very feeble CH, whether hapten was painted epicutaneously or injected i.c. at the irradiated site. Based on these and other recent reported results, it is concluded that (1) BALB/c mice rely partially upon dermal, F4/80+ cells as a source of APC when hapten is applied epicutaneously, whereas C57BL/10 mice rely almost exclusively upon epidermal Langerhans' cells in this circumstance; and (2) after UVB radiation of skin, BALB/c mice can use F4/80+ dermal cells as the source of APC function when hapten is painted epicutaneously. These findings are discussed with respect to the cellular basis for the differential susceptibilities of genetically defined strains of mice to the deleterious effects of UVB radiation on CH induction. PMID- 7558159 TI - Functional studies of major histocompatibility class II-positive dendritic cells and resident tissue macrophages isolated from the rat iris. AB - Recent immunomorphological studies have demonstrated the presence of distinct populations of resident tissue macrophages and major histocompatibility complex (MHC) class II+ dendritic cells within tissues lining the anterior chamber of rat, mouse and human eyes. The location of these cells in sites of potential contact with the aqueous humour-filled anterior chamber suggests that either of these cells may perform a role in immunosurveillance of this 'immune-privileged site'. The aim of the present study was to isolate highly purified dendritic cells and tissue macrophages from enzymatically disaggregated rat irides and to compare their relative capacity to stimulate unprimed T lymphocytes in vitro in a mixed leucocyte reaction assay. Dendritic cells freshly isolated from iris tissue exhibited a moderate ability to stimulate unprimed T lymphocytes. However, following 48 hr of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF)-supplemented medium, MHC class II+ dendritic cells demonstrated a markedly enhanced stimulatory capacity that was identical to that of Langerhans' cells isolated from skin. Tissue macrophages isolated from rat iris, however, demonstrated little allostimulatory capacity, either when freshly isolated or following 48 hr of culture in GM-CSF. This study provides the first definitive evidence that MHC class II+ cells within tissues lining the anterior chamber are functionally equivalent to dendritic cells described in other tissues. These findings have important implications for our understanding of the mechanisms of immune surveillance within the anterior chamber of the eye. PMID- 7558160 TI - Natural killer cell activation-associated induction of target cell DNA fragmentation in a spontaneously regressing rat histiocytoma. AB - The spontaneous regression of AK-5 histiocytoma is mediated by natural killer (NK) cells through antibody-dependent cell-mediated cellular cytotoxicity (ADCC) and the target cell death involves necrosis and apoptosis. We have studied the NK cell activation and the associated induction of apoptosis in the AK-5 tumour in rats. NK cells from immune animals expressed very low levels of CD16 and CD25 surface receptors, as revealed by Northern hybridization and flow cytometry. Interaction between NK cells and antibody-tagged AK-5 cells triggered the expression of these receptors to a higher level and affected AK-5 killing. Treatment of naive NK cells in vitro with interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-12 also enhanced the expression of these activation markers. Co-culture of NK cells from immune animals with antibody-tagged AK-5 cells induced formation of nuclear bodies in AK-5 and extensive fragmentation of AK-5 cell DNA. NK-mediated apoptosis was inhibited by zinc, actinomycin D and cycloheximide. The in vitro treatment of NK cells with cytokines enhanced their ability to induce apoptosis in AK-5 tumour. These results suggest that the NK cells acquire their ability to induce apoptosis in AK-5 tumour in association with their optimal activation. PMID- 7558162 TI - Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition. AB - Collectin-43 (CL-43) is a C-type serum lectin and a member of the collectin family of soluble proteins that are effector molecules in innate immunity. We have investigated the binding specificity of CL-43 using as model systems a panel of structurally defined oligosaccharides in the form of neoglycolipids, and several glycoproteins derived from the complement glycoprotein C3 during activation of the complement cascade. A specificity is revealed towards fucose as part of the Lea oligosaccharide sequence, and towards mannose as found on high mannose-type chains. These are features shared with other serum collectins, conglutinin and mannan-binding proteins; a major difference is the lack of detectable binding by CL-43 to N-glycosidic oligosaccharides terminating in N acetylglucosamine. CL-43 has a unique pattern of reactivity towards high mannose type oligosaccharides on the two glycosylation sites of C3 and derived glycoproteins: it binds to C3c (not bound by conglutinin and mannan-binding protein) but not to hydrolysed C3 [C3(H2O)], C3b or iC3b immobilized on microwells (all bound by conglutinin but not by mannan-binding protein). When these glycoproteins are sodium dodecyl sulphate (SDS)-treated and immobilized on nitrocellulose, CL-43 (but not conglutinin nor mannan-binding protein) binds strongly to C3(H2O), iC3b and C3c. The salient conclusions are, first, that there are remarkable positive or negative effects of carrier protein on oligosaccharide presentation and these differ for the individual collectins. Second, the different though partially overlapping binding patterns among the collectins may be important for their function as circulating effector molecules with broad surveillance capabilities. PMID- 7558156 TI - Modulation of cell-bound and soluble CD23, spontaneous and ongoing IgE synthesis of human peripheral blood mononuclear cells by soluble IL-4 receptors and the partial antagonistic IL-4 mutant protein IL-4 (Y124D). AB - We studied the influence of human recombinant soluble interleukin-4 receptors (sIL-4R) and a partial antagonistic mutant IL-4 protein, IL-4(Y124D), on the in vitro CD23 expression, soluble (s)CD23 release and IgE synthesis of human peripheral blood mononuclear cells (PBMC). The data show that sIL-4R suppressed the IL-4-induced IgE synthesis of PBMC. sIL-4R fusion protein stabilized with human Fc gamma fragments showed a more pronounced effect than unconjugated sIL 4R. IL-4(Y124D) also suppressed the IL-4-induced IgE synthesis. The IL-4-induced antigen CD23 and its soluble fragments were suppressed by sIL-4R and IL-4(Y124D). PBMC of atopic donors with a spontaneous in vitro IgE synthesis showed a partial suppression of the IgE production after sIL-4R or IL-4(Y124D) application. The IgE synthesis and sCD23 release of normal donor PBMC were suppressed when the substances were applied 0-4 days after IL-4 treatment. After 4 days of IL-4 stimulation, sIL-4R and IL-4(Y124D) enhanced the IgE synthesis. These data demonstrate that sIL-4R and IL-4(Y124D) are suppressive for the primary IgE synthesis induced by IL-4. In contrast, the ongoing IgE synthesis was only partially modulated by sIL-4R and IL-4(Y124D), and in some conditions even an enhancement of the IgE production was observed. These data suggest a differential function for IL-4 in the early and late phase of PBMC IgE production. PMID- 7558166 TI - The majority of Fc gamma RIII-positive gamma delta T cells do not express HLA-DR in patients with primary Sjogren's syndrome. AB - The percentages of circulating gamma delta T cells and the proportions of these expressing Fc gamma RIII (CD16) or HLA-DR in patients with primary Sjogren's syndrome (pSS) and controls were determined using monoclonal antibodies and flow cytometry. There was no significant difference in the percentages of gamma delta T cells in the pSS patients compared with controls. There was, however, a significant increase in the proportions of both CD16+ and HLA-DR+ gamma delta T cells in pSS patients. A 3-colour immunofluorescence technique demonstrated that these two markers were mutually exclusive and therefore may identify either subpopulations of gamma delta T cells or different stages of the activation process. PMID- 7558165 TI - Immunogenicity of immunoliposomes: reactivity against species-specific IgG and liposomal phospholipids. AB - Immunoliposomes with surface-linked avidin-biotinylated mouse IgG2a were prepared from dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG) and biotinylated dipalmitoylphosphatidylethanolamine (DPPE-biotin) in the molar ratio 10:1:0.1 with or without 5 mol% poly(ethylene glycol) dipalmitate (PEG-(C18)2). The ability of IgG2a immunoliposomes to elicit anti-IgG2a antibodies in mice was compared with alum and N-acetylmuramyl-L-alanyl-D isoglutamine (MDP). IgG2a 5 microgram/mouse) did not elicit an IgG1 antibody response after 4 s.c. injections. Alum-adsorbed IgG2a elicited 2.1 +/- microgram IgG1 antibody/ml serum, whereas MDP elicited 24.3 +/- microgram/ml serum. IgG2a immunoliposomes elicited 12.4 +/- 3.7 microgram IgG1 antibody/ml serum, while immunoliposomes containing lipophilic PEG-(C18)2 elicited 21.4 +/- 5.1 microgram IgG1 antibody/ml serum. Elicited antibodies were specific for IgG2a, with no cross-reactivity with IgG2b. Anti-DPPC or anti-DMPG IgG antibody levels did not change during immunization. Anti-DPPE IgG antibody levels were slightly but significantly elevated during immunization, and there was a significant increase in the level of anti-DPPE-biotin antibodies. These results demonstrate that immunoliposomes prepared with species-specific antibody are immunogenic and induce significant levels of isotypespecific antibody upon repeated injection. PMID- 7558163 TI - Distinct physicochemical characteristics of human mannose binding protein expressed by individuals of differing genotype. AB - Mannose binding protein (MBP) is a serum collectin (collagenous lectin) believed to be of importance in innate immunity. Three point mutations, in codons 52, 54 and 57 of exon 1 of the human MBP gene, have been predicted to affect the tertiary structure of the collagenous region of the protein, and are known to be associated with low serum concentrations of MBP. However, other groups working with recombinant mutant proteins have claimed that the proteins are expressed and assembled normally. The aim of the present investigation was to characterize the effects of these mutations on the physicochemical nature of MBP that is present in the circulation in vivo, and for this we used polyacrylamide gel electrophoresis, gel filtration and sucrose density gradient centrifugation, followed by immunoblotting and enhanced chemiluminescence. The circulating wild type MBP appeared to comprise a mixture of polymers formed from two to eight subunits (each based on three identical 32,000 MW polypeptide chains) of apparent molecular weights 200,000-700,000, with dimers and trimers constituting the predominant forms. Individuals homozygous for the codon 54 or 57 mutation had dramatically reduced concentrations of serum MBP, mainly comprising material of an apparent molecular weight of 120,000-130,000. Heterozygous individuals showed characteristics of both phenotypes. In contrast to the results obtained with artificial expression systems, our data suggest that individuals homozygous for the MBP mutations have very little circulating protein and that this comprises mainly low molecular weight material. PMID- 7558167 TI - Counteracting effect of interferon-alpha and -beta on interferon- gamma-induced production of nitric oxide which is suppressive for antibody response. AB - We investigated the nitric oxide (NO)-mediated immunosuppressive activity of macrophages and the regulatory effect of type I (alpha and beta) interferons (IFNs) on IFN-gamma-induced NO production by macrophages. In the sheep red blood cell (SRBC)-specific secondary antibody response in vitro, the addition of macrophages to the culture of primed spleen cells substantially decreased the number of plaque-forming cells. The concentration of nitrite in the culture supernatant showed a positive correlation with the number of macrophages added in a dose- dependent manner, and the addition of NMMA, a potent inhibitor of NO production, resulted in the restoration of of the response. In addition, anti-IFN gamma antibody abolished the ability of the immune culture supernatant to stimulate macrophages to produce NO. Therefore, the macrophage-dependent immunosuppression was due primarily to NO that was produced by macrophages in response to IFN-gamma derived from responding lymphocytes. The pretreatment of macrophages with IFN-alpha or -beta gave rise to a dramatic reduction of IFN gamma-mediated NO production. In addition, the suppressive activity of these macrophages was much lower than that of untreated macrophages. These results indicate that type I IFNs can regulate the immune response by modulating IFN gamma-induced production of immunosuppressive NO. PMID- 7558164 TI - Comprehensive pharmacokinetics of a humanized antibody and analysis of residual anti-idiotypic responses. AB - A murine antibody to human tumour necrosis factor-alpha (TNF-alpha) (CB0010) was complementarity-determining region (CDR)-grafted using human IgG4 heavy and kappa light chain constant regions. In cynomolgus monkeys, the grafted antibody (CDP571) was eliminated with a half-life of 40-90 hr, two to three times longer than CB0010, and immunogenicity was reduced by > 90%. Responses to the constant regions were almost entirely eliminated and responses to the CDR loop (anti idiotype) were lowered. CDP571 was given to 24 human volunteers in doses from 0.1 to 10.0 mg/kg. It was well tolerated, with a half-life of approximately 13 days. Anti-CDP571 antibodies were low or undetectable at higher doses. At lower doses, anti-CDP571 peaked at 2 weeks and then declined. The response was primarily IgM (in contrast to the cynomolgus monkey, where by 5 weeks IgG predominated) and was against a conformational epitope comprising heavy and light chain CDR loops. No antibodies were detected against the gamma 4/kappa domains or frameworks. The response had little or no effect on CDP571 binding to TNF-alpha or on plasma clearance. PMID- 7558168 TI - Identification and characterization of antimitochondrial autoantibodies in sera of patients with monoclonal gammopathies. AB - Monoclonal gammopathies (MG) are defined by the accumulation of monoclonal immunoglobulins, a result of monoclonal B lymphocytes or plasma cell proliferative disorder. Only rarely do these antibodies cause an overt disease by binding to a specific autoantigen (e.g., factor VIII). In the present study, sera from 100 patients with MG were screened for the presence of antibodies against the mitochondrial pyruvate dehydrogenase complex (PDH) -- autoantibodies that are the hallmark primary biliary cirrhosis (PBC). Anti-PDH antibodies were found in 6 patients, all asymptomatic. Using ELISA and immunoblotting methods, it was found that the titre of the anti-PDH antibodies was relatively low (average OD +/- SD: 0.744 +/- 0.529; PBC patients: 1.225 +/- 0.291; P = 0.02). In each patient the autoantibodies were of both kappa and lambda chains, suggesting that they are of polyclonal origin and implying that in MG there is a significant production of polyclonal autoantibodies, in addition to monoclonal proliferation. Furthermore, in 5 of the 6 patients (83%) the anti-PDH antibodies did not recognize the E2 component of PDH (which is the major autoantigen in PBC) an did not inhibit the activity of PDH (which was inhibited by PBC autoantibodies). This is in concert with the fact that none of the patients developed liver disease and emphasizes the specificity of the anti-PDH autoantibodies associated with PBC. PMID- 7558169 TI - The effects of cell number, concentrations of mitogen and glutamine and time of culture on [3H]thymidine incorporation into cervical lymph node lymphocytes stimulated by concanavalin-A. AB - The amount of [3H]thymidine incorporated into DNA in lymphocytes stimulated with Concanavalin-A increases exponentially with time at different concentrations of glutamine, reaches a peak value, then gradually decreases. When the value (log10 thymidine incorporation glutamine present -log10 thymidine incorporation glutamine absent) obtained from the exponential phase is plotted against time, a linear plot is obtained for each glutamine concentration. When these linear rates of incorporation are plotted against glutamine concentration, hyperbolic curves are obtained for different times of culture. The peak value of incorporation (which reflects the final number of cells which entered the cell cycle) is determined by the concentration of mitogen and occurs at an earlier time as the number of cells in culture is increased and as the concentration of glutamine is increased. These findings suggest that increasing the plasma glutamine concentration above the normal physiological level may be of value in increasing the proliferation of lymphocytes in conditions of lymphopenia. Adenosine, a fuel of purine nucleotide synthesis, which may affect the lymphoproliferative response also via specific adenosine receptors, increases the rate of incorporation of [3H]thymidine but this effect depends upon the concentration of glutamine; at low concentrations of glutamine, the stimulation by adenosine is apparent whereas at high concentrations of glutamine adenosine appeared to inhibit proliferation. PMID- 7558171 TI - Age-related changes in T gamma delta cells of NOD mice. AB - T gamma delta cells have been reported to recognize both mycobacterial and human heat-shock proteins (HSP), and a possible role of 65 kDa HSP has been suggested also in the pathogenesis of insulin-dependent diabetes mellitus. The aim of this study was to investigate age-related changes of T gamma delta cells during diabetes development in non-obese diabetic (NOD) mice. Using FACS analysis relative numbers of T gamma delta + cells from thymus, blood and spleen were determined in a 3-week-old non-diabetic, at onset of diabetes, and 1-week diabetic NOD mice and corresponding BALB/cJ controls. In comparison to BALB/cJ mice, higher values (2.4 +/- 0.2% vs. 1.1 +/- 0.1%) were found in the thymus of 3 week-old NOD mice (P < 0.01) as well as spleens of 22-week-old littermates (1.1 +/- 0.1% vs. 0.6 +/- 0.1%, P < 0.01). In addition, a higher proportion of T gamma delta cells was observed in blood samples of all age groups of NOD as compared to BALB/cJ mice, with values 3.5 +/- 0.7% (P < 0.05) in 3-week-old to 4.4 +/- 0.9% and 3.7 +/- 0.3% (P < 0.01) in 16- and 22-week-old NOD littermates. Differences in TCR gamma delta expression did not influence the whole CD3+ subset of mononuclear cells. Thus, our results show relatively higher numbers of T gamma delta cells in NOD mice and their increase in the periphery at onset of diabetes and later may suggest that T gamma delta cells participate in beta-cell destruction. PMID- 7558172 TI - In vitro modulation of preleukemic AKR mouse macrophage function by bacterial immunomodulators. AB - Spleen macrophages from 1- and 4-month-old preleukemic AKR mice were stimulated in vitro with the bacterial immunomodulators lipopolysaccharide (LPS), peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), in order to study their migration ability and mitochondrial enzyme activity. Macrophages from 1-month-old AKR mice, characterized by higher functional activity, failed to demonstrate any changes in the parameters studied after in vitro stimulation with the employed compounds. Conversely, the depressed macrophage function, spontaneously developed in 4-month-old AKR mice, most probably related to the preleukemic state, improved significantly and to about the same extent with all three immunomodulators. PMID- 7558170 TI - The diversity of T-cell receptor repertoire of peripheral CD4+ lymphocytes in LEC mutant rats. AB - A novel mutant strain of rats, LEC, exhibits an arrest of T-cell maturation from CD4+CD8+ to CD4+CD8- but not the CD4-CD8+ cells in the thymus. Nevertheless, CD4+T cells arise in lymph nodes of LEC rats, implying extrathymic T-cell maturation. We analyzed the variable (V) and junctional region diversity of T cell receptor (TCR) beta- chains in this population, since several reports have provided evidence that extrathymically matured T cells exhibit a biased expression of the TCR repertoire. CD4+ T cells in LEC rat lymph nodes exhibited a polymorphic V beta gene expression pattern. However, biased V beta gene expression was not identified in peripheral CD4+ T cells of LEC rats when compared with V beta gene expression in their thymocytes. Furthermore, junctional regions of TCR beta chains exhibited a vast diversity created by non-germline- encoded nucleotide (N nucleotide) additions. These results indicate that peripheral CD4+ T cells in LEC rats possess a vast diversity in the TCR repertoire as do thymocytes of the same rat, suggesting that they derive from the thymus. PMID- 7558161 TI - Effects of sodium fusidate in animal models of insulin-dependent diabetes mellitus and septic shock. AB - We have evaluated the effects of the novel immunosuppressant sodium fusidate (fusidin) in the non-obese diabetic (NOD) mouse and in D-galactosamine (D-Gal) presensitized BALB/c mice challenged with the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB) or with the endotoxin, Escherichia coli lipopolysaccharide (LPS). The NOD mouse model has clinical and histoimmunological features similar to those of human insulin-dependent diabetes mellitus (IDDM). The SEB- and LPS-treated BALB/c mouse models exhibit pathogenic similarities with human septic shock conditions. In the NOD mouse, fusidin suppressed the spontaneous development of insulitis (mean inhibition 73%) and hyperglycaemia (IDDM incidence 25% versus 0%) when administered at 40 mg/kg five times weekly for 8 consecutive weeks from the fourth week of age; concurrently treated animals exhibited reduced percentages of splenic T lymphocytes. This anti-diabetogenic effect was confirmed in the accelerated model of diabetes induced in the NOD mouse with cyclophosphamide (CY) (IDDM incidence 55% versus 21-6% using dosages of fusidin from 40 to 80 mg/kg five times weekly); protection from IDDM development was achieved even when the drug (80 mg/kg/day) was first administered 7 days after CY challenge. In contrast, fusidin did not reverse hyperglycaemia when administered to CY-treated animals within 3 days of IDDM development. In the two models of septic shock, prophylactic treatment with fusidin, 80 mg/kg given three times for 2 days prior to D-Gal/SEB or D-Gal/LPS challenge, drastically reduced the lethality compared with D-Gal/buffer-treated mice. This effect may depend on the inhibitory action of fusidin on the secretion of cytokines such as interferon-gamma and tumour necrosis factor-alpha, the serum levels of which were greatly diminished in the fusidin-treated mice (mean inhibition 50-90%). These results demonstrate that fusidin may have a role in the treatment of cell mediated autoimmune diseases and cytokine-mediated infectious diseases in humans. PMID- 7558173 TI - Proliferation and autoantibody production by mouse thyroglobulin (MTg)-specific B cells activated in vitro by MTg and MTg-specific T cells. AB - Immunization of genetically susceptible strains of mice with mouse thyroglobulin (MTg) and adjuvant results in sensitization of MTg-specific CD4+ T cells that can induce experimental autoimmune thyroiditis (EAT) after in vitro activation with MTg. These MTg specific CD4+ T cells also provide help to MTg-specific B cells which produce anti-MTg autoantibodies in vivo. This study was undertaken to develop a system with which to measure MTg-specific helper function in vitro. MTg activated T cells were shown to provide help to MTg-primed B cells in vitro as assessed by B-cell proliferation and anti-MTg autoantibody production. These responses were MTg-specific and required MTg priming of both the T- and B-cell populations. An MTg-specific CD4+ T-cell line also induced MTg-specific B-cell proliferation and was capable of inducing IgG1, IgG2a and IgG2b synthesis in MTg- primed B cells. PMID- 7558176 TI - Differential regulation of surface immunoglobulin expression by various muramyl dipeptides in a murine pre-B cell line. AB - The murine pre-B cell line 70Z/3 responds to lipopolysaccharide (LPS), interleukin-1 (IL-1) or interferon-gamma (IGN gamma) by kappa gene transcription and expression of surface IgM (sIg). We found that muramyl dipeptide (MDP), a synthetic immunoadjuvant analog of a bacterial membrane structure, produced a weak increase in the number of sIg-positive 70Z/3 cells as measured by immunofluorescence staining. This number was significantly increased after exposure to MDP. Moreover, when MDP was used in combination with LPS, IL-1 or IFN gamma, an enhancement of sIg expression was observed showing an early influence of MDP in the presence of a second stimulant. Unexpectedly, two adjuvant-active analogs of MDP did not share its capacity to stimulate differentiation of the cell line when used alone or associated with other agents, indicating that adjuvanticity of MDP was not the only requirement. Two other products of bacterial origin, a Staphylococcus aureus cell extract (SAC) and the Toxic Shock Syndrome Toxin TSST-1 could neither enhance the kappa gene expression in 70Z/3 cells nor increase the MDP effect. The stimulating effect displayed by MDP could by related to NF-kappa B activation. PMID- 7558174 TI - Anti-CD40 monoclonal antibody induces the proliferation of murine B cells as a B cell mitogen through a distinct pathway from receptors for antigens or lipopolysaccharide. AB - To study the activation and differentiation of murine B cells, we prepared a hybridoma secreting monoclonal antibody, LB429, which can directly induce the proliferation of murine B cells in vitro. LB429 recognizes a B cell specific surface molecule of 45 kDa. It recognizes an epitope of murine CD40 produced as a soluble fusion protein with glutathione S-transferase. LB429 stains COS-7 transfectant with murine CD40 cDNA and mature B-cell lines but does not stain pre B cell lines. Two color staining demonstrated that the epitope recognized with LB429 appears on the surface of B220+ cells of spleen and bone marrow. LB429 can induce a strong proliferation of murine B cells from spleen in the absence of initial triggering with anti-IgM antibody or with anti-IgM antibody + IL-4. LB429 induced the cell size enlargement and the cell cycle transition of resting B cells as well as lipopolysaccharide (LPS). LB429 and LPS stimulate B cells synergistically in vitro by accumulating 44.7% of cells in S/G2/M phases of cell cycle. However, stimulation of spleen B cells with LB429 resulted in the increase of sIgM high+ sIgD(high)+ B cells, in contrast LPS showed the proliferation of both sIgM(high)+ sIgD(high)+ B cells and sIgM(low)+ sIgD(high)+ B cells. These results suggested that LB429 and LPS cause the proliferation of B cells through different stimulatory pathways. This anti-mouse CD40 antibody (LB429) is a very useful reagent to study the activation and differentiation of B cells in vitro. PMID- 7558175 TI - Effects of N-acetyl-L-cysteine on T-cell apoptosis are not mediated by increased cellular glutathione. AB - Thiol-containing antioxidants such as N-acetyl-L-cysteine (NAC) are known to inhibit apoptosis, although it is unclear whether this effect is direct or mediated through modulation of intracellular glutathione (GSH). In the present study, NAC treatment of the murine T-cell hybridoma DO-11.10 was found to inhibit apoptosis triggered by anti-CD3 antibody but enhance the process when induced by 6-alpha- methylprednisolone. HPLC measurements showed that these effects were not correlated with the levels of GSH or glutathione disulfide (GSSG) in the cells. Similar effects on DNA fragmentation were obtained when the experiments were repeated in the presence either of a specific inhibitor of GSH biosynthesis (buthionine sulfoximine) or the isomer N-acetyl-D-cysteine which cannot be enzymatically converted into GSH. We conclude that NAC can have divergent effects on apoptosis independent of changes in either the amount or redox state of intracellular GSH. PMID- 7558177 TI - SCID mice reconstituted with Oct-2-deficient lymphocytes can cure Leishmania major infection and generate normal antigen-specific T cells. PMID- 7558178 TI - Increased survival of nasal polyp eosinophils. PMID- 7558179 TI - Birch pollen allergens fail to evoke IgG1 responses in non-atopic individuals. PMID- 7558180 TI - Nutritional stress-induced implantation failure in laboratory mice: inhibition by continuous illumination. AB - The implantation failure in newly inseminated mice induced by food deprivation for 48 hr, beginning at 0900 hrs on day 4 post coitum, was prevented by simultaneous exposure to light continuously for 48 or 36 hr. Food-deprived females that were exposed to continuous light for 36 hr showed a significant increase in fetal resorption as compared with food-deprived females exposed to continuous light for 48 hr. Since failure of hypophysial prolactin release appears to be the primary endocrine cause of the inanition-induced implantation failure, the results suggest that exposure to continuous light protects implantation in food-deprived females by stimulating luteotrophic activity. PMID- 7558181 TI - Effect of chilli (Capsicum frutescens) extract on proliferation of oral mucosal fibroblasts. AB - Various concentrations of chilli extract were tested in 2 stages on the growth of a human buccal mucosa fibroblast cell line. Firstly, concentrations of 25, 75, 150 and 300 micrograms/ml were tested on the cell line for 6 days followed by a repeat which included concentrations of 400 and 500 micrograms/ml. In the latter, cell growth was monitored for 18 days. From day 3 (72 hr) suppression of cell growth was evident with concentrations 300-500 micrograms/ml. Total cell death occurred at 16 days with 300 micrograms/ml and at 6 days with 400-500 micrograms/ml. With the lower concentrations, 25-150 micrograms/ml, the daily counts were lower than the control but the difference was not statistically significant. Growth continued unabated. It is therefore concluded that cytopathic effect of chilli extract to fibroblasts is concentration dependent. PMID- 7558182 TI - Hepatoprotective studies on Phyllanthus emblica Linn. and quercetin. AB - Phyllanthus emblica is a constituent of many hepatoprotective formulations available in market. 50% alcoholic extract of P. emblica and quercetin isolated from it were studied for hepatoprotective effect against country made liquor (CML) and paracetamol challenge in albino rats and mice respectively. The extract at the dose of 100 mg/100 g [corrected], po and quercetin at the dose of 15 mg/100 g, po, produced significant hepatoprotection. PMID- 7558183 TI - In vivo effects of acetaminophen on rat RBC and role of vitamin E. AB - Non-therapeutic toxic dose (250 mg/kg) of acetaminophen (paracetamol), in vivo to albino rats significantly decreased red cell reduced glutathione (GSH) content and activity of (Na+, K+)-ATPase enzyme, whereas osmotic fragility (O.F.) was increased. However, no change was observed in the activity of glutathione reductase (GR) after acetaminophen treatment, while acetaminophen plus vitamin E treated rats showed significant increase in GR activity. Supplementation of vitamin E to the drug treated rats effectively brought the GSH content, (Na+, K+) ATPase activity and O.F. back to almost normal. The results suggest that acetaminophen toxic dose treatment induces metabolic and membranal alterations making red cells prone to hemolysis, while vitamin E which is an antioxidant shows its ameliorating role to these changes. PMID- 7558185 TI - Inhibition of lysyl oxidase by isoniazid and its effect on wound healing. AB - Isoniazid, an antitubercular drug, is known to be a potent inhibitor of monoamine oxidases. Effects of this drug, on lysyl oxidase (also a monoamine oxidase) and other wound healing parameters were studied in albino rats, in presence and absence of pyridoxal phosphate, using a dead space wound model. Tensile strength, collagen and glycosaminoglycan contents as well as lysyl oxidase activity were estimated in the granuloma tissue harvested from 10 day old dead space wounds. Isoniazid inhibited lysyl oxidase activity and a decrease in tensile strength as well as collagen content were observed. The effects were reversed on administration of a stoichiometric amount of pyridoxal phosphate. Hexosamine level was increased and hexuronic acid level decreased in the drug treated animals. Therefore, isoniazid may decrease the mechanical strength of collagen by inhibiting lysyl oxidase, by competiting for its obligatory cofactor pyridoxal phosphate, as well as by interfering in electrostatic interactions between collagen and the ground substance. PMID- 7558184 TI - Possible involvement of superoxide radical in biochemical lesions induced by nitrous oxide. AB - Inactivation of methionine synthase (MS) by nitrous oxide (N2O) administration to animals and man has been postulated to be mediated by hydroxyl radical (OH). An alternate mechanism has been proposed which involves superoxide radical (O2.-) originating from N2O in the inactivation of MS by OH that may arise from O2.- through Fenton/Haber-Weiss reaction. Rats inhaling a mixture of N2O:O2 (1:1) for 5 hr showed inactivation of MS in liver to nearly 90% which could not be reversed by prior administration of either dimethyl sulfoxide (DMSO) or sodium benzoate. Pretreatment of rats with superoxide dismutase (SOD) at two doses, 0.1 mg and 3.5 mg/100 g body weight, retarded the in vivo inactivation of MS by N2O, to 76% and 47% respectively. In vitro inactivation of MS with N2O could not be retarded by DMSO or Na-benzoate, or either exogenous SOD or catalase. PMID- 7558187 TI - Effect of felodipine on cholinergic responses of the colonic smooth muscle of streptozotocin induced diabetic rats. AB - In streptozotocin induced diabetic rats, irrespective of felodipine treatment (5 mg/kg/day po for 4 weeks), a reduction in contractile response of colonic smooth muscle in vitro was observed. Similarly, in both control and diabetic rats treated with felodipine, contractile response was reduced. However, in felodipine treated diabetic rats there was a significant increase in response to exogenous acetylcholine. It may be of interest to study the effect of felodipine, on gastro intestinal motility in vivo in diabetic rats, to enable extrapolation of the present results to the effect of felodipine on gastrointestinal complications of diabetes mellitus. PMID- 7558186 TI - Effect of cisplatin on physiological status of normal rat testis. AB - Effects of cisplatin with or without testosterone on the physiology of normal adult rats were studied. The weights of epididymis, seminal vesicles and ventral prostate showed the decreased level of testosterone in cisplatin-treated rats. There was no change in DNA, RNA and protein contents in both cisplatin and cisplatin-testosterone treated animals. Hence it is suggested that the administration of cisplatin does not inhibit DNA, RNA and protein synthesis in the testis of normal rats. It could be due to either the normal cells are able to oppose the cross links formation or their removal at faster rate than in tumor cells by repair mechanisms or proteins which the tumor cells lack. PMID- 7558188 TI - Jaundice in new born and erythrocyte and plasma antioxidant defence system. AB - Alteration in the antioxidant defence mechanism of erythrocytes with rise in serum unconjugated bilirubin level in neonates has been observed. The cellular glutathione level was found to be significantly low. The activity of glutathione reductase in erythrocyte increased to combat the cellular loss of reduced glutathione in neonatal jaundice. In plasma fraction the level of glutathione (reduced) was found to be significantly higher with lowering of glutathione reductase level. Glutathione peroxidase was reduced in cellular level whereas an increase was observed in plasma fraction. Gamma glutamyl transpeptidase level was barely detectable in erythrocyte whereas an increase was observed in plasma fraction. In all the cases erythrocyte G-6-PDH activity level was found within normal limits. PMID- 7558189 TI - Prolonged stability of R plasmids KR61 and KR61-A in Salmonella host. AB - The R plasmids, KR61 and KR61-A, that were originally isolated from a clinical strain of Aerobacter aerogenes in 1971 and determined resistance to kanamycin (Km), neomycin (Nm), streptomycin (Sm), tetracycline (Tc); and ampicillin (Ap) respectively were found stable in Salmonella typhimurium LT2 even after 22 years of cultivation on antibiotic free media. KR61, carrying resistance to KmNmSmTc, not only maintained all its resistances but also maintained its conjugal transferability (RTF) as indicated by its subsequent transfer to Escherichia and Salmonella hosts. KR61-A that carried resistance to Ap and lacked an RTF could be mobilized by KR61 from S. typhimurium LT2, constructed to bear KR61-A and KR61, to E. coli recipients. S. typhimurium LT2 carrying KR61-A + KR61 (ApKmNmSmTc), showed the characteristic conjugal transfer of resistances in following three patterns: (i) Ap, (ii) KmNmSmTc and (iii) ApKmNmSmTc. The findings reported here are based on conjugal isolation of plasmids. Physical isolation of KR61 and KR61 A was never made. PMID- 7558190 TI - Cholesterol mediated changes in beta-glucuronidase activities of rat theca interstitial cells and granulosa cells. AB - Effects of steroid hormones on beta-glucuronidase activities of granulosa cells and theca interstitial cells were studied in vitro in the presence and absence of cholesterol in minimum essential medium (MEM with Hank's salts). Conspicuous fall in the enzyme activities of both these cells were noticed during first 10 min of incubation in MEM without cholesterol and remained lower throughout the experiment. Addition of cholesterol to incubation medium maintained beta glucuronidase activities of both the cells as observed in the cells of immature ovary immediately after isolation. 17 beta-estradiol did not affect beta glucuronidase activities of these cells, while testosterone and progesterone suppressed the enzyme activities of these cells in the presence of cholesterol. PMID- 7558191 TI - Binding characteristics of bovine serum albumin-aflatoxin B1 to polystyrene microtiter plates: importance of hapten to carrier protein molar ratio. AB - Binding characteristics of bovine serum albumin-aflatoxin B1 conjugates with high (1:54), medium (2:25) and low (1:9) hapten to carrier molar ratios, to the polystyrene microtiter plates are influenced by the stoichiometry of the hapten (Aflatoxin B1) to the carrier protein (bovine serum albumin). Conjugates with optimal hapten to carrier molar ratios (1:25) showed a better binding capacity to the polystyrene microtiter plate as compared to the conjugates with the high molar ratios in a non-competitive ELISA for aflatoxin B1. Denaturation of the conjugate with molar ratio of 1:54 in order to enhance its binding capacity, however, did not result in any significant improvement. PMID- 7558192 TI - Antiperoxide effects of S-allyl cysteine sulphoxide isolated from Allium sativum Linn and gugulipid in cholesterol diet fed rats. AB - Cholesterol containing diet significantly increased not only the body weight, but also the weight of liver and adipose tissue of rats. This is accompanied by a significant increase in blood lipids, atherogenic index and lipid peroxidation and a significant decrease in reduced glutathione level, superoxide dismutase and catalase activities in tissues. Treatment with S-allyl cysteine sulphoxide reverses the deleterious effects of cholesterol diet significantly and almost as effectively as gugulipid. PMID- 7558193 TI - Influence of Ca2+ on microsomal lipid peroxidation. AB - Lipid peroxidation in microsomes prepared from liver of mice was initiated by NADPH, ascorbic acid and ferrous ions. The presence of Ca2+ modulated the lipid peroxidation in all these three systems. The mode and magnitude depend on the system and concentration of cofactors used for initiation of lipid peroxidation. In ascorbate system, Ca2+ enhanced the lipid peroxidation up to 30 microM concentration of ascorbic acid and beyond 30 microM concentration it inhibited. Ca2+ increased NADPH-dependent lipid peroxidation at all concentrations. Depending on concentration of Fe2+, lipid peroxidation was either decreased or increased in presence of Ca2+. It suggested that the in vitro findings may be cautiously extrapolated to the animal systems. In absence of cofactors, Ca2+ enhanced lipid peroxidation. EGTA inhibited Ca2+-enhanced lipid peroxidation. However in presence of ionophore A23187, Ca2+ potentiated lipid peroxidation. Since Ca2+ has a closed-shell electronic state and lacks electronic transitions, it may not participate directly in lipid peroxidation process. The effect of Ca2+ on lipid peroxidation may be through some biochemical processes or its interactions with membranes leading to various changes in their characteristics. PMID- 7558194 TI - Mechanism of histamine induced dispersal response in the isolated web melanophores of a frog, Rana tigerina (Daud.). AB - Effects of histamine and specific H1, H2 receptor agonists have been investigated on the isolated web skin melanophores of the frog, R. tigerina. Histamine, 2 methyl-histamine and 4-methyl-histamine all induced dose dependent dispersion in the frog melanophores. Reserpine treatment of the frog skin, either in vivo, or in vitro potentiated the dispersal response of the frog skin melanophores elicited by histamine. Histamine induced dispersal responses in the frog integumentary melanophores were partially blocked by mepyramine and ranitidine. Atropine failed to inhibit the dispersal response in any degree. Propranolol partially inhibited the dispersion of frog melanophores induced by histamine and 2-methyl-histamine. However, propranolol completely blocked the dispersal response of the mealnophores to 4-methyl-histamine. It is suggested that histamine induced dispersion in the frog skin melanophores in vitro is mediated partially through specific H1, H2 and beta adrenergic receptors. PMID- 7558195 TI - Degradation of naphthalene by a Pseudomonas strain NGK1. AB - A Pseudomonas strain NGK1 capable of degrading naphthalene as a sole carbon source was isolated from biological waste treatment effluent. After 48 hr of incubation in 0.1% naphthalene mineral salts medium, the bacterial culture showed irregular clumped cells and salicylic acid (68 micrograms per ml) in the medium (pH 3.8). The strain degraded naphthalene through salicylate and catechol as was evidenced by metabolite characterization, oxygen uptake and enzymatic studies. PMID- 7558197 TI - Centrophenoxine activates acetylcholinesterase activity in hippocampus of aged rats. AB - Age-related changes in the acetylcholinesterase activity were measured in the hippocampus, brain stem and cerebellum of rats (aged 4, 8, 16 and 24 months). The age-dependent decrease in the enzyme activity first appeared in the hippocampus; the brain stem was affected later while the cerebellum remained unaffected. Centrophenoxine, usually considered as an ageing reversal drug and also regarded as a neuroenergeticum in human therapy, increased the acetylcholinesterase activity in the hippocampus of aged rats, the activity was also elevated in the brain stem but no in the cerebellum. The acetylcholinesterase-stimulating influence of the drug is likely to be implicated in the pharmacological reversal of the age related decline of the cholinergic system. This effect of the drug may also mediate its effects on cognitive and neuronal synaptic functions. PMID- 7558196 TI - Effect of cerebellar modulation on gastroduodenal PGE2 and 5-HT content of rat. AB - Nodular cerebellar lesion decreased PGE2 and 5-HT content of gastroduodenal tissue along with a decrease in enterochromaffin (EC) cell count. On the other hand, when nodular lesioned rats were subjected to vestibular stimulation, both PGE2 and 5-HT content of gastroduodenal tissue and EC cell count increased, suggesting nodular cerebellar influence on PGE2 and 5-HT content of gastroduodenal tissue. PMID- 7558199 TI - A rapid method for the preparation of plasmid DNA. AB - A rapid method was developed to prepare plasmid DNA to be used for in vitro transcription, subcloning and sequencing. Clean plasmid DNA can be prepared within 90 min using this method and the quality of the plasmid DNA was found to be suitable for sequencing, preparation of in vitro RNA transcript and electroporation. Using this method plasmid DNA can be prepared from 2 ml to 100 ml culture. The yield of the recombinant plasmids was found to be in the range of 50-100 micrograms from 10 ml culture depending on the low and high copy number plasmids. The advantage of this method is that one can easily and rapidly prepare large quantities of plasmid DNA of high quality to be used for various purposes. PMID- 7558200 TI - Survivability of Salmonella paratyphi B var Java on experimentally infected cockroaches. AB - Cockroaches are inhabitant of sewers and frequent visitors of kitchen and stores in the night to feed on left over. It is liable to disseminate a number of pathogens by contaminating kitchen surface, feeding vessels and food items left open. Salmonella paratyphi B var Java a common pathogen of man and animal was used in the study to evaluate its survival and excretion in cockroaches. The host when fed on semisolid feed containing 1 x 10(7) CFU of S. paratyphi B var Java g 1, it was found that the pathogen was eliminated earlier from the live than euthanized cockroaches. PMID- 7558198 TI - Protection of UV-radiation induced skin toxicity by reactive oxygen scavengers. AB - Although, photoaddition reactions of psoralens with DNA and membrane lipid are regarded as the major photochemical events responsible for skin photosensitization and photochemotherapeutic properties, their O2-dependent reactive oxygen specie (ROS) generating potential is also responsible for biologic photooxidation reactions and cutaneous phototoxicity. We have investigated the skin-sensitization reactions of psoralen in presence of selected free radical scavengers. The results confirm that sodium azide, DABCO and beta carotene inhibited considerably the 1O2 generation reactions in a chemical system (determined by monitoring the bleaching of N,N-dimethyl-p-nitrosoaniline in the presence of histidine used as a selective acceptor of 1O2) as well as skin sensitization in vivo (epilated guinea pig skin). These observation suggest that the O2-dependent photodynamic action of psoralen contributes significantly to the development of cutaneous toxicity which can be inhibited by selective scavengers of 1O2. PMID- 7558203 TI - Transmission of dengue virus-specific suppressor signal depends on the presence of calcium. AB - The present study was undertaken to investigate the role of calcium in the production of dengue type 2 virus (DV) induced suppressor cytokine (SF) and in transmission of the suppressor signal via syngeneic macrophage (M phi) to recruit the second subpopulation of suppressor T(TS2) cell. Effect of calcium channel blocking drugs, Verapamil and Nifedipine, on the production and activity of SF was investigated. The production of SF was inhibited in a dose dependent manner by the calcium channel blockers. The suppressor activity of SF was also inhibited by calcium channel blocking drugs. SF could not be produced when spleen cells were cultured in a calcium free medium, the production being restored on substitution with calcium chloride. Treatment of M phi with the calcium channel blockers inhibited the transmission of the suppressor signal from TS1 to TS2 cells in a dose dependent manner. The influx of calcium during transmission of the suppressor signal was studied by measuring the 45Ca uptake. Calcium channel blocking drugs inhibited the 45Ca uptake by T lymphocytes. Thus, presence of calcium is obligatory for the production and suppressor activity of SF and it plays a critical role in transmission of the suppressor signal. PMID- 7558201 TI - Alternative method for complete immobilization of insect for surgical operations. PMID- 7558202 TI - Effect of ethylene bis isobutyl xanthate isolated from a marine green alga Dictyosphaeria favulosa on mosquito Aedes aegypti. PMID- 7558204 TI - In vitro antifungal susceptibility of Candida. AB - To develop a simple, standardised procedure for antifungal susceptibility of yeasts, disc diffusion test was evaluated in comparison to agar dilution and the macrobroth dilution procedure of the National Committee for Clinical Laboratory Standards USA. One hundred Candida strains isolated from clinical material were used against four antifungal agents (amphotericin B, 5-flucytosine, ketoconazole and fluconazole). Inoculum size of 10(6) yeasts/ml just failed to produce confluent colonies at 37 degrees C after 48 h on yeast nitrogen-glucose-agar with or without asparagine medium. The zones of inhibition were correlated with both broth and agar dilution minimum inhibitory concentrations (MICs). The results were found to correlate well using amphotericin B, 5-flucytosine and fluconazole (with broth dilution only). But there was no significant correlation using ketoconazole. Therefore, disc diffusion susceptibility testing of Candida strains appears to be generally applicable except for azoles where broth dilution test would confirm the resistant isolates. PMID- 7558205 TI - Comparative activity of three repellents against bedbugs Cimex hemipterus (Fabr.). AB - Comparative activity of three repellents viz., diethyl-m-toluamide (DEET), diethyl phenyl-acetamide (DEPA) and dimethylphthalate (DMP) against bedbugs Cimex hemipterus (Fabr) was studied by applying repellent solution on the shaven skin of rabbits. DEET (Tech. grade) 20mgai/cm2 exhibited 93 and 83 per cent repellency after 2 and 8 h respectively, while DEPA (Tech. grade) at this concentration exhibited 90 and 71 per cent repellency. DEET and DEPA at 75 per cent concentration (15mgai/cm2) showed 85 and 78 per cent repellency up to 2 h and 52 and 42 per cent repellency 6 h after treatment respectively. The repellent activity was also found to be concentration and time of treatment dependent in respect of DEET and DEPA. DMP (Tech. grade) did not show any appreciable repellent activity at any concentration tested. Further, the blood intake by C. hemipterus following treatment with various concentrations of repellents was found to decrease with increase in the concentration of repellents. PMID- 7558207 TI - An epidemiological study of prevalence of pain in head & neck cancers. AB - This study was undertaken on 117 consecutive patients referred to a head and neck oncology clinic at a major hospital in New Delhi. 89.5 per cent patients were in clinical stage III or IV at the time of referral. The prevalence of pain was 83.8 per cent with a mean pain duration of 6.2 +/- 5.9 months and a mean pain intensity of 5.0 +/- 2.7 on a modified numerical rating scale ranging from 0 to 10. The mean intensity of pain was not significantly different for age groups or stage of the disease but patients with oro-pharyngeal cancers had significantly higher pain scores than patients with hypopharyngeal cancers (4.6 +/- 3.0 vs 2.9 +/- 2.5, P < 0.05). PMID- 7558206 TI - Prognostic significance of DNA index by flowcytometry in acute lymphoblastic leukaemia. AB - DNA index (DI) is considered an important prognostic factor in acute lymphoblastic leukaemia (ALL). We undertook this study to correlate DI with other presenting features and response to therapy. Of the 30 patients of ALL treated at our hospital and entered in this study, 15 were put on the aggressive MCP (multi center protocol) 841 protocol and equal number on the Alternate protocol. Eighteen achieved complete remission (13/15 on the former protocol and 5/15 on the later). DI was less than 0.8 in 8 (27%) patients, between 0.8 and 1.2 in 18 (60%) and more than 1.2 in 4 patients (13%). These figures are different from those reported in Caucasians. On multivariate regression analysis, the DI significantly correlated with percentage of blasts in peripheral blood (P = 0.0035). There was no correlation with outcome or response to treatment. PMID- 7558208 TI - Public awareness, understanding & attitudes toward epilepsy. AB - The public awareness, understanding and attitudes towards epilepsy were evaluated in a north Indian population in 1992 by personal interview method. The study revealed that 92 per cent of the respondents had read or heard about epilepsy. More than 55 per cent knew someone and had seen a case of seizure. Eighty five per cent of the respondents were not aware of the cause of epilepsy or had wrong beliefs. Eighteen and 15 per cent thought epilepsy to be a hereditary disorder and a form of insanity respectively. About 40 per cent of the respondents felt that children with epilepsy should not be sent to school and also objected to their children's contact with epileptics at school or at play. Two-thirds of the respondents objected to their children marrying a person who had ever had epilepsy. Twenty per cent were ignorant about the manifestation of epilepsy and an equal number were unable to recommend any therapy in case their relatives or friends had epilepsy. Fifty seven per cent did not know what kind of first aid should be given during the epileptic attack. Although the awareness of epilepsy among Indian people was comparable to that of individuals in Western countries, the attitudes of the Indians were much more negative. Better educated people belonging to higher occupational groups were less prejudiced against social contact and schooling of their children with epileptic children compared to low educational and occupational groups. PMID- 7558209 TI - Comparison of HPLC & EMIT methods of cyclosporine assay in blood after bone marrow transplantation. AB - Cyclosporine (CsA) analysis in blood from patients who had undergone bone marrow transplantation for various haematological disorders was done both by high performance liquid chromatography (HPLC) and enzyme multiplied immunoassay technique (EMIT) and the results were compared. HPLC kit from Biorad Laboratories, USA, and EMIT kit from SYVA, UK, were used. The procedure for EMIT was slightly modified in-house to suit the Hitachi 704 discrete selective analyser. The CsA values obtained by these two methods correlated well within the therapeutic range (r value 0.96), HPLC method being most suitable outside the therapeutic range. Although HPLC is the ideal method for CsA, EMIT is quite suitable and can be adopted by any laboratory with an autoanalyser incorporating our modified procedure. PMID- 7558210 TI - Stereological analysis of the sexually mature rat thymus after orchidectomy. AB - To assess the significance of gonadal function in the maintenance of thymic structure, male, sexually mature, 60 days old rats were bilaterally orchidectomized and the volumes of both the thymic compartments, total cellularity of each of them, as well as density (Nv) and mean diameter (D) of thymocytes within the specific thymic regions were stereologically analyzed 3, 7 and 15 days later. Three days after the orchidectomy a decrease in the thymic weight, and a reduction of both the cortical and medullary volumes, were observed. The reduction in cortical volume was followed by a decrease in overall number of the cortical thymocytes, suggesting an enhanced process of thymocyte death. In the same animals, the total number of the medullary thymocytes as well as D remained unchanged. Seven days after orchidectomy both volume of the cortex and total number of the cortical thymocytes reached the control values, while all the changes observed in the medulla of rats 3 days post castration, persisted. In addition, 7 days post castration, the volume of the interlobular connective tissue was increased and this persisted at day 15 post castration. The results indicate that orchidectomy can affect the thymic structure, and possibly the process of intrathymic T cell maturation; the extent of changes depending on the duration of the gonadal deprivation. PMID- 7558211 TI - Outbreak of leptospirosis with pulmonary involvement in north Andaman. AB - An outbreak of acute febrile illness with haemorrhagic manifestations and pulmonary involvement occurred in Diglipur of North Andaman during October November 1993. Investigations were carried out to see whether leptospires were responsible for this outbreak. Serum samples were collected from suspected cases and tested for presence of antibodies to leptospires by microscopic agglutination test (MAT) using a battery of 19 antigens representing 16 serogroups. 66.7 per cent of the specimens showed significant titres of antibodies against leptospires, 18 of 23 paired sera (78.3%) showed sero-conversion or four-fold rise in antibody titres. The commonest serovar involved was Leptospira grippotyphosa followed by L. canicola and L. JEZ bratislava. In 7 patients L. grippotyphosa was the sole serovar against which antibodies were detected. Clinical and epidemiological observations of this outbreak were similar with that of earlier seasonal outbreaks of acute febrile illness with haemorrhagic manifestations occurring in the same area, indicating that the past outbreaks may also have been due to leptospires. This is the first report of pulmonary leptospirosis from India. PMID- 7558212 TI - Influence of intraumbilical oxytocin on the third stage of labour. AB - In this study the effect of intraumbilical oxytocin on duration and amount of blood loss in third stage of labour was studied. Pregnant women were randomized into 2 groups of 50 each. Group I was managed actively with 10 units of oxytocin diluted in 20 ml saline given through umbilical vein immediately after cord claming and Group II managed traditionally with oxytocin infusion 10 units in 250 ml of dexrose saline at rate of 125 ml/hr given after delivery of baby. In the study group there was a statistically significant decrease in duration of third stage of labour <1.48 min vs 3.27 min>, fall in haemoglobin <1.2 g/dl vs 1.96 g/dl> and fall in haematocrit <3.88% Vs 7.20%<. It was concluded that intraumbilical oxytocin appears to be a useful, safe and practical method for active management of third stage. PMID- 7558213 TI - Myocarditis in enteric fever. AB - 1. Myocarditis in the course of enteric fever is a common occurrence. In a series of 100 bacteriologically or serologically proved enteric fever, we found 7 cases with clinical evidence of myocarditis and 46 cases with ECG evidence of myocarditis. 2. Commonest ECG abnormality was Q-Tc prolongation (29%) followed by ST-T changes (20%) bundle branch block (7%) first degree A-V Block (%) and arrhythmia (2%). 3. All the ECG changes were transient except bundle branch block which persisted in 3 cases. 4. Those with other systemic complications had a higher chance of having myocarditis (P < 0.01). 5. Autopsy evidence of myocarditis was found in 2 cases. With our results, it is obvious that ECT must be recorded in all cases of enteric fever. Those with ECG changes must be observed carefully for clinical evidence of myocarditis. All these patients must have absolute bed rest. Judicious use of corticosteroids is indicated in selected cases of selected cases of severe myocarditis. Diuretics are indicated in cases with evidence of congestive cardiac failure. PMID- 7558215 TI - Arsenic adulteration in Indian tobacco. AB - Sixty samples of smoking and chewing tobacco from subjects in and around Ludhiana, Punjab were tested for arsenic content. No significant difference between the two groups could be detected. The probable role of arsenicosis in neuropathy and liver disease in the Indian population is discussed. PMID- 7558216 TI - Leukemoid reaction in amoebic liver abscess. AB - A case of amoebic liver abscess with leukemoid reaction is described. This patient also had jaundice as the presenting feature. Therapy with metronidazole resulted in improved general condition and both blood picture and liver function tests also became normal. PMID- 7558214 TI - Gall stones in children (non hemolytic) (case reports). AB - Gall bladder disease is considered rare in child hood and hence is frequently over looked in the differential diagnosis as the classical clinical picture and laboratory confirmation in adults does not apply to children. Attention must be paid to the possibility of gall stones in the evaluation of children with non specific abdominal symptoms. Ultrasonography has become a very accurate tool for the evaluation of these children. Once the diagnosis of cholelithiasis is established, elective cholecystectomy is the treatment of choice. PMID- 7558217 TI - Recent advances in the management of Rh hemolytic disease of the newborn (HDN). PMID- 7558218 TI - Investigation of the phenylethanolamine N-methyltransferase gene as a candidate gene for hypertension. AB - Genetic mapping studies have located a gene, Bp1, that accounts for approximately 30% of the genetic variation in the stroke-prone spontaneously hypertensive rat (SHRSP) to a region on chromosome 10 containing the angiotensin-converting enzyme gene. In humans, the gene encoding phenylethanolamine N-methyltransferase (PNMT) was localized near the angiotensin-converting enzyme gene on human chromosome 17. Since most of human chromosome 17 is known to be homologous to rat chromosome 10 and PNMT is known to play a role in blood pressure homeostasis, we reasoned (1) that the rat gene encoding PNMT (Pnmt) may reside on chromosome 10 within the confidence interval containing Bp1 and (2) that Pnmt is a good candidate gene for Bp1. With the use of a somatic cell hybrid panel and genetic mapping techniques, Pnmt mapped within the confidence interval that contains Bp1. To examine further this possibility of Pnmt as a candidate for Bp1, we cloned and characterized Pnmts of the original parental strains, the Wistar-Kyoto rat and SHRSP from the Heidelberg colony. We did not identify any sequence differences between the Wistar-Kyoto rats and SHRSP in the primary structure, in 1077 bp of the 5' flanking region, or in the 256-bp 3'-end region, making Pnmt an unlikely gene for the genetic basis of salt-loaded hypertension. PMID- 7558219 TI - Renal circulation and blockade of the renin-angiotensin system. Is angiotensin converting enzyme inhibition the last word? AB - The mechanism by which angiotensin-converting enzyme (ACE) inhibition influences renal perfusion and function has assumed growing importance as alternatives for blocking the system have emerged. Neither renin inhibitors nor angiotensin II (Ang II) antagonists are likely to trigger responses similar to ACE inhibitor induced involvement of kinins, prostaglandins, or nitric oxide. Several observations suggest species variation in the contribution of these pathways to the renal response to ACE inhibition. In humans, recent investigation suggests that virtually all of the renal response is due to a fall in Ang II formation. Perhaps most persuasive is the surprising observation that the renal hemodynamic response to renin inhibitors exceeds by more than 50% the response to ACE inhibition in healthy humans. To the extent that kinins or prostaglandins contribute to the renal response to ACE inhibition, one would anticipate a smaller response to renin inhibition. Possible explanations include an unanticipated additional action of renin inhibitors, better tissue penetration of these highly lipophilic agents, or more effective blockade of Ang II formation through an action at the rate-limiting step or non-ACE-dependent Ang II generation. Substantial evidence favors the latter two possibilities. Whatever the explanation, these observations raise the intriguing possibility that the undoubted therapeutic efficacy of ACE inhibition in renal injury, documented most rigorously for type I diabetes mellitus, might be exceeded with the newer classes of agent. PMID- 7558221 TI - Weight threshold and blood pressure in a lean black population. AB - Hypertension is virtually absent in very lean rural African populations but is becoming more common in higher-weight urban African populations and is very common in predominantly obese Westernized black populations. This implies that there is a threshold above which weight is related to blood pressure. We studied urban Nigerian civil servants, a lean population in transition toward a more Westernized lifestyle. Blood pressure, fat-related measurements, fasting insulin, physical activity, alcohol intake, macronutrient intake, and electrolyte excretion were measured in 500 male and 299 female civil servants in Benin City, Nigeria, in 1992. Median body mass index (BMI) was 21.5 kg/m2 in men and 24.0 kg/m2 in women. Examination of age-adjusted mean blood pressure across quantiles of BMI in men and women suggested a threshold of 21.5 kg/m2 below which blood pressure was not correlated with BMI. Above this threshold blood pressure was correlated with BMI. Comparison of groups above and below the lower BMI threshold found that differences in blood pressure-BMI covariation were not explained by differences in alcohol intake, caloric or macronutrient intake, or electrolyte excretion. Physical activity was higher in men below the threshold. Fasting insulin and waist-hip ratio were strongly correlated with BMI even in this very lean population but neither was independently related to blood pressure. We conclude that there is a threshold below which little relationship between blood pressure and weight is observed. Above this threshold even at levels considered lean in US blacks, weight is a major determinant of blood pressure in this population of African blacks, which shares ancestry with US blacks. PMID- 7558220 TI - Combined renal effects of overweight and hypertension. AB - The existence of a direct relationship between body mass and arterial pressure is well recognized; however, the effect of obesity on known target organs of hypertension is not clearly understood. We undertook the present studies to assess the influence of obesity on renal function and urinary albumin excretion in 40 normotensive subjects and 80 nevertreated hypertensive patients matched for age, sex, arterial pressure level, and known duration of hypertension in whom an oral glucose tolerance test was within normal limits. Glomerular filtration rate and effective renal plasma flow (expressed as absolute values or values normalized for height) were increased in overweight compared with lean subjects whether normotensive or hypertensive. Glomerular filtration rate was positively correlated with protein intake (as assessed from urinary excretion of urea) and fasting serum insulin level. Urinary excretion of albumin but not IgG and beta 2 microglobulin was higher in hypertensive patients compared with normotensive subjects. The overweight condition clearly enhanced the influence of arterial pressure on albuminuria; in fact, a steeper regression line between albumin excretion rate and arterial pressure was found in overweight compared with lean subjects. These results indicate that the overweight condition is associated with renal hyperfiltration and hyperperfusion, irrespective of the presence of hypertension, and that obesity magnifies the effect of hypertension on albuminuria, thus raising the possibility of an increased susceptibility of obese hypertensive patients to the development of renal damage. PMID- 7558222 TI - Reduced renal extraction of atrial natriuretic peptide in primary aldosteronism. AB - We investigated renal and peripheral forearm extraction of atrial natriuretic peptide in patients with primary aldosteronism to determine whether alterations in extraction may contribute to the elevated levels of circulating atrial natriuretic peptide observed in primary aldosteronism. We obtained simultaneous venous blood samples from the left renal vein and a peripheral vein and from the radial artery in 28 patients with primary aldosteronism and 10 patients with essential hypertension. Renal extraction of atrial natriuretic peptide was significantly (P < .001) reduced (40 +/- 2%) in primary aldosteronism compared with essential hypertensive patients (62 +/- 3%). Peripheral forearm extraction was also reduced (P < .01) in primary aldosteronism compared with essential hypertensive patients (24 +/- 3% versus 38 +/- 4%). These findings are consistent with widespread downregulation of atrial natriuretic peptide receptors in primary aldosteronism. Consistent with reports that marked reduction in glomerular filtration rate is required before the renal extraction of atrial natriuretic peptide is reduced, no significant relationship between renal extraction of atrial natriuretic peptide and plasma creatinine was seen in primary aldosteronism or essential hypertension. Although the major regulators of atrial natriuretic peptide secretion in primary aldosteronism are presumably alterations in arterial blood pressure and plasma volume, reduced renal and peripheral extraction of atrial natriuretic peptide in primary aldosteronism may also contribute significantly to the elevated circulating levels observed. PMID- 7558223 TI - Acute effects of physiological increments of brain natriuretic peptide in humans. AB - To investigate the effects of physiological increases in plasma brain natriuretic peptide concentration in humans, we studied six healthy volunteers who received incremental infusions (0.25 pmol/kg per minute in the first hour and 0.50 pmol/kg per minute in the second) of synthetic human brain natriuretic peptide-32 in a placebo-controlled, crossover study. Peptide plasma levels were 1.69 +/- 0.39 pmol/L at baseline and rose 1.5- and 3-fold with the lower and higher doses, respectively. These values were within the normal range and also comparable to those reported in patients with mild essential hypertension. The urinary excretion rate of cGMP also increased during brain natriuretic peptide infusion, indicating stimulation of natriuretic peptide receptors. Peptide administration induced a significant 1.7-fold increase in urinary sodium excretion without affecting renal plasma flow (para-aminohippurate clearance), glomerular filtration rate (creatinine clearance), and urine flow rate. Fractional proximal sodium reabsorption (lithium clearance method) was unchanged, and fractional distal sodium reabsorption significantly decreased. Brain natriuretic peptide caused no changes in arterial pressure, heart rate, hematocrit, and serum proteins, but it exerted an inhibitory effect on the renin-aldosterone axis, as indicated by the significant 50% or more decrease of plasma renin activity and urinary excretion rate of aldosterone. These results suggest that brain natriuretic peptide may be involved in the overall regulation of body fluid and cardiovascular homeostasis in humans, mainly through its natriuretic and endocrine effects. PMID- 7558225 TI - Apoptosis in target organs of hypertension. AB - Apoptosis or programmed cell death frequently parallels abnormalities in cell proliferation and differentiation. As hypertrophy/hyperplasia or remodeling occurs in organs affected by hypertension, we evaluated the degree of apoptosis in the heart, kidney, and brain in situ in genetically hypertensive mice and rats as well as in cultured vascular smooth muscle cells. Apoptosis was characterized by morphological features, DNA fragmentation, and laddering as well as by terminal deoxynucleotidyl transferase labeling of the 3' OH ends of both extracted DNA and tissue sections. The present report provides the first evidence of increased apoptosis in whole organs of genetically hypertensive rat and mouse strains: in the heart of spontaneously hypertensive rats (SHR) and in the heart (ventricular cardiomyocytes), kidney (inner cortex and medulla), and brain (cortex, striatum, hippocampus, and thalamus) of spontaneously hypertensive mice, with a higher effect of apoptotic inducers in cultured aortic smooth muscle cells derived from SHR. Both types of known apoptotic processes, oligonucleosomal cleavage and large DNA fragmentation, were observed in vascular smooth muscle cells, but only the former appeared to be increased in SHR. This study underlines the importance of cell death dysregulation in hypertension, reveals a new route for investigation of the pathogenesis of hypertension, and suggests novel targets of therapeutic intervention. PMID- 7558224 TI - Atrial natriuretic peptide in chronically hypertensive dogs. AB - We determined the renal and depressor activities of 10, 50, and 100 pmol/kg per minute i.v. of human atrial natriuretic peptide-(99-126) in conscious one-kidney, one clip dogs with chronic hypertension and modest renal dysfunction, as indicated by mild proteinuria. Atrial natriuretic peptide increased fractional sodium excretion by 0.009 +/- 0.002, 0.042 +/- 0.005, and 0.049 +/- 0.007, respectively; urinary excretion of atrial natriuretic peptide by -0.4 +/- 0.8, 3.3 +/- 1.4, and 15.8 +/- 7.4 fmol/min; and cGMP excretion by 0.65 +/- 0.06, 1.65 +/- 0.08, and 4.88 +/- 0.85 nmol/min in one-kidney shams. The changes in fractional sodium excretion were significantly attenuated in the hypertensive dogs (0.005 +/- 0.002, 0.018 +/- 0.003, and 0.022 +/- 0.004, respectively) despite exaggerated increases in atrial natriuretic peptide excretion (3.3 +/- 1.6, 22.0 +/- 5.0, and 46.6 +/- 10.8 fmol/min) and cGMP excretion (0.96 +/- 0.47, 4.51 +/- 1.27, and 7.06 +/- 1.38 nmol/min). The slope of the line relating urinary atrial natriuretic peptide to cGMP was significantly suppressed in the hypertensive dogs, suggesting a downregulation of the guanylate cyclase-linked receptors. The slope of the relationship between cGMP excretion and the natriuretic response was also depressed in the hypertensive dogs, indicating possible impairment of cGMP signal transduction. The differences between sham and hypertensive dogs were diminished when urinary levels of atrial natriuretic peptide were maximized by prior treatment with SQ 28603, an inhibitor of neutral endopeptidase EC 3.4.24.11. Atrial natriuretic peptide caused comparable decreases in mean arterial pressure and increases in glomerular filtration rate in sham and hypertensive dogs, suggesting similar vascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558226 TI - Sodium-proton exchange and primary hypertension. An update. AB - An enhancement of sodium-proton exchange in blood cells of patients with primary hypertension has been described by various investigators. The present review summarizes some of the most recent findings regarding the enhanced sodium-proton exchanger activity in primary hypertension and discusses the potential mechanisms that may contribute to or explain these findings. Novel evidence has been accumulated on the in vivo regulation of the sodium-proton exchanger in humans, and recent findings suggest that metabolic acidosis, high NaCl intake, and circulating hormones (eg, insulin) can enhance sodium-proton exchanger activity in blood cells. However, the relative roles of such exogenous factors in the stimulation of sodium-proton exchanger activity in primary hypertension remain questionable because enhanced sodium-proton exchanger activity persists in immortalized lymphoblasts from patients with primary hypertension after prolonged cell culture. Therefore, at least in a certain group of hypertensive subjects this abnormality cannot be due to metabolic or hormonal alterations of the "hypertensive" in vivo milieu but appears to be under genetic control. Available evidence strongly argues against intrinsic changes of the sodium-proton exchanger protein itself in primary hypertension, for example, a mutation in the encoding gene. Interestingly, immortalized cells from hypertensive subjects with enhanced sodium-proton exchanger activity display a distinctly enhanced proliferation pattern that appears to be independent of this ion transport. At present we speculate that enhanced sodium-proton exchanger activity and proliferation may represent indicators of a genetically fixed enhanced intracellular signal transduction in primary hypertension that may be caused by an increased activation of pertussis toxin-sensitive G proteins. PMID- 7558227 TI - Renin mRNA, quantified by polymerase chain reaction, in renal hypertensive rat tissues. AB - To determine responses in renin gene expression in different tissues of two kidney, one clip hypertensive Sprague-Dawley rats and the effect of NaCl loading, we developed a novel, highly sensitive quantitative polymerase chain reaction technique and measured renin mRNA at 19 and 40 days after clipping. Basal renin mRNA concentrations were 1575 +/- 127 fg/micrograms total RNA in kidney, 52 +/- 7 in hypothalamus, 7.9 +/- 0.7 in adrenal, and 4.7 +/- 0.5 in atria. In two-kidney, one clip rats, renin mRNA in the clipped kidney was increased 5.4-fold (P = .00001) and 2.3-fold (P = .001) on each respective day after clipping and in the unclipped kidney was decreased by 27% (P = .01) and 38% (P = .04). In adrenal, 3.9-fold (P = .004) and 1.7-fold (P = .02) increases were seen on days 19 and 40, respectively, and a decrease of 57% (P = .02) was found in a hypothalamic block at day 19 but not at day 40. The decrease in hypothalamus was abolished by 1% oral NaCl, which reduced renin mRNA by 37% in the clipped kidney and by 30% in the adrenal but did not lead to any change in the unclipped kidney or hypothalamus at day 40. Hypothalamic renin mRNA was also decreased by enalapril compared with increases of sixfold to ninefold in other tissues. In conclusion, we have quantified a decrease in hypothalamic renin mRNA in two-kidney, one clip rats 19 days after clipping that can be abolished by NaCl loading, whereas in the adrenal, renin mRNA was increased. Similar relative tissue-specific changes were also seen in enalapril-treated rats. PMID- 7558229 TI - Effects of hypertension and dyslipidemia on the decline in renal function. AB - Experimental evidence suggests that in addition to hypertension, serum lipids might also accelerate the decline in renal function. We tested this hypothesis in 2702 dyslipidemic middle-aged men without renal disease participating in the Helsinki Heart Study, a coronary primary prevention trial. The decline in renal function was estimated from linear regression slopes based on reciprocals of 10 serum creatinine determinations over the study period. Renal function deteriorated 3% on average during the 5-year study, and hypertension accelerated this change. Subjects with an elevated ratio of low- to high-density lipoprotein cholesterol ( > 4.4) had a 20% faster decline than those with a ratio less than 3.2. Both the contribution of the lipoprotein ratio and the protective effect of high-density lipoprotein cholesterol alone remained significant in multiple regression analyses. In the study of joint effects the contribution of lipids was confined to subjects with simultaneous elevation of blood pressure and lipids. The results suggest that in addition to hypertension, blood lipids also modify the decline in renal function. PMID- 7558228 TI - Glucocorticoids regulate V1a vasopressin receptor expression by increasing mRNA stability in vascular smooth muscle cells. AB - Enhancement of vascular responsiveness is considered to be one of the major contributing factors observed in glucocorticoid-induced hypertension. We examined the effects of glucocorticoids on V1a arginine vasopressin receptor mRNA and protein levels in vascular smooth muscle cells. Dexamethasone (1 mumol/L) produced a 1.8-fold increase in V1a receptor density without changing its affinity. Steady-state values of V1a receptor mRNA, analyzed by Northern blotting, increased 2.7-fold after a 12-hour exposure to dexamethasone. This effect of dexamethasone was blocked by the glucocorticoid antagonist RU38486 and did not occur in the presence of the protein synthesis inhibitor cycloheximide. The V1a receptor gene transcription rate, determined by nuclear run-off assays, was unchanged in cells treated with dexamethasone for 12 hours. Dexamethasone increased the half-life of V1a receptor mRNA by 2.2-fold. These findings suggest that dexamethasone upregulates the expression of the V1a receptor by increasing mRNA stability rather than by gene transcription and that de novo protein synthesis is involved in this regulation. PMID- 7558230 TI - Acute exercise attenuates cardiac autonomic regulation in hypertensive rats. AB - Dynamic exercise may be used as a safe, therapeutic approach to reduce sympathetic nerve activity at rest and thus may be beneficial for individuals with hypertension. Therefore, we tested the hypothesis that a single bout of mild to moderate dynamic exercise would decrease cardiac sympathetic tonus at rest. We designed two experimental protocols to test this hypothesis in male spontaneously hypertensive rats. In protocol 1 (n = 6) cardiac sympathetic tonus and parasympathetic tonus were determined before and after a single bout of dynamic exercise. We developed protocol 2 (n = 5) to determine the component of the autonomic nervous system responsible for the postexercise reduction in heart rate. Rats were instrumented with catheters inserted into the descending aorta for measurements of arterial pressure, mean arterial pressure, and heart rate and into the jugular vein for infusion of drugs. A single bout of mild to moderate dynamic treadmill exercise (12 m/min, 10% grade for 42 +/- 1 minutes, representing approximately 74% to 79% of maximal heart rate) resulted in a postexercise reduction in mean arterial pressure (163 +/- 7 to 149 +/- 5 mm Hg; P < .05). Associated with the postexercise hypotension was a reduction in sympathetic and parasympathetic tonus (47 +/- 12% and 71 +/- 12%, respectively). The reduction in heart rate during the early recovery phase was due to a withdrawal of sympathetic tonus, because beta 1-adrenergic receptor blockade significantly enhanced the postexercise reduction in heart rate, and muscarinic cholinergic receptor blockade did not affect the postexercise decrease in heart rate until 20 minutes after exercise. PMID- 7558232 TI - Sympathetically mediated hypertension caused by chronic inhibition of nitric oxide. AB - Pharmacological inhibition of nitric oxide synthase causes sustained hypertension in many animal species. Although this hypertension has been attributed to inhibition of endothelium-dependent vasodilation, short-term studies in anesthetized preparations have advanced the hypothesis that there could be a sympathetic component to this hypertension. To test this hypothesis we measured intra-arterial pressure directly before and after 1 week of treatment with the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, approximately 80 mg/kg per day in drinking water) in conscious unrestrained rats with or without chronic guanethidine-induced sympathectomy. The major new finding is that the hypertensive response to L-NAME was greatly attenuated by sympathectomy. With L-NAME, mean arterial pressure increased from 101 +/- 3 to 152 +/- 6 mm Hg in rats without sympathectomy (n = 11) but only from 96 +/- 2 to 122 +/- 3 mm Hg in rats with sympathectomy (n = 15, +52 +/- 5 versus +27 +/- 4 mm Hg, P < .01). Sympathectomy did not alter maximal endothelium-dependent vasodilation assessed by femoral vascular responses to intra-arterial acetylcholine or bradykinin, indicating that the differing hypertensive responses to L-NAME in rats with versus without sympathectomy could be related to inhibition of neuronal rather than endothelial nitric oxide synthesis. We also found that L-NAME-induced hypertension, once developed, is completely reversed by acute ganglionic blockade. In conclusion, these findings identify an important sympathetic neural component to the sustained hypertension produced by pharmacological inhibition of nitric oxide in the rat. PMID- 7558231 TI - Relationship of arachidonic acid release to porcine coronary artery relaxation. AB - In porcine coronary artery endothelium-dependent relaxation to bradykinin is in part attributed to a chemically unidentified factor, termed endothelium-derived hyperpolarizing factor (EDHF). We hypothesize that arachidonic acid, acting through a cyclooxygenase-independent mechanism, is responsible for EDHF production. To define the relationship between EDHF production and arachidonic acid release, we investigated the role of phospholipase C in bradykinin-induced relaxation and prostaglandin I2 production (an index of arachidonic acid release) in porcine coronary artery. The phospholipase C inhibitor U73122 (1 mumol/L) abolished bradykinin-induced, nitric oxide-mediated relaxation but did not inhibit either bradykinin-induced, EDHF-mediated relaxation or prostaglandin I2 production. However, when given at a larger dose (20 mumol/L) U73122 abolished both bradykinin-induced, EDHF-mediated relaxation and prostaglandin I2 production. Similarly, the calcium-ATPase inhibitor thapsigargin, given at a dose (1 mumol/L) that abolished bradykinin-induced increases in intracellular calcium concentration in cultured porcine coronary artery endothelial cells, eliminated both bradykinin-induced. EDHF-mediated relaxation and prostaglandin I2 production. Although thapsigargin abolished bradykinin-induced prostaglandin I2 production, the basal production of prostaglandin I2 was enhanced and contraction of endothelium-intact rings was attenuated. These latter responses are most likely related to enhanced basal arachidonic acid release and associated EDHF production. These observations suggest that phospholipase C activation and increased intracellular calcium concentration are required for both bradykinin induced arachidonic acid release and EDHF production in porcine coronary artery.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558233 TI - Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. AB - Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 mumol/L isobutylmethyl-xanthine or 0.01 to 10 mumol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase-polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both alpha1- and beta1-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC alpha1 subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both. PMID- 7558234 TI - Cyclosporine impairs vasodilation without increased sympathetic activity in humans. AB - Hypertension and nephrotoxicity frequently complicate treatment with cyclosporine; two suggested mechanisms are increased sympathetic activity and altered vascular reactivity. It is difficult to assess these mechanisms in patients receiving cyclosporine after transplantation because of the accompanying major physiological alterations. Therefore, we studied 12 patients with rheumatoid arthritis twice--while they were taking and not taking cyclosporine. We measured vascular response in the dorsal hand vein using the linear variable differential transformer technique. Cyclosporine treatment significantly attenuated vasodilation induced by 60 ng/min isoproterenol (no cyclosporine, 19.8 +/- 3.5% versus cyclosporine, 7.9 +/- 2.2%; P = .02) and prostaglandin E1 at 1000 pg/min (no cyclosporine, 72.6 +/- 10.2% versus cyclosporine 45.6 +/- 9.0%) and 2000 pg/min (no cyclosporine, 100.8 +/- 14.7% versus cyclosporine, 68.6 +/- 8.0%; F = 5.47, P = .047). However, neither vascular response to phenylephrine or nitroglycerin nor sympathetic activity assessed by measurement of norepinephrine spillover with a radioisotope dilution technique was affected by cyclosporine (no cyclosporine, 516.1 +/- 47.9 ng/min versus cyclosporine, 476.6 +/- 51.8 ng/min; P = .42). Cyclosporine impaired venodilation in response to two agonists that act through adenylate cyclase without altering alpha-agonist-induced venoconstriction or sympathetic activity. Therefore, in humans impaired vasodilation rather than sympathetic activation or enhanced vasoconstriction may be an important mechanism for the alterations of vascular tone that occur after long-term cyclosporine administration. PMID- 7558235 TI - Effects of chronic estrogen replacement therapy on beat-to-beat blood pressure dynamics in healthy postmenopausal women. AB - Recent data showing gender differences in autonomic control of heart rate and acute estrogen effects on vasodilatation suggest that estrogen may influence autonomic regulation of heart rate and blood pressure. We aimed to determine the effect of postmenopausal estrogen replacement therapy on autonomic control of beat-to-beat heart rate and blood pressure dynamics. Subjects included 20 healthy postmenopausal women aged 60 to 75 years with normal exercise tolerance tests, 10 of whom were taking oral estrogen for 13 +/- 3 (+/- SEM) years. Six healthy premenopausal women were also studied. Continuous electrocardiographic and noninvasive radial artery blood pressure measurements and intermittent forearm blood flow recordings (by venous-occlusion plethysmography) were obtained before and after a 20-minute, 60 degrees head-up tilt and a 420-kcal meal during periods of spontaneous and metronomic breathing (at 0.25 Hz). Low-frequency (0.01- to 0.15-Hz) and high-frequency (0.15- to 0.50-Hz) heart rate and blood pressure spectral powers were compared with a fast Fourier transform. Cardiovascular and heart rate spectral power responses to upright tilt and meal digestion were the same in postmenopausal estrogen users and nonusers. However, during spontaneous breathing the blood pressure spectral power responses to upright tilt and meal ingestion were significantly different between the two groups of women. The low frequency systolic pressure power response to upright tilt was smaller in estrogen users than nonusers (P = .01). After meal ingestion nonusers had an early postprandial fall (20 to 30 minutes after the meal) and late rise (50 to 60 minutes) in low-frequency systolic and diastolic pressure powers, which were significantly attenuated in estrogen users (P < .02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558236 TI - Effect of antihypertensive therapy on small artery structure in hypertensive patients. PMID- 7558238 TI - Multiply myelinated axons in the optic nerve of mice deficient for the myelin associated glycoprotein. AB - We recently reported that some retinal ganglion cell axons in mice deficient for the myelin-associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon-to fiber ratio. PMID- 7558237 TI - AMPA-type glutamate receptors in glial precursor cells of the rat corpus callosum: ionic and pharmacological properties. AB - Glial cells in fiber tracts express various functional transmitter receptors, e.g., got glutamate. However, little is known about their biophysical and pharmacological profile. Using the in situ patch-clamp technique, kainate- and AMPA-induced conductance changes of glial precursor cells in the rat corpus callosum were investigated to study these aspects. Precursor cells were identified by their voltage-gated currents and were easily discernable from astrocytes and oligodendrocytes. Kainate induced two overlying effects in these cells: the activation of a cationic current and the block of potassium conductances. Cesium in the pipette solution blocked potassium conductances nearly completely and the ionic profile of the kainate-induced cationic current could be studied in detail. Full replacement of the sodium in the bath by calcium resulted only in a small kainate-induced (calcium) inward current flow, but the kainate-induced outward current carried by Cs+ was less affected reflecting a weak calcium permeability. The kainate response could be blocked by 6,7 dinitroquinoxaline-2,3-dione (DNQX) and millimolar zinc concentrations. Co application of micromolar concentrations of zinc slightly enhanced the kainate induced current, while Evans Blue was without any significant effect. Cyclothiazide increased the kainate response by a factor of x6 while concanavalin A did not enlarge it. The AMPA-induced current was amplified by a factor of x39 by cyclothiazide. The present data suggested the expression of weakly calcium permeable AMPA receptors on glial precursor cells in the rat corpus callosum. Only a small fraction of the agonist-induced current could be seen without the appropriate blockers of receptor desensitization. An additional expression of kainate-preferring glutamate receptors could not be shown. PMID- 7558241 TI - Chondroitin sulfate proteoglycan staining in astrocyte-Schwann cell co-cultures. AB - Transplantation of Schwann cells (SCs) in the central nervous system (CNS) for remyelination in pathological situations has been considered a promising approach. However, numerous studies have indicated that astrocytes have a restrictive effect on SC migration within the CNS. We have previously established an in vitro model which demonstrates the restrictive effect of astrocytes on SCs (Ghirnikar and Eng, Glia 4:367-377, 1994). Using this culture model, in the present study, we have characterized the molecular basis underlying astrocyte-SC interaction and demonstrated chondroitin sulfate proteoglycan (CSP) staining in the co-cultures. Following 1-2 weeks of incubation, CSP staining was specifically associated with SCs co-cultured with astrocytes. Staining with antibodies specific for the different chondroitin sulfate isomers revealed the presence of both, chondroitin-4- and 6-sulfates in SCs. In contrast, SCs when cultured alone, or in the presence of astrocytes conditioned medium did not show CSP staining. These data suggest that CSP staining is associated with SCs following co-culture with astrocytes and mediated by cell to cell contact. We hypothesize that the CSP, alone or in combination with other molecules expressed by astrocytes and/or SCs, may be involved in the restrictive effects of astrocytes on SCs. Identification of molecules involved in the unfavorable interaction between astrocytes and SCs will have an important bearing on efforts to remyelinate demyelinated axons by SC transplantation within the damaged CNS. PMID- 7558242 TI - Intracellular acidification and Ca2+ transients in cultured rat cerebellar astrocytes evoked by glutamate agonists and noradrenaline. AB - The effect of different neurotransmitters on the intracellular pH (pHi) and intracellular calcium (Ca2+i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 microM) in a HEPES-buffered, CO2/HCO3(-) -free saline induced a decrease in pHi and an increase in Ca2+i. Amplitude and time course of the pHi and Ca2+i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 +/- 0.05 (n = 29) and 0.20 +/- 0.09 (n = 12) pH units, respectively. The changes in pHi and Ca2+i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7 dinitroquinozalin-2,3-dion (CNQX; 50 microM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pHi changes were secondary to the Ca2+ rises was tested. In the absence of extracellular Ca2+, the kainate induced Ca2+i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca2+ reduced the glutamate-induced pHi change by 8%, while the second component of the Ca2+i transient was abolished. Application of trans-( +/- )-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 microM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 microM) evoked a Ca2+i increase, but no change of pHi. D aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca2+i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca2+i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system. PMID- 7558243 TI - The Na(+)-Ca2+ exchange system in rat glial cells in culture: activation by external monovalent cations. AB - Cultured rat glial cells display a Na(+)-Ca2+ exchange system located at the plasma membrane levels. This was evidenced by the Na+ (i)-dependency of a Na+ (o) inhibitable influx of Ca2+, or reversal exchange mode. This antiporter has an external site where monovalent cations (K+, Li+, and Na+ were investigated) stimulate the exchange by a chemical action. The monovalent cation is not transported during the exchange cycle. The mechanism of that stimulation agrees with an increase in the apparent affinity of the carrier for Ca2+(o) without effect on the maximal translocation rate. Two models can equally well account for the data: i) the formation of ECa(o) is essential for the binding of the monovalent cation, or ii) the activating cation can bind even when the carrier is free of Ca2+(o). The cations K+ and Li+ produced only stimulation, although that of K+ seem to require actions other than the chemical effect. The response to Na+ was biphasic; this can be fully explained considering that at low concentrations, Na+(o) binds preferentially to the activating monovalent site while at high concentrations it displaces Ca2+ from its external transporting site. Pure type I astrocytes displayed the same Na(+)-Ca2+ exchange mechanism. PMID- 7558240 TI - Isoform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations. AB - The expression of sodium channel alpha-subunit isoforms in astrocytes cultured from P-0 rat spinal cord and P-7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform-specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11-I, which is directed against a subtype-specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11-II, which is directed against a subtype-specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32-III, which is directed against a subtype-sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11-II, and SP32-III staining persisted in stellate spinal cord astrocytes through 14-21 DIV, while SP20 and SP11-II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11-I, SP11-II, and SP32-III. Flat optic nerve astrocytes lacked or displayed very low SP11-II staining, and SP11-I and SP32-III staining was not detectable. These observations demonstrate that cultures astrocytes are immunoreactive to antibodies generated against conserved and isotype-specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS. PMID- 7558239 TI - Amoeboid microglia expressing GD3 ganglioside are concentrated in regions of oligodendrogenesis during development of the rat corpus callosum. AB - In recent study we demonstrated expression of the platelet-derived growth factor alpha receptor (PDGFR alpha) in cells of the early oligodendrocyte lineage that were identified as either GD3 ganglioside + oligodendrocyte progenitors or O4 sulfatide+ preoligodendrocytes. We also identified a subpopulation of GD3 immunoreactive cells that did not express mRNA for the PDGF receptor. The distinct large amoeboid morphology of these cells was characteristic of cells in the macrophage lineage rather than in the oligodendrocyte lineage. To determine if the GD3-positive but PDGFR alpha mRNA-negative cells were in the macrophage lineage, we compared the spatial and temporal expression patterns of GD3 ganglioside and ED1, a macrophage-specific antigen. Analysis prenatally indicated that at embryonic day 15, ED1+ and GD3+ cell populations resided in the subpial connective tissue. At embryonic day 21, these two populations were seen in a region extending from the lateral ventricle through the subventricular and intermediate zones. In this study we report that these large, round, GD3 immunoreactive cells have the same cell morphology and anatomical distribution as the ED1 immunoreactive cells. Both cell populations contained pyknotic nuclei within their cytoplasm. Furthermore, the GD3+/PDGFR alpha- cells appear to be involved in clearing cellular debris in regions of gliogenesis. These data suggest that this subpopulation of GD3 immunoreactive cells belongs to the microglia/macrophage lineage. PMID- 7558244 TI - Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures. AB - Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O2) VEGF expression in astrocytes in barely detectable by northern analysis. However, after exposure to 0.2% O2 for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl2 or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme-containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia-induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC-dependent and -independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia-mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin-sensitive tyrosine kinase may serve as negative regulators of the hypoxia-activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway. PMID- 7558245 TI - Interferon-gamma and lipopolysaccharide reduce cAMP responses in cultured glial cells: reversal by a type IV phosphodiesterase inhibitor. AB - The aim of the present study was to determine whether two classical macrophage activators, bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) could affect the accumulation of the second messenger cAMP in cultured rat microglia and astrocytes. Purified microglia and astrocyte secondary cultures obtained from the neonatal rat were grown for 3 days in basal medium Eagle (BME) + 10% fetal calf serum (FCS). Exposure of microglia to LPS resulted into a dose- and time-dependent decrease in the accumulation of cAMP induced by receptor mediated (isoproterenol or prostaglandin E2) or direct (forskolin) activation of adenylate cyclase. The inhibitory effect of LPS was rapid (a 10 min preincubation was sufficient to approach a maximal effect), occurred at low doses (IC50 = 1.2 ng/ml), and was not abrogated by pertussis toxin. A selective inhibitor of type IV phosphodiesterase (rolipram, 100 nM) prevented the effect of LPS on cAMP accumulation, while inhibitors of other forms of phosphodiesterase were unable to do so. IFN-gamma (100 u/ml) also caused a depression of the evoked cAMP accumulation in microglia after a 10 min preincubation, and its effect was prevented by rolipram, as in the case of LPS. Astrocytes differed from microglia in that LPS (1-100 ng/ml) did not inhibit the accumulation of cAMP induced by either isoproterenol or forskolin; on the other hand, IFN-gamma did have an inhibitory effect (though less pronounced than in microglia) that could be prevented by rolipram.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558247 TI - Production of reactive nitrogen intermediates (RNI) by peritoneal macrophages from rats with experimental autoimmune prostatitis (EAP). AB - Peritoneal macrophages from experimental autoimmune prostatitis (EAP) rats were examined for their capacity to secrete reactive nitrogen intermediates (RNI), measured by the release of nitrite (NO2-). Under basal conditions, there was a significant increase of NO2- secretion by cells from autoimmune rats in relation to resident cells. After stimulation in vitro with lipopolysaccharide (LPS), the NO2- production was higher in cells from autoimmune rats compared to treated and nontreated controls. The NO2- production was dependent upon the presence of L arginine in the culture medium. The addition of L-NG-monomethyl arginine, an inhibitor of nitric oxide synthesis, to the medium reduced the amount of measurable NO2-. Kinetic studies in cells from EAP rats showed that in basal conditions there was an significant release of NO2- at day 7 of immunization that was maintained during the whole period studied. After LPS stimulation, there was a similar behavior and maximum values were reached at day 28 of immunization. These results, together with the lesion observed in the prostate gland, suggest that RNI may be of pathogenic importance in the development of early tissue inflammation and autoimmune disease of the prostate. PMID- 7558249 TI - Transmucosal potential difference in experimental colitis in rats. AB - Colon transmucosal potential difference (TPD), macro- and microscopic lesions, myeloperoxidase activity, and leukotriene levels were studied after the induction of experimental colitis in the rat. Forty-three male Wistar rats were subjected to the instillation of 200 mg/ml 2,4,6-trinitrobenzenesulfonic acid (TNB) solution through a rectal cannula. TPD measurements were made at different distances from the anus before and 24 h and one, two, three, and four weeks after lesion induction. Leukotriene B4 levels were assayed by intracolonic dialysis 24 h and one, two, three and four weeks after lesion induction. Macro- and microscopic evaluations were made of the bowel lesions, and myeloperoxidase activity was assayed. The mean basal TPD was -46.06 mV at 1 cm from the anus, and +10.86 mV in the proximal colon. Twenty-four hours after lesion induction the values proved markedly positive. This was correlated with an abrupt increase in LTB4 levels and myeloperoxidase activity. After one week the TPD values exhibited a greater electronegativity, returning to basal values by the fourth week after lesion induction. This coincided with an improved macroscopic lesion index, LTB4 levels, and myeloperoxidase activity. In conclusion, TPD is a useful indicator of acute colonic lesions and correlates well with LTB4 and myeloperoxidase assays. Moreover, the parameter is able to delimit lesion evolution, reflecting possible ad integrum restoration of the bowel mucosa. PMID- 7558246 TI - Plasma levels of beta-carotene are inversely correlated with circulating neutrophil counts in young male cigarette smokers. AB - Relationships between plasma levels of beta-carotene (BC) and the numbers and oxidant-generating activities of circulating neutrophils have been investigated in a group of asymptomatic young male cigarette smokers (N = 40) and in a group of nonsmoking control subjects. Plasma BC levels were measured using HPLC, while oxidant generation was measured using a phorbol myristate acetate (PMA) activated whole blood luminol-enhanced chemiluminescence (LECL) method. Relative to nonsmokers, the numbers of circulating neutrophils, as well as the LECL responses of these cells, were increased by 41% (p = 0.0001) and 47% (p = 0.004), respectively, while plasma BC levels were decreased by 24% (p = 0.01). In cigarette smokers, but not in nonsmokers, the numbers of circulating neutrophils, as well as the LECL responses of these cells, were significantly and inversely correlated with plasma BC levels (r = -0.36, p = 0.02; and r = -0.33, P = 0.04 respectively). Diminished plasma levels of BC in cigarette smokers probably reflect the increased numbers and prooxidative activities of circulating neutrophils. Intake of this antioxidant nutrient may be a determinant of susceptibility to smoking-related pulmonary dysfunction mediated by oxidants derived from smoke-activated phagocytes. PMID- 7558248 TI - Differentiation of human osteoblastic cells in culture: modulation of proteases by extracellular matrix and tumor necrosis factor-alpha. AB - We have followed the synthesis and secretion of urokinase-type plasminogen activator (u-PA) and its inhibitor, PAI-1, and matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP-1) during differentiation of a human osteoblastic cell line, HOS TE85, and the effect of TNF-alpha on this process. Our results show that the ratio of u-PA/PAI-1 associated with the cell matrix components increases during differentiation of these cells over a 14-day period. Although TNF-alpha suppresses the induced increase in steady-state mRNA levels of u-PA and PAI-1 during maturation of extracellular matrix (ECM), the u PA/PAI-1 ratio is altered in such a way that PA activity associated with the ECM is higher than control cells. The expression of MMP-1 is low and remains essentially invariant over a culture period of 14 days. TNF-alpha enhances MMP-1 transcription nearly 12-fold initially, after which mRNA levels drop off but remain significantly higher than the controls. Activities and steady-state mRNA levels of MMP-2 and MMP-9 increase nearly 15-fold during maturation of the ECM, but the level of TIMP-1 mRNA is not appreciably altered. The presence of TNF alpha suppresses maturation-induced transcription of MMP-2, enhances TIMP-1 transcription, but has little effect on MMP-9 mRNA levels. The data show that chronic exposure to TNF-alpha alters the balance between u-PA/PAI-1 and MMPs/TIMP 1, which favors higher activity of proteinases. Accordingly, the presence of TNF alpha in chronic inflammatory episodes would be expected to alter bone remodeling by inhibiting maturation of ECM and formation of bone. PMID- 7558250 TI - Transient endotoxemia during burn wound revision causes leukocyte beta 2 integrin up-regulation and cytokine release. AB - After severe burns, a wound revision is often done to remove devitalized tissue and minimize bacterial growth. After such revision, the patient may show signs of sepsis. In a group of burned patients we found a transient endotoxemia, and a subsequent leukocyte activation, monitored as increased expression of the beta 2 integrin CD11b, after such wound revision. In most patients we could detect elevated levels of plasma TNF-alpha before the operation, with no increases in these levels after the operation. Plasma levels of IL-6 were elevated in all patients and increased after the wound revision in all patients. They also had elevated plasma levels of soluble E-selectin, indicating systemic inflammation. The close relation between endotoxin levels and CD11b expression, and lack of evidence for additional production of TNF-alpha, suggests that up-regulation of the beta 2 adhesion protein during wound revision is mainly caused by endotoxin interaction with the leukocyte. PMID- 7558252 TI - Flow cytometric approach to human polymorphonuclear leukocyte activation induced by gingival crevicular fluid in periodontal disease. AB - In gingival pockets of patients with periodontal disease, polymorphonuclear leukocytes (PMN) are in contact with a peculiar exudate, the gingival crevicular fluid (GCF). Because of the pivotal role played by PMN in periodontal disease, we evaluated the ability of GCF in modulating normal human PMN. GCF was obtained from two gingival sites with severe periodontitis (SP) and two gingival sites with only mild periodontitis (MP) in 12 patients. Purified PMN were exposed to GCF from SP and MP sites and, as a control, to sterile culture medium. GCF activity was evaluated by monitoring the modulation of membrane molecules relevant to cell function. Compared to control medium, GCF from SP and MP sites was able to induce an activation status in PMN evidenced by an increased CD11b (62 +/- 9% and 28 +/- 7%, respectively) and f-Met-Leu-Phe (56 +/- 5% and 31 +/- 7%, respectively) receptor expression, with a concomitant reduction of CD62L expression (56 +/- 8% and 23 +/- 7%, respectively). Thus, reflecting the clinical status, GCF from SP sites was significantly more efficient in affecting PMN than GCF from MP sites. Cell size modifications, evaluated as an additional indicator of PMN activation, were consistent with membrane molecule modulation. The difference in PMN-activating capacity between SP and MP was abrogated by the successful completion of an appropriate periodontal therapy that dramatically improved clinical status. This is the first direct demonstration that GCF from periodontitis has the capacity to activate normal resting PMN and that this capacity reflects the magnitude of the inflammatory process that takes place in the gingiva. PMID- 7558254 TI - Blast injury of the ear: an overview and guide to management. AB - Blast injury is uncommon in many parts of the world but sporadic cases occur. The ear is particularly susceptible to damage and easily forgotten in patients with multiple injuries. The aim of this review article is to draw together the more important aspects of blast ear injury for those who are unfamiliar with it and to serve as a reminder of the problems to others. It covers the interactions of blast waves with the ear giving a summary of the mechanisms and types of injury. The management of blast-related injuries is discussed. PMID- 7558251 TI - Role of xanthine oxidase and eicosanoids in development of pancreatic ischemia reperfusion injury. AB - The implication of different eicosanoids and oxygen free radicals in the development of pancreatic injury after an ischemia-reperfusion process has been evaluated. For this purpose we have compared the effect of allopurinol and indomethacin administration on the pancreatic levels of eicosanoids in a rat model of pancreatic ischemia-reperfusion. After 60 min of pancreatic ischemia and 2 h of reperfusion, significant increases in 6-keto-PGF1 alpha, PGE2, and LTB4 in pancreas tissue were detected. Allopurinol before the ischemic period reduced 6 keto-PGF1 alpha, PGE2, and LTB4 levels to the range of basal values, while prior indomethacin treatment significantly reduced 6-keto-PGF1 alpha and PGE2 levels, with LTB4 remaining unmodified. Increased postischemic plasma lipases were also significantly reduced by allopurinol to the range of sham-operated animals whereas indomethacin did not modify these levels. The data suggest a role for lipoxygenase metabolites in the development of pancreatic injury and the importance of the enzyme xanthine oxidase as an inductor of eicosanoid biosynthesis. PMID- 7558253 TI - Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture. AB - Rabbit tracheal epithelial cells were cultured in a serum-free and hormone supplemented medium with and without retinoic acid. The cells showed time dependent mucin gene expression when cultured in the medium with retinoic acid. In the absence of retinoic acid, however, mucin mRNA was barely detectable in the cells. When retinoic acid was added back to the medium, the mucin message was prominent again. Actinomycin D and cycloheximide did not inhibit mucin gene expression. The mucin message was slightly elevated by cAMP agonists. A mucin antisense oligomer inhibited the retinoic acid-induced mucin mRNA expression and secretion, thus offering an alternate approach in the management of mucus hypersecretion in upper airway respiratory diseases such as chronic bronchitis, asthma, and cystic fibrosis. PMID- 7558255 TI - Unstable intertrochanteric fractures treated with the Variwall reconstruction nail. AB - Forty consecutive patients with unstable intertrochanteric fractures were treated with the Variwall reconstruction nail. Twelve of these had subtrochanteric extensions to their fractures, while most were highly comminuted grade 5 fractures. This nail was found to be relatively easy to use with a mean operative time of 58 min. Mean follow up was 5.8 months. Clinically, 86 per cent had occasional pain or none at all. There were no external rotation deformities, and only one case of shortening. Radiologically there were no non-unions and no case of shaft fractures. Failure occurred in one case, but in the remaining 39 no varus collapse occurred. In conclusion this method of fixation in unstable intertrochanteric fractures provides the necessary stability to give good results. PMID- 7558257 TI - Femoral shaft fractures treated by intramedullary nailing. A follow-up study focusing on problems related to the method. AB - One hundred and sixteen patients with 120 femoral fractures treated by reamed intramedullary nailing were reviewed. All fractures but one healed without additional procedures. One comminuted fracture had a bone transplant after 8 months to enhance bone remodelling in the lateral part of the fracture area. Three patients developed adult respiratory distress syndrome; all patients survived. Deep infection complicated one osteosynthesis. Thromboembolism was recorded in five cases. Twenty-three patients had a true torsional deformity (anteversion difference of 15 degrees or more), but only nine had complaints. Four of these patients needed a corrective osteotomy. Shortening of 10 mm or more was revealed in 11 patients; only one was above 20 mm. Prior to nail removal, hip and knee pain was present in 26 and 20 per cent, respectively. Few patients had such pain after nail removal. We conclude that reamed IM nailing of femoral fractures gives excellent fracture healing, rapid patient recovery and few complications. Some problems are, however, related to the method: torsional deformity occurs frequently, but will not always cause complaints. Shortening is a potential problem, but dramatic shortenings can be avoided when static locking is used. Hip and knee pain occurs frequently, but will usually disappear after nail removal. PMID- 7558256 TI - Post-traumatic multiple organ dysfunction syndrome--infection is an uncommon antecedent risk factor. AB - A pattern of multiple organ dysfunction syndrome (MODS) and risk factors following blunt trauma was identified, based on analyses of clinical data from 3611 patients who were admitted directly to a level I trauma centre and had hospital stays > or = 3 days. Five system dysfunctions were simultaneously associated (P < 0.05) with death (adjusted odds ratio): adult respiratory distress syndrome (ARDS) (4.9), renal failure (6.7), hyperglycaemia (3.6), recurrent acidosis (4.8) and hypoalbuminaemia (1.8). Mortality increased with the number of system dysfunctions. For the 336 patients with MODS (> or = 2 dysfunctions), mortality was 32.4 per cent compared with 1.3 per cent in the non MODS group (P = 0.0001). Of the 254 with MODS occurring within 72 hours, mortality was 27 per cent compared with 49 per cent in those manifesting MODS later (P < 0.001). The 175 (52.1 per cent) with organ failure (renal failure and/or ARDS) also had metabolic dysfunction. Seven admission risk factors were independently associated (P < 0.003) with MODS [adjusted odds ratio]: pre existing condition (3.4), age > 50 (3.1), Injury Severity Score > or = 25 (6.4), hypotension (2.8), acidaemia (2.2), 24 h blood loss > 1 l (3.7), and major base deficit (1.6). Only 13 per cent with MODS had an infection in the 5 days before or at initiation of MODS. Haemodynamic instability, acidosis, blood loss, pre existing condition, age and serious injury were risk factors independently related to life-threatening MODS, but infection was an uncommon precursor except in late MODS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558258 TI - Isolated sternal fracture: an audit of 10 years' experience. AB - We have reviewed 162 consecutive cases of sternal fracture admitted to the Leicester Royal Infirmary over a 10 year period. There were no incidences of cardiogenic shock or arrhythmia developing in patients who had sustained an isolated sternal fracture, irrespective of the aetiology. There were three deaths, three ITU admissions and one arrhythmia, all occurring in patients with severe thoracic injuries, or other associated injury. Our series confirms the observations of other authors, that patients with isolated sternal fractures, especially those sustained by car occupants wearing seatbelts, do not develop myocardial pump failure or arrhythmias as a late or occult phenomenon and can often be discharged home if there is no clinical evidence of cardiac failure and a 12-lead ECG is normal. PMID- 7558259 TI - Use of the Russell-Taylor reconstruction nail in femoral shaft fractures. AB - Of 242 patients with injuries treated by locked intramedullary nailing of the femur, 52 (22 percent) were considered suitable for insertion of the reconstruction nail. The Russell-Taylor reconstruction nail was found to be useful in a greater variety of fractures than those for which it was originally designed. In addition to ipsilateral neck and shaft fractures, the Russell-Taylor reconstruction nail proved successful in high-energy comminuted subtrochanteric fractures, elderly subtrochanteric fractures and pathological lesions. In two cases, problems were encountered due to the relative straightness of the nail, but in the remaining patients there were no technical problems. In the 43 elderly patients and those with pathological lesions, seven complications occurred: a number which compares favourably with published reports for extramedullary devices. There were no cases of implant failure and in all patients the device supported the fracture to union or death. PMID- 7558260 TI - Morbidity of negative coeliotomy in trauma. AB - Exploratory coeliotomy is essential in the care of abdominal trauma, but negative operation has a reported morbidity rate as high as 18 per cent. Ancillary studies such as computerized tomography, diagnostic peritoneal lavage and abdominal ultrasound have improved both sensitivity and specificity of evaluation in blunt and penetrating trauma, thus decreasing the rate of negative coeliotomy. A retrospective study of 50 consecutive negative laparotomies (10.5 per cent of all trauma laparotomies) at our Trauma Center revealed a morbidity rate of 22 per cent and mortality of 6 per cent. Although the negative coeliotomy rate was lower for blunt than penetrating trauma, morbidity was significantly higher for blunt trauma. Extra-abdominal injury alone could not account for this difference. We conclude that negative coeliotomy in penetrating trauma does not carry excessive morbidity. Negative coeliotomy in blunt trauma is accompanied by high morbidity and mortality, so adjunct diagnostic procedures should be utilized in this population in an effort to minimize negative laparotomies. PMID- 7558261 TI - Early management of severe head injury in Northern Ireland. AB - As part of a study into the management of major trauma, the early management of severe head injury in Northern Ireland was evaluated over a 12-month period. The injury severity score was used to define those patients considered to have severe head injury. There were 131 patients with severe head injury; 27 per cent were hypoxic and 18 per cent were hypotensive on admission to the primary hospital. Almost half had severe multiple injuries. Early endotracheal intubation was performed in 92 per cent of comatose patients, and adequate resuscitation (including laparotomy in some) was performed in 87 per cent of shocked patients transferred to the neurosurgical unit (NSU). Eighty patients were transferred to the NSU; 60 per cent were comatose, 68 per cent were intubated and ventilated, and 74 per cent were transferred by an anaesthetist. Two patients were in hypovolaemic shock after transfer and required laparotomy. Thirty-eight had evacuation of an acute intracranial haematoma; 26 of these would not have been classified as severe head injury by the Glasgow coma scale. The overall mortality rate was 38 per cent, and 24 per cent for those transferred to the NSU. We conclude that the early management of head injury in Northern Ireland is good, but there are problems, including pre-hospital oxygenation and delays in transfer. PMID- 7558263 TI - Closure of an open high below-knee guillotine amputation wound using a skin stretching device. AB - A skin-stretching device (SSD) which is designed to harness the viscoelastic properties of the skin using incremental traction is described. It has proved to be of value in securing closure, within 72 h, of an open high below-knee guillotine stump wound which did not heal during a period of 2 months after amputation. The skin-stretching device provided healthy, durable and sensate skin for stump coverage. Hospitalization time was shortened and an early post-surgical prosthetic fitting and ambulation was achieved. PMID- 7558262 TI - Use of the Injury Severity Score in head injury. AB - As part of a study of the early management of severe head injury, the use of the Glasgow Coma Score (GCS), Injury Severity Score (ISS) and TRISS was investigated. These injury scores were compared in correlating with outcome at one year as assessed by the Glasgow Outcome Score (GOS) and mortality. One hundred and thirty one patients had a severe head injury, as defined by an ISS of 16 or higher, in whom the Abbreviated Injury Score (AIS) for craniocerebral injury was 3 or higher. Seventy-eight of these also fulfilled the accepted GCS definition of severe head injury (GCS less than 8 with no eye-opening). Thirty-eight had evacuation of an acute intracranial haematoma; 26 of these patients would not have been classified as severe head injury by GCS. The overall mortality rate was 38 per cent, and 24 per cent for those transferred to the neurosurgical unit. TRISS was slightly better than GCS for predicting outcome based on both GOS and mortality, however this difference was not significant. TRISS identified patients who died that are not considered as severe head injury by GCS. Use of TRISS allows the effects of systemic factors and other injuries to be taken into account when assessing severity of head injury. PMID- 7558268 TI - Bungee jumping causing a comminuted proximal femoral fracture. PMID- 7558264 TI - Multiple cranial nerve palsies in a motorcyclist caused by the helmet. PMID- 7558266 TI - An avulsion fracture of the calcaneal apophysis in a young gymnast. PMID- 7558265 TI - An unusual forearm fracture in a child suggesting a mechanism for the Monteggia injury. PMID- 7558267 TI - Stress fracture of os ischium. PMID- 7558270 TI - Isolated trans-trapezoid axial-radial carpal dislocation. PMID- 7558269 TI - Severely comminuted forearm fracture treated with acute shortening. PMID- 7558271 TI - Treatment of diaphyseal tibial stress fractures associated with knee osteoarthrosis. PMID- 7558272 TI - Subcapital stress fractures of the hip complicating total knee replacement. AB - Subcapital stress fracture of the femoral neck may complicate total knee replacement particularly if a large deformity has been corrected in a poorly mobile patient. This complication must be suspected in a patient presenting with ipsilateral hip pain following a total knee replacement as prompt diagnosis and internal fixation prior to fracture displacement is more likely to allow preservation of the patient's own femoral head. PMID- 7558274 TI - Reconstruction nailing for subtrochanteric fractures in the Pagetic femur. PMID- 7558273 TI - Chest tube insertion in the ventilated patient. PMID- 7558275 TI - Management of penetrating injuries of the anterior triangle of the neck. PMID- 7558277 TI - Relationship between virulence of Mycobacterium avium strains and induction of tumor necrosis factor alpha production in infected mice and in in vitro-cultured mouse macrophages. AB - We studied the ability of two Mycobacterium avium strains with different virulences to induce tumor necrosis factor alpha (TNF) synthesis by mouse resident peritoneal macrophages (RPM phi) in vitro in an experiment to look for a possible correlation between virulence and this TNF-inducing capacity. The low virulence strain, 1983, induced significantly higher production of TNF by RPM phi than did the high-virulence strain, ATCC 25291. TNF neutralization during culture of infected RPM phi resulted in enhancement of growth of strain 1983 and had no effect on growth of strain ATCC 25291; TNF treatment of strain ATCC 25291 infected macrophages had no effect on mycobacterial growth. The extent of M. avium growth and the amount of TNF synthesis were independent of the presence of contaminating T cells or NK cells in the macrophage monolayers. Intraperitoneal administration of anti-TNF monoclonal antibodies to BALB/c mice infected intravenously with M. avium 1983 abrogated the elimination of the bacteria in the liver and caused a slight increase in bacterial growth in the spleen. Neutralization of TNF led to a minor increase in the proliferation of M. avium ATCC 25291 in the liver and spleen of BALB/c mice late in infection. Anti-TNF treatment did not affect the growth of the two M. avium strains in BALB/c.Bcgr (C.D2) mice, suggesting that restriction of M. avium strains to induce TNF production by macrophages may limit their ability to proliferate both in vitro and in vivo. PMID- 7558276 TI - Immunogenicity of synthetic peptides of Haemophilus influenzae type b outer membrane protein P1. AB - To identify the B- and T-cell epitopes of P1 of Haemophilus influenzae type b, 13 peptides covering 90% of the protein were chemically synthesized. Mouse, guinea pig, and rabbit antisera raised against purified native P1 were tested for their reactivities against the peptides in peptide-specific enzyme-linked immunosorbent assays (ELISAs). Six immunodominant linear B-cell epitopes were mapped to residues 103 to 137, 189 to 218, 248 to 283, 307 to 331, 384 to 412, and 400 to 437 of the mature P1 protein. When P1 peptides were screened for their reactivities with three human convalescent-phase serum specimens, peptides corresponding to residues 39 to 64, 226 to 253, and 400 to 437 reacted strongly with the antisera. Four regions (residues 39 to 64, 226 to 253, 339 to 370, and 400 to 437) contained murine T-cell epitopes. Rabbit antipeptide antisera were tested for their reactivities with the immunizing peptides and P1 protein by ELISA and immunoblots. All anti-P1 peptide antisera except those raised against peptide HIBP1-8 (residues 279 to 312) or HIBP1-8-keyhole limpet hemocyanin conjugate were shown to be specific for their respective immunizing peptides by ELISA. In addition, rabbit antisera raised against the synthetic peptides corresponding to residues 1 to 29, 39 to 64, 103 to 137, 189 to 218, 226 to 253, 248 to 283, 307 to 331, and 400 to 437 of the mature P1 protein recognized the P1 protein from both typeable and nontypeable isolates. These results suggest that these peptides contain epitopes highly conserved among typeable and nontypeable strains of H. influenzae. However, none of the antipeptide antisera have bactericidal activity, nor were they protective against H. influenzae type b in the infant rat model of bacteremia. PMID- 7558278 TI - Human T-cell activation by 14- and 18-kilodalton nuclear proteins of Leishmania infantum. AB - Leishmanial antigens which stimulate T lymphocytes from primed individuals may be candidates for a vaccine. We recently found a significant concordance between the humoral response specific for two proteins from Leishmania infantum promastigotes, p14 and p18, and a positive leishmanin delayed-type hypersensitivity reaction, testifying to the occurrence of cell-mediated immunity. In this communication, we describe a partial characterization of these antigens and an in vitro analysis of their capacity to activate primed human T cells. We showed, by immunofluorescent staining and through analysis of subcellular fractions by Western immunoblotting, that in stationary-phase promastigotes, p14 and p18 were located only in the parasite nuclei; in the middle of the log phase, a transitory and only weak expression outside the nucleus was detected. We then showed that p14 and p18 antigens shared a common epitope(s). Finally, we analyzed the in vitro proliferation and interleukin-2 production induced by leishmanial proteins in human peripheral blood mononuclear cells from sensitized subjects. We showed that in some individuals who have been exposed to L. infantum the specific response to the whole lysate was mostly due to the nuclear antigens. We demonstrated directly the capacity of nitrocellulose bound p14 and p18 to activate in vitro all of the tested primed peripheral blood mononuclear cells, which contrasted with a lack of stimulatory activity of other membrane-bound leishmanial proteins. Taken together, our results suggest that an antigenic determinant(s) dominant for some individuals might exist on both antigens. PMID- 7558279 TI - Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2. AB - Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2 dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease. PMID- 7558280 TI - Identification of two bvg-repressed surface proteins of Bordetella pertussis. AB - Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen. PMID- 7558281 TI - Contribution of urease to acid tolerance in Yersinia enterocolitica. AB - The stomach serves as a barrier to enteric infection because of the antibacterial effect of the hydrochloric acid in gastric juice. In this study, we tested the ability of the enteric pathogen Yersinia enterocolitica to tolerate a pH range of 2.0 to 6.0 and found that under the conditions of a normal human fasting stomach (pH < 3 and a gastric emptying time of 2 h), Y. enterocolitica is highly acid resistant, showing approximately 85% survival. The resistance of Y. enterocolitica to acid in vitro depended on the bacterial growth phase and the concentration of urea in the medium, being maximal during stationary phase in the presence of at least 0.3 mM urea. Urease-negative mutants of Y. enterocolitica were constructed by disrupting the urease gene complex of a virulent strain of serogroup O9. Compared with the wild type, these mutants showed an approximately 1,000-fold decrease in the ability to tolerate acid in vitro (< 0.08% survival) and a 10-fold reduction in viability after passage through the stomachs of mice. Complementation of the disrupted urease genes in trans restored the ability of urease-negative mutants to tolerate low pH in vitro and gastric acidity to approximately wild-type levels. These findings indicate that urease is responsible for acid resistance in Y. enterocolitica and suggest that urease contributes to the virulence of Y. enterocolitica by enhancing the likelihood of bacterial survival during passage through the stomach. PMID- 7558283 TI - Isolation and characterization of fibronectin-binding sites of Borrelia garinii N34. AB - Adherence of bacteria to host cell membranes is one of the initial steps of microbial pathogenicity. Numerous studies have suggested that fibronectin promotes this interaction in some bacterial species. In this study, we have examined the ability of Borrelia garinii to bind fibronectin. The binding of fibronectin to the spirochete was specific and saturable. Scatchard plot analysis of the binding data revealed two types of ligands on the spirochetal surface, one with high affinity and one with low affinity for fibronectin. The fibronectin binding sites were solubilized from the surface of B. garinii N34 by lysozyme treatment. Fast protein liquid chromatography (FPLC) purification of the solubilized binding sites resulted in one band with a high fibronectin-binding activity and a molecular weight of ca. 147,000. FPLC-purified binding sites, fibronectin, and antibodies to fibronectin inhibited the adherence of the spirochete to epithelial cells competitively. These data provide strong support for the hypothesis that fibronectin-binding sites on the surface of B. garinii are involved in the adherence of the spirochete to their respective host cells. PMID- 7558285 TI - Differential effects of monoclonal antibody blockade of adhesion molecules on in vivo susceptibility to soft tissue infection. AB - Leukocyte adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury as well as in host defense against various infectious microorganisms. Administration of monoclonal antibodies directed against the beta chain of the leukocyte integrins inhibits leukocyte-endothelial-cell adherence and has been reported to modulate ischemia-reperfusion and inflammatory injury. However, such inhibition of adhesion molecule function adversely affects resistance to infection. The following studies were carried out to determine whether monoclonal antibodies to other adhesion molecules, including L-selectin (CD62L), and CD11a (the alpha chain of LFA-1), also increase susceptibility to infection. New Zealand White rabbits were shaved and given subcutaneous injections on their dorsa with 10(9) CFU of Staphylococcus aureus ATCC 25923 at two sites and with 10(8) CFU at two sites. A second set of rabbits were given subcutaneous injections with 10(8) CFU of P. aeruginosa ATCC 27853 at two sites and with 10(7) CFUs at two sites. The animals were monitored for 1 week. There were three blinded experimental groups: controls given saline and two groups given blocking monoclonal antibodies to either L-selectin (Dreg-200) or CD11a (R7.1). In contrast to monoclonal antibodies to CD18, none of the monoclonal antibodies significantly increased the risk of abscess formation by S. aureus, although inhibition of CD11a increased the rate of abscess formation by P. aeruginosa. PMID- 7558284 TI - Characterization of transferrin binding proteins 1 and 2 in invasive type b and nontypeable strains of Haemophilus influenzae. AB - Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa. PMID- 7558286 TI - Proteolytic activity of the Bacteroides fragilis enterotoxin causes fluid secretion and intestinal damage in vivo. AB - Strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in farm animals and humans during the past decade. Our laboratory has purified and characterized this enterotoxin as a single polypeptide (M(r), approximately 20,000). Recently, we used PCR to clone and sequence the enterotoxin gene from B. fragilis and showed that it exhibits significant homology with extracellular metalloproteases. Further studies showed that the purified enterotoxin has protease activity. To further characterize the role of this enterotoxin in diarrheal disease, we studied the histological and pathological effects of highly purified B. fragilis enterotoxin in lamb, rabbit, and rat ligated intestinal loops. When the enterotoxin was injected into ligated ileal and colonic loops, there was significant tissue damage and subsequent fluid accumulation. The fluid response in the ileum was greater in lambs than in rabbits and rats, whereas the fluid response in the colon was greater in rabbits than in lambs and rats. Analysis of the intestinal fluid elicited by the enterotoxin revealed an accumulation of chloride and sodium as well as albumin and total protein. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to the chelated enterotoxin. Our results indicate that the enterotoxin elicits a significant fluid response subsequent to tissue damage in the small and large intestine. These data further support the idea that this enterotoxin is an important virulence factor in B. fragilis associated diarrhea. PMID- 7558282 TI - Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung. AB - Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p nitrophenoxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced. The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein. The amino acid sequence of A. fumigatus aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively. The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases. Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli. Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A. fumigatus. The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene. Immunogold electron microscopy showed that the aspartic proteinase was secreted by A. fumigatus invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae. PMID- 7558289 TI - A Cryptosporidium parvum genomic region encoding hemolytic activity. AB - Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5 kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle. PMID- 7558287 TI - Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract. AB - Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract to extraintestinal sites, such as the mesenteric lymph node (MLN), spleen, liver, kidneys, and blood. Previously, we reported that depletion of CD4+ and/or CD8+ T cells promotes bacterial translocation from the GI tract to the MLN. In the present study, CD4+ and/or CD8+ T cells, harvested from donor mice, were adoptively transferred to mice previously depleted of T cells by thymectomy plus intraperitoneal injection of rat anti-mouse T-cell monoclonal antibodies. The adoptively transferred CD4+ and/or CD8+ T cells inhibited the translocation of Escherichia coli from the GI tract. Migration of the adoptively transferred T cells to the spleens and MLNs of the recipient mice was determined by utilizing Thy 1.1+ donor cells adoptively transferred into mice whose cells express the Thy 1.2 marker. These results provide further evidence of the importance of T cells in the host immune defense against bacterial translocation from the GI tract. PMID- 7558292 TI - Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes. AB - Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes. PMID- 7558290 TI - Mapping of functional regions on the transferrin-binding protein (TfbA) of Actinobacillus pleuropneumoniae. AB - Actinobacillus pleuropneumoniae can use porcine transferrin as the sole source of iron. Two proteins with molecular masses of approximately 60 kDa (TfbA) and 110 kDa have been shown to specifically bind porcine transferrin; from the TfbA protein, three isoforms from A. pleuropneumoniae serotypes 1, 5, and 7 have been identified and characterized by nucleotide sequence analysis. Here we defined the transferrin-binding region(s) of the TfbA protein of A. pleuropneumoniae serotype 7 by TnphoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino acid sequence identity among the three isoforms, was shown to be responsible for transferrin binding by TnphoA mutagenesis. This result was confirmed by analysis of six random mutants with decreased transferrin binding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed three domains of 13 or 14 amino acids in length with transferrin-binding activity. They overlapped, or were very close to, point mutations decreasing transferrin-binding ability. The first and third domains were unique to the TfbA protein of A. pleuropneumoniae serotype 7. In contrast, the sequence of the second domain was present in almost identical forms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae serotypes 1 and 5; in addition, a sequence consisting of functionally homologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N. meningitidis (Tbp2), and Haemophilus influenzae (Tbp2). PMID- 7558288 TI - Potential role of nitric oxide in the pathophysiology of experimental bacterial meningitis in rats. AB - We have investigated the possible role of nitric oxide (NO) in the pathophysiology of bacterial meningitis (BM) by using the rat model of experimental BM. The nitrite concentration in cerebrospinal fluid (CSF) was used as a measure of NO production in vivo since NO rapidly degrades to nitrite and nitrate. Rats were inoculated intracisternally with live bacteria (5 x 10(6) CFU of Haemophilus influenzae type b strain DL42 or Rd-/b+/O2), with bacterial endotoxin (20 ng of DL42 lipooligosaccharide [LOS] or 200 ng of Escherichia coli lipopolysaccharide), or with a saline control vehicle. CSF samples were collected preinoculation and at the time of maximal alteration in blood-brain barrier permeability (BBBP). CSF [nitrite] was quantified by measuring A550 after addition of the Greiss reagent and comparison to a standard curve of sodium nitrite. Rats inoculated with either DL42, Rd-/b+/O2, LOS, or lipopolysaccharide demonstrated a significantly elevated mean peak CSF [nitrite] (8.34, 15.62, 10.75, and 10.44 mM, respectively) versus the concentration prior to treatment and/or those in saline-treated animals (5.29 and 5.33 mM, respectively; P < 0.05 for each comparison). We then determined if there was a correlation between CSF [nitrite] and percent BBBP (%BBBP) at various time points postinoculation with Rd /b+/O2. %BBBP was defined as the concentration of systemically administered 125I labeled bovine serum albumin in the CSF divided by the level of 125I-labeled bovine serum albumin in serum multiplied by 100. The mean %BBBP increased in tandem with the mean CSF [nitrite] (R = 0.84, P = 0.018), which peaked at 18 h in the absence of a change in the serum [nitrite]. Systemic administration of the NO synthase inhibitor N-nitro-L-arginine methyl ester demonstrated a significant reduction of mean CSF nitrite production (0.95 versus 6.0 mM in controls; P = 0.02) when administered intravenously to animals which had been inoculated intracisternally with 20 ng of LOS. Suppression of mean leukocyte pleocytosis (3,117 versus 11,590 leukocytes per mm3 in control LOS-challenged rats; P = 0.03) and mean alterations of BBBP (2.11 versus 6.49% in control LOS-challenged rats; P = 0.009) was observed concomitantly with decreased CSF [nitrite]. These results support the hypothesis that NO contributes to increased %BBBP in experimental BM. PMID- 7558291 TI - Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells. AB - The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo. PMID- 7558294 TI - Chemokine response in mice infected with Mycobacterium tuberculosis. AB - We show here that infection of murine macrophages with various strains of Mycobacterium tuberculosis induces the rapid in vitro expression of genes encoding chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 2, which recruit neutrophils to sites of infection, and macrophage-recruiting chemokines 10-kDa, interferon-inducible protein (IP-10) and macrophage chemotactic protein 1. Three strains of M. tuberculosis, Erdman and the clinical isolates CSU 22 and CSU 46, induced similar levels of secretion of macrophage chemotactic protein 1 from infected macrophage monolayers; however, the Erdman strain failed to induce levels of secretion of tumor necrosis factor alpha similar to those induced by either CSU 22 or CSU 46. Using a low-dose aerosol infection model, we also found that while the Erdman strain induced negligible increases in chemokine mRNA levels in the lungs, infection with either CSU 22 or CSU 46 resulted in greater levels of mRNA production for all four chemokines tested. The growth of these strains in the lungs was, however, equally well contained by acquired host immunity. These data allow us to hypothesize that the chemokine response in the lungs probably does not control the protective granulomatous response and that perhaps other T-cell- or macrophage-associated cytokines such as tumor necrosis factor alpha or interleukin 12 may be involved in this process. PMID- 7558293 TI - Generation of interleukin-8 from human monocytes in response to Trichomonas vaginalis stimulation. AB - Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients with Trichomonas vaginalis infection. We have investigated the possible role of interleukin-8 (IL-8) in the inflammatory response elicited by T. vaginalis infection. This study has shown that T. vaginalis induces blood monocytes to produce large amounts of bioactive IL-8, mainly by membrane components of T. vaginalis (MTV). Monocyte-derived IL-8 induced by MTV was dose and time dependent. The peak level of IL-8 was 102 +/- 11 ng/ml of conditioned media (mean +/- standard error; n = 5) obtained from MTV-stimulated monocytes (MTVCM) at 36 h of cultivation. With a multichamber chemotactic assay, we found an optimal neutrophil chemotaxis (177 +/- 14 migrated cells) induced by MTVCM collected at 16 h of cultivation when the level of IL-8 was 42 +/- 8 ng/ml. A neutralizing monoclonal antibody directed against IL-8, but not the irrelevant antibodies, significantly blocked the neutrophil chemotactic activity (decreased from 153 +/- 6 to 23 +/- 3 migrated cells; n = 3 [P < 0.001]) induced by MTVCM. Moreover, the maximum increase of the IL-8 mRNA level from MTV-treated monocytes was observed after a 5-h cultivation and decreased thereafter. Monocytes cocultured with MTV in the presence of a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, but not against IL-1 beta, decreased IL-8 production by 25% (P < 0.05), indicating that the release of IL-8 in MTV-stimulated monocytes is partially dependent on tumor necrosis factor alpha. The capacity of MTV-induced monocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in T. vaginalis infection. PMID- 7558295 TI - Porphyromonas gingivalis invasion of gingival epithelial cells. AB - Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases. PMID- 7558296 TI - Nitric oxide production during murine Lyme disease: lack of involvement in host resistance or pathology. AB - The murine model of Lyme disease was used to determine the role of inflammatory induced nitric oxide (NO) during infection by the spirochete Borrelia burgdorferi. The outer surface lipoproteins of B. burgdorferi are potent stimulators of inflammatory cytokines and NO production by cultured macrophages in vitro. The addition of NO to cultures of B. burgdorferi prevents growth, suggesting a protective role of NO for the infected host. NO is also a crucial effector in some models of arthritis. Therefore, the involvement of NO in controlling B. burgdorferi infection and its participation in pathological development of arthritis were investigated. Both mildly arthritic (BALB/c) and severely arthritic (C3H/HeJ) strains of mice systemically produced high levels of NO 1 week after infection with B. burgdorferi, as determined by urinary nitrate. NO production remained high throughout the infection in BALB/c mice, while in C3H/HeJ mice NO production returned rapidly to uninfected levels. The in vivo inhibitor of the NO synthase enzyme NG-L-monomethyl arginine (LMMA) was given to mice to investigate whether decreasing NO production would alter the course of disease. LMMA effectively blocked NO production in infected mice; however, there was no significant difference in arthritis development, spirochete infection of tissues, or production of specific antibody in LMMA-treated mice. These results indicate that B. burgdorferi is able to persist in the host even in the presence of high levels of NO. Furthermore, NO is not involved in the control of spirochete infection of tissues, nor is it involved in the development of arthritis. The potent activity of NO against intracellular pathogens and the in vivo resistance of B. burgdorferi to NO suggest that this organism is not located in an intracellular compartment during an essential portion of its infection of the mammalian host. PMID- 7558297 TI - Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and independent mechanisms. AB - Internalization of Listeria monocytogenes into nonphagocytic cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA dependent and -independent mechanisms. Tissue culture invasion assays employing various epithelial cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence. PMID- 7558300 TI - Pertussis toxin export genes are regulated by the ptx promoter and may be required for efficient translation of ptx mRNA in Bordetella pertussis. AB - The gene products from an 8-kb region adjacent to the 3' end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin. At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space. ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space. Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain. The transcription of ptl is subject to modulation by MgSO4 in the same manner as ptx is; however, in B. pertussis strains containing an E. coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO4. Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site. Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a > 11-kb mRNA. PMID- 7558298 TI - Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. AB - We have employed a germfree mouse model to study the development and persistence of a humoral mucosal immune response to a gram-negative murine commensal organism, Morganella morganii. M. morganii bacteria rapidly colonize the gut, resulting in hypertrophy of Peyer's patches (PP), including germinal center reactions (GCR), and the development of specific immunoglobulin A (IgA) responses detected in vitro in PP fragment cultures and by ELISPOT assays of lamina propria cells. The GCR peaks 14 days after infection and begins to wane thereafter. Upon colonization, the organisms successfully translocate to the mesenteric lymph node and spleen, but the number of translocating bacteria begins to drop with the onset of a specific IgA response. A clonal B-cell microculture technique was used to determine the frequency of specific IgA plasmablasts and IgA memory cells. The frequencies of preplasmablasts were seen to be higher in the earlier stages of germinal center development, whereas the frequencies of antigen-specific memory cells appeared to remain at a relatively constant level even after 193 days postmonoassociation. We suggest that a successful secretory IgA response can attenuate chronic stimulation of GCR even though the bacteria persist in the gut. The observed developing hyporesponsiveness to a chronically present commensal organism may be relevant to the use of bacterial vectors for mucosal immunization. PMID- 7558301 TI - Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a. AB - The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection. PMID- 7558302 TI - Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase. AB - Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification. PMID- 7558299 TI - Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection. AB - The gram-negative bacterium Actinobacillus actinomycetemcomitans is considered an important etiological agent in periodontal diseases. In this study, we show that A. actinomycetemcomitans strains are cytotoxic for the murine macrophage cell line J774.1. On the other hand, Porphyromonas gingivalis strains, other gram negative oral species implicated in adult periodontitis, showed weak cytotoxic effects. For this to occur, A. actinomycetemcomitans had to gain entry into the macrophages, since cytotoxicity was prevented by cytochalasin D. We demonstrate that cell death induced by A. actinomycetemcomitans Y4 occurs through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether the cytotoxicity induced by A. actinomycetemcomitans Y4 could be modulated by the protein kinase inhibitors H7 and HA1004. Apoptotic cell death induced by A. actinomycetemcomitans Y4 was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals of protein kinases may regulate apoptosis induced by A. actinomycetemcomitans Y4. The ability of A. actinomycetemcomitans to promote the apoptosis of macrophages may be important for the initiation of infection and the development of periodontal diseases. PMID- 7558305 TI - Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei. AB - Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb 3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be > 150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined. PMID- 7558304 TI - T-cell suppression and selective in vivo activation of TH2 subpopulation by the Entamoeba histolytica 220-kilodalton lectin. AB - A 220-kDa surface protein (L220) with lectin activity from Entamoeba histolytica trophozoites has been characterized previously (J. L. Rosales-Encina, I. Meza, A. Lopez-de-Leon, P. Talamas-Rohana, and M. Rojkind, J. Infect. Dis. 156:790-797, 1987). This molecule is involved in the adhesion process (I. Meza, F. Cazares, J. L. Rosales-Encina, P. Talamas-Rohana, and M. Rojkind, J. Infect. Dis. 156:798 805, 1987) and is very immunogenic. In this work, we studied both the humoral and the cellular immune responses to L220. We compared L220 with L220-derived components, such as a fusion peptide (M-11) and chemically obtained peptides (by treating the 220-kDa molecule with N-chlorosuccinimide-urea). Spleen cells from L220-immunized mice were unable to proliferate in vitro when stimulated with the protein. However, a proliferative response was obtained when mice were immunized with the L220-derived fusion peptide or the cleaved lectin. To find out if there was a correlation between the observed responses and TH1 or TH2 activation, we analyzed patterns of cytokine secretion (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon). Cells from mice immunized with peptides that induced cell proliferation (100, 80, and 47 kDa) with the peptides (P < 0.01) and with the intact molecule secreted IL-2 and gamma interferon, showing a TH1-subset pattern. Conversely, cells from mice immunized with the intact 220-kDa molecule secreted IL-4 and IL-10, typical of a TH2 subpopulation; however, antibodies from each group recognized the 220-kDa molecule as determined by Western blotting (immunoblotting). These results suggest that various epitopes in the 220-kDa molecule generate different response patterns, suppressing or activating T-cell responses. PMID- 7558306 TI - A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. AB - Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice. PMID- 7558303 TI - Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry. AB - Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis. PMID- 7558308 TI - T-lymphocyte responsiveness in murine schistosomiasis mansoni is dependent upon the adhesion molecules intercellular adhesion molecule-1, lymphocyte function associated antigen-1, and very late antigen-4. AB - Granuloma formation in murine schistosomiasis is dependent on CD4+ Th lymphocytes and requires recruitment and accumulation of inflammatory cells at the site of egg deposition. The present study examined the role of three adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), and very late antigen-4 (VLA-4), that participate in cellular recruitment, interaction, and lymphocyte activation during in vitro activation of acutely and chronically infected spleen and liver granuloma lymphocytes. Blockade of ICAM-1, LFA-1, or VLA-4 by rat monoclonal antibody inhibited spleen and granuloma lymphocyte interleukin-2 (IL-2) and IL-4 production as well as lymphoproliferative responses at similar levels (66 to 87%). The down-modulated cytokine and proliferative responses of chronically infected lymphocytes were inhibited to the same extent as their acutely infected counterparts. Cell sorting analysis demonstrated that acutely and chronically infected splenic and granuloma lymphocytes expressed similar levels of LFA-1, ICAM-1, and VLA-4 and that more ICAM-1 was expressed on infected than on uninfected mouse lymphocytes. By exposure of cells to paired monoclonal antibodies at suboptimal doses, it was determined that whereas all three adhesion molecules may participate, only ICAM-1 and LFA-1 showed synergistic interactions in determining lymphocyte responsiveness. These data suggest that spleen and liver granuloma lymphocytes are equally well armed with functional adhesion receptors. Thus, ICAM-1, LFA-1, and VLA-4 play an important accessory role in inflammatory cytokine production and lymphocyte proliferation, and therefore these adhesion molecules may participate in the initiation and maintenance of the granulomatous inflammation. PMID- 7558307 TI - Proteases of Treponema denticola outer sheath and extracellular vesicles. AB - Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases. PMID- 7558309 TI - Cellular mechanisms in the immune response to malaria in Plasmodium vinckei infected mice. AB - Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified. PMID- 7558310 TI - Molecular and biochemical characterization of a Coccidioides immitis-specific antigen. AB - Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19 kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections. PMID- 7558312 TI - Endogenous interleukin-12 is involved in resistance of mice to Mycobacterium avium complex infection. AB - Acquired cellular resistance against Mycobacterium avium complex (MAC) infections involves the induction of Th1 type gamma interferon (IFN-gamma)-producing T cells. Interleukin-12 (IL-12) is a cytokine involved in the control of IFN-gamma production by T cells and NK cells. The role of IL-12 in the response to MAC infection was investigated. Depletion of endogenous IL-12 by injection of monoclonal antibody prior to and during intranasal infection with MAC resulted in an 150- to 550-fold increase in bacterial load in lung, spleen, and liver homogenates by 10 weeks postinfection. Depletion of IL-12 abrogated the ability of spleen cell cultures to produce IFN-gamma in response to stimulus with live MAC. IL-12-depleted mice showed a 75% decrease in the number of inflammatory cells entering the lungs following intranasal infection with MAC, with significant reductions in cytotoxic activity and nitric oxide production by lung cells. This work suggests that IL-12 plays a major role in the activation of IFN gamma-producing cells during MAC infection. PMID- 7558311 TI - Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex. AB - Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061 nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7558313 TI - Association of lysogenic bacteriophage MAV1 with virulence of Mycoplasma arthritidis. AB - Mycoplasma arthritidis causes a severe polyarthritis under natural conditions in rats and under experimental conditions in both rats and mice. Although the disease itself has been extensively studied, M. arthritidis virulence factors remain uncharacterized. Comparison of relative arthritogenicity of 20 strains of M. arthritidis revealed that the strains tended to fall into two groups, a highly arthritogenic group, inducing maximum arthritis scores of > or = 11 in rats, and a low-virulence group, inducing maximum scores of < 6. Chromosomal DNA from the more highly arthritogenic strains possessed sequences that hybridized by Southern analysis with a probe prepared from lysogenic M. arthritidis bacteriophage MAV1, while DNA from low-virulence strains did not. One of the low-virulence strains, 158, was experimentally lysogenized with MAV1. Lysogenized 158 showed a significant increase in arthritogenicity over nonlysogenized 158. These data suggest that MAV1 carries a factor that is important in pathogenesis of M. arthritidis-induced arthritis of rats. PMID- 7558314 TI - Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ. AB - Contact of Salmonella typhimurium with cultured epithelial cells results in the assembly of surface appendages termed invasomes which are presumably required for the internalization of these organisms into host cells. The assembly of these structures requires the function of a dedicated protein secretion system encoded in the inv locus. We show in this report that contact of wild-type S. typhimurium with cultured Henle-407 cells stimulated the secretion of InvJ, a recently identified target of the inv-encoded type III protein secretion system. Stimulation of InvJ secretion also occurred upon bacterial contact with bovine calf serum-coated culture dishes but did not occur upon S. typhimurium contact with glutaraldehyde-fixed Henle-407 cells. The stimulation of InvJ secretion did not require de novo protein synthesis. Invasion-defective invC and invG mutants of S. typhimurium failed to secrete InvJ upon contact with live Henle-407 cells. In contrast, contact-dependent secretion of InvJ in S. typhimurium invE mutants occurred at levels equivalent to those of the wild type. These results indicate that the presence of Henle-407 cells and/or serum is capable of activating the type III secretion system encoded in the inv locus, further supporting the notion that Salmonella entry into cultured cells is the result of a biochemical cross talk between the bacteria and the host cells. PMID- 7558315 TI - Lack of a role for natural killer cells in early control of Brucella abortus 2308 infections in mice. AB - Studies were conducted to determine if natural killer (NK) cells are important for early control of the virulent strain Brucella abortus 2308 following infection of mice with high or low challenge doses. Splenocytes from C57BL/10 and BALB/c mice that had been infected with the lower dose of B. abortus displayed increased cytotoxicity against YAC-1 cells during the first week after infection, while infection of C57BL/10 mice with the higher challenge dose either did not alter the level of NK cytotoxic activity or decreased it, depending upon the time postinfection. In vivo depletion of NK cells by monoclonal antibody anti-NK1.1 or polyclonal anti-asialoGM1 antiserum did not result in an increase in the number of brucellae recovered from the spleens or livers of the brucella-resistant C57BL/10 mice or from the spleens of the susceptible BALB/c mice during the first week after infection. Treatment of control mice with the NK-reactive antibodies, however, decreased killing of the NK-sensitive target YAC-1, indicating that the NK cell depletion regimes were effective. Our results suggest that NK cells are not crucial for early control of B. abortus 2308 even though they may be activated following infection. Further experiments indicated that treatment of C57BL/10 mice with poly(A:U) did not decrease the number of brucellae recovered from their spleens although it did decrease the CFU in livers of mice infected with the high challenge dose. PMID- 7558317 TI - Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans. AB - By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest M(r) (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE- plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents. PMID- 7558316 TI - Relationship between maternally derived anti-Plasmodium falciparum antibodies and risk of infection and disease in infants living in an area of Liberia, west Africa, in which malaria is highly endemic. AB - In areas where Plasmodium falciparum is endemic, immunoglobulin G is acquired by the fetus in utero, mainly during the third trimester of pregnancy. The potential protective effect of transferred anti-P. falciparum maternal antibodies was examined in a longitudinal study of 100 infants from birth to 1 year of age. The probability of acquiring a P. falciparum infection and developing an episode of clinical malaria was determined in relation to the P. falciparum-specific antibody level of the infant at birth against P. falciparum schizont antigen or recombinant merozoite surface protein MSP1(19) antigen. The risk of acquiring an episode of clinical malaria increased from birth to 6 months of age, after which it decreased. The overall prevalence of P. falciparum parasitemia was highest (48.9%) in the 6-month-old infants. The age-specific hematocrit value showed the lowest mean value (30.2) from 6 to 9 months, and the spleen rate was the highest (69.8%) at the same age. There was a lower risk of developing an episode of clinical malaria during the first year of life in the infants with high levels of anti-MSP1(19) antibodies at birth. The level of maternally derived overall anti schizont antigen antibodies did not seem to play a role in the relative risk of developing malaria infection or disease during the first year of life, though the level of specific anti-MSP1(19) antibodies may be associated with protection. PMID- 7558319 TI - Escherichia coli induces transuroepithelial neutrophil migration by an intercellular adhesion molecule-1-dependent mechanism. AB - During bacterial infections at mucosal sites, neutrophils migrate to the mucosa and cross the epithelial barrier. We have examined neutrophil migration across Escherichia coli-stimulated uroepithelial cell layers in an attempt to more fully understand this process. Stimulation of uroepithelial cells with E. coli or interleukin-1 alpha (IL-1 alpha) induced transepithelial neutrophil migration in a time- and stimulant dose-dependent manner. Uroepithelial cell lines and nontransformed uroepithelial cells expressed intercellular adhesion molecule-1 (ICAM-1) but not ICAM-2, E-selectin, or P-selectin. Epithelial ICAM-1 expression was enhanced after stimulation with E. coli or IL-1 alpha. Anti-ICAM-1 antibody reduced transepithelial neutrophil migration by 61 to 85%, indicating that neutrophils bound ICAM-1 on the epithelial surface. Antibodies to CD18 and CD11b reduced migration by 70 to 79%, suggesting that CD11b/CD18 (Mac-1) was acting as the neutrophil receptor for ICAM-1 in this process. Anti-CD11a antibodies had no effect on neutrophil migration. In conclusion, E. coli induced ICAM-1- and Mac-1 dependent transepithelial neutrophil migration. Previous studies have shown that urinary tract epithelial cells secrete IL-8 when exposed to E. coli or IL-1 alpha. These observations suggest that epithelial cells play an active role in neutrophil migration during urinary tract infections. PMID- 7558318 TI - Activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble CD14. AB - In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14 dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway. PMID- 7558320 TI - Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin. AB - The bacterial pathogen Clostridium difficle synthesizes two high-molecular-weight toxins (A and B), which exhibit toxic effects in vivo and in vitro. Here, we present evidence that the major intracellular targets of these two toxins are the Rho GTPases. Overexpression of RhoA, RhoB, or RhoC GTPases in transfected HeLa cells conferred an increased resistance to toxins A and B, indicating that these toxins cause their cytopathic effects primarily by affecting Rho proteins. In addition, toxin A and B treatment appeared to result in modification of Rho, since Rho isolated from toxin-treated cells had a decreased ability to be ADP ribosylated by Clostridium botulinum C3 exoenzyme. In contrast, the lethal toxin (LT) of Clostridium sordellii, although structurally and immunologically related to C. difficile toxin B, appeared to induce cytopathic effects independently of the Rho GTPases. Overexpression of RhoA in transfected HeLa cells did not protect them from the effect of LT, and Rho isolated from lysates of LT-treated cells was not resistant to modification by C3. Immunofluorescence studies showed that LT treatment caused a cytopathic effect that was very different from those described for C. difficile toxins A and B, resulting in an increase in cortical F-actin, with a concomitant decrease in the number of stress fibers, and in the formation of numerous microvilli containing the actin-bundling protein fimbrin/plastin. PMID- 7558321 TI - Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro. AB - Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection. PMID- 7558322 TI - Candida albicans cell walls contain the fluorescent cross-linking amino acid dityrosine. AB - Several clinical and laboratory isolates of Candida albicans have a natural blue surface fluorescence when cultured and observed with sensitive optics. The localization and color of the fluorescence are similar to those of the natural fluorescence of sporulated Saccharomyces cerevisiae which is caused by the generation and surface deposition of the cross-linking amino acid dityrosine. In S. cerevisiae, dityrosine production results from the direct action of at least two genes and is responsible for resistance of the ascospores to lytic enzymes and physicochemical trauma. Among the criteria for the identification of dityrosine is pH sensitivity of the fluorescence intensity and a highly characteristic shift of the fluorescence excitation maximum with a change in pH. Video microscopy of whole Candida organisms revealed the characteristic dityrosine intensity maximum at pH approximately 10 and the intensity minimum at pH approximately 2. Separation of an acid hydrolysate of Candida cell walls by reverse-phase high-performance liquid chromatography revealed a fluorescence peak that coelutes with the reagent dityrosine. At pH approximately 10, this peak has a fluorescence excitation maximum of 320 to 325 nm, while at pH approximately 2, the excitation maximum is 285 to 290 nm. This excitation maximum shift and the observed emission maximum of approximately 410 nm are characteristic of dityrosine. Two separate strains of C. albicans were injected intraperitoneally into mice and harvested at 24 h. Blue surface fluorescence was observed, suggesting that dityrosine generation occurs in vivo as well as in vitro. This is the first report of the presence of dityrosine in a human fungal pathogen. PMID- 7558323 TI - Gene expression and production of tumor necrosis factor alpha, interleukin 1, interleukin 6, and gamma interferon in C3H/HeN and C57BL/6N mice in acute Mycoplasma pulmonis disease. AB - Studies were conducted to determine whether the production of various cytokines is associated with Mycoplasma pulmonis disease expression. Susceptible C3H/HeN and resistant C57BL/6N mice were inoculated intranasally with 10(7) CFU of virulent M. pulmonis UAB CT or avirulent M. pulmonis UAB T. Expression of genes for tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), IL 1 beta, IL-6, and gamma interferon (IFN-gamma) in whole lung tissue and TNF-alpha gene expression in bronchoalveolar lavage (BAL) cells was determined by reverse transcription-PCR using specific cytokine primers at various times postinoculation. In addition, concentrations of TNF-alpha, IL-1, IL-6, and IFN gamma were determined in BAL fluid and serum samples at various times postinoculation. Our results showed that there was a sequential appearance of cytokines in the lungs of infected mice: TNF-alpha, produced primarily by BAL cells, appeared first, followed by IL-1 and IL-6, which were followed by IFN gamma. Susceptible C3H/HeN mice had higher and more persistent concentrations of TNF-alpha and IL-6 in BAL fluid than did resistant C57BL/6N mice, indicating that TNF-alpha and possibly IL-6 are important factors in pathogenesis of acute M. pulmonis disease in mice. Serum concentrations of IL-6 were elevated in C3H/HeN mice, but not C57BL/6N mice, following infection with M. pulmonis, suggesting that IL-6 has both local and systemic effects in M. pulmonis disease. PMID- 7558324 TI - Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae. AB - The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis. PMID- 7558325 TI - Interleukin-12-stimulated natural killer cells can activate human macrophages to inhibit growth of Mycobacterium avium. AB - Interleukin-12 (IL-12) is a critical cytokine that affects many of the biological functions of NK cells and T cells. We have previously shown that both human and murine NK cells are important in host defense against Mycobacterium avium complex and act by secreting cytokines that induce macrophages to inhibit the growth of intracellular M. avium. To define the role of IL-12 in M. avium complex infection, we stimulated human NK cells with recombinant human IL-12 at 0.01 to 1 ng/ml for 24 h and used the tissue culture supernatant to treat human monocyte derived macrophage monolayers infected with M. avium. IL-12 had no direct effect on M. avium-infected macrophages, but culture supernatant from IL-12-treated NK cells activated macrophages to inhibit the growth of intracellular M. avium in a dose-dependent manner. Stimulation of NK cells with IL-12 in combination with tumor necrosis factor alpha (TNF-alpha) or IL-1 increased the ability of supernatant from NK-cell culture to limit M. avium growth within macrophages, compared with that of culture supernatant from IL-12-treated NK cells. Results with supernatant from nonstimulated NK cells were similar to those with supernatant from untreated controls. Treatment of supernatant from IL-12 stimulated NK cells with anti-TNF-alpha, anti-granulocyte-macrophage colony stimulating factor, but not anti-gamma interferon antibodies decreased the ability of NK-cell supernatant to induce anti-M. avium activity in infected macrophages. Treatment of macrophage monolayers with anti-transforming growth factor beta antibody before adding supernatant from IL-12-stimulated NK cells was associated with an increase of anti-M. avium activity compared with that of supernatant from IL-12-treated NK cells. These results suggest that IL-12 has a role in host defense against M. avium and that the effect of IL-12 is dependent chiefly on TNF-alpha and granulocyte-macrophage colony-stimulating factor. PMID- 7558326 TI - Immune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans. AB - The course of human infection with Leishmania braziliensis is variable, ranging from self-healing infection to chronic disease. It is therefore a useful system in which to study immunoregulatory aspects of leishmaniasis, including the effects of parasite antigens on host responses. In the present study, we report on the cloning of, expression of, and comparative analyses of patient immune response to two different L. braziliensis genes homologous to the genes for the eukaryotic 83- and 70-kDa heat shock proteins. rLbhsp83 contains a potent T-cell epitope(s) which stimulated peripheral blood mononuclear cells (PBMC) from all L. braziliensis-infected individuals to proliferate and to produce interleukin-2 (IL 2) gamma interferon, and tumor necrosis factor alpha. The elicitation of IL-4 and IL-10 mRNAs was found to differ depending on the portion of the rLbhsp83 used to stimulate PBMC. rLbhsp83a, which represents the nearly full-length protein, stimulated IL-10 but not IL-4 mRNA. In contrast, a approximately 43-kDa protein representing the C-terminal region of Lbhsp83 stimulated the production of IL-4 but not IL-10 mRNA. rLbhsp70 stimulated PBMC proliferation from patients with mucosal disease but, unlike rLbhsp83, did not stimulate PBMC from self-healing individuals. PBMC from mucosal patients were not stimulated by rHuhsp70 to either proliferate or produce cytokines. This suggests that the hyperresponsiveness of mucosal patient PBMC to Leishmania heat shock proteins does not involve an auto immune phenomenon resulting from cross-reactivity with self hsp70. In general, although the cytokine profile of patient PBMC in response to both of these Leishmania heat shock proteins represents a mixed Th1-Th2 pattern, the levels of gamma interferon and IL-2 were significantly higher than those of the Th2 cytokines IL-4 and IL-10. Patients with active mucosal and cutaneous disease but not self-healing individuals had significant anti-immunoglobulin G antibody titers to both rLbhsp83 and rLbhsp70 but not to the homologous rHuhsp70. It therefore appears that differential patient immune responses to Leishmania hsp83 and hsp70 may be of particular significance in the induction of protective immune responses as well as in the development of tissue damage in cases with particularly strong hypersensitive reactions. PMID- 7558327 TI - Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae. AB - Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. NTHi releases lipooligosaccharide (LOS) as outer membrane fragments during its growth. The release of LOS may play an important role in the pathogenicity of otitis media caused by this organism. The amounts of LOS in bacterial cells and growth media for five NTHi strains were determined by quantitative silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains were estimated to have 1.6 x 10(6) to 4.8 x 10(6) LOS molecules per bacterium. During a 3-day growth period, these NTHi strains released variable but significant amounts of LOS into the growth medium. Cells started to release detectable amounts of LOS into the medium at 2 to 5 h and continued to do so for up to 48 or 72 h. The concentrations of LOS in the culture supernatants released by these five strains were 10 to 55 micrograms/ml at 24 h and 40 to 100 micrograms/ml at 72 h, which was 34 to 189% of the cell-bound LOS concentration. The biological properties of released and cell-bound LOSs from two representative strains were compared. Released LOS showed an approximately 10 fold increase in inducing human monocytes to produce tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, a 13- to 28-fold increase in mouse lethal toxicity, and a 16- to 37-fold increase in the clotting of Limulus amebocyte lysate. These results suggested that released LOS or its inflammatory mediators play a more important role than the LOS in bacteria in the pathogenicity of otitis media caused by this organism. PMID- 7558328 TI - Panton-Valentine leucocidin and gamma-hemolysin from Staphylococcus aureus ATCC 49775 are encoded by distinct genetic loci and have different biological activities. AB - Staphylococcus aureus ATCC 49775 produces three proteins recognized by affinity purified antibodies against the S component of Panton-Valentine leucocidin (LukS PV) and two proteins recognized by affinity-purified antibodies against the F component of this toxin (LukF-PV). Purification of these proteins and cloning of the corresponding genes provided evidence for the presence of two loci. The first one, encoding Panton-Valentine leucocidin, consisted of two cotranscribed open reading frames, lukS-PV and lukF-PV, coding the class S and the class F components, respectively. The second one coded for a gamma-hemolysin and consisted of two transcription units, the first one encoding an HlgA-like protein, a class S component, and the second one encoding two cotranscribed open reading frames identical to HlgC and HlgB, class S and class F components, respectively, from gamma-hemolysin from the reference strain Smith 5R. It appears that the Panton-Valentine leucocidin from S. aureus ATCC 49775 (V8 strain) should not be confused with leucocidin from ATCC 27733 (another isolate of V8 strain), which had 95% identity with HlgC and HlgB from gamma-hemolysin. The cosecretion of these five proteins led to six possible synergistic combinations between F and S components. Two of these combinations (LukS-PV-LukF-PV and HlgA-LukF-PV) had dermonecrotic activity on rabbit skin, and all six were leukocytolytic on glass adsorbed leukocytes. Only three were hemolytic on rabbit erythrocytes, the two gamma-hemolysin combinations and the combination LukF-PV-HlgA. PMID- 7558329 TI - Experimental immunization with Borrelia burgdorferi induces development of antibodies to gangliosides. AB - Patients with neuroborreliosis produce antibodies, mostly of the immunoglobulin M (IgM) class, to gangliosides, particularly to those with Gal(beta 1-3)GalNac terminal sequences. Lewis rats were immunized with a nonpathogenic strain of Borrelia burgdorferi and with a chloroform-methanol extract (nonprotein) of this organism (CM) to determine whether antibodies to B. burgdorferi also recognized gangliosides. Rats were also immunized with asialo-GM1 to determine whether the elicited antibodies recognized antigens in B. burgdorferi. Rats immunized with B. burgdorferi produced low levels of IgM antibodies that cross-reacted with asialo GM1 and GM1. Rats immunized with CM had marked IgM reactivity to asialo-GM1 and GM1. Immunization with asialo-GM1 resulted in antibodies that cross-reacted with B. burgdorferi antigens. Although antibodies to B. burgdorferi were of both the IgM and IgG classes, those to CM and to asialo-GM1 and GM1 were predominantly in the IgM fraction. Reactivity of the IgM antibodies decreased after adsorption with the heterologous and the homologous antigens, indicating bidirectional cross reactivity between CM, asialo-GM1, and GM1 and that immunization with one produces antibodies to the other. There was no in vivo deposition of Ig in peripheral nerves, nor was there nerve pathology as a result of immunizations, but IgM antibodies to asialo-GM1 and CM recognized homologous antigens in the nodes of Ranvier of peripheral nerves from nonimmunized rats. This immunization model suggests that antibodies to gangliosides in Lyme disease have a microbial origin and are potentially relevant in pathogenesis. PMID- 7558331 TI - Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99. AB - The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH. Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene. Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons. fanC was the most highly expressed, and fanD was expressed at very low levels. The expression of TnlacZ fusions in fanA and fanB fusions was high. Deletion of fanA, fanB, and part of fanC abolished the expression of fanD but had no effect on the distal genes fanE to fanH. To locate the DNA regions required for expression of fanE to fanH, deletion mutations were prepared and the effects on expression of fanE to fanH were determined. The deletion of a segment between fanD and fanE abolished fanE and fanF expression but did not affect fanG and fanH. The deletion of a portion of fanF (approximately 1 kb proximal to fanG) abolished the expression of fanG and fanH. These results indicate the presence of regulatory elements proximal to fanE and to fanG. Putative promoters were identified in these regions by DNA homology and by primer extension. A stem-loop structure that may act as a transcriptional attenuator of fanF was also found at the beginning of fanF. These data confirm our previous model of K99 transcriptional organization. PMID- 7558330 TI - Chronic respiratory mycoplasmosis in C3H/HeN and C57BL/6N mice: lesion severity and antibody response. AB - Mycoplasma pneumoniae is a leading, worldwide cause of death and disability due to pneumonia. Mycoplasma pulmonis infection in mice is an invaluable model for the study of host defenses against respiratory mycoplasmas in vivo. C3H/HeN mice are much more susceptible to acute inflammatory lung disease due to M. pulmonis than C57BL/6N mice, but little is known about the chronic disease in these mouse strains. We infected C3H/HeN and C57BL/6N mice with 10(4) CFU of M. pulmonis UAB CT and evaluated them at weekly intervals by quantitative mycoplasma culture of nasal passages, trachea, and lungs, assessment of lesion severity in nasal passages, trachea, and lungs, and determination of serum immunoglobulin classes and subclasses by enzyme-linked immunosorbent assay. We found that C3H/HeN mice had 2 to 5 logs more organisms in their lungs and far more severe lung disease than C57BL/6N mice through 63 days postinfection. Although both strains of mice developed the same classes of antibody, C3H/HeN mice had much greater anti-M. pulmonis immunoglobulin G (IgG) responses in the IgG1 and IgG2a subclasses than C57BL/6N mice. These results suggest that adaptive immunity does not effect resolution of chronic mycoplasma infection and disease in the lungs. PMID- 7558332 TI - Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis. AB - The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes. PMID- 7558333 TI - Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. AB - The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response. PMID- 7558334 TI - Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake. AB - Helicobacter pylori is known to be a causative agent of gastritis and peptic ulcer disease in humans. The acquisition of iron from the human host may contribute greatly to the virulence of this organism. To study this, H. pylori was cultured under iron-restrictive conditions to induce synthesis of possible iron-regulated outer membrane proteins. This was achieved by the addition of 20% (vol/vol) heat-inactivated newborn calf serum, which contains iron-binding proteins like transferrin and albumin, and no free iron. The newborn calf serum was able to bind free ionic iron in brucella broth culture medium. Electrophoretic analysis of outer membrane preparations from H. pylori cultured under conditions of iron restriction showed several proteins to be present at elevated levels. These appeared to be iron-repressible outer membrane proteins (IROMPs). In addition, IROMPs with molecular sizes of 77, 50, and 48 kDa were isolated by use of hemin-agarose affinity chromatography. These three heme binding IROMPs might be involved in the uptake of heme from the host and might therefore be important virulence factors of H. pylori. PMID- 7558335 TI - Avirulence of a Pseudomonas aeruginosa algC mutant in a burned-mouse model of infection. AB - The virulence of wild-type Pseudomonas aeruginosa PAO1 and that of a genetically defined algC mutant, PAO1 algC::tet, were compared in a burned-mouse model of infection. Unlike PAO1, PAO1 algC::tet was avirulent, grew less well in the eschar, and did not disseminate to the liver of challenged animals. We have previously shown that the P. aeruginosa algC gene is required for biosynthesis of alginate and lipopolysaccharide (M.J. Coyne, Jr., K.S. Russell, C.L. Coyle, and J.B. Goldberg, J. Bacteriol. 176:3500-3507, 1994). In order to determine whether the alginate or lipopolysaccharide (LPS) defect was responsible for the avirulence of this strain, we constructed a strain with a mutation in an alginate specific gene, algD. PAO1-algD was virulent in the burned-mouse model, thus implicating the LPS defect in PAO1 algC::tet as the relevant alteration responsible for the avirulence of this strain. PMID- 7558338 TI - Production of tumor necrosis factor alpha, interleukin-1 alpha, and interleukin-6 during murine coccidioidomycosis. AB - The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease. PMID- 7558336 TI - Influence of the Bcg locus on macrophage response to the dimorphic fungus Candida albicans. AB - The Bcg/Ity/Lsh gene (candidate Nramp) controls natural resistance to several parasites, such as Mycobacterium bovis, Leishmania donovani, and Salmonella typhimurium. Using two macrophage (M phi) cell lines (B10R and B10S) derived from mouse strains congenic at Bcg, we found that M phi s from resistant mice (B10R M phi s) act more effectively against the two morphogenetic forms of the dimorphic fungus Candida albicans compared with M phi s from susceptible mice (B10S M phi s). Moreover, when assessed for tumor necrosis factor secretion in response to the hyphal form of C. albicans, B10R M phi s are significantly more effective at expressing this secretory function than are B10S M phi s, closely resembling the trend of response to lipopolysaccharide. Overall, these results provide insight into the influence of the Bcg locus on the M phi response to C. albicans. PMID- 7558337 TI - AIDS-associated mycoplasmas possess phospholipases C in the membrane. AB - Phosphatidylinositol- and phosphatidylcholine-hydrolyzing activities existed in the cell lysates of AIDS-associated Mycoplasma species, including Mycoplasma fermentans IID 812, M. fermentans incognitus, and M. penetrans GTU-54-6A1. These activities were found to be attributable to phospholipases C, because the water soluble product from phosphatidylcholine digested by the cell lysate of M. fermentans IID 812 was phosphorylcholine. M. fermentans IID 812 was examined for localization of these enzymes, and it was found that they were associated with the membrane. PMID- 7558339 TI - Molecular characterization of FrpB, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis. AB - The structural gene encoding the 70-kDa outer membrane protein FrpB of Neisseria meningitidis was cloned and sequenced. A mutant lacking FrpB was constructed. No difference in iron utilization between the mutant and the parental strain was observed. A minor effect of the mutation on serum resistance was observed. A topology model for FrpB in the outer membrane is proposed. PMID- 7558340 TI - Preferential induction of septic arthritis and mortality by superantigen producing staphylococci. AB - Staphylococcal enterotoxins A through D (SEA through SED) and toxic shock syndrome toxin-1 display superantigen properties, i.e., they stimulate a great fraction of T cells expressing certain T-cell receptor V beta sequences. Using a newly established rat model of septic Staphylococcus aureus arthritis, we have recently shown that an S. aureus strain producing SEA showed marked arthritogenic properties. In the present study we decided to employ another five S. aureus strains, each one producing a distinct exotoxin. Almost all rats injected with superantigen-producing strains developed arthritis. In contrast, only 20% of rats injected with an S. aureus strain lacking superantigen production displayed mild and transient arthritis. Mortality was preferentially induced among the rats inoculated with the S. aureus strains producing SEB and SED. This study emphasizes that superantigen production is an important virulence factor in the development of septic S. aureus arthritis. Differences concerning mortality between staphylococcal strains producing different exotoxins may be dependent on the degree of activation of the immune system. PMID- 7558341 TI - Adenovirus serotype 1 does not act synergistically with Moraxella (Branhamella) catarrhalis to induce otitis media in the chinchilla. AB - A chinchilla model of otitis media in which adenovirus compromise of the tubotympanum facilitates the subsequent induction of middle ear disease was used to investigate Moraxella (Branhamella) catarrhalis pathogenesis. Intranasally inoculated M. catarrhalis did readily colonize the nasopharynx of this host; however, despite evidence of viral infection and tubotympanal compromise, M. catarrhalis did not induce culture-positive otitis media in this model. PMID- 7558343 TI - Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli. AB - Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC. PMID- 7558344 TI - Ail expression in Yersinia enterocolitica is affected by oxygen tension. AB - We investigated several environmental factors for their abilities to regulate ail gene expression and found that ail transcript levels are regulated by oxygen tension. Bacteria growing under anaerobiosis at 37 degrees C repress ail mRNA and Ail expression, resulting in a loss of Ail-mediated serum resistance and cell invasion. PMID- 7558342 TI - Effects of systemic cell-mediated immunity on vaginal candidiasis in mice resistant and susceptible to Candida albicans infections. AB - Studies to date with CBA/J mice suggest a limited role for systemic cell-mediated immunity (CMI) against vaginal Candida albicans infections. The results of the present study show that preinduced Candida-specific systemic CMI was equally nonprotective against C. albicans vaginal infections in mice with high (BALB/cJ), low (DBA/2), or intermediate (CBA/J) resistance to C. albicans infections. Similarly, the locally acquired partial protection against a second C. albicans vaginal infection was equally observed with BALB/cJ, DBA/2, and CBA/J mice. These results indicate that observations made previously with CBA/J mice were not murine strain specific and provide additional support for the hypothesis that systemic CMI does not represent a dominant host defense mechanism at the vaginal mucosa. PMID- 7558345 TI - Salmonella strain secreting active listeriolysin changes its intracellular localization. AB - We describe the construction of an attenuated Salmonella dublin aroA strain which secretes via the Escherichia coli hemolysin secretion machinery an active hybrid cytolysin consisting of listeriolysin from Listeria monocytogenes and the C terminal secretion signal of E. coli hemolysin. This hemolytic S. dublin strain is partially released into the cytoplasm of the host cell following uptake by J774 macrophage cells, whereas the nonhemolytic control S. dublin aroA strain remains in the phagosome. PMID- 7558347 TI - The structure and scope of dental quality assurance systems in member countries of the Federation Dentaire Internationale. FDI Working Group. PMID- 7558346 TI - Interleukin-8 production by human mesothelial cells after direct stimulation with staphylococci. AB - Mesothelial cells (MC) are able to produce interleukin-8 (IL-8) after stimulation with IL-1 beta or tumor necrosis factor alpha. The aim of our study was to investigate whether MC are able to produce IL-8 after direct stimulation with clinically relevant bacteria. We observed a significant IL-8 response by the MC which were directly stimulated with viable staphylococci. PMID- 7558348 TI - Marginal ridge strength of teeth with tunnel preparations. AB - The aim of this study was to investigate how the cavity design of tunnel preparations influences the strength of the marginal ridge and to assess the ability of a restoration to support it. Extracted upper premolars were divided into eight experimental groups and one control group of intact teeth. The size and the position of the tunnel preparation in relation to the marginal ridge differed in four of the groups, the preparations remaining unfilled, while teeth in four corresponding groups were filled. The teeth were subjected to incremental dynamic forces until fracture of the marginal ridge occurred at which point a 'maximum energy-index' was calculated. The relationship between this index and the experimental variables was analysed by the Cox proportional hazards model. The results for both filled and unfilled teeth indicated that the distance from the preparation to the marginal ridge is more influential on weakening the ridge than is the buccopalatinal size of the opening. A conservative tunnel restoration situated 2 mm from the marginal ridge, does not significantly weaken an otherwise intact tooth. PMID- 7558349 TI - The relevance of health state utilities to lower third molar surgery. AB - A study of the attitudes of 102 arts and science undergraduate students was carried out to establish relative utility values, that is the participants own assessments, for possible outcomes of surgical intervention and non-intervention in lower third molar management. Using a standardised visual analogue scale, respondents were asked to rate 17 outcomes of treatment presented to them by means of short descriptions (vignettes). Ranking of mean utility values showed that post operative complications were considered to be a greater handicap than the sequelae of leaving third molars in situ. The respondents considered that the effects of irreversible lingual and inferior dental nerve damage reduced health to a major degree, and that normally encountered post operative complications such as moderate pain, swelling and trismus were a greater handicap than recurrent mild/moderate pericoronitis. PMID- 7558350 TI - Research directions and output in European dental schools. AB - Information pertaining to the research directions and output of dental schools in different countries is of importance in understanding intra- and inter- national differences amongst groups of dental practitioners. The research output of European dental schools has been assessed quantitatively and qualitatively on the basis of published abstracts of research presentations at IADR-meetings during 1993. These data are compared with comparable data for 1990/91. The results indicate that European dental schools are substantially different in terms of research output relative to total population, with the UK at one extreme and Italy at the other. The UK, Denmark, The Netherlands, Sweden and Norway are highly ranked while Portugal, Spain, Italy, Germany and France have low rankings, according to the method used. Periodontology, plaque, dental materials, and restorative dentistry would appear to be the principal foci of research. Possible ways of strengthening the coordination and the efficiency and effectiveness of dental research at the European level are discussed. It is suggested that the creation of centres of excellence and research networks are approaches to be considered. An unresolved problem will be the contradictory requirements from educational and research perspectives in relation to the training of dental practitioners. PMID- 7558354 TI - Trends in publications on clinical techniques/materials 1971-1991. PMID- 7558355 TI - Oral health and dental care in Hong Kong. AB - With a population of six million people and situated at the South-eastern corner of mainland China, Hong Kong is considered as the gateway between East and West, often playing a role disproportionate to its territorial or population size. The FDI World Dental Congress 1995 provides an impetus to familiarise colleagues worldwide with the oral health and dental care situation in Hong Kong. Water fluoridation was introduced in 1961, which has achieved a continuous low and stable caries prevalence in children as well as in adults. Edentulousness among adults is usually not apparent until after age 55, but at much lower rates than in comparable western industrialised populations. Periodontal conditions in adults are dominated by calculus and shallow pockets. Overall, treatment needs are mainly oral hygiene and simple restorative treatment. Dental awareness is considered low with only a minority of the population seeking dental care on a regular basis. Traditional Chinese health beliefs are common and may influence health behaviour in terms of self- or professional care. Dental care is mainly provided by around 1000 private dental practitioners but special government dental programmes are established for primary school children, who are mainly treated by dental therapists, and for civil servants and their dependents. Dental hygienists are trained in very limited numbers. Dental specialties are under development as part of a newly established Academy of Medicine. Developments are under way to introduce new preventive oriented programmes for pre-school and secondary school age groups. PMID- 7558352 TI - Preregistration and post graduate practice training period. FDI Working Group. AB - Working Group 14 has had formal meetings at FDI Congresses in Milan 1991, Berlin 1992 and Goteborg 1993 and this report was approved by the Commission at the Vancouver FDI World Dental Congress in 1994. At all stages the subject has attracted great interest and working group meetings have been well attended. The agreed terms of reference were: To collect information on practice training systems. To prepare an analytical report to assist those countries that would like to introduce a practice training period. During the early meetings it was quickly realised that no single system could be identified as the desirable global model and that an illustration of some systems currently operating would assist FDI member countries contemplating the commencement of a practice training scheme. To this extent the terms of reference have been fulfilled. This descriptive report makes no attempt to be definitive and there are, of necessity, some anecdotal elements within it. PMID- 7558353 TI - A five year follow up into changes in caries experience amongst a sample of 12 year old children from Athens. AB - A five year follow up study of the dental health of 12 year old children in Athens has recorded a significant (P < 0.01) 32.3 per cent reduction in DMFT. In 1988 the mean DMFT score was 2.41 compared with 1.63 in 1993. The caries reduction may be due in part to increased marketing of fluoride dentifrice by a number of competing national and international companies. PMID- 7558356 TI - A reader's and writer's guide to the publication of clinical trials. Reports of agents and procedures to prevent caries and periodontal diseases. FDI Working Group. Federation Dentaire Internationale. PMID- 7558357 TI - Evaluation of clinical trials of agents and procedures to prevent caries and periodontal disease: choosing products and recommending procedures. AB - The prevention of caries, gingivitis and periodontal diseases has been, and still is, a major objective for the dental profession. A large number of products are available to the general public, either over the counter or on prescription, formulated to prevent these diseases. Formulations are also used by professionals for the prevention and treatment of dental diseases. In recommending or using agents for prevention or therapy it is necessary for the practitioner to appreciate whether proof of efficacy is established. Preventive or therapeutic roles of formulations are frequently confused and there is a need to define the often misleading terminology used to describe products. Guidelines are required to enable the dental professional to understand research data and what constitutes proof of efficacy for preventive and/or therapeutic formulations. The guidelines should describe the research methodologies used today, including study design and analysis, ethics, the format of research papers and the relevance of laboratory data to clinical efficacy. PMID- 7558351 TI - Some physical and biological properties of glass ionomer cement. AB - It has become apparent through both clinical use and laboratory experiments that the glass ionomer cements have several highly desirable properties. They show a continuing fluoride release and the ability to take up further fluoride under favourable conditions. The presence of fluoride also helps to inhibit plaque formation. The adhesion between tooth structure and cement also results in almost complete prevention of the bacterial micro-leakage. Also, the cement itself is so highly bio-compatible that it is now being used as a bone substitute and it has become apparent that there is no need to place a sub-lining under a glass ionomer restoration. Recent research is leading to the development of self curing cements with enhanced physical properties so that, in the presence of the above advantages, their use in clinical dentistry is rapidly expanding. Glass ionomer cements are of great value for any restoration which is not under undue occlusal stress and they work well also as a long term temporary restoration in the presence of a high caries rate, where zinc oxide and eugenol used to be the material of choice. PMID- 7558358 TI - National survey of periodontal status and treatment need among Nigerians. AB - A national pathfinder survey of periodontal conditions and treatment needs was carried out among 4631 male and female Nigerians drawn from all four Primary Health Care (PHC) Administrative zones. The survey was conducted according to WHO Basic Methods. Differences between PHC zones, and rural and urban communities were examined. The Chi-Square test was used to analyse differences in frequencies between groups. The percentage of healthy subjects was very small, nearly zero, in all age groups and in all zones. A majority of subjects had calculus. The prevalence of shallow and deep pockets were relatively high; prevalence of '4 or 5mm pockets' increased from 39 per cent among 15 year olds to 57 per cent among those 25-29 years before declining to 17 per cent among those 65 years of age and older. Prevalence of deep pockets increased steadily with age. Few sextants had '4 or 5mm pockets' or deep pockets. There were also very few edentulous sextants. Differences between the PHC zones, and rural/urban areas were mainly limited to number of sextants with '4 or 5mm pockets'. Virtually all subjects needed one form of periodontal treatment as assessed by the CPITN. PMID- 7558359 TI - Periodontal treatment needs and oral hygiene among Ethiopian immigrants. AB - Periodontal disease studies in developing countries over the past four decades indicate periodontitis to be a major problem, even in populations with relatively low caries rates. The aim of this study was to document levels of periodontal treatment needs, as well as oral hygiene levels for a large sample covering all ages of an Ethiopian Jewish population recently immigrated into Israel. Over 800 subjects from the total of 15,000 Ethiopians who immigrated were examined utilising the PTNS Index and the OHI-S Index. Results indicate that all the examined population was in need of oral hygiene instruction. Almost 80 per cent are in need of scaling and approximately 20 per cent are in need of more complex treatment such as surgery. Significant differences were found according to age. This could be attributed to the high OHI-S scores, ranging from 2.53 at age 0-12 to 4.82 at the 51+ age group, with a mean Debris Index of 2.12 for the total population. It is suggested that this population should be exposed immediately to preventive and treatment programmes to improve oral hygiene and decrease needs for future, costly periodontal treatment. PMID- 7558360 TI - Corrosion evaluation of recasting non-precious dental alloys. AB - The effect of recasting, up to four times, non precious Ni-Cr and Co-Cr commercial dental alloys on their corrosion behaviour in saliva and saline media was carried out. A potentiokinetic technique was utilised to analyse the electrochemical characteristics of the anodic polarisation curves of these alloys. A considerable anodic polarisation range (about 2.0 V vs SCE) was used to investigate the possibility of developing a passive regime at such high potential range. The most important feature noted was a rapid rise in current density above a certain critical potential called pitting potential, Ep. The experimental data show that the open circuit potential, Eoc does not enable differentiation between the corrosion resistance of the four alloys used in this study. However, Ep and the rupture potential Er does distinguish between them. Increasing the number of the successive recastings of Wirolloy leads to enhancing the pitting potential, thus, the corrosion resistance of Wirolloy (Ni-Cr) improves after remelting and recasting. It has been found that Wirolloy corrodes 26 times faster than Wironit alloy under the same solution. The alloys containing cobalt and molybdenum show higher corrosion resistance than those containing nickel. Additionally, their corrosion resistance was not affected by successive melting and recasting. In chloride solutions Ni-Cr alloys show a high susceptibility to pitting corrosion, while Co-Cr alloys show a noble behaviour. The corrosion resistance of the four non precious alloys were in the following order: Biocast > Wironit > Cobond > Wirolloy. PMID- 7558362 TI - Genetics in oral-facial growth and diseases. AB - Much debate has taken place over the relative importance of heredity and environment in the development of various diseases and conditions. Dental problems are not often easily categorised into either genetic or environmental, because most anomalies involve the interaction of both factors. Since many genetic disorders involve the orofacial region, the importance of the subject is paramount in dentistry to enable a proper understanding of the development of the various conditions and the treatment of patients presenting with those conditions. The incidence of newborn children with any congenital anomaly is estimated to be as high as 2 to 3 per cent, with more than half of these abnormalities, including minor manifestations, found in the head and neck region. It is therefore not unusual for dentists to be the first to recognise such conditions in their patients and be responsible for providing the necessary treatment. This paper describes many conditions, genetically or partly genetically mediated, which affect the oro-facial tissues, and offers descriptions by which the clinician can start to identify those conditions. PMID- 7558361 TI - The effect of the extract of the miswak (chewing sticks) used in Jordan and the Middle East on oral bacteria. AB - Chewing sticks are commonly used in Jordan, Saudi Arabia and the United Arab Emirates in particular, and the Middle East, Asia, and Africa in general, in addition to many other areas for oral hygiene, religious and social purposes. Recently, the World Health Organisation (WHO) has recommended and encouraged the use of these sticks as an effective tool for oral hygiene. The antibacterial activity of one of these sticks has been tested against some oral aerobic and anaerobic bacteria. Three methods of antibacterial activity were carried out: streaked plate method, ditch plate method, and tube dilution test for minimum inhibitory concentration (MIC). It was found that the extract of these sticks had a drastic effect on the growth of Staphylococcus aureus with MIC values of 69 mg/100 cc, while a variable effect on other bacterial species was noted. It is concluded that using chewing sticks twice a day on a regular basis may reduce the incidence of gingivitis and possibly dental caries. Apart from their antibacterial activity which may help control the formation and activity of dental plaque, they can be used effectively as a natural toothbrush for teeth cleaning. Such sticks are effective, inexpensive, common, available, and contain many medical properties. PMID- 7558364 TI - Educational and practice standards for dental auxiliaries. FDI Commission, 1994. AB - This study examines the education and practice standards of the two most commonly employed dental auxiliaries in North America. Canada and the United States of America have very similar educational standards for the dental hygienist, whereas the dental assistants' educational programmes are less similar. The specific education requirements and legal provisions for employment of these dental auxiliaries is reviewed for both North American counties. PMID- 7558365 TI - Osseointegrated implants: a review of the literature. AB - The success of osseointegrated implants as a treatment option is extremely high. In recent years the emphasis in implant development has moved towards improved aesthetics. This paper reviews some of the recent advances associated with aesthetics and implant supported restorations. The techniques of bone grafting and guided tissue regeneration have allowed more ideal placement of implant fixtures in many situations. Implant companies continue to improve their components making implant supported prostheses possible for a wider variety of patients. Further advances in the management of the soft tissues, particularly the interdental papillae, are required to optimise aesthetic results. PMID- 7558363 TI - A study into the prevention of fissure caries using an antimicrobial varnish. AB - The aim of this study, performed in Bangkok, was to evaluate the possibility of reducing fissure caries development using an antimicrobial varnish, Cervitec. Children aged 7-8 years and 12-13 years, 251 in each age group, with at least 2 sound contra-lateral permanent molars, were selected. A split mouth method was used with one test and one control tooth within the same jaw. At baseline and after two years all children were investigated for DMFS and DMFT. In addition, the size of any cavities was estimated. From 200 children, plaque samples of test and control occlusal surfaces were collected at baseline and after one year and processed to estimate the number of mutans streptococci. Mutans streptococci in saliva were estimated by the Strip mutans method. Cervitec varnish, containing 1 per cent chlorhexidine and thymol was applied at baseline, after 3-4 and after 8 9 months. The results showed that: Cervitec varnish reduced fissure caries development significantly; the levels of salivary mutans streptococci at baseline were significantly correlated with caries status at baseline and with total caries increment over the two-year period; caries development in a fissure was significantly correlated to the level of plaque mutans streptococci at that same site; three months after the last varnish application, a certain reduction of mutans streptococci in plaque could be seen in the test teeth; comparing the size of the lesions, more large cavities were found in the untreated teeth. It is concluded that varnishes should be considered as further options for prevention of fissure caries, possibly in more individualised programmes or in combination with already established methods. PMID- 7558366 TI - Specific serum IgA titres as realistic indicators of treatment prognoses. AB - Some patients with adult periodontitis (AP), as defined by the American Academy of Periodontology, experience better healing than others after scaling and root planing. It is well documented that AP evolves by exacerbation and remission stages and that treatment during one of these stages influences the healing of periodontal pockets. Antibodies directed against major periodontal pathogens involved in AP could be used as indicators of active tissue destruction in AP. The levels of anti-Porphyromonas gingivalis (Pg) antibodies in the sera, saliva and crevicular fluid of ten patients with moderate AP were determined before and after root planing. The results suggest that testing serum IgA levels before scaling and root planing could help improve treatment prognoses. PMID- 7558367 TI - The biological evaluation of medical devices used in dentistry. The influence of the European Union on the preclinical screening of dental materials. AB - The advent of the European Union requires the biological evaluation of all medical devices used in dentistry prior to their general availability for clinical use. Biological evaluation is one component of the 'Essential Requirements' with which all devices need to conform. Such conformity will result in the issue of the CE mark. To facilitate this process, harmonised European standards are required. It is the policy of The European Committee for Standardisation (CEN) that, whenever possible, the relevant International standards will be adopted as European standards. PMID- 7558370 TI - Age and responses to the Love Attitudes Scale: consistency in structure, differences in scores. AB - Two separate issues concerning the relation between age and love are addressed in this article. The first issue is concerned with the age generalizability of the factor structure produced by responses to the Love Attitudes Scale. The data presented indicate that the factor structure of responses to the Love Attitudes Scale is highly similar in college-aged and middle-aged participants. The second issue concerns the relation between age and actual scores on the sub-scales of the Love Attitude Scales. The data indicate that age is related to responses on the Mania and Agape sub-scales, particularly for females. Collectively, these data suggest that general structural conceptions of love remain relatively constant into middle-age, but that there is a relation between age and some specific love styles. PMID- 7558369 TI - Embodiment and emplacement: identities, representation and landscape in Sun City retirement communities. AB - This article focuses on the embodiment, emplacement and representation of aged identities and suggests that more attention be paid to the spatiality of age relations. Identities are both externally imposed and self nominated. Using the example of retirees I explored the ways in which particular places are involved in the creation of identities. Sun City retirement communities are shown to 1) have a material form that serves the retiree identity, 2) be represented in particular ways that contribute to that identity and 3) simultaneously act as a representation. The article asks whether or not these communities are indicative of a shift to postmodernism, characterized by increased emphasis on surveillance, security, simulations, consumerism, and fragmentation of identities, in both life courses and landscapes. PMID- 7558368 TI - A pilot survey on compliance with recommended infection control procedures in ninety dental practices in New Zealand. PMID- 7558371 TI - Older adults' expectations about mental health counseling: a multivariate and discriminant analysis. AB - The present study aimed at identifying significant predictors of expectations about mental health counseling in a sample of highly educated elderly. This task was achieved by administering the full version of the Expectations About Counseling (EAC) questionnaire to fifty-seven retired professors. We first addressed the issue of the current elderly's under-utilization of formal counseling services, then conducted a literature review on the relationship between elderly's characteristics and their views of counseling. Specific hypotheses were formulated for each of the seven possible predictors of EAC scale scores. Previous counseling experience and marital status were significant predictors of EAC scale scores. Young-old adults (i.e., younger than 75 years of age) had received counseling experience significantly more than their older counterpart; their EAC scores, however, were not significantly different than those of old-old participants. Gender, area of residence, income and religiosity did not predict expectations about counseling significantly. Cell size heterogeneity for some predictors might have been responsible for lack of significance on additional factors. The article ends with a discussion of several clinical implications of the findings. PMID- 7558372 TI - Preliminary typology of couplehood for community-dwelling wives: "I" versus "we". AB - A qualitative analysis of women whose husbands reside in nursing homes was conducted to understand the concept of couplehood. Couplehood is defined here as the extent to which one person perceives herself as married to another person (feeling like an "I" or like part of a "We"). An "I"--"We" continuum was presented to each woman, and noticeable patterns emerged. Some women identified themselves as perceiving no couplehood with their institutionalized husbands ("Unmarried Marrieds"). A second group of women perceived a high degree of couplehood ("'Til Death Do Us Parts"), while a third group perceived low couplehood ("Husbandless Wives"). Differences seem to exist in terms of the degree to which couplehood is perceived (no, low, high). Implications of such a typology are offered for researchers, clinicians, and community-dwelling wives. PMID- 7558374 TI - Effect of testicular temperature on vasomotion and blood flow. AB - Vasomotion (spontaneous rhythmic variations in blood flow) has been demonstrated in the parenchyma of the testes of anaesthetized rats, using a laser-Doppler flow probe. As the temperature of the testis was increased, mean blood flow showed no change, but the frequency of vasomotion increased and its amplitude decreased, until vasomotion disappeared between 36 degrees C and 42 degrees C. As the testis was then cooled, vasomotion reappeared, and increased in amplitude and decreased in frequency as the temperature fell. In control rats, in which the temperature of the testes was maintained at normal scrotal temperature of about 33 degrees C, there no changes in vasomotion over an equivalent period. In both groups of rats, when the temperature of the testes was then allowed to fall below normal scrotal temperature, the amplitude of vasomotion increased and its frequency decreased even further, without any change in mean blood flow. Capillary blood flow was also measured with microspheres at the end of the experiment, when testicular temperature was between 19 degrees C and 27 degrees C, and there was no difference between the cooled and control testes or epididymides. PMID- 7558375 TI - Androgen regulation of intra-and extra-thymic T cells and its effect on sex differences in the immune system. AB - Male predominance of malignant disease and female predominance of autoimmune diseases is widely known in humans. To elucidate one of the underlying mechanisms, we examined whether sex differences exist at the level of extra thymic T cells in various organs of mice under physiological conditions. Effects of castration and testosterone administration were also examined. Intra- and extra-thymic T cells with T cell receptors (TCR) of bright and intermediate intensities, respectively, and with nil and high levels of the interleukin-2 receptor beta-chain respectively, were identified by immunofluorescence. Of the three strains tested, it was demonstrated that intermediate TCR cells were predominant in the liver of female mice. In the liver of male mice after bilateral orchidectomy, mononuclear cells increased and intermediate TCR cells predominated as if they were female; testosterone treatment prevented this immunological consequence of orchidectomy. As extra-thymic T cells comprise a considerable proportion of self-reactive forbidden clones and possess cytotoxic activity against syngeneic tumour cells, the present results suggest a possible relationship between (a) physiologically low levels of extra-thymic T cells in males and the male predominance of malignancy, and (b) the high levels of extra thymic T cells in females and female predominance of autoimmune diseases. PMID- 7558373 TI - Phosphomannosyl receptors on the surface of spermatozoa from the cauda epididymis of the rat. AB - This study demonstrates that beta-glucuronidase from rat preputial glands binds with high affinity to spermatozoa from the cauda epididymis. The binding was calcium-independent and was inhibited by mannose-6-phosphate, but not by other phosphorylated or non-phosphorylated sugars. Binding was also inhibited by alpha mannosidase from Dictyostelium discoideum, an enzyme known to have mannose-6 phosphate as the ligand. From solubilized sperm membranes, a protein of > 200 kDa and one of 45 kDa, were absorbed to a column of D. discoideum enzyme and to a phosphomannan column respectively, and eluted with mannose-6-phosphate. According to histochemical observations at the light and the electron microscopic level, gold particles coated with the enzyme became bound to the external surface of the plasmalemma in the acrosomal region of caudal spermatozoa. Similar labelling was observed using gold particles coated with antibodies against the rat 300 kDa phosphomannosyl receptor. The existence of phosphomannosyl receptors on the sperm plasma membrane, and our previous demonstration of the presence of affinity sites for epididymal beta-galactosidase on these gametes which is inhibited by phosphofructosyl derivatives, suggest strongly that maturing spermatozoa could be a target for glycosidases secreted into the lumen of the cauda epididymis, which then become bound to these cells via different ligand-receptor systems. PMID- 7558376 TI - Tight underpants and trousers and risk of dyspermia. AB - The association between the wearing of tight underpants and trousers and risk of dyspermia has been analysed using data from a case--control study. Cases included infertile men with a diagnosis of unexplained dyspermia. Normospermic men of infertile couples were eligible as controls. In comparison with men usually wearing loose underpants, the odds ratio (OR) of dyspermia was 1.9 (95% confidence interval, CI, 0.9-4.1) in those wearing tight underpants. Likewise, the OR of dyspermia was 1.6 (95% CI 0.9-3.0) in men reporting usually wearing tight trousers (including jeans), in comparison with those wearing loose trousers. PMID- 7558380 TI - Rates of testosterone-induced suppression to severe oligozoospermia or azoospermia in two multinational clinical studies. World Health Organization Task force on Methods for The Regulations of Male Fertility. AB - Two multicentre studies of the contraceptive efficacy of azoospermia and severe oligozoospermia were conducted in 16 centres in 10 countries. They used a common protocol of weekly testosterone injections for sperm suppression, the patterns and degrees of which were compared among men from different population groups. Six hundred and seventy normal, health volunteers, of whom 205 were Asian (mostly Chinese) and 465 of non-Asian origin, were given weekly injections of testosterone enanthate, 200 mg IM, during the suppression (6-month) and efficacy (12-month) phases. Patterns of sperm suppression were assessed by semen analysis at monthly or 2-weekly intervals. Sperm counts suppressed more slowly in the Asian than in the non-Asian men in the first 2 months of injections but subsequently suppressed to lower sperm concentrations below 5 million/ml, 3 million/ml or azoospermia at 6 months were 97.1% 95.6%, and 66.7%, respectively for non-Asian men, compared to 99.4%, 98.4% and 89.2%, respectively for Asian men. In conclusion, a hormonal contraceptive method based on regular testosterone injections can suppress spermatogenesis to azoospermia or severe oligozoospermia in 97% of men, regardless of their ethnic origin. PMID- 7558377 TI - Tobacco smoke disrupts testicular microcirculation in the rat. AB - Anaesthetized adult rats were exposed repeatedly to cigarette smoke for 2 s interspersed with exposure to fresh air for either 10, 15 or 30 s using a smoking apparatus. The acute effects of this treatment on testicular microcirculation were studied using laser Doppler flowmetry. Peripheral tissue O2 saturation was measured continuously in the foot during the experiment. Exposure to cigarette smoke caused a moderate acute increase in the testicular blood flow and it inhibited vasomotion. Blood flow and the pattern of vasomotion were normalized after termination of smoke exposure. The time between the last smoke exposure and flow normalization was dependent on the smoke/air dose. Peripheral tissue O2 saturation did not change during the experiments. The effects on testicular microcirculation of local injections of an aqueous extract of cigarette smoke and passive smoking were also studied. Local injection of smoke extract increased blood flow and inhibited vasomotion. Passive smoking caused a moderate decrease in the frequency of vasomotion and amplitude. The observation that cigarette smoke influences testicular microcirculation acutely is discussed in relation to possible adverse effects on spermatogenesis. PMID- 7558378 TI - In vitro fertilization and the effect of progesterone and 17 alpha hydroxyprogesterone on acrosome reaction of mouse epididymal spermatozoa. AB - This study assessed the effect of progesterone and 17 alpha-hydroxyprogesterone (17 alpha-OH-progesterone) at concentrations of 0.01-10 micrograms/ml, on the acrosome reaction and in vitro fertilizing ability of mouse epididymal spermatozoa. Cumulus masses containing oocytes were cultured in Brinster's medium, to which were added capacitated epididymal spermatozoa which had been incubated in medium with various concentrations of progesterone or 17 alpha-OH progesterone for 90 min. IVF success rate was assessed 20-24 h following insemination. Progesterone was found to increase the fertilization rate at the 1 microgram/ml and 10 microgram/ml concentrations while lower concentrations had no effect. However, 17 alpha-OH-progesterone failed to show any effect on fertilizing ability. Incubation of epididymal spermatozoa in medium containing 1 microgram/ml and 10 micrograms/ml progesterone significantly increased the acrosome reaction as monitored by a chlortetracycline fluorescence assay. 17 alpha-OH-progesterone, however, failed to show any effect on the acrosome reaction. The results suggest direct effects of progesterone, but not of 17 alpha OH-progesterone, on fertilization and the acrosome reaction of mouse spermatozoa. PMID- 7558381 TI - Functional motion changes during sperm transit to the site of fertilization and in-vitro applications: a review. AB - Continued research to define the parameters of sperm function should aid the evaluation of various approaches in infertility as well as the efficacy of contraceptives for men which do not necessarily achieve azoospermia. Many treatment forms have been advocated for male factor infertility but have yielded little effect. These included, for example, gonadotrophins, clomiphene citrate, the weakly androgenic steroid, mesterolone. Often, improvements in oligoasthenozoospermia that are not related to genital infection, do not attain normozoospermic levels. Owing to lack of success with various treatment modalities, assisted reproductive technology encompassing artificial insemination by husband or donor following in vitro enhancement of sperm function have assumed an important role in male infertility. Agents that have been shown to induce and support sperm capacitation processes such as hyperactivation, could serve an important role. These include human follicular fluid (HFF), maternal serum, fetal cord serum and methyl xanthine derivatives. PMID- 7558382 TI - Assessment of the vitality and acrosomal status of human spermatozoa using fluorescent probes. AB - The acrosomal status and vitality of human spermatozoa are generally assessed simultaneously using the lectins Pisums sativum agglutinin (PSA), Peanut agglutinin (PNA) and Concanavalin A (Con A) in conjunction with either Hoechst 33258 (H258; a fluorescent DNA-binding supravital stain with limited membrane permeability) or a hypo-osmotic swelling (HOS) test. In the present study, sperm vitality was assessed using H258 under different staining conditions and the HOS test. The sensitivity of PNA- and Con A-labelling methods were also compared by evaluating the acrosomal status of motile human spermatozoa after capacitation (1 and 4 h) in vitro followed by exposure to dimethylsulphoxide (DMSO) and a calcium ionophore. The E-N method employing eosin-staining for 15 sec, rather than for 30 sec, provided a more reliable estimate of sperm vitality. H258, as used in the H258/Con A-labelling method (with and without ethanol fixation) rather than under the staining conditions equivalent of H258/PNA-labelling, was as good for vitality assessment as the E-N method employing eosin-staining for 15 sec. However, the HOS test overestimated the proportion of dead spermatozoa compared to those obtained using different H258 and E-N methods. Further, the Con A labelling method consistently scored a significantly lower percentage of spermatozoa undergoing the acrosome reaction compared to those estimated by the PNA-labelling method. It is concluded that the different sensitivity of these methods can be attributed to the different binding specificity of the lectins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558379 TI - Cholesterol, phospholipids and markers of the function of the accessory sex glands in the semen of men with hypercholesterolaemia. AB - The effect of hypercholesterolaemia on the cholesterol and phospholipid content of spermatozoa and seminal plasma was studied. Testosterone and specific markers of the accessory sex glands were also measured. Semen samples from 11 hypercholesterolaemic patients (plasma cholesterol > 6.42 mmol/l, plasma triglycerides < 2 mmol/l) were compared with those of 11 normocholesterolaemic controls (plasma cholesterol < 5.14 mmol/l, plasma triglycerides < 2 mmol/l). Cholesterol, phospholipids and the molar ratio of cholesterol: phospholipids were not significantly different between the two groups of patients either in spermatozoa or in seminal plasma. In hypercholesterolaemic patients the total amount of carnitine in the ejaculate was significantly higher, but there were no significant differences in the levels of acid phosphatase or fructose. There were no significant differences in seminal plasma levels of testosterone in the two groups of subjects. These results suggest that hypercholesterolaemia has no effect on cholesterol and phospholipid levels in spermatozoa and does not cause gross modification of the secretory function of the accessory sex glands. PMID- 7558383 TI - Application of the hypo-osmotic swelling test to spermatozoa prepared by swim-up and discontinuous Percoll separation. AB - The quality of spermatozoa prepared by washing and swim-up or by discontinuous Percoll centrifugation, was compared by applying the hypo-osmotic swelling (HOS) test to semen samples from 116 men of infertile couples. The HOS test performed on 95 normal semen samples showed that the percentage of swollen spermatozoa separated by both techniques was significantly higher than in the initial ejaculate (p < 0.001). The percentage of HOS-positive spermatozoa separated by the Percoll gradient technique was significantly higher than that separated by the swim-up technique (p < 0.001). On the contrary, in 21 abnormal semen samples, there was no significant difference in the percentage of spermatozoa which were positive in the HOS test between the Percoll gradient and the swim-up technique (p = 0.44). It is suggested that the Percoll gradient technique appears to be preferable to the swim-up technique when semen parameters are normal, but there is no significant difference between these two technique in abnormal semen. PMID- 7558386 TI - A strategy for identifying candidate sperm antigens for immunocontraception: isolation of human testis cDNA clones using polyclonal antisera directed against hamster acrosomal membrane preparation. AB - Components of the mammalian sperm acrosome that have been conserved during evolution are probably essential for fertilization and are therefore potential antigens for the development of an immunocontraceptive vaccine. In order to identify such protein components, a series of specific polyclonal antisera were generated by immunizing rabbits with purified acrosomal membrane fractions from hamster epididymal spermatozoa. Antisera were finally selected using immunological and in-vitro fertilization assays, and used to then screen a human testis lambda gt11 cDNA library. As a result of this screening over 70 clones were identified, selected and purified. The cDNAs were amplified by polymerase chain reaction (PCR) and the inserts characterized by restriction enzyme digestion and oligonucleotide probing techniques. The functional activity beta galactosidase fusion proteins expressed by these clones (HA5-2, HA6-2 and HB4-1) inhibited significantly fertilization and reduced spermatozoa binding compared to controls. To date, sequence data has been obtained from HB4-1 (1.75 kb). The first 1132 nucleotides displayed > 96% homology to human testis-specific lactate dehydrogenase (LDH-C4) gene, the product of which is a known candidate antigen for a contraceptive vaccine. This finding suggests that a strategy involving the screening across species for conserved moieties of the mammalian acrosome may be useful for identifying candidate antigens for immunocontraception. PMID- 7558385 TI - Testicular size in young adult Sinhalese. AB - Height, body weight, arm span and testicular size were measured in 200 Sinhalese men aged 21-31 years. The mean +/- SD right testicular size was 17.2 + 4.1 ml and that of the left was 16.1 + 3.9 ml. The testes were of equal size in 75% and the right testis was larger in 20% of the men. The prevalence of undescended testes was 2%. There as a significant correlation of testicular size with weight (p < 0.05) and a significant correlation of right testicular size with span (p < 0.01). Testicular size was not correlated with height or body mass index. Although the Sinhalese are ethnically closer to the Caucasians, their testicular size is similar to that reported by previously for Thai men. PMID- 7558384 TI - A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures. AB - Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking. PMID- 7558388 TI - Intravasal injection of formed-in-place medical grade silicone rubber for vas occlusion. AB - This paper describes two consecutive studies: a volume study and an efficacy study. The volume study determined the appropriated volume of Medical Grade Silicone Rubber (MSR) needed to achieve complete occlusion of the vas deferens. This was done by in-vitro testing of 130 human vas specimens containing plugs of MSR formed in vivo. The volume of MSR needed to occlude the vas was 0.1531 +/- 0.0059 ml injected by five to six turns of the applicator handwheel. There was a correlation between MSR volume, weight, and the number of turns of the applicator handwheel. An influence of body height upon the volume and weight of the MSR was also observed. An oval shaped clamp (15 mm long) was more effective in producing secure vas occlusion than was a round clamp (10 mm long). The mean outer diameter of the vas specimens was 1.80 +/0 0.15 mm, and the mean maximum dilated inner diameter was 0.93 +/- 0.11 mm. The efficacy study was a clinical trial to compare MSR was occlusion (using an oval 15 mm clamp and the appropriate volume derived from the volume study, n = 58) with no-scalpel vasectomy as the standard procedure (n = 64). The azoospermia rate following MSR vas occlusion was not significantly different from that following no-scalpel vasectomy, and was achieved in 3-6 months. PMID- 7558387 TI - Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial. AB - To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP binding ratio of test and control spermatozoa averaged 0.82 (range 0.51 1.13).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558390 TI - Contribution of the male factor to unexplained infertility: a review. AB - The more exhaustive the evaluation of couples with unexplained infertility, the more likely is the opportunity for detecting the aetiological factor responsible for infertility. Transport of spermatozoa through the upper genital tract and their ability to fertilize the oocyte are two obscure areas for the conventional evaluation of infertility. Although research in the former area is limited, there is indirect evidence that impaired sperm transport could be one of the causes of infertility in some couples with otherwise unexplained infertility. on the other hand, the availability of sperm function tests and the correlation of their results with in-vitro fertilization rates have allowed the detection of a previously hidden male factor in couples with unexplained infertility. It has been demonstrated that couples suffering unexplained infertility have significantly lower in-vitro fertilization rates in comparison with patients with tubal problems. These results can be explained because of several case control studies in patients with unexplained infertility have reported defects in capacitation and sperm motion characteristics, binding of the spermatozoa to the zone pellucida, acrosome reaction, acrosin activity of the spermatozoa, and the ability of the spermatozoa to penetrate zona-free hamster oocytes. These observations suggest that methods for assessing the fertilizing capacity of the spermatozoa have to incorporated in the evaluation of couples with unexplained infertility in order to amplify the scope of the workup and to better decide the appropriate treatment for these couples. PMID- 7558391 TI - Intrauterine insemination of washed spermatozoa for treatment of oligozoospermia. AB - Efficacy of intrauterine insemination (IUI) using washed spermatozoa for treatment of oligozoospermia was evaluated by a prospective randomized study in 50 couples, using LH-timed natural intercourse in the alternate menstrual cycles as a control. The quality of spermatozoa in terms of their concentration and motility before and after sperm washing was compared. Sperm motility increased significantly after sperm preparation but the number of sperm was reduced. Eight pregnancies occurred in 253 cycles of IUI with washed spermatozoa and clomiphene citrate-stimulated cycles (3.16% per cycle). Only one patient conceived in 242 LH timed natural intercourse cycles (0.41% per cycle). Compared with LH-timed natural intercourse, IUI provided a significantly improved pregnancy rate. When the sperm count was < 5 x 10(6) per ml, no pregnancy occurred with the IUI method. Therefore, IUI is a of rather limited usefulness when the sperm quality is very poor. Few complications occurred after IUI, but included slight cervical contact bleeding and mild abdominal discomfort and/or cramps. In conclusion, IUI should be considered as a useful and relatively non-invasive therapeutic modality for treating caused by moderate oligozoospermia (> 5 x 10(6)/ml), when sexual intercourse fails. PMID- 7558389 TI - Subcapsular intratesticular assay: a preliminary screening method for putative male antifertility drugs. AB - The subcapsular intratesticular assay was developed for the preliminary screening of male antifertility agents. In the assay, small amounts of the test sample are injected twice, 1 week apart just beneath the tunica albuginea of the testes of rats. The rats were killed within 3 weeks of treatment to examine epididymal spermatozoa and the histology of the testis and epididymis. There is an excellent agreement between this method and the modified MB-50 assay developed by the World Health Organization. To date, more than 300 samples have been assayed, of which the results of 10 plant-derived compounds are reported here. Not one active sample has been missed as confirmed by the modified MB-50 method, although one compound which was positive in the subcapsular assay did not show antifertility action in the in-vivo assay. Compared with the latter assay, the advantages of this method are: simplicity, time-saving, rat-saving and the need for far smaller amounts of test sample. The absence of false-negative results makes the subcapsular intratesticular assay applicable as a preliminary screening method to test potential male fertility regulating agents. PMID- 7558392 TI - Severe asthenozoospermia: a structural and functional study. AB - Ejaculates from 25 patients with severe asthenozoospermia (all spermatozoa immotile or only non-progressively motile) were studied to identify individually the cause of impaired motility. Multiple tests were performed, viz. light and electron microscopic studies and sperm function tests. An objective scoring was applied to both the routine and the functional analyses. Three categories of samples were identified: (1) necrozoospermia (n = 9), where sperm viability was very poor; (2) structural tail abnormality as seen by light microscopy (n = 4); and (3) ultrastructural abnormality (n = 12). In the last category, one case showed absence of dynein arms; this was associated with mitochondrial abnormalities. Mitochondrial abnormality with normal tail components was observed in the majority (n = 7) and accessory fibre abnormality in four cases. The scoring system revealed that, functionally, all samples were abnormal whereas routine analysis showed 15 samples to be subnormal and 10 to be abnormal, which indicates the need for functional analysis. Because of the multiple defects seen in these samples, there is a need for a battery of sperm function tests. This study indicates that mitochondrial defects are one of the causes that may account for the loss of sperm motility in the patient population. PMID- 7558396 TI - Reading the changes: new frontiers in dialysis. PMID- 7558397 TI - The future of artificial organs. PMID- 7558395 TI - Acidic, pyruvate-based peritoneal dialysis solutions: relationship between low titratable acidity and biocompatibility. PMID- 7558394 TI - Treatment of homozygous and double heterozygous familial hypercholesterolemic children with LDL-apheresis. AB - Within the framework of a seven-year clinical experience on treatment of severe hyperlipoproteinemia with/without associated coronary heart disease, with therapeutic plasmapheresis (APO B-100-containing lipoprotein-apheresis), we focused the present report on two young patients aged 7 and 11 years, respectively. The older patient is a boy treated since 1990 by plasma-exchange, cascade filtration-low density lipoprotein apheresis (LDL-apheresis), and dextrane sulphate-LDL apheresis. Over the treatment period the patient was submitted to three consecutive coronary angiographies. The second is a girl first submitted to a coronary angiography and then treated with dextrane sulphate-LDL apheresis. Up to now, a total of one-hundred therapeutic plasmaphereses have been performed. The interval of treatment was of fifteen days, and a volume of 2-3000 ml of plasma was processed at each session. The systems used were the following: DIDECO Vivacell BT 798-A, DIDECO Vivacell BT 798-A + BT 803, DIDECO BT 985 (Dideco, Mirandola, Italy), KANEKA MA-01 (Kanegafuchi, Osaka, Japan). Mean (SD) plasma apo B-100-containing major lipoprotein-LDL, Lp(a)-levels during treatment, are reported below: [table: see text] The treatment was very well tolerated. Rare, moderate hypotensive events occurred. Nevertheless, all procedures were regularly completed. A mild hypochromic anemia, regressed using drug treatment, was observed in the boy. Along with the improvement of plasma atherogenic profile, a regression of skin xanthomas and unchanged favourable coronary angiograms, were obtained in the above mentioned patient. PMID- 7558393 TI - Sperm binding to the human zona pellucida after migration through human cervical mucus. AB - During lactational amenorrhea a special type of cervical mucus, similar to that found during the luteal phase, is produced. This mucus, however, is able to support sperm migration. In the study described, the ability of spermatozoa to bind to the human zona pellucida (hZP) after migration through periovulatory and post-partum mucus was studied. Mucus was obtained from exclusively breastfeeding women in amenorrhea at 30, 60, 120 and 180 days post-partum. Periovulatory mucus samples from normally cycling women were used as a control. Flat capillary tubes were filled with BWW culture medium at the top and cervical mucus at the bottom. The tubes were immersed in a semen reservoir and the spermatozoa allowed to migrate through the mucus for 3 h into the culture media. Then the spermatozoa were coincubated with 3-4 hZP for 30 min and the number of bound spermatozoa per zona was counted. Periovulatory cervical mucus had an average Insler score of 14 +/- 0.5 as compared to 4.6 +/- 0.4 for post-partum mucus. Spermatozoa recovered from periovulatory mucus were always able to bind to the hZP in only 68 +/- 7% of the cases. Moreover, spermatozoa recovered from post-partum mucus bound to the ZP in lower numbers than did spermatozoa recovered fro periovulatory mucus (p < 0.03). These results suggest a greater ability of sperm-hZP binding after migration through periovulatory mucus and they also indicate that sperm binding to the ZP is possible even after sperm migration through a low quality mucus. PMID- 7558398 TI - Neutrophil intracellular pH after exposure of neutrophils to a euhydric, lactate based peritoneal dialysis solution and its euhydric, bicarbonate-based counterpart. AB - The effects of euhydric, lactate-based peritoneal dialysis solutions and those of their euhydric, bicarbonate-based counterparts, on neutrophil intracellular pH are comparable. This similarity in effects on intracellular acidity may have bearings on the influences of these solutions on cellular functions in general. PMID- 7558399 TI - Markers of hepatitis C infection among hemodialysis patients with acute and chronic infection: implications for infection control strategies in hemodialysis units. AB - To evaluate strategies for screening patients on hemodialysis (HD) for markers of acute and chronic hepatitis C virus (HCV) infection, we studied sixty-nine patients at a single center over a 36-month period. Serum samples were tested for alanine aminotransferase (ALT) levels, anti-HCV and HCV RNA at 3-4 month intervals. Anti-HCV was tested for by EIA1, EIA2, and RIBA2. HCV RNA was detected by polymerase chain reaction (PCR). In addition, IgM antibody to the c33 antigen of HCV was detected by an experimental EIA. Of the 43 HD patients at the start of the study, anti-HCV was detected by EIA1 in 13 (30%). All EIA1 positive patients and 14 (47%) of the 30 EIA1 negative patients tested positive by EIA2. Thus, at the start of the study 27 (63%) of 43 patients tested positive for anti-HCV by EIA2. The presence of anti-HCV among EIA2 positive patients was confirmed by RIBA2 in all patients. Based on the PCR results, the sensitivity, specificity, positive predictive value and negative predictive value for EIA1 were 48%, 100%, 53% and 100%, respectively, and for EIA2 were 100%, 100%, 100% and 100%, respectively. During follow-up, 26 EIA2 negative patients began HD in the unit. Of the 42 EIA2 negative patients, five (12%) seroconverted for anti-HCV during follow-up. All five patients with new HCV infection tested positive for HCV RNA three months prior to the detection of anti-HCV by EIA2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558400 TI - De novo membranous glomerulopathy in renal allografts with unusual histology. AB - We describe two cases of de novo membranous glomerulopathy in the renal allograft, with unusual histologic findings. In one patient the allograft nephrectomy specimen showed numerous foam cells in the intima of hyperplastic arteries along with prominent features of chronic rejection. In the other patient, prominent IgM deposits were seen in the glomerular mesangium without chronic rejection. PMID- 7558402 TI - On line echocardiography in evaluating left atrial changes in acute mitral regurgitation and in monitoring a catheter-mounted balloon for its correction. AB - The purpose of this study was to evaluate changes in the left atrial size in acute mitral regurgitation (AMR) and monitor its correction by a catheter-mounted balloon (B). In 16 dogs, pressure changes in the left atrium (LA) were related to LA size and regurgitant mitral flow after the production of AMR by severing the mitral cusps. TEE was used for evaluating LA area (ALA) and the mitral regurgitation flow area (AMRF). TEE was also used in monitoring the position of a catheter-mounted B inserted through the LV apex and positioned on the mitral ring to relieve AMR. The B was inflated during systole and deflated during diastole. The ALA increased by 4.89 +/- 1.21 cm2 (mean +/- 1 SD) (p < 0.001) after AMR, the AMRF increased by 3.01 +/- 0.85 cm2 (p < 0.001) and the mean atrial pressure (LAP) by 9.38 +/- 2.43 mmHg (p < 0.001). In all experiments the position of the B could be confirmed in the 2D image and in 4 the reduction of AMRF by the function of the B was documented by the colour flow Doppler. It is concluded that after AMR the size of the LA increases along with the increase in LA pressure and a phasically inflated balloon and its effect on AMR can be easily identified and monitored by TEE. PMID- 7558401 TI - Closed loop control of PaCO2 during ECC. AB - A prototype for the control of blood gases during extracorporeal circulation (ECC) has been built. It is composed of a CDI300 continuous gas blood analyzer, a programmable gas blender and an IBM PC. The air blender is composed of three mass flow controllers (air, oxygen and total flow rate). The microcomputer commands these controllers in order to obtain the desired mixture of air and oxygen. The system acquires the data sent by the blood gas analyzer every 6 seconds and commands the gas blender in order to maintain the desired arterial dioxide partial pressure (PaCO2) in the arterial line. The first experimental results on sheep show that if blood gas parameters are stable when beginning the closed loop control, then the desired PaCO2 value may be obtained. Otherwise, the value of PaCO2 under control is slightly different but a stable value is obtained after 10 minutes. More experiments must be done in order to establish the real limits of such a system and optimize the gain of the control system. PMID- 7558403 TI - Collagen and fibronectin immobilization on PHEMA microcarriers for hepatocyte attachment. AB - Polyhydroxyethylmethacrylate (PHEMA) microcarriers in a size range of 150-250 microns were prepared by a suspension polymerization in an aqueous phase containing magnesium oxide. The hydroxyl groups on the PHEMA microcarriers were activated by cyanogen bromide. In order to improve cell attachment, cell-adhesive proteins, namely, collagen and fibronectin were immobilized onto PHEMA microcarriers. The nonspecific adsorption values for collagen and fibronectin were 0.10 mg collagen/g PHEMA and 0.044 mg fibronectin/g PHEMA, respectively. Collagen and fibronectin immobilization on PHEMA microcarriers were studied at different pH by using single protein solutions containing different amounts of proteins, at a constant temperature of 20 degrees C. The maximum immobilizations were 0.85 mg collagen/g PHEMA (at pH: 9.5) and 0.52 mg fibronectin/g PHEMA (at pH: 7.4). Hepatocyte attachment onto these biologically modified PHEMA microcarriers was studied. Hydrophilic PHEMA microcarriers did not support cell attachment. High hepatocyte attachment yields (up to 75% surface coverage) were observed on collagen and fibronectin immobilized PHEMA microcarriers. PMID- 7558404 TI - Evaluation of new small barium alginate microcapsules. AB - Microencapsulation of islets of Langerhans has been proposed in order to prevent immune rejection and possible recurrence of autoimmune disease. This study introduces a fast simple one-step microencapsulation procedure which allows the production of small sized barium-alginate beads. The volume of the microcapsules produced was approximately that of the encapsulated islets. Consequently, the insulin kinetics and the oxygen diffusion were favoured, while the transplanted tissue volume was reduced. Electron microscopy and immunoisolating testing were performed to evaluate the molecular cut-off, the physical and chemical characteristics of these microcapsules. Immunohistochemical staining and perifusion experiments of microencapsulated pancreatic islets showed their viability after the encapsulation procedure as well as in vivo experiments. In fact, microencapsulated porcine islets were implanted intraperitoneally into streptozotocin-diabetic rats. The xenografts reversed the hyperglycemic state and functioned for a period ranging from 9 to 385 days. The low mannuronic acid concentration and the purity grade of the alginate, exerted a combined influence on the capsule biocompatibility as in vivo studies showed. PMID- 7558409 TI - The human bioclimates of Western and South Pacific islands and climate change. AB - The current bioclimates of equatorial Western and tropical South Pacific have been assessed, using the concepts of effective temperature and relative strain as a basis for evaluating likely changes in human comfort regimes as a consequence of global warming. Current bioclimates may be considered marginally stressful for indigenous populations. Global warming will result in changes in the frequency, duration and intensity of physioclimatically stressful conditions. PMID- 7558405 TI - Significant correlations between certain spectra of atmospherics and daily periodic activities of Mongolian gerbils (Meriones unguiculatus L.). PMID- 7558408 TI - Meaningful wind chill indicators derived from heat transfer principles. AB - The wind chill index (WCI) and the more widely used wind chill equivalent temperature represent an attempt to combine several weather-related variables (temperature, wind velocity and solar radiation) into a single index which can indicate human comfort. Since its introduction in 1945, the WCI has been criticized mainly on the ground that the underlying model does not comply with modern heat transfer theory. In spite of that, the WCI, "calibrated" to human comfort, has proven to be successful in predicting discomfort and tolerance of man to the cold. Nevertheless, neither the WCI nor the wind chill equivalent temperature can be actually measured and, therefore, without the additional 'calibration' they are meaningless. In this study we have shown that the WCI represents the instantaneous rate of heat loss from bare skin at the moment of exposure to the cold, and as such, it correlates reasonably well with measurable variables that represent a feeling of cold. Two new wind chill indicators have been introduced: exposed skin temperature and maximum exposure time. These indicators yield more information than the WCI provides, are measurable, have physical meaning and are based on established heat transfer principles. PMID- 7558406 TI - Free and total thyroid hormones in humans at extreme altitude. AB - Alterations in circulatory levels of total T4 (TT4), total T3 (TT3), free T4 (FT4), free T3 (FT3), thyrotropin (TSH) and T3 uptake (T3U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized low landers, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT3 and FT4 occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T3U was found to be higher at high altitudes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558407 TI - Cardiac output variations in supine resting subjects during head-out cold water immersion. AB - Five men, aged 31.2 years (SD 2.3), under semi-nude conditions and resting in a dorsal reclining position, were exposed to thermoneutral air for 30 min, followed immediately by a cold water (15 degrees C) immersion for 60 min. Cardiac output was measured using a dual-beam Doppler flow meter. During immersion in cold water, cardiac frequency (fc) showed an initial bradycardia. The lowest values were reached at about 10 min after immersion, 58.3 (SD 2.5) to 48.3 (SD 7.8) beats min-1 (P < 0.05). By the 20th min of exposure, fc had gradually risen to 70.0 beats min-1 (SD 6.6, P < 0.05). This change could be due to the inhibition of the initial vagal reflex by increased catecholamine concentration. Stroke volume (Vs) was significantly increased (P < 0.05) during the whole cold immersion period. Cardiac output, increased from 3.57 (SD 0.50) to 6.26 (SD 1.33) l min-1 (P < 0.05) and its change with time was a function of both Vs and fc. On the other hand, systolic flow acceleration was unchanged during the period of immersion. The changes in the respiratory variables (ventilation, oxygen uptake, carbon dioxide output and respiratory exchange ratio) during immersion showed an initial hyperventilation followed, as immersion proceeded, by a slower metabolic increase due to shivering. PMID- 7558410 TI - Occupational cancer in developed and developing countries. PMID- 7558412 TI - Analysis of the antibody response to immunization with purified O-acetyl GD3 gangliosides in patients with malignant melanoma. AB - Gangliosides expressed in malignant melanoma are potential targets for immunotherapy. Immunization of melanoma patients with vaccines containing purified GM2 ganglioside has resulted in induction of GM2 antibodies, and high titers of GM2 antibodies have correlated with increased survival. Melanoma ganglioside 9-O-acetyl GD3 is another candidate for ganglioside vaccine construction because of its limited expression in normal human tissues. As purification of 9-O-acetyl GD3 from human melanoma (9-O-acetylated on the terminal sialic acid) is not practical for broad application, we investigated the antibody response of melanoma patients to O-acetyl GD3 from several additional sources: hamster melanoma (7-O-acetyl GD3), bovine buttermilk (mixture of 7-O acetyl GD3, 9-O-acetyl GD3 and 7,9-di-O-acetyl GD3) and chemically modified GD3 from bovine brain (9-O-acetylated on the subterminal sialic acid). Only immunization with the buttermilk-derived O-acetyl GD3 preparation resulted in consistent production of IgM antibodies. However, the induced antibodies reacted with the immunogen and with 7-O-acetyl GD3 derived from hamster melanoma but not with 9-O-acetyl GD3 or human melanoma cells expressing 9-O-acetyl GD3 on their cell surface. In contrast, all O-acetyl GD3 derivatives used for immunization were recognized by murine MAbs that reacted with 9-O-acetyl GD3, and immunization of mice with buttermilk-derived O-acetyl GD3 resulted in the production of antibodies that reacted with human melanoma cells expressing 9-O-acetyl GD3. Apparently, the human and murine immune systems preferentially recognize different epitopes on these molecules. PMID- 7558413 TI - Detection of an Epstein-Barr-virus variant in T-cell-lymphoma tissues identical to the distinct strain observed in nasopharyngeal carcinoma in the Taiwanese population. AB - An EBV variant has been identified in NPC tissues in Taiwan. This EBV variant contains a point mutation in exon I of the LMP I gene. This mutation results in the loss of an XhoI site at nt 169,426, which is present in strain B95-8. In addition, this variant contains a 30-bp deletion in exon 3 of the gene. The recent demonstration of the prevalence of EBV-containing nasal and peripheral T cell lymphoma in this region drove us to evaluate the presence of this NPC-EBV strain in 7 cases of T-cell lymphoma, as well as in 48 NPC tissues, 2 cases of Hodgkin's disease and I B-cell lymphoma. Four samples of normal lymph node tissue, 40 of normal nasopharynx tissue and 78 throat washings of healthy individuals were included for comparison. We used sequence-specific primers and the polymerase chain reaction (PCR) method to amplify LMP I gene fragments containing these variations. Mutations were then confirmed by restriction-enzyme digestion and the DNA sequencing analysis. Our results showed that 57 of 58 tumor tissues samples were EBV-positive. Among them, 56, including 6 T-cell-lymphoma samples, belonged to the NPC strain. This strain of EBV was also present in 92% of EBV-positive normal nasopharynx tissues and in 84% of EBV-positive throat washings of the healthy individuals tested. These results suggest that the NPC EBV strain is prominently present in Taiwan. PMID- 7558414 TI - Reproductive and other factors and risk of epithelial ovarian cancer: an Australian case-control study. Survey of Women's Health Study Group. AB - Of the few factors known to be associated with epithelial ovarian cancer, the most consistently observed relate to women's reproductive function, although even here uncertainties remain. We have undertaken a case-control study involving personal interviews with over 1,600 women, the largest of its kind to date, to investigate further the associations between women's reproductive histories and other factors and the development of ovarian cancer. Cases were drawn from women diagnosed with epithelial ovarian cancer in 3 Australian states, Queensland, New South Wales and Victoria, between August 1990 and December 1993, and controls were drawn at random from the electoral roll, stratified by age and geographic region. Trained interviewers administered standard questionnaires to obtain detailed information about women's reproductive and contraceptive histories and other factors of interest, such as smoking and family history of ovarian or other cancer. Findings were based on data from 824 cases and 860 controls and confirmed the reduced risk of ovarian cancer associated with increasing parity and duration of use of the oral contraceptive pill (OCP), hysterectomy and tubal ligation. The strongest association of all was seen with use of the OCP for 10 years or more. An inverse association between ovarian cancer and age at first birth was observed, but this was not statistically significant. There were no associations between development of ovarian cancer and number of incomplete pregnancies, use of hormone replacement therapy or menstrual history. Among other factors considered, education after leaving school was negatively associated and high body mass index, family history of ovarian cancer, use of talc in the abdominal or perineal region and smoking were positively associated with occurrence of ovarian cancer. PMID- 7558411 TI - Epidermal growth factor expression in human colon and colon carcinomas: anti sense epidermal growth factor receptor RNA down-regulates the proliferation of human colon cancer cells. AB - Human colon cancer cell lines express epidermal growth factor (EGF) mRNA, secrete EGF and may respond to it via the cell-surface EGF receptor (EGFR). Expression of these molecules in human colon and colon tumor, however, is not clear. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of RNA prepared from paired normal human colon and colon tumor samples from 12 individuals followed by Southern blotting analyses of the RT-PCR products revealed a major fragment of 527 bp and a minor fragment of 404 bp that hybridized to a human EGF cDNA probe under stringent conditions. Identical results were obtained from 8 human colon cancer cell lines. Cloning and sequencing of PCR products confirmed that both fragments were from the human EGF gene; the 527-bp fragment corresponded exactly to nucleotides 2,891 to 3,417 of the human EGF mRNA reported by others. A deletion of 123 nucleotides (nucleotides 3,172 to 3,294) was found in the 404-bp fragment. Immunohistochemical studies using cyostat sections of human colon specimens showed that EGF was expressed in the human colon and that expression was restricted to the epithelial colonic crypt cells and epithelium-derived cancer cells. Since EGF and EGF-related molecules are potent mitogens that mediated their effect through the EGFR, we also determined the efficacy of anti sense EGFR RNA in circumventing the EGFR-related pathway of proliferation. Expression of anti-sense EGFR RNA, by transfection with an inducible anti-sense EGFR expression vector, down-regulated cell-surface EGFR expression and proliferation of these cells and their ability to grow in soft agar. Anti-sense EGFR RNA was found to be an anti-proliferative agent in both relatively non aggressive and highly aggressive human colon cancer cells. PMID- 7558415 TI - Familial aggregation of tumors and detection of hereditary non-polyposis colorectal cancer in 3-year experience of 2 population-based colorectal-cancer registries. AB - The clinical data of 2 population-based registries, located in areas with different incidence rates of colorectal cancer, were used in order to assess the role of familial factors in the pathogenesis of these tumors. The occurrence of tumors in family members was investigated in 389 subjects with colorectal cancer registered in Modena (Northern Italy, an area characterized by a high incidence of colorectal malignancies) between 1984 and 1986; similar information was obtained in 213 patients with tumors of the large bowel registered in Ragusa (Sicily, Southern Italy, an area of similar magnitude and with low incidence rates for these tumors) in the 3-year period 1988 to 1990. In both series, colorectal cancer occurred significantly more often among relatives of patients. Controls were patients of the same sex and age (+/- 5 years) hospitalized during the study periods, but not for gastrointestinal or neoplastic diseases. There were 89 cancer cases (3.1%) among 2,851 relatives of patients in Modena, vs. 17 cases among 1,744 relatives (1.0%) in Ragusa (p < 0.01). Apart from colorectal cancer, there was no excess of other types of tumors in patients' families (in both series). During the 3 years of registration, 17 cases of hereditary non polyposis colorectal cancer (HNPCC, or Lynch syndrome) were diagnosed in Modena; in contrast, this syndrome was more rare in Ragusa (one case only during 3 years of observation). Similarly, many more families with clinical suspicion of HNPCC were recorded in Northern regions (44 vs. 10). Although incidence rates of colorectal cancer are appreciably higher in Northern than in Southern Italian regions, the excess of this cancer type among close relatives is similar. However, full-blown HNPCC or suspected Lynch syndrome were significantly more frequent in Northern Italy. PMID- 7558416 TI - Expression of urokinase receptor in various stromal-cell populations in human colon cancer: immunoelectron microscopical analysis. AB - The host reaction is an important factor in the biological behavior of cancers. In human colon adenocarcinoma, stromal cells and some cancer cells express the urokinase receptor (uPAR), a molecule involved in the regulation of extracellular proteolysis. The present study reveals the identity of uPAR-expressing cell types and the subcellular localization of this molecule by immunoelectron microscopy in colon cancer. uPAR-positive cells were most abundant at the invasive margin of colon cancer and were identified as macrophages, fibroblasts, neutrophilic and eosinophilic granulocytes, endothelial cells and cancer cells. Of these, the most numerous were macrophages with uPAR detected along the plasma membrane, in accordance with its function in plasminogen activation on the cell surface. Fibroblasts were labeled in the lumen of rough endoplasmic reticulum, indicating its intracellular synthesis. Some granulocytes and endothelial cells expressed immunoreactivity along the plasma membrane. uPAR-positive cancer cells were stained along the plasma membrane and in rough endoplasmic reticulum. These findings suggested that a variety of non-malignant host cells play an important role in the plasmin-mediated breakdown of the extracellular matrix at the invasive margin. PMID- 7558417 TI - Among 17 inducers of differentiation only sodium butyrate causes a permanent down regulation of c-myc in Burkitt's lymphoma. AB - De-regulation of c-myc by chromosomal translocation is one crucial step for the development of Burkitt's lymphoma. The de-regulation is caused through juxtaposition of c-myc with one of the 3 immunoglobulin loci. We have reported earlier that treatment of Burkitt's lymphoma cells with n-butyrate causes transcriptional down-regulation of c-myc expression. Because of the possible therapeutic implication of this result, we looked for other compounds which, on the one hand, might be applicable in vivo and, on the other hand, might cause down-regulation of c-myc expression in Burkitt's lymphoma cells. Since n-butyrate is known to induce differentiation, we have examined other differentiation inducers of different chemical nature for their ability to reduce c-myc expression in Burkitt's lymphoma cells. Many of the substances tested caused down regulation of c-myc expression, which, however, was transient except for n butyrate. Three types of compounds proved to be particularly active: polar planar compounds (e.g., dimethylsulfoxide), heterocyclic compounds (e.g., hypoxanthine), and short-chain fatty acids (e.g., n-butyric acid). The action of n-butyrate on c myc suppression was exceptional not only in not being transient, but also in being allele-specific: it down-regulated the translocated allele without affecting the normal one. Medium transfer experiments revealed that neither degradation of the active compound nor an intracellular resistance mechanism can fully account for the reversibility of c-myc down-regulation after treatment with the transiently acting polar planar compounds. PMID- 7558419 TI - Growth control of human colon-adenocarcinoma-derived Caco-2 cells by vitamin-D compounds and extracellular calcium in vitro: relation to c-myc-oncogene and vitamin-D-receptor expression. AB - The human colon-cancer cell line Caco-2, though of malignant origin, is still able to express the c-myc proto-oncogene in a regulable fashion. Transition from the logarithmic growth phase into the quiescent, i.e., confluent state, is accompanied by a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a concomitant reduction of c-myc mRNA and of nuclear association of c-myc protein. Conversely, growth stimulation by lowering extracellular [Ca++]0 to 0.25 mM results in up-regulation of c-myc expression levels and consequently inhibition of re-entry of Caco-2 cells into the G0/G1 phase. In contrast, regulation of c-myc in Caco-2 cells is completely resistant to vitamin-D sterols, since the anti-mitogenic action of I alpha, 25 dihdroxyvitamin D3 (I alpha, 25(OH)2D3) and of 2 synthetic analogs, I alpha, 25(OH)2-16-ene-23-yne-D3 and I alpha, 25(OH)2-26, 27-F6-16-ene-23-yne-D3, occurred independently of any change in c-myc mRNA and nuclear protein levels. Although the antiproliferative effect of the vitamin-D sterols requires high affinity binding to the cytoplasmic vitamin-D receptor (VDR), vitamin-D sterols have no effect on VDR mRNA levels in Caco-2 cells. However, VDR mRNA expression changed in an antiparallel fashion to c-myc regulation upon transition between different growth states. This suggests that VDR mRNA abundance could nevertheless be important for vitamin-D-related c-myc-independent growth control in Caco-2 cells. PMID- 7558418 TI - Characterization of a prostate carcinoma mucin-like antigen (PMA). AB - A recently identified, high m.w. human tumor antigen, reactive with monoclonal antibody (MAb) PD41 and designated prostate mucin antigen (PMA), was found to have expression highly restricted to prostate carcinomas. Both biochemical and immunological characteristics of this antigen and its relationship to other tumor associated mucins and various species of submaxillary mucins were evaluated. Immunohistochemical examination of submaxillary tissues revealed differential expression of this antigen and other human tumor-associated mucins, with MAb PD41 expression found only in bovine submaxillary gland serous cells. Neuraminidase treatment enhanced antibody binding by 50%, suggesting partial masking of the PD41 epitope by sialic acid. Antigenicity was reduced by treatment with alkaline borohydride, sodium metaperiodate, beta-galactosidase, O-glycanase, and various proteolytic enzymes. MAb PD41 antibody binding also could be significantly reduced by selected lectins and sugars suggesting that the immunodominant carbohydrate involved in the epitope was an O-linked oligosaccharide containing N acetyl galactosamine as a major component. This antigen was further distinguished from T, Tn, or Sialyl-Tn antigens and blood group carbohydrate antigens by radioimmunometric analyses. Cross-blocking and double determinant RIA experiments also showed a distinction between the PD41 epitope and several well-characterized tumor-associated mucin antigens such as MUCI, CEA, M344, OCI25, and AR3 as well as bovine submaxillary core protein. Our results indicate that the PD41-reactive epitope is a non-acidic O-linked carbohydrate or glycopeptide epitope with restricted expression in prostate carcinomas and bovine submaxillary glands. This expression is distinct from other mucin-like tumor-associated antigens identified in human prostate carcinomas. PMID- 7558423 TI - In vivo effects of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) and alpha-difluoromethylornithine (DFMO) on L1210 growth, cell-cycle phase distribution and polyamine contents. AB - We studied the in vivo effects of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A), alpha-difluoromethylornithine (DFMO) and a combination of CGP 48664A DFMO on tumor growth, cell-cycle phase distribution and polyamine contents. DBA-2 mice were inoculated i.p. with 10(5) L1210 cells on day 0, treated i.p. on days 1 4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect cell-cycle phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G0/G1- and G2/M-phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G0/G1- and G2/M-phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition. PMID- 7558420 TI - DNA-binding properties of novel cis- and trans platinum-based anticancer agents in 2 human ovarian carcinoma cell lines. AB - We have investigated the comparative initial DNA binding properties of 7 platinum based anticancer drugs: 5 cis-oriented compounds, cisplatin, tetraplatin (Ormaplatin), JM118 [cis ammine dichloro (cyclohexylamine) platinum (II)], JM216 [bisacetato cis ammine dichloro (cyclohexylamine) platinum (IV)] and JM149 [cis ammine dichloro (cyclohexylamine) trans dihydroxo platinum (IV)], and 2 trans oriented compounds, transplatin and JM335 [trans ammine dichloro (cyclohexylamine) dihydroxo platinum (IV)] in SKOV-3 and CHI human ovarian carcinoma cells. Unlike transplatin, the trans complex JM335 was comparably cytotoxic to its cis isomer JM149 and cisplatin. No significant correlation was observed between levels of total platinum bound to DNA after exposure to the 7 drugs and cytotoxicity in either cell line. Using a competitive enzyme-linked immunoabsorbent assay, DNA extracted from CH1 cells exposed to the 5 cis platinum drugs was recognized by the monoclonal antibody ICR4 (raised against DNA platinated by cisplatin) in the order JM118 > cisplatin > JM216 > tetraplatin > JM149; a strong positive correlation which just attained statistical significance was observed between recognition by ICR4 and cytotoxicity. In contrast, DNA extracted from CH1 cells exposed to the trans platinum drugs transplatin and JM335 was no more immunoreactive than control DNA. Using alkaline elution, interstrand cross-link levels after exposure to drug did not correlate with cytotoxicity in either cell line. The 5 cis drugs formed interstrand cross-links in both cell lines, whereas transplatin formed very low levels in SKOV-3 and undetectable levels in CH1. JM335 was efficient at forming interstrand cross links in SKOV-3 but, notably, none were observed in CH1. In contrast, in the CH1 cells, single-strand breaks were observed with JM335 (but not with any other drug). The novel trans complex JM335 was unique, among the platinum drugs studied, in its ability to form both DNA interstrand cross-links and single strand breaks (DNA lesion formation being cell line dependent), a property which may account for its cytotoxicity. PMID- 7558422 TI - Interleukin-6 inhibits the potent stimulatory action of androgens, glucocorticoids and interleukin-1 alpha on apolipoprotein D and GCDFP-15 expression in human breast cancer cells. AB - Our study was designed to investigate the potential interaction between steroid hormones and interleukin-6 (IL-6) in the regulation of apolipoprotein D (apo-D) and gross cystic disease fluid protein 15 (GCDFP-15) expression in ZR-75-1 human breast cancer cells. We first observed that exposure to IL-6 for 6-14 days decreased basal apo-D and GCDFP-15 secretion by 50% and 23%, respectively. In the same experiment, such treatment with IL-6 decreased cell proliferation by approximately 40% after 6 and 14 days of incubation. Exposure to IL-6 markedly decreased dihydrotestosterone (DHT)-induced apo-D and GCDFP-15 release, with a half-maximal effect measured at 13 U/ml. A similar inhibitory action of IL-6 was observed on the glucocorticoid dexamethasone (DEX)-induced apo-D and GC-DFP-15 secretion. The sensitivity of the apo-D and GCDFP-15 response to the stimulatory action of DHT or DEX was, however, not changed by concomitant exposure to IL-6. The inhibitory effect of IL-6 on the secretion of these two biochemical markers was additive to that of 17 beta-estradiol. In addition, IL-6 blocked the stimulatory effect of interleukin-1 alpha (IL-1 alpha) on apo-D and GCDFP-15 secretion. Our results show that IL-6 is a potent inhibitory of basal as well as androgen-, glucocorticoid- and IL-1 alpha-induced apo-D and GCDFP-15 secretion in ZR-75-1 human breast cancer cells, while cell proliferation is inhibited by this cytokine. PMID- 7558421 TI - Recurrent cytogenetic alterations of prostate carcinoma and amplification of c myc or epidermal growth factor receptor in subclones of immortalized PNT1 human prostate epithelial cell line. AB - To develop an experimental prostate cancer model, we immortalized normal human prostate adult epithelial cells with SV40 large-T antigen. Two sublines were derived in culture, namely PNT1A and PNT1B. They retained the characteristics of prostate epithelial cells, but did not clone in soft agarose. PNT1A occasionally formed undifferentiated adenocarcinoma tumors in nude mice, but only in the presence of matrigel. PNT1A and PNT1B displayed common cytogenetic alterations: a 10q arm deletion, which is a recurrent alteration in prostate carcinoma, chromosome losses and a translocation involving chromosome 5. An extensive study of oncogenic alterations occurring in these cells showed that PNT1A displayed c myc gene amplification, forming an hsr on chromosome 4, as well as gene amplification, forming an hsr on chromosome 4, as well as c-myc mRNA overexpression, with a faster doubling time (25 hr); moreover, it seemed less sensitive to EGF than PNT1B. PNT1B had a doubling time identical to that of normal cells (48 hr) but displayed EGF receptor gene amplification accompanied by an increased number of EGF binding sites and sensitivity to EGF. Because both cell lines displayed cytogenetic and oncogenic alterations found in prostate cancer, as well as differing malignant potentials, they represent an interesting model for studying the progression of prostate tumors. PMID- 7558424 TI - Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell derived inhibition of nitric-oxide synthesis in cerebral endothelial cells. AB - Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta. PMID- 7558426 TI - Modulation of heparan-sulphate/chondroitin-sulphate ratio by glycosaminoglycan biosynthesis inhibitors affects liver metastatic potential of tumor cells. AB - Previous data have indicated that the proteoglycan (PG) pattern is different on tumor cells with different liver metastatic potential. We selected "conventional" glycosaminoglycan (GAG) biosynthesis inhibitors, beta-D-xyloside (BX), 2-deoxy-D glucose (2-DG), ethane-l-hydroxy-l,l-diphosphonate (ETDP) and the newly discovered 5-hexyl-2-deoxyuridine (HUdR), to modulate PGs on highly metastatic/liver-specific 3LL-HH murine carcinoma and HT168 human melanoma cells and to influence their liver colonization potential. These compounds all induced remarkable changes in GAG biosynthesis, but to varying degrees: glucosamine labelling was affected mainly by 2-DG, and HUdR and sulphation by BX and HUdR. Furthermore, the ratio of heparan sulphate/chondroitin sulphate (HS/CS) of PGs was increased by ETDP and decreased after treatment by HUdR. In addition to changes in PG metabolism, tumor-cell proliferation and adhesion to fibronectin were affected; BX and 2-DG stimulated cell proliferation and adhesion, while HUdR inhibited both proliferation and adhesion. Most interestingly, HUdR, the most effective inhibitor of HS/HSPG, depressed the formation of liver colonies, while ETDP, the most effective inhibitor of CS/CSPG, stimulated the appearance of liver colonies. These observations indicated that, at least in these experimental systems, tumor cells with a high HS/CS ratio are more likely to form liver metastases; consequently, anti-HS agents could also be anti-metastatic. PMID- 7558425 TI - Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes. AB - Aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily, and participate in the reduction of a wide range of carbonyl compounds. We have purified aldose reductase from rat lens and raised antiserum against it in rabbits. Immunoblot analyses using this antibody showed that a significant amount of aldose reductase was expressed in cell lines derived from hepatomas while it was negligible in normal hepatocytes. Elevated expression of aldose reductase was also observed in cancerous lesions of 3'-methyl-4-dimethyl aminoazobenzene (3'-Me-DAB)-induced hepatocarcinomas. Expression of aldose reductase mRNA was confirmed in these cells by Northern-blot analysis, suggesting that the induction occurred at the stage of gene transcription. The level of aldehyde reductase, however, did not change in cancerous tissue or in the cell lines. The viability of hepatoma cells in the presence of 3-deoxyglucosone and glyceraldehyde was decreased by an aldose reductase inhibitor, ONO-2235 (5 [1Z,2E)-2-methyl-3-phenylpropenylidene]-4-oxo-2-thioxo -3- thiazolidineacetic acid). Taken together, induction of aldose reductase gene expression during hepatocarcinogenesis may render cancer cells resistant to various toxic carbonyl compounds produced during metabolism or administered as anti-cancer drugs. PMID- 7558428 TI - Migratory patterns of lac-z transfected human glioma cells in the rat brain. AB - Malignant brain tumors are characterized by extensive tumor-cell infiltration into the normal brain tissue. The present work describes the migratory behavior of human glioma cells transplanted into the adult rat brain with the aim of exploiting the extent of active cell migration and passive cell displacement within the central nervous system. To detect every transplanted tumor cell, a stably bacterial beta-galactosidase (lac-z) transfected human glioma cell line was used. To distinguish between an active cell migration process and passive cell displacement, rat brains were also implanted with inert fluorescent polystyrene microspheres and the distribution of tumor cells and microspheres was studied 1 hr and 3 days after implantation. One hour after implantation the tumor cells were strictly localized at the implantation site. However, 3 days after implantation, both tumor cells and microspheres showed an extensive distribution within the brain. Confirming earlier neuropathological and experimental studies, it is shown that the lac-z-transfected glioma cells had the capacity to move within the Virchow-Robin and subarachnoid spaces. However, since fluorescent microspheres were also found in these areas, this spread of tumor cells may be primarily mediated by the extensive cerebrospinal fluid flow that exists within the brain. Three days after implantation, the glioma cells also showed an active migration over the corpus callosum. In comparison, the fluorescent microspheres showed only limited spread along the callosal body. It is concluded that the bacterial lac-z gene can be stably transfected into human glioma cells and, since every tumor cell can be visualized within the brain, this model provides a tool for studying the mechanisms behind tumor-cell invasion of the brain. PMID- 7558427 TI - Amphiregulin anti-sense oligodeoxynucleotides inhibit growth and transformation of a human colon carcinoma cell line. AB - Amphiregulin (AR) is a secreted heparin-binding growth factor that is structurally and functionally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha). GEO cells are from a human colon cancer cell line that expresses high levels of AR protein and mRNA. To assess the role of AR in colon-cancer cell proliferation and transformation, 2 different anti-sense 20-mer phosphorothioate oligodeoxynucleotides (AR AS-1 and AR AS-2 S oligos) complementary to the 5' sequence of AR mRNA were synthesized. Both AR AS S-oligos were able to inhibit the anchorage-dependent growth (ADG) of GEO cells. The 2 AR AS S-oligos were equipotent when used in equimolar concentrations. In particular, a 40% growth inhibition was observed at a concentration of 10 microM, while a mis-sense S-oligo used as control had no effect on GEO cell growth. The AR AS-1 S-oligo used at the same concentration also inhibited by 40% the 3H thymidine incorporation by DNA of GEO cells. The anchorage-independent growth (AIG) of GEO cells was even more significantly affected by AR AS S-oligo treatment. In fact, up to 80% inhibition of the AIG of GEO cells was observed when cells were treated with 10 microM of both AR AS S-oligos. Finally, the AR AS S-oligos were able to specifically inhibit AR protein expression in GEO cells, as assessed by immunocytochemistry. These data suggest that AR is involved in colon cancer cell transformation, and that AR may represent a suitable target for gene therapy in human colon carcinomas. PMID- 7558430 TI - Cell-cell and cell-collagen interactions influence gelatinase production by human breast-carcinoma cell line 8701-BC. AB - We previously produced evidence that the human mammary-carcinoma cell line 8701 BC expresses several metalloproteinases (MMP-1, -2, -9, and -10) and their tissue inhibitors). In order to obtain a better understanding of the environmental control over gelatinolytic activities, we have tested the enzyme production of 8701-BC cells, at time intervals after plating on different collagen substrates, i.e., types I, III, IV, V and OF/LB, used as films in culture dishes. Proteinase activities, released in the conditioned culture media, were tested by zymography on SDS-PAGE, and by quantificative analyses, using 14C carboxymethylated transferrin as substrate in a liquid incubation medium. Enzymatic activities varied with time and were inversely related to cell densities, with minimum values at cell confluence. The enzymatic activity was positively supported by collagen substrates, with a maximal increase in activity when OF/LB collagen was used. In addition to the known MMPs, we found a proteinase with an M(r) of about 20 kDa, which displayed higher activity at 48 hr after cell plating and gradually decreased with cell increment. In contrast to the other MMPs, this proteinase is inhibited by soybean trypsin inhibitor, but it does not display a complete identity with trypsin, since it does not digest casein and is not inhibited by other serine proteinase inhibitors. PMID- 7558429 TI - Estrogen-induced anchorage-independence in human endometrial stromal cells. AB - Estrogens are important etiologic agents for most gynecologic malignancies, and chronic exposure to estrogen that is unopposed by progestins conveys the greatest risk. Treatments with estrogen facilitate the process of malignant transformation in rodents, but relatively few studies of estrogen-induced carcinogenesis have been performed using human cells. Most malignancies in estrogen-responsive tissues arise from epithelial cells, but an increasing body of evidence emphasizes the role of stromal cells as mediators of the effects of estrogens on epithelial cells. Our studies were designed to assess estrogens as carcinogens for human endometrial stromal cells and to provide a basis for studies of the role of stroma in estrogen-induced carcinogenesis in humans. Acute treatments with the estrogens diethylstilbestrol (DES), 17 beta-estradiol (E2) and beta dienestrol enhance anchorage-independent proliferation (AIP) of SV40-immortalized human endometrial stromal cells in the rank order of DES > E2 > beta-dienestrol. The anti-estrogenic compound tamoxifen inhibits DES-induced AIP. The magnitude of DES-induced AIP increases with prolonged duration of treatment. After 11 months of chronic treatment with 0.1 nM DES, AIP was 20-fold higher than in vehicle treated control cultures. Expression of the estrogen receptor was altered by treatments with DES in parallel with increased capacity for AIP. These conditionally immortal human endometrial stromal cells appear to be a good model for estrogen-induced transformation of human cells. PMID- 7558431 TI - Paradoxical effects of bleomycin and heavy water (D2O) in mice. AB - Bleomycin (BLM) lacks many side effects of other cytostatic drugs. Pulmonary toxicity is the major dose-limiting effect of BLM. This is based in part on generation of free radicals. It is conceivable that deuterium in body fluids lessens the production of free radicals, thus preventing or diminishing the morphologic expression of pulmonary BLM toxicity. We therefore studied the effect of moderate deuteration of body fluids on BLM-induced lung damage in BALB/c-mice. In addition to conventional histopathological methods, we used a vertical sectioning design for stereological estimation of pulmonary volumes and surface areas. BLM (low/medium/high dose: 25/50/75 IU/kg body weight) was injected i.p. once a week for 6 weeks. Half the mice drank deuterated water before, during and after BLM treatment. Three weeks after the last injection, the lungs were fixed by airway instillation. Deuterated animals treated with BLM lacked signs of irreversible BLM-induced pulmonary damage. Conversely, focal sub-pleural fibrosis and fibrosing alveolitis were present in BLM-treated mice drinking tap water. Deuterated mice had stereological values for almost all lung parameters that were lower than in non-deuterated mice. The organ-specific advantage of deuteration was offset by marked enhancement of systemic toxicity of BLM. We conclude that (1) moderate concentrations of deuterium may prevent the development of fibrosing alveolitis in BLM-treated mice, possibly by reducing proliferation of alveolar fibroblasts, and, less probably, by impairing generation or enhancing capture of free radicals; (2) the toxicity of BLM was enhanced by ingestion of deuterium, resulting in morphological liver alterations and increased mortality. PMID- 7558433 TI - Myf-3 hypermethylation in malignant lymphoproliferative disorders. PMID- 7558432 TI - Irradiation induces up-regulation of E9 protein (CD105) in human vascular endothelial cells. AB - The use of MAb E-9 raised against tissue-cultured endothelial cells (EC) has shown marked heterogeneity in vascular EC lining the blood vessels of normal and tumour tissues. MAb E-9 is human EC-specific and the protein recognized by it is a homodimer with a molecular mass of 97 kDa. The E-9 protein is resistant to treatment by 3 mM sodium periodate, but is sensitive to 10% trichloroacetic acid and 70% ethanol. E-9 protein has been assigned to a new cluster, CD105, and mapped to human chromosome 9q3. It has approximately 70% homology with type-III cell-surface receptor for transforming growth factor beta (TGF-beta). Recently CD105 has been reported to be the gene in patients with hereditary haemorrhagic telangiectasia. We have examined the effects of radiation on its expression in normal human umbilical-vein endothelial cells (HUVEC) and brain-tumour-derived endothelial cells (BTEC). Irradiation induced dose- and time-dependent up regulation in the expression of the E-9 protein on the plasma membranes of EC, and also resulted in greater increase in the expression of the E-9 protein in semi-confluent (proliferating) as compared with confluent (non-proliferating) EC. It may well be that, following radiotherapy in cancer patients, E-9 protein is also up-regulated. The presence of increased amounts of E-9 protein in EC makes it an attractive target in the control of angiogenesis, especially after radiotherapy in cancer patients. The time scale involved in the up-regulation of E-9 protein following irradiation has led us to suggest that it may be a secondary event, the primary being the production and release of mitogenic factors (such as basic fibroblast growth factor) from irradiated EC. PMID- 7558434 TI - Bladder tumours following chemotherapy and radiotherapy for ovarian cancer: a case-control study. AB - A collaborative group of cancer registries and hospitals carried out a case control study of tumours of the bladder in women who had previously been treated for ovarian cancer. A total of 63 cases of bladder tumours were identified, and 188 controls were selected matching for age, year of ovarian cancer diagnosis and survival time. Full details of the treatment for ovarian cancer were sought for both cases and for controls. The risk of bladder tumours was increased for patients who had been treated by radiotherapy alone (1.9; 95% confidence interval, 0.77-4.9), by chemotherapy alone (3.2; 0.97-10), and by chemotherapy and radiotherapy (5.2; 1.6-16), when comparison was made with patients treated only by surgery. Patients treated by chemotherapy were separated into 2 groups according to whether they had received cyclophosphamide. Among those who had, there was a clear increase in risk (approximately 4-fold) regardless of whether or not they had also received radiotherapy. For those who received only other drugs, risk was increased substantially among patients who had also been treated by radiation, as compared with patients treated by surgery alone, and those who had received radiotherapy only. Both melphalan and thiotepa were implicated as potential bladder carcinogens on the basis of these results. The estimated risk of bladder tumours due to cyclophosphamide was more than twice the risk following radiation to the bladder, and it appeared substantially earlier. For both agents, the risk continued to increase more than 10 years after treatment began. PMID- 7558435 TI - Loss of cell-contact regulation and altered responses to autocrine motility factor correlate with increased malignancy in prostate cancer cells. AB - Tumor cell migration and proliferation in new organ environments are critical steps in cancer progression and can be modulated by tumor- and host-secreted molecules. Autocrine motility factor (AMF) is a tumor-secreted cytokine which regulates growth and motility by a receptor-mediated pathway. The AMF receptor, a 78-kDa cell surface glycoprotein (gp78), is regulated by cell contact in normal fibroblastic and bladder cells; however, this mechanism is disrupted during tumor progression. A prostatic carcinoma cell line which is low- to non-metastatic in nude mice (PC-3) and a derived metastatic variant (PC-3M) were examined to determine if gp78 cell density regulation is involved in prostate cancer progression. Both cell lines expressed gp78 and, although the basal migration of the parental PC-3 cells was higher than that of the metastatic variant, only the PC-3M cells were capable of responding to tumor-derived AMF with increased motility. Furthermore, these cells exhibited differential patterns of wound closure in an experimental system whereby the low-metastatic PC-3 cells migrated primarily along the wound edge while individual high-metastatic PC-3M cells entered the cell-free wound area directly. Cell surface gp78 distribution distinguished the cell populations with a markedly concentrated display of gp78 in polarized capped regions on the surface of the metastatic cells. Cell-cell contact down-regulated gp78 expression in the parental, but not the metastatic, cells, and mitogenic responses to exogenous AMF differed between these cell lines as well. In this model, metastasis appears to be associated with aberrant regulation of gp78 expression and distribution, coupled with enhanced exploitation of AMF's locomotory and proliferative effects. PMID- 7558436 TI - In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by oestrone-3-O-sulphamate. AB - Many tumours in endocrine-sensitive tissues, such as the breast and endometrium, are hormone-dependent and the hydrolysis of oestrone sulphate (EIS) to oestrone by oestrone sulphatase (EI-STS) is a major source of oestrogen in such tumours. Oestrone-3-O-sulphamate (EMATE) has been shown to be a potent EI-STS inhibitor in vitro, and in this study its ability to inhibit enzyme activity in vivo was examined. EMATE was initially administered to female rats for 7 days, after which liver EI-STS activity was measured. As EMATE also inhibits a related sulphatase in vitro, dehydroepiandrosterone sulphatase (DHA-STS), its effect on the activity of this enzyme in vivo was also investigated. DHA-STS has a pivotal role in regulating the synthesis of another steroid with potent oestrogenic properties, androstenediol. Administration of EMATE almost completely inhibited liver EI-STS (99%) and DHA-STS (99%) activities and was active when given by the oral or subcutaneous routes. After a single dose of EMATE or following the cessation of multiple doses for 10 days, liver EI-STS activity remained inhibited ( > 95%) for up to 7 and 10 days, respectively. Other compounds, such as 4-hydroxytamoxifen and the "pure" antioestrogen ICI 182,780, which are reported to inhibit EI-STS activity in vitro, did not inhibit activity in vivo. In a preliminary study, EMATE, when injected over a 12-day period, effectively reduced the growth of EIS stimulated nitrosomethyl-urea-induced mammary tumours in ovariectomised rats and inhibited tumour sulphatase activity in treated animals. PMID- 7558438 TI - Anti-vascular approaches to solid tumour therapy: evaluation of vinblastine and flavone acetic acid. AB - Several agents have now been identified which exert their anti-tumour effects in large part via the tumour vasculature; these include TNF alpha and flavone acetic acid (FAA). More recently, Vincristine and Vinblastine have also been shown to cause a prolonged and selective decrease in tumour perfusion. Vinblastine, unlike, FAA, causes no increase in plasma TNF alpha levels in mice bearing the CaNT tumour, suggesting 2 distinct mechanisms of anti-vascular activity for these structurally diverse agents. Since FAA and Vinblastine also show quite different normal tissue toxicities, which are separately dose-limiting, we have examined the strategy of combining these 2 agents. When Vinblastine preceded FAA by 24 hr or less, tumour growth delay was significantly enhanced without a concomitant increase in toxicity. The level of enhancement was not significantly reduced by a 5-fold decrease in Vinblastine dose, though any reduction in the dose of FAA caused a rapid reduction in treatment effectiveness. Investigation of the functional vasculature of treated tumours suggested that increased anti-vascular effects may contribute to the enhanced growth inhibition of the combined treatment. Our results demonstrate the potential benefit of combining 2 different classes of anti-vascular agent, using Vinblastine and FAA (or 5,6-MeXAA) as prototype drugs. PMID- 7558437 TI - Enhancement of in vitro tumor-cell transcellular migration by tumor-cell-secreted endothelial-cell-retraction factor. AB - To investigate the factors affecting endothelial-cell retraction, we have studied the interaction of tumor cells with endothelial cells in 2 human pancreatic cancer cell lines, PSN-1 and MiaPaca-2. The extent of endothelial-cell retraction measured by the amount of intercellular junctional transport of FITC-dextran through an endothelial monolayer was increased by the addition of a conditioned medium (CM) from both cell lines, while CM from PSN-1 cells was 2 to 3 times more potent than that from MiaPaca-2 cells. After the treatment of endothelial monolayer with CM of PSN-1 cells, the ability of both PSN-1 cells and MiaPaca cells to adhere to or invade the monolayer increased. The addition of CM from PSN 1 cells did not affect the growth rate of either the endothelial or the tumor cells. The activity in the CM was heat-stable and bound to heparin-Sepharose, but was inactivated when treated by 0.5% trypsin. Protease inhibitors did not influence the activity. Pre-treatment of PSN-1 cells by an inhibitor of protein synthesis, cycloheximide, or of protein processing, benzyl-N-acetyl-alpha-D galactosaminide, reduced endothelial-cell-retraction activity in the CM. The active substance in the CM fractionated in the molecular-weight range of 10,000 to 50,000. These results suggest that PSN-1 cells produce and secrete (a) soluble factor(s) that can induce endothelial-cell retraction, thus facilitating tumor cell invasion. PMID- 7558439 TI - Increased susceptibility of ras-transformed cells to phenylacetate is associated with inhibition of p21ras isoprenylation and phenotypic reversion. AB - Alterations in the expression of ras oncogenes are characteristic of a wide variety of human neoplasms. Accumulating evidence has linked elevated ras expression with disease progression and with failure of tumors to respond to conventional therapies, including radiotherapy and certain chemotherapies. These observations led us to investigate the response of ras-transformed cells to the differentiation-inducer phenylacetate (PA). Using gene transfer models, we show that PA caused cytostasis in ras-transformed mesenchymal cells, associated with increased expression of 2',5'-oligoadenylate synthetase, an enzyme implicated in negative growth control. PA also induced phenotypic reversion characterized by loss of anchorage-independent growth, reduced invasiveness and increased expression of collagen alpha type I, a marker of cell differentiation. The anti tumor activity of PA was observed in cases involving either Ha- or Ki-ras and was independent of the mode of oncogene activation. Interestingly, in contrast to their relative resistance to radiation and doxorubicin, ras-transformed cells were significantly more sensitive to PA than their parental cells. The profound changes in tumor cell and molecular biology were associated with reduced isoprenylation of the ras-encoded p21. Our results indicate that PA can suppress the growth of ras-transformed cells, resistant otherwise to free-radical based therapies, through interference with p21ras isoprenylation, critical to signal transduction and maintenance of the malignant phenotype. PMID- 7558440 TI - Dietary fat and the risk of breast cancer: a prospective study of 25,892 Norwegian women. AB - In this prospective study, the relationship between energy and fat consumption and the risk of breast cancer was examined. Between 1977 and 1983, 31,209 Norwegian women, 20 to 54 years of age attended a health screening. The attendees were given a food-frequency questionnaire to be completed at home, and this was returned by 25,892 (83%). During the 7 to 13 years of follow-up, 248 cases of breast cancer were identified for analysis by linkage to the Norwegian Cancer Registry. The relative risk of women who ate meat more than 5 times a week, as compared with those who consumed meat twice or less than twice a week, was 2.44. Consumers of 0.75 litres or more of full-fat milk daily had a relative risk of 2.91 compared with those who consumed 0.15 litres or less. A positive relationship was found between those reporting the highest quartile of mono unsaturated fat consumption and the risk of breast cancer. The main foods contributing to the mono-unsaturated fat index were edible fats, meat and milk. PMID- 7558441 TI - Efficacy of TNF-alpha gene-transduced tumor cells in treatment of established in vivo tumor. AB - The therapeutic effect of TNF gene-transduced mouse fibrosarcoma cells (Meth-A: C5) on pre-inoculated parental cells (Meth-A: M0) was studied. Subcutaneous (s.c.) transplantation of M0 cells into one flank of syngeneic BALB/c mice was followed by s.c. injection of irradiated MO or C5 into the opposite flank 1 week later. The initial M0 tumor (T-MO) completely regressed in C5-vaccinated mice, whereas in M0-vaccinated mice continuous growth of T-M0 was observed. When a similar experiment was carried out in SCID mice, no regression of T-MO was observed, suggesting that the tumor regression in BALB/c mice was not due to direct anti-tumor activity of TNF secreted from C5, but to systemic immunity. Regression of the rechallenged M0 tumor was observed in mice which had shown T-MO regression by C5 vaccination, but rechallenged Colon 26 cells (syngeneic to BALB/c mice) continued to grow, indicating a specific immunity to Meth-A cells). The systemic immunity evoked in C5-vaccinated mice was directly demonstrated by enhanced killer activities of LAK and CTL with a proliferation of T-cell population in their splenocytes. Abrogation of the therapeutic effect of C5 vaccination with anti-Thy 1 and anti-Lyt 2 also demonstrates the involvement of cellular immunity in tumor regression. PMID- 7558442 TI - Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts. AB - Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8 hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected. PMID- 7558443 TI - Malignant transformation of human fibroblast strain MSU-1.1 by v-fes requires an additional genetic change. AB - To determine whether the v-fes oncogene can malignantly transform human fibroblasts that have acquired an infinite life span and are partially growth factor-independent, we transfected cell strain MSU-1.1 with a plasmid containing the v-fes oncogene and a bacterial histidinol dehydrogenase gene. Of the 60 independent histidinol-resistant clones isolated and assayed for v-fes expression using a fes-specific monoclonal antibody, 6 were found to express the v-fes protein at a detectable level. When progeny cells from these 6 clones were further characterized, 3 of the 6 clonal populations exhibited a significant increase in the ability to form medium-sized colonies in agarose, but none were tumorigenic in athymic mice. However, when the 6 populations were propagated for many generations, the same 3 populations acquired the ability to form very large colonies in agarose ( > or = 100 microM in diameter) at a frequency of 2% to 17%, and formed malignant tumors in athymic mice. This suggests that an additional genetic change required for malignant transformation had been spontaneously acquired in 3 of the v-fes -transformed cell strains. To determine whether the change or changes were the equivalent of an activated sis or ras proto-oncogene, we transfected the v-fes oncogene into derivative strains of MSU-1.1 that express a transfected v-sis, c-H-ras or c-N-ras oncogene, but that do not form tumors, and assayed the v-fes-expressing transfectants for tumorigenicity. The results showed that when complemented either by a ras oncogene expressed at a somewhat enhanced level or by the v-sis oncogene, v-fes can supply the additional change required for malignant transformation. PMID- 7558444 TI - Differential inactivation of O6-methylguanine-DNA methyltransferase activity by O6-arylmethylguanines. AB - Activity of O6-methylguanine-DNA methyltransferase (MGMT) is well related with drug resistance of tumor cells to chloroethylnitrosoureas (CENUs), because MGMT removes CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. Inactivation of MGMT is a feasible way to overcome MGMT-related resistance of tumor cells to CENUs. O6-Benzylguanine is known to be a strong depleter of MGMT. We previously reported the potentiation effect of O6 arylmethylguanine derivatives on cytotoxicity of 1-(4-amino-2-methyl-5 pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), a CENU, for HeLa S3 cells. In this study, we tested the activity of these O6 arylmethylguanine derivatives as depleters of MGMT using HeLa S3 cell-free extract. The O6-arylmethylguanine derivatives tested were O6-benzylguanine (1), O6-(4-, 3-, and 2-fluorobenzyl)guanines (2-4), O6-(4, 3-, and 2 trifluoromethylbenzyl)guanines (5-7), O6-(4-, 3-, and 2-pyridylmethyl)guanines (8 10), and O6-(2- and 1-naphtylmethyl)guanines (11,12). Among these, compounds 1-3, 5, 8, 9 and 11 showed a strong MGMT depletion activity, whereas compounds 4, 6, 7, 10 and 12 were inactive. These inactive compounds, except for 6, have a substituent at the alpha position of the benzyl group (4, 7, 12) or are an alpha nitrogen analogue of 1 (10). There was a good relation (r = -0.856, p < 0.001) between the MGMT depletion activity of O6-arylmethylguanine derivatives and their potentiation activity of ACNU cytotoxicity. These results suggest that the alpha position of the benzyl group plays an important role in the interaction of O6 arylmethylguanine derivatives with MGMT to result in the inactivation of MGMT. Potent MGMT inactivators (1-3, 5, 8, 9, 11) sensitize tumor cells to CENU chemotherapy. PMID- 7558447 TI - Cancer in the European population of Harare, Zimbabwe, 1990-1992. AB - The data presented from the population-based cancer registry in Harare, Zimbabwe, represent the first information on the incidence of cancer in a population of European origin living in Southern Africa for over 30 years. Their cancer pattern is more or less typical of white populations of high socio-economic status living in Europe or North America, with elevated incidence rates of breast cancer, large bowel cancer and, in women, lung cancer. However, there are also several unusual features, with extremely high incidence rates of skin cancers, including melanomas, and higher rates of liver and bladder cancer than normally seen in white populations. PMID- 7558446 TI - An ecological study of diet and lung cancer in the South Pacific. AB - Incidence rates of lung cancer have been markedly lower for Fiji than for other South Pacific countries, despite similar rates of smoking. We conducted population-based surveys in several island nations of the South Pacific (Cook Islands, Fiji, Tahiti and New Caledonia) and used data from Caucasian, Japanese, Hawaiian, Filipino and Chinese controls in a case-control study of lung cancer in Hawaii to investigate the role of diet in explaining differences in lung cancer incidence among 20 ethnic-sex groups. In a stepwise linear regression of lung cancer rates on smoking, diet and other variables, smoking, as expected, explained the majority (61%) of the variability in incidence. However, several dietary components also explained significant portions of the variance. Lutein intake explained 14% and vitamin E intake, cholesterol intake and height explained 5-7% each of the remaining variance in incidence. Associations with lutein and vitamin E were inverse, whereas those with cholesterol and height were direct. Dietary beta-carotene intake was not associated with lung cancer incidence. These ecological data provide evidence for a protective effect of lutein against lung cancer. A protective effect of dietary vitamin E and a risk enhancing effect of dietary cholesterol are also suggested. PMID- 7558448 TI - Cancer in the African population of Harare, Zimbabwe, 1990-1992. AB - The data presented from the population-based cancer registry in Harare, Zimbabwe, represent the first information on the incidence of cancer in Southern Africa for almost 20 years. In the African population in Zimbabwe there are several features in common with other countries in sub-Saharan Africa: high rates of liver, prostate and cervix cancer, low rates of large-bowel cancer and breast cancer. Also, as reported from southern and south-eastern Africa, there are relatively high incidence rates of cancers of the oesophagus, bladder and (in men) lung. The AIDS epidemic has given rise to a striking increase in incidence of Kaposi's sarcoma (now the commonest cancer of African men), but there is not much evidence for an increase in incidence of non-Hodgkin lymphomas nor, although rates are very high, of cervical cancer. PMID- 7558449 TI - Descriptive epidemiology of lymphoid and haemopoietic malignancies in Bangalore, India. AB - Lymphoid and haemopoietic malignancies as a group constitute one of the important cancers in India, as elsewhere in the world. While information on incidence and mortality of these cancers, and that on survival, are available from most developed countries, there are very few reports describing this experience in developing ones. Population-based cancer registration commenced in Bangalore, India, in January 1982, under the auspices of the Indian Council of Medical Research. This source provides fairly complete and reliable incidence data, but, in order to obtain mortality and survival information, active follow-up involving visits of homes of patients was undertaken. Between 1982 and 1989, 1397 cases of lymphoid and haemopoietic malignancies were registered in the Bangalore cancer registry, giving an age-adjusted incidence rate of 7.7 and 4.8 per 100,000 in males and females respectively. Active follow-up provided mortality/survival information in 1267 or 90.7% of these cases. The overall observed 5-year survival for these cancers combined (both sexes) was 26%, and relative survival 28.4%. The 5-year survival rate was lower in all the individual lymphomas and leukaemias as compared with similar reports from the developed countries. Survival in Hodgkin's disease was influenced by clinical stage and age at presentation. PMID- 7558445 TI - Novel muteins of human tumor necrosis factor with potent antitumor activity and less lethal toxicity in mice. AB - Eight muteins of recombinant human tumor necrosis factor-alpha (rhTNF; 1SSSRTP...29RR...155L), in which 29Arg was replaced by another amino acid, were prepared and their anti-tumor effects in BALB/c mice bearing Meth A fibrosarcoma were evaluated. The therapeutic indices, which mark the extent of the therapeutically effective dose, of V29 (29Arg-->Val) and D29 (-->Asp) were 3.5 and 3.2, respectively, whereas that of rhTNF was 1.4. Clearly, the therapeutically effective range of these muteins was extended along with a decrease in lethal toxicity. V29 did not produce hypotension in the rat system, but D29 did. In addition, V29 showed potent anti-tumor activity (Tumor Volume Inhibition Rate = 81% on day 15 after implantation) in 3 consecutive injection schedules despite the decreases in toxicity compared with rhTNF. The relative receptor binding constant was determined using HEp-2 cells (expressing mainly 55 kDa-TNF receptor; p55R) and HL60 cells (expressing mainly 75-kDa-TNF receptor; p75R), and revealed that the reduced toxicity of V29 in mice was due to the reduced binding to p55R (34% of rhTNF). On the other hand, the ratio of the constants HEp-2/HL60 of V29 was 11 in comparison with the value of 1.0 for rhTNF, suggesting that this mutein binds preferentially to p55R. The biological activities in human cell lines (HEp-2 and HL60 cells) correlated well with the binding activities to each receptor in vitro. Therefore, the much lower toxicity and the potent anti-tumor activity of this mutein suggest that V29 merits further investigation in pre-clinical and clinical trials. PMID- 7558451 TI - The prognostic value of DNA ploidy and S-phase estimate in primary breast cancer: a prospective study. AB - In this prospective study, the independent prognostic value of DNA ploidy in combination with the major clinico-pathological characteristics (histological grade, nodal status, tumor size, estrogen and progesterone receptor status, number of tumors, multicentricity, lympho-vascular infiltration) was evaluated in a series of 399 breast-cancer patients. The mean follow-up time was 4.5 years. The DNA content was measured using image cytometry on fresh tumor samples. The overall survival of tetraploid and slowly proliferating diploid cases was significantly different compared with that of aneuploid and rapidly proliferating diploid cases (p = 0.0002). Thus, DNA ploidy combined with S-phase estimate (DNA histogram type) appeared to be good prognostic factors. In a multivariate survival analysis, DNA histogram type was not an independent prognostic factor unless the histological grade was excluded. This effect of DNA histogram type on survival was also observed among patients with grade-I or -II tumors and patients with small tumors. In conclusion, DNA histogram type was a valuable prognostic factor in univariate analysis, and provided independent complementary information for patients considered at low or intermediate risk by classical pathological findings. PMID- 7558450 TI - Comparisons of colon-cancer survival among European countries: The Eurocare Study. AB - Under the aegis of EUROCARE, a European Union project to assemble survival data from population-based cancer registries and analyze them according to standardized procedures, we have investigated and compared colon-cancer survival in 10 European countries. We analyzed 68,283 colon-cancer cases diagnosed between 1978 and 1985 and followed for at least 6 years. After calculating relative survival, putative factors prognostic for survival were investigated by univariate and multiple-regression analyses. Important intercountry colon-cancer survival differences exist within Europe, which are not explained by methodological differences, nor by demographic confounders. In patients aged 60 to 69, the mean European 5-year cumulative relative survival was 40%. Switzerland, Finland and The Netherlands had significantly higher 5-year relative survival, while one area in the UK and Cracow in Poland had significantly lower survival than this European estimate. Prognosis improved over time: from 1978 to 1985, the risk of death was reduced by about 4% per year in all countries studied. Age at diagnosis is inversely related to prognosis. Differences in health provision and hence in quality of care and stage at presentation seem largely responsible for the differences in colon-cancer survival found in the EUROCARE countries. PMID- 7558452 TI - Effectiveness of screening for breast cancer in women under 50 years at entry: the Kotka pilot project in Finland. AB - It is still uncertain whether mammography is beneficial to women under 50 years of age. We report a follow-up of 4 1-year birth cohorts in 13 Finnish municipalities subjected to service screening. The women in these cohorts were born in even calendar years and they were 40-47 years old at the time of first screening, which was repeated every other year. At the beginning, 4,163 women were invited and 86% attended. Women were screened with mammography, physical breast examination and information on detection and early diagnosis of breast cancer by breast self-examination (BSE) was given. Incident cases of breast cancer and breast-cancer deaths were recorded. The expected numbers of incident breast-cancer cases and deaths from breast cancer were derived from control women who were residents of the same municipalities but who were born in adjacent calendar years (odd years) to those screened. In the screened cohorts 13 cancers were screen-detected, 32 cancers were interval cases and 8 cases were diagnosed among the non-attenders, while in the controls 85 cancers were detected during a 9-year follow-up from 1982 to 1990. One death from breast cancer occurred in the screened cohorts among cases diagnosed between 1982 and 1990, against 10 expected breast-cancer deaths during a 10-year follow-up from 1982 to 1991 of women initially free of breast cancer. Breast-cancer mortality in the screened cohorts was significantly lower than in the controls (Rate Ratio 0.11), but it is not clear how much of the reduction in mortality could be accounted for by the mammography itself and how much to other activities included in the screening programme (especially training in BSE) or to chance variation. It is unlikely that a programme sensitivity of 25% (13/53) would have resulted in 89% (100-11) effectiveness or reduction in the risk of death from breast cancer. PMID- 7558453 TI - Association of herpesvirus infection with the development of genital cancer. AB - Clinical observations and epidemiological studies on genital cancer have revealed an association with sexual behavior, thus motivating research into sexually transmitted agents which may be responsible for the neoplasia. In this study, we used the PCR technique to examine the presence of CMV, HSV and EBV viruses in 187 cases of human genital lesions and found that infection with CMV or HSV was associated with cervical cancer. When we stratified according to HPV status this association was found only for HPV-DNA-negative cases. These findings indicate that past infection with CMV or HSV could be interpreted as a surrogate marker of HPV infection. However, these viruses may play an important role themselves in cervical cancer. PMID- 7558455 TI - Effect of gastrointestinal hormones and synthetic analogues on the growth of pancreatic cancer. AB - The effects of hormones and synthetic analogues have been examined on the growth of 2 human pancreatic cancer cell lines, MiaPaCa2 a well-established cell line and PANI which was derived in our own laboratories from a tumour specimen. The hormones/growth factors included gastrin (G-17), epidermal growth factor (EGF) and bombesin, while the synthetic analogues used were a gastrin receptor antagonist (CR 1718), a somatostatin analogue (RC-160) and a bombesin receptor antagonist (ICI 216,140). Cell proliferation was assessed by the [75Se]selenomethionine uptake method which has been shown to correlate with cell counts. The effect of each hormone or growth factor on growth was expressed as a percentage of the untreated control. There were 5 replicates in each experiment, and each one was repeated at least 3 times. In vitro growth of both cell lines was unaffected by gastrin, bombesin or the respective antagonists (CR1718 and ICI 216140). The somatostatin analogue RC-160 also had no effect on basal growth. Significant growth stimulation of both MiaPaCa2 and PANI was seen with epidermal growth factor. We tested the hypothesis that somatostatin analogues may inhibit EGF-stimulated growth on both MiaPaCa2, a somatostatin receptor positive cell line, and on PANI which is negative for somatostatin receptors. RC-160 did not inhibit EGF-stimulated growth of either MiaPaCA2 or PANI. Both cell lines were established in vivo as xenografts in nude mice. The effect of RC-160 on tumour growth was measured. RC-160 inhibited the growth of MiaPaCa2, the somatostatin receptor-positive cell line, but not of PANI. PMID- 7558454 TI - Clonal heterogeneity in breast cancer: karyotypic comparisons of multiple intra- and extra-tumorous samples from 3 patients. AB - Intratumor phenotypic heterogeneity is one of the characteristics of breast carcinomas, and genetic mechanisms are likely to contribute to it. We have studied breast cancer clonal heterogeneity by cytogenetic analysis of multiple tumor samples (one from each tumor quadrant) as well as samples of macroscopically normal surrounding breast tissue from 3 patients with this disease. Clonal chromosome aberrations were found in all 8 successfully analyzed samples from the carcinomas. Two to 6 cytogenetically unrelated clones were detected in each case, unevenly distributed among the tumor quadrants. Karyotypic abnormalities were also found in 4 out of 9 macroscopically tumor-free samples from the surrounding tissue; in 2 of these samples, a ductal carcinoma in situ was detected histologically, and the cytogenetic evidence suggests that the remaining 2 samples also contained neoplastic cells. Quantitative analysis of the findings revealed a statistically significant higher frequency of karyotypically abnormal cells in samples with a histologic diagnosis of carcinoma vs. samples without any detected malignancy. That cells bearing cytogenetic evidence that they belong to the tumor parenchyma are left behind during breast-conserving surgery for carcinoma of the breast may account for the relatively high long-term local relapse rates seen in this disease. PMID- 7558456 TI - Different risk groups in node-negative breast cancer: prognostic value of cytophotometrically assessed DNA, morphometry and texture. AB - Feulgen-stained imprints and fine-needle aspirates from 528 lymph-node-negative breast cancers were investigated by means of an image analysis system. Several DNA, morphometric and textural parameters were evaluated for each patient. The prognostic value of the parameters was investigated by multivariate Cox regression analysis. As prognostic criteria, a distant recurrence-free survival of 5 years and an overall survival of 8 years were considered. In multivariate analyses the anisokaryosis (standard deviation of nuclear radius, Rad-SD) was the strongest parameter in predicting the clinical course of node-negative patients. This was followed by a textural parameter (run-length, NR2 M) and the tumor size (pT). The DNA histogram type could also add prognostic information concerning distant recurrence-free survival, but not overall survival. In both approaches a multivariate prognostic factor was calculated for each of the node-negative patients by a linear combination of the selected variables. Using this factor, patients could be split into 5 subgroups with significantly different risks of distant metastases. Thus, a low-risk subgroup, with a 5-year distant recurrence rate of only 3%, and a subgroup with a considerably higher risk and a distant recurrence rate of 35%, could be distinguished. In survival analysis the low-risk group of node-negative patients showed an 8-year death rate of only 3%, whereas in the high-risk group 30% of the patients had died at 8 years. Thus DNA, morphometric and textural parameters can provide powerful prognostic information in node-negative breast carcinomas. The multivariate combination of the relevant variables may allow a better selection of those node-negative patients with a proven good prognosis, and of those who are at risk of distant recurrence and therefore may benefit from adjuvant treatment. PMID- 7558457 TI - Human chromosome 11 suppresses the tumorigenicity of adenovirus transformed baby rat kidney cells: involvement of the Wilms' tumor 1 gene. AB - Human chromosome 11 was introduced into adenovirus-transformed baby rat kidney (BRK) cells by microcell-mediated chromosome transfer. The resulting microcell hybrids (MCHs) showed a reduced ability to form tumors upon s.c. injection into athymic mice. Further analysis, with the use of defined deletion chromosomes of 11p, indicated that the presence of region 11p13-p12 is necessary for the suppression of tumorigenicity. In contrast, the presence of region 11p15-14.1 appeared to increase the rate of tumor growth. Expression studies on the human Wilms' tumor I (WTI) and the insulin-like growth factor II (IGF-II) genes, which lie in regions 11p13 and 11p15, respectively, suggested the involvement of both genes in determining the degree of suppression of tumorigenicity. Finally, stable expression of a murine WTI protein in the adenovirus-transformed cells resulted in almost complete suppression of tumorigenicity, establishing the WTI protein as a tumor suppressor in this cell system. PMID- 7558459 TI - Differences in expression of metalloproteinases and plasminogen activators in murine melanocytes and B16 melanoma variants: lack of association with in vitro invasion. AB - We investigated in vitro chemotactic responses to fibronectin and laminin, invasion through reconstituted basement membrane (Matrigel) and secretion of matrix metalloproteinases and plasminogen activators by non-tumorigenic Mel-ab melanocytes; B16 melanoma; and the metastatic sublines, B16F1, B16F10 and B16BL6. In vitro chemotactic and invasive ability were not associated with in vivo metastatic potential. Secretion of various matrix-degrading enzymes was not related to in vitro invasion. Conditioned media from all B16 melanoma sublines, but not from Mel-ab cells, contained the M(r) 92,000 progelatinase. The activated M(r) 85,000 species was present only in conditioned media from Mel-ab, B16 and B16F1 cells. Mel-ab cells secreted copious amounts of the M(r) 72,000 progelatinase, and the M(r) 66,000 active form was also present in conditioned media. Secretion of the M(r) 72,000 progelatinase by B16 melanoma sublines was markedly lower, and only conditioned media from B16 cells contained the activated M(r) 66,000 form. Furthermore, cell lysates of Mel-ab cells contained a M(r) 67,000 metalloproteinase which was absent in the tumor cells. All cells secreted tissue plasminogen activator; however, the metastatic B16F1, B16F10 and B16-BL6 cells also secreted urokinase plasminogen activator. Our results indicate that matrix metalloproteinase secretion by itself is not associated with tumorigenicity or metastatic potential. Secretion of urokinase plasminogen activator, and not tissue plasminogen activator, reflected the metastatic characteristics of the B16 melanoma tumor sublines. PMID- 7558460 TI - Echocardiographic diagnosis of subclinical carditis in acute rheumatic fever. AB - The diagnosis of carditis in acute rheumatic fever traditionally depends on characteristic auscultatory findings. The advent of pulsed and colour Doppler echocardiography provides a method of detecting minor degrees of pathological regurgitation without characteristic clinical signs. Using strict criteria, pathological left heart regurgitation can be differentiated from physiological regurgitation: colour Doppler must show a substantial colour jet in two planes extending well beyond the valve leaflets; pulsed Doppler must confirm a high velocity signal, holosystolic for mitral regurgitation, or holodiastolic for aortic regurgitation. Several centres have observed subclinical carditis in children with acute rheumatic fever. We are confident that we are not overdiagnosing valvulitis, having tested this in a blinded fashion. Subclinical valvulitis should be accepted as evidence of carditis, a major diagnostic criterion for acute rheumatic fever. PMID- 7558458 TI - Increased risk of cancer in the descendants of Syrian hamsters exposed prenatally to diethylnitrosamine (DEN). AB - Transmission of site-specific tumorigenicity (papillomas in larynx and trachea) of diethylnitrosamine (DEN) to the 2 subsequent generations (F1 and F2) was studied using an outbred strain (Han:AURA) of pregnant Syrian golden hamsters (P generation), which were treated i.p. with 10 mg/kg b.w. of DEN on day 12, 13 or 14 of gestation. Laryngotracheal papillomas were induced by DEN in the P and F1 generations only, while these tumours did not occur in the F2 generation. Spontaneously occurring tumours, including uterine adenocarcinomas, lymphomas, and laryngotracheal neuro-endocrine cell tumours, were observed at higher incidences among the F2 animals derived from the P generation hamsters treated with DEN only on day 13 or 14 of gestation. In the same animals, the ratio of malignant to benign tumours was considerably higher than in controls. In addition, the F2 hamsters derived from the DEN-treated P generation showed more frequent multiple organ involvement in tumorigenesis than the F2 controls. Several uncommon malignant tumours were detected in the F2 offspring, possibly the result of damage caused to germ cells by the prenatal exposure of F1 Syrian hamsters to DEN. PMID- 7558461 TI - Double-chambered right ventricle: experience with 52 cases. AB - The presence of anomalous muscle bundles may produce a pressure gradient between the inflow and outflow portions of the right ventricle, thus resulting in double chambered right ventricle bearing troublesome clinically in its diagnosis. The aim of the present study was to review the diagnostic criteria. Fifty-two patients with a double-chambered right ventricle were seen during an 8-year period. They ranged in age at the catheterization from 4 months to 17 years (mean 7.5 +/- 4.4 years). Diagnosis was confirmed in 51 patients at cardiac catheterization and in other one on operation. The majority of the patients had associated cardiac anomalies: there were 33 ventricular septal defect (63%), 21 pulmonary valve stenosis (40%), nine atrial septal defect (17%), and four double outlet right ventricle. The electrocardiograms revealed upright T waves alone in right precordial leads suggesting right ventricular hypertrophy in 33% of the patients. At cardiac catheterization, there was a pressure gradient of 20-160 mmHg between the right ventricular inflow and outflow portions. Forty patients have had surgery and four have undergone balloon pulmonary valvuloplasty. Surgical treatment was planned for two patients and other six had no indication for treatment. PMID- 7558462 TI - Is diastolic dysfunction associated with thrombogenesis? A study of circulating markers of a prothrombotic state in patients with coronary artery disease. AB - Clinical data on the contributory role of heart failure to thromboembolic risk does not differentiate between systolic and diastolic left ventricular dysfunction. We therefore conducted a population-controlled cross-sectional study to determine levels of plasma fibrinogen (associated with thromboembolism), fibrin D-dimer (a marker of fibrin turnover) and von Willebrand factor (a marker of endothelial dysfunction) in patients with ischaemic heart disease (a common cause of diastolic dysfunction) in whom left ventricular diastolic function was defined by echocardiography. We studied 106 patients: those with normal left ventricular function (n = 42, Group 1); those with left ventricular dysfunction but without aneurysms (n = 34, Group 2); and those with left ventricular aneurysm formation (n = 30, Group 3). Each of these groups was subdivided into those with (a) and without (b) diastolic dysfunction. Diastolic dysfunction was present in over 60% of patients, irrespective of left ventricular systolic impairment. There were no significant differences in median levels of plasma fibrinogen, fibrin D dimer or von Willebrand factor in each group of patients with ischaemic heart disease, whether or not left ventricular diastolic dysfunction was present (Mann Whitney test; P = N.S.). Systolic (rather than diastolic) dysfunction was the main correlate of these (analysis of variance, general linear model--ANOVA-GLM--P < 0.05) and the greatest abnormalities of fibrinogen, endothelial dysfunction and intravascular fibrin turnover were seen in patients with left ventricular aneurysms whether or not diastolic dysfunction was present. This study demonstrates that there is no evidence of a significant additional contribution to thrombotic risk (as assessed by plasma fibrinogen, von Willebrand factor and fibrin D-dimer) for patients with left ventricular diastolic dysfunction. A relationship is noted between some prothrombotic factors and Doppler indices of flow, which suggests a possible association between cardiac haemodynamics and thrombogenesis. PMID- 7558463 TI - Electromechanical effects of caffeine in failing human ventricular myocardium. AB - We studied, using standard microelectrode technique, the effects of caffeine alone and in conjunction with epinephrine in ventricular myocardial fibers obtained from the failing hearts of 12 recipients of heart transplant. Results revealed that caffeine (1-3 mM) prolonged slightly the duration of fast response action potential near final repolarization and the twitch curve but slightly increased, or even decreased, the twitch force. Epinephrine (3 microM) induced a submaximal positive inotropic effect in myocardial fibers obtained from the failing hearts. Caffeine at 1 mM significantly potentiated the force of contraction and accelerated the rate of twitch relaxation increased by epinephrine. Increasing concentration of caffeine to 3 mM in the presence of epinephrine did not enhance further the twitch force but rather induced the delayed after-depolarization in two of eight experiments. In a preparation from one patient with dilated cardiomyopathy, the combination of caffeine and epinephrine induced repetitive triggered rhythms. The present findings suggest that, in human ventricular myocardium obtained from certain patients transplanted for congestive heart failure, caffeine could induce positive inotropy and triggered automaticity through a potentiation of the actions of catecholamines. PMID- 7558464 TI - Dobutamine stress echocardiography for the identification of multivessel coronary artery disease after uncomplicated myocardial infarction: the importance of test end-point. AB - Our aim was to verify whether the sensitivity of pharmachological stress echocardiography for multivessel disease after acute myocardial infarction may be improved by a more aggressive protocol, i.e. not considering the appearance of the first wall motion abnormality as the absolute end-point if it occurs in the infarcted area without clinical or instrumental markers of extensive ischemia or left ventricular dysfunction. One-hundred twenty-one consecutive patients (age 32 71 years) prospectively underwent dobutamine-atropine stress echo (dobutamine infusion up to 40 micrograms/kg/min with additional atropine 1 mg) 11.8 +/- 4.8 days after uncomplicated myocardial infarction and coronary angiography within 6 weeks. Criteria for stopping the test were: significant ST depression or elevation, typical chest pain, major arrhythmias and left ventricular dysfunction. The test was considered as positive if a deterioration of basal wall motion pattern was observed: it was defined homozonally positive (the deterioration occurred in the myocardial area fed by the culprit vessel) or heterozonally positive (the deterioration occurred in a different vascular area). A coronary stenosis > 70% of vessel lumen was defined as critical. Thirty-four patients showed a negative test result. Among the 87 patients with positive test, 65 had no further wall motion deterioration from the first-induced wall motion abnormality (WMA) to peak test (Group A), whereas nine patients showed further homozonal (Group B) and 13 further heterozonal (Group C) asynergies. Sensitivity, specificity and accuracy of dobutamine stress echocardiography for multivessel disease were, respectively, 63%, 96% and 82% using the first-induced wall motion abnormality as test end-point, whilst they were 84% (P < 0.01), 93% and 89% according to the aggressive approach previously described. Dobutamine stress time of patients with multivessel disease was higher in Groups B and C (13.1 +/- 3.6 min) than in Group A (9.8 +/- 3.7 min, P < 0.01) and, finally, the mean obstruction of non-culprit vessel was higher in Group A (62.2%) than in Group C (47.4%, P < 0.05). No major complications were found. We conclude that the sensitivity of dobutamine stress echocardiography for multivessel disease following recent myocardial infarction is critically dependent on the test end point. It may be improved by a more aggressive approach capable to identify less severe heterozonal coronary lesions. PMID- 7558468 TI - Silent myocardial rupture in a patient with diabetes and Addison's disease. AB - We present a case of left ventricular rupture and formation of a pseudoaneurysm after silent myocardial infarction in a patient with Schmidt syndrome (polyglandular deficiency syndrome including Addison's disease, lymphocytic thyroiditis and diabetes mellitus). This case illustrates the possibility of myocardial rupture without hemodynamic collapse and a possible role of chronic steroid substitution. PMID- 7558467 TI - The Interdisciplinary Heart Failure and Transplant Program Munster: a 5-year experience. AB - The variety and complexity of therapeutic options available for the increasing number of patients with advanced chronic heart failure has lead to the institution of specialized heart failure/heart transplant programs. The central task consists of carefully evaluating and selecting potential transplant recipients, based on the comparative benefit rationale, i.e. the expected gain in life expectancy and quality of life by cardiac transplantation compared with all other treatment options. Further tasks include a comprehensive chronic heart failure management, high risk conventional cardiac surgery, mechanical and antiarrhythmic bridging and dedicated posttransplant care. This concept is described on the basis of the 5-year experience of the Interdisciplinary Heart Failure and Transplant Program Munster. PMID- 7558466 TI - Increased sympathetic nervous system activity in Chinese hypertensive patients with type II diabetes mellitus. AB - To evaluate the coexistence of sympathetic overactivity and hypertension in type 2 diabetic patients, a population-based study was conducted of newly-diagnosed type 2 diabetic patients recruited from a single community located at northern Taiwan. This study included 2877 (male 1382, female 1495) middle-aged ethnic Chinese adults, aged 45-65 years. Of the 1382 males, 67 had newly-diagnosed type 2 diabetes mellitus, whereas 75 of the 1495 females had type 2 diabetes. The data showed that about 39% of diabetic patients had borderline hypertension (mean blood pressure 141/91 mmHg) whereas the average incidence in non-diabetic subjects was 15.5%. The borderline hypertensive diabetic patients had significantly higher heart rates (mean 78.8 vs. 69.3 beats/min; P < 0.001) than control subjects. However, the cardiac index was similar in both control and diabetic subjects (mean 2.48 vs. 2.53 l/min/m2; P > 0.05). Our data show that sympathetic overactivity and increased incidence of hypertension actually existed in these type 2 diabetic patients of Chinese origin. PMID- 7558465 TI - Signal-averaged electrocardiography in myotonic dystrophy. AB - We performed signal-averaged electrocardiography and 24-h ambulatory electrocardiographic monitoring in 53 patients with myotonic dystrophy to determine the incidence and clinical significance of ventricular late potentials. Patients were followed up for a mean period of 31 +/- 17 months (range 11-68 months). At entry, none of the patients had bundle branch block on 12-lead electrocardiogram and none had wall motion abnormalities on routine echocardiogram. Also, no patient had history of syncope or clinical evidence of ischemic heart disease or a documented sustained ventricular tachycardia. A group of 47 healthy subjects matched for age and sex also underwent signal-averaged electrocardiography for comparison with the patient group. Late potentials were diagnosed in the presence of at least two of the following measures: duration of the filtered QRS > 114 ms, root-mean-square voltage of the terminal 40 ms of the filtered QRS < 20 microV, and duration of the low-amplitude (< 40 microV) signals of terminal filtered QRS > 38 ms. Late potentials were more frequent in patients than in controls: 18 of the 53 patients (34%) showed late potentials compared with four of the 47 controls (8.5%) (P < 0.01). In 45 patients (85%) no ventricular ectopy (40 cases) or infrequent premature ventricular complexes (five cases) were detected on Holter monitoring. Complex ventricular arrhythmias were traced in the remaining eight patients. These were six of the 18 patients with, and two of the 45 patients without late potentials (33% vs. 6%, respectively; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558469 TI - Lipoprotein lipase does not affect lipoprotein (a) levels in normotriglyceridemic patients. PMID- 7558470 TI - The role of gender and acculturation on determining the consumption of alcoholic beverages among Mexican-Americans and Central Americans in the United States. AB - This study analyzed the responses of 391 Mexican-Americans (44.9% males) and 531 Central Americans (40.2% males) who were between 21 and 65 years of age and resided in San Francisco, California. In general, Mexican-Americans were found to have a lower proportion of abstainers (56.8%) than Central Americans (64.4%). Mexican-Americans reported drinking more often and in greater quantities than Central Americans, and the proportion of "high" drinkers was higher among Mexican American men and women than among the Central American respondents. Despite this diversity in the topography of alcoholic beverage consumption between Mexican Americans and Central Americans, the role of gender and acculturation on shaping those variables was fairly consistent across groups. The acculturation level of the respondents was found to significantly affect the proportion of abstainers in both groups. Furthermore, gender was an important determinant of frequency, total number of drinks, and volume of drinking for Mexican-Americans and for Central Americans. PMID- 7558474 TI - A critical incident model for considering issues in cross-cultural research. Failures in cultural sensitivity. AB - This paper consider anecdotes obtained from researchers which relate to issues in cross-cultural studies. The anecdotes are described and the relevant cross cultural issues are outlined and discussed. Recommendations are made to help researchers conduct studies which are more culturally sensitive. PMID- 7558471 TI - Adolescent drug misuse treatment and use of medical care services. AB - Research on adults has documented that use of medical services decreases after initiation of treatment for alcohol problems, but little is known about this relationship among adolescents. We studied utilization and costs of care following participation in the Adolescent Chemical Health Program (ACHP) of Kaiser Permanente, Northwest Region, in 1986-88. Three groups of adolescents (and their parents) were identified: adolescents who were assessed and initiated treatment in ACHP (n = 561), adolescents who were assessed and recommended for treatment but did not return for treatment (n = 278), and adolescents with no known substance use problems (n = 381). Medical records were reviewed for 1 year pre- and 1.5 years postassessment. After adjusting for preassessment medical visits, severity of alcohol and drug use, gender, and age, analyses suggested that substance user treatment was not associated with reduced use of medical services or costs by either adolescents or parents. PMID- 7558473 TI - Psychological symptomatology in spouses and adult children of alcoholics: an examination of the hypothesized personality characteristics of codependency. AB - The concept of codependency has been advanced to explain certain psychological traits purported to be characteristic of spouses and adult children of alcoholics. To test the validity of this hypothesized syndrome, 97 female subjects living with either an alcoholic (SA), a psychiatric patient (SP), or dentistry patient (SD) were studied; approximately 50% of subjects had a positive family history (FH+) for alcoholism. All subjects were administered an extensive battery of psychological tests. The results revealed significantly greater levels of psychological symptomatology among the SA and FH+ subjects, in part consistent with the hypothesized symptomatology of codependency. PMID- 7558472 TI - Normative data for a brief neuropsychologic test battery in a cohort of injecting drug users. AB - Recent epidemiologic studies of the cognitive performance of injecting drug users have demonstrated the need to establish appropriate test norms for this population. This report provides normative data from a group of 150 injecting drug users on a battery of standardized tests of cognitive performance stratified by age group (range 20 to 49 years) and educational level (mean 11.6, standard deviation 2.0). The analysis also includes estimation of partial correlations between neuropsychologic test scores and age and education. The analysis demonstrates that age and education are important determinants of performance for several of these tests, and provides norms that may be of use as a reference for clinical evaluation and research in drug user populations. PMID- 7558476 TI - A treatment model for women substance users. AB - This paper sets forth a multifaceted program which consists of drug user women centered counseling plus ancillary services based on the five basic steps of Freire's empowerment theory. The program is designed to increase self-awareness, skills, and knowledge acquisition, to reduce feelings of isolation by broadening interpersonal networks, includes activities to enhance assertiveness and communication skills, and parenting education to counteract inappropriate mother child interaction as well as to positively influence role modeling. Both transportation and child care are recommended ancillary services. PMID- 7558477 TI - The validity of behavioral data reported by injection drug users on a clinical risk assessment. AB - The validity of drug and sexual behavior data collected by drug user treatment staff using a short clinical risk assessment among 387 injection drug users is evaluated using in-depth, confidential interview process. Moderate to high agreement levels were found for most, but not all, variables. Participants consistently reported less risky behaviors on the clinical risk assessment than on the in-depth interview. More easily recalled information can be accurately gathered through a short clinical risk assessment. However, in-depth, confidential interviewing is important when gathering self-reports of the frequency of drug and sexual behaviors. PMID- 7558478 TI - Submission and rebellion: excessive drinking of women in problematic heterosexual partner relationships. AB - Functionality of alcohol use for coping with partner relationship problems was explored in a group of 45 alcohol-dependent women with semistructured interviews. Six functions were categorized, three of them referring to adjustment and three to opposition to the partner. Respondents who had started excessive drinking mainly in response to problems with the partner reported more partner-related functions than respondents for whom a problematic partner relationship was not an important factor in the development of excessive drinking. Respondents of the first group also reported other characteristics of their relationship, notably they said more often that their partner was dominant. It was concluded that for a subgroup of women with alcohol problems, alcohol use may be a way of coping with a situation of powerlessness toward the partner. PMID- 7558479 TI - The Drinker's Partner Distress Scale: an instrument for measuring the distress caused by drinkers to their partners. AB - Although it is generally accepted that the spouses of excessive drinkers experience a range of problems as a result of their partner's alcohol consumption, there have been few attempts at measuring the hardship experienced. Previous attempts have counted only the number of categories of hardship experienced over the previous year and not taken into account the frequency with which these occur or the degree of inconvenience suffered. The Drinker's Partner Distress Scale (DPDS) is proposed as a measure which overcomes these limitations. Two dimensions of alcohol-induced problems are measured; depression and marital discord. Both subscales demonstrated internal consistency, and predictions of convergent and discriminant validity were supported in relation to both subscales. PMID- 7558475 TI - Issues in treatment process and services research. PMID- 7558480 TI - When social problems converge: homeless, mentally ill, chemical misusing men in New York City. AB - Despite their pervasiveness in large American cities, homeless, mentally ill, chemical misusing (HMICM) people have rarely been studied as a sociological phenomenon. In this study, 518 HMICM men who sought community-based treatment were interviewed at length on their psychosocial characteristics, family background, and childhood history. The findings show these HMICM men to have severe psychiatric and substance misuse problems. Examination of their family background reveals a common pattern represented by the absence of their fathers and often of their mothers from very early age. Fatherlessness and social rootlessness are hypothesized as related to the HMICM syndrome. PMID- 7558482 TI - The prevalence of drug use with reference to problem use in Finland. AB - The first general population mail survey of the prevalence of drug use in Finland was conducted in 1992. The data consisted of 3,457 responses with a response rate of 70.7%. The persons surveyed were from 18 to 74 years old. These data are supplemented with data about "problem use" of drugs in Finland, which is mostly characterized by the mixed use of medicines (also medicines containing opiates) and alcohol. There were clear signs of an increase in the mixed use during the last few years. During the last year cannabis was used by 1% of the population. The use of opiates and amphetamines was very limited, and there had not been any special changes in their use. According to the statistics of social and health welfare sources, the need for services caused by the use of drugs had been only about 1% of the whole intoxicant (mostly alcohol) problem during the past few years. PMID- 7558481 TI - "Unselling" drugs: the marketing of prevention. AB - By borrowing from the field of social marketing, the examination and execution of drug prevention strategies should be more rigorous and disciplined by the year 2000. This article briefly describes marketing strategies and techniques which can assist in the maximization of drug prevention communications and programs. PMID- 7558483 TI - The efficacy of treatments in reducing alcohol consumption: a meta-analysis. AB - Meta-analysis was used to assess the relative efficacy of various treatments in reducing alcohol consumption over the short-term, 6 months, and 12 months. All the treatments were administered in well-controlled studies. In the short-term and 1-year follow-up studies, patients in the experimental group drank much less than the control group. However, between group consumption differences were negligible in the 6-month studies. When the studies were pooled, regardless of the follow-up assessment periods, the experimental group drank significantly less than the control group. These results suggest that, in general, patients who received experimental treatments consumed much less alcohol than patients in the control groups. PMID- 7558484 TI - Prevalence of abuse and dependency in chronic pain patients. AB - A series of 414 chronic pain patients referred to Are Hospital, Are, Sweden, for evaluation and rehabilitation were administered a structured diagnostic interview to detect alcohol and drug misuse and dependence according to DSM-III-R criteria. A total of 97 (23.4%) met criteria for active alcohol, analgesic, or sedative misuse or dependency; an additional 39 (9.4%) met criteria for a remission diagnosis. Current dependency was most common for analgesics (12.6%) followed by alcohol (9.7%) and sedatives (7.0%). PMID- 7558485 TI - Gambling among methadone patients. AB - In this paper we assess participation in various forms of gambling activities and establish the prevalence of pathological gambling in a sample of patients (N = 117) enrolled in a large methadone maintenance treatment program in New York City. Respondents were interviewed with a protocol that incorporates the South Oaks Gambling Screen. We found that gambling was a common part of the regular activities of many patients, that 15% of the patients had some problem with gambling, and that an additional 16% were probable pathological gamblers. The implications of our findings are discussed. PMID- 7558486 TI - Quality of alcohol use histories collected at intake to substance user treatment. AB - Many survey questions on alcohol require complex cognitive tasks, such as long term recall, shifting reference periods, and numeric calculation. Moreover, alcohol-related impairment is known to affect cognitive ability. To assess the quality of data on self-reported alcohol use, internal consistency analyses were conducted as part of a comprehensive multisite prospective study of drug user treatment outcome undertaken in 11 cities throughout the United States (DATOS). Contrary to expectation, analyses found high levels of internal consistency. For questions on age of initiation of different types of alcohol use, over 99% of respondents (N = 2,842) reported consistent answers for each pair of logically related questions. Reports of being drunk and of quantity of alcohol consumed were similarly consistent. PMID- 7558487 TI - Controlled heroin availability in Australia? How and to what end? AB - The general public, police, service providers, and users/ex-users were asked their views about options for trial design and trial outcomes with regard to a proposal for experimental controlled heroin availability. There was substantial agreement between the samples on issues concerning trial design. In general, the samples from the community, service providers and users/ex-users were more likely to report that a trial would result in positive outcomes, whereas the police sample was more likely to report that a trial would result in negative outcomes. This study illustrates the value of systematic consultation of key groups in exploring the options for change, raising potential difficulties, and highlighting different interests. PMID- 7558489 TI - Long term results of uncemented Judet hip endoprostheses. AB - We reviewed 94 patients in whom 113 Judet uncemented total hip endoprostheses had been inserted; 83 had a mean follow up of 10.8 years (range 6 to 13 years). The Merle dAubigne and Harris hip scores were used for clinical evaluation. In the long term follow up, severe or moderate pain was present in 38.6% of hips. Radiographs were studied and radiolucent lines, and other evidence of loosening, were recorded. Twenty hips (24.1%) showed radiological signs of loosening of the acetabular cup and a displacement of more than 5 mm in 37.3%. The loosening rate of femoral stems was 32.5%, but only 8.4% showed subsidence. The cumulative survival rate at 6 years was 84.1%, decreasing to 55.1% after the eleventh year. Complications and other aspects of the outcome are discussed. Our results were on the whole not satisfactory and we cannot recommend this type of uncemented endoprosthesis for routine total hip replacement. PMID- 7558491 TI - Biodegradable implants for potential use in bone infection. An in vitro study of antibiotic-loaded calcium sulphate. AB - Local antibiotic therapy by diffusion from plaster of Paris beads has proved promising in bone surgery. Sustained local delivery depends on thermostability, so we tested the antibacterial activity of 11 antibiotic solutions after storage at 37 degrees C using a microbiological method. Cephalosporins and penicillins were unstable, but aminoglycosides remained fully stable with 100% activity after 2 weeks. About 60% of the initial bactericidal activity of quinolone, glycopeptides and sodium fusidate were still detectable after 2 weeks. Release of these antibiotics from plaster of Paris beads was evaluated in vitro. Even those in the same family differed in their release rate. Plaster beads with sodium fusidate were the most effective association. A therapeutic level of glycopeptides, aminoglycosides and amoxicillin was leached for about 3 weeks. Cephalosporins and sodium amoxicillin were released in 2 to 3 days, and quinolone beads were too brittle to be used. Plaster of Paris, which is cheap, biocompatible and biodegradable, is an excellent carrier for sodium fusidate, aminoglycosides and glycopeptides. PMID- 7558490 TI - [Replacement of infected total hip prosthesis in two stages]. AB - Since 1975 we have treated 36 infected hip replacements by a two stage procedure in which the prosthesis and cement were first removed. Gentamicin impregnated beads were left at the sites of the prosthesis. Not less than 45 days later a fresh prosthesis was inserted into the hip. In two hips revision was not undertaken because of persistence of infection. The remaining 34 hips received a fresh prosthesis when the site was bacteriologically sterile. Gentamicin impregnated cement was used and antibiotics were given for a further three months. The functional results were satisfactory at an average of five years of follow up. Incomplete removal of methylmethacrylate was a factor in the persistence of infection and of pain in the absence of infection. The bacteriological efficacy of Gentamicin impregnated beads is uncertain, but their use gave a better functional result. PMID- 7558492 TI - Alteration in the pattern of macrophage subtypes in chronic osteomyelitis compared with acute joint infection. AB - Macrophage subtypes were detected in cryostat sections of biopsies from patients with chronic osteomyelitis, acute joint infections and normal bone marrow, using monoclonal antibodies against different macrophage populations. The resident macrophage subtype 25F9, the gluco-corticoid-inducible macrophage RM 3/1 and the inflammatory type 27E10 were found in abundance in acute infections. They were also present in tissue sections of uninflamed bone marrow. By contrast, in about 50% of the biopsies from patients with chronic osteomyelitis a reduced number of macrophage subtypes, or even the lack of one or more macrophage subpopulations was found. The unusual absence of macrophage phenotypes seems to be restricted to the area of osteomyelitis because in the tissues of inflamed sinuses in these patients, the macrophage subtypes were present. These findings suggest a disturbance at the level of the macrophages which may contribute to the persistence of the inflammatory process in osteomyelitis. PMID- 7558488 TI - Endoprostheses for osteonecrosis of the femoral head. A comparison of four models in young patients. AB - Eighty-eight hip arthroplasties were carried out in 75 patients, all aged 50 years or less, with osteonecrosis. All the operations were carried out by one surgeon. Four different methods were used: a standard cemented arthroplasty; a cemented THARIES surface replacement; an uncemented surface replacement; a cemented titanium femoral surface hemiarthroplasty. Comparable clinical improvement occurred in all 4 groups initially. Aseptic loosening, with intersurface degradation and osteolytic lesions, was the most common cause of failure in the 3 types which had a polythene bearing. Polyethylene is the major contributor to bone loss around the endoprosthesis and loosening. Fixation of the surface hemiarthroplasty remained intact in every patient and failure was secondary to wear of the acetabular cartilage. We recommend surface hemiarthroplasty as an interim solution for young patients with stage III or early stage IV osteonecrosis because this procedure conserves bone stock, there is little osteolysis and it can easily be revised. PMID- 7558494 TI - A radiological sign in pulled elbows. AB - A new radiological sign of distal shift of the radius compared to the ulna is described in the condition of pulled elbow. This measurement has been termed the proximal radial length and was altered in 21 out of 25 cases of pulled elbow diagnosed clinically. The normal relationship was restored after manipulation. PMID- 7558493 TI - Hydroxyapatite-glass composite as a bone substitute in large metaphyseal cavities in rabbits. AB - This study evaluated hydroxyapatite-glass (HA-G) composite as a filler material when implanted into a proximal metaphyseal tibial cavity consisting of medullary tissue and cancellous bone in rabbits. The repair process was compared with that of autogenous bone grafts and untreated controls. Scanning electron microscopy, energy dispersive x-ray studies, radiography, histology with tetracycline fluorescence and morphometric bone measurements were used for evaluation. Equal amounts of mineralised bone were produced in both HA-G granule and autograft groups at 3 weeks; thereafter, autografts mostly resorbed, whereas there was appositional lamellar bone formation on the early woven bone in the HA-group. A relatively small proportion of the cavities was repaired by new bone. The HA-G granules were biocompatible without foreign body reaction and showed good osteoconductive potential. Mesenchymal cells were able to differentiate into osteoblasts on bioactive surfaces of the composite. Although HA-G was able to form Ca-P-rich and Si-rich layers, most of the granules underwent degradation through its glass matrix, resulting in fewer bony contacts than granules with no surface degradation. PMID- 7558502 TI - The Howard University Hospital Transplant and Dialysis Support Group: twenty years and going strong. AB - This article discusses group therapy as part of a multidisciplinary approach to the management of the various psychological and physical rehabilitation concerns posed by kidney and liver transplant candidates and recipients at Howard University Hospital in Washington, DC. The group's history, format, intervention foci, and roles of clinicians and patients attending the group are described and evaluated. Given the relative paucity of research literature in this area, the authors offer recommendations for empirical evaluation of the benefits of multidisciplinary group psychotherapy in ameliorating physical and emotional suffering and prolonging life among transplant patients. PMID- 7558498 TI - False aneurysm of the femoral artery as a late complication of an intertrochanteric fracture. A case report. AB - False aneurysms of the femoral vessels are an important, but rare, complication after an intertrochanteric fracture of the femur. The diagnosis is usually delayed, because pain, a haematoma and unexplained anaemia are the nonspecific clinical signs. Early diagnosis by duplex and colour Doppler ultrasound is a good alternative to arteriography or magnetic resonance imaging. PMID- 7558500 TI - H. Baba, Q. Chen, K. Kamitani, S. Imura, and K. Tomita: Revision surgery for lumbar disc herniation--an analysis of 45 patients. PMID- 7558501 TI - The spirit of Jungian group psychotherapy: from taboo to totem. AB - Practitioners of analytical psychology were late in coming to the practice of group psychotherapy because Carl Jung effectively forbade the treatment of individuals in stranger groups. This article explores Jung's objections to group therapy and, by way of a conceptual review of the literature, expands on the practice that grew up proximate to his death. It is argued that Jungian theory is especially conducive to collective treatment because it is concerned with the relationship between oppositions (whether in persons or between people) and uses synthetic and symbolic processes to bring about an integration of the one with the many. For Jungians who espouse a theory of symbolic transformation, archetype, and myth, the group is embodied in individuals and can be accessed by working with individuals in groups. PMID- 7558497 TI - Malignant squamous cell carcinoma arising in a lumbar dermoid cyst. A case report. AB - A case of malignant transformation to a squamous cell carcinoma in a long standing lumbar dermoid cyst is described. Progress was slow during 6 years. This type of transformation in a dermoid cyst is extremely uncommon and has never been recorded at this site. PMID- 7558495 TI - Lumbar radiculopathy associated with anterior intraspinal bony lesions. AB - Thirty-one patients underwent posterior decompression for anterior intraspinal bony lesions which were causing symptoms. The diagnoses were a displaced apophyseal endplate, vertebral corner bulge, and an ossified posterior longitudinal ligament or disc. Symptoms were caused by associated lumbar disc herniations or by gradual impingement by the bony lesion on neural tissue. The success rate after operation was 84%. Anterior spinal canal compromise by bony lesions can be treated by decompression through a posterior approach with minimal destruction of the laminae and facets. PMID- 7558503 TI - Cultivating the observing ego in the group setting. AB - Group therapy provides the ideal arena for helping individuals cultivate the observing ego--that part of the sensorium that has the power of witnessing oneself in the world. The essence of our method is to employ the group as a temporary observing ego, out of which members may develop their own observing egos. A five-step process is presented to (1) help the members see how they are affecting others when they do not see this themselves, (2) help the members see how they produce this effect, (3) help the members see the self-injurious nature of their behavior, (4) resolve resistance to seeing any of this, and (5) help the members arrive at new modes of behavior. PMID- 7558499 TI - [Bone metastasis for choriocarcinoma]. AB - Metastasis to bone in the pelvis occurred in a woman with a choriocarcinoma. The diagnosis was made on the basis of high levels of HCG and a bone biopsy. The presence of bone metastases in a young woman requires vigorous systemic search for a primary lesion. PMID- 7558496 TI - Intrathoracic dural ectasia mimicking neurofibroma and scoliosis. A case report. AB - Angular thoracic scoliosis combined with dural ectasia and foraminal enlargement is reported in a patient with neurofibromatosis. The dural cyst was resected through a high thoracic approach and the scoliosis dealt with by posterior spinal fixation with Modulock instrumentation. Follow up after 12 months showed no neurological deficit and no progress of the scoliosis. PMID- 7558505 TI - Countertransference in group psychotherapy: waking a sleeping dog. AB - This paper reviews and critiques the literature on countertransference (CT) in group psychotherapy. The literature review is organized within a framework that calls attention to the origins, triggers, manifestations, and effects of CT in groups, as well as CT management factors. An overriding critique is that the present literature lacks a research foundation of any kind. An argument is made for beginning research in this domain, and guidelines for doing so are presented. PMID- 7558504 TI - Reflections and suggestions on leadership of psychotherapy groups. AB - This paper attempts to provide a helpful, "hands-on" guide to leaders of psychotherapy groups in a manner that is free of any one particular theoretical orientation. It focuses on characteristics that can be applied to groups emphasizing life skills, cognitive skills, or interpersonal skills in a variety of settings and types of groups--inpatient or outpatient, short or long term. Issues covered include the importance of structure as it relates to anxiety, group composition, skill requirements, resistance, and symbolic factors. The major theme is that leaders need to make choices and that these choices can be made easier with an awareness of the major factors involved in group psychotherapy. PMID- 7558506 TI - Group supervision: focus on countertransference. AB - This paper examines the advantages of a particular way of supervising psychotherapy, namely, in a group setting with a special focus on the supervisee's countertransference experience. Group supervision is conceptualized as much more than presenting a case and getting feedback. Rather, the group is used in all its interactive complexity as it resonates in a myriad of ways to aspects of the case being presented. Furthermore, because of the complexity of conscious and unconscious interactions and reverberations during this process, it is often helpful to have a focus in the supervision. One helpful possibility is to center on the supervisee's countertransference experience and use the group to reflect, amplify, and process that experience. This can be a highly valuable way of helping the therapists increase their understanding of the case and enhance the quality of therapeutic interventions. PMID- 7558507 TI - A group therapy program for individuals identified as autistic who are without speech and use facilitated communication. AB - The first author directed a group-therapy program of 20 sessions for clients without speech, diagnosed with autism who communicate using facilitated communication. An average of five clients and their facilitators, the leader, and an assistant leader comprised the group. The themes that emerged and the group development process observed paralleled regular verbal groups in many respects. The success of the project challenges accepted views of person labeled autistic as intractably and inevitably isolated and unreachable. PMID- 7558508 TI - Group psychotherapy in the managed-care marketplace: further comments following Helfman and Mone on MacKenzie. PMID- 7558509 TI - Differences in the effects of dexamethasone on macrophage nitrite production: dependence on exposure regimen (in vivo or in vitro) and activation stimuli. AB - Exposure to glucocorticoids in vitro is known to suppress the production of reactive nitrogen intermediates (RNI) by macrophages, and it has been suggested that this contributes to the anti-inflammatory action of glucocorticoids in vivo. However, the effects of glucocorticoid administration in vivo on subsequent RNI production as measured in vitro are not known. In the present study, dexamethasone was administered in vivo and was also used to treat macrophages in vitro prior to, and during, stimulation of nitrite production by interferon-gamma (IFN-gamma) and/or bacterial lipopolysaccharide (LPS). Macrophages were isolated 24 h after daily administration of dexamethasone (0.1-30 mg/kg/day) to female B6C3F1 mice for 3, 6, or 16 days. In most cases, these cells produced an equal or greater concentration of nitrite in response to IFN-gamma, LPS, or IFN-gamma plus LPS, than cells from vehicle control mice. In contrast, continuous exposure of macrophages to dexamethasone during stimulation in vitro caused dose-dependent inhibition of nitrite production. However, the inhibition was much less pronounced when LPS or IFN-gamma together were used to stimulate the macrophages than when either was used separately. Similar results were noted when macrophages were exposed to dexamethasone for 24 or 72 h in vitro followed by a 0-24 h recovery period after removal of dexamethasone. Thus, immunosuppressive doses of dexamethasone in vivo do not decrease the induction of nitrite production 24 h after the last dose, whereas significant decreases are noted 24 h after termination of dexamethasone exposure in vitro. The basis for this difference is not clear, but there was no indication that administration of dexamethasone in vivo selects for a "glucocorticoid resistant" population of macrophages. These observations have implications with regard to the mechanisms of glucocorticoid mediated anti-inflammatory and immunosuppressive action in vivo. PMID- 7558510 TI - Effects of postirradiation carboxymethylglucan administration in mice. AB - The hemopoiesis-enhancing ability of a soluble glucan derivative, i.e. carboxymethylglucan (CMG), was investigated in gamma-irradiated mice. Attention was focused on the usefulness of its single or repeated postirradiation administration. CMG was administered i.p. at (a) single dose of 6 mg 2 h postirradiation, (b) four 6 mg doses in the first 4 days postirradiation, (c) four 1.5 mg doses at the same time intervals. Indices of granulopoiesis and inflammatory side effects (liver weight increase and hepatic granulomas) were investigated in mice irradiated with a sublethal dose of 7 Gy. All three CMG treated groups of mice were found to exhibit enhanced hemopoietic recovery in comparison with the controls. Although the mice repeatedly given the 6 mg CMG doses showed the most rapid recoveries of all the evaluated parameters of granulopoiesis, the most pronounced hepatic side effects were found in these mice, too. When survival of mice was recorded in lethally (9 Gy) irradiated animals, the best protective response were obtained following the repeated administration of the 1.5 mg CMG dose, the survival by day 30 in this group being significantly higher not only in comparison with the controls but also with the mice repeatedly given the 6 mg dose of CMG. The results suggest that the postirradiation CMG administration can be useful for enhancing radiation suppressed hemopoiesis. However, repeated larger CMG doses may produce side effects which compromise the overall survival of irradiated mice. PMID- 7558511 TI - The effect of organic acids on phytohaemagglutinin-activated proliferation of human lymphocytes in vitro. AB - The present study assesses the effects of 25 organic acids on in vitro proliferation of human peripheral lymphocyte stimulated with phytohaemagglutinin (PHA). Lymphocytes were cultured in flat-bottomed 96-well microplates at 37 degrees C for 96 h in the presence of the mitogen and of one acid. The concentrations of organic acids tested in the cultures were from 1 to 5 mM, corresponding to those usually found in the blood of patients with organic acidaemias. Cellular growth was measured by the incorporation of 6[3H]-thymidine into cellular DNA. We observed that tiglic (2-methylcrotonic), alpha-keto-beta methylvaleric, aminoadipic, sebacic and alpha-ketoisocaproic acids strongly inhibited lymphocyte DNA synthesis, whereas alpha-ketoisovaleric, propionic, alpha-hydroxy-beta-methylvaleric, alpha-methylbutyric and isobutyric acids moderately suppressed DNA synthesis. Lactic and ethylmalonic acids, however, stimulated DNA synthesis. The most inhibitory acids were added to cultures at different times after the beginning of the incubation period. Except for tiglic acid, whose action persisted even after 48 h from the onset of cultures, the others acted only when added during the first 24 h. The present study demonstrated that organic acids modulate DNA synthesis in mitogen-stimulated human lymphocytes. PMID- 7558512 TI - Nitric oxide production by chicken macrophages activated by Acemannan, a complex carbohydrate extracted from Aloe vera. AB - Cultures of normal chicken spleen cells and HD11 line cells produce nitric oxide (NO) in response to Acemannan, a complex carbohydrate derived from the Aloe vera plant. Neither cell type produced detectable amounts of NO in response to similar concentrations of yeast mannan, another complex carbohydrate. Nitric oxide production was dose dependent and inhibitable by the nitric oxide synthase inhibitor NG-methyl-L-arginine. In addition, the production of NO was inhibited by preincubation of ACM with concanavalin A in a dose-dependent manner. These results suggest that ACM-induced NO synthesis may be mediated through macrophage mannose receptors, and macrophage activation may be accountable for some of the immunomodulatory effects of ACM in chickens. PMID- 7558516 TI - Immunomodulating effects of the new anti-rheumatic drug tenidap on collagen induced arthritis. AB - We investigated the in vivo action of the newly developed anti-rheumatic agent tenidap, CP-66,248 (Pfizer Inc., New York), on arthritis in collagen-induced arthritic mice. The inhibitory effect of tenidap on the development of arthritis was statistically more significant than piroxicam. The serum anti-type II collagen antibody titer was markedly inhibited in the mice treated by tenidap. These results suggest that, unlike NSAIDs, tenidap inhibits the progress of collagen-induced arthritis through its immunomodulating effect. PMID- 7558513 TI - Different T-cell activation by streptozotocin and Freund's adjuvant in popliteal lymph node (PLN). AB - A popliteal lymph node (PLN) test was further validated for predictive screening of autoimmunity-inducing drugs. Autoimmune-like T-cell activation of streptozotocin (STZ) was compared with the effect of Freund's complete adjuvant (FCA), injected locally into the foot pad of BALB/c mice. Early cell activation in enlarged PLN was monitored by flow cytometry. Injection of both STZ and FCA markedly increased the absolute PLN cell number as well as specific T-helper (CD4+), T-suppressor/cytotoxic (CD8+), and B (Ig+) subsets. However, quantitative analysis of early T-cell activation revealed important differences between STZ induced PLN reaction and FCA-related lymphoproliferation. At 72 h, the number of cells stained with anti-early activation marker (EAM+; CD69+) increased over 10 times in STZ-enlarged nodes and only 3 times in the FCA-inflamed nodes. Furthermore, different cytometric profiles were noted for STZ-activated and FCA activated cells stained with anti-interleukin-2 receptor (IL-2R) (CD25+). The data suggest the applicability of early cytometric screening of enlarged PLN for predictive analysis detection of chemicals inducing an autoimmune-like reaction. PMID- 7558514 TI - Adrenocorticotropic hormone as a potential enhancer of T-lymphocyte function in the rat mixed lymphocyte reaction. AB - The effects of adrenocorticotropic hormone (ACTH) (10(-6) to 1 U/ml) on T lymphocyte proliferation and function were studied in the rat mixed lymphocyte reaction (MLR). ACTH produced a modest increase in lymphocyte proliferation on day 3 in lymph node (LN) cells and on day 5 in spleen cells. In addition, LN MLR cells, activated in the presence of ACTH, showed a higher proliferative response to restimulation on day 5 and on day 11 of the primary culture. Cytotoxic activity and the number of IL-2R- cells were increased in ACTH-treated LN MLR cultures in experiments where control MLR levels were low. ACTH also overcame the generation of low levels of suppressor activity in spleen MLR cells. These findings indicate that ACTH could play a role in increasing the priming of T lymphocytes and enhancing, in particular, suboptimal primary responses. PMID- 7558515 TI - Immunomodulatory activity of met-enkephalin and its two potent analogs. AB - The effects of Met-enkephalin (Met-Enk), a delta receptor binding opioid peptide, and its more stable synthetic analogs, Tyr-D-Ala-Gly-MePhe-Met-NHC3H7-iso (1), Tyr-D-Ala-Gly-MePhe-Gly-NHC3H7-iso (2) and Tyr-D-Ala-Gly-MePhe-Gly-NHCH2C6H5 (3), on human T-cell transformation and natural killer (NK) cell cytotoxicity have been evaluated. Analogs 1 and 2 have been found to be as potent as Met-Enk in stimulating T-cell transformation and augmenting NK cell cytotoxicity. Analog 3 had no effect on T-cell transformation and NK cell cytotoxicity. Proliferative response was measured by 3H-thymidine uptake after 5 days of incubation. The kinetics of the T-cell transformation response (peak 5th day) is similar to those for in vitro T-cell responses to specific antigens rather than via polyclonal activation. PMID- 7558517 TI - Inhibition of LPS-induced TNF alpha production in human monocytes by adenosine (A2) receptor selective agonists. AB - We examined the effects of adenosine A1 and A2 receptor agonists on LPS stimulated TNF alpha production by human monocytes isolated from peripheral blood. We have demonstrated that CGS-21680, a highly selective A2 agonist inhibited production of TNF alpha at the protein level by 75% whereas the A1 selective agonist N6 reduced TNF alpha production by only 25%. The action of CGS 21680 was mediated via the A2 receptors since its effect on TNF production was blocked by 3,7-dimethyl-1-propargylxanthine (DMPX) but not by xanthine amine cogener (XAC), antagonists selective for the A2 and A1 receptors, respectively. Thus intervention with A2-selective agonists or compounds that can elevate endogenously released adenosine may be beneficial in TNF alpha-mediated diseases. PMID- 7558518 TI - Muramyl dipeptide mimicry in the regulation of murine macrophage activation by serotonin. AB - Muramyl peptides (MPs) are regulators of macrophage function. That the activities of MPs may be mediated by serotonin (5-HT) is supported by earlier work that demonstrated specific binding sites for MPs on macrophages that competitively bind 5-HT. Both mediators were also shown to enhance the production of superoxide anion (an antibacterial agent) by these cells. We now report on two additional macrophage activation phenomena affected by 5-HT: phagocytosis and induction of tumor necrosis factor alpha (TNF) mRNA. Serotonin acts as a muramyl peptide-like agonist by increasing phagocytosis of tubercle bacilli by murine peritoneal macrophages, and as a partial agonist/antagonist in the induction of mRNA for tumor necrosis factor. These observations provide further evidence for a serotonergic involvement in some of the physiological responses to MPs. PMID- 7558520 TI - Prenatal alcohol exposure selectively suppresses cell-mediated but not humoral immune responsiveness. AB - The present study examined effects of fetal alcohol exposure (FAE) on the ability of peripubertal male and female rats to mount a humoral immune response against T cell-dependent as well as independent antigens. The appropriate pair-fed (PF) and control (C) cohort rats were included. Serum immunoglobulins (Ig) levels were determined following a primary or secondary immune response. In addition, plasma corticosterone levels were measured in conscious, freely moving FAE, PF and C rats following sensitization with the T-cell-dependent antigen sheep red blood cells (SRBC). The study demonstrates that, in response to primary or secondary immunization, serum Ig levels in FAE rats were not significantly different from those in the PF or C cohorts. On the other hand, a marked reduction in mitogen induced T-cell proliferative response was observed in FAE male rats in the same age group. Plasma corticosterone concentrations were increased almost four-fold 7 days after the primary immunization with SRBC, but there were no significant differences among the FAE, PF or C groups. Taken together, evidence from in vivo and in vitro studies indicates that FAE is associated with a selective impairment of cell-mediated immune function, including mitogen-induced T-cell proliferation, graft versus host as well as contact sensitization responses, but not of humoral immune function. PMID- 7558526 TI - Salivary cortisol and testosterone variations during an official and a simulated weight-lifting competition. AB - This study was undertaken to compare salivary levels of cortisol (C), testosterone (T) and T/C ratio during an official and a simulated weight-lifting competition. Thirteen top-level weight-lifters (French team) including 7 national level athletes (NG) and 6 international level athletes (IG) entered the study. The performance levels corrected with the IWF index (an index which relates the performance to body weight) obtained during the competition were higher than those obtained during the simulation and were higher in IG than in NG. Salivary samples were collected for each session after weighing, snatch and clean-and jerk. The mean T-levels of these athletes were the same as those obtained in the laboratory on sedentary adults; they did not vary throughout the competition, nor between the competition and the simulation. Conversely the C-levels were statistically higher in the competition than in the simulation (p < 0.05). These C-levels are correlated with the performance levels corrected with the IWF index obtained during the competition (r = 0.67; p < 0.05; n = 13). Moreover, C-levels noted at the time of the competition were higher in the international athletes than in the national ones. Therefore, we concluded that the cortisol level may be considered as a performance factor in weight-lifting. PMID- 7558519 TI - Decreased interleukin-2 receptor and cell cycle changes in murine lymphocytes exposed in vitro to low doses of cadmium chloride. AB - Relationships between in vitro cadmium-related cell cytotoxicity, ultrastructural changes and altered cell cycle were determined at 21-72 h after mitogenic stimulation of C57BL/6 mouse spleen lymphocytes with concanavalin A (Con A). Relatively low doses, 0.6-10 microM cadmium (Cd), added at 4 h after the mitogen activation, induced a significant cell cytotoxicity and reduced the lymphoblastic activity of the cells. Cytometric analysis of the lymphoid cell cycle at 72 h revealed that at concentrations > or = 0.6 microM Cd, the number of cells arrested in G0 + G1 phase increased, whereas the proportions of cells of the S and G2 + M phases were substantially reduced. Staining of cells with fluorescent anti-CD25 monoclonal antibody showed a cadmium-related decreased number and relative mean fluorescence of CD25+ cells, demonstrating a decreased level of interleukin-2 receptor (IL-2R). Furthermore, immunogold ultramicroscopic assay was developed for determination of intracellular interleukin-2 (IL-2) in cadmium treated lymphocytes. The level of cytoplasmic and nuclear IL-2, localized in situ by colloidal gold ultraimmunocytochemical technique, has been estimated as markedly decreased in cells treated with > or = 1.2 microM Cd, as compared with the untreated controls. Disorganization/fragmentation of mitochondrion cristae and dilatation of cisternae of the Golgi apparatus appeared as the major ultrastructural change in 1.2 microM Cd-treated lymphocytes. Interestingly, addition of cadmium in the incubation medium, up to 4 h after mitogen activation, also interacted with lymphoproliferative mechanisms of cells in G0 + G1, S and G2 + M phase. Overall, multiple ultrastructural changes of Cd-treated lymphoid cells were clearly related with the reduced cell viability and reduced number of activated lymphoblasts. PMID- 7558529 TI - Response to resistance training in young women and men. AB - Six women and 6 men trained the elbow flexors 3 days per week for 20 wks, one arm performing in each session 3-5 sets of 10 maximal concentric actions on an accommodating resistance device, the other arm 3-5 sets of 8-12 coupled eccentric/concentric actions on a weight training device. With results collapsed across the two training modes, the women made significantly (p < 0.05) greater relative increases than men in strength measured on the weight (116 vs. 46%) and accommodating (99 vs. 46%) resistance devices, and greater absolute (3.5 vs. -1.3 N.m) and relative (13.7 vs. -3.2%) increases in strength measured on an isokinetic dynamometer. Absolute (cm2) and relative (%) biceps, brachialis, and total elbow flexor cross-sectional area (from CT scans) increased significantly; however, the women's vs. men's respective relative and absolute increases did not differ significantly: biceps (13 vs. 7%, 0.9 vs. 1.0 cm2), brachialis (53 vs. 31%, 2.1 vs. 2.3 cm2), and total (26 vs. 15%, 3.1 vs. 3.3 cm2) flexor area. Biceps type I and II fiber area, and the II/I area ratio did not increase significantly. The data indicate that in response to the same short-term training program, muscle size increases similarly in women and men but women make greater relative increases in strength. PMID- 7558527 TI - Physiological responses of speed skaters to treadmill low walking and cycle ergometry. AB - Speed skaters have previously relied on cycle ergometry for physiological testing. Current evidence suggests skate-specific testing might be more appropriate. Unlike cycling, skating and off-ice low walk training involves a 'crouched' posture, placing the quadriceps in static contraction. This may compromise blood flow to working muscles and influence VO2. We compared physiological variables between skate-specific treadmill low walking (LW) and cycle ergometry (BK). Skaters (N = 8) performed LW and BK to fatigue in randomized order. No difference existed for peak HR. Peak VO2 was lower for LW (4.13 +/- 0.34 vs 4.43 +/- 0.35, p < 0.05). Peak VE was lower during LW (146 +/- 13 vs 180 +/- 31, p < 0.05). R was significantly lower for LW, although no difference in peak lactate (LA) was evident. At submaximal heart rates, VO2 was lower during LW (p < 0.05), and submaximal LA was higher when expressed as percent peak VO2 (p < 0.05). These results are consistent with the hypothesis that skating posture limits O2 delivery to the lower extremities, and thus accounts for a greater dependence upon anaerobic energy production. PMID- 7558525 TI - Training-induced protection and effect of terminated training on exercise-induced damage and water content in mouse skeletal muscles. AB - Muscle swelling in connection with training-induced protection against exercise induced damage and the disappearance of the protective adaptation after termination of training was studied in male NMRI-mice, aged 8 weeks at the beginning of the experiment. Mice were randomly assigned to several different treatment groups (training, training-exercise, exercise, detraining, detraining exercise, together with their respective controls). Training and prolonged exercise sessions were performed by running uphill (6 degrees) on a motor-driven treadmill. Muscle damage was estimated by the total activity of beta glucuronidase and water content from the red parts of m. quadriceps femoris (MQF), m. soleus (MS), m. rectus femoris (MRF), and from m. triceps brachii (MTB). Training-induced protection was observed in MS after only one week of training. In MQF the protection was incomplete after 1 and 4 weeks' training. In MS the protection disappeared already one week after the cessation of training. In MQF the training-induced protection persisted in part for one week but after four-week's detraining no protection was observed. The present results suggest that diminution of muscle swelling forms part of the adaptation of skeletal muscle in resisting exercise damage, and, vice versa, increased susceptibility to exercise damage, which follows the termination of training, is linked to muscle reswelling. PMID- 7558521 TI - The effects of different treadmill running programs on the muscle morphology of adult rats. AB - The biochemical adaptations of different muscle fiber types to endurance training of various intensities and durations have previously been investigated. The objective of this study was to determine the effects of two different endurance training programs on muscle fiber morphology. Twenty-four male Sprague-Dawley rats were randomly assigned to three groups: high intensity/low duration endurance trained (HILD), low intensity/high duration endurance trained (LIHD), or untrained (controls). Following the twelve week training period, muscle fibers of the gastrocnemius muscle of the rats were histochemically classified as type I, type IIa, or type IIb following myosin ATPase staining with pre-incubation at pH 4.6. Muscle fiber type distribution and cross-sectional areas were then determined. Neither HILD nor LIHD rats demonstrated significant differences in fiber type distribution compared to controls. When all fiber types were pooled together and analyzed, there were no differences between the three groups with respect to fiber size. However, when the three fiber types were analyzed individually, HILD animals demonstrated a significant reduction in the size of type IIa fibers while LIHD rats experienced a significant diminution in the size of type I and type IIb fibers. Thus, the morphological adaptations of the muscle fibers in the HILD and LIHD groups were fiber type specific. PMID- 7558528 TI - Effect of prolonged exercise at 65 and 80% of VO2max on running economy. AB - The purpose of this study was to investigate the effect of prolonged exercise at 65 and 80% of VO2max on running economy. Fourteen male long distance runners performed two 90-min runs on an outdoor 400 m track at velocities of 65 and 80% of VO2max. Prior to and following each 90-min run, running economy was measured as the steady-state VO2 during treadmill runs at speeds of 188 and 228 m/min. During the 90-min run at 65% of VO2max, the mean weight loss was 1.3 kg. HR increased from 143 bpm between minutes 5-10 to 150 bpm between minutes 85-90. During the 90-min run at 80% of VO2max, the mean weight loss was 1.4 kg. The HR was 161 bpm between minutes 5-10 and increased to 165 bpm between minutes 85-90. When the post running economy test was conducted following each 90-min run, there was a significant increase in VO2 expressed in both l/min and ml/kg min. The increase in VO2 following the 90-min run at 80% of VO2max. These results suggest that 90-min runs at 65 and 80% of VO2max alter running economy with a significant increase in oxygen cost. PMID- 7558531 TI - Immune function in athletes versus nonathletes. AB - The purpose of this study was to compare natural killer cell cytotoxic activity (NKCA) and Con A-induced lymphocyte proliferation (T cell function) in athletes versus nonathletes, with measurement of natural killer (NK) and T cells to allow a comparison on a "per-cell" adjusted basis. Eighteen young male endurance athletes (10 runners and 8 cyclists) with a mean VO2max of 70.7 +/- 1.3 ml.kg 1.min-1 and 6.6 +/- 0.8 years of competitive experience were compared with 11 nonathletic male adults (47.6 +/- 3.1 ml.kg-1.min-1). Concentrations of circulating leukocyte and lymphocyte subsets, including NK and T cells, were not significantly different between groups. NKCA and T cell function also did not differ between groups, whether expressed unadjusted or adjusted on a per-cell basis. For all subjects combined, both NKCA and T cell function were unrelated to VO2max (r = 0.005, p = 0.98; r = 0.007, p = 0.97, respectively). These data do not support the contention that immune function, as measured in this study, is altered in endurance athletes. PMID- 7558530 TI - The acute immune response to exhaustive resistance exercise. AB - Ten young male adults (mean age 46.9 +/- 1.2 yrs) with 9.2 +/- 1.4 years of weight training experience and the ability to parallel squat at least 1.5 times their body mass were selected as subjects. The exercise session consisted of sets of 10 repetitions at 65% 1-RM of the parallel leg squat, with a cadence of one rep every 6 sec and 3 min rest between sets, to muscular failure. The average subject lifted a total of 9711 +/- 1576 kg during 98 +/- 14 reps for a total work output of 72.5 +/- 10.5 kJ before muscular failure occurred. Mean oxygen consumption during exercise was 1.58 +/- 0.06 l/min at 42.5 +/- 2.0% peak VO2. A strong leukocytosis, lymphocytosis, and lymphocytopenia, similar to what has been reported following high-intensity cardiorespiratory exercise, were measured following leg squat exercise. Con A-stimulated lymphocyte proliferation (unadjusted) rose 50% above preexercise levels (p = 0.07), but when these data were adjusted on a per T cell (CD3+) basis, no change from rest was observed. Natural killer cell cytotoxic activity (NKCA), when adjusted on a per NK cell (CD56+) basis, was decreased about 40% below preexercise levels for at least 2 h post-exercise. No significant increase in cortisol was seen after exercise, although norepinephrine and epinephrine increased moderately (465% and 133%, respectively), immediately following exercise. The data demonstrate that leg squat exercise to muscular failure results in a very similar immune response to that associated with intense endurance exercise, despite a lower mean oxygen consumption and only a moderate hormonal response. PMID- 7558524 TI - Light concentric exercise and heavy eccentric muscle loading: effects on CK, MRI and markers of inflammation. AB - The consequences of a single bout of heavy eccentric exercise with and without repeated concentric exercises on MRI images, serum CK levels and markers of inflammation were studied. Two groups (ECC and ECCON), each consisting of 18 male volunteers, performed 70 eccentic contractions of the quadriceps femoris muscle. The study group (ECCON) performed additional concentric contractions on a dynamometer (Cybex II+) one day before and two hours, 1, 2, 3, 6 and 9 days after eccentric loading. Serum levels of creatine kinase (CK) were examined as a function of time, and correlated with measurements of magnetic resonance imaging (MRI) of the involved muscle groups. T2-weighted images of the thigh muscles were studied. Serum C-reactive protein, complement factors C3c and C4, haptoglobin and transferrin were measured as markers of inflammation. Additional concentric contractions (ECCON group) significantly increased CK, compared to the ECC group. However, it has no apparent effect on MRI signal intensity changes, which were of equal magnitude in the loaded vastus intermedius and deep parts of the vastus lateralis in both groups. Likewise, the serum markers of inflammation of the exercised muscles appeared to be absent. Based on MRI-images, additional concentric contractions had no statistically significant effect on muscle damage and breakdown of connective tissue. The five-fold increase in CK in the ECCON group could be a reflection of "massaging out" of the CK from the muscles into the circulation by additional concentric exercises. However, it could also be an indication for a superior sensitivity of assessing muscle fiber damage in comparison to the MRI. PMID- 7558533 TI - Inappropriate use of the correlation coefficient. PMID- 7558532 TI - Moderate exercise training and natural killer cell cytotoxic activity in breast cancer patients. AB - Sixteen female breast cancer patients who had been diagnosed (3.0 +/- 1.2 years previous to the study) and undergone surgery, chemotherapy, and/or radiation treatment were randomly assigned to exercise and nonexercise groups. Pre- and post-study measurements were taken for aerobic performance, leg strength, and concentrations of circulating lymphocyte subsets and natural killer cell cytotoxic activity (NKCA). Exercise training consisted of 60 minutes of supervised weight training and aerobic activity three times each week for eight weeks. Although subjects in the exercise groups demonstrated some modest improvement in the various aerobic and strength tests, NKCA and concentrations of circulating T and NK cells were not significantly altered relative to the nonexercise group. This study suggests that moderate exercise over an eight-week period has no significant effect on the function of in vitro natural killer cells in breast cancer patients. PMID- 7558522 TI - The effect of running on serum and red cell ferritin. A longitudinal comparison. AB - It is unclear whether running can affect iron stores. Results using the serum ferritin assay (SER FER) have been conflicting. Decreased red cell ferritin (RBC FER) values (< or = 4 ag/RBC) occur in iron depleted or inflammatory states. We compared the longitudinal changes of hemoglobin (Hb), SER FER, RBC FER, % saturation of total iron binding capacity (% sat TIBC), and daily dietary intake in 27 runners during a training program. These parameters were measured at days 0, 49 (range 48-52), and 115 (range 85-120). No significant changes occurred in the SER FER, % sat TIBC and Hb determinations throughout the study. Overall the RBC FER values trended down (mean values 11.7 ag/RBC to 7.7 ag/RBC; p = 0.06). Fifteen runners (56%) acquired RBC FER values in the iron deficient range (mean 6.8 ag/RBC to 2.4 ag/RBC; p < 0.05). These values differed significantly from the remaining 12 runners (mean 17.3 ag/RBC to 14.7 ag/RBC). The decline in RBC FER into the iron deficient range was primarily seen in a subset of runners who began with a RBC FER value < or = 10 ag/RBC (positive predictive value 0.79) and was independent of iron intake. We conclude that ferritin can be affected by running as recognized by the red cell ferritin assay. Moreover our results suggest that this decrease in red cell ferritin is likely a function of defective iron utilization rather than total body iron deficiency. A potential consideration is that this fall may occur as a result of repetitive running-associated injury and inflammation. PMID- 7558535 TI - Cellular basis for gender-dependent differences in growth hormone secretion in young chickens: analysis using reverse hemolytic plaque assays. AB - In young broiler chickens, males exhibit a greater body weight than females. This gender difference in body weight coincides with higher serum growth hormone (GH) concentrations in young males than in juvenile females. The cellular basis for this gender-dependent difference in GH secretion was addressed in the present study. Anterior pituitary cells from 4- to 5-week-old male and female broiler chickens were subjected to reverse hemolytic plaque assays for GH. The percentage of cells that formed GH plaques and average areas of plaques formed were compared between the genders. Maximal plaque formation was found to occur by 4 h of incubation regardless of sex or treatment. Data analysis identified a significant (p < 0.05) gender by treatment interaction. In the presence of GHRH, there was an increased percentage in plaque formation in males (p < 0.05), but GHRH did not affect the percentage of GH secreting cells in cultures from females (p = 0.26). Males were found to have a higher (p < 0.05; n = 6) percentage of somatotrophs than females (e.g. 20.8 +/- 1.2% vs. 13.2 +/- 2.6% of all cells, respectively, after 4 h of detection in the presence of GHRH). Moreover, high levels of GH Releasing Hormone induced formation of 1.5-fold larger (p < 0.01) plaques around somatotrophs derived from males than those derived from females. The data presented here suggest that gender-dependent differences in GH secretion in young broiler chickens result from differences in the percentage of pituitary cells that release GH, the relative secretory capacity of each individual somatotroph and the responsiveness of the somatotroph populations to GHRH. PMID- 7558536 TI - Effect of somatotropin on rate and composition of gain and feed intake in young, pituitary intact female rats. AB - The effects of recombinant porcine somatotropin on rate and composition of gain in the carcass, and feed intake, and the consequences of somatotropin treatment withdrawal in young, growing, pituitary intact rats were investigated. Female Sprague-Dawley rats were assigned to one of two groups for a 21 day treatment phase, and a 14 day withdrawal phase: 1) Control, saline injected; 2) Somatotropin, 2 mg porcine somatotropin/d. During the 21 day treatment phase, somatotropin increased rate of body weight gain (46%), average daily feed intake (11%), and feed conversion efficiency (30%), relative to the control group. Upon withdrawal of somatotropin, there was a reversal of these effects relative to the treatment phase and to the controls. Somatotropin increased carcass protein accretion rate by 82% during the treatment phase. Upon cessation of treatment, protein accretion was decreased by 67% relative to the control group. The rates of carcass lipid accretion (g/d) were increased 44% over the control group during the treatment phase, but were decreased (49%) upon withdrawal. This data demonstrates that somatotropin is able to stimulate greater than normal growth in rapidly growing young female rats, but upon withdrawal of exogenous somatotropin, many of the benefits are not sustained. PMID- 7558538 TI - Small angle dislocations of the newt limb axes can test the validity of several regeneration models. AB - We describe two experiments on the regenerating forelimbs of the urodele Triturus cristatus. In the first, a contralateral grafting is performed where the anteroposterior axis of the regenerating blastema coincides with the dorsoventral axis of the host stump. In the second, the regenerating blastema is ipsilaterally rotated on the stump at angles 90 degrees or 270 degrees. For these experimental setups several regeneration models, each based on different reference frames (cartesian versus polar coordinates), have diverging predictions for the resulting supernumerary outgrowths. We have analyzed these outgrowths morphologically and histologically and we conclude that both experiments are well described by a hierarchical extension of the Polar Coordinate Model. PMID- 7558539 TI - Age-related changes in D-aspartic acid of rat teeth. AB - In addition to L-aspartic acid, D-aspartic acid was detected in the protein (collagen) of rat teeth. The content of D-aspartic acid in rat molars increased significantly with age, showing a close correlation with age (r = 0.965-0.993), whereas in rat incisors D-aspartic acid did not increase. This suggests that there is little metabolic activity in rat molars after their formation. The amount of D-aspartic acid present in molars of a single rat was in the order first molars > second molars > third molars. This indicates that the amount of D aspartic acid is higher in teeth formed earlier, corresponding to the time of their formation. The rate of racemization in rat molars was about ten-fold higher than that in human dentin, suggesting the importance of body temperature in the rate of racemization. These findings also suggest that D-aspartic acid is present in the teeth of other animals. PMID- 7558537 TI - Genotype and sexual influences on growth and muscle development of chicken embryos. AB - The influence of sex and genotype on growth of chick embryos was studied using eggs from commercial broiler hens mated with broiler strain males (BrBr) or with bantam males (BaBr) and using eggs from bantam females mated to bantam males (BaBa) or to broiler strain males (BrBa). Male BrBr embryos were first significantly heavier than females at 9 days of incubation. The influence of sire's genotype was expressed between 6 and 16 days of incubation but not at 20 days. Dam's genotype, and the associated difference in egg weight, significantly affected embryo weight by 12 days of incubation and by 20 days it alone influenced embryo weight. Plasma insulin-like growth factor-I (IGF-I) levels of BrBr and BaBr embryos did not differ at 8, 10, 12, or 14 days of incubation, nor were there sex differences. There were no genotype differences in whole body protein concentration, DNA concentration or protein:DNA ratios at 4 or 8 days of incubation. At 12 days the pectoralis major DNA concentration of BaBa embryos was significantly higher than that of all other groups. Pectoralis muscle protein concentration of BrBa embryos was significantly lower than those of BaBr embryos at 12 days of incubation, but by 16 days it was highest. The protein:DNA ratios of embryos developing in bantam eggs were significantly lower than those in broiler eggs at 12 days. In summary, genotypic and sex differences in body weight and muscularity that develop after hatching appear to be predetermined by differences in embryonic growth patterns, but there is no clear relationship between embryonic growth, plasma IGF-I concentration or the concentration of DNA or protein in pectoralis muscle. PMID- 7558523 TI - Circulating gastrointestinal hormone changes in marathon running. AB - The purpose of this study was to observe the effect of marathon running on the release of gastrointestinal hormones and whether these might be related to gastrointestinal disturbances in marathon runners. Vasoactive intestinal polypeptide, gastrin, secretin, pancreatic polypeptide, neurokinin A, pancreastatin, insulin and glucagon-like peptide 1 were measured before, immediately upon finishing and 30 min after the race. Twenty-six competitors of the 1992 Belfast Marathon volunteered for this study. They had a mean age of 37 years and a mean finishing time of 239 min. Eight of the subjects complained of gastrointestinal distress during the race. The circulating concentration of all the GI hormones measured, except insulin were significantly elevated after the race. There was no significant change in glucose levels at the finish of the race. Statistical analysis revealed no direct relationship between the large increases in hormone levels and the occurrence of GI symptoms. These results show that GI hormone concentrations are affected by marathon running. Mechanisms of release and possible roles are discussed. PMID- 7558534 TI - Food restriction and lysine supplementation alter growth, RNA, DNA, and protein contents of skeletal muscle. AB - Efficacy of supplementing total protein or a limiting amino acid to maintain muscle development during food restriction was examined in growing rats. Male rats weighing 108 +/- 8 g were assigned to one of five diet groups plus an initial group. Animals were fed either a wheat gluten-based diet or the wheat gluten-based diet supplemented with adequate levels of lysine. These diets were fed ad libitum or at a 75% restricted level. One restricted group was fed a high gluten diet designed to meet lysine requirements but at the restricted energy level. Rats were fed these diets for 6 weeks. Lysine supplementation resulted in higher levels of protein, RNA, and DNA in skeletal muscle and liver of animals fed ad libitum. Food restriction resulted in loss of protein and RNA from liver and skeletal muscle and lower ratios of protein/DNA. Initial DNA contents of plantaris and soleus muscles were not affected by food restriction; however, hepatic DNA was reduced. Supplementation of lysine to animals restricted in food intake failed to improve growth of skeletal muscle or liver, and resulted in lower protein contents in liver (11%), plantaris (6%), and soleus (38%). Increasing total protein intake for the animals with the restricted intake resulted in a higher RNA/DNA ratio without a parallel increase in protein in liver or muscle. This study demonstrates that during severe food restriction skeletal muscle DNA is preserved which maintains high potential for growth recovery. This study also indicates that during severe, prolonged food restriction supplementation of protein or limiting amino acids results in lower tissue protein contents. PMID- 7558541 TI - Essays in honour of Professor Walter W Holland. PMID- 7558540 TI - Decreased birth weight and attainment of postnatal catch-up growth in offspring of rats exposed to cigarette smoke during gestation. AB - Pregnant rats were exposed to cigarette smoke daily for a 2-hour period throughout gestation and the body weights of offspring were monitored for 5 weeks postnatally. The average birth weight of the cigarette smoke exposed pups was significantly smaller compared to both pair-fed and ad libitum control groups. However, the body weights of the pups exposed to smoke were no longer significantly different from those in the pair-fed and ad libitum control groups at weeks 1 and 2 after birth, respectively. This study indicates that fetal growth retardation caused by exposure to cigarette smoke during pregnancy does not persist after birth. PMID- 7558542 TI - Ways of seeing: explaining variations in use of acute hospital services. AB - BACKGROUND: In the US Medicare programme, hospitals are paid directly by activity. To provide incentives for efficiency, the US Federal Government has sought objective measures of inpatients' need for resources. In the UK National Health Service, resources are allocated for acute hospital services as part of a global budget to purchasers, who then contract with hospitals. To provide equity in resource allocation, the Department of Health in England, has sought objective measures of populations' need for resources. METHODS: Examination of policy and technology that has used variations in utilization of resources to derive objective measures of efficiency and equity. RESULTS: The technology of developing empirical measures of resources needed by patients has lacked information on outcomes, which is vital for measures of efficiency. The technology of developing empirical measures of resources needed by populations has relied on aggregate data. Analyses of specific procedures and conditions consistently find that these variations are explained by differences in medical practice and not by need. CONCLUSIONS: There is scope for multidisciplinary research to explain small area variations for specific procedures and conditions in resources used by populations. It seems unlikely, however, that governments will be interested in findings from this research. PMID- 7558543 TI - The contribution of epidemiology to the definition of health goals and targets for Australia. AB - Health goals and targets may be used to improve the efficiency of investment of resources for health care and the promotion of health the better to fulfil the values and expectations of society. Australia has been developing health goals and targets since 1985. The first step was the establishment of the Better Health Commission that year. Its proposals led to the states and territories undertaking concerted health promotion activities through the National Better Health Program. Subsequently the health-care system as a whole, through the 1993 Medicare agreement, endorsed the goals and targets approach. Epidemiology has played a major role in every step in this process. Initially it was used to establish health status baselines or to point to serious data deficits which stopped a baseline being set. It provided data about the burden and distribution of illness in the community. It was used critically to appraise evidence about aetiology and the fraction of illness attributable to modifiable risk factors and the effectiveness of interventions, and to provide estimates of potential years of life lost through illness and injury. This has contributed, along with economic analysis which is a critically important corollary, to the definition of health care priorities. Epidemiology, while being necessary for these developments, however, has not been sufficient. This 'insufficiency' has three elements. First, existing epidemiological knowledge and methods have been inadequately applied or the data have been missing, or distorted by inappropriate interpretation. Second, there are now goals and targets in health literacy, the health care system, and the environment where epidemiological methods (and hence data) are poorly developed or non-existent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558544 TI - The Multifund and outcome research. AB - The UK health reforms, among other things, have allowed some family practitioners ('fundholders') to control part of the budget used to purchase secondary care for the patients registered with their practices. Some feared that this change in organization might threaten the equity of the National Health Service (NHS) by giving advantages to the patients of fundholding general practitioners (GPs). This has led to the formation of the first large GP 'Multifund' in the UK. Whilst the first small fundholding co-operatives were conceived simply as a way of improving the business efficiency of ordinary fundholding, the much larger Kingston and Richmond (K&R) Multifund is a professional co-operative of fundholding family practitioners who share a common management allowance, which controls pounds 32.6 million per annum, about 20% of the public funds devoted to health care in its locality, and has a broader agenda. It sets out to end any two tier system discriminating between the patients of fundholding and other practitioners within a locality by making it possible for all GPs to become fundholders within an open, democratic doctors' co-operative. Its declared aims are to maintain equity and clinical integrity, to plan health care and to undertake scientific evaluation. Its overall goal is to preserve and improve upon the economic and ethical success of the British NHS. A brief evaluation is included which suggests that Multifund members believe their organization is currently meeting most of these aims. The essay argues for GP-led Multifund systems centred on outcome evidence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558547 TI - Interpreting excess mortality in a prevention trial for older adults. AB - BACKGROUND: In a randomized trial evaluating preventive services for older adults excess mortality was observed in the treatment group. We examined four explanations: unbalance of baseline characteristics, unintended effects of the intervention, consequence of an autonomy intervention (including increased number of living wills in the treatment group), and chance. We focus here on the effects of the autonomy intervention. METHODS: Preparation of living wills in the treatment and control groups was compared both at baseline and follow-up. A linear predictor of mortality was used to identify participants at high risk of dying. Charts of these 200 participants were reviewed for evidence of serious medical events and resuscitation decisions. Rates of life-sustaining treatment were compared between treatment and controls using logistic regression. RESULTS: More living wills (65%) were noted for the treatment group than control group (47%) at follow-up. Thirty-six per cent of participants were identified as having a serious medical event; of these, participants in the treatment group were over twice as likely not to receive life-sustaining treatment. CONCLUSIONS: Advance directives contributed to excess deaths, indicating the success of the autonomy intervention. PMID- 7558545 TI - An investigation assessing the fraction of human immunodeficiency virus infection attributable to ethnic group variations in risk. AB - Considerable speculation and a recent publication has suggested that much of the human immunodeficiency virus (HIV-1) infection in the subset of the UK population represented by women having babies in Metropolitan London is substantially imported from Africa. Other data are supportive of this hypothesis. Ades et al. (1992) concluded that the fraction of HIV seroprevalence, identified in a large London-based study using neonatal blood from the dried blood spots on Guthrie cards, attributable to mothers born in Africa was 92.4% with 95% CI: 82-100%. This is an important observation which requires confirmation. This paper reports on an attempt to do this with closely similar methodology using the Unlinked Anonymous Survey (UAS) of HIV-1 seroprevalence in pregnant women attending antenatal centres, together with data from the Office of Population Censuses and Surveys (OPCS) on the country of origins of mothers delivering in 1991 at the 15 London centres included in the UAS. As in Ades' analysis there appeared to be a strong association between centre prevalences and the proportions of women of African origin delivering at those centres. This was consistent with a high fraction of seroprevalence attributable to such women, but it was also clear that the assumptions required for the estimation of the attributable fractions were not fully met. Analyses modified to justify the assumptions produced attributable fraction estimates ranging from 44% to 96% with lower 95% confidence limits 20% or less.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558548 TI - Walter W Holland. PMID- 7558549 TI - Outbreak investigation: the need for 'quick and clean' epidemiology. AB - Epidemiology investigations of outbreaks of infectious disease have to be carried out rapidly but must be methodologically sound. In 14 of 25 consecutive Salmonella outbreaks in Wales from 1986 to 1990 case-control or cohort studies were undertaken. Food vehicles of infection were identified by statistically significant associations with cases in 13 of the 14, including three outbreaks where there were less than 10 cases. The particular problems of epidemiological field investigations are discussed. PMID- 7558546 TI - Infant feeding and mental and motor development at 18 months of age in first born singletons. AB - OBJECTIVE: To determine the relationship between type of infant feeding and mental and psychomotor development at age 18 months. METHOD: A follow-up study of children born to primigravidae living in Dundee and booked into antenatal clinics in the City of Dundee (Local Authority District) from 1 May 1985 to 30 April 1986. The study population was 846 first born singletons, of whom 592 attended for developmental assessment at age 18 months. The main outcome measures were the Bayley Scales of Infant Mental and Motor Development. RESULTS: Higher mental development was significantly related to breast feeding on discharge from hospital and according to the health visitors' notes at about 2 weeks after discharge after allowing for partner's social class, mother's education, height, alcohol and cigarette consumption; placental weight and the child's sex, birth weight and gestational age at birth. After adjustment for statistically significant variables, the difference in Bayley mental development index between breast and bottle fed infants was between 3.7 and 5.7 units depending on the source of feeding data. No differences were found for psychomotor development or behaviour. CONCLUSION: The study provides further evidence of a robust statistical association between type of feeding and child intelligence. However, the literature is replete with suggestions for potential confounding variables which offer alternative causal explanations. To unravel what is an important clinical and public health question, further research should concentrate on randomized trials of supplemented formula feeds for children of mothers opting for bottle feeding and on epidemiological studies designed to disentangle the relation between method of feeding, parental intelligence and social environment. PMID- 7558550 TI - Early detection of cutaneous malignant melanoma in Britain. AB - A multidisciplinary approach to research has been one of the hallmarks of Walter Holland's outstanding career in medical research. Studies arising from evaluation of the Cancer Research Campaign's health education programme for the early detection of cutaneous malignant melanoma were reviewed at Walter's Festschrift because they illustrate the importance of the multidisciplinary approach and raise issues about medical screening which has been a special interest of Walter's The programme took place in 1987 in seven areas of England and Scotland with a target population of 3.6 million. The main aim of the evaluation study is to investigate the effect of the programme on mortality from melanoma. Interim effects on awareness about melanoma, workload and incidence rates of melanoma by stage are reviewed. The importance of collaboration between dermatologists, epidemiologists, histopathologists and health education specialists has been essential. However, the effectiveness of high profile campaigns alone to promote early detection of melanoma is uncertain and future initiatives should be evaluated in randomized controlled trials. PMID- 7558551 TI - Trends and patterns in suicide in England and Wales. AB - BACKGROUND: Suicides among young men have been rising since the early 1970s while at the same time the rates for women of all ages and for men over age 44 been falling. Byu 1992 the highest risk group was men aged 25-44. In all age groups female rates have declined relative to those for men. METHODS: A case-control analysis was undertaken comparing suicide death with deaths from natural causes fo the years 1990-1992, based on data collected on the death certificate, and 1991 census data relating to the ward of residence of the person who died. Data for men and women aged 16-64 were analysed separately, with allowance for the fact that the effects of the factors analysed could be different above and 45. RESULTS: For men, different models were necessary for the two age groups, but or women a single model sufficed. For men and women occupations with access to effective methods of suicide, such as veterinarians, medical practitioners, nurses and pharmacists had much higher risks than other professions. Being widowed/divorced or marital status not stated was also an important risk factor, as was being single for men aged 45 and over and women of all ages. Country of birth was also significantly related to suicide risk. For older men, living in a ward with unemployment rates below 5% was a risk factor, as was living in a ward with high own occupancy rates. Younger men living in wards with owner occupancy rates below 55% were at reduced risk. For women owner occupancy and unemployment rates had little association with suicide rates.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558552 TI - Controlling non-insulin-dependent diabetes mellitus in developing countries. AB - The epidemiological transition has brought an increasing burden of chronic non communicable disorders to middle- and even low-income countries. This paper reviews the problem with particular reference to non-insulin-dependent diabetes mellitus (NIDDM) in the English-speaking Caribbean region. Surveys conducted over the last three decades have documented a high prevalence of NIDDM in a number of communities and evidence has accumulated to support the control of obesity and physical inactivity in the primary prevention of non-insulin-dependent diabetes. The problem of introducing and monitoring suitable interventions on a long-term basis in high-risk populations in different cultures has yet to be addressed. The impact of diabetes on health status in developing countries has not been well documented but it is clear that there are high levels of acute illness from disorders of glycaemic control, long-term disability from blindness and limb amputation and premature mortality from stroke, coronary heart disease and renal disease. Present evidence suggests that improving the quality of preventive clinical management can be the most immediately productive approach to controlling health problems from diabetes. Achieving this objective within the social, organizational and resource constraints of the Caribbean presents a range of problems. Identifying the most cost-effective means of improving existing services is therefore the most immediate research priority for NIDDM in the English-speaking countries of the Caribbean. PMID- 7558554 TI - Population laboratories and health needs assessment. AB - In the 1960s Walter Holland established a series of epidemiological studies in a defined geographical population in southeast London. This endeavour was an example of the ambition held by generations of epidemiologists to study human populations under actual conditions of community life. Early examples of American population laboratories were mainly concerned with studies of aetiology whereas p.c. (post-Cochrane) studies have aimed to identify both aetiological factors and to assess need for effective and efficient health services. The case is made for the value of a population laboratory in facilitating mortality follow-up studies, undertaking health care needs assessments and evaluating new technologies. PMID- 7558553 TI - Studies of death and disability from stroke: how can they effect change in service provision? AB - BACKGROUND: Although stroke is a major public health issue little is known about the need for stroke services or the most cost-effective methods of treating stroke patients. This paper discusses how stroke registry data can be used to inform and promote health service changes to meet these needs. METHODS AND RESULTS: Stroke registers were established in three districts of southern England. The initial study findings were that incidence rates reflected mortality rates in these districts, with the higher rates being observed in inner city districts. Hospitalization rates were high with significant use of health service resources but with only 5% being spent on rehabilitation. As a result of these findings a stroke steering group was established in the district with the highest incidence. Standards for care were developed and are currently being audited. A multidisciplinary rehabilitation team was set up in collaboration with the stroke registry. The local purchasers of health care have commissioned an evaluation of a community rehabilitation team to address the problem of low rehabilitation rates in the community. CONCLUSION: Although it is not possible to control for external influences within a health service this paper illustrates how research findings can influence local health service provision in a tangible way. Epidemiological research can only hope to effect such change if the original studies are undertaken rigorously and the researchers are proactive in the dissemination process, creating opportunities for further research to resolve questions raised by the studies. PMID- 7558555 TI - The National Study of Health and Growth (NSHG): 23 years on the road. AB - This paper aims to give a record of the National Study of Health and Growth (NSHG) in terms of structure, process, its scientific contribution, outcome measurements, and the commissioning and funding of the study. The NSHG is a nutritional monitoring system of primary school children. The study included three samples: representative English and Scottish samples, and an inner city sample. There were 56 study areas and 50% were surveyed every year. The study was started in 1972 and ran without interruption for 23 years. Measurements were taken for height, weight and skinfold thickness for more than 95% of the children and the response rate to the questionnaire was approximately 85%. This level of collaboration did not diminish over time. More than 80 papers have been published on the study. Results from the study have documented an increase of height and obesity in England and Scotland, and an increase in asthma. The NSHG has shown that research units in a university environment can marry the scientific and administrative tasks of a large survey carried out over a long-term period. PMID- 7558557 TI - Monitoring the growth of children: conclusions from a long-term study. AB - BACKGROUND: The National Study of Health and Growth (NSHG) was set up in 1972 to monitor the growth of primary school children. Areas were selected in England and Scotland by stratified random sampling. Schools within these areas were visited annually until 1982, biennially thereafter, resulting in a mixed longitudinal design. The reasons for the original design and the study as it has operated are reviewed, with advantages and disadvantages compared to the monitoring system now to be implemented by the Department of Health. METHODS: Description of the statistical and interpretive problems of monitoring rates of growth and a comparison of the response rates achieved in the two types of monitoring. RESULTS: Although the design of the NSHG was selected in order to monitor rates of growth this presents statistical problems. The usable information is contained in trends in attained height rather than in rates of growth. This study has achieved an average response rate of over 95%; less than 78% can be expected from the proposed survey of households. CONCLUSIONS: The small loss of efficiency of the mixed longitudinal design compared with repeated cross-sectional studies is more than compensated for by its high response rate and the comparability of data over time, neither of which can be guaranteed by the proposed survey. PMID- 7558556 TI - Comparing two methods of clinical measurement: a personal history. AB - Despite the fundamental importance of measurement in medicine, studies which compare methods of clinical measurement are often analysed inappropriately and may thus come to incorrect conclusions. We explain the problems with the main methods used, notably correlation and regression, and describe the limits of agreement approach which we have proposed. We consider why incorrect methods are so widely used and describe our own experience in bringing this issue to the attention of medical researchers. PMID- 7558559 TI - Health outcomes of oral disorders. AB - While there is a substantial body of data on patterns of dental disease in both adult and child populations, there is relatively little information concerning the consequences of oral disorders for well-being and the quality of life. In addition, dentistry lacks measures of health outcomes for use in dental health surveys and clinical trials. However, interest in this area is growing and a number of oral-condition specific health status measures have been developed over the last 10 years. The most sophisticated is the Oral Health Impact Profile (OHIP) which is being developed and tested by research teams in Australia, Canada and the US. This consists of 49 items organized into seven sub-scales which address the way in which oral conditions compromise functioning and social and psychological well-being. A study of older adults in Canada which used OHIP found that it had good measurement properties. Nevertheless, there was only a weak association between scores on this measure and clinical indicators of oral disease. Further analysis revealed that social factors were as important as clinical factors in explaining the health outcome of oral disorders. This finding is consistent with contemporary concepts of health and provides some evidence to support the view that the social context in which we live is important in shaping responses to disease and the experience of health and illness. PMID- 7558558 TI - The significance of ethnicity for health promotion: patients' use of anti hypertensive drugs in inner London. AB - Afro-Caribbeans are the second largest ethnic minority in the UK and are concentrated in some inner London areas where they comprise over 12% of the population. Standardized mortality ratios for stroke are relatively high among the Afro-Caribbeans, for whom the control of high blood pressure is thus of particular significance. Semi-structured interviews were conducted with matched groups of 'white' and Afro-Caribbean hypertensive patients attending 15 general practices in an inner London area to examine their beliefs and practices regarding the prescribed drugs. Adherence was high among 'white' patients, but less than half the Afro-Caribbeans took the drugs regularly as prescribed with many having poorly controlled blood pressures. Non-adherence was influenced by traditional cultural beliefs and practices which often strengthened concerns about the long-term harmful effects of drugs and provided an alternative resource in terms of herbal remedies. There was also evidence of a cultural gulf and lack of communication between Afro-Caribbean patients and their general practitioners, thus reducing the effectiveness of this preventive strategy. PMID- 7558560 TI - Using operational research modelling to improve the provision of health services: the case of DNA technology. AB - Operational research (OR) analysis provides information and systems to support decision making. In health care there are examples of OR being used to support decisions surrounding both the organization of health services and the treatment of individual patients. However, its uptake is currently low in spite of the increase in potential areas of application. In practice there is a lack of awareness amongst health service staff about what OR is and how it can help. This paper addresses this issue by exposing the contribution that OR made to a programme of research relating to DNA technology. Examples are given of the way it was used to provide information on the costs and outcomes of services and the ways these evolve over time. These examples demonstrate the way OR methods increase the understanding of both analysts and service providers about a problem area. This helps ensure that appropriate and valid approaches to tackling problem areas are developed. When developing these approaches, the problem orientated philosophy of OR means analysts are willing to use a range of methodologies, some originating in OR and some in other disciplines. The broad focus of OR also means that its findings enhance those provided by other disciplines which might seem to be competitors. The conclusion of the paper is that OR has a crucial role to play in the improvement of health services. PMID- 7558561 TI - Geographical resource allocation in the English National Health Service, 1971 1994: the tension between normative and empirical approaches. AB - The policy response to the problem of developing a geographical resource allocation formula sensitive to relative population needs for hospital and community health services resources in the National Health Service demonstrates a continuing tension between normative and empirical solutions. Since 1988, the balance has shifted in favour of a more empirical approach to identifying and weighting population needs indicators in response to concerns about the theoretically justified, but essentially approximate, nature of the Resource Allocation Working Party formula introduced in 1977-1978. However, judgements and assumptions about the nature of 'need' have still to be made in order to construct a usable resource allocation formula since empirical data on what is cannot provide a complete guide to what ought to be a fair distribution of resources in relation to need. PMID- 7558562 TI - Multiple protease activities in Giardia intestinalis trophozoites. AB - Azocasein and a wide range of chromogenic and fluorogenic peptidyl substrates, representing 21 different peptide sequences, were degraded by the intracellular proteases present in lysates of trophozoites of Giardia intestinalis (syn lamblia) Portland 1 strain. Total protease activity differed considerably with the substrate with the p-nitroanilide derivatives Bz-pro-phe-arg-pNA and Bz-phe val-arg-pNA being most rapidly hydrolysed (specific activities 4.2 +/- 0.4 and 1.1 +/- 0.6 U/mg protein, respectively). Activities were increased (16-72%) by addition of 1 mM dithiothreitol and were maximal, for the substrates monitored, in the range pH 5.5-7.0. These data and the inhibitor susceptibilities of the trophozoite proteases confirmed that the activity was predominantly due to cysteine proteases, although the presence of some serine protease, aspartic protease and aminopeptidase activity in the lysates was also indicated. The multiplicity of the protease activities was confirmed by gelatin-polyacrylamide electrophoretic analysis. Eighteen proteolytic activities with Mr values in the range 30,000 to > 211,000 were detected. These gelatinase activities were enhanced by dithiothreitol, were maximal at or close to pH 6, and were inhibited by cysteine proteinase and some serine proteinase inhibitors. Four of the proteases present in the gels exhibited activity towards fluorogenic amidomethylcoumarin peptides, but with different substrate preferences. The results show that G. intestinalis contains multiple proteases, many of which are of the cysteine type. PMID- 7558564 TI - Opisthorchis viverrini: the tegumental cytoskeleton. AB - The tegumental cytoskeleton of Opisthorchis viverrini was observed using both conventional transmission electron microscopy and Triton X-100 extraction. The cytoskeletal elements of the newly excysted juveniles, first-week and adult stages are composed of 2 components: firstly, the network of knobbed fibres designated as microtrabeculae which form the principal scaffold of the cytoplasm; and secondly, the microtubules. The microtrabeculae are more densely packed in the newly excysted juveniles and become less densely packed later in the first week and adult stages. Generally, their compactness in the tegument of each stage is higher in the apical and middle zones than in the basal zone. The results from extraction by Triton X-100 suggest that the microtrabeculae may be composed, at the primary level, of thin and straight fibres, partly coiled up to form knobbed fibres, which are highly cross-linked at the secondary level. At the tertiary level, these knobbed fibres may be coiled up further and form closely aggregated globules that appear as dense dots in cross-section. Most microtubules are confined within the tegumental cells' processes and splay out in the basal zone of the tegument. In addition, there are condensed laminae of cytoplasm with intermittent dense plaques underlining the outer membrane, with microtrabecular fibres inserting into them. This organization may help to stabilize the outer membrane and preserve the surface contour. Along the inner membrane of the tegument, there are hemidesmosomes distributed at regular intervals, with fine fibres radiating out from them to intertwine with the microtrabecular network, which may help to anchor the tegument to the basal lamina. Spines, which exist mainly in the newly excysted juveniles, appear as a crystalline lattice structure whose bases are firmly fused to the inner membrane. PMID- 7558563 TI - Transhydrogenase activities and malate dismutation linked to fumarate reductase system in the filarial parasite Setaria digitata. AB - Setaria digitata, a cattle filarial parasite, similar to human filarial parasites, possesses significant activities of the 4 transhydrogenases namely NADH-NAD+, NADPH-NAD+, NADH-NADP+, and NADPH-NADP+ in the sonicated mitochondria like particles. The transhydrogenases appear to regulate the metabolic pathways of the parasite in response to the presence of adenyl nucleotides and are non energy linked. Observations on the transhydrogenase and fumarate reductase activities show the existence of a protein bound NAD in the MLP and a linkage between the fumarate reductase system and malic enzyme through transhydrogenases. The malate dismutation reaction is the result of malic and fumarase enzyme activities. Fumarase and fumarate reductase activities result in succinate formation under anaerobic conditions showing major energy production at the fumarate reductase site. The existence of acetate kinase, phosphotransacetylase, pyruvate carboxylase, propionyl CoA carboxylase and CoA transferase enzymes in the mitochondrial system shows the presence of other energy producing sites in the parasite. The transhydrogenase system, NAD+/NADP+ malic enzyme, fumarase and fumarate reductase are the key enzymes of, production of reducing power for synthetic reactions and regulation of oxidative and reductive stages of the mitochondrial system. Hence, specific drugs targeted against this interconnected complex enzyme system, will be very effective in the control of filarial parasites. PMID- 7558565 TI - A study of the composition of the sclerites of Gyrodactylus Nordmann, 1832 (Monogenea) using X-ray elemental analysis. AB - The elemental compositions of the hamuli, marginal hooks and ventral bars were examined following their release from the opisthaptor of the monogenean parasites Gyrodactylus salaris, G. caledoniensis and G. colemanensis. Their compositions were determined using X-ray elemental analysis on an analytical scanning electron microscope, and the variation between each species investigated. Hamuli and marginal hooks exhibited a major peak for sulphur, whilst the ventral bar had high peaks of both sulphur and calcium. The high sulphur content and structure of the hamuli suggest they are composed of a keratin-like protein, while the nature of the ventral bar is less clear. The hamuli of G. colemanensis were differentiated from those of G. caledoniensis by virtue of their calcium fraction. Low levels of several other elements, including vanadium, were also detected in some sclerites. The composition of the hooks of Gyrodactylus and their relationship to the sclerites of monopisthocotyleans and polyopisthocotyleans is commented upon. PMID- 7558566 TI - Transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in Uruguay. AB - A base-line survey was carried out on the transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in the Department of Florida, Uruguay. Mean life expectancy at birth of the sheep population in this Department was estimated at 3.5 and 4.8 years for male and female sheep, respectively. Both intensity and prevalence of E. granulosus infection increased with age providing no evidence that immunity was acquired in the population. This revealed that E. granulosus was only relatively stable and in an equilibrium endemic steady state with an estimate of the basic reproduction ratio (Ro) of about 1.2. The mature larval population in sheep was over-dispersed with parameter k, from fitting a negative binomial, being 0.199 and 0.556 in 42- and 54-month-old sheep, respectively. The mean number of cysts increased by 1.05 cysts per year and the infection pressure was calculated as 0.174 infections per year, implying that each infection consisted of 693.6 eggs to produce 6.03 cysts. Neither intensity nor prevalence of infection with T. hydatigena or T. ovis increased with age. This indicated that immunity was acquired to reinfection by both parasites and they were in an equilibrium hyperenedemic steady state with an estimated Ro of 4.2-5.8. Viability and fertility of E. granulosus, and viability of T. hydatigena cysts in the older age groups were low. The effects of the different transmission levels of the parasites on potential control strategies are discussed. PMID- 7558568 TI - Genetic factors affecting antibody responses to four species of nematode parasite in Romney ewe lambs. AB - Serum levels of antibodies (Ab) and immunoglobulin G1 (IgG1) to the larval (L3) stage of the internal parasites Cooperia curticei and Trichostrongylus colubriformis and levels of Ab to the L3 stages of Haemonchus contortus and Ostertagia circumcincta were determined in 1432 Romney ewe lambs which were born on one farm in 1990 and 1991 and were the progeny of 63 rams. The objectives were to estimate heritabilities of, and genetic correlations among, the serum concentrations in newly weaned lambs under commercial conditions and to estimate genetic correlations of Ab and IgG1 with production traits. Lambs were exposed to a natural parasite challenge on pasture, following an anthelmintic drench at weaning. Blood and faecal samples from 4- to 6-month-old lambs were then taken when the mean faecal nematode egg count of a monitor group reached 800-1500 eggs g-1. Heritabilities for the serum levels of the four Abs ranged from 0.25 +/- 0.05 to 0.37 +/- 0.06. Heritabilities for the level of IgG1 developed against C. curticei was 0.19 +/- 0.04 and against T. colubriformis, 0.18 +/- 0.05. Genetic correlations between Abs for the 4 species were high, averaging 0.84, and between the two IgG1s it was 0.82. The genetic correlations between Ab or IgG1 levels and weight or gain traits were negative (for the 6 significant values out of 18), with yearling fleece weight positive (for the 2 significant values out of 6), whilst those with loge faecal egg count were all negative (average 0.15).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558567 TI - An ovine major histocompatibility complex DRB1 allele is associated with low faecal egg counts following natural, predominantly Ostertagia circumcincta infection. AB - Infection with Ostertagia circumcincta is a major constraint on sheep production in temperate areas of the world. A potential control strategy is the use of genetically resistant sheep. Therefore we examined the association between MHC DRB1 alleles and faecal egg counts following natural, predominately O. circumcincta infection in a flock of Scottish Blackface sheep. Nineteen DRB1 alleles were identified by a combination of variation in the length of simple repetitive sequences within the intron between exons 2 and 3 and hybridisation of selected oligonucleotides to polymorphisms within exon 2. Faecal samples were taken from 200 lambs from one to six months of age at intervals of 4 weeks. Genetic effects were strongest at 6 months of age. Least-squares analysis indicated that substitution of the most common allele (I) by allele G2 would result in a 58-fold reduction in faecal egg counts in 6-month-old lambs and a 22 fold reduction in 5-month-old lambs. These results suggest that the major histocompatibility complex plays an important role in the development of resistance to O. circumcincta. PMID- 7558572 TI - Attempted immunisation of sheep against Fasciola hepatica using gamma-irradiated metacercariae. AB - The potential of gamma-irradiated Fasciola hepatica metacercariae to vaccinate sheep against fascioliasis was examined. The effect of the size of the inocula of irradiated metacercariae and the level of gamma-irradiation on the recovery of non-irradiated fluke was assessed following homologous challenge. Groups of Merino wethers were vaccinated with a single infection of either 500 or 2000 metacercariae, previously exposed to either 30, 100 or 400 Gy of gamma irradiation. No significant reduction of fluke burdens were observed in any group, although a nonsignificant 20% reduction was observed in sheep vaccinated with 2000 metacercariae irradiated with 100 Gy. A second trial was conducted in which groups of sheep were vaccinated with 2 doses, given 4 weeks apart, of 2000 metacercariae, previously irradiated at either 70, 100 or 150 Gy. In both trials parasite viability was severely affected by doses of gamma-irradiation of 30 Gy or greater and no mature flukes were recovered from control sheep given metacercariae attenuated with 70 Gy or greater. A strong humoral immune response to somatic F. hepatica antigens was observed in all sheep. Only sera from sheep receiving 70 Gy irradiated metacercariae recognised the 2 candidate liver fluke vaccine molecules, F. hepatica glutathione S-transferase and cathepsin-L proteases. No reduction was observed in either the number of flukes or the production of fluke eggs in any vaccinated group. Vaccination appeared to affect the development of the challenge fluke population, resulting in reduced hepatic damage during migration, as measured by levels of serum glutamate dehydrogenase, and an increase in mean fluke weight. PMID- 7558569 TI - Immunisation of guinea pigs against Dictyocaulus viviparus using adult ES products enriched for acetylcholinesterases. AB - The adult ES products of Dictyocaulus viviparus are a source of protective antigens against challenge in the guinea pig laboratory model. High levels of acetylcholinesterase (AChE) activity are present in these products and these enzymes are immunogenic in infected cattle. Here, the potential role of these enzymes in protective immunity was investigated using a fraction enriched for AChE to immunise guinea pigs. The antibody response stimulated by immunisation with AChE-enriched ES products and the worm burdens obtained following challenge with infective larvae were compared with those in animals immunised with whole ES products and challenge controls. The AChE-enriched preparation stimulated high levels of enzyme-specific antibody in immunised animals, which was not the case for those which received unfractionaed ES products. Worm burdens of guinea pigs which received the AChE-enriched fraction were significantly lower than those obtained in adjuvant controls. The animals which received the unfractionated ES products were not significantly protected against challenge. These results suggest that AChEs may be potential candidates for incorporation in a sub-unit vaccine against D. viviparus. PMID- 7558570 TI - Vaccination of sheep against haemonchosis with H11, a gut membrane-derived protective antigen from the adult parasite: prevention of the periparturient rise and colostral transfer of protective immunity. AB - Pregnant ewes were immunised with a fraction highly enriched in the membrane glycoprotein antigen H11, isolated from the intestinal brush border of adult Haemonchus contortus. Immunity induced by immunisation was able to abolish almost completely (98-99%) the worm egg output from pregnant ewes challenged with ca. 10,000 infective larvae of H. contortus during the last trimester. Furthermore, lambs born and reared on vaccinated ewes had substantial antibody levels to H11 derived from maternal transfer. This antibody conferred moderate protection against a bolus challenge of ca. 3000 infective larvae of H. contortus in 5-week old lambs. PMID- 7558574 TI - Evolutionary and ecological parasitology: a changing of the guard? PMID- 7558571 TI - Antigens in phenotypes of Heligmosomoides polygyrus raised selectively from different strains of mice. AB - Antigens from Heligmosomoides polygyrus, which had been passaged selectively for over 40 generations through naive (Hpn) and immune (Hpa) mice, were extracted as whole worm (WWA) and membrane bound antigens (MBA), soluble adult worm homogenates (AWH), and excretory/secretory (ES) products. The antigen complexes were separated by SDS-PAGE, and 2-dimensional electrophoresis and assayed by western blot. Qualitative and quantitative differences were observed between profiles and antigenic reactivity of the constituents from selected parasites, which reflected their genetic heterogeneity. The survival of Hpa parasite phenotypes was improved compared with that of their Hpn counterparts in homologous strains of immunized mice but this did not correlate strongly with the antigenic differences observed. Three small molecules at 18, 21 and 23 kDa, respectively, dominated the somatic and ES components of all worms but they were of low immunogenicity during natural infection in mice and after vaccination in rabbits; and their role in immunomodulation is discussed. PMID- 7558575 TI - Misconceptions about the measurement of aggregation: a reply to Ploeger. PMID- 7558573 TI - Immune responses of sheep to microdissected parts of Haemonchus contortus. AB - Adult specimens of Haemonchus contortus were microdissected into four body fragments: oesophagus, cuticle of oesophagus, gut and cuticle with adjacent muscle layer. The antigenicity of these different body fragments was analysed in comparison to total (whole) worm extracts with immunoblotting and ELISA using sera of H. contortus-infected sheep. In particular, oesophagus-derived antigens appeared to be specifically recognized and may prove valuable in diagnostic assays. PMID- 7558576 TI - Secretory nematode SOD--offensive or defensive? PMID- 7558577 TI - Chagas disease--a recent meeting on practical aspects. Applied meeting of Chagas disease 4-6 November 1993, Uberaba, Brazil. PMID- 7558578 TI - Solid-phase synthesis of peptide haptens containing a cysteine-sulfur mustard adduct. AB - Solid-phase synthesis of peptide haptens containing 2-[2-(S-cysteinyl)ethanol has been achieved by solution-phase synthesis of a properly protected S-alkylated cysteine derivative and subsequent solid-phase incorporation. PMID- 7558579 TI - Peptides containing the sulfonamide transition-state isostere: synthesis and structure of N-acetyl-tauryl-L-proline methylamide. AB - The structure of the sulfonamide isostere-containing peptide N-acetyl-tauryl proline methylamide 4 was compared to information on the structure of the peptide N-acetyl-beta-alanyl-proline methylamide 6. NMR measurements of the beta-alanine containing peptide 6 showed the presence of two conformations due to cis/trans isomerism of the beta-Ala-Pro amide bond, whereas the sulfonamide-containing peptide 4 appeared in only one conformation. The crystal structure of N-acetyl tauryl-proline methylamide 4 gave additional evidence for the absence of cis/trans isomerism. The crystals are orthorhombic, space group P2(1)2(1)2(1), Z = 4, F(000) = 592, a = 7.5919(3), b = 10.3822(2), c = 17.1908(7) A, V = 1354.99(8) A3, Dx = 1.359 g cm-3. The oxygen atoms connected to the sulfur take positions similar to both the cis and trans positions of the carbonyl oxygen of an amide. Consequently the tauryl part is placed perpendicular to the proline alpha-C-C(O) bond, giving it an extended conformation in contrast to the cis/trans isomers of N-acetyl-beta-alanyl-proline methylamide 6. PMID- 7558581 TI - Self-activation of N-phosphoamino acids and N-phosphodipeptides in oligopeptide formation. AB - N-Phosphorylamino acids and N-phosphoryldipeptides can self-activate to give the oligopeptide. An intramolecular mixed anhydride intermediate might exist during the self-activating process. A peptide bond was formed from the N-terminus to the C-terminus by the '1 + n' mechanism, which was similar to the rRNA-peptide biosynthesis pathway. PMID- 7558580 TI - Synthesis of potent agonists of substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of substance P. AB - The analogues [Glp6,Glu(OBzl)11]SP(6-11) and [Glp5,Glu(OBzl)11]SP(5-11) of the C terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH2C6H5 ester group has replaced the SCH3 group of the Met11 side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI+At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. PMID- 7558582 TI - NMR conformational study of a model tetradecapeptide mimicking the RXVRG consensus cleavage site of a Xenopus laevis skin endoprotease. AB - A model tetradecapeptide, used for the purification of the RXVRG-endoprotease from Xenopus laevis skin exudate, has been studied by two-dimensional NMR, correlation (COSY) and NOE (NOESY) spectroscopy. This peptide has the 5-9 consensus sequence (RXVRG), along with an acidic moiety (1-4) and a hydrophobic domain (10-14). Variations with temperature of NH chemical shifts in a dimethyl sulfoxide solution (low thermal coefficients at residues 6, 7 and 8) and quantified NOE values from four spectra at different mixing times clearly showed a structural organization in the consensus domain with psi-angles around [-40, 10 degrees] for residues 7 and 8, and two NOE correlations of alpha HiNHi + 2 type (5-7 and 6-8). Moreover, a privileged rotamer in the side chain is established for three residues (Val2, Asp3 and Val7) and limited possibilities are discussed for seven others. Most of the folding trends were not observed in the [Ser7] derivative, underlying the relationship between the conformations and a full consensus sequence. In the model tetradecapeptide an equilibrium between two beta-turns of type I, fragments 4-7 and 5-8, seems the most probable. Comparison between this tetradecapeptide and its 4-14 fragment, also a substrate for RXVRG-endoprotease, shows that the 1-3 moiety (DVD) influences the consensus domain structure(s) and clearly stabilizes the folded one(s). Finally, two analytical methods are developed in order to determine: (1) the trifluoroacetic acid content of the peptide samples, on the basis of 19F NMR spectroscopy; (2) the mean phi- and psi-angles of each residue, from the whole set of NH/alpha H coupling constants (3JN alpha) and NOE data at a local level. PMID- 7558585 TI - Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides. AB - Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH- leaving group on the side chain carboxylic acid of the aspartic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558584 TI - Synthesis of a new dipeptide template, its X-ray structure, and modeling studies on its conformational features. AB - The diastereoselective synthesis is reported of a dipeptide template which is closely related to H-Gly-Trp-OH. Intramolecular bond formation between alpha-C of Gly and ring position 2 of the Trp unit has been achieved by a Pictet-Spengler type electrophilic aromatic substitution. The absolute configuration of the N-Moc protected dipeptide template 9.2H2O was determined by single-crystal X-ray crystallography and found to be (2S,5S). The cis orientation of the amino and carboxy termini prompted us to investigate the potential of 9 as a beta-turn mimic. MD calculations on the model pseudopeptide Ac-Ala-Gly-Trp-Ala-NHMe 11 suggest that an unusually tight turn should be favoured rather than a beta-turn. The proper protective situation as a pre-requisite for the incorporation of the template into a peptide has been established, and comments about its chemical properties are given. PMID- 7558583 TI - Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn. AB - Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation. PMID- 7558586 TI - 1H and 13C NMR assignments and molecular modelling of a minor groove DNA-binding peptide from the HMG-I protein. AB - The HMG-I subfamily of high mobility group (HMG) chromatin proteins consists of DNA-binding proteins that preferentially bind to stretches of A.T-rich sequence both in vitro and in vivo. Recently, members of the HMG-I family have been suggested to bind in vitro to the narrow minor groove of A.T-DNA by means of an 11 amino acid peptide binding domain (BD) which, because of its predicted structure, is called the 'A.T-hook motif' [Reeves, R. & Nissen, M. (1990) J. Biol. Chem. 265, 8573-8582], and would appear to be crescent-shaped. A BD peptide with 13 amino-acid residues was synthesized and examined by proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. The peptide contains four proline residues, and on the basis of NOEs and 13C chemical shifts was found to exist in an all-trans conformation. Molecular modelling based on this result provides evidence for a dynamic equilibrium between turn-like conformations in solution, the most populated of which is likely to be an S-shaped conformer, on the basis of amide exchange data. PMID- 7558587 TI - Development of a radioimmunoassay for a pseudononapeptide bombesin/GRP antagonist with antitumor activity. AB - Bombesin-like and GRP-like peptides may act as autocrine growth factors in the proliferation of some cancers. A pseudononapeptide bombesin antagonist, [D Tpi6,Leu13 psi(CH2NH)-Leu14]bombesin(6-14), and related analogs synthesized in our laboratory significantly inhibit tumor growth in various cancer models. A radio-immunoassay (RIA), suitable for determination of RC-3095 and its congeners in unextracted serum, was developed in order to facilitate further experimental and clinical evaluation of this bombesin/GRP receptor antagonist for the treatment of various tumors. Antibodies were generated against RC-3095 and Des Tpi1-RC-3095, conjugated to bovine serum albumin with glutaraldehyde. Antiserum JH-631b was selected for further experiments based on the antibody characterization. At an antiserum dilution of 1:189,000, this antibody bound approximately 50% of 7 fmol of added radiolabeled Tyr1-RC-3095. The antibody crossreacted with C-terminal fragments of RC-3095. Fragments without the C terminus and naturally existing peptides of the bombesin family or structurally unrelated peptides did not cross-react. The minimum detectable dose of RC-3095 was 0.4 pg/tube. Intra- and interassay coefficients of variation ranged from 3.2 to 4.4% and from 5.6 to 12.8%, respectively. The RIA is suitable for direct determination of RC-3095 in serum. The RIA should be of value for monitoring levels of this analog in serum during long-term therapy. PMID- 7558588 TI - Acid catalysis in the formation of dioxopiperazines from peptides containing tetrahydroisoquinoline-3-carboxylic acid at position 2. AB - The kinetics of the spontaneous formation of 2,5-dioxopiperazines from peptides containing the Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) residue in the 2-position of the sequence has been studied in DMSO and water solution. The reaction is first order in Tic-peptide and subject to general-acid catalysis. Moreover, only the fraction of peptide having the amino terminal group in the deprotonated state reacts with appreciable rate. In pure organic solvent, and in aqueous solution with low buffer concentration, the degradation reaction of Tic peptides is very low; at 20 degrees C for the peptide H-Tyr-Tic-Phe-Phe-NH2, in DMSO and in neutral water in the absence of buffer, the half-lives (t1/2) are 3 x 10(4) and 1.2 x 10(4) h, respectively. The addition of carboxylic acids or buffers to the reaction solutions markedly increases the reaction rate; in 0.01 m HAc in DMSO and in 0.1 M phosphate buffer in water, pH 7.1, t1/2 values for the tetrapeptide are 61 and 121 h, respectively. PMID- 7558589 TI - Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR, CD and stimulated annealing study. AB - The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by 1H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with alpha-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 phi-angle constraints obtained from 3JNH alpha coupling constants, as well as about 10 N ... O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu 15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin. PMID- 7558590 TI - Identification and characterization of a novel synthetic peptide substrate specific for Src-family protein tyrosine kinases. AB - Using a random combinatorial peptide library method [Wu, J., Ma, Q. N. & Lam, K. S. (1994) Biochemistry 33, 14825-14833] a novel peptide, YIYGSFK, was identified as a substrate for p60c-src protein tyrosine kinase. Mass spectrometric analysis showed that tyrosine-3 from the N-terminus was the phosphorylation site. Kinetic studies showed that the Km of YIYGSFK for p60c-src was 55 microM, about 6.4-fold lower than a peptide derived from p34cdc2 [cdc2(6-20), KVEKIGEGTYGVVYK], which had been reported to be a specific and efficient substrate for the Src-family protein tyrosine kinases. Comparison of the specificity of YIYGSFK and cdc2(6-20) as a substrate for various Src-family and non-Src-family protein tyrosine kinases suggests that YIYGSFK is a much more specific and efficient substrate for the Src family protein tyrosine kinases. PMID- 7558592 TI - The delineation of hydrogen-bonding patterns in supramolecular self-assembly of several core oxalo retro-peptides and crystal structure of MeO-Ser-Leu-COCO-Leu Ser-OMe. AB - Pseudo-C5-type intramolecular hydrogen bonding, arising from the C2 disposition of the core NH-CO-CO-NH unit in oxalo retro-peptides, is formed in addition to extended hydrogen bonding with neighbors forming sheets in MeO-Aib-COCO-Aib-OMe (1), helices in MeO-Leu-COCO-Leu-OMe (2) and ribbons in MeO-Ser-Leu-COCO-Leu-Ser OMe (3). In 3 the OH in the Ser side-chains form hydrogen bonds with the backbone carbonyl of Leu groups from neighboring molecules. Crystal parameters for 3 are: C22H38N4O10, monoclinic, space group P2(1), a = 7.374(1), b = 18.218(4), c = 10.661(3) A, beta = 95.72(2) degrees, R = 0.063 for 1738 observed reflections with [formula: see text]. PMID- 7558591 TI - Interaction of 3'-O-caffeoyl D-quinic acid with human serum albumin. AB - The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the view-point of thermodynamics and mechanism of binding at pH 6.0. The association constants (Ka) for the HSA-CGA interaction at 10, 25 and 40 degrees C were 6.0 x 10(4), 9.0 x 10(3) and 2 x 10(4) M-1, resulting in delta G of -6.21, -5.80, -6.32 kcal/mol, respectively. These high Ka-values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 nm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the alpha-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4 degrees C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 +/- 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 x 10(4) M-1 and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA >> CGA >>> QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding. PMID- 7558593 TI - Polymethylene spacer linked bis(Ala) peptides form modified beta-sheet structures. Crystal structure and self-assembly pattern of adipoyl and suberoyl analogues. AB - The adipoyl- and suberoyl-linked bis(Ala) peptides have an extended backbone between the two C alpha atoms in each molecule. They self-assemble, through intermolecular hydrogen bonds and stacking of parallel strands, into highly ordered modified beta-sheet-like structures. Crystals data for adipoyl bis(Ala)diester are as follows: C14H24N2O6, monoclinic space group P2(1), a = 4.900(1), b = 29.093(10), c = 6.021(2) A, beta = 104.20(2) degrees, R = 0.053 for 1100 data > 3 sigma (F); for suberoyl bis(Ala)diester: C16H28N2O6, monoclinic space group P2(1), a = 4.887(2), b = 32.650(9), c = 6.004(2) A, beta = 103.79(3), R = 0.070 for 1065 data > 3 sigma (F). PMID- 7558595 TI - Solution conformation of mu-selective dermorphin and delta-selective deltorphin-I in phospholipid micelles, studied by NMR spectroscopy and molecular dynamics simulations. AB - Complete proton resonance assignments of the naturally occurring mu-selective dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and delta-selective deltorphin-I (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) were carried out by two-dimensional 1H-NMR techniques to investigate the conformational features in the membrane-mimetic micelles of perdeuterated dodecylphosphocholine. Fifty possible three-dimensional structures for respective peptides were generated by means of distance geometry calculations, all of which satisfy the proton-proton distances derived from NOE measurements within the allowable range, and 25 of them were subjected to the molecular dynamics simulations for 10 ps, in which the NOE distances were included as the energetic constraints. Although conformers simulated for dermorphin showed relatively large conformational variations because of the limited NOE data, most of them were characterized as an entirely folded structure bent at the Gly4 residue, where each of the N- and C-terminal tetrapeptides took an extended conformation. On the other hand, most conformations of deltorphin-I showed the common feature that the N-terminal Tyr-D-Ala-Phe-Asp and C-terminal Val-Val-Gly-NH2 sequences took respective folded conformations, and these were almost at right angles on the border of the Asp-Val sequence. These conformational characteristics are discussed in terms of the possible relationship with the mu/delta-opioid receptor selectivity. PMID- 7558597 TI - Solution conformational dynamics of the C-terminal residues in endothelin-1 and some analogues: a time-resolved fluorescence study. AB - The rotational relaxation times of the single tryptophan residues in endothelin 1, [Ala1,3,11,15]endothelin-1, human pro-endothelin-1, the linear hexapeptide Ac His-Leu-Asp-Ile-Ile-Trp which corresponds to the C-terminal residues 16-21 in endothelin-1, the cyclic pentapeptide BQ123, and several di- and tri-peptides possessing C-terminal tryptophan residues have been determined from time-resolved fluorescence anisotropy decays obtained by phase/modulation techniques. Fluorescence lifetime distribution widths have also been examined as predictors of conformational heterogeneity/restriction. A significant contribution from a slow rotational component supports either the persistence, on the nano-second timescale at least, of a non-flexible alpha-helical structure for the C-terminal tail residues of endothelin-1 in water as solvent, as seen in the X-ray crystallographic structure, or the interaction of the C-terminal tail residues 16 21 with the constrained disulfide-bridged core residues 1-15. This slow rotational contribution is less evident in the linear, acyclic tetraalanine analogue but greatly increased in pro-endothelin-1. In BQ123 the fluorescence characteristics support the occurrence of a dominant rotameric form involving the indole sidechain of the D-tryptophan residue (C alpha-C beta torsion angle chi 1 of 60 degrees, as previously determined by NMR. PMID- 7558594 TI - Peptic proteolysis of esterified beta-casein and beta-lactoglobulin. AB - Moderate esterification induces slight secondary structure changes in two major milk proteins, beta-lactoglobulin and beta-casein. Esterification of beta lactoglobulin prompts its tertiary structure 'melting', opening it to peptic cleavage. Twenty-two new cleavage sites were characterised in beta-lactoglobulin and five in beta-casein. Some of them are due to esterification-improved peptide bond accessibility, some to the bias of pepsin specificity by glutamate and aspartate esters. The resulting fragmentation yields original and partially amphiphilic peptide populations. PMID- 7558598 TI - Methanol-mediated, modular self-assembly of antiparallel beta-dimers. Crystal structure of C alpha backbone-modified tripeptide Bz-Aib-NHCOCO-Aib-OMe. AB - The backbone-modified tripeptide Bz-Aib-NHCOCO-Aib-OMe, in which the central C alpha has been replaced with CO, self-assembles in the solid state into highly ordered two-dimensional arrays through MeOH mediated intermolecular stacking of dimeric 'disk' modules formed by an antiparallel beta-sheet-type arrangement of tripeptide molecules. The C alpha(C'O)N(C'O)(C')NC alpha segment is nearly coplanar (average deviation from a mean least-square plane is +/- 0.037 A). The oxalyl moiety forms two pseudo-C5 intramolecular hydrogen bonds. Crystal parameters are as follows: C18H23N3)6.CH3OH, triclinic space group P1-, alpha = 9.796(4), b = 10.348(3), c = 11.836(4) A, alpha = 78.28(3), beta = 73.72(3), gamma = 69.45(3), R = 0.082 for 1747 reflections measured with [formula: see text]. PMID- 7558596 TI - Structure-activity relationships of dermorphin analogues containing N-substituted amino acids in the 2-position of the peptide sequence. AB - A series of dermorphin analogues containing an N-alkylated amino-acid residue Xaa in the 2-position of the peptide sequence was synthesized (Xaa = N-methylalanine, proline, pipecolic acid, N-methylphenylalanine, 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid [Tic]). These peptides have the potential of assuming a cis Tyr1 Xaa2 peptide bond. Their in vitro opioid activity profiles were determined in mu- and delta-receptor-representative binding assays and bioassays. Aside from [D Pro2]dermorphin, all analogues showed high affinity for mu- and/or delta-opioid receptors. Whereas most compounds were found to be full mu-agonists in the guinea pig ileum (GPI) assay, [Tic2]dermorphin (compound 7) was a partial mu-agonist. Replacement of Gly4 in 7 with Phe resulted in an analogue (8) with weak mu antagonist activity. Furthermore, analogues 7 and 8 both were potent delta antagonists (Ke = 3-40 nM) against the delta-agonists Leu-enkephalin, DPDPE and deltorphin I in the mouse vas deferens (MVD) assay. Compound 3, containing L-Pro in the 2-position, turned out to be one of the most mu-receptor-selective linear dermorphin analogues reported to date. Low-temperature HPLC experiments using micropellicular octadecyl silica as stationary phase revealed conformational heterogeneity of the dermorphin analogues which was ascribed to cis-trans isomerization around the Tyr1-Xaa2- and Tyr5-Pro6 peptide bonds. In the case of analogue 7 four separate peaks corresponding to the four possible isomers were apparent at -5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558599 TI - Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian cobra) venom. AB - The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. PMID- 7558600 TI - CoMFA investigations on two series of artificial peptide inhibitors of the serine protease thermitase. Synthesis of an inhibitor of predicted greater potency. AB - Comparative molecular field analysis (CoMFA) is shown to be a very useful tool in treating two series of artificial substrate analogue inhibitors, peptide methyl ketones and chloromethyl ketones, of the serine protease thermitase. The backbone structure of the native polypeptide eglin c found in the X-ray structure of its complex with the enzyme served as a template for the alignment of the inhibitors, which could be shown to be the key for success. Restricted only by the relatively small number of different amino acids representing the peptide sequence we were able to determine the regions of the compounds that are important for the value of the inhibitor constant K(i). On the basis of these results we have suggested some new structures with possibly increased inhibitory activity. One such structure was synthesized and is shown to be the most active compound tested up to now, with an experimental K(i)-value in the predicted range. PMID- 7558603 TI - NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH). AB - The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-c(Asp5-D-Arg6-Leu7- Lys8)-Pro9- D-Ala10-NH2 (I), Ac-D-Phe(p-Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-c(Glu5-D-Arg6-Leu7- Lys8)-Pro9-D-Ala10-NH2 (II) and their linear analogues, Ac-D-Phe(p-Cl)1-D- Phe(p Cl)2-D-Trp3-Ser4-Asp5-D-Arg6-Leu7-Lys8-Pro9-D-++ +Ala10-NH2 (III) and Ac-D-Phe(p Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-Glu5-D-Arg6-Leu7-++ +Lys8-Pro9-D-Ala10-NH2 (IV), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II' beta-turn involving residues 3-6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a beta turn between residues 5 and 8. PMID- 7558604 TI - Hermeneutics or Hermes-mess? PMID- 7558602 TI - Conformational studies of a synthetic cyclic decapeptide fragment of rat transforming growth factor-alpha. AB - The solution conformation of a synthetic cyclic decapeptide [with sequence mimicking the third disulfide loop of rat transforming growth factor-alpha (rTGF alpha)] in deuterated dimethyl sulfoxide was studied by 2D NMR. The determination of solution structures was based on NOE interproton distances, using a combination of distance geometry and simulated annealing protocols. The convergence of the selected structures was evident from the small atomic pairwise root-mean-square deviation values among them. Good agreement was noted between the experimental and simulated NOESY spectra, thereby reflecting the accuracy of the calculated solution structures. Analysis of the structures indicates that the residues Tyr5 and Arg9 exhibit similar side chain orientation as that in the corresponding disulfide loop of human transforming growth factor-alpha. PMID- 7558601 TI - Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors. AB - A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural L-amino acids (except for cysteine), plus the non-natural L-amino acids ornithine and norleucine, The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN', alpha-chymotrypsin, trypsin, anhydro-alpha-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to alpha-chymotrypsin, two of which also bind to anhydro-alpha-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K(i)) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K(i) values. In the case of the alpha-chymotrypsin inhibitors, the peptides with the lowest K(i) (0.1-0.05 mM) were the only peptides that bound to anhydro-alpha-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that alpha chymotrypsin and trypsin are inhibited by these cyclic peptides. PMID- 7558605 TI - The three 'W's: what, where and when: the rationale of interpretation. AB - The author considers the work of interpreting to be a continuous diagnostic process in which the analyst uses both emotional and intellectual activity. In this paper she discusses three fundamental factors in the mind of the analyst when making interpretations--'what', 'where' and 'when'. In 'What' she speaks of the transference relationship as the central core of the interpretation. She understands the transference as an expression of past internal object relationships that for some reason could not develop, and that repeat themselves with their specific affects, anxieties and defences in the patient-analyst relationship. In 'Where' she examines where the patient feels the problem resides: with the analyst or with himself. This depends on the patient's level of functioning. If he is functioning predominantly in a paranoid-schizoid level, the use of projective mechanisms will be considerable and interpretations should be centred in the analyst. When the mode of functioning corresponds mainly to depressive-position level, the patient can feel the problems as his; therefore interpretations should be centred on him. Finally, in 'When' the author addresses herself to the factor(s) that help the analyst to integrate the different elements of the patient's communication into a new formulation. A whole session from the analysis of a patient is fully reported and used to illustrate and argue the views espoused in the paper. PMID- 7558607 TI - Countertransference: the emerging common ground. AB - In the last decade or so, the understanding of countertransference has become an emerging area of common ground among psychoanalysts of diverse theoretical perspectives. This convergence can be traced to the development of two key concepts--projective identification and countertransference enactment. Projective identification has evolved from a patient's intrapsychic fantasy in Klein's original work to an interpersonal interaction between patient and analyst. The notion of countertransference enactment has been widely used to capture clinical situations in which a countertransference reaction in the analyst corresponds to the patient's attempt to actualise a transference fantasy. These ideas, in conjunction with the contributions of social constructivists and relational theorists, as well as Sandler's conceptualisation of role-responsiveness, have led to an understanding of countertransference as a 'joint creation' by analyst and patient. The relative contributions of analyst and patient vary somewhat according to the theoretical perspective espoused by the analyst. This common ground is best regarded as comprising a gradient or continuum in which more weight is given to the analyst's contribution on one end of the continuum and more emphasis to the patient's contribution on the other. While countertransference enactments are widely regarded as inevitable, their role in creating intrapsychic change is more controversial. PMID- 7558606 TI - The concept of 'early triangulation' as a key to a unified model of hysteria. AB - On the basis of a subtle and critical use of the concepts of separation and triangulation, this paper develops a psychodynamically unified model of the multi faceted phenomenology of hysteria in both women and men. It offers an explanation for the manner in which pre-oedipal and oedipal factors interact in hysteria. The fundamental conflict in malignant and benign hysterias is assumed to have the same content, the severity of the disturbance being determined by qualitative and quantitative factors (specific relational constellations, specific forms of traumatisation, and the subject's age, maturity and ability to cope with the relevant problems). The fundamental conflict common to all hysterias is deemed to lie in the subject's attempt to utilise oedipal triangulation in the context of a sexualised form of relationship with the parent of opposite sex, in order to obtain by force the missing early (pre-oedipal) triangulation (in the case of female hysterics) or to secure a substitute for it (in male hysterics), and thereby to achieve the separation from the mother that could not originally be negotiated. Traumatisations and typical defensive constellations associated with this neurotic attempt at resolution are described. The same theoretical approach makes it possible to understand the interactional function of conversion symptoms, and at the same time to consider their significance for this specific neurotic configuration. PMID- 7558608 TI - Understanding the violent patient: the use of the body and the role of the father. AB - We offer some thoughts about the roots of habitual violence in patients who are not part of the 'cycle of abuse'. We suggest that both self-harm and mindless assaults on others may reflect inadequate capacity to mentalise. Poor functioning of this capacity tends to lead to mental states being experienced as physical, in both the self and others, and the violence is seen as an attempt to obliterate intolerable psychic experience. This experience is felt to belong to somebody else, originally to mother or father. The problem can be traced back to a crucial stage of the development of the self when the child searches the face of his primary object for a representation of his own states of mind. Failure to find this forces him into pathological solutions to achieve an containing organisation. We explore the meaning of the gender difference in the direction of aggression, and the way in which the child has a second chance to foster a secure psychological self through his relation to the father, even when the mother has been unable to support this and to separate successfully. These issues and others of technique are explored in the treatment of a violent young man. PMID- 7558610 TI - Explanation in the hermeneutic science. AB - Using clinical vignettes, we first examine the idea that psychoanalysis is, in Gill's (1991) phrase, a hermeneutic science--hermeneutic because it interprets meanings, scientific because the meaning connections it deals with are also usually causal connections. Next we explore some aspects of the structure of the kinds of explanation distinctive to psychoanalysis. Three kinds are distinguished: conveyance of content explanations, which have been central in the accounts of some recent philosophers of psychoanalysis, and two kinds of explanation in terms of intentionality, in terms, that is to say, of what the psychic states in question are about. One deals with forward-looking states such as wishes, in which the state is about some future, sought-after state of affairs such as a wish-fulfilment; the other deals with backward-looking psychic states such as memories, in which the state to be explained is about some earlier experience, fantasy, or whatever, one often quite unknown to the analysand prior to analysis. The paper concludes by making a quick case for the idea that good explanations in terms of meaning or intentionality often also identify the cause of the psychic states being explained. PMID- 7558609 TI - Affirmation in psychoanalysis. AB - It is argued that recent developments in psychoanalytic theory have prepared the theoretical space for a concept of affirmation and rendered necessary a discussion of the justification of affirmative interventions in clinical practice. Exemplified by a scene from one of Ingmar Bergman's plays, an affirmative intervention is defined as a communication which removes doubt about the experience of reality and thereby re-establishes a feeling of identity. This definition is compared with Freud's explanation of affirmation. Clinically, affirmation may function both as a silent background and as a delimited intervention in the foreground. Affirmative interventions are illustrated by a clinical vignette. The intonation of the voice of the analyst seems to be especially potent in mediating affirmative messages. Affirmation and interpretation address different experiential modes. They are complementary kinds of interventions, and the analyst has to oscillate between the two. In conclusion, the question raised is whether affirmation can repair structural defects ensuing from early affect deficit. PMID- 7558612 TI - How secure are Grunbaum's Foundations? AB - As exemplified in Freud's cases, behaviour becomes meaningful as it is situated within a history (narrative). Operating from a Unity of Science orientation, Grunbaum emphasises causal rather than meaningful connections, and selectively follows early Freud: when (and only when) the repressed historical cause is uncovered, the symptom disappears. Since that consequential connection has proven to be not causally invariable Grunbaum discredits psychoanalysis as unscientific. Counterposed to his causal approach is that of philosopher MacIntyre, who contends that identity and moral responsibility must be situated within personal history. Also counterposed is sociologist Max Weber who struggled with parallel issues in the German 'Geisteswissenschaften' of Freud's time. The oedipal complex is akin to Weber's ideal types, as are the various models implicit in analytic discourse. Psychoanalysis must be understood as a normative science. In so far as contemporary psychology and psychiatry emphasise causal relationships, their procedures (experimental, epidemiological) can never hope to encompass psychoanalysis. Like other grand theoretical systems, psychoanalysis has applications in wide arenas--personal, developmental, familial, cultural, religious, historical, ethnological; its development during the last century has come from the interaction of findings from those arenas with those from the consulting room. PMID- 7558611 TI - The specificity of hermeneutics in psychoanalysis: leaps on the path from construction to recollection. AB - Psychoanalysis is the search for and revelation, through interpretation and construction, of unknown, unconscious implications. But the question is raised: are the meanings being uncovered or are we providing them? This basic realist idealist contraposition is at the heart of current discussions about the hermeneutic trend in psychoanalysis. Ricoeur's often misrepresented contribution serves as a basis for understanding Freud's method as forming a 'hermeneutic constellation', evolving within a 'moderate', in contrast to a 'radical-idealist' hermeneutic psychoanalysis. Ahumada's recent charge of 'verbal creationism' and Laplanche's re-framing of interpretation between determinism and hermeneutics are discussed in order to specify the more moderate position taken in this paper. It is argued that although they are correct in pointing to potential excesses, both authors misrepresent the hermeneuticist trend as unnecessarily idealistic. A case example from a session illustrates the discussion. PMID- 7558614 TI - Unprovoked assaults--making sense of apparently random violence. AB - An unprovoked assault occurs when a total stranger assaults a totally innocent victim he has never met. Presenting findings from three such cases, representative of a series of ten seen as part of his work as a psychoanalyst at a special forensic hospital in the UK, the author attempts to tie together the victim and his attacker in the attacker's mind, setting out to explain how the victim fits the need of a psychotic man's fantasy, at a particular time. A provisional hypothesis about the internal situation of such patients and its diagnosis, treatment and management is put forward and discussed, in the context of the implications for the treatment of such offenders and the psychoanalytic theory of projection and symbolisation. PMID- 7558613 TI - Towards a definition of the term and concept of interaction. AB - Although often used descriptively, the concept 'interaction' has not been an inherently psychoanalytic term. It suggests an external or social purview, rather than an intrapsychic one. If, however, the term were qualified to convey 'experienced' interaction and a clear delineation were maintained between the point-of-view of the patient and that of the analyst or other outside observer, 'interaction' might then be credibly employed within the psychoanalytic lexicon. For in this way the investigative terrain remains the patient's psychical reality -with its conscious and unconscious, conflictual, defensive and imaginative expressions still our fundamental purview. Clinical examples are offered to illustrate a sharpened focus on interaction as seen from the patient's point-of view to highlight the implications of this position in furthering our psychoanalytic inquiry. PMID- 7558615 TI - The mutual search for self in the analytic dyad. AB - Clinical material is presented from seven consecutive sessions in the third year of an analysis to show the way in which patient and analyst worked at the task of allowing each of their separate self and object representations to emerge. These emergent representations are ones in which different aspects of the self, both 'good' and 'bad', are gradually integrated. The continued tension in both participants between an infantile narcissistic view of self and object and a realistic integrated one is highlighted. The way in which the analyst's psychic work on the acceptance of her own narcissism aids the patient's integration of disowned aspects of self is discussed. This material is presented within the framework of ideas about the development of a theory of mind and of reflective self function proposed by Fonagy et al. (1993a, b) and in the context of recent writing on the analyst's unconscious participation in the analytic process, e.g. Levine (1994). PMID- 7558616 TI - The importance of facial expressions in dreams. PMID- 7558617 TI - The effects of trauma on young children: a case of 2-year-old twins. AB - Trauma and its effects on early intrapsychic development are issues that need to be better understood as young children are exposed to ever increasing levels of violence. In this paper, we have considered trauma from the perspective of its effects on young children in the context of chronic community and family violence. Characteristic features of adults with post traumatic stress disorder have been identified in children under 4 years old who have been exposed to extreme trauma. In understanding trauma and its effects on children, it is important to consider how external events may impact on intrapsychic structure and the development of the self. A case is presented of identical 2 year, 11 month old twin boys who were referred after having witnessed the shooting death of their mother by their father seven months earlier. The course of psychoanalytically informed play psychotherapy is discussed with a major goal of the therapy being to help the children work through the trauma and return to a healthy developmental course. While the twins responded well to treatment and were able to work through traumatic material, systematic follow-up is needed to determine the later developmental course. PMID- 7558618 TI - Resistance to awareness of the supervisor's transferences with special reference to the parallel process. AB - Supervision is an essential part of psychoanalytic education. Although not taken for granted, it is not studied with the same critical eye as is the analytic process. This paper examines the supervision specifically with a focus on the supervisor's transference towards the supervisee. The point is made, in the context of clinical examples, that one of the ways these transference reactions may be rationalised is within the setting of the parallel process so often encountered in supervision. Parallel process, a very familiar term, is used frequently and easily when discussing supervision. It may be used also as a resistance to awareness of transference phenomena within the supervisor in relation to the supervisee, particularly because of its clinical presentation. It is an enactment between supervisor and supervisee, thus ripe with possibilities for disguise, displacement and gratification. While transference reactions of the supervisee are often discussed, those of the supervisor are notably missing in our literature. PMID- 7558619 TI - Interactions between sensory perivascular nerves and the endothelium in brain microvessels. AB - The purpose of our study was to see if selective denervation of sensory perivascular nerves leads to changes in release of vasoactive substances of endothelial origin from brain microvessels during increased flow. Male rats pups were treated with the neurotoxin, capsaicin, to destroy primary afferent sensory nerves. Three months later, brain microvessels were isolated, placed on a micropore filter and perfused with Krebs buffer. During the course of a 30-min experiment, the flow rate was increased from 0.5 to 3.0 ml/min for two 3-min periods. The levels of ATP, substance P, vasopressin and endothelin in the effluent were measured. Microvessels from 5 rats were pooled for each perfusion experiment. Data from 5 perfusion experiments showed that the release of substance P was significantly higher in the capsaicin-treated rats during the second period of increased flow (3.02 +/- 1.04 pmol/min/mg protein) compared to the vehicle-treated controls (0.30 +/- 0.21 pmol/min/mg protein; p < 0.02). In contrast, the release of ATP at low flow and during the second period of increased flow was significantly lower in the capsaicin-treated rats (0.21 +/- 0.04 compared to 0.50 +/- 0.03 pmol/min/mg protein for controls at low flow, p < 0.001; and 1.19 +/- 0.15 compared to 2.11 +/- 0.26 pmol/min/mg protein for controls during the second period of increased flow, p < 0.01). The release patterns of endothelin and vasopressin were similar in capsaicin-treated and vehicle-treated controls. In conclusion, chronic depletion of sensory innervation leads to altered release of two endothelium-dependent vasodilators, ATP and substance P from the brain microvasculature.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558620 TI - Effects of subcutaneous insulin-like growth factor-I infusion on skin microcirculation. AB - When healthy volunteers were treated with human insulin-like growth factor-I (IGF I), mild generalized edema often developed. In the present study, the effect of IGF-I on cutaneous capillary permeability and microvascular skin blood flow was investigated using fluorescence videomicroscopy and laser Doppler fluxmetry. Transcapillary diffusion of intravenously injected sodium fluorescein (NaF) was quantitated by videodensitometry in terms of fluorescent light intensities (FLIs) 5, 10, 20, 30, 60, 120, 180 and 300 s after the first appearance of the dye. Laser Doppler fluxmetry was performed at rest (LDFrest) and during postocclusive reactive hyperemia (LDFpeak). Eight healthy subjects (3 women, 5 men; mean age 28 years, range 24-30 years) were investigated. The sensing site was the skin on the right dorsal wrist. Measurements were performed after 4 days of subcutaneous infusion of 0.9% saline (control) and of IGF-I. Mean values for the FLI were significantly higher after IGF-I than after saline infusion (p < 0.05), when the FLIs were expressed in arbitrary units. As percentages of their individual maxima, the differences were significant (p = 0.05) after 60 s (23.6 +/- 6.6% with NaCl and 31.9 +/- 7.6% with IGF-I). LDFrest and LDFpeak tended to be higher after IGF-I treatment without achieving statistical significance (p = 0.176). The mean appearance time of the dye after injection was significantly shorter (p = 0.016) in the IGF-I group than in the control group (32.0 +/- 8.4 s with IGF-I, 42.4 +/- 8.3 s with NaCl).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558621 TI - Prevalence of cyclic changes in limb volume (volumotion) of male patients with knee injury and the effects of ischemia/reperfusion due to tourniquet. AB - During surgery of limbs tourniquet up to a maximum of 2 h is frequently applied which may cause ischemia/reperfusion injury (IRI). During this condition the presence of vasomotion may have consequences for the perfusion and nutritive state of the tissues. We used a noninvasive plethysmographic method to investigate periodic changes in limb circumference (volumotion) in healthy male patients (n = 24) undergoing surgery for knee injury. To facilitate surgery a tourniquet was applied to the thigh, which caused an IRI of the leg. Results are given as mean of all values +/- SEM. Immediately after tourniquet release (duration 57.75 +/- 5.19 min) blood lactate levels in the femoral vein increased significantly from 1.40 +/- 0.08 to 2.59 +/- 0.20 mmol/l (p < 0.001) and pH fell from 7.39 +/- 0.01 to 7.32 +/- 0.01 (p < 0.001). Preoperatively 10 out of 24 patients (42%) showed signs of volumotion on the injured leg with a periodicity ranging from 0.8 to 6.9 cycles/min, whereas none showed volumotion in the control leg (p < 0.001). In the second measurement, taken after surgery and reperfusion while peripheral sympathetic nerves were blocked, 7 out of 18 patients (39%) showed volumotion on the injured leg and 0 on the control leg (p < 0.004). 6 h after IRI, volumotion was observed in 11 out of 17 patients (65%) on the injured leg and in 1 patient (6%) on the control leg (p < 0.001). The mean volume change in the patients with volumotion on the injured leg was 0.057 +/- 0.007 ml/100 ml tissues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558623 TI - Induced periodic hemodynamics in skeletal muscle of anesthetized rabbits, studied with multiple laser Doppler flow probes. AB - Periodic fluctuations, regular slow-wave flowmotion, were induced in the skeletal muscle of six mature anesthetized New Zealand White rabbits by acute femoral artery pressure reduction from a median control value of 78 to 36 mm Hg. This phenomenon was monitored simultaneously with four laser Doppler flowmetry (LDF) probes placed over the gastrocnemius muscle in a linear array with a spacing of 5 mm. The median relative peak-to-trough amplitude of the oscillatory flow patterns was 47%, white a frequency of approximately 2.5 cycles per minute (cpm), which remained relatively stable over an observation period of 1 h (+/- 15-20%). Application of two frequency analysis methods, Welch's FFT method and Prony Spectral Line Estimation yielded similar results and showed a correlation coefficient of r = +0.84. Spectral coherence between pairs of regular slow-wave flowmotion records decreased with increasing probe separation (0.28 at 5 mm; 0.01 at 15 mm). However, patterns of frequency fluctuation over time were significantly correlated between concurrent record pairs regardless of probe separation. These results suggest that the regular slow-wave flowmotion signal originates in regions that are independently regulated by local vasoactive sites, which may be called pacemakers. These sites may also be influenced by an additional common control mechanism, which may be myogenic/metabolic or central in nature. PMID- 7558622 TI - Abnormal peripheral microcirculation in seemingly normal subjects living in blackfoot-disease-hyperendemic villages in Taiwan. AB - Blackfoot disease (BFD) is an endemic peripheral arterial disease confined to the southwestern coast of Taiwan. The cause of the disease has been ascribed to the high-arsenic artesian well water. The purpose of this study was to examine the possible association between the long-term exposure to artesian well water and the change in microvascular circulation in the absence of peripheral arterial insufficiency. A total of 45 men living in the BFD-hyperendemic villages and another 51 age- sex- body-mass index-matched men who lived in nonendemic villages nearby were recruited into this study. All subjects were free from peripheral vascular disease (resting ankle-brachial index > 1.00), clinical claudication, cigarette smoking, diabetes mellitus, hypertension, ischemic heart disease, cerebral infarction and obesity. Laser Doppler flowmetry was used to measure the peripheral microcirculation on the big toes both at 36 degrees C (basal perfusion, Pb) and after a hyperthermic test at 42 degrees C (Ph). The time required to reach Ph (T), and the average rate (R) of increase from Pb to Ph measured by (Ph-Pb)/T were also calculated. Results showed that those living in the BFD-hyperendemic area had a lower Pb [32.8 +/- 6.0 perfusion units (PU) vs. 67.0 +/- 4.3 PU, p < 0.001], a lower Ph (193.2 +/- 13.6 vs. 231.1 +/- 6.3 PU, p < 0.005), a longer T (3.04 +/- 0.19 vs. 1.31 +/- 0.08 min, p < 0.001) and a slower rate of increase from Pb to Ph (48.0 +/- 4.8 vs. 76.2 +/- 5.4 PU/min, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558624 TI - On the number of measurements necessary to assess regional cerebral blood flow by local laser Doppler recordings: a simulation study with data from 45 rabbits. AB - Laser Doppler fluxmetry may improve the monitoring of cortical blood flow in neurosurgical patients. So far, however, the variability of laser Doppler readings found in the cerebral cortex has prevented a consequent usage of the technique in clinical practice. The current report analyzes the regional variability of laser Doppler readings from experimental animals. Typical frequency histograms of observed flow values display non-Gaussian distributions. A simulation technique is used to assess the number of measuring sites required for valid estimates of regional cortical flow. From a total of 990 local flow measurements from 45 rabbits random samples of sizes between 5 and 100 were repeatedly drawn to estimate the variability of median flow thus determined. The study underlines that small sample sizes below n = 20 are associated with a large variability (95th percentile: +38%) which decreases in a biphasic manner. Sample sizes above n = 25 are necessary to obtain more reliable information on regional cerebral blood flow: the 95th percentiles remain between 24 and 28% up to n = 45, and are still at 15% with n = 99. PMID- 7558625 TI - Early ultrastructural changes of myocardial endothelial cells in traumatic haemorrhagic shock in rats. AB - The aim of this study was to assess early traumatic haemorrhagic shock (THS) induced changes in myocardial endothelial cells (mECs) both morphologically and morphometrically. The mECs of capillaries of the left ventricular myocardium were investigated by electron microscopy 15, 30, 60, and 120 min after a standardised THS and compared to a control group. With the use of a computer-assisted image analysis system, we measured the following morphometric parameters--height, area, circumference, cytoplasmic processes, and interendothelial junctions--and determined the number and distribution of cytoplasmic vesicles in every mEC. THS induced a significantly increased formation of cytoplasmic processes and a redistribution of cytoplasmic vesicles towards the cell centre, with the changes peaking between 15 and 60 min. There was no evidence of an increase in mEC height or area/circumference ratio used as indicators for early mEC oedema, although the latter dropped significantly after 15 min. No increase in damaged mitochondria was observed and the interendothelial junctions remained close. The THS-induced ultrastructural changes represent early mEC activation. Irreversible damage of mECs does not occur. PMID- 7558627 TI - [Gaceta Sanitaria 1993-94: balance and perspectives]. PMID- 7558626 TI - Response of hamster cheek pouch microcirculation to endothelin and endothelin receptor antagonists. AB - Our goal was to examine the effects of endothelin-1 and endothelin-3 on hamster cheek pouch arterioles in vivo in the absence and presence of endothelin receptor antagonists. Endothelin-1 (0.1, 1.0 and 10 pM) and endothelin-3 (0.1 and 1.0 nM) produced dose-related constriction of cheek pouch arterioles which was inhibited by the endothelin ETAB receptor antagonist PD 142893 (Ac-D-Dip-Leu-Asp-Ile-Ile Trp) and endothelin ETA receptor antagonist PD 147953 [FR 139317: (hexahydro-1H azepinyl)carbonyl-Leu-(N-Me)-D-Trp- D-2-Pya]. The thromboxane analogue, U-46619 (0.1 and 1.0 nM) also produced dose-related constriction of cheek pouch arterioles, but in contrast to endothelin-1 and endothelin-3 this vasoconstriction was not abolished by endothelin receptor antagonists. These findings suggest that endothelin-1 and endothelin-3 produce marked constriction of cheek pouch arterioles which appears to be mediated by activation of endothelin ETA receptors. PMID- 7558629 TI - [Validity of death certificates for cancer in Mallorca]. AB - We assessed the quality and completeness of the information of death certificates (CMD) for cancer in Mallorca during 1989. Likewise we studied the possible bias introduced by the excluded cases for lack of clinical information (DCO cases). We compared the information of the death certificates with the clinical records and data of the Population-based Cancer Registry. A global concordance of 77.2% was obtained out of 1,173 certificates. This concordance varied according to sex (males: 79.8%, women: 73.1%; p < 0.01), place of death (hospital: 83.1%, home: 69.6%; p < 0.001) and age ( < 50 years: 77.8%; > 70 years: 74.2%; p < 0.05). The best notified sites were: breast, lung, and leukemias and the worst, unknown and poorly defined sites, bones and soft tissue tumours. We detected that the death certificates carry a subnotification of at least 5% in cancer deaths. Even so, they result a valid source of information for the Population-based Cancer Registry. The cases excluded for lack of clinical documentation present a more advanced age and a greater proportion of hepatic cancers and unknown and poorly defined sites. PMID- 7558628 TI - [Evolution of the epidemiologic pattern of the cases of AIDS in Spain]. AB - Based on cases reported to the National AIDS Register by end June 1994, we analysed changes in the epidemiological pattern of AIDS in Spain for the periods 1981-1989 and 1990-1992. Cases diagnosed in 1981-1989 were mainly males (83.1%), subjects under 30 years of age (55.9%), and intravenous drug users (63.8%) or homo/bisexual males (16.8%). In 1990-1992 there was an increase in the proportion of females (18.5%), cases older than 29 (55.0%), and mean age diagnosis. Proportions of homo/bisexual, blood components recipients, and mother-to-child transmission decreased, while heterosexual transmission increased from 4.3% to 8.0%. In spite of large inter-regional differences in 1981-1989, some regions, such as La Rioja, Navarre, Murcia, Galicia and Aragon, rose more than Spain as a whole. Although the epidemiological pattern in both periods is similar, significant and independent changes in analysed variables are to be observed. PMID- 7558630 TI - [Validity of a questionnaire on recent physical activity]. AB - This study aims to validate a frequency questionnaire for categorically ranking people according to recent physical activity (PA) (previous two weeks), applicable to both sexes. Seven one-day PA records were collected for 30 males and 32 females during two weeks. After, the frequency questionnaire was administered covering the same period. The differences (paired-test) between the means obtained by both methods were globally significant for females and for some subgroups of activities in both sexes. The Pearson's correlation coefficient between both methods was 0.78 for males and 0.65 for females. The degree of agreement among methods classifying the subjects on terciles of total PA was lower in males (weighted kappa kw = 0.35, p < 0.06) than in females (KW = 0.52, p < 0.004). Although the study size prevents to infer definitive conclusions, the questionnaire performs better when measuring basic, occupational and total PA than leisure time PA. Its use as a way of ranking individuals in three categories (terciles) of total PA will produce an approximated degree of misclassification of 30% in the lowest tercile of males meanwhile this degree will not go far beyond than 10% for other categories in both sexes. PMID- 7558632 TI - [The use of statistical methods in 2 medical journals with high impact factor]. AB - This paper intends to contribute to the discussion about the use of statistical techniques within the current biomedical and health investigation. We hypothesized that statistical methods necessary to go ahead with most high level research were rather small. Two high impact factor journals about epidemiology and health sciences were selected: New England Journal of Medicine and American Journal of Epidemiology. Both represent what we have called "successful research". The 2386 original and special articles which were published along the 1986-1990 period were carefully examined. Each statistical technique was classified into one of four complexity levels. Each paper was eventually classified on the same grounds. The frequency of each method was used within each level was then accounted. Statistical cited literature was also registered for each article. Finally, a descriptive examination of this information was developed. The results show that techniques widely dominant are those classified as from lower levels. Only the 12% for New England Journal of Medicine, and 17% for American Journal of Epidemiology belong to the most complex type. It was shown that current biomedical research dismisses, to a large extent both, complex statistical techniques and specialized statistical literature. In general, it limits itself to resources no more ahead than elementary multivariate procedures. It was concluded that simpler statistical methods are useful enough not only to understand what is currently published in journals like the ones studied but to produce high level results as well. PMID- 7558631 TI - [Comparative study of a neonatology unit by means of 2 patient grouping technics]. AB - In this study we make a measurement of the case-mix of the neonatology section in Hospital Puerta del mar in Cadiz during the year 1990, through two methods: Diagnosis Related Groups (GRDs), and Patient Management Categories (PMCs). We compare this case-mix with other hospitals. We compare this case mix with other hospitals. We also compare both methods between themselves through a multiple lineal regression. The results point out that the complexity of casuistry attended in Cadiz was lower to those attended in other control hospitals, and that these ones worked more efficiently. As for comparison of both technics, the GRDs demonstrated to have a superior capacity than the PMCs to discriminate the casuistry. PMID- 7558633 TI - [Public policies of research]. AB - With its present configuration, the Spanish public device of research in health sciences has limited possibilities to achieve properly the aims of the Ley General de Sanidad, due to its reduced financial importance as well as its thematic and lack of mechanisms of interterritorial compensation. These limitations are effective according to its small capacity to provide information for the development of a health system, oriented to the overcoming of territorial and social imbalance as well as the promotion of health and prevention of diseases. The capacity of public policies of research in Spain to affect the work of researchers is very small due to the importance of the widespread practice of not using funds which are not specifically dedicated to research of the institutions employing the researchers, among other reasons. Most of the public resources of research are concentrated in Madrid and Barcelona. This situation can seriously jeopardize in a near future the development and quality of the attention given in Regional Health Services transferred to Autonomic Communities without big cities. The funds are mostly used to finance researches of basic sciences, medical specialties and clinic laboratories. Investigation in Public Health is only 0.8% of the research budgets, and the funds dedicated to research in Health Primary Care are also very small. The present predominant thematic and methodological orientation of health research in our country, with medicalized research aims, subindividual observation units, experimental designs, and analysis which are basically quantitative, can endanger the possibilities of Spain to achieve the health aims established by the OMS. PMID- 7558634 TI - [The debate on the separation of financing functions from service delivery in the welfare state and its relevance to Spain]. PMID- 7558635 TI - [Gaceta Sanitaria]. PMID- 7558636 TI - [The 1993 bibliographic impact factor. Public health and epidemiology]. PMID- 7558638 TI - Transient evoked otoacoustic emission-based screening in typical nurseries: a response to Jacobson and Jacobson. AB - Jacobson and Jacobson (Int. J. Pediatr. Otorhinolaryngol. 29 (1994) 235-248) recently questioned whether TEOAE-based newborn hearing screening similar to what was recommended by the National Institutes of Health could be implemented in a typical nursery setting. They concluded that, 'the theoretical advantage of TEOAEs as a method for screening newborn babies at risk for hearing loss may not be realized in acute practice.' This article presents data based on dozens of currently operational TEOAE-based newborn hearing screening programs which demonstrate that the concerns raised by Jacobson and Jacobson are not representative of what is being experienced by operational newborn hearing screening programs. PMID- 7558637 TI - Otitis media in a population of black American and white American infants, 0-2 years of age. AB - To determine the incidence of otitis media (OM) and the bacteriology of acute otitis media (AOM) in a clinic population of young children in Pittsburgh, 138 black infants and 60 white infants were followed from birth to 2 years of age, examined at monthly intervals and whenever an upper respiratory tract infection (URI) or OM intervened. By 24 months of age the cumulative incidence of episodes of AOM was 43% and 42%, and of episodes of middle-ear effusion (MEE) was 86% and 85% in black and white infants, respectively. The average rate of episodes of AOM was 0.41 and 0.39 and of episodes of MEE was 1.68 and 1.70 in black and white infants, respectively. Tympanocentesis was performed for episodes of AOM and the following organisms were isolated from black and white infants, respectively: Streptococcus pneumoniae 43% and 43% of episodes; Moraxella catarrhalis 24% and 24%; non-typable Haemophilus influenzae 18% and 24%; and Haemophilus influenzae type b 5% and 0%. In both black and white infants first born children had less ear disease. We found no difference in the incidence of otitis media during the first 2 years of life between black and white infants. PMID- 7558639 TI - Clinical manifestations of CHARGE Association. AB - The clinical literature regarding CHARGE Association is mostly retrospective in nature and deals largely with non-auditory issues related to the care and management of these patients with multisystem involvements. In this paper, we describe the clinical findings in 24 patients evaluated in the Division of Audiology and Electrophysiology at the University of Michigan Medical Center from 1983 to 1993. We report on the clinical manifestations of CHARGE Association in these patients with particular attention paid to their audiologic status. We discuss the relationships between auditory, ear, and craniofacial anomalies. Our review of these previously unreported cases suggests the following: (1) a variety of audiologic outcomes is possible, however, if a sensorineural or mixed hearing loss exists, it tends to be severe in degree; (2) progressive hearing loss does not appear to occur, but recurring otitis media is a probable confounding factor in the early identification of hearing loss; (3) congenital unresolved facial weakness may serve as a reliable predictor of sensorineural hearing loss; and (4) amplification use may be poor due to a number of factors. We hope to offer guidance to the professionals from assorted disciplines who participate in the care of these children. PMID- 7558640 TI - Long-term airway considerations after treatment of severe pediatric laryngotracheal stenosis in five children. AB - After initial treatment of severe laryngotracheal stenosis (LTS), we evaluated subsequent airway function. Five children between 2 and 11 years were treated previously for severe LTS by T-tube stenting. One case underwent subsequent laryngotracheal reconstruction. All patients demonstrated dyspnea, the severity of which increased with age and duration of time after completion of stenosis treatment. In the most severe cases, magnetic resonance imaging and endoscopy revealed secondary subglottic stenosis. Ventilatory function tests disclosed obstruction of both extrathoracic and pulmonary origin. These findings raise questions regarding the treatment of the initial stenosing tissue and of the secondary stenoses. PMID- 7558641 TI - Lingual pressures induced by mouthgags. AB - Airway obstruction from tongue edema following intraoral procedures is uncommon. However, the insidious nature of postoperative lingual edema and the gravity of acute airway obstruction requires diligent monitoring by the surgeon. The etiology of lingual edema is likely related to tissue ischemia, secondary to venous or arterial obstruction. This study shows that during normal usage the mouthgag can easily generate pressures which typically exceed venous pressure and often exceeds arterial pressure. Suspension of the mouthgag almost always exceeds arterial pressure. Whether that pressure is transferred to the respective arteries and veins is likely highly variable and relates to the patient's individual dentofacial anatomy. Avoidance of these high pressures should minimize the risk of postoperative lingual edema. The U.C. Davis Mouthgag Usage Protocol is presented. PMID- 7558642 TI - Fundamental voice frequency and jitter in girls and boys measured with electroglottography: influence of age and height. AB - The fundamental frequency and jitter of the voice was measured by electroglottography in 71 children between the age of 7 and 15 years. In this series of children the fundamental frequency and jitter did not depend on the gender. The median (range) fundamental frequency was 244 (182-331) Hz in girls and 250 (205-293) Hz in boys. It decreased with increasing height (r = -0.59; P < 0.0005) and age (r = -0.57; P < 0.001). The median jitter ratio was 9.7 (1.6 33.3) in girls and 10.3 (2.0-4.3) in boys. The jitter ratio was negatively related to height (r = -0.31; P < 0.05), but not to age. PMID- 7558643 TI - Ligation of the common carotid artery for the management of a mycotic pseudoaneurysm of an extracranial internal carotid artery. A case report and review of the literature. AB - Although vascular involvement by deep neck space infections occurs very rarely today with the widespread use of antibiotics, they often pose a significant challenge to the modern otolaryngologist, who most likely will have had no previous experience with either the diagnosis or treatment of these potentially life-threatening infections. We describe the case of a young female presenting with fevers, dysphagia, and blood-tinged sputum, who was diagnosed by contrast enhanced computerized tomography, to possess a mycotic pseudoaneurysm of her right extracranial internal carotid artery, for which ligation of her common carotid artery was required. We also discuss both the clinical findings which should lead one to suspect that a neck infection may be involving the extracranial carotid arteries, and the English literature, on the morbidity and mortality of ligating a common carotid artery. PMID- 7558644 TI - Dapsone-induced methemoglobinemia: an anesthetic risk. AB - Dapsone is used to treat several systemic inflammatory diseases, many of which have head and neck manifestations, such as leprosy, systemic lupus erythematosus, rhinosporidiosis, relapsing polychondritis, dermatitis herpetiformis, pemphigus vulgaris and bullous pemphigoid. It has also been recently used prophylactically alone or in combination against malaria and in AIDS patients against Pneumocystis carinii infections. This is significant to the otolaryngologist-head and neck surgeon since approximately 40% of AIDS patients will have head and neck manifestations. Thus, the likelihood that otolaryngologists will be treating patients who are taking dapsone regularly is significant. We present a case of a 16-year-old female who presented with a presumptive diagnosis of discoid lupus for biopsy confirmation of her disease. Induction of general anesthesia was complicated by methemoglobinemia, an uncommon side effect of dapsone. We will discuss recognition and prevention of this side effect, its potential anesthetic implications, complications and treatment. PMID- 7558646 TI - Aging and performance on implicit memory tasks: a brief review. AB - Older adults exhibit poorer performance than younger adults on tests of recall and recognition. Recall and recognition are labeled explicit memory tasks; they require conscious recollection of a previous learning experience. This paper deals with the performance of elderly people on tasks that do not require intentional recollection--so-called implicit memory tasks. It appears that older subjects perform normally on perceptual implicit tasks, but show somewhat reduced priming in conceptual implicit tasks. This finding is in accordance with the neuroanatomical changes in old age. Implications for theories of implicit memory are discussed. PMID- 7558647 TI - Increase of REM duration and decrease of REM latency after a prolonged test of visual attention. AB - Following one adaptation night, the nocturnal sleep of nine female college students was recorded for two baseline and two experimental nights. In both experimental nights, prior to sleep the subjects performed a 4 hour long visual task requiring the recognition of letters tachistoscopically presented in the right visual hemifield. In the first experimental night eye movements were permitted, while in the second subjects were allowed only covert movements of attention while keeping their gaze on a fixation point. Results show that, as a consequence of the experimental manipulation, there is an increase of REM duration and a shortening of REM latency without any concomitant significant change in REM density and in the duration and latency of the other sleep stages. The increase of REM duration was greater in the night in which only attentional "movements" were permitted (i.e., when there is a covert orienting of attention), suggesting that a load of spatial attention might have a role in increasing REM pressure. PMID- 7558648 TI - Mood recognition across the ages. AB - A cross-sectional study was conducted in which level of mood recognition was assessed from early childhood through old age. Asymptotic performance was obtained on facial affect recognitions tasks from ages 3-34 yrs. There was a progressive decline in facial affect recognitions beginning at age 45, with the principal problem that of recognizing angry faces. With respect to auditory prosody, there was an improvement in recognition level from ages 3-12 yrs. Performance remained asymptotic through age 43 and then began to decline increasingly beyond age 45. PMID- 7558645 TI - Malignant schwannoma located in the retroauricular region. AB - There have been no reports about malignant Schwannoma located retroauricularly in the medical literature we reviewed. Malignant Schwannoma is a very malignant tumor, which is rare. For diagnosis, microscopic and immunohistochemical studies are needed. It is often seen together with neurofibromatosis. The prognosis is very poor and the only available treatment is surgical excision. Although it is known that both radiotherapy and chemotherapy are not effective, we achieved good outcome with this combination applied postoperatively. PMID- 7558649 TI - Demography of DSM-III borderline personality disorder (PD): a comparison with Axis II PDs, affective illness and schizophrenia convergent and discriminant validation. AB - Demographic characteristics of borderline personality disorder (PD) defined according to DSM-III criteria were compared with those found for schizophrenia, affective illness, and other Axis II PDs. Borderline PD, unlike affective illness and most other Axis II PDs, usually occurs before the age of 30. By contrast to antisocial PD and schizophrenia, borderline PD usually occurs after the age of 25. For borderline PD (N = 280) average age was significantly more homogeneous compared with affective illness (N = 157) and Axis II PDs (N = 71) across 9 studies. By contrast, variability for 63 predominantly male schizophrenics in 3 studies was significantly less, reflecting the younger age at admission compared with borderline PD (N = 84). According to predictions based upon an age-of-risk hypothesis (Dahl, 1985) for 106 borderline PD patients, a significantly lower percentage were > or = 40 years of age than diagnostic controls (N = 181) predominantly with DSM-III affective illness. Borderline PD is predominantly diagnosed in females either single or who have been divorced compared with Axis II PDs and affective illness, to a lesser extent. Unlike antisocial PD, as well as schizophrenia, the preponderance of male and single/divorced patients usually occur significantly less than for borderline PD. Borderline PD usually occur significantly less than Axis II PDs, affective illness and schizophrenia and ethnic minorities, particularly Afro-Americans. These differences in ethnic/racial distribution are explained in terms of two hypotheses. From the perspective of demographic variables, borderline PD closely converges with neither (a) schizophrenia, (b) antisocial PC, (c) other Axis II PDs, nor (d) affective illness. Evidence for discriminant and convergent validation of these data is provided by (a) cluster analyses and intersample pairwise contrasts, as well as comparisons with (b) clinical samples selected on the basis of DSM-III-R and criteria of the (c) Diagnostic Interview for Borderline Patients, (d) a longitudinal case registry study conducted in Denmark, and (e) prospective surveys conducted among (i) North Carolina community residents and (ii) first degree (nonpatients) relatives of psychiatric patients in Iowa. PMID- 7558650 TI - Hemispheric semantics: effects on pictorial organization of patients with unilateral brain damage. AB - Memory for incongruous scenes (violating "knowledge-of-the-world") versus unorganized (jumbled) arrays was tested in war veterans with unilateral penetrating missile wounds of the brain in order to assess hemispheric differences in semantic schemas in long-term memory. Left brain-damaged (LBD) patients were selectively impaired in remembering unorganized scenes compared to right brain-damage (RBD) patients. There was no significant LBD-RBD difference in remembering incongruous scenes. The results confirm the presence of hemispheric asymmetry for semantic schemas in long-term memory, even in pictorial material. However, some features in the organization of semantic schemas are common to both hemispheres and suggests presence of similar knowledge systems. The asymmetry is discussed in terms of intact left hemisphere specialization for assigning meaning to a visual scene when obvious meaning appears to be absent. PMID- 7558651 TI - Chronic relapsing multiple sclerosis: a case of rapid recovery by application of weak electromagnetic fields. AB - A 54 year-old woman was diagnosed with multiple sclerosis (MS) in 1985 at the age of 45 after she developed diplopia, slurred speech, and weakness in the right leg. A Magnetic Resonance Imaging (MRI) scan obtained in 1985 showed several areas of plaque formation distributed in the periventricular white matter and centrum semiovale bilaterally. Coincident with slow deterioration in her condition since 1990 a second MRI scan was obtained in 1991 which showed a considerable increase in the number and size of plaques throughout both cerebral hemispheres, subcortical white matter, periventricularly and brainstem. In 1994, the patient received treatment with Interferon beta- 1b (Betaseron) for 6 months with no improvement in symptoms. However, following two successive extracranial applications of pulsed electromagnetic fields (EMFs) in the picotesla (pT) range each of 20 minutes duration the patient experienced an immediate improvement in symptoms most dramatically in gait, balance, speech, level of energy, swallowing, mood, and vision. On a maintenance program of 3 treatments per month the patient's only symptom is mild right foot and leg weakness. The report points to the unique efficacy of externally applied pT range EMFs in the symptomatic treatment of MS, indicates a lack of an association between the extent of demyelinating plaques on MRI scan and rate and extent of recovery in response to EMFs, and supports the notion that dysfunction of synaptic conductivity due to neurotransmitter deficiency particularly of serotonin (5-HT) contributes more significantly to the development of MS symptoms than the process of demyelination which clinically seems to represent an epiphenomenon of the disease. PMID- 7558653 TI - Reversal of visuospatial deficit on the Clock Drawing Test in Parkinson's disease by treatment with weak electromagnetic fields. AB - Visuospatial deficits are among the most frequently encountered abnormalities in neuropsychological testing of patients with Parkinson's disease, being present in up to 90% of cases. Clinically, impairment of visuospatial functions may not be noted by Parkinsonian patients but may contribute to various functional disabilities including frequent falls, difficulties operating a vehicle, ambulating, and dressing. I have reported recently that treatment with external electromagnetic fields (EMFs) in the picotesla (pT) range intensity is an effective nonpharmacological modality in the management of the motor and various cognitive deficits of Parkinsonism including visuoperceptive and visuospatial functions. The present communication concerns four fully medicated Parkinsonian patients who, in response to treatment with EMFs, exhibited reversal of visuospatial impairments as demonstrated on the Clock Drawing Test. Specifically, prior to treatment with EMFs these patients demonstrated a visuospatial deficit which was evident by the placement of the numbers on the clock distant from the periphery. Following a series of treatments with EMFs this visuospatial deficit was corrected. The report supports prior observations demonstrating that externally applied pT range intensity EMFs may bring about reversal of visuospatial deficits in Parkinsonian patients which usually are not improved by treatment with dopaminergic or anticholinergic drugs. PMID- 7558652 TI - Biochemical, neuropathological and behavioral studies in hens induced by acute exposure of tri-ortho-cresyl phosphate. AB - A "hen model" or organophosphorus induced delayed neuropathy (OPIDN) has been developed using white leghorn exposed acutely to one of five dosages of tri-ortho cresyl phosphate (TOCP), between 300 to 700 mg/Kg. Neuropathy target esterase was studied in brain and peripheral nerve 24 and 48 hrs following exposure. Behavioral symptoms abnormality was assessed from days 1 through 20 after exposure using a 7 point rating scale and neuropathological examination was conducted on sample collected from animals on days 0, 7, 14 and 21. Neuropathological abnormalities were indicated by damage scores between 0 (no damage) and 4 (gliosis of brain tissue, myelin loss, appearance of axonal foci etc and more than 55% degeneration of peripheral nerve fibres). TOCP (600 and 700 mg/Kg, orally) was able to inhibit NTE more than 75% in brain and peripheral nerves. TOCP at the same dosage was also capable of resulting maximal levels of neuropathological score at 4. After exposure to doses weight loss was observed abruptly in a greater extent at the beginning leading to a change in weight gain till the end of the experiment. Behavioral signs were also dose dependent. Symptoms (gain abnormality, ataxia, paresis) were noted on the early stage of experiment. Inhibition of NTE was 65% could not be reached in hens given TOCP without causing lethality and no significant ataxia or lesions developed in those birds. Behavioral signs were also observed to be late onset. These data indicate that more than 75% inhibition of peripheral nerve NTE after 24 hr exposure was predictive of severe behavioral abnormalities and pathology in the hen whereas less peripheral NTE inhibition was indicative of less severe behavioral abnormalities and a lower score for neuropathological damage. PMID- 7558654 TI - Improvement of body image perception in Parkinson's disease by treatment with weak electromagnetic fields. AB - Neuropsychological studies have demonstrated that Parkinson's disease (PD) is associated with various cognitive deficits ultimately leading in about 30% of patients to the development of dementia. These studies have demonstrated also a greater decrement of right hemispheric functions which are manifested by visuospatial deficits occurring in up to 90% of PD patients. The Human Figure Drawing Test has been employed in the assessment of generalized intellectual deterioration and specifically in the evaluation of visuperceptive, visuospatial and visuoconstructional abilities in brain injured patients. I have demonstrated recently, on the basis of various drawing tests, that external application of electromagnetic fields (EMFs) in the picotesla (pT) range intensity improved visuoperceptive and visuospatial functions in Parkinsonian patients. In the present communication I present 4 fully medicated nondemented Parkinsonian patients who were administered the Human Figure Drawing Test before and after a series of treatments with EMFs. The Human Figure Drawing Test was selected for the study specifically since it was shown to be sensitive to the effects of surgery to the basal ganglia in Parkinsonian patients. Prior to application of EMFs these patients' drawings showed distortion, poor perspective, impoverished facial expression, and lack of attention to details suggested poor body image perception related to right posterior hemispheric dysfunction. In response to the administration of EMFs the group demonstrated a striking improvement in the drawings particularly the depiction of the face the perception of which is localized to the right parietal lobe. These findings demonstrate that treatment with pT EMFs improves body image perception in Parkinsonian patients, a deficit which may remain unaffected by treatment with standard dopaminergic pharmacotherapy. PMID- 7558655 TI - Weak electromagnetic fields improve body image perception in patients with multiple sclerosis. AB - Neuropsychological studies have demonstrated that multiple sclerosis (MS) is associated with various cognitive deficits and it has been suggested that it be considered a form of subcortical dementia. It is now recognized that visuoperceptive and visuomotor deficits commonly occur in MS patients particularly in those with chronic progressive course of the disease. The Human Figure Drawing Test has been employed in the assessment of generalized intellectual deterioration and specifically in the evaluation of visuoperceptive, visuospatial and visuoconstructional abilities in brain injured patients. I have demonstrated recently, on the basis of various drawing test, the external application of electromagnetic fields (EMFs) in the picotesla (pT) range intensity improved visuoperceptive and visuospatial functions in patients with MS. In the present communication I present five MS patients who were administered the Human Figure Drawing Test before and after a series of treatments with EMFs. Prior to application of EMFs four of these patients' drawings showed distortions, poor perspectives, impoverished facial expression, and lack of attention to details suggesting poor body image perception related to right posterior hemispheric dysfunction. In response to the administration of EMFs the group demonstrated improvement in motor disability which was associated with a striking improvement in the drawing particularly the drawings of the face the perception of which is localized to the right parietal lobe. These findings demonstrate that treatment with pT EMFs improves body image perception in MS patients thus corroborating previous observations which demonstrated this treatment modality to exert beneficial effects on cognitive functions in patients with MS. PMID- 7558656 TI - The case for ostectomy--a time-tested therapeutic modality in selected periodontitis sites. AB - Although ostectomy has been proven in numerous studies to be an effective means of reducing pocket depth, some clinicians still question its merits. To determine the efficacy of ostectomy for the treatment of periodontitis, 870 pairs of presurgical and postsurgical radiographs of sites treated by ostectomy were examined. The postsurgical radiographs, taken 5 to 30 years postoperatively, revealed distinct lamina dura and demonstrated that ostectomy is an effective and predictable technique for intercepting the progressive loss of attachment in selected sites with periodontitis. PMID- 7558657 TI - An 11-year reevaluation of resin-bonded fixed partial dentures. AB - Clinical success of resin-bonded fixed partial dentures is primarily dependent on their long-term retention. Retention rates of resin-bonded prostheses placed in 66 patients treated by three dentists in private practice were evaluated in the present study. Seventy-seven resin-bonded fixed partial dentures were followed over a period of 1 year to 11 years. Thirty (39%) became dislodged, but nine were successfully rebonded; thus there were 21 failures (27%). Prosthesis location and operator expertise minimally influenced the longevity of these restorations, but the luting agents tested in this sample had a profound impact on retention. Debonding rates were excessive for some materials tested, whereas other agents proved to be predictable. Modifications in preparation design that enhanced resistance form were also likely contributors to improved retention rates. PMID- 7558658 TI - The histology of new attachment utilizing a thick autogenous soft tissue graft in an area of deep recession: a case report. AB - An area of deep, long-standing recession on a mandibular first premolar was treated for root coverage in a 40-year-old woman. The recession was 6.0 mm deep with a probing depth of 2.0 mm, and there was no attached gingiva. A thick (1.5 mm) free autogenous epithelium and connective tissue graft from the palate was placed to the cementoenamel junction of the tooth after instrumentation and tetracycline conditioning of the root surface. The tooth and facial soft tissues were removed in block section 10.5 months later. At the time of extraction, there had been a gain of 5.0 mm of root coverage, and there was 5.0 mm of keratinized gingiva on the facial aspect. The probing depth was 1 mm. Histologic measurement showed 4.4 mm of new attachment and 4.0 mm of new bone growth. The coronal extent of the new attachment and new bone were in an area previously exposed by recession. PMID- 7558659 TI - Resorbable membrane in the treatment of human buccal recession: a nine-case report. AB - Recent studies have reported the successful use of guided tissue regeneration procedures with nonresorbable barrier membranes to treat buccal recession in humans. Nonresorbable membranes, however, require a reentry procedure for removal, disturbing the delicate healing process. Resorbable membranes were used in a guided tissue regeneration procedure in nine patients with one site of buccal recession each. The resorbable barrier yielded satisfactory clinical results, providing significant gains in probing attachment and root coverage. However, both the surgical technique and the design of the barrier used require improvement for application at sites of buccal recession. PMID- 7558660 TI - The accuracy of dental radiographic techniques used for evaluation of implant fixture placement. AB - Periapical, panoramic, linear tomographic, and computerized tomographic radiographs were made of a partially dentate human mandible with four implants in place. Measurements taken from the radiographs and computer-generated images were compared to measurements made directly on the cross-sectioned test specimen. Periapical radiographs produced the most accurate measurements. Computerized and linear tomographic images provided the unique advantage of cross-sectional views of anatomic structures, but image blurring inherent to linear tomography and volume-averaging error inherent to computerized tomography affected the accuracy of measurements made from these images. The use of a dense dimensional reference object helped to compensate for radiographic distortion and is appropriate when projection radiographic techniques are used. The CT reference scale provided the most accurate method for interpreting measurements made from the computerized tomogram. The use of a dense dimensional reference object is inappropriate with CT because of its volume-averaging error. PMID- 7558661 TI - New dimensions in guided tissue regeneration treatment modalities for profound marginal periodontitis. AB - This article presents two methods of guided tissue regeneration with polytetrafluoroethylene membranes that promote extensive regeneration of periodontal supporting tissue around several adjacent teeth. These techniques are effective in patients with profound marginal periodontitis and advanced horizontal and vertical bony defects affecting large areas of the mouth. In method A, the goal of regeneration is reached by covering the defect with a row of several overlapping membranes. However, the favorable results obtained with this method had to be weighed against various problems concerning the surgical procedure and wound healing. These unsolved difficulties prompted the development of method B, in which only one large membrane is used. After appropriate relieving incisions are made in the membrane, it is placed into the interdental space and thus able to cover an extensive periodontal defect. During the last 4 years, favorable results have been obtained with both new methods of guided tissue regeneration. PMID- 7558663 TI - Mechanical loading affects the synthesis of decorin and biglycan in intact immature articular cartilage in vitro. AB - Sesamoid bone cartilage from the metacarpophalangeal (MCP) joints of six-month old calves was cultured intact on its bone support. The sesamoid bones for the experiment were subjected to loading with 0.2 MPa at 0.2 Hz for a week. Controls were cultured for 0 or 7 days, labelled with [35S] sulfate for 17 h, harvested, and extracted in 4 M guanidine HCI with proteinase inhibitors, for analysis on Sepharose CL-2B columns under dissociative conditions. It was found that the population of labelled small proteoglycans was significantly larger in the loaded cartilage than in the cultured controls. This population was pooled and further purified on dissociative Sepharose CL-4B columns. The resulting profiles showed two peaks. The material eluting at Kav 0.80 contained only chondroitin sulfate and keratan sulfate chains. The peak at Kav 0.48 was analysed by SDS polyacrylamide gel electrophoresis. It consisted of the dermatan sulfate proteoglycans decorin and biglycan. The amounts of newly synthesized decorin and biglycan in the cultured control cartilage were lower than in the control cartilage that had been labelled on day 0. The synthesis of decorin, but not of biglycan, was significantly higher in the loaded cartilage than in the cultured control cartilage. Through its interaction with collagen type II, decorin may aid the adaptation of articular cartilage to increased loading. PMID- 7558664 TI - Inhibitory effect of morphine on the control of rat fibroblast cell proliferation. AB - The G0/G1 to S-phase transition in quiescent EL2 rat fibroblast cells stimulated by mitogen (such as the epidermal growth factor) can be blocked by the addition of morphine to the system. The drug must be actively present when quiescent EL2 cells are induced to enter the proliferative state. Even when morphine is added after mitogenic stimulus, an exposure only within 6 hours is required to inhibit DNA synthesis. These results suggest that morphine can directly influence the proliferation of the nonlymphoid cell system, particularly during the establishment of a competence state (i.e. G0/S phase transition). PMID- 7558662 TI - Coronally positioned palatal sliding flap. AB - A new flap design, the coronally positioned palatal sliding flap, was used to cover barrier membrane placed over implants in one patient, and to provide localized ridge augmentation around implants in another patient. The method is a valid surgical approach because of the favorable risk-benefit ratio. The surgical technique is easy to to perform, and it is possible to obtain a sufficient sliding position of the palatal tissue. This new palatal flap design may be indicated for a variety of periodontal surgical procedures, including guided tissue regeneration and implant surgery. PMID- 7558665 TI - Accumulation, release and subcellular localization of azithromycin in phagocytic and non-phagocytic cells in culture. AB - The authors have examined the pharmacokinetic parameters of azithromycin in phagocytic (J774 macrophages) and non-phagocytic (rat embryo fibroblasts and NRK cells) cultured cells. Azithromycin demonstrates an exceptionally large accumulation in all the cell types tested (perhaps in two functionally and structurally distinct compartments) and a slow release of the cell-associated drug. Azithromycin probably accumulates in cells by a non-specific transport process following the model of diffusion/segregation. The cell-associated drug distributes mostly in the lysosomal compartment (50-70%) and the remaining part is freely soluble in the cytosol. In fibroblasts, and to a lesser extent in NRK cells, azithromycin (10mg/l) induces a decrease of the buoyant density of the lysosomes which may be brought about by the drug itself together with osmotically bound water and/or by the accumulation of low-density materials within these organelles. These observations open important questions with respect to the potential toxicity of azithromycin. The significance of such alterations and of their biological consequences are at present under investigation. PMID- 7558667 TI - The aging process of skin and the increase in size of subcutaneous adipocytes. AB - The aging of skin has been associated with an increase in size of the adipocytes located within the subcutaneous tissue. This topic is the subject of our study conducted on rats clinically treated with L-acetyl-carnitine (LAC) at 4, 8, 16, and 21 months of age and on a control group. Normal rats showed a significant increase in adipocyte diameter between four and eight months, and between sixteen and twenty-one months of age. Rats treated with L-acetyl-carnitine did not show significant changes up to the age of sixteen months, but did so between sixteen and twenty-one months of age. Four-month-old rats, both those under treatment and controls, did not show a significant change in adipocyte diameter. On the other hand, rats receiving L-acetyl-carnitine showed significantly smaller adipocyte diameters than those of the control group. Our results demonstrate that the long term administration of L-acetyl-carnitine blocks the progressive increase in size of the subcutaneous adipocytes present in the rat's aging skin. We hypothesize that L-acetyl-carnitine reequilibrates the catabolic deficit of fats in the skin of the elderly. PMID- 7558668 TI - Effects of dopamine on isolated failing rat heart. AB - Dopamine has been used for many years to treat patients with severe heart failure. It is not clear whether improvements of cardiac function may be due to a direct action on heart. This study was aimed to investigate the direct action of dopamine on failing heart. we chose male Wistar rats which had undergone uninephrectomy under ether anaesthesia to induce hypertension to result in heart failure. After 5 weeks the hearts were excised and perfused according to Langerdoff's technique. Heart rate, systolic and diastolic ventricular pressures, the derivative of the intraventricular pressure time ratio, and coronary flow were measured at baseline, at 2 and 5 min and then every 5 min during a 30-min period. Rat hearts were divided into 4 groups of 5 hearts: group 1, perfused without drug; group 2, perfused with dopamine at 4 micrograms/kg/min; group 3, perfused with dopamine at 8 micrograms/kg/min; group 4, perfused with dopamine at 8 micrograms/kg/min and with 100 nM I.C.I. 118.551 (beta 2-ant: beta-2 receptors antagonist) at the same time. Our results show that dopamine induced a negative inotropic effect and a reduction of coronary flow. Moreover, there was a significant chronotropic action even when dopamine was administered at high concentrations. So we found no positive dopamine effect on isolated failured hearts of rat. This might be explained by both alpha-1-induced vasoconstriction and the stimulation of alpha-1B receptors. We conclude that the favourable effects of dopamine in heart failure could be due to DA1 vasodilation rather than to a direct inotropic action on the heart. PMID- 7558666 TI - Protective effect of some calcium antagonists against mitochondrial calcium injury. AB - Ischaemia induces an increase in calcium cytosolic concentration, leading to a mitochondrial Ca2+ overload. As Ca2+ uptake and storage into mitochondria are the alternative route to oxidative phosphorylation, the Ca2+ overload induces a decrease in ATP synthesis. We have tested in vitro the ability of certain calcium antagonists to restore the ATP synthesis inhibited by mitochondrial calcium overload. Our results showed that diltiazem and bepridil, clinically used as antiischaemic agents, each can restore the ATP synthesis. It is suggested that our in-vitro results might serve to explain the antiischaemic properties of some calcium antagonists. PMID- 7558669 TI - Immunocytochemical localization of plasminogen activators in carcinomas in the cervix and vulva. AB - The presence of plasminogen activators (PA) in a variety of solid tumours appears to correlate, in a number of instances, with enhanced invasive and/or metastatic ability. Urokinase and tissue-type plasminogen activators (u-PA and t-PA) in normal and neoplastic tissues of cervix and of vulva were immunohistochemically identified by means of polycyclonal antibodies. In addition, frozen sections were analysed for u-PA and t-PA activity by in-situ zymography technique. Data collected in our study showed that in invasive cancer u-PA increased more in malignant cells as compared to normal cells in both the inactive and active enzymatic forms. The t-PA distribution pattern was related to angiogenesis while it did not relate to the degree of tumor differentiation. A synergic interaction between proteolytic tumoral activity and proteolytic inflammatory action could be hypothesized. PMID- 7558671 TI - Linking quality improvement with primary health care. AB - To improve the quality of health care, the PHC strategy can be linked to continuous quality improvement (CQI)--also referred to as total quality management (TQM). The linkup--with emphasis on consumer participation--is explained below. PMID- 7558670 TI - Letter from The Netherlands. PMID- 7558672 TI - Primary health care progress in nursing citations. PMID- 7558673 TI - Measuring quality of life in Thai and US communities. AB - Assuring that the quality of life is continually improved for community residents is an important outcome of primary health care (PHC) programmes. Yet, how can it be measured. Below, the authors describe a tool developed to elicit client perceptions of PHC conditions in two communities in two different cultures: one in Thailand, the other in the USA. Such a tool can help communities prioritize their concerns and monitor the effect of PHC interventions. PMID- 7558674 TI - Worldwide survey of nursing issues. AB - Taking the international nursing pulse rate via questionnaire has been a biennial task at ICN since 1991. By learning about its members' more pressing concerns, ICN can better respond to their needs in way of specific programmes, documentation, workshops or even a special task force. Although efforts have been made to increase the response rate, it continues to be under half of total ICN members (112 in 1994). Below, the responses from 46 members, or 43% of membership, representing seven regions worldwide, regarding nursing issues, priorities and activities during the 1993-94 period. PMID- 7558675 TI - An audit study of defaulters of regular psychiatric outpatient appointments in Hong Kong. AB - This paper describes an audit study of general psychiatric outpatient defaulters in Hong Kong. Defaulters were increased among those who were married, unemployed, housewives, seen within one year, receiving medications and previously admitted to hospital. Clinical diagnoses (ICD-10 Axis-1) of the F1, F2, F3 and F7 groups were also associated with increased defaulting behaviour. However, fewer defaulters were found among those who were aged below 19, single, employed, students, and had diagnostic groups of F4 and F9. These results added new information on the non-attendance behavioral pattern of Chinese patients. PMID- 7558676 TI - Acculturation and psychiatric morbidity among Cambodian refugees in New Zealand. AB - A community survey examining the relationship between acculturation and minor psychiatric morbidity among 223 Cambodians living in Dunedin, New Zealand was conducted. Most subjects would like to retain their parent culture but would also like to assimilate into the host culture, indicating a preference for an integrated mode of acculturation. Those who were older, widowed, less educated, had shorter duration of stay in New Zealand, and of lower socioeconomic status, were less acculturated. Overall, the least acculturated were found to have the highest rate of psychiatric morbidity. When age and sex were controlled, the association between acculturation and psychiatric morbidity remained significant only for women aged 31 to 50. Overall, the associations between psychiatric morbidity and acculturation held true for marital status, duration of stay in N.Z., educational level, and socioeconomic status. The relevant conceptual and methodological issues in acculturation studies were discussed. PMID- 7558677 TI - Day-program-based treatment in the Amsterdam City Center. AB - The Social Psychiatric Services Center (SPDC C/OW) is the acute psychiatric clinic for the Amsterdam city center. The unit, originally an integrated part of a General Psychiatric Hospital located outside Amsterdam, was transferred to the city center in 1988. Since then, the unit has been transformed into a fully operational institute for day-program-based treatment, both as a substitute for 24 hours hospitalization and as an extension of community treatment programs. The SPDC's organization, the format and contents of the treatment program and the integrated cooperation with the district mental health care partners proceed directly from the consistently applied key concepts: day-program-based treatment, continuity of care and tailor-made care. After an overview of some relevant developments in the Netherlands in general and in Amsterdam more specifically the SPDC is described in detail. Some conclusions, based on the experience of 6 years work in this organisation, are drawn, focussing on long term treatment strategies and the differences and merits of community-based versus hospital-based care. PMID- 7558678 TI - Eating disorders: an Indian perspective. AB - Anorexia nervosa and related eating disorders are rare in non-western cultures. In India the information regarding these disorders is very limited. The authors describe five cases of young women who chiefly presented with refusal to eat, persistent vomiting, marked weight loss, amenorrhea and other somatic symptoms. They did not show overactivity or disturbances in body image seen characteristically in anorexia nervosa. Though finally diagnosed and treated as cases of eating disorder, they presented considerable difficulty in diagnosis. The paper discusses the reasons for the seeming rarity of anorexia nervosa in India and sociocultural reasons for its atypical presentation. PMID- 7558679 TI - The pastoral myth of the mental hospital: a personal account. PMID- 7558680 TI - Attributions for admission to Zomba Mental Hospital: implications for the development of mental health services in Malawi. AB - Within Malawi, as in many other African countries, a variety of traditional and modern attributions exist regarding the cause of a person's mental disturbance, or their admission to a 'mental' hospital. It is argued that a good mental health service should consider the beliefs of the patients it seeks to serve. Consequently we studied 103 consecutive admissions to Zomba Mental Hospital in order to find out how patients explained their own admission to the hospital. Traditional attributions were the most common, followed by medical and then psychological attributions. Some patients explained their admission to the hospital by combining traditional, medical or psychological ideas. Content analysis of traditional attributions identified examples of "Tropical Tolerance" and the "Pull Down" phenomenon. The possible interactive nature of traditional, medical and psychological processes is discussed and it is suggested that traditional healers should be incorporated into 'modern' Malawian mental health services. PMID- 7558681 TI - Factor structure of social support and its relationship to minor psychiatric disorders among Japanese adolescents. AB - Although social support has been one of the preferred topics in health psychology for the past two decades, there still remains controversy about its construct and the measurement model for it. The People In Your Life (PIYL) scale, a self-report adaptation of Henderson et al.'s Interview Schedule for Social Interactions (1980), appears to be an outgrowth of the recent developments in this domain. We administered PIYL to two samples, 550 and 434 each, of Japanese adolescents and examined its factor structure, its psychometric properties and its concurrent relationship to minor psychiatric disorders. Of the original four subscales, the presence of two subscales called availability of social integration and availability of attachment was confirmed, while the remaining subscales called adequacy of social integration and adequacy of attachment were found to form one general factor of perceived adequacy. Such a three-factor model appeared to fit the data better, psychometrically more sound and externally more valid in accounting for the minor psychiatric disorders. PMID- 7558683 TI - The comparative cell biology of accessory somatic (or Sertoli) cells in the animal testis. AB - A comparative account is given of recent advances in the cell biology of testicular accessory somatic (or Sertoli) cells in mammals, nonmammalian vertebrates, and invertebrates by comparing and contrasting their structure and function. Their structure is discussed in relation to the nucleus, cytoplasmic organelles, and inclusions (lipids, the cytoskeleton, junctional complexes, and blood-testis barrier, which show great diversity and a variable testicular architecture), and mode of spermatogenesis. A very limited somatic cell-germinal association or its complete absence is observed in some groups of invertebrates. Wherever the somatic accessory cells are present, their comparative functions are discussed in relation to (1) mechanical support and nutrition; (2) translocation of germ cells; (3) paracrine regulation and a combination of male germ cell proliferation and differentiation by secretion of regulatory proteins, including peptide growth factors and hormones; (4) phagocytosis; (5) steroid hormone synthesis and metabolism; and (6) spermiation. Comparative cellular and molecular aspects of Sertoli cell-germ cell and peritubular cell interactions and the regulatory (hormonal) mechanisms involved as well as gaps in our knowledge about the molecular aspects of these interactions are emphasized for a better understanding of diversity in the patterns and regulation of spermatogenesis in animals. PMID- 7558682 TI - Sterility in neonatally androgenized female rats and the decidual cell reaction. AB - In female rats, administration of androgen during early postnatal life results in anovulatory sterility characterized by polyfollicular ovaries and persistent vaginal cornification in adulthood. In these androgen-sterilized rats, the acyclic male pattern of gonadotropin secretion results from permanent damage to hypothalamic centers normally responsive to steroid feedback. The capacity of the endometrium to differentiate into deciduoma in response to endometrial stimulation is markedly reduced in the uterus of the androgenized rats receiving an appropriate regimen of progesterone and estrogen injections. The hypothesis is presented that the lowered uterine responsiveness in the androgenized rats is largely ascribable to the effect of androgen given neonatally rather than to the influence of continued exposure to ovarian estrogen. This review deals with the nature of the uterine response to ovarian hormones and the deciduogenic stimulus in androgenized rats in order to verify the mechanism involved in androgen action on the neonatal uterus. PMID- 7558684 TI - Factors controlling growth, motility, and morphogenesis of normal and malignant epithelial cells. AB - Factors that control epithelial growth, motility, and morphogenesis play important roles in malignancy and in normal development. Here we discuss the molecular nature and the function of two types of molecules that control the development and maintenance of epithelia: Components that regulate epithelial cell adhesion; and soluble factors and their receptors that regulate growth, motility, differentiation, and morphogenesis. In development, the establishment of epithelial cell characteristics and organization is crucially dependent on cell adhesion and the formation of functional adherens junctions. The integrity of adherens junctions is frequently disturbed late in tumor progression, and the resulting loss of epithelial characteristics correlates with the metastatic potential of carcinoma cells. Various soluble factors that induce epithelial growth, motility, or differentiation in cell culture, function via tyrosine kinase receptors. We concentrate here on receptors that are expressed exclusively or predominantly on epithelia, and on ligands that are derived from the mesenchyme. In development, these receptors and their ligands function in mesenchymal-epithelial interactions, which are known to govern growth, morphogenesis, and differentiation of epithelia. During tumor development, mutations or overexpression of the receptors are frequently observed; these alterations contribute to the development and progression of carcinomas. PMID- 7558685 TI - Dynamics of the seminal vesicle epithelium. AB - In newborn rodents, seminal vesicle epithelium (SVEP) cells display a poorly developed rough endoplasmic reticulum (RER) and Golgi complex, and they show no signs of secretion. From puberty onward, secretory material starts to appear, and the RER and Golgi complex progressively develop and reorganize until the adult ultrastructure is established around 40-60 days of age. Multivesicular bodies and lysosomes follow in this development but lysosomes evolve to lipofucsin granules with aging. The duration of the secretory cycle in SVEP cells is shorter than in other exocrine cells and the secretory protein pattern depends on the animal species, androgen status, and sexual activity. SVEP cells are also involved in endocytosis, which is coupled to exocytosis, and their endocytic pathway intersects the exocytic pathway in Golgi cisterns. The structure and function of SVEP cells depends mainly on testosterone, but other hormones and factors, such as the neuropeptide VIP, also influence their activity. Castration leads to programmed death and regression of SVEP cells to an extent that depends on the animal species. In addition, castration induces changes in the secretory protein pattern and delays its intracellular transport. Endocytic kinetics is also delayed following castration. Primary cultures of SVEP cells in a bicameral system are proposed as a model to investigate the activities of SVEP cells further. PMID- 7558686 TI - Molecular organization of hepatocyte peroxisomes. AB - The matrix of peroxisomes has been considered to be homogeneous. However, a fine network of tubules is visible in electron micrographs at very high magnification. This substructure becomes more positive in a high-contrast photocopy and with an imaging-plate method. Clofibrate, bezafibrate, and aspirin increase peroxisomes. In proliferated peroxisomes, the density of matrix is low and the fine network is more visible. The effect of proliferators is more significant in males than in females. This sex difference may involve the action of estrogen, growth hormone, cytochrome P-450 and thyroxine. Mg-ATPase is localized on the limiting membrane of peroxisomes. Even on the membrane of irregular projections of proliferated peroxisomes, Mg-ATPase is evident cytochemically. Carnitine acetyltransferase is detectable in the matrix of proliferated peroxisomes. Withdrawal of proliferators results in a rapid decrease of peroxisomes. This may indicate the existence of peroxisome suppressors. Alternatively, dynamic transformation of vesicular to tubular types in peroxisome reticulum may occur. Such transformation has been described in lysosomes and mitochondria. PMID- 7558687 TI - Mechanistic and developmental aspects of genetic imprinting in mammals. AB - Genetic imprinting in mammals allows the recognition and differential expression of maternal and paternal alleles of certain genes. Recent results from a number of laboratories indicate that, at least for some genes, gametic imprints, which must exist in order to mark chromosomes or genes as having been transmitted via sperm or ovum, are not by themselves sufficient to determine allele expression. Other postfertilization events are required, and these events are subject to both tissue-specific and developmental stage-specific regulation. Changes in imprinted gene methylation during preimplantation and fetal life indicate that the establishment of additional allele-specific modifications is likely to contribute to imprinted regulation. Disruptions in imprinting processes, loss of imprints, and loss of nonimprinted alleles through uniparental disomy are likely to contribute to a variety of developmental abnormalities and pathological conditions in both mice and humans. PMID- 7558688 TI - Molecular mechanisms for passive and active transport of water. AB - Water crosses cell membranes by passive transport and by secondary active cotransport along with ions. While the first concept is well established, the second is new. The two modes of transport allow cellular H2O homeostasis to be viewed as a balance between H2O leaks and H2O pumps. Consequently, cells can be hyperosmolar relative to their surroundings during steady states. Under physiological conditions, cells from leaky epithelia may be hyperosmolar by roughly 5 mosm liter-1, under dilute conditions, hyperosmolarities up to 40 mosm liter-1 have been recorded. Most intracellular H2O is free to serve as solvent for small inorganic ions. The mechanism of transport across the membrane depends on how H2O interacts with the proteinaceous or lipoid pathways. Osmotic transport of H2O through specific H2O channels such as CHIP 28 is hydraulic if the pore is impermeable to the solute and diffusive if the pore is permeable. Cotransport of ions and H2O can be a result of conformational changes in proteins, which in addition to ion transport also translocate H2O bound to or occlude in the protein. A cellular model of a leaky epithelium based on H2O leaks and H2O pumps quantitatively predicts a number of so-far unexplained observations of H2O transport. PMID- 7558689 TI - The role of growth factors in vascular cell development and differentiation. AB - The control of vascular growth and differentiation is a complex system of activity and interaction between positive and negative modulators of these processes. A number of important stimulators and inhibitors of both smooth muscle cells and endothelial cells have now been purified and biochemically characterized. Imbalances in the activity of these factors can result in serious pathologies. In this chapter, we briefly discuss the biology of blood vessel development and growth, review the current literature which describes these stimulators and inhibitors, and discuss current therapeutic strategies designed around these growth modulators. PMID- 7558690 TI - Analyzing renal glomeruli with the new stereology. AB - The highly specialized architecture of the renal glomerulus is altered in a variety of disease states. Morphometric methods, including stereological methods, have been widely used to analyze these changes in both animal and human glomeruli. However, many of the methods available until recently were biased and provided incomplete information. The past few years have witnessed the development of a new generation of unbiased stereological methods. Another advantage of these new methods and strategies is that they are less influenced by technical artifacts than the traditional methods. This chapter describes how these new stereological methods can be used to quantify glomerular morphology. Parameters considered include glomerular number and volume; glomerular cell number and size; and the length, surface area, and number of glomerular capillaries. Methods for obtaining data for average glomeruli as well as individual glomeruli are described. Technical details are included wherever possible. PMID- 7558691 TI - Membrane mechanisms and intracellular signalling in cell volume regulation. AB - Recent work on selected aspects of the cellular and molecular physiology of cell volume regulation is reviewed. First, the physiological significance of the regulation of cell volume is discussed. Membrane transporters involved in cell volume regulation are reviewed, including volume-sensitive K+ and Cl- channels, K+, Cl- and Na+, K+, 2Cl- cotransporters, and the Na+, H+, Cl-, HCO3-, and K+, H+ exchangers. The role of amino acids, particularly taurine, as cellular osmolytes is discussed. Possible mechanisms by which cells sense their volumes, along with the sensors of these signals, are discussed. The signals are mechanical changes in the membrane and changes in macromolecular crowding. Sensors of these signals include stretch-activated channels, the cytoskeleton, and specific membrane or cytoplasmic enzymes. Mechanisms for transduction of the signal from sensors to transporters are reviewed. These include the Ca(2+)-calmodulin system, phospholipases, polyphosphoinositide metabolism, eicosanoid metabolism, and protein kinases and phosphatases. A detailed model is presented for the swelling initiated signal transduction pathway in Ehrlich ascites tumor cells. Finally, the coordinated control of volume-regulatory transport processes and changes in the expression of organic osmolyte transporters with long-term adaptation to osmotic stress are reviewed briefly. PMID- 7558692 TI - Bacterial stimulators of macrophages. AB - Our current understanding of the interaction between bacteria and macrophages, cells of the immune system that play a major role in the defense against infection, is summarized. Cell-surface structures of Gram-negative and Gram positive bacteria that account for these interactions are described in detail. Besides surface structures, soluble bacterial molecules, toxins that are derived from pathogenic bacteria, are also shown to modulate macrophage functions. In order to affect macrophage functions, bacterial surface structures have to be recognized by the macrophage and toxins have to be taken up. Subsequently, signal transduction mechanisms are initiated that enable the macrophage to respond to the invading bacteria. To destroy bacteria, macrophages employ many strategies, among which antigen processing and presentation to T cells, phagocytosis, chemotaxis, and different bactericidal mechanisms are considered to be the main weapons. PMID- 7558693 TI - Sexuality of mitochondria: fusion, recombination, and plasmids. AB - Mitochondrial fusion, recombination, and mobile genetic elements, which are essential for mitochondrial sexuality, are well established in various organisms. The recombination of mitochondrial DNA (mtDNA) depends upon fusion between parental mitochondria, and between their mtDNA-containing areas (mt-nuclei), to allow pairing between the parental mtDNAs. Such mitochondrial fusion followed by recombination may be called "mitochondrial sex." We have identified a novel mitochondrial plasmid named mF. This plasmid is apparently responsible for promoting mitochondrial fusion and crosses over with mtDNA in successive sexual crosses with mF- strains. Only in mF+ strains carrying the mF plasmid did small spherical mitochondria fuse which subsequently underwent fusion between the mt nuclei that contained the mtDNA derived from individual mitochondria. Several successive mitochondrial divisions followed, accompanied by mt-nuclear divisions. The resulting mitochondria contained recombinant mtDNA with the mF plasmid. Such features remind us also of the bacterial conjugative plasmids such as F plasmid. Therefore, in the final part of this chapter, we discuss the origin of sex and its relationship to the sexuality of mitochondria. PMID- 7558695 TI - [Airway occlusion as an emergency. Recognition and treatment]. PMID- 7558694 TI - [Circulatory shock. Extrapulmonary etiology of respiratory insufficiency]. PMID- 7558696 TI - [Respiratory regulation and compensatory mechanisms in ventilatory insufficiency]. PMID- 7558698 TI - [ARDS. Intensive therapy of acute lung failure]. PMID- 7558697 TI - [Severe life threatening asthma attack]. PMID- 7558699 TI - [Biphasic positive "airway pressure" (BIPAP). Principles and clinical applications of a new ventilatory method in acute respiratory insufficiency]. PMID- 7558700 TI - [Respirator dependency. Intensive care unit or home ventilation?]. PMID- 7558701 TI - [Respiratory insufficiency. Role of lung and heart/lung transplantation. Indications and results]. PMID- 7558702 TI - [Epigastric symptoms and alcohol drinking in a mechanic]. PMID- 7558704 TI - [Drug indications and the physician's treatment choice]. PMID- 7558703 TI - [Pelvic symptoms 6 months after implantation of an automatic cardioverter defibrillator]. AB - Very few complications after implantation of an automatic cardioverter defibrillator (ICD) have been observed. Most complications are caused by infections, electrode problems and inappropriate ICD shocks caused by supraventricular tachyarrythmia. We report on a 64-year-old patient who received an ICD after successful resuscitation due to ventricular fibrillation. Six months after ICD implant the patient suffered from abdominal pain. Abdominal X-ray and ultrasound revealed perforation of the ICD into the pelvis. After operative revision and implantation into a new pulse generator pouch, the patient was fine up to 7 months later. Then incarceration of an epigastric hernia was present without any signs that the defibrillator pouch was involved. PMID- 7558705 TI - [Ventricular extrasystole with R-on-T phenomenon in long-term ECG--prognostic relevance, subsequent diagnosis and therapeutic consequences?]. PMID- 7558706 TI - [Diagnosis and therapy of ascites]. PMID- 7558707 TI - Optic disc diameter measurement using planimeter and scanning laser ophthalmoscope. PMID- 7558708 TI - Influence of cerebrospinal fluid pressure on the lamina cribrosa tissue pressure gradient. PMID- 7558709 TI - Acquired retinochoroiditis in hamsters inoculated with ME 49 strain Toxoplasma. AB - PURPOSE: These studies were undertaken to establish an animal model for use in studies of ocular toxoplasmosis. An animal model is needed to examine the development, progression, and resolution of ocular Toxoplasma infections and to study the effects on the disease of currently used and experimental therapies. METHODS: Cysts of the ME 49 strain of Toxoplasma gondii were injected intraperitoneally into each of 60 golden hamsters. The hamsters' eyes were examined before inoculation and at intervals after inoculation, and fundus photographs were taken. Histologic sections were analyzed and photographed to document the ocular effects of the infection. RESULTS: Retinochoroiditis was found in both eyes of all hamsters within 2 to 3 weeks of inoculation. The disease resolved spontaneously without treatment and was quiescent in most cases at 12 weeks after inoculation. The animals remained in good general health, and those tested had high antibody titers to Toxoplasma (1:256 to 1:32,000) at 6 months after the infection. The discovery of cysts and lesions in the retina confirmed the diagnosis. CONCLUSIONS: Although the lesions were not identical to those of human disease, this animal model of ocular toxoplasmosis offers several advantages: reproducibility, short incubation time, spontaneous resolution without treatment, consistent production of cysts, and ease of inoculation intraperitoneally without intraocular injection. PMID- 7558710 TI - Fate of orthotopic corneal allografts in eyes that cannot support anterior chamber-associated immune deviation induction. AB - PURPOSE: Corneal allografts placed in human eyes at high risk often fail, and immune rejection is thought to be a major pathogenic factor. To understand the immunologic factors responsible for rejection in this instance, the authors have created "high-risk" eyes in mice by inducing corneal neovascularization. The authors then examined the fate of orthotopic corneal grafts placed in these beds and assessed the development of donor-specific delayed hypersensitivity (DH) in recipient mice. METHODS: Three interrupted sutures were placed in the central cornea of recipient BALB/c mice to induce corneal neovascularization. Two weeks later, when corneal vessels occupied more than two quadrants of the cornea, mice received orthotopic corneal grafts from donor mice expressing alloantigens encoded by major and minor histocompatibility loci. Corneal allografts were evaluated by slit-lamp examination after grafting, and recipient mice were examined at the time of the rejection to determine whether they had acquired DH to alloantigens expressed on the corneal grafts. RESULTS: Compared to grafts placed in normal eyes, a much higher incidence of rejection was observed among corneal allografts placed in neovascularized eyes (96.7% versus 46.7%). Moreover, grafts in neovascularized beds were rejected much more swiftly (2 weeks versus > 3 to 4 weeks). Rejection of corneal allografts in high-risk eyes coincided temporally with development of intense donor-specific DH, and the specificity of this immune response was directed solely at minor H antigens (not major histocompatibility complex-encoded antigens) on the graft. CONCLUSIONS: These results indicate that eyes rendered high risk by virtue of corneal neovascularization fail to provide immune privilege for orthotopic corneal allografts. In this circumstance, the grafts rapidly induce intense donor specific DH that is readily detectable within 2 weeks of engraftment, at which time the grafts are acutely and universally rejected. The recipient DH response is directed exclusively at minor H antigens on the graft, which is consistent with the view that neovascularization creates graft beds in which recipient antigen-presenting cells infiltrate the graft and carry antigenic information by lymphatics to draining lymph nodes. In this manner, anterior chamber-associated immune deviation is avoided, and potentially allodestructive DH is promoted. PMID- 7558711 TI - Low incidence of retinitis pigmentosa among heterozygous carriers of a specific rhodopsin splice site mutation. AB - PURPOSE: To determine whether a rhodopsin splice donor site mutation at the 5' end of intron 4 is a cause of autosomal dominant retinitis pigmentosa. METHODS: Heterozygous carriers of the same rhodopsin splice site mutation in two pedigrees were identified using single-strand conformation polymorphism analysis. Twelve heterozygous carriers were evaluated by ophthalmoscopy. Goldmann kinetic visual fields, dark adaptation thresholds, and full-field electroretinograms including rod intensity-response functions. Clinical findings from the heterozygous carriers of the splice site mutation were compared with those from heterozygous carriers from a separate family with a known recessive rhodopsin null mutation, Glu249X. RESULTS: Analysis of DNA from 48 members of two pedigrees revealed 25 heterozygous carriers of the splice site mutation, ranging in age from 14 to 82 years. There were no homozygotes with the rhodopsin splice site mutation. Of the 25 heterozygous carriers, 24 were asymptomatic. Eleven asymptomatic heterozygotes were examined, including four older than 65 years of age. They were found to have normal fundi, full visual fields, and slightly elevated final rod dark adaptation thresholds. Their rod electroretinographic b-wave amplitudes were slightly diminished over the full range of blue light intensities. Rod a-wave implicit times were slightly but significantly prolonged in response to the brightest blue flash of light. These subtle abnormalities in rod function were similar to those found in asymptomatic heterozygous carriers of the recessive Glu249X mutation. Only one of the 25 heterozygous carriers of the splice site mutation had symptoms and signs of retinitis pigmentosa. CONCLUSIONS: Because 96% of these heterozygous carriers do not have retinitis pigmentosa, it is unlikely that this mutation in intron 4 is a dominant allele. The subtle abnormalities of rod function found in asymptomatic carriers are similar to those found in heterozygous carriers of a recessive rhodopsin allele. The one heterozygous carrier with retinitis pigmentosa probably has a second mutation in the rhodopsin gene or has a defect or defects in another gene that causes his disease. PMID- 7558713 TI - Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues. AB - PURPOSE: Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication deficient adenovirus human HO-1 cDNA (Adv-HHO). METHODS: Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. RESULTS: Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. CONCLUSIONS: These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of ocular diseases such as cataract, light-induced injury, age-related macular degeneration, and diabetic retinopathy. PMID- 7558715 TI - Frequency and luminance-dependent blood flow and K+ ion changes during flicker stimuli in cat optic nerve head. AB - PURPOSE: The purpose of this study was to investigate whether blood flow in the cat optic nerve head (ONH) is related to increased neuronal activity elicited by diffuse luminance flickering light stimulation. METHODS: ONH blood flow was measured by laser Doppler flowmetry in anesthetized cats during 1 to 3 minutes of flickering light stimulation at controlled luminance and frequency (n = 227 measurements in 18 cats) using either a conventional visual stimulator (repetitive short flashes) or a sinusoidally varying light stimulator. Potassium ion concentration ([K+]) changes in the vitreous humor immediately in front of the optic disk were measured with neutral carrier K+ ionophore liquid membrane microelectrodes. Effects of varying flicker frequency (2 to 80 Hz) at constant luminance were quantified. Effects of luminance were quantified by varying the modulation depth of the stimulus at constant frequency. RESULTS: Both ONH blood flow and [K+] increased during flicker stimulus with an average slope of 0.305% +/- 0.064% (SE)/microM [K+] (257 measurements in 18 cats). The peak ONH blood flow increase was 59% +/- 11% above baseline at 33.3 +/- 3.1 Hz. The peak [K+] increase was 188 +/- 42 microM above baseline at 38.3 +/- 3.3 Hz. Both ONH blood flow and [K+] changes had similar bandpass characteristics with frequency, first increasing, then dropping off at higher frequencies (122 measurements in 10 cats). Both frequency responses were described by power law functions (y = af"). Luminance responses for both ONH blood flow and [K+] changes could be fit by a modified Hill model and were 50% of maximum at light modulation depths of 21.2% +/- 4.6% and 22.5% +/- 3.7%, respectively (53 measurements in 5 cats). CONCLUSIONS: Increases in ONH blood flow were correlated with changes in [K+]. Both responses were remarkably similar, with no significant differences in the frequency for peak responses in ONH blood flow or [K+], in low- and high frequency power law exponents of the two responses, or in the 50% response to light modulation. The results are consistent with close coupling of neuronal activity and ONH blood flow. PMID- 7558712 TI - Photoreceptor cells in the vitiligo mouse die by apoptosis. TRPM-2/clusterin expression is increased in the neural retina and in the retinal pigment epithelium. AB - PURPOSE: To determine the mechanism of photoreceptor cell death in the vitiligo mouse, a model of retinal degeneration in which the genetic defect is not retina specific but is instead caused by single point mutation in the microphthalmia (mi) gene that codes for a basic helix-loop-helix DNA transcription factor. METHODS: Detection of apoptotic cells was performed in fixed retinal tissue using the TUNEL assay in animals 1, 2, 4, 6, 8, 16, 32, 40, and 52 weeks. Electron microscopic analysis was used to confirm the morphologic hallmarks of apoptosis, and Southern blot analysis was used to detect internucleosomal DNA fragmentation. Additionally, the expression of a gene associated with apoptosis, TRPM 2/clusterin, was examined. RESULTS: At ages beyond the time of normal retinal programmed cell death, vitiligo retinas had significantly more TUNEL-positive photoreceptor cells and more photoreceptor cells with condensed chromatin than controls. DNA internucleosomal fragmentation ladders were present in vitiligo retinas even as late as 15 weeks, a time well beyond developmental apoptosis in controls. TRPM-2/clusterin mRNA levels in vitiligo neural retinas were similar to controls initially but were two times greater than controls by 12 weeks. Surprisingly, TRPM-2/clusterin mRNA levels were elevated in the retinal pigment epithelium in the mutant; the expression at one week was two times greater than normals and remained elevated for many months, even though retinal pigment epithelial cells showed no morphologic evidence of apoptosis. CONCLUSIONS: The morphologic and biochemical data suggest that photoreceptor cells die by apoptosis in vitiligo mice. The increased retinal TRPM-2/clusterin mRNA levels may be a direct response to these events. The increased expression of this gene in the retinal pigment epithelium, however, may reflect its role in tissue regression and membrane remodeling. Mechanisms by which the mi gene defect might result in the vitiligo retinopathy are proposed. PMID- 7558714 TI - In vivo gene transfer into murine corneal endothelial and trabecular meshwork cells. AB - PURPOSE: To determine whether a reporter gene can be introduced into adult mammalian corneal endothelial and trabecular meshwork cells in vivo using a recombinant replication-deficient adenovirus. METHODS: Purified replication deficient adenovirus containing the cytomegalovirus-promoted Escherichia coli reporter gene, lacZ, was injected into the vitreous cavities or anterior chambers of 30 adult CD-1 mice using the contralateral eyes as controls. LacZ expression was assessed histochemically in enucleated eyes from 2 to 21 days after injection using the beta-Galactosidase (beta-Gal) assay. RESULTS: LacZ expression was demonstrated in corneal endothelial and trabecular meshwork cells for as long as 14 days after injection. beta-Gal activity was also observed in lens and iris epithelial cells. There was no toxicity of the adenoviral vector demonstrated histologically, and no nonocular tissues expressed lacZ as measured by beta-Gal assay. CONCLUSIONS: A functional gene can be transferred in vivo into adult mammalian corneal endothelial and trabecular meshwork cells using a replication defective adenoviral vector. Gene expression is relatively short-lived compared to that demonstrated previously in other ocular tissues (photoreceptors and retinal pigment epithelium). Adenoviral vectors may be a viable means for short term delivery of therapeutic genes in vivo to cells in the anterior segment of the eye. PMID- 7558716 TI - Nitric oxide controls arteriolar tone in the retina of the miniature pig. AB - PURPOSE: Experimental evidence indicates that the retinal microcirculation is mainly controlled by factors released from the tissue surrounding the arterioles. This study explores whether nitric oxide (NO), a possible factor, is released in the retina and controls the arteriolar tone. METHODS: Using a NO microprobe, the authors measured [NO] in the preretinal vitreous of miniature pigs as a function of distance from the retinal surface. Additionally, the NO-synthase inhibitor nitro-L-arginine was pressure injected. Finally, the retinal pool size of arginine and its biosynthesis from 14C(U)-glucose were biochemically assessed on retinal tissue and acutely isolated Muller cells. RESULTS: At the retinal surface, [NO] measured 6 to 9 microM, and, in the vitreous, it fell to zero approximately 180 microns away from the retina. Therefore, NO is degraded faster in the vitreous (65 to 80 microM.minute-1) than in aqueous solution. Light flicker stimulation of the dark-adapted retina induced a reversible increase of [NO] (approximately 1.6 microM). Preretinal juxta-arteriolar microinjections of nitro-L-arginine (0.6 mM) induced a segmental and reversible arteriolar vasoconstriction of 45%; in contrast, intravenous infusion of nitro-L-arginine had no measurable effect on arteriolar diameter. The retinal pool size of arginine was small (< or = 200 microM), but there was an important rate of arginine biosynthesis in Muller cells. CONCLUSIONS: These results strongly suggest that cells in the retina, other than endothelial cells, produce and release NO, which in turn controls the basal dilating arteriolar tone in the inner retina. PMID- 7558717 TI - Characteristics of [3H]5-hydroxytryptamine binding to iris-ciliary body tissue of the rabbit. AB - PURPOSE: This study sought to identify, characterize, and localize subtypes of 5 hydroxytryptamine (HT) receptors in rabbit iris-ciliary body. METHODS: Radioligand binding assays were performed with [3H]5-hydroxytryptamine on membranes prepared from rabbit iris-ciliary bodies and on tissue sections subsequently developed by autoradiography. RESULTS: [3H]5-HT appeared to bind to a single population of receptors in membrane preparations of rabbit iris-ciliary body. The apparent affinity of the ligand (KD) was 2.19 nM, and the density of binding sites was 58.3 fmol/mg protein. Binding of [3H] 5-HT exhibited guanosine 5-triphosphate sensitivity. Competitive inhibition experiments were performed to differentiate between 5-HT receptor subtypes. A relative potency order of 5-CT > 5-HT = 8-OH-DPAT > ipsapirone > RU24969 > sumatriptan > ritanserin > ketanserin was demonstrated. The apparent inhibitory constants for the ligands tested fit with the profile expected of binding to 5-HT1A receptors. Inhibition studies with [3H] 5-HT plus 100 nM 8-OH-DPAT (which inhibits binding to 5-HT1A receptors only) representing total binding, indicated that no further displacement occurred when ligands preferentially selective for 5-HT1B, 5-HT1D alpha,1D beta, or 5-HT2C were tested. Total binding of [3H] 5-HT in tissue sections developed by autoradiography was displaced completely by 100 nM 8-OH-DPAT. Melatonin showed little affinity for the [3H] 5-HT binding sites. CONCLUSIONS: A population of 5 HT1A receptors is present in rabbit ciliary processes. There is no evidence to suggest the presence of 5-HT1D alpha, 5-HT1D beta, 5-HT2B, or 5-HT2C receptors in the iris-ciliary body. PMID- 7558718 TI - Lymphocyte adhesive interactions with lacrimal gland acinar epithelial cells in primary culture. AB - PURPOSE: In lacrimal glands, cell-cell interactions control the localization of lymphocyte populations that play a role in immune defense at the ocular surface. This study describes lymphocyte adhesive interactions with cultured lacrimal gland acinar epithelial cells. METHODS: Primary cultures of lacrimal gland epithelial cells were used as targets for in vitro lymphocyte binding assays. The relative adherence of lymphocyte populations was determined. Various physiologically active agents and putative ligand analogs were tested for their effect in the binding assay. RESULTS: Thoracic duct lymphocytes (TDL) bound to cultured lacrimal acinar epithelial cells in greater numbers than did thymocytes (54 cells/mm2 versus 8 cells/mm2). B cells showed preferential adherence compared with T cells (75% sIg+, 14% W3/13+). Thoracic duct lymphocyte binding required intact metabolic and membrane-cytoskeletal function and was inhibited by treating the lymphocytes with sodium azide, formaldehyde, or cytochalasin B (23%, 12%, and 10% of control binding, respectively). Further, adherence was dependent on divalent cations. Ethylenediaminetetraacetic acid-mediated inhibition (42% of untreated) was restored by replacing calcium (89%) but not magnesium (41%). Lymphocyte adherence was inhibited in the presence of fucoidin or phosphomannan polysaccharides (36% and 48% of control binding, respectively). Fibronectin peptides, which are involved in certain types of integrin-mediated adherence, had no effect in this system. Lacrimal culture supernatants contained a factor that was inhibitory for TDL adherence (more than 50% inhibition when concentrated 5 or 10 times). CONCLUSIONS: Thoracic duct lymphocyte adherence to cultured lacrimal gland acinar epithelial cells shows good correlation with previously reported adherence to lacrimal gland frozen sections. Further, lacrimal cell culture supernatants contain soluble factors that inhibit TDL adherence to epithelial cells. These findings suggest that the lacrimal molecules involved in lymphocyte localization are shed and that lacrimal epithelial cell cultures will be useful for ligand isolation and characterization. PMID- 7558719 TI - Thrombin increases expression of urokinase receptor by activation of the thrombin receptor. AB - PURPOSE: To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. METHODS: The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. RESULTS: Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expression in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. CONCLUSION: Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor. PMID- 7558720 TI - Cytokine modulation of adhesion molecule expression on human retinal pigment epithelial cells. AB - PURPOSE: The purpose of this study was to assess qualitatively the expression of adhesion molecules by human retinal pigment epithelium (RPE) and to study their regulation by inflammatory cytokines. These molecular events and the role played by inflammatory cytokines are important for selective lymphocyte trafficking into the eye during uveitis. METHODS: Expression of intracellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular adhesion molecule (VCAM-1) by early passage human RPE cells was assessed by flow cytometry. In addition, the regulation of the expression of these molecules by the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) was determined. Reverse transcription polymerase chain reaction (RT-PCR) was used to characterize further adhesion molecule expression. RESULTS: Flow cytometric analysis determined that ICAM-1 was constitutively expressed on RPE cell lines and that in the presence of TNF alpha, IFN gamma, and IL-1 beta, there was a median fold increase in expression of 4.4, 5.4, and 4.4, respectively. In contrast, flow cytometric analysis of ELAM-1, PECAM-1, and VCAM-1 indicated that these adhesion molecules were not constitutively expressed at the cell surface; only the expression of VCAM-1 was upregulated by the presence of cytokines. The results of RT-PCR on RPE cells indicated that mRNA for all the adhesion molecules was present constitutively in some RPE cultures. After activation with IFN gamma, TNF alpha, and IL-1 beta, RT PCR analysis showed that the number of RPE cell lines expressing all the adhesion molecules increased. CONCLUSIONS: ICAM-1 expression is markedly upregulated by inflammatory cytokines. Although mRNA for other adhesion molecules is expressed in RPE cells and is enhanced by inflammatory cytokines, this does not necessarily reflect the cell surface protein expression. Thus, the expression of adhesion molecules by RPE cells, and the subsequent recruitment of specific leukocytes, may be determined by the local cytokine environment. PMID- 7558721 TI - Trabecular aspiration. A new mode to treat pseudoexfoliation glaucoma. AB - PURPOSE: The primary cause of intraocular pressure (IOP) elevation in pseudoexfoliation glaucoma is obstruction of the intertrabecular spaces by exfoliation material. The aim of the current study was to evaluate the efficacy of a novel surgical approach--trabecular aspiration--for the management of pseudoexfoliation glaucoma. METHODS: Twelve patients characterized by medically uncontrolled IOP level in pseudoexfoliation glaucoma underwent trabecular aspiration as a primary antiglaucomatous procedure. Aspiration treatment of the meshwork was performed in the inferior circumference of the chamber angle using a specially designed irrigation-aspiration device to fit the chamber angle. Trabecular debris and pigment were cleared with a suction force of 100 to 200 mm Hg. RESULTS: Before surgery, IOP ranged from 24 mm Hg to 52 mm Hg (mean, 37.4 mm Hg). Fifteen months after surgery, IOP ranged from 15 mm Hg to 23 mm Hg (mean, 18.3 mm Hg), which is equivalent to an absolute decrease of 18.7 mm Hg (50%). After surgery, the IOP of 10 eyes was less than 21 mm Hg, although 6 of these eyes still required topical medication. Two eyes attained a final IOP of 24 mm Hg. All treated eyes were aspirated once. Mean antiglaucomatous medication decreased from 4.3 medications daily before surgery to 1.39 medications daily at 15 months after surgery. CONCLUSIONS: This new surgical modality, removing intertrabecular and pretrabecular debris of the trabecular meshwork, can be effective in the management of pseudoexfoliation glaucoma. However, long-term follow-up and extended sample size must be prospected for further clinical evaluation. PMID- 7558723 TI - Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses. AB - PURPOSE: The sulfhydryl complexing agent p-chloromercuri-phenylsulfonate (pCMPS) has been shown to increase lens membrane permeability, Na+ and Ca2+ content, and light scatter in the rat lens in vitro. This study aimed to investigate the ultrastructural changes accompanying the increase in light scatter. In addition, high-resolution histochemistry was used to study the cellular distribution of Ca2+ in normal and cataractous lenses. METHODS: Rat lenses were incubated for 4 hours in normal (1 mM) and high (5 mM) Ca2+ containing media supplemented with 40 microM pCMPS. Control lenses were incubated in 1 mM Ca2+ containing medium. They were prepared for scanning and transmission electron microscopy and for Ca2+ localization using the oxalate-pyroantimonate procedure. RESULTS: Control lenses incubated for 4 hours had normal morphology and showed no evidence of light scatter. Calcium distribution as observed with the oxalate-pyroantimonate precipitation method was low in superficial fibers, high in the membranes of intermediate fibers, and declined again toward the nucleus. In the deeper cortex, there also were small vacuoles of calcium accumulation. pCMPS treatment (in 1 and 5 mM Ca2+) induced a significant influx of calcium into the lens cytoplasm. Calcium-containing extracellular vacuoles also were seen in the intermediate cortex in both cases. The presence of these vacuoles appeared to correlate with the major areas of light scatter in the lens. In 5 mM Ca2+, intracellular vacuoles were observed throughout the superficial cortex. CONCLUSIONS: Most of the calcium observed by oxalate-pyroantimonate in the normal lens is located at the membrane, and the staining appears strongest in the intermediate cortex. In pCMPS treatment, large extracellular vacuoles are present in this intermediate zone and appear to be the major source of light scatter. This zone may be the initiation site of many different types of cataract, including some described in human lenses. PMID- 7558722 TI - Alpha-2,3 sialylation differentiate the limbal and corneal epithelial cell phenotypes. AB - PURPOSE: The initial differentiation event for the corneal epithelial cell lineage occurs as the limbally localized stem cells yield, through mitosis, the highly proliferative, transiently amplifying corneal peripheral cells. This differentiation is characterized by the expression of tissue-specific cytokeratins, as well as the loss of alpha-enolase and pigmentation. All these are intracellular events. The aim of this study was to identify and characterize, through lectin analysis, changes in cell surface properties associated with differentiation. METHODS: Cryostat sections of the limbo-corneal area from freshly dissected pigmented rabbit corneas were stained with fluorescent lectins. RESULTS: Peanut lectin (PNA; binds to Ser/Threo-GalNAc-beta-1,3-Gal, if the Gal residue is not sialylated) stained the plasma membrane of all layers of the conjunctiva and limbus but was excluded from corneal cell membranes. Maakia amurensis agglutinin (MAA; binds to sialic acid attached to galactose through alpha-2,3 bonds in either N-glycans or O-glycans) stained exclusively corneal cell plasma membrane. After complete tissue desialylation, all corneal plasma membranes became PNA positive with equal stain intensity across both sides of the limbo-corneal margin. The binding of the agglutinins from Limax flavus (binds unselectively to sialic acid) and Sambucus nigra (binds to sialic acid attached through alpha-2,6 bonds) to the basement membrane displayed a large increase at the corneal side of limbo-corneal demarcation. CONCLUSIONS: Limbal (stem) cells express on the cell surface unsialylated galactose residues that are recognized by PNA and that lack any sialic acid bound through alpha-2,3 bonds. The initial differentiation involves sialylation of these residues and the concurrent appearance of alpha-2,3 sialic acid residues, suggesting expression or activation of alpha-2-3 sialytransferase. Changes in basement membrane composition, charge, or both may underpin this expression. PMID- 7558726 TI - The 'light scattering factor'. Importance of stimulus geometry, contrast definition, and adaptation. AB - PURPOSE: Paulsson and Sjostrand have suggested that the light scattering factor (LSF) can be estimated by using the equation: LSF = L/E (M2/M1-1). Here L is the space average luminance of the target, E is the illuminance of the glare source, and M2 and M1 are modulation contrast thresholds in the presence and absence of the glare source. To compensate for change of adaptation. Abrahamsson and Sjostrand later modified the above equation by introducing a correction factor (CF): LSF = L/E ((CF) (M2/M1-1). The purpose of this study is to analyze the validity of the above equations. METHODS: The importance of stimulus geometry, contrast definition, background luminance, and glare illumination is studied through theoretical analysis and comparison with earlier studies. Stimulus geometry and contrast definition are studied through optical modeling. Adaptation is modeled according to the laws of Weber and DeVries-Rose. RESULTS: The choice of contrast definition may corrupt the result by a factor of 2. At background luminance levels above approximately 10 cd/m2, the Paulsson-Sjostrand equation agrees well with theory. At lower background levels, the Abrahamsson-Sjostrand equation is used with correction factors derived from adaptation measurements. Using this equation and earlier published data from glare testing performed at 2 cd/m2, the results are found to be in fair agreement with the light scattering theory. CONCLUSIONS: Glare testing using the Paulsson-Sjostrand equation is found to be valid as long as the measurements are performed at high luminance levels (above 10 cd/m2), with targets of low spatiotemporal frequencies (e.g., 2 cpd and 1 Hz) and with the use of a properly chosen definition of contrast. At lower luminance levels, the Abrahamsson-Sjostrand equation may be used with well derived correction factors. PMID- 7558725 TI - Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture. AB - PURPOSE: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures. METHODS: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin. RESULTS: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin. CONCLUSIONS: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures. PMID- 7558724 TI - Sequence, molecular properties, and chromosomal mapping of mouse lumican. AB - PURPOSE: Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS: Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS: Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS: This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene. PMID- 7558731 TI - Computed tomography of the large bowel wall. Choice of slice thickness and intraluminal contrast medium. AB - RATIONALE AND OBJECTIVES: To determine the relative contribution of slice thickness and intraluminal contrast density to imaging of the large bowel wall. METHODS: The authors used phantom experiments to evaluate the partial volume averaging of 2-, 4-, and 8-mm slices on imaging a single density interface (320/ 100 HU) and 5 dual-density interfaces (320/50/-100 HU, 160/50/-100 HU, 0/50/-100 HU, -100/50/100 HU, and -1000/50/100 HU). For the experiments with a dual-density interface, which simulated the bowel wall, the phantom was scanned at an angle 45 degrees to the scan plane. RESULTS: The most accurate display of interfaces was obtained with a slice thickness of 2 mm. When using 8-mm slices, walls can appear to be much thinner or thicker than normal or not even recognizable as a wall depending on the densities in the interface. Adjusting the density of the intraluminal contrast medium to within the range of the mean and window setting will improve the representation of the bowel wall. CONCLUSIONS: Slice thickness has a significant influence on the true representation of the bowel wall using computed tomography. This effect is greater than the effect of the intraluminal contrast medium density. PMID- 7558730 TI - Postmortem metabolic and morphologic alterations of the dog brain thalamus with use of in vivo 1H magnetic resonance spectroscopy and electron microscopy. AB - RATIONALE AND OBJECTIVES: To investigate postmortem metabolic and morphologic alterations in the dog brain thalamus and to contribute diagnostic aids in medicolegal science. METHODS: A homogeneous group of 17 dogs was used for in vivo 1H magnetic resonance (MR) spectroscopy and electron microscopic studies, which were performed under intravenous and intramuscular anesthesia before and after the dogs were killed. All 1H MR spectroscopy and electron microscopy examinations were performed on a 1.5 T MR imaging/MR spectroscopy system using a stimulated echo acquisition mode pulse sequence and an electron microscope, respectively. RESULTS: The lactate resonance signal, which could be used as a criterion for tissue survival, appeared during the entire postmortem period. The lactate: creatine ratio significantly increased in the early postmortem metabolic process. The inositol: creatine ratio showed a mild gradual increase. However, the N acetyl-aspartate: creatine and choline/creatine ratios generally were unchanged. The decomposition of proton metabolites was observed in the MR spectrum 48 hours postmortem. In the postmortem period, high power field (x10,000) electron microscopic results showed significant morphologic alterations of the thalamus, revealing nuclear pyknosis and perikaryal condensation, cytoplasmic vacuoles, clumping of nuclear chromatin, and destruction of cellular organelles and nuclear membrane. CONCLUSIONS: Results suggest that the combination of 1H MR spectroscopy and electron microscopy may simultaneously provide good quality metabolic and morphologic information of cerebral tissue in the field of thanatochronology. PMID- 7558727 TI - Dystrophin localization at presynapse in rat retina revealed by immunoelectron microscopy. AB - PURPOSE: It has been accepted that retinal dystrophin is localized in the outer plexiform layer (OPL) of the retina, but no electron microscopic evidence has been reported until now. Using immunoelectron microscopy, the authors investigate whether retinal dystrophin localizes at the presynaptic or postsynaptic membrane of synaptic regions. METHODS: Monoclonal antibody raised against human dystrophin C-terminus was used. Immunoblotting analysis was used for testing dystrophin protein in retinal tissues. Tissue preparations were stained with the immunofluorescence or immunoperoxidase method and were observed with light and electron microscopy. RESULTS: Immunoblotting analysis showed a molecular weight of band 420 kDa in the sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the retinal tissues. Immunoelectron microscopy disclosed that retinal dystrophin was localized at the presynaptic membrane of synaptic regions in the OPL. CONCLUSIONS: Rat retinal dystrophin localizes at the presynaptic membrane in the OPL, suggesting that it may play some role in the neurotransmitter release of the photoreceptor cell. PMID- 7558729 TI - In vivo measurement of lipofuscin in Stargardt's disease--Fundus flavimaculatus. AB - PURPOSE: Several histopathologic studies have concluded that Stargardt's disease (Fundus flavimaculatus) is associated with abnormally high levels of lipofuscin like material in the retinal pigment epithelium. The purpose of this study was to determine whether this material has the same fluorescence characteristics as lipofuscin in vivo and whether noninvasive measurements identify a significant elevation in this material. METHODS: Five patients with autosomal recessive Stargardt's disease were included in this study, as were 45 healthy controls. All patients had the angiographic dark choroid sign. The intensity and emission spectra of lipofuscin fluorescence were measured by noninvasive fundus spectrophotometry at 7 degrees temporal to the fovea. RESULTS: The fluorescence intensities in the five patients with Stargardt's disease were significantly higher (P < 0.0001) than those observed in normal subjects of the same age. The emission spectra in the patients are similar in shape to those measured in normals, but flecks appear to shift the spectra toward shorter wavelengths. CONCLUSIONS: The spectral characteristics of the fluorophore observed in patients with Stargardt's disease are consistent with those of retinal pigment epithelial lipofuscin. These patients have abnormally high levels of lipofuscin, confirming previous histopathologic observations. Noninvasive retinal pigment epithelial lipofuscin measurements may be a useful adjunct in the diagnosis of Stargardt's disease.-F. flavimaculatus. PMID- 7558732 TI - Allergic-type adverse reactions to low osmolality contrast media in patients with a history of allergy or asthma. AB - RATIONALE AND OBJECTIVES: The author evaluated the hypothesis that the incidence of allergic-type adverse events among atopic and asthmatic cardiac angiography patients would be lower in patients randomized to receive nonionic versus ionic lower osmolal contrast medium. METHODS: Enrollment criteria included a history of food allergy, asthma, atopic dermatitis or pollinosis but not prior contrast media-induced adverse events. One hundred forty-two patients enrolled were randomized to receive either the ionic agent ioxaglate or nonionic iopamidol. Patients and investigators were masked. Sixty-eight patients received iopamidol, and 74 received ioxaglate. Group means were compared using the unpaired Student's t test. Contingency tables were analyzed using chi-square or Fisher's exact test. RESULTS: Allergic-type adverse events (excluding flushing, warmth, and cardiac events) were recorded in 8 of 68 patients in the iopamidol group versus 19 of 74 in the ioxaglate group. Ioxaglate was more likely to be associated with a reaction than iopamidol (P < 0.05, chi-square test). Iopamidol was more likely to be associated with flushing when considered independently (38 of 68) than use ioxaglate (24 of 74)(P < 0.005, chi-square test). There was no difference in the incidence of cardiac events, or any combination of allergic-type adverse events, cardiac events, and flushing in patients who received iopamidol or ioxaglate. CONCLUSION: In this double-masked study, iopamidol caused fewer allergic-type adverse events (excluding flushing and cardiac events) but more flushing than ioxaglate in patients with a history of asthma or atopic disease. When cardiac events also were considered, there was no difference in the incidence of reactions after iopamidol and ioxaglate. PMID- 7558728 TI - Intraocular viral replication after systemic murine cytomegalovirus infection requires immunosuppression. AB - PURPOSE: Human cytomegalovirus retinitis is the most common blinding complication of acquired immune deficiency syndrome. However, the pathogenesis of the disease is poorly understood. The authors sought to characterize intraocular viral replication after systemic murine cytomegalovirus (MCMV) infection in the normal and immunosuppressed Balb/c mouse. METHODS: Normal or immunosuppressed mice (400 rads radiation plus antilymphocyte serum) were infected intravenously with a recombinant MCMV (RM408) that carries an MCMV IE1 promoter--LacZ insert. In vivo MCMV replication and its tissue distribution were monitored by beta-gal activity with x-gal staining on frozen tissue sections of multiple organs harvested from infected mice at different time points after inoculation. RESULTS: MCMV replication within the eye can be detected in the immunosuppressed Balb/c mouse but not in the normal host. Intraocular viral replication was noted first, and most frequently, in the ciliary body and was mainly restricted to the uveal tract. Intraocular viral replication coincided with the peak of systemic viral replication; however, the neurosensory retina was spared. In contrast, supraciliary inoculation of MCMV in the immunosuppressed Balb/c mouse resulted in massive viral replication and destruction of the neurosensory retina. CONCLUSIONS: This study demonstrated that intraocular MCMV replication after systemic infection requires systemic immunosuppression. Furthermore, the ciliary body is the portal of entry for the virus within the eye. MCMV can replicate in the epithelium of the uvea and retinal pigment epithelium, but it does not replicate within the neurosensory retina. The absence of MCMV replication within the neurosensory retina is not caused by either a defect in the recombinant virus or the inability of the host tissue to support viral replication. PMID- 7558733 TI - A comparative study on contrast enhancement between 0.5- and 2.0-Tesla field strength in rabbit VX2 carcinomas. AB - RATIONALE AND OBJECTIVES: Theoretically, the degree of contrast enhancement in magnetic resonance (MR) is affected by magnetic field strength. The authors conducted this study to compare the degree of MR contrast enhancement between 0.5 Tesla (T) and 2.0 T in VX carcinomas of rabbits. METHODS: In 12 rabbits with VX2 tumors, both 0.5 T and 2.0 T MR imaging was performed within 1 day before and after intravenous injection of gadopentetate dimeglumine. Each rabbit received an initial standard dose (0.1 mmol/KG) followed by and additional standard dose (total, double dose) during the MR study at each field strength. The degree of contrast enhancement in the VX2 tumors was assessed quantitatively. RESULTS: With standard and double dose studies, overall mean tumor and muscle contrast-to-noise ratio was higher at 2.0 T than 0.5 T by 95% and 81%, respectively. The double dose study showed higher contrast-to-noise ratio than the standard dose study at both field strengths. CONCLUSIONS: Results indicate that the effect of contrast enhancement increases with field strength. PMID- 7558735 TI - Digital chest imaging using a selenium detector. A simulated diffuse interstitial pulmonary disease detection study. AB - RATIONALE AND OBJECTIVES: To assess the diagnostic performance of a new digital chest imaging system based on a selenium detector in the detection of simulated diffuse interstitial pulmonary disease (DIPD). METHODS: Diffuse interstitial pulmonary disease was simulated by superimposing plastic sheets containing small radio-paque objects (birdseed) on an anthropomorphic chest phantom. the number of sheets was varied from 0 to 4 to simulate the degree of pathology. Eighty conventional radiographs and 80 images with the selenium detector were obtained under comparable conditions. Six observers were asked to indicate the presence of DIPD using a five-level scale of confidence. The total 160 chest images were analyzed using receiver operating characteristic curves. RESULTS: There was no significant difference in detection performance between the conventional and the digital images (95% confidence interval). CONCLUSIONS: None of the observers assessed the diagnostic performance of the digital chest images as significantly different from that of the conventional system. The interobserver differences were higher than the differences between digital and conventional chest images. PMID- 7558734 TI - Plasma levels and urinary excretion of iodine after oral administration of iohexol in dogs and cats. AB - RATIONALE AND OBJECTIVES: Plasma and urinary iodine concentrations were assessed after oral administration of iohexol in five dogs and five cats. MATERIALS AND METHODS: Ventrodorsal and right lateral radiographs were taken; a 3-mL blood sample was collected 30 minutes and 1, 2, 4, and 6 hours after dose in each animal; and the bladder was emptied at 2 and 6 hours in dogs and at 6 hours in cats after oral administration of 700 mg I/kg iohexol (300 mg I/mL). Iodine concentration was measured using a sequential inductively coupled plasma atomic emission spectrometer system. RESULTS: Peak concentrations of plasma iodine were detected in samples taken from both dogs (0.33 mg I/mL) and cats (0.28 mg I/mL) 30 minutes after iohexol administration, with a gradual decline at 1, 2, 4, and 6 hours. Total urinary excretion during the 6 hours after the administration does of iohexol was higher (4.3%) in cats than in dogs (2.0%). In each species, there was a relationship between gastrointestinal motility observed radiographically and the subsequent excretion of iodine in the urine. CONCLUSIONS: Results indicate that approximately 2% of the oral dose is absorbed and excreted in the urine of dogs within 6 hours, and approximately 4% of the dose is absorbed and excreted in cats. PMID- 7558737 TI - Behavior of bubbles of slowly permeating gas used for ultrasonic imaging contrast. AB - RATIONALE AND OBJECTIVES: The authors predict behavior of blood cell-sized bubbles containing a foreign gas that slowly crosses a gas-liquid interface. Such bubbles are being developed for ultrasonic contrast. METHODS: Using appropriate coefficients for N2, O2, CO2, and foreign gases, the authors simulate diffusion between bubbles and blood with a numerically solved equation system. RESULTS: Within 30 seconds after intravenous injection of bubbles, entrance of endogenous gases more than doubles the radius. In vivo size and duration of the bubbles are quantitatively related to the amount of the foreign gas inside. Size and ultrasonic imaging effectiveness of the circulating bubbles become larger in lungs than in the rest of the circulation. As the foreign gas is lost, imaging effectiveness diminishes more rapidly than bubble radius. CONCLUSIONS: Bubbles of slowly permeating gas change size and composition after injection and also during their passage through different parts of the circulation. PMID- 7558738 TI - Effects of iodinated X-ray contrast media on renal epithelial cells in culture. PMID- 7558736 TI - Significance of magnetic resonance signal enhancement in evaluation of myocardial infarction in cats. AB - RATIONALE AND OBJECTIVES: To correlate magnetic resonance (MR) signal enhancement with pathophysiologic changes occurring during ischemia and reperfusion in evaluation of myocardial infarction in cats. METHODS: Seven cats were subjected to 150 minutes of occlusion of the left anterior descending coronary artery followed by 90 minutes of reperfusion. Gadolinium (Gd) diethylenetriaminepentaacetic acid-polylysine (molecular weight [mw] = 40 to 50 kd, DP230) contrast-enhanced MR images were acquired with coinjection of poly-L lysine-fluorescein isothiocyanate (mW = 40 to 50 kd, DP219) as a fluorescent tracer molecule of the contrast agent. Signal intensities on the MR images and fluorescence activities on the resected cardiac specimens were measured. Pathologic examinations by electron and light microscopes and 2,3,5 triphenyltetrazolium chloride histochemical staining were performed on the specimens. RESULTS: Magnetic resonance signal intensity was lower in the center (3.49 +/- 0.36) than in the periphery (4.71 +/- 0.89) of the enhanced area. Fluorescence activities were absent in the normal myocardium; they were scant as nonspecific discrete dots in the center and numerous as specific interstitial distribution in the periphery of the ischemic myocardium. Electron and light microscopic examinations showed severely damaged ultrastructures of the center and moderately distorted ultrastructures of the periphery. CONCLUSIONS: The center of the MR signal-enhanced area is the infarct center with severe ultrastructural damages, which also might be an area of little or absent of blood reflow. Conversely, the peripheral-enhanced area is the infarct periphery with moderate myocardial damages. PMID- 7558739 TI - Principles of Medicare reform. PMID- 7558741 TI - Medicare under a microscope at AMA. PMID- 7558740 TI - Organized medicine: it's for students, too. PMID- 7558743 TI - IMS recommendations on Medicaid program. PMID- 7558742 TI - IMS staying involved in the CHMIS process. PMID- 7558744 TI - Prior authorization for Medicaid drugs. PMID- 7558746 TI - Air pellet gun injury. PMID- 7558745 TI - Physicians on the front line. PMID- 7558747 TI - Obstetrical stays--IMS, AMA policy. PMID- 7558748 TI - Medical supervision of student athletes. PMID- 7558749 TI - Managed care in Iowa. A difficult transition. AB - On March 1, Medco Behavioral Care began managing mental health services for Iowa's Title 19 population. The state says Medco can improve access and save money, but many providers and other patient advocates have grave concerns about Iowa's first major experience with managed care. PMID- 7558750 TI - Metastasis of adenocarcinoma of breast to gluteus medius. PMID- 7558751 TI - Drive-thru delivery. PMID- 7558753 TI - "Typhoid Mary" strikes again. The social and the scientific in the making of modern public health. PMID- 7558752 TI - Addiction and the laboratory. The work of the National Research Council's Committee on Drug Addiction, 1928-1939. PMID- 7558754 TI - Problems in implementing the 1991 Treatment of Mental Patients Law in Israel. AB - The application of the new Treatment of Mental Patients Law, 1991, has been responsible for significant changes in the work of mental health workers, in particular those involved in the often complex process of civil commitment for examination/hospitalization. The experience of implementing this new law shows that some of the clauses related to this process contain controversial concepts. These concepts will be the focus of this paper. PMID- 7558755 TI - Partial insanity: when the judiciary and the psychiatric world collide. AB - The psychiatric and judicial concepts of mental disorders are similar but not always identical. The gap between them creates confusion, a lack of unity and inconsistencies in decision making. This gap needs to be bridged. In the hinterland of these concepts lies a range of psychiatric disturbances such as encapsulated over organized delusional disorders (Paranoia Vera) and life endangering eating disorders (Anorexia Nervosa). When life threatening or criminal responsibility circumstances arise, the dilemma presented to the psychiatrists on the one hand and the judicial system on the other reaches its peak. Presented in the article are aspects of the gray areas of involuntary commitment and criminal responsibility. PMID- 7558756 TI - Enforced psychiatric treatment of minors in Israel: the interface between the Mental Health Act and the Youth Law. AB - Proposed changes in the current Youth Law will provide enforced psychiatric treatment, including civil commitment, for "at risk" minors. Such minors are considered at significant risk of future developmental damage without such treatment. The potential exists for a clash between the proposed criteria for enforced psychiatric treatment in the Youth Law and the existing criteria in the current Mental Health Act: "need for treatment" in the former versus "immediate and physical danger to self or others as a result of psychotic illness" in the latter. Proposed changes in the Mental Health Act include broadening the criteria for enforced psychiatric treatment of minors. The revised criteria would include non-psychotic emotional disorders (such as recurrent suicidal behavior) which endanger the minor's physical and emotional development. These changes are welcomed. The authors suggest the adoption of "need for treatment" criteria, as against the current "physical danger to self or others" criteria for enforced psychiatric treatment of minors. PMID- 7558757 TI - Sigmund Freud and the countenance of Moses. AB - Sigmund Freud, as is well known, was overwhelmed by the person of Moses. This profound impact is revealed in his "The Moses of Michelangelo," first published in 1914, and "Moses and Monotheism," issued in 1933, the final work of his career. His biographers, in the light of these writings, have sought to discover the sources of this strong influence of Moses. A few claim that Moses arrived late on Freud's life stage. Most recognize that Freud was attracted to Moses early in his personal development. Their explanations to account for Freud's attachment to Moses, however, seem inadequate. The present article explores this issue and attributes the fascination and powerful imprint of Moses as stemming from a frequently viewed frontispiece found in each volume of the Philippson Bible, a lithograph of Moses displaying the Ten Commandments to the people of Israel. From childhood on, Sigmund gazed upon the face of Moses with his piercing eyes as his father related biblical stories to him, particularly the deeds of Moses, during their studies. The role of Jacob Freud with respect to the Freud Moses relationship is herein discussed. The general conclusion emphasizes, as does Sigmund Freud himself, the enduring power of childhood experiences and their lifelong effect. PMID- 7558758 TI - Divergences and convergences between strategic and psychodynamic therapy. AB - Although psychodynamic and strategic therapy have very different starting points, they have evolved in converging directions under the Zeitgeist which favors self actualization and have met similar dilemmas concerning the therapist's attitude towards the issues of power and knowledge. The Winnicottian concept of "therapy as play" is suggested as a solution to the therapist's dilemma on the use of power in both dynamic and strategic models and as a conceptual bridge between them. PMID- 7558759 TI - The nightmare of returning home: a case of acute onset nightmare disorder treated by lucid dreaming. AB - Nightmare disorder with acute onset involves the sudden appearance of frightening and disruptive dreams. In severe cases it may involve high levels of anxiety, fear of falling asleep, cognitive deficits secondary to sleep deprivation and so may pose a psychiatric emergency. Standard techniques of dream interpretation appear limited in dealing with such a crisis. Lucid dreaming, the experience of dreaming and simultaneously being aware that one is dreaming is an easily learned technique that may provide effective and dramatic relief. The usefulness of lucid dreaming is illustrated by a case history. PMID- 7558760 TI - Ultra-orthodox men and psychiatric treatment. PMID- 7558761 TI - Conditions for research in biological psychiatry. PMID- 7558762 TI - Forensic psychiatry in Israel: involuntary out-patient treatment. PMID- 7558764 TI - The future of forensic psychiatry: the suggested Israeli model. AB - The expansion of knowledge in medicine lead to the emergence of specialties and to sub-specialties limited in breadth and expanded in depth. The increasing health-care costs created a three-level model: general mental health workers (general practitioners and non-psychiatric mental health professionals), primary psychiatrists and the subspecialist experts. The paper defines forensic psychiatry as a subspecialty, describes its development and the pros and cons concerning its independent existence. We describe the state of forensic psychiatry in Europe and in Israel. In Israel there are no binding rules regulating the forensic services. The first steps of professional development were taken in the late 1980s by introducing lectures in forensic psychiatry at the Tel Aviv University, creating the Forum of District Psychiatrists and founding the Israeli Forensic Psychiatry Association. We discuss the problematic relationship of law and psychiatry. Most of the requirements for making forensic psychiatry an independent discipline have already been fulfilled in Israel. In order to gain full formal acknowledgement we have to establish appropriate recruitment, licensing and training procedures. The authors present five different models concerning the training of forensic psychiatrists, and describe in details the model chosen by the Forensic Psychiatric Association to be submitted for approval to the Medical Association. PMID- 7558763 TI - The implementation of mental health care reform in Israel. PMID- 7558765 TI - Rights of the patient vs. rights of the public in the "Treatment of the Mentally Ill Law--1991". AB - The new Israeli "Treatment of the Mentally Ill Law--1991" pays much attention to the protection of patients from unnecessary hospitalization in mental hospitals and to defining the rights and duties of patients who have been hospitalized, whether voluntarily or involuntarily. The question, however, is whether the rights of the public to be protected from dangerous patients were not sacrificed for the sake of the protection of the rights of the individual patient. The new law protects the rights of the patient in two main ways. First, it defines strictly and clearly the grounds for which involuntary hospitalization is permitted and lists the rights of the hospitalized patient. Secondly, it specifies clearly the procedure for the issue of a hospitalization order and distributes the power to issue such an order among various administrative bodies. The psychiatric committee is mainly a controller body to which appeals can be made on decisions of the District Psychiatrist or of directors of mental hospitals. Entrusting such a body with original administrative functions, such as the issuing of hospitalization orders for long periods, is in our opinion a conceptual mistake. Hospitalization orders through judicial proceedings are then discussed. Whenever the conditions specified in section 9 are fulfilled, the District Psychiatrist is entitled to issue a hospitalization order. The District Psychiatrist is not obliged, however, according to the new law, to do it. On this point we have some reservations. PMID- 7558766 TI - Fluctuations in levels of various gangliosides in the different tracts of the oviduct of the frog (Rana esculenta) during the reproductive cycle. AB - The ganglioside content and pattern have been followed in the different tracts (rectus, convoluted and uterine) of the frog oviduct during the reproductive cycle. The main variations we observed are: a) average higher levels of ganglioside sialic acid in the preovulatory phase, with two peaks in March and April for the convoluted and rectus tract, respectively, and a more homogenous behaviour for the uterine tract; in all three tracts of the oviduct a minimum coincident with the ovulation has been found; b) a balanced presence of sulfolipids and gangliosides in the uterine tract: in fact sulfolipids, whose variations have been determined in a previous work, are higher when gangliosides are lower and vice versa, maintaining nearly constant the total negative charge due to these glycolipids; c) an alternate fluctuation of monosialo- and disialo gangliosides in the preovulatory phase and a net trend toward the increase of monosialo- and the decrease of disialogangliosides in the postovulatory phase; trisialo-gangliosides are in general less represented and show less marked variations; d) the presence of particular gangliosides in particular moments of the reproductive cycle: Fuc-GM1, a fucosylated ganglioside, is higher than the more represented GM1 during the ovulation, while GD1 alpha, a ganglioside with a sialic acid residue linked to GalNAc, is steadily present in all three tracts after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558767 TI - Pavia memory project: study design and first results. AB - This paper describes the design and first results of the cross-sectional phase of a study on memory impairment in elderly subjects (Pavia Memory Project). The target population consisted of the 1,046 subjects born in 1925 and currently living in Pavia. Four hundred and thirty-six subjects (plus 287 interviewed at home) participated in the first stage, which consisted of a semi-structured anamnestic interview. The 400 interviewed subjects with none of the exclusion criteria participated in the second stage, which consisted of a memory test battery, a standardized neurological examination and screening procedures for depression (GDS) and dementia (MMSE). On the basis of the memory scores, three groups were defined: memory impairment (MI: 8.8%), mild memory impairment (MMI: 39.8%); normal (N: 51.3%). There were more failures on the visuo-spatial memory tests. Depression was equally distributed in the N and MI groups (about 15%), but was more frequent in the MMI group (27%). Abnormal neurological signs were largely independent of memory performance. Two cohorts (of MI and N subjects) underwent neuropsychological and instrumental (CT-scan, EEG, ERPs, eye movements) assessment, which will be regularly repeated in the longitudinal phase. PMID- 7558768 TI - Brain MRI and SPET in thrombotic thrombocytopenic purpura. AB - We describe 5 cases of thrombotic thrombocytopenic purpura (TTP) with neurological manifestations. All of the patients underwent brain magnetic resonance imaging (MRI) following recovery; two underwent single photon emission tomography (SPET) during the acute phase of the disease. SPET showed reduced cerebral blood flow, whereas the results of brain MRI were normal in all patients. Plasma exchange (PE) treatment was promptly instituted in all cases. Our findings show that prompt treatment with PE may avoid permanent brain damage even when the neurological signs and symptoms are relate to brain ischemia. PMID- 7558769 TI - Neurological diagnoses in psychiatric patients. The uncertain boundaries between neurology and psychiatry. AB - The study highlights the high prevalence of neurological diagnoses in a population of psychiatric in-patients admitted to 16 private clinics and 6 private institutes because of their mental and behavioral disorders. Neurological diseases affected 13.05% of acute and 68.09% of chronic psychiatric patients. These results emphasize the need for specific neurological competence in the treatment of many psychiatric patients and better integration between neurological and psychiatric departments. The close relationship between neurology and psychiatry, as well as the renaissance of neuropsychiatry are discussed. PMID- 7558770 TI - Twitch response of striated muscle in patients with progressive external ophthalmoplegia, mitochondrial myopathy and focal cytochrome c-oxidase deficiency. AB - Some aspects of the contractile properties of skeletal muscle in patients with progressive external ophthalmoplegia (PEO), mitochondrial myopathy and focal cytochrome c-oxidase deficiency were investigated by studying the twitch response (TR) of the tibialis anterior muscle both at rest and after maximum isometric contraction. The results of needle electromyography were normal in four of the six examined patients, and myopathic in the remaining two. A slowing in muscle relaxation was the most frequently observed abnormality; significantly prolonged muscle contraction times and reduced twitch torque potentiation values after isometric contraction were also detected. TR abnormalities in PEO patients may be due either to a dysfunction of the contractile machinery depending upon impaired muscle energy supply or to altered muscle fiber characterized by the predominance of type I slow fiber. In addition to conventional electromyographic investigations, TR study may be a useful diagnostic tool in PEO patients. PMID- 7558771 TI - The clinical and paraclinical profile of optic neuritis: a prospective study. AB - To define the clinical and paraclinical profile of optic neuritis (ON), patients with a suspicion of ON among a population of 1.5 million were examined over 2.5 years. A diagnosis of monosymptomatic ON was established in 74 patients. Cerebrospinal fluid (CSF) studies in 73 patients revealed oligoclonal IgG bands in 67% and 32 of 60 patients examined (53%) had three or more high signal lesions on magnetic resonance imaging (MRI). A strong correlation was found between oligoclonal bands and abnormal MRI. In 52 patients, two or more CSF examinations revealed strong variations in individual patients for mononuclear cell count and IgG index. In contrast, of 39 patients with oligoclonal bands in the first sample, none showed the disappearance of bands, and of 13 patients initially negative for bands, only one developed bands. There was no correlation between exacerbation or remission and CSF findings. During a short-term follow-up of 6-40 months, 19 patients converted to MS. PMID- 7558772 TI - Aging and memory: corrections for age, sex and education for three widely used memory tests. AB - The associate learning subtest from the Wechsler Memory Scale; Benton's Visual Retention test and a Controlled Word Association Task (FAS) were administered to a random sample of normal, healthy individuals whose age ranged from 20 to 79 years, recruited within the Italian peninsula. The neuropsychological examination took place on a mobile unit and the tests were given by the same team of neuropsychologists to reduce variability among examiners. The Research Project was known as Progetto Memoria. Corrections to the scores of these tests were calculated for age, sex, and education. These corrected values will allow clinicians to screen for memory impairment with greater precision among normally aging individuals, thus improving differential diagnosis between physiologic and pathologic deterioration of cognitive functions. PMID- 7558774 TI - The Proustian itinerary from sensation to memory. AB - Information processing streams that proceed through the sensory unimodal and multimodal areas of the cerebral cortex, and hence the limbic system, bring from perception to memory. This itinerary through the neural networks underlying perception and cognition is briefly outlined through famous passages from Marcel Proust's "Swann's Way", in which the flavour of the "petites madeleines" triggers vivid recollection. PMID- 7558773 TI - Partial seizures, cerebral calcifications and celiac disease. AB - We describe the case of a 25 year old woman who has been clinically and instrumentally examined over a period of about 20 years. A diagnosis of celiac disease was made when she was four years old and, ten years later, CAT revealed the presence of bilateral cerebral calcifications. The partial occipital seizures were controlled by adopting a gluten-free diet, which is still being followed four years after the discontinuation of anti-epileptic treatment. PMID- 7558775 TI - The last days of Alessandro Manzoni. AB - Alessandro Manzoni died on 22 May 1873 at the age of 88. On the morning of 6 January 1873, on his way to Mass in the Milanese church of San Fedele, he fell and hit his head on the church steps. From the newspaper accounts of the day, it appears that this apparently banal accident was fatal for the old writer. It seems likely that his death was caused by a chronic subdural hematoma. PMID- 7558776 TI - Is obstructive sleep apnea a common cause of essential hypertension? AB - Obstructive sleep apnea (OSA) occurs in about 10% of the middle-aged population but in about 30% of the hypertensive population of the same age. About 20% of the middle-aged population has hypertension but about 50% of patients with OSA have hypertension. Despite this close relationship between these two entities, previous attempts to determine whether the respiratory abnormalities in OSA were responsible for the hypertension were inconclusive, particularly because of the confounding effect of obesity which is common to both conditions. Data from recent observational and intervention studies, however, have succeeded in avoiding many of the pitfalls of earlier studies and it is now becoming evident that OSA may be a major cause of hypertension--responsible for about 30% of all cases. Successful treatment of OSA by any means has been shown in most studies to cause significant reductions in blood pressure throughout the 24 h period, while at the same time alleviating the vast array of symptoms and clinical abnormalities associated with this common and serious condition. Despite the encouraging results of these recent data, more studies are urgently required which should include larger numbers of patients and controls in order to clarify further the relationship between OSA and hypertension. PMID- 7558777 TI - Is there a need for diagnostic upper gastrointestinal endoscopy before cholecystectomy? AB - Cholecystectomy is recommended for symptomatic gallstone disease, however atypical abdominal symptoms may be incorrectly attributed to gallstones found by chance at ultrasound. High rates of post-cholecystectomy symptoms confirm that surgery is often performed inappropriately in patients whose complaints do not derive from the biliary tract. We therefore performed esophagogastroduodenoscopy (EGD) in 56 symptomatic patients with gallstones to determine if there was an alternative explanation for patient symptoms. A surgeon then assessed the patient in the light of these findings and decided whether to operate. Of 41 patients who were questioned 12.4 +/- 7.3 months after entry, 1 of 22 patients who underwent surgery, and 5 of 19 patients treated conservatively, were symptomatic (P < 0.05). In the surgical group, one of seven patients with an endoscopic finding remained symptomatic, and in the nonsurgical group one of nine patients remained symptomatic (NS). However, in patients without endoscopic findings, none of the 15 surgical patients but 4 of 10 nonsurgical patients (40.0%) remained symptomatic (P < 0.01). We conclude that patients with gallstone disease and negative EGD who undergo cholecystectomy are less likely to remain symptomatic than those treated conservatively. Thus, endoscopy of the upper gastrointestinal tract in patients with gallstones may provide important information for the surgeon, prior to the making of therapeutic decisions. PMID- 7558779 TI - Strategy of biological monitoring and setting of biological threshold limits (BAT values) in Germany. AB - Biological monitoring is of fundamental importance in Germany. The successful strategy of biological monitoring is based on four factors. Recommendations for the pre-analytical phase have been issued by official societies. For the analyses in blood and urine specimens around 100 methods have been recommended and standardized. All measurements must be carried out under a quality control scheme issued by the German Ministry of Labor and organized by the German Society of Occupational and Environmental Medicine. The occupational medical judgement of the biological monitoring results take place by "Biological Tolerance Values for Working Materials" (BAT values) and "Exposure Equivalents for Carcinogenic Working Materials" (EKA values). A BAT value is, by definition, the maximum allowable concentration for an individual which should be exceeded; 53 BAT values for 37 agents or groups of agents and 18 EKA values for 10 compounds have been defined. These values have been published since 1982 in an annual list of occupational standards. Every BAT or EKA value is based on extensive scientific documentation. PMID- 7558778 TI - The association of occupational asbestos dust exposure and laryngeal carcinoma. AB - Controversy exists as to whether occupational asbestos dust exposure is responsible, or at least partially responsible, for the development of laryngeal carcinoma. The aim of this study is to present the current scientific knowledge on this subject based on a comprehensive literature research and to critically review the epidemiological investigations published. The results of 31 cohort studies and 24 case-control studies, and the conclusions in 11 more recent reviews are contradictory. In most studies there was no statistically significant indication of a casual relationship. It is noteworthy that an increased risk of laryngeal carcinoma among persons exposed to asbestos dust was observed mostly in older studies in which smoking habits and/or alcohol consumption as the most important nonoccupational risk factors were usually not taken into account. In addition, since most of the calculated positive associations are very weak, no reliable conclusion can be reached. While there may be a causal relationship between occupational exposure to asbestos dust and the occurrence of laryngeal carcinoma, this cannot be regarded as certain on the basis of the evidence reviewed. PMID- 7558780 TI - Neonatal mastitis--diagnosis and treatment. AB - Neonatal mastitis is an uncommon infection. Twenty-one neonates with mastitis were treated at the Bnai Zion Medical Center and Hillel Yaffe Hospital during the years 1985-92. Half of them presented with mastitis, and the other half with breast abscess. The most common pathogen was Staphylococcus aureus, which was isolated in 85% of cases. Antibiotic therapy was the initial treatment in all cases except one, and included i.v. orbenin or augmentin. Puncture of six breast abscesses followed the initial antibiotic course, and another five abscesses were treated surgically by incision and drainage. About half the neonates (10 of 21) recovered after antibiotic treatment alone, indicating that aggressive antibiotic therapy is effective in about 50% of cases and, if started immediately upon diagnosis, no additional surgical treatment is necessary. When an abscess was formed, needle aspiration was as effective as incision and drainage. PMID- 7558782 TI - Workers at risk: the need for biological monitoring. PMID- 7558783 TI - Soluble cytokine receptors in disease. PMID- 7558784 TI - Are we managing the infertile couple effectively? AB - The purpose of our study was to construct a management sequence allowing classification of infertile couples into specific groups according to the available treatment modalities. We achieved this by the following: a search of the existing medical literature; critical evaluation of personal experience gained by extensive practice in treating infertile couples; and perusal of methods and modalities for diagnosis and treatment of infertility available at present. We present a new concise classification of infertility, permitting efficient use of existing techniques and treatment modalities and conforming to the expectations of a modern consumer society. PMID- 7558781 TI - The inter-relationship between sleep apnea syndrome and hypertension. PMID- 7558785 TI - Bactericidal UV-C lamp misuse. PMID- 7558786 TI - Screening for prostate cancer. PMID- 7558787 TI - Human experimentation and clinical trials [correction of trails]. PMID- 7558788 TI - Gems from the Talmud: hygiene and public health V--nutrition. PMID- 7558789 TI - Julius Tandler--professor of anatomy, and social reformer. PMID- 7558792 TI - The status of psychiatric nursing: characteristics and perceptions of nurses in the state of Michigan. AB - Concerned about the status of both mental health services and the role of professional nursing in the delivery of those services, the Michigan Nurses' Association Psychiatric-Mental Health Nursing Practice Section initiated a survey of psychiatric nurses in the state. One hundred forty psychiatric nurses returned questionnaires that explored their professional characteristics and activities, the characteristics of their clients, their perceptions of major issues confronting mental health services and psychiatric nursing and recommended actions, the perceived strengths of psychiatric nursing, and the skills and knowledge they believed are necessary for the future. Respondents are working with a variety of patients in difficult circumstances. Responses indicate the need for specialized knowledge and skills to provide care to mentally ill clients with increasingly complex problems, additional nursing involvement in political and health care arenas, and more nursing leadership and support within nursing. PMID- 7558790 TI - The nurse as tour guide: a metaphor for debriefing students in mental health nursing. AB - This article responds to the call by Heinrich (1992) to harness the power of metaphor to reenergize nursing classrooms. An overview of the power of metaphor is provided, as well as a description of a learning activity that prompted students to think metaphorically about a nursing experience. Students reflected on the image of nurse as tour guide and discovered theory being lived out in practice, new ways of understanding the role of the nurse, appreciation for the individual approach in nursing, and a new confidence in sharing and creating. PMID- 7558793 TI - Patient and staff views of factors influencing assaults on psychiatric hospital employees. AB - This study compares the viewpoints of psychiatric hospital employees and patients as to situational and interactional factors that are related to patient assaults on staff. Sixty-nine patients from eight high-assault wards in two state psychiatric hospitals were interviewed to identify hospital practices and aspects of the physical environment that they believed to be related to assaultive behavior. One hundred thirty-seven nursing staff members on the same wards were surveyed. Patients and staff had many concerns in common, including restrictions on patient smoking and access to the outdoors, staff clinical skills and patients' being treated with respect, and the use of seclusion and restraint on the wards. Additionally, patients were concerned about rules not being explained. Staff believed that the single most important issue was an adequate number of personnel. A considerable portion of assaults may relate to circumstances that are amenable to intervention, and assaults may be reduced in severity or number by changes in hospital practices. PMID- 7558791 TI - Use of RN case management and costs and utilization of outpatient mental health services: a pilot study. AB - The majority of outcome studies on case management seem to have been studied on the chronically mentally ill patients. Little is known regarding the use of a case management approach with general psychiatric populations, which include non chronically mentally ill patients in an outpatient setting. This study examined the effect of a registered nurse case management (RNCM) model on the delivery and cost of mental health services during an 18-month period for clients in an outpatient psychiatric setting. Sixty clients met the criteria for sample selection. The data indicated that the use of the RNCM model resulted in more nursing than psychiatric care. Despite more frequent visits to the RNCM and longer RNCM visits, RNCM services appeared cost effective and were related to decreased length of hospital stay. A subgroup of high-risk clients was identified, and a significantly greater use of services by this group was substantiated. PMID- 7558795 TI - Suicidality correlates in Mexican American teens. AB - This study was conducted to determine the relationships between suicidality and life stress, coping, depression, and family dysfunction in Mexican American teens. The research question was: What are the magnitude of the relationships between suicidality and life stress, coping, depression, and family dysfunction in Mexican American teenagers? The investigators discuss the findings of the study from the perspective of the Mexican American culture, followed by specific conclusions and recommendations drawn from the results of the study. In this article, the term teen is used interchangeably with the terms teenager, youth, and adolescent; suicidality is used interchangeably with suicide risk. PMID- 7558796 TI - Manifestations of sexual abuse in preschool-aged children. AB - All adults who interact with preschool children need to be aware of possible indicators of sexual abuse. This information is especially important to advanced practice psychiatric/mental health nurses who interact with preschool-aged children in clinical, therapeutic, educational, research, legal, and community settings and to other nurses working in pediatric settings. Because there are few, if any, absolute physical indicators of child sexual abuse, the identification of empirically based emotional and behavioral indicators is important. This article reviews six studies that sought to identify such indicators for the preschool population. Studies reviewed confirm that not all sexually abused children are equally traumatized. When sexually abused children were compared to groups of non-sexually abused children receiving psychiatric services only one discriminating variable consistently arose. When overt sexual behavior, inappropriate for age, is manifested by a preschool-aged child, sexual abuse should be suspected. The identification of manifestations of sexual abuse in preschool-aged children is an appropriate topic for nursing research. PMID- 7558797 TI - Pediatric asthma--a correlation of clinical treatment and oxygen saturation. AB - To determine the relationship between changes in room air oxygen saturation (SaO2) and changes in the clinical signs of pediatric asthma patients after treatment with nebulized albuterol, a 9-month prospective observational study was conducted. Eighty-two patients from 2 to 15 years of age who had exacerbations of asthma were studied when they presented to a military community hospital emergency department with an annual census of 62,500. For each patient, the change in SaO2 30 minutes after administration of nebulized albuterol was compared to the change in an ordinal clinical scoring system for asthma. Physicians were blinded to SaO2 measurements. Data are reported as mean values with differences between groups analyzed using the paired t-test. Patients with an initial SaO2 < 95% who clinically improved after treatment had a mean increase in their SaO2 of 2.6%. Patients with an initial SaO2 < 95% who did not clinically improve after treatment had a mean decrease in SaO2 of 1.1%, but this was not statistically significant (p = 0.14). The positive predictive value for improved SaO2 indicating clinical improvement is 98%. Patients with an initial SaO2 > 95% did not have significant changes in SaO2 after treatment regardless of clinical response. For pediatric asthma patients with an initial SaO2 < 95%, increased SaO2 after treatment with inhaled albuterol is predictive of clinical improvement. Patients with an initial SaO2 < 95% who do not have improved SaO2 after treatment require further evaluation. PMID- 7558794 TI - Affectivity and mental health among elderly religious. AB - Fifty elderly Catholic nuns from the Midwest were interviewed in a descriptive study to obtain information regarding affectivity and mental health. Lawton's Philadelphia Geriatric Center affectivity instrument was used to determine frequency of positive versus negative emotions and 10 dimensions of affectivity. Overall, these subjects reported positive emotional experiences to be more common than negative ones. The nuns were shown to be strongest in the affectivity dimensions of duration of feelings, affective dissimulation, and emotional adaptation. They scored lowest on sensation seeking, variability of feelings, and emotional maturity. The mean scores of these subjects demonstrated statistically stronger emotional experiences than did Lawton's subjects. In response to an open ended question on definition of health, evidence of a multidimensional perception of health was present among these elderly nuns. This research provides the foundation for further work designing and implementing interventions aimed at promoting optimal mental health among members of cultural minority groups. PMID- 7558798 TI - Acute anticholinergic syndrome following ingestion of Angel's Trumpet tea. PMID- 7558799 TI - Survey of lead levels in patients presenting to child and adolescent psychiatry. PMID- 7558803 TI - HMSA's HealthPass--a strategy for delivery of preventive services. AB - Several groups have recommended standards for preventive health care services. Despite this, our health care system contains many obstacles that impede their delivery. Recognizing the need to provide members with appropriate, meaningful, and cost-effective health promotion and disease prevention services, HMSA has created HealthPass. This benefit is now available to 40% of the membership. It offers health promotion, education, and screening services through a formalized network across the state. HealthPass services are integrated with individual physician providers to offer selected periodic evaluations for adults. HMSA's HealthPass was created to promote public health and further enable members to obtain preventive services. HMSA encourages its physician providers to participate in this statewide effort at primary health promotion. PMID- 7558802 TI - Testicular cysts: a case report and review of a recent Tripler experience. PMID- 7558800 TI - Cutaneous melanoma in an active duty soldier. PMID- 7558801 TI - Conservative therapy for gastroesophageal reflux in infants with obstructive pulmonary disease. AB - Gastroesophageal reflux (GER) is a commonly recognized problem in infants. However, it is difficult to demonstrate a causal relationship between GER and recurrent obstructive pulmonary disease (ROPD) in infants. In this review, 3 infants with GER and severe ROPD experienced dramatic improvement with conservative GER therapy. PMID- 7558804 TI - Squamous cell carcinoma of the ovary. AB - Benign cystic teratoma is a very common ovarian lesion; and it commonly occurs during a woman's reproductive years and most often is benign. In approximately 1% to 2% of cases, however, it can undergo a malignant transformation with a very poor prognosis. This is especially the case when disseminated disease is present. Usually the associated malignancy is squamous cell carcinoma, and radical surgery is recommended. An American Samoan woman was air-evacuated to Tripler Army Medical Center for further evaluation and therapy after having undergone an exploratory laparotomy and right ovarian cystectomy. Her pathology at the time of her initial procedure revealed a mature cystic teratoma with a malignant degenerative component. Her diagnostic evaluation upon arrival was unremarkable except for her physical exam and pelvic CT. She subsequently underwent a radical surgical procedure to include a surgical staging procedure, revealing disseminated squamous cell carcinoma with FIGO stage III disease. Whereas malignant transformation of a benign cystic teratoma is a rare occurrence, a high index of suspicion should be maintained whenever a preoperative diagnosis is encountered; a radical surgical approach with en bloc resection should be employed. Adjuvant therapy with radiation or chemotherapeutic agents in general has not been shown to improve the outcome, especially in disseminated disease. PMID- 7558808 TI - Supraomohyoid neck dissection as a staging procedure for squamous cell carcinomas of the oral cavity and oropharynx. AB - BACKGROUND: A multi-institutional retrospective study was performed to evaluate the efficacy of the supraomohyoid neck dissection (SOHND) for detection of occult cervical metastasis in squamous cell carcinoma of the oral cavity and oropharynx. METHODS: Seventy-five previously untreated patients with clinically negative necks were studied. Seventeen (23%) neck specimens revealed occult metastatic disease, and 58 (77%) were histologically negative. Postoperative irradiation was received by 94% of the patients with positive specimens and 22% with negative specimens. Patients were followed until recurrence of neck disease, or for a period of 2 years or longer. RESULTS: Cervical metastasis subsequently developed in 25% of treated positive specimen patients, none of the untreated positive specimen patients, 8% of the treated negative specimen patients, and 11% of the untreated negative specimen patients. The sensitivity of SOHND for cervical metastasis was 82%, negative predictive value 91%, and accuracy 94%. CONCLUSIONS: The authors conclude that SOHND is a useful procedure for detection of occult cervical metastatic disease in cancer of the oral cavity and oropharynx. PMID- 7558805 TI - Cytogenetic analysis of tissues from patients with familial paragangliomas of the head and neck. AB - BACKGROUND: Paragangliomas of the head and neck are slow-growing tumors that originate from neural crest cells. Between 7% and 9% of these tumors have a familial occurrence. The suspected gene for familial paragangliomas (FP) is transmitted with an autosomal dominant mode of inheritance with incomplete penetrance, and appears to exhibit genomic imprinting. It has been demonstrated by family studies that individuals who inherit the gene(s) from their father will develop the disease. Through linkage analysis, the gene(s) for FP has been postulated to be located on the long arm of chromosome 11. The discovery of many different genes has been elucidated through the cytogenetic analysis of affected individuals who carry specific chromosome aberrations. This project was designed to look for chromosome abnormalities in several second-generation family members to further assist in the localization of the gene(s) for FP. METHODS: This study involved the cytogenetic evaluation of lymphocytes, fibroblasts, and tumor cells of several second-generation family members from a three-generation family with FP of the head and neck to look for chromosome abnormalities generally, and for abnormalities of chromosome 11 specifically. Standard cytogenetic techniques were used for lymphocyte and fibroblast cultures. Tumor cells were cultured in a collagen matrix with F12 medium supplemented with 3% L-glutamine and 10% fetal calf serum. RESULTS: There were no detectable abnormalities of chromosome 11 in any of the cells. However, nonrandom abnormalities of chromosomes 5 and 7 were seen in some of the tumor cells of one FP patient. To our knowledge, this is the first article which demonstrated the ability to successfully culture FP of the head and neck. PMID- 7558806 TI - Prognostic significance of prostaglandin E2 production in fresh tissues of head and neck cancer patients. AB - BACKGROUND: Immunosuppressive prostaglandins may play a role in the biologic behavior of head and neck cancer. Increased levels of prostaglandin E2 (PGE2) have been measured in squamous cell carcinoma of the head and neck (SCCHN). METHODS: To address this question, tissue levels of PGE2 were measured in tumor tissues, normal mucosa, and lymph nodes of 37 patients undergoing tumor resections. Tissue specimens were placed in culture media, and levels of PGE2 released into the supernatant were measured by radioimmunoassay. RESULTS: Tissue levels of PGE2 were significantly greater in tumor and normal mucosal tissues compared to lymph nodes (p = 0.0003). There was no difference between metastatic and tumor-free lymph nodes. Although tumor tissue levels of PGE2 were not associated with tumor stage, increased levels of PGE2 were associated with increased 2-year disease-free survival (p = 0.02). CONCLUSIONS: Although PGE2 may have adverse effects on local immune function in tumor tissues, improved survival of patients with increased local PGE2 production may be indicative of an enhanced immunologic response to the tumor which has a favorable impact on outcome. PMID- 7558807 TI - Nonsquamous cell malignant neoplasms of the nasal cavities and paranasal sinuses. AB - BACKGROUND: Nonsquamous cell malignant tumors of the nasal cavity and paranasal sinuses are an uncommon and diverse group of neoplasms. Our goal was to define the relative frequency and distribution of these neoplasms, as well as treatment and outcome. METHODS: This retrospective study analyzed 106 patients with nonsquamous cell malignant neoplasms of the nasal cavity and paranasal sinuses treated at our institution between 1966 and 1982. RESULTS: Pathology included: salivary type carcinoma (33 patients); sarcoma (25 patients); melanoma (18 patients); esthesioneuroblastoma (11 patients); lymphoma (11 patients); and anaplastic cancer (9 patients). Most tumors arose in the nasal cavity (50%), followed by the antrum (39%), ethmoid sinus (9%), and frontal sinus (2%). Fifty four percent of all patients were treated with surgery alone. Determinate 5-year and 10-year cure rates were: esthesioneuroblastoma, 70% and 50%; lymphoma, 45% and 30%; anaplastic, 33% and 25%; salivary, 31% and 18%; sarcoma, 25% and 21%; and melanoma, 19% and 0%. CONCLUSIONS: Overall survival for patients with nonsquamous cell malignant neoplasms of the nasal cavity and paranasal sinuses is poor, particularly with follow-up extended to 10 years. Local recurrence is the major source of treatment failure. We were unable to demonstrate any survival advantage in the group of patients who received surgery and radiotherapy in combination. PMID- 7558810 TI - Surgical access to tumors of the cervicothoracic junction. AB - BACKGROUND: Surgical resection of tumors of the cervicothoracic junction is often problematic due to the limitations imposed by the thoracic cage and adjacent neurovascular structures. The majority of surgical approaches to this region have been designed with the intent of providing adequate exposure for vertebrectomy with tumor resection and vertebral column stabilization. These approaches do not provide adequate exposure for a heterogeneous group of tumors which also involve the cervicothoracic junction. We used a combined cervicothoracic surgical approach to determine its efficacy in tumor resection. METHOD: Seventeen patients with a heterogeneous group of malignancies arising in a variety of soft tissues underwent combined cervicothoracic resection. The approach consisted of anterior cervical access, median sternotomy, and anterior thoracotomy. RESULTS: Complete gross tumor resection was accomplished in all 17 patients, 15 of whom had negative microscopic margins. Extensive reconstruction was employed in 6 patients. Three patients received intraoperative brachytherapy implants and 5 patients received external-beam postoperative radiotherapy. Local tumor control was obtained in 12 patients, and 10 patients are currently alive, free of disease (median: 12 months; range: 3-47 months). There was no inadvertent sacrifice of neurovascular structures. The sternoclavicular joint was maintained in all patients. There were 4 major complications, and no perioperative mortality associated with the surgical procedure. CONCLUSION: The combined "trap door" technique provides sufficient exposure for resection of cervicothoracic tumors. Surgery is performed with limited morbidity with the sparing of uninvolved neurovascular structures. The sterno-clavicular joint was maintained in all patients. Preliminary results using this approach for resections of tumors of the cervicothoracic junction are encouraging. PMID- 7558809 TI - Parapharyngeal space neoplasms. AB - BACKGROUND: Primary parapharyngeal space neoplasms are rare. Tumor distribution characteristics and long-term outcome have been quite variable in the recent literature. We report the results of a retrospective review of 172 patients with primary parapharyngeal space neoplasms treated surgically from 1960 to 1990 at a large multispecialty clinic and teaching hospital. METHODS: Initially, 195 patients seen at our institution were found to have parapharyngeal space neoplasms. Twenty-three patients previously treated elsewhere were excluded. The remaining 172 patients (96 female and 76 male) were from 11.7 months to 91.5 years of age. All patients were treated surgically; the transparotid-cervical approach was most widely used. RESULTS: One hundred thirty-seven tumors (80%) were benign and 35 (20%) were malignant. High-resolution computed tomography or magnetic resonance imaging was essential in the diagnosis and presurgical planning. Fine-needle aspiration specimens were accurate in the diagnosis of 8 or 9 cases. Pleomorphic adenoma was the most common neoplasm (40%), followed by paraganglioma (20%), neurogenic tumor (14%), malignant salivary gland tumor (13%), miscellaneous malignant tumors (7%), and miscellaneous benign tumors (6%). Mandibulotomy was required for only 6% of all tumors; its use was reserved for malignant lesions and large skull base tumors when risk of tumor rupture was high. Recurrent or persistent disease was recorded in 27 patients with malignant tumors; of these, 24 (89%) are likely to die of disease. CONCLUSIONS: The transparotid-cervical approach is the preferred procedure for most parapharyngeal tumors and can be combined with midline mandibulotomy for large vascular or selected malignant tumors. Recurrence after removal of pleomorphic adenomas is only 4%. Recurrent or persistent malignant disease is nearly always fatal. Perioperative mortality is zero, and morbidity is most often associated with cranial nerve neuropathy. PMID- 7558811 TI - Incidence of cervical metastasis from uveal melanoma: implications for treatment. AB - BACKGROUND: Although the literature has focused on the propensity of uveal melanoma to metastasize to the liver, little attention has been directed to its spread in regional, cervical lymph nodes. METHODS: We reviewed records of 77 patients who underwent primary resection of uveal melanoma during a 15-year period at our institution. RESULTS: A total of only 6.5% of patients developed cervical metastasis. Factors such as age, race, sex, tumor location, and histology were not influential in identifying these patients. Tumor size and local orbital recurrence were, however, associated with a higher risk of cervical lymphadenopathy (p < 0.05). Cervical metastases were also associated with the presence of distant metastatic disease. CONCLUSION: Neck dissection should be reserved for patients with clinically demonstrable lymphadenopathy only after distant disease has been excluded. PMID- 7558812 TI - Cell adhesion and regulatory molecules involved in tumor formation, hemostasis, and wound healing. AB - BACKGROUND: Dynamic changes in cell adhesion and migration are fundamental properties of the cells that are important in hemostasis, vascularization, and wound repair. Changes in cell adhesion and migration are very important in the formation of tumors, and invasion and metastasis by neoplasms. METHODS: Using immunolocalization and immunopurification techniques, expression of integrin molecules involved in cell adhesion has been characterized during tumor development and wound healing. The ability of cytokines to regulate expression and antibody and peptide inhibitors to inhibit function of integrins has been explored to better understand the role of integrins in pathogenesis. RESULTS: Increased suprabasilar expression of the integrins alpha 6 beta 4, alpha 2 beta 1, and alpha 3 beta 1 accompanies development of squamous cell neoplasms of the head and neck. Integrin alpha II beta 3 is an integrin receptor which is important in platelet aggregation in hemostasis and formation of tumor metastases. Increased expression of integrin alpha v beta 3 has been identified in angiogenesis in response to growth factors and tumor angiogenic factors. Cytokine TGF-beta and arachadonic acid metabolite 12(S)-HETE are important signals in regulation of integrins and affect cell aggregation and migration in these processes. Ligands and inhibitors for some of these receptors have been identified which will be useful in determining their role during tumor development and progression. CONCLUSION: Recent advances in understanding the functions of the integrin cell adhesion molecules may soon add new diagnostic methods and therapies to the armamentarium of the head and neck oncologic, microvascular, plastic, and reconstructive surgeon. Therapies directed at controlling integrin cell-adhesion molecule expression and function are being explored to improve scar formation, bone synthesis, inhibition of vascular occlusion, and inhibition of tumor invasion and metastasis. PMID- 7558813 TI - Management of inverted papilloma. PMID- 7558814 TI - Tension pneumocephalus following craniofacial resection. AB - BACKGROUND: The craniofacial approach is a reliable method for excising tumors involving the anterior skull base. Advances in technique have minimized complications. Although cerebrospinal fluid leaks and meningitis are well-known complications, tension pneumocephalus is not well described. We review two cases and discuss the pathophysiology, clinical manifestations, radiographic features, and treatment of tension pneumocephalus. METHODS: Case study. We reviewed the records of all patients who underwent anterior craniofacial resection at our institution, a tertiary care center, from 1976 to 1993. Among 45 patients identified, 2 had tension pneumocephalus. RESULTS: Neurologic deterioration after anterior craniofacial resection occurred in both patients in the immediate postoperative period. Both patients had extradural intracranial air under pressure and were diagnosed with tension pneumocephalus. In one patient, this was treated by needle aspiration followed by catheter drainage, and the second patient was treated with needle aspiration followed by airway diversion. The first patient recovered fully and was discharged on postoperative day 14; the second patient's mental status did not return to the preoperative level, and he was discharged on postoperative day 23 to a rehabilitative facility. Approximately 3 months later, his level of mentation returned to baseline. CONCLUSIONS: Tension pneumocephalus is a potentially devastating complication that may occur after craniofacial resection. It requires prompt recognition and treatment to minimize morbidity. PMID- 7558817 TI - Free jejunal interposition reconstruction after pharyngolaryngectomy: 201 consecutive cases. AB - BACKGROUND: Reconstruction of tubular defects following pharyngolaryngectomy has required complicated surgery with high perioperative morbidity and mortality. Free jejunal interposition provides an excellent reconstruction with potential for lower immediate complications and better long-term results than other procedures. METHODS: A total of 201 consecutive free jejunal interpositions were performed following pharyngolaryngectomy between 1977 and 1993. Operative details, complications, and outcome were prospectively documented. RESULTS: Perioperative mortality was low (4.5%) and microvascular success rate high (97%), although a small number of late failures were recorded. Average time until swallowing postoperatively was 11 days, and 92% of patients could maintain full nutrition. Voice rehabilitation was mentioned, and increasingly good results are being obtained. Complication rates for the neck (17%) and the abdomen (2.5%) were also low. There were no problems with excess mucus production or reflux. Radiation effect on the jejunal conduit was not detrimental to long-term patency of the vascular anastomoses or to function as a conduit. CONCLUSIONS: Comparison with other published techniques permits the contention that a free jejunal interposition is the reconstruction of choice after pharyngolaryngectomy. PMID- 7558820 TI - [Intestinal colonization with Candida albicans and its effect on chronic inflammatory dermatoses]. PMID- 7558818 TI - Histocytologic grading of mucoepidermoid carcinoma of major salivary glands in prognosis and survival: a clinicopathologic and flow cytometric investigation. AB - BACKGROUND: Controversy exists regarding the role of a 3-tiered grading system for mucoepidermoid carcinoma (MEC) of salivary glands in prognosis and survival. This retrospective investigation evaluated a 3-tiered grading system modified from Healey by Batsakis and Luna and compared various clinical, pathologic, and flow cytometric parameters and overall survival among MECs of differing grades. METHODS: Forty-eight patients with 7 low-grade (LG), 23 intermediate-grade (IG), and 18 high-grade (HG) MECs of parotid (n = 43) and submandibular (n = 5) glands were studied. Data were analyzed using categorical statistics (Wilcoxon, Kruskal Wallis and Chi-squared tests where appropriate). RESULTS: Mean ages were 42 years for patients with LG tumors; 47 years, IG; and 59 years, HG (p = 0.02). Gender ratio (p < 0.001) changed from female predominance in LG (6 F:1 M) and IG (2.1 F:1 M) to male predominance in HG (3.5 M:1 F). Mean tumor stage was 1.4 LG, 2.4 IG, and 3.6 HG (p < 0.005). Tumor size increased from 2.1 cm for LG to 3.8 cm for HG (p = 0.01). Margins were involved by tumor in 0% LG, 44% IG, and 61% HG (p < 0.001). Lymph node involvement was 0% LG, 22% IG, and 72% HG (p < 0.001). DNA aneuploidy (DNA index < 0.9 or > 1.1) was present in 0% LG, 13% IG, and 28% HG (p = 0.05). Proliferative fraction (S + G2M) was 5% LG, 7% IG, and 13% HG (p = 0.008). Radiotherapy was administered in 14% LG, 35% IG, and 61% HG (p = 0.03). Recurrences (local and/or metastatic) occurred in 0% LG, 39% IG, and 61% HG (p = 0.009). Survival was decreased significantly (p < 0.0001) with increasing tumor grade (100% LG, 70% IG, and 22% HG). CONCLUSION: Histologic grading of mucoepidermoid carcinomas of major salivary glands, using the modified Healey 3 tiered system, correlates well with clinical, pathologic, and flow cytometric factors which influence the prognosis and overall survival in affected individuals. PMID- 7558815 TI - Papillary thyroid carcinoma mistaken for malignant melanoma: pitfalls in diagnosis. AB - BACKGROUND: Immunohistochemical methods and aspiration cytology are used with increasing frequency in the diagnosis of head and neck histopathology. Magnetic resonance imaging is also gaining popularity as a diagnostic tool. METHODS: We report a case of papillary thyroid carcinoma which was initially misdiagnosed as malignant melanoma due to several confounding diagnostic test results. RESULTS: False-positive immunostaining of a cytologic preparation with a commercial preparation of HMB45 melanoma stain and characteristic MRI findings contributed to the erroneous diagnosis of malignant melanoma. CONCLUSIONS: Potential pitfalls in test interpretation are discussed, and recommendations for the judicious use of immunostaining, MRI, and other diagnostic studies are presented. PMID- 7558819 TI - Radiotherapy for Merkel cell carcinoma of the skin of the head and neck. AB - BACKGROUND: Merkel cell carcinoma is a relatively rare neuroendocrine carcinoma of the skin. It arises in the head and neck region in approximately 50% of cases. Its aggressive behavior predisposes patients to local-regional recurrence and distant metastases after surgical excision alone. In this article, we describe our experience with Merkel cell carcinoma of the head and neck. METHODS: Of 18 patients with Merkel cell carcinoma treated in the Department of Radiation Oncology at the University of Florida, 12 patients who had primary tumors in the head and neck region are reported. Eight patients were treated at initial diagnosis (group A), and four were treated at the time of local-regional recurrence (group B). RESULTS: Local-regional control was achieved in seven of eight patients in group A and all four patients in group B. One patient in group A and all patients in group B developed distant metastases and eventually died of their disease. Bone exposure developed in one patient, requiring surgical debridement and hyperbaric oxygen treatment. CONCLUSION: Patients with Merkel cell carcinoma of the head and neck should be treated aggressively. Our data suggest that local-regional recurrence is a harbinger of distant metastases. We recommend that these patients receive treatment to both the primary site and draining lymphatics at initial presentation. The role of chemotherapy remains unclear. PMID- 7558816 TI - Breast carcinoma metastatic to paranasal sinuses. AB - BACKGROUND: Breast cancer affects 150,000 women a year in the United States. Breast carcinoma that is metastatic to the paranasal sinus (PNS) is rare. A patient with breast cancer treated recently at the University of Texas M. D. Anderson Cancer Center developed bilateral metastasis to the ethmoid sinuses. This case prompted a review of the incidence of this disease, its treatment, and outcome. METHODS: A review of the published reports dating back to 1939 of breast carcinoma that was metastatic to the PNS was performed. In addition, autopsy series in the breast oncology literature were reviewed for cases of breast carcinoma that was metastatic to the PNS. RESULTS: From this review, we identified only eight cases of breast carcinoma that was metastatic to the PNS. All cases were unilateral and presented as mass lesions. Despite treatment, all patients died soon after diagnosis. CONCLUSIONS: We conclude that breast carcinoma metastatic to the PNS is rare and has been uniformly fatal because, as disseminated disease, it does not respond well to conventional systemic therapies. PMID- 7558824 TI - [Use of keratinocytes in therapy of ulcera crurum]. AB - Freshly isolated keratinocytes were used for the treatment of non-healing leg ulcers (21 cases). For application, keratinocytes were suspended in fibrin (Tissucol Duo S). One or more applications of keratinocytes initiated granulation and reepithelialization of nearly all leg ulcers (20 cases). The method is compared with the established therapy entailing transplantation of allogenic keratinocyte sheets onto leg ulcers. Our method shows the advantages: autologous cells that are immediately and easily available, freedom from pain, and short time immobilization (48 h) after application. PMID- 7558825 TI - [Successful treatment of a case of cutaneous Langerhans cell granulomatosis with 2-chlorodeoxyadenosine and thalidomide]. AB - We describe the case of a 65-year-old female patient with cutaneous Langerhans' cell granulomatosis without any signs of disease in other organs. She also had systemic lupus erythematosus that had been diagnosed several years before. The coexistence of these two diseases has not been described before as far as we know. The purine analogue 2-chlorodeoxyadenosine (Cladribin), which has been used successfully in the treatment of hairy cell leukaemia, induced complete remission in our patient after 1 week of treatment. After 2 months, however, the patient had a relapse; this was successfully treated with thalidomide. A new understanding of Langerhans' cells granulomatosis as a reactive but not cancerous disease has emerged as a result of recent investigations showing that tumour necrosis factor-alpha (TNF-alpha) plays an important part in the induction of Langerhans' cells from their immature precursors. Because thalidomide has been shown to inhibit TNF-alpha production, down-modulation of this cytokine seems to be a useful treatment strategy in Langerhans' cell granulomatosis. Some asspects of the diagnosis and therapy of this disease are briefly reviewed. PMID- 7558822 TI - [Treatment of naevi flammei in adulthood with a flashlamp pumped pulsed dye laser]. AB - Since 1993 almost 400 patients with port wine stains have been treated with the flashlamp pumped pulsed dye laser. The therapeutic effect on 61 adult patients was evaluated. After a mean of 2.16 treatments the colour lightened by between 33% and 100% in 70% of these patients. The best results were obtained in patients with plain pink port wine stains. In this kind of port wine stains the argon laser has proved to be far less successful. The rate of adverse effects amounted to only 5%. The results are expected to be improved by further laser sessions. The flashlamp pumped pulsed dye laser is an important advance in the treatment of port wine stains. PMID- 7558826 TI - [Generalized granuloma annulare or diffuse dermal histiocytosis?]. AB - Generalized granuloma annulare is a rare variant of granuloma annulare affecting the trunk and extremities with a multitude of lesions. In contrast to localized granuloma annulare, generalized granuloma annulare occurs in older patients, shows a stronger association with diabetes, and is characteristically chronic. Like our 55-year-old patient, most patients present with papules and annular plaques; less often, macular or non-annular lesions may be encountered. Histology often fails to show necrobiotic or necrotic connective tissue changes demarcated by a palisading granuloma. Instead, there are diffuse dermal, band-like or nodular aggregations of histiocytes intermingled with some multinucleated giant cells and a predominantly lymphocytic infiltrate in the periphery. Because of its special characteristics, it has been suggested that generalized granuloma annulare might constitute a separate disease entity and that it should be classed among the primary cutaneous histiocytoses as a diffuse dermal histiocytosis. Using immunohistochemistry to determine the macrophage phenotype of the lesional histiocytes, we have shown that generalized granuloma annulare is not a cutaneous histiocytosis. Neither MS-1 high-molecular-weight protein, a new specific marker for cutaneous non-Langerhans cell histiocytoses, nor CD1a, the well-known marker for Langerhans cells and Langerhans cell histiocytoses, is expressed by the lesional histiocytes of our patient. In contrast, the antigen expression pattern was diagnostic for non-infectious granulomas and was highly similar to that in localized granuloma annulare. In contrast to the successful treatment of localized granuloma annulare reported with intralesional interferon beta-1, systemic treatment with interferon alpha-2b (9 x 10(6) units three times a week) was ineffective. PMID- 7558821 TI - [Lindane]. AB - In Europe and in the US ectoparasitic diseases are increasingly diagnosed in all classes of society. Modern means of transportation promote the spreading from endemic areas into new regions and therefore questions of therapy are becoming increasingly important. A series of preparations in various galenic formulations and in various concentrations are available to treat the ectoparasitic diseases. Due to reports on Lindane poisonings the clinical use of Lindane preparations for the treatment of scabies and pediculosis has become somewhat controversial. In this article we review older and more recent data on pharmacology, pharmacokinetics and toxicology of gamma-1,2,3,4,5,6-Hexachlorcyclohexan [Lindane]. It is pointed out that in the USA Lindane is offered in 1% preparations whereas in the German speaking Europe only 0.3% preparations are available. This latter concentration is considered as sufficient to treat the ectoparasitosis. Furthermore the use of this remedy and aspects of Lindane resistance are discussed. It is concluded that only through additional in depth education of the patients and their folks successful treatment is possible and intoxication can be prevented. PMID- 7558827 TI - [Periarteritis nodosa cutanea. 20 years follow-up with leg pain, Raynaud syndrome and nail changes]. AB - Polyarteritis nodosa is a multi-systemic vasculitic disease process involving small and medium-sized arteries throughout the body. Cutaneous polyarteritis nodosa has been described as a limited form involving the skin, skeletal muscles, and peripheral nerves. A protracted course of 20 years or longer, with few cases eventually developing visceral involvement, has been emphasized by many authors. A case of polyarteritis with a 20-year history of exacerbations and spontaneous remissions that never seriously compromised the patient's health is presented. The initial and predominant symptoms were leg pains, Raynaud's phenomenon, and nail defects (onychoschizia). PMID- 7558829 TI - [Axillary accessory breast tissue]. AB - A 34-year-old women had a hen's egg-sized supernumerary mammary gland with no nipple or areola in the left anterior axillary line for the last 8 years. The main complaint about the axillary "lump" was that it was aesthetically displeasing, and it was excised. The importance of an axillary breast lies in the possibility that pathologic changes could develop in its substance and that clinically it could be confused with a lipoma. Early diagnosis and correct excision are usually recommended. PMID- 7558831 TI - [Macular amyloidosis with systemic involvement?]. PMID- 7558828 TI - [Anaphylactic shock after lipid exchange chromatography in a patient with congenital IgA deficiency]. AB - We report on a 59-year-old woman patient with selective IgA deficiency and familial hypercholesterinaemia. To lower elevated LDL levels in the patient's blood, immunoadsorption therapy with sheep-anti-human polyclonal antibodies coupled to sepharose columns was administered. During the procedure, the patient developed an anaphylactic shock requiring intensive care treatment. The patient's history revealed a fresh cell therapy with fetal sheep cells 10 years previously. Intracutaneous testing confirmed sensitization to sheep immunoglobulin, which was the most likely reason for the anaphylactic shock. PMID- 7558832 TI - [Systemic scleroderma]. PMID- 7558823 TI - [Pulsed direct current iontophoresis as a possible new treatment for hyperhidrosis]. AB - Tap water iontophoresis with direct current represents the therapy of choice in palmoplantar hyperhidrosis. Side effects are minor discomfort and skin irritations. Improper use may induce iontophoretic burns at sites of minor skin injuries. The aim of this study was to find ways of minimizing side effects, increasing safety standards and reducing the technical complications of tap water iontophoresis without loss of efficacy. In a blind study, 30 patients with palmar hyperhidrosis were treated with tap water iontophoresis using pulsed direct current of 4.3 kHz or 10.0 kHz. Efficacy and side effects were compared with those of the conventional direct current method as a control. Normal sweat secretion rates of palms were found after an average of ten treatment sessions with the conventional direct current method and after twelve with pulsed direct current of 4.3 or 10 kHz. Treatment with pulsed direct current of 4.3 kHz failed to inhibit palmar hyperhidrosis in two of ten patients. Occasionally, such side effects as discomfort, skin irritation, and mild electric shock occurred when direct current was applied. Using pulsed direct current subjective sensations of discomfort and skin irritation were rare (4.3 kHz) or very rare (10 kHz). Electric shock was completely prevented. Because of the minimal side effects, despite minor loss of efficacy tap water iontophoresis with pulsed direct current can be a valuable alternative treatment for palmar hyperhidrosis. PMID- 7558830 TI - [Metastatic aspergillus panniculitis in blast transformation of a myelodysplastic syndrome and agranulocytosis]. AB - We present a 58-year-old patient with disseminated aspergillosis and metastatic panniculitis. He suffered from agranulocytosis and blastic transformation of a myelodysplastic syndrome. The skin lesion showed a single subcutaneous inflammatory nodule, which was clearly shown by histolog to be of embolic origin. This clinical presentation has not previously been described. Disseminated aspergillosis is found mainly in immunocompromised and neutropenic patients and the mortality is high. The present case demonstrates how histological examination together with skin biopsy culture can allow the diagnosis. PMID- 7558833 TI - Certainty and uncertainty in radiation research: the 1995 Lauriston S. Taylor Lecture. PMID- 7558834 TI - ICRP recommendations applicable to the mining and minerals processing industries and to natural sources. International Commission on Radiological Protection. AB - The current views of the International Commission on Radiological Protection on exposure to natural radiation are presented in the new recommendations (ICRP Publication 60) where the concepts of practice and intervention are both used to establish mechanisms of control. The history of the present recommendations is reviewed, and the latest guidance on the control of 222Rn at home and at work (ICRP Publication 65) is outlined. The new model of the respiratory tract, which has been adopted by ICRP, is discussed in the context of its predictions of the doses from radon and from particulate natural radionuclides. Consideration is given finally to criteria for the restoration of sites previously contaminated by natural radionuclides. PMID- 7558835 TI - Measurement of neutron and gamma radiation in a mixed field. AB - This paper describes a study of dosimeters with a range of 0 to 0.2 mGy that were developed by the authors and built by the Federal Emergency Management Agency (FEMA). These instruments are a type of air-filled ion chamber that is self reading by means of an internal carbon fiber electrometer. Two types of these dosimeters were constructed: one with an ion chamber wall made of a conductive hydrogenous material, and the other device made with a conductive wall lining of non-hydrogenous material. Both types of dosimeters have the same sensitivity for gamma radiation, but greatly different sensitivities for fast neutrons, thus making it possible to measure gamma radiation and neutron doses separately in a mixed radiation field. The results indicate that such pairs of dosimeters can be used for the first time to accurately monitor personnel for gamma ray and neutron doses in real time. Since the difference in neutron sensitivities is due to the properties of wall materials, periodic calibrations of the dosimeter system can be accomplished using only gamma rays after the material constants are measured. The absolute number of neutron induced transmutations in sulfur was required for this work. Methods and techniques which were applied to determine this quantity are described in the text. This approach was one of several dosimetric procedures utilized in this investigation. PMID- 7558836 TI - 210Po and 210Pb intake by the Portuguese population: the contribution of seafood in the dietary intake of 210Po and 210Pb. AB - Through analysis of 210Po and 210Pb in the diet, the average ingestion rate for the Portuguese population is estimated at 1.2 and 0.47 Bq d-1 per capita for 210Po and 210Pb, respectively. Detailed analysis of foods indicate that seafood alone contributes up to 70% of the 210Po ingestion rate, whereas cereals, vegetables, and meat altogether contribute 79% of the 210Pb ingestion rate. Consumption of seafood, both in terms of quantities (kg d-1 per person) and preferential consumption of certain marine species, is the cause of the relatively high intake of 210Po and high 210Po:210Pb ratio in the diet in comparison with other countries. Other 210Po and 210Pb sources, namely inhalation of surface air and cigarette smoke, contribute only a small percentage of the absorption of these radionuclides in the blood. Estimated total body burdens of 210Po and 210Pb in adult men, 70 Bq, are 3.5 times higher than estimates for humans living in normal radioactivity regions and consuming a reference diet. Average whole body effective doses for the adult from the Portuguese population are estimated at about 85 microSv y-1 from 210Po and 170 microS y-1 from 210Pb absorbed with the diet. Effective dose from 210Po in the diet may vary from 25 microSv y-1 in a person consuming no seafood to 120 microSv y-1 in an heavy consumer of sardines, to 1,000 microSv y-1 in an hypothetical heavy consumer of molluscs. PMID- 7558837 TI - Lung dose estimates from air sampling and bioassay data--a comparison. AB - Occupational monitoring data are typically in the form of air samples or biological samples. Air samples are more frequently available and often have been used to characterize personnel exposure in epidemiological studies. Air samples that are not specific to individual employees are easier and cheaper to procure than biological samples such as urinalyses. However, the correlation between concurrent air samples and urinalyses has not always been found to be strong. The purpose of this paper is to compare internal radiation doses for uranium workers estimated from air sample results with those estimated from urine sample results. The comparison was made on results associated with individuals who worked in a uranium processing and fabrication facility between 1950 and 1956. Independent lung dose estimates were made for individuals for whom both types of data were available for periods of 300-365 d. Plots of the data and statistical analyses failed to show evidence of correlation of any practical significance between the data generated by the two methods. A number of unquantifiable variables were enumerated for both types of estimates. It is concluded that within this study (1) only minimal correlation was demonstrable between lung doses based on air monitoring and doses based on urinalysis data because of the number of such variables affecting the measurements; and (2) general air data would not be the most useful measure of exposure upon which to base lung dose estimates. PMID- 7558838 TI - Surveillance monitoring of soils for radioactivity: Lawrence Livermore National Laboratory 1976 to 1992. AB - Environmental monitoring of soils at the U.S. Department of Energy's Lawrence Livermore National Laboratory in California has been conducted for more than 20 years. The purposes of the program are to determine the effects, if any, of LLNL's operations on the surrounding areas near its two separate sites and to determine if there are any long-term trends. Data from 1976 to 1992 were analyzed to determine their appropriate statistical distribution, and differences among locations and changes with time were evaluated. The data generally followed lognormal distributions, and results for 239 + 240Pu and 137Cs data were consistent with previously reported values for world-wide fallout. Although all values for 239 + 240Pu were small, 239 + 240Pu data for locations downwind from the Livermore site were found to be statistically significantly higher than for upwind locations. Results for 238U at Site 300 locations near where 238U is used were significantly higher than background locations. Results for 137Cs and 40K were not significantly different between sites, whereas results for 232Th were significantly higher for Site 300 than for the Livermore site. Doses for all radionuclides were below acceptable levels, and doses for most radionuclides were negligible. Evaluation of trends with time yielded small or statistically insignificant changes for all radionuclides. PMID- 7558839 TI - Radon-exposed underground miners and inverse dose-rate (protraction enhancement) effects. AB - Recent models for radon-induced lung cancer assume that at high levels of cumulative exposure, as experienced historically by many underground miners of uranium and other ores, the risk of lung cancer follows an inverse dose-rate (protraction enhancement) pattern. That is, for equal total dose, a greater risk is incurred by those whose total dose is accumulated at a lower rate over a longer duration than at a higher rate over a shorter duration. This inverse dose rate effect is hypothesized to be the consequence of multiple traversals of the nucleus of a target cell by alpha particles. It has recently been concluded, however, that for low total doses, as in most residential settings, the inverse dose-rate effect should diminish and perhaps even disappear, since at very low doses the probability that more than one alpha particle would traverse a cell is small and there would be no possibility for interactions from multiple hits. Pooling original data from 11 cohort studies of underground miners, including nearly 1.2 million person-y of observation and 2,701 lung cancer deaths, we evaluate the presence of an inverse dose-rate effect and its modification by total dose. An inverse dose-rate effect was confirmed in each cohort, except one, and overall in the pooled data. There also appears to be a diminution of the inverse dose-rate effect below 50 Working Level Months (WLM), although analyses were necessarily hampered by a limited range of exposure rates at low total WLM. These data support both the presence of an inverse dose-rate effect, as well as its diminution at low total dose. As a consequence, assessment of risks of radon progeny exposure in homes (on average 15-20 WLM for a lifetime) using miner-based models should not assume an ever-increasing risk per unit dose. Rather, it is more appropriate to apply risk models that take into account protraction enhancement and its diminution. PMID- 7558841 TI - Some problems on the measurement of 222Rn concentrations by passive cup method. AB - A passive cup monitor has been constructed by using a 50 mm radius stainless steel hemisphere. The conductive housing has reduced the scatter in track densities. In dwellings with high 220Rn concentrations, it is particularly necessary to measure 222Rn concentration with a monitor having a radon exchange rate less than 0.1 h-1. PMID- 7558840 TI - On the reduction of error in alpha track detector measurements of indoor 222Rn. AB - Small plastic chambers containing alpha track detectors are often used for measuring indoor concentrations of 222Rn. This paper reports an experimental assessment of sources of error in measurements made with alpha track detector containing chambers. The results demonstrate the feasibility of a nondestructive test for the identification of alpha track detector-containing chambers that are packaged in a way that does not effectively prevent ambient 222Rn from inducing tracks prior to field exposure. Results also indicate that there is statistically significant variation among manufacturing lots and among chemical processing batches. Error from these sources can be avoided or compensated if appropriate control measures are implemented as part of a quality assurance program during any survey using alpha track detector-containing chambers. PMID- 7558842 TI - Radon emanation coefficients for phosphogypsum. AB - Phosphogypsum is a by-product of the phosphate fertilizer industry which is stockpiled in large quantities world-wide. Phosphogypsum consists mainly of dihydrate gypsum (CaSO42H2O) but also contains elevated concentrations of 226Ra and other inorganic species which originate from the processing of phosphate rock. 222Rn gas is the first decay product of 226Ra and has been identified as one of the major environmental concerns associated with phosphogypsum. This study was conducted to determine effects of particle size, weathering, and moisture content on the 222Rn emanation coefficient (epsilon) for phosphogypsum. Average epsilon for air-dry, unfractionated phosphogypsums derived from Togo, Florida, or Idaho rock was approximately 12%. Average epsilon for fine fraction phosphogypsum (< 20 microns diameter) was greater than for unfractionated phosphogypsum by a factor of 4.6, 1.4, and 4.4 for samples derived from Idaho rock, Togo rock, and Florida rock, respectively. Phosphogypsum samples subjected to an artificial weathering procedure lost 40% mass, with no change in epsilon. Increasing water content was found to first slightly decrease, then to increase epsilon compared to air-dry samples; epsilon for 100% saturated phosphogypsum was 1.9-fold greater than in air-dry phosphogypsum. Particle size sorting could account for variability of 222Rn exhalation at repositories. Very high moisture contents could slightly increase 222Rn emanation, but exhalation would likely be reduced due to slow diffusion through porosity of saturated phosphogypsum. PMID- 7558843 TI - The influence of extracorporeal clearance techniques on elimination of radiocesium after internal contamination. AB - Radiocesium, an isotope released after nuclear accidents such as Chernobyl, causes damage to the health of humans after internal contamination. As a result of an internal deposit of radiocesium these persons are continuously irradiated and noxious effects may occur. Removal of this internal radiation source will reduce immediate (short-term) and future damage (long-term). In order to obtain data with respect to cesium kinetics in vivo, data obtained in dogs by Nold et al. were fitted by a computer program. On the basis of these data, simulations were carried out to evaluate the influences of extracorporeal clearance on cesium kinetics. The influence of various treatments on the committed effective dose [E(50)] as a measure of radiation harm was simulated. For this purpose an equivalence between the committed effective dose and the area under the curve, a kinetic parameter, was derived. This equivalence only holds when comparisons are made for different treatments of one subject contaminated with one isotope. Treatment with orally administered Prussian Blue salts reduces the committed effective dose by 29% (50 y). This can be insufficient to prevent deterministic effects as a result of a severe internal contamination with radiocesium. For this purpose other methods are evaluated. In simulations, extracorporeal clearance (e.g. hemoperfusion or hemodialysis) proved to be more effective in reducing E(50) (> 50%, 50 y). Extracorporeal clearance also seems to be effective in the early dose reduction and its consequent deterministic effects. Simulations revealed that effectiveness is improved when the treatment is started earlier and continued for a longer period. Effective extracorporeal clearance may be considered to be a promising method to treat victims of nuclear accidents internally contaminated with radiocesium. PMID- 7558845 TI - Health physics and aviation: 1990-1994. AB - For several years, the U.S. Federal Aviation Administration has explicitly recognized flight crew members as workers who are occupationally exposed to radiation. To help these workers assess their radiation risks, the FAA has developed a computer program that will estimate cosmic-ray exposures from individual flights. They have published documents which inform crew members of the statistical risks associated with their radiation exposure, and they have issued an advisory to the commercial air carriers suggesting that they institute an appropriate educational program for their employees. These developments represent a significant change from ten years ago when the Federal Aviation Administration concluded that, in the area of in-flight radiation exposure, any regulatory involvement on their part would be unwarranted because the vast majority of crew members would not exceed the recommended maximum permissible dose limits then applicable to members of the general public. PMID- 7558844 TI - Soft tissue tumors induced by monomeric 239Pu. AB - Individual records of soft tissue tumor occurrence (lifetime incidence) among 236 beagles injected with 239Pu citrate as young adults and 131 comparable control beagles given no radioactivity enabled us to analyze the possible effects on soft tissue tumor induction resulting from internal exposure to 239Pu. A significant trend was identified in the proportion of animals having malignant liver tumors with increasing radiation dose from 239Pu. There was also a significant difference in the relative numbers of both malignant liver tumors (3.2 expected, 22 observed) and benign liver tumors (18.1 expected, 66 observed). Malignant tumors of the mouth, pancreas, and skin were more frequent among controls than among the dogs given 239Pu as were all tumors (malignant plus benign) of the mouth, pancreas, testis, and vagina. For all other tumor sites or types, there was no significant difference for both malignant and all (malignant plus benign) tumors. Mammary tumor occurrence appeared not to be associated with 239Pu incorporation. We conclude that the only soft-tissue neoplasia induced by the intake of 239Pu directly into blood is probably a liver tumor. PMID- 7558846 TI - Hemodialysis as a potential method for the decontamination of persons exposed to radiocesium. AB - Radiocesium may be deposited in the environment as a result of accidents in nuclear installations, for example, as in Chernobyl. Significant internal contamination with radiocesium poses a serious risk to human health, and, therefore, expedient removal is essential to reduce the radiation body burden. In vitro hemodialysis was tested as a potential method to remove radiocesium from a pasteurized plasma solution of bovine or human blood. Clearance values were calculated by a flow independent method. Hemodialysis appears to be a good method to remove radiocesium from blood: within 4 h more than 90% of the administered radiocesium is removed from blood or plasma. Radiocesium in dialysis fluid can be concentrated on Prussian Blue coated columns that were tested previously for hemoperfusion. Radioactive waste disposal problems can be solved by concentration of radiocesium on these columns. In vivo experiments are necessary to confirm these in vitro results. PMID- 7558847 TI - Is measurement of transportability class of inhaled material by in vitro dissolution satisfactory? AB - Much of the published data on in vitro dissolution of uranium is based on the batch replacement technique. Recent papers have suggested that this technique, though convenient, may produce results that are determined more by the experimental design than by the transportability (solubility) of the material being tested. Re-examination of some of the published data appears to support this suggestion. PMID- 7558848 TI - Alpha self-absorption in monazite dusts. AB - Measurements have been made of the self-absorption effects in monazite of alpha particles of the 232Th decay series. Samples of six size fractions of monazite were deposited on filters at different dust concentrations and then the gross alpha activity determined. Self-absorption effects were negligible in monazite particles up to 8 microns diameter provided dust concentrations were less than 1 mg cm-2. Significant self-absorption effects occurred for both larger particle sizes and higher dust loadings. As reported AMAD values in the mineral sands industry range up to 15 microns, which is equivalent to an actual mean size of 8 microns diameter monazite particle, minimal self-absorption occurs in samples collected in air monitoring programs conducted in the industry provided that dust concentrations on the filters are less than 1 mg cm-2. PMID- 7558849 TI - Thermoluminescence dosimetry of the Hiroshima atomic-bomb gamma rays between 1.59 km and 1.63 km from the hypocenter. AB - Gamma-ray kermas from the Hiroshima atomic bomb were measured by a thermoluminescence (TL) method using tile specimens collected from five locations on the rooftop of a building (Postal Saving Bureau), 1,591-1,635 m from the hypocenter. Four tile specimens each from five locations were sampled and quartz grains were extracted from them. TL of these grains were analyzed by the high temperature TL method and gamma-ray kermas were obtained. The results in tissue kerma, on average, are 21% higher (standard errors are 4.3-7.3%) than the DS86 estimation. The present data and the reported TL results indicate that the measured gamma-ray kermas begin to exceed the DS86 values at about 1.3 km and the discrepancy between them increases with ground distance. This discrepancy is attributed to errors in the DS86 neutron source spectrum and is supported by previous neutron activation measurements. PMID- 7558850 TI - The Canadian whole body counting intercomparison program: a summary report for 1989-1993. AB - The Human Monitoring Laboratory, which is the Canadian National Calibration Reference Centre for In-Vivo Measurements, has been conducting an annual intercomparison program for whole body counting facilities since 1989. Each year a number of phantoms are prepared for participants to measure the accuracy, precision, size dependency, and identification capabilities of their whole body counters. Other tests are designed to measure the sensitivity of the whole body counter to geometry factors. Some phantoms are prepared with the same radionuclide from year to year so that time dependent data can be acquired, whereas other phantoms are prepared as unknowns. This article describes the tests that were performed, gives the results for Canadian participants, and compares the results to interim performance criteria. Measurement results from a human volunteer who ingested 137Cs contaminated caribou meat and was counted at a number of facilities in 1990 are also presented. PMID- 7558851 TI - Automated smear counting and data processing using a notebook computer in a biomedical research facility. AB - An automated smear counting and data processing system for a life science laboratory was developed to facilitate routine surveys and eliminate human errors by using a notebook computer. This system was composed of a personal computer, a liquid scintillation counter and a well-type NaI(Tl) scintillation counter. The radioactivity of smear samples was automatically measured by these counters. The personal computer received raw signals from the counters through an interface of RS-232C. The software for the computer evaluated the surface density of each radioisotope and printed out that value along with other items as a report. The software was programmed in Pascal language. This system was successfully applied to routine surveys for contamination in our facility. PMID- 7558852 TI - Criteria for performing multiple dosimetry. AB - When radiation fields vary spatially as a result of job- or location-specific conditions, the use of more than one dosimeter may be necessary for a variety of radiation protection purposes. This paper contains a methodology for when and how to use multiple dosimeters under conditions incident to routine activities in the presence of ionizing radiation. It also describes a methodology for determining the effective dose equivalent when the use of multiple dosimeters has been deemed necessary by radiation protection professionals. The methodology is based upon application of compartments and compartment factors. Included herein are examples demonstrating when multiple dosimeters should be used and how effective dose equivalent should be calculated using the compartment factors. PMID- 7558854 TI - Sensitivity of a mixed field dosimetry algorithm to uncertainties in thermoluminescent element readings. PMID- 7558853 TI - Test of the linear no-threshold theory. PMID- 7558855 TI - ICRU 50 revisited. PMID- 7558856 TI - Special issue: Medical uses of radiation--the first 100 years in review. PMID- 7558857 TI - X rays in diagnostic radiology. AB - An overview of the technical developments in and growth of the diagnostic use of x rays over the century since their discovery is presented. The major developments described include the introduction of the hot cathode x-ray tube, the Potter-Bucky grid, the image intensifier, the computerized tomographic scanner, and the adoption of the digital computer for image processing, display, and storage. Other significant but smaller advances discussed include improved film/screen technology, the rotating anode tube, linear tomography, and some recent advances in image receptors. PMID- 7558858 TI - History and status of x-ray mammography. AB - Mammography is a relatively recent addition to the array of x-ray services available for the detection, diagnosis, and staging of cancer. Technological advances over the past two decades have led to substantial improvements in the quality of mammographic images and reductions in patient doses of more than an order of magnitude. Today x-ray mammography is used not only to characterize suspicious regions of breast tissue discovered by physical examination, but also to screen asymptomatic women for the early detection of breast cancer. National programs have been established to help ensure high quality images, accurate interpretations, and efficient utilization of x-radiation in the detection and diagnosis of breast cancer. In all of the advances that have lead to widespread utilization of mammography, physicists have played a substantial role. PMID- 7558860 TI - Uses of therapeutic x rays in medicine. AB - Treatment of diseases with x rays began within months of Roentgen's discovery, and within four years x rays were being used successfully for the treatment of skin cancers. Deep-seated cancers began to be treated successfully in the 1920's with the advent of "deep" x-ray units and, especially, once supervoltage therapy machines became available in the 1930's. The 1940's and 1950's saw significant growth of megavoltage therapy, initially with Van de Graaff generators and betatrons, and later with linear accelerators. Linear accelerators became popular during the 1960's and 1970's and, by the 1980's they began to replace 60Co units as the most common form of treatment machine. With high-energy linear accelerators, computerized treatment planning, and ingenious fractionation schemes, modern radiotherapy has become a vital component of cancer treatment. PMID- 7558859 TI - Use of diagnostic radionuclides in medicine. AB - The purpose of this paper is to follow the course of historical development in the use of radiopharmaceuticals as a diagnostic tool in nuclear medicine. This course has been a series of plateaus and growth spurts throughout its history. This article is designed to identify the different phases of the development of nuclear medicine, pointing out the events which most shaped its history along the way. Those events included such things as the discovery of radioactivity, the development of the cyclotron and nuclear reactor as a method of producing high specific activity radioactive material, the development of imaging equipment such as the rectilinear scanner, scintillation camera, PET and SPECT, the application of computers, and the discovery of 99mTc and the development of associated kits designed to image many organs and processes in the human body. PMID- 7558862 TI - One hundred years of medical diagnostic imaging technology. AB - Imaging technology for medical diagnostic purposes had its beginning with the discovery of x rays in 1895. Over the past 100 years, the technological advances of x-ray tubes, power generation, imaging detectors, imaging techniques, nuclear medicine, magnetic resonance, and ultrasound have been astounding. A look back at where we have been, where we are, and in some instances where we are going with respect to a range of imaging technologies is the focus of this paper. PMID- 7558861 TI - Use of therapeutic radionuclides in medicine. AB - The purpose of this paper is to follow the course of historical development in the use of radiopharmaceuticals as a therapeutic tool in nuclear medicine. This chapter is designed to point out the different phases of the development of therapeutic nuclear medicine, pointing out the events which most shaped its history along the way. Those events included the discovery of radioactivity, the development of the cyclotron and nuclear reactor as a method of delivering high specific activity radioactive sources, and a few significant therapeutic radionuclides such as 131I and 32P. The most significant therapeutic radionuclide was radium, which is treated very extensively in this paper from an historical viewpoint. It is only recently that attention of the nuclear medicine community turned to new therapeutic agents, such as bone pain palliation agents, monoclonal antibodies, and others. It may be that the next growth phase of nuclear medicine will revolve around therapy with these radionuclides. PMID- 7558863 TI - History of medical radionuclide production. AB - Radionuclide production for medical use originally was incidental to isotope discoveries by physicists and chemists. Once the available radionuclides were identified they were evaluated for potential medical use. Hevesy first used 32P in 1935 to study phosphorous metabolism in rats. Since that time, the development of cyclotrons, linear accelerators, and nuclear reactors have produced hundreds of radionuclides for potential medical use. The history of medical radionuclide production represents an evolutionary, interdisciplinary development of applied nuclear technology. Today the technology is represented by a mature industry and provides medical benefits to millions of patients annually. PMID- 7558865 TI - History of the shielding of diagnostic x-ray facilities. AB - The results of inadequate protection of radiation workers against the harmful effects of diagnostic x-rays became appallingly apparent shortly after the turn of this century. After these effects (which included erythema, malignancy, and even death) became widely known, efforts were begun to implement recommendations to reduce the hazards to radiologic workers and members of the general public. This work will trace the development of diagnostic radiation shielding standards from the earliest days to the present time. PMID- 7558864 TI - Radiation monitoring with reference to the medical environment. AB - This paper discusses the general principles of monitoring with some examples taken from the medical environment. The discussion is divided into two sections. The first section deals with the principles of personnel monitoring as well as the most common types of dosimeters used. Specific techniques applicable to the various areas of radiology are delineated. The second section deals with the use of portable monitors in a medical environment. The types of instruments commonly encountered and their limitations are detailed. This section also discusses the use of portable monitors as applied to the various areas of radiology. In each section some brief historical background has been added where possible. PMID- 7558866 TI - A history of radiation shielding of x-ray therapy rooms. AB - In this report the history of shielding for radiation treatment rooms is traced from the time of the discovery of x-rays to the present. During the early part of the twentieth century the hazards from ionizing radiation were recognized and the use of lead and other materials became commonplace for shielding against x-rays. Techniques for the calculation of the shield thickness needed for x-ray protection were developed in the 1920's and shielding materials were characterized in terms of the half value layer or simple exponential factors. At the same time, better knowledge of the interaction between radiation and matter was acquired. With the development of high energy medical accelerators after 1940, new and more complex shielding problems had to be addressed. Recently, shielding requirements have become more stringent as standards for exposure of personnel and the general public have been reduced. The art of shielding of radiation treatment facilities is still being developed and the need for a revision of the reports on shielding of medical accelerators from the National Council on Radiation Protection and Measurements is emphasized in this article. PMID- 7558867 TI - A history of medical internal dosimetry. AB - This paper presents a short history of the development of medical internal dosimetry. It reviews the evolution of the equations and discusses the development of various mathematical models used to improve radiation absorbed dose estimates. The contributions of Leonidas Marinelli, Edith Quimby, William Mayneord, Robert Loevinger, Walter Snyder, and others are emphasized. PMID- 7558869 TI - History of standards, certification, and licensure in medical health physics. AB - Immediately following the discovery of x rays they were applied to medical diagnosis and therapy. These early medical applications were soon shown to be accompanied by rather severe biologic responses and injuries. This resulted in the development of standards. Radiation standards were developed to properly control the application of x rays in medicine by controlling equipment and how it was used. Control of personnel occurred later with first certification and then licensure. PMID- 7558870 TI - Radiation protection and medical practice with special reference to health physicists and the Health Physics Society. AB - This paper broadly examines the historical development of radiation protection in medical practice through an examination of the interactions of those concerned with radiation protection and medical practice with emphasis on the last half century and on the role played by health physicists and the Health Physics Society. Included is a brief discussion of the relevant activities of various federal agencies and other organizations including the Atomic Energy Commission and successors and the U.S. Public Health Service; the National Council on Radiation Protection and Measurements and International Commission on Radiological Protection; and the Health Physics Society, American Board of Health Physics, American Association of Physicists in Medicine, Society of Nuclear Medicine, and Conference of Radiation Control Program Directors and related scientific and professional bodies. PMID- 7558868 TI - History of the medical uses of radiation: regulatory and voluntary standards of protection. AB - The main focus of this paper is on the historical development of safety standards in the use of radiation or radioactive materials in medicine. However, to provide better understanding and perspective on this history, it must be interwoven with major events and advancements in the development and use of radiation, particularly in the field of medicine. Since this history, as well as that of major events that stimulated the development of radiation protection standards, is extensive, only a very brief overview can be given here. Thus, a sufficient list of references is also provided to allow further examination of detailed historical documentation, and to provide an easier entry into further research. Also, some identification of individuals who have made important contributions to the development of standards, but who are not widely identified in either the relevant standards or the historical literature, is included. This will aid the serious historian in examining files of organizations to uncover facts or rationale that could better explain historical events or developments. PMID- 7558871 TI - Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2 in a Caucasian family from Alabama. AB - Several members of a large Caucasian family who presented with a congenital Heinz body hemolytic anemia were found to be carriers of the unstable Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2. Identification by protein analysis was hampered by the instability of the variant which complicated its isolation from shipped blood samples. Moreover, the detection of the CTG-->CCG mutation at codon 136 of the alpha 2 gene required the substitution of dGTP by dITP during the DNA sequencing process to prevent the occurrence of secondary structures and compressions in the sequencing gel. The first Hb Bibba heterozygote, characterized in 1968 (1), is believed to be a member of this family. The clinical expression of the disease is surprisingly variable. PMID- 7558872 TI - Hb Hakkari or alpha 2 beta 2 31(B13)Leu-->Arg, a severely unstable hemoglobin variant associated with numerous intra-erythroblastic inclusions and erythroid hyperplasia of the bone marrow. AB - A severely unstable hemoglobin variant, Hb Hakkari or alpha 2 beta 2 31 (B13)Leu- >Arg, has been observed in a 5-year-old Turkish girl with a severe hemolytic anemia without Heinz body formation. A modest increase in liver and spleen size was present and the level of Hb F was a high 33%. The variant could not be observed in red cells and was only detected through sequencing of the amplified beta-globin gene and also by hybridization with specific oligonucleotide probes. The parents were normal, and it is assumed that the variant occurred as a de novo mutation. Smears from bone marrow aspirates showed numerous inclusion bodies in the erythroblast and, as a result, a erythroid hyperplasia. It is suggested that the hemoglobin variant which is unstable and is readily losing its heme group because one of the heme binding sites has been lost, precipitates in the erythroblasts, thus interfering with the maturation process and causing the severe anemia. PMID- 7558874 TI - A novel frameshift mutation causing beta-thalassemia in a Sikh. AB - During our survey of beta-thalassemia mutations among residents of the United Arab Emirates, we came across a Sikh family who had two new beta-thalassemia mutations. The father had a frameshift mutation at codons 47/48 (+ATCT), and the mother another frameshift mutation at codons 57/58 (+C). The offspring of this family were two daughters with beta-thalassemia trait and a boy with a compound heterozygosity. The boy, who was transfusion-dependent from the age of 7 months, had a successful bone marrow transplant from his eldest sister at the age of 13 months. PMID- 7558873 TI - Two fetal hemoglobin variants affecting the same residue: Hb F-Emirates [G gamma 59(E3)Lys-->Glu] and Hb F-Sacromonte [G gamma 59(E3)Lys-->Gln]. AB - Two fast-moving fetal hemoglobin variants were discovered in hematologically normal newborn babies; the first originated in the United Arab Emirates and the second in France. The structural study, carried out by miniaturized techniques of protein chemistry, showed that these two mutations affected the same residue of the G gamma chain, the lysine at position 59(E3) was replaced by glutamic acid in Hb F-Emirates, and by glutamine in Hb F-Sacromonte. PMID- 7558876 TI - Hb Kurosaki [alpha 7(A5)Lys-->Glu]: a new alpha chain variant found in a Japanese woman. PMID- 7558875 TI - Binding specificity of a monoclonal antibody to human Hb A. AB - HemoCardTM kits use monoclonal antibody-conjugated metal sol particles in an immuno-concentration assay. The HemoCard Hbs S, C, and E kits detect the three most common hemoglobin variants in both adult and newborn samples. The HemoCard Hb A kit can be used in conjunction with HemoCard Hbs S and C (but not HemoCard Hb E) to distinguish heterozygotes from homozygotes. We examined the antigen binding domain of a monoclonal antibody to Hb A by testing samples from human and animal sources. We confirmed that the Hb A conjugate recognizes an epitope in which glutamic acid at beta 6 is necessary to provide the proper conformation for binding. This binding is not affected by substitutions at positions directly adjacent to beta 6. However, extensive substitutions in the primary sequence (e.g. in chicken, beta 6: glutamic acid, substitutions at beta 3, beta 5, beta 9, beta 10, beta 11) may produce conformational changes, resulting in diminished binding. PMID- 7558878 TI - A newly discovered beta O-thalassemia (IVS-II-850, G-->A) mutation in a north European family. PMID- 7558877 TI - HB Mizuho or alpha 2 beta 2 68(E12)Leu-->Pro in a young Dutch boy. PMID- 7558879 TI - The beta- and delta-thalassemia repository (eighth edition). PMID- 7558880 TI - Molecular analysis of an XY mare with gonadal dysgenesis. AB - In this study, cytogenetic analysis of an infertile mare revealed a 64, XY karyotype. The XY sex-reversed animal had a female phenotype with gonadal dysgenesis. Using Southern blot analysis, we tested for the presence of two Y specific genes SRY and ZFY by using DNA isolated from peripheral blood leukocytes. The results showed that at least the DNA-binding domain of the SRY gene was deleted from the Y chromosome of the XY mare but that the ZFY gene was present on this chromosome. PMID- 7558881 TI - Colcemid resistance in murine SEWA cells: non-Pgy gene amplification at low levels of resistance and preferential Pgy2 gene amplification at high levels of resistance. AB - Mammalian cell lines often become multidrug-resistant to cytotoxic drugs by amplification and/or overexpression of the P-glycoprotein (Pgy) genes. However, several malignant cell lines seem to acquire low levels of drug resistance by non P-glycoprotein mediated mechanisms. We report here on cytogenetical signs of non Pgy gene amplification in murine SEWA cells during the early steps of selection in Colcemid (COL). In line TC13COL0.01, rare cells exhibited a homogeneously staining region (HSR) distally in chromosome 16. As the COL-concentration was raised the HSR-chromosome was retained and, in addition, the cells developed numerous double minutes (DMs). The DMs, but not the HSR, contained amplified Pgy genes. The HSR may correspond to amplified heat shock protein 70 (Hsp70) genes, detected by Southern analysis. A second low-level COL-resistant line, TC13D70.01, contained DMs but showed no amplification of Pgy, Hsp70, Hsp90, alpha- or beta tubulin genes. In higher COL-concentration, P-glycoprotein mediated drug resistance was induced. In contrast to actinomycin D-resistant SEWA cells, in which higher amplification levels of Pgy1 than of Pgy2 are regularly present, the COL-resistant lines showed a preference for Pgy2 gene amplification. These results are in line with the suggestion that the murine Pgy1 and Pgy2 genes have overlapping but distinct drug specificities. PMID- 7558882 TI - Polymorphism and genetic relatedness among wild and cultivated rice species determined by AP-PCR analysis. AB - We have applied the arbitrarily primed polymerase chain reaction (AP-PCR) technique to the analysis of the relationships among six japonica and indica cultivars, and four wild species of rice. Chosen were four primers of arbitrary sequence that gave multiple amplification products when rice DNA was used as template. Among a total of 50 bands scored, 44 were polymorphic, which was sufficient to distinguish the species used in this study. It is apparent from the comparisons of genetic distances that cultivated rice (Oryza sativa) exhibits closest molecular affinity to wild O. rufipogon, suggesting that the origin of cultivated rice is from O. rufipogon. In a dendrogram of cultivated rice and O. rufipogon from different regions, japonica and indica rice were most closely clustered with O. rufipogon from China and India, respectively. Japonica and indica subspecies showed closer affinity with O. rufipogon from different origins than with each other, supporting a hypothesis of multi-centers of the origin of rice cultivation. PMID- 7558884 TI - The XY pair of the mink (Mustela vision) during different periods of testicular activity. AB - Synaptonemal complexes of the mink (Mustela vison) were examined during different stages of testicular activity to determine whether the distribution of prophase substages and the configuration of the sex complement are altered during pre quiescent and regenerative phases compared to those detected during the breeding period. Spermatocytes obtained during pre-quiescence showed no differences from those of breeding season in terms of substage distribution, whereas those from regenerating testes were mainly in zygotene and early pachytene substages, reflecting the high mitotic activity of spermatogonia and their subsequent transit to meiosis. Based on the location of kinetochores on the sex complement, the synapsed segments were identified as the short arm of the X (Xp) and the long arm of the Y (Yq), although pairing of the X and Y beyond the "pseudoautosomal region" was frequently observed. In some spermatocytes, the entire Y chromosome synapsed with the X or split into two strands with only one strand "paired" with the X while the other remained unpaired. It is not clear at present whether the Y chromosome splitting is part of the mechanisms that prevent crossing over in the non-homologous segments of the sex complement that often undergo synapsis or a post-crossover phenomenon unrelated to pairing mechanisms. PMID- 7558883 TI - Localization of fragile sites in the karyotype of Sus scrofa domestica: present status. AB - Fragile sites were induced to expression in pig lymphocyte chromosomes by in vitro exposure to folate antagonists and BUdR-Hoechst 33258. Expressed fragility was localized according to the presented idiograms and compared with the location of known spontaneous translocations. PMID- 7558886 TI - Recombination in single and double heterozygotes for two partially overlapping inversions in chromosome 1 of the house mouse. AB - Recombination frequency, chiasmata and chromatid interference were studied by means of analysis of meiotic configurations in diakinesis and metaphase II in single and double heterozygotes for the partially overlapping inversions In(1)1Rk and In(1)12Rk in chromosome 1 of the house mouse. Recombination frequency in the inverted regions was decreased in single heterozygotes and increased in the double heterozygotes as compared to normal homozygotes. Chiasmata in the inverted regions in the double heterozygotes did not interfere with each other. A significant excess of 4-chromatid double exchanges in the inverted regions was detected in the double heterozygotes. PMID- 7558887 TI - Genotype, environment and the asymmetry phenotype. Dieldrin-resistance in Lucilia cuprina (the Australian sheep blowfly). AB - Dieldrin-resistant (Rdl/Rdl and Rdl/+) and susceptible (+/+) phenotypes of Lucilia cuprina were scored for departures from bilateral symmetry for bristle characters after development at different temperatures, larval densities or concentrations of dieldrin. The asymmetry phenotype of resistant flies was dominant and independent of developmental temperature and larval density. The asymmetry of susceptibles increased for temperatures and larval densities above and below standard rearing conditions. A positive correlation was observed between asymmetry score and dieldrin concentration for all genotypes. The susceptible phenotype did not attain the asymmetry score of resistant in any environment. Resistant phenotypes showed an antisymmetric pattern in each environment; fluctuating asymmetry was observed for susceptibles. The relevance of the results of genetic and general or specific environmental stresses to estimates of developmental perturbation is discussed. PMID- 7558885 TI - A 4/21 tandem fusion in cattle. AB - A tandem fusion involving chromosome 4 and chromosome 21 of the bovine karyotype, is described in a newborn Holstein-Friesian heifer which was also a blood chimera exhibiting female cells of normal bovine karyotype and male cells with a 59, XY + tan (4:21). The rearranged chromosome was a dicentric and the longest acrocentric in the male cells. Apart from the features characteristic of the freemartin condition, no other phenotypic abnormalities were detected in the heifer calf. Since the heifer and other calves of the herd were not eartagged at birth, 20 newborn bull calves of the same age in the herd were analyzed in an attempt to identify the male born co-twin to the heifer. However, this effort proved unsuccessful, probably because the co-twin had died in utero or was erroneously included among the calves sent to the slaughter house. PMID- 7558889 TI - Inherited stress resistance and longevity: a stress theory of ageing. AB - Ageing is considered in the context of the abiotic stresses to which free-living organisms are normally exposed. Assuming that the primary target of selection of stress is at the level of energy carriers, trade-offs under the rate-of-living theory of ageing predict increased longevity from selection for stress resistance. Changes in longevity then become incidental to selection for stress resistance. I therefore suggest the reformulation of the rate-of-living theory to become a stress theory of ageing. This directly incorporates the characteristics of habitats in nature. Under this theory, the primary trait inherited is resistance to stress. Consequently, at extreme ages those with inherited resistance to abiotic stress should dominate. Furthermore, the reduction in homeostasis manifested by deteriorating ability to adapt to abiotic stress as ageing proceeds, should be slowest in those surviving longest. PMID- 7558888 TI - Genetic aspects of communication during male-male competition in the Madagascar hissing cockroach: honest signalling of size. AB - Male Madagascar hissing cockroaches, Gromphadorhina portentosa, engage in agonistic contests with other males and produce audible sounds or 'hisses' during these interactions. Hisses are used to maintain, rather than to establish, social relationships among males. The agonistic hisses of males are variable and could be used as signals to communicate size or competitive ability of an individual. In this study we examined how size influences male-male competition, as well as the genetic variation and covariation of male body size and components of the agonistic hiss. We found that male size affected the outcome of agonistic interactions between pairs of males: a male that dominated in a pair was significantly larger than the male that was subordinate. However, we found no differences in the hisses produced by dominant and subordinate males after controlling for male weight. We estimated heritabilities, evolvability and genetic correlations for male size and characteristics of the hiss from a full sib analysis of brothers. The patterns of heritabilities and evolvabilities were very similar. The heritabilities of both male weight and duration of the hiss were significantly greater than zero. There was a significant positive genetic correlation between duration of the agonistic hiss and male weight, and a significant negative genetic correlation between hiss duration and the beginning dominant frequency. There was also a positive phenotypic correlation and a negative environmental correlation between male weight and hiss duration. Thus, hiss duration can signal the present influence of the environment on male size, whereas information from hiss duration and beginning dominant frequency can signal the male's ability to transmit genetic influence for size.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558891 TI - Wolbachia and cytoplasmic incompatibility in mycophagous Drosophila and their relatives. AB - Bacterial symbionts belonging to the genus Wolbachia are associated with postzygotic reproductive incompatibility in a number of insect species. Using polymerase chain reaction (PCR) amplification of bacterial gene sequences, strains from 10 species belonging to the closely related quinaria, testacea and tripunctata groups of Drosophila were screened for the presence of Wolbachia in their reproductive tissues. Those screened included the mycophagous species D. falleni, D. recens, D. phalerata, D. testacea, D. neotestacea, D. orientacea, D. putrida and D. tripunctata, and the nonmycophagous species D. palustris and D. quinaria. Two species, D. recens and D. orientacea, were found to be infected with Wolbachia. Subsequent tests of four additional strains of D. recens found all to be infected with the bacteria. It was established that these bacteria cause partial cytoplasmic incompatibility in D. recens by antibiotic curing followed by crosses between cured and uncured strains. Curing was confirmed by a PCR assay. Although most species of insects shown to be infected with Wolbachia are cosmopolitan and/or have undergone recent range expansion in association with human activity, D. recens and D. orientacea are endemic species with specialized ecological habits. Preliminary molecular phylogenetic analysis indicates that, among the species we examined, D. quinaria is most closely related to D. recens. To determine whether the bacteria are involved in reproductive isolation between these two species, reciprocal crosses were carried out between D. quinaria and both infected and uninfected (cured) strains of D. recens. Although these species did mate with each other, all interspecific crosses failed to yield hybrid progeny, indicating that the bacteria are not responsible for reproductive incompatibility between these species. PMID- 7558890 TI - Evolution of the rDNA spacer, ITS 2, in the ticks Ixodes scapularis and I. pacificus (Acari: Ixodidae). AB - Evolution of the rDNA spacer, ITS 2, is examined by comparing 17 DNA sequences of the ticks, Ixodes scapularis and I. pacificus. The distribution of fixed interspecific differences and the relative frequency of base changes vs. insertions/deletions (indels) matches the distribution and relative frequency for intraspecifically variable sites. This suggests that most intraspecific variation is not effectively selected against. The base composition of the ITS 2 transcript is G- and U-biased. But, 5-base regions enriched (> 80 per cent) for A or U occur more frequently than expected while G- and C-enriched regions occur less frequently than expected. Enriched sequences may be prone to replication slippage, accounting for the A/T bias in insertions. Slippage-mediated gains and losses of A/T-rich tandem repeats apparently account for most indels. Minimum energy conformations of the two species' folded transcripts share major structural features. Structural inertia arises from intramolecular base pairing within stems that allows most mutations to be absorbed as new bulges off stems. Yet, there is evidence of selection to maintain the conformation. First, intraspecifically variable sites are concentrated at the ends of stems in loops and intersections, structures that do not contribute to intramolecular base pairing. Moreover, some indels that have become fixed in one species compensate for the presence of conformation-destabilizing indels. However, high rates of sequence evolution within stems and absence of compensatory base evolution contraindicates selective constraint. Degenerate dispersed and tandem copies of two subrepeats, each approximately 20 bases long, may account for much of the ITS 2 sequence. These are approximately inverses of each other and are, consequently, capable of significant intramolecular hydrogen bonding to produce folded transcripts of low energy. Evolution of the ITS 2 sequence may largely entail replication slippage-mediated gains and losses of these repeats or their composite subrepeats. PMID- 7558893 TI - Accumulation of cells containing factor XIII subunit a around the foci of intense fibrosis in human epulides. AB - On the basis of clinical and biochemical findings, Factor XIII subunit a (FXIII A) has been conjectured to play an important role in fibrotic processes. Epulis samples at different stages of fibrotic tissue formation were used as a model system for studying the localization and tissue distribution of FXIII A during the course of connective tissue generation. Marker characteristics of cells containing FXIII A (FXIII A+ cells) were determined as well. In double immunofluorescent labelling systems, FXIII A was localized in monocyte-derived (CD-14+), activated (HLA-DR+), and phagocytosing (Ki-M7+) tissue macrophages, which are widely distributed homogeneously in granulation tissues, but start to accumulate around foci of fibrosis as soon as the foci appear. During the relatively long process of fibrosis, FXIII A+ macrophages continuously decrease in number, and their morphological appearance changes from stellate to spindle shaped. The nuclei of these cells were not labelled by Ki-67 monoclonal antibody; this indicating that they represent a non-proliferating cell population in the connective tissue stroma. The present findings may help to link theories concerning the role of FXIII A and those of macrophages in the connective tissue formation so far found separately in the literature. PMID- 7558892 TI - Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium. AB - The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat ductus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria-but not in the digestive vacuoles-of some clear cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558900 TI - [Stapes-plasty and diving sports. Condition of the ear and diving capacity]. PMID- 7558899 TI - An immunohistochemical method for the detection of tumour necrosis factor alpha in cytospins of human bronchoalveolar lavage cells. AB - An immunohistochemical method for assessing the level of tumour necrosis factor alpha in alveolar macrophages obtained by brochoalveolar lavage is described. Cytospins of mixed populations of lung cells were incubated first with a monoclonal antibody to CD68 and then with a specific peroxidase-labelled second antibody in a two-step reaction for the detection of the macrophage marker CD68. A second similarly based two-step reaction for the detection of tumour necrosis factor-alpha followed. Both reactions were visualized, on completion, using different coloured peroxidase substrates which produced a third colour in the event of dual deposition of the substrates. Dual substrate deposition was indicative of alveolar macrophages positive for tumour necrosis factor-alpha. This method has provided a specific and reproducible semi-quantitative test for the presence of tumour necrosis factor-alpha in human activated alveolar macrophages, which can be performed retrospectively on clinical material. A range of concentrations of the cytokine has been demonstrated in individual samples. This dual detection method has the potential for detection of any cell-associated protein product by minor modification of the described method. PMID- 7558896 TI - The thickness of the mucus layer in different segments of the rat intestine. AB - The thickness of the pre-epithelial mucus layer has been measured in different gut segments of rats kept under normal (ad libitum) feeding conditions, and after 48 h of fasting, using cryostat sections and celloidin stabilization from samples containing luminal contents. The mucus layer of the stomach, duodenum, jejunum, ileum, caecum, proximal colon, colon transversum, distal colon and rectum was studied in five groups of male rats (10, 40, 70 and 150 days of age, and older). Under ad libitum feeding conditions, a distinct and continuous mucus layer, with a thickness of more than 3 microns, was only observed in the colon transversum, in the distal colon, in the rectum and in the stomach. No pre-epithelial mucus layer was observed in the duodenum and jejunum where the glycocalix from the apical membrane of the superficial cells appeared to be in a direct contact with the luminal ingesta. In the ileum, caecum and the proximal colon, the surface epithelium of the mucosa was only partly covered by a mucus layer of highly variable thickness. After 48 h of fasting, a mucus layer of 28.8 +/- 25.6 microns and 93.3 +/- 59.4 microns thickness, respectively, was found in the duodenum and jejunum of adult rats, but no increase in the thickness of the mucus layer was observed in the rat hind gut. PMID- 7558894 TI - Characterization of cells containing factor XIII subunit a in benign and malignant buccal lesions. AB - In the present study, the distribution pattern and characteristics of cells containing Factor XIII subunit a (FXIII A) have been studied in benign and malignant lesions of human buccal mucosa. Tissues from four irritation fibromas and three squamous cell carcinomas were studied by means of double immunofluorescent staining techniques in which the detection of FXIII A was combined with a reaction with CD14 (recognizing a monocyte/macrophage differentiation marker antigen), Mac 387 (reacting with a special subset of macrophages), anti-HLA-DR, Ki-M7 (labelling phagocytosing macrophages) or Ki-67 (visualizing a nuclear antigen associated with cell proliferation) monoclonal antibodies. FXIII A was detected in cells of the connective tissue stroma in both benign and malignant buccal lesions. The number of these FXIII A-reactive cells (FXIII A+ cells) increased considerably in the tumour tissues, in particular in those surrounding tumour cell clusters. FXIII A+ cells scattered in the fibromatous tissues were spindle-shaped, whereas in the tumour stroma, large stellate cells predominated, and round cells were likewise labelled around blood vessels. FXIII A+ cells were labelled with CD14 and Ki-M7 in both fibromatous and tumoural buccal mucosa; however, they failed to show any reaction with Ki-67. FXIII A+ cells accumulated in the tumour stroma reacted for HLA-DR as well. These results indicate that in both the benign and malignant buccal lesions FXIII A is contained in a subpopulation of tissue macrophages, which represents a monocyte derived (CD14+) and phagocytosing (Ki-M7+) cell population. The accumulation of the FXIII A+ cells in the tumour stroma is believed to be a result of direct migration from the circulating blood.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558901 TI - [Stapes-plasty and diving sports. Is diving after stapes-plasty really indicated?]. PMID- 7558898 TI - Endocrine cells of the human gastrointestinal tract have no proliferative capacity. AB - There is compelling evidence that the epithelial cell lineage of the gastrointestinal tract are derived from a common stem cell precursor, but the details of the subsequent cellular hierarchies remain uncertain. In this context, it is important to know the arrangement of cell proliferation that gives rise to the final cell populations. In rodents, a number of studies have been performed examining the possible proliferative capacity of endocrine cells, but a wide range of technical problems makes interpretation of these data difficult. Continuous labelling studies suggest there is potential for proliferation in endocrine cells but flash labelling studies have not been conclusive. In man there are no data on this issue. We have taken advantage of the ability to perform double immunostaining for operational markers of proliferation (Ki67 antigen) and endocrine cell phenotype (chromogranin expression). We demonstrate that there are no double-labelled cells in the normal stomach, small intestine or colon of fetal, neonatal or adult humans. Moreover, no double-labelled cells are found in pathological states associated with endocrine cell hyperplasia (gastritis, ulcerative colitis). These data indicate that the normal endocrine cells of the human gut have no proliferative capacity and that, in this cell lineage, population expansion precedes differentiation. PMID- 7558895 TI - Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy. AB - The ability of tannic acid to enhance binding of glycosaminoglycans to purified collagen was analysed in an in vitro system using amino sugar analysis on an amino acid analyser, transmission electron microscopy, and scanning electron microscopy. Collagen was purified by digestion with trypsin, papain, and hyaluronidase. Purified collagen was incubated with hyaluronic acid or with chondroitin sulphate glycosaminoglycan and then treated with tannic acid. Tannic acid was found to enhance retention during preparation for electron microscopy of either of the glycosaminoglycans onto collagen fibres. The ability of tannic acid to enhance binding of collagen and glycosaminoglycans might explain, at least in part, its structural reinforcement effect on resected synovial joint-apposing surfaces during preparation for scanning electron microscopy. PMID- 7558897 TI - Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues. AB - An antiserum raised against a synthetic peptide derived from the primary amino sequence of rat secretogranin II (chromogranin C) was used for immunological (quantitative radioimmunoassay analysis) and immunohistochemical studies of normal human endocrine and nervous tissues. This antibody recognized a novel and biologically active neuropeptide which was coined as secretoneurin. In endocrine tissues, secretoneurin was mainly co-localized with chromogranin A and B with some exceptions (e.g., parathyroid gland). Secretoneurin was demonstrated immunohistochemically in the adrenal medulla, thyroid C cells, TSH- and FSH/LH producing cells of the anterior pituitary, A and B cells of pancreatic islets, in endocrine cells of the gastrointestinal tract and the bronchial mucosa, and the prostate. Immunoreactivity determined by radioimmunoassay analysis revealed high secretoneurin levels in the anterior and posterior pituitary and lower levels in pancreatic and thyroid tissue. A strong secretoneurin immunoreactivity was also found in ganglion cells of the submucosal and myenteric plexus of the gastrointestinal tract, and in ganglionic cells of dorsal root ganglia, peripheral nerves, and ganglion cells of the adrenal medulla. Thus, secretoneurin may serve as a useful marker of gangliocytic/neuronal differentiation. PMID- 7558903 TI - [Risk of hearing loss caused by loud music."Social Acoustic (Civilization Hearing Loss)" Committee, Institute for Water, Soil and Air Public Health]. PMID- 7558902 TI - [Reports from the German ORL congress in Karlsruhe]. PMID- 7558904 TI - [Fatal complications of otitis 60 years ago]. AB - The temporal bone collection of the ENT Department of the Charite Hospital in Berlin contains 128 cases from 1924 to 1941 of fatal complications of acute otitis media and cholesteatoma. In 1927 alone, one patient died on average every 2 weeks at Charite Hospital from fatal otitic disease. Lesions include labyrinthitis, meningitis, sinus thrombosis and brain abscess. Each case is documented by clinical history, autopsy findings and temporal bone sections. The reasons for the various features of each illness are briefly discussed. PMID- 7558905 TI - [Current aspects of frontal sinus surgery. IV: On therapy of frontal sinus osteoma]. AB - Osteomas are the most common benign tumors of frontal sinus. We evaluated 15 patients with osteomas of the frontal sinus who were managed between 1979 and 1992. The average duration of follow-up was 33 months. In 4 cases osteomas were removed completely via the endonasal route using a microscope and endoscope. We recommend performing a frontal sinus drainage-type 3 primarily. Indications are osteomas of the posterior wall of the frontal sinus located close to the infundibulum. For osteomas of the anterior wall and those located laterally or for very large osteomas we prefer the osteoplastic approach. Apart from the excellent exposure this latter procedure also provides good aesthetic results after carefully placed incisions and precise replacement of the bone flap. We recommend the bicoronal incision in patients with large frontal sinuses, women, or in the presence of good hair growth. Incisions are best placed in a frontal crease in patients with hair loss or a, small frontal sinus. PMID- 7558906 TI - [The value of clinical examination methods in diagnosis of acoustic neuroma]. AB - The diagnostic value of routine clinical tests for predicting the presence of an acoustic neuroma (AN) was assessed retrospectively in a group of 391 patients having magnetic resonance imaging (MRI) scans because of a suspected retrolabyrinthine lesion. An AN was found by MRI only in 9% of these patients. The positive predictive value of unilateral sensorineural hearing loss in a pure tone audiogram (PTA) and unilaterally impaired caloric response was 9% and 12%, respectively. In contrast, AN was found in 36% of patients with pathological brainstem evoked response audiometry (BE-RA). A normal PTA or normal caloric response did not exclude the presence of AN (specificity 90% and 71%, respectively). A normal BERA was found in less than 5% of the patients with AN. According to these results, the following guidelines should be followed for the diagnosis of AN: (1) patients with a high suspicion for AN from history, PTA and caloric responses should be sent directly for MRI; (2) patients with low suspicion for AN from screening tests should have BERA performed to exclude a retrolabyrinthine lesion. PMID- 7558907 TI - [Glutathione-SH as protection from cytotoxic side-effects of gentamycin. Studies with isolated outer hair cells]. AB - Hearing loss with irreversible cochlear damage can be the consequence of aminoglycoside treatment. The present investigation found that metabolized gentamicin (mG) but not native gentamicin (G) was cytotoxic for isolated guinea pig outer hair cells (OHC). Glutathione-SH (GSH), a detoxifying agent, completely inhibited the cytotoxic effects of mG. Using FURA-2 fluorescence, both G and mG were found to reduce intracellular Ca2+ concentrations in unstimulated OHC and inhibit increases in Ca2+ concentrations during K(+)-induced depolarization. GSH did not interfere with G and mG effects on intracellular Ca2+ concentrations. In unstimulated OHC, mG but not G induced pathological OHC depolarization, indicating opening of transduction channels to allow influx of K+ ions. GSH inhibited this effect, suggesting that GSH probably inhibits mG-induced pathological opening of transduction channels. In so doing GSH may inhibit ototoxic side-effects of mG. PMID- 7558908 TI - [Dystonia as the cause of pharyngolaryngeal motility disorders]. AB - Dystonia as cause of pharyngo-laryngeal motility disorders has not been adequately considered in most clinical ENT practices. This case study of a patient with spasmodic torticollis, Meige's syndrome and pharyngo-laryngeal motility disorder was found to be due to dystonia as the underlying cause. The possibility of local symptomatic therapy with botulinum toxin injections has currently provided the physician with an effective means for alleviating the disorder. PMID- 7558909 TI - [Lightening strike injuries of the ear]. AB - Although lightning injuries are quite common, lightning-induced damage to the middle or inner ear and to the vestibular nerve has been reported in only a very few cases. Two case reports are presented: a 22-year-old woman and a 26-year-old man who experienced lightning injuries to several body parts including the ear. Pathophysiology, diagnostic criteria and treatment are discussed. PMID- 7558910 TI - [Lyme borreliosis in childhood]. PMID- 7558911 TI - The cytotoxic HLA-DQ3 reactive human hybridoma antibody 4166 that may distinguish DQ7 + 8 from DQ9. AB - The human cytotoxic hybridoma antibody 4166 (IgM kappa) was generated by fusing an in vitro EBV-transformed B-LCL from a multiparous woman with the mouse-human heteromyeloma fusion partner CB-F7. In microcytotoxicity and IIF tests with B LCLs as target cells, the mAb 4166 was specific for DQ3 (= DQ7 + 8 + 9). However, when used for DQ typing of class-II-positive PBMCs, 4166 could be rendered functionally specific for DQ7 + 8 and did not react with DQ9+ PBMCs. Binding of mAb 4166 to DQ8-positive cells was efficiently blocked by several allotype specific mAbs recognizing DQ8. Other HLA class-II-specific mAbs were unable to inhibit. With the use of mAb 4166, it is possible to discriminate DQ7 + 8 from DQ9 in serologic DQ typing. PMID- 7558913 TI - HLA-linked genes acting as additive susceptibility factors in celiac disease. AB - Susceptibility to developing CD is widely accepted to be primarily associated with a particular HLA-DQ alpha beta heterodimer encoded by the DQA1*0501 and DQB1*0201 alleles in cis position on the DR3,DQ2 haplotype or in trans position by DR5,DQ7/DR7,DQ2 heterozygotes. We performed genomic HLA-DR and -DQ typing of 100 unrelated Spanish celiac children and 180 ethnically matched controls. As expected, most (92 out of 100) celiac patients carried the HLA-DQ alpha beta heterodimer, and we selected these individuals for further studies. The results corroborate that although the DQA1*0501 and DQB1*0201 genes in single dosage appear sufficient for conferring disease susceptibility, individuals homozygotes for DQB1*0201 show an increased risk. Furthermore, our data also show that those carrying the genotype DR5,DQ7/DR7,DQ2 have a significantly increased risk of developing CD as compared to those that are non-DR7 positive, also carrying the CD-associated HLA-DQ alpha beta heterodimer. This strongly suggests that there is an MHC linked non-HLA-DQ gene primarily associated with CD present on DR7,DQ2 haplotype, which should either be DR7 or in strong linkage disequilibrium with it. Our data also indicate that, as has already been suggested, another HLA associated CD susceptibility gene may be present on some DR4-carrying haplotypes. PMID- 7558914 TI - Identification of the epitope recognized by the human V beta 5-specific monoclonal antibody 42/1C1. Potential implications for disease therapy. AB - A panel of T-cell clones was generated that was specific for amino acid residues 4-13 of the mycobacterial 65-kd stress protein. All the clones were found to express a member of the V beta 5 family, as defined by PCR. However, the clones could be differentiated on the basis of different staining characteristics with the mAb 42/1C1. This antibody is known to recognize both V beta 5.2 and V beta 5.3, as was the PCR primer pair used in the analysis. Sequencing of the TCRs revealed that those clones which were not stained by 42/1C1 expressed a previously unidentified member of the V beta 5 family. By comparing the sequences of the V beta 5 family members that are recognized by 42/1C1 with those that are not, we were able to identify a probable epitope for the antibody. It is also clear from our data that the TCRs of T cells recognizing identical MHC-peptide combinations, although very similar, may be differentiated by mAbs, thereby posing potential problems in any proposed disease therapy involving treatment with monoclonals. PMID- 7558912 TI - T-cell activation in HLA-B8, DR3-positive individuals. Early antigen expression defect in vitro. AB - The HLA-B8, DR3 haplotype is overrepresented in several autoimmune diseases, implying that genes predisposing to these disorders are linked to this haplotype. In the patients affected by these diseases, as well as in healthy HLA-B8, DR3 individuals, various dysfunctions reflecting an impairment of T-cell activation have been found. To better characterize T-cell impairment of HLA-B8, DR3-positive healthy individuals, we analyzed the surface expression of early (CD69) and late (CD71) activation phenotypes. MNC cultures were stimulated with PHA and used for T-cell phenotyping by flow cytometry analysis. The results showed that the percentage of CD69+ T cells was significantly decreased in MNC from HLA-B8, DR3+ subjects. This defect was detected in cell cultures from all subjects studied, but it attained significance only in females in the early hours after stimulation. The difference in CD69 expression between HLA-B8, DR3-positive individuals and -negative ones was not due to differences in CD4 and CD8 ratios in the HLA-B8, DR3 cells that underwent activation, as following activation the pattern of CD4 and CD8 antigen expression was the same in both groups of subjects. Concerning the late antigen CD71, no significant difference in percentage was observed between T lymphocytes from HLA-B8, DR3+ and HLA-B8, DR3- subjects at all the times studied. The analysis of the requirements for CD69 expression has suggested that sustained PKC activation and an increase of intracellular CA2+ could be responsible for TCR/CD3-mediated CD69 induction. Thus, present data suggest a defect in the signal transduction pathway of the TCR/CD3 complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558916 TI - Sequence-based typing of HLA-A2 alleles using a primer with an extra base mismatch. AB - A PCR-SBT method using genomic DNA for HLA-A2 alleles was established. To achieve specific amplification for detecting a single base difference between A2 alleles and other HLA-A alleles, a primer having one extra mismatch at the second position from its 3'-end was designed. The primer exhibited a high specificity with annealing temperatures from 64 degrees C to 68 degrees C. Thirty-eight Japanese samples were screened using this method. The majority of Japanese A2 antigens were coded by A*0201. A*0206 and A*0207 were observed at relatively high frequencies. Serologically defined split antigens, A2S and A2AK, which we have recently identified, corresponded to A*0203 and A*0210, respectively. Moreover, A*0207 was strongly associated with B46 and DR8.1. PMID- 7558915 TI - High-resolution HLA-DQB1 typing by combination of group-specific amplification and restriction fragment length polymorphism. AB - A reliable method for high-resolution HLA-DQB1 typing using the combination of group-specific amplification and RFLP analysis is described. Group-specific amplification was carried out for the alleles of two groups using the two primer pairs under the same PCR conditions. One group contains DQ5 and DQ6 specificities and the other DQ2, DQ3, and DQ4 specificities. Computer analysis on cleavage patterns for 19 alleles of the DQB1 gene showed that the 11 alleles of the former group could be distinguished with five restriction enzymes and the eight alleles of the latter group could be distinguished with four enzymes. We could reduce the number of restriction endonucleases required compared with the number used in previous studies because we selected appropriate restriction enzymes which had at least one recognition site in almost all DQB1 alleles as a form of internal control. Moreover, DQB1*0602 and 0603, which were indistinguishable using the previously reported PCR-RFLP methods, could be distinguished by the present method. The results of typing of 100 samples from Japanese individuals by this method showed no discrepancy with the results obtained by serologic methods. The calculated allele frequencies showed good agreement with those reported at the 11th International Histocompatibility Workshop. PMID- 7558919 TI - T-cell receptor variable region of the beta-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis. AB - In order to look for a site-specific T-cell response in RA SM, PCR analyses using oligonucleotide primers specific for 24 TCRBV (V beta) families were performed to compare the respective usage of each TCRBV gene by T cells present in PB and SM of 13 patients with RA. In four patients, SM cells from two or three sites of inflammation were subjected to analysis. In one patient, synovial tissue was studied at two different phases of the disease, resulting in a total number of 19 samples of SM cells, which were compared with paired samples of PB cells. The results showed that whereas all 24 TCRBV gene families could be detected in both PB and SM cells, there was some skewing of increased or decreased usage frequencies of particular TCR V beta genes among SM cells. Three TCRBV families were often overexpressed in SM: V beta 3, V beta 17, and V beta 22. Moreover, V beta 4 was often decreased in SM (7 out of 13). This decrease was statistically significant in the RA population studied. SM from different joints of a given patient showed similar variations of T-cell repertoire compared to PB, even 6 months later in the course of the disease. These results demonstrate a biased TCRBV gene utilization in RA SM. This bias appears to be similar in different joints and at different times in the course of the disease. No correlation was found between the bias of TCR repertoire in SM and the HLA typing of these patients. PMID- 7558917 TI - Characterization of an HLA-DQ2-specific monoclonal antibody. Influence of amino acid substitutions in DQ beta 1*0202. AB - The HLA-DQ(alpha 1*0501, beta 1*0201) and-DQ(alpha 1*0501, beta 1*0202) (i.e., DQ2) heterodimers are probably involved in the pathogenesis of celiac disease and several other HLA-DQ-associated diseases. To obtain a tool for studies of these molecules, a mAb of the IgG1 isotype, 2.12.E11, was produced by immunization with purified DQ(alpha 1*0501, beta 1*0201) molecules and murine NIH 3T3 cells transfected with both DQA1*05011 and DQB1*0202. Panel cell studies with HLA homozygous B-lymphoblastoid cells and HLA-transfected murine cells demonstrated that 2.12.E11 bound only to cells expressing HLA-DQ beta 1*0201 or 0202, irrespective of the accompanying DQ alpha chain (i.e., DQ alpha 1*0501 or DQ alpha 1*0201). Another DQ2-specific mAb (XIII 358.4) and the broadly HLA class-II reactive mAb Tu39 strongly inhibited binding of 2.12.E11. Epitope mapping employing mutants with single aa substitutions of DQ beta 1*0202 indicated that position 37 may be of some importance for 2.12.E11 binding. A triple mutant (45G- >E, 46E-->V, 47F-->Y) failed to bind 2.12.E11, suggesting a crucial role for one or more of these residues in the epitope. However, the expression of the mutant beta chain could not be formally proved, as none of the DQ2-reactive mAbs recognized this transfectant. PMID- 7558920 TI - Nomenclature for factors of the HLA system, update January 1995. PMID- 7558922 TI - HLA, trans-Pacific contacts, and retrovirus. PMID- 7558918 TI - A subset of Sjogren's syndrome associates with the TCRBV13S2 locus but not the TCRBV2S1 locus. AB - HGPSS associates with the TCRBV6S7 locus within the TCR beta-chain gene complex. However, V beta 6.7 T cells, encoded by this locus, have never been implicated in the salivary gland destruction that characterizes primary Sjogren's syndrome. Both V beta 13 and V beta 2 T cells have been implicated in glandular destruction. We therefore analyzed the association of HGPSS with both TCRBV2S1, the only TCRBV2 locus, and the TCRBV13S2 locus (the TCRBV13 family member which lies closest to TCRBV6S7). Our results show that the prevalence of TCRBV13S2*2 homozygotes is significantly increased in HGPSS and that there is a high degree of linkage disequilibrium between this locus and TCRBV6S7 not previously described across the TCR beta-chain gene complex. However, HGPSS does not associate with the TCRBV2S1 locus. These results suggest that it is the V beta 13.2 T cell which may be responsible for the autoimmune destruction that characterizes HGPSS and that the previous association of this condition with the TCRBV6S7 locus is primary due to the linkage disequilibrium that exists between it and TCRBV13S2. PMID- 7558921 TI - Interaction between HLA-DR and HLA-DP, and between HLA and interleukin 1 alpha in juvenile rheumatoid arthritis indicates heterogeneity of pathogenic mechanisms of the disease. AB - EOP-JRA is an autoimmune disease that displays associations with DPB1*0201, DR8, DR5, and DR6, as well as an association with IL1A2 (a variant of IL1 alpha gene, not HLA linked). The purpose of this study was to analyze interactions between these genetic factors. We studied 103 EOP-JRA patients, 181 random controls, and 69 DR8-positive controls. We found a positive interaction between DPB1*0201 and the DRB1 alleles encoding DR3, DR5, or DR6, but not DR8. In addition, we found evidence for an interaction between IL1A2 and DR(3, 5, or 6) and DP2, but not DR8. We interpret the data to suggest heterogeneity in the HLA-associated pathogenic mechanisms of EOP-JRA. PMID- 7558923 TI - Detailed motifs for peptide binding to HLA-A*0201 derived from large random sets of peptides using a cellular binding assay. AB - Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A*0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A*0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at positions 4 for binding, and the deleterious effect of R at position 5 (in 9 mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A*0201 by computer analysis (see accompanying paper [56]). PMID- 7558924 TI - A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs. AB - Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths. PMID- 7558926 TI - HLA-DRB, -DQA, and -DQB polymorphism in celiac disease and enteropathy-associated T-cell lymphoma. Common features and additional risk factors for malignancy. AB - CD is a gluten-sensitive enteropathy, strongly associated with expression of the DQA1*0501, DQB1*0201 genotype. CD patients have an increased risk of malignancy, particularly EATCL. However, it is controversial as to whether adults with EATCL represent a subgroup of patients with CD or should be regarded as a distinct entity. To investigate the genetic relationship between CD and EATCL, HLA class II DRB1, DQA1, and DQB1 typing of peripheral blood, frozen or paraffin-embedded biopsy tissue obtained from Caucasian patients with CD (n = 91) or EATCL (n = 47) was performed by PCR-SSOP typing. Genotype frequencies were compared with those observed in 151 unrelated control individuals. A total of 83 (91%) of 91 CD patients were of DQA1*0501, DQB1*0201 genotype (pc < 10(-6), RR = 522.2), compared with 40 (93%) of 43 EATCL patients (pc < 10(-6), RR = 44.2) with amplifiable DNA versus 35 (23%) of 151 controls. DRB1*03 frequencies were also elevated in both patient groups (79 of 91 in CD [87%; pc < 10(-6), RR = 24.5] and 38 of 40 in EATCL [95%; pc < 10(-6), RR = 70.7]) compared with controls (32 of 151, 21%). These results confirm previous studies of HLA associations in CD and also suggest that EATCL arises in individuals with the DQA1*0501, DQB1*0201 CD predisposing genotype. However, the frequency of DRB1*03,04 heterozygotes was significantly increased in the EATCL group (16 of 40, 40%) compared with both control individuals (3 of 151, 2%; pc < 10(-6), RR = 32.9) and uncomplicated CD patients (6 of 91, 7%; pc = 0.04, RR = 9.4).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7558928 TI - Expression of HLA-DR in granulocytes of polytraumatized patients treated with recombinant human granulocyte macrophage-colony-stimulating factor. AB - MHC class II determinants are the restriction elements involved in antigen specific activation of helper T lymphocytes and interaction with CD4 molecules. They are typically expressed on a limited number of cell types, mostly endowed with antigen-presenting capacity. Recently, expression of HLA-DR has been detected on granulocytes stimulated "in vitro" with GM-CSF. However, no evidence of "in vivo" expression in humans has been presented so far. We report here that class II determinant expression is detectable in vivo on peripheral blood granulocytes of polytraumatized patients upon intravenous administration of rhGM CSF. Expression of these molecules appears to be an early effect of rhGM-CSF treatment, independent from endotoxemia or endogenous production of IL-6 or TNF alpha, and rapidly declining upon discontinuation of therapy. Thus, this treatment might increase the number of cells potentially capable of presenting class-II-restricted antigens in these patients. PMID- 7558925 TI - Cytokine-regulated production of the major histocompatibility complex class-III encoded complement proteins factor B and C4 by human glomerular mesangial cells. AB - Local production of complement within normal or diseased kidneys could be of importance during local inflammatory reactions. In the present study, we demonstrate that human MCs are able to synthesize the MHC class-III-encoded complement proteins factor B and C4 in vitro. This synthesis is strongly upregulated following stimulation with cytokine-containing supernatants of activated peripheral blood mononuclear cells. All primary cell lines tested so far are able to synthesize factor B and C4 after stimulation. To determine more specifically whether defined cytokines are able to enhance factor B and C4 complement production, MCs were stimulated with IL-1 alpha, IFN-gamma, and TNF alpha. Factor B synthesis was increased in a dose-dependent fashion by IL-1 alpha, TNF-alpha, and IFN-gamma, whereas C4 synthesis was only upregulated by IFN gamma. Furthermore, factor B synthesis was upregulated after stimulation with IFN alpha, -beta, and -gamma and C4 synthesis only by IFN-gamma. The synthesis of factor B and C4 was inhibited by cycloheximide, suggesting de novo protein synthesis. The cytoplasmic localization of both components was shown by immunofluorescence studies. Northern and dot blot analysis revealed induction of factor B and C4 mRNA after stimulation with cytokines. PMID- 7558927 TI - Nonradioactive alternative to clinical mixed lymphocyte reaction. AB - The MLR is used clinically as a functional assay for genetic HLA disparity. Traditionally this test relies on [3H]thymidine incorporation to detect T-cell proliferation as an indicator of alloantigenicity. Environmental and administrative concerns regarding radioisotope use and disposal have encouraged the development of sensitive, nonradioactive assay for T-cell alloactivation. We describe a nonradioactive alternative to the clinical MLR which uses an IL-2 dependent cell line, CTLL20.3, and MTT reduction to detect IL-2 accumulation in MLC SNs as an index of T-cell alloactivation. We first determined (a) the optimal number of CTLL20.3 cells for the assay, (b) the optimal time of SN analysis for IL-2, and (c) additional manipulations that significantly increase the assay sensitivity for IL-2. Using this assay system with patient lymphocyte combinations, we demonstrated that the CTLL20.3-MTT assays correlate well with the [3H]thymidine assays of T-cell proliferation for the detection of MHC incompatibility. Indeed, the CTLL20.3-MTT assay may be slightly more sensitive than the traditional clinical MLR. PMID- 7558929 TI - Both HLA-B*1301 and B*1302 exist in Asian populations and are associated with different haplotypes. AB - A B13 split antigen was newly identified with three alloantisera in Japanese, and two B13 split antigens were found in a Thai family. To confirm the variation of B13 and understand the correspondence between the serologic splits and the published B13 alleles, B*1301 and B*1302, we determined the sequences of genes coding for these B13 splits. The common Japanese B13 allele was found to be B*1301, whereas another split antigen was shown to be coded by B*1302. Two B13 variants identified in a Thai individual corresponded to B*1301 and B*1302. Moreover, 57 B13-positive samples from several ethnic groups were examined using the PCR-SSO method. Differing from previous reports, both B*1301 and B*1302 were found in samples from Asian populations. These two alleles were separately associated with different antigens: HLA-B*1301 exhibited a strong association with A2, Cw10, DR12, and DQ7 antigens, whereas HLA-B*1302 was strongly associated with A30, Cw6, DR7, and DQ2 antigens. In addition, applying the PCR-SSCP method, B*1301 and B*1302 could also be simply distinguished from each other. PMID- 7558930 TI - TAP2 association with insulin-dependent diabetes mellitus is secondary to HLA DQB1. AB - IDDM is known to be associated with genes of HLA complex, particularly alleles of HLA-DQ. The 40-kb TAP gene complex is located approximately 150 kb centrometric to the DQB1 locus. The TAP1-TAP2 protein heterodimer is required for normal expression levels of class I, molecules on the surface of cells. While present evidence implicates HLA-DQ as the major susceptibility locus in IDDM, as class I expression apparently plays a role in the progression of disease, the possibility exists that the association attributed to HLA-DQ is in fact due to an association with the TAP genes. Several studies have concluded that the alleles of TAP1 are not significantly associated with IDDM; this report concentrates on the more telomeric TAP2 locus. During this investigation, six previously described TAP2 alleles were identified in 208 normal Caucasians and 241 Caucasian diabetics. Sequence analysis of cDNA clones identified a seventh allele of TAP2, TAP2*F, which contains an arginine-to-cystine interchange at amino acid position 651. Overall, our results indicate only a modest association of IDDM with TAP2; however, the newly described TAP2*F allele was found to be significantly increased in a modest subset of our large diabetic population. These data, generated from the same population of controls and diabetics we previously studied at all other relevant MHC loci, provide additional evidence that the HLA susceptibility to IDDM maps to HLA-DQ. PMID- 7558931 TI - A rare neutral polymorphism in 21-hydroxylase genes as HLA haplotype marker. Evidence for strong founder effect in the Finnish population. AB - The usefulness of rare neutral gene polymorphisms as an HLA haplotype marker and as a probe for founder effect in small populations was tested by determining the frequency and MHC associations of an NcoI polymorphism in the P450c21 genes in the Finnish population. In the general population, 13% (9 of 70) of samples had the NcoI site. A very strong association with the HLA-B62, Bf*F, C4A*3, C4B*Q0, DRB1*13, DQA1*0103, DQB1*0603 alleles was observed. P450c21A and P450c21B gene specific amplifications mapped the polymorphic site to both P450c21A pseudogene and P450c21B functional gene of this haplotype in all cases. The majority of haplotypes with the NcoI cutting site found in this population may thus have derived from a single ancestral haplotype. The HLA homozygous cell lines with the NcoI site showed heterogeneous HLA associations. Our results suggest that in small populations the variety of MHC haplotypes may be surprisingly low and rare polymorphisms can serve as informative markers. PMID- 7558932 TI - Biochemical and molecular characterization of rhesus monkey major histocompatibility complex class II DR. AB - Rapid, simple techniques for MHC class II typing were explored to facilitate the use of a wide variety of nonhuman primate species as models of human diseases and therapies. We demonstrate that radioimmunoprecipitation and 1-D IEF or 1-D NEPHGE can be employed for characterizing MHC class II DR alleles in rhesus monkeys. Complementary molecular analyses have yielded the first full-length nonhuman primate DRA sequence and the first full-length rhesus macaque DRB sequences. In this way it has also been possible to determine which subset of the DRB sequences amplified from a B-LCL are actually expressed. PMID- 7558933 TI - Polymerase chain reaction analysis of the Xba I polymorphism of the human complement C4 genes provides evidence for strong haplotype conservation. AB - The genes coding for the two isotypes of the fourth component of human complement, C4A and C4B, are located between the HLA-B and -DR loci of the MHC. We studied the linkage relationship of the previously described XbaI RFLP to obtain further insight into the evolution of the tandemly arranged C4 genes. Using exon-specific PCR amplification followed by restriction analysis and direct DNA sequencing, the polymorphic site could be located in exon 40 of the C4 gene (cDNA position 5095). The polymorphism does not change an amino acid residue. Using nested PCR amplification with isotype-specific primers to amplify either C4A or C4B alleles the haplotype arrangement of the XbaI sites in both isotypic C4 genes was analyzed independently. It was observed that the XbaI restriction site was either present or absent in both C4 genes of a given haplotype. In a study of 106 Caucasian haplotypes, only two different haplotypes could be identified carrying a C4A gene with and a C4B gene without the XbaI restriction site. Also, the XbaI site could only be detected in long C4 genes possessing the 6.5-kb insertion in intron 9. Our findings provide evidence that the mutation creating the XbaI polymorphism occurred in an ancestral C4 gene already carrying the long intron 9. The duplicating resulting in the presence of two isotypic genes, C4A and C4B, must have taken place subsequently giving rise to haplotypes with or without the XbaI site. PMID- 7558935 TI - Interleukin-7 rescues human activated T lymphocytes from apoptosis induced by glucocorticoesteroids and regulates bcl-2 and CD25 expression. AB - We studied the ability of several interleukins to inhibit the cellular death of IL-2-dependent human T cells deprived of IL-2 testing viability, DNA integrity, and expression of bcl-2 gene product. Our in vitro results showed that the addition of IL-7, and in a far less efficient manner IL-4, augmented the viability of IL-2-dependent T-cell clones of different origin, specificity, and phenotype. Furthermore, IL-7 reduced the percentage of apoptotic T cells inhibiting DNA fragmentation. In addition, IL-7 but not IL-4 was consistently able to suppress the cell death of IL-2-dependent T cells triggered by DEX, a synthetic GC. The suppression of T-cell death triggered by IL-7 was not affected by the addition of anti-IL-2 antibody. Interestingly, IL-7 inhibited the downregulation of bcl-2 gene product expression that appeared on TCCs after IL-2 withdrawal and also shared with IL-2 the ability to induce the upregulation of CD25 antigen on activated T lymphocytes in the presence of DEX. These experiments establish a novel role for IL-7 in regulating viability and GC-induced apoptosis on activated human T cells and suggest that the maintenance of bcl-2 levels is a general mechanism by which interleukins preserve activated T cells from undergoing apoptosis. PMID- 7558934 TI - Characterization of B27 haplotypes by oligotyping and genomic sequencing in the Mexican Mestizo population with ankylosing spondylitis: juvenile and adult onset. AB - The aim of this study was to investigate the contribution of the different B27 subtypes in the Mexican Mestizo population with juvenile and adult AS. No differences in the distribution of B27 subtypes were found between both populations, B*2705 being the predominant subtype followed by B*2702. Transracial gene mapping was performed in order to find out the origin of the B27 alleles of the Mexican Mestizos. A PCR with SSOPs was used to analyze the polymorphism in exons 2 and 3 of HLA-B27 and HLA-C related alleles. This population shares with the Spanish Caucasians B*2705 and B*2702, which are absent in Central and South American Indians. AS and healthy Mexican mestizo donors were analyzed to ascertain B27/Cw haplotypes. The B27/Cw linkage arrangements seen in mestizos are similar to those reported for Caucasian Spaniards with three different haplotypes positively associated with AS in both populations, B*2705/Cw*0102, B*2705/Cw*02022, and B*2702/Cw*02022, suggesting that B27 in Mexicans may be due to a recent Caucasoid admixture with the Spanish genes. Finally, a strategy for sequence analysis of exons 2 and 3 from genomic DNA of HLA-B27 alleles was developed. A novel HLA-B27-like allele typed serologically as B27 was identified and sequenced by this method in a healthy Mexican mestizo, corresponding to the B*7301 variant found with low frequency in different populations. Analysis of the association of B*7301 to AS would require an extensive study in different populations and could provide insights into the molecular structure of the alleles involved in the disease. PMID- 7558936 TI - Reevaluation of the relative risk for susceptibility to celiac disease of HLA DRB1, -DQA1, -DQB1, -DPB1, and -TAP2 alleles in a French population. AB - In a population of 46 children with CD recruited in the Paris area of France, an excess of DRB1*03 and DRB1*07 alleles and of DR3/DR7, DR3/DR3 and DR11(or 12)/DR7 phenotypes was found (RRs of 6.3, 9.3, 24.6, 15, and 15.1, respectively), which is reminiscent of the markers of susceptibility observed in southern rather than in northern European celiac patients. More importantly, the highest association with CD was not found in individuals expressing the DQA1*0501-DQB1*0201 heterodimer in single dosage (RR = 24.9) or in homozygous state, but in people co expressing one copy of DQA1*0501-DQB1*0201 on one haplotype and a second copy of DQB1*0201 on the second haplotype (RR = 35.7). This suggests that in our population either DQB1*0201 or a gene closely linked to DQB1*0201 influences the susceptibility to CD conferred by the DQA1*0501-DQB1*0201 heterodimer. Significant positive or negative RRs conferred by some TAP2 or DPB1 alleles were found. However, they were moderate compared to the RR conferred by the expression of a second copy of DQB1*0201. Moreover, they were no longer significant when patients were compared with HLA-DR matched controls. This suggests that associations of CD with TAP2 and DPB1 alleles are secondary to linkage disequilibria and argues against the contribution of these alleles in resistance and/or susceptibility to CD. Thus the "raison d'etre" of a "DQB1*0201 second haplotype effect" in susceptibility to CD remains to be elucidated. PMID- 7558937 TI - Serologic subtyping of HLA-DR8 by means of the cytotoxic human monoclonal antibody 5643. AB - EBV-transformed B cells from a multiparous woman with anti-HLA antibodies were hybridized with CB-F7 heteromyeloma cells. The resulting hybridoma produced the cytotoxic human IgM(kappa) mAb 5643. Testing of the hybridoma supernatant against HLA homozygous cell lines demonstrated positive reactions (titer range 128-512) only with three cell lines that carried the DR (alpha, beta 1*0801) subtype of DR8. There was no reaction with cells expressing other variants of DR8; i.e., carrying the DR beta 1*0802, 0803, or 0804 chains. The only difference between the DR beta 1*0801 and DR beta 1*0802/0804 chains and between the DR beta 1*0801 and 0803 chains is a single aa substitution, a Ser-->Asp substitution at residue 57 and a Phe-->Ile substitution at residue 67, respectively. Residues 57 and 67 are both situated on the alpha-helix of the DR beta chain. The distance between residues 57 and 67 is two full turns of the alpha-helix; i.e., about 1 nm. It is possible that both 57 Ser and 67 Phe are directly involved in the epitope recognition by mAb 5643. PMID- 7558938 TI - Healthy human T-cell receptor beta-chain repertoire. Quantitative analysis and evidence for J beta-related effects on CDR3 structure and diversity. AB - Analysis of TCR repertoire usage and clonality is of potential value in understanding the pathogenesis of a number of human immune-mediated diseases. In diseases that are likely to be dependent on antigen-driven T cells, it has been suggested that particular TCR junctional region or CDR3 sequences may be critical. Rigorous methods for TCR analysis which are both quantitative and qualitative are therefore required. Of those commonly available, only anchor and inverse PCR are capable of giving high-quality information on V, D, N, and J region usage, but it has not been established whether both methods are quantitatively or analytically equivalent. We show here that both methods detected considerable variability in the usage of V beta and J beta segments in the peripheral blood repertoire of a normal individual. No preferential V-J pairing could be demonstrated. An excess usage of J beta 2 family members was indicated by both methods, although the relative usage of different J beta 2 families differed between the two techniques. The predominantly used V beta usage showed that for some families, estimates of their frequency in the repertoire differed significantly between the anchor and inverse libraries. When sampling relatively few clones the variation between V beta families estimated using the two methods can be considerable. This is likely to be a result of sampling error from a large gene family. Large-scale screening of several thousand clones is recommended to confirm the absolute values obtained from sequencing. Variation in CDR3 length appeared to be normally distributed, suggesting that a statistically optimal junctional region length may have been selected for contact with antigen. CDR3 length distribution differs significantly between receptors, which have rearranged to J beta 1 versus J beta 2 families, with the J beta 2-associated CDR3 on average between 0.5 and 1.2 of an amino acid longer. Thus the TCR beta junctional region repertoire of humans is subject to structural constraints associated with J beta usage. It will be important to establish whether variation in CDR3 length and J beta usage exists between subsets of human T cells in order to interpret TCR repertoire data from disease and control tissues. PMID- 7558939 TI - The role of HLA-DP beta residue 69 in the definition of antibody-binding epitopes. AB - Residue 69 of the DP beta chain has been previously identified as being involved in T-cell recognition as well as in the susceptibility to certain autoimmune diseases. The codon for Glu 69 in the DPB1*02012 allele was changed to the codon for Lys found in DPB1*0402, and transfectant L cells expressing wild-type or mutant HLA-DP molecule were obtained. The binding of a large panel of mAbs to these transfectants was tested by flow cytometry. Glu to Lys 69 substitution decreased the binding to the DPB1*02012 allele of some of the DP mAbs and completely eliminated the binding of four of the antibodies tested. These results clearly showed that this residue is involved in the formation of DP antibody binding epitopes. Because this residue should be located in the alpha-helix of the DP beta polypeptide with the side chain pointing into the peptide-binding groove, its implication in the definition in some DP antibody-binding epitopes should be (a) defining conformational epitopes through effects on the conformation of adjacent regions of the molecule, and (b) determining the binding of peptides to the DP cleft which is directly or indirectly involved in these epitopes. PMID- 7558940 TI - TCRBV12 genes are polymorphic but the protein products encoded by each gene are conserved. AB - The human TCRBV gene complex contains three BV12 genes and all are polymorphic. There are two alleles of BV12S4; one allele contains a stop codon in the coding region which precludes its expression. There are four alleles of BV12S2 and two alleles of BV12S3 genes; however, the substitutions present in both genes are conservative. Although allelic variants exist for each of the three BV12 genes, only one protein product is encoded by each gene. PMID- 7558941 TI - Soluble HLA-G molecule. An alternatively spliced HLA-G mRNA form candidate to encode it in peripheral blood mononuclear cells and human trophoblasts. AB - The HLA-G nonclassic MHC class I gene expressed at the maternal-fetal interface may be involved in cell protection against NK cell lysis. HLA-G mRNA is observed in different adult or fetal human cells and exhibits four alternative forms: HLA G1, HLA-G2, HLA-G3, and HLA-G4 lacking, respectively, exon 7; exons 7 and 3, exons 7, 3, and 4; exons 7 and 4. Because exon 5 encodes the transmembrane domain of the HLA-G antigen, none of these transcripts could give a soluble form as detected in supernatant of trophoblasts. In this report, we describe an additional alternatively spliced for of HLA-G transcript (HLA-G5) present in adult PBMCs and first-trimester trophoblasts that contains intron 4. Moreover, as with all other transcripts, HLA-G5, is devoid of exon 7. Its relative frequency is, respectively, approximately 1:8 and approximately 1:26 in adult PBMCs and first-trimester trophoblasts. The presence of intron 4 generates a stop codon that excludes transmembrane region (exon 5) of the HLA-G molecule and therefore might produce a soluble antigen. These results are discussed with regard to data on soluble forms of classic HLA antigens and the possible role of HLA-G. PMID- 7558948 TI - Second occurrence of symptomatic metastatic spinal cord compression and findings of multiple spinal epidural metastases. AB - PURPOSE: To examine the frequency of initial multiple epidural metastases, and the occurrence of secondary spinal cord compression (SCC). METHODS AND MATERIALS: To evaluate the frequency of a recurrent SCC after radiotherapy, and to compare among patients with single and multiple intraspinal metastases the risk of having a second SCC, we followed 107 patients with SCC from a histologically verified solid tumor prospectively with regular neurological examinations until death. RESULTS: Multiple metastases were demonstrated in 37 (35%). Eight (7.5%) patients developed a second occurrence of SCC all in a new location within the spinal canal. The second occurrence of SCC was found with the same frequency in patients with single metastases (7.1%) compared to patients with multiple metastases (8.1%). The median survival time after the diagnosis of spinal cord compression was 3.4 months, while in the group of patients who developed a second occurrence of SCC the median survival time was 9.2 months. CONCLUSION: Only symptomatic epidural metastases should be irradiated, and that all patients treated for SCC should be followed regularly and observed for development of a second SCC. PMID- 7558943 TI - Local control of carcinoma of the tonsil by radiation therapy: an analysis of patterns of fractionation in nine institutions. AB - PURPOSE: To investigate the importance to outcome of treatment for squamous cell carcinomas of the tonsillar fossa, of dose per fraction, overall treatment duration, and total dose. METHODS AND MATERIALS: A collaborative retrospective study was undertaken in nine centers that used widely different dose fractionation patterns for external beam radiation therapy. RESULTS: There were 676 eligible cases treated only with photon beams during the years 1976-1985. The probability of local control (of the tonsillar fossa primary) was influenced by both T-stage and N-stage. Significant treatment parameters were total dose and overall treatment duration, but not dose per fraction. Over the range of about 40 to 90% and for a constant overall treatment duration, local tumor control probability increased by nearly 2% for each 1 Gy increase in total dose. For a constant total dose there was a decrease in the probability of local control associated with prolongation of overall treatment duration, presumed to result from accelerated regrowth of surviving tumor clonogens during the course of treatment. If it is assumed that accelerated regrowth occurred at a constant rate and began within 9 days of the start of treatment, an average of 0.53 Gy extra dose per day's extension of treatment would be required to maintain a constant probability of local control. Correspondingly, the probability of local control from a constant dose would be lowered by an average of at least 1% for each day's extension of treatment duration. However, the data are slightly more consistent with an average delay of as long as 30 days before onset of accelerated repopulation, with a consequent increase to an average of 0.73 Gy per day for the value of the compensatory dose. The alpha/beta ratio for this tumor is high enough that the effect of fraction size on the probability of local control can be ignored; a precise estimate is not possible because the best value for beta was close to zero. After accounting for the significant variables studied (treatment time, T-stage, N-stage), the dose-response curves for tumor control were still shallow, suggesting that there are additional causes for heterogeneity of responses among these tumors. CONCLUSIONS: Total dose is important to treatment outcome: After accounting for other treatment variables, there is about a 2% per Gy increase in probability of tumor control over the ranges of control commonly achieved. Overall treatment duration is important. There is at least a 1% per day decrease in tumor control probability if delivery of a constant total dose is prolonged, requiring a compensatory increase in dose by 0.5-0.7 Gy per day to achieve a constant rate of tumor control. Fraction size is not, of itself, an important factor in the response of primary carcinoma of the tonsil. If a tumor has demonstrated a capacity for metastatic spread to lymph nodes, a higher total dose should be considered to achieve control rates at the primary site equivalent to those in node negative patients. Even after accounting for variables such as tumor stage, total dose, and overall treatment duration, there is sufficient heterogeneity in other undocumented determinants of tumor control to cause the tumor control probability curve to be a shallow function of dose. PMID- 7558947 TI - Adult soft tissue sarcomas of the head and neck treated by radiation and surgery or radiation alone: patterns of failure and prognostic factors. AB - PURPOSE: To analyze our experience treating soft tissue sarcomas of the head and neck in adults, and to identify patterns of failure and prognostic factors. METHODS AND MATERIALS: The records of 57 patients with Stage M0 disease treated by radiation with or without surgery between 1972 and 1993 were reviewed. Median follow-up time was 4.3 years (range, 1.1-16.8 years). A group of potential prognostic factors was evaluated, including age at diagnosis, sex, initial tumor presentation (primary vs. recurrent), grade, T-stage, direct tumor extension, tumor depth, duration of treatment, and radiation dose. RESULTS: The subset of angiosarcomas (11 out of 57 patients) had a considerably adverse effect on treatment outcome for the total group of sarcomas, with actuarial 5-year overall survival (OS), locoregional control (LRC), and freedom from distant metastasis (FDM) rates being 31%, 24%, and 42%, respectively. In contrast, for the remaining 46 patients with other histopathological tumor types, OS, LRC, and FDM rates were significantly higher (74%, 69%, and 83%, respectively). For this group of patients, significant prognostic factors identified by uni- and multivariate analysis included tumor grade as a predictor of OS and T-stage as a predictor of LRC (p < or = 0.050). Those patients who experienced a locoregional recurrence were at a significantly increased risk of dying (p = 0.004 in a multivariate model). All 17 patients without direct tumor extension to neurovascular structures, bone, contiguous organs, or skin remained free from distant failure. In contrast, 27% of 29 patients with direct extension had developed distant metastases at 5 years. In multivariate analysis, the absence of direct extension was a positive predictor of FDM (p = 0.007) and of OS (p = 0.034). CONCLUSIONS: 1) Angiosarcomas of the head and neck have a considerably poorer prognosis than other soft tissue sarcomas of this site. 2) In addition to tumor grade and size, direct tumor extension may be a useful additional staging parameter. 3) High rates of locoregional failure in the head and neck area, a potential cause of morbidity and death, indicate a need for improved treatment strategies. PMID- 7558946 TI - Radiation therapy for chordomas of the base of skull and cervical spine: patterns of failure and outcome after relapse. AB - PURPOSE: To determine the patterns of failure and outcome following relapse of chordomas of the base of skull and cervical spine. METHODS AND MATERIALS: Between November 1975 and October 1993, 204 patients were treated for chordoma of the base of skull or cervical spine, of which 63 have developed relapse. These 63 patients constitute the main focus of this study. Forty-five patients presented with base of skull and 18 with cervical spine tumors. All patients received combined proton and photon beam radiation. The median prescribed dose was 70.1 cobalt-Gray equivalent (CGE) (range 66.6-77.4). There were 25 males and 38 females, with a median age of 41 years (range 7-66). Median follow-up was 54 months (range 8-158). RESULTS: Sixty-three of the 204 patients treated (31%) had treatment failure. Among the 63 patients who relapsed, 60 (95%) experienced local recurrence, and in 49 patients (78%), this was the only site of failure. Two of 63 patients (3%) developed regional lymph node relapse and 3 of 63 (5%) developed surgical pathway recurrence (1 left neck, 1 palate and 1 nasal cavity). Thirteen of 204 patients relapsed in distant sites, accounting for 20% (13 of 63) of all patients with recurrence in this series. The most common metastatic sites were lungs and bones presenting in 7 of 13 and 6 of 13 patients, respectively. Only 2 of 13 patients failed with isolated distant metastasis. The actuarial 3- and 5 year survival rates after local relapse (60 patients) were 44 and 5%, respectively. Following distant failure (13 patients), the 3- and 5-year survival rates were 25 and 12%, respectively. After any relapse (63 patients) the corresponding survival rates were 43 and 7%. Following local relapse, 49 of 60 patients underwent salvage therapy consisting of subtotal resection in most patients (46 of 49). The remaining 11 of 60 patients received supportive care only. Salvage therapy resulted in stable or improved status without subsequent disease progression in 26 of 49 (53%), and progressive disease in 16 of 49 patients (33%). The actuarial 2- and 5-year overall survival rates following relapse for the 49 patients who underwent salvage treatment were 63 and 6%, which favorably compared to the 2-year survival rate of 21% for those who received supportive care only (p = 0.001). CONCLUSION: Local relapse is the predominant type of treatment failure for chordomas of the base of skull and cervical spine. Salvage treatment may relieve symptoms; however, most patients will ultimately succumb to their disease. Poor long-term survival rates following relapse emphasize the importance of a combined treatment approach with experienced surgeons and radiation oncologists at the time of primary treatment. For most patients, only permanent local tumor control will offer a chance of cure. PMID- 7558945 TI - A prospective randomized study of chemotherapy adjunctive to definitive radiotherapy in advanced nasopharyngeal carcinoma. AB - PURPOSE: A prospective randomized trial was conducted to compare chemoradiotherapy against radiotherapy alone in the treatment of locoregionally advanced nasopharyngeal carcinoma. METHODS AND MATERIALS: Eighty-two patients with histologically proven nasopharyngeal carcinoma who had either Ho's N3 staging or any N stage with a nodal diameter of > or = 4 cm were entered. Seventy seven patients were evaluated for tumor response and survival. The patients were randomized to receive two cycles of cisplatin 100 mg/m2 Day 1,5-fluorouracil 1000 mg/m2 24-h infusion Days 2, 3, and 4 before radical radiotherapy, and four cycles of postradiotherapy chemotherapy (37 patients) or radiotherapy alone (40 patients). All patients received radical radiotherapy to the nasopharynx and neck. The nasopharynx and upper neck were treated to 66 Gy by conventional fractionation and the lower neck to 58 Gy. Booster radiotherapy (7.5 Gy/two fractions/week) was given to any residual nodes after standard radiotherapy. RESULTS: The patient characteristics, including staging, were similar in both arms. The overall response rate to neoadjuvant chemotherapy was 81% (19% complete response, 62% partial response). The rates of radiotherapy for boosting parapharyngeal disease or residual lymph nodes were not significantly different in the two arms. The overall complete response rate to chemoradiotherapy was 100%, and to radiotherapy alone, 95%. Toxicities in the chemoradiotherapy arm were mainly myelosuppression, nephrotoxicity, and nausea and vomiting. The degree of mucositis was not significantly different in the two arms. There was no treatment-related death. The median follow up was 28.5 months. The 2-year overall survival was 80% in the chemoradiotherapy arm and 80.5% in the radiotherapy arm. The 2-year disease-free survival was 68% in the chemoradiotherapy arm and 72% in the radiotherapy arm, without significant difference between the two arms. The locoregional relapse rate, distant metastatic rate, and median time to relapse were also not significantly different between the two arms. CONCLUSION: Despite promising tumor response rates from Phase II trials, this prospective randomized trial has demonstrated no benefit from adjunctive chemotherapy to radiotherapy in the treatment of locoregionally advanced nasopharyngeal carcinoma. PMID- 7558944 TI - Late normal tissue sequelae from radiation therapy for carcinoma of the tonsil: patterns of fractionation study of radiobiology. AB - PURPOSE: To evaluate the influence of dose fractionation and other factors on the development of late complications in mandibular bone, muscle, and mucosa of the oral cavity after external beam radiation therapy for carcinoma of the tonsil. METHODS AND MATERIALS: A retrospective analysis was made of the results in 676 patients treated with a spectrum of fractionation regimens in nine centers during the years 1976-1985. Only severe (Grades 3-4) late complications were analyzed. RESULTS: With more than 5 years follow-up, it was found that total dose was a factor for all three types of complications, but that in other respects, the radiobiology of late-(> 3 months) developing mucosal ulcerations was different from that for mandibular necrosis and muscle injury. Dose per fraction was a significant factor for bone and muscle (estimated alpha/beta values of 0.85 Gy and 3.1 Gy, respectively). By contrast, mucosa showed no influence on response from change in fraction size over the range of approximately 1.0-3.5 Gy. Complications in bone and muscle were not related to overall treatment duration, whereas there was a significant inverse relationship for mucosa breakdown. The rate of development of complications was fastest in mucosa and slowest in bone. The appearance of complications by 4 years after treatment was about 80% of those developing by 8 years in the mucosa, 66% in muscle, and about 50% in bone. The high alpha/beta ratio, inverse relationship with overall treatment duration, and faster development of mucosal complications suggests that they may develop as a consequence of earlier mucosal injury. As anticipated, adequate retrospective analysis of acute complications could not be made even when objective criteria such as weight loss, unplanned delays in completing treatment, or hospitalization during treatment were the measures. Field size was a significant factor for mandible complications, but not for muscle or mucosa. CONCLUSION: The radiobiological characteristics of bone and muscle were those characteristic of other late-responding tissues, whereas late sequelae in mucosa had radiobiological parameters similar to those for acute responses. Field size was a significant factor for bone complications but not for others. PMID- 7558949 TI - Long-term visual changes following pituitary irradiation. AB - PURPOSE: To analyze possible long-term effects of pituitary irradiation on visual fields and acuity. METHODS AND MATERIALS: Eighty-six patients were treated with radiotherapy for pituitary tumors at the National Cancer Institute between 1980 and 1991. Twenty-one patients had baseline preradiation and long-term follow-up visual fields. Eyes were followed with serial Goldmann or Humphrey visual field testing. Neuroradiologic correlation was made with the available brain scans. There were 12 females and 9 males with an median age of 44. Eighteen patients had hormone-secreting tumors and three had chromophobe adenomas. All but one patient with an inoperable invasive macroadenoma were irradiated after one or more transphenoidal resections or a craniotomy. The indications for radiation in the operable patients were: nine patients, partial tumor resection; nine patients, tumor recurrence; and two patients, persistent hormonal elevation after surgery. The median dose delivered was 50 Gy (45-59.4 Gy). The average field size was 6 x 6 cm (5 x 5 cm to 10 x 12.5 cm). RESULTS: With a median follow-up of 48 months (14-128) after radiotherapy, 1 out of 21 patients has recurred (at 8 months) and all patients are alive. Of the 38 sighted eyes, 27 had normal visual fields before and after radiation, 7 eyes showed improvement, and 4 eyes had a stable defect, mostly in the superior temporal region. There were no cases of radiation induced visual field or acuity deterioration. Six out of 21 patients (29%) had neurologic symptoms in follow-up, most of which appeared vascular in nature. Four patients complained of atypical migranous-like headaches that first began 1.5-3 years following treatment. One patient complained of recurrent vertical diplopia and one patient had a cerebral vascular accident 7 years following therapy. A dose-related association with these neurovascular symptoms approached statistical significance. Only 1 out of 11 (9%) patients who received doses less than or equal to 50 Gy developed these symptoms, whereas 5 out of 10 (50%) patients who received doses greater than 50 Gy developed symptoms (p2 = 0.064). CONCLUSION: Postoperative radiation for partially resected or recurrent pituitary adenomas using megavoltage radiation, as well as modern field arrangements and fractionation, is extremely effective and safe. Ninety-five percent of patients are free of recurrence with not deterioration in the visual fields or acuities. Some patients experienced neurovascular symptoms (mostly vascular headaches) following surgery and radiation. There is a trend (p2 = 0.064) toward increased symptoms following higher radiation doses. PMID- 7558942 TI - Evidence for the presence of the alternatively spliced HLA-G mRNA forms in human mononuclear cells from peripheral blood and umbilical cord blood. AB - The HLA-G monomorphic, nonclassic HLA class I gene encodes the molecule that is the only major histocompatibility complex antigen expressed on cytotrophoblasts of placenta. This restricted expression on fetal tissue that is in contact with maternal tissue suggests that HLA-G products may play a role in maternofetal tolerance. We previously have demonstrated in first-trimester human trophoblasts a new alternatively spliced form of HLA-G mRNA lacking exon 4 (HLA-G4) and weak expression of HLA-G1 copy mRNA in adult peripheral blood lymphocytes. By using exon-specific HLA-G primers, we demonstrate in this work the presence of three additional alternatively spliced forms of HLA-G mRNA in human peripheral mononuclear cells (HLA-G2, HLA-G3, and HLA-G4). In umbilical cord blood, HLA-G transcription was observed at very low level and only three alternatively spliced forms were detected (HLA-G1, HLA-G2, and HLA-G3). In contrast, we did not revealed any HLA-G transcript in CD34+ fraction of cord blood cells. PMID- 7558950 TI - Analysis of weekly complete blood counts in patients receiving standard fractionated partial body radiation therapy. AB - PURPOSE: Hematopoiesis is among the most sensitive systems in the body to radiation. Routine complete blood counts (CBCs) are common in clinical radiotherapy practice. Only a few studies have attempted to characterize the behavior of peripheral blood levels during partial body radiation therapy with field sizes smaller than those used in hemibody or total nodal irradiation. Such information is needed to identify which patients are at risk for cytopenia and require close monitoring. METHODS AND MATERIALS: In 1993, 412 new patients were seen at Michael Reese Hospital for radiotherapy. A total of 972 weekly CBCs were identified for 155 patients receiving a minimum of 5 weeks of treatment for breast, prostate, lung, gynecological, or head and neck malignancies. Linear regression models were fitted to the weekly CBC values for those patients who had pretreatment CBC values recorded. Factors affecting starting levels, rates of decline, and nadirs during treatment were determined for leukocytes, platelets, and hemoglobin. RESULTS: Leukocytes declined most dramatically during the first week of treatment (16% from pretreatment to Week 1 levels) and then at a rate of 3.3% per week from Week 1 to Week 7 (p < 0.001). Total mean leukocyte decrease over 7 weeks of therapy was 30%. Platelets declined 9% on average during the first week of therapy and then at a mean rate of 1.4% per week (p < 0.02). A statistically significant decrease in hemoglobin levels could not be detected. No difference in the rate of decrease could be found for different disease sites, age groups, or amount of marrow irradiated. The effects of chemotherapy were variable, depending on blood element and whether therapy was sequential or concomitant. The odds of a nadir < 2000 counts/mm3 for white blood count (WBC), < 50,000 counts/mm3 for platelets, and < 8.0 g/dl for hemoglobin were all well below 5%. A strong correlation existed between starting CBC values and nadirs; patients with lower Week 1 CBC levels were most likely to have the lowest nadirs. CONCLUSIONS: Low CBC levels during radiation therapy are likely to be the result of other medical problems that cancer patients face. Regional irradiation with small field sizes (< 40% of total body marrow) typically used in clinical radiotherapy is unlikely to be the cause of marrow depression significant enough to warrant medical intervention. Blood levels taken during the first week of treatment (Week 1) can be used to determine risks of developing critical nadirs. Localized breast and prostate cancer patients are unlikely to require routine CBCs if initial levels are normal. Routine CBC levels on all radiation oncology patients without other reasons for hematopoietic depression requires reevaluation, as millions of dollars are spent on unnecessary testing. If weekly CBC blood levels are avoided in localized breast and prostate cancer patients, this alone could potentially result in a savings of as much as $40 million a year nationally. PMID- 7558953 TI - Proliferation and clonal survival of human lung cancer cells treated with fractionated irradiation in combination with paclitaxel. AB - PURPOSE: This study was performed to determine the effects of a continuous exposure to paclitaxel (taxol) in combination with fractionated irradiation on cell proliferation and survival. METHODS AND MATERIALS: Human lung carcinoma cells (SW1573) were given a daily treatment with 3 Gy of x-rays during 5 days in the continuous presence of 5 nM taxol. The surviving fraction and the total number of cells were determined every 24 h before and immediately after irradiation. RESULTS: Irradiation with 5 x 3 Gy and 5 nM taxol cause approximately the same inhibition of cell proliferation. In combination these treatments have an additional effect and the cell population increases no further after the first 24 h. Whereas the cells become more resistant to taxol after the first 24 h with a minimum survival of 42%, taxol progressively reduces the population of surviving cells in combination with x-rays when the number of fractions increases, up to 25-fold relative to irradiation alone. The enhancement effect of 5 nM taxol is likely to be attributed to an inhibition of the repopulation during fractionated irradiation and not to an increased radiosensitivity. Only after treatment with 10 or 100 nM taxol for 24 h, which is attended with a high cytotoxicity, is moderate radiosensitization observed. CONCLUSION: Taxol, continuously present at a low concentration with little cytotoxicity, causes a progressive reduction of the surviving cell population in combination with fractionated irradiation, mainly by an inhibition of the repopulation of surviving cells between the dose fractions. PMID- 7558952 TI - Inhibition of radiation-induced up-regulation of leukocyte adhesion to endothelial cells with the platelet-activating factor inhibitor, BN52021. AB - PURPOSE: The inflammatory process is likely involved in normal tissue damage after radiation exposure, yet few studies have directly evaluated the factors that might be involved in the regulation of inflammation after irradiation in vivo. We tested the hypothesis that platelet-activating factor, a neutrophil agonist synthesized by endothelial cells, is involved in the upregulation of radiation-induced leukocyte-endothelial cell interactions by using an inhibitor of its receptor, BN52021. METHODS AND MATERIALS: Fischer-344 rats with dorsal skin-fold window chambers were randomized to three experimental groups: control (sham irradiation); 6 Gy radiation; and 6 Gy + BN52021. BN52021 (0.5 mg/kg) was administered 5 min prior to 6 Gy radiation. Leukocytes were stained in vivo with i.v. acridine orange for visualization with fluorescent microscopy. Venous vessel diameters were measured and number of rolling leukocytes were counted per 30-s period. The number of adhering leukocytes per unit surface area was also determined. Differences among the three experimental groups for rolling and adhering leukocytes were analyzed using a mixed-effects linear model with vessel shear rate used as a covariate. Results are reported as means +/- standard errors. RESULTS: Irradiation caused upregulation of leukocyte rolling, as compared with sham-treated controls (p = 0.04): the BN compound in addition to radiation did not downregulate this effect. Irradiation also upregulated leukocyte adhesion (p < 0.001), but the addition of BN52021 prior to irradiation blocked this effect. The drug did not affect heart rate or blood pressure. CONCLUSIONS: These results support the hypothesis that radiation-induced upregulation of leukocyte adhesion is mediated by platelet-activating factor. These results are consistent with prior reports that platelet-activating factor is not involved in leukocyte rolling, which involves separate families of adhesion molecules from those that regulate adhesion. BN52021, a ginkolide, or other related drugs might provide a useful pharmacological means to prevent or ameliorate inflammatory pathways that are invoked after radiation exposure. PMID- 7558951 TI - Induction of acute phase gene expression by brain irradiation. AB - PURPOSE: To investigate the in vivo acute phase molecular response of the brain to ionizing radiation. METHODS AND MATERIALS: C3Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-gamma), tumor necrosis factors (TNF-alpha and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), alpha 1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined. RESULTS: Levels of TNF-alpha, IL-1 beta, ICAM-1, EB22/5.3 and to a lesser extent IL-1 alpha and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen. CONCLUSIONS: The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances. PMID- 7558954 TI - Irradiation enhances cellular uptake of carboplatin. AB - PURPOSE: Because radiation is known to damage cellular membranes, the purpose of this study was to determine whether irradiation of cultured cells might modify the cellular uptake of the chemotherapy agent carboplatin. METHODS AND MATERIALS: Total intracellular platinum was measured using atomic absorption spectrometry in cultured V79 cells and in four Chinese hamster ovary (CHO) cell lines. RESULTS: Intracellular carboplatin concentrations increased linearly with radiation dose (10-50 Gy) under both hypoxic and oxic irradiation conditions. Similar doses of radiation did not significantly increase the uptake of a nontoxic platinum compound [Pt(NH3)4Cl2.H2O] (p > 0.5). Compared to unirradiated controls, there was no increase in intracellular carboplatin concentrations when carboplatin was irradiated prior to administration to the cell cultures (p > 0.5). Within the 32.5 min or less required to deliver the radiation, a dose of 50 Gy produced approximately a 50% increase in intracellular platinum in V79 cells and approximately an increase of a factor of 1.3-1.6 in the CHO cell lines. Although the increase in drug uptake would be expected to be less than 10% for most cell lines at the doses of radiation used to investigate radiosensitization by carboplatin, this level of increase may play a significant role in the radioenhancement observed in UV41 cells because these excision-repair--deficient cells are much more sensitive to carboplatin as measured by cytotoxicity. CONCLUSION: These results suggest that some of the enhanced cell killing that results when cells are exposed to carboplatin in combination with radiation may be attributed to an increased cellular uptake. One mechanism of radiopotentiation may be an enhanced chemotoxicity resulting from a radiation-induced increase in carboplatin uptake. PMID- 7558955 TI - Treatment planning optimization for multiple arcs stereotactic radiosurgery using a linear accelerator. AB - PURPOSE: Multiarc stereotactic radiosurgery is a technique used to irradiate an intracranial tumor with minimal damage to the surrounding normal tissue. The purpose of this paper is to present a method for and the results from optimizing three dimensional (3D) treatment dose for multiarc stereotactic radiosurgery. METHODS AND MATERIALS: The normal procedure for a physician-physicist team designing a treatment plan for multiarc stereotactic radiosurgery is the trial and-error approach of changing the collimator size and the isocenter of radiation by viewing the isodose curves on a two dimensional (2D) computed tomography (CT) or magnetic resonance imaging (MRI) image plane. Not only is this time consuming, but the resulting treatment plan is not optimal in most, if not all, cases. One reason for such nonconformal isodose curves is that the same collimator size is used for all arcs. However, it is very difficult to determine manually the different collimator sizes for different arcs. A derivative free optimization method is used to optimize the collimator size for each arc, as well as the 3D coordinates of the isocenter(s). RESULTS: One spherical and two ellipsoidal artificial tumors, and one actual tumor, were used to show the utilities of the optimization process. The 90% isodose curves resulting from optimization conform very well with the tumor; whereas the 90% isodose curves from the conventional method either do not envelop the entire tumor when the collimator size is too small, or a large volume of normal tissue is also irradiated by the 90% dose when the next larger collimator size is used. CONCLUSIONS: When the collimator size for each arc and the location of the isocenters(s) are optimized in a multiarc stereotactic surgery treatment plan, the 90% isodose curve conforms to the tumor much better than when the same collimator size is used for all arcs. PMID- 7558956 TI - Thermoradiotherapy of intraocular tumors in an animal model: concurrent vs. sequential brachytherapy and ferromagnetic hyperthermia. AB - PURPOSE: To compare concurrent vs. sequential ferromagnetic thermoradiotherapy in vivo. METHODS AND MATERIALS: Greene melanomas were implanted subretinally in rabbits and observed until they were 3-5 mm in diameter. Episcleral plaques were assembled with 125I seeds for radiation therapy, or with ferromagnetic (FM) thermoseeds and nonradioactive I seeds for hyperthermia. Rabbits were implanted by centering a plaque over the intraocular melanoma. After a given dose of radiation had been delivered, the plaque was removed and a nonradioactive plaque containing FM thermoseeds was inserted into the same extrascleral space. One hour later, hyperthermia (46-47 degrees C at the plaque-scleral interface) was initiated and continued for a period of 1 h by placing the rabbits in a magnetic induction coil powered to 1200 W. Tumor size was determined at 1- to 2-week intervals by indirect ophthalmoscopy and by ultrasound. RESULTS: Dose-response analysis of 27 treated eye melanomas showed 50% local tumor control at 43 Gy for 125I alone and 29.4 Gy for 125I followed by FM hyperthermia. The thermal enhancement ratio was 1.4. CONCLUSION: Comparison with a previously published thermal enhancement ratio of 4.4 (for concurrent 125I and FM hyperthermia) leads us to conclude that thermal enhancement of 125I brachytherapy is more efficient in this tumor model system when hyperthermia is delivered during, rather than after, the irradiation process. PMID- 7558957 TI - Primary central nervous system non-Hodgkin's lymphoma: survival advantages with combined initial therapy? AB - PURPOSE: Results of multiple radiation, chemotherapy, and combined treatment trials have shown that the fate of primary central nervous system lymphoma (PCNSL) patients is very different from that of patients with similarly treated systemic IE non-Hodgkin's lymphoma. This study was designed to improve the survival of PCNSL patients by the use of combined initial therapy. METHODS AND MATERIALS: Forty-six eligible primary PCNSL patients were treated with whole brain irradiation and adjuvant chemotherapy consisting of preirradiation cyclophosphamide-adriamycin-vincristine-prednisone (CHOP) and postirradiation high-dose cytosine arabinoside (HDAC) as part of an ongoing Phase II Mayo/North Central Cancer Treatment Group/Eastern Cooperative Oncology Group (M/NCCTG/ECOG) intergroup effort, which opened in April 1986. RESULTS: This cohort consisted of 23 men and 23 women with median age 63.5 years (range 24 to 75 years). Only 5% were under age 40; 36% were age 40 to 59, 37% were age 60 to 69, and 22% were age 70 and over. Forty-six percent had good performance scores of ECOG 0-1 at time of study entry. Forty-six patients were evaluable for treatment outcome as of October 6, 1993. Of these, 10 were still alive. Estimated median survival and 21 month survival were 45.3 weeks and 29%, respectively. There were four early deaths ranging from Day 9 to Day 15 (three drug-related, one from other complications), and two CHOP responders died at 32 and 35 days, soon after Cycle 2 of CHOP (one probably drug-related, one from other complications). There was no significant difference in survival according to baseline performance status. However, survival was consistently worse for patients > 60 years old than for the younger patients (< or = 60 years). With deaths recorded for 21 of 21 older patients, but only 9 of the 14 younger patients, 21-month survival for older vs. younger was 14 vs. 50% based on the 35 patients who entered the study at least 21 months ago (p = 0.0365). Of the 46 patients evaluable for response, 63% had objective remissions on CHOP and another 20% remained stable. CONCLUSION: Combined modality therapy in this study did not produce an overall survival advantage in treating PCNSL. The 50% 21-month survival of younger patients may be a reflection of age only. PMID- 7558959 TI - Using simulation data to predict lung geometry for inhomogeneity corrections in breast cancer treatments. AB - PURPOSE: To develop a statistical model based only on simulation measurement data, to predict the lung geometry in the central slice of the tangential radiation treatment fields for breast cancer. METHODS AND MATERIALS: A linear regression analysis was performed on 22 patients to determine the shape of lung in the central axis plane of the tangential radiation fields. Data collected include the greatest perpendicular distance (GPD) measured from the chest wall to the field border on computed tomography (CT) images, the central lung distance (CLD) measured from the posterior field border to the chest wall on the simulation portal images, and the lung contours digitized at 1 cm intervals. The lung contours of these patients were fitted to a parabolic curve through a polynomial regression model. A lung template based on the regression model is used to construct a "generic lung" contour on patients' external body contours for treatment planning. The accuracy of this technique was tested on another group of 15 patients for its ability to predict the shape of lung on the central axis plane and the accuracy of dose to the prescription point. RESULTS: The polynomial regression indicates that all the patients' lung contours in the tangential fields follow a parabolic curve: Y = -0.0808 X2 + 0.0096 X + 0.0326. The maximum lung involvement (GPD) can be determined from the value of CLD measured on the simulation film by the linear regression model with a determination coefficient of 0.712. The 15-patient test results indicate that our model predicts the lung separation on the central axis with an average deviation of 1.35 cm, and the average absolute dose deviation to the dose prescription point is 1.46%. CONCLUSION: The model presented in this article provides an efficient method to estimate the lung geometry for breast cancer treatment planning without the requirement of CT data. The lung contour predicted by our model is useful for calculating dose distributions with inhomogeneity correction and may potentially benefit patients at higher risk of pulmonary toxicity. PMID- 7558960 TI - Long-term follow-up of efficacy and safety of megavoltage radiotherapy in high risk giant cell tumors of bone. AB - PURPOSE: Giant cell tumors of the bone are rare and have variable presentations and natural history. There may be significant functional sequelae as a result of their locally aggressive nature or as a result of treatment. We reviewed the long term results of radiotherapy for high risk giant cell tumors to assess: the efficacy of radiotherapy and the potential late toxicity of treatment, and to determine indications for radiation treatment. METHODS AND MATERIALS: This report is a retrospective review of 21 localized giant cell tumors of the bone treated with radiotherapy between 1959 and 1991. Radiation was used in the primary management of 13 cases and for recurrent disease in eight cases. In the primary cases, two received radiotherapy as the sole modality (including a massive pelvic lesion and an advanced maxillary sinus tumor with orbital and pterygoid invasion), and in the other 11, 3 had gross residual disease following surgery and 8 had microscopic residual disease. Of eight recurrent cases, five were treated with radiotherapy alone and three with combined surgery and radiation. Sites of origin included extremity bones in nine cases, pelvis in six, spine in four, and skull in two. Extraosseous disease was apparent in 18 tumors and extended to contiguous structures in 14 (including four cases of spinal cord compression, three cases of sacral plexopathy, and one patient with temporal lobe invasion). The most common dose regimen was 35 Gy/15 fractions/3 weeks (14 cases), with varying schedules for the remainder. RESULTS: Mean follow-up time was 15.4 years (2 to 35 years). Local control was achieved in 19 of 21 patients with radiotherapy. The two failures were subsequently salvaged for an ultimate control rate of 100%. One of the two radiotherapy failures was a marginal failure and was subsequently salvaged with combined surgery and radiotherapy. No patient died of giant cell tumor. Radiotherapy was well tolerated, with no serious late toxicity. There were no cases of malignant transformation or radiation-induced cancer. CONCLUSION: Long-term results in this series indicate that radiotherapy in modest doses (35 Gy in 15 fractions or equivalent) is a safe and effective option for primary and recurrent giant cell tumors of the bone. Radiotherapy should be used if surgery would result in significant functional morbidity and should be considered in select sites where the probability of recurrence is high and there is potential for significant morbidity from tumor relapse or subsequent surgery. PMID- 7558958 TI - Split hyperfractionated accelerated radiation therapy and concomitant cisplatin for locally advanced head and neck carcinomas: a preliminary report. AB - PURPOSE: The feasibility and activity of an intensive chemoradiotherapeutic scheme for patients with locally advanced squamous cell head and neck cancers were tested in a single institution Phase II pilot study. METHODS AND MATERIALS: Between January 1990 and February 1992, 40 patients were entered into this trial. The treatment protocol consisted of split hyperfractionated accelerated radiation therapy (SHART), 1.6 Gy per fraction given twice per day to a total dose of 64 67.2 Gy for a total of 6 weeks with a 2-week gap, and cisplatin (20 mg/sqm/Days 1 to 5, in continuous perfusion) concomitantly. RESULTS: All of the 40 patients are evaluable for response and survival. Toxicity was significant, but tolerable. A complete tumor response to this treatment was achieved by 37 patients (92.5%). With a minimal follow-up of 22 months (median 30 months) there have been 16 local relapses and 19 patients have died, 2 without tumor. The projected 2- and 3-year overall survival rates are 64% (confidence interval (CI) 95%, 49-79%) and 47%, respectively. The 2-year local control probability has been 56% (CI 95%, 39-73%). CONCLUSION: This treatment obtains a high rate of complete responses with increased acute toxicity but tolerable late effects. Preliminary results are encouraging for laryngeal neoplasms. A longer follow-up is needed to evaluate the impact of this treatment on patient survival. PMID- 7558961 TI - Harvesting backscatter electrons for radiation therapy. AB - PURPOSE: An innovative technique is used to harvest backscatter electrons for the treatment of superficial small lesions of skin, oral cavity, and rectum where a significant dose gradient and maximum surface dose is desired. METHODS AND MATERIALS: Backscatter electrons are harvested out of the primary electron beams from the linear accelerators. The design consists of a short cylindrical cone that fits snugly over a long cylindrical electron cone. The short cylindrical cone has a thick circular plate of high atomic number medium (Pb) attached to the distal end, and a lateral slit of variable length and width. The width of the slit could be closed as desired by rotating the two cones and the length can be increased by lowering the short cylindrical cone. Primary electrons strike the Pb plate perpendicularly and produce backscatter electrons that pass through the lateral slit for treatment. Using film and a parallel plate ion chamber, backscattered electron dose characteristics are studied. RESULTS: The depth dose characteristic of the backscatter electron is very similar to that of the 0.2 mm Al half-value layer x-ray beam that is commonly used for the intracavitary and superficial lesions. The backscatter electron energy is nearly constant and effectively < or = 1 MeV from the clinical megavoltage beams. The backscatter electron dose rate of 0.32-0.8 Gy/min could be achieved from modern accelerators without any modification. The beam flatness is dependent on the slit size and the depth of treatment, but is satisfactory to treat small lesions. CONCLUSIONS: The measured data for backscatter electron energy, fluence, depth dose, flatness, dose rate, and absolute dose indicates that the harvested backscattered electrons are suitable for clinical use. PMID- 7558962 TI - Ocular globe topography in radiotherapy. AB - PURPOSE: Ocular lens, retina, and olfactory bulb exposure are common concerns in contemporary radiotherapy practice. Methods to clinically localize soft tissue structures (i.e., lens and retina) are varied and often imprecise. We hypothesized that eyelid markers constituted a better reference point than the commonly used lateral canthus marker for lateral beam simulations, unless diagnostic computed tomography or ultrasound examinations were available and/or used. METHODS AND MATERIALS: Sixty-six pre-Magnetic Resonance Image, normal, orbital computed tomography scans from adult patients were used to measure (a) sagittal distances from eyelid to posterior lens surface, from lateral canthus to posterior lens surface and to the globe's posterior pole, (b) supero-inferior distances in the lateral projection from the lens to the cribriform plate, and (c) common dimensions to establish internal validity of the measurements. RESULTS: The eyelid to lens and retina topography is individually more constant than that from the canthus. There is little if any supero-inferior separation between the lens and the cribriform plate lateral projections. CONCLUSIONS: The lateral canthus does not specify lens or retina locations. Eyelid markers of known size provide more accurate anatomical information. Lateral beam ocular globe shielding has to be individualized. Lens shielding is questionable if the olfactory bulb needs to be irradiated by a lateral beam. PMID- 7558963 TI - Intra-arterial 90Y brachytherapy: preliminary dosimetric study using a specially modified angioplasty balloon. AB - PURPOSE: Irradiation has been shown to be effective in preventing restenosis after dilatation in human peripheral arteries. We have developed a dedicated system for coronary intraarterial irradiation using a 90Y pure beta-emitting source inside a specially modified angioplasty balloon. This paper presents a preliminary dosimetric evaluation of this system. METHODS AND MATERIALS: Specially fabricated titanium-covered and activated yttrium wires (outer diameter 0.32 mm) were used for these studies. Dosimetry was performed using small thermoluminescent dosimeters (TLDs) placed on the surface of the 2-cm long angioplasty balloons, inflated with contrast medium to a diameter of 2.5, 3, 3.5, and 4 mm. Radioactive 90Y wires were left in the inner balloon catheter and the surface dose rate was measured and extrapolated to 72 h after activation to allow a comparison between the values obtained. After observing the poor centering of the source within the standard angioplasty balloon, a new centering balloon was developed. A conventional balloon was subdivided into four evenly spaced interconnecting chambers, thus assuring adequate centering of the inner catheter. Thermoluminescent dosimetric measurements were performed with a 3.5 mm centering balloon to evaluate the homogeneity of the surface doses compared to those measured with the conventional balloon. RESULTS: Thermoluminescent dosimetric measurements using the standard balloons filled with contrast medium were plotted semilogarithmically as a function of distance from the balloon surface. The logarithms of the measured doses fit a straight line as a function of depth. The doses at 1 mm and 3 mm are approximately 50 and 10% of the surface dose, respectively. Due to the poor centering of the source in the conventional balloons, the dispersion and standard deviations (SDs) of the measured surface doses increased proportionally to the balloon diameter (SDs are 1.89, 5.52, 5.79, and 6.46 Gy for 2.5, 3, 3.5, and 4 mm balloon diameters, respectively). For the 3.5 mm centering and conventional balloons the respective mean, minimum, and maximum surface doses were 8.41 Gy (min. 7.26; max. 9.46) and 7.89 Gy (min. 2.18; max. 16.06) and their standard deviations were 0.66 and 5.79 Gy, respectively. CONCLUSIONS: Conventional angioplasty balloons cannot ensure a homogeneous dose delivery to an arterial wall with an intralumenal 90Y beta source. Preliminary dosimetric results using a modified centering balloon show that it permits improved surface dose distribution (axial and circumferential homogeneity), making it suitable for clinical applications. PMID- 7558964 TI - Radiation-induced changes in human brain metabolites as studied by 1H nuclear magnetic resonance spectroscopy in vivo. AB - PURPOSE: External radiation therapy for brain tumors exposes healthy areas of brain to considerable doses of radiation. This may cause cognitive and psychological impairment, which indicate neuronal dysfunction. 1H-magnetic resonance spectroscopy (MRS) was used to study brain metabolites in the adjacent regions 0.5-13 years after exposure to therapeutic irradiation. METHODS AND MATERIALS: Eight patients with irradiated brain tumors were examined by means of in vivo 1H-MRS using a point-resolved spectroscopy (PRESS) sequence with echo times of 60 or 270 ms. The metabolites were quantified by using brain water concentration as internal reference. The volume of interest (VOI) was positioned in irradiated brain areas excluding, however, scar and recurrent tumor. The respective radiation doses were measured based on radiation therapy plans, simulator films, and localization MR images. RESULTS: The concentration of the neuron-specific metabolite N-acetyl-L-aspartate (NAA) was 13.2 +/- 1.4 mmol/l in controls, whereas it was reduced in the brains of treated patients to 8.6 +/- 0.9 mmol/l (total radiation dose 59-62 Gy). Concentrations of creatine and choline containing compounds were unchanged. The T2 of water was longer in irradiated than in unexposed brain areas. CONCLUSION: Therapeutic brain irradiation causes neuronal damage, which is reflected by reduction of N-acetyl-L-aspartate (NAA) concentrations. 1H-MRS could serve clinically as a means of evaluating adverse effects in the central nervous system, enabling intervention and rehabilitation. PMID- 7558965 TI - Intraoperative electron beam radiation therapy: technique, dosimetry, and dose specification: report of task force 48 of the Radiation Therapy Committee, American Association of Physicists in Medicine. AB - Intraoperative radiation therapy (IORT) is a treatment modality whereby a large single dose of radiation is delivered to a surgically open, exposed cancer site. Typically, a beam of megavoltage electrons is directed at an exposed tumor or tumor bed through a specially designed applicator system. In the last few years, IORT facilities have proliferated around the world. The IORT technique and the applicator systems used at these facilities vary greatly in sophistication and design philosophy. The IORT beam characteristics vary for different designs of applicator systems. It is necessary to document the existing techniques of IORT, to detail the dosimetry data required for accurate delivery of the prescribed dose, and to have a uniform method of dose specification for cooperative clinical trials. The specific charge to the task group includes the following: (a) identify the multidisciplinary IORT team, (b) outline special considerations that must be addressed by an IORT program, (c) review currently available IORT techniques, (d) describe dosimetric measurements necessary for accurate delivery of prescribed dose, (e) describe dosimetric measurements necessary in documenting doses to the surrounding normal tissues, (f) recommend quality assurance procedures for IORT, (g) review methods of treatment documentation and verification, and (h) recommend methods of dose specification and recording for cooperative clinical trials. PMID- 7558966 TI - Evolution and accomplishments of the Radiation Therapy Oncology Group. AB - PURPOSE: The Radiation Therapy Oncology Group (RTOG) recently completed its first quarter century as a cooperative clinical cancer research organization. It is timely and appropriate to document its origins, evolution, and accomplishments. METHODS AND MATERIALS: The historical review of the RTOG called upon written and oral documentation. RESULTS: The RTOG is the most enduring product of the Committee for Radiation Therapy Studies (CRTS). Although not one of the original 17 clinical trials groups developed by the National Cancer Institute in 1956, the RTOG has pursued trials suggested by laboratory findings including the oxygen effect, intrinsic radiosensitivity, proliferation kinetics of normal and tumor cells, and interactions with other cytotoxic agents. Improvements in survival have been demonstrated for patients with carcinoma of the esophagus and cervix, and nonsmall cell carcinomas of the lung. The national and international radiation oncology communities have benefitted from standards and quality control/assurance guidelines for established and new modalities. A growing number of institutions in North America participate in RTOG trials. CONCLUSION: The RTOG is an important clinical research resource, which has contributed to improved outcome for patients with many forms of cancer. It has become increasingly productive and widely adopted and endorsed by oncologists throughout North America. PMID- 7558968 TI - Late normal tissue complications: new insights. PMID- 7558967 TI - Time factor for tonsillar carcinoma. PMID- 7558969 TI - Nasopharynx carcinoma: choice of treatment. PMID- 7558970 TI - High efficacy without visual damage: the current status of pituitary radiotherapy. PMID- 7558972 TI - Intraluminal brachytherapy in the treatment of pancreas and bile duct carcinoma: regarding Montemaggi et al., IJROBP 32:437; 1995. PMID- 7558971 TI - Central nervous system lymphomas: progress in chemotherapy and radiotherapy. PMID- 7558973 TI - Clinical results of radiotherapy in 140 elderly patients treated at Basel University Hospital between 1980 and 1985. PMID- 7558974 TI - Introduction to Fromm-Reichmann's "female psychosexuality" and "Jewish food rituals". PMID- 7558975 TI - "Madness" in the countertransference: evocative encounters in hospital treatment. PMID- 7558978 TI - Female psychosexuality. PMID- 7558977 TI - Successful and unsuccessful marriages in borderline patients. PMID- 7558979 TI - The myth of masochism. AB - The personality disorder called "Masochism," which traditionally has been regarded as a preference for and pursuit of suffering is dynamically misconceived. There presently is no fundamental agreement on a diagnostic category for this personality disorder. There are aspects of what is called masochism, such as sexual arousal associated with pain, which have not been considered in this study. I feel that the essential nature of the pain in those designated as masochists is low self-esteem and that it develops in a childhood context of inadequate loving nurturance. I strongly believe that low self-esteem evolves from and is maintained by the competitive social milieu and a resultant personal functioning, which is diffusely competitive. The competitive activity is often associated unawarely with cynicism (Bonime, 1989). The gratification of victory substitutes for the gratifications of intimacy and growth, but is relatively brief, and although it produces some self acceptance, it is never enough to overcome the basic low feeling about oneself. Here was Marjorie, a woman who had miserably low self-esteem as a child, as low as being "just a crack in the door." As an adult, as shown in her dream, she felt like a woman full of shit that comes out involuntarily. She struggled to keep from being exposed as such and ached with humiliation or inwardly--and sometimes openly--raged when some distasteful quality or inadequacy showed or threatened to show. This pained person strove to overcome her sense of inadequacy as a person. She overcame it by succeeding in outdoing others according to her private, competitive standards. Her criteria of value were success in evoking signs of approval and sustaining herself while suffering, while "taking it." Her highest value was being able to take it, to suffer intensely without breaking. She was cynical and never sensed for long that she was genuinely regarded as an acceptable human being. She didn't believe in the existence of any genuine caring. She dreamt of an ecstatic evening dining, dancing, and having wonderful sex with "Bill" who she felt was an exceptional man. Later in the dream, she expected the same ecstasy the following evening with him. However, he said to her they couldn't go on because she was cruel. Almost always the prospect of an enjoyable situation "scared" her because it required her to abandon her protective cynicism, which made her a defeatable, exploitable target for the cynicism and competitiveness of others.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7558980 TI - Poststructural psychoanalysis or wild analysis? PMID- 7558976 TI - One analyst's journey into darkness: countertransference resistance to recognizing sexual abuse, ritual abuse, and multiple personality disorders. PMID- 7558982 TI - The place of the signifier in psychoanalytic object relations theory. PMID- 7558984 TI - Internalized homophobia and the negative therapeutic reaction. PMID- 7558981 TI - Jewish food rituals.1927. PMID- 7558983 TI - A Latino perspective on the role of ethnicity in the development of moral values: implications for psychoanalytic theory and practice. PMID- 7558985 TI - An ERP study on the specificity of facial expression processing. AB - Facial expression processing specificity was investigated by means of event related potentials (ERPs). Stimuli consisted on happiness facial expressions (H) along with three other visual stimuli with different physical and affective characteristics: neutral facial expressions, landscapes and grey slides. The 32 subjects had to rate the stimuli with respect to two affective dimensions: 'valence' and 'arousal'. EEG was recorded at T7, T8, Fz and Cz, and the epoch was of 1000 ms (100 ms prior to stimulus onset). Analyses revealed that the variability of N200 and P200 components, which were not able to detect a differential activity in response to H stimuli, could be explained in terms of physical complexity and relevance of the stimuli. N300 did detect a differentiation between H and the rest of stimuli, sharing some characteristics of previous and later components. With respect to late components, P300 seemed to reflect the trends of 'arousal' ratings and SW results suggest that its amplitude was influenced by the stimulus recognition effort. Not a clear right or left hemisphere advantage in facial expression processing was found. These ERP results suggest that the specificity of facial expression processing is not strictly circumscribed to a particular latency or to a specific neural area, but it resides in a particular combination of discrete processes. PMID- 7558986 TI - Effects of music on physiological and affective responses to graded treadmill exercise in trained and untrained runners. AB - The influence of music on physiological and affective exercise responses was studied in 8 trained and 8 untrained runners under three music conditions ("no", "sedative", and "fast") during low, moderate, and high intensity exercise. Repeated measures ANOVA revealed increased respiratory frequency (FR) during fast music as compared to the no music and sedative music conditions (p < 0.01). Plasma cortisol levels did not differ at baseline across the music conditions; however, following high intensity exercise, higher cortisol levels were associated with fast music as compared to no music and sedative music (music x intensity interaction, p < 0.01). Affective measures during exercise (FEELING scale) showed no overall training group differences; however, there was a music x group x intensity interaction (p < 0.05) in which untrained subjects reported more positive affect compared to trained subjects while listening to fast music during low and high intensity exercise. Data collected at voluntary exhaustion revealed significantly more positive affect and higher skin temperature (p values < 0.01) in untrained compared to trained subjects. Collectively, these results suggest listening to fast, upbeat music during exercise may be beneficial for untrained runners but counterproductive for trained runners. PMID- 7558987 TI - Brainstem frequency-following and behavioral responses during selective attention to pure tone and missing fundamental stimuli. AB - Reaction time (RT), discrimination sensitivity (d'), and the brainstem frequency following response (FFR) were recorded in 32 subjects performing a selective attention task. Auditory stimuli were a 400 Hz pure tone and a complex "missing fundamental" (MF) presented dichotically to separate ears (channels). In two tasks, infrequent target stimuli were either of lower intensity or greater duration than standard stimuli. Behavioral results showed consistently better performance (faster RTs and higher d' scores) in the duration task, and better overall detection of MF targets. FFR attention effects were evidenced by differing amplitudes in attend and ignore conditions. Amplitudes in the attended channel were larger to MF stimuli in both tasks, and to the tone stimulus in the duration task. Responses to tone in the intensity task, however, were lowest when the channel was attended, perhaps reflecting some property of greater task difficulty. The demonstration of FFR amplitude differences between attended and ignored channels suggests that selective attention can modify brainstem evoked responses in humans. PMID- 7558988 TI - Cognitive influences on electrocortical and heart rate activity in obsessive compulsive disorder. AB - The present study examined patterns of electrocortical and heart rate activity in obsessive-compulsive (OC) patients, and both high and low trait anxious control groups. Physiological patterns were examined in light of an intake-rejection attentional paradigm. For each group, electrocortical and cardiac activity were recorded during conditions of mental rest and mental activity requiring the internal versus external processing of information. The pattern of heart rate activity observed in the present study validated the choice of tasks used to examine intake and rejection attentional processes. Overall, the study reported three major findings. First, at baseline, both OC patients and high trait anxious subjects showed a pattern of activity in their EEG spectrum that differed significantly from low trait anxious subjects. Second, similar heart rate differences for the intake and rejection tasks were detected in all three groups. Third, EEG patterns unique to OCs were evidenced in the frontal region during intake and rejection tasks. PMID- 7558989 TI - Cynical hostility influences anger, but not cardiovascular reactivity during competition with harassment. AB - Cynical hostility has been linked to coronary heart disease (CHD), and there is mixed support for the hypothesis that cynical hostility may contribute to CHD through exaggerated cardiovascular responses to anger-provoking stressors. The present study tested the influences of cynical hostility on affective and cardiovascular responses to provocation in 68 undergraduate men. Subjects were divided into high and low cynical hostility groups by a median split on Cook Medley Hostility Scale scores, and half of the subjects in each group were harassed during competition on a video game. High hostile subjects reported greater anger than low hostile subjects during the competition, independently of harassment, and harassment produced stronger feelings of mistreatment, independently of hostility. Harassed subjects experienced larger systolic blood pressure responses only during an affect rating period after the competition, but the responses were not influenced by hostility. These findings provide further evidence that cynical hostility, anger, and cardiovascular reactivity are not simply nor consistently related. PMID- 7558990 TI - Modulation of semantic processing using word length and complexity: an ERP study. AB - The objective of this study was to assess changes in semantic priming in two experiments which separately manipulated word length and the location of a semantic incongruence within spoken compound words. All words began 1500 ms after the presentation of related, partially unrelated, or totally unrelated picture primes. Using a picture of a dogbone as an example, either the spoken words DOGBONE, WISHBONE, DOGHOUSE, or MAILBOX could occur as targets (underscores indicate semantically unrelated parts). In practice, only one of the four possible targets occurred with each prime. Subjects made speeded responses by pressing one of two buttons (related/unrelated) while event-related brain potentials (ERPs) were collected from various scalp locations. The behavioral analyses of response time and accuracy were sensitive to changes in word length, but inconclusive for semantic relatedness. However, repeated-measures ANOVAs of peak amplitude, latency, and mean area amplitude of the N400 ERP revealed significant effects of word length and semantic priming. Comparison of related compounds with partially unrelated word-initial violations of context (e.g., WISHBONE) revealed diminished activity following the peak of the N400. Comparison of partially unrelated word-final contextual violations (e.g., DOGHOUSE) revealed an N400 effect delayed by approximately the mean length of the word-initial component. These results demonstrate the dynamic sensitivity of the cortical semantic processor. PMID- 7558991 TI - Smoking and EEG power spectra: effects of differences in arousal seeking. AB - Reversal theory, a general theory of motivation, emotion and action, has recently been shown to predict lapses in smoking cessation. Individuals are less likely to lapse if they are in the telic (serious-minded, arousal avoidant, goal-oriented) state than when they are in the paratelic (playful, arousal seeking, spontaneous) state. The literature indicates that people can smoke in such a way as to either increase or decrease central nervous system arousal; smoking in the telic and paratelic states might therefore differentially affect the resting electroencephalograph, as quantified by Fast Fourier Transform analysis. The basic hypothesis was supported. Theta power was decreased when subjects in the telic state smoked, while beta 2 power was increased when subjects in the paratelic state smoked; the latter finding was, however, true only for men. The results have important implications for research on changing health behaviors and for smoking cessation programs. PMID- 7558992 TI - Temporal localization of the response selection processing stage. AB - Response choice is one of the stages in the information processing model proposed by Sanders. It is influenced by stimulus-response (S-R) compatibility. Segmentation of the processing window in intervals between RT and peak latencies and between peak latencies, was used to test the assumption that the decisional processes would be concomitant with the N200 rather than the P300 component. ERPs were recorded in ten subjects during a spatial S-R compatibility auditory task. The S-R compatibility effect is observed on P300 latencies but is only a trend on the N200 component. An effect also observed on the interval between RT and N200 and especially between N200 and P300 while no effect is observed on the interval between RT and P300. These results support the idea that the selection processes ending with P300 occurrence could start as early as the N200 peak component. PMID- 7558993 TI - Human EEG, behavioral stillness and biofeedback. AB - This theoretical synthesis affirms that the normal human EEG is: (1) an indicator of movements of behavior; (2) an undifferentiated indicator of cortical work; but (3) not an indicator of mental processes. A majority of cortical work for an awake person is the mobilization and regulation of all the processes involved in the production, control and prediction of movements of behavior. Abundant synchronous slow waves (alpha, mu, sensory-motor rhythm) indicate a demobilization of voluntary and reflexive, phasic neuromuscular processes which predict, initiate, regulate, and terminate voluntary behavior and movement with a corresponding reduction of afferent feedback associated with sensory data capture, sensory motor integration and behavior. Cortical theta in association with drowsiness indicates that there is further demobilization of reflexive, synergistic, neuromuscular process (as for gait, defensive responses, anti gravity support), and a reduction of tonic processes which maintain muscle tone or tension with concomitant further reduction of afferent feedback. These various states of behavioral stillness are the catalyst of beneficial psychological and behavioral processes which have been observed to follow biofeedback training to increase synchronous EEG rhythms, and may provide a therapeutic context for psychotherapeutic interventions. PMID- 7558994 TI - Temporal stability of children's cardiovascular (CV) reactivity: role of ethnicity, gender and family history of myocardial infarction. AB - Cardiovascular responses to three laboratory stressors (i.e., postural change, treadmill exercise, forehead cold) were evaluated in 106 children (72 Whites, 34 Blacks) who varied in family history (FH) of early myocardial infarction (53 positive FH, 53 negative FH). Subjects were evaluated on two occasions separated by one year. In general, regardless of ethnicity, gender or FH, stability of resting blood pressures (BP) and heart rates were comparable to existing data. Resting cardiac output (CO) and total peripheral resistance (TPR) stability estimates were comparable or higher than the BP estimates across groups except for the +FH subjects and males who showed poor stability to one or both parameters. Moderate stability was observed for all parameters in response to forehead cold and low to moderate stability was observed in HR, CO, and TPR postural change reactivity. The only consistent pattern of significant differences in stability estimates involved ethnicity. African-Americans exhibited significantly higher coefficients compared to Whites in TPR at rest and during postural change and forehead cold. Reasons for the low to moderate resting and reactivity stability estimates are discussed. PMID- 7558995 TI - Temporal stability of acute stressor-induced changes in cellular immunity. AB - This study examined the temporal stability of enumerative immune responses to acute psychosocial stress. Lymphocyte subsets were measured in 24 healthy male subjects at rest and following a speaking stressor on two occasions approximately six weeks apart. The speaking task caused significant increases in T suppressor/cytotoxic cells, natural killer cells, T-cells, and total WBC and decreases in the T-helper/suppressor ratio. Baseline test-retest correlation's were statistically significant for all variables (r values = 0.40-0.90). With two exceptions (T-cells and T-suppressor/cytotoxic cells), speaking task values (absolute reactivity scores) were also statistically significant (r values = 0.48 0.92). Baseline adjusted test-retest correlations were however generally less reliable, with only natural killer cells (r values > 0.40), the T helper/suppressor ratio (r = 0.60), and total WBC (r = 0.48) showing statistical significance. The findings suggest that certain but not all cellular immune responses to acute stress are moderately stable over time. PMID- 7558996 TI - Licensure by endorsement model reaches to Canada, US territories. PMID- 7558997 TI - National disciplinary database facilitates mobility, protects public. PMID- 7558999 TI - Washington's Aquatic Animal Health Program: serving needs of growing industry and educating. PMID- 7558998 TI - FDA reaffirms position on extra-label use of ionophore drugs. PMID- 7559000 TI - Well-being of zoo and aquarium animals explored. PMID- 7559001 TI - Cautions use of firm stomach tubes in horses. PMID- 7559002 TI - Proper use of the term model. PMID- 7559003 TI - What is your diagnosis? Blindness and ataxia in a foal caused by an abscess with gas-forming bacteria. PMID- 7559004 TI - Antimicrobial agents for treatment of infectious keratitis in horses. PMID- 7559005 TI - Vicarious liability. PMID- 7559006 TI - Review of the first interactive workshop on professional flexible labeling. The Center for Veterinary Medicine perspective. PMID- 7559007 TI - Review of the first interactive workshop on professional flexible labeling. An Animal Health Institute perspective. PMID- 7559009 TI - American Academy of Veterinary Pharmacology and Therapeutics: perspectives on the utility of flexible drug labeling. An AAVPT perspective: Part I. PMID- 7559008 TI - Review of the first interactive workshop on professional flexible labeling. An American Veterinary Medical Association perspective. PMID- 7559010 TI - American Academy of Veterinary Pharmacology and Therapeutics: perspectives on the utility of flexible drug labeling. An AAVPT perspective: Part II. PMID- 7559011 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Animal Hospital Association (AAHA). PMID- 7559012 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Association of Avian Pathologists (AAAP). PMID- 7559013 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Association of Bovine Practitioners (AABP). PMID- 7559014 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Association of Equine Practitioners (AAEP). PMID- 7559016 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Association of Swine Practitioners (AASP). PMID- 7559015 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the American Association of Small Ruminant Practitioners (AASRP). PMID- 7559017 TI - AVMA/Practice Group perspectives: use of drug labels in the prescription of antimicrobial therapy. Representing the Aquaculture and Seafood Safety Advisory Committee (ASSAC). PMID- 7559020 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. A clinical pharmacologist's perspective. PMID- 7559021 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. Liability issues. PMID- 7559018 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. Practicality of clinical development. PMID- 7559019 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. In vitro microbiological aspects of flexible labeling. PMID- 7559022 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. Practical aspects of flexible labeling in feedlots. PMID- 7559023 TI - AHI perspectives: flexible drug labeling and its impact on the drug development process. Break-out session. PMID- 7559026 TI - Fracture of the distal sesamoid bone in horses: 17 cases (1982-1992). AB - Medical records of 17 horses in which a distal sesamoid bone fracture was diagnosed between 1982 and 1992 were reviewed. There were 8 Standardbreds, 6 Quarter Horses, 2 Thoroughbreds, and 1 Arabian. Mean age was 4.7 years. A forelimb was affected in 15 horses, and a hind limb was affected in 2. All horses were lame, and most were grade III/V lame at the trot. In all horses, the diagnosis was confirmed by means of radiography. Five horses were treated with stall rest alone; 5 underwent neurectomy; 4 were treated with stall rest and corrective shoeing; and 1 was treated with stall rest and external coaptation. The other 2 horses were euthanatized. Two Quarter Horses, 1 treated with stall rest and corrective shoeing and the other treated with stall rest and external coaptation, returned to use as halter horses, and 2 Standardbreds treated with stall rest alone returned to racing, but at a lower level than they had raced prior to injury. One horse that underwent neurectomy could be used for pleasure riding. Long-term rest may be important in achieving a successful outcome. Pleasure horses would appear to have a better prognosis for return to use after a distal sesamoid bone fracture than do performance horses. PMID- 7559025 TI - Ataxia associated with lymphosarcoma in a dog. AB - A 4-year-old English Springer Spaniel with ataxia was examined because of progression of neurologic signs. Complete physical examination and results of CBC and serum biochemical analysis revealed no other abnormalities. The dog developed respiratory arrest and was euthanatized during recovery from general anesthesia, which had been performed to obtain a CSF sample. Results of CSF analysis were within reference limits. Necropsy revealed a single, well-circumscribed mass in the cerebellum, which was diagnosed as lymphosarcoma. The dog had no peripheral lymphadenopathy or organomegaly suggestive of the generalized form of the disease. PMID- 7559028 TI - Vertebral fracture associated with trauma during movement and restraint of cattle. AB - Two female Limousin calves in a group of 68 calves suffered fractures of a lumbar vertebra subsequent to moving through a chute and being restrained to facilitate administration of vaccines. One calf collapsed as it exited the chute, whereas the second calf collapsed 10 minutes after it was released from the chute. Both heifers were euthanatized. The fractures, of the first lumbar and of the third lumbar vertebral bodies, respectively, were not associated with mineral imbalances. Fractures were determined to be the result of calves attempting to escape through a gap formed by erosion of the area beneath a gate in the holding pen of the corral system. By moving cattle in such a way as to bypass the gate, further injuries were avoided. The calves described here underscore the importance of the use of corral systems appropriate for cattle of various sizes and the necessity of maintaining properly repaired corral systems to prevent injuries to cattle. PMID- 7559027 TI - Antibacterial susceptibility patterns for microbial isolates associated with infectious keratitis in horses: 63 cases (1986-1994). AB - Seventy-three aerobic bacterial isolates were cultured from 64 eyes of 63 horses with infectious keratitis. Forty-two (58%) of the organisms isolated initially were gram-positive (g+, 10 genera) and 31 (42%) were gram-negative (g-, 5 genera). After local antimicrobial treatment, repeat cultures from samples obtained from 15 eyes of hospitalized horses yielded 21 secondary bacterial isolates. Staphylococci spp and Streptococci spp were the most common g(+) isolates and accounted for 79% of g(+) organisms isolated initially. Antibiograms revealed ticarcillin to be the most efficacious antibiotic tested on g(+) organisms, with 28 of 30 (93%) being susceptible. Of commercially available topical ophthalmic antibiotics tested on g(+) organisms, erythromycin was the most efficacious, with 32 of 35 (91%) isolates being susceptible. Pseudomonas spp, Escherichia coli, and Acinetobacter spp accounted for 68% of g(-) organisms isolated initially. Gentamicin, tobramycin, polymyxin B, and neomycin were highly effective in vitro against initial g(-) isolates. Chloramphenicol was ineffective against g(+) and g(-) organisms isolated initially. A significantly (P < 0.05) higher frequency of g(-) organisms was noticed on repeat cultures after intensive topical antimicrobial treatments as compared to organisms isolated at initial examination. Pseudomonas organisms isolated from second cultures were resistant to gentamicin, but susceptible to ciprofloxacin. Overall, secondary g(-) isolates were more susceptible to ciprofloxacin, neomycin, tobramycin, or amikacin than to gentamicin. Fungi were isolated in 24 of 63 (38%) horses in the study. Twenty five filamentous fungi and 2 yeasts were identified from 24 eyes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559024 TI - Genetic test for pyruvate kinase deficiency of Basenjis. AB - Pyruvate kinase (PK) deficiency is an autosomal, recessive, inherited disease of Basenjis that causes chronic, regenerative, hemolytic anemia. Diagnostic methods currently used to identify carrier animals rely on measurement of erythrocyte PK activity and frequently give equivocal results. A genetic test incorporating polymerase chain reaction amplification of genomic DNA and restriction fragment length polymorphism has been developed to determine the PK genotype of Basenjis. To determine whether results of this genetic test compared with results of standard tests for PK deficiency, erythrocyte PK activity, hematocrit, and reticulocyte counts were determined in, and the genetic test was performed on, 24 dogs. The genetic test accurately identified the 11 dogs whose PK genotype was known prior to this study, and results were consistent with results of measuring erythrocyte PK activity in the remaining 13 dogs. The genetic test may be of value in determining PK genotype of Basenjis. PMID- 7559032 TI - Pets play role in child development. PMID- 7559030 TI - Repeat laparotomy for gastrointestinal disorders in cattle: 57 cases (1968-1992). AB - A study was conducted to investigate indications for, typical findings during, and outcome of repeat laparotomies in 57 cattle with gastrointestinal disorders. Cattle were grouped according to the reason the initial laparotomy had been performed. Group-1 cattle (n = 46) had had left or right displacement of the abomasum (LDA or RDA) or right volvulus of the abomasum. Group-2 cattle (n = 10) had had ruminal, reticular, or abomasal obstruction or perforation. Group-3 cattle (n = 7) had had obstruction, volvulus, distention, or perforation of the small or large intestine. Median interval between laparotomies was 1 month (range, 1 day to 38 months). Intraoperative findings during repeat laparotomy in group-1 cattle included LDA (n = 18), RDA (9), right volvulus of the abdomen (5), pneumoperitoneum (1), diffuse abdominal adhesions (1), cecal adhesions (1), adhesions of the rumen to the body wall (1), abscess in the cranial portion of the abdomen (1), reticular adhesions (1), localized necrosis (1), and idiopathic rumen distention (1). Intraoperative findings during repeat laparotomy in group-2 cattle included abscess in the cranial portion of the abdomen (4), no abnormalities (1), distended rumen (1), ruptured abomasum (1), recurrent abomasal impaction (1), diffuse abdominal adhesions (1), and pyloric fat necrosis (1). Intraoperative findings during repeat laparotomy in group-3 cattle included LDA (3), RDA (1), cecal volvulus (1), anastomotic rupture (1), and impacted colocolostomy (1). Seven cows and 1 calf underwent additional repeat laparotomies. Forty-four (77%) of the 57 cattle were discharged following final laparotomy. Fourteen of the 20 group-1 cattle, 1 of the 6 group-2 cattle, and 2 of the 3 group-3 cattle for which follow-up information was available were productive after repeat laparotomy. PMID- 7559031 TI - Keeping screwworms at bay. PMID- 7559029 TI - Myositis, lameness, and paraparesis associated with use of an oil-adjuvant bacterin in beef cows. AB - Right hind limb lameness, progressing to bilateral paraparesis, was observed in 56 of 610 (9%) beef cows. Lameness began 6 days to 4 weeks after vaccination in the right longissimus lumborum (loin) muscle with an Escherichia coli/Campylobacter bacterin in an oil adjuvant. Postmortem examination of 5 affected cows revealed a large inflammatory mass at the site of vaccination. In each cow, the mass spread through adjacent intervertebral foramina into the vertebral canal and compressed the lumbar portion of the spinal cord. Microbiologic procedures did not reveal a microbial agent in affected tissues or in an unopened bottle of bacterin from the same lot used in the herd. Histologic examination revealed pyogranulomatous inflammation of the vaccination site and adjacent epidural tissue, with inflammatory nodules centered around large clear spaces that probably represented remnant emulsion from the oil adjuvant in the bacterin. As evident in these cows, IM injection of irritating products may cause severe myositis. Vaccination into paravertebral muscles is risky because of possible extension of inflammation through intervertebral foramina. PMID- 7559033 TI - Competing with changes superstores bring. PMID- 7559034 TI - Comments about frequency of vaccination. PMID- 7559036 TI - Comments about frequency of vaccination. PMID- 7559037 TI - Pathophysiologic mechanisms of noncardiogenic pulmonary edema. PMID- 7559035 TI - Comments about frequency of vaccination. PMID- 7559038 TI - Recommends legislation for some pet handlers. PMID- 7559039 TI - The natural wrongs about animal rights and animal liberation. PMID- 7559042 TI - Comparison of veterinary medical degree enrollment for academic years 1993-1994 and 1994-1995. PMID- 7559040 TI - Exciting times for beef practitioners. PMID- 7559043 TI - Long-term renal and hematologic effects of uninephrectomy in healthy feline kidney donors. AB - To assess long-term hematologic and renal effects associated with a solitary kidney, 16 healthy cats undergoing uninephrectomy for kidney donation between May 1987 and January 1991 were evaluated by use of physical examination, CBC, serum biochemical analysis, urinalysis, and urine protein:creatinine ratio. Results of preoperative CBC, serum biochemical analysis, and urinalysis were within reference limits in all donors. Median age at surgery and at follow-up evaluation was 34 and 72 months, respectively. Mean (+/- SEM) interval between follow-up and uninephrectomy was 39.3 +/- 14.6 months. Postuninephrectomy hematocrit and RBC indices were within reference limits in 15 donors. One cat with chronic renal insufficiency had normocytic, normochromic, nonregenerative anemia. In 15 clinically normal donor cats, mean (+/- SEM) serum creatinine concentrations pre- and post-uninephrectomy were 1.36 +/- 0.20 and 1.71 +/- 0.33 mg/dl, respectively (P = 0.0002); however, the clinical relevance of this statistical difference in serum creatinine is uncertain, because all values were within reference limits. In addition, urine-concentrating ability was maintained in 14 donors, with urine specific gravity > or = 1.040. Two donors, including the cat with chronic renal insufficiency, produced dilute urine (specific gravity < or = 1.020) and had substantial proteinuria, with urine protein:creatinine ratios of 2.16 and 3.62, respectively. Mean urine protein:creatinine ratio in donor cats was not significantly different from that in an age- and sex-matched comparison group. Renal and erythropoetic function was clinically preserved in the group of donor cats within 2 to 5 years after uninephrectomy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559044 TI - Measurement of serum bile acids concentrations for diagnosis of hepatobiliary disease in cats. AB - Serum bile acid concentrations were measured after food had been withheld for 12 hours (fasting serum bile acid [FSBA] concentration) and 2 hours after a meal (post-prandial serum bile acid [PSBA] concentration) using a direct enzymatic procedure in 108 cats clinically suspected of having hepatobiliary disease. In all cats, liver tissue was examined histologically to confirm the diagnosis. Twenty-six cats did not have histologic evidence of hepatobiliary disease and served as controls. The remaining 82 cats had hepatobiliary disease including hepatic lipidosis (n = 20), portosystemic vascular anomaly (n = 24), hepatic necrosis (n =13), hepatic neoplasia (n = 8), or cholestatic hepatic disease(n = 17). Sensitivity and specificity of measuring FSBA and PSBA concentrations were calculated for each test alone and when results were interpreted in combination (ie, in series and in parallel), and were compared with sensitivity and specificity of routinely used serum biochemical tests, including measuring serum activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase, and measuring serum concentrations of cholesterol, BUN, and total bilirubin. When tests were considered individually, determination of FSBA and PSBA concentrations had higher specificity than did the other tests (using a cutoff of 15 mumol/L for FSBA concentration and of 20 mumol/L for PSBA concentration). Determination of PSBA concentration had the highest sensitivity of all single tests in cats with hepatic lipidosis, portosystemic vascular anomaly, or cholestasis; determination of alanine aminotransferase activity or PSBA concentration had the highest sensitivity for cats with hepatic necrosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559045 TI - Response to high-dose radioactive iodine administration in cats with thyroid carcinoma that had previously undergone surgery. AB - Seven cats with thyroid carcinomas that had previously undergone surgical removal of neoplastic tissue were treated with 30 mCi of radioactive iodine (131I). Six of the cats had clinical signs of hyperthyroidism; 1 did not. There were no complications associated with 131I treatment, and clinical signs resolved in all cats. Technetium scans of 4 cats made after treatment did not have evidence of isotope uptake. In the remaining 3 cats, small areas of isotope uptake, the intensity of which was equal to or less than the intensity of uptake in the salivary glands, were seen. All 7 cats became hypothyroid after treatment; 4 required L-thyroxine supplementation. One cat was alive 33 months after treatment. The other 6 cats were euthanatized because of unrelated diseases 10 to 41 months after treatment. PMID- 7559041 TI - What is your diagnosis? Multiple pelvic fractures right sacroiliac luxation, abnormal gas densities surrounding the descending colon, and loss of abdominal detail. PMID- 7559046 TI - Progressive lymphangiectasis and recurrent chylothorax in a dog after thoracic duct ligation. AB - A 2-year-old Bernese Mountain Dog was examined to determine the cause of bilateral pleural effusion. Torsion was diagnosed, and a lobectomy of a lung lobe was performed. Chylothorax developed 12 days after lung lobectomy. Mesenteric lymphangiography revealed lymphangiectasis Lymphangiography immediately after surgical thoracic duct was completely obstructed, but chylothorax persisted after thoracic duct ligation. Lymphangiography was repeated 50 days after ligation of the thoracic duct and revealed multiple patent thoracic duct branches and progressive lymphangiectasis. A second attempt to ligate the thoracic duct caused the effusion to become serosanguineous. A pleuroperitoneal shunt with a manually operated pump chamber was used to remove the pleural effusion. Chylothorax was again detected 50 weeks after placement of shunt. Mesenteric lymphangiography revealed multiple patent thoracic duct branches and a lymphatic plexus that extended across the thoracic cavity. PMID- 7559047 TI - Granulosa cell tumor in an ovariohysterectomized dog. AB - A granulosa cell tumor was found in a dog with clinical signs of persistent estrus that began 6 years after ovariohysterectomy had been performed. The tumor was diagnosed by use of ultrasonography, provocative testing with human chorionic gonadotropin, and exploratory laparotomy. Hyperestrogenism from functional tumor cells caused bone marrow suppression and endocrine alopecia. Successful treatment included tumor removal, blood transfusions, and antibiotic administration. PMID- 7559049 TI - Treatment of dogs with wounds of the limbs caused by shearing forces: 98 cases (1975-1993). AB - Medical records of 98 dogs with wounds of the limbs caused by shearing forces (shearing wounds) were reviewed. Shearing wounds were reported most often in young dogs of many breeds, and most often resulted from trauma received during an encounter with an automobile. Wounds were located mainly on the distal portion of the limbs, with the most common site being the medial aspect of the tarsus and metatarsus. All injuries resulted in exposure of subcutaneous soft tissues, about three fourths of all injuries resulted in bone or joint exposure, and slightly more than half of the dogs had joint instability. All wounds were lavaged and debrided. Wounds were sutured, using primary closure techniques or methods that allowed the wound to remain partially or totally open to heal by second intention. Bandages were applied in all dogs. Most bandages were reinforced with a splint device. Grafting procedures or internal fixation devices were not used in initial treatment of the injuries. In the study reported here, healing time ranged from 2.0 to 8.7 weeks, depending on the size and depth of the wound and type of closure used. Mean number of anesthetic and surgical procedures was 1.7 per dog, and the mean number of rechecks after discharge from the hospital was 5.5 per dog. Outcome was considered excellent or good in 89 of 98 (91%) dogs; these dogs were clinically normal or had only minor functional abnormalities after their injuries healed. Outcome was poor in 9 of 60 (15%) dogs. PMID- 7559048 TI - Adrenalectomy for treatment of hyperadrenocorticism in cats: 10 cases (1988 1992). AB - Outcome of and complications associated with bilateral adrenalectomy in 8 cats with pituitary-dependent hyperadrenocorticism and bilateral adrenocortical hyperplasia and outcome of and complications associated with unilateral adrenalectomy in 2 cats with adrenocortical tumor (adrenocortical adenoma, 1 cat; adrenocortical carcinoma, 1 cat) and unilateral adrenomegaly were determined. Glucocorticoids were administered to all cats at the time of surgery, and mineralocorticoids were administered to the 8 cats that underwent bilateral adrenalectomy. A ventral midline celiotomy was performed in all cats. Intraoperative complications did not develop in any cat. Postoperative complications developed in all cats and included abnormal serum electrolyte concentrations (n = 8), skin lacerations (n = 5), pancreatitis (n = 3), hypoglycemia (n = 2), pneumonia (n = 1), and venous thrombosis (n = 1). Three cats died within 5 weeks after surgery of complications associated with sepsis (n = 2) or thromboembolism (n = 1). Clinical signs and physical abnormalities caused by hyperadrenocorticism resolved in the remaining 7 cats 2 to 4 months after adrenalectomy. Insulin treatment was discontinued in 4 of 6 cats with diabetes mellitus. Median survival time for these 7 cats was 12 months (range, 3 to > 30 months). Two cats died of acute adrenocortical insufficiency 3 and 6 months after bilateral adrenalectomy, 2 cats were euthanatized because of chronic renal failure 3 and 12 months after bilateral (n = 1) or unilateral (n = 1) adrenalectomy, and 2 cats were alive 9 and 14 months after bilateral adrenalectomy. In the remaining cat, clinical signs recurred 10 months after the cat had undergone unilateral adrenalectomy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559050 TI - Histologic appearance of axial osteochondral fragments from the proximoplantar/proximopalmar aspect of the proximal phalanx in horses. AB - Osteochondral fragments from the axial proximoplantar/proximopalmar region of the proximal phalanx were removed from 38 joints in 30 horses. Ninety-three percent of the horses were Standardbreds, and 28 of the 30 had a low-grade lameness. All but 1 of the horses had hind limb involvement. A total of 43 fragments were removed. Most (71%) of the fragments involved the medial aspect of the joint and had to be dissected from a covering of synovial tissue. Histologically, the circumference of most fragments consisted of a transition zone at the attachment of the joint capsule, a region of nonarticular, non-weight-bearing cartilage, a region where organized, dense connective tissue, presumably remnants of the short sesamoidean ligament were attached, and a region consisting of irregular truncated bony surfaces covered by healing of chronic fracture. There were several areas of degenerate hyaline cartilage, but no areas of normal hyaline cartilage or areas containing related cartilage cores or other evidence of delayed endochondral ossification. Immunohistochemical staining of 4 segments from 1 horse revealed sensory substance P immunoreactive nerves in the fibrous tissue surrounding the bony fragments and within the central cancellous spaces. The histologic appearance suggests that these osteochondral fragments may be a result of fracture, rather than a manifestation of osteochondrosis. PMID- 7559052 TI - Use of tension band wires in horses with fractures of the ulna: 22 cases (1980 1992). AB - Twenty-two horses with ulnar fractures were treated, using tension band wires alone or tension band wires in combination with pins or cortical bone screws. Age of the horses ranged from 2 weeks to 12 years (median, 4 months), and body weight ranged from 68 to 477 kg (median, 181 kg). Fractures were classified according to the Donecker and Bramlage ulnar fracture classification and included type 1-a (4 horses), type 1-b (4), type-2 (6), type-3 (1), type-4 (3), and type-5 (4), fractures. Tension band wires alone were used in 7 horses. Tension band wires were used in conjunction with unthreaded pins in 10 horses. In 3 horses, 5.5-mm cortical bone screws were inserted longitudinally instead of pins. A combination of a 5.5-mm cortical bone screw and a pin was used in 2 horses. In addition to pins and tension band wires, 4.5-mm cortical bone screws were placed in lag fashion to aid reduction of comminuted or oblique fractures in 7 of the 22 horses. Fractures healed in 18 (82%) horses. Four horses were euthanatized because of complications that included catastrophic failure of fixation during recovery from general anesthesia in 1 foal, septic arthritis and hyperextension of the contralateral metacarpophalangeal joint in 1 foal, and wound infection with partial disruption of repair in 2 horses. Nonfatal complications developed in 6 horses and included incision infection, partial wound dehiscence, carpal contraction, carpus varus of the contralateral forelimb, slight distraction of proximal fragments of the fractures, bent implants, and distal migration of pins. Long-term monitoring was performed on 17 horses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559051 TI - Endoscopy of the auditory tube diverticula in four horses with otitis media/interna. AB - Endoscopic examination of the auditory tube diverticula was a diagnostic aid in the evaluation of 4 horses with otitis media/interna and associated osseous changes of the stylohyoid and petrous temporal. One of the horses was examined because of persistent head shaking; the other 3 were examined because of an acute onset of facial and vestibulocochlear nerve dysfunction. Proliferative lesions involving the petrous temporal bone and proximal portion of the stylohyoid bone were identified endoscopically in all 4 horses. Endoscopy is a noninvasive procedure that provides an alternative to skull radiography and tympanocentesis in the diagnosis of otitis media/interna in horses. In addition, risks associated with general anesthesia are avoided. PMID- 7559053 TI - Wildlife and zoo veterinarians take responsibility for ecosystem. PMID- 7559054 TI - Veterinary teams federally activated to St Thomas. PMID- 7559056 TI - Food safety equation begins on farm. PMID- 7559055 TI - Tattoo identification eliminates possibility of initiating repeat spay/neuter surgery. PMID- 7559057 TI - Opinion on ear cropping and tail docking. PMID- 7559058 TI - Testing for hyperadrenocorticism. PMID- 7559059 TI - Unlicensed practice of veterinary medicine. PMID- 7559060 TI - What is your diagnosis? Bilateral hip dysplasia associated with medial luxation of the left patella in a dog. PMID- 7559061 TI - What is your neurologic diagnosis? Nerve root neoplasm. PMID- 7559063 TI - Valuation of a veterinary medical practice. PMID- 7559062 TI - Animal behavior case of the month. Dogs were evaluated because of aggression. PMID- 7559064 TI - Bacteriologic culture and histologic examination of samples collected from recumbent cattle at slaughter. AB - OBJECTIVE: To evaluate the potential food safety risks constituted by recumbent cattle that are slaughtered for edible beef. DESIGN: Prospective case series. ANIMALS: Thirty cattle in recumbency that passed a routine antemortem inspection at a US federally inspected abattoir. PROCEDURE: Aerobic, bacteriologic culture of blood samples taken immediately prior to slaughter and of spleens taken during viscera inspection. Gross lesions were recorded, and samples of liver, lung, kidney, and heart were collected from each animal for routine light microscopic examination. RESULTS: Bacteremia caused by Salmonella dublin was documented in 1 cow that had arthritis. Two other cows were condemned after postmortem inspection: 1 because of pneumonia and pleuritis and the other because of vegetative endocarditis. Three carcasses were retained and later condemned because of antibiotic residues in tissues; 1 of these cows had mastitis, 1 had liver abscesses, and 1 was the cow with vegetative endocarditis. Sarcocystosis was found in 27 of 30 hearts, but other clinically important histologic lesions were observed only in liver samples. In 11 of the 30 cows, multifocal, microscopic foci of hepatitis were observed, suggesting that terminal embolic bacterial showering of the liver had occurred in these animals. Liver samples were not submitted for bacteriologic culture. CLINICAL IMPLICATIONS: Most recumbent cows slaughtered for edible beef are not contaminated by bacteria; however, the viscera from these animals may present a food safety danger. Efforts should be made to develop rapid tests to identify bacteremic animals at slaughter and to more fully evaluate terminal showering of viscera by bacteria in cattle at slaughter. PMID- 7559065 TI - Ultrasonographic evaluation of the adrenal glands in dogs. AB - OBJECTIVE: To determine normal adrenal gland size by means of ultrasonography in dogs and to determine the value of ultrasonography in the diagnosis of pituitary dependent hyperadrenocorticism (PDH) in dogs. DESIGN: Prospective observational study. ANIMALS: 62 dogs: 20 healthy dogs, 20 dogs with non-endocrine disease, and 22 dogs with untreated PDH. PROCEDURE: Length and maximum and minimum diameter of the adrenal glands were measured ultrasonographically. Multiple regression and correlation analyses were used to determine whether body weight, kidney length, aortic diameter, or age was related to adrenal gland size. Two-tailed t-tests and multiple linear regression analysis were used to compare values between groups. Sensitivity and specificity of using ultrasonographic measurement of adrenal gland size as a diagnostic test for PDH were determined. RESULTS: There was a significant linear relationship between adrenal gland length, but not maximum and minimum diameters, and body weight, aortic diameter, and kidney length in healthy dogs and in dogs with nonendocrine diseases. Length, maximum diameter, and minimum diameter of the right adrenal gland and maximum and minimum diameters of the left adrenal gland were significantly greater in dogs with PDH than in healthy dogs and dogs with nonendocrine diseases. As a diagnostic test for PDH, ultrasonographic measurement of maximum or minimum diameter of the left adrenal gland gave the best combination of sensitivity and specificity. For maximum diameter of the left adrenal gland, sensitivity was 77% and specificity was 80%. For minimum diameter of the left adrenal gland, sensitivity was 73% and specificity was 85%. CLINICAL IMPLICATION: Ultrasonography of the adrenal glands is a valuable diagnostic procedure in dogs suspected of having pituitary dependent hyperadrenocorticism. PMID- 7559066 TI - Use of a low-profile gastrostomy device for administering nutrients in two dogs. AB - Percutaneous endoscopic gastrostomy (PEG) tubes are an excellent means of providing nutritional support via the enteral route. Most problems with PEG tubes are associated with their maintenance. A low-profile feeding device has been developed for use in human beings to replace standard PEG tubes in situations in which long-term nutritional support is necessary. We adapted a low-profile feeding device for use in 2 dogs. The device offered the advantage of being easier to maintain than standard PEG tubes. It appeared to have been well tolerated. Its low-profile design is likely to result in few problems resulting from an animal chewing on the device or from accidental dislodgment. PMID- 7559068 TI - Correlation of ultrasonographic findings with surgical, portographic, and necropsy findings in dogs and cats with portosystemic shunts: 63 cases (1987 1993). AB - Results of abdominal ultrasonography in 63 dogs and cats with suspected congenital portosystemic shunts (PSS) were compared with surgical, portographic, and necropsy findings. True-positive ultrasonographic results were found in 33 animals, and 2 animals had false-positive results for detection of single extrahepatic PSS. In 4 animals, results were true negative and in 8 animals, were false-negative for detection of extrahepatic PSS. The false-negative rate decreased substantially with operator experience. The sensitivity of ultrasonography for detection of extrahepatic PSS in these animals was 80.5%, and the specificity was 66.7%. In 5 other animals, ultrasonographic examination was strongly suggestive of the existence of a shunt, but the vessel could not be definitively imaged. All 5 animals had single extrahepatic shunts. Eleven animals had true-positive results for detection of intrahepatic PSS. One animal had false positive ultrasonographic results for intrahepatic PSS, but an extrahepatic shunt was diagnosed at surgery. The sensitivity of ultrasonography for detection of intrahepatic PSS was 100%. PMID- 7559067 TI - Furunculoid myiasis in a dog caused by Cordylobia anthropophaga. AB - Two days after returning with its owners from a 60-day visit to Liberia, an 8 year-old 6.5-kg spayed female Miniature Schnauzer was examined by a veterinarian in the United States. A 1.0 x 1.0-cm raised erythematous nodule was noticed on the medial aspect of the right pinna. In the center of the nodule was a 1.0 x 1.0 mm pore from which a 0.5 x 0.5 x 1.0-cm white larva was extracted. The larva was identified as a third-stage larva of Cordylobia anthropophaga, the African Tumbu fly, a fly restricted to sub-Saharan Africa. Cordylobia anthropophaga does have zoonotic potential, but the owners did not have any skin lesions. Detection of C anthropophaga in the United States warranted reports to state and federal authorities and personnel at the Centers for Disease Control and Prevention. The situation described here illustrated the potential of tourists' pets to import foreign diseases. PMID- 7559070 TI - Gunshot injuries in horses: 22 cases (1971-1993) AB - Twenty-two horses were examined because of firearm injuries. Nine had been shot with .22-caliber bullets, 2 with BB pellets, 6 with buckshot, 1 with a .35 caliber bullet, and 1 with an airgun pellet. Injury was confined to the skin or skeletal muscles in 8 horses. Of these, 7 returned to their previous use. In 14 horses, injuries to additional structures were incurred, including the sinus and pharynx (n = 2), mandible (n = 1), tooth (n = 1), aorta (n = 1), eye (n = 3), tibia (n = 1), gastrointestinal tract (n = 3), joint (n = 1), and trachea (n = 1). The 3 horses that had only eye injuries were discharged to their owners. Of the other 11 horses with injuries to deep/vital structures, 3 died, 5 were euthanatized, and 3 survived. PMID- 7559069 TI - Endoscopic and surgical retrieval of fishhooks from the stomach and esophagus in dogs and cats: 75 cases (1977-1993) AB - Medical records of 3 cats and 72 dogs that had a fishhook endoscopically or surgically retrieved from the stomach or esophagus were reviewed. Endoscopic retrieval was successful in 41 of 62 (66%) animals, and retrieval time and hospitalization time for endoscopic retrieval were significantly shorter than times for surgical retrieval. Rate of failure of endoscopic retrieval was higher for animals with treble-barb, rather than single-barb, fishhooks. Whether a fishhook could be successfully retrieved endoscopically was independent of body weight, amount of time the fishhook had been present, location of the hook, and orientation within the esophagus. PMID- 7559071 TI - Loop colostomy for treatment of grade-3 rectal tears in horses: seven cases (1983 1994) AB - OBJECTIVE: To determine the feasibility of performing a single-incision loop colostomy for treatment of grade-3 rectal tears in horses. DESIGN: Retrospective case series. ANIMALS: Seven adult horses with grade-3 rectal tears. PROCEDURE: A single-incision loop colostomy was performed with horses under general anesthesia (n = 6) or while restrained in standing stocks (n = 1). The rectal tear was lavaged via an endoscope. The colostomy was resected after the rectal tear healed. RESULTS: Rectal tears ranged from 4 to 10 cm in diameter and were > 25 cm proximal to the anus. All horses survived colostomy surgery. One horse was euthanatized at the request of the owner 1 day after surgery. Six horses underwent colostomy resection 13 to 30 days after colostomy. All horses had evidence of atrophy of the distal portion of the small colon, predisposing to impaction at the small colon anastomosis in 2 horses. One horse was euthanatized while hospitalized because of severe recurrent colic. Five horses were discharged from the hospital 31 to 45 days after admission. One horse was euthanatized 60 months after discharge from the hospital because of severe colic, and 4 horses were alive at the time of follow-up evaluation (3 to 12 months after discharge). CLINICAL IMPLICATIONS: The prognosis for horses with grade-3 rectal tears treated by colostomy appears to be favorable. PMID- 7559073 TI - Allergic rhinitis in a herd of cattle. AB - Allergic rhinitis was identified in a herd of Hereford cattle. Affected cattle had clinical signs of rhinitis (eg, nasal discharge, sneezing, nasal irritation, and nasal pruritus) and multiple small proliferative lesions in the nasal passages. Eosinophils were the predominant cell type in nasal discharges, and histologic examination of nasal mucosa biopsy specimens revealed chronic proliferative eosinophilic rhinitis. Results of CBC were normal; plasma fibrinogen concentrations were within reference limits. Results of intradermal allergen sensitivity testing and an ELISA for allergen-specific IgE only suggested an exaggerated IgE-mediated response to environmental allergens. Allergen-specific IgG may have acted as a competitive blocking antibody and limited clinical signs of disease in some cattle. PMID- 7559074 TI - Microsurgical excision of an abdominal mass in a gourami. AB - A female, sunset, thick-lipped gourami (Colisa labiosa) that weighed 8 g and was 5.4 cm from the snout to the end of the vertebral column was examined because of a 5-mm-diameter, midventrally located mass that had developed suddenly 1 month earlier. Cytologic examination of a sample obtained by use of fine-needle aspiration, survey radiography, positive-contrast radiography of the gastrointestinal tract, and Doppler ultrasonography were performed to evaluate the mass. These procedures were not able to provide a definitive diagnosis, but did facilitate surgical planning. The mass was excised, and the abdominal musculature was repaired, using microsurgical techniques. Redevelopment of the mass was not detected during the 5-month period after surgery. Histologic evaluation of the mass revealed an organized hematoma of undetermined cause. Successful management of this gourami illustrated that size should not be a deterrent to diagnostic evaluation and surgical intervention in diminutive fish. PMID- 7559072 TI - Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. AB - OBJECTIVE: To estimate the minimum rate of abortion attributable to infection with Neospora sp in selected California dairy herds. DESIGN: Prospective study. ANIMALS: Twenty-six dairy herds containing 19,708 cows were studied. Fourteen herds had a history of abortions attributable to neosporosis, and 12 were herds in which neosporosis had not been identified as a cause of abortions. PROCEDURE: During a 1-year period, all available aborted fetuses were submitted to veterinary diagnostic laboratories to determine the cause of abortion. Reproductive records of cows that aborted were reviewed. RESULTS: Neospora sp infection was the major cause of abortion identified (113/266 abortions, 42.5%). The majority (232/266, 87.2%) of the aborted fetuses were submitted from herds with a history of abortions attributable to neosporosis, and Neospora sp infection was identified as the causative agent in 101 of 232 (43.5%) of the abortions from these herds. Fewer aborted fetuses were submitted from the 12 herds that did not have a history of abortion attributable to Neospora sp; however, neosporosis was confirmed as a cause of abortion in 6 of these 12 herds and was identified as the causative agent in 12 of 34 (35.3%) abortions from these herds. The disease was widespread throughout the state (19/26 herds in our study). Available reproductive histories of cows that had abortions attributed to neosporosis were evaluated, and 4 cows were identified that twice aborted Neospora-infected fetuses. CLINICAL IMPLICATIONS: Abortion attributable to Neospora sp infections can be expected to be a continuing major cause of abortion in dairy herds with a history of neosporosis as well as in dairy herds that have a history of sporadic abortions, but for which Neospora sp infections have not been previously identified as a cause of abortion. Subsequent pregnancies in cows that abort a Neospora sp-infected fetus also are at risk of infection, suggesting that the immunity provided by an initial infection is inadequate to prevent repeat infection or that cows can be persistently infected with Neospora sp. PMID- 7559075 TI - VEGF-FLT1 receptor system: a new ligand-receptor system involved in normal and tumor angiogenesis. PMID- 7559076 TI - Frequent amplification of the cyclin E gene in human gastric carcinomas. AB - We searched for genetic alterations of the cyclin D1 and cyclin E genes in 45 human gastric carcinoma tissues. Expression of cyclin E mRNA and protein was also analyzed in eight of them by Northern and Western blots and immunohistochemical staining. The cyclin E gene was amplified 3-10 fold in seven gastric cancer tissues (15.6%), of which six were advanced gastric cancers. All of the cases with the cyclin E gene amplification displayed lymph node metastasis. Moreover, the case with the gene amplification overexpressed the cyclin E mRNA and protein. One of eight gastric cancer cell lines, MKN-7, shared the cyclin E gene amplification, and all of the gastric cancer cell lines expressed high levels of the cyclin E mRNA and protein even without gene amplification. Amplification of the cyclin D1 gene was not observed in any of the gastric carcinoma tissues or gastric carcinoma cell lines. These results suggest that the gene amplification and overexpression of cyclin E play an important role in the abnormal growth and progression of gastric carcinoma. PMID- 7559077 TI - Mutational analysis of CDKN2 (CDK4I/MTS1) gene in tissues and cell lines of human prostate cancer. AB - To study mutation of the CDKN2 gene in prostate cancer, samples from 51 Japanese patients and four human prostate cancer cell lines were examined by single-strand conformation polymorphism analysis and direct sequencing. Only one out of 51 (2%) patients revealed a mutation, which was a 24 bp deletion from the 5'-untranslated region to codon 3, resulting in loss of the initiation site. One of the four cell lines revealed a missense mutation, a GAC-->TAC (Asp-->Tyr) at codon 84. These results indicate that mutation of the CDKN2 gene is rare in prostate cancer and thus does not contribute significantly to the pathogenesis of human prostate cancer. Prostate cancer cell lines may acquire more frequent abnormality of the CDKN2 gene than tumor tissues. PMID- 7559078 TI - Detection of hepatic micrometastasis in pancreatic adenocarcinoma patients by two stage polymerase chain reaction/restriction fragment length polymorphism analysis. AB - Hepatic metastasis and retroperitoneal recurrence generally are considered to be the two primary modes of recurrence in pancreatic cancer. The goal of this study was to determine if patients with pancreatic adenocarcinoma have hepatic and peritoneal micrometastasis at operation. Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Liver tissue specimens were obtained from 30 patients (17 with pancreatic adenocarcinoma and 13 with other diseases) with a biopsy needle at operation. Peritoneal washings were obtained during operation from 20 patients with pancreatic adenocarcinoma. Two-stage polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to detect K-ras oncogene mutation at codon 12. Thirteen of 17 pancreatic adenocarcinoma patients had K-ras gene mutations in the liver, whereas all 13 patients with other diseases did not. However, only two of 20 pancreatic adenocarcinoma patients revealed K-ras gene mutation in peritoneal lavage fluids. These results indicate the feasibility of detecting hepatic micrometastasis in patients with pancreatic adenocarcinoma, and imply that PCR/RFLP analysis may be of value in the diagnosis, treatment and follow-up of hepatic metastasis of pancreatic adenocarcinoma. PMID- 7559079 TI - Elevated serum growth hormone accelerates gastric tumorigenesis in F344 rats after treatment with N-methyl-N-nitrosourea in their drinking water. AB - We examined the effects of growth hormone on tumorigenesis in F344 rats treated with N-methyl-N-nitrosourea (MNU). Four-week-old male F344 rats were exposed to 100 ppm MNU in their drinking water for 15 weeks. Thereafter Group II animals received 100 microCi/100 g body weight of 131I (radiothyroidectomy, Tx) injected i.p. and Group III rats were implanted with pituitary tumors (MtT) secreting growth hormone while Group I received no further treatment after MNU. Non carcinogen control animals received MtT, Tx or no treatment. Animals were killed at 39 weeks after starting MNU administration. Gastric tumors were present in 13 of 31 (43%), 15 of 32 (47%) and 17 of 32 (53%) rats in Groups I to III, respectively. All tumors were of well-differentiated type. Spinal cord tumors appeared in 15 of 31 (47%) in Group I, 10 of 32 (32%) in Group II and 10 of 32 (32%) in Group III, most being malignant schwannomas. Thymic lymphomas also appeared in 10 of 31 (32%), 5 of 32 (16%) and 6 of 32 (19%) animals in Groups I to III, respectively. There were no significant differences among the groups. However, tumors in Group III developed significantly earlier than in Groups I or II. This was mainly due to gastric tumors, and cumulative incidence curves for spinal cord tumors or thymic lymphomas were similar in all groups. The results indicate that gastric tumors induced by MNU in F344 male rats are influenced by elevated levels of growth hormone. PMID- 7559081 TI - Site-specific effects of testosterone propionate on the prostate of rat pretreated with 3,2'-dimethyl-4-aminobiphenyl: dose-dependent induction of invasive carcinomas. AB - It has been shown that testosterone propionate (TP) strongly promotes induction of invasive carcinomas in previously initiated accessory sex organs. In this study, in order to clarify the dose-dependence of this promotion, TP was given at 3 different levels (high, medium or low doses) using different sizes (2, 1 and 0.5 cm long) of Silastic tube for 40 weeks after administration of 3,2'-dimethyl 4-aminobiphenyl to male F344 rats. The data showed development of invasive carcinomas in the dorso-lateral and anterior prostate and in the seminal vesicle to be dose-dependent with the high dose of TP being most effective for tumor induction. Average levels of serum testosterone were approximately 800, 600, 300 and 150 ng/dl in rats given the high to low doses and in control rats, respectively. Development of neoplastic lesions in the ventral prostate demonstrated an inverse dependence on the dose of TP. These findings, together with previous data, suggest that the tumor-promoting potential of TP on rat prostate is unlikely to be simply due to its androgenic action and other factors should also be considered. PMID- 7559083 TI - Increased midkine gene expression in human gastrointestinal cancers. AB - Midkine (MK) is a product of a retinoic acid-responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non-cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas. PMID- 7559080 TI - Genetic regulation of development of thymic lymphomas induced by N-propyl-N nitrosourea in the rat. AB - To clarify the linkage between Hbb and Tls-1 (thymic lymphoma susceptible-1) loci and to investigate other loci concerned in thymic lymphomagenesis, the BUF/Mna rat, which is highly sensitive to the lymphomagenic activity of N-propyl-N nitrosourea (PNU), the WKY/NCrj rat, reported to be resistant, and their cross offspring were subjected to genetic analysis. F1 hybrid and backcross generations were raised from the 2 strains, and 6 genetic markers including Hbb were analyzed in individuals of the backcross generation. However, no linkage between Hbb and Tls-1 loci could be demonstrated since WKY rats also developed a high incidence of thymic lymphomas in response to PNU. Nevertheless, thymic lymphomas developed more rapidly and reached a larger size in the BUF rats. F1 rats expressed a rather rapid and large tumor growth phenotype, while the [(WKY X BUF) X WKY] backcross generation consisted of rats with either rapidly growing or slowly growing tumors. It was thus concluded that rapid development of thymic lymphomas is determined by a gene, provisionally designated Tls-3. Analysis of the relationship between 6 genetic markers and development of thymic lymphoma in the backcross generation demonstrated that the Tls-3 locus is loosely linked to the Gc locus, suggesting a possible location on rat chromosome 14. Tls-3 may not be identical with Tls-1 and other genes known to be relevant to thymic tumors, but its relationship with Tls-2 remains obscure. PMID- 7559082 TI - A maternal risk factor for mother-to-child HTLV-I transmission: viral antigen producing capacities in culture of peripheral blood and breast milk cells. AB - We examined the relationship between productivity of HTLV-I antigen-positive cells in cultured peripheral blood mononuclear cells (PBMC) and breast milk mononuclear cells (BMMC) and the incidence of mother-to-child transmission of HTLV-I. Among 61 cases of HTLV-I carrier mothers, 17 cases were revealed to produce large numbers of HTLV-I antigen-positive cells (high HTLV-I antigen producing mothers) whose positive rate was 9.6% in PBMC and 10.2% in BMMC, while the remaining 44 cases produced small numbers of HTLV-I antigen-positive cells (low HTLV-I antigen-producing mothers) whose positive rate was 0.3% in PBMC and 0.5% in BMMC. The HTLV-I transmission rate among children born to the high HTLV-I antigen-producing mothers was 37.5% (6/16 children from 11 mothers), while that of the low HTLV-I antigen-producing mothers was 3.2% (1/31 children from 20 mothers). The transmission rate of HTLV-I was significantly different between high and low HTLV-I antigen-producing mothers (P < 0.05). However, there was no positive relationship between anti-HTLV-I antibody titers and productivity of HTLV-I antigen-positive cells (P = 0.11). These results suggested that mother-to child transmission of HTLV-I might be influenced by a maternally determined factor to produce HTLV-I antigen-positive cells in PBMC and BMMC of HTLV-I carrier mothers. PMID- 7559084 TI - Induction of invasive growth in a gallbladder cancer cell line by hepatocyte growth factor in vitro. AB - To study the mechanism of invasion and metastasis of gallbladder cancer cells, we established a cancer cell line, GB-d1, from a metastatic lymphnode of poorly differentiated adenocarcinoma of the gallbladder. GB-d1 cells proliferate well in a dish culture and form small cystic cell clusters in a collagen gel containing 10% fetal bovine serum. A conditioned medium of human embryonic lung fibroblasts (HEL) stimulated the proliferation of GB-d1 cells and induced cell scattering in the dish culture. In the gel culture, the conditioned medium induced a transformation of the spherical clusters to arborizating colonies with tubular projections that mimicked an invasion of cancer cells into the surrounding tissue. Similar results were obtained when 10 ng/ml of human recombinant hepatocyte growth factor (h-rHGF) was added to the culture medium. The proliferative and morphological changes induced by the conditioned medium were inhibited by antiserum against h-HGF. HEL and human gallbladder stromal fibroblast-like cells produced substantial levels of HGF in the culture media, while GB-d1 did not produce any detectable level of HGF. These results suggest that HGF promotes the invasive growth of gallbladder cancer cells in vitro, and it was also suggested that stromal fibroblasts may play an important role in the invasive progression of gallbladder cancer in a paracrine fashion. PMID- 7559085 TI - Stimulatory effect of interleukin-1 alpha on proliferation through a Ca2+/calmodulin-dependent pathway of a human thyroid carcinoma cell line, NIM 1. AB - NIM 1 cells, a human thyroid cell line established from a patient with thyroid papillary adenocarcinoma, produce cytokines such as interleukin-1 alpha (IL-1 alpha) and granulocyte-colony stimulating factor. In the present study, we investigated the signal transduction pathway in the proliferation of NIM 1 cells evoked by IL-1 alpha. Incubation of NIM 1 cells with IL-1 alpha for 48 h increased the incorporation of 3H-thymidine (3H-TdR). The stimulatory effect of IL-1 alpha was evident at 0.01 ng/ml and the maximal effect was seen at 10 ng/ml. IL-1 alpha evoked an influx of 45Ca into NIM 1 cells within 3 min in a concentration-dependent manner (0.01-1 ng/ml). These stimulatory effects of IL-1 alpha on both 3H-TdR incorporation and 45Ca influx were similarly inhibited by nicardipine, an inhibitor of voltage-dependent Ca2+ channels, in a concentration dependent manner (10-1000 nM). The stimulatory effect of IL-1 alpha on 3H-TdR incorporation was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), an antagonist of calmodulin, but not by 1-(5-isoquinoline sulfonyl)-2 methylpiperazine (H-7), an inhibitor of protein kinase C. While the culture medium initially contained 0.75 mM Ca2+, inhibition of 3H-TdR incorporation by nicardipine and W-7 under these baseline conditions was also recognized. These results suggest that IL-1 alpha stimulates cell proliferation through a Ca2+/calmodulin-dependent pathway in NIM 1 cells. PMID- 7559086 TI - Enhancement of Ca(2+)-dependent endonuclease activity in L1210 cells during apoptosis induced by 1-beta-D-arabinofuranosylcytosine: possible involvement of activating factor(s). AB - Internucleosomal DNA fragmentation and morphological changes in nuclei typical of apoptosis were observed in L1210 cells incubated with 1.0 micrograms/ml of 1-beta D-arabinofuranosylcytosine (ara-C). To investigate the mechanisms involved, we examined the activities of endogenous endonucleases in nuclei and cytoplasm. Both fractions of control cells contained Ca(2+)-dependent endonuclease which was capable of mediating internucleosomal DNA fragmentation. The assay system using two kinds of target substrates, i.e., nuclear chromatin of CCRF-CEM cells and naked DNA purified from the same cells, revealed that the activity of Ca(2+) dependent endonuclease was enhanced in the crude nuclear extracts of cells treated with 1.0 microgram/ml of ara-C for 24 h or 48 h. The activity was extracted more easily from ara-C-treated cells than control cells without sonication of the nuclear fraction. On the other hand, in the cytoplasmic fraction of the cells, the activity towards naked DNA was unchanged, whereas that towards nuclear chromatin was clearly enhanced. These results suggest that internucleosomal DNA fragmentation induced by ara-C treatment is associated with enhancement and activation of constitutively expressed Ca(2+)-dependent endonuclease in L1210 cells. PMID- 7559090 TI - Interventional trial for colorectal cancer prevention in Osaka: an introduction to the protocol. AB - We established a protocol for an interventional randomized controlled trial for prevention of colorectal cancer by attaching special importance to feasibility. The subjects were patients with multiple colorectal tumors. Two regimens were formulated for prevention of colorectal cancer. One was dietary guidance alone (Regimen I), and the other was dietary guidance plus eating wheat bran biscuits (Regimen II). The main end points of the trial were examinations for recurrence of colorectal tumors after 2 and 4 years. The target number of patients was 200 in total, i.e. 100 for each group. During the 18 months from the beginning of recruiting of subjects (up to November 1994), 28 (97%) of the 29 patients recruited for Regimen I and 32 (97%) of the 33 patients recruited for Regimen II agreed to participate in the trial. The trial progressing well. PMID- 7559087 TI - Tumor cell attachment to laminin promotes degradation of the extracellular matrix and cell migration in high-metastatic clone cells of RCT sarcoma in vitro. AB - We investigated the roles of extracellular matrix proteins, laminin and fibronectin, in promoting invasiveness through the extracellular matrix in high metastatic [RCT(+)] clone cells established from poorly differentiated murine RCT sarcoma in C3H/He mice. Laminin stimulated the type IV collagenolytic activity of RCT(+) cells. After more than 6 h of incubation, the type IV collagenolysis of the cell-conditioned medium was significantly higher in laminin-treated groups compared with the control. The migration activity of RCT(+) cells was stimulated by laminin. However, fibronectin did not influence the type IV collagenolysis or cell migration in this clone cell. The amino acid sequence YIGSR, which is derived from laminin, inhibited the laminin-mediated cell attachment and the laminin-promoted type IV collagenolysis, as well as cell migration of RCT(+) cells. RGD derived from fibronectin did not influence the cell attachment to laminin or Matrigel in this clone. In the invasion assay employing a Matrigel coated filter in a Boyden chamber, YIGSR showed greater inhibition of invasion through the Matrigel than did RGD with RCT(+) cells. YIGSR might inhibit the promoted-matrix degradation and cell migration in response to the cell attachment to laminin by competing with laminin for binding to cell surface laminin receptor. We suggest that laminin-mediated cell attachment to the extracellular matrix may play a role in promoting the matrix degradation and cell migration during metastatic cascades. PMID- 7559089 TI - Enzyme-linked immunosorbent assay of pro-gastrin-releasing peptide for small cell lung cancer patients in comparison with neuron-specific enolase measurement. AB - Our previous study demonstrated that pro-gastrin-releasing peptide(31-98), or ProGRP, is a specific tumor marker in patients with small cell lung carcinoma (SCLC). Using a newly developed, highly sensitive enzyme-linked immunosorbent assay (ELISA) for ProGRP, we analyzed 1,446 samples including those obtained from 478 lung cancer patients to evaluate the clinical usefulness of this ELISA. Several properties indicated that ProGRP is a useful tumor marker for SCLC. First, ProGRP was specifically elevated in SCLC patients. In non-SCLC patients and patients with non-tumorous lung diseases, its serum level was very rarely elevated. Secondly, ProGRP was a reliable marker, in terms of the marked elevation of serum ProGRP levels in SCLC patients. Thirdly, serum ProGRP levels were elevated in SCLC patients even at a relatively early stage of this disease. Fourthly, changes in the serum ProGRP level showed an excellent correlation with the therapeutic responses in SCLC patients. Neuron-specific enolase (NSE) is accepted as a tumor marker of SCLC patients. With the aim of comparing ProGRP and NSE as tumor markers for SCLC patients, we measured serum NSE levels in all samples collected in the present study. We found that ProGRP was superior to NSE in terms of sensitivity, specificity and reliability. Therefore, we consider that ProGRP can play a major role as a clinical tumor marker for SCLC patients. PMID- 7559088 TI - T-cell receptor gene structures of HLA-A26-restricted cytotoxic T lymphocyte lines against human autologous pancreatic adenocarcinoma. AB - We isolated two cytotoxic T lymphocyte (CTL) lines, which were independently obtained by mixed lymphocyte-tumor cell culture from tumor-infiltrating lymphocytes of a patient with pancreatic adenocarcinoma. Both lines behaved identically in all the functional aspects tested and appeared to be HLA-A26 restricted. We analyzed their T cell receptor (TCR) gene structures, including V (D)-J junctional sequences, which are unique to each T-cell clonotype and contribute to TCR diversity. Each line consisted of a clonal T-cell expressing V alpha 18 and V beta 7. The alpha chain gene was composed of V alpha 18/J alpha F/C alpha and the beta-chain gene, of V beta 7.1/D beta/J beta 1.4/C beta 2. The sequences were all in-frame and therefore should yield functional transcripts. The junctional sequences were identical between the two lines. These data suggested that the two CTL clones having the same CDR3 had descended from a common precursor lymphocyte. The clonal expansion of CTL lines with the identical CDR3 implies that they are directed against the same tumor antigen, which seemed to be immunologically dominant in the specific interaction between the CTL and the autologous pancreatic adenocarcinoma. PMID- 7559091 TI - Changes in radiation sensitivity of human osteosarcoma cells after p53 introduction. AB - Human osteosarcoma SAOS-2 cells, which have a deletion in p53 gene, were transfected with plasmid pMSVneop53 containing human p53 cDNA and neomycin resistance gene. Three clones (SAOS-MC10, SAOS-MC11 and SAOS-MC43) among 60 clones expressed p53 mRNA. No p53 protein was observed in SAOS-MC10, while SAOS MC11 and SAOS-MC43 produced p53 protein. The molecular weight of p53 protein in SAOS-MC43 was lower than that in SAOS-MC11, SAOS-MC11 and SAOS-MC43 were more sensitive and more resistant, respectively, to ionizing radiation than the parental SAOS-2. We suggest that exogenous p53 protein might be one of the factors determining cellular radiosensitivity. PMID- 7559092 TI - Expression of MAGE-1 gene by esophageal carcinomas. AB - Expression of the MAGE genes encoding tumor-rejection antigens on HLA-A1 and Cw1601 recognized by cytotoxic T lymphocytes was investigated in esophageal carcinomas at the mRNA level by the semiquantitative reverse transcription polymerase chain reaction method. MAGE-1 and -2 genes, but not MAGE-3, -3/-6 and 4a/-4b genes, were expressed in substantial proportions of the primary esophageal carcinomas and their metastatic lymph nodes. The proportion of MAGE-positive samples in the primary esophageal carcinomas correlated with the T factor of the TNM classification (pT1: 2 of 12 tumors, pT2: 1 of 6, pT3: 12 of 29, and pT4: 7 of 18). These results have important implications for specific immunotherapy of esophageal carcinomas using MAGE-1 gene product. PMID- 7559094 TI - Inhibitory effects of medroxyprogesterone acetate on mouse endometrial carcinogenesis. AB - The present study was undertaken to examine the effects of cyclic administration of low-dose progestogen on endometrial carcinogenesis in mice. A total of 115 female ICR mice, 10 weeks of age, were divided into four experimental and control groups. Mice in groups 1-3 received laparotomy and were injected with N-methyl-N nitrosourea (MNU) solution at a dose of 1 mg/100 g body weight to the left uterine tube and with normal saline to the right uterine tube. From one week after the MNU exposure, groups 1 and 2 were given 5 ppm 17 beta-estradiol (E2) containing diet throughout the experiment. Mice in group 1 received 5 s.c. injections of medroxyprogesterone acetate (MPA) (2 mg/mouse) at intervals of 4 weeks from week 7. Group 3 was treated with MNU/normal saline alone. Group 4 consisted of mice treated with MPA alone. At the termination of the experiment (week 30), all animals were killed and autopsied for pathological examinations. It was found that adenocarcinomas and preneoplastic lesions developed in the bilateral uterine corpora in mice of groups 1-3. MPA treatment significantly decreased the weight of the uterine corpus (P < 0.05) and the incidences of endometrial adenocarcinoma and atypical or adenomatous (P < 0.001) but not cystic glandular hyperplasias in the MNU/E2-treated groups. Additionally, MPA treatment tended to decrease the proliferating cell nuclear antigen-labeling index in endometrial glandular cells. These data indicate that MPA, even at low dose, has an inhibitory effect on mouse endometrial carcinogenesis induced by MNU and E2. PMID- 7559093 TI - Hepatocyte growth factor enhancement of preneoplastic hepatic foci development in rats treated with diethylnitrosamine and N-ethyl-N-hydroxyethylnitrosamine. AB - Effects of hepatocyte growth factor were investigated in a two-stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N ethyl-N-hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 micrograms/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S-transferase placental form-positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two-thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor. PMID- 7559095 TI - Allelic frequency of p53 gene codon 72 polymorphism in urologic cancers. AB - Alterations in the p53 tumor suppressor gene appear to be important in the development of many human tumors. The wild-type p53 gene has a polymorphism at codon 72 that presents the arginine (CGC) or proline (CCC) genotype, which recently has been reported to be associated with genetically determined susceptibility to smoking-related lung cancers. To determine whether this p53 genotype influences individual risk of urologic cancer and/or its progression, we used polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) analysis to assay the allelic frequencies of this polymorphism in 85 renal cell carcinoma patients, 151 urothelial cancer patients, 33 testicular cancer patients, 28 prostatic cancer patients and 56 patients without neoplastic disease. The allelic distributions of the three genotypes (Arg/Arg, Arg/Pro, Pro/Pro) in patients with renal cell carcinoma (29.4%, 55.3%, 15.3%), urothelial cancers (45.7%, 39.7%, 14.6%), testicular cancer (45.4%, 48.5%, 6.1%) or prostate cancer (42.9%, 50.0%, 7.1%) did not differ significantly from those in the normal controls. However, Pro/Pro genotype in renal cell carcinoma and urothelial cancer (smoking-related cancers) was more frequent than that in prostate cancer and testicular cancer (smoking-unrelated cancers) with borderline significance (P = 0.0881). There was no particular correlation between frequency of the three genotypes and grade or stage of each type of tumor. The association of genetic predisposition to urologic cancers with p53 gene codon 72 polymorphism is not so clear as the previous study of Japanese lung cancer patients, but this polymorphism may play some role in urothelial cancers and renal cell carcinoma, in which smoking is an epidemiological risk factor. PMID- 7559096 TI - Point mutations of ras and Gs alpha subunit genes in thyroid tumors. AB - We studied 43 thyroid tumors including 5 adenomatous goiters, 7 follicular adenomas, 22 papillary carcinomas, and 9 medullary carcinomas with regard to the presence of point mutations in the genes of Gs alpha subunit (Gs alpha), Gi2 alpha subunit (Gi2 alpha), H-ras, K-ras, and N-ras by a polymerase chain reaction direct sequencing method. An adenomatous goiter and a follicular adenoma showed double mutations at codon 227 and 231, and 4 papillary carcinomas showed mutation at codon 231 of the Gs alpha gene. An adenomatous goiter, a follicular adenoma, and a papillary carcinoma showed a missense mutation in codon 13 of the K-ras gene. There were no such missense mutations of these G-protein or ras genes in medullary carcinomas. These data indicate that the genetic events involved in the oncogenesis of parafollicular C-cells are different from those of thyroid follicular cells, in which missense mutations of Gs alpha and ras genes seem to play important roles in tumorigenesis. PMID- 7559097 TI - Apoptosis in gastric carcinomas and its association with cell proliferation and differentiation. AB - The significance of apoptosis in human gastric carcinomas was investigated in comparison with proliferative activity and p53 accumulation, using an in situ DNA nick end labeling method and immunohistochemistry for both Ki-67 antigen and p53 protein. Apoptotic labeling indices (LI) of 51 differentiated carcinomas (21 of early and 22 of advanced stage) were significantly lower than for 33 undifferentiated tumors (9 of early and 24 of advanced stage) (P < 0.05). In both types, apoptotic LI of advanced stage lesions were significantly higher than for the early stage cases (P < 0.005, P < 0.03). The distribution of apoptotic cells was different from that of Ki-67-positive cells, generally exhibiting an inverse correlation for areas of predominance. In contrast, there was no significant correlation between p53 immunoreactivity and either apoptotic LI or Ki-67 LI. It is concluded that in human gastric carcinomas the susceptibility to apoptosis is related to tumor cell differentiation and depth of invasion, and may play a role in selection of clonal subpopulations with high growth potential. PMID- 7559098 TI - Activation of protein kinase C by mycobacterial cord factor, trehalose 6 monomycolate, resulting in tumor necrosis factor-alpha release in mouse lung tissues. AB - Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed. PMID- 7559099 TI - Flat serrated adenomas and flat tubular adenomas of the colorectal mucosa: differences in the pattern of cell proliferation. AB - In the present work we have investigated the cell proliferation pattern of flat serrated adenomas and flat tubular adenomas. For this purpose tissue sections from 23 consecutive flat serrated adenomas and 22 consecutive flat tubular adenomas of the colorectal mucosa were challenged with MIB1, a monoclonal antibody directed against a proliferation-related antigen. The results (including semi-quantitative studies) demonstrated that, whereas flat serrated adenomas had a high cell proliferation at the lower part of the crypts, flat tubular adenomas had a high cell proliferation in the upper part of the crypts. In serrated adenomas with invasive adenocarcinoma, high cell proliferation was demonstrated both at the lower portion of the crypts and in the subjacent submucosa. This suggests that the cells of the lower portion of the crypts in serrated adenomas are truly neoplastic, with the capacity to evolve into invasive growth. The difference in cell proliferation between the two types of flat lesions reported here is a new argument in favor of the classification of flat serrated adenomas as a novel and independent type of neoplastic change of the colorectal mucosa. PMID- 7559101 TI - In vivo antitumor activity of hexamethylmelamine against human breast, stomach and colon carcinoma xenografts. AB - We have evaluated the antitumor activity of Altretamine (hexamethylmelamine, HMM) on human carcinoma xenografts serially transplanted in nude mice. Five human breast carcinoma xenografts, MX-1, T-61, MCF-7, R-27 and Br-10, were inoculated subcutaneously into female nude mice. Two human stomach carcinoma xenografts, SC 1-NU and St-4, and three human colon carcinoma xenografts, Co-3, Co-4 and Co-6, were inoculated subcutaneously into male nude mice. One pellet of 17 beta estradiol (0.1 mg/mouse) was inoculated subcutaneously in the mice transplanted with MCF-7 when the tumors were inoculated. HMM was administered per os daily for 4 weeks. MX-1 and T-61 tumors regressed completely after treatment with HMM at a dose of 75 mg/kg (the maximum tolerated dose: MTD) for MX-1 and 25 mg/kg for T 61. Br-10 was sensitive, whereas MCF-7 and R-27 were resistant to HMM at its MTD. HMM exerted the most potent antitumor effect against T-61. Against MX-1, it exerted an antitumor effect equivalent to that of cisplatin or cyclophosphamide. In addition, this agent was effective against all stomach and colon carcinoma xenografts, in particular St-4 (T/C% = 10.7: the mean tumor weight of treated group/the mean tumor weight of control group) and Co-3 (T/C% = 31.5%) which are insensitive to presently available agents. HMM seems worthy of further clinical investigation as a candidate agent to treat breast, stomach, colon and other carcinomas. PMID- 7559100 TI - Dipyridamole combined with tumor necrosis factor-alpha enhances inhibition of proliferation in human tumor cell lines. AB - In the search for cytokines whose antiproliferative action could be enhanced by combination with dipyridamole, 2,6-bis(diethanolamino)-4,8 dipiperidinopyrimido[5,4-d]pyrim idine, the combination of tumor necrosis factor alpha (TNF-alpha) with this agent was evaluated in various human tumor cell lines. Inhibition of the proliferation of human melanoma cell lines MM-1CB and HMV-1 by TNF-alpha (1-10(2) U/ml) was enhanced in culture dishes by combination treatment with dipyridamole (0.1-10 microM). The enhancement effect was also detected in other tumor cell lines: T98 (glioma), SCC-1CB (squamous cell carcinoma), HAC-2 (ovarian clear-cell carcinoma), HLE (hepatoma), HEC-1 (endometrial adenocarcinoma) and HOC-21 (ovarian serous cystadenocarcinoma). The incorporation of [14C]amino acids and [3H]uridine into acid-insoluble cell materials in the combination-treated cells was not significantly different from that in cells treated with TNF-alpha or dipyridamole. However, the incorporation of [3H]thymidine was specifically inhibited in all cell lines examined after more than 12 h of the TNF-alpha and dipyridamole combination treatment, although neither agent alone inhibited this incorporation. On the other hand, the growth of tumors induced by the injection of MM-1CB and HMV-1 cells into nude mice was more markedly inhibited by the subcutaneous administration of TNF-alpha in combination with orally administered dipyridamole than by either agent alone. The results presented suggested that dipyridamole is beneficial in assuring the effectiveness of anti-cancer cytokine therapy. PMID- 7559102 TI - A new water-soluble camptothecin derivative, DX-8951f, exhibits potent antitumor activity against human tumors in vitro and in vivo. AB - CPT-11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a pro-drug that is converted to an active metabolite, SN-38, in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble and non-pro-drug analog of camptothecin, DX-8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti-proliferative activity of DX 8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN-38 or SK&F 10486-A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX-8951f administered i.v. at 4-day intervals against human gastric adenocarcinoma SC-6 xenografts was greater than that of CPT-11 or SK&F 10486-A. Moreover, it overcame P-glycoprotein-mediated multi-drug resistance. These data suggest that DX-8951f has a high antitumor activity and is a potential therapeutic agent. PMID- 7559105 TI - Quantitative morphology of the subchondral plate of the tibial plateau. AB - The object of the present investigation was to measure the thickness distribution of the subchondral plate of the tibial plateau. The data were obtained by computerised image analysis of serial sections. The measured values revealed a marked difference in the thickness between the various regions of the joint surface. Thinner zones (100-300 microns) are found in the peripheral region near the margin of the tibial plateau. Thickness maxima (up to 1500 microns and more) are to be seen at the centres of the joint surfaces. The relationship between the thickness distribution of the subchondral plate and information about the stress distribution of this particular joint surface support the conclusion that the morphology of the subchondral plate of the tibial plateau is determined by the function of the joint. PMID- 7559106 TI - The specialised structure of crypt epithelium in the human palatine tonsil and its functional significance. AB - Material from 25 human palatine tonsils was studied by light microscopy, immunocytochemistry, scanning and transmission electron microscopy. Special attention was focused on the structure of the epithelium lining the tonsillar crypts in the context of its ascribed immunological functions. This epithelium was not uniform and contained patches of stratified squamous nonkeratinising epithelium and patches of reticulated sponge-like epithelium. The degree of reticulation of the epithelial cells and the infiltration of nonepithelial cells varied. Reticulated patches were associated with disruptions in the continuity of basement membrane, and often also with desquamation of the upper cell layers, and contained numerous small blood vessels. The epithelial cells showed considerable variation in their morphology when surrounded by infiltrating cells. The rearrangement of their cytoskeleton and redistribution of desmosomal contacts indicate the responsiveness and dynamic nature of such epithelium. Cytoplasmic glycogen granules, located in the upper strata, suggest the possibility of energy demanding functions such as absorption and secretion. The numerous membrane coating granules may have contributed to cell membrane thickening and possibly also to tonsillar mucosal protection. Some areas contained a few keratohyalin granules but there was little evidence of keratinisation. The presence, and sometimes the predominance, of nonepithelial cells was characteristic of the reticulated epithelium. T and B cells often infiltrated the whole epithelial thickness, and many plasma cells were located around intraepithelial vessels, while macrophages and interdigitating cells showed a patchy distribution. It is proposed that the major functions of the reticulated epithelium are: (1) to provide a favourable environment for the intimate contact between the effector cells of immune responses; (2) to facilitate direct transport of antigens; (3) to synthesise the secretory component continually; and (4) to contain a pool of immunoglobulins. Thus the reticulated epithelium lining the tonsillar crypts represents a specialised compartment, important in the immunological functions of the tonsil as a whole. PMID- 7559103 TI - Combination assay of urinary beta-core fragment of human chorionic gonadotropin with serum tumor markers in gynecologic cancers. AB - Ectopic production of the immunoreactive beta-subunit of human chorionic gonadotropin (IR-hCG beta) by gynecologic malignancies has been well recognized, but IR-hCG beta has not yet been established as a clinically useful tumor marker, except for germ cell tumors. We measured the concentrations of IR-hCG beta related molecules, intact hCG, free hCG beta, and beta-CF, in the sera and urine of patients with various gynecologic cancers (cervical, endometrial, and ovarian cancers) to assess their clinical usefulness as a tumor marker in comparison with serum tumor markers such as CEA, SCC, CA125, and CA19-9. The highest incidence of IR-hCG beta was obtained in the assay for beta-CF in the urine, with positive rates of 47.7% (94 of 197) for cervical, 37.8% (14 of 37) for endometrial, and 84.4% (38 of 45) for ovarian cancers with a cut-off value of 0.2 ng/mg of creatinine. In cervical cancer, there was no significant correlation between the concentrations of urinary beta-CF and serum SCC, and 57.9% (114 of 197) of the patients were detected by the combination assay of these tumor markers. Serial determination in 22 cervical cancer patients with elevated urinary beta-CF level prior to therapy showed that its level decreased after successful treatment, but 4 of 5 patients with persistent or recurrent disease had elevated levels of urinary beta-CF. All of the ovarian cancer patients examined were detected by the combination assay of urinary beta-CF and serum CA125. The levels of urinary beta CF showed little correlation with those of the serum tumor markers, indicating the usefulness of the combination assay of urinary beta-CF with serum tumor markers for detecting cervical and ovarian cancers. PMID- 7559107 TI - The arrangement of gut-associated lymphoid tissues and lymph pathways in the koala (Phascolarctos cinereus). AB - Gut-associated lymphoid tissues are poorly developed in koalas. They comprise paired caecocolic lymphoid patches, and a few small mesenteric lymph nodes. The patches lie opposite one another in the lateral gut wall at the junction of the ileum, caecum and proximal colon. The lymphoid parenchyma of the patches consists of a layer of nodules and internodular parenchyma in the submucosa. Apoptosis is common in the nodules. The mucosa and lymphoid tissue of each patch is continuous over a caecocolic recess, formed by the coalescence of laminae which extend along the large intestine. Lymph sinuses between and beneath the lymphoid nodules are continuous with efferent lymph vessels in the submucosa. These then enter 2-4 small lymph nodes at the root of the mesentery. The paucity of lymphoid tissue associated with the gut may be related to germicidal activity in the Eucalypt leaves eaten by the koala. PMID- 7559108 TI - Growth of secondary hair follicles of the Cashmere goat in vitro and their response to prolactin and melatonin. AB - The isolation and viability in vitro of anagen secondary hair follicles of the Cashmere goat were studied. Isolated hair follicles were used to determine the effects on hair shaft elongation, of prolactin and melatonin, hormones considered to influence hair follicle growth and activity in vivo. Intact hair follicles were isolated from the dermal layer of the skin singly or in groups using watchmakers' forceps under a dissecting microscope. The isolated follicles were maintained floating in Williams E medium. The medium was supplemented with 1 of 6 concentrations of ovine prolactin (0, 50, 200, 400, 800 and 4000 micrograms/l) for the culture of hair follicles isolated during July and August, and with 1 of 5 concentrations of melatonin (0, 50, 150, 300, 600 ng/l) for the culture of hair follicles isolated during September and October. There was clear evidence of DNA synthesis, observed by autoradiography, in matrix cells of freshly isolated follicles incubated for 6 h in the presence of [methyl-3H]-thymidine. Similar measurements after 96 h of maintenance indicated a marked reduction in the incorporation of [methyl-3H]-thymidine in matrix cells of the follicles studied. Prolactin and melatonin were shown to have a stimulating effect on hair shaft elongation of secondary follicles during 24 h periods of measurement and cumulatively over 120 h. Maximum hair follicle growth was observed in follicles exposed to 400 micrograms/l of prolactin and follicles exposed to 300 ng/l of melatonin. The number of follicles remaining viable during each 24 h measuring period was not affected by prolactin, but was significantly reduced by melatonin treatment after 96 h of maintenance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559110 TI - Intrafusal motor innervation: a quantitative histological analysis of tenuissimus muscle spindles in the cat. AB - A quantitative analysis of the motor innervation of intrafusal muscle fibres is described, based on teased silver-impregnated spindles of the tenuissimus muscle of the cat. Included in the analysis are the number and distribution of intrafusal branches of both skeletofusimotor (beta) and purely fusimotor (gamma) axons, and the form of their endings. The number of axonal branches per spindle was found to follow binomial probability distributions, as had previously been shown for the afferent axons. There was a strong correlation between the numbers of gamma intrafusal branches and afferent axons, but none for the intrafusal branches of beta axons. The degree of segregation of gamma input to bag2 and chain fibres was assessed and was found, among other things, to be related to the presence of secondary sensory endings in the same pole. In this and other respects it did not appear to have the properties that would be expected if independent activation of the bag2 and chain fibres were to be functionally important. Morphometric analysis of the motor endings supplied to bag2 or chain fibres by gamma axons revealed some differences between those of intrafusal branches with segregated as opposed to unsegregated distributions, but this cannot be taken as evidence of more than one type of static gamma motoneuron because of the likely contribution of other influential factors such as fibre size. Finally, the relevance of studies on intrafusal motor innervation to the concept of the motor unit and its development are discussed. PMID- 7559109 TI - Continuous darkness and continuous light induce structural changes in the rat thymus. AB - It is known that neuroendocrine responses to environmental stimuli, such as light, can influence immune responses through the pineal gland. It is also known that periods of constant darkness and constant light cause stimulation and inhibition of melatonin secretion from the pineal gland, successively. In this study, we provide experimental evidence that changes in the rhythm of the photoperiod have considerable effects on thymic structure of the rat. Male albino Wistar rats were divided into 3 groups. Group 1 rats were kept in a dark room, group 2 in a room under a bright artificial light and group 3 (control) animals were exposed to a 12:12 h light: dark cycle. All animals were killed after 4 wk. In group 1, thymus weight increased by 315%, the increase in volume affecting the medulla (cortex 190%, medulla 655%). The absolute number of epithelial cells and lymphocytes increased both in the cortex and medulla. Thymic cortical epithelial cells were hypertrophied and contained numerous large clear vesicles. Perivascular spaces were enlarged. In group 2 thymus weight decreased by 53%, the reduction in volume affecting mainly the cortex (cortex 61%, medulla 27%). The absolute numbers of cortical epithelial cells and lymphocytes were decreased, and pyknotic lymphocyte nuclei were frequent both in the cortex and medulla. It is concluded that constant darkness causes hypertrophy and increased cellularity of the thymus, while constant light causes involution of the thymus and death of lymphocytes. These changes possibly reflect the well known immunostimulatory effects of melatonin acting directly or indirectly, on the thymic lymphocytes and epithelial cells. PMID- 7559104 TI - The harderian gland: a tercentennial review. AB - The harderian gland was first described in 1694 by Johann Jacob Harder (1656 1711). It occurs in most terrestrial vertebrates and is located within the orbit where, in some species, it is the largest structure. It may be compound tubular or compound tubuloalveolar, and its secretory duct is usually morphologically distinct only after leaving the substance of the gland to open on the surface of the nictitating membrane. The tubules of the gland are formed of a single layer of columnar epithelial cells surrounded by myoepithelial cells. The chief product(s) of the gland varies between different groups of vertebrates, and epithelial cells possess granules or vacuoles whose contents may be mucous, serous or lipid. In rodents, the gland synthesises lipids, porphyrins and indoles. In the case of lipid vacuoles, the gland is unusual in releasing these by an exocytotic mechanism. It is unclear whether the gland can act both as an exocrine and endocrine organ. There is control of gland structure and synthesis through a variety of humoral agents, including gonadal, thyroid and pituitary hormones; in addition there is a rich autonomic innervation and many neuropeptides have been identified. The proposed functions of the gland are remarkably diverse and include the gland being (1) a source of 'saliva', (2) a site of immune response, (3) a photoprotective organ, (4) part of a retinal pineal axis, (5) a source of pheromones, (6) a source of thermoregulatory lipids, (7) a site of osmoregulation, and (8) a source of growth factors. The gland is discussed in terms of its embryology and phylogeny, and in relation to ecological variables. Several goals of future research are identified. PMID- 7559111 TI - The fibre type composition of the rabbit latissimus dorsi muscle. AB - The fibre type distribution has been mapped in the latissimus dorsi muscle of the Dutch rabbit. Using the myosin ATPase stain, a distinct border was found to run in a cranial to caudal direction, which effectively divided the muscle into 2 segments of different fibre type proportions. Although both segments contained mostly fast twitch fibres, the medial areas were found to contain approximately 10-20% slow (i.e. type I) fibres while the lateral portions contained very few, if any, slow fibres. Significantly fewer type IIa fibres were also found in the lateral areas of the muscle. These histochemical findings were confirmed by the use of the reverse transcriptase polymerase chain reaction, which demonstrated that more messenger RNA of the slow myosin heavy chain was found in the medial regions compared with the lateral segment. These results demonstrate the importance of choosing well defined sampling sites when evaluating regimes designed to transform this heterogeneous muscle for use in subsequent myoplasty procedures. PMID- 7559112 TI - p185neu is expressed in yolk sac during rat postimplantation development. AB - We have shown that the neu oncogene product (p185neu) is not present in the rat embryo before organogenesis. However, coincident with the onset of organogenesis, p185neu was detected in neural and connective tissue as well as in the secretory epithelium as was described by Kokai et al. (1987). In addition, p185neu is also expressed in the rat visceral yolk sac (VYS) endodermal cells but not in the mesenchymal and mesothelial layers of the same structure nor in the amnion. The first detectable sign of p185neu expression in VYS was found at d 11 of gestation and the levels of protein increased towards the end of pregnancy. In the yolk sac carcinoma (YSC), which is considered to be the malignant counterpart of the rat yolk sac, p185neu was observed only within columnar epithelial cells (the visceral component of the neoplasm) while parietal endoderm-like cells were devoid of detectable protein. From d 9 of pregnancy up to delivery some of the trophoblastic giant cells also showed a faint to moderate immunoreactivity. Results are presented which would indicate a possible role of p185neu in rat embryogenesis. PMID- 7559114 TI - Spinalis capitis, or an accessory paraspinous muscle? AB - A unilateral muscle, the location and dimensions of which do not exactly conform to existing descriptions, was found during dissection of the suboccipital region. The muscle in question extended from the spine and transverse process of the 6th cervical vertebra to the base of the skull. At its rostral attachment it blended with the insertion of the left rectus capitis posterior minor muscle on the inferior nuchal line. The caudal attachment arched over the semispinalis cervicis, separated from that muscle by an extensive venous complex. Medially, along the length of the muscle, weak fascial attachments to the ligamentum nuchae were present. Arterial branches from the occipital artery entered the muscle near its rostral end and nerve fibres and vascular channels from the lower cervical region entered the deep surface of the muscle. PMID- 7559113 TI - Variations in the thoracic duct of the Japanese monkey (Macaca fuscata). AB - Variations in the thoracic duct of Japanese monkeys were compared with previous reports on the human (Japanese) thoracic duct. The most striking difference between man and the Japanese monkey was that in the majority of the monkeys examined the right thoracic duct ascended without crossing to the left side whereas in man the upper portion of the right duct crosses over to the left. Although most thoracic ducts in man, other primates and domestic animals reportedly enter the left venous angle, about half of the ducts of Japanese monkeys enter the right venous angle. A ring configuration in the thoracic duct was found in 24.4% of Japanese monkeys. This was less frequent than human Japanese. Three out of 41 monkeys had an intercalated lymph node, situated between the 10th and 11th and 12th thoracic vertebrae; this has only occasionally been reported in man. PMID- 7559116 TI - Studies of the lymphatic vessel-associated neurons in the intestine of the guinea pig. AB - A unique group of neurons in the submucous plexus of the gastrointestinal tract in guinea pigs was studied using (1) Nissl staining and an enzyme histochemical technique for acetylcholinesterase (AChE), (2) immunohistochemical methods for the localisation of neuron specific enolase (NSE) and neuropeptides, including vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), calcitonin gene-related peptide (CGRP), leu-enkephalin (leu-ENK), neuropeptide (NPY) and cholecystokinin (CCK), (3) a fluorescence tracer technique involving the intraperitoneal (i.p.) injection of fluorogold, and (4) normal electron microscopy. The results showed that these neurons were distributed singly or in groups in the submucosa. They were closely adherent to the outer walls of lymphatic vessels, some appearing to protrude into the lumen. Ultrastructurally, only a thin layer of basal lamina and some collagen fibrils intervened between the endothelia of the lymphatic vessels and these neurons. Based on their synaptic contacts and the features of their content of synaptic vesicles, at least 4 types of axon terminal forming synaptic contacts with the 'lymphatic vessel-associated neurons' (LV-AN) were identified. The sources of origin of these terminals remains uncertain although it is speculated that they may be derived from vagal efferents or of intrinsic origin from the neighbouring neurons. All the LV-AN showed AChE and NSE positive reactions, but only a varying number were positive for VIP, SP, SOM, ENK, CGRP, CCK or NPY. The LV-AN were labelled with fluorogold injected i.p.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559115 TI - The arterial pattern at the base of arhinencephalic and holoprosencephalic brains. AB - The mechanisms by which the anatomical variations of the circle of Willis develop is considered to be related to haemodynamic factors, i.e. the differential growth of the various parts of the brain will continuously change the haemodynamic demands and consequently the flow patterns in the cerebral arteries. It is therefore to be expected that, if a selected part of the brain does not develop, the change in the haemodynamic demand will affect the development of some cerebral arteries. Consequently the arteries at the base of 2 arhinencephalic and 8 holoprosencephalic brains were studied in conjunction with the brain malformations. The defects of holoprosencephaly are believed to arise from a failure of the prosencephalon to separate fully into the telencephalon and diencephalon and become manifest at the time that the prosencephalon normally starts to separate into the hemispheres, i.e. 28-34 d p.c. Arhinencephalic brains are fully diverticulated. There is only a partial or complete agenesis of the olfactory tracts and bulbs. The defect causing arhinencephaly starts at 43 d p.c. In the arhinencephalic brains no particular vascular abnormalities were found. However, at the base of the holoprosencephalic brains no complete circle of Willis was present; the anterior part was lacking and was replaced by anterior branches which emerged unilaterally or bilaterally from the internal carotid artery. The choroidal arteries were of very large calibre and ran to the highly vascularised wall of the dorsal cyst which is usually present in holoprosencephalic brains. In contrast to the anterior part, the posterior arterial pattern was almost identical to the posterior part of the circle of Willis of normal brains. The basic vascular patterns found in the holoprosencephalic brains displayed the features of Padget's developmental stages 2 and 3 of the cerebral vasculature, i.e. the pattern that has normally developed within 28-40 d p.c. The further modification of this pattern could largely be understood from the functional demand imposed on the circulation by the enlarged anterior choroidal arteries. Because the development of the anterior part of the circle of Willis precedes the developmental derangement causing arhinencephaly, a complete circle was found in these brains. PMID- 7559117 TI - Immunophenotypic features of epiplexus cells and their response to interferon gamma injected intraperitoneally in postnatal rats. AB - The expression of major histocompatibility complex (MHC) antigens (class I and II), type 3 complement receptor (CR3) and leucocyte common antigen (LCA) was examined in epiplexus cells in rats of different ages. The cells exhibited intense immunoreactivity with the monoclonal antibody OX-42 which recognizes CR3 receptors. In early postnatal rats (1 d), the immunolabelled cells were mostly round but with increasing age (7 wk), they assumed a ramified or elongated form. The expression of LCA marked by the monoclonal antibody OX-1 followed a similar staining pattern. Class I MHC antigen expression was also demonstrated in some epiplexus cells using the monoclonal antibody OX-18 but they were less numerous than the OX-42 or OX-1 positive cells. Only sporadic OX-6 positive cells were observed in postnatal rats but they showed a marked increase in number in adult rats, suggesting an upregulation of class II MHC antigens with age. The expression of MHC class II antigens was vigorously elevated in postnatal rats receiving 6 successive intraperitoneal (i.p.) injections of interferon gamma (IFN gamma). In these animals, a large number of intensely stained OX-6 positive epiplexus cells were observed. These were mostly elongated or ramified with long processes. The immunostaining of epiplexus cells with OX-18 was also enhanced after IFN-gamma injections but the expression of CR3 and LCA appeared to be unaffected. It is concluded that the expression of MHC class I and II antigens on epiplexus cells is upregulated and induced respectively after successive i.p. injections of IFN-gamma into postnatal rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559118 TI - Sialylation of terminal saccharides of glycoconjugates expressed by murine molar tooth germs developing in vitro and in vivo. AB - During development of the mammalian tooth germ the pattern of terminal saccharides of glycoconjugates changes, with many structures losing lectin reactivity in a consistent pattern. This study investigated whether the epitopes are lost or become masked by terminal sialylation, using a combination of neuraminidase treatment of sections and sialic acid-reactive lectins. The results suggested that most of the terminal galactosamine and fucose sites in the epithelial enamel organ were removed during morphogenesis. Conversely, during condensation of the dental mesenchyme, masked peanut agglutinin (PNA)-reactive galactose epitopes appeared. During differentiation and organisation of the mesenchyme into odontoblasts and a subodontoblastic layer the PNA-reactive sites became masked again. These regions also specifically expressed sialylated glucosamine. However, at the proliferating epithelial cervical loop galactose sites appeared to be masked. This was more pronounced during in vitro development when abnormal expression of PNA-reactive sites was found at the cervical loop. Additionally, fucosylated sites persisted in the enamel organ, further indicating that the expression of terminal saccharides was disrupted during development in the organ culture system. These data suggest that loss of terminal galactose and galactosamine is related to differentiation of the cells. However, whether this loss occurs by removal or sialic acid masking is not dependent either on the origin of the cells or the epitope being lost. PMID- 7559119 TI - Fibre type composition of rabbit tibialis anterior and extensor digitorum longus muscles. AB - Rabbit tibialis anterior (TA) and extensor digitorum longus (EDL) muscles are used extensively in studies of stimulation-induced fibre type transformation, but the proportions and sizes of the 2 main fibre types, and the way in which they are distributed within the muscles, have never been described in any detail. In this study, transverse sections were processed by enzyme histochemical and immunohistochemical techniques and assessed morphometrically. The data were analysed by multivariate methods. In both TA and EDL muscles, the proportion of type 1 fibres varied significantly, and to a similar extent, within a cross section, from lateral to medial and from superficial to deep parts. The fibre density, an indirect estimate of the mean muscle fibre area, also varied significantly, but not systematically, within a cross-section. For the EDL muscle, the proportion of type 1 fibres was consistently higher in the distal than in the proximal part of the muscle. The proportion of type 1 fibres was also significantly higher in the EDL than in the TA muscle for each of the 6 rabbits. There was no systematic variation between muscles from left and right limbs. The type proportions and fibre densities for both TA and EDL muscles differed significantly between individual rabbits, but not between sexes. The study provides a database that has hitherto been lacking on normal fibre type composition and its variation within and between these experimentally important muscles. PMID- 7559120 TI - The role of the diaphragm in lymphatic absorption from the peritoneal cavity. AB - Lymphatics in the diaphragm form a specialised system draining fluid from the peritoneal cavity and returning it to the vascular system. Fluid enters subperitoneal lymphatic lacunae, between muscle fibres of the diaphragm, the lacunae being separated from the peritoneal cavity by a barrier comprising, successively, lymphatic endothelium, a layer of collagenous fibres, a thin fenestrated layer of elastic tissue, and the peritoneal mesothelium. To reach the lacunae, peritoneal fluid passes through stomata located between cuboidal mesothelial cells of the lacunar roof. Whilst the distribution of mesothelial stomata and subjacent lymphatic lacunae varies in different species, stomata appear to be exclusive to the diaphragm and may serve as the main drainage channels for absorption from the peritoneal cavity. Clinically, they may provide escape for tumour cells, pathogens and toxins from the peritoneal cavity. They could provide access for blood transfusions, for intraperitoneal chemotherapy to treat malignancies, and for peritoneal dialysis in treating chronic renal failure. From the lacunae, fluid traverses the diaphragm via intrinsic lymphatics to reach collecting lymphatics beneath the diaphragmatic pleura. Both intrinsic and collecting lymphatics contain valves. The collecting lymphatics drain principally into retrosternal (parasternal) lymphatic trunks that carry lymph to the great veins after it filters through mediastinal lymph nodes. PMID- 7559121 TI - Colour thresholding in video imaging. AB - The basic aspects of video imaging are reviewed as they relate to measurements of histological and anatomical features, with particular emphasis on the advantages and disadvantages of colour and black-and-white imaging modes. In black-and-white imaging, calculations are based on the manipulation of picture elements (pixels) that contain 0-255 levels of information. Black is represented by the absence of light (0) and white by 255 grades of light. In colour imaging, the pixels contain variation of hues for the primary (red, green and blue) and secondary (magenta, yellow, cyan, pink) colours. Manipulation of pixels with colour information is more computer intense than that for black-and-white pixels, because there are over 16 million possible combinations of colour in a system with a 24-bit resolution. The narrow 128 possible grades of separation in black and white often makes distinction between pixels with overlapping intensities difficult. Such difficulty is greatly reduced by colour thresholding of systems that base the representation of colour on a combination of hue-saturation-intensity (HSI) format. PMID- 7559124 TI - Functional relationship between the abductor pollicis longus and abductor pollicis brevis muscles: an EMG analysis. AB - This study examined the anatomical and functional relationships between the abductor pollicis longus (APL) and the abductor pollicis brevis (APB) muscles. The APL has 2 divisions, a distal superficial division and a more proximal deep one. A direct anatomical connection between the extrinsic and intrinsic muscles of the thumb is made up by the deep division of the APL, the APB and the trapezium. Electromyographic (EMG) recordings were made from these muscles to investigate differences and similarities in muscle activation. The EMG activity was recorded with intramuscular wire electrodes during isometric as well as dynamic contractions in different directions, both for the thumb and for the hand. The EMG activity of the right hand of 8 subjects was scaled relative to the mean EMG value at the maximum voluntary isometric contraction in order to compare relative muscle activity in various directions for different subjects. APB and APL were activated in a number of directions for the thumb as well as for the hand. Cooperation between these muscles is necessary to stabilise the trapezium to the carpus in movements of the thumb. APB is activated in movements of the hand to maintain the tension in the deep APL. The deep APL is activated in movements of the thumb to prevent undesired movements of the hand and the forearm. The APB and superficial APL are prime movers of the thumb. PMID- 7559123 TI - Regenerative and other responses to injury in the retinal stump of the optic nerve in adult albino rats: transection of the intracranial optic nerve. AB - The response to injury of the proximal (retinal) stump of the intracranially transected optic nerve in adult rats has been studied by electron microscopy. The central part of the retinal stump of the optic nerve underwent severe ischaemic damage resulting in the formation by 3 days postoperation (dpo) of a cone-shaped region of necrotic tissue which extended from a base occupying most of the cross sectional area of the nerve at the cut end to an apex within the intraorbital part of the nerve and only 2-3 mm from the eyeball. A mixture of apparently viable and dead or dying cells and axons was present in an intermediate zone surrounding the ischaemic core. Apparently intact nerve fibres occupied most of the periphery of the optic nerve. Small bundles of sprout-like axons were seen in the intermediate zone at 3 dpo, and by 5 dpo such sprouts were present at the periphery of the degenerative core. By 7 dpo, the sprouts were also found in the centre of the degenerative core, accompanied by astrocyte processes. The number of axonal sprouts present in the degenerative core and intermediate zone was much higher at 2 and 4 wk postoperation (wpo) than at 7 dpo, then declined gradually by 6 and 8 wpo. These results show that intracranial transection of the rat optic nerve produces extensive degeneration in the proximal stump and effectively produces an intraorbital axotomy of many retinal ganglion cells. Nevertheless, surviving axons display the ability to produce regenerative sprouts which persist for considerably longer than those produced after intraorbital injury. PMID- 7559122 TI - Molecular anatomy and genetics of myelin proteins in the peripheral nervous system. AB - Myelin contains a number of proteins, the major examples of which are protein zero (Po), P2 protein, peripheral myelin protein 22 (PMP22), myelin basic proteins (MBPs), myelin-associated glycoprotein (MAG) and the recently described connexin 32 (Cx32). This list is probably still incomplete. The localisation and possible functions of these proteins are reviewed. In the past few years a number of inherited demyelinating neuropathies in mice and the human have been shown to be due to mutations affecting the genes PMP22, Po and Cx32 so that it has become possible to characterise the molecular pathology of the majority of these disorders. This has provided important insights into the relationships between the structure of myelin and the function of its constituent proteins. PMID- 7559127 TI - Alterations to mandibular form following motor denervation of the masseter muscle. An experimental study in the rat. AB - The form of the mandible in young rats was studied following denervation of the masseter muscle. Wistar-derived male rats aged 27 d were randomly assigned either to experimental, sham-operated or control groups. The main trunk and initial branches of the masseteric nerve were resected on one side in each experimental animal. The nerve was exposed, but not resected, in the sham-operated group. No surgery was performed on the control group. All animals were killed at 69 d of age, examined and standardised contact radiographs taken of the cleaned and dried mandibles. Radiographic measurements describing the form and size of the mandibles were compared. On the operated side in the experimental animals the masseter muscle was smaller and the temporalis muscle larger as compared with the unoperated side. The angular process and the subcondylar incisure were also smaller and the coronoid process was positioned more rostrally. The most striking feature was that relatively little change accompanied denervation of masseter in the growing experimental rats. It is concluded that some of the changes previously attributed to muscle denervation may be due to other causes. PMID- 7559125 TI - Lymph pathways of the medial retropharyngeal lymph node in dogs. AB - In dogs, lymph drains from tissues throughout the head, including the tonsils, along lymphatic vessels to the facial, parotid, lateral retropharyngeal and mandibular lymph nodes. From the mandibular lymph nodes, lymph may flow to the ipsilateral medial retropharyngeal lymph nodes, or along anastomotic connections to the contralateral node. Afferent lymphatics convey lymph from these nodes to defined areas in the medial retropharyngeal nodes. They divide over the surface of the node, and within trabeculae. Terminal afferent lymphatics are connected to the subcapsular and trabecular sinuses either through circular or oval holes in the vessel wall, or terminate at the sinus where the vessel contains a valve adjacent to the point of entry. The subcapsular sinus surrounds the entire node, and is continuous with an interconnecting network of trabecular and cortical sinuses which convey lymph through the cortex. Connective tissue septa extend through the sinuses and lymph flows freely between adjacent sinuses through holes in the septal walls. Initial efferent lymphatic vessels, which arise from the medullary sinuses between medullary cords, converge towards and unite within the network of medullary trabeculae. Other vessels, which contain valve-like flaps, drain lymph from the subcapsular sinus. Efferent vessels emerge along the hilus and coalesce to form the tracheal trunk. The tracheal trunk has several layers of smooth muscle cells, well developed elastic laminae and connective tissue, surrounding the lymphatic endothelium. PMID- 7559128 TI - Multiple origins of cerebellar cholinergic afferents from the lower brainstem in the gerbil. AB - The possible origins of cerebellar cholinergic afferents from the lower brainstem of the gerbil were examined using immunohistochemistry combined with retrograde neuronal labelling techniques. Choline acetyltransferase (ChAT) monoclonal antibody was used in conjunction with a retrogradely transported tracer, horseradish peroxidase (HRP). The use of this technique allowed an unequivocal localisation of cholinergic neurons in different parts of the lower brainstem projecting to the cerebellum. In addition, single labelling of acetylcholinesterase (AChE), ChAT and HRP was carried out to elucidate the efferent projection from the lower brainstem to the cerebellum as well as the cholinergic distribution in these two areas. Our results showed the presence of HRP/ChAT double-labelled neurons in (1) the midline medulla: the periventricular gray beneath the 4th ventricle, C3 adrenergic area, raphe obscurus nucleus and medial longitudinal fasciculus, (2) the reticular formation: the medullary, lateral, intermediate, gigantocellular, lateral paragigantocellular and dorsal paragigantocellular reticular nuclei and gigantocellular reticular nucleus ventralis, and (3) sensory nuclei: the gracile nucleus, cuneate nucleus, external cuneate nucleus, spinal trigeminal nucleus interpolaris, prepositus hypoglossal nucleus and medial vestibular nucleus. In the cerebellum, AChE-positive mossy fibres were chiefly localised in the vermian lobules VIb,c, VII and X, paramedian lobule, crura I and II, paraflocculus and flocculus, and they were distributed in the white matter and granular layer of the cortex. The 3 above-mentioned cerebellar cholinergic afferent systems associated with the unique AChE distribution pattern in the cerebellum may be of important functional significance. PMID- 7559126 TI - Teratogenic effects of amniotic sac puncture: a mouse model. AB - The possibility of an association between chorionic villus sampling (cvs) and limb abnormalities has prompted a review of the relevant experimental data. Although a vascular aetiology is favoured by many at present, the possibility exists that a proportion of cases may be caused by oligohydramnios secondary to inadvertent amniotic sac puncture. A mouse model of amniotic puncture syndrome has been developed to study the craniofacial and limb abnormalities produced by this procedure. Pregnant mice were anaesthetised and a laparotomy performed. One uterine horn was exteriorised, and the amniotic sacs punctured through the wall of the uterus with either a 21 gauge or a 25 gauge needle. The conceptuses in the contralateral uterine horn acted as controls. The mice were all killed on d 19 of pregnancy (day of finding a vaginal plug = d 1 of pregnancy) by cervical dislocation, and the morphological features of the embryos examined in detail. In a preliminary study, amniotic sac puncture was carried out on d 12, 13, 14, 15 or 16 of pregnancy, with either a 21 or a 25 gauge needle. Since the highest rates of palatal defects and limb deformities were observed following amniotic sac puncture using a 21 gauge needle, when this procedure was carried out on either d 13 or 14 of pregnancy, the main study was undertaken using a 21 gauge needle on these two days of pregnancy. Of 102 embryos in which amniotic sac puncture was carried out on d 13, 53% survived to d 19. Of the latter, 35% had a cleft palate, 61% had one or more morphologically abnormal limbs, and 43% had an abnormal tail. When amniotic sac puncture was carried out on d 14 of pregnancy, of 83 embryos subjected to this procedure, 81% survived to d 19. Of the latter, 27% had a cleft palate, 39% had one or more morphologically abnormal limbs, and 19% had an abnormal tail. In the controls, of 86 and 61 embryos isolated respectively from the d 13 and 14 mice, the survival rates were 97 and 90%, respectively. Palatal, limb and tail abnormalities were not observed in the control series. Tentative relationships are drawn between cvs and amniocentesis-associated abnormalities, the Robin sequence and their rodent phenocopies. PMID- 7559130 TI - Disposition of collagen fibrils in human tendons. AB - Fixed and unfixed human tendons originating from cadavers and postoperation specimens were examined using inclined parallel beams of light in a reflecting mode. Along the tendon, numerous planes, constantly inclined to the axis, were observed edge-on at the surface and within the interior. Their angle of inclination, with respect to the distal end was very nearly +/- 50 degrees. The planes consisted of individual segments arranged in steps which were on average 190 x 50 microns. Similar configurations were also observed with the scanning electron microscope. Using this technique, the segments were identified with collagen bundles turning at a sharp angle with respect to the axis of the tendon at the level of the inclined plane. Crimped planes were found to be irregularly distributed along the tendons. On longer flatter tendons the average distance between planes was in the range of 1-12 mm. On stretching, the inclined pattern disappeared and was rapidly reestablished in the previously observed position when the strain was released. It is suggested that the observed structure forms a mechanism which is responsible for the appearance of the first part of 'foot' region of the tendon's stress-strain diagram. PMID- 7559129 TI - Electron microscopy of the seminiferous epithelium in the triploid (ZZZ and ZZW) fowl, Gallus domesticus. AB - The ultrastructure of cells of the seminiferous epithelium is described in both triploid ZZZ and ZZW fowls, especially cytological aberrations influencing the development of morphologically abnormal spermatids and spermatozoa. Mitotic divisions in triploid fowls are similar to those in diploid fowls, but meiosis in both the ZZZ and ZZW triploid fowl is abnormal and many defective gametes are produced. The majority of spermatids have structural abnormalities and this is attributed to the aneuploidy expected from meiotic chromosome pairing in a triploid. Some spermatids were observed to possess an irregularly shaped head, 2 centriolar complexes, 2 acrosomes and 2 tails and this type differentiated into large-headed spermatoza. Such spermatids and spermatozoa are suggested to be near diploid due to their large nuclear size and 2 sets of organelles. Other spermatids, observed with only 1 centriolar complex, 1 acrosome and 1 tail, are suggested to be near haploid. Differentiation of these spermatids is similar to the process observed in diploid fowls except that there is a high percentage of abnormally shaped heads. The fact that morphologically normal spermatozoa were rare is consistent with the expectation that very few spermatozoa would be exactly haploid. In both ZZZ and ZZW triploid fowls, triple pairing of chromosomes is observed in synaptonemal complexes (SC) during meiosis and recombination nodules are distributed at random in the central regions of the triple pairing SC. In ZZW triploid fowls gamete formation is more severely affected than in ZZZ triploid fowls and it is suggested that many of the degenerative changes occurring in ZZW fowls could be attributed to those spermatids that possess a W sex chromosome, since normal male fowls are the homogametic sex (ZZ) and normal spermatids and spermatozoa contain only a Z sex chromosome. PMID- 7559132 TI - The effect of NGF depletion on the neurotropic influence exerted by the distal stump following nerve transection. AB - Following nerve section, regenerating axons from the proximal stump grow preferentially towards the distal stump. It has been postulated that this may result from the release of a neurotropic factor. To investigate whether the protein nerve growth factor (NGF) plays such a role, we immunised adult rats against NGF and examined the effect on regeneration of sectioned nerves through Y shaped silastic tubes towards either the distal stump or an empty arm. Regeneration through the tubes was assessed electrophysiologically and the number of myelinated and nonmyelinated fibres at different sites was quantified using electron microscopy. There was electrophysiological evidence of regeneration towards the distal nerve stump in all the animals and there was no significant difference between the immunised and control animals in the size of compound action potential (CAP) ratios. Histologically, the majority of axons were found to have regenerated towards the distal nerve stump in 9/10 of the control animals and 7/9 of the immunised animals and there was no significant difference between the two groups in the numbers of regenerating myelinated or unmyelinated axons. However, in the immunised animals both myelinated and unmyelinated axons were slightly but significantly smaller and the myelin sheaths were thinner than in the control animals. In 2 immunised animals and none of the controls a small CAP was recorded while stimulating distal to the 'empty arm' and the presence of a small number of myelinated and unmyelinated axons was confirmed histologically. We conclude that as depletion of NGF does not block the preferential growth of regenerating axons towards the distal nerve stump it does not play the major neurotropic role in nerve regeneration. The reduction in size and myelin thickness of the regenerated axons after immunisation confirms the neurotrophic effects of NGF. PMID- 7559131 TI - Ultrastructural features of left ventricular myocytes in active and torpid hamsters compared with rats: a morphometric study. AB - Myocytes from the midmyocardium of the left ventricle of rats and hamsters were examined by transmission electron microscopy. The volume fraction of lipid droplets in such myocytes was about 6 times greater in the active hamster than in the rat, but it became progressively reduced during cold exposure and entry into hibernation to values similar to those of the rat. The volume fraction of the T system as well as the surface density of its membranes were each found to be twice as large in hamster myocytes as in the rat but there was no difference in these parameters between control, cold-exposed and torpid hamsters. The surface density of the junctional sarcoplasmic reticulum coupled with elements of the T system was greater in active hamsters when compared with those of the rat, and greater still in torpid hamsters. There was no significant difference in the surface density of free sarcoplasmic reticulum between control hamsters, cold exposed hamsters and rats but it was almost doubled in torpid hamsters. It is proposed that these features represent inherent differences in the ultrastructure of the left ventricle between the rat and hamster that may facilitate entry into hibernation. Additionally, further structural modifications during entry into hibernation may be related to alterations in lipid metabolism and modifications of calcium handling. PMID- 7559134 TI - The composition of trigeminal nerve branches in normal adult chickens and after debeaking at different ages. AB - The long term effects of amputation of the tip of the beak were studied in adult hens that were debeaked on the day of hatching, at the age of 8 d and at 6 wk, by EM analysis of fibre spectra of the medial branch of the ophthalmic nerve and of the intramandibular nerve. Three categories of fibre were distinguished for further analysis, i.e. unmyelinated axons, small myelinated fibres and large myelinated fibres. In normal birds the ophthalmic nerve contains relatively more large fibres than the intramandibular nerve. Amputation consistently results in a reduction of the number of large fibres and a substantial increase in the number of small myelinated fibres. The proportion of unmyelinated axons is rather variable, but is not affected by beak trimming. Age at debeaking has no effect. The observations are inconclusive concerning the possibility of heightened nociception. PMID- 7559136 TI - A morphometric study of the expansion of the chick area vasculosa in shell-less culture. AB - A morphometric evaluation, utilising a computerised system, was undertaken to quantify the growth of blood vessels in the chick area vasculosa, between the 2nd and the 5th incubation days. In this phase of embryonic growth the total length of the vascular tree for the arterial and venous systems alone, increases more (2.7 times) than the area occupied by the area vasculosa. Two main mechanisms might regulate the growth of vessels: the release of collaterals from the vessels 1.01-2 mm long and the lengthening of the vessels by 2.01-7 mm. PMID- 7559135 TI - An immunocytochemical and morphometric study of the rat pancreatic islets. AB - The rat pancreas has frequently been used as an animal model to study changes in islet cells in pathological conditions, such as diabetes mellitus and islet cell tumours, but detailed quantitative data on the islets are not available. This study was therefore undertaken to investigate (1) the volume density of pancreatic islets, (2) islet diameter, islet volume and islet cell number and (3) islet cell pattern, i.e. the distribution, volume and number of each cell type per islet. The study also investigated the possibility of differences in various pancreatic regions derived from the dorsal primordium. The rat pancreas was divided into 4 regions: lower duodenal (derived from the ventral primordium) and upper duodenal, gastric and splenic regions (derived from the dorsal primordium). Sections were stained immunocytochemically with anti-insulin (B cells), antiglucagon (A cells), antisomatostatin (D cells) and antipancreatic polypeptide (PP cells) antibodies, and were used for morphometric analysis. A total of 1292 islets was examined, 328 from the lower duodenal, 245 from the upper duodenal, 314 from the gastric and 405 from the splenic regions. The mean volume density of the islets per pancreatic tissue was found to be 2.6 +/- 0.1%, 2.3 +/- 0.1%, 2.9 +/- 0.2% and 3.3 +/- 0.2%, in the lower duodenal, upper duodenal, gastric and splenic regions, respectively. The size-frequency distribution of the profile diameters of the islets showed an overall shift of all the size classes towards smaller sizes in the upper duodenal region, and towards larger sizes in the splenic region, as compared with the corresponding classes of the other regions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559133 TI - Craniofacial abnormalities in homozygous Small eye (Sey/Sey) embryos and newborn mice. AB - The Small eye (Sey) gene in the mouse is lethal in the homozygous state. It is located on chromosome 2, is a mutation in the Pax-6 gene, and is genetically homologous with the human aniridia 2 (AN2) gene mutation. Numerous studies over the last few years, using genetic and molecular biological approaches, have investigated both the location of the gene as well as its possible mode of action. In the homozygous state, the primary defect appears to be limited to the failure of differentiation of the presumptive lens and nasal placodes. Such mice therefore display a characteristic phenotype; they possess neither eyes nor any nasal derivatives. Their heterozygous (Sey/+) and normal (+/+) littermates may be distinguished before birth only by a detailed examination of their eyes. Few detailed morphological/histological studies have been undertaken to date in the Sey/Sey embryos and newborn, and in the present study we describe a variety of craniofacial abnormalities that have not previously been reported. We observed, with one exception, delayed closure of the palate, and the presence in 80% of mice of an abnormal complement of upper incisor teeth, so that 35% possessed 1 supernumerary tooth while 45% possessed 2 supernumerary teeth. In these mice, a total of either 3 or 4, rather than the normal complement of 2, upper incisor teeth were present. Possibly the most unexpected finding, however, was the presence of a median cartilaginous rod-like structure which protruded between the 2 maxillae to give the Alizarin red S and Alcian blue-stained 'cleared' skulls of the newborn mice a characteristic 'unicorn-like' appearance. While this structure appeared to be a rostral extension of the chondrocranium, its exact derivation is unclear. PMID- 7559137 TI - Incidence of the coracoclavicular joint in South African populations. AB - The presence of a diarthrotic coracoclavicular joint, as represented by an articular facet on the conoid tubercle of the clavicle and the superior surface of the coracoid process of the scapula, was investigated. The sample consisted of 60 white and 180 black South African (60 Sotho, 60 Xhosa and 60 Zulu) skeletons. Each group consisted of 30 male and 30 female skeletons. The presence of the articular facet was recorded as either bilateral, unilateral left or unilateral right. The effect of clavicular length, scapular size and first rib angle on the presence of the coracoclavicular joint was also investigated. The presence of the articular facet was noted in 23 (9.6%) of the 240 individuals studied. Of these 23 individuals, 6 (26.1%) were white and 17 (73.9%) were black. Males (56.5%) presented a higher incidence of this anomaly than females (43.5%). The articular facet occurred bilaterally in 47.9% (11/23), unilaterally on the left in 30.4% (7/23) and unilaterally on the right in 21.7% (5/23). Sexual, racial and tribal differences were not statistically significant. Individuals possessing the joint showed statistically significantly (P < 0.01) larger scapulae (increased border lengths and superior angles), longer clavicles and longer first ribs. No statistically significant differences in the first rib angles were observed between individuals who possessed the joint and those who did not, thus implying similar thoracic inlet size. It is proposed that the aforementioned morphometry of the scapulae, clavicles and first ribs may restrict associated movements of the scapulae, resulting in the development of the coracoclavicular joint. PMID- 7559138 TI - Age-related bone resorption in the normal incus: a case of maladaptive remodelling? AB - The changes that occur in the normal human incus with age have been investigated. Evidence for age-related changes in this ossicle, especially in the region of the long process, has been accumulating over the last 30-40 years and yet they have neither been confirmed quantitatively nor explained satisfactorily. In this study the results of a morphometric study of the long processes of a series of normal incudes are presented. These demonstrate that the lenticular and long processes undergo progressive symmetric resorption with advancing age. We consider these findings in the light of previous considerations of incudal remodelling and propose that these remodelling changes may reflect a normal adaptive response to the biomechanical milieu of the human middle ear. PMID- 7559139 TI - Anti-androgen effects of the aromatase inhibitor, atamestane. AB - Prostatic hyperplasia can be induced in both intact and castrated dogs and in intact cynomolgus monkeys by the administration of androgenic steroids. Estrogenic steroids potentiate this effect in dogs. These changes also can be induced by androstenedione, which increases androgen and estrogen levels. Atamestane (ATA; 1-methyl-3,17-dione-androsta-1,4-diene), a potent aromatase inhibitor, inhibits some of the androstendione-induced effects; however, the nonsteroidal aromatase inhibitor, CGS-16949A, has been reported to decrease serum estradiol levels in adult rats but to have no effect on androgen-dependent organ weights. To examine the mechanisms by which ATA affects the rat prostate, in vivo and in vitro studies were conducted using adult rat ventral prostate (VP). Intact Sprague-Dawley rats were injected daily for 14 days with sesame seed oil, ATA (70 mg/kg/day), finasteride (FIN; 5 mg/kg/day), a 5 alpha-reductase inhibitor, or the combination of FIN plus ATA. A fifth group was castrated (CASTR) on day 1. The mean +/- standard error VP weight of the controls was 350 +/- 19 mg. It was reduced 17% (P < 0.05) by ATA, 29% (P < 0.001) by FIN, 48% (P < 0.001) by FIN plus ATA, and 86% (P < 0.001) by CASTR. The DNA/VP was reduced 22% (not significant) by ATA, 18% by FIN (not significant), 35% (P < 0.01) by FIN plus ATA, and 60% (P < 0.001) by CASTR. More significant changes were observed in RNA and protein. The mRNA for prostatein C3 was reduced by each of the treatments, but only CASTR increased the mRNA for TRPM-2, a marker of apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559140 TI - Gene expression in the aging brown Norway rat epididymis. AB - The mammalian epididymis is the site where spermatozoa are matured and then stored. Though many studies have described epididymal functions and their regulation, little is known about how aging affects this tissue. The Brown Norway rat, which does not show the many age-related pathologies common to other rat strains, was used as a model to study aging of the epididymis. The present study was designed to determine the effect of aging on the mRNA levels for selected markers of epididymal function. Brown Norway rats ranging in age from 6 to 30 months were examined at 6-month intervals; epididymides were sectioned into caput corpus and cauda regions. Relative mRNA concentrations were assessed using Northern blot analysis and specific cDNAs for the rat 5 alpha-reductase isozymes, types 1 and 2; proenkephalin; the androgen receptor; epididymal proteins B/C and D/E; and sulfated glycoprotein-2 (SGP-2, clusterin). Northern blots were quantitated by densitometric scanning. In the caput-corpus epididymidis, 5 alpha reductase type 1 and type 2 mRNA levels decreased significantly by 43% and 33%, respectively, between 6 and 12 months and by 64% and 40%, respectively, between 6 and 30 months. No significant change, however, was found in the expression of the 5 alpha-reductase mRNAs in the cauda epididymidis. Interestingly, proenkephalin mRNA was only detected in the caput-corpus epididymidis of 6-month-old rats. In marked contrast to the 5 alpha-reductase isozymes and proenkephalin, no significant age-related changes were observed in the mRNA levels for the androgen receptor, protein B/C, or protein D/E. No age-related changes in mRNA expression for SGP-2 occurred in the caput-corpus epididymidis. However, in the cauda epididymidis, SGP-2 mRNA levels rose by twofold between 6 and 18 months and then decreased sharply by 75% between 18 and 30 months. We conclude that as the epididymis ages, the expression of genes for certain specific markers of epididymal function is affected in a region-specific manner. Further, the decrease in the concentrations of the mRNAs for the 5 alpha-reductase isozymes and proenkephalin in the epididymis between 6 and 12 months is thus far the earliest marker for aging in the male reproductive tract of the Brown Norway rat. PMID- 7559141 TI - The effects of aging on basement membrane in the testis. AB - Age-related increases in basement membrane thickness have been noted in many tissues, including the testis. The current investigation examined the morphology of the basement membrane in the aged Brown Norway rat and sought to determine whether the accumulation of basement membrane was the result of an increase in the expression of the basement membrane genes. The aged testis was characterized by atrophy of the seminiferous tubules. Closer examination of the degenerated tubules revealed that the seminiferous epithelium was completely devoid of germ cells and that the basement membrane of these tubules was thickened and highly convoluted. In some animals, there was a measurable increase in basement membrane thickness in tubules of normal diameter together with an apparently normal epithelium, suggesting that the thickening is not solely due to a shrinkage of the tubules. To determine whether an increase in basement membrane synthesis was responsible for the thickening, the expression of the genes for laminin, collagen IV, heparan sulfate proteoglycan, and fibronectin was analyzed by Northern blot. There were no changes in the expression of the genes for the laminin B1 and B2 chains, heparan sulfate proteoglycan, or fibronectin that could be correlated with increasing age. Surprisingly, however, the levels of mRNA for the laminin A chain and collagen IV decreased with age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559143 TI - Differential responses of human sperm to varying concentrations of pentoxyfylline with demonstration of toxicity. AB - Pentoxyfylline (PF), a methylxanthine derivative, is an inhibitor of the cAMP phosphodiesterase enzyme, and is known to stimulate the motility of fresh and post-thaw human sperm. The purpose of this study was to examine the effects of different concentrations of PF on motility (MOT), path (curvilinear) velocity (PV), and hyperactivation (HA) of fresh sperm from patients (n = 24) and donors (n = 6) and post-thaw donor sperm (n = 5). For cryopreservation, the donor semen was frozen in liquid nitrogen using test-yolk-glycerol cryopreservative, stored for a minimum of 48 hours, then thawed at room temperature prior to assay. Aliquots of all samples to equal 10 x 10(6)/ml were diluted in 1 ml of the following: medium (human tubal fluid) only (control), or 2.5, 5, 10, or 20 mg/ml PF in medium. Specimens were incubated at 37 degrees C, and all were assessed by computer-assisted motion analysis at 0, 0.5, 1, and 2 hours. The patient specimens were divided into two groups: group 1, mean percent (standard deviation [SD]) MOT < 20% (12.8 +/- 5.8); group 2, mean percent (SD) MOT > 20% (37.8 +/- 14). For fresh donor sperm, 2.5 mg/ml PF significantly stimulated PV and HA at 0, 1, and 2 hours, and MOT at 0, 0.5, and 2 hours. PF at 5 mg/ml resulted in a decreased PV and HA, whereas MOT was decreased by 10 mg/ml. In the < 20% MOT group, 2.5 mg/ml PF significantly stimulated MOT at 0.5, 1, and 2 hours, and HA at 0 and 2 hours. There was no effect on PV.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559144 TI - Human chorionic gonadotropin but not the calcitonin gene-related peptide induces postnatal testicular descent in mice. AB - The androgen-regulated paracrine factor, calcitonin gene-related peptide (CGRP), has been proposed as a possible mediator of testicular descent. This peptide has been found to increase rhythmic contractions of gubernaculae and is known to be released by the genitofemoral nerve. We have investigated the ability of CGRP to induce premature testicular descent. CGRP was administered alone, or in combination with human chorionic gonadotropin (hCG) to C57BL/6 male mice postnatally. The extent of testicular descent at 18 days postpartum was then ascertained. The potential relationship between testicular weight and descent was also examined. Our results show that testes of mice treated with either hCG alone, or in combination with 500 ng CGRP, were at a significantly lower position than those of controls by 16% and 17%, respectively. In contrast, mice treated with 500 ng of CGRP alone had testes at a higher position when compared to those of controls, by 19%. In mice treated with 50 ng of CGRP alone or in combination with hCG, testes were at a position similar to those in controls. Furthermore, testicular descent was analyzed in relation to testicular weight, and we found that significantly smaller testes per gram of body weight than those of controls were at a significantly lower position compared to those of controls. Our data demonstrate that CGRP had no effect on postnatal testicular descent and that there is no relationship between postnatal descent and testicular weight. PMID- 7559142 TI - The duration of the cycle of the seminiferous epithelium is altered by administration of 2,5-hexanedione in the adult Sprague-Dawley rat. AB - The duration of the cycle of the seminiferous epithelium is believed to be under genetic control rather than being influenced by other factors. However, the frequencies of certain spermatogenic stages--reflecting their relative durations- can be altered under various conditions including treatment with the n-hexane metabolite, 2,5-hexanedione (HD). To investigate whether HD administration alters the duration of the spermatogenic process, adult Sprague-Dawley rats were exposed to vehicle (n = 20) or 1% HD dissolved in the drinking water (n = 40) throughout a period of 29 days. On day 17, 100 mg/kg of 5-bromodeoxyuridine (BrdU) was given intraperitoneally; 3 hours later one testis was removed, and the remaining testis was excised 12 days later. BrdU was localized in Bouin's-fixed, Paraplast embedded testes using immunogold-silver-staining, and sections were counterstained with periodic acid--Schiff and hematoxylin to permit identification of the spermatogenic stages. HD treatment reduced weights of body, testis, and epididymis (P < 0.05) but had no effects on serum levels of follicle stimulating hormone or testosterone. The seminiferous epithelium was vacuolized, and spermatogenesis was affected to various degrees. HD significantly reduced the frequency of stage VII tubules (P < 0.01) and increased the frequency of stage IX and stage X tubules (P < 0.05). Furthermore, HD reduced the stage-dependent progression of BrdU-labeled germ cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559146 TI - Testis factors that may regulate gene expression: evidence from a patient with Kartagener's syndrome. AB - Patients with Kartagener's syndrome characteristically have immotile cilia and flagella. Ultrastructural analysis of the efferent ducts of the patient described in this report revealed that the cilia totally lacked dynein arms. Based on clinical findings his respiratory cilia also were not functional. In contrast, this patient's spermatozoa were motile and displayed normal ultrastructure, including dynein arms. This suggests that two genes exist that code for slightly different proteins with analogous functions; one of these genes functions in somatic tissue and the other in the male germ tissue. Differential gene expression in somatic and male germ tissue may be related to the syncytial nature of male germ cells, low scrotal temperature, or haploid gene expression. PMID- 7559145 TI - Acute effects of spinal cord injury on the pituitary-testicular hormone axis and Sertoli cell functions: a time course study. AB - The present study investigated the time course of the onset of the abnormalities in spermatogenesis following spinal cord injury, and their relationship to changes in the pituitary testicular hormonal axis and Sertoli cell function. Spinal cord injury (SCI) was induced in adult male rats by surgical transection of the spinal cord at the level of T9 and L1 vertebrae. Animals were killed 3, 7, and 14 days after the operation. As early as 3 days following SCI, abnormalities in spermatogenesis, including delayed spermiation and vacuolization of the nucleus of spermatids, were noted in both the T9 and L1 animals. By 14 days, other lesions, including phagocytosis of mature spermatids, incomplete cellular associations, and total regression of seminiferous epithelium, became apparent. Concurrently a transient but significant (P < 0.05) suppression of serum follicle stimulating hormone (FSH) occurred in the T9 animals, and a suppression of serum luteinizing hormone (LH) occurred in both the T9 and the L1 animals 3 days after the surgery. This was accompanied by a suppression of testicular and serum testosterone levels (P < 0.05, P < 0.01, respectively). Most of the hormonal parameters had recovered and were not different from those of sham-operated animals by 14 days (P > 0.10). Northern blot analysis of testicular poly(A)+ RNA revealed a transient but significant reduction in the steady-state level of the 2.7-kilobase (kb) Sertoli cell transferrin mRNA transcript in both the T9 and the L1 animals 3 days after the operation (P < 0.05). On the other hand, the 1.7-kb androgen binding protein (ABP) mRNA remained unaffected during the 2-week study period. The steady-state level of mRNA transcripts for spermatogenic cell specific hemiferrin and spermatid specific transition protein 2 and protamine 1 also remained unchanged. These results suggest that spinal cord injury will result in a temporary, but profound, effect on the pituitary-testicular hormone axis. These changes may impair certain aspects of Sertoli cell function that could render these cells incapable of supporting normal spermatogenesis. However, the severity of spermatogenic lesions and the disparate responses of the two major Sertoli cell proteins make it unlikely that hormone deficiency is the only mechanism responsible for the impaired spermatogenesis following spinal cord injury. PMID- 7559147 TI - Enhanced pharmacological testing in patients with erectile dysfunction. AB - This study was designed to evaluate the practical use of the combination of a low dose intracavernous injection test (ICI) and visual erotic stimulation (VES) in the assessment of patients with erectile dysfunction. Additionally, a postinvestigation questionnaire (PIQ) was used to rate the erection following the investigation when the patient had left the office. It was hypothesized that the combination (ICIVES) and the PIQ would yield a higher erectile response rate than ICI or VES alone and would minimize the occurrence of prolonged erection. Of the 90 patients studied, 17 (19%) showed adequate erections to VES, 10 (11%) to ICI, and 60 (67%) to ICIVES. Of the 30 non-responders, 7 (23%) reported adequate erections on the PIQ. Overall, in 75% of the patients adequate erections were achieved. No prolonged erections were encountered. It is concluded that the combination of a low-dose ICI and VES, and the application of a postinvestigation questionnaire, is a powerful and safe test to select patients for autoinjection therapy. PMID- 7559149 TI - Comparative study of boar sperm coming from the caput, corpus, and cauda regions of the epididymis. AB - Sperm quality in the caput, corpus, and cauda regions of the epididymis of healthy and sexually mature Landrace boars was studied. Epididymal sperm characteristics were examined by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sperm vitality decreased very slightly although progressively with the transport of sperm through the epididymis. Osmotic resistance of acrosomes was very low in the sperm from the caput and approximately 100% in the corpus and cauda. The incidence of spermatozoa with the head detached from the tail remained stable in the first two regions of the epididymis, increasing notably in the cauda. Sperm agglutination increased progressively as sperm progressed along the epididymal duct. The percent of mature spermatozoa and aberrant spermatozoa increased from the caput to the cauda, whereas the percent of immature spermatozoa decreased. In the caput and corpus the percent of immature spermatozoa was similar, although in the caput they were characterized by the presence of a proximal cytoplasmic droplet; in the corpus the cytoplasmic droplet was distal. The acrosomal protuberance was highly developed in spermatozoa from the epididymal caput, but its volume was considerably reduced in those from the epididymal cauda. The electron density of the acrosomal content was lower in spermatozoa from the caput than in those from the epididymal cauda. The mitochondrial sheath of spermatozoa from the caput was made of voluminous mitochondria of unequal size, with a low electron-dense matrix. In the cauda region, the mitochondria were smaller in diameter, homogeneous in size, and with greater matrix electron density. This last fact is related to the loss of the capacity of spermatozoa to fold their tail by the midpiece as they progress along the epididymal duct. The complex epididymal maturation process of the sperm results in quantitative and qualitative changes that can be characterized in each of the three epididymal regions. The presence in the ejaculate of one or more gamete forms belonging to the epididymal caput, corpus, or cauda will allow workers to better establish the intensity of stress produced by a high frequency of semen collection. PMID- 7559150 TI - Expression and androgenic regulation of human prostate-specific kallikreins. AB - In vitro studies suggest that the protease activity of PSA might play a functional role to facilitate the growth and spreading of cancerous prostatic cells. hK2 may have similar properties. Further investigation to prove their in vivo effects is required. Regulation of PSA and hKLK2 gene expression is mediated not only by androgens, but also by a number of autocrine and paracrine factors, suggesting that the control mechanisms for expression of these genes are complex and multifaceted. Such factors may also be integral for the growth and differentiation of prostate cells. Thus PSA and hKLK2 may serve as useful markers to study the regulation of gene expression during cell growth and differentiation of the prostate. PMID- 7559148 TI - The contraceptive effects of etoprine on male mice and rats. AB - We had previously found that 2,4-diaminopyrimidines affected spermatogenesis, possibly through the inhibition of testicular dihydrofolate reductase (DHFR). The current study examined the effects of etoprine, a highly lipophilic 2,4 diaminopyrimidine that is also a potent DHFR inhibitor, on the fertility of male mice at various dosages (0.1-50 mg/kg/day) for 55 days and male rats at 5 mg/kg/day for 65 days. Two other substituted diaminopyrimidines were tested at dosages of 50 mg/kg/day for 55 days. Results of breeding trials along with assessment of various parameters indicative of male fertility were noted. We found that of the compounds tested, etoprine is a potent antifertility agent that causes complete infertility at doses of > or = 5 mg/kg/day in mice with a threshold of effectiveness occurring between 1 and 5 mg/kg/day. The antifertility action of etoprine may be related to its capacity to inhibit testicular DHFR and its high degree of lipophilicity. PMID- 7559152 TI - Localization of androgen receptor and cell-specific cytokeratins in basal cells of rat ventral prostate. AB - Immunocytochemical techniques labeled androgen receptor and cell-specific cytokeratins in the basal cells of rat ventral prostate. In addition, nonradioactive in situ hybridization verified the production of androgen receptor transcripts in the basal cells. Androgen receptors and transcripts were localized in the nuclei and cytoplasm of the adult basal cells using these two techniques. Monoclonal anti-cytokeratin antibodies identified temporal changes in the expression of basal cell-specific intermediate filaments of fetal, neonatal, normal adult, orchidectomized adult, and testosterone-treated orchidectomized adult prostates. Labeling intensity of the basal cells was elevated during development when compared to the staining in normal adult tissue. Orchidectomized adults exhibited the greatest intensity of labeling, which decreased after testosterone treatment. The detection of androgen receptor and its transcripts in the basal cells supports the hypothesis that these cells are androgen responsive. The observed change in the anti-cytokeratin staining patterns of these cells during development, growth, and regression is an indirect measure of androgenic influence. The androgen-repressed cytokeratin expression in basal cells is similar to that found in prostatic luminal cells. PMID- 7559151 TI - Regulation of Leydig cell function by insulin-like growth factor-I and binding proteins. PMID- 7559153 TI - Cloning and partial sequence of the rabbit androgen receptor: expression in fetal urogenital tissues. AB - Genetic studies indicate that the actions of testosterone and dihydrotestosterone are mediated by a single androgen receptor (AR). The basis for the differential effects of these hormones during development is not known. To address this problem, we have turned to the rabbit, a species in which the endocrine and metabolic events in male sexual differentiation have been studied in detail. As a first step, we have cloned a partial cDNA encoding the rabbit prostate AR and have analyzed the expression of the cDNA in tissues of rabbit embryos at the time during which male phenotypic differentiation takes place. The coding sequence of the rabbit AR cDNA reveals a high degree of conservation with the sequences of the human, mouse, and rat ARs. By Northern analysis the principal transcript expressed in the mesonephros and the anlage of the external genitalia on gestation day 18 appeared to be identical to the mRNA expressed in adult prostate and epididymis. PMID- 7559154 TI - Endothelin-1 and its receptors in human testis. AB - We have previously found the presence of endothelin (ET) receptor and ET-like immunoreactivity in rat testis. We now extend our studies from rat to human testis. We found expression of a specific transcript for ET-1 and ET-1-like immunoreactivity in human testis. Positive staining was confined to the Sertoli cells of the tubular compartment, although few peritubular and interstitial cells were also stained. We also identified specific ETA and ETB receptor transcripts in human testis; ETA expression was more abundant than the ETB expression. Mathematical analysis of multiple self- and cross-competition studies among [125I]ET-1, [125I]ET-3, and analogues confirmed the presence of the ETA and ETB isoreceptors. In testicular homogenates, the ETA receptor was sevenfold more concentrated than the ETB receptor. In order to localize the receptors, we performed [125I]ET-1 autoradiography. Binding sites were mostly concentrated into the seminiferous tubules, although interstitial and peritubular myoid cells were also positive. Within the seminiferous tubules, [125I]ET-1 binding sites were confined to primary and secondary spermatocytes and early spermatids, whereas Sertoli cells were negative. We were unable to demonstrate the presence of functional ET receptors in ejaculated spermatozoa. Because ET-like immunoreactivity was present in Sertoli cells, we next asked whether authentic ET 1 is present in human seminal fluid and represents a good index for Sertoli cell function. Reverse-phase high-performance liquid chromatography analysis of ET like immunoreactivity in seminal fluid indicated that most of the detected peptides correspond to the ET-1 precursor, big-ET-1. The seminal concentration of ET-like immunoreactivity was similar in normospermic, oligospermic, azoospermic, and vasectomized men, indicating that ETs are produced in different parts of the male genital tract and that they do not represent an useful tool for the diagnosis of male reproductive diseases. In conclusion, this study demonstrated, for the first time, the presence of ET-1 and its receptors in human testis. PMID- 7559155 TI - Effects of two non-steroidal antiandrogens on testicular function in prepubertal rats. AB - The effects of two non-steroidal antiandrogens, flutamide and casodex, were evaluated in prepubertal male rats. Animals (23 days old) were subcutaneously administered vehicle or 1, 2, 5, or 10 mg/day of flutamide or casodex for 10 days. Testis weights were diminished at the 10 mg/day dose of both antiandrogens. A significant increase in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels was detected. Notwithstanding, flutamide influenced LH/FSH levels more severely than casodex. No changes were observed in serum prolactin. Serum testosterone, dihydrotestosterone, and 3 alpha-androstanediol levels were increased in flutamide-treated rats from the 2 mg/day dose, whereas only 3 alpha androstanediol was modified at 10 mg/day of casodex, suggesting a differential effect on androgen metabolism. An elevation of testicular concentration and basal production of androgens was found, indicating that flutamide and casodex administration are capable of stimulating testicular steroidogenesis, as well as 5 alpha-reduction. However, the in vitro maximal responsiveness of the gonads to human chorionic gonadotropin was preserved. Antiandrogen administration did not modify testicular androgen binding protein concentration. In conclusion, the blockade of androgen action during sexual maturation caused profound changes on the pituitary-gonadal axis in male rats. PMID- 7559156 TI - Development of male urogenital epithelia elicited by soluble mesenchymal factors. AB - Androgen-dependent development of male secondary sexual glands is mediated by paracrine mesenchymal-epithelial interactions that regulate a complex array of biological events such as epithelial morphogenesis, growth, and cytodifferentiation. It is not known whether the action of mesenchyme on epithelial development in the male genital tract requires cell-cell contact or whether soluble, diffusible mediators are involved. To examine paracrine effects of urogenital sinus mesenchyme (UGM) on epithelial development, conditioned media (CM) from embryonic mouse UGM of normal (wild-type) and androgen-insensitive Tfm (testicular feminization) mice were tested for growth and morphogenetic effects on heterotypic tissue recombinants composed of rat or mouse bladder mesenchyme plus neonatal mouse seminal vesicle epithelium (BLM+SVE) or rat or mouse bladder mesenchyme plus neonatal mouse bulbourethral gland epithelium (BLM+BUG-E). Addition of a concentrate of CM from wild-type UGM grown in the presence of dihydrotestosterone (DHT) induced epithelial growth and complex epithelial morphogenesis in BLM+SVE recombinants, whereas CM from DHT-treated Tfm UGM or a saline control were without effect. CM from wild-type UGM elicited similar trophic effects in BLM+BUG-E recombinants, but in addition induced precocious mucous epithelial differentiation in BLM+BUG-E recombinants. These results suggest that the normally androgen-dependent epithelial growth and branching morphogenesis in developing male urogenital organs is elicited by soluble mesenchymal factors. Two-dimensional gel electrophoresis of proteins synthesized and secreted by wild-type UGM revealed several androgen-dependent proteins with molecular weights of approximately 30 kDa that are absent in CM of Tfm UGM either in the presence or absence of DHT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559158 TI - Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat. AB - Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole. PMID- 7559160 TI - Importance of bicarbonate to the progesterone-initiated human sperm acrosome reaction. AB - Progesterone, a putative in vivo initiator of the human sperm acrosome reaction (AR), has previously been shown to act at the sperm plasma membrane to initiate the AR in vitro. Here, we have investigated whether bicarbonate (HCO3-) was required for the progesterone-initiated human AR and whether HCO3(-)-dependent cAMP activation might be involved. Capacitated human sperm were suspended in the presence of high (25 mM) or low (1 mM) HCO3- media. The AR was assayed using fluorescein isothiocyanate (FITC)-conconavalin A with sperm fixed 5 minutes after progesterone or solvent control addition. Progesterone initiated the AR in both high and low HCO3- media, but the percentage of AR was significantly lower in the latter medium. In the presence of high HCO3-, 20-minute preincubation with 4,4' diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a blocker of HCO3- transport, inhibited the progesterone-initiated AR in a dose-dependent manner. The maximum inhibition (85%) was obtained with 18 microM DIDS. Inhibition by DIDS was reversed by washing sperm after treatment. Preincubation of sperm with dibutyryl cAMP (0.1 microM-1 mM) plus DIDS almost completely eliminated the inhibition of the progesterone-initiated AR by DIDS. Dibutyryl cAMP alone did not have a stimulatory effect on the progesterone-initiated AR, when high HCO3- was present, but it was able to partially overcome the reduction of AR by low HCO3-. These results are the first to demonstrate the importance of HCO3- to: 1) mammalian AR initiation by the putative physiological initiator progesterone, and 2) the human sperm AR.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559159 TI - Release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation and acrosome reaction. AB - The present study examines the release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation conditions and in correlation to acrosome reaction and cell death. The ACE content of spermatozoa was measured by treating the cells with different detergents. Glass wool-filtered and washed human spermatozoa were incubated for 3 hours at 37 degrees C. Percentages of acrosome-reacted and dead spermatozoa did not change significantly, but the ACE release increased from 0 to 2.93 +/- 0.44 mU/100 x 10(6) spermatozoa after 180 minutes (P < 0.001). In order to study the influence of acrosome reaction on ACE release, the acrosome reaction of noncapacitated spermatozoa was induced by 10 microM calcium ionophore A23187. The percentages of acrosome reaction and viability in noncapacitated spermatozoa as well as the ACE release were compared to corresponding data from experiments using capacitated spermatozoa (3 hours, 37 degrees C) from the same donors. Although the number of living acrosome-reacted spermatozoa after ionophore treatment (30.5 +/- 4.0%) was significantly higher than after capacitation (13.3 +/- 2.8%, P < 0.001), ACE release from ionophore treated, noncapacitated spermatozoa was lower (P < 0.05). The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction. Measurement of ACE release may be a clinically useful assay for human sperm capacitation. PMID- 7559163 TI - OHC response recruitment and its correlation with loudness recruitment. AB - After proper noise exposure, Hensen's cells, which have been shown to follow closely the response characteristics of the outer hair cells, suffered a loss of sensitivity at low and moderate SPLs. The lower the stimulus level, the greater was the loss. When the low-SPL loss did not exceed about 40 dB, input-output functions showed an increased rate of amplitude growth, so that the post-exposure response caught up with its pre-exposure counterpart between 60 and 90 dB SPL, depending on the severity of the loss. These results, together with preceding clinical observations, led us to the conclusion that loudness recruitment occurs at least in part at the hair cell level and is basically a local event as opposed to a pathological spread of excitation. The response recruitment we have discovered appears to result from a decreased effect of the active feedback when the passive cochlear mechanisms are intact. Evidence for these relationships is presented and an explanation is offered for previous experimental successes and failures in observing a steepening of rate-intensity functions in auditory nerve fibers after noise exposures or administration of ototoxic drugs. PMID- 7559161 TI - Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF. AB - Acrosin activity was determined using a gelatinolysis technique in 100-microliter semen aliquots of 114 patients (normozoospermia, n = 90; asthenozoospermia, n = 12; oligozoospermia, n = 10; polyzoospermia, n = 2) attending an in vitro fertilization (IVF) program. Halo diameter, halo formation rate, and a calculated acrosin activity index correlated significantly with the IVF rates (P = 0.0054, r = 0.396; P = 0.0009, r = 0.401; and P = 0.0003, r = 0.428, respectively). In cases where the halo diameter was < 10 microns and halo formation rate was < 60%, all patients were subfertile or infertile, that is, they showed poor or no fertilization in vitro, respectively. The assay demonstrated a relatively low sensitivity: 25.7% for halo diameter, 37.1% for halo formation rate, and 25.7% for acrosin activity index, respectively. This might be attributed to other sperm functional aspects, such as disturbed acrosome reaction or impaired zona binding. PMID- 7559162 TI - Cryopreservation extenders affect calcium flux in bovine spermatozoa during a temperature challenge. AB - Bovine spermatozoa are commercially cryopreserved by diluting the cells in media, known as extenders, followed by slow cooling and freezing. Previous work has shown that this process of cryopreservation alters the cells' ability to control divalent calcium (Ca2+) movement. This study evaluated the effect of a brief exposure to common extenders on bovine spermatozoa during subsequent cooling and rewarming. Three fresh ejaculates from each of three bulls were each split and incubated for 30 minutes at 25 degrees C in milk extender or phosphate-buffered saline (PBS) (control); three other fresh ejaculates from each of three bulls were similarly incubated in egg yolk-Tris extender (EYT) or PBS. Spermatozoa were washed and the fluorescent Ca2+ indicator, indo-1 acetoxymethyl ester, was used to monitor the internal Ca2+ in the spermatozoa in Ca(2+)-free PBS over a continuous temperature gradient of 25 degrees C (15 minutes), cooling to 5 degrees C (32 minutes), at 5 degrees C (15 minutes), rewarming to 25 degrees C (25 minutes), and at 25 degrees C (15 minutes). Milk exposure reduced the initial percentage of missing acrosomes and EYT exposure improved the initial viability and acrosome morphology compared to the controls; only milk immediatetly increased internal Ca2+. The initial rate of Ca2+ uptake at 25 degrees C was greater for milk or EYT-exposed spermatozoa than controls (P < 0.05). During cooling, the rate of Ca2+ uptake in all spermatozoa increased (P < 0.01), and it continued to increase during the 15 minutes at 5 degrees C. During rewarming to 25 degrees C, the internal Ca2+ in all spermatozoa declined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559157 TI - Increased expression of a 68-kDa protein in the corpus cavernosum of some men with erectile dysfunction. AB - Erectile dysfunction (ED) may be caused by abnormalities of intracavernous penile structures. In order to investigate whether specific proteins could be identified that might be related to ED, the composition of structural proteins in cavernous tissues of patients with ED was compared to that of normal cavernous tissues by gel electrophoresis. Increased expression of a 68-kDa nonionic detergent extraction-resistant protein was demonstrated in tissues of more than half of the patients with vasculogenic ED, whereas only one out of nine normal cavernous tissues showed the same phenomenon. Increased expression was not related to a specific type of vascular insufficiency, aging, or diabetic constituency. Histochemical and immunochemical studies revealed that the increased amount of the 68-kDa protein is not merely the result of a surplus of nervous, smooth muscle, or elastic tissues. Furthermore, antibodies specific for 68-kDa neurofilament and 62- to 67.5-kDa tropoelastin did not recognize the 68-kDa protein on Western blots. The possibility that the 68-kDa protein may help us understand the etiology of certain cases of erectile dysfunction is discussed. PMID- 7559165 TI - A two-filter model for frequency discrimination. AB - In this paper, we explore a two-filter model, the simplest version of multi channel models for frequency discrimination of simple tones. According to this model, frequency discrimination is based on a change in the relative output levels of two auditory filters, one centered below and the other above the frequency of the tone. This idea can explain the experimental results that frequency discrimination is relatively unaffected by randomization of stimulus level. Moreover, it suggests a close relationship between the ability of listeners to perform frequency discrimination of simple tones and spectral-shape discrimination of two-tone complexes. The ability of three listeners to perform these two tasks was measured at six frequencies (from 0.25 to 8 kHz). The results from the spectral-shape-discrimination task were used to predict frequency difference limens. There was a high correlation between obtained and predicted values. PMID- 7559164 TI - Retrograde axonal transport of different fluorescent tracers from the neocortex to the suprageniculate nucleus in the rat. AB - The retrograde fluorescent tracers in a combination of two different dyes (Fluoro gold/Nuclear yellow or Fast blue/Fluoro gold) were used for the study of the projections from the medial geniculate body to the frontal and temporal cortices in rats. There were only single-labeled cells, no double-labeled ones in the medial geniculate body (MGB). The suprageniculate nucleus (SG) was considered to be the origin of the rat direct pathway to the frontal cortex. The present results suggest that projections from the suprageniculate nucleus to the frontal cortex and the temporal cortex consist of separate neuronal groups in the rat MGB and SG. The inputs to the SG from the auditory and oculomotor system may be processed in different ways. PMID- 7559166 TI - The effect of protein kinase C stimulator and inhibitor on cochlear potentials in the guinea pig. AB - To determine a possible role of protein kinase C (PKC) in the cochlear, the effects of a PKC stimulator (phorbol-12-myristate-13-acetate; PMA), an inactive analogue of PKC stimulator (4 alpha-phorbol-12,13-didecanoate; 4 alpha-PDD) and a PKC inhibitor (D-sphingosine) on cochlear potentials were examined in the guinea pig. The perilymphatic perfusion with PMA (3 x 10(-6) M) produced an increase in compound action potential (CAP) amplitude and no change in N1 latency, the amplitudes of negative summating potential (-SP), cochlear microphonics (CM) and endocochlear potential (EP). The perfusion with 4 alpha-PDD (3 x 10(-6) M) did not change the sound-evoked cochlear potentials and the EP. The perfusion with D sphingosine (10(-5) M) produced a decrease in CAP amplitude and no change in N1 latency and the amplitudes of -SP, CM and EP. The results suggest that PKC may be involved in the mechanism underlying the CAP generation. PMID- 7559167 TI - Atomic force microscope observations of otoconia in the newt. AB - Calcitic and aragonitic otoconia from the Japanese red-bellied newt, Cynops pyrrhogaster, were examined using an atomic force microscope. The surface structure of both otoconial polymorphs consisted of arrays of elements approximately 50 nm in diameter. Elements were generally round and were separated by shallow depressions of no more than 20 nm. The elements are suggested to be single crystals of calcium carbonate. The relationship of these observations to theories of otoconial genesis is discussed. PMID- 7559168 TI - Cochlear effects of indacrinone are not altered by penicillin. AB - Indacrinone is a loop diuretic structurally related to ethacrynic acid. Indacrinone is a racemic mixture. Previous studies have shown that the (-) enantiomer caused reduction of endocochlear potential (EP) and elevation of compound action potential (CAP) threshold (Rybak and Whitworth, 1987a). It has been demonstrated that organic acids such as penicillin, probenecid and sodium salicylate prevent the reduction of EP normally observed after furosemide administration (Rybak et al., 1992a). The present study was designed to determine whether penicillin pretreatment could prevent changes in EP and CAP threshold in (-)-indacrinone treated chinchillas. Adult chinchillas were anesthetized with ketamine and pentobarbital. A microelectrode was advanced into the scala media using the round window approach, and CAP responses to clicks were measured. One group was treated with (-)-indacrinone 100 mg/kg via the jugular vein. A second group of animals received penicillin 50 mg/kg i.v. thirty minutes before (-) indacrinone. The mean EP change in the indacrinone-treated animals was 38.38 +/- 19.32 millivolts (mv). The reduction of EP in the group receiving penicillin was 24.43 +/- 20.74 mv (P > 0.09). The mean CAP threshold changes in animals receiving indacrinone was 20 +/- 14.14 dB whereas those pretreated with penicillin showed a threshold shift of 21.43 +/- 20.35 dB (P > 0.05). These findings are consistent with previous studies which showed that the effect of ethacrynic acid on the EP and CAP was not changed by the pretreatment with penicillin (Rybak et al., 1990). PMID- 7559169 TI - Responses of neurons in the inferior colliculus of the rat to interaural time and intensity differences in transient stimuli: Implications for the latency hypotheses. AB - Although the sensitivity to interaural intensity differences (IIDs) of neurons receiving excitatory - inhibitory binaural input (EI neurons) has been examined in numerous studies, the mechanisms underlying this sensitivity remain unclear. According to the 'latency hypotheses' neuronal sensitivity to IIDs reflects sensitivity to differences in the timing of ipsilateral and contralateral inputs that are produced as a consequence of the effects of intensity upon latency. If the latency hypothesis is correct, a neuron's responses over any given IID range should be predicted by its responses to the interaural time differences (ITDs) that are 'equivalent' to the IIDs tested, in the sense that they produce the same changes in the relative timing of inputs. This prediction from the latency hypotheses were examined by determining the sensitivity of EI neurons in the inferior colliculus of anesthetized rats to IIDs and ITDs in click stimuli, under conditions that allowed 'equivalent' ITDs to be estimated. In approximately 10% of the 41 neurons tested, the IID-sensitivity function was a perfect or near perfect match to the equivalent-ITD function, indicating that IID sensitivity could be entirely accounted for in terms of sensitivity to intensity-produced neural time differences, as asserted by the latency hypothesis. For the majority of neurons, however, sensitivity to equivalent ITDs accounted only partially for the characteristics of the IID-sensitivity function; other features of the function in these cases appeared to reflect the operation of an additional factor, most probably the relative magnitude of the inputs from the two ears. Although the conclusions are qualified by the fact that one of the assumptions on which the estimation of equivalent ITDs was based was probably not satisfied for some neurons, the results suggest that intensity-produced changes in both the magnitude and the timing of excitatory and inhibitory inputs shape the IID sensitivity of most EI neurons. PMID- 7559174 TI - On the heritability of spontaneous otoacoustic emissions: A twins study. AB - Spontaneous otoacoustic emissions (SOAEs) were measured in human monozygotic (MZ) and dizygotic (DZ) twins and in a sample of non-twins. The number of SOAEs exhibited was more highly correlated in MZ co-twins than in same-sex DZ co-twins. Model-fitting to the correlations suggested that about three-quarters of the individual variation in the expression of SOAEs is attributable to genes. There was no convincing evidence for the heritability of specific SOAE frequencies. In accord with past surveys, SOAEs were more numerous in right than left ears, and in female than male subjects. Also investigated were the numbers of SOAEs exhibited by dark-versus light-eyed people and by MZ versus DZ twins. Those differences in our data were small and not statistically significant, but they were in a direction consistent with other studies: more SOAEs in dark-eyed individuals and in MZ twins. The view presented here is that SOAEs themselves are unlikely objects for natural selection, and probably are epiphenomena resulting from selection for those cochlear mechanisms that contribute to good hearing sensitivity - which is related to SOAE expression. It is argued that, in addition to genetics, other factors have the potential to affect the specific numbers of SOAEs that are expressed. For example, some aspects of the complex prenatal process of producing a male fetus are presumed to be responsible for the smaller number of SOAEs seen in males than females. PMID- 7559173 TI - Scaling of the mammalian middle ear. AB - This study considers the general question how animal size limits the size and information receiving capacity of sense organs. To clarify this in the case of the mammalian middle ear, I studied 63 mammalian species, ranging from a small bat to the Indian elephant. I determined the skull mass and the masses of the ossicles malleus, incus and stapes (M, I and S), and measured the tympanic membrane area, A1. The ossicular mass (in mg) is generally negatively allometric to skull mass (in g), the regression equation for the whole material (excluding true seals) being y = 1.373 x(0.513). However, for very small mammals the allometry approaches isometry. Within a group of large mammals no distinct allometry can be discerned. The true seals (Phocidae) are exceptional by having massive ossicles. The size relations within the middle ear are generally rather constant. However, the I/M relation is slightly positively allometric, y = 0.554 x(1.162). Two particularly isometric relations were found; the S/(M + I) relation for the ossicles characterized by the regression equation y = 0.054 x(0.993), and the relation between a two-dimensional measure of the ossicles and the tympanic membrane ares, (M + I)2/3 /A1. As in isometric ears the sound energy collected by the tympanic membrane is linearly related to its area, the latter isometry suggests that, regardless of animal size, a given ossicular cross-sectional area is exposed to a similar sound-induced stress. Possible morphological middle ear adaptations to particular acoustic environments are discussed. PMID- 7559171 TI - Responses of pontine neurons of the big brown bat, Eptesicus fuscus, to temporally patterned sound pulses. AB - Under free field stimulation conditions, this study examined the responses of pontine neurons of Eptesicus fucus to temporally patterned sound pulses by means of repetitive single pulses and pulse trains. Among 93 pontine neurons isolated, 90 always discharged less than 5 impulses to sound pulses presented during this study and 3 discharged impulses throughout the whole duration of each presented pulse. Responses to sound pulses at different repetition rates were examined in 65 neurons. The number of impulses of individual neurons discharged to each pulse varied within a given repetition rate and among different repetition rates. Although these pontine neurons showed different degrees of habituation to high repetition rates, more than 25% could follow the highest repetition rate tested (100 pps). However, they did not always discharge maximal number of impulses to this repetition rate. The total number of impulses discharged by a neuron was also affected by pulse duration. Thus, each pontine neuron discharged maximally to a specific combination of pulse repetition rate and duration. Using a 50% difference between the maximal and minimal responses as a criterion, the function with respect to repetition rate and duration can be described as band-pass, high pass, all-pass and irregular. These response properties reflect more those of inferior collicular neurons than auditory cortical neurons. This study also showed that response latencies of pontine neurons examined were lengthened by increasing pulse repetition rate and duration. In addition, whereas minimum thresholds of pontine neurons were elevated by increasing repetition rate, they were lowered by increasing pulse duration. PMID- 7559177 TI - What middle ear parameters tell about impedance matching and high frequency hearing. AB - Acoustic energy enters the mammalian cochlea aided by an anatomical impedance matching performed by the middle ear. The purpose of this paper is to analyse the functional consequences of changes in scale of the middle ear when going from the smallest mammals to the largest. Our anatomical measurements in mammals of different sizes ranging from bats to elephants indicate that middle ear proportions are largely isometric. Thus the calculated transformer ratio is basically independent of animal size, a typical value lying between 30 and 80. Similarly, the calculated specific acoustic input impedance of the inner ear is independent of animal size, the average value being about 140 kPa s/m. We show that if the high frequency hearing limit of isometric ears is limited by ossicle inertia, it should be inversely proportional to the cubic root of the ossicular mass. This prediction is in reasonable agreement with published audiogram data. We then present a three-parameter model of the middle ear where some obvious deviations from perfect isometry are taken into account. The high frequency hearing limits of different species generally agree well with the predictions of this simple model. However, the hearing limits of small rodents clearly deviate from the model calculation. We interpret this observation as indicating that the hearing limit towards very high frequencies may be set by cochlear transduction mechanisms. Further we discuss the exceptional high frequency hearing of the cat and the amphibious hearing of seals. PMID- 7559170 TI - Time-varying alterations in the f2-f1 DPOAE response to continuous primary stimulation. I: Response characterization and contribution of the olivocochlear efferents. AB - The f2-f1 distortion product otoacoustic emission (DPOAE) can be observed to undergo gradual alterations in amplitude during continuous ipsilateral stimulation with primary tones. In the present experiments, we characterized the dependence of these amplitude alterations on several stimulus variables (intensity, duration, frequency) and on DPOAE type (quadratic vs cubic) and tested the hypothesis that such alterations are mediated by the olivocochlear (OC) efferents. Responses were recorded in urethane-anesthetized guinea pigs with sectioned middle ear muscles before and after intracochlear application of antagonists (curare, 1 microM; bicuculline, 10 microM; tetrodotoxin, 1 microM) or before and after OC efferent section at the midline of the floor of the IVth ventricle. We confirm previous reports of continuous stimulation-related alterations in the amplitude of the quadratic distortion product, f2-f1, and report a novel, suppressive 'off-effect' apparent in f2-f1 amplitude following a short rest from such stimulation. Response alterations were sensitive to primary intensity and to duration of rest from continuous stimulation, but were not clearly frequency-dependent over the ranges tested. Corresponding alterations in the amplitude of the cubic nonlinearity, 2f1-f2 were very small or absent. Amplitude alterations in f2-f1 were reduced but not blocked by OC efferent antagonists (curare, bicuculline) and were largely unaffected by TTX or by midline brainstem section. All of these manipulations, however prevented completely the known efferent-mediated contralateral sound suppression of both f2 f1 and 2f1-f2 DPOAEs. Taken together, these results do not provide support for efferent control of the f2-f1 amplitude alterations observed during continuous ipsilateral stimulation. PMID- 7559175 TI - The junctions of the spindle-shaped cells of the stria vascularis: a link that completes the barrier between perilymph and endolymph. AB - It is current opinion that the intercellular spaces of the stria vascularis represent a closed compartment isolated from the endolymph by the tight junctions of the marginal cells and from the perilymph by the junctional complexes of the basel cells. However, it has not yet been investigated whether these two barriers meet at the stria margins toward Reissner's membrane and the spiral prominence. Possible candidates for this sealing could be junctions between the spindle shaped cells. In the present study freeze-fracture replicas of guinea pig specimens fixed in the presence of filipin were used in order to investigate the junctions of the spindle-shaped cells and to localize the cholesterol in their plasma membrane. Replicas reveal that, below the belt-like apical zonula occludens, the basolateral plasma membranes of the spindle-shaped cells adjacent to each other and to the basal cells are joined over their entire extension by a large number of junctional strands intermingled with numerous filipin-cholesterol complexes. Gap junctions are present in the meshes formed by these junctional strands. Thus, the plasma membrane of the spindle-shaped cells shows morphological and cytochemical characteristics which indicate that they are the anatomical components completing the barrier isolating the intrastrial compartment from the surrounding fluids. PMID- 7559178 TI - Acoustic features and acoustic changes are represented by different central pathways. AB - The central processing of acoustic stimulus changes can be observed neurophysiologically in the mismatch negativity auditory evoked potential (MMN). Stimuli differing in interaural phase were used to investigate the contributions of the primary and non-primary auditory pathways to the encoding of binaural stimuli and to investigate passively elicited measures of binaural processing in experimental animals. In guinea pigs, the MMN was obtained in response to 1000 Hz tones embedded in white noise (S:N = 2 dB). Using a modified oddball paradigm (that is, two stimuli presented in a series, each with a different probability of occurrence), stimuli were presented binaurally with both the tone and noise in phase to the two ears (S0N0) as the standard stimulus ans the tone 180 degrees out-of-phase (S(PI)N0) as the deviant stimulus. The MMN, by definition, should occur only in response to a change, or 'mismatch,' between the standard and deviant stimuli. The response to the deviant stimulus in the oddball paradigm was compared to the response to the same stimulus when presented in a series alone. The responses to S0N0 and S(PI)N0 collected in a series alone, termed the intrinsic responses, were also compared. Responses were recorded from two surface epidural electrodes - one at the posterior midline and one over the left temporal lobe. AEPs from these locations have been shown to reflect the activity of primary and non-primary thalamo-cortical pathways respectively. A significant MMN was observed at the midline electrode, but no MMN was observed over the temporal lobe.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559172 TI - Distribution and features of melanocytes during inner ear development in pigmented and albino rats. AB - In this developmental study, the distribution and features of melanocytes in the inner ear of pigmented and albino rats was investigated with the use of an antibody, which specifically reacts with a melanocyte differentiation antigen present in the membranes of (pre)melanosomes. Melanocyte precursors could be traced from 13 days post conception onwards and the course was followed to their targets in the inner ear. Melanocytes which settle in the modiolus appeared to reach their target along another pathway than strial and vestibular melanocytes. No difference was observed in the melanocyte distribution between pigmented and albino rats. The integration of melanocytes into the stria vascularis was associated with an increased rate of melanosome production in both strains, but in the albinos far fewer melanosomes were produced. After the stria had reached maturity, melanosome production was arrested and melanosomes were subject to lysosomal digestion. In the stria of the pigmented rats, cells with aggregations of disintegrating melanosomes appeared and persisted into adulthood. In the adult, the majority of the intermediate cells contained only a few scattered melanosomes, while melanosomes could only rarely be detected in the albinos. These observations indicate that there is a close relationship between melanosome production and the process of interdigitation of melanocytes with the marginal cells. It seems unlikely that melanosomes or melanin make any important contribution to the function of the adult stria vascularis. Outside the stria, the features of melanocytes in both strains were similar to skin melanocytes. PMID- 7559176 TI - Osteopontin expression detected in adult cochleae and inner ear fluids. AB - Localization of protein epitopes and mRNA expression showed that there was a wide spread distribution of osteopontin (OPN) within the membranous labyrinth of the adult mammalian cochleae. Immunoreaction product and mRNA were found within the stria vascularis, VIIIth cranial nerve, spiral ligament and limbus. Only specific cell types within these regions contained abundant OPN mRNA or protein, the main cell type being fibrocytes that populate the spiral limbus and spiral ligament. Epithelial cells that line the luminal surface of the stria vascularis (marginal cells) and neurons that compose the vestibular and auditory ganglia also showed high opn expression. The pattern of anti-OPN staining within membranous labyrinth was comparable to that observed in tissues such as gall bladder, breast and kidney. In those tissues, luminal epithelial cells, corresponding to the marginal cells of the stria vascularis, may be responsible for manufacturing and secreting OPN into the luminal fluids. consistent with those observations, we detected OPN epitopes in cochlear fluids withdrawn from the scalae media and tympani of the cochlea. We found that the protein species in cochlear fluid differed from those present in cerebrospinal fluid (CSF) suggesting that OPN exists in tissue specific isoforms that may correspond to particular cellular functions. PMID- 7559180 TI - Middle latency auditory evoked potentials to tones of different frequency. AB - The scalp distributions of middle latency auditory evoked potentials (MAEPs) elicited by tone bursts of 250 and 4000 Hz were compared in two experiments. Na (19.9 ms), Pa (29.8 ms), and Pb (51.4 ms) components elicited by tones of either frequency had fronto-central distributions, whereas the Nb component (38.4 ms) was maximal at parietal sites. Although the distributions of MAEP components varied as a function of the ear of stimulation, no significant differences were found as a function of tone frequency. The results are consistent with suggestions that MAEPs reflect activation of non-tonotopically organized generators. PMID- 7559179 TI - Distribution of calbindin D-28k immunoreactivity in the cochlear nucleus of the young adult chinchilla. AB - Calbindin is a 28 kD calcium-binding protein found in neural tissue. Although its functional role in nerve cell physiological processing is still uncertain, previous investigations have suggested that because of its intracellular calcium buffering and regulation properties, it could influence temporal precision of neuronal firing to subserve temporal processing in the auditory brainstem, or could mediate monaural versus binaural coding, or be involved in synaptic plasticity (learning). The present study demonstrates differential calbindin immunoreactivity in the cochlear nuclear complex of the chinchilla, a rodent with exceptionally good low-frequency hearing. The most intense labeling in the cochlear cochlear nucleus was in somata of cartwheel and fusiform cells of the fusiform cell layer, and somata and process of the molecular layer of the dorsal cochlear nucleus (DCN). Only a relatively few scattered neurons were stained in the deep layers of DCN. In contrast, moderate labeling of neurons and neuropil throughout the ventral cochlear nucleus was seen. For instance, moderately stained spherical and elongate cells of the anteroventral cochlear nucleus were observed in contact with labeled puncta and amidst stained fibers. In the cochlear nerve root region, stained auditory nerve fibers and global cells were noted. In the posteroventral cochlear nucleus, principal cells of elongate and octopus shape were observed, in contact with labeled swellings and surrounded by labeled neuropil. PMID- 7559181 TI - Effects of electrode configuration on threshold functions for electrical stimulation of the cochlea. AB - Psychophysical detection threshold vs frequency functions for sinusoidal electrical stimulation of the deafened cochlea were measured in 18 nonhuman primate subjects. Functions for monopolar or widely-spaced ( > 2.5 mm) bipolar stimulation were lower and usually had steeper slopes than those for more narrowly-spaced ( < 2.0 mm) bipolar stimulation. In 56% of the cases the difference between thresholds for narrowly-spaced bipolar of monopolar stimulation was greater for low frequency stimuli (63 or 100 Hz) than for high frequency stimuli (800 or 1,000 Hz) by 5 dB or more. Two cases were compared in more detail using pulsatile stimuli. For sinusoidal stimuli, one of these cases showed a moderate frequency dependent effect of electrode configuration and the other did not. The case with the frequency dependent effect of electrode configuration for sinusoids also showed a phase-duration dependent effect of electrode configuration for detection of single biphasic pulses: strength duration curves (detection threshold in decibels vs pulse duration in ms/phase) were steeper for monopolar stimulation than for narrowly-spaced (0.7 mm) bipolar stimulation. This effect was not seen in the case that showed little or no frequency dependence in the effect of electrode configuration for sinusoidal stimuli. Slopes of threshold vs pulse rate functions where pulse duration was held constant at 2 ms/phase were not affected by electrode configuration in either subject. PMID- 7559182 TI - Developmental expression of proteoglycans in the tectorial and basilar membrane of the gerbil cochlea. AB - The appearance and distribution of specific proteoglycans (PGs) was assessed during development and maturation of the tectorial (TM) and basilar membranes (BM) in the gerbil cochlea. At birth, monoclonal antibodies against keratan sulfate (KSPG) and chondroitin 4- or 6-sulfate (4S CSPG, 6S CSPG) reacted with the upper fibrous layer of the TM with staining for anti-KSPG predominating. Reactivity for 4S CSPG remained constant whereas that for 6S CSPG increased through day 20 when it exceeded that of 4S CSPG. The region of Kollikers organ near the developing tunnel of Corti stained positively with all three PG antibodies from birth through day 8. In contrast, cells in the developing inner spiral sulcus lacked immunoreactive KSPG but expressed CSPG. PGs were first detectable in the BM of the basal turn at day 8 and increased to near adult levels by 16 days after birth. Anti-KSPG again showed the strongest staining with labeling density for 4S and 6S CSPG being about equal at maturity. Staining with all three antibodies was localized along the margins of the BM. Reactivity of the TM and BM in the upper turns lagged behind that of the basal turns by 24-48 h. Our results show that the TM is relatively mature at birth, needing only minor changes in its PG content to reach adult levels. In contrast, the BM showed a marked increase in its content of PGs during a period corresponding to the onset and rapid development of auditory function. PMID- 7559184 TI - Treatment of infection caused by toxigenic and non-toxigenic strains of Corynebacterium diphtheriae. PMID- 7559183 TI - Masking and pitch shift of tone bursts and clicks by low-frequency tones. AB - From experiments in animals and investigations in humans it is known that the normally phase-dependent masking of a short stimulus by a low-frequency continuous tone does not occur in the case of endolymphatic hydrops. The recording of the masked threshold of short tone stimuli in a loud tone of 30 Hz is to be evaluated for the clinical diagnostics of Meniere's disease. To this purpose, the main parameters of the measurements (type, frequency, duration of the stimulus, and intensity of the masker) and their effect of phase-dependent masking and pitch-shift are investigated. Stimuli above 2 kHz are masked less than those of lower frequencies. Wide-band stimuli are less useful, since only the low-frequency component of their spectrum is masked. The tone stimuli should be short (1 - 2 ms) in order to make the measurement of the phase dependence more accurate. With increasing masker level the masking at phase 0 degree corresponds to the increase in level, at phase 270 degrees the amount is twice as much. The pitch shift which is perceived in low-tone masking depends on the phase of the stimulus, and on the levels of the stimulus and the masking tone. The use of brain stem recordings in the investigation of phase-dependent low tone masking is problematic since well-synchronizing stimuli with high frequency spectral components are masked poorly. PMID- 7559185 TI - The Garrod Lecture. Selective toxicity and concordant pharmacodynamics of antibiotics and other drugs. PMID- 7559186 TI - Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI and RPMI-2% glucose on the selection of endpoint criteria. AB - We have compared amphotericin B, flucytosine, ketoconazole and fluconazole susceptibilities of 40 clinical isolates of Candida albicans by broth microdilution in two different media: RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per L (RPMI-2% glucose). Preparation of media, drugs and inocula, as well as incubation temperature, followed the recommendations of the National Committee for Clinical Laboratory Standards (Villanova, PA, USA) antifungal agent working group for broth macrodilution tests with antifungal agents. Microtitre plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader. MIC endpoints were defined in three different ways: (i) MIC-100% for amphotericin B and flucytosine; (ii) MIC-80% for azole-drugs and also for flucytosine; (iii) IC1/2 for azole-drugs. The MIC endpoints were compared between the two different media in order to ascertain which was the best criterion. For amphotericin B, 100% of strains had a maximum difference of a twofold dilution in both media. For flucytosine, MIC values were very similar in both media, irrespective of the MIC endpoint chosen, MIC-100% or MIC-80%. For azole-drugs the (MIC-80%)50 and (MIC 80%)90 in RPMI were higher than those in RPMI-2% glucose. However, (IC1/2)50 and (IC1/2)90 were identical in both media as well as (MIC-80%)50 and (MIC-80%)90 in RPMI-2% glucose, The limited growth of yeasts in RPMI precludes the selection of an azole-MIC-80% endpoint, although the MIC determination was performed with an objective turbidimetric method (spectrophotometric reading plus mathematical MIC calculation). The use of RPMI-2% glucose produces the same MIC whatever methods was used, IC1/2 or MIC-80%. However, some minor discrepancies can be expected between IC1/2 and MIC-80% when strains with higher "trailing effect" are being tested. Therefore, we recommended IC1/2 in RPMI-2% glucose as the method of choice for MIC calculation, until more studies correlating in-vitro results with clinical outcome have been performed. PMID- 7559187 TI - Redox potential and oxygen concentration as factors in the susceptibility of Helicobacter pylori to nitroheterocyclic drugs. AB - Metronidazole sensitive (MtzS) and resistant (MtzR) strains of Helicobacter pylori were tested for their sensitivities to eleven nitroheterocyclic drugs of known reduction potential under a wide range of environmental conditions. Under microaerophilic conditions, MtzR strains were sensitive to all the 2 nitroimidazoles, resistant to every 5-nitroimidazole, and slightly sensitive to the nitrothiazole, niridazole. MtzS strains were sensitive to all the nitroimidazoles except for 4(5)-nitroimidazole which has the lowest redox potential of all the drugs investigated. MtzS strains displayed the greatest sensitivity towards niridazole and satranidazole, the latter having the highest redox potential of the 5-nitroimidazoles. High and low oxygen tensions had no effect on the activity of the drugs but periods of anaerobic incubation caused resistant strains to become sensitive to the 5-nitroimidazoles. Superoxide dismutase and catalase were not induced by metronidazole and enzyme levels showed no correlation with resistance patterns. The results show that futile cycling does not occur in H. pylori and that the mechanism of action of the nitroimidazoles is related to their redox potential. Anaerobiosis abolishes resistance to metronidazole which suggests that it may be mediated through the activation of anaerobic metabolic pathways which function less under microaerophilic conditions or not at all. PMID- 7559188 TI - Activity of cefepime against ceftazidime-resistant gram-negative bacilli using low and high inocula. AB - Cefepime is a broad-spectrum cephalosporin that is reported to have enhanced activity against ceftazidime-resistant Gram-negative bacilli. In this study the effects of varying inoculum size on in-vitro susceptibility to cefepime and other selected antimicrobial agents were determined by agar dilution MICs and in time kill studies. Among strains of Pseudomonas aeruginosa (n = 55) and Enterobacter spp (n = 56) that had previously been identified as ceftazidime-resistant, 73% and 96% were susceptible to cefepime (MIC < or = 16 mg/L), respectively, when tested with an inoculum of 10(4) cfu. However, with an inoculum of 10(7) cfu, 98% and 100% of strains were resistant, respectively. Furthermore, the bactericidal activity of cefepime against ceftazidime-resistant isolates was also inoculum dependent. In time-kill studies, bactericidal action was obtained only at the lowest concentration of organisms (10(4) cfu/mL). beta-Lactamase extracted from an isolate of P. aeruginosa that demonstrated an inoculum effect had a lower affinity for cefepime than for ceftazidime. Overall, cefepime proved to be more resistant to hydrolysis by the beta-lactamase. However, differences in kinetics of the beta-lactamase against cefepime or ceftazidime do not appear to be of consequence in determining susceptibility of P. aeruginosa and Enterobacter spp. at high bacterial densities, since most strains with chromosomally-mediated beta lactamase are highly resistant. PMID- 7559189 TI - The in-vitro susceptibilities of vaginal lactobacilli to four broad-spectrum antibiotics, as determined by the agar dilution and E test methods. AB - The minimum inhibitory concentrations (MICs) of cefaclor, co-amoxiclav, clarithromycin and ciprofloxacin for 59 strains of vaginal lactobacilli were determined by both the reference agar dilution and E test methods. With the exception of clarithromycin, there was poor correlation between the results obtained by the two techniques. This was most apparent for the beta-lactams studied, the MICs of cefaclor as determined by the E test being particularly difficult to define. The stability of antimicrobial gradients in the E test may cause problems when testing slow-growing bacteria and/or organisms which grow only under anaerobic conditions. Accordingly, only those MICs determined by the agar dilution method are reported. The percentages of susceptible isolates were as follows: clarithromycin, 100; co-amoxiclav, 100; cefaclor, 20; and ciprofloxacin, 4. The administration of antimicrobials, such as ciprofloxacin and cefaclor, which have poor activities in vitro against lactobacilli, may therefore be advantageous to the host because it allows the protective effects of the normal vaginal flora to be preserved. PMID- 7559190 TI - Prevalence of penicillin resistant bacteria in acute suppurative oral infection. AB - Pus aspirated from acute suppurative oral infections in 78 patients (age range 13 76 years) yielded a total of 331 bacterial strains consisting of 143 facultative anaerobes (predominantly Streptococcus spp.) and 188 strict anaerobes (predominantly Prevotella spp.). Seventy-five isolates (23%) were resistant to penicillin (MIC > 1 mg/L), 37 (11%) were resistant to ampicillin (MIC > 2 mg/L) and 16 (5%) isolates were resistant to amoxycillin/clavulanic acid (MIC > 2 mg/L). Samples from 43 (55%) of the patients yielded at least one penicillin resistant isolate and within this group 30 samples (73%) contained at least one strain which produced beta-lactamase. A history of antibiotic therapy during the 6 months before enrollment in the study did not influence the isolation of penicillin resistant bacteria. It is concluded that penicillin resistant bacteria are often present in the microflora of acute dental infection. PMID- 7559191 TI - Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. AB - The in-vitro susceptibilities of 40 clinical isolates of Candida albicans to ketoconazole and fluconazole were determined and an attempt was made to correlate these data with the clinical responses of the patients from whom the strains were originally isolated to treatment with these agents. Of 40 patients with the acquired immunodeficiency syndrome (AIDS) with oropharyngeal and/or oesophageal candidosis, 21 received ketoconazole and 19 fluconazole. Susceptibility testing was performed by a microbroth dilution method with RPMI-2% glucose medium according to the recommendations of the National Committee for Clinical Laboratory Standards; growth inhibition was estimated spectrophotometrically and the MIC endpoint was defined in terms of the IC1/2. The MICs of 236 additional strains of C. albicans, which were also isolated from AIDS patients, were used to establish a susceptibility profile for this species. On the basis of the susceptibility test results and the clinical responses of the 40 patients, the following tentative breakpoints for ketoconazole and fluconazole are proposed: patients with infections caused by C. albicans strains with MICs of ketoconazole and fluconazole or < or = 0.001 and < or = 0.25 mg/L respectively would be expected to respond to treatment with these agents and isolates with MICs which meet these criteria are therefore classified as susceptible; patients with infections caused by strains with MICs of ketoconazole and fluconazole of > or = 0.06 and > or = 16.0 mg/L respectively would not be expected to respond to treatment with these agents and isolates with MICs which meet these criteria are therefore classified as resistant; the response of patients with infections caused by strains with MICs of ketoconazole and fluconazole of 0.003-0.03 and 0.5 8.0 mg/L respectively cannot be reliably predicted and isolates with MICs which fall within these ranges are therefore classified as being of indeterminate susceptibility. The present study demonstrates that the results of in-vitro susceptibility testing with RPMI-2% glucose broth correlate with the clinical response to therapy and can be used to facilitate optimal treatment in AIDS patients with oropharyngeal and/or oesophageal candidosis. PMID- 7559193 TI - In-vitro and in-vivo activities of roxithromycin in combination with pyrimethamine or sulphadiazine against Toxoplasma gondii. AB - The efficacy of roxithromycin alone or in combination with pyrimethamine or sulphadiazine was examined in vitro and in a murine model of acute toxoplasmosis. In-vitro studies were performed with MRC5 fibroblast tissue cultures, with quantification of toxoplasma growth by an enzyme-linked immunosorbent assay. For in-vivo studies, mice were infected with 10(4) tachyzoites of the virulent RH strain and then treated perorally for 10 days from day 1 after infection. The efficacy of each drug regimen was assessed by determination of survival rates and sequential titration of parasites in blood, brain and lungs, using a tissue culture method. In vitro, roxithromycin inhibited toxoplasma growth at a concentration of > or = 0.02 mg/L; the 50% inhibitory concentration was estimated to be 1.34 mg/L. No synergistic effect was observed when it was combined with pyrimethamine or sulphadiazine. In vivo, roxithromycin alone at 50 or 200 mg/kg/day slightly prolonged survival compared with untreated mice, but a striking synergistic effect was observed when roxithromycin was administered in combination with pyrimethamine or sulphadiazine at subtherapeutic doses, i.e., 12.5 and 100 mg/kg/day, respectively. Combination regimens consistently resulted in a marked reduction fo the parasite burdens in blood and tissue, compared with those in mice treated with any of the agents alone. These results suggest that in vivo activities of either pyrimethamine or sulphadiazine against T. gondii are reinforced by roxithromycin and such combinations should be considered in development of alternative treatments for human toxoplasmosis. PMID- 7559192 TI - Chemotherapeutic activity of levofloxacin (HR 355, DR-3355) against systemic and localized infections in laboratory animals. AB - Ofloxacin, its optical isomers levofloxacin (HR 355, DR-3355) and D-ofloxacin (DR 3354) and ciprofloxacin were administered orally to mice and rats which had systemic and localized infections. Both levofloxacin and ciprofloxacin were equally effective in treating systemic murine infections caused by staphylococci. Enterobacteriaceae or Pseudomonas aeruginosa with ED50s ranging from 0.18 to 15.8 mg/kg and 0.42 to 16.3 mg/kg respectively and both these agents were twice as effective as ofloxacin which had an ED50 0.41 to 39.7 mg/kg. In contrast, D ofloxacin was either inactive or exhibited only modest chemotherapeutic activity against the staphylococci and the Gram-negative organisms tested. When given to mice to treat staphylococcal abscesses and lung infections due to Klebsiella pneumoniae DT-S levofloxacin was up to four times more effective and produced a more pronounced bactericidal effect against the pathogens in vivo than the reference compounds. Despite possessing a similar, if not lesser, in-vitro activity against the infecting pathogens, levofloxacin was more effective than ofloxacin and ciprofloxacin in rats with localized infections caused by Enterobacteriaceae and P. aeruginosa. PMID- 7559194 TI - Absorption of clindamycin after intravaginal application of clindamycin phosphate 2% cream. AB - In recent clinical studies, clindamycin phosphate cream administered intravaginally has been shown to be efficacious in the treatment of bacterial vaginosis (BV). In support of the safety profile for this dosage form, a study assessing the systemic absorption of clindamycin was undertaken in five BV patients and six healthy volunteers who were dosed with 5 mL (100 mg) clindamycin phosphate 2% cream intravaginally once daily in the evening for 7 days. Two weeks later, a single 100 mg dose of clindamycin phosphate sterile solution was administered as a 4 min iv infusion. Serial blood samples were collected on days 1, 4, and 7 after dosing with the cream and over a 16 h period after the iv treatment. Clindamycin concentrations in serum samples were assayed by capillary gas chromatography. After treatment with the cream, steady-state serum clindamycin concentrations were reached by 24 h in all but one subject in each group. Mean absolute bioavailability was 2.7%-4.3% in the BV group and 2.7%-4.7% in the healthy group. These results indicate that systemic exposure to clindamycin from intravaginal administration of clindamycin phosphate cream is minimal. PMID- 7559195 TI - A double-blind randomized trial comparing the efficacy and safety of a 5-day course of cefotiam hexetil with that of a 10-day course of penicillin V in adult patients with pharyngitis caused by group A beta-haemolytic streptococci. AB - A 10-day course of penicillin is the antibiotic regimen currently recommended by the American Heart Association (AHA) as treatment for patients with tonsillitis caused by group A beta-haemolytic streptococci (GABHS), with the aim of preventing both the suppurative and non-suppurative complications of this infection. This prospective, multicentre, randomized, double-blind, double-dummy clinical trial was undertaken in order to compare the efficacy of, tolerability of and compliance with a 5-day course of cefotiam hexetil (CTM) 200 mg bd with that of a 10-day course of penicillin V (PEV) 1 megaunit (600 mg) tds, to investigate the significance of recovering GABHS during or after treatment and to evaluate the potential economic advantages of short-term regimens. Two hundred and fifty ambulatory adult patients with a presumptive diagnosis (based on a positive rapid antigen detection test) of GABHS tonsillitis were recruited in 60 centres; the diagnosis was subsequently confirmed by a positive culture of a throat swab. At the time of entry into the trial there was no statistically significant difference between the groups in terms of clinical symptoms. In an intention-to-treat analysis, both the clinical and bacteriological response rates at days 10 and 30 were comparable for each group i.e. 106 of 119 (89.1%) patients and 90 of 109 (82.6%) patients respectively in the CTM group and 103 of 117 (88.0%) patients and 92 of 107 (86.0%) patients respectively in the PEV group. The times until defervescence and resolution of symptoms were also similar. Of the 115 patients in each group who were assessed at day 90, there were three clinical relapses in the CTM group and seven in the PEV group. No non-suppurative complications of GABHS infection were detected. Tolerance was significantly better in the CTM group than in the PEV group, 14 of 119 (11.8%) patients and 26 of 117 (22.2%) patients in the former and latter groups respectively reporting adverse events. In three cases in each group treatment was discontinued prematurely because of adverse events; none of these in the CTM group was serious but one patient in the PEV group experienced a severe allergic reaction. Compliance in both groups was good during the first 5 days of therapy but, by the end of each course, 93.6% of patients in the CTM group had completed treatment, compared with 73.0% in the PEV group.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559196 TI - Intravenous ciprofloxacin as treatment for patients with acute suppurative cholangitis: a randomized, controlled clinical trial. AB - One hundred consecutive patients with acute suppurative cholangitis were randomized in a prospective, controlled clinical trial to receive either ciprofloxacin (200 mg bd iv) or triple therapy comprising ceftazidime (1 g bd iv), ampicillin (500 mg qds iv) and metronidazole (500 mg tds iv); 46 and 44 patients in the ciprofloxacin and triple therapy groups respectively were suitable for inclusion in the analysis of efficacy. In two-thirds of the patients biliary obstruction was caused by ductal calculi and in one-third by malignant or benign strictures of the biliary tract. Bacteraemia was documented in 38% of patients in the ciprofloxacin group and in 34% of patients in the triple therapy group, while bile cultures were positive in 87% and 92% of patients in the ciprofloxacin and triple therapy groups respectively. Escherichia coli, Klebsiella spp. and Enterococcus spp. were the most common biliary isolates. Eighty-five per cent of evaluable patients in the ciprofloxacin group and 77% of those in the triple therapy group responded to therapy. The mean durations of fever, septicaemic shock and hospitalization were also similar in the two treatment groups. Six (13%) patients in the ciprofloxacin group and seven (16%) in the triple therapy group required urgent endoscopy or surgery for uncontrolled infection. Recurrence of fever after an initial response was documented in one (2%) patient receiving ciprofloxacin and in three (7%) patients receiving triple therapy. The incidences of mortality were 4% in the ciprofloxacin group and 2% in the triple therapy group. The results of this study suggest that ciprofloxacin alone is adequate empirical therapy for patients with cholangitis. PMID- 7559198 TI - The in-vitro activity of CP 99,219, a new naphthyridone antimicrobial agent: a comparison with fluoroquinolone agents. AB - The in-vitro activity of a new fluoroquinolone, CP 99,219 was compared with that of ciprofloxacin, DU 6859, sparfloxacin and levofloxacin. CP 99,219 showed generally similar in-vitro activity to the other compounds tested against the Enterobacteriaceae (MIC90 of all agents < 0.5 mg/L except Morganella morganii). It was found to be more active than ciprofloxacin, sparfloxacin and levofloxacin against the strains of Acinetobacter calcoaceticus tested (MIC90 1 mg/L). CP 99,219 was found to be four-fold more active against strains of Stenotrophomonas maltophilia than ciprofloxacin (MIC90 1 and 8 mg/L, respectively). Haemophilus influenzae and Neisseria spp. were highly susceptible to all the agents tested. CP 99,219 was more active than ciprofloxacin, sparfloxacin and levofloxacin against Gram-positive organisms, but less active than DU 6859. The enhanced anti Gram-positive activity of CP 99,219 was most marked against the nine strains of methicillin-resistant Staphylococcus aureus tested. The MIC90 of CP 99,219 of these strains was 1.0 mg/L compared with 64 mg/L for ciprofloxacin. Against Bacteroides fragilis, CP 99,219 (MIC90 0.25 mg/L) and DU 6859 (MIC90 0.03 mg/L) were more active than the other quinolones. Chlamydia spp. were susceptible to < or = 0.12 mg/L of CP 99,219. Mycobacterial strains tested were less susceptible to CP 99,219 than to ciprofloxacin. The protein binding of CP 99,219 was 87.9% at 1 mg/L. PMID- 7559197 TI - Activity of the semi-synthetic kanamycin B derivative, arbekacin against methicillin-resistant Staphylococcus aureus. AB - Arbekacin (1-N-((S)-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin B) was active against 54 strains of methicillin-resistant Staphylococcus aureus from 16 different countries, all of which were resistant to > or = 32 mg/L amikacin. MICs of arbekacin ranged from 1 to 16 mg/L, the MIC50 was 3.2 mg/L and the mode geometric mean MICs were 4 and 4.5 mg/L respectively. PMID- 7559199 TI - In-vitro activity of two glycylcyclines against enterococci resistant to other agents. AB - Two new glycylcyclines, CL 329,998 and CL 331,002, were tested for in-vitro activity against 178 clinical strains of enterococci which were resistant to one or more of the commonly used agents (ampicillin, high level gentamicin, vancomycin and teicoplanin). Both glycylcyclines demonstrated good activity (MICs < or = 0.5 mg/L) against all isolates tested, including those strains which demonstrated multiple resistance. As resistant enterococci are increasing in importance, new agents with activity against these strains are urgently required. Glycylcyclines have in-vitro activity which suggests they may be useful agents for treatment of infections caused by these organisms. PMID- 7559200 TI - Comparative activity of ampicillin, amoxycillin, amoxycillin/clavulanate and cefotaxime against 189 penicillin-susceptible and -resistant pneumococci. AB - Agar dilution and E-test MICs of penicillin G, ampicillin, amoxycillin, amoxycillin/clavulanate and cefotaxime were determined against 189 pneumococci. Addition of clavulanate did not influence amoxycillin MICs, MIC50/MIC90 (mg/L) for penicillin G, ampicillin, amoxycillin and cefotaxime were: Penicillin susceptible strains (62): 0.03/0.03; 0.03/0.06; 0.015/0.03; 0.015/0.06. Penicillin intermediate strains (69): 0.25/1.0; 0.25/4.0; 0.125/1.0; 0.125/1.0. Penicillin resistant strains (58): 2.0/4.0; 4.0/4.0; 1.0/2.0; 1.0/2.0. E-test MICs correlated well with those obtained by agar dilution. PMID- 7559201 TI - Combined therapy with co-amoxiclav and erythromycin against intracellular Legionella pneumophila in cell culture and in an experimental respiratory infection in neutropenic rats. PMID- 7559203 TI - Empiric antibiotic therapy for acute infective diarrhoea in the developed world. PMID- 7559202 TI - Comparison of rufloxacin and norfloxacin effects on faecal flora. AB - The suppression of faecal Escherichia coli by quinolones was studied in a randomized cross-over phase I trial with single oral doses of 400 mg rufloxacin or norfloxacin. Both drugs caused > 70% decontamination in a similar number of subjects in the 72 h post-dose period (83% vs 100% respectively), but suppression of E. coli at 168 h was significantly (P < 0.05) higher after rufloxacin (83.3 vs 40%). PMID- 7559204 TI - Susceptibility testing of Streptococcus pneumoniae to trimethoprim. PMID- 7559205 TI - Phorbol ester potentiation of canine pulmonary vasoreactivity to histamine. AB - The effect of phorbol myristate acetate (PMA) on canine pulmonary vasoreactivity to histamine was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured by using vascular occlusion techniques. Histamine (10(-5) M) significantly increased postcapillary resistance by venoconstriction and significantly attenuated total vascular compliance by decreasing large-vessel compliance and middle-compartment compliance. Pretreatment with the phorbol ester PMA (10(-7) M) significantly potentiated the vasoactive response to histamine and elicited an edemagenic effect in the isolated dog lung through modulation of the histaminergic vasoconstrictor effect on precapillary resistance, postcapillary resistance, and pulmonary vascular compliance. Pretreatment with the protein kinase C inhibitors staurosporine (10( 7) M) and calphostin C (10(-6) M) and the dihydropyridine Ca2+ channel blocker nifedipine (10(-5) M) significantly attenuated the effect of PMA on histaminergic mediated vasoconstriction. The results of this study indicate that phorbol esters may exert their effect on canine pulmonary vasoreactivity predominantly through activation of protein kinase C and influx of Ca2+ through voltage-dependent Ca2+ channels. PMID- 7559207 TI - Influence of cardiac action on gas mixing in closed-chest dogs. AB - We performed single-breath tests in closed-chest, paralyzed, and anesthetized dogs (6 with bilateral vagotomy and 6 with intact vagi) with the heart beating and during cardiac arrest. Repeated cardiac arrest was achieved by ventricular fibrillation and subsequent defibrillation. Twenty-four single-breath tests per dog were performed in combinations of three inspiratory volumes (VI; 0.2, 0.5, and 0.8 liter) and four postinspiratory pauses (0, 5, 10, and 30 s), either with or without cardiac arrest. The test gas contained four inert relatively insoluble gases (He, Ne, Ar, and SF6) with a sixfold range in diffusivity. Series dead space (VD) decreased with increasing postinspiratory pause, increasing gas diffusivity, or decreasing VI. In vagotomized animals, VD was smaller with the heart beating than during cardiac arrest, but this relationship was reversed in animals with intact vagi. The decrease in VD due to cardiogenic mixing accounted for only 10.8% of the total decrease in VD occurring during a 30-s postinspiratory pause. The slope of phase III decreased with increasing postinspiratory pause except at VI of 0.2 liter. No significant differences were noted in the slope of phase III between experiments performed with the heart beating or arrested. Tracer gas retained in the residual volume after expiration increased with increasing inspiratory pause. Gas retention was greater for He than for SF6 but was not affected by cardiac action. These findings indicate that cardiac action mainly affects the interface between inspired and alveolar gas and has little effect on mixing in the alveolar compartment. PMID- 7559206 TI - Role of endothelial factors in active hyperemic responses in contracting canine muscle. AB - We investigated whether endothelium-derived relaxing factor (EDRF) and prostaglandins, which may be released under conditions of increased blood flow, contribute to the active hyperemia in contracting muscle of anesthetized dogs. The venous outflow from the left gastrocnemius muscle was isolated and measured. The tendon was cut and placed in a force transducer. One group served as a control (Con; n = 9); EDRF synthesis was inhibited using N omega-nitro-L-arginine methyl ester (L-NAME) in a second group (n = 9), and a third group (n = 7) received L-NAME and indomethacin (L-NAME+Indo) to inhibit prostaglandin synthesis. After resting measurements, the distal end of the cut sciatic nerve was stimulated to produce isometric contractions at 1, 2, 4, and 6 twitches/s for 6-8 min, separated by 25-min recovery periods. Blood flow and O2 uptake increased linearly from resting values of 11.8 +/- 2.4 and 0.3 +/- 0.05 ml.100 g-1.min-1, respectively, to maximal values of 84.2 +/- 5.1 and 11.1 +/- 0.7 ml.100 g-1.min-1 in the Con group; neither these values nor those for tension development were different from values observed at comparable contraction frequencies in the L NAME and L-NAME+Indo groups. At rest, resistance was greater (P < 0.05) in both the L-NAME and L-NAME+Indo groups compared with Con, the highest value (P < 0.05) occurring in the L-NAME+Indo group. Muscle resistance decreased (P < 0.05) in all groups at all contraction frequencies; the values were not different among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559208 TI - Lipocortin-1 distribution in bronchoalveolar lavage from healthy human lung: effect of prednisolone. AB - Lipocortin-1 (LC-1; annexin-1) may mediate some anti-inflammatory actions of the glucocorticoids, probably after binding to specific cell surface binding sites. We have quantified LC-1 levels in bronchoalveolar lavage (BAL) fluid and cells collected from seven healthy volunteers before and after 7 days of treatment with an oral glucocorticoid, prednisolone (30 mg/day). Extracellular BAL LC-1 was higher and cellular LC-1 was lower after prednisolone than before [extracellular: before, median 98 ng/mg albumin (range 48-350 ng/mg albumin); after, 236 ng/mg albumin (19-414 ng/mg albumin); P < 0.05. Cellular: before, 23.3 ng/10(6) cells (14.6-26.9 ng/10(6) cells); after, 18.0 ng/10(6) cells (122-268 ng/10(6) cells); P < 0.05]. The distribution of LC-1 within BAL cells ex vivo (cell surface = 25%, cytosol = 50%, membrane = 25%) was unaffected by prednisolone treatment. However, in adherent cells that had been cultured for 4 h, 70-80% of the LC-1 was on the cell surface. In summary, prednisolone appears to promote cellular release of LC 1. The difference in distribution of cellular LC-1 in BAL cells ex vivo and in vitro may reflect adherence and/or activation. PMID- 7559209 TI - Superoxide dismutase derivative reduces oxidative damage in skeletal muscle of rats during exhaustive exercise. AB - A superoxide dismutase derivative (SM-SOD) that circulates and is bound to albumin with a half-life of 6 h was injected intraperitoneally into rats before exhaustive treadmill running to study its antioxidant scavenging capacity in the plasma and soleus and tibialis muscles. The exercise induced a marked increase in xanthine oxidase activity in plasma and an increase in thiobarbituric acid reactive substances in the plasma as well as in the soleus and tibialis muscles of nonadministered rats immediately after the exercise. The immunoreactive content and activity of both SOD isoenzymes (Cu,Zn-SOD and Mn-SOD) of the nonadministered rats increased in the soleus and tibialis muscles immediately after running. SM-SOD treatment definitely attenuated the degree of the increase in thiobarbituric acid-reactive substances and xanthine oxidase in all samples examined immediately after exercise. Glutathione peroxidase activity significantly increased in the soleus muscle of nonadministered rats 1 day after running, whereas catalase activity remained unchanged throughout the experimental period. These results suggest that a single bout of exhaustive exercise induces oxidative stress in skeletal muscle of rats and that this oxidative stress can be attenuated by exogenous SM-SOD. PMID- 7559212 TI - Effects of glutamine on the immune system: influence of muscular exercise and HIV infection. AB - Glutamine increased the proliferative response and the lymphokine-activated killer cell activity of blood mononuclear cells isolated from normal healthy subjects (n = 6) in a dose-dependent manner, with optimum at 0.3-1.0 mM. The relative fraction of CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells was not changed by glutamine at a concentration of 0.6 mM, except in the phytohemagglutinin-stimulated proliferation experiment where the fraction of CD4+, and therefore CD3+ cells, increased. The natural killer cell activity was not influenced by glutamine. Human immunodeficiency virus (HIV)-seropositive subjects (n = 8) who performed concentric bicycle exercise for 1 h at 75% of maximal O2 consumption had an overall lower phytohemagglutinin-stimulated proliferative response, compared with the HIV-seronegative control group (n = 7). The proliferation during exercise was lower in both the HIV-seropositive and the HIV-seronegative group. Addition of glutamine in vitro did not normalize the lower proliferation in the HIV-seropositive group or the attenuated proliferation seen during exercise in both groups. PMID- 7559210 TI - Brain glucose metabolism in hypobaric hypoxia. AB - Hypobaric hypoxia at one-half atmospheric pressure for 3 wk was reported to increase the brain capillary density and glucose transport at the blood-brain barrier in the adult rat. We examined the metabolic concomitants of these alterations in rats subjected to the same hypoxic insult. Hypoxic rats increased brain glucose and lactate concentrations and decreased brain glycogen. However, hypoxia had no significant effects on regional brain levels of ATP and phosphocreatine or on intracellular pH, indicating successful adaptation to the hypoxic insult. 2-Deoxyglucose studies showed that hypoxia increased the regional metabolic rate for glucose by 10-40%. These results indicate increased glycolysis in the hypoxic rat brain, which probably underlies the increased density of glucose transporters in brain microvessels and the increased blood-to-brain glucose influx in hypoxia. PMID- 7559211 TI - Circulating endothelin-1 reduces splanchnic and renal blood flow and splanchnic glucose production in humans. AB - The effect of minimal changes in circulating plasma endothelin-1 (ET-1) was studied in 12 healthy subjects receiving either 60 min of ET-1 (0.2 pmol.kg-1.min 1) or physiological saline intravenously. Blood was drawn from arterial, renal, and central hepatic vein catheters. Arterial ET-1-like immunoreactivity (ET-1-LI) increased from 4.7 +/- 0.4 (SE) to 8.6 +/- 1.0 pmol/l during ET-1 infusion. After 10 min, plasma ET-1-LI had increased to 6.12 +/- 0.29 pmol/l. For comparison the plasma ET-1-LI level was 12.9 +/- 4.2 pmol/in five patients with sepsis syndrome. Mean arterial blood pressure rose from 92 +/- 3 to 99 +/- 4 mmHg. Estimated splanchnic and renal blood flows fell by 18 +/- 5 and 10 +/- 3%, respectively, and splanchnic glucose production fell by 42 +/- 6% within 10 min of the ET-1 infusion and differed compared with corresponding control values. Only estimated splanchnic blood flow had increased 60 min after the ET-1 infusion. No change in splanchnic uptake of lactate or glycerol was seen. In conclusion, we suggest that circulating ET-1 with small or no demonstrable change in plasma concentration interferes with vasoactivity and splanchnic glycogenolyses in health and possibly pathophysiological conditions. PMID- 7559215 TI - Fiber type-specific effects of clenbuterol and exercise training on insulin resistant muscle. AB - The interrelationships among glucose uptake, GLUT-4 protein, and citrate synthase activity in insulin-resistant skeletal muscle were investigated. Female obese (fa/fa) Zucker rats were randomly assigned to treadmill training, ingestion of the selective beta 2-adrenergic agonist clenbuterol, or sedentary control groups. After 7-8 wk of treatment, hindlimbs were perfused to determine maximal insulin stimulated (10 mU/ml) 2-[3H]deoxy-D-glucose (2-DG) uptake. Exercise training significantly enhanced 2-DG uptake and GLUT-4 protein in red gastrocnemius and plantaris. Alternatively, 2-DG uptake was not altered in soleus after exercise training despite a 52% increase in GLUT-4 protein. The increases in GLUT-4 protein in red gastrocnemius, plantaris, and soleus of the trained rats were accompanied by increases in citrate synthase activity. In contrast to exercise training, clenbuterol administration decreased citrate synthase activity in red and white gastrocnemius, yet had no effect on GLUT-4 protein levels or maximal insulin-stimulated 2-DG uptake. Clenbuterol treatment did, however, increase citrate synthase activity and GLUT-4 protein in soleus. These findings indicate that total GLUT-4 protein largely determines the maximal rate of insulin stimulated glucose uptake in fast-twitch muscle, whereas in slow-twitch muscle it does not. In addition, the results demonstrate that coordination of proteins governing glucose uptake and disposal may be disrupted in a fiber type-specific manner. Overall, the findings raise important questions as to whether regulation of proteins governing glucose uptake and disposal differs significantly among fiber types. PMID- 7559213 TI - Alterations in uteroplacental blood flow precede hypertension in preeclampsia at high altitude. AB - High-altitude residence during pregnancy is associated with an increased incidence of preeclampsia. To determine whether uteroplacental blood flow was reduced and pelvic blood flow distribution altered before the onset of hypertension, we measured common iliac (CI), uterine (UA), and external iliac (EI) artery flow velocities (FV), indexes of flow distribution, and blood volume (BV) at week 12, 24, and 36 of pregnancy and 6 mo postpartum in 23 normotensive, 7 preeclamptic, 5 transiently hypertensive, and 3 chronically hypertensive residents of 3,100 m. Normotensive women had a progressive increase in CIFV and UAFV, decrease in EIFV, redistribution of CIFV from the EI to the UA, and increase in BV with advancing pregnancy. Preeclamptic women attained maximal UAFV and redistribution of CIFV from the EI to the UA well before the onset of hypertension and, unlike normotensive women, showed no further increases near term. Plasma volume increment with pregnancy related to the fall in the EIFV/CIFV ratio. Transiently hypertensive women resembled normotensive subjects in the parameters measured, whereas chronically hypertensive subjects resembled preeclamptic subjects. We concluded that preeclamptic vs. normotensive pregnant residents of high altitude had less redistribution of CI flow to the UA and no increase in UA blood flow near term. That these differences were present before the onset of hypertension supports the concept that preeclampsia is characterized by an incomplete vascular adjustment to pregnancy. PMID- 7559216 TI - Effect of unweighting on skeletal muscle use during exercise. AB - Exercise-induced spin-spin relaxation time (T2) shifts in magnetic resonance (MR) images were used to test the hypothesis that more muscle would be used to perform a given submaximal task after 5 wk of unweighting. Before and after unilateral lower limb suspension (ULLS), 7 subjects performed 5 sets of 10 unilateral concentric actions with the quadriceps femoris muscle group (QF) at each of 4 loads: 25, 40, 55, and 70% of maximum. T2-weighted MR images of the thigh were collected at rest and after each relative load. ULLS elicited a 20% decrease in strength of the left unweighted QF and a 14% decrease in average cross-sectional area (CSA) with no changes in the right weight-bearing QF. Average CSA of the left or right QF showing exercise-induced T2 shift increased as a function of exercise intensity both before and after ULLS. On average, 12 +/- 1, 15 +/- 2, 18 +/- 2, and 22 +/- 1 cm2 of either QF showed elevated T2 for the 25, 40, 55, and 70% loads, respectively, before ULLS. Average CSA of the left but not the right QF, showing elevated T2 after ULLS, was increased to 16 +/- 2, 23 +/- 3, 31 +/- 7, and 39 +/- 5 cm2, respectively. The results indicated that unweighting increased exercise-induced T2 shift in MR images, presumably due to greater muscle mass involvement in exercise after than before unweighting, suggesting a change in motor control. PMID- 7559217 TI - Effects of nasal positive-pressure hyperventilation on the glottis in normal awake subjects. AB - We have recently observed obstructive apneas during nasal intermittent positive pressure ventilation (nIPPV) and suggested that they were due to hypocapnia induced glottic closure. To confirm this hypothesis, we studied seven healthy subjects and submitted them to nIPPV while their glottis was continuously monitored through a fiber-optic bronchoscope. During wakefulness, we measured breath by breath the widest inspiratory angle formed by the vocal cords at the anterior commissure along with several other indexes. Mechanical ventilation was progressively increased up to 30 l/min. In the absence of diaphragmatic activity, increases in delivered minute ventilation resulted in progressive narrowing of the vocal cords, with an increase in inspiratory resistance and a progressive reduction in the percentage of the delivered tidal volume effectively reaching the lungs. Adding CO2 to the inspired gas led to partial widening of the glottis in two of three subjects. Moreover, activation of the diaphragmatic muscle was always associated with a significant inspiratory abduction of the vocal cords. Sporadically, complete adduction of the vocal cords was directly responsible for obstructive laryngeal apneas and cyclic changes in the glottic aperture resulted in waxing and waning of tidal volume. We conclude that in awake humans passive ventilation with nIPPV results in vocal cord adduction that depends partly on hypocapnia, but our results suggest that other factors may also influence glottic width. PMID- 7559218 TI - Effects of nasal positive-pressure hyperventilation on the glottis in normal sleeping subjects. AB - We have previously observed that, in normal awake subjects passively hyperventilated with intermittent positive-pressure ventilation delivered through nasal access (nIPPV), the glottis could interfere with the ventilation. We report on data obtained in the same subjects during stable sleep. In all cases, the glottis was continuously observed through a fiber-optic bronchoscope, and other indexes were also continuously recorded. Mechanical ventilation was progressively increased up to 30 l/min. We have observed during passive nIPPV in stable sleep that increases in delivered minute ventilation (VEd) resulted in progressive narrowing of the glottic aperture, with increases in inspiratory resistance and progressive reductions in the percentage of the delivered tidal volume effectively reaching the lungs. For a given level of VEd, comparisons showed that the glottis was significantly narrower during sleep than during wakefulness and that the glottis was significantly narrower during stage 2 than during stages 3/4 non-rapid-eye-movement sleep. Moreover, when CO2 is added to the inspired air, glottic aperture increased in five of nine trials without changes in sleep stage. We also observed a significant negative correlation between glottic width and the VED, independent of the CO2 level. We conclude that during nIPPV glottis narrowing results in a decrease in the proportion of the delivered tidal volume reaching the lungs. PMID- 7559220 TI - Monitoring urine oxygen tension during acute change in cardiac output in dogs. AB - To evaluate whether renal blood flow (RBF) can be monitored during acute change in cardiac index, ureter urine oxygen tension (PuO2) and bladder urine oxygen tension (PbO2) were measured in six mongrel dogs. PuO2, cardiac index, and RBF increased after dobutamine infusion and decreased after propranolol infusion. PuO2 had an excellent correlation with RBF (r = 0.94) and a fair correlation with cardiac index (r = 0.50) and mean blood pressure (r = 0.56); RBF had a fair correlation with mean blood pressure (r = 0.52, P < 0.05) but was not related to cardiac index. With multiple-regression analysis, PuO2 was found to be the significant factor related to RBF. PbO2 had a good correlation with PuO2 (r = 0.94) at control levels. Furthermore, when two dogs were added to evaluate relationships among PbO2, PuO2, and RBF, PbO2 had an excellent correlation with PuO2 (r = 0.92) and RBF (r = 0.91). These data indicate that PuO2 is a more sensitive predictor of RBF than cardiac index and mean blood pressure and that PbO2 can be a noninvasive indicator reflecting RBF during acute circulatory change in dogs. PMID- 7559219 TI - Exercise training-induced changes in anterior pituitary gonadotrope of the female rat. AB - Chronic exercise training may alter the hypothalamic-pituitary-ovarian axis by affecting gonadotropin secretion and structural indexes of the anterior pituitary gonadotropes. Young female Harlan Sprague-Dawley rats were randomly assigned to either a 12-wk intensive treadmill-running program (n = 16) or to a sedentary untrained group [untrained-proestrus (UTP), n = 16]. After training for 3 mo, animals were killed on proestrous morning, and individual pituitary glands were enzymatically dispersed into single-cell suspensions. Trained rats maintained either predominantly regular estrous cycles [trained-proestrus (TP), n = 11] or exhibited anestrous vaginal cytology [trained-anestrus (TA), n = 5]. At death, plasma estradiol concentrations for TP (57.4 +/- 6 pg/ml, mean +/- SE) and UTP (54.6 +/- 5 pg/ml) were similar. However, TA had plasma estradiol levels (2.7 +/- 0.2 pg/ml; P < 0.05) reflecting their anestrous status. Basal luteinizing hormone (LH) secretion (20-h static incubation) was significantly reduced in gonadotropes from TA (7.8 +/- 1.1 ng/pooled wells) compared with TP (13.6 +/- 1.6 ng/pooled wells) and UTP (12.6 +/- 1.0 ng/pooled wells). Immunofluorescence LH staining and cytoplasmic granularity were significantly lower within the TA gonadotrope population compared with those of TP and UTP rats. Evidence of impaired basal LH secretion, reduced gonadotrope LH-specific fluorescein content, and reduced gonadotrope cytoplasmic granularity in TA rats suggests that, concomitant with proposed changes in the central neural drive, there are important exercise training-induced alterations of the anterior pituitary gonadotrope that influence rats' estrous cycles and affect their reproductive status. PMID- 7559214 TI - Immediate effects of arousal from sleep on cardiac autonomic outflow in the absence of breathing in dogs. AB - To determine the immediate effects of arousal from non-rapid-eye-movement (non REM) sleep on cardiac sympathetic and parasympathetic activities, six dogs were studied breathing through an endotracheal tube inserted into a chronic tracheostomy. Mean heart rates (HRs) during non-REM sleep were compared with 1) awake periods immediately after spontaneous arousals (ARs) and 2) later periods of stable relaxed wakefulness (RW). During spontaneous breathing, HR increased after AR (mean = 31.0%; P < 0.001) and in RW (mean = 7.6%; P < 0.001). To avoid the confounding influence of changes in breathing pattern, lung volume, and blood gases accompanying AR on HR, further studies were performed during constant mechanical hyperventilation that eliminated spontaneous breathing. In this condition, HR still increased after AR (mean = 29.9%; P < 0.001) and in RW (mean = 5.7%; P < 0.001), suggesting that the HR increases could be mediated by an effect of the state change per se on autonomic activity. This interpretation was confirmed when the HR increases were essentially abolished by combined cardiac sympathetic and parasympathetic block. In contrast, parasympathetic block alone did not prevent the HR increases after AR (mean = 12.2%; P < 0.001) or in RW (mean = 12.3%; P < 0.001), whereas sympathetic block alone almost abolished the HR increases in RW (mean = 3.6%) but did not prevent the HR increases during AR (mean = 30.2%; P < 0.001). The results show that, compared with non-REM sleep, AR is associated with acute cardiac sympathetic activation and parasympathetic withdrawal, whereas stable RW is associated mainly with sympathetic activation. These effects may have clinical relevance to the cardiovascular sequelae of breathing disorders that cause repetitive arousals from sleep. PMID- 7559221 TI - Combined hypoxia and hypercapnia evokes long-lasting sympathetic activation in humans. AB - We studied ventilatory and neurocirculatory responses to combined hypoxia (arterial O2 saturation 80%) and hypercapnia (end-tidal CO2 + 5 Torr) in awake humans. This asphyxic stimulus produced a substantial increase in minute ventilation (6.9 +/- 0.4 to 20.0 +/- 1.5 l/min) that promptly subsided on return to room air breathing. During asphyxia, muscle sympathetic nerve activity (intraneural microelectrodes) increased to 220 +/- 28% of the room air baseline. Approximately two-thirds of this sympathetic activation persisted after return to room air breathing for the duration of our measurements (20 min in 8 subjects, 1 h in 2 subjects). In contrast, neither ventilation nor sympathetic outflow changed during time control experiments. A 20-min exposure to hyperoxic hypercapnia also caused a sustained increase in sympathetic activity, but, unlike the aftereffect of asphyxia, this effect was short lived and coincident with continued hyperpnea. In summary, relatively brief periods of asphyxic stimulation cause substantial increases in sympathetic vasomotor outflow that outlast the chemical stimuli. These findings provide a potential explanation for the chronically elevated sympathetic nervous system activity that accompanies sleep apnea syndrome. PMID- 7559222 TI - Pressure-flow relationships in in vitro model of compartment syndrome. AB - Compartment syndrome is a condition in which an increase in intramuscular pressure decreases blood flow to skeletal muscle. According to the Starling resistor (i.e., vascular waterfall) model of blood flow, the decrease in flow could occur through an increase in arterial resistance (Rart) or an increase in the critical closing pressure (Pcrit). To determine which explains the decrease in flow, we pump perfused a canine gastrocnemius muscle placed within an airtight box, controlled box pressures (Pbox) so that flow ranged from 100 to 50%, and measured Pcrit, Rart, arterial compliance, small venular pressure (measured by the double-occlusion technique), and venous pressure. An increase in Pbox limited flow mainly through an increase in Pcrit (75-85%), with only small changes in Rart (15-25%) and no change in arterial compliance. Increases in Pbox also produced a vascular waterfall in the venous circulation, but small venular transmural pressure always remained less than control levels. We conclude that increases in Pbox mostly limit blood flow through increases in Pcrit and that Rart plays a minor role. Transmural pressure across the small venules decreases with increases in intramuscular pressure, which contradicts the currently held belief that compartment syndrome is due to a cycle of swelling-ischemia-swelling. PMID- 7559224 TI - Pulmonary vascular adaptations to augmented polycythemia during chronic hypoxia. AB - We previously found that augmentation of polycythemia by exogenous human recombinant erythropoietin (EPO) failed to worsen the severity of hypoxic pulmonary hypertension in rats. We asked whether this unexpected finding was related to reductions in cardiac output, left ventricular end-diastolic pressure, pulmonary vascular resistance, or some combination of these factors. Four groups of Sprague-Dawley rats were studied over a 3-wk period: hypoxic (0.5 ATM) and normoxic animals each injected with EPO (500 U/kg sc thrice weekly) or saline (control animals). As observed previously, we found that pulmonary arterial (PA) pressures and right ventricular hypertrophy were not increased in EPO-treated rats despite significant increases in hematocrit and blood viscosity. Cardiac outputs, blood volumes, and left ventricular end-diastolic pressures were similar in EPO-treated and control rats. Acute PA pressure responses to acute normoxia in hypoxic rats and to acute hypoxia in normoxic rats were similar, suggesting no differences in vasoreactivity. However, lungs isolated from EPO-treated hypoxic rats had lower pulmonary vascular resistance than saline-treated hypoxic rats when perfused with blood from normocythemic donor rats. PA medial thickness and the percentage of muscularized small PAs were significantly lower in EPO-treated hypoxic rats. These results indicate that augmented polycythemia fails to worsen hypoxic pulmonary hypertension in rats because of a decrease in the severity of structural remodeling. PMID- 7559223 TI - Enhanced cardiac metabolism of plasma glucose in high-altitude natives: adaptation against chronic hypoxia. AB - The metabolism of glucose in mammalian heart is 25-50% more O2 efficient than the metabolism of free fatty acids. To assess the role of substrate preference in adaptations to chronic hypoxia, positron emission tomographic measurements of heart regional glucose uptake rates after an overnight fast were made in volunteer Quechua subjects and in Sherpa subjects, both indigenous to altitudes of over 3,000 m, and in a group of lowlander volunteers. Highest uptake rates were found in the Quechuas on arrival and in the Sherpas after a 3-wk period at low altitude, intermediate rates in Quechuas after a 3-wk period at low altitude and in the lowlanders, and lowest rates in Sherpas on arrival. These low values were probably related to the stress of travel to the site of the experiments. Measured plasma catecholamines, hormones, and substrates indicated that glucose concentrations correlated best with observed variations in glucose uptake, with a negative correlation for the control subjects and a positive correlation for the Quechuas and Sherpas. Uptake values in Quechuas declined significantly after a 3 wk period at low altitude, but the positive correlation with glucose levels persisted. We conclude that an elevated glucose preference in heart is a true metabolic adaptation in humans adapted over generations to chronic hypoxia. PMID- 7559225 TI - Effects of muscle fiber type and size on EMG median frequency and conduction velocity. AB - This paper describes an in vitro method for comparing surface-detected electromyographic median frequency (MF) and conduction velocity (CV) parameters with histochemical measurements of muscle fiber type composition and cross sectional area (CSA). Electromyographic signals were recorded during electrically elicited tetanic contractions from rat soleus, extensor digitorum longus, and diaphragm muscles placed in an oxygenated Krebs bath. Fibers were typed as slow oxidative, fast oxidative glycolytic, and fast glycolytic based on histochemical enzyme stains. Muscles with a greater percentage of fast glycolytic and fast oxidative glycolytic fibers exhibited greater initial values of MF and CV as well as a greater reduction in these variables over the course of the contraction. Regression indicated that fiber type composition could be predicted based on two MF parameters. A weighted measure of muscle fiber CSA was found to be linearly related to both initial MF and CV. The results of this study suggest that MF and CV parameters recorded during a muscular contraction are related to muscle fiber type composition and muscle fiber CSA. PMID- 7559226 TI - Limitations of the efficacy of hemoglobin-based oxygen-carrying solutions. AB - Improvements in hemoglobin-based oxygen-carrying (HBOCs) solutions have overcome the toxicities that plagued earlier efforts. However, limitations of the efficacy of HBOCs are emerging. The potential limitations of an HBOC were studied in an ovine model (n = 6) of exchange transfusion. Hemodynamic, oxygen transport, and hemoglobin kinetic parameters were examined during isovolumic blood exchange to a final hematocrit of 3.2 +/- 0.7% and a plasma hemoglobin concentration of 8.1 +/- 0.4 g/dl while sheep were awake and breathing room air. However, the infusion of HBOC was associated with immediate increases in systemic and pulmonary arterial pressures. Despite hemodilution with HBOC, systemic and pulmonary vascular resistance increased 43.9% (P < 0.001) and 204.2% (P < 0.001), respectively, after HBOC infusion. After blood exchange, the plasma hemoglobin level exhibited a circulatory half-life of 52.7 +/- 18.0 h. The formation of methemoglobin was significant, accounting for 33.0 +/- 7.1% of the total circulating plasma hemoglobin at 24 h; the half-life of HBOC capable of carrying oxygen was 30.1 +/- 5.4 h. This relatively short period of oxygen-carrying efficacy and the observed vasoconstriction properties may constrain the potential applications of HBOC solutions. PMID- 7559227 TI - Intra-airway gas transport during high-frequency chest vibration with tracheal insufflation in dogs. AB - High-frequency external chest vibration with tracheal insufflation (high frequency vibration ventilation) has previously been shown to be an effective mode of artificial ventilation in experimental animals. To investigate the intra airway gas mixing during high-frequency vibration ventilation (frequency 30 Hz, amplitude 0.4 cm), we used an analysis of the single-breath washout curve that gives the vibration-induced mixing coefficient distribution relative to the no vibration situation. Data from four anesthetized dogs were collected during constant-flow insufflation at six rates (0.05-0.4 l.min-1.kg-1), at three insufflation durations (2, 4, and 7 s), and with the insufflation catheter outlet at three positions (carina, trachea, and a bronchus) while the vibration was on and off. Vibration enhanced intra-airway gas mixing 14.1 +/- 3.9-fold, with the peak of the enhancement distribution located 125 +/- 29 ml from the airway opening and a distribution width of 121 +/- 29 ml. As insufflation flow increased, the position of the peak enhancement shifted toward the alveolar zone and diminished in peak amplitude. Changing the insufflation duration and the catheter position did not affect the intra-airway mixing induced by vibration. External chest vibration causes a substantial increase of intra-airway gas mixing, bringing alveolar gas to central airways. This leads to overall increased pulmonary gas transport when fresh gas is insufflating the tracheal carina area. PMID- 7559228 TI - Intrinsic myocardial function and oxidative stress after exhaustive exercise. AB - The purposes of this study were to determine the effect of an exhaustive running bout on intrinsic myocardial function by using the isolated working rat heart and to determine whether exhaustive exercise resulted in measurable oxidative stress in the myocardium. Untrained familiarized male rats were run at 18 m/min on a 0% grade until exhausted. Run time to exhaustion was approximately 75 min. Postexhaustion isolated heart measurements of cardiac output, rate-pressure product at low and high workloads, maximum left ventricular pressure, or 50-min performance at 85% of peak rate-pressure product were not different from those of nonexercised perfused control hearts. Exhaustive exercise resulted in a significant decline (174 vs. 224 nmol/g wet wt; P < 0.05) in nonprotein nonglutathione sulfhydryls, a thiol fraction indicative of oxidative stress. However, the magnitude of this measure of oxidative stress appears insufficient to cause alterations in intrinsic myocardial performance. We conclude that healthy untrained rats subjected to exhaustive exercise fail to demonstrate accumulation of a functionally significant level of myocardial oxidative stress. PMID- 7559229 TI - Ventilatory and metabolic responses to hypoxia during moderate hypothermia in anesthetized rats. AB - In resting euthermic mammals, hypoxia elicits a hyperventilation that results from a combination of hyperpnea and hypometabolism. Often accompanying the hypoxia-induced hypometabolism is a drop in body temperature. To separate the synergic effects of hypothermia per se from the direct effects of hypoxia on metabolic rate, ventilation (VE), and O2 consumption (VO2) were measured in anesthetized rats fitted with abdominal heat exchangers and maintained at either normothermic (37.5 degrees C) or hypothermic (35 degrees C) body temperatures while exposed to either normoxia or hypoxia (7% O2). Hypothermia induced parallel decreases in VE and VO2, thereby maintaining VE/VO2. Hypoxia resulted in a hyperventilation achieved with the same relative decrease in VO2 and increase in VE in both normothermic and hypothermic rats. The results suggest that 1) the changes in metabolic rate and VE during hypothermia reflect a direct effect of cold and, 2) because of similar levels of hypoxic hyperventilation in the hypothermic and normothermic rats, relative to metabolic rate, respiratory gain has not been depressed in hypothermic rats. PMID- 7559230 TI - Arterial, capillary, and venous transit times and dispersion measured in isolated rabbit lungs. AB - Transit time and relative dispersion of the arterial, capillary, and venous segments of the pulmonary circulation were measured in isolated perfused rabbit lungs. Fluorescence videomicroscopy was used to record the passage of dye through the main pulmonary artery, subpleural microcirculation, and venous outflow. Dye dilution curves were obtained at the main pulmonary artery, subpleural arterioles and venules, and pulmonary vein. Measurements were made at 5-cmH2O airway pressure, at blood flows of approximately 80, 50, and 25 ml.min-1.kg-1, and at left atrial pressures of approximately 0 cmH2O (zone 2) and approximately 12 cmH2O (zone 3). The dye dilution curves were modeled as lagged normal density curves that were used to calculate transit time and relative dispersion between the pulmonary artery and arteriole (artery), arteriole and venule (capillary), venule and pulmonary vein (vein), and pulmonary artery and pulmonary vein (whole lung). In open-chest anesthetized dogs, the passage of dye was recorded from the subpleural arterioles and venules between the seventh and eighth ribs in the left lateral position. At comparable blood flows, capillary transit time was larger in the dog than in the rabbit lung [3.4 +/- 2.4 (SD) vs. 0.87 +/- 0.47 s]. In the rabbit lung, relative dispersion was greater in pulmonary capillaries (average values 0.83-1.6) and veins (0.91-1.6) than in arteries (0.39-0.50), which was similar to the whole lung dispersion (0.47-0.52). A similarly high dispersion (0.93) was measured in the dog's pulmonary capillaries. Thus high dispersion in pulmonary capillaries and veins cannot be detected by whole lung dispersion measurements. PMID- 7559231 TI - Measurement of the EMG-force relationship in a human upper airway muscle. AB - The upper airway muscles play an important role in breathing, swallowing, and speaking, but little is known about the electromyogram (EMG)-force relationship of these muscles. We have measured the peak integrated EMG activity (iEMG) and force of human nasal dilator muscles (NDM) with a custom-designed headpiece that was attached via the forehead and upper lip. The headpiece contains a micromanipulator that holds a rod with a load cell mounted on its tip. The reproducibility of the force measurements was examined by measuring the lateral or "flaring" force of the NDM in multiple trials on two separate occasions in 13 subjects. For these studies the subjects were instructed to perform maximal voluntary contractions (MVCs). Test-retest reproducibility averaged 8.3% (coefficient of variation) for within-day comparisons and 13.7% between days. We also measured iEMG and NDM force during an incremental exercise test in nine of the subjects; they were instructed to breathe nasally throughout one 30-s epoch at rest and at each workload. The iEMG and force during peak exercise (175-275 W) averaged 81 +/- 26% (SD) MVC and 235 +/- 127 mN (approximately 75% MVC), respectively. The iEMG during incremental exercise was linearly related to the peak force (r = 0.90, P < 0.001). Contractile properties were measured in seven of the subjects by application of single supramaximal shocks (0.1-ms pulse) to the facial nerve. Twitch force averaged 9 +/- 6% MVC, and the time to peak force was 62 +/- 13 ms, which is considerably faster than that in human diaphragm or elbow flexors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559233 TI - Vascular adaptations in rat hindlimb skeletal muscle after voluntary running wheel exercise. AB - To test the hypothesis that voluntary running-wheel exercise would elicit vascular adaptations in rat skeletal muscle, male Sprague-Dawley rats (202 +/- 5 g) were cage confined (C, n = 11) or housed in cages with free access to running wheels (R, n = 13) for 12 wk. Vascular transport capacity was determined in maximally vasodilated (papaverine) hindquarters of C and R rats with measurements of total and regional (radiolabeled microspheres) flow capacity and capillary filtration coefficient. R rats voluntarily ran 29 +/- 4 km/wk over the 12-wk period; however, performance of individual rats varied greatly (range 4-74 km/wk). Citrate synthase activity was increased in the medial head (81%, P < 0.001) and the red long head (88%, P < 0.001) of the triceps brachii muscle in R rats but not in the white long head (25%, P = 0.06). Capillary filtration coefficient was 27% greater in R compared with C rats (0.040 +/- 0.003 vs. 0.031 +/- 0.002 ml.min-1.100 g-1.mmHg-1, respectively, P < 0.001) suggesting that there was an increase in microvascular surface area available for fluid exchange. Total hindquarters flow was increased in R rats (P < 0.05) at all perfusion pressures examined, indicative of an increased flow capacity. Regional flows revealed that skin flow was unchanged in R rats and that the increase in total flow was due to increased skeletal muscle flow capacity. These results indicate that voluntary running-wheel exercise elicits adaptive increases in skeletal muscle vascular transport capacity and oxidative capacity comparable to those seen in treadmill trained rats and support the use of voluntary running-wheel exercise as a less stressful training modality in exercise studies using rats. PMID- 7559232 TI - Changes in serum lipids and apolipoproteins after exercise in men with high cholesterol: influence of intensity. AB - The purpose of this study was to characterize the short-term changes in blood lipid and apolipoprotein concentrations in healthy hypercholesterolemic men after high-intensity [80% maximal O2 uptake (VO2max); n = 20] or moderate-intensity (50% VO2max; n = 19) cycle ergometer exercise balanced for caloric expenditure (350 kcal). The men's age, height, weight, %fat, and VO2max were 46 +/- 2 yr, 173 +/- 7 cm, 82.7 +/- 2.2 kg, 28 +/- 1%, and 31.1 +/- 1.0 ml O2.kg-1.min-1, respectively. Blood samples were drawn before exercise, immediately after exercise, then 24 and 48 h later, and concentrations of all variables were adjusted for changes in plasma volume. Significant changes (P < 0.0016) were as follows: total and low-density lipoprotein cholesterol fell by 4% immediately after exercise and then rose by 5-8% by 48 h. Triglycerides were 18 and 15% lower at 24 and 48 h, respectively. HDL-cholesterol, high-density lipoprotein3 cholesterol, and apolipoprotein B rose 8-9% by 24 h and remained elevated. High density lipoprotein2-cholesterol rose by 27% by 48 h after exercise, but this change was not significant. Apolipoprotein A-I did not change with exercise. The response patterns were not affected by exercise intensity. These data show that a single session of exercise performed by untrained hypercholesterolemic men alters blood lipid and apolipoprotein concentrations. Furthermore, the postexercise response patterns are not influenced by exercise intensity, as long as caloric expenditure is held constant. PMID- 7559234 TI - Maximal skin vascular conductance in subjects aged 5-85 yr. AB - This study examined maximal forearm skin vascular conductance (FVCmax) as a function of age in 74 healthy male and female subjects ranging in age from 5 to 85 yr. The skin temperature of the left forearm was uniformly clamped at 42 degrees C by spraying a fine mist of water over the surface. Forearm blood flow (FBF) was measured by venous occlusion plethysmography (Hg-in-Silastic strain gauge). After 60 min of heating, a reactive hyperemia maneuver was performed to verify that forearm skin blood flow was maximal by using laser Doppler flowmetry to isolate the skin component of FBF. The maximal FBF of each subject was then divided by mean arterial pressure to yield FVCmax (in ml.100 ml-1.min-1.100 mmHg 1), i.e., minimal resistance. The model that best fits the data was curvilinear, as described by FVCmax = 13.1 + 86.9 (age-0.75) (r2 = 0.52, P < 0.001). The exclusion of subjects younger than 18 yr of age simplified the model to a linear fit with a slope of -0.16 conductance units/yr for adults. Interindividual variability remained constant across the entire age span. Once the age effect was considered, there were no significant effects of gender, adiposity, resting blood pressure, physical activity level, or body surface area on FVCmax. PMID- 7559235 TI - Is the manuscript original or redundant? Judgment calls, scientific integrity, and the law. PMID- 7559237 TI - Neuromechanical regulation of respiratory motor output in ventilator-dependent C1 C3 quadriplegics. AB - To evaluate the role of phrenic and sternocleidomastoid afferents as alternate sources of inhibitory feedback during mechanical ventilation, we studied five C2 C3 quadriplegics with sensory denervation of the rib cage and diaphragm, six C1 C2 quadriplegics with additional loss of sensory feedback from the neck muscles, and seven normal subjects. We compared the return of inspiratory muscle activity [the recruitment threshold (PCO2RT)] during mechanical ventilation between subject groups after stepwise increases in end-tidal PCO2 (PETCO2) either by increasing the inspired fraction of CO2 (FICO2), decreasing tidal volume (VT; 50 ml/min), or decreasing frequency (f; 1 breath/2 min). Normal subjects were mechanically hyperventilated via a nasal mask until inspiratory activity was undetectable. Efferent input to the sternocleidomastoid was intact at both levels of spinal cord injury, but phasic activity was not evident at the quadriplegics' baseline resting ventilation. The PCO2RT was defined as the level of PETCO2 at which phasic activity of the diaphragm in normal subjects and of the sternocleidomastoid in C1-C2 and C2-C3 quadriplegics recurred. The mean PCO2RT (in response to raising PETCO2 via increased FICO2 while maintaining a high VT and f) was not significantly different (P = 0.6) between normal subjects (43 +/- 3 Torr) and C2-C3 quadriplegics (38 +/- 5 Torr). Both subject groups demonstrated a frequency- and volume-related inhibition, as evidenced by a substantially lower PCO2RT when PETCO2 was raised by reducing either VT or f. In contrast to the C2 C3 quadriplegics, the C1-C2 quadriplegics responded with a similar PCO2RT among the three different mechanical ventilation trials, independent of whether PETCO2 was raised with high VT and f, with reduced VT, or with reduced f. We conclude that feedback from at least some part of the chest wall is required to produce a volume- and frequency-dependent inhibition of inspiratory muscle activity observed during mechanical ventilation. PMID- 7559238 TI - Acute systemic blood pressure elevation in obstructive and nonobstructive breath hold in primates. AB - Phasic blood pressure (BP) response during obstructive apnea (OA) in human sleep has been previously described as consisting of a slow incremental increase in BP to the point of apnea termination followed by a rapid rise and then fall in BP at the resumption of respiration. This rise in BP has been attributed to postapneic augmentation of cardiac output resulting after release of the marked negative intrathoracic pressure (NIP) of obstructed inspiration. Via an endotracheal tube, we created obstructed and nonobstructed breath hold (apnea) in chloralose anesthesized baboons consisting of fixed-duration (30, 45, and 60 s) single OAs (mechanical obstruction) and nonobstructive (paralysis, ventilator cessation) apneas of matched duration and arterial desaturation. Systemic BP was measured before apnea (T0), during the last 5 s of apnea (T1), and during the first 5 s after resumption of respiration (T2). Despite wide fluctuations in NIP and BP during the T0 to T1 phase of OA, BP elevation in OA and nonobstructive apnea at T0, T1, or T2 did not differ for any duration apnea. At the release of obstruction, when resolution of NIP changes could theoretically increase cardiac output and accentuate BP, there was no difference in T1 and T2 pressures between the two conditions. We conclude that in this anesthesized animal model, mechanical (NIP) changes do not play a major role in overall maximum BP response to OA. Because of physiological differences between natural sleep in humans and the anesthetized state in animals, care must be taken in extrapolating these results to human sleep apnea. PMID- 7559236 TI - Effect of multiple denervations on the exercise hyperpnea in awake ponies. AB - In three previously reported studies, we had documented that the normal exercise hyperventilation in ponies is accentuated by carotid body denervation (CBD), not affected by hilar nerve pulmonary vagal denervation (HND), and mildly attenuated by spinal cord ablation of the dorsal lateral columns at L2 (SA). In the present study, we hypothesized that if redundancy of control existed in exercising ponies, then multiple denervations of theoretically important pathways in the same animal might attenuate the ventilatory response to exercise in a way not predictable by the individual lesion experiments alone. There were three major findings in the various combinations of CBD, HND, and SA in ponies during treadmill exercise. First, the combination of CBD with HND or SA resulted generally in an accentuation of the hypocapnia during exercise that was predictable on the basis of CBD alone. However, in one pony that showed a hypercapnic exercise response after SA alone, CBD subsequently caused a greater exercise hypercapnia. Second, HND in a CBD or SA pony did not affect the exercise arterial PCO2 response, which is consistent with previous data showing the lack of an HND effect in otherwise intact ponies. Third, in ponies with all three denervations together, the predominant response was an increase, not a decrease, in the exercise hyperventilation; this increase was greater than that predicted from the individual lesions. We conclude that these data do not provide evidence of redundancy in mechanism for the exercise hyperpnea other than instances of carotid chemoreceptor error sensing when hypercapnia occurs during exercise. PMID- 7559240 TI - Maximal rate of blood lactate accumulation during exercise at altitude in humans. AB - The lower peak lactate accumulation in blood ([La(b)]p) at altitude may be associated with a reduced maximal glycolytic flux. Based on certain assumptions, the latter can be indirectly evaluated in vivo, during short supramaximal exercises, by measuring the maximal rate of lactate accumulation in blood (delta [La(b)]max). delta [La(b)]max was determined on six white subjects at sea level (SL1), after approximately 1 wk (Alt1) and 4 wk (Alt2) of a 35-day sojourn at 5,050 m, and 1 wk after return to sea level (SL2). The subjects performed exercises of increasing duration (5, 15, 25, 35, 45 s or until exhaustion) on a bicycle ergometer at loads = 200% of the individual Wmax. The latter was previously determined in each condition as the greatest work rate that could be sustained for 2-4 min during an incremental exercise. Net [La(b)] accumulation (delta [La(b)]) was measured after each exercise bout. delta [La(b)] resulted to be linearly related to exercise duration. The slopes of the individual delta [La(b)] vs. exercise duration lines were taken as delta [La(b)]max. Exhaustion times were approximately 30-45 s in all conditions. [La(b)]p (in mM) during recovery after the exhaustive load was higher at SL1 (10.22 +/- 1.09; means +/- SD) than at Alt1 (5.08 +/- 0.82), Alt2 (8.13 +/- 2.67), and SL2 (8.18 +/- 1.43). delta [La(b)]max was lower at Alt1 (0.09 +/- 0.02) and at Alt2 (0.17 +/- 0.05) than at SL1 (0.25 +/- 0.05) and SL2 (0.23 +/- 0.06). Both [La(b)]p and delta [La(b)]max increased during acclimatization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559239 TI - Endothelium-dependent vasodilation of proximal coronary arteries from exercise trained pigs. AB - We recently reported that alpha-adrenergic vasoconstriction is blunted and adenosine-induced vasodilation is enhanced in proximal coronary arteries of exercise-trained miniature swine [C. L. Oltman, J. L. Parker, H. R. Adams, and M. H. Laughlin. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H372-H382, 1992]. The purpose of the present study was to determine whether this model of exercise training also alters endothelium-dependent vasodilator responses of proximal coronary arteries. Female Yucatan miniature swine were exercise trained (ET) on a motor-driven treadmill or were cage confined (Sed) for 13-20 wk. Exercise tolerance, heart weight-to-body weight ratios, and skeletal muscle oxidative capacity were all significantly greater in ET than in Sed animals. Vasodilator responses were evaluated in vitro by determining concentration-response curves by using vascular rings (3.5-4 mm in axial length) isolated from right and left coronary arteries. Vasorelaxation responses were determined, after tone had been produced with either 30 microM prostaglandin F2 alpha, 30 mM KCl, or 30 nM endothelin. Concentration-response curves were obtained to endothelium-dependent vasodilators including bradykinin (10(-9)-10(-6) M), substance P (10(-12)-10(-6) M), clonidine (10(-9)-10(-6) M), serotonin (10(-10)-10(-5) M), and the Ca2+ ionophore A-23187 (10(-10)-10(-6) M). Endothelium-independent vasodilator responses to sodium nitroprusside (10(-9)-10(-4) M) were not different between arteries from Sed and ET. Bradykinin, substance P, and A-23187 were potent vasodilators in arteries from both groups, whereas serotonin and clonidine did not consistently produce vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559242 TI - Dynamic viscoelastic nonlinearity of lung parenchymal tissue. AB - To investigate the contribution of nonlinear tissue viscoelasticity to the dynamic behavior of lung, time and frequency responses of isolated parenchymal strips of degassed dog lungs were investigated. The strips were subjected to loading and unloading stretch steps for 60 s and to sinusoidal oscillations (0.03 3 Hz) of different stretch amplitudes (delta lambda = 0.05, 0.1, and 0.2) and at different operating stresses (T(o) = 0.5, 1, and 2 kPa). Elastance (E) increased linearly with the logarithm of frequency (approximately 10% per frequency decade), and resistance (R) decreased hyperbolically with frequency. Both E and R varied little with delta lambda but they increased proportionally with T(o). Hysteresivity (eta = R x 2 pi x frequency/E) ranged from 0.07 to 0.10. In agreement with the frequency response, the magnitude of the unit step response increased with T(o) and was higher when loading than when unloading, and the stress relaxation ratio (approximately 0.10) did not vary greatly with T(o) or with delta lambda. The time and frequency behavior of the strips were interpreted in terms of the quasilinear viscoelastic model of Navajas et al. (J. Appl. Physiol. 73:2681-2692, 1992). The model explains most of the dependencies of step and oscillatory responses on the measurement conditions, in particular the proportional dependence of E and R on T(o). According to the model, about two thirds of energy dissipated during oscillation arises from tissue viscoelasticity. The remaining dissipated energy could be accounted for by plasticity. Thus the effect of nonlinear elasticity on the dynamic behavior of lung tissue can be empirically described by a simple quasilinear model characterized by only two parameters. PMID- 7559243 TI - A computer simulation of pulmonary perfusion in three dimensions. AB - Pulmonary perfusion is spatially correlated with neighboring regions of lung having similar magnitudes of flow and distant pieces exhibiting negative correlation. Although local correlation has been noted in a wide variety of natural processes, negative correlation has not and it may be unique to organ blood flow. We investigate the regional perfusion predicted by a three dimensional branching vascular model to determine whether such a model can create negative correlation of perfusion. The distribution of flows is modeled by a dichotomously branching tree in which the fraction of flow from parent to daughter branches is gamma and 1-gamma at each bifurcation. The flow asymmetry parameter (gamma) is randomly chosen for each bifurcation from a normal distribution with a mean of 0.5 with an SD of sigma. The branches branch along one of three orthogonal directions to assure a space-filling structure. This model produces flow distributions similar to those observed in experimental animals, with perfusion being positively correlated locally and negatively correlated at distance. The model is refined by incorporating an effect of gravity, which redirects a fraction (delta), of the flow against gravity to the companion daughter branch in the gravitational direction. A flow bias in the "dorsal" direction is also introduced to account for differences in supine-prone perfusion gradients. In its final form, this three-dimensional branching model accounts for previously observed 1) spatial correlation of regional perfusion with negative correlation over distance, 2) isogravitational perfusion heterogeneity, 3) differences in supine and prone perfusion gradients, 4) positive correlation of flows between supine and prone postures, 5) relatively small contributions of gravity to perfusion heterogeneity, and 6) fractal distributions of flow. This three-dimensional branching vascular model relates the function and structure of the pulmonary vascular tree, offering an explanation for both heterogeneous and spatially correlated regional flows. PMID- 7559241 TI - N-acetylcysteine administration and loaded breathing. AB - Recent work has shown that loaded breathing produces alterations in diaphragmatic glutathione metabolism. Moreover, it has been suggested that alterations in glutathione levels may be related to the development of respiratory muscle fatigue and respiratory failure during loading. The purpose of this study was to determine whether it was possible to augment diaphragmatic stores of reduced glutathione (GSH) and thereby delay the development of respiratory failure during loaded breathing by administering N-acetylcysteine (NAC), a glutathione precursor. We compared the effects of massive inspiratory loading on saline- and NAC-treated groups of decerebrate unanesthetized rats with loading continuing until respiratory arrest occurred. As controls, we also studied unloaded saline- and NAC-treated animals. After arrest, diaphragms were excised, measurement was made of diaphragmatic GSH and oxidized glutathione (GSSG) concentrations, and assessment was made of in vitro diaphragmatic contractility (i.e., the force frequency relationship and in vitro fatigability). We found that loading of saline-treated animals produced reductions in the diaphragmatic force-frequency curve, reductions in GSH, and increases in GSSG levels. NAC administration blunted loading-induced decreases in diaphragmatic GSH levels and reduced the in vitro fatigability of excised diaphragm muscle strips. NAC did not significantly alter the time to respiratory arrest, however, and also failed to alter the effect of loaded breathing on the diaphragmatic force-frequency relationship. These findings suggest that free radical-mediated GSH depletion is not the limiting factor determining the development of respiratory failure in this model of loaded breathing. PMID- 7559244 TI - Mucociliary function in the mouse measured in explanted lung tissue. AB - To develop a method for the study of mucociliary clearance in small-caliber airways, we investigated ciliary function in an in vitro lung tissue culture technique in mice. Lungs were excised from 45 anesthetized mice [weight 30.9 +/- 6.2 (SD) g] and inflated with 2% liquid agarose at 37 degrees C via the trachea. After cooling to 4 degrees C, the lungs were cut into 0.5- to 1.0-mm thick slices and cultured overnight. Ciliary beat frequency (CBF) was measured in airways cut in cross section using a computerized image processing system. In some experiments, charcoal particle transport (PT) in tangentially cut airways was also measured. Airway diameter ranged from 0.3 to 0.8 mm. In this preparation CBF was stable over a 3-h period and unaffected by minor pH changes. Both CBF and PT exhibited a linear dependency on temperature. CBF and PT were significantly correlated with each other. CBF at 37 degrees C (18.7 +/- 2.93 Hz) was almost twofold higher than values at 22 degrees C (9.74 +/- 3.11 Hz). Isoproterenol increased CBF in a dose-dependent fashion (50% effective concentration of 10( 6.75) M); the effect of isoproterenol could be blocked by propranolol. Administration of forskolin (10 microM) also increased both CBF and PT significantly. These findings demonstrate the feasibility of measuring the major aspects of mucociliary clearance in this system. This approach holds promise as a technique suitable to the investigation of both the small airways of humans and other large animals as well as of airways in murine genetic models of respiratory disease. PMID- 7559245 TI - Climate-related corrections for improved estimation of energy expenditure from heart rate in children. AB - The purpose of this study was to determine the relationship between children's heart rate (HR) and climatic heat stress at several O2 uptake (Vo2) levels and to construct equations and nomograms for an improved estimation of energy expenditure (EE) from HR monitoring. On four occasions 12 boys and 8 girls (8-11 yr) cycled for 5 min each at 35, 55, and 75% of peak Vo2 (random order), with rest periods in between, in a climatic chamber. The randomly assigned conditions were 22 degrees C dry bulb temperature, 50% relative humidity (RH); 28 degrees C, 55% RH; 32 degrees C, 52% RH; or 35 degrees C, 58% RH. HR and Vo2 were determined during the final 2 min of rest and exercise bouts. HR measured at a dry bulb temperature (T) (HRmeas) was then related to a HR at 22 degrees C that would have been expected to occur at an identical Vo2 (HRcalc22) by using individual 22 degrees C HR-Vo2 regression lines. HRcalc22 could be estimated from HRmeas.[1.175032-(0.007956.T)], with a mean error of prediction for the entire group < 1.5 beats/min for each temperature. The following conclusions were drawn: 1) children's HR at a given Vo2 increases linearly with ambient temperature at 22 35 degrees C, 50-60% RH; and 2) HR can be corrected for the influence of climate in groups of children resting and exercising at the above conditions, thereby reducing the error of estimating EE from HR. PMID- 7559246 TI - Invited editorial on "effect of altitude on uterine artery blood flow during normal pregnancy" and "alterations in uteroplacental blood flow precede hypertension in preeclampsia at high altitude". PMID- 7559247 TI - Inhomogeneous activation of the parasternal intercostals during breathing. AB - Recent computations of the mechanical advantage of the canine intercostal muscles have suggested that the inspiratory advantage of the parasternal intercostals is not uniform. In the present studies, we have initially tested this hypothesis. Using a caliper and markers implanted in the costal cartilages, we have thus measured, in four supine paralyzed dogs, the length of the medial, middle, and lateral parasternal fibers at functional residual capacity and after a 1-liter mechanical inflation. With inflation, the medial fibers always shortened more than did the middle fibers (-9.8 +/- 0.8 vs. -6.0 +/- 0.8%; P < 0.001), whereas the lateral fibers remained virtually constant in length (-0.2 +/- 0.8%). This gradient of mechanical advantage agreed well with the gradient of orientation of the muscle fibers. Therefore, we have also recorded the electromyograms of the medial, middle, and lateral parasternal bundles during spontaneous breathing in nine anesthetized animals (20 interspaces); each activity was expressed as a percentage of the activity recorded during tetanic, supramaximal stimulation of the internal intercostal nerve (maximal activity). The medial bundle was invariably more active than was the middle bundle during resting breathing (57.3 +/- 3.3 vs. 25.5 +/- 3.4% of maximum; P < 0.001), and in 10 interspaces, medial activity consistently preceded middle activity at the onset of inspiration. These differences persisted during hypercapnia, during inspiratory resistive loading, as well as after phrenicotomy. Activity was never recorded from the lateral bundle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559248 TI - Effects of neuromuscular blockade on fetal heart rate variability: a power spectrum analysis. AB - Spectral analysis of fetal heart rate variability allows quantitative determination of the main components that affect this variability. The physiological significance of these components is unclear; however, movements appear to contribute to variability. We studied six fetuses in which immobility required for in utero magnetic resonance or invasive fetal procedures was achieved by fetal intravascular injection of curare between 32 and 36 amenorrhea weeks. For each fetus, we compared spectral density parameters of heart rate variability. After curare administration, mean spectrum power was halved. We did not observe a larger significant decrease in any (very low, low, or high) frequency band. The other parameters of spectral analysis of variability were unaltered. Fetal movements accounted for a significant proportion of human fetal heart rate variability but did not constitute a unique frequency component. PMID- 7559249 TI - Does the anatomic makeup of parenchymal lung strips affect oscillatory mechanics during induced constriction? AB - Parenchymal tissue strips have been used to investigate the mechanical behavior of the lung parenchyma. We questioned whether the relative amounts of alveolar, blood vessel, and bronchial walls would be important when the contractile response of parenchymal strips from Sprague-Dawley rats was studied. One group of strips was cut from the subpleural edge and another from between 1 and 3 mm proximal to the pleura. Strips were suspended in an organ bath filled with Krebs solution (37 degrees C, pH 7.4) bubbled with 95% O2-5% CO2. Resting tension (T) was set at 1.1 g, and sinusoidal oscillations of 2.5% resting length at a frequency of 1 Hz were applied. Measurements of length and T were recorded during baseline conditions and after acetylcholine (10(-3) M) was added to the bath. Elastance, resistance, and hysteresivity (the ratio of the energy dissipated to that conserved) were calculated. Strips were fixed in Formalin at a T of 1 g, histological sections were prepared, and the fractional areas of alveolar, blood vessel, and bronchial walls were measured by using point counting. Significant differences were found between the two groups of strips in the acetylcholine response and anatomic makeup. The magnitude of the changes of all the mechanical parameters were correlated with the volume proportions of the different anatomic constituents when all the strips were plotted together but not when the subpleural strips were considered alone. We conclude that subpleural parenchymal strips are a sound model of parenchymal lung behavior. When more proximal strips are studied, the amount of bronchial wall may play an important role in determining the hysteretic response. PMID- 7559251 TI - Early adaptations in collateral and microvascular resistances after ligation of the rat femoral artery. AB - Collateral and microvascular (including feed artery) resistances in the rat hindlimb were determined immediately or 1 wk after ligation of the femoral artery. Collateral-to-microvascular resistance ratios were determined from in vivo pressure measurements proximal and distal to the ligation. Microvascular resistance was 32 +/- 2.5 and 41 +/- 1.5% of the total collateral-dependent vasculature in acutely and chronically ligated limbs, respectively, and decreased 20% in both groups during reactive hyperemia. Minimum resistances of collateral vessels and the microcirculation arising from arterial branches proximal and distal to the ligation were determined by using a modification of the standard hindquarter perfusion technique for determining maximum vascular conductance. One week postligation, minimum total hindquarter resistance was decreased by a reduction in the resistance of the collaterals (approximately 50%) and microcirculation (approximately 33%) proximal to the ligation. The results suggest that the microvasculature distal to the occlusion is able to increase flow by dilation both initially and at 1 wk postligation but that collateral adaptations are primarily responsible for decreases in the minimum total resistance of the collateral-dependent region. PMID- 7559250 TI - Effect of altitude on uterine artery blood flow during normal pregnancy. AB - To determine whether uterine blood flow was reduced and indexes of pelvic blood flow distribution altered in normotensive pregnancy at high (3,100 m) compared with low altitude (1,600 m), we measured uterine, common iliac, and external iliac artery blood flow velocities and diameters in women during pregnancy and again postpartum. Pregnancy increased uterine artery diameter, blood flow velocity, and volumetric flow at both altitudes. Uterine artery blood flow velocity was greater (69.0 +/- 2.2 vs. 59.4 +/- 3.0 cm/s; P < 0.005) but diameter was smaller at 3,100 m than at 1,600 m (2.5 +/- 0.3 mm vs. 3.4 +/- 0.2 mm; P < 0.005), resulting in volumetric flow that was one-third lower at week 36 of pregnancy (203 +/- 48 vs. 312 +/- 22 ml/min, respectively; P < 0.01). Pregnancy increased common iliac blood flow velocity and decreased external iliac artery blood flow velocity at both altitudes. The uterine artery received a smaller percent of common iliac flow at 3,100 than at 1,600 m (46 +/- 7 vs. 74 +/- 6%; P < 0.005). Gestational age was similar but birth weight was lower at 3,100 m than at 1,600 m. Among subjects at 1,600 m, variation in uterine blood flow velocity correlated positively with infant birth weight. We concluded that reduced uterine blood flow and altered pelvic blood flow distribution during pregnancy at high altitude likely contributed to the altitude-associated reduction in infant birth weight. PMID- 7559253 TI - Relative expansion of extracellular water in obese vs. normal children. AB - This preliminary communication reports data regarding the distribution between intracellular (ICW) and extracellular (ECW) water compartments in a group of 21 prepubertal young obese children of both sexes in comparison with a group of 18 normal children weight matched for age. Our data indicate that obesity is associated with a highly significant relative expansion of extracellular water (ECW/ICW = 0.61 +/- 0.19 and 0.76 +/- 0.09 in control and obese subjects, respectively; P < 0.0015). This observation, which has been already reported in adult women, suggests that some disturbances of water homeostasis have an early onset and stress the need for an early control of energy imbalance in children. These findings are of great concern also in the field of human body composition, suggesting the opportunity for a critical reevaluation of the assumed constancy of some human body characteristics. Body composition methodologies developed for "normal" populations would require adjustment for use in the obese population, since a considerable error would be introduced. PMID- 7559254 TI - Truncal vagotomy does not affect postabsorptive glucose metabolism in humans. AB - To evaluate the effects of hepatic vagal denervation on the adaptation of glucose metabolism to short-term starvation (i.e., < 24 h), glucose metabolism was studied after 16 and again after 22 h of fasting in postsurgical patients with truncal vagotomy (n = 9; radical resection of esophageal carcinoma) and without truncal vagotomy (n = 5; partial resection of the large bowel for carcinoma). Glucose metabolism was studied 3-7.5 mo postoperatively by [3-3H]glucose turnover and by indirect calorimetry. Basal glucose production, plasma glucose concentration, glucose oxidation, serum free fatty acid concentration, and fat oxidation were not different between groups; neither were plasma concentrations of glucoregulatory hormones. The adaptation to prolongation of the fast by 6 h was not different for any of these parameters between both groups. In conclusion, truncal vagotomy does not affect the adaptation of glucose metabolism to the postabsorptive state (16-22 h of fasting). PMID- 7559255 TI - Topobiology in hematopoiesis. PMID- 7559252 TI - Effect of perception of mechanical loading on human respiratory pattern regulation. AB - We applied external flow resistive (R) and elastic (E) mechanical loads over the entire respiratory cycle to five normal subjects by using a pseudorandom loading protocol. Loads ranged in magnitude from imperceptible (R0/E0) through just perceptible (R1/E1) to large (R2/E2) and resulted in respiratory pattern responses that were due to reflex responses alone (R0/E0) or to a combination of reflex responses and behavioral reactions to the perception of impeded breathing (R1/E1 and R2/E2). Pattern regulation dynamics were estimated from the computed impulse responses of tidal volume and inspiratory and expiratory durations. We anticipated that emergence of behavioral contributions would be marked by increased variability in response strategies and by increased nonlinearity in the observed responses. Regarding the immediate pattern response to loading, there was a tendency for increased qualitative variation across subjects as the load size increased, but the within-subject variability (coefficient of variation) was unaffected. We found no evidence for increased nonlinearity as loads became perceptible. The emergence of behavioral control in some instances seemed to be marked by reduction of complexity of the impulse response to one dominated by the zeroth-order lag, leading to dynamically simpler responses compared to control. PMID- 7559256 TI - Progress in antisense therapeutics. AB - Antisense technology offers the potential to create compounds specific enough to support highly isotypically selective pharmacology (i.e., pharmacologic agents specific enough to affect only one isotype in a multigene family of related proteins). However, enthusiasm about the potential of the technology has been appropriately tempered by questions about its ability to deliver on its promise. The fundamental issue is: Can oligonucleotides that have acceptable drug properties be created? This review discusses recent progress that helps to define the pharmacokinetic, pharmacologic, and toxicologic properties of phosphorothioate oligonucleotides. Progress in the medicinal chemistry of oligonucleotides that has resulted in a wide range of new chemical classes is also described. PMID- 7559257 TI - Ex vivo expansion of hematopoietic progenitor cells in human cord blood: an effect enhanced by cord blood serum. AB - The concept of ex vivo expansion of the human stem/progenitor cell population was tested in this study. Cord blood mononuclear cells were isolated and cultured in the presence of SCF, IL-3, and either cord blood serum (CBS) or fetal calf serum (FCS). After 8 days of incubation with CBS or FCS, the number of CFU-GM was increased ninefold and four-and-a-half-fold, respectively. However, the enhancing effect lasted for only a short period. This study demonstrates that CBS could contain growth factors expanding progenitor cells. Whether the ability of CBS to enhance the CFU-GM expanding capacity is attributable to a novel factor or factors, or represents effects of other known cytokines, remains to be determined. PMID- 7559258 TI - Lineage identification of acute leukemias: relevance of immunologic and ultrastructural techniques. AB - This study assesses the value of immunologic and ultrastructural methods in disclosing the lineage commitment of cells from acute leukemias (ALs). Two hundred and fifty-one ALs were characterized morphologically, cytochemically, and immunologically. Myeloperoxidase (MPO) positivity in > 3% of blasts was regarded as evidence of the myeloid origin of leukemic cells, cytoplasmic CD22 (cCD22) expression was taken as an indication for B-lineage acute lymphoblastic leukemia (ALL), and CD3+ (membrane or cytoplasmic) cases were classified as T-ALL. Diagnosis of minimally differentiated acute myeloid leukemia (AML-M0) was made when blast cells had undifferentiated features by light microscopy, reacted with at least one of the antibodies to myeloid-specific antigens (CD13, CD33, MPO), and lacked CD19, cCD22, and c/mCD3. Megakaryoblastic differentiation was demonstrated by the expression of CD41 and/or CD61. Following these criteria, 209 cases were classified as acute myeloid leukemia (AML) and 39 as ALL. Expression of lymphoid antigens was detected in 45% of AML cases and 30% of ALLs showed myeloid antigens. One case was regarded as a true biphenotypic leukemia because of the combined expression of MPO and CD33 for the myeloid lineage, and cCD3, CD2, and CD5 for the T-cell lineage. Two cases lacked signs of myeloid or lymphoid differentiation and were studied by electron microscopy methods. One displayed platelet peroxidase (PPO) activity and was classified as a megakaryoblastic variant, one other reacted with anti-CD33 and was considered AML M0. We conclude that light microscopy and standard immunologic methods can accurately demonstrate the lineage orientation in greater than 99% of ALs. Integration with ultrastructural analysis can define the cell nature of virtually all cases of AL. PMID- 7559259 TI - Bone marrow morphology during induction phase of therapy for acute myeloid leukemia (AML). AB - Sequential bone marrow aspirates and sections from patients with acute myeloid leukemia (AML) were examined to determine if a correlation exists between bone marrow morphology during induction phase of therapy and outcome. Of 95 patients of AML diagnosed between July 1987 and December 1991, 53 uniformly treated patients (induction therapy with cytosine arabinoside and daunorubicin) had sequential bone marrow examinations performed in the 2- to 5-week period following initiation of induction therapy. Four morphologic patterns were recognized in these 53 patients: Group I (22 patients)--hypocellularity or normal regeneration (> or = 15% cellularity and < 5% blasts) on the initial 2-week marrow followed by marrows showing normal regeneration; Group II (10 patients)- hypocellularity followed by "reactive myeloblastosis" (> or = 15% cellularity, 5% to 34% blasts, with promyelocytes = or > blasts); Group III (12 patients)- residual blasts (> or = 5% blasts with blasts >> promyelocytes) in the initial posttherapy marrow; Group IV (9 patients)--atypical patterns not fitting any of the other categories. Complete remission was achieved in all 32 patients in Groups I and II without additional induction therapy, but was achieved eventually in only 10 of 21 patients in Groups III and IV combined (p < 0.005), 15 of whom received additional induction therapy. Remission duration and actuarial survival for each group were as follows: Group I: 344/596 days; Group II: 443 days/> 660 days; Group III and IV combined: 351/311 days (p = 0.017 for actuarial survival). Seven of 21 patients in Groups III and IV had unfavorable initial morphology (MO, hypoplastic AML and AML preceded by myelodysplasia) compared to only 3 of 32 patients in Groups I and II (p = 0.039). It was thus observed that "reactive myeloblastosis" with up to 34% blasts on the third or fourth week bone marrow following an initial hypocellular marrow does not require additional induction therapy to achieve durable remissions or favorable survival. Also, residual blasts that outnumber promyelocytes, and atypical patterns of regeneration correlate with lower remission induction rates, shortened survival, and unfavorable morphology on the initial diagnostic bone marrow. PMID- 7559261 TI - Pet ownership in the UAE: its effect on allergy and respiratory symptoms. AB - The aim of this paper was to study the effect of pets and other domestic animals on bronchial asthma among United Arab Emirates (UAE) schoolchildren aged 6-14 years. A cross-sectional study of 850 schoolchildren living in both urban and rural areas (average age 9.36 +/- 2.11 years, 46.8% boys and 53.2% girls) was conducted using self-administered questionnaires between October 1992 and May 1993. Prevalence rate for asthma, rhinitis, wheeze, cough, and eczema in children from families with and without animals were investigated. A total of 40.7% of families studied were found to keep animals in their homes. Children from families with animals were found to have a significantly higher prevalence rate of respiratory symptoms than those without. The prevalence rate for asthma in children with animals was found to be twice that of children without (RR: 2.03; 95% CI: 1.40-2.95). The risk of having chronic cough (RR: 1.93; 95% CI: 1.21 3.10), breathlessness/chest tightness (RR: 2.53; 95% CI: 1.59-4.02), chronic wheeze (RR: 2.10; 95% CI: 1.20-3.67), allergic rhinitis (RR: 1.53; 95% CI: 1.17 2.00) was significantly higher in children with animals than in children without. Similarly, the risk of having eczema (RR: 2.55; 95%, CI: 1.74-3.75) was significantly higher among children with animals than among those without. Overall, there was a highly statistically significant difference in the prevalence of asthma, wheeze, nocturnal cough, eczema, and rhinitis between children in families with animals and those without (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559260 TI - Zaditen SRO permits once-daily dosing with superior efficacy in the prophylaxis of asthma. AB - This international, multicenter clinical trial was designed to compare the efficacy and safety of two different formulations of ketotifen: Zaditen SRO and Zaditen Standard Form. In a randomized double-blind study over a 12-week treatment period, 3 parallel groups of asthmatic subjects received Zaditen SRO (2 mg once daily), Zaditen SRO (4 mg once daily), or Zaditen Standard Form (1 mg twice daily). Asthmatic subjects (362 evaluable cases, aged 6-29 years) kept daily records of clinical symptoms, use of concomitant medication, and peak flow recordings and were examined at 2-week intervals up to the end of the study. Zaditen SRO 4 mg administered once a day at night showed a statistically significant faster onset of action and was more clinically effective than Zaditen Standard Form. The Zaditen SRO 4-mg and 2-mg formulations were at least as well tolerated as the standard form, with somnolence occurring equally after both formulations. In conclusion, Zaditen SRO (4 mg once daily) was found to be equally safe and more effective in the prophylactic treatment of mild and moderate bronchial asthma than Zaditen Standard Form (1 mg twice daily). PMID- 7559262 TI - A beta 2-agonist, procaterol, inhibits basophil migration. AB - Beta 2-receptor agonists have recently been reported to be effective on allergen induced late-phase reaction (LPR) in addition to their inhibitory effect on immediate-phase reaction, although the precise mechanism is not fully understood. In this study, we tested the effect of a selective beta 2-agonist, procaterol, on human basophil migration, which may be an important characteristic of LPR. Procaterol inhibited IL-8- and C5a-induced basophil migration in a dose-dependent fashion; 10(-7) M of procaterol reduced 30% of migration induced by both factors. The action of procaterol was rapid since the inhibition of migration was detected without preincubation and was not via the toxic effect on basophils as assessed by trypan blue test. The results of this study extend the repertoire of anti inflammatory actions of beta 2-agonists. PMID- 7559263 TI - Physicians' prevention-related practice behaviors in treating adult patients with asthma: results of a national survey. AB - To improve the health outcome of adults with asthma, it is important to understand the current practice behaviors of physicians related to the prevention and treatment of asthma. A national survey was conducted to ascertain the practice behaviors of physicians in five specialty areas: internal medicine, pulmonary, allergy/immunology, occupational health, and family health. Similarities and differences in practice among the specialty areas are indicated. The data provide a basis for recommendations to improve the management of asthma by standardizing history taking, increasing the use of pulmonary function testing, and using effective counseling and patient education strategies. PMID- 7559264 TI - Duration of action of a single dose of azelastine in patients with chronic asthma. AB - The aim of this double-blind, multicenter trial was to determine the duration of bronchodilating activity of a single 4-mg dose of azelastine in chronic asthmatics. Eligible patients had an FEV1 of 40-79% of predicted value with demonstrated reversibility of airway obstruction. A single dose of azelastine 4 mg resulted in rapid onset of bronchodilating activity with a 12-hr duration of action as demonstrated by a clinically and statistically significant mean percent improvement in FEV1, from hours 2-12. As an overall measure of improvement in FEV1, the area under the curve was statistically significantly greater for azelastine than for placebo. Mean improvements in symptom severity in the azelastine group exceeded those for placebo at all observation points. No serious adverse experiences were associated with azelastine. PMID- 7559265 TI - An epidemiological investigation into nocturnal asthma. AB - The prevalence of nocturnal asthma and its relationship with clinical features of the disease were studied in 150 consecutive patients. Nearly three-fourths of patients had nocturnal symptoms of asthma, either currently or in the past. There was no relation between the tendency to develop nocturnal symptoms and age, sex, atopic status, or periodicity of the disease. However, those with more severe asthma had a greater tendency to develop nocturnal symptoms. Patients currently having nocturnal symptoms had a poorer lung function. PMID- 7559267 TI - Anaphylaxis to kiwi fruit in a 12-year-old boy. PMID- 7559268 TI - Care of asthmatics on discharge from hospital: a hospital audit. AB - The objective of this study was to audit the care of patients after discharge from hospital, following admission for acute severe asthma, using the British Thoracic Society guidelines (1) as a standard. Discharge prescriptions and questionnaires sent to patients at home were analyzed for 51 patients who had been admitted to a teaching hospital with acute severe asthma. Main outcome measures were Peak flow measurements, written instructions, prescription of steroids, and outpatient follow-up. Of the 34 patients responding to the questionnaires, 15 (44%) had no peak flow meter, 23 (68%) had no written instructions, 13 (38%) had no supply of oral steroids at home, and 32 (94%) were prescribed a beta-agonist regularly of whom 12 (35%) were not on an inhaled steroid. Four (9%) patients were not followed up as outpatients; appointments ranged from 2 to 56 days following discharge. In over 60% of asthmatic patients discharged from hospital the guidelines recommended by the British Thoracic Society were not followed. The method used is an inexpensive, efficient way of auditing hospital practice. PMID- 7559266 TI - Serum levels of soluble interleukin-2 receptor in asthma patients. AB - We measured the serum concentration of soluble interleukin-2 receptor (sIL-2R) in asthma patients and healthy controls to evaluate T-lymphocyte activation in this disease. The SIL-2R concentrations in patients with asthma irrespective of presence of acute attacks were significantly higher than those of the controls (p < 0.001). Although the sIL-2R concentrations were similar in the patients with acute asthma and those in remission, levels in patients with moderate and severe asthma attacks were significantly higher than levels for patients in remission (p < 0.001). These results suggest that T-lymphocyte activation occurs in asthma patients and becomes more prominent during moderate and severe acute attacks. PMID- 7559272 TI - Asthma and managed care. PMID- 7559270 TI - Two new roles for theophylline in the asthmatic? PMID- 7559269 TI - Theophylline--down but not yet out. PMID- 7559271 TI - Risk factors for intubation of adult asthmatic patients. AB - Our object was to describe demographic data from a population of adult asthmatics admitted to a regional tertiary medical center to identify risk factors for intubation. We performed a retrospective cohort study of all asthma admissions (International Classification of Diseases, Ninth Revision, Code 493.9) excluding cases with chronic obstructive pulmonary disease. This included all patients with asthma 20 years and above admitted to the University of California Davis Medical Center, Sacramento, from January 1, 1990 to June 30, 1993. A total of 375 asthma admissions were reviewed. There were 244 women (29 intubated) and 131 men (13 intubated) with a mean age of 40.7 (range 20-72) years. Of this group, 131 people were white, 140 black, 56 Hispanic, 42 Asian, and 6 American Indian. By National Heart, Lung, and Blood Institute Guidelines, there were 101 mild, 181 moderate, and 93 severe cases. Significant risk parameters identified for intubation were psychosocial problems [odds ratio (O.R.) 9.3; 95% confidence interval (C.I.) 6.8, 12.7], low socioeconomic group (O.R. 2.9; 95% C.I. 1.5, 5.8), little formal education (O.R. 5.4; 95% C.I. 2.8, 10.2), atopic allergy (O.R. 11.7; 95% C.I. 5.7, 23.7), duration of asthma > or = 15 years (O.R. 2.6; 95% C.I. 1.3, 5.3), previous intubation (O.R. 14.0; 95% C.I. 7.6, 25.6), upper respiratory infection (O.R. 4.0; 95% C.I. 2.2, 7.5), hospital admission for asthma within the last year (O.R. 5.3; 95% C.I. 2.7, 10.4), emergency room visit within the last year (O.R. 8.8; 95% C.I. 3.9, 20.1), and steroid dependency (O.R. 5.5; 95% C.I. 3.0, 10.2).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559273 TI - Managed care and the treatment of asthma. AB - Few diagnoses offer managed care more return for the investment than asthma. Physicians are beset by mandates to use more anti-inflammatory therapy, peak flow meters, spacer devices, and written step care plans. The managed care system offers the organization and resources to meet this challenge to support physicians on the front line. Economies of scale and computer data bases now make asthma care outcome measures available for evaluation and revision. The current review focuses on five areas for development of successful asthma intervention: (1) physician education, (2) the comanagement concept, (3) patient education, (4) cost-effectiveness, and (5) implementation. PMID- 7559274 TI - Effectiveness of a specialized asthma clinic in reducing asthma morbidity in an inner-city minority population. AB - Asthma is the most common chronic disease of childhood and a leading cause of morbidity in adults. Despite significant advances in medical therapy, asthma morbidity and mortality rates have risen dramatically over the past two decades, especially in minority and socioeconomically disadvantaged populations. Numerous intervention programs have been designed in an attempt to reduce asthma morbidity but few have targeted poor or minority populations. The purpose of this study was to assess whether an outpatient intervention program specifically targeted at a high-minority population in East Harlem, in New York City, was successful in reducing asthma morbidity. A retrospective chart review of 84 patient records was conducted. The patients were divided into two groups, an intervention group (n = 45), who were followed by an asthma specialist (allergist/immunologist), and a nonintervention group, followed by a general internist or pediatrician. Outcome variables including clinic walk-in visits, emergency room visits, and hospitalizations were determined and compared in the pre- and postintervention period in both groups. Patients in the intervention group had reduced total walk in visits (73 vs. 27, p < 0.001), emergency room visits (30 vs. 5, p < 0.001), and hospitalizations (16 vs. 2, p < 0.001). In contrast, patients in the nonintervention group had no change in total walk-in visits (88 vs. 72), increased emergency visits (7 vs. 22, p < 0.05), and no change in hospitalizations (5 vs. 2), respectively. The outpatient intervention program has been successful in reducing asthma morbidity in the high-risk minority community of East Harlem. Future larger studies are warranted to extend this pilot program to other high-risk minority populations. PMID- 7559275 TI - Psychosocial adjustment and the role of functional status for children with asthma. AB - This study examined the psychosocial adjustment of children with asthma compared to children with diabetes, with cancer, and healthy children and the role of functional status in psychosocial adjustment. The total sample included 100 children, aged 8-16 years, (mean = 11.5 years), consisting of 48 boys and 52 girls. Children with asthma scored significantly higher on measures of affective adjustment (depression and internalizing behavior), significantly lower on self esteem, and evidenced significantly greater functional impairment. Children with cancer missed significantly more school days. After controlling for functional status, no significant differences remained in affective adjustment but absences remained significantly higher for the children with cancer. PMID- 7559276 TI - Patients' opinions on respiratory care: do doctors fulfill their needs? AB - To date, guidelines and consensus reports on quality care for asthma and chronic obstructive pulmonary disease (COPD) are mainly based on research and opinions of care providers. Patients' viewpoints on good medical care have rarely been studied. We designed a postal questionnaire to study the needs of patients with asthma or COPD for medical care provided by general practitioners and lung physicians. A total of 121 patients filled out the questionnaire, which included 111 items about needs. Although generally satisfied, patients reported several unfulfilled needs. Main topics were the need for information about diagnostic tests, prognosis, and long-term use of medication. In addition, patients wanted more written information about the nature of their disease. One-third of patients wanted more participation in decisions about their treatment. These results suggest the quality of medical care for patients with asthma or COPD can be improved by adjusting provided care to needs expressed by patients. PMID- 7559277 TI - Characteristics of hospitalization for asthma among persons less than 35 years of age in Chicago. AB - Mortality from asthma in Chicago is among the highest in the nation for 5-34-year olds. Data for all hospitalizations among Chicago residents less than 35 years of age were examined to define characteristics that may be contributing to morbidity and mortality from asthma. From 1987 through 1989, the average annual age adjusted hospitalization rate among persons less than 35 years of age in Chicago was 3.57 per 1000 persons. There were significant associations of community hospitalization rate with median income (r = -0.61, p < 0.001) and with proportion of community asthma hospitalizations using Medicaid, Medicare, or self payment (r = 0.69, p < 0.001). Among 18-34-year-old men, asthma hospitalization rates for Medicaid recipients were 17.4-34.1-fold higher than among men using other forms of insurance. Asthma admissions using Medicaid, Medicare, or self payment were more likely than those using other forms of insurance to present through the emergency department (79.3% and 66.4%, respectively, p < 0.001) and be discharged against medical advice (1.8% vs. 0.7%, respectively, p < 0.001). These data suggest that differential access to or utilization of health care may be contributing to asthma morbidity in Chicago. PMID- 7559279 TI - Risk factors for asthmatic patients requiring intubation. II. Observations in teenagers. AB - The asthma mortality rate has increased steadily over the past 15 years in the United States and has only recently shown signs of leveling off. It is widely believed, although unproven, that many asthma deaths may be preventable. We have addressed one critical factor in severe asthma by attempting a definition of risk factors for intubation using demographic data and a retrospective cohort study of hospitalized asthmatic teenagers. This study included all asthmatics aged 13-19 years admitted over a 10-year period (1984-1994) to the University of California Davis Medical Center, Sacramento, California. A total of 143 such asthma admissions were reviewed, involving 68 females and 75 males, mean age 16.4 +/- 2.3 years. Of this group, 85 teenagers were black, 34 were Caucasian, 14 were Hispanic, and 10 were Asian. By National Heart, Lung, and Blood Institute guidelines, there were 42 mild, 85 moderate, and 16 severe cases. Ten of the 143 teenagers studied required intubation for their asthma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559278 TI - Clinical features, management, and outcome of patients with severe asthma admitted to the intensive care unit. AB - The objective of this study was to retrospectively review the medical records of 38 consecutive admissions to the medical intensive care unit of a tertiary-care university hospital of patients with severe asthma, and to determine the clinical characteristics of these patients, treatment regimens, and ultimate outcome. The 38 patients presented with severe asthma accompanied by hypoxemia, hypercapnia (mean pCO2 of 54.3 +/- 4.5 mm Hg), and decreased peak flow rates (125.5 +/- 12.4 L/min). The patients spent a mean of 60.1 +/- 9.7 hr in the medical intensive care unit. Seventeen of the 38 patients required intubation and mechanical ventilation. Overall, there were no deaths or significant complications. All 38 patients were discharged from the hospital. We conclude that severe, life threatening asthma can be appropriately managed in the medical intensive care unit with a low incidence of complications and death. Prolonged mechanical ventilation is rarely required and most patients respond while the relatively simple management strategies. PMID- 7559280 TI - Generativity in the play of young people with autism. AB - Examines the ability of young people with autism to generate ideas for play. Young people with autism, children with learning difficulties, and younger normal children were asked to generate 12 different actions and follow 12 instructions with a car and a doll. The young people with autism were impaired, relative to the controls, at generating original actions with the car, but were as able as the controls to follow the instructions. However, the young people with autism were not impaired at generating original actions with the doll. All three groups produced similar amounts of symbolic play. Possible explanations for the difference in results for the two toys are discussed. PMID- 7559281 TI - Teaching symbolic play skills to children with autism using pivotal response training. AB - Used Pivotal Response Training (PRT) to teach 7 children with autism to engage in symbolic play behaviors. Symbolic play, complexity of play behavior, and creativity of play were assessed. In addition, generalization measures were obtained across settings, toys, and play partners. Interaction with the play partners and comparison with typical controls were also examined. Results indicated that children with autism rarely exhibited symbolic play before training or after a control condition. After specific symbolic play training using PRT, all of the children learned to perform complex and creative symbolic play actions at levels similar to that of language-matched typical controls. In most cases the children generalized their play to new toys, environments, and play partners and continued to engage in symbolic play behavior after a 3-month follow-up period. In addition, interaction skills improved after training. Treatment implications for these findings are discussed. PMID- 7559285 TI - Questionnaire screening for comorbid pervasive developmental disorders in congenitally blind children: a pilot study. AB - Teachers and parents completed the Autism Behavior Checklist (ABC) on a clinical sample of 17 congenitally blind children. Of the 17 children, 4 had a definite or likely pervasive developmental disorder (PDD) as judged by independent case note review. The ABC was administered both in its original format and in a slightly modified format. Only teacher-completed ABCs detected group differences and had satisfactory test-retest reliability. The modified-format ABC completed by teachers detected 3 of the 4 children with PDDs without any false positives. Screening questionnaires may have a limited but useful role in locating subjects with blindness plus putative PDDs for further study. PMID- 7559283 TI - Psychiatric and behavioral disorders in hospitalized preschoolers with developmental disabilities. AB - Describes the types of psychiatric and behavioral disturbance present in 169 preschoolers with developmental disabilities admitted to a specialized psychiatric inpatient unit. Differences in the proportion of some diagnoses and behavior problems across cognitive functioning level and across age were found. Seventy-two percent of the sample had one or more medical diagnosis. Similarities and differences with earlier reports in the literature are discussed. PMID- 7559284 TI - Cerebrospinal fluid biopterin and biogenic amine metabolites during oral R-THBP therapy for infantile autism. AB - Treatment with 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP) has been suggested to improve autistic behavior. Cerebrospinal fluid (CSF) levels of total biopterin, oxidized and reduced forms of biopterin, homovanillic acid, and 5 hydroxyindoleacetic acid were measured in 14 autistic children and 18 controls to clarify the mechanism of action of R-THBP. The 14 autistic children received R THBP orally at 1 mg/kg per day; 7 children showed clinical improvement (responders) and the other 7 patients did not (nonresponders). There were no significant differences between responders, nonresponders, and controls in the CSF levels of the metabolites before R-THBP administration. When lumbar puncture was repeated in 6 autistic children in the 24th week of R-THBP therapy, there was no significant change in the CSF levels of any metabolites. PMID- 7559282 TI - Issues in the taxonomy of psychopathology in mental retardation. AB - Several factors appear to impede the development of a valid taxonomy of psychopathology in children and adolescents with mental retardation. These include (a) the lack of a widely accepted definition of psychopathology in mental retardation, (b) disagreement on the nature of the relationship between mental retardation and psychopathology, and (c) insufficient evidence for the reliability and validity of current DSM or ICD systems in this population. In this article, we offer a definition of psychopathology in children with mental retardation; review concepts of the relationship between psychopathology and mental retardation; argue that in moving toward a valid taxonomy factors to be considered should include data from multivariate studies, findings related to behavior phenotypes, and diagnostic considerations with stereotypic behavior and self-injury, organic brain syndromes and pervasive developmental disorders. Finally, we outline a research strategy that may serve as a useful framework for developing a valid taxonomy of psychopathology in this population. PMID- 7559286 TI - An evaluation of facilitated communication in a group of nonverbal individuals with mental retardation. PMID- 7559288 TI - Stealth virus isolated from an autistic child. PMID- 7559287 TI - Brief report: fostering environmental control in a young child with Rett syndrome: a case study. PMID- 7559289 TI - The Developmental Behavior Checklist: the development and validation of an instrument to assess behavioral and emotional disturbance in children and adolescents with mental retardation. AB - Describes the development and validation of the Developmental Behavior Checklist (DBC), a standardized instrument completed by lay informants to assess behavioral and emotional disturbance in children and adolescents with mental retardation (MR). Items describing common behavioral and emotional problems in this population were generated by extracting descriptions from 664 case files of children and adolescents with behavior disorders seen at a specialist developmental assessment service over 12 years. These items were reduced to a set of 96 items administered to a sample of 1,093 children and adolescents with mental retardation and then submitted to a principal components analysis. Six interpretable and partly validated subscales were obtained which explained 36% of the total variance and had satisfactory internal consistency. Interrater and test retest agreement were satisfactory for both total scale score and for scores on each of the subscales. Good evidence of concurrent validity was provided by substantial positive correlations between total scores on the DBC completed by lay informants and the ratings of experienced psychiatrists based upon interviews and scores on two standardized instruments that must be completed by health professionals. The discriminative validity of the total score as assessed by area under the ROC curve was excellent (92%). Standardized norms for the DBC are derived from an epidemiological study of behavior problems in children and adolescents with mental retardation undertaken in two Australian States. Norms are available for the mild, moderate, severe, and profound MR groups and for the MR population as a whole. PMID- 7559290 TI - The abilities of a musical savant and his family. AB - The ability of a male autistic savant (TR) to play two unfamiliar piano pieces after listening to a tape-recording was tested, closely following the procedures of Sloboda, Hermelin, and O'Connor (1985). Other components of TR's musical ability--pitch recognition, improvisation, and ability to provide harmonic accompaniment--were also examined. TR's musical precocity was examined in relation to his general level of intellectual functioning as indexed by a battery of standardized psychological tests of intelligence, memory, reading, visual organization, and creativity. His parents and two male siblings also completed tests of intelligence. Results from psychometric testing indicated that TR has idiosyncratic levels of cognitive functioning with difficulties in verbal reasoning but high levels of concentration and memory. His speed of information processing, as indicated by Inspection Time, and was better than average. TR demonstrated perfect pitch recognition and other family members also demonstrated excellent relative pitch. TR's ability to recall and perform structured music within both the diatonic and whole-tone systems was exceptional but dependent upon his familiarity with musical structure and was therefore organized and rule driven. Furthermore, TR demonstrated competence in improvisation and composition, albeit restricted by his adherence to structural representations of familiar musical rules. PMID- 7559292 TI - Effects of sociodramatic play training on children with autism. AB - We assessed the effects of teaching sociodramatic play to three children with autism. The training was conducted using a variation of Pivotal Response Training (PRT), a program traditionally used to teach language to children with autism. Measures of play skills, social behavior, and language skills were obtained before treatment, after treatment, and at a follow-up period. The correlation between language and pretend play was explored, as was the relationship between sociodramatic play and social competence. Positive changes were observed in play, language, and social skills. These changes generalized across toys and settings, although little generalization to other play partners occurred. Effects of play training with children with autism and maintenance of behavior change is discussed. PMID- 7559291 TI - Disorders of regulation of cognitive activity in autistic children. AB - Infantile autism is a pervasive developmental disorder characterized by disturbances concerning not only the areas of socialization and communication ("aloneness") but also the ability to modify and change behavior ("need for sameness"). In most recent studies, various abnormal and deviant cognitive activities, such as the ability to regulate one's behavior, were considered as accounting for these signs. In this report, we examined the regulation of cognitive activity, from a developmental perspective in comparing autistic with mentally retarded children matched in a pairwise manner by global, verbal, and nonverbal developmental ages. All children were tested with tasks adapted from the Object Permanence Test which corresponds to Piaget's sensorimotor development Stages IV to VI. Results showed that autistic children had a pervasive difficulty in maintenance set, made more perseverative errors when the abstraction degree of task was higher, and were more variable in their behavioral strategies. Discussion is focused on the interests and limits of these tasks for the examination of regulation activity from diagnostic and developmental perspectives. Finally, interpretations about recent neuropsychological and neurophysiological works, and additional interdisciplinary studies are suggested. PMID- 7559293 TI - Use of methylphenidate in the treatment of children with autistic disorder. AB - The use of psychostimulants in autistic disorder has not received extensive evaluation. Furthermore, their use for the symptomatic control of autistic disorder has been felt to be contraindicated. This study investigates the use of methylphenidate (MPH) for the treatment of selected symptoms of autistic disorder. Ten children, ages 7-11, with a DSM-III-R diagnosis of autistic disorder participated in a double-blind crossover study using placebo and two MPH doses (10 mg or 20 mg bid). Subjects showed modest but statistically significant improvement on MPH over placebo. No significant side effects including worsening stereotypic movements occurred on either dose. Improvement in hyperactivity and lack of adverse effects suggest that MPH may be useful in the treatment of hyperactive autistic children. PMID- 7559295 TI - Brief report: the impact of regression on language development in autism. PMID- 7559296 TI - Brief report: thought disorder in Asperger syndrome: comparison with high functioning autism. AB - Asperger syndrome (AS) is a pervasive developmental disorder generally regarded as a variant of autism. While it has been included in the ICD-10 and DSM-IV as a distinct diagnostic entity, it is still unclear to what extent it differs from high-functioning autism (HFA). Persons with HFA have been reported to show a variety of deficits of thought processes. Abnormalities such as poor reality testing, perceptual distortions, and areas of cognitive slippage have been described using the Rorschach inkblot test (Dykens, Volkmar, & Glick, 1991). Since AS has been conceptualized as a mild variant of autism, we hypothesized that persons with AS will have fewer abnormalities on the Rorschach test compared to persons with HFA. To test this hypothesis, we compared 12 subjects with AS (ICD-10, 10 male, mean age = 12.2 +/- 3.3 years, mean full-scale IQ = 99.6) with 8 subjects with HFA (ICD-10/DSM-III-R, 7 male, mean age = 12.2 +/- 3.8 years, mean full-scale IQ = 83.4) on the Rorschach test. AS subjects demonstrated a trend towards greater levels of disorganized thinking than the HFA group. They were also more likely to be classified as "Introversive" suggesting that AS subjects may have more complex inner lives involving elaborate fantasies, Also, AS subjects tended to be more focused on their internal experiences. However, overall, the Rorschach test was not found to differentiate the two diagnostic groups on the majority of structural variables. Implications of these findings are discussed with regard to the diagnostic validity of Asperger syndrome. PMID- 7559297 TI - Brief report: assessing allegations of sexual molestation made through facilitated communication. PMID- 7559298 TI - Brief report: allergological evaluation of children with autism. PMID- 7559300 TI - Distractibility as a symptom of ADHD. PMID- 7559299 TI - Exercise prescription for autistic populations. PMID- 7559301 TI - Children traumatized by war. PMID- 7559294 TI - Plasma androgens in autism. AB - Plasma levels of testosterone and the adrenal androgen dehydroepiandrosterone sulfate (DHEA-S) were measured in male autistic subjects (31 prepubertal, 8 postpubertal), mentally retarded/cognitively impaired subjects (MR, 12 prepubertal), and normal control subjects (NC, 10 prepubertal, 11 postpubertal). Mean levels of plasma testosterone were similar in the postpubertal autistic (4.54 +/- 1.12 ng/ml) and postpubertal NC (5.02 +/- 1.87 ng/ml) groups. Plasma DHEA-S levels in postpubertal autistic (2170 +/- 1020 ng/ml) and postpubertal NC (1850 +/- 777 ng/ml) groups also were not significantly different. Similarly, no significant group differences were seen for testosterone or DHEA-S in the prepubertal autistic, MR, or NC individuals, although prepubertal MR individuals with cerebral palsy did have increased plasma DHEA-S levels compared to age matched MR or NC individuals. Significant negative correlations were found between testosterone and whole blood serotonin (5-HT) levels in the combined (all subjects, all ages) groups and in the autistic group, suggesting that the effect of puberty on whole blood 5-HT may deserve further study. Data indicate that altered secretion of the androgens is not a common feature of autism. However, abnormalities of adrenal androgen secretion may be present in individuals with cerebral palsy. PMID- 7559303 TI - Early predictors of conduct disorder. PMID- 7559302 TI - Triple suicide pact. PMID- 7559304 TI - Managed care: income to outcome. PMID- 7559305 TI - Psychopharmacology in child and adolescent psychiatry: a review of the past seven years. Part I. AB - OBJECTIVE: To present a critical overview of the literature published in the past 7 years on the efficacy and safety of psychoactive agents in mental retardation with associated psychiatric disorders, autistic disorder, Tourett's disorder, and attention-deficit/hyperactivity disorder. METHOD: Double-blind and placebo controlled clinical trials and open studies were reviewed and selected reports presented. RESULTS: The literature review reveals that progress has been made in the psychopharmacological treatment of the above conditions. This is partly because more studies use larger sample sizes and a narrower age range of diagnostically homogeneous patients and use a more sophisticated methodology than in previous years. Greater attention is being paid to a critical assessment of psychoactive agents and to their safety, to the efficacy as well as to the effectiveness of drugs. The 5-year National Plan for Research on Child and Adolescent Mental Disorders (1991) based on the Institute of Medicine Report (1989) already has had a significant impact on psychophamacology research. CONCLUSIONS: Advances in methodology, initiatives of the National Institute of Mental Health, and the advent of DSM-IV should continue to enhance research and improve pharmacotherapy in clinical practice. PMID- 7559306 TI - The relationship between tic disorders and Tourette's syndrome revisited. AB - OBJECTIVE: The relationship between Tourette's syndrome (TS) and chronic tic disorder is of great clinical and scientific importance because of uncertainties in both prognosis and pharmacotherapeutic strategies. One approach to evaluating the relationship between TS and chronic tics is to examine whether they share similar neuropsychological and psychiatric correlates. METHOD: Children with TS (n = 32) and children with chronic tics (n = 39) were ascertained from an unselected sample of the children referred for psychopharmacological treatment and examined using standardized diagnostic assessments and testing procedures. RESULTS: Children with TS and those with chronic tics were similar to each other and different from controls in clinical correlates that included psychiatric comorbidity, as well as school, neuropsychological, and psychosocial impairments. Patients with TS also had higher rates of obsessive-compulsive disorder, oppositional defiant disorder, and simple phobia than did patients with chronic tic disorder. CONCLUSIONS: These findings indicate that TS and chronic tic disorder are part of the same disease entity, with TS being a more severe form of tic disorder. PMID- 7559307 TI - Guanfacine treatment of comorbid attention-deficit hyperactivity disorder and Tourette's syndrome: preliminary clinical experience. AB - OBJECTIVE: Many children with Tourette's syndrome (TS) are handicapped more by difficulties with inattention, impulsivity, and hyperactivity than by their tics. However, stimulant medications used to treat attention-deficit hyperactivity disorder (ADHD) can exacerbate tics. Guanfacine is an alpha 2-adrenergic agonist that may have beneficial effects on attention, without the hypotensive or sedative effects of clonidine, which is often used as an alternative to stimulants. METHOD: An open-label study of guanfacine was performed in 10 children with TS+ADHD, aged 8 to 16 years. The duration of follow-up was 4 to 20 weeks, and the majority of subjects were treated with 1.5 mg/day. Ratings of tic severity and ADHD symptoms were obtained using the Yale Global Tic Severity Scale (YGTSS), the Tic Symptom Self Report (TSSR), and the Conners Parent Rating Scale. In addition, blind Continuous Performance Tests (CPTs) were performed at baseline and at two follow-up intervals in eight subjects. RESULTS: Guanfacine was associated with significant decreases in both commission errors (p < .02) and omission errors (p < .01) on the CPT. In addition, guanfacine caused a significant decrease in severity of motor (p < .02) and phonic (p < .02) tics as measured by the TSSR and the YGTSS, respectively. The most common side effects were transient sedation and headaches. CONCLUSION: Guanfacine may provide a safe alternative therapy for children with ADHD in the presence of tics. Future double blind, controlled trials should be undertaken. PMID- 7559308 TI - Risperidone treatment of children and adolescents with chronic tic disorders: a preliminary report. AB - OBJECTIVE: The purpose of this trial was to investigate the short-term safety and efficacy of risperidone in the treatment of chronic tic disorders in children and adolescents. METHOD: This was an 11-week open-label trial and included seven subjects (five boys and two girls) with a mean age of 12.9 +/- 1.9 years. The sample included five patients with Tourette's syndrome and two with chronic motor tic disorder. The children were seen at baseline and for two follow-up visits. Three children had a comorbid diagnosis of obsessive-compulsive disorder (OCD). RESULTS: Clinical response, as measured by the Yale Global Tic Severity Scale and the Children's version of the Yale-Brown Obsessive Compulsive Scale, revealed a statistically significant reduction in tic scores ranging from 26% [corrected] to 66%. One of three children with comorbid OCD showed substantial improvement; the other two subjects showed no change. The most frequent side effect was weight gain, which ranged from 8 to 14 lb. CONCLUSIONS: Risperidone, a neuroleptic with both serotonin- and dopamine-blocking properties, appears to be effective in reducing tic frequency and intensity in children and adolescents with chronic tic disorders. PMID- 7559309 TI - Adolescent survivors of "ethnic cleansing": observations on the first year in America. AB - OBJECTIVE: To describe the psychiatric assessments and trauma testimonies of 12 Bosnian adolescents newly resettled in America. METHOD: Twelve Bosnian adolescents who experienced the massive psychic trauma of "ethnic cleansing" were assessed during the first year after their resettlement in the United States. Assessments consisted of systematic, trauma-focused, clinical interviews that included standard assessment scales of posttraumatic stress disorder (PTSD) and depression, as well as the opportunity to give testimony about their experiences. RESULTS: PTSD was diagnosed in 25% of subjects and depressive disorders in 17%. Reexperiencing cluster symptoms were present in 50%, avoidance cluster symptoms in 31%, and hyperarousal cluster symptoms in 29%. CONCLUSIONS: The relatively low rate of PTSD in this sample (in comparison with adult survivors of "ethnic cleansing" and with Cambodian adolescent survivors) may be attributable to normal prior development, time-limited adversity, lack of physical or sexual traumas, rejoining nuclear families, or insufficient time for the development of delayed onset PTSD. It may also be a reflection of the resiliency of adolescence. PMID- 7559310 TI - Posttraumatic stress disorder across two generations of Cambodian refugees. AB - OBJECTIVE: To examine the expression of war-related trauma as manifested by DSM III-R rates of posttraumatic stress disorder (PTSD) and major depressive disorder in two generations of Cambodian refugees living in the western United States. METHOD: A probability sample of 209 Khmer adolescents and one of their parents were interviewed using portions of the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Epidemiologic Version and the PTSD section of the Diagnostic Interview for Children and Adolescents. Interviews were conducted in English by a master's-level clinician with a Khmer interpreter. RESULTS: PTSD was found to be significantly related across parent-child generations. A nonsignificant generational trend was also found for depressive disorders. A number of environmental variables measured in the study (amount of reported war trauma, loss, living arrangements, treatment received, socioeconomic status) were not related to these findings. Parents were more likely to report an earlier onset of PTSD symptoms. CONCLUSIONS: This study suggests that PTSD in refugees may cluster in families. Whether this phenomenon is caused by a genetic susceptibility to trauma awaits further research. PTSD and depressive disorders in refugee populations, while often comorbid, appear to follow different courses over time. PMID- 7559311 TI - Comorbidity of psychiatric diagnoses with posttraumatic stress disorder in survivors of childhood trauma. AB - OBJECTIVE: This study examines posttraumatic stress disorder (PTSD) symptoms, trauma exposure, gender, and diagnostic comorbidity in a sample of 59 Cambodian young adults (29 male and 30 female) who survived massive trauma as children. METHOD: Psychiatric diagnoses were made using the Structured Clinical Interview for DSM-III-R-Non-Patient version, a structured diagnostic interview, and trauma exposure was measured with a Traumatic Life Events Questionnaire. RESULTS: A significant number of those with PTSD (59%) had one or more additional DSM-III-R Axis I disorders. Major depression and generalized anxiety disorder were the most common comorbid disorders. Somatoform pain disorder was also found to coexist with PTSD but only among females. Women were also found to have higher levels of both current and lifetime PTSD symptoms. CONCLUSION: Trauma symptoms were related to exposure and exposure was related to age, but age was not related to symptoms. The findings suggest that the significant levels of comorbid diagnoses previously found to exist with PTSD in people traumatized as adults can be found among survivors of massive childhood trauma. Also, the rate of PTSD diagnoses found in this sample 15 years after the trauma of Pol Pot is comparable to findings previously reported in studies of Cambodian youths and shows that the effects of trauma experienced in childhood persist into early adulthood. PMID- 7559312 TI - Psychiatric comorbidity in children after the 1988 earthquake in Armenia. AB - OBJECTIVE: To determine current rates of posttraumatic stress disorder (PTSD), depressive disorder, and separation anxiety disorder (SAD) among children 1 1/2 years after the 1988 earthquake in Armenia; to determine current rates of comorbid PTSD and depressive disorder; and to assess the contribution of exposure, gender, loss of family members, and loss of residence. METHOD: Two hundred eighteen school-age children from three cities at increasing distances from the epicenter were evaluated using the Child Posttraumatic Stress Disorder Reaction Index, the Depression Self-Rating Scale, and the section on SAD from the Diagnostic Interview for Children and Adolescents. RESULTS: On the basis of these evaluations, high rates of current PTSD, depressive disorder, and their co occurrence were found among victims residing in the two heavily impacted cities. SAD was comparatively less frequent, although symptoms of SAD had been pervasive throughout the region. Severity of posttraumatic stress and depressive reactions were highly correlated. Extent of loss of family members was independently correlated with each. CONCLUSION: After a catastrophic natural disaster, children are at risk for comorbid PTSD and secondary depression. Based on the findings, an interactive model is proposed of postdisaster psychopathology. Early clinical intervention is recommended to prevent chronic posttraumatic stress reactions and secondary depression. PMID- 7559313 TI - Psychological effects of Hurricane Andrew on an elementary school population. AB - OBJECTIVE: To explore the prevalence and progression of posttraumatic symptomatology (PTS), using emotional and behavioral indices of psychopathology in school-age children in the pathway of Hurricane Andrew (HI-IMPACT) and in a comparison group north of Miami (LO-IMPACT). METHOD: Pynoos' Posttraumatic Stress Disorder Reaction Index and Achenbach's Teacher's Report Form (TRF) were administered 8 weeks and 32 weeks after the hurricane. In addition, 21 measures of disruptive behavior cataloged by Dade County Public Schools were aggregated and compared by grading period between pre- and posthurricane school years. RESULTS: There were no statistically significant differences between the two schools in PTS at 8 weeks after the hurricane, although the children in the HI IMPACT school were more likely to have severe PTS. TRF findings at 8 weeks revealed that children in the HI-IMPACT school evidenced lower means on the eight TRF scales and on the broader Internalizing and Externalizing measures. Analysis of the disruptive behavior revealed a drop in the marking period immediately after the hurricane in the HI-IMPACT area, but an opposite effect was observed in the LO-IMPACT area. CONCLUSIONS: After the hurricane there was an initial increase in PTS and a concomitant decrease in other measures of behavior and psychopathology. PTS remained relatively high throughout the school year, but there was a rebound and subsequent normalization of the measures of disruptive behavior. PMID- 7559314 TI - Posttraumatic stress disorder in adolescents after Hurricane Andrew. AB - OBJECTIVE: To examine rates and correlates of posttraumatic stress disorder (PTSD) in adolescents after Hurricane Andrew. METHOD: A random-digit dialing sample of 158 Hispanic, 116 black, and 104 white adolescent-parent pairs were surveyed in high- and low-impact areas within Dade County, Florida, 6 months after Hurricane Andrew. Subjects completed a structured telephone interview focused on within-disaster experiences and emotional reaction, disaster-related losses, lifetime exposure to violent or traumatic events, recent stressful experiences, and psychiatric symptomatology. RESULTS: Approximately 3% of males (95% confidence interval 0.4 to 5.3) and 9% of females (95% confidence interval 4.6 to 13.7) met the criteria for PTSD. Rates were highest among blacks (8.3%, 95% confidence interval 2.3 to 14.2) and Hispanics (6.1%, 95% confidence interval 2.2 to 9.9) and increased with age (odds ratio of 1.34, 95% confidence interval 1.04 to 1.72) and the number of undesirable events reported (odds ratio of 1.38, 95% confidence interval 1.21 to 1.57). CONCLUSIONS: While only a relatively small percentage of adolescents reported symptoms consistent with a diagnosis of PTSD, most reported some posttraumatic symptoms. Postdisaster planning should recognize that common stressful events occurring after disasters may be more strongly associated with PTSD than magnitude of contact with the actual disaster. PMID- 7559315 TI - Frequency of phobic disorder in a community sample of young adolescents. AB - OBJECTIVE: To investigate the frequency and phenomenology of clinical, subsyndromal, and subthreshold phobias in young adolescents. METHODS: A two-stage epidemiological study originally designed to investigate adolescent depression was conducted between 1986 and 1988 in the southeastern United States. In the first stage, a self-report depressive symptom questionnaire was administered to a community sample of 3,283 adolescents. In the diagnostic stage, the Schedule for Affective Disorders and Schizophrenia for School-Age Children and the Children's Global Assessment Scale were administered to 487 mother-child pairs. RESULTS: Prevalence rates of clinical, subsyndromal, and subthreshold phobia were 2.3%, 14.5%, and 22.2%, respectively. One-year incidence rates were 0.4%, 8.0%, and 16.9%, with 43.0% of phobic subjects categorized at the same or a more severe level after a year. Females, blacks, subjects not living with both biological parents, and older adolescents were more likely to meet the diagnostic criteria for clinical phobia. The majority (77%) of subjects with clinical phobia experienced multiple phobias. Subsyndromal (52%) and subthreshold (74%) phobics were more likely to experience simple phobias only. CONCLUSIONS: Phobic symptoms are relatively common at a moderate level and in the majority of adolescents are somewhat transitory in nature. Characteristic symptomatology and comorbidity may facilitate earlier identification of subjects at risk of persistent symptomatology and in need of treatment. PMID- 7559317 TI - Case study: body dysmorphic disorder in adolescents. AB - Body dysmorphic disorder, an often-secret preoccupation with an imagined or slight defect in appearance, is an underrecognized disorder that is unknown to many clinicians. This disorder has gone virtually unmentioned in the adolescent literature, despite the fact that it often occurs during adolescence. Body dysmorphic disorder is more common than is realized and causes significant distress and impairment in functioning. This report presents four cases of adolescents with body dysmorphic disorder, all of who responded to a serotonin reuptake inhibitor. The clinical features of body dysmorphic disorder are reviewed, as are available data on the treatment of this distressing and often disabling disorder. PMID- 7559316 TI - An epidemiological study of trichotillomania in Israeli adolescents. AB - OBJECTIVE: To determine the prevalence of trichotillomania and comorbid psychopathology in nonreferred adolescents. METHOD: Using a questionnaire and interview, 794 Israeli 17-year-olds were screened for current and past hair pulling and comorbid psychopathology. RESULTS: Eight current or past hair-pullers (5 male, 3 female) were identified, yielding a lifetime prevalence of hair pulling of 1%. Four subjects reported current hair-pulling (point prevalence of 0.5%). None of these reported alopecia, distress, or tension before pulling; only two reported relief after pulling. Thus, none met the full DSM-III-R criteria for trichotillomania. Four subjects reported past but not current hair-pulling, with bald spots in two cases. Three of the four current hair-pullers had significant obsessive-compulsive symptoms, a significantly elevated rate compared to the entire screened population. Two subjects with obsessive-compulsive disorder also had generalized anxiety disorder and, in one case, chronic simple vocal tics. Hair-pullers did not differ significantly from non-hair-pullers in IQ, physical fitness, and overall competency, or prevalence of other comorbid disorders. CONCLUSIONS: In a community adolescent sample, only 25% of hair-pullers reported resulting bare spots and none endorsed both rising tension and subsequent relief. The prevalence of obsessive-compulsive symptoms was significantly elevated in these nonreferred hair-pullers. PMID- 7559318 TI - Adolescent psychopathology: IV. Specificity of psychosocial risk factors for depression and substance abuse in older adolescents. AB - OBJECTIVE: To determine the specificity to major depressive disorder (MDD) of a wide array of psychosocial risk factors in older adolescents (aged 14 through 18 years). METHOD: Diagnostic and psychosocial assessments were conducted with 1,507 randomly selected high school students at T1 and after approximately 1 year (T2). Three diagnostic groups were compared: those who had an episode of MDD during that year (n = 90), those who had an episode of substance use disorder during that year (SUD) (n = 42), and a control group with no disorder (n = 1,189). RESULTS: Risk factors specific to MDD were stress (minor and major events), emotional reliance, physical symptoms and disease, history of suicide attempt, and a past episode of depression or anxiety disorder. Risk factors specific to SUD were tobacco use, academic difficulties, and a past episode of SUD. Risk factors that were shared were current depression symptoms, internalizing and externalizing behavior problems, coping skills, interpersonal conflict with parents, and dissatisfaction with grades. CONCLUSIONS: By determining the number of risk factors for MDD, for SUD, or those that are general to both disorders, clinicians can make informed predictions concerning the probable future onset of a full-fledged episode of MDD and/or SUD in individual cases. The results of this study allow for the identification of adolescents who are at elevated risk for MDD and SUD. The results also have implications for the design of interventions aimed at preventing the occurrence of these disorders. Such interventions should target change on risk factors of the type identified in this study. PMID- 7559319 TI - Longitudinally predicting late adolescent and young adult drug use: childhood and adolescent precursors. AB - OBJECTIVE: To examine the childhood and adolescent personality determinants of young adult drug use. METHOD: Data were obtained on children when they were approximately 5.5 (time 1; T1), 14 (T2), 16 (T3), and 22 (T4) years of age. T2-T4 interviews of subjects and their mothers assessed child personality and behavior. At T1, 976 mothers were interviewed. The analysis was based on 734 subjects. RESULTS: Specific childhood and adolescent personality traits are related to stage of drug use in young adulthood. Regressions showed that (1) traits at T2 and T3 mediated the effect of traits at earlier ages on T4 drug use and (2) stage of drug use was stable from T3 to T4 despite controlling for personality. Significant interaction revealed two buffers weakening the effect of T3 drug use on T4 drug use. Many more T1-T3 personality traits, particularly low aggression, enhanced the effect of low T3 use on T4 use. CONCLUSIONS: Earlier findings that childhood personality is related to adolescent personality and then to drug use were extended to young adulthood. This mediational model indicates the stability of personality across development. Despite this stability, other results suggest ways to modify drug use. PMID- 7559322 TI - Five promoters integrate control of the cob/pdu regulon in Salmonella typhimurium. AB - Propanediol is degraded by a B12-dependent pathway in Salmonella typhimurium. The enzymes for this pathway are encoded in a small region (minute 41) that includes the pdu operon (controlling B12-dependent degradation of propanediol) and the divergent cob operon (controlling synthesis of cobalamin, B12). Expression of both operons is induced by propanediol and globally controlled by the ArcA and Crp systems. The region between the two operons encodes two proteins, PduF, a transporter of propanediol, and PocR, which mediates the induction of the regulon by propanediol. Insertion mutations between the pdu and cob operons have been characterized, and their exact positions have been correlated with mutant phenotypes. The region includes five promoters, four of which are controlled by the PocR protein and induced by propanediol. The cob and pdu operons each have one regulated promoter; the pduF gene is expressed from two regulated promoters (P1 and P2). The P1 and P2 transcripts extend beyond pduF to include the pocR gene; thus the PocR protein autoregulates its expression from these promoters. The fifth promoter, PPoc, is adjacent to the pocR gene and associated with a Crp binding site. We suggest that all global control of the regulon is exerted by regulating the level of PocR protein at the P1, P2, and PPoc promoters. A putative binding site for the PocR protein has been identified by computer analysis. Eight close matches to this proposed site were found in regions near the four promoters known to be regulated by PocR protein: PPdu, P1, P2, and PCob. A three-state model is proposed in which the regulon uses all five of its promoters to control expression. PMID- 7559321 TI - The gene for the longest known Escherichia coli protein is a member of helicase superfamily II. AB - The Escherichia coli rnt gene, which encodes the RNA-processing enzyme RNase T, is cotranscribed with a downstream gene. Complete sequencing of this gene indicates that its coding region encompasses 1,538 amino acids, making it the longest known protein in E. coli. The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II. An approximately 170-kDa protein is observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled extracts prepared from cells in which lhr is under the control of an induced T7 promoter. This protein is absent when lhr is interrupted or when no plasmid is present. Downstream of lhr is the C-terminal region of a convergent gene with homology to glutaredoxin. Interruptions of chromosomal lhr at two different positions within the gene do not affect the growth of E. coli at various temperatures in rich or minimal medium, indicating that lhr is not essential for usual laboratory growth. lhr interruption also has no effect on anaerobic growth. In addition, cells lacking Lhr recover normally from starvation, plate phage normally, and display normal sensitivities to UV irradiation and H2O2. Southern analysis showed that no other gene closely related to lhr is present on the E. coli chromosome. These data expand the known size range of E. coli proteins and suggest that very large helicases are present in this organism. PMID- 7559320 TI - Resolved: cardiac arrhythmias make desipramine an unacceptable choice in children. PMID- 7559323 TI - Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1. AB - The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfbKpO1) contains six genes whose products are required for the biosynthesis of a lipopolysaccharide O antigen with the following repeating unit structure: -->3 beta-D-Galf-1-->3-alpha-D-Galp-1-->(D-galactan I). rfbFKpO1 is the last gene in the cluster, and its gene product is required for the initiation of D-galactan I synthesis. Escherichia coli K-12 strains expressing the RfbFKpO1 polypeptide contain dual galactopyranosyl and galactofuranosyl transferase activity. This activity modifies the host lipopolysaccharide core by adding the disaccharide beta-D-Galf-1-->3-alpha-D-Galp, representing a single repeating unit of D galactan I. The formation of the lipopolysaccharide substituted either with the disaccharide or with authentic polymeric D-galactan I is dependent on the activity of the Rfe enzyme. Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1 phosphate transferase) catalyzes the formation of the lipid-linked biosynthetic intermediate to which galactosyl residues are transferred during the initial steps of D-galactan I synthesis. The rfbFKpO1 gene comprises 1,131 nucleotides, and the predicted polypeptide consists of 373 amino acid residues with a predicted M(r) of 42,600. A polypeptide with an M(r) of 42,000 was evident in sodium dodecyl sulfate-polyacrylamide gels when rfbKpO1 was expressed behind the T7 promoter. The carboxy-terminal region of RfbFKpO1 shares similarity with the carboxy terminus of RfpB, a galactopyranosyl transferase which is involved in the synthesis of the type 1 O antigen of Shigella dysenteriae. PMID- 7559324 TI - Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7. AB - Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction. PMID- 7559326 TI - Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. AB - Transcription of the cob/pdu regulon of Salmonella typhimurium is activated by the PocR regulatory protein in response to 1,2-propanediol (1,2-PDL) in the environment. Nutritional analysis and DNA sequencing confirmed that a strain defective in expression of the cob/pdu regulon in response to 1,2-PDL lacked a functional gshA gene. gshA encodes gamma-glutamylcysteine synthetase (L glutamate:L-cysteine gamma-ligase [ADP forming]; EC 6.3.2.2), the enzyme that catalyzes the first step in the synthesis of glutathione (GSH). The DNA sequence of gshA was partially determined, and the location of gshA in the chromosome was established by two-factor crosses. P22 cotransduction of gshA with nearby markers showed 21% linkage to srl and 1% linkage to hyd; srl was 9% cotransducible with hyd. In light of these data, the gene order gshA srl hyd is suggested. The level of reduced thiols in the gshA strain was 87% lower than the levels measured in the wild-type strain in both aerobically and anaerobically grown cells. 1,2-PDL dependent transcription of cob/pdu was studied by using M. Casadaban's Mu-lacZ fusions. In aerobically grown cells, transcription of a cbi-lacZ fusion (the cbi genes are the subset of cob genes that encode functions needed for the synthesis of the corrin ring) was 4-fold lower and transcription of a pdu-lacZ fusion was 10-fold lower in a gshA mutant than in the wild-type strain. Expression of the cob/pdu regulon in response to 1,2-PDL was restored when GSH was included in the medium. In anaerobically grown cells, cbi-lacZ transcription was only 0.4-fold lower than in the gshA+ strain; pdu-lacZ transcription was reduced only by 0.34 fold, despite the lower thiol levels in the mutant. cobA-lacZ transcription was used as negative control of gene whose transcription is not controlled by the PocR/1,2-PDL system; under both conditions, cobA transcription remained unaffected. The gshA mutant strain was unable to utilize 1,2-PDL, ethanolamine, or propionate as a carbon and energy source. The defect in ethanolamine utilization appears to be at the level of ethanolamine ammonia-lyase activity, not at the transcriptional level. Possible roles for GSH in ethanolamine, 1,2 PDL, and propionate catabolism are proposed and discussed. PMID- 7559325 TI - Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). AB - To study the regulatory mechanism controlling the heat-inducible expression of Bacillus subtilis groE, two regulatory elements, the sigma A-like promoter and the inverted repeat (IR [CIRCE]) in the control region, were characterized. The groE promoter was shown to be transcribed by the major RNA polymerase under both heat shock and non-heat shock conditions. The IR was found to have two functions. (i) It ensures the fast turnover of the groE transcript, and (ii) it serves as an operator. This IR acts as a negative heat shock regulatory element, since deletion of this sequence resulted in high-level expression of groE even at 37 degrees C. Although this IR is present in the 5' untranslated region of the groE transcript, groE transcripts under heat shock and non-heat shock conditions showed similar in vivo half-lives of 5 min. This rapid turnover at 37 degrees C requires the presence of the IR. Without the IR, the groE transcript showed a longer half-life of 17 min. Increasing the distance between the groE transcription start site and the IR systematically by inserting nucleotide sequences from 5 to 21 bp in length resulted in a gradual abolition of the negative regulatory effect mediated by the IR. This effect was not due to a significant change in transcript stability or the transcription start site and is consistent with the model that this IR serves as an operator. PMID- 7559327 TI - Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes. AB - For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20 fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested. PMID- 7559328 TI - Isolation and characterization of a conserved porin protein from Helicobacter pylori. AB - Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed. PMID- 7559329 TI - Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region. AB - pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants. PMID- 7559330 TI - Horizontal transference of S-layer genes within Thermus thermophilus. AB - The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments. PMID- 7559332 TI - Motility and thermotactic responses of Thermotoga maritima. AB - Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C. The cells are propelled by a single flagellum which most of the time spins clockwise. Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation. The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C. The cells show a thermotactic response to temporal temperature changes. When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate. PMID- 7559331 TI - Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization. AB - The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription. PMID- 7559333 TI - Physical map of the genome of the green phototrophic bacterium Chlorobium tepidum. AB - A physical restriction map of the chromosome of the green sulfur bacterium Chlorobium tepidum was generated by determining the order of the fragments obtained after digestion with the restriction endonucleases XbaI and PacI and subsequent separation of the fragments by pulsed-field gel electrophoresis. The size of the chromosome is estimated to be 2.1 Mb. Fifteen genes and operons, mainly encoding proteins involved in photosynthesis, have been placed on this map by hybridization to fragments obtained after single- and double-restriction digestions. PMID- 7559334 TI - Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species. AB - Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum. PMID- 7559335 TI - Interchromosomal recombination in the extremely radioresistant bacterium Deinococcus radiodurans. AB - Deinococcus radiodurans and other members of the genus Deinococcus are remarkable for their extreme resistance to ionizing radiation and many other agents that damage DNA. We have recently shown that recombinational processes participate in interplasmidic repair following in vivo irradiation. We now present direct studies on interchromosomal recombination among chromosomes irradiated in vivo during stationary phase (four chromosomes per cell). Following an exposure to 1.75 Mrad (the dose required to achieve a survival of 37%, which degrades the cells' four chromosomes into about 500 fragments), we determined that there may be as many as 175 crossovers per chromosome (700 crossovers per nucleoid) undergoing repair. In addition, these studies suggest that many of the crossovers occurring during repair are nonreciprocal. PMID- 7559336 TI - Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli. AB - We have characterized four genes from the 52-min region on the Escherichia coli linkage map. Three of these genes are directly involved in the metabolism of xanthosine, whereas the function of the fourth gene is unknown. One of the genes (xapA) encodes xanthosine phosphorylase. The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG. The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB. The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer xanthosine. Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins. In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of xanthosine. PMID- 7559337 TI - Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese. AB - A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2 mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation. PMID- 7559339 TI - Repression of the Klebsiella aerogenes nac promoter. AB - Transcription of the nitrogen-regulated nac promoter of Klebsiella aerogenes requires sigma54 RNA polymerase, is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), and is repressed by the product of the nac gene, Nac. Nac protects a large portion of the nac control region, extending from positions -130 to -70, from digestion by DNase I. This site(s) lies immediately upstream from the site at which sigma 54 RNA polymerase binds, is downstream of a high-affinity binding site for the transcriptional activator NRI approximately P, and partially overlaps a low-affinity NRI approximately P-binding site. Binding of Nac to the DNA resulted in bending of the DNA but did not interfere with the binding of sigma 54 RNA polymerase to the promoter or with the binding of NRI approximately P to either the high-affinity site or low-affinity site. Furthermore, transcription assays with various wild type and mutant templates suggested that Nac did not exclude NRI approximately P from either the low- or high-affinity sites, nor did Nac interfere with the ability of the polymerase to form the open complex when the binding sites for NRI approximately P were moved to different locations upstream from the promoter. Rather, Nac seemed to repress by an antiactivation mechanism in which the interaction of the NRI approximately P, bound at its normal sites, with sigma 54 RNA polymerase, bound to the promoter, was prevented. PMID- 7559338 TI - Activation of transcription initiation from the nac promoter of Klebsiella aerogenes. AB - The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRI approximately P than did the activation of the Escherichia coli glnAp2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma 54 RNA polymerase than did glnAp2, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp2 promoter. The nac enhancer, consisting of a single high affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp2 promoter was decreased at low concentrations of NRI approximately P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI approximately P binding site was strongly affected by subtle alterations in the position of the NRI approximately P binding site(s), indicating a face-of-the helix dependency for activation. PMID- 7559341 TI - Comparative characterization of release factor RF-3 genes of Escherichia coli, Salmonella typhimurium, and Dichelobacter nodosus. AB - The termination of protein synthesis in bacteria requires two codon-specific release factors, RF-1 and RF-2. A gene for a third factor, RF-3, that stimulates the RF-1 and RF-2 activities has been isolated from the gram-negative bacteria Escherichia coli and Dichelobacter nodosus. In this work, we isolated the RF-3 gene from Salmonella typhimurium and compared the three encoded RF-3 proteins by immunoblotting and intergeneric complementation and suppression. A murine polyclonal antibody against E. coli RF-3 reacted with both S. typhimurium and D. nodosus RF-3 proteins. The heterologous RF-3 genes complemented a null RF-3 mutation of E. coli regardless of having different sequence identities at the protein level. Additionally, multicopy expression of either of these RF-3 genes suppressed temperature-sensitive RF-2 mutations of E. coli and S. typhimurium by restoring adequate peptide chain release. These findings strongly suggest that the RF-3 proteins of these gram-negative bacteria share common structural and functional domains necessary for RF-3 activity and support the notion that RF-3 interacts functionally and/or physically with RF-2 during translation termination. PMID- 7559340 TI - Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene. AB - The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster. PMID- 7559343 TI - Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase encoding gene from the archaebacterium Sulfolobus solfataricus. AB - Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme. PMID- 7559342 TI - Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics. AB - We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth. PMID- 7559345 TI - Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response. AB - The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10. PMID- 7559344 TI - Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes. AB - Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown. PMID- 7559347 TI - Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis. AB - The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions. The bglP gene encodes an aryl-beta glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase. Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism. Distinct deletions of the promoter region were created to determine cis-acting sites for regulation. An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon. The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B. subtilis. Catabolite repression is abolished in both ccpA and ptsH1 mutants. The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP. This structure is very similar to that of transcriptional terminators which regulate the induction of the B. subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon. The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process. Expression of bglP is under the negative control of its own gene product. The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression. Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression. PMID- 7559346 TI - Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. . AB - DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level. PMID- 7559349 TI - Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803. AB - The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803. PMID- 7559348 TI - Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis. AB - A sigma B-dependent stress gene of Bacillus subtilis was localized downstream of the licS gene. The predicted amino acid sequence exhibited a significant similarity to the sequence of the katE-encoded catalase HPII of Escherichia coli, and we designated it the open reading frame katE. In a B. subtilis katE mutant, catalase 2 could not be detected. The amount of katE-specific mRNA was increased after heat, salt, or ethanol stress or after glucose starvation in a sigma B dependent manner. As in E. coli, the transcription of the katE gene in B. subtilis was unaffected by the addition of H2O2 to exponentially growing cells. In contrast, the katA gene encoding catalase 1 of B. subtilis showed an induction pattern different from that of katE; katA expression was strongly increased by oxidative stress. The similarity between E. coli sigma S-dependent genes and B. subtilis sigma B-dependent genes suggests that both may confer multiple stress resistance to stationary-phase cells. PMID- 7559351 TI - Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis. AB - The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane. PMID- 7559350 TI - Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. AB - Site-directed mutagenesis has been employed to address the functional significance of the highly conserved aspartic and glutamic acid residues present in the Walker B (also called motif II) sequence in Escherichia coli DNA helicase II. Two mutant proteins, UvrDE221Q and UvrDD220NE221Q, were expressed and purified to apparent homogeneity. Biochemical characterization of the DNA dependent ATPase activity of each mutant protein demonstrated a kcat that was < 0.5% of that of the wild-type protein, with no significant change in the apparent Km for ATP. The E221Q mutant protein exhibited no detectable unwinding of either partial duplex or blunt duplex DNA substrates. The D220NE221Q mutant, however, catalyzed unwinding of both partial duplex and blunt duplex substrates, but at a greatly reduced rate compared with that of the wild-type enzyme. Both mutants were able to bind DNA. Thus, the motif II mutants E221Q and D220NE221Q were able to bind ATP and DNA to the same extent as wild-type helicase II but demonstrate a significant reduction in ATP hydrolysis and helicase functions. The mutant uvrD alleles were also characterized by examining their abilities to complement the mutator and UV light-sensitive phenotypes of a uvrD deletion mutant. Neither the uvrDE221Q nor the uvrDD220NE221Q allele, supplied on a plasmid, was able to complement either phenotype. Further genetic characterization of the mutant uvrD alleles demonstrated that uvrDE221Q confers a dominant negative growth phenotype; the uvrDD220NE221Q allele does not exhibit this effect. The observed difference in effect on viability may reflect the gene products' dissimilar kinetics for unwinding duplex DNA substrates in vitro. PMID- 7559354 TI - Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction. AB - Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G. PMID- 7559352 TI - Identification and characterization of sporulation gene spoVS from Bacillus subtilis. AB - We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H. We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum. PMID- 7559353 TI - Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis. AB - Variable lipoproteins (Vlp) constitute the major coat protein of Mycoplasma hyorhinis. They are products of multiple, divergent, single-copy genes organized in a chromosomal cluster. Three genes, vlpA, vlpB, and vlpC, have been previously identified in clonal isolates of M. hyorhinis SK76. Each is linked to a characteristic promoter region containing a homopolymeric tract of adenine residues [poly(A) tract], subject to hypermutation, that transcriptionally controls phase variation of vlp genes and leads to combinatorial surface mosaics of distinct Vlp products. The size of the natural vlp gene repertoire is unknown but may critically determine the degree of structural and combinatorial diversity available in this species. In this study, the vlp repertoire of M. hyorhinis GDL 1 was characterized and shown to contain three additional genes, vlpD, vlpE, and vlpF, clustered with other known vlp genes in the order 5'-vlpD-vlpE-vlpF-IS-vlpA IS-vlpB-vlpC+ ++-3', where IS represents copies of the IS1221 element of M. hyorhinis. The 5' boundary of this expanded family was identical to that of the more limited family 5'-vlpA-IS-vlpB-vlpC-3' previously described in a clonal isolate of strain SK76. A recombinant construct containing vlpD, vlpE, and vlpF expressed antigenically distinguishable products corresponding to each gene. These genes encode characteristic C-terminal repetitive regions that are subject to size variation by insertion or deletion of intragenic repeats but maintain an extended, charged structure. Each vlp gene also contained characteristic alternative open reading frames, which provide a potential reservoir of coding sequence for Vlp diversity, possibly recruited through insertion and/or deletion mutations. These findings demonstrate a vastly expanded potential for structural diversity and combinatorial display of surface mosaics on this organism and suggest that modulation of the vlp repertoire, possibly in conjunction with mobile elements, may determine the capacity for surface variation in natural populations and laboratory strains of this mycoplasma species. PMID- 7559355 TI - Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function. AB - Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determine by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype. PMID- 7559356 TI - The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene. AB - Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN. PMID- 7559357 TI - Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa. AB - Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the 35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions. PMID- 7559361 TI - Self-electrophoresis is not the mechanism for motility in swimming cyanobacteria. AB - Swimming cyanobacteria do not have flagella. In principle, they could be propelled by streams of ions flowing from head to tail, i.e., by a self electrophoretic mechanism. We have ruled out this possibility by showing that cells of a swimming Synechococcus species fail to drift in an external electric field. PMID- 7559358 TI - Rapid expansion of the physical and genetic map of the chromosome of Clostridium perfringens CPN50. AB - The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship. PMID- 7559360 TI - Mutations in GltC that increase Bacillus subtilis gltA expression. AB - Mutants with altered forms of GltC, a positive LysR-type regulator of Bacillus subtilis glutamate synthase gene expression, were isolated. The mutant GltC proteins stimulated expression from the wild-type gltA promoter region 1.5- to 2.0-fold and from mutant promoter regions up to 80-fold. Moreover, expression of gltA became much less dependent on a nitrogen source-associated signal. PMID- 7559362 TI - Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system. AB - In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose. PMID- 7559363 TI - Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis. AB - The DNA-binding protein IHF was found to be required for starvation survival and for the induction of 14 proteins of the glucose starvation stimulon. Many of these proteins have been shown previously to be general responders to diverse stress conditions. Overexpression of IHF during balanced growth was not sufficient to induce these proteins, but it resulted in an increased synthesis of rpoH-dependent heat shock proteins. PMID- 7559364 TI - Replication through the terminus region of the Bacillus subtilis chromosome is not essential for the formation of a division septum that partitions the DNA. AB - Germinated and outgrowing spores of a temperature-sensitive DNA initiation mutant of Bacillus subtilis were allowed to initiate a single round of replication by being shifted from 34 to 47 degrees C at the appropriate time. The DNA replication inhibitor 6-(parahydroxyphenylazo)-uracil was added to separate portions of the culture at various times during the round. Samples were collected from each around the time of the first division septation for measurements of the extent of the round completed, the level of division septation, the position of the septum within the outgrown cell, and the distribution of DNA (nucleoid) in relation to the septum. The extent of replication was measured directly through a hybridization approach. The results show clearly that a central division septum can close down onto a chromosome that is only partially replicated (to a minimum extent of about 60% of the round) such that DNA appears on both sides of the septum and frequently very close to it. It is concluded, as claimed previously on the basis of a less direct approach (T. McGinness and R.G. Wake, J. Mol. Biol. 134:251-264, 1979), that replication through the terminus region of the chromosome is not essential for the formation of a division septum that partitions the DNA. PMID- 7559365 TI - Identification of channel-forming activity in the cell wall of Corynebacterium glutamicum. AB - The cell wall of the gram-positive Corynebacterium glutamicum was prepared. It contained an ion-permeable channel with a single-channel conductance of about 6 nS in 1 M KCl. The mobility sequence of the ions in the channel is similar to that in the aqueous phase, suggesting that it is a water-filled channel wide enough to allow unhindered diffusion of ions. The results indicate that we have identified the hydrophilic pathway through the mycolic acid layer of C. glutamicum. PMID- 7559359 TI - Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression. AB - The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position -64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the -35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7- to 40-fold under nonactivating conditions and about 2 fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn. PMID- 7559366 TI - Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis. AB - We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis. PMID- 7559367 TI - Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4. AB - The gene encoding the major autolysin of Staphylococcus aureus 8325/4 has been cloned, sequenced, and insertionally inactivated. The three-domain, 137,384-Da protein has a C-terminal glucosaminidase active site and is involved in cell separation, generalized cell lysis, and release of wall material at the cell surface. Expression occurs throughout growth and is stimulated by low temperatures and in the presence of 1 M NaCl. PMID- 7559369 TI - Threats and promises. PMID- 7559370 TI - Effectiveness of venlafaxine in patients hospitalized for major depression and melancholia. AB - BACKGROUND: This study was undertaken to compare the antidepressant efficacy and short-term safety of venlafaxine with those of placebo in hospitalized patients with major depression and melancholia. METHOD: Ninety-three inpatients with a minimum prestudy Montgomery-Asberg Depression Rating Scale (MADRS) score of 25 were treated for up to 4 weeks with either venlafaxine or placebo. Dosage averaged approximately 350 mg/day during Weeks 2 to 4. Efficacy and safety were assessed throughout the study. Efficacy was evaluated using the MADRS, the 21 item Hamilton Rating Scale for Depression (HAM-D), and the Clinical Global Impressions (CGI) scale. Recorded study events, vital signs and body weight measurements, laboratory determinations, physical examinations, and ECG recordings were used to assess safety. RESULTS: Venlafaxine provided significantly greater improvement in the MADRS scores after 4 days and in the HAM D scores after 1 week than did placebo. Response rate (based on a 50% decrease in MADRS scores) was 65% (30 of 46 patients) for venlafaxine and 28% (13 of 47 patients) for placebo. Significantly more placebo-treated patients (40%; N = 19) than venlafaxine-treated patients (9%; N = 4) discontinued treatment early because of lack of efficacy. Nausea and sweating were the most common events, occurring at a significantly higher rate in the venlafaxine group. CONCLUSION: Venlafaxine is an effective and well-tolerated antidepressant in hospitalized patients with major depression and melancholia. PMID- 7559368 TI - The experts converse. Special considerations in switching antidepressants. PMID- 7559371 TI - Disabling compulsions in 11 mentally retarded adults: an open trial of clomipramine SR. AB - BACKGROUND: Clinicians and researchers who work with mentally retarded subjects have reported on the frequent exhibition of mental disorders and behavioral problems in this population. Rituals are often among the disturbances described. We decided to treat disabling cleaning and collecting compulsions in mentally retarded adults with clomipramine 75 mg/day, sustained release (SR) preparation. METHOD: The 8-week trial included 11 subjects, mean age of 24.1 years (range, 21 27) with a mean +/- SD I.Q. of 65.0 +/- 11.0. The majority of subjects (N = 9) were occupied with washing rituals that took hours to complete. Subjects were started on a schedule of 32.5 mg of clomipramine SR once daily for 1 week, then received 75 mg SR once daily for an additional 7 weeks. RESULTS: Improvement was assessed by using the National Institute of Mental Health Global Obsessive Compulsive Scale and the Global Improvement score of the Yale-Brown Obsessive Compulsive Scale. Statistically significant reduction in severity of rituals (p < .05) was found at the trial's completion. CONCLUSION: Once-daily clomipramine SR 75 mg is effective in treating adults with mental retardation and disabling rituals. PMID- 7559372 TI - Command hallucinations in outpatients with schizophrenia. AB - BACKGROUND: The presence of command hallucinations in individuals with schizophrenia may result in an increase in clinical monitoring to reduce the perceived risk of violent behavior. However, the issue of whether command hallucinations hold any clinical relevance in relatively stable outpatient samples has not been established. METHOD: The clinical and research records of individuals with schizophrenia who participated in outpatient research protocols at the University of California, San Diego were reviewed for the presence of command hallucinations. Information on clinical characteristics was collected in a detailed chart review from 106 patient records. RESULTS: Command hallucinations were reported by one half of all patients with auditory hallucinations, and these hallucinations often were violent in content. Yet, in over a third of the patients, these hallucinations had not been documented in their clinical charts, but instead were uncovered during a secondary source review. Patients with command hallucinations generally did not differ on prognostic or clinical course variables. However, the 2 patients who committed suicide during the study were patients with command hallucinations. CONCLUSION: Although command hallucinations may be more frequent than clinicians generally note, in most cases they have minimal influence on the outcome of schizophrenia. However, in outpatients with schizophrenia who have a history of suicide attempts, suicidal command hallucinations should be taken seriously. PMID- 7559376 TI - The use of fluoxetine and buspirone for treatment-refractory depersonalization disorder. PMID- 7559374 TI - Addition of monoamine oxidase inhibitors to carbamazepine: preliminary evidence of safety and antidepressant efficacy in treatment-resistant depression. AB - BACKGROUND: Depression is often resistant to treatment with mood stabilizers. The antidepressant effects of carbamazepine can be potentiated by lithium supplementation, but some patients fail to respond to the combination. Although monoamine oxidase inhibitors (MAOIs) appear useful in atypical and bipolar depressions, concerns have been raised regarding safety and pharmacokinetic interactions when they are combined with carbamazepine. METHOD: Ten inpatients (7 bipolar, 3 unipolar) with refractory DSM-III-R major depression, also resistant to double-blind treatment with carbamazepine, plus lithium augmentation in 8, received double-blind MAOI augmentation (phenelzine in 4, tranylcypromine in 6). RESULTS: All 10 patients tolerated the addition of an MAOI well, and mean self rated side effect scores did not change significantly. Four of 10 patients improved substantially and became euthymic, allowing discharge from hospital on the carbamazepine +/- lithium plus MAOI combination. These 4 patients improved in spite of prior inadequate responses to the same MAOI without carbamazepine and carbamazepine without an MAOI. CONCLUSION: This preliminary evidence suggests that the addition of MAOIs to carbamazepine +/- lithium may be well tolerated, may not affect carbamazepine and lithium pharmacokinetics, and may provide relief of refractory depressive symptoms in some patients. Further studies are needed to establish the safety and efficacy of combining carbamazepine with MAOIs. PMID- 7559377 TI - Risperidone and tardive dyskinesia: a case report. PMID- 7559375 TI - Is there a rationale for iron supplementation in the treatment of akathisia? A review of the evidence. AB - BACKGROUND: An association found between akathisia and iron deficiency led to the suggestion that iron supplementation might be a useful therapeutic intervention for patients with akathisia. There is, however, a body of literature on the abnormal deposition of iron in the brain in several degenerative diseases like Hallervorden-Spatz syndrome, Parkinson's disease, and Alzheimer's disease. Given the ability of neuroleptics to chelate iron and promote its deposition in the brain, we questioned whether peripheral measures of iron are an accurate reflection of central iron levels and thus whether there was a rationale for iron supplementation in akathisia. METHOD: A MEDLINE search for literature relating to iron and akathisia, tardive dyskinesia, and Parkinson's disease was carried out and critically reviewed. RESULTS: Evidence is presented for the ability of neuroleptics to chelate iron, mobilize it from peripheral stores, and deposit it in the basal ganglia. The effect of iron on dopaminergic receptor activity in brain and the potential role of iron in degenerative and neuroleptic-induced movement disorders are reviewed. The preponderance of the evidence shows a relationship between iron excess in the basal ganglia and the movement disorders. We found no studies that have examined the regulation of central levels of iron in patients with akathisia. CONCLUSION: The rationale for iron supplementation in the treatment of akathisia is relatively weak, and there are potentially adverse long-term consequences as outlined in our review. More research is required to directly measure the level of iron in the brain of patients with akathisia, e.g., using magnetic resonance imaging, before such therapeutic intervention can be recommended. PMID- 7559379 TI - Photosensitivity reaction to fluoxetine. PMID- 7559378 TI - Antipsychotic effect of cannabidiol. PMID- 7559380 TI - Highlight of the 1995 Psychiatry Global Medical Conference. PMID- 7559373 TI - Clinical predictors of acute risperidone response in schizophrenia, schizoaffective disorder, and psychotic mood disorders. AB - BACKGROUND: In studies of patients with schizophrenia, the atypical antipsychotic risperidone has been shown to be comparable in efficacy to haloperidol and, at dosages of 4 to 8 mg/day, to have a lower rate of extrapyramidal side effects. However, little is known about the efficacy of risperidone in patients with schizophrenia refractory to treatment with typical antipsychotics, schizoaffective disorder, and psychotic mood disorders. The purpose of this study was to assess the efficacy of risperidone in the treatment of these disorders and to identify clinical factors associated with risperidone response. METHOD: By surveying treating clinicians and chart data, we assessed response to risperidone and factors associated with response to risperidone in 144 consecutive patients treated with the drug for at least 2 weeks at a regional state psychiatric hospital. RESULTS: Patients displaying a moderate-to-marked response to risperidone were more likely to be younger; receive diagnoses of bipolar disorder or schizoaffective disorder, depressive type; and have a shorter duration of illness and shorter length of stay prior to risperidone treatment. Response to risperidone was sufficient to allow discharge in 26% of patients with treatment refractory schizophrenia hospitalized for at least 10 weeks prior to risperidone and in 11% of patients with treatment-refractory schizophrenia hospitalized for greater than 1 year. CONCLUSION: Risperidone may be a useful alternative or adjunctive treatment for patients with schizophrenia refractory to treatment with standard antipsychotic agents, schizoaffective disorder (especially the depressive type), and bipolar disorder when used in conjunction with mood stabilizers. PMID- 7559382 TI - Purification and characterization of the XPF-ERCC1 complex of human DNA repair excision nuclease. AB - A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1 complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with a "bubble" structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication protein A when the DNA contains UV damage. PMID- 7559381 TI - Interleukin-3 regulates development of the 5-lipoxygenase/leukotriene C4 synthase pathway in mouse mast cells. AB - To study cytokine regulation of the 5-lipoxygenase (5-LO)/leukotriene (LT) synthase pathway we have developed mouse bone marrow-derived mast cells (BMMC) that minimally express each protein of the pathway by using a novel culture system, lacking interleukin (IL)-3. When mouse bone marrow cells were cultured for 5 weeks with 100 ng/ml c-kit ligand (KL) and 10 units/ml IL-10, a population of > 95% mast cells was obtained. These cells generated 8.3 +/- 4.5 ng of LTC4/10(6) cells and 8.1 +/- 2.4 ng of prostaglandin (PG) D2/10(6) cells after IgE-dependent activation. When these BMMC were cultured for 2-5 weeks more with 100 units/ml IL-3 in the continued presence of KL and IL-10, the IgE-dependent generation of LTC4 and PGD2 increased to 212 +/- 36 and 25.5 +/- 8.6 ng/10(6) cells, respectively. The dramatic increase in the IgE-dependent generation of LTC4 in response to IL-3 was accompanied by a concomitant increase in expression of 5-LO and 5-LO-activating protein and preceded the increased expression of cytosolic phospholipase A2 and LTC4 synthase. The recognition that IL-3 up regulates the expression of each protein of the 5-LO pathway for the generation of LTC4 contrasts with our recent finding that KL up-regulates the expression of cytosolic phospholipase A2, prostaglandin endoperoxide synthase-1, and hematopoietic PGD2 synthase and increases the IgE-dependent generation of PGD2 in BMMC developed from bone marrow with IL-3. Thus, developmentally segregated regulation of the prostanoid and cysteinyl leukotriene pathways in lineage related committed mast cell progenitors reveals the pleiotropism of this effector cell of allergic inflammation, a cytokine/growth factor basis for preferential expression of pathways of eicosanoid biosynthesis, and the particular role of IL 3 in regulating the expression of the proteins of the 5-LO/LTC4 synthase pathway. PMID- 7559384 TI - The interaction of TFIIIA with specific RNA-DNA heteroduplexes. AB - We examine the association of transcription factor TFIIIA with RNA-DNA heteroduplexes containing sequences from the Xenopus borealis 5 S rRNA gene. Under conditions where TFIIIA selectively binds to 5 S rRNA or the internal control region of the 5 S rRNA gene, no specific association of TFIIIA with DNA RNA heteroduplexes containing either strand of 5 S DNA could be detected. We discuss our results with respect to specific models of TFIIIA recognition of the internal control region and of DNA-RNA hybrids by zinc finger proteins. PMID- 7559385 TI - A novel function of Escherichia coli chaperone DnaJ. Protein-disulfide isomerase. AB - Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes. The DnaJ protein of Escherichia coli and the DnaJ-like proteins of eukaryotes are known as molecular chaperones and specific regulators of DnaK-like proteins and are involved in protein folding and renaturation after stress. In this study we show that DnaJ, like thioredoxin, protein-disulfide isomerase, and DsbA, possesses an active dithiol/disulfide group and catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). These results suggest that, in addition to its known function as a chaperone, DnaJ might be involved in controlling the redox state of cytoplasmic, membrane, or exported proteins. PMID- 7559386 TI - Activation of the p21 pathway of growth arrest and apoptosis by the beta 4 integrin cytoplasmic domain. AB - The integrin alpha 6 beta 4, a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study alpha 6 beta 4-mediated functions in more detail and to assess the contribution of the beta 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta 4 integrin subunit. This cell line, termed RKO, expresses alpha 6 beta 1 but not alpha 6 beta 4, and it interacts with laminin-1 less avidly than similar cell lines that express alpha 6 beta 4. We expressed a full-length beta 4 cDNA, as well as a mutant cDNA that lacks the beta 4 cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full length beta 4 cDNA in association with endogenous alpha 6 exhibited partial G1 arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha 6 beta 4 integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the beta 4 integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta 4 transfected RKO cells. PMID- 7559387 TI - Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin. AB - P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein on leukocytes that is a high affinity ligand for P-selectin. Previous studies have shown that sialylation and fucosylation of PSGL-1 are required for its binding to P-selectin, but other post-translational modifications of PSGL-1 may also be important. We demonstrate that PSGL-1 synthesized in human HL-60 cells can be metabolically labeled with [35S]sulfate that is incorporated primarily into tyrosine sulfate. Treatment of PSGL-1 with a bacterial arylsulfatase releases sulfate from tyrosine, resulting in a concordant decrease in binding to P selectin. These studies demonstrate that tyrosine sulfate on PSGL-1 functions in conjunction with sialylated and fucosylated glycans to mediate high affinity binding to P-selectin. PMID- 7559383 TI - Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor. AB - Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems. PMID- 7559388 TI - Stimulation of sky receptor tyrosine kinase by the product of growth arrest specific gene 6. AB - Sky (also called Rse, Brt, and Tyro3) is a member of a subfamily of related receptor tyrosine kinases, including Axl/Ufo/Ark and c-Eyk/Mer. We obtained evidence that Gas6 (the product of growth arrest-specific gene 6) is a ligand of the Sky receptor tyrosine kinase. Gas6, but not protein S (an anticoagulant protein structurally similar to Gas6), specifically bound to the soluble form of Sky (Sky-Fc), composed of the extracellular domain of Sky fused to the Fc domain of human immunoglobulin G1. The native and recombinant Gas6, but not protein S, stimulated tyrosine phosphorylation of Sky ectopically expressed in Chinese hamster ovary cells. Stimulation of Sky in response to Gas6 was inhibited by Sky Fc. The half-maximal concentration of Gas6 that stimulated Sky was about 1 nM. Thus, Gas6 as a ligand for Sky specifically binds to and stimulates Sky receptor tyrosine kinase. PMID- 7559389 TI - A human STX cDNA confers polysialic acid expression in mammalian cells. AB - Polysialic acid, or PSA, is a term used to refer to linear homopolymers of alpha(2,8)-sialic acid residues displayed at the surface of some mammalian cells. PSA is typically linked to the neural cell adhesion molecule N-CAM, where it can modulate the homotypic adhesive properties of this polypeptide. PSA expression is developmentally regulated, presumably through mechanisms involving regulated expression of sialyltransferases involved in PSA biosynthesis. Several different sialytransferase sequences have been implicated in PSA expression, although the precise roles of these enzymes in this context remain unclear. One such sequence, termed STX, maintains approximately 59% amino acid sequence identity with another sialyltransferase (PST-1, from hamster; PST, human) that is known to participate in PSA expression. While a murine STX fusion protein can catalyze the synthesis of a single alpha(2,8)-sialic acid linkage in vitro, the ability of STX to participate in PSA expression in vivo has not been demonstrated. We show here that STX transcripts are present in a PSA-positive, N-CAM-positive human small cell carcinoma line (NCI-H69/F3), but are absent in a variant of this line (NCI H69/E2) selected to be PSA-negative and N-CAM-positive. To functionally confirm this correlation, we have cloned a human cDNA encoding the human STX sequence, and show, by transfection studies, that human STX can restore PSA expression when expressed in the PSA-negative, N-CAM-positive small cell carcinoma variant. We furthermore show that STX can confer PSA expression when expressed in a PSA negative, N-CAM-positive murine cell line (NIH-3T3 cells), or when expressed in PSA-negative, N-CAM-negative COS-7 cells. These observations imply that STX, like PST-1/PST, can determine PSA expression in vivo. When considered together with the correlation between STX expression and PSA expression in vivo in the brain, these results suggest a regulatory role for STX in PSA expression in the developing central nervous system and small cell lung carcinoma. PMID- 7559392 TI - Activation of the herpes simplex virus type 1 protease. AB - The catalytic efficiency of the mature HSV-1 protease has been examined as a function of solvent composition. With the peptide substrate HTYLQASEKFKMWG-amide, the specificity constant (kcat/Km) at pH 7.5 for cleavage is 5.2 M-1 s-1. This value increases to 38 M-1 s-1 when 25% glycerol is present in the reaction mixture. It was found that glycerol activation is but one case of the general phenomenon of HSV-1 protease activation by kosmotropes, or water structure forming cosolvents. For example, an 860-fold increase in the protease activity (kcat/Km = 4500 M-1 s-1) occurs in the presence of 0.8 M sodium citrate. Similarly, the presence of 0.8 M sodium phosphate activates the catalytic efficiency by 420-fold (kcat/Km = 2200 M-1 s-1). The extent of HSV-1 protease activation by various anions correlates with the Hofmeister series. Both the susceptibility to proteolysis by trypsin and the protein fluorescence spectra of the HSV-1 protease change in the presence of activating solvents, suggesting a conformational change accompanying activation. PMID- 7559391 TI - Isolation of inactive and G protein-resistant adenylyl cyclase mutants using random mutagenesis. AB - We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattractant receptor and guanosine 5'-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis and regulation of this important class of enzymes. PMID- 7559390 TI - Ceramide activates the stress-activated protein kinases. AB - Tumor necrosis factor alpha (TNF alpha) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JNKs) resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NF kappa B to the nucleus resulting in the stimulation of NF kappa B-dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNF alpha activates sphingomyelinases. We show that treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A2, C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C2-ceramide (5 microM) or TNF alpha (10 ng/ml). The prolonged stimulation of this kinase activity by C2-ceramide is similar to that previously reported for TNF alpha. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNF alpha treatment (1.5-fold) and are inhibited by C2 ceramide treatment. TNF alpha also potently stimulates NF-kappa B DNA binding activity and transcriptional activity, but these effects are not mimicked by addition of C2-ceramide or sphingomyelinase to intact cells. Furthermore, TNF alpha, sphingomyelinase, and C2-ceramide induce c-jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNF alpha's biochemical and biological effects. PMID- 7559393 TI - Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. AB - We have fluorescently labeled one of the eight genomic segments of influenza virus RNA and a recombinant influenza viral protein, the nucleoprotein (NP), to investigate the requirement for their uptake into nuclei of digitonin permeabilized cells. We found that the influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein. Thus, at 0 degrees C it docks at the nuclear envelope only in the presence of the heterodimeric karyopherin (either karyopherin alpha 1 beta or karyopherin alpha 2 beta), and docking is competitively inhibited by an unlabeled NLS containing substrate. Like other NLS containing proteins, at 20 degrees C NP is imported into the nucleus after further addition of the GTPase Ran and of p10. In contrast, the fluorescently labeled, 890-nucleotide-long viral RNA segment does not dock to the nuclear envelope or enter the nucleus either in the presence of exogenous cytosol or of karyopherin heterodimer, Ran, and p10. However, in the presence of NP the RNA is able to dock and enter the nucleus with transport requirements indistinguishable from those for docking and entry of NP. These data indicate that uptake of the influenza virus RNA segment is not via a signal in the RNA but via an NLS of a viral protein such as NP. PMID- 7559394 TI - CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte-mediated apoptosis. AB - Cytotoxic T-lymphocytes (CTLs), by virtue of their ability to recognize and induce apoptotic death of virus-infected cells, comprise a major antiviral defense mechanism. The induction of apoptosis by CTLs can be completely accounted for by two mechanisms: (i) a Ca(2+)-dependent component that involves the exocytotic release of serine proteases known as granzymes from CTL granules and their subsequent insertion into the target cell to induce apoptosis and (ii) a Ca(2+)-independent component that involves the activation of Fas, a receptor on the target cell membrane that triggers apoptosis. Although viruses have evolved several indirect mechanisms for evading the CTL response, direct inhibition of the apoptotic cascade has never been described. We now show for the first time that the cowpox virus protein CrmA, a protease inhibitor of the serpin family, is capable of inhibiting CTL-mediated cytolysis. The inhibitory effect is largely the result of blockade of the Ca(2+)-independent (i.e. Fas-mediated) component of CTL killing. CrmA thus represents the first example of a viral gene product capable of directly blocking CTL-mediated cell death. PMID- 7559395 TI - Protonophoric activity of ellipticine and isomers across the energy-transducing membrane of mitochondria. AB - Ellipticine is an antitumor alkaloid capable of uncoupling mitochondrial oxidative phosphorylation. It behaves as a lipophilic weak base with pK = 7.40. We have investigated its molecular mode of action using several of its isomers with pK ranging between 5.8 and 7.7 and ellipticinium, which is a permanent cationic derivative. The effects of these molecules on mitochondrial oxygen uptake and transmembrane potential were compared at different pHs. Ellipticinium exhibited very low effects on both respiratory rate and membrane potential. By contrast, protonable derivatives showed maximal stimulation of oxygen uptake and depolarizing effects when the pH of the medium was close to the drug pK. These effects were lowered when the transmembrane delta pH was dissipated, which indicates that the neutral form of the drug is implicated in the uncoupling mechanism. In addition, protonable derivatives of ellipticine display a linear relationship between oxidation rate and transmembrane potential, which suggests that the uncoupling properties of these molecules result from a protonophoric mechanism. From these results, the following cyclic protonophoric mechanism is proposed for protonable ellipticines: (i) electrophoretical accumulation of the protonated form; (ii) deprotonation at the matrix interface; (iii) diffusion outwards; and (iv) reprotonation at the external interface. PMID- 7559396 TI - Genomic structure and expression of the human gene encoding cytochrome b561, an integral protein of the chromaffin granule membrane. AB - Cytochrome b561 is an electron transfer protein unique to neuroendocrine secretory vesicles. The Southern blot hybridization shows that it is a single copy gene highly conserved throughout phylogeny. The transcription unit spans approximately 11 kilobases, and heterologous transcription sites are located 404 bases 5' to the translation initiation codon. The sequence of the 5'-flanking region is GC-rich and lacks a typical TATA box at the usual position. However, it has a CAAT sequence at -132 and potential recognition sequences for several transcription factors including SP1, GR-PR-MMTV, AP4, gERE, JCV repeat, AP2, and NF-kappa B. Each of the five transmembrane segments are encoded by five consecutive exons. This corroborates the five-transmembrane model proposed for human, mouse, and Xenopus rather than six proposed for bovine. The cytochrome was found to be highly expressed in colon cancer cell lines, T cell lymphomas, and K 562 cell lines. However, in B-cell lymphomas such as Burkitt's and Daudi, the cytochrome b561 expression was completely shut down. The results in this report are the first to demonstrate the structural organization and regulatory sequences of the cytochrome b561 gene encoding an integral membrane protein of neuroendocrine storage vesicles of neurotransmitters and peptide hormones. Unexpected results on cytochrome b561 expression in cells of lymphocytic origin and its complex regulation in tumor cells provide new insights into cytochrome b561 gene regulation. PMID- 7559397 TI - Fos leucine zipper variants with increased association capacity. AB - The Fos wild-type leucine zipper is unable to support homodimerization. This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers. Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface. If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization. If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions. The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein. In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e. Ile Leu > Val > Phe > Met. However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e. on the local environment of the a residue. This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met. Most likely the a1 residue modulates the interhelical repulsion between two glutamic acid side chains in positions g1 and e2. Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer. A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation. PMID- 7559398 TI - Identification of a mouse p21Cdc42/Rac activated kinase. AB - We have isolated a novel member of the mammalian PAK (p21 activated kinase) and yeast Ste20 serine/threonine kinase family from a mouse fibroblast cDNA library, designated mPAK-3. Expression of mPAK-3 in Saccharomyces cerevisiae partially restores mating function in ste20 null cells. Like other PAKs, mPAK-3 contains a putative Cdc42Hs/Rac binding sequence and when transiently expressed in COS cells, full-length mPAK-3 binds activated (GTP gamma S (guanosine 5'-3-O-(thio triphosphate)-bound) glutathione S-transferase (GST)-Cdc42Hs and GST-Rac1 but not GST-RhoA. As expected for a putative target molecule, mPAK-3 does not bind to an effector domain mutant of Cdc42Hs. Furthermore, activated His-tagged Cdc42Hs and His-tagged Rac stimulate mPAK-3 autophosphorylation and phosphorylation of myelin basic protein by mPAK-3 in vitro. Interestingly, the amino-terminal region of mPAK-3 contains potential SH3-binding sites and we find that mPAK-3, expressed in vitro and in vivo, shows highly specific binding to the SH3 domain of phospholipase C-gamma and at least one SH3 domain in the adapter protein Nck. These results raise the possibility of an additional level of regulation of the PAK family in vivo. PMID- 7559400 TI - NAD(+)-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes. AB - Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+) dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase. Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows. When intact rat lymphocytes expressing RT6.1 mRNA were incubated with [alpha-32P]NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000. The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum. The radioactivity incorporated into the 31-kDa protein was recovered as 5'-[32P]AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment. These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue. When intact lymphocytes, in which RT6.1 had been once modified by [32P]ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the [32P]ADP-ribosylated RT6.1. The reduced radioactivity was recovered from the incubation medium as [32P]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture. Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes. PMID- 7559401 TI - The NH2-terminal half of the Tn10-specified tetracycline efflux protein TetA contains a dimerization domain. AB - The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented. PMID- 7559402 TI - Cloning of a fibroblast growth factor receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity. AB - A cDNA clone, predicted to encode a variant form of the type 1 fibroblast growth factor receptor (FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located within the juxtamembrane region, was isolated from a Xenopus embryo (stage 8 blastula) library. Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region demonstrated that this variant form arises from use of an alternative 5' splice donor site. RNase protection analysis revealed that both VT- and VT+ forms of the FGFR1 were expressed throughout embryonic development, the VT+ being the major form. Amino acid position 424 is located within a consensus sequence for phosphorylation by a number of Ser/Thr kinases. We demonstrate that a VT+ peptide was specifically phosphorylated by protein kinase C (PKC) in vitro, but not by protein kinase A (PKA). A VT- peptide, on the other hand, was not a substrate for either enzyme. Phosphorylation levels of in vitro synthesized FGFR-VT+ protein by PKC were twice that of FGFR-VT- protein. In a functional assay, Xenopus oocytes expressing FGFR VT- or FGFR-VT+ protein were equally able to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However, pretreatment with phorbol 12-myristate 13-acetate significantly reduced this mobilization in oocytes expressing FGFR-VT+ while having little effect on oocytes expressing FGFR VT-. These findings demonstrate that alternative splicing of Val423-Thr424 generates isoforms which differ in their ability to be regulated by phosphorylation and thus represents an important mechanism for regulating FGFR activity. PMID- 7559403 TI - Substitutions of serine 775 in the alpha subunit of the Na,K-ATPase selectively disrupt K+ high affinity activation without affecting Na+ interaction. AB - The functional role of serine 775, predicted to be located in the fifth transmembrane segment of the alpha subunit of the Na,K-ATPase (YTLTSNIPE), was studied using site-directed mutagenesis, expression, and kinetic analysis. Substitutions S775A, S775C, and S775Y were introduced into an ouabain-resistant alpha 1 sheep isoform and expressed in HeLa cells. cDNAs carrying substitutions S775C and S775A produced ouabain-resistant colonies only when extracellular K+ was increased from 5.4 mM to 10 or 20 mM, respectively. No ouabain-resistant colonies were obtained for substitutions S775Y at any tested K+ concentration. Kinetic characterization of S775C and S775A substituted enzymes showed expression levels higher than control enzyme, reduced Vmax and turnover, and normal phosphorylation and high affinity ATP binding. Dephosphorylation experiments indicated that S775A substituted enzyme is insensitive to ADP but readily dephosphorylated by K+. The K+ K1/2 values for the activation of the Na,K-ATPase were markedly altered, with S775C displaying a 13-fold increase and S775A exhibiting a 31-fold increase. These large changes in the Na,K-ATPase affinity for K+ are consistent with the participation of this amino acid in binding K+ during the translocation of this cation. Substitutions of Ser775 did not change Na+ affinity, indicating that this residue is likely not involved in Na+ binding and occlusion. These data show that the electronegative oxygen and the small side chain of Ser775 are required for efficient enzyme function. Moreover, these results suggest Ser775 plays a distinct role in K+ transport and not in Na+ interactions, revealing a possible mechanism for the enzymatic differentiation of these cations by the Na,K-ATPase. PMID- 7559399 TI - Phosphatidylcholines with sn-1 saturated and sn-2 cis-monounsaturated acyl chains. Their melting behavior and structures. AB - Recently, we have shown by high resolution differential scanning calorimetry that the position of a cis double bond (delta-bond) in a series of 1-stearoyl-2 octadecenoyl- phosphatidylcholines can affect the phase transition temperature (Tm) or enthalpy (delta H) of the gel-to-liquid crystalline phase transition of this series of lipids in the following manner. The value of Tm (or delta H) is minimal when the delta-bond is positioned at C(11) in the sn-2 acyl chain; in addition, this value increases steadily as the delta-bond migrates toward either end of the acyl chain, resulting in a symmetrical, inverted bell-shaped profile (Wang, Z.-q., Lin, H.-n., Li, S., and Huang, C. (1995) J. Biol. Chem. 270, 2014 2023). In this communication, we have further demonstrated the inverted bell shaped profile of Tm using 1-arachidoyl-2-eicosenoyl-phosphatidylcholines. In addition, we have extended the lipid series of 1-stearoyl-2-octadecenoyl phosphatidylcholines to include 1-arachidoyl-2-octadecenoyl- phosphatidylcholines and 1-behenoyl-2-octadecenoyl-phosphatidylcholine, each series with a delta-bond at varying carbon position of 6, 7, 9, 11, 12, and 13. Calorimetric results obtained with these three series of lipids indicate that the inverted bell-shaped curve shifts toward higher temperatures in a nonuniform manner as the saturated sn-1 acyl chain length increases from 17 to 19 and then to 21 C-C bond lengths. Specifically, the Tm (or delta H) values are nearly identical for these cis monoenoic lipids when their delta-bonds are positioned at C(13). Based on the height of the rotational energy barrier obtained with molecular mechanics calculations, it is evident that the rotational flexibility of the single C-C bond adjacent to the delta-bond in 1-stearoyl-2-octadecenoyl-phosphatidylcholine increases as the delta-bond migrates from C(9) to C(13). The differential scanning calorimetry results obtained with the three series of lipids can thus be attributed to an increase in the rotational flexibility of the short chain segment succeeding the C(14) atom in the sn-2 octadecenoyl chain. In this communication, we also propose that in the gel-state bilayer of sn-1 saturated/sn 2 cis-monounsaturated phosphatidylcholine the entire length of the shorter segment of the sn-2 acyl chain acts as a structural perturbing element; hence, it is mainly responsible for the large lower Tm of the monoenoic lipid relative to the saturated counterpart. Finally, two general equations relating Tm with the structural parameters of cis-monoenoic phosphatidylcholines are presented.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559404 TI - Identification of dynorphins as endogenous ligands for an opioid receptor-like orphan receptor. AB - To identify the endogenous ligands for a cloned orphan receptor that shares high degrees of sequence homology with opioid receptors, this orphan receptor was expressed in Xenopus oocytes and in mammalian cell lines CHO-K1 and HEK-293. The coupling of the receptor to a G protein-activated K+ channel was used as a functional assay in oocytes. Endogenous opioid peptide dynorphins were found to activate the K+ channel by stimulating the orphan receptor. This activation was dose-dependent, with EC50 values at 45 and 37 nM for dynorphin A and dynorphin A (1-13), respectively. The dynorphin effect was antagonized by the non-selective opioid antagonist naloxone but at rather high concentrations in the micromolar range. Naloxone also caused a rightward shift of the dose-response curve for dynorphin A, suggesting a competitive antagonism mechanism. In transiently transfected cells, 5 microM dynorphin A-(1-13) inhibited the forskolin-stimulated cyclic AMP increase by 51 and 35% in CHO-K1 and HEK-293 cells, respectively. Other classes of endogenous opioids, i.e. enkephalins and endorphins, caused very little activation of this receptor. These results suggest that this orphan receptor is a member of the opioid receptor family and that dynorphins are endogenous ligands for this receptor. PMID- 7559405 TI - Identification of a new subclass of Alu DNA repeats which can function as estrogen receptor-dependent transcriptional enhancers. AB - We have utilized a genetic selection system in yeast to identify novel estrogen responsive genes within the human genome and to define the sequences in the BRCA 1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells. PMID- 7559406 TI - alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, but not N-methyl-D aspartate, activates mitogen-activated protein kinase through G-protein beta gamma subunits in rat cortical neurons. AB - alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels play important roles in plasticity, neurotransmission, and neurotoxicity in the central nervous system. AMPA, but not N-methyl-D-aspartate (NMDA), receptor signaling in rat cortical neurons was found to involve a G-protein coupled to a protein kinase cascade. While both NMDA and AMPA activated p42 mitogen-activated protein kinase (MAPK) in neurons, only AMPA-induced MAPK was inhibited by pertussis toxin. AMPA, but not NMDA, caused an association of a G-protein beta subunit with a Ras, Raf kinase, and MAP/ERK kinase (MEK)-1 complex. The evidence indicates that AMPA triggers MAPK activation via a novel mechanism in which G protein beta gamma dimers released from G alpha bind to a Ras protein complex causing the activation of Ras, Raf kinase, MEK-1, and finally MAPK. PMID- 7559408 TI - ADP-ribosylation factor translocation correlates with potentiation of GTP gamma S stimulated phospholipase D activity in membrane fractions of HL-60 cells. AB - Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP gamma S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP gamma S-stimulated PLD activity in the membrane fractions of these cells. Inactive phorbol esters induced no such potentiation. Both fMLP and active phorbol esters induced a 2-3-fold increase in GTP gamma S stimulated PLD in HL-60 membranes. Membranes derived from stimulated HL-60 cells contained 60-70% more ARF as compared with membranes derived from control cells. Membrane contents of ARF were assessed by Western blotting with the anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation. Therefore, ARF translocation correlates with the potentiation of the GTP gamma S-stimulated PLD activity. The effect on PLD activity and ARF membrane content achieved through fMLP stimulation was greatly enhanced by prior treatment of the cells with cytochalasin B. Membranes derived from control and fMLP-stimulated cells were assayed for PLD activity in the presence of exogenous ARF and a 50-kDa fraction known to contain elements implicated in PLD activation. The ability of ARF and the 50-kDa fraction to enhance GTP gamma S-sensitive PLD activity was significantly reduced when the membranes were derived from fMLP-stimulated cells. The data indicate that, in addition to ARF, elements of the 50-kDa PLD-inducing factors were likely already translocated to the membranes upon stimulation. We propose that ARF, upon stimulation with agonists such as fMLP or phorbol esters, is translocated to the membrane and in concert with other protein components of the 50-kDa fraction enhances PLD activity. PMID- 7559411 TI - Escherichia coli chorismate synthase catalyzes the conversion of (6S)-6-fluoro-5 enolpyruvylshikimate-3-phosphate to 6-fluorochorismate. Implications for the enzyme mechanism and the antimicrobial action of (6S)-6-fluoroshikimate. AB - Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3 phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6 fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406). PMID- 7559413 TI - Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. AB - The gonadotropin-releasing hormone (GnRH) receptor belongs to the superfamily of heptahelical G protein coupled receptors, most of which have a highly conserved DRYXXV/IXXPL sequence in the second intracellular (2i) loop that has been implicated in G protein coupling. The predicted 2i loop of the GnRH receptor contains serine rather than tyrosine in the DRY sequence but retains the conserved hydrophobic Leu residue, which is required for G protein coupling and internalization of muscarinic receptors. The present study examined the effects of mutating the unique Ser140 to the conserved Tyr, and the conserved Leu147 to Ala or Asp, on agonist binding, internalization, and signal transduction. The S140Y mutant showed a 100% increase in agonist binding affinity, and its internalization was increased by 60% above that of the wild-type receptor. The binding characteristics of the Leu147 mutants were indistinguishable from those of the wild-type receptor, but their internalization was reduced by about 50%. The L147A and L147D mutants also showed significant impairment of GnRH-stimulated inositol phosphate production. These findings demonstrate that substitution of Ser140 by Tyr does not affect G protein coupling but significantly increases receptor affinity and internalization rate. In contrast, replacement of a conserved aliphatic residue (Leu147) impairs both G protein coupling and agonist induced receptor internalization. PMID- 7559407 TI - Characterization of yeast translation initiation factor 1A and cloning of its essential gene. AB - Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It functions primarily by dissociating ribosomes and stabilizing 40 S preinitiation complexes. To better elucidate its precise role in promoting the translation initiation process, the yeast form of eIF1A has been identified in Saccharomyces cerevisiae and purified to homogeneity on the basis of its cross-reaction with antibodies prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22 kDa) resembles that of the mammalian homolog (20 kDa), and the yeast factor is active in stimulating methionyl puromycin synthesis in an assay composed of mammalian components. The gene encoding yeast eIF1A, named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the deduced amino acid sequence exhibits 65% identity with the sequence of human eIF1A. Both human and yeast eIF1A contain clusters of positive residues at the N terminus and negative residues at the C terminus. Deletion/disruption of TIF11 demonstrates that eIF1A is essential for cell growth. Expression of human eIF1A cDNA rescues the growth defect of TIF11-disrupted cells, indicating that the structure/function of yeast and mammalian eIF1A is highly conserved. PMID- 7559409 TI - Codon pair utilization biases influence translational elongation step times. AB - Two independent assays capable of measuring the relative in vivo translational step times across a selected codon pair in a growing polypeptide in the bacterium Escherichia coli have been employed to demonstrate that codon pairs observed in protein coding sequences more frequently than predicted (over-represented codon pairs) are translated slower than pairs observed less frequently than expected (under-represented codon pairs). These results are consistent with the findings that translational step times are influenced by codon context and that these context effects are related to the compatabilities of adjacent tRNA isoacceptor molecules on the surface of a translating ribosome. These results also support our previous suggestion that the frequency of one codon next to another has co evolved with the structure and abundance of tRNA isoacceptors in order to control the rates of translational step times without imposing additional constraints on amino acid sequences or protein structures. PMID- 7559412 TI - Mechanisms of nucleobase transport in rabbit choroid plexus. Evidence for a Na(+) dependent nucleobase transporter with broad substrate selectivity. AB - The overall goal of this study was to determine the mechanisms by which nucleobases are transported in the choroid plexus. Choroid plexus tissue slices were obtained from the lateral ventricles of rabbit brains and depleted of ATP with 2,4-dinitrophenol. In the presence of an initial inwardly directed Na+ gradient, hypoxanthine accumulated in the tissue slices against a concentration gradient. Na(+)-stimulated hypoxanthine uptake was saturable with a Km of 31.1 +/ 9.71 microM and a Vmax of 2.69 +/- 0.941 nmol/g/s (mean +/- S.E.). Na(+) stimulated hypoxanthine uptake was inhibited by (100) microM naturally occurring purine and pyrimidine nucleobases (adenine, cytosine, guanine, hypoxanthine, thymine, uracil, and xanthine) as well as by the nucleoside analog, dideoxyadenosine. The stoichiometric coupling ratio between Na+ and hypoxanthine was 1.7:1. The data demonstrate the presence of a novel Na(+)-dependent nucleobase transporter in the choroid plexus, which is distinct from the previously described Na(+)-nucleoside transporter in choroid plexus and from Na(+)-dependent nucleobase transporters in other tissues in terms of its kinetics, substrate selectivity, and Na(+)-nucleobase stoichiometry. This transporter may play a role in the targeting of both salvageable nucleobases and therapeutic nucleoside analogs to the central nervous system. PMID- 7559410 TI - Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets. AB - We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4) bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating beta-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C-->diacylglycerol-->protein kinase C. PMID- 7559414 TI - Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor. AB - The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP. PMID- 7559415 TI - Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB. AB - The Escherichia coli SecB protein binds newly synthesized precursor maltose binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site. PMID- 7559418 TI - Beta-gamma subunit interaction is required for catalysis by H(+)-ATPase (ATP synthase). Beta subunit amino acid replacements suppress a gamma subunit mutation having a long unrelated carboxyl terminus. AB - The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150- >Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme. PMID- 7559416 TI - 3'-Azidothymidine (zidovudine) inhibits glycosylation and dramatically alters glycosphingolipid synthesis in whole cells at clinically relevant concentrations. AB - Recent in vitro work with Golgi-enriched membranes showed that 3'-azidothymidine 5'-monophosphate (AZTMP), the primary intracellular metabolite of 3' azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al. (1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 microM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication. PMID- 7559420 TI - Characterization of prenylcysteines that interact with P-glycoprotein and inhibit drug transport in tumor cells. AB - Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976). The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates. We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein. Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate. Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated. The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein. The structure at the nitrogen atom can, however, influence the type of interaction. Acetylation of the free amino group of prenylcysteine/results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity. Instead, certain acetylated prenylcysteines behave as inhibitors of this activity. In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the steady-state accumulation of [3H]vinblastine, [3H]colchicine, and [3H]taxol. These inhibitors do not, however, affect drug accumulation in parental cells. These studies provide a novel approach for designing P-glycoprotein inhibitors that could prove effective in reversing the phenotype of multidrug resistance in tumor cells. PMID- 7559417 TI - Biogenesis of mitochondrial heme lyases in yeast. Import and folding in the intermembrane space. AB - Heme lyases are components of the mitochondrial intermembrane space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are synthesized in the cytosol without the typical N terminal mitochondrial targeting signal. Here, we have analyzed the mode of import and folding of the two heme lyases of the yeast Saccharomyces cerevisiae, namely of cytochrome c heme lyase and of cytochrome c1 heme lyase. For transport into mitochondria, both proteins use the general protein import machinery of the outer membrane. Import occurred independently of a membrane potential, delta psi, across the inner membrane and ATP in the matrix space, suggesting that the inner membrane is not required for transport along this direct sorting pathway. The presence of a large folded domain in heme lyases was utilized to study their folding in the intermembrane space. Formation of this domain occurred at the same rate as import, indicating that heme lyases fold either during or immediately after their transfer across the membrane. Folding was not affected by depletion of ATP and delta psi or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve homologs of known folding factors (like Hsp60 and Hsp70). The energy derived from folding cannot be regarded as a major driving force for import, since the folded domain could be imported into mitochondria with the same efficiency as the intact protein. We conclude that protein folding in the intermembrane space obeys principles different from those established for other subcellular compartments. PMID- 7559421 TI - Identification of structural determinants controlling human and mouse stromelysin 3 proteolytic activities. AB - Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling processes, including those associated with cancer progression. Stromelysin-3, which is expressed in most invasive human carcinomas, is a matrix metalloproteinase with unusual functional properties. In particular, its mature form does not cleave any of the major extracellular matrix components. To define critical structural determinants involved in controlling stromelysin-3 proteolytic activity, we have used site directed mutagenesis. We show that the deletion of at least 175 C-terminal amino acids is sufficient to endow mouse stromelysin-3 with activities against casein, laminin, and type IV collagen. In the case of the human enzyme, however, a further and single Ala-235-->Pro substitution is necessary to observe similar activities. Ala-235, which characterizes human stromelysin-3 among matrixins, is located immediately after the C terminus of the "Met-turn," which forms a hydrophobic basis for the catalytic zinc atom in the metzincin family. We conclude that human stromelysin-3 has gained specific functional properties during evolution by amino acid substitution in the catalytic zinc environment, and that it represents an attractive target for specific inhibitors that may be used to prevent cancer progression. PMID- 7559419 TI - Substrate requirements for transglutaminases. Influence of the amino acid residue preceding the amine donor lysine in a native protein. AB - Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacity of these mutants to be cross linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes. It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases. This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect. This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases. It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins. PMID- 7559422 TI - The human COL11A2 gene structure indicates that the gene has not evolved with the genes for the major fibrillar collagens. AB - The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5'-end of the gene were in general larger than the introns at the 3'-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor beta in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the alpha 2(XI) chain suggested that the chain can be incorporated into the same molecule as alpha 1(XI) and alpha 1(V) chains but not into the same molecule as the alpha 3(XI)/alpha 1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl terminal propeptides of the pro alpha 1(XI) chain and pro alpha chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids. PMID- 7559423 TI - Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120. AB - Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5' flanking region and 430 base pairs of the 3'-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription. PMID- 7559424 TI - Direct kinetic evidence for triplet state energy transfer from Escherichia coli alkaline phosphatase tryptophan 109 to bound terbium. AB - The addition of excess Tb3+ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum. Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen. The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium. The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (kET approximately 360 and 120 s-1) from the triplet state of tryptophan to terbium followed by terbium decay. This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp109) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9-10 A. PMID- 7559425 TI - Probing the roles of active site residues in D-xylose isomerase. AB - The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate. PMID- 7559426 TI - cDNA cloning, gene organization, and chromosomal localization of a human mercurial insensitive water channel. Evidence for distinct transcriptional units. AB - Two distinct cDNAs encoding a human mercurial insensitive water channel (hMIWC) were cloned from a fetal brain cDNA library. The longest open reading frame of cDNA clone hMIWC1 encoded 301 amino acids with 94% identity to rat MIWC (Hasegawa, H., Ma, T., Skach, W., Matthay, M. M., and Verkman, A. S. (1994) J. Biol. Chem. 269, 5497-5500). A second cDNA (hMIWC2) had a distinct 5'-sequence upstream from base pair (bp) -34 in clone hMIWC1 and contained two additional inframe translation start codons. Expression of hMIWC cRNAs in Xenopus oocytes increased osmotic water permeability by 10-20-fold in a mercurial insensitive manner. Cell-free translation in a reticulocyte lysate/microsome system generated single protein bands at 30 kDa (hMIWC1) and 32-34 kDa (hMIWC2) without glycosylation. Northern blot and polymerase chain reaction/Southern blot analysis showed expression of mRNA encoding hMIWC in human brain - muscle >> heart, kidney, lung, and trachea. Analysis of hMIWC genomic clones indicated two distinct but overlapping transcription units from which multiple hMIWC mRNAs are transcribed. The promoter region of hMIWC1 was identified and contained TATA, CAAT, AP-1, and other regulatory elements. Primer extension revealed hMIWC1 transcription initiation at 46 bp downstream from the TATA box. There were three introns (lengths 0.9, 0.2, and 6 kilobases) in the hMIWC1 coding sequence at bp 381, 546, and 627. A distinct 5'-sequence in clone hMIWC2 suggested an alternative upstream transcription initiation site. Two alternatively spliced, nonfunctional hMIWC transcripts with exon 3 deletion and partial exon 4 deletion were identified. A poly(A)+ signal sequence was identified at 138 bp downstream of the translation stop codon. Genomic Southern blot analysis indicated the presence of a single copy hMIWC gene; chromosome-specific polymerase chain reaction and in situ hybridization localized hMIWC to human chromosome 18q22. The structural organization of the hMIWC gene represents a first step in definition of hMIWC differential expression, regulation, and possible role in human disease. PMID- 7559427 TI - Structure determination of the octa- and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestinal heparin. AB - Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G. (1992) J. Biol. Chem. 267, 1528-1533). In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only. IV was successfully isolated only after subsequent digestion with glycuronate-2 sulfatase. Their structures were determined by chemical and enzymatic analyses and 1H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: delta HexA(2S)alpha 1-4GlcN(NS,6S)alpha 1-4GlcA beta 1-4GlcNAc alpha 1-4- GlcA beta 1 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (I), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (II), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1- 4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcNAc-alpha 1- 4GlcA beta 1-3Gal beta 1 3Gal beta 1-4Xyl beta 1-O-Ser (III), delta HexA alpha 1-4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc(6S)alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O Ser (IV) (delta HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II contained 1 mol of Gly in addition to Ser. The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures. The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin. PMID- 7559429 TI - Transcytosis of aminopeptidase N in Caco-2 cells is mediated by a non-cytoplasmic signal. AB - In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N was observed. This indicates that transcytosis of aminopeptidase N from the basolateral to the apical membrane does not occur by default transport but involves an active sorting mechanism. PMID- 7559428 TI - Interleukin 4-inducible phosphorylation of HMG-I(Y) is inhibited by rapamycin. AB - The non-histone chromosomal protein HMG-I(Y) participates in repression of transcription directed by a promoter which confers interleukin 4 (IL-4)-inducible activation in transfected B cell lines. Metabolic labeling, phosphoamino acid analyses, and in vitro phosphorylation studies demonstrate that IL-4 induces serine phosphorylation of HMG-I(Y) in B lymphocytes. Phosphopeptide mapping shows that the predominant site of phosphorylation contains a casein kinase II consensus motif. The immunosuppressive agent rapamycin has been shown preferentially to inhibit IgE production by IL-4-treated human B cells. It is shown here that rapamycin inhibits both activation of the human germ line epsilon promoter by IL-4 and IL-4-inducible phosphorylation of HMG-I(Y). These findings demonstrate a rapamycin-sensitive pathway that transduces signals from the IL-4 receptor to nuclear factors that regulate inducible transcription. The affinity of normal nuclear HMG-I(Y) for DNA is increased by dephosphorylation in vitro, whereas in vitro kinase reactions using casein kinase II decrease recombinant HMG I(Y) binding to DNA. These data further suggest a novel mechanism in which phosphorylation triggered by IL-4 or other cytokines could regulate the effects of HMG-I(Y) on gene transcription. PMID- 7559430 TI - Messenger RNA recognition by fragments of ribosomal protein S4. AB - Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the alpha operon mRNA, where it represses its own synthesis. No similarity between the two RNA binding sites has been detected. To find out whether separate protein regions are responsible for rRNA and mRNA recognition, proteins with N-terminal or C-terminal deletions have been overexpressed and purified. Protein-mRNA interactions were detected by (i) a nitrocellulose filter binding assay, (ii) inhibition of primer extension by reverse transcriptase, and (iii) a gel shift assay. Circular dichroism spectra were taken to determine whether the proteins adopted stable secondary structures. From these studies it is concluded that amino acids 48-104 make specific contacts with the mRNA, although residues 105-177 (out of 205) are required to observe the same toeprint pattern as full-length protein and may stabilize a specific portion of the mRNA structure. These results parallel ribosomal RNA binding properties of similar fragments (Conrad, R. C., and Craven, G. R. (1987) Nucleic Acids Res. 15, 10331-10343, and references therein). It appears that the same protein domain is responsible for both mRNA and rRNA binding activities. PMID- 7559431 TI - Structures of the glycosyl-phosphatidylinositol anchors of porcine and human renal membrane dipeptidase. Comprehensive structural studies on the porcine anchor and interspecies comparison of the glycan core structures. AB - The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose alpha (1-2) linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl phosphatidylinositol with a minor amount of stearoyl-palmitoyl phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein. PMID- 7559433 TI - Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound peptides. AB - For the first time, it has been shown that GroEL can be converted from tetradecamers (14-mers) to monomers under conditions commonly used for the preparation of this chaperonin. The essential requirements are the simultaneous presence of nucleotides such as MgATP or MgADP and a solid-phase anion-exchange medium. The monomers that are formed are metastable in that they only reassemble to a small degree in the absence of additives. These results are in keeping with previous studies on high pressure dissociation that showed the separated monomers display conformational plasticity and can undergo conformational relaxation when relieved of the constraints of the quaternary structure in the oligomer (Gorovits, B., Raman, C. S., and Horowitz, P. M. (1995) J. Biol. Chem. 270, 2061 2066). The monomers display greatly enhanced hydrophobic exposure to the probe 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, although they are not active in folding functions, and they are unable to form complexes with partially folded rhodanese. The monomers can be completely reassembled to 14-mers by incubation in 1 M ammonium sulfate. There is no evidence of intermediates in the reassembly process. Compared with the original oligomers, the reassembled 14-mers have (a) very low levels of polypeptide contaminants and tryptophan-like fluorescence, two problems that previously hampered spectroscopic studies of GroEL structure and function; (b) functional properties that are very similar to the original material; (c) considerably decreased hydrophobic exposure in the native state; and (d) a similar triggered exposure of hydrophobic surfaces after treatment with urea or spermidine. This study demonstrates that the quaternary structure of GroEL is more labile than previously thought. These results are consistent with suggestions that nucleotides can loosen subunit interactions and show that changes in quaternary structure can operate under conditions where GroEL function has been demonstrated. PMID- 7559432 TI - Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity. AB - The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 microM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide binding domains may need to bind ATP to couple drug binding to ATPase activity. PMID- 7559436 TI - Refined solution structure of a DNA heteroduplex containing an aldehydic abasic site. AB - The solution structure of the DNA duplex d(C1G2C3G4A5D6A7C8G9C10C11)-d (G22C21G20C19T18A17T16G15C14G13-G12), with D indicating a deoxyribose aldehyde abasic site and numbering from 5' to 3', has been determined by the combined use of NMR and restrained molecular dynamics. The 31P and 31P-1H correlation data indicate that the backbones of these duplex DNAs are regular. One- and two dimensional 1H NMR data indicate that the duplexes are right-handed and B-form. Conformational changes due to the presence of the abasic site extends to the base pairs adjacent to the lesion site with the local conformation of the DNA being dependent on whether the abasic site is in the alpha or beta configuration. When the sugar of the abasic site is in the beta configuration the deoxyribose is within the helix, whereas when the sugar is in the alpha configuration the deoxyribose is out of the helix. The base of residue A17 in the position opposite the abasic site is predominantly stacked in the helix in both cases. A water molecule can apparently form a hydrogen bond bridge between the beta abasic site and A17. PMID- 7559434 TI - Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors. AB - Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS. PMID- 7559435 TI - Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line. AB - This laboratory previously described an L1210 leukemia cell line (MTXrA) selected for resistance to methotrexate by virtue of impaired transport due to a functional defect in the translocation process. We now report on the sequence analysis of cDNAs encoding the reduced folate carrier from this line and identify a single mutation that results in the substitution of a proline for an alanine in a highly conserved transmembrane region of the protein. Transfection of the parental reduced folate carrier into MTXrA cells resulted in a cell line which exhibited a complete restoration of methotrexate uptake and an enhanced sensitivity to methotrexate. Northern analysis and specific [3H]MTX cell surface binding indicated that expression of the reduced folate carrier was elevated approximately 5-fold in the transfectant compared to parental and MTXrA cells. The MTX influx properties of the transfectant cell line were identical to those of the well characterized reduced folate carrier from parental L1210 cells in terms of: 1) patterns of sensitivity to competing folates, 2) sensitivity to the organic anion sulfobromophthalein, 3) lack of energy dependence, and 4) capacity for trans-stimulation. We also provide new data which suggests that the nucleotide sequence 5' of the predicted ATG initiation codon may encode additional protein information in the form of a leader sequence. Finally, we demonstrate that the MTXrA line has both the mutant and the parental reduced folate carrier alleles but that expression appears to be restricted to the mutant allele. Thus, the methotrexate transport phenotype and resultant drug resistance in this cell line result from genetic/regulatory events at both alleles. PMID- 7559437 TI - Orphan nuclear receptor HNF-4 binds to the human coagulation factor VII promoter. AB - The human coagulation protease factor VII plays a pivotal role in the initiation of the coagulation cascade by both the extrinsic and the intrinsic pathway. Although the gene, encoding factor VII, is expressed predominantly in the liver, the mechanisms underlying this tissue-specific expression have not been elucidated. In this study, we have analyzed the contribution of 5 kilobases upstream of the ATG translational initiation codon upon hepatic factor VII gene transcription. Transient transfection assays of a set of nested deletions in both liver and non-liver cell lines, HepG2 and HeLa respectively, indicate that several regions are involved in liver-specific expression. A slight negative effect on factor VII promoter activity in HepG2 cells is mediated by sequences upstream of position -1212. DNase I protection experiments reveal six footprints, FPVII1 through FPVII6, within the proximal 714 base pairs but a minimal promoter of 165 base pairs containing only FPVII3-6 is sufficient to confer liver-specific expression in HepG2 cells. Interestingly, FPVII6, at position -14 to +10 on the sense strand, would indicate that an as yet unknown transcription factor covers the ATG translational initiation codon. Gel retardation experiments show that the liver-enriched transcription factor HNF-4 binds specifically to footprint FPVII4 at position -71 to -49. Furthermore, a T --> A transversion, that in the HNF-4 binding site of factor IX causes a severe bleeding disorder, was introduced into the HNF-4-binding site of factor VII and reduced promoter activity by 20-50%. Coordinate HNF-4-mediated regulation of several blood protease genes as well as genes involved in lipid metabolism might account for the positive correlation of these factors with increased risk of occlusive heart diseases. PMID- 7559439 TI - Involvement of a mitogen-activated protein kinase signaling pathway in the regulation of urokinase promoter activity by c-Ha-ras. AB - The expression of the urokinase-type plasminogen activator, which plays a crucial role in tissue remodeling by controlling the synthesis of the broadly acting plasmin serine protease, is regulated by several tyrosine kinases. Since the actions of these tyrosine kinases is dependent on the activation of ras proteins, we undertook a study to identify signaling events downstream of ras responsible for the stimulation of urokinase promoter activity. Transient expression of an activated c-Ha-ras in OVCAR-3 cells, which do not harbor the mutated oncogene, led to a dose-dependent trans-activation of the urokinase promoter. A sequence residing between -2109 and -1964 was critical for the stimulation of the urokinase promoter by c-Ha-ras. Mutation of an AP-1 and a PEA3 site at -1967 and 1973, respectively, or the co-expression of a transactivation domain-lacking c jun substantially impaired the ability of c-Ha-ras to stimulate urokinase promoter activity. The induction of the urokinase promoter by ras was completely blocked by expression of a dominant negative c-raf expression vector and substantially reduced in cells made to co-express a catalytically inactive mitogen-activated protein kinase kinase. Further, the expression of an ERK1/ERK2 inactivating phosphatase (CL100) abrogated the stimulation of the urokinase promoter by c-Ha-ras. These data argue for a role of a mitogen-activated protein kinase-dependent signaling pathway in the regulation of urokinase promoter activity by ras. PMID- 7559438 TI - Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. AB - Neuronal NO synthase (NOS) is a flavin-containing hemeprotein that generates NO from L-arginine, NADPH, and O2. NO has recently been proposed to autoinhibit NOS. We have investigated whether a NOS heme-NO complex forms during aerobic steady state catalysis. Visible and resonance Raman spectra recorded during steady-state NO synthesis by NOS showed that the majority of enzyme (70-90%) was present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O2. Its level remained constant during NO synthesis until the NADPH was exhausted, after which the complex decayed to regenerate ferric resting NOS. Stopped-flow measurements revealed that the buildup of the ferrous-NO complex was rapid (< 2 s) and caused a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay could occur several times with no adverse affect on its subsequent formation or on NOS catalytic activity. Neither enzyme dilution nor NO scavengers (superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl NOS or prevented the catalytic inhibition attributed to its formation. The ferrous-nitrosyl complex also formed in unfractionated cell cytosol containing neuronal NOS upon initiating NO synthesis. We conclude that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrous-nitrosyl complex during NO synthesis independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS. PMID- 7559440 TI - Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. AB - Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; Takino, T., Sato, H., Yamamoto, E., and Seiki, M. (1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT MMP, MT-MMP-1. MT-MMP-1 and -2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68 kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT MMP-1 and -2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls. PMID- 7559441 TI - Point mutations in awdKpn which revert the prune/Killer of prune lethal interaction affect conserved residues that are involved in nucleoside diphosphate kinase substrate binding and catalysis. AB - The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awdKpn mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awdKpn gene. Three revertants have deletions of the awdKpn protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awdKpn mutation. PMID- 7559444 TI - Cleavage-dependent ligation by the FLP recombinase. Characterization of a mutant FLP protein with an alteration in a catalytic amino acid. AB - The FLP recombinase of the 2 microM plasmid of Saccharomyces cerevisiae belongs to the integrase family of recombinases whose members have in common four absolutely conserved residues (Arg-191, His-305, Arg-308, and Tyr-343). We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine. Although FLP R308K was previously reported to be defective in ligation of certain substrates (Pan, G., Luetke, K., and Sadowski, P.D., Mol. Cell. Biol. 13, 3167-3175, 1993b), we show in this work that the protein is able to ligate those substrates that it can cleave (cleavage-dependent ligation activity). FLP R308K is defective in in vitro recombination and in strand exchange. It is able to carry out strand exchange at one of the two cleavage sites of the FLP recognition target site (FRT site), but is defective in strand exchange at the other cleavage site. These results are consistent with a model in which wild-type FLP initiates recombination only at one of the two cleavage sites. FLP R308K may be defective in the initiation of recombination. PMID- 7559446 TI - Peptide environment of the peptidyl transferase center from Escherichia coli 70 S ribosomes as determined by thermoaffinity labeling with dihydrospiramycin. AB - In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6 Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level. PMID- 7559445 TI - L- and D-enantiomers of 2',3'-dideoxycytidine 5'-triphosphate analogs as substrates for human DNA polymerases. Implications for the mechanism of toxicity. AB - 5'-Triphosphates of beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3 dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases alpha, beta, gamma, delta, and epsilon. L-ddCTP was not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA polymerases and was a less potent inhibitor of DNA polymerases gamma and beta than its D-enantiomer by 2 orders of magnitude. In contrast, all L-dioxolane analogs were potent inhibitors and chain terminators for all cellular DNA polymerases studied. The Ki values of their 5' triphosphates for DNA polymerase gamma were found to be in the following order: D ddC < D-FddC L-OddC D-FOddC < L-FOddC << L-FddC. The Ki values of L-OddCTP for the reactions catalyzed by DNA polymerases alpha, delta, epsilon, beta, and gamma were 6.0, 1.9, 0.4, 3.0, and 0.014 microM, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 microM, respectively. The Km values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 microM for DNA polymerases alpha, delta, epsilon, beta, and gamma, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity. PMID- 7559443 TI - Molecular cloning of three distinct cDNAs, each encoding a different adipokinetic hormone precursor, of the migratory locust, Locusta migratoria. Differential expression of the distinct adipokinetic hormone precursor genes during flight activity. AB - Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (approximately 570 bases), AKH II precursor (approximately 600 bases), and AKH III precursor (approximately 670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (approximately 2.0 times each) and the AKH III mRNA (approximately 4.2 times) in the corpora cardiaca. PMID- 7559442 TI - Constitutive overexpression of CDK2 inhibits neuronal differentiation of rat pheochromocytoma PC12 cells. AB - Changes in the levels of cyclins A, D, and E, p21, and cyclin-dependent kinase 2 (CDK2) were examined in rat pheochromocytoma PC12 cells during neuronal differentiation induced by nerve growth factor (NGF). Expression of cyclin A decreased to an undetectable level after 5 days of exposure to NGF, while expression of CDK2 decreased gradually after day 3. In contrast, the levels of cyclins D1 and E increased gradually through day 10, yet the amount of cyclin E associated with CDK2 decreased concomitant with a decrease in the CDK2 protein level. p21 expression increased gradually after day 7, while the level of CDK2 associated p21 remained unchanged. When human cDNAs encoding cyclins and CDK2 were introduced into PC12 cells, only CDK2 overexpression inhibited NGF-induced differentiation. The cell lines overexpressing CDK2 showed stable and high levels of CDK2 kinase activity during differentiation, whereas parental and vector transfected cell lines displayed a marked decline in CDK2 kinase activity 1 day after NGF treatment. In cell lines overexpressing cyclins A, D, and E, this reduction of the kinase activity was not apparent until day 3. These results suggest that down-regulation of CDK2 activity is a crucial event for the neuronal differentiation of PC12 cells. PMID- 7559447 TI - Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. AB - The alpha-hemolysin (alpha HL) polypeptide is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into lipid bilayers to form cylindrical heptameric pores 1-2 nm in effective internal diameter. We have individually replaced each charged residue (79 of 293 amino acids) and four neutral residues in alpha HL with cysteine, which is not found in the wild-type protein. The properties of these mutants have been examined before and after modification with the 450-Da dianionic sulfhydryl reagent 4-acetamido-4' ((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). This modification was highly informative as 28 of 83 modified polypeptides showed substantially reduced pore forming activity on rabbit erythrocytes (rRBC), while only five of the unmodified cysteine mutants were markedly affected. Through detailed examination of the phenotypes of the mutant and modified hemolysins, we have pinpointed residues and regions in the alpha HL polypeptide chain that are important for binding to rRBC, oligomer formation and pore activity. Residues in both the N-terminal (Arg-66 and Glu-70) and C-terminal (Arg-200, Asp-254, Asp-255, and Asp-276) thirds of the protein are implicated in binding to cells. The His-35 replacement mutant modified with IASD was the only polypeptide in this study that failed to form SDS resistant oligomers on rRBC. Altered hemolysins that formed oligomers but failed to lyse rRBC represented the most common defect. These alterations were clustered in the central glycine-rich loop, which has previously been implicated as a component of the lumen of the membrane-spanning channel, and in the regions flanking the loop. Alterations in mutant and modified hemolysins with the same defect were also scattered between the N terminus and His-48, in keeping with previous suggestions that an N-terminal segment and the central loop cooperate in the final step of pore assembly. PMID- 7559448 TI - Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis. AB - alpha-Hemolysin (alpha HL), a pore-forming polypeptide of 293 amino acids, is secreted by Staphylococcus aureus as a water-soluble monomer. Residues that play key roles in the formation of functional heptameric pores on rabbit red blood cells (rRBC) have been identified previously by site-directed mutagenesis. alpha HL-H35N, in which the histidine at position 35 of the wild-type sequence is replaced with asparagine, is nonlytic and is arrested in assembly as a heptameric prepore. In this study, second-site revertants of H35N that have the ability to lyse rRBC were generated by error-prone PCR under conditions designed to produce single base changes. The analysis of 22 revertants revealed new codons clustered predominantly in three distinct regions of the H35N gene. One cluster includes amino acids 107-111 (four revertants) and another residues 144-155 (five revertants). These two clusters flank the central glycine-rich loop of alpha HL, which previously has been implicated in formation of the transmembrane channel, and encompass residues Lys-110 and Asp-152 that, like His-35, are crucial for lytic activity. The third cluster lies in the region spanning amino acids 217-228 (eight revertants), a region previously unexplored by mutagenesis. Single revertants were found at amino acid positions 84 and 169. When compared with H35N, the heptameric prepores formed by the revertants underwent more rapid conversion to fully assembled pores, as determined by conformational analysis by limited proteolysis. The rate of conversion to the fully assembled pore was strongly correlated with hemolytic activity. Previous work has suggested that the N terminus of alpha HL and the central loop cooperate in the final step of assembly. The present study suggests that the key N-terminal residue His-35 operates in conjunction with residues flanking the loop and C-terminal residues in the region 217-228. Hence, reversion mutagenesis extends the linear analysis that has been provided by direct point mutagenesis. PMID- 7559450 TI - Characterization of active and inactive forms of the JAK2 protein-tyrosine kinase produced via the baculovirus expression vector system. AB - Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results. PMID- 7559451 TI - Acid-facilitated supramolecular assembly of G-quadruplexes in d(CGG)4. AB - Molar [K+] induces aggregate formation in d(CGG)4, as evidenced by absorbance, circular dichroic (CD), and gel measurements. The kinetics of this transformation are extremely slow at pH 8 but are found to be greatly facilitated in acidic conditions. Kinetic profiles via absorbance or CD monitoring at single wavelength resemble those of autocatalytic reacting systems with characteristic induction periods. More than 0.8 M KCl is needed to observe the onset of aggregation at 20 degrees C and pH 5.4 within the time span of 1 day. Time-dependent CD spectral characteristics indicate the formation of parallel G-tetraplexes prior to the onset of aggregation. Despite the evidence of K(+)-induced parallel G-quadruplex and higher molecular weight complex formation, both d(TGG)4 and d(CGG)4T fail to exhibit the observed phenomenon, thus strongly implicating the crucial roles played by the terminal G and base protonation of cytosines. A plausible mechanism for the formation of a novel self-assembled structure is speculated. Aided by the C+.C base pair formation, parallel quadruplexes are initially formed and subsequently converted to quadruplexes with contiguous G-tetrads and looped-out cytosines due to high [K+]. These quadruplexes then vertically stack as well as horizontally expand via interquadruplex C+.C base pairing to result in dendrimer type self-assembled super structures. PMID- 7559452 TI - Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D. Implications for the origin of lipocalins. AB - We report a novel outer membrane lipoprotein of Escherichia coli. DNA sequencing between ampC and sugE at the 94.5 min region of the E. coli chromosome revealed an open reading frame specifying 177 amino acid residues. Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of beta galactosidase activity from a lacZ translational fusion. The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins. The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein. However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus. Overexpression, membrane fractionation, and metabolic labeling with [3H]palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein. Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E. coli. PMID- 7559449 TI - Regulation of the human P-selectin promoter by Bcl-3 and specific homodimeric members of the NF-kappa B/Rel family. AB - P-selectin, an adhesion receptor for leukocytes, is constitutively expressed by megakaryocytes and endothelial cells. Synthesis of P-selectin is also increased by some inflammatory mediators. We characterized a previously identified kappa B site (-218GGGGGTGACCCC-207) in the promoter of the human P-selectin gene. The kappa B site was unique in that it bound constitutive nuclear protein complexes containing p50 or p52, but not inducible nuclear protein complexes containing p65. Furthermore, the element bound recombinant p50 or p52 homodimers, but not p65 homodimers. Methylation interference analysis indicated that p50 or p52 homodimers contacted the guanines at positions -218 to -214 on the coding strand and at -210 to -207 on the noncoding strand. Changes in the three central residues at -213 to -211 altered binding specificity for members of the NF-kappa B/Rel family. Mutations that eliminated binding to NF-kappa B/Rel proteins reduced by approximately 40% the expression of a reporter gene driven by the P selectin promoter in transfected bovine aortic endothelial cells. Overexpression of p52 enhanced P-selectin promoter activity, and co-overexpression of Bcl-3 further induced promoter activity in a kappa B site-dependent manner. In contrast, overexpression of p50 repressed promoter activity; this repression was prevented by co-overexpression of Bcl-3. Similar phenomena were observed with reporter gene constructs driven by two tandem P-selectin kappa B sequences linked to the SV40 minimal promoter. These data suggest that Bcl-3 differentially regulates the effects of p50 and p52 homodimers bound to the kappa B site of the P-selectin promoter. This site may be a prototype for kappa B elements in other genes that bind specifically to p50 and/or p52 homodimers. PMID- 7559453 TI - Heterodimeric neurotrophins induce phosphorylation of Trk receptors and promote neuronal differentiation in PC12 cells. AB - Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer. PMID- 7559456 TI - Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon. AB - Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase. PMID- 7559454 TI - Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. AB - KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5' of the initiation methionine codon. The 5'-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that approximately 4 kilobases of KDR/flk-1 5'-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells. PMID- 7559455 TI - Phosphorylation of Gz alpha by protein kinase C blocks interaction with the beta gamma complex. AB - Gz alpha is a G protein alpha subunit with biochemical properties that distinguish it from other members of the G protein alpha subunit family. One such property is its ability to be stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation has been mapped to a region near the N terminus of Gz alpha, but no functional significance of the modification has been established. To investigate this question, we have developed a baculovirus/Sf9 cell expression system to produce Gz alpha. The protein purified from Sf9 cells is functional as assessed by its ability both to bind guanine nucleotide in a Mg(2+)-sensitive fashion and to serve as a substrate for phosphorylation by PKC. Furthermore, addition of the G protein beta gamma complex purified from bovine brain inhibits phosphorylation of Gz alpha in a dose-dependent manner. Conversely, phosphorylation of Gz alpha inhibits its ability to interact with beta gamma subunits. These results establish a functional consequence for PKC-catalyzed phosphorylation of Gz alpha and suggest a mechanism for regulation of signaling through Gz by preventing reassociation of its subunits. PMID- 7559457 TI - Inducible interaction of integrin alpha 2 beta 1 with calreticulin. Dependence on the activation state of the integrin. AB - We have previously demonstrated an interaction between the highly conserved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domains and calreticulin. Since this highly conserved sequence motif has been implicated in the regulation of the integrin affinity state, we wanted to determine whether the calreticulin-integrin interaction also depended on the integrin affinity state, and whether calreticulin occupation of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state. We now demonstrate that anti-integrin antibody- or phorbol 12-myristate 13-acetate (PMA)-induced activation of the alpha 2 beta 1 integrin on Jurkat cells, as determined by stimulation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha 2 beta 1 activation with either anti beta 1 or anti-alpha 2 monoclonal antibodies resulted in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abrogated the calreticulin-integrin interaction. A correlation was also found between PMA-induced alpha 2 beta 1 activation and the amount of calreticulin bound to this integrin. Furthermore, pretreatment of streptolysin O-permeablized Jurkat cells with an anti calreticulin antibody resulted in a significant and specific inhibition of the adhesion to collagen type I that could be induced by antibodies to alpha 2 beta 1 or by PMA. These data suggest that the active, high affinity form of alpha 2 beta 1 binds calreticulin and that calreticulin binding to the alpha 2 cytoplasmic domain may be required for stabilizing the high affinity state of this integrin. The data presented here also demonstrate, for the first time, an inducible interaction of an integrin with an intracellular protein that occurs via the alpha subunit of the integrin. PMID- 7559459 TI - Cross-talk between the histidine protein kinase VanS and the response regulator PhoB. Characterization and identification of a VanS domain that inhibits activation of PhoB. AB - VanS is a two-component transmembrane sensory kinase that, together with its response regulator VanR, activates the expression of genes responsible for vancomycin resistance in Enterococcus faecium BM4147. In this report, we demonstrate that the cytoplasmic domain of VanS (including residues Met95 to Ser384) is capable of high level activation (> 500 fold) of the Escherichia coli response regulator PhoB in vivo in the absence of its signaling kinases PhoR, CreC (PhoM), or acetyl phosphate synthesis. In vitro experiments carried out on the purified proteins confirmed that the activation is due to efficient cross talk between VanS and PhoB, since phospho-VanS catalyzed transfer of its phosphoryl group to PhoB with approximately 90% transfer in 5 min at a 1:4 VanS/PhoB stoichiometry. However, the rate of transfer was at least 100-fold slower than that observed between phospho-VanS and VanR. The in vivo activation of PhoB was used as a reporter system to identify peptide fragments of VanS capable of interfering with activation by VanS(Met95-Ser384), in order to identify an interaction domain. A library of plasmids encoding fragments of VanS(Met95-Ser384) was constructed using transposon mutagenesis, and a subpopulation of these plasmids encoded peptides that interfered with activation of PhoB by VanS(Met95-Ser384). A minimal size fragment (Met95-Ile174) was shown to be both necessary and sufficient for potent inhibition (85%) of this activation. PMID- 7559458 TI - Function of the homeo and paired domain proteins TTF-1 and Pax-8 in thyroid cell proliferation. AB - The thyroid transcription factors TTF-1 and Pax-8 are homeobox- and paired box containing genes, respectively, that are responsible for thyroid-specific gene expression, thyroid development, and thyroid cell differentiation. However, it is not clear if such factors play a role in thyroid cell proliferation. The antisense oligonucleotide strategy was used in order to clarify this point. Treatment of quiescent FRTL-5 thyroid cells with TTF-1 or Pax-8 antisense oligonucleotides caused a significant reduction in thyroid-stimulating hormone and insulin-like growth factor-I-stimulated cell proliferation, measured by DNA synthesis and cell counting. The same results were obtained with forskolin indicating that the TTF-1 or Pax-8 role in mediating the thyroid-stimulating hormone growth effect occurred via the cAMP pathway. The effect was higher with TTF-1 as the blockage by this factor caused a 65% decrease in cell proliferation compared to the control. Pax-8 blocking lead only to a 30% decrease. The blocking of both thyroid transcription factors together did not result in an additive effect. These data provide direct evidence that both homeo and paired box gene expression is essential for FRTL-5 thyroid cell proliferation, with each one possibly playing a different regulatory role. PMID- 7559461 TI - Stimulation of a Ca(2+)-calmodulin-activated histone 3 arginine kinase in quiescent rat heart endothelial cells compared to actively dividing cells. AB - A Ca(2+)-calmodulin-activated histone 3 kinase was partially purified from nuclear extracts of dividing and quiescent rat heart endothelial cells. The histone 3 phosphorylating activity was 20-100-fold higher in quiescent than in dividing cells. Base hydrolysis followed by amino acid analysis revealed that histone 3 was phosphorylated on arginine. Further investigations were conducted to determine whether phosphorylation of histone 3 also occurred in vivo. Cells were incubated for 3 h in a phosphate-free medium supplemented with [32P]phosphoric acid. It was observed that the nuclear content of arginine phosphorylated histone 3 was considerably higher in quiescent than in dividing rat heart endothelial cells. The histone 3 arginine kinase is a component of a complex containing a Ca(2+)-dependent calmodulin-binding protein of apparent molecular mass of 85 kDa. Using polyclonal antibodies to an 85-kDa protein, also the major Ca(2+)-dependent calmodulin-binding component of the histone 3 arginine kinase from calf thymus, an immunoreactive protein of identical apparent molecular mass was found to be present in equal amounts both in dividing and quiescent cells. We propose that the 85-kDa protein is either the histone 3 arginine kinase or one of its subunits and that phosphorylation of histone 3 is involved with cell cycle exit in eukaryotes. PMID- 7559462 TI - Modulation of interleukin-6-induced plasma protein secretion in hepatoma cells by p53 species. AB - The ability of p53 species (wild-type and mutant) to modulate the "differentiated" response of human hepatoma cell lines Hep3B and HepG2 to interleukin-6 (IL-6) was investigated. Transient transfection experiments were carried out in Hep3B and HepG2 cell cultures in which IL-6 was used to activate a beta-fibrinogen (beta Fib) enhancer/reporter construct containing two copies of the 36-base pair IL-6-response element (IL-6RE) (p beta FibCAT). Cotransfection with constitutive expression vectors for wild-type (wt) human or murine p53 inhibited the activation of the p beta FibCAT reporter by IL-6 in both Hep3B and HepG2 cells. Several mutant p53 species either did not inhibit the activation of p beta FibCAT or up-regulated the response. Hepatoma cell lines stably expressing the Val-135 temperature-sensitive mutant of murine p53 (wt-like at 32.5 degrees C and mutant-like at 37 degrees C) were derived from Hep3B cells and tested for the temperature-sensitive phenotype of their ability to synthesize and secrete fibrinogen and alpha 1-antichymotrypsin in response to IL-6. In an experimental protocol in which the parental Hep3B cells did not show a significant difference in plasma protein secretion at the two temperatures, hepatoma line 3 (p53Val 135+) had a greater response to IL-6 at 37 degrees C than parental Hep3B cells, while line 3 cells had a reduced response to IL-6 at 32.5 degrees C. Similarly, hepatoma lines 1 and 2 (both p53Val-135+) had reduced IL-6 responsiveness at 32.5 degrees C, whereas line 22 (transfected with pSVneo alone) and the parental Hep3B cells did not. These data indicate that mutations in p53 contained in tumor cells can modulate the "differentiated" response of these cells to cytokines. PMID- 7559460 TI - The role of coiled-coil alpha-helices and disulfide bonds in the assembly and stabilization of cartilage matrix protein subunits. A mutational analysis. AB - Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP A domain form an intradomain disulfide bond. PMID- 7559463 TI - Parathyroid hormone and parathyroid hormone-related peptide activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic cells (UMR-106) via a cAMP-dependent pathway. AB - Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na+/H+ exchanger activity in osteoblastic cells, although the signaling components involved are not precisely defined. Since these peptide hormones can stimulate production of diverse second messengers (i.e. cAMP and diacylglycerol) that activate protein kinase A (PKA) and protein kinase C (PKC) in target cells, it is conceivable that either one or both of these pathways can participate in modulating exchanger activity. To discriminate among these possibilities, a series of synthetic PTH and PTHRP fragments were used that stimulate adenylate cyclase and/or PKC. In the osteoblastic cell line UMR-106, human PTH(1-34) and PTHRP(1-34) augmented adenylate cyclase activity, whereas PTH(3-34), PTH(28-42), and PTH(28-48) had no effect. Nevertheless, all these peptide fragments were found to enhance PKC translocation from the cytosol to the membrane in a dose dependent (10(-11) to 10(-7) M) manner. PTHRP(1-16), a biologically inert fragment, was incapable of influencing either the PKA or PKC pathway. PTH(1-34) and PTHRP(1-34), but not PTH(3-34), PTH(28-42), PTH(28-48), or PTHRP(1-16), elevated Na+/H+ exchanger activity, implicating cAMP as the transducing signal. In accordance with this observation, forskolin (10 microM), which directly stimulates adenylate cyclase, also activated Na+/H+ exchanger activity. The involvement of PKA was verified when the highly specific PKA inhibitor, H-89, completely abolished the stimulatory effect of PTH(1-34) and forskolin on Na+/H+ exchange. In addition, Northern blot analysis revealed the presence of only the NHE-1 isoform of the Na+/H+ exchanger in UMR-106 cells. In summary, these results indicated that PTH and PTHRP activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic UMR-106 cells exclusively via a cAMP-dependent pathway. PMID- 7559464 TI - Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. AB - p21ras has been implicated in the hypertrophic response of cultured cardiac myocytes to defined growth stimuli. To determine if activation of ras-dependent intracellular signaling pathways is sufficient to induce in vivo hypertrophy, transgenic mice were created that express oncogenic ras in the cardiac ventricular chamber. Mice homozygous for the transgene displayed morphological, physiological, and genetic markers of marked cardiac muscle hypertrophy. Miniaturized catheterization technology documented a selective prolongation of cardiac relaxation, similar to that seen in early human hypertrophic heart disease. An increase in left atrial mass, in the absence of transgene expression in that chamber, further supported physiologically abnormal left ventricular diastolic function. Histological analysis revealed myofibrillar disarray, indistinguishable from that in hypertrophic cardiomyopathy in man. These studies establish a ras-dependent pathway for hypertrophic heart disease and document the feasibility of mapping in vivo signaling pathways for cardiac hypertrophy and dysfunction by applying in vivo microphysiological assays to genetically manipulated mice. ras-dependent pathways may also be a rational target for developing new approaches to inhibit the genesis of hypertrophy in certain pathological settings. PMID- 7559465 TI - Retina-specifically expressed novel subtypes of bovine cyclophilin. AB - The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retina cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well s other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina-specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins. PMID- 7559467 TI - The human integrin alpha 8 beta 1 functions as a receptor for tenascin, fibronectin, and vitronectin. AB - The integrin family of adhesion receptors consists of at least 21 heterodimeric transmembrane proteins that differ in their tissue distribution and ligand specificity. The recently identified alpha 8 integrin subunit associates with beta 1 and is predominantly expressed in smooth muscle and other contractile cells in adult tissues, and in mesenchymal and neural cells during development. We now show that alpha 8 beta 1 specifically localizes to focal contacts in cells plated on the extracellular matrix proteins fibronectin or vitronectin. In addition we show that human embryonic kidney cells (293), transfected with alpha 8 cDNA, express alpha 8 beta 1 on their surface and use this receptor for adhesion to fibronectin and vitronectin. Furthermore, alpha 8 beta 1 binds to both fibronectin- and vitronectin-Sepharose and can be specifically eluted from either matrix protein by the arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP. Because fibronectin and vitronectin adhesion appeared to be mediated by RGD, we examined additional RGD-containing proteins, including tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I. We found that only tenascin was able to mediate adhesion of alpha 8-transfected 293 cells. By using recombinant fragments of tenascin in adhesion assays, we were able to localize the alpha 8 beta 1 binding domain of tenascin to the RGD containing third fibronectin type III repeat. These data strongly suggest that tenascin, fibronectin, and vitronectin are ligands for alpha 8 beta 1 and that this integrin binds to the RGD site in each of these ligands through mechanisms that are distinct and separate from alpha 5- and alpha v-containing integrins. PMID- 7559466 TI - Identification of critical determinants of alpha 1-adrenergic receptor subtype selective agonist binding. AB - alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids. PMID- 7559468 TI - Functional roles of individual recombinase monomers in strand breakage and strand union during site-specific DNA recombination. AB - The site-specific recombinase Flp from Saccharomyces cerevisiae accomplishes recombination between two target DNA sites by executing a pair of strand exchanges at either end of the strand exchange region. One round of recombination requires the cooperative action of four recombinase monomers. We demonstrate here that, in the presence of the appropriate nucleophiles, a single Flp monomer associated with its binding element can mediate strand cleavage and strand joining at the exchange site phosphate adjacent to it. Our results support a model of recombination in which pairs of Flp monomers reverse catalytic roles to mediate the first and second sets of strand breakage/union reactions. They disfavor a model that involves a relay of recombinase monomers between binding elements to assemble separate active sites for strand cleavage and strand joining. Our data are consistent with the breakage and joining reactions being carried out by a single composite active site in which some residues contribute to both reactions while others contribute to one of the two reactions. PMID- 7559469 TI - Role of N-linked glycosylation in human osteonectin. Effect of carbohydrate removal by N-glycanase and site-directed mutagenesis on structure and binding of type V collagen. AB - In this study we demonstrate that the binding region of recombinant truncated human bone osteonectin (tHON) for type V collagen resides between amino acids 1 and 146. After removal of oligosaccharide chain structures from tHON, bovine bone osteonectin (BBON) and human platelet osteonectin (HPON) by N-glycanase, their ability to bind to type V collagen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a direct or regulatory effect on osteonectin binding to collagen V and that the increase in tHON binding upon removal of carbohydrate is the result of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of each N-glycosylation site in tHON, Asn71 and Asn99 were mutated to Gln (N71Q, N99Q) and Thr73 and Thr101 mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment. The binding affinity of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubled mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn99-X-Thr101) in tHON is important in the reduction of binding of osteonectin to collagen V. Consistent with the binding data is the observation that both the N71Q and T73A mutant proteins migrate on SDS-polyacrylamide gel electrophoresis gels identically to wild-type tHON, suggesting that there is little or no N-glycosylation of residue 71 in wild-type osteonectin. PMID- 7559471 TI - Identification and characterization of multiple forms of bovine brain N myristoyltransferase. AB - N-Myristoyltransferase (NMT) catalyzes the co-translational addition of myristic acid to the N-terminal glycine of many cellular, viral, and fungal proteins which are essential to normal cell functioning and/or are potential therapeutic targets. We have found that bovine brain NMT exists as a heterogeneous mixture of interconvertible high molecular mass multimers involving approximately 60-kDa NMT subunit(s). Gel filtration chromatography of partially purified NMT at low to moderate ionic strength yields NMT activity eluting as 391 +/- 52 and 126 +/- 17 kDa peaks as well as activity which profiles the protein fractions and likely results from NMT nonspecifically associating with background proteins and/or column matrix. Chromatography in 1 M NaCl causes 100% of this activity to elute as a single peak of approximately 391 kDa. Subsequent treatment of the approximately 391 kDa activity peak with an NMT peptide reaction product (i.e. N myristoyl-peptide) results in approximately 75% of the activity re-eluting as a approximately 126-kDa peak in 1 M NaCl. Rechromatography also yields small amounts of a approximately 50-kDa NMT monomer which increases with prior storage at 4 degrees C. Up to 5 NMT subunits were identified by SDS-polyacrylamide gel electrophoresis and specific immunoblotting with a human NMT peptide antibody and by cofactor-dependent chemical cross-linking with an 125I-peptide substrate of NMT. The prominent 60 kDa and minor 57-, 53-, 49-, and 47-kDa NMT immunoblotted subunits co-migrate with five of nine silver-stained proteins in an enzyme preparation purified > 7,000-fold with approximately 50% yield by selective elution from octyl-agarose with the myristoyl-CoA analog, S-(2-ketopentadecyl) CoA. Storage at 4 degrees C also leads to conversion of the larger NMT subunit(s) into 49 and 47 kDa forms with no loss of NMT activity. These results identify two interconvertible forms of NMT in bovine brain that result from NMT subunit multimerization and/or complex formation with other cellular proteins. The data also identify a fully active NMT monomer which arises from subunit proteolysis. This study thus reveals a previously unappreciated level of NMT complexity which may have important mechanistic and/or regulatory significance for N myristoylation in mammalian cells. PMID- 7559472 TI - Metabolic regulation of gene transcription in mammals. PMID- 7559470 TI - Mutations in the elongation factor 2 gene which confer resistance to diphtheria toxin and Pseudomonas exotoxin A. Genetic and biochemical analyses. AB - Both diphtheria toxin and Pseudomonas exotoxin A inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a posttranslationally modified histidine residue present in the elongation factor 2 (EF-2) protein. Elongation factor 2 cannot be ADP-ribosylated by the toxins unless this histidine is modified. In this report we identify three new point mutations in toxin-resistant alleles of the Chinese hamster ovary cell elongation factor 2 gene. The mutations resulted in amino acid substitutions at positions 584 (serine to glycine), 714 (isoleucine to asparagine), and 719 (glycine to aspartic acid). All three amino acid substitutions prevented the biosynthesis of diphthamide. The amount by which the toxins reduced protein synthesis in each of these mutant cell strains suggested that all three mutations also either impaired the function of EF-2 or reduced its steady state level in the cytoplasm. Western blot analysis showed that equal amounts of EF-2 were present in each of the cell strains, indicating that the mutations impaired the catalytic function of EF-2. PMID- 7559473 TI - A novel matrix attachment region DNA binding motif identified using a random phage peptide library. AB - SATB1 is a nuclear matrix attachment DNA (MAR)-binding protein which is predominantly expressed in thymocytes. This protein binds to the minor groove specifically recognizing an unusual DNA context exhibited by a specific MAR region with strong base-unpairing propensity. A phage library displaying nonamer random peptides without any built-in structure was used to identify a MAR binding motif of SATB1. One predominant cyclic peptide C1 of CRQNWGLEGC selected by a MAR affinity column showed 50% identity with a segment in SATB1 (amino acids 355 363). Replacement of the C1 similarity segment in SATB1 by a random amino acid sequence or its truncation resulted in more than 80% reduction in MAR binding. In contrast, replacement of the same SATB1 segment with the C1 peptide restored full MAR binding activity and specificity as the wild-type protein. Single amino acid mutation of the conserved Arg or Glu residue to Ala greatly reduced MAR binding. Taken together our data show that a nine amino acid sequence in SATB1 represents a key MAR binding motif. Phage display may provide a general tool for rapid identification of DNA binding peptide motifs. PMID- 7559474 TI - Regulated formation of Golgi secretory vesicles containing Alzheimer beta-amyloid precursor protein. AB - Phorbol esters, activators of protein kinase C (PKC), regulate the relative utilization of alternative processing pathways for the Alzheimer beta-amyloid precursor protein (beta-APP) in intact cells, increasing the production of nonamyloidogenic soluble beta-APP (s beta-APP) and decreasing that of neurotoxic beta-amyloid (A beta) peptide. The molecular and cellular bases of PKC-regulated beta-APP cleavage are poorly understood. Here we demonstrate in a reconstituted cell-free system that activation of endogenous PKC increases formation from the trans-Golgi network of secretory vesicles containing beta-APP and that this effect can be mimicked by purified PKC. The results demonstrate directly that PKC is involved in regulation of secretory vesicle formation and provide a mechanism by which PKC may reduce the formation of the A beta peptide characteristic of Alzheimer disease. PMID- 7559475 TI - Determination of the consensus binding site for MEF2 expressed in muscle and brain reveals tissue-specific sequence constraints. AB - The myocyte-specific enhancer factor-2 (MEF2) proteins are expressed in the three major types of muscle (skeletal, cardiac, and smooth) and function as transcriptional activators of muscle-specific and growth factor-regulated genes through binding to a canonical A/T-rich cis-element. Although MEF2 proteins are also expressed in brain, MEF2-regulated muscle-specific gene products are not detected in this tissue. To gain insight into the regulation of MEF2 function in vivo, we have selected its optimal DNA targets from a library of degenerate oligonucleotides using anti-MEF2A antibodies and cell extracts from skeletal muscle, heart, and brain. The consensus binding site in these three tissues contains an indistinguishable core motif, 5'-CT(A/t)(a/t)AAATAG-3'. However, the optimal target for MEF2 expressed in the brain shows additional sequence constraints (5'-TGTTACT(A/t)(a/t)AAATAGA(A/t)-3') that are not observed in the sequences selected with skeletal and cardiac muscle extracts. Thus, differences in DNA binding preferences of MEF2 proteins in muscle and brain may contribute to tissue-specific gene expression during myogenesis and neurogenesis. PMID- 7559476 TI - Electrophoretic and spectral characterization of wild type and mutant adenovirus protease. AB - The P137L mutation in the adenovirus type 2 protease results in a temperature sensitive protein-trafficking phenotype expressed during infection but not in vitro. Homology-derived secondary structure prediction placed the mutation within an externally disposed loop. Circular dichroism and urea gradient gel electrophoresis suggested that, unlike other thiol proteases, the Ad2 protease is comprised of a single conformational domain. The -0.32-kcal difference in the free energy of folding and the temperature-independent CD spectra of the mutant and wild type enzymes point to a very subtle structural change as the cause of the in vivo phenotype. PMID- 7559477 TI - STAT3 and STAT5B are targets of two different signal pathways activated by hematopoietin receptors and control transcription via separate cytokine response elements. AB - Transient transfection of expression vectors for various members of the hematopoietin receptor family and STAT proteins into COS-1 cells indicated that each receptor was capable of stimulating the DNA binding activity of STAT1, STAT3, and STAT5B. However, gp130 preferentially activated STAT1 and STAT3. Activation of STAT5B differed from that of the other two in that the box 3 sequence motif in the cytoplasmic domain of gp130 was not required. Moreover, STAT5B and STAT3 enhanced gene transcription via separate regulatory elements. This study has identified two potential signal transduction pathways by which hematopoietin receptors, including the interleukin-6 receptor, control transcription of acute phase plasma protein genes in hepatic cells. PMID- 7559478 TI - Distinct modes of interaction of SHC and insulin receptor substrate-1 with the insulin receptor NPEY region via non-SH2 domains. AB - Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr 960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated. PMID- 7559479 TI - Transcriptional repression by human adenovirus E1A N terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis. AB - The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors. PMID- 7559481 TI - Sec-dependent thylakoid protein translocation. Delta pH requirement is dictated by passenger protein and ATP concentration. AB - A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal delta pH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the delta pH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually delta pH-independent whereas others are absolutely dependent. delta pH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between delta pH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 microM. The results indicate that the roles of the delta pH and ATP overlap and suggest that the delta pH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved. PMID- 7559480 TI - Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants. AB - The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake. PMID- 7559483 TI - Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis. AB - Cellular responses initiated by tumor necrosis factor (TNF) are mediated by two different cell surface receptors with respective molecular masses of 55 kDa (p55) and 75 kDa (p75). p55 is functional in almost every cell type and can independently transmit most biological activities of TNF. In contrast, TNF signaling via p75 seems so far largely restricted to cells of lymphoid origin, where it can induce proliferation, cytokine production, and/or apoptosis. The mechanisms that regulate TNF receptor activity are largely unknown. Here we report that the p75 of unstimulated p75-responsive PC60 T cells is phosphorylated on serine by a kinase activity present in p75 immune complexes. Several lines of evidence indicate that the latter kinase is casein kinase-1 (CK-1). Previous results have shown that the p75 TNF receptor is constitutively phosphorylated in vivo. Our data show that the latter in vivo phosphorylation is also at least partially due to CK-1. Pretreatment of cells with TNF had no detectable effect on p75 phosphorylation in vitro or in vivo. However, a specific CK-1 inhibitor potentiated TNF-induced apoptosis mediated by p75, suggesting an inhibitory role for phosphorylation by CK-1. Although in vivo p75 phosphorylation could be seen in both p75-unresponsive and p75-responsive cell lines, in vitro p75 phosphorylation in p75 coimmunoprecipitates could not be observed in cell lines that were biologically unresponsive to p75 stimulation. The latter observation further indicates a regulatory role for p75 phosphorylation in p75-mediated signaling. Taken together, our data demonstrate that the p75 TNF receptor is phosphorylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling. PMID- 7559482 TI - High activity suppression of myeloid progenitor proliferation by chimeric mutants of interleukin 8 and platelet factor 4. AB - The proliferation of human myeloid progenitor cells is negatively regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting of domains of either PF4 and IL-8, Gro-alpha and PF4, or Gro-beta and PF4 were observed to inhibit myeloid cell proliferation at concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal "ELR" motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 6. Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines. PMID- 7559484 TI - Alanine-scanning mutagenesis of the epsilon subunit of the F1-F0 ATP synthase from Escherichia coli reveals two classes of mutants. AB - Alanine-scanning mutagenesis was applied to the epsilon subunit of the F1-F0 ATP synthase from E. coli. Nineteen amino acid residues were changed to alanine, either singly or in pairs, between residues 10 and 93. All mutants, when expressed in the epsilon deletion strain XH1, were able to grow on succinate minimal medium. Membranes were prepared from all mutants and assayed for ATP driven proton translocation, ATP hydrolysis +/- lauryldiethylamine oxide, and sensitivity of ATPase activity to N,N'-dicyclohexylcarbodiimide (DCCD). Most of the mutants fell into 2 distinct classes. The first group had inhibited ATPase activity, with near normal levels of membrane-bound F1, but decreased sensitivity to DCCD. The second group had stimulated ATPase activity, with a reduced level of membrane-bound F1, but normal sensitivity to DCCD. Membranes from all mutants were further characterized by immunoblotting using 2 monoclonal antibodies. A model for the secondary structure of epsilon and its role in the function of the ATP synthase has been developed. Some residues are important for the binding of epsilon to F1 and therefore for inhibition. Other residues, from Glu-59 through Glu-70, are important for the release of inhibition by epsilon that is part of the normal enzyme cycle. PMID- 7559485 TI - Differential regulation of sphingomyelinase and ceramidase activities by growth factors and cytokines. Implications for cellular proliferation and differentiation. AB - Sphingosine is a product of sphingolipid metabolism that has been linked to a protein kinase C-independent mitogenic response. In previously published data, utilizing an in vitro model system for platelet-derived growth factor (PDGF) induced vascular smooth muscle proliferation, we have demonstrated that sphingosine is increased at the expense of a concomitant decrease in ceramide formation, implicating an altered ceramidase activity. To explore mechanisms of growth factor-stimulated sphingosine formation, we have developed and investigated a cell-free model system assessing ceramidase activity. We now report that an alkaline, membrane-associated, ceramidase activity in the rat glomerular mesangial cell, a smooth muscle-like pericyte, is up-regulated by growth factors, apparently via a tyrosine kinase phosphorylation mechanism. PDGF also stimulated sphingomyelinase activity which generates sufficient substrate to drive the subsequent ceramidase reaction. Inflammatory cytokines, including interleukin-1, and tumor necrosis factor-alpha, stimulated sphingomyelinase but not ceramidase activity, a result consistent with the cellular accumulation of the ceramide, apoptidic, differentiating second messenger. Mitogenic vasoconstrictor peptides such as endothelin-1 stimulated neither sphingomyelinase nor ceramidase activities. An inhibitor of ceramidase activity, N oleoylethanolamine, reduced PDGF- but not endothelin-1-stimulated proliferation. Thus, we conclude that, in mesangial cells, growth factors but not vasoconstrictor peptides or cytokines induce mitogenesis, in part, through ceramidase-mediated sphingosine formation. PMID- 7559486 TI - Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells. AB - We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP3R) in WB rat liver epithelial cell extracts, when the immunoprecipitation is carried out with a type-I isoform specific antibody (Joseph, S. K. (1994) J. Biol. Chem. 269, 5673-5679). Utilizing isoform-specific antibodies raised to unique sequences within the COOH-terminal region of IP3 receptors, we now report that the co immunoprecipitating 222-kDa polypeptide is the type-III IP3R isoform and that type-III IP3R antibodies (Abs) can co-immunoprecipitate the type-I IP3R isoform. Co-immunoprecipitation of IP3R isoforms was not due to cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react with type-I IP3R and vice versa; (b) inclusion of the COOH terminal type-III peptide had no effect on the ability of type-I IP3R Ab to co immunoprecipitate the type-III IP3R but blocked the ability of type-III IP3R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type-III IP3R, and immunoprecipitation with type III IP3R Ab does not co-immunoprecipitate any other isoforms. However, type-I and type-II IP3R isoforms were co-immunoprecipitated by their respective antibodies in hepatocyte lysates. Sucrose density gradient analysis of WB cell lysates indicated that the co-immunoprecipitating fraction is exclusively located at the density expected for tetrameric receptors, suggesting that co-immunoprecipitation was not a reflection of the nonspecific aggregation of IP3R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP3R could be phosphorylated in vitro. Fractionation of WB cell membranes and immunofluorescence studies showed that the type-I and type-III isoforms have very similar sub-cellular localizations. We conclude that the WB cell contains both type-I and type-III IP3R isoforms and that a proportion of these receptors exist as heterotetramers. PMID- 7559487 TI - Protein kinase C regulates pleckstrin by phosphorylation of sites adjacent to the N-terminal pleckstrin homology domain. AB - Pleckstrin is a substrate for protein kinase C in activated platelets that contains at its N and C termini two of the pleckstrin homology (PH) domains that have been proposed to mediate protein-protein and protein-lipid interactions. We have recently shown that pleckstrin can inhibit agonist-induced phosphoinositide hydrolysis and that this inhibition requires an intact N-terminal PH domain (residues 6 to 99). In the present studies, we have identified the sites of phosphorylation in pleckstrin and examined their contribution to pleckstrin function. In human platelets activated with thrombin or phorbol esters, and in COS-1 cells expressing pleckstrin, a combination of phosphopeptide analysis and site-directed mutagenesis shows that three residues in the intervening sequence between the two pleckstrin PH domains become phosphorylated: Ser113, Thr114, and Ser117. Replacing all three of these sites with glycine decreased phosphorylation by > 90% and reduced pleckstrin's ability to inhibit phosphoinositide hydrolysis by as much as 80%. Replacing the phosphorylation sites with alanine residues had a similar effect, while substitution with aspartate, glutamate, or lysine residues produced pleckstrin variants that were fully active even in the absence of phosphorylation. These results suggest that phosphorylation enhances pleckstrin's activity by introducing a cluster of charges into a region adjacent to, but not within, the N-terminal PH domain. This may have an allosteric effect on the N-terminal PH domain, regulating its interaction with other molecules necessary for the inhibition of phosphoinositide hydrolysis. PMID- 7559488 TI - Yeast two-hybrid system demonstrates that estrogen receptor dimerization is ligand-dependent in vivo. AB - Previous studies using in vitro procedures have not clearly established whether the estrogen receptor (ER) acts as a monomer or dimer in the cell. We have used the yeast two-hybrid system as an in vivo approach to investigate the dimerization of the estrogen receptor in the absence and presence of estrogen and anti-estrogens. This system is independent of ER binding to the estrogen response element. Two vectors, expressing GAL4 DNA binding domain-human ER and GAL4 transactivation domain-human ER, were constructed. Control experiments showed that each fusion protein had a high affinity binding site for estradiol-17 beta and could transactivate an ERE-LacZ reporter gene in yeast similar to the wild type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were expressed in the yeast strain, PCY2, which carries a GAL1 promoter-lacZ reporter. ER dimerization was measured via reconstitution of GAL4 through interaction of the fusion proteins, which transactivates LacZ through the GAL1 promoter. When both ER fusion proteins were expressed, beta-galactosidase activity was estradiol-17 beta-inducible. Furthermore, we showed that both tamoxifen and ICI 182,780 also induced beta-galactosidase activity, albeit lower than that induced by estradiol 17 beta. These results strongly argue that ER dimerization is ligand-dependent and the dimer can be induced by estradiol-17 beta, tamoxifen, or ICI 182,780. We also treated the yeast containing the two fusion proteins with estradiol-17 beta and tamoxifen or ICI 182,780 simultaneously to determine the effects on ER dimerization. beta-Galactosidase activity was lower when the yeast was treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen than estradiol-17 beta alone. Taken together, we conclude that ER dimerization is ligand (estradiol 17 beta, tamoxifen, or ICI 182, 780)-dependent, and we suggest that estradiol-17 beta-induced dimers are destabilized when estradiol-17 beta is used with tamoxifen or ICI 182,780 simultaneously. PMID- 7559490 TI - Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells. AB - A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr766, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by "far-Western" analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway. PMID- 7559489 TI - Transcription termination at the thr attenuator. Evidence that the adenine residues upstream of the stem and loop structure are not required for termination. AB - The Escherichia coli thr operon attenuator has a structure similar to other Rho independent terminators. The DNA sequence immediately 5' to the termination site is dG+dC-rich and contains a region of dyad symmetry that, when transcribed into RNA, encodes a hairpin structure in the transcript. It also contains a stretch of 9 consecutive dA-dT residues immediately distal to the region of dyad symmetry which encode uridine residues at the 3' end of the terminated transcript. In addition, the thr attenuator has a stretch of 6 dA-dT residues immediately upstream of the region of dyad symmetry which encode 6 adenines. These adenines could potentially pair with the distal uridines to form a hairpin structure extended by as much as 6 A-U base pairs. In this report we have examined the role of the upstream adenines in transcription termination. We used templates that specify mismatches or create new base pairs in the potential A-U secondary structure of the transcript as well as templates that delete segments of the A residues upstream of the hairpin. We conclude that A-U pairing is not required for efficient transcription termination at the thr attenuator. This conclusion is likely to apply to other Rho-independent terminators that contain hairpin proximal dA-dT residues. PMID- 7559491 TI - Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures. AB - A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins. PMID- 7559492 TI - Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Willebrand factor to platelets. AB - We have studied the mechanism of interaction between soluble von Willebrand factor (vWF), labeled with fluorescein isothiocyanate (FITC), and platelets exposed to shear in a cone-and-plate viscometer. A flow cytometer calibrated with fluorescent bead standards was used to calculate the number of molecules associated with each platelet in suspension. To validate the methods and reagents used, binding of the same labeled vWF was assessed in the presence of ristocetin or alpha-thrombin and found to be saturable, with a narrow and symmetric distribution on > 90% of the platelets. As expected, essentially all bound ligand interacted exclusively with platelet membrane glycoprotein (GP) Ib alpha in the presence of ristocetin and with GP IIb-IIIa after stimulation with alpha thrombin. In contrast, only a minor proportion (< 20%) of the platelets exposed to shear were found to bind vWF, with no evidence for saturation and markedly decreased interaction when the platelet count was below 100,000 microliters. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ib alpha or GP IIb-IIIa or against the respective binding sites in vWF. Thus, both receptors are involved in the process, possibly through initial transient interactions mediated by GP Ib alpha that lead to platelet activation and subsequent irreversible binding supported by GP IIb-IIIa. While the levels of shear stress theoretically applied to platelets in these experiments are above those thought to occur in the normal circulation, our findings demonstrate a unique vWF binding mechanism that is not mimicked by other known modulators and correlates with platelet aggregation. Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus formation. PMID- 7559494 TI - The structure of a 19-residue fragment from the C-loop of the fourth epidermal growth factor-like domain of thrombomodulin. AB - The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin, a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic interactions, which in turn stabilize the two beta turns in the structure. The first beta-turn in the C-loop represents a conserved motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of Phe376 in the stabilization of the first beta-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for further folding. PMID- 7559493 TI - Temporal activation of nontransmembrane protein-tyrosine kinases following mast cell Fc epsilon RI engagement. AB - One of the primary responses observed following antigen-induced cross-linking in mast cells is an increase in the phosphorylation of certain cellular proteins on tyrosine residues. Stimulation of protein-tyrosine kinase activity appears to be necessary for induction of downstream responses such as degranulation. The role of nonreceptor protein-tyrosine kinases in the signal transduction pathway initiated by Fc epsilon RI engagement in an interleukin-3-dependent mast cell line has been examined. The results presented here show that the enzymatic activity of Lyn is increased within seconds of receptor engagement. Syk activity also undergoes a rapid and transient increase, reaching a peak at approximately 30 s. Similarly, the activity of Fer, representing a third class of nontransmembrane protein-tyrosine kinase increases as well, with its activity peak reached at 1 min poststimulation. The enzymatic activities of Syk and Fer were found to correspond to anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine residues of the beta and gamma chains of Fc epsilon RI increased concomitant with increased protein-tyrosine kinase activity. These results indicate that at least three classes of nontransmembrane protein-tyrosine kinases are involved in mast cell FceRI signaling and that the activation of these classes of enzymes is temporally regulated. PMID- 7559495 TI - Identification of residues that stabilize the single-chain Fv of monoclonal antibodies B3. AB - B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv portion of the B3 antibody in a single-chain form, which serves as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety. B3(Fv)-PE38 is specifically cytotoxic to many human cancer cell lines and is currently evaluated in a clinical trial. Monoclonal antibodies B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were previously cloned and expressed as the single-chain Fv-immunotoxins B3(Fv)-PE38 and B5(Fv)-PE38, respectively. The B3(Fv)-PE38 immunotoxin binds to antigen-positive cancer cells with a higher affinity than B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38. However, it is less stable and rapidly aggregates upon incubation at 37 degrees C. The VL domains of the two Fvs are very similar, differing by only three residues, the fourth and seventh Fr1 residues and the fifth CDR1 residue. The VH domains of the two Fvs vary considerably. To investigate whether any of the different VL residues may influence the stability of the B3(Fv), we constructed a chimeric immunotoxin containing the B3VH and the B5VL. This chimera had an improved stability and a higher apparent antigen binding affinity and cytotoxic activity when compared with B3(Fv)-PE38. Site-specific mutagenesis was used to show that the VL M4L mutation has an important role in stabilizing B3(Fv), although residues VL Ser-7 and VL Ile-28 also play a role in the increased stability. When tested in an in vivo model system, the chimera containing the B3VH and the B5VL had an improved antitumor activity in a human xenograft mouse model. These studies indicate that the common use of degenerate ("family specific") primers to clone Fv fragments may introduce destabilizing mutations. PMID- 7559496 TI - The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. AB - The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways. PMID- 7559498 TI - Tethered ligand library for discovery of peptide agonists. AB - We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptor's amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a "tethered ligand library," which displayed the thrombin receptor's amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors. PMID- 7559497 TI - The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa cell transcription extract that comigrate with transcription factors IIE and IIF. AB - The C-terminal domain (CTD) of RNA polymerase II (RNAP II) is essential for the assembly of RNAP II into preinitiation complexes on some promoters such as the dihydrofolate reductase (DHFR) promoter. In addition, during the transition from a preinitiation complex to a stable elongation complex, the CTD becomes heavily phosphorylated. In this report, interactions involving the CTD have been examined by protein-protein cross-linking. As a prelude to the study of CTD interactions, the effect of recombinant CTD on in vitro transcription was examined. The presence of recombinant CTD inhibits in vitro transcription from both the DHFR and adenovirus 2 major late promoters, suggesting that the CTD is involved in essential interactions with a general transcription factor(s). Factors in the transcription extract that interact with the CTD were identified by protein protein cross-linking. Recombinant CTD was phosphorylated at its casein kinase II site, at the C terminus of the CTD, in the presence of [35S]adenosine 5'-O (thiotriphosphate) and alkylated with azidophenacyl bromide. Incubation of azido modified 35S-labeled CTD with a HeLa transcription extract followed by ultraviolet irradiation results in the covalent cross-linking of the CTD to proteins in contact with the CTD at the time of irradiation. Subsequent incubation with phenylmercuric acetate results in the transfer of 35S from the CTD to the protein to which it was cross-linked. The two major photolabeled bands have a M(r) of 34,000 and 74,000. The specificity of CTD interactions was demonstrated by a reduction in photolabeling in the presence of unmodified CTD or RNAP II containing an intact CTD (RNAP IIA) but not in the presence of a CTD-less RNAP II (RNAP IIB). The 35S-labeled 34- and 74-kDa proteins comigrate on SDS polyacrylamide gel electrophoresis with the beta subunit of transcription factor IIE and the 74-kDa subunit of transcription factor IIF, respectively. Moreover, some of the minor 35S-labeled bands comigrate with other subunits of the general transcription factors. PMID- 7559500 TI - A protein dissociation step limits turnover in FLP recombinase-mediated site specific recombination. AB - When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional dissassembly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro. PMID- 7559501 TI - Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification. AB - The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates. PMID- 7559502 TI - Divergent mechanisms for homologous desensitization of p21ras by insulin and growth factors. AB - Previous work suggested that desensitization of p21ras in response to growth factors such as epidermal growth factor (EGF) results from receptor down regulation. Here we show that p21ras is desensitized by insulin in 3T3-L1 adipocytes in the continued presence of activated insulin receptors, while loss of epidermal growth factor and platelet-derived growth factor (PDGF) receptors in response to their ligands correlates with p21ras desensitization. Furthermore, elevated amounts of Grb2/Shc complexes persisted throughout p21ras desensitization by insulin. However, immunoblotting of anti-Son-of-sevenless (Sos) 1 and 2 immunoprecipitates with anti-Grb2 antisera revealed that p21ras desensitization in response to insulin and PDGF, but not EGF, is associated with a marked decrease in cellular complexes containing Sos and Grb2 proteins. Nonetheless, the desensitization of p21ras in response to these stimuli was homologous, in that each peptide could reactivate [32P]GTP loading of p21ras after desensitization by any of the others. Taken together, these data indicate that insulin, EGF, and PDGF all cause disassembly of Sos proteins from signaling complexes during p21ras desensitization, but at least two mechanisms are involved. Insulin elicits dissociation of Sos from Grb2 SH3 domains, whereas EGF signaling is reversed by receptor down-regulation and Shc dephosphorylation, releasing Grb2 SH2 domains. PDGF action triggers both mechanisms of Grb2 disassembly, which probably operate in concert with GAP to attenuate p21ras signaling. PMID- 7559499 TI - Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR associated PI 3-kinase activity. AB - We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr503 within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3 kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR. PMID- 7559504 TI - The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin. AB - Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1 antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes. PMID- 7559505 TI - Variants of tissue-type plasminogen activator with substantially enhanced response and selectivity toward fibrin co-factors. AB - Unlike most proteases, tissue-type plasminogen activator (t-PA) is not synthesized as an inactive precursor or zymogen. Instead, the single-chain "proenzyme" form of t-PA possesses very significant catalytic activity. Recent investigations of the molecular basis of the unusually high enzymatic activity of single-chain t-PA have focused attention upon Asp-194, a residue that is invariant among chymotrypsin-like enzymes. The critical role of this residue in securing the active conformation of mature chymotrypsin-like enzymes has been discussed extensively. Subsequent work, however, has indicated that this conserved residue can also form interactions that dramatically influence the catalytic activity of serine protease zymogens. While Asp-194 forms interactions that suppress the activity of the zymogen chymotrypsinogen, it may, by contrast, directly promote the catalytically active conformation of single-chain t-PA. To test the hypothesis that Asp-194 promotes the activity of both single- and two chain t-PA and therefore plays opposing roles in single-chain t-PA and chymotrypsinogen, and also to examine whether this invariant residue plays an essential role in the stimulation of t-PA by fibrin, we used site-directed mutagenesis to construct the following variants of t-PA: t-PA/D194E, t-PA/D194N, t-PA/R15E,D194E, and t-PA/R15E,D194N. In the absence of fibrin, the activity of enzymes carrying a mutation at position 194 was reduced by factors of 1000-2000 compared to wild type t-PA. Similar reductions of activity were observed for both single- and two-chain variants, suggesting an important role for Asp-194 in both forms of the enzyme. The mutated enzymes, however, displayed a dramatically enhanced response to fibrin monomers. While the activity of wild type t-PA was stimulated by fibrin monomers by a factor of 960, the corresponding stimulation factor for the mutated enzymes varied from 498,000-1,050,000. PMID- 7559506 TI - The glutamine hydrolysis function of human GMP synthetase. Identification of an essential active site cysteine. AB - GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The "glutamine amide transfer" or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated. Mass spectrometry and Edman sequence analysis show that Cys104 is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys104 is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP. PMID- 7559503 TI - Characterization of novel costimulatory molecules. A protein of 38-42 kDa from B cell surface is concerned with T cell activation and differentiation. AB - Optimal activation of T cells often requires signals delivered by the ligation of T cell receptor (TcR) and those resulting from costimulatory interaction between certain T cell surface accessory molecules and their respective counter receptors on antigen presenting cells. The molecular events underlying the co-stimulatory activity are still not understood fully. Here we describe a 38-42-kDa (B3) protein, present on the surface of lipopolysaccharide-activated B cells, which can provide co-stimulation to resting T cells leading to a predominant release of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2 and interferon-gamma. Binding assay and electron microscopic autoradiography data suggest that this molecule binds T cells, and the same can be competed by unlabeled B3. Characterization experiments point out that B3 shows up as a single prominent peak on reverse phase-high performance liquid chromatography, runs as a single spot in reducing two-dimensional gel electrophoresis, and is a phosphoglycoprotein. The Western analysis indicate that it does not cross-react with antibodies directed against murine ICAM-1, LFA-1 alpha, VCAM-1, HSA, and B7 suggesting the novelty of the protein. The internal amino acid sequence of this molecule suggests that it does not belong to a known category of murine B cell surface molecules. PMID- 7559507 TI - Evidence for a differential interaction of SHC and the insulin receptor substrate 1 (IRS-1) with the insulin-like growth factor-I (IGF-I) receptor in the yeast two hybrid system. AB - Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr 1131, -1135, and -1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR. PMID- 7559508 TI - 1,4-Dihydropyridine binding sites in moss plasma membranes. Properties of receptors for a calcium channel antagonist. AB - An increase in cytoplasmic calcium is an early event in hormone (cytokinin) induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. To understand the molecular nature of the dihydropyridine-sensitive calcium channel, we have established conditions for the binding of the arylazide 1,4-dihydropyridine, [3H]azidopine, to its receptor in moss plasma membranes. [3H]Azidopine bound specifically in a saturable and reversible manner. The KD for [3H]azidopine binding was 5.2 nM and the Bmax was 35.6 pmol/mg of protein. Association and dissociation of the receptor and [3H]azidopine were temperature-dependent, and association varied as a function of pH. Binding was inhibited by dihydropyridine, phenylalkylamine, and benzothiazepine calcium channel blockers, bepridil, lanthanum, and N-ethylmaleimide. [3H]Azidopine binding was stimulated by cations including calcium, strontium, manganese, and barium. [3H]Azidopine binding was also stimulated by cytokinin with a Km value for kinetin of 0.13 nM. These studies utilize a simple plant system to provide a biochemical framework for understanding calcium regulation during development and have implications for understanding mechanisms of signal transduction in plants. PMID- 7559509 TI - Protease nexin-2/amyloid beta-protein precursor inhibits factor Xa in the prothrombinase complex. AB - Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of coagulation factors XIa and IXa. Here we show that PN-2/A beta PP and its KPI domain also inhibited isolated factor Xa with a Ki of 10(-8) M. On a solid phase binding assay, PN-2/A beta PP formed a complex with factor Xa. Incubation of molar excess factor Xa to PN-2/A beta PP produced a single cleavage within PN-2/A beta PP's heparin binding domain liberating a 8.2-kDa amino-terminal peptide. PN 2/A beta PP and its KPI domain equally inhibited factor Xa in the prothrombinase complex with a Ki of 1.9 x 10(-8) M and 1.3 x 10(-8) M, respectively. A beta PP695 which does not contain the KPI domain was a substrate of factor Xa but did not inhibit it, indicating the PN-2/A beta PP inhibition of factor Xa was not substrate inhibition. All of the factor Xa inhibition in the prothrombinase complex by PN-2/A beta PP and its KPI domain on the chromogenic assay was accounted for by inhibition of release of prothrombin fragment F1+2 as determined on immunochemical assay. In the prothrombinase complex, PN-2/A beta PP inhibited factor Xa with a kassoc = 1.8 +/- 0.7 x 10(6) M-1 min-1 similar to antithrombin III and heparin inhibition (kassoc of 3.0 +/- 0.2 x 10(6) M-1 min-1). These studies indicated that PN-2/A beta PP in the assembled prothrombinase complex inhibited factor Xa comparable to antithrombin III in the presence of heparin. PN 2/A beta PP's factor Xa inhibitory activity along with its known inhibition of factors XIa and IXa suggest that this protease inhibitor and related proteins could be regulators of hemostatic reactions on membranes of cells in the intravascular compartment. PMID- 7559510 TI - Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate. AB - Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465 13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18 kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate. PMID- 7559511 TI - FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli. AB - The FtsH (HflB) protein of Escherichia coli is integrated into the membrane with two N-terminally located transmembrane segments, while its large cytoplasmic domain is homologous to the AAA family of ATPases. The previous studies on dominant negative ftsH mutants raised a possibility that FtsH functions in multimeric states. We found that FtsH was eluted at fractions corresponding to a larger molecular weight than expected from monomeric structure in size-exclusion chromatography. Moreover, treatment of membranes or their detergent extracts with a cross-linker, dithiobis(succinimidyl propionate), yielded cross-linked products of FtsH. To dissect possible FtsH complex, we constructed an FtsH derivative with c-Myc epitope at its C terminus (FtsH-His6-Myc). When membranes prepared from cells in which FtsH-His6-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His6-Myc without the tag were cross-linked with the tagged FtsH protein. Co immunoprecipitation experiments confirmed the interaction between the FtsH molecules. To identify regions of FtsH required or sufficient for this interaction, we constructed chimeric proteins between FtsH and EnvZ, a protein with a similar topological arrangement, by exchanging their corresponding domains. We found that only the FtsH-EnvZ hybrid protein with an FtsH-derived membrane anchoring domain and an EnvZ-derived cytoplasmic domain caused a dominant ftsH phenotype and was cross-linked with FtsH. We suggest that the N terminal transmembrane region of FtsH mediates directly the interaction between the FtsH subunits. PMID- 7559512 TI - Transcriptional regulation of the human GLUT4 gene promoter in diabetic transgenic mice. AB - We previously reported that 2400 base pairs (bp) of 5'-flanking DNA is sufficient for tissue-specific and hormonal/metabolic regulation of the human GLUT4 gene in transgenic mice (Liu, M.-L., Olson, A. L., Moye-Rowley, W. S., Buse, J. B., Bell, G. I., and Pessin, J. E. (1992) J. Biol. Chem. 267, 11673-11676). To further define the DNA sequences required for GLUT4 expression, we generated transgenic mice carrying 1975, 1639, 1154, 730, and 412 bp of the GLUT4 5'-flank (hG4) fused to the chloramphenicol acetyltransferase (CAT) reporter gene. The 1975-hG4-CAT, 1639-hG4-CAT, and 1154-hG4-CAT constructs were expressed in a tissue-specific manner identical to the endogenous murine GLUT4 mRNA. Regulation of these reporter gene constructs in insulin-deficient diabetes also paralleled the endogenous gene. In contrast, 730-hG4-CAT was expressed at high levels only in skeletal muscle and at low levels in all of the other tissues examined. Additionally, expression of 412-hG4-CAT was completely unrestricted. Neither the 730-hG4-CAT nor the 412-hG4-CAT reporter genes displayed any insulin-dependent regulation. These data demonstrate that a skeletal muscle-specific DNA element is located within 730 bp of the GLUT4 5'-flanking DNA but that 1154 bp is necessary to direct the full extent of tissue-specific and insulin-dependent regulation of the human GLUT4 gene in transgenic mice. PMID- 7559513 TI - The assembly of laminin-5 subunits. AB - Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC 25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24 h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion. PMID- 7559514 TI - Degradation of plastocyanin in copper-deficient Chlamydomonas reinhardtii. Evidence for a protease-susceptible conformation of the apoprotein and regulated proteolysis. AB - In the green alga Chlamydomonas reinhardtii, the copper-dependent accumulation of plastocyanin is effected via the altered stability of the protein in copper deficient versus copper-sufficient medium (t1/2) < 20 min versus several hours). To understand the mechanism of plastocyanin degradation in vivo, the purified apoprotein was characterized relative to the holoprotein with respect to conformation and protease susceptibility. Circular dichroism spectroscopy revealed that the apoprotein in solution did not display the characteristic secondary structure displayed by the native or reconstituted holoprotein. The apoprotein was also susceptible to digestion in vitro by chymotrypsin whereas the holoprotein was resistant. High ionic conditions, which stabilize the folded structure of apoplastocyanin, also inhibit its degradation by chymotrypsin. These results suggest that one explanation for plastocyanin degradation in copper deficient cells in vivo might be the increased susceptibility of the apo form to a lumenal protease. Since apoplastocyanin is a normal biosynthetic intermediate for the formation of holoplastocyanin, the increased susceptibility of apoplastocyanin to proteolysis implies that degradative and biosynthetic activities would compete for the same substrate. However, characterization of an apoplastocyanin-accumulating mutant suggests that a plastocyanin-degrading protease is active only in copper-deficient cells. Thus, apoplastocyanin is rapidly degraded in copper-deficient cells, whereas its major fate in copper supplemented cells is holoplastocyanin formation. PMID- 7559515 TI - Transcription factor repression and activation of the human acetylcholinesterase gene. AB - Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2 containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription. PMID- 7559516 TI - Chemical structure and translation inhibition studies of the antibiotic microcin C7. AB - Escherichia coli microcin C7 (MccC7) is an antibiotic that inhibits protein synthesis in vivo. It is a heptapeptide containing unknown modifications at the N and C termini (Garcia-Bustos, J. F., Pezzi, N., and Mendez, E. (1985) Antimicrob. Agents Chemoth. 27, 791-797). The chemical structure of MccC7 has been characterized by use of 1H homonuclear and heteronuclear (13C, 15N, 31P) nuclear magnetic resonance spectroscopy as well as mass spectrometry (1177 +/- 1 Da). The heptapeptide Met-Arg-Thr-Gly-Asn-Ala-Asp is substituted at the N terminus by a N formyl group. The C-terminal substituent consists of the phosphodiester of 5' adenylic acid and n-aminopropanol (AMPap), which is linked via the phosphorus atom to an amide group, thus forming a phosphoramide. The main chain carbonyl of the C-terminal aspartic acid residue is connected via this amide bond to the modified nucleotide unit. MccC7 and the peptide unit inhibit protein translation in vitro while a synthetic analog of the AMPap substituent is not active. Neither the peptide nor the AMPap molecule has an effect on the growth of MccC7-sensible cells. Our results strongly suggest that the peptide is responsible for MccC7 antibiotic activity while the C-terminal substituent is needed for MccC7 transport. Implications of the structure determined in this work for MccC7 synthesis and mode of action are discussed. PMID- 7559518 TI - L-tryptophan 2',3'-oxidase from Chromobacterium violaceum. Substrate specificity and mechanistic implications. AB - L-Tryptophan 2',3'-oxidase, an amino acid alpha,beta-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C alpha and C beta carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an alpha-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic beta-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of alpha,beta-dehydrotryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct alpha,beta-dehydrogenation of the tryptophan side chain is proposed. PMID- 7559517 TI - Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties. AB - Biosynthesis of lantibiotics such as nisin and subtilin involves post translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (SL), fused to nisin subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide. PMID- 7559519 TI - Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor. AB - An abundant 95-kDa protein belonging to the low density lipoprotein receptor supergene family is essential for chicken oocyte growth by mediating the uptake of multiple plasma-borne yolk precursors. This receptor harbors at the amino terminus a cluster of eight tandemly arranged repeats typical of the ligand binding domains of members of this family and is designated low density lipoprotein receptor relative with 8 repeats (LR8). Here, we demonstrate by reverse transcriptase-polymerase chain reaction, Northern, and Western blot analyses that the chicken expresses two forms of LR8, which are generated by differential splicing of an exon encoding a serine- and threonine-rich region characteristic of LRs, termed O-linked sugar domain. The female germ cell of the chicken expresses extremely high levels of the short form of LR8 (LR8-), i.e. the 95-kDa protein; in contrast, somatic cells express lower but detectable levels of the form containing the O-linked sugar domain (LR8+). The main sites of LR8+ expression in the chicken are the heart and skeletal muscle, i.e. the same tissues were LR8 mRNAs predominate in mammals; in addition, in situ hybridization demonstrates that a significant amount of LR8+ is produced in the hen's ovarian follicular granulosa cells. We found no apparent functional difference between the two receptor forms; however, cell type-specific targeting of the multiple ligands of these receptors possibly relates to their respective expression on the cell surface. PMID- 7559520 TI - Activation function 1 of retinoic acid receptor beta 2 is an acidic activator resembling VP16. AB - The mechanisms underlying transcriptional activation are not very well understood, and knowledge is based on experiments with a small number of mostly viral activators. We have investigated the mechanism underlying transactivation by the activation domain present in the N-terminal part of retinoic acid receptor (RAR) beta 2 (AF-1). We show that RAR beta 2 phosphorylation is not crucial for its activity although it may modulate AF-1 activity. Sequential mutation of the negatively charged residues (Asp) resulted in a stepwise decrease in activity, while mutation of all aspartic acid residues resulted in complete loss of activity. Comparison of the critical region for activation with other activators revealed moderate homology with the viral activator VP16. The hydrophobic amino acids surrounding the negatively charged residues reported to be critical for activation by VP16 are all conserved in AF-1. The hydrophobic residues are required for AF-1, since mutation of these residues resulted in a decrease in activity. Furthermore, the activity of this activator, VP16 and TA1 of RelA, is squelched by overexpression of an AF-1-containing expression construct, indicating that AF-1 is an acidic activator. Squelching experiments further indicate that AF-1 and AF-2 function by different mechanisms. Comparison of activation functions present in the AB region of other members of the steroid/thyroid hormone receptor family: RAR alpha 2, RAR gamma 2, and GR suggested that also these receptors contain an acidic activation domain. The mechanism underlying activation by AF-1 is discussed. PMID- 7559521 TI - Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate. AB - The CobU protein of Salmonella typhimurium was overexpressed and purified to approximately 94% homogeneity. N-terminal sequencing of purified CobU confirmed the first 22 amino acids. In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamide-phosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme guanylyl intermediate. In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. Treatment of inactive CobU with dithiothreitol restored approximately 20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5'-dithiobis(2-nitrobenzoic acid) and N ethylmaleimide abolished both CobU activities. Native CobU protein was a dimer (approximately 40 kDa) that functioned optimally at pH 8.8-9.0 and 37 degrees C. Substrates and kinetic parameters for both activities were determined. The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen. PMID- 7559522 TI - Circular structures in retroviral and cellular genomes. AB - A computer program for predicting DNA bending from nucleotide sequence was used to identify circular structures in retroviral and cellular genomes. An 830-base pair circular structure was located in a control region near the center of the genome of the human immunodeficiency virus type I (HIV-I). This unusual structure displayed relatively smooth planar bending throughout its length. The structure is conserved in diverse isolates of HIV-I, HIV-II, and simian immunodeficiency viruses, which implies that it is under selective constraints. A search of all sequences in the GenBank data base was carried out in order to identify similar circular structures in cellular DNA. The results revealed that the structures are associated with a wide range of sequences that undergo recombination, including most known examples of DNA inversion and subtelomeric translocation systems. Circular structures were also associated with replication and transposition systems where DNA looping has been implicated in the generation of large protein DNA complexes. Experimental evidence for the structures was provided by studies which demonstrated that two sequences detected as circular by computer preferentially formed covalently closed circles during ligation reactions in vitro when compared to nonbent fragments, bent fragments with noncircular shapes, and total genomic DNA. In addition, a single T-->C substitution in one of these sequences rendered it less planar as seen by computer analysis and significantly reduced its rate of ligase-catalyzed cyclization. These results permit us to speculate that intrinsically circular structures facilitate DNA looping during formation of the large protein-DNA complexes that are involved in site- and region-specific recombination and in other genomic processes. PMID- 7559523 TI - DNA determinants and substrate specificities of Escherichia coli MutY. AB - Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro 8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein. PMID- 7559524 TI - Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. AB - Several different oncogenes and growth factors promote G1 phase progression. Cyclin D1, the regulatory subunit of several cyclin-dependent kinases, is required for, and capable of shortening, the G1 phase of the cell cycle. The present study demonstrates that transforming mutants of p21ras (Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter in human trophoblasts (JEG-3), mink lung epithelial (Mv1.Lu), and in Chinese hamster ovary fibroblast cell lines. Site directed mutagenesis of AP-1-like sequences at -954 abolished p21ras-dependent activation of cyclin D1 expression. The AP-1-like sequences were also required for activation of the cyclin D1 promoter by c-Jun. In electrophoretic mobility shift assays using nuclear extracts from cultured cells and primary tissues, several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bound the cyclin D1 -954 region. Cyclin D1 promoter activity was stimulated by overexpression of mitogen activated protein kinase (p41MAPK) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK (p41MAPKi) or dominant negatives of ETS activation (Ets-LacZ), antagonized MAPK-dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, was antagonized by either p41MAPKi or Ets-LacZ, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21ras provides evidence for cross-talk between the p21ras and cell cycle regulatory pathways. PMID- 7559525 TI - Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta adrenoceptor agonists. AB - Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2. PMID- 7559526 TI - Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside 5'-triphosphates. AB - A set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV 1), which confers on the virus a reduced sensitivity to multiple therapeutic dideoxynucleosides (ddNs), has been identified. In this study, we defined the biochemical properties of RT with such mutations by using site-directed mutagenesis, overproduction of recombinant RTs, and steady-state kinetic analyses. A single mutation, Q151M, which developed first among the five mutations in patients receiving therapy, most profoundly reduced the sensitivity of RT to multiple ddN 5'-triphosphate (ddNTPs). Addition of other mutations to Q151M further reduced the sensitivity of RT to ddNTPs. RT with the five mutations proved to be resistant by 65-fold to 3'-azido-2',3'-dideoxythymidine 5' triphosphate (AZTTP), 12-fold to ddCTP, 8.8-fold to ddATP, and 3.3-fold to 2',3' dideoxyguanosine 5'-triphosphate (ddGTP), compared with wild-type RT (RTwt). Steady-state kinetic studies revealed comparable catalytic efficiency (kcat/Km) of RTs carrying combined mutations as compared with that of RTwt (< 3-fold), although a marked difference was noted in inhibition constants (Ki) (e.g. Ki of a mutant RT carrying the five mutations was 62-fold higher for AZTTP than that of RTwt). Thus, we conclude that the alteration of RT's substrate recognition, caused by these mutations, accounts for the observed multi-ddN resistance of HIV 1. The features of multi-ddNTP-resistant RTs should provide insights into the molecular mechanism of RT discriminating ddNTPs from natural substrates. PMID- 7559527 TI - Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles. AB - The insulin-regulated glucose transporter isotype GlutT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle. PMID- 7559528 TI - Construction and characterization of arginine-specific cysteine proteinase (Arg gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. AB - Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arg-gingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain encoding genes designated rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpA mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arg-gingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis. PMID- 7559530 TI - Single-chain recombinant human cytomegalovirus protease. Activity against its natural protein substrate and fluorogenic peptide substrates. AB - We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a continuous assay for this protease. In order to produce the human cytomegalovirus (HCMV) protease for structural studies and accurate kinetic analysis, mutation of alanine 143 at an internal cleavage site was introduced to prevent auto proteolysis. The resulting soluble 29-kDa A143Q protease was purified to homogeneity as a stable single-chain protein by hydrophobic interaction and ionic exchange chromatography. The in vivo protein substrate, assembly protein precursor, was also expressed and purified for activity studies. To develop a continuous protease assay, fluorescent synthetic peptide substrates similar to the cleavage sequence P5 to P5' of the maturation site containing anthranilic acid and nitrotyrosine as a resonance energy transfer donor-acceptor pair were designed. Purified HCMV A143Q protease cleaved the recombinant assembly protein precursor with Km and kcat values of 3.0 +/- 1.0 microM and 13.3 +/- 1.6 min-1. The Km for peptide substrates is at least 45-fold higher than for the natural protein substrate, but the kcat values are similar. A sensitive assay was developed using fluorescent peptide substrates, which can detect nM HCMV protease activity. PMID- 7559529 TI - GABP and PU.1 compete for binding, yet cooperate to increase CD18 (beta 2 leukocyte integrin) transcription. AB - CD18 (beta 2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABP alpha, and ets factor, and GABP beta, a subunit with homology to Drosophila Notch. GABP alpha competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABP alpha and GABP beta together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription. PMID- 7559531 TI - Sorting and intracellular trafficking of a glycosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin Darby canine kidney epithelial cells. AB - We compared the trafficking of the glycosylphosphatidylinositol (GPI)-anchored placental alkaline phosphatase (PLAP) and two chimeric transmembrane proteins containing the PLAP ectodomain in stably transfected Madin-Darby canine kidney epithelial cells to determine whether different mechanisms might be used in apical sorting of GPI-anchored and transmembrane proteins. PLAP-G, which contained the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein, was delivered directly to the basolateral surface. PLAP-HA contained the transmembrane and cytoplasmic domains of influenza hemagglutinin. Both PLAP and PLAP-HA were delivered directly to the apical membrane. PLAP becomes insoluble in Triton X-100 during biosynthetic transport, as it associates with detergent-resistant membranes. Neither hybrid protein was detergent insoluble, though the small amount of PLAP that was missorted to the basolateral surface was insoluble. We examined the effects of three drugs known to interfere with membrane trafficking on sorting and delivery of PLAP and the hybrid proteins. Monensin had no effect on sorting or surface expression of any of the proteins. Nocodazole affected the sorting of both PLAP and PLAP-HA but not of PLAP-G. Brefeldin A appeared to disrupt the sorting of PLAP and PLAP-HA but not of PLAP-G. This conclusion was tempered by the observation that this drug affected the distribution of proteins at the cell surface. Thus, sorting and transport of GPI-anchored and apical transmembrane proteins are similar in a number of respects. PMID- 7559533 TI - Flavinylation of monoamine oxidase B. AB - Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397. PMID- 7559532 TI - The alpha 6A beta 1 and alpha 6B beta 1 integrin variants signal differences in the tyrosine phosphorylation of paxillin and other proteins. AB - Integrin receptors can mediate transmembrane signaling in response to ligand binding. To further examine the role of the integrin alpha subunit in these signaling functions, we assessed the contribution of the alpha 6 cytoplasmic domain variants to the signaling properties of the alpha 6 beta 1 integrin using P388D1 cells that had been transfected with either the alpha 6A or the alpha 6B cDNA. The alpha 6A beta 1 and alpha 6B beta 1 receptors induced marked quantitative differences in the tyrosine phosphorylation of several proteins after binding to laminin. Specifically, the alpha 6A cytoplasmic domain was more effective than the alpha 6B cytoplasmic domain in inducing the tyrosine phosphorylation of three major proteins (molecular mass, 120, 110, and 76 kDa). In addition to these proteins, we also observed that the tyrosine phosphorylation of the cytoskeletal protein paxillin was increased significantly more by alpha 6A beta 1 integrin-mediated adhesion to laminin than by that of alpha 6B beta 1. This differential pattern of tyrosine phosphorylation induction does not appear to be a secondary event initiated by cell shape changes. Also, differences in tyrosine phosphorylation in the alpha 6 transfectants were not evident in response to attachment to other substrates. These findings provide biochemical evidence for functional differences between alpha subunit cytoplasmic domain variants of the same integrin. PMID- 7559534 TI - Transcriptional regulation of the chicken caldesmon gene. Activation of gizzard type caldesmon promoter requires a CArG box-like motif. AB - Caldesmon, which plays a vital role in the actomyosin system, is distributed in smooth muscle and non-muscle cells, and its isoformal interconversion between a high M(r) form and low M(r) form is a favorable molecular event for studying phenotypic modulation of smooth muscle cells. Genomic analysis reveals two promoters, of which the gizzard-type promoter displays much higher activity than the brain-type promoter. Here, we have characterized transcriptional regulation of the gizzard-type promoter. Transient transfection assays in chick gizzard smooth muscle cells, chick embryo fibroblasts, mouse skeletal muscle cell line (C2C12), and HeLa cells revealed that the promoter activity was high in smooth muscle cells and fibroblasts, but was extremely low in other cells. Cell type specific promoter activity depended on an element, CArG1, containing a unique CArG box-like motif (CCAAAAAAGG) at -315, while multiple E boxes were not directly involved in this event. Gel shift assays showed the specific interaction between the CArG1 and nuclear protein factors in smooth muscle cells and fibroblasts. These results suggest that the CArG1 is an essential cis-element for cell type-specific expression of caldesmon and that the function of CArG1 might be controlled under phenotypic modulation of smooth muscle cells. PMID- 7559535 TI - Ca2+ regulates the interaction between synaptotagmin and syntaxin 1. AB - While there is compelling evidence that the synaptic vesicle protein synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis, it is not known how Ca2+ binding initiates membrane fusion. Here we report that Ca2+ increases the affinity, by approximately 2 orders of magnitude, between synaptotagmin and syntaxin 1, a component of the synaptic fusion apparatus. This effect is specific for divalent cations which can stimulate exocytosis of synaptic vesicles (Ca2+ > Ba2+, Sr2+ >> Mg2+). The Ca(2+)-dependence of the interaction was composed of two components with EC50 values of 0.7 and 180 microM Ca2+. The interaction is mediated by the carboxyl-terminal region of syntaxin 1 (residues 194-288) and is regulated by a novel Ca(2+)-binding site(s) which does not require phospholipids and is not disrupted by mutations that abolish Ca(2+) dependent phospholipid binding to synaptotagmin. We propose that this interaction constitutes an essential step in excitation-secretion coupling. PMID- 7559536 TI - Alternative splicing of a specific cytoplasmic exon alters the binding characteristics of murine platelet/endothelial cell adhesion molecule-1 (PECAM 1). AB - Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Analysis of PECAM-1 expression in the developing mouse embryo has revealed the presence of multiple isoforms of murine PECAM-1 (muPECAM-1) that appeared to result from the alternative splicing of exons encoding cytoplasmic domain sequences (exons 10-16) (Baldwin, H. S., Shen, H. M., Yan, H., DeLisser, H. M., Chung, A., Mickanin, C., Trask, T., Kirschbaum, N. E. Newman, P. J., Albelda, S., and Buck, C. A. (1994) Development 120, 2539-2553). To investigate the functional consequences of alternatively spliced muPECAM-1 cytoplasmic domains, L-cells were transfected with cDNA for each variant and their ability to promote cell aggregation was compared. In this assay, full-length muPECAM-1 and all three isoforms containing exon 14 behaved like human PECAM-1 in that they mediated calcium- and heparin dependent heterophilic aggregation. In contrast, three muPECAM-1 variants, all missing exon 14, mediated calcium- and heparin-independent homophilic aggregation. Exon 14 thus appears to modulate the ligand and adhesive interactions of the extracellular domain of PECAM-1. These findings suggest that alternative splicing may represent a mode of regulating the adhesive function of PECAM-1 in vivo and provides direct evidence that alternative splicing involving the cytoplasmic domain affects the ligand specificity and binding properties of a cell adhesion receptor. PMID- 7559537 TI - Identification of the 14.3.3 zeta domains important for self-association and Raf binding. AB - The 14.3.3 zeta protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A2, which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably 1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 zeta polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 zeta binds primarily to Raf noncatalytic sequences distinct from those that bind Ras GTP, and in vitro 14.3.3 zeta binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 zeta (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 zeta polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 zeta in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 zeta binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 zeta, are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c Raf. Overexpression of 14.3.3 zeta enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated. PMID- 7559538 TI - In vitro processing of human tumor necrosis factor-alpha. AB - Tumor necrosis factor (TNF)-alpha is initially synthesized as a membrane-bound, cell-associated 26-kDa protein that is further cleaved to yield the soluble 17 kDa form. By using a radiolabeled in vitro translated TNF-alpha precursor we detected a serine proteinase processing activity present in crude membrane preparations of monocytic cells able to generate a 17-kDa active protein. A similar processing pattern was obtained using purified neutral serine proteinase proteinase-3 (PR-3). Moreover, while a secretory leukocyte proteinase inhibitor (a natural serine anti-proteinase) did not affect the in vitro TNF-alpha processing, IgG preparations containing high titers of anti-PR-3 autoantibodies completely blocked this activity. The NH2-terminal sequencing of the reaction products obtained with either membrane preparations or PR-3 showed that cleavage occurs in both cases between Val77 and Arg78. These results together with cellular expression and localization of PR-3 suggest a potential role for this enzyme as an accessory TNF-alpha processing enzyme. PMID- 7559539 TI - Photoaffinity labeling of human lysosomal beta-hexosaminidase B. Identification of Glu-355 at the substrate binding site. AB - The carbene precursor 3-azi-1-[([6-3H]-2-acetamido-2-deoxy-1-beta-D galactopyranosyl)thi o -butane (also designated [3H]-1-ATB-GalNAc) has been used as a photoaffinity label for human lysosomal beta-hexosaminidase B (Hex B, EC 3.2.1.52) purified to apparent homogeneity from postmortal liver. [3H]-1-ATB GalNAc behaved as an active site-directed inhibitor, which bound covalently to Hex B upon photolysis at 350 nm and resulted in 15% inactivation of enzyme activity. Up to 75% of the inactivation of Hex B was prevented by including the competitive inhibitor 2-acetamido-2-deoxy-D-glucono-1,5-lactone in the photoaffinity experiment. Incubation of [3H]-1-ATB-GalNAc with the enzyme followed by irradiation and subsequent separation of the three polypeptides composing the beta-subunit led mainly to labeling of the beta a-polypeptide. Subsequent proteolysis of beta a with trypsin and separation of the resulting peptides by high pressure liquid chromatography yielded one prominently labeled peptide fraction. Edman degradation resulted in the sequence E339ISEVFPDQFIHLGGD EVEFK359. However, no modified amino acid was detected, indicating that the photoaffinity label was presumably bound to the peptide by a labile ester linkage. This was proven when the radiolabel was almost completely released from the peptide by treatment with aqueous ammonium hydroxide. Simultaneously, Glu-355 was converted into Gln-355, which is located within a region of Hex B that shows considerable homology with the alpha-subunit of human hexosaminidase A and other hexosaminidases from various species. PMID- 7559540 TI - Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site. AB - The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site. PMID- 7559543 TI - Translocation of ricin A-chain into proteoliposomes reconstituted from Golgi and endoplasmic reticulum. AB - Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment of concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation. PMID- 7559541 TI - Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. AB - We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na+, whereas the intracellular accumulation of ascorbic acid was found to be a Na(+)-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na(+)-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation. PMID- 7559542 TI - Analysis of ligand binding to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that lipoprotein lipase and the carboxyl-terminal domain of the receptor-associated protein bind to the same site. AB - The endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor related protein (alpha 2MR/LRP) binds several classes of extracellular ligands at independent sites. In addition, alpha 2MR/LRP can bind multiple copies of the 39 40-kDa receptor-associated protein (RAP). Both amino-terminal and carboxyl terminal fragments of RAP exhibit affinity, and the fragments apparently bind to different sites on the receptor. RAP completely inhibits the binding of all presently known extracellular ligands, whereas several ligands such as alpha 2 macroglobulin and tissue-type plasminogen activator are poor inhibitors of RAP binding. Since RAP is largely an intracellular molecule that normally does not occupy alpha 2MR/LRP at the cell surface, we hypothesized that an established extracellular ligand might bind to those sites on the receptor capable of binding the RAP fragments. We found complete cross-competition between carboxyl-terminal RAP fragments and fragments of lipoprotein lipase containing the recently identified binding domain for alpha 2MR/LRP (Nykjaer, A., Nielsen, M., Lookene, A., Meyer, N., Roigaard, H., Etzerodt, M., Beisiegel, U., Olivecrona, G., and Gliemann, J. (1994) J. Biol. Chem. 269, 31747-31755). Moreover, the lipoprotein lipase fragment completely inhibited the binding of several alpha 2MR/LRP ligands in a pattern similar to that of carboxyl-terminal RAP fragments. On the other hand, the amino-terminal RAP fragment was a poor competitor of binding of the lipoprotein lipase fragment, whereas it competed effectively with pro-uPA for binding to the receptor. The results provide evidence that lipoprotein lipase binds to the site on alpha2MR/LRP also available for binding of the carboxyl terminal domain of RAP and suggest that pro-uPA may bind to or overlap the site available for the amino-terminal domain of RAP. PMID- 7559544 TI - Molecular cloning and expression of a 26 S protease subunit enriched in dileucine repeats. AB - The 26 S protease is a multisubunit enzyme required for ubiquitin-dependent proteolysis. Recently, we identified a 50-kDa subunit (S5) of this enzyme that binds ubiquitin polymers (Deveraux, Q., Ustrell, V., Pickart, C., and Rechsteiner, M. (1994) J. Biol. Chem. 269, 7059-7061). We have now isolated, sequenced, and expressed a cDNA encoding a novel 50-kDa subunit of the 26 S protease. The recombinant protein does not bind ubiquitin polymers. Two dimensional electrophoresis reveals that two subunits of the 26 S protease have apparent molecular masses of 50 kDa. Antibodies specific for the recombinant protein recognize the more basic of the two subunits (S5b), whereas the more acidic subunit (S5a) binds ubiquitin chains. Thus, the 26 S protease contains at least two distinct subunits with apparent molecular masses of 50 kDa. PMID- 7559545 TI - Thrombin induces the activation of progelatinase A in vascular endothelial cells. Physiologic regulation of angiogenesis. AB - Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor. PMID- 7559546 TI - Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes. AB - Viral infection results in transcriptional activation of the cellular interferon alpha/beta-stimulated genes (ISGs) independent of the autocrine action of interferon alpha/beta (IFN-alpha/beta). Induction of ISG expression by virus appears to be mediated through production of viral double-stranded RNA (dsRNA). Previously, we identified two novel dsRNA-activated factors (DRAFs) that bind to the interferon-stimulated response element (ISRE), the DNA sequence that mediates transcriptional activation by IFN-alpha/beta. In this report we define sequences that flank the classical ISRE to be necessary for DRAF1 binding. More significantly, it is shown that the sequences required to bind DRAF1 correlate with the ability to mediate ISG induction by virus. These results strongly suggest that DRAF1 is a positive regulator of ISG transcription. DRAF1 is shown to bind selectively to the promoters of those ISGs which are strongly induced by viral infection, again suggesting the functional significance of this factor. UV cross-linking experiments indicate that DRAF1 and DRAF2 share a common DNA binding subunit of approximately 70 kDa which is referred to as the DRAF binding component (DRAFB). DRAFB is shown to preexist in the cytoplasm of unstimulated cells. Consistent with this observation, both DRAF1 and DRAF2 are activated in the cytoplasm prior to nuclear translocation. PMID- 7559548 TI - Saturation mutagenesis of human interleukin-3. AB - A deletion variant of human interleukin-3, hIL-3(15-125), was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3(15-125). Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5 26-fold greater than that of native hIL-3. The majority of amino acids in hIL 3(15-125) could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor. PMID- 7559547 TI - Thrombospondin mediates calcium mobilization in fibroblasts via its Arg-Gly-Asp and carboxyl-terminal domains. AB - Thrombospondin is a matrix glycoprotein found in various cells that can modulate cell attachment, migration, and proliferation. We now show that intact soluble thrombospondin causes a transient [Ca2+]i increase in IMR-90 fibroblasts. This [Ca2+]i increase is mediated partly by the RGD-containing domain of thrombospondin that binds to the integrin alpha v beta 3 as demonstrated by inhibitor studies using anti-alpha v beta 3 antibody and RGD-containing peptides. A non-RGD and non-alpha v beta 3 component of this [Ca2+]i increase is mediated by the carboxyl-terminal domain of thrombospondin through an unidentified receptor on fibroblasts as shown by the antibody to the carboxyl-terminal of thrombospondin, C6.7. In addition, the carboxyl-terminal derived peptide, RFYVVMWK, also triggers [Ca2+]i increase in approximately 35% of fibroblasts. Both EGTA and Ni2+ block the entire [Ca2+]i increase indicating that this is due to an influx of extracellular Ca2+. B6H12, an antibody to the integrin-associated protein, blocks this [Ca2+]i increase by 50%, suggesting that some of the Ca2+ might be entering through an integrin-associated calcium channel. The current findings demonstrate that multiple domains on thrombospondin can trigger signal transduction events by increasing [Ca2+]i through their interactions with different cell receptors. PMID- 7559550 TI - The alpha chain of the AP-2 adaptor is a clathrin binding subunit. AB - We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta 2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation renaturation. Translated alpha a exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha a bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approximately 29-kDa alpha a clathrin-binding fragment that remained tightly cage-associated. Translated alpha a also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta 2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H. (1987) Cell Biol. 105, 1989). It also raises the possibility that the alpha clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions. PMID- 7559549 TI - Secretion of biologically active recombinant fibrinogen by yeast. AB - Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal 1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation. PMID- 7559552 TI - Tumor necrosis factor alpha-induced phosphorylation of insulin receptor substrate 1 (IRS-1). Possible mechanism for suppression of insulin-stimulated tyrosine phosphorylation of IRS-1. AB - Tumor necrosis factor-alpha (TNF) has been suggested to be the mediator of insulin resistance in infection, tumor cachexia, and obesity. We have previously shown that TNF diminishes insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). The current work examines potential mechanisms that mediate this event. TNF effect on IRS-1 in Fao hepatoma cells was not associated with a significant reduction in insulin receptor tyrosine kinase activity as measured in vitro but impaired the association of IRS-1 with phosphatidylinositol 3-kinase, localizing TNF impact to IRS-1. TNF did not increase protein-tyrosine phosphatase activity and protein-tyrosine phosphatase inhibition by vanadate did not change TNF effect on IRS-1 tyrosine phosphorylation, suggesting that protein tyrosine phosphatases are not involved in this TNF effect. In contrast, TNF increased IRS-1 phosphorylation on serine residues, leading to a decrease in its electrophoretic mobility. TNF effect on IRS-1 tyrosine phosphorylation was not abolished by inhibiting protein kinase C using staurosporine, while inactivation of Ser/Thr phosphatases by calyculin A and okadaic acid mimicked it. Our data suggest that TNF induces serine phosphorylation of IRS-1 through inhibition of serine phosphatases or activation of serine kinases other than protein kinase C. This increased serine phosphorylation interferes with insulin-induced tyrosine phosphorylation of IRS-1 and impairs insulin action. PMID- 7559551 TI - Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. AB - We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID- 7559554 TI - The function of the NC1 domains in type IV collagen. AB - At its C terminus, the collagen IV molecule bears a globular NC1 domain, to which two functions have been assigned. In the macromolecular network of collagen IV, two molecules are connected via their NC1 domains, which form a hexameric complex, stabilized by intermolecular disulfide bonds. In addition, the NC1 domains are thought to be responsible for chain selection and assembly. In order to understand the role of the NC1 domains during these steps, hexameric complexes were isolated and further investigated. SDS-polyacrylamide gel electrophoresis and Western blot revealed disulfide-linked alpha 1 (IV)NC1 and alpha 2(IV)NC1 homodimers but no heterodimers. The hexamers were dissociated at low pH, separated into monomers and dimers, and submitted to reconstitution experiments. Only alpha 1(IV)NC1 dimers were able to reconstitute a hexameric complex. alpha(IV)-NC1 and alpha 2(IV)NC1 monomers as well as the alpha 2(IV)NC1 dimers showed only a low tendency to form complexes. It is assumed that during formation of the collagen IV network, lateral aggregation of the molecules via the triple helical domains brings the C termini of two molecules into close vicinity and that subsequently the weak interactions observed between the NC1 subdomains provide the correct alignment for a disulfide exchange. It is, however, questionable whether the low affinity between the NC1 subdomains alone is sufficient for chain assembly and alignment of the alpha(IV) chains before molecule formation. PMID- 7559555 TI - ERM, a PEA3 subfamily of Ets transcription factors, can cooperate with c-Jun. AB - A human cDNA clone for ERM, a member of the ets gene family, has been obtained by polymerase chain reaction with degenerate primers corresponding to highly conserved regions within an Ets DNA binding domain. ERM mRNA is expressed ubiquitously. The gene was mapped to chromosome 3q27. In in vivo transient expression assays, ERM induced transcription more efficiently from a synthetic element containing both an ets-binding site (EBS) and a cyclic AMP response element (CRE) than from one containing an EBS alone. The activation of a synthetic EBS-CRE site by ERM was likely to involve a leucine zipper protein capable of dimerizing with CRE-BP1 leucine zipper. Indeed, ERM and c-Jun synergistically activated the EBS-CRE without making an apparent ternary complex. The synergy between c-Jun and ERM may be attributed to the enhancing effect of c Jun on the transcription activity of ERM, because c-Jun increased ERM transcription activity by more than 20-fold in an assay system using a variety of fusion proteins between a Gal4 DNA-binding domain and a portion of ERM. This enhancing effect of c-Jun required the amino-terminal portion of ERM. PMID- 7559553 TI - Repression of the c-myb gene by WT1 protein in T and B cell lines. AB - The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c myb expression in both T and B cell lines. PMID- 7559557 TI - The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro. AB - Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25 mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells. PMID- 7559556 TI - The Saccharomyces cerevisiae RIB4 gene codes for 6,7-dimethyl-8-ribityllumazine synthase involved in riboflavin biosynthesis. Molecular characterization of the gene and purification of the encoded protein. AB - 6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6 ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity. Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein. Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV. Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure. Initial structural characterization of 6,7 dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the beta subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. PMID- 7559558 TI - Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3 kinase rather than receptor-operated Ca2+ influx. AB - Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from non-treated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca(2+)-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils. PMID- 7559559 TI - Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide. AB - Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein. PMID- 7559560 TI - Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene. AB - In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions. PMID- 7559561 TI - Studies on the influence of cytosine methylation on DNA recombination and end joining in mammalian cells. AB - To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase. Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end joining or homologous recombination. Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells. These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure. PMID- 7559562 TI - Purification and characterization of a dynorphin-processing endopeptidase. AB - Dynorphin B (Dyn B-13, also known as rimorphin) is generated from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An enzymatic activity capable of processing at this monobasic site has been previously reported in neurosecretory vesicles of the bovine pituitary and pituitary-derived cell lines. This enzyme termed "the dynorphin-converting enzyme" (DCE) has been purified to apparent homogeneity from the neurointermediate lobe of the bovine pituitary using hydrophobic chromatography on phenyl-Sepharose, preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5, and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under reducing conditions. DCE is a metallopeptidase and exhibits a neutral pH optimum. Specific Inhibitors of soluble metallopeptidases such as enkephalinase (EC 3.4.24.11) or enkephalin generating neutral endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating that DCE is distinct from these two enzymes. Cleavage site determination with matrix-assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29 N terminus to the Arg14 generating Dyn B-13 and Dyn B-(14-29). Among other peptides derived from Dyn B-29, DCE cleaves only those peptides that fit the predicted "consensus motif" for monobasic processing. These data are consistent with a broader role for the dynorphin converting enzyme in the biosynthesis of many peptide hormones and neuropeptides by processing at monobasic sites. PMID- 7559565 TI - Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1. AB - Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA molecule that are required for interaction with the RT protein remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified RT to mutant forms of tRNA Lys3. The anticodon loop could be mutated without eliminating RT recognition. However, mutations in the T psi C stem were found to partially interfere with RT binding, and D arm mutants were completely inactive in RT binding. Interestingly, binding of the RT protein to tRNA Lys3 facilitates the subsequent annealing of template strand to the 3'-terminus of the tRNA molecule. Consistent with this finding, we demonstrate that mutant HIV-1 virions lacking the RT protein do contain a viral RNA genome without an associated tRNA Lys3 primer. We also found that a preformed primer tRNA-template complex is efficiently recognized by RT protein in vitro. Extension of the template molecule over the T psi C loop did result in complete inhibition of RT binding, suggesting the presence of additional recognition elements in the T psi C loop. These results, combined with a comparative sequence analysis of tRNA species present in HIV-1 virions and RNA motifs selected in vitro for high affinity RT binding, suggest that RT recognizes the central domain of the tRNA tertiary structure, which is formed by interaction of the D and T psi C loops. PMID- 7559566 TI - Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides. AB - One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution. PMID- 7559564 TI - Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. AB - Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals. PMID- 7559567 TI - Unusual reverse transcriptases. PMID- 7559563 TI - The regulatory region of calcium/calmodulin-dependent protein kinase I contains closely associated autoinhibitory and calmodulin-binding domains. AB - The mechanism for the regulation of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was investigated using a series of COOH-terminal truncated mutants. These mutants were expressed in bacteria as fusion proteins with glutathione S-transferase and purified by affinity chromatography using glutathione Sepharose 4B. A mutant (residues 1-332) showed complete Ca2+/CaM dependent activity. Truncation mutants (residues 1-321, 1-314, and 1-309) exhibited decreasing affinities for Ca2+/CaM and also exhibited decreasing Ca2+/CaM-dependent activities. Truncation mutants (residues 1-305 or 1-299) were unable to bind Ca2+/CaM and were inactive. In contrast, truncation mutants (residues 1-293 or 1-277) were constitutively active at a slightly higher level (2-fold) than fully active CaM kinase I. These results indicate the location of the Ca2+/CaM-binding domain on CaM kinase I (residues 294-321) and predict the existence of an autoinhibitory domain near, or overlapping, the Ca2+/CaM-binding domain. These conclusions were supported by studies which showed that a synthetic peptide (CaM kinase I (294-321)) corresponding to residues 294-321 of CaM kinase I inhibited the fully active kinase in a manner that was competitive with Ca2+/CaM and also inhibited the constitutively active mutant (residues 1-293) in a manner that was competitive with Syntide-2, a peptide substrate, (Ki = 1.2 microM) but was non-competitive with ATP. Thus, these results suggest that CaM kinase I is regulated through an intrasteric mechanism common to other members of the family of Ca2+/CaM-dependent protein kinases. PMID- 7559568 TI - Interaction of the transcriptional activator Stat-2 with the type I interferon receptor. AB - Binding of interferon-alpha (IFN alpha) to the multisubunit type I IFN receptor (IFNR) induces activation of the Tyk-2 and Jak-1 kinases and tyrosine phosphorylation of multiple signaling elements, including the Stat proteins that form the ISGF3 alpha complex. Although Jak kinases are required for IFN alpha dependent activation of Stats, the mechanisms by which Stats interact with these kinases are not known. We report that Stat-2 associates with beta s subunit of the type I IFN receptor in an interferon-dependent manner. This association is rapid, occurring within 1 min of interferon treatment of cells, and is inducible by various type I (alpha, beta, omega) but not type II (gamma) IFNs. The kinetics of Stat-2-IFNR association are similar to the kinetics of phosphorylation of Stat 2, suggesting that during its binding to the type I IFNR, Stat 2 acts as a substrate for interferon-dependent tyrosine kinase activity. These findings support the hypothesis that the type I IFNR acts as an adaptor, linking Stat proteins to Jak kinases. Interaction of Stat-2 with the beta s subunit of the type I IFNR may be a critical signaling event, required for the formation of the ISGF3 alpha complex and downstream transcription of interferon-stimulated genes. PMID- 7559569 TI - G alpha 12 and G alpha 13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly. AB - Rho, a member of the Ras superfamily of GTP-binding proteins, regulates actin polymerization resulting in the formation of stress fibers and the assembly of focal adhesions. In Swiss 3T3 cells, heterotrimeric G protein-coupled receptors for lysophosphatidic acid and gastrin releasing peptide stimulate Rho-dependent stress fiber and focal adhesion formation. The specific heterotrimeric G protein subunits mediating Rho-dependent stress fiber and focal adhesion formation have not been defined previously. We have expressed GTPase-deficient, constitutively activated G protein alpha subunits and mixtures of beta and gamma subunits in Swiss 3T3 cells. Measurement of actin polymerization and focal adhesion formation indicated that GTPase-deficient alpha 12 and alpha 13, but not the activated forms of alpha 12 or alpha q stimulated stress fiber and focal adhesion assembly. Combinations of beta and gamma subunits were unable to stimulate stress fiber or focal adhesion formation. G alpha 12- and alpha 13-mediated stress fiber and focal adhesion assembly was inhibited by botulinum C3 exoenzyme, which ADP ribosylates and inactivates Rho, indicating that alpha 12 and alpha 13, but not other G protein alpha subunits or beta gamma complexes, regulate Rho-dependent responses. The results define the integration of G12 and G13 with the regulation of the actin cytoskeleton. PMID- 7559570 TI - Protein-tyrosine phosphatase 1D modulates its own state of tyrosine phosphorylation. AB - The insulin receptor-mediated signal transduction pathway involves insulin receptor substrate 1 and a variety of proteins containing Src homology-2 (SH2) domains, such as phosphatidylinositol 3-kinase, Grb2, and protein-tyrosine phosphatase 1D (PTP1D). Upon insulin stimulation of baby hamster kidney cells overexpressing the IR, the catalytically inactive mutant of PTP1D, C463A, becomes tyrosine-phosphorylated and coprecipitates with Grb2. Tyrosine phosphorylation of this mutant is significantly reduced when wild type PTP1D is coexpressed. Substitution of tyrosine residues 546 and 584 with phenylalanine abrogates tyrosine phosphorylation of the catalytically inactive mutant and abolishes its interaction with Grb2. PMID- 7559571 TI - Role of the Rad1 and Rad10 proteins in nucleotide excision repair and recombination. AB - In Saccharomyces cerevisiae, the RAD1 and RAD10 genes are involved in DNA nucleotide excision repair (NER) and in a pathway of mitotic recombination that occurs between direct repeat DNA sequences. In this paper, we show that purified Rad1 and Rad10 interact with a synthetic bubble structure and incise the DNA at the 5'-side of the centrally unpaired region. When Rad1-Rad10 and purified XPG protein (the human homolog of yeast Rad2 protein) were co-incubated with the DNA substrate, we observed incisions at both ends of the bubble. This reaction mimics the dual incision step in nucleotide excision repair in vivo. In addition, the recent suggestion that Rad1 can act to resolve Holliday junctions (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) Nature 371, 531-534), explaining the recombination defect observed in rad1 mutants, has been further investigated. However, using proteins purified in two different laboratories we were unable to show any interaction between Rad1 and synthetic Holliday junctions. The role that Rad1-Rad10 plays in recombination is likely to resemble its activity in NER by acting upon partially unpaired DNA intermediates such as those formed by recombination mechanisms involving single-strand DNA annealing. PMID- 7559574 TI - Complex-type asparagine-linked oligosaccharides on phosphacan and protein tyrosine phosphatase-zeta/beta mediate their binding to neural cell adhesion molecules and tenascin. AB - Phosphacan, a soluble nervous tissue-specific chondroitin sulfate proteoglycan, is an alternative splicing product representing the entire extracellular domain of a transmembrane receptor-type protein-tyrosine phosphatase (RPTP zeta/beta) that also occurs as a chondroitin sulfate proteoglycan in brain. We have previously demonstrated that phosphacan binds with high affinity to neural cell adhesion molecules (Ng-CAM/L1 and N-CAM) and to the extracellular matrix protein tenascin and that it is a potent inhibitor of cell adhesion and neurite outgrowth. Tryptic digests of 125I-labeled phosphacan contain two glycopeptides that bind to Ng-CAM/L1, N-CAM, and tenascin. The larger of these (17 kDa) begins at Gln-209 near the end of the carbonic anhydrase-like domain of phosphacan/RPTP zeta/beta, whereas a 13-kDa glycopeptide begins at His-361 located in the middle of the fibronectin type III-like domain. Treatment of phosphacan with peptide N glycosidase under nondenaturing conditions reduced its binding the neural cell adhesion molecules and tenascin by 65-75%, whereas endo-beta-N acetylglucosaminidase H had no effect, and peptide N-glycosidase treatment both decreased the molecular sizes of the tryptic peptides to congruent to 11 kDa and abolished their binding. Based on the amino acid sequence of phosphacan, it can be concluded that each of the tryptic peptides contains one potential N glycosylation site (at Asn-232 and Asn-381), and analyses of the isolated glycopeptides demonstrated the presence of sialylated complex-type oligosaccharides. Our results therefore indicate that the interactions of phosphacan/RPTP zeta/beta with neural cell adhesion molecules and tenascin are mediated by asparagine-linked oligosaccharides present in their carbonic anhydrase- and fibronectin type III-like domains. PMID- 7559573 TI - Restoration of phosphorylation-dependent regulation to the skeletal muscle myosin regulatory light chain. AB - Regulation of the ATPase activity of smooth and nonmuscle myosin II involves reversible phosphorylation of the regulatory light chain (RLC). The RLC from skeletal muscle myosin (skRLC) is unable to confer regulation (myosin is locked in an inactive state) to smooth muscle myosin when substituted for the endogenous smooth RLC (smRLC). Studies of chimeric light chains comprised of the N- or C terminal half of each skRLC and smRLC suggest that the structural basis for the loss of this regulation is within the C-terminal half of the RLC (Trybus, K.M., and Chatman, T.A. (1993) J. Biol. Chem. 268, 4412-4419). The purpose of this study is to delineate the structural elements within the C-terminal half of the smRLC that are absent in the skRLC and are necessary for regulation. By sequence comparison, six residues, Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg 160, which are conserved in regulated myosin RLCs but missing in nonregulated myosin RLCs, were identified in smRLC. To test whether these amino acids provide the missing structural elements necessary for phosphorylation-mediated regulation, a skRLC was engineered that replaced the corresponding skRLC amino acids (positions 100, 120, 126, 127, 140, and 157, respectively) with their smRLC counterparts. Using a newly developed RLC exchange procedure, the purified mutant protein was evaluated for its ability to regulate chicken gizzard smooth muscle myosin. Substitution of the six conserved amino acids into the skRLC completely restored phosphorylation-mediated regulation. Thus, a subset of these amino acids, including four basic arginine residues located in the E, F, G, and H helices which are missing in skRLC, may be the structural coordinates for the phosphorylserine in the N terminus. Based on this result, the regulation of glycogen phosphorylase is discussed as a model for the regulation of smooth muscle myosin. PMID- 7559575 TI - Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene. AB - The sequence encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the choline acetyltransferase (ChAT) gene in various species. In rat, we previously identified a class of VAChT mRNAs that may originate from the same promoter region as two ChAT mRNAs. Here, we demonstrate by a detailed analysis of the 5'-noncoding region of the VAChT gene, that two specific VAChT promoters lie within the first intron of the ChAT gene. Two VAChT mRNAs are generated from these promoters. These results demonstrate that the promoter regions of these two genes are intermingled, which highlight the unique organization of the ChAT/VAChT gene locus. PMID- 7559576 TI - Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. AB - Holo-acyl carrier protein synthase (ACPS) transfers the 4'-phosphopantetheine (4' PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli. This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4'-PP. We have purified E. coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions. N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS. We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E. coli. PMID- 7559572 TI - Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2. AB - Defects in the c-ret proto-oncogene, a member of the protein tyrosine kinase receptor family, have recently been linked to two types of genetic syndromes, Hirschsprung's disease and the multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is the product of a papillary thyroid carcinoma translocation event between the genes coding for c-ret and the type I alpha regulatory subunit of protein kinase A (RI alpha) (Lanzi, C., Borrello, M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M., Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M. (1992) Oncogene 7, 2189-2194). The resulting 596-residue protein contains the first two-thirds of RI alpha and the entire tyrosine kinase domain of c-ret (RETtk). An in vivo assay of growth stimulatory effects was developed, which consisted of microinjecting a RET/ptc2 expression plasmid into the nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of 5-bromodeoxyuridine. This assay was used to determine that only the dimerization domain of RI alpha fused to RETtk is required for RET/ptc2's mitogenic activity. In addition, all of the reported Hirschsprung's disease point mutations in the RETtk (S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay, confirming that these are loss of function mutations. Two tyrosines outside the conserved kinase core were also identified that are essential for full mitogenic activity of RET/ptc2. These two tyrosines, Tyr-350 and Tyr-586, are potential sites for Src homology 2 and phosphotyrosine binding domain interactions. PMID- 7559577 TI - Gating effects of component B on oxygen activation by the methane monooxygenase hydroxylase component. AB - Component B (MMOB) of the soluble methane monooxygenase (MMO) system accelerates the initial velocity of methane oxidation by up to 150-fold by an unknown mechanism. The active site of MMO contains a diferric, hydroxo-bridged diiron cluster located on the hydroxylase component (MMOH). This cluster is reduced by the NAD(P)H-coupled reductase component to the diferrous state, which then reacts with O2 to yield two reaction cycle intermediates sequentially termed compounds P and Q. The rate of compound P formation is shown here to be independent of O2 concentration, suggesting that an MMOH-O2 complex (compound O) is (congruent to irreversibly) formed before compound P. Compound Q is capable of reacting with hydrocarbons to yield the MMOH-product complex, compound T. It is shown here that MMOB accelerates catalysis by increasing congruent to 1000-fold the rate of O2 association and reaction with diferrous MMOH leading to compound P. Modeling of the single turnover reaction in the presence of substoichiometric MMOB suggests that MMOB also accelerates the compound P to Q conversion by congruent to 40 fold. Due to this O2-gating effect of MMOB, either compound Q or T becomes the dominant species during turnover, depending upon the substrate concentration and type. Because these are the species that either react with substrate (Q) or release product (T), their buildup maximizes the turnover rate. This is the first direct role in catalysis to be recognized for MMOB and represents a novel method for oxygenase regulation. PMID- 7559578 TI - The novel structural motif Gln795-Gln802 in the integrin beta 1C cytoplasmic domain regulates cell proliferation. AB - Alternative splicing of the integrin beta 1 subunit mRNA generates a variant form, beta 1C, with a unique cytoplasmic domain that differs from beta 1 for a 48 amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1 cells transiently transfected with cDNAs coding for human integrin beta 1C or beta 1 subunits or mutants containing truncated forms of the beta 1C cytoplasmic domain. A differential effect of beta 1C and beta 1 on cell proliferation was observed. Expression of wild type beta 1 was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of beta 1C. Cells expressing the beta 1C mutant truncated at Leu794 and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta 1C deletion mutants, lacking the COOH terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802 of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of beta 1C expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta 1C, full-length or deletion mutants, or beta 1 equally adhered to a functionally inhibitory monoclonal antibody against human beta 1 integrin. Expression of beta 1C inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta 1C was induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo. PMID- 7559579 TI - 4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice. AB - Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J., Wang, L. M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W.S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1 deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling. PMID- 7559584 TI - Effect of pH on the conformation and backbone dynamics of a 27-residue peptide in trifluoroethanol. An NMR and CD Study. AB - The C-terminal fragment, residues 385-411, from human fibrinogen gamma-chain, i.e. KIIPFNRLTIGEGQQHHLG-GAKQAGDV, shows multiple turn conformations in aqueous solution (Mayo, K. H., Burke, C., Lindon, J. N., and Kloczewiak, M. (1990) Biochemistry 29, 3277-3286). The present study investigates the effect of pH and trifluoroethanol on the conformation and backbone dynamics of this 27-residue peptide. Both circular dichroism (CD) and 1H-NMR data indicate the normally observed increased helical conformations as a function of increasing trifluoroethanol. 1H-NMR structural studies done in the presence of 40% trifluoroethanol, pH 5.3, yield a network of nuclear Overhauser effects consistent with significant populations of helix-like conformation. Distance geometry calculations based on nuclear Overhauser effect-derived distance constraints yield a family of structures with relatively well defined N- and C terminal conformations and an ill defined mid-peptide region from Gly397 to Gly403. Similar conformational populations are observed at pH 2.5. CD studies, however, indicate an increase in average alpha-helix content on decreasing the pH from 6 to 2. This apparent conflict between CD and NMR results may be explained by a transition from multiple beta-turn character at pH 5.3 to increased alpha helix structure at pH 2.5. 13C alpha NMR relaxation data analyzed with the Lipari Szabo model-free approach provide order parameters that demonstrate little if any influence of pH on backbone motional restrictions within the more flexible mid peptide domain. At low pH, however, motions become less restricted within N terminal residues Lys385-Phe389 and more restricted within C-terminal residues Ala405-Val411. PMID- 7559581 TI - The insulin-dependent biosynthesis of GLUT1 and GLUT3 glucose transporters in L6 muscle cells is mediated by distinct pathways. Roles of p21ras and pp70 S6 kinase. AB - Insulin binding results in rapid phosphorylation of insulin receptor substrate-1 to activate p21ras and mitogen-activated protein kinase. Insulin also activates the ribosomal protein S6 kinase (pp70 S6 kinase) independently of the Ras pathway. Chronic (18 h) treatment of L6 muscle cells with insulin increases glucose transport activity severalfold due to biosynthetic elevation of the GLUT1 and GLUT3 but not the GLUT4 glucose transporters. Here we investigate the roles of p21ras and pp70 S6 kinase in the insulin-mediated increases in GLUT1 and GLUT3 expression. L6 cells were transfected with the dominant negative Ras(S17N) under the control of a dexamethasone-inducible promoter. Induction of Ras(S17N) failed to block the insulin-mediated increase in GLUT1 glucose transporter protein and mRNA; however, it abrogated the insulin-mediated increase in GLUT3 glucose transporter protein and mRNA. Inhibition of pp70 S6 kinase by rapamycin, on the other hand, eliminated the insulin-mediated increase in GLUT1 but had no effect on that of GLUT3 in both parental and Ras(S17N) transfected L6 cells. These results suggest that the biosynthetic regulation of glucose transporters is differentially determined, with pp70 S6 kinase and p21ras playing active roles in the insulin-stimulated increases in GLUT1 and GLUT3, respectively. PMID- 7559582 TI - A disulfide bond between conserved extracellular cysteines in the thyrotropin releasing hormone receptor is critical for binding. AB - The assumption that a disulfide bond is present between two highly conserved cysteines in the extracellular loops of G protein-coupled receptors and is critical for receptor function has been cast in doubt. We undertook to determine whether a disulfide bond important for binding or activation is present in the thyrotropin-releasing hormone (TRH) receptor (TRH-R). Studies were performed with cells expressing wild-type (WT) and mutant receptors in the absence or presence of the reducing agent dithiothreitol (DTT). The affinity of WT TRH-R was 16-22 fold lower in the presence of DTT than in the absence of DTT. Mutant receptors were constructed in which Ala was substituted for conserved Cys-98 and Cys-179 of extracellular loops 1 and 2, respectively, and for the nonconserved Cys-100. C98A and C179A TRH-Rs did not exhibit high affinity binding. These mutant receptors were capable of stimulating inositol phosphate second messenger formation to the same extent as WT TRH-Rs but with a markedly lower potency. The affinities of C98A and C179A TRH-Rs, estimated from their potencies, were 4400- and 640-fold lower, respectively, than WT TRH-R. The estimated affinities of neither C98A nor C179A TRH-R were decreased by DTT. In contrast, the estimated affinity of C100A TRH-R was not different from WT TRH-R and was DTT sensitive. Moreover, the effect of mutating both Cys-98 and Cys-179 was not additive with the effects of the individual mutations. These data provide strong evidence that Cys-98 and Cys-179 form a disulfide bond. This interaction is not involved in receptor activation but is critical for maintaining the high affinity conformation of TRH-R. PMID- 7559585 TI - Mutational analysis of PC1 (SPC3) in PC12 cells. 66-kDa PC1 is fully functional. AB - The proteinase mPC1, a neuroendocrine member of the mammalian family of subtilisin-like enzymes, has previously been shown to be converted to a carboxyl terminally truncated 66-kDa form during transport through the secretory pathway. The cleavage site and the function of this carboxyl-terminal truncation event are unknown. We have performed site-directed mutagenesis of two paried basic sites in the mPC1 carboxyl-terminal tail and expressed these constructs in PC12 cells, a rat pheochromocytoma known to lack endogenous PC1. We found that the most likely site for the truncation event was at Arg590-Arg591 since mutation of this site to Lys-His prevented processing of 87-kDa PC1. A PC1 mutant carboxyl-terminally truncated at this site and expressed in PC12 cells was efficiently routed to the secretory pathway and stored in secretory granules, indicating that the carboxyl terminal extension is not required for sorting of this enzyme. The function of the various PC1 constructs was assessed by analyzing proneurotensin cleavage to various forms. The carboxyl-terminally truncated PC1 mutant was found to perform most of the cleavages of this precursor as well as wild-type PC1; however, the blockade mutant processed proneurotensin much less efficiently. Differences between the site preferences of the various enzymes were noted. Our results support the notion that carboxyl-terminal processing of PC1 serves to regulate PC1 activity. PMID- 7559580 TI - Identification of a gene encoding a yeast histone H4 acetyltransferase. AB - A collection of yeast temperature-sensitive mutants was screened by an enzymatic assay to find a mutant defective in the acetylation of histone H4. The assay used a fractionated cell extract and measured acetylation of a peptide corresponding to amino acids 1-28 of H4. There are at least two activities in this fraction that acetylate the peptide. A mutation, hat1-1, that eliminates one of the activities was identified and mapped to a locus near the centromere of chromosome XVI. The HAT1 gene was cloned and found to encode a protein of 374 amino acids. Analysis of the peptide used in the assay demonstrated that the HAT1 enzyme acetylates lysine 12 of histone H4. hat1 mutants have no obvious growth defects or phenotypes other than the enzyme defect itself. The HAT1 protein expressed in Escherichia coli gave histone acetyltransferase activity in vitro, demonstrating that HAT1 is the structural gene for the enzyme. PMID- 7559586 TI - The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5. AB - The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5) requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction. PMID- 7559583 TI - Kinetic mechanism of aminoglycoside phosphotransferase type IIIa. Evidence for a Theorell-Chance mechanism. AB - Bacterial resistance to aminoglycoside-aminocyclitol antibiotics is mediated primarily by covalent modification of the drugs by a variety of enzymes. One such modifying enzyme, the 3'-aminoglycoside phosphotransferase, which is produced by Gram-positive cocci such as Enterococcus and Streptococcus inactivates a broad range of aminoglycosides by ATP-dependent phosphorylation of specific hydroxyl residues on the antibiotics. Through the use of dead-end and product inhibitor studies, we present the first detailed examination of the kinetic mechanism for the 3'-aminoglycoside phosphotransferase-IIIa. Initial velocity patterns deduced from steady-state kinetics indicate a sequential mechanism with ordered binding of ATP first followed by aminoglycoside. Dead-end inhibition by AMP and adenylyl imidodiphosphate is competitive versus ATP and noncompetitive versus kanamycin A. Dead-end inhibition by tobramycin, a kanamycin analogue lacking a 3'-OH, is competitive versus both kanamycin A and uncompetitive versus ATP, indicative of ordered substrate binding where ATP must add prior to aminoglycoside addition. Product inhibition by kanamycin phosphate is noncompetitive versus ATP when kanamycin A is held at subsaturating concentrations (Km(kanA)), whereas no inhibition is observed when the concentration of kanamycin A is held at 10Km(kanA). This is consistent with kanamycin phosphate being the first product released followed by ADP release. The patterns of inhibition observed support a mechanism where ATP binding precedes aminoglycoside binding followed by a rapid catalytic step. Product release proceeds in an ordered fashion where kanamycin phosphate is released quickly followed by a slow release of ADP. Aminoglycoside substrates, such as kanamycin A, show substrate inhibition that is uncompetitive versus ATP. This indicates binding of the aminoglycosides to the slowly dissociating (E-ADP) complex at high drug concentrations. These experiments are consistent with a Theorell-Chance kinetic mechanism for 3'-aminoglycoside phosphotransferase-IIIa. PMID- 7559587 TI - Irreversible binding kinetics of Bacillus thuringiensis CryIA delta-endotoxins to gypsy moth brush border membrane vesicles is directly correlated to toxicity. AB - To examine the binding of Bacillus thuringiensis delta-endotoxins, CryIAa, CryIAb, and CryIAc, to Lymantria dispar (gypsy moth) brush border membrane vesicles (BBMV), saturation kinetic analyses were conducted according to a two step interaction scheme [formula: see text] for delta-endotoxin binding to BBMV, rather than the one-step reversible binding presented in prior reports. The order of toxicity of the delta-endotoxins, as measured by the dose required for a 50% inhibition of weight gain (ID50), was CryIAa (77.3 ng) > CryIAb (157 ng) > CryIAc (187 ng). While both the maximum extent of binding, Bmax, and the half-maximum insertion rate concentration, K1/2, was observed to be indirectly related to toxicity, the rate constant of irreversible binding, k2, was found to be directly correlated to toxicity. PMID- 7559589 TI - Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70. AB - The precursor (pmAspAT) and mature (mAspAT) forms of mitochondrial aspartate aminotransferase interact with hsp70 very early during translation when synthesized in either rabbit reticulocyte lysate or wheat germ extract (Lain, B., Iriarte, A., and Martinez-Carrion. (1994) J. Biol. Chem. 269, 15588-15596). The nature of the structural elements responsible for recognition and binding of this protein to hsp70 has been studied by examining the folding and potential association with the chaperone of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT bind hsp70 very early during translation, the cytosolic form of this enzyme (cAspAT) does not interact with hsp70. A fusion protein consisting of the mitochondrial presequence peptide attached to the amino terminus of cAspAT associates with hsp70 only after the protein has acquired its native-like conformation, apparently through binding to the presequence exposed on the surface of the folded protein. Deletion of the amino-terminal segment of mAspAT or its replacement with the corresponding domain from the cytosolic isozyme eliminates the cotranslational binding of hsp70 to the mitochondrial protein. We conclude that both the presequence and NH2-terminal region of pmAspAT represent recognition signals for binding of hsp70 to the newly synthesized mitochondrial precursor. Results from competition studies with synthetic peptides support this conclusion. The ability of hsp70 to discriminate between these two highly homologous proteins probably involves the recognition of specific sequence elements in the NH2-terminal portion of the mitochondrial protein and may relate to their separate localization in the cell. A slower folding rate and higher affinity for cytosolic chaperones may represent evolutionary adaptations of translocated mitochondrial proteins to ensure their efficient importation into the organelle. PMID- 7559588 TI - Selective regulation of TrkA and TrkB receptors by retinoic acid and interferon gamma in human neuroblastoma cell lines. AB - Trk receptors are a family of genes implicated in the survival, differentiation, and growth of certain neurons and tumors of the nervous system. A better understanding of the regulation of Trk receptors is relevant for developmental and oncological studies. Human neuroblastoma (NB) cell lines constitutively express low levels of TrkA mRNA, while TrkB mRNA is not readily detectable. Differentiation of NB cells is accompanied by a differential modulation of Trk expression in human NB cells. Nanomolar concentrations of RA induce a stable increase of TrkB mRNA. A transient induction of TrkA mRNA levels requires micromolar concentrations of RA. Induction of both TrkA and TrkB mRNA does not require new protein synthesis. However, RA-induced TrkB mRNA expression is transcriptionally regulated, while the transient RA-induced increase of TrkA mRNA is a consequence of extended mRNA stability. Interferon gamma (IFN-gamma) selectively increases TrkA mRNA without affecting TrkB mRNA levels. Similar to RA, IFN-gamma does not modify the transcriptional rate of TrkA mRNA, but rather increases TrkA mRNA stability. Thus, RA and IFN-gamma differentially regulate TrkA or TrkB expression in the same cell type by predominantly transcriptional (TrkB) or post transcriptional (TrkA) mechanisms. Such experiments indicate the complexity of Trk mRNA regulation and also indicate compounds that may affect neurotrophin responsiveness in developing neural cells. PMID- 7559590 TI - Stimulation of immediate early gene expression in striatal neurons by nitric oxide. AB - Exposure of primary cultures of embryonic rat striatal neurons to agents releasing nitric oxide (NO), including sin-1 molsidomine, S-nitroso-n-acetyl penicillamine (SNAP), and S-nitrosoglutathione, resulted in an increase in the levels of expression of the immediate early genes c-fos and zif/268 in the cultured neurons. The membrane-permeable cGMP analogue, 8-bromo-cGMP, did not significantly affect c-fos and zif/268 mRNA levels, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the elevation in c-fos and zif/268 mRNA levels induced by SNAP. The induction of c fos by the calcium ionophore A23187 was reduced by treatment with SNAP or 8-bromo cGMP. Inhibitors of ADP-ribosyltransferases attenuated the stimulation of c-fos expression by SNAP. These results demonstrate for the first time that NO can induce immediate early gene expression in neurons, suggesting that NO may act as a mediator of neuronal plasticity via alterations in the expression of downstream genes. In addition, the results suggest that NO may exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase. PMID- 7559593 TI - Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between the alpha-subunits. AB - Voltage-gated potassium (K+) channels are assembled by four identical or homologous alpha-subunits to form a tetrameric complex with a central conduction pore for potassium ions. Most of the cloned genes for the alpha-subunits are classified into four subfamilies: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal). Subfamily-specific assembly of heteromeric K+ channel complexes has been observed in vitro and in vivo, which contributes to the diversity of K+ currents. However, the molecular codes that mediate the subfamily-specific association remain unknown. To understand the molecular basis of the subfamily-specific assembly, we tested the protein-protein interactions of different regions of alpha-subunits. We report here that the cytoplasmic NH2-terminal domains of Kv1, Kv2, Kv3, and Kv4 subfamilies each associate to form homomultimers. Using the yeast two-hybrid system and eight K+ channel genes, two genes (one isolated from rat and one from Drosophila) from each subfamily, we demonstrated that the associations to form heteromultimers by the NH2-terminal domains are strictly subfamily-specific. These subfamily-specific associations suggest a molecular basis for the selective formation of heteromultimeric channels in vivo. PMID- 7559592 TI - Identification of a Gs coupling domain in the amino terminus of the third intracellular loop of the alpha 2A-adrenergic receptor. Evidence for distinct structural determinants that confer Gs versus Gi coupling. AB - alpha2-Adrenergic receptors (alpha 2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human alpha 2AAR (alpha 2C10) for potential Gs coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the alpha 2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the beta 2AR (a Gs-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation. In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated alpha 2C10-mediated stimulation of adenylyl cyclase activity, while alpha 2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218 235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 +/- 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha 2C10. In contrast, Gi coupling for this mutant remained fully intact (57 +/- 2% versus 52 +/- 1% inhibition for wild-type alpha 2C10). Similar substitution with beta 2AR sequence had no effect on Gi coupling but did reduce Gs coupling. Two additional mutated alpha 2C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While Gi coupling remained intact with both mutants, Gs coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display alpha 2AR-Gi coupling) and COS-7 cells (which exclusively display alpha 2AR-Gs coupling). Thus, a critical determinant for Gs coupling is contained within 11 amino acids (218-228) of the amino terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain. Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted alpha 2AR-Gs coupling, which is distinct and separable from the structural requirements for alpha 2AR-Gi coupling. PMID- 7559595 TI - The alpha-dendrotoxin footprint on a mammalian potassium channel. AB - alpha-Dendrotoxin, a 59-amino acid basic peptide from the venom of Dendroaspis angusticeps (green mamba snake), potently blocks some but not all voltage dependent potassium channels. Here we have investigated the relative contribution of the individual alpha-subunits constituting functional Kv1.1 potassium channels to alpha-dentroxin binding. Three residues critical for alpha-dentrotoxin binding and located in the loop between domains S5 and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs were constructed encoding homo- and heterotetrameric channels composed of all possible ratios of wild-type and mutant alpha-subunits. Complete mutant channels were about 200-fold less sensitive for the alpha dendrotoxin block than complete wild-type channels, which is attributable to a smaller association rate. Analysis of the bimolecular reaction between alpha dendrotoxin and the different homo- and heteromeric channel constructs revealed that the association rate depends on the number of wild-type alpha-subunits in the functional channel. Furthermore, we observed a linear relationship between the number of wild-type alpha-subunits in functional channels and the free energy for alpha-dendrotoxin binding, providing evidence that all four alpha-subunits must interact with alpha-dendrotoxin to produce a high affinity binding site. PMID- 7559594 TI - Multiple deletions are detectable in mitochondrial DNA of aging mice. AB - Mutational damage to human mitochondrial DNA (mtDNA) can cause disorders in oxidative phosphorylation; speculation that such damage is involved in degenerative diseases and aging is common. We have detected deletions in mouse mtDNA which resemble those found in elderly humans or patients with certain mtDNA disorders. Five different mtDNA deletions, predicted from the positions of short, direct DNA repeats, were present in aged, but not young, mice. Deleted regions were surrounded by either exact or inexact repeats and occurred in both the major and minor regions of the mtDNA genome. The abundance of a particular deletion was generally related to the thermodynamic stability of the bounding repeat sequence. Deletions in aged mice were present at low levels (less than 0.01% of total mtDNA). However, in contrast to results from aged humans, deletions were more abundant in liver than in brain, heart, or skeletal muscle. These results make it possible to predict the location and relative abundance of deletions in any sequenced mtDNA, including inbred mouse strains differing in inherent natural lifespan. The inbred mouse model will allow a critical examination of the relationship between the presence and abundance of mtDNA deletions and the aging process. PMID- 7559591 TI - Characterization of the gamma protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii. AB - Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (alpha 2 beta 2) and contains the iron-molybdenum cofactor (FeMo co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of alpha 2 beta 2 gamma 2. The gamma subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro. Characterization of gamma protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. These are three major forms of gamma protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo co. A second form of gamma protein is unassociated with other proteins and exists as a homodimer. Both of these forms of gamma protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into gamma protein. Crude extracts that contain this form of gamma protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which gamma protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex. PMID- 7559597 TI - Binding of fibrinogen A alpha 1-50-beta-galactosidase fusion protein to thrombin stabilizes the slow form. AB - The interaction of fibrinogen A alpha1-50-beta-galactosidase fusion protein with the slow and fast forms of thrombin was studied and compared to thrombin fibrinogen interaction under identical solution conditions. At equilibrium, the affinity of the fusion protein for the slow form of thrombin is 3 times higher than its affinity for the fast form. The fusion protein and fibrinogen have the same affinity for the fast form. On the other hand, the affinity of the fusion protein for the slow form of thrombin is 40 times tighter than that of fibrinogen. In the transition state, binding of the fusion protein has the same properties as fibrinogen, with the fast form showing higher specificity. The N terminal fragment of the fibrinogen A alpha chain thus contains residues that are responsible for the preferential binding of the fusion protein to the slow form at equilibrium and to the fast form in the transition state. If this fragment binds to thrombin in a similar way for fibrinogen and the fusion protein, then the N-terminal domains of the B beta and gamma chains of fibrinogen, that are not present in the fusion protein, must play a key role in the binding of fibrinogen to thrombin at equilibrium. These chains may destabilize binding to the slow form by nearly 2.4 kcal/mol, thereby favoring binding of fibrinogen to the fast form. We propose that the three chains of fibrinogen play different roles in the thrombin-fibrinogen interaction, with the A alpha chain containing residues for preferential binding to the fast form in the transition state and the B beta and gamma chains containing residues that destabilize binding to the slow form at equilibrium. PMID- 7559596 TI - Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. AB - The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration. PMID- 7559598 TI - Regulation of calcineurin gene expression in Schizosaccharomyces pombe. Dependence on the ste11 transcription factor. AB - Calmodulin and its target enzymes are important regulators of numerous cellular processes, including reversible protein phosphorylation. The calmodulin-dependent protein phosphatase (calcineurin) has been suggested to play roles in activation of T cells and in the mating response of yeast. Recently, studies have shown it to be the target of immunosuppressant drugs such as cyclosporin and FK-506. In this study, we have cloned the gene for the catalytic subunit of calcineurin, CnA, from the yeast Schizosaccharomyces pombe. The gene (named ppb1+) has been mapped to chromosome II by analysis of the hybridization of a genomic DNA probe to an ordered library. The gene produces a single mRNA species of 2.5 kilobases, which varies during the cell cycle in exponentially growing cells. In addition, expression of ppb1+ mRA is induced by nitrogen starvation, a condition that favors mating in S. pombe. The ppb1+ gene promoter contains a cis-acting element for the ste11 transcription factor, and we have shown that induction of the ppb1+ mRNA during nitrogen starvation is dependent on the ste11 gene product. Together with earlier studies showing that disruption of the ppb1+ gene in S. pombe results in sterility (Yoshida, T., Toda, T., and Yanagida, M. (1994) J. Cell Sci., 107, 1725-1735), our studies suggest that the ppb1+ gene plays a role in the gene expression cascade that is essential for mating and sporulation in S. pombe. PMID- 7559600 TI - Steady-state in vivo glutamate dehydrogenase activity in rat brain measured by 15N NMR. AB - The in vivo activity of glutamate dehydrogenase (GDH) in the direction of reductive amination was measured in rat brain at steady-state concentrations of brain ammonia and glutamate after intravenous infusion of the substrate 15NH4+. The in vivo rate was determined from the steady-state fractional 15N enrichment of brain ammonia, measured by selective observation of 15NH4+ protons in brain extract by 1H-15N heteronuclear multiple-quantum coherence transfer NMR, and the rate of increase of brain [15N]glutamate and [2-15N]glutamine measured by 15N NMR. The in vivo GDH activity was 0.76-1.17 mumol/h/g, and 1.1-1.2 mumol/h/g at 1.0 +/- 0.17 mumol/g. Comparison of the observed in vivo GDH activity with the in vivo rates of glutamine synthesis and of phosphate-activated glutaminase suggests that, under mild hyperammonemia, GDH-catalyzed de novo synthesis can provide a minimum of 19% of the glutamate pool that is recycled from neurons to astrocytes through the glutamate-glutamine cycle. PMID- 7559599 TI - Two receptor systems are involved in the plasma clearance of tissue factor pathway inhibitor in vivo. AB - Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the blood coagulation factor VIIa-tissue factor complex, as well as a direct inhibitor of factor Xa. Intravenously administered TFPI is rapidly cleared from circulation predominantly via liver. We previously reported that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the uptake and degradation of TFPI in hepatoma cells. This process is inhibited by a 39-kDa receptor-associated protein which binds to LRP and regulates its ligand binding activity. However, a distinct, low affinity binding site (perhaps heparin sulfate proteoglycans, HSPGs) on the endothelium and liver is thought to be responsible for the majority of TFPI cell surface binding. In the current study, we investigated the role of LRP and this second binding site in the clearance of 125I-TFPI in vivo using competitors and inhibitors of the receptors. Mice overexpressing the 39-kDa protein via adenoviral-mediated gene transfer displayed diminished plasma clearance of 125I-TFPI. Blockade of cell surface HSPGs sites by incubation with the positively charged molecule, protamine, inhibited 125I-TFPI binding to the hepatoma cells in vitro. In addition, preadministration of protamine in vivo prolonged the plasma clearance of 125I TFPI in a dose-dependent manner. However, a dramatic increase of the plasma half life of 125I-TFPI and virtual elimination of 125I-TFPI clearance was observed in mice overexpressing the 39-kDa protein and administered protamine. Taken together, our results suggest that two receptor mechanisms are involved in the clearance of TFPI in vivo. PMID- 7559601 TI - N- and O-glycosylation and phosphorylation of the bar secretion leader derived from the barrier protease of Saccharomyces cerevisiae. AB - A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure. PMID- 7559602 TI - MIDA1, a protein associated with Id, regulates cell growth. AB - We have isolated cDNA clone encoding a protein that can associate with Id, a helix-loop-helix (HLH) protein. This protein is named MIDA1 (Mouse Id Associate 1), and its predicted amino acid sequence consists of Zuotin (a putative Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats similar to c-Myb oncoprotein. MIDA1 associates with the HLH region of Id with the conserved region adjacent to eukaryotic DnaJ conserved motif within the Zuotin homology region, although it does not have any canonical HLH motif. The addition of antisense oligonucleotide of MIDA1 inhibited growth of murine erythroleukemia cells without interfering with erythroid differentiation, indicating that it regulates cell growth. PMID- 7559603 TI - Syp associates with gp130 and Janus kinase 2 in response to interleukin-11 in 3T3 L1 mouse preadipocytes. AB - Protein tyrosine phosphorylation and thus dephosphorylation are part of the interleukin (IL)-11 response in mouse 3T3-L1 cells. We report here for the first time the involvement and interactions of the SH2-containing protein tyrosine phosphatase Syp in the IL-11 signal transduction pathway. Addition of IL-11 to 3T3-L1 cells resulted in an increase in the tyrosine phosphorylation of Syp. When cell lysates were precipitated with glutathione S-transferase fusion products of Syp, the C-terminal SH2 domain of Syp was shown to precipitate several proteins of 70, 130, 150, and 200 kDa that were tyrosine phosphorylated in response to IL 11. Reciprocal immunoprecipitation experiments showed that Syp was inducibly associated with both gp130 and Janus kinase 2 (JAK2). A phosphopeptide containing the sequence for a potential Syp binding site (YXXV) was used to compete with the associations of Syp with gp130 and JAK2. The phosphopeptide reduced the Syp association with both gp130 and JAK2. To summarize, Syp has multiple interactions in IL-11 signal transduction. In addition to the IL-11-induced tyrosine phosphorylation of Syp, Syp coprecipitated with gp130, JAK2, and other tyrosine phosphorylated proteins in response to IL-11. These findings may have extensive significance to IL-11 and related cytokine signal transduction, suggesting new pathways and mechanisms. PMID- 7559604 TI - Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae. AB - Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [3H]myristate when expressed in yeast. Both wild type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex. PMID- 7559606 TI - Transcription factor AP-2 regulates human insulin-like growth factor binding protein-5 gene expression. AB - Insulin-like growth factor binding protein-5 (IG-FBP-5) is an important modulator of IGF actions. IG-FBP-5 mRNA is abundant in human fibroblasts and is regulated by cAMP. To understand the molecular mechanism underlying this cell type-specific expression and regulation, we isolated the 5'-flanking region of the human IGFBP 5 gene and fused it to a promoter-less reporter plasmid encoding luciferase. Transient transfection of the construct into fibroblasts displayed both constitutive and cAMP-induced promoter activity in an orientation-specific manner. Sequence analysis revealed the existence of distal and proximal consensus AP-2 recognition sites located 5' from the TATA box. Both sequences bound specifically to human AP-2 in vitro by gel shift mobility assay. The possible role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with the IGFBP-5 promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated IGFBP-5 promoter activity. This trans-activation was IGFBP-5 promoter and AP-2 specific. In AP-2 abundant fibroblasts, expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed IGFBP-5 promoter activity. In HepG2 cells, AP-2B alone had no significant effect, but the AP-2-induced activation of promoter activity was inhibited by AP-2B in a dose-dependent manner. The relative functional importance of the putative AP-2 binding sites was examined using a number of deletion mutants and point mutations. When the first two distal CCCCACCC-like putative AP 2 sites were deleted or mutated, there was no change in AP-2-induced trans activation. Deletion or mutation of the proximal GCCNNNGGC-like sequences, however, abolished the AP-2-induced activation. These results suggest that AP-2 regulates the IGFBP-5 gene expression through the proximal GCCNNNGGC-like sequences. This AP-2-mediated trans-activation contributes at least in part to the constitutively high expression of IGFBP-5 in fibroblasts and to the cAMP responsiveness of this gene. PMID- 7559605 TI - Molecular cloning and pattern of expression of an alpha-L-fucosidase gene from pea seedlings. AB - alpha-L-Fucosidase is a cell wall protein purified from pea (Pisum sativum) epicotyls. The alpha-L-fucosidase hydrolyzes terminal fucosyl residues from oligosaccharides of plant cell wall xyloglucan. alpha-L-Fucosidase may be an important factor in plant growth regulation, as it inactivates fucose-containing xyloglucan oligosaccharides that inhibit growth of pea stem segments. The amino acid sequences of the NH2-terminal region and one internal peptide were used to design redundant oligonucleotides that were utilized as primers in a polymerase chain reaction (PCR) with cDNA, generated from pea mRNA, as the template. A specific PCR amplification product containing 357 base pairs was isolated, cloned, and sequenced. The deduced amino acid sequence included the two peptides used to design the primers for PCR plus two other peptides obtained by proteinase digestion of alpha-L-fucosidase. No sequence homology to other alpha-L fucosidases was apparent, although the NH2-terminal region is strongly homologous to Kunitz-type trypsin inhibitors. cDNA and genomic copies were isolated and sequenced. In pea, the gene is present in two or three copies. Its mRNA is present in roots, leaves, and elongating shoots. The spatial pattern of expression of the alpha-L-fucosidase was determined by in situ hybridization. PMID- 7559608 TI - The role of the leader sequence coding region in expression and assembly of bacteriorhodopsin. AB - Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane. PMID- 7559607 TI - Upstream enhancer activity in the human surfactant protein B gene is mediated by thyroid transcription factor 1. AB - Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5'-flanking region of the human SP-B gene (-439 to -331 base pair, hSP-B(-439/ 331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter luciferase reporter gene in a human pulmonary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to bhe region of hSP-B(-439/-331). Co-transfection of H441 4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (-80 to -110) and distal (-439 to -331) cis-acting elements than regulate lung epithelial cell-specific transcription of the human SP-B gene. PMID- 7559610 TI - Functional domains of soluble guanylyl cyclase. AB - Soluble guanylyl cyclase is a heterodimer consisting of an alpha and beta subunit and stimulation occurs upon binding of NO to a prosthetic group. Little is known about the localization of catalytic and regulatory domains within the subunits of soluble guanylyl cyclase. We used deletion mutagenesis to identify the regions of alpha 1 and beta 1 subunits that are responsible for cGMP production or NO-heme mediated activation. The amino terminus of the beta 1 subunit was necessary for NO stimulation since deletion of the 64 NH2-terminal amino acids resulted in a mutant with intact basal activity but complete loss of NO activation. The amino terminus of the alpha 1 subunit also appeared to be essential for NO sensitivity since deletion of 131 NH2-terminal amino acids of alpha 1 led to markedly reduced NO activation. These results suggest that NH2-terminal regions of alpha 1 and beta 1 are involved in NO-heme-mediated signal transduction. The NH2 terminally truncated beta 1 subunit exerted a dominant negative effect exclusively on the NO stimulated activity of the wild type enzyme, further underlining that the regulatory domain is located within the NH2 terminus of the enzyme. Aside for the structural implications, the mutant represents a powerful tool to investigate nitric oxide-sensitive signaling pathways. Coexpression of the COOH-terminal halves of alpha 1 and beta 1 were sufficient for basal cGMP production while either of the halves expressed alone was inactive. Therefore the COOH-terminal regions appear to contain sufficient information for dimerization and basal enzymatic activity. Thus, we provide the first evidence that the regulatory and catalytic properties of soluble guanylyl cyclase can be attributed to different regions of the subunits and that the catalytic domain can be functionally expressed separately from the NH2-terminal regulatory domain. Taken together with findings on the membrane bound enzyme form, guanylyl cyclases, appear to resemble fusion proteins where different regulatory domains have been joined with a common cGMP-forming segment. PMID- 7559612 TI - The ligand binding domain of the human retinoic acid receptor gamma is predominantly alpha-helical with a Trp residue in the ligand binding site. AB - Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR). The details of this interaction are unknown due mainly to the lack of sufficient quantities of pure functional receptor protein for biochemical and structural studies. We have recently subcloned the D and E domains of human RAR gamma for expression in Escherichia coli. Using nickel-chelation affinity chromatography with a polyhistidine amino terminal tail, purification of the DE peptide with a pI of 5.18 was accomplished to greater than 98% purity. Scatchard analysis and fluorescence quenching techniques using the purified protein indicate a very high percentage of functional molecules ( > 95%) with a Kd for retinoic acid (t-RA) of 0.6 +/- 0.1 nM. Circular dichroism spectra of the purified domains predict a predominantly alpha-helical structure (approximately 56%) with little beta sheet present. No significant changes in these structural characteristics were observed upon binding of t-RA. Inspection of the amino acid sequence within these domains identified a single tryptophan residue at position 227. Modeling the amino acid sequence in this region as an alpha-helical structure indicates that this tryptophan is adjacent to alanine 234, which corresponds to alanine 225 in RAR beta that has previously been linked to the ligand binding site. Fluorescence of this tryptophan was quenched in a dose-dependent manner on the addition of t-RA, confirming that Trp-227 is within the ligand binding site. Tryptophan fluorescence quenching analysis also demonstrates that a single retinoic acid molecule is bound per receptor and suggests that receptor-ligand interactions occur within the amino-terminal portion of the predominantly alpha-helical ligand binding domain. PMID- 7559611 TI - Heme degradation in the presence of glutathione. A proposed mechanism to account for the high levels of non-heme iron found in the membranes of hemoglobinopathic red blood cells. AB - Unstable hemoglobins and oxidative conditions tend to produce hemichromes which demonstrably release their heme to the erythrocyte membrane, with consequent lipid peroxidation and cell lysis. High levels of non-heme iron are also found in such circumstances, but the origin of this iron is uncertain. In the present work, we show that reduced glutathione (GSH) is able to degrade heme in solution with a pH optimum of 7. Degradation depended on the presence of oxygen and on heme and GSH concentrations. It was inhibited by catalase and superoxide dismutase, implicating the involvement of perferryl reactive species in the process of heme degradation. Heme degradation at pH 7 and 37 degrees C is rapid (t1/2 = 70 s) and results in the release of iron from heme. Heme that was dissolved in red blood cell ghosts is also degraded by GSH with a concomitant increase in non-heme iron, most of which (75%) remains associated with the cell membrane. Loading of intact erythrocytes with heme was followed by time-dependent decrease of membrane-associated heme and caused an acceleration of the hexose monophosphate shunt due to the production of H2O2 and the oxidation of intracellular GSH. Most of the activation of the hexose monophosphate pathway was due to redox cycling of iron, since iron chelators inhibited it considerably. These results explain the origin of non-heme iron found in the membrane of sickle cells and the oxidative stress that is observed in these and other abnormal erythrocytes. PMID- 7559609 TI - Cationic liposome-mediated intravenous gene delivery. AB - Systemic gene transfer provides new opportunities for the analysis of gene function and gene regulation in vivo, as well as for human gene therapy. We used the chloramphenicol acetyltransferase reporter gene to examine several parameters important for the development of efficient, cationic liposome-mediated, intravenous (IV) gene transfer in mice. We then demonstrated that this approach can produce high level expression of biologically important genes. Specifically, we assessed the relationship of expression vector design to the level of systemic gene expression produced, and compared transfection levels produced by intravenously injecting DNA alone versus DNA-liposome complexes. We found that both the position of the heterologous intron, and the promoter element used in the expression plasmid, significantly affected the level of systemic gene expression produced. Although intravenous injection of plasmid DNA alone transfected every tissue analyzed, liposome-mediated delivery was much more efficient. We also established that repeated i.v. injection of DNA-liposome complexes produced high level systemic transfection. The second injection of DNA liposome complexes produced levels of gene expression at least as high as those following a single i.v. injection. Thus, unlike some viral vectors, a neutralizing host-immune response does not limit re-expression, following reinjection of DNA-liposome complexes. Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, i.v. injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophage-colony stimulating factor cDNA or the human granulocyte CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant. PMID- 7559614 TI - Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release. AB - Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component. PMID- 7559613 TI - Atrial natriuretic peptide suppresses the transcription of its guanylyl cyclase linked receptor. AB - Atrial natriuretic peptide (ANP) treatment of rat aortic smooth muscle cells suppressed both 125I-ANP binding and ANP-dependent cGMP accumulation, suggesting reductions in the type C (NPR-C) and type A (NPR-A) natriuretic peptide receptor populations, respectively. NPR-A, but not NPR-C, mRNA levels were reduced in a dose-dependent fashion by ANP. The latter effect appeared to be due, at least in part, to suppression of NPR-A gene promoter activity. ANP effected a dose- and time-dependent reduction in a transiently transfected NPR-A luciferase reporter ( 1575LUC). Analysis of 5' deletion mutants of the NPR-A promoter demonstrated that the ANP-dependent sequence lies between -1575 and -1290 relative to the transcription start site. Inhibition of the ANP promoter was also effected by brain natriuretic peptide, type C natriuretic peptide, and 8-bromo-cGMP, but not by the NPR-C-selective ligand cANF. In the case of 8-bromo-cGMP, the responsive element(s) was localized to the same 285-base pair region linked to the ANP effect above. These findings indicate that ANP autoregulates its own receptors in these cells and, at least in the case of NPR-A, it does so through suppression of receptor gene expression and receptor synthesis. This suppression may operate through a cGMP-dependent element located more than a kilobase upstream from the transcription start site. PMID- 7559615 TI - Growth hormone induction of hepatic serine protease inhibitor 2.1 transcription is mediated by a Stat5-related factor binding synergistically to two gamma activated sites. AB - A growth hormone (GH)-inducible nuclear factor (GHINF) from rat liver has been purified to near homogeneity. On SDS-polyacrylamide gel electrophoresis and UV cross-linking, a major band of mass approximately 93 kDa and a minor band of approximately 70 kDa are detected in the purified fraction. DNase I footprinting using purified GHINF yields a protected region of -149/-115 on the rat serine protease inhibitor 2.1 (Spi 2.1) promoter encompassed within the growth hormone response element (GHRE). Mutational analysis demonstrated that GHINF binds synergistically to two gamma-interferon-activated sites (GAS) within the GHRE, with the 3' element being the pivotal binding domain. Functional assays show that both GAS elements are necessary for full GH response. GHINF has no immunoreactivity with either a C-terminal Stat1 antibody or an N-terminal Stat3 antibody, while cross-reacting with a C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS elements from the Stat5 binding region of the beta-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription. PMID- 7559616 TI - Ca2+ x calmodulin prevents myristoylated alanine-rich kinase C substrate protein phosphorylation by protein kinase Cs in C6 rat glioma cells. AB - Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level. PMID- 7559617 TI - Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region. AB - Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF I gene transcription associated with diabetes mellitus. PMID- 7559618 TI - Degradation of apolipoprotein B in cultured rat hepatocytes occurs in a post endoplasmic reticulum compartment. AB - The site of apolipoprotein B (apoB) degradation was investigated in cultured rat hepatocytes. Brefeldin A plus nocodazole completely blocked apoB degradation suggesting the involvement of a post-endoplasmic reticulum (ER) compartment. Monensin inhibited apoB degradation by 40% implying that a post-Golgi compartment could be involved in degradation of apoB. Ammonium chloride or chloroquine inhibited partially the degradation of apoB100 and apoB48, indicating some degradation in lysosomes, or in an acidic compartment such as trans-Golgi or endosomes. The degradations of apoB100 and apoB48 were blocked completely by (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (EST) during a chase of 90 min demonstrating that a cysteine protease was responsible for apoB degradation. Chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and aprotinin had no significant effect on the degradation of apoB48. However, leupeptin and pepstatin decreased the degradation of apoB100 by 20-30%. Degradation of apoB100 and apoB48 occurred in isolated Golgi fractions with little degradation in heavy or light ER. Degradation of apoB in Golgi fractions was inhibited by EST and by preincubating hepatocytes with 10 nM dexamethasone. Immunofluorescent microscopy revealed that apoB accumulated in the Golgi region after EST treatment. It is concluded that a major part of apoB degradation in rat hepatocytes occurs in a post-ER compartment via the action of a cysteine protease that is regulated by glucocorticoids. PMID- 7559620 TI - Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. AB - Heparan sulfate proteoglycans are thought to be obligatory for receptor binding and subsequent mitogenic activity of basic fibroblast growth factor (FGF-2). In a previous study (Nurcombe V., Ford, M. D., Wildschut, J., Bartlett, P. F. (1993) Science 260, 103-106) we have shown that primary cultures of mouse neuroepithelial cells and a cell line derived from then, 2.3D, secrete a heparan sulfate proteoglycan with a high affinity for FGF-2. In this study, a combination of affinity chromatography and gel chromatography was used to further isolate heparan sulfate side chains with high affinity for FGF-2. These active chains had an average molecular weight of 18,000-20,000. In order to determine whether heparan sulfate chains with specificity for FGF-2 also displayed selectivity for the different FGF receptors, peptides designed to the heparin-binding region of the receptors were used in competitive inhibition studies. The structure of the predicted heparin-binding domain of the FGF receptor 1 was modeled on the basis of its presumed secondary and tertiary structure homology with immunoglobulin loops. These results suggested that many of the basic residues within the second immunoglobulin loop of the FGF receptor 1 form a basic domain in the molecule and therefore form part of a heparin-binding site. Peptides homologous to this region of FGF receptor 1 were shown to inhibit mitogenesis in 2.3D cells, while those to FGF receptor types 2, 3, and 4 did not. A reverse transcriptase-polymerase chain reaction assay designed to detect expression of the four FGF receptors types demonstrated that FGF receptors 1 and 3 were present on the 2.3D cell line but that receptors 2 and 4 were not. These findings indicate that unique heparan sulfate domains interact with specific cell-surface receptors to direct cellular responses. PMID- 7559619 TI - The far-upstream enhancer of the carbamoyl-phosphate synthetase I gene is responsible for the tissue specificity and hormone inducibility of its expression. AB - The role of the proximal promoter and the far-upstream enhancer in the hepatocyte specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5'-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity. PMID- 7559621 TI - Distinct isoforms of the CD45 protein-tyrosine phosphatase differentially regulate interleukin 2 secretion and activation signal pathways involving Vav in T cells. AB - The CD45 family of transmembrane protein-tyrosine phosphatases plays a crucial role in the regulation of lymphocyte activation by coupling activation signals from antigen receptors to the signal transduction apparatus. Multiple CD45 isoforms, generated through regulated alternative mRNA splicing, differ only in the length and glycosylation of their extracellular domains. Differential distribution of these isoforms defines subsets of T cells having distinct functions and activation requirements. While the requirement for the intracellular protein-tyrosine phosphatase domains has been documented, the physiological role of the extracellular domains remains elusive. Here we report the generation of CD45-antisense transfected Jurkat T cell clones that lack CD45 or have been reconstituted to uniquely express either the smallest, CD45(0), or the largest, CD45(ABC), isoform. These cells exhibited marked isoform-dependent differences in IL-2 production and tyrosine phosphorylation of cellular proteins, including Vav after anti-CD3 stimulation. These results demonstrate that the distinct CD45 extracellular domains differentially regulate T cell receptor mediated signaling pathways. Furthermore, these findings suggest that alterations in CD45 isoform expression by individual T cells during thymic ontogeny and after antigen exposure in the periphery directly affects the signaling pathways utilized. PMID- 7559623 TI - Enthalpy and entropy of hydration of bovine crystallins. AB - Transparency of the lens of the eye is the result of a short range order in the packing of crystallin molecules within the fiber cells. Short range order depends on crystallin-crystallin as well as water-crystallin interactions. Light scattering measurements can provide information on the hydration of crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration, and temperature on dilute solutions of beta H, beta L, and gamma fractions of bovine lens crystallins. The temperature dependence of the second virial coefficient was negative for the beta crystallin fractions and positive for the gamma fraction as well as that for alpha crystallin (Wang, X., and Bettelheim, F. A. (1989) Proteins Struct. Funct. Genet. 5, 166-169). The partial molar enthalpy values of the solutions were negative for the beta crystallin fractions, indicating a tendency for homo- and heterodimer and -oligomer association. The enthalpy values were positive for the alpha and gamma fractions. The negative values of the enthalpy of solutions differentiate the beta crystallins from the other crystallins. The partial molar entropy values of solutions of beta L and gamma fractions were identical, those of the oligomeric beta H fraction were higher, whereas those of alpha crystallin were a magnitude larger than those of the smaller crystallin molecules. PMID- 7559622 TI - The effects of streptozotocin-induced diabetes and insulin supplementation on expression of the glycogen phosphorylase gene in rat liver. AB - We have previously observed that the chronic effects of streptozotocin-induced diabetes cause a decrease in the total hepatic glycogen phosphorylase activity with a corresponding reduction in the phosphorylase protein levels. These effects were normalized by insulin administration to diabetic rats. There was no change in the total glycogen synthase activity as a result of diabetes or insulin supplementation. These results are extended to examine the effects of diabetes and insulin administration to diabetic animals on the expression of phosphorylase and glycogen synthase enzymes. The expression (i.e. mRNA levels) of phosphorylase was down-regulated (45% of normal levels) in diabetic livers, and this was normalized by insulin supplementation to diabetic animals. Diabetes or insulin supplementation to diabetic rats showed no effect on the transcription rate of phosphorylase. As expected, diabetes (or insulin administration to diabetic animals) did not cause any alteration in the mRNA levels or in the transcription rate of hepatic glycogen synthase. The stability of phosphorylase mRNA was then examined using hepatocytes prepared from normal and diabetic rats. Diabetes caused a decrease in the half-life of phosphorylase mRNA from 14 h in normal hepatocytes to 6.5 h in diabetic hepatocytes. Insulin supplementation to the medium of diabetic hepatocytes increased the half-life of phosphorylase mRNA to a level comparable with normal values. This study indicates that the chronic effect of insulin on the activation of the total hepatic phosphorylase activity (and protein) is mediated through the stabilization of its mRNA levels. PMID- 7559625 TI - Insulin resistance is mediated by a proteolytic fragment of the insulin receptor. AB - Insulin resistance is a common clinical feature of obesity and non-insulin dependent diabetes mellitus, and is characterized by elevated serum levels of glucose, insulin, and lipids. The mechanism by which insulin resistance is acquired is unknown. We have previously demonstrated that upon chronic treatment of fibroblasts with insulin, conditions that mimic the hyperinsulinemia associated with insulin resistance, the membrane-associated insulin receptor beta subunit is proteolytically cleaved, resulting in the generation of a cytosolic fragment of the beta subunit, beta', and that the generation of beta' is inhibited by the thiol protease inhibitor E64 (Knutson, V. P. (1991) J. Biol. Chem. 266, 15656-15662). In this report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic beta' is generated by chronic insulin administration to the cells, and that E64 inhibits the production of beta'; 2) chronic administration of insulin to the adipocytes leads to an insulin-resistant state, as measured by lipogenesis and glycogen synthesis, and E64 totally prevents the generation of this insulin-induced cellular insulin resistance; 3) E64 has no effect on the insulin-induced down-regulation of insulin receptor substrate-1, and therefore insulin resistance is not mediated by the down-regulation of insulin receptor substrate-1; 4) under in vitro conditions, partially purified beta' stoichiometrically inhibits the insulin-induced autophosphorylation of the insulin receptor beta subunit; and 5) administration of E64 to obese Zucker fatty rats improves the insulin resistance of the rats compared to saline-treated animals. These data indicate that beta' is a mediator of insulin resistance, and the mechanism of action of beta' is the inhibition of the insulin-induced autophosphorylation of the beta subunit of the insulin receptor. PMID- 7559624 TI - Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element. AB - There exist two distinct isozymes of prostaglandin-endoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-O tetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5'-flanking region of the human PES 2 gene (nucleotides -327 to +59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides -1010 to +69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides -132 to -124) and the cyclic AMP response element (CRE) (nucleotides -59 to -53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein delta (C/EBP delta), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP delta mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP delta functions as one of the trans-acting factors. PMID- 7559626 TI - Differential biochemical regulation of the URA7- and URA8-encoded CTP synthetases from Saccharomyces cerevisiae. AB - The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae. URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. The subunit molecular mass (67 kDa) of purified URA8 encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product. Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase. Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5. The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM). GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold. CTP potently inhibited (IC50 = 85 microM) URA8 encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative. The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP. The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA. Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth. PMID- 7559627 TI - Functional analyses of the transcription factor Sp4 reveal properties distinct from Sp1 and Sp3. AB - Sp4 is a human sequence-specific DNA binding protein with structural features similar to those described for the transcription factors Sp1 and Sp3. These three proteins contain two glutamine-rich regions and a highly conserved DNA binding domain composed of three zinc fingers. Consistently, Sp1, Sp3, and Sp4 do have the same DNA binding specificities. In this report, we have embarked on a detailed analysis of the transcriptional properties of Sp4 in direct comparison to Sp1 and Sp3. Cotransfection experiments into Drosophila SL2 cells lacking endogenous Sp factors demonstrate that Sp4 is an activator protein like Sp1. However, in contrast to Sp1, Sp4 is not able to act synergistically through adjacent binding sites. The transactivation function of Sp4 resides, like that of Sp1, in the N-terminal glutamine-rich region. Sp4 can function as a target for the Sp1 activation domains in a superactivation assay, suggesting that the activation domains of Sp1 and Sp4 are functionally related. Furthermore, we show that Sp4-mediated transcriptional activation can be repressed by Sp3. Taken together, our results demonstrate that the transcription factor Sp4 exhibits specific functional properties distinct from Sp1 and Sp3. PMID- 7559628 TI - Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane) [corrected]. AB - When activated, NF-kappa B, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-kappa B family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-kappa B activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-kappa B, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding factor was unaffected. Besides TNF, curcumin also blocked phorbol ester- and hydrogen peroxide-mediated activation of NF-kappa B. The TNF-dependent phosphorylation and degradation of I kappa B alpha was not observed in curcumin treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-kappa B activation pathway at a step before I kappa B alpha phosphorylation but after the convergence of various stimuli. PMID- 7559631 TI - Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. AB - Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation deficient mutant type. PMID- 7559630 TI - Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. AB - Carboxypeptidase E (CPE) is involved in the biosynthesis of most neuropeptides and peptide hormones. Until recently, CPE was the only intracellular carboxypeptidase thought to be involved in neuroendocrine peptide processing. However, the finding that fat/fat mice, which have a mutation within the CPE gene that inactivates the enzyme, are capable of a reduced amount of insulin processing suggests that another carboxypeptidase is present within the secretory pathway. We have detected a CPE-like enzyme, designated CPD, which has many properties in common with those of CPE. Like CPE, CPD is a metallocarboxypeptidase that has a pH optimum of 5.5-6. The Km and Kcat values for a series of short peptide substrates show only minor differences between CPD and CPE. Several active site-directed inhibitors also show generally similar potency toward the two enzymes, although guanidinoethylmercaptosuccinic acid is approximately 10-fold more potent, and hippuryl-Arg is approximately 100-fold more potent as an inhibitor of CPD than of CPE. A major difference between the two enzymes is the molecular masses; CPE is 50,000-56,000, whereas CPD is approximately 180,000. Also, CPD does not elute from a substrate affinity column when the pH is raised to 8, which elutes CPE, although CPD can subsequently be eluted by arginine. Both CPE and CPD are present in purified bovine anterior pituitary secretory vesicles, but the tissue distribution of CPD is more uniform than that of CPE. Antisera to the N- and C-terminal regions of CPE do not recognize CPD. The partial N-terminal amino acid sequence of bovine CPD shows 30 40% homology with an N-terminal region of bovine and rat CPE and 70% homology with a duck protein known as gp180, a hepatitis B virus particle binding protein that shows 47% homology to CPE. Taken together, these results suggest that CPD is a novel secretory pathway enzyme that may be the bovine homologue of gp180. PMID- 7559632 TI - Identification by in Organello footprinting of protein contact sites and of single-stranded DNA sequences in the regulatory region of rat mitochondrial DNA. Protein binding sites and single-stranded DNA regions in isolated rat liver mitochondria. AB - Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB 1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria. PMID- 7559629 TI - Zinc ions inhibit the QP center of bovine heart mitochondrial bc1 complex by blocking a protonatable group. AB - Bovine heart bc1 complex is reversibly inhibited by zinc ions with an inhibition constant KI of 10(-7) M at pH > or = 7.0. Binding of zinc is at least a factor of 10 tighter than binding of any other metal ion tested. Essentially complete inhibition of ubihydroquinone:cytochrome c oxidoreductase activity is observed at concentrations of [Zn2+] > 5 microM. Zinc does not affect the Km for the substrates, ubihydroquinone or cytochrome c, but zinc inhibits reduction of the cytochromes by ubihydroquinone through the QP center. A radioactive binding assay using 65Zn revealed one high affinity binding site per bc1 complex with KD < or = 10(-7) M at pH = 7.0 and 3-4 additional low affinity binding sites (KD > 2 x 10( 6) M). Zinc binding does not depend on the redox state of the high potential chain (iron-sulfur protein and cytochrome c1). Zinc binds 3 times tighter to Fe-S depleted bc1 complex indicating that the zinc binding site is not on the "Rieske" iron-sulfur protein in contrast to a recent report by Lorusso et al. (Lorusso, M., Cocco, T., Sardanella, A.M., Minuto, M., Bonomi, F., and Papa, S. (1991) Eur. J. Biochem. 197, 555-561). Zinc binds to a site which has the same affinity for zinc as for protons. We conclude that the zinc binding site is close to a protonatable group of the bc1 complex with pKa = 7.2 which has not been identified previously. We propose that this group is part of the proton channel at the hydroquinone oxidation center of the bc1 complex. PMID- 7559633 TI - A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells. AB - Signal transduction of cytokine receptors is mediated by the JAK family of tyrosine kinases. Recently, the kinase partners for the interleukin (IL)-2 receptor have been identified as JAK1 and JAK3. In this study, we report the identification of splice variants that may modulate JAK3 signaling. Three splice variants were isolated from different mRNA sources: breast (B), spleen (S), and activated monocytes (M). Sequence analysis revealed that the splice variants contain identical NH2-terminal regions but diverge at the COOH termini. Analyses of expression of the JAK3 splice isoforms by reverse transcriptase-polymerase chain reaction on a panel of cell lines show splice preferences in different cell lines: the S-form is more commonly seen in hematopoietic lines, whereas the B- and M-forms are detected in cells both of hematopoietic and epithelial origins. Antibodies raised against peptides to the B-form splice variant confirmed that the 125-kDa JAK3B protein product is found abundantly in hematopoietic as well as epithelial cells, including primary breast cancers. The lack of subdomain XI in the tyrosine kinase core of the B-form JAK3 protein suggests that it is a defective kinase. This is supported by the lack of detected autokinase activity of the B-form JAK3. Intriguingly, both the S and B splice isoforms of JAK3 appear to co-immunoprecipitate with the IL-2 receptor from HUT-78 cell lysates. This and the presence of multiple COOH-terminal splice variants coexpressed in the same cells suggest that the JAK3 splice isoforms are functional in JAK3 signaling and may enrich the complexity of the intracellular responses functional in IL-2 or cytokine signaling. PMID- 7559636 TI - Evidence for recycling of the resident medial/trans Golgi enzyme, N acetylglucosaminyltransferase I, in ldlD cells. AB - ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc Glc-NAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-Glc-NAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs. PMID- 7559635 TI - Molecular cloning, expression, chromosomal assignment, and tissue-specific expression of a murine alpha-(1,3)-fucosyltransferase locus corresponding to the human ELAM-1 ligand fucosyl transferase. AB - Terminal Fuc alpha 1-3GlcNAc moieties are displayed by mammalian cell surface glycoconjugates in a tissue-specific manner. These oligosaccharides participate in selectin-dependent leukocyte adhesion and have been implicated in adhesive events during murine embryogenesis. Other functions for these molecules remain to be defined, as do the tissue-specific expression patterns of the corresponding alpha-(1-3)-fucosyltransferase (alpha 1-3FT) genes. This report characterizes a murine alpha 1-3FT that shares 77% amino acid sequence identity with human ELAM ligand fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene maps to mouse chromosome 9 in a region of homology with the Fuc-TIV locus on human chromosome 11q. In vitro, the murine alpha 1-3FT can efficiently fucosylate the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc (apparent Km of 0.71 mM) to form an unusual tetrasaccharide (Gal alpha 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) described in periimplantation mouse tissues. The enzyme can also form the Lewis x determinant from Gal beta 1-4GlcNAc (Km = 2.05 mM), and the sialyl Lewis x determinant from NeuNAc alpha 2-3Gal beta 1-4GlcNAc (Km = 1.78mM). However, it does not yield sialyl Lewis x determinants when expressed in a mammalian cell line that maintains sialyl Lewis x precursors. Transcripts from this gene accumulate to low levels in hematopoietic organs, but are unexpectedly abundant in epithelia that line the stomach, small intestine, colon, and epididymus. Epithelial cell-specific expression of this gene suggests function(s) in addition to, and distinct from, its proposed role in selectin ligand synthesis. PMID- 7559634 TI - Efflux of newly synthesized cholesterol and biosynthetic sterol intermediates from cells. Dependence on acceptor type and on enrichment of cells with cholesterol. AB - Previous studies suggest that during sterol synthesis in cells, cholesterol and precusor sterols are transported to the plasma membrane and that this transport is stimulated by the binding of high density lipoprotein (HDL) to its putative cell surface receptor, leading to enhanced sterol efflux. Little is known about the identities of synthesized sterols subject to efflux or whether efflux of cholesterol and precursor sterols are stimulated equally by HDL. To address these issues, cells were incubated with [3H]acetate or [3H]mevalonate and sterol acceptors, and then the labeled sterols in cells and efflux media were analyzed by high pressure liquid chromatography methods that resolved cholesterol and precursor sterols. In non-hepatic cells (Chinese hamster ovary (CHO), fibroblasts, and smooth muscle), cholesterol and multiple precursor sterols accumulated. In CHO cells, the major products were cholesterol and desmosterol, which together constituted 50% of labeled nonsaponifiable lipids. When media contained human HDL3 (1 mg of protein/ml), the molar efflux of synthesized desmosterol was four times that of cholesterol, and the 8-h efflux of these sterols, each normalized to its own production, averaged 48 and 16%, respectively. When media contained egg phosphatidylcholine vesicles (1 mg/ml), the efflux of these sterols averaged 18 and 2.4%, respectively. Thus, with both acceptors, desmosterol was the major synthesized sterol released from cells, and its efflux was substantially greater than that of synthesized cholesterol. High relative efflux of desmosterol (or a desmosterol-like sterol) occurred in all cell types and in both cholesterol-enriched and unenriched cells. These results demonstrated qualitatively similar efflux of synthesized sterols in the presence of HDL3 and phospholipid vesicles, arguing against an absolute requirement for acceptors that interact with the HDL receptor. To probe for possible quantitative differences in the capabilities of these two acceptors, the ratios of (efflux to HDL3)/(efflux to phosphatidylcholine vesicles) were calculated for synthesized cholesterol and desmosterol, plasma membrane cholesterol, and lysosomal cholesterol. In comparison to plasma membrane cholesterol, there was little or no HDL selectivity for lysosomal cholesterol or synthesized desmosterol, whereas there was a 2-3-fold selectivity for synthesized cholesterol, suggesting that the ability of HDL to enhance the efflux of synthesized sterols is a modest quantitative effect and confined to cholesterol. PMID- 7559637 TI - Regulation of placenta-specific expression of the aromatase cytochrome P-450 gene. Involvement of the trophoblast-specific element binding protein. AB - The aromatase (cytochrome P-450AROM) gene contains multiple untranslated exons I that are differentially transcribed in a tissue-specific manner. DNA sequences within the initial -301 upstream of placenta-specific exon I (exon Ia) are sufficient for placenta-specific expression of aromatase. In gel mobility shift assay, three separate domains in this region form specific binding complexes with proteins extracted from choriocarcinoma JEG-3 nuclei. A fragment containing these domains activates transcription driven by a heterologous promoter in a cell type specific manner. Two of the binding domains that form major complexes in gel shift assay compete with each other and with a DNA fragment containing the trophoblast-specific element (TSE), which is derived from the enhancer region of the human chorionic gonadotropin alpha-subunit gene and is believed to confer placenta-specific expression of the gene. The core sequence RNCCTNNRG is sufficient for recognition of the TSE-binding protein, which is detected only in nuclear extracts prepared from placenta and choriocarcinoma. A mutation introduced in the distal TSE core in aromatase promoter resulted in marked reduction of transcriptional activity, although TSE region by itself did not show enhancer activity as that in human chorionic gonadotropin alpha-subunit gene. PMID- 7559638 TI - Molecular cloning of a new member of the p21-Cdc42/Rac-activated kinase (PAK) family. AB - A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40 46). The purified serine/threonine kinase p65PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform beta-PAK, which shares 79% amino acid identity with the previously described alpha-isoform. Their mRNAs are differentially expressed in the brain, with alpha-PAK mRNA being particularly abundant in motor associated regions. In vitro translation products of the alpha- and beta-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific beta-PAK peptide sequence was obtained from rat brain-purified p65PAK. Recombinant alpha- and beta-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of alpha-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant beta-PAK and brain p65PAK, which is observed upon kinase activation does not occur with recombinant alpha-PAK. PMID- 7559640 TI - Functional characterization of an inositol-sensitive upstream activation sequence in yeast. A cis-regulatory element responsible for inositol-choline mediated regulation of phospholipid biosynthesis. AB - A repeated element, the inositol-sensitive upstream activation sequence (UASINO), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UASINO element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UASINO element are homologous with canonical binding motif for proteins of the basic helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UASINO function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UASINO function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10 base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5' CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p x Ino4p binding. The strength of any given sequence as a UASINO element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p x Ino4p binding. We also tested a subset of the modified UASINO elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UASINO activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559639 TI - Mutated yeast heat shock transcription factor exhibits elevated basal transcriptional activation and confers metal resistance. AB - Cadmium-resistant Saccharomyces cerevisiae strain 301N exhibits high basal as well as cadmium-induced expression of the CUP1 metallothionein gene. Since regulation of CUP1 is usually restricted to copper ions, our goal was to identify the factor responsible for the high metallothionein levels in strain 301N. The gene responsible for the observed phenotype is a spontaneously mutated heat shock transcription factor gene (HSF1). A double, semidominant HSF1 mutant with substitutions at codons 206 and 256 within the DNA-binding domain of the heat shock factor (HSF) confers two phenotypes. The first phenotype is elevated transcriptional activity of the HSF mutant (HSF301), which results in constitutive thermotolerance. A second HSF301 phenotype is enhanced binding affinity for the heat shock element (HSE) within the CUP1 5'-sequences, resulting in high basal transcription of metallothionein. The CUP1 HSE is a minimal heat shock element containing only two perfectly spaced inverted repeats of the basic nGAAn block. Cells containing HSF301 are resistant to cadmium salts. The single R206S mutation is responsible for the high affinity binding to the CUP1 HSE. In addition, the R206S HSF substitution exhibits constitutive transcriptional activation from a consensus HSE (HSE2). The F256Y substitution in HSF attenuates the effects of R206S on the consensus HSE2, but not on the CUP1 HSE. PMID- 7559641 TI - Angiotensin II-induced hypertrophy of rat vascular smooth muscle is associated with increased 18 S rRNA synthesis and phosphorylation of the rRNA transcription factor, upstream binding factor. AB - Hypertrophy of vascular smooth muscle cells (VSMC) is an important adaptive response of hypertension. Drug intervention studies have implicated a role for angiotensin II (A-II) in the mediation of VSMC hypertrophy in vivo, and A-II is a potent hypertrophic agent for VSMC in culture. Our laboratory has previously shown that A-II-induced hypertrophy of cultured VSMC is due in part to generalized increases in protein synthesis and increased content of rRNA. The aim of the present study was to determine if A-II stimulates rRNA gene synthesis and whether the rRNA transcription factor, upstream binding factor (UBF), is involved. Nuclear run-on analysis demonstrated that A-II induced a greater than 5 fold increase in rRNA gene synthesis within 6 h of stimulation. A-II also stimulated a rapid increase in UBF phosphorylation as well as nucleolar localization, but no changes in the content of UBF. Phosphoamino acid analysis showed that phosphorylation occurred only on serine residue(s). Results demonstrate that increased transcription of ribosomal DNA contributes to the A-II induced increase in protein synthesis and VSMC hypertrophy, and suggest that an important regulatory event in this pathway may be the phosphorylation and/or nucleolar localization of UBF. PMID- 7559642 TI - Effects of lovastatin on trafficking of cystic fibrosis transmembrane conductance regulator in human tracheal epithelium. AB - Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, cause cystic fibrosis. Most defective forms of CFTR show improper intracellular trafficking. Because isoprenylated, small GTP-binding proteins are involved in the vesicular trafficking of other integral membrane proteins, we have investigated the role of isoprenylation in the trafficking of CFTR to the apical membranes of primary cultures of human airway epithelium and of Calu-3 cells, a human lung carcinoma cell line. CFTR function was measured as short circuit current, 125I efflux, and conductance of cell sheets with permeabilized basolateral membranes. Lovastatin, an inhibitor of isoprenyl lipid biosynthesis, markedly inhibited all measures of CFTR function. The lovastatin-induced declines in CFTR function were corrected by the simultaneous addition of mevalonate or the isoprenyl lipids geranylgeranyl and farnesyl but not cholesterol. Lovastatin reduced total cellular CFTR as assessed by immunoprecipitation. Mevalonate or isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for isoprenyl lipids, presumably through the actions of small GTP-binding proteins, in the trafficking of CFTR to the apical membrane of human airway epithelium. PMID- 7559644 TI - The role of the calpain-calpastatin system in thyrotropin-releasing hormone induced selective down-regulation of a protein kinase C isozyme, nPKC epsilon, in rat pituitary GH4C1 cells. AB - We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals. PMID- 7559643 TI - Cytotoxic T lymphocyte-associated molecule-4, a high-avidity receptor for CD80 and CD86, contains an intracellular localization motif in its cytoplasmic tail. AB - CD28 and CTLA-4, T cell receptors for B7-1 (CD80) and B7-2 (CD86) molecules on antigen-presenting cells, transmit costimulatory signals important for optimal T cell activation. Despite sharing sequence homology and common ligands, these receptors have distinct binding properties and patterns of expression. The function of CTLA-4 during T cell activation is not well understood, although an important role is suggested by complete amino acid sequence conservation of its cytoplasmic tail in all species studied to date. We report here a role of the cytoplasmic tail of CTLA-4 in regulating its subcellular localization and cell surface expression. In activated human peripheral blood T cells, or in several transfected or transduced cell types, CTLA-4 is not primarily a cell surface protein, but rather is localized intracellularly in a region which overlaps the Golgi apparatus. Transfer of 11 cytoplasmic residues, 161TTGVYVKMPPT, from the CTLA-4 cytoplasmic tail to the homologous position in CD28 was sufficient to confer intracellular localization. Mutation of the tyrosine residue (Tyr165) in this motif to phenylalanine resulted in increased surface expression of CTLA-4. Thus, the subcellular localization of CTLA-4 is controlled by a tyrosine containing motif within its cytoplasmic domain. Contained within this motif is a binding site for SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase. PMID- 7559645 TI - Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels. AB - New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine. PMID- 7559647 TI - Low resolution X-ray structure of human methylamine-treated alpha 2 macroglobulin. AB - The structure of methylamine-treated human alpha 2-macroglobulin (alpha 2M-Ma), a 720-kDa tetrameric inactivated proteinase inhibitor from plasma, has been determined to a resolution of 10 A. Data were collected with synchrotron radiation at 120 K, and phases were calculated by multiple isomorphous replacement and solvent flattening. A novel feature of the structure of alpha 2-M is present in its proteinase-binding cavity, dividing it into two compartments. The potential sites for proteinase entrapment in these compartments are sterically restricted. The positions of the thiol groups appearing from the functional important thiol esters upon their cleavage have been determined. They are found at the walls of the compartments at the center of the structure. The overall structure of alpha 2M-MA is much more sphere-like than previously inferred from electron microscopy studies. However, several aspects of the structure are well described by recent three-dimensional reconstructions. Possible models for the monomer, the disulfide bridged dimer, and native alpha 2M are discussed. PMID- 7559646 TI - Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants. AB - The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme. Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M. (1992) J. Mol. Biol. 226, 931-933). Here we report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile-67-->Val, Ile-48-->Thr, Gly-44-->Asp, Lys-36-->Glu, Ala-30-->Thr, and Pro-15-->Leu) and a nonsense mutation (N59-mer) at the codon for Lys-18. These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity. Surprisingly, Lys-36- >Glu, Ala-30-->Thr and Pro-15-->Leu were found to still function as a chaperone for in vitro refolding of denatured subtilisin BPN' with 60, 80, and 54% efficiency compared to the wild-type propeptide, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively, almost identical to the Ki value exhibited by the wild-type propeptide (1.4 x 10(-9) M). In contrast, Ile-67-->Val and Gly-44-->Asp were able to refold denatured subtilisin BPN' with only 18 and13% efficiencies and had Ki values of 10 and 11 x 10(-9) M, respectively. The Ile-48-->Thr mutant propeptide was unable to refold denatured subtilisin BPN' and gave a 100-fold higher Ki (118 x 10(-9) M) than the wild-type propeptide. The N59-mer propeptide extending from Leu-19 to Met-78 was unable to function as a chaperone. Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra. The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme. This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme. PMID- 7559649 TI - Expression of a Drosophila GATA transcription factor in multiple tissues in the developing embryos. Identification of homozygous lethal mutants with P-element insertion at the promoter region. AB - GATA transcription factors are DNA-binding proteins that recognize the core consensus sequence, WGATAR. Previous studies indicated that GATA factors play ann important role in the development of tissue-specific functions in vertebrates. Here we report the identification of a new Drosophila melanogaster GATA factor, dGATAc, which displays a distinct expression pattern in embryos. The local concentration of dGATAc transcripts varies at different stages, being most prominent in the procephalic region at stages 6-10 and in the posterior spiracles, the gut, and the central nervous system at stages 11-13. On the basis of its predicted sequence, DNA-binding assays were performed to confirm that the dGATAc gene encodes a zinc finger protein that can bind the GATA consensus motif with predicted specificity. Two independent mutants carrying a P-element insertion at the dGATAc gene promoter region were identified that are homozygous lethal at the embryonic stage. Using a genetic scheme, it was demonstrated that the lack of dGATAc function can block normal embryonic development. Our results suggest that the dGATAc protein is a tissue-specific transcription factor that is vital to the development of multiple organ systems in D. melanogaster. PMID- 7559648 TI - Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon. AB - Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria. PMID- 7559650 TI - Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development. AB - We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism. PMID- 7559651 TI - Unexpected consequences of deletion of the first two repeats of the ligand binding domain from the low density lipoprotein receptor. Evidence from a human mutation. AB - Heterozygosity for a 5-kilobase (kb) deletion of the first two ligand-binding repeats (exons 2 and 3) of the low density lipoprotein (LDL) receptor (R) gene (LDL-R delta 5kb) confers familial hypercholesterolemia (FH). The FH phenotype is unexpected based on previous site-directed mutagenesis showing that deletion of exons 2 and 3 resulted in little or no defect in LDL-R activity. In the present study, we took unique advantage of the ability to distinguish the LDL-R delta 5kb from the normal receptor on the basis of size, in order to resolve this apparent discrepancy. Fibroblasts from heterozygotes for the LDL-R delta 5kb displayed 50% of normal capacity to bind LDL and beta-VLDL, apparently due to lower receptor number. Cellular mRNA for the delta 5kb allele was at least as abundant as that for the normal allele. Immunoblotting and cell binding assays with anti-LDL-R antibody IgG-4A4 demonstrated normal synthesis and transport of the delta 5kb receptor. Ligand blotting demonstrated that the delta 5kb receptor displayed minimal or no ability to bind LDL or beta-VLDL. Thus, in contrast to transfected cell lines, in human fibroblasts, the first two cysteine rich repeats of the LDL R appear functionally necessary. These characteristics of the LDL-R delta 5kb in human fibroblasts explain the in vivo phenotype of carriers. PMID- 7559654 TI - The yeast immunophilin Fpr3 is a physiological substrate of the tyrosine-specific phosphoprotein phosphatase Ptp1. AB - The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo. The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo. Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells. One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1. p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family. The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1. Site directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3. Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine. Fpr3 represents the first phosphotyrosyl protein identified in S. cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1. PMID- 7559652 TI - The ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments. AB - The low molecular weight GTP-binding proteins RhoA, RhoB, and RhoC are characterized as specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum and are supposed to be involved in the organization of the microfilamental network and transformation. rhoB is known to be immediate-early inducible by growth factors and protein-tyrosine kinases. Since increasing evidence indicates overlapping of growth factor- and UV-induced signal pathways, we studied the effect of UV light and other genotoxic agents on early rhoB transcription. Within 30 min after UV irradiation of NIH3T3 cells, the amount of rhoB mRNA increased 3-4-fold. Elevated rhoB mRNA was accompanied by an increase in RhoB protein, as detected by C3-mediated [32P]ADP-ribosylation. The transcription inhibitor actinomycin D prevented the UV-induced increase in rhoB mRNA and proved rhoB mRNA to be unstable with a half-life of approximately 20 min. Transcriptional activation of rhoB by UV light was confirmed by run-on analysis. The increase in rhoB mRNA after UV irradiation was prevented by inhibitors of protein kinase A (H9) and C (H7, Go18). The tyrosine kinase inhibitor genistein did not affect UV induction of rhoB. In addition to UV, N methyl-N-nitrosourea and the cytostatic drug cisplatin evoked rhoB response. Cycloheximide was likewise effective in increasing the amount of rhoB mRNA, whereas Bt2cAMP, 12-O-tetradecanoylphorbol-13-acetate, and retinoic acid were without effect. Prior down-regulation of signaling by 12-O-tetradecanoylphorbol 13-acetate and serum pretreatment reduced UV-stimulated rhoB expression. The data indicate that rhoB represents a novel DNA damage-inducible function involved in early steps of signal transduction upon genotoxic stress. PMID- 7559653 TI - The role of N-glycosylation for functional expression of the human platelet activating factor receptor. Glycosylation is required for efficient membrane trafficking. AB - Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane. PMID- 7559655 TI - Functional analyses of the human metallothionein-IG gene. In vitro and in vivo studies. AB - We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region ( 174 to +5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box binding protein as observed using DNase I protection. PMID- 7559657 TI - Functional analysis of the high affinity, Na(+)-dependent glutamate transporter GLAST-1 by site-directed mutagenesis. AB - The reuptake of excitatory amino acids, such as glutamate, terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the first member of the recently discovered glutamate transporter family, which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is structurally closely related to this new family of membrane proteins. Transmembrane transport of neutral amino acids is expected to differ in its binding site from that of the acidic excitatory amino acids glutamate and aspartate. Three positively charged amino acid residues, Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all glutamate transporters. They are replaced by apolar amino acid residues in the ASCT1 sequence. We exchanged these residues in the GLAST-1 specific cDNA by site-directed mutagenesis. cRNAs of these mutants were expressed in the Xenopus oocyte system. The functional characterization of the mutants R122I and R280V and the double mutant R122I, R280V revealed that the mutations have no influence on the intrinsic properties and kinetics of glutamate transport but alter the Km-values for L-aspartate and the competitive inhibitor D,L-threo-3 hydroxy aspartate. Substitutions of Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate the glutamate transporter. Immunoprecipitations of [35S]methionine-labeled transporter molecules indicate similar expression levels of wild-type and mutant transporters. Immunostaining of oocyte sections clearly proves the correct targeting to and integration of the mutant GLAST-1 proteins in the plasma membrane. Our results suggest the pivotal function of the hydroxy group of the highly conserved Tyr-405 and the positively charged Arg-479 in the binding of the negatively charged acidic neurotransmitter glutamate. PMID- 7559656 TI - Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase. AB - Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+) sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, E.V., Laura, R.P., and Hurley, J.B. (1994) Neuron 12, 1345-1352; Lowe, D.G., Dizhoor, A.M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as GCAP-1. PMID- 7559658 TI - Ke 6 gene. Sequence and organization and aberrant regulation in murine polycystic kidney disease. AB - Ke 6 gene is a newly identified gene located in the major histocompatibility complex and is a candidate steroid dehydrogenase gene because of structural homology and regulatory similarities with mammalian steroid dehydrogenases. We report here the complete nucleotide sequence and intron-exon organization of the Ke 6 gene and cloning of the alternatively spliced Ke 6b transcript. We find that Ke 6 gene expression is down-regulated in pcy mice which is a murine model of polycystic kidney disease (PKD). Thus far, Ke 6 gene expression is down-regulated in all murine models of PKD we have examined. Abnormal steroid metabolism as a possible cause of PKD is discussed. PMID- 7559659 TI - Apoprotein B100, an inefficiently translocated secretory protein, is bound to the cytosolic chaperone, heat shock protein 70. AB - Apoprotein B100 (apoB) is a secretory protein that appears to be constitutively translated but inefficiently translocated into the lumen of the endoplasmic reticulum. Using several experimental approaches, we found that apoB is bound to the cytosolic chaperone protein, heat shock protein 72/73 (commonly referred to as Hsp70). Similar to other chaperone-protein interactions, this binding was transient and ATP-sensitive. The binding of apoB to Hsp70 in HepG2 cells was decreased by treatment with oleic acid, which increases both translocation and secretion of apoB, and was increased by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor which efficiently protects apoB from cellular degradation without affecting translocation. The N-terminal 16% of apoB, which is efficiently translocated into the endoplasmic reticulum lumen in stably transfected Chinese hamster ovary (CHO) cells, showed minimal, if any, binding to Hsp70. The N terminal 50% of apoB, which is very poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggest that domains of nascent apoB localized on the C-terminal regions of the molecule are transiently exposed to the cytosol during translation and/or translocation, and that Hsp70 functions as a molecular chaperone to maintain apoB in a translocational competent conformation until translocation is completed. PMID- 7559660 TI - Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation. Role of plastoquinol bound to the reduced cytochrome bf complex. AB - Signal transduction via light-dependent redox control of reversible thylakoid protein phosphorylation has evolved in plants as a unique mechanism for controlling events related to light energy utilization. Here we report for the first time that protein phosphorylation can be activated without light or the addition of reducing agents by a transient exposure of isolated thylakoid membranes to low pH in darkness. The activation of the kinase after incubation of dark-adapted thylakoids at pH 4.3 coincides with an increase in the plastoquinol: plastoquinone ratio up to 0.25. However, rapid plastoquinol reoxidation ( < 1 min) at pH 7.4 contrasts with the slow kinase deactivation (t 1/2 = 4 min), which indicates that the redox control is not directly dependent on the plastoquinone pool. Use of inhibitors and a cytochrome bf-deficient mutant of Lemna demonstrate the involvement of the cytochrome bf complex in the low-pH induced protein phosphorylation. EPR spectroscopy shows that subsequent to the transient low pH treatment and transfer of the thylakoids to pH 7.4, the Rieske Fe-S center, and plastocyanin become reduced and are not reoxidized while the kinase is slowly deactivated. However, the deactivation correlates with a decrease of the EPR gz signal of the reduced Rieske Fe-S center, which is also affected by quinone analogues that inhibit the kinase. Our data point to an activation mechanism of thylakoid protein phosphorylation that involves the binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center. PMID- 7559661 TI - Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes. AB - Interleukin-1 triggers the down-regulation of several hepatic cytochrome P450 gene products, but the cellular signaling pathways involved are not known. We have examined the role of sphingomyelin hydrolysis to ceramide in the suppression of CYP2C11, a major constitutive form of cytochrome P450, by interleukin-1. Treatment of rat hepatocytes cultured on matrigel with interleukin-1 beta caused a rapid turnover of sphingomyelin and an increase in cellular ceramide, with no change in cellular phosphatidylcholine. The ceramide was composed mainly of a D erythro-sphingosine backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of CYP2C11 and induced the expression of the interleukin-1-responsive alpha 1-acid glycoprotein mRNA. In contrast, the acute-phase gene beta-fibrinogen, which is induced by interleukin-6 but not by interleukin-1, did not respond to C2-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the effect of C2-ceramide on CYP2C11, but not on alpha 1-acid glycoprotein expression. These results are consistent with a role for ceramide or a related sphingolipid in mediating the down-regulation of CYP2C11, the induction of alpha 1-acid glycoprotein, and perhaps other cellular effects of interleukin-1 in hepatocytes. PMID- 7559662 TI - Role of a proximal NF-Y binding promoter element in S phase-specific expression of mouse ribonucleotide reductase R2 gene. AB - Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains. PMID- 7559663 TI - Effects of 12-O-tetradecanoylphorbol-13-acetate on estrogen receptor activity in MCF-7 cells. AB - The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor. PMID- 7559664 TI - Involvement of direct phosphorylation in the regulation of the rat parotid Na(+) K(+)-2Cl- cotransporter. AB - We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na(+)-K(+)-2Cl- cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na(+)-K(+)-2Cl- cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the beta adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (approximately 20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF(-)4 treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (approximately 6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (approximately 3 fold and approximately 6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca(2+)-mobilizing agent thapsigargin, which is able to fully mimic the up regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na(+)-K(+)-2Cl- cotransporter. PMID- 7559665 TI - Dual effect of beta-adrenergic receptors on mitogen-activated protein kinase. Evidence for a beta gamma-dependent activation and a G alpha s-cAMP-mediated inhibition. AB - The enzymatic activity of mitogen-activated protein kinases (MAP kinases) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins of the Gi and Gq family. Recently, it has been shown that stimulation of beta-adrenergic receptors, which are typical of those that act through Gs to activate adenylyl cyclases, potently activates MAP kinases in the heart, resulting in the hypertrophy of the cardiac muscle (Lazou, A., Bogoyevitch, M.A., Clerk, A., Fuller, S.J., Marshall, C.J., and Sudgen, P.H. (1994) Circ. Res. 75, 938-941). We have observed that exposure of COS-7 cells to a beta-adrenergic agonist, isoproterenol, raises intracellular levels of cAMP and effectively activates protein kinase A (PKA) and an epitope tagged MAP kinase. However, MAP kinase stimulation by isoproterenol was neither mimicked by expression of an activated mutant of G alpha s, nor by treatment with PKA-stimulating agents. Moreover, pretreatment of COS-7 with a permeable cAMP analog, 8-Br-cAMP, markedly decreased MAP kinase activation by either isoproterenol or epidermal growth factor. Thus, in COS-7 cells cAMP and PKA do not appear to mediate MAP kinase activation by beta-adrenergic receptors. Signaling from beta-adrenergic receptors to MAP kinase was inhibited by transfection of a chimeric molecule consisting of the CD8 receptor and the carboxyl terminus of the beta-adrenergic receptor kinase, which includes the beta gamma-binding domain. MAP kinase activation by isoproterenol was not affected by depletion of protein kinase C, but it was completely abolished by expression of Ras-inhibiting molecules. We conclude that signaling from beta-adrenergic receptors to MAP kinase involves an activating signal mediated by beta gamma subunits acting on a Ras-dependent pathway and a G alpha s-induced inhibitory signal mediated by cAMP and PKA. The balance between these two opposing mechanisms of regulation would be expected to control the MAP kinase response to beta-adrenergic agonists as well as to other biologically active agents known to act on Gs coupled receptors, including a number of hormones, neurotransmitters, and lipid mediators. PMID- 7559666 TI - Promoter analysis of Zfp-36, the mitogen-inducible gene encoding the zinc finger protein tristetraprolin. AB - The gene encoding the putative zinc finger protein tristetraprolin (TTP), Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show here that 77 base pairs 5' of the transcription start site are sufficient for full serum inducibility of the mouse Zfp-36 promoter. This region of the promoter includes consensus sequences for the binding of the transcription factors EGR-1, AP2, and Sp1. In addition, we have identified a previously undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence includes a palindrome and is identical in the human, bovine, and mouse genes. Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the serum induction of Zfp-36 and can confer serum-inducible expression on a heterologous minimal promoter. Gel mobility shift assays demonstrated the formation of complexes consisting of this region of the promoter and cellular nuclear proteins and demonstrated that the extent of complex formation could be altered by treatment of the cells with serum or insulin. These results suggest that the response of Zfp-36 to serum and other mitogens is mediated by a series of cis-acting elements acting in concert to confer full inducible transcription of this gene. PMID- 7559667 TI - Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. AB - The unc-13 gene in Caenorhabditis elegans is essential for normal presynaptic function and encodes a large protein with C1- and C2-domains. In protein kinase C and synaptotagmin, C1- and/or C2-domains are regulatory domains for Ca2+, phospholipids, and diacylglycerol, suggesting a role for unc-13 in regulating neurotransmitter release. To determine if a similar protein is a component of the presynaptic machinery for neurotransmitter release in vertebrates, we studied unc 13 homologues in rat. Molecular cloning revealed that three homologues of unc-13 called Munc13-1, -13-2, and -13-3 are expressed in rat brain. Munc13s are large, brain-specific proteins with divergent N termini but conserved C termini containing C1- and C2-domains. Specific antibodies demonstrated that Munc13-1 is a peripheral membrane protein that is enriched in synaptosomes and localized to plasma membranes but absent from synaptic vesicles. Our data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as plasma membrane proteins in nerve terminals. The presence of C1- and C2-domains in these proteins and the phenotype of the C. elegans mutants raise the possibility that Munc13s may have an essential signaling role during neurotransmitter release. PMID- 7559668 TI - Cloning and characterization of three new murine genes encoding short homologues of RNase P RNA. AB - Three novel genes encoding small RNAs homologous to human and mouse RNase P RNA have been isolated from a mouse genomic library. As assessed by Northern blot analysis and nuclease protection assays, transcripts derived from one or more of these genes are expressed in murine cells and tissues. The RNA products of these RNase P RNA-homologous genes are smaller in size (238-248 nucleotides) than the 305-nucleotide transcript previously identified. These smaller transcripts are uniformly less abundant than the larger RNase P RNA, but their expression varies severalfold among different mouse tissues. Similar short homologues of RNase P RNA also are expressed in rat, rabbit, and human cells. We conclude that higher vertebrates express multiple isoforms of RNase P RNA. PMID- 7559669 TI - The interaction of Escherichia coli topoisomerase IV with DNA. AB - The two type II topoisomerases in Escherichia coli, DNA gyrase and topoisomerase (Topo) IV, share considerable amino acid sequence similarity, yet they have distinctive topoisomerization activities. Only DNA gyrase can supercoil relaxed DNA, whereas during oriC DNA replication in vitro, only Topo IV can support the final stages of replication, processing of the late intermediate and decatenation of the daughter molecules. In order to develop an understanding for the basis of the differential activities of these two enzymes, we have initiated a characterization of Topo IV binding to DNA. We find that unlike gyrase, Topo IV neither constrains DNA in a positive supercoil when it binds nor protects a 150 base pair region of DNA from digestion with micro-coccal nuclease. Consistent with this, DNase I footprinting experiments showed that Topo IV protected a 34 base pair region roughly centered about the topoisomerase-induced cleavage site. In addition, Topo IV preferentially bound supercoiled rather than relaxed DNA. Thus, the DNA binding characteristics of Topo IV are more akin to those of the type II eukaryotic enzymes rather than those of its prokaryotic partner. PMID- 7559670 TI - Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity. AB - The dioxin receptor is a cytoplasmic basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocyclic amines, and halogenated aromatic hydrocarbons, e.g. dioxins. Ligand-induced activation of the dioxin receptor initiates a process whereby the receptor is transformed into a nuclear transcription factor complex with a specific bHLH/PAS partner protein, Arnt. In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a minimal ligand binding domain of the dioxin receptor from the central PAS region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand binding domain in wheat germ lysates or bacteria, systems which harbor hsp90 homologs unable to interact with the glucocorticoid or dioxin receptors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain for dioxin was similar to the affinity inherent in the full-length dioxin receptor, and a profile of ligand structures which specifically bound the minimal ligand binding domain was found to be conserved between this domain and the native receptor. These experiments show that the minimal ligand binding domain maintains the quantitative and qualitative aspects of ligand binding exhibited by the full-length receptor, implying that the central ligand binding pocket may exist to accommodate all classes of specific dioxin receptor ligands, and that this pocket is critically dependent upon hsp90 for its ligand binding conformation. PMID- 7559671 TI - A back-propagation neural network model of lumbar muscle recruitment during moderate static exertions. AB - A model employing artificial neural networks (ANNs) is developed for the prediction of lumbar muscle activity in response to steady-state static external moment loads. The model is constructed using standard feedforward networks and trained with available data using the standard back-propagation algorithm. Training with a limited set of exemplars allowed accurate prediction of muscle activity for novel moment loads (generalization). Sensitivity analyses during training and testing phases showed that the choice of specific network parameters was not critical except at extreme values of those parameters. Model predictions were better correlated with experimental data than predictions made using two optimization-based methods (average r2 = 0.83 using ANNs and 0.65 using optimization). The results suggest that lumbar muscle response varies smoothly and consistently with respect to the magnitude and orientation of external moments, and they also imply an upper limit on the accuracy of muscle activity prediction using only moment loads as input. ANNs present a useful alternative to EMG- and optimization-based approaches by being both 'reality-based' and predictive. PMID- 7559672 TI - Role of plantar fascia in the load bearing capacity of the human foot. AB - Plantar fascia release is an accepted and widely used surgical way to reduce heel pain, however its effect of the load bearing characteristics of the foot is not well studied. A simple biomechanical model is developed here to analyze load bearing mechanism of the foot during the stance phase of the gait cycle. Quasilinearization is used for the system identification, and all model's parameters are determined from the in vivo tests. The model is used to compare the load bearing mechanism of different pathological situations. The results of the study suggest that the plantar fascia carries as much as 14% of the total load on the foot. Its surgical release decreases dynamic loading on the ankle by only 10%. It is also found that the lowering of the arch degenerates the load bearing capacity of the foot. Thus, the plantar fascia plays an important part in the load bearing by the foot and its surgical release should be carefully considered. PMID- 7559673 TI - The force-velocity curve in passive whole muscle is asymmetric about zero velocity. AB - The force-velocity property of passive muscle was investigated to determine if a discontinuity of slope occurred at zero velocity. Isolated, unstimulated whole frog sartorius muscles were subjected to constant-velocity stretches and releases using a servo-controlled lever. The force due to damping (delta T) was calculated by subtracting the tension measured at a very low speed (1.0 mm s-1) from the tension measured at the same length while the muscle was shortening or lengthening at a particular test speed. The experiments were performed over a range of speeds at each of several lengths and at two temperatures. For comparison, the same experiments were performed using a strip of pure latex rubber and a steel spring. Curves showing the magnitude of delta T vs velocity were nearly symmetric about the zero-velocity axis for the steel spring and the rubber strip, but were markedly asymmetric for passive muscle, showing a positive delta T for lengthening at all speeds that was between four and 11 times the negative delta T for shortening at the same speed, depending on the temperature and initial stretch length. The force due to damping at a given speed increased with extension above the rest length in passive muscle but decreased with increasing length in experiments using the latex strip. Predictions obtained from a mathematical model based on a damping element in series with a lightly damped spring were fitted to the experimental measurements of delta T vs velocity. The damping parameter provisionally representing interfilamentary sliding was between six and 12 times larger for lengthening than for shortening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559674 TI - A theory of aneurysm sounds. AB - Narrow-band sounds are known to be associated with some intracranial aneurysms. Previously proposed theories for the mechanism of aneurysm sounds do not satisfactorily explain the small spectral widths of the sounds. A simple theory is proposed here which gives quantitatively correct predictions of the spectral widths and which also explains other salient features of aneurysm sounds. The physical features of the aneurysm are described in terms of lumped mechanical elements, and the interaction between the aneurysm vibration and the blood flow is recognized as having the characteristic features of a nonlinear feedback system. The resulting model, with the application of the method of describing function analysis commonly used in nonlinear control theory, yields predictions of steady oscillation frequencies and predictions of the ranges of arterial flow velocities for which substantial oscillations can be excited. An analysis of radiation losses associated with peristaltic waves indicates that aneurysms, in the absence of any nonlinearity, behave as low-quality factor resonators with resonator quality factors on the order of 1-10, much lower than those that would be inferred from the observed spectral widths of aneurysm sounds. Aneurysm sounds are predicted by the present nonlinear theory to have center frequencies on the order of 400 Hz and bandwidths corresponding to quality factors on the order of 40, in good agreement with in vivo observations. It is concluded that linear resonance theories are incapable of fully describing aneurysm sounds.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559675 TI - The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. AB - The effects of streaming potential, fluid flow and hydrostatic pressure on chondrocyte biosynthesis were studied by comparing the spatial profiles of these physical stimuli to the profiles of biosynthesis within cartilage disks subjected to dynamic unconfined compression. The radial streaming potential was measured using compression frequencies and disk sizes relevant to studies of physical modulation of cartilage metabolism; a general analytical solution to the unconfined compression of a poroelastic cylinder with impermeable, rigid, adhesive platens was derived using potential theory. The solution with adhesive platen boundary conditions, using measured values of cartilage material properties, predicted streaming potentials that were much closer to experimental results between 0.001 and 1 Hz than a solution using frictionless platen boundary conditions. The predicted radial profiles of streaming potential gradient and fluid velocity (but not hydrostatic pressure) were similar to the previously reported radial dependence of proteoglycan synthesis induced by dynamic unconfined compression. Changes in stiffness associated with reduction of disk diameter suggested that the relative contributions of collagen and proteoglycans to cartilage mechanical properties may be a function of loading frequency in unconfined compression; such anisotropies may explain the remaining discrepencies between measured stiffness and stiffness predicted by the present model. PMID- 7559677 TI - Simulation of 'smart' pole vaulting. AB - The process of pole vaulting is simulated using a finite element two-dimensional model of the pole and the vaulter. The pole is modelled with 20 beam elements and the vaulter with seven such elements linked together by pin joints. 'Smart' behaviour is achieved through control of limited muscle torques at the joints according to a given strategy. This control strategy is such that the vaulter strives to carry through a prescribed sequence of motions, corresponding to a given style, during a vault. The optimum pole length is determined for a vaulter with given initial velocity, strength and style. When a pole of optimum length is used, the maximum increase of the potential energy of the vaulter is 1.27 times the initial kinetic energy of the vaulter and the pole. This shows that the contribution from muscle work to the increase in potential energy during a vault may be significant. The simulation method should be a useful tool for optimization of pole design. It might also be useful for optimization of vaulting style or for judging the importance of vaulter strength. PMID- 7559678 TI - Stress in the Achilles tendon during a topple-over movement in the ankle joint. AB - The achilles tendon is one of the strongest tendons in the human body, and a healthy tendon will only rupture under very severe conditions. Experiments and statistics show a high probability of tendon rupture in the case of a person trying to make a quick start and thereby toppling over in the ankle joint. Toppling over means a quick overturning in the lower ankle joint as it may occur when starting out of the sagittal plane. Based on a verified model of the achilles tendon, of M. Gastrocnemius and M. Soleus (Hatze, H. (1980) IEEE Trans. Automatic Control 25, 375-385; Hoy et al. (1990) J. Biomechanics 23, 1185-1198.), the stress in different tendon fibres during such a movement is investigated. The results of this simulation show that the expected extreme stresses in the lateral tendon fibres are a possible starting point for a rupture. PMID- 7559676 TI - Coulomb frictional interfaces in modeling cemented total hip replacements: a more realistic model. AB - Loosening of cemented femoral hip stems could be initiated by failure of the cement mantle due to high cement stresses. The goals of this study were to determine if realistic stem-cement interface characteristics could result in high cement stresses when compared to a bonded stem-cement interface and to determine if stem design parameters could be chosen to reduce peak cement stresses. Three dimensional finite-element models of cemented femoral hip components were studied with bonded or realistic Coulomb friction stem-cement interfaces. The results showed that the use of a non-bonded, non-linear Coulomb friction interface resulted in substantially different stress fields in the cement when compared to a bonded stem-cement interface. Tensile stresses in the proximal cement mantel for the Coulomb friction interface case (10.8 MPa) were greater than the fatigue strength of the cement. In contrast, the tensile stresses in the cement mantle were not greater than the fatigue strength for the bonded case (7.5 MPa). Failure of the cement mantle in the proximal femur could therefore be initiated by a lack of a bond at the stem-cement interface. The effect of different cross-sectional stem geometries (medial radii of 3.0, 4.9 and 5.5 mm and antero-posterior widths of 9.8 and 13.7 mm) and different elastic moduli (cobalt chromium alloy and titanium alloy) for the stem material were also evaluated for models with a Coulomb friction interface. Changes in the stem cross-section and elastic modulus had only limited effects on the stress distributions in the cement. Of the parameters evaluated in this study, the characteristics of the stem-cement interface had the largest effect on cement mantle stresses. PMID- 7559679 TI - Force and moment distributions among osseointegrated dental implants. AB - Distributions of force, bending moment and torque are determined by structural analysis for an osseointegrated dental implant system. The system is a dental prosthesis rigidly connected to bone by implants. Since the implants have the lowest flexural rigidity of the structural components, they are considered to be the only elastic components of the system. In the analysis, the number and position of the implants are variable and the magnitude, direction and location of the applied load are arbitrary. The distributions found by force and moment balances are in the form of simple algebraic equations, a form which is useful for clinicians in determining the number and location of implants so that forces and moments are shared equitably. One immediate result of the structural analysis is that the bending moment due to the vertical component of the applied load--a moment which has previously been neglected--can produce stresses in the implant which are an order of magnitude larger than the direct axial stresses. PMID- 7559680 TI - Human pendulum approach to simulate and quantify locomotor impact loading. AB - The understanding of impact mechanics during locomotion is important for research within the fields of injury prevention and footwear design. Instrumented missiles offer a worthy solution to the lack of control inherent in in vivo activities and to the isolated nature of tissue studies. However, missiles cannot mimic the magnitude and temporal characteristics of locomotion impacts. A human pendulum approach employed the subject's own body as the missile to impart controlled impacts to the lower extremity. The subject is swung toward a force platform instrumented wall while lying supine on a suspended lightweight bed. The ability of the pendulum to reproduce locomotor impact loading was assessed for heel-toe running. Axial reaction force and shank acceleration patterns recorded during pendulum tests in ten subjects were found to closely resemble running patterns and they were obtained without discomfort to the subjects. This new approach relies upon one's own body to impart impacts representative of locomotion. It should prove useful to study human impact loading in a controlled manner. PMID- 7559681 TI - Visual guidance to force plates does not influence ground reaction force variability. AB - Gait analysis methods commonly require the subject to avoid visual guidance, that is, targeting, the force plate upon which a foot-strike is necessary. The putative rationale underlying the preference for not using targeting is the unsubstantiated contention that gait is altered. This study evaluated the influence of force plate targeting on the variability of ground reaction forces (GRF) in 15 normal subjects. Secondary factors of single vs multiple steps to the force plate and whether or not subjects were informed of the study's primary purpose were also examined. ANOVA main effects revealed that targeting did not significantly affect GRF variability (p > 0.05). A significant main effect of the number of steps required to reach the force plate on AP force variability was found (p = 0.002). Prior knowledge of the purpose of the study did not significantly affect GRF variability (p > 0.05). It was concluded that the variability of ground reaction forces is not significantly affected by targeting the force plate. Thus, targeting would not be expected to influence the variability of calculated kinetic variables that are subservient to GRF. PMID- 7559682 TI - Active force in rabbit ventricular myocytes. AB - Although recent technical advances have established the feasibility of force measurements in single cardiac myocytes, the physiological relevance of this model has not been fully evaluated. We measured active force and sarcomere length in single rabbit left ventricular myocytes and compared their physiological responses to changes in stimulus interval, calcium concentration and sarcomere length to results from isolated papillary muscles. Myocytes were attached to two poly-L-lysine-coated glass plates and force was measured with a capacitive force transducer (Cambridge 406A). Stable recordings from a continuously contracting myocyte could be maintained for over 1 h. In five cells, increasing stimulus interval significantly decreased active force development. This force-stimulus interval relation was similar to that obtained from papillary muscles. In one cell, we obtained a force-length relation that was similar to force-length relations from multicellular preparations. Active stresses (active forces normalized by cross-sectional area) were of similar magnitude when comparing myocytes (at slack length) and papillary muscles (at 85% of Lmax). These results confirm the physiological relevance of force measurements obtained from intact mammalian cardiac myocytes. PMID- 7559683 TI - Experimental and mathematical methods for representing relative surface elongation of the ACL. AB - The common approach to assess the stabilizing role of the ACL in the knee has been to measure the elongation of a few marked fibers in the ligament. A comparison of the relative elongation (RE) of these marked fibers between different specimens and studies is delicate due to the difficulty of marking the same fibers. More consistent comparisons would be achieved if the RE of the whole ligament surface was presented. Hence, we developed a mathematical method leading to a continuous description of the relative elongation of the ligament's surface based on experimental measurements of the RE of five fibers. The ligament fibers of two knee specimens were marked by radiopaque markers and a Roentgen Stereophotogrammetric Analysis system was used to reconstruct the three dimensional positions of these artificial landmarks. The mathematical procedure used isoparametric cubic splines to interpolate the contours of the insertion sites. The results showed that the general pattern of the RE for both specimens was similar, characterized by an undulation near full flexion. In fact, close to full flexion all the RE of the fibers increased. Such a representation describes the changes in the RE for a given fiber during knee flexion and at the same time characterizes the RE distribution at a given flexion angle. PMID- 7559684 TI - Computational method for determination of bone and joint loads using bone density distributions. AB - Because bone structure is influenced by mechanical loading during ontogeny, the geometry and density distribution of bones contain information about their loading histories. Based on a mathematical theory relating stress history to bone remodeling, we have developed a method to determine dominant bone loading conditions using an optimization procedure. We applied this load determination method using a simplified two-dimensional bone-end finite element model, for which a standard density distribution had been calculated under a given set of loading conditions. With this density distribution, the optimization procedure was used to determine the original loads from a broad set of many plausible basic load distributions and locations. The optimization procedure adjusted the magnitude of each basic load to achieve the desired tissue level attractor stress stimulus throughout the model. The results show that the density-based bone load determination method yields accurate results for basic test cases and, thus, may have potential for estimating in vivo bone loads for both extant and extinct animals. PMID- 7559685 TI - Optimizing the determination of the body center of mass. AB - The position or trajectory of the body center of mass (COM) is often a parameter of interest when studying posture or movement. For instance, in balance control studies the body COM can be related to the ground reaction force or to the base of support. Since small displacements of the body COM are important in balance control studies, it is essential to obtain valid estimates of the body COM. The main source of error in the determination of the body COM is the estimation of the masses and centers of mass of the body segments. Especially the determination of the trunk COM is prone to error. In the current study five subjects maintained three postures, differing in trunk angle, during a few seconds. The relation between the center of pressure of the ground reaction force and the vertical projection of the body COM during the postures was used to optimize the trunk COM position. Additionally the subjects performed two lifting movements. The validity of the body COM trajectory estimation during the lifting movements, both with and without optimized trunk COM, was checked by relating the external moment of the ground reaction force with respect to the body COM to the rate of change of the angular momentum of the whole body. It was shown that the correspondence between the external moment and the rate of change of the angular momentum improved after optimization of the trunk COM. This suggests that the body COM trajectory estimation can be improved by the proposed optimization procedure. PMID- 7559687 TI - The Ilizarov technique: a method criticised but valued. PMID- 7559686 TI - Paget's disease of bone. PMID- 7559688 TI - The pathology of frozen shoulder. A Dupuytren-like disease. AB - Of 935 consecutive patients referred with shoulder pain, 50 fitted the criteria for primary frozen shoulder. Twelve patients who failed to improve after conservative treatment and manipulation had excision of the coracohumeral ligament and the rotator interval of the capsule. The specimens were examined histologically, using special stains for collagen. Immunocytochemistry was performed with monoclonal antibodies against leucocyte common antigen (LCA, CD45) and a macrophage/synovial antigen (PGMI, CD68) to assess the inflammatory component, and vimentin and smooth-muscle actin to evaluate fibroblasts and myofibroblasts. Our histological and immunocytochemical findings show that the pathological process is active fibroblastic proliferation, accompanied by some transformation to a smooth muscle phenotype (myofibroblasts). The fibroblasts lay down collagen which appears as a thick nodular band or fleshy mass. These appearances are very similar to those in Dupuytren's disease of the hand, with no inflammation and no synovial involvement. The contracture acts as a check-rein against external rotation, causing loss of both active and passive movement. PMID- 7559689 TI - Frozen shoulder and lipids. AB - We prospectively studied 50 patients with the diagnosis of primary frozen shoulder. The serum lipid levels were measured in 43 of these patients and compared with those in 43 age-matched and sex-matched control subjects. The fasting serum triglyceride and cholesterol levels were significantly elevated in the frozen-shoulder group (cholesterol p < 0.01; triglyceride p < 0.02). PMID- 7559690 TI - Humeral head torsion in recurrent anterior dislocation of the shoulder. AB - We measured torsion of the humeral head in 38 patients (40 shoulders) with recurrent anterior dislocation of the shoulder (RADS) and in 40 normal subjects. We found a reduced mean retroversion in the patients with RADS at 4.3 +/- 10.56 degrees (17 degrees anteversion to 32 degrees retroversion) as compared with 16.1 +/- 11.07 degrees in the control group (0 degrees to 49 degrees) (p = 0.0001). There was anteversion in 11 of the 40 shoulders in the RADS group (27.5%) and in none of the control group. The first dislocation had occurred after minimal force in 18 of 25 patients with less than 10 degrees retroversion, but in only three of 15 with over 10 degrees retroversion. We conclude that decreased retroversion of the humeral head is often associated with RADS and with first dislocation of the shoulder caused by minimal force. PMID- 7559691 TI - Revision of failed total elbow arthroplasty. AB - We reviewed 25 patients with rheumatoid arthritis who had failure of 26 primary total elbow arthroplasties causing pain and loss of function. Most revision cases required special custom implants to treat varying bone loss and soft-tissue disruption. Assessment showed satisfactory functional results in the patients treated by revision at a mean follow-up period of 35 months. Our review suggests that revision surgery produces short- to medium-term painfree function, and is the treatment of choice for a failed total elbow arthroplasty in the absence of infection. PMID- 7559692 TI - The midline posterior elbow incision. An anatomical appraisal. AB - The formation of a painful neuroma after operations on the medial or lateral sides of the elbow is a common problem. Our aim was to determine the relationship of the cutaneous nerves to the three usual skin incisions around the elbow. In 18 freshly frozen cadaver arms we made three standard 16 cm incisions in the skin medially, laterally, and posteriorly and explored them using loupe magnification. The number of nerves crossing each incision was determined by gross observation and their diameter measured by electronic microcallipers. In ten arms, biopsies of the nerves in each incision were sent for histological examination. We found significantly more cutaneous nerves crossing the medial and lateral incisions than the posterior. The diameter of the nerves crossing the posterior incision was significantly smaller than those crossing the lateral incision. Cutaneous nerves are at considerable risk of injury when medial or lateral incisions are used to approach the elbow, but the posterior approach carries less hazard. The routine use of the posterior incision may reduce the incidence of symptomatic paraesthesia and the formation of a painful neuroma after operation. PMID- 7559693 TI - Wrist arthrodesis using a dynamic compression plate. AB - We performed 45 wrist arthrodeses in 43 patients by a modification of the AO technique using the dynamic compression plate. Radiological follow-up was obtained in 41 wrists; all had united at a mean of ten weeks. Clinical follow-up was obtained in 32 wrists. Subjectively, the surgical outcome was satisfactory in 26, marginally satisfactory in two and unsatisfactory in four. This method is safe and reliable. The plate can be contoured to allow a variety of positions of fusion, and gives rigid immobilisation. The rate of union is higher than that for other techniques. PMID- 7559694 TI - Prediction of clinical outcome of THR from migration measurements on standard radiographs. A study of cemented Charnley and Stanmore femoral stems. AB - We report the theoretical basis of a method to measure axial migration of femoral components of total hip replacements (THR). The use of the top of the greater trochanter and a lateral point on the collar of the stem, allowing for variations of up to 10 degrees rotation of the femur in any direction between successive radiographs, gave a maximum error of 0.37 mm. At a more realistic 5 degrees rotational variation, the error was only 0.13 mm. These data were confirmed in an experimental study using digitisation of points and special software. We also showed that the centre of the femoral head, the stem tip, and the lesser trochanter provided less accurate landmarks. In a second study we digitised a series of radiographs of 51 Charnley and 57 Stanmore THRs; the mean migration rates were found to be identical. We then studied 46 successful stems with a minimum follow-up of eight years and 46 stems which had failed by aseptic loosening at different times. At two years, the successful stems had migrated by a mean of 1.45 +/- 0.68 mm, but the failed cases had a mean migration of 4.32 +/- 2.58 mm (p < 0.0001). Of the successful cases 76% had migrated less than 2 mm, while in the failed group 84% had migrated more than 2 mm. For any particular case migration of more than 2.6 mm at two years had only a 5% chance of continuing success and would therefore merit special follow-up. Only 24% of the eventually successful stems showed migration at the stem-cement interface, but this had happened in every failed stem. We conclude that it would be possible to evaluate a new cemented design of femoral stem over a two-year period by the use of our method and to compare its performance against the reported known standard of the Charnley and Stanmore designs. PMID- 7559695 TI - Perioperative low-molecular-weight heparin. Is it effective and safe. AB - In previous randomised clinical trials of thromboprophylaxis after total hip replacement, low-molecular-weight heparin has been given for an arbitrary 7 to 14 days. The risk factors are mainly perioperative and it is possible that a shorter course may be adequate. We assessed the safety and effectiveness of a three-day course. We assessed 156 primary THR patients after randomisation to either a control group or to receive enoxaparin at 12 hours preoperatively and 12 and 36 hours postoperatively. Thrombosis was diagnosed by routine venography. Haemorrhagic side-effects were assessed by measurement of blood loss, and soft tissue side-effects by descriptive scores for wound discharge and bruising of the leg. The prevalence of calf thrombosis was 15.4% in the enoxaparin group and 32.1% in the control group (p = 0.01); the prevalence of proximal thrombosis was 15.4% and 17.9% respectively (not significant). There was no difference in haemorrhagic side-effects or wound discharge, but there was more bruising in the enoxaparin group. PMID- 7559696 TI - Can Ilizarov joint distraction delay the need for an arthrodesis of the ankle? A preliminary report. AB - We applied joint distraction using an Ilizarov apparatus in 11 patients with post traumatic osteoarthritis of the ankle to try to delay the need for an arthrodesis. Distraction for three months resulted in clinical improvement in pain and mobility for a mean of two years, with an increase in the joint space. We considered that these effects may be produced by the absence of mechanical stress on the cartilage combined with the intra-articular hydrostatic pressures during distraction. We measured these pressures during walking with distraction, and found levels very similar to those reported to improve osteoarthritic cartilage when applied in vitro. PMID- 7559697 TI - Duplication of medial erosion in unicompartmental knee arthroplasties. AB - Osteoarthritis of the medial compartment of the knee often shows a specific pattern of anterior wear. Review of our revisions from a series of medial metal backed Brigham unicondylar knee replacements performed between 1983 and 1989 showed that this wear pattern was common on the tibial polyethylene surface. We reviewed these cases retrospectively to compare the pattern of preoperative erosion with the wear of the prosthesis. In all 14 knees with severe anterior wear in a unicompartmental replacement, the prearthroplasty radiographs showed similar patterns, suggesting that the implanted tibial component may continue to be subjected to the same localised stresses that precipitated the failure of the original articular cartilage. Many tibial components implanted during the 1980s had an unacceptably thin anterior rim of polyethylene and it seems that greater thickness is essential at the anterior and peripheral margins of the tibial plateau. PMID- 7559698 TI - Development of the clinical tibiofemoral angle in normal adolescents. A study of 427 normal subjects from 10 to 16 years of age. AB - We measured the clinical tibiofemoral (TF) angle and the intercondylar (IC) or intermalleolar (IM) distance in 427 normal European children (212 male and 215 female) aged from 10 to 16 years. In our study, girls had a constant valgus (5.5 degrees) and displayed an IM distance of < 8 cm or an IC distance of < 4 cm. By contrast, boys had a varus evolution (4.4 degrees) during the last two years of growth and displayed an IM distance of < 4 cm or an IC distance of < 5 cm. Values above these for genu varum or genu valgum may require careful follow-up and evaluation. PMID- 7559699 TI - Medial physeal stapling for primary and secondary genu valgum in late childhood and adolescence. AB - We report the results of medial physeal stapling in 16 knees with primary genu valgum and 27 with secondary genu valgum. In the primary group, stapling was undertaken at a mean chronological age of 12 years in girls and 13 years in boys. The medial femoral physis was stapled in ten knees and the medial femoral and tibial physes in six knees. At skeletal maturity, all patients had excellent or good leg alignment. Secondary genu valgum is due to skeletal dysplasia, haematological or endocrine disorders, or to juvenile chronic arthritis. Stapling was at a mean chronological age of 11 years in girls and 14 years in boys. The medial femoral physis was stapled in 13 knees, the medial tibial physis in three and both in 11 knees. At skeletal maturity, 85% had excellent or good leg alignment, and correction had occurred within one year. Two of the poor results were due to staple extrusion from osteoporotic bone, and two to overcorrection. Rebound growth was minimal and unpredictable after the removal of staples. Medial physeal stapling is a suitable method of treatment for both primary and secondary genu valgum in late childhood and in adolescence. At least one year of knee growth is required to achieve correction, and care is needed to avoid overcorrection of the secondary genu valgum. PMID- 7559700 TI - Physeal slope in Perthes disease. AB - In 40 children with unilateral Perthes' disease, we measured the physeal slope, the angle between the physeal plane and the axis of the femoral shaft, from radiographs taken early in the disease. Thirty-seven of the 40 hips were classified as Catterall grades III and IV. Heat-at-risk signs were present in 23. We found no statistically significant difference in the physeal slope between the involved and normal hips (p = 0.20), those with or without head-at-risk signs (p = 0.96), those with or without lateral epiphyseal subluxation (p = 0.82), and different Catterall (p = 0.56) or lateral pillar (p = 0.67) gradings. PMID- 7559701 TI - The Herring lateral pillar classification for prognosis in Perthes disease. Late results in 49 patients treated conservatively. AB - We reviewed the radiographs of 49 patients with Perthes' disease at the stage of fragmentation and also after the end of skeletal growth to assess the value of the lateral pillar classification of Herring. The average age of the patients at diagnosis was 7 years 6 months and the mean follow-up was 24 years. Ten of the 11 Herring group-A hips showed good reconstruction of the femoral head. There were good results in group-B hips when the patients were less than nine years of age at diagnosis. All 11 group-C patients showed hip deformity at follow-up. The Herring classification provides a valid long-term prognosis in Perthes' disease, although age at diagnosis is also an important prognostic factor. The classification is relatively easy to apply, is reliable, and requires only an anteroposterior radiograph taken during the fragmentation stage of the disease. PMID- 7559702 TI - A graphic method for timing the correction of leg-length discrepancy. AB - We have developed a clinical method for the graphic recording, analysis and planning of treatment of leg-length discrepancy during growth. Initially, the clinically determined discrepancy is plotted against the chronological age yearly, and then in late childhood at six-monthly intervals. CT and measurements of skeletal age are made in middle and late childhood to confirm the clinical findings. In a prospective study in 20 children, we observed that only eight had a linear increase in discrepancy. The observed pattern of increase was therefore used to estimate the mature discrepancy. Epiphyseodesis reference slopes were used to determine the most appropriate time and type of epiphyseodesis. In all children, the leg-length discrepancy at maturity was within 1 cm of the predicted amount. Changes in discrepancy due to leg lengthening or correction of deformity were also plotted graphically. We conclude that the clinical graphic method is simple to use, takes into account the varying patterns of discrepancy, and minimises radiation dosage. PMID- 7559703 TI - Recurrent irritable hip in childhood. AB - Between 1989 and 1992 we admitted 426 children with an irritable hip, 363 (85.2%) once and 63 (14.8%) on 143 occasions. We assessed the records retrospectively to determine whether the groups differed and in particular whether recurrence was followed by pathological sequelae. We identified no feature which distinguished between them at either presentation. The use of bone isotope scans was greatly increased in recurrent cases, without clinical benefit. No relationship between recurrence and subsequent abnormality was identified, with 22 (42%) of the recurrences taking place in the opposite hip. The only difference was a higher incidence of 'psychosocial factors' recorded in the notes of children who presented on more than two occasions. The incidence of recurrent irritable hip is larger than previously indicated and in the presence of normal radiographs and low-grade clinical signs, more detailed investigation on subsequent admission is unlikely to be helpful. PMID- 7559704 TI - Slipped capital femoral epiphysis. Incidence and clinical assessment of physeal instability. AB - In an unselected series of 55 cases of slipped capital femoral epiphysis (SCFE) we observed an incidence of 25% of epiphyseal reduction, mostly unintentional. Reduction indicated physeal instability and was associated with an effusion, detected by sonography on admission, and inability to bear weight. The true prevalence of instability may be higher since an effusion was noted in 33 cases (60%) on the initial sonographic assessment. Serial radiographs showed reduction in 12 (22%), with an average change of 15.1 degrees in the head-neck angle. Serial sonography showed reduction in 7 out of 20 cases (35%), with an average change of 3.7 mm in displacement. In two cases reduction was seen on sonography but not on radiography. Of the hips which showed subsequent reduction, 12 had had a bone scan on admission; three showed initial epiphyseal avascularity but only one progressed to symptomatic avascular necrosis. All stable hips had normal epiphyseal vascularity on the initial bone scan. This indicates the importance of injury from the initial displacement in causing avascular necrosis, rather than effusion, vascular compromise or iatrogenic injury from gentle repositioning. Physeal instability in SCFE is common and should be assessed clinically on admission. It is indicated by joint effusion or inability to bear weight. A slip is very unlikely to be unstable in a child able to bear weight and with no sonographic effusion. PMID- 7559705 TI - Varus derotation osteotomy for persistent dysplasia in congenital dislocation of the hip. Proximal femoral growth and alignment changes in the leg. AB - We studied the morphological changes in the proximal femur in 42 patients (42 hips) who had had varus derotation osteotomy of the upper femur for residual acetabular dysplasia and congenital subluxation of the hip and who did not show deformity of the head of the femur. In 19 patients the alignment of the whole leg was examined. The femoral neck-shaft angle (FNSA) at the final examination was unrelated to that immediately after operation or to the state of the acetabulum at that time. The postoperative FNSA was not related to the final result but the CE angle obtained at surgery influenced the outcome. The femoral length did not differ significantly between the unaffected and affected sides. Significant differences were found in the femorotibial angle (176.6 degrees v 174.5 degrees) and in the point of intersection between the mechanical axis and knee (65.0% v 57.2%). PMID- 7559706 TI - Absent superficial abdominal reflexes in children with scoliosis. An early indicator of syringomyelia. AB - We describe 12 children with idiopathic scoliosis who had a persistent absent superficial abdominal reflex (SAR) on routine neurological examination. MRI showed syringomyelia to be present in ten. The average age at detection of the scoliosis was 4.3 years and at diagnosis of syringomyelia 6.6 years. In all ten children the SAR was consistently absent on the same side as the convexity of the curve. In two it was the only abnormal neurological sign. An absent SAR in patients with scoliosis is an indication for investigation for underlying syringomyelia. In the children with syringomyelia, six had thoracic and four thoracolumbar curves. The clinical features differed in the two groups. Patients with thoracic curves were generally asymptomatic. Their neurological signs were subtle and none had any motor signs. By contrast, patients with thoracolumbar curves had symptoms and neurological signs. Abnormal gait was present in all four patients with thoracolumbar curves. In three this was due to considerable motor weakness. In eight children syringomyelia was associated with a Chiari-I malformation. In seven the syrinx was treated surgically by decompression of the foramen magnum. PMID- 7559709 TI - Neurological injury in thoracolumbar burst fractures. AB - Many authors recommend surgery to remove retropulsed bone fragments from the canal in burst fractures to 'decompress' the spinal canal. We believe, however, that neurological damage occurs at the moment of injury when the anatomy is most distorted, and is not due to impingement in the resting positions observed afterwards. We studied 20 consecutive patients admitted to our spinal injuries unit over a two-year period with a T12 or L1 burst fracture. There was no correlation between bony or canal disruption and the degree of neurological compromise sustained but there was a significant correlation between the energy of the injury (as gauged by the Injury Severity Score) and the neurological status (p < 0.001). This suggests that neurological injury occurs at the time of trauma rather than being a result of pressure from fragments in the canal afterwards and questions the need to operate simply to remove these fragments. PMID- 7559708 TI - Vertebral slip in lumbar spondylolysis and spondylolisthesis. Long-term follow-up of 22 adult patients. AB - We studied the aetiology of vertebral slip in a long-term follow-up of 22 adult patients with isthmic spondylolysis or spondylolisthesis of L5. Of the 18 with spondylolysis without slip, 13 showed no slip after ten years, but five developed displacement of over 5%. All four patients with spondylolisthesis showed progression of the slip. We found that the vertical thickness of the transverse process of L5 was significantly greater (p < 0.01) in the 13 patients with no slip than in the other two groups. The relationship of vertebral slip to the shape of the transverse processes of L5 may be explained by differences in the bulk or physiological strength of the posterior bands of the iliolumbar ligament. PMID- 7559707 TI - Variability in Cobb angle measurements in children with congenital scoliosis. AB - The variability in measurement of angles in congenital scoliosis is not known, but it is postulated that it is larger than that in adolescent idiopathic scoliosis due to skeletal immaturity, incomplete ossification, and anomalous development of the end-vertebrae. To determine this variability, we selected 54 radiographs of adequate quality showing 67 scoliotic curves from children with congenital scoliosis. The end-vertebrae were preselected. Each curve was measured by the Cobb method on two separate occasions by six different observers, using the same goniometer and marker. The intraobserver variability was +/- 9.6 degrees and the interobserver variability +/- 11.8 degrees. If 'significant progression' is to be used as a criterion for surgical fusion in congenital scoliosis, there should be at least a 23 degrees increase, the entire range of the interobserver variability, in the curvature to ensure that the perceived increase is not due to variability in measurement. PMID- 7559712 TI - Soft-tissue interposition of femoral fractures. Detection by ultrasonography during closed nailing. AB - We have compared the sonographic findings of six femoral fractures with soft tissue interposition which required open reduction with those of a control group of 40 other femoral fractures. Ultrasound assessment before operation showed that the fractured end of the proximal fragment had penetrated the quadriceps muscle anteriorly while the distal fragment lay beneath it. Transverse scans showed less soft-tissue thickness over the end of the proximal fragment in the problem cases. Radiographic image intensification did not provide any additional information. Ultrasound is of value in demonstrating soft-tissue interposition at the fracture site before femoral nailing. PMID- 7559710 TI - Partial excision of the clavicle for nonunion in National Hunt Jockeys. AB - National Hunt jockeys suffer a disproportionate number of clavicular fractures and their return to riding may be considerably delayed by refracture and symptomatic nonunion, with obvious implications. We report six such cases in which excision of the clavicular fragment distal to the fracture was associated with an early return to work and no recurrent injury to the shoulder. PMID- 7559711 TI - Distal metaphyseal fractures of the tibia with minimal involvement of the ankle. Classification and treatment by locked intramedullary nailing. AB - We reviewed 63 patients with fractures of the distal tibial metaphysis, with or without minimally displaced extension into the ankle joint. The fractures had been caused by two distinct mechanisms, either a direct bending force or a twisting injury. This influenced the pattern of the fracture and its time to union. All fractures were managed by statically locked intramedullary nailing, with some modifications of the procedure used for diaphyseal fractures. There were few intra-operative complications. At a mean of 46 months, all but five patients had a satisfactory functional outcome. The poor outcomes were associated with either technical error or the presence of other injuries. We conclude that closed intramedullary nailing is a safe and effective method of managing these fractures. PMID- 7559715 TI - Changing crystallinity of polyethylene in the acetabular cups of Weller hip prostheses. AB - We performed thermal analysis of polyethylene samples obtained from 73 new cups of the Weller hip prosthesis. There were marked individual differences in the degree of their crystallinity, ranging from 37.8% to 67.2% with a mean of 53%. Analysis of polyethylene from removed cups also showed differences in crystallinity in individual cups, with the mean value being higher in the removed implants than in the new ones. This difference in crystallinity between the groups was statistically significant. Marked individual differences in the degree of cup wear may result from these changes. PMID- 7559714 TI - Percutaneous Kirschner-wire pinning for severely displaced distal radial fractures in children. A report of 157 cases. AB - Distal radial fractures are common in children. Recent outcome studies have cast doubt on the success of treatment by closed reduction and application of plaster. The most important risk factor for poor outcome is translation of the fracture. If a distal radial fracture is displaced by more than half the diameter of the bone at the fracture site it should be classified as high risk. We performed percutaneous Kirschner-wire pinning on 157 such high-risk distal radial fractures in children under 16 years of age. The predicted early and late failure rate was reduced from 60% to 14% and only 1.5% of patients had significant limitation of forearm movement of more than 15 degrees in the final assessment at a mean of 31 months after operation. There were no cases of early physeal closure or deep infection. PMID- 7559713 TI - The 'floating elbow' in children. Simultaneous supracondylar fractures of the humerus and of the forearm in the same upper limb. AB - During a six-year period we prospectively studied eight children who presented with supracondylar fractures of the humerus and of the forearm on the same side. They were treated by prompt closed reduction, percutaneous fixation with Kirschner wires, and appropriate management of neurovascular and soft-tissue injuries. The results were assessed clinically and radiographically at a minimum of 12 months after injury. According to a clinical scoring system they were acceptable in seven children and poor in one. PMID- 7559716 TI - Microvascular transplantation of physeal allografts. AB - We compared growth in vascularised allograft transplants, autografts and in non operated physes in rabbits immunosuppressed with cyclosporin A and in non immunosuppressed animals. Molecular haplotyping was undertaken before operation to ensure allogenicity. Postoperative bone scans and fluorochrome labelling were used to confirm physeal vascularity. The animals were killed at three or five weeks. Proximal tibial physeal autografts, with or without cyclosporin A, or allografts with cyclosporin A, grew at similar rates to the physes of non operated rabbits. All the operated physes grew at rates significantly greater than their contralateral controls. 99mTc-MDP bone scans accurately predicted the viability of the epiphyseal plate. Quantitative histomorphological analysis of the heights of the physeal proliferative and hypertrophic zones showed that successful physeal transplants have a normal appearance, but when unsuccessful have thickened hypertrophic zones compatible with physeal ischaemia. We discuss the significance of these results in relation to the transplantation of physes in children. PMID- 7559717 TI - Bone remodelling in the proximal femur after Charnley total hip arthroplasty. AB - We measured bone mineral density (BMD) in the proximal femur by dual-energy X-ray absorptiometry (DEXA) in 20 patients after cemented total hip arthroplasty over a period of one year. We found a statistically significant reduction in periprosthetic BMD after six months on the medial side and on the lateral side adjacent to the mid and distal thirds of the prosthesis. At one year after operation there was a mean 6.7% reduction in BMD in the region of the calcar and a mean 5.3% increase in BMD in the femoral shaft distal to the tip of the implant. These changes reflect a pattern of reduced stress in the proximal femur and increased stress around the tip of the prosthesis. They support current concepts of bone remodelling in the proximal femur in response to prosthetic implantation. PMID- 7559718 TI - Fibrogenesis imperfecta ossium. AB - The clinical features, investigation, treatment and outcome of two adults with fibrogenesis imperfecta ossium are described. In this rare acquired disorder of bone, normal lamellar collagen is replaced by structurally unsound collagen deficient tissue, which leads to extreme bone fragility and ununited fractures. Transmission microscopy and SEM showed striking ultrastructural changes in bone structure and mineralisation. Both patients had monoclonal IgG paraproteins in the plasma and one excreted monoclonal lambda light chains in the urine. No abnormal plasma cells were found in the bone marrow and there was no evidence of amyloid deposition in the tissues. In both patients initial treatment with 1 alpha-hydroxycholecalciferol appeared to be ineffective, but in one, repeated courses of melphalan and corticosteroids over three years together with 1 alpha hydroxycholecalciferol produced striking clinical and histological improvement. The findings in these and other patients strongly suggest that paraproteinaemia is an integral feature of fibrogenesis imperfecta ossium, and this needs further investigation. PMID- 7559719 TI - Percutaneous autogenous bone-marrow grafting in congenital tibial pseudarthrosis. PMID- 7559721 TI - Tenotomy of the ligamentum patellae in spina bifida: management of limited flexion range at the knee. PMID- 7559720 TI - Dislocation of the coccyx: a case report. PMID- 7559722 TI - Dural ectasia in von Recklinghausen's disease of the lumbar spine: a case report. PMID- 7559723 TI - Amputation for reflex sympathetic dystrophy. PMID- 7559724 TI - Second-look arthroscopy after meniscal repair. PMID- 7559725 TI - Issue dedicated to Dr. Haruo Sugano in honor of his 70th birthday. PMID- 7559726 TI - Molecular targets in the National Cancer Institute drug screen. PMID- 7559727 TI - Cancer chemotherapy 1973-present: a personal perspective. PMID- 7559728 TI - Antitumor antibiotics discovered and studied at the Institute of Microbial Chemistry. AB - In 1951, we were pioneers in initiating screening of antitumor agents from microbial metabolites. We discovered bleomycin in 1962 and aclacinomycin in 1975. Peplomycin, a derivative of bleomycin, and pirarubicin, a tetrahydropyranyl derivative of doxorubicin, were also studied. All of them have been clinically used for the treatment of cancer. Using new screening methods, we isolated spergualin in 1982. This agent exhibited immunosuppressive activity as well as antitumor activity. Deoxyspergualin, a derivative of spergualin, is now clinically used for the treatment of acute rejection after kidney transplantation. Bestatin was screened as an inhibitor of aminopeptidase B in 1976. It binds to the aminopeptidases located on the cell membrane of immunocompetent cells and modulates immune responses. It is now used for the treatment of acute non-lymphocytic leukemia. Microbial metabolites will become more important as a source of anticancer drugs in the future. PMID- 7559729 TI - Hepatocarcinogenesis in rodents and humans. AB - In hepatocarcinogenesis in rodents, induction of foci and nodules comprising clonally proliferated initiated cells is considered to be essential for the future development of carcinomas. Nodules in human cirrhotic liver, though known to be associated with a high hepatocellular carcinoma risk, have generally been regarded as regenerative in nature, and not the result of clonal or neoplastic cell proliferation, on a morphological basis. However, when we analyzed 83 cirrhotic nodules from 11 HBV carrier patients, utilizing hepatitis B virus (HBV) integration as a marker for clonal proliferation, we found the existence of clonal populations of more than 10(5) hepatocytes in 26 (31.3%) of them. Although such clonal cell populations are morphologically not discernible from neighboring hepatocytes, they may have particular histogenetic significance in human hepatocarcinogenesis and clearly deserve further investigation. Allelotype analysis of mouse hepatocellular carcinomas (HCC), induced by a single dose of diethyl nitrosaminine in C3H/MSM F1 hybrids, revealed no remarkable alterations in the original tumors when microsatellite probes were used, but loss of heterozygosity of chromosome 4 at extremely high frequency (95%) in cultured cell lines derived from these HCC. The shortest common region was about 10 cM distal to the interferon alpha gene, in which the p16 gene is located. The results indicated that loss of gene function, most probably including that of the p16 gene, may be essential for immortalization of cultured hepatocytes but that it may not play any role in initiation or early events in mouse hepatocarcinogenesis in vivo. The mouse HCC used for analysis in this study may be comparable with human HCC at an early stage, for which only very limited genetic alterations have so far been identified. PMID- 7559730 TI - Regulation of the interferon system and cell growth by the IRF transcription factors. AB - Induction of gene transcription in response to extracellular stimuli constitutes an essential aspect of host defense mechanisms. Interferons (IFN) are families of cytokines that have been discovered and extensively characterized in the context of host defense against viral infections. In elucidating the mechanism of transcriptional induction of the IFN genes by viruses, we have discovered two structurally related transcription factors, Interferon regulatory factor 1 (IRF 1) and IRF-2. These two factors, however, function not only as regulators of the IFN system, but are also key transcription factors in the regulation of cell growth and apoptosis. Thus, these studies uncover a complex gene transcription network in which the fate of cellular responses is determined by how the IRF transcription factors function in conjunction with other factors on the promoters of distinct genes under different conditions of the cells. PMID- 7559733 TI - Chronological changes of characteristics of early gastric cancer and therapy: experience in the Cancer Institute Hospital of Tokyo, 1950-1994. AB - Gastric cancer, the leading cause of death from cancer in Japan, has long been studied. We received our first patient with early gastric cancer in 1950 and have since treated 2382 patients with this cancer up to 1990. The percentage of early gastric cancers diagnosed has been on the increase following the improvement in diagnostic skills and the establishment of mass screening. At present, more than half of the gastric cancers presenting are in the early stages. Chronological changes in diagnoses of early gastric cancer are characterized by the increased findings of (a) small tumors less than 4 cm in diameter (b) depressed-type carcinoma (c) lesions of the upper part of the stomach, and (d) undifferentiated type adenocarcinoma. The standard method of treatment for early gastric cancer was standard radical operation in the 1970s. In the 1980s endoscopic mucosal resection and limited operation were adopted and their use has been increasing annually. The prognosis for early gastric cancer is quite favorable (the 5-year survival rate is more than 90%), and it is regarded as a disease with good prognosis. To obtain still better therapeutic results, it is essential to increase the proportion of early gastric cancers where endoscopic mucosal resection or limited operation is indicated, and improve the techniques of those procedures. PMID- 7559731 TI - HTLV-1 oncoprotein Tax deregulates transcription of cellular genes through multiple mechanisms. AB - Infection of a human retrovirus HTLV-1 induces adult T cell leukemia and a neurological disease, HAM/TSP. Regulatory protein Tax of HTLV-1 is thought to contribute to the pathogenesis. We have studied the mechanism of transcriptional activation induced by Tax protein and identified two independent mechanisms: (a) binding to the enhancer-binding proteins, CREB, CREM, NF-kappa B and SRF, resulting in the activation of these factors through indirect binding to each specific DNA sequence, and (b) binding to I kappa B protein resulting in the suppression of the negative regulator I kappa B, which binds to NF-kappa B. In addition to these factors, a new protein GLI/THP is also involved in the transactivation. On the basis of these mechanisms, gene regulations in vitro and in vivo in HTLV-1-infected cells are discussed. PMID- 7559734 TI - Bone metabolic markers in bone metastases. AB - The efficacy and cost/performance benefit of radionuclide bone scintigraphy in monitoring metastatic bone activity remain controversial. Recently developed bone metabolic markers are expected to play an additional role in the diagnosis of bone metastasis. We measured osteoclastic and osteoblastic markers in 267 patients with breast cancer (100 with bone metastasis), 38 patients with prostatic cancer (25 with bone metastasis), 50 patients with lung cancer (12 with bone metastasis) and 33 patients with miscellaneous cancers (13 with bone metastasis) and compared the values in the presence and absence of bone metastasis. Bone metabolic markers, both osteoclastic and osteoblastic, increased significantly in patients with bone metastasis. In breast cancer (bone metastasis is mostly of the mixed type), osteoclastic markers were good in detecting bone metastasis. In prostatic cancer (bone metastasis is mostly osteoblastic), osteoclastic and osteoblastic markers were equally effective in detecting bone metastasis. In lung cancer (bone metastasis is mostly osteolytic), osteoclastic markers were elevated preferentially in bone metastasis. Over all, osteoclastic markers were more sensitive in the diagnosis of bone metastasis, and among osteoclastic markers, serum pyridionoline-cross-linked carboxyterminal telopeptide was the most efficient in both specificity (91.0%) and sensitivity (48.6%) for detecting bone metastasis. PMID- 7559735 TI - Experience of quadrantectomy with axillary dissection without radiotherapy sustained by serial pathological examination for stage I breast cancer. AB - Breast conserving treatment usually consists of lumpectomy and axillary dissection followed by a limited dose of irradiation so that no significant side effects occur. However, the precision of lumpectomy depends on the surgical maneuver and pathological evaluation performed at each institution. For this reason, post-operative irradiation to the preserved breast and for the occult carcinoma in the same breast is absolutely mandatory, and effectively becomes a routine step. In 1986, we started to adopt the new breast-conservation method of quadranectomy with axillary dissection for restricted stage I breast cancer without using radiotherapy, at the Cancer Institute Hospital, Tokyo, Japan. As an alternative to irradiation to ensure safety, we chose to administer an elaborate pathological examination on serial sections. The pathological proof has saved troublesome post-operative irradiation, and the results have shown this method to be safe and clear-cut compared to the traditional breast-conserving treatment cited in the literature. From July 1986 to December 1994, we performed 321 cases of quadranectomy and axillary dissection (Q+Ax). If the detailed pathological examination of 5-mm serial sections revealed the stump to be negative, we did not treat the preserved breast with radiotherapy. Out of 321 cases, 247 were analyzed as being stump-negative and of these 235 did not receive radiotherapy at all. During a 5 year 4 month observation period, we have not yet encountered any local recurrence. However, we have experienced 4 cases (1.70%) in which a second cancer developed in the conserved breast. The annual incidence rate was 0.32%. These results are the best so far compared to other published world reports. PMID- 7559736 TI - New method of evaluating the surgical margin and safety margin for musculoskeletal sarcoma, analysed on the basis of 457 surgical cases. AB - The evaluation of surgical margin is useful in determining the curative success of surgical treatment of musculoskeletal sarcoma and the degree to which later surgery will be reduced by the preoperative therapy. However, until recently no reliable evaluation method has been developed for these purposes. In this paper we propose a new method for evaluating the surgical margin as drafted in 1989 by the Bone and Soft Tissue Tumor Committee of the Japanese Orthopaedic Association (JOA). In this method, surgical margin was classified into four types based on the distance between the surgical margin and the reactive zone of the tumour. These classifications of surgical margin (in order to surgical extent) are curative wide margin (curative margin), wide margin, marginal margin, and intralesional margin. The surgical margin is said to be curative if the margin is more than 5 cm outside the reactive zone. It is referred to as wide if the margin is less than 5 cm. Similarly, a margin that is in the reactive zone is considered as marginal, and a margin passing through a tumour as intralesional. Moreover, wide margin is classified as adequate (at least 2 cm outside the reactive zone) or inadequate (1 cm). In our evaluation, a "thin" barrier is considered to be a 2 cm thickness of normal tissue, a "thick" barrier as a 3-cm thickness, and joint cartilage is said to be equivalent to a 5-cm thickness. In addition, a surgical margin that is outside a barrier, with normal tissue between the barrier and the reactive zone of the tumour, is considered to be curative. This method was applied in 457 cases (involving 499 surgeries) at the Cancer Institute Hospital to determine clinical significance in the treatment of musculoskeletal sarcoma (1979-1994). From the results of this study we were able to conclude that this evaluation method can be highly useful in clinical practice for establishing optimum surgery. Moreover, we found that the safety margin for high-grade musculoskeletal sarcoma is a curative margin providing an adequate wide margin of 3 cm or more when preoperative therapy is not performed or is not effective, while the safety margin for high-grade sarcoma that responds to preoperative chemo- or radiotherapy seems to be an adequate wide margin of 2 cm. Here, radiotherapy is more effective compared to chemotherapy for reducing surgical margin. An inadequate wide margin, however, combined with radiotherapy, is not enough to ensure local curative success following surgery for musculoskeletal sarcoma.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559732 TI - The adenomatous polyposis coli gene and human cancers. AB - The APC (adenomatous polyposis coli) gene was isolated as a gene responsible for familial polyposis coli, an autosomal-dominant disease, characterized by development of hundreds to thousands of adenomatous polyps in the colon and rectum. However, recent studies revealed that inactivation of the APC gene also plays a significant role in development of sporadic forms of colorectal adenoma and carcinoma. Furthermore, somatic mutations have also been detected in pancreatic carcinomas as well as some type of gastric carcinomas, suggesting that APC has a critical function in regulation of cell growth in digestive tissues. PMID- 7559739 TI - p53 overexpression in flat serrated adenomas and flat tubular adenomas of the colorectal mucosa. AB - The expression of the p53 protein was investigated in flat serrated neoplasias as well as in other histological phenotypes of flat or exophytic hyperplasias or neoplasias of the colorectal mucosa. A total of 104 such lesions were analyzed: 24 were flat serrated neoplasias (22 flat serrated adenomas and 2 flat serrated adenocarcinomas), 26 flat tubular adenomas, 17 flat hyperplastic polyps, 29 exophytic tubular and/or villous neoplasias (23 adenomas and 6 exophytic adenocarcinomas) and the remaining 8, exophytic hyperplastic polyps. Deparaffinized, rehydrated sections were treated immunohistochemically to detect those overexpressing the p53 protein. Lesions having slight (+), moderate (++) or intense ( ) staining were considered immunoreactive. The results showed that 50% of the flat serrated adenomas with low-grade dysplasia (LGD) and 66.7% of those with high-grade dysplasia (HGD) had p53 immunoreactivity. None of the flat tubular or of the exophytic adenomas with LGD expressed p53, but immunoreactivity was present in 61.5% of the flat tubular adenomas with HGD and in 52.3% of the exophytic adenomas with HGD. All adenocarcinomas had an intense p53 reaction. Weak p53 expression was demonstrated by 11.7% of the flat hyperplastic polyps but none of the exophytic polyps reacted. The occurrence of p53 expression in flat serrated adenomas with LGD suggested that, despite its low histological profile, one-half of those lesions could be biologically already committed to independent growth. The occurrence of p53 expression in nearly 12% of the flat hyperplastic polyps was totally unexpected and deserves further investigation. Flat serrated adenoma emerges as a novel, independent histological entity among the various phenotypes of flat neoplasias of the colorectal mucosa. PMID- 7559737 TI - Incidence of latent breast cancer in Japanese women. AB - Detection of small or early neoplastic lesions is essential for the secondary prevention of cancer, and for this purpose information concerning their incidence and characteristics is required. Although data for latent or early cancers are available for various organs, details are limited in the case of the breast. The present investigation was carried out to cast light on the incidence of latent cancers, considered as lesions, in women with no symptoms and no palpable mass of the breast. Out of 2126 women visiting our hospital for initial breast examination during July to December in 1987, 542 (25.5%) had no complaint and neither a detectable mass on careful palpation nor a tumor shadow on mammography. However, 21 (3.9%) demonstrated microcalcification, 7 giving the impression of a possible malignancy. Subsequent biopsy of the 7 cases revealed 3 carcinomas. The detection rate of breast cancer in this population was thus 0.6% (3/542). Adopting the frequency (36%) of microcalcification detectable on mammography in 185 breast cancers diagnosed in this period as a base, the incidence of latent breast cancer among women visiting our hospital was calculated to be 1.5%. PMID- 7559738 TI - Lack of apparent excess of malignant mesothelioma but increased overall malignancies of peritoneal cavity in Japanese autopsies with Thorotrast injection into blood vessels. AB - The carcinogenicity of thorium dioxide sol (Thorotrast), an X-ray contrast medium used in 1930-1955, in the liver and bone marrow has been established and agrees well with the effects of a high dosage of alpha radiation in the organs. Recently, however, German and Danish epidemiologic studies have shown excess mesotheliomas in the pleura and peritoneum that are unlikely to have been heavily irradiated by alpha particles. To confirm these observations, we examined the incidence of the cancer in those who underwent Thorotrast injections into blood vessels (n = 370) by using autopsy files of the Japanese Thorotrast study. Only one malignant mesothelioma of the peritoneum was registered, whereas three peritoneal or retroperitoneal sarcomas were observed. Thus, our study did not find any increment of mesothelioma in Thorotrast patients. However, when we took the pleuroperitoneal and retroperitoneal malignancies altogether, the incidence (4/370 = 1.1%) was five times more frequent than that (344/162,000 = 0.2%) in the controls (P < 0.005). Clinicopathological data of the four cases are also presented. PMID- 7559740 TI - High K-ras mutation rates in goblet-cell-type adenocarcinomas of the lungs. AB - Adenocarcinomas of the lungs show a variable histology. We have subclassified such lesions into five cell types: hobnail, columnar, polygonal, mixed and goblet cell types, and investigated their relationships with K-ras mutations. Codons 12, 13 and 61 of the K-ras gene in 120 surgically resected pulmonary adenocarcinomas were examined by the mutation-allele-specific amplification method. Point mutations were observed in 10% of the adenocarcinomas limited to K-ras codon 12 and the commonest base substitution (nine cases) was a G to T transversion. Of the five types, goblet cell lesions demonstrated the highest mutation index, which at 100% (6/6) was significantly different from that of all other cell types. No relationship between K-ras mutation and cigarette smoking was observed. From these findings, it appears that development of goblet-cell-type adenocarcinomas of the lung may involve different carcinogenic mechanisms from adenocarcinomas of other subtypes. PMID- 7559741 TI - Reconstitution of purified P-glycoprotein into liposomes. AB - To study the molecular function of the multidrug-resistance gene product P glycoprotein, we purified and reconstituted it into liposomes. Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein. We found that transport activity was effective reconstituted after solubilization with cholate, glycocholate and taurocholate. Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity. The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The reconstituted P-glycoprotein specifically bound [3H]azidopine and had an ATPase activity that was slightly stimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine. PMID- 7559742 TI - Antitumor effect induced by granulocyte/macrophage-colony-stimulating factor gene modified tumor vaccination: comparison of adenovirus- and retrovirus-mediated genetic transduction. AB - Irradiated tumor cells genetically modified to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF tumor vaccine) are potent stimulators of systemic antitumor immunity. For the preparation of a GM-CSF gene-modified tumor vaccine, it is important to achieve efficient genetic transduction of tumor cells, leading to an appropriate expression of the induced gene. In this report, with a view to developing a protocol for an effective cancer vaccination therapy, we examined the vaccination efficacies of tumor cells secreting GM-CSF by either adenovirus- or retrovirus-mediated genetic transduction. By using an adenoviral vector, Adex1CAmGMCSF, a highly efficient gene delivery and a high-level expression of the GM-CSF gene were achieved. Unexpectedly, animal vaccination studies showed that the GM-CSF tumor vaccine transduced with the Adex1CAmGMCSF recombinant adenovirus (adenoviral GM-CSF tumor vaccine) was less efficacious than that transduced with the MFGmGMCSF recombinant retrovirus (retroviral GM-CSF tumor vaccine). The GM-CSF serum concentration attained by the adenoviral GM-CSF tumor vaccine was much higher than that obtained by the retroviral GM-CSF tumor vaccine. Our findings indicate that an optimal level of GM-CSF production is important for the tumor vaccine to elicit an adequate response in the host antitumor immunity. PMID- 7559743 TI - Disruption of the function of tumor-suppressor gene p53 by the hepatitis B virus X protein and hepatocarcinogenesis. AB - The X gene of the hepatitis B virus codes for a small basic protein and is able to transactivate viral and cellular genes, although the X protein exhibits no DNA binding activity. The mechanism of transactivation by X protein has been suggested to be via protein-protein interaction(s). We first demonstrated that X protein had amino acid sequences homologous to the functionally essential domain of Kunitz-type serine protease inhibitors and that those sequences were indispensable for the transactivation function. We demonstrated that X protein exhibited an inhibitor activity against hepatic serine proteases, and subsequently found that the protein activated X gene transcription in HepG2 cells and that the X responsive element was localized in the minimal promoter of the X gene. In contrast, the tumor-suppressor gene p53, but not mutant p53, remarkably reduced transcription from the minimal promoter. This p53 repression on the X gene promoter was cancelled by X gene co-expression, probably indicating that the X protein disrupts the p53 tumor suppressor function in the nucleus. All data suggest that X protein leads to transactivation of cellular oncogenes by preventing an interaction between p53 and cellular transcription factor(s) consisting of the basal transcriptional machinery. PMID- 7559744 TI - Renal carcinogenesis in the Eker rat. AB - The Eker rat hereditary renal carcinoma is an excellent example of a Mendelian dominant predisposition to a specific cancer in an experimental animal. We recently reported that a germline insertion in the rat homologue of the human tuberous sclerosis (TSC2) gene gives rise to the dominantly inherited cancer in the Eker rat model. The function of the TSC2/Tsc2 gene product (called "tuberine" in the human case) is not yet understood, although it contains a short amino acid sequence homologous to the ras family GTPase-activating proteins (GAP3). In the study, we isolated subtracted cDNA clones having increased expression in Eker renal carcinoma cells, using a modified representational difference analysis method to search for additional genes specifically involved in renal carcinogenesis. Here we identified four genes: the third component of the complement (C3) gene, the fos-related antigen I (fra-1) gene, an unknown gene (designated as being expressed in renal carcinoma: erc) and the calpactine I heavy-chain (Annexin II) gene. PMID- 7559745 TI - Tissue-specific activation of tumor marker glutathione transferase P transgenes in transgenic rats. AB - By means of transgenic rats, we have recently shown that the GPEI enhancer of the glutathione transferase P (GST-P) gene, which has two one-base-missmatched AP-1 sites locating palindromically with three-base spacing in between, is sufficient for conferring tumor-specific activation of the gene in vivo. It is noted that there is another consensus AP-1 site near the promoter of this gene. By using seven independent transgenic rats, bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase (CAT) coding sequence, we analyzed CAT expression in various tissues (brain, lung, liver, kidney, spleen) in these transgenic rats. We found that the ECAT gene, which has sufficient of the upstream regulatory region (approx. 2.9 kb) of the gene containing GPEI, is trans-activated in the kidney and lung of transgenic rats in a similar manner to endogenous GST-P. When either the GPEI core sequence or the AP-1 site near the promoter is deleted, CAT expression decreases to almost background level. Substitution of the GPEI core or the AP-1 site near the promoter to this silent construct (5CATGPEIcore) reconstituted CAT expression in the transgenic rats. In these rats, CAT was expressed in the brain and lung rather than in the kidney, showing a somewhat different pattern from the endogenous GST-P. In the brain tissue of the 5CATGPEIcore transgenic rat, CAT was demonstrated in the glia cells, which is consistent with endogenous GST-P expression. These results suggest that a relatively long upstream region (approx. 2.9 kb) is required for tissue-specific expression of the GST-P gene and that GST-P expression in the brain may be regulated differently from its expression in other organs. PMID- 7559747 TI - Tautomycin: an inhibitor of protein phosphatases 1 and 2A but not a tumor promoter on mouse skin and in rat glandular stomach. AB - Tautomycin isolated from Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Tautomycin induced hyperphosphorylation of cytokeratin peptides in human keratinocytes (PHK 16-I cells) 30 times less strongly than did okadaic acid. Repeated applications of tautomycin (30 micrograms, 40 nmol/application) did not induce tumor promotion in a two-stage carcinogenesis experiment on mouse skin initiated with 7,12 dimethylbenz[a]anthracene, whereas okadaic acid (1 microgram, 1.2 nmol/application) as a control induced tumor promotion strongly. As for mucosa of rat glandular stomach, tautomycin induced ornithine decarboxylase 4 h after intubation into the stomach. The tumor-promoting activity of tautomycin was next studied in the glandular stomach initiated with N-methyl-N'-nitro-N nitrosoguanidine (MNNG). Administration of tautomycin in the diet (1 mg rat-1 day 1), from week 9 to week 52 of the experiment, inhibited rather than enhanced tumor development in the glandular stomach initiated with MNNG. The percentages of tumor-bearing rats of the groups treated with MNNG plus tautomycin, MNNG alone, and tautomycin alone were 20.0%, 40.6%, and 0% respectively in week 52. The reason for the absence of tumor-promoting activity of tautomycin was studied in relation to tumor necrosis factor alpha (TNF alpha), an endogenous tumor promoter. We found that tautomycin neither enhanced TNF alpha mRNA expression in mouse skin nor induced TNF alpha release in a human stomach cancer cell line (KATO III cells), whereas okadaic acid did both. These results indicate that not all inhibitors of protein phosphatases are tumor promoters, and suggest that tumor promotion of the okadaic acid class of compounds is mediated by TNF alpha. PMID- 7559748 TI - Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus. AB - We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation. PMID- 7559746 TI - Transcriptional activation domains of the Ah receptor and Ah receptor nuclear translocator. AB - The Ah receptor (AhR) and Ah receptor nuclear translocator (Arnt) heterodimer bind the xenobiotic-responsive element (XRE) sequence in the upstream region of the genes for some drug-metabolizing enzymes, such as P4501A1 and glutathione S transferase Ya, to activate their transcription. This paper describes transcriptional activation domains of the AhR and Arnt as examined in vivo by DNA transfection experiments using GAL4-AhR or GAL4-Arnt chimeric plasmids and a reporter plasmid containing five GAL4 DNA binding sites. The major activation domain of Arnt was localized in a short segment of the C-terminal 34 amino acids, while the glutamine-rich domain of Arnt showed no transcriptional activity. This activation domain of Arnt could be further divided into two subdomains with some sequence similarity. Point mutation analysis of one of the subdomains revealed that bulky hydrophobic amino acids and neighboring acidic amino acids were necessary for the transcription-enhancing activity of Arnt. The C-terminal half of the AhR showed a strong transcription-stimulating activity, apparently five times as strong as that of Arnt. Further analysis of the activity revealed that the C-terminal transcriptional activity was distributed in several activation domains, one of which is rich in glutamine residues. These results indicate that the glutamine-rich domains of the AhR and Arnt function differently in the heterodimer regulatory complex. Previously, we showed that the enhancer activity of XRE was repressed by E1A proteins, especially the 12S form of E1A. Cotransfection experiments using an E1A12S expression plasmid and a GAL4-AhR or GAL4-Arnt expression plasmid demonstrated that E1A protein rather predominantly inhibited the transcriptional activity of Arnt. PMID- 7559749 TI - Proenkephalin is a nuclear protein responsive to growth arrest and differentiation signals. AB - Neuropeptide precursors are traditionally viewed as molecules destined to be cleaved into bioactive peptides, which are then released from the cell to act on target cell surface receptors. In this report we demonstrate nuclear localization of the enkephalin precursor, proenkephalin, in rodent and human embryonic fibroblasts (Swiss 3T3 and MRC-5 cells) and in rodent myoblasts (C2C12 cells). Nuclear proenkephalin, detected by immunofluorescence with a panel of antiproenkephalin monoclonal antibodies, is distributed predominantly in three patterns. Selective abolition of these patterns with salt, nuclease, or methanol is associated with liberation of immunoprecipitable proenkephalin into the extraction supernatant. Proenkephalin antigenic domains, mapped using phage display libraries and synthetic peptides, are differentially revealed in the three distribution patterns. Selective epitope revelation may reflect different conformational forms of proenkephalin or its existence in complexes with other nuclear proteins, forms which therefore have different biochemical associations with the nuclear substructure. In fibroblast cell populations in transition to growth arrest, nuclear proenkephalin responds promptly to mitogen withdrawal and cell-cell contact by transient, virtually synchronous unmasking of multiple antigenic domains in a fine punctate distribution. A similar phenomenon is observed in myoblasts undergoing differentiation. The acknowledgment of growth arrest and differentiation signals by nuclear proenkephalin suggests its integration with transduction pathways mediating these signals. To begin to address the mechanism of nuclear targeting, we have transfected mutated and nonmutated proenkephalin into COS (African green monkey kidney) cells. Nonmutated proenkephalin is localized exclusively in the cytoplasm; however, proenkephalin mutated at the first ATG codon, or devoid of its signal peptide sequence, is targeted to the nucleus as well as to the cytoplasm. From this we speculate that nuclear proenkephalin arises from a primary translation product that lacks a signal peptide sequence because of initiation at a different site. PMID- 7559751 TI - NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export. AB - We have isolated and characterized the gene encoding a novel essential nucleoporin of 82 kD, termed NUP82. Indirect immunofluorescence of cells containing an epitope tagged copy of the NUP82 localized it to the nuclear pore complex (NPC). Primary structure analysis indicates that the COOH-terminal 195 amino acids contain a putative coiled-coil domain. Deletion of the COOH-terminal 87 amino acids of this domain causes slower cell growth; deletion of the COOH terminal 108 amino acids results in slower growth at 30 degrees C and lethality at 37 degrees C. Cells in which the last 108 amino acids of NUP82 have been deleted, when shifted to 37 degrees C, do not display any gross morphological defects in their nuclear pore complexes or nuclear envelopes. They do, however, accumulate poly(A)+ RNA in their nuclei at 37 degrees C. We propose that NUP82 acts as a linker to tether nucleoporins directly involved in nuclear transport to pore scaffolding via its coiled-coil domain. PMID- 7559750 TI - A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. AB - Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures. PMID- 7559752 TI - Fus2 localizes near the site of cell fusion and is required for both cell fusion and nuclear alignment during zygote formation. AB - Zygote formation occurs through tightly coordinated cell and nuclear fusion events. Genetic evidence suggests that the FUS2 gene product promotes cell fusion during zygote formation in Saccharomyces cerevisiae, functioning with the Fus1 plasma membrane protein at or before cell wall and plasma membrane fusion. Here we report the sequence of the FUS2 gene, localization of Fus2 protein, and show that fus1 and fus2 mutants have distinct defects in cell fusion. FUS2 encodes a unique open reading frame of 617 residues that only is expressed in haploid cells in response to mating pheromone. Consistent with a role in cell fusion, Fus2 protein localizes with discrete structures that could be of cytoskeletal or vesicular origin that accumulate at the tip of pheromone-induced shmoos and at the junction of paired cells in zygotes. Fus2 is predicted to be a coiled-coil protein and fractionates with a 100,000 g pellet, suggesting that it is associated with cytoskeleton, membranes, or other macromolecular structures. Fus2 may interact with structures involved in the alignment of the nuclei during cell fusion, because fus2 mutants have strong defects in karyogamy and fail to orient microtubules between parental nuclei in zygotes. In contrast, fus1 mutants show no karyogamy defects. These, and other results suggest that Fus2 defines a novel cell fusion function and subcellular structure that is also required for the alignment of parental nuclei before nuclear fusion. PMID- 7559753 TI - A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes. AB - The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function. PMID- 7559754 TI - Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells. AB - Vacuolar matrix proteins in plant cells are sorted from the secretory pathway to the vacuoles at the Golgi apparatus. Previously, we reported that the NH2 terminal propeptide (NTPP) of the sporamin precursor and the COOH-terminal propeptide (CTPP) of the barley lectin precursor contain information for vacuolar sorting. To analyze whether these propeptides are interchangeable, we expressed constructs consisting of wild-type or mutated NTPP with the mature part of barley lectin and sporamin with CTPP and mutated NTPP in tobacco BY-2 cells. The vacuolar localization of these constructs indicated that the signals were interchangeable. We next analyzed the effect of wortmannin, a specific inhibitor of mammalian phosphatidylinositol (PI) 3-kinase on vacuolar delivery by NTPP and CTPP in tobacco cells. Pulse-chase analysis indicated that 33 microM wortmannin caused almost complete inhibition of CTPP-mediated transport to the vacuoles, while NTPP-mediated transport displayed almost no sensitivity to wortmannin at this concentration. This indicates that there are at least two different mechanisms for vacuolar sorting in tobacco cells, and the CTPP-mediated pathway is sensitive to wortmannin. We compared the dose dependencies of wortmannin on the inhibition of CTPP-mediated vacuolar delivery of proteins and on the inhibition of the synthesis of phospholipids in tobacco cells. Wortmannin inhibited PI 3- and PI 4-kinase activities and phospholipid synthesis. Missorting caused by wortmannin displays a dose dependency that is similar to the dose dependency for the inhibition of synthesis of PI 4-phosphate and major phospholipids. This is different, however, than the inhibition of synthesis of PI 3-phosphate. Thus, the synthesis of phospholipids could be involved in CTPP mediated vacuolar transport. PMID- 7559755 TI - Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes. AB - To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Go 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Go 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C. PMID- 7559756 TI - Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae. AB - Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins. alpha-Agglutinin is a GPI-anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor. Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C. 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9. Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses. One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins. When labeled with [2-3H]inositol, mutants accumulate reduced amounts of radiolabeled GPI anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains. On the other hand, invertase and acid phosphatase are secreted by all but one mutant. All mutants show an increased sensitivity to calcofluor white and hygromycin B. This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall. PMID- 7559757 TI - Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. AB - Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate. PMID- 7559758 TI - Phragmoplast of the green alga Spirogyra is functionally distinct from the higher plant phragmoplast. AB - Cytokinesis in the green alga Spirogyra (Zygnemataceae) is characterized by centripetal growth of a septum, which impinges on a persistent, centrifugally expanding telophase spindle, leading to a phragmoplast-like structure of potential phylogenetic significance (Fowke, L. C., and J. D. Pickett-Heaps. 1969. J. Phycol. 5:273-281). Combining fluorescent tagging of the cytoskeleton in situ and video-enhanced differential interference contrast microscopy of live cells, the process of cytokinesis was investigated with emphasis on cytoskeletal reorganization and concomitant redistribution of organelles. Based on a sequence of cytoskeletal arrangements and the effects of cytoskeletal inhibitors thereon, cytokinetic progression could be divided into three functional stages with respect to the contribution of microfilaments (MFs) and microtubules (MTs): (1) Initiation: in early prophase, a cross wall initial was formed independently of MFs and MTs at the presumptive site of wall growth. (2) Septum ingrowth: numerous organelles accumulated at the cross wall initial concomitant with reorganization of the extensive peripheral interphase MF array into a distinct circumferential MF array. This array guided the ingrowing septum until it contacted the expanding interzonal MT array. (3) Cross wall closure: MFs at the growing edge of the septum coaligned with and extended along the interzonal MTs toward the daughter nuclei. Thus, actin-based transportation of small organelles during this third stage occurred, in part, along a scaffold previously deployed in space by MTs. Displacement of the nuclei-associated interzonal MT array by centrifugation and depolymerization of the phragmoplast-like structure showed that the success of cytokinesis at the third stage depends on the interaction of both MF and MT cytoskeletons. Important features of the phragmoplast-like structure in Spirogyra were different from the higher plant phragmoplast: in particular, MFs were responsible for the positioning of organelles at the fusion site, contrary to the proposed role of MTs in the higher plant phragmoplast. PMID- 7559762 TI - Increasing neurofilament subunit NF-M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons. AB - The carboxy-terminal tail domains of neurofilament subunits neurofilament NF-M and NF-H have been postulated to be responsible for the modulation of axonal caliber. To test how subunit composition affects caliber, transgenic mice were generated to increase axonal NF-M. Total neurofilament subunit content in motor and sensory axons remained essentially unchanged, but increases in NF-M were offset by proportionate decreases in both NF-H and axonal cross-sectional area. Increase in NF-M did not affect the level of phosphorylation of NF-H. This indicates that (a) in vivo NF-H and NF-M compete either for coassembly with a limiting amount of NF-L or as substrates for axonal transport, and (b) NF-H abundance is a primary determinant of axonal caliber. Despite inhibition of radial growth, increase in NF-M and reduction in axonal NF-H did not affect nearest neighbor spacing between neurofilaments, indicating that cross-bridging between nearest neighbors does not play a crucial role in radial growth. Increase in NF-M did not result in an overt phenotype or neuronal loss, although filamentous swellings in perikarya and proximal axons of motor neurons were frequently found. PMID- 7559760 TI - KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport. AB - We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo. PMID- 7559761 TI - Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization. AB - The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins. PMID- 7559759 TI - Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae. AB - In many eucaryotic cells, the midzone of the mitotic spindle forms a distinct structure containing a specific set of proteins. We have isolated ASE1, a gene encoding a component of the Saccharomyces cerevisiae spindle midzone. Strains lacking both ASE1 and BIK1, which encodes an S. cerevisiae microtubule-associated protein, are inviable. The analysis of the phenotype of a bik1 ase1 conditional double mutant suggests that BIK1 and ASE1 are not required for the assembly of a bipolar spindle, but are essential for anaphase spindle elongation. The steady state levels of Ase1p are regulated in a manner that is consistent with a function during anaphase: they are low in G1, accumulate to maximal levels after S phase and then drop as cells exit mitosis. Components of the spindle midzone may therefore be required in vivo for anaphase spindle movement. Additionally, anaphase spindle movement may depend on a dedicated set of genes whose expression is induced at G2/M. PMID- 7559763 TI - ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction. AB - ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses. PMID- 7559764 TI - Regulation of nerve growth factor receptor gene expression in sympathetic neurons during development. AB - We used quantitative reverse transcription (RT)/PCR to study the regulation of p75 mRNA and trkA mRNA expression in the developing sympathetic neurons of the mouse superior cervical sympathetic ganglion (SCG) in vivo and in vitro. At E13, the SCG contains proliferating cells that express many features of differentiated neurons. These immature neurons survived in culture without NGF, and NGF did not induce c-fos expression. Low levels of p75 and trkA mRNAs were expressed at this stage in vivo. There was no significant increase in the level of either trkA mRNA or p75 mRNA in E13 control cultures up to 72 h in vitro, and neither NGF nor depolarizing levels of K+ ions (40 mM KC1) affected the expression of trkA mRNA. In E14 cultures, NGF induced c-fos expression in 10-15% of the neurons and enhanced the survival of a similar percentage of neurons. The proportion of neurons responding to NGF increased with age, reaching 90% in E18 cultures. The in vivo level of trkA mRNA increased markedly from E14 onward, but in contrast to sensory neurons (in which p75 and trkA mRNA levels increase in parallel), the level of trkA mRNA initially increased far more rapidly than that of p75 mRNA. After E17, the level of p75 mRNA increased rapidly and approached that of trkA mRNA postnatally, but at no stage did this exceed the level of trkA mRNA. In E14 cultures, the level of trkA mRNA increased in the absence of neurotrophins or 40 mM KC1. The level of p75 mRNA in E14 cultures was enhanced by NGF but was unaffected by 40 mM KC1. Our findings show that NGF receptor expression during the earliest stages of sympathetic neuron development is not affected by depolarization but indicate that by an early developmental stage (between E13 and E14 in vivo), sympathetic neurons become specified to upregulate trkA mRNA in culture independently of added factors. In addition, our findings reveal several distinctive features of p75 mRNA and trkA mRNA expression in sympathetic neurons compared with sensory neurons and provide a plausible explanation for previously observed differences in the effects of a p75 null mutation on the response of sensory and sympathetic neurons during embryonic and postnatal development. PMID- 7559765 TI - Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture. AB - Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role. PMID- 7559768 TI - Fly division. PMID- 7559767 TI - Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1). AB - Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1 expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac 1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction. PMID- 7559769 TI - Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2. AB - Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus. PMID- 7559766 TI - Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways. AB - The teratocarcinoma-derived C1 clone behaves as a mesodermal tripotential progenitor cell whose choice of fate, either osteoblast, chondroblast, or adipoblast, is strictly dependent on the spatial organization of the cells and the nature of the induction. In the absence of cell contact before the addition of inducers, the C1 cells maintain a stable undifferentiated phenotype while expressing potential regulators of embryonic mesodermal stem cell fate such a M twist and Id1. Upon establishment of cell contacts before the induction of differentiation, the early genes characteristic of the three fates become expressed. In the presence of beta glycerophosphate and ascorbate, provided the cells have formed aggregates, 95% of the C1 cells mineralize with a kinetics of gene expression close to that of osteoblasts (Poliard, A., D. Lamblin, P. J. Marie, M. H. Buc, and O. Kellerman. 1993. J. Cell Sci. 106:503-512). With 10(-6)M dexamethasone, 80% of the same aggregates differentiate into foci of chondroblast like cells. The kinetics of expression of the genes encoding type II, IX, X, and XI collagens, aggrecan and link protein during the conversion toward cartilage hypertrophy resembles that accompanying in vivo chondrogenesis. The synergistic action of dexamethasone and insulin convert most confluent C1 cells into functional adipocytes and induce a pattern of gene expression close to that reported for adipoblast cell lines. The C1 clone with its capacity to differentiate along three alternative pathways with high frequency, therefore appears as a valid in vitro model for deciphering the molecular basis of mesoblast ontogeny. PMID- 7559770 TI - Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells. AB - A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction. PMID- 7559771 TI - Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. AB - We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte. PMID- 7559773 TI - Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. AB - We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton. PMID- 7559772 TI - CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium. AB - Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types. PMID- 7559775 TI - Isolation and characterization of nuclear envelopes from the yeast Saccharomyces. AB - We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems. PMID- 7559776 TI - Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery. AB - The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis. PMID- 7559777 TI - gamma-tubulin is a minus end-specific microtubule binding protein. AB - The role of microtubules in mediating chromosome segregation during mitosis is well-recognized. In addition, interphase cells depend upon a radial and uniform orientation of microtubules, which are intrinsically asymmetric polymers, for the directional transport of many cytoplasmic components and for the maintenance of the structural integrity of certain organelles. The slow growing minus ends of microtubules are linked to the centrosome ensuring extension of the fast growing plus ends toward the cell periphery. However, the molecular mechanism of this linkage is not clear. One hypothesis is that gamma-tubulin, located at the centrosome, binds to the minus ends of microtubules. To test this model, we synthesized radiolabeled gamma-tubulin in vitro. We demonstrate here biochemically a specific, saturable, and tight (Kd = 10(-10) M) interaction of gamma-tubulin and microtubule ends with a stoichiometry of 12.6 +/- 4.9 molecules of gamma-tubulin per microtubule. In addition, we designed an in vitro assay to visualize gamma-tubulin at the minus ends of axonemal microtubules. These data show that gamma-tubulin represents the first protein to bind microtubule minus ends and might be responsible for mediating the link between microtubules and the centrosome. PMID- 7559774 TI - Myosin is involved in postmitotic cell spreading. AB - We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II. PMID- 7559778 TI - Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. AB - We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal-epithelial interactions mediated by HGF and NRG. PMID- 7559779 TI - Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells. AB - Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation. PMID- 7559781 TI - E-cadherin is functionally involved in the maturation of the erythroid lineage. AB - Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage. PMID- 7559782 TI - Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact. AB - RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts. PMID- 7559780 TI - Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition. AB - Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431 F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells. PMID- 7559783 TI - Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules. AB - Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope. PMID- 7559786 TI - Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway. AB - ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC 53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant. PMID- 7559788 TI - A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes. AB - Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved. PMID- 7559784 TI - A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones. AB - Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396-430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo. PMID- 7559785 TI - Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies. AB - The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes. PMID- 7559787 TI - Clathrin-independent pinocytosis is induced in cells overexpressing a temperature sensitive mutant of dynamin. AB - A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway. PMID- 7559789 TI - Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe. AB - Elevated levels of certain membrane proteins, including the sterol biosynthetic enzyme HMG-CoA reductase, induce proliferation of the endoplasmic reticulum. When the amounts of these proteins return to basal levels, the proliferated membranes are degraded, but the molecular details of this degradation remain unknown. We have examined the degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe. In this yeast, increased levels of the Saccharomyces cerevisiae HMG-CoA reductase isozyme encoded by HMG1 induced several types of membranes, including karmellae, which formed a cap of stacked membranes that partially surrounded the nucleus. When expression of HMG1 was repressed, the karmellae detached from the nucleus and formed concentric, multilayered membrane whorls that were then degraded. During the degradation process, CDCFDA-stained compartments distinct from preexisting vacuoles formed within the interior of the whorls. In addition to these compartments, particles that contained neutral lipids also formed within the whorl. As the thickness of the whorl decreased, the lipid particle became larger. When degradation was complete, only the lipid particle remained. Cycloheximide treatment did not prevent the formation of whorls. Thus, new protein synthesis was not needed for the initial stages of karmellae degradation. On the contrary, cycloheximide promoted the detachment of karmellae to form whorls, suggesting that a short lived protein may be involved in maintaining karmellae integrity. Taken together, these results demonstrate that karmellae membranes differentiated into self degradative organelles. This process may be a common pathway by which ER membranes are turned over in cells. PMID- 7559791 TI - Adaptation of Ehrlich ascites carcinoma cells to energy deprivation in vivo can be associated with heat shock protein accumulation. AB - Tumor adaptation to chronic energy starvation in vivo was studied on Ehrlich ascites carcinoma (EAC) cells. EAC cells were isolated from mice and incubated in a glucose-free medium containing blocators of mitochondrial ATP generation (rotenone, 2,4-dinitrophenol, or oligomycin). ATP level in the treated cells decreased to 3-4% of the initial during 30 min of the incubation. The aggregation of cytoskeletal proteins, blebbing, and necrotic death within 2-3 h were observed in ATP-depleted EAC which were isolated and treated in the exponential phase of growth (5 days after inoculation), whereas stationary EAC (8 days after inoculation) were considerably more resistant to ATP depletion, and actin aggregation as well as bleb formation were suppressed in these cells despite the ATP loss. In contrast to the exponentially growing cells, thermotolerance and unexpected expression of inducible HSP68 and HSP27 as well as an elevated level of HSP90 were found in stationary EAC. Since the stationary cells had decreased content of ATP, ATP/ADP ratio, and energy charge, we suggest that this energy dysbalance may be conducive to HSP induction within the ascites tumor in vivo, and, at the same time, EAC cells with elevated content of HSPs acquire resistance to chronic energy starvation occurring in late stages of the tumor growth. PMID- 7559790 TI - Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1. AB - Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-->Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT. PMID- 7559793 TI - Concanavalin A induced apoptosis in fibroblasts: the role of cell surface carbohydrates in lectin mediated cytotoxicity. AB - Cell surface carbohydrates play important regulatory roles during development and in tissue homeostasis by mediating cellular signalling. Concanavalin A (con A) cross links mannose residues on cell surfaces. We used con A to examine the role of cell surface carbohydrates in apoptosis. Balb/c 3T3 (3T3) and diploid human gingival fibroblasts (HGF) incubated with con A (50 micrograms/mL) exhibited rounding, reduction of cell size, loss of cytoskeletal definition, nuclear condensation, and ultrastructural changes consistent with apoptotic cell death. However oligonucleosomal DNA fragmentation was not observed. The sugar methyl alpha-D-mannopyranoside (MADM) competes for binding sites with con A but failed to induce apoptosis. Cell survival assays after con A treatment demonstrated a concentration dependent (5-500 micrograms/mL) loss of cell viability in 3T3 and HGF cells which was blocked by MADM. The relationship between cell surface binding of con A and cell death was studied in 3T3 cells labelled with varying concentrations of FITC-con A. Flow cytometry and cell survival analyses revealed that smaller cells with lower total cell surface FITC-con A binding sites were approximately 8 times more susceptible to cell death than larger cells with higher number of binding sites. This suggests a positive relationship between cell size, number of con A binding sites, and cell death. Flow cytometric cell cycle analysis of HGF cells showed a 46% reduction in the proportion of G1 phase cells but there was little change in the relative proportions of S and G2+M phase cells. 3H-thymidine labelling of con A treated cells showed a five-fold decrease (chi sq; P < 0.05) in the percentage of labelled cells, indicating blockade of cell cycle transit into S. Thus, cell death occurred predominantly in G1, possibly due to inhibition of protein synthesis which in turn prevented entry of cells into S phase. 35S-methionine uptake in con A treated cells was significantly reduced (approximately 42% con A 50 micrograms/mL; approximately 67% con A 500 micrograms/mL; P < 0.05) compared to untreated controls indicating inhibition of de novo protein synthesis. SDS-PAGE of total cellular proteins confirmed reduction of predominantly lower molecular mass proteins after con A treatment. Con A treatment (50 micrograms/mL) induced a 25% reduction of free intracellular calcium ion concentration over 30 min suggesting that calcium dependent enzymes and translational mechanisms may be inhibited. Collectively, these results indicate that con A binding of cell surface carbohydrates can induce apoptotic cell death in fibroblastic cells due in part to protein synthesis inhibition. PMID- 7559792 TI - Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways. AB - Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. PMID- 7559794 TI - Lithium-stimulated proliferation and alteration of phosphoinositide metabolites in MCF-7 human breast cancer cells. AB - Lithium, which is used to treat bipolar psychiatric disorders, can stimulate proliferation of a number of cells in tissue culture. Proliferation of MCF-7 human breast cancer cells, which also respond to EGF and estrogens, was stimulated by LiCl (1-5 mM) within the concentration range that is encountered during human therapy with lithium. Stimulation of growth was specific for lithium; rubidium, potassium, and sodium showed no such effect. In the presence of antiestrogen, lithium stimulated the growth of hormone-dependent breast cancer cells MCF-7, ZR-75-1, and T47D but not hormone-independent MDA-MB-231 cells or an estrogen-independent clone of MCF-7 cells. Lithium-stimulated proliferation was limited by cytotoxicity which could be moderated by added potassium chloride (5 20 mM) in the medium. Each of the mitogens lithium, 17 beta-estradiol, and EGF increased the rate of uptake of myo-inositol into MCF-7 cells. Whether normalized to inositol lipids, to protein, or to DNA, steady-state levels of inositol phosphates were elevated by each of the mitogens including lithium, which inhibits the breakdown of inositol phosphates in the phosphoinositide signaling pathway. These data indicate that therapeutic concentrations of lithium can stimulate the proliferation of human breast cancer cells by a mechanism that may involve the phosphoinositide pathway. PMID- 7559796 TI - Increased tumorigenicity in the human pancreatic cell line MIA PaCa-2 is associated with an aberrant regulation of an IGF-1 autocrine loop and lack of expression of the TGF-beta type RII receptor. AB - The growth characteristics associated with tumorigenicity were determined in clones of MIA PaCa-2 and PANC-1 pancreatic carcinoma cells. MIA PaCa-2 cells differed from PANC-1 cells in that they rapidly formed tumors in nude mice, formed colonies more rapidly and formed larger colonies in soft agar, and were cloned more efficiently when seeded at low density. MIA PaCa-2 cells but not PANC 1 cells were stimulated to escape quiescence and undergo DNA synthesis with nutrient media lacking growth factors. Both cell lines were stimulated to proliferate with serum-free media containing EGF, transferrin, and insulin. Antibody neutralization assays indicated that an IGF-1 autocrine loop was required for the nutrient stimulation of growth in MIA PaCa-2 cells and for the growth-factor stimulation in both MIA PaCa-2 and PANC-1 cells. Both cell lines were stimulated to proliferate with exogenous IGF-1 in basal media; this stimulation was specifically blocked by antibodies to IGF-1 or its receptor. MIA PaCa-2 and PANC-1 cells expressed similar levels of IGF-1 receptor mRNA and showed similar binding kinetics in receptor binding assays. In contrast to PANC-1 cells, MIA PaCa-2 cells were insensitive to TGF-beta 1 and did not express TGF beta receptor type II. The results suggest that the growth-factor independence is representative of a more tumorigenic phenotype. We hypothesize that growth-factor independence of MIA PaCa-2 cells is mediated by an aberrant regulation of an IGF 1 autocrine loop. A decreased regulation of this IGF-1 loop may be potentiated by loss of response to TGF-beta. PMID- 7559797 TI - Regulation of parathyroid hormone/parathyroid hormone-related protein receptor expression by osteoblast-deposited extracellular matrix in a human osteoblast like cell line. AB - Parathyroid hormone (PTH) receptors and the biological response to PTH in osteoblasts have been shown to be influenced by glucocorticoids, growth factors, cytokines or PTH itself. Furthermore, components of extracellular matrix (ECM) appear to regulate the response to PTH as well. We investigated the effects of osteoblast-deposited ECM on PTH-related protein (PTHrP)-stimulated cAMP production, PTHrP binding and PTH/PTHrP receptor mRNA in the human osteoblast like cell line SaOS-2. ECM was laid down by the human osteoblastic cell line MG 63. At confluence, maximal cAMP stimulation induced by 100 nmol/l PTHrP (1-34) was decreased in SaOS-2 cells grown on ECM as compared with cultures on plastic dishes, without any change in PTHrP concentration producing half-maximal stimulation. In contrast, cAMP production stimulated by PGE2 was increased in cells on ECM. Saturable 125I-PTHrP binding (as evaluated by Scatchard plot analysis) was markedly diminished in cells grown on ECM (5,600 +/- 2,010 vs. 20,700 +/- 1,710 binding sites/cell, x +/- S.E.M., P < 0.01, n = 4 experiments), without any significant change in affinity (1.3 +/- 0.4 vs. 2.5 +/- 0.5 nmol/l (NS), in cells on ECM and plastic, respectively). This apparent decrease in membrane receptor density was associated with markedly lower steady state PTH/PTHrP receptor mRNA levels as assessed by Northern blot analysis (ECM/control: 0.4 +/- 0.1). A difference in PTH/PTHrP receptor mRNA levels between cells on ECM or on plastic dishes was detectable by 8 hours but not by 4 hours, after seeding the cells at high density. By 24 hours after plating, PTH/PTHrP receptor mRNA levels were maximally decreased in cells on ECM. These results in the human osteoblast-like cell line SaOS-2 indicate that PTH/PTHrP receptors are down-regulated by growth on ECM. Thus, attachment of bone cells to bone surface could influence differentiation and function of osteoblasts. PMID- 7559795 TI - Influences of silicates and carnitine-silicate mixtures on the inhibition of aggregation of erythrocytes elicited by the presence of fibrinogen. AB - Carnitine and acylcarnitine derivatives have been reported to inhibit cell aggregation (Fritz and Burdzy, 1989, J. Cell. Physiol., 140:18-28). A follow-up of these observations showed that whereas the previously described effects of long-chain acylcarnitines were well replicated, those of carnitine on erythrocytes showed marked variability. The latter phenomenon was traced to the presence of silicates in carnitine solutions derived from the use of sodium hydroxide solutions stored in glass containers for the neutralization of carnitine. The present experiments have led to the discovery that oligomeric forms of silicates are powerful inhibitors of red blood cell aggregation which otherwise occurs in the presence of fibrinogen alone. The active form(s) of silicates in this assay, which appear to be generated by polymerization of silicates in metasilicate solutions on neutralization, are unstable and therefore transient under usual conditions. We estimate that the active oligomeric forms contain between 4 to 18 silicon atoms per molecule. When maintained at -18 degrees C in the presence of carnitine, but not in its absence, the active forms of oligomeric silicates remained stable for months, judging from their ability to inhibit cell aggregation. We conclude that carnitine stabilized the oligomeric form(s) of silicate, or that the species stabilized is an oligomeric silicate carnitine complex. Comparable concentrations of choline, deoxycarnitine, or gamma aminobutyrate were less effective in stabilizing the active silicate oligomers. The active forms of the silicate oligomers had Ki values of about 10 microM, calculated as the monomeric form, in inhibiting red blood cell aggregation. The data indicate that free carnitine does not directly inhibit erythrocyte inhibition, as previously interpreted, whereas long-chain acylcarnitine derivatives are active in the absence of silicates. Possible mechanism of actions of silicate oligomers on membranes are discussed. PMID- 7559798 TI - Secretin potentiates cholecystokinin-stimulated amylase release by AR4-2J cells via a stimulation of phospholipase C. AB - Alterations in the activity of phospholipase C (PLC) are thought to be the primary intracellular events leading to pancreatic acinar cell exocytosis of zymogen granules. When multiple hormones, each of which may stimulate different signal transduction pathways, bind to cell surface receptors, the cell must integrate these signals into a common response through communication (cross-talk) among intracellular second messengers. We show that cholecystokinin (CCK) induces amylase secretion from AR4-2J pancreatic acinar cells via stimulation of PLC activity. Secretin indirectly stimulated the PLC pathway through cross-talk of the activated cAMP pathway to potentiate the CCK-stimulated amylase secretion. Therefore, secretin potentiated the acinar cell secretory response to CCK by cAMP mediated cross-talk with the PLC signal transduction pathway. PMID- 7559802 TI - Transforming growth factor-beta receptor expression on endothelial cells: heterogeneity of type III receptor expression. AB - Recent studies of whole animal responses have defined a role for circulating TGF beta in the preservation and stabilization of microvascular endothelial function (Lefer et al. [1993] Proc. Natl. Acad. Sci. U.S.A., 90:1018-1022; Pfister et al. [1992] J. Exp. Med., 176:265-269). In order to determine which TGF-beta receptor types are responsible for this endothelial cell responsiveness, we used an affinity-labeling technique with 125I-TGF-beta 1 and -beta 2 to characterize TGF beta receptors on five different endothelial cell cultures: early passage bovine lung and rat epididymal fat pad microvascular endothelial cells (BLMEC and REEC), established endothelial cell lines from bovine adrenal medulla capillaries (EJG), fetal bovine heart (FBHE), and bovine pulmonary artery (CPAE). Since it is known that endothelial cells from different parts of the vasculature vary with respect to cell surface antigen expression (McCarthy et al. [1991] Trends Pharmacol. Sci., 12:462-467; Augustin et al. [1994] Bioessays, 16:901-906), it is important to compare TGF-beta receptor expression on microvascular and macrovascular endothelial cells. We observed 85 kDa and 200-400 kDa labeled receptor bands and analyzed their relationship to the cloned Type II and III receptors using peptide antibodies. We used dithiothreitol and phosphoinositol-phospholipase C pretreatments to establish whether the 65 kDa labeled band which we observed corresponded to the Type I receptor or a glycophosphotidylinositol-linked binding protein. The results demonstrated that microvascular but not macrovascular endothelial cells express high levels of the Type III receptor. This differential expression of the Type III receptor indicates that distinct anatomical segments of the vasculature have distinct TGF-beta receptor profiles. The presence of the Type III receptor on micro- but not macrovascular endothelial cells may account for the reportedly different potency of TGF-beta 1 and TGF-beta 2 on these two endothelial cell types. Analysis of the 85 kDa and 65 kDa affinity-labeled bands revealed that all the endothelial cells express the Type II receptor and a band consistent with the presence of a dithiothreitol-sensitive Type I receptor. Two isoform-specific phosphoinositol-phospholipase C releasable TGF-beta binding proteins were also detected: a 60 kDa protein on one micro- (EJG) and one macro- (FBHE) vascular endothelial cell line and a 150/180 kDa protein on the macrovascular cell lines (FBHE and CPAE). These studies emphasize the heterogeneous nature of endothelial cells and underline the importance of using microvascular endothelial cells when examining TGF-beta responses related to microvascular function. PMID- 7559799 TI - Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle. AB - We previously reported that MCF-7 cells were arrested in the G0/G1 phase of the cell cycle by agents known to block the activity of ATP-sensitive potassium channels (Woodfork et al., 1995, J. Cell Physiol. 162:163-171). The goal of our current study was to determine if MCF-7 cells undergo changes in membrane potential during the cell cycle that might be linked to changes in K permeability. The resting membrane potentials of unsynchronized MCF-7 cells during exponential growth phase were measured using sharp glass microelectrodes, and they ranged from -58.6 mV to -2.7 mV. The distribution of membrane potentials was best fitted by the sum of four Gaussian distributions with means of -9.0 mV, 17.4 mV, -24.6 mV, and -40.4 mV. These membrane potential groups were designated D (depolarized), ID (intermediate depolarized), IH (intermediate hyperpolarized), and H (hyperpolarized), respectively. The membrane potential was sensitive to the substitution of external K and Na but not Cl. The K:Na permeability ratio increased in proportion to the negativity of the membrane potential. MCF-7 cells pharmacologically arrested in G0/G1 phase were depolarized compared to control, with cells shifted from the H and IH groups to the D group. Tamoxifen-arrested cells chased from G0/G1 into S phase by the addition of mitogenic concentrations of 17 beta-estradiol were not depolarized, and these cells were shifted from the D group back to the IH and H groups. We conclude that MCF-7 cells hyperpolarize during passage through G0/G1 and into S phase, and this hyperpolarization probably results from an increase in the relative permeability of the plasma membrane to K. PMID- 7559800 TI - Tenascin-C induction by the diffusible factor epidermal growth factor in stromal epithelial interactions. AB - Tenascin-C, a six-armed extracellular matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis and invasion or metastasis of carcinoma cells in association with stromal-epithelial interactions. The human epidermoid carcinoma-derived cell lines, A431 and HEp-2, which do not express tenascin-C by themselves in vitro, do express tenascin-C after transplantation into nude mice, and transforming growth factor beta 1 (TGF beta 1) induces them to express tenascin-C in vitro. Epidermal growth factor (EGF) induced tenascin-C in these cells more effectively (about 3.5-fold greater) than did TGF-beta 1. Hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) had little effect on the induction of tenascin-C. EGF also induced other extracellular matrix components, fibronectin and laminin. Tenascin-C was also induced when the carcinoma cells were co-cultured with embryonic fibroblasts from mice which were homozygous for a null mutation in the tenascin-C gene, or when the conditioned medium from these cells was added. The induction of tenascin C in the co-culture was reduced by treating the cells with antibodies against EGF or its receptor. The addition of EGF caused both cell types to disrupt their cytoskeleton and focal contacts as evidenced by the loss of stress fibers and vinculin plaques. EGF did neither induce tenascin-C nor affect the morphology in tenascin-C-nonproducing A549 carcinoma cells, which did not produce tenascin-C after transplantation. Thus, EGF induces tenascin-C in tenascin-C-nonproducing human carcinoma cells through EGF receptors. Furthermore, in stromalepithelial interactions, the diffusible factor EGF participates in the induction of human tenascin-C in these cells through EGF receptors. PMID- 7559801 TI - Short circuiting stress protein expression via a tyrosine kinase inhibitor, herbimycin A. AB - We set out to identify pharmacological means by which to activate the so-called heat shock or stress response and thereby harness the protective effect afforded to the cell by its acquisition of a thermotolerant phenotype. An earlier report by Murakami et al. (1991, Exp. Cell Res., 195: 338-344) described the increased expression of the 70 kDa heat shock proteins in human A431 cells exposed to Herbimycin A (HA), a benzoquinoid ansamycin antibiotic. We show here that treatment of cells with HA results in the increased expression of all of the constitutively expressed stress proteins and confers upon the cells a thermotolerant-like phenotype. Increases in the expression of the stress proteins continued for as long as the cells were exposed to the drug and was independent of the pre-existing levels of the stress proteins. Unlike heat shock or other metabolic stressors, we did not observe any adverse cellular effects following HA exposure. For example, unlike most agents/treatments that elicit the stress response HA-treated cells exhibited no obvious abnormalities with respect to protein maturation, protein insolubility, the integrity of the intermediate filament cytoskeleton, or overall cell viability. In addition, unlike other metabolic stressors, HA treatment did not result in the translocation of hsp 73 into the nucleus/nucleolus. Finally, for at least rodent cells, HA exposure did not result in any obvious activation of the heat shock transcription factor. Based on these findings, we suggest that HA treatment of cells results in a "short-circuiting" of the pathway(s) that normally regulates the expression of the stress proteins. These results are discussed as they pertain to the potential use of HA in animals as a way to harness the protective effects afforded by the stress response. PMID- 7559803 TI - Insulin-like growth factors modulate the growth inhibitory effects of retinoic acid on MCF-7 breast cancer cells. AB - Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I, IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (> or = 10(-6) M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10(-7) M RA, and that 10(-9) - 10(-7) M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10(-9) - 10(-7) M RA. Treatment with the IGF I receptor blocking antibody, alpha IR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth inhibited cultures and in IGF-II expressing clones that are resistant to RA induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10(-9) - 10(-7) M RA enhancing cell proliferation and > or = 10(-6) M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies. PMID- 7559804 TI - Protein kinase A regulates the degradation rate of Rs acetylcholine receptors. AB - Acetylcholine receptors at the neuromuscular junction of innervated vertebrate muscle (called Rs AChRs) have a stable degradation rate (t1/2 approximately 8-12 days) which accelerates after denervation to a half-life of approximately 3 days, but can be restabilized by reinnervation or by cAMP. We examined the mechanism by which cAMP regulates the Rs degradation rate. When dibutyryl cAMP (DB-cAMP) was applied to denervated mouse diaphragms in organ culture, it stabilized the accelerated degradation rate of the Rs. We found that this stabilization is reversible upon removal of the DB-cAMP, is cAMP specific and is mediated by intracellular cAMP. A major observation of this study is that the cAMP-induced stabilization of Rs AChRs is via protein kinase A (PKA), since H89, a PKA inhibitor, blocked the DB-cAMP induced stabilization of Rs, and H85, an analog of H89, which does not inhibit PKA but does inhibit other kinases as efficiently as H89, did not prevent the DB-cAMP-induced stabilization of Rs degradation. These results suggest that the cAMP messenger system via a PKA-dependent pathway could be among the mechanisms whereby the nerve regulates AChR degradation. PMID- 7559805 TI - Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression. AB - To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as early as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was > 90% neutralized by antibody to tumor necrosis factor alpha (TNF-alpha), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-alpha; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM 1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. PMID- 7559806 TI - Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan containing coats around normal human mesothelial cells. AB - The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of 3H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80-90% could be ascribed to hyaluronan and only 3-5% to chondroitin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1-3.0 microns, as well as erythrocytes of diameter 7 microns. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 microns or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer. PMID- 7559808 TI - Members of the 70 kDa heat shock protein family specifically recognize sulfoglycolipids: role in gamete recognition and mycoplasma-related infertility. AB - We have previously shown that several mycoplasma species associated with infertility bind specifically to sulfated glycolipids isolated from the mammalian reproductive tract. We now show that a germ cell-specific sulfoglycolipid binding protein (SLIP 1), which is a potent inhibitor of sperm/egg binding in vitro, is immunologically related to the heat shock protein(Hsp) 70 family of stress proteins and that Hsps are surface antigens in male germ cells. Our present data demonstrate that several mycoplasma and mammalian Hsps share this glycolipid binding specificity in vitro, and suggest that surface Hsps can function as adhesins which mediate sulfoglycolipid recognition in infectious disease and normal reproductive physiology. PMID- 7559807 TI - Inhibition of the arachidonic acid pathway prevents induction of IL-8 mRNA by phorbol ester and changes the release of IL-8 from HL 60 cells: differential inhibition of induced expression of IL-8, TNF-alpha, IL-1 alpha, and IL-1 beta. AB - The promyelocytic HL 60 cell line can be used as an in vitro model system to study hematopoetic cell differentiation and inflammatory events. We studied the signal transduction pathway of induced interleukin (IL)-8 expression and compared it with those of tumor necrosis factor alpha (TNF-alpha), IL-1 alpha, and IL-1 beta. The differentiation of HL 60 cells to macrophage-like cells by PMA resulted in a rapid and marked induction of these inflammatory cytokines. The up regulation occurred in the absence of ongoing protein synthesis, but cycloheximide-sensitive gene products modulated their induction kinetics. Staurosporine, a potent inhibitor of protein kinases, strongly inhibited their gene expression. Phosphorylation may not act directly on latent transcription factors, since bromophenacyl bromide, an inhibitor for the release of arachidonic acid from phorbol-12 myristate 13-acetate (PMA)-stimulated HL 60 cells, markedly depressed the induced mRNAs for IL-8, TNF-alpha, and IL-1 alpha and -beta. Similarly, 5,8,11,14 eicosatetraynoic acid (ETYA), another inhibitor of the arachidonic acid pathway, blocked the induction of transcripts for TNF-alpha, and both IL-1 genes in phorbol ester-stimulated HL 60 cells. In contrast, ETYA increased the induced IL-8 RNA levels and stimulated the release for IL-8. Also, ketoconazole, an inhibitor of 5-lipoxygenase and indomethacin, an inhibitor of cyclooxygenases did not block the induction of IL-8 mRNA. However, the release of IL-8 protein was regulated by indomethacin and ketoconazole. Our results indicate that arachidonic acid metabolites are mediators in the signal transduction pathway of IL-8 expression and that the involved second messengers are different from those which are important for the induction of TNF-alpha and IL-1 beta expression. PMID- 7559809 TI - Significant induction of spermidine/spermine N1-acetyltransferase without cytotoxicity by the growth-supporting polyamine analogue 1,12-dimethylspermine. AB - The superinduction of the polyamine catabolic enzyme spermidine/spermine N1 acetyltransferase (SSAT) has been implicated in the cell type-specific cytotoxic activity of some polyamine analogues. We now report that one polyamine analogue, 1,12-dimethylspermine (DMSpm), produces a large induction of SSAT with no significant effects on growth in the human large cell lung carcinoma line, NCl H157. This cell line has been demonstrated to respond to other analogues with SSAT superinduction and cell death. Treatment of the lung cancer cell line with DMSpm produces a rapid increase in SSAT activity and a near complete depletion of the natural polyamines. Additionally, DMSpm supports cell growth in cells which have been depleted of their natural polyamines by the ornithine decarboxylase inhibitor, 2-difluoromethylornithine. The current results suggest that significant induction of SSAT can occur in the absence of cytotoxicity when the inducing polyamine analogue can support growth and that increased SSAT activity alone is not sufficient for cytotoxicity to occur. PMID- 7559811 TI - Insulin-like growth factor-1 stimulates amino acid uptake by the cultured human placental trophoblast. AB - Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory characterized the uptake of alpha-aminoisobutyric acid (AIB) stimulated by insulin in the cultured human placental trophoblast. The current study evaluated the effect of insulin-like growth factor-1 (IGF-1) on AIB uptake in cultured human placental trophoblasts. Na(+)-dependent AIB uptake was significantly stimulated by IGF-I in a time dependent manner, as early as 30 min after hormone exposure. The maximum effect was at 2-4 hr of continuous exposure to IGF-I and the stimulation was dependent upon IGF-1 concentration approaching maximal stimulation at 50 ng.ml-1. AIB uptake was inhibited by increasing concentrations of alpha (methylamino)isobutyric acid (MeAIB). Approximately 75% of basal (unstimulated) Na(+)-dependent AIB uptake was inhibited by MeAIB. The IGF-1-stimulated increment above basal AIB uptake was completely inhibited by MeAIB. IGF-1 increased the maximum uptake velocity but not Km. Using equimolar concentrations, stimulation was greater with IGF-1 than with IGF-2. Stimulation by IGF-1, but not insulin, was inhibited by anti-IGF-1 receptor antibody, indicating mediation via the IGF-1 receptor. H7, a nonspecific inhibitor of serine-threonine kinase, inhibited IGF-1 dependent stimulation of AIB uptake. In addition, calphostin C (a specific inhibitor of protein kinase C), but not H89 (a specific inhibitor of protein kinase A), inhibited the IGF-1 action. This study further characterizes regulated amino acid uptake by the human placental trophoblasts and demonstrates that the Na(+)-dependent component of AIB uptake is stimulated by physiologic concentrations of IGF-1. PMID- 7559810 TI - Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages. AB - Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E2 (PGE2). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 micrograms/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE2 and prostaglandin H synthase 2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL 4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE2, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. PMID- 7559814 TI - Anatomy of the extensor mechanism of the hand and wrist. AB - This article describes extensor muscle and tendon anatomy from origin to insertion. As usual, the use of appropriate terminology is the building block for understanding the function and disorders of the extensor mechanism. PMID- 7559815 TI - Extensor physiology in the hand and wrist. AB - The Physiology of finger extension is complex because of the various components of the extensor tendon mechanism. This article discusses the role each component plays in finger extension. Tendon excursion, strength, and electromyographic activity of the extensors are discussed. PMID- 7559812 TI - Ca2+ influx, phosphoinositide hydrolysis, and histamine release induced by lysophosphatidylserine in mast cells. AB - We have previously demonstrated that snake venom phospholipases A2 (PLA2s) and mammalian PLA2s induced inflammatory processes. This effect was correlated with the activity of the enzymes and the release of lipid mediators. We have now determined the role of lysophosphatidylserine (LysoPS) as an inflammatory lipid mediator. Thus, we have studied the possibility that intracellular calcium concentration, phosphoinositide hydrolysis, and the subsequent histamine release in mast cells is due to the action of lysophosphatidylserine. Lysophosphatidylserine-stimulated release of histamine was significantly higher than release by other lysophospholipids. The contribution of increased phospholipase C activity and the intracellular Ca2+ influx were therefore examined. LysoPS increased mast cell calcium concentration, and this increment was associated with phospholipase C activation and release of inositol phosphates. The increase in intracellular calcium and histamine degranulation induced by LysoPS were inhibited by apomorphine. Pretreatment of mast cells with pertussis toxin decreased the secretagogic effect of LysoPS and compound 48/80 without modifying the effect of the ionophore A23187. These results suggest that pertussis toxin-sensitive G-protein might be involved in the mast cell degranulation produced by lysophosphatidylserine and allow the increase in phospholipase C activity, thus enhancing intracellular calcium concentration, which then induces exocytosis of histamine. PMID- 7559813 TI - Serglycin and betaglycan proteoglycans are expressed in the megakaryocytic cell line CHRF 288-11 and normal human megakaryocytes. AB - This study has characterized the proteoglycans from the megakaryocytic tumor cell line CHRF 288-11 and the effect of the differentiation-inducing agents phorbol-12 myristate-13-acetate (PMA) and dimethylsulfoxide (DMSO) on proteoglycan synthesis in these cells. There appeared to be two classes of proteoglycans. One, serglycin, was recognized to have a core protein of 31 kDa, an overall molecular mass of 200-300 kDa, and glycosaminoglycan chains of mean size < 25 kDa. The size of this proteoglycan was increased by both PMA and DMSO. Synthesis was increased by PMA and reduced by DMSO. mRNA for serglycin was increased at 24 to 72 hr following PMA treatment. In addition, the cells contained a core protein triplet at 96, 110, and 120 kDa, and the medium only the bands at 96 and 110 kDa, suggesting the presence of betaglycan. Synthesis of this proteoglycan was enhanced by PMA. This proteoglycan had an overall size of 130-150 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in control cells, but in the presence of PMA, a component > 250 kDa was present. Probes for Northern blot analysis were prepared by polymerase chain reaction (PCR) based on the sequences of human serglycin and betaglycan. The serglycin probe recognized a 1.4 kb band, and the betaglycan probe recognized a 4.1 kb band, on blots prepared from RNA from CHRF cells and cultured normal human megakaryocytes. Both proteoglycans in their intact form adhered to peptides derived from fibronectin and collagen, but the free GAGs released by alkaline borohydride digestion did not adhere. Synthesis of two proteoglycans appears to be a part of the differentiation process of megakaryocytic tumor cells and normal megakaryocytes. PMID- 7559816 TI - Extensor tendon injuries at the distal interphalangeal joint. AB - The treatment options for the soft-tissue mallet finger, in both its acute and chronic forms, continue to generate some degree of controversy. Priority always should be given to nonoperative management of these injuries. This translates into a 6- to 8-week period of uninterrupted immobilization of the DIP joint with an external splint. Splinting has been shown to be highly effective, safe, and reproducible for both acute and chronic lesions. Even in the presence of an open injury, the value of splinting must be appreciated by the practitioner. The conversion of an acute closed soft-tissue injury to an open one is to be discouraged because of unacceptable complication rates. When surgery is contemplated, our first option would be the placement of a transarticular Kirschner wire at the DIP joint. If external splinting fails in an acute injury, an argument certainly can be made for a second trial of conservative management, and we offer this alternative to such patients. We find that some patients will not tolerate a second period of immobilization, and in most cases, we offer surgery in these failed cases. Surgical choice, again, would be a transarticular Kirschner wire. Another choice would be one of the salvage procedures, such as central slip tenotomy, with the expectation of a good result. In summary, mallet injuries are treated using closed, nonoperative techniques. The period of time after injury that this nonoperative treatment can be delayed and still be effective is being extended and the absolute outside time limit still is not known. When surgery is done, we prefer the simple placement of a transarticular Kirschner wire for 6 to 8 weeks.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7559817 TI - Boutonniere deformity. AB - The boutonniere (French for button hole) deformity is a result of an injury to the finger extensor mechanism that causes a characteristic flexion attitude of the proximal interphalangeal joint and distal interphalangeal joint hyperextension. This article reviews the pertinent anatomy, the mechanisms of injury, the diagnosis, and a systematic approach to the prevention and treatment of the boutonniere deformity. PMID- 7559818 TI - Acute injuries of the extensor tendons proximal to the metacarpophalangeal joints. AB - The results of acute repair of the extensor tendons proximal to the metacarpophalangeal joint vary with the degree of associated injuries. Shortening should be kept to a minimum at the time of repair. The Kleinert modification of the Bunnell technique affords the greatest tensile strength. A 3-0 or 4-0 nonabsorbable suture on a small tapered needle should be used. The extensor retinaculum should be resected or transposed for injuries in zones VII and T V. Sensory branches of the ulnar and radial nerves should be repaired primarily, if possible. The most frequent complication is loss of metacarpophalangeal joint flexion secondary to tendon adhesions. The more complex the wound, the greater the indication for controlled mobilization. PMID- 7559820 TI - Extensor tendon grafting on the dorsum of the hand in massive tendon loss. AB - After severe injury to the dorsum of the hand, it may not be possible to directly repair the extensor tendons. Patients in this article were treated by initial skin replacement with pedicle flaps for coverage prior to extensor tendon grafting. Multiple operative procedures were necessary, but restoration was uniformly successful. PMID- 7559819 TI - Tendon transfer for rupture of the extensor pollicis longus. AB - Reviewing the history and etiology of extensor pollicis longus tendon rupture shows the most compelling mechanism of rupture apparently is interruption of the tendon's vascularity secondary to hemorrhage and pressure, which causes the damaged tendon to be more susceptible to rupture secondary to late ischemic necrosis and attrition. Treatment options tried have included direct repair, tendon grafting, and tendon transfer. The authors recommend the extensor indicis proprius tendon transfer as the most predictable procedure to restore the original function of the EPL. This technique can be performed reliably, requires little postoperative re-education, and has few associated complications. PMID- 7559822 TI - Treatment of the chronic boutonniere deformity by extensor tenotomy. AB - A patient with a chronic boutonniere deformity often experiences more difficulty because of the hyperextension deformity at the distal joint, rather than the lack of extension at the proximal interphalangeal joint. It is in these cases that restoration of flexion at the terminal joint by tenotomy of the terminal extensor tendon is of most value. PMID- 7559824 TI - Salvage of extensor tendon function with tenolysis and joint release. AB - Extensor tendon adhesions following laceration, fracture, or crush injuries will prevent normal joint function. Tenolysis often will not address the resulting stiffness. A systematic release of the extensors, joint capsule, collateral ligaments, and occasionally the flexor tendons is needed to restore function. PMID- 7559823 TI - Extensor tendon ruptures in rheumatoid arthritis. AB - The best treatment for extensor tendon rupture is prevention, either by medical management or surgical tenosynovectomy before tendon ruptures occur. Once a rupture has occurred, tendon transfer or free tendon grafting can provide acceptable restoration of extensor function. Communication with the rheumatologist is necessary to provide timely treatment for chronic dorsal tenosynovitis (and impending tendon rupture) or for single finger extension loss before the disease progresses to multiple finger extension loss. Consideration always must be given to associated joint involvement when planning surgical treatment. PMID- 7559825 TI - Microsurgical reconstruction of the extensor system. AB - Single stage transfer using a compound vascularized flap is sometimes necessary for severe dorsal defects. The use of vascularized tendon grafts in conjunction with the radial artery forearm flap and the dorsalis pedis flap is described. PMID- 7559821 TI - The operative repair of chronic nontraumatic extensor tendon subluxations in the hand. AB - The authors study the literature and the surgical treatment in chronic nonrheumatoid cases. Their technique employs one-half of the extensor digitorum communis tendon as a tether on the central extensor tendon, recentralizing it over the metacarpophalangeal joint. PMID- 7559827 TI - Ficus. PMID- 7559826 TI - Immediate active short arc motion following extensor tendon repair. AB - Postoperative management of the extensor tendon injury has been altered over the past 15 years from treatment with 4 to 6 weeks of immobilization to controlled passive motion and, now, to active SAM with minimal tension and wrist tenodesis programs. The concepts of immediate motion are supported biochemically in experimental studies, and biomechanically through excursion studies, mathematical analysis of tendon excursion and force application, and study of repair tensile strengths. The results cited in this article and those reported by others demonstrate that early motion for extensor tendons in zones III, IV, V, VI, VII, T IV, and T V is safe and effective if force application is precise. Early referral to therapy, meticulous care in the control of edema, precise positions of postoperative splinting, and controlled motion programs will greatly improve the results of both simple and complex extensor tendon injuries, both in terms of function achieved and in terms of time and expense. PMID- 7559828 TI - Deep T-wave inversion in an elderly man. PMID- 7559829 TI - Barbiturates in alcohol withdrawal. PMID- 7559830 TI - The once and future hospital. PMID- 7559831 TI - Multiple chemical sensitivity syndrome. PMID- 7559832 TI - Hematemesis in a woman with skin, eye, and heart abnormalities. PMID- 7559833 TI - Headache, nausea, and vomiting in a heavy drinker and smoker. PMID- 7559834 TI - Case in point. Aortic root abscess caused by Staphylococcus aureus. PMID- 7559835 TI - Renal sodium retention in liver disease. AB - In many patients with cirrhosis, salt retention eventually culminates in ascites and edema. The pathogenesis probably involves several hormonal and neural effectors acting in concert. When conservative therapy with diet and diuretics fails to mobilize fluid, options for more intensive treatment include large volume paracentesis and the transjugular intrahepatic portosystemic shunt. PMID- 7559836 TI - Intervening in GERD: the phases of management. AB - The discomforts and histologic changes of gastroesophageal reflux disease call for a stepped approach consisting of four phases: lifestyle modification, initiation of pharmacologic therapy, intensification of drug therapy, and in some cases antireflux surgery. Decisions are guided by symptom patterns, which serve to suggest the predominant pathophysiologic mechanisms. PMID- 7559837 TI - Oral tolerance: mobilizing the gut. AB - Immunologically active lymphoid tissue in gut mucosa is preferentially programmed to inhibit autoimmune reactions, a property that can be exploited by oral administration of self-antigen. Identifying the target autoantigens to use in oral preparations may not be necessary. The trigger for oral tolerization is antigen-specific, but suppressive events in the target organ are nonspecific. PMID- 7559838 TI - Pelvic inflammatory disease: an ongoing epidemic. AB - In the absence of a practical screening test, diagnosis is based on physical and laboratory findings, a strategy compromised by low sensitivity and specificity- and by asymptomatic cases. Antibiotic combinations must be directed against a wide range of pathogens, including Neisseria gonorrhoeae and Chlamydia trachomatis. PMID- 7559839 TI - Sara Josephine Baker: crusader for women and children's health. PMID- 7559840 TI - New directions in treating insulin resistance. PMID- 7559843 TI - Security of attachment in 8-12-year-olds: a revised version of the Separation Anxiety Test, its psychometric properties and clinical interpretation. AB - This study examined the reliability and validity of an adapted version of the Separation Anxiety Test (SAT) for 8-12-year-old children. Clinical (N = 21) and nonclinical (N = 21) participants were compared on responses to the SAT coded using the Seattle scoring indices. Inter-rater reliability was established at an acceptable level, however, test-retest reliability and internal test construction were less satisfactory. Sample comparisons found the clinical participants described themselves as less securely attached and more avoidant of emotional expression than the control participants. The indices of attachment security were also related to various socioemotional aspects of functioning. In addition, the results indicated that describing "self" as different from a similar "other" child was significantly related to parental reports of poor social competence. Results are discussed in relation to an Attachment Theory framework as well as from a clinical perspective. PMID- 7559841 TI - Annotation: aspects of pain in children and adolescents. PMID- 7559842 TI - Child and adolescent sex abuse perpetrators: a review of the research literature. AB - This paper reviews the existing literature on selected issues in relation to child and adolescent sex abuse perpetrators. The relatively recent recognition of this phenomenon and the multiple problems of definition in this field may have affected the figures for incidence and prevalence. Among known cases a high proportion of young sex abuse perpetrators have suffered earlier adversities such as sexual and physical victimisation, dysfunctional family relationships and peer isolation. Despite a slender research data base on which to draw, treatment programmes for this client group have proliferated and many of these have not been adequately evaluated. There is a pressing need for the adoption of agreed definitions of outcome targets and measures and establishing longterm outcome studies with this group of children and adolescents. PMID- 7559847 TI - The stability and prediction of fears in Chinese children and adolescents: a one year follow-up. AB - The stability in number, level, content, and pattern of fears over a one-year period was examined in Chinese children and adolescents aged 7-17. In addition, perceptions of fear in friends and parents as well as perceptions of the controllability and modifiability of fears were explored as predictors of subsequent fear level. Evidence for moderate stability of fears was obtained. However, stability was qualified by age and sex interaction effects. A developmental-cultural hypothesis was put forth to account for those findings. Hierarchical regression analyses revealed that perceptions of fears in friends and parents, along with perceptions of the controllability and modifiability of fears, were predictive of fear level at follow-up. Limitations of the study are noted. PMID- 7559844 TI - A twin study of competence and problem behavior in childhood and early adolescence. AB - The Child Behavior Checklist (CBCL) was completed by parents of 181 pairs of same sex twins ages 7-15 (mean age = 11.0 years). Correlations between scores on the CBCL scales for 99 pairs of monozygotic twins and 82 pairs of dizygotic twins indicated significant genetic influences that varied according to the specific area of competence and problem behavior. Model-fitting estimates derived from multiple regression analyses indicated significant genetic influence on competence in school and on all areas of problem behavior. In addition, significant shared environmental influence was detected for amount and quality of participation in activities, quality of social relationships, performance in school, anxiety/depression, and delinquent behaviour. Implications for future work on the mechanisms underlying these effects are discussed. PMID- 7559848 TI - Nine-month changes of maternal expressed emotion in conduct and emotional disorders of childhood: a follow-up study. AB - In this follow-up study, mothers of 28 children with conduct disorders (CD) and 29 children with emotional disorders (ED) of 6-11 years of age, were interviewed and rated for expressed emotion 9 months after their first clinical assessment. Mothers of children with CD expressed significantly less criticism and higher warmth after 9 months. Initial externalizing scores in the CD group and initial internalizing scores in the ED group at the first assessment predicted symptomatic change, in contrast with EE scales. Initial externalizing scores and low levels of maternal warmth predicted presence of conduct disorder at the last contact with the clinician. The implications of the findings on the development of treatment techniques are discussed. PMID- 7559846 TI - Schizoid personality in girls: a follow-up study--what are the links with Asperger's syndrome? AB - Child psychiatric records of 33 girls given a diagnosis of "schizoid" personality in childhood, were compared with records of a control group of other referred girls and with those of 32 pairs of "schizoid" and control boys. Seventeen "schizoid" girls were seen again in adult life and compared with 32 "schizoid" boys previously followed up at the same age. The features of "schizoid" girls in childhood and adult life were very similar to those of the boys. A striking finding, possible due to referral bias, was the high rate of antisocial conduct in "schizoid" girls, both in childhood and later life. The dilemmas of diagnostic classification of this group of patients are discussed. PMID- 7559845 TI - Inter-rater reliability of global assessment of functioning in a clinical setting. AB - Four studies on the inter-rater reliability of a proposed Axis V version for DSM IV and of the CGAS involving 162 child and adolescent patients and 20 clinicians showed moderate agreement (intraclass correlation: 0.53-0.66). This was comparable to previous versions of Axis V, but lower than that reported for the CGAS. More detailed description of anchor points did not increase reliability nor there were differences in agreement when rating current or previous functioning. PMID- 7559851 TI - ADHD and thyroid abnormalities: a research note. AB - Contradictory findings have been reported on associations between ADHD and thyroid abnormalities including the syndrome of generalized resistance to thyroid hormone. We systematically reviewed thyroid function in a large group of children and adolescents with ADHD (N = 132). We failed to find evidence of generalized resistance to thyroid hormone. Although mild laboratory abnormalities in thyroid function were observed in a minority of ADHD subjects, they were not different than rates reported in the literature for normal children. PMID- 7559849 TI - Family structure, family functioning and adolescent well-being: the transcendent influence of parental style. AB - This study assessed the association between parental style, family functioning and adolescent well being, contrasting intact families with those of changed configuration. Eight hundred and one grade 10 general level teenagers in 11 high schools of a single educational system were the subjects. Results indicated that the configuration of the family was not the key determinant of effectiveness of family functioning. Instead the style of parenting turned out to be the main determinant of both family functioning and well being of the adolescents. While both "parents" were judged to have contributed to these outcomes cross gender effects were found. PMID- 7559850 TI - Clinical correlates of enuresis in ADHD and non-ADHD children. AB - Enuresis and attention deficit hyperactivity disorder (ADHD) are common childhood disorders that often co-occur. Although each has been linked to neurodevelopmental immaturity and increased risk for psychopathology, the clinical correlates of enuresis remain unclear. Subjects were 140 6-17-year-old boys with DSM-III-R ADHD and 120 non-ADHD controls. Information on enuresis and psychiatric diagnoses was obtained in a standardized manner blind to the child's clinical status. Our results show that (1) enuresis did not increase the risk for psychopathology in children with or without ADHD; (2) enuresis was not associated with psychosocial adversity or developmental immaturity; (3) enuresis was associated with increased risk for learning disability, impaired intellectual functioning, and impaired school achievement in normal control children but not in children with ADHD; and (4) the same pattern of findings was obtained after stratifying children with enuresis by primary versus secondary and by nocturnal versus diurnal subtypes. These results suggest that the clinical implications of enuresis may differ for ADHD and non-ADHD children. PMID- 7559852 TI - Teacher ratings of children's behaviors and teachers' management styles: a research note. AB - The purpose of this study was to explore how changes in teacher ratings of children's behavior problems from kindergarten to first grade might vary with teachers' styles of managing behavior problems. Familial and socioeconomical variables were controlled because of their potential modulating effects. Subjects were from a community sample of 1,573 boys and girls rated by teachers, mothers and peers (for one part of the sample) on aggressiveness-hyperactivity, anxiety withdrawal and social skills deficits. Teacher ratings of aggressive-hyperactive behaviors significantly changed from kindergarten to grade one when the teachers had different management styles. Mother and peer ratings, however, did not change. The importance of considering teachers' management styles when assessing behavior problems in school is stressed. PMID- 7559855 TI - A rapid antiviral in situ enzyme-linked immunosorbent assay for feline immunodeficiency virus. AB - An in situ enzyme-linked immunosorbent assay (ELISA) was developed as a rapid alternative to the focal infectivity assay (FIA) for screening potential anti retroviral molecules. The assay utilizes 96-well microtiter plates to allow for determination of antiviral effect and cytotoxicity of multiple compounds simultaneously. In contrast to the FIA which requires visual scoring of foci under low-power microscopy, the 96-well ELISA is read spectrophotometrically based on the soluble alkaline phosphatase substrate, p-nitrophenyl phosphate. The IC50 and CC50 values for several antiretroviral compounds were determined using the ELISA and results were confirmed by FIA. In all cases, compounds assayed by the newly described ELISA exhibited IC50 values in agreement with literature values derived from either the FIA or reverse transcriptase assays. PMID- 7559853 TI - Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus. AB - A recombinant glutathione-S-transferase fusion protein expressing amino acids 55 98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rGL 55-98 as a diagnostic antigen. PMID- 7559854 TI - Detection of beet necrotic yellow vein virus using reverse transcription and polymerase chain reaction. AB - A diagnostic test based on reverse transcription followed by the polymerase chain reaction (RT-PCR) was developed for the detection of beet necrotic yellow vein virus (BNYVV). A specific 500-base-pair fragment was amplified from the read through region of the coat protein gene located on RNA-2. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having 94.5% homology with published sequence data for BNYVV. The assay gave a sensitivity of 800 times that of a TAS-ELISA and 50 times that of an amplified TAS-ELISA method. A range of BNYVV isolates from the UK and worldwide could be detected by this test, either as mechanically inoculated Chenopodium quinoa leaves or infected sugar beet roots. Use of the assay in routine diagnostic tests allowed a reduction of time needed for the detection of Rhizomania in soils from 7 to 4 weeks. PMID- 7559856 TI - A mouse model for testing the pathogenicity of equine herpes virus-1 strains. AB - A mouse model was developed for testing the pathogenicity of equine herpes virus 1 (EHV-1) strains. The model was validated with EHV-1 strains that are known to be of a low or high pathogenicity in horses. From all parameters tested, the safety index, which was calculated from the body weights of the mice after infection, proved to be the best predictive parameter. When this parameter was used, good and reliable correlations were found with the pathogenicity of the EHV 1 strains in horses. This method enabled the differentiation between the two experimental EHV-1 strains whose genetic backgrounds were supposedly equal. PMID- 7559858 TI - Synthesis of PCR-derived, digoxigenin-labeled DNA probes for in situ detection of Epstein-Barr early RNAs in Epstein-Barr virus-infected cells. AB - A PCR-derived digoxigenin-labeled DNA probe was used for for Epstein-Barr early RNA (EBER) in situ hybridization in formalin-fixed paraffin-embedded tissues. The results showed that the hybridization signal was morphologically distinct and the intensity of signal was comparable with those by RNA riboprobe. The advantages of using PCR-derived DNA probes for EBER in situ hybridization include: (1) the synthesis of digoxigenin-labeled DNA probes is easy and simple by PCR; (2) the labeled amplification product can be used as a probe without further purification; (3) DNA probes are potentially more stable than RNA probes; and (4) the preparation of DNA probes is relatively efficient and rapid. It is concluded that this technique is an ideal candidate for detection of EBER expression in clinical specimens. PMID- 7559857 TI - Methods to remove inhibitors in sewage and other fecal wastes for enterovirus detection by the polymerase chain reaction. AB - Typical environmental sample concentration procedures developed to purify virions are not always compatible with reverse transcription-polymerase chain reaction (RT-PCR). The processing steps of polyethylene glycol (PEG) precipitation and ultrafiltration were found to enrich not only virions but also certain RT-PCR inhibitors. Inhibitors were eliminated by a single-step guanidinium isothiocyanate (GIT) extraction to purify and precipitate viral genomic RNAs immediately prior to RT-PCR. The detection sensitivity of GIT extraction--RT-PCR was found to be 0.6-0.003 50% tissue culture infectious doses (TCID50) for 9 enteroviruses in infected cell extracts. When sewage, concentrated up to 385,000 fold and seeded with 1-3 plaque-forming units of poliovirus, was extracted with GIT solution, viruses were detectable in samples originally judged negative by direct RT-PCR without GIT extraction. Eleven waste samples (3 sewage, 5 latrine solids, 2 gauze pads extracts and 1 stool) processed by a series of steps that included PEG precipitation, solvent extractions, and Sephadex G-200 chromatography were examined for enteroviruses by RT-PCR, both with and without GIT extraction. GIT extraction eliminated sample inhibitory substances and increased the proportion of enterovirus positive samples from 3 to 7 of 11. GIT extraction in conjunction with virion concentration improves RT-PCR detection of viruses in sewage and other fecal wastes. PMID- 7559859 TI - Application of the NASBA nucleic acid amplification method for the detection of human papillomavirus type 16 E6-E7 transcripts. AB - Using a human papillomavirus type 16 (HPV-16) E6-E7 specific primer set in a nucleic acid sequence-based amplification (NASBA) reaction, detection of HPV-16 transcripts was accomplished in a single enzymatic reaction at 41 degrees C. The NASBA reaction product was visualized either by Northern bolt analysis with an HPV-16 E6-E7-specific 32P-labelled oligonucleotide probe or by a non-radioactive enzyme-linked gel assay (ELGA). In combination with a rapid nucleic acid extraction procedure this method appears to be very suitable for the sensitive and specific detection of HPV-16 transcripts on small amounts of HPV-16 expressing cells of various sources, including cervical smears. PMID- 7559860 TI - Strong bones don't break! PMID- 7559861 TI - Estrogens do not affect whole-body protein metabolism in the prepubertal female. AB - The increase in bone mass, muscle bulk, and linear growth that occur during puberty are mediated, at least in part, through the action of sex steroids. By using infusions of nonradioactive tracers of leucine, we have recently shown a significant protein anabolic effect of testosterone in prepubertal boys. The present study was designed to determine whether estrogens can cause similar changes in protein metabolism in females. Seven prepubertal girls (Turner's syndrome, n = 6; hypogonadotropism, n = 1; mean age 12.2 +/- 0.3 yr) were studied. A 4-h infusion of L(-)[1-(13C)]leucine was given, and the isotopic enrichment of [13C]ketoisocaproic acid and 13CO2 were measured in plasma and in breath with use of gas chromatography/mass spectroscopy and an isotope ratio mass spectrometer, respectively. The reciprocal pool model was used for analysis of leucine kinetics. Subjects were then started on ethinyl estradiol orally (n = 5) or depot estradiol parenterally (n = 2). An identical study was repeated 4 weeks later. There were comparable changes in leucine kinetics in all girls studied, hence their data were grouped for analysis. After administration of ethinyl estradiol, there were insignificant changes in the rate of appearance of leucine, an index of proteolysis (+6% +/- 8%); leucine oxidation (-9% +/- 11%); and nonoxidative leucine disposal, an index of whole body protein synthesis (+10% +/- 8%). This is in sharp contrast to the changes found in boys studied similarly after treatment with testosterone (rate of appearance: 17 +/- 6%, P = .036; leucine oxidation -49 +/- 5%, P = 0.004; and nonoxidative leucine disposal +35 +/ 8%, P = 0.009). The lack of anabolic effect on whole-body protein in the girls reported here was observed despite significant increases in plasma insulin-like growth factor I concentrations during 4 h of sampling when comparing the 2 study days, similar to that of the prepubertal boys who were treated with testosterone. In summary, we observed that contrary to androgens, estrogens do not alter estimates of whole-body protein turnover and anabolism in prepubertal humans, despite significant increases in circulating insulin-like growth factor I concentrations. In conclusion, the impact of testosterone on protein metabolism seems to be a direct effect of androgens, independent of aromatization. These findings correlate with the significant differences in muscle bulk between the sexes as children go through puberty. PMID- 7559862 TI - Modeling Graves' disease in the mouse--work still in progress. PMID- 7559863 TI - Graves' disease in severe combined immunodeficient mice. AB - Graves' disease (GD) is an autoimmune thyroid disorder involving an antibody (TSAb) directed against the TSH receptor (TSHR) producing thyroid stimulation. We have developed an animal model of GD by engrafting peripheral blood mononuclear cells or T cell lines plus autologous thyroid tissue into severe combined immunodeficient (SCID) mice. We xenografted Graves' thyroid tissue from six patients into six groups of SCID mice. Autologous PBMC and T cell lines reactive to recombinant human TSHR extracellular domain and non-TSHR lines were injected ip into the designated groups. In some of the studies, thyroid tissue was irradiated with 2000 rads before xenografting. Irradiation of xenografts induced thyroid tissue damage and release of thyroid antigens and hormones. Mice reconstituted with peripheral blood mononuclear cells or nonspecific T cell lines did not simulate GD. However, we achieved production of TSAb, elevation of serum T3, and TSAb-dependent survival and function of human Graves' thyroid tissue in SCID mice reconstituted with TSHR-specific T cell lines. We reconstituted SCID mice with PBMC and TSHR-specific T cell lines that recognized TSHR peptide 158 176. This may be in vivo evidence of the importance of peptide 158-176 as an immunodominant epitope on the TSHR extracellular domain. PMID- 7559864 TI - Skeletal recovery after treatment of Cushing's: still room for improvement. PMID- 7559866 TI - Management of a functioning mediastinal parathyroid cyst. PMID- 7559867 TI - Adrenoleukodystrophy: a link between adrenal insufficiency and school performance. AB - The combination of neurodevelopmental regression and adrenal insufficiency should alert practitioners or emergency room physicians about ALD. Although still unproven, early medical intervention with either gene therapy or bone marrow transplantation may offer more promise to these patients. PMID- 7559865 TI - Bone mineral density and bone turnover before and after surgical cure of Cushing's syndrome. AB - We measured bone mineral density (BMD) using dual-energy x-ray absorptiometry in 20 patients with Cushing's syndrome (CS) (14 pre- and 2 postmenopausal women, 4 men) before and in 18 of them also at regular intervals after surgical cure (median duration of follow-up, 36 months). In addition, in the premenopausal women with CS, fasting blood samples and 2-h fasting urine samples for measurement of biochemical parameters of bone and collagen metabolism were collected before and in 9 of them also at regular intervals during the first 2 yr after surgery. Marked osteopenia was present in most patients with active CS (Z scores: lumbar spine -1.45 +/- 1.44 and femoral neck -1.50 +/- 1.02; mean +/- SD). No consistent change in BMD was observed at 3 and 6 months after surgery. Thereafter BMD increased considerably in almost all patients. For the 15 patients with a follow-up of at least 1 yr, Z-scores at the last evaluation were -0.65 +/- 1.27 for the lumbar spine and -0.98 +/- 1.02 for the femoral neck (both P < 0.002 compared with pretreatment values). In the premenopausal patients, the increase in BMD both in the lumbar spine and in the femoral neck at 24 months was inversely correlated with age (r = -0.733, P < 0.03, and r = -0.667, P < 0.05, respectively). Serum levels of osteocalcin, bone alkaline phosphatase, carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen, and the cross-linked telopeptide of type I collagen were not significantly different between the group of 14 premenopausal patients with active CS and a control group of 18 age-matched healthy premenopausal women. However, the urinary hydroxyproline/creatinine ratio was significantly higher in patients with CS (24.6 +/- 9.6 vs. 16.2 +/- 3.5 mumol/mmol, P < 0.01). In all 9 premenopausal patients, serum levels of osteocalcin increased considerably between 0 and 3 months (from 1.04 +/- 0.20 to 3.82 +/- 0.30 nmol/L) (mean +/- SEM, P < 0.0001), indicating a prompt increase of osteoblast activity. Also serum levels of carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen, and cross-linked telopeptide of type I collagen, and the urinary hydroxyproline/creatinine ratio increased significantly between 0 and 3 months. Thereafter these levels decreased gradually. We conclude that marked osteopenia in the lumbar spine and femoral neck is present in most patients with active Cushing's syndrome.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559868 TI - A therapeutic controversy. Thyroid hormone treatment: when and what? PMID- 7559869 TI - Plasma proinsulin-like component in insulinoma: a 25-year experience. PMID- 7559872 TI - Integrin alpha 6 is a differentiation antigen of human granulosa cells. AB - We previously reported a new murine monoclonal antibody (mAb), OG-1, raised against human granulosa cells (GC) and showed that the OG-1 antigen is a cell surface differentiation-related molecule expressed on GC of follicles and on large luteal cells in the early to midluteal phase. In this study, we further characterized the OG-1 antigen. An immunohistochemical study showed that the OG-1 antigen was also expressed on endometrial glandular and stromal cells and on first trimester and term placental trophoblasts. The antigen purified from endometrium, chorionic villi, or placenta by immunoaffinity chromatography using the mAb OG-1 consisted of a 120-kilodalton (kDa) protein. Partial amino acid sequencing of the 120-kDa protein purified from placenta revealed that the 16 amino acids from the N-terminus were identical to those of the heavy chain of human integrin alpha 6, the molecular mass of which is reportedly 118-126 kDa. We examined the tissue distribution of integrins alpha 6, beta 1, and beta 4, because integrin alpha 6 forms heterodimers with integrins beta 1 or beta 4. Indirect immunofluorescence staining of various follicles and corpora lutea using two known antiintegrin alpha 6 monoclonal antibodies (450-30A1 and GoH3) showed that integrin alpha 6 was expressed on granulosa and large luteal cells in a profile similar to that of the OG-1 antigen. Integrin beta 1 was expressed on granulosa and large luteal cells, but integrin beta 4 was undetectable. Integrin beta 1 was also expressed on thecal and stromal cells. In term placentas, the OG 1 antigen and integrins alpha 6 and beta 4 were expressed on trophoblasts, whereas integrin beta 1 was expressed on villous capillary endothelia. These results indicate that the mAb OG-1 recognizes integrin alpha 6, and that integrin alpha 6 is a differentiation-related antigen of human GC, suggesting the involvement of integrins in human folliculogenesis and luteal formation. PMID- 7559871 TI - Preserved counterregulatory hormone release and symptoms after short term hypoglycemic episodes in normal men. AB - To test the hypothesis that subsequent neuroendocrine and symptomatic responses are sustained after short term hypoglycemic episodes of less than 1-h duration, we studied hypoglycemia on 4 consecutive days and after an 8-day pause in 10 nondiabetic men. Highly reproducible decreases in plasma glucose (< 2.8 mmol/L) occurred on study days 1, 2, 3, 4, and 12 after iv insulin boluses (0.04 U/kg). Levels of the counterregulatory hormones rose during the hypoglycemic episodes in all instances, but maximal concentrations on study day 4 were not attenuated: glucagon (peaks on day 1 vs. day 4), 150 +/- 10 vs. 180 +/- 20 ng/L; cortisol, 400 +/- 30 vs. 420 +/- 40 nmol/L; ACTH, 12 +/- 2 vs. 13 +/- 2 pmol/L; GH, 11.1 +/ 1.8 vs. 12.5 +/- 2.2 micrograms/L; norepinephrine, 1.68 +/- 0.17 vs. 1.65 +/- 0.13 nmol/L; and epinephrine, 1370 +/- 440 vs. 1520 +/- 480 pmol. On each study day, symptoms of hypoglycemia were produced after induction of hypoglycemia, and there was no decrease in the degree of symptomatology on subsequent days. The multivariate analysis of variance showed no day to day differences in plasma glucose, counterregulatory hormones, or hypoglycemic symptoms. We conclude, firstly, that after short term hypoglycemic episodes, the neuroendocrine and symptomatic responses remain completely intact in normal individuals and, secondly, that short term periods of hypoglycemia are fundamentally different from prolonged periods, as described previously. PMID- 7559870 TI - Corticotropin-releasing hormone-induced vasodilatation in the human fetal placental circulation: involvement of the nitric oxide-cyclic guanosine 3',5' monophosphate-mediated pathway. AB - This study has used an in vitro perfusion method to investigate the mechanism by which CRH causes vasodilatation in the human fetal-placental circulation. In normal term placentas, vasodilatory responses to human CRH (24-7000 pmol/L) were examined during submaximal vasoconstriction (100-120 mm Hg) of the fetal placental vasculature induced by prostaglandin F2 alpha (0.7-2 mumol/L), KCl (50 100 mmol/L), or the thromboxane A2 mimetic, U46619 (0.05-0.5 mumol/L). Infusion of CRH caused a concentration-dependent vasodilatation that was similar in the presence of each constrictor agent (P > 0.05). The CRH antagonist, alpha-helical CRH-(9-41) (200 pmol/L), and a polyclonal CRH antiserum significantly inhibited CRH-induced vasodilatation during constriction with prostaglandin F2 alpha (P < 0.05). Vasodilatory responses to CRH were attenuated by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (100 mumol/L; P < 0.05), and the guanylate cyclase inhibitor, LY 83583 (1 mumol/L; P < 0.05), but not by the cyclooxygenase inhibitor, indomethacin (3 mumol/L; P > 0.05). In placentas of women with increased fetal vascular resistance, as demonstrated by Doppler ultrasound waveforms in vivo, CRH-induced vasodilatation was significantly reduced (P < 0.05). These results indicate that in the human fetal-placental circulation, CRH causes a vasodilatory response via a nitric oxide-/cGMP-dependent pathway. CRH may play a role in the control of vascular resistance to blood flow in the normal human placenta, and there may be a deficiency in the CRH signaling pathway of placentas with increased fetal vascular resistance. PMID- 7559873 TI - Hyponatremia and osmoregulation of vasopressin secretion in patients with intracranial bleeding. AB - To clarify the cause and pathophysiology of hyponatremia after intracranial bleeding, we analyzed the possible causative factors, and examined the response of vasopressin (AVP) secretion to osmotic stimulus in six patients. Despite hyponatremia, urinary sodium excretion persisted, with urinary osmolality exceeding plasma osmolality. Serum levels of urea nitrogen, creatinine, and uric acid were not elevated in any of them. PRA was normal or subnormal in four patients, and all had normal adrenocortical and thyroid functions. Although these laboratory findings may support the diagnosis of the syndrome of inappropriate antidiuretic hormone secretion, the cause of hyponatremia in our patients was attributed to excessive renal excretion of sodium, because water load performed in an euvolemic state showed no impairment in diuresis, and replenishment of sodium without water restriction improved hyponatremia as well as clinical conditions. Plasma AVP levels relative to plasma osmolality in these patients were constantly elevated. When challenged by an osmotic stimulus, AVP secretion increased with increasing plasma osmolality in one patient, but no consistent pattern of AVP secretion was observed in others. The potentiating effect of hypovolemia on osmotic secretion of AVP was not demonstrated in any of the patients. These results show that hyponatremia after intracranial bleeding with clinical features almost indistinguishable from those of syndrome of inappropriate antidiuretic hormone secretion may result from an impaired renal sodium-conserving mechanism of unknown cause. Persistent AVP secretion without an alteration in the sensitivity of the osmostat in this pathological state may be due to an incomplete suppression by plasma hypotonicity per se of the baroreceptor-mediated stimulation of AVP release. PMID- 7559874 TI - Soluble interleukin-6 (IL-6) receptor in the sera of pregnant women forms a complex with IL-6 and augments human chorionic gonadotropin production by normal human trophoblasts through binding to the IL-6 signal transducer. AB - To study the role of soluble interleukin-6 receptor (sIL-6R) during pregnancy, sIL-6R levels in the sera of pregnant women in the first, second, and third trimesters were determined and found to remain unchanged during pregnancy, but were significantly higher than those in nonpregnant women in the follicular, ovulatory, and luteal phases of the menstrual cycle (P < 0.001). IL-6 levels, however, in the sera of pregnant women at all trimesters showed no difference from those in nonpregnant women at any stage of the menstrual cycle. Recombinant sIL-6R (rsIL-6R) augmented hCG production by rIL-6-stimulated trophoblasts dose dependently, but failed to enhance hCG production by unstimulated trophoblasts. rIL-6- and rsIL-6R-induced hCG production was significantly blocked by anti-IL-6R antibody, PM1; antisignal transducing glycoprotein 130 (gp130) antibody, GPX7; or a tyrosine kinase inhibitor, genistein. Thus, sIL-6R in serum from pregnant women forms a complex with placental and decidual IL-6 in a manner similar to trophoblast membrane-bound IL-6R. These two discrete types of IL-6R and IL-6 complex might act cooperatively by binding to gp130 and subsequently evoking tyrosine kinase activity in the trophoblasts to produce hCG in vivo. PMID- 7559875 TI - Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. AB - Genetic and environmental factors can modulate the level of sensitivity to various hormones, including estrogens. Enhanced sensitivity to estradiol (E2) has been demonstrated in several biological conditions, such as in sheep during the nonbreeding season, in untreated patients with Turner's syndrome, and in the prepubertal state in normal girls. We postulated that secondary responses to hormonal therapy in patients with breast cancer could also result from enhanced E2 sensitivity, developing as an adaptive mechanism to E2 deprivation. The present study used the MCF-7 human breast cancer cell line as a model system to test the concept that enhanced sensitivity to E2 may occur as a result of adaptation to low E2 levels. After depriving MCF-7 cells of estrogens in tissue culture medium for periods of 1-6 months, we established conditions under which replication could be stimulated maximally by 10(-14)-10(-15) mol/L E2. In contrast, wild-type cells not exposed to estrogen deprivation required 10(-10) mol/L E2 to grow at the same rate. Further, the concentration of the antiestrogen, ICI 164384, needed to inhibit growth by 50% in estrogen-deprived cells was much lower than that required in wild-type cells (i.e. 10(-15) vs. 10( 9) mol/L). Nude mice implanted with these estrogen-deprived cells demonstrated an earlier appearance of palpable tumors in response to E2 than animals bearing wild type cells. Reexposure to 10(-10)-10(-9) mol/L E2, either in vivo or in vitro, returned these cells to the level of estrogen sensitivity observed in wild-type cells. Taken together, these observations suggest that breast cancer cells can adapt to low levels of estrogens by enhancing their sensitivity to E2. PMID- 7559876 TI - Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. AB - Precursor forms of the alpha-subunit of inhibin are abundant in human follicular fluid and possibly plasma, although their function is uncertain. We now describe the development of a new enzyme-linked immunosorbent assay to measure inhibin forms containing both the pro and alpha C regions of the alpha-subunit. The assay has a detection limit for purified human pro-alpha C of 0.5 pg/mL and less than 0.02% cross-reaction with recombinant forms of inhibin, activin, and follistatin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of follicular fluid extracts demonstrated that the assay is likely to detect pro containing precursor forms of both the free alpha-subunit and intact dimeric inhibin. The serum concentration was measured in normal men (446 +/- 28 pg/mL), postmenopausal women (45.8 +/- 3.8 pg/mL), and women treated with FSH before in vitro fertilization (1827 pg/mL). Pooled human follicular fluid contained 488 ng/mL. The mean serum concentration in the female menstrual cycle rose from 150.6 +/- 26.1 pg/mL in the early follicular phase to 692.2 +/- 113 pg/mL in the midluteal phase. This assay offers a useful tool for investigation of the role of inhibin-related proteins in human reproduction. There may be particular clinical value under circumstances in which other assays for inhibin forms have insufficient sensitivity. PMID- 7559878 TI - Insulin-like growth factors (IGFs) and IGF-binding proteins in human skin interstitial fluid. AB - Despite extensive investigation of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in the circulation and body fluids, there is no information on this in interstitial fluid. We have compared the IGF/IGFBP system in the circulation with that in fluid obtained from blisters artificially raised by negative pressure in 10 healthy volunteers. IGFBP-1, -2, -3, and -4 were all found in blister fluid, but in concentrations much lower than those in matched serum. The IGF-I, IGF-II, and IGFBP-3 levels measured by RIA were 18%, 14%, and 16% of those in serum, respectively. Fast protein liquid chromoatography showed that both IGF-I and IGFBP-3 in 150- and 50-kilodalton complexes were approximately 13% and 37%, respectively, of the corresponding peaks found in matched serum. Compared to that in serum, the IGFBP-3 in the blister fluid was predominantly in a modified 29-kilodalton form, and there was increased activity of an IGFBP-3 protease. Therefore, although IGF concentrations are much lower in interstitial fluid than in the circulation, a greater proportion of this IGF is in forms more readily available for interaction with tissue receptors. The blister fluid appears to represent physiological interstitial fluid and may provide a model for studying the physiology and pathophysiology of growth factors in the interstitial environment. PMID- 7559879 TI - Morbidity and mortality in follicular thyroid cancer. AB - The natural history and results of treatment have been analyzed in a group of 49 patients with follicular thyroid carcinoma who were followed for an average of 10.7 yr. Striking differences between the course of follicular thyroid carcinoma and the course of papillary carcinoma are evident. Deaths from cancer were double (16% for follicular), age at diagnosis was older, and age at death was younger. All deaths and recurrences happened within 13 yr, in contrast to the continued experience of deaths and recurrences in papillary cancer, even through 40 yr of observation. Adverse outcome correlated with extent of disease at diagnosis and with size of primary tumor, but did not correlate with vessel invasion, extent of capsule invasion, degree of dedifferentiation, extent of primary surgery, or radioactive iodide ablation. These observations are again in striking contrast to experience with papillary cancer. No patient with intrathyroidal disease who was under age 45 at diagnosis and with a primary tumor of less than 2.5 cm died. Our observations suggest that follicular cancer, even if apparently intrathyroidal, carries a high mortality rate in patients over age 45 or in those with tumors larger than 2.5 cm at the time of diagnosis and suggest that we must consider additional therapeutic measures in this group of patients, including larger radioiodine doses for initial therapy, external radiotherapy, and even possibly prophylactic chemotherapy. PMID- 7559880 TI - Suppression of hypothalmic-pituitary-adrenal axis responses to stress in lactating women. AB - In the rat, lactation suppresses a variety of physiological responses to stress. We investigated whether stress-responsive neurohormonal systems are also restrained during breast feeding in humans. We chose treadmill exercise as a stressor because this stimulus produces an exercise intensity-dependent activation of the hypothalamic-pituitary-adrenal axis and the sympathomedullary system that is independent of differences in physical conditioning among subjects. Ten lactating and ten nonlactating women who were between 7 and 18 weeks postpartum performed 20 min of graded treadmill exercise. The final 5 min of exercise was set to elicit 90% of the maximal oxygen uptake of each subject. Plasma ACTH, cortisol, and glucose responses to exercise were significantly attenuated in lactating women (P < 0.001, P < 0.05, and P < 0.001, respectively). Basal norepinephrine levels were also reduced in lactating women (P < 0.05). These results indicate that stress-responsive neurohormonal systems are restrained in lactating women. PMID- 7559877 TI - Identification of alternatively spliced messenger ribonucleic acid encoding truncated growth hormone-releasing hormone receptor in human pituitary adenomas. AB - The expression of GHRH receptor (GHRH-R) messenger ribonucleic acid (mRNA) was studied in 22 pituitary adenomas and 2 normal anterior pituitaries. Northern blot analysis revealed that GHRH-R mRNA were expressed in all 14 GH-producing adenomas, 1 of 3 ACTH-producing adenomas, the 1 PRL-producing adenoma, 2 of 4 nonfunctioning adenomas, and the 2 normal anterior pituitaries. Their expression levels varied among GH-producing adenomas and were relatively low in GH nonproducing adenomas. In addition to the major transcript with a molecular mass of 2.0 kilobases (kb), the transcripts were identified at 2.8 and 4.5 kb in some GH-producing adenomas. To examine the structural variations in GHRH-R mRNA in pituitary adenomas, we amplified the complementary DNA fragment encompassing the region from the third cytoplasmic loop to the sixth transmembrane domain of GHRH R. This region was selected because this region of the G protein-coupled receptor has been known to interact with G protein. Two amplified fragments with the molecular masses of 250 and 810 base pairs were identified by the reverse transcriptase-polymerase chain reaction method. The nucleotide sequence of a smaller fragment, which was the expected size, revealed that no mutations were found in this region in 10 GH-producing adenomas examined. However, a larger fragment contained the currently unidentified insertion. Compared with the genomic DNA sequence, this insertion was found to be generated through alternative splicing. In addition, this variant form contained the premature stop codon in-frame, indicating that it encodes the truncated GHRH-R. This insertion specific probe could hybridize with 2.8- and 4.5-kb species of GHRH-R mRNA on Northern blot analysis, and these transcripts were expressed mainly in GH producing adenomas. Finally, study of cell transfection and cAMP measurement revealed that this truncated GHRH-R was unable to transmit GHRH signals. These results suggest that some GH-producing adenomas preferentially express the truncated GHRH-R as a nonfunctioning receptor through alternative splicing. PMID- 7559881 TI - Evidence for normal vitamin D receptor messenger ribonucleic acid and genotype in absorptive hypercalciuria. AB - Absorptive hypercalciuria (a stone-forming condition) is characterized by gut hyperabsorption of calcium, hypercalciuria, and reduced bone density. Inasmuch as these features implicate enhanced calcitriol action in gut and bone, we analyzed the vitamin D receptor (VDR) gene to ascertain whether an abnormality of this gene marks patients with intestinal hyperabsorption of calcium. We have compared the frequency of a restriction fragment length polymorphism (Bsm I) associated with different alleles of the VDR gene in a group of 33 well characterized absorptive hypercalciuric patients and a group of 36 normal race- and age-matched control subjects. There was no difference between the distribution of the VDR alleles in the patient population when compared with the normal population. The coding region of VDR messenger RNA was also normal, as determined by both DNA sequence analysis and chemical mismatch cleavage analysis of copy DNA from 11 index absorptive hypercalciuric patients. On the basis of these results, we propose that the enhanced intestinal calcium absorption invariably seen in absorptive hypercalciuria and attendant symptoms of this disorder are not attributable to mutations of the VDR and are not linked to a common VDR genotype. PMID- 7559882 TI - Metabolic features of polycystic ovary syndrome are found in adolescent girls with hyperandrogenism. AB - Recently, we reported that hyperandrogenism in adolescent girls is accompanied by augmented LH pulsatility and elevated LH/FSH ratio with increased ovarian volume. Together with higher concentrations of 17-hydroxyprogesterone, androstenedione, testosterone, and estrone that are ovarian in origin, these neuroendocrine features are identical to those seen in adult women with polycystic ovary syndrome. In the present study, we report the metabolic characteristics of these hyperandrogenic adolescent girls. The GH insulin-like growth factor I (IGF-I) binding protein (BP)-3 axis, insulin sensitivity, and insulin-IGFBP-1/insulin sex hormone binding globulin axes were evaluated in 13 adolescent girls (ages 11-18 yr) with mild to moderate signs of hyperandrogenism (HA) and 28 age-matched normal girls. Insulin sensitivity was assessed by a frequent-sample iv glucose tolerance test (ivGTT, 0.3 g/kg). Twenty-four hour blood samples were obtained at 10-min intervals and were used to determine GH pulsatility (20-min samples), IGFBP-3 levels (0800-0900 h), and fluctuations of insulin, IGFBP-1, and IGF-I (hourly samples) during feeding and fasting phases of the day. In addition, GH responses to GHRH stimulation (1 microgram/kg) were assessed. Fasting insulin concentrations, but not plasma glucose levels, were significantly elevated in the HA group compared with those in the normal group (256 +/- 35 vs. 103 +/- 24 pmol/L, P = 0.0008), as were insulin responses to ivGTT and meals (P < 0.01) and 24-h mean insulin concentrations (P < 0.01). Thus, hyperinsulinemia with normal fasting glucose levels in HA girls may reflect insulin resistance, as suggested by the increased ratio of insulin and glucose (P < 0.001). All measures of insulin were correlated with body mass index (BMI); however, insulin remained significantly higher in the HA group after correcting for BMI, suggesting that decreased insulin sensitivity was related to other factors in addition to BMI. Twenty-four hour IGFBP-1 concentrations showed a diurnal pattern with an inverse relationship to insulin, and 24-h mean concentrations were lower in the HA group (0.35 +/- 0.13 vs. 0.76 +/- 0.09 micrograms/L, P = 0.02). Reduced sex hormone binding globulin levels were also inversely related to insulin levels (P = 0.0007). In contrast, GH pulsatile characteristics and IGF-I/IGFBP-3 levels, as well as GH responses to GHRH, were similar between the groups.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559884 TI - Gonadotropin releasing hormone agonist (nafarelin) test to differentiate gonadotropin deficiency from constitutionally delayed puberty in teen-age boys--a clinical research center study. AB - The objective of this study was to determine whether the hormonal response to a GnRH agonist (nafarelin) challenge differentiates hypogonadotropinism from delayed puberty as well as the sleep test does. We studied boys ages 13.25-17.6 yr with prepubertal constitutional delay of puberty (CDP, n = 11), prepubertal gonadotropin deficiency (GnD, n = 10), pubertal CDP (PCDP, n = 11) and partial GnD (PGnD, n = 2). These disorders were defined on the basis of the following independent criteria: CDP = isolated delayed puberty with documentation of subsequent pubertal progression; GnD = panhypopituitarism or anosmia with absence of subsequent pubertal progression; PCDP = isolated delayed puberty in an early pubertal child; and PGnD = arrest of puberty in boys with partial hypopituitarism. CDP was compared with GnD and PCDP was compared with PGnD by analysis of variance and two-tailed t tests. Each patient had a nafarelin test with measurement of LH, FSH, and testosterone responses at intervals after nafarelin administration. Most patients had a sleep test with measurement of LH and testosterone levels at intervals overnight. CDP and GnD patients could not be distinguished by pubertal staging criteria. All but 1 patient with CDP had an LH response higher than that of GnD patients 4 h postnafarelin (P = 0.003). An incremental response to nafarelin of LH (delta LH at 4 h) below 4.8 IU/L was the best discriminant; it distinguished GnD from CDP in 95% of the cases and PGnD from PCDP completely. During the sleep test, all patients with CDP and 2 of 8 with GnD exhibited a significant increase in plasma LH. An incremental increase in LH during sleep (mean LH asleep minus mean LH awake) of less than 0.35 IU/L, near the limit of sensitivity of the method, differentiated GnD from CDP similarly to the nafarelin test. We conclude that the LH response to nafarelin distinguished gonadotropin deficiency from constitutional delay of puberty as well as the sleep test did and with certain advantages. The diagnostic reliability of the GnRH agonist test deserves to be determined prospectively in teen-agers with isolated GnD and partial hypopituitarism. PMID- 7559885 TI - Diagnostic studies with intravenous and intranasal growth hormone-releasing peptide-2 in children of short stature. AB - GH secretion is primarily regulated by the hypothalamic-releasing hormones GHRH and somatostatin. Additionally, several neurotransmitters act at the hypothalamus and pituitary to modulate GH release. The agents commonly used in clinical practice to diagnose GH deficiency, such as arginine, insulin and L-dopa, act through the neural GH network. Many children with a poor GH response to conventional agents have a significant serum GH response to iv GHRH. GH-releasing peptides (GHRPs) are synthetic peptides that like GHRH act directly on pituitary somatotrophs to stimulate GH release. GHRP-2, an investigational drug, is one of the most potent members of the GHRP family. It has been shown to be effective in adults via the oral and intranasal as well as the iv route of administration. In this study, GH responses to GHRP-2 were compared with GH responses to other provocative agents in children of short stature. GHRP-2 was administered iv or intranasally to children with short stature. In the same subjects, GHRP-2 was administered iv in combination with GHRH. Twenty-four children undergoing evaluation for GH deficiency received at least one conventional agent (arginine, L-dopa/exercise, insulin) in addition to iv GHRH and GHRP-2. The GH responses to GHRH or GHRP-2 were similar in each child, and both were equally reliable predictors of pituitary reserve. The conventional agents used in GH testing were less likely to predict the capacity of the pituitary to release GH than were either GHRH or GHRP-2. There was no correlation between maximal GH response to standard tests with GH responses to GHRH or GHRP-2. A subset of the group of 21 children who had a robust response to iv GHRP-2 were later administered GHRH+GHRP 2 simultaneously. The GH response to GHRH+GHRP-2 was synergistic in this group of 12 children, similar to previously reported observations in adults of normal stature. Fifteen of the 21 children who had a robust response to the iv GH releasing factors also received intranasal GHRP-2. All 15 of these children had a significant GH response to intranasal GHRP-2 over a dose range of 5-20 micrograms/kg per dose. The mean peak GH response to 15 micrograms/kg was 31.3 micrograms/L. The intranasal preparation was well tolerated.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559883 TI - Expression of somatostatin receptor subtypes in breast carcinoma, carcinoid tumor, and renal cell carcinoma. AB - The presence of transcripts for somatostatin receptor (SSTR) subtypes 1, 2, 3, and 4 was probed by reverse transcription and polymerase chain reaction in ribonucleic acid isolated from 46 malignant and 9 nonmalignant breast tissues, 15 carcinoid tumor tissues, and 13 renal cell carcinoma tissues. The transcripts for SSTR2 were amplified in all but 2 tissue samples, whereas transcripts for SSTR1, SSTR3, and SSTR4 were detected sporadically. In renal cell tumors, SSTR3 transcripts were completely absent. In breast cancer tissue, SSTR subtypes were transcribed independently of patient age, menstrual status, diagnosis, histological grade, and levels of estrogen receptor and progesterone receptor. The probability of finding transcripts for SSTR subtypes, P, was ranked differently for the three types of tumor tissues. For breast cancer, P2 > P3 = P1 > P4; for carcinoid tumors, P2 > P1 > P3 = P4; and for renal cell tumors, P2 > P1 > P4 > P3. PMID- 7559886 TI - Up-regulation of high-affinity dehydroepiandrosterone binding activity by dehydroepiandrosterone in activated human T lymphocytes. AB - Although evidence indicates that dehydroepiandrosterone (DHEA) exerts direct physiological effects, its mechanism of action remains unknown. DHEA binding sites were examined using a whole-cell binding assay in a human T lymphoid cell line, PEER, revealing that a single class of high-affinity binding sites for DHEA (dissociation constant = 7.4 +/- 0.53 nmol/L, mean +/- SE, n = 4) was greatly increased when treated with DHEA, phorbol-12-myristate-13-acetate, and the Ca2+ ionophore A23187. Bound [3H]DHEA was displaced sensitively by DHEA and secondarily by dihydrotestosterone, but not effectively by other steroids, including DHEA sulfate. These results not only indicate the existence of a DHEA receptor, but also suggest that T cells become susceptible to regulation by DHEA during the process of signal-induced activation. PMID- 7559888 TI - Alterations in cortisol metabolism in insulin-dependent diabetes mellitus: relationship with metabolic control and estimated blood volume and effect of angiotensin-converting enzyme inhibition. AB - 11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the interconversion of cortisol and its inactive metabolite, cortisone, and protects the mineralocorticoid receptor from activation by cortisol. Sodium and fluid retention is a well documented phenomenon in insulin-dependent diabetes mellitus (IDDM), but it is not known whether diabetes-associated alterations in cortisol metabolism contribute to its pathogenesis. Therefore, we evaluated some aspects of cortisol metabolism by measuring urinary metabolites of cortisol and cortisone in eight microalbuminuric and eight normoalbuminuric IDDM patients and eight matched control subjects. In both IDDM groups, the overnight excretion of tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), and tetrahydrocortisone (THE) was lower than that in the control group (P < 0.05 to P < 0.01). Both the allo-THF/THF ratio, a parameter of 5 alpha/5 beta-reduction of cortisol, and the cortisol to cortisone metabolite ratio (THF+allo-THF/THE), which reflects the overall direction of the cortisol to cortisone interconversion, were lower in the IDDM groups (P < 0.05 to P < 0.01). In the combined subjects (n = 24), allo-THF, allo-THF/THF, and THF+allo-THF/THE were inversely correlated with hemoglobin A1c (r = -0.69, P < 0.001; r = -0.61, P < 0.01; and r = -0.58, P < 0.01, respectively). Upper arm segmental blood volume, estimated by an electrical impedance technique, was positively correlated with the cortisol to cortisone metabolite ratio in both the control subjects (r = 0.77; P < 0.05) and the IDDM patients in whom it was measured (r = 0.56; P < 0.05; n = 13), whereas the regression line was shifted leftward in IDDM (i.e. a lower ratio at the same blood volume; P < 0.03, by analysis of covariance). In seven microalbuminuric IDDM patients, the angiotensin-converting enzyme inhibitor, enalapril (10 mg daily for 6-12 weeks), resulted in a moderate further lowering of the cortisol to cortisone metabolite ratio (P < 0.05). The present data suggest a chronic hyperglycemia-related impairment in the reduction of corticoids to tetrahydro metabolites and an imbalance in 11 beta HSD. Altered 11 beta HSD activity is unlikely to be primarily responsible for the sodium and fluid retention in IDDM. Moreover, an additional mechanism of action of angiotensin-converting enzyme inhibition might be provided by an effect on 11 beta HSD activity. PMID- 7559889 TI - Regional release and removal of catecholamines and extraneuronal metabolism to metanephrines. AB - Norepinephrine (NE) and epinephrine (E) are metabolized extraneuronally by catechol-O-methyl-transferase to the metanephrines (MNs), normetanephrine (NMN) and metanephrine (MN). Subjects in this study received infusions of tritium labeled NE and E. Concentrations of MNs and catecholamines were measured in plasma flowing into and out of the heart, forearm, lungs, kidneys, mesenteric organs (gastrointestinal tract, spleen, and pancreas), liver, and adrenals to examine the regional production of MNs from circulating and locally released catecholamines. NE spillover from mesenteric organs and kidneys accounted for 64% of the spillover from all tissues. There was detectable spillover of E from most extraadrenal tissues, but 91% was from the adrenals. The production of MNs from locally released and circulating catecholamines varied widely among tissues. The liver made the largest contribution to removal of circulating NE (57%) and E (32%) and the largest contribution to the production of NMN (54%) and MN (37%) from metabolism of circulating catecholamines. In all other tissues more NMN was produced from locally released than from circulating NE. Thus, the metabolism of circulating NE was responsible for only 19% of the total production of NMN. An even smaller portion (6%) of plasma MN was derived from metabolism of circulating E. Most plasma MN (91%) was produced within the adrenals, which also provided the largest single source (23%) of NMN. The regional variation in extraneuronal production of MNs indicates considerable heterogeneity in how circulating and locally released catecholamines are handled by different tissues. The substantial contribution of the adrenals to the production of MNs explains the extraordinary sensitivity of these metabolites for the diagnosis of pheochromocytoma. PMID- 7559891 TI - Amplified nocturnal luteinizing hormone (LH) secretory burst frequency with selective attenuation of pulsatile (but not basal) testosterone secretion in healthy aged men: possible Leydig cell desensitization to endogenous LH signaling -a clinical research center study. AB - The specific mechanisms underlying the relative hypogonadism of aging remain to be elucidated fully. We used frequent venous sampling (every 2.5 min), sensitive and specific LH and testosterone assays, and deconvolution analysis of the endocrine time series to delineate the differences between healthy young (n = 10, age 21-34 yr) and aged (n = 8, age 62-74 yr) men in the nocturnal secretion of LH and testosterone and their half-lives. We found that elderly men vs. young men had more frequent bursts of LH secretion (1.4 vs. 0.9/h, P = 0.003), less testosterone secreted per testosterone secretory burst (9.2 vs. 17.0 nmol/L, P = 0.021), and less testosterone secreted per hour (10.7 vs. 25.0 nmol/L.h, P = 0.05). As the frequency of nocturnal LH secretory bursts increased, the frequency of testosterone bursts decreased (r = -0.746, P = 0.034). We conclude that healthy aging is associated with diminished nocturnal testosterone production attributable to 1) a decrement in the mass of testosterone released per burst and 2) an inadequate response by the pituitary gland to enhance testosterone production via an accelerated LH secretory pulse frequency. We speculate that this may reflect a partial desensitization of Leydig cells to LH. PMID- 7559892 TI - Glucose, growth hormone, cortisol, and insulin responses to glucagon injection in normal infants, aged 0.5-12 months. AB - We performed glucagon stimulation tests in 59 normally growing siblings of children who died from sudden infant death syndrome. These investigations were performed to exclude a possible metabolic disorder (found in 4 siblings) as an underlying cause of sudden infant death syndrome. The remaining 55 siblings (32 boys and 23 girls) provide control data for this age range. Testing was performed at 0800 h after a 15-h fast. The median age was 98 days (range, 13-349 days). Plasma glucose and serum cortisol, insulin, and GH were determined before and 30, 60, 90, 120, 150, and 180 min after im injection of 0.1 mg/kg glucagon. No side effects were observed during the procedure. Asymptomatic hypoglycemia was noted in 11% of the infants at least once between 120-180 min. Basal and peak GH concentrations were greater than 10 micrograms/L in 31% and 80% of the infants, respectively. There was a significant negative correlation between age and basal GH concentration [Spearman's rank correlation coefficient (rs) = -0.37; P < 0.01]. There was a significant correlation between age and glucagon-stimulated cortisol at 120, 150, and 180 min (rs) = 0.41; P < 0.005), but not between age and changes in glucose levels. There was no significant correlation between age and basal cortisol or peak GH concentrations and no difference between boys and girls for any of the variables studied. In conclusion, the glucagon stimulation test is well tolerated in very young subjects. The peak GH response to glucagon injection is independent of age between 0.5-12 months. The age-related increase in the glucagon-stimulated cortisol response despite a similar decrease in glucose suggests the existence of a postnatal maturation in the response of the pituitary-adrenal axis to stress. PMID- 7559887 TI - The effects of beta 1-adrenergic blockade on the growth response to growth hormone (GH)-releasing hormone therapy in GH-deficient children. AB - Acute suppression of SRIH secretion with a beta-adrenergic antagonist can increase the GH response to GHRH. To determine whether chronic beta-blockade could enhance the growth-promoting effects of GHRH therapy, we conducted a double blind, placebo-controlled, randomized, cross-over trial of coadministration of the selective beta 1-antagonist atenolol together with GHRH in 11 GH-deficient children. In randomly chosen order, each patient received two 12-month treatment periods with a single daily injection of GHRH (20 micrograms/kg, sc, at bedtime), plus daily oral administration of either atenolol (1 mg/kg) or placebo. The growth velocity increased, rising from a mean +/- SD of 2.6 +/- 0.4 cm/yr before treatment to 5.4 +/- 1.0 cm/yr during the first year of treatment with GHRH plus placebo and to 6.8 +/- 1.2 cm/yr during the first year of treatment with GHRH plus atenolol. The mean growth velocity during treatment with GHRH plus atenolol was significantly greater than that observed during GHRH plus placebo (P < 0.05). After cross-over, however, during the second year of therapy, we did not observe any significant differences in growth velocity between the two groups (4.2 +/- 1.4 vs. 3.9 +/- 0.8 cm/yr during treatment with GHRH plus placebo and GHRH plus atenolol, respectively). The mean 24-h serum GH levels were 1.4 +/- 0.9 micrograms/L during the baseline period, 1.3 +/- 0.2 and 2.0 +/- 1.4 micrograms/L during the first year of GHRH plus placebo and GHRH plus atenolol, respectively (P = NS), and 2.7 +/- 1.4 and 1.4 +/- 0.4 micrograms/L during the second year of GHRH plus placebo and GHRH plus atenolol, respectively (P < 0.05). This is the first demonstration that alteration of neurotransmitter action can enhance the therapeutic response to a hypothalamic releasing factor. PMID- 7559895 TI - Factitious hypercortisoluria. AB - A case of factitious hypercortisoluria due to the presumed addition of glucocorticoid to the urine collections is presented. The discrepancy between urine and blood steroid hormone levels first suggested that the patient had tampered with the urine collections. Plasma steroid hormone levels were normal, whereas the urinary free cortisol level fluctuated in a totally random fashion. Urinary 17-hydroxycorticosteroid and urinary free corticosterone levels were normal. Partition chromatography of the urine indicated that the cortisol immunoactivity coeluted with authentic cortisol. This study illustrates the value of multiple urinary and plasma steroid determinations, especially urinary or plasma corticosterone measurements, in suspected cases of factitious Cushing's syndrome. PMID- 7559890 TI - Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures. AB - In continuation of earlier observations on the involvement of interleukin-1 (IL 1) in ovarian function, we examined the ability of IL-1 to modulate plasminogen activator (PA) activity and prostaglandin (PG) synthesis in human granulosa lutein cells (GLCs). Toward this goal, GLCs were obtained from women undergoing in vitro fertilization, preincubated with 10% fetal calf serum for 48 h, and subsequently cultured for 48 h in serum-free media in the absence or presence of IL-1 beta (10 ng/mL). Cellular PA activity was measured by plasminogen-dependent cleavage of the chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Prostaglandin E (PGE) levels were assayed by conventional RIA. Exposure of GLCs to IL-1 resulted in a 50% increase in PGE production, a 33% suppression of PA activity, and a 75% increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. The inhibitory capacity was attributable to an IL-1-mediated increase in PA inhibitor type-1 (PAI-1) production, inasmuch as urokinase inhibition could be abolished by the administration of a polyclonal antihuman PAI-1 immunoglobulin G. IL-1 treatment had no effect on plasmin or trypsin inhibition. Exposure of GLCs to IL-1 receptor antagonist abolished the ability of IL-1 to enhance PA inhibitory activity and PGE production, thereby establishing specific IL-1 receptor-mediated effects. The ability of IL-1 to suppress PA activity and to produce PAI-1 persisted in the presence of indomethacin, a potent inhibitor of PG synthesis. Likewise, transforming growth factor-beta 1 suppressed the ability of IL-1 to stimulate PGE production without affecting the IL-1-induced effects on the PA system. The present findings suggest a pluripotent response of GLCs to IL-1, characterized by the induction of PAI-1 and the suppression of PA occurring concurrent with, but independent of, PG production. These observations support the potential involvement of IL-1 in the regulation of human ovulatory processes. PMID- 7559893 TI - Hyperprolactinemia caused by lactation and pituitary adenomas is associated with altered serum calcium, phosphate, parathyroid hormone (PTH), and PTH-related peptide levels. AB - PRL stimulates systemic release of PTH-related peptide (PTHrP) in animals. To determine whether hyperprolactinemia causes PTHrP release in humans, we studied the relationship between PRL and PTHrP in lactating women and patients with PRL producing pituitary adenomas. Thirty-three lactating women and 16 patients with pituitary adenomas were paired with healthy age- and sex-matched controls. Serum total calcium, albumin, phosphate, PRL, intact PTH, and PTHrP were measured. Mean calcium and phosphate levels were higher in lactating women than in control subjects [2.39 +/- 0.01 vs. 2.35 +/- 0.01 mmol/L (P < 0.01) and 1.33 +/- 0.03 vs. 1.13 +/- 0.02 mmol/L (P << 0.001), respectively]. Mean PTH was lower (2.49 +/- 0.24 vs. 3.17 +/- 0.23 pmol/L; P < 0.04) and mean PTHrP was higher than control values (0.93 + 0.08 vs. 0.38 +/- 0.04 pmol/L; P << 0.001). PRL correlated negatively with PTH (P < 0.02) and positively with PTHrP (P < 0.05). Mean calcium, phosphate, and PTH levels were not different between patients with pituitary adenomas and control subjects. The mean PTHrP level was higher in patients with pituitary adenomas (0.75 +/- 0.10 vs. 0.39 +/- 0.07 pmol/L; P < 0.006) and fell significantly with therapy to normalize PRL (P < 0.03). We conclude that PTHrP levels are increased in hyperprolactinemia caused by lactation and pituitary adenomas. In lactating women, the increased PTHrP was associated with higher mean calcium and phosphate and lower PTH levels. The metabolic consequences of these abnormalities in hyperprolactinemia require further elucidation. PMID- 7559898 TI - Calcium absorption responses to calcitriol in black and white premenopausal women. AB - On the basis of recent findings that adult black women had similar calcium absorption but higher levels of 1,25-dihydroxyvitamin D [1,25-(OH)2D] than white women, we hypothesized that blacks have a gut resistance to the action of calcitriol. To test this, we studied 11 black [age, 32.4 +/- 5.7 (+/- SD) yr] and 12 white women (28.4 +/- 5.5 yr). The women were maintained on a constant 500-mg calcium diet for 4 weeks, and each received calcitriol (0.25 microgram) four times daily for the last 2 weeks. After 2 and 4 weeks, each subject had measurements of fractional 45Ca absorption index and blood and urine tests. At 2 weeks, the black women had similar calcium absorption indexes [18.7 +/- 1.9% (+/- SEM)/L vs. 20.0 +/- 1.8%/L; age adjusted], borderline higher 1,25-(OH)2D levels [95.7 +/- 6.4 (+/- SEM) vs. 78.2 +/- 6.2 pmol/L; P = 0.071; age adjusted], higher serum PTH levels, and lower urinary calcium excretion. Calcitriol therapy induced similar increments in plasma 1,25-(OH)2D levels in the two groups, but a smaller increment in calcium absorption in the black women (18.4 +/- 8.6% vs. 44.6 +/- 7.8%; P = 0.043; means adjusted for age and initial absorption index). These findings support the hypothesis that, compared with whites, healthy premenopausal black women have gut resistance to the action of calcitriol. PMID- 7559896 TI - Aromatase activity and interleukin-6 production by normal and malignant breast tissues. AB - The aromatase enzyme complex, which regulates the conversion of androstenedione to estrone, may have an important role in regulating estrogen synthesis in breast tissues. In this study the effect of tumor location on aromatase activity in adjacent tissue was examined and related to interleukin-6 (IL-6) production, which has been shown to stimulate aromatase activity in breast cancer cells. Samples of normal and malignant breast tissues were obtained from 11 women undergoing mastectomy. In 7 patients, aromatase activity was highest in the quadrant in which the tumor was located or on which the tumor impinged. Aromatase activity in tumor-bearing quadrants was significantly higher than that in adjacent and opposite quadrants. Aromatase activity and IL-6 production, expressed in terms of tissue weight, were significantly higher for tumor tissue compared with normal breast adipose tissue. A significant correlation was found between aromatase activity and IL-6 production for breast tumor tissue (rs = 0.56; P < 0.05), but not for adipose tissue from the breast quadrants. Aromatase activity and IL-6 production were also measured in tissue obtained from a normal woman undergoing reduction mammoplasty who had previously had breast augmentation by silicone injection, not contained within a capsule. In tissue from this patient there was evidence of chronic inflammation and a marked macrophage response. Aromatase activity in this tissue was considerably higher than that detected in mastectomy adipose tissue samples, and a significant correlation was found between aromatase activity and IL-6 production (rs = 0.77; P < 0.05). A preliminary study to examine the potential role of cells of the immune system in regulating breast tissue aromatase activity revealed that conditioned medium collected from macrophages and lymphocytes could markedly stimulate aromatase activity in tumor-derived fibroblasts. The results of this study confirmed that breast tumor location can influence aromatase activity in adjacent tissues and showed that aromatase activity is increased in tumor-bearing quadrants. The increased production of IL-6 by tumor tissue and its correlation with aromatase activity suggest that tumors may be the major source of IL-6, which is able to influence aromatase activity in adjacent tissues. PMID- 7559894 TI - Extragonadal alpha-inhibin precursor proteins circulate in human male serum. AB - The role of inhibin as a negative feedback regulator of pituitary FSH secretion in men remains controversial inasmuch as serum inhibin and FSH levels are often not correlated, in part because of the known alpha-inhibin subunit cross reactivity of the most widely used inhibin antiserum (Monash #1989). The objective of this study was to identify the nature of these alpha-inhibin proteins in male serum using antisera specific for the alpha-inhibin precursor. Three polyclonal antisera were raised against synthetic peptide fragments from the proregion (amino acids 21-35 = precursor alpha inhibin (PIN) 1 and 42-56 = PIN 2) and alpha-N segment (113-127 = PIN 3) of the human alpha-inhibin precursor protein. These antisera were then used in individual RIAs with the homologous peptide as both standard and radioligand. Because pure human alpha-inhibin subunit proteins are not available, recombinant alpha-inhibin medium and porcine follicular fluid were used as reference preparations in the PIN assays. All assays were specific for alpha-inhibin proteins, i.e. 1) they showed no significant cross-reactivity with other alpha-inhibin peptide fragments, dimeric 32-kDa inhibin, recombinant activin, FSH, human albumin, or the inhibin binding proteins alpha-2 macroglobulin and follistatin, and 2) they recognized native protein in alpha-inhibin precursor-containing porcine follicular fluid and in medium from a recombinant alpha-inhibin-only secreting cell line. Furthermore, immunoreactivity of all serum proteins seen by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting with the PIN antisera was abolished or significantly reduced (40-100%) by preincubation of each PIN antiserum with the homologous peptide. Serial dilutions of serum from normal, GnRH-deficient, and castrate males exhibited equivalent displacement curves in each precursor (PIN) assay, and there were no significant group differences in PIN 2 immunoreactivity between normal (n = 14), GnRH-deficient (n = 8), and castrate men (n = 3). Western blotting of serum samples from a normal and a GnRH deficient male revealed immunoreactive proteins of approximately 57 and 29 kDa under reducing conditions with all three PIN antisera. An additional 40-kDa protein was observed with the pro-alpha-inhibin antisera, PIN 1 and 2, and a protein of more than 97 kDa was seen with the PIN 2 antiserum.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559900 TI - Postmortem findings in congenital generalized lipodystrophy. PMID- 7559901 TI - Clinical review 74: Dermatological manifestations of endocrine disorders. PMID- 7559899 TI - Effect of corticotropin-releasing factor-binding protein on prostaglandin release from cultured maternal decidua and on contractile activity of human myometrium in vitro. AB - Human placenta and uterine tissues are sites of production and local action of corticotropin-releasing factor (CRF). The recent evidence that CRF-binding protein (CRF-BP), a protein that blocks CRF-induced pituitary ACTH release, is produced by placental tissues suggested the present study to investigate the effects of CRF-BP on prostaglandin release and contractile activity of myometrial strips. Primary cultures of decidual cells were prepared using tissue collected from healthy women undergoing cesarean delivery at term. Mechanical and enzymatic cell dispersions were carried out, and experiments were performed 24-28 h after cell plating. The prostaglandin E2 (PGE2) concentration in cultured medium was measured by RIA. Myometrial strips were obtained from the upper edge of the uterine incision during elective cesarean section at term. Dissected free of connective tissue, strips were mounted in a 30-mL two-chamber organ bath containing oxygenated Tyrode's buffer (37 C) and connected to a two-channel isometric smooth muscle transducer. Cultured decidual cells collected at term significantly increased the release of PGE2 in the presence of CRF (P < 0.01). The addition of CRF-BP did not significantly modify PGE2 release, but completely reversed the effect of CRF. When human myometrial strips were incubated in the presence of CRF and PGF2 alpha, a significant increase in contractile activity was observed (P < 0.01); preincubation with CRF-BP prevented the increased contractile activity induced by CRF. The present data show that CRF-BP is able to counteract the biological effect of CRF on human pregnancy endometrium and myometrium and suggest that CRF-BP may be a regulatory protein that plays a role in the local function of uterine tissues during pregnancy. PMID- 7559902 TI - Mutations of codon 918 in the RET proto-oncogene correlate to poor prognosis in sporadic medullary thyroid carcinomas. AB - The hereditary multiple endocrine neoplasia syndromes types 2A and B (MEN 2A and B) were recently linked to germline mutations in the RET proto-oncogene, altering one of five cysteine residues in exon 10 or 11 (MEN 2A), or substituting a methionine for a threonine at codon 918 in exon 16 (MEN 2B). The latter mutation also occurs somatically in some sporadic medullary thyroid carcinomas (MTC), and has in a previous study been correlated with a less favorable clinical outcome. In the present study, 46 MTCs were selected for investigation of the codon 918 mutation. The mutation was found in 29 tumors (63%), and was significantly correlated with a poor outcome, with regard to distant metastasis or tumor recurrence (p < 10(-4)). Two tumors showed multifocal growth and C-cell hyperplasia, and these patients were therefore also investigated for germline mutations in exons 10, 11 and 16. The codon 918 mutation was found only in the tumors, thus of somatic origin. The RET codon 918 mutation may have prognostic impact, and therefore preoperative assessment may influence decision-making in the treatment of patients suffering from MTC. PMID- 7559903 TI - Atrial natriuretic factor production by the human placenta. AB - Atrial natriuretic factor (ANF), a 28 amino acid peptide, is primarily produced and secreted by cardiac atrial myocytes to modulate cardiovascular and renal functions. Although ANF is also produced in tissues other than the heart, it remains uncertain whether the peptide is synthesised by human placentae. We provide here evidence from in vitro studies suggesting de novo production of ANF by the human placenta. Placental tissues were collected from normal term pregnancies by elective Cesarean section and acid extracted for ANF radioimmunoassay. The level of placental immunoreactive (ir) ANF was 186 +/- 33 pg/gm wet tissues (mean +/- SE, n = 6). Perfusates from in vitro perfusion of the fetoplacental compartment of placental lobules yielded 26 +/- 6 pg/min/gm wet tissue (n = 3) of irANF. Furthermore, pro-ANF mRNA signals were localised by colorimetric in situ hybridization in a subpopulation of placental cytotrophoblast-like cells, but not in syncytiotrophoblasts nor in chorionic cells of placental sections. Northern blot analysis of placental tissue extracts showed a single band of pro-ANF mRNA signals (-0.85 Kb) similar in size to that found in the rat heart. Our findings suggest that ANF is expressed and produced by a small population of human placental cytotrophoblast-like cells. The possibility that placental ANF may be secreted locally or into the fetoplacental circulation to exert paracrine, autocrine or both effects now needs to be considered. PMID- 7559905 TI - The diagnosis of growth hormone deficiency (GHD) in adults. PMID- 7559897 TI - Serum insulin-like growth factor I (IGF-I) and IGF-binding protein 3 levels are increased in central precocious puberty: effects of two different treatment regimens with gonadotropin-releasing hormone agonists, without or in combination with an antiandrogen (cyproterone acetate). AB - Central precocious puberty (CPP) is characterized by early activation of the pituitary-gonadal axis, which leads to increased growth velocity and development of secondary sexual characteristics. It is generally believed that gonadal sex steroids stimulate pulsatile GH secretion, which, in turn, stimulates insulin like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) production. However, little is known about GH, IGF-I, and IGFBP-3 serum levels in children with precocious puberty. Treatment of CPP by GnRH agonists has become the treatment of choice. However, the effect of long term treatment with GnRH in combination with an antiandrogen (cyproterone acetate) to block the possible effect of adrenal androgens has not previously been evaluated. We, therefore, studied 40 patients with idiopathic CPP that were treated for 24 months with either GnRH analog (Buserelin) in combination with cyproterone acetate (Androcur; n = 23) or with long acting GnRH analog (Decapeptyl Depot; n = 17). We found that serum IGF-I levels were increased before treatment in both groups (mean +/- SE, 446 +/- 35 and 391 +/- 35 micrograms/L; P < 0.0001, respectively) compared to those in normal age-matched prepubertal children. Similarly, IGFBP-3 levels were significantly elevated (4675 +/- 209 and 4305 +/- 162 micrograms/L, respectively; P < 0.0001) in the two groups. Treatment with GnRH analog in combination with cyproterone acetate significantly decreased height velocity and serum IGF-I and IGFBP-3 levels to normal levels after 2 yr of treatment (P < 0.0001). Serum IGF-I levels remained unchanged during monthly im treatment with long acting GnRH analog, whereas IGFBP-3 levels significantly increased during the first year of this treatment despite unmeasurable estradiol levels. Thus, in both groups, the molar ratio between IGF-I and IGFBP-3 (i.e. free biologically active IGF-I) declined concomitantly with a decrease in growth velocity. Serum levels of IGF-I and IGFBP-3 (expressed as the SD score for bone age), but not those of estradiol, correlated with height velocity before and during treatment (r = 0.34; P < 0.0001 and r = 0.24; P = 0.003, respectively). Six of the patients with a subnormal GH response to clonidine had similar IGF-I and IGFBP-3 serum levels and growth velocity compared to the other 34 girls with CPP and a normal GH response.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7559904 TI - Expression of the gene encoding growth hormone in the human mammary gland. AB - Progestins cause a syndrome of growth hormone (GH) excess and enhanced mammary tumorigenesis in the dog. This has been regarded as being specific for the dog. Recently we reported that progestin-induced GH excess originates from foci of hyperplastic ductular epithelium of the mammary gland in the dog. In the present report we demonstrate by reverse-transcriptase PCR and immunohistochemistry that a main factor involved in tissue growth, i.e. GH, is also expressed in normal and neoplastic human mammary glands. The gene expressed in the human mammary gland proved to be identical to the gene encoding GH in the pituitary gland. The role of progesterone in the GH expression of the human mammary gland needs, however, to be proven. It is hypothesized that this locally produced hGH may play a pathogenetic role in breast cancer. PMID- 7559906 TI - Human testicular aromatase: clinical experience. PMID- 7559908 TI - Surveillance of adverse reactions in patients self-infusing intravenous immunoglobulin at home. AB - This is the first prospective study of adverse reactions occurring in primary antibody-deficient patients self-infusing intravenous immunoglobulin at home. One hundred nineteen patients, already self-infusing at home, were entered into the study. They had been fully instructed about all aspects of adverse reactions, including their prevention and treatment. In the event of any adverse reaction, a form was completed detailing the product, the dose, the symptoms that occurred and the time they lasted, the rate of infusion, and other relative information such as predisposing factors and any medication taken. The severity of reactions were classified as mild, moderate, or severe. The total number of reactions documented was 19 in 2031 infusions, and all resolved without medical aid. No serious reactions occurred. Excluding those reactions in which predisposing factors were identified, the overall reaction rate was 0.7%. In conclusion the study showed the reaction rate in patients self-infusing intravenous immunoglobulin at home was low following formal training of selected patients at recognized training centers. PMID- 7559907 TI - IL-4 synthesis by in vivo-primed memory CD4+ T cells: II. Presence of IL-4 is not required for IL-4 synthesis in primed CD4+ T cells. AB - Previous studies have shown that the presence of IL-4 is required for the development of IL-4 synthesis in naive CD4+ T cells. The purpose of our current studies was to investigate the role of IL-4 in the development of IL-4 synthesis in primed memory T cells. We therefore examined CD4+ T cells taken from lymph nodes of BALB/c mice immunized with keyhole limpet hemocyanin (KLH) and restimulated in vitro with KLH. Our results with such primed resting CD4+ T cells programmed to produce IL-4 indicated that the production of IL-4 did not require the presence of IL-4 (although the presence of IL-2 was absolutely necessary), and was only slightly limited by the presence of anti-IL-4 MAb. These results with resting memory T cells were not biased by the presence of activated T cells already producing substantial quantities of IL-4, since we demonstrated that high density memory T cells could produce IL-4 in the absence of IL-4, and because T cells that actively produce IL-4 do not persist in vivo very long after antigen exposure. These results indicate that IL-4 synthesis in T cells committed to IL-4 production can indeed occur in the absence of IL-4 when culture conditions have been optimized and suggest that therapies with anti-IL-4 MAb or with soluble IL-4 receptors designed to control the development of IL-4 synthesis in memory T cells from individuals exhibiting excessive IL-4 synthesis will be unsuccessful. Therefore, other therapies, for example, utilizing IL-12, will be required to modulate the relatively fixed programs in memory T cells that direct the development of cytokine synthesis. PMID- 7559909 TI - HIV-mediated B-lymphocyte activation and lymphomagenesis. AB - Non-Hodgkin's (1ii)lymphoma is an AIDS-defining event in a significant percent of U.S. patients infected with the human immunodeficiency virus (HIV). Advances in anti-retroviral treatment and management of opportunistic infection have been accompanied by an increase in the incidence of these lymphomas. In the immunocompromised state of patients late in the course of HIV infection, these lymphomas represent a complication of HIV infection that is associated with an extremely poor prognosis. Currently, there is little understanding of the pathogenesis of HIV-associated lymphomas, which are nearly exclusively of B-cell origin. Experimental data do not support HIV infection in these lymphomas. While some lymphomas show evidence of EBV infection, the majority do not. Polyclonal B cell hyperactivity during the early phases of HIV infection argues that chronic B cell stimulation may be the major process predisposing B-cells in the HIV infected individual to malignant transformation. The mechanism of this stimulation of normal B cells and its relationship to AIDS-associated lymphomas remain poorly understood. In this review, we will summarize current information on HIV-associated B lymphoma and then discuss our views on the association and regulation of HIV-related hyperactivity on the pathogenesis of this lymphoma. PMID- 7559910 TI - Decreased serum levels of TGF-beta in patients with acute Plasmodium falciparum malaria. AB - Apart from cellular immunity and immunopathology, various cytokines have been implicated in malaria-associated immunosuppression. In this study, serum levels of transforming growth factor-beta (TGF-beta) were determined with an enzyme linked immunosorbent assay in 37 patients with acute Plasmodium falciparum malaria prior to, during, and after therapy and in 17 healthy controls in Bangkok, Thailand. Patients were treated with artesunate and mefloquine. TGF-beta serum levels were found decreased prior to treatment (14 +/- 11 pg/ml versus 63 +/- 15 pg/ml in healthy controls; P < 0.05). The serum concentrations of TGF-beta increased after initiation of treatment and were within normal range on day 21. Serum levels of both tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptor 55 kDa were inversely correlated to serum levels of TGF-beta (r = -0.667 and r = -0.592, n = 37; respectively, P < 0.05 for both). No correlation between parasitemia and serum levels of TGF-beta could be found. The results are compatible with a decreased production and release, an enhanced clearance or utilization, or tissue accumulation of TGF-beta in acute P. falciparum malaria. PMID- 7559911 TI - Autologous human B-cell immune response to pulmonary adenocarcinomatous polymorphic epithelial mucin. AB - In order to investigate whether a B-cell immune response to the polymorphic epithelial mucin (PEM) is present in adenocarcinomatous patients, peripheral blood lymphocytes from 20 patients with adenocarcinomas from various sites were fused with the mouse-human heteromyeloma cell line SHM-D33. One IgM(K) monoclonal antibody derived from peripheral blood lymphocytes of a patient with a lung adenocarcinoma was generated with binding reactivity for bovine submaxillary mucin (BSM). The latter is one of the PEMs, and it contains a large amount of the tumor-associated sialosyl-Tn epitope. The generated antibody was designated KMD 2. Immunohistological studies on various tissues showed that KMD-2 reacted with 6/6 colorectal adenocarcinomas and 4/6 pulmonary adenocarcinomas. The antibody also displayed a slight degree of cross-reactivity with a limited number of normal tissues, especially those that elaborate mucin. Our data show that an autologous antibody response in the form of IgM to the immunosuppressive PEM is present in some adenocarcinomatous patients. The data also suggest that the KMD-2 (IgM/K) antibody may be of clinical importance for diagnostic purposes of adenocarcinomatous disease. PMID- 7559912 TI - Characterization of human thymic epithelial cell surface antigens: phenotypic similarity of thymic epithelial cells to epidermal keratinocytes. AB - Cellular interactions between developing thymocytes and cells of the thymic microenvironment are necessary for maturation of thymocytes into mature T cells. While much is known about the molecules on developing T cells that mediate these interactions, little is known about the surface molecules of human thymic epithelial (TE) cells. In this study, using a panel of 276 MAb including 255 MAb from the 5th International Workshop on Human Leukocyte Differentiation Antigens (HLDA-V), we have determined the expression of CD1 through CDw130 and other surface molecules on resting and IFN-gamma-activated cultured human TE cells and on resting epidermal keratinocytes (EK). We demonstrate the surface expression of 50 of the 161 molecules assayed for on TE cells, including a number of adhesion molecules, cytokine receptors, Apo-1, and MHC-encoded molecules. While activation of TE cells with IFN-gamma for 48 hr induced a greater than fivefold increase in the expression of four surface molecules (CD38, CD54, MHC class I, and MHC class II), it also induced a greater than 50% increase in the expression of 14 other surface molecules (CD12, CD29, CD40, CD44, CD47, CD49b, CD49c, CD49e, CD55, CD66, CD87, CD104, TE4, and STE3) and a decrease in the expression of three molecules (CDw65, CDw109, and STE2). In comparing the phenotype of TE cells to 83 other cell lines studied in HLDA-V, we found that TE cells were strikingly more similar to EK than to any of the other cell types tested. PMID- 7559913 TI - Altered IL-10 levels in trauma patients' M phi and T lymphocytes. AB - Trauma results in concomitant immunosuppression and elevated monocyte (M phi) inflammatory cytokine levels. The augmenting or ameliorating effect of IL-10 in septic complications after trauma is controversial. Here, IL-10 levels of trauma patients' and normals' PBMC, isolated M phi, and isolated T cells were assessed and correlated to their PBMC mitogen responses, their T-cell proliferation in an APC independent system, and their M phi production of elevated TNF-alpha levels. Trauma patients with depressed PBMC responses to PHA stimulation also had significantly decreased IL-10 levels in their stimulated PBMC supernates (P = 0.0022) and their MDP-stimulated isolated M phi population (P = 0.0004). However, patients with depressed PHA responses could have either normal or depressed T cell proliferation in an anti-CD3-, anti-CD4-stimulated system. If APC independent T-cell proliferation was depressed, induced IL-10 levels were suppressed (P = 0.007). However, if APC-independent T-cell proliferation was normal or elevated, IL-10 levels could be normal or elevated (P = 0.018). Decreased IL-10 levels correlated with depressed mitogen responses and depressed T-cell proliferation. IL-10, therefore, could not be inducing trauma patients' immunosuppression. Patients with elevated M phi TNF-alpha levels had depressed M phi IL-10 levels. PMID- 7559915 TI - CD8+ T lymphocytes are recruited to neoplastic cervix. AB - ToliicIV distinguish normal cervical lymphocyte populations from phenotypes recruited to the cervix in response to cervical neoplasia, lymphocytes were isolated from normal and neoplastic cervix. A portion of the cervical transformation zone was obtained from 19 patients with pathologically confirmed cervical intraepithelial neoplasia and from 20 patients with normal cervices undergoing hysterectomy for benign indications. Mononuclear cells were harvested from cervical tissue using a serial, multienzymatic digestion procedure and enriched by density gradient centrifugation. Isolated cell populations were stained with surface marker-specific monoclonal antibodies and analyzed by fluorescent activated cell sorter to determine the percentage of B cells, total T cells, CD4+ T cells, CD8+ T cells, and natural killer (NK) cells. The distribution of circulating peripheral blood lymphocyte phenotypes was similar for both patients with neoplasia and normal controls. A marked disparity in the proportions of NK cells and T cells was demonstrated among lymphocyte phenotypes infiltrating the cervix. The percentage of CD4+ T cells and NK cells was significantly depressed (P = 0.04, P = 0.03, respectively) in dysplastic tissue as compared to normal cervical tissue. In contrast, the proportion of CD8+ T cells was significantly increased in the dysplastic tissue (P = 0.0001). Analysis of immunocompetent cells in the circulation appears to have little correlation with immunocytes present in the dysplastic epithelium. The depression in the proportion of CD4+ T lymphocytes and NK cells at the cervical squamocolumnar junction reflects a local recruitment of CD8+ T cells to the site of neoplasia in the cervix. PMID- 7559914 TI - Biology of human TH1 and TH2 cells. AB - Evidence is accumulating to suggest the existence of polarized human T-cell responses, reminiscent of TH1 and TH2 subsets described for mouse T cells. Human TH1 cells preferentially develop during infections by intracellular bacteria and trigger phagocyte-mediated host defense, whereas TH2 cells, which predominate during helminthic infestations and in response to common environmental allergens, are responsible for phagocyte-independent host response. Human TH1 and TH2 cells exhibit not only different functional properties but probably also distinct surface markers; TH2, but not TH1, clones express membrane CD30 and release the soluble form of CD30, a member of the TNF receptor superfamily. The cytokine profile of "natural immunity" evoked by different offending agents in the context of different host genetic backgrounds appears to be the most critical factor in determining the phenotype of the subsequent specific response. IL-12 and IFN alpha and gamma produced by macrophages and NK cells favor the development of TH1 cells, whereas the early production of IL-4 by a still-unidentified cell type favors the development of TH2 cells. Clearly, polarized human TH1 and TH2 responses not only play different roles in protection, they can also promote different immunopathological reactions. Strong and persistent TH1 responses seen to be involved in organ-specific autoimmunity, contact dermatitis, and some chronic inflammatory disorders of unknown etiology. In contrast, polarized TH2 responses favor a reduced protection against the majority of infectious agents (including HIV) and, in genetically predisposed hosts, are responsible for triggering of allergic atopic disorders. PMID- 7559919 TI - [Helicobacter pylori infection in peptic ulcer and gastric cancer diseases]. PMID- 7559916 TI - Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation. AB - Fifty-three pediatric patients given an allogeneic or an autologous bone marrow transplantation (BMT) were immunized with a polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax II). Vaccine was administered six months or more after BMT and the pneumococcal IgM, total IgG, and IgG subclasses levels were evaluated before and three weeks after immunization. Immunization promoted a significant rise in antibody serum levels (P < 0.000001), and all children vaccinated more than two years after transplantation responded to pneumococcal polysaccharides, whereas only 20-30% and 50% of patients given BMT between six months and one year and one and two years, respectively, mounted an effective antibody production (P < 0.0001). In univariate analysis, lapse of time from BMT to vaccination, chronic graft-versus-host disease occurrence, and female sex influenced the response rate. However, in multivariate analysis, only time between marrow transplant and immunization was a powerful predictor of response. Interestingly, four of 11 patients with IgG2 deficiency before immunization normalized serum levels of this IgG subclass after the pneumococcal antigenic challenge. Our study suggests that time after transplant is the major factor influencing the recovery of immune reactivity to polysaccharide antigens. This seems to confirm the hypothesis that ontogeny of the B-cell repertoire follows a predetermined sequential program in which polysaccharide antigens are some of the last to evoke an antibody response. PMID- 7559918 TI - Inhibition of cell-mediated cytolysis and P-glycoprotein function in natural killer cells by verapamil isomers and cyclosporine A analogs. AB - We have previously shown that among normal leukocytes, CD56+ and CD8+ cells express relatively high levels of P-glycoprotein (P-gp), a transmembrane efflux pump. While the physiologic significance of P-gp expression in leukocytes is unknown, the relatively high levels of P-gp in CD56+ and CD8+ cells suggest that P-gp may function in cell-mediated cytolysis. To explore this possibility we examined the effect of four inhibitors of P-gp efflux [(R)-verapamil (R-ver), (S) verapamil (S-ver), cyclosporine A (CsA), and PSC833 (PSC)] on both the inhibition of natural killer cell (NK) function and on P-gp efflux. NK function was assayed by measuring the lysis of 51Cr-labeled K562 target cells in the presence and absence of inhibitors. All four P-gp efflux inhibitors inhibited NK-mediated cytolysis in a dose-dependent manner. The stereoisomers of verapamil were more potent inhibitors of cell-mediated cytolysis than the cyclosporines CsA and PSC. In contrast, CsA and PSC were more potent as inhibitors of P-gp-mediated rhodamine 123 dye efflux than the verapamil isomers. Both CsA and PSC maximally inhibited P-gp efflux at 3 microM, but only minimally inhibited cell-mediated cytolysis. The verapamil compounds demonstrated closer correlation between efflux inhibition of NK-mediated cytolysis. The data support a role for P-gp in NK mediated cytolysis; however, these studies also suggest that the NK cytolytic process is multifaceted and that inhibition of the P-gp-mediated efflux mechanism only partially abrogates this process. PMID- 7559917 TI - Staphylococcus aureus Cowan I-induced human immunoglobulin responses: preferential IgM rheumatoid factor production and VH3 mRNA expression by protein A-binding B cells. AB - Protein A (PA), a cell wall component of SAC, activates human B cells by cross linking the Fabs of membrane immunoglobulins. Recent data indicate that PA binds only to Fabs that use VH3 heavy chains, and thus it has been designated as a B cell superantigen. We previously reported that Staphylococcus aureus Cowan I (SAC)-induced IgM rheumatoid factor (RF) by human PBMC was mediated by PA. Therefore, we sought to determine if SAC-induced IgMRF production was confined to PA-binding B cells and if these B cells were enriched for the expression of VH3 heavy chains. We observed that the elicitation of IgMRF in response to SAC was limited to a subset of B cells that bind PA and that this subset was enriched for VH3 mRNA expression. Taken together, these results suggest that IgMRFs produced in response to SAC are enriched for usage of VH3 heavy chains. Thus, this SAC induced autoantibody response appears to represent a new B-cell superantigenic property of PA. PMID- 7559921 TI - [The mode of excitation of cat motoneurons]. AB - 1. Effects of afferent nerve stimulation on the waveform of the IS-spike were studied intracellularly in 101 motoneurons of cats which were either anesthetized with sodium pentobarbitone or decerebrated at the intercollicular level. 2. In 78 motoneurons repetitive or double shock stimulation of afferent nerves (the L 7 or S 1 dorsal root, biceps-semitendinosus nerve and tibial nerve) could produce 2-4 kinds of IS-spikes, as distinguished by differences in waveform. In 34 motoneurons 2-3 small potentials were superimposed on the smallest IS-spike with progressively longer latencies to form a larger IS-spike. These potentials were thought as dendritic in origin because only the dendrites are known to be capable of exhibiting multiple spikes and in 14 motoneurons dendritic spikes were actually shown to exist. 3. In 13 motoneurons following a repetitive stimulation of a dorsal root there occurred an increase in excitability of the membrane generating the IS-spike (the IS-membrane) in the absence of any appreciable changes in the resting membrane potential. This would indicate that the IS membrane is subject to plastic changes or the IS-spike itself also originates in dendrites which are known to exhibit such changes. PMID- 7559920 TI - [Effect of anticancer drugs on invasive capacity of human small-cell lung cancer cells in vitro]. AB - The effect of anticancer drugs on invasive capacity of Lu 135 human small-cell lung cancer cells was studied in vitro. the invasive capacity decreased in a dose dependent manner when tumor cells were treated with the anticancer drugs cisplatin and etopside. The inhibition of tumor cell invasion was almost parallel with the inhibition of tumor cell growth. The anticancer drugs also suppressed tumor cell migration and the activity of the matrix-degrading protease, type IV collagenase, activity. But they did not suppress adhesion to basement membrane protein such as laminin, fibronectin, or type IV collagen. Because tumor cells express adhesion molecules on the cell surface, the effect of the anticancer drugs on the expression of one such molecule, integrin, was also studied. Lu 135 cells expressed alpha 4/beta 1 integrin on the cell surface, and the pattern of integrin expression did not change with exposure with the anticancer drugs. These data suggest that the anticancer drugs inhibit the invasive capacity by suppression of migration and type IV collagenase activity of tumor cells, and that they do not modulate adhesion to the extracellular matrix or cell surface adhesion molecules such as integrin. Furthermore, these findings indicate that anticancer drugs may be useful for anti-metastatic therapy in patients with small cell lung cancer. PMID- 7559922 TI - [Biochemical changes in lens, aqueous humor and vitreous body and effects of aldose reductase inhibitor (TAT) on rats with experimental diabetes]. AB - The etiology of diabetic cataract is usually explained by the following process; the conversion of glucose and galactose to polyol by aldose reductase, then the accumulation of polyol in lens, and the opacity of lens. Another explanation is that the hyperoxidation of lens membrane due to an increase of active oxygen and lipid peroxide in lens induces diabetic cataract. The experimental animals used in the present study were rats with galactose cataract and streptozotocin cataract. We measured the levels of antioxidants (glutathione, ascorbic acid) and lipid peroxide (malonodialdehyde) in lens, aqueous humor and vitreous body. Furthermore we studied the effects of aldose reductase inhibitor (TAT) on these levels. In streptozotocin diabetes rats, the increased malonodialdehyde levels in lens, aqueous humor and serum were suppressed by TAT administration. In galactose and streptozotocin diabetes rats, the decreased levels of glutathione and ascorbic acid were suppressed by TAT administration. PMID- 7559923 TI - [Effect of argatroban on flap survival length]. AB - The effect of argatroban, an anti-thrombin agent, on a 1.5 x 6.0 cm caudally based flap raised on the backs of Wistar female rats was studied. Argatroban was administered to one group via an implanted osmotic pump, and nothing was administered to a control group. In experiment 1, the effect was evaluated by survival length (ratio of length to width) and a statistically significant difference (p < 0.01) was found between the argatroban group and the control group. In experiment 2, at 3 spots along the midline (M1, M2, M3) in each group, ISO2 (a parameter of oxygen saturation) and IHb (a parameter of hemogrobin concentration) were measured with a reflectance spectrophotometer, and FLOW (a parameter of blood flow) and MASS (a parameter of blood mass) were measured with a laser Doppler flowmeter. M1 was a distal spot of flap, M2 was a proximal spot on the flap and M3 was a spot beside the flap used as a control. At M1, flaps in both groups became necrotic. At M2, flaps in the argatroban group survived, whereas those in the control group became necrotic, and each parameter indicated improvement in blood circulation only in the argatroban group. Thus, argatroban, which antagonizes thrombin, can improve microvascular circulation in congestive tissue and consequently help the flap survive. Argatroban has advantages over other agents that have been given to increase the length of the surviving experimented flap. In comparison with heparin, the coagulation time is easier to control. Also argatroban does not dilate peripheral vessels, which may be a demerit of alprostadil. Argatroban also promotes the reaction of plasminogen activator. The serum concentration of argatroban in this study was almost equal to that used clinically. Therefore, argatroban may be useful when elevating a flap clinically. PMID- 7559925 TI - [Radiological studies of the cervical spine after laminoplasty by longitudinal splitting of the spinous process]. AB - This study involved a clinical evaluation and radiological follow-up of patients who underwent laminoplasty by longitudinal splitting of the spinous process for compressive myelopathy. The subjects were 47 patients with myelopathy caused by cervical spondylosis or ossification of the posterior longitudinal ligament in the cervical spine. The average age at the time of surgery was 61, and the average follow-up period was 3 years. Overall results: the preoperative score, by the Japanese Orthopaedic Association scoring system, was 9.8, and the postoperative score was 13.9; the average recovery rate was 54.3%. Lateral roentgenograms showed a decreased lordosis in the cervical curvature in 50% of the patients after laminoplasty, especially in those patients where stripping of the attachments of the semispinalis cervices to the spinous process of C2 occurred. The recovery rate was good in patients with postoperative lordotic or straightened necks (61.1% and 55.0%, respectively), but it was poor in those with kyphotic or S-shaped necks (average: 36.1%). The postoperative range of motion of the cervical spine had decreased to 43% of the preoperative range. PMID- 7559924 TI - [Biomechanical properties of the anterior and posterior longitudinal ligament in the cervical spine]. AB - The purpose of this study is to investigate the biomechanical properties of the anterior longitudinal ligament (ALL) and the posterior longitudinal ligament (PLL) of the human cervical spine. Twenty C3-C4 and twenty C5-C6 motion segments are dissected from the spines of human cadavers. Experimental specimens of the bone-ligament-bone complex are prepared for mounting on a testing jig. Static tensile tests are performed with an Instron-type material test system. Dynamic biomechanical properties were tested with a biomechanical spectrometer. The failure load and failure stress of the PLL tend to be higher than those of the ALL, but there are no statistically significant differences between them. The tan delta of the ALL is higher than that of the PLL (p < 0.01). The dynamic stiffness of the PLL is higher than that of the ALL (p < 0.01), but there was no statistically significant difference in the dynamic elastic modulus between the ALL and the PLL. These results imply that the PLL has better biomechanical properties as a stabilizer for cervical motion than the ALL in the lower cervical spine. PMID- 7559927 TI - Effects of an HMG-CoA reductase inhibitor on cytokine production by human monocytes/macrophages. AB - It has been reported that the HMG-CoA reductase inhibitor simvastatin does not always effectively lower plasma LDL. This drug acts to monocytes/macrophages directly and inhibits cholesterol ester accumulation in these cells. However cytokine production in macrophages when simvastatin was administrated has not been described. In this study, we examined whether simvastatin affects cytokine production in human monocyte-derived macrophages. Simvastatin at doses ranging from 10(-9) to 10(-5) M did not affect the synthesis of proinflammatory cytokines (IL-1 beta, IL-6, IL-8) from human peripheral mononuclear cells. In addition, any changes in cytokine-induced cytokine production (IL-1-induced IL-8 synthesis) were not detected after the addition of simvastatin. The present results suggest that simvastatin suppresses foam cell formation in monocyte/macrophage, without affecting the immunological or inflammatory functions of these cells. PMID- 7559926 TI - [Visco-elasticity of transplanted menisci in rabbits. A correlative histological and hydrodynamical studies]. AB - The purpose of this study is to investigate the visco-elasticity, histology and hydrodynamics of transplanted menisci following transplantation. Experiments to examine visco-elasticity are performed on 30 rabbits, to examine NMR on 20 rabbits and on 12 rabbits respectively for histological study. Under intravenous general anesthesia the medial meniscus was completely resected, and a cryopreserved allograft meniscus was implanted. The rabbits are sacrificed 1, 2, 3 and 5 months post-operatively, and the implanted menisci are removed for examination. One month after surgery, dynamic stiffness is markedly lower in the implanted menisci than in the controls. Thereafter, it gradually increased. Three months after surgery, there is no significant difference between the transplanted menisci and the controls. However the value of tan delta which serves as indicator of dynamic visco-elasticity is larger in the implanted menisci than in the control specimens throughout the experiment. The water content was higher in the transplanted menisci throughout. The 1H-NMR relaxation time (T2) was markedly prolonged 1 month post-operatively, but shortened rapidly after that. Nevertheless, it is still longer than in the control specimen after 5 months. In the histological study, no chondrocytes are observed in the transplanted menisci, and the matrix was poorly stained with Eosin 1 month after surgery. Thereafter, granulomatous tissues proliferated in the menisci, and by 5 months after surgery, the chondrocytes appear to be almost normal. However, matrix was weakly stained with Eosin. These results suggest that the interactions between high molecular weight matrix and water in transplanted menisci are different from those in normal menisci, and that therefore the viscosity of transplanted menisci is higher. PMID- 7559929 TI - [Prognosis and outcome of rheumatoid arthritis. Influence of anti-rheumatic therapy on natural course of disease and objectives in daily treatment]. PMID- 7559931 TI - 11th Annual meeting of the European Society of Human Reproduction and Embryology. Hamburg, June 28-July 1, 1995. Abstracts. PMID- 7559928 TI - [Two autopsy cases of malignant mixed mullerian tumor of the uterus with metastasis to alimentary tract and liver]. AB - Two autopsy cases (69 and 87-year-old women) of malignant mixed mullerian tumor (MMMT) following radiation therapy for uterine cervical cancer sixteen and twenty years ago respectively were reported. They were admitted due to abdominal pain and diagnosed as ileus. In the first case, CT examination revealed a tumor measuring about 8 x 10 cm in size in the uterine posterior wall. Recurrence of the uterine cancer was suspected and hysterooophorectomy combined with sigmoidectomy was performed. In the second case, artificial anus formation was performed because of sigmoid stricture by the invasion of the tumor. Histologically, tumors in both cases were composed of carcinomatous and sarcomatous components including heterologous elements such as cartilage. The patients died of extensive spreads and metastasis in the liver and alimentary tract. PMID- 7559930 TI - [Practical application of DNA-diagnosis in the Nippon Medical School Hospital]. PMID- 7559933 TI - Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung. AB - The presence of clamp connections on hyphae and the development of fruiting bodies in culture are primary characters which allow identification of the basidiomycete Schizophyllum commune in cases of human infection. The diagnostic problems presented by a nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described. Several features of the isolated fungus, including rapid growth rate and white, dense, cottony colonies, tolerance to the fungicide benomyl at a concentration of 10 micrograms/ml, and susceptibility to cycloheximide at 400 micrograms/ml, suggested that it might be a basidiomycete. Transmission electron microscopy showed the presence of a dolipore septum with perforate pore cap characteristic of fungi in the class Holobasidiomycetes. However, species identification remained elusive until compatibility tests with known single-basidiospore isolates confirmed the identification of the sterile lung isolate as S. commune. Sequence analysis of the 5' internal transcribed spacer region of ribosomal DNA further supported conspecificity. PMID- 7559932 TI - Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay. AB - A gene amplification method that combines PCR with an enzyme immunoassay (PCR EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR amplified HPV DNA from common genital HPV types. PMID- 7559938 TI - Expression of Sendai virus nucleocapsid protein in a baculovirus expression system and application to diagnostic assays for Sendai virus infection. AB - The most common diagnostic technique for the detection of Sendai virus infection in rodents is serological evaluation by enzyme-linked immunosorbent assay (ELISA) with semipurified preparations of whole virions as antigens. This assay often suffers from a lack of specificity. The goal of the present project was to develop more specific antigens for use in diagnostic testing by producing recombinant antigens in insect cells. To identify viral proteins immunoreactive in multiple laboratory rodent species, Western blots (immunoblots) of viral polypeptides were probed with immune sera from mice, rats, and hamsters. The nucleocapsid protein (NP) reacted with immune sera from all species tested. Therefore, the NP gene was selected for cloning and expression in a baculovirus. To construct the recombinant, complementary DNA was synthesized by reverse transcription PCR from Sendai virus RNA with primers from the 5' and 3' termini of the NP-coding region. Amplified DNA was cloned into a baculovirus transfer vector (pBlueBacHis A) and was cotransfected with wild-type baculovirus into insect cells. Baculovirus recombinants containing the NP gene were identified by PCR. Evaluation of the recombinant proteins expressed in insect cells by Western blot analysis revealed specific reactivity with immune sera. In comparison with conventional ELISAs that use whole virions as the antigen, ELISAs that use recombinant NP were more specific. PMID- 7559935 TI - Characterization of Finnish Borrelia burgdorferi sensu lato isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with monoclonal antibodies. AB - Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis. PMID- 7559936 TI - Evaluation of commercial antisera for Shigella serogrouping. AB - Shigella serogrouping antisera from six companies (Becton Dickinson, Denka, Difco, Murex, Roach, and Sanofi-Pasteur) intended for the slide agglutination test and those of the Wellcolex Colour Shigella latex agglutination test were evaluated to identify quality products for Shigella identification. Forty-six reference Shigella strains (one for each serotype and species), 50 clinical strains (21 S. flexneri, 21 S. sonnei, 4 S. dysenteriae, 4 S. boydii) representing the most prevalent species and serotypes encountered in Quebec, and 9 non-Shigella strains were tested according to the manufacturers' instructions. A 3+ reaction (> or = 75% agglutination) was considered positive for the slide agglutination tests. Sensitivity varied from 47% (Roach) to 94% (Difco). For the 105 strains tested, accuracy ranged from 53% (Roach) to 91% (Wellcolex). Specificity varied from 97 to 100% for group A antisera, from 96 to 100% for group B antisera, from 88 to 100% for group C antisera, and from 95 to 99% for group D antisera. The costs of reagents required to test one strain varied from $3.50 to $13.20 (in Canadian dollars). In conclusion, Roach reagents proved to be unsatisfactory for Shigella serogrouping. Among those from the remaining companies, the Denka, Difco, and Wellcolex reagents met a performance standard of 90% accuracy. PMID- 7559937 TI - Human Bordetella bronchiseptica infection related to contact with infected animals: persistence of bacteria in host. AB - Within a period of 2 1/2 years, Bordetella bronchiseptica was isolated four times from a 79-year-old woman with bronchopneumonia. We have demonstrated by pulsed field gel electrophoresis that this infection was related to contact with infected rabbits. The initial human B. bronchiseptica isolate had a phenotype characteristic of usual B. bronchiseptica clinical isolates; it produced toxin and adhesins, such as adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin, and was able to induce lethality in a murine respiratory model. By contrast, although the three successive human isolates produced adhesins, they did not express adenylate cyclase-hemolysin and were unable to induce lethality. This implies that adenylate cyclase-hemolysin is required to induce lethality. We suggest that B. bronchiseptica may persist in the host, with expression of adenylate cyclase-hemolysin being essential for the initiation of infection and expression of adhesins being essential for persistence. PMID- 7559934 TI - Evaluation of cleaving agents other than trypsin in direct agglutination test for further improving diagnosis of visceral leishmaniasis. AB - Trypsin treatment of Leishmania promastigote antigen has proved to be indispensible in the direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). In the present study four antigen batches were prepared with pronase (400 micrograms/ml), lipase (0.45% [wt/vol]), pancreatin (0.3% [wt/vol]), or 2 mercaptoethanol (2-ME) (1.2% [vol/vol]) at a ratio of 20:1 versus promastigote packed cell volume or a density of 10(8)/ml. Batches prepared in this way performed satisfactorily when compared with the performance of the initial trypsinated antigen. Even higher was the sensitivity and specificity of the 2-ME processed antigen, scoring a minimum DAT titer of 1:102,400 in the VL and CVL group and a maximum of 1:400 in the negative control group. Corresponding titers ranging from 1:6,400 to 1:12,800 and 1:800 to 1:1,600 were obtained with the antigen variants processed with pronase, lipase, pancreatin, or trypsin. By combining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in the antigen processing step, a threefold increase in titer was attained in sera from the respective areas where VL is endemic. 2-ME-processed antigen suspensions maintained stability at 4 degrees C for up to 9 months, as evidenced by the absence of autoagglutination and the reproducibility of DAT readings with standard sera. The specificity of DAT was further improved by supplementation of the sample diluent with 0.03 M urea and incubation of the test plates at 37 degrees C for 1 h. Titers ranging from 1:200 to 1:12,800 in the sera of patients and laboratory animals infected with various trypanosoma species were significantly reduce (/=1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT. PMID- 7559939 TI - Effect of PANTA on growth of Mycobacterium kansasii in BACTEC 12B medium. AB - Mycobacterium kansasii isolates from two patients showed relatively slow growth in BACTEC 12B medium (12B) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) compared with the more rapid growth of these isolates on solid media. This finding prompted an evaluation of the effect of PANTA (Becton Dickinson) on the growth rate of these isolates. Suspensions of one isolate from each of these two patients (A and B), six additional isolates from six other patients (C through H), and one M. kansasii American Type Culture Collection isolate were inoculated into 12B with PANTA, 12B with reconstituting fluid only, and Middlebrook 7H11 agar plates (Remel, Lenexa, Kans.). For the isolates from patients A and B, the average times to detection for 12B with PANTA, 12B with reconstituting fluid, and Middlebrook 7H11 agar were 12.3, 7.4, and 9.0 days, respectively. For the remaining six patient isolates and the American Type Culture Collection strain, the average times to detection for these media were 9.2, 8.1, and 9.6 days. Susceptibility tests performed with the isolates from patients A and B with the individual component antibiotics of PANTA and testing of four of the other isolates with nalidixic acid alone suggested that nalidixic acid exerts some degree of inhibition on the growth of M. kansasii. The eight patient isolates were also inoculated onto Lowenstein Jensen medium (Remel) and onto a variety of selective mycobacterial media containing nalidixic acid and other antimicrobial agents. All isolates showed some degree of inhibition on at least one of these selective media. PMID- 7559941 TI - In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods. AB - We evaluated test discriminatory power and DNA type alterations among methicillin resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality. PMID- 7559940 TI - PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital. AB - Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and not the birth canal is the major source of C. difficile acquisition by neonates in this maternity hospital setting. Furthermore, AP-PCR appears to be a fast and useful method for epidemiologically relevant typing of C. difficile isolates. PMID- 7559944 TI - Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe. AB - We have developed a rapid, pseudohomogeneous assay for the detection of PCR amplicons, based on the use of electrochemiluminescence generated from a Tris bipyridine ruthenium(II) label. PCR amplification of highly conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequences was performed with SK38 and SK39 primers, the latter of which was 5' biotinylated. Post-PCR reaction mixtures were combined with 10(12) copies of the SK19 probe-Tris-bipyridine ruthenium(II) conjugate, denatured by heating at 100 degrees C for 5 min, and hybridized at 55 degrees C for an additional 15 min. Hybridization to the biotinylated strand of the amplified DNA was determined by the addition of streptavidin-conjugated magnetic particles and analyzed by using an Origen-1 electrochemiluminescence analyzer. Our results demonstrated a sensitivity of fewer than five copies of HIV-1 (pre-PCR), by using either purified plasmid DNA containing one complete copy of the HIV-1 cDNA genome or lysed, proteinase K treated 8E5 cells as the starting material. In an evaluation of actual clinical specimens (peripheral blood monocytes from both healthy and HIV-1-infected children), the electrochemiluminescent detection assay correlated 100% with both our standard method (solution hybridization with a radiolabeled probe followed by polyacrylamide gel electrophoresis [PAGE] and autoradiography) and a commercial method (Roche Amplicor). The electrochemiluminescent method was substantially easier to perform than either the PAGE or microtiter plate assays and was considerable faster to perform than either of these alternative formats. PMID- 7559942 TI - Turnover of nonencapsulated Haemophilus influenzae in the nasopharynges of otitis prone children. AB - Restriction enzyme analysis of total genomic DNA was applied to study the epidemiology of nontypeable Haemophilus influenzae (NTHI) isolated from the nasopharynges of children with recurrent acute otitis media (AOM). The turnover of strains, as judged from genetic fingerprinting of a total of 213 H. influenzae isolates collected prospectively during a 2-year study period from 38 children under 3 years of age, was examined in relation to episodes of AOM as well as to courses of antibiotic treatment. The children were selected if they had had at least one episode of AOM before 1 year of age and if more than two nasopharyngeal isolates of H. influenzae were recovered. The 213 H. influenzae isolates (90% NTHI) recovered corresponded to 128 different DNA fingerprints. Fifty-eight percent of the fingerprints were observed only once, whereas 42% appeared on two or more occasions in isolates from the same individual or in close relatives, i.e., brothers and sisters. Sixty-seven percent of these strains had a minimum colonization period of 2 months or less. Intermittent nasopharyngeal colonization periods longer than 5 months could be demonstrated for 13% of the strains. The present data suggest that intermittent colonization is due to endogenous reinfections. Genetically identical NTHI strains from unrelated individuals were never identified. As expected from the observation of a relatively high proportion of persistent colonizations, no correlation was found between episodes of AOM and the acquisition of new strains of H. influenzae, nor was any direct relation between antimicrobial therapy and the elimination of nasopharyngeal colonization with a particular strain of H. influenzae observed. PMID- 7559943 TI - Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital. AB - Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid 1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefania and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade. PMID- 7559945 TI - Detection of Chlamydia trachomatis infection in urine samples from men and women by ligase chain reaction. AB - The suitability of urine specimens from women and men for the detection of Chlamydia trachomatis infection by a ligase chain reaction (LCR)-based assay with plasmid primers was examined with a group of patients attending a sexually transmitted disease clinic in Amsterdam, The Netherlands. Cervical specimens from 15 of 237 (6.3%) women tested positive for C. trachomatis by cell culture. Of the 25 (10.5%) female urine samples that tested positive by the plasmid-LCR assay, 13 were obtained from cervical culture-positive women. Nine of the 12 plasmid-LCR positive urine samples from cervical culture-negative women were confirmed to be positive by a second LCR assay with primers based on chromosomal DNA. Urethral specimens from 24 of 258 (9.3%) men were positive for C. trachomatis infection by cell culture. Of the 25 (9.7%) urine samples that tested positive by plasmid-LCR, 20 were from culture-positive men. All five of the LCR-positive urine samples from culture-negative men were confirmed to be positive by the LCR with chromosomal DNA primers. Relative to cell culture, testing by plasmid-LCR analysis of male urine samples had a sensitivity of 83.3% and a specificity of 97.9%; after resolution of discordant samples, these values were 86.2 and 100%, respectively. In the study with women, the sensitivities of plasmid-LCR analysis of cervical and urine specimens in comparison with cervical cell culture were 93.3 and 86.7%, respectively. After resolution of discrepant samples, the sensitivities of the plasmid-LCR test for cervical swabs and female urine samples were 96.3 and 92.6%, respectively. These results indicate that the plasmid-LCR based assay is a very reliable, sensitive, convenient test for the detection of C. trachomatis infection in female and male urine specimens. PMID- 7559946 TI - Investigation of sheep-associated malignant catarrhal fever virus infection in ruminants by PCR and competitive inhibition enzyme-linked immunosorbent assay. AB - Development of control measures for the gammaherpesviral disease of cattle known as sheep-associated malignant catarrhal fever (SA-MCF) has been hampered by a lack of accurate diagnostic tests either for the causative virus or for antibody against that virus. A recently developed competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) for the detection of antibody to malignant catarrhal fever (MCF) virus (MCFV) in ruminants based on a monoclonal antibody to a widely conserved epitope of MCFV (H. Li, D. T. Shen, D. P. Knowles, J. R. Gorham, and T. B. Crawford, J. Clin. Microbiol. 32:1674-1679, 1994) and a PCR assay based on previously reported primers (S. I. F. Baxter, I. Pow, A. Bridgen, and H. W. Reid, Arch. Virol. 132:145-159, 1993) were used to detect anti-MCFV antibody and SA-MCFV DNA in sheep and other ruminants. The PCR amplified a specific 238-bp SA-MCFV genomic DNA fragment from peripheral blood lymphocytes of adult sheep and other ruminants with clinical MCF. Of 144 samples from randomly selected healthy adult sheep, 143 (99%) were positive by PCR and 136 (94%) were positive by CI-ELISA. The agreement between the two assays exceeded 95%. Of nine samples collected from cattle and deer with clinical MCF of apparent sheep origin, seven were CI-ELISA positive and all 9 were PCR positive. Among 59 serum samples from presuckling lambs, none contained antibody detectable by CI-ELISA. After suckling, maternal anti-MCFV antibody was detectable for about 10 +/- 3 weeks. Although all colostrum and milk samples from infected ewes were strongly PCR positive, the appearance of detectable SA-MCFV DNA in lambs was correlated generally with antibody patterns, which suggests that the natural infection event in sheep may not occur during the perinatal period but occurs sometime later in life. PMID- 7559948 TI - Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts. AB - Thirty-nine patients with condylomas (12 women and 27 men) attending a dermatology clinic were tested for genital human papillomavirus (HPV) DNA and for seroprevalence to HPV type 6 (HPV6) L1 virus-like particles. The L1 consensus PCR system (with primers MY09 and MY11) was used to determine the presence and types of HPV in sample specimens. All 37 (100%) patients with sufficient DNA specimens were positive for HPV DNA, and 35 (94%) had HPV6 DNA detected at the wart site. Three patients (8%) had HPV11 detected at the wart site, and one patient had both HPV6 and -11 detected at the wart site. Thirteen additional HPV types were detected among the patients; the most frequent were HPV54 (8%) and HPV58 (8%). Baculovirus-expressed HPV6 L1 virus-like particles were used in enzyme-linked immunosorbent assays to determine seroprevalence among the patients with warts. Seronegativity was defined by a control group of 21 women who were consistently PCR negative for HPV DNA. Seroprevalence was also determined for reference groups that included cytologically normal women who had detectable DNA from either HPV6 or HPV16 and women with HPV16-associated cervical intraepithelial neoplasia. Among the asymptomatic women with HPV6, only 2 of 9 (22%) were seropositive, compared with 12 of 12 (100%) female patients with warts. A similar trend in increased HPV6 seropositivity with increased grade of disease was found with the HPV16 DNA-positive women, whose seroprevalence increased from 1 in 11 (9%) in cytologically normal women to 6 in 15 (40%) among women with cervical intraepithelial neoplasia 1 or 3. However, only 4 of 25 (16%) male patients were seropositive. No factors examined, such as age, sexual behavior, or a history of warts, were found to definitively account for the gender difference in seroresponse. PMID- 7559949 TI - Genetic and biochemical characterization of Citrobacter rodentium sp. nov. AB - An unusual bacterial pathogen of laboratory mice has been previously classified as an atypical biotype of Citrobacter freundii. Designated C. freundii biotype 4280, this bacterium is the etiologic agent of transmissible murine clonic hyperplasia. An eaeA gene has been shown to be present in this organism and to be necessary for virulence in laboratory mice. However, other biotypes of C. freundii lack DNA homology with the eaeA gene. Because of the recent reclassification in which five named species and three unnamed species, all previously considered C. freundii, were described, we determined the taxonomic status of C. freundii biotype 4280. With a battery of biochemical tests and DNA relatedness studies, three isolates of C. freundii biotype 4280 were shown to be members of an unnamed Citrobacter species, designated species 9. In total, six isolates of Citrobacter species 9, but none of the type strains of the other eight named species or of the two remaining unnamed species of Citrobacter, were shown to possess DNA homology with both the eaeA and the eaeB genes. Species 9 was named Citrobacter rodentium sp. nov. PMID- 7559947 TI - Inactivation of human immunodeficiency virus type 1 by the amine oxidase peroxidase system. AB - Human immunodeficiency virus type 1 (HIV-1) is rapidly inactivated by exposure to a naturally occurring antimicrobial system consisting of peroxidase, H2O2, and a halide. Among the potential sources of H2O2 is the amine oxidase system in which mono-, di-, and polyamines are oxidatively deaminated with the formation of H2O2. The polyamine spermine is present at exceptionally high concentrations in semen. We report here that spermine, spermidine, and, to a lesser degree, the synthetic polyamine 15-deoxyspergualin are viricidal to HIV-1 when combined with amine oxidase and myeloperoxidase. Antiviral activity required each component of the spermine-amine oxidase-peroxidase system and was inhibited by azide (a peroxidase inhibitor) and by catalase but not by superoxide dismutase. Heat treatment of catalase largely abolished its inhibitory effect. These findings implicate H2O2 formed by the amine oxidase system in the antiviral effect and raise the possibility that the polyamine-amine oxidase-peroxidase system influences the survival of HIV-1 in semen and in the vaginal canal. PMID- 7559951 TI - Molecular epidemiology of tuberculosis in Denmark in 1992. AB - The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark. PMID- 7559952 TI - Utility of recombinant flagellar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection. AB - The protozoan Trypanosoma cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America and of growing concern in the United States as the number of infected immigrants increases. There is currently no testing of U.S. blood products for T. cruzi infection, and the best tests available, although highly sensitive, are not of high enough specificity to be useful for widespread screening of the blood supply in this country. Among the parasite antigens detected by sera of infected humans and mice, those in the range of 24 to 26 kDa are particularly reactive. With an aim of developing a sensitive, specific, recombinant antigen-based serologic test for T. cruzi infection, we used two antibody reagents specific for these 24- to 26-kDa antigens to isolate cDNA clones from a T. cruzi expression library. One clone was found to encode a previously characterized T. cruzi antigen, a 24-kDa flagellar calcium-binding protein (FCaBP). Recombinant FCaBP was found to be a sensitive, specific reagent for distinguishing T. cruzi-infected individuals from uninfected persons, and it therefore could potentially be used for screening purposes, especially if combined with other recombinant T. cruzi antigens that have similarly high degrees of diagnostic sensitivity and specificity. PMID- 7559950 TI - PCR strategy for identification and differentiation of small pox and other orthopoxviruses. AB - Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species. PMID- 7559954 TI - Identification of some clinical strains of CDC coryneform group A-3 and A-4 bacteria as Cellulomonas species and proposal of Cellulomonas hominis sp. nov. for some group A-3 strains. AB - CDC coryneform group A-3 and A-4 bacteria were defined by Hollis and Weaver in 1981, but their taxonomic position is still unclear. By using biochemical and chemotaxonomical methods, four clinical strains belonging to CDC coryneform groups A-3 (n = 2) and A-4 (n = 2) were studied and could be assigned to the genus Cellulomonas, resulting in the first description of Cellulomonas strains isolated from clinical specimens. CDC coryneform group A-3 and A-4 strains were compared with the type strains of the seven species constituting the genus Cellulomonas at present as well as with the closely related species Oerskovia turbata, Oerskovia xanthineolytica, and Jonesia denitrificans, but their biochemical patterns were not compatible with the patterns of any of those species. Almost the entire sequences of the 16S rRNA genes of one representative strain of both CDC taxa were determined, and comparative sequence analysis confirmed the placement of the CDC coryneform group A-3 and A-4 strains studied in the Cellulomonas-Oerskovia subbranch of the actinomycetes. Both CDC taxa exhibited > 99% base pair homology within their 16S rDNAs. On the basis of phenotypic and molecular data, we formally propose a new species, Cellulomonas hominis sp. nov., for the CDC coryneform group A-3 bacteria examined. The type strain is DSM 9581. The precise taxonomic status of the CDC coryneform group A-4 strains studied remains to be established by quantitative DNA-DNA hybridizations. PMID- 7559953 TI - Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease. AB - A 24-kDa recombinant protein from Trypanosoma cruzi (rTc24) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) tests to identify treated chagasic patients considered parasitologically cured on the basis of persistently negative tests of hemocultures and lytic antibodies. Some of these patients were termed dissociated because their sera, although negative by the complement-mediated lysis test, were positive by conventional serology. The negative lysis test indicates the absence of active infection after specific treatment, but this assay requires live and infectious parasites and cannot be used easily in a laboratory routine. Here we tested rTc24 by ELISA and Western blotting as an alternative for the complement-mediated lysis test. For the group of patients with active infection despite the treatment (uncured patients), all the sera tested recognized rTc24 in both tests. For the dissociated patients, approximately 80% of the sera did not react with rTc24 in the ELISA or in Western blots, in agreement with the negative complement-mediated lysis tests. Thus, the 24-kDa T. cruzi recombinant antigen, when used for initial trials to evaluate cure of chagasic patients submitted to specific treatment, will allow the identification of most, but not all, cases. PMID- 7559955 TI - Detection kinetics for positive blood culture bottles by using the VITAL automated system. AB - The VITAL system principle is based on homogeneous fluorescence technology. During an 11-month period, a total of 19,706 blood cultures from adult patients hospitalized in various establishments of the Montpellier Teaching Hospital were collected in VITAL bottles, of which 1,939 were declared positive. Only 204 bottles (1.04%) were false positives. The 1,735 true-positive bottles were collected from 130 patients. The final visual control permitted the detection of 10 falsely negative bottles (0.05%), of which 5 contained clinically significant microorganisms from four patients. The kinetics of detection for all microorganisms showed that 66.6% were detected within 24 h, 83.1% within 48 h, 95.5% within 120 h, and 100% within 150 h. No clinical episode would have been missed had a 5-day protocol been used instead of a 7-day protocol. Among the positive bottles, 65.7% were detected by the SLOPE algorithm, 20.1% by the DELTA algorithm, and 14.2% by the THRESHOLD algorithm. This retrospective study of our results shows that a 5-day protocol is sufficient for the detection of septic episodes using the VITAL system. PMID- 7559956 TI - Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare. AB - The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex. PMID- 7559958 TI - Report of cases of and taxonomic considerations for large-colony-forming Lancefield group C streptococcal bacteremia. AB - Traditionally, group C streptococci include four species: Streptococcus equisimilis, S. zooepidemicus, S. equi, and S. dysgalactiae, the first three of which are group C beta-hemolytic streptococci (GCBHS). However, many of the beta hemolytic streptococci carrying Lancefield group C antigen isolated from clinical specimens are S. milleri. These organisms can be differentiated by colony size. We retrospectively collected data concerning large-colony-forming GCBHS bacteremia that occurred during a period of 8 years at the Massachusetts General Hospital. A total of 222 cases of beta-hemolytic streptococcal bacteremia were identified; data on the Lancefield grouping were available in 192 cases: 45 cases (23.6%) were group A, 96 cases (50%) were group B, 7 cases (3.6%) were group C (large colony forming), and 44 cases (22.9%) were group G. The medical records for cases of large-colony-forming GCBHS bacteremia were reviewed. In one case, the isolate was thought to be a contaminant; the other six cases are reported (five males and one female; mean age, 55 years). All patients had severe underlying conditions, and none had a history of exposure to animals. The clinical syndromes included two cases of cellulitis and one case each of endocarditis, myocardial infarction complicated by infection, pneumonia, and myofasciitis. The diagnoses for two patients with endovascular infections were delayed. Three of the six patients had fatal outcomes, and other two, after prolonged hospitalization, were transferred to a long-term rehabilitation center. We concluded that the severe outcomes reflect delay in diagnosis and treatment as well as the severity of the underlying diseases. The taxonomy of GCBHS is discussed. More reports differentiating large- and small-colony-forming GCBHS are needed. PMID- 7559957 TI - Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. AB - We wished to develop a cost-effective, rapid strategy to detect and identify Bartonella species in the clinical laboratory and to determine the prevalence of Bartonella infection in the Houston veteran population. Bartonella colonies were identified by colony morphology, Gram stain, RapID ANA, repetitive extragenic palindromic-PCR (REP-PCR) and whole-cell fatty acid (CFA) analysis, and these methods were compared for their usefulness. A new test order for "Rochalimaea culture" (the genus Bartonella was previously known as the genus Rochalimaea) was instituted, and in addition, all blood specimens submitted for fungal culture (obtained in an isolator tube) were processed for Bartonella culture. Over a 16 month period we isolated Bartonella henselae from only 0.4% (2 of 533) of total cultures but from 1% (2 of 204) of human immunodeficiency virus-positive patients. After sufficient growth, identification of the Bartonella isolates to the species level could be obtained in 2 days. The REP-PCR allowed discrimination of all known species, whereas CFA analysis distinguished all except B. henselae and Bartonella quintana. The RapID ANA results failed to differentiate between B. henselae and B. quintana, and results for other species differed by only one or two tests. Blood obtained from a kitten which had been introduced into the household of one patient 2 months before the onset of fever yielded a Bartonella strain which was shown to be different from the strain from the patient and distinct from other Bartonella species by a combination of REP-PCR, CFA, and growth characteristics. Subsequent analysis of the citrate synthase gene sequence showed only an 86% similarity with any of the other known Bartonella species, suggesting that this isolate represents a distinct, previously uncharacterized species of Bartonella. PMID- 7559962 TI - Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei. AB - The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells. PMID- 7559959 TI - Comparison of culture methods for monitoring Legionella species in hospital potable water systems and recommendations for standardization of such methods. AB - A lack of standardization of environmental monitoring techniques for Legionella spp. complicates the interpretation of results and comparisons of results from different institutions. A comparative assessment of techniques recommended by the Centers for Disease Control and Prevention, the Hygiene Institute (Graz, Austria), and our laboratory was performed. Variables investigated were sampling method (swabbing and collection of water samples [250 ml] before and after swabbing), method of concentration (none, filtration, and centrifugation), acid buffer treatment (no acid treatment, treatment for 3 min, and treatment for 15 min), and choice of medium (five formulations of buffered charcoal yeast extract agar with glycine, vancomycin, polymyxin B, anisomycin, or cycloheximide). Thirty three sites in seven hospital buildings were studied. Recovery by swab correlated with recovery from water after swabbing (P < 0.05). However, the quantity of Legionella spp. recovered from swab specimens (mean, 3.0 x 10(4) CFU per swab) was greater than that recovered from water (mean, 4.7 x 10(3) CFU/250 ml). Filtration resulted in recovery rates (mean, 5.2 x 10(3) CFU/250 ml) higher than those by centrifugation (mean, 2.3 x 10(3) CFU/250 ml). Three minutes of acid buffer treatment to reduce overgrowth by commensal flora did not improve selectivity or sensitivity for Legionella spp. if glycine-containing selective media were used. Fifteen minutes of acid buffer treatment reduced recovery compared with that after a 3-min treatment. All glycine-containing media tested effectively inhibited background flora, but one selective medium containing dyes, glycine, vancomycin, and polymyxin B (DGVP) resulted in the greatest quantitative recovery of Legionella pneumophila. Use of buffered charcoal yeast extract agar and the acid buffer treatment gave the greatest recovery of non-pneumophila species. A standardized protocol with an emphasis on the culturing of swab samples is presented. PMID- 7559963 TI - Microbiology of secondary bacterial infection in scabies lesions. AB - Aerobic and anaerobic bacteria were grown from specimens obtained from 30 children with secondarily infected scabies lesions. Aerobic or facultative bacteria only were present in 14 (47%) patients, anaerobic bacteria only were present in 6 (20%) patients, and a mixed anaerobic-aerobic flora was present in 10 (33%) patients. Fifty isolates were recovered (1.7 per specimen); 27 were aerobic or facultative bacteria and 23 were strict anaerobes. The predominant aerobic and facultative bacteria were Staphylococcus aureus (nine isolates), group A streptococci (five isolates), and Pseudomonas aeruginosa (three isolates). The predominant anaerobes were Peptostreptococcus sp. (nine isolates) and pigmented Prevotella and Porphyromonas spp. (four isolates). Single bacterial isolates were recovered from nine (30%) patients; five of these were S. aureus. Sixteen organisms isolated from 12 (40%) patients produced the enzyme beta lactamase. Organisms that resided in the mucous membranes close to or in contact with the lesions predominated in those infections. Enteric gram-negative rods were recovered in leg and trunk lesions. Group A streptococci and S. aureus predominated in finger and hand lesions. Bacteroides fragilis group and Clostridium sp. were isolated from leg lesions, and pigmented Prevotella sp. and Porphyromonas and Fusobacterium spp. were recovered from finger lesions. The polymicrobial etiology of secondarily infected scabies lesions in children and the association of bacterial flora with the anatomical sites of the lesions are demonstrated. PMID- 7559961 TI - Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization. AB - Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic. PMID- 7559960 TI - Quantitative PCR for human herpesviruses 6 and 7. AB - A quantitative PCR assay for the detection of human herpesvirus 6 (HHV-6) (variants A and B) and HHV-7 DNAs in clinical samples was developed. The assay uses a nonhomologous internal standard (IS) for each virus that is coamplified with the wild-type target sequence in the same vial and with the same pair of primers. This method allows for a correction of the variability of efficiency of the PCR technique. A standard curve is constructed for each experiment by coamplification of known quantities of the cloned HHV-6 or HHV-7 target templates with the respective IS. Absolute quantitation of the test samples is then achieved by determining the viral target/IS ratio of the hybridization signals of the amplification products and plotting this value against the standard curve. Using this assay, we quantitated the amount of HHV-6 or HHV-7 DNA in infected cell cultures and demonstrated an inhibitory effect of phosphonoformic acid on the replication of HHV-6 and HHV-7 in vitro. As the first clinical application of this procedure, we performed preliminary measurements of the loads of HHV-6 and HHV-7 in lymph nodes from patients with Hodgkin's disease and AIDS. Application of this quantitative PCR method should be helpful for elucidating the pathogenic roles of HHV-6 and HHV-7. PMID- 7559964 TI - Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR. AB - Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections. PMID- 7559967 TI - Molecular epidemiology of a fast-food restaurant-associated outbreak of Escherichia coli O157:H7 in Washington State. AB - We studied the molecular epidemiology of the recent fast-food restaurant chain associated Escherichia coli O157:H7 outbreak in Washington State. Genomic DNAs prepared from strains isolated from 433 patients were probed with radiolabelled Shiga-like toxin (SLT) I and SLT II genes and bacteriophage lambda DNA and were subsequently analyzed for their restriction fragment length polymorphism (RFLP) patterns. The SLT RFLP and lambda RFLP profiles of an E. coli O157:H7 strain isolated from the incriminated beef and prototype patient were compared with those of the patient isolates for determination of the concordance between patterns. Of the 377 patients with primary and secondary cases of infection epidemiologically linked to the outbreak, isolates from 367 (97.3%) of the patients displayed SLT RFLP and lambda RFLP profiles identical to those of the outbreak strains. Isolates from 10 of the 377 (2.6%) patients possessed SLT RFLP and lambda RFLP profiles different from those of the outbreak strains, and the patients from whom those isolates were obtained were subsequently characterized as having non-outbreak-related infections. The E. coli O157:H7 strains isolated from 31 of 44 (70.4%) patients who were epidemiologically excluded from the outbreak were linked to the outbreak by RFLP typing. Our results indicate that SLT RFLP and lambda RFLP analyses are stable and sensitive methods, and when they are used in conjunction with an epidemiological investigation they could result in an earlier recognition of outbreaks and their sources, hence prompting measures to prevent the continued transmission of E. coli O157:H7. PMID- 7559966 TI - Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes. AB - The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results. PMID- 7559968 TI - Antigenic differences between a field isolate and vaccine strains of bovine viral diarrhea virus. AB - Antigenic diversity among the bovine viral diarrhea virus (BVDV) cytopathic strains 87-2552 (field isolate) and NADL and Singer (prototype strains) was demonstrated with monoclonal antibodies (MAbs) in enzyme-linked immunosorbent, immunofluorescence, virus neutralization, and immunoprecipitation assays. Two MAbs against BVDV strain 87-2552, designated D11 and B7, strongly neutralized this field strain and were specific for the 48-kDa glycoprotein of the virus. These two MAbs have different subisotypes, immunoglobulin G1 for D11 and immunoglobulin G3 for B7. MAbs against BVDV strains 87-2552 and NADL were specific for their respective strains in virus neutralization assays. The results indicated significant antigenic differences between BVDV strain 87-2552 and the NADL and Singer strains. PMID- 7559965 TI - Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae. AB - Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7. PMID- 7559969 TI - Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens. AB - The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique. PMID- 7559971 TI - Genetic diversity in clinical isolates of the dimorphic fungus Blastomyces dermatitidis detected by a PCR-based random amplified polymorphic DNA assay. AB - Blastomyces dermatitidis is a dimorphic fungus causing localized or systemic infection in areas where the organism is endemic in the central and southeastern United States. In this study, 19 independent isolates of B. dermatitidis from Little Rock, Ark., were grouped into three classes based on restriction fragment length polymorphism patterns in mitochondrial DNA with a heterologous probe from Histoplasma capsulatum. One large class of 15 isolates and two smaller classes (classes 2 and 3), each consisting of two isolates, were observed in BglII digests. Strain-specific arrays of PCR-amplified DNA products were obtained with arbitrarily selected primers (18 to 29 nucleotides long; G+C contents, 33 to 56%). In the large class 1 group, 13 isolates could be differentiated by the random amplified polymorphic DNA (RAPD) method with various primers. The two remaining class 1 isolates were obtained from the same patients and produced identical RAPD arrays. Dissimilar RAPD patterns were obtained from the smaller class 2 group but not from the class 3 isolates. Significant genetic diversity in clinical isolates of B. dermatitidis was observed; this may underscore a similar environmental diversification. Further application of the typing techniques may provide significant insight into the epidemiology of blastomycosis and aid in the assessment of specific virulence phenotypes. PMID- 7559970 TI - Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis. AB - The diagnosis of brucellosis in cattle and small ruminants requires the use of more than one serological test. The complement fixation test (CFT), the rose bengal test (RBT), and the serum agglutination test (SAT) are among the most useful tests for routine diagnosis. The microagglutination test (MAT) was developed as a simpler and more efficient test than the SAT. The relative efficacy of this test compared with that of the SAT was evaluated by using brucella-free sheep and goats prior to and after vaccination treatment. The specificities of the MAT and the SAT were 100%. Of the ewes and goats with a vaccination history, one ewe, expectedly a negative responder, had reactions in the MAT, the complement fixation test, and the rose bengal test but not in the SAT, suggesting a lower sensitivity of the SAT in this case. The calculated sensitivities of the MAT and the SAT were 93.9%. The agreement between MAT and SAT results from nonresponders was examined by using sera from unvaccinated lambs and kids (95.2% agreement), unvaccinated ewes and goats (84.4%), and ewes and goats with a vaccination history (43.9%). For the latter group higher levels of agglutination units were observed by the MAT than by the SAT in 51.5% of the samples. In testing sera from positive reactors after vaccination neither method was superior (MAT values were greater than SAT values for 23.5% of the samples, and MAT values were less than SAT values for 21.9% of the samples). Comparison of the methods on the individual sample level revealed a significant correlation between the MAT and the SAT (r = 0.96 +/- 0.005; P < 0.001). Since the MAT is simpler to perform than the SAT and can potentially be automated, the inclusion of the MAT as a supplementary test in brucellosis control programs is recommended. PMID- 7559972 TI - Comparison of preservation media for storage of stool samples. AB - Transportation of clinical samples and long-term recoverability of pathogens are critical to epidemiological studies, particularly when conditions do not permit immediate processing. This study confirms that Cary-Blair medium (CB) is suitable for the preservation of Salmonella and Shigella isolates for more than 2 weeks at 25, 4, or -70 degrees C. Campylobacter jejuni was not recovered after 2 days of storage in CB at 25 degrees C when an inoculum of 12 x 10(8) cells per ml was used. Lower temperatures supported the recovery of this organism for 6 days. When individual pathogens were preserved with stools in CB and incubated at 25, 4, or 70 degrees C, the Salmonella and Shigella concentrations dropped from 12 x 10(8) cells to 1 x 10(3) or 1 x 10(4) cells per ml within 2 days and then remained stable for the rest of the observation period (15 days). C. jejuni survived preservation with stools for 5 to 9 days. The addition of blood and glycerol to CB improved the recoverability of all enteropathogens, particularly C. jejuni, which was consistently detected for 7 to 9 days at the different preservation temperatures used. When trypticase soy broth-glycerol (freezing medium), with or without blood, was used, there was little or no decrease in the Salmonella and Shigella concentrations during 2 weeks of preservation with stools at -70 degrees C. C. jejuni demonstrated a relatively sustained high concentration in Trypticase soy broth-glycerol with 5% blood. The use of defibrinated, laked sheep blood as a long-term freezing medium supported the recovery of low concentrations of Salmonella and Shigella spp. (10(2) to 10(3) cells per ml) for more than 14 weeks. Recovery of C. jejuni was consistent for 7 weeks when an initial concentration of 10(6) cells per ml present in stools. Laked blood provided a simple, sterile, and inexpensive medium for the preservation of individual isolates and clinical samples. PMID- 7559974 TI - Rapid detection of methicillin resistance in coagulase-negative staphylococci by commercially available fluorescence test. AB - A commercially available fluorescence test (Crystal MRSA ID System; BBL) for the rapid detection of methicillin resistance in Staphylococcus aureus was evaluated for the detection of methicillin resistance in coagulase-negative staphylococci. The assay was compared with the agar dilution method and mecA detection by PCR. Provided that one uses an inoculum equivalent to a no. 2 McFarland standard instead of a no. 0.5 McFarland standard, which is used for S. aureus, the Crystal MRSA ID System detects methicillin resistance among coagulase-negative staphylococci with sensitivity, specificity, and positive and negative predictive values of 98.5, 95.4, 96.3, and 98.1%, respectively. PMID- 7559973 TI - Detection of the Staphylococcal mecA gene by chemiluminescent DNA hybridization. AB - A new solution-phase DNA hybridization capture assay for the rapid detection of the mecA gene in clinical isolates of Staphylococcus was compared with multiplex PCR and disk diffusion methods. The assay uses a DNA capture probe immobilized on paramagnetic particles and a second DNA probe labeled with an acridinium ester. Bacteria from 24-h cultures are lysed, and the lysates are hybridized with the DNA probes. After magnetic separation to remove unhybridized labeled probe, the mecA gene is detected by the chemiluminescence of the hybridized probe. Four hundred consecutive staphylococcal isolates were assayed, including 147 S. aureus and 253 coagulase-negative Staphylococcus isolates. Among the S. aureus isolates, 14 of 147 were MecA+ by both the hybridization capture assay and PCR; 133 of 147 were MecA negative by both assays (positive and negative predictive values, 100%). Comparison of disk diffusion results with those obtained by genotypic methods indicated that 14 of 147 S. aureus isolates judged to be resistant were positive by both methods; 119 of 147 were Oxs and negative by both genotypic methods (positive and negative predictive values, 50 and 100%, respectively). The remaining 14 S. aureus isolates were MecA- Oxr; among these, 13 were beta lactamase hyperproducers. For coagulase-negative staphylococci, 130 of 253 were MecA+ by the hybridization capture assay; 129 of 130 of these isolates were positive by PCR (positive and negative predictive values, 99.2 and 100%, respectively). Comparison with the disk diffusion assay showed that 128 of the coagulase-negative MecA+ isolates were Oxr; 111 of 253 were MecA- and Oxs (positive and negative predictive values, 90.8 and 99.1%, respectively). Thirteen coagulase-negative isolates were MecA-Oxr; among these, three were beta-lactamase hyperproducers. Comparison of the hybridization capture assay results with PCR indicates that the DNA hybridization assay is a sensitive and specific test for the detection of the mecA gene in clinical isolates of Staphylococcus. PMID- 7559975 TI - Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal. AB - Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139. PMID- 7559977 TI - Auxotrophy of Burkholderia (Pseudomonas) cepacia from cystic fibrosis patients. AB - The nutritional status of 89 isolates of Burkholderia cepacia from 81 cystic fibrosis (CF) patients was evaluated. Forty of the isolates, from 38 patients, were not able to grow in a minimal medium containing glucose and mineral salts only and were thus auxotrophs. In contrast, all of 29 isolates from non-CF (clinical and environmental) sources were prototrophic. Addition of a pool of amino acids to the minimal medium was sufficient to promote growth of all tested CF auxotrophic isolates. Indeed, phenylalanine, tyrosine, cysteine, methionine, and histidine alone or in combination were required for growth by the majority of the nutritionally deficient B. cepacia isolates. Furthermore, extracts of sputum from CF patients, when added to minimal medium, promoted growth of 29 auxotrophic B. cepacia isolates regardless of their amino acid requirements. Finally, auxotrophic and prototrophic isolates from the same patient exhibited a conserved genotype, as determined by macrorestriction analysis of chromosomal DNA. These results suggest that the auxotrophic mutants are selected from the prototrophic population and maintained by the nutritionally rich environment of the CF airways. PMID- 7559976 TI - Detection of Escherichia coli O157:H7 by multiplex PCR. AB - In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60 MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157. PMID- 7559978 TI - Molecular typing of Stenotrophomonas (Xanthomonas) maltophilia by DNA macrorestriction analysis and random amplified polymorphic DNA analysis. AB - Stenotrophomonas (Xanthomonas) maltophilia is a multidrug-resistant, nosocomial pathogen for which optimal typing methods in epidemiologic investigations of nosocomial outbreaks have not been defined. We compared DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) with random amplified polymorphic DNA (RAPD) analysis by arbitrarily primed PCR for molecular typing of 109 multidrug-resistant strains of S. maltophilia from multiple outbreaks at our institution over a 10-month period in 1993. PFGE after digestion with restriction endonuclease DraI revealed 62 unique DNA restriction profiles among the 109 strains, with 23, 11, 6, 6, and 3 strains having concordant profiles in each of five types. There were four concordant profiles among 8 strains (2 strains with each profile), while unique profiles were present in each of the remaining 52 strains. Further RAPD analysis with a decanucleotide primer showed the same number of distinct strain types as PFGE but more subtype diversity within each clonal type. We concluded that DNA macrorestriction analysis and RAPD analysis are sufficiently discriminatory and useful for differentiation of S. maltophilia strains in epidemiologic investigations of nosocomial outbreaks. However, RAPD analysis by arbitrarily primed PCR is faster and less laborious method of molecular typing. PMID- 7559979 TI - Sorbitol-negative phenotype among enterohemorrhagic Escherichia coli strains of different serotypes and from different sources. AB - Enterohemorrhagic Escherichia coli (EHEC) strains detected with DNA probes (for virulence plasmid and Shiga-like toxins) from subjects with hemolytic-uremic syndrome (n = 19) or diarrhea (n = 41) or asymptomatic carriers (n = 29) were examined for sorbitol fermentability, as were enterotoxigenic (n = 40), enteropathogenic (n = 40), and enteroinvasive (n = 40) E. coli and urinary tract infection (n = 40) strains and normal flora E. coli strains (n = 40). Sorbitol negativity was common only in EHEC, particularly among strains from severe clinical infections. All 19 EHEC strains from patients with hemolytic-uremic syndrome, irrespective of O:H serotype or Shiga-like toxin genotype, were sorbitol negative. PMID- 7559980 TI - Tear fluid of hepatitis C virus carriers could be infectious. AB - For up to 20 to 40% of patients chronically infected with hepatitis C virus (HCV), the mode of transmission is still unknown. We demonstrate that tear fluid contains HCV RNA-carrying material with the properties of infectious virus and conclude that smear infection with tear fluid may play a role in HCV transmission. PMID- 7559981 TI - Evaluation of a simplified HEp-2 cell adherence assay for Escherichia coli isolated from south Indian children with acute diarrhea and controls. AB - A simplified HEp-2 cell adhesion assay was performed with stored Escherichia coli isolates from 761 children with acute diarrhea and 531 matched controls, and the results were evaluated by means of fluorescent actin staining and hybridization with DNA probes. The prevalence rates of localized adherence and aggregative adherence were significantly higher for patients (9.9 and 7.6%, respectively) than for controls (3.7 and 3.9%, respectively). PMID- 7559982 TI - Detection of anti-toxoplasma immunoglobulin A antibodies by Platelia-Toxo IgA directed against P30 and by IMx Toxo IgA for diagnosis of acquired and congenital toxoplasmosis. AB - Platelia-Toxo IgA and IMx Toxo IgA assays were used with 260 serum samples, of which 93 were from seroconverted patients, 58 were from 21 congenitally infected children, and 109 were from uninfected patients, to detect anti-P30 immunoglobulin A antibodies. Because of its enhanced sensitivity, Platelia-Toxo IgA is more efficient in diagnosing acute or congenital toxoplasmosis. IMx Toxo IgA must not be used to diagnose congenital toxoplasmosis. PMID- 7559983 TI - Molecular epidemiology of an outbreak of meningococcal disease in a university community. AB - Over a 2-month period, five cases of serogroup C meningococcal disease occurred in Iowa City, Iowa. Two patients were unacquainted university students who had independently visited another university with endemic meningococcal disease. Isolates from these patients had DNA fingerprints identical to those of the isolates responsible for infections on the other campus. Three cases for which the patients' isolates had a different DNA fingerprint were linked to visiting a local tavern. To disrupt the outbreak, the University of Iowa offered free meningococcal vaccine to all students. This report demonstrates that outbreaks of meningococcal disease may be due to more than one circulating strain and illustrates the utility of pulsed-field gel electrophoresis in defining the molecular epidemiology of meningococcal infections. PMID- 7559984 TI - Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis. AB - Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species. PMID- 7559985 TI - Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients. AB - Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency. PMID- 7559986 TI - Agglutination of Streptococcus suis by sialic acid-binding lectins. AB - The 35 Streptococcus suis capsular-type reference strains as well as 45 field strains of type 2 were tested with sialic acid-binding lectins from Sambucus nigra (SNA I), Triticum vulgaris, Maackia amurensis, Homarus americanus, and Limax flavus. Only types 1, 1/2, 2, 14, 15, and 16 agglutinated with SNA I and/or the T. vulgaris lectin. All field strains agglutinated only with SNA I. Reaction with SNA I was probably due to the sialic acid moiety since it disappeared after sialidase treatment. These results confirm the presence of sialic acid in S. suis with the possible terminal sequence N-acetylneuraminic acid-alpha(2,6)GalNAc. PMID- 7559987 TI - Symptomatic Shigella sonnei urinary tract infection. AB - The clinical course for a patient with symptomatic urinary tract infection due to Shigella sonnei is described. The role of Shigella spp. as urinary pathogens is reviewed. PMID- 7559990 TI - Comparison of ViraPap, southern hybridization, and polymerase chain reaction methods for human papillomavirus identification in an epidemiological investigation of cervical cancer. PMID- 7559989 TI - Evaluation of Albicans ID plates. PMID- 7559991 TI - Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients. PMID- 7559988 TI - Genotypic analysis of pseudoepidemic due to contamination of Hanks' balanced salt solution with Candida parapsilosis. AB - A cluster of isolates of Candida parapsilosis recovered from clinical specimens was demonstrated, by both classical and molecular epidemiological techniques, to have resulted from contamination in the laboratory. The source of the pseudoepidemic was a repeatedly utilized contaminated container of Hanks' balanced salt solution used in specimen processing. The patterns of restriction fragment length polymorphisms of DNA extracted from the clinical and environmental isolates were identical to each other but composed a newly identified unique C. parapsilosis DNA type. PMID- 7559992 TI - Survey of rotavirus G and P types associated with human gastroenteritis in Sao Paulo, Brazil, from 1986 to 1992. PMID- 7559993 TI - Weakly-coupled models for motor enzyme function. AB - In strongly-coupled models for motor enzyme function, such as the original Huxley (1957) model for muscle, ATP binding and subsequent hydrolysis are required for the detachment and reattachment of every force-producing cross-bridge. In weakly coupled models, cross-bridges can be 'mechanically detached' without ATP binding when they have been pushed far beyond their free energy minimum and have accumulated so much strain that the attached state is less stable than the detached state. Weakly-coupled models assume that these mechanically detached cross-bridges can rejoin the pool of detached molecules that can reattach as force-producing cross-bridges, without going through an ATP hydrolysis cycle. This paper bases this assumption on a thermodynamically rigorous model for interaction between a motor enzyme molecule and binding sites on a cytoskeletal protein filament, equivalent to other examples of ligand binding interactions. It attempts to identify more clearly the features that must be added to the idea of ligand binding equilibrium to simulate a weakly-coupled motor enzyme model. Models that assume a vectorial conformational change and a longitudinal series elastic element appear to be incompatible with the assumptions of weakly-coupled cross-bridge models. A stochastic computational method has been used to examine the properties of these models. The computations have examined the behaviour of a model containing a four-state ATPase cycle, but the model is computationally a nine-state model because a force-generating attached state is allowed to equilibrate with different detached states at negative and at positive distortions, and because three adjacent sites are considered as possible attachment sites for each of the two attached states of the ATPase cycle. PMID- 7559995 TI - Differentiation and growth of muscle in the fish Sparus aurata (L): II. Hyperplastic and hypertrophic growth of lateral muscle from hatching to adult. AB - Post-hatching growth of lateral muscle in a teleost fish, Sparus aurata (L) was studied morphometrically to identify and quantify muscle fibre hyperplasia and hypertrophy, and by in vivo nuclear labelling with 5-bromo-deoxyuridine to identify areas of myoblast proliferation. Muscle fibre types were identified principally by myosin ATPase histochemistry and immunostaining, and labelled nuclei were identified at light and electronmicroscope level by immunostaining with a specific monoclonal antibody. Hyperplastic growth was slow at hatching, but then increased to a maximum at the mid-point of larval life. Larval hyperplastic growth occurred by apposition of new fibres along proliferation zones, principally just under the lateral line and in the apical regions of the myotome, but also just under the superficial monolayer at intermediate positions. The first of these zones gave rise to slow and pink muscle fibres, in a process which continued through into postlarval life. The other zones added new fibres to the fast-white muscle layer in a process which was exhausted by the end of larval life. Post-larvally, between 60 and 90 days posthatching, a new hyperplastic process started in the fast-white muscle as nuclei proliferated and new muscle fibres were formed throughout the whole layer. This process resulted in a several fold increase in the number of fast-white fibres over a few weeks, and then waned to very low levels in juveniles. Hyperplasia by apposition continued for some time postlarvally on the deep surface of the superficial monolayer, but at this stage gave rise to slow fibres only. Hypertrophic growth occurred at all ages, but was the dominant mechanism of muscle growth only in the juvenile and adult stages. Mechanisms giving rise to these different growth processes in fish muscle are discussed, and compared with muscle development in higher vertebrates. PMID- 7559994 TI - Differentiation and growth of muscle in the fish Sparus aurata (L): I. Myosin expression and organization of fibre types in lateral muscle from hatching to adult. AB - Post-hatching development of lateral muscle in a teleost fish, Sparus aurata (L) was examined. At hatching only two fibre types were present, several layers of mitochondria-poor, myofibril-rich deep muscle fibres surrounded the notochord and were covered by a superficial monolayer of mitochondria-rich, myofibril-poor A third ultrastructurally distinct fibre type first appeared as one or two fibres located just under the lateral line at 6 days post-hatching. This type, which gradually increased in number during larval life, contained a slow isoform of myosin, identified by mATPase staining and immunostaining with myosin isoform specific antibodies. Deep muscle fibres--the presumptive fast-white type- contained a fast myosin, and superficial monolayer fibres an isoform similar but not identical to that in adult pink muscle fibres. The only fibres present during larval life which showed a clear change in myosin expression were the superficial monolayer fibres, which gradually transformed into the slow type post-larvally. Pink muscle fibres first appeared near the end of larval life. Both slow and pink muscle fibres remained concentrated around the horizontal septum under the lateral line during larval life, expanding outwards towards the apices of the myotomes only after metamorphosis. Between 60 and 90 days very small diameter fibres with a distinct mATPase profile appeared scattered throughout the deep, fast-white muscle layer, giving it a 'mosaic' appearance, which persisted into adult life. A marked expansion in the slow muscle layer began at the same time, partly by transformation of superficial monolayer fibres, but mainly by addition of new fibres both on the deep surface of the superficial monolayer and close to the lateral line. The order of appearance of these fibre types, their myosin composition, and the significance of the superficial monolayer layer are discussed and compared to muscle fibre type development in higher vertebrates. PMID- 7559996 TI - Isomyosin patterns of single type IIB, IID and IIA fibres from rabbit skeletal muscle. AB - The present study demonstrates for the first time isomyosin patterns of the three fast-twitch fibre types IIB, IID/X, and IIA. Single muscle fibres were dissected from freeze-dried fibre bundles of rabbit adductor magnus, extensor digitorum longus, and psoas muscles. Pure fibre types, expressing only one myosin heavy chain isoform (MHCIIb, MHCIId/x, MHCIIa), were delineated by electrophoresis of fibre fragments under denaturing conditions. Pieces of the same fibres were then subjected to electrophoresis under non-denaturing conditions. A three-band pattern of fast isomyosins, representing the LC3f homodimer (FM1), the LC1f/LC3f heterodimer (FM2), and the LC1f homodimer (FM3), was detected in each of the three pure fibre types. Therefore, three isomyosins, different in their light chain complement, coexist in each pure fibre. The relative mobilities of the three bands, migrating in the order FM1 > FM2 > FM3, were the same in the three fibre types. The absolute electrophoretic mobilities of the MHCIIb-, MHCIId- and MHCIIa-based isomyosin triplets differed in the order MHCIIb triplets > MHCIId triplets > MHCIIa triplets. The proportions of FM1, FM2, and FM3 varied in type IIB, IID, and IIA fibres. FM2 was the dominant isomyosin in all three fibre types, but fibre type-related differences existed in the FM1 to FM3 ratio. This ratio was lowest in IIA fibres and highest in IIB fibres which agrees with our previous observations that the LC3f/(LC1f + LC3f) fraction is lowest in type IIA and highest in type IIB fibres. PMID- 7559999 TI - Two mechanically distinct types of fast twitch muscle fibres of the frog and their temperature sensitivity, as detected by sinusoidal analysis. AB - The mechanical responses to sinusoidal oscillations were recorded from tetanically contracting fast twitch fibres from the anterior tibialis muscle of the frog, Rana japonica. Two distinct fibre types were recognized. One type of fibres (tentatively referred to as 'worker') were similar to skinned rabbit skeletal muscle fibres in that three exponential processes (each represents a single exponential viscoelastic decay) were resolved. The second fastest process (process b) had a negative polarity and caused the fibres to produce net work during oscillations in a range of frequencies. In the other type of fibres (tentatively referred to as 'idler'), this negative process was much smaller, and no net work was produced at any frequency. No intermediate fibre has been found so far. In the 'worker' type of fibres, the tension response to oscillation had a modest amount of harmonic components at frequencies just above the range for work production. In the 'idler' type, the corresponding harmonic components were much greater. In spite of these large apparent differences, the two types of fibres showed similar temperature sensitivity, raising a possibility that the basic contractile mechanisms and their rate constants are common to both types of fibres. In the 'idler' type, the rate constant for the fastest process (process c) is possibly distributed over a wide range, thus masking the work-producing process (process b). PMID- 7559997 TI - Cloning and in vivo expression of the pig MyoD gene. AB - The complete sequence of the pig MyoD gene has been determined through the isolation and characterization of a 17 kb genomic clone. The deduced amino acid sequence shows relative conservation of about 90% with the MyoD sequences determined in other mammalian species, though there are several non-conservative changes scattered throughout the C-terminus. In situ hybridization on pig embryos (20-30 days post coitum) showed that MyoD expression was confined to myotomes and skeletal muscle masses. PMID- 7560000 TI - Characterization of ultrastructural and contractile activation properties of crustacean (Cherax destructor) muscle fibres during claw regeneration and moulting. AB - Long-(SL > 6 microns) and short-sarcomere (SL < 4 microns) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca2+ or Sr2+ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca2+ or Sr2+. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism(s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca2+ and Sr2+ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominantly of fibres with short-sarcomeres. PMID- 7559998 TI - Simple modelling of linear and nonlinear mechanical responses to sinusoidal oscillations during muscle contraction. AB - The mechanical responses of muscle to sinusoidal oscillations were analysed using a contraction model based on Huxley's 1957 model. To reproduce the three well documented exponential processes resolved in the course of sinusoidal analysis, a few modifications had to be made on the original Huxley's model: (1) Each myosin cross-bridge should support substantial force at its position where the rate of detachment (g) abruptly changes. (2) Each cross-bridge should have the capability of stress-relaxation while attached to actin. This stress-relaxation should occur with a rate constant greater than the overall turnover rate of cross-bridges. After these modifications, the model reproduces not only the three exponential processes, but also the nonlinearity in the mechanical response, which is derived from the asymmetrical dependence of the rate constants on the cross-bridge strain. The present study provides a simplest model which reproduces the linear and nonlinear responses to sinusoidal oscillations, and help relate the exponential processes to the underlying attachment/detachment steps in the cross bridge cycle. PMID- 7560001 TI - The effect of temperature on contractile activation of intact and chemically skinned 'catch' muscle fibre bundles of Mytilus edulis. AB - The effect of temperature (5-35 degrees C) on maximum force production was examined in intact and chemically skinned muscle fibre bundles (10-25 fibres) from the anterior byssus retractor muscle of Mytilus edulis. In intact fibre bundles, 10 microM acetylcholine induced a tonic contraction which had a magnitude of 65.4 +/- 4.0 N cm-2 (n = 30) at 23 degrees C. Activation by caffeine (20 mM) produced a force response which was 157.1 +/- 7.9% (n = 16) of the acetylcholine response at 23 degrees C and acetylcholine and caffeine together produced force which was not significantly different from the response to caffeine alone. At 5 degrees C the acetylcholine and caffeine responses were decreased by 9.6 +/- 3.4% (n = 6) and 14.6 +/- 2.8% (n = 8) compared with the respective responses at 23 degrees C. However, there was no significant reduction of the response induced by the combined action of acetylcholine and caffeine when the temperature was decreased from 23 degrees C to 5 degrees C. The 20-80% of peak force activation time increased by about one order of magnitude for all acetylcholine, caffeine and combined acetylcholine-caffeine-induced responses when the temperature was decreased from 23-5 degrees C. Repeated exposure of the intact preparation to caffeine caused a marked decrease in the caffeine-induced response (complete abolition of force after the third exposure to caffeine), but the response to caffeine could be fully restored following one acetylcholine induced activation. The maximum Ca(2+)-activated force after skinning the preparation with saponin was not significantly different from the caffeine or combined acetylcholine-caffeine-induced responses before skinning. In the saponin skinned fibre preparation a drop in temperature from 23 degrees C to 15 degrees C or 5 degrees C decreased the maximum Ca(2+)-activated force by 13.2 +/- 1.4% (n = 8) and 41.4 +/- 3.1% (n = 5) respectively. The activation time between 20-80% of the peak Ca(2+)-activated force increased at 15 degrees C and 5 degrees C by a factor of 1.5 +/- 0.1 (n = 5) and 6.8 +/- 1.1 (n = 5) respectively when compared to corresponding values at 23 degrees C. The relaxation half-time decreased by a factor of 1.7 +/- 0.2 (n = 5) and 3.0 +/- 0.2 (n = 5) at 15 degrees C and 5 degrees C respectively compared with that at 23 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7560002 TI - Stereological methods based on point counting and unbiased counting frames for two-dimensional measurements in muscles: comparison with manual and image analysis methods. AB - Stereological methods, using the principle of point counting and unbiased counting frames, for the estimation of muscle area, total fibre area, number of muscle fibres and mean fibre area are described in detail. Their practical application is demonstrated on cross-sections of the rat soleus muscles. It is shown that the efficiency of these methods is high and their results are comparable with those achieved by the conventional manual and image analysis methods. The main advantages of two-dimensional stereological methods in muscle morphometry are pointed out: measurements are made directly on specimens under the microscope and in the simplest implementation do not require sophisticated and expensive technical equipment. Furthermore, unbiased results are obtained, no segmentation and edge effect problems arise and the quantity of work invested in stereological estimation is reasonable. Based upon the study of the efficiency of used stereological methods, a suitable test system for muscle morphometry is proposed. PMID- 7560006 TI - The annual meeting on Muscle Contraction and Cell Motility, Osaka, Japan, 5-7 January 1995. PMID- 7560007 TI - In vitro cytokine, sCD23 and IgG secretion in multiple sclerosis. AB - Synthesis of IgG by peripheral blood mononuclear cells from patients with multiple sclerosis (MS) and with other neurological diseases and from healthy controls was induced by Pokeweed mitogen (PWM) in short-term cultures. As expected, MS patients produced more immunoglobulin (Ig) G and had a higher percentage of 'high responders' to PWM stimulation as compared to controls. Interleukin (IL)-4 was undetectable in all samples. IL-6 and tumor necrosis factor (TNF)-alpha synthesis was induced by PWM stimulation in all groups, but MS patients showed the most significant increase of both cytokines. Interestingly, only MS patients showed a significant increase of the soluble form of CD23 receptor (sCD23). Moreover, only sCD23 levels correlated with in vitro IgG production in MS patients. The levels of IL-6, TNF-alpha, sCD23 were greater in high responders compared to low responders in all groups. The mean value of each molecule, however, did not differ significantly among overall groups. A highly significant difference was reported for sCD23 in MS patients. We suggest that sCD23, also known as B cell growth factor, may play a role in the well-documented phenomenon of in vitro IgG hypersynthesis in MS patients, adding support to the concept of B cell up-regulation in the peripheral blood of these patients. PMID- 7560004 TI - Alternate disposition of tetrads in peripheral couplings of skeletal muscle. AB - The sarcoplasmic reticulum forms junctions with the surface membrane (peripheral couplings), which are structurally and functionally equivalent to the junctions between sarcoplasmic reticulum and transverse tubules (triads and dyads). Feet (ryanodine receptors, or sarcoplasmic reticulum calcium release channels) are disposed in arrays in the junctional sarcoplasmic reticulum membrane. Tetrads (groups of four dihydropyridine receptors, each called a unit) are disposed in ordered arrays in junctional domains of transverse tubules and surface membrane. We measured three parameters of tetrad arrays in peripheral couplings from three different species: (1) the centre-to-centre distances between tetrads (intertetrad spacing); (2) the angle between lines joining tetrad units and those joining the centres of tetrads (skew angle); (3) the centre-to-centre distance between tetrad units (intratetrad spacing). These measurements are compared with those predicted from models of feet and tetrad arrays. Intratetrad spacings and skew angles are consistent with an interaction of tetrads with alternate feet and with a location of tetrad units over feet subunits. The slightly larger size of the intratetrad spacing relative to the distance between feet subunits indicates that tetrads may be larger than feet, despite the fact that the molecular weight of DHPRs is less than that of feet subunits. This is offered as a possible explanation for the association of tetrads with alternate feet. Arrays of tetrads tend to be incomplete in images from freeze-fractures, due to lack of some of the units composing the tetrads.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560003 TI - Evaluation of freeze substitution in rabbit skeletal muscle. Comparison of electron microscopy to X-ray diffraction. AB - Rabbit psoas muscle fibres, relaxed and in rigor, have been freeze substituted for electron microscopy. Fourier transforms and average density maps of micrographs of transverse sections have been obtained and compared to X-ray diffraction data. The Fourier amplitudes from rigor and relaxed muscle are comparable to equatorial data from X-ray diffraction of muscle if there is more disorder in the electron micrographs which can be described by a 'temperature' factor. The phases of reflections out to the 3,2 have been determined; those reflections at the same radius and therefore not separable in the X-ray patterns, such as the 2,1 and the 1,2, are separated in the transforms of sections through the A band. In transforms from both rigor and relaxed muscle they have the same phase. In rigor muscle they have different amplitudes. All the phases are positive or negative showing that the lattice is centrosymmetric at the resolution obtained. The phases obtained generally support those suggested by model building studies using X-ray diffraction data. In rigor muscle, areas where the cross-bridges are regularly attached are clearly seen in thin transverse sections. A handedness to this structure is indicated by a lack of mirror symmetry, in both the Fourier transform of thick sections, and in the averaged density map. This correlates well with the arrangement where the myosin head is bound as in the acto-S1 structure but only to actin monomers within a limited azimuthal range. PMID- 7560008 TI - Interferon beta-1b reduces interferon gamma-induced antigen-presenting capacity of human glial and B cells. AB - Interferon (IFN) beta-1b has been shown to alter the course of multiple sclerosis and to inhibit MHC class II expression, but its effect on antigen presentation has not been examined in a functional assay (Neurology 43 (1993) 655-661). The effect of IFN beta-1b on alloantigen presentation by human antigen-presenting cells (APC) including human fetal astrocytes (HFA) and microglia was examined. The effect of IFN beta-1b on the ability of B cells to present tetanus toxoid (TT) to TT-specific T cell lines was also examined. APC were pre-treated with IFN gamma (100 units/ml), IFN beta-1b (10-2000 units/ml), or a combination of IFN gamma and IFN beta-1b for 3 days and washed thoroughly prior to culture with allogeneic peripheral blood lymphocytes (PBL) for a period of 6 days. Lymphocyte proliferation was then measured by tritiated thymidine uptake. Treatment of the APC with IFN beta-1b resulted in a reduction in the IFN gamma-enhanced alloantigen-induced T cell responses. This reduction ranged between 50 and 70%, was associated with a 30-50% reduction in HLA class II (DR) and 35-40% reduction in ICAM-1 expression on the HFA used as APC. IFN beta-1b pretreatment of B cells reduced their constitutive and IFN gamma enhanced capacity to present TT to TT specific T cell lines by 50-80%. This was associated with a 30 +/- 11% mean reduction in class II (DR) expression and approximately 50 +/- 1% reduction in ICAM-1 expression in IFN beta-1b + IFN gamma-treated B cells compared to IFN gamma-treated B cells (mean of three experiments).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560005 TI - Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein. AB - The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography. PMID- 7560009 TI - Interferon effects on interleukin-10 secretion. Mononuclear cell response to interleukin-10 is normal in multiple sclerosis patients. AB - The mechanism of action of recombinant interferon beta 1b (rIFN beta 1b/IFN beta 1b), the approved therapy for multiple sclerosis (MS), is still unclear. Here we present evidence that part of the therapeutic effects of rIFN beta 1b in MS might result from the induction of the secretion of interleukin (IL)-10, a cytokine previously designated cytokine synthesis inhibitory factor (CSIF). We observed that rIFN beta 1b stimulated significant IL-10 secretion by monocytes from MS patients after brief incubation (18 h), whereas rIFN gamma, an inducer of MS exacerbations, was unable to stimulate IL-10 production in similar conditions. To determine the role of IL-10 as CSIF in the disease, we have also investigated its effects on TNF alpha and IL-6 secretion by peripheral blood mononuclear cells from MS patients. Recombinant human IL-10 significantly inhibited tumor necrosis factor alpha and IL-6 secretion induced by rIFN gamma, lipopolysaccharide (LPS), and rIFN gamma + LPS in MS patients and in control subjects. The induction of IL 10 secretion by rIFN beta 1b and the IL-10 inhibitory activity on pro inflammatory cytokine secretion induced by rIFN gamma in MS make this cytokine a potential candidate to treat the disease. PMID- 7560010 TI - Impairment of electrophysiological function of astrocytes by cerebrospinal fluid from a patient with Waldenstrom's macroglobulinemia. AB - Patients with the Bing Neel type of Waldenstrom's macroglobulinemia often present with global neurological symptoms. In this case report, we investigated the effects of cerebrospinal fluid (CSF) of a such a patients (CSF-WM), who presented with seizures and psychomotor slowing, on the electrophysiological properties of cultured rat neurons and astrocytes. Membrane potential and Na+ and K+ currents of neurons were unaffected. Astrocytes, however, were significantly depolarized from -77.6 +/- 8.2 mV to -48.0 +/- 7.6 mV (38%) by CSF-WM. The depolarization was markedly reduced after CSF-WM heat inactivation or after pre-incubation of astrocytes with dexamethasone (1 microM). Astrocytes are electrophysiologically active cells, which control local ionic micro-environment. Therefore, we conclude that global neurological symptoms in the Bing Neel type of Waldenstrom's macroglobulinemia like generalized seizures can result from an impairment of glial cells electrophysiological functions. PMID- 7560011 TI - The role of herpes simplex thymidine kinase expression in neurovirulence and latency in newborn vs. adult mice. AB - Infection by standard thymidine kinase-positive (TK+) and TK- mutant herpes simplex virus (HSV) was performed in order to evaluate the role of HSV TK expression in neurovirulence and in HSV latency. In newborn mice, mortality and trigeminal ganglion (TG) HSV titer correlated (both were high) for TK+ and TK- HSV. In adult mice after TK- HSV infection they also correlated (both were low). After TK+ infection of adult mice, correlation was not present; mortality was low while HSV titer was moderately high. During the period of HSV latent infection (> 28 days after HSV infection), the number of neurons expressing HSV latency associated transcript (LAT) was much greater for TK- HSV newborn-inoculated mice (average of 943/ganglion) than adult-inoculated mice (average of 138/ganglion). In addition, total amount of TG LAT was greater in the former than the latter. Reactivation from latency was restricted, however, for both groups. This result supported the important role of HSV TK expression in HSV reactivation, even when the number of LAT-positive neurons was greatly increased. The following conclusions were drawn from the study of TK- HSV in newborn mice: (i) HSV TK expression was of limited importance for neurovirulence and in vivo HSV TG infection (but was of importance in adult mice); (ii) increased in vivo HSV TG infection correlated with increased number of LAT-positive neurons, so that HSV replication and establishment of latency were not completely separable; and (iii) even with greatly increased numbers of latently infected neurons, HSV TK expression was important for reactivation from latency. Results in newborn mice suggested that the role of HSV TK expression in reactivation from latency and in neurovirulence were separable. To further investigate HSV replication in newborn and adult mice, ganglia were infected with HSV in vitro and either maintained in vitro or transplanted beneath the renal capsule of adult recipients. In both of these studies, HSV titers in ganglia were much higher in newborn than adult ganglia. This suggested that in addition to the well-know role of the immune system in HSV neurovirulence in newborn mice, it is likely that HSV replication per se in neural tissue is greater in newborn than adult mice. This may be related to the high level of HSV neurovirulence in newborn mice. PMID- 7560012 TI - In vitro superfusion method for the investigation of nerve-immune cell interaction in murine spleen. AB - Immune defence mechanisms can be modulated by brain function. To study such interactions, an in vitro method was developed to examine the release of cytokines and norepinephrine (NE) after electrical stimulation. Slices of mouse spleen were placed in chambers with a volume of 80 microliters and superfused with culture medium. To characterize electrically evoked NE release and cell viability a suitable stimulation protocol was developed using of [3H]NE. As parameter for immune function, modulation of interleukin-6 (IL-6) release by the spleen cells brought about by electrical stimulation was investigated. Splenic [3H]NE overflow was calcium-dependent, tetrodotoxin-sensitive and elicited by 54 mM potassium. Electrically evoked [3H]NE release at 22 h was about 80% of the release at 5.3 h. Electrical stimulation substantially reduced IL-6 secretion at 6 h (control: 143.4 +/- 14.3 vs. electrical: 71.3 +/- 7.9 pg/ml/10(6) leukocytes, P = 0.0001). This effect was inhibited in a concentration-dependent manner by the beta-adrenergic antagonist propranolol (P = 0.0298, EC50 approx. 10(-7) M). In conclusion, this new technique allows long-term investigation of cell function in slices of murine spleen. In addition, these are the first in vitro data indicating the presence of a functional neuroimmunological link in murine lymphoid tissue. PMID- 7560016 TI - Antibodies against the amino-terminal portion of pro-enkephalin inhibit DNA synthesis in human peripheral mononuclear cells. AB - Pro-enkephalin (PENK) mRNA and PENK-derived peptides have been reported in lymphocytes, monocytes, and macrophages. Met-enkephalin (ME) and/or synenkephalin (SYN)-containing peptides are produced and released by human peripheral blood lymphocytes (HPBL) activated with phytohemagglutinin (PHA). Furthermore, SYN (PENK 1-70) was cleaved to low-molecular-mass peptides in HPBL. In this work we studied the effect of a mouse monoclonal antibody (mAb) and a rabbit antiserum (pAb) against the C-terminal portion of SYN on DNA synthesis in PHA-activated HPBL. [3H]Thymidine incorporation into HPBL incubated with 0.1 microgram/ml of PHA was tested in the presence of different concentrations of mAb immunoglobulin (Ig) G or different dilutions of pAb. mAb induced a concentration-dependent decrease of [3H]thymidine incorporation into HPBL: 7%, 19%, 28%, and 35% of inhibition was observed with 0.1, 1, 1.5, and 2 micrograms IgG, respectively, reaching values of 65% with 10 micrograms IgG. Similarly, pAb dilutions of 1/500, 1/1000, 1/2000 and 1/4000 inhibited DNA synthesis by 63%, 61%, 43%, and 30%, respectively. The inhibitory effect of mAb and pAb was specific since it was not produced by non-immune mouse IgG or several non-immune rabbit sera and was completely reversed by 1 microM of the synthetic peptide [Tyr63](syn 63-70) synenkephalin. These results suggest that low-molecular-mass SYN-derived peptides released by PHA-activated HPBL may participate in the proliferative response of these cells. This is further evidence that the non-opioid portion of PENK--that is, SYN-derived peptides--may be involved in tissue development. PMID- 7560013 TI - Regulation of macrophage-derived tumor necrosis factor production by modification of adrenergic receptor sensitivity. AB - Catecholamines and prostaglandins are among the many diverse mediators which participate in an interactive communication between the nervous and immune systems. We have examined the response of murine peritoneal macrophages (M luminal diameter) to prostaglandin-E2 (PGE2) and the beta-adrenergic agonist isoproterenol. In the present study we found a relationship between the response elicited by PGE2 and a beta-adrenergic agonist, which in a fashion similar to the response of PGE2 on M luminal diameters suppresses lipopolysaccharide (LPS) induced tumor necrosis factor (TNF) production. It has been established that exposure of M luminal diameters to PGE2 desensitizes the suppressive function of PGE2. In this study, prior exposure of M luminal diameters to a beta-adrenergic agonist and the effects on subsequent beta-adrenergic responses, as well as the relationship to PGE2 sensitivity was determined. Complete Freund's adjuvant elicited M luminal diameters were incubated with or without either a beta adrenergic agonist or antagonist. All groups of cells were then extensively washed, followed by incubation with LPS (100 ng/ml) with or without graded concentrations of PGE2 or the beta-adrenergic agonist isoproterenol. Supernatants were collected to determine TNF concentrations by a fibroblast cytolytic assay, and Northern blot analysis was used to determine changes in the regulation of TNF mRNA accumulation. Both isoproterenol and PGE2 inhibited LPS-stimulated TNF release and TNF mRNA accumulation. We have established M luminal diameters regulation of sensitivity to isoproterenol-induced suppression of TNF production. The isoproterenol concentration-effect curve was shifted to the right after pre exposure of M luminal diameter to the beta-agonist, suggesting a desensitized beta-adrenergic receptor population. Further studies demonstrated that M luminal diameters pre-exposed to the beta-adrenergic antagonist, ICI 118.551, washed, and then challenged with LPS show an increased sensitivity for isoproterenol-induced suppression of TNF production. In addition, a decreased sensitivity of M luminal diameters to exogenous PGE2 was observed during the desensitization to the beta adrenergic agonist. Although concomitant addition of isoproterenol increased PGE2 induced suppression of LPS-stimulated TNF production, M luminal diameter pre exposed to isoproterenol (10(-6) M) demonstrated a decreased sensitivity for PGE2 induced suppression of LPS-stimulated TNF production and TNF mRNA accumulation. Our results show that the effects observed after acute administration of a mediator may be different when M luminal diameters have been previously exposed to that or other mediators. These investigations support a role for mediators released from the nervous system to regulate the release of a cytokine needed to maintain inflammatory responses. PMID- 7560014 TI - Immune cell infiltration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes simplex virus type 1. AB - Following inoculation of the mouse cornea with herpes simplex virus type 1 (HSV 1), the spread of virus was investigated and the types of immune cell infiltrating the trigeminal ganglion (TG) were identified in low temperature paraffin wax sections. Virus antigen was first found on day 3 and was absent after day 14. Early presentation of antigen to T cells may occur since increased expression of major histocompatibility complex (MHC) class II antigens, including de novo expression on satellite and Schwann cells, was detected in foci of such antigen on day 3. A second large peak of such expression was detected on day 10 together with increasing numbers of B and T cells. Large numbers of these lymphocytes and extensive expression of MHC class II were seen in the TG well into the phase of virus latency; the significance of this is discussed. PMID- 7560015 TI - Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia. AB - The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system. PMID- 7560017 TI - HLA and anti-GQ1b IgG antibody in Miller Fisher syndrome and Guillain-Barre syndrome. AB - We investigated serological human leukocyte antigen (HLA) types in patients with histories of Miller Fisher syndrome (MFS) and Guillain-Barre syndrome (GBS) with ophthalmoplegia, in whom serum anti-GQ1b IgG antibody was present during the acute phase. We examined class I antigens (A, B and C) in 32 patients and class II antigens (DR and DQ) in 30, but found no association. We conclude that particular serologically defined HLA types are not preferred for the immunoresponse of anti-GQ1b IgG antibody in MFS and GBS. PMID- 7560018 TI - Macrophage recruitment to acutely injured central nervous system is inhibited by a resident factor: a basis for an immune-brain barrier. AB - Compared to the peripheral nervous system (PNS), the central nervous system (CNS) of mammals has a poor prospect for regeneration. Accumulating evidence suggests that this is due, in part, to differences in how the immune and nervous systems communicate in response to injury. The macrophage is one of the central cells in this communication with the capacity to respond in a variety of ways depending on the conditions of stimulation. After injury, macrophages enter the CNS much later and in fewer numbers than they do the PNS. It is possible that this late and reduced response is not sufficient to modify the CNS environment to one that is conducive to successful regeneration. In the present study we investigated whether the limited macrophage invasion of injured CNS is due to the presence of an endogenous inhibitory factor that is persistent after injury. Using an in vitro migration assay, we show that rat optic nerve (CNS) is deficient in its ability to attract monocytes as compared to rat sciatic nerve (PNS). We further demonstrate that this deficiency is due to the presence of a soluble inhibitory factor in the CNS. This factor may also cause a subsequent effective difference in those macrophages that are recruited, as is shown by morphological data. The brain-resident factor that inhibits macrophage migration may be the physiological basis of an immune-brain barrier underlying the known phenomenon of immune privilege. PMID- 7560020 TI - Epidemiology of status epilepticus. AB - This study presents a review of the epidemiology of status epilepticus (SE) in Richmond, Virginia, U.S.A. The data summarize some of the first population-based information on the natural presentation of SE in a controlled community setting. SE occurred with an absolute incidence rate of 41 patients per 100,000 residents per year in Richmond. The frequency of total SE occurrences was 50 patients per 100,000 residents per year. Overall mortality in this population was 22%. Absolute incidence and occurrences of SE in this population were shown to be underestimates due to the inability, for multiple reasons, to document all cases of SE. Based on the Richmond data, the number of SE cases, frequencies of occurrence, and deaths per year occurring in the United States were estimated to be 102,000-152,000, 125,000-195,000, and 22,000-42,000, respectively. In Richmond, nonwhites had a much higher incidence of SE than did whites. Partial SE was the most common form of seizure initiating SE. Age and etiology were also found to contribute to mortality. Infants <1 year of age had the highest incidence of SE, but the elderly population represented the largest number of SE cases. This study provides a review of the first prospective, population-based, epidemiological data on SE and shows that SE is a major medical and neurological emergency in both academic and community hospital settings. PMID- 7560021 TI - Pathophysiology of status epilepticus. AB - The cellular and molecular pathophysiology of status epilepticus (SE) provides a conceptual framework for understanding clinical scenarios and prospectively designing logical therapies. SE is a dynamic process that evolves over time in a predictable manner with an established sequence of EEG, motor, physiologic, and cellular changes. Neuronal injury and death are the result of processes intrinsic to the brain, mediated by a complex neurotoxic cascade consisting of multiple serial and parallel processes. The risk of cell injury depends also on the overall pathophysiologic profile, including the presence of alterations resulting from SE and occurring independent of SE. On neurophysiologic grounds, we divide SE into "spike-wave" and "nonspike-wave" forms. Spike-wave "absence" status epilepticus carries a low risk of epileptic brain damage, and therapy should be adjusted accordingly. All nonspike-wave SE has a theoretical basis for epileptic brain damage, but the actual risk is variable. There is a significant known risk of cell injury during generalized convulsive SE, a variety of nonspike-wave SE, so aggressive treatment is warranted to prevent sequelae. There is also a theoretical basis for epileptic brain damage in nonspike-wave nonconvulsive SE, but prospective studies are needed to determine which of these patients warrant aggressive therapy. Based on pathophysiologic principles, future treatment of nonspike-wave SE may use a combination of anti-ictal agents, including gamma aminobutyric acid agonists and N-methyl-D-aspartate antagonists, as well as various neuroprotectants. PMID- 7560019 TI - beta-Endorphin enhances the replication of neurotropic human immunodeficiency virus in fetal perivascular microglia. AB - The effect of an endogenous opiate, beta-endorphin, on the replication of HIV was investigated in brain perivascular microglia. Beta-endorphin enhanced the synthesis of p-24 antigen and transactivation of HIV promoter. Dialysed culture supernatants of endorphin-treated microglia re-activated latent HIV infection. These culture supernatants showed elevated levels of interleukin-1 beta, IL-6 and tumor necrosis factor alpha. Sub-optimal concentration of beta-endorphin potentiated GP-120-induced synthesis of these cytokines. Nalaxone reversed beta endorphin-induced, but not GP-120-induced, cytokine production and enhanced HIV replication. These results suggest that endogenous opiates may contribute to the progression of AIDS dementia complex. PMID- 7560022 TI - Electroclinical features of status epilepticus. AB - Status epilepticus (SE) is a condition wherein epileptic seizure discharges are sufficiently prolonged or repetitive so as to produce persistent alterations in neurologic function and in the underlying physiologic and neurochemical activities of the brain. Thus, the definition of SE now includes any disorder in which there is sustained and prolonged excitation of neurons. Electroencephalographic (EEG) patterns associated with specific types of SE are important components in their classification. Like epileptic seizures, SE can be divided into partial onset SE and primarily generalized SE. Partial onset SE includes secondarily generalized convulsive SE (GCSE), complex partial SE (CPSE), simple partial SE (SPSE), and the syndromes of epilepsia partialis continua (EPC) and rolandic SE (RSE). Primarily generalized SE includes primarily GCSE, absence SE, atypical absence SE, generalized myoclonic SE, generalized clonic SE, generalized tonic SE, atonic SE, and the syndromes of electrical SE of sleep (ESES) and minor epileptic SE of Brett. SE is a dynamic disorder. Behavioral and electrical manifestations change over time if seizure activity is allowed to persist without successful treatment A progression from overt to subtle convulsive activity occurs in secondarily GCSE and there is also a progression of predictable EEG changes in prolonged GCSE. CPSE begins as discrete complex partial seizures but also progresses behaviorally and electrically through a sequence similar to that observed in GCSE. Progressive behavioral and electrical changes have not been reported in primarily generalized forms of SE. EEG is an important tool for verifying successful treatment of SE if the patient does not immediately recover neurologic function. EEG recordings also contribute substantially to understanding the mechanisms of, and development of better treatments for, human SE through their use in the study of experimental SE in the laboratory. PMID- 7560025 TI - Regarding "Sympathetic skin response". PMID- 7560024 TI - Quantitative topographic electroencephalographic mapping during drowsiness and sleep onset. AB - The topographic EEG features of drowsiness and sleep onset are much less well documented than are their temporal aspects. A commercial topographical mapping system was used to assess the main EEG features employing all 19 international 10/20 system electrode sites referenced to linked ears during wakefulness, stages 1A and 1B drowsiness, stage 2 sleep, and sleep onset REM periods in 19 patients. All patients had been referred for a diagnostic EEG or a Multiple Sleep Latency Test and had essentially normal EEGs. Anterior alpha of drowsiness seldom represented frontal spread of the occipital alpha rhythm but usually was a distinct activity of apparent separate origin. Theta activities of drowsiness were maximum at CZ and FZ. Vertex sharp waves and sawtooth waves of rapid eye movement (REM) sleep had similar fields maximum at the midline with a steep decrease laterally. Isolated anterior mainly negative waves were identified. Sleep spindles were usually maximal in fronto central, occasionally centro parietal, or even parietal areas. PMID- 7560023 TI - Use of frameless stereotaxy with location of electroencephalographic electrodes on three-dimensional computed tomographic images in epilepsy surgery. AB - Epileptiform activity corresponding to structural lesions was identified by three dimensional (3D) imaging using computed tomographic (CT) scan data concurrently with scalp EEG electrodes. The electrodes, placed according to the international 10-20 system, were used to record interictal and ictal epileptogenic foci in eight patients. Electrodes placed where marked or moderate epileptiform activity was detected were replaced with markers detectable on CT. Scans with these markers on the skin were obtained and the data transferred to a 3D imaging system, and correlated with underlying cerebral structures. The reformatted images were used to assess the relation among intracranial lesions, brain surface structures, and epileptogenic zones depicted by the markers. The images help the surgeon plan a craniotomy with enough space for both lesionectomy and resection of the epileptogenic zone. In the central regions where crucial motor functions are located, the markers indicate the central fissure or postcentral gyrus. An intraoperative 3D frameless stereotactic pointing device helps in directing further examination of the epileptogenic zone. This system improves on the precision available through intraoperative electrocorticographic recording in the extratemporal lobes, thus avoiding only approximate excision of lesion and epileptogenic zone and enabling the neurosurgeon to perform epilepsy surgery with greater confidence. PMID- 7560026 TI - Advances in children's elbow fractures. PMID- 7560027 TI - Nondisplaced and minimally displaced fractures of the lateral humeral condyle in children: a prospective radiographic investigation of fracture stability. AB - Subsequent displacement of nondisplaced or minimally displaced fractures of the lateral humeral condyle while immobilized in plaster may contribute to severe complications. The possibility of assessing the stability of such fractures may be a help in planning the initial treatment. Our prospective investigation of 112 children aged 1-11 years aimed at describing radiographic criteria for prognosticating the stability of the fractures. According to the radiographic findings, the fractures were allocated to one of three groups representing stable fractures, fractures with undefinable risk, and fractures with high risk of later displacement. All children were treated with splinting only. Sixty-five fractures were classified as stable and turned out to be so without exception. Displacement occurred in six of 35 (17%) of the fractures judged uncertain and in five of 12 (42%) of those judged unstable. The subsequent displacement was 1 or 2 mm, and in one case, 3 mm. The defined criteria were found to be efficient in separating the stable and the high-risk fractures with acceptable confidence. The group of fractures with undecided risk of subsequent displacement was fairly large--one third of the total material. The implication the findings may bear to the treatment strategy of fractures is analyzed in a work in progress. PMID- 7560028 TI - Epiphyseal separation of the distal end of the humeral epiphysis: a follow-up note. AB - Twenty-one children with fracture-separations of the distal end of the humeral epiphysis were treated. Fifteen of the 21 children developed cubitus varus deformity after treatment. Thirteen children were treated at our hospital, and seven of these children developed cubitus varus deformity. Eight children were treated elsewhere initially and were referred to our institution after development of cubitus varus deformity. Nine patients underwent osteotomies for correction of cubitus varus deformities. Ages at injury ranged from 1-10 years. Fourteen patients were < 6 years of age. Fractures were classified according to Ogden; one was type IA, 16 were type IIA, and four were type IIC. Follow-up was from 10 months to 17 years. Most of the patients who were initially treated at our institution regained nearly normal function. All nine patients who were treated with closing wedge osteotomies regained nearly normal carrying angles. The carrying angles after treatment of the injury and osteotomy for the deformity did not change except in one patient. PMID- 7560029 TI - Clinical evaluation of crossed-pin versus lateral-pin fixation in displaced supracondylar humerus fractures. AB - The radiographs and patient charts of 47 children treated with closed reduction and percutaneous pin fixation of displaced supracondylar humerus fractures were reviewed. Twenty-seven fractures were fixed with crossed medial and lateral pins. Twenty fractures were treated with two parallel laterally placed pins. Baumann's angle on the anteroposterior elbow film and the humerocapitellar angle on the lateral elbow film were independently measured by the three authors on initial postoperative films and on films taken at the time of pin removal. No statistically significant differences regarding maintenance of reduction were found when comparing the two fixation groups. There were two complications in the medial pin group (one cubitus varus and one ulnar nerve injury) and none in the lateral-pin group. We conclude that crossed-pin fixation offers no clinically significant advantage over two laterally placed pins in the treatment of supracondylar humerus fractures. PMID- 7560030 TI - Traumatic and iatrogenic neurological complications after supracondylar humerus fractures in children. AB - A retrospective review of 162 displaced supracondylar fractures in children at Los Angeles County Harbor-UCLA Medical Center from 1975-1981 revealed 23 neural injuries. These injuries occurred in 19 patients whose ages ranged from 5-11 years of age. There were 12 radial, six ulnar, and five median neuropathies detected. Four of the ulnar nerve injuries and one radial nerve injury were iatrogenic, resulting from both percutaneous pinning and open reduction and internal fixation. All of the deficits resolved spontaneously within a range of 2 6 months. The average time to resolution for traumatic injuries was 2.3 months. Fifty-eight percent of these injuries were found to be associated with a Holmberg type III fracture pattern and 42% with a type IV supracondylar fracture. PMID- 7560031 TI - Supracondylar elbow fractures with impaction of the medial condyle in children. AB - We reviewed the cases of 13 children with supracondylar elbow fractures with impaction of the medial wall. The displacement was underestimated in two patients and treated as a minimally displaced fracture by simple immobilization without reduction, resulting in a cubitus varus deformity. In one patient, the correct diagnosis was made, but treatment by reduction was refused by the parents. A loss of carrying angle of 10 degrees resulted from this. Two patients referred for treatment of cubitus varus were treated elsewhere for the initial injury: one by reduction and plaster cast immobilization and the other by simple immobilization without reduction. Two children with a supracondylar fracture with buckling of the medial condyle without prominent deformity were not reduced and showed no deformity at follow-up. Six children with collapse of the medial condyle, treated by closed reduction and percutaneous pinning, all had a normal carrying angle at follow-up. We believe that this method is safe to prevent varus angulation. PMID- 7560032 TI - Evaluation of the role of comparison radiographs in the diagnosis of traumatic elbow injuries. AB - This study attempted to determine whether comparison radiographic views of the uninjured elbow result in increased diagnostic accuracy. Physicians (one junior and one senior orthopaedic resident and an orthopaedic surgeon) were provided with a short clinical summary and asked to interpret radiographs of the injured elbow (IE) or both the injured and uninjured elbow (UE) in a randomized fashion from 25 children with elbow injuries. The overall percentage of correct diagnoses (one vs. two elbow radiographs) were as follows: orthopaedic surgeon, 80 versus 96%; for junior resident, 80 versus 84%; and for senior resident, 84 versus 88% (p > 0.05). Kappa scores for interobserver variability and intrarater agreement were in the high range (0.756 to 0.904, kappa = 0.08). Clinically relevant diagnoses were missed by trainees (not the orthopaedic surgeon) whether radiographs of IE or both IE and UE were interpreted. Incorrect radiograph interpretations were due to false positives in 39 of 40 cases. Comparison radiographs of the UE were not useful in improving diagnostic accuracy in elbow trauma. However, although they may be necessary in some instances, routine radiographic examination of the UE is not warranted. PMID- 7560033 TI - Acute annular ligament interposition into the radiocapitellar joint in children (nursemaid's elbow). AB - Acute annular ligament interposition into the radiocapitellar joint ("nursemaid's elbow") is a common injury in children younger than 5 years. The injury occurs when axial traction is applied to an extended, pronated arm. There are no abnormal radiographic findings associated with this condition. We recommend that children with a classic history and clinical presentation of an acute annular ligament interposition into the radiocapitellar joint be treated without obtaining radiographs. PMID- 7560034 TI - Pediatric femur fractures: effects of spica cast treatment on family and community. AB - Twenty-three consecutive children with isolated closed femur fractures treated with early spica casts were studied to determine the impact of this treatment on family, school, and other support systems. Mobility was identified by families as the major problem. In families with two working parents, a mean of 3 weeks time off work was needed. None of the children was accepted into school in cast. Home tutoring for a mean of 8 weeks was used, with 48 tutor-hours per child. Despite this, none of the children fell permanently behind their class, and only two temporarily. No child required physical therapy beyond instruction in walking, and 12 had no physical therapy at all. Mean time to independent walking was 5 days and to running, 25 days; skills returned faster in younger children. All aspects of spica treatment were easier for preschool children. This study highlights some of the adaptations necessary with this treatment method and aids in advance planning. PMID- 7560035 TI - Femur fractures in infants: a new therapeutic approach. AB - Fourteen patients with 16 femur fractures sustained between birth and 18 months of age were treated with a Pavlik harness rather than traditional casting methods. All of the fractures went on to stable union within 5 weeks. Eleven fractures have been followed-up for > 12 months, with a range of 12-30 months, and a mean of 20.1 months. All of the fractures healed in good alignment, with leg-length discrepancies < 1 cm. There have been no adverse results or complications as a result of treatment with the Pavlik harness to treat femur fractures, including fractures of the proximal and middle thirds of the femur; nonambulatory infants; < 4 months old at the start of treatment or small size in selected infants up to 6 months old; and shortening of < 2 cm. Advantages of the use of the Pavlik harness include ease of application without general anesthesia, minimal hospitalization, ease of reduction, ability to adjust the harness (and therefore the fracture alignment) if the reduction is lost, minimal cost, and ease of nursing, bonding with, and changing diapers on the infant. PMID- 7560036 TI - The use of modified Neufeld's skeletal traction in children and adolescents. AB - A modified form of Neufeld's skeletal traction is described. This technique is a useful method of treating children and adolescents with femoral or pelvic fractures. The advantage over standard femoral skeletal traction is early mobilization of the patient to a sitting position. Additionally, the patient can be placed in this form of traction at home, allowing earlier discharge from the hospital. PMID- 7560037 TI - Femoral neck stress fractures in children and adolescents. AB - Five new cases of femoral neck stress fracture in children and adolescents are added to seven cases in the literature. All patients were involved in increased repetitive activity without evidence of violent trauma. Most fractures healed uneventfully with appropriate treatment. However, increased morbidity from displacement of the fracture, delayed union, nonunion, or avascular necrosis does occur in this age group and needs to be avoided. A delayed union healed after treatment in a spica cast; a nonunion and a displaced fracture healed after internal fixation. All patients returned to their previous level of activity. Our treatment algorithm addresses the unique differences associated with this injury in immature patients and allows for early diagnosis and treatment to prevent complications. PMID- 7560039 TI - Extra-articular triplane fracture of the distal tibial epiphysis. AB - We describe an extra-articular triplane fracture of the distal tibia in the skeletally immature patient. This variant of the triplane fracture has been largely ignored in the literature. The clinical significance of recognizing this fracture is that, although it constitutes an epiphyseal fracture, it remains extra-articular. Unlike the standard triplane fracture that exists through the tibiotalar joint, this variant can be treated acceptably with less than an anatomical reduction, therefore often avoiding the need for surgical management. PMID- 7560038 TI - Fractures of the distal femoral epiphyseal plate. AB - A retrospective analysis of 30 consecutive fractures of the distal femoral epiphyseal plate showed the best results occurred when fractures were anatomically reduced and fixed with pins. No fractures with internal fixation displaced, whereas 43% of fractures reduced without fixation displaced during cast treatment. Complications were more frequent in displaced than nondisplaced fractures. We were unable to demonstrate that gentle reduction under general anesthesia offered protection against subsequent physeal arrest when compared with closed reduction in the emergency room; however, reductions in the operating room were more likely to be anatomic. PMID- 7560042 TI - Premature closure of the distal radial physis after fracture of the distal radial metaphysis. AB - Two fractures of the distal radius seemingly with lack of involvement of the distal radial physis are presented. Nevertheless, complete arrest of the adjacent physis occurred, suggesting a Salter-Harris V mechanism of injury to the vulnerable distal radial physis. Ulnar "overgrowth" produced impingement of the carpus and functional complaints sufficient to warrant treatment. In this clinical setting, an ulnar-shortening osteotomy done at skeletal maturity resulted in marked improvement in the range of motion at the radiocarpal joint and corresponding improvement in function for both patients so treated. PMID- 7560041 TI - Tibia valga after proximal metaphyseal fractures in childhood: a normal biologic response. AB - Seventeen children with 19 proximal tibial metaphyseal fractures were followed-up between 2 and 7 years after injury. Detailed measurements of the metaphyseal/diaphyseal/metaphyseal distances medially and laterally on the injured and noninjured sides demonstrated overgrowth. In four patients, the medial distance of the injured tibia was longer than the lateral distance, which was the same distance as the uninjured tibia. In 11 patients, there was an overgrowth of both the medial and lateral sides of the injured tibia, compared to the unijured tibia, and in each instance, the medial distance of the injured tibia was always longer than the lateral. In a patient with bilateral metaphyseal fractures, the medial length exceeded the lateral length in both tibias. In the child with metaphyseal and diaphyseal fractures, the medial side of the tibia with the metaphyseal fracture was the longest of the four measurements. In five of six patients with Harris lines, there was distal as well as proximal tibial metaphyseal overgrowth, but the distal line was always parallel to the physis and did not contribute to the valgus angulation. Thus there was not only a generalized increased growth proximally and distally, but there also was an eccentric proximal medial overgrowth in every patient. PMID- 7560040 TI - A review of open tibia fractures in children. AB - Fifty-six open tibia fractures in 55 children were studied retrospectively. The overall case fatality rate was 7% (four patients). The most significant factor affecting death rate was injury to the chest and abdomen. Four amputations were performed in four patients with five injuries. Statistically, the presence of neurovascular compromise was significant, with four of eight compromised extremities (in seven patients) requiring amputation. Infection occurred in eight injuries, for an incidence of 14% overall (50% deep). The most important variables were presence of neurovascular injury and delay in getting the patient to surgery. A delay of > 6 h was correlated with a 25% infection rate compared with a 12% rate for those operated on within 6 h. The average time to union was approximately 5 +/- 4 months (range, 1.5-24.8 months). The most significant factor affecting union time was the age of the patient. PMID- 7560043 TI - The use of methylmethacrylate as an external fixator in children and adolescents. AB - Methylmethacrylate has been used as an external fixator on 52 bones in 36 children and adolescents. There were 36 osteotomies and 16 selected fractures of long bones. The application of the acrylic cement onto 229 half-pins was performed with the use of flexible plastic tubes, in all but two patients. Union in the desired position was achieved in 51 bones. Delayed union occurred in one and required internal fixation. In the rest of the cases, there were only a few minor complications. The methylmethacrylate appears to be a stable fixator for osteotomized and fractured bones in young patients. The method is practical, easily performed, and of low cost. PMID- 7560044 TI - Lawn mower injuries of the pediatric foot and ankle: observations on prevention and management. AB - We reviewed 32 children with lower extremity injuries caused by power lawn mowers. Functional outcome of 21 patients was evaluated. Anatomical injury patterns provide some guidelines in management and prediction of functional outcome. Consistently, the most severe injuries result from ride-on mowers and wounds to the posterior/plantar foot and ankle. Our experience with pediatric foot and ankle lawn mower injuries permits recommendations for maximum functional outcome with minimal intervention. Public awareness and mower safety devices may be required to decrease the rate of accidents in the future. PMID- 7560045 TI - Ambulation in children and adolescents with spinal cord injuries. AB - The ambulatory status of 76 children and adolescents with spinal cord injuries was studied to delineate the natural history of ambulation and to identify factors predictive of successful ambulation. The type of orthotic used and the degree and duration of ambulation depended on age, completeness and level of the neurologic deficit, and lower extremity contractures. Younger age, L3 or better neurologic levels, incomplete lesions with motor preservation, and absence of lower extremity contractures were associated with functional walking. The psychological advantages of ambulation must be weighed against the cost of orthotics and associated training and the nonfunctional nature of ambulation in the vast majority of individuals with spinal cord injuries. In addition, the developmental aspects of pediatric spinal cord injuries require changing therapeutic goals at different developmental stages. PMID- 7560046 TI - Latex allergy in children and adolescents with spinal cord injuries. AB - The incidence of latex allergy in children and adolescents with spinal cord injuries was investigated using history and laboratory testing. Among 67 individuals with spinal cord injuries, four had histories of latex allergy, and two of these also had positive laboratory tests. There were eight individuals without a history of latex allergy who had positive laboratory tests. Defining latex allergy as a positive history or a positive laboratory test, 12 of the 67 subjects (18%) would be considered allergic to latex. The incidence of latex allergy increased with time since the spinal cord injury. Recommendations are made for the diagnosis, management, and prevention of latex allergy in individuals with spinal cord injuries. PMID- 7560047 TI - Lumbar compression fractures secondary to lap-belt use in children. AB - The correlation between flexion-distraction injuries and lap-belt use has been well documented. Over a 10-year period, we identified seven children admitted to Children's National Medical Center, Washington, DC, with compression fractures of the lumbar spine secondary to lap-belt use. Four were rear seat passengers, and three were in the front seat. The average age was 7 years. Four of the seven (57%) suffered associated abdominal injuries. One died of an associated head injury. We hypothesize that the mechanism of injury in these cases was similar to that in flexion-distraction injuries. The increased elasticity in the posterior ligamentous complex in children may be responsible for the occurrence of these compression fractures rather than the expected flexion-distraction-type injuries. PMID- 7560048 TI - Boston brace in the treatment of idiopathic scoliosis. AB - In a retrospective follow-up study of 64 patients with adolescent idiopathic scoliosis, the initial correction of Boston brace with straight lumbar profile strongly predicted good treatment results at follow-up (mean, 4.8 years). If the initial correction was > 50%, the curve was permanently reduced with an average of 7.2 degrees. The average initial correction was 62%. This initial brace effect was preserved in another comparable group of 60 patients treated with a Boston brace with 15 degrees lumbar lordosis. It is concluded that the Boston brace is effective in the conservative treatment of idiopathic scoliosis, and the corrective ability seemingly does not deteriorate with a change in the brace design from 0 to 15 degrees lordosis. PMID- 7560050 TI - An evaluation of the Adams forward bend test and the scoliometer in a scoliosis school screening setting. AB - We examined the ability of the Adams forward bend test and the scoliometer to detect truncal rotation or asymmetry in a school screening setting. Of 954 sixth graders examined with each test independently, 123 and 13 [using an angle of trunk rotation (ATR) of > 5 and 7 degrees, respectively] were found to be abnormal on scoliometer examination but appeared normal on the Adams test. Selecting children for scoliometer examination on the basis of the Adams test is not supported by our data and may not be consistent with generally accepted principles of public health screening. PMID- 7560049 TI - Rotation of the spine in congenital scoliosis. AB - We reviewed the progression of spinal rotation in 100 consecutive patients with 119 curves. Eighty-four patients had a single curve and composed this study. Thirty-nine cases were due to failure of segmentation, 38 cases were due to failure of formation, and seven cases were unclassifiable. Sixty-five of the patients eventually underwent spinal arthrodesis, and 19 of the patients were treated with nonoperative means, such as bracing or observation. Cases of unilateral bar, hemivertebra, and wedge vertebra showed progressive rotation, regardless of the type of treatment; however, progression of curve magnitude was limited by spinal arthrodesis. Rotation of the spine was not seen in patients with block vertebrae. Rotation of the spine and progression of curve magnitude varied in cases of complex vertebral deformities. Because of the uncertainty of remaining growth potential in congenitally dysplastic vertebrae, future growth in the spine should be considered before the undertaking of operative procedures. PMID- 7560051 TI - Occipitocervical fusion by Luque loop rod instrumentation in Down syndrome. AB - Four children with Down syndrome who had developed atlantoaxial dislocation and myelopathy underwent occipitocervical fusion with Luque loop rod instrumentation and decompressive laminectomy of C1. The postoperative results are presented and the indications discussed. This procedure provides many advantages in the surgical treatment of Down syndrome compared with the conventional procedures, because of the associated mental retardation that makes the postoperative management very complicated. PMID- 7560053 TI - The role of public health in improving the health of America. PMID- 7560052 TI - Posterior spinal arthrodesis for atlantoaxial instability in Down syndrome. AB - Nine children with Down syndrome who had atlantoaxial instability underwent posterior spinal fusion. At follow-up, all patients had stabilization or improvement of their neurologic symptoms. Evaluation of the spine using flexion and lateral radiographs, as well as selective cineradiography, showed no instability over the fused area or adjacent motion segments. Stable fibrous union with no clinical significance was noted in three of the nine patients. We recommend posterior spinal fusion in situ with external immobilization as the safest and most effective means of surgical stabilization in the patient with Down syndrome who has symptomatic atlantoaxial instability. PMID- 7560054 TI - Health department administration of the Canadian Health Care Program. AB - Public administration of the Canadian health care program has been very successful in preserving a single-tiered system, ensuring accessibility and accountability, reducing administrative costs and the need for regulation, and fairly successful in achieving orderly planning and cost control. Although the provincial programs are administered by health departments, failure to link personal to public health services has robbed them of a population perspective, resulting in insufficient emphasis on prevention and on the effectiveness of interventions, and has hampered the development of comprehensive primary care. The program supports insufficient research and innovation, and has failed to link payments to performance. Four lessons for a national health care program are drawn from the Canadian experience: (1) keep it public; (2) use a single payer; (3) link personal health services to public health; and (4) provide health departments with policy expertise. Despite its overall success, the Canadian program can no longer be afforded. Many of the proposals for its reform challenge the values upon which the program is built. PMID- 7560055 TI - Regulation of accountable health plans in rural areas. AB - Monitoring newly emerging "accountable health plans" is a public health responsibility related to Certificate-of-Need regulation. AHPs should use locally based primary care providers, assure access to high quality affordable care to rural communities, contract with essential community providers, be governed by local boards of health, and report information necessary to evaluate their performance. Performance should be measured epidemiologically, using population based rates such as infant mortality, incidence of late-stage breast and cervical cancer, and avoidable hospitalizations. Supplemental indicators include travel time to care, primary care personnel per capita, volume in volume-sensitive procedures, EMS response time, and client satisfaction. PMID- 7560056 TI - Tobacco and alcohol billboards in 50 Chicago neighborhoods: market segmentation to sell dangerous products to the poor. AB - This paper describes a study of billboard advertising of tobacco and alcohol products in the city of Chicago. All billboards were counted and their advertising themes noted. These data were matched with information on population and race from the 1990 census in order to document which geographic areas of the city, if any, had excess tobacco or alcohol billboards. The data revealed that minority wards were burdened with three times as many tobacco billboards and five times as many alcohol billboards when compared to white wards. The findings are congruent with studies conducted in other urban areas, which demonstrate a consistent pattern of tobacco and alcohol advertisers targeting poor and minority neighborhoods for outdoor advertising of their dangerous products. Chicago legislative initiatives based on the billboard study are described. PMID- 7560057 TI - MADD's position on alcohol advertising: a response to Marshall and Oleson. PMID- 7560058 TI - Late-onset hereditary anemia: why the delay? PMID- 7560059 TI - Starve a tumor: therapeutic advice for breast cancer? PMID- 7560060 TI - Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. AB - We have previously shown that absence of the mouse mdr1a (also called mdr3) P glycoprotein in mdr1a (-/-) "knockout" mice has a profound effect on the tissue distribution and elimination of vinblastine and ivermectin, and hence on the toxicity of these compounds. We show here that the mouse mdr1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. Injection of these radio-labeled drugs in mdr1a ( /-) and wild-type mice resulted in markedly (20- to 50-fold) higher levels of radioactivity in mdr1a (-/-) brain for digoxin and cyclosporin A, with more moderate effects for dexamethasone (2- to 3-fold) and morphine (1.7-fold). Digoxin and cyclosporin A were also more slowly eliminated from mdr1a (-/-) mice. Our findings show that P-glycoprotein can be a major determinant for the pharmacology of several medically important drugs other than anti-cancer agents, especially in the blood-brain barrier. These results may explain a range of pharmacological interactions observed between various drugs in patients. PMID- 7560061 TI - Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi OspA or B. AB - The evolution of Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface proteins (Osps) A or B was assessed to investigate the role of immunity to OspA or B in infection and pathogenesis of Lyme disease. Antibodies to OspA or B protect immunocompetent C3H/HeJ or C.B.17 severe combined immunodeficient (scid) mice from challenge with B. burgdorferi. Moreover, arthritis in infected C3H mice resolves with the rise of high titers of B. burgdorferi specific antibodies, including OspA and B, whereas disease persists in scid mice--suggesting that the regression of arthritis may be due to the development of borreliacidal OspA or B antibodies. To evaluate the course of Lyme borreliosis in OspA or B tolerant mice we developed transgenic mice that expressed OspA or B under control of the major histocompatibility complex (MHC) class I promoter. Mice carrying OspA or B transgenes on a C3H/HeJ (C3H, disease susceptible) or C57BL/6 (B6, disease-resistant) background, immunized with OspA or B, did not mount a humoral or cellular immune response to OspA or B, respectively, but responded normally to other B. burgdorferi antigens. The evolution of Lyme borreliosis, including infection and the development of arthritis and carditis, was similar in transgenic and nontransgenic littermates suggesting that an OspA or B immune response is not singularly involved in either the genesis or regression of Lyme disease in C3H or B6 mice. PMID- 7560062 TI - Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity. AB - Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in E. coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general PMID- 7560063 TI - Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms. AB - Insulin inhibits proteolysis in human muscle thereby increasing protein anabolism. In contrast, IGF-I promotes muscle protein anabolism principally by stimulating protein synthesis. As increases or decreases of plasma amino acids may affect protein turnover in muscle and also alter the muscle's response to insulin and/or IGF-I, this study was designed to examine the effects of insulin and IGF-I on human muscle protein turnover during hyperaminoacidemia. We measured phenylalanine balance and [3H]-phenylalanine kinetics in both forearms of 22 postabsorptive adults during a continuous [3H] phenylalanine infusion. Measurements were made basally and at 3 and 6 h after beginning a systemic infusion of a balanced amino acid mixture that raised arterial phenylalanine concentration about twofold. Throughout the 6 h, 10 subjects received insulin locally (0.035 mU/min per kg) into one brachial artery while 12 other subjects were given intraaterial IGF-I (100 ng/min per kg) to raise insulin or IGF-I concentrations, respectively, in the infused arm. The contralateral arm in each study served as a simultaneous control for the effects of amino acids (aa) alone. Glucose uptake and lactate release increased in the insulin- and IGF-I-infused forearms (P < 0.01) but did not change in the contralateral (aa alone) forearm in either study. In the aa alone arm in both studies, hyperaminoacidemia reversed the postabsorptive net phenylalanine release by muscle to a net uptake (P < 0.025, for each) due to a stimulation of muscle protein synthesis. In the hormone infused arms, the addition of either insulin or IGF-I promoted greater positive shifts in phenylalanine balance than the aa alone arm (P < 0.01). With insulin, the enhanced anabolism was due to inhibition of protein degradation (P < 0.02), whereas IGF-I augmented anabolism by a further stimulation of protein synthesis above aa alone (P < 0.02). We conclude that: (a) hyperaminoacidemia specifically stimulates muscle protein synthesis; (b) insulin, even with hyperaminoacidemia, improves muscle protein balance solely by inhibiting proteolysis; and (c) hyperaminoacidemia combined with IGF-I enhances protein synthesis more than either alone. PMID- 7560064 TI - Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen. AB - Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature. PMID- 7560065 TI - Possible links between glucose-induced changes in the energy state of pancreatic B cells and insulin release. Unmasking by decreasing a stable pool of adenine nucleotides in mouse islets. AB - Whether adenine nucleotides in pancreatic B cells serve as second messengers during glucose stimulation of insulin secretion remains disputed. Our hypothesis was that the actual changes in ATP and ADP are obscured by the large pool of adenine nucleotides (ATP/ADP ratio close to 1) in insulin granules. Therefore, mouse islets were degranulated acutely with a cocktail of glucose, KCl, forskolin, and phorbol ester or during overnight culture in RPMI-1640 medium containing 10 mM glucose. When these islets were then incubated in 0 glucose + azide (to minimize cytoplasmic and mitochondrial adenine nucleotides), their content in ATP + ADP + AMP was decreased in proportion to the decrease in insulin stores. After incubation in 10 mM glucose (no azide), the ATP/ADP ratio increased from 2.4 to > 8 in cultured islets, and only from 2 to < 4 in fresh islets. These differences were not explained by changes in glucose oxidation. The glucose dependency (0-30 mM) of the changes in insulin secretion and in the ATP/ADP ratio were then compared in the same islets. In nondegranulated, fresh islets, the ATP/ADP ratio increased between 0 and 10 mM glucose and then stabilized although insulin release kept increasing. In degranulated islets, the ATP/ADP ratio also increased between 0 and 10 mM glucose, but a further increase still occurred between 10 and 20 mM glucose, in parallel with the stimulation of insulin release. In conclusion, decreasing the granular pool of ATP and ADP unmasks large changes in the ATP/ADP ratio and a glucose dependency which persists within the range of stimulatory concentrations. The ATP/ADP ratio might thus serve as a coupling factor between glucose metabolism and insulin release. PMID- 7560068 TI - Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. AB - We investigated immunohistochemical localization of V2 vasopressin receptor along the nephron using a specific polyclonal antibody. Staining was observed in some of thick ascending limbs and all of principal and inner medullary collecting duct (IMCD) cells. Not only basolateral but also luminal membrane was stained in collecting ducts, especially in terminal IMCD (tIMCD). To learn the functional role of luminal V2 receptor in tIMCD, we studied the luminal effects of arginine vasopressin (AVP) on osmotic water permeability (Pf), urea permeability (Pu), and cAMP accumulation using isolated perfused rat tIMCD. In the absence of bath AVP, luminal AVP caused a small increase in cAMP accumulation, Pf and Pu, confirming the presence of V2 receptor in the lumen of tIMCD. In contrast, luminal AVP inhibited Pf and Pu by 30-65% in the presence of bath AVP by decreasing cAMP accumulation via V1a or oxytocin receptors and by an unknown mechanism via V2 receptors in the luminal membrane of tIMCD. These data show that V2 receptors are localized not only in the basolateral membrane but also in the luminal membrane of the distal nephron. Luminal AVP acts as a negative feedback system upon the basolateral action of AVP in tIMCD. PMID- 7560066 TI - Transfer of rheumatoid arthritis into severe combined immunodeficient mice. The pathogenetic implications of T cell populations oligoclonally expanding in the rheumatoid joints. AB - To investigate the pathogenicity of T cells infiltrating in the rheumatoid joints, mononuclear cells (MNC), predominantly T cells, isolated from either synovial fluid or synovial tissues of the patients with RA were transferred into severe combined immunodeficient (SCID) mice by intraarticular injections. According to our observations in this experimental system, patients with RA could be classified into at least two groups. In one group of patients, the infiltrating MNC induced synovial hyperplasia in the recipient SCID mice (the positive group). Whereas, in the other group no synovial hyperplasia was observed (the negative group). The induction of synovial hyperplasia observed in the positive group was prevented by an anti-human CD3 antibody (OKT3), indicating T cell mediation. Analysis of T cell receptor (TCR) V beta usage by reverse transcriptase polymerase chain reaction in the infiltrating MNC transferred into SCID mice revealed a marked skew towards the preferential use of certain V beta genes, which was not seen in the peripheral blood MNC, in only the positive group. The patterns of TCR/V beta skew were not uniform among the patients. The analysis of the PCR-amplified genes of such skewed TCR/ V beta by single strand conformational polymorphism showed distinct bands, indicating that the T cell populations expanding in rheumatoid joints of the positive group were oligoclonal. Furthermore, the enrichment of the T cell populations expressing such skewed TCR/V beta by in vitro stimulation of peripheral blood MNC of the patients with the relevant superantigen enabled the induction of synovial hyperplasia in the SCID mice. These results suggest that the pathogenic T cells could be activated locally in rheumatoid joints by certain antigens in some, but not in all patients with RA. PMID- 7560069 TI - Markedly inhibited 7-dehydrocholesterol-delta 7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes. AB - We investigated the enzyme defect in late cholesterol biosynthesis in the Smith Lemli-Opitz syndrome, a recessively inherited developmental disorder characterized by facial dysmorphism, mental retardation, and multiple organ congenital anomalies. Reduced plasma and tissue cholesterol with increased 7 dehydrocholesterol concentrations are biochemical features diagnostic of the inherited enzyme defect. Using isotope incorporation assays, we measured the transformation of the precursors, [3 alpha- 3H]lathosterol and [1,2-3H]7 dehydrocholesterol into cholesterol by liver microsomes from seven controls and four Smith-Lemli-Opitz homozygous subjects. The introduction of the double bond in lathosterol at C-5[6] to form 7-dehydrocholesterol that is catalyzed by lathosterol-5-dehydrogenase was equally rapid in controls and homozygotes liver microsomes (120 +/- 8 vs 100 +/- 7 pmol/mg protein per min, P = NS). In distinction, the reduction of the double bond at C-7 [8] in 7-dehydrocholesterol to yield cholesterol catalyzed by 7-dehydrocholesterol-delta 7-reductase was nine times greater in controls than homozygotes microsomes (365 +/- 23 vs 40 +/- 4 pmol/mg protein per min, P < 0.0001). These results demonstrate that the pathway of lathosterol to cholesterol in human liver includes 7-dehydrocholesterol as a key intermediate. In Smith-Lemli-Opitz homozygotes, the transformation of 7 dehydrocholesterol to cholesterol by hepatic microsomes was blocked although 7 dehydrocholesterol was produced abundantly from lathosterol. Thus, lathosterol 5 dehydrogenase is equally active which indicates that homozygotes liver microsomes are viable. Accordingly, microsomal 7-dehydrocholesterol-delta 7-reductase is inherited abnormally in Smith-Lemli-Opitz homozygotes. PMID- 7560070 TI - Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. AB - Secondary hyperparathyroidism is characterized by an increase in parathyroid (PT) cell number, and parathyroid hormone (PTH) synthesis and secretion. It is still unknown as to what stimuli regulate PT cell proliferation and how they do this. We have studied rats with dietary-induced secondary hyper- and hypoparathyroidism, rats given 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and rats after 5/6 nephrectomy for the presence of PT cell proliferation and apoptosis. PT cell proliferation has been measured by staining for proliferating cell nuclear antigen (PCNA) and apoptosis by in situ detection of nuclear DNA fragmentation and correlated with serum biochemistry and PTH mRNA levels. A low calcium diet led to increased levels of PTH mRNA and a 10-fold increase in PT cell proliferation. A low phosphate diet led to decreased levels of PTH mRNA and the complete absence of PT cell proliferation. 1,25 (OH)2D3 (25 pmol/d x 3) led to a decrease in PTH mRNA levels and unlike the hypophosphatemic rats there was no decrease in cell proliferation. There were no cells undergoing apoptosis in any of the experimental conditions. The secondary hyperparathyroidism of 5/6 nephrectomized rats was characterized by an increase in PTH mRNA levels and PT cell proliferation which were both markedly decreased by a low phosphate diet. The number of PCNA positive cells was increased by a high phosphate diet. Therefore hypocalcemia, hyperphosphatemia and uremia lead to PT cell proliferation, and hypophosphatemia completely abolishes this effect. Injected 1,25 (OH)2D3 had no effect. These findings emphasize the importance of a normal phosphate and calcium in the prevention of PT cell hyperplasia. PMID- 7560067 TI - Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue. AB - Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats. PMID- 7560071 TI - Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+) ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells. AB - Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+) dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells. PMID- 7560072 TI - Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype. AB - An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT1 gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial beta-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis. PMID- 7560073 TI - Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. AB - Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer. PMID- 7560074 TI - Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. AB - Previous studies suggest that while lung angiotensin converting enzyme (ACE) activity is reduced during chronic hypoxia, inhibitors of ACE attenuate hypoxic pulmonary hypertension. In an attempt to explain this paradox we investigated the possibility that whole lung ACE activity may not reflect local pulmonary vascular ACE expression. The experimental approach combined in vivo hemodynamic studies in control and chronically hypoxic rats, measurement of whole lung ACE activity, and evaluation of local pulmonary vascular ACE expression by in situ hybridization and immunohistochemistry. Total lung ACE activity was reduced to 50% of control activity by 5 d of hypoxia and remained low for the duration of the study. Immunohistochemistry showed a marked reduction of ACE staining in alveolar capillary endothelium. However, an increase in ACE staining was observed in the walls of small newly muscularized pulmonary arteries at the level of alveolar ducts and walls. In situ hybridization studies showed increased signal for ACE mRNA in the same vessels. Inhibition of ACE by captopril during chronic hypoxia attenuated pulmonary hypertension and markedly reduced distal muscularization of small pulmonary arteries. In addition, we demonstrated marked longitudinal variation in ACE expression along the normal pulmonary vasculature with the highest levels found in small muscular arteries associated with terminal and respiratory bronchioles. We conclude that local ACE expression is increased in the walls of small pulmonary arteries during the development of hypoxic pulmonary hypertension, despite a generalized reduction in alveolar capillary ACE expression, and we speculate that local arteriolar ACE may play a role in the vascular remodeling associated with pulmonary hypertension. PMID- 7560075 TI - Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels. AB - Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2 water channels. PMID- 7560076 TI - Genetic factors in lipoprotein metabolism. Analysis of a genetic cross between inbred mouse strains NZB/BINJ and SM/J using a complete linkage map approach. AB - A genetic cross was constructed from two parental inbred strains of mice, NZB/BINJ and SM/J, which differ markedly in their plasma lipoprotein levels. Plasma lipid and apolipoprotein values were measured in 184 F2 progeny on a normal chow diet and on an atherogenic diet. Genetic markers were typed at 126 loci spanning all chromosomes except the Y. Statistical analysis revealed significant linkage or suggestive linkage of lipoprotein levels with markers on a number of chromosomes. Chromosome 1 markers were linked to levels of total cholesterol (lod 5.9) and high density lipoprotein (HDL) cholesterol (lod 8.1), chromosome 5 markers were linked to levels of total cholesterol (lod 6.7) and HDL cholesterol (lod 5.6), and chromosome 7 markers were linked to levels of total plasma triglycerides (lod 5.1) and free fatty acids (lod 5.6). Plasma apoAII levels were linked to the apoAII gene (lod score 19.6) and were highly correlated with plasma HDL cholesterol levels (r = 0.63, P = 0.0001), indicating that apoAII expression influences HDL cholesterol levels. Molecular studies suggested that structural differences in the apoAII polypeptide of the two strains may contribute to differences in clearance of the protein. PMID- 7560077 TI - Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. AB - To clarify the physiological roles of CD36 as an oxidized low density lipoprotein (OxLDL) receptor, we analyzed the monocyte-derived macrophages from normal and two CD36-deficient subjects, since we identified the molecular abnormalities (Kashiwagi, H., Y. Tomiyama, Y. Kosugi, M. Shiraga, R. H. Lipsky, Y. Kanayama, Y. Kurata, and Y. Matsuzawa 1994. Blood. 83:3545-3552; and Kashiwagi, H., Y. Tomiyama, S. Honda, S. Kosugi, M. Shiraga, N. Nagao, S. Sekiguchi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1995. J. Clin. Invest. 95:1040-1046). Scatchard analysis of 125I-OxLDL binding showed a linear plot and the maximum binding was lower by approximately 40% in the macrophages from subjects with CD36 deficiency than those from normal controls. Competition studies showed that the uptake of 125I-OxLDL was suppressed by OKM5, an antibody against CD36, by 53% in normal control macrophages, but not in the CD36-deficient macrophages. After incubation with OxLDL for 24 h, cholesteryl ester mass accumulation was reduced by approximately 40% in the macrophages from CD36-deficient subjects than those from normal controls. These results suggest that CD36 is one of the physiological receptors for OxLDL. Since specific binding of OxLDL was only reduced by approximately 40% in spite of the complete deficiency of CD36, several other receptors also may have some role in OxLDL uptake. Further studies will be needed to assess the quantitative role of CD36 in foam cell formation in vivo. PMID- 7560078 TI - In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals. AB - Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID- 7560080 TI - Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions. AB - Both P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin are localized on the microvilli of neutrophils and have been implicated in the attachment of neutrophils to P-selectin or E-selectin. We directly compared the attachment and rolling of neutrophils on P-selectin and E-selectin under flow, with emphasis on the functions of PSGL-1 and L-selectin. Flowing neutrophils attached more avidly and rolled at lower velocities on P-selectin than on E-selectin at matched densities. Studies with purified molecules indicated that P-selectin and E selectin bound to a related site on PSGL-1 that overlapped the epitope for the anti-PSGL-1 mAb PL1. E-selectin bound with lower affinity than P-selectin to this site and also bound to an additional site(s) on PSGL-1.PL1 abolished adhesion of neutrophils to P-selectin under shear or static conditions, whereas DREG-56, a mAb to L-selectin, had no effect on adhesion to P-selectin. PL1 inhibited attachment of neutrophils to E-selectin under flow but not static conditions. DREG-56 also inhibited attachment of flowing neutrophils to E-selectin, and a combination of DREG-56 and PL1 nearly eliminated attachment to E-selectin under flow. These data suggest that PSGL-1 functions cooperatively with L-selectin to mediate optimal attachment of flowing neutrophils to E-selectin but not to P selectin. Neutrophils attach more efficiently and with greater strength to P selectin, perhaps because of the higher affinity of P-selectin for the PL1 defined site on PSGL-1. PMID- 7560079 TI - Phorbol esters induce death in MCF-7 breast cancer cells with altered expression of protein kinase C isoforms. Role for p53-independent induction of gadd-45 in initiating death. AB - Protein kinase C (PKC) modulates growth, differentiation and apoptosis in a cell specific fashion. Overexpression of PKC-alpha in MCF-7 breast cancer cells (MCF-7 PKC-alpha cell) leads to expression of a more transformed phenotype. The response of MCF-7 and MCF-7-PKC-alpha cells to phorbol esters (TPA) was examined. TPA treated MCF-7 cells demonstrated a modest cytostatic response associated with a G1 arrest that was accompanied by Cip1 expression and retinoblastoma hypophosphorylation. While p53 was detected in MCF-7 cells, evidence for TPA induced stimulation of p53 transcriptional activity was not evident. In contrast, TPA treatment induced death of MCF-7-PKC-alpha cells. Bryostatin 1, another PKC activator, exerted modest cytostatic effects on MCF-7 cells while producing a cytotoxic response at low doses in MCF-7-PKC-alpha cells that waned at higher concentrations. TPA-treated MCF-7-PKC-alpha cells accumulated in G2/M, did not express p53, displayed decreased Cip1 expression, and demonstrated a reduction in retinoblastoma hypophosphorylation. TPA-treated MCF-7-PKC-alpha cells expressed gadd-45 which occurred before the onset of apoptosis. Thus, alterations in the PKC pathway can modulate the decision of a breast cancer cell to undergo death or differentiation. In addition, these data show that PKC activation can induce expression of gadd45 in a p53-independent fashion. PMID- 7560081 TI - Sympathetic neural mechanisms in obstructive sleep apnea. AB - Blood pressure, heart rate, sympathetic nerve activity, and polysomnography were recorded during wakefulness and sleep in 10 patients with obstructive sleep apnea. Measurements were also obtained after treatment with continuous positive airway pressure (CPAP) in four patients. Awake sympathetic activity was also measured in 10 age- and sex-matched control subjects and in 5 obese subjects without a history of sleep apnea. Patients with sleep apnea had high levels of nerve activity even when awake (P < 0.001). Blood pressure and sympathetic nerve activity did not fall during any stage of sleep. Mean blood pressure was 92 +/- 4.5 mmHg when awake and reached peak levels of 116 +/- 5 and 127 +/- 7 mmHg during stage II sleep (n = 10) and rapid eye movement (REM) sleep (n = 5), respectively (P < 0.001). Sympathetic activity increased during sleep (P = 0.01) especially during stage II (133 +/- 9% above wakefulness; P = 0.006) and REM (141 +/- 13%; P = 0.007). Peak sympathetic activity (measured over the last 10 s of each apneic event) increased to 299 +/- 96% during stage II sleep and to 246 +/- 36% during REM sleep (both P < 0.001). CPAP decreased sympathetic activity and blood pressure during sleep (P < 0.03). We conclude that patients with obstructive sleep apnea have high sympathetic activity when awake, with further increases in blood pressure and sympathetic activity during sleep. These increases are attenuated by treatment with CPAP. PMID- 7560082 TI - Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet derived growth factor. AB - Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration. PMID- 7560083 TI - Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. AB - The assumption that cellular oxygen pressure (PO2) is close to zero in maximally exercising muscle is essential for the hypothesis that O2 transport between blood and mitochondria has a finite conductance that determines maximum O2 consumption. The unique combination of isolated human quadriceps exercise, direct measures of arterial, femoral venous PO2, and 1H nuclear magnetic resonance spectroscopy to detect myoglobin desaturation enabled this assumption to be tested in six trained men while breathing room air (normoxic, N) and 12% O2 (hypoxic, H). Within 20 s of exercise onset partial myoglobin desaturation was evident even at 50% of maximum O2 consumption, was significantly greater in H than N, and was then constant at an average of 51 +/- 3% (N) and 60 +/- 3% (H) throughout the incremental exercise protocol to maximum work rate. Assuming a myoglobin PO2 where 50% of myoglobin binding sites are bound with O2 of 3.2 mmHg, myoglobin associated PO2 averaged 3.1 +/- .3 (N) and 2.1 +/- .2 mmHg (H). At maximal exercise, measurements of arterial PO2 (115 +/- 4 [N] and 46 +/- 1 mmHg [H]) and femoral venous PO2 (22 +/- 1.6 [N] and 17 +/- 1.3 mmHg [H]) resulted in calculated mean capillary PO2 values of 38 +/- 2 (N) and 30 +/- 2 mmHg(H). Thus, for the first time, large differences in PO2 between blood and intracellular tissue have been demonstrated in intact normal human muscle and are found over a wide range of exercise intensities. These data are consistent with an O2 diffusion limitation across the 1-5-microns path-length from red cell to the sarcolemma that plays a role in determining maximal muscle O2 uptake in normal humans. PMID- 7560084 TI - Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes. AB - To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell. PMID- 7560085 TI - Identification of a new susceptibility locus for insulin-dependent diabetes mellitus by ancestral haplotype congenic mapping. AB - The number and exact locations of the major histocompatibility complex (MHC) linked diabetogenic genes (Idd-1) are unknown because of strong linkage disequilibrium within the MHC. By using a congenic NOD mouse strain that possesses a recombinant MHC from a diabetes-resistant sister strain, we have now shown that Idd-1 consists of at least two components, one in and one outside the class II A and E regions. A new susceptibility gene (Idd-16) was mapped to the < 11-centiMorgan segment of chromosome 17 adjacent to, but distinct from, previously known Idd-1 candidates, class II A, E, and Tap genes. The coding sequences and splicing donor and acceptor sequences of the Tnfa gene, a candidate gene for Idd-16, were identical in the NOD, CTS, and BALB/c alleles, ruling out amino acid changes in the TNF molecule as a determinant of insulin-dependent diabetes mellitus susceptibility. Our results not only map a new MHC-linked diabetogenic gene(s) but also suggest a new way to fine map disease susceptibility genes within a region where strong linkage disequilibrium exists. PMID- 7560086 TI - Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency. AB - Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus. PMID- 7560087 TI - Comparative distribution of the alpha 1(IV), alpha 5(IV), and alpha 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X linked Alport syndrome patients. AB - We have shown previously that the 5' ends of the genes for the alpha 5(IV) and alpha 6(IV) collagen chains lie head-to-head on Xq22 and are deleted in patients with Alport syndrome (AS)-associated diffuse leiomyomatosis. In this study, we raised a rabbit anti-human alpha 6(IV)chain antibody, demonstrated its specificity by the analysis of recombinant NC1 domains af all six type IV chains, and studied the distribution of the alpha 6(IV) chain in relation to the alpha 1(IV) and alpha 5(IV) chains in human adult and fetal tissues involved in AS and diffuse leiomyomatosis. The alpha 6(IV) chain colocalizes with the alpha 5(IV) chain in basement membranes (BMs) of many tissues, but not in glomerular BM. These data exclude the alpha 6(IV) chain as a site for AS mutations. The head-to head genomic pairing of the alpha 5(IV) and alpha 6 (IV) genes implies coordinate transcription of the two genes. Differential localization of the alpha 5(IV) and alpha 6(IV) chains shows that the two chains are not always coordinately regulated. The alpha 6(IV) chain, together with the alpha 3(IV)-alpha 5(IV) chains, was absent from all renal BMs in eight patients with X-linked AS while the alpha 1(IV) and alpha 2(IV) chains were increased. The data support the existence of two independent collagen networks, one for the alpha 3(IV)-alpha 6(IV) chains and one for the alpha 1(IV) and alpha 2(IV) chains. PMID- 7560089 TI - Loss of hepatic autoregulation after carbohydrate overfeeding in normal man. AB - To determine the effect of increased glycogen stores on hepatic carbohydrate metabolism, 15 nondiabetic volunteers were studied before and after 4 d of progressive overfeeding. Glucose production and gluconeogenesis were assessed with [2-3H] glucose and [6-14C] glucose (Study I, n = 6) or [3-3H] glucose and [U 14C]-alanine (Study II, n = 9) and substrate oxidation was determined by indirect calorimetry. Overfeeding was associated with significant (P < 0.01) increases in plasma glucose (4.97 +/- 0.10 to 5.09 +/- 0.11 mmol/liter), insulin (18.8 +/- 1.5 to 46.6 +/- 10.0 pmol/liter) and carbohydrate oxidation (4.7 +/- 1.4 to 18.0 +/- 1.5 mumol.kg-1.min-1) and a decrease in lipid oxidation (1.2 +/- 0.2 to 0.3 +/- 0.1 mumol.kg-1.min-1). Hepatic glucose output (HGO) increased in Study I (10.2 +/ 0.5 to 13.1 +/- 0.9 mumol.kg-1.min-1, P < 0.01) and Study II (11.17 +/- 0.67 to 13.33 +/- 0.83 mumol.kg-1.min-1, P < 0.01), and gluconeogenesis decreased (57.6 +/- 6.4 to 33.4 +/- 4.9 mumol/min, P < 0.01), indicating an increase in glycogenolysis. The increase in glycogenolysis was only partly compensated by an increase in glucose cycle activity (2.2 +/- 0.2 to 3.4 +/- 0.4 mumol.kg-1.min-1, P < 0.01) and the fall in gluconeogenesis, thus resulting in increased HGO. The suppression of gluconeogenesis despite increased lactate and alanine (glycerol was decreased) was associated with decreased free fatty acid (FFA) oxidation and negligible FFA enhanced gluconeogenesis. These studies suggest that increased liver glycogen stores alone can overwhelm normal intrahepatic mechanisms regulating carbohydrate metabolism resulting in increased HGO in nondiabetic man. PMID- 7560090 TI - Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. AB - In the HXB and BXH recombinant inbred strains derived from the spontaneously hypertensive rat and the normotensive Brown Norway rat, we determined the strain distribution patterns of 500 genetic markers to scan the rodent genome for quantitative trait loci regulating cardiac mass and blood pressure. The markers spanned approximately 1,139 cM of the genome and were tested for correlations with left ventricular mass adjusted for body weight, and with systolic, diastolic, and mean arterial pressures. The marker for the dopamine 1A receptor (Drd1a) on chromosome 17 showed the strongest correlation with left ventricular heart weight (P = .00038, r = -0.59) and the relationship to heart weight was independent of blood pressure. The markers showing the strongest correlations with systolic, diastolic, and mean arterial pressure were D19Mit7 on chromosome 19 (P = .0012, r = .55), D2N35 on chromosome 2 (P = .0008, r = .56), and Il6 on chromosome 4 (P = .0018, r = .53), respectively. These studies demonstrate that the HXB and BXH strains can be effectively used for genome scanning studies of complex traits and have revealed several chromosome regions that may be involved in the genetic control of blood pressure and cardiac mass in the rat. PMID- 7560088 TI - Human suction blister interstitial fluid prevents metal ion-dependent oxidation of low density lipoprotein by macrophages and in cell-free systems. AB - LDL in the circulation is well protected against oxidation by the highly efficient antioxidant defense mechanisms of human plasma. LDL oxidation contributing to atherosclerosis, therefore, has been hypothesized to take place in the interstitial fluid of the arterial wall. We investigated the antioxidant composition and the capacity to inhibit LDL oxidation of human suction blister interstitial fluid (SBIF), a suitable representative of interstitial fluid. We found that the concentrations in SBIF of the aqueous small-molecule antioxidants ascorbate and urate were, respectively, significantly higher (P < 0.05) and identical to plasma concentrations. In contrast, lipoprotein-associated lipids and lipid-soluble antioxidants (alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene) were present at only 8-23% of the concentrations in plasma. No lipid hydroperoxides could be detected ( < 5 nM) in either fluid. The capacity of serum and SBIF to protect LDL from oxidation was investigated in three metal ion dependent systems: copper, iron, and murine macrophages in Ham's F-10 medium. In all three systems, addition of > or = 6% (vol/vol) of either serum or SBIF inhibited LDL oxidation by > 90%. The concentration that inhibited macrophage mediated LDL oxidation by 50% was as low as 0.3% serum and 0.7% SBIF. The enzymatic or physical removal of ascorbate or urate and other low molecular weight components did not affect the ability of either fluid to prevent LDL oxidation, and the high molecular weight fraction was as protective as whole serum or SBIF. These data demonstrate that both serum and SBIF very effectively protect LDL from metal ion-dependent oxidation, most probably because of a cumulative metal-binding effect of several proteins. Our data suggest that LDL in the interstitial fluid of the arterial wall is very unlikely to get modified by metal ion-mediated oxidation. PMID- 7560091 TI - Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma. AB - Adenosine potentiates mast cell activation, but the receptor type and molecular mechanisms involved have not been defined. We, therefore, investigated the effects of adenosine on the human mast cell line HMC-1. Both the A2a selective agonist CGS21680 and the A2a/A2b nonselective agonist 5'-N ethylcarboxamidoadenosine (NECA) increased cAMP, but NECA was fourfold more efficacious and had a Hill coefficient of 0.55, suggesting the presence of both A2a and A2b receptors. NECA 10 microM evoked IL-8 release from HMC-1, but CGS21680 10 microM had no effect. In separate studies we found that enprofylline, an antiasthmatic previously thought to lack adenosine antagonistic properties, is as effective as theophylline as an antagonist of A2b receptors at concentrations achieved clinically. Both theophylline and enprofylline 300 micro completely blocked the release of IL-8 by NECA. NECA, but not CGS21680, increases inositol phosphate formation and intracellular calcium mobilization through a cholera and pertussis toxin-insensitive mechanism. In conclusion, both A2a and A2b receptors are present in HMC-1 cells and are coupled to adenylate cyclase. In addition, A2b receptors are coupled to phospholipase C and evoke IL-8 release. This effect is blocked by theophylline and enprofylline, raising the possibility that this mechanism contributes to their antiasthmatic effects. PMID- 7560092 TI - Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5. AB - The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects. PMID- 7560093 TI - Functional expression and segmental localization of rat colonic K-adenosine triphosphatase. AB - A putative cDNA for the colonic K-ATPase has recently been cloned (Crowson, M.S., and G. E. Shull. 1992. J. Biol. Chem. 267:13740-13748). Considerable evidence exists that there are two K-ATPases and active K absorptive processes in the rat distal colon: one that is ouabain sensitive and the other ouabain insensitive. The present study used the baculovirus expression system to express K-ATPase activity in insect Spodoptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), developed against a fusion protein produced from the 327 nucleotide fragment from 5' coding region of the putative K-ATPase cDNA, to identify the specific localization of the K-ATPase protein. K-ATPase activity (28.7 +/- 1.2 nmol inorganic phosphate/mg protein min) was expressed in plasma membranes isolated from Sf 9 cells infected with baculovirus containing recombinant DNA with the putative K-ATPase cDNA. Km for K for the K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibited by ouabain (1 mM); while the Ki for vanadate inhibition was 8.3 microM. Western blot analysis with the M-1 antibody identified a 100-kD protein in apical membranes prepared from distal, but not proximal, rat colon. Immunohistochemical studies with M-1 antibody localized K ATPase only in the apical membrane of surface cells, while an mAb (c464.6) against Na,K-ATPase localized basolateral membranes of both surface and crypt cells of rat distal colon. In conclusion, the putative K-ATPase cDNA encodes an ouabain-insensitive K-ATPase that is present only in the apical membrane of surface cells of rat distal colon. PMID- 7560094 TI - Impairment of selectin-mediated leukocyte adhesion to venular endothelium in spontaneously hypertensive rats. AB - The present study was designed to elucidate whether molecular mechanisms for leukocyte adhesion to microvascular endothelium may differ between spontaneously hypertensive rats and Wistar Kyoto rats. Leukocyte rolling and adhesion were investigated while monitoring venular wall shear rates in the mesenteric microcirculation stimulated with histamine or tert-butyl hydroperoxide in the two strains. In Wistar Kyoto rats, 10 microM histamine as well as 500 microM tertbutyl hydroperoxide promoted a significant reduction of venular leukocyte rolling velocity and subsequent adhesion. These changes in leukocyte behavior were blocked by monoclonal antibodies against P-selectin (PB 1.3) and against sialyl Lewis X-like carbohydrates (2H5). However, spontaneously hypertensive rats exhibited a blunted response of the stimulus-elicited leukocyte rolling, which was associated with impairment of venular P-selectin expression as well as a decrease in the expression of sialyl Lewis X-like carbohydrates on circulating neutrophils. No significant differences were detected between the two strains not only in the surface CD11b/CD18 expression but also in the CD18-mediated adhesivity of neutrophils to intracellular adhesion molecule-1 transfectants in vitro. These results suggest that impairment of selectin-mediated leukocyte adhesion is an event responsible for disorders of inflammatory responses in spontaneously hypertensive rats. PMID- 7560097 TI - Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. AB - This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals. PMID- 7560095 TI - Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth. AB - In addition to its well-known homoeostatic actions in the cardiovascular system, ET-1 has been shown to constitute a potent growth regulatory peptide in various tissues. We have studied the expression of ET-1 and its receptors (ET-Ar and ET Br) in human meningiomas (n = 35) as well as their involvement in cellular growth. By PCR of reverse-transcribed RNA we detected ET-1 mRNA in 91% (32 of 35), ET-Ar mRNA in 82% (29 of 35), and ET-Br mRNA in 42% (15 of 35) of human meningiomas examined. The localization of ET-1 mRNA, ET-Ar mRNA, and ET-1 peptide in tumoral cells was observed by in situ hybridization and immunohistochemistry, whereas ET-Br mRNA was expressed at low level only in cells belonging to blood vessels. In addition, we found that ET-1 stimulated [3H] thymidine incorporation in primary cell cultures of 20 meningiomas and that this effect could be blocked by BQ-123, a specific antagonist for ET-Ar. In contrast, RES-701-3, an antagonist of ET-Br, did not block the proliferative effect of ET-1. In conclusion, our data provide evidence that ET-1 constitutes an important growth factor for meningiomas acting via ET-Ar. We can hypothesize that ET-1, acting in concert with other growth factors and cytokines, is involved in the meningioma tumorigenesis. PMID- 7560098 TI - Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus. AB - Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder. PMID- 7560100 TI - Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. AB - To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content. PMID- 7560096 TI - Intratracheal instillation of keratinocyte growth factor decreases hyperoxia induced mortality in rats. AB - Alveolar type II cell proliferation occurs after many forms of lung injury and is thought to play a critical role in alveolar epithelial repair. Keratinocyte growth factor/fibroblast growth factor 7 (KGF) has been shown to promote alveolar type II cell growth in primary culture and alveolar epithelial hyperplasia in vivo. In this study, we used immunohistochemical analysis to determine the intrapulmonary distribution and cellular localization of recombinant human KGF (rhKGF) instilled into the trachea of rats. 6 h after administration, immunoreactive KGF was observed within the lung parenchyma and along alveolar epithelial cell membranes. By 18-24 h, KGF was detected intracellularly in alveolar epithelial cells and intraalveolar macrophages. Immunoreactive KGF was not demonstrable 48 h after delivery or in lung sections from PBS-treated animals. Intratracheal instillation of 5 mg/kg rhKGF stimulated a marked, time dependent increase in the alveolar type II cell specific labeling index to a maximum level of 33 +/- 3% 48 h after rhKGF administration compared with 1.3 +/- 0.3% after PBS instillation. In addition, this increase in type II cell proliferation in vivo was documented by flow cytometric analysis of isolated type II cells which revealed a nearly fivefold increase in the proportion of cells traversing through the S and G2/M phases of the cell cycle. To test the hypothesis that KGFs effects on type II cells in vivo might affect the response to lung injury, rats were treated with rhKGF and exposed to hyperoxia. Animals that received 1 or 5 mg/kg rhKGF exhibited dramatically reduced mortality (P < 0.001, for both doses). Survival for animals treated with 0.1 mg/kg rhKGF was not significantly different from either untreated rats or animals treated with heat denatured rhKGF. The lungs of rhKGF-treated animals that survived hyperoxia exposure had minimal hemorrhage and no exudate within the intraalveolar space. These experiments established that intratracheal administration of rhKGF stimulated alveolar type II cell proliferation in vivo and reduced hyperoxia induced lung injury in rats. Directed delivery of KGF to the lungs may provide a therapeutic strategy to preserve or restore the alveolar epithelium during exposure to hyperoxia or other injurious agents. PMID- 7560099 TI - A mouse model for the delta F508 allele of cystic fibrosis. AB - The most common cause of cystic fibrosis is a mutation that deletes phenylalanine 508 in cystic fibrosis transmembrane conductance regulator (CFTR). The delta F508 protein is misprocessed and degraded rather than traveling to the apical membrane. We used a novel strategy to introduce the delta F508 mutation into the mouse CFTR gene. Affected epithelia from homozygous delta F508 mice lacked CFTR in the apical membrane and were Cl-impermeable. These abnormalities are the same as those observed in patients with delta F508 and suggest that these mice have the same cellular defect. 40% of homozygous delta F508 animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. These animals should provide an excellent model to investigate pathogenesis and to examine therapies directed at correcting the delta F508 defect. PMID- 7560101 TI - Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene. AB - The human cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins. The activity of CETP results in a reduction in HDL cholesterol levels, but CETP may also promote reverse cholesterol transport. Thus, the net impact of CETP expression on atherogenesis is uncertain. The influence of hypertriglyceridemia and CETP on the development of atherosclerotic lesions in the proximal aorta was assessed by feeding transgenic mice a high cholesterol diet for 16 wk. 13 out of 14 (93%) hypertriglyceridemic human apo CIII (HuCIII) transgenic (Tg) mice developed atherosclerotic lesions, compared to 18 out of 29 (62%) controls. In HuCIII/CETPTg, human apo AI/CIIITg and HuAI/CIII/CETPTg mice, 7 of 13 (54%), 5 of 10 (50%), and 5 of 13 (38%), respectively, developed lesions in the proximal aorta (P < .05 compared to HuCIIITg). The average number of aortic lesions per mouse in HuCIIITg and controls was 3.4 +/- 0.8 and 2.7 +/- 0.6, respectively in HuCIII/CETPTg, HuAI/CIIIg, and HuAI/CIII/CETPTg mice the number of lesions was significantly lower than in HuCIIITg and control mice: 0.9 +/- 0.4, 1.5 +/- 0.5, and 0.9 +/- 0.4, respectively. There were parallel reductions in mean lesion area. In a separate study, we found an increased susceptibility to dietary atherosclerosis in nonhypertriglyceridemic CETP transgenic mice compared to controls. We conclude that CETP expression inhibits the development of early atherosclerotic lesions but only in hypertriglyceridemic mice. PMID- 7560102 TI - Beta-carotene inhibits atherosclerosis in hypercholesterolemic rabbits. AB - Oxidatively damaged LDL may be of central importance in atherogenesis. Epidemiological evidence suggests that high dietary intakes of beta-carotene and vitamin E decreases the risk for atherosclerotic vascular disease, raising the possibility that lipid-soluble antioxidants slow vascular disease by protecting LDL from oxidation. To test this hypothesis, we fed male New Zealand White rabbits a high-cholesterol diet or the same diet supplemented with either 1% probucol, 0.01% vitamin E, 0.01% all-trans beta-carotene, or 0.01% 9-cis beta carotene; then we assessed both the susceptibility of LDL to oxidation ex vivo and the extent of aortic atherosclerosis. As in earlier studies, probucol protected LDL from oxidation and inhibited lesion formation. In contrast, vitamin E modestly inhibited LDL oxidation but did not prevent atherosclerosis. While beta-carotene had no effect on LDL oxidation ex vivo, the all-trans isomer inhibited lesion formation to the same degree as probucol. Moreover, all-trans beta-carotene was undetectable in LDL isolated from rabbits fed the compound, although tissue levels of retinyl palmitate were increased. The effect of all trans beta-carotene on atherogenesis can thus be separated from the resistance of LDL to oxidation, indicating that other mechanisms may account for the ability of this compound to prevent vascular disease. Our results suggest that metabolites derived from all-trans beta-carotene inhibit atherosclerosis in hypercholesterolemic rabbits, possibly via stereospecific interactions with retinoic acid receptors in the artery wall. PMID- 7560103 TI - Sheep lung cytochrome P4501A1 (CYP1A1): cDNA cloning and transcriptional regulation by oxygen tension. AB - Lung cytochrome P450 activity has been linked to neoplasia and may produce reactive oxidant species and potent arachidonic acid metabolites. In lamb lung, oxygen breathing increases lung P450 activity, and inhibition of lung cytochrome P450 activity reduces oxygen-induced lung injury. The P4501A1 (CYP1A1) isozyme is present in many lung cells, including endothelial cells, and may therefore be involved in the pathogenesis of hyperoxic injury to microvascular endothelium. Therefore, to test the hypothesis that oxygen regulates P4501A1 gene expression in the lung, we cloned the sheep P4501A1 cDNA, and examined its regulation by oxygen breathing significantly increased lung P4501A1 RNA levels and that this increase preceded the increase in isozyme activity. Oxygen exposure also promptly increased P4501A1 RNA levels in cultured lamb lung microvascular endothelial cells but not in endothelial cells isolated from the main pulmonary artery or in lung smooth muscle cells. The oxygen-stimulated increase in P4501A1 RNA levels was not serum dependent, was unaffected by cycloheximide treatment, and could not be mimicked by treatment of the cells with oxygenated medium, conditioned medium, or by chemical oxidants. By nuclear run-on assay in cultured lung endothelial cells, oxygen increased the transcription rate of P4501A1 by almost fourfold after 90 min of oxygen exposure but had no significant effect on P4501A1 RNA stability. We conclude that oxygen tension, but not chemical oxidants, increases P4501A1 gene expression pretranslationally in lung microvascular endothelial cells. We speculate that oxygen induction of P450 activity in these cells may contribute to microvascular injury during oxygen breathing. PMID- 7560105 TI - The use of the temporalis muscle flap in facial and craniofacial reconstructive surgery. A review of 182 cases. AB - The authors report on their 16-year experience of reconstruction with the temporalis myofascial flap in 182 cases. All aspects of reconstructive cranio maxillofacial surgery are covered: trauma, deformities, tumours, TMJ ankylosis, facial paralysis. The temporalis myofascial flap was used both as a single and as a composite flap with cranial bone, coronoid process or skin island. Major complications were not observed. On the basis of their experience, the authors confirm the reliability, versatility and reproducibility of the use of this flap. This is due both to its rich blood supply and to its proximity to the reconstruction site. It is suggested that the use of the temporalis muscle flap should be taken into consideration before deciding on more extensive reconstructive procedures. PMID- 7560104 TI - Late-onset X-linked sideroblastic anemia. Missense mutations in the erythroid delta-aminolevulinate synthase (ALAS2) gene in two pyridoxine-responsive patients initially diagnosed with acquired refractory anemia and ringed sideroblasts. AB - X-linked sideroblastic anemia (XLSA) is caused by mutations of the erythroid specific delta-aminolevulinate synthase gene (ALAS2) resulting in deficient heme synthesis. The characteristic hypochromic, microcytic anemia typically becomes manifest in the first three decades of life. Hematologic response to pyridoxine is variable and rarely complete. We report two unrelated cases of highly pyridoxine-responsive XLSA in geriatric patients previously diagnosed with refractory anemia and ringed sideroblasts. A previously unaffected 77-yr-old male and an 81-yr-old female were each found to have developed severe hypochromic, microcytic anemia with ringed sideroblasts in the bone marrow, which responded dramatically to pyridoxine with normalization of hemoglobin values. Sequence analysis identified an A to C transversion in exon 7 (K299Q) of the ALAS2 gene in the male proband and his daughter. In the female proband a G to A transition was identified in exon 5 (A172T). This mutation resulted in decreased in vitro stability of bone marrow delta-aminolevulinate synthase activity. Each patient's recombinant mutant ALAS2 enzyme had marked thermolability. Addition of pyridoxal 5'-phosphate in vitro stabilized the mutant enzymes, consistent with the observed dramatic response to pyridoxine in vivo. This late-onset form of XLSA can be distinguished from refractory anemia and ringed sideroblasts by microcytosis, pyridoxine-responsiveness, and ALAS2 mutations. These findings emphasize the need to consider all elderly patients with microcytic sideroblastic anemia as candidates for XLSA, especially if pyridoxine responsiveness is demonstrated. PMID- 7560106 TI - Loose bodies in the temporomandibular joint. The advantages of arthroscopy. AB - Loose bodies are a rare cause of temporomandibular joint symptoms. Their main source is synovial chondromatosis. We report on clinical findings, diagnostic methods, treatment choices and outcome following the removal of loose bodies in 10 patients. Seven patients were evaluated and treated by means of arthroscopy, while in three patients open arthrotomy was performed. In five patients, no diagnostic imaging technique had demonstrated the presence of loose bodies prior to arthroscopy. In six patients, histology revealed synovial chondromatosis. In four patients, osteochondral fragments alone were found. Until now, the recommended treatment of choice for the removal of all loose bodies and of affected synovial tissue required open arthrotomy. We conclude that the advantages of arthroscopy consist in locating loose bodies that are not detectable radiologically and in reducing operative trauma. PMID- 7560107 TI - Surgically assisted, rapid maxillary expansion in adults. A retrospective long term follow-up study. AB - This study presents a technique for surgically assisted, rapid maxillary expansion performed on 20 patients and the results after orthodontic treatment. The mean age of the patients was 36.3 years and on average they had been followed up for 3 years and 6 months. The results seemed to be reliable long-term. The definitive expansion in the first molar region was 7.1 mm +/- 2.4 and in the canine region 4.8 mm +/- 2.7. The relapse measured after the observation period, was in the corresponding regions 1.2 mm +/- 1.3 and 0.2 mm +/- 2.1. The study also seemed to support the theory that the suture, anterior to the incisive canal, never ossifies until very late in life. PMID- 7560108 TI - Free fat transplantation in the face. AB - Several operative procedures have been described for correction of soft tissue defects in the face. Free fat transplantation has been criticised many times. In a twelve-year period, we have carried out 19 free fat transfers from the gluteal fold, of a size ranging from the palm of a child's hand to the size of an adult hand. Follow-up has been possible in 12 patients with 14 such transfers. Initially, grafts tended to be too large and subsequent fat reduction was required. It is apparent that the tendency to resorption of non-vascularised fat can only be estimated to a limited extent. Ultrasound examination in the course of long-term follow-up showed structural changes within the grafts but none was particularly striking. The fat has a good consistency on palpation particularly for the cheeks and remains in situ after healing. This technique still has a role in suitable cases. PMID- 7560109 TI - Computed tomographic evaluation of lymph node metastasis in head and neck carcinomas. AB - A retrospective study of 53 patients was undertaken to evaluate the efficacy of computer tomography (CT) in the detection of nodal metastases from carcinomas of the head and neck. The CT findings of 53 patients with head and neck carcinomas who underwent a total of 57 neck dissections were compared with the findings of physical examination (PE) and histopathological examination. Using node size larger than 10 mm in the short-axis diameter or the presence of central lucency as the criteria of nodal metastasis CT scanning staged correctly 52 of 57 necks, providing an accuracy of 91%, a sensitivity of 86% and a specificity of 100% in the detection of nodal metastases. There was agreement of PE findings with histology in 43 (74%) of 57 necks, with a sensitivity of 97% and a specificity of 38%, respectively. Because CT scanning was superior to PE in the pre-operative staging of head and neck carcinomas, it should be used for pre-operative evaluation of metastatic neck disease. PMID- 7560110 TI - Interstitial high-dose rate brachytherapy with iridium-192 in patients with oral squamous cell carcinoma. AB - Thirty-four patients with recurrent oral and oropharyngeal carcinomas were treated over a period of 4 years, by interstitial high-dose rate (HDR) brachytherapy (BT) using an iridium-192 source (Gammamed 2i and 12i equipment, Sauerwein, Germany) and fractionated application (1 up to 3 times, weekly recovery phases, single maximum dose 10 Gy). Pretreatment characteristics of patients in terms of irradiation (RT) and surgery differed (22 had external RT alone, with a total dose between 60.0 and 75.6 Gy; RT and surgery: 7; surgery alone: 1). The initial TNM-stages (UICC, Hermanek et al., 1987) of patients were: I = 2, II =3, III = 7, IV = 22. In the majority of cases, clinical indications for HDR-BT included tumour recurrence or progression following external RT, and second primary tumours of the oral cavity. Therapy was successful in most cases, i.e. complete remission: 11, partial remission: 16, no change: 2, progression: 5. Local control and overall survival rates, including patients surgically treated after BT, were at 6 months 58% and 62%, and 44% and 53% at 12 months, respectively. This type of treatment is recommended in patients with local recurrence or second primary tumours after previous external RT in the head and neck region. However, the benefit of interstitial HDR-BT remains questionable, particularly in patients with large tumours and lymph node metastases. PMID- 7560111 TI - Long-term quality of life after ablative intraoral tumour surgery. AB - The aim of the present retrospective study was to determine the long-term quality of life of patients who had undergone intraoral tumour resection. 135 patients with a malignant tumour located in the floor of the mouth and the adjacent area were enrolled in the study. A standard questionnaire was used to determine the physical functional status, the psychological status and social functioning of cancer patients (Schipper et al., 1984). The results were related to the T-stage, the size and the location of the intraoral soft tissue defect, the mode of reconstruction and the postoperative interval. The results showed a significant correlation of the Functional Living Index-Cancer (FLIC) score with the Karnowsky Index. The values were significantly lower in the higher T-stages. The location of the soft tissue defect, the type of soft tissue reconstruction and discontinuity resections of the mandible were crucial for postoperative quality of life, inasmuch as bilateral defects with loss of mandibular continuity and myocutaneous flap reconstructions showed significantly lower FLIC values. Reconstruction of mandibular continuity did not contribute to an increase in FLIC values. Dysphagia, reflux of food through the lips and nose during meals, decreased appetite and persistent pain significantly decreased the FLIC scores. It is concluded, that the FLIC is suitable for the determination of life quality in cancer patients since the score has shown the potential to reflect differences in postoperative life quality with regard to surgical procedures and functional sequelae. PMID- 7560112 TI - The innervation of the trapezius muscle: a cervical motor supply. AB - Controversy surrounds the nerve supply to the trapezius muscle. Anatomical studies have revealed independent connections from the C3 and C4 roots of the cervical plexus directly to the trapezius muscle. The objective of this study was to determine if these nerve bundles contain motor fibres and thus constitute an additional motor supply to trapezius. From a study of 32 cadavers, a representative example was chosen. Using a high powered operating microscope it was seen that such fibres were traceable to the ventral roots of the spinal cord. It is generally accepted that fibres in the ventral roots of the spinal cord are motor and thus, in this specimen, the trapezius muscle appeared to receive a direct motor supply from the cervical plexus independent of the accessory nerve. PMID- 7560113 TI - Pathogenesis of bromodeoxyuridine-induced cleft palate in mice. AB - This study was designed to examine the pathogenesis of bromodeoxyuridine-induced (BrdU) clefts of the secondary palate in mice. Intraperitoneal injections of BrdU (500 mg/kg body weight) were given on days 11 and 12 to some pregnant mice and on days 12 and 13, and days 11, 12 and 13 to others. Evaluation of craniofacial relations and palate development in BrdU-treated mice revealed inhibition of vertical development of the palatal shelves, mandibular hypoplasia which led to failure of downward displacement of the tongue and the creation of an obstacle to reorientation of the palatal shelves. The results of this study demonstrate a strong correlation between induction of cleft palate and the presence of structural alterations in the mandible, and the mechanism of BrdU-induced cleft palate resembles the defect in the Pierre Robin anomaly. PMID- 7560114 TI - Pulpal and apical changes secondary to segmental osteotomy in the mandible--an experimental study. AB - Posterior segmental osteotomies were performed on 7 dogs to investigate postoperative changes in the pulpal and periodontal tissue. Osteotomy cuts were made at various distances from the apices of roots. After 3 to 6 months, results were evaluated clinically, radiographically and histopathologically. Clinical and radiographic healing of the osteotomy sites was uneventful. Histological evaluation revealed that when the osteotomy cuts were made at a safe distance (3 5 mm) from the apices of roots, neither important pulpal degeneration nor loss of teeth occurred. Even two of the teeth whose apices were cut off had completely healthy pulpal and periapical tissues. The periodontium in almost all of the cases was also found to be healthy. PMID- 7560115 TI - Orthognathic surgery in osteogenesis imperfecta: a case report with management considerations. AB - Osteogenesis imperfecta is a disease of connective tissues with additional metabolic defects. It is associated with recognisable facial disproportion and sometimes warrants surgical intervention for aesthetic, functional and psychological reasons. A severe case of osteogenesis imperfecta type 3 is presented to illustrate the feasibility of bimaxillary surgery using a mandibular body step osteotomy and maxillary down grafting at the Le Fort I level. The medical, anaesthetic, surgical and specific maxillofacial implications of surgery in these patients are discussed. PMID- 7560116 TI - Simulation and counterconditioning as adjuncts to pharmacotherapy for invasive pediatric procedures. AB - Behavioral counterconditioning was conducted during simulated medical routines to supplement medical management of five children's distress during invasive procedures (bone marrow aspiration, lumbar puncture, tracheostomy care, venipuncture, and finger pricks). Preferred activities were paired in vivo with medical stimuli, and differential positive reinforcement was provided contingent on engagement with the preferred activities and on compliance with adult instructions, first during simulations and later during actual procedures. Data on cooperation, escape/avoidance, and negative vocalizations were evaluated using both single-subject experimental methods and a baseline-treatment group statistical comparison. Results support the benefits of this approach as an adjunctive intervention to decrease behavioral distress in some children requiring repeated invasive procedures. Discussion emphasizes the potential for training medical personnel to implement these techniques preventively, for decreasing or discontinuing sedation for some children treated under local anesthesia, and for preparing children with developmental disabilities for invasive procedures. PMID- 7560117 TI - The role of genetic factors in conduct disorder based on studies of Tourette syndrome and attention-deficit hyperactivity disorder probands and their relatives. AB - To examine the role of genetic factors in oppositional defiant disorder (ODD) and conduct disorder (CD), 38 variables relating to the relevant DSM-III-R criteria, as well as other angry and aggressive behaviors, were examined in 1177 Tourette syndrome (TS) and attention-deficit hyperactivity disorder (ADHD) probands, their first-degree relatives, and controls. Two techniques were used: (1) a genetic loading technique comparing the frequency of symptoms in groups with progressively less genetic loading for Gts and ADHD genes, and (2) comparison of the frequency of symptoms in relatives with, versus relatives without, TS or ADHD. When significant, the latter rules out ascertainment bias and inappropriate controls. For TS, the results were significant with most p values less than 10( 8). The same trends were seen in the smaller number of ADHD families. A polygenic model is proposed in which TS and ADHD alone represent lesser degrees of genetic loading and expression, and TS + CD not equal to ADHD represents a higher degree of genetic loading and expression of genes common to all three disorders. These studies emphasize the important role of genetic factors in ODD and CD. The therapeutic implications are discussed. PMID- 7560118 TI - Specificity of preventative pediatric intervention effects in early infancy. AB - The efficacy of two contrasting short term preventative interventions administered to a heterogeneous sample of new mothers during the perinatal period was evaluated. The first was infant-centered and used the Brazelton Neonatal Behavioral Assessment Scale (NBAS) as a method of highlighting newborn behavior to new mothers. The second was mother-centered and consisted of an in-depth interview focused on the mothers' concerns about parenting. Mothers were randomized into groups and were administered intervention by experienced clinicians at 3 days in the hospital and again at 14 and 30 days at home. Effects of intervention on maternal reports of parenting stress, mother-infant interactive behavior, and infant developmental quotient were evaluated at 4 months infant age. It was hypothesized that participation in the infant-centered intervention would be related to more positive maternal and infant outcomes at 4 months. It was also expected that the impact of each intervention would be moderated by differences in maternal and infant risk and parity. Hierarchical multiple regressions controlling for risk and parity yielded no significant main effects of intervention at 4 months. However, significant interactions of intervention with parity and risk were observed, indicating that intervention was beneficial for specific groups of mothers. These data suggest that early intervention should be tailored to the needs of individual groups of mothers. PMID- 7560119 TI - School observations of children with attention-deficit hyperactivity disorder and comorbid tic disorder: effects of methylphenidate treatment. AB - Although the findings from recent controlled studies suggest that methylphenidate is a safe and effective treatment for many children with attention-deficit hyperactivity disorder (ADHD) and comorbid tic disorder, relatively little is known about drug effects on school behavior. Thirty-four prepubertal children with ADHD and tic disorder received placebo and 3 doses of methylphenidate (0.1, 0.3, and 0.5 mg/kg) twice daily for 2 weeks each under double-blind conditions. Treatment effects were assessed using direct observations of child behavior in classroom, lunchroom, and playground settings. Treatment with methylphenidate resulted in marked reductions of hyperactive, disruptive, and aggressive behavior, which was evident even for the 0.1 mg/kg dose. There were no "nonresponders." The only observed changes in tics were a small but statistically significant increase in the frequency of motor tics (classroom, 0.1 mg/kg dose) and a tendency for fewer vocal tics (lunchroom). However, these changes in motor tic frequency were not perceived by care providers as a worsening in the severity of the child's tic disorder. Most dose-response relationships were linear, but the mean (operationally defined) minimal effective dose (MED) was 0.3 mg/kg. In summary, the findings support the conclusions that (1) methylphenidate suppresses ADHD behaviors in the classroom and aggressive behavior in all settings and that (2) a low dose may have a weak exacerbation effect on the frequency of motor tics; but, in general, the majority of youngsters do not experience clinically significant tic worsening with a MED. PMID- 7560120 TI - An examination of reliability in developmental research. AB - The purpose of this investigation was to examine quantitative methods used to determine reliability in developmental research. Procedures used to compute reliability estimates in 30 studies published in three developmental journals were examined. Four types of reliability studies were identified and analyzed. These included interrater reliability, stability (test-retest and intrarater reliability), equivalence reliability, and internal consistency. Interrater reliability investigations were the most frequently reported in the developmental literature reviewed (45%). The Pearson product moment correlation (r) was the most commonly reported reliability statistic. The findings reveal that researchers in developmental pediatrics frequently analyze reliability data using the Pearson product moment correlation and interpret the results as indicating consensus (agreement) among raters or across instruments. The Pearson product moment correlation (r) provides information on covariation among variables but does not indicate agreement. Thus, the findings suggest that developmental researchers may be misinterpreting the statistical results of reliability investigations. The argument is made that the intraclass correlation coefficient (ICC) is a more appropriate method of analysis when the purpose of the research is to examine consensus. PMID- 7560122 TI - Homesickness and the use of a camp infirmary: a preliminary report. AB - Homesickness is a problem that is well known to parents but is little discussed in the medical literature. The goal of this study was to describe the prevalence of homesickness at one summer camp and to assess the hypothesis that children who are homesick are more frequent users of the camp infirmary than children who are not homesick. Homesickness was assessed by a questionnaire devised by the authors that was completed by the child's counselor at the end of camp. Homesickness was found in 20% of the campers. Two of three measures of homesickness derived from the questionnaire were significantly associated with multiple visits to the infirmary, and the third measure showed a trend in the same direction. Our findings led us to the conclusion that homesickness is a common problem and that multiple visits to a camp infirmary might be a warning signal of lack of adjustment to the camp environment. PMID- 7560121 TI - Infant-mother attachment and nutrition in children with cystic fibrosis. AB - The association between nutritional status (percentage of weight for height) and infant-mother relationship was studied over 4 years in 38 children diagnosed with cystic fibrosis in the first year of life. Infant-mother relationship was assessed in a standardized laboratory observation. Although they could not be distinguished medically from the others at the time of diagnosis, infants showing a specific form of insecure relationship with their mothers (insecure-avoidant) differed from the others in: (1) failure to improve in nutritional status in the first year; (2) continuing decline in weight for height in the first 3 years; and (3) significantly lower weight for height at 1, 2, and 3 years of age. These data suggest that attention to mother-infant relationships, particularly feeding interactions, may improve nutritional status in children with cystic fibrosis. PMID- 7560124 TI - The discovery of marijuana use by a parent of an early adolescent. PMID- 7560125 TI - Developmental delay and psychopathology in young children. Introduction. PMID- 7560123 TI - Attention-deficit hyperactivity disorders in adolescence. PMID- 7560126 TI - Attachment and risk for psychopathology: the early years. PMID- 7560128 TI - Family functioning and child rearing in an urban environment. PMID- 7560129 TI - The role of comorbidity in the differential diagnosis of young children with deviant behavior. PMID- 7560127 TI - Intervention with children with developmental disabilities and attachment disorders. PMID- 7560130 TI - Multiplex developmental disorder. PMID- 7560132 TI - Hopelessness predicts future depressive symptoms: a prospective analysis of cognitive vulnerability and cognitive content specificity. AB - Beck's content specificity hypothesis predicts distinct cognitive content within specific psychological disorders. We evaluated whether the third component of the "cognitive triad", negative view of the future (hopelessness), would be related 4 weeks later to depressive symptoms, but not to anxiety. University student participants (83 females, 71 males) were tested on the Beck Depression Inventory and the Beck Anxiety Inventory at two points in time, separated by 4 weeks. The Hopelessness Scale was administered at time 1 and a Life Events Inventory at time 2. Concurrent cognitive content specificity was replicated. For males only, hopelessness predicted future depression severity scores, but not anxiety. Hopelessness predicted depressive symptoms over and above life event stressors, but not vice versa. PMID- 7560131 TI - Development of an instrument for assessing self-efficacy in schizophrenic spectrum disorders. AB - Self-efficacy constructs recently have been incorporated into a model of social functioning for schizophrenics. This model proposes that self-efficacy mediates patients' coping efforts. To assess this hypothesis accurately, it is critical to have a scale that measures self-efficacy reliably and validly. Such a scale was developed in this research, which used two independent samples of schizophrenic spectrum patients. Results indicate that this scale is both valid and reliable. The data support the validity of two underlying subscales, one that measures positive symptom self-efficacy and one that measures negative symptom/social interaction self-efficacy. Future directions of psychosocial constructs in schizophrenic-symptom development are discussed, including the need for concurrent tracking of symptom development and self-efficacy. PMID- 7560133 TI - A meta-analysis of antidepressant outcome studies that involved children and adolescents. AB - A meta-analysis was performed of available studies that document response to antidepressant medications in the pediatric population, based upon guidelines developed in a methodological review of adult antidepressant response (Greenberg, Borstein, Greenberg, & Fisher, 1992). The 27 studies accessed through standard medical and psychological data bases were assessed against the following criteria: degree of blindness, placebo group design, rating issues, sample size, inclusion criteria, and other methodological issues. Serious methodological flaws emerged in all areas, as well as an inverse relationship between positive findings and the presence of adequate experimental controls. Recommendations are made for improvements in experimental methodology, and caution is urged in the absence of well-controlled studies. PMID- 7560134 TI - Eating problems and interpersonal functioning among several groups of women. AB - This paper examines the relationship between eating disorders and problems in interpersonal functioning. Questionnaires that measure eating problems, interpersonal adjustment, and general neuroticism were completed by several groups of women: anorexic patients, persons whose interests or occupations involved a concern about bodily shape or condition (dancers, models, and athletes), and members of the general public. A substantial and significant relationship was found between eating symptomatology and interpersonal functioning in all groups. However, this association survived only in the anorexic patient group when general neuroticism was partialled out. We take this to imply that psychosocial difficulties are unlikely to play a strong role in the initial development of eating problems. PMID- 7560137 TI - Differential sensitivity of the MMPI-2 depression scales and subscales. AB - The MMPI-2 was administered to 122 psychiatric inpatients (62 female, 60 male, mean age 37 years). The various MMPI-2 depression scales and subscales were examined to compare their relative sensitivity. Using the 65T cut-off, scales DEP and D identified approximately 60% and 62%, respectively, as suffering depressotypal symptoms. Using a 70T cut-off, scales D1, D2, D3, D4, D5, D Obvious, and D-Subtle identified 53%, 24%, 37%, 57%, 45%, 53%, and 3%, respectively, as suffering depressotypal symptoms. These findings are discussed in terms of their implications for clinical practice. PMID- 7560136 TI - Development of a three-scale MMPI: the MMPI-TRI. AB - A 60-item short form of the MMPI with very high content validity and items that appear on both the MMPI and MMPI-2 was developed and named the MMPI-TRI. It contains three 20-item scales--the Subjective Distress, Acting-Out, and Psychosis scales. These three scales have excellent internal consistency and sufficient independence from each other. An anxiety and depression group of patients, prison inmate group, and a schizophrenic and other psychotic group had the highest mean scores on Subjective Distress, Acting-Out, and Psychosis, respectively. Correlations with the 13 regular scales of the MMPI and MMPI-2, their content and supplementary scales, and four other psychometric instruments provided very strong evidence for validity. Norms are provided. PMID- 7560139 TI - Dimensional characteristics of the SCL-90-R: evaluation of gender differences in dually diagnosed inpatients. AB - The dimensional characteristics of the SCL-90-R were assessed for both male and female dually diagnosed inpatients. Subjects included 402 males and 227 females who presented with concurrent psychiatric and substance use disorders at a private psychiatric hospital. Confirmatory factor analyses resulted in the rejection of four previous measurement models for both genders including the original nine symptom dimensions. Subsequent principal component analyses resulted in four- and seven-component solutions for males and females, respectively; the majority of item covariation was accounted for by the first component. These results are consistent with previous research that has suggested a primary global distress factor and are in contrast to studies that have demonstrated factorial invariance across gender. PMID- 7560138 TI - Assessment of self-control behavior: validity analyses on a Japanese version of the self-control schedule. AB - To investigate the validity of a Japanese translation of Rosenbaum's Self-Control Schedule (SCS), 338 Japanese undergraduates were administered the scale. Construct validity of the SCS subscales, Constructive Self-Control and Ineffective Self-Control, in this study was achieved by correlations with coping responses. In addition, multiple regression analyses showed that Constructive Self-Control contributed to a negative prediction of future depression and psychosomatic symptoms, and Ineffective Self-Control predicted future depression and psychosomatic symptoms. These findings indicated that the Japanese version of the SCS employed in this study appeared to be a valid and promising measure of self-control. PMID- 7560140 TI - Event-related potentials during delayed recognition of Wechsler Memory Scale-R paired associate learning. AB - The P300 is a component of cortical event related-potentials that has been linked to memory and recognition. This study examined P300 amplitude and latency during recognition of pairs of words from the WMS-R Verbal Paired Associate Learning subscale. After 24 subjects learned the pairs of words, they were shown pairs of words taken directly from the subscale that were combined to form matching or nonmatching pairs. This parallels the Delayed Recall subtest of the WMS-R. P300 amplitude was greater during recognition of matching word pairs and most prominent at central and parietal recording sites. P300 latency was shorter for matching than for nonmatching pairs. This points to a degree of psychophysiological validity of the WMS-R paired associate learning delayed recall task. PMID- 7560141 TI - Clinical utility of Kaufman's "amazingly" short forms of the WAIS-R with educable mentally retarded adolescents. AB - This study investigated the comparability between three "amazingly" short forms of the WAIS-R (a dyad, triad, and tetrad) suggested by Kaufman, Ishikuma, and Kaufman-Packer (1991) and the complete battery among a sample of 114 educable mentally handicapped adolescents. Results indicate that the short forms are highly correlated with the complete battery Full Scale IQs, and all three short forms yielded scores, on average, within 2 points of the complete battery. Analyses at an individual level revealed that more than 90% of the sample fell within 3 SEM (9 points) of the FSIQ for all three short forms. Overall results are consistent with previous studies and support the use of these "amazingly" short forms. PMID- 7560142 TI - Chance performance in the absence of norms for neuropsychological tests--a clinical note. AB - Some widely used neuropsychological tests have few normative data for interpretation. Chance bases for performance are a neglected baseline. Below chance performance also may be diagnostic, e.g., malingering. PMID- 7560135 TI - Clinical diagnoses and MCMI codetypes. AB - From a comprehensive literature review of all papers published on the Millon Clinical Multiaxial Inventory (I/II) from 1980-1993, MCMI test codetypes associated with various personality and clinical disorders were derived from the empirical literature. They are presented in an Appendix for clinical and research interests. PMID- 7560143 TI - The effects of cognitive pretraining on cohesion and self-disclosure in small groups: an analog study. AB - This study examined the effects of written, cognitive pretraining on the development of cohesion and self-disclosure in interpersonal learning groups. Subjects in the experimental condition received a written introduction to the group experience, and subjects in the control condition received written information on the history of group psychotherapy. Results demonstrated that the experimental groups had a higher level of group cohesion than the control groups, p < .0001, but were not different on self-disclosure. The correlation between cohesion and self-disclosure was not statistically significant. The results were discussed as an extension of the empirical support for cognitive pretraining. PMID- 7560144 TI - Test-retest evaluation of a four-subtest WAIS-R short form with young offenders. AB - The validity of a four-subtest short form (SF4) of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) was evaluated in a sample of incarcerated Caucasian and Native youth 16 to 18 years of age. A test-retest methodology was used in which 21 subjects received SF4 first and 26 subjects received the full WAIS-R first. Results showed that SF4 has validity as an estimate of Full Scale IQ, but with accuracy limitations. In addition to the IQ overestimates and misclassification rates that have been found in other short form research, this study showed that a large overestimate of about 11 points occurred on average when SF4 followed the standard administration. PMID- 7560145 TI - Issues in the assessment of personality disorder and substance abuse using the Millon Clinical Multiaxial Inventory (MCMI-II). AB - Substance abuse treatment clients often present other severe mental health problems that affect treatment outcomes. Hence, screening and assessment for psychological distress and personality disorder are an important part of effective treatment, discharge, and aftercare planning. The Millon Clinical Multiaxial Inventory-II (MCMI-II) frequently is used for this purpose. In this paper, several issues of concern to MCMI-II users are addressed. These include the extent to which MCMI-II scales correspond to DSM-III-R disorders; overdiagnosis of disorders using the MCMI-II; accuracy of MCMI-II diagnostic cut off scores; and the clinical utility of MCMI-II diagnostic algorithms. Approaches to addressing these issues are offered. PMID- 7560147 TI - Responding to graduate students' professional deficiencies: a national survey. AB - Seventy-one doctoral programs in professional psychology were surveyed with respect to the incidence of students' professional deficiencies and related procedures. Professional deficiencies, particularly limited clinical skills and personality/emotional problems, were reported by 89% of the respondents, twice the number that have official policies for dealing with such problems. Incidence of limited clinical skills was related positively to interviewing applicants and use of practicum grades for evaluation and related negatively to using recommendation letters and reliance on clinical supervisor's assessment. Responses to deficiencies reflected appropriate due process concepts and were most likely to include terminating students and psychotherapy referral. Recommendations are made for definitions of professional competencies, development of policies, and research on the reliability and validity of screening and evaluation mechanisms. PMID- 7560146 TI - Prevalence estimates of alcohol problems in a Veterans Administration outpatient population: AUDIT vs. MAST. AB - Prior reports have indicated that the prevalence of alcohol-related problems in VA patients is significantly higher than that found in the general population. Prevalence rates, however, are likely to be affected by the screening instrument employed. A sample of 722 VA outpatients awaiting general medical and urgent care treatment was asked to complete two screening questionnaires: the World Health Organization's AUDIT and the brief MAST. Of 508 completed AUDITs, 55 (11%) scored above the cut-offs of 11 for harmful consumption, and 85 (17%) above the more liberal cut-off score of 8; whereas of 501 completed MASTs, 104 (21%) scored above the cut-off. Differences in identification rates are discussed. PMID- 7560149 TI - Career pathways and current practice patterns of clinical and counseling psychologists: a follow-up study of former interns. AB - The present study examined career pathways and current work patterns for 110 graduates of an internship program. Results show that the group trained after 1980 went into private practice more rapidly than those trained prior to 1980. Both groups do extensive psychological testing. Implications are discussed. PMID- 7560148 TI - Differences in family of origin functioning among graduate students of different disciplines. AB - Compared the degree of dysfunction in the family of origin of psychology graduate students to that of graduate students in the disciplines of business, education, engineering, and health sciences. Students were accessed through University of Alaska Anchorage graduate departments. Family background was assessed with the Index of Family Relations (IFR; Hudson, 1990a), the Self-Report Family Inventory (SFI; Beavers, Hampson, & Hulgus, 1990), and relevant demographics obtained from a biographical questionnaire. Results indicated significant differences among the student groups; psychology graduate students demonstrated higher degrees of family dysfunction than students in other disciplines. Implications of these findings for psychology graduate training programs are discussed. PMID- 7560150 TI - Clinical interventions by psychologists with lesbians and gay men. AB - A questionnaire that assesses methods of intervention with, and attitudes toward, lesbians and gay men was completed by 139 clinical psychologists. None claimed to use aversion techniques to change sexual orientation. Eleven percent reported use of alternative methods (e.g., psychoanalytic techniques) to change sexual orientation, whereas 5.8% supported the use of aversion therapy for this purpose. Viewing homosexuality as "unacceptable" predicted both the use of alternative therapies and support of aversion therapy to change sexual orientation. PMID- 7560152 TI - Changes in transmitral and pulmonary venous flow patterns in the first day of life. AB - The purpose of this study was to see the relationship between changes in pulmonary venous flow velocities and those in transmitral flow velocities during the first day of life. A serial Doppler echocardiography was performed in 24 normal neonates at 2, 12, and 24 hours of age. The size of the ductus arteriosus was 4.3 +/- 0.5 mm at 2 hours and 2.1 +/- 0.7 mm at 12 hours, and closed in 21 of 24 neonates at 24 hours. The peak systolic pulmonary venous flow (peak S), peak diastolic pulmonary venous flow (peak D), peak D/S, peak velocity at early diastolic filling (peak E), and peak E/A, which were high at 2 hours, decreased significantly at 12 hours but remained constant thereafter. The size of the ductus arteriosus was found to be correlated with peak S, peak D, and peak D/S. There was a direct correlation between peaks E and D (r: 0.64, p < 0.01) as well as a direct correlation between peaks E/A and D/S (r: 0.36, p < 0.05). These results indicate that the diastolic pulmonary venous flow is determined by the same factors that influence the transmitral flow. These waveforms may reflect the increase in pulmonary circulatory volume by left-to-right shunting through the ductus arteriosus. PMID- 7560151 TI - Rey 15-item memorization and Dot counting scores in a "stress" claim worker's compensation population: relationship to personality (MCMI) scores. AB - The relationship between apparent faking of cognitive impairment (as detected by such "malingering" tests as 15-item Memorization and Dot Counting) and faking of psychiatric symptoms has not been investigated formally. In addition, no empirical literature is available on the relationship between personality traits and faking of cognitive symptoms. Of 154 subjects who claimed "stress" psychiatric injury, 12% appeared to be faking cognitive impairment; 4.5% failed the 15-item Memorization Test, and 10% failed the Dot Counting task. Faking of cognitive symptoms occurred in only 23% of subjects who were faking/exaggerating psychological symptoms on the MCMI. Malingering test failure was associated with significant elevations on MCMI personality scales, although this appeared to be an artifact of attempts to fake/exaggerate on the MCMI, rather than a reflection of "true" personality traits. PMID- 7560153 TI - Hypertensive intra-arterial chemotherapy for endometrial carcinoma assessed by transvaginal Doppler ultrasound and magnetic resonance imaging. AB - We examined the effectiveness of hypertensive intra-arterial chemotherapy for endometrial carcinoma using transvaginal Doppler ultrasound and magnetic resonance imaging. Angiotensin II, 100 mg cisplatin, and 40 mg doxorubicin were prescribed for 8 patients with endometrial carcinoma (3 stage Ia; 3 stage Ib; 2 stage II). The resistance index (RI) was obtained for intratumoral blood flow velocity waveforms by transvaginal Doppler ultrasound and changes in RI (delta RI: differences before and after chemotherapy) were calculated. The tumor volume (TV) was also evaluated, based on the T2-weighted image of magnetic resonance imaging (MRI). The decrease in tumor size [DR-T: (TV before chemotherapy--TV after chemotherapy)/TV before chemotherapy x 100] was determined. RI measurements did not correlate with TV, either before or after chemotherapy. The delta RI varied from 0.007 to 0.615 (mean: 0.207) and DR-T varied from 20.1% to 65.0% (mean: 45.5%). The correlation between delta RI and DR-T [DR-T = 23.5 + 167.2 (delta RI)-165.6 (delta RI)2; R2 = 0.772, p < 0.05] was significant. Therefore, we confirmed the effectiveness of hypertensive intra-arterial chemotherapy for endometrial carcinoma using both transvaginal Doppler ultrasound and MRI. PMID- 7560157 TI - Flow reversal in the right anterior branch of the portal vein: "bicolor portal vein" color Doppler analysis. AB - The purpose of this study is to describe a phenomenon of bidirectional flow, "bicolor portal vein" (BPV), within the right anterior branch of the portal vein (RAPV), with color Doppler imaging (CDI). We prospectively studied with CDI the intrahepatic portal vein and its branches in 316 consecutive patients in search of areas of nonlaminar flow within the RAPV. Forty patients were excluded from the study due to varied conditions that precluded an accurate CDI study. A prospective design allowed us to record the frequency of appearance, systematically measure a set of parameters in each patient, and establish different groups for comparison. We found an area of BPV within the RAPV in six patients. Five of them had a history of liver disease (8.2% of the patients with known liver disease). The BPV was consistently portrayed as a well-defined blue oval-shaped area of flow reversal attached to the medial side of the RAPV immediately before its bifurcation, suggesting a rotary motion of blood. All six patients presented a transverse diameter of the RAPV significantly larger than the rest of the patients (p < 0.05). Furthermore, blood flow measurements within the RAPV of the six patients with BPV were found to be significantly higher when compared with the rest of the patients (< 0.05). Knowledge of the possible presence of a BPV within the RAPV can prevent diagnostic confusion with reversal of flow, a truly pathologic condition related to liver disease. PMID- 7560154 TI - Abdominal tuberculosis: comparison of sonography and computed tomography. AB - Sonographic and computed tomography findings were retrospectively compared in 13 patients with proven abdominal tuberculosis who had both diagnostic modalities in their workup. The patients were treated over 9 years in two general hospitals in Kuwait. Ultrasound could detect the same findings as CT in 9 of 13 patients. Both had the same accuracy in detecting the type of ascites (in 6 patients) and solid organ tuberculosis (four lesions). Ultrasound gave useful information but could not completely replace CT as it detected less bowel thickening (3 of 5 lesions) and abdominal tuberculous lymphadenopathy (1 of 4 lesions) than CT scanning. PMID- 7560158 TI - Ultrasound appearance of primary carcinoid tumor of the gallbladder associated with carcinoid syndrome. PMID- 7560156 TI - Pylorus subjacent to the gallbladder: an additional finding in hypertrophic pyloric stenosis. AB - We have noted that sonographically positive cases of hypertrophic pyloric stenosis (HPS) demonstrate a distortion of normal anatomy in which the elongated pylorus displaces the duodenum and lies adjacent to and below the gallbladder. Ten consecutive cases of HPS diagnosed by ultrasound were reviewed. In each case the pylorus was imaged subjacent to the gallbladder. Sonograms from 100 infants from a different institution were then reviewed. There were 13 cases with the pylorus subjacent to the gallbladder, 12 of these met criteria for HPS. Recognition of this anatomic relationship may aid in the diagnosis of HPS. PMID- 7560155 TI - Antenatal sonographic detection of the proximal esophageal segment: specific evidence for congenital esophageal atresia. AB - To determine the biologic significance of a transient anechoic area observed in the midline of the fetal neck when attempting an antenatal ultrasound diagnosis of congenital esophageal atresia (CEA), a prospective study was made in 10 cases presenting both polyhydramnios and an unusually small stomach size due to a decrease in fetal stomach fluid. There were 8 cases indicating a transient anechoic area in the fetal neck, all of which were diagnosed as having CEA postnatally by plain roentgenogram, neonatal surgery, or autopsy findings. The remaining 2 cases had no CEA; one had Nager's syndrome and the other, a disorder involving neuronal migration in the central nervous system. These results suggest that an anechoic area in the middle of the fetal neck can be used as an indication of CEA and also for differentiating this condition from diseases with possible swallowing impairment. PMID- 7560159 TI - Complex lesion of the gastric wall: an unusual presentation of ectopic pancreas. PMID- 7560160 TI - Antenatal sonographic findings of fetal adrenal hemorrhage. PMID- 7560161 TI - Breast sparganosis: mammographic and ultrasound features. PMID- 7560162 TI - Meckel-Gruber syndrome associated with Rokitansky-Kuster-Hauser syndrome. PMID- 7560163 TI - Recent advances in muscular dystrophies and myopathies. PMID- 7560164 TI - ACP Broadsheet No. 145: July 1995. Investigation of patients with autoimmune haemolytic anaemia and provision of blood for transfusion. PMID- 7560166 TI - Interobserver variation in cell selection for DNA image cytometry. AB - AIMS: To describe a systematic investigation of interobserver differences in interpretation of nuclear morphology in preparations of small cell lung cancer (SCLC). METHODS: The screening/reviewing facility on the highly optimised microscope environment was used to individually tag 127 nuclei, chosen to reflect the spectrum of morphological appearances in nuclear preparations from three biopsy specimens of SCLC. Each nucleus was reviewed and labelled as control (lymphocyte), malignant or unsatisfactory by each of four observers. DNA histograms were plotted for each specimen using the nuclei identified as malignant by each participant. The histograms were compared in terms of identification of DNA stemlines and by calculation of a 5c exceeding rate (5cER). RESULTS: Interobserver variation in assessment of morphology was seen in 55.1% of nuclei. Disagreement occurred most frequently in the malignant/unsatisfactory category. Differences in morphological classification had little influence on histogram assessment by means of visual inspection but did show an effect on 5cER. CONCLUSIONS: There are significant interobserver differences in subjective assessment of nuclear morphology in cytometric preparations. This effect may seriously influence cytometric measurements. PMID- 7560165 TI - Atypical ductal hyperplasia of the breast: clonal proliferation with loss of heterozygosity on chromosomes 16q and 17p. AB - AIMS: To determine if allelic loss on chromosomes 16q and 17p, commonly encountered in in situ and invasive ductal carcinomas, is present in atypical ductal hyperplasia (ADH); to determine whether ADH is a neoplastic (clonal) or hyperplastic (polyclonal) proliferation. METHODS: Fourteen cases of ADH were examined for allele loss at loci on chromosome 16q and 17p using a microdissection technique, polymorphic DNA markers and the polymerase chain reaction (PCR). RESULTS: Loss of heterozygosity (LOH) was detected in five of nine informative cases on chromosome 16q at the microsatellite D16S413 and two of eight informative cases on chromosome 17p at D17S796. CONCLUSIONS: The incidence of LOH at these loci is similar to that previously observed in ductal carcinoma in situ and in invasive ductal carcinoma. Because of the nature of the technique used, our findings also demonstrate that ADH is a monoclonal, and hence, neoplastic proliferation rather than a hyperplastic (polyclonal) condition as its name suggests. There is thus a case for including ADH, as presently defined, within the spectrum of ductal carcinoma in situ. PMID- 7560167 TI - Quantification of proliferative activity in colorectal adenomas by mitotic counts: relationship to degree of dysplasia and histological type. AB - AIM: Proliferative activity of tumours reflects their malignant potential. In colorectal adenomas, a subjective impression of the number of mitoses is a criterion often used to assess the degree of dysplasia. Since these subjective impressions of mitotic activity may lack reproducibility, the aim of this study was to perform an objective analysis. METHODS: Mitotic counts were conducted in tissue sections of 59 colorectal adenomas. Of these, 20 showed mild, 20 moderate, and 19 severe dysplasia, according to blind duplicate assessments by two pathologists. Forty three were classified as tubular adenomas and 16 as "villous" adenomas (tubulo-villous and villous). The number of mitoses, both per unit area of epithelium (area weighted mitotic counts, AWMC) and per colonic crypt (mitotic counts per colonic crypt MCCC), was scored in the most dysplastic area within the adenoma. Mitotic figures were counted using a light microscope (ocular x 10, objective x 40, NA 0.75), and the area of the glandular epithelium was measured using an interactive video overlay measurement system. Twenty glands per specimen were assessed. In the intra-observer reproducibility tests, the coefficients of error for the AWMC and MCCC were 4.5% and 7.4% respectively. RESULTS: For the AWMC a significant difference was found between mild and moderate as well as between mild and severe dysplasia, but not between moderate and severe dysplasia. The results of the MCCC showed the same trend, but the differences did not reach a significant level. Furthermore, cases classified as mild dysplasia were found that showed numerous mitoses, while cases classified as severe dysplasia were found with only very few mitoses. No significant difference in AWMC was found between tubular and villous adenomas. Thus the different malignant potential of tubular and villous adenomas was not reflected by a difference in AWMC. A seemingly strong difference for MCCC between tubular and "villous" adenomas appeared to depend completely on the difference in crypt size between these two groups. CONCLUSIONS: The area weighted mitotic count, rather than the mitotic count per colonic crypt, may be useful for assessing the proliferation rate in colorectal adenomas. PMID- 7560168 TI - OV 632 and MOC 31 in the diagnosis of mesothelioma and adenocarcinoma: an assessment of their use in formalin fixed and paraffin wax embedded material. AB - AIMS: To investigate the reaction of antibodies OV 632 and MOC 31 in paraffin wax sections as opposed to frozen sections and cytological preparations; to evaluate their usefulness in the differential diagnosis of malignant mesothelioma and secondary adenocarcinoma of the pleura; and to assess the efficacy of microwave pretreatment of sections in unmasking their associated epitopes. METHODS: Immunohistochemistry, using a standard avidin-biotin technique, with microwave pretreatment and trypsinisation in a certain proportion of cases. The material comprised 43 mesotheliomas, 44 adenocarcinomas and five reactive pleuras. RESULTS: Epithelial mesotheliomas and the hyperplastic mesothelial cells reacted strongly with OV 632, the reaction with sarcomatoid and desmoplastic tumours was weak, and the reaction with adenocarcinomas was variable. An unequivocal but sometimes patchy positive reaction was obtained with MOC 31 in all but one of the adenocarcinomas; all but one of the mesotheliomas and all the reactive pleuras were negative. Review of the two apparently anomalous cases revealed that the original diagnoses had probably been incorrect. Reactions to both antibodies were abolished by microwave pretreatment, and also by prior trypsinisation in the case of OV 632. CONCLUSIONS: OV 632 is unsuitable for routine clinical use in paraffin wax embedded material. MOC 31, however, would be a useful addition to a panel of antibodies in the differential diagnosis of mesothelioma and adenocarcinoma in large biopsy and resection specimens and necropsy material. Its value in small biopsy specimens remains to be assessed. Microwave pretreatment does not enhance the reactions with either antibody. PMID- 7560169 TI - Distribution of OV-TL 3 and MOv18 in normal and malignant ovarian tissue. AB - AIMS: To analyse the distribution of OV-TL 3 and MOv18 in normal ovarian tissue to determine which antibody is most suitable for (radio)immunotherapy of ovarian carcinoma. METHODS: The distribution of OV-TL 3 and MOv18 was determined using immunohistochemistry and flow cytometry. RESULTS: Epithelial and other cells in many tissues, and leucocytes in peripheral blood, bone marrow and spleen stained positively with OV-TL 3. The staining pattern of MOv18 in normal tissues was more restricted and was confined to epithelial cells in the lung, kidney, pancreas, salivary gland, ovary, Fallopian tubes, and cervix. Reactivity was also observed with pneumocytes in the lung, tubuli in the kidney, acinar cells in the salivary gland and pancreas, in the placenta, and with Kupffer cells in the liver. The staining pattern of chimeric MOv18 was identical with the murine form. OV-TL 3 and MOv18 reacted with 100% and 98% (45/46) of the 46 tested epithelial ovarian cancers, respectively. In ovarian carcinoma tissue homogeneous staining of epithelial cells was observed with OV-TL 3 and more heterogeneous staining with MOv18. In 12 and nine patients, respectively, a difference in staining intensity for OV-TL 3 and MOv18 was observed between various tumour samples from the same patient. CONCLUSION: MOv18 has greater therapeutic potential because of its restricted reactivity with normal tissues and especially, in contrast to OV-TL 3, its lack of reactivity with haematopoietic cells. PMID- 7560170 TI - A necropsy and histomorphometric study of abnormalities in the course of the vertebral artery associated with ossified stylohyoid ligaments. AB - AIMS: To establish whether abnormalities in the course of the vertebral artery occur and whether they are relevant to arterial injury associated with head and neck movements. METHODS: Twenty vertebral arteries were carefully dissected at necropsy and abnormalities in course were noted, along with any other bony or cartilaginous cervical anomalies. The effect of head and neck movement on these vessels was studied before a detailed histomorphometric examination was undertaken on sections of the excised arteries. RESULTS: Five vessels had an abnormal course. One vessel entered the transverse foramina of the fifth cervical vertebra rather than the sixth, but was otherwise normal. In two subjects both vertebral arteries were abnormal in the upper cervical portion with, in each case, a straight left vertebral artery and a right vertebral artery with a deficient loop, closely applied to the atlanto-axial joint. Both of these subjects also had completely ossified stylohyoid ligaments and the arteries visibly stretched with modest head and neck movements. Histology revealed variable degrees of smooth muscle disarray in the tunica media of two of the arteries with loop deficiencies. The circumference of one of the straight arteries was smaller than expected but in all other measured histomorphometric parameters these vessels appeared normal. CONCLUSIONS: Vertebral artery loops are deficient in a number of subjects. This finding is important given the recently described biomechanical susceptibility of the vertebral artery to longitudinal extension and may explain the smooth muscle changes, in that this may represent attempts at arterial wall remodelling. Subjects with such loop deficiencies may be more susceptible to a variety of head and neck insults and such abnormalities should be sought at necropsy in subjects who die as a result of fatal vertebral artery injury. PMID- 7560171 TI - Thiol methyltransferase activity in colonocytes and erythrocyte membranes. AB - AIMS: To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects. METHODS: High performance liquid chromatography with radioactivity detection was used to measure 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine. RESULTS: Verification of radiolabelled 14C-methylmercaptoethanol was by exogenous addition of methylmercaptoethanol and simultaneous ultraviolet detection at 214 nm. Using a substrate concentration of 10 mM mercaptoethanol, the Km for S adenosylmethionine was 25 microM. The sensitivity of the radioactive method was 2 pmol, with coefficients of variation of 7% within assay and 6.4% between assay. TMT activities (mean +/- SE; n = 17) were 471 +/- 64 pmol/hour/mg protein for colonocytes and 73 +/- 7 pmol/hour/mg protein for erythrocyte membranes. CONCLUSIONS: The direct assay of TMT activity is sensitive, specific and eliminates concern over non-enzymatic methylation of thiol compounds. High activities in colonic epithelial cells deserve evaluation in disease states. PMID- 7560172 TI - Effect of proctocolectomy on serum antineutrophil cytoplasmic antibodies in patients with chronic ulcerative colitis. AB - AIMS: To study the effect of proctocolectomy on the antineutrophil cytoplasmic antibody (ANCA) titres in association with ulcerative colitis. METHODS: Serum samples were taken from 15 patients with ulcerative colitis immediately before and at a mean of 24 months after proctocolectomy. Indirect immunofluorescence for ANCA and enzyme immunoassays for myeloperoxidase and proteinase-3 antibodies were employed. A liver biopsy was taken from every patient during the proctocolectomy, and serum liver enzyme activities were also determined. RESULTS: Before proctocolectomy, 13 of the 15 patients had perinuclear antineutrophil cytoplasmic antibodies (p-ANCA). Additionally, one patient had a low tire of classical cytoplasmic ANCA and one had granulocyte specific antinuclear antibodies. After proctocolectomy, the ANCA titres decreased in 10 patients, in two of whom they became negative. The titres remained the same in four patients with positive ANCA and increased twofold in one patient. Only one patient was proteinase-3 antibody positive and all 15 patients were myeloperoxidase antibody negative. The clinical condition improved in all patients, irrespective of the ANCA status after proctocolectomy. Seven patients, all of whom were positive for p-ANCA before proctocolectomy, had histological liver abnormalities. No correlation was observed between serum liver enzyme levels and ANCA staining patterns or titres. CONCLUSIONS: Proctocolectomy decreased the ANCA titres in the majority of our patients, suggesting that reduction of the inflammation or the available antigenic material modifies the immune disturbance related to ulcerative colitis. PMID- 7560173 TI - Variation in serum electrolytes and enzyme concentrations in patients with sickle cell disease. AB - AIM: To assess levels of some biochemical variables in sickle cell disease patients from eastern Saudi Arabia during steady state and in crises states, with a view to comparing biochemical and clinical manifestations of the disease with those in other geographical locations. METHODS: Serum calcium, uric acid, total bilirubin, lactate dehydrogenase, hydroxybutyrate dehydrogenase, and haemoglobin were measured in 110 sickle cell patients when in steady state. The same variables were measured on 30 of the patients when they went into crisis. RESULTS: Serum calcium tended to be lower in sickle cell patients than in healthy controls, while uric acid tended to be in the high normal range. Crises did not make any difference to serum calcium but they increased the uric acid level significantly. All the other variables measured were significantly abnormal and more so during crises. CONCLUSIONS: Although the abnormal levels obtained for these biochemical variables in patients with sickle cell disease from eastern Saudi Arabia were similar to those from other geographical locations, there were noticeable differences in the severity of the abnormalities, which probably explains the differences in the clinical manifestations of the disease between geographical locations. Values of some of these variables could be adapted for use to monitor crises. PMID- 7560174 TI - Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. AB - AIMS: To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS: Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS: Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS: The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID- 7560178 TI - Growth and detection of filamentous fungi in the BacT/Alert blood culture system. AB - Little is known about the behaviour of filamentous fungi in most blood culture systems, despite their increasingly recognised role in infections of immunocompromised hosts. The ability of the BacT/Alert system (Organon Teknika, Durham, North Carolina, USA) to detect the growth of 19 such fungi was examined. Eleven species grew and were detected rapidly; two species did not grow. Six species grew slowly, and were generally only recovered with terminal subculture after prolonged incubation. The CO2 production graph for some of these fungi showed a slow but steady rise, insufficient to cause the apparatus to signal positive. These results show that the BacT/Alert system may miss some fungi, either because of no growth in the medium or undetected slow growth. The latter problem could be overcome by prolonged incubation and terminal subculture when fungal infection is considered likely. Alteration of the signalling mechanism might permit earlier detection of some slow growing fungi. PMID- 7560177 TI - Documentation of blood culture results. AB - AIMS: To evaluate the adequacy of documentation of blood culture results in patients' medical notes. METHODS: A pro-forma was completed following review of medical notes at 24 and 48 hours after a blood culture had been reported as positive. The study was performed on blood cultures received at the Department of Microbiology, Royal Hallamshire Hospital, Sheffield, from two local hospitals. Two periods were studied: (A) May to June 1993 and (B) September to October 1993. RESULTS: There were 43 results studied in period A and 79 in period B, giving a total of 122 results studied. Overall, 72 (59%) of 122 results were recorded in the medical notes at 24 hours. Of those results deemed highly significant, 40 (63%) of 63 were recorded. There was no significant difference in the documentation of results if the result was given personally or via the telephone. Nor was there any difference in documentation between different medical grades. Throughout the study there were six inaccurate records. The cumulative documentation over 48 hours of positive results was 54 (86%) of 63 of highly significant, 27 (69%) of 39 of uncertain significance, and 11 (55%) of 20 probable contaminant results. CONCLUSIONS: Documentation of blood culture results is currently suboptimal. PMID- 7560179 TI - Longstanding proliferation of CD3 negative large granular lymphocytes preceding the development of high grade non-Hodgkin's lymphoma. AB - A patient with a persistent CD3 negative large granular lymphocyte (LGL) proliferation with immunophenotypic and functional characteristics of natural killer cells is described. The LGL proliferation persisted and six years after diagnosis the patient developed a high grade B cell non-Hodgkin's lymphoma. Molecular studies demonstrated clonal B cell populations in the peripheral blood, distinct from that identified in the lymphoma, both at presentation with non Hodgkin's lymphoma and at complete remission following combination chemotherapy. It is postulated that T cell dysregulation associated with the CD3 negative LGL proliferation may have led to B cell dysfunction and loss of normal B cell control, with the subsequent development of a clonal B cell lymphoproliferative disorder. PMID- 7560176 TI - High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay. AB - AIMS: To investigate the prevalence of Helicobacter pylori in the saliva of patients infected with this bacterium. METHODS: A novel polymerase chain reaction (PCR) assay was developed to detect H pylori in saliva and gastric biopsy specimens from patients undergoing endoscopy. RESULTS: Our PCR assay amplified a 417 base pair fragment of DNA from all 21 DNAs derived from H pylori clinical isolates but did not amplify DNA from 23 non-H pylori strains. Sixty three frozen gastric biopsy and 56 saliva specimens were tested. H pylori specific DNA was detected by PCR in all 39 culture positive biopsy specimens and was also identified from another seven biopsy specimens which were negative by culture but positive by histology. H pylori specific DNA was identified by PCR in saliva specimens from 30 (75%) of 40 patients with H pylori infection demonstrated by culture or histological examination, or both, and in three patients without H pylori infection in the stomach. CONCLUSION: The results indicate that the oral cavity harbours H pylori and may be the source of infection and transmission. PMID- 7560183 TI - Microscopic thymoma and myasthenia gravis. AB - A rare case of microscopically sized thymoma is described in a 56 year old man suffering from myasthenia gravis. Histological examination of the surgically removed thymus showed the presence of several epithelial thymoma-like islands. As controls, 100 thymuses obtained from consecutive necropsies were sampled: 4% of these cases showed epithelial islands. This case is further proof that "microscopic thymoma" is a true pathological entity and suggests that every thymus removed from myasthenic patients in which there is no macroscopic evidence of thymoma should be examined microscopically on serial sections. PMID- 7560182 TI - P-glycoprotein positive, drug resistant invasive lymphoepithelial thymoma: treatment response to chemotherapy with cyclosporin and quinine. AB - A case of invasive drug resistant thymoma, expressing P-glycoprotein, which showed noticeable clinical response to chemotherapy and the multidrug resistance modulating agents cyclosporin and quinine is reported. A 46 year old man presented with severe left shoulder pain and a diagnosis of invasive lymphoepithelial thymoma was made following chest x ray and a computed tomography scan. The patient underwent extensive chemotherapy without resolution of the tumour. More than 90% of the malignant epithelial cells were strongly positive for P-glycoprotein and based on this observation, cyclosporin and quinine were added to the chemotherapy regimen. The mediastinal mass completely resolved and the size of the pleural metastasis decreased substantially. The patient, however, died of an intercurrent infection. This case report highlights the feasibility and efficacy of using cyclosporin and quinine in combination with VAD chemotherapy in the treatment of invasive thymoma. PMID- 7560181 TI - Brenner tumour of the vagina. AB - Polyps of the vagina are rare and are either of inflammatory/reactive or neoplastic origin. A case of extraovarian Brenner tumour of the vagina in a postmenopausal woman, who presented with a vaginal polyp, is described. The polyp was excised and on histological examination, had the triphasic pattern (transitional, glandular and stromal) characteristic of Brenner tumour. The histogenesis of Brenner tumour is discussed in the context of this unusual location and the controversy of its origin. PMID- 7560175 TI - Value of screening for oro-pharyngeal Chlamydia trachomatis infection. AB - AIMS: To determine whether oro-pharyngeal colonisation by Chlamydia trachomatis occurs in patients at risk of genital chlamydia infection; to determine whether screening pharyngeal specimens by polymerase chain reaction (PCR) increases detection of C trachomatis compared with isolation and the immune dot blot test; and to correlate the detection of C trachomatis and Neisseria gonorrhoeae in the pharynx with a history of oro-genital contact. METHODS: Thirteen homosexuals and 11 heterosexuals were included in the study. Urogenital and pharyngeal specimens were tested for C trachomatis and N gonorrhoeae using standard clinical diagnostic procedures. Two different PCR methodologies were also used to detect C trachomatis in the pharyngeal specimens. Results were correlated with the mode of sexual practice. RESULTS: Oro-genital sexual contact was practised by 64.9% (72/111) of heterosexuals in addition to penetrative penovaginal intercourse. Additionally, 62.1% (77/124) of all patients did not use any form of barrier protection. Of those who admitted to oro-genital sexual contact, 17.6% of patients with a genital chlamydial infection and 36.4% of those with genital gonorrhoea also had asymptomatic pharyngeal colonisation. C trachomatis was detected in three of 124 (2.4%) pharyngeal specimens by PCR which were reported as negative by chlamydial culture; one was positive by the immune dot blot test. CONCLUSION: The majority of patients practised unprotected oro-genital contact and significant pharyngeal colonisation by C trachomatis and N gonorrhoeae occurred if genital infection was present. Despite the use of PCR in a population at high risk of sexually transmitted disease, the prevalence of chlamydia in the pharynx was very low. This indicates that transmission of C trachomatis to the oro-pharynx does not pose a serious health risk and that screening of patients for oro-pharyngeal C trachomatis is not worthwhile. PMID- 7560180 TI - Nuclear pseudoinclusions in melanocytic naevi and melanomas. AB - Melanocytes in melanocytic naevi and melanomas can display great variation. The presence of nuclear pseudoinclusions (NPI) is said to be useful in the histological and cytological differential diagnosis of malignant melanoma. The prevalence and characteristics of NPI in a series of 493 naevi and 50 melanomas are described. NPI were found in 31% of adult naevi, 30% of congenital naevi from children, 42% of Spitz naevi, 20% of dysplastic naevi, and 56% of melanomas. The presence of NPI is not a reliable criterion for differentiating melanoma from benign melanocytic lesions, although it is useful in distinguishing melanocytic from non-melanocytic tumours. PMID- 7560184 TI - Evaluation of the API-campy system in the biochemical identification of hippurate negative campylobacter strains isolated from faeces. AB - The aim was to evaluate the efficacy of the API-Campy system in the biochemical identification of 62 hippurate negative campylobacter strains isolated from the faeces. The strains were identified manually as 34 nalidixic acid susceptible C coli (NAS), 20 nalidixic acid resistant C coli (NAR), and eight C lari. The 34 strains of NAS C coli were identified as such by the API-Campy system. Of the 20 strains of NAR C coli, 15 (75%) were correctly identified by the commercial system. None of the five NAR C coli strains which were also erythromycin resistant was identified as such by the system. The eight C lari strains could not be identified by the API-Campy system because the bionumber obtained does not exist in the database of the computer system. The API-Campy system could be very useful for the identification of NAS C coli. However, failure to allow for a higher percentage of resistance to nalidixic acid in this species does not permit good identification of NAR strains. More important discrepancies are observed in C lari strains. In order to improve the identification of NAR C coli and C lari stains, it is advisable to include, or recommend as complementary, the indoxyl acetate hydrolysis test. PMID- 7560185 TI - A new, fast and convenient method for layering blood or bone marrow over density gradient medium. AB - A density gradient dispenser has been developed, providing a fast, easy and convenient method for layering peripheral blood or bone marrow over a density gradient solution for the isolation of lymphocytes and mononuclear cells. The present approach is just the opposite of the conventional method of layering blood over a density gradient solution. In the conventional method a required amount of density gradient solution is first placed in a centrifuge tube and then blood or marrow is layered on top using a pasteur pipette. In the present method a required amount of blood or marrow is first placed in a centrifuge tube. The required amount of density gradient solution is then gently delivered underneath the blood sample using the newly devised density gradient dispenser. Because of the difference in densities, the released density gradient solution lifts the sample of blood upwards and produces exactly the same blood and density gradient suspension as that produced by the conventional method but is less cumbersome, faster and more convenient. PMID- 7560186 TI - Systemic absorption of vancomycin. PMID- 7560187 TI - The value of Toxoplasma specific IgA in diagnosis. PMID- 7560188 TI - A rapid and safe method to fix india ink on specimen resection margins. PMID- 7560189 TI - Audit of tumour pathology reviewed by a regional oncology centre. PMID- 7560190 TI - Necrobiotic granulomas of the urogenital system. PMID- 7560193 TI - Detection of mycobacteria in bone marrow biopsy specimens taken to investigate pyrexia of unknown origin. AB - AIMS: To investigate the value of bone marrow biopsy in the diagnosis of mycobacterial infection. METHODS: The culture results of 433 bone marrow samples taken between 1983 and 1992 were reviewed. The histopathology reports on bone marrow trephine specimens of culture positive samples and all those on HIV positive patients sent in 1992 were also reviewed. RESULTS: Fifty one specimens yielded Mycobacterium spp, 47 were obtained from HIV positive patients. Of the isolates, 42 were Mycobacterium avium-intracellulare (MAI), five were M tuberculosis (MTB), and the remaining four comprised a variety of atypical mycobacteria. All MAI positive samples were obtained from HIV positive patients, with the bone marrow being the only culture positive specimen in one third. Bone marrow yielded MTB only in patients from whom it was also isolated in other specimens. Eleven of 47 trephine specimens from positive bone marrow showed granulomata and nine showed acid-fast bacilli. No acid-fast bacilli were seen in the absence of granulomata. CONCLUSION: Bone marrow biopsy for mycobacterial culture should be reserved for severely immunosuppressed patients and should not be advocated for immunocompetent patients with suspected tuberculosis. Bone marrow biopsy still has a role in the investigation of pyrexia of unknown origin in HIV positive patients, despite the advent of mycobacterial blood culture techniques, particularly if these can be processed safely in automated systems. PMID- 7560192 TI - Lesser known mycobacteria. PMID- 7560191 TI - ACP Broadsheet No 146: August 1995. Macroscopic examination of prostatic specimens. PMID- 7560195 TI - Comparison of agar based media for primary isolation of Helicobacter pylori. AB - AIMS: To determine the best medium for the primary isolation of Helicobacter pylori. METHODS: Sixty six gastric mucosal biopsy specimens frozen in 1 ml Cysteine Albimi media with 20% glycerol from 22 histologically proven H pylori infected patients were cultured on brain heart infusion agar (BHIA) with 7% fresh whole defibrinated horse blood, egg yolk agar (EYA), Columbia blood agar cyclodextrin agar (CBA-Cd), and commercial trypticase soy agar (TSA) supplemented with 5% sheep blood. RESULTS: Successful primary isolation of H pylori was 96% with BHIA, 78% with TSA, 64% for EYA, and 32% with CBA-Cd. Colonies appeared earlier on BHIA (4.7 +/- 0.1 days, 5.3 +/- 0.4 days, 5.3 +/- 0.4 days, and 7.1 +/ 0.9 days for BHIA, TSA, EYA, and CBA-Cd) and there were more colonies on BHIA than on CBA-Cd, EYA or TSA (599 +/- 88, 104 +/- 66, 260 +/- 107, and 358 +/- 89, respectively). CONCLUSIONS: Success of a medium for passage of isolates apparently does not reliably predict usefulness for primary isolation. Freshly made BHIA with 7% horse blood medium is recommended for primary isolation. However, the easily obtainable TSA media would be the best alternative for routine clinical laboratories with no access to BHIA. PMID- 7560194 TI - Evaluation of the Cult-Dip Plus dip slide method for urinary tract infection. AB - AIM: To evaluate the Cult-Dip Plus (Merck, Germany), a bacteriological culture test for detecting uropathogens. METHODS: Cult-Dip Plus consists of Brolacin (CLED) and MacConkey agar, each containing methylumbelliferylglucuronide (MUG). Using 1022 urine samples, this product was compared with the routine method of calibrated loop inoculated CLED and blood agar for screening urine for uropathogens. The MUG test for identifying Escherichia coli was also evaluated. RESULTS: Compared with the routine method, Cult-Dip Plus has a sensitivity, specificity, positive predictive value and negative predictive value of 88.3%, 98.0%, 91.9%, and 97.1%, respectively. The MUG test correctly identified 92% of E coli isolates with a sensitivity, specificity and positive predictive value of 91.6%, 95.2%, and 93.6%, respectively. CONCLUSION: Cult-Dip Plus appears to be an alternative method to the calibrate loop method for detecting uropathogens. The MUG test permits rapid, reliable and inexpensive identification of E coli. PMID- 7560196 TI - Rate of growth of Pseudomonas fluorescens in donated blood. AB - AIMS: To examine how delayed refrigeration of blood affects the growth of Pseudomonas fluorescens, one of the two most important causes of sepsis resulting from transfusion of contaminated blood. METHODS: Two donations of whole blood were each divided into three aliquots and inoculated with 5-10 cfu/ml of a P fluorescens strain from a case of transfusion associated sepsis. From each donation, one aliquot was placed at 4 degrees C, one was held at 20 degrees C for six hours prior to refrigeration and the third was held at 20 degrees C for 24 hours prior to refrigeration. Samples were aseptically withdrawn over 17 days and bacterial counts were determined using a pour plate technique. RESULTS: The rate of growth of P fluorescens in blood at 20 degrees C was increased compared with blood at 4 degrees C. At 24 hours the aliquots held at 20 degrees C for six and 24 hours had, respectively, 174 and 29,000 cfu/ml compared with 15 cfu/ml in aliquots held at 4 degrees C. There was no evidence of increased killing of P fluorescens at the higher temperature. CONCLUSIONS: These results suggest that blood for transfusion should be refrigerated as soon as possible after collection. PMID- 7560197 TI - Simple method for quantifying viable bacterial numbers in sputum. AB - AIMS: To establish a simple method of quantitative culture for determining the viable bacterial numbers present in expectorated sputum samples. METHODS: Sputum samples were homogenised with dithiothreitol, sterile saline or glass beads to determine which method recovered the greatest number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacterial viability. Sputum samples homogenised with dithiothreitol were diluted in sterile saline and sampled using either standard bacteriological loops or a precision pipette to determine which method resulted in the least variation. RESULTS: Homogenisation of sputum using dithiothreitol increased the recovery of viable bacteria compared with sterile glass beads and/or saline, with no apparent effect on bacterial viability when incubated with culture broths. By inoculating agar plates with 10(-3), 10(-4) and 10(-5) dilutions of the homogenised sputum sample, all potential pathogens could easily be identified. A 10 microliter sample volume dispensed by precision pipette and spread with a "hockey stick" resulted in the least variation between plates (less than 16%) and an even distribution of bacterial colonies. Numbers of viable bacteria recovered from different aliquots of individual sputum samples were generally of the same order of magnitude. CONCLUSIONS: This method represents a relatively quick and simple technique for accurately quantifying viable bacteria present in sputum samples. The use of a small portion appears to be representative of the sample as a whole. PMID- 7560198 TI - Detection and identification of gastrointestinal microsporidia using non-invasive techniques. AB - AIMS: To detect enteric microsporidia in faecal specimens from patients with the acquired immunodeficiency syndrome (AIDS), and to identify the spores to species level without using invasive procedures. METHODS: Formalised faecal preparations were examined using a modification of the strong trichrome staining method to demonstrate microsporidian spores. Six positive specimens were prepared for electron microscopy by emulsification and separation using a 9% Ficoll gradient. RESULTS: The modified staining technique readily identified microsporidian spores. Spores of different species showed variation in size. Identification using electron microscopy was successful for five of the six positive specimens examined. It was unsuccessful for one specimen in which spores were less abundant on initial staining. CONCLUSIONS: The modified strong trichrome staining method is a useful way of detecting spores of intestinal microsporidia in faecal specimens. Variation in spore size may permit provisional identification by light microscopy. Electron microscopic examination of faecal preparations is useful for identifying spores to species level. PMID- 7560199 TI - Human papillomavirus in false negative archival cervical smears: implications for screening for cervical cancer. AB - AIM: To assess the value of detecting human papillomavirus (HPV) DNA in false negative archival cervical smears in population based screening programmes for cervical cancer. METHODS: Cytomorphologically classified false negative archival Pap smears (n = 27) taken from 18 women up to six years before cervical cancer was diagnosed were blindly mixed with 89 smears from hospital patients with a variety of gynaecological complaints and tested for HPV by the polymerase chain reaction (PCR). Corresponding cervical cancer biopsy specimens were also available for HPV analysis. Neither the examining cytopathologist nor the molecular biologist was aware of the study design. RESULTS: HPV DNA was detected in the smears of 16 patients with cervical cancer missed previously by cytology. HPV 16 and 18 were found predominantly in those smears taken up to six years before the diagnosis of cervical cancer. The smears of the two remaining patients were reclassified as inadequate for cytology or contained no suitable DNA for PCR. In 15 patients the same HPV type could be found in the smears and the cervical cancer biopsy specimens. CONCLUSIONS: The results indicate that high risk HPV types can be detected in archival smears classified as false negative on cytology and that cytological screening errors may be reduced if combined with PCR testing for HPV. PMID- 7560200 TI - Retrospective determination of HIV-1 status by a PCR method on paraffin wax embedded sections. AB - AIM: To develop a simple but reliable polymerase chain reaction (PCR) method to determine the HIV-1 status of patients on formalin fixed, paraffin wax embedded lymph node tissue. METHODS: Fifty lymph node specimens, 20 from HIV-1 seropositive and 30 from HIV-1 seronegative patients, were analysed. Lymph nodes with a variety of disease conditions were included in the study. Tissue sections were treated with a DNA extraction buffer containing proteinase K and the crude cell lysate was used in PCR analysis. Nested primers were used to amplify HIV-1 DNA sequences coding for gag, pol and env proteins. PCR products were demonstrated by polyacrylamide gel electrophoresis. Results were then compared with HIV-1 serology of the patients from whom the tissue was obtained. RESULTS: The PCR method yielded a specificity of 100%, a sensitivity of 95%, a positive predictive value of 100%, and a negative predictive value of 97% when compared with HIV-1 serology. The kappa statistic (0.958) showed an excellent agreement between the PCR method and serology. Furthermore, HIV-1 DNA was demonstrated in lymph node tissue from a serologically unconfirmed acquired immunodeficiency syndrome case necropsied in 1982. CONCLUSION: This PCR method is a simple and reliable means of retrospectively determining the HIV-1 status of patients using formalin fixed, paraffin wax embedded lymph node tissue. PMID- 7560204 TI - Chromogranin positive cells in colorectal carcinoma and transitional mucosa. AB - AIMS: Immunostaining of chromogranin identifies gastrointestinal mucosal endocrine cells. The detailed distribution and significance of chromogranin positive cells in colorectal carcinomas and in transitional mucosa remain unclear. The aim of this study was to clarify these aspects. METHODS: The distribution of chromogranin positive cells was studied by immunohistochemical methods in normal epithelium remote from carcinoma, in transitional mucosa, and in carcinomas of the colorectum. In selected cases northern or western blot analyses were performed. RESULTS: Chromogranin positive cells were seen in the lower third of the normal crypts and less frequently in transitional mucosa. Thirty five per cent (n = 38) of colorectal carcinomas showed immunohistochemically positive carcinoma cells in the tumour tissue. Northern and western blot analyses showed similar results. There was no difference in clinicopathological factors, including prognosis, between chromogranin positive cases of colorectal carcinoma (n = 38) and chromogranin negative cases (n = 70). CONCLUSIONS: Neuroendocrine cell differentiation is controlled in transitional mucosa and the presence of chromogranin positive cells in carcinoma tissue does not influence the patient's prognosis. PMID- 7560201 TI - Apocrine adenosis: a precursor of aggressive breast cancer? AB - AIM: To investigate overexpression of c-erbB2, expression of the p53 protein product and proliferation rates in benign breast lesions with specific reference to apocrine adenosis. METHODS: Twenty one cases of apocrine adenosis were stained with monoclonal antibodies to p185, the protein product of the c-erbB2 oncogene, the protein product of the p53 tumour suppressor gene and to the cell cycle related protein Ki67. Three cases were associated with concomitant ductal carcinoma in situ of large cell type and two were associated with invasive tubular or cribriform carcinoma. RESULTS: Twelve (57.1%) cases showed membrane staining for c-erbB2 oncoprotein of apocrine cells within sclerosing adenosis and six (28.6%) had occasional p53 protein positive cells. One case not associated with carcinoma showed extensive staining of apocrine metaplasia outside the area of apocrine adenosis. The proliferation rate, as measured by Ki67 staining, was increased in some of the lesions and all lesions showed at least some of the cells to be in the cell cycle. CONCLUSIONS: The expression of abnormal oncogene products and increased proliferation in some of these apocrine lesions questions the supposed degenerative nature of the atypia seen in such cases and suggests that there may be an association between these lesions and large cell ductal carcinoma in situ and hence invasive carcinoma. PMID- 7560205 TI - S100, alpha-smooth muscle actin and cytokeratin 19 immunohistochemistry in odontogenic and soft tissue myxomas. AB - AIMS: To compare the expression of S100 protein, alpha-smooth muscle actin (alpha SMA) and keratin 19 in odontogenic myxomas and non-odontogenic myxoid lesions. METHODS: Formalin fixed, paraffin wax embedded tissue from seven odontogenic myxomas, three soft tissue myxomas, six hyperplastic myxoid dental follicles, two intramuscular myxomas, 12 cardiac myxomas, and seven normal dental follicles were examined immunocytochemically for S100 protein, alpha-SMA and cytokeratin 19 using the Streptavidin-biotin method. RESULTS: A minority of odontogenic myxomas (three of seven) were positive for S100 and the staining was of moderate intensity and in all myxofibroblasts. Soft tissue myxomas, normal dental follicles, intramuscular myxomas, and most enlarged myxoid follicles were negative. In the cardiac myxomas the cells forming cords and islands were positive in approximately half (seven of 12), but the dispersed stellate myxoblasts were positive in only two cases. A population of cells in all the odontogenic myxomas and hyperplastic dental follicles contained alpha-SMA, but such cells were sparse in cardiac myxomas and present in only four cases. Cytokeratin 19 was present in odontogenic epithelium of odontogenic myxoma and follicles. CONCLUSIONS: A minority of odontogenic myxomas, but not other oral myxoid lesions, may express S100 protein and this could cause difficulty distinguishing myxoma from myxoid nerve sheath tumours. Sparse myofibroblastic cells occurred in all types of myxoma tested. The epithelium sometimes found within jaw myxomas expresses cytokeratin 19 and this is consistent with an odontogenic origin. PMID- 7560206 TI - Postmortem blood ferritin concentrations in sudden infant death syndrome. AB - AIMS--To confirm the observation of extremely high concentrations of ferritin in postmortem serum samples in sudden infant death syndrome (SIDS); to examine the factors influencing blood ferritin concentrations postmortem; to determine whether or not these high blood ferritin concentrations are characteristic of SIDS. METHODS--Postmortem samples of cardiac blood were obtained from 58 full term infants who died of SIDS and 14 full-term infants who died of a variety of other causes. Whole blood and serum ferritin concentrations were determined and compared with age at death, liver iron concentration, serum iron concentration, and serum lactate dehydrogenase activity. RESULTS--The median postmortem blood ferritin concentration for all infants was 18,600 micrograms/l, which is about 200 times the concentration found in the serum of normal, live infants. Serum iron concentrations were high and there was a highly significant correlation between serum ferritin and iron concentrations suggesting that much of the serum iron was contributed by ferritin. There was no significant difference between serum and whole blood ferritin concentrations. H to L type ferritin ratios were higher in blood from the left than the right ventricle of the heart but the ferritin was always predominantly L type. Blood ferritin concentrations rose rapidly after death but in samples collected at postmortem examination there was a significant correlation with liver iron concentration and an inverse correlation with age. Median values for blood ferritin were higher in SIDS (22,500; n = 58) than in control cases (6900; n = 7) dying under one year of age; however, in both groups ferritin concentrations decreased with age. CONCLUSIONS- Release of ferritin into the blood postmortem seems to be characteristic of infants dying before the age of one year rather than characteristic of SIDS. Two factors may cause such ferritin release postmortem: tissue breakdown and the high level of storage iron in cells of the reticuloendothelial system (including endothelial cells lining vessel walls). SIDS occurs when tissue iron concentrations are higher than at any other time of life. It is possible that the ready availability of iron enhances free radical damage which might be implicated in SIDS. PMID- 7560203 TI - Histological discrimination of idiopathic inflammatory bowel disease from other types of colitis. AB - AIMS: To assess the value of histology in diagnosing inflammatory bowel disease (IBD) in colorectal biopsy specimens. METHODS: Retrospective, double blind evaluation of colorectal biopsy specimens from 41 patients with colitis (28 with ischaemic colitis and 13 with acute self-limited colitis) and 84 patients with IBD (42 with Crohn's disease and 42 with ulcerative colitis). RESULTS: The features distinguishing IBD from other forms of colitis included distorted architecture, lymphocyte and plasma cell infiltrate, excess of polymorphonuclear leucocytes, polymorphonuclear cryptitis, crypt abscesses, and basal lymphoid aggregates. The features discriminating between Crohn's disease and ulcerative colitis included an irregular or villous surface, distorted architecture, decrease in mucus content, and polymorphonuclear cryptitis. Using multivariate analysis, 90% of patients with Crohn's disease and 71% of those with ulcerative colitis were correctly classified, the former being strongly defined by epithelioid granulomas, microgranulomas and isolated giant cells, and the latter best defined by an irregular or villous surface, decrease in mucus content and crypt atrophy. CONCLUSIONS: Examination of colorectal biopsy specimens is a reliable method for diagnosing IBD. In the absence of epithelioid granulomas, microgranulomas and isolated giant cells a diagnosis of Crohn's disease is based on the absence of histological criteria favouring ulcerative colitis. The histological spectrum of indeterminate colitis remains to be clarified. PMID- 7560202 TI - Intraepithelial and lamina propria leucocyte subsets in inflammatory bowel disease: an immunohistochemical study of colon and rectal biopsy specimens. AB - AIMS: To gain new insights into the pathogenesis and differential diagnosis of ulcerative colitis and colonic Crohn's disease. METHODS: Immunohistochemistry for different leucocyte subsets was performed in biopsy specimens of the sigmoid colon and rectum from 55 patients with inflammatory bowel disease and 11 healthy controls. RESULTS: Colonic biopsy specimens from patients with active ulcerative colitis had significantly higher numbers of CD45+ and CD3+ leucocytes compared with those from patients with inactive disease, and higher numbers of total leucocytes and macrophages than those from patients with Crohn's disease. Rectal biopsy specimens from patients with Crohn's disease had greater numbers of intraepithelial leucocytes (CD45, CD3 and CD8 cells) than specimens from patients with active or inactive ulcerative colitis, or from healthy controls. CONCLUSIONS: Because of the phenotypic differences in the inflammatory infiltrate in the mucosa from the sigmoid colon and the rectum, the segment of the intestine to be biopsied should be specified. Assessment of the leucocytic component of the intraepithelial infiltrate in rectal biopsy specimens was more useful than examination of colonic biopsy specimens in the differential diagnosis of ulcerative colitis and Crohn's disease. PMID- 7560208 TI - Serum glutathione S-transferase B1 activity as an index of liver function in cystic fibrosis. AB - AIMS: To evaluate serum glutathione S-transferase B1 (GST B1), a highly sensitive test of hepatocellular function, as a means of identifying liver disease in patients with cystic fibrosis (CF). METHODS: The presence of liver disease was sought over a three year period in 60 children with CF, using a combination of clinical assessment, ultrasound examination, conventional biochemical tests of liver function (LFTs), and measurement of GST B1. RESULTS: Reference ranges for serum GST B1 were established in a paediatric control population. The 95% value (4.55 micrograms/l) was similar to the upper limit of normal previously derived in adults. Mean (SE) serum GST B1 activities were higher in the CF population (9.0 (1.14) micrograms/l) than in age matched controls (2.4 (0.15) micrograms/l). Ten patients with CF showed clinical signs of liver dysfunction. All but one had a serum GST B1 > 4.55 micrograms/l. Twelve other patients had elevated LFTs without clinically evident liver dysfunction, six had abnormal ultrasound scans and two showed both of these anomalies. Thirty patients with CF had neither biochemical, ultrasonographic nor clinical signs of liver disease. On review three years later, clinically important liver disease was reaffirmed in eight of the 10 index cases and had become apparent in a further eight, all of whom had elevated GST B1 activities. Five (36%) of the patients with elevated LFTs and two (33%) with isolated ultrasound changes continued to show these abnormalities. CONCLUSIONS: The limitations of conventional LFTs and ultrasound scans were evident from this study. The results suggest that elevated GST B1 activities may be a better predictor of hepatic dysfunction in CF than conventional LFTs. PMID- 7560207 TI - Organophosphates and monocyte esterase deficiency. AB - AIMS: To examine the possibility that monocyte esterase deficiency (MED) could be caused by exposure to organophosphates. METHODS: Pseudocholinesterase, paraoxonase and arylesterase activities were measured in the serum and acetylcholinesterase activity was measured in the red cells of a group of monocyte esterase deficient subjects and compared with the enzyme activities of a control group of monocyte esterase positive subjects. RESULTS: No significant difference was found between the enzyme activities of the monocyte esterase deficient group and the control group for any of the esterases investigated. CONCLUSION: Current or recent exposure to organophosphorus is not the cause of MED. PMID- 7560209 TI - Waterhouse-Friderichsen syndrome complicating primary biliary sepsis due to Pasteurella multocida in a patient with cirrhosis. AB - Pasteurella multocida is an opportunistic pathogen causing bacteraemia in patients with liver dysfunction. A fulminant case of acute cholecystitis and septicaemia caused by P multocida, complicated by Waterhouse-Friderichsen syndrome without skin haemorrhage, is reported in a previously healthy 64 year old Chinese woman. The patient presented with a six hour history of sudden onset epigastric pain, vomiting, chills, and rigors. A presumptive diagnosis of cholangitis with septicaemic shock was made. Disease progression was rapid and the patient died within eight hours of symptom onset. This case is further proof that skin and mucosal haemorrhages are not an essential feature of Waterhouse Friderichsen syndrome and this condition should be suspected in all patients presenting with sudden illness and fulminant septicaemia. PMID- 7560210 TI - Microbiological investigation of polyarthralgia. AB - Results of serological investigations on patients with joint pain, arthralgia or polyarthritis were analysed and this information was used to develop a diagnostic algorithm to ensure optimal utilisation of laboratory resources. Accordingly, all cases are now examined for parvovirus IgM, mycoplasma IgM and streptococcal antibodies. Further tests are undertaken by following the algorithm after obtaining supplementary information from a questionnaire. This approach is put forward as a preliminary standard which other laboratories may like to evaluate and develop according to local requirements. PMID- 7560211 TI - Fatal adenovirus 32 infection in a bone marrow transplant recipient. AB - A case of disseminated adenovirus type 32 infection causing severe hepatitis, gastrointestinal ulceration and also with respiratory involvement is reported in a bone marrow transplant recipient. Typical viral inclusions were seen in the postmortem histological sections and adenovirus infection was confirmed using in situ hybridisation and isolation of adenovirus type 32 from separate organs at necropsy. This is the first case in which adenovirus 32 was the cause of fatal disseminated disease in a bone marrow transplant recipient. PMID- 7560213 TI - Multifactorial audit of invasive cervical cancer. PMID- 7560212 TI - Sudden death from coronary artery dissection. AB - Spontaneous dissection of the coronary arteries is an uncommon condition that may lead to sudden, fatal coronary artery occlusion. Three cases of sudden death attributable to coronary artery occlusion are presented. Dissection was associated with Marfan's syndrome in the first case, and occurred three weeks postpartum in the second case. In case 1, dissection occurred within the intima, and was not associated with an inflammatory cell infiltrate. In cases 2 and 3, dissection occurred between the tunica media and the external elastic lamina, and was associated with a mixed inflammatory infiltrate, rich in eosinophils, T lymphocytes, and histiocytes. The spatial limitation of the inflammatory infiltrate to the adventitial compartment, together with the absence of inflammation in case 1, suggests a reactive origin rather than a causative role for the inflammatory cells. Detailed examination of serial blocks of any coronary artery occlusion is essential in young patients. PMID- 7560214 TI - Multifactorial audit of invasive cervical cancer. PMID- 7560215 TI - Detection of autoantibodies to neutrophil cytoplasmic antigens. PMID- 7560216 TI - Physical state of human papillomavirus using non-isotopic in situ hybridisation. PMID- 7560217 TI - Inaccurate haemoglobin estimation in Waldenstrom's macroglobulinaemia. PMID- 7560218 TI - Pitfalls in diagnosing coeliac disease. PMID- 7560219 TI - Triclosan inhibits histamine-induced inflammation in human skin. AB - Previous studies indicate that triclosan reduces the pain and other symptoms after chemically-induced inflammation in the oral mucosa and skin when sodium lauryl sulfate (SLS) is used as an irritant. The aim of the present study was to examine whether triclosan has an effect on the inflammation in human skin caused by intradermal administration of histamine. 9 female volunteers participated in a double-blind study, and skin patch tests were performed in 2 series. In the 1st, the skin was pre-treated for 1 h with triclosan before the histamine was applied. In the 2nd, the histamine reaction was elicited first and triclosan applied subsequently. The effect of triclosan on the weals formed in the skin after histamine application was measured. It was found that triclosan reduced the size of the weals markedly when triclosan was applied after the weals were formed, and that pre-treatment of the skin had only a slight effect. It is assumed that triclosan has an effect on the cascade reactions of inflammation elicited by histamine. 2 other phenols tested in the same study had similar effects. PMID- 7560221 TI - Citric acid demineralization of cementum and dentin: the effect of application pressure. AB - Previous work has shown that a 25-30% citric acid solution was the most effective concentration with which to demineralize dentin. The purpose of this investigation was to study the topography, using a scanning electron microscope, of root surfaces treated with a 30% citric acid solution using various application pressures. 20 freshly extracted human teeth were collected and stored in physiologic saline at room temperature. 3 root specimens, approximately 3x5x5 mm in size, were prepared from the coronal periodontally healthy area of each tooth. 30 specimens were root planed to expose dentin (dentin group) while the remaining 30 specimens were lightly scaled to remove periodontal soft tissue (cementum group). A freshly made 30% citric acid (CA) solution, (pH = 1.60), was applied to each of the experimental areas. Cotton pellets soaked in the citric acid solution were either placed (CAP), lightly rubbed (CAR) or heavily burnished (CAB) on the prepared root surface for 3 min. Pellets were resoaked every 30 s. The root sections were rinsed, fixed in glutaraldehyde, dehydrated in graded ethanol, critically point dried in liquid CO2 and sputter coated in gold. The treated surfaces were assessed for fibril tufting using scanning electron microscopy. Assessment was made of: (i) the % of surface area tufted; (ii) tufting depth (0-3); (iii) tufting density (1-3). Results of the study showed significantly more tufted surface area and greater tufting depth on both dentin and cementum for CAR and CAB compared to CAP. CAP produced a flat/mat fibril surface with no evidence of tufting.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560220 TI - Adjunctive supragingival irrigation with acetylsalicylic acid in periodontal supportive therapy. AB - To assess the clinical efficacy of adjunctive supragingival irrigation with buffered 0.3% acetylsalicylic acid (ASA), 60 patients with periodontitis receiving supportive periodontal therapy were randomly assigned to 1 of 3 home regimens: (1) 1x daily adjunctive supragingival irrigation with 300 ml water immediately followed by 200 ml of buffered 0.3% ASA; (2) 1x daily adjunctive supragingival irrigation with 500 ml water; or (3) normal oral hygiene alone. Clinical parameters were assessed at baseline and 6 months. Irrigator use was measured by timers built into the irrigator units. Results at 6 months showed that both supragingival irrigation with buffered 0.3% ASA and supragingival irrigation with water significantly reduced gingival index scores (median 0.1 and 0.35, respectively) and pocket probing depths (both median 0.26 mm) compared to the control group. In addition, irrigation with water resulted in a significant reduction in bleeding on probing (median 0.13), whereas irrigation with buffered 0.3% ASA had no significant effect on bleeding on probing compared to the control group. The clinical efficacy of irrigation with either ASA or water was found to be positively correlated to initial disease severity and irrigator use. Thus, frequent supragingival irrigation with either 0.3% ASA or water in addition to regular oral hygiene appears to be a beneficial adjunct to periodontal supportive therapy in patients with moderate to severe signs of periodontitis. However, the use of buffered 0.3% ASA as an irrigant does not seem to enhance the clinical efficacy of supragingival irrigation on periodontal health. PMID- 7560222 TI - The effect of triclosan on developing gingivitis. AB - The aim of the study was to examine whether triclosan has an effect on developing gingival inflammation. 10 volunteers, with clinically healthy gingivae were enrolled. The study was performed as a 2-week, double-blind, cross-over, experimental gingivitis trial. Between each plaque accumulation period, there was a wash-out phase of 4 weeks. A baseline examination was performed which included assessment of plaque and gingivitis. The volunteers were asked to refrain from mechanical oral hygiene measures for 2 weeks. During this period, they rinsed 2x daily with one of the randomly assigned mouthrinse preparations. Solution A (period A): 0.06% triclosan+ 2%tween 80. Solution B (period B): 0.06% triclosan+ 0.25% sodium lauryl sulphate. Re-examinations were performed on days 4, 7, 11 and 14. The mean plaque score increased during period A to 2.2 (day 4), 2.8 (day 7), 3.1 (day 11) and 3.1 (day 14). The corresponding scores for period B were significantly lower; 1.2 (day 4), 1.8 (day 7), 2.0 (day 11) and 2.2 (day 14). The mean gingivitis scores at baseline were 0.17 (periods A and B). The mean gingivitis scores increased to 0.45 (day 4), 0.69 (day 7), 0.83 (day 11) and 0.96 (day 14) when the subjects rinsed with solution A and 0.42 (day 4), 0.64 (day 7), 0.78 (day 11) and 0.92 (day 14) in period B. There were no statistically significant differences between periods A and B with respect to gingivitis. Thus, although significantly more plaque formed during period A than period B, no differences could be found between the gingivitis scores in the 2 periods. PMID- 7560223 TI - Some suspected periodontopathogens and serum antibody response in adult long duration insulin-dependent diabetics. AB - The subgingival microflora and serum antibody response were examined in long duration insulin-dependent diabetics and age- and sex-matched non-diabetics. The material consisted of 9 diabetics aged 40-49 years and 19 aged 50-59 years, 13 non-diabetics aged 40-49 years and 21 aged 50-59 years. The bacterial species studied (Actinobacillus actinomycetemcomitans, Campylobacter rectus, Capnocytophaga spp, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia) were recovered in diabetics as well as in non diabetics. Significantly more diabetics in both age groups harboured P. gingivalis compared to non-diabetics. Prevalence of P. gingivalis was associated with deepened periodontal pockets among non-diabetics but not among diabetics. In diabetics and non-diabetics, the serum antibody titres for most antigens were similar. PMID- 7560224 TI - Long-time etching at low pH jeopardizes periodontal healing. AB - The purpose of the present investigation was to examine whether the immediate necrotizing effects of low pH etching agents also impair periodontal healing in the long-term perspective. It was concluded that long-time etching (3 min) at low pH of root surfaces surrounded by vital periodontal issues resulted in impaired periodontal healing. In contrast, short-time etching appeared to promote connective tissue formation by preventing epithelialization of the periodontal wound despite its low pH. However, clinical inferences should be drawn with caution from the present results, since healing results recorded at 8 weeks may not prove to be stable, especially for those cases sensitive to the immediate influence of marginal infection such as connective tissue attachments and epithelial junctions, unless optimal oral hygiene is maintained. Furthermore, the choice of acid may be of importance. PMID- 7560226 TI - Periodontal pathogens on polytetrafluoroethylene membrane for guided tissue regeneration inhibit healing. AB - This study determined the microbial composition of the apical parts of the expanded polytetrafluoroethylene membrane surfaces facing the gingiva and the tooth in guided tissue regeneration. Microbial and clinical features of 2-to-3 wall periodontal bony defects treated with membranes with and without concomitant use of systemic Augmentin therapy were also determined. 18 patients with 18 study sites participated. 9 patients received systemic 500 mg Augmentin 1 h prior to surgery, and 500 mg TID for 8 days thereafter. 9 patients received no systemic antimicrobial therapy. Microbiological examination was performed 1 h prior to surgery, at the time of membrane removal at week 6, and at 6 months post-surgery. Microbial morphotypes, total viable counts, and the occurrence of selected microbial species were determined by phase-contrast microscopy, selective and non selective culture, and DNA probes. Study sites were examined for probing pocket depths and attachment levels. At baseline, no microbial or clinical parameter showed statistical differences between groups. At 6 months, the Augmentin group demonstrated a significantly higher (P = 0.032; Student t-test) mean probing attachment gain (36.5% of potential gain to the cemento-enamel junction) than the 9 control patients (22.4% of potential gain). At the time of removal, membranes in the Augmentin group showed significantly fewer organisms than membranes in the control group (52.2 x 10(6) versus 488.6 x 10(6)). Sites free of pathogens on the membrane surface toward the tooth gained the most clinical attachment, even in the presence of various pathogens on the gingiva-facing membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560225 TI - Periodontal tissue-vitality after different etching modalities. AB - The purpose of the present study was to investigate possible immediate necrotizing effects on periodontal tissues of etching agents operating either at low or neutral pH. Phosphoric and citric acids, both of which operate at low pH, exerted immediate (within 20 s) necrotizing effects on both mucosal flaps and periodontal tissues. The penetration depth increased with time up to 1/4 of the circumference of the root after 3 min. This was in sharp contrast to EDTA which operates at neutral pH. EDTA failed to induce any detectable necrosis during the experimental periods. Whether or not the immediate necrotizing effects of low pH etching agents also impair periodontal healing in the long-term perspective, needs to be evaluated with controlled experimental studies in vivo as well as clinically in order to determine whether etching at neutral pH is preferable. PMID- 7560227 TI - Resolution of ligature-induced peri-implantitis lesions in the dog. AB - The present experiment in the Labrador dog was performed to study tissue alterations that occurred in a peri-implantitis lesion following ligature removal. The study was carried out in 5, 1-year old Labrador dogs. The mandibular right and left 1st molars and 4th and 3rd premolars were extracted to establish recipient sites for implants. After 3 months of healing, 4 titanium fixtures, 2 in each jaw quadrant, were installed and abutment connection carried out in a 2nd stage procedure after another 3 months. After a 6-month period of healing, cotton floss ligatures were placed in a submarginal position around the neck of the fixture abutments. The ligatures were forced into a position "apical" of the margin of the peri-implant mucosa and secured. When the tissue destruction after 4-6 weeks was found to amount to about 25% of the original bone height at each individual implant, the ligatures were removed. 2 dogs were sacrificed 1 month and 3 dogs 3 months after ligature removal. The animals were perfused with a fixative and block biopsies were obtained from the implant sites. The biopsies were prepared for histometric and morphometric analyses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560229 TI - Triclosan reduces the clinical symptoms of the allergic patch test reaction (APR) elicited with 1% nickel sulphate in sensitised patients. AB - It has previously been shown that triclosan reduces the clinical skin reactions caused by sodium lauryl sulphate (SLS) in an irritant patch test reaction (IPR) model. Furthermore, triclosan reduces the inflammatory reactions on skin after subcutaneous injection of histamine. The aim of the present study was to examine whether triclosan reduces the clinical symptoms on skin after exposure to nickel in an allergic patch test reaction (APR) model. 1% nickel sulphate was used for APR in 10 nickel-allergic females. The results showed that application of triclosan on skin reduced the APR symptoms from nickel in sensitised patients significantly (p < 0.05) compared to the saline and alcohol solutions. It is suggested that inflammatory mediators produced by the sensitised lymphocytes are inhibited by triclosan. PMID- 7560228 TI - The effect of triclosan on mediators of gingival inflammation. AB - Triclosan (2,4,4',-trichloro-2'-hydroxydiphenylether) is a well-known and widely used nonionic antibacterial agent which has recently been introduced in toothpastes and mouthrinses. The efficacy of triclosan-containing toothpaste and mouthrinse to reduce both plaque and gingivitis in long-term clinical trials has been well documented. Until recently, it was generally assumed that triclosan's effect on gingival inflammation was due to its antimicrobial and anti-plaque effect. It has now become apparent that triclosan may have a direct anti inflammatory effect on the gingival tissues. Several in vitro studies were conducted to evaluate the effect of triclosan on 4 primary enzymes of the pathways of arachidonic acid metabolism, cyclo-oxygenase 1, cyclo-oxygenase 2, 5 lipoxygenase and 15-lipoxygenase. These pathways lead to the production of known mediators of inflammation such as the prostaglandins, leukotrienes and lipoxins. Triclosan inhibited both cyclooxygenase 1 and cyclo-oxygenase 2 with IC-50 values of 43 microM and 227 microM, respectively. Triclosan also inhibited 5 lipoxygenase with an IC-50 of 43 microM. The 15-lipoxygenase was similarly inhibited by triclosan with an IC-50 of 61 microM. Hence, triclosan has the ability to inhibit both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism with similar efficacy. In cell culture experiments, it was found that triclosan inhibited IL-1 beta induced prostaglandin E2 production by human gingival fibroblasts in a concentration dependent manner, and at relatively low concentrations. These data, taken together, indicate that triclosan can inhibit formation of several important mediators of gingival inflammation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560230 TI - Effects of triclosan on the rat phrenic nerve-diaphragm preparation. AB - It has been reported that the lipid soluble, anti-inflammatory drug triclosan, which is currently used in toothpastes and mouthrinses, may reduce pain. This may be an aspect of the anti-inflammatory effect of triclosan, which probably reduces the production of prostaglandin PGE2. However, triclosan may also exhibit a direct analgesic effect through an effect on excitable membranes. The aim of the present study was to examine the possible effect of triclosan on the nerve action potential, on the neuromuscular transmission and on the excitation-contraction coupling of skeletal muscle. Phrenic nerve-hemidiaphragm preparations, as well as isolated phrenic nerves, were dissected from Wistar albino rats and mounted by standard methods in Tyrode solution. Both during indirect and direct stimulation, the preparations were irreversibly inhibited by triclosan at concentrations higher than 5.0 x 10(-6)M. At low concentrations, the inhibition of the twitches during indirect stimulation and of the compound action potential was probably caused by a threshold increase for excitation of the phrenic nerve. At high concentrations, an additional inhibitory effect at the neuromuscular junction was disclosed. The directly stimulated preparation was also probably inhibited by an increase of the threshold for initiation of the muscle action potentials. In addition, a reduced KCl contracture and an acceleration of the caffeine contracture indicated an interaction with the sarcolemma. These results suggest that the analgesic effect of triclosan may be due to a direct interaction with excitable membranes. PMID- 7560234 TI - Psychosocial factors in inflammatory periodontal diseases. A review. AB - Reviewing the literature concerning the possible role of psychosocial factors in the aetiology of inflammatory periodontal diseases, it may be concluded that there is evidence which strongly suggests that emotional stress is one of the predisposing factors to ANUG. On the other hand, it is not clear that the scientific evidence is sufficient to substantiate the hypothesis that psychosocial factors are of aetiological importance in periodontitis. The proposed mechanisms which may mediate the putative relationship between psychosocial conditions and inflammatory periodontal diseases remain to be tested. However, psychoneuroimmunologic studies make lowered host resistance especially interesting as a possible mechanism. Although available studies do not definitively support causal relationships, they suggest that psychosocial factors may be involved in the aetiology of inflammatory periodontal diseases, which, in turn, would relate to clinical management of these conditions. PMID- 7560231 TI - The effects on chronic periodontitis of a subgingivally-placed redox agent in a slow release device. AB - Adjunctive chemical agents can reduce the need for meticulous plaque control. The aim of this investigation was to evaluate the periodontal treatment potential of subgingival application of the redox agent methylene blue in a slow-release device. This randomized, single-blind, split-mouth study included 18 patients aged 35-57 years, with chronic adult periodontitis, pocketing of at least 5 mm and radiographic evidence of regular bone loss. All experimental sites received subgingival debridement at day 0. Test sites received 32% w/w methylene blue in the slow release device at days 0 and 28. Clinical examination and microbiological sampling were performed at days 0, 7, 28, 56 and 84. Clinical improvements were seen in both groups, but test sites showed consistently greater improvements, some of which were statistically significant (as determined by between-group comparisons utilising SNDs). Significant between-group differences in relation to baseline levels were seen in bleeding index at days 7 and 56, in probeable pocket depth at day 56 and for the Perioscan BANA test at day 7. This pilot study thus showed that adjunctive methylene blue in a slow-release device can produce greater clinical and microbiological improvements than subgingival debridement alone. PMID- 7560233 TI - Effect of periodontal treatments on serum IgG antibody titers against periodontopathic bacteria. AB - Serum IgG antibody titers to 7 periodontopathic bacteria in periodontitis patients were measured at the 1st visit and after various periodontal treatments with clinically successful improvement, in order to evaluate what kind of factors are associated with changes of serum antibody titers. 20 patients (10 male and 10 female from 23 to 61 years old) with adult, rapidly progressive periodontitis were enrolled in this study. All patients received initial preparation and most of them also underwent surgical procedure. After the treatments, the mean probing pocket depths decreased from 3.72 mm to 1.56 mm. Serum samples were collected from patients at the initial and final examinations. Serum IgG antibody titers against sonicated antigens of Porphyromonas gingivalis FDC 381, Prevotella intermedia ATCC 25611, Prevotella loescheii ATCC 15930, Fusobacterium nucleatum subspecies nucleatum ATCC 25586, Actinobacillus actinomycetemcomitans FDC Y4, Eikenella corrodens FDC 1073 and Capnocytophaga ochracea # M 12 were determined by enzyme-linked immunosorbent assay. The mean antibody titers to P. gingivalis and P. intermedia decreased significantly after the treatment as compared to their pretreatment levels. The antibody titer to P. gingivalis, especially, decreased in all of the patients examined. A significant relationship was found between the decreased antibody titer to P. gingivalis and the number of teeth which received periodontal surgery, as well as treatment length, and the relationship between the decreased antibody titer to P. intermedia and the number of extracted teeth was also significant. These results suggest that the changes of serum IgG titers against P. gingivalis and P. intermedia are related to the suppression of such pathogens in subgingival plague. PMID- 7560232 TI - Gingival crevicular stromelysin, collagenase and tissue inhibitor of metalloproteinases levels in healthy and diseased sites. AB - The ability of stromelysin (SL), fibroblast-type collagenase (FIB-CL) and tissue inhibitor of metalloproteinases (TIMP), to differentiate between healthy, gingivitis and periodontitis sites was investigated. SL and FIB-CL are members of a family of enzymes which are capable of degrading most of the extracellular matrix macromolecules. Extracellular control of these enzymes is performed by TIMP. 40 patients each provided 3 GCF samples from healthy, gingivitis and periodontitis sites. GCF samples were collected by means of sterile paper strips. GCF samples were eluted into 500 microliters of assay buffer and assays for SL, FIB-CL and TIMP were performed by a sandwich ELISA. The mean amounts of SL and TIMP in diseased sites (gingivitis and periodontitis) were significantly higher than the mean amount of these GCF components in healthy sites (MANOVA p values were: 0.006 for SL and 0.001 for TIMP). GCF SL and TIMP differentiated healthy from diseased sites. Both SL and TIMP showed moderate correlation with clinical indices. FIB-CL was detectable in only 20.8% of all sites and did not correlate with disease status. PMID- 7560235 TI - A 6-month home-usage trial of 0.1% and 0.2% delmopinol mouthwashes (II). Effects on the plaque microflora. AB - The effects of 0.1% and 0.2% delmopinol mouthwashes on supragingival plaque flora were investigated in a 6-month home-use study. 141 subjects were studied from whom plaque was collected at baseline, 12, 24, and 36 weeks. Overall, there were no consistent effects on microscopic or total counts. However, there was a significant reduction in the proportion of dextran-producing streptococci in the active groups compared to the control group throughout treatment. There was no colonisation by Candida or Gram-negative aerobic bacilli in the active groups nor was there any decrease in susceptibility to delmopinol. Delmopinol appears to mediate its anti-plaque effect without causing a major shift in bacterial populations, although dextran-producing bacteria appear to be affected, which may have relevance to this agent's mode of action. PMID- 7560236 TI - Plaque formation and gingivitis after mouthrinsing with 0.2% delmopinol hydrochloride, 0.2% chlorhexidine digluconate and placebo for 4 weeks, following an initial professional tooth cleaning. AB - A double-blind, randomised, 4-week clinical trial with parallel group design in 57 patients with gingivitis was conducted for studying the antibacterial efficacy and safety of a delmopinol HCl aqueous solution 2 mg/ml (0.2% w/v), which was used for unsupervised mouth-rinsing and compared with placebo and chlorhexidine digluconate 2 mg/ml (0.2% w/v, Hibitane Dental, ICI Pharmaceuticals, UK). The plaque index and plaque wet weight were used to measure plaque formation, and gingival fluid flow and bleeding on probing to measure gingivitis. According to the reduction from baseline, chlorhexidine showed a significantly better effect on plaque formation than the placebo after 4 weeks treatment for both plaque measurements. Delmopinol exhibited significantly lower plaque index scores than placebo. The difference between chlorhexidine and delmopinol was not statistically significant for any of the plaque measurements. For gingivitis, no statistically significant differences were obtained between the effects of delmopinol, chlorhexidine and placebo. A transient anaesthetic sensation in the oral mucosa was experienced more clearly by the patients in the delmopinol group than by those using chlorhexidine or placebo rinses. Rinsing with chlorhexidine resulted in more staining of the teeth and tongue than did delmopinol and placebo. The placebo solution tasted better than the 2 active solutions. The results showed that rinsing with either delmopinol HCl aqueous solution 2 mg/ml or chlorhexidine digluconate 2 mg/ml 2x daily for 60 as a supplement to normal oral hygiene, following an initial professional tooth cleaning, leads to a lower plaque formation than rinsing with placebo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560237 TI - The use of plaque area and plaque index to measure the effect of fluoride and chlorhexidine toothpastes on 24-h plaque regrowth. AB - Short-term methodologies have been developed to screen chemical agents for plaque inhibitory effects. Most measure inhibition of plaque regrowth over several days. A method was described to study agents over 16-24 h periods, although some difficulties with the index used were reported. The aim of this study was to determine whether more conventional plaque scoring methods could be used. These 2 crossover studies measured plaque regrowth over 24 h in 20 subjects and in response to single brushings with either a chlorhexidine and minus active toothpaste or to a fluoride toothpaste product and water. Significantly less plaque had reformed after brushing with the chlorhexidine toothpaste compared with its control and with the fluoride toothpaste compared with water. 18 subjects participated in both studies and using their data for an interstudy comparison, less plaque developed with the chlorhexidine compared with the fluoride toothpaste. These studies had the advantage that longer-term trials already demonstrated the same differences. This permits one to conclude with some certainty that the 24-h plaque regrowth study design, using conventional measures of plaque accumulation, could be a useful and rapid method of screening potential plaque inhibitory agents and formulations. PMID- 7560239 TI - Effects of citric acid treatment on the Ca, P and Mg contents of human dental roots. AB - An electron microprobe analyser associated with a scanning electronic microscope was used to measure Ca, P and Mg contents of the human dental root hard tissues, before and after a citric acid treatment (pH = 1). The measurements were made on transverse sections through the cervical 1/3 of the molar roots. The measurements were performed at the following 8 levels: the internal cementum, the cementum dentine junction, cementum-related dentine, 4 external dentine levels located at 220 microns, 420 microns, 620 microns and 820 microns from the cementum-dentine junction, and finally the juxta-pulpal dentine. After the citric acid treatment, the losses in Ca and P, but not in Mg, varied significantly with the level; an acid-resistant dentine layer of approximately 600 microns was found under the cementum-dentine junction. An increase in the Ca/P ratio was also observed in this layer. Since this external dentine zone is less demineralized by the citric acid than the underlying dentine, the collagen matrix in this region may also be less exposed. These differences in the response to citric acid that depend on the distance from the root surface may explain the unpredictability of periodontal healing after citric acid treatment of diseased root surfaces, because the amount of tissue removed during root planing is not constant. PMID- 7560240 TI - Comparison of periodontal disease in HIV seropositive subjects and controls (I). Clinical features. AB - The aims of this study were to investigate the prevalence and severity of periodontal diseases in HIV seropositive and assumed HIV seronegative individuals in the Edinburgh area. 29 HIV seropositive subjects were examined at baseline and at 3-monthly intervals. Attachment loss and dichotomous indicators of periodontal disease were recorded. 27 control subjects were seen at baseline only. Although there was no significant difference between the 2 groups in terms of mean attachment loss at baseline, HIV seropositive subjects had experienced more severe attachment loss localised to the lower incisor region. HIV seropositive subjects had a significantly higher mean % of sites exhibiting suppuration and redness. A distinct subgroup of 9 HIV seropositive subjects with widespread attachment loss was detected, which included those subjects with severely affected lower incisors. Site-specific attachment loss of > or = 3 mm was observed in only 20 sites out of 2814 sites in HIV+subjects observed longitudinally. The incidence of aggressive periodontal disease in this study was lower than anticipated, despite the inclusion of patients with advanced HIV infection or AIDS. PMID- 7560238 TI - A comparative clinical study: the use of human type I collagen with and without the addition of metronidazole in the GTR method of treatment of periodontal disease. AB - This investigation was undertaken to evaluate cross-linked human type I collagen, with and without added metronidazole, when used as a barrier membrane in the guided tissue regeneration (GTR) principle of treatment for periodontal disease. 16 patients suffering from moderate to severe periodontitis with 78 bilaterally matched periodontal defects underwent similar contralateral surgical flap procedures after preliminary scaling, polishing and oral hygiene instruction. At the experimental sites, which were selected at random, the flap was closed over metronidazole impregnated collagen as a GTR membrane, the contralateral sites receiving a plain collagen barrier as control. The plaque index (PLI), gingival index (GI), bleeding index (BI), probing pocket depth (PPD) and probing attachment level (PAL) were recorded at baseline, 6, 12 and 26 weeks post operatively. The bony defects were classified and furcation involvement noted. The clinical parameters were recorded by an examiner, other than the surgeon, who had been previously assessed for accurate reproducibility of measurements and was unaware of the experimental sites. PPD and PAL were measured with a constant pressure probe, localised by a soft stent. Post-operative discomfort was evaluated by means of a questionnaire. PLI, GI and BI were significantly improved compared to baseline for both test and control sites at 6, 12 and 26 weeks post surgery (p < 0.001) but there was no significant difference between these sites (p > 0.05). There was a reduction in PPD at 6 weeks which was significant at 12 and 26 weeks post-operatively (p < 0.001) for both test and control sites, but no difference between these sites was evident (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560241 TI - Comparison of periodontal disease in HIV seropositive subjects and controls (II). Microbiology, immunology and predictors of disease progression. AB - The aims of this study were to compare the prevalence of suspected periodontal pathogens in subgingival plaque from 29 HIV seropositive and 27 control subjects and to determine the association of these bacteria with periodontal destruction. Subgingival plaque was collected from the mesiobuccal sites of all teeth, except 3rd molars. Bacteria were identified and enumerated using non-isotopic whole chromosomal DNA probes and a colony lift method. At baseline, HIV seropositive subjects had significantly higher mean % of Porphyromonas gingivalis than control subjects. This difference could be attributed to a subgroup of HIV seropositive subjects with widespread attachment loss. No correlations were observed between the mean %s of DNA probe species and mean attachment loss, CD4 and CD8 T lymphocyte counts or CD4: CD8 ratio. No significant microbiological differences were detected between active and control sites in HIV seropositive subjects on a longitudinal basis. There appeared to be an inverse relationship between the mean %s of P. gingivalis and V. parvula, with respect to progression of HIV infection. The ability of microbiological parameters to predict site-specific breakdown in HIV seropositive subjects requires further investigation. PMID- 7560244 TI - Another perspective on the cocktail strategy: "two glasses of wine or a six pack". PMID- 7560243 TI - Helicobacter pylori and gastrointestinal disease. AB - New paradigms are few and far between. The discovery that peptic ulcer disease is the result of an infection by a previously unrecognized microorganism rather than the consequence of excess secretion of gastric acid aptly qualifies as a paradigm shift. Eradication of Helicobacter pylori from the stomach by antibacterial agents heals ulcers at least as effectively as H2-blockers. Further, eradication of the organism results in long-term cures of peptic ulcers. In contrast, the relapse rate in patients successfully treated with H2-blockers exceeds 50% in the first year after healing. Also of interest is the role of H. pylori in gastric cancer. PMID- 7560245 TI - Pharmacokinetic-based ticarcillin/clavulanic acid dose recommendations for infants and children. AB - The pharmacokinetic characteristics of ticarcillin and clavulanic acid were determined after the first dose (n = 22) and again under steady-state conditions (n = 16) in a group of infants and children. Study subjects ranged in age from 1 month to 9.3 years; all but 3 study patients were 6 months of age or older. Each patient received 50 mg of ticarcillin and 1.7 mg of clavulanic acid (30:1 ratio) per kg of body weight given intravenously every 4 hours. Elimination half-life, steady-state volume of distribution, and body clearance averaged 1.1 hours, 0.22 L/kg, and 2.7 mL/min/kg, respectively, for ticarcillin, and 0.9 hours, 0.4 L/kg, and 6.2 mL/min/kg, respectively, for clavulanic acid. A total of 71% of the ticarcillin and 50% of the clavulanic acid dose were excreted unchanged in the urine over the 4-hour sampling period. Corresponding renal clearances averaged 2.1 and 3.2 mL/min/kg for ticarcillin and clavulanic acid, respectively. No differences were observed between first dose and steady-state evaluations in the pharmacokinetic behavior of either agent. In contrast, the pharmacokinetic behavior of clavulanic acid was significantly different from that observed for ticarcillin. These pharmacokinetic data combined with known in vitro susceptibilities of important clinical pathogens support a dose of 80 mg of ticarcillin and 2.7 mg/kg clavulanic acid per kg body weight given as a fixed dose combination every 8 hours for the treatment of most systemic infections that occur outside the central nervous system. PMID- 7560246 TI - Steady-state pharmacokinetic evaluation of a novel, controlled-release morphine suppository and subcutaneous morphine in cancer pain. AB - Although the oral route is the preferred method for morphine administration for cancer pain, many patients will require an alternate route of administration at some point during their illness. The authors studied the steady-state pharmacokinetics of morphine after administration of a novel, controlled-release suppository (MS-CRS) and subcutaneous morphine in a randomized, double-blind, two way crossover evaluation in 10 patients with cancer pain. When administered at a 2.5:1 analgesic ratio, MS-CRS given every 12 hours showed an equivalent extent of absorption compared with subcutaneous morphine given every 4 hours (AUC0-12, 132.5 +/- 30.1 versus 123.8 +/- 27.3 ng.h.mL-1, P = not significant [NS]). Peak morphine concentrations were lower, time of peak was later, and percent fluctuation less after MS-CRS than after subcutaneous morphine (Cmax, 14.7 +/- 2.9 versus 29.9 +/- 5.4 ng/mL, P = .0110; tmax, 3.33 +/- 0.75 versus 2.22 +/- 0.15 hours, P = .0160; fluctuation, 122 +/- 71 versus 356 +/- 123%, P = .00160). Relative bioavailability of MS-CRS using the 2.5:1 analgesic ratio was 105%, and bioavailability from data dose normalized without regard to route specificity in metabolism was 42%. For both routes of administration there was a significant linear relationship between morphine dose and AUC (MS-CRS, r = .8568, P = .0032; subcutaneous morphine, r = .8314, P = .0055). MS-CRS morphine provides a pharmacokinetic profile consistent with dosing every 12 hours; at steady state, the extent of absorption is comparable with that of subcutaneous morphine when administered at a 2.5:1 dose ratio.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560242 TI - Influence of gingival crevicular washing on the expression of polymorphonuclear leukocyte membrane receptors before and after periodontal therapy. AB - Extensive data demonstrate that polymorphonuclear leukocytes (PMN) are the predominant cell type involved in periodontal disease and that gingival crevicular fluid constituents are influenced by the inflamed gingiva. The aim of the present study was to evaluate the ability of gingival crevicular washing (GCW) (a dilution of gingival crevicular fluid) from periodontal sites in different clinical conditions of modulating the PMN membrane receptors involved in motility, adhesion and phagocytosis before and after periodontal treatment. 10 patients affected by adult periodontitis (AP) were selected. From each patient, 2 test sites (TS) were chosen on the basis of a probing depth > 5 mm and attachment loss, and 2 control sites (CS) with probing depth < 3 mm without. Modifications of membrane receptor density of PMN from healthy donors incubated with GCW harvested from TS and CS was evaluated using fluorescent probes and flow cytometry. Compared to CS-GCW, TS-GCW before therapy increased the expression of the beta 2 integrin CD11b and the chemotactic receptor for the oligopeptide N formyl methionyl leucyl phenylalanine (FMLP-R) while it reduced the expression of L-selectin. GCW collected from the same TS after the successful completion of periodontal treatment did not influence PMN receptors, indicating that the clinical improvement paralleled the disappearance of the PMN modulating capability contained in TS-GCW before therapy. In conclusion, the present data illustrate the relevant modifications occurring at PMN membrane in chronic adult periodontitis exerted by GCW obtained by a simple fluid collection technique. Thus, monitoring gingival crevicular fluid PMN activating capability may help disclose the presence of chronic periodontitis and may be useful in assessing successful treatment. PMID- 7560247 TI - Lisinopril versus placebo in the treatment of heart failure: the Lisinopril Heart Failure Study Group. AB - Lisinopril, a long-acting, angiotensin-converting enzyme inhibitor, was compared with placebo in a randomized, parallel, double-blind, 12-week study of 193 patients with heart failure. All patients were New York Heart Association Functional Class II, III, or IV and had remained symptomatic despite optimal dosing with digoxin and diuretics. After 12 weeks of therapy, the improvement in treadmill exercise duration was greater in the lisinopril group (113 seconds) compared with the placebo group (86 seconds). This improvement in exercise duration was particularly evident in patients with left ventricular ejection fractions less than 35% (lisinopril = 130 seconds; placebo = 94 seconds). In patients receiving lisinopril, the increase in exercise duration was accompanied by an improvement in quality of life as measured by the Yale Scale Dyspnea/Fatigue Index and in signs and symptoms of heart failure. In addition, the lisinopril group had a larger mean increase (3.7%) in left ventricular ejection fraction when compared with the placebo group (1.3%). Thus, lisinopril, administered once daily for 12 weeks, was well tolerated and efficacious in the treatment of heart failure when used concomitantly with diuretics and digoxin. PMID- 7560248 TI - Pharmacokinetics of orally administered cyclosporine in patients with Crohn's disease. AB - Cyclosporine pharmacokinetics were reported to be influenced in patients with Crohn's disease. To explore the relationship between Crohn's disease and cyclosporine pharmacokinetics, this investigation was performed in 20 patients with varying Crohn's Disease Activity Index (CDAI). A single oral dose of 300 mg of cyclosporine was given and serial blood samples were obtained over 52 hours. Cyclosporine whole blood concentrations were determined by a specific monoclonal radioimmunoassay. Pharmacokinetic parameters were comparable with those of healthy volunteers. No statistically significant difference between the pharmacokinetic parameters of the patients with CDAI values less than 150 and those with CDAI values 150 or greater could be shown. Although several factors associated with the pathology of Crohn's disease theoretically could influence the pharmacokinetics of orally administered cyclosporine, this investigation did not identify statistically significant differences in cyclosporine pharmacokinetics in Crohn's disease patients with different disease activities or different localization of inflammation as compared with healthy volunteers. However, if large parts of the small bowel were removed, a decrease of absorption of cyclosporine could be observed. In any case, it is important to be aware of the clinical variability of Crohn's disease and its potential implications in cyclosporine absorption. PMID- 7560250 TI - Impact of a four-day head-down tilt (-6 degrees) on lidocaine pharmacokinetics used as probe to evaluate hepatic blood flow. AB - The impact of a microgravity simulation using a head-down tilt (-6 degrees) on lidocaine pharmacokinetics used as a probe to evaluate hepatic blood flow is discussed. Eight healthy male subjects were selected for a 7-day study, including a 4-day head-down tilt from day 2 to day 5. Subjects were given 1 mg/kg of lidocaine on days 1 through 5 and 7. Blood sampling, cardiac output, and hepatic artery blood flow velocity measurements were done within 6 hours after administration. Cardiac output increased significantly during head-down tilt, and returned to basal values during the recovery period. Blood flow velocity in the hepatic artery increased during the first day of the down tilt. Slight side effects (buzzing noise in the ears and sleepy feeling) were reported within minutes after the injection of lidocaine. Lidocaine disposition was modified during head-down tilt: a significant decrease in maximal concentration (1.47 +/- 0.26 mg/L on day 1 and 0.96 +/- 0.30 mg/L on day 2); an increase in elimination clearance from 8.24 +/- 3.22 mL/kg.minutes-1 to 11.63 +/- 3.00 mL/kg.minutes-1; an increase in volume of distribution on day 2 and a decrease to lower than basal value on the other days (2.77 +/- 1.73 L/kg on day 1 and 2.33 +/- 0.48 L/kg on day 7). Half-life regularly decreased from 264 +/- 210 minutes to 160 +/- 60 minutes between day 1 and day 7.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560249 TI - The pharmacokinetics and cardiovascular pharmacodynamics of HP 749 (besipirdine HCl) and metabolite P86-7480 in the conscious monkey. AB - HP 749 was absorbed slowly in the conscious monkey after single oral doses (10, 20, and 40 mg/kg), with gradual metabolism to the N-despropyl metabolite, P86 7480. The tmax was 2 to 4 hours after dosing, with nonlinear increases in Cmax and the AUC0-4th for HP 749. The calculated elimination half life (t1/2) after oral administration was 7.4 +/- 2.1 hours; however, absorption appeared to influence the terminal phase because the t1/2 after intravenous administration of 10 mg/kg was 1.5 hours. Plasma concentration of HP 749 2 minutes after intravenous bolus was 26.08 micrograms/mL. The HP 749 was rapidly distributed (t1/2 alpha = 0.064 +/- 0.033 hours) after intravenous administration, and displayed a VZ of 2.6 +/- 0.85 L/kg. The CL of HP 749 was 20.8 +/- 6.9 mL/min/kg, whereas renal clearance (CLR) of unchanged drug was only 0.13 +/- 0.04 mL/min/kg. Thus, only about 1% of the administered dose was excreted unchanged by the kidney. The P86-7480 also was rapidly distributed and eliminated after an intravenous bolus, but was less extensively distributed than HP 749. HP 749 administered either as an intravenous bolus or orally caused a significant pressor effect soon after dosing. A significant tachycardia resulted from intravenous administration, but not after oral administration of the drug. An intravenous bolus of P86-7480 (0.1 mg/kg) resulted in an immediate increase in MAP and decreased heart rate. The duration of these cardiovascular events was significantly shorter after intravenous administration of P86-7480 than with intravenous or oral administration of the parent drug.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560251 TI - A double-blind, placebo-controlled, dose-ranging safety evaluation of single-dose intravenous dolasetron in healthy male volunteers. AB - The safety and tolerability of dolasetron mesylate, a potent and selective 5-HT3 receptor antagonist, were evaluated after single intravenous doses in healthy male volunteers. In this double-blind, placebo-controlled, randomized, phase I study, 80 subjects received either placebo or dolasetron in escalating doses (0.6 to 5.0 mg/k). Subjects were monitored for adverse events, vital sign and laboratory alterations, and changes in electrocardiographic (ECG) intervals and electroencephalographic (EEG) patterns. Overall, the percentage of subjects reporting adverse events was similar in those receiving dolasetron (44/64; 68.8%) or placebo (10/16; 62.5%); most adverse events were mild in severity. Subjects receiving dolasetron reported a higher incidence of central nervous system (headache and dizziness/lightheadedness), gastrointestinal (increased appetite and nausea), and visual adverse events and taste alterations. No clinically significant changes in laboratory variables were observed. Transient and asymptomatic ECG changes (small mean increases in PR interval and QRS complex duration versus baseline) were noted in several subjects at 1 to 2 hours after infusion at doses > or = 3.0 mg/kg. Transient, mild blood pressure decreases were observed in five subjects, including one on placebo. Dolastron mesylate was well tolerated in single intravenous doses up to 5.0 mg/kg in healthy male volunteers. Clinical studies of the drug are ongoing for antiemetic indications. PMID- 7560252 TI - Safety, tolerability, and effect of food on the pharmacokinetics of iloperidone (HP 873), a potential atypical antipsychotic. AB - Iloperidone (HP 873) is a D2 and 5-HT2 receptor-antagonist that is under development as a potential atypical antipsychotic agent. Two studies on iloperidone evaluated its safety and tolerability, made a preliminary pharmacokinetic assessment of single 3- and 5-mg doses, and determined the effect of food on its tolerability and pharmacokinetics in healthy volunteers after single 3-mg doses. Iloperidone was well absorbed orally in fasted subjects. The Cmax occurred approximately 2 to 3 hours after administration of a single 3- or 5 mg dose. The pharmacokinetic parameters increased with the dose between 3 and 5 mg (from 2.2 to 5.2 ng/mL for Cmax, and 16 to 50 ng/mL.h for AUC). Iloperidone was eliminated slowly, with a mean t1/2 of 13.5 to 14.0 hours. Coadministration with food did not significantly affect AUC, tmax, or Cmax. These results indicate that the rate of iloperidone's absorption is decreased, but the overall bioavailability is unchanged, when the drug is taken with food. Orthostatic hypotension, dizziness, and somnolence were the most commonly reported adverse events. Coadministration of food reduced the incidence and severity of these events. PMID- 7560253 TI - Single-dose interaction study of diprafenone HCl and propranolol HCl in healthy volunteers. AB - Using a 3 x 3 Latin Square design, a possible interaction between diprafenone HCl a class IC antiarrhythmic drug with nonspecific beta-antagonist activity and propranolol HCl was investigated in nine young, healthy, caucasian, male volunteers. The volunteers randomly received 3 single-dose treatments: (A) 200 mg DHCl, (B) 80 mg PHCl, and (C) 200 mg DHCl and 80 mg PHCl. Scheduled blood samples were taken and plasma concentrations of both diprafenone and propranolol were measured by sensitive and specific assay methods. Lead II electrocardiogram intervals at rest, heart rate during erect bicycle ergometry, and echocardiographic variables at rest and shortly after exercise were recorded. The data analysis used compartment model independent methods. There was no evidence for a pharmacokinetic interaction between the two drugs. With DHCl, two of the nine subjects showed greatly increased areas under the plasma concentration-time curves and apparent disposition half-lives in the presence and absence of PHCl, indicating that metabolism of diprafenone may be subject to pharmacogenetic polymorphism. There was evidence for a pharmacodynamic interaction between DHCl and PHCl regarding the negative chronotropic effect at rest and during exercise. There was no difference in the pharmacodynamics and tolerability of the three treatments in suspected "poor" and "extensive metabolizers" of DHCl. PMID- 7560256 TI - Decision making in the treatment of school-age children who stutter. AB - The purpose of this article is to provide clinicians information on some of the key issues in making decisions about the treatment process for children who stutter. Ten decisions that should be considered prior to, during, and at the time of dismissal are discussed. These decisions relate to a number of issues regarding treatment such as: increasing clinicians' confidence in treating stuttering in children, setting long- and short-term goals, selecting an approach to treatment, documenting progress, involving parents and teachers in the treatment process, and determining when the child is ready to be dismissed from treatment. Additionally, questions clinicians should ask themselves are presented with each of the ten decisions. The intent of this article is to show how an analysis of clinical information systematically collected over a period of time will help a clinician make accurate decisions about the treatment of children who stutter. PMID- 7560255 TI - Glyburide protein binding and the effect of albumin glycation in children, young adults, and older adults with diabetes. AB - Because glyburide is a weak acid that is more than 98% bound to albumin, the authors evaluated the binding in vitro and the influence of albumin glycation in children, young adults, and older adults with diabetes mellitus. Glyburide binding to non-glycated albumin was greater than 98%, and remained constant over a total concentration range of 50 to 1000 ng/mL. Increasing the albumin concentration from 0.5 to 5.0 g/dL was logarithmically related to the free fraction of glyburide. After the in vitro glycation of albumin (range, 5.7 15.6%), mean (+/- SD) glyburide binding was 99.05 +/- 0.082%, a value in agreement with that obtained from control serum. Serum samples from 57 subjects with type I and 16 patients with type II diabetes were incubated with 300 ng/mL of unlabeled glyburide and 200 ng/mL of 14C-glyburide. The extent of albumin glycation varied from 5 to 22% for the type I subjects and 5 to 14% for the type II subjects. The free fraction from these groups ranged from 1.07 to 1.75% and 0.66 to 0.88% for the type I and type II subjects, respectively. Although these values did not differ significantly from those of the control samples, the glyburide free fraction in patients with type I diabetes (1.39 +/- 0.85%) was significantly greater than that found for the 16 elderly patients with type II diabetes (0.78 +/- 0.05%). A significant linear relationship was not found between glyburide free fraction and the degree of albumin glycation for either group. Glyburide protein binding did not appear to be influenced by the extent of albumin glycation. PMID- 7560254 TI - Pharmacokinetic and pharmacodynamic evaluation of warfarin and nefazodone coadministration in healthy subjects. AB - Nefazodone, an antidepressant with serotonin and norepinephrine receptor modulating activity, is highly protein bound and eliminated by oxidative metabolism. This study evaluated the potential for clinically significant drug interactions with warfarin and nefazodone coadministration. Eighteen subjects received warfarin daily for 14 days, achieving steady-state warfarin concentrations and a stable prothrombin ratio. Nefazodone 200 mg every 12 hours (n = 12) or placebo every 12 hours (n = 6) was then added to the daily warfarin dose for the next 7 days in a double-blind, randomized design. No serious or unexpected adverse events or events suggestive of abnormal bleeding occurred during coadministration. The addition of nefazodone had no effect on the unbound fraction of total warfarin in plasma or on the steady-state pharmacokinetics of R warfarin based on within-subject or comparison to placebo-treated subjects. The steady-state AUCTAU over the dosing interval and Cmax of S-warfarin decreased by 12%; however, this change is clinically insignificant because the prothrombin ratio and bleeding time remained unchanged. The steady-state minimum concentrations for nefazodone and metabolites, achieved on coadministration day 3, were typical of healthy men treated with this nefazodone dosage. In conclusion, warfarin and nefazodone coadministration was safe and well-tolerated with no clinically significant interactions. PMID- 7560257 TI - Treating fluency disordered adolescents. AB - This paper presents fluency disorders in three types of stuttering syndromes with etiological considerations. The syndromes consist of affective, behavioral and cognitive components useful for fluency assessment and establishment of therapy goals with adolescents who stutter. The ultimate goal of the treatment program is a feeling of fluency control rather than actual fluency. A therapy process for fluency disordered adolescents is suggested which outlines four stages with goals and objectives for each stage. Behavior techniques for eliciting a feeling of fluency control are also included. Finally, a case history illustrates the therapy process for an adolescent with a fluency disorder. PMID- 7560258 TI - Exploring the attitudes of adults who stutter. AB - In an effort to increase clinicians' confidence and competence in addressing attitudes of those adults who stutter, the following explores the nature of attitudes, attitudinal domains meriting consideration throughout the therapy process, and assessment issues. Included in this discussion are the results of a series of studies that examined communication attitudes and profiles of adults. Finally, some thoughts on intervention and specific research needs are presented. PMID- 7560259 TI - A behavioral-cognitive therapy program for adults who stutter: computers and counseling. AB - This paper evaluates the efficacy of a behavioral-cognitive treatment program for adults who stutter. The program combines a commercially available computer assisted biofeedback program for the reduction of stuttering and a relapse management program for counseling and attitude change. Four adults who stutter, between the ages of 20 and 25 years, participated in a study with multiple baseline across individuals. The initial treatment was conducted in an intensive time block, followed by extended treatment sessions. Results show that subjects reduced their disfluencies to below 3% stuttered syllables and maintained those changes at the 6 and 12 month follow-up. Measures of attitude changes were also assessed and showed that increases in positive feelings and attitudes were maintained at follow-up. PMID- 7560260 TI - The topography of beginning stuttering. AB - Presently, there are a number of protocols available to assist speech-language pathologists in the identification of early stuttering (Gordon and Luper, 1992). While such protocols offer guidelines for differentiating children who stutter from those who are normally disfluent, the criteria for doing so is frequently based on clinical observation, and often suffers from limited empirical support. The purpose of this paper is to provide an update and "recalibration" of what we currently know to be the salient behaviors of beginning stuttering, and how specific features of these behaviors distinguish stuttering children form their normally disfluent counterparts. Specifically, studies examining (1) frequency, (2) type, and (3) duration of disfluency, including number of repeated units and additional temporal aspects of instances of sound, syllable, and whole-word repetition, as well as (4) associated speech and nonspeech behaviors produced by children who stutter will be reviewed, and comparisons to normally speaking children will be made when possible. With a few exceptions, discussion will be limited to those studies which specifically examined the speech of preschool children, or which purported to include children close to the onset of stuttering, regardless of age. PMID- 7560262 TI - Substance P-immunoreactive neurons in the human retina. AB - Substance P (SP) is a neuropeptide that acts as a neurotransmitter or a neuromodulator in the retina. The aim of this study was to identify the type(s) and the distribution of the SP-immunoreactive (SP-IR) cells in the human retina. We have used an antiserum to SP to immunostain neurons in postmortem human retinae. Immunostained retinae were processed with the avidin-biotin complex (ABC) to visualize the cells either whole mounted in glycerol or embedded in plastic. Some retinae were also sectioned at 20 microns in order to obtain radial views of stained cells. SP-IR amacrine cells stain intensely and appear to be of a single type in the human retina. They are large-field cells with large cell bodies (16 microns diameter) lying in normal or displaced positions on either side of the inner plexiform layer (IPL). Their sturdy, spiny, and appendage bearing dendrites stratify in stratum 3 (S3) of the IPL, where many overlapping, fine dendrites intermingle to form a plexus of stained processes. Either cell bodies or primary dendrites emit an "axon-like" process that, typically, divides into two long, fine processes, which run in opposite directions for hundreds of micrometers in S5 and S3 before disappearing as distinct entities in the stained plexus in S3. Long, fine dendrites also pass from the dendritic plexus to run in S5 and down to the nerve fiber layer to end as large varicosities at blood vessel walls. In addition, fine processes are emitted from the dendritic plexus that runs in S1, and some pass up to the outer plexiform layer (OPL) to run therein for short distances. The SP-IR amacrine cell has many similarities to the thorny, type 2 amacrine cells described from Golgi studies. In addition to the SP-IR amacrine cells, a presumed ganglion cell type is faintly immunoreactive. Its 20 22 microns cell body gives rise to a radiate, sparsely branched, wide-spreading dendritic tree running in S3. Its dendrites and cell body become enveloped by the more intensely SP-IR processes and boutons from the SP-IR amacrine cell type. The SP-IR ganglion cell type most resembles G21 from a Golgi study. PMID- 7560263 TI - Immunocytochemical localization of the renal neutral and basic amino acid transporter in rat adrenal gland, brainstem, and spinal cord. AB - A neutral and basic amino acid transporter (NBAT) cloned from rat kidney was recently localized to enteroendocrine cells and enteric neurons. We used an antibody directed against a synthetic peptide representing a putative extracellular domain of NBAT to determine whether this transporter was also present in other endocrine and neural tissues, including rat adrenal gland, brainstem, and spinal cord. Abundant, highly granular labeling for NBAT was observed in the cytoplasm of chromaffin and ganglion cells in the adrenal medulla. A small population of intensely labeled varicose processes was also seen in both the cortex and the medulla of the adrenal gland. More numerous, intensely labeled varicose processes were detected in brainstem and spinal cord nuclei, including the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, dorsal motor nucleus of the vagus, and intermediolateral cell column of the thoracic spinal cord. Significant perikaryal labeling for NBAT was only detected in brainstem and spinal cord following intraventricular colchicine treatment, which increased the number, distribution, and intensity of NBAT immunolabeled cells. These NBAT-immunoreactive perikarya were most numerous in the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, and raphe nuclei. Ultrastructural examination of the nuclei of the solitary tract of normal rats showed that NBAT was localized predominantly to axon terminals. Within these labeled terminals, NBAT was associated with large dense core vesicles and discrete segments of plasma membrane. The observed localization of NBAT suggests that this renal specific amino acid transporter subserves a role as a vesicular or plasmalemmal transporter in monoamine-containing cells, including chromaffin cells and autonomic neurons. PMID- 7560264 TI - Effects of target deprivation on the morphology and survival of adult dorsal column nuclei neurons. AB - During development, interaction with target cells plays a critical role in the regulation of survival of afferent neurons. In an attempt to define the role of target cells in the adult central nervous system, the somatodendritic morphology and survival of adult cuneate neurons deprived of their targets by in situ injection of kainic acid in the rat thalamus were studied. In neuron-specific, enolase-immunostained sections, a 20% decrease in the mean longest diameter of the labeled cells was detected at 4 months postlesion. This somatic atrophy was accompanied by a loss of distal dendritic arborizations as observed after labeling by intracellular diffusion of horseradish peroxidase. Cytochrome oxidase staining did not reveal detectable alterations of the metabolic activity of these neurons, and an ultrastructural study also failed to demonstrate major changes in the neuronal somata. Cell counts indicated a much delayed death of 25% of the neurons at 10 months postlesion, whereas no neuronal death was detected at 7 months. The glial cells appeared unaltered both in number and in immunolabeling when using OX-42 antibodies or antiglial fibrillary acidic protein (anti-GFAP) antibodies. Results obtained in this time-course study indicate that neuronal death and alteration of the somatodendritic morphology are much delayed events after excitotoxic loss of targets. Somatodendritic atrophy occurs several months postlesion, and neuronal death occurs close to 1 year after lesion. These results suggest that the hypothesis of a necessary continuous trophic support by target cells does not hold as firmly for the adult central nervous system as during development. PMID- 7560265 TI - Neuropeptide tyrosine in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization. AB - Lungfishes, which share similarities with both fishes and amphibians, represent an interesting group in which to investigate the evolutionary transition from fishes to tetrapods. In the present study, we have investigated the localization and biochemical characteristics of neuropeptide Y (NPY)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. NPY immunoreactive cell bodies were found in various regions of the brain, most notably in the telencephalon (septal area, ventral striatum, and nucleus accumbens), in the diencephalon (preoptic nucleus, periventricular region of the hypothalamus, and ventral thalamus), and in the tegmentum of the mesencephalon. A strong immunoreaction was also detected in cell bodies of the nervus terminalis. Immunoreactive nerve fibers were particularly abundant in the ventral striatum, the nucleus accumbens, the diagonal band of Broca, the hypothalamus, and the mesencephalic tegmentum. Positive fibers were also seen in the median eminence and in the neural lobe of the pituitary. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high performance liquid chromatography (HPLC) analysis and radioimmunological quantitation. The displacement curves obtained with synthetic porcine and frog NPY and serial dilutions of brain and pituitary extracts were parallel. Reversed phase HPLC analysis of telencephalon, diencephalon, and pituitary extracts resolved a major NPY-immunoreactive peak that coeluted with frog NPY. The similarity between the distribution of NPY-containing neurons and the biochemical characteristics of the immunoreactive peptide in the brain of lungfish and frog strongly favors a close phylogenetic relationship between dipnoans and amphibians. PMID- 7560261 TI - Parents as partners: including mothers and fathers in the treatment of children who stutter. AB - Parents are frequently included in our intervention methods with children who stutter. However, if we examine the research and clinical literature, we find that we do not distinguish between the roles of mothers and fathers in our diagnosis and intervention procedures. Evidence that mothers and fathers may differ in their interactions with children, in general, and children who stutter, in particular, will be presented. Implications of these findings for our clinical practices will be discussed. By doing so, a case will be made for including both mothers and fathers as partners in the treatment of children who stutter. PMID- 7560267 TI - Premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles: differential distribution in the lower brainstem of the rat. AB - The distribution of premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles was examined in the lower brainstem of the rat by using retrograde and anterograde labeling techniques. First, Fluorogold, a fluorescent retrograde tracer, was injected into the dorsolateral or ventromedial division of the trigeminal motor nucleus, each of which contains motoneurons innervating the jaw-closing or jaw-opening muscles, respectively. Second, Phaseolus vulgaris-leucoagglutinin, an anterograde tracer, was injected into each of the lower brainstem sites, where clusters of retrogradely labeled premotor neurons had been seen in the first set of experiments. Third, after injection of the anterograde tracer into a lower brainstem site, followed by injection of the retrograde tracer cholera toxin B subunit into a masticatory muscle, termination of anterogradely labeled axons onto retrogradely labeled motoneurons was confirmed with the aid of a confocal laser-scanning microscope. It was found that the premotor neurons distributed in the mesencephalic trigeminal nucleus, medial part of the parabrachial region, supratrigeminal region, and dorsal parts of the principal sensory, oral spinal and interpolar spinal trigeminal nuclei project preferentially to the dorsolateral division of the trigeminal motor nucleus, whereas those in the lateral part of the parabrachial region, intermediate parts of the principal sensory, oral spinal and interpolar spinal trigeminal nuclei, and alpha part of the gigantocellular reticular nucleus project preferentially to the ventromedial division of the trigeminal motor nucleus. The dorsal and lateral parts of the medullary reticular formation and the medullary raphe nuclei contain premotor neurons of both types. Group k motoneurons, a cluster of trigeminal motoneurons that innervate the tensor tympani muscle, receive projection fibers predominantly from the dorsolateral part of the oral pontine reticular formation. PMID- 7560268 TI - Morphometric and electrical properties of reconstructed hippocampal CA3 neurons recorded in vivo. AB - CA3 pyramidal neurons were stained with biocytin during intracellular recording in rat hippocampus in vivo and reconstructed using a computer-based system. The in vivo CA3 neurons were characterized primarily according to their proximity to the hilus and secondarily with respect to the septotemporal location. Neurons measured in CA3a (n = 4), in CA3b (n = 4), and in posterior/ventral locations (n = 3) had the greatest dendritic lengths (19.8, 19.1, and 26.8 mm on average, respectively). Cells closer to the hilus showed much shorter dendritic lengths, averaging 10.4 mm for CA3c neurons (n = 4) and 11.6 mm for zone 3 neurons (n = 2). Half of the cells showed more than one major apical dendrite, and dendritic trees were highly variable even within CA3 subregions. The mean electronic length for these cell groups averaged between 0.30 lambda (CA3c) and 0.45 lambda (posterior/ventral), assuming a constant specific-membrane resistivity of 60 K omega-cm2. These CA3 neurons form a database of reconstructed neurons for further morphometric and electrical modelling studies. The large degree of variability between individual CA3 neurons indicates that both dendritic and electrical properties should be specifically calculated for each cell rather than assuming a "typical" morphology. PMID- 7560266 TI - Circuit reorganization in granuloprival cerebellar cultures in the absence of neuronal activity. AB - Neonatal mouse cerebellar cultures exposed to cytosine arabinoside for the first 5 days in vitro to destroy granule cells and compromise glia undergo a circuit reorganization featured by profuse sprouting of Purkinje cell recurrent axon collaterals, which hyperinnervate the somata of other Purkinje cells and project to Purkinje cell dendritic spines. Such granuloprival cultures were exposed continuously from explanation to tetrodotoxin and elevated levels of magnesium to block neuronal activity. A similar circuit reorganization occurred, except that there was a reduction in the number of axospinous synapses and Purkinje cell axosomatic synapses, which in this case were all inhibitory. Functionally, after recovery from the blockade, granuloprival cultures developed sustained cortical hyperactivity, which was consistent with the reduction of inhibitory synapses. While the absence of neuronal activity did not prevent reorganizational changes following granule cell loss, the full development of the inhibitory circuitry was not attained. These results further support the concept that spontaneous neuronal activity is necessary for the complete development of inhibitory synapses. PMID- 7560270 TI - Developmental changes in the expression of gamma-aminobutyric acidA/benzodiazepine receptor subunit mRNAs in the murine inferior olivary complex. AB - The pharmacological and physiological properties of ligand-gated ion channels are dependent on their subunit composition; spontaneously occurring changes in subunit composition during neuronal development may result in dramatic functional differences between embryonic and adult forms of the receptor complex. In the present study, in situ hybridization with antisense cRNA probes was used to examine the subunit composition of the gamma-aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor in the developing inferior olivary complex. This receptor is thought to be a pentameric chloride channel comprised of selected alpha, beta, gamma, delta, and rho subunits, the majority of which have several isoforms: alpha 1-6, beta 1-4, gamma 1-4, and rho 1,2. Among the 13 subunit variants present in the mammalian central nervous system, alpha 2-5, beta 3, and gamma 1,2 mRNAs are expressed at significant levels in the inferior olivary complex. Two clearly different temporal patterns of GABAA/BZ receptor subunit mRNA expression were observed: The expression of alpha 3, alpha 5, beta 3, and gamma 2 mRNAs was at a peak during embryonic and early postnatal development followed by rapid down regulation thereafter. Conversely, alpha 2, alpha 4, and gamma 1 mRNA expression was very low or absent during early development, and a pronounced increase was observed at the end of postnatal week 1. These studies suggest that there are developmental changes in the subunit composition of the GABAA/BZ receptor in inferior olivary neurons. These changes in subunit expression, which occur during a period of major alterations in afferent and efferent synaptic connections, may subserve a change in the role of GABA from its function as a neurotrophic factor to that of an inhibitory neurotransmitter. PMID- 7560271 TI - Expression of polysialylated N-CAM in the central nervous system of adult canaries and its possible relation to function. AB - Polysialylated neuronal cell adhesion molecule (PSA-N-CAM) is a cell surface molecule associated with neurons that undergo changes in configuration or spatial translocation. In both cases, this molecule is thought to reduce the adhesivity of these cells or of their processes, which can thereby insinuate themselves into the existing parenchyma. We used a monoclonal antibody specific to PSA to offer what we believe is the first account of the distribution of PSA-N-CAM in the adult songbird brain. This antibody stained a diversity of cell classes and processes, as follows: 1) a subset of ventricular zone cells; 2) migrating cells thought to be neuroblasts; 3) a subset of differentiated neurons; 4) some brain surface astrocytes; 5) some tanycytes; 6) the neuropil of some regions; 7) some axonal fibers; and 8) possibly some synapses. Our results demonstrate also, for the first time, the wide distribution of a very numerous population of migrating cells in the telencephalon and the seasonal regulation of PSA-N-CAM expression in a part of the adult brain known to undergo seasonal changes in cell recruitment and function. However, we did not find PSA-N-CAM associated with young migrating cells in the high vocal center (HVC), nor was there PSA-N-CAM in the robust nucleus of the archistriatum (RA), which is known to receive new axonal endings from HVC. In these instances spatial translocation may occur with the assistance of other surface molecules. PMID- 7560269 TI - Fine structure of the dorsal lateral geniculate nucleus of the turtle, Emys orbicularis: a Golgi, combined HRP tracing and GABA immunocytochemical study. AB - The afferent and efferent cortical projections of the dorsal lateral geniculate nucleus (GLD) of adult specimens of the turtle Emys orbicularis were investigated after intraocular or intracortical injections of horseradish peroxidase (HRP), and the distribution of gamma aminobutyric acid (GABA) immunoreactivity in the nucleus was carried out by immunocytochemical techniques, both techniques being combined with light and electron microscopy. In addition, some specimens were prepared for double-labeling of HRP and GABA immunoreactivity, and additional samples impregnated by a rapid Golgi technique. On purely morphological grounds, four types of neurons can be distinguished by light microscopy: two types of large cells in the cell plate which project to the cortex, and two types of smaller cells in the neuropil and optic tract which do not. The small cells are consistently GABA-immunoreactive, while the former are, with extremely rare exceptions, immunonegative for GABA. The supposition that the small neurons of the neuropil are interneurons is supported by electron microscopic observations; these strongly GABA-immunoreactive cells have large plicated nuclei surrounded by a thin layer of cytoplasm poorly endowed with organelles. The dendrites of these cells may contain pleomorphic synaptic vesicles (DCSVs) and appear to be presynaptic to other dendritic profiles. These DCSVs are occasionally contacted by GABA-immunoreactive axon terminals, and more frequently by retinal terminals consistently immunonegative for GABA. The latter, frequently organized in glomeruli, also make synaptic contacts with immunonegative dendrites arising from corticopetal neurons of the cell plate. Two major categories of GABA immunoreactive axon terminals can be distinguished, and we are led to the conclusion that one of these represents an intrinsic GABAergic innervation of the GLD, while the second is tentatively interpreted as an extrinsic source of GABA to the nucleus, possibly from ventral thalamic structures. The fine structure of the dorsal lateral geniculate nucleus of Emys orbicularis thus shows many similarities with that of mammals. PMID- 7560272 TI - The organization of the turtle inner retina. I. ON- and OFF-center pathways. AB - Intracellular recordings and dye injections of Lucifer yellow, horseradish peroxidase, or Neurobiotin were made in bipolar, amacrine, and ganglion cells of the Pseudemys turtle retina. By using a standard light-stimulation protocol in a sample of 375 labeled neurons, we were able to identify morphological and physiological characteristics of 11 types of bipolar cell, 37 types of amacrine cell, and 24 types of ganglion cell. To make sense of these data, we have chosen to group the 72 essentially different neuron types into traditional, functionally significant pathways. In this paper we look at the neuronal types in the inner plexiform layer (IPL) in terms of their contribution to generalized luminosity responses such as sustained ON- or OFF-center and transient ON-OFF ganglion cells; in the companion paper (J. Ammermuller, J.F. Muller, and H. Kolb, 1995, J. Comp. Neurol. 358:35-62) we look at them in terms of their involvement in color opponency and directional selectivity. A functional organization of the turtle IPL into OFF sublaminae (strata 1 and 2) and ON sublaminae (strata 3, 4, and 5), as has been described for other vertebrate retinas, was quite clear for two varieties of OFF-center bipolar cells (B4 and B5) and for all four types of sustained ON-center bipolar cell (B1, B2, B6, and B7). Thus, we found no sustained ON-center bipolar cell terminating in strata 1 and 2. We did, however, see three varieties of sustained OFF-center bipolar cells (B3, B9, and B10) having axon terminals in strata 3-5 (the ON sublamina) in addition to their terminations in stratum 1 or 2 (the OFF sublamina). Monostratified sustained ON- and OFF-center amacrine and ganglion cells rigidly obeyed the border of ON and OFF sublaminae. However, multistratified and diffuse sustained amacrine and ganglion cells could be either ON-center or OFF-center, and they did not strictly obey the border: such ON-center cells always had processes in one of the ON sublaminae (strata 3-5), and the equivalent OFF-center cells always had processes in one of the OFF sublaminae (strata 1 and 2). Monostratified transient amacrine and ganglion cells were concentrated in the middle of the IPL (around stratum 3), whereas bi-, tri-, or multistratified transient amacrine or ganglion cells always had processes in both the ON and the OFF sublaminae.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7560273 TI - Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus. AB - In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing. PMID- 7560274 TI - Differential synaptic innervation of neurons in the internal and external segments of the globus pallidus by the GABA- and glutamate-containing terminals in the squirrel monkey. AB - The present study aimed at comparing the pattern of synaptic innervation of neurons in the external (GPe) and internal (GPi) pallidum by gamma-aminobutyric acid (GABA)- and glutamate-immunoreactive terminals in the squirrel monkey. Four major populations of terminals were encountered in GPe and GPi. Our findings combined with those obtained in previous tract-tracing studies reveal that the synaptic innervation of perikarya in GPe is strikingly different from that in GPi. Although the GABA-positive type I boutons (from the striatum) represent 85% of the terminals in contact with somata in GPe, only 32% of the axosomatic synapses involve this type of terminal in GPi. However, the type II terminals (from GPe), which display a moderate level of GABA and glutamate immunoreactivities, account for 48% of the boutons in contact with perikarya in GPi but only 10% in GPe. In both pallidal segments, less than 10% of the axosomatic synapses involve the glutamate-immunoreactive type III terminals (from the subthalamic nucleus). Finally, the type IIa boutons (unknown source), which show levels of immunoreactivities similar to the type II terminals, account for 12% of the boutons in contact with perikarya in GPi but only 4% in GPe. In contrast to perikarya, the innervation of dendritic shafts is similar in both GPe and GPi; more than 80% of the axodendritic synapses involve the type I terminals, 10-15% involve the type III terminals, less than 5% are formed by the type II boutons, and less than 1% involve the type IIa terminals. Three other categories of boutons (types IV, V, VI) account for less than 1% of the total population of terminals in GPe and GPi. In conclusion, our findings demonstrate a differential synaptic innervation of neuronal perikarya in GPe and GPi in primates. These data suggest that the two pallidal segments are separate functional entities of which the neuronal activity is largely controlled by extrinsic inputs that are differentially distributed at the level of single cells. PMID- 7560275 TI - Developmental time course of acidic and basic fibroblast growth factors' expression in distinct cellular populations of the rat central nervous system. AB - Acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) are expressed in high levels in adult central nervous system (CNS). We report the time course of developmental appearance and distribution of these factors and of two FGF receptors, FGFR-1 and FGFR-2, in the CNS of rats ranging in age from embryonic day 16 to adult. Immunohistochemical analysis showed that sensory neurons in the midbrain were the first cells to contain detectable aFGF immunoreactivity at embryonic day 18. The next cell group to contain aFGF were motor neurons, which were found to be aFGF-positive at the day of birth. A number of other subcortical neuronal populations were observed to contain aFGF immunoreactivity after postnatal day 7. Adult levels and distribution patterns of aFGF were reached in all CNS areas by postnatal day 28. Basic FGF immunoreactivity was observed at postnatal day 0 in neurons in the CA2 subfield of hippocampus. Astrocytes contained detectable bFGF immunoreactivity, starting at postnatal day 7. Adult levels and patterns of distribution of bFGF were reached in all CNS areas by postnatal day 28. These immunohistochemical observations were confirmed by using bioassay and Western blot techniques. FGFR-1 and FGFR-2 mRNA were expressed in significant levels in all CNS areas at all time points analyzed. The observation that aFGF and bFGF appear in specific and distinct cellular populations at relatively late developmental times suggests that these FGFs may be involved in specific mechanisms of CNS maturation, maintenance, and repair. PMID- 7560276 TI - The organization of the turtle inner retina. II. Analysis of color-coded and directionally selective cells. AB - Color coding and directional selectivity (DS) of retinal neurons were studied in the Pseudemys turtle by using similar intracellular recording and staining techniques as in the preceding paper (J. Ammermuller and H. Kolb, 1995, J. Comp. Neuronal. 358:1-34). Color-coded responses were elicited by red (621 or 694 nm), green (525 or 514 nm), and blue (455 nm) light flashes. In addition to red/green and yellow/blue types of chromaticity horizontal cells, in our sample of 305 identified cells we found that 17% of bipolar cells, 6.5% of amacrine cells, and 18% of ganglion cells exhibit color-coded responses. DS responses were found in 37% of the tested ganglion cells and 41% of the tested amacrine cells. Two morphologically identified bipolar cell types, B10 and B11, were red-ON/blue-OFF and red-OFF/green, blue-ON, respectively. Of five identified amacrine cell types, three were red-OFF/blue-ON center (A1, A3, A23b), one was red-OFF/green-ON center (A32), and one (A33) was double color-opponent of red-ON/blue-OFF center:red OFF/blue-ON surround. Five ganglion cell types had variously color-coded centers (G14 and G24) or surrounds (G3 and G18), including one type, G6, that was double color-opponent (red-OFF/green-ON center:red-ON/green-OFF surround). Responses to colors were found primarily in sustained responses of bipolar and ganglion cells. However, in amacrine cells, transient components of the response also showed color dependence. Red-OFF-center responses were found in ganglion cells that were in a position to make connections at the strata 2/3 border with the red-OFF bipolar cell (B11); red-ON-center responses occurred in ganglion cells with branches in stratum 4 of the IPL where the red-ON-center bipolar (B10) ended. Blue-ON-center signals appeared to be processed mainly in strata 1-2/3, and blue OFF-center signals in strata 3-5 of the IPL, with contributions of amacrine cells and bipolar cells. Labeled DS amacrine cells could be identified as A9, A20, and A22, and ganglion cells as G19, G20, and G24. The latter type (G24) showed DS and color coding. All response types (ON-center, OFF-center, ON-OFF) were encountered. DS amacrine cells were monostratified near the middle of the IPL, whereas DS ganglion cells were mono-, bi-, and multistratified, although all DS ganglion cells had one feature in common: they had dendrites in stratum 1 of the IPL.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 7560277 TI - Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide. AB - Intracellular staining with biotinamide was used to study the axonal projection and synaptic morphology of rat jaw-muscle spindle afferents. Intracellular recordings in the mesencephalic trigeminal nucleus (Vme) were identified as spindle afferent responses by their increased firing during stretching of the jaw elevator muscles. Biotinamide-stained axon collaterals with boutons were found in the trigeminal motor nucleus (Vmo), Vme, the region dorsal to Vmo including the supratrigeminal region, the dorsomedial portion of the trigeminal principal sensory nucleus, and the dorsomedial part of the rostral spinal trigeminal subnucleus oralis. Additional, previously undescribed projections of jaw-muscle spindle afferents were found to the dorsomedial portion of the caudal spinal trigeminal subnucleus oralis (Vodm), the dorsomedial part of the spinal trigeminal subnucleus interpolaris (Vidm), the caudal parvicellular reticular formation, laminae IV and V of the spinal trigeminal subnucleus caudalis (Vc), and the dorsal division of the medullary reticular field. Labeled spindle boutons in Vodm formed predominately axodendritic synapses. Some of these boutons received presynaptic inputs from unlabeled P-type boutons containing clear, spherical, or flattened vesicles. In Vidm, labeled collaterals and boutons were densely clustered into glomerular-like structures. Labeled boutons in Vidm made axodendritic, axosomatic, and axoaxonic synapses and received synaptic contacts from unlabeled boutons containing clear, spherical, or flat and pleomorphic vesicles. Unlabeled presynaptic boutons in Vidm occasionally contained dense core vesicles. Labeled boutons in Vc mainly formed synaptic contacts with large diameter dendrites. This projection of jaw-muscle spindle afferents to caudal brainstem regions may play a significant role in masticatory-muscle stretch reflexes and in the integration of trigeminal proprioceptive information and its transmission to higher centers. PMID- 7560279 TI - Development of muscarinic receptor subtypes in the forebrain of the mouse. AB - Cholinergic mechanisms are involved in the regulation of developmental events in the nervous system. Muscarinic cholinergic receptors are thought to be the predominant mediator of cholinergic neurotransmission in the forebrain; however, their developmental role is less well understood. The present study takes advantage of subtype-specific antibodies to muscarinic receptor proteins to investigate the cellular localization of the subtypes in developing mouse forebrain. Receptor protein expression was assessed between postnatal day (PND) 5 and adulthood by immunocytochemical methods with antibodies to m1, m2, and m4 receptors, the most abundant subtypes in rodent brain. We have found dramatic developmental changes in the distribution of all three receptors. In the adult mouse, m1 and m2 receptor immunoreactivity displayed complementary staining patterns in most forebrain areas with m4 sharing similarities in pattern with both m1 and m2. Furthermore, each receptor was expressed transiently in gray matter areas or fiber bundles at various developmental stages. The m4 receptor was also expressed in developing blood vessels. Such transient immunoreactivity was usually associated with times and areas of dynamic morphogenesis, thus suggesting distinct roles for the receptor subtypes in ontogenetic events. PMID- 7560278 TI - Locus coeruleus cell loss in the aging human brain: a non-random process. AB - Quantitative neuroanatomical techniques were used to determine whether with aging there is random or systematic loss of locus coeruleus (LC) neurons in the human brain. The cells were identified by immunohistochemical staining for the catecholaminergic enzyme tyrosine hydroxylase and/or by neuromelanin pigment content. Cell locations were mapped, using computer imaging procedures, in horizontal sections spaced 0.5 to 0.8 mm throughout the rostrocaudal extent of the nucleus in 17 cases, from 1 to 104 years of age. Neuromelanin pigment accumulated within the neurons with aging. In brains less than 25 years of age there were many fewer pigment-containing neurons than tyrosine hydroxylase containing neurons; however, by the fifth decade the number of cells identified by the two markers was comparable. From the first to the tenth decade of life there is over a 50% loss of LC neurons: in four cases from "young" individuals (1 28 years of age) there were 21,084 +/- 653 tyrosine hydroxylase immunostained cells (mean +/- standard error of the mean) on one side of the brain; in seven cases from "old" individuals (60-82 years of age) there were 16,502 +/- 921 pigment-containing cells; and in the three cases from the "oldest" individuals (103-104 years of age) there were 9,493 +/- 1,236 pigment-containing neurons. In both the "old" and "oldest" groups, compared to the "young," there was significantly greater loss of rostral cells than caudal cells. These data indicate a systematic loss of cells such that the rostral, forebrain-projecting neurons decrease in number with aging to a greater extent than do the caudal, spinal cord-projecting neurons.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560280 TI - Ultrastructural and immunocytochemical observations on the superior olivary complex of the mustached bat. AB - This study investigates the functional organization of the superior olivary complex of the mustached bat with classical transmission electron microscopy and postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) and glycine antisera in semithin serial sections. The ultrastructure and distribution of terminal types in the lateral superior olive (LSO) and the medial nucleus of the trapezoid body (MNTB) closely resemble that of other mammals; the organization within the medial superior olive (MSO) differs significantly. The differences concern the relative proportion of putatively inhibitory boutons, which appear as symmetrical synapses with flattened vesicles on MSO somata. In the bat, inhibitory boutons comprised 75-100% of perisomatic boutons, a value identical to that observed in the LSO. These terminals most likely arise from the MNTB. In other species, putatively inhibitory terminals form a much smaller proportion of perisomatic boutons in MSO. This difference suggests that in the bat MSO excitatory input to cell somata is considerably reduced and outweighed by inhibitory input. This suggestion is corroborated by immunocytochemical data. Glycine-immunoreactive puncta encrust somata of LSO and MSO cells to a similar degree and in rather homogeneous patterns throughout these nuclei. Putatively GABAergic terminals are located mainly on distal dendrites of MSO and LSO cells. Regional variations in the density of GABA-immunoreactive puncta in LSO suggest that different tonotopic zones are under differential modulatory influence. Both the LSO and MSO of the mustached bat contain significant amounts of putatively inhibitory projection cells. Coexistence of both antigens was commonly observed in subsets of cells. Quantitative analyses of labeling patterns and comparisons with other mammals suggest that the mix of neurotransmitters in projection cells of LSO and MSO is phylogenetically flexible, and thus the details of the functions of ascending pathways are species specific. In contrast to other mammals, the bat MSO forms parallel output pathways with excitatory and inhibitory components. Data are discussed in relation to specialized physiological response features. PMID- 7560281 TI - Variation and evolution of mammalian corticospinal somata with special reference to primates. AB - The morphology of the somata originating the corticospinal tract was examined in 24 species of mammals to identify commonalities and major sources of variation among the different species. Horseradish peroxidase was applied to a hemisection of the spinal cord at the C1-C2 junction. After tetramethylbenzidine processing, the labeled somata throughout the cerebral cortex were plotted and counted. Then, 23 morphological characteristics of the corticospinal somata were examined, including their number, size, and density across the cortical surface. The results show that morphological characteristics of corticospinal somata are closely related to an animal's body, brain, and cerebral cortex size. That is, mammals with large neocortical surfaces tend to have larger as well as more corticospinal somata; mammals with large bodies tend to have corticospinal somata that are less densely distributed. Moreover, the probable increase in the ratio of local noncorticospinal somata to corticospinal somata implies that the evolution of the corticospinal tract was accomplished by an increase in "support" or "server" cells as well as an increase in the size of the tract itself. The results also show that several characteristics are reliably related to an animal's taxonomic classification and hence its ancestry. Comparisons among three mammalian lineages indicate that some characteristics may have changed uniquely in the anthropoid primate lineage, and thus, presumably, in the human lineage. The results suggest that if morphological characteristics of the corticospinal tract important in the evolution of the specialized motor abilities in anthropoid primates are sought, then examination of the role of changes in soma diameter, rostral (motor)/caudal (sensory) ratios of density, concentration, surface density, and volume density may be more instructive than examination of the total number of corticospinal neurons alone. PMID- 7560282 TI - Glial environment in the developing superior colliculus of hamsters in relation to the timing of retinal axon ingrowth. AB - We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In contrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time retinotectal projections are being fully established. These raphe glia may be involved in maintaining the laterality of the retinotectal projection. PMID- 7560284 TI - Organization of connections between the thalamic reticular and the anterior thalamic nuclei in the rat. AB - The thalamic reticular nucleus (TRN) receives topographically organized input from specific sensory nuclei such as the lateral geniculate nucleus. The present study shows this in the rat. However, the pattern of thalamic connections to the limbic reticular sector is unknown. Injecting biocytin into the ventral parts of anteroventral and anteromedial nuclei labeled neurons and axons in the rostral TRN. Filled axon collaterals and their terminals occupied a rectangular sheet in a plane close to the horizontal, and were confined to the inner zone (the medial portion) of the limbic TRN. Retrogradely filled cells were in the middle of the rostral pole in the same horizontal plane, receiving synapses from surrounding labeled boutons. In electron micrographs, thalamic terminals were found to contain round, densely packed synaptic vesicles and formed asymmetrical synapses onto reticular somata and dendritic profiles. Displacing the injection site along the dorso-ventral and rostro-caudal axis in the anterior nuclei produced corresponding shifts of antero- and retrograde labeling within the inner reticular zone. Projections from the dorsal portions of the anterior nuclei did not follow this pattern. Axons from the anterodorsal nucleus occupied the rostralmost tip of both inner and outer zones of the dorsal limbic sector. In accordance with earlier reports, the limbic sector was found to represent several dorsal thalamic nuclei parallel to each other medio-laterally. A topography is described for the limbic reticulo-thalamic connections, suggesting that the rostral TRN is able to influence circumscribed areas of the limbic thalamus. PMID- 7560283 TI - Brainstem afferents to the lateral mesencephalic tegmental region of the cat. AB - The lateral mesencephalic tegmental region (LTR) is a part of the midbrain reticular formation characterized by the presence of neurons exhibiting head movement-related discharge modulation. In addition, the LTR contains directionally selective visual units. Possible sources for these vestibular and visual signals were studied by retrograde axonal transport of horseradish peroxidase and three different fluorescent tracers (rhodamine, fast blue, and fluorogold) injected into various parts of the LTR. All injections into the LTR traced afferents from the vestibular nuclei and from the nucleus prepositus hypoglossi. Predominant projections were derived from the ipsilateral nucleus prepositus hypoglossi and the ipsilateral medial vestibular nucleus, whereas the observed inputs from the inferior, lateral, and superior vestibular nuclei were much weaker. Further inputs to the LTR originated in the deep and intermediate layers of the ipsilateral superior colliculus and the ipsilateral periaqueductal gray, the contralateral LTR, and the contralateral marginal nucleus of the brachium conjunctivum. Tracer deposits in medial parts of the tegmentum neighboring the LTR never produced the pattern of afferents observed after injections into the LTR. Our results suggest that afferents from the deeper layers of the superior colliculus are probably the source of visual signals in the LTR and that head movement-related responses are likely to be derived from the nucleus prepositus hypoglossi and the medial vestibular nucleus. PMID- 7560285 TI - Organization of the olfactory system in the adult zebrafish: histological, immunohistochemical, and quantitative analysis. AB - The zebrafish, Danio rerio, is becoming an important model system for developmental studies. We have used a variety of histological techniques to characterize the adult structure of the olfactory system in this teleost to form a base for future developmental work. The olfactory epithelium in this fish contains ciliated and microvillar sensory neurons, microvillar supporting cells, secretory goblet cells, and basal cells, and the adjacent nonsensory epithelium contains ciliated supporting cells. The olfactory bulb is a diffusely organized structure with four laminae: olfactory nerve, glomerular, mixed mitral cell/plexiform, and granule cell layers. These structures and the synapses observed in the olfactory bulb are typical of what is found in other vertebrates. We also examined the distribution of several neurotransmitter markers (tyrosine hydroxylase, neuropeptide Y, dopamine-beta-hydroxylase, and serotonin) in the olfactory bulb. Antibodies to neuropeptide Y, dopamine-beta-hydroxylase, and serotonin labeled fibers in the olfactory bulb and cell bodies in caudal regions of the brain in distributions comparable to other species. Tyrosine hydroxylase immunoreactivity was observed in a set of intrinsic bulb neurons with extensive processes in the glomerular layer. In addition, the structural proteins glial fibrillary acidic protein and vimentin have distributions similar to those in the olfactory bulbs of other animals. Thus, the adult olfactory structures are analogous to the structures in other vertebrate animals in morphology and chemical neuroanatomy. This similarity, along with its numerous advantages for developmental studies, makes the zebrafish a good model for studies of olfaction and forebrain maturation. PMID- 7560286 TI - Topographic organization of a forebrain pathway involved with vocal learning in zebra finches. AB - A serial pathway from a thalamic nucleus (DLM; the medial portion of the dorsolateral nucleus of the anterior thalamus) to a cortical region (lMAN; the lateral magnocellular nucleus of the anterior neostriatum) to a motor-cortical region (RA; the robust nucleus of the archistriatum) is necessary for vocal production during song learning in juvenile zebra finches but not for the recitation of a song already learned by adults. To obtain new information about the possible function of the DLM-->lMAN-->RA pathway in vocal learning, we used anterograde and retrograde tract-tracing techniques (pressure injections of DiI and DiA) to map the pattern of axonal connections between these brain regions in adult male zebra finches. Results revealed two topographically organized pathways that traverse the songbird forebrain in parallel. An oval-shaped dorsal/lateral portion of DLM projects solely to the central core of lMAN (lMANcore), whereas a crescent-shaped region, including ventral and medial DLM, projects exclusively to a parvicellular shell that encircles lMANcore (lMANshell). In turn, lMANshell neurons project solely to an arc-shaped region of dorsal archistriatum just lateral to RA (Ad; archistriatum, pars dorsalis), whereas lMANcore neurons project exclusively to RA. We also identified crossed and reciprocal pathways between lMANcore/shell and the lateral portion of the ventral archistriatum, which may contribute to interhemispheric coordination of vocal behavior. A robust topographic organization was observed in the axonal projections from dorsal/lateral-DLM-->lMANcore-->RA and from ventral/medial-DLM-->lMANshell-->Ad, raising the question of what is being mapped within these two forebrain pathways. Because RA projection neurons are organized myotopically with respect to the major vocal (syringeal) muscles (D.S. Vicario, 1991, J. Comp. Neurol. 309:486 494), one possibility is that a mapping of vocal/expiratory musculature is preserved "upstream" within these pathways. Similarly, the presence of song selective auditory neurons in DLM, lMAN, and RA (A.J. Doupe and M. Konishi, 1991, Soc. Neurosci. Abstr. 18:527) suggests that these pathways might subserve some form of auditory or auditory-motor mapping. PMID- 7560287 TI - Distribution of Big tau in the central nervous system of the adult and developing rat. AB - The diversity of neuronal morphology and function is correlated with specific expression of various microtubule associated proteins (MAPs). One of the major neuronal MAPs, tau, has multiple isoforms formed as a result of alternative splicing and phosphorylation that are differentially expressed during development. Big tau is a high molecular weight isoform that contains an additional large exon (4a) and is expressed primarily by neurons in the peripheral nervous system (PNS). We cloned the complete 4a exon in an expression vector, isolated the recombinant protein and produced antibodies specific to Big tau that were used to localize Big tau in the developing spinal cord and in the adult central nervous system (CNS). In developing spinal cord, Big tau is first expressed in the central projections of the dorsal root ganglia neurons and in motor neurons at embryonic day 18 and postnatal day 2, respectively. In the adult rat CNS, almost all neurons that extend processes into the PNS express Big tau, including all cranial nerve motor nuclei and central processes of most sensory ganglia; of these ganglia, only the bipolar neurons of the olfactory, vestibular and spiral ganglia did not express Big tau. Retinal ganglion cells are the only CNS neurons, whose processes remain entirely within the CNS, that express high levels of Big tau. The limited and specific distribution of Big tau is consistent with a role in stabilizing microtubules in axons that are subjected to great shear forces. PMID- 7560289 TI - Acutely isolated and cultured cells from the electrosensory lateral line lobe of a gymnotiform teleost. AB - The present study established the morphological and immunocytochemical criteria necessary to identify neuronal and nonneuronal cells after dissociating select regions of the medullary electrosensory lateral line lobe of adult weakly electric fish (Apteronotus leptorhynchus). Cells dissociated from the pyramidal cell body layers of the centromedial and lateral segments exhibited similar characteristics in the acutely dissociated preparation and up to 14 days in culture. Basilar and nonbasilar pyramidal cells were tentatively identified according to a bipolar or monopolar process extension, and polymorphic cells by the extension of three or more processes and positive immunoreactivity for gamma aminobutyric acid. Nonneuronal cells were identified by the pattern of process arborization and positive immunolabel for gamma-aminobutyric acid or glial fibrillary acidic protein. Neuronal cells increased in total number over the first 4 days and could appear for the first time on any day in culture. Individual pyramidal cells could maintain their morphology from the time of dissociation and over several days in culture. Pyramidal cell processes were phenotypically similar to apical and basal dendrites found in situ but were reduced in size and in the degree of process branching. These results indicate that dissociated adult apteronotid neurons can maintain a morphology sufficiently similar to that found in situ as to allow tentative identification, opening up a wide range of possibilities for studying the electrophysiological and regenerative properties of electrosensory neurons. PMID- 7560288 TI - An anatomical substrate for the inhibitory gradient in the VLVp of the owl. AB - The interaural difference in the level of sounds is an important cue for the localization of the sound's source. In the barn owl, a keen auditory predator, this binaural cue is first computed in the nucleus ventralis lemnisci laterale, pars posterior (VLVp), a cell group found within the fibers of the lateral lemniscus. Its neurons are excited by inputs from the contralateral ear and inhibited by inputs to the ipsilateral ear and are therefore sensitive indicators of interaural level difference. The excitation arrives by a direct input from the contralateral nucleus angularis, a cochlear nucleus, and the inhibition is mediated by a commissural projection that interconnects the VLVps of the two sides. The dorsally located neurons in the VLVp are more heavily inhibited than those found more ventrally, thus giving rise to a gradient of inhibition. This inhibitory gradient plays a central role in recent models of VLVp function. We present evidence based on standard anterograde tracing methods that this gradient of inhibition is mediated by a dorsoventral gradient in the density of synaptic inputs from the contralateral VLVp, the source of inhibition. Specifically, injection of tracers into one VLVp, regardless of the position of the injection within the nucleus, produced a vertically oriented field of label that was densest along the dorsal margin of the contralateral VLVp and became sparser a more ventral levels. Furthermore, we found that injections into the medial and lateral aspects of the nucleus produced this dorsoventrally graded field of label along the medial and lateral aspects of the contralateral VLVp, respectively. Finally, we confirmed an earlier observation suggesting that the anterior and posterior aspects of one VLVp project to the anterior and posterior aspects of the contralateral nucleus, respectively. PMID- 7560290 TI - Ultrastructural heterogeneity of enkephalin-containing terminals in the rat hippocampal formation. AB - Opioid peptides, including leu-enkephalin (LE), are important neuromodulators in the hippocampal formation where they may play a role in learning and memory as well as epileptogenesis. We examined the cellular substrates that underlie the function of LE in each lamina of the rat hippocampal formation by immunocytochemistry at the electron microscopic level in single section analysis. LE-like immunoreactivity (LE-LI) was primarily associated with large dense-core vesicles (80-100 nm), usually found in axons and axon terminals, but was also observed in perikarya and occasionally in dendrites. The morphology and synaptic associations of LE-LI-containing terminals were strikingly distinct in each region of the hippocampal formation. In the molecular layer of the dentate gyrus, terminals with LE-LI were typically small (0.6 microns) and formed primarily asymmetric (excitatory type) synapses on single dendritic spines, which is consistent with the presence of LE in the lateral perforant path. In the hilus of the dentate gyrus, two types of LE-containing terminals were present: (1) small round terminals that were heterogeneous in size (0.4-1 microns) and in type of contact formed and (2) larger (3-5 microns) terminals exhibiting the characteristic morphology of mossy fiber boutons that formed asymmetric synapses on spines. This variation in morphology and the type of contact suggests LE may have a heterogeneous influence on diverse hilar interneurons. In the CA3 region of the hippocampus, LE-LI was localized to large mossy fiber boutons (3-7 microns) that formed multiple asymmetric synapses on complex spiny dendritic processes and often formed puncta adherentia with the shafts of large CA3 pyramidal cell dendrites, indicating that this peptide may be directly released onto pyramidal cells. At the border of stratum radiatum and lacunosum moleculare in the CA1 region of the hippocampus, LE-labeled terminals averaged 0.8 microns in diameter and often formed symmetric (inhibitory type) synapses on dendritic shafts, which is consistent with a role in disinhibition. In conclusion, these heterogeneous cellular interactions indicate that LE has diverse functional roles and mechanisms of action within each lamina of the hippocampal formation and may directly and indirectly modulate hippocampal cell activity. PMID- 7560291 TI - Colocalization of gamma-aminobutyric acid with vasopressin, vasoactive intestinal peptide, and somatostatin in the rat suprachiasmatic nucleus. AB - The seemingly contradictory observations in previous publications that gamma aminobutyric acid (GABA) is detected in all cell bodies of the suprachiasmatic nucleus (SCN) and that terminals originating from the SCN are only 20-30% GABA positive prompted us to investigate whether this might be explained by a preference of colocalization in terminals of certain peptidergic neurons in the SCN or by a day/night rhythm in GABA synthesis. At three different circadian times, animals were perfusion fixed, and their SCNs were stained for vasopressin (VP), somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). Subsequently, the number of GABA peptide-positive terminals was determined using GABA postembedding staining in ultrathin sections. It appeared that the highest percentage of colocalization with GABA was detected in VIP terminals (38%) and the lowest in VP terminals (15%). No differences in colocalization percentages could be observed in any parameter at any circadian time. In the dorsomedial hypothalamus, one of the target areas of the VP and VIP fibers from the SCN, a colocalization of GABA within VP and VIP terminals was found similar to that in the SCN. In the region of the somatostatin-containing neurons in the SCN, a number of axoaxonal contacts could be observed that sometimes exhibited synaptic specializations. In nearly all cases, the axoaxonic terminals contained GABA and/or SOM. The conclusion is that the high level of intrinsic GABAergic connections in the SCN represents a putatively powerful mechanism to synchronize or shut down the activity of the SCN. We discuss the possibility that, depending on the firing frequency of the neurons, the colocalization of GABA with all peptides under investigation allows for the selection of which transmitter is released, the peptidergic one or the amino acid. PMID- 7560292 TI - Histochemical and immunocytochemical localization of nitric oxide synthase in the central nervous system of the goldfish, carassius auratus. AB - The distribution of the neuronal type of nitric oxide synthase in the goldfish brain and spinal cord was investigated via NADPH-diaphorase histochemistry and immunocytochemistry using an antiserum raised against the purified mammalian enzyme. Many structures, including magnocellular neurosecretory cells, motoneurons, mesencephalic trigeminal neurons, and radial glial fibers, were stained by the NADPH-diaphorase reaction but were not immunoreactive. This nonspecific NADPH-diaphorase activity was strongly reduced after preincubation of the sections. Therefore, when sections were first reacted for immunofluorescence and, thereafter, stained for NADPH-diaphorase, a corresponding staining pattern was obtained that allowed the reliable localization of neuronal nitric oxide synthase based on both complementary staining methods. In the telencephalon, positive neurons were concentrated in the ventral and posterior parts of the area ventralis. Many intensely stained neurons were present in various diencephalic nuclei, including the nucleus centralis posterior and the ventromedial nucleus of the thalamus, the nucleus tori lateralis, the nucleus recessus lateralis, the nucleus tuberis posterior, and the central nucleus of the inferior lobe. In the midbrain, neurons containing nitric oxide synthase were located in the periventricular zone of the optic tectum, the nucleus vermiformis, and the nucleus reticularis mesencephali. Specific staining in the cerebellum was concentrated in Golgi cells. In the hindbrain, nitroxergic neurons were numerous in all four sensory nuclei of the trigeminus, in the facial lobe, the superior olive, the inferior reticular formation, and the medial general visceral nucleus of the vagus. The dorsal horn of the spinal cord was enriched with positive neurons. A few strongly stained cells were also present in the ventral horn. In conclusion, neurons capable of synthesizing nitric oxide occur throughout the teleost central nervous system. The presence of nitric oxide synthase in projection areas of most afferent nerves suggests a widespread involvement of nitric oxide in sensory information processing. The distribution of nitric oxide synthase-containing neurons in certain areas, e.g., the tectum opticum and the spinal cord, indicates an evolutionarily conserved pattern. Similar to the case in other vertebrates, there appears to be no comprehensive overlap between the distribution of nitric oxide synthase and that of any other chemically characterized neuronal population described thus far. However, strongly positive cell groups in the mesencephalic reticular formation suggest the idea of an evolutionarily conserved mesopontine cholinergic system coexpressing nitric oxide synthase. PMID- 7560295 TI - Raphe nuclei in three cartilaginous fishes, Hydrolagus colliei, Heterodontus francisci, and Squalus acanthias. AB - The vertebrate reticular formation, containing over 30 nuclei in mammals, is a core brainstem area with a long evolutionary history. However, not all reticular nuclei are equally old. Nuclei that are widespread among the vertebrate classes are probably ones that evolved early. We describe raphe nuclei in the reticular formation of three cartilaginous fishes that diverged from a common ancestor over 350 million years ago. These fishes are Hydrolagus colliei, a holocephalan, Squalus acanthias, a small-brained shark, and Heterodontus francisci, a large brained shark. Nuclear identification was based on immunohistochemical localization of serotonin and leu-enkephalin, on brainstem location, and on cytoarchitectonics. Raphe nuclei are clustered in inferior and superior cell groups, but within these groups individual nuclei can be identified: raphe pallidus, raphe obscurus, and raphe magnus in the inferior group and raphe pontis, raphe dorsalis, raphe centralis superior, and raphe linearis in the superior group. Hydrolagus lacked a dorsal raphe nucleus, but the nucleus was present in the sharks. The majority of immunoreactive cells are found in the superior group, especially in raphe centralis superior, but immunoreactive cells are present from spinal cord to caudal mesencephalon. The distribution and cytoarchitectonics of serotoninergic and enkephalinergic cells are similar to each other, but raphe nuclei contain fewer enkephalinergic than serotoninergic cells. The cytoarchitectonics of immunoreactive raphe cells in cartilaginous fishes are remarkably similar to those described for raphe nuclei in mammals; however, the lack of a raphe dorsalis in Hydrolagus indicates that either it evolved later than the other raphe nuclei or it was lost in holocephalan fishes. PMID- 7560294 TI - Laminar organization of receptive field properties in the dorsal lateral geniculate nucleus of the tree shrew (Tupaiaglis belangeri). AB - A laminar analysis of the receptive field properties of relay cells in the binocular region of the tree shrew dorsal lateral geniculate nucleus (LGN) found three main subdivisions. Lamina 1 (receiving ipsilateral eye input) and lamina 2 (contralateral) comprise a pair of layers that contain only ON-center neurons. Laminae 4 (contralateral) and 5 (ipsilateral) comprise a pair of layers with mostly OFF-center cells (86%). Laminae 3 and 6 (both contralaterally innervated) also form a distinct pair, although lamina 3 contains a mixture of cells with ON centers (43%) or OFF-centers (57%), and lamina 6 contains mostly cells with ON OFF centers and suppressive surrounds (81%). Cells located in the interlaminar zones resembled neurons in laminae 3 and 6. In comparison with the cells in the OFF-center laminae 4 and 5, the ON-center cells in laminae 1 and 2 had smaller, more elliptical receptive field centers with stronger responses to flashed visual stimuli. In addition, cells in the ipsilateral eye laminae 1 and 5 showed a greater change in center diameter, with eccentricity from the area centralis, than cells in the contralateral eye laminae 2 and 4. Principal components analysis using six receptive field properties (latency to optic chiasm stimulation, receptive field center diameter, maintained discharge rate, response onset latency, peak spike density, and phasic-tonic index) suggested that the cells in laminae 3 and 6 and the interlaminar zones are W-like. Principal components analysis of the same receptive field properties in laminae 1, 2, 4, and 5 did not reveal differences clearly related to X-like (parvocellular) and Y like (magnocellular) categories. Ninety-seven percent of the cells tested for linearity of spatial summation in laminae 1, 2, 4, and 5 were linear. We conclude that the dominant organizational features of the tree shrew LGN are the ON center, OFF-center, and W pairs of layers that project to different regions within the striate cortex. PMID- 7560293 TI - Postnatal maturation of the dopaminergic innervation of monkey prefrontal and motor cortices: a tyrosine hydroxylase immunohistochemical analysis. AB - The mature functional architecture of the primate prefrontal cortex arises during a protracted period of postnatal development. Although catecholaminergic afferents arrive in the primate cortex quite early during fetal development, several lines of evidence suggest that substantial changes in the dopaminergic innervation of prefrontal cortex may occur during postnatal development. In this study, we used immunocytochemical techniques and antibodies against tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, to examine the precise time course from birth to adulthood of the maturational changes of tyrosine hydroxylase-labeled axons in prefrontal cortical areas 9 and 46 and primary motor cortex (area 4) of rhesus monkeys. In area 9, the densities of tyrosine hydroxylase-labeled axons and varicosities in the superficial and deep cortical layers remained relatively constant during postnatal development. In contrast, marked developmental changes in innervation density occurred in the middle cortical layers. For example, in deep layer III, the density of tyrosine hydroxylase-positive varicosities was relatively low and uniform in animals under 1 month of age but then increased by a factor of three in animals 2-3 months of age. The density of labeled varicosities continued to increase, reaching a peak (sixfold greater than in the youngest animals) in animals 2-3 years of age before declining to stable adult levels. Similar laminar-specific patterns of change also occurred in areas 46 and 4, although regional differences were present in the magnitude and precise time course of these developmental changes. These findings demonstrate that the innervation of monkey frontal cortex by tyrosine hydroxylase-immunoreactive axons undergoes a protracted, laminar-specific pattern of change during postnatal development that continues through adolescence and into early adulthood. These developmental refinements may interact with other modifications of cortical circuitry that underlie the functional maturation of these regions. PMID- 7560296 TI - Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach. AB - Recent pharmacological evidence suggests that the nonadrenergic, noncholinergic (NANC) vagal inhibitory input responsible for receptive relaxation of the fundic stomach is mediated by nitric oxide-synthesizing enteric neurons. To demonstrate anatomically such direct vagal inputs to neurochemically identified enteric neurons, we utilized the nicotinamide acetamide dinucleotide phosphate (NADPH) diaphorase histochemical reaction in conjunction with selective anterograde labeling of vagal efferents or afferents. Approximately 30% of all myenteric neurons of the fundic myenteric plexus stained positive for NADPH diaphorase, and the principal recipient of axonal projections from NADPH diaphorase-positive neurons was the circular muscle layer. In a group of animals showing the most complete labeling of vagal efferent preganglionics with the carbocyanine dye DiA, quantitative analysis of the half of the ventral fundic wall closer to the greater curvature revealed that 46.8% +/- 4.4% of all myenteric neurons received some degree of vagal contacts and that 30.5% +/- 6.6% of such vagally contacted neurons were also NADPH diaphorase positive. In another group of rats with the most successful selective labeling of vagal afferents through DiI injections into the left nodose ganglion, analysis of select ganglia throughout the ventral fundic wall revealed that, of a total of 454 neurons with vagal afferent contacts, 34.8% +/- 2.8% were NADPH diaphorase positive. These findings support the view that, in the fundic stomach, some vagal preganglionic efferents terminate on nitric oxide-synthesizing neurons that, in turn, project to and relax the external smooth muscle layers. Furthermore, vagal afferent endings also contact NADPH diaphorase-positive neurons, suggesting the possibility of local axon reflexes originating from smooth muscular in-series tension receptors and terminating on nitrergic neurons of the myenteric plexus. PMID- 7560298 TI - Pestivirus diversity. PMID- 7560299 TI - Leiomyosarcoma of soft tissue in a cow. AB - A 4-year-old Holstein cow developed a large neoplastic mass, which was formed in the deep soft tissue of the neck and removed surgically. The neoplasm was shown to be a smooth muscle tumour by the presence of alpha smooth muscle actin and fine intracytoplasmic filaments with focal densities. This malignant neoplasm showed extensive areas of necrosis and haemorrhage, high cellularity, marked pleomorphism, atypical giant cells and numerous mitoses with occasional abnormal figures. PMID- 7560300 TI - Neuroaxonal dystrophy in experimental Creutzfeldt-Jakob disease: electron microscopical and immunohistochemical demonstration of neurofilament accumulations within affected neurites. AB - Neuroaxonal dystrophy is a feature of neuronal degeneration encountered in all subacute spongiform "virus" encephalopathies, including scrapie and Creutzfeldt Jakob disease (CJD). By immunohistochemical techniques, the accumulation of 200 kDa neurofilament protein was demonstrated in affected neurites in murine CJD. These neurites exhibited the ultrastructural features of dystrophic neurites encountered in other neurodegenerative disorders, particularly Alzheimer's disease. These findings support the hypothesis that impairment of slow axoplasmic transport is a common pathogenetic mechanism for CJD and many other neurodegenerative conditions. PMID- 7560297 TI - Reduced nicotinamide adenine dinucleotide phosphate-diaphorase/nitric oxide synthase profiles in the human hippocampal formation and perirhinal cortex. AB - Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-stained profiles were evaluated throughout the human hippocampal formation (i.e., dentate gyrus, Ammon's horn, subicular complex, entorhinal cortex) and perirhinal cortex. NADPH d staining revealed pleomorphic cells, fibers, and blood vessels. Within the entorhinal and the perirhinal cortices, darkly stained (type 1) NADPH-d pyramidal, fusiform, bipolar, and multipolar neurons with extensive dendrites were scattered mainly within deep layers and subjacent white matter. Moderately stained (type 2) NADPH-d round or oval neurons were seen mainly in layers II and III of the entorhinal and perirhinal cortices, in the dentate gyrus polymorphic layer, in the CA fields stratum pyramidal and radiatum, and in the subicular complex. The distribution of type 2 cells was more abundant in the perirhinal cortex compared to the hippocampal formation. Lightly stained (type 3) NADPH-d pyramidal and oval neurons were distributed in CA4, the entorhinal cortex medial subfields, and the amygdalohippocampal transition area. Sections concurrently stained for NADPH-d and nitric oxide synthase (NOS) revealed that all type 1 neurons coexpressed NOS, whereas types 2 and 3 were NOS immunonegative. NADPH-d fibers were heterogeneously distributed within the different regions examined and were frequently in close apposition to reactive blood vessels. The greatest concentration of fibers was in layers III and V-VI of the entorhinal and perirhinal cortices, dentate gyrus polymorphic and molecular layers, and CA1 and CA4. A band of fibers coursing within CA1 divided into dorsal and ventral bundles to reach the presubiculum and entorhinal cortex, respectively. Although the distribution of NADPH-d fibers was conserved across all ages examined (28-98 years), we observed an increase in the density of fiber staining in the aged cases. These results may be relevant to our understanding of selective vulnerability of neuronal systems within the human hippocampal formation in aging and in neurodegenerative diseases. PMID- 7560301 TI - Non-neoplastic lesions in beluga whales (Delphinapterus leucas) and other marine mammals from the St Lawrence Estuary. AB - In a 3-year (1988-1990) pathological study, 24 carcasses of beluga whales from the St Lawrence Estuary, Quebec, Canada, showed numerous severe lesions, many of which had never been reported in cetaceans. The most common lesions were found in the digestive tract (21 animals) and consisted mainly of periodontitis and of erosions and ulcers in the oesophagus and the first two gastric compartments. Pneumonia, usually of parasitic origin, was also a common finding (12 animals). The adrenal glands often contained nodules (five animals) or cysts (seven animals), and mastitis was observed in five females. Overall, the incidence of degenerative, infectious, hyperplastic or necrotic lesions, in addition to numerous neoplasms described in another paper, was considerably higher than that found in marine mammals elsewhere or in other species of marine mammal from the same waters. PMID- 7560303 TI - Pathological changes in the renal interstitial capillaries of pigs inoculated with two different strains of African swine fever virus. AB - African swine fever is a viral disease of pigs characterized predominantly by haemorrhagic lesions. This paper reports the lesions observed in the renal interstitial capillaries of pigs inoculated with African swine fever virus strains of differing virulence: the Malawi'83 strain (haemadsorbent and highly virulent) and the Dominican Republic'78 strain (haemadsorbent and moderately virulent). In pigs infected with the Malawi'83 strain, petechial haemorrhages and microhaemorrhages were observed 5 days after inoculation and lesions were evident in the renal capillaries. Signs of phagocyte activation were noticeable in endothelial cells, with enlarged fenestrations and even loss of endothelium, leaving the basement membrane of the vessels exposed. Platelet plugs and microthrombi were also observed in these vessels. At 7 days after inoculation these lesions had intensified, and were accompanied by virus replication in the endothelial cells. In pigs infected with the Dominican Republic'78 strain, haemorrhages were more abundant and more extensive, and although no endothelial cell lesions were observed, there was intense vasodilation with diapedesis of erythrocytes. PMID- 7560302 TI - Granulomatous lesions caused by Pseudomonas aeruginosa in the ostrich (Struthio camelus). AB - Granulomatous lesions were observed in imported ostriches aged 3 months. Clinically, the birds showed lassitude, incoordination, and inappetence. At necropsy, yellowish white nodules often accompanied by a pseudodiphtheritic membrane were found in the oral, pharyngeal, tracheal and air sac mucosae, the lungs, oesophageal serosa, and abdominal peritoneum. Histopathological examination revealed purulent granulomatous lesions containing central bacterial colonies with an outer shell and club formation. The bacteria were small Gram negative bacilli, which showed positive immunohistochemical staining for Pseudomonas aeruginosa. The bacterial colonies were positive for chicken IgM. Clubs around the colonies were negative for P. aeruginosa and chicken IgM. Such findings have not previously been reported in the ostrich. PMID- 7560304 TI - Muscle fibre expression of transforming growth factor-beta 1 and latent transforming growth factor-beta binding protein in canine masticatory muscle myositis. AB - Masticatory muscle myositis (MMM) is presumed to be an immunologically mediated canine myopathy but is of unknown origin. Severe atrophy and degeneration of masticatory muscle fibres, infiltration of eosinophilic granulocytes, and proliferation of the fibrous interstitial tissue are the hallmarks of MMM. Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide controlling myogenesis, inflammation and tissue repair. We investigated immunocytochemically the expression of TGF-beta 1 and latent transforming growth factor-beta binding protein (LTBP), a TGF-beta modulator protein, in cases of MMM. The study demonstrated the presence of TGF-beta and LTBP in muscle fibres. infiltrating leucocytes and extracellular matrix in MMM, and suggested that TGF beta and LTBP play a role in muscle tissue repair, inflammation and fibrogenesis in MMM. PMID- 7560305 TI - Helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats. AB - Gastric biopsies from the fundic gland region of 122 dogs and 127 cats were subjected to histopathological examination. The aim of the study was to determine infection rates and degrees of colonization by Helicobacter-like organisms (HLOs), and to ascertain their possible relationship to histopathological changes. In all, 82% of the dogs and 76% of the cats had an HLO infection. The most striking histopathological changes were glandular degeneration with accumulation of lymphocytes and neutrophilic granulocytes (dogs, 21%; cats, 39%), fibrosis of the lamina propria mucosae (dogs, 41%; cats, 58%), oedema in the lamina propria mucosae (dogs, 54%; cats, 23%), lymphoid follicles (dogs, 17%; cats, 19%) and lymphoplasmacytic infiltrates. A relation between the degree of colonization by HLOs and the extent of histopathological changes could only be discovered in the cats. It was not possible to ascertain whether these bacteria, irrespective of the degree of colonization, were responsible for the histopathological changes in the dogs. PMID- 7560308 TI - Solar heat gain in a desert rodent: unexpected increases with wind speed and implications for estimating the heat balance of free-living animals. AB - We quantified metabolic power consumption as a function of wind speed in the presence and absence of simulated solar radiation in rock squirrels, Spermophilus variegatus, a diurnal rodent inhabiting arid regions of Mexico and the western United States. In the absence of solar radiation, metabolic rate increased 2.2 fold as wind speed increased from 0.25 to 4.0m.s-1. Whole-body thermal resistance declined 56% as wind speed increased over this range, indicating that body insulation in this species is much more sensitive to wind disruption than in other mammals. In the presence of 950W.m-2 simulated solar radiation, metabolic rate increased 2.3-fold as wind speed was elevated from 0.25 to 4.0m.s-1. Solar heat gain, calculated as the reduction in metabolic heat production associated with the addition of solar radiation, increased with wind speed from 1.26mW.g-1 at 0.25m.s-1 to 2.92mW.g-1 at 4.0m.s-1. This increase is opposite to theoretical expectations. Both the unexpected increase in solar heat gain at elevated wind speeds and the large-scale reduction of coat insulation suggests that assumptions often used in heat-transfer analyses of animals can produce important errors. PMID- 7560306 TI - Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata, identification and specificity of a branching beta 1-->6 galactosyltransferase. AB - Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-3H]-uridine 5'diphosphogalactose the incorporation of labelled D-galactose into the polysaccharide can be traced in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltransferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient beta-(1-->6) galactosyltransferase was detected when methyl 3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by 1H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked beta 1-->3. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer D-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(beta-D galactopyranosyl)-D-galactopyranose, but without any for methyl-3-O-(beta-D galactopyranosyl)-beta-D-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan. PMID- 7560307 TI - Reduction of metabolic rate and thermoregulation during daily torpor. AB - Physiological mechanisms causing reduction of metabolic rate during torpor in heterothermic endotherms are controversial. The original view that metabolic rate is reduced below the basal metabolic rate because the lowered body temperature reduces tissue metabolism has been challenged by a recent hypothesis which claims that metabolic rate during torpor is actively downregulated and is a function of the differential between body temperature and ambient temperature, rather than body temperature per se. In the present study, both the steady-state metabolic rate and body temperature of torpid stripe-faced dunnarts, Sminthopsis macroura (Dasyuridae: Marsupialia), showed two clearly different phases in response to change of air temperature. At air temperatures between 14 and 30 degrees C, metabolic rate and body temperature decreased with air temperature, and metabolic rate showed an exponential relationship with body temperature (r2 = 0.74). The Q10 for metabolic rate was between 2 and 3 over the body temperature range of 16 to 32 degrees C. The difference between body temperature and air temperature over this temperature range did not change significantly, and the metabolic rate was not related to the difference between body temperature and air temperature (P = 0.35). However, the apparent conductance decreased with air temperature. At air temperatures below 14 degrees C, metabolic rate increased linearly with the decrease of air temperature (r2 = 0.58) and body temperature was maintained above 16 degrees C, largely independent of air temperature. Over this air temperature range, metabolic rate was positively correlated with the difference between body temperature and air temperature (r2 = 0.61). Nevertheless, the Q10 for metabolic rate between normothermic and torpid thermoregulating animals at the same air temperature was also in the range of 2-3.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 7560309 TI - Effects of physical exercise and aging on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs. AB - The relationship of physical activity and aging, two processes with a high production of oxygen-free radicals to the ascorbate and superoxide anion (O2-) contents of peritoneal macrophages was studied in two animals species: guinea-pig (in which ascorbic acid is a vitamin) and mouse (in which ascorbic acid is not a vitamin). The effects of exhaustive exercise were examined in young and old animals. The results show that macrophages from old animals have a lower ascorbate content than those from young ones, whereas with exercise the ascorbate content increased in both old and young animals. This increase was higher in young than in old animals, and more evident in mice than in guinea-pigs. Aging also resulted in an increase in the O2- levels of macrophages. With exercise these levels decreased in young mice but increased in young guinea-pigs. In old animals the exhaustive exercise did not change the O2- levels. The results suggest in general a lack of correlation between the intracellular ascorbate and O2- levels in relation to both physical exercise and aging. PMID- 7560310 TI - Kidney structure and function of obligate and facultative hibernators: the white tailed prairie dog (Cynomys leucurus) and the black-tailed prairie dog (Cynomys ludovicianus). AB - The white-tailed prairie dog is an obligate hibernator that enters a heterothermic phase when maintained in the cold with low intensity light and ad libitum food and water. The black-tailed prairie dog (a facultative hibernator) will not hibernate under similar conditions. It has been suggested that the black tailed prairie dog remains active during the winter because it can conserve water more effectively due to a more efficient kidney. The present study revealed no significant differences between the species in renal morphology: relative medullary thickness, nephron heterogeneity, renal vasculature, or fornix dimensions, all of which are structures associated with the urinary concentrating mechanism. In addition, there was no difference in number of nephrons between the two species. The black-tailed prairie dog does produce a more concentrated urine when food and water deprived. However, this difference was not observed when the animals were salt loaded. The water-deprivation and salt-loading experiments suggest that the higher urine osmolality produced by the back-tailed prairie dog during fasting is a result of a higher urea load due to a greater protein catabolism and not because of a differential capacity to concentrate urine. PMID- 7560312 TI - Factors influencing flow-induced signal loss in MR angiography: an in vitro study. AB - OBJECTIVE: Signal loss due to poststenotic turbulence is one remaining limitation in the clinical use of MRA. The objective of this study was to determine the factors influencing poststenotic signal loss (PSL) in a stenotic tube system. MATERIALS AND METHODS: With use of a two-dimensional gradient echo sequence (FLASH), the influence of (a) the degree of stenosis (50, 75, and 91% cross sectional area reduction), (b) flow velocity (12, 47, 71, and 94 cm/s), (c) TE (3, 6, 13, and 20 ms), and (d) flip angle (5, 10, 20, ... , 90 degrees) on the length of PSL was measured in a nonpulsatile stenotic tube system. To quantify the effect of first-order gradient motion refocusing (GMR), the signal intensity ratio from flow-compensated and -uncompensated images was calculated. For statistical analysis, multiple regression analysis and unpaired Student t test were used. RESULTS: In the flow model used, the length of PSL increased significantly with the degree of stenosis (beta ST' = 0.483 mm/%, beta ST" = 0.447 mm/%, p < 0.0001), flow velocity (beta v' = 0.297 s, beta v" = 0.213 s, p < 0.0001), and TE (beta TE = 1.88 mm/ms, p < 0.0001), respectively, whereas no correlation emerged for varying flip angles (beta alpha = 0.0214, p = 0.29). First-order GMR reduced significantly PSL in through-plane measurements (p < 0.0001). Maximal signal-enhancing effects of first-order GMR were observed 1-3 cm distal to the stenosis. CONCLUSION: Flow model parameters (i.e., degree of stenosis, flow velocity) markedly influenced the length of PSL that could be compensate for by use orf shortened TEs and first-order GMR. Varying flip angles had no significant influence on PSL. PMID- 7560311 TI - Detection of subtle brain changes using subvoxel registration and subtraction of serial MR images. AB - OBJECTIVE: The aim of this study was to examine the potential of accurate image registration for detecting subtle changes in the brain. MATERIALS AND METHODS: Isotropic T1-weighted volume images were obtained in 10 normal subjects and five patients on two or more occasions (including pre- and postcontrast studies). The images were segmented and a 3D rigid body translation and rotation technique was used with sinc interpolation to precisely match the images using a chi 2-test. The registered images and the subtraction images produced from them were used to detect changes in signal intensity, sit, shape, and size of the brain. RESULTS: Small changes due to differences in orientation of the head, growth, and development as well as inhalation of oxygen and carbogen (95% O2/5% CO2) were observed in normal subjects. Changes were also observed in patients with minor head trauma, a meningioma, an astrocytoma, and multiple sclerosis. Differences due to contrast enhancement and surgery and/or anesthesia were also seen. CONCLUSION: With use of subvoxel registration, subtle changes in the brain were detected in a variety of physiological and clinical situations where differences have hitherto been difficult or impossible to detect. PMID- 7560313 TI - High resolution, magnetization transfer saturation, variable flip angle, time-of flight MRA in the detection of intracranial vascular stenoses. AB - OBJECTIVE: Factors that restrict 3D TOF MRA are limited resolution, saturation of flow, and degree of background suppression. We evaluated MRA for intracranial stenoses by using a 3D TOF technique that minimizes these factors. MATERIALS AND METHODS: Twenty-nine patients underwent MRA and intraarterial digital subtraction angiography (DSA). The MRA studies were performed on a 1.5 T Siemens SP 4000 system. Integrated techniques applied to the conventional 3D TOF acquisition included the following: (a) 256 x 256 matrix with a 140 mm FOV and 0.9 mm slice thickness, yielding a 0.54 x 0.54 x 0.9 mm3 voxel; (b) tilted optimized nonsaturating excitation (TONE); and (c) magnetization transfer saturation (MTS). The intraarterial DSA was performed on a Siemens Angiostar system with a 1,024 x 1,024 noninterpolated matrix. The MRAs were reviewed by two neuroradiologists. Two hundred seventy-seven vessels were evaluated for a total of 806 segments. Vessel segments were evaluated with a 5 point scale. RESULTS: The estimated accuracy of MRA for detecting stenosis over all intracranial vessel segments was 0.88 +/- 0.03 and 0.89 +/- 0.02 for the two readers, respectively. The estimated accuracy ranged from 0.94 +/- 0.02 and 0.93 +/- 0.02 for detecting internal carotid artery stenosis by the two readers, respectively, to 0.65 +/- 0.17 and 0.71 +/- 0.15 for detecting distal vertebral artery stenosis. In vessels determined by catheter angiography to be stenosis-free, reader confidence at the proximal versus distal segments was similar for the internal carotid, basilar, and posterior cerebral arteries. However, for the anterior and middle cerebral arteries, one or both readers were more confident in diagnosing the proximal segment. CONCLUSION: High resolution MTS TONE 3D TOF MRA is an accurate technique for the screening of medium and large vessel intracranial stenoses. PMID- 7560314 TI - Evaluation of large intracranial aneurysm with three-dimensional MRI. AB - OBJECTIVE: The purpose of this study was to evaluate large intracranial aneurysms by contrast-enhanced 3D MRI. MATERIALS AND METHODS: Eleven patients, nine women and two men, were examined. The aneurysms involved the internal carotid artery in seven patients, the middle cerebral artery in one, the basilar artery in two, and the posterior cerebral artery in one. Contrast-enhanced 3D MR images were obtained using FLASH (fast low angle shot) on a 1.5 T system. We obtained axial images first, then sagittal and coronal images by multiplanar reconstruction. Those images were reviewed for comparison with conventional and MR angiography. RESULTS: We could easily evaluate the true aneurysmal size, the intraaneurysmal constitution (patent lumen and intraluminal thrombus), and the detailed relationship of the aneurysm to the surrounding vascular and neural structures. Except for the cavernous internal carotid artery, we could identify the aneurysmal neck by carefully observing axial, sagittal, and coronal images. In addition, the progression of intraaneurysmal thrombosis and the patency of the parent arterial lumen after the endovascular treatment were well appreciated. CONCLUSION: We consider that 3D MRI appears to be useful for evaluation of the large intracranial aneurysm. PMID- 7560315 TI - Postcontrast MRI of cranial meninges: leptomeningitis versus pachymeningitis. AB - OBJECTIVE: Our goal was to characterize the patterns of meningeal enhancement in postcontrast MR images and correlate these patterns with the clinical disorders. MATERIALS AND METHODS: The MR scans, medical records, and laboratory findings of 83 patients, whose postcontrast MR studies of the head demonstrated meningeal enhancement, were reviewed retrospectively. The patterns of enhancement of the different layers of the meninges were divided into two types: leptomeningeal (pia and arachnoid), when enhancement of the meninges followed the convolutions of the gyri and/or involved the meninges around the basal cisterns; and pachymeningeal (dura), when the enhancement was thick and linear or nodular along the inner surface of the calvarium, falx, or tentorium without extension into the cortical gyri or basal cistern involvement. Enhancement around the basal cistern was considered leptomeningeal, since the dura-arachnoid is widely separated from the pia-arachnoid in this region. Further, the meningeal enhancement was divided into five etiologic subgroups, i.e., carcinomatous, infectious, inflammatory, reactive, and chemical. The medical history, clinical presentation, and findings on CSF analysis were used to distinguish infectious from carcinomatous meningitis. Meningeal enhancement due to surgery, shunt, or trauma was considered reactive, while ruptured cysts (dermoid or cysticercoid) or intrathecal chemotherapy were classified as chemical meningitis. Meningitis secondary to involvement by collagen vascular disease or sarcoidosis was considered to be inflammatory. RESULTS: Thirty of the 83 subjects had carcinomatous, 28 infectious, 14 reactive, 8 chemical, and 3 inflammatory etiology for meningitis. Twenty-five cases (83%) of the carcinomatous, 14 (100%) of the reactive, 3 (100%) of the inflammatory, and 1 (12%) of the chemical meningitis subgroups demonstrated pachymeningeal enhancement, while 28 cases (100%) of the infectious meningitis and 7 (78%) of the chemical meningitis subgroups had leptomeningeal enhancement. Only five cases (17%) of the carcinomatous meningitis subgroup showed leptomeningeal enhancement. Four of these five cases were as a result of direct spread of intraparenchymal tumors or through perineural extension, rather than hematogenous involvement. Only one patient with carcinomatous meningitis demonstrated leptomeningeal enhancement without clear intraparenchymal lesion. CONCLUSION: The recognition of various patterns of meningeal enhancement (leptomeningitis versus pachymeningitis) may help in differentiating between infectious and carcinomatous meningitis. This study demonstrated that infectious meningitis presents mostly as leptomeningitis, while carcinomatous meningitis presents as pachymeningitis. PMID- 7560317 TI - CT features of pulmonary nocardiosis. AB - OBJECTIVE: Our goal was to identify the CT features of pulmonary nocardiosis, specifically in AIDS patients versus other immunocompromised hosts. MATERIALS AND METHODS: Twenty-four patients with pulmonary nocardiosis were identified; medical records, CT scans, and chest radiographs were reviewed. Five patients had HIV disease, 17 had other causes of immunosuppression, and 2 had normal immunity. RESULTS: CT features of pulmonary nocardiosis included one or more nodules/masses (20/24 cases, 83%), cavitation (8, 33%), consolidation/infiltrates (8, 33%), and pleural thickening (7, 29%). All AIDS patients with nocardiosis had multiple (four or more) nodules detected on CT and 80% (4/5) had cavitation compared with 21% each of non-AIDS patients (p < 0.01 for multiple nodules, p < 0.045 for cavitation). CONCLUSION: CT features of pulmonary nocardiosis are varied, and AIDS patients are more likely to display cavitation and multiple nodules. Although not widely recognized, Nocardia infection should be included in the differential diagnosis of an unexplained nodule or progressive cavitary process in HIV-positive patients. PMID- 7560316 TI - Three-dimensional fast spin echo T1-weighted imaging of the pediatric spine. AB - OBJECTIVE: We evaluated the feasibility of using a three-dimensional (3D) fast SE (FSE) pulse sequence to acquire T1-weighted (T1W) images of the pediatric spine. MATERIALS AND METHODS: The 3D FSE T1W images were acquired in nine pediatric patients undergoing spine MRI for different clinical indications. The 3D FSE images were compared with our standard 2D SE T1W images. RESULTS: Image contrast was comparable between the two sequences. The 3D FSE sequence produced contiguous thin sections that allowed multiplanar reformations not possible with the 2D SE sequence. CONCLUSION: 3D T1W spine imaging with conventional SE-like contrast is feasible with 3D FSE. PMID- 7560318 TI - Pulmonary zygomycosis: CT appearance. AB - OBJECTIVE: We describe the CT appearance of pulmonary zygomycosis (mucormycosis), an opportunistic infection typically occurring in immunocompromised patients. MATERIALS AND METHODS: Eight patients with pulmonary zygomycosis imaged with CT were reviewed, seven at initial diagnosis and one with a subsequent complication. The appearance, number, and location of pulmonary lesions and the presence of pleural effusions and extrapulmonary involvement were assessed. Rim enhancement, air bronchograms, the halo sign, air crescent sign, cavitation, and central low attenuation suggesting necrosis were recorded. RESULTS: There were 14 nodules and 5 areas of mass-like or wedge-shaped consolidation. Pleural effusion was present in five patients, halo sign in three, central low attenuation in two, and cavitation in one. In the affected lobe 13 of 14 nodules and all consolidations were posterior. Of 19 lesions 16 (84%) were confined to the upper lobes, with 3 in the superior segment of a lower lobe. Endobronchial disease with lobar collapse was the only manifestation in one patient. Major complications were direct spinal invasion in one patient and multiple pulmonary artery pseudoaneurysms in another patient. CONCLUSION: In the appropriate clinical circumstance, nodules or mass-like or wedge-shaped consolidation, especially posteriorly in the upper lobes of the lung, should suggest zygomycosis. Endobronchial zygomycosis is less common. PMID- 7560319 TI - Pancreatic carcinoma: MR assessment of tumor invasion of the peripancreatic vessels. AB - OBJECTIVE: Our goal was to determine the value of MRI in the assessment of vascular invasion in the preoperative staging of pancreatic carcinoma. MATERIALS AND METHODS: In 53 consecutive patients with an established diagnosis of pancreatic ductal adenocarcinoma, SE T1-weighted and breath-hold gradient echo images at 0.5 T were obtained before and after bolus injection of Gd-DTPA. Major peripancreatic vessels were evaluated for the presence of (a) no tumor invasion, (b) contiguity with tumor, and (c) tumor encasement. All patients subsequently underwent surgery. Results of unenhanced and contrast-enhanced MRI studies were compared with the histologic findings in the resected specimens in 34 cases and with the surgical findings in 19 nonresected cases. RESULTS: In six patients, pathologic examination showed the presence of tumor confined to the pancreas with no vascular invasion. With MRI, five of these cases were correctly evaluated; in the remaining case, tumor-vessel contiguity was erroneously diagnosed. In 21 patients, tumor contiguity with adjacent vessels was found at pathologic examination. At MRI, 16 of these cases were correctly assessed, and 5 were understaged as tumors confined to the pancreas. Of the remaining 26 patients, 7 had vascular encasement by tumor at pathologic examination of the resected specimen; the other 19 patients had unresectable tumors encasing the adjacent vessels at surgical evaluation. MRI detected vascular encasement in 21 of these 26 cases; in the other 5, tumor contiguity was incorrectly diagnosed. The overall accuracy of MRI for determining vascular invasion was 79%. CONCLUSION: MRI is a helpful method for preoperative assessment of vascular involvement due to pancreatic carcinoma. PMID- 7560322 TI - Dynamic MRI of small renal cell carcinoma. AB - OBJECTIVE: To evaluate the appearance of contrast-enhanced dynamic MRI of small renal cell carcinoma (RCC) (< 3 cm diameter). MATERIALS AND METHODS: Twenty-seven patients with 28 small RCCs were studied by both conventional spin echo and contrast-enhanced dynamic MRI for tumor detection and characterization. All tumors were detected by contrast-enhanced CT and confirmed by operation. Dynamic MRI was performed using a breath-hold fast low angle shot technique after rapid injection of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). Characterization of a renal mass was done by the visual analysis of tumor vascularity as well as quantitative assessment of the contrast noise ratio (CNR) and degree of enhancement. RESULTS: With conventional spin echo MR and dynamic MR, 18 and 26 of 28 small RCCs were detected, respectively. In the dynamic study the tumor/renal medulla contrast was marked at 90-120 s after administration of Gd-DTPA (CNR:15.1). The tumor/renal medulla contrast in the dynamic study was more marked than that of postcontrast T1-weighted imaging (CNR, -0.25); T2 weighted imaging (CNR, 3.1); and T1-weighted imaging (CNR, -0.76). CONCLUSION: Contrast-enhanced dynamic MRI is a useful technique for the diagnosis of small RCC especially in a patient with renal insufficiency or allergy to iodinated contrast material. PMID- 7560320 TI - MRI of the anal sphincter. AB - OBJECTIVE: The anal sphincter was imaged with MR using an internal coil to demonstrate its anatomy, contrast enhancement patterns, and appearance in disease. MATERIALS AND METHODS: A cylindrical saddle geometry coil was placed in the anal canal. Sixteen volunteers and 18 patients were examined. Imaging was performed on a 0.5 T Picker Asset MRI scanner in all the volunteers and nine patients and on a 1.0 T Picker HPQ Vista in nine patients. Then T1- and T2 weighted SE, T1-weighted GE and STIR images transverse to the sphincter, and T1 weighted SE images parallel to the sphincter in the coronal oblique plane were obtained. Intravenous gadopentetate dimeglumine (0.1 mmol/kg) was given to 2 normal subjects for dynamic studies and 10 patients for conventional postcontrast imaging. RESULTS: The coils were easy to insert and well tolerated and provided high spatial resolution. The internal sphincter had a higher signal intensity than the external sphincter on all sequences but particularly on STIR images. Brisk contrast enhancement of the internal sphincter was seen. Sphincteric abscesses and fistulous tracks were identified in three patients and confirmed at surgery. Sphincter defects were seen in three patients with past obstetric trauma, and these were also confirmed at surgery. Sphincter atrophy was seen in three patients with idiopathic fecal soiling. CONCLUSION: MRI of the anal sphincter with an internal coil provides excellent visualization of normal anatomy and may be of considerable value in diagnosis. PMID- 7560323 TI - Detection of deep myometrial invasion in endometrial carcinoma: comparison of transvaginal ultrasound, CT, and MRI. AB - OBJECTIVE: The aim of this study was to compare the accuracy of transvaginal US (TVUS), CT, and MRI in detecting deep myometrial invasion in endometrial carcinoma. MATERIALS AND METHODS: Twenty-six patients with endometrial carcinoma were evaluated with TVUS, CT, and MRI. The depth of myometrial invasion was estimated in each examination. The invasion depth was categorized into no/superficial and deep and was compared to surgical and pathologic findings. RESULTS: At pathologic examination, the depth of myometrial invasion was no/superficial in 16 patients and deep in 10. The accuracy/sensitivity/specificity of TVUS, CT, and MRI in detecting deep myometrial invasion were 69/50/81, 61/40/75, and 89%/90%/88%, respectively. The sensitivity and accuracy of MRI in detecting deep myometrial invasion were significantly higher than those of TVUS and CT (p = 0.049 for both sensitivity and accuracy). CONCLUSION: We recommend MRI instead of CT or TVUS for the evaluation of the depth of myometrial invasion in endometrial carcinoma. PMID- 7560321 TI - Staging of rectal carcinoma using MR double surface coil, MR endorectal coil, and intrarectal ultrasound: correlation with histopathologic findings. AB - OBJECTIVE: Our goal was assessment of the preoperative staging of rectal carcinoma with MR with double surface coil, MR with endorectal coil, and intrarectal ultrasound (IUS) as correlated with histopathologic findings. MATERIALS AND METHODS: Fifteen patients with rectal carcinoma had preoperative evaluation using intrarectal ultrasound (all 15 patients), MR with double surface coil alone (6 patients), and MR with double surface coil combined with endorectal surface coil (9 patients). The results of the preoperative staging were correlated with the histopathologic findings. RESULTS: IUS correctly staged the depth of bowel wall invasion in 10 of 15 patients, understaged 4, and overstaged 1. MRI correctly staged 10 of 15 patients. Without the endorectal surface coil, three of six were correct, and with endorectal surface coil seven of nine. MR with the endorectal surface coil is able to show the rectal wall in more detail than the double surface coil. CONCLUSION: Endorectal surface coil MRI provides increased detail of the rectal wall, leading to better delineation of its different layers. This may lead to better staging results than with other MR techniques. The results with endorectal MRI probably equal those of IUS for staging small tumors in the rectal wall. MR with the double surface coil gives additional information about tumor spread in more advanced cases. PMID- 7560324 TI - Comparison of bone contusion seen by MRI in partial and complete tears of the anterior cruciate ligament. AB - OBJECTIVE: Bone contusions are frequently found in association with complete tears of the anterior cruciate ligament (ACL) and can be a helpful secondary sign in diagnosis. We compare the frequency, location, and significance of bone contusions in complete and partial ACL tears. MATERIALS AND METHODS: Twenty-nine patients with complete and 42 patients with incomplete tears of the ACL were examined by MRI for the presence of accompanying bone contusions within 1 month of injury. RESULTS: Bone contusions were found in 72% of the complete ACL tears but in only 12% of the partial tears. Of the partial ACL tears with accompanying contusions, 80% were high grade injuries that eventually went on to complete rupture within 6 months. Only 16% of the partial ACL injuries without bone contusion progressed to complete rupture at 1-2 year follow-up. There was no difference between the bone contusions of partial and complete tears in terms of general appearance and location. They were predominantly in the lateral compartment (90%) and had a specific predilection for the mid portion of the lateral femoral condyle and the posterior portion of the lateral tibial plateau, often occurring in tandem. CONCLUSION: Bone contusions occur with much less frequency in partial ACL tears than in complete tears but their presence in partial rupture favors a high grade tear that is likely to become complete. PMID- 7560326 TI - Different biologic features of desmoid tumors in adult and juvenile patients: MR demonstration. AB - OBJECTIVE: To demonstrate different biologic features of desmoid tumors on MRI between juvenile and adult patients. MATERIALS AND METHODS: We have retrospectively analyzed clinical records and 121 MRI findings in 40 patients (8 juveniles and 32 adults) with proven desmoid tumors. The Fisher exact test and Kaplan-Meier curve were utilized for statistical analysis. RESULTS: Recurrences in the juvenile patients were more multiple (50 vs 12%) and appeared significantly earlier than in the adult patients. Adult patients demonstrate a much greater recurrence-free rate (p = 0.0001). Infiltrative pattern was significantly predominant in the juvenile patients (63%) whereas the nodular pattern was more frequent in the adult patients (81%). Low-signal intensity zones on T1- and T2-weighted imaging as well as the type of contrast enhancement did not show any significant relationship with biological behavior. Four cases with no significant contrast enhancement showed low signal intensities on T2-weighted imaging. CONCLUSION: Magnetic resonance demonstrates different biologic features between juvenile and adult patients with histologically same desmoid tumors. These differences may be useful in consideration of MRI follow-up studies. PMID- 7560325 TI - Tibial shaft fractures: assessment of fracture healing with computed tomography. AB - OBJECTIVE: To assess the capability of CT in assessing stability of tibial shaft fractures with planimetry. MATERIALS AND METHODS: Eighteen patients with fracture of the tibial shaft were treated by external fixator. All patients underwent CT 1, 6, 12, and 18 weeks postoperatively to assess the fracture healing. The callus formation and compacta were determined with planimetry. Fractometry, a noninvasive method to measure stability, was used as the gold standard to determine the stability of the fracture. RESULTS: The patients were divided into three groups according to different periods of time for removal of external fixation. Twelve patients with stable fractures showed a steady increase of callus, which was 50% higher after 12 weeks. Three patients with delayed fracture healing demonstrated an increase of callus of > 50% after 18 weeks. Three patients with unstable fractures obtained only callus formation of < 20% after 15 18 weeks. The external fixator was removed and endomedullary nails were used for stabilization. CONCLUSION: The CT data allow quantification of callus and compacta formation and are able to determine the stability of tibial shaft fractures. PMID- 7560327 TI - Estimating smoothness in statistical parametric maps: variability of p values. AB - OBJECTIVE: The smoothness parameter that characterises the spatial dependence of pixel values in functional brain images is usually estimated empirically from the data. Since this parameter is essential for the assessment of significant changes in brain activity, it is important to know (a) the variance of its estimator and (b) how this variability affects the results of the ensuing statistical analysis. MATERIALS AND METHODS: In this article, we derive an approximate expression for the variance of the smoothness estimator and investigate the effects of this variability on assessing the significance of cerebral activation in statistical parametric maps using a verbal fluency PET activation experiment. RESULTS: Our results suggest that, for p values around 0.05, the variability in the p value (due to smoothness estimation) is approximately 20%. CONCLUSION: The effect of the assessment of the spatial dependency of the data is far from being negligible, and this suggests a more comprehensive methodology for functional imaging than the one used so far. This work provides a simple tool for taking into account this effect. PMID- 7560330 TI - CT findings of malignant change in recurrent respiratory papillomatosis. AB - Recurrent respiratory papillomatosis is a chronic disease of children and young adults associated with various complications, one of which is malignant change. We report CT findings of malignant change in a case of recurrent respiratory papillomatosis. PMID- 7560329 TI - Cine MRI in the evaluation of the Proplast-Teflon TMJ interpositional implant. AB - Proplast-Teflon (Vitek Inc., Houston, TX, U.S.A.) interpositional implants (PTIPIs) have been removed from the market due to complications that include severe bony destruction of both condyle and fossa as a result of exuberant foreign body giant cell reaction. As per Food and Drug Administration recommendations, the radiologist will often be called to evaluate the status of these implants in the large population that received them. We present a case of bilateral PTIPIs with nonreducing anterior displacement of the meniscal prosthesis made more apparent by cine MRI. Extensive granulomatous reaction in the temporal bone exhibited mobility with cine. PMID- 7560328 TI - CT cisternography in congenital perilymphatic fistula of the inner ear. AB - Perilymphatic fistulas of the inner ear constitute abnormal leaks of perilymphatic fluid into the middle ear or mastoid air cell system and represent a rare cause of otorrhea. We report the case of a 5-month-old child presenting with sudden otorrhea. High resolution CT cisternography showed a malformation of the middle ear and a passage of contrast-enhanced CSF into the tympanic cavity through the left oval window. Surgery confirmed a tear of the tympanic membrane as well as a stapes malformation with aplasia of the crura and an associated perilymphatic fistula through a defect in the stapes footplate. The fistula was closed with adipose tissue from the ear lobe. PMID- 7560331 TI - CT findings in acute necrotizing Meckel diverticulitis due to obstructing enterolith. PMID- 7560333 TI - CT of thrombosed renal vein presenting as a hyperdense mass. AB - We report a patient with focal renal vein thrombosis that presented as a hyperdense mass mimicking a hyperdense cyst or a pelvic or parenchymal tumor. The lesion was described as a well-defined round hyperdense mass in the right renal sinus on noncontrast CT and was surgically proven to be acute thrombosis of the renal vein. Renal vein thrombosis should be considered in the differential diagnosis of a hyperdense mass in the renal sinus even if the patient has no underlying renal abnormality. PMID- 7560334 TI - Renal capsular hemangioma: unusual MR findings. PMID- 7560332 TI - MRI of hepatic schistosomiasis mansoni. AB - Magnetic resonance imaging (MRI) was performed in a patient with hepatic schistosomiasis mansoni. On T1-weighted images periportal zones appeared isointense to the surrounding liver, but strongly enhanced after injection of Gd DTPA. This finding equals periportal enhancement in postcontrast CT studies and does not discriminate between periportal inflammation in early stages of the disease and portal venous and hepatic artery collaterals in late stages of periportal fibrosis. However, on T2-weighted images periportal zones appeared as high signal bands throughout the liver suggesting periportal inflammatory changes with edema. Thus T2-weighted spin echo sequences should be performed and may be helpful to assess activity of periportal inflammation. PMID- 7560336 TI - The "pituitary pseudoaneurysm" artefact with 3D TOF MRA of the circle of Willis. PMID- 7560335 TI - MR findings in congenital glenoid dysplasia. AB - The MR features of congenital glenoid dysplasia are described. Osseous changes include both dysplasia of the glenoid and hypoplasia of the glenoid process. In addition, there are cartilaginous changes with hypertrophy of both the articular cartilage and the glenoid labra. PMID- 7560337 TI - CT of multiple subcapsular pseudocysts of the kidney complicating acute pancreatitis. PMID- 7560338 TI - Placenta percreta post evacuation: an unusual uterine mass on MRI. PMID- 7560339 TI - Retained placenta accreta: MRI and pathologic correlation. PMID- 7560340 TI - A knowledge-based approach to deconvolve the water component in in vivo proton MR spectroscopy. AB - OBJECTIVE: The water component dominates in in vivo proton (1H) spectroscopy. This consists of a prominent central peak with large "wings," and consequently metabolites that lie on the "wings" become difficult to quantitate. A method has been developed to deconvolve the water component in in vivo 1H spectroscopy, hence highlighting the metabolite information. MATERIALS AND METHODS: Spectra were acquired from volunteers and patients using 4D chemical shift imaging. The water deconvolution procedure employed knowledge-based data processing in the frequency domain and was fully automated. This involved describing the water component as a coarse function consisting of a "bulk" region and "wing" areas. Points were identified in the spectrum that fit this description and then linked together to produce the water component. The latter was smoothed and then subtracted from the original spectra to produce good water deconvolution. RESULTS: Over 2,000 in vivo 1H spectra have been subjected to this algorithm. The method took approximately 5 s to execute per spectrum consisting of 2,048 data points. CONCLUSION: Knowledge-based data processing has provided a fast, efficient, and robust procedure to deconvolve the water component. PMID- 7560342 TI - Cutaneous cysts of Gardner's syndrome are similar to follicular stem cells. AB - Cutaneous cysts from a patient with Gardner's syndrome were histopathologically studied in detail. The cysts were, by and large, indistinguishable from ordinary epidermal cysts. However, several distinctive features were found: 1) epidermal or trichilemmal keratinization, 2) mature sebaceous glands connected with the cyst wall, 3) hair matrix-like structures associated with dermal papilla cells, 4) pilomatricoma-like changes, 5) intraluminal masses or pericystic deposits of shadow cells variably accompanied with foreign body reaction, 6) foreign body reaction or masses of shadow cells lining completely eroded cysts, 7) the presumptive bulge area, and 8) epithelial islands adjacent to the cyst. Each cutaneous cyst showed a variable combination of the findings described above. Foci of the basal layer of some cyst walls or epithelial islands were immunohistochemically stained with CK19, where CK20-reactive Merkel cells were also present. These findings were consistent with those of the bulge area. Unexpectedly, desmin-reactive muscle bundles, presumably indicating arrector pili muscle, were observed along the cyst wall. Our observations suggest that Gardner's cysts may be derived from putative follicular stem cells which reside in the bulge area. PMID- 7560341 TI - Determination of spiral CT slice sensitivity profiles using a point response phantom. AB - OBJECTIVE: This study was done to compare the slice sensitivity profiles (SSP) for combinations of collimation, pitch, and table speed for spiral CT using a point response phantom. The goal was to determine the optimal combination of parameters to reduce partial volume averaging without compromising z-axis coverage. MATERIALS AND METHODS: A copper ball bearing measuring 0.4 mm was embedded in a closed-cell air-foam background to create a point response input phantom. The phantom was scanned at pitches from 0.1 to 2.0 for collimations of 5, 8, and 10 mm. The full width half maximums (FWHMs) and full width tenth maximums (FWTMs) were estimated from SSP curves generated by plotting the maximum pixel value in HU for each reconstructed image against table position. FWHMs and FWTMs were compared separately for constant collimation and increasing table speed and for constant table speed and decreasing collimation using either a two tailed z-test or chi-square test. RESULTS: Differences between FWHMs and between FWTMs for comparisons made between different collimations at constant table speeds of 8 and 10 mm/s were significantly different (p < or = 0.0001). Differences between FWHMs and between FWTMs showed a linear trend, increasing with increasing pitch for constant collimation (p < or = 0.0013). CONCLUSION: Scanning at narrower collimation but higher pitch provides a narrower SSP when scanning at equivalent table speeds without compromising z-axis coverage. PMID- 7560344 TI - Approach to diagnostic image analysis of melanocytic tumors. AB - Numerous attempts have been made to apply image analysis in dermatopathology. The technics used comprise measurement of nuclear size, shape, chromatin content, and texture, evaluation of immunohistological slides, assessment of proliferation, pattern analysis, and tumor volume estimation. For commonly accepted routine use, however, image analysis research has to be extended to large numbers of cases, using straightforward and reproducible measuring procedures, and to the development of ready-to-use equipment for specific tasks. In this way, image analysis in dermatopathology might supply useful diagnostic tools in addition to conventional microscopy, and may increase our understanding of morphology as a whole. PMID- 7560343 TI - Immunohistochemical assessment of tumor vascularity in recurrent Clark II melanomas using antibody to type IV collagen. AB - Thin melanomas, measuring 0.76 mm or less, are generally associated with an excellent prognosis. However, a certain subset of these seemingly innocuous lesions have been reported to develop recurrences. Therefore, the predictive values of currently accepted prognostic indicators have been questioned in thin melanomas. Several studies concerning tumor vascularity in melanoma and certain non-melanocytic malignancies suggest that the degree of vascularization correlates with growth rate and biologic aggressiveness. In the present study, we determined the vascularity of a small group of Clark level II melanomas that resulted in recurrence, and compared these results to an equal number of nonrecurrent lesions with similar prognostic indicators. Blood vessels were labeled by immunoperoxidase staining techniques for Type IV collagen, and quantified by image analysis. No statistical difference was found between the two groups when mean blood vessel counts and percent vascular area were measured. The recurrent tumors had a mean PVA of 4.68 compared to 4.34 for the nonrecurrent group (p = 0.677). The mean blood vessel count beneath the recurrent group was 29.6 per 400 x field, and the corresponding value for the nonrecurrent group was 31.8 (p = 0.681). Our data is preliminary within this limited group of tumors, yet it suggests that tumor vascularity is not a distinctive prognostic indicator by which eventual outcome can be predicted in thin Clark level II malignant melanomas. PMID- 7560346 TI - Ultrastructural spectrum of cutaneous nerve sheath myxoma/cellular neurothekeoma. AB - The histogenesis of cutaneous nerve sheath myxoma (NSM)/cellular neurothekeoma (CNT) is still controversial. In this study, we examined the ultrastructural features of 16 NSM (3 classical, 11 CNT, and 2 mixed NSM/CNT). We classified the cells into 4 groups ultrastructurally. Type I cells were undifferentiated polygonal cells with ovoid nuclei, cytoplasmic microfilaments, and occassionally with microfilament-associated dense bodies. In most cells, the cytoplasmic membrane showed focal membranous densities and occasional basal-lamina-like material. This cell type comprised approximately 90% of CNT. Type II cells were more differentiated, had ovoid or spindled shapes, were rich in intracytoplasmic filaments, and were surrounded by continuous basal lamina. These cells were consistent with Schwann cells and were present in the classical and mixed forms of NSM, and in a single case of CNT. Type III cells had features of perineurial cells and were relatively rare in classical NSM. Type IV cells resembled fibroblasts and were encountered in all variants of NSM. These results support the view that 1) the classical NSM has neural (mainly Schwann cell) differentiation, 2) CNT is predominantly composed of undifferentiated cells with partial features of Schwann cells, smooth muscle cells, myofibroblasts and fibroblasts, suggesting a divergent differentiation, and 3) CNT and NSM represent a histologic spectrum, but in CNT, the neural features are not fully expressed. PMID- 7560345 TI - Nucleolar organizer regions and image analysis nuclear morphometry of small cell (nevoid) melanoma. AB - Small cell (nevoid) melanomas may provide difficulties in diagnosis as their constituent cell type resembles a benign nevoid melanocyte. In the present study, 10 small cell melanomas were analyzed for the silver staining of their nucleolar organizing regions (AgNORs), and their nuclear area and perimeter were measured by computerized digital image analysis and compared with 10 superficial spreading melanomas lacking small cell differentiation and 10 dermal nevi. The average number of AgNORs per nucleus was 5.83 (SD +/- 1.69) for small cell melanomas and was significantly different when compared with 8.49 (SD +/- 1.58) for superficial spreading melanomas (p < 0.05) and 2.71 (SD +/- 0.50) for dermal nevi (p < 0.05). Digital image analysis confirmed that the nuclear perimeter and nuclear area of cells in nevoid melanomas did not significantly differ from those of ordinary dermal nevi (p > 0.05), but both group were significantly different from superficial spreading melanomas lacking a small cell morphology (p < 0.05). Counting AgNOR numbers may be useful in evaluating small cell (nevoid) melanomas and provides a technique for differentiating their constituent cell from ordinary nevus cells. Nuclear morphometry determined by digital image analysis may help better define the nuclear size in small cell melanomas. PMID- 7560347 TI - Expression of alpha subunit of guanine nucleotide-binding protein Go in Merkel cell carcinoma. AB - The alpha subunit of guanine nucleotide-binding protein Go (Go alpha), which was initially isolated from bovine brain, interacts with muscarinic cholinergic receptors and regulates neuronal calcium channels. Go alpha is known to be localized in neural tissues, some endocrine cells, and neuroendocrine tumors. We have immunohistochemically investigated the expression of Go alpha in 4 cases of Merkel cell carcinoma using the method of microwave treatment. In all cases of Merkel cell carcinoma, Go alpha was consistently detected on the plasma membrane and cytoplasm of the tumor cells. Nerve fibers in the skin were also positive for Go alpha, but other epidermal or dermal components such as keratinocytes, melanocytes, fibroblasts, or lymphoid cells were negative. Tumor cells of squamous cell carcinoma, cutaneous lymphoma, sweat gland carcinoma, and malignant melanoma were negative for Go alpha. The present study indicates that Go alpha may be a useful immunohistochemical marker of Merkel cell carcinoma. PMID- 7560348 TI - Expression of androgen receptors in skin appendage tumors: an immunohistochemical study. AB - Androgen receptor (AR) expression was examined in normal skin and in 52 cases of various skin appendage tumors using a monoclonal antibody (F39.4.1) raised against the N-terminal domain of human AR. Microwave oven heating in citrate buffer solution followed by immunostaining with the labeled streptavidin biotin (LSAB) method was applied to formalin-fixed paraffin-embedded sections. Immunoreactive AR was restricted to the nuclei. In normal skin, AR was consistently localized in seboblasts and in some differentiated sebocytes, and variable expression was seen in luminal epithelial cells of eccrine and apocrine glands in the secretory portion. Hair follicles and epidermis showed no reactivity. In sweat gland tumors, AR was identified focally in inner layer cells of the tubuloglandular component of ten of thirteen chondroid syringomas but the remaining tumors were nonreactive. In sebaceous gland tumors, benign tumors with mature sebaceous elements (sebaceous nevi and sebaceous adenomas) showed AR expression, but the sebaceous epitheliomas and sebaceous carcinomas lost their expression. No AR expression was observed in hair follicle tumors, except in AR positive mature sebaceous glands incorporated into the cyst wall of steatocystomas. PMID- 7560350 TI - Outcome of facial rashes with non-specific histological features: a long-term follow-up of 64 cases. AB - Facial inflammatory dermatoses can be difficult to diagnose in their early stages; skin biopsy may help but often shows indeterminate features. The aims of our study were to identify patients presenting with a facial dermatosis requiring skin biopsy, in whom the initial histological features were classified as "non specific", to determine the eventual diagnostic outcome, and to attempt to identify subtle early histologic features. We located 79 such cases with indeterminate histology from 45,000 dermatopathology reports 1972-1987. The original biopsies were assessed and scored using a check-list of 20 features and the patients' case-records up to 1992 were examined. Follow-up information was available in 64 cases and an eventual clinical diagnosis was made in 54 (84%) of these; the commonest diagnoses were rosacea (26%), chronic discoid lupus erythematosus (24%) and seborrheic or unclassified dermatitis (17%). Histological differences were seen in the original biopsies of these three diagnostic categories, although none was predictive. We conclude that clinical follow-up leads to the diagnosis of most facial rashes initially showing "non-specific" histology. PMID- 7560352 TI - Human parvovirus B19 infection: immunohistochemical and electron microscopic studies of skin lesions. AB - Erythema infectiosum is known to be caused by human parvovirus B19 and shows characteristic clinical skin manifestations in children, although adult cases of human parvovirus B19 infection do not always show such characteristic features. Recently, we experienced an epidemic adult cases of human parvovirus B19 infection and examined the erythematous skin lesion by immunohistochemistry and electron microscopy to clarify the pathogenesis of the skin manifestations. Light microscopic examination showed slightly irregular-shaped vessels in the dermis. By immunohistochemistry, using anti-human parvovirus B19 monoclonal antibody, positive reactions were found in endothelial cells. No immunoglobulins were found, but C3 deposits were present in the perivascular areas. By electron microscopy, virus particles were found in the cytoplasm of endothelial cells. An inflammatory reaction due to the direct human parvovirus B19 infection in dermal vessels seems to be an important factor in the pathogenesis of the skin manifestations. PMID- 7560351 TI - Acquired progressive lymphangioma of the skin following radiotherapy for breast carcinoma. AB - A case of a vascular tumor clinically and pathologically consistent with acquired progressive lymphangioma (benign lymphangioendothelioma) in a 48-year-old woman is reported. The lesion appeared in the skin close to a mastectomy scar 3 years after surgery and radiotherapy for invasive ductal carcinoma. On histologic examination, it mimicked an aggressive vascular neoplasm because of its infiltrative pattern. However, follow-up studies confirmed the benign nature of the lesion, clinically and histologically. This case indicates that acquired progressive lymphangioma may follow radiotherapy and must be considered in the differential diagnosis of other vascular proliferations occurring in the skin of the breast, especially of low-grade postradiation angiosarcoma, a recently described neoplastic entity. PMID- 7560353 TI - Spindle cell hemangioendothelioma: a neoplasm associated with Maffucci's syndrome. AB - A case of a 21-year-old male with Maffucci's syndrome is presented in which the patient developed multiple cutaneous spindle cell hemangioendotheliomas (SHE). Recent reports suggest an association between spindle cell hemangioendotheliomas (SHE) and Maffucci's syndrome. Our case supports the interpretation that SHE is associated with Maffucci's syndrome. PMID- 7560349 TI - Photoaging versus intrinsic aging: a morphologic assessment of facial skin. AB - Histologic studies have become increasingly important in recognizing morphologic differences in photoaged versus intrinsically aged skin. Earlier histologic studies have attempted to evaluate these changes by examining anatomical sites which are not comparable, such as face and buttocks. As part of a multicenter study, we have quantitatively examined a panel of 16 histologic features in baseline facial skin biopsies from 158 women with moderate to severe photodamage. When compared to the postauricular area (photo protected), biopsies of the crow's feet area (photo exposed) had a twofold increase in melanocytes and a statistically significant increase in melanocytic atypia (p < .0001) and epidermal melanin (p < .0001). Other epidermal changes included reduced epidermal thickness (p < .01), more compact stratum corneum (p < .0001) and increased granular layer thickness (p < .0001) in the crow's feet skin. There was increased solar elastosis (p < .0001), dermal elastic tissue (p < .0001), melanophages (p < .0001), perivascular inflammation (p < .05) and perifollicular fibrosis (p < .01) but no change in the number of mast cells or dermal mucin in the photo exposed skin. Our data document quantitative differences in photoaged versus intrinsically aged facial skin and provides the groundwork for future studies to evaluate the efficacy of new treatments for photoaged skin. PMID- 7560356 TI - Sebaceous adenoma with a cutaneous horn. AB - Cutaneous horns are common lesions that usually represent actinic keratoses, verrucae, seborrheic keratoses, and squamous cell carcinomas. We present a remarkable example of a sebaceous adenoma with cutaneous horn formation. The solitary lesion occurred over the left clavicle of a 71-year-old man without evidence of the Muir-Torre syndrome. There have been only three previous reports of sebaceous tumors (one sebaceous adenoma and two sebaceous carcinomas) producing cutaneous horns, the latter of which are actually pseudohorns, exophytic extensions of tumor without hyperkeratosis. PMID- 7560354 TI - Solitary reticulohistiocytoma in pregnancy: immunohistochemical and ultrastructural study of a case with unusual immunophenotype. AB - A case of a solitary reticulohistiocytoma during pregnancy is reported. The tumor arose on the right thigh of a 31-year-old woman during the 2nd month of gestation. Physical examination 6 months later revealed a 9 x 6 mm yellowish white papule. A biopsy showed a dermal proliferation of spindle to polygonal cells embedded in fibrous tissue with admixed neutrophils, eosinophils, and lymphocytes. Immunohistochemistry was positive for S-100, factor XIIIa, HLA-DR, KP-1, vimentin, and neuron-specific enolase. Ultrastructurally, the cells showed abundant lipid, lysozomes, and endoplasmic reticulum. No Birbeck granules could be identified. The results suggest an aberrant immunophenotype intermediate between indeterminate cells and dermal dendritic cells. The relationship of these lineages to reticulohistiocytoma and other dermal proliferative disorders is reviewed. PMID- 7560357 TI - Cystic clear cell acanthoma. AB - An histologically typical clear cell acanthoma was identified forming a cystic structure. The finding of clear cell acanthoma within a cyst is histologically unique. PMID- 7560355 TI - Massive papillary dermal fatty infiltration in a patient with psoriasis. AB - A 36-year-old Caucasian man presented with chronic, slightly erythematous, scaly plaques of 6 years duration. Some improvement was noticed after treatment with topical steroids. Differential clinical diagnoses included psoriasis and eczema. A biopsy revealed psoriasiform changes with "fatty vacuoles" replacing the papillary dermis. The histogenesis of these findings is unknown. Even though small foci of fatty vacuolization have been observed in association with heavy inflammatory infiltrates in skin, this is the first reported case of massive fatty infiltrates in a non-neoplastic disorder of the skin associated with only sparse inflammatory infiltrate.